The ACE-DD genotype is associated with endothelial dysfunction in postmenopausal women.

PubMed

Méthot, Julie; Hamelin, Bettina A; Arsenault, Marie; Bogaty, Peter; Plante, Sylvain; Poirier, Paul

2006-01-01

To evaluate the effects of the angiotensin-converting enzyme (ACE) insertion/deletion (I/D), the angiotensinogen M235T and the angiotensin II type 1 receptor A1166C polymorphisms, and hormone therapy used on endothelial function in postmenopausal women without manifestation of coronary artery disease. Sixty-four postmenopausal women (42 hormone therapy users and 22 hormone therapy nonusers) without clinical manifestation of coronary artery disease were evaluated using external vascular ultrasonography to measure endothelium-dependent (hyperemic response, flow-mediated dilatation) and -independent (nitroglycerin) dilatation. Genotypes were determined by polymerase chain reaction amplification. Women with the ACE-DD genotype displayed a lower flow-mediated dilatation compared to those with the ACE-II genotype (8.4% +/- 3.9% vs 12.6% +/- 5.4%, P = 0.04). Endothelial function was not associated with the angiotensinogen M235T and anglotensin II type 1 receptor A1166C polymorphisms. ACE polymorphism seems to modulate endothelial function among postmenopausal women without hormone therapy (8.2% +/- 5.1% vs 18.4% +/- 5.9% for the DD and the II genotype, respectively, P = 0.02). However, in hormone therapy users, flow-mediated dilatation was similar according to the ACE genotypes. Our findings suggest that ACE-I/D polymorphism is related to endothelial dysfunction in postmenopausal women. Furthermore, a potential interaction between estrogen users and ACE polymorphism on endothelial function may be present.

Protein kinases as mediators of fluid shear stress stimulated signal transduction in endothelial cells: a hypothesis for calcium-dependent and calcium-independent events activated by flow.

PubMed

Berk, B C; Corson, M A; Peterson, T E; Tseng, H

1995-12-01

Fluid shear stress regulates endothelial cell function, but the signal transduction mechanisms involved in mechanotransduction remain unclear. Recent findings demonstrate that several intracellular kinases are activated by mechanical forces. In particular, members of the mitogen-activated protein (MAP) kinase family are stimulated by hyperosmolarity, stretch, and stress such as heat shock. We propose a model for mechanotransduction in endothelial cells involving calcium-dependent and calcium-independent protein kinase pathways. The calcium-dependent pathway involves activation of phospholipase C, hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2), increases in intracellular calcium and stimulation of kinases such as calcium-calmodulin and C kinases (PKC). The calcium-independent pathway involves activation of a small GTP-binding protein and stimulation of calcium-independent PKC and MAP kinases. The calcium-dependent pathway mediates the rapid, transient response to fluid shear stress including activation of nitric oxide synthase (NOS) and ion transport. In contrast, the calcium-independent pathway mediates a slower response including the sustained activation of NOS and changes in cell morphology and gene expression. We propose that focal adhesion complexes link the calcium-dependent and calcium-independent pathways by regulating activity of phosphatidylinositol 4-phosphate (PIP) 5-kinase (which regulates PIP2 levels) and p125 focal adhesion kinase (FAK, which phosphorylates paxillin and interacts with cytoskeletal proteins). This model predicts that dynamic interactions between integrin molecules present in focal adhesion complexes and membrane events involved in mechanotransduction will be integrated by calcium-dependent and calcium-independent kinases to generate intracellular signals involved in the endothelial cell response to flow.

Apicobasal polarity of brain endothelial cells

PubMed Central

Worzfeld, Thomas

2015-01-01

Normal brain homeostasis depends on the integrity of the blood–brain barrier that controls the access of nutrients, humoral factors, and immune cells to the CNS. The blood–brain barrier is composed mainly of brain endothelial cells. Forming the interface between two compartments, they are highly polarized. Apical/luminal and basolateral/abluminal membranes differ in their lipid and (glyco-)protein composition, allowing brain endothelial cells to secrete or transport soluble factors in a polarized manner and to maintain blood flow. Here, we summarize the basic concepts of apicobasal cell polarity in brain endothelial cells. To address potential molecular mechanisms underlying apicobasal polarity in brain endothelial cells, we draw on investigations in epithelial cells and discuss how polarity may go awry in neurological diseases. PMID:26661193

Effects of Aged Stored Autologous Red Blood Cells on Human Endothelial Function

PubMed Central

Kanias, Tamir; Triulzi, Darrel; Donadee, Chenell; Barge, Suchitra; Badlam, Jessica; Jain, Shilpa; Belanger, Andrea M.; Kim-Shapiro, Daniel B.

2015-01-01

Rationale: A major abnormality that characterizes the red cell “storage lesion” is increased hemolysis and reduced red cell lifespan after infusion. Low levels of intravascular hemolysis after transfusion of aged stored red cells disrupt nitric oxide (NO) bioavailabity, via accelerated NO scavenging reaction with cell-free plasma hemoglobin. The degree of intravascular hemolysis post-transfusion and effects on endothelial-dependent vasodilation responses to acetylcholine have not been fully characterized in humans. Objectives: To evaluate the effects of blood aged to the limits of Food and Drug Administration–approved storage time on the human microcirculation and endothelial function. Methods: Eighteen healthy individuals donated 1 U of leukopheresed red cells, divided and autologously transfused into the forearm brachial artery 5 and 42 days after blood donation. Blood samples were obtained from stored blood bag supernatants and the antecubital vein of the infusion arm. Forearm blood flow measurements were performed using strain-gauge plethysmography during transfusion, followed by testing of endothelium-dependent blood flow with increasing doses of intraarterial acetylcholine. Measurements and Main Results: We demonstrate that aged stored blood has higher levels of arginase-1 and cell-free plasma hemoglobin. Compared with 5-day blood, the transfusion of 42-day packed red cells decreases acetylcholine-dependent forearm blood flows. Intravascular venous levels of arginase-1 and cell-free plasma hemoglobin increase immediately after red cell transfusion, with more significant increases observed after infusion of 42-day-old blood. Conclusions: We demonstrate that the transfusion of blood at the limits of Food and Drug Administration–approved storage has a significant effect on the forearm circulation and impairs endothelial function. Clinical trial registered with www.clinicaltrials.gov (NCT 01137656) PMID:26222884

KV7 channels contribute to paracrine, but not metabolic or ischemic, regulation of coronary vascular reactivity in swine

PubMed Central

Goodwill, Adam G.; Fu, Lijuan; Noblet, Jillian N.; Casalini, Eli D.; Berwick, Zachary C.; Kassab, Ghassan S.; Tune, Johnathan D.

2016-01-01

Hydrogen peroxide (H2O2) and voltage-dependent K+ (KV) channels play key roles in regulating coronary blood flow in response to metabolic, ischemic, and paracrine stimuli. The KV channels responsible have not been identified, but KV7 channels are possible candidates. Existing data regarding KV7 channel function in the coronary circulation (limited to ex vivo assessments) are mixed. Thus we examined the hypothesis that KV7 channels are present in cells of the coronary vascular wall and regulate vasodilation in swine. We performed a variety of molecular, biochemical, and functional (in vivo and ex vivo) studies. Coronary arteries expressed KCNQ genes (quantitative PCR) and KV7.4 protein (Western blot). Immunostaining demonstrated KV7.4 expression in conduit and resistance vessels, perhaps most prominently in the endothelial and adventitial layers. Flupirtine, a KV7 opener, relaxed coronary artery rings, and this was attenuated by linopirdine, a KV7 blocker. Endothelial denudation inhibited the flupirtine-induced and linopirdine-sensitive relaxation of coronary artery rings. Moreover, linopirdine diminished bradykinin-induced endothelial-dependent relaxation of coronary artery rings. There was no effect of intracoronary flupirtine or linopirdine on coronary blood flow at the resting heart rate in vivo. Linopirdine had no effect on coronary vasodilation in vivo elicited by ischemia, H2O2, or tachycardia. However, bradykinin increased coronary blood flow in vivo, and this was attenuated by linopirdine. These data indicate that KV7 channels are expressed in some coronary cell type(s) and influence endothelial function. Other physiological functions of coronary vascular KV7 channels remain unclear, but they do appear to contribute to endothelium-dependent responses to paracrine stimuli. PMID:26825518

KV7 channels contribute to paracrine, but not metabolic or ischemic, regulation of coronary vascular reactivity in swine.

PubMed

Goodwill, Adam G; Fu, Lijuan; Noblet, Jillian N; Casalini, Eli D; Sassoon, Daniel; Berwick, Zachary C; Kassab, Ghassan S; Tune, Johnathan D; Dick, Gregory M

2016-03-15

Hydrogen peroxide (H2O2) and voltage-dependent K(+) (KV) channels play key roles in regulating coronary blood flow in response to metabolic, ischemic, and paracrine stimuli. The KV channels responsible have not been identified, but KV7 channels are possible candidates. Existing data regarding KV7 channel function in the coronary circulation (limited to ex vivo assessments) are mixed. Thus we examined the hypothesis that KV7 channels are present in cells of the coronary vascular wall and regulate vasodilation in swine. We performed a variety of molecular, biochemical, and functional (in vivo and ex vivo) studies. Coronary arteries expressed KCNQ genes (quantitative PCR) and KV7.4 protein (Western blot). Immunostaining demonstrated KV7.4 expression in conduit and resistance vessels, perhaps most prominently in the endothelial and adventitial layers. Flupirtine, a KV7 opener, relaxed coronary artery rings, and this was attenuated by linopirdine, a KV7 blocker. Endothelial denudation inhibited the flupirtine-induced and linopirdine-sensitive relaxation of coronary artery rings. Moreover, linopirdine diminished bradykinin-induced endothelial-dependent relaxation of coronary artery rings. There was no effect of intracoronary flupirtine or linopirdine on coronary blood flow at the resting heart rate in vivo. Linopirdine had no effect on coronary vasodilation in vivo elicited by ischemia, H2O2, or tachycardia. However, bradykinin increased coronary blood flow in vivo, and this was attenuated by linopirdine. These data indicate that KV7 channels are expressed in some coronary cell type(s) and influence endothelial function. Other physiological functions of coronary vascular KV7 channels remain unclear, but they do appear to contribute to endothelium-dependent responses to paracrine stimuli. Copyright © 2016 the American Physiological Society.

Endothelial and kidney function in women with a history of preeclampsia and healthy parous controls: A case control study.

PubMed

Lopes van Balen, Veronica A; Spaan, Julia J; Cornelis, Tom; Heidema, Wieteke M; Scholten, Ralph R; Spaanderman, Marc E A

2018-03-01

Preeclampsia (PE) is a pregnancy related endothelial disease characterized by hypertension and albuminuria. Postpartum endothelial dysfunction often persists in these women. We postulate that in women with a history of PE reduced endothelial dependent vasodilation coincides with attenuated kidney function, as both reflect endothelial dysfunction. We assessed endothelial and kidney function in women with a history of PE (n=79) and uncomplicated pregnancies (n=49) at least 4years postpartum. Women with hypertension, diabetes or kidney disease prior to pregnancy were excluded. Brachial artery flow mediated dilatation (FMD) was measured and analysed by a custom designed edge-detection and wall-tracking software. We measured albumin and creatinine levels in a 24-h urine sample and calculated glomerular filtration rate (GFR) by CKD-EPI. Women with a history of PE had lower FMD but comparable GFR and albumin creatinine ratio (ACR) compared with controls. Independent of obstetric history, in both controls and women with a history of PE respectively, GFR (r=0.19, p=0.17 and r=0.12, p=0.29) and albumin creatinine ratio (r=0.07, p=0.62 and r=0.06 p=0.57) did not correlate with FMD. At least 4years after pregnancy, women with a history of PE demonstrated decreased flow mediated dilatation when compared to healthy parous controls. In this study, decreased flow mediated dilation however did not coincide with decreased kidney function. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Selective endothelin ETA and dual ET(A)/ET(B) receptor blockade improve endothelium-dependent vasodilatation in patients with type 2 diabetes and coronary artery disease.

PubMed

Rafnsson, Arnar; Shemyakin, Alexey; Pernow, John

2014-11-24

Endothelin-1 contributes to endothelial dysfunction in patients with atherosclerosis and type 2 diabetes. In healthy arteries the ETA receptor mediates the main part of the vasoconstriction induced by endothelin-1 whilst the ETB receptor mediates vasodilatation. The ETB receptor expression is upregulated on vascular smooth muscle cells in atherosclerosis and may contribute to the increased vasoconstrictor tone and endothelial dysfunction observed in this condition. Due to these opposing effects of the ETB receptor it remains unclear whether ETB blockade together with ETA blockade may be detrimental or beneficial. The aim was therefore to compare the effects of selective ETA and dual ETA/ETB blockade on endothelial function in patients with type 2 diabetes and coronary artery disease. Forearm endothelium-dependent and endothelium-independent vasodilatation was assessed by venous occlusion plethysmography in 12 patients before and after selective ETA or dual ETA/ETB receptor blockade. Dual ETA/ETB receptor blockade increased baseline forearm blood flow by 30±14% (P<0.01) whereas selective ETA blockade did not (14±8%). Both selective ETA blockade and dual ETA/ETB blockade significantly improved endothelium-dependent vasodilatation. The improvement did not differ between the two treatments. There was also an increase in endothelium-independent vasodilatation with both treatments. Dual ETA/ETB blockade did not significantly increase microvascular flow but improved transcutaneous pO2. Both selective ETA and dual ETA/ETB improve endothelium-dependent vasodilatation in patients with type 2 diabetes and coronary artery disease. ETB blockade increases basal blood flow but does not additionally improve endothelium-dependent vasodilatation. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Analysis of temporal shear stress gradients during the onset phase of flow over a backward-facing step.

PubMed

Haidekker, M A; White, C R; Frangos, J A

2001-10-01

Endothelial cells in blood vessels are exposed to bloodflow and thus fluid shear stress. In arterial bifurcations and stenoses, disturbed flow causes zones of recirculation and stagnation, which are associated with both spatial and temporal gradients of shear stress. Such gradients have been linked to the generation of atherosclerotic plaques. For in-vitro studies of endothelial cell responses, the sudden-expansion flow chamber has been widely used and described. A two-dimensional numerical simulation of the onset phase of flow through the chamber was performed. The wall shear stress action on the bottom plate was computed as a function of time and distance from the sudden expansion. The results showed that depending on the time for the flow to be established, significant temporal gradients occurred close to the second stagnation point of flow. Slowly ramping the flow over 15 s instead of 200 ms reduces the temporal gradients by a factor of 300, while spatial gradients are reduced by 23 percent. Thus, the effects of spatial and temporal gradients can be observed separately. In experiments on endothelial cells, disturbed flow stimulated cell proliferation only when flow onset was sudden. The spatial patterns of proliferation rate match the exposure to temporal gradients. This study provides information on the dynamics of spatial and temporal gradients to which the cells are exposed in a sudden-expansion flow chamber and relates them to changes in the onset phase of flow.

DD angiotensin-converting enzyme gene polymorphism is associated with endothelial dysfunction in normal humans.

PubMed

Butler, R; Morris, A D; Burchell, B; Struthers, A D

1999-05-01

A polymorphism within the angiotensin-converting enzyme (ACE) gene may increase the risk of myocardial infarction in individuals previously thought to be at low cardiovascular risk. The mechanism through which it exerts this effect is unknown but may be due to increased angiotensin II-induced nitric oxide (NO) breakdown and/or reduced bradykinin-mediated NO release. We investigated whether endothelial function was different between different ACE genotypes. We performed a cross-sectional study comparing the endothelial function of the 3 genotypes (II: n=25; ID: n=31; DD: n=12). Mean+/-SD ages of the subjects were 24+/-4 (II), 25+/-6 (ID), and 25+/-6 (DD) years. We assessed the impact of the genotypes on endothelial function and found that the DD genotype was associated with a significant blunting in endothelial-dependent vasodilatation (forearm blood flow data are presented as mean+/-SD ratio of blood flow in response to 3 incrementally increasing doses of each vasoactive agent in the test arm to blood flow in the control arm; the comparison is between DD versus ID versus II; the P value is an expression of an overall difference by ANOVA, and the 95% CIs are of a pairwise comparison between genotypes): acetylcholine, 2.88+/-1.45 versus 3.81+/-1.93 versus 4.23+/-2.37 (P=0.002; 95% CI [II versus ID], -0.19 to 0.91; 95% CI [II versus DD], 0.36 to 1.80; 95% CI [ID versus DD], 0.02 to 1.42). There was also a significant difference with the endothelial-independent vasodilator sodium nitroprusside, with values of 2.11+/-1.00 versus 2.55+/-1.36 versus 2.75+/-1.18 (P<0.05; 95% CI [II versus ID], -0.15 to 0.51; 95% CI [II versus DD], 0.03 to 0.89; 95% CI [ID versus DD], -0.13 to 0.71), but not with verapamil. There was no effect of the ACE genotype on endothelial-dependent or -independent vasoconstrictors NG-monomethyl-L-arginine or norepinephrine. Investigating the effects of cigarette smoking on each genotype demonstrated that for II and DD genotypes, acetylcholine responses were further blunted if subjects smoked. These data demonstrate that the DD ACE genotype in a young population is associated with a blunting of stimulated endothelial NO and donated NO responses but not to non-NO vasodilators or vasoconstrictors.

[Role of endothelium-derived nitric oxide in sustained flow-dependent dilatation of human peripheral conduit arteries].

PubMed

Bellien, J; Joannidès, R; Iacob, M; Eltchaninoff, H; Thuillez, Ch

2003-01-01

Endothelial dysfunction is involved in the pathogenesis of cardiovascular diseases and is generally associated to the decrease in arterial nitric oxide (NO) availability. In humans, endothelial function can be evaluated by the post-ischaemic flow-dependent dilatation (FDD) of peripheral conduit arteries which is mainly mediated by the NO release when short duration of reactive hyperaemia are used (3 to 5 min ischaemia). However, recent studies suggest that the role of NO in this response decreases as the duration of the hyperaemic stimulation increases. The aim of the present study was thus, to evaluate, in healthy subjects, the role of NO in the FDD of conduct arteries in response to a sustained stimulation. Radial artery diameter (echotracking) and flow (Doppler) were measured, 7 cm under the elbow line, at baseline and during post-ischaemic hyperaemia (10 min wrist cuff inflation) in 10 healthy subjects (age: 24 +/- 1 years) in control period and after acute blockade of the endothelial NO-synthase by local infusion of NG-monomethyl L-arginine (L-NMMA, brachial artery, 8 mumol/min, 7 min). Endothelium-independent dilatation was studied by mean of sodium nitroprusside infusion (SNP: 5, 10 and 20 nmol/min, 3 min each dose before and after L-NMMA). L-NMMA administration decreased radial artery blood flow at base (Control: 14 +/- 2 vs L-NMMA: 10 +/- 1 ml/min, P < 0.05) and increased radial artery vasodilatation in response to SNP (P < 0.05) thus, demonstrating NO-synthase inhibition. Therefore, after L-NMMA there was a small decrease in radial FDD (Control: base: 2.52 +/- 0.05 mm, FDD: 11.3 +/- 0.6% vs L-NMMA: base: 2.51 +/- 0.04 mm: FDD: 9.0 +/- 0.9%; p < 0.05) without change in hyperaemia. In conclusion, our results demonstrate, in contrast to those obtained after short duration of hyperaemia, that the relative implication of NO in the flow-dependent vasodilatation of peripheral conduit arteries in humans decreases in response to sustained stimulation and suggest, in these experimental conditions, an associated flow-dependent vasodilating mechanism that is unaffected by the NO-synthase inhibition.

In situ tissue engineering: endothelial growth patterns as a function of flow diverter design.

PubMed

Marosfoi, Miklos; Langan, Erin T; Strittmatter, Lara; van der Marel, Kajo; Vedantham, Srinivasan; Arends, Jennifer; Lylyk, Ivan R; Loganathan, Siddharth; Hendricks, Gregory M; Szikora, Istvan; Puri, Ajit S; Wakhloo, Ajay K; Gounis, Matthew J

2017-10-01

Vascular remodeling in response to implantation of a tissue engineering scaffold such as a flow diverter (FD) leads to the cure of intracranial aneurysms. We hypothesize that the vascular response is dependent on FD design, and CD34+ progenitor cells play an important role in the endothelialization of the implant. Sixteen rabbit aneurysms were randomly treated with two different single-layer braided FDs made of cobalt-chrome alloys. The FD-48 and FD-72 devices had 48 and 72 wires, respectively. Aneurysm occlusion rate was assessed during the final digital subtraction angiogram at 10, 20, 30, and 60 days (n=2 per device per time point). Implanted vessels were analyzed with scanning electron microscopy for tissue coverage, endothelialization, and immuno-gold labeling for CD34+ cells. Complete aneurysm occlusion rates were similar between the devices; however, complete or near complete occlusion was more frequently observed in aneurysms with neck ≤4.2 mm (p=0.008). Total tissue coverage at 10 days over the surface of the FD-48 and FD-72 devices was 56.4±11.6% and 76.6±3.6%, respectively. Endothelial cell growth over the surface was time-dependent for the FD-72 device (Spearman's r=0.86, p=0.013) but not for the FD-48 device (Spearman's r=-0.59, p=0.094). The endothelialization score was marginally correlated with the distance from the aneurysm neck for the FD-48 device (Spearman's r=1, p=0.083) but not for the FD-72 device (Spearman's r=0.8, p=0.33). CD34+ cells were present along the entirety of both devices at all time points. This study gives preliminary evidence that temporal and spatial endothelialization is dependent on FD design. Circulating CD34+ progenitor cells contribute to endothelialization throughout the healing process. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Age-related endothelial dysfunction in human skeletal muscle feed arteries: the role of free radicals derived from mitochondria in the vasculature.

PubMed

Park, S-Y; Kwon, O S; Andtbacka, R H I; Hyngstrom, J R; Reese, V; Murphy, M P; Richardson, R S

2018-01-01

This study sought to determine the role of free radicals derived from mitochondria in the vasculature in the recognized age-related endothelial dysfunction of human skeletal muscle feed arteries (SMFAs). A total of 44 SMFAs were studied with and without acute exposure to the mitochondria-targeted antioxidant MitoQ and nitric oxide synthase (NOS) blockade. The relative abundance of proteins from the electron transport chain, phosphorylated (p-) to endothelial (e) NOS ratio, manganese superoxide dismutase (MnSOD) and the mitochondria-derived superoxide (O2-) levels were assessed in SMFA. Endothelium-dependent and endothelium-independent SMFA vasodilation was assessed in response to flow-induced shear stress, acetylcholine (ACh) and sodium nitroprusside (SNP). MitoQ restored endothelium-dependent vasodilation in the old to that of the young when stimulated by both flow (young: 68 ± 5; old: 25 ± 7; old + MitoQ 65 ± 9%) and ACh (young: 97 ± 4; old: 59 ± 10; old + MitoQ: 98 ± 5%), but did not alter the initially uncompromised, endothelium-independent vasodilation (SNP). Compared to the young, MitoQ in the old diminished the initially elevated mitochondria-derived O2- levels and appeared to attenuate the breakdown of MnSOD. Furthermore, MitoQ increased the ratio of p-eNOS to NOS and the restoration of endothelium-dependent vasodilation in the old by MitoQ was ablated by NOS blockade. This study demonstrated that MitoQ reverses age-related vascular dysfunction by what appears to be an NO-dependent mechanism in human SMFAs. These findings suggest that mitochondria-targeted antioxidants may have utility in terms of counteracting the attenuated blood flow and vascular dysfunction associated with advancing age. © 2017 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

Elevated 20-HETE impairs coronary collateral growth in metabolic syndrome via endothelial dysfunction.

PubMed

Joseph, Gregory; Soler, Amanda; Hutcheson, Rebecca; Hunter, Ian; Bradford, Chastity; Hutcheson, Brenda; Gotlinger, Katherine H; Jiang, Houli; Falck, John R; Proctor, Spencer; Schwartzman, Michal Laniado; Rocic, Petra

2017-03-01

Coronary collateral growth (CCG) is impaired in metabolic syndrome (MetS). microRNA-145 (miR-145-Adv) delivery to our rat model of MetS (JCR) completely restored and neutrophil depletion significantly improved CCG. We determined whether low endogenous levels of miR-145 in MetS allowed for elevated production of 20-hydroxyeicosatetraenoic acid (20-HETE), which, in turn, resulted in excessive neutrophil accumulation and endothelial dysfunction leading to impaired CCG. Rats underwent 0-9 days of repetitive ischemia (RI). RI-induced cardiac CYP4F (neutrophil-specific 20-HETE synthase) expression and 20-HETE levels were increased (4-fold) in JCR vs. normal rats. miR-145-Adv and 20-HETE antagonists abolished and neutrophil depletion (blocking antibodies) reduced (~60%) RI-induced increases in CYP4F expression and 20-HETE production in JCR rats. Impaired CCG in JCR rats (collateral-dependent blood flow using microspheres) was completely restored by 20-HETE antagonists [collateral-dependent zone (CZ)/normal zone (NZ) flow ratio was 0.76 ± 0.07 in JCR + 20-SOLA, 0.84 ± 0.05 in JCR + 20-HEDGE vs. 0.11 ± 0.02 in JCR vs. 0.84 ± 0.03 in normal rats]. In JCR rats, elevated 20-HETE was associated with excessive expression of endothelial adhesion molecules and neutrophil infiltration, which were reversed by miR-145-Adv. Endothelium-dependent vasodilation of coronary arteries, endothelial nitric oxide synthase (eNOS) Ser1179 phosphorylation, eNOS-dependent NO ·- production and endothelial cell survival were compromised in JCR rats. These parameters of endothelial dysfunction were completely reversed by 20-HETE antagonism or miR-145-Adv delivery, whereas neutrophil depletion resulted in partial reversal (~70%). We conclude that low miR-145 in MetS allows for increased 20-HETE, mainly from neutrophils, which compromises endothelial cell survival and function leading to impaired CCG. 20-HETE antagonists could provide viable therapy for restoration of CCG in MetS. NEW & NOTEWORTHY Elevated 20-hydroxyeicosatetraenoic acid (20-HETE) impairs coronary collateral growth (CCG) in metabolic syndrome by eliciting endothelial dysfunction and apoptosis via excessive neutrophil infiltration. 20-HETE antagonists completely restore coronary collateral growth in metabolic syndrome. microRNA-145 (miR-145) is an upstream regulator of 20-HETE production in metabolic syndrome; low expression of miR-145 in metabolic syndrome promotes elevated production of 20-HETE. Copyright © 2017 the American Physiological Society.

Non-canonical Wnt signalling modulates the endothelial shear stress flow sensor in vascular remodelling

PubMed Central

Franco, Claudio A; Jones, Martin L; Bernabeu, Miguel O; Vion, Anne-Clemence; Barbacena, Pedro; Fan, Jieqing; Mathivet, Thomas; Fonseca, Catarina G; Ragab, Anan; Yamaguchi, Terry P; Coveney, Peter V; Lang, Richard A; Gerhardt, Holger

2016-01-01

Endothelial cells respond to molecular and physical forces in development and vascular homeostasis. Deregulation of endothelial responses to flow-induced shear is believed to contribute to many aspects of cardiovascular diseases including atherosclerosis. However, how molecular signals and shear-mediated physical forces integrate to regulate vascular patterning is poorly understood. Here we show that endothelial non-canonical Wnt signalling regulates endothelial sensitivity to shear forces. Loss of Wnt5a/Wnt11 renders endothelial cells more sensitive to shear, resulting in axial polarization and migration against flow at lower shear levels. Integration of flow modelling and polarity analysis in entire vascular networks demonstrates that polarization against flow is achieved differentially in artery, vein, capillaries and the primitive sprouting front. Collectively our data suggest that non-canonical Wnt signalling stabilizes forming vascular networks by reducing endothelial shear sensitivity, thus keeping vessels open under low flow conditions that prevail in the primitive plexus. DOI: http://dx.doi.org/10.7554/eLife.07727.001 PMID:26845523

Hypoxic pulmonary vasoconstriction requires connexin 40–mediated endothelial signal conduction

PubMed Central

Wang, Liming; Yin, Jun; Nickles, Hannah T.; Ranke, Hannes; Tabuchi, Arata; Hoffmann, Julia; Tabeling, Christoph; Barbosa-Sicard, Eduardo; Chanson, Marc; Kwak, Brenda R.; Shin, Hee-Sup; Wu, Songwei; Isakson, Brant E.; Witzenrath, Martin; de Wit, Cor; Fleming, Ingrid; Kuppe, Hermann; Kuebler, Wolfgang M.

2012-01-01

Hypoxic pulmonary vasoconstriction (HPV) is a physiological mechanism by which pulmonary arteries constrict in hypoxic lung areas in order to redirect blood flow to areas with greater oxygen supply. Both oxygen sensing and the contractile response are thought to be intrinsic to pulmonary arterial smooth muscle cells. Here we speculated that the ideal site for oxygen sensing might instead be at the alveolocapillary level, with subsequent retrograde propagation to upstream arterioles via connexin 40 (Cx40) endothelial gap junctions. HPV was largely attenuated by Cx40-specific and nonspecific gap junction uncouplers in the lungs of wild-type mice and in lungs from mice lacking Cx40 (Cx40–/–). In vivo, hypoxemia was more severe in Cx40–/– mice than in wild-type mice. Real-time fluorescence imaging revealed that hypoxia caused endothelial membrane depolarization in alveolar capillaries that propagated to upstream arterioles in wild-type, but not Cx40–/–, mice. Transformation of endothelial depolarization into vasoconstriction involved endothelial voltage-dependent α1G subtype Ca2+ channels, cytosolic phospholipase A2, and epoxyeicosatrienoic acids. Based on these data, we propose that HPV originates at the alveolocapillary level, from which the hypoxic signal is propagated as endothelial membrane depolarization to upstream arterioles in a Cx40-dependent manner. PMID:23093775

EGCG protects against homocysteine-induced human umbilical vein endothelial cells apoptosis by modulating mitochondrial-dependent apoptotic signaling and PI3K/Akt/eNOS signaling pathways.

PubMed

Liu, Shumin; Sun, Zhengwu; Chu, Peng; Li, Hailong; Ahsan, Anil; Zhou, Ziru; Zhang, Zonghui; Sun, Bin; Wu, Jingjun; Xi, Yalin; Han, Guozhu; Lin, Yuan; Peng, Jinyong; Tang, Zeyao

2017-05-01

Homocysteine (Hcy) induced vascular endothelial injury leads to the progression of endothelial dysfunction in atherosclerosis. Epigallocatechin gallate (EGCG), a natural dietary antioxidant, has been applied to protect against atherosclerosis. However, the underlying protective mechanism of EGCG has not been clarified. The present study investigated the mechanism of EGCG protected against Hcy-induced human umbilical vein endothelial cells (HUVECs) apoptosis. Methyl thiazolyl tetrazolium assay (MTT), transmission electron microscope, fluorescent staining, flow cytometry, western blot were used in this study. The study has demonstrated that EGCG suppressed Hcy-induced endothelial cell morphological changes and reactive oxygen species (ROS) generation. Moreover, EGCG dose-dependently prevented Hcy-induced HUVECs cytotoxicity and apoptotic biochemical changes such as reducing mitochondrial membrane potential (MMP), decreasing Bcl-2/Bax protein ratio and activating caspase-9 and 3. In addition, EGCG enhanced the protein ratio of p-Akt/Akt, endothelial nitric oxide synthase (eNOS) activation and nitric oxide (NO) formation in injured cells. In conclusion, the present study shows that EGCG prevents Hcy-induced HUVECs apoptosis via modulating mitochondrial apoptotic and PI3K/Akt/eNOS signaling pathways. Furthermore, the results indicate that EGCG is likely to represent a potential therapeutic strategy for atherosclerosis associated with Hyperhomocysteinemia (HHcy).

Exercise training improves in vivo endothelial repair capacity of early endothelial progenitor cells in subjects with metabolic syndrome.

PubMed

Sonnenschein, Kristina; Horváth, Tibor; Mueller, Maja; Markowski, Andrea; Siegmund, Tina; Jacob, Christian; Drexler, Helmut; Landmesser, Ulf

2011-06-01

Endothelial dysfunction and injury are considered to contribute considerably to the development and progression of atherosclerosis. It has been suggested that intense exercise training can increase the number and angiogenic properties of early endothelial progenitor cells (EPCs). However, whether exercise training stimulates the capacity of early EPCs to promote repair of endothelial damage and potential underlying mechanisms remain to be determined. The present study was designed to evaluate the effects of moderate exercise training on in vivo endothelial repair capacity of early EPCs, and their nitric oxide and superoxide production as characterized by electron spin resonance spectroscopy analysis in subjects with metabolic syndrome. Twenty-four subjects with metabolic syndrome were randomized to an 8 weeks exercise training or a control group. Superoxide production and nitric oxide (NO) availability of early EPCs were characterized by using electron spin resonance (ESR) spectroscopy analysis. In vivo endothelial repair capacity of EPCs was examined by transplantation into nude mice with defined carotid endothelial injury. Endothelium-dependent, flow-mediated vasodilation was analysed using high-resolution ultrasound. Importantly, exercise training resulted in a substantially improved in vivo endothelial repair capacity of early EPCs (24.0 vs 12.7%; p < 0.05) and improved endothelium-dependent vasodilation. Nitric oxide production of EPCs was substantially increased after exercise training, but not in the control group. Moreover, exercise training reduced superoxide production of EPCs, which was not observed in the control group. The present study suggests for the first time that moderate exercise training increases nitric oxide production of early endothelial progenitor cells and reduces their superoxide production. Importantly, this is associated with a marked beneficial effect on the in vivo endothelial repair capacity of early EPCs in subjects with metabolic syndrome.

Endothelial dysfunction in patients with obstructive sleep apnoea independent of metabolic syndrome.

PubMed

Amra, Babak; Karbasi, Elaheh; Hashemi, Mohammad; Hoffmann-Castendiek, Birgit; Golshan, Mohammad

2009-05-01

Obstructive sleep apnoea syndrome (OSAS), characterised by intermittent hypoxia/re-oxygenation, has been identified as an independent risk factor for cardiovascular diseases and endothelial dysfunction. Our aim was to investigate flow-mediated dilatation (FMD) in patients with obstructive sleep apnoea with and without metabolic syndrome. Fifty-two subjects with OSAS diagnosed by polysomnography were classified into 2 groups according to the presence and absence of the metabolic syndrome and also according to the severity: mild to moderate OSAS group and severe OSAS group. Endothelial function of the brachial artery was evaluated by using high-resolution vascular ultrasound. Endothelial-dependent dilatation (EDD) was assessed by establishing reactive hyperaemia and endothelial-independent dilatation (EID) was determined by using sublingual isosorbide dinitrate. Spearman correlation and regression analysis were performed. EDD was not significantly different in patients with OSAS and metabolic syndrome as compared with OSAS without metabolic syndrome (4.62 +/- 0.69 versus 4.49 +/- 0.93, P >0.05). Endothelial dysfunction in OSA may be independent of metabolic syndrome.

Endothelial Cell Autonomous Role of Akt1: Regulation of Vascular Tone and Ischemia-Induced Arteriogenesis.

PubMed

Lee, Monica Y; Gamez-Mendez, Ana; Zhang, Jiasheng; Zhuang, Zhenwu; Vinyard, David J; Kraehling, Jan; Velazquez, Heino; Brudvig, Gary W; Kyriakides, Themis R; Simons, Michael; Sessa, William C

2018-04-01

The importance of PI3K/Akt signaling in the vasculature has been demonstrated in several models, as global loss of Akt1 results in impaired postnatal ischemia- and VEGF-induced angiogenesis. The ubiquitous expression of Akt1, however, raises the possibility of cell-type-dependent Akt1-driven actions, thereby necessitating tissue-specific characterization. Herein, we used an inducible, endothelial-specific Akt1-deleted adult mouse model (Akt1iECKO) to characterize the endothelial cell autonomous functions of Akt1 in the vascular system. Endothelial-targeted ablation of Akt1 reduces eNOS (endothelial nitric oxide synthase) phosphorylation and promotes both increased vascular contractility in isolated vessels and elevated diastolic blood pressures throughout the diurnal cycle in vivo. Furthermore, Akt1iECKO mice subject to the hindlimb ischemia model display impaired blood flow and decreased arteriogenesis. Endothelial Akt1 signaling is necessary for ischemic resolution post-injury and likely reflects the consequence of NO insufficiency critical for vascular repair. © 2018 American Heart Association, Inc.

Shear stress reduces protease activated receptor-1 expression in human endothelial cells

NASA Technical Reports Server (NTRS)

Nguyen, K. T.; Eskin, S. G.; Patterson, C.; Runge, M. S.; McIntire, L. V.

2001-01-01

Shear stress has been shown to regulate several genes involved in the thrombotic and proliferative functions of endothelial cells. Thrombin receptor (protease-activated receptor-1: PAR-1) increases at sites of vascular injury, which suggests an important role for PAR-1 in vascular diseases. However, the effect of shear stress on PAR-1 expression has not been previously studied. This work investigates effects of shear stress on PAR-1 gene expression in both human umbilical vein endothelial cells (HUVECs) and microvascular endothelial cells (HMECs). Cells were exposed to different shear stresses using a parallel plate flow system. Northern blot and flow cytometry analysis showed that shear stress down-regulated PAR-1 messenger RNA (mRNA) and protein levels in both HUVECs and HMECs but with different thresholds. Furthermore, shear-reduced PAR-1 mRNA was due to a decrease of transcription rate, not increased mRNA degradation. Postshear stress release of endothelin-1 in response to thrombin was reduced in HUVECs and HMECs. Moreover, inhibitors of potential signaling pathways applied during shear stress indicated mediation of the shear-decreased PAR-1 expression by protein kinases. In conclusion, shear stress exposure reduces PAR-1 gene expression in HMECs and HUVECs through a mechanism dependent in part on protein kinases, leading to altered endothelial cell functional responses to thrombin.

Coordinated Endothelial Nitric Oxide Synthase activation by translocation and phosphorylation determines flow-induced NO production in resistance vessels

PubMed Central

Figueroa, Xavier F.; González, Daniel R.; Puebla, Mariela; Acevedo, Juan P.; Rojas-Libano, Daniel; Durán, Walter N.; Boric, Mauricio P.

2013-01-01

Background/Aims Endothelial nitric oxide synthase (eNOS) is associated with caveolin-1 (Cav-1) in plasma membrane. We tested the hypothesis that eNOS activation by shear stress in resistance vessels depends on synchronized phosphorylation, dissociation from Cav-1 and translocation of the membrane-bound enzyme to Golgi and cytosol. Methods In isolated, perfused rat arterial mesenteric beds, we evaluated the effect of changes in flow rate (2–10 mL/min), on NO production, eNOS phosphorylation at serine 1177, eNOS subcellular distribution and co-immunoprecipitation with Cav-1, in the presence or absence of extracellular Ca2+. Results Increases in flow induced a biphasic rise in NO production: a rapid transient phase (3–5-min) that peaked during the first 15-sec, followed by a sustained phase, which lasted until the end of stimulation. Concomitantly, flow caused a rapid translocation of eNOS from the microsomal compartment to the cytosol and Golgi, paralleled by an increase in eNOS phosphorylation and a reduction in eNOS-Cav-1 association. Transient NO production, eNOS translocation, and dissociation from Cav-1 depended on extracellular Ca2+, while sustained NO production was abolished by the PI3K-Akt blocker wortmannin. Conclusions In intact resistance vessels, changes in flow induce NO production by transient Ca2+-dependent eNOS translocation from membrane to intracellular compartments and sustained Ca2+-independent PI3K-Akt-mediated phosphorylation. PMID:24217770

Cilostazol improves high glucose-induced impaired angiogenesis in human endothelial progenitor cells and vascular endothelial cells as well as enhances vasculoangiogenesis in hyperglycemic mice mediated by the adenosine monophosphate-activated protein kinase pathway.

PubMed

Tseng, Shih-Ya; Chao, Ting-Hsing; Li, Yi-Heng; Liu, Ping-Yen; Lee, Cheng-Han; Cho, Chung-Lung; Wu, Hua-Lin; Chen, Jyh-Hong

2016-04-01

Cilostazol is an antiplatelet agent with vasodilatory effects that works by increasing intracellular concentrations of cyclic adenosine monophosphate (cAMP). This study investigated the effects of cilostazol in preventing high glucose (HG)-induced impaired angiogenesis and examined the potential mechanisms involving activation of AMP-activated protein kinase (AMPK). Assays for colony formation, adhesion, proliferation, migration, and vascular tube formation were used to determine the effect of cilostazol in HG-treated endothelial progenitor cells (EPCs) or human umbilical vein endothelial cells (HUVECs). Animal-based assays were performed in hyperglycemic ICR mice undergoing hind limb ischemia. An immnunoblotting assay was used to identify the expression and activation of signaling molecules in vitro and in vivo. Cilostazol treatment significantly restored endothelial function in EPCs and HUVECs through activation of AMPK/acetyl-coenzyme A carboxylase (ACC)-dependent pathways and cAMP/protein kinase A (PKA)-dependent pathways. Recovery of blood flow in the ischemic hind limb and the population of circulating CD34(+) cells were significantly improved in cilostazol-treated mice, and these effects were abolished by local AMPK knockdown. Cilostazol increased the phosphorylation of AMPK/ACC and Akt/endothelial nitric oxide synthase signaling molecules in parallel with or downstream of the cAMP/PKA-dependent signaling pathway in vitro and in vivo. Cilostazol prevents HG-induced endothelial dysfunction in EPCs and HUVECs and enhances angiogenesis in hyperglycemic mice by interactions with a broad signaling network, including activation of AMPK/ACC and probably cAMP/PKA pathways. Copyright © 2016 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.

What is going on between defibrotide and endothelial cells? Snapshots reveal the hot spots of their romance

PubMed Central

Mir, Enrique; Rovira, Montse; Escolar, Ginés; Carreras, Enric; Diaz-Ricart, Maribel

2016-01-01

Defibrotide (DF) has received European Medicines Agency authorization to treat sinusoidal obstruction syndrome, an early complication after hematopoietic cell transplantation. DF has a recognized role as an endothelial protective agent, although its precise mechanism of action remains to be elucidated. The aim of the present study was to investigate the interaction of DF with endothelial cells (ECs). A human hepatic EC line was exposed to different DF concentrations, previously labeled. Using inhibitory assays and flow cytometry techniques along with confocal microscopy, we explored: DF-EC interaction, endocytic pathways, and internalization kinetics. Moreover, we evaluated the potential role of adenosine receptors in DF-EC interaction and if DF effects on endothelium were dependent of its internalization. Confocal microscopy showed interaction of DF with EC membranes followed by internalization, though DF did not reach the cell nucleus even after 24 hours. Flow cytometry revealed concentration, temperature, and time dependent uptake of DF in 2 EC models but not in other cell types. Moreover, inhibitory assays indicated that entrance of DF into ECs occurs primarily through macropinocytosis. Our experimental approach did not show any evidence of the involvement of adenosine receptors in DF-EC interaction. The antiinflammatory and antioxidant properties of DF seem to be caused by the interaction of the drug with the cell membrane. Our findings contribute to a better understanding of the precise mechanisms of action of DF as a therapeutic and potential preventive agent on the endothelial damage underlying different pathologic situations. PMID:26755708

A novel role of thrombopoietin as a physiological modulator of coronary flow.

PubMed

Ramella, Roberta; Gallo, Maria Pia; Spatola, Tiziana; Lupia, Enrico; Alloatti, Giuseppe

2011-02-25

Thrombopoietin (TPO) is known for its ability to stimulate platelet production. However, little is currently known whether TPO plays a physiological function in the heart. The potential vasodilatory role of TPO was tested on the isolated rat heart. The expression of TPO receptor (c-mpl) and the TPO-dependent eNOS phosphorylation (P(Ser1179)) were studied on Cardiac-derived normal Human Micro Vascular Endothelial Cells (HMVEC-C) by Western blot analysis. While TPO (10-200 pg/mL) did not modify coronary flow (CF) under basal conditions, it reduced the coronary constriction caused by endothelin-1 (ET-1; 10nM) in a dose-dependent manner. This effect was blocked by both Wortmannin (100 nM) and L-NAME (100 nM); on HMVEC-C, TPO induced eNOS phosphorylation through a Wortmannin sensitive mechanism. Taken together, our data suggest a potential role of TPO as a physiological regulator of CF. By acting on specific receptors present on endothelial cells, TPO may induce PI3K/Akt-dependent eNOS phosphorylation and NO release. Copyright © 2011 Elsevier B.V. All rights reserved.

The chemokine receptor CX3CR1 coordinates monocyte recruitment and endothelial regeneration after arterial injury.

PubMed

Getzin, Tobias; Krishnasamy, Kashyap; Gamrekelashvili, Jaba; Kapanadze, Tamar; Limbourg, Anne; Häger, Christine; Napp, L Christian; Bauersachs, Johann; Haller, Hermann; Limbourg, Florian P

2018-02-01

Regeneration of arterial endothelium after injury is critical for the maintenance of normal blood flow, cell trafficking, and vascular function. Using mouse models of carotid injury, we show that the transition from a static to a dynamic phase of endothelial regeneration is marked by a strong increase in endothelial proliferation, which is accompanied by induction of the chemokine CX 3 CL1 in endothelial cells near the wound edge, leading to progressive recruitment of Ly6C lo monocytes expressing high levels of the cognate CX 3 CR1 chemokine receptor. In Cx3cr1 -deficient mice recruitment of Ly6C lo monocytes, endothelial proliferation and regeneration of the endothelial monolayer after carotid injury are impaired, which is rescued by acute transfer of normal Ly6C lo monocytes. Furthermore, human non-classical monocytes induce proliferation of endothelial cells in co-culture experiments in a VEGFA-dependent manner, and monocyte transfer following carotid injury promotes endothelial wound closure in a hybrid mouse model in vivo Thus, CX 3 CR1 coordinates recruitment of specific monocyte subsets to sites of endothelial regeneration, which promote endothelial proliferation and arterial regeneration. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Substrate stiffness-dependent exacerbation of endothelial permeability and inflammation: mechanisms and potential implications in ALI and PH (2017 Grover Conference Series)

PubMed Central

Karki, Pratap; Birukova, Anna A.

2018-01-01

The maintenance of endothelial barrier integrity is absolutely essential to prevent the vascular leak associated with pneumonia, pulmonary edema resulting from inhalation of toxins, acute elevation to high altitude, traumatic and septic lung injury, acute lung injury (ALI), and its life-threatening complication, acute respiratory distress syndrome (ARDS). In addition to the long-known edemagenic and inflammatory agonists, emerging evidences suggest that factors of endothelial cell (EC) mechanical microenvironment such as blood flow, mechanical strain of the vessel, or extracellular matrix stiffness also play an essential role in the control of endothelial permeability and inflammation. Recent studies from our group and others have demonstrated that substrate stiffening causes endothelial barrier disruption and renders EC more susceptible to agonist-induced cytoskeletal rearrangement and inflammation. Further in vivo studies have provided direct evidence that proinflammatory stimuli increase lung microvascular stiffness which in turn exacerbates endothelial permeability and inflammation and perpetuates a vicious circle of lung inflammation. Accumulating evidence suggests a key role for RhoA GTPases signaling in stiffness-dependent mechanotransduction mechanisms defining EC permeability and inflammatory responses. Vascular stiffening is also known to be a key contributor to other cardiovascular diseases such as arterial pulmonary hypertension (PH), although the precise role of stiffness in the development and progression of PH remains to be elucidated. This review summarizes the current understanding of stiffness-dependent regulation of pulmonary EC permeability and inflammation, and discusses potential implication of pulmonary vascular stiffness alterations at macro- and microscale in development and modulation of ALI and PH. PMID:29714090

Short-term increases in pressure and shear stress attenuate age-related declines in endothelial function in skeletal muscle feed arteries.

PubMed

Seawright, John W; Luttrell, Meredith; Trache, Andreea; Woodman, Christopher R

2016-07-01

We tested the hypothesis that exposure to a short-term (1 h) increase in intraluminal pressure and shear stress (SS), to mimic two mechanical signals associated with a bout of exercise, improves nitric oxide (NO)-mediated endothelium-dependent dilation in aged soleus muscle feed arteries (SFA). In addition, we hypothesized that pressure and SS would interact to produce greater improvements in endothelial function than pressure alone. SFA from young (4 months) and old (24 months) Fischer 344 rats were cannulated and pressurized at 90 (P90) or 130 (P130) cmH2O and exposed to no SS (0 dyn/cm(2)) or high SS (~65 dyn/cm(2)) for 1 h. At the end of the 1 h treatment period, pressure in all P130 SFA was set to 90 cmH2O and no SS (0 dyn/cm(2)) for examination of endothelium-dependent [flow and acetylcholine (ACh)] and endothelium-independent [sodium nitroprusside (SNP)] dilation. To evaluate the contribution of NO, vasodilator responses were assessed in the presence of N(ω)-nitro- l -arginine (L-NNA). Flow- and ACh-induced dilations were impaired in Old P90 SFA. Treatment with increased pressure + SS for 1 h improved flow- and ACh-induced dilations in old SFA. The beneficial effect of pressure + SS was abolished in the presence of L-NNA and was not greater than treatment with increased pressure alone. These results indicate that short-duration increases in pressure + SS improve NO-mediated endothelium-dependent dilation in aged SFA; however, pressure and SS do not interact to produce greater improvements in endothelial function than pressure alone.

Macro- and microscale variables regulate stent haemodynamics, fibrin deposition and thrombomodulin expression

PubMed Central

Jiménez, Juan M.; Prasad, Varesh; Yu, Michael D.; Kampmeyer, Christopher P.; Kaakour, Abdul-Hadi; Wang, Pei-Jiang; Maloney, Sean F.; Wright, Nathan; Johnston, Ian; Jiang, Yi-Zhou; Davies, Peter F.

2014-01-01

Drug eluting stents are associated with late stent thrombosis (LST), delayed healing and prolonged exposure of stent struts to blood flow. Using macroscale disturbed and undisturbed fluid flow waveforms, we numerically and experimentally determined the effects of microscale model strut geometries upon the generation of prothrombotic conditions that are mediated by flow perturbations. Rectangular cross-sectional stent strut geometries of varying heights and corresponding streamlined versions were studied in the presence of disturbed and undisturbed bulk fluid flow. Numerical simulations and particle flow visualization experiments demonstrated that the interaction of bulk fluid flow and stent struts regulated the generation, size and dynamics of the peristrut flow recirculation zones. In the absence of endothelial cells, deposition of thrombin-generated fibrin occurred primarily in the recirculation zones. When endothelium was present, peristrut expression of anticoagulant thrombomodulin (TM) was dependent on strut height and geometry. Thinner and streamlined strut geometries reduced peristrut flow recirculation zones decreasing prothrombotic fibrin deposition and increasing endothelial anticoagulant TM expression. The studies define physical and functional consequences of macro- and microscale variables that relate to thrombogenicity associated with the most current stent designs, and particularly to LST. PMID:24554575

Peroxynitrite Disrupts Endothelial Caveolae Leading to eNOS Uncoupling and Diminished Flow-Mediated Dilation in Coronary Arterioles of Diabetic Patients

PubMed Central

Cassuto, James; Dou, Huijuan; Czikora, Istvan; Szabo, Andras; Patel, Vijay S.; Kamath, Vinayak; Belin de Chantemele, Eric; Feher, Attila; Romero, Maritza J.; Bagi, Zsolt

2014-01-01

Peroxynitrite (ONOO−) contributes to coronary microvascular dysfunction in diabetes mellitus (DM). We hypothesized that in DM, ONOO− interferes with the function of coronary endothelial caveolae, which plays an important role in nitric oxide (NO)-dependent vasomotor regulation. Flow-mediated dilation (FMD) of coronary arterioles was investigated in DM (n = 41) and non-DM (n = 37) patients undergoing heart surgery. NO-mediated coronary FMD was significantly reduced in DM patients, which was restored by ONOO− scavenger, iron-(III)-tetrakis(N-methyl-4'pyridyl)porphyrin-pentachloride, or uric acid, whereas exogenous ONOO− reduced FMD in non-DM subjects. Immunoelectron microscopy demonstrated an increased 3-nitrotyrosine formation (ONOO−-specific protein nitration) in endothelial plasma membrane in DM, which colocalized with caveolin-1 (Cav-1), the key structural protein of caveolae. The membrane-localized Cav-1 was significantly reduced in DM and also in high glucose–exposed coronary endothelial cells. We also found that DM patients exhibited a decreased number of endothelial caveolae, whereas exogenous ONOO− reduced caveolae number. Correspondingly, pharmacological (methyl-β-cyclodextrin) or genetic disruption of caveolae (Cav-1 knockout mice) abolished coronary FMD, which was rescued by sepiapterin, the stable precursor of NO synthase (NOS) cofactor, tetrahydrobiopterin. Sepiapterin also restored coronary FMD in DM patients. Thus, we propose that ONOO− selectively targets and disrupts endothelial caveolae, which contributes to NOS uncoupling, and, hence, reduced NO-mediated coronary vasodilation in DM patients. PMID:24353182

In vitro effects of ATG-Fresenius on immune cell adhesion.

PubMed

Kanzler, I; Seitz-Merwald, I; Schleger, S; Kaczmarek, I; Kur, F; Beiras-Fernandez, A

2013-06-01

ATG-Fresenius, a purified rabbit polyclonal anti-human T-lymphocyte immunoglobulin is used for induction immunosuppression as well as prevention and treatment of acute rejection episodes among patients receiving solid organ transplants. The aim of this study was to investigate the in vitro activity of ATG-Fresenius upon immune cell adhesion, which may explain its activity to mitigate ischemia-reperfusion injury. Human vascular endothelial cells (HUVEC) and peripheral blood mononuclear cells (PBMCs) isolated from umbilical vein or peripheral blood were incubated 20 to 24 hours before analysis. HUVEC were incubated with 10 and 100 μg/mL ATG-Fresenius or reference polyclonal rabbit immunoglobulin G. Analysis of immune cell adhesion to endothelial cells was studied in cocultures of PBMCs and adherent HUVEC. Endothelial cell expression of adhesion molecules CD62E and CD54 was determined by flow cytometry. The numbers of T-, B- and natural killer cells attached to HUVEC were also determined by flow cytometry. Groups were compared using one-way analysis of variance. We showed that ATG-Fresenius binds to endothelial cells particularly activated ones expressing increased levels of E-selectin and ICAM-1. The increased binding of ATG-Fresenius to activated endothelial cells was consistent with its known binding to Intercellular Adhesion Molecule 1 (ICAM-1) and selectins. We also showed that ATG-Fresenius inhibited adhesion of prestimulated immune cells to activated endothelium. We demonstrated dose-dependent binding of ATG-Fresenius to activated endothelial cells. Copyright © 2013 Elsevier Inc. All rights reserved.

Flow-mediated dilation and cardiovascular event prediction: does nitric oxide matter?

PubMed

Green, Daniel J; Jones, Helen; Thijssen, Dick; Cable, N T; Atkinson, Greg

2011-03-01

Endothelial dysfunction is an early atherosclerotic event that precedes clinical symptoms and may also render established plaque vulnerable to rupture. Noninvasive assessment of endothelial function is commonly undertaken using the flow-mediated dilation (FMD) technique. Some studies indicate that FMD possesses independent prognostic value to predict future cardiovascular events that may exceed that associated with traditional risk factor assessment. It has been assumed that this association is related to the proposal that FMD provides an index of endothelium-derived nitric oxide (NO) function. Interestingly, placement of the occlusion cuff during the FMD procedure alters the shear stress stimulus and NO dependency of the resulting dilation: cuff placement distal to the imaged artery leads to a largely NO-mediated response, whereas proximal cuff placement leads to dilation which is less NO dependent. We used this physiological observation and the knowledge that prognostic studies have used both approaches to examine whether the prognostic capacity of FMD is related to its role as a putative index of NO function. In a meta-analysis of 14 studies (>8300 subjects), we found that FMD derived using a proximal cuff was at least as predictive as that derived using distal cuff placement, despite the latter being more NO dependent. This suggests that, whilst FMD is strongly predictive of future cardiovascular events, this may not solely be related to its assumed NO dependency. Although this finding should be confirmed with more and larger studies, we suggest that any direct measure of vascular (endothelial) function may provide independent prognostic information in humans.

Visualization of three pathways for macromolecule transport across cultured endothelium and their modification by flow.

PubMed

Ghim, Mean; Alpresa, Paola; Yang, Sung-Wook; Braakman, Sietse T; Gray, Stephen G; Sherwin, Spencer J; van Reeuwijk, Maarten; Weinberg, Peter D

2017-11-01

Transport of macromolecules across vascular endothelium and its modification by fluid mechanical forces are important for normal tissue function and in the development of atherosclerosis. However, the routes by which macromolecules cross endothelium, the hemodynamic stresses that maintain endothelial physiology or trigger disease, and the dependence of transendothelial transport on hemodynamic stresses are controversial. We visualized pathways for macromolecule transport and determined the effect on these pathways of different types of flow. Endothelial monolayers were cultured under static conditions or on an orbital shaker producing different flow profiles in different parts of the wells. Fluorescent tracers that bound to the substrate after crossing the endothelium were used to identify transport pathways. Maps of tracer distribution were compared with numerical simulations of flow to determine effects of different shear stress metrics on permeability. Albumin-sized tracers dominantly crossed the cultured endothelium via junctions between neighboring cells, high-density lipoprotein-sized tracers crossed at tricellular junctions, and low-density lipoprotein-sized tracers crossed through cells. Cells aligned close to the angle that minimized shear stresses across their long axis. The rate of paracellular transport under flow correlated with the magnitude of these minimized transverse stresses, whereas transport across cells was uniformly reduced by all types of flow. These results contradict the long-standing two-pore theory of solute transport across microvessel walls and the consensus view that endothelial cells align with the mean shear vector. They suggest that endothelial cells minimize transverse shear, supporting its postulated proatherogenic role. Preliminary data show that similar tracer techniques are practicable in vivo. NEW & NOTEWORTHY Solutes of increasing size crossed cultured endothelium through intercellular junctions, through tricellular junctions, or transcellularly. Cells aligned to minimize the shear stress acting across their long axis. Paracellular transport correlated with the level of this minimized shear, but transcellular transport was reduced uniformly by flow regardless of the shear profile. Copyright © 2017 the American Physiological Society.

Parallel-plate Flow Chamber and Continuous Flow Circuit to Evaluate Endothelial Progenitor Cells under Laminar Flow Shear Stress

PubMed Central

Lane, Whitney O.; Jantzen, Alexandra E.; Carlon, Tim A.; Jamiolkowski, Ryan M.; Grenet, Justin E.; Ley, Melissa M.; Haseltine, Justin M.; Galinat, Lauren J.; Lin, Fu-Hsiung; Allen, Jason D.; Truskey, George A.; Achneck, Hardean E.

2012-01-01

The overall goal of this method is to describe a technique to subject adherent cells to laminar flow conditions and evaluate their response to well quantifiable fluid shear stresses1. Our flow chamber design and flow circuit (Fig. 1) contains a transparent viewing region that enables testing of cell adhesion and imaging of cell morphology immediately before flow (Fig. 11A, B), at various time points during flow (Fig. 11C), and after flow (Fig. 11D). These experiments are illustrated with human umbilical cord blood-derived endothelial progenitor cells (EPCs) and porcine EPCs2,3. This method is also applicable to other adherent cell types, e.g. smooth muscle cells (SMCs) or fibroblasts. The chamber and all parts of the circuit are easily sterilized with steam autoclaving. In contrast to other chambers, e.g. microfluidic chambers, large numbers of cells (> 1 million depending on cell size) can be recovered after the flow experiment under sterile conditions for cell culture or other experiments, e.g. DNA or RNA extraction, or immunohistochemistry (Fig. 11E), or scanning electron microscopy5. The shear stress can be adjusted by varying the flow rate of the perfusate, the fluid viscosity, or the channel height and width. The latter can reduce fluid volume or cell needs while ensuring that one-dimensional flow is maintained. It is not necessary to measure chamber height between experiments, since the chamber height does not depend on the use of gaskets, which greatly increases the ease of multiple experiments. Furthermore, the circuit design easily enables the collection of perfusate samples for analysis and/or quantification of metabolites secreted by cells under fluid shear stress exposure, e.g. nitric oxide (Fig. 12)6. PMID:22297325

Crucial importance of the endothelial K+ channel SK3 and connexin40 in arteriolar dilations during skeletal muscle contraction.

PubMed

Milkau, Malte; Köhler, Ralf; de Wit, Cor

2010-09-01

Skeletal muscle activity requires substantial increases in blood flow, and the underlying vasodilation involves endothelial activity, but the contribution of the endothelium-dependent hyperpolarizing factor (EDHF) is only poorly defined. In EDHF signaling, endothelial hyperpolarization mediated by the Ca(2+)-activated K(+) channels SK3 and IK1 is a key step and also initiates gap junction-dependent conducted dilations. We assessed the role of SK3, IK1, and connexin40 (Cx40) in muscular contraction-induced dilations in the microcirculation in vivo. Hitherto, arterioles were observed in the electrically stimulated cremaster skeletal muscle of anesthetized mice lacking SK3, IK1, or Cx40 using intravital microscopy. Genetic deficiency of SK3, but not of IK1, strongly attenuated dilations to muscular contraction. Similarly, pharmacologic blockade of SK3 by the specific blocker UCL1684 impaired such dilations in wild-type and IK1-deficient mice. In contrast, IK1 was required for acetylcholine-induced dilations. Genetic deficiency of Cx40 also attenuated dilations induced by muscular contraction but not by acetylcholine. These data support the concept that endothelial hyperpolarization through activation of SK3 contributes to exercise hyperemia and the hyperpolarization ascends the vascular tree through gap junctions formed by Cx40 to orchestrate dilation. The differential impact of SK3- and IK1-deficiency on dilations to distinct stimuli suggests stimulus-dependent activation of these endothelial channels.

Mechanisms of Amplified Arteriogenesis in Collateral Artery Segments Exposed to Flow Direction Reversal

PubMed Central

Heuslein, Joshua L.; Meisner, Joshua K.; Li, Xuanyue; Song, Ji; Vincentelli, Helena; Leiphart, Ryan J.; Ames, Elizabeth G.; Price, Richard J.

2015-01-01

Objective Collateral arteriogenesis, the growth of existing arterial vessels to a larger diameter, is a fundamental adaptive response that is often critical for the perfusion and survival of tissues downstream of chronic arterial occlusion(s). Shear stress regulates arteriogenesis; however, the arteriogenic significance of flow direction reversal, occurring in numerous collateral artery segments after femoral artery ligation (FAL), is unknown. Our objective was to determine if flow direction reversal in collateral artery segments differentially regulates endothelial cell signaling and arteriogenesis. Approach and Results Collateral segments experiencing flow reversal after FAL in C57BL/6 mice exhibit increased pericollateral macrophage recruitment, amplified arteriogenesis (30% diameter and 2.8-fold conductance increases), and remarkably permanent (12 weeks post-FAL) remodeling. Genome-wide transcriptional analyses on HUVECs exposed to flow reversal conditions mimicking those occurring in-vivo yielded 10-fold more significantly regulated transcripts, as well as enhanced activation of upstream regulators (NFκB, VEGF, FGF2, TGFβ) and arteriogenic canonical pathways (PKA, PDE, MAPK). Augmented expression of key pro-arteriogenic molecules (KLF2, ICAM-1, eNOS) was also verified by qRT-PCR, leading us to test whether ICAM-1 and/or eNOS regulate amplified arteriogenesis in flow-reversed collateral segments in-vivo. Interestingly, enhanced pericollateral macrophage recruitment and amplified arteriogenesis was attenuated in flow-reversed collateral segments after FAL in ICAM-1−/− mice; however, eNOS−/− mice showed no such differences. Conclusions Flow reversal leads to a broad amplification of pro-arteriogenic endothelial signaling and a sustained ICAM-1-dependent augmentation of arteriogenesis. Further investigation of the endothelial mechanotransduction pathways activated by flow reversal may lead to more effective and durable therapeutic options for arterial occlusive diseases. PMID:26338297

Mechanotransduction Effects on Endothelial Cell Proliferation via CD31 and VEGFR2: Implications for Immunomagnetic Separation.

PubMed

Mahajan, Kalpesh D; Nabar, Gauri M; Xue, Wei; Anghelina, Mirela; Moldovan, Nicanor I; Chalmers, Jeffrey J; Winter, Jessica O

2017-09-01

Immunomagnetic separation is used to isolate circulating endothelial cells (ECs) and endothelial progenitor cells (EPCs) for diagnostics and tissue engineering. However, potentially detrimental changes in cell properties have been observed post-separation. Here, the effect of mechanical force, which is naturally applied during immunomagnetic separation, on proliferation of human umbilical vein endothelial cells (HUVEC), kinase insert domain-positive receptor (KDR) cells, and peripheral blood mononuclear cells (PBMCs). Cells are exposed to CD31 or Vascular Endothelial Growth Factor Receptor-2 (VEGFR2) targeted MACSi beads at varying bead to cell ratios and compared to free antibody and unconjugated beads. A vertical magnetic gradient is applied to static 2D cultures, and a magnetic cell sorter is used to analyze cells in dynamic flow. No significant difference in EC proliferation is observed for controls or VEGFR2-targeting beads, whereas CD31-conjugated beads increase proliferation in a dose dependent manner in static 2-D cultures. This effect occurs in the absence of magnetic field, but is more pronounced with magnetic force. After flow sorting, similar increases in proliferation are seen for CD31 targeting beads. Thus, the effects of targeting antibody and magnetic force applied should be considered when designing immunomagnetic separation protocols for ECs. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Abrupt reflow enhances cytokine-induced proinflammatory activation of endothelial cells during simulated shock and resuscitation.

PubMed

Li, Ranran; Zijlstra, Jan G; Kamps, Jan A A M; van Meurs, Matijs; Molema, Grietje

2014-10-01

Circulatory shock and resuscitation are associated with systemic hemodynamic changes, which may contribute to the development of MODS (multiple organ dysfunction syndrome). In this study, we used an in vitro flow system to simulate the consecutive changes in blood flow as occurring during hemorrhagic shock and resuscitation in vivo. We examined the kinetic responses of different endothelial genes in human umbilical vein endothelial cells preconditioned to 20 dyne/cm unidirectional laminar shear stress for 48 h to flow cessation and abrupt reflow, respectively, as well as the effect of flow cessation and reflow on tumor necrosis factor-α (TNF-α)-induced endothelial proinflammatory activation. Endothelial CD31 and VE-cadherin were not affected by the changes in flow in the absence or presence of TNF-α. The messenger RNA levels of proinflammatory molecules E-selectin, VCAM-1 (vascular cell adhesion molecule 1), and IL-8 (interleukin 8) were significantly induced by flow cessation respectively acute reflow, whereas ICAM-1 (intercellular adhesion molecule 1) was downregulated on flow cessation and induced by subsequent acute reflow. Flow cessation also affected the Ang/Tie2 (Angiopoietin/Tie2 receptor tyrosine kinase) system by downregulating Tie2 and inducing its endothelial ligand Ang2, an effect that was further extended on acute reflow. Furthermore, the induction of proinflammatory adhesion molecules by TNF-α under flow cessation was significantly enhanced on subsequent acute reflow. This study demonstrated that flow alterations per se during shock and resuscitation contribute to endothelial activation and that these alterations interact with proinflammatory factors coexisting in vivo such as TNF-α. The abrupt reflow-related enhancement of cytokine-induced endothelial proinflammatory activation supports the concept that sudden regain of flow during resuscitation has an aggravating effect on endothelial activation, which may play a significant role in vascular dysfunction and consequent organ injury. This study implies that the improvement of resuscitation strategies and the pharmacological interference with proinflammatory signaling cascades at the right time of resuscitation of shock patients may be beneficial to regain and/or maintain organ function in patients after circulatory shock.

Age-related changes in endothelial function and blood flow regulation.

PubMed

Toda, Noboru

2012-02-01

Vascular endothelial dysfunction is regarded as a primary phenotypic expression of normal human aging. This senescence-induced disorder is the likely culprit underlying the increased cardiovascular and metabolic disease risks associated with aging. The rate of this age-dependent deterioration is largely influenced by the poor-quality lifestyle choice, such as smoking, sedentary daily life, chronic alcohol ingestion, high salt intake, unbalanced diet, and mental stress; and it is accelerated by cardiovascular and metabolic diseases. Although minimizing these detrimental factors is the best course of action, nonetheless chronological age steadily impairs endothelial function through reduced endothelial nitric oxide synthase (eNOS) expression/action, accelerated nitric oxide (NO) degradation, increased phosphodiesterase activity, inhibition of NOS activity by endogenous NOS inhibitors, increased production of reactive oxygen species, inflammatory reactions, decreased endothelial progenitor cell number and function, and impaired telomerase activity or telomere shortening. Endothelial dysfunction in regional vasculatures results in cerebral hypoperfusion triggering cognitive dysfunction and Alzheimer's disease, coronary artery insufficiency, penile erectile dysfunction, and circulatory failures in other organs and tissues. Possible prophylactic measures to minimize age-related endothelial dysfunction are also summarized in this review. Copyright © 2011 Elsevier Inc. All rights reserved.

Human endothelial progenitor cells-derived exosomes accelerate cutaneous wound healing in diabetic rats by promoting endothelial function.

PubMed

Li, Xiaocong; Jiang, Chunyu; Zhao, Jungong

2016-08-01

Wound healing is deeply dependent on neovascularization to restore blood flow. The neovascularization of endothelial progenitor cells (EPCs) through paracrine secretion has been reported in various tissue repair models. Exosomes, key components of cell paracrine mechanism, have been rarely reported in wound healing. Exosomes were isolated from the media of EPCs obtained from human umbilical cord blood. Diabetic rats wound model was established and treated with exosomes. The in vitro effects of exosomes on the proliferation, migration and angiogenic tubule formation of endothelial cells were investigated. We revealed that human umbilical cord blood EPCs derived exosomes transplantation could accelerate cutaneous wound healing in diabetic rats. We also showed that exosomes enhanced the proliferation, migration and tube formation of vascular endothelial cells in vitro. Furthermore, we found that endothelial cells stimulated with these exosomes would increase expression of angiogenesis-related molecules, including FGF-1, VEGFA, VEGFR-2, ANG-1, E-selectin, CXCL-16, eNOS and IL-8. Taken together, our findings indicated that EPCs-derived exosomes facilitate wound healing by positively modulating vascular endothelial cells function. Copyright © 2016 Elsevier Inc. All rights reserved.

What is going on between defibrotide and endothelial cells? Snapshots reveal the hot spots of their romance.

PubMed

Palomo, Marta; Mir, Enrique; Rovira, Montse; Escolar, Ginés; Carreras, Enric; Diaz-Ricart, Maribel

2016-03-31

Defibrotide (DF) has received European Medicines Agency authorization to treat sinusoidal obstruction syndrome, an early complication after hematopoietic cell transplantation. DF has a recognized role as an endothelial protective agent, although its precise mechanism of action remains to be elucidated. The aim of the present study was to investigate the interaction of DF with endothelial cells (ECs). A human hepatic EC line was exposed to different DF concentrations, previously labeled. Using inhibitory assays and flow cytometry techniques along with confocal microscopy, we explored: DF-EC interaction, endocytic pathways, and internalization kinetics. Moreover, we evaluated the potential role of adenosine receptors in DF-EC interaction and if DF effects on endothelium were dependent of its internalization. Confocal microscopy showed interaction of DF with EC membranes followed by internalization, though DF did not reach the cell nucleus even after 24 hours. Flow cytometry revealed concentration, temperature, and time dependent uptake of DF in 2 EC models but not in other cell types. Moreover, inhibitory assays indicated that entrance of DF into ECs occurs primarily through macropinocytosis. Our experimental approach did not show any evidence of the involvement of adenosine receptors in DF-EC interaction. The antiinflammatory and antioxidant properties of DF seem to be caused by the interaction of the drug with the cell membrane. Our findings contribute to a better understanding of the precise mechanisms of action of DF as a therapeutic and potential preventive agent on the endothelial damage underlying different pathologic situations. © 2016 by The American Society of Hematology.

Effects of High Intensity Training and High Volume Training on Endothelial Microparticles and Angiogenic Growth Factors

PubMed Central

Achtzehn, Silvia; Schmitz, Theresa; Bloch, Wilhelm; Mester, Joachim; Werner, Nikos

2014-01-01

Aims Endothelial microparticles (EMP) are complex vesicular structures shed from activated or apoptotic endothelial cells. As endurance exercise affects the endothelium, the objective of the study was to examine levels of EMP and angiogenic growth factors following different endurance exercise protocols. Methods 12 subjects performed 3 different endurance exercise protocols: 1. High volume training (HVT; 130 min at 55% peak power output (PPO); 2. 4×4 min at 95% PPO; 3. 4×30 sec all-out. EMPs were quantified using flow cytometry after staining platelet-poor-plasma. Events positive for Annexin-V and CD31, and negative for CD42b, were classified as EMPs. Vascular endothelial growth factor (VEGF), migratory inhibiting factor (MIF) and hepatocyte growth factor (HGF) were determined by ELISA technique. For all these measurements venous blood samples were taken pre, 0′, 30′, 60′ and 180′ after each intervention. Furthermore, in vitro experiments were performed to explore the effect of collected sera on target endothelial functions and MP uptake capacities. Results VEGF and HGF significantly increased after HIT interventions. All three interventions caused a significant decrease in EMP levels post exercise compared to pre values. The sera taken after exercise increased the uptake of EMP in target endothelial cells compared to sera taken under resting conditions, which was shown to be phosphatidylserin-dependent. Increased EMP uptake was associated with an improved protection of target cells against apoptosis. Sera taken prior and after exercise promoted target endothelial cell migration, which was abrogated after inhibition of VEGF. Conclusion Physical exercise leads to decreased EMP levels and promotes a phosphatidylserin-dependent uptake of EMP into target endothelial cells, which is associated with a protection of target cells against apoptosis. PMID:24770423

Dietary sodium restriction reverses vascular endothelial dysfunction in middle-aged/older adults with moderately elevated systolic blood pressure

PubMed Central

Jablonski, Kristen L.; Racine, Matthew L.; Geolfos, Candace J.; Gates, Phillip E.; Chonchol, Michel; McQueen, Matthew B.; Seals, Douglas R.

2013-01-01

Objectives We determined the efficacy of dietary sodium restriction (DSR) for improving vascular endothelial dysfunction in middle-aged/older adults with moderately elevated systolic blood pressure (SBP; 130–159 mmHg) and the associated physiological mechanisms. Background Vascular endothelial dysfunction develops with advancing age and elevated SBP, contributing to increased cardiovascular risk. DSR lowers BP, but its effect on vascular endothelial function and mechanisms involved are unknown. Methods Seventeen subjects (11M/6F; 62±7 yrs, mean±S.D.) completed a randomized, crossover study of 4 weeks of both low and normal sodium intake. Vascular endothelial function (endothelium-dependent dilation; EDD), nitric oxide (NO)/tetrahydrobiopterin (BH4) bioavailability and oxidative stress-associated mechanisms were assessed following each condition. Results Urinary sodium excretion was reduced by ~50% (to 70±30 mmol/day), and conduit (brachial artery flow-mediated dilation [FMDBA]) and resistance (forearm blood flow responses to acetylcholine [FBFACh]) artery EDD were 68% and 42% (peak FBFACh) higher following the low sodium diet (p<0.005). Low sodium markedly enhanced NO- mediated EDD (greater ΔFBFACh with endothelial NO synthase [eNOS] inhibition) without changing eNOS expression/activation (Ser1177 phosphorylation), restored BH4 bioactivity (less ΔFMDBA with acute BH4), abolished tonic superoxide suppression of EDD (less ΔFMDBA and ΔFBFACh with ascorbic acid infusion), and increased circulating superoxide dismutase activity (p<0.05). These effects were independent of ΔSBP. Other subject characteristics/dietary factors and endothelium-independent dilation were unchanged. Conclusions DSR largely reverses both macro- and microvascular endothelial dysfunction by enhancing NO and BH4 bioavailability and reducing oxidative stress. Our findings support the emerging concept that DSR induces “vascular protection” beyond that attributable to its BP-lowering effects. PMID:23141486

In vitro and in vivo assessment of cellular permeability and pharmacodynamics of S-nitrosylated Captopril, a nitric oxide donor

PubMed Central

Jia, Lee; Wong, Hong

2001-01-01

The present studies were aimed at testing the hypothesis that S-nitrosylated captopril (CapNO), a novel crystalline nitric oxide (NO) donor, readily permeates both in vitro and in vivo endothelial monolayers, resulting in its pharmacodynamic effects. CapNO and Captopril (Cap) were added to apical side of endothelial monolayers formed on microporous membranes, and the permeated drugs were collected from basolateral side and detected by a HPLC method. The permeability coefficient (Papp; cm sec−1) of CapNO across the endothelial monolayers was 6.0×10−5, higher than that of Cap (3.13×10−5), indicating the enhancement effect of the attached NO group in CapNO on cellular permeability. The Papp of CapNO and Cap across Caco-2 cells were 3.15×10−5 and 1.53×10−5, respectively. The low Papp of CapNO to Caco-2 cells may be attributed to the high membrane resistance of Caco-2 cells. A bolus injection of CapNO to epicardial coronary artery of chronically-instrumented awake dogs caused significant increases in coronary blood flow and coronary diameters dose-dependently without significant changes in aortic pressure. In contrast, the equimolar doses of Cap did not produce haemodynamic responses. Intravenous CapNO caused an instant increase in the regional cerebral blood flow determined by H2-clearance, whereas the equimolar doses of Cap did not enhance the cerebral blood flow. These results conclude that the NO group, an active component of CapNO, enhances both in vitro and in vivo endothelial permeability to the entire compound, resulting in instant increases in blood flow and vascular diameters. In contrast, the equimolar Cap does not have the instant vascular effects. PMID:11739246

In vitro and in vivo assessment of cellular permeability and pharmacodynamics of S-nitrosylated captopril, a nitric oxide donor.

PubMed

Jia, L; Wong, H

2001-12-01

1. The present studies were aimed at testing the hypothesis that S-nitrosylated captopril (CapNO), a novel crystalline nitric oxide (NO) donor, readily permeates both in vitro and in vivo endothelial monolayers, resulting in its pharmacodynamic effects. 2. CapNO and Captopril (Cap) were added to apical side of endothelial monolayers formed on microporous membranes, and the permeated drugs were collected from basolateral side and detected by a HPLC method. The permeability coefficient (P(app); cm sec(-1)) of CapNO across the endothelial monolayers was 6.0 x 10(-5), higher than that of Cap (3.13 x 10(-5)), indicating the enhancement effect of the attached NO group in CapNO on cellular permeability. The P(app) of CapNO and Cap across Caco-2 cells were 3.15 x 10(-5) and 1.53 x 10(-5), respectively. The low P(app) of CapNO to Caco-2 cells may be attributed to the high membrane resistance of Caco-2 cells. 3. A bolus injection of CapNO to epicardial coronary artery of chronically-instrumented awake dogs caused significant increases in coronary blood flow and coronary diameters dose-dependently without significant changes in aortic pressure. In contrast, the equimolar doses of Cap did not produce haemodynamic responses. 4. Intravenous CapNO caused an instant increase in the regional cerebral blood flow determined by H(2)-clearance, whereas the equimolar doses of Cap did not enhance the cerebral blood flow. 5. These results conclude that the NO group, an active component of CapNO, enhances both in vitro and in vivo endothelial permeability to the entire compound, resulting in instant increases in blood flow and vascular diameters. In contrast, the equimolar Cap does not have the instant vascular effects.

Detergent sclerosants at sub-lytic concentrations induce endothelial cell apoptosis through a caspase dependent pathway.

PubMed

Cooley-Andrade, Osvaldo; Cheung, Kelvin; Chew, An-Ning; Connor, David Ewan; Parsi, Kurosh

2016-07-01

To investigate the apoptotic effects of detergent sclerosants sodium tetradecylsulphate (STS) and polidocanol (POL) on endothelial cells at sub-lytic concentrations. Human umbilical vein endothelial cells (HUVECs) were isolated and labelled with antibodies to assess for apoptosis and examined with confocal microscopy and flow cytometry. Isolated HUVECs viability was assessed using propidium iodide staining. Early apoptosis was determined by increased phosphatidylserine exposure by lactadherin binding. Caspase 3, 8, 9 and Bax activation as well as inhibitory assays with Pan Caspase (Z-VAD-FMK) and Bax (BI-6C9) were assessed to identify apoptotic pathways. Porimin activation was used to assess cell membrane permeability. Cell lysis reached almost 100 % with STS at 0.3 % and with POL at 0.6 %. Apoptosis was seen with both STS and POL at concentrations ranging from 0.075 to 0.15 %. PS exposure increased with both STS and POL and exhibited a dose-dependent trend. Active Caspase 3, 8 and 9 but not Bax were increased in HUVECs stimulated with low concentrations of both STS and POL. Inhibitory assays demonstrated Caspase 3, 8, 9 inhibition at low concentrations (0.075 to 0.6 %) with both STS and POL. Both agents increased the activation of porimin at all concentrations. Both sclerosants induced endothelial cell (EC) apoptosis at sub-lytic concentrations through a caspase-dependant pathway. Both agents induced EC oncosis.

Comparison of skin microvascular reactivity with hemostatic markers of endothelial dysfunction and damage in type 2 diabetes

PubMed Central

Beer, Sandra; Feihl, François; Ruiz, Juan; Juhan-Vague, Irène; Aillaud, Marie-Françoise; Wetzel, Sandrine Golay; Liaudet, Lucas; Gaillard, Rolf C; Waeber, Bernard

2008-01-01

Aim: Patients with non-insulin-dependent diabetes mellitus (NIDDM) are at increased cardiovascular risk due to an accelerated atherosclerotic process. The present study aimed to compare skin microvascular function, pulse wave velocity (PWV), and a variety of hemostatic markers of endothelium injury [von Willebrand factor (vWF), plasminogen activator inhibitor-1 (PAI-1), tissue plasminogen activator (t-PA), tissue factor pathway inhibitor (TFPI), and the soluble form of thrombomodulin (s-TM)] in patients with NIDDM. Methods: 54 patients with NIDDM and 38 sex- and age-matched controls were studied. 27 diabetics had no overt micro- and/or macrovascular complications, while the remainder had either or both. The forearm skin blood flow was assessed by laser-Doppler imaging, which allowed the measurement of the response to iontophoretically applied acetylcholine (endothelium-dependent vasodilation) and sodium nitroprusside (endothelium-independent vasodilation), as well as the reactive hyperemia triggered by the transient occlusion of the circulation. Results: Both endothelial and non-endothelial reactivity were significantly blunted in diabetics, regardless of the presence or the absence of vascular complications. Plasma vWF, TFPI and s-TM levels were significantly increased compared with controls only in patients exhibiting vascular complications. Concentrations of t-PA and PAI-1 were significantly increased in the two groups of diabetics versus controls. Conclusion: In NIDDM, both endothelium-dependent and -independent microvascular skin reactivity are impaired, whether or not underlying vascular complications exist. It also appears that microvascular endothelial dysfunction is not necessarily associated in NIDDM with increased circulating levels of hemostatic markers of endothelial damage known to reflect a hypercoagulable state. PMID:19337558

Augmentation of Muscle Blood Flow by Ultrasound Cavitation Is Mediated by ATP and Purinergic Signaling.

PubMed

Belcik, J Todd; Davidson, Brian P; Xie, Aris; Wu, Melinda D; Yadava, Mrinal; Qi, Yue; Liang, Sherry; Chon, Chae Ryung; Ammi, Azzdine Y; Field, Joshua; Harmann, Leanne; Chilian, William M; Linden, Joel; Lindner, Jonathan R

2017-03-28

Augmentation of tissue blood flow by therapeutic ultrasound is thought to rely on convective shear. Microbubble contrast agents that undergo ultrasound-mediated cavitation markedly amplify these effects. We hypothesized that purinergic signaling is responsible for shear-dependent increases in muscle perfusion during therapeutic cavitation. Unilateral exposure of the proximal hindlimb of mice (with or without ischemia produced by iliac ligation) to therapeutic ultrasound (1.3 MHz, mechanical index 1.3) was performed for 10 minutes after intravenous injection of 2×10 8 lipid microbubbles. Microvascular perfusion was evaluated by low-power contrast ultrasound perfusion imaging. In vivo muscle ATP release and in vitro ATP release from endothelial cells or erythrocytes were assessed by a luciferin-luciferase assay. Purinergic signaling pathways were assessed by studying interventions that (1) accelerated ATP degradation; (2) inhibited P2Y receptors, adenosine receptors, or K ATP channels; or (3) inhibited downstream signaling pathways involving endothelial nitric oxide synthase or prostanoid production (indomethacin). Augmentation in muscle perfusion by ultrasound cavitation was assessed in a proof-of-concept clinical trial in 12 subjects with stable sickle cell disease. Therapeutic ultrasound cavitation increased muscle perfusion by 7-fold in normal mice, reversed tissue ischemia for up to 24 hours in the murine model of peripheral artery disease, and doubled muscle perfusion in patients with sickle cell disease. Augmentation in flow extended well beyond the region of ultrasound exposure. Ultrasound cavitation produced an ≈40-fold focal and sustained increase in ATP, the source of which included both endothelial cells and erythrocytes. Inhibitory studies indicated that ATP was a critical mediator of flow augmentation that acts primarily through either P2Y receptors or adenosine produced by ectonucleotidase activity. Combined indomethacin and inhibition of endothelial nitric oxide synthase abolished the effects of therapeutic ultrasound, indicating downstream signaling through both nitric oxide and prostaglandins. Therapeutic ultrasound using microbubble cavitation to increase muscle perfusion relies on shear-dependent increases in ATP, which can act through a diverse portfolio of purinergic signaling pathways. These events can reverse hindlimb ischemia in mice for >24 hours and increase muscle blood flow in patients with sickle cell disease. URL: http://clinicaltrials.gov. Unique identifier: NCT01566890. © 2017 American Heart Association, Inc.

Agglucetin, a tetrameric C-type lectin-like venom protein, regulates endothelial cell survival and promotes angiogenesis by activating integrin {alpha}v{beta}3 signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, W.-J.

2008-05-02

Agglucetin, a platelet glycoprotein (GP)Ib binding protein from Formosan Agkistrodon acutus (A. acutus) venom, could sustain human umbilical vein endothelial cell (HUVEC) proliferation and HUVEC adhering to immobilized agglucetin showed extensive spreading, which was strongly abrogated by integrin antagonists 7E3 and triflavin. Flow cytometric analyses confirmed the expression of GPIb complex on HUVEC is absent and fluorescein isothiocyanate (FITC)-agglucetin binds to HUVEC in a dose-dependent and saturable manner. Furthermore, native agglucetin specifically and dose-dependently inhibited the binding of FITC-23C6, an anti-{alpha}v{beta}3 monoclonal antibody (mAb), but not antibodies against {alpha}2 and {alpha}5, toward HUVEC and purified {alpha}v{beta}3 also bound to immobilizedmore » agglucetin-{beta} in a dose-dependent manner. Moreover, agglucetin exhibited a pro-angiogenic effect in vitro, as well as the focal adhesion kinase (FAK)-associated signaling molecules responsible for HUVEC activation were initiated by agglucetin. In conclusion, agglucetin, acting as a survival factor, promotes endothelial adhesion and angiogenesis by triggering {alpha}v{beta}3 signaling through FAK/phosphatidylinositol 3-kinase (PI3K)/Akt pathway.« less

Short- and long-term black tea consumption reverses endothelial dysfunction in patients with coronary artery disease.

PubMed

Duffy, S J; Keaney , J F; Holbrook, M; Gokce, N; Swerdloff, P L; Frei, B; Vita, J A

2001-07-10

Epidemiological studies suggest that tea consumption decreases cardiovascular risk, but the mechanisms of benefit remain undefined. Endothelial dysfunction has been associated with coronary artery disease and increased oxidative stress. Some antioxidants have been shown to reverse endothelial dysfunction, and tea contains antioxidant flavonoids. Methods and Results-- To test the hypothesis that tea consumption will reverse endothelial dysfunction, we randomized 66 patients with proven coronary artery disease to consume black tea and water in a crossover design. Short-term effects were examined 2 hours after consumption of 450 mL tea or water. Long-term effects were examined after consumption of 900 mL tea or water daily for 4 weeks. Vasomotor function of the brachial artery was examined at baseline and after each intervention with vascular ultrasound. Fifty patients completed the protocol and had technically suitable ultrasound measurements. Both short- and long-term tea consumption improved endothelium- dependent flow-mediated dilation of the brachial artery, whereas consumption of water had no effect (P<0.001 by repeated-measures ANOVA). Tea consumption had no effect on endothelium-independent nitroglycerin-induced dilation. An equivalent oral dose of caffeine (200 mg) had no short-term effect on flow-mediated dilation. Plasma flavonoids increased after short- and long-term tea consumption. Short- and long-term black tea consumption reverses endothelial vasomotor dysfunction in patients with coronary artery disease. This finding may partly explain the association between tea intake and decreased cardiovascular disease events.

Oxidative stress activates endothelial innate immunity via sterol regulatory element binding protein 2 (SREBP2) transactivation of microRNA-92a.

PubMed

Chen, Zhen; Wen, Liang; Martin, Marcy; Hsu, Chien-Yi; Fang, Longhou; Lin, Feng-Mao; Lin, Ting-Yang; Geary, McKenna J; Geary, Greg G; Zhao, Yongli; Johnson, David A; Chen, Jaw-Wen; Lin, Shing-Jong; Chien, Shu; Huang, Hsien-Da; Miller, Yury I; Huang, Po-Hsun; Shyy, John Y-J

2015-03-03

Oxidative stress activates endothelial innate immunity and disrupts endothelial functions, including endothelial nitric oxide synthase-derived nitric oxide bioavailability. Here, we postulated that oxidative stress induces sterol regulatory element-binding protein 2 (SREBP2) and microRNA-92a (miR-92a), which in turn activate endothelial innate immune response, leading to dysfunctional endothelium. Using cultured endothelial cells challenged by diverse oxidative stresses, hypercholesterolemic zebrafish, and angiotensin II-infused or aged mice, we demonstrated that SREBP2 transactivation of microRNA-92a (miR-92a) is oxidative stress inducible. The SREBP2-induced miR-92a targets key molecules in endothelial homeostasis, including sirtuin 1, Krüppel-like factor 2, and Krüppel-like factor 4, leading to NOD-like receptor family pyrin domain-containing 3 inflammasome activation and endothelial nitric oxide synthase inhibition. In endothelial cell-specific SREBP2 transgenic mice, locked nucleic acid-modified antisense miR-92a attenuates inflammasome, improves vasodilation, and ameliorates angiotensin II-induced and aging-related atherogenesis. In patients with coronary artery disease, the level of circulating miR-92a is inversely correlated with endothelial cell-dependent, flow-mediated vasodilation and is positively correlated with serum level of interleukin-1β. Our findings suggest that SREBP2-miR-92a-inflammasome exacerbates endothelial dysfunction during oxidative stress. Identification of this mechanism may help in the diagnosis or treatment of disorders associated with oxidative stress, innate immune activation, and endothelial dysfunction. © 2014 American Heart Association, Inc.

Factors Released from Endothelial Cells Exposed to Flow Impact Adhesion, Proliferation, and Fate Choice in the Adult Neural Stem Cell Lineage.

PubMed

Dumont, Courtney M; Piselli, Jennifer M; Kazi, Nadeem; Bowman, Evan; Li, Guoyun; Linhardt, Robert J; Temple, Sally; Dai, Guohao; Thompson, Deanna M

2017-08-15

The microvasculature within the neural stem cell (NSC) niche promotes self-renewal and regulates lineage progression. Previous work identified endothelial-produced soluble factors as key regulators of neural progenitor cell (NPC) fate and proliferation; however, endothelial cells (ECs) are sensitive to local hemodynamics, and the effect of this key physiological process has not been defined. In this study, we evaluated adult mouse NPC response to soluble factors isolated from static or dynamic (flow) EC cultures. Endothelial factors generated under dynamic conditions significantly increased neuronal differentiation, while those released under static conditions stimulated oligodendrocyte differentiation. Flow increases EC release of neurogenic factors and of heparin sulfate glycosaminoglycans that increase their bioactivity, likely underlying the enhanced neuronal differentiation. Additionally, endothelial factors, especially from static conditions, promoted adherent growth. Together, our data suggest that blood flow may impact proliferation, adhesion, and the neuron-glial fate choice of adult NPCs, with implications for diseases and aging that reduce flow.

Skin grafting impairs postsynaptic cutaneous vasodilator and sweating responses.

PubMed

Davis, Scott L; Shibasaki, Manabu; Low, David A; Cui, Jian; Keller, David M; Purdue, Gary F; Hunt, John L; Arnoldo, Brett D; Kowalske, Karen J; Crandall, Craig G

2007-01-01

This study tested the hypothesis that postsynaptic cutaneous vascular responses to endothelial-dependent and -independent vasodilators, as well as sweat gland function, are impaired in split-thickness grafted skin 5 to 9 months after surgery. Intradermal microdialysis membranes were placed in grafted and adjacent control skin, thereby allowing local delivery of the endothelial-dependent vasodilator, acetylcholine (ACh; 1 x 10(-7) to 1 x 10(-1) M at 10-fold increments) and the endothelial-independent nitric oxide donor, sodium nitroprusside (SNP; 5 x 10(-8) to 5 x 10(-2) M at 10-fold increments). Skin blood flow and sweat rate were simultaneously assessed over the semipermeable portion of the membrane. Cutaneous vascular conductance (CVC) was calculated from the ratio of laser Doppler-derived skin blood flow to mean arterial blood pressure. deltaCVC responses from baseline to these drugs were modeled via nonlinear regression curve fitting to identify the dose of ACh and SNP causing 50% of the maximal vasodilator response (EC50). A rightward shift in the CVC dose response curve for ACh was observed in grafted (EC50 = -2.61 +/- 0.44 log M) compared to adjacent control skin (EC50 = -3.34 +/- 0.46 log M; P = .003), whereas the mean EC50 for SNP was similar between grafted (EC50 = -4.21 +/- 0.94 log M) and adjacent control skin (EC50 = -3.87 +/- 0.65 log M; P = 0.332). Only minimal sweating to exogenous ACh was observed in grafted skin whereas normal sweating was observed in control skin. Increased EC50 and decreased maximal CVC responses to the exogenous administration of ACh suggest impairment of endothelial-dependent cutaneous vasodilator responses in grafted skin 5 to 9 months after surgery. Greatly attenuated sweating responses to ACh suggests either abnormal or an absence of functional sweat glands in the grafted skin.

Verocytotoxin-induced apoptosis of human microvascular endothelial cells.

PubMed

Pijpers, A H; van Setten, P A; van den Heuvel, L P; Assmann, K J; Dijkman, H B; Pennings, A H; Monnens, L A; van Hinsbergh, V W

2001-04-01

The pathogenesis of the epidemic form of hemolytic uremic syndrome is characterized by endothelial cell damage. In this study, the role of apoptosis in verocytotoxin (VT)-mediated endothelial cell death in human glomerular microvascular endothelial cells (GMVEC), human umbilical vein endothelial cells, and foreskin microvascular endothelial cells (FMVEC) was investigated. VT induced apoptosis in GMVEC and human umbilical vein endothelial cells when the cells were prestimulated with the inflammatory mediator tumor necrosis factor-alpha (TNF-alpha). FMVEC displayed strong binding of VT and high susceptibility to VT under basal conditions, which made them suitable for the study of VT-induced apoptosis without TNF-alpha interference. On the basis of functional (flow cytometry and immunofluorescence microscopy using FITC-conjugated annexin V and propidium iodide), morphologic (transmission electron microscopy), and molecular (agarose gel electrophoresis of cellular DNA fragments) criteria, it was documented that VT induced programmed cell death in microvascular endothelial cells in a dose- and time-dependent manner. Furthermore, whereas partial inhibition of protein synthesis by VT was associated with a considerable number of apoptotic cells, comparable inhibition of protein synthesis by cycloheximide was not. This suggests that additional pathways, independent of protein synthesis inhibition, may be involved in VT-mediated apoptosis in microvascular endothelial cells. Specific inhibition of caspases by Ac-Asp-Glu-Val-Asp-CHO, but not by Ac-Tyr-Val-Ala-Asp-CHO, was accompanied by inhibition of VT-induced apoptosis in FMVEC and TNF-alpha-treated GMVEC. These data indicate that VT can induce apoptosis in human microvascular endothelial cells.

Flow shear stress differentially regulates endothelial uptake of nanocarriers targeted to distinct epitopes of PECAM-1.

PubMed

Han, Jingyan; Shuvaev, Vladimir V; Davies, Peter F; Eckmann, David M; Muro, Silvia; Muzykantov, Vladimir R

2015-07-28

Targeting nanocarriers (NC) to endothelial cell adhesion molecules including Platelet-Endothelial Cell Adhesion Molecule-1 (PECAM-1 or CD31) improves drug delivery and pharmacotherapy of inflammation, oxidative stress, thrombosis and ischemia in animal models. Recent studies unveiled that hydrodynamic conditions modulate endothelial endocytosis of NC targeted to PECAM-1, but the specificity and mechanism of effects of flow remain unknown. Here we studied the effect of flow on endocytosis by human endothelial cells of NC targeted by monoclonal antibodies Ab62 and Ab37 to distinct epitopes on the distal extracellular domain of PECAM. Flow in the range of 1-8dyn/cm(2), typical for venous vasculature, stimulated the uptake of spherical Ab/NC (~180nm diameter) carrying ~50 vs 200 Ab62 and Ab37 per NC, respectively. Effect of flow was inhibited by disruption of cholesterol-rich plasmalemma domains and deletion of PECAM-1 cytosolic tail. Flow stimulated endocytosis of Ab62/NC and Ab37/NC via eliciting distinct signaling pathways mediated by RhoA/ROCK and Src Family Kinases, respectively. Therefore, flow stimulates endothelial endocytosis of Ab/NC in a PECAM-1 epitope specific manner. Using ligands of binding to distinct epitopes on the same target molecule may enable fine-tuning of intracellular delivery based on the hemodynamic conditions in the vascular area of interest. Copyright © 2015 Elsevier B.V. All rights reserved.

Insulin regulates its own delivery to skeletal muscle by feed-forward actions on the vasculature

PubMed Central

Wang, Hong; Upchurch, Charles T.; Liu, Zhenqi

2011-01-01

Insulin, at physiological concentrations, regulates the volume of microvasculature perfused within skeletal and cardiac muscle. It can also, by relaxing the larger resistance vessels, increase total muscle blood flow. Both of these effects require endothelial cell nitric oxide generation and smooth muscle cell relaxation, and each could increase delivery of insulin and nutrients to muscle. The capillary microvasculature possesses the greatest endothelial surface area of the body. Yet, whether insulin acts on the capillary endothelial cell is not known. Here, we review insulin's actions at each of three levels of the arterial vasculature as well as recent data suggesting that insulin can regulate a vesicular transport system within the endothelial cell. This latter action, if it occurs at the capillary level, could enhance insulin delivery to muscle interstitium and thereby complement insulin's actions on arteriolar endothelium to increase insulin delivery. We also review work that suggests that this action of insulin on vesicle transport depends on endothelial cell nitric oxide generation and that insulin's ability to regulate this vesicular transport system is impaired by inflammatory cytokines that provoke insulin resistance. PMID:21610226

Vitamin D and endothelial vasodilation in older individuals: data from the PIVUS study.

PubMed

Maggio, Marcello; De Vita, Francesca; Lauretani, Fulvio; Ceda, Gian Paolo; Volpi, Elena; Giallauria, Francesco; De Cicco, Giuseppe; Cattabiani, Chiara; Melhus, Håkan; Michaëlsson, Karl; Cederholm, Tommy; Lind, Lars

2014-09-01

Vitamin D plays a role in a wide range of extraskeletal processes, including vascular function. Endothelial dysfunction is a predictor of cardiovascular disease, especially in older subjects. However, the relationship between vitamin D levels and indexes of endothelial vasodilation has never been fully addressed in older individuals. The objective of this study was to examine the association between vitamin D and endothelial function in a large community-based sample of older subjects. This cross-sectional study involved 852 community-dwelling men and women aged 70 years from the Prospective Study of the Vasculature in Uppsala Seniors (PIVUS), with complete data on vascular function and 25-hydroxyvitamin D. We evaluated endothelium-dependent vasodilation by an invasive forearm technique with acetylcholine, endothelium-independent vasodilation by sodium nitroprussiate, flow-mediated vasodilation, and the pulse wave analysis (reflectance index). Vitamin D levels were measured by chemiluminescence. We used multivariate regression models adjusted for body mass index (model 1) and for multiple confounders (high-sensitivity C-reactive protein, insulin, total cholesterol, high-density lipoprotein-cholesterol, low-density lipoprotein-cholesterol, smoking, sex hormones, season of blood collection, hypertension, diabetes, cardiovascular medications and diseases, statin usage, plasma calcium and calcium intake, PTH, physical exercise, liver and kidney function tests, albumin; model 2). In women, but not in men, vitamin D levels were positively associated with endothelium-independent vasodilation in both model 1 (β ± SE = 1.41 ± 0.54; P = .001), and model 2 (β ± SE = 2.01 ± 0.68; P = .003).We found no significant relationship between vitamin D levels and endothelium-dependent vasodilation, flow-mediated vasodilation, and reflectance index in both sexes. In older women, but not in men, vitamin D is positively and independently associated with EIDV.

Vitamin D and Endothelial Vasodilation in Older Individuals: Data From the PIVUS Study

PubMed Central

De Vita, Francesca; Lauretani, Fulvio; Ceda, Gian Paolo; Volpi, Elena; Giallauria, Francesco; De Cicco, Giuseppe; Cattabiani, Chiara; Melhus, Håkan; Michaëlsson, Karl; Cederholm, Tommy; Lind, Lars

2014-01-01

Context: Vitamin D plays a role in a wide range of extraskeletal processes, including vascular function. Endothelial dysfunction is a predictor of cardiovascular disease, especially in older subjects. However, the relationship between vitamin D levels and indexes of endothelial vasodilation has never been fully addressed in older individuals. Objective: The objective of this study was to examine the association between vitamin D and endothelial function in a large community-based sample of older subjects. Methods: This cross-sectional study involved 852 community-dwelling men and women aged 70 years from the Prospective Study of the Vasculature in Uppsala Seniors (PIVUS), with complete data on vascular function and 25-hydroxyvitamin D. We evaluated endothelium-dependent vasodilation by an invasive forearm technique with acetylcholine, endothelium-independent vasodilation by sodium nitroprussiate, flow-mediated vasodilation, and the pulse wave analysis (reflectance index). Vitamin D levels were measured by chemiluminescence. We used multivariate regression models adjusted for body mass index (model 1) and for multiple confounders (high-sensitivity C-reactive protein, insulin, total cholesterol, high-density lipoprotein-cholesterol, low-density lipoprotein-cholesterol, smoking, sex hormones, season of blood collection, hypertension, diabetes, cardiovascular medications and diseases, statin usage, plasma calcium and calcium intake, PTH, physical exercise, liver and kidney function tests, albumin; model 2). Results: In women, but not in men, vitamin D levels were positively associated with endothelium-independent vasodilation in both model 1 (β ± SE = 1.41 ± 0.54; P = .001), and model 2 (β ± SE = 2.01 ± 0.68; P = .003).We found no significant relationship between vitamin D levels and endothelium-dependent vasodilation, flow-mediated vasodilation, and reflectance index in both sexes. Conclusions: In older women, but not in men, vitamin D is positively and independently associated with EIDV. PMID:24892991

Kisspeptin-10 induces endothelial cellular senescence and impaired endothelial cell growth.

PubMed

Usui, Sayaka; Iso, Yoshitaka; Sasai, Masahiro; Mizukami, Takuya; Mori, Hiroyoshi; Watanabe, Takuya; Shioda, Seiji; Suzuki, Hiroshi

2014-07-01

The KPs (kisspeptins) are a family of multifunctional peptides with established roles in cancer metastasis, puberty and vasoconstriction. The effects of KPs on endothelial cells have yet to be determined. The aim of the present study was to investigate the effects of KP-10 on endothelial cell growth and the mechanisms underlying those effects. The administration of recombinant KP-10 into the hindlimbs of rats with ischaemia significantly impaired blood flow recovery, as shown by laser Doppler, and capillary growth, as shown using histology, compared with the controls. HUVECs (human umbilical vein endothelial cells) express the KP receptor and were treated with KP-10 in culture studies. KP-10 inhibited endothelial cell tube formation and proliferation in a significant and dose-dependent manner. The HUVECs treated with KP exhibited the senescent phenotype, as determined using a senescence-associated β-galactosidase assay, cell morphology analysis, and decreased Sirt1 (sirtuin 1) expression and increased p53 expression shown by Western blot analysis. Intriguingly, a pharmacological Rho kinase inhibitor, Y-27632, was found to increase the proliferation of HUVECs and to reduce the number of senescent phenotype cells affected by KP-10. In conclusion, KP-10 suppressed endothelial cells growth both in vivo and in vitro in the present study. The adverse effect of KP on endothelial cells was attributable, at least in part, to the induction of cellular senescence.

In-vivo cell tracking to quantify endothelial cell migration during zebrafish angiogenesis

NASA Astrophysics Data System (ADS)

Menon, Prahlad G.; Rochon, Elizabeth R.; Roman, Beth L.

2016-03-01

The mechanism of endothelial cell migration as individual cells or collectively while remaining an integral component of a functional blood vessel has not been well characterized. In this study, our overarching goal is to define an image processing workflow to facilitate quantification of how endothelial cells within the first aortic arch and are proximal to the zebrafish heart behave in response to the onset of flow (i.e. onset of heart beating). Endothelial cell imaging was conducted at this developmental time-point i.e. ~24-28 hours post fertilization (hpf) when flow first begins, using 3D+time two-photon confocal microscopy of a live, wild-type, transgenic, zebrafish expressing green fluorescent protein (GFP) in endothelial cell nuclei. An image processing pipeline comprised of image signal enhancement, median filtering for speckle noise reduction, automated identification of the nuclei positions, extraction of the relative movement of nuclei between consecutive time instances, and finally tracking of nuclei, was designed for achieving the tracking of endothelial cell nuclei and the identification of their movement towards or away from the heart. Pilot results lead to a hypothesis that upon the onset of heart beat and blood flow, endothelial cells migrate collectively towards the heart (by 21.51+/-10.35 μm) in opposition to blood flow (i.e. subtending 142.170+/-21.170 with the flow direction).

Domain-Specific Partitioning of Uterine Artery Endothelial Connexin43 and Caveolin-1.

PubMed

Ampey, Bryan C; Morschauser, Timothy J; Ramadoss, Jayanth; Magness, Ronald R

2016-10-01

Uterine vascular adaptations facilitate rises in uterine blood flow during pregnancy, which are associated with gap junction connexin (Cx) proteins and endothelial nitric oxide synthase. In uterine artery endothelial cells (UAECs), ATP activates endothelial nitric oxide synthase in a pregnancy (P)-specific manner that is dependent on Cx43 function. Caveolar subcellular domain partitioning plays key roles in ATP-induced endothelial nitric oxide synthase activation and nitric oxide production. Little is known regarding the partitioning of Cx proteins to caveolar domains or their dynamics with ATP treatment. We observed that Cx43-mediated gap junction function with ATP stimulation is associated with Cx43 repartitioning between the noncaveolar and caveolar domains. Compared with UAECs from nonpregnant (NP) ewes, levels of ATP, PGI2, cAMP, NOx, and cGMP were 2-fold higher (P<0.05) in pregnant UAECs. In pregnant UAECs, ATP increased Lucifer yellow dye transfer, a response abrogated by Gap27, but not Gap 26, indicating involvement of Cx43, but not Cx37. Confocal microscopy revealed domain partitioning of Cx43 and caveolin-1. In pregnant UAECs, LC/MS/MS analysis revealed only Cx43 in the caveolar domain. In contrast, Cx37 was located only in the noncaveolar pool. Western analysis revealed that ATP increased Cx43 distribution (1.7-fold; P=0.013) to the caveolar domain, but had no effect on Cx37. These data demonstrate rapid ATP-stimulated repartitioning of Cx43 to the caveolae, where endothelial nitric oxide synthase resides and plays an important role in nitric oxide-mediated increasing uterine blood flow during pregnancy. © 2016 American Heart Association, Inc.

Endothelial deletion of Ino80 disrupts coronary angiogenesis and causes congenital heart disease.

PubMed

Rhee, Siyeon; Chung, Jae I; King, Devin A; D'amato, Gaetano; Paik, David T; Duan, Anna; Chang, Andrew; Nagelberg, Danielle; Sharma, Bikram; Jeong, Youngtae; Diehn, Maximilian; Wu, Joseph C; Morrison, Ashby J; Red-Horse, Kristy

2018-01-25

During development, the formation of a mature, well-functioning heart requires transformation of the ventricular wall from a loose trabecular network into a dense compact myocardium at mid-gestation. Failure to compact is associated in humans with congenital diseases such as left ventricular non-compaction (LVNC). The mechanisms regulating myocardial compaction are however still poorly understood. Here, we show that deletion of the Ino80 chromatin remodeler in vascular endothelial cells prevents ventricular compaction in the developing mouse heart. This correlates with defective coronary vascularization, and specific deletion of Ino80 in the two major coronary progenitor tissues-sinus venosus and endocardium-causes intermediate phenotypes. In vitro, endothelial cells promote myocardial expansion independently of blood flow in an Ino80-dependent manner. Ino80 deletion increases the expression of E2F-activated genes and endothelial cell S-phase occupancy. Thus, Ino80 is essential for coronary angiogenesis and allows coronary vessels to support proper compaction of the heart wall.

CCK receptors-related signaling involved in nitric oxide production caused by gastrin 17 in porcine coronary endothelial cells.

PubMed

Grossini, Elena; Caimmi, Philippe; Molinari, Claudio; Uberti, Francesca; Mary, David; Vacca, Giovanni

2012-03-05

In anesthetized pigs gastrin-17 increased coronary blood flow through CCK1/CCK2 receptors and β(2)-adrenoceptors-related nitric oxide (NO) release. Since the intracellular pathway has not been investigated the purpose of this study was to examine in coronary endothelial cells the CCK1/CCK2 receptors-related signaling involved in the effects of gastrin-17 on NO release. Gastrin-17 caused a concentration-dependent increase of NO production (17.3-62.6%; p<0.05), which was augmented by CCK1/CCK2 receptors agonists (p<0.05). The effect of gastrin-17 was amplified by the adenylyl-cyclase activator and β(2)-adrenoceptors agonist (p<0.05), abolished by cAMP/PKA and β(2)-adrenoceptors and CCK1/CCK2 receptors blockers, and reduced by PLC/PKC inhibitor. Finally, Western-blot revealed the preferential involvement of PKA vs. PKC as downstream effectors of CCK1/CCK2 receptors activation leading to Akt, ERK, p38 and endothelial NOS (eNOS) phosphorylation. In conclusion, in coronary endothelial cells, gastrin-17 induced eNOS-dependent NO production through CCK1/CCK2 receptors- and β(2)-adrenoceptors-related pathway. The intracellular signaling involved a preferential PKA pathway over PKC. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

Downregulation of endothelial adhesion molecules by dimethylfumarate, but not monomethylfumarate, and impairment of dynamic lymphocyte-endothelial cell interactions.

PubMed

Wallbrecht, Katrin; Drick, Nora; Hund, Anna-Carina; Schön, Michael P

2011-12-01

Although fumaric acid esters (FAE) have a decade-long firm place in the therapeutic armamentarium for psoriasis, their pleiotropic mode of action is not yet fully understood. While most previous studies have focused on the effects of FAE on leucocytes, we have addressed their activity on macro- and microvascular endothelial cells. As detected both on mRNA and protein levels, dimethylfumarate effected a profound reduction of TNFα-induced expression of E-selectin (CD62E), ICAM-1 (CD54) and VCAM-1 (CD106) on two different endothelial cell populations in a concentration-dependent manner. This reduction of several endothelial adhesion molecules was accompanied by a dramatic diminution of both rolling and firm adhesive interactions between endothelial cells and lymphocytes in a dynamic flow chamber system. Dimethylfumarate, at a concentration of 50 μm, reduced lymphocyte rolling on endothelial cells by 85.9% (P<0.001 compared to untreated controls), and it diminished the number of adherent cells by 88% (P<0.001). In contrast, monomethylfumarate (MMF) influenced neither surface expression of adhesion molecules nor interactions between endothelial cells and lymphocytes. These observations demonstrate that endothelial cells, in addition to the known effects on leucocytes, undergo profound functional changes in response to dimethylfumarate. These changes are accompanied by severely impaired dynamic interactions with lymphocytes, which constitute the critical initial step of leucocyte recruitment to inflamed tissues in psoriasis and other TNF-related inflammatory disorders. © 2011 John Wiley & Sons A/S.

Differential response of endothelial cells to simvastatin when conditioned with steady, non-reversing pulsatile or oscillating shear stress.

PubMed

Rossi, Joanna; Jonak, Paul; Rouleau, Leonie; Danielczak, Lisa; Tardif, Jean-Claude; Leask, Richard L

2011-01-01

Few studies have investigated whether fluid mechanics can impair or enhance endothelial cell response to pharmacological agents such as statin drugs. We evaluated and compared Kruppel-like factor 2 (KLF2), endothelial nitric oxide synthase (eNOS), and thrombomodulin (TM) expression in human abdominal aortic endothelial cells (HAAEC) treated with increasing simvastatin concentrations (0.1, 1 or 10 μM) under static culture and shear stress (steady, non-reversing pulsatile, and oscillating). Simvastatin, steady flow, and non-reversing pulsatile flow each separately upregulated KLF2, eNOS, and TM mRNA. At lower simvastatin concentrations (0.1 and 1 μM), the combination of statin and unidirectional steady or pulsatile flow produced an overall additive increase in mRNA levels. At higher simvastatin concentration (10 μM), a synergistic increase in eNOS and TM mRNA expression was observed. In contrast, oscillating flow impaired KLF2 and TM, but not eNOS expression by simvastatin at 1 μM. A higher simvastatin concentration of 10 μM overcame the inhibitory effect of oscillating flow. Our findings suggest that oscillating shear stress renders the endothelial cells less responsive to simvastatin than cells exposed to unidirectional steady or pulsatile flow. Consequently, the pleiotropic effects of statins in vivo may be less effective in endothelial cells exposed to atheroprone hemodynamics.

Gαq/11-mediated intracellular calcium responses to retrograde flow in endothelial cells.

PubMed

Melchior, Benoît; Frangos, John A

2012-08-15

Disturbed flow patterns, including reversal in flow direction, are key factors in the development of dysfunctional endothelial cells (ECs) and atherosclerotic lesions. An almost immediate response of ECs to fluid shear stress is the increase in cytosolic calcium concentration ([Ca(2+)](i)). Whether the source of [Ca(2+)](i) is extracellular, released from Ca(2+) intracellular stores, or both is still undefined, though it is likely dependent on the nature of forces involved. We have previously shown that a change in flow direction (retrograde flow) on a flow-adapted endothelial monolayer induces the remodeling of the cell-cell junction along with a dramatic [Ca(2+)](i) burst compared with cells exposed to unidirectional or orthograde flow. The heterotrimeric G protein-α q and 11 subunit (Gα(q/11)) is a likely candidate in effecting shear-induced increases in [Ca(2+)](i) since its expression is enriched at the junction and has been previously shown to be activated within seconds after onset of flow. In flow-adapted human ECs, we have investigated to what extent the Gα(q/11) pathway mediates calcium dynamics after reversal in flow direction. We observed that the elapsed time to peak [Ca(2+)](i) response to a 10 dyn/cm(2) retrograde shear stress was increased by 11 s in cells silenced with small interfering RNA directed against Gα(q/11). A similar lag in [Ca(2+)](i) transient was observed after cells were treated with the phospholipase C (PLC)-βγ inhibitor, U-73122, or the phosphatidylinositol-specific PLC inhibitor, edelfosine, compared with controls. Lower levels of inositol 1,4,5-trisphosphate accumulation seconds after the onset of flow correlated with the increased lag in [Ca(2+)](i) responses observed with the different treatments. In addition, inhibition of the inositol 1,4,5-trisphosphate receptor entirely abrogated flow-induced [Ca(2+)](i). Taken together, our results identify the Gα(q/11)-PLC pathway as the initial trigger for retrograde flow-induced endoplasmic reticulum calcium store release, thereby offering a novel approach to regulating EC dysfunctions in regions subjected to the reversal of blood flow.

Melatonin protects cardiac microvasculature against ischemia/reperfusion injury via suppression of mitochondrial fission-VDAC1-HK2-mPTP-mitophagy axis.

PubMed

Zhou, Hao; Zhang, Ying; Hu, Shunying; Shi, Chen; Zhu, Pingjun; Ma, Qiang; Jin, Qinhua; Cao, Feng; Tian, Feng; Chen, Yundai

2017-08-01

The cardiac microvascular system, which is primarily composed of monolayer endothelial cells, is the site of blood supply and nutrient exchange to cardiomyocytes. However, microvascular ischemia/reperfusion injury (IRI) following percutaneous coronary intervention is a woefully neglected topic, and few strategies are available to reverse such pathologies. Here, we studied the effects of melatonin on microcirculation IRI and elucidated the underlying mechanism. Melatonin markedly reduced infarcted area, improved cardiac function, restored blood flow, and lower microcirculation perfusion defects. Histological analysis showed that cardiac microcirculation endothelial cells (CMEC) in melatonin-treated mice had an unbroken endothelial barrier, increased endothelial nitric oxide synthase expression, unobstructed lumen, reduced inflammatory cell infiltration, and less endothelial damage. In contrast, AMP-activated protein kinase α (AMPKα) deficiency abolished the beneficial effects of melatonin on microvasculature. In vitro, IRI activated dynamin-related protein 1 (Drp1)-dependent mitochondrial fission, which subsequently induced voltage-dependent anion channel 1 (VDAC1) oligomerization, hexokinase 2 (HK2) liberation, mitochondrial permeability transition pore (mPTP) opening, PINK1/Parkin upregulation, and ultimately mitophagy-mediated CMEC death. However, melatonin strengthened CMEC survival via activation of AMPKα, followed by p-Drp1 S616 downregulation and p-Drp1 S37 upregulation, which blunted Drp1-dependent mitochondrial fission. Suppression of mitochondrial fission by melatonin recovered VDAC1-HK2 interaction that prevented mPTP opening and PINK1/Parkin activation, eventually blocking mitophagy-mediated cellular death. In summary, this study confirmed that melatonin protects cardiac microvasculature against IRI. The underlying mechanism may be attributed to the inhibitory effects of melatonin on mitochondrial fission-VDAC1-HK2-mPTP-mitophagy axis via activation of AMPKα. © 2017 The Authors. Journal of Pineal Research Published by John Wiley & Sons Ltd.

Flow Perturbation Mediates Neutrophil Recruitment and Potentiates Endothelial Injury via TLR2 in Mice: Implications for Superficial Erosion.

PubMed

Franck, Grégory; Mawson, Thomas; Sausen, Grasiele; Salinas, Manuel; Masson, Gustavo Santos; Cole, Andrew; Beltrami-Moreira, Marina; Chatzizisis, Yiannis; Quillard, Thibault; Tesmenitsky, Yevgenia; Shvartz, Eugenia; Sukhova, Galina K; Swirski, Filip K; Nahrendorf, Matthias; Aikawa, Elena; Croce, Kevin J; Libby, Peter

2017-06-23

Superficial erosion currently causes up to a third of acute coronary syndromes; yet, we lack understanding of its mechanisms. Thrombi because of superficial intimal erosion characteristically complicate matrix-rich atheromata in regions of flow perturbation. This study tested in vivo the involvement of disturbed flow and of neutrophils, hyaluronan, and Toll-like receptor 2 ligation in superficial intimal injury, a process implicated in superficial erosion. In mouse carotid arteries with established intimal lesions tailored to resemble the substrate of human eroded plaques, acute flow perturbation promoted downstream endothelial cell activation, neutrophil accumulation, endothelial cell death and desquamation, and mural thrombosis. Neutrophil loss-of-function limited these findings. Toll-like receptor 2 agonism activated luminal endothelial cells, and deficiency of this innate immune receptor decreased intimal neutrophil adherence in regions of local flow disturbance, reducing endothelial cell injury and local thrombosis ( P <0.05). These results implicate flow disturbance, neutrophils, and Toll-like receptor 2 signaling as mechanisms that contribute to superficial erosion, a cause of acute coronary syndrome of likely growing importance in the statin era. © 2017 American Heart Association, Inc.

Atorvastatin Restores Endothelial Function in Normocholesterolemic Smokers Independent of Changes in Low-Density Lipoprotein

PubMed Central

Beckman, Joshua A.; Liao, James K.; Hurley, Shauna; Garrett, Leslie A.; Chui, Daoshan; Mitra, Debi; Creager, Mark A.

2009-01-01

Cigarette smoking impairs endothelial function. Hydroxymethylglutaryl (HMG) CoA reductase inhibitors (statins) may favorably affect endothelial function via nonlipid mechanisms. We tested the hypothesis that statins would improve endothelial function independent of changes in lipids in cigarette smokers. Twenty normocholesterolemic cigarette smokers and 20 matched healthy control subjects were randomized to atorvastatin 40 mg daily or placebo for 4 weeks, washed out for 4 weeks, and then crossed-over to the other treatment. Baseline low-density lipoprotein (LDL) levels were similar in smokers and healthy subjects, 103±22 versus 95±27 mg/dL, respectively (P=NS) and were reduced similarly in smokers and control subjects by atorvastatin, to 55±30 and 58±20 mg/dL, respectively (P=NS). Vascular ultrasonography was used to determine brachial artery, flow-mediated, endothelium-dependent, and nitroglycerin-mediated, endothelium-independent vasodilation. To elucidate potential molecular mechanisms that may account for changes in endothelial function, skin biopsy specimens were assayed for eNOS mRNA, eNOS activity, and nitrotyrosine. Endothelium-dependent vasodilation was less in smokers than nonsmoking control subjects during placebo treatment, 8.0±0.6% versus 12.1±1.1%, (P=0.003). Atorvastatin increased endothelium-dependent vasodilation in smokers to 10.5±1.3% (P=0.017 versus placebo) but did not change endothelium-dependent vasodilation in control subjects (to 11.0±0.8%, P=NS). Endothelium-independent vasodilation did not differ between groups during placebo treatment and was not significantly affected by atorvastatin. Multivariate analysis did not demonstrate any association between baseline lipid levels or the change in lipid levels and endothelium-dependent vasodilation. Cutaneous nitrotyrosine levels and skin microvessel eNOS mRNA, but not ENOS activity, were increased in smokers compared with controls but unaffected by atorvastatin treatment. Atorvastatin restores endothelium-dependent vasodilation in normocholesterolemic cigarette smokers independent of changes in lipids. These results are consistent with a lipid-independent vascular benefit of statins but could not be explained by changes in eNOS message and tissue oxidative stress. These findings implicate a potential role for statin therapy to restore endothelial function and thereby investigate vascular disease in cigarette smokers. PMID:15178637

HSP27 Inhibits Homocysteine-Induced Endothelial Apoptosis by Modulation of ROS Production and Mitochondrial Caspase-Dependent Apoptotic Pathway.

PubMed

Tian, Xin; Zhao, Lei; Song, Xianjing; Yan, Youyou; Liu, Ning; Li, Tianyi; Yan, Bingdi; Liu, Bin

2016-01-01

Objectives. Elevated plasma homocysteine (Hcy) could lead to endothelial dysfunction and is viewed as an independent risk factor for atherosclerosis. Heat shock protein 27 (HSP27), a small heat shock protein, is reported to exert protective effect against atherosclerosis. This study aims to investigate the protective effect of HSP27 against Hcy-induced endothelial cell apoptosis in human umbilical vein endothelial cells (HUVECs) and to determine the underlying mechanisms. Methods. Apoptosis, reactive oxygen species (ROS), and mitochondrial membrane potential (MMP) of normal or HSP27-overexpressing HUVECs in the presence of Hcy were analyzed by flow cytometry. The mRNA and protein expression levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Results. We found that Hcy could induce cell apoptosis with corresponding decrease of nitric oxide (NO) level, increase of endothelin-1 (ET-1), intracellular adhesion molecule-1 (ICAM-1), vascular cellular adhesion molecule-1 (VCAM-1), and monocyte chemoattractant protein-1 (MCP-1) levels, elevation of ROS, and dissipation of MMP. In addition, HSP27 could protect the cell against Hcy-induced apoptosis and inhibit the effect of Hcy on HUVECs. Furthermore, HSP27 could increase the ratio of Bcl-2/Bax and inhibit caspase-3 activity. Conclusions. Therefore, we concluded that HSP27 played a protective role against Hcy-induced endothelial apoptosis through modulation of ROS production and the mitochondrial caspase-dependent apoptotic pathway.

Bevacizumab inhibits proliferation of choroidal endothelial cells by regulation of the cell cycle.

PubMed

Rusovici, Raluca; Patel, Chirag J; Chalam, Kakarla V

2013-01-01

The purpose of this study was to evaluate cell cycle changes in choroidal endothelial cells treated with varying doses of bevacizumab in the presence of a range of concentrations of vascular endothelial growth factor (VEGF). Bevacizumab, a drug widely used in the treatment of neovascular age-related macular degeneration, choroidal neovascularization, and proliferative diabetic retinopathy, neutralizes all isoforms of VEGF. However, the effect of intravitreal administration of bevacizumab on the choroidal endothelial cell cycle has not been established. Monkey choroidal endothelial (RF/6A) cells were treated with VEGF 50 ng/mL and escalating doses of bevacizumab 0.1-2 mg/mL for 72 hours. Cell cycle changes in response to bevacizumab were analyzed by flow cytometry and propidium iodide staining. Cell proliferation was measured using the WST-1 assay. Morphological changes were recorded by bright field cell microscopy. Bevacizumab inhibited proliferation of choroidal endothelial cells by stabilization of the cell cycle in G0/G1 phase. Cell cycle analysis of VEGF-enriched choroidal endothelial cells revealed a predominant increase in the G2/M population (21.84%, P, 0.01) and a decrease in the G0/G1 phase population (55.08%, P, 0.01). Addition of escalating doses of bevacizumab stabilized VEGF-enriched cells in the G0/G1 phase (55.08%, 54.49%, 56.3%, and 64% [P, 0.01]) and arrested proliferation by inhibiting the G2/M phase (21.84%, 21.46%, 20.59%, 20.94%, and 16.1% [P, 0.01]). The increase in G0/G1 subpopulation in VEGF-enriched and bevacizumab-treated cells compared with VEGF-enriched cells alone was dose-dependent. Bevacizumab arrests proliferation of VEGF-enriched choroidal endothelial cells by stabilizing the cell cycle in the G0/G1 phase and inhibiting the G2/M phase in a dose-dependent fashion.

Abacavir and didanosine induce the interaction between human leukocytes and endothelial cells through Mac-1 upregulation.

PubMed

De Pablo, Carmen; Orden, Samuel; Apostolova, Nadezda; Blanquer, Amando; Esplugues, Juan V; Alvarez, Angeles

2010-06-01

Abacavir and didanosine are nucleoside reverse transcriptase inhibitors (NRTI) widely used in therapy for HIV-infection but which have been linked to cardiovascular complications. The objective of this study was to analyze the effects of clinically relevant doses of abacavir and didanosine on human leukocyte-endothelium interactions and to compare them with those of other NRTIs. The interactions between human leukocytes - specifically peripheral blood polymorphonuclear (PMN) or mononuclear (PBMC) cells - and human umbilical vein endothelial cells were evaluated in a flow chamber system that reproduces conditions in vivo. The expression of adhesion molecules was analyzed by flow cytometry. Abacavir induced a dose-dependent increase in PMN and PBMC rolling and adhesion. This was reproduced by didanosine but not by lamivudine or zidovudine. Both abacavir and didanosine increased Mac-1 expression in neutrophils and monocytes, but produced no effects on either lymphocytes or the expression of endothelial adhesion molecules. The PMN/PBMC rolling and adhesion induced by abacavir or didanosine did not occur when antibodies against Mac-1 or its ligand ICAM-1 were blocked. Abacavir induces significant human leukocyte accumulation through the activation of Mac-1, which in turn interacts with its endothelial ligand ICAM-1. The fact that didanosine exhibits similar effects and that lamivudine and zidovudine do not points to a relationship between the chemical structure of NRTIs and the induction of leukocyte/endothelial cell interactions. This mechanism may be especially relevant to the progression of the vascular damage associated with atherosclerosis and myocardial infarction in abacavir and didanosine-treated patients.

Propionyl-L-carnitine improves endothelial function, microcirculation and pain management in critical limb ischemia.

PubMed

De Marchi, S; Zecchetto, S; Rigoni, A; Prior, M; Fondrieschi, L; Scuro, A; Rulfo, F; Arosio, E

2012-10-01

Chronic critical limb ischemia (CLI) is a severe condition of hypo-perfusion of lower limbs, which is associated with inflammation and a pro-coagulative state. It is a disease at high risk of amputation and cardiovascular death. Propionyl-L-carnitine (PLC) is efficacious in improving pain free walking distance in peripheral arterial disease with claudication; it also exerts favorable effects on the arterial wall and on endothelial function. The purpose of this study was to evaluate the effects of PLC on microcirculation, endothelial function and pain relief in patients affected by CLI not suitable for surgical intervention. We enrolled 48 patients with CLI. Patients were randomized into two groups: the first group was treated with PLC, the second was treated with saline solution. All of them underwent the following tests: laser Doppler flowmetry at the forefoot at rest and after ischemia, trans cutaneous oxygen partial pressure and carbon dioxide partial pressure at the forefoot at rest and after ischemia, endothelium dependent dilation of the brachial artery. All tests were repeated after treatments. Pain was assessed by visual analog pain scale. Endothelium dependent dilation increased after PLC (9.5 ± 3.2 vs 4.9 ± 1.4 %; p < 0.05). Post-ischemic peak flow with laser-Doppler flow increased after PLC. TcPO2 increased, while TcPCO2 decreased after PLC; CO2 production decreased after PLC. VAS showed a significant reduction in pain perception after active treatment. In CLI patients, PLC can improve microcirculation (post ischemic hyperemia, TcPO2 and TcPCO2 production). PLC also enhances endothelium dependent dilation and reduces analgesic consumption and pain perception.

Endothelial-dependent flow-mediated dilation in African Americans with masked-hypertension.

PubMed

Veerabhadrappa, Praveen; Diaz, Keith M; Feairheller, Deborah L; Sturgeon, Katie M; Williamson, Sheara T; Crabbe, Deborah L; Kashem, Abul M; Brown, Michael D

2011-10-01

Office-blood pressure (BP) measurements alone overlook a significant number of individuals with masked-hypertension (office-BP: 120/80-139/89 mm Hg and 24-h ambulatory BP monitoring (ABPM) daytime ≥135/85 mm Hg or night-time ≥120/70 mm Hg). Diminished endothelial function contributes to the pathogenesis of hypertension. To better understand the pathophysiology involved in the increased cardiovascular (CV) disease risk associated with masked-hypertension, we estimated the occurrence, assessed the endothelial function, compared plasma levels of inflammatory markers, white blood cell count (WBC count), tumor necrosis factor-α (TNF-α), and high sensitivity C-reactive protein (hsCRP) and examined the possible relationship between endothelial function and inflammatory markers in apparently healthy prehypertensive (office-BP: 120/80-139/89 mm Hg) African Americans. Fifty African Americans who were sedentary, nondiabetic, nonsmoking, devoid of CV disease were recruited. Office-BP was measured according to JNC-7 guidelines to identify prehypertensives in whom ABPM was then assessed. Fasting plasma samples were assayed for inflammatory markers. Brachial artery flow-mediated dilation (FMD) at rest and during reactive hyperemia was measured in a subset of prehypertensives. Subjects in the masked-hypertension sub-group had a higher hsCRP (P = 0.04) and diminished endothelial function (P = 0.03) compared to the true-prehypertensive sub-group (office-BP: 120/80-139/89 mm Hg and ABPM: daytime <135/85 mm Hg or night-time <120/70 mm Hg). Regression analysis showed that endothelial function was inversely related to hsCRP amongst the masked-hypertensive sub-group (R(2) = 0.160; P = 0.04). Masked-hypertension was identified in 58% of African Americans which suggests that a masking phenomenon may exist in a sub-group of prehypertensives who also seem to have a diminished endothelial function that could be mediated by an elevated subclinical inflammation leading to the increased CV disease.

[Finite Element Analysis of Intravascular Stent Based on ANSYS Software].

PubMed

Shi, Gengqiang; Song, Xiaobing

2015-10-01

This paper adopted UG8.0 to bulid the stent and blood vessel models. The models were then imported into the finite element analysis software ANSYS. The simulation results of ANSYS software showed that after endothelial stent implantation, the velocity of the blood was slow and the fluctuation of velocity was small, which meant the flow was relatively stable. When blood flowed through the endothelial stent, the pressure gradually became smaller, and the range of the pressure was not wide. The endothelial shear stress basically unchanged. In general, it can be concluded that the endothelial stents have little impact on the flow of blood and can fully realize its function.

Comprehensive assessment of impaired peripheral and coronary artery endothelial functions in smokers using brachial artery ultrasound and oxygen-15-labeled water PET.

PubMed

Ochi, Noriki; Yoshinaga, Keiichiro; Ito, Yoichi M; Tomiyama, Yuuki; Inoue, Mamiko; Nishida, Mutsumi; Manabe, Osamu; Shibuya, Hitoshi; Shimizu, Chikara; Suzuki, Eriko; Fujii, Satoshi; Katoh, Chietsugu; Tamaki, Nagara

2016-10-01

Comprehensive evaluation of endothelium-dependent and endothelium-independent vascular functions in peripheral arteries and coronary arteries in smokers has never been performed previously. Through the use of brachial artery ultrasound and oxygen-15-labeled water positron emission tomography (PET), we sought to investigate peripheral and coronary vascular dysfunctions in smokers. Eight smokers and 10 healthy individuals underwent brachial artery ultrasound at rest, during reactive hyperemia [250mmHg cuff occlusion (flow-mediated dilatation (FMD)], and following sublingual nitroglycerin (NTG) administration. Myocardial blood flow (MBF) was assessed through O-15-labeled water PET at rest, during adenosine triphosphate (ATP) administration, and during a cold pressor test (CPT). Through ultrasound, smokers were shown to have significantly reduced %FMD compared to controls (6.62±2.28% vs. 11.29±2.75%, p=0.0014). As assessed by O-15-labeled water PET, smokers were shown to have a significantly lower CPT response than were controls (21.1±9.5% vs. 50.9±16.9%, p=0.0004). There was no relationship between %FMD and CPT response (r=0.40, p=0.097). Endothelium-independent vascular dilatation was similar for both groups in terms of coronary flow reserve with PET (p=0.19). Smokers tended to have lower %NTG in the brachial artery (p=0.055). Smokers exhibited impaired coronary endothelial function as well as peripheral brachial artery endothelial function. In addition, there was no correlation between PET and ultrasound measurements, possibly implying that while smokers may have systemic vascular endothelial dysfunction, the characteristics of that dysfunction may be different in peripheral arteries and coronary arteries. Copyright © 2016. Published by Elsevier Ltd.

The effect of bioresorbable vascular scaffold implantation on distal coronary endothelial function in dyslipidemic swine with and without diabetes.

PubMed

van den Heuvel, Mieke; Sorop, Oana; van Ditzhuijzen, Nienke S; de Vries, René; van Duin, Richard W B; Peters, Ilona; van Loon, Janine E; de Maat, Moniek P; van Beusekom, Heleen M; van der Giessen, Wim J; Jan Danser, A H; Duncker, Dirk J

2018-02-01

We studied the effect of bioresorbable vascular scaffold (BVS) implantation on distal coronary endothelial function, in swine on a high fat diet without (HFD) or with diabetes (DM+HFD). Five DM+HFD and five HFD swine underwent BVS implantation on top of coronary plaques, and were studied six months later. Conduit artery segments >5mm proximal and distal to the scaffold and corresponding segments of non-scaffolded coronary arteries, and segments of small arteries within the flow-territory of scaffolded and non-scaffolded arteries were harvested for in vitro vasoreactivity studies. Conduit segments proximal and distal of the BVS edges showed reduced endothelium-dependent vasodilation as compared to control vessels (p≤0.01), with distal segments being most prominently affected(p≤0.01). Endothelial dysfunction was only observed in DM±HFD swine and was principally due to a loss of NO. Endothelium-independent vasodilation and vasoconstriction were unaffected. Surprisingly, segments from the microcirculation distal to the BVS showed enhanced endothelium-dependent vasodilation (p<0.01), whereas endothelium-independent vasodilation and vasoconstriction were unaltered. This enhanced vasorelaxation was only observed in DM+HFD swine, and did not appear to be either NO- or EDHF-mediated. Six months of BVS implantation in DM+HFD swine causes NO-mediated endothelial dysfunction in nearby coronary segments, which is accompanied by a, possibly compensatory, increase in endothelial function of the distal microcirculation. Endothelial dysfunction extending into coronary conduit segments beyond the implantation-site, is in agreement with recent reports expressing concern for late scaffold thrombosis and of early BVS failure in diabetic patients. Copyright © 2017. Published by Elsevier B.V.

Gender differences in response to cold pressor test assessed with velocity-encoded cardiovascular magnetic resonance of the coronary sinus.

PubMed

Moro, Pierre-Julien; Flavian, Antonin; Jacquier, Alexis; Kober, Frank; Quilici, Jacques; Gaborit, Bénédicte; Bonnet, Jean-Louis; Moulin, Guy; Cozzone, Patrick J; Bernard, Monique

2011-09-23

Gender-specific differences in cardiovascular risk are well known, and current evidence supports an existing role of endothelium in these differences. The purpose of this study was to assess non invasively coronary endothelial function in male and female young volunteers by myocardial blood flow (MBF) measurement using coronary sinus (CS) flow quantification by velocity encoded cine cardiovascular magnetic resonance (CMR) at rest and during cold pressor test (CPT). Twenty-four healthy volunteers (12 men, 12 women) underwent CMR in a 3 Tesla MR imager. Coronary sinus flow was measured at rest and during CPT using non breath-hold velocity encoded phase contrast cine-CMR. Myocardial function and morphology were acquired using a cine steady-state free precession sequence. At baseline, mean MBF was 0.63 ± 0.23 mL·g⁻¹·min⁻¹ in men and 0.79 ± 0.21 mL·g⁻¹·min⁻¹ in women. During CPT, the rate pressure product in men significantly increased by 49 ± 36% (p < 0.0001) and in women by 52 ± 22% (p < 0.0001). MBF increased significantly in both men and women by 0.22 ± 0.19 mL·g⁻¹·min⁻¹ (p = 0.0022) and by 0.73 ± 0.43 mL·g⁻¹·min⁻¹ (p = 0.0001), respectively. The increase in MBF was significantly higher in women than in men (p = 0.0012). CMR coronary sinus flow quantification for measuring myocardial blood flow revealed a higher response of MBF to CPT in women than in men. This finding may reflect gender differences in endothelial-dependent vasodilatation in these young subjects. This non invasive rest/stress protocol may become helpful to study endothelial function in normal physiology and in physiopathology.

Augmentation of limb perfusion and reversal of tissue ischemia produced by ultrasound-mediated microbubble cavitation.

PubMed

Belcik, J Todd; Mott, Brian H; Xie, Aris; Zhao, Yan; Kim, Sajeevani; Lindner, Nathan J; Ammi, Azzdine; Linden, Joel M; Lindner, Jonathan R

2015-04-01

Ultrasound can increase tissue blood flow, in part, through the intravascular shear produced by oscillatory pressure fluctuations. We hypothesized that ultrasound-mediated increases in perfusion can be augmented by microbubble contrast agents that undergo ultrasound-mediated cavitation and sought to characterize the biological mediators. Contrast ultrasound perfusion imaging of hindlimb skeletal muscle and femoral artery diameter measurement were performed in nonischemic mice after unilateral 10-minute exposure to intermittent ultrasound alone (mechanical index, 0.6 or 1.3) or ultrasound with lipid microbubbles (2×10(8) IV). Studies were also performed after inhibiting shear- or pressure-dependent vasodilator pathways, and in mice with hindlimb ischemia. Ultrasound alone produced a 2-fold increase (P<0.05) in muscle perfusion regardless of ultrasound power. Ultrasound-mediated augmentation in flow was greater with microbubbles (3- and 10-fold higher than control for mechanical index 0.6 and 1.3, respectively; P<0.05), as was femoral artery dilation. Inhibition of endothelial nitric oxide synthase attenuated flow augmentation produced by ultrasound and microbubbles by 70% (P<0.01), whereas inhibition of adenosine-A2a receptors and epoxyeicosatrienoic acids had minimal effect. Limb nitric oxide production and muscle phospho-endothelial nitric oxide synthase increased in a stepwise fashion by ultrasound and ultrasound with microbubbles. In mice with unilateral hindlimb ischemia (40%-50% reduction in flow), ultrasound (mechanical index, 1.3) with microbubbles increased perfusion by 2-fold to a degree that was greater than the control nonischemic limb. Increases in muscle blood flow during high-power ultrasound are markedly amplified by the intravascular presence of microbubbles and can reverse tissue ischemia. These effects are most likely mediated by cavitation-related increases in shear and activation of endothelial nitric oxide synthase. © 2015 American Heart Association, Inc.

Effect of streptavidin-biotin on endothelial vasoregulation and leukocyte adhesion.

PubMed

Chan, Bernard P; Reichert, William M; Truskey, George A

2004-08-01

The current study examines whether the adhesion promoting arginine-glycine-aspartate-streptavidin mutant (RGD-SA) also affects two important endothelial cell (EC) functions in vitro: vasoregulation and leukocyte adhesion. EC adherent to surfaces via fibronectin (Fn) or Fn plus RGD-SA were subjected to laminar shear flow and media samples were collected over a period of 4h to measure the concentration of nitric oxide (NO), prostacyclin (PGI(2)), and endothelin-1 (ET-1). Western blot analysis was used to quantify the levels of endothelial-derived nitric oxide synthase (eNOS) and cyclooxygenase II (COX II). In a separate set of experiments, fluorescent polymorphonuclear leukocyte (PMN) adhesion to EC was quantified for EC with and without exposure to flow preconditioning. When cell adhesion was supplemented with the SA-biotin system, flow-induced production of NO and PGI(2) increased significantly relative to cells adherent on Fn alone. Previous exposure of EC to shear flow also significantly decreased PMN attachment to SA-biotin supplemented EC, but only after 2h of exposure to shear flow. The observed decrease in PMN-EC adhesion was negated by NG-nitro-L-arginine methyl ester (L-NAME), an antagonist of NO synthesis, but not by indomethacin, an inhibitor to PGI(2) synthesis, indicating the induced effect of PMN-EC interaction is primarily NO-dependent. Results from this study suggest that the use of SA-biotin to supplement EC adhesion encourages vasodilation and PMN adhesion in vitro under physiological shear-stress conditions. We postulate that the presence of SA-biotin more efficiently transmits the shear-stress signal and amplifies the downstream events including the NO and PGI(2) release and leukocyte-EC inhibition. These results may have ramifications for reducing thrombus-induced vascular graft failure.

Therapeutic potential of pro-angiogenic BPC157 is associated with VEGFR2 activation and up-regulation.

PubMed

Hsieh, Ming-Jer; Liu, Hsien-Ta; Wang, Chao-Nin; Huang, Hsiu-Yun; Lin, Yuling; Ko, Yu-Shien; Wang, Jong-Shyan; Chang, Vincent Hung-Shu; Pang, Jong-Hwei S

2017-03-01

BPC 157, a pentadecapeptide with extensive healing effects, has recently been suggested to contribute to angiogenesis. However, the underlying mechanism is not yet clear. The present study aimed to explore the potential therapeutic effect and pro-angiogenic mechanism of BPC 157. As demonstrated by the chick chorioallantoic membrane (CAM) assay and endothelial tube formation assay, BPC 157 could increase the vessel density both in vivo and in vitro, respectively. BPC 157 could also accelerate the recovery of blood flow in the ischemic muscle of the rat hind limb as detected by laser Doppler scanning, indicating the promotion of angiogenesis. Histological analysis of the hind limb muscle confirmed the increased number of vessels and the enhanced vascular expression of vascular endothelial growth factor receptor 2 (VEGFR2) in rat with BPC 157 treatment. In vitro study using human vascular endothelial cells further confirmed the increased mRNA and protein expressions of VEGFR2 but not VEGF-A by BPC 157. In addition, BPC 157 could promote VEGFR2 internalization in vascular endothelial cells which was blocked in the presence of dynasore, an inhibitor of endocytosis. BPC 157 time dependently activated the VEGFR2-Akt-eNOS signaling pathway which could also be suppressed by dynasore. The increase of endothelial tube formation induced by BPC 157 was also inhibited by dynasore. This study demonstrates the pro-angiogenic effects of BPC 157 that is associated with the increased expression, internalization of VEGFR2, and the activation of VEGFR2-Akt-eNOS signaling pathway. BPC 157 promotes angiogenesis in CAM assay and tube formation assay. BPC 157 accelerates the blood flow recovery and vessel number in rats with hind limb ischemia. BPC 157 up-regulates VEGFR2 expression in rats with hind limb ischemia and endothelial cell culture. BPC 157 promotes VEGFR2 internalization in association with VEGFR2-Akt-eNOS activation. BPC 157 promotes angiogenesis in CAM assay and tube formation assay. BPC 157 accelerates the blood flow recovery and vessel number in rats with hind limb ischemia. BPC 157 up-regulates VEGFR2 expression in rats with hind limb ischemia and endothelial cell culture. BPC 157 promotes VEGFR2 internalization in association with VEGFR2-Akt-eNOS activation.

PECAM1 regulates flow-mediated Gab1 tyrosine phosphorylation and signaling

PubMed Central

Xu, Suowen; Ha, Chang Hoon; Wang, Weiye; Xu, Xiangbin; Yin, Meimei; Jin, Felix Q.; Mastrangelo, Michael; Koroleva, Marina; Fujiwara, Keigi; Jin, Zheng Gen

2016-01-01

Endothelial dysfunction, characterized by impaired activation of endothelial nitric oxide (NO) synthase (eNOS) and ensued decrease of NO production, is a common mechanism of various cardiovascular pathologies, including hypertension and atherosclerosis. Laminar blood flow-mediated specific signaling cascades modulate vascular endothelial cells (ECs) structure and functions. We have previously shown that flow-stimulated Gab1 (Grb2-associated binder-1) tyrosine phosphorylation mediates eNOS activation in ECs, which in part confers laminar flow atheroprotective action. However, the molecular mechanisms whereby flow regulates Gab1 tyrosine phosphorylation and its downstream signaling events remain unclear. Here we show that platelet endothelial cell adhesion molecule-1 (PECAM1), a key molecule in an endothelial mechanosensing complex, specifically mediates Gab1 tyrosine phosphorylation and its downstream Akt and eNOS activation in ECs upon flow rather than hepatocyte growth factor (HGF) stimulation. Small interfering RNA (siRNA) targeting PECAM1 abolished flow- but not HGF-induced Gab1 tyrosine phosphorylation and Akt, eNOS activation as well as Gab1 membrane translocation. Protein-tyrosine phosphatase SHP2, which has been shown to interact with Gab1, was involved in flow signaling and HGF signaling, as SHP2 siRNA diminished the flow- and HGF-induced Gab1 tyrosine phosphorylation, membrane localization and downstream signaling. Pharmacological inhibition of PI3K decreased flow-, but not HGF-mediated Gab1 phosphorylation and membrane localization as well as eNOS activation. Finally, we observed that flow-mediated Gab1 and eNOS phosphorylation in vivo induced by voluntary wheel running was reduced in PECAM1 knockout mice. These results demonstrate a specific role of PECAM1 in flow-mediated Gab1 tyrosine phosphorylation and eNOS signaling in ECs. PMID:26706435

Shear-induced endothelial mechanotransduction: the interplay between reactive oxygen species (ROS) and nitric oxide (NO) and the pathophysiological implications

PubMed Central

2014-01-01

Hemodynamic shear stress, the blood flow-generated frictional force acting on the vascular endothelial cells, is essential for endothelial homeostasis under normal physiological conditions. Mechanosensors on endothelial cells detect shear stress and transduce it into biochemical signals to trigger vascular adaptive responses. Among the various shear-induced signaling molecules, reactive oxygen species (ROS) and nitric oxide (NO) have been implicated in vascular homeostasis and diseases. In this review, we explore the molecular, cellular, and vascular processes arising from shear-induced signaling (mechanotransduction) with emphasis on the roles of ROS and NO, and also discuss the mechanisms that may lead to excessive vascular remodeling and thus drive pathobiologic processes responsible for atherosclerosis. Current evidence suggests that NADPH oxidase is one of main cellular sources of ROS generation in endothelial cells under flow condition. Flow patterns and magnitude of shear determine the amount of ROS produced by endothelial cells, usually an irregular flow pattern (disturbed or oscillatory) producing higher levels of ROS than a regular flow pattern (steady or pulsatile). ROS production is closely linked to NO generation and elevated levels of ROS lead to low NO bioavailability, as is often observed in endothelial cells exposed to irregular flow. The low NO bioavailability is partly caused by the reaction of ROS with NO to form peroxynitrite, a key molecule which may initiate many pro-atherogenic events. This differential production of ROS and RNS (reactive nitrogen species) under various flow patterns and conditions modulates endothelial gene expression and thus results in differential vascular responses. Moreover, ROS/RNS are able to promote specific post-translational modifications in regulatory proteins (including S-glutathionylation, S-nitrosylation and tyrosine nitration), which constitute chemical signals that are relevant in cardiovascular pathophysiology. Overall, the dynamic interplay between local hemodynamic milieu and the resulting oxidative and S-nitrosative modification of regulatory proteins is important for ensuing vascular homeostasis. Based on available evidence, it is proposed that a regular flow pattern produces lower levels of ROS and higher NO bioavailability, creating an anti-atherogenic environment. On the other hand, an irregular flow pattern results in higher levels of ROS and yet lower NO bioavailability, thus triggering pro-atherogenic effects. PMID:24410814

Expression of endothelin-1 and constitutional nitric oxide synthase messenger RNA in saphenous vein endothelial cells exposed to arterial flow shear stress.

PubMed

Zhu, Z G; Li, H H; Zhang, B R

1997-11-01

It has long been speculated that increased blood flow shear stress might be one of the major factors affecting the patency of grafted saphenous vein in coronary artery bypass operations. The underlying cellular and molecular mechanisms for so-called "shear stress damage" have not yet been well elucidated. Endothelial cells harvested from human saphenous vein were cultured in vitro and then exposed to a high arterial level flow shear stress in the parallel flow chamber. The expression levels of endothelin-1 and constitutional nitric oxide synthase by the endothelial cells were evaluated semiquantitatively at the gene transcription (messenger RNA) level using reverse transcription polymerase chain reaction. After 7 hours of exposure to arterial level shear stress, the expression of constitutional nitric oxide synthase messenger RNA by saphenous vein endothelial cells was significantly reduced, whereas the expression of endothelin-1 messenger RNA was substantially increased. These changes were more predominant at 24 hours. Arterial level flow shear stress could cause important changes in the gene transcription level in saphenous vein endothelial cells within a short period of time. The functional alterations of saphenous vein endothelial cells, as manifested by the increased expression of endothelin-1 and decreased expression of nitric oxide synthase messenger RNA, might play a crucial role in the vein graft remodeling process.

Effect of flow on endothelial endocytosis of nanocarriers targeted to ICAM-1

PubMed Central

Bhowmick, Tridib; Berk, Erik; Cui, Xiumin; Muzykantov, Vladimir R.; Muro, Silvia

2011-01-01

Delivery of drugs into the endothelium by nanocarriers targeted to endothelial determinants may improve treatment of vascular maladies. This is the case for intercellular adhesion molecule 1 (ICAM-1), a glycoprotein overexpressed on endothelial cells (ECs) in many pathologies. ICAM-1-targeted nanocarriers bind to and are internalized by ECs via a non-classical pathway, CAM-mediated endocytosis. In this work we studied the effects of endothelial adaptation to physiological flow on the endocytosis of model polymer nanocarriers targeted to ICAM-1 (anti-ICAM/NCs, ~180-nm diameter). Culturing established endothelial-like cells (EAhy926 cells) and primary human umbilical vein ECs (HUVECs) under 4 dyn/cm2 laminar shear stress for 24 h resulted in flow adaptation: cell elongation and formation of actin stress fibers aligned to the flow direction. Fluorescence microscopy showed that flow-adapted cells internalized anti-ICAM/NCs under flow, although at slower rate versus non flow-adapted cells under static incubation (~35% reduction). Uptake was inhibited by amiloride, whereas marginally affected by filipin and cadaverine, implicating that CAM-endocytosis accounts for anti-ICAM/NC uptake under flow. Internalization under flow was more modestly affected by inhibiting protein kinase C, which regulates actin remodeling during CAM-endocytosis. Actin recruitment to stress fibers that maintain the cell shape under flow may delay uptake of anti-ICAM/NCs under this condition by interfering with actin reorganization needed for CAM-endocytosis. Electron microscopy revealed somewhat slow, yet effective endocytosis of anti-ICAM/NCs by pulmonary endothelium after i.v. injection in mice, similar to that of flow-adapted cell cultures: ~40% (30 min) and 80% (3 h) internalization. Similar to cell culture data, uptake was slightly faster in capillaries with lower shear stress. Further, LPS treatment accelerated internalization of anti-ICAM/NCs in mice. Therefore, regulation of endocytosis of ICAM-1-targeted nanocarriers by flow and endothelial status may modulate drug delivery into ECs exposed to different physiological (capillaries vs. arterioles/venules) or pathological (ischemia, inflammation) levels and patterns of blood flow. PMID:21951807

Effect of flow on endothelial endocytosis of nanocarriers targeted to ICAM-1.

PubMed

Bhowmick, Tridib; Berk, Erik; Cui, Xiumin; Muzykantov, Vladimir R; Muro, Silvia

2012-02-10

Delivery of drugs into the endothelium by nanocarriers targeted to endothelial determinants may improve treatment of vascular maladies. This is the case for intercellular adhesion molecule 1 (ICAM-1), a glycoprotein overexpressed on endothelial cells (ECs) in many pathologies. ICAM-1-targeted nanocarriers bind to and are internalized by ECs via a non-classical pathway, CAM-mediated endocytosis. In this work we studied the effects of endothelial adaptation to physiological flow on the endocytosis of model polymer nanocarriers targeted to ICAM-1 (anti-ICAM/NCs, ~180 nm diameter). Culturing established endothelial-like cells (EAhy926 cells) and primary human umbilical vein ECs (HUVECs) under 4 dyn/cm(2) laminar shear stress for 24 h resulted in flow adaptation: cell elongation and formation of actin stress fibers aligned to the flow direction. Fluorescence microscopy showed that flow-adapted cells internalized anti-ICAM/NCs under flow, although at slower rate versus non flow-adapted cells under static incubation (~35% reduction). Uptake was inhibited by amiloride, whereas marginally affected by filipin and cadaverine, implicating that CAM-endocytosis accounts for anti-ICAM/NC uptake under flow. Internalization under flow was more modestly affected by inhibiting protein kinase C, which regulates actin remodeling during CAM-endocytosis. Actin recruitment to stress fibers that maintain the cell shape under flow may delay uptake of anti-ICAM/NCs under this condition by interfering with actin reorganization needed for CAM-endocytosis. Electron microscopy revealed somewhat slow, yet effective endocytosis of anti-ICAM/NCs by pulmonary endothelium after i.v. injection in mice, similar to that of flow-adapted cell cultures: ~40% (30 min) and 80% (3 h) internalization. Similar to cell culture data, uptake was slightly faster in capillaries with lower shear stress. Further, LPS treatment accelerated internalization of anti-ICAM/NCs in mice. Therefore, regulation of endocytosis of ICAM-1-targeted nanocarriers by flow and endothelial status may modulate drug delivery into ECs exposed to different physiological (capillaries vs. arterioles/venules) or pathological (ischemia, inflammation) levels and patterns of blood flow. Copyright © 2011 Elsevier B.V. All rights reserved.

Mechanical Injury Induces Brain Endothelial-Derived Microvesicle Release: Implications for Cerebral Vascular Injury during Traumatic Brain Injury.

PubMed

Andrews, Allison M; Lutton, Evan M; Merkel, Steven F; Razmpour, Roshanak; Ramirez, Servio H

2016-01-01

It is well established that the endothelium responds to mechanical forces induced by changes in shear stress and strain. However, our understanding of vascular remodeling following traumatic brain injury (TBI) remains incomplete. Recently published studies have revealed that lung and umbilical endothelial cells produce extracellular microvesicles (eMVs), such as microparticles, in response to changes in mechanical forces (blood flow and mechanical injury). Yet, to date, no studies have shown whether brain endothelial cells produce eMVs following TBI. The brain endothelium is highly specialized and forms the blood-brain barrier (BBB), which regulates diffusion and transport of solutes into the brain. This specialization is largely due to the presence of tight junction proteins (TJPs) between neighboring endothelial cells. Following TBI, a breakdown in tight junction complexes at the BBB leads to increased permeability, which greatly contributes to the secondary phase of injury. We have therefore tested the hypothesis that brain endothelium responds to mechanical injury, by producing eMVs that contain brain endothelial proteins, specifically TJPs. In our study, primary human adult brain microvascular endothelial cells (BMVEC) were subjected to rapid mechanical injury to simulate the abrupt endothelial disruption that can occur in the primary injury phase of TBI. eMVs were isolated from the media following injury at 2, 6, 24, and 48 h. Western blot analysis of eMVs demonstrated a time-dependent increase in TJP occludin, PECAM-1 and ICAM-1 following mechanical injury. In addition, activation of ARF6, a small GTPase linked to extracellular vesicle production, was increased after injury. To confirm these results in vivo, mice were subjected to sham surgery or TBI and blood plasma was collected 24 h post-injury. Isolation and analysis of eMVs from blood plasma using cryo-EM and flow cytometry revealed elevated levels of vesicles containing occludin following brain trauma. These results indicate that following TBI, the cerebral endothelium undergoes vascular remodeling through shedding of eMVs containing TJPs and endothelial markers. The detection of this shedding potentially allows for a novel methodology for real-time monitoring of cerebral vascular health (remodeling), BBB status and neuroinflammation following a TBI event.

Mechanical property quantification of endothelial cells using scanning acoustic microscopy

NASA Astrophysics Data System (ADS)

Shelke, A.; Brand, S.; Kundu, T.; Bereiter-Hahn, J.; Blase, C.

2012-04-01

The mechanical properties of cells reflect dynamic changes of cellular organization which occur during physiologic activities like cell movement, cell volume regulation or cell division. Thus the study of cell mechanical properties can yield important information for understanding these physiologic activities. Endothelial cells form the thin inner lining of blood vessels in the cardiovascular system and are thus exposed to shear stress as well as tensile stress caused by the pulsatile blood flow. Endothelial dysfunction might occur due to reduced resistance to mechanical stress and is an initial step in the development of cardiovascular disease like, e.g., atherosclerosis. Therefore we investigated the mechanical properties of primary human endothelial cells (HUVEC) of different age using scanning acoustic microscopy at 1.2 GHz. The HUVECs are classified as young (tD < 90 h) and old (tD > 90 h) cells depending upon the generation time for the population doubling of the culture (tD). Longitudinal sound velocity and geometrical properties of cells (thickness) were determined using the material signature curve V(z) method for variable culture condition along spatial coordinates. The plane wave technique with normal incidence is assumed to solve two-dimensional wave equation. The size of the cells is modeled using multilayered (solid-fluid) system. The propagation of transversal wave and surface acoustic wave are neglected in soft matter analysis. The biomechanical properties of HUVEC cells are quantified in an age dependent manner.

Effect of Flow on Gene Regulation in Smooth Muscle Cells and Macromolecular Transport Across Endothelial Cell Monolayers

NASA Technical Reports Server (NTRS)

McIntire, Larry V.; Wagner, John E.; Papadaki, Maria; Whitson, Peggy A.; Eskin, Suzanne G.

1996-01-01

Endothelial cells line all of the vessels of the circulatory system, providing a non-thrombogenic conduit for blood flow; they regulate many complex functions in the vasculature, such as coagulation, fibrinolysis, platelet aggregation, vessel tone and growth, and leukocyte traffic; and they form the principal barrier to transport of substances between the blood and the surrounding tissue space. The permeability of endothelial cell changes with environmental stimuli; shear stress, in particular, applied either in vivo, or in vitro, induces changes in protein expression and secretion of vasoactive factors by endothelial cells. The ability to study the effects of shear on the macromolecular permeability of the cerebral vasculature is particularly important, since in no other place is the barrier function of the endothelium more important than in the brain. The endothelial cells of this organ have developed special barrier properties that keep the cerebral system from experiencing any drastic change in composition; together with glial cells, they form the blood brain barrier (BBB). We have studied the effect of flow on bovine BBB using flow chambers and tissue culture systems.

Albumin infusion improves renal blood flow autoregulation in patients with acute decompensation of cirrhosis and acute kidney injury.

PubMed

Garcia-Martinez, Rita; Noiret, Lorette; Sen, Sambit; Mookerjee, Rajeshwar; Jalan, Rajiv

2015-02-01

In cirrhotic patients with renal failure, renal blood flow autoregulation curve is shifted to the right, which is consequent upon sympathetic nervous system activation and endothelial dysfunction. Albumin infusion improves renal function in cirrhosis by mechanisms that are incompletely understood. We aimed to determine the effect of albumin infusion on systemic haemodynamics, renal blood flow, renal function and endothelial function in patients with acute decompensation of cirrhosis and acute kidney injury. Twelve patients with refractory ascites and 10 patients with acute decompensation of cirrhosis and acute kidney injury were studied. Both groups were treated with intravenous albumin infusion, 40-60 g/days over 3-4 days. Cardiac and renal haemodynamics were measured. Endothelial activation/dysfunction was assessed using von Willebrand factor and serum nitrite levels. F2α Isoprostanes, resting neutrophil burst and noradrenaline levels were quantified as markers of oxidative stress, endotoxemia and sympathetic activation respectively. Albumin infusion leads to a shift in the renal blood flow autoregulation curve towards normalization, which resulted in a significant increase in renal blood flow. Accordingly, improvement of renal function was observed. In parallel, a significant decrease in sympathetic activation, inflammation/oxidative stress and endothelial activation/dysfunction was documented. Improvement of renal blood flow correlated with improvement in endothelial activation (r = 0.741, P < 0.001). The data suggest that albumin infusion improves renal function in acutely decompensated cirrhotic patients with acute kidney injury by impacting on renal blood flow autoregulation. This is possibly achieved through endothelial stabilization and a reduction in the sympathetic tone, endotoxemia and oxidative stress. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Are there Race-Dependent Endothelial Cell Responses to Exercise?

PubMed Central

Brown, Michael D.; Feairheller, Deborah L.

2013-01-01

African Americans have endothelial dysfunction which likely contributes to their high prevalence of hypertension. Endothelial cell responses to stimuli could play a role in the development of endothelial dysfunction and hypertension. High physiological levels of vascular laminar shear stress can profoundly alter endothelial cell phenotype. It is not known whether there are race-dependent endothelial cell responses to laminar shear stress. PMID:23262464

Impact of competitive flow on wall shear stress in coronary surgery: computational fluid dynamics of a LIMA-LAD model.

PubMed

Nordgaard, Håvard; Swillens, Abigail; Nordhaug, Dag; Kirkeby-Garstad, Idar; Van Loo, Denis; Vitale, Nicola; Segers, Patrick; Haaverstad, Rune; Lovstakken, Lasse

2010-12-01

Competitive flow from native coronary vessels is considered a major factor in the failure of coronary bypass grafts. However, the pathophysiological effects are not fully understood. Low and oscillatory wall shear stress (WSS) is known to induce endothelial dysfunction and vascular disease, like atherosclerosis and intimal hyperplasia. The aim was to investigate the impact of competitive flow on WSS in mammary artery bypass grafts. Using computational fluid dynamics, WSS was calculated in a left internal mammary artery (LIMA) graft to the left anterior descending artery in a three-dimensional in vivo porcine coronary artery bypass graft model. The following conditions were investigated: high competitive flow (non-significant coronary lesion), partial competitive flow (significant coronary lesion), and no competitive flow (totally occluded coronary vessel). Time-averaged WSS of LIMA at high, partial, and no competitive flow were 0.3-0.6, 0.6-3.0, and 0.9-3.0 Pa, respectively. Further, oscillatory WSS quantified as the oscillatory shear index (OSI) ranged from (maximum OSI = 0.5 equals zero net WSS) 0.15 to 0.35, <0.05, and <0.05, respectively. Thus, high competitive flow resulted in substantial oscillatory and low WSS. Moderate competitive flow resulted in WSS and OSI similar to the no competitive flow condition. Graft flow is highly dependent on the degree of competitive flow. High competitive flow was found to produce unfavourable WSS consistent with endothelial dysfunction and subsequent graft narrowing and failure. Partial competitive flow, however, may be better tolerated as it was found to be similar to the ideal condition of no competitive flow.

Moon Dust may Simulate Vascular Hazards of Urban Pollution

NASA Astrophysics Data System (ADS)

Rowe, W. J.

A long duration mission to the moon presents several potential cardiovascular complications. To the risks of microgravity and hypokinesia, and the fact that pharmaceuticals cannot be always depended upon in the space fight conditions, there is a possible additional risk due to inhalation in the lunar module of ultra-fine dust (<100 nm). This may trigger endothelial dysfunction by mechanisms similar to those shown to precipitate endothelial insults complicating ultra-fine urban dust exposure. Vascular constriction and a significant increase in diastolic blood pressures have been found in subjects inhaling urban dust within just two hours, possibly triggered by oxidative stress, inflammatory effects, and calcium overload with a potential magnesium ion deficit playing an important contributing role. Both Irwin and Scott on Apollo 15, experienced arrhythmias, and in Irwin's case associated with syncope and severe dyspnea with angina during reentry. After the mission both had impairment in cardiac function, and delay in cardiovascular recovery, with Irwin in addition having stress test- induced extremely high blood pressures, with no available stress test results in Scott's case for comparison. It is conceivable that the chemical nature or particle size of the lunar dust is sufficiently variable to account for these complications, which were not described on the other Apollo missions. This could be determined by non-invasive endothelial-dependent flow-mediated dilatation studies in the lunar environment at various sites, thereby determining the site with the least endothelial vulnerability to dysfunction. These studies could be used also to demonstrate possible intensification of endothelial dysfunction from inhalation of ultra-fine moon dust in the lunar module.

The effects of spatial inhomogeneities on flow through the endothelial surface layer.

PubMed

Leiderman, Karin M; Miller, Laura A; Fogelson, Aaron L

2008-05-21

Flow through the endothelial surface layer (the glycocalyx and adsorbed plasma proteins) plays an important but poorly understood role in cell signaling through a process known as mechanotransduction. Characterizing the flow rates and shear stresses throughout this layer is critical for understanding how flow-induced ionic currents, deformations of transmembrane proteins, and the convection of extracellular molecules signal biochemical events within the cell, including cytoskeletal rearrangements, gene activation, and the release of vasodilators. Previous mathematical models of flow through the endothelial surface layer are based upon the assumptions that the layer is of constant hydraulic permeability and constant height. These models also assume that the layer is continuous across the endothelium and that the layer extends into only a small portion of the vessel lumen. Results of these models predict that fluid shear stress is dissipated through the surface layer and is thus negligible near endothelial cell membranes. In this paper, such assumptions are removed, and the resultant flow rates and shear stresses through the layer are described. The endothelial surface layer is modeled as clumps of a Brinkman medium immersed in a Newtonian fluid. The width and spacing of each clump, hydraulic permeability, and fraction of the vessel lumen occupied by the layer are varied. The two-dimensional Navier-Stokes equations with an additional Brinkman resistance term are solved using a projection method. Several fluid shear stress transitions in which the stress at the membrane shifts from low to high values are described. These transitions could be significant to cell signaling since the endothelial surface layer is likely dynamic in its composition, density, and height.

Effects of six-month supplementation with beta-hydroxy-beta-methylbutyrate, glutamine, and arginine on vascular endothelial function of older adults

PubMed Central

Ellis, Amy; Patterson, Morgan; Dudenbostel, Tanja; Calhoun, David; Gower, Barbara

2015-01-01

Background Vascular endothelial function declines with advancing age, due in part to increased oxidative stress and inflammation, and this age-related vascular dysfunction has been identified as an independent risk factor for cardiovascular diseases (CVD). This double-blind, placebo-controlled trial investigated the effects of a dietary supplement containing β-hydroxy-β-methylbutyrate (HMB), glutamine, and arginine on endothelial-dependent vasodilation of older adults. Subjects/Methods Thirty-one community-dwelling men and women aged 65-87 years were randomly assigned to two groups. The treatment group received two doses of the supplement daily (totaling 3g HMB, 14g glutamine, 14g arginine) for six months while the control group received an isocaloric placebo. At baseline and week 24, vascular endothelial function was measured by flow-mediated dilation of the brachial artery, and fasting blood samples were obtained to measure high-sensitivity C-reactive protein (hsCRP) and tumor necrosis factor-α (TNF-α). Results Paired samples t-tests revealed a 27% increase in flow-mediated dilation among the treatment group (p=0.003) while no change was observed in the placebo group (p=0.651). Repeated-measures ANOVA verified a significant time by group interaction (p=0.038). Although no significant changes were observed for hsCRP or TNF-α, a trend was observed for increasing hsCRP among the placebo group only (p=0.059). Conclusions These results suggest that dietary supplementation of HMB, glutamine, and arginine may favorably impact vascular endothelial function in older adults. Additional studies are needed to elucidate whether reduced inflammation or other mechanisms may underlie the benefits of supplementation. PMID:26306566

Effects of 6-month supplementation with β-hydroxy-β-methylbutyrate, glutamine and arginine on vascular endothelial function of older adults.

PubMed

Ellis, A C; Patterson, M; Dudenbostel, T; Calhoun, D; Gower, B

2016-02-01

Vascular endothelial function declines with advancing age, due in part to increased oxidative stress and inflammation, and this age-related vascular dysfunction has been identified as an independent risk factor for cardiovascular diseases. This double-blind, placebo-controlled trial investigated the effects of a dietary supplement containing β-hydroxy-β-methylbutyrate (HMB), glutamine and arginine on endothelial-dependent vasodilation of older adults. A total of 31 community-dwelling men and women aged 65-87 years were randomly assigned to two groups. The treatment group received two doses of the supplement daily (totaling 3 g HMB, 14 g glutamine and 14 g arginine) for 6 months, whereas the control group received an isocaloric placebo. At baseline and week 24, vascular endothelial function was measured by flow-mediated dilation of the brachial artery, and fasting blood samples were obtained to measure high-sensitivity C-reactive protein (hsCRP) and tumor necrosis factor-α (TNF-α). Paired sample t-tests revealed a 27% increase in flow-mediated dilation among the treatment group (P=0.003), whereas no change was observed in the placebo group (P=0.651). Repeated-measures analysis of variance verified a significant time by group interaction (P=0.038). Although no significant changes were observed for hsCRP or TNF-α, a trend was observed for increasing hsCRP among the placebo group only (P=0.059). These results suggest that dietary supplementation of HMB, glutamine and arginine may favorably affect vascular endothelial function in older adults. Additional studies are needed to elucidate whether reduced inflammation or other mechanisms may underlie the benefits of supplementation.

Effect of repaglinide on endothelial dysfunction during a glucose tolerance test in subjects with impaired glucose tolerance

PubMed Central

Schmoelzer, Isabella; Wascher, Thomas C

2006-01-01

Background Impaired glucose tolerance (IGT) is associated with increased cardiovascular risk. The pathophysiological mechanisms linking post-challenge hyperglycemia to accelerated atherosclerosis, however remain to be elucidated. Methods A prospective, open, randomised, cross-over study was performed to investigate the effect of 2 mg repaglinide on hyperglycemia and endothelial function during an oral glucose tolerance test (75 g glucose) in 12 subjects with diagnosed IGT. Blood samples for determination of plasma glucose were drawn fasting, 1 and 2 hours after glucose ingestion. Endothelial function was assessed by measuring flow-mediated dilatation (FMD) of the brachial artery with high-resolution ultrasound. Results Administration of repaglinide resulted in a significant reduction of plasma glucose at 2 hours (172.8+/-48.4 vs. 138.3+/-41.2 mg/dl; p < 0.001). The flow-mediated dilatation (FMD) 2 hours after the glucose-load was significantly reduced in comparison to fasting in the control group (6.21+/-2.69 vs. 7.98+/-2.24 %; p = 0.028), whereas after theadministration of repaglinide the FMD was not significantly different to fasting values (7.24+/-2.57 vs. 8.18+/-2.93 %; p = n.s.). Linear and logistic regression analysis revealed that only the change of glucose was significantly correlated to the change of FMD observed (p < 0.001). Regression analysis after grouping for treatment and time confirmed the strong negative association of the changes of plasma glucose and FMD and indicate that the effect of repaglinide observed is based on the reduction glycemia. Conclusion In subjects with IGT, the endothelial dysfunction observed after a glucose challenge is related to the extent of hyperglycemia. Reduction of hyperglycemia by repaglinide reduces endothelial dysfunction in a glucose dependent manner. PMID:16606452

Effect of repaglinide on endothelial dysfunction during a glucose tolerance test in subjects with impaired glucose tolerance.

PubMed

Schmoelzer, Isabella; Wascher, Thomas C

2006-04-10

Impaired glucose tolerance (IGT) is associated with increased cardiovascular risk. The pathophysiological mechanisms linking post-challenge hyperglycemia to accelerated atherosclerosis, however remain to be elucidated. A prospective, open, randomised, cross-over study was performed to investigate the effect of 2 mg repaglinide on hyperglycemia and endothelial function during an oral glucose tolerance test (75 g glucose) in 12 subjects with diagnosed IGT. Blood samples for determination of plasma glucose were drawn fasting, 1 and 2 hours after glucose ingestion. Endothelial function was assessed by measuring flow-mediated dilatation (FMD) of the brachial artery with high-resolution ultrasound. Administration of repaglinide resulted in a significant reduction of plasma glucose at 2 hours (172.8+/-48.4 vs. 138.3+/-41.2 mg/dl; p < 0.001). The flow-mediated dilatation (FMD) 2 hours after the glucose-load was significantly reduced in comparison to fasting in the control group (6.21+/-2.69 vs. 7.98+/-2.24 %; p = 0.028), whereas after theadministration of repaglinide the FMD was not significantly different to fasting values (7.24+/-2.57 vs. 8.18+/-2.93 %; p = n.s.). Linear and logistic regression analysis revealed that only the change of glucose was significantly correlated to the change of FMD observed (p < 0.001). Regression analysis after grouping for treatment and time confirmed the strong negative association of the changes of plasma glucose and FMD and indicate that the effect of repaglinide observed is based on the reduction glycemia. In subjects with IGT, the endothelial dysfunction observed after a glucose challenge is related to the extent of hyperglycemia. Reduction of hyperglycemia by repaglinide reduces endothelial dysfunction in a glucose dependent manner.

Nitric Oxide-Mediated Coronary Flow Regulation in Patients with Coronary Artery Disease: Recent Advances

PubMed Central

Toda, Noboru; Tanabe, Shinichi; Nakanishi, Sadanobu

2011-01-01

Nitric oxide (NO) formed via endothelial NO synthase (eNOS) plays crucial roles in the regulation of coronary blood flow through vasodilatation and decreased vascular resistance, and in inhibition of platelet aggregation and adhesion, leading to the prevention of coronary circulatory failure, thrombosis, and atherosclerosis. Endothelial function is impaired by several pathogenic factors including smoking, chronic alcohol intake, hypercholesterolemia, obesity, hyperglycemia, and hypertension. The mechanisms underlying endothelial dysfunction include reduced NO synthase (NOS) expression and activity, decreased NO bioavailability, and increased production of oxygen radicals and endogenous NOS inhibitors. Atrial fibrillation appears to be a risk factor for endothelial dysfunction. Endothelial dysfunction is an important predictor of coronary artery disease (CAD) in humans. Penile erectile dysfunction, associated with impaired bioavailability of NO produced by eNOS and neuronal NOS, is also considered to be highly predictive of ischemic heart disease. There is evidence suggesting an important role of nitrergic innervation in coronary blood flow regulation. Prophylactic and therapeutic measures to eliminate pathogenic factors inducing endothelial and nitrergic nerve dysfunction would be quite important in preventing the genesis and development of CAD. PMID:22942627

No Evidence of Racial Differences in Endothelial Function and Exercise Blood Flow in Young, Healthy Males Following Acute Antioxidant Supplementation.

PubMed

Kappus, Rebecca M; Bunsawat, Kanokwan; Rosenberg, Alexander J; Fernhall, Bo

2017-03-01

This study investigated the effects of acute antioxidant supplementation on endothelial function, exercise blood flow and oxidative stress biomarkers in 9 young African American compared to 10 Caucasian males (25.7±1.2 years). We hypothesized that African American males would have lower exercise blood flow and endothelial responsiveness compared to Caucasian males, and these responses would be improved following antioxidant supplementation. Ultrasonography was used to measure blood flow during handgrip exercise. Endothelial function was assessed using flow-mediated dilation, and lipid peroxidation was assessed by measuring levels of malondialdehyde-thiobarbituric acid reactive substances. African American males exhibited lower endothelial function than Caucasians at baseline (8.3±1.7 vs. 12.2±1.7%) and the difference was ameliorated with antioxidant supplementation (10.7±1.9% vs. 10.8±1.8%), but the interaction was not significant (p=0.10). There were no significant changes in malondialdehyde-thiobarbituric acid reactive substances following antioxidant supplementation. There was a significant increase in brachial blood flow and forearm vascular conductance with exercise but no differences with antioxidant supplementation. There were no group differences in exercise responses and no differences with antioxidant supplementation, suggesting a lack of influence of oxidative stress during exercise in this cohort. © Georg Thieme Verlag KG Stuttgart · New York.

Intimal cushions and endothelial nuclear elongation around mouse aortic branches and their spatial correspondence with patterns of lipid deposition

PubMed Central

Bond, Andrew R.; Ni, Chih-Wen; Jo, Hanjoong

2010-01-01

Spatial variation in hemodynamic stresses acting on the arterial wall may explain the nonuniform distribution of atherosclerosis. In thoracic aortas of LDL receptor/apolipoprotein E double knockout mice, lesions develop preferentially around the entire circumference of intercostal branch ostia, regardless of age, with the highest prevalence occurring upstream. Additional chevron-shaped lesions occur further upstream of the ostia. This pattern differs from the age-related ones occurring in people and rabbits. In the present study, patterns of near-wall blood flow around intercostal ostia in wild-type mice were estimated from the morphology of endothelial nuclei, which were shown in vitro to elongate in response to elevated shear stress and to align with the flow, and wall structure was assessed from confocal and scanning electron microscopy. A triangular intimal cushion surrounded the upstream part of most ostia. Nuclear length-to-width ratios were lowest over this cushion and highest at the sides of branches, regardless of age. Nuclear orientations were consistent with flow diverging around the branch. The pattern of nuclear morphology differed from the age-related ones observed in rabbits. The intimal cushion and the distribution of shear stress inferred from these observations can partly account for the pattern of lesions observed in knockout mice. Nuclear elongation in nonbranch regions was approximately constant across animals of different size, demonstrating the existence of a mechanism by which endothelial cells compensate for the dependence of mean aortic wall shear stress on body mass. PMID:19933414

In Vitro Activation of eNOS by Mangifera indica (Careless™) and Determination of an Effective Dosage in a Randomized, Double-Blind, Human Pilot Study on Microcirculation.

PubMed

Gerstgrasser, Alexandra; Röchter, Sigrid; Dressler, Dirk; Schön, Christiane; Reule, Claudia; Buchwald-Werner, Sybille

2016-03-01

Mangifera indica fruit preparation (Careless™) activates the evolutionary conserved metabolic sensors sirtuin 1 and adenosine monophosphate-activated protein kinase, which have been identified as playing a key role in microcirculation and endothelial function. Here, an acute effect of a single dose of 100 mg or 300 mg Careless™ on microcirculation was investigated in a randomized, double-blind, crossover pilot study in ten healthy women to determine the effective dosage. Microcirculation and endothelial function were assessed by the Oxygen-to-see system and pulse amplitude tonometry (EndoPAT™), respectively. Cutaneous blood flow was increased over time by 100 mg (54% over pre-values, p = 0.0157) and 300 mg (35% over pre-value, p = 0.209) Careless™. The EndoPAT™ reactive hyperemia response was slightly improved 3 h after intake compared to pretesting with 300 mg Careless™. Furthermore, activation of endothelial nitric oxide synthase, as an important regulator for endothelial function, was tested in vitro in primary human umbilical vein endothelial cells. Careless™, after simulation of digestion, increased the activated form of endothelial nitric oxide synthase dose-dependently by 23% (300 µg/mL), 42% (1500 µg/mL), and 60% (3000 µg/mL) compared to the untreated control. In conclusion, the study suggests moderate beneficial effects of Careless™ on microcirculation, which is at least partly mediated by endothelial nitric oxide synthase activation. Georg Thieme Verlag KG Stuttgart · New York.

Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo. Potential Consequences in Chronic Obstructive Pulmonary Disease

PubMed Central

Marques, Patrice; Collado, Aida; Escudero, Paula; Rius, Cristina; González, Cruz; Servera, Emilio; Piqueras, Laura; Sanz, Maria-Jesus

2017-01-01

Cardiovascular disease (CVD) is a major comorbidity in chronic obstructive pulmonary disease (COPD). Although the mechanism of its development remains largely unknown, it appears to be associated with cigarette consumption and reduced lung function. Therefore, the aim of this study was to investigate the potential link between water-soluble cigarette smoke extract (CSE)-induced endothelial dysfunction and the function of CXCL16/CXCR6 axis on the initial attachment of leukocytes, in addition to its possible impact on COPD-associated systemic inflammation. To do this, we employed several experimental approaches, including RNA silencing and flow cytometry analysis, the dynamic flow chamber technique, and intravital microscopy in the cremasteric arterioles of animals exposed to cigarette smoke (CS). CSE-induced arterial CXCL16 expression, leading to increased platelet–leukocyte and mononuclear cell adhesiveness. CSE-induced CXCL16 expression was dependent on Nox5 expression and subsequent RhoA/p38 MAPK/NF-κB activation. Flow cytometry analysis revealed that COPD patients (n = 35) presented greater numbers of activated circulating platelets (PAC-1+ and P-selectin+) expressing CXCL16 and CXCR6 as compared with age-matched controls (n = 17), with a higher number of CXCR6+-platelets in the smoking COPD group than in ex-smokers. This correlated with enhanced circulating CXCR6+-platelet–leukocyte aggregates in COPD patients. The increase in circulating numbers of CXCR6-expressing platelets and mononuclear cells resulted in enhanced platelet–leukocyte and leukocyte adhesiveness to CSE-stimulated arterial endothelium, which was greater than that found in age-matched controls and was partly dependent on endothelial CXCL16 upregulation. Furthermore, CS exposure provoked CXCL16-dependent leukocyte–arteriolar adhesion in cremasteric arterioles, which was significantly reduced in animals with a nonfunctional CXCR6 receptor. In conclusion, we provide the first evidence that increased numbers of CXCR6-expressing circulating platelets and mononuclear leukocytes from patients with COPD might be a marker of systemic inflammation with potential consequences in CVD development. Accordingly, CXCL16/CXCR6 axis blockade might constitute a new therapeutic approach for decreasing the risk of CVD in COPD patients. PMID:29326688

Cigarette Smoke Increases Endothelial CXCL16-Leukocyte CXCR6 Adhesion In Vitro and In Vivo. Potential Consequences in Chronic Obstructive Pulmonary Disease.

PubMed

Marques, Patrice; Collado, Aida; Escudero, Paula; Rius, Cristina; González, Cruz; Servera, Emilio; Piqueras, Laura; Sanz, Maria-Jesus

2017-01-01

Cardiovascular disease (CVD) is a major comorbidity in chronic obstructive pulmonary disease (COPD). Although the mechanism of its development remains largely unknown, it appears to be associated with cigarette consumption and reduced lung function. Therefore, the aim of this study was to investigate the potential link between water-soluble cigarette smoke extract (CSE)-induced endothelial dysfunction and the function of CXCL16/CXCR6 axis on the initial attachment of leukocytes, in addition to its possible impact on COPD-associated systemic inflammation. To do this, we employed several experimental approaches, including RNA silencing and flow cytometry analysis, the dynamic flow chamber technique, and intravital microscopy in the cremasteric arterioles of animals exposed to cigarette smoke (CS). CSE-induced arterial CXCL16 expression, leading to increased platelet-leukocyte and mononuclear cell adhesiveness. CSE-induced CXCL16 expression was dependent on Nox5 expression and subsequent RhoA/p38 MAPK/NF-κB activation. Flow cytometry analysis revealed that COPD patients ( n  = 35) presented greater numbers of activated circulating platelets (PAC-1 + and P-selectin + ) expressing CXCL16 and CXCR6 as compared with age-matched controls ( n  = 17), with a higher number of CXCR6 + -platelets in the smoking COPD group than in ex-smokers. This correlated with enhanced circulating CXCR6 + -platelet-leukocyte aggregates in COPD patients. The increase in circulating numbers of CXCR6-expressing platelets and mononuclear cells resulted in enhanced platelet-leukocyte and leukocyte adhesiveness to CSE-stimulated arterial endothelium, which was greater than that found in age-matched controls and was partly dependent on endothelial CXCL16 upregulation. Furthermore, CS exposure provoked CXCL16-dependent leukocyte-arteriolar adhesion in cremasteric arterioles, which was significantly reduced in animals with a nonfunctional CXCR6 receptor. In conclusion, we provide the first evidence that increased numbers of CXCR6-expressing circulating platelets and mononuclear leukocytes from patients with COPD might be a marker of systemic inflammation with potential consequences in CVD development. Accordingly, CXCL16/CXCR6 axis blockade might constitute a new therapeutic approach for decreasing the risk of CVD in COPD patients.

In vitro Flow Adhesion Assay for Analyzing Shear-resistant Adhesion of Metastatic Cancer Cells to Endothelial Cells.

PubMed

Kang, Shin-Ae; Bajana, Sandra; Tanaka, Takemi

2016-02-20

Hematogenous metastasis is a primary cause of mortality from metastatic cancer. The shear-resistant adhesion of circulating tumor cells to the vascular endothelial cell surface under blood flow is an essential step in cell extravasation and further tissue invasion. This is similar to a process exploited by leukocytes for adhesion to inflamed blood vessels (leukocyte mimicry). The shear resistant adhesion is mediated by high affinity interactions between endothelial adhesion molecules and their counter receptor ligand expressed on circulating cells. Thus, weak interaction results in a rapid detachment of circulating cells from endothelium. Despite the critical role of vascular adhesion of cancer cells in hematogenous metastasis, our knowledge regarding this process has been limited due to the difficulty of mimicking dynamic flow conditions in vitro . In order to gain better insight into the shear-resistant adhesion of cancer cells to the endothelium, we developed a protocol for measuring the shear resistant adhesion of circulating tumor cells to endothelial cells under physiologic flow conditions by adapting a well established flow adhesion assay for inflammatory cells. This technique is useful to evaluate 1) the shear resistant adhesion competency of cancer cells and 2) the endothelial adhesion molecules necessary to support cancer cell adhesion (Kang et al. , 2015).

Non-nucleoside reverse transcriptase inhibitor efavirenz increases monolayer permeability of human coronary artery endothelial cells

PubMed Central

Jamaluddin, Md Saha; Lin, Peter H.; Yao, Qizhi; Chen, Changyi

2009-01-01

Highly active antiretroviral therapy (HAART) is often associated with endothelial dysfunction and cardiovascular complications. In this study, we determined whether HIV non-nucleoside reverse transcriptase inhibitor efavirenz (EFV) could increase endothelial permeability. Human coronary artery endothelial cells (HCAECs) were treated with EFV (1, 5 and 10 µg/ml) and endothelial permeability was determined by a transwell system with a fluorescence-labeled dextran tracer. HCAECs treated with EFV showed a significant increase of endothelial permeability in a concentration-dependent manner. With real time PCR analysis, EFV significantly reduced the mRNA levels of tight junction proteins claudin-1, occludin, zonula occluden-1 and junctional adhesion molecule-1 compared with controls (P < 0.05). Protein levels of these tight junction molecules were also reduced substantially in the EFV-treated cells by western blot and flow cytometry analyses. In addition, EFV also increased superoxide anion production with dihydroethidium and cellular glutathione assays, while it decreased mitochondrial membrane potential with JC-staining. Antioxidants (ginkgolide B and MnTBAP) effectively blocked EFV-induced endothelial permeability and mitochondrial dysfunction. Furthermore, EFV increased the phosphorylation of MAPK JNK and IκBα, thereby increasing NFκB translocation to the nucleus. Chemical JNK inhibitor and dominant negative mutant JNK and IkBa adenoviruses effectively blocked the effects of EFV on HCAECs. Thus, EFV increases endothelial permeability which may be due to the decrease of tight junction proteins and the increase of superoxide anion. JNK and NFκB activation may be directly involved in the signal transduction pathway of EFV action in HCAECs. PMID:19674747

Regional heterogeneity of endothelial cells in the porcine vortex vein system.

PubMed

Tan, Priscilla Ern Zhi; Yu, Paula K; Cringle, Stephen J; Morgan, William H; Yu, Dao-Yi

2013-09-01

The aim of this study was to investigate whether region-dependent endothelial heterogeneity is present within the porcine vortex vein system. The superior temporal vortex vein in young adult pig eyes were dissected out and cannulated. The intact vortex vein system down to the choroidal veins was then perfused with labels for f-actin and nucleic acid. The endothelial cells within the choroidal veins, pre-ampulla, anterior portion of the ampulla, mid-ampulla, posterior portion of the ampulla, post-ampulla, intra-scleral canal and the extra-ocular vortex vein regions were studied in detail using a confocal microscopy technique. The endothelial cell and nuclei length, width, area and perimeter were measured and compared between the different regions. Significant regional differences in the endothelial cell and nuclei length, width, area and perimeter were observed throughout the porcine vortex vein system. Most notably, very narrow and elongated endothelia were found in the post-ampulla region. A lack of smooth muscle cells was noted in the ampulla region compared to other regions. Heterogeneity in endothelial cell morphology is present throughout the porcine vortex vein system and there is a lack of smooth muscle cells in the ampulla region. This likely reflects the highly varied haemodynamic conditions and potential blood flow control mechanisms in different regions of the vortex vein system. Copyright © 2013 Elsevier Inc. All rights reserved.

Shear stress-induced mitochondrial biogenesis decreases the release of microparticles from endothelial cells.

PubMed

Kim, Ji-Seok; Kim, Boa; Lee, Hojun; Thakkar, Sunny; Babbitt, Dianne M; Eguchi, Satoru; Brown, Michael D; Park, Joon-Young

2015-08-01

The concept of enhancing structural integrity of mitochondria has emerged as a novel therapeutic option for cardiovascular disease. Flow-induced increase in laminar shear stress is a potent physiological stimulant associated with exercise, which exerts atheroprotective effects in the vasculature. However, the effect of laminar shear stress on mitochondrial remodeling within the vascular endothelium and its related functional consequences remain largely unknown. Using in vitro and in vivo complementary studies, here, we report that aerobic exercise alleviates the release of endothelial microparticles in prehypertensive individuals and that these salutary effects are, in part, mediated by shear stress-induced mitochondrial biogenesis. Circulating levels of total (CD31(+)/CD42a(-)) and activated (CD62E(+)) microparticles released by endothelial cells were significantly decreased (∼40% for both) after a 6-mo supervised aerobic exercise training program in individuals with prehypertension. In cultured human endothelial cells, laminar shear stress reduced the release of endothelial microparticles, which was accompanied by an increase in mitochondrial biogenesis through a sirtuin 1 (SIRT1)-dependent mechanism. Resveratrol, a SIRT1 activator, treatment showed similar effects. SIRT1 knockdown using small-interfering RNA completely abolished the protective effect of shear stress. Disruption of mitochondrial integrity by either antimycin A or peroxisome proliferator-activated receptor-γ coactivator-1α small-interfering RNA significantly increased the number of total, and activated, released endothelial microparticles, and shear stress restored these back to basal levels. Collectively, these data demonstrate a critical role of endothelial mitochondrial integrity in preserving endothelial homeostasis. Moreover, prolonged laminar shear stress, which is systemically elevated during aerobic exercise in the vessel wall, mitigates endothelial dysfunction by promoting mitochondrial biogenesis. Copyright © 2015 the American Physiological Society.

Platelet-independent adhesion of calcium-loaded erythrocytes to von Willebrand factor

PubMed Central

Bierings, Ruben; Meems, Henriet; Mul, Frederik P. J.; Geerts, Dirk; Vlaar, Alexander P. J.; Voorberg, Jan; Hordijk, Peter L.

2017-01-01

Adhesion of erythrocytes to endothelial cells lining the vascular wall can cause vaso-occlusive events that impair blood flow which in turn may result in ischemia and tissue damage. Adhesion of erythrocytes to vascular endothelial cells has been described in multiple hemolytic disorders, especially in sickle cell disease, but the adhesion of normal erythrocytes to endothelial cells has hardly been described. It was shown that calcium-loaded erythrocytes can adhere to endothelial cells. Because sickle erythrocyte adhesion to ECs can be enhanced by ultra-large von Willebrand factor multimers, we investigated whether calcium loading of erythrocytes could promote binding to endothelial cells via ultra-large von Willebrand factor multimers. We used (immunofluorescent) live-cell imaging of washed erythrocytes perfused over primary endothelial cells at venular flow rate. Using this approach, we show that calcium-loaded erythrocytes strongly adhere to histamine-stimulated primary human endothelial cells. This adhesion is mediated by ultra-large von Willebrand factor multimers. Von Willebrand factor knockdown or ADAMTS13 cleavage abolished the binding of erythrocytes to activated endothelial cells under flow. Platelet depletion did not interfere with erythrocyte binding to von Willebrand factor. Our results reveal platelet-independent adhesion of calcium-loaded erythrocytes to endothelium-derived von Willebrand factor. Erythrocyte adhesion to von Willebrand factor may be particularly relevant for venous thrombosis, which is characterized by the formation of erythrocyte-rich thrombi. PMID:28249049

Endothelial progenitor cells in chronic obstructive pulmonary disease and emphysema

PubMed Central

Tracy, Russell P.; Parikh, Megha A.; Hoffman, Eric A.; Shimbo, Daichi; Austin, John H. M.; Smith, Benjamin M.; Hueper, Katja; Vogel-Claussen, Jens; Lima, Joao; Gomes, Antoinette; Watson, Karol; Kawut, Steven; Barr, R. Graham

2017-01-01

Endothelial injury is implicated in the pathogenesis of COPD and emphysema; however the role of endothelial progenitor cells (EPCs), a marker of endothelial cell repair, and circulating endothelial cells (CECs), a marker of endothelial cell injury, in COPD and its subphenotypes is unresolved. We hypothesized that endothelial progenitor cell populations would be decreased in COPD and emphysema and that circulating endothelial cells would be increased. Associations with other subphenotypes were examined. The Multi-Ethnic Study of Atherosclerosis COPD Study recruited smokers with COPD and controls age 50–79 years without clinical cardiovascular disease. Endothelial progenitor cell populations (CD34+KDR+ and CD34+KDR+CD133+ cells) and circulating endothelial cells (CD45dimCD31+CD146+CD133-) were measured by flow cytometry. COPD was defined by standard spirometric criteria. Emphysema was assessed qualitatively and quantitatively on CT. Full pulmonary function testing and expiratory CTs were measured in a subset. Among 257 participants, both endothelial progenitor cell populations, and particularly CD34+KDR+ endothelial progenitor cells, were reduced in COPD. The CD34+KDR+CD133+ endothelial progenitor cells were associated inversely with emphysema extent. Both endothelial progenitor cell populations were associated inversely with extent of panlobular emphysema and positively with diffusing capacity. Circulating endothelial cells were not significantly altered in COPD but were inversely associated with pulmonary microvascular blood flow on MRI. There was no consistent association of endothelial progenitor cells or circulating endothelial cells with measures of gas trapping. These data provide evidence that endothelial repair is impaired in COPD and suggest that this pathological process is specific to emphysema. PMID:28291826

Quantum dot interactions and flow effects in angiogenic zebrafish (Danio rerio) vessels and human endothelial cells.

PubMed

Jiang, Xiao-Yu; Sarsons, Christopher D; Gomez-Garcia, M Juliana; Cramb, David T; Rinker, Kristina D; Childs, Sarah J

2017-04-01

Nanoparticle (NP) interactions with biological tissues are affected by the size, shape and surface chemistry of the NPs. Here we use in vivo (zebrafish) and in vitro (HUVEC) models to investigate association of quantum dots (QDs) with endothelial cells and the effect of fluid flow. After injection into the developing zebrafish, circulating QDs associate with endothelium and penetrate surrounding tissue parenchyma over time. Amino-functionalized QDs cluster, interact with cells, and clear more rapidly than carboxy-functionalized QDs in vivo, highlighting charge influences. QDs show stronger accumulation in slow-flowing, small caliber venous vessels than in fast-flowing high caliber arterial vessels. Parallel-plate flow experiments with HUVEC support these findings, showing reduced QD-EC association with increasing flow. In vivo, flow arrest after nanoparticle injection still results in venous accumulation at 18 h. Overall our results suggest that both QD charge and blood flow modulate particle-endothelial cell interactions. Copyright © 2016 Elsevier Inc. All rights reserved.

Terrestrosin D, a steroidal saponin from Tribulus terrestris L., inhibits growth and angiogenesis of human prostate cancer in vitro and in vivo.

PubMed

Wei, Shihu; Fukuhara, Hideo; Chen, Guang; Kawada, Chiaki; Kurabayashi, Atsushi; Furihata, Mutsuo; Inoue, Keiji; Shuin, Taro

2014-01-01

The aim of this study was to investigate whether terrestrosin D (TED) inhibits the progression of castration-resistant prostate cancer and consider its mechanism. Cell cycle, mitochondrial membrane potential (ΔΨm) and apoptosis were determined by flow cytometry. Caspase-3 activity and vascular endothelial growth factor secretion were detected by a caspase-3 assay and human vascular endothelial growth factor kit, respectively. A PC-3 xenograft mouse model was used to evaluate the anticancer effect of TED in vivo. In vitro, TED strongly suppressed the growth of prostate cancer cells and endothelial cells in a dose-dependent manner. TED induced cell cycle arrest and apoptosis in PC-3 cells and human umbilical vascular endothelial cells (HUVECs). TED-induced apoptosis did not involve the caspase pathway. TED also decreased ΔΨm in PC-3 cells and HUVECs. In vivo, TED significantly suppressed tumor growth in nude mice bearing PC-3 cells, without any overt toxicity. Immunohistochemical analysis showed TED induced apoptotic cell death and inhibited angiogenesis in xenograft tumor cells. Cell cycle arrest and induction of apoptosis in cancer cells and endothelial cells might be plausible mechanisms of actions for the observed antitumor and antiangiogenic activities of TED. © 2014 S. Karger AG, Basel.

Beer elicits vasculoprotective effects through Akt/eNOS activation.

PubMed

Vilahur, Gemma; Casani, Laura; Mendieta, Guiomar; Lamuela-Raventos, Rosa M; Estruch, Ramon; Badimon, Lina

2014-12-01

There is controversy regarding the effect of alcohol beverage intake in vascular vasodilatory function in peripheral arteries. The effects of beer intake in coronary vasodilation remain unknown. We investigated whether regular beer intake (alcohol and alcohol-free) protects against hypercholesterolaemia-induced coronary endothelial dysfunction and the mechanisms behind this effect. Pigs were fed 10 days: (i) a Western-type hypercholesterolaemic diet (WD); (ii) WD+low-dose beer (12·5 g alcohol/day); (iii) WD+moderate-dose beer (25 g alcohol/day); or (iv) WD+moderate-dose alcohol-free-beer (0·0 g alcohol/day). Coronary responses to endothelium-dependent vasoactive drugs (acetylcholine: receptor mediated; calcium ionophore-A23189: nonreceptor mediated), endothelium-independent vasoactive drug (SNP) and L-NMMA (NOS-antagonist) were evaluated in the LAD coronary artery by flow Doppler. Coronary Akt/eNOS activation, MCP-1 expression, oxidative DNA damage and superoxide production were assessed. Lipid profile, lipoproteins resistance to oxidation and urinary isoxanthohumol concentration were evaluated. Alcoholic and nonalcoholic beer intake prevented WD-induced impairment of receptor- and non-receptor-operated endothelial-dependent coronary vasodilation. All animals displayed a similar vasodilatory response to SNP and L-NMMA blunted all endothelial-dependent vasorelaxation responses. Haemodynamic parameters remained unchanged. Coronary arteries showed lower DNA damage and increased Akt/eNOS axis activation in beer-fed animals. Animals taking beer showed HDL with higher antioxidant capacity, higher LDL resistance to oxidation and increased isoxanthohumol levels. Weight, lipids levels, liver enzymes and MCP-1 expression were not affected by beer intake. Non-alcoholic-related beer components protect against hyperlipemia-induced coronary endothelial dysfunction by counteracting vascular oxidative damage and preserving the Akt/eNOS pathway. Light-to-moderate beer consumption prevents and/or reduces the endothelial dysfunction associated with cardiovascular risk factors. © 2014 Stichting European Society for Clinical Investigation Journal Foundation.

Drag reduction by polyethylene glycol in the tail arterial bed of normotensive and hypertensive rats.

PubMed

Bessa, K L; Belletati, J F; Santos, L; Rossoni, L V; Ortiz, J P

2011-08-01

This study was designed to evaluate the effect of drag reducer polymers (DRP) on arteries from normotensive (Wistar) and spontaneously hypertensive rats (SHR). Polyethylene glycol (PEG 4000 at 5000 ppm) was perfused in the tail arterial bed with (E+) and without endothelium (E-) from male, adult Wistar (N = 14) and SHR (N = 13) animals under basal conditions (constant flow at 2.5 mL/min). In these preparations, flow-pressure curves (1.5 to 10 mL/min) were constructed before and 1 h after PEG 4000 perfusion. Afterwards, the tail arterial bed was fixed and the internal diameters of the arteries were then measured by microscopy and drag reduction was assessed based on the values of wall shear stress (WSS) by computational simulation. In Wistar and SHR groups, perfusion of PEG 4000 significantly reduced pulsatile pressure (Wistar/E+: 17.5 ± 2.8; SHR/E+: 16.3 ± 2.7%), WSS (Wistar/E+: 36; SHR/E+: 40%) and the flow-pressure response. The E- reduced the effects of PEG 4000 on arteries from both groups, suggesting that endothelial damage decreased the effect of PEG 4000 as a DRP. Moreover, the effects of PEG 4000 were more pronounced in the tail arterial bed from SHR compared to Wistar rats. In conclusion, these data demonstrated for the first time that PEG 4000 was more effective in reducing the pressure-flow response as well as WSS in the tail arterial bed of hypertensive than of normotensive rats and these effects were amplified by, but not dependent on, endothelial integrity. Thus, these results show an additional mechanism of action of this polymer besides its mechanical effect through the release and/or bioavailability of endothelial factors.

Sirtuin1 protects endothelial Caveolin-1 expression and preserves endothelial function via suppressing miR-204 and endoplasmic reticulum stress.

PubMed

Kassan, M; Vikram, A; Kim, Y R; Li, Q; Kassan, A; Patel, H H; Kumar, S; Gabani, M; Liu, J; Jacobs, J S; Irani, K

2017-02-09

Sirtuin1 (Sirt1) is a class III histone deacetylase that regulates a variety of physiological processes, including endothelial function. Caveolin1 (Cav1) is also an important determinant of endothelial function. We asked if Sirt1 governs endothelial Cav1 and endothelial function by regulating miR-204 expression and endoplasmic reticulum (ER) stress. Knockdown of Sirt1 in endothelial cells, and in vivo deletion of endothelial Sirt1, induced endothelial ER stress and miR-204 expression, reduced Cav1, and impaired endothelium-dependent vasorelaxation. All of these effects were reversed by a miR-204 inhibitor (miR-204 I) or with overexpression of Cav1. A miR-204 mimic (miR-204 M) decreased Cav1 in endothelial cells. In addition, high-fat diet (HFD) feeding induced vascular miR-204 and reduced endothelial Cav1. MiR-204-I protected against HFD-induced downregulation of endothelial Cav1. Moreover, pharmacologic induction of ER stress with tunicamycin downregulated endothelial Cav1 and impaired endothelium-dependent vasorelaxation that was rescued by overexpressing Cav1. In conclusion, Sirt1 preserves Cav1-dependent endothelial function by mitigating miR-204-mediated vascular ER stress.

Blood flow patterns during incremental and steady-state aerobic exercise.

PubMed

Coovert, Daniel; Evans, LeVisa D; Jarrett, Steven; Lima, Carla; Lima, Natalia; Gurovich, Alvaro N

2017-05-30

Endothelial shear stress (ESS) is a physiological stimulus for vascular homeostasis, highly dependent on blood flow patterns. Exercise-induced ESS might be beneficial on vascular health. However, it is unclear what type of ESS aerobic exercise (AX) produces. The aims of this study are to characterize exercise-induced blood flow patterns during incremental and steady-state AX. We expect blood flow pattern during exercise will be intensity-dependent and bidirectional. Six college-aged students (2 males and 4 females) were recruited to perform 2 exercise tests on cycleergometer. First, an 8-12-min incremental test (Test 1) where oxygen uptake (VO2), heart rate (HR), blood pressure (BP), and blood lactate (La) were measured at rest and after each 2-min step. Then, at least 48-hr. after the first test, a 3-step steady state exercise test (Test 2) was performed measuring VO2, HR, BP, and La. The three steps were performed at the following exercise intensities according to La: 0-2 mmol/L, 2-4 mmol/L, and 4-6 mmol/L. During both tests, blood flow patterns were determined by high-definition ultrasound and Doppler on the brachial artery. These measurements allowed to determine blood flow velocities and directions during exercise. On Test 1 VO2, HR, BP, La, and antegrade blood flow velocity significantly increased in an intensity-dependent manner (repeated measures ANOVA, p<0.05). Retrograde blood flow velocity did not significantly change during Test 1. On Test 2 all the previous variables significantly increased in an intensity-dependent manner (repeated measures ANOVA, p<0.05). These results support the hypothesis that exercise induced ESS might be increased in an intensity-dependent way and blood flow patterns during incremental and steady-state exercises include both antegrade and retrograde blood flows.

Glu298Asp polymorphism influences the beneficial effects of fish oil fatty acids on postprandial vascular function[S

PubMed Central

Thompson, Abby K.; Newens, Katie J.; Jackson, Kim G.; Wright, John; Williams, Christine M.

2012-01-01

Our objective was to determine whether the endothelial nitric oxide synthase (eNOS) Glu298Asp polymorphism influences vascular response to raised NEFA enriched with saturated fatty acids (SFA) or long-chain (LC) n-3 polyunsaturated fatty acids (PUFA). Subjects were prospectively recruited for genotype (Glu298, n = 30 and Asp298, n = 29; balanced for age and gender) consumed SFA on two occasions, with and without the substitution of 0.07 g fat/kg body weight with LC n-3 PUFA, and with heparin infusion to elevate NEFA. Endothelial function was measured before and after NEFA elevation (240 min), with blood samples taken every 30 min. Flow-mediated dilation (FMD) decreased following SFA alone and increased following SFA+LC n-3 PUFA. There were 2-fold differences in the change in FMD response to the different fat loads between the Asp298 and Glu298 genotypes (P = 0.002) and between genders (P < 0.02). Sodium nitroprusside-induced reactivity, measured by laser Doppler imaging with iontophoresis, was significantly greater with SFA+LC n-3 PUFA in all female subjects (P < 0.001) but not in males. Elevated NEFA influences both endothelial-dependent and endothelial-independent vasodilation during the postprandial phase. Effects of fat composition appear to be genotype and gender dependent, with the greatest difference in vasodilatory response to the two fat loads seen in the Asp298 females. PMID:22847178

The Effects of Endurance Exercise Training on the Coronary Vascular Responsiveness to Intracoronary Acetylcholine in Swine

DTIC Science & Technology

1993-04-09

systems. The mechanisms of sympathet ic innervation involve a-adrenergic-mediated coronary vascular smooth muscle contraction, and (1- adrenergic-mediated...may cause muscarinic-mediated relaxation or contraction of vascular smooth muscle , depending on the animal species and presence of endothelial...both cardiac muscle layers receive equal flows over a cardiac cycle, regardless of the differences from systo lic compression (Buckberg and Kattus

Microfluidically supported biochip design for culture of endothelial cell layers with improved perfusion conditions.

PubMed

Raasch, Martin; Rennert, Knut; Jahn, Tobias; Peters, Sven; Henkel, Thomas; Huber, Otmar; Schulz, Ingo; Becker, Holger; Lorkowski, Stefan; Funke, Harald; Mosig, Alexander

2015-03-02

Hemodynamic forces generated by the blood flow are of central importance for the function of endothelial cells (ECs), which form a biologically active cellular monolayer in blood vessels and serve as a selective barrier for macromolecular permeability. Mechanical stimulation of the endothelial monolayer induces morphological remodeling in its cytoskeleton. For in vitro studies on EC biology culture devices are desirable that simulate conditions of flow in blood vessels and allow flow-based adhesion/permeability assays under optimal perfusion conditions. With this aim we designed a biochip comprising a perfusable membrane that serves as cell culture platform multi-organ-tissue-flow (MOTiF biochip). This biochip allows an effective supply with nutrition medium, discharge of catabolic cell metabolites and defined application of shear stress to ECs under laminar flow conditions. To characterize EC layers cultured in the MOTiF biochip we investigated cell viability, expression of EC marker proteins and cell adhesion molecules of ECs dynamically cultured under low and high shear stress, and compared them with an endothelial culture in established two-dimensionally perfused flow chambers and under static conditions. We show that ECs cultured in the MOTiF biochip form a tight EC monolayer with increased cellular density, enhanced cell layer thickness, presumably as the result of a rapid and effective adaption to shear stress by remodeling of the cytoskeleton. Moreover, endothelial layers in the MOTiF biochip express higher amounts of EC marker proteins von-Willebrand-factor and PECAM-1. EC layers were highly responsive to stimulation with TNFα as detected at the level of ICAM-1, VCAM-1 and E-selectin expression and modulation of endothelial permeability in response to TNFα/IFNγ treatment under flow conditions. Compared to static and two-dimensionally perfused cell culture condition we consider MOTiF biochips as a valuable tool for studying EC biology in vitro under advanced culture conditions more closely resembling the in vivo situation.

Coffee bean polyphenols ameliorate postprandial endothelial dysfunction in healthy male adults.

PubMed

Ochiai, Ryuji; Sugiura, Yoko; Otsuka, Kazuhiro; Katsuragi, Yoshihisa; Hashiguchi, Teruto

2015-05-01

To reveal the effect of coffee bean polyphenols (CBPs) on blood vessels, this study aimed to investigate the effect of CBPs on acute postprandial endothelial dysfunction. Thirteen healthy non-diabetic men (mean age, 44.9 ± 1.4 years) consumed a test beverage (active: containing CBPs, placebo: no CBPs) before a 554-kcal test meal containing 14 g of protein, 30 g of fat and 58 g of carbohydrates. Then, a crossover analysis was performed to investigate the time-dependent changes in flow-mediated dilation (FMD) in the brachial artery. In the active group, the postprandial impairment of FMD was significantly improved, the two-hour postprandial nitric oxide metabolite levels were significantly increased and the six-hour postprandial urinary 8-epi-prostaglandin F2α levels were significantly reduced compared to the placebo group. The test meal increased the levels of blood glucose, insulin and triglycerides in both groups with no significant intergroup differences. These findings indicate that CBPs intake ameliorates postprandial endothelial dysfunction in healthy men.

Traumatic Brain Injury Causes Endothelial Dysfunction in the Systemic Microcirculation through Arginase-1-Dependent Uncoupling of Endothelial Nitric Oxide Synthase.

PubMed

Villalba, Nuria; Sackheim, Adrian M; Nunez, Ivette A; Hill-Eubanks, David C; Nelson, Mark T; Wellman, George C; Freeman, Kalev

2017-01-01

Endothelial dysfunction is a hallmark of many chronic diseases, including diabetes and long-term hypertension. We show that acute traumatic brain injury (TBI) leads to endothelial dysfunction in rat mesenteric arteries. Endothelial-dependent dilation was greatly diminished 24 h after TBI because of impaired nitric oxide (NO) production. The activity of arginase, which competes with endothelial NO synthase (eNOS) for the common substrate l-arginine, were also significantly increased in arteries, suggesting that arginase-mediated depletion of l-arginine underlies diminished NO production. Consistent with this, substrate restoration by exogenous application of l-arginine or inhibition of arginase recovered endothelial function. Moreover, evidence for increased reactive oxygen species production, a consequence of l-arginine starvation-dependent eNOS uncoupling, was detected in endothelium and plasma. Collectively, our findings demonstrate endothelial dysfunction in a remote vascular bed after TBI, manifesting as impaired endothelial-dependent vasodilation, with increased arginase activity, decreased generation of NO, and increased O 2 - production. We conclude that blood vessels have a "molecular memory" of neurotrauma, 24 h after injury, because of functional changes in vascular endothelial cells; these effects are pertinent to understanding the systemic inflammatory response that occurs after TBI even in the absence of polytrauma.

RECOMBINANT FACTOR XIII DIMINISHES MULTIPLE ORGAN DYSFUNCTION IN RATS CAUSED BY GUT ISCHEMIA-REPERFUSION INJURY

PubMed Central

Zaets, Sergey B.; Xu, Da-Zhong; Lu, Qi; Feketova, Eleonora; Berezina, Tamara L.; Gruda, Maryann; Malinina, Inga V.; Deitch, Edwin A.; Olsen, Eva H. N.

2010-01-01

Plasma factor XIII (FXIII) is responsible for stabilization of fibrin clot at the final stage of blood coagulation. Because FXIII has also been shown to modulate inflammation and endothelial permeability, we hypothesized that FXIII diminishes multiple organ dysfunction caused by gut I/R injury. A model of superior mesenteric artery occlusion (SMAO) was used to induce gut I/R injury. Rats were subjected to 45-min SMAO or sham SMAO and treated with recombinant human FXIII A2 subunit (rFXIII) or placebo at the beginning of the reperfusion period. Lung permeability, lung and gut myeloperoxidase activity, gut histology, neutrophil respiratory burst, and microvascular blood flow in the liver and muscles were measured after a 3-h reperfusion period. The effect of activated rFXIII on transendothelial resistance of human umbilical vein endothelial cells was tested in vitro. Superior mesenteric artery occlusion–induced lung permeability as well as lung and gut myeloperoxidase activity was significantly lower in rFXIII-treated versus untreated animals. Similarly, rFXIII-treated rats had lower neutrophil respiratory burst activity and ileal mucosal injury. Rats treated with rFXIII also had higher liver microvascular blood flow compared with the placebo group. Superior mesenteric artery occlusion did not cause FXIII consumption during the study period. In vitro, activated rFXIII caused a dose-dependent increase in human umbilical vein endothelial cell monolayer resistance to thrombin-induced injury. Thus, administration of rFXIII diminishes SMAO-induced multiple organ dysfunction in rats, presumably by preservation of endothelial barrier function and the limitation of polymorphonuclear leukocyte activation. PMID:18948851

Normal endothelial function after meals rich in olive or safflower oil previously used for deep frying.

PubMed

Williams, M J; Sutherland, W H; McCormick, M P; Yeoman, D; de Jong, S A; Walker, R J

2001-06-01

Polyunsaturated fats are more susceptible to oxidation during heating than monounsaturated fats but their effects on endothelial function when heated are unknown. The aim of this study was to compare the effect of meals rich in heat-modified safflower and olive oils on postprandial flow-mediated endothelium-dependent dilation (EDD) in healthy men. Flow-mediated EDD and glyceryltrinitrate-induced endothelium-independent dilation of the brachial artery were investigated in 14 subjects before and 4 hours after meals rich in olive oil and safflower oil used hourly for deep-frying for 8 hours in a double-blind crossover study design. There were high levels of lipid oxidation products (peroxides and carbonyls) in both heated oils. Plasma triglycerides were markedly increased at 4 hours after heated olive oil (1.26 +/- 0.43 vs 2.06 +/- 0.97 mmol/L) and heated safflower oil (1.44 +/- 0.63 vs 1.99 +/- 0.88 mmol/L). There was no change in EDD between fasting and postprandial studies and the response during the postprandial period was not significantly (p = 0.51) different between the meals (heated olive oil: 4.9 +/- 2.2% vs 4.9 +/- 2.5%; heated safflower oil: 5.1 +/- 3.1% vs 5.6 +/- 3.4%). Meals rich in olive and safflower oils previously used for deep frying and containing high levels of lipid oxidation products increase postprandial serum triglycerides without affecting endothelial function. These findings suggest that relatively short-term use of these vegetable oils for frying may not adversely affect postprandial endothelial function when foods containing the heat-modified oils are consumed.

Endothelial microparticle uptake in target cells is annexin I/phosphatidylserine receptor dependent and prevents apoptosis.

PubMed

Jansen, Felix; Yang, Xiaoyan; Hoyer, Friedrich Felix; Paul, Kathrin; Heiermann, Nadine; Becher, Marc Ulrich; Abu Hussein, Nebal; Kebschull, Moritz; Bedorf, Jörg; Franklin, Bernardo S; Latz, Eicke; Nickenig, Georg; Werner, Nikos

2012-08-01

Endothelial microparticles (EMP) are released from activated or apoptotic cells, but their effect on target cells and the exact way of incorporation are largely unknown. We sought to determine the uptake mechanism and the biological effect of EMP on endothelial and endothelial-regenerating cells. EMP were generated from starved endothelial cells and isolated by ultracentrifugation. Caspase 3 activity assay and terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that EMP protect target endothelial cells against apoptosis in a dose-dependent manner. Proteomic analysis was performed to identify molecules contained in EMP, which might be involved in EMP uptake. Expression of annexin I in EMP was found and confirmed by Western blot, whereas the corresponding receptor phosphatidylserine receptor was present on endothelial target cells. Silencing either annexin I on EMP or phosphatidylserine receptor on target cells using small interfering RNA showed that the uptake of EMP by human coronary artery endothelial cells is annexin I/phosphatidylserine receptor dependent. Annexin I-downregulated EMP abrogated the EMP-mediated protection against apoptosis of endothelial target cells. p38 activation was found to mediate camptothecin-induced apoptosis. Finally, human coronary artery endothelial cells pretreated with EMP inhibited camptothecin-induced p38 activation. EMP are incorporated by endothelial cells in an annexin I/phosphatidylserine receptor-dependent manner and protect target cells against apoptosis. Inhibition of p38 activity is involved in EMP-mediated protection against apoptosis.

AUGMENTATION OF MUSCLE BLOOD FLOW BY ULTRASOUND CAVITATION IS MEDIATED BY ATP AND PURINERGIC SIGNALING

PubMed Central

Belcik, J. Todd; Davidson, Brian P.; Xie, Aris; Wu, Melinda D.; Yadava, Mrinal; Qi, Yue; Liang, Sherry; Chon, Chae Ryung; Ammi, Azzdine Y.; Field, Joshua; Harmann, Leanne; Chilian, William M.; Linden, Joel; Lindner, Jonathan R.

2017-01-01

Background Augmentation of tissue blood flow by therapeutic ultrasound is thought to rely on convective shear. Microbubble contrast agents that undergo ultrasound-mediated cavitation markedly amplify these effects. We hypothesized that purinergic signalling is responsible for shear-dependent increases in muscle perfusion during therapeutic cavitation. Methods Unilateral exposure of the proximal hindlimb of mice (with or without ischemia produced by iliac ligation) to therapeutic ultrasound (1.3 MHz, mechanical index 1.3) was performed for ten minutes after intravenous injection of 2×108 lipid microbubbles. Microvascular perfusion was evaluated by low-power contrast ultrasound perfusion imaging. In vivo muscle ATP release and in vitro ATP release from endothelial cells or erythrocytes were assessed by a luciferin-luciferase assay. Purinergic signalling pathways were assessed by studying interventions that either (1) accelerated ATP degradation; (2) inhibited P2Y receptors, adenosine receptors, or KATP channels; or (3) inhibited downstream signalling pathways involving endothelial nitric oxide synthase (eNOS) or prostanoid production (indomethacin). Augmentation in muscle perfusion by ultrasound cavitation was assessed in a proof-of-concept clinical trial in 12 subjects with stable sickle cell disease (SCD). Results Therapeutic ultrasound cavitation increased muscle perfusion by 7-fold in normal mice, reversed tissue ischemia for up to 24 hrs in the murine model of peripheral artery disease, and doubled muscle perfusion in patients with SCD. Augmentation in flow extended well beyond the region of ultrasound exposure. Ultrasound cavitation produced a nearly 40-fold focal and sustained increase in ATP, the source of which included both endothelial cells and erythrocytes. Inhibitory studies indicated that ATP was a critical mediator of flow augmentation that acts primarily through either P2Y receptors or through adenosine produced by ectonucleotidase activity. Combined indomethacin and inhibition of eNOS abolished the effects of therapeutic ultrasound, indicating downstream signalling through both NO and prostaglandins. Conclusions Therapeutic ultrasound using microbubble cavitation to increase muscle perfusion relies on shear-dependent increases in ATP which can act through a diverse portfolio of purinergic signalling pathways. These events can reverse hindlimb ischemia in mice for >24 hours, and increase muscle blood flow in patients with sickle cell disease. Clinical Trial Registration NCT01566890 (https://clinicaltrials.gov/ct2/show/NCT01566890) PMID:28174191

Laminar shear stress modulates endothelial luminal surface stiffness in a tissue-specific manner.

PubMed

Merna, Nick; Wong, Andrew K; Barahona, Victor; Llanos, Pierre; Kunar, Balvir; Palikuqi, Brisa; Ginsberg, Michael; Rafii, Shahin; Rabbany, Sina Y

2018-04-17

Endothelial cells form vascular beds in all organs and are exposed to a range of mechanical forces that regulate cellular phenotype. We sought to determine the role of endothelial luminal surface stiffness in tissue-specific mechanotransduction of laminar shear stress in microvascular mouse cells and the role of arachidonic acid in mediating this response. Microvascular mouse endothelial cells were subjected to laminar shear stress at 4 dynes/cm 2 for 12 hours in parallel plate flow chambers that enabled real-time optical microscopy and atomic force microscopy measurements of cell stiffness. Lung endothelial cells aligned parallel to flow, while cardiac endothelial cells did not. This rapid alignment was accompanied by increased cell stiffness. The addition of arachidonic acid to cardiac endothelial cells increased alignment and stiffness in response to shear stress. Inhibition of arachidonic acid in lung endothelial cells and embryonic stem cell-derived endothelial cells prevented cellular alignment and decreased cell stiffness. Our findings suggest that increased endothelial luminal surface stiffness in microvascular cells may facilitate mechanotransduction and alignment in response to laminar shear stress. Furthermore, the arachidonic acid pathway may mediate this tissue-specific process. An improved understanding of this response will aid in the treatment of organ-specific vascular disease. © 2018 John Wiley & Sons Ltd.

Iron ion irradiation increases promotes adhesion of monocytic cells to arterial vascular endothelium

NASA Astrophysics Data System (ADS)

Kucik, Dennis; Khaled, Saman; Gupta, Kiran; Wu, Xing; Yu, Tao; Chang, Polly; Kabarowski, Janusz

Radiation causes inflammation, and chronic, low-level vascular inflammation is a risk factor for atherosclerosis. Consistent with this, exposure to radiation from a variety of sources is associated with increased risk of heart disease and stroke. Part of the inflammatory response to radiation is a change in the adhesiveness of the endothelial cells that line the blood vessels, triggering inappropriate accumulation of leukocytes, leading to later, damaging effects of inflammation. Although some studies have been done on the effects of gamma irradiation on vascular endothelium, the response of endothelium to heavy ion radiation likely to be encountered in prolonged space flight has not been determined. We investigated how irradiation of aortic endothelial cells with iron ions affects adhesiveness of cultured aortic endothelial cells for monocytic cells and the consequences of this for development of atherosclerosis. Aortic endothelial cells were irradiated with 600 MeV iron ions at Brookhaven National Laboratory and adhesion-related changes were measured. Cells remained viable for at least 72 hours, and were even able to repair acute damage to cell junctions. We found that iron ion irradiation altered expression levels of specific endothelial cell adhesion molecules. Further, these changes had functional consequences. Using a flow chamber adhesion assay to measure adhesion of monocytic cells to endothelial cells under physiological shear stress, we found that adhesivity of vascular endothelium was enhanced in as little as 24 hours after irradiation. Further, the radiation dose dependence was not monotonic, suggesting that it was not simply the result of endothelial cell damage. We also irradiated aortic arches and carotid arteries of Apolipoprotein-E-deficient mice. Histologic analysis of these mice will be conducted to determine whether effects of radiation on endothelial adhesiveness result in consequences for development of atherosclerosis. (Supported by NSBRI: NCC-9-58-162)

Bone morphogenic protein 4 produced in endothelial cells by oscillatory shear stress stimulates an inflammatory response

NASA Technical Reports Server (NTRS)

Sorescu, George P.; Sykes, Michelle; Weiss, Daiana; Platt, Manu O.; Saha, Aniket; Hwang, Jinah; Boyd, Nolan; Boo, Yong C.; Vega, J. David; Taylor, W. Robert;

Endothelial C-type natriuretic peptide maintains vascular homeostasis

PubMed Central

Moyes, Amie J.; Khambata, Rayomand S.; Villar, Inmaculada; Bubb, Kristen J.; Baliga, Reshma S.; Lumsden, Natalie G.; Xiao, Fang; Gane, Paul J.; Rebstock, Anne-Sophie; Worthington, Roberta J.; Simone, Michela I.; Mota, Filipa; Rivilla, Fernando; Vallejo, Susana; Peiró, Concepción; Sánchez Ferrer, Carlos F.; Djordjevic, Snezana; Caulfield, Mark J.; MacAllister, Raymond J.; Selwood, David L.; Ahluwalia, Amrita; Hobbs, Adrian J.

2014-01-01

The endothelium plays a fundamental role in maintaining vascular homeostasis by releasing factors that regulate local blood flow, systemic blood pressure, and the reactivity of leukocytes and platelets. Accordingly, endothelial dysfunction underpins many cardiovascular diseases, including hypertension, myocardial infarction, and stroke. Herein, we evaluated mice with endothelial-specific deletion of Nppc, which encodes C-type natriuretic peptide (CNP), and determined that this mediator is essential for multiple aspects of vascular regulation. Specifically, disruption of CNP leads to endothelial dysfunction, hypertension, atherogenesis, and aneurysm. Moreover, we identified natriuretic peptide receptor–C (NPR-C) as the cognate receptor that primarily underlies CNP-dependent vasoprotective functions and developed small-molecule NPR-C agonists to target this pathway. Administration of NPR-C agonists promotes a vasorelaxation of isolated resistance arteries and a reduction in blood pressure in wild-type animals that is diminished in mice lacking NPR-C. This work provides a mechanistic explanation for genome-wide association studies that have linked the NPR-C (Npr3) locus with hypertension by demonstrating the importance of CNP/NPR-C signaling in preserving vascular homoeostasis. Furthermore, these results suggest that the CNP/NPR-C pathway has potential as a disease-modifying therapeutic target for cardiovascular disorders. PMID:25105365

Mechanisms of erosion of atherosclerotic plaques.

PubMed

Quillard, Thibaut; Franck, Grégory; Mawson, Thomas; Folco, Eduardo; Libby, Peter

2017-10-01

The present review explores the mechanisms of superficial intimal erosion, a common cause of thrombotic complications of atherosclerosis. Human coronary artery atheroma that give rise to thrombosis because of erosion differ diametrically from those associated with fibrous cap rupture. Eroded lesions characteristically contain few inflammatory cells, abundant extracellular matrix, and neutrophil extracellular traps (NETs). Innate immune mechanisms such as engagement of Toll-like receptor 2 (TLR2) on cultured endothelial cells can impair their viability, attachment, and ability to recover a wound. Hyaluronan fragments may serve as endogenous TLR2 ligands. Mouse experiments demonstrate that flow disturbance in arteries with neointimas tailored to resemble features of human eroded plaques disturbs endothelial cell barrier function, impairs endothelial cell viability, recruits neutrophils, and provokes endothelial cells desquamation, NET formation, and thrombosis in a TLR2-dependent manner. Mechanisms of erosion have received much less attention than those that provoke plaque rupture. Intensive statin treatment changes the characteristic of plaques that render them less susceptible to rupture. Thus, erosion may contribute importantly to the current residual burden of risk. Understanding the mechanisms of erosion may inform the development and deployment of novel therapies to combat the remaining atherothrombotic risk in the statin era.

A diet enriched with mackerel (Scomber scombrus)-derived products improves the endothelial function in a senior population (Prevención de las Enfermedades Cardiovasculares: Estudio Santoña--PECES project).

PubMed

de Berrazueta, J R; Gómez de Berrazueta, J M; Amado Señarís, J A; Peña Sarabia, N; Fernández Viadero, C; García-Unzueta, M T; Sáez de Adana, M; Sanchez Ovejero, C J; Llorca, J

2009-03-01

Regular consumption of fish reduces cardiovascular risks. Here, we investigate if the consumption of products with mackerel (Scomber scombrus) with 8.82 g of eicosapentaenoic acid (EPA) + docosahexaenoic acid (DHA) content per 100 g of product improves parameters of endothelial function in a controlled population. Subjects maintained a 12-week diet with products with mackerel. The population consisted of 58 senior subjects (12 withdrawals, 25 women), aged 82.08 +/- 8.13 years (Group A). Twenty-three senior subjects (13 women) on a regular diet were used as the control group (Group B). Subjects of Group A received 57 portions throughout 12 weeks (four to five portions a week of products with a mean EPA + DHA content of 2.5 g a day). A continuous follow-up and a final evaluation were performed to determine the level of consumption. Plasma samples were stored at -70 degrees C for a biochemical study. Endothelial function was analysed by reactive hyperemia with a mercury strain gauge plethysmography with measurement of blood flow in the forearm, both baseline and at the end of the 12-week diet. Endothelium-dependent vasodilatation significantly increased in Group A subjects (P < 0.001). No changes were found in Group B. The subgroup analyses showed that improvements were produced in Group A subjects without cardiovascular disease (P < 0.001). Nitrites/nitrates and von Willebrand factor plasma concentrations were higher in participants after the 12-week diet. The consumption of mackerel meat products improves endothelium-dependent, flow-mediated vasodilatation in a senior population. This finding might explain some of the cardioprotective effects of fish consumption.

Insulin resistance adds to endothelial dysfunction in hypertensive patients and in normotensive offspring of subjects with essential hypertension.

PubMed

Zizek, B; Poredos, P

2001-02-01

To evaluate whether endothelium-dependent (nitric oxide-mediated) dilation of the brachial artery (BA) is impaired in patients being treated for essential hypertension (EH), and whether this abnormality can be detected in normotensive offspring of subjects with EH (familial trait, FT); and to investigate the interrelationship between flow-mediated vasodilation (FMD) and hyperinsulinaemia/insulin resistance. Cross-sectional study. Angiology department at a teaching hospital. The study encompassed 172 subjects, of whom 46 were treated hypertonics aged 40-55 (49) years, and 44 age-matched, normotensive volunteers as controls. We also investigated 41 normotonics with FT aged 20-30 (25) years and 41 age-and sex-matched controls without FT. Using high-resolution ultrasound, BA diameters at rest, during reactive hyperaemia (endothelium-dependent dilation) and after sublingual glyceryl trinitrate (GTN) application (endothelium-independent dilation) were measured. In hypertonics FMD was significantly lower than in controls [2.4 (2.9) vs. 7.4 (2.5)%; P < 0.00005], as was GTN-induced dilation [12.1 (4.3) vs. 16.1 (4.6)%; P=0.0007]. In subjects with FT, FMD was also decreased compared with the control group [5.8 (4.1) vs. 10.0 (3.0)%; P < 0.00005]. The response to GTN was comparable in both groups of young subjects. FMD was negatively related to insulin concentration in all subjects studied (P < 0.00005). In treated patients with EH, flow-mediated dilation of the BA as well as endothelium-independent dilation are decreased. In individuals with FT the endothelial function of the peripheral arteries is also altered in the absence of elevated blood pressure. Endothelial dysfunction is related to hyperinsulinaemia/insulin resistance, which could be one of the pathogenetic determinants of EH and its complications.

Flow-dependent vascular heat transfer during microwave thermal ablation.

PubMed

Chiang, Jason; Hynes, Kieran; Brace, Christopher L

2012-01-01

Microwave tumor ablation is an attractive option for thermal ablation because of its inherent benefits over radiofrequency ablation (RFA) in the treatment of solid tumors such as hepatocellular carcinoma (HCC). Microwave energy heats tissue to higher temperatures and at a faster rate than RFA, creating larger, more homogenous ablation zones. In this study, we investigate microwave heating near large vasculature using coupled fluid-flow and thermal analysis. Low-flow conditions are predicted to be more likely to cause cytotoxic heating and, therefore, vessel thrombosis and endothelial damage of downstream tissues. Such conditions may be more prevalent in patient with severe cirrhosis or compromised blood flow. High-flow conditions create the more familiar heat-sink effect that can protect perivascular tissues from the intended thermal damage. These results may help guide placement and use of microwave ablation technologies in future studies.

Oxygen dependence of endothelium-dependent vasodilation: importance in chronic obstructive pulmonary disease.

PubMed

Keymel, Stefanie; Schueller, Benedikt; Sansone, Roberto; Wagstaff, Rabea; Steiner, Stephan; Kelm, Malte; Heiss, Christian

2018-03-01

Epidemiological studies have shown increased morbidity and mortality in patients with coronary artery disease (CAD) and chronic obstructive pulmonary disease (COPD). We aimed to characterize the oxygen dependence of endothelial function in patients with CAD and coexisting COPD. In CAD patients with and without COPD ( n = 33), we non-invasively measured flow-mediated dilation (FMD) and intima-media thickness (IMT) of the brachial artery (BA), forearm blood flow (FBF), and perfusion of the cutaneous microcirculation with laser Doppler perfusion imaging (LDPI). In an experimental setup, vascular function was assessed in healthy volunteers ( n = 5) breathing 12% oxygen or 100% oxygen in comparison to room air. COPD was associated with impaired FMD (3.4 ±0.5 vs. 4.2 ±0.6%; p < 0.001) and increased IMT (0.49 ±0.04 vs. 0.44 ±0.04 mm; p <0.01), indicating functional and structural alterations of the BA in COPD. Forearm blood flow and LDPI were comparable between the groups. Flow-mediated dilation correlated with capillary oxygen pressure (pO 2 , r = 0.608). Subgroup analysis in COPD patients with pO 2 > 65 mm Hg and pO 2 ≤ 65 mm Hg revealed even lower FMD in patients with lower pO 2 (3.0 ±0.5 vs. 3.7 ±0.4%; p < 0.01). Multivariate analysis showed that pO 2 was a predictor of FMD independent of the forced expiratory volume and pack years. Exposure to hypoxic air led to an acute decrease in FMD, whereby exposure to 100% oxygen did not change vascular function. Our data suggest that in CAD patients with COPD, decreased systemic oxygen levels lead to endothelial dysfunction, underlining the relevance of cardiopulmonary interaction and the potential importance of pulmonary treatment in secondary prevention of vascular disease.

Cytotoxicity of lidocaine to human corneal endothelial cells in vitro.

PubMed

Yu, Hao-Ze; Li, Yi-Han; Wang, Rui-Xin; Zhou, Xin; Yu, Miao-Miao; Ge, Yuan; Zhao, Jun; Fan, Ting-Jun

2014-04-01

Lidocaine has been reported to induce apoptosis on rabbit corneal endothelial cells. However, the apoptotic effect and exact mechanism involved in cytotoxicity of lidocaine are not well-established in human corneal endothelial (HCE) cells. In this study, we investigated the apoptosis-inducing effect of lidocaine on HCE cells in vitro. After HCE cells were treated with lidocaine at concentrations of 0.15625-10.0 g/l, the morphology and ultrastructure of the cells were observed by inverted light microscope and transmission electron microscope (TEM). Cell viability was measured by MTT assay, and the apoptotic ratio was evaluated with flow cytometry and fluorescent microscopic counting after FITC-Annexin V/PI and AO/EB staining. DNA fragmentation was detected by electrophoresis, and the activation of caspases was evaluated by ELISA. In addition, changes in mitochondrial membrane potential were determined by JC-1 staining. Results suggest that lidocaine above 1.25 g/l reduced cellular viability and triggered apoptosis in HCE cells in a time- and dose-dependent manner. Diminishment of ΔΨm and the activation of caspases indicate that lidocaine-induced apoptosis was caspase dependent and may be related to mitochondrial pathway. © 2013 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

Epoetin beta pegol ameliorates flow-mediated dilation with improving endothelial nitric oxide synthase coupling state in nonobese diabetic rats.

PubMed

Serizawa, Kenichi; Yogo, Kenji; Tashiro, Yoshihito; Kawasaki, Ryohei; Endo, Koichi; Shimonaka, Yasushi; Hirata, Michinori

2017-04-01

Patients with diabetic nephropathy have a high cardiovascular mortality. Epoetin beta pegol (continuous erythropoietin receptor activator, C.E.R.A.) is a drug for the treatment of renal anemia. In this study, we investigated the effect of C.E.R.A. on vascular endothelial function as evaluated by flow-mediated dilation (FMD) and the relationship between hematopoiesis and FMD in diabetic nephropathy rats. Male Spontaneously Diabetic Torii rats (SDT, 22 weeks old) were used. C.E.R.A. (0.6, 1.2 μg/kg) was administered subcutaneously once every 2 weeks for 8 weeks. At 1 week after last administration (31 weeks old), we assessed FMD in the femoral arteries of anesthetized rats using a high-resolution ultrasound system. FMD was also measured 1 week after single C.E.R.A. treatment (5.0 μg/kg) to examine the influence of hematopoiesis. Flow-mediated dilation was significantly decreased in SDT rats before the start of C.E.R.A. treatment (22 weeks old). Repeated administration of C.E.R.A. dose-dependently improved FMD in SDT rats (31 weeks old) without changing blood glucose, nitroglycerin-induced vasodilation, or kidney function. Long-term administration of C.E.R.A. improved the state of endothelial nitric oxide synthase uncoupling in the femoral arteries of SDT rats, which showed a positive correlation with FMD. On the other hand, there was no correlation between FMD and Hb or Hct in SDT rats. Furthermore, at 1 week after single administration of C.E.R.A., FMD was not significantly improved although hemoglobin levels were comparable with levels following long-term C.E.R.A. Long-term treatment with C.E.R.A. improved FMD in SDT rats even after onset of endothelial dysfunction. © 2017 The Authors. Cardiovascular Therapeutics Published by John Wiley & Sons Ltd.

Autophagy is required for endothelial cell alignment and atheroprotection under physiological blood flow

PubMed Central

Vion, Anne-Clemence; Hammoutene, Adel; Poisson, Johanne; Lasselin, Juliette; Devue, Cecile; Pic, Isabelle; Dupont, Nicolas; Busse, Johanna; Stark, Konstantin; Lafaurie-Janvore, Julie; Barakat, Abdul I.; Loyer, Xavier; Souyri, Michele; Viollet, Benoit; Julia, Pierre; Tedgui, Alain; Codogno, Patrice; Rautou, Pierre-Emmanuel

2017-01-01

It has been known for some time that atherosclerotic lesions preferentially develop in areas exposed to low SS and are characterized by a proinflammatory, apoptotic, and senescent endothelial phenotype. Conversely, areas exposed to high SS are protected from plaque development, but the mechanisms have remained elusive. Autophagy is a protective mechanism that allows recycling of defective organelles and proteins to maintain cellular homeostasis. We aimed to understand the role of endothelial autophagy in the atheroprotective effect of high SS. Atheroprotective high SS stimulated endothelial autophagic flux in human and murine arteries. On the contrary, endothelial cells exposed to atheroprone low SS were characterized by inefficient autophagy as a result of mammalian target of rapamycin (mTOR) activation, AMPKα inhibition, and blockade of the autophagic flux. In hypercholesterolemic mice, deficiency in endothelial autophagy increased plaque burden only in the atheroresistant areas exposed to high SS; plaque size was unchanged in atheroprone areas, in which endothelial autophagy flux is already blocked. In cultured cells and in transgenic mice, deficiency in endothelial autophagy was characterized by defects in endothelial alignment with flow direction, a hallmark of endothelial cell health. This effect was associated with an increase in endothelial apoptosis and senescence in high-SS regions. Deficiency in endothelial autophagy also increased TNF-α–induced inflammation under high-SS conditions and decreased expression of the antiinflammatory factor KLF-2. Altogether, these results show that adequate endothelial autophagic flux under high SS limits atherosclerotic plaque formation by preventing endothelial apoptosis, senescence, and inflammation. PMID:28973855

d(-) Lactic Acid-Induced Adhesion of Bovine Neutrophils onto Endothelial Cells Is Dependent on Neutrophils Extracellular Traps Formation and CD11b Expression.

PubMed

Alarcón, Pablo; Manosalva, Carolina; Conejeros, Ivan; Carretta, María D; Muñoz-Caro, Tamara; Silva, Liliana M R; Taubert, Anja; Hermosilla, Carlos; Hidalgo, María A; Burgos, Rafael A

2017-01-01

Bovine ruminal acidosis is of economic importance as it contributes to reduced milk and meat production. This phenomenon is mainly attributed to an overload of highly fermentable carbohydrate, resulting in increased d(-) lactic acid levels in serum and plasma. Ruminal acidosis correlates with elevated acute phase proteins in blood, along with neutrophil activation and infiltration into various tissues leading to laminitis and aseptic polysynovitis. Previous studies in bovine neutrophils indicated that d(-) lactic acid decreased expression of L-selectin and increased expression of CD11b to concentrations higher than 6 mM, suggesting a potential role in neutrophil adhesion onto endothelia. The two aims of this study were to evaluate whether d(-) lactic acid influenced neutrophil and endothelial adhesion and to trigger neutrophil extracellular trap (NET) production (NETosis) in exposed neutrophils. Exposure of bovine neutrophils to 5 mM d(-) lactic acid elevated NET release compared to unstimulated neutrophil negative controls. Moreover, this NET contains CD11b and histone H 4 citrullinated, the latter was dependent on PAD4 activation, a critical enzyme in DNA decondensation and NETosis. Furthermore, NET formation was dependent on d(-) lactic acid plasma membrane transport through monocarboxylate transporter 1 (MCT1). d(-) lactic acid enhanced neutrophil adhesion onto endothelial sheets as demonstrated by in vitro neutrophil adhesion assays under continuous physiological flow conditions, indicating that cell adhesion was a NET- and a CD11b/ICAM-1-dependent process. Finally, d(-) lactic acid was demonstrated for the first time to trigger NETosis in a PAD4- and MCT1-dependent manner. Thus, d(-) lactic acid-mediated neutrophil activation may contribute to neutrophil-derived pro-inflammatory processes, such as aseptic laminitis and/or polysynovitis in animals suffering acute ruminal acidosis.

Association Between the Female Athlete Triad and Endothelial Dysfunction in Dancers

PubMed Central

Hoch, Anne Z.; Papanek, Paula; Szabo, Aniko; Widlansky, Michael E.; Schimke, Jane E.; Gutterman, David D.

2013-01-01

Objective To determine the prevalence of the 3 components of the female athlete triad [disordered eating, menstrual dysfunction, low bone mineral density (BMD)] and their relationships with brachial artery flow-mediated dilation in professional dancers. Design Prospective study. Setting Academic institution in the Midwest. Participants Twenty-two professional ballet dancers volunteered for this study. Interventions The prevalence of the female athlete triad and its relationship to endothelial dysfunction. Main Outcome Measures Subjects completed questionnaires to assess disordered eating and menstrual status/history. They also completed a 3-day food record and wore an accelerometer for 3 days to determine energy availability. Serum baseline thyrotropin, prolactin, and hormonal concentrations were obtained. Bone mineral density and body composition were measured with a GE Lunar Prodigy dual-energy X-ray absorptiometry. Endothelial function was determined as flow-mediated vasodilation measured by high-frequency ultrasound in the brachial artery. An increase in brachial diameter <5% to hyperemic flow stimulus was defined a priori as endothelial dysfunction. Results Seventeen dancers (77%) had evidence of low/negative energy availability. Thirty-two percent had disordered eating (EDE-Q score). Thirty-six percent had menstrual dysfunction and 14% were currently using hormone contraception. Twenty-three percent had evidence of low bone density (Z-score < −1.0). Sixty-four percent had abnormal brachial artery flow-mediated dilation (<5%). Flow-mediated dilation values were significantly correlated with serum estrogen and whole-body and lumbar BMD. All the 3 components of the triad plus endothelial dysfunction were present in 14% of the subjects. Conclusions Endothelial dysfunction was correlated with reduced BMD, menstrual dysfunction, and low serum estrogen. These findings may have profound implications for cardiovascular and bone health in professional women dancers. PMID:21358502

Mild Hyperthermia Downregulates Receptor-dependent Neutrophil Function

PubMed Central

Fröhlich, Dieter; Wittmann, Sigrid; Rothe, Gregor; Sessler, Daniel I.; Vogel, Peter; Taeger, Kai

2005-01-01

Mild hypothermia impairs resistance to infection and, reportedly, impairs phagocytosis and oxidative killing of un-opsonized bacteria. We evaluated various functions at 33 to 41°C in neutrophils taken from volunteers. Adhesion on endothelial cells was determined using light microscopy. Adhesion molecules expression and receptors, phagocytosis, and release of reactive oxidants were assessed using flow cytometric assays. Adhesion protein CD11b expression on resting neutrophils was temperature independent. However, upregulation of CD11b with TNF-α was increased by hypothermia and decreased with hyperthermia. Neutrophil adhesion to either resting or activated endothelial cells was not temperature dependent. Bacterial uptake was inversely related to temperature, more so with E. coli than S. aureus. Temperature dependence of phagocytosis occurred only with opsonized bacteria. Hypothermia slightly increased N-Formyl-L-methionyl-L-leucyl-phenylalanine (FMLP) receptors on neutrophils: hyperthermia decreased expression, especially with TNF-α. FMLP-induced H2O2 production was inversely related to temperature, especially in the presence of TNF-α. Conversely, phorbol-13-myristate-12-acetate, an activator of protein kinase C, induced an extreme and homogenous release of reactive oxidants that increased with temperature. In contrast to non-receptor dependent phagocytosis and oxidative killing, several crucial receptor-dependent neutrophil activities show temperature-dependent regulation, with hypothermia increasing function. The temperature dependence of neutrophil function is thus more complicated than previously appreciated. PMID:15281545

Rapid flow-induced responses in endothelial cells

NASA Technical Reports Server (NTRS)

Stamatas, G. N.; McIntire, L. V.

2001-01-01

Endothelial cells alter their morphology, growth rate, and metabolism in response to fluid shear stress. To study rapid flow-induced responses in the 3D endothelial cell morphology and calcium distribution, coupled fluorescence microscopy with optical sectioning, digital imaging, and numerical deconvolution techniques have been utilized. Results demonstrate that within the first minutes of flow application nuclear calcium is increasing. In the same time frame whole cell height and nuclear height are reduced by about 1 microm. Whole cell height changes may facilitate reduction of shear stress gradients on the luminal surface, whereas nuclear structural changes may be important for modulating endothelial growth rate and metabolism. To study the role of the cytoskeleton in these responses, endothelial cells have been treated with specific disrupters (acrylamide, cytochalasin D, and colchicine) of each of the cytoskeleton elements (intermediate filaments, microfilaments, and microtubules, respectively). None of these compounds had any effect on the shear-induced calcium response. Cytochalasin D and acrylamide did not affect the shear-induced nuclear morphology changes. Colchicine, however, completely abrogated the response, indicating that microtubules may be implicated in force transmission from the plasma membrane to the nucleus. A pedagogical model based on tensegrity theory principles is presented that is consistent with the results on the 3D endothelial morphology.

Exogenous L-Arginine Attenuates the Effects of Angiotensin II on Renal Hemodynamics and the Pressure Natriuresis-Diuresis Relationship

PubMed Central

Das, Satarupa; Mattson, David L.

2014-01-01

SUMMARY Administration of exogenous L-Arginine (L-Arg) attenuates Angiotensin II (AngII)-mediated hypertension and kidney disease in rats. The present study assessed renal hemodynamics and pressure-diuresis-natriuresis in anesthetized rats infused with vehicle, AngII (20 ng/kg/min, iv) or AngII + L-Arg (300 µg/kg/min, iv). Increasing renal perfusion pressure (RPP) from approximately 100 to 140 mmHg resulted in a 9–10 fold increase in urine flow and sodium excretion rate in control animals. In comparison, AngII infusion significantly reduced renal blood flow (RBF) and glomerular filtration rate (GFR) by 40–42% and blunted the pressure-dependent increase in urine flow and sodium excretion rate by 54–58% at elevated RPP. Supplementation of L-Arg reversed the vasoconstrictor effects of AngII and restored pressure-dependent diuresis to levels not significantly different from control rats. Experiments in isolated aortic rings were performed to assess L-Arg effects on the vasculature. Dose-dependent contraction to AngII (10−10M to 10−7M) was observed with a maximal force equal to 27±3% of the response to 10−5M phenylephrine. Contraction to 10−7M AngII was blunted by 75±3% with 10−4M L-Arg. The influence of L-Arg to blunt AngII mediated contraction was eliminated by endothelial denudation or incubation with nitric oxide synthase inhibitors. Moreover, the addition of 10−3M cationic or neutral amino acids, which compete with L-Arg for cellular uptake, blocked the effect of L-Arg. Anionic amino acids did not influence the effects of L-Arg on AngII-mediated contraction. These studies indicate that L-Arg blunts AngII-mediated vascular contraction by an endothelial- and NOS-dependent mechanism involving cellular uptake of L-Arg. PMID:24472006

Are endothelial cell bioeffects from acoustic droplet vaporization proximity dependent?

NASA Astrophysics Data System (ADS)

Seda, Robinson; Li, David; Fowlkes, J. Brian; Bull, Joseph

2013-11-01

Acoustic droplet vaporization (ADV) produces gas microbubbles that provide a means of selective occlusion in gas embolotherapy. Vaporization and subsequent occlusion occur inside blood vessels supplying the targeted tissue, such as tumors. Theoretical and computational studies showed that ADV within a vessel can impart high fluid mechanical stresses on the vessel wall. Previous in vitro studies have demonstrated that vaporization at an endothelial layer may affect cell attachment and viability. The current study is aimed at investigating the role of vaporization distance away from the endothelial layer. HUVECs were cultured in OptiCell™ chambers until reaching confluence. Dodecafluoropentane microdroplets were added, attaining a 10:1 droplet to cell ratio. A single ultrasound pulse (7.5 MHz) consisting of 16 cycles (~ 2 μs) and a 5 MPa peak rarefactional pressure was used to produce ADV while varying the vaporization distance from the endothelial layer (0 μm, 500 μm, 1000 μm). Results indicated that cell attachment and viability was significantly different if the distance was 0 μm (at the endothelial layer). Other distances were not significantly different from the control. ADV will significantly affect the endothelium if droplets are in direct contact with the cells. Droplet concentration and flow conditions inside blood vessels may play an important role. This work was supported by NIH grant R01EB006476.

Endothelial cell expression of haemoglobin α regulates nitric oxide signalling.

PubMed

Straub, Adam C; Lohman, Alexander W; Billaud, Marie; Johnstone, Scott R; Dwyer, Scott T; Lee, Monica Y; Bortz, Pamela Schoppee; Best, Angela K; Columbus, Linda; Gaston, Benjamin; Isakson, Brant E

2012-11-15

Models of unregulated nitric oxide (NO) diffusion do not consistently account for the biochemistry of NO synthase (NOS)-dependent signalling in many cell systems. For example, endothelial NOS controls blood pressure, blood flow and oxygen delivery through its effect on vascular smooth muscle tone, but the regulation of these processes is not adequately explained by simple NO diffusion from endothelium to smooth muscle. Here we report a new model for the regulation of NO signalling by demonstrating that haemoglobin (Hb) α (encoded by the HBA1 and HBA2 genes in humans) is expressed in human and mouse arterial endothelial cells and enriched at the myoendothelial junction, where it regulates the effects of NO on vascular reactivity. Notably, this function is unique to Hb α and is abrogated by its genetic depletion. Mechanistically, endothelial Hb α haem iron in the Fe(3+) state permits NO signalling, and this signalling is shut off when Hb α is reduced to the Fe(2+) state by endothelial cytochrome b5 reductase 3 (CYB5R3, also known as diaphorase 1). Genetic and pharmacological inhibition of CYB5R3 increases NO bioactivity in small arteries. These data reveal a new mechanism by which the regulation of the intracellular Hb α oxidation state controls NOS signalling in non-erythroid cells. This model may be relevant to haem-containing globins in a broad range of NOS-containing somatic cells.

Suppression of complement regulatory protein C1 inhibitor in vascular endothelial activation by inhibiting vascular cell adhesion molecule-1 action

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Haimou; Qin, Gangjian; Liang, Gang

Increased expression of adhesion molecules by activated endothelium is a critical feature of vascular inflammation associated with the several diseases such as endotoxin shock and sepsis/septic shock. Our data demonstrated complement regulatory protein C1 inhibitor (C1INH) prevents endothelial cell injury. We hypothesized that C1INH has the ability of an anti-endothelial activation associated with suppression of expression of adhesion molecule(s). C1INH blocked leukocyte adhesion to endothelial cell monolayer in both static assay and flow conditions. In inflammatory condition, C1INH reduced vascular cell adhesion molecule (VCAM-1) expression associated with its cytoplasmic mRNA destabilization and nuclear transcription level. Studies exploring the underlying mechanismmore » of C1INH-mediated suppression in VCAM-1 expression were related to reduction of NF-{kappa}B activation and nuclear translocation in an I{kappa}B{alpha}-dependent manner. The inhibitory effects were associated with reduction of inhibitor I{kappa}B kinase activity and stabilization of the NF-{kappa}B inhibitor I{kappa}B. These findings indicate a novel role for C1INH in inhibition of vascular endothelial activation. These observations could provide the basis for new therapeutic application of C1INH to target inflammatory processes in different pathologic situations.« less

Folic acid inhibits homocysteine-induced cell apoptosis in human umbilical vein endothelial cells.

PubMed

Cui, Shanshan; Li, Wen; Wang, Pengyan; Lv, Xin; Gao, Yuxia; Huang, Guowei

2017-12-18

Homocysteine may be responsible for vascular endothelial cell injury, which occurs early in the pathology of cardiovascular disease. Homocysteine metabolism requires enzymatic interaction with vitamins such as folic acid, vitamin B12, and vitamin B6. We hypothesized that folic acid alleviated homocysteine-induced vascular injury by regulating the metabolic pathway of apoptosis. Human umbilical vein endothelial cells were incubated for 48 h with folic acid at the concentrations of 0-1000 nmol/L, in combination with either 1000 μmol/L homocysteine or vehicle for the first 24 h. We then assessed cell viability and apoptosis by methyl thiazolyl tetrazolium assay and flow cytometry, respectively. To further investigate how folic acid influenced cell apoptosis, we also analyzed the activities of caspase-3/7 and the mRNA and protein expressions of BCL2, BAX, TP53, CASP3, and CASP8 in human umbilical vein endothelial cells. We showed that folic acid increased cell viability and decreased apoptosis in a dose-dependent manner, and that this effect was mediated by decreased caspase-3/7 activity, upregulated BCL2/BAX ratio, and downregulated TP53, CASP3, and CASP8 expressions. Thus, we conclude that folic acid inhibits cell apoptosis and ameliorates homocysteine toxicity by regulating the expression of apoptosis-related genes in human umbilical vein endothelial cells.

Time of correlation of low-frequency fluctuations in the regional laser Doppler flow signal from human skin

NASA Astrophysics Data System (ADS)

Folgosi-Correa, M. S.; Nogueira, G. E. C.

2012-06-01

The laser Doppler flowmetry allows the non-invasive assessment of the skin perfusion in real-time, being an attractive technique to study the human microcirculation in clinical settings. Low-frequency oscillations in the laser Doppler blood flow signal from the skin have been related to the endothelial, endothelial-metabolic, neurogenic and myogenic mechanisms of microvascular flow control, in the range 0.005-0.0095 Hz, 0.0095-0.021 Hz, 0.021-0.052 Hz and 0.052- 0.145 Hz respectively. The mean Amplitude (A) of the periodic fluctuations in the laser Doppler blood flow signal, in each frequency range, derived from the respective wavelet-transformed coefficients, has been used to assess the function and dysfunctions of each mechanism of flow control. Known sources of flow signal variances include spatial and temporal variability, diminishing the discriminatory capability of the technique. Here a new time domain method of analysis is proposed, based on the Time of Correlation (TC) of flow fluctuations between two adjacent sites. Registers of blood flow from two adjacent regions, for skin temperature at 32 0C (basal) and thermally stimulated (42 0C) of volar forearms from 20 healthy volunteers were collected and analyzed. The results obtained revealed high time of correlation between two adjacent regions when thermally stimulated, for signals in the endothelial, endothelial-metabolic, neurogenic and myogenic frequency ranges. Experimental data also indicate lower variability for TC when compared to A, when thermally stimulated, suggesting a new promising parameter for assessment of the microvascular flow control.

Resveratrol recruits rat muscle microvasculature via a nitric oxide-dependent mechanism that is blocked by TNFα

PubMed Central

Wang, Nasui; Ko, Seung-Hyun; Chai, Weidong; Li, Guolian; Barrett, Eugene J.; Tao, Lijian; Cao, Wenhong

2011-01-01

Resveratrol, a polyphenol found in many plants, has antioxidant and anti-inflammatory actions. It also improves endothelial function and may be cardioprotective. Tumor necrosis factor-α (TNFα) causes oxidative stress and microvascular endothelial dysfunction. Whether resveratrol affects microvascular function in vivo and, if so, whether inflammatory cytokines antagonize its microvascular action are not clear. In cultured bovine aortic endothelial cells (BAECs), reserveratrol (100 nM) increased the phosphorylation of protein kinase B (Akt), endothelial nitric oxide (NO) synthase (eNOS), and ERK1/2 within 15 min by more than twofold, and this effect lasted for at least 2 h. Treatment of BAECs with TNFα (10 ng/ml) significantly increased the NADPH oxidase activity and the production of hydrogen peroxide and superoxide. Pretreatment of cells with resveratrol (100 nM) prevented each of these. Injection (ip) of resveratrol in rats potently increased muscle microvascular blood volume (MBV; P = 0.007) and flow (MBF; P < 0.02) within 30 min, and this was sustained for at least 2 h. The phosphorylation of Akt in liver or muscle was unchanged. Superimposed systemic infusion of l-NAME (NOS inhibitor) completely abolished resveratrol-induced increases in MBV and MBF. Similarly, systemic infusion of TNFα prevented resveratrol-induced muscle microvascular recruitment. In conclusion, resveratrol activates eNOS and increases muscle microvascular recruitment via an NO-dependent mechanism. Despite the potent antioxidant effect of resveratrol, TNFα at concentrations that block insulin-mediated muscle microvascular recruitment completely neutralized resveratrol's microvascular action. Thus, chronic inflammation, as seen in type 2 diabetes, may limit resveratrol's vasodilatory actions on muscle microvasculature. PMID:20978231

Exercise Training Stimulates Ischemia-Induced Neovascularization via Phosphatidylinositol 3-Kinase/Akt-Dependent Hypoxia-Induced Factor-1α Reactivation in Mice of Advanced Age

PubMed Central

Cheng, Xian Wu; Kuzuya, Masafumi; Kim, Weon; Song, Haizhen; Hu, Lina; Inoue, Aiko; Nakamura, Kae; Di, Qun; Sasaki, Takeshi; Tsuzuki, Michitaka; Shi, Guo-Ping; Okumura, Kenji; Murohara, Toyoaki

2011-01-01

Background Exercise stimulates the vascular response in pathological conditions, including ischemia; however, the molecular mechanisms by which exercise improves the impaired hypoxia-induced factor (HIF)-1α–mediated response to hypoxia associated with aging are poorly understood. Here, we report that swimming training (ST) modulates the vascular response to ischemia in aged (24-month-old) mice. Methods and Results Aged wild-type mice (MMP-2+/+) that maintained ST (swimming 1 h/d) from day 1 after surgery were randomly assigned to 4 groups that were treated with either vehicle, LY294002, or deferoxamine for 14 days. Mice that were maintained in a sedentary condition served as controls. ST increased blood flow, capillary density, and levels of p-Akt, HIF-1α, vascular endothelial growth factor, Fit-1, and matrix metalloproteinase-2 (MMP-2) in MMP-2+/+ mice. ST also increased the numbers of circulating endothelial progenitor cells and their function associated with activation of HIF-1α. All of these effects were diminished by LY294002, an inhibitor of phosphatidylinositol 3-kinase; enhanced by deferoxamine, an HIF-1α stabilizer; and impaired by knockout of MMP-2. Finally, bone marrow transplantation confirmed that ST enhanced endothelial progenitor cell homing to ischemic sites in aged mice. Conclusions ST can improve neovascularization in response to hypoxia via a phosphatidylinositol 3-kinase–dependent mechanism that is mediated by the HIF-1α/vascular endothelial growth factor/MMP-2 pathway in advanced age. PMID:20679550

A multi-component parallel-plate flow chamber system for studying the effect of exercise-induced wall shear stress on endothelial cells.

PubMed

Wang, Yan-Xia; Xiang, Cheng; Liu, Bo; Zhu, Yong; Luan, Yong; Liu, Shu-Tian; Qin, Kai-Rong

2016-12-28

In vivo studies have demonstrated that reasonable exercise training can improve endothelial function. To confirm the key role of wall shear stress induced by exercise on endothelial cells, and to understand how wall shear stress affects the structure and the function of endothelial cells, it is crucial to design and fabricate an in vitro multi-component parallel-plate flow chamber system which can closely replicate exercise-induced wall shear stress waveforms in artery. The in vivo wall shear stress waveforms from the common carotid artery of a healthy volunteer in resting and immediately after 30 min acute aerobic cycling exercise were first calculated by measuring the inner diameter and the center-line blood flow velocity with a color Doppler ultrasound. According to the above in vivo wall shear stress waveforms, we designed and fabricated a parallel-plate flow chamber system with appropriate components based on a lumped parameter hemodynamics model. To validate the feasibility of this system, human umbilical vein endothelial cells (HUVECs) line were cultured within the parallel-plate flow chamber under abovementioned two types of wall shear stress waveforms and the intracellular actin microfilaments and nitric oxide (NO) production level were evaluated using fluorescence microscope. Our results show that the trends of resting and exercise-induced wall shear stress waveforms, especially the maximal, minimal and mean wall shear stress as well as oscillatory shear index, generated by the parallel-plate flow chamber system are similar to those acquired from the common carotid artery. In addition, the cellular experiments demonstrate that the actin microfilaments and the production of NO within cells exposed to the two different wall shear stress waveforms exhibit different dynamic behaviors; there are larger numbers of actin microfilaments and higher level NO in cells exposed in exercise-induced wall shear stress condition than resting wall shear stress condition. The parallel-plate flow chamber system can well reproduce wall shear stress waveforms acquired from the common carotid artery in resting and immediately after exercise states. Furthermore, it can be used for studying the endothelial cells responses under resting and exercise-induced wall shear stress environments in vitro.

Effect of black tea consumption on brachial artery flow-mediated dilation and ischaemia-reperfusion in humans.

PubMed

Schreuder, Tim H A; Eijsvogels, Thijs M H; Greyling, Arno; Draijer, Richard; Hopman, Maria T E; Thijssen, Dick H J

2014-02-01

Tea consumption is associated with reduced cardiovascular risk. Previous studies found that tea flavonoids work through direct effects on the vasculature, leading to dose-dependent improvements in endothelial function. Cardioprotective effects of regular tea consumption may relate to the prevention of endothelial ischaemia-reperfusion (IR) injury. Therefore, we examined the effect of black tea consumption on endothelial function and the ability of tea to prevent IR injury. In a randomized, crossover study, 20 healthy subjects underwent 7 days of tea consumption (3 cups per day) or abstinence from tea. We examined brachial artery (BA) endothelial function via flow-mediated dilation (FMD), using high resolution echo-Doppler, before and 90 min after tea or hot water consumption. Subsequently, we followed a 20-min ischaemia and 20-min reperfusion protocol of the BA after which we measured FMD to examine the potential of tea consumption to protect against IR injury. Tea consumption resulted in an immediate increase in FMD% (pre-consumption: 5.8 ± 2.5; post-consumption: 7.2 ± 3.2; p < 0.01), whilst no such change occurred after ingestion of hot water. The IR protocol resulted in a significant decrease in FMD (p < 0.005), which was also present after tea consumption (p < 0.001). This decline was accompanied by an increase in the post-IR baseline diameter. In conclusion, these data indicate that tea ingestion improves BA FMD. However, the impact of the IR protocol on FMD was not influenced by tea consumption. Therefore, the cardioprotective association of tea ingestion relates to a direct effect of tea on the endothelium in humans in vivo.

Flow through internal elastic lamina affects shear stress on smooth muscle cells (3D simulations).

PubMed

Tada, Shigeru; Tarbell, John M

2002-02-01

We describe a three-dimensional numerical simulation of interstitial flow through the medial layer of an artery accounting for the complex entrance condition associated with fenestral pores in the internal elastic lamina (IEL) to investigate the fluid mechanical environment around the smooth muscle cells (SMCs) right beneath the IEL. The IEL was modeled as an impermeable barrier to water flow except for the fenestral pores, which were assumed to be uniformly distributed over the IEL. The medial layer was modeled as a heterogeneous medium composed of a periodic array of cylindrical SMCs embedded in a continuous porous medium representing the interstitial proteoglycan and collagen matrix. Depending on the distance between the IEL bottom surface and the upstream end of the proximal layer of SMCs, the local shear stress on SMCs right beneath the fenestral pore could be more than 10 times higher than that on the cells far removed from the IEL under the conditions that the fenestral pore diameter and area fraction of pores were kept constant at 1.4 microm and 0.05, respectively. Thus these proximal SMCs may experience shear stress levels that are even higher than endothelial cells exposed to normal blood flow (order of 10 dyn/cm(2)). Furthermore, entrance flow through fenestral pores alters considerably the interstitial flow field in the medial layer over a spatial length scale of the order of the fenestral pore diameter. Thus the spatial gradient of shear stress on the most superficial SMC is noticeably higher than computed for endothelial cell surfaces.

Endothelial C-Type Natriuretic Peptide Acts on Pericytes to Regulate Microcirculatory Flow and Blood Pressure.

PubMed

Špiranec, Katarina; Chen, Wen; Werner, Franziska; Nikolaev, Viacheslav O; Naruke, Takashi; Koch, Franziska; Werner, Andrea; Eder-Negrin, Petra; Diéguez-Hurtado, Rodrigo; Adams, Ralf H; Baba, Hideo A; Schmidt, Hannes; Schuh, Kai; Skryabin, Boris V; Movahedi, Kiavash; Schweda, Frank; Kuhn, Michaela

2018-04-06

Background -Peripheral vascular resistance has a major impact on arterial blood pressure levels. Endothelial C-type natriuretic peptide (CNP) participates in the local regulation of vascular tone but the target cells remain controversial. The cGMP-producing guanylyl cyclase-B (GC-B) receptor for CNP is expressed in vascular smooth muscle cells (VSMC). However, whereas endothelial cell-specific CNP knockout mice are hypertensive, mice with deletion of GC-B in VSMC have unaltered blood pressure. Methods -We analyzed whether the vasodilating response to CNP changes along the vascular tree, i.e. whether the GC-B receptor is expressed in microvascular types of cells. Mice with a floxed GC-B ( Npr2 ) gene were interbred with Tie2-Cre or PDGF-Rβ-Cre ERT2 lines to develop mice lacking GC-B in endothelial cells or in precapillary arteriolar SMC and capillary pericytes. Intravital microscopy, (non)invasive hemodynamics, fluorescence energy transfer studies of pericyte's cAMP levels in situ and renal physiology were combined to dissect whether and how CNP/GC-B/cGMP signaling modulates microcirculatory tone and blood pressure. Results -Intravital microscopy studies revealed that the vasodilatatory effect of CNP increases towards small-diameter arterioles and capillaries. Consistently, CNP did not prevent endothelin-1-induced acute constrictions of proximal arterioles but fully reversed endothelin effects in precapillary arterioles and capillaries. Here, the GC-B receptor is expressed both in endothelial and mural cells, i.e. in pericytes. Notably, the vasodilatatory effects of CNP were preserved in mice with endothelial GC-B deletion but abolished in mice lacking GC-B in microcirculatory SMC and pericytes. CNP, via GC-B/cGMP signaling modulates two signaling cascades in pericytes: it activates cGMP-dependent protein kinase I to phosphorylate downstream targets such as the cytoskeleton-associated vasodilator activated phosphoprotein; and it inhibits phosphodiesterase 3A, thereby enhancing pericyte's cAMP levels. Ultimately these pathways prevent endothelin-induced increases of pericyte calcium levels and pericyte contraction. Mice with deletion of GC-B in microcirculatory SMC and pericytes have elevated peripheral resistance and chronic arterial hypertension without a change in renal function. Conclusions -Our studies indicate that endothelial CNP regulates distal arteriolar and capillary blood flow. CNP-induced GC-B/cGMP signaling in microvascular SMC and pericytes is essential for the maintenance of normal microvascular resistance and blood pressure.

Cerebral vascular structure in the motor cortex of adult mice is stable and is not altered by voluntary exercise.

PubMed

Cudmore, Robert H; Dougherty, Sarah E; Linden, David J

2017-12-01

The cerebral vasculature provides blood flow throughout the brain, and local changes in blood flow are regulated to match the metabolic demands of the active brain regions. This neurovascular coupling is mediated by real-time changes in vessel diameter and depends on the underlying vascular network structure. Neurovascular structure is configured during development by genetic and activity-dependent factors. In adulthood, it can be altered by experiences such as prolonged hypoxia, sensory deprivation and seizure. Here, we have sought to determine whether exercise could alter cerebral vascular structure in the adult mouse. We performed repeated in vivo two-photon imaging in the motor cortex of adult transgenic mice expressing membrane-anchored green fluorescent protein in endothelial cells (tyrosine endothelial kinase 2 receptor (Tie2)-Cre:mTmG). This strategy allows for high-resolution imaging of the vessel walls throughout the lifespan. Vascular structure, as measured by capillary branch point number and position, segment diameter and length remained stable over a time scale of months as did pericyte number and position. Furthermore, we compared the vascular structure before, during, and after periods of voluntary wheel running and found no alterations in these same parameters. In both running and control mice, we observed a low rate of capillary segment subtraction. Interestingly, these rare subtraction events preferentially remove short vascular loops.

Indigo naturalis and its component tryptanthrin exert anti-angiogenic effect by arresting cell cycle and inhibiting Akt and FAK signaling in human vascular endothelial cells.

PubMed

Chang, Hsin-Ning; Huang, Sheng-Teng; Yeh, Yuan-Chieh; Wang, Hsin-Shih; Wang, Tzu-Hao; Wu, Yi-Hong; Pang, Jong-Hwei S

2015-11-04

Indigo naturalis has been used to treat inflammatory diseases and dermatosis, including psoriasis, since thousands of years in China. It has been proven effective in our previous clinical studies on treating psoriasis, but the active component and the mechanism of how indigo naturalis working still needs to be clarified. Since the dysregulated angiogenesis is known to play an important role in the pathogenesis of psoriasis, the anti-angiogenic effect of indigo naturalis and tryptanthrin, a pure component of indigo naturalis, was investigated. The in vivo angiogenesis was studied by chick chorioallantoic membrane assay. The in vitro studies were performed using human vascular endothelial cells. Cell viability was determined by MTT assay. Cell cycle distribution was revealed by flow cytometry. The cellular messenger (m)RNA or protein expression level was analyzed by real-time RT-PCR or Western blot, respectively. Transwell filter migration assay and matrix gel-induced tube formation method were applied to examine the angiogenic potential. Indigo naturalis significantly inhibited the in vivo vascular endothelial growth factor (VEGF)-induced angiogenesis, as well as tryptanthrin. In vitro studies confirmed that indigo naturalis and tryptanthrin reduced the number of viable vascular endothelial cells. Tryptanthrin resulted in a cell cycle arrest and dose-dependently decreased the expressions of cyclin A, cyclin B, cyclin dependent kinase(CDK) 1 and 2, but not cyclin D and cyclin E, at both the mRNA and protein levels. The migration and tube formation of vascular endothelial cells were significantly inhibited by tryptanthrin in a dose-dependent manner. Result also showed that tryptanthrin could reduce the phosphorylated levels of both protein kinase B (PKB or Akt) and focal adhesion kinase (FAK). All together, these results demonstrated the anti-angiogenic effect of tryptanthrin, the acting component of indigo naturalis and revealed the underlying mechanism by inhibiting the cell cycle progression, cell migration and tube formation, likely mediated through blocking the Akt and FAK pathways. Copyright © 2015 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Endothelial MMP14 is required for endothelial-dependent growth support of human airway basal cells

PubMed Central

Ding, Bi-Sen; Gomi, Kazunori; Rafii, Shahin; Crystal, Ronald G.; Walters, Matthew S.

2015-01-01

ABSTRACT Human airway basal cells are the stem (or progenitor) population of the airway epithelium, and play a central role in anchoring the epithelium to the basement membrane. The anatomic position of basal cells allows for potential paracrine signaling between them and the underlying non-epithelial stromal cells. In support of this, we have previously demonstrated that endothelial cells support growth of basal cells during co-culture through vascular endothelial growth factor A (VEGFA)-mediated signaling. Building on these findings, we found, by RNA sequencing analysis, that basal cells expressed multiple fibroblast growth factor (FGF) ligands (FGF2, FGF5, FGF11 and FGF13) and that only FGF2 and FGF5 were capable of functioning in a paracrine manner to activate classical FGF receptor (FGFR) signaling. Antibody-mediated blocking of FGFR1 during basal-cell–endothelial-cell co-culture significantly reduced the endothelial-cell-dependent basal cell growth. Stimulation of endothelial cells with basal-cell-derived growth factors induced endothelial cell expression of matrix metallopeptidase 14 (MMP14), and short hairpin RNA (shRNA)-mediated knockdown of endothelial cell MMP14 significantly reduced the endothelial-cell-dependent growth of basal cells. Overall, these data characterize a new growth-factor-mediated reciprocal ‘crosstalk’ between human airway basal cells and endothelial cells that regulates proliferation of basal cells. PMID:26116571

Influence of homeobox B2 antisense oligodeoxynucleotides on the biological characteristics of in vitro cultured primary human umbilical vein endothelial cells.

PubMed

Liu, X S; Zhang, X Q; Tian, T; Liu, L; Ming, J

2008-01-01

This study aims to explore the influence of homeobox B2 (HOXB2) antisense oligodeoxynucleotides (asodn) on the biological characteristics of in vitro cultured primary human umbilical vein endothelial cells (HUVECs). The distribution of HOXB2 asodn in the HUVECs was observed by fluorescent labelling, and the influence of different concentrations of HOXB2 asodn on the DNA synthesis of HUVECs was assessed. Flow cytometry and a reverse transcriptase-polymerase chain reaction (RT- PCR) method were employed to observe the influence of HOXB2 asodn on HOXB2 expression and the HUVEC cell cycle. After the induction of liposome, the nuclear fluorescent staining of HOXB2 asodn was weaker 15 min after transfection and the staining reached the strongest level at 4-8 h but then weakened and disappeared by 16 h after transfection. This indicated that endothelial DNA synthesis could be inhibited by HOXB2 asodn in a dose-dependent manner. Furthermore, the HUVECs could be delayed in their passage from G1 to S. Simultaneously, expression of HOXB2 mRNA had decreased significantly by 24-48 h after transfection. Clearly, HOXB2 plays important roles in the proliferation of endothelial cells and also affects the cell cycle.

Theoretical estimates of mechanical properties of the endothelial cell cytoskeleton.

PubMed Central

Satcher, R L; Dewey, C F

1996-01-01

Current modeling of endothelial cell mechanics does not account for the network of F-actin that permeates the cytoplasm. This network, the distributed cytoplasmic structural actin (DCSA), extends from apical to basal membranes, with frequent attachments. Stress fibers are intercalated within the network, with similar frequent attachments. The microscopic structure of the DCSA resembles a foam, so that the mechanical properties can be estimated with analogy to these well-studied systems. The moduli of shear and elastic deformations are estimated to be on the order of 10(5) dynes/cm2. This prediction agrees with experimental measurements of the properties of cytoplasm and endothelial cells reported elsewhere. Stress fibers can potentially increase the modulus by a factor of 2-10, depending on whether they act in series or parallel to the network in transmitting surface forces. The deformations produced by physiological flow fields are of insufficient magnitude to disrupt cell-to-cell or DCSA cross-linkages. The questions raised by this paradox, and the ramifications of implicating the previously unreported DCSA as the primary force transmission element are discussed. Images FIGURE 2 PMID:8804594

Brief Exposure to Secondhand Smoke Reversibly Impairs Endothelial Vasodilatory Function

PubMed Central

2014-01-01

Introduction: We sought to determine the effects of brief exposures to low concentrations of tobacco secondhand smoke (SHS) on arterial flow-mediated dilation (FMD, a nitric oxide-dependent measure of vascular endothelial function), in a controlled animal model never before exposed to smoke. In humans, SHS exposure for 30min impairs FMD. It is important to gain a better understanding of the acute effects of exposure to SHS at low concentrations and for brief periods of time. Methods: We measured changes in FMD in rats exposed to a range of real-world levels of SHS for durations of 30min, 10min, 1min, and 4 breaths (roughly 15 s). Results: We observed a dose-response relationship between SHS particle concentration over 30min and post-exposure impairment of FMD, which was linear through the range typically encountered in smoky restaurants and then saturated at higher concentrations. One min of exposure to SHS at moderate concentrations was sufficient to impair FMD. Conclusions: Brief SHS exposure at real-world levels reversibly impairs FMD. Even 1min of SHS exposure can cause reduction of endothelial function. PMID:24302638

Astrocytes Can Adopt Endothelial Cell Fates in a p53-Dependent Manner.

PubMed

Brumm, Andrew J; Nunez, Stefanie; Doroudchi, Mehdi M; Kawaguchi, Riki; Duan, Jinhzu; Pellegrini, Matteo; Lam, Larry; Carmichael, S Thomas; Deb, Arjun; Hinman, Jason D

2017-08-01

Astrocytes respond to a variety of CNS injuries by cellular enlargement, process outgrowth, and upregulation of extracellular matrix proteins that function to prevent expansion of the injured region. This astrocytic response, though critical to the acute injury response, results in the formation of a glial scar that inhibits neural repair. Scar-forming cells (fibroblasts) in the heart can undergo mesenchymal-endothelial transition into endothelial cell fates following cardiac injury in a process dependent on p53 that can be modulated to augment cardiac repair. Here, we sought to determine whether astrocytes, as the primary scar-forming cell of the CNS, are able to undergo a similar cellular phenotypic transition and adopt endothelial cell fates. Serum deprivation of differentiated astrocytes resulted in a change in cellular morphology and upregulation of endothelial cell marker genes. In a tube formation assay, serum-deprived astrocytes showed a substantial increase in vessel-like morphology that was comparable to human umbilical vein endothelial cells and dependent on p53. RNA sequencing of serum-deprived astrocytes demonstrated an expression profile that mimicked an endothelial rather than astrocyte transcriptome and identified p53 and angiogenic pathways as specifically upregulated. Inhibition of p53 with genetic or pharmacologic strategies inhibited astrocyte-endothelial transition. Astrocyte-endothelial cell transition could also be modulated by miR-194, a microRNA downstream of p53 that affects expression of genes regulating angiogenesis. Together, these studies demonstrate that differentiated astrocytes retain a stimulus-dependent mechanism for cellular transition into an endothelial phenotype that may modulate formation of the glial scar and promote injury-induced angiogenesis.

Crataegus special extract WS 1442 causes endothelium-dependent relaxation via a redox-sensitive Src- and Akt-dependent activation of endothelial NO synthase but not via activation of estrogen receptors.

PubMed

Anselm, Eric; Socorro, Vanesca Frota Madeira; Dal-Ros, Stéphanie; Schott, Christa; Bronner, Christian; Schini-Kerth, Valérie B

2009-03-01

This study determined whether the Crataegus (Hawthorn species) special extract WS 1442 stimulates the endothelial formation of nitric oxide (NO), a vasoprotective factor, and characterized the underlying mechanism. Vascular reactivity was assessed in porcine coronary artery rings, reactive oxygen species (ROS) formation in artery sections by microscopy, and phosphorylation of Akt and endothelial NO synthase (eNOS) in endothelial cells by Western blot analysis. WS 1442 caused endothelium-dependent relaxations in coronary artery rings, which were reduced by N-nitro-L-arginine (a competitive inhibitor of NO synthase) and by charybdotoxin plus apamin (two inhibitors of endothelium-derived hyperpolarizing factor-mediated responses). Relaxations to WS 1442 were inhibited by intracellular ROS scavengers and inhibitors of Src and PI3-kinase, but not by an estrogen receptor antagonist. WS 1442 stimulated the endothelial formation of ROS in artery sections, and a redox-sensitive phosphorylation of Akt and eNOS in endothelial cells. WS 1442 induced endothelium-dependent NO-mediated relaxations of coronary artery rings through the redox-sensitive Src/PI3-kinase/Akt-dependent phosphorylation of eNOS.

The role of platelet and endothelial GARP in thrombosis and hemostasis.

PubMed

Vermeersch, Elien; Denorme, Frederik; Maes, Wim; De Meyer, Simon F; Vanhoorelbeke, Karen; Edwards, Justin; Shevach, Ethan M; Unutmaz, Derya; Fujii, Hodaka; Deckmyn, Hans; Tersteeg, Claudia

2017-01-01

Glycoprotein-A Repetitions Predominant protein (GARP or LRRC32) is present on among others human platelets and endothelial cells. Evidence for its involvement in thrombus formation was suggested by full knockout of GARP in zebrafish. To evaluate the role of GARP in platelet physiology and in thrombus formation using platelet and endothelial conditional GARP knock out mice. Platelet and endothelial specific GARP knockout mice were generated using the Cre-loxP recombination system. The function of platelets without GARP was measured by flow cytometry, spreading analysis and aggregometry using PAR4-activating peptide and collagen related peptide. Additionally, clot retraction and collagen-induced platelet adhesion and aggregation under flow were analyzed. Finally, in vivo tail bleeding time, occlusion time of the mesenteric and carotid artery after FeCl3-induced thrombosis were determined in platelet and endothelial specific GARP knock out mice. Platelet specific GARP knockout mice had normal surface GPIb, GPVI and integrin αIIb glycoprotein expression. Although GARP expression was increased upon platelet activation, platelets without GARP displayed normal agonist induced activation, spreading on fibrinogen and aggregation responses. Furthermore, absence of GARP on platelets did not influence clot retraction and had no impact on thrombus formation on collagen-coated surfaces under flow. In line with this, neither the tail bleeding time nor the occlusion time in the carotid- and mesenteric artery after FeCl3-induced thrombus formation in platelet or endothelial specific GARP knock out mice were affected. Evidence is provided that platelet and endothelial GARP are not important in hemostasis and thrombosis in mice.

The role of platelet and endothelial GARP in thrombosis and hemostasis

PubMed Central

Vermeersch, Elien; Denorme, Frederik; Maes, Wim; De Meyer, Simon F.; Vanhoorelbeke, Karen; Edwards, Justin; Shevach, Ethan M.; Unutmaz, Derya; Fujii, Hodaka; Deckmyn, Hans; Tersteeg, Claudia

2017-01-01

Background Glycoprotein-A Repetitions Predominant protein (GARP or LRRC32) is present on among others human platelets and endothelial cells. Evidence for its involvement in thrombus formation was suggested by full knockout of GARP in zebrafish. Objectives To evaluate the role of GARP in platelet physiology and in thrombus formation using platelet and endothelial conditional GARP knock out mice. Methods Platelet and endothelial specific GARP knockout mice were generated using the Cre-loxP recombination system. The function of platelets without GARP was measured by flow cytometry, spreading analysis and aggregometry using PAR4-activating peptide and collagen related peptide. Additionally, clot retraction and collagen-induced platelet adhesion and aggregation under flow were analyzed. Finally, in vivo tail bleeding time, occlusion time of the mesenteric and carotid artery after FeCl3-induced thrombosis were determined in platelet and endothelial specific GARP knock out mice. Results Platelet specific GARP knockout mice had normal surface GPIb, GPVI and integrin αIIb glycoprotein expression. Although GARP expression was increased upon platelet activation, platelets without GARP displayed normal agonist induced activation, spreading on fibrinogen and aggregation responses. Furthermore, absence of GARP on platelets did not influence clot retraction and had no impact on thrombus formation on collagen-coated surfaces under flow. In line with this, neither the tail bleeding time nor the occlusion time in the carotid- and mesenteric artery after FeCl3-induced thrombus formation in platelet or endothelial specific GARP knock out mice were affected. Conclusions Evidence is provided that platelet and endothelial GARP are not important in hemostasis and thrombosis in mice. PMID:28278197

Phosphodiesterase-4 inhibition as a therapeutic approach to treat capillary leakage in systemic inflammation.

PubMed

Schick, Martin Alexander; Wunder, Christian; Wollborn, Jakob; Roewer, Norbert; Waschke, Jens; Germer, Christoph-Thomas; Schlegel, Nicolas

2012-06-01

In sepsis and systemic inflammation, increased microvascular permeability and consecutive breakdown of microcirculatory flow significantly contribute to organ failure and death. Evidence points to a critical role of cAMP levels in endothelial cells to maintain capillary endothelial barrier properties in acute inflammation. However, approaches to verify this observation in systemic models are rare. Therefore we tested here whether systemic application of the phosphodiesterase-4-inhibitors (PD-4-Is) rolipram or roflumilast to increase endothelial cAMP was effective to attenuate capillary leakage and breakdown of microcirculatory flow in severe lipopolysaccharide (LPS)-induced systemic inflammation in rats. Measurements of cAMP in mesenteric microvessels demonstrated significant LPS-induced loss of cAMP levels which was blocked by application of rolipram. Increased endothelial cAMP by application of either PD-4-I rolipram or roflumilast led to stabilization of endothelial barrier properties as revealed by measurements of extravasated FITC-albumin in postcapillary mesenteric venules. Accordingly, microcirculatory flow in mesenteric venules was significantly increased following PD-4-I treatment and blood gas analyses indicated improved metabolism. Furthermore application of PD-4-I after manifestation of LPS-induced systemic inflammation and capillary leakage therapeutically stabilized endothelial barrier properties as revealed by significantly reduced volume resuscitation for haemodynamic stabilization. Accordingly microcirculation was significantly improved following treatment with PD-4-Is. Our results demonstrate that inflammation-derived loss of endothelial cAMP contributes to capillary leakage which was blocked by systemic PD-4-I treatment. Therefore these data suggest a highly clinically relevant and applicable approach to stabilize capillary leakage in sepsis and systemic inflammation.

Polyphenolic compounds from red grapes acutely improve endothelial function in patients with coronary heart disease.

PubMed

Lekakis, John; Rallidis, Loukianos S; Andreadou, Ioanna; Vamvakou, Georgia; Kazantzoglou, Georgios; Magiatis, Prokopios; Skaltsounis, Alexios-Leandros; Kremastinos, Dimitrios T

2005-12-01

It has been shown that acute intake of red wine improves endothelial-dependent vasodilatation. It is not clear, however, which constituents of red wine are responsible for this effect. We examined whether acute intake of a red grape polyphenol extract has a positive effect on brachial artery flow-mediated dilatation. We recruited 30 male patients with coronary heart disease. They were randomly assigned either to a red grape polyphenol extract (600 mg) dissolved in 20 ml of water (n = 15) or 20 ml of water (placebo) (n = 15). The extract of grapes contained 4.32 mg epicatechin, 2.72 mg catechin, 2.07 mg gallic acid, 0.9 mg trans-resveratrol, 0.47 mg rutin, 0.42 mg epsilon-viniferin, 0.28 mg, p-coumaric acid, 0.14 mg ferulic acid and 0.04 mg quercetin per gram. Flow-mediated dilatation of the brachial artery was evaluated after reactive hyperemia induced by cuff obstruction of the forearm, using high-resolution ultasonography. Particularly, flow-mediated dilatation was measured after fasting and 30, 60 and 120 min after the intake of the grape extract or placebo. Intake of the red grape polyphenol extract caused an increase in flow-mediated dilatation, peaking at 60 min, which was significantly higher than the baseline values (4.52+/-1.34 versus 2.6+/-1.5%; P < 0.001) and the corresponding values at 60 min after the intake of placebo (4.52+/-1.34 versus 2.64+/-1.8%, P < 0.001). There was no change in FMD values after the intake of placebo throughout the whole duration of the study. Polyphenolic compounds from red grapes acutely improve endothelial function in patients with coronary heart disease. These results could probably, at least partly, explain the favorable effects of red wine on the cardiovascular system.

Vascular endothelial function and oxidative stress mechanisms in patients with Behçet's syndrome.

PubMed

Chambers, J C; Haskard, D O; Kooner, J S

2001-02-01

We sought to test the hypothesis that vascular endothelial function is impaired in Behçet's syndrome and reflects increased levels of oxidative stress. Behçet's syndrome is a multisystem inflammatory disorder commonly complicated by vascular thrombosis and arterial aneurysm formation. The precise mechanisms underlying vascular disease in Behçet's syndrome are not known. We studied 19 patients with Behçet's syndrome (18 to 50 years old, 9 men) and 21 healthy volunteers (18 to 50 years old, 10 men). Brachial artery flow-mediated dilation (endothelium-dependent), and nitroglycerin (NTG)-induced dilation (endothelium-independent) were measured. To investigate oxidative stress mechanisms, vascular studies were repeated 1 h after administration of vitamin C (1 g, intravenous) in 12 patients and 12 control subjects. Flow-mediated dilation was reduced in patients with Behcet's syndrome as compared with control subjects (0.7 +/- 0.9% vs. 5.7 +/- 0.9%, p = 0.001). In contrast, there were no significant differences in the brachial artery diameter (4.2 +/- 0.2 vs. 4.0 +/- 0.2 mm, p = 0.47) or NTG-induced dilation (19.7 +/- 1.9% vs. 19.7 +/- 1.2%, p = 0.98). In regression analysis, Behçet's syndrome was associated with impaired flow-mediated dilation independent of age, gender, brachial artery diameter, blood pressure, cholesterol and glucose. Vitamin C increased flow-mediated dilation in Behçet's syndrome (0.2 +/- 0.7% to 3.5 +/- 1.0%, p = 0.002), but not in control subjects (4.3 +/- 0.6% to 4.7 +/- 0.4%, p = 0.51). In both groups, NTG-induced dilation and brachial artery diameter were unchanged after vitamin C treatment. Vascular endothelial function is impaired in Behcet's syndrome and can be rapidly improved by vitamin C treatment. Our results support a role for oxidative stress in the pathophysiology of Behçet's syndrome and provide a rationale for therapeutic studies aimed at reducing vascular complications in this disorder.

Factor X/Xa elicits protective signaling responses in endothelial cells directly via PAR-2 and indirectly via endothelial protein C receptor-dependent recruitment of PAR-1.

PubMed

Bae, Jong-Sup; Yang, Likui; Rezaie, Alireza R

2010-11-05

We recently demonstrated that the Gla domain-dependent interaction of protein C with endothelial protein C receptor (EPCR) leads to dissociation of the receptor from caveolin-1 and recruitment of PAR-1 to a protective signaling pathway. Thus, the activation of PAR-1 by either thrombin or PAR-1 agonist peptide elicited a barrier-protective response if endothelial cells were preincubated with protein C. In this study, we examined whether other vitamin K-dependent coagulation protease zymogens can modulate PAR-dependent signaling responses in endothelial cells. We discovered that the activation of both PAR-1 and PAR-2 in endothelial cells pretreated with factor FX (FX)-S195A, but not other procoagulant protease zymogens, also results in initiation of protective intracellular responses. Interestingly, similar to protein C, FX interaction with endothelial cells leads to dissociation of EPCR from caveolin-1 and recruitment of PAR-1 to a protective pathway. Further studies revealed that, FX activated by factor VIIa on tissue factor bearing endothelial cells also initiates protective signaling responses through the activation of PAR-2 independent of EPCR mobilization. All results could be recapitulated by the receptor agonist peptides to both PAR-1 and PAR-2. These results suggest that a cross-talk between EPCR and an unknown FX/FXa receptor, which does not require interaction with the Gla domain of FX, recruits PAR-1 to protective signaling pathways in endothelial cells.

Physiologically assessed hot flashes and endothelial function among midlife women.

PubMed

Thurston, Rebecca C; Chang, Yuefang; Barinas-Mitchell, Emma; Jennings, J Richard; von Känel, Roland; Landsittel, Doug P; Matthews, Karen A

2017-08-01

Hot flashes are experienced by most midlife women. Emerging data indicate that they may be associated with endothelial dysfunction. No studies have tested whether hot flashes are associated with endothelial function using physiologic measures of hot flashes. We tested whether physiologically assessed hot flashes were associated with poorer endothelial function. We also considered whether age modified associations. Two hundred seventy-two nonsmoking women reporting either daily hot flashes or no hot flashes, aged 40 to 60 years, and free of clinical cardiovascular disease, underwent ambulatory physiologic hot flash and diary hot flash monitoring; a blood draw; and ultrasound measurement of brachial artery flow-mediated dilation to assess endothelial function. Associations between hot flashes and flow-mediated dilation were tested in linear regression models controlling for lumen diameter, demographics, cardiovascular disease risk factors, and estradiol. In multivariable models incorporating cardiovascular disease risk factors, significant interactions by age (P < 0.05) indicated that among the younger tertile of women in the sample (age 40-53 years), the presence of hot flashes (beta [standard error] = -2.07 [0.79], P = 0.01), and more frequent physiologic hot flashes (for each hot flash: beta [standard error] = -0.10 [0.05], P = 0.03, multivariable) were associated with lower flow-mediated dilation. Associations were not accounted for by estradiol. Associations were not observed among the older women (age 54-60 years) or for self-reported hot flash frequency, severity, or bother. Among the younger women, hot flashes explained more variance in flow-mediated dilation than standard cardiovascular disease risk factors or estradiol. Among younger midlife women, frequent hot flashes were associated with poorer endothelial function and may provide information about women's vascular status beyond cardiovascular disease risk factors and estradiol.

Posttraumatic Stress Disorder Is Associated With Worse Endothelial Function Among Veterans.

PubMed

Grenon, S Marlene; Owens, Christopher D; Alley, Hugh; Perez, Sandra; Whooley, Mary A; Neylan, Thomas C; Aschbacher, Kirstin; Gasper, Warren J; Hilton, Joan F; Cohen, Beth E

2016-03-23

Current research in behavioral cardiology reveals a significant association between posttraumatic stress disorder (PTSD) and increased risk for cardiovascular disease and mortality; however, the underlying mechanisms remain poorly understood. We hypothesized that patients with PTSD would exhibit endothelial dysfunction, a potential mechanism involved in the development and progression of cardiovascular disease. A total of 214 outpatients treated at the San Francisco Veterans Affairs Medical Center underwent tests of endothelial function and evaluation for PTSD. Flow-mediated vasodilation of the brachial artery was performed to assess endothelial function, and current PTSD status was defined by the PTSD Checklist, based on the Diagnostic and Statistical Manual of Mental Disorders (Fourth Edition), with a score ≥40. Multivariable linear regression models were used to estimate the association between PTSD status and endothelial function. Patients with PTSD (n=67) were more likely to be male (99% versus 91%, P=0.04) and to have depression (58% versus 8%, P<0.0001) and were less likely to be on an angiotensin-converting enzyme inhibitor (17% versus 36%, P=0.007) or β-blocker treatment (25% versus 41%, P=0.03). Univariate analysis demonstrated that patients with PTSD had significantly lower flow-mediated vasodilation (5.8±3.4% versus 7.5±3.7%; P=0.003); furthermore, lower flow-mediated vasodilation was associated with increasing age (P=0.008), decreasing estimated glomerular filtration rate (P=0.003), hypertension (P=0.002), aspirin (P=0.03), and β-blocker treatments (P=0.01). In multivariable analysis, PTSD remained independently associated with lower flow-mediated vasodilation (P=0.0005). After adjusting for demographic, comorbidity, and treatment characteristics, PTSD remained associated with worse endothelial function in an outpatient population. Whether poor endothelial function contributes to the higher risk of cardiovascular disease in patients with PTSD deserves further study. © 2016 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Shear stress induced stimulation of mammalian cell metabolism

NASA Technical Reports Server (NTRS)

Mcintire, L. V.; Frangos, J. A.; Eskin, S. G.

1988-01-01

A flow apparatus was developed for the study of the metabolic response of anchorage dependent cells to a wide range of steady and pulsatile shear stresses under well controlled conditions. Human umbilical vein endothelial cell monolayers were subjected to steady shear stresses of up to 24 dynes/sq cm, and the production of prostacyclin was determined. The onset of flow led to a burst in prostacyclin production which decayed to a long term steady state rate (SSR). The SSR of cells exposed to flow was greater than the basal release level, and increased linearly with increasing shear stress. It is demonstrated that shear stresses in certain ranges may not be detrimental to mammalian cell metabolism. In fact, throughout the range of shear stresses studied, metabolite production is maximized by maximizing shear stress.

Vascular Reactivity Profile of Novel KCa3.1-Selective Positive-Gating Modulators in the Coronary Vascular Bed

PubMed Central

Oliván-Viguera, Aida; Valero, Marta Sofía; Pinilla, Estéfano; Amor, Sara; García-Villalón, Ángel Luis; Coleman, Nichole; Laría, Celia; Calvín-Tienza, Víctor; García-Otín, Ángel-Luis; Fernández-Fernández, José M.; Murillo, Ma Divina; Gálvez, José A.; Díaz-de-Villegas, María D.; Badorrey, Ramón; Simonsen, Ulf; Rivera, Luis; Wulff, Heike; Köhler, Ralf

2017-01-01

Opening of intermediate-conductance calcium-activated potassium channels (KCa3.1) produces membrane hyperpolarization in the vascular endothelium. Here, we studied the ability of two new KCa3.1-selective positive-gating modulators, SKA-111 and SKA-121, to (1) evoke porcine endothelial cell KCa3.1 membrane hyperpolarization, (2) induce endothelium-dependent and, particularly, endothelium-derived hyperpolarization (EDH)-type relaxation in porcine coronary arteries (PCA) and (3) influence coronary artery tone in isolated rat hearts. In whole-cell patch-clamp experiments on endothelial cells of PCA (PCAEC), KCa currents evoked by bradykinin (BK) were potentiated ≈7-fold by either SKA-111 or SKA-121 (both at 1 μM) and were blocked by a KCa3.1 blocker, TRAM-34. In membrane potential measurements, SKA-111 and SKA-121 augmented bradykinin-induced hyperpolarization. Isometric tension measurements in large- and small-calibre PCA showed that SKA-111 and SKA-121 potentiated endothelium-dependent relaxation with intact NO synthesis and EDH-type relaxation to BK by ≈2-fold. Potentiation of the BK response was prevented by KCa3.1 inhibition. In Langendorff-perfused rat hearts, SKA-111 potentiated coronary vasodilation elicited by BK. In conclusion, our data show that positive-gating modulation of KCa3.1 channels improves BK-induced membrane hyperpolarization and endothelium-dependent relaxation in small and large PCA as well as in the coronary circulation of rats. Positive-gating modulators of KCa3.1 could be therapeutically useful to improve coronary blood flow and counteract impaired coronary endothelial dysfunction in cardiovascular disease. PMID:26821335

Soluble intercellular adhesion molecule-1 and interleukin-6 levels reflect endothelial dysfunction in patients with primary hypercholesterolaemia treated with atorvastatin.

PubMed

Nawawi, H; Osman, N S; Annuar, R; Khalid, B A K; Yusoff, K

2003-08-01

Adhesion molecules and cytokines are involved in the pathogenesis of intimal injury in atherosclerosis but their relationship with endothelial function remains unclear. The objectives of this study were to examine the effects of atorvastatin on soluble adhesion molecules, interleukin-6 (IL-6) and brachial artery endothelial-dependent flow mediated dilatation (FMD) in patients with familial (FH) and non-familial hypercholesterolaemia (NFH). A total of 74 patients (27 FH and 47 NFH) were recruited. Fasting lipid profiles, soluble intercellular adhesion molecule-1 (sICAM-1), soluble vascular-cellular adhesion molecule-1 (sVCAM-1), E-selectin, IL-6 and FMD were measured at baseline, 2 weeks, 3 and 9 months post-atorvastatin treatment (FH--80 mg/day, NFH--10 mg/day). In both groups, compared to baseline, sICAM-1 levels were significantly reduced at 2 weeks, further reduced at 3 months and maintained at 9 months (P<0.0001). The IL-6 levels were significantly reduced at 3 months and 9 months compared to baseline for FH (P<0.005) and NFH (P<0.0001). In both groups, the FMD at 2 weeks was higher than baseline (P<0.005), with progressive improvement up to 9 months. FMD was negatively correlated with sICAM-1 and IL-6. In conclusion, both low and high doses of atorvastatin lead to early progressive improvement in endothelial function in patients with primary hypercholesterolaemia. sICAM-1 and IL-6 levels reflect endothelial dysfunction in these patients.

Hydrodynamic shear stress and mass transport modulation of endothelial cell metabolism.

PubMed

Nollert, M U; Diamond, S L; McIntire, L V

1991-09-01

Mammalian cells responds to physical forces by altering their growth rate, morphology, metabolism, and genetic expression. We have studied the mechanism by which these cells detect the presence of mechanical stress and convert this force into intracellular signals. As our model systems, we have studied cultured human endothelial cells, which line the blood vessels and forms the interface between the blood and the vessel wall. These cell responds within minutes to the initiation of flow by increasing their arachidonic acid metabolism and increasing the level of the intracellular second messengers inositol trisphosphate and calcium ion concentration. With continued exposure to arterial levels of wall shear stress for up to 24 h, endothelial cells increase the expression of tissue plasminogen activator (tPA) and tPA messenger RNA (mRNA) and decrease the expression of endothelin peptide and endothelin mRNA. Since the initiation of flow also causes enhanced convective mass transfer to the endothelial cell monolayer, we have investigated the role of enhanced convection of adenosine trisphosphate (ATP) to the cell surface in eliciting a cellular response by monitoring cytosolic calcium concentrations on the single cell level and by computing the concentration profile of ATP in a parallel-plate flow geometry. Our result demonstrate that endothelial cells respond in very specific ways to the initiation of flow and that mass transfer and fluid shear stress can both play a role in the modulation of intracellular signal transduction and metabolism.

Difference in protective effects of GIP and GLP-1 on endothelial cells according to cyclic adenosine monophosphate response.

PubMed

Lim, Dong-Mee; Park, Keun-Young; Hwang, Won-Min; Kim, Ju-Young; Kim, Byung-Joon

2017-05-01

Receptors for glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) are present in vascular endothelial cells. Previous studies investigating euglycemic status have demonstrated that GIP is directly involved in the physiology of blood vessels by controlling the blood flow rate of portal veins and that GLP-1 has a protective effect on blood vessels by acting on endothelial cells. However, to the best of our knowledge, the effects of GIP and GLP-1 on endothelial cells in patients with hyperglycemia remain unknown. Therefore, the present study investigated whether the effect of the incretin hormones GLP-1 and GIP differed with regards to the reversal of endothelial cell dysfunction caused by hyperglycemia. The production of nitric oxide (NO) was measured using the Griess reagent system kit and the expression of cyclic adenosine monophosphate (cAMP) in the cell was measured at a wavelength of 405 nm with the ELISA reader using the cyclic AMP EIA kit. Exposure of human umbilical vein endothelial cells (HUVEC) to a high glucose concentration decreased NO and endothelial nitric oxide synthase (eNOS) levels but increased inducible NOS (iNOS) levels. However, when HUVECs were pretreated with GLP-1, a reduction of iNOS expression was observed and the expression of eNOS and NO were increased, as opposed to pretreatment with GIP. The results differed according to the response of cAMP, the second messenger of incretin hormones: The GIP pretreatment group did not exhibit an increase in cAMP levels while the GLP-1 pretreatment group did. The results of the present study provide evidence that GLP-1, but not GIP, has a protective effect on endothelial function associated with cardiovascular disease, as it is associated with increased eNOS expression and the levels of NO. This effect may be due to an increase in the cAMP concentration during hyperglycemic events.

Electroacupuncture prevents endothelial dysfunction induced by ischemia-reperfusion injury via a cyclooxygenase-2-dependent mechanism: A randomized controlled crossover trial

PubMed Central

Park, Jimin; Woo, Jong Shin; Leem, Jungtae; Park, Jun Hyeong; Lee, Sanghoon; Chung, Hyemoon; Lee, Jung Myung; Kim, Jin-Bae; Kim, Woo-Shik; Kim, Kwon Sam; Kim, Weon

2017-01-01

Objective Exploring clinically effective methods to reduce ischemia-reperfusion (IR) injury in humans is critical. Several drugs have shown protective effects, but studies using other interventions have been rare. Electroacupuncture (EA) has induced similar protection in several animal studies but no study has investigated how the effects could be translated and reproduced in humans. This study aimed to explore the potential effect and mechanisms of EA in IR-induced endothelial dysfunction in humans. Methods This is a prospective, randomized, crossover, sham-controlled trial consisting of two protocols. Protocol 1 was a crossover study to investigate the effect of EA on IR-induced endothelial dysfunction. Twenty healthy volunteers were randomly assigned to EA or sham EA (sham). Flow mediated dilation (FMD) of the brachial artery (BA), nitroglycerin-mediated endothelial independent dilation, blood pressure before and after IR were measured. In protocol 2, seven volunteers were administered COX-2 inhibitor celecoxib (200 mg orally twice daily) for five days. After consumption, volunteers underwent FMD before and after IR identical to protocol 1. Results In protocol 1, baseline BA diameter, Pre-IR BA diameter and FMD were similar between the two groups (p = NS). After IR, sham group showed significantly blunted FMD (Pre-IR: 11.41 ± 3.10%, Post-IR: 4.49 ± 2.04%, p < 0.001). However, EA protected this blunted FMD (Pre-IR: 10.96 ± 5.30%, Post-IR: 9.47 ± 5.23%, p = NS, p < 0.05 compared with sham EA after IR). In protocol 2, this protective effect was completely abolished by pre-treatment with celecoxib (Pre-IR: 11.05 ± 3.27%; Post-IR: 4.20 ± 1.68%, p = 0.001). Conclusion EA may prevent IR-induced endothelial dysfunction via a COX-2 dependent mechanism. PMID:28591155

Regional Coronary Endothelial Function is Closely Related to Local Early Coronary Atherosclerosis in Patients with Mild Coronary Artery Disease: A Pilot Study

PubMed Central

Hays, Allison G.; Kelle, Sebastian; Hirsch, Glenn A.; Soleimanifard, Sahar; Yu, Jing; Agarwal, Harsh K.; Gerstenblith, Gary; Schär, Michael; Stuber, Matthias; Weiss, Robert G.

2012-01-01

Background Coronary endothelial function (endoFx) is abnormal in patients with established coronary artery disease (CAD) and was recently shown by MRI to relate to the severity of luminal stenosis. Recent advances in MRI now allow the non-invasive assessment of both anatomic and functional (endoFx) changes that previously required invasive studies. We tested the hypothesis that abnormal coronary endoFx is related to measures of early atherosclerosis such as increased coronary wall thickness (CWT). Methods and Results Seventeen arteries in fourteen healthy adults and seventeen arteries in fourteen patients with non-obstructive CAD were studied. To measure endoFx, coronary MRI was performed before and during isometric handgrip exercise, an endothelial-dependent stressor and changes in coronary cross-sectional area (CSA) and flow were measured. Black blood imaging was performed to quantify CWT and other indices of arterial remodeling. The mean stress-induced change in CSA was significantly higher in healthy adults (13.5%±12.8%, mean±SD, n=17) than in those with mildly diseased arteries (-2.2±6.8%, p<0.0001, n=17). Mean CWT was lower in healthy subjects (0.9±0.2mm) than in CAD patients (1.4±0.3mm, p<0.0001). In contrast to healthy subjects, stress-induced changes in CSA, a measure of coronary endoFx, correlated inversely with CWT in CAD patients (r= -0.73, p=0.0008). Conclusions There is an inverse relationship between coronary endothelial function and local CWT in CAD patients but not in healthy adults. These findings demonstrate that local endothelial-dependent functional changes are related to the extent of early anatomic atherosclerosis in mildly diseased arteries. This combined MRI approach enables the anatomic and functional investigation of early coronary disease. PMID:22492483

The human prothrombin kringle-2 derived peptide, NSA9, is internalized into bovine capillary endothelial cells through endocytosis and energy-dependent pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hwang, Hyun Sook; Kim, Soung Soo

Human prothrombin kringle-2 and its partial peptide, NSA9 (NSAVQLVEN), have been reported to have potent anti-angiogenic activities. Here, the internalization mechanism of NSA9 into bovine capillary endothelial (BCE) cells was examined using lactate dehydrogenase (LDH) release assay, fluorescence microscopy, and flow cytometry. LDH release assay results suggested that the integrity of the BCE cell membrane was unaffected by NSA9. Fluorescence microscopy indicated that internalized NSA9 was localized in the cytoplasm around the nucleus, and showed a punctuated fluorescence pattern, which is indicative of endocytic vesicles. Also, the cellular internalization of NSA9 is significantly inhibited by depletion of the cellular ATPmore » pool, endocytosis inhibitors such as chloroquine and nocodazole, and incubation at low temperature (4 deg C). In addition, the anti-proliferative activity of NSA9 against BCE cells was diminished in the presence of endocytosis or metabolic inhibitors. In conclusion, these results strongly suggest that NSA9 might exert its anti-proliferative activity through internalization into BCE cells by endocytosis and energy-dependent pathways.« less

Cigarette smoking impairs nitric oxide-mediated cerebral blood flow increase: Implications for Alzheimer's disease.

PubMed

Toda, Noboru; Okamura, Tomio

2016-08-01

Cerebral blood flow is mainly regulated by nitrergic (parasympathetic, postganglionic) nerves and nitric oxide (NO) liberated from endothelial cells in response to shear stress and stretch of vasculature, whereas sympathetic vasoconstrictor control is quite weak. On the other hand, peripheral vascular resistance and blood flow are mainly controlled by adrenergic vasoconstrictor nerves; endothelium-derived NO and nitrergic nerves play some roles as vasodilator factors. Cigarette smoking impairs NO synthesis in cerebral vascular endothelial cells and nitrergic nerves leading to interference with cerebral blood flow and glucose metabolism in the brain. Smoking-induced cerebral hypoperfusion is induced by impairment of synthesis and actions of NO via endothelial nitric oxide synthase (eNOS)/neuronal NOS (nNOS) inhibition and by increased production of oxygen radicals, resulting in decreased actions of NO on vascular smooth muscle. Nicotine acutely and chronically impairs the action of endothelial NO and also inhibits nitrergic nerve function in chronic use. Impaired cerebral blood supply promotes the synthesis of amyloid β that accelerates blood flow decrease. This vicious cycle is thought to be one of the important factors involving in Alzheimer's disease (AD). Quitting smoking is undoubtedly one of the important ways to prevent and delay the genesis or slow the progress of impaired cognitive function and AD. Copyright © 2016 The Authors. Production and hosting by Elsevier B.V. All rights reserved.

The effect of α1 -adrenergic blockade on post-exercise brachial artery flow-mediated dilatation at sea level and high altitude.

PubMed

Tymko, Michael M; Tremblay, Joshua C; Hansen, Alex B; Howe, Connor A; Willie, Chris K; Stembridge, Mike; Green, Daniel J; Hoiland, Ryan L; Subedi, Prajan; Anholm, James D; Ainslie, Philip N

2017-03-01

Our objective was to quantify endothelial function (via brachial artery flow-mediated dilatation) at sea level (344 m) and high altitude (3800 m) at rest and following both maximal exercise and 30 min of moderate-intensity cycling exercise with and without administration of an α 1 -adrenergic blockade. Brachial endothelial function did not differ between sea level and high altitude at rest, nor following maximal exercise. At sea level, endothelial function decreased following 30 min of moderate-intensity exercise, and this decrease was abolished with α 1 -adrenergic blockade. At high altitude, endothelial function did not decrease immediately after 30 min of moderate-intensity exercise, and administration of α 1 -adrenergic blockade resulted in an increase in flow-mediated dilatation. Our data indicate that post-exercise endothelial function is modified at high altitude (i.e. prolonged hypoxaemia). The current study helps to elucidate the physiological mechanisms associated with high-altitude acclimatization, and provides insight into the relationship between sympathetic nervous activity and vascular endothelial function. We examined the hypotheses that (1) at rest, endothelial function would be impaired at high altitude compared to sea level, (2) endothelial function would be reduced to a greater extent at sea level compared to high altitude after maximal exercise, and (3) reductions in endothelial function following moderate-intensity exercise at both sea level and high altitude are mediated via an α 1 -adrenergic pathway. In a double-blinded, counterbalanced, randomized and placebo-controlled design, nine healthy participants performed a maximal-exercise test, and two 30 min sessions of semi-recumbent cycling exercise at 50% peak output following either placebo or α 1 -adrenergic blockade (prazosin; 0.05 mg kg  -1 ). These experiments were completed at both sea-level (344 m) and high altitude (3800 m). Blood pressure (finger photoplethysmography), heart rate (electrocardiogram), oxygen saturation (pulse oximetry), and brachial artery blood flow and shear rate (ultrasound) were recorded before, during and following exercise. Endothelial function assessed by brachial artery flow-mediated dilatation (FMD) was measured before, immediately following and 60 min after exercise. Our findings were: (1) at rest, FMD remained unchanged between sea level and high altitude (placebo P = 0.287; prazosin: P = 0.110); (2) FMD remained unchanged after maximal exercise at sea level and high altitude (P = 0.244); and (3) the 2.9 ± 0.8% (P = 0.043) reduction in FMD immediately after moderate-intensity exercise at sea level was abolished via α 1 -adrenergic blockade. Conversely, at high altitude, FMD was unaltered following moderate-intensity exercise, and administration of α 1 -adrenergic blockade elevated FMD (P = 0.032). Our results suggest endothelial function is differentially affected by exercise when exposed to hypobaric hypoxia. These findings have implications for understanding the chronic impacts of hypoxaemia on exercise, and the interactions between the α 1 -adrenergic pathway and endothelial function. © 2016 The Authors. The Journal of Physiology © 2016 The Physiological Society.

Lipopolysaccharide-induced pulmonary endothelial barrier disruption and lung edema: critical role for bicarbonate stimulation of AC10.

PubMed

Nickols, Jordan; Obiako, Boniface; Ramila, K C; Putinta, Kevin; Schilling, Sarah; Sayner, Sarah L

2015-12-15

Bacteria-induced sepsis is a common cause of pulmonary endothelial barrier dysfunction and can progress toward acute respiratory distress syndrome. Elevations in intracellular cAMP tightly regulate pulmonary endothelial barrier integrity; however, cAMP signals are highly compartmentalized: whether cAMP is barrier-protective or -disruptive depends on the compartment (plasma membrane or cytosol, respectively) in which the signal is generated. The mammalian soluble adenylyl cyclase isoform 10 (AC10) is uniquely stimulated by bicarbonate and is expressed in pulmonary microvascular endothelial cells (PMVECs). Elevated extracellular bicarbonate increases cAMP in PMVECs to disrupt the endothelial barrier and increase the filtration coefficient (Kf) in the isolated lung. We tested the hypothesis that sepsis-induced endothelial barrier disruption and increased permeability are dependent on extracellular bicarbonate and activation of AC10. Our findings reveal that LPS-induced endothelial barrier disruption is dependent on extracellular bicarbonate: LPS-induced barrier failure and increased permeability are exacerbated in elevated bicarbonate compared with low extracellular bicarbonate. The AC10 inhibitor KH7 attenuated the bicarbonate-dependent LPS-induced barrier disruption. In the isolated lung, LPS failed to increase Kf in the presence of minimal perfusate bicarbonate. An increase in perfusate bicarbonate to the physiological range (24 mM) revealed the LPS-induced increase in Kf, which was attenuated by KH7. Furthermore, in PMVECs treated with LPS for 6 h, there was a dose-dependent increase in AC10 expression. Thus these findings reveal that LPS-induced pulmonary endothelial barrier failure requires bicarbonate activation of AC10. Copyright © 2015 the American Physiological Society.

Lipopolysaccharide-induced pulmonary endothelial barrier disruption and lung edema: critical role for bicarbonate stimulation of AC10

PubMed Central

Nickols, Jordan; Obiako, Boniface; Ramila, K. C.; Putinta, Kevin; Schilling, Sarah

2015-01-01

Bacteria-induced sepsis is a common cause of pulmonary endothelial barrier dysfunction and can progress toward acute respiratory distress syndrome. Elevations in intracellular cAMP tightly regulate pulmonary endothelial barrier integrity; however, cAMP signals are highly compartmentalized: whether cAMP is barrier-protective or -disruptive depends on the compartment (plasma membrane or cytosol, respectively) in which the signal is generated. The mammalian soluble adenylyl cyclase isoform 10 (AC10) is uniquely stimulated by bicarbonate and is expressed in pulmonary microvascular endothelial cells (PMVECs). Elevated extracellular bicarbonate increases cAMP in PMVECs to disrupt the endothelial barrier and increase the filtration coefficient (Kf) in the isolated lung. We tested the hypothesis that sepsis-induced endothelial barrier disruption and increased permeability are dependent on extracellular bicarbonate and activation of AC10. Our findings reveal that LPS-induced endothelial barrier disruption is dependent on extracellular bicarbonate: LPS-induced barrier failure and increased permeability are exacerbated in elevated bicarbonate compared with low extracellular bicarbonate. The AC10 inhibitor KH7 attenuated the bicarbonate-dependent LPS-induced barrier disruption. In the isolated lung, LPS failed to increase Kf in the presence of minimal perfusate bicarbonate. An increase in perfusate bicarbonate to the physiological range (24 mM) revealed the LPS-induced increase in Kf, which was attenuated by KH7. Furthermore, in PMVECs treated with LPS for 6 h, there was a dose-dependent increase in AC10 expression. Thus these findings reveal that LPS-induced pulmonary endothelial barrier failure requires bicarbonate activation of AC10. PMID:26475732

Connexin43high prostate cancer cells induce endothelial connexin43 up-regulation through the activation of intercellular ERK1/2-dependent signaling axis.

PubMed

Piwowarczyk, Katarzyna; Paw, Milena; Ryszawy, Damian; Rutkowska-Zapała, Magdalena; Madeja, Zbigniew; Siedlar, Maciej; Czyż, Jarosław

2017-06-01

Connexin(Cx)43 regulates the invasive potential of prostate cancer cells and participates in their extravasation. To address the role of endothelial Cx43 in this process, we analyzed Cx43 regulation in human umbilical vein endothelial cells in the proximity of Cx43 high (DU-145 and MAT-LyLu) and Cx43 low prostate cancer cells (PC-3 and AT-2). Endothelial Cx43 up-regulation was observed during the diapedesis of DU-145 and MAT-LyLu cells. This process was attenuated by transient Cx43 silencing in cancer cells and by chemical inhibition of ERK1/2-dependent signaling in endothelial cells. Cx43 expression in endothelial cells was insensitive to the inhibition of gap junctional intercellular coupling between Cx43 high prostate cancer and endothelial cells by 18α-glycyrrhetinic acid. Instead, endothelial Cx43 up-regulation was correlated with the local contraction of endothelial cells and with their activation in the proximity of Cx43 high DU-145 and MAT-LyLu cells. It was also sensitive to pro-inflammatory factors secreted by peripheral blood monocytes, such as TNFα. In contrast to Cx43 low AT-2 cells, Cx43 low PC-3 cells produced angioactive factors that locally activated the endothelial cells in the absence of endothelial Cx43 up-regulation. Collectively, these data show that Cx43 low and Cx43 high prostate cancer cells can adapt discrete, Cx43-independent and Cx43-dependent strategies of diapedesis. Our observations identify a novel strategy of prostate cancer cell diapedesis, which depends on the activation of intercellular Cx43/ERK1/2/Cx43 signaling axis at the interfaces between Cx43 high prostate cancer and endothelial cells. Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.

Allopurinol improves endothelial dysfunction in chronic heart failure.

PubMed

Farquharson, Colin A J; Butler, Robert; Hill, Alexander; Belch, Jill J F; Struthers, Allan D

2002-07-09

Increased oxidative stress in chronic heart failure is thought to contribute to endothelial dysfunction. Xanthine oxidase produces oxidative stress and therefore we examined whether allopurinol improved endothelial dysfunction in chronic heart failure. We performed a randomized, placebo-controlled, double-blind crossover study on 11 patients with New York Heart Association class II-III chronic heart failure, comparing 300 mg allopurinol daily (1 month) versus placebo. Endothelial function was assessed by standard forearm venous occlusion plethysmography with acetylcholine, nitroprusside, and verapamil. Plasma malondialdehyde levels were also compared to assess significant changes in oxidative stress. Allopurinol significantly increased the forearm blood flow response to acetylcholine (percentage change in forearm blood flow [mean+/-SEM]: 181+/-19% versus 120+/-22% allopurinol versus placebo; P=0.003). There were no significant differences in the forearm blood flow changes between the placebo and allopurinol treatment arms with regard to sodium nitroprusside or verapamil. Plasma malondialdehyde was significantly reduced with allopurinol treatment (346+/-128 nmol/L versus 461+/-101 nmol/L, allopurinol versus placebo; P=0.03), consistent with reduced oxidative stress with allopurinol therapy. We have shown that allopurinol improves endothelial dysfunction in chronic heart failure. This raises the distinct possibility that allopurinol might reduce cardiovascular events and even improve exercise capacity in chronic heart failure.

Endothelial cell senescence with aging in healthy humans: prevention by habitual exercise and relation to vascular endothelial function.

PubMed

Rossman, Matthew J; Kaplon, Rachelle E; Hill, Sierra D; McNamara, Molly N; Santos-Parker, Jessica R; Pierce, Gary L; Seals, Douglas R; Donato, Anthony J

2017-11-01

Cellular senescence is emerging as a key mechanism of age-related vascular endothelial dysfunction, but evidence in healthy humans is lacking. Moreover, the influence of lifestyle factors such as habitual exercise on endothelial cell (EC) senescence is unknown. We tested the hypothesis that EC senescence increases with sedentary, but not physically active, aging and is associated with vascular endothelial dysfunction. Protein expression (quantitative immunofluorescence) of p53, a transcription factor related to increased cellular senescence, and the cyclin-dependent kinase inhibitors p21 and p16 were 116%, 119%, and 128% greater (all P < 0.05), respectively, in ECs obtained from antecubital veins of older sedentary (60 ± 1 yr, n = 12) versus young sedentary (22 ± 1 yr, n = 9) adults. These age-related differences were not present (all P > 0.05) in venous ECs from older exercising adults (57 ± 1 yr, n = 13). Furthermore, venous EC protein levels of p53 ( r  = -0.49, P = 0.003), p21 ( r  = -0.38, P = 0.03), and p16 ( r  = -0.58, P = 0.002) were inversely associated with vascular endothelial function (brachial artery flow-mediated dilation). Similarly, protein expression of p53 and p21 was 26% and 23% higher (both P < 0.05), respectively, in ECs sampled from brachial arteries of healthy older sedentary (63 ± 1 yr, n = 18) versus young sedentary (25 ± 1 yr, n = 9) adults; age-related changes in arterial EC p53 and p21 expression were not observed ( P > 0.05) in older habitually exercising adults (59 ± 1 yr, n = 14). These data indicate that EC senescence is associated with sedentary aging and is linked to endothelial dysfunction. Moreover, these data suggest that prevention of EC senescence may be one mechanism by which aerobic exercise protects against endothelial dysfunction with age. NEW & NOTEWORTHY Our study provides novel evidence in humans of increased endothelial cell senescence with sedentary aging, which is associated with impaired vascular endothelial function. Furthermore, our data suggest an absence of age-related increases in endothelial cell senescence in older exercising adults, which is linked with preserved vascular endothelial function. Copyright © 2017 the American Physiological Society.

Functional dilatation and medial remodeling of the renal artery in response to chronic increased blood flow.

PubMed

Roan, Jun-Neng; Yeh, Chin-Yi; Chiu, Wen-Cheng; Lee, Chou-Hwei; Chang, Shih-Wei; Jiangshieh, Ya-Fen; Tsai, Yu-Chuan; Lam, Chen-Fuh

2011-01-01

Renal blood flow (RBF) is tightly regulated by several intrinsic pathways in maintaining optimal kidney blood supply. Using a rat model of aortocaval (AC) fistula, we investigated remodeling of the renal artery following prolonged increased blood flow. An AC fistula was created in the infrarenal aorta of anesthetized rats, and changes of blood flow in the renal artery were assessed using an ultrasonic flow probe. Morphological changes and expression of endothelial nitric oxide synthase and matrix metalloproteinase-2 in the remodeled renal artery were analyzed. Blood flow in the renal artery increased immediately after creation of AC fistula, but normal RBF was restored 8 weeks later. The renal artery dilated significantly 8 weeks after operation. Expression of endothelial nitric oxide synthase and matrix metalloproteinase-2 was upregulated shortly after blood flow increase, and returned to baseline levels after 3 weeks. Histological sections showed luminal dilatation with medial thickening and endothelial cell-to-smooth muscle cell attachments in the remodeled renal artery. Increased RBF was accommodated by functional dilatation and remodeling in the medial layer of the renal artery in order to restore normal blood flow. Our results provide important mechanistic insight into the intrinsic regulation of the renal artery in response to increased RBF. Copyright © 2011 S. Karger AG, Basel.

Aldosterone affects blood flow and vascular tone regulated by endothelium-derived NO: therapeutic implications

PubMed Central

Toda, Noboru; Nakanishi, Sadanobu; Tanabe, Shinichi

2013-01-01

Aldosterone, in doses inappropriate to the salt status, plays an important role in the development of cardiovascular injury, including endothelial dysfunction, independent of its hypertensive effects. Acute non-genomic effects of aldosterone acting on mineralocorticoid receptors are inconsistent in healthy humans: vasoconstriction or forearm blood flow decrease via endothelial dysfunction, vasodilatation mediated by increased NO actions, or no effects. However, in studies with experimental animals, aldosterone mostly enhances vasodilatation mediated by endothelium-derived NO. Chronic exposure to aldosterone, which induces genomic responses, results in impairments of endothelial function through decreased NO synthesis and action in healthy individuals, experimental animals and isolated endothelial cells. Chronic aldosterone reduces NO release from isolated human endothelial cells only when extracellular sodium is raised. Oxidative stress is involved in the impairment of endothelial function by promoting NO degradation. Aldosterone liberates endothelin-1 (ET-1) from endothelial cells, which elicits ETA receptor–mediated vasoconstriction by inhibiting endothelial NO synthesis and action and through its own direct vasoconstrictor action. Ca2+ flux through T-type Ca2+ channels activates aldosterone synthesis and thus enhances unwanted effects of aldosterone on the endothelium. Mineralocorticoid receptor inhibitors, ETA receptor antagonists and T-type Ca2+ channel blockers appear to diminish the pathophysiological participation of aldosterone in cardiovascular disease and exert beneficial actions on bioavailability of endothelium-derived NO, particularly in resistant hypertension and aldosteronism. PMID:23190073

The immunosuppressive drug mycophenolate mofetil impairs the adhesion capacity of gastrointestinal tumour cells

PubMed Central

LECKEL, K; BEECKEN, W-D; JONAS, D; OPPERMANN, E; COMAN, M C; BECK, K-F; CINATL, J; HAILER, N P; AUTH, M K H; BECHSTEIN, W O; SHIPKOVA, M; BLAHETA, R A

2003-01-01

Immunosuppression correlates with the development and recurrence of cancer. Mycophenolate mofetil (MMF) has been shown to reduce adhesion molecule expression and leucocyte recruitment into the donor organ. We have hypothesized that MMF might also prevent receptor-dependent tumour dissemination. Therefore, we have investigated the effects of MMF on tumour cell adhesion to human umbilical vein endothelial cells (HUVEC) and compared them with the effects on T cell–endothelial cell interactions. Influence of MMF on cellular adhesion to HUVEC was analysed using isolated CD4+ and CD8+ T cells, or WiDr colon adenocarcinoma cells as the model tumour. HUVEC receptors ICAM-1, VCAM-1, E-selectin and P-selectin were detected by flow cytometry, Western blot or Northern blot analysis. Binding activity of T cells or WiDr cells in the presence of MMF were measured using immobilized receptor globulin chimeras. MMF potently blocked both T cell and WiDr cell binding to endothelium by 80%. Surface expression of the endothelial cell receptors was reduced by MMF in a dose-dependent manner. E-selectin mRNA was concurrently reduced with a maximum effect at 1 µm. Interestingly, MMF acted differently on T cells and WiDr cells. Maximum efficacy of MMF was reached at 10 and 1 µm, respectively. Furthermore, MMF specifically suppressed T cell attachment to ICAM-1, VCAM-1 and P-selectin. In contrast, MMF prevented WiDr cell attachment to E-selectin. In conclusion, our data reveal distinct effects of MMF on both T cell adhesion and tumour cell adhesion to endothelial cells. This suggests that MMF not only interferes with the invasion of alloactivated T cells, but might also be of value in managing post-transplantation malignancy. PMID:14616783

The immunosuppressive drug mycophenolate mofetil impairs the adhesion capacity of gastrointestinal tumour cells.

PubMed

Leckel, K; Beecken, W-D; Jonas, D; Oppermann, E; Coman, M C; Beck, K-F; Cinatl, J; Hailer, N P; Auth, M K H; Bechstein, W O; Shipkova, M; Blaheta, R A

2003-11-01

Immunosuppression correlates with the development and recurrence of cancer. Mycophenolate mofetil (MMF) has been shown to reduce adhesion molecule expression and leucocyte recruitment into the donor organ. We have hypothesized that MMF might also prevent receptor-dependent tumour dissemination. Therefore, we have investigated the effects of MMF on tumour cell adhesion to human umbilical vein endothelial cells (HUVEC) and compared them with the effects on T cell-endothelial cell interactions. Influence of MMF on cellular adhesion to HUVEC was analysed using isolated CD4+ and CD8+ T cells, or WiDr colon adenocarcinoma cells as the model tumour. HUVEC receptors ICAM-1, VCAM-1, E-selectin and P-selectin were detected by flow cytometry, Western blot or Northern blot analysis. Binding activity of T cells or WiDr cells in the presence of MMF were measured using immobilized receptor globulin chimeras. MMF potently blocked both T cell and WiDr cell binding to endothelium by 80%. Surface expression of the endothelial cell receptors was reduced by MMF in a dose-dependent manner. E-selectin mRNA was concurrently reduced with a maximum effect at 1 microm. Interestingly, MMF acted differently on T cells and WiDr cells. Maximum efficacy of MMF was reached at 10 and 1 microm, respectively. Furthermore, MMF specifically suppressed T cell attachment to ICAM-1, VCAM-1 and P-selectin. In contrast, MMF prevented WiDr cell attachment to E-selectin. In conclusion, our data reveal distinct effects of MMF on both T cell adhesion and tumour cell adhesion to endothelial cells. This suggests that MMF not only interferes with the invasion of alloactivated T cells, but might also be of value in managing post-transplantation malignancy.

Steatosis Grade is the Most Important Risk Factor for Development of Endothelial Dysfunction in NAFLD

PubMed Central

Sapmaz, Ferdane; Uzman, Metin; Basyigit, Sebahat; Ozkan, Selcuk; Yavuz, Bunyamin; Yeniova, Abdullah; Kefeli, Ayse; Asilturk, Zeliha; Nazligül, Yasar

2016-01-01

Abstract It is shown that there are strong associations between nonalcoholic fatty liver disease (NAFLD) and endothelial dysfunction. The aim of our study was to reveal whether steatosis or fibrosis score is more important in the development of endothelial dysfunction in patients with NAFLD in a prospective manner. This cross-sectional study included 266 subjects. These subjects were divided into 2 groups depending on presence of hepatosteatosis sonographically. Patients with hepatosteatosis were also divided into 3 subgroups depending on degree of steatosis: grade 1, 2, and 3. In all patients, Aspartate aminotransferase-to-Platelet Ratio Index and Fibrosis-4 (FIB4) scores were calculated. In addition, flow-mediated dilatation (FMD) measurements were recorded. There was NAFLD in 176 (66.2%) of 266 patients included. There were no significant differences in sex and age distributions between patients with NAFLD (group 1) and controls without NAFLD (group 2) (P = 0.05). Mean Aspartate aminotransferase-to-Platelet Ratio Index score was significantly higher in group 1 compared with the control group (P = 0.001), whereas no significant difference was detected regarding FIB4 scores between groups (P = 0.4). Mean FMD value was found to be significantly lower in group 1 (P = 0.008). Patients with grade 3 hepatosteatosis had significantly lower FMD values than those with grade 1 steatosis and controls (P = 0.001). In univariate and multivariate analyses in group 1, no significant difference was detected regarding mean FMD measurements (P = 0.03). Again, no significant difference was detected in mean FMD measurement between FIB4 subgroups among patients with NAFLD and the whole study group (P = 0.09). The endothelial dysfunction is associated with steatosis in patients with NAFLD. PMID:27057890

Purple grape juice improves endothelial function and reduces the susceptibility of LDL cholesterol to oxidation in patients with coronary artery disease.

PubMed

Stein, J H; Keevil, J G; Wiebe, D A; Aeschlimann, S; Folts, J D

1999-09-07

In vitro, the flavonoid components of red wine and purple grape juice are powerful antioxidants that induce endothelium-dependent vasodilation of vascular rings derived from rat aortas and human coronary arteries. Although improved endothelial function and inhibition of LDL oxidation may be potential mechanisms by which red wine and flavonoids reduce cardiovascular risk, the in vivo effects of grape products on endothelial function and LDL oxidation have not been investigated. This study assessed the effects of ingesting purple grape juice on endothelial function and LDL susceptibility to oxidation in patients with coronary artery disease (CAD). Fifteen adults with angiographically documented CAD ingested 7.7+/-1.2 mL. kg(-1). d(-1) of purple grape juice for 14 days. Flow-mediated vasodilation (FMD) was measured using high-resolution brachial artery ultrasonography. Susceptibility of LDL particles to oxidation was determined from the rate of conjugated diene formation after exposure to copper chloride. At baseline, FMD was impaired (2.2+/-2. 9%). After ingestion of grape juice, FMD increased to 6.4+/-4.7% (P=0.003). In a linear regression model that included age, artery diameter, lipid values, and use of lipid-lowering and antioxidant therapies, the effect of grape juice on FMD remained significant (mean change 4.2+/-4.4%, P<0.001). After ingestion of grape juice, lag time increased by 34.5% (P=0.015). Short-term ingestion of purple grape juice improves FMD and reduces LDL susceptibility to oxidation in CAD patients. Improved endothelium-dependent vasodilation and prevention of LDL oxidation are potential mechanisms by which flavonoids in purple grape products may prevent cardiovascular events, independent of alcohol content.

Mechanisms for the control of local tissue blood flow during thermal interventions: influence of temperature‐dependent ATP release from human blood and endothelial cells

PubMed Central

Chiesa, Scott T.; Trangmar, Steven J.; Ali, Leena; Lotlikar, Makrand D.; González‐Alonso, José

2017-01-01

New Findings What is the central question of this study? Skin and muscle blood flow increases with heating and decreases with cooling, but the temperature‐sensitive mechanisms underlying these responses are not fully elucidated. What is the main finding and its importance? We found that local tissue hyperaemia was related to elevations in ATP release from erythrocytes. Increasing intravascular ATP augmented skin and tissue perfusion to levels equal or above thermal hyperaemia. ATP release from isolated erythrocytes was altered by heating and cooling. Our findings suggest that erythrocytes are involved in thermal regulation of blood flow via modulation of ATP release. Local tissue perfusion changes with alterations in temperature during heating and cooling, but the thermosensitivity of the vascular ATP signalling mechanisms for control of blood flow during thermal interventions remains unknown. Here, we tested the hypotheses that the release of the vasodilator mediator ATP from human erythrocytes, but not from endothelial cells or other blood constituents, is sensitive to both increases and reductions in temperature and that increasing intravascular ATP availability with ATP infusion would potentiate thermal hyperaemia in limb tissues. We first measured blood temperature, brachial artery blood flow and plasma [ATP] during passive arm heating and cooling in healthy men and found that they increased by 3.0 ± 1.2°C, 105 ± 25 ml min−1 °C−1 and twofold, respectively, (all P < 0.05) with heating, but decreased or remained unchanged with cooling. In additional men, infusion of ATP into the brachial artery increased skin and deep tissue perfusion to levels equal or above thermal hyperaemia. In isolated erythrocyte samples exposed to different temperatures, ATP release increased 1.9‐fold from 33 to 39°C (P < 0.05) and declined by ∼50% at 20°C (P < 0.05), but no changes were observed in cultured human endothelial cells, plasma or serum samples. In conclusion, increases in plasma [ATP] and skin and deep tissue perfusion with limb heating are associated with elevations in ATP release from erythrocytes, but not from endothelial cells or other blood constituents. Erythrocyte ATP release is also sensitive to temperature reductions, suggesting that erythrocytes may function as thermal sensors and ATP signalling generators for control of tissue perfusion during thermal interventions. PMID:27859767

Increased plasma and endothelial cell expression of chemokines and adhesion molecules in chronic kidney disease.

PubMed

Stinghen, A E M; Gonçalves, S M; Martines, E G; Nakao, L S; Riella, M C; Aita, C A; Pecoits-Filho, R

2009-01-01

Chemokines and adhesion molecules are involved in early events of atherogenesis. In the present study, we investigated the effects of the uremic milieu on the expression of monocyte chemoattractant protein-1 (MCP-1), interleukin-8 (IL-8), soluble vascular adhesion molecule-1 (sVCAM-1) and soluble intercellular adhesion molecule-1 (sICAM-1) and their relationship to cardiovascular status. Plasma samples were obtained from patients in different stages of chronic kidney disease (CKD). Cardiovascular status was evaluated by intima-media thickness and endothelial dysfunction by flow mediation dilatation and proteinuria. In vitro studies were performed using human umbilical endothelial cells exposed to uremic plasma or plasma from healthy subjects. MCP-1, IL-8, sVCAM-1 and sICAM-1 levels in plasma and in supernatant were analyzed by enzyme-linked immunosorbent assay. The population consisted of 73 (mean age 57 years; 48% males) CKD patients with glomerular filtration rate (GFR) of 37 +/- 2 ml/min. MCP-1 and sVCAM-1 plasma levels were negatively correlated with GFR (rho = -0.40, p < 0.0005 and rho = -0.42, p < 0.0005, respectively). Fibrinogen was positively correlated with MCP-1, sICAM-1 and sVCAM-1 (rho = 0.33, p < 0.005, rho = 0.32, p < 0.05 and rho = 0.25, p < 0.05, respectively) and ultra-high-sensitivity C-reactive protein was positively correlated with sICAM-1 (rho = 0.25, p < 0.0005). Plasma IL-8 had a significant positive correlation with proteinuria (rho = 0.31, p < 0.01). There was a time- and CKD-stage-dependent MCP-1, IL-8 and sVCAM-1 endothelial expression (p < 0.05). In summary, plasma levels of markers of endothelial cell activation (MCP-1 and sVCAM-1) are increased in more advanced CKD. Exposure of endothelial cells to uremic plasma results in a time- and CKD-stage-dependent increased expression of MCP-1, IL-8 and sVCAM-1, suggesting a link between vascular activation, systemic inflammation and uremic toxicity. Future studies are necessary to investigate whether these biomarkers add predictive value in comparison to the previously described ones. Also, endothelial response to uremic toxicity should be viewed as a potential target for intervention in order to reduce morbidity and mortality in CKD-related cardiovascular disease. Copyright 2009 S. Karger AG, Basel.

Can regurgitant flow damage the left atrial endothelium in patients with prosthetic mechanical heart valves?

PubMed

Milo, Simcha; Zarandi, Mehrdad; Gutfinger, Chaim; Gharib, Morteza

2005-05-01

Previous in-vitro studies of mechanical heart valves (MHVs) in the closed position demonstrated the formation of regurgitant flows, with bubbles and jets forming vortices during each systole. The study aim was to determine whether the regurgitant flow observed in patients with MHVs can damage the left atrial endothelium, due to shear stresses exerted on the endothelial layers. This objective has been accomplished by appropriate in-vitro simulation experiments. In these experiments, leakage flow through several commercial MHVs was investigated. The geometry of the set-up closely resembled that of the left atrial anatomy. Water was forced through the slit of a closed MHV and directed toward the hemispherical cup coated with fluorescent paint. The flow field between the valve and the cup was photographed using high-speed videography, from which local velocities were measured, using digital particle imaging velocimetry. Qualitative damage to the surface of the cup was assessed from the amount of fluorescent paint removed from the cup. The experimental results and calculations indicated that flows through the gaps of the closed valves were sufficient to generate strong vortices, with velocities near the atrial wall in the range of 0.5 to 4.0 m/s, depending on the valve. This led to high shear stresses on the left atrial wall, which far exceeded physiologically acceptable levels. The calculated shear stresses exceeded by orders of magnitude the maximum physiologically tolerated stresses. This suggests that shear stresses associated with regurgitant jets in MHVs may damage the endothelial cells, leading to the activation of the inflammatory reaction, enhanced procoagulation, platelet activation and aggregation, and mechanical cell denudation.

ACh-induced endothelial NO synthase translocation, NO release and vasodilatation in the hamster microcirculation in vivo

PubMed Central

Figueroa, Xavier F; González, Daniel R; Martínez, Agustín D; Durán, Walter N; Boric, Mauricio P

2002-01-01

Studies in cultured cells show that activation of endothelial nitric oxide (NO) synthase (eNOS) requires the dissociation of this enzyme from its inhibitory association with caveolin-1 (Cav-1), and perhaps its translocation from plasma membrane caveolae to other cellular compartments. We investigated the hypothesis that in vivo NO-dependent vasodilatation is associated with the translocation of eNOS from the cell membrane. To this end, we applied ACh topically (10-100 μm for 10 min) to the hamster cheek pouch microcirculation and measured NO production, blood flow and vessel diameter, and assessed subcellular eNOS distribution by Western blotting. Baseline NO production was 54.4 ± 5.2 pmol min−1 (n = 16). ACh increased NO release, caused arteriolar and venular dilatation and elevated microvascular flow. These responses were inhibited by NG-nitro-L-arginine (30 μm). The maximal increase in NO production induced by 10 μm and 100 μm ACh was 45 ± 20 % and 111 ± 33 %, respectively; the corresponding blood flow increases were 50 ± 10 % and 130 ± 24 %, respectively (n = 4-6). Both responses followed a similar time course, although increases in NO preceded flow changes. In non-stimulated tissues, eNOS was distributed mainly in the microsomal fraction. ACh-induced vasodilatation was associated with eNOS translocation to the cytosolic and Golgi-enriched fractions. After 1.5, 3.0 or 6.0 min of application, 10 μm ACh decreased the level of membrane-bound eNOS by -13 ± 4 %, -60 ± 4 % and -19 ± 17 %, respectively; at the same time points, 100 μm ACh reduced microsomal eNOS content by -38 ± 9 %, -61 ± 16 % and -40 ± 18 %, respectively (n = 4-5). In all cases, microsomal Cav-1 content did not change. The close ACh concentration dependence and the concomitance between eNOS subcellular redistribution and NO release support the concept that eNOS translocation from the plasma membrane is part of an activation mechanism that induces NO-dependent vasodilatation in vivo. PMID:12411531

PolNet: A Tool to Quantify Network-Level Cell Polarity and Blood Flow in Vascular Remodeling.

PubMed

Bernabeu, Miguel O; Jones, Martin L; Nash, Rupert W; Pezzarossa, Anna; Coveney, Peter V; Gerhardt, Holger; Franco, Claudio A

2018-05-08

In this article, we present PolNet, an open-source software tool for the study of blood flow and cell-level biological activity during vessel morphogenesis. We provide an image acquisition, segmentation, and analysis protocol to quantify endothelial cell polarity in entire in vivo vascular networks. In combination, we use computational fluid dynamics to characterize the hemodynamics of the vascular networks under study. The tool enables, to our knowledge for the first time, a network-level analysis of polarity and flow for individual endothelial cells. To date, PolNet has proven invaluable for the study of endothelial cell polarization and migration during vascular patterning, as demonstrated by two recent publications. Additionally, the tool can be easily extended to correlate blood flow with other experimental observations at the cellular/molecular level. We release the source code of our tool under the Lesser General Public License. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Human endothelial cell responses to cardiovascular inspired pulsatile shear stress

NASA Astrophysics Data System (ADS)

Watson, Matthew; Baugh, Lauren; Black, Lauren, III; Kemmerling, Erica

2016-11-01

It is well established that hemodynamic shear stress regulates blood vessel structure and the development of vascular pathology. This process can be studied via in vitro models of endothelial cell responses to pulsatile shear stress. In this study, a macro-scale cone and plate viscometer was designed to mimic various shear stress waveforms found in the body and apply these stresses to human endothelial cells. The device was actuated by a PID-controlled DC gear-motor. Cells were exposed to 24 hours of pulsatile shear and then imaged and stained to track their morphology and secretions. These measurements were compared with control groups of cells exposed to constant shear and no shear. The results showed that flow pulsatility influenced levels of secreted proteins such as VE-cadherin and neuroregulin IHC. Cell morphology was also influenced by flow pulsatility; in general cells exposed to pulsatile shear stress developed a higher aspect ratio than cells exposed to no flow but a lower aspect ratio than cells exposed to steady flow.

Effect of diabetes on the cutaneous microcirculation of the feet in patients with intermittent claudication.

PubMed

Klonizakis, M; Manning, G; Lingam, K; Donnelly, R; Yeung, J M C

2015-01-01

To evaluate endothelial-dependent and - independent cutaneous vasodilator responses in the feet of patients with peripheral arterial disease (PAD) with or without Type 2 diabetes. Cutaneous microvascular responses in the dorsum of both lower limbs were measured in the supine position using Laser Doppler Fluximetry combined with iontophoretic administration of endothelial-dependent (acetylcholine, Ach) and -independent (sodium nitroprusside, SNP) vasodilators in diabetic (n = 19) and non diabetic (n = 17) patients with PAD (presenting as unilateral calf intermittent claudication (IC). In patients with diabetes and IC, endothelial-dependent vasodilation was significantly impaired in the symptomatic limb [74 (57,105) vs 68 (24,81) PU, Z =-2.79, p = 0.005] compared to the asymptomatic limb. Patients without diabetes showed no impairment of vasodilation. Resting ankle-brachial pressure index did not identify the presence of abnormalities in microvascular function. The combination of diabetes and PAD is associated with a reduction in endothelial-dependent cutaneous vasodilation in the feet without an associated reduction in endothelial independent vasodilation.

Endothelial dysfunction, vascular disease and stroke: the ARTICO study.

PubMed

Roquer, J; Segura, T; Serena, J; Castillo, J

2009-01-01

Endothelial dysfunction is a fundamental step in the atherosclerotic disease process. Its presence is a risk factor for the development of clinical events, and may represent a marker of atherothrombotic burden. Also, endothelial dysfunction contributes to enhanced plaque vulnerability, may trigger plaque rupture, and favors thrombus formation. The assessment of endothelial vasomotion is a useful marker of atherosclerotic vascular disease. There are different methods to assess endothelial function: endothelium-dependent vasodilatation brachial flow-mediated dilation, cerebrovascular reactivity to L-arginine, and the determination of some biomarkers such as microalbuminuria, platelet function, and C-reactive protein. Endothelial dysfunction has been observed in stroke patients and has been related to stroke physiopathology, stroke subtypes, clinical severity and outcome. Resting ankle-brachial index (ABI) is also considered an indicator of generalized atherosclerosis, and a low ABI is associated with an increase in stroke incidence in the elderly. Despite all these data, there are no studies analyzing the predictive value of ABI for new cardiovascular events in patients after suffering an acute ischemic stroke. ARTICO is an ongoing prospective, observational, multicenter study being performed in 50 Spanish hospitals. The aim of the ARTICO study is to evaluate the prognostic value of a pathological ABI (

Shear stress regulates endothelial microparticle release.

PubMed

Vion, Anne-Clémence; Ramkhelawon, Bhama; Loyer, Xavier; Chironi, Gilles; Devue, Cecile; Loirand, Gervaise; Tedgui, Alain; Lehoux, Stéphanie; Boulanger, Chantal M

2013-05-10

Endothelial activation and apoptosis release membrane-shed microparticles (EMP) that emerge as important biological effectors. Because laminar shear stress (SS) is a major physiological regulator of endothelial survival, we tested the hypothesis that SS regulates EMP release. EMP levels were quantified by flow cytometry in medium of endothelial cells subjected to low or high SS (2 and 20 dyne/cm(2)). EMP levels augmented with time in low SS conditions compared with high SS conditions. This effect was sensitive to extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and Rho kinases inhibitors but unaffected by caspase inhibitors. Low SS-stimulated EMP release was associated with increased endothelial Rho kinases and ERK1/2 activities and cytoskeletal reorganization. Overexpression of constitutively active RhoA stimulated EMP release under high SS. We also examined the effect of nitric oxide (NO) in mediating SS effects. L-NG-nitroarginine methyl ester (L-NAME), but not D-NG-nitroarginine methyl ester, increased high SS-induced EMP levels by 3-fold, whereas the NO donor S-nitroso-N-acetyl-D,L-penicillamine (SNAP) decreased it. L-NAME and SNAP did not affect Rho kinases and ERK1/2 activities. Then, we investigated NO effect on membrane remodeling because microparticle release is abolished in ABCA1-deficient cells. ABCA1 expression, which was greater under low SS than under high SS, was augmented by L-NAME under high SS and decreased by SNAP under low SS conditions. Altogether, these results demonstrate that sustained atheroprone low SS stimulates EMP release through activation of Rho kinases and ERK1/2 pathways, whereas atheroprotective high SS limits EMP release in a NO-dependent regulation of ABCA1 expression and of cytoskeletal reorganization. These findings, therefore, identify endothelial SS as a physiological regulator of microparticle release.

Functional role of endothelial CXCL16/CXCR6-platelet-leukocyte axis in angiotensin II-associated metabolic disorders.

PubMed

Collado, Aida; Marques, Patrice; Escudero, Paula; Rius, Cristina; Domingo, Elena; Martinez-Hervás, Sergio; Real, José T; Ascaso, Juan F; Piqueras, Laura; Sanz, Maria-Jesus

2018-05-23

Angiotensin-II (Ang-II) is the main effector peptide of the renin-angiotensin system (RAS) and promotes leukocyte adhesion to the stimulated endothelium. Because RAS activation and Ang-II signaling are implicated in metabolic syndrome (MS) and abdominal aortic aneurysm (AAA), we investigated the effect of Ang-II on CXCL16 arterial expression, the underlying mechanisms, and the functional role of the CXCL16/CXCR6 axis in these cardiometabolic disorders. Results from in vitro chamber assays revealed that CXCL16 neutralization significantly inhibited mononuclear leukocyte adhesion to arterial but not to venous endothelial cells. Flow cytometry and immunofluorescence studies confirmed that Ang-II induced enhanced endothelial CXCL16 expression, which was dependent on Nox5 up-regulation and subsequent RhoA/p38-MAPK/NFκB activation. Flow cytometry analysis further showed that MS patients had higher levels of platelet activation and a higher percentage of circulating CXCR6-expressing platelets, CXCR6-expressing-platelet-bound neutrophils, monocytes and CD8+ lymphocytes than age-matched controls, leading to enhanced CXCR6/CXCL16-dependent adhesion to the dysfunctional (Ang-II- and TNFα-stimulated) arterial endothelium. Ang-II-challenged apolipoprotein E-deficient (apoE-/-) mice had a higher incidence of AAA, macrophage, CD3+ and CXCR6+ cell infiltration and neovascularization than unchallenged animals, which was accompanied by greater CCL2, CXCL16 and VEGF mRNA expression within the lesion together with elevated levels of circulating soluble CXCL16. Significant reductions in these parameters were found in animals co-treated with the AT1 receptor antagonist losartan or in apoE-/- mice lacking functional CXCR6 receptor (CXCR6GFP/GFP). CXCR6 expression on platelet-bound monocytes and CD8+ lymphocytes may constitute a new membrane-associated biomarker for adverse cardiovascular events. Moreover, pharmacological modulation of this axis may positively affect cardiovascular outcome in metabolic disorders linked to Ang-II.

Insufficient sleep is associated with impaired nitric oxide-mediated endothelium-dependent vasodilation.

PubMed

Bain, Anthony R; Weil, Brian R; Diehl, Kyle J; Greiner, Jared J; Stauffer, Brian L; DeSouza, Christopher A

2017-10-01

Habitual short nightly sleep duration is associated with increased atherosclerotic cardiovascular disease risk and morbidity. Vascular endothelial dysfunction represents an important mechanism that may underlie this heightened cardiovascular risk. Impaired endothelium-dependent vasodilation, particularly NO-mediated vasodilation, contributes to the development and progression of atherosclerotic vascular disease and acute vascular events. We tested the hypothesis that chronic insufficient sleep is associated with impaired NO-mediated endothelium-dependent vasodilation in middle-aged adults. Thirty adult men were studied: 15 with normal nightly sleep duration (age: 58 ± 2 y; sleep duration: 7.7 ± 0.2 h/night) and 15 with short nightly sleep duration (55 ± 2 y; 6.1 ± 0.2 h/night). Forearm blood flow (FBF) responses to intra-arterial infusion of acetylcholine, in the absence and presence of the endothelial NO synthase inhibitor N G -monomethyl-L-arginine (L-NMMA), as well as responses to sodium nitroprusside, were determined by strain-gauge venous occlusion plethysmography. The FBF response to acetylcholine was lower (∼20%; p<0.05) in the short sleep duration group (from 4.6 ± 0.3 to 11.7 ± 1.0 ml/100 ml tissue/min) compared with normal sleep duration group (from 4.4 ± 0.3 to 14.5 ± 0.5 ml/100 ml tissue/min). L-NMMA significantly reduced the FBF response to acetylcholine in the normal sleep duration group (∼40%), but not the short sleep duration group. There were no group differences in the vasodilator response to sodium nitroprusside. These data indicate that short nightly sleep duration is associated with endothelial-dependent vasodilator dysfunction due, in part, to diminished NO bioavailability. Impaired NO-mediated endothelium-dependent vasodilation may contribute to the increased cardiovascular risk with insufficient sleep. Copyright © 2017 Elsevier B.V. All rights reserved.

d(−) Lactic Acid-Induced Adhesion of Bovine Neutrophils onto Endothelial Cells Is Dependent on Neutrophils Extracellular Traps Formation and CD11b Expression

PubMed Central

Alarcón, Pablo; Manosalva, Carolina; Conejeros, Ivan; Carretta, María D.; Muñoz-Caro, Tamara; Silva, Liliana M. R.; Taubert, Anja; Hermosilla, Carlos; Hidalgo, María A.; Burgos, Rafael A.

2017-01-01

Bovine ruminal acidosis is of economic importance as it contributes to reduced milk and meat production. This phenomenon is mainly attributed to an overload of highly fermentable carbohydrate, resulting in increased d(−) lactic acid levels in serum and plasma. Ruminal acidosis correlates with elevated acute phase proteins in blood, along with neutrophil activation and infiltration into various tissues leading to laminitis and aseptic polysynovitis. Previous studies in bovine neutrophils indicated that d(−) lactic acid decreased expression of L-selectin and increased expression of CD11b to concentrations higher than 6 mM, suggesting a potential role in neutrophil adhesion onto endothelia. The two aims of this study were to evaluate whether d(−) lactic acid influenced neutrophil and endothelial adhesion and to trigger neutrophil extracellular trap (NET) production (NETosis) in exposed neutrophils. Exposure of bovine neutrophils to 5 mM d(−) lactic acid elevated NET release compared to unstimulated neutrophil negative controls. Moreover, this NET contains CD11b and histone H4 citrullinated, the latter was dependent on PAD4 activation, a critical enzyme in DNA decondensation and NETosis. Furthermore, NET formation was dependent on d(−) lactic acid plasma membrane transport through monocarboxylate transporter 1 (MCT1). d(−) lactic acid enhanced neutrophil adhesion onto endothelial sheets as demonstrated by in vitro neutrophil adhesion assays under continuous physiological flow conditions, indicating that cell adhesion was a NET- and a CD11b/ICAM-1-dependent process. Finally, d(−) lactic acid was demonstrated for the first time to trigger NETosis in a PAD4- and MCT1-dependent manner. Thus, d(−) lactic acid-mediated neutrophil activation may contribute to neutrophil-derived pro-inflammatory processes, such as aseptic laminitis and/or polysynovitis in animals suffering acute ruminal acidosis. PMID:28861083

Sex differences in salt sensitivity to nitric oxide dependent vasodilation in healthy young adults

PubMed Central

Gullixson, Leah R.; Kost, Susan L.; Joyner, Michael J.; Turner, Stephen T.; Nicholson, Wayne T.

2012-01-01

Dietary sodium and blood pressure regulation differs between normotensive men and women, an effect which may involve endothelial production of nitric oxide (NO). Therefore, we tested the hypothesis that differences in the NO component of endothelium-dependent vasodilation between low and high dietary sodium intake depend on sex. For 5 days prior to study, healthy adults consumed a controlled low-sodium diet (10 mmol/day, n = 30, mean age ± SE: 30 ± 1 yr, 16 men) or high-sodium diet (400 mmol/day, n = 36, age 23 ± 1 yr, 13 men). Forearm blood flow (FBF, plethysmography) responses to brachial artery administration of acetylcholine (ACh, 4 μg·100 ml tissue−1·min−1) were measured before and after endothelial NO synthase inhibition with NG-monomethyl-l-arginine (l-NMMA, 50 mg bolus + 1 mg/min infusion). The NO component of endothelium-dependent dilation was calculated as the response to ACh before and after l-NMMA accounting for changes in baseline FBF: [(FBF ACh − FBF baseline) − (FBF AChL-NMMA − FBF baselineL-NMMA)]. This value was 5.7 ± 1.3 and 2.5 ± 0.8 ml·100 ml forearm tissue−1·min−1 for the low- and high-sodium diets, respectively (main effect of sodium, P = 0.019). The sodium effect was larger for the men, with values of 7.9 ± 2.0 and 2.2 ± 1.4 for men vs. 3.1 ± 1.3 and 2.7 ± 1.0 ml·100 ml forearm tissue−1·min−1 for the women (P = 0.034, sex-by-sodium interaction). We conclude that the NO component of endothelium-dependent vasodilation is altered by dietary sodium intake based on sex, suggesting that endothelial NO production is sensitive to dietary sodium in healthy young men but not women. PMID:22194324

Endotoxin-Induced Endothelial Fibrosis Is Dependent on Expression of Transforming Growth Factors β1 and β2

PubMed Central

Echeverría, César; Montorfano, Ignacio; Tapia, Pablo; Riedel, Claudia; Cabello-Verrugio, Claudio

2014-01-01

During endotoxemia-induced inflammatory disease, bacterial endotoxins circulate in the bloodstream and interact with endothelial cells (ECs), inducing dysfunction of the ECs. We previously reported that endotoxins induce the conversion of ECs into activated fibroblasts. Through endotoxin-induced endothelial fibrosis, ECs change their morphology and their protein expression pattern, thereby suppressing endothelial markers and upregulating fibrotic proteins. The most commonly used fibrotic inducers are transforming growth factor β1 (TGF-β1) and TGF-β2. However, whether TGF-β1 and TGF-β2 participate in endotoxin-induced endothelial fibrosis remains unknown. We have shown that the endotoxin-induced endothelial fibrosis process is dependent on the TGF-β receptor, ALK5, and the activation of Smad3, a protein that is activated by ALK5 activation, thus suggesting that endotoxin elicits TGF-β production to mediate endotoxin-induced endothelial fibrosis. Therefore, we investigated the dependence of endotoxin-induced endothelial fibrosis on the expression of TGF-β1 and TGF-β2. Endotoxin-treated ECs induced the expression and secretion of TGF-β1 and TGF-β2. TGF-β1 and TGF-β2 downregulation inhibited the endotoxin-induced changes in the endothelial marker VE-cadherin and in the fibrotic proteins α-SMA and fibronectin. Thus, endotoxin induces the production of TGF-β1 and TGF-β2 as a mechanism to promote endotoxin-induced endothelial fibrosis. To the best of our knowledge, this is the first report showing that endotoxin induces endothelial fibrosis via TGF-β secretion, which represents an emerging source of vascular dysfunction. These findings contribute to understanding the molecular mechanism of endotoxin-induced endothelial fibrosis, which could be useful in the treatment of inflammatory diseases. PMID:24935972

Endotoxin-induced endothelial fibrosis is dependent on expression of transforming growth factors β1 and β2.

PubMed

Echeverría, César; Montorfano, Ignacio; Tapia, Pablo; Riedel, Claudia; Cabello-Verrugio, Claudio; Simon, Felipe

2014-09-01

During endotoxemia-induced inflammatory disease, bacterial endotoxins circulate in the bloodstream and interact with endothelial cells (ECs), inducing dysfunction of the ECs. We previously reported that endotoxins induce the conversion of ECs into activated fibroblasts. Through endotoxin-induced endothelial fibrosis, ECs change their morphology and their protein expression pattern, thereby suppressing endothelial markers and upregulating fibrotic proteins. The most commonly used fibrotic inducers are transforming growth factor β1 (TGF-β1) and TGF-β2. However, whether TGF-β1 and TGF-β2 participate in endotoxin-induced endothelial fibrosis remains unknown. We have shown that the endotoxin-induced endothelial fibrosis process is dependent on the TGF-β receptor, ALK5, and the activation of Smad3, a protein that is activated by ALK5 activation, thus suggesting that endotoxin elicits TGF-β production to mediate endotoxin-induced endothelial fibrosis. Therefore, we investigated the dependence of endotoxin-induced endothelial fibrosis on the expression of TGF-β1 and TGF-β2. Endotoxin-treated ECs induced the expression and secretion of TGF-β1 and TGF-β2. TGF-β1 and TGF-β2 downregulation inhibited the endotoxin-induced changes in the endothelial marker VE-cadherin and in the fibrotic proteins α-SMA and fibronectin. Thus, endotoxin induces the production of TGF-β1 and TGF-β2 as a mechanism to promote endotoxin-induced endothelial fibrosis. To the best of our knowledge, this is the first report showing that endotoxin induces endothelial fibrosis via TGF-β secretion, which represents an emerging source of vascular dysfunction. These findings contribute to understanding the molecular mechanism of endotoxin-induced endothelial fibrosis, which could be useful in the treatment of inflammatory diseases. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Sustained expression of MCP-1 by low wall shear stress loading concomitant with turbulent flow on endothelial cells of intracranial aneurysm.

PubMed

Aoki, Tomohiro; Yamamoto, Kimiko; Fukuda, Miyuki; Shimogonya, Yuji; Fukuda, Shunichi; Narumiya, Shuh

2016-05-09

Enlargement of a pre-existing intracranial aneurysm is a well-established risk factor of rupture. Excessive low wall shear stress concomitant with turbulent flow in the dome of an aneurysm may contribute to progression and rupture. However, how stress conditions regulate enlargement of a pre-existing aneurysm remains to be elucidated. Wall shear stress was calculated with 3D-computational fluid dynamics simulation using three cases of unruptured intracranial aneurysm. The resulting value, 0.017 Pa at the dome, was much lower than that in the parent artery. We loaded wall shear stress corresponding to the value and also turbulent flow to the primary culture of endothelial cells. We then obtained gene expression profiles by RNA sequence analysis. RNA sequence analysis detected hundreds of differentially expressed genes among groups. Gene ontology and pathway analysis identified signaling related with cell division/proliferation as overrepresented in the low wall shear stress-loaded group, which was further augmented by the addition of turbulent flow. Moreover, expression of some chemoattractants for inflammatory cells, including MCP-1, was upregulated under low wall shear stress with concomitant turbulent flow. We further examined the temporal sequence of expressions of factors identified in an in vitro study using a rat model. No proliferative cells were detected, but MCP-1 expression was induced and sustained in the endothelial cell layer. Low wall shear stress concomitant with turbulent flow contributes to sustained expression of MCP-1 in endothelial cells and presumably plays a role in facilitating macrophage infiltration and exacerbating inflammation, which leads to enlargement or rupture.

Engineered living blood vessels: functional endothelia generated from human umbilical cord-derived progenitors.

PubMed

Schmidt, Dörthe; Asmis, Lars M; Odermatt, Bernhard; Kelm, Jens; Breymann, Christian; Gössi, Matthias; Genoni, Michele; Zund, Gregor; Hoerstrup, Simon P

2006-10-01

Tissue-engineered living blood vessels (TEBV) with growth capacity represent a promising new option for the repair of congenital malformations. We investigate the functionality of TEBV with endothelia generated from human umbilical cord blood-derived endothelial progenitor cells. Tissue-engineered living blood vessels were generated from human umbilical cord-derived myofibroblasts seeded on biodegradable vascular scaffolds, followed by endothelialization with differentiated cord blood-derived endothelial progenitor cells. During in vitro maturation the TEBV were exposed to physiologic conditioning in a flow bioreactor. For functional assessment, a subgroup of TEBV was stimulated with tumor necrosis factor-alpha. Control vessels endothelialized with standard vascular endothelial cells were treated in parallel. Analysis of the TEBV included histology, immunohistochemistry, biochemistry (extracellular matrix analysis, DNA), and biomechanical testing. Endothelia were analyzed by flow cytometry and immunohistochemistry (CD31, von Willebrand factor, thrombomodulin, tissue factor, endothelial nitric oxide synthase). Histologically, a three-layered tissue organization of the TEBV analogous to native vessels was observed, and biochemistry revealed the major matrix constituents (collagen, proteoglycans) of blood vessels. Biomechanical properties (Young's modulus, 2.03 +/- 0.65 MPa) showed profiles resembling those of native tissue. Endothelial progenitor cells expressed typical endothelial cell markers CD31, von Willebrand factor, and endothelial nitric oxide synthase comparable to standard vascular endothelial cells. Stimulation with tumor necrosis factor-alpha resulted in physiologic upregulation of tissue factor and downregulation of thrombomodulin expression. These results indicate that TEBV with tissue architecture and functional endothelia similar to native blood vessels can be successfully generated from human umbilical cord progenitor cells. Thus, blood-derived progenitor cells obtained before or at birth may enable the clinical realization of tissue engineering constructs for pediatric applications.

Flow-Mediated Endothelial Mechanotransduction

PubMed Central

Davies, Peter F.

2011-01-01

Mechanical forces associated with blood flow play important roles in the acute control of vascular tone, the regulation of arterial structure and remodeling, and the localization of atherosclerotic lesions. Major regulation of the blood vessel responses occurs by the action of hemodynamic shear stresses on the endothelium. The transmission of hemodynamic forces throughout the endothelium and the mechanotransduction mechanisms that lead to biophysical, biochemical, and gene regulatory responses of endothelial cells to hemodynamic shear stresses are reviewed. PMID:7624393

Successful silencing of plasminogen activator inhibitor-1 in human vascular endothelial cells using small interfering RNA.

PubMed

Hecke, Anneke; Brooks, Hilary; Meryet-Figuière, Matthieu; Minne, Stephanie; Konstantinides, Stavros; Hasenfuss, Gerd; Lebleu, Bernard; Schäfer, Katrin

2006-05-01

Clinical as well as experimental evidence suggests that vascular overexpression of plasminogen activator inhibitor (PAI)-1, the primary physiological inhibitor of both urokinase and tissue-type plasminogen activator, may be involved in the pathophysiology of atherosclerosis and cardiovascular disease. We investigated the feasibility, efficacy and functional effects of PAI-1 gene silencing in human vascular endothelial cells using small interfering RNA. Double-stranded 21 bp-RNA molecules targeted at sequences within the human PAI-1 gene were constructed. Successful siRNA transfection of HUVEC was confirmed using fluorescence microscopy and flow cytometry. One of five candidate siRNA sequences reduced PAI-1 mRNA and protein in a concentration- and time-dependent manner. Suppression of PAI-1 mRNA was detected up to 72 hours after transfection. Moreover, siRNA treatment reduced the activity of PAI-1 released from HUVEC, and prevented the oxLDL- or LPS-induced upregulation of PAI-1 secretion. Importantly, siRNA treatment did not affect the expression of other endothelial-cell markers. Moreover, downregulation of PAI-1 significantly enhanced the ability of endothelial cells to adhere to vitronectin, and this effect could be reversed upon addition of recombinant PAI-1. SiRNA-mediated reduction of PAI-1 expression may be a promising strategy for dissecting the effects of PAI-1 on vascular homeostasis.

Accumulation of Multipotent Progenitor Cells on Polymethylpentene Membranes During Extracorporeal Membrane Oxygenation.

PubMed

Lehle, Karla; Friedl, Lucas; Wilm, Julius; Philipp, Alois; Müller, Thomas; Lubnow, Matthias; Schmid, Christof

2016-06-01

Multipotent progenitor cells were mobilized during pediatric extracorporeal membrane oxygenation (ECMO). We hypothesize that these cells also adhered onto polymethylpentene (PMP) fibers within the membrane oxygenator (MO) during adult ECMO support. Mononuclear cells were removed from the surface of explanted PMP-MOs (n = 16). Endothelial-like outgrowth and mesenchymal-like cells were characterized by flow cytometric analysis using different surface markers. Spindle-shaped attaching cells were identified early, but without proliferative activity. After long-term cultivation palisading type or cobblestone-type outgrowth cells with high proliferative activity appeared and were characterized as (i) leukocytoid CD45+/CD31+ (CD133+/VEGFR-II+/CD90+/CD14+/CD146dim/CD105dim); (ii) endothelial-like CD45-/CD31+ (VEGF-RII+/CD146+/CD105+/CD133-/CD14-/CD90-); and (iii) mesenchymal-like cells CD45-/CD31- (CD105+/CD90+/CD133dim/VEGFR-II-/CD146-/CD14-). The distribution of the cell populations depended on the MO and cultivation time. Endothelial-like cells formed capillary-like structures and did uptake Dil-acetylated low-density lipoprotein. Endothelial- and mesenchymal-like cells adhered on the surface of PMP-MOs. Further research is needed to identify the clinical relevance of these cells. Copyright © 2015 The Authors. Artificial Organs published by Wiley Periodicals, Inc. on behalf of International Center for Artificial Organs and Transplantation (ICAOT).

Evidence for a role of platelet endothelial cell adhesion molecule-1 in endothelial cell mechanosignal transduction: is it a mechanoresponsive molecule?

PubMed

Osawa, Masaki; Masuda, Michitaka; Kusano, Ken-ichi; Fujiwara, Keigi

2002-08-19

Fluid shear stress (FSS) induces many forms of responses, including phosphorylation of extracellular signal-regulated kinase (ERK) in endothelial cells (ECs). We have earlier reported rapid tyrosine phosphorylation of platelet endothelial cell adhesion molecule-1 (PECAM-1) in ECs exposed to FSS. Osmotic changes also induced similar PECAM-1 and ERK phosphorylation with nearly identical kinetics. Because both FSS and osmotic changes should mechanically perturb the cell membrane, they might activate the same mechanosignaling cascade. When PECAM-1 is tyrosine phosphorylated by FSS or osmotic changes, SHP-2 binds to it. Here we show that ERK phosphorylation by FSS or osmotic changes depends on PECAM-1 tyrosine phosphorylation, SHP-2 binding to phospho-PECAM-1, and SHP-2 phosphatase activity. In ECs under flow, detectable amounts of SHP-2 and Gab1 translocated from the cytoplasm to the EC junction. When magnetic beads coated with antibodies against the extracellular domain of PECAM-1 were attached to ECs and tugged by magnetic force for 10 min, PECAM-1 associated with the beads was tyrosine phosphorylated. ERK was also phosphorylated in these cells. Binding of the beads by itself or pulling on the cell surface using poly-l-coated beads did not induce phosphorylation of PECAM-1 and ERK. These results suggest that PECAM-1 is a mechanotransduction molecule.

Porcine endothelium induces DNA-histone complex formation in human whole blood: a harmful effect of histone on coagulation and endothelial activation.

PubMed

Yoo, Hyun Ju; Kim, Ji-Eun; Gu, Ja Yoon; Lee, Sae Bom; Lee, Hyun Joo; Hwang, Ho Young; Hwang, Yoohwa; Kim, Young Tae; Kim, Hyun Kyung

2016-11-01

Neutrophils play a role in xenograft rejection. When neutrophils are stimulated, they eject the DNA-histone complex into the extracellular space, called neutrophil extracellular traps (NET). We investigated whether NET formation actively occurs in the xenograft and contributes to coagulation and endothelial activation. Human whole blood was incubated with porcine aortic endothelial cells (pEC) from wild-type or α1,3-galactosyltransferase gene-knockout (GTKO) pigs. In the supernatant plasma from human blood, the level of the DNA-histone complex was measured by ELISA, and thrombin generation was measured using a calibrated automated thrombogram. Histone-induced tissue factor and adhesion molecule expression were measured by flow cytometry. pEC from both wild-type and GTKO pigs significantly induced DNA-histone complex formation in human whole blood. The DNA-histone complex produced shortened the thrombin generation time and clotting time. Histone alone dose-dependently induced tissue factor and adhesion molecule expression in pEC. Aurintricarboxylic acid pretreatment partially inhibited pEC-induced DNA-histone complex formation. DNA-histone complex actively generated upon xenotransplantation is a novel target to inhibit coagulation and endothelial activation. To prevent tissue factor and adhesion molecule expression, a strategy to block soluble histone may be required in xenotransplantation. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Biological factors in plasma from diabetes mellitus patients enhance hyperglycaemia and pulsatile shear stress-induced endothelial cell apoptosis.

PubMed

Liu, X F; Yu, J Q; Dalan, R; Liu, A Q; Luo, K Q

2014-05-01

People suffering from Diabetes Mellitus (DM) are prone to an array of vascular complications leading to end organ damage. The hallmark of these vascular complications is endothelium dysfunction, which is caused by endothelial cell (EC) apoptosis. Although the endothelial cell (EC) dysfunction induced by hyperglycaemia and fluid shear stress has been studied, the effects of biological factors in the blood of DM patients on EC integrity have not been reported in the in vitro models that mimic the physiological pulsatile nature of the vascular system. This study reports the development of a hemodynamic lab-on-a-chip system to investigate this issue. The pulsatile flow was applied to a monolayer of endothelial cells expressing a fluorescence resonance energy transfer (FRET)-based biosensor that changes colour from green to blue in response to caspase-3 activation during apoptosis. Plasma samples from healthy volunteers and DM patients were compared to identify biological factors that are critical to endothelial disruption. Three types of microchannels were designed to simulate the blood vessels under healthy and partially blocked pathological conditions. The results showed that EC apoptosis rates increased with increasing glucose concentration and levels of shear stress. The rates of apoptosis further increased by a factor of 1.4-2.3 for hyperglycaemic plasma under all dynamic conditions. Under static conditions, little difference was detected in the rate of EC apoptosis between experiments using plasma from DM patients and glucose medium, suggesting that the effects of hyperglycaemia and biological factors on the induction of EC apoptosis are all shear flow-dependent. A proteomics study was then conducted to identify biological factors, demonstrating that the levels of eight proteins, including haptoglobin and clusterin, were significantly down-regulated, while six proteins, including apolipoprotein C-III, were significantly up-regulated in the plasma of DM patients compared to healthy volunteers. This hemodynamic lab-on-a-chip system can serve as a high throughput platform to assess the risk of vascular complications of DM patients and to determine the effects of therapeutics or other interventions on EC apoptosis.

Endothelial Dysfunction in Human Diabetes Is Mediated by Wnt5a-JNK Signaling.

PubMed

Bretón-Romero, Rosa; Feng, Bihua; Holbrook, Monika; Farb, Melissa G; Fetterman, Jessica L; Linder, Erika A; Berk, Brittany D; Masaki, Nobuyuki; Weisbrod, Robert M; Inagaki, Elica; Gokce, Noyan; Fuster, Jose J; Walsh, Kenneth; Hamburg, Naomi M

2016-03-01

Endothelial dysfunction is linked to insulin resistance, inflammatory activation, and increased cardiovascular risk in diabetes mellitus; however, the mechanisms remain incompletely understood. Recent studies have identified proinflammatory signaling of wingless-type family member (Wnt) 5a through c-jun N-terminal kinase (JNK) as a regulator of metabolic dysfunction with potential relevance to vascular function. We sought to gain evidence that increased activation of Wnt5a-JNK signaling contributes to impaired endothelial function in patients with diabetes mellitus. We measured flow-mediated dilation of the brachial artery and characterized freshly isolated endothelial cells by protein expression, eNOS activation, and nitric oxide production in 85 subjects with type 2 diabetes mellitus (n=42) and age- and sex-matched nondiabetic controls (n=43) and in human aortic endothelial cells treated with Wnt5a. Endothelial cells from patients with diabetes mellitus displayed 1.3-fold higher Wnt5a levels (P=0.01) along with 1.4-fold higher JNK activation (P<0.01) without a difference in total JNK levels. Higher JNK activation was associated with lower flow-mediated dilation, consistent with endothelial dysfunction (r=0.53, P=0.02). Inhibition of Wnt5a and JNK signaling restored insulin and A23187-mediated eNOS activation and improved nitric oxide production in endothelial cells from patients with diabetes mellitus. In endothelial cells from nondiabetic controls, rWnt5a treatment inhibited eNOS activation replicating the diabetic endothelial phenotype. In human aortic endothelial cells, Wnt5a-induced impairment of eNOS activation and nitric oxide production was reversed by Wnt5a and JNK inhibition. Our findings demonstrate that noncanonical Wnt5a signaling and JNK activity contribute to vascular insulin resistance and endothelial dysfunction and may represent a novel therapeutic opportunity to protect the vasculature in patients with diabetes mellitus. © 2016 American Heart Association, Inc.

Folic Acid Supplementation Improves Vascular Function in Professional Dancers With Endothelial Dysfunction

PubMed Central

Hoch, Anne Z.; Papanek, Paula; Szabo, Aniko; Widlansky, Michael E.; Gutterman, David D.

2012-01-01

Objective To determine if folic acid supplementation improves vascular function (brachial artery flow-mediated dilation [FMD]) in professional dancers with known endothelial dysfunction. Design Prospective cross-sectional study. Setting Academic institution in the Midwestern United States. Subjects Twenty-two professional ballet dancers volunteered for this study. Main Outcome Measures Subjects completed a 3-day food record to determine caloric and micronutrient intake. Menstrual status was determined by interview and questionnaire. Endothelial function was determined as flow-induced vasodilation measured by high-frequency ultrasound of the brachial artery. A change in brachial diameter of <5% to hyperemic flow stimulus was defined a priori as endothelial dysfunction. Subjects with abnormal FMD took 10 mg of folic acid daily for 4 weeks, and FMD testing was then repeated. Serum whole blood was measured for folic acid levels before and after supplementation. Results Sixty-four percent of dancers (n = 14) had abnormal brachial artery FMD (<5%) (mean ± standard deviation, 2.9% ± 1.5%). After 4 weeks of folic acid supplementation (10 mg/day), FMD improved in all the subjects (7.1% ± 2.3%; P < .0001). Conclusions This study reveals that vascular endothelial function improves in dancers after supplementation with folic acid (10 mg/day) for at least 4 weeks. This finding may have clinically important implications for future cardiovascular disease risk prevention. PMID:21715240

Implications Enzymatic Degradation of the Endothelial Glycocalyx on the Microvascular Hemodynamics and the Arteriolar Red Cell Free Layer of the Rat Cremaster Muscle.

PubMed

Yalcin, Ozlem; Jani, Vivek P; Johnson, Paul C; Cabrales, Pedro

2018-01-01

The endothelial glycocalyx is a complex network of glycoproteins, proteoglycans, and glycosaminoglycans; it lines the vascular endothelial cells facing the lumen of blood vessels forming the endothelial glycocalyx layer (EGL). This study aims to investigate the microvascular hemodynamics implications of the EGL by quantifying changes in blood flow hydrodynamics post-enzymatic degradation of the glycocalyx layer. High-speed intravital microscopy videos of small arteries (around 35 μm) of the rat cremaster muscle were recorded at various time points after enzymatic degradation of the EGL. The thickness of the cell free layer (CFL), blood flow velocity profiles, and volumetric flow rates were quantified. Hydrodynamic effects of the presence of the EGL were observed in the differences between the thickness of CFL in microvessels with an intact EGL and glass tubes of similar diameters. Maximal changes in the thickness of CFL were observed 40 min post-enzymatic degradation of the EGL. Analysis of the frequency distribution of the thickness of CFL allows for estimation of the thickness of the endothelial surface layer (ESL), the plasma layer, and the glycocalyx. Peak flow, maximum velocity, and mean velocity were found to statistically increase by 24, 27, and 25%, respectively, after enzymatic degradation of the glycocalyx. The change in peak-to-peak maximum velocity and mean velocity were found to statistically increase by 39 and 32%, respectively, after 40 min post-enzymatic degradation of the EGL. The bluntness of blood flow velocity profiles was found to be reduced post-degradation of the EGL, as the exclusion volume occupied by the EGL increased the effective volume impermeable to RBCs in microvessels. This study presents the effects of the EGL on microvascular hemodynamics. Enzymatic degradation of the EGL resulted in a decrease in the thickness of CFL, an increase in blood velocity, blood flow, and decrease of the bluntness of the blood flow velocity profile in small arterioles. In summary, the EGL functions as a molecular sieve to solute transport and as a lubrication layer to protect the endothelium from red blood cell (RBC) motion near the vessel wall, determining wall shear stress.

Endothelial Barrier Protection by Local Anesthetics: Ropivacaine and Lidocaine Block Tumor Necrosis Factor-α–induced Endothelial Cell Src Activation

PubMed Central

Piegeler, Tobias; Votta-Velis, E. Gina; Bakhshi, Farnaz R.; Mao, Mao; Carnegie, Graeme; Bonini, Marcelo G.; Schwartz, David E.; Borgeat, Alain; Beck-Schimmer, Beatrice; Minshall, Richard D.

2014-01-01

Background Pulmonary endothelial barrier dysfunction mediated in part by Src-kinase activation plays a crucial role in acute inflammatory disease. Proinflammatory cytokines, such as tumor necrosis factor-α (TNFα), activate Src via phosphatidylinositide 3-kinase/Akt-dependent nitric oxide generation, a process initiated by recruitment of phosphatidylinositide 3-kinase regulatory subunit p85 to TNF-receptor-1. Because amide-linked local anesthetics have well-established anti-inflammatory effects, the authors hypothesized that ropivacaine and lidocaine attenuate inflammatory Src signaling by disrupting the phosphatidylinositide 3-kinase–Akt–nitric oxide pathway, thus blocking Src-dependent neutrophil adhesion and endothelial hyperpermeability. Methods Human lung microvascular endothelial cells, incubated with TNFα in the absence or presence of clinically relevant concentrations of ropivacaine and lidocaine, were analyzed by Western blot, probing for phosphorylated/activated Src, endothelial nitric oxide synthase, Akt, intercellular adhesion molecule-1, and caveolin-1. The effect of ropivacaine on TNFα-induced nitric oxide generation, co-immunoprecipitation of TNF-receptor-1 with p85, neutrophil adhesion, and endothelial barrier disruption were assessed. Results Ropivacaine and lidocaine attenuated TNFα-induced Src activation (half-maximal inhibitory concentration [IC50] = 8.611 × 10−10 M for ropivacaine; IC50 = 5.864 × 10−10 M for lidocaine) and endothelial nitric oxide synthase phosphorylation (IC50 = 7.572 × 10−10 M for ropivacaine; IC50 = 6.377 × 10−10 M for lidocaine). Akt activation (n = 7; P = 0.006) and stimulus-dependent binding of TNF-receptor-1 and p85 (n = 6; P = 0.043) were blocked by 1 nM of ropivacaine. TNFα-induced neutrophil adhesion and disruption of endothelial monolayers via Src-dependent intercellular adhesion molecule-1- and caveolin-1-phosphorylation, respectively, were also attenuated. Conclusions Ropivacaine and lidocaine effectively blocked inflammatory TNFα signaling in endothelial cells by attenuating p85 recruitment to TNF-receptor-1. The resultant decrease in Akt, endothelial nitric oxide synthase, and Src phosphorylation reduced neutrophil adhesion and endothelial hyperpermeability. This novel anti-inflammatory “side-effect” of ropivacaine and lidocaine may provide therapeutic benefit in acute inflammatory disease. PMID:24525631

Endothelial barrier protection by local anesthetics: ropivacaine and lidocaine block tumor necrosis factor-α-induced endothelial cell Src activation.

PubMed

Piegeler, Tobias; Votta-Velis, E Gina; Bakhshi, Farnaz R; Mao, Mao; Carnegie, Graeme; Bonini, Marcelo G; Schwartz, David E; Borgeat, Alain; Beck-Schimmer, Beatrice; Minshall, Richard D

2014-06-01

Pulmonary endothelial barrier dysfunction mediated in part by Src-kinase activation plays a crucial role in acute inflammatory disease. Proinflammatory cytokines, such as tumor necrosis factor-α (TNFα), activate Src via phosphatidylinositide 3-kinase/Akt-dependent nitric oxide generation, a process initiated by recruitment of phosphatidylinositide 3-kinase regulatory subunit p85 to TNF-receptor-1. Because amide-linked local anesthetics have well-established anti-inflammatory effects, the authors hypothesized that ropivacaine and lidocaine attenuate inflammatory Src signaling by disrupting the phosphatidylinositide 3-kinase-Akt-nitric oxide pathway, thus blocking Src-dependent neutrophil adhesion and endothelial hyperpermeability. Human lung microvascular endothelial cells, incubated with TNFα in the absence or presence of clinically relevant concentrations of ropivacaine and lidocaine, were analyzed by Western blot, probing for phosphorylated/activated Src, endothelial nitric oxide synthase, Akt, intercellular adhesion molecule-1, and caveolin-1. The effect of ropivacaine on TNFα-induced nitric oxide generation, co-immunoprecipitation of TNF-receptor-1 with p85, neutrophil adhesion, and endothelial barrier disruption were assessed. Ropivacaine and lidocaine attenuated TNFα-induced Src activation (half-maximal inhibitory concentration [IC50] = 8.611 × 10 M for ropivacaine; IC50 = 5.864 × 10 M for lidocaine) and endothelial nitric oxide synthase phosphorylation (IC50 = 7.572 × 10 M for ropivacaine; IC50 = 6.377 × 10 M for lidocaine). Akt activation (n = 7; P = 0.006) and stimulus-dependent binding of TNF-receptor-1 and p85 (n = 6; P = 0.043) were blocked by 1 nM of ropivacaine. TNFα-induced neutrophil adhesion and disruption of endothelial monolayers via Src-dependent intercellular adhesion molecule-1- and caveolin-1-phosphorylation, respectively, were also attenuated. Ropivacaine and lidocaine effectively blocked inflammatory TNFα signaling in endothelial cells by attenuating p85 recruitment to TNF-receptor-1. The resultant decrease in Akt, endothelial nitric oxide synthase, and Src phosphorylation reduced neutrophil adhesion and endothelial hyperpermeability. This novel anti-inflammatory "side-effect" of ropivacaine and lidocaine may provide therapeutic benefit in acute inflammatory disease.

Endothelial Activation by Platelets from Sickle Cell Anemia Patients

PubMed Central

Proença-Ferreira, Renata; Brugnerotto, Ana Flávia; Garrido, Vanessa Tonin; Dominical, Venina Marcela; Vital, Daiana Morelli; Ribeiro, Marilene de Fátima Reis; dos Santos, Melissa Ercolin; Traina, Fabíola; Olalla-Saad, Sara T.; Costa, Fernando Ferreira; Conran, Nicola

2014-01-01

Sickle cell anemia (SCA) is associated with a hypercoagulable state. Increased platelet activation is reported in SCA and SCA platelets may present augmented adhesion to the vascular endothelium, potentially contributing to the vaso-occlusive process. We sought to observe the effects of platelets (PLTs) from healthy control (CON) individuals and SCA individuals on endothelial activation, in vitro. Human umbilical vein endothelial cells (HUVEC) were cultured, in the presence, or not, of washed PLTs from CON or steady-state SCA individuals. Supernatants were reserved for cytokine quantification, and endothelial adhesion molecules (EAM) were analyzed by flow cytometry; gene expressions of ICAM1 and genes of the NF-κB pathway were analyzed by qPCR. SCA PLTs were found to be more inflammatory, displaying increased adhesive properties, an increased production of IL-1β and a tendency towards elevated expressions of P-selectin and activated αIIbβ3. Following culture in the presence of SCA PLTs, HUVEC presented significant augmentations in the expressions of the EAM, ICAM-1 and E-selectin, as well as increased IL-8 production and increased ICAM1 and NFKB1 (encodes p50 subunit of NF-κB) gene expressions. Interestingly, transwell inserts abolished the effects of SCA PLTs on EAM expression. Furthermore, an inhibitor of the NF-κB pathway, BAY 11-7082, also prevented the induction of EAM expression on the HUVEC surface by SCA PLTs. In conclusion, we find further evidence to indicate that platelets circulate in an activated state in sickle cell disease and are capable of stimulating endothelial cell activation. This effect appears to be mediated by direct contact, or even adhesion, between the platelets and endothelial cells and via NFκB-dependent signaling. As such, activated platelets in SCD may contribute to endothelial activation and, therefore, to the vaso-occlusive process. Results provide further evidence to support the use of anti-platelet approaches in association with other therapies for SCD. PMID:24551209

MicroRNA-101 mediates the suppressive effect of laminar shear stress on mTOR expression in vascular endothelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Kui; Fan, Wendong; Wang, Xing

Highlights: Black-Right-Pointing-Pointer Laminar shear stress upregulates miR-101 expression in vascular endothelial cells. Black-Right-Pointing-Pointer miR-101 represses mTOR expression through a specific 3 Prime UTR binding site. Black-Right-Pointing-Pointer Overexpression of miR-101 inhibits G1/S transition and endothelial cell proliferation. Black-Right-Pointing-Pointer Blockade of miR-101 attenuates the suppressive effect of laminar flow on mTOR expression. -- Abstract: Shear stress associated with blood flow plays an important role in regulating gene expression and cell function in endothelial cells (ECs). MicroRNAs (miRNAs) are highly conserved, small non-coding RNAs that negatively regulate the expression of target genes by binding to the mRNA 3 Prime -untranslated region (3 Primemore » UTR) at the posttranscriptional level involved in diverse cellular processes. This study demonstrates that microRNA-101 in response to laminar shear stress (LSS) is involved in the flow regulation of gene expression in ECs. qRT-PCR analysis showed that miR-101 expression was significantly upregulated in human umbilical vein endothelial cells (HUVECs) exposed to 12 dyn/cm{sup 2} laminar shear stress for 12 h. We found that transfection of miR-101 significantly decreased the luciferase activity of plasmid reporter containing the 3 Prime UTR of mammalian target of rapamycin (mTOR) gene. Western analysis revealed that the protein level of mTOR was significantly reduced in ECs transfected with miR-101. Furthermore, miR-101 overexpression induced cell cycle arrest at the G1/S transition and suppressed endothelial cell proliferation. Finally, transfection of miR-101 inhibitors attenuated the suppressive effects of LSS on mTOR expression, which identified the efficacy of loss-of-function of miR-101 in laminar flow-treated ECs. In conclusion, we have demonstrated that upregulation of miR-101 in response to LSS contributes to the suppressive effects of LSS on mTOR expression and EC proliferation. These studies advance our understanding of the posttranscriptional mechanisms by which shear stress modulates endothelial homeostasis.« less

Short-term therapy with relatively low-dose cerivastatin improves endothelial function independently of its lipid-lowering effect: Evaluation of brachial artery vasodilatation using B-mode ultrasound imaging.

PubMed

Sakabe, Koichi; Fukuda, Nobuo; Nada, Teru; Onose, Yukiko; Soeki, Takeshi; Shinohara, Hisanori; Tamura, Yoshiyuki

2002-12-01

Administration of 0.4 to 0.8 mg of cerivastatin per day for 2 weeks has been reported to have pleiotropic effects and improve endothelial function. Whether low-dose cerivastatin would produce these rapid pleiotropic effects in the clinical setting remains uncertain, however. We investigated the effect of short-term therapy with relatively low-dose cerivastatin (0.15 mg/day) on endothelial function, thrombostatic parameters, and C-reactive protein (CRP) levels in hypercholesterolemic patients. Thirteen patients with LDL-cholesterol>160 mg/dl were treated with daily doses of 0.15 mg of cerivastatin for 2 weeks. Endothelial function, thrombostatic parameters (tissue-type plasminogen activator [t-PA], plasminogen activator inhibitor type 1 [PAI-1], and CRP were estimated at baseline and again after 2 weeks of treatment. Endothelial function was measured as flow-mediated vasodilation. Flow-mediated vasodilatation was assessed by measuring the percent change in the diameter of the brachial artery in response to reactive hyperemia using high-resolution ultrasound. Endothelium-independent vasodilatation was also measured using sublingual nitroglycerin. No major complications developed after the treatment. Total cholesterol decreased significantly, from 258±32 to 211±21 mg/dl, and LDL-cholesterol also decreased from 171±15 to 133±16 mg/dl after the treatment. Flow-mediated vasodilatation increased significantly, from 4.6±1.3 percent to 8.7±3.5 percent after 2 weeks of therapy, although endothelium-independent vasodilatation was not affected (9.5±2.4% vs 8.8±3.1%). No relation was found between percent change in flow-mediated vasodilatation and improvement in levels of LDL-cholesterol after therapy (r=0.07). PAI-1, t-PA, and CRP were not significantly changed by 2 weeks of therapy. (1) Evaluating vasodilation of the brachial artery with B-mode ultrasound imaging was useful in investigating the effect of statin on endothelial function. (2) Although no effect was detected in PAI-1, t-PA, or CRP, relatively low-dose cerivastatin therapy for 2 weeks improved endothelial function and lipid level independently and safely in hypercholesterolemic patients.

Influence of methylenetetrahydrofolate reductase genotype, exercise and other risk factors on endothelial function in healthy individuals.

PubMed

Pullin, Catherine H; Wilson, John F; Ashfield-Watt, Pauline A L; Clark, Zoë E; Whiting, Jenny M; Lewis, Malcolm J; McDowell, Ian F W

2002-01-01

Cardiovascular disease has a multifactorial aetiology that is influenced by both genetic and environmental factors. Endothelial dysfunction is a key event in the pathogenesis of vascular disease that occurs before structural vascular changes or clinical symptoms are evident. Conventional risk factors, for example hypertension and diabetes mellitus, are associated with endothelial dysfunction, but the influence of other putative risk factors is not clear. The methylenetetrahydrofolate reductase (MTHFR) C677T genotype, a common polymorphism that induces hyperhomocysteinaemia, has been proposed as being a genetic risk factor for cardiovascular disease. A total of 126 healthy adults recruited by MTHFR C677T genotype (42 of each genotype, i.e. CC, CT and TT) underwent assessment of endothelial function. Brachial artery endothelium-dependent flow-mediated dilatation (FMD) was measured using high-resolution ultrasonic vessel "wall-tracking". Using multiple regression analysis, MTHFR genotype and 21 other subject and subject-lifestyle variables were investigated as potential predictors of endothelial function. FMD was influenced positively by frequency of aerobic exercise and by hormone replacement therapy, and negatively by increases in systolic blood pressure. MTHFR C677T genotype and the associated variation in plasma homocysteine levels did not influence FMD. Additionally, other factors, including plasma cholesterol and self-supplementation with either antioxidant vitamins or cod liver oil, showed no significant relationship with FMD, although these findings are compromised by the narrow range studied for cholesterol and the small number of subjects taking supplements. These observations have implications for risk factor management in the primary prevention of cardiovascular disease in healthy individuals.

Cell cycle progression in irradiated endothelial cells cultured from bovine aorta

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rubin, D.B.; Drab, E.A.; Ward, W.F.

1988-11-01

Logarithmically growing endothelial cells from bovine aortas were exposed to single doses of 0-10 Gy of 60Co gamma rays, and cell cycle phase distribution and progression were examined by flow cytometry and autoradiography. In some experiments, cells were synchronized in the cell cycle with hydroxyurea (1 mM). Cell number in sham-irradiated control cultures doubled in approximately 24 h. Estimated cycle stage times for control cells were 14.4 h for G1 phase, 7.2 h for S phase, and 2.4 h for G2 + M phase. Irradiated cells demonstrated a reduced distribution at the G1/S phase border at 4 h, and anmore » increased distribution in G2 + M phase at 24 h postirradiation. Autoradiographs of irradiated cells after continuous (3H)thymidine labeling indicated a block in G1 phase or at the G1/S-phase border. The duration of the block was dose dependent (2-3 min/cGy). Progression of the endothelial cells through S phase after removal of the hydroxyurea block also was retarded by irradiation, as demonstrated by increased distribution in early S phase and decreased distribution in late S phase. These results indicate that progression of asynchronous cultured bovine aortic endothelial cells through the DNA synthetic cycle is susceptible to radiation inhibition at specific sites in the cycle, resulting in redistribution and partial synchronization of the population. Thus aortic endothelial cells, diploid cells from a normal tissue, resemble many immortal cell types that have been examined in this regard in vitro.« less

Effect of cigarette smoke extract and nicotine on the expression of thrombomodulin and endothelial protein C receptor in cultured human umbilical vein endothelial cells

PubMed Central

Wei, Yujie; Lai, Bin; Liu, Huiliang; Li, Yi; Zhen, Wang; Fu, Ling

2018-01-01

The present study investigated the influence of cigarette smoke extract (CSE) and nicotine on the expression of thrombomodulin (TM) and endothelial protein C receptor (EPCR) in human umbilical vein endothelial cells (HUVECs). Smoking is associated with intravascular thrombosis. As a vital anticoagulation cofactor, TM is located on the endothelial cell surface and regulates intravascular coagulation by binding to thrombin, hence activating protein C. Activated protein C is a natural anticoagulant that interacts with EPCR to enhance the function of anticoagulation system. The effects of CSE (0.5–5%) and nicotine (10-3-10-9 mol/l) on the expression of TM and EPCR in HUVECs were observed. Reverse transcription-quantitative polymerase chain reaction and flow cytometric analysis techniques were used for detecting TM and EPCR mRNA and protein expression levels, respectively. After 6-h exposure, TM protein and mRNA expression levels decreased in a dose-dependent manner. Stimulation with 5% CSE for 0, 6, 10, 12 and 24 h led to a decrease in the levels of TM mRNA and protein over time, which reached a peak at 12 h. The levels were significantly reduced compared with the control group (P<0.001). However, CSE had no effect on EPCR. Furthermore, nicotine had no influence on TM and EPCR. In conclusion, the present study supports a novel molecular mechanism of cigarette smoking-associated thrombosis by the decreased expression of TM. Further studies are required to identify specific components in CSE responsible for decreasing TM expression and its associated consequences. PMID:29257196

Vascular Reactivity Profile of Novel KCa 3.1-Selective Positive-Gating Modulators in the Coronary Vascular Bed.

PubMed

Oliván-Viguera, Aida; Valero, Marta Sofía; Pinilla, Estéfano; Amor, Sara; García-Villalón, Ángel Luis; Coleman, Nichole; Laría, Celia; Calvín-Tienza, Víctor; García-Otín, Ángel-Luis; Fernández-Fernández, José M; Murillo, M Divina; Gálvez, José A; Díaz-de-Villegas, María D; Badorrey, Ramón; Simonsen, Ulf; Rivera, Luis; Wulff, Heike; Köhler, Ralf

2016-08-01

Opening of intermediate-conductance calcium-activated potassium channels (KC a 3.1) produces membrane hyperpolarization in the vascular endothelium. Here, we studied the ability of two new KC a 3.1-selective positive-gating modulators, SKA-111 and SKA-121, to (1) evoke porcine endothelial cell KC a 3.1 membrane hyperpolarization, (2) induce endothelium-dependent and, particularly, endothelium-derived hyperpolarization (EDH)-type relaxation in porcine coronary arteries (PCA) and (3) influence coronary artery tone in isolated rat hearts. In whole-cell patch-clamp experiments on endothelial cells of PCA (PCAEC), KC a currents evoked by bradykinin (BK) were potentiated ≈7-fold by either SKA-111 or SKA-121 (both at 1 μM) and were blocked by a KC a 3.1 blocker, TRAM-34. In membrane potential measurements, SKA-111 and SKA-121 augmented bradykinin-induced hyperpolarization. Isometric tension measurements in large- and small-calibre PCA showed that SKA-111 and SKA-121 potentiated endothelium-dependent relaxation with intact NO synthesis and EDH-type relaxation to BK by ≈2-fold. Potentiation of the BK response was prevented by KC a 3.1 inhibition. In Langendorff-perfused rat hearts, SKA-111 potentiated coronary vasodilation elicited by BK. In conclusion, our data show that positive-gating modulation of KC a 3.1 channels improves BK-induced membrane hyperpolarization and endothelium-dependent relaxation in small and large PCA as well as in the coronary circulation of rats. Positive-gating modulators of KC a 3.1 could be therapeutically useful to improve coronary blood flow and counteract impaired coronary endothelial dysfunction in cardiovascular disease. © 2016 Nordic Association for the Publication of BCPT (former Nordic Pharmacological Society).

Altered skin flowmotion in hypertensive humans

PubMed Central

Bruning, R.S.; Kenney, W.L.; Alexander, L.M.

2017-01-01

Essential hypertensive humans exhibit attenuated cutaneous nitric oxide (NO)-dependent vasodilation. Using spectral analysis (fast Fourier transformation) we aimed to characterize the skin flowmotion contained in the laser-Doppler flowmetry recordings during local heating-induced vasodilation before and after concurrent pharmacological inhibition of nitric oxide synthase (NOS) in hypertensive and age-matched normotensive men and women. We hypothesized that hypertensive subjects would have lower total power spectral densities (PSD), specifically in the frequency intervals associated with intrinsic endothelial and neurogenic control of the microvasculature. Furthermore, we hypothesized that NOS inhibition would attenuate the endothelial frequency interval. Laser-Doppler flowmetry recordings during local heating experiments from 18 hypertensive (MAP: 108±2mmHg) and 18 normotensive (MAP: 88±2mmHg) men and women were analyzed. Within site NO-dependent vasodilation was assessed by perfusion of a non-specific NOS inhibitor (NG-nitro-L-arginine methyl ester; L-NAME) through intradermal microdialysis during the heating-induced plateau in skin blood flow. Local heating-induced vasodilation increased total PSD for all frequency intervals (all p<0.001). Hypertensives had a lower total PSD (p=0.03) and absolute neurogenic frequency intervals (p<0.01) compared to the normotensives. When normalized as a percentage of total PSD, hypertensives had reduced neurogenic (p<0.001) and augmented myogenic contributions (p=0.04) to the total spectrum. NOS inhibition decreased total PSD (p<0.001) for both groups, but hypertensives exhibited lower absolute endothelial (p<0.01), neurogenic (p<0.05), and total PSD (p<0.001) frequency intervals compared to normotensives. These data suggest that essential hypertension results in altered neurogenic and NOS-dependent control of skin flowmotion and support the use of spectral analysis as a non-invasive technique to study vasoreactivity. PMID:24418051

Analysis of cell flux in the parallel plate flow chamber: implications for cell capture studies.

PubMed Central

Munn, L L; Melder, R J; Jain, R K

1994-01-01

The parallel plate flow chamber provides a controlled environment for determinations of the shear stress at which cells in suspension can bind to endothelial cell monolayers. By decreasing the flow rate of cell-containing media over the monolayer and assessing the number of cells bound at each wall shear stress, the relationship between shear force and binding efficiency can be determined. The rate of binding should depend on the delivery of cells to the surface as well as the intrinsic cell-surface interactions; thus, only if the cell flux to the surface is known can the resulting binding curves be interpreted correctly. We present the development and validation of a mathematical model based on the sedimentation rate and velocity profile in the chamber for the delivery of cells from a flowing suspension to the chamber surface. Our results show that the flux depends on the bulk cell concentration, the distance from the entrance point, and the flow rate of the cell-containing medium. The model was then used in a normalization procedure for experiments in which T cells attach to TNF-alpha-stimulated HUVEC monolayers, showing that a threshold for adhesion occurs at a shear stress of about 3 dyn/cm2. Images FIGURE 1 FIGURE 2 PMID:7948702

Human vascular tissue models formed from human induced pluripotent stem cell derived endothelial cells

PubMed Central

Belair, David G.; Whisler, Jordan A.; Valdez, Jorge; Velazquez, Jeremy; Molenda, James A.; Vickerman, Vernella; Lewis, Rachel; Daigh, Christine; Hansen, Tyler D.; Mann, David A.; Thomson, James A.; Griffith, Linda G.; Kamm, Roger D.; Schwartz, Michael P.; Murphy, William L.

2015-01-01

Here we describe a strategy to model blood vessel development using a well-defined iPSC-derived endothelial cell type (iPSC-EC) cultured within engineered platforms that mimic the 3D microenvironment. The iPSC-ECs used here were first characterized by expression of endothelial markers and functional properties that included VEGF responsiveness, TNF-α-induced upregulation of cell adhesion molecules (MCAM/CD146; ICAM1/CD54), thrombin-dependent barrier function, shear stress-induced alignment, and 2D and 3D capillary-like network formation in Matrigel. The iPSC-ECs also formed 3D vascular networks in a variety of engineering contexts, yielded perfusable, interconnected lumen when co-cultured with primary human fibroblasts, and aligned with flow in microfluidics devices. iPSC-EC function during tubule network formation, barrier formation, and sprouting was consistent with that of primary ECs, and the results suggest a VEGF-independent mechanism for sprouting, which is relevant to therapeutic anti-angiogenesis strategies. Our combined results demonstrate the feasibility of using a well-defined, stable source of iPSC-ECs to model blood vessel formation within a variety of contexts using standard in vitro formats. PMID:25190668

Microgravity Effects on Transendothelial Transport

NASA Technical Reports Server (NTRS)

Tarbell, John M.

1996-01-01

The Endothelial Cell (EC) layer which lines blood vessels from the aorta to the capillaries provides the principal barrier to transport of water and solutes between blood and underlying tissue. Endothelial cells are continuously exposed to the mechanical shearing force (shear stress) and normal force (pressure) imposed by flowing blood on their surface, and they are adapted to this mechanical environment. When the cardiovascular system is exposed to microgravity, the mechanical environmental of endothelial cells is perturbed drastically and the transport properties of EC layers are altered in response. We have shown recently that step changes in shear stress have an acute effect on transport properties of EC layers in a cell culture model, and several recent studies in different vessels of live animals have confirmed the shear-dependent transport properties of the endothelium. We hypothesize that alterations in mechanical forces induced by microgravity and their resultant influence on transendothelial transport of water and solutes are, in large measure, responsible for the characteristic cephalad fluid shift observed in humans experiencing microgravity. To study the effects of altered mechanical forces on transendothelial transport and to test pharmacologic agents as counter measures to microgravity induced fluid shifts we have proposed ground-based studies using well defined cell culture models.

Geraniol improves endothelial function by inhibiting NOX-2 derived oxidative stress in high fat diet fed mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Xiaoyu; Zhao, Shiqi; Su, Mengqi

Endothelial dysfunction occurs in obese patients and high-fat diet (HFD) fed experimental animals. While geraniol has been reported to ameliorate inflammation and oxidative stress, inhibit tumor cell proliferation, and improve atherosclerosis, its direct effect on endothelial function remains uncharacterized. The present study therefore investigated the effect of geraniol on endothelial function in HFD mice and its underlying mechanisms. C57 BL/6 mice were fed an HFD (n = 40) or a normal diet (n = 20) for 8 weeks. HFD fed mice then were randomized to intraperitoneal treatment with geraniol (n = 20) or vehicle (n = 20) for another 6 weeks. Acetylcholine (Ach)-induced endothelial dependent vasorelaxation was measuredmore » on wire myography; reactive oxygen species (ROS) generation was assessed by fluorescence imaging, and NADPH oxidases (NOXs) and adhesive molecules VCAM-1 and ICAM-1 protein expression by western blotting. Geraniol improved endothelial function in HFD fed mice, as evidenced by its: 1. restoring endothelial dependent vasorelaxation induced by Ach, and reversing increased VCAM-1 and ICAM-1 expression; 2. attenuating HFD induced increased serum TBARS and aortic ROS generation; and 3. downregulating aortic NOX-2 expression in both HFD fed mice and in palmitic acid treated endothelial cells. Geraniol therefore protects against endothelial dysfunction induced by HFD through reducing NOX-2 associated ROS generation. -- Highlights: •Geraniol improved endothelial dependent relaxation in high fat diet fed mice. •Geraniol alleviated vascular injury in high fat diet fed mice. •Geraniol inhibited ROS generation through downregulating NOX-2 expression.« less

Microfluidic perfusion culture chip providing different strengths of shear stress for analysis of vascular endothelial function.

PubMed

Hattori, Koji; Munehira, Yoichi; Kobayashi, Hideki; Satoh, Taku; Sugiura, Shinji; Kanamori, Toshiyuki

2014-09-01

We developed a microfluidic perfusion cell culture chip that provides three different shear stress strengths and a large cell culture area for the analysis of vascular endothelial functions. The microfluidic network was composed of shallow flow-control channels of three different depths and deep cell culture channels. The flow-control channels with high fluidic resistances created shear stress strengths ranging from 1.0 to 10.0 dyn/cm(2) in the cell culture channels. The large surface area of the culture channels enabled cultivation of a large number (approximately 6.0 × 10(5)) of cells. We cultured human umbilical vein endothelial cells (HUVECs) and evaluated the changes in cellular morphology and gene expression in response to applied shear stress. The HUVECs were aligned in the direction of flow when exposed to a shear stress of 10.0 dyn/cm(2). Compared with conditions of no shear stress, endothelial nitric oxide synthase mRNA expression increased by 50% and thrombomodulin mRNA expression increased by 8-fold under a shear stress of 9.5 dyn/cm(2). Copyright © 2014 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Evaluating endothelial function of the common carotid artery: an in vivo human model.

PubMed

Mazzucco, S; Bifari, F; Trombetta, M; Guidi, G C; Mazzi, M; Anzola, G P; Rizzuto, N; Bonadonna, R

2009-03-01

Flow mediated dilation (FMD) of peripheral conduit arteries is a well-established tool to evaluate endothelial function. The aims of this study are to apply the FMD model to cerebral circulation by using acetazolamide (ACZ)-induced intracranial vasodilation as a stimulus to increase common carotid artery (CCA) diameter in response to a local increase of blood flow velocity (BFV). In 15 healthy subjects, CCA end-diastolic diameter and BFV, middle cerebral artery (MCA) BFV and mean arterial blood pressure (MBP) were measured at basal conditions, after an intravenous bolus of 1g ACZ, and after placebo (saline) sublingual administration at the 15th and 20th minute. In a separate session, the same parameters were evaluated after placebo (saline) infusion instead of ACZ and after 10 microg/m(2) bs and 300 microg of glyceryl trinitrate (GTN), administered sublingually, at the 15th and 20th minute, respectively. After ACZ bolus, there was a 35% maximal MCA mean BFV increment (14th minute), together with a 22% increase of mean CCA end-diastolic BFV and a CCA diameter increment of 3.9% at the 3rd minute (p=0.024). There were no MBP significant variations up to the 15th minute (p=0.35). After GTN administration, there was a significant increment in CCA diameter (p<0.00001). ACZ causes a detectable CCA dilation in healthy individuals concomitantly with an increase in BFV. Upon demonstration that this phenomenon is endothelium dependent, this experimental model might become a valuable tool to assess endothelial function in the carotid artery.

Effect of genistein on endothelial function in postmenopausal women: a randomized, double-blind, controlled study.

PubMed

Squadrito, Francesco; Altavilla, Domenica; Crisafulli, Alessandra; Saitta, Antonino; Cucinotta, Domenico; Morabito, Nunziata; D'Anna, Rosario; Corrado, Francesco; Ruggeri, Pietro; Frisina, Nicola; Squadrito, Giovanni

2003-04-15

Genistein, a phytoestrogen found in soybeans, corrects endothelial dysfunction induced by oophorectomy in animals. Using a double-blind, controlled, randomized design, we evaluated its effects on endothelial function in women. We enrolled 79 healthy postmenopausal women (mean [+/- SD] age, 56 +/- 4 years) and randomly assigned them to receive continuous estrogen/progestin therapy (n = 26; 17beta-estradiol [1 mg/d] combined with norethisterone acetate [0.5 mg/d]), genistein (n = 27; 54 mg/d), or placebo (n = 26). Brachial artery flow-mediated, endothelium-dependent vasodilation and plasma levels of nitrites/nitrates (a marker of nitric oxide metabolism) and endothelin-1 were measured at baseline and after 1 year of therapy. Treatment with genistein increased levels of nitrites/nitrates (mean increase, 21 micromol/L; 95% confidence interval [CI]: 15 to 26 micromol/L; P <0.001 vs. placebo); estrogen/progestin therapy caused similar changes (P <0.001 vs. placebo). Plasma endothelin-1 levels decreased following 12 months of genistein (mean decrease, 7 pg/mL; 95% CI: 3 to 10 pg/mL; P <0.001 vs. placebo) and after 12 months of estrogen/progestin (P <0.001 vs. placebo). When compared with placebo, brachial artery flow-mediated dilation was improved by genistein (mean increase, 5.5%; 95% CI: 3.9% to 7.0%; P <0.001) and by estrogen/progestin (P <0.001). There were no significant differences between estrogen and genistein for any of these parameters (all P >0.4). One year of genistein therapy improves endothelium function in postmenopausal women to a similar extent as does an estrogen/progestin regimen.

Endothelial cells respond to the direction of mechanical stimuli through SMAD signaling to regulate coronary artery size.

PubMed

Poduri, Aruna; Chang, Andrew H; Raftrey, Brian; Rhee, Siyeon; Van, Mike; Red-Horse, Kristy

2017-09-15

How mechanotransduction intersects with chemical and transcriptional factors to shape organogenesis is an important question in developmental biology. This is particularly relevant to the cardiovascular system, which uses mechanical signals from flowing blood to stimulate cytoskeletal and transcriptional responses that form a highly efficient vascular network. Using this system, artery size and structure are tightly regulated, but the underlying mechanisms are poorly understood. Here, we demonstrate that deletion of Smad4 increased the diameter of coronary arteries during mouse embryonic development, a phenotype that followed the initiation of blood flow. At the same time, the BMP signal transducers SMAD1/5/8 were activated in developing coronary arteries. In a culture model of blood flow-induced shear stress, human coronary artery endothelial cells failed to align when either BMPs were inhibited or SMAD4 was depleted. In contrast to control cells, SMAD4- deficient cells did not migrate against the direction of shear stress and increased proliferation rates specifically under flow. Similar alterations were seen in coronary arteries in vivo Thus, endothelial cells perceive the direction of blood flow and respond through SMAD signaling to regulate artery size. © 2017. Published by The Company of Biologists Ltd.

Direct evidence for the role of caveolin-1 and caveolae in mechanotransduction and remodeling of blood vessels

PubMed Central

Yu, Jun; Bergaya, Sonia; Murata, Takahisa; Alp, Ilkay F.; Bauer, Michael P.; Lin, Michelle I.; Drab, Marek; Kurzchalia, Teymuras V.; Stan, Radu V.; Sessa, William C.

2006-01-01

Caveolae in endothelial cells have been implicated as plasma membrane microdomains that sense or transduce hemodynamic changes into biochemical signals that regulate vascular function. Therefore we compared long- and short-term flow-mediated mechanotransduction in vessels from WT mice, caveolin-1 knockout (Cav-1 KO) mice, and Cav-1 KO mice reconstituted with a transgene expressing Cav-1 specifically in endothelial cells (Cav-1 RC mice). Arterial remodeling during chronic changes in flow and shear stress were initially examined in these mice. Ligation of the left external carotid for 14 days to lower blood flow in the common carotid artery reduced the lumen diameter of carotid arteries from WT and Cav-1 RC mice. In Cav-1 KO mice, the decrease in blood flow did not reduce the lumen diameter but paradoxically increased wall thickness and cellular proliferation. In addition, in isolated pressurized carotid arteries, flow-mediated dilation was markedly reduced in Cav-1 KO arteries compared with those of WT mice. This impairment in response to flow was rescued by reconstituting Cav-1 into the endothelium. In conclusion, these results showed that endothelial Cav-1 and caveolae are necessary for both rapid and long-term mechanotransduction in intact blood vessels. PMID:16670769

Relationship between S-adenosylmethionine, S-adenosylhomocysteine, asymmetric dimethylarginine, and endothelial function in healthy human subjects during experimental hyper- and hypohomocysteinemia.

PubMed

Doshi, Sagar; McDowell, Ian; Goodfellow, Jonathan; Stabler, Sally; Boger, Rainer; Allen, Robert; Newcombe, Robert; Lewis, Malcolm; Moat, Stuart

2005-03-01

Experimental hyperhomocysteinemia after an oral methionine or homocysteine load is associated with impaired nitric oxide-dependent vasodilatation in healthy human beings. However, it remains unproven that this effect is mediated by elevations in plasma homocysteine. There is evidence that an increase in plasma homocysteine may increase the formation of asymmetric dimethylarginine (ADMA), an inhibitor of nitric oxide synthase. The methyl groups within ADMA are derived from the conversion of S -adenosylmethionine to S -adenosylhomocysteine intermediates in the methionine/homocysteine pathway. No previous study has assessed the role of methylation status, its impact on ADMA formation, and their association with endothelial function in healthy human beings. In a randomized, placebo-controlled, crossover study, 10 healthy subjects (mean age, 29.1 +/- 3.9 years) were administered an oral dose of methionine (0.1 g/kg), l -homocysteine (0.01 g/kg), N-acetylcysteine (NAC) (0.1 g/kg), or placebo. Endothelial function as assessed by flow-mediated dilatation (FMD) of the brachial artery was impaired after both the methionine and homocysteine load compared with placebo at 4 hours (36 +/- 15, 67 +/- 23 vs 219 +/- 26 microm, respectively, P < .001). N-Acetylcysteine had no effect on flow-mediated dilatation. Plasma total homocysteine was significantly elevated at 4 hours after methionine (23.1 +/- 6.2) and homocysteine (41.5 +/- 8.9) loading, but significantly reduced after NAC 2.4 +/- 0.6 vs 7.1 +/- 2.1 micromol/L in the placebo (P < .001). Plasma S-adenosylmethionine/S-adenosylhomocysteine ratio was significantly (P < .001) increased at 4 hours after methionine (10.9 +/- 0.7) compared with homocysteine (5.4 +/- 0.4), NAC (5.0 +/- 0.3), and placebo (6.0 +/- 0.5). Plasma ADMA concentrations were not altered by any intervention. Our results suggest that endothelial dysfunction due to methionine or homocysteine loading is not associated with an increase in plasma ADMA or a disruption in methylation status.

Hemodynamically driven stent strut design.

PubMed

Jiménez, Juan M; Davies, Peter F

2009-08-01

Stents are deployed to physically reopen stenotic regions of arteries and to restore blood flow. However, inflammation and localized stent thrombosis remain a risk for all current commercial stent designs. Computational fluid dynamics results predict that nonstreamlined stent struts deployed at the arterial surface in contact with flowing blood, regardless of the strut height, promote the creation of proximal and distal flow conditions that are characterized by flow recirculation, low flow (shear) rates, and prolonged particle residence time. Furthermore, low shear rates yield an environment less conducive for endothelialization, while local flow recirculation zones can serve as micro-reaction chambers where procoagulant and pro-inflammatory elements from the blood and vessel wall accumulate. By merging aerodynamic theory with local hemodynamic conditions we propose a streamlined stent strut design that promotes the development of a local flow field free of recirculation zones, which is predicted to inhibit thrombosis and is more conducive for endothelialization.

Impact of endothelin blockade on acute exercise-induced changes in blood flow and endothelial function in type 2 diabetes mellitus.

PubMed

Schreuder, Tim H A; van Lotringen, Jaap H; Hopman, Maria T E; Thijssen, Dick H J

2014-09-01

Positive vascular effects of exercise training are mediated by acute increases in blood flow. Type 2 diabetes patients show attenuated exercise-induced increases in blood flow, possibly mediated by the endothelin pathway, preventing an optimal stimulus for vascular adaptation. We examined the impact of endothelin receptor blockade (bosentan) on exercise-induced blood flow in the brachial artery and on pre- and postexercise endothelial function in type 2 diabetes patients (n = 9, 60 ± 7 years old) and control subjects (n = 10, 60 ± 5 years old). Subjects reported twice to the laboratory to perform hand-grip exercise in the presence of endothelin receptor blockade or placebo. We examined brachial artery endothelial function (via flow-mediated dilatation) before and after exercise, as well as blood flow during exercise. Endothelin receptor blockade resulted in a larger increase in blood flow during exercise in type 2 diabetes patients (P = 0.046), but not in control subjects (P = 0.309). Exercise increased shear rate across the exercise protocol, unaffected by endothelin receptor blockade. Exercise did not alter brachial artery diameter in either group, but endothelin receptor blockade resulted in a larger brachial artery diameter in type 2 diabetes patients (P = 0.033). Exercise significantly increased brachial artery flow-mediated dilatation in both groups, unaffected by endothelin receptor blockade. Endothelin receptor blockade increased exercise-induced brachial artery blood flow in type 2 diabetes patients, but not in control subjects. Despite this effect of endothelin receptor blockade on blood flow, we found no impact on baseline or post-exercise endothelial function in type 2 diabetes patients or control subjects, possibly related to normalization of the shear stimulus during exercise. The successful increase in blood flow during exercise in type 2 diabetes patients through endothelin receptor blockade may have beneficial effects in repeated exercise training. © 2014 The Authors. Experimental Physiology © 2014 The Physiological Society.

Periodontal treatment improves endothelial dysfunction in patients with severe periodontitis.

PubMed

Seinost, Gerald; Wimmer, Gernot; Skerget, Martina; Thaller, Erik; Brodmann, Marianne; Gasser, Robert; Bratschko, Rudolf O; Pilger, Ernst

2005-06-01

Because epidemiological studies provide evidence that periodontal infections are associated with an increased risk of progression of cardiovascular and cerebrovascular disease, we postulated that endothelial dysfunction, a critical element in the pathogenesis of atherosclerosis, would be present in patients with periodontal disease. We tested endothelial function in 30 patients with severe periodontitis and 31 control subjects using flow-mediated dilation (FMD) of the brachial artery. The groups were matched for age, sex, and cardiovascular risk factors. Three months after periodontal treatment, including both mechanical and pharmacological therapy, endothelial function was reassessed by brachial artery FMD. Markers of systemic inflammation were measured at baseline and at follow up. Flow-mediated dilation was significantly lower in patients with periodontitis than in control subjects (6.1% +/- 4.4% vs 8.5% +/- 3.4%, P = .002). Successful periodontal treatment resulted in a significant improvement in FMD (9.8% +/- 5.7%; P = .003 compared to baseline) accompanied by a significant decrease in C-reactive protein concentrations (1.1 +/- 1.9 vs 0.8 +/- 0.8 at baseline, P = .026). Endothelium-independent nitro-induced vasodilation did not differ between the study groups at baseline or after periodontal therapy. These results indicate that treatment of severe periodontitis reverses endothelial dysfunction. Whether improved endothelial function will translate into a beneficial effect on atherogenesis and cardiovascular events needs further investigation.

Ethnic differences in macrovascular and microvascular function in systolic heart failure.

PubMed

Shantsila, Eduard; Wrigley, Benjamin; Shantsila, Alena; Tapp, Luke D; Blann, Andrew D; Gill, Paramjit S; Lip, Gregory Y H

2011-11-01

Endothelial dysfunction is implicated in the pathophysiological features of heart failure (HF), and ethnic differences in the presentation of cardiovascular disease are evident, with an excess seen among South Asians (SAs). However, data on ethnic differences in endothelial function in HF are limited. In a cross-sectional study, we recruited 128 subjects with systolic HF: 50 SAs, 50 whites, and 28 African Caribbeans (ACs). In addition, SAs with systolic HF were compared with 40 SAs with coronary artery disease without HF ("disease controls") and 40 SA healthy controls. Macrovascular endothelial function was assessed by measurement of flow-mediated dilation (FMD) in response to hyperemia, arterial stiffness was assessed by the pulse-wave velocity, and microvascular endothelial function was assessed by forearm laser Doppler flowmetry. CD144-expressing endothelial microparticles were measured by flow cytometry. When compared with disease controls and healthy controls, SAs with HF had an impaired microvascular response to acetylcholine (P=0.001) and reduced FMD (P<0.001). In comparing ethnic groups, SAs with HF had an impaired response to acetylcholine (123±95.5%) compared with whites (258±156%) and ACs (286±173%, P<0.001 for both). Whites had a higher FMD (8.49±4.63%) than SAs (4.76±4.78%, P<0.001) and ACs (4.55±3.56%, P=0.01). No difference in endothelial-independent response was observed between study groups or in pulse-wave velocity. Ethnicity remained associated with microvascular endothelial function even after adjustment for age, presence of hypertension and diabetes mellitus, blood pressure, and glucose levels (P=0.003). There were no differences in numbers of endothelial microparticles. The SAs with HF have impaired microvascular and macrovascular endothelial function but preserved arterial elastic properties. Significant ethnic differences in endothelial function are evident in subjects with HF, with ethnicity being associated with microvascular endothelial dysfunction in this disorder.

Endothelial Dysfunction in Human Diabetes is mediated by Wnt5a-JNK Signaling

PubMed Central

Bretón-Romero, Rosa; Feng, Bihua; Holbrook, Monika; Farb, Melissa G.; Fetterman, Jessica L.; Linder, Erika A.; Berk, Brittany D.; Masaki, Nobuyuki; Weisbrod, Robert M.; Inagaki, Elica; Gokce, Noyan; Fuster, Jose J.; Walsh, Kenneth; Hamburg, Naomi M.

2016-01-01

Objectives Endothelial dysfunction is linked to insulin resistance, inflammatory activation and increased cardiovascular risk in diabetes mellitus; however the mechanisms remain incompletely understood. Recent studies have identified pro-inflammatory signaling of Wnt5a through JNK as a regulator of metabolic dysfunction with potential relevance to vascular function. We sought to gain evidence that increased activation of Wnt5a-JNK signaling contributes to impaired endothelial function in patients with diabetes mellitus. Approach We measured flow-mediated dilation of the brachial artery and characterized freshly isolated endothelial cells by protein expression, eNOS activation, and nitric oxide production in from 85 subjects with Type 2 diabetes mellitus (n=42) and age- and sex-matched non-diabetic controls (n=43) and in human aortic endothelial cells treated with Wnt5a. Results Endothelial cells from patients with diabetes displayed 1.3-fold higher Wnt5a levels (P=0.01) along with 1.4-fold higher JNK activation (P<0.01) without a difference in total JNK levels. Higher JNK activation was associated with lower flow-mediated dilation, consistent with endothelial dysfunction (r=0.53, P=0.02). Inhibition of Wnt5a and JNK signaling restored insulin and A23187-mediated eNOS activation and improved nitric oxide production in endothelial cells from patients with diabetes. In endothelial cells from non-diabetic controls, rWnt5a treatment inhibited eNOS activation replicating the diabetic endothelial phenotype. In HAECs, Wnt5a-induced impairment of eNOS activation and nitric oxide production was reversed by Wnt5a and JNK inhibition. Conclusions Our findings demonstrate that non-canonical Wnt5a signaling and JNK activity contributes to vascular insulin resistance and endothelial dysfunction and may represent a novel therapeutic opportunity to protect the vasculature in patients with diabetes. PMID:26800561

Asbestos-induced endothelial cell activation and injury. Demonstration of fiber phagocytosis and oxidant-dependent toxicity.

PubMed

Garcia, J G; Gray, L D; Dodson, R F; Callahan, K S

1988-10-01

Vascular endothelial cell injury is important in the development of a variety of chronic interstitial lung disorders. However, the involvement of such injury in the inflammatory response associated with the inhalation of asbestos fibers is unclear and the mechanism of asbestos fiber cytotoxicity remains unknown. In the present study, human umbilical vein endothelial cells were challenged with amosite asbestos and several parameters of cellular function were examined. Electron microscopic examination revealed that endothelial cell exposure to asbestos resulted in active phagocytosis of these particulates. Biochemical evidence of dose-dependent asbestos-mediated endothelial cell activation was indicated by increased metabolism of arachidonic acid. For example, amosite asbestos (500 micrograms/ml) produced a ninefold increase in prostacyclin (PGI2) levels over those levels in non-exposed cells. Incubation of human endothelial cells with asbestos fibers induced specific 51Cr release in both a dose- and time-dependent fashion indicative of cellular injury. Injury induced by amosite asbestos was not significantly attenuated by treatment of the endothelial cell monolayer with either the iron chelator deferoxamine, which prevents hydroxyl radical (.OH) formation, or by the superoxide anion (O2-) scavenger, superoxide dismutase. However, significant dose-dependent protection was observed with the hydrogen peroxide (H2O2) scavenger, catalase. Chelation of elemental iron present within amosite asbestos fibers by deferoxamine produced a 33% reduction in asbestos cytotoxicity, suggesting a potential role for hydroxyl radical-mediated injury via the iron-catalyzed Haber-Weiss reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of thyroxine replacement on endothelial function and carotid artery intima-media thickness in female patients with mild subclinical hypothyroidism

PubMed Central

Cabral, Monica Dias; Teixeira, Patricia; Soares, Debora; Leite, Sandra; Salles, Elizabeth; Waisman, Mario

2011-01-01

BACKGROUND: Previous studies have suggested an association between subclinical hypothyroidism and coronary artery disease that could be related to changes in serum lipids or endothelial dysfunction. METHODS: Thirty-two female subclinical hypothyroidism patients were randomly assigned to 12 months of L-thyroxine replacement or no treatment. Endothelial function was measured by the flow-mediated vasodilatation of the brachial artery, as well as mean carotid artery intima-media thickness, and lipid profiles were studied at baseline and after 12 months of follow-up. RESULTS: The mean (±SD) serum thyroid-stimulating hormone levels in the L-thyroxine replacement and control groups were 6.09±1.32 and 6.27±1.39 µUI/ml, respectively. No relationship between carotid artery intima-media thickness or brachial flow-mediated vasodilatation and free T4 and serum thyroid-stimulating hormone was found. The median L-T4 dose was 44.23±18.13 µg/day. After 12 months, there was a significant decrease in the flow-mediated vasodilatation in the subclinical hypothyroidism control group (before: 17.33±7.88 to after: 13.1±4.75%, p = 0.03), but there were no significant differences in flow-mediated vasodilatation in the L-thyroxine treated group (before: 16.81±7.0 to after: 18.52±7.44%, p = 0.39). We did not find any significant change in mean carotid intima-media thickness after 12 months of L-thyroxine treatment. CONCLUSION: Replacement therapy prevents a decline in flow-mediated vasodilatation with continuation of the subclinical hypothyroidism state. Large prospective multicenter placebo-controlled trials are necessary to investigate endothelial physiology further in subclinical hypothyroidism patients and to define the role of L-thyroxine therapy in improving endothelial function in these patients. PMID:21915478

Angiogenesis mediated by soluble forms of E-selectin and vascular cell adhesion molecule-1

NASA Astrophysics Data System (ADS)

Koch, Alisa E.; Halloran, Margaret M.; Haskell, Catherine J.; Shah, Manisha R.; Polverini, Peter J.

1995-08-01

ENDOTHELIAL adhesion molecules facilitate the entry of leukocytes into inflamed tissues. This in turn promotes neovascularization, a process central to the progression of rheumatoid arthritis, tumour growth and wound repair1. Here we test the hypothesis that soluble endothelial adhesion molecules promote angiogenesis2á¤-4. Human recombinant soluble E-selectin and soluble vascular cell adhesion molecule-1 induced chemotaxis of human endothelial cells in vitro and were angiogenic in rat cornea. Soluble E-selectin acted on endothelial cells in part through a sialyl Lewis-X-dependent mechanism, while soluble vascular cell adhesion molecule-1 acted on endothelial cells in part through a very late antigen (VLA)-4 dependent mechanism. The chemotactic activity of rheumatoid synovial fluid for endothelial cells, and also its angiogenic activity, were blocked by antibodies to either soluble E-selectin or soluble vascular cell adhesion molecule-1. These results suggest a novel function for soluble endothelial adhesion molecules as mediators of angiogenesis.

Endothelial atheroprotective and anti-inflammatory mechanisms.

PubMed

Berk, B C; Abe, J I; Min, W; Surapisitchat, J; Yan, C

2001-12-01

Atherosclerosis preferentially occurs in areas of turbulent flow and low fluid shear stress, whereas laminar flow and high shear stress are atheroprotective. Inflammatory cytokines, such as tumor necrosis factor-alpha (TNF), have been shown to stimulate expression of endothelial cell (EC) genes that may promote atherosclerosis. Recent data suggest that steady laminar flow decreases EC apoptosis and blocks TNF-mediated EC activation. EC apoptosis is likely important in the process termed "plaque erosion" that leads to platelet aggregation. Steady laminar flow inhibits EC apoptosis by preventing cell cycle entry, by increasing antioxidant mechanisms (e.g., superoxide dismutase), and by stimulating nitric oxide-dependent protective pathways that involve enzymes PI3-kinase and Akt. Conversely, our laboratory has identified nitric oxide-independent mechanisms that limit TNF signal transduction. TNF regulates gene expression in EC, in part, by stimulating mitogen-activated protein kinases (MAPK) which phosphorylate transcription factors. We hypothesized that fluid shear stress modulates TNF effects on EC by inhibiting TNF-mediated activation of MAP kinases. To test this hypothesis, we determined the effects of steady laminar flow (shear stress = 12 dynes/cm2) on TNF-stimulated activity of two MAP kinases: extracellular signal regulated kinase (ERK1/2) and c-Jun N-terminal kinase (JNK). Flow alone stimulated ERK1/2 activity, but decreased JNK activity compared to static controls. TNF (10 ng/ml) alone activated both ERK1/2 and JNK maximally at 15 minutes in human umbilical vein EC (HUVEC). Pre-exposing HUVEC for 10 minutes to flow inhibited TNF activation of JNK by 46%, but it had no significant effect on ERK1/2 activation. Incubation of EC with PD98059, a specific mitogen-activated protein kinase kinase inhibitor, blocked the flow-mediated inhibition of TNF activation of JNK. Flow-mediated inhibition of JNK was unaffected by 0.1 mM L-nitroarginine, 100 pM 8-bromo-cyclic GMP, or 100 microM 8-bromo-cyclic AMP. Transfection studies with dominant negative constructs of the protein kinase MEK1 and MEK5 suggested an important role for BMK1 in flow-mediated regulation of TNF signals. In summary, the atheroprotective effects of steady laminar flow on the endothelium involve multiple synergistic mechanisms.

Aquaporin-1 facilitates pressure-driven water flow across the aortic endothelium.

PubMed

Nguyen, Tieuvi; Toussaint, Jimmy; Xue, Yan; Raval, Chirag; Cancel, Limary; Russell, Stewart; Shou, Yixin; Sedes, Omer; Sun, Yu; Yakobov, Roman; Tarbell, John M; Jan, Kung-ming; Rumschitzki, David S

2015-05-01

Aquaporin-1, a ubiquitous water channel membrane protein, is a major contributor to cell membrane osmotic water permeability. Arteries are the physiological system where hydrostatic dominates osmotic pressure differences. In the present study, we show that the walls of large conduit arteries constitute the first example where hydrostatic pressure drives aquaporin-1-mediated transcellular/transendothelial flow. We studied cultured aortic endothelial cell monolayers and excised whole aortas of male Sprague-Dawley rats with intact and inhibited aquaporin-1 activity and with normal and knocked down aquaporin-1 expression. We subjected these systems to transmural hydrostatic pressure differences at zero osmotic pressure differences. Impaired aquaporin-1 endothelia consistently showed reduced engineering flow metrics (transendothelial water flux and hydraulic conductivity). In vitro experiments with tracers that only cross the endothelium paracellularly showed that changes in junctional transport cannot explain these reductions. Percent reductions in whole aortic wall hydraulic conductivity with either chemical blocking or knockdown of aquaporin-1 differed at low and high transmural pressures. This observation highlights how aquaporin-1 expression likely directly influences aortic wall mechanics by changing the critical transmural pressure at which its sparse subendothelial intima compresses. Such compression increases transwall flow resistance. Our endothelial and historic erythrocyte membrane aquaporin density estimates were consistent. In conclusion, aquaporin-1 significantly contributes to hydrostatic pressure-driven water transport across aortic endothelial monolayers, both in culture and in whole rat aortas. This transport, and parallel junctional flow, can dilute solutes that entered the wall paracellularly or through endothelial monolayer disruptions. Lower atherogenic precursor solute concentrations may slow their intimal entrainment kinetics. Copyright © 2015 the American Physiological Society.

Aquaporin-1 facilitates pressure-driven water flow across the aortic endothelium

PubMed Central

Nguyen, Tieuvi; Toussaint, Jimmy; Xue, Yan; Raval, Chirag; Cancel, Limary; Russell, Stewart; Shou, Yixin; Sedes, Omer; Sun, Yu; Yakobov, Roman; Tarbell, John M.; Jan, Kung-ming

2015-01-01

Aquaporin-1, a ubiquitous water channel membrane protein, is a major contributor to cell membrane osmotic water permeability. Arteries are the physiological system where hydrostatic dominates osmotic pressure differences. In the present study, we show that the walls of large conduit arteries constitute the first example where hydrostatic pressure drives aquaporin-1-mediated transcellular/transendothelial flow. We studied cultured aortic endothelial cell monolayers and excised whole aortas of male Sprague-Dawley rats with intact and inhibited aquaporin-1 activity and with normal and knocked down aquaporin-1 expression. We subjected these systems to transmural hydrostatic pressure differences at zero osmotic pressure differences. Impaired aquaporin-1 endothelia consistently showed reduced engineering flow metrics (transendothelial water flux and hydraulic conductivity). In vitro experiments with tracers that only cross the endothelium paracellularly showed that changes in junctional transport cannot explain these reductions. Percent reductions in whole aortic wall hydraulic conductivity with either chemical blocking or knockdown of aquaporin-1 differed at low and high transmural pressures. This observation highlights how aquaporin-1 expression likely directly influences aortic wall mechanics by changing the critical transmural pressure at which its sparse subendothelial intima compresses. Such compression increases transwall flow resistance. Our endothelial and historic erythrocyte membrane aquaporin density estimates were consistent. In conclusion, aquaporin-1 significantly contributes to hydrostatic pressure-driven water transport across aortic endothelial monolayers, both in culture and in whole rat aortas. This transport, and parallel junctional flow, can dilute solutes that entered the wall paracellularly or through endothelial monolayer disruptions. Lower atherogenic precursor solute concentrations may slow their intimal entrainment kinetics. PMID:25659484

Nitric oxide-mediated blood flow regulation as affected by smoking and nicotine.

PubMed

Toda, Noboru; Toda, Hiroshi

2010-12-15

Cigarette smoking is a major risk factor for atherosclerosis, cerebral and coronary vascular diseases, hypertension, and diabetes mellitus. Chronic smoking impairs endothelial function by decreasing the formation of nitric oxide and increasing the degradation of nitric oxide via generation of oxygen free radicals. Nitric oxide liberated from efferent nitrergic nerves is also involved in vasodilatation, increased regional blood flow, and hypotension that are impaired through nitric oxide sequestering by smoking-induced factors. Influence of smoking on nitric oxide-induced blood flow regulation is not necessarily the same in all organs and tissues. However, human studies are limited mainly to the forearm blood flow measurement that assesses endothelial function under basal and stimulated conditions and also determination of penile tumescence and erection in response to endothelial and neuronal nitric oxide. Therefore, information about blood flow regulation in other organs, such as the brain and placenta, has been provided mainly from studies on experimental animals. Nicotine, a major constituent of cigarette smoke, acutely dilates cerebral arteries and arterioles through nitric oxide liberated from nitrergic neurons, but chronically interferes with endothelial function in various vasculatures, both being noted in studies on experimental animals. Cigarette smoke constituents other than nicotine also have some vascular actions. Not only active but also passive smoking is undoubtedly harmful for both the smokers themselves and their neighbors, who should bear in mind that they can face serious diseases in the future, which may result in lengthy hospitalization, and a shortened lifespan. Copyright © 2010 Elsevier B.V. All rights reserved.

Flow-mediated dilation and peripheral arterial tonometry are disturbed in preeclampsia and reflect different aspects of endothelial function.

PubMed

Mannaerts, Dominique; Faes, Ellen; Goovaerts, Inge; Stoop, Tibor; Cornette, Jerome; Gyselaers, Wilfried; Spaanderman, Marc; Van Craenenbroeck, Emeline M; Jacquemyn, Yves

2017-11-01

Endothelial function and arterial stiffness are known to be altered in preeclamptic pregnancies. Previous studies have shown conflicting results regarding the best technique for assessing vascular function in pregnancy. In this study, we made a comprehensive evaluation of in vivo vascular function [including flow-mediated dilatation (FMD), peripheral arterial tonometry (PAT), and arterial stiffness] in preeclamptic patients and compared them with normal pregnancies. In addition, we assessed the relation between vascular function and systemic inflammation. Fourteen patients with preeclampsia (PE) and 14 healthy pregnant controls were included. Endothelial function was determined by FMD and PAT and arterial stiffness by carotid-femoral pulse-wave velocity and augmentation index. Systemic inflammation was assessed using mean platelet volume (MPV) and neutrophil-lymphocyte ratio (NLR). The reactive hyperemia index, assessed using PAT, is decreased at the third trimester compared with the first trimester in a normal, uncomplicated pregnancy ( P = 0.001). Arterial stiffness is significantly higher in PE versus normal pregnancy ( P < 0.001). Endothelial function, obtained by FMD, is deteriorated in PE versus normal pregnancy ( P = 0.015), whereas endothelial function assessment by PAT is improved in PE versus normal pregnancy ( P = 0.001). Systemic inflammation (MPV and NLR) increases during normal pregnancy. FMD and PAT are disturbed in PE. Endothelial function, assessed by FMD and PAT, shows distinct results. This may indicate that measurements with FMD and PAT reflect different aspects of endothelial function and that PAT should not be used as a substitute for FMD as a measure of endothelial function in pregnancy. Copyright © 2017 the American Physiological Society.

The role of endothelial cell attachment to elastic fibre molecules in the enhancement of monolayer formation and retention, and the inhibition of smooth muscle cell recruitment.

PubMed

Williamson, Matthew R; Shuttleworth, Adrian; Canfield, Ann E; Black, Richard A; Kielty, Cay M

2007-12-01

The endothelium is an essential modulator of vascular tone and thrombogenicity and a critical barrier between the vessel wall and blood components. In tissue-engineered small-diameter vascular constructs, endothelial cell detachment in flow can lead to thrombosis and graft failure. The subendothelial extracellular matrix provides stable endothelial cell anchorage through interactions with cell surface receptors, and influences the proliferation, migration, and survival of both endothelial cells and smooth muscle cells. We have tested the hypothesis that these desired physiological characteristics can be conferred by surface coatings of natural vascular matrix components, focusing on the elastic fiber molecules, fibrillin-1, fibulin-5 and tropoelastin. On fibrillin-1 or fibulin-5-coated surfaces, endothelial cells exhibited strong integrin-mediated attachment in static conditions (82% and 76% attachment, respectively) and flow conditions (67% and 78% cell retention on fibrillin-1 or fibulin-5, respectively, at 25 dynes/cm2), confluent monolayer formation, and stable functional characteristics. Adhesion to these two molecules also strongly inhibited smooth muscle cell migration to the endothelial monolayer. In contrast, on elastin, endothelial cells attached poorly, did not spread, and had markedly impaired functional properties. Thus, fibrillin-1 and fibulin-5, but not elastin, can be exploited to enhance endothelial stability, and to inhibit SMC migration within vascular graft scaffolds. These findings have important implications for the design of vascular graft scaffolds, the clinical performance of which may be enhanced by exploiting natural cell-matrix biology to regulate cell attachment and function.

Flow cytometric detection of endothelial microparticles (EMP): effects of centrifugation and storage alter with the phenotype studied.

PubMed

van Ierssel, Sabrina H; Van Craenenbroeck, Emeline M; Conraads, Viviane M; Van Tendeloo, Viggo F; Vrints, Christiaan J; Jorens, Philippe G; Hoymans, Vicky Y

2010-04-01

Endothelial microparticles (EMP) are released into the circulation in case of endothelial disturbance, and are therefore increasingly investigated as a biomarker reflecting disease activity. Numerous pre-analytic methods have been proposed for their flow cytometric enumeration, but standardization is still lacking. In this study we evaluated the influence of centrifugation and storage conditions on EMP quantification. Platelet-poor plasma (PPP) from 10 healthy volunteers was prepared by centrifugation at 1,550 g for 20 minutes twice. A first aliquot of PPP was analyzed immediately, a second after storage at 4 degrees C for 7 hours. A third and fourth aliquot were snap-frozen and stored at -80 degrees C for 7 and 28 days. A final aliquot was further centrifuged at 10,000g for 10 minutes and analyzed immediately. EMP were defined as CD31+CD42b-, CD62E+, CD144+ or CD144+CD105+ particles, smaller than 1.0 microm. High speed centrifugation led to a significant loss of CD31+CD42b- EMP (p=0.004). A good correlation between PPP and high speed centrifuged PPP was only found for CD144+ EMP (Kendall tau b=0.611, p=0.025). Storage at 4 degrees C did not affect EMP quantification. However, freezing at -80 degrees C increased CD31+CD42b- and CD62E+ EMP counts, and lowered CD144+ EMP (p<0.05). Nevertheless, the agreement among the different storage conditions was relatively good (Kendall coefficient of concordance >0.487; p<0.05). The flow cytometric detection of EMP varies with the centrifugation protocol and the storage method used, and these changes also depend on the phenotype studied. The results of this study caution against comparing study results gathered with different EMP laboratory protocols. (c) 2010 Elsevier Ltd. All rights reserved.

Visit-to-visit and 24-h blood pressure variability: association with endothelial and smooth muscle function in African Americans.

PubMed

Diaz, K M; Veerabhadrappa, P; Kashem, M A; Thakkar, S R; Feairheller, D L; Sturgeon, K M; Ling, C; Williamson, S T; Kretzschmar, J; Lee, H; Grimm, H; Babbitt, D M; Vin, C; Fan, X; Crabbe, D L; Brown, M D

2013-11-01

The purpose of this study was to investigate the association of visit-to-visit and 24-h blood pressure (BP) variability with markers of endothelial injury and vascular function. We recruited 72 African Americans who were non-diabetic, non-smoking and free of cardiovascular (CV) and renal disease. Office BP was measured at three visits and 24-h ambulatory BP monitoring was conducted to measure visit-to-visit and 24-h BP variability, respectively. The 5-min time-course of brachial artery flow-mediated dilation and nitroglycerin-mediated dilation were assessed as measures of endothelial and smooth muscle function. Fasted blood samples were analyzed for circulating endothelial microparticles (EMPs). Significantly lower CD31+CD42- EMPs were found in participants with high visit-to-visit systolic blood pressure (SBP) variability or high 24-h diastolic blood pressure (DBP) variability. Participants with high visit-to-visit DBP variability had significantly lower flow-mediated dilation and higher nitroglycerin-mediated dilation at multiple time-points. When analyzed as continuous variables, 24-h mean arterial pressure variability was inversely associated with CD62+ EMPs; visit-to-visit DBP variability was inversely associated with flow-mediated dilation normalized by smooth muscle function and was positively associated with nitroglycerin-mediated dilation; and 24-h DBP variability was positively associated with nitroglycerin-mediated dilation. All associations were independent of age, gender, body mass index and mean BP. In conclusion, in this cohort of African Americans visit-to-visit and 24-h BP variability were associated with measures of endothelial injury, endothelial function and smooth muscle function. These results suggest that BP variability may influence the pathogenesis of CV disease, in part, through influences on vascular health.

Extracellular signal-regulated kinase activation and endothelin-1 production in human endothelial cells exposed to vibration

PubMed Central

White, Charles R; Haidekker, Mark A; Stevens, Hazel Y; Frangos, John A

2004-01-01

Hand–arm vibration syndrome is a vascular disease of occupational origin and a form of secondary Raynaud's phenomenon. Chronic exposure to hand-held vibrating tools may cause endothelial injury. This study investigates the biomechanical forces involved in the transduction of fluid vibration in the endothelium. Human endothelial cells were exposed to direct vibration and rapid low-volume fluid oscillation. Rapid low-volume fluid oscillation was used to simulate the effects of vibration by generating defined temporal gradients in fluid shear stress across an endothelial monolayer. Extracellular signal-regulated kinase (ERK1/2) phosphorylation and endothelin-1 (ET-1) release were monitored as specific biochemical markers for temporal gradients and endothelial response, respectively. Both vibrational methods were found to phosphorylate ERK1/2 in a similar pattern. At a fixed frequency of fluid oscillation where the duration of each pulse cycle remained constant, ERK1/2 phosphorylation increased with the increasing magnitude of the applied temporal gradient. However, when the frequency of flow oscillation was increased (thus decreasing the duration of each pulse cycle), ERK1/2 phosphorylation was attenuated across all temporal gradient flow profiles. Fluid oscillation significantly stimulated ET-1 release compared to steady flow, and endothelin-1 was also attenuated with the increase in oscillation frequency. Taken together, these results show that both the absolute magnitude of the temporal gradient and the frequency/duration of each pulse cycle play a role in the biomechanical transduction of fluid vibrational forces in endothelial cells. Furthermore, this study reports for the first time a link between the ERK1/2 signal transduction pathway and transmission of vibrational forces in the endothelium. PMID:14724194

Endotoxin induces fibrosis in vascular endothelial cells through a mechanism dependent on transient receptor protein melastatin 7 activity.

PubMed

Echeverría, Cesar; Montorfano, Ignacio; Hermosilla, Tamara; Armisén, Ricardo; Velásquez, Luis A; Cabello-Verrugio, Claudio; Varela, Diego; Simon, Felipe

2014-01-01

The pathogenesis of systemic inflammatory diseases, including endotoxemia-derived sepsis syndrome, is characterized by endothelial dysfunction. It has been demonstrated that the endotoxin lipopolysaccharide (LPS) induces the conversion of endothelial cells (ECs) into activated fibroblasts through endothelial-to-mesenchymal transition mechanism. Fibrogenesis is highly dependent on intracellular Ca2+ concentration increases through the participation of calcium channels. However, the specific molecular identity of the calcium channel that mediates the Ca2+ influx during endotoxin-induced endothelial fibrosis is still unknown. Transient receptor potential melastatin 7 (TRPM7) is a calcium channel that is expressed in many cell types, including ECs. TRPM7 is involved in a number of crucial processes such as the conversion of fibroblasts into activated fibroblasts, or myofibroblasts, being responsible for the development of several characteristics of them. However, the role of the TRPM7 ion channel in endotoxin-induced endothelial fibrosis is unknown. Thus, our aim was to study whether the TRPM7 calcium channel participates in endotoxin-induced endothelial fibrosis. Using primary cultures of ECs, we demonstrated that TRPM7 is a crucial protein involved in endotoxin-induced endothelial fibrosis. Suppression of TRPM7 expression protected ECs from the fibrogenic process stimulated by endotoxin. Downregulation of TRPM7 prevented the endotoxin-induced endothelial markers decrease and fibrotic genes increase in ECs. In addition, TRPM7 downregulation abolished the endotoxin-induced increase in ECM proteins in ECs. Furthermore, we showed that intracellular Ca2+ levels were greatly increased upon LPS challenge in a mechanism dependent on TRPM7 expression. These results demonstrate that TRPM7 is a key protein involved in the mechanism underlying endotoxin-induced endothelial fibrosis.

Plasma levels of endothelial and B-cell-derived microparticles are restored by fingolimod treatment in multiple sclerosis patients.

PubMed

Zinger, Anna; Latham, Sharissa L; Combes, Valery; Byrne, Scott; Barnett, Michael H; Hawke, Simon; Grau, Georges E

2016-12-01

No molecular marker can monitor disease progression and treatment efficacy in multiple sclerosis (MS). Circulating microparticles represent a potential snapshot of disease activity at the blood brain barrier. To profile plasma microparticles by flow cytometry in MS and determine how fingolimod could impact endothelial microparticles production. In non-treated MS patients compared to healthy and fingolimod-treated patients, endothelial microparticles were higher, while B-cell-microparticle numbers were lower. Fingolimod dramatically reduced tumour necrosis factor (TNF)-induced endothelial microparticle release in vitro. Fingolimod restored dysregulated endothelial and B-cell-microparticle numbers, which could serve as a biomarker in MS. © The Author(s), 2016.

Endothelial miR-17∼92 cluster negatively regulates arteriogenesis via miRNA-19 repression of WNT signaling.

PubMed

Landskroner-Eiger, Shira; Qiu, Cong; Perrotta, Paola; Siragusa, Mauro; Lee, Monica Y; Ulrich, Victoria; Luciano, Amelia K; Zhuang, Zhen W; Corti, Federico; Simons, Michael; Montgomery, Rusty L; Wu, Dianqing; Yu, Jun; Sessa, William C

2015-10-13

The contribution of endothelial-derived miR-17∼92 to ischemia-induced arteriogenesis has not been investigated in an in vivo model. In the present study, we demonstrate a critical role for the endothelial-derived miR-17∼92 cluster in shaping physiological and ischemia-triggered arteriogenesis. Endothelial-specific deletion of miR-17∼92 results in an increase in collateral density limbs and hearts and in ischemic limbs compared with control mice, and consequently improves blood flow recovery. Individual cluster components positively or negatively regulate endothelial cell (EC) functions in vitro, and, remarkably, ECs lacking the cluster spontaneously form cords in a manner rescued by miR-17a, -18a, and -19a. Using both in vitro and in vivo analyses, we identified FZD4 and LRP6 as targets of miR-19a/b. Both of these targets were up-regulated in 17∼92 KO ECs compared with control ECs, and both were shown to be targeted by miR-19 using luciferase assays. We demonstrate that miR-19a negatively regulates FZD4, its coreceptor LRP6, and WNT signaling, and that antagonism of miR-19a/b in aged mice improves blood flow recovery after ischemia and reduces repression of these targets. Collectively, these data provide insights into miRNA regulation of arterialization and highlight the importance of vascular WNT signaling in maintaining arterial blood flow.

Mechanism study of endothelial protection and inhibits platelet activation of low molecular weight fucoidan from Laminaria japonica

NASA Astrophysics Data System (ADS)

Chen, Anjin; Zhang, Fang; Shi, Jie; Zhao, Xue; Yan, Meixing

2016-10-01

Several studies have indicated that fucoidan fractions with low molecular weight and different sulfate content from Laminaria japonica could inhibit the activation of platelets directly by reducing the platelet aggregation. To explore the direct effect of LMW fucoidan on the platelet system furthermore and examine the possible mechanism, the endothelial protection and inhibits platelet activation effects of two LMW fucoidan were investigated. In the present study, Endothelial injury model of rats was made by injection of adrenaline (0.4 mg kg-1) and human umbilical vein endothelial cells were cultured. vWF level was be investigated in vivo and in vitro as an important index of endothelial injury. LMW fucoidan could significantly reduce vWF level in vascular endothelial injury rats and also significantly reduce vWF level in vitro. The number of EMPs was be detected as another important index of endothelial injury. The results showed that LMW fucoidan reduced EMPs stimulated by tumor necrosis factor. In this study, it was found that by inhibiting platelet adhesion, LMW fucoidan played a role in anti-thrombosis and the specific mechanism of action is to inhibit the flow of extracellular Ca2+. All in a word, LMW fucoidan could inhibit the activation of platelets indirectly by reducing the concentration of EMPs and vWF, at the same time; LMW fucoidan inhibited the activation of platelets directly by inhibiting the flow of extracellular Ca2+.

Endothelial microparticles released by activated protein C protect beta cells through EPCR/PAR1 and annexin A1/FPR2 pathways in islets.

PubMed

Kreutter, Guillaume; Kassem, Mohamad; El Habhab, Ali; Baltzinger, Philippe; Abbas, Malak; Boisrame-Helms, Julie; Amoura, Lamia; Peluso, Jean; Yver, Blandine; Fatiha, Zobairi; Ubeaud-Sequier, Geneviève; Kessler, Laurence; Toti, Florence

2017-11-01

Islet transplantation is associated with early ischaemia/reperfusion, localized coagulation and redox-sensitive endothelial dysfunction. In animal models, islet cytoprotection by activated protein C (aPC) restores islet vascularization and protects graft function, suggesting that aPC triggers various lineages. aPC also prompts the release of endothelial MP that bear EPCR, its specific receptor. Microparticles (MP) are plasma membrane procoagulant vesicles, surrogate markers of stress and cellular effectors. We measured the cytoprotective effects of aPC on endothelial and insulin-secreting Rin-m5f β-cells and its role in autocrine and paracrine MP-mediated cell crosstalk under conditions of oxidative stress. MP from aPC-treated primary endothelial (EC) or β-cells were applied to H 2 O 2 -treated Rin-m5f. aPC activity was measured by enzymatic assay and ROS species by dihydroethidium. The capture of PKH26-stained MP and the expression of EPCR were probed by fluorescence microscopy and apoptosis by flow cytometry. aPC treatment enhanced both annexin A1 (ANXA1) and PAR-1 expression in EC and to a lesser extent in β-cells. MP from aPC-treated EC (eM aPC ) exhibited high EPCR and annexin A1 content, protected β-cells, restored insulin secretion and were captured by 80% of β cells in a phosphatidylserine and ANXA1-dependent mechanism. eMP activated EPCR/PAR-1 and ANXA1/FPR2-dependent pathways and up-regulated the expression of EPCR, and of FPR2/ALX, the ANXA1 receptor. Cytoprotection was confirmed in H 2 O 2 -treated rat islets with increased viability (62% versus 48% H 2 O 2 ), reduced apoptosis and preserved insulin secretion in response to glucose elevation (16 versus 5 ng/ml insulin per 10 islets). MP may prove a promising therapeutic tool in the protection of transplanted islets. © 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

The effect of 48 weeks of aerobic exercise training on cutaneous vasodilator function in post-menopausal females.

PubMed

Hodges, Gary J; Sharp, Lisa; Stephenson, Claire; Patwala, Ashish Y; George, Keith P; Goldspink, David F; Tim Cable, N

2010-04-01

Skin blood flow (SkBF) and endothelial-dependent vasodilatation decline with ageing and can be reversed with exercise training. We tested whether 48 weeks of training could improve SkBF and endothelial function in post-menopausal females; 20 post-menopausal subjects completed the study. SkBF was measured by laser-Doppler flowmetry (LDF). Cutaneous vascular conductance (CVC) was calculated as LDF/blood pressure. Resting CVC was measured at 32 degrees C and peak CVC at 42 degrees C. Cutaneous endothelial-dependent and -independent vasodilatations were determined by the iontophoresis of acetylcholine (ACh) and sodium nitroprusside (SNP), respectively. All assessments described were performed at entry (week 0), and after 6, 12, 24, 36, and 48 weeks of training. Resting CVC measures did not change (P > 0.05) throughout the study. Peak CVC increased (P < 0.05) after 24 weeks (7.2 +/- 1.2 vs. 11.6 +/- 1.4 AU mmHg(-1)) and at the 36- and 48-week assessments (13.0 +/- 1.7 and 14.9 +/- 2.1 AU mmHg(-1), respectively). Responses to ACh also increased (P < 0.05) at the 24-week assessment (5.1 +/- 2.1 vs. 8.55 +/- 2.3 AU mmHg(-1)) and increased further at the 36 and 48-week assessments (11.6 +/- 3.7 and 13.2 +/- 3.9 AU mmHg(-1), respectively). Cutaneous responses to SNP increased (P < 0.05) after 36 weeks (8.7 +/- 2.1 vs. 13.02 +/- 2.23 AU mmHg(-1) at 36 weeks). VO(2max) increased after 12 weeks (23.5 +/- 0.7 vs. 25.4 +/- 0.9 ml kg(-1) min(-1)) and improved (P < 0.05) further throughout the study (31.6 +/- 1.8 ml kg(-1) min(-1) at week 48). Aerobic exercise produces positive adaptations in the cutaneous vasodilator function to local heating as well as in cutaneous endothelial and endothelial-independent vasodilator mechanisms. Aerobic capacity was also significantly improved. These adaptations were further enhanced with progressive increases in exercise intensity.

Gata4-Dependent Differentiation of c-Kit+ Derived Endothelial Cells Underlies Artefactual Cardiomyocyte Regeneration in the Heart.

PubMed

Maliken, Bryan D; Kanisicak, Onur; Karch, Jason; Khalil, Hadi; Fu, Xing; Boyer, Justin G; Prasad, Vikram; Zheng, Yi; Molkentin, Jeffery D

2018-04-17

Background -While c-Kit + adult progenitor cells were initially reported to produce new cardiomyocytes in the heart, recent genetic evidence suggests that such events are exceedingly rare. However, to determine if these rare events represent true de novo cardiomyocyte formation we deleted the necessary cardiogenic transcription factors Gata4 and Gata6 from c-Kit-expressing cardiac progenitor cells (CPCs). Methods - Kit allele-dependent lineage tracing and fusion analysis was performed in mice following simultaneous Gata4 and Gata6 cell-type specific deletion to examine rates of putative de novo cardiomyocyte formation from c-Kit + cells. Bone marrow transplantation experiments were used to define the contribution of Kit allele-derived hematopoietic cells versus Kit lineage-dependent cells endogenous to the heart in contributing to apparent de novo lineage-traced cardiomyocytes. A Tie2 CreERT2 transgene was also used to examine the global impact of Gata4 deletion on the mature cardiac endothelial cell network, which was further evaluated with select angiogenesis assays. Results -Deletion of Gata4 in Kit lineage-derived endothelial cells or in total endothelial cells using the Tie2 CreERT2 transgene, but not from bone morrow cells, resulted in profound endothelial cell expansion, defective endothelial cell differentiation, leukocyte infiltration into the heart and a dramatic increase in Kit allele-dependent lineage-traced cardiomyocytes. However, this increase in labeled cardiomyocytes was an artefact of greater leukocyte-cardiomyocyte cellular fusion due to defective endothelial cell differentiation in the absence of Gata4 Conclusions -Past identification of presumed de novo cardiomyocyte formation in the heart from c-Kit + cells using Kit allele lineage tracing appears to be an artefact of labeled leukocyte fusion with cardiomyocytes. Deletion of Gata4 from c-Kit + endothelial progenitor cells or adult endothelial cells negatively impacted angiogenesis and capillary network integrity.

Monoamine oxidases are mediators of endothelial dysfunction in the mouse aorta.

PubMed

Sturza, Adrian; Leisegang, Matthias S; Babelova, Andrea; Schröder, Katrin; Benkhoff, Sebastian; Loot, Annemarieke E; Fleming, Ingrid; Schulz, Rainer; Muntean, Danina M; Brandes, Ralf P

2013-07-01

Monoamine oxidases (MAOs) generate H(2)O(2) as a by-product of their catalytic cycle. Whether MAOs are mediators of endothelial dysfunction is unknown and was determined here in the angiotensin II and lipopolysaccharide-models of vascular dysfunction in mice. Quantitative real-time polymerase chain reaction revealed that mouse aortas contain enzymes involved in catecholamine generation and MAO-A and MAO-B mRNA. MAO-A and -B proteins could be detected by Western blot not only in mouse aortas but also in human umbilical vein endothelial cells. Ex vivo incubation of mouse aorta with recombinant MAO-A increased H(2)O(2) formation and induced endothelial dysfunction that was attenuated by polyethylene glycol-catalase and MAO inhibitors. In vivo lipopolysaccharide (8 mg/kg IP overnight) or angiotensin II (1 mg/kg per day, 2 weeks, minipump) treatment induced vascular MAO-A and -B expressions and resulted in attenuated endothelium-dependent relaxation of the aorta in response to acetylcholine. MAO inhibitors reduced the lipopolysaccharide- and angiotensin II-induced aortic reactive oxygen species formation by 50% (ferrous oxidation xylenol orange assay) and partially normalized endothelium-dependent relaxation. MAO-A and MAO-B inhibitors had an additive effect; combined application completely restored endothelium-dependent relaxation. To determine how MAO-dependent H(2)O(2) formation induces endothelial dysfunction, cyclic GMP was measured. Histamine stimulation of human umbilical vein endothelial cells to activate endothelial NO synthase resulted in an increase in cyclic GMP, which was almost abrogated by MAO-A exposure. MAO inhibition prevented this effect, suggesting that MAO-induced H(2)O(2) formation is sufficient to attenuate endothelial NO release. Thus, MAO-A and MAO-B are both expressed in the mouse aorta, induced by in vivo lipopolysaccharide and angiotensin II treatment and contribute via the generation of H(2)O(2) to endothelial dysfunction in vascular disease models.

Ischemia-reperfusion injury in the isolated rat lung. Role of flow and endogenous leukocytes.

PubMed

Seibert, A F; Haynes, J; Taylor, A

1993-02-01

Microvascular lung injury caused by ischemia-reperfusion (IR) may occur via leukocyte-dependent and leukocyte-independent pathways. Leukocyte-endothelial adhesion may be a rate-limiting step in IR lung injury. Leukocyte adhesion to microvascular endothelium occurs when the attractant forces between leukocyte and endothelium are greater than the kinetic energy of the leukocyte and the vascular wall shear rate. We hypothesized (1) that isolated, buffer-perfused rat lungs are not free of endogenous leukocytes, (2) that endogenous leukocytes contribute to IR-induced microvascular injury as measured by the capillary filtration coefficient (Kfc), and (3) that a reduction of perfusate flow rate would potentiate leukocyte-dependent IR injury. Sixty lungs were divided into four groups: (1) low-flow controls, (2) high-flow controls, (3) low-flow IR, and (4) high-flow IR. Microvascular injury was linearly related to baseline perfusate leukocyte concentrations at both low (r = 0.78) and high (r = 0.82) flow rates. Kfc in the high-flow IR group (0.58 +/- 0.03 ml/min/cm H2O/100 g) was less (p < 0.05) than Kfc in the low-flow IR group (0.82 +/- 0.07), and in both groups Kfc values were significantly greater than low-flow (0.34 +/- 0.03) and high-flow (0.31 +/- 0.01) control Kfc values after 75 min. Retention of leukocytes in the lung, evaluated by a tissue myeloperoxidase assay, was greatest in the low-flow IR group. We conclude (1) that isolated, buffer-perfused rat lungs contain significant quantities of leukocytes and that these leukocytes contribute to IR lung injury, and (2) that IR-induced microvascular injury is potentiated by low flow.

Targeting Endothelial Cells with Multifunctional GaN/Fe Nanoparticles

NASA Astrophysics Data System (ADS)

Braniste, Tudor; Tiginyanu, Ion; Horvath, Tibor; Raevschi, Simion; Andrée, Birgit; Cebotari, Serghei; Boyle, Erin C.; Haverich, Axel; Hilfiker, Andres

2017-08-01

In this paper, we report on the interaction of multifunctional nanoparticles with living endothelial cells. The nanoparticles were synthesized using direct growth of gallium nitride on zinc oxide nanoparticles alloyed with iron oxide followed by core decomposition in hydrogen flow at high temperature. Using transmission electron microscopy, we demonstrate that porcine aortic endothelial cells take up GaN-based nanoparticles suspended in the growth medium. The nanoparticles are deposited in vesicles and the endothelial cells show no sign of cellular damage. Intracellular inert nanoparticles are used as guiding elements for controlled transportation or designed spatial distribution of cells in external magnetic fields.

Regimes of Flow over Complex Structures of Endothelial Glycocalyx: A Molecular Dynamics Simulation Study.

PubMed

Jiang, Xi Zhuo; Feng, Muye; Ventikos, Yiannis; Luo, Kai H

2018-04-10

Flow patterns on surfaces grafted with complex structures play a pivotal role in many engineering and biomedical applications. In this research, large-scale molecular dynamics (MD) simulations are conducted to study the flow over complex surface structures of an endothelial glycocalyx layer. A detailed structure of glycocalyx has been adopted and the flow/glycocalyx system comprises about 5,800,000 atoms. Four cases involving varying external forces and modified glycocalyx configurations are constructed to reveal intricate fluid behaviour. Flow profiles including temporal evolutions and spatial distributions of velocity are illustrated. Moreover, streamline length and vorticity distributions under the four scenarios are compared and discussed to elucidate the effects of external forces and glycocalyx configurations on flow patterns. Results show that sugar chain configurations affect streamline length distributions but their impact on vorticity distributions is statistically insignificant, whilst the influence of the external forces on both streamline length and vorticity distributions are trivial. Finally, a regime diagram for flow over complex surface structures is proposed to categorise flow patterns.

Dynamic regulation of canonical TGFβ signalling by endothelial transcription factor ERG protects from liver fibrogenesis.

PubMed

Dufton, Neil P; Peghaire, Claire R; Osuna-Almagro, Lourdes; Raimondi, Claudio; Kalna, Viktoria; Chuahan, Abhishek; Webb, Gwilym; Yang, Youwen; Birdsey, Graeme M; Lalor, Patricia; Mason, Justin C; Adams, David H; Randi, Anna M

2017-10-12

The role of the endothelium in protecting from chronic liver disease and TGFβ-mediated fibrosis remains unclear. Here we describe how the endothelial transcription factor ETS-related gene (ERG) promotes liver homoeostasis by controlling canonical TGFβ-SMAD signalling, driving the SMAD1 pathway while repressing SMAD3 activity. Molecular analysis shows that ERG binds to SMAD3, restricting its access to DNA. Ablation of ERG expression results in endothelial-to-mesenchymal transition (EndMT) and spontaneous liver fibrogenesis in EC-specific constitutive hemi-deficient (Erg cEC-Het ) and inducible homozygous deficient mice (Erg iEC-KO ), in a SMAD3-dependent manner. Acute administration of the TNF-α inhibitor etanercept inhibits carbon tetrachloride (CCL 4 )-induced fibrogenesis in an ERG-dependent manner in mice. Decreased ERG expression also correlates with EndMT in tissues from patients with end-stage liver fibrosis. These studies identify a pathogenic mechanism where loss of ERG causes endothelial-dependent liver fibrogenesis via regulation of SMAD2/3. Moreover, ERG represents a promising candidate biomarker for assessing EndMT in liver disease.The transcription factor ERG is key to endothelial lineage specification and vascular homeostasis. Here the authors show that ERG balances TGFβ signalling through the SMAD1 and SMAD3 pathways, protecting the endothelium from endothelial-to-mesenchymal transition and consequent liver fibrosis in mice via a SMAD3-dependent mechanism.

FGF-dependent metabolic control of vascular development

PubMed Central

Yu, Pengchun; Alves, Tiago C.; Fang, Jennifer S.; Xie, Yi; Zhu, Jie; Chen, Zehua; De Smet, Frederik; Zhang, Jiasheng; Jin, Suk-Won; Sun, Lele; Sun, Hongye; Kibbey, Richard G.; Hirschi, Karen K.; Hay, Nissim; Carmeliet, Peter; Chittenden, Thomas W.; Eichmann, Anne; Potente, Michael; Simons, Michael

2017-01-01

Blood and lymphatic vasculatures are intimately involved in tissue oxygenation and fluid homeostasis maintenance. Assembly of these vascular networks involves sprouting, migration and proliferation of endothelial cells. Recent studies have suggested that changes in cellular metabolism are of importance to these processes1. While much is known about vascular endothelial growth factor (VEGF)-dependent regulation of vascular development and metabolism2,3, little is understood about the role of fibroblast growth factors (FGFs) in this context4. Here we identify FGF receptor (FGFR) signaling as a critical regulator of vascular development. This is achieved by FGF-dependent control of c-MYC (MYC) expression that, in turn, regulates expression of the glycolytic enzyme hexokinase 2 (HK2). A decrease in HK2 levels in the absence of FGF signaling inputs results in decreased glycolysis leading to impaired endothelial cell proliferation and migration. Pan-endothelial- and lymphatic-specific Hk2 knockouts phenocopy blood and/or lymphatic vascular defects seen in Fgfr1/r3 double mutant mice while HK2 overexpression partially rescues the defects caused by suppression of FGF signaling. Thus, FGF-dependent regulation of endothelial glycolysis is a pivotal process in developmental and adult vascular growth and development. PMID:28467822

Electrophysiological and mechanical effects of substance P and acetylcholine on rabbit aorta.

PubMed Central

Bény, J L; Brunet, P C

1988-01-01

1. The mechanical and electrical properties of smooth muscle cells of the rabbit aorta were recorded simultaneously using respectively a force transducer and a 3 M-KCl-filled glass microelectrode. 2. Acetylcholine had two effects depending on concentration. At low concentration, it caused a persistent endothelium-dependent relaxation and hyperpolarization. At higher concentrations the acetylcholine endothelium-dependent relaxation summed with an endothelium-independent contraction. 3. Substance P caused a transient endothelium-dependent relaxation and hyperpolarization. 4. Acetylcholine and substance P depolarized and contracted de-endothelialized smooth muscle. When the de-endothelialized strip was pre-contracted by noradrenaline, acetylcholine depolarized the muscle but substance P did not. 5. In a 'cascade' experiment, the perfusate from an upstream intact aorta passed over a downstream de-endothelialized strip. Acetylcholine and substance P relaxed the downstream strip showing that they released an endothelial humoral factor which relaxes smooth muscle. 6. The results suggest a constant release of a factor from the endothelial cells which hyperpolarizes the smooth muscle cells in the media. Activation of acetylcholine and substance P receptors on the endothelium accelerates the release of this factor and causes vasodilatation. PMID:2455799

FGF-dependent metabolic control of vascular development.

PubMed

Yu, Pengchun; Wilhelm, Kerstin; Dubrac, Alexandre; Tung, Joe K; Alves, Tiago C; Fang, Jennifer S; Xie, Yi; Zhu, Jie; Chen, Zehua; De Smet, Frederik; Zhang, Jiasheng; Jin, Suk-Won; Sun, Lele; Sun, Hongye; Kibbey, Richard G; Hirschi, Karen K; Hay, Nissim; Carmeliet, Peter; Chittenden, Thomas W; Eichmann, Anne; Potente, Michael; Simons, Michael

2017-05-11

Blood and lymphatic vasculatures are intimately involved in tissue oxygenation and fluid homeostasis maintenance. Assembly of these vascular networks involves sprouting, migration and proliferation of endothelial cells. Recent studies have suggested that changes in cellular metabolism are important to these processes. Although much is known about vascular endothelial growth factor (VEGF)-dependent regulation of vascular development and metabolism, little is understood about the role of fibroblast growth factors (FGFs) in this context. Here we identify FGF receptor (FGFR) signalling as a critical regulator of vascular development. This is achieved by FGF-dependent control of c-MYC (MYC) expression that, in turn, regulates expression of the glycolytic enzyme hexokinase 2 (HK2). A decrease in HK2 levels in the absence of FGF signalling inputs results in decreased glycolysis, leading to impaired endothelial cell proliferation and migration. Pan-endothelial- and lymphatic-specific Hk2 knockouts phenocopy blood and/or lymphatic vascular defects seen in Fgfr1/Fgfr3 double mutant mice, while HK2 overexpression partly rescues the defects caused by suppression of FGF signalling. Thus, FGF-dependent regulation of endothelial glycolysis is a pivotal process in developmental and adult vascular growth and development.

A standalone perfusion platform for drug testing and target validation in micro-vessel networks

PubMed Central

Zhang, Boyang; Peticone, Carlotta; Murthy, Shashi K.; Radisic, Milica

2013-01-01

Studying the effects of pharmacological agents on human endothelium includes the routine use of cell monolayers cultivated in multi-well plates. This configuration fails to recapitulate the complex architecture of vascular networks in vivo and does not capture the relationship between shear stress (i.e. flow) experienced by the cells and dose of the applied pharmacological agents. Microfluidic platforms have been applied extensively to create vascular systems in vitro; however, they rely on bulky external hardware to operate, which hinders the wide application of microfluidic chips by non-microfluidic experts. Here, we have developed a standalone perfusion platform where multiple devices were perfused at a time with a single miniaturized peristaltic pump. Using the platform, multiple micro-vessel networks, that contained three levels of branching structures, were created by culturing endothelial cells within circular micro-channel networks mimicking the geometrical configuration of natural blood vessels. To demonstrate the feasibility of our platform for drug testing and validation assays, a drug induced nitric oxide assay was performed on the engineered micro-vessel network using a panel of vaso-active drugs (acetylcholine, phenylephrine, atorvastatin, and sildenafil), showing both flow and drug dose dependent responses. The interactive effects between flow and drug dose for sildenafil could not be captured by a simple straight rectangular channel coated with endothelial cells, but it was captured in a more physiological branching circular network. A monocyte adhesion assay was also demonstrated with and without stimulation by an inflammatory cytokine, tumor necrosis factor-α. PMID:24404058

Propionyl-L-Carnitine Enhances Wound Healing and Counteracts Microvascular Endothelial Cell Dysfunction

PubMed Central

Scioli, Maria Giovanna; Lo Giudice, Pietro; Bielli, Alessandra; Tarallo, Valeria; De Rosa, Alfonso; De Falco, Sandro; Orlandi, Augusto

2015-01-01

Background Impaired wound healing represents a high cost for health care systems. Endothelial dysfunction characterizes dermal microangiopathy and contributes to delayed wound healing and chronic ulcers. Endothelial dysfunction impairs cutaneous microvascular blood flow by inducing an imbalance between vasorelaxation and vasoconstriction as a consequence of reduced nitric oxide (NO) production and the increase of oxidative stress and inflammation. Propionyl-L-carnitine (PLC) is a natural derivative of carnitine that has been reported to ameliorate post-ischemic blood flow recovery. Methods and Results We investigated the effects of PLC in rat skin flap and cutaneous wound healing. A daily oral PLC treatment improved skin flap viability and associated with reactive oxygen species (ROS) reduction, inducible nitric oxide synthase (iNOS) and NO up-regulation, accelerated wound healing and increased capillary density, likely favoring dermal angiogenesis by up-regulation for iNOS, vascular endothelial growth factor (VEGF), placental growth factor (PlGF) and reduction of NADPH-oxidase 4 (Nox4) expression. In serum-deprived human dermal microvascular endothelial cell cultures, PLC ameliorated endothelial dysfunction by increasing iNOS, PlGF, VEGF receptors 1 and 2 expression and NO level. In addition, PLC counteracted serum deprivation-induced impairment of mitochondrial β-oxidation, Nox4 and cellular adhesion molecule (CAM) expression, ROS generation and leukocyte adhesion. Moreover, dermal microvascular endothelial cell dysfunction was prevented by Nox4 inhibition. Interestingly, inhibition of β-oxidation counteracted the beneficial effects of PLC on oxidative stress and endothelial dysfunction. Conclusion PLC treatment improved rat skin flap viability, accelerated wound healing and dermal angiogenesis. The beneficial effects of PLC likely derived from improvement of mitochondrial β-oxidation and reduction of Nox4-mediated oxidative stress and endothelial dysfunction. Antioxidant therapy and pharmacological targeting of endothelial dysfunction may represent a promising tool for the treatment of delayed wound healing or chronic ulcers. PMID:26473356

Platelet-free shear flow assay facilitates analysis of shear-dependent functions of VWF and ADAMTS13.

PubMed

Kraus, Emma; Kraus, Kristina; Obser, Tobias; Oyen, Florian; Klemm, Ulrike; Schneppenheim, Reinhard; Brehm, Maria A

2014-12-01

The multimeric form of von Willebrand factor (VWF), is the largest soluble protein in mammals and exhibits a multidomain structure resulting in multiple functions. Upon agonist stimulation endothelial cells secrete VWF multimers from Weibel-Palade bodies into the blood stream where VWF plays an essential role in platelet-dependent primary hemostasis. Elongation of VWF strings on the cells' surface leads to accessibility of VWF binding sites for proteins, such as platelet membrane glycoprotein Ib. The prothrombotic strings are size-regulated by the metalloprotease ADAMTS13 by shear force-activated proteolytic cleavage. VWF string formation was induced by histamine stimulation of HUVEC cells under unidirectional shear flow and VWF strings were detected employing the VWF binding peptide of platelet glycoprotein Ib coupled to latex beads. VWF strings were then used as substrate for kinetic studies of recombinant and plasma ADAMTS13. To investigate specific aspects of the shear-dependent functions of VWF and ADAMTS13, we developed a shear flow assay that allows observation of VWF string formation and their degradation by ADAMTS13 without the need for isolated platelets. Our assay specifically detects VWF strings, can be coupled with fluorescent applications and allows semi-automated, quantitative assessment of recombinant and plasma ADAMTS13 activity. Our assay may serve as a valuable research tool to investigate the biochemical characteristics of VWF and ADAMTS13 under shear flow and could complement diagnostics of von Willebrand Disease and Thrombotic Thrombocytopenic Purpura as it allows detection of shear flow-dependent dysfunction of VWD-associated VWF mutants as well as TTP-associated ADAMTS13 mutants. Copyright © 2014 Elsevier Ltd. All rights reserved.

Hydrostatic pressure and shear stress affect endothelin-1 and nitric oxide release by endothelial cells in bioreactors.

PubMed

Vozzi, Federico; Bianchi, Francesca; Ahluwalia, Arti; Domenici, Claudio

2014-01-01

Abundant experimental evidence demonstrates that endothelial cells are sensitive to flow; however, the effect of fluid pressure or pressure gradients that are used to drive viscous flow is not well understood. There are two principal physical forces exerted on the blood vessel wall by the passage of intra-luminal blood: pressure and shear. To analyze the effects of pressure and shear independently, these two stresses were applied to cultured cells in two different types of bioreactors: a pressure-controlled bioreactor and a laminar flow bioreactor, in which controlled levels of pressure or shear stress, respectively, can be generated. Using these bioreactor systems, endothelin-1 (ET-1) and nitric oxide (NO) release from human umbilical vein endothelial cells were measured under various shear stress and pressure conditions. Compared to the controls, a decrease of ET-1 production by the cells cultured in both bioreactors was observed, whereas NO synthesis was up-regulated in cells under shear stress, but was not modulated by hydrostatic pressure. These results show that the two hemodynamic forces acting on blood vessels affect endothelial cell function in different ways, and that both should be considered when planning in vitro experiments in the presence of flow. Understanding the individual and synergic effects of the two forces could provide important insights into physiological and pathological processes involved in vascular remodeling and adaptation. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Hypothyroidism Is Associated With Coronary Endothelial Dysfunction in Women

PubMed Central

Sara, Jaskanwal D; Zhang, Ming; Gharib, Hossein; Lerman, Lilach O; Lerman, Amir

2015-01-01

Background Hypothyroidism is associated with an increased risk of coronary artery disease, beyond that which can be explained by its association with conventional cardiovascular risk factors. Coronary endothelial dysfunction precedes atherosclerosis, has been linked to adverse cardiovascular events, and may account for some of the increased risk in patients with hypothyroidism. The aim of this study was to determine whether there is an association between epicardial and microvascular coronary endothelial dysfunction and hypothyroidism. Methods and Results In 1388 patients (mean age 50.5 [12.3] years, 34% male) presenting with stable chest pain to Mayo Clinic, Rochester, MN for diagnostic coronary angiography, and who were found to have nonobstructive coronary artery disease (<40% stenosis), we invasively assessed coronary artery endothelial-dependent microvascular and epicardial function by evaluating changes in coronary blood flow (% Δ CBF Ach) and diameter (% Δ CAD Ach), respectively, in response to intracoronary infusions of acetylcholine. Patients were divided into 2 groups: hypothyroidism, defined as a documented history of hypothyroidism or a thyroid-stimulating hormone (TSH) >10.0 mU/mL, n=188, and euthyroidism, defined as an absence of a history of hypothyroidism in the clinical record and/or 0.3

The Relationship between Magnesium and Endothelial Function in End-Stage Renal Disease Patients on Hemodialysis.

PubMed

Lee, Shina; Ryu, Jung Hwa; Kim, Seung Jung; Ryu, Dong Ryeol; Kang, Duk Hee; Choi, Kyu Bok

2016-11-01

Chronic kidney disease (CKD) patients tend to have higher serum magnesium values than healthy population due to their positive balance of magnesium in kidney. Recent studies found that magnesium level is positively correlated with endothelial function. Therefore, this study was conducted to define the relationship between magnesium level and endothelial dysfunction in end stage renal disease (ESRD) patients on hemodialysis (HD). A total of 27 patients were included in this cross-sectional study. Iontophoresis with laser-Doppler flowmetry, flow mediated dilation (FMD), and carotid intima-media thickness were measured. Patients' average serum magnesium levels were measured over previous three months, including the examination month. Pearson's correlation coefficient analysis and multivariate regression model were used to define the association between magnesium and endothelial function. In the univariate analysis, higher magnesium levels were associated with better endothelium-dependent vasodilation (EDV) of the FMD in ESRD patients on HD (r=0.516, p=0.007). When the participants were divided into two groups according to the median magnesium level (3.47 mg/dL), there was a significant difference in EDV of FMD (less than 3.47 mg/dL, 2.8±1.7%; more than 3.47 mg/dL, 5.1±2.0%, p=0.004). In multivariate analysis, magnesium and albumin were identified as independent factors for FMD (β=1.794, p=0.030 for serum magnesium; β=3.642, p=0.012 for albumin). This study demonstrated that higher serum magnesium level may be associated with better endothelial function in ESRD patients on HD. In the future, a large, prospective study is needed to elucidate optimal range of serum magnesium levels in ESRD on HD patients.

Acid Sphingomyelinase-Derived Ceramide Regulates ICAM-1 Function during T Cell Transmigration across Brain Endothelial Cells.

PubMed

Lopes Pinheiro, Melissa A; Kroon, Jeffrey; Hoogenboezem, Mark; Geerts, Dirk; van Het Hof, Bert; van der Pol, Susanne M A; van Buul, Jaap D; de Vries, Helga E

2016-01-01

Multiple sclerosis (MS) is a chronic demyelinating disorder of the CNS characterized by immune cell infiltration across the brain vasculature into the brain, a process not yet fully understood. We previously demonstrated that the sphingolipid metabolism is altered in MS lesions. In particular, acid sphingomyelinase (ASM), a critical enzyme in the production of the bioactive lipid ceramide, is involved in the pathogenesis of MS; however, its role in the brain vasculature remains unknown. Transmigration of T lymphocytes is highly dependent on adhesion molecules in the vasculature such as intercellular adhesion molecule-1 (ICAM-1). In this article, we hypothesize that ASM controls T cell migration by regulating ICAM-1 function. To study the role of endothelial ASM in transmigration, we generated brain endothelial cells lacking ASM activity using a lentiviral shRNA approach. Interestingly, although ICAM-1 expression was increased in cells lacking ASM activity, we measured a significant decrease in T lymphocyte adhesion and consequently transmigration both in static and under flow conditions. As an underlying mechanism, we revealed that upon lack of endothelial ASM activity, the phosphorylation of ezrin was perturbed as well as the interaction between filamin and ICAM-1 upon ICAM-1 clustering. Functionally this resulted in reduced microvilli formation and impaired transendothelial migration of T cells. In conclusion, in this article, we show that ASM coordinates ICAM-1 function in brain endothelial cells by regulating its interaction with filamin and phosphorylation of ezrin. The understanding of these underlying mechanisms of T lymphocyte transmigration is of great value to develop new strategies against MS lesion formation. Copyright © 2015 by The American Association of Immunologists, Inc.

Evaluation of the in vitro and in vivo angiogenic effects of exendin-4

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kang, Hye-Min; Kang, Yujung; Chun, Hyung J.

2013-04-26

Highlights: •We investigated the effects of exendin-4 on the angiogenic process. •Exendin-4 increased migration, sprouting, and tube formation by HUVECs in in vitro. •Exendin-4 increased sprouts in aortic rings and induced new vessels in Matrigel in in vivo. •Exendin-4 may be of potential use for the treatment of vascular complications of diabetes. -- Abstract: Exendin-4, an analog of glucagon-like peptide (GLP)-1, has beneficial effects on cardiovascular disease induced by diabetes mellitus (DM). Recently, exendin-4 was reported to induce the proliferation of endothelial cells. However, its angiogenic effect on endothelial cells has not been clearly evaluated. Therefore, we investigated the effectsmore » of exendin-4 on the angiogenic process with respect to migration, sprouting, and neovascularization using in vitro and in vivo assays. Treatment with exendin-4 increased the migration of human umbilical vein endothelial cells (HUVECs) in in vitro scratch wound assays, as well as the number of lumenized vessels sprouting from HUVECs in in vitro 3D bead assays. These responses were abolished by co-treatment with exendin (9–39), a GLP-1 receptor antagonist, which suggests that exendin-4 regulates endothelial cell migration and tube formation in a GLP-1 receptor-dependent manner. In an ex vivo assay, treatment of aortic rings with exendin-4 increased the sprouting of endothelial cells. Exendin-4 also significantly increased the number of new vessels and induced blood flow in Matrigel plugs in in vivo assays. Our results provide clear evidence for the angiogenic effect of exendin-4 in in vitro and in vivo assays and provide a mechanism underlying the cardioprotective effects of exendin-4.« less

The Relationship between Magnesium and Endothelial Function in End-Stage Renal Disease Patients on Hemodialysis

PubMed Central

Lee, Shina; Ryu, Jung-Hwa; Kim, Seung-Jung; Ryu, Dong-Ryeol; Kang, Duk-Hee

2016-01-01

Purpose Chronic kidney disease (CKD) patients tend to have higher serum magnesium values than healthy population due to their positive balance of magnesium in kidney. Recent studies found that magnesium level is positively correlated with endothelial function. Therefore, this study was conducted to define the relationship between magnesium level and endothelial dysfunction in end stage renal disease (ESRD) patients on hemodialysis (HD). Materials and Methods A total of 27 patients were included in this cross-sectional study. Iontophoresis with laser-Doppler flowmetry, flow mediated dilation (FMD), and carotid intima-media thickness were measured. Patients' average serum magnesium levels were measured over previous three months, including the examination month. Pearson's correlation coefficient analysis and multivariate regression model were used to define the association between magnesium and endothelial function. Results In the univariate analysis, higher magnesium levels were associated with better endothelium-dependent vasodilation (EDV) of the FMD in ESRD patients on HD (r=0.516, p=0.007). When the participants were divided into two groups according to the median magnesium level (3.47 mg/dL), there was a significant difference in EDV of FMD (less than 3.47 mg/dL, 2.8±1.7%; more than 3.47 mg/dL, 5.1±2.0%, p=0.004). In multivariate analysis, magnesium and albumin were identified as independent factors for FMD (β=1.794, p=0.030 for serum magnesium; β=3.642, p=0.012 for albumin). Conclusion This study demonstrated that higher serum magnesium level may be associated with better endothelial function in ESRD patients on HD. In the future, a large, prospective study is needed to elucidate optimal range of serum magnesium levels in ESRD on HD patients. PMID:27593873

Is reversal of endothelial dysfunction by tea related to flavonoid metabolism?

PubMed

Hodgson, Jonathan M; Puddey, Ian B; Burke, Valerie; Croft, Kevin D

2006-01-01

Dietary flavonoids can improve endothelial function, but the response varies between individuals. Wide variability is also seen in flavonoid O-methylation, a major pathway of flavonoid metabolism. The O-methylation of flavonoids could alter activity, and thus influence any effect on endothelial function. The objective of the current analysis was to investigate whether variability in the endothelial function response to ingestion of tea, a rich source of flavonoids, is related to the degree of O-methylation of flavonoids. This relationship was investigated in two studies in which endothelium-dependent flow-mediated dilatation (FMD) of the brachial artery was assessed and urinary 4-O-methylgallic acid (4OMGA) excretion was used as a marker of the O-methylation of tea-derived flavonoids. In the first study, amongst participants consuming five cups of tea per day for 4 weeks, the degree of increase in 4OMGA excretion was inversely associated with the change in FMD responses (r -078, P=0.008). In the second study, there was a significant difference in the FMD responses to acute ingestion of three cups of tea between individuals with a low (median) 4OMGA response (1.94 (sem 0.79) % and -0.25 (sem 0.53) %, respectively; P=0.03). That is, any improvement in FMD following ingestion of tea may be enhanced in individuals who O-methylate less of the absorbed flavonoids. The present results are consistent with the suggestion that differences in flavonoid metabolism may influence their effect on endothelial function. Thus, differences in flavonoid metabolism could be related to the level of benefit of dietary flavonoids on the risk of CVD.

Post-challenge hyperglycaemia, nitric oxide production and endothelial dysfunction: the putative role of asymmetric dimethylarginine (ADMA).

PubMed

Siervo, M; Corander, M; Stranges, S; Bluck, L

2011-01-01

The endothelium is a thin layer of cells at the internal surface of blood vessels in continuous contact with the circulating fluids. The endothelial cells represent the primary barrier for the transport of glucose from the vascular conduits into the interstitial space. Insulin and nitric oxide have an important role in the regulation of glucose transport and metabolism. Hyperglycaemia is the main criteria for the diagnosis of diabetes and is responsible for the micro- and macro-vascular pathology seen in diabetic patients. Recent evidence suggests that post-challenge hyperglycaemia is a better predictor of cardiovascular risk than fasting glucose. Acute glucose elevations have been associated with a reduced endothelial-dependent flow mediated dilation indicating a decrease in nitric oxide production. Post-prandial hyperglycaemic peaks have been directly associated with increased intima media thickness in type 2 diabetic patients indicative of an increased atherosclerotic risk. The increase in intra-cellular glucose concentrations in the endothelial cells induces a hyper-generation of reactive oxygen species via the activation of different pathways (polyol-sorbitol, hexosamine, advanced glycated end products, activation of PKC, asymmetric dimethylarginine (ADMA)). These mechanisms influence the expression of genes and release of signalling and structural molecules involved in several functions (inflammation, angiogenesis, coagulation, vascular tone and permeability, cellular migration, nutrient metabolism). ADMA is considered as a biomarker of endothelial dysfunction and it has been associated with an increased risk of atherosclerosis and cardiovascular diseases. The increased generation of ADMA and reactive oxygen species in subjects with persistent hyperglycaemia could lead to an impairment of nitric oxide synthesis. Copyright © 2010 Elsevier B.V. All rights reserved.

A six-colour flow cytometric method for simultaneous detection of cell phenotype and apoptosis of circulating endothelial cells.

PubMed

Mariucci, S; Rovati, B; Chatzileontiadou, S; Bencardino, K; Manzoni, M; Delfanti, S; Danova, M

2009-01-01

Blood circulating endothelial cells (CECs), with their resting and activated subsets, (rCECs and aCECs) and circulating progenitors cells (CEPs) are two extremely rare cell populations that are important in tissue vascularization. Their number and function are modulated in diseases involving vascular injury, such as human tumours. Although a consensus on the phenotypic definition of endothelial cells, as well as on the optimal enumeration technique, is still lacking, the number of clinical studies based on assessment of these cells is rapidly expanding, as well as the analytical methods employed. The present study aimed to develop a rapid and sensitive flow cytometric method of quantifying and characterizing CECs (with both their subsets and the apoptotic fraction) and CEPs. We analysed peripheral blood samples from 21 subjects with a six-colour flow cytometric approach allowing detection of the cell phenotype of CECs and CEPs using a monoclonal antibodies panel and a dedicated gating strategy. Apoptotic CECs were detected with Annexin V and dead cells with 7-amino-actinomycin D staining. The described technique proved to be a new, reliable, tool increasing our knowledge of the biology of CECs and CEPs and can readily be applied in the study of many pathological conditions characterized by endothelial damage.

Is endothelial microvascular function equally impaired among patients with chronic Chagas and ischemic cardiomyopathy?

PubMed

Borges, Juliana Pereira; Mendes, Fernanda de Souza Nogueira Sardinha; Lopes, Gabriella de Oliveira; Sousa, Andréa Silvestre de; Mediano, Mauro Felippe Felix; Tibiriçá, Eduardo

2018-08-15

Chronic Chagas cardiomyopathy (CCC) and cardiomyopathies due to other etiologies involve differences in pathophysiological pathways that are still unclear. Systemic microvascular abnormalities are associated with the pathogenesis of ischemic heart disease. However, systemic microvascular endothelial function in CCC remains to be elucidated. Thus, we compared the microvascular endothelial function of patients presenting with CCC to those with ischemic cardiomyopathy disease. Microvascular reactivity was assessed in 21 patients with cardiomyopathy secondary to Chagas disease, 21 patients with cardiomyopathy secondary to ischemic disease and 21 healthy controls. Microvascular blood flow was assessed in the skin of the forearm using laser speckle contrast imaging coupled with iontophoresis of acetylcholine (ACh). Peak increase in forearm blood flow with ACh iontophoresis in relation to baseline was greater in healthy controls than in patients with heart disease (controls: 162.7 ± 58.4% vs. ischemic heart disease: 74.1 ± 48.3% and Chagas: 85.1 ± 68.1%; p < 0.0001). Patients with Chagas and ischemic cardiomyopathy presented similar ACh-induced changes from baseline in skin blood flow (p = 0.55). Endothelial microvascular function was equally impaired among patients with CCC and ischemic cardiomyopathy. Copyright © 2018 Elsevier B.V. All rights reserved.

The antiangiogenic activity of cleaved high molecular weight kininogen is mediated through binding to endothelial cell tropomyosin

PubMed Central

Zhang, Jing-Chuan; Doñate, Fernando; Qi, Xiaoping; Ziats, Nicholas P.; Juarez, Jose C.; Mazar, Andrew P.; Pang, Yuan-Ping; McCrae, Keith R.

2002-01-01

Conformationally altered proteins and protein fragments derived from the extracellular matrix and hemostatic system may function as naturally occurring angiogenesis inhibitors. One example of such a protein is cleaved high molecular weight kininogen (HKa). HKa inhibits angiogenesis by inducing apoptosis of proliferating endothelial cells, effects mediated largely by HKa domain 5. However, the mechanisms underlying the antiangiogenic activity of HKa have not been characterized, and its binding site on proliferating endothelial cells has not been defined. Here, we report that the induction of endothelial cell apoptosis by HKa, as well as the antiangiogenic activity of HKa in the chick chorioallantoic membrane, was inhibited completely by antitropomyosin monoclonal antibody TM-311. TM-311 also blocked the high-affinity Zn2+-dependent binding of HKa to both purified tropomyosin and proliferating endothelial cells. Confocal microscopic analysis of endothelial cells stained with monoclonal antibody TM-311, as well as biotin labeling of cell surface proteins on intact endothelial cells, revealed that tropomyosin exposure was enhanced on the surface of proliferating cells. These studies demonstrate that the antiangiogenic effects of HKa depend on high-affinity binding to endothelial cell tropomyosin. PMID:12196635

De Novo Lipogenesis Maintains Vascular Homeostasis through Endothelial Nitric-oxide Synthase (eNOS) Palmitoylation*♦

PubMed Central

Wei, Xiaochao; Schneider, Jochen G.; Shenouda, Sherene M.; Lee, Ada; Towler, Dwight A.; Chakravarthy, Manu V.; Vita, Joseph A.; Semenkovich, Clay F.

2011-01-01

Endothelial dysfunction leads to lethal vascular complications in diabetes and related metabolic disorders. Here, we demonstrate that de novo lipogenesis, an insulin-dependent process driven by the multifunctional enzyme fatty-acid synthase (FAS), maintains endothelial function by targeting endothelial nitric-oxide synthase (eNOS) to the plasma membrane. In mice with endothelial inactivation of FAS (FASTie mice), eNOS membrane content and activity were decreased. eNOS and FAS were physically associated; eNOS palmitoylation was decreased in FAS-deficient cells, and incorporation of labeled carbon into eNOS-associated palmitate was FAS-dependent. FASTie mice manifested a proinflammatory state reflected as increases in vascular permeability, endothelial inflammatory markers, leukocyte migration, and susceptibility to LPS-induced death that was reversed with an NO donor. FAS-deficient endothelial cells showed deficient migratory capacity, and angiogenesis was decreased in FASTie mice subjected to hindlimb ischemia. Insulin induced FAS in endothelial cells freshly isolated from humans, and eNOS palmitoylation was decreased in mice with insulin-deficient or insulin-resistant diabetes. Thus, disrupting eNOS bioavailability through impaired lipogenesis identifies a novel mechanism coordinating nutritional status and tissue repair that may contribute to diabetic vascular disease. PMID:21098489

What is the Efficiency of ATP Signaling from Erythrocytes to Regulate Distribution of O2 Supply within the Microvasculature?

PubMed Central

C.G., Ellis; S., Milkovich; D., Goldman

2012-01-01

Erythrocytes appear to be ideal sensors for regulating microvascular O2 supply since they release the potent vasodilator adenosine 5′-triphosphate (ATP) in an O2 saturation dependent manner. Whether erythrocytes play a significant role in regulating O2 supply in the complex environment of diffusional O2 exchange among capillaries, arterioles and venules, depends on the efficiency with which erythrocytes signal the vascular endothelium. If one assumes that the distribution of purinergic receptors is uniform throughout the microvasculature, then the most efficient site for signaling should occur in capillaries, where the erythrocyte membrane is in close proximity to the endothelium. ATP released from erythrocytes would diffuse a short distance to P2y receptors inducing an increase in blood flow possibly the result of endothelial hyperpolarization. We hypothesize that this hyperpolarization varies across the capillary bed dependent upon erythrocyte supply rate and the flux of O2 from these erythrocytes to support O2 metabolism. This would suggest that the capillary bed would be the most effective site for erythrocytes to communicate tissue oxygen needs. Electrically coupled endothelial cells conduct the integrated signal upstream where arterioles adjust vascular resistance, thus enabling ATP released from erythrocytes to regulate the magnitude and distribution of O2 supply to individual capillary networks. PMID:22587367

Protein kinase Cα phosphorylates a novel argininosuccinate synthase site at serine 328 during calcium-dependent stimulation of endothelial nitric-oxide synthase in vascular endothelial cells.

PubMed

Haines, Ricci J; Corbin, Karen D; Pendleton, Laura C; Eichler, Duane C

2012-07-27

Endothelial nitric-oxide synthase (eNOS) utilizes l-arginine as its principal substrate, converting it to l-citrulline and nitric oxide (NO). l-Citrulline is recycled to l-arginine by two enzymes, argininosuccinate synthase (AS) and argininosuccinate lyase, providing the substrate arginine for eNOS and NO production in endothelial cells. Together, these three enzymes, eNOS, AS, and argininosuccinate lyase, make up the citrulline-NO cycle. Although AS catalyzes the rate-limiting step in NO production, little is known about the regulation of AS in endothelial cells beyond the level of transcription. In this study, we showed that AS Ser-328 phosphorylation was coordinately regulated with eNOS Ser-1179 phosphorylation when bovine aortic endothelial cells were stimulated by either a calcium ionophore or thapsigargin to produce NO. Furthermore, using in vitro kinase assay, kinase inhibition studies, as well as protein kinase Cα (PKCα) knockdown experiments, we demonstrate that the calcium-dependent phosphorylation of AS Ser-328 is mediated by PKCα. Collectively, these findings suggest that phosphorylation of AS at Ser-328 is regulated in accordance with the calcium-dependent regulation of eNOS under conditions that promote NO production and are in keeping with the rate-limiting role of AS in the citrulline-NO cycle of vascular endothelial cells.

Dengue virus NS1 cytokine-independent vascular leak is dependent on endothelial glycocalyx components

PubMed Central

Beatty, P. Robert

2017-01-01

Dengue virus (DENV) is the most prevalent, medically important mosquito-borne virus. Disease ranges from uncomplicated dengue to life-threatening disease, characterized by endothelial dysfunction and vascular leakage. Previously, we demonstrated that DENV nonstructural protein 1 (NS1) induces endothelial hyperpermeability in a systemic mouse model and human pulmonary endothelial cells, where NS1 disrupts the endothelial glycocalyx-like layer. NS1 also triggers release of inflammatory cytokines from PBMCs via TLR4. Here, we examined the relative contributions of inflammatory mediators and endothelial cell-intrinsic pathways. In vivo, we demonstrated that DENV NS1 but not the closely-related West Nile virus NS1 triggers localized vascular leak in the dorsal dermis of wild-type C57BL/6 mice. In vitro, we showed that human dermal endothelial cells exposed to DENV NS1 do not produce inflammatory cytokines (TNF-α, IL-6, IL-8) and that blocking these cytokines does not affect DENV NS1-induced endothelial hyperpermeability. Further, we demonstrated that DENV NS1 induces vascular leak in TLR4- or TNF-α receptor-deficient mice at similar levels to wild-type animals. Finally, we blocked DENV NS1-induced vascular leak in vivo using inhibitors targeting molecules involved in glycocalyx disruption. Taken together, these data indicate that DENV NS1-induced endothelial cell-intrinsic vascular leak is independent of inflammatory cytokines but dependent on endothelial glycocalyx components. PMID:29121099

Human severe sepsis cytokine mixture increases β2-integrin-dependent polymorphonuclear leukocyte adhesion to cerebral microvascular endothelial cells in vitro.

PubMed

Blom, Chris; Deller, Brittany L; Fraser, Douglas D; Patterson, Eric K; Martin, Claudio M; Young, Bryan; Liaw, Patricia C; Yazdan-Ashoori, Payam; Ortiz, Angelica; Webb, Brian; Kilmer, Greg; Carter, David E; Cepinskas, Gediminas

2015-04-07

Sepsis-associated encephalopathy (SAE) is a state of acute brain dysfunction in response to a systemic infection. We propose that systemic inflammation during sepsis causes increased adhesion of leukocytes to the brain microvasculature, resulting in blood-brain barrier dysfunction. Thus, our objectives were to measure inflammatory analytes in plasma of severe sepsis patients to create an experimental cytokine mixture (CM), and to use this CM to investigate the activation and interactions of polymorphonuclear leukocytes (PMN) and human cerebrovascular endothelial cells (hCMEC/D3) in vitro. The concentrations of 41 inflammatory analytes were quantified in plasma obtained from 20 severe sepsis patients and 20 age- and sex-matched healthy controls employing an antibody microarray. Two CMs were prepared to mimic severe sepsis (SSCM) and control (CCM), and these CMs were then used for PMN and hCMEC/D3 stimulation in vitro. PMN adhesion to hCMEC/D3 was assessed under conditions of flow (shear stress 0.7 dyn/cm(2)). Eight inflammatory analytes elevated in plasma obtained from severe sepsis patients were used to prepare SSCM and CCM. Stimulation of PMN with SSCM led to a marked increase in PMN adhesion to hCMEC/D3, as compared to CCM. PMN adhesion was abolished with neutralizing antibodies to either β2 (CD18), αL/β2 (CD11α/CD18; LFA-1) or αM/β2 (CD11β/CD18; Mac-1) integrins. In addition, immune-neutralization of the endothelial (hCMEC/D3) cell adhesion molecule, ICAM-1 (CD54) also suppressed PMN adhesion. Human SSCM up-regulates PMN pro-adhesive phenotype and promotes PMN adhesion to cerebrovascular endothelial cells through a β2-integrin-ICAM-1-dependent mechanism. PMN adhesion to the brain microvasculature may contribute to SAE.

Glutathione adducts on sarcoplasmic/endoplasmic reticulum Ca2+ ATPase Cys-674 regulate endothelial cell calcium stores and angiogenic function as well as promote ischemic blood flow recovery.

PubMed

Thompson, Melissa D; Mei, Yu; Weisbrod, Robert M; Silver, Marcy; Shukla, Praphulla C; Bolotina, Victoria M; Cohen, Richard A; Tong, Xiaoyong

2014-07-18

The sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) is key to Ca(2+) homeostasis and is redox-regulated by reversible glutathione (GSH) adducts on the cysteine (C) 674 thiol that stimulate Ca(2+) uptake activity and endothelial cell angiogenic responses in vitro. We found that mouse hind limb muscle ischemia induced S-glutathione adducts on SERCA in both whole muscle tissue and endothelial cells. To determine the role of S-glutathiolation, we used a SERCA 2 C674S heterozygote knock-in (SKI) mouse lacking half the key thiol. Following hind limb ischemia, SKI animals had decreased SERCA S-glutathione adducts and impaired blood flow recovery. We studied SKI microvascular endothelial cells in which total SERCA 2 expression was unchanged. Cultured SKI microvascular endothelial cells showed impaired migration and network formation compared with wild type (WT). Ca(2+) studies showed decreased nitric oxide (·NO)-induced (45)Ca(2+) uptake into the endoplasmic reticulum (ER) of SKI cells, while Fura-2 studies revealed lower Ca(2+) stores and decreased vascular endothelial growth factor (VEGF)- and ·NO-induced Ca(2+) influx. Adenoviral overexpression of calreticulin, an ER Ca(2+) binding protein, increased ionomycin-releasable stores, VEGF-induced Ca(2+) influx and endothelial cell migration. Taken together, these data indicate that the redox-sensitive Cys-674 thiol on SERCA 2 is required for normal endothelial cell Ca(2+) homeostasis and ischemia-induced angiogenic responses, revealing a novel redox control of angiogenesis via Ca(2+) stores. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Citrus Polyphenol Hesperidin Stimulates Production of Nitric Oxide in Endothelial Cells while Improving Endothelial Function and Reducing Inflammatory Markers in Patients with Metabolic Syndrome

PubMed Central

Rizza, Stefano; Muniyappa, Ranganath; Iantorno, Micaela; Kim, Jeong-a; Chen, Hui; Pullikotil, Philomena; Senese, Nicoletta; Tesauro, Manfredi; Lauro, Davide; Cardillo, Carmine

2011-01-01

Context: Hesperidin, a citrus flavonoid, and its metabolite hesperetin may have vascular actions relevant to their health benefits. Molecular and physiological mechanisms of hesperetin actions are unknown. Objective: We tested whether hesperetin stimulates production of nitric oxide (NO) from vascular endothelium and evaluated endothelial function in subjects with metabolic syndrome on oral hesperidin therapy. Design, Setting, and Interventions: Cellular mechanisms of action of hesperetin were evaluated in bovine aortic endothelial cells (BAEC) in primary culture. A randomized, placebo-controlled, double-blind, crossover trial examined whether oral hesperidin administration (500 mg once daily for 3 wk) improves endothelial function in individuals with metabolic syndrome (n = 24). Main Outcome Measure: We measured the difference in brachial artery flow-mediated dilation between placebo and hesperidin treatment periods. Results: Treatment of BAEC with hesperetin acutely stimulated phosphorylation of Src, Akt, AMP kinase, and endothelial NO synthase to produce NO; this required generation of H2O2. Increased adhesion of monocytes to BAEC and expression of vascular cell adhesion molecule-1 in response to TNF-α treatment was reduced by pretreatment with hesperetin. In the clinical study, when compared with placebo, hesperidin treatment increased flow-mediated dilation (10.26 ± 1.19 vs. 7.78 ± 0.76%; P = 0.02) and reduced concentrations of circulating inflammatory biomarkers (high-sensitivity C-reactive protein, serum amyloid A protein, soluble E-selectin). Conclusions: Novel mechanisms for hesperetin action in endothelial cells inform effects of oral hesperidin treatment to improve endothelial dysfunction and reduce circulating markers of inflammation in our exploratory clinical trial. Hesperetin has vasculoprotective actions that may explain beneficial cardiovascular effects of citrus consumption. PMID:21346065

High glucose increases the formation and pro-oxidative activity of endothelial microparticles.

PubMed

Burger, Dylan; Turner, Maddison; Xiao, Fengxia; Munkonda, Mercedes N; Akbari, Shareef; Burns, Kevin D

2017-09-01

Individuals with diabetes exhibit increases in circulating endothelial microparticles (eMPs, also referred to as endothelial microvesicles), which are associated with endothelial dysfunction and a heightened risk of cardiovascular complications. We have shown that eMPs are markers and mediators of vascular injury although their role in diabetes is unclear. We hypothesised that the composition and biological activity of eMPs are altered in response to high glucose exposure. We assessed the effects of high glucose on eMP formation, composition and signalling in cultured HUVECs. eMPs were isolated from the media of HUVECs cultured under conditions of normal glucose (eMP NG ), high glucose (eMP HG ) or osmotic control of L-glucose (eMP LG ). eMP size, concentration and surface charge were assessed by nanoparticle tracking analysis and flow cytometry. eMP protein composition was assessed by liquid chromatography-tandem mass spectrometry, and eMP-mediated effects on coagulation, reactive oxygen species (ROS) production and vessel function were assessed. Exposure of HUVECs to high glucose for 24 h caused a threefold increase in eMP formation, increased mean particle size (269 ± 18 nm vs 226 ± 11 nm) and decreased surface charge. Compared with eMP NG or eMP LG , eMP HG possessed approximately threefold greater pro-coagulant activity, stimulated HUVEC ROS production to a greater extent (~250% of eMP NG ) and were more potent inhibitors of endothelial-dependent relaxation. Proteomic analysis of eMPs identified 1212 independent proteins of which 68 were exclusively found in eMP HG . Gene ontology analysis revealed that eMP HG -exclusive proteins were associated with signalling pathways related to blood coagulation, cell signalling and immune cell activation. Our results indicate that elevated glucose is a potent stimulus for eMP formation that also alters their molecular composition leading to increased bioactivity. Such effects may contribute to progressive endothelial injury and subsequent cardiovascular complications in diabetes.

Vascular endothelial growth factor c/vascular endothelial growth factor receptor 3 signaling regulates chemokine gradients and lymphocyte migration from tissues to lymphatics.

PubMed

Iwami, Daiki; Brinkman, C Colin; Bromberg, Jonathan S

2015-04-01

Circulation of leukocytes via blood, tissue and lymph is integral to adaptive immunity. Afferent lymphatics form CCL21 gradients to guide dendritic cells and T cells to lymphatics and then to draining lymph nodes (dLN). Vascular endothelial growth factor C and vascular endothelial growth factor receptor 3 (VEGFR-3) are the major lymphatic growth factor and receptor. We hypothesized these molecules also regulate chemokine gradients and lymphatic migration. CD4 T cells were injected into the foot pad or ear pinnae, and migration to afferent lymphatics and dLN quantified by flow cytometry or whole mount immunohistochemistry. Vascular endothelial growth factor receptor 3 or its signaling or downstream actions were modified with blocking monoclonal antibodies (mAbs) or other reagents. Anti-VEGFR-3 prevented migration of CD4 T cells into lymphatic lumen and significantly decreased the number that migrated to dLN. Anti-VEGFR-3 abolished CCL21 gradients around lymphatics, although CCL21 production was not inhibited. Heparan sulfate (HS), critical to establish CCL21 gradients, was down-regulated around lymphatics by anti-VEGFR-3 and this was dependent on heparanase-mediated degradation. Moreover, a Phosphoinositide 3-kinase (PI3K)α inhibitor disrupted HS and CCL21 gradients, whereas a PI3K activator prevented the effects of anti-VEGFR-3. During contact hypersensitivity, VEGFR-3, CCL21, and HS expression were all attenuated, and anti-heparanase or PI3K activator reversed these effects. Vascular endothelial growth factor C/VEGFR-3 signaling through PI3Kα regulates the activity of heparanase, which modifies HS and CCL21 gradients around lymphatics. The functional and physical linkages of these molecules regulate lymphatic migration from tissues to dLN. These represent new therapeutic targets to influence immunity and inflammation.

Impaired postprandial endothelial function depends on the type of fat consumed by healthy men.

PubMed

Berry, Sarah E E; Tucker, Sally; Banerji, Radhika; Jiang, Benyu; Chowienczyk, Phillip J; Charles, Sonia M; Sanders, Thomas A B

2008-10-01

Postprandial lipemia impairs endothelial function possibly via an oxidative stress mechanism. A stearic acid-rich triacylglycerol (TAG) (shea butter) results in a blunted postprandial increase in plasma TAG compared with an oleic acid-rich TAG; however, its acute effects on endothelial function and oxidative stress are unknown. A randomized crossover trial (n = 17 men) compared the effects of 50 g fat, rich in stearic acid [shea butter blend (SA)] or oleic acid [high oleic sunflower oil (HO)], on changes in endothelial function [brachial artery flow-mediated dilatation (FMD)], arterial tone [pulse wave analysis (PWA), and carotid-femoral pulse wave velocity (PWV(c-f))], and oxidative stress (plasma 8-isoprostane F2alpha) at fasting and 3 h following the test meals. The postprandial increase in plasma TAG was lower (66% lower incremental area under curve) following the SA meal [28.3 (9.7, 46.9)] than after the HO meal [83.4 (57.0, 109.8); P < 0.001] (geometric means with 95% CI, arbitary units). Following the HO meal, there was a decrease in FMD [-3.0% (-4.4, -1.6); P < 0.001] and an increase in plasma 8-isoprostane F2alpha [10.4ng/L (3.8, 16.9); P = 0.005] compared with fasting values, but no changes followed the SA meal. The changes in 8-isoprostane F2alpha and FMD differed between meals and were 14.0 ng/L (6.4, 21.6; P = 0.001) and 1.75% (0.10, 3.39; P = 0.02), respectively. The reductions in PWA and PWV c-f did not differ between meals. This study demonstrates that a stearic acid-rich fat attenuates the postprandial impairment in endothelial function compared with an oleic acid-rich fat and supports the hypothesis that postprandial lipemia impairs endothelial function via an increase in oxidative stress.

Adhesion behavior of endothelial progenitor cells to endothelial cells in simple shear flow

NASA Astrophysics Data System (ADS)

Gong, Xiao-Bo; Li, Yu-Qing; Gao, Quan-Chao; Cheng, Bin-Bin; Shen, Bao-Rong; Yan, Zhi-Qiang; Jiang, Zong-Lai

2011-12-01

The adhesion of endothelial progenitor cells (EPCs) on endothelial cells (ECs) is one of the critical physiological processes for the regenesis of vascular vessels and the prevention of serious cardiovascular diseases. Here, the rolling and adhesion behavior of EPCs on ECs was studied numerically. A two-dimensional numerical model was developed based on the immersed boundary method for simulating the rolling and adhesion of cells in a channel flow. The binding force arising from the catch bond of a receptor and ligand pair was modeled with stochastic Monte Carlo method and Hookean spring model. The effect of tumor necrosis factor alpha (TNF- α) on the expression of the number of adhesion molecules in ECs was analyzed experimentally. A flow chamber system with CCD camera was set up to observe the top view of the rolling of EPCs on the substrate cultivated with ECs. Numerical results prove that the adhesion of EPC on ECs is closely related to membrane stiffness of the cell and shear rate of the flow. It also suggests that the adhesion force between EPC and EC by P-selectin glycoprotein ligand-1 only is not strong enough to bond the cell onto vessel walls unless contributions of other catch bond are considered. Experimental results demonstrate that TNF- α enhanced the expressions of VCAM, ICAM, P-selectin and E-selectin in ECs, which supports the numerical results that the rolling velocity of EPC on TNF- α treated EC substrate decreases obviously compared with its velocity on the untreated one. It is found that because the adhesion is affected by both the rolling velocity and the deformability of the cell, an optimal stiffness of EPC may exist at a given shear rate of flow for achieving maximum adhesion rates.

Endothelial dysfunction in normoglycaemic first-degree relatives of type 2 diabetes mellitus complicated with hyperuricaemia.

PubMed

Zhang, Junxia; Xiang, Lin; Zhang, Bilin; Cheng, Yangyang

2017-03-01

To reveal the effect of hyperuricaemia on endothelial function in normoglycaemic first-degree relatives of type 2 diabetes mellitus. In all, 40 first-degree relatives of type 2 diabetes mellitus with hyperuricaemia, 40 first-degree relatives of type 2 diabetes mellitus with normouricaemia and 35 healthy subjects without diabetic family history were recruited in this study. Anthropometric parameters as well as blood pressure, blood lipids, fasting blood glucose, fasting insulin, C-reactive protein, tumour necrosis factor-α and interleukin-6 were measured. Insulin resistance was assessed with homoeostasis model assessment index-insulin resistance index. To assess endothelial function, high-resolution ultrasonography was used for measuring flow- and nitroglycerine-mediated brachial artery vasodilation. When compared with control, flow-mediated dilation was lower in first-degree relatives with or without hyperuricaemia (both p < 0.001). When compared with first-degree relative subjects with normouricaemia, there were lower flow-mediated dilation ( p < 0.001) and higher levels of uric acid ( p < 0.001), fasting blood glucose ( p < 0.001), C-reactive protein ( p = 0.001), tumour necrosis factor-α ( p < 0.001) and interleukin-6 ( p < 0.001) in first-degree relative subjects with hyperuricaemia. Flow-mediated dilation was found to be negatively related to uric acid ( r = -0.597, p < 0.001). Stepwise multiple regressions demonstrated that uric acid was a significant determinant of flow-mediated dilation independent of other variables in first-degree relatives of type 2 diabetes mellitus (β = -0.677, p < 0.001; confidence interval: -0.010 to -0.006). Further endothelial dysfunction is found in normoglycaemic first-degree relatives of type 2 diabetes mellitus complicated with hyperuricaemia.

Longitudinal assessment of maternal endothelial function and markers of inflammation and placental function throughout pregnancy in lean and obese mothers.

PubMed

Stewart, Frances M; Freeman, Dilys J; Ramsay, Jane E; Greer, Ian A; Caslake, Muriel; Ferrell, William R

2007-03-01

Obesity in pregnancy is increasing and is a risk factor for metabolic pathology such as preeclampsia. In the nonpregnant, obesity is associated with dyslipidemia, vascular dysfunction, and low-grade chronic inflammation. Our aim was to measure microvascular endothelial function in lean and obese pregnant women at intervals throughout their pregnancies and at 4 months after delivery. Plasma markers of endothelial function, inflammation, and placental function and their association with microvascular function were also assessed. Women in the 1st trimester of pregnancy were recruited, 30 with a body mass index (BMI) less than 30 kg/m(2) and 30 with a BMI more than or equal to 30 kg/m(2) matched for age, parity, and smoking status. In vivo endothelial-dependent and -independent microvascular function was measured using laser Doppler imaging in the 1st, 2nd, and 3rd trimesters of pregnancy and at 4 months postnatal. Plasma markers of endothelial activation [soluble intercellular cell adhesion molecule-1 (sVCAM-1), soluble vascular cell adhesion molecule-1 (sVCAM-1), von Willebrand factor (vWF), and plasminogen activator inhibitor (PAI)-1], inflammation (IL-6, TNFalpha, C-reactive protein, and IL-10), and placental function (PAI-1/PAI-2 ratio) were also assessed at each time point. The pattern of improving endothelial function during pregnancy was the same for lean and obese, but endothelial-dependent vasodilation was significantly lower (P < 0.05) in the obese women at each trimester (51, 41, and 39%, respectively). In the postpartum period, the improvement in endothelial-dependent vasodilation persisted in the lean women but declined to near 1st trimester levels in the obese (lean/obese difference, 115%; P < 0.01). There was a small but significant difference in endothelial-independent vasodilation between the two groups, lean response being greater than obese (P = 0.021), and response declined in both groups in the postpartum period. In multivariate analysis, time of sampling had the most impact on endothelial-independent function [18.5% (adjusted sum of squares expressed as a percentage of total means squared), P < 0.001 for sodium nitroprusside response; 9.8%, P < 0.001 for acetylcholine response], and obesity had the most impact on endothelial-dependent microvascular function (1.7%, P = 0.046 for sodium nitroprusside response; 19.3%, P < 0.001 for acetylcholine response). Time of sampling (11.2%, P < 0.001), IL-6 (4.0%, P = 0.002), and IL-10 (2.4%, P = 0.018) were significant independent contributors to variation in endothelial-dependent microvascular function. When obesity was entered into the model, the association with IL-6 and IL-10 was no longer significant, and obesity explained 6.8% (P < 0.001) of the variability in endothelial-dependent microvascular function. In the 1st trimester, obese women had a significantly higher PAI-1/PAI-2 ratio [obese median (interquartile range), 0.87 (0.54-1.21) vs. lean 0.30 (0.21-0.47), P < 0.001), reflecting the lower PAI-2 levels in obese pregnant women. In a multivariate analysis, 1st trimester BMI (7.6%, P = 0.012), IL-10 (8.2%, P < 0.001), and sVCAM-1 (0.73%, P = 0.007) contributed to the 1st trimester PAI-1/PAI-2 ratio. Obese mothers have a lower endothelium-dependent and -independent vasodilation when compared with lean counterparts. There was a higher PAI-1/ PAI-2 ratio in the 1st trimester in obese women, which improved later in pregnancy. Obese pregnancy is associated with chronic preexisting endothelial activation and impairment of endothelial function secondary to increased production of inflammatory T-helper cells-2 cytokines.

Influence of shear stress and size on viability of endothelial cells exposed to gold nanoparticles

NASA Astrophysics Data System (ADS)

Fede, C.; Albertin, Giovanna; Petrelli, L.; De Caro, R.; Fortunati, I.; Weber, V.; Ferrante, Camilla

2017-09-01

Screening nanoparticle toxicity directly on cell culture can be a fast and cheap technique. Nevertheless, to obtain results in accordance with those observed in live animals, the conditions in which cells are cultivated should resemble the one encountered in live systems. Microfluidic devices offer the possibility to satisfy this requirement, in particular with endothelial cell lines, because they are capable to reproduce the flowing media and shear stress experienced by these cell lines in vivo. In this work, we exploit a microfluidic device to observe how human umbilical vein endothelial cells (HUVEC) viability changes when subject to a continuous flow of culture medium, in which spherical citrate-stabilized gold nanoparticles of different sizes and at varying doses are investigated. For comparison, the same experiments are also run in multiwells where the cells do not experience the shear stress induced by the flowing medium. We discuss the results considering the influence of mode of exposure and nanoparticle size (24 and 13 nm). We observed that gold nanoparticles show a lower toxicity under flow conditions with respect to static and the HUVEC viability decreases as the nanoparticle surface area per unit volume increases, regardless of size.

SALT LOADING HAS A MORE DELETERIOUS EFFECT ON FLOW-MEDIATED DILATION IN SALT-RESISTANT MEN THAN WOMEN

PubMed Central

Lennon-Edwards, S.; Ramick, M.G.; Matthews, E.L.; Brian, M.S.; Farquhar, W.B.; Edwards, D.G.

2014-01-01

Background and Aims Dietary sodium loading has been shown to adversely impact endothelial function independently of blood pressure (BP). However, it is unknown whether dietary sodium loading impacts endothelial function differently in men as compared to women. The aim of this study was to test the hypothesis that endothelial-dependent dilation (EDD) would be lower in men as compared to women in response to a high sodium diet. Methods and Results Thirty subjects (14F, 31±2y; 16M, 29±2y) underwent a randomized, crossover, controlled diet study consisting of 7 days of low sodium (LS; 20 mmol/day) and 7 days of high sodium (HS; 300–350 mmol/day). Salt-resistance was determined by a change in 24-hr mean arterial pressure (MAP)≤ 5 mmHg between HS and LS as assessed on day 7 of each diet. Blood and 24-hr urine were also collected and EDD was assessed by brachial artery flow-mediated dilation(FMD). By design, MAP was not different between LS and HS conditions and urinary sodium excretion increased on HS diet (p<0.01). FMD did not differ between men and women on the LS diet (10.2±0.65 vs. 10.7±0.83; p>0.05) and declined in both men and women on HS (p<0.001). However, FMD was lower in men as compared to women on HS (5.7±0.5 vs. 8.6±0.86; p<0.01). Conclusions HS reduced FMD in both men and women. In response to a HS diet, FMD was lower in men compared to women suggesting a greater sensitivity of the vasculature to high sodium in men. PMID:24989702

Endothelial cell respiration is affected by the oxygen tension during shear exposure: role of mitochondrial peroxynitrite.

PubMed

Jones, Charles I; Han, Zhaosheng; Presley, Tennille; Varadharaj, Saradhadevi; Zweier, Jay L; Ilangovan, Govindasamy; Alevriadou, B Rita

2008-07-01

Cultured vascular endothelial cell (EC) exposure to steady laminar shear stress results in peroxynitrite (ONOO(-)) formation intramitochondrially and inactivation of the electron transport chain. We examined whether the "hyperoxic state" of 21% O(2), compared with more physiological O(2) tensions (Po(2)), increases the shear-induced nitric oxide (NO) synthesis and mitochondrial superoxide (O(2)(*-)) generation leading to ONOO(-) formation and suppression of respiration. Electron paramagnetic resonance oximetry was used to measure O(2) consumption rates of bovine aortic ECs sheared (10 dyn/cm(2), 30 min) at 5%, 10%, or 21% O(2) or left static at 5% or 21% O(2). Respiration was inhibited to a greater extent when ECs were sheared at 21% O(2) than at lower Po(2) or left static at different Po(2). Flow in the presence of an endothelial NO synthase (eNOS) inhibitor or a ONOO(-) scavenger abolished the inhibitory effect. EC transfection with an adenovirus that expresses manganese superoxide dismutase in mitochondria, and not a control virus, blocked the inhibitory effect. Intracellular and mitochondrial O(2)(*-) production was higher in ECs sheared at 21% than at 5% O(2), as determined by dihydroethidium and MitoSOX red fluorescence, respectively, and the latter was, at least in part, NO-dependent. Accumulation of NO metabolites in media of ECs sheared at 21% O(2) was modestly increased compared with ECs sheared at lower Po(2), suggesting that eNOS activity may be higher at 21% O(2). Hence, the hyperoxia of in vitro EC flow studies, via increased NO and mitochondrial O(2)(*-) production, leads to enhanced ONOO(-) formation intramitochondrially and suppression of respiration.

ASSOCIATIONS OF MACRO- AND MICROVASCULAR ENDOTHELIAL DYSFUNCTION WITH SUBCLINICAL VENTRICULAR DYSFUNCTION IN END-STAGE RENAL DISEASE

PubMed Central

Dubin, Ruth F; Guajardo, Isabella; Ayer, Amrita; Mills, Claire; Donovan, Catherine; Beussink, Lauren; Scherzer, Rebecca; Ganz, Peter; Shah, Sanjiv J

2016-01-01

Patients with end-stage renal disease (ESRD) suffer high rates of heart failure and cardiovascular mortality, and we lack a thorough understanding of what, if any, modifiable factors contribute to cardiac dysfunction in these high-risk patients. In order to evaluate endothelial function as a potentially modifiable cause of cardiac dysfunction in ESRD, we investigated cross-sectional associations of macro- and microvascular dysfunction with left and right ventricular dysfunction in a well-controlled ESRD cohort. We performed comprehensive echocardiography, including tissue Doppler imaging and speckle tracking echocardiography of the left and right ventricle, in 149 ESRD patients enrolled in an ongoing prospective, observational study. Of these participants, 123 also underwent endothelium-dependent flow-mediated dilation (FMD) of the brachial artery (macrovascular function). Microvascular function was measured as the velocity time integral (VTI) of hyperemic blood flow following cuff deflation. Impaired FMD was associated with higher LV mass, independently of age and blood pressure: per two-fold lower FMD, LV mass was 4.1% higher (95%CI [0.49, 7.7], p=0.03). After adjustment for demographics, blood pressure, comorbidities and medications, a two-fold lower VTI was associated with 9.5% higher E/e’ ratio (95% CI [1.0, 16], p=0.03) and 6.7% lower absolute RV longitudinal strain (95% CI [2.0, 12], p=0.003). Endothelial dysfunction is a major correlate of cardiac dysfunction in ESRD, particularly diastolic and right ventricular dysfunction, in patients whose volume status is well-controlled. Future investigations are needed to determine whether therapies targeting the vascular endothelium could improve cardiac outcomes in ESRD. PMID:27550915

Alternative RNA splicing in the endothelium mediated in part by Rbfox2 regulates the arterial response to low flow

PubMed Central

Butty, Vincent L; Boutz, Paul L; Begum, Shahinoor; Kimble, Amy L; Sharp, Phillip A; Burge, Christopher B

2018-01-01

Low and disturbed blood flow drives the progression of arterial diseases including atherosclerosis and aneurysms. The endothelial response to flow and its interactions with recruited platelets and leukocytes determine disease progression. Here, we report widespread changes in alternative splicing of pre-mRNA in the flow-activated murine arterial endothelium in vivo. Alternative splicing was suppressed by depletion of platelets and macrophages recruited to the arterial endothelium under low and disturbed flow. Binding motifs for the Rbfox-family are enriched adjacent to many of the regulated exons. Endothelial deletion of Rbfox2, the only family member expressed in arterial endothelium, suppresses a subset of the changes in transcription and RNA splicing induced by low flow. Our data reveal an alternative splicing program activated by Rbfox2 in the endothelium on recruitment of platelets and macrophages and demonstrate its relevance in transcriptional responses during flow-driven vascular inflammation. PMID:29293084

Disruption of astrocyte-vascular coupling and the blood-brain barrier by invading glioma cells

PubMed Central

Watkins, Stacey; Robel, Stefanie; Kimbrough, Ian F.; Robert, Stephanie M.; Ellis-Davies, Graham; Sontheimer, Harald

2014-01-01

Astrocytic endfeet cover the entire cerebral vasculature and serve as exchange sites for ions, metabolites, and energy substrates from the blood to the brain. They maintain endothelial tight junctions that form the blood-brain barrier (BBB) and release vasoactive molecules that regulate vascular tone. Malignant gliomas are highly invasive tumors that use the perivascular space for invasion and co-opt existing vessels as satellite tumors form. Here we use a clinically relevant mouse model of glioma and find that glioma cells, as they populate the perivascular space of pre-existing vessels, displace astrocytic endfeet from endothelial or vascular smooth muscle cells. This causes a focal breach in the BBB. Furthermore, astrocyte-mediated gliovascular coupling is lost, and glioma cells seize control over regulation of vascular tone through Ca2+-dependent release of K+. These findings have important clinical implications regarding blood flow in the tumor-associated brain and the ability to locally deliver chemotherapeutic drugs in disease. PMID:24943270

Endothelial cell response to biomechanical forces under simulated vascular loading conditions.

PubMed

Punchard, M A; Stenson-Cox, C; O'cearbhaill, E D; Lyons, E; Gundy, S; Murphy, L; Pandit, A; McHugh, P E; Barron, V

2007-01-01

In vivo, endothelial cells (EC) are constantly exposed to the haemodynamic forces (HF) of pressure, wall shear stress and hoop stress. The main aim of this study was to design, create and validate a novel perfusion bioreactor capable of delivering shear stress and intravascular pressure to EC in vitro and to characterise their morphology, orientation and gene expression. Here we report the creation and validation of such a simulator and the dual application of pressure (120/60 mmHg) and low shear stress (5 dyn/cm(2)) to a monolayer of EC established on a non-compliant silicone tube. Under these conditions, EC elongated and realigned obliquely to the direction of applied shear stress in a time-dependent manner. Furthermore, randomly distributed F-actin microfilaments reorganised into long, dense stress fibres crossing the cells in a direction perpendicular to that of flow. Finally, combinatorial biomechanical conditioning of EC induced the expression of the inflammatory-associated E-selectin gene.

Bioeffects due to acoustic droplet vaporization

NASA Astrophysics Data System (ADS)

Bull, Joseph

2015-11-01

Encapsulated micro- and nano-droplets can be vaporized via ultrasound, a process termed acoustic droplet vaporization. Our interest is primarily motivated by a developmental gas embolotherapy technique for cancer treatment. In this methodology, infarction of tumors is induced by selectively formed vascular gas bubbles that arise from the acoustic vaporization of vascular microdroplets. Additionally, the microdroplets may be used as vehicles for localized drug delivery, with or without flow occlusion. In this talk, we examine the dynamics of acoustic droplet vaporization through experiments and theoretical/computational fluid mechanics models, and investigate the bioeffects of acoustic droplet vaporization on endothelial cells and in vivo. Early timescale vaporization events, including phase change, are directly visualized using ultra-high speed imaging, and the influence of acoustic parameters on droplet/bubble dynamics is discussed. Acoustic and fluid mechanics parameters affecting the severity of endothelial cell bioeffects are explored. These findings suggest parameter spaces for which bioeffects may be reduced or enhanced, depending on the objective of the therapy. This work was supported by NIH grant R01EB006476.

β2-glycoprotein I, lipopolysaccharide and endothelial TLR4: three players in the two hit theory for anti-phospholipid-mediated thrombosis.

PubMed

Raschi, Elena; Chighizola, Cecilia B; Grossi, Claudia; Ronda, Nicoletta; Gatti, Rita; Meroni, Pier Luigi; Borghi, M Orietta

2014-12-01

The thrombogenic effect of β2-glycoprotein I (β2GPI)-dependent anti-phospholipid antibodies (aPL) in animal models was found to be LPS dependent. Since β2GPI behaves as LPS scavenger, LPS/β2GPI complex was suggested to account for in vitro cell activation through LPS/TLR4 involvement being LPS the actual bridge ligand between β2GPI and TLR4 at least in monocytes/macrophages. However, no definite information is available on the interaction among β2GPI, LPS and endothelial TLR4 in spite of the main role of endothelial cells (EC) in clotting. To analyse at the endothelial level the need of LPS, we investigated the in vitro interaction of β2GPI with endothelial TLR4 and we assessed the role of LPS in such an interaction. To do this, we evaluated the direct binding and internalization of β2GPI by confocal microscopy in living TLR4-MD2 transfected CHO cells (CHO/TLR4-MD2) and β2GPI binding to CHO/TLR4-MD2 cells and human umbilical cord vein EC (HUVEC) by flow cytometry and cell-ELISA using anti-β2GPI monoclonal antibodies in the absence or presence of various concentrations of exogenous LPS. To further investigate the role of TLR4, we performed anti-β2GPI antibody binding and adhesion molecule up-regulation in TLR4-silenced HUVEC. Confocal microscopy studies show that β2GPI does interact with TLR4 at the cell membrane and is internalized in cytoplasmic granules in CHO/TLR4-MD2 cells. β2GPI binding to CHO/TLR4-MD2 cells and HUVEC is also confirmed by flow cytometry and cell-ELISA, respectively. The interaction between β2GPI and TLR4 is confirmed by the reduction of anti-β2GPI antibody binding and by the up-regulation of E-selectin or ICAM-1 by TLR4 silencing in HUVEC. β2GPI binding is not affected by LPS at concentrations comparable to those found in both β2GPI and antibody preparations. Only higher amount of LPS that can activate EC and up-regulate TLR4 expression are found to increase the binding. Our findings demonstrate that β2GPI interacts directly with TLR4 expressed on EC, and that such interaction may contribute to β2GPI-dependent aPL-mediated EC activation. At variance of monocytic cells, we also showed a threshold effect for the action of LPS, that is able to enhance anti-β2GPI antibody EC binding only at cell activating concentrations, shown to increase TLR4 expression. This in vitro model may explain why LPS behaves as a second hit increasing the expression of β2GPI in vascular tissues and triggering aPL-mediated thrombosis in experimental animals. Copyright © 2014 Elsevier Ltd. All rights reserved.

Long Non-Coding RNA Malat1 Regulates Angiogenesis in Hindlimb Ischemia.

PubMed

Zhang, Xuejing; Tang, Xuelian; Hamblin, Milton H; Yin, Ke-Jie

2018-06-11

Angiogenesis is a complex process that depends on the delicate regulation of gene expression. Dysregulation of transcription during angiogenesis often leads to various human diseases. Emerging evidence has recently begun to show that long non-coding RNAs (lncRNAs) may mediate angiogenesis in both physiological and pathological conditions; concurrently, underlying molecular mechanisms are largely unexplored. Previously, our lab identified metastasis associates lung adenocarcinoma transcript 1 ( Malat1 ) as an oxygen-glucose deprivation (OGD)-responsive endothelial lncRNA. Here we reported that genetic deficiency of Malat1 leads to reduced blood vessel formation and local blood flow perfusion in mouse hind limbs at one to four weeks after hindlimb ischemia. Malat1 and vascular endothelial growth factor receptor 2 ( VEGFR2 ) levels were found to be increased in both cultured mouse primary skeletal muscle microvascular endothelial cells (SMMECs) after 16 h OGD followed by 24 h reperfusion and in mouse gastrocnemius muscle that underwent hindlimb ischemia followed by 28 days of reperfusion. Moreover, Malat1 silencing by locked nucleic acid (LNA)-GapmeRs significantly reduced tube formation, cell migration, and cell proliferation in SMMEC cultures. Mechanistically, RNA subcellular isolation and RNA-immunoprecipitation experiments demonstrate that Malat1 directly targets VEGFR2 to facilitate angiogenesis. The results suggest that Malat1 regulates cell-autonomous angiogenesis through direct regulation of VEGFR2.

Short-term tibolone does not interfere with endothelial function: a double-blinded, randomized, controlled trial.

PubMed

Celani, M F S; Hurtado, R; Brandão, A H F; Maciel da Fonseca, A M R; Geber, S

2016-06-01

Objective To evaluate the effect of short-term hormone replacement therapy with tibolone 2.5 mg daily on endothelial function of healthy postmenopausal women, using flow-mediated dilation (FMD) of the brachial artery. Methods We performed a randomized, double-blinded, placebo-controlled study. A total of 100 healthy postmenopausal women were randomly allocated to receive tibolone (n = 50) or placebo (n = 50) for 28 days. Measurement of the FMD of the brachial artery was performed before and after the use of tibolone and placebo. Results A total of 31 women completed the study in the tibolone group, and 32 women completed the study in the control group. The results of the FMD measurements obtained from the women in the two groups before treatment were similar (0.018 and 0.091, for tibolone and placebo, p = 0.57). The values of the FMD in women who used tibolone and placebo, before and after the treatment, were similar in both groups. The numbers of women who presented an increase in the values of the FMD in both groups were also similar. Conclusion Our results demonstrate that the administration of 2.5 mg tibolone to healthy postmenopausal women for 28 days does not promote endothelial-dependent vasodilation, measured by FMD of the brachial artery.

Calycosin and Formononetin Induce Endothelium-Dependent Vasodilation by the Activation of Large-Conductance Ca2+-Activated K+ Channels (BKCa).

PubMed

Tseng, Hisa Hui Ling; Vong, Chi Teng; Leung, George Pak-Heng; Seto, Sai Wang; Kwan, Yiu Wa; Lee, Simon Ming-Yuen; Hoi, Maggie Pui Man

2016-01-01

Calycosin and formononetin are two structurally similar isoflavonoids that have been shown to induce vasodilation in aorta and conduit arteries, but study of their actions on endothelial functions is lacking. Here, we demonstrated that both isoflavonoids relaxed rat mesenteric resistance arteries in a concentration-dependent manner, which was reduced by endothelial disruption and nitric oxide synthase (NOS) inhibition, indicating the involvement of both endothelium and vascular smooth muscle. In addition, the endothelium-dependent vasodilation, but not the endothelium-independent vasodilation, was blocked by BK Ca inhibitor iberiotoxin (IbTX). Using human umbilical vein endothelial cells (HUVECs) as a model, we showed calycosin and formononetin induced dose-dependent outwardly rectifying K + currents using whole cell patch clamp. These currents were blocked by tetraethylammonium chloride (TEACl), charybdotoxin (ChTX), or IbTX, but not apamin. We further demonstrated that both isoflavonoids significantly increased nitric oxide (NO) production and upregulated the activities and expressions of endothelial NOS (eNOS) and neuronal NOS (nNOS). These results suggested that calycosin and formononetin act as endothelial BK Ca activators for mediating endothelium-dependent vasodilation through enhancing endothelium hyperpolarization and NO production. Since activation of BK Ca plays a role in improving behavioral and cognitive disorders, we suggested that these two isoflavonoids could provide beneficial effects to cognitive disorders through vascular regulation.

Calycosin and Formononetin Induce Endothelium-Dependent Vasodilation by the Activation of Large-Conductance Ca2+-Activated K+ Channels (BKCa)

PubMed Central

Tseng, Hisa Hui Ling; Vong, Chi Teng; Leung, George Pak-Heng; Seto, Sai Wang; Lee, Simon Ming-Yuen

2016-01-01

Calycosin and formononetin are two structurally similar isoflavonoids that have been shown to induce vasodilation in aorta and conduit arteries, but study of their actions on endothelial functions is lacking. Here, we demonstrated that both isoflavonoids relaxed rat mesenteric resistance arteries in a concentration-dependent manner, which was reduced by endothelial disruption and nitric oxide synthase (NOS) inhibition, indicating the involvement of both endothelium and vascular smooth muscle. In addition, the endothelium-dependent vasodilation, but not the endothelium-independent vasodilation, was blocked by BKCa inhibitor iberiotoxin (IbTX). Using human umbilical vein endothelial cells (HUVECs) as a model, we showed calycosin and formononetin induced dose-dependent outwardly rectifying K+ currents using whole cell patch clamp. These currents were blocked by tetraethylammonium chloride (TEACl), charybdotoxin (ChTX), or IbTX, but not apamin. We further demonstrated that both isoflavonoids significantly increased nitric oxide (NO) production and upregulated the activities and expressions of endothelial NOS (eNOS) and neuronal NOS (nNOS). These results suggested that calycosin and formononetin act as endothelial BKCa activators for mediating endothelium-dependent vasodilation through enhancing endothelium hyperpolarization and NO production. Since activation of BKCa plays a role in improving behavioral and cognitive disorders, we suggested that these two isoflavonoids could provide beneficial effects to cognitive disorders through vascular regulation. PMID:27994632

Effect of electrocautery on endothelial integrity of the internal thoracic artery: ultrastructural analysis with transmission electron microscopy.

PubMed

Onan, Burak; Yeniterzi, Mehmet; Onan, Ismihan Selen; Ersoy, Burak; Gonca, Suheyla; Gelenli, Elif; Solakoglu, Seyhun; Bakir, Ihsan

2014-10-01

The internal thoracic artery (ITA) is typically harvested from the chest wall by means of conventional electrocautery. We investigated the effects of electrocautery on endothelial-cell and vessel-wall morphology at the ultrastructural level during ITA harvesting. Internal thoracic artery specimens from 20 patients who underwent elective coronary artery bypass grafting were investigated in 2 groups. The ITA grafts were sharply dissected with use of a scalpel and clips in the control group (n=10) and were harvested by means of electrocautery in the study group (n=10). Each sample was evaluated for intimal, elastic-tissue, muscular-layer, and adventitial changes. Free flow was measured intraoperatively. Light microscopic examinations were performed after hematoxylin-eosin and Masson's trichrome staining. Transmission electron microscopy was used to evaluate ultrastructural changes in the endothelial cells and vessel walls of each ITA. In the sharp-dissection group, the endothelial surfaces were lined with normal amounts of original endothelium, endothelial cells were distinctly attached to the basal lamina, cytoplasmic organelles were evident, and intercellular junctional complexes were intact. Conversely, in the electrocautery group, the morphologic integrity of endothelial cells was distorted, with some cell separations and splits, contracted cells, numerous large cytoplasmic vacuoles, and no visible cytoplasmic organelles. The subendothelial layer exhibited disintegration. Free ITA flow was higher in the sharp-dissection group (P=0.04). The integrity of endothelial cells can be better preserved when the ITA is mobilized by means of sharp dissection, rather than solely by electrocautery; we recommend a combined approach.

Effect of Electrocautery on Endothelial Integrity of the Internal Thoracic Artery: Ultrastructural Analysis with Transmission Electron Microscopy

PubMed Central

Onan, Burak; Yeniterzi, Mehmet; Onan, Ismihan Selen; Ersoy, Burak; Gonca, Suheyla; Gelenli, Elif; Solakoglu, Seyhun

2014-01-01

The internal thoracic artery (ITA) is typically harvested from the chest wall by means of conventional electrocautery. We investigated the effects of electrocautery on endothelial-cell and vessel-wall morphology at the ultrastructural level during ITA harvesting. Internal thoracic artery specimens from 20 patients who underwent elective coronary artery bypass grafting were investigated in 2 groups. The ITA grafts were sharply dissected with use of a scalpel and clips in the control group (n=10) and were harvested by means of electrocautery in the study group (n=10). Each sample was evaluated for intimal, elastic-tissue, muscular-layer, and adventitial changes. Free flow was measured intraoperatively. Light microscopic examinations were performed after hematoxylin-eosin and Masson's trichrome staining. Transmission electron microscopy was used to evaluate ultrastructural changes in the endothelial cells and vessel walls of each ITA. In the sharp-dissection group, the endothelial surfaces were lined with normal amounts of original endothelium, endothelial cells were distinctly attached to the basal lamina, cytoplasmic organelles were evident, and intercellular junctional complexes were intact. Conversely, in the electrocautery group, the morphologic integrity of endothelial cells was distorted, with some cell separations and splits, contracted cells, numerous large cytoplasmic vacuoles, and no visible cytoplasmic organelles. The subendothelial layer exhibited disintegration. Free ITA flow was higher in the sharp-dissection group (P=0.04). The integrity of endothelial cells can be better preserved when the ITA is mobilized by means of sharp dissection, rather than solely by electrocautery; we recommend a combined approach. PMID:25425979

N-acetylcysteine neither lowers plasma homocysteine concentrations nor improves brachial artery endothelial function in cardiac transplant recipients.

PubMed

Miner, S E S; Cole, D E C; Evrovski, J; Forrest, Q; Hutchison, S J; Holmes, K; Ross, H J

2002-05-01

N-acetylcysteine is a novel antioxidant that has been reported to reduce plasma homocysteine concentrations and improve endothelial function. Cardiac transplant recipients have a high incidence of coronary endothelial dysfunction and hyperhomocysteinemia, both of which may lead to the development of transplantation coronary artery disease. It was hypothesized that N-acetylcysteine would reduce plasma homocysteine concentrations and improve brachial endothelial function in cardiac transplant recipients. A cohort of stable cardiac transplant recipients was recruited from the outpatient clinic at the Toronto General Hospital, Toronto, Ontario. Brachial artery endothelial functions were studied according to standard techniques to determine flow-mediated dilation of the brachial artery. Plasma homocysteine concentrations were assayed using high performance liquid chromatography with electrochemical detection and pulsed integrated amperometry. After baseline testing, patients were treated in an unblinded fashion with N-acetylcysteine 500 mg/day. After 10 weeks of therapy, patients returned for follow-up endothelial function and homocysteine testing. Thirty-one patients were initially enrolled. Two patients withdrew due to excessive gastrointestinal upset. Two patients did not return for follow-up testing. The remaining 27 patients tolerated the treatment well. At baseline, 85% of the patients had hyperhomocysteinemia (greater than 15 mol/L) with a mean plasma concentration of 18.6 4.7 mol/L. No changes in homocysteine concentrations were seen at follow-up. At baseline, the average flow-mediated dilation was only 4.7 6.3%. No changes were seen at follow-up. Hyperhomocysteinemia and brachial endothelial dysfunction are common in stable cardiac transplant recipients and are unaffected by supplementation with N-acetylcysteine.

Effect of onion peel extract on endothelial function and endothelial progenitor cells in overweight and obese individuals.

PubMed

Choi, Eun-Yong; Lee, Hansongyi; Woo, Jong Shin; Jang, Hyun Hee; Hwang, Seung Joon; Kim, Hyun Soo; Kim, Woo-Sik; Kim, Young-Seol; Choue, Ryowon; Cha, Yong-Jun; Yim, Jung-Eun; Kim, Weon

2015-09-01

Acute or chronic intake of polyphenol-rich foods has been reported to improve endothelial function. Quercetin, found abundantly in onion, is a potent antioxidant flavonoid. The aim of this study was to investigate whether consumption of onion peel extract (OPE) improves endothelial function in healthy overweight and obese individuals. This was a randomized double-blind, placebo-controlled study. Seventy-two healthy overweight and obese participants were randomly assigned to receive a red, soft capsule of OPE (100 mg quercetin/d, 50 mg quercetin twice daily; n = 36 participants) or an identical placebo capsule (n = 36) for 12 wk. Endothelial function, defined by flow-mediated dilation (FMD), circulating endothelial progenitor cells (EPCs) by flow cytometry, and laboratory test were determined at baseline and after treatment. Baseline characteristics and laboratory findings did not significantly differ between the two groups. Compared with baseline values, the OPE group showed significantly improved FMD at 12 wk (from 12.5 ± 5.2 to 15.2 ± 6.1; P = 0.002), whereas the placebo group showed no difference. Nitroglycerin-mediated dilation did not change in either group. EPC counts (44.2 ± 25.6 versus 52.3 ± 18.6; P = 0.005) and the percentage of EPCs were significantly increased in the OPE group. When FMD was divided into quartiles, rate of patients with endothelial dysfunction defined as lowest quartile (cutoff value, 8.6%) of FMD improved from 26% to 9% by OPE. Medium-term administration of OPE an improvement in FMD and circulating EPCs. Copyright © 2015 Elsevier Inc. All rights reserved.

Molecular imaging of the paracrine proangiogenic effects of progenitor cell therapy in limb ischemia.

PubMed

Ryu, Jae Choon; Davidson, Brian P; Xie, Aris; Qi, Yue; Zha, Daogang; Belcik, J Todd; Caplan, Evan S; Woda, Juliana M; Hedrick, Catherine C; Hanna, Richard N; Lehman, Nicholas; Zhao, Yan; Ting, Anthony; Lindner, Jonathan R

2013-02-12

Stem cells are thought to enhance vascular remodeling in ischemic tissue in part through paracrine effects. Using molecular imaging, we tested the hypothesis that treatment of limb ischemia with multipotential adult progenitor cells (MAPCs) promotes recovery of blood flow through the recruitment of proangiogenic monocytes. Hind-limb ischemia was produced in mice by iliac artery ligation, and MAPCs were administered intramuscularly on day 1. Optical imaging of luciferase-transfected MAPCs indicated that cells survived for 1 week. Contrast-enhanced ultrasound on days 3, 7, and 21 showed a more complete recovery of blood flow and greater expansion of microvascular blood volume in MAPC-treated mice than in controls. Fluorescent microangiography demonstrated more complete distribution of flow to microvascular units in MAPC-treated mice. On ultrasound molecular imaging, expression of endothelial P-selectin and intravascular recruitment of CX(3)CR-1-positive monocytes were significantly higher in MAPC-treated mice than in the control groups at days 3 and 7 after arterial ligation. Muscle immunohistology showed a >10-fold-greater infiltration of monocytes in MAPC-treated than control-treated ischemic limbs at all time points. Intravital microscopy of ischemic or tumor necrosis factor-α-treated cremaster muscle demonstrated that MAPCs migrate to perimicrovascular locations and potentiate selectin-dependent leukocyte rolling. In vitro migration of human CD14(+) monocytes was 10-fold greater in response to MAPC-conditioned than basal media. In limb ischemia, MAPCs stimulate the recruitment of proangiogenic monocytes through endothelial activation and enhanced chemotaxis. These responses are sustained beyond the MAPC lifespan, suggesting that paracrine effects promote flow recovery by rebalancing the immune response toward a more regenerative phenotype.

Molecular Imaging of the Paracrine Proangiogenic Effects of Progenitor Cell Therapy in Limb Ischemia

PubMed Central

Ryu, Jae Choon; Davidson, Brian P.; Xie, Aris; Qi, Yue; Zha, Daogang; Belcik, J. Todd; Caplan, Evan S.; Woda, Juliana M.; Hedrick, Catherine C.; Hanna, Richard N.; Lehman, Nicholas; Zhao, Yan; Ting, Anthony; Lindner, Jonathan R.

2013-01-01

Background Stem cells are thought to enhance vascular remodeling in ischemic tissue in part through paracrine effects. Using molecular imaging, we tested the hypothesis that treatment of limb ischemia with multipotential adult progenitor cells (MAPC) promotes recovery of blood flow through the recruitment of pro-angiogenic monocytes. Methods and Results Hindlimb ischemia was produced in mice by iliac artery ligation and MAPC were administered intramuscularly on day 1. Optical imaging of luciferase-transfected MAPC indicated that cells survived for 1 week. Contrast-enhanced ultrasound on day 3, 7 and 21 showed a more complete recovery of blood flow and greater expansion of microvascular blood volume in MAPC-treated mice than in controls. Fluorescent microangiography demonstrated more complete distribution of flow to microvascular units in MAPC-treated mice. On ultrasound molecular imaging, expression of endothelial P-selectin and intravascular recruitment of CX3CR-1-positive monocytes was significantly higher in MAPC-treated than control groups at day 3 and 7 after arterial ligation. Muscle immunohistology showed a >10-fold greater infiltration of monocytes in MAPC-treated than control-treated ischemic limbs at all time points. Intravital microscopy of ischemic or TNF-α-treated cremaster muscle demonstrated that MAPC migrate to peri-microvascular locations and potentiate selectin-dependent leukocyte rolling. In vitro migration of human CD14+ monocytes was 10-fold greater in response to MAPC-conditioned than basal media. Conclusions In limb ischemia, MAPC stimulate the recruitment of pro-angiogenic monocytes through endothelial activation and enhanced chemotaxis. These responses are sustained beyond MAPC lifespan suggesting that paracrine effects promote flow recovery by rebalancing the immune response toward a more regenerative phenotype. PMID:23307829

Structural and functional changes in the microcirculation of lepromatous leprosy patients - Observation using orthogonal polarization spectral imaging and laser Doppler flowmetry iontophoresis

PubMed Central

Treu, Curt; de Souza, Maria das Graças Coelho; Lupi, Omar; Sicuro, Fernando Lencastre; Maranhão, Priscila Alves; Kraemer-Aguiar, Luiz Guilherme; Bouskela, Eliete

2017-01-01

Leprosy is a chronic granulomatous infection of skin and peripheral nerves caused by Mycobacterium leprae and is considered the main infectious cause of disability worldwide. Despite the several studies regarding leprosy, little is known about its effects on microvascular structure and function in vivo. Thus, we have aimed to compare skin capillary structure and functional density, cutaneous vasomotion (spontaneous oscillations of arteriolar diameter), which ensures optimal blood flow distribution to skin capillaries) and cutaneous microvascular blood flow and reactivity between ten men with lepromatous leprosy (without any other comorbidity) and ten age- and gender-matched healthy controls. Orthogonal polarization spectral imaging was used to evaluate skin capillary morphology and functional density and laser Doppler flowmetry to evaluate blood flow, vasomotion and spectral analysis of flowmotion (oscillations of blood flow generated by vasomotion) and microvascular reactivity, in response to iontophoresis of acetylcholine and sodium nitroprusside. The contribution of different frequency components of flowmotion (endothelial, neurogenic, myogenic, respiratory and cardiac) was not statistically different between groups. However, endothelial-dependent and -independent vasodilatations elicited by acetylcholine and sodium nitroprusside iontophoresis, respectively, were significantly reduced in lepromatous leprosy patients compared to controls, characterizing the existence of microvascular dysfunction. These patients also presented a significant increase in the number of capillaries with morphological abnormalities and in the diameters of the dermal papilla and capillary bulk when compared to controls. Our results suggest that lepromatous leprosy causes severe microvascular dysfunction and significant alterations in capillary structure. These structural and functional changes are probably induced by exposure of the microvascular bed to chronic inflammation evoked by the Mycobacterium leprae. PMID:28419120

Exploring the effects of cell seeding density on the differentiation of human pluripotent stem cells to brain microvascular endothelial cells.

PubMed

Wilson, Hannah K; Canfield, Scott G; Hjortness, Michael K; Palecek, Sean P; Shusta, Eric V

2015-05-21

Brain microvascular-like endothelial cells (BMECs) derived from human pluripotent stem cells (hPSCs) have significant promise as tools for drug screening and studying the structure and function of the BBB in health and disease. The density of hPSCs is a key factor in regulating cell fate and yield during differentiation. Prior reports of hPSC differentiation to BMECs have seeded hPSCs in aggregates, leading to non-uniform cell densities that may result in differentiation heterogeneity. Here we report a singularized-cell seeding approach compatible with hPSC-derived BMEC differentiation protocols and evaluate the effects of initial hPSC seeding density on the subsequent differentiation, yield, and blood-brain barrier (BBB) phenotype. A range of densities of hPSCs was seeded and differentiated, with the resultant endothelial cell yield quantified via VE-cadherin flow cytometry. Barrier phenotype of purified hPSC-derived BMECs was measured via transendothelial electrical resistance (TEER), and purification protocols were subsequently optimized to maximize TEER. Expression of characteristic vascular markers, tight junction proteins, and transporters was confirmed by immunocytochemistry and quantified by flow cytometry. P-glycoprotein and MRP-family transporter activity was assessed by intracellular accumulation assay. The initial hPSC seeding density of approximately 30,000 cells/cm(2) served to maximize the yield of VE-cadherin+ BMECs per input hPSC. BMECs displayed the highest TEER (>2,000 Ω × cm(2)) within this same range of initial seeding densities, although optimization of the BMEC purification method could minimize the seeding density dependence for some lines. Localization and expression levels of tight junction proteins as well as efflux transporter activity were largely independent of hPSC seeding density. Finally, the utility of the singularized-cell seeding approach was demonstrated by scaling the differentiation and purification process down from 6-well to 96-well culture without impacting BBB phenotype. Given the yield and barrier dependence on initial seeding density, the singularized-cell seeding approach reported here should enhance the reproducibility and scalability of hPSC-derived BBB models, particularly for the application to new pluripotent stem cell lines.

Local over-expression of VEGF-DΔNΔC in the uterine arteries of pregnant sheep results in long-term changes in uterine artery contractility and angiogenesis.

PubMed

Mehta, Vedanta; Abi-Nader, Khalil N; Shangaris, Panicos; Shaw, S W Steven; Filippi, Elisa; Benjamin, Elizabeth; Boyd, Michael; Peebles, Donald M; Martin, John; Zachary, Ian; David, Anna L

2014-01-01

The normal development of the uteroplacental circulation in pregnancy depends on angiogenic and vasodilatory factors such as vascular endothelial growth factor (VEGF). Reduced uterine artery blood flow (UABF) is a common cause of fetal growth restriction; abnormalities in angiogenic factors are implicated. Previously we showed that adenovirus (Ad)-mediated VEGF-A165 expression in the pregnant sheep uterine artery (UtA) increased nitric oxide synthase (NOS) expression, altered vascular reactivity and increased UABF. VEGF-D is a VEGF family member that promotes angiogenesis and vasodilatation but, in contrast to VEGF-A, does not increase vascular permeability. Here we examined the effect of Ad.VEGF-DΔNΔC vector encoding a fully processed form of VEGF-D, on the uteroplacental circulation. UtA transit-time flow probes and carotid artery catheters were implanted in mid-gestation pregnant sheep (n = 5) to measure baseline UABF and maternal haemodynamics respectively. 7-14 days later, after injection of Ad.VEGF-DΔNΔC vector (5×10(11) particles) into one UtA and an Ad vector encoding β-galactosidase (Ad.LacZ) contralaterally, UABF was measured daily until scheduled post-mortem examination at term. UtAs were assessed for vascular reactivity, NOS expression and endothelial cell proliferation; NOS expression was studied in ex vivo transduced UtA endothelial cells (UAECs). At 4 weeks post-injection, Ad.VEGF-DΔNΔC treated UtAs showed significantly lesser vasoconstriction (Emax144.0 v/s 184.2, p = 0.002). There was a tendency to higher UABF in Ad.VEGF-DΔNΔC compared to Ad.LacZ transduced UtAs (50.58% v/s 26.94%, p = 0.152). There was no significant effect on maternal haemodynamics. An increased number of proliferating endothelial cells and adventitial blood vessels were observed in immunohistochemistry. Ad.VEGF-DΔNΔC expression in cultured UAECs upregulated eNOS and iNOS expression. Local over-expression of VEGF-DΔNΔC in the UtAs of pregnant mid-gestation sheep reduced vasoconstriction, promoted endothelial cell proliferation and showed a trend towards increased UABF. Studies in cultured UAECs indicate that VEGF-DΔNΔC may act in part through upregulation of eNOS and iNOS.

Matrix metalloproteinase-10 is upregulated by thrombin in endothelial cells and increased in patients with enhanced thrombin generation.

PubMed

Orbe, Josune; Rodríguez, José A; Calvayrac, Olivier; Rodríguez-Calvo, Ricardo; Rodríguez, Cristina; Roncal, Carmen; Martínez de Lizarrondo, Sara; Barrenetxe, Jaione; Reverter, Juan C; Martínez-González, José; Páramo, José A

2009-12-01

Thrombin is a multifunctional serine protease that promotes vascular proinflammatory responses whose effect on endothelial MMP-10 expression has not previously been evaluated. Thrombin induced endothelial MMP-10 mRNA and protein levels, through a protease-activated receptor-1 (PAR-1)-dependent mechanism, in a dose- and time-dependent manner. This effect was mimicked by a PAR-1 agonist peptide (TRAP-1) and antagonized by an anti-PAR-1 blocking antibody. MMP-10 induction was dependent on extracellular regulated kinase1/2 (ERK1/2) and c-jun N-terminal kinase (JNK) pathways. By serial deletion analysis, site-directed mutagenesis and electrophoretic mobility shift assay an AP-1 site in the proximal region of MMP-10 promoter was found to be critical for thrombin-induced MMP-10 transcriptional activity. Thrombin and TRAP-1 upregulated MMP-10 in murine endothelial cells in culture and in vivo in mouse aorta. This effect of thrombin was not observed in PAR-1-deficient mice. Interestingly, circulating MMP-10 levels (P<0.01) were augmented in patients with endothelial activation associated with high (disseminated intravascular coagulation) and moderate (previous acute myocardial infarction) systemic thrombin generation. Thrombin induces MMP-10 through a PAR-1-dependent mechanism mediated by ERK1/2, JNK, and AP-1 activation. Endothelial MMP-10 upregulation could be regarded as a new proinflammatory effect of thrombin whose pathological consequences in thrombin-related disorders and plaque stability deserve further investigation.

Anti-TNFα therapy transiently improves high density lipoprotein cholesterol levels and microvascular endothelial function in patients with rheumatoid arthritis: a Pilot Study

PubMed Central

2012-01-01

Background Rheumatoid arthritis (RA) is associated with increased morbidity and mortality from cardiovascular disease (CVD). This can be only partially attributed to traditional CVD risk factors such as dyslipidaemia and their downstream effects on endothelial function. The most common lipid abnormality in RA is reduced levels of high-density lipoprotein (HDL) cholesterol, probably due to active inflammation. In this longitudinal study we hypothesised that anti-tumor necrosis factor-α (anti-TNFα) therapy in patients with active RA improves HDL cholesterol, microvascular and macrovascular endothelial function. Methods Twenty-three RA patients starting on anti-TNFα treatment were assessed for HDL cholesterol level, and endothelial-dependent and -independent function of microvessels and macrovessels at baseline, 2-weeks and 3 months of treatment. Results Disease activity (CRP, fibrinogen, DAS28) significantly decreased during the follow-up period. There was an increase in HDL cholesterol levels at 2 weeks (p < 0.05) which was paralleled by a significant increase in microvascular endothelial-dependent function (p < 0.05). However, both parameters returned towards baseline at 12 weeks. Conclusion Anti-TNFα therapy in RA patients appears to be accompanied by transient but significant improvements in HDL cholesterol levels, which coexists with an improvement in microvascular endothelial-dependent function. PMID:22824166

The role of shear stress and altered tissue properties on endothelial to mesenchymal transformation and tumor-endothelial cell interaction.

PubMed

Mina, Sara G; Huang, Peter; Murray, Bruce T; Mahler, Gretchen J

2017-07-01

Tumor development is influenced by stromal cells in aspects including invasion, growth, angiogenesis, and metastasis. Activated fibroblasts are one group of stromal cells involved in cancer metastasis, and one source of activated fibroblasts is endothelial to mesenchymal transformation (EndMT). EndMT begins when the endothelial cells delaminate from the cell monolayer, lose cell-cell contacts, lose endothelial markers such as vascular endothelial-cadherin (VE-cadherin), gain mesenchymal markers like alpha-smooth muscle actin (α-SMA), and acquire mesenchymal cell-like properties. A three-dimensional (3D) culture microfluidic device was developed for investigating the role of steady low shear stress (1 dyne/cm 2 ) and altered extracellular matrix (ECM) composition and stiffness on EndMT. Shear stresses resulting from fluid flow within tumor tissue are relevant to both cancer metastasis and treatment effectiveness. Low and oscillatory shear stress rates have been shown to enhance the invasion of metastatic cancer cells through specific changes in actin and tubulin remodeling. The 3D ECM within the device was composed of type I collagen and glycosaminoglycans (GAGs), hyaluronic acid and chondroitin sulfate. An increase in collagen and GAGs has been observed in the solid tumor microenvironment and has been correlated with poor prognosis in many different cancer types. In this study, it was found that ECM composition and low shear stress upregulated EndMT, including upregulation of mesenchymal-like markers (α-SMA and Snail) and downregulated endothelial marker protein and gene expression (VE-cadherin). Furthermore, this novel model was utilized to investigate the role of EndMT in breast cancer cell proliferation and migration. Cancer cell spheroids were embedded within the 3D ECM of the microfluidic device. The results using this device show for the first time that the breast cancer spheroid size is dependent on shear stress and that the cancer cell migration rate, distance, and proliferation are induced by EndMT-derived activated fibroblasts. This model can be used to explore new therapeutics in a tumor microenvironment.

Functional Characterization of S100A8 and S100A9 in Altering Monolayer Permeability of Human Umbilical Endothelial Cells

PubMed Central

Wang, Liqun; Luo, Haihua; Chen, Xiaohuan; Jiang, Yong; Huang, Qiaobing

2014-01-01

S100A8, S100A9 and S100A8/A9 complexes have been known as important endogenous damage-associated molecular pattern (DAMP) proteins. But the pathophysiological roles of S100A8, S100A9 and S100A8/A9 in cardiovascular diseases are incompletely explained. In this present study, the effects of homo S100A8, S100A9 and their hetero-complex S100A8/A9 on endothelial barrier function were tested respectively in cultured human umbilical venous endothelial cells (HUVECs). The involvement of TLR4 and RAGE were observed by using inhibitor of TLR4 and blocking antibody of RAGE. The clarification of different MAPK subtypes in S100A8/A9-induced endothelial response was implemented by using specific inhibitors. The calcium-dependency was detected in the absence of Ca2+ or in the presence of gradient-dose Ca2+. The results showed that S100A8, S100A9 and S100A8/A9 could induce F-actin and ZO-1 disorganization in HUVECs and evoked the increases of HUVEC monolayer permeability in a dose- and time-dependent manner. The effects of S100A8, S100A9 and S100A8/A9 on endothelial barrier function depended on the activation of p38 and ERK1/2 signal pathways through receptors TLR4 and RAGE. Most importantly, we revealed the preference of S100A8 on TLR4 and S100A9 on RAGE in HUVECs. The results also showed the calcium dependency in S100A8- and S100A9-evoked endothelial response, indicating that calcium dependency on formation of S100A8 or A9 dimmers might be the prerequisite for this endothelial functional alteration. PMID:24595267

Heat Shock Protein 70 Prevents Hyperoxia-Induced Disruption of Lung Endothelial Barrier via Caspase-Dependent and AIF-Dependent Pathways

PubMed Central

Kondrikov, Dmitry; Fulton, David; Dong, Zheng; Su, Yunchao

2015-01-01

Exposure of pulmonary artery endothelial cells (PAECs) to hyperoxia results in a compromise in endothelial monolayer integrity, an increase in caspase-3 activity, and nuclear translocation of apoptosis-inducing factor (AIF), a marker of caspase-independent apoptosis. In an endeavor to identify proteins involved in hyperoxic endothelial injury, we found that the protein expression of heat-shock protein 70 (Hsp70) was increased in hyperoxic PAECs. The hyperoxia-induced Hsp70 protein expression is from hspA1B gene. Neither inhibition nor overexpression of Hsp70 affected the first phase barrier disruption of endothelial monolayer. Nevertheless, inhibition of Hsp70 by using the Hsp70 inhibitor KNK437 or knock down Hsp70 using siRNA exaggerated and overexpression of Hsp70 prevented the second phase disruption of lung endothelial integrity. Moreover, inhibition of Hsp70 exacerbated and overexpression of Hsp70 prevented hyperoxia-induced apoptosis, caspase-3 activation, and increase in nuclear AIF protein level in PAECs. Furthermore, we found that Hsp70 interacted with AIF in the cytosol in hyperoxic PAECs. Inhibition of Hsp70/AIF association by KNK437 correlated with increased nuclear AIF level and apoptosis in KNK437-treated PAECs. Finally, the ROS scavenger NAC prevented the hyperoxia-induced increase in Hsp70 expression and reduced the interaction of Hsp70 with AIF in hyperoxic PAECs. Together, these data indicate that increased expression of Hsp70 plays a protective role against hyperoxia-induced lung endothelial barrier disruption through caspase-dependent and AIF-dependent apoptotic pathways. Association of Hsp70 with AIF prevents AIF nuclear translocation, contributing to the protective effect of Hsp70 on hyperoxia-induced endothelial apoptosis. The hyperoxia-induced increase in Hsp70 expression and Hsp70/AIF interaction is contributed to ROS formation. PMID:26066050

Cancer Cells Regulate Biomechanical Properties of Human Microvascular Endothelial Cells*

PubMed Central

Mierke, Claudia Tanja

2011-01-01

Metastasis is a key event of malignant tumor progression. The capability to metastasize depends on the ability of the cancer cell to migrate into connective tissue, adhere, and possibly transmigrate through the endothelium. Previously we reported that the endothelium does not generally act as barrier for cancer cells to migrate in three-dimensional extracellular matrices (3D-ECMs). Instead, the endothelium acts as an enhancer or a promoter for the invasiveness of certain cancer cells. How invasive cancer cells diminish the endothelial barrier function still remains elusive. Therefore, this study investigates whether invasive cancer cells can decrease the endothelial barrier function through alterations of endothelial biomechanical properties. To address this, MDA-MB-231 breast cancer cells were used that invade deeper and more numerous into 3D-ECMs when co-cultured with microvascular endothelial cells. Using magnetic tweezer measurements, MDA-MB-231 cells were found to alter the mechanical properties of endothelial cells by reducing endothelial cell stiffness. Using spontaneous bead diffusion, actin cytoskeletal remodeling dynamics were shown to be increased in endothelial cells co-cultured with MDA-MB-231 cells compared with mono-cultured endothelial cells. In addition, knockdown of the α5 integrin subunit in highly transmigrating α5β1high cells derived from breast, bladder, and kidney cancer cells abolished the endothelial invasion-enhancing effect comparable with the inhibition of myosin light chain kinase. These results indicate that the endothelial invasion-enhancing effect is α5β1 integrin-dependent. Moreover, inhibition of Rac-1, Rho kinase, MEK kinase, and PI3K reduced the endothelial invasion-enhancing effect, indicating that signaling via small GTPases may play a role in the endothelial facilitated increased invasiveness of cancer cells. In conclusion, decreased stiffness and increased cytoskeletal remodeling dynamics of endothelial cells may account for the breakdown of endothelial barrier function, suggesting that biomechanical alterations are sufficient to facilitate the transmigration and invasion of invasive cancer cells into 3D-ECMs. PMID:21940631

Febuxostat improves endothelial function in hemodialysis patients with hyperuricemia: A randomized controlled study.

PubMed

Tsuruta, Yuki; Kikuchi, Kan; Tsuruta, Yukio; Sasaki, Yuko; Moriyama, Takahito; Itabashi, Mitsuyo; Takei, Takashi; Uchida, Keiko; Akiba, Takashi; Tsuchiya, Ken; Nitta, Kosaku

2015-10-01

Endothelial dysfunction is often found in both hyperuricemia and hemodialysis patients. Recent studies have shown that treating hyperuricemia with allopurinol improves endothelial dysfunction. This study is performed to assess the effect of febuxostat on endothelial dysfunction in hemodialysis patients with hyperuricemia. We randomly assigned 53 hemodialysis patients with hyperuricemia to a febuxostat (10 mg daily) group and a control group and measured flow-mediated dilation, serum uric acid (UA) levels, systolic and diastolic blood pressure, malondialdehyde-modified low-density lipoprotein (MDA-LDL), and highly sensitive C-reactive protein (hsCRP) at baseline and at the end of a 4-week study period. Flow-mediated dilation increased from 5.3% ± 2.4% to 8.9% ± 3.6% in the febuxostat group but did not change significantly in the control group. Treatment with febuxostat resulted in a significant decrease in serum UA level and a significant decrease in MDA-LDL compared with baseline, but no significant difference was observed in hsCRP level or blood pressure. No significant differences were observed in the control group. Febuxostat improved endothelial dysfunction and reduced serum UA levels and oxidative stress in hemodialysis patients with hyperuricemia. © 2015 International Society for Hemodialysis.

Capture of endothelial cells under flow using immobilized vascular endothelial growth factor

PubMed Central

Smith, Randall J.; Koobatian, Maxwell T.; Shahini, Aref; Swartz, Daniel D.; Andreadis, Stelios T.

2015-01-01

We demonstrate the ability of immobilized vascular endothelial growth factor (VEGF) to capture endothelial cells (EC) with high specificity under fluid flow. To this end, we engineered a surface consisting of heparin bound to poly-L-lysine to permit immobilization of VEGF through the C-terminal heparin-binding domain. The immobilized growth factor retained its biological activity as shown by proliferation of EC and prolonged activation of KDR signaling. Using a microfluidic device we assessed the ability to capture EC under a range of shear stresses from low (0.5 dyne/cm2) to physiological (15 dyne/cm2). Capture was significant for all shear stresses tested. Immobilized VEGF was highly selective for EC as evidenced by significant capture of human umbilical vein and ovine pulmonary artery EC but no capture of human dermal fibroblasts, human hair follicle derived mesenchymal stem cells, or mouse fibroblasts. Further, VEGF could capture EC from mixtures with non-EC under low and high shear conditions as well as from complex fluids like whole human blood under high shear. Our findings may have far reaching implications, as they suggest that VEGF could be used to promote endothelialization of vascular grafts or neovascularization of implanted tissues by rare but continuously circulating EC. PMID:25771020

Geraniol improves endothelial function by inhibiting NOX-2 derived oxidative stress in high fat diet fed mice.

PubMed

Wang, Xiaoyu; Zhao, Shiqi; Su, Mengqi; Sun, Li; Zhang, Song; Wang, Dingyu; Liu, Zhaorui; Yuan, Yue; Liu, Yang; Li, Yue

2016-05-20

Endothelial dysfunction occurs in obese patients and high-fat diet (HFD) fed experimental animals. While geraniol has been reported to ameliorate inflammation and oxidative stress, inhibit tumor cell proliferation, and improve atherosclerosis, its direct effect on endothelial function remains uncharacterized. The present study therefore investigated the effect of geraniol on endothelial function in HFD mice and its underlying mechanisms. C57 BL/6 mice were fed an HFD (n = 40) or a normal diet (n = 20) for 8 weeks. HFD fed mice then were randomized to intraperitoneal treatment with geraniol (n = 20) or vehicle (n = 20) for another 6 weeks. Acetylcholine (Ach)-induced endothelial dependent vasorelaxation was measured on wire myography; reactive oxygen species (ROS) generation was assessed by fluorescence imaging, and NADPH oxidases (NOXs) and adhesive molecules VCAM-1 and ICAM-1 protein expression by western blotting. Geraniol improved endothelial function in HFD fed mice, as evidenced by its: 1. restoring endothelial dependent vasorelaxation induced by Ach, and reversing increased VCAM-1 and ICAM-1 expression; 2. attenuating HFD induced increased serum TBARS and aortic ROS generation; and 3. downregulating aortic NOX-2 expression in both HFD fed mice and in palmitic acid treated endothelial cells. Geraniol therefore protects against endothelial dysfunction induced by HFD through reducing NOX-2 associated ROS generation. Copyright © 2016 Elsevier Inc. All rights reserved.

Dobesilate enhances endothelial nitric oxide synthase-activity in macro- and microvascular endothelial cells

PubMed Central

Suschek, Christoph; Kolb, Hubert; Kolb-Bachofen, Victoria

1997-01-01

Dobesilate is used for normalizing vascular dysfunction in a number of diseases. In search for an effect on endothelial NO production, macrovascular endothelial cells from rat aorta, microvascular endothelial cells from rat exocrine pancreatic tissue, and capillary endothelial cells from rat islets, were cultured in the presence or absence of Mg-Dobesilate. The activity of constitutive nitric oxide synthase (ecNOS) in resident cells as well as of inducible nitric oxide synthase (iNOS) in cytokine-activated cells was measured indirectly by recording the citrulline concentrations in culture supernatants.In each of the different endothelial cells Mg-Dobesilate incubation (0.25–1 mM) for 24 h led to a significant and concentration-dependent increase in ecNOS-activities. With cytokine-activated endothelial cell cultures only moderate effects were seen with little or no concentration-dependency. Addition of the NOS-inhibitor NG-monomethyl-L-arginine led to a significant suppression of citrulline formation in all cultures as an evidence for the enzyme specificity of these effects.iNOS- and ecNOS-specific reverse transcription and semi-quantitative polymerase chain reaction (RT–PCR) with RNA from resident or cytokine-activated endothelial cells gave no evidence for an increase in NOS-specific mRNA after Mg-Dobesilate-treatment. Furthermore, Dobesilate-mediated enhancement of NO synthesis in resting endothelial cells was not due to iNOS induction in these cells, as no iNOS-specific signal was found by RT–PCR. PMID:9421302

Inhibitive Effects of Quercetin on Myeloperoxidase-Dependent Hypochlorous Acid Formation and Vascular Endothelial Injury.

PubMed

Lu, Naihao; Sui, Yinhua; Tian, Rong; Peng, Yi-Yuan

2018-05-16

Myeloperoxidase (MPO) from activated neutrophils plays important roles in multiple human inflammatory diseases by catalyzing the formation of powerful oxidant hypochlorous acid (HOCl). As a major flavonoid in the human diet, quercetin has been suggested to act as antioxidant and anti-inflammatory agent in vitro and in vivo. In this study, we showed that quercetin inhibited MPO-mediated HOCl formation (75.0 ± 6.2% for 10 μM quercetin versus 100 ± 5.2% for control group, P < 0.01) and cytotoxicity to endothelial cells in vitro, while this flavonoid was nontoxic to endothelial cell cultures ( P > 0.05, all cases). Moreover, quercetin inhibited HOCl generation by stimulated neutrophils (a rich source of MPO) and protected endothelial cells from neutrophils-induced injury. Furthermore, quercetin could inhibit HOCl-induced endothelial dysfunction such as loss of cell viability, and decrease of nitric oxide formation in endothelial cells ( P < 0.05, all cases). Consistent with these in vitro data, quercetin attenuated lipopolysaccharide-induced endothelial dysfunction and increase of MPO activity in mouse aortas, while this flavonoid could protect against HOCl-mediated endothelial dysfunction in isolated aortas ( P < 0.05). Therefore, it was proposed that quercetin attenuated endothelial injury in inflammatory vasculature via inhibition of vascular-bound MPO-mediated HOCl formation or scavenging of HOCl. These data indicate that quercetin is a nontoxic inhibitor of MPO activity and MPO/neutrophils-induced cytotoxicity in endothelial cells and may be useful for targeting MPO-dependent vascular disease and inflammation.

Preservation of endothelium-dependent relaxation in atherosclerotic mice with endothelium-restricted endothelin-1 overexpression.

PubMed

Mian, Muhammad Oneeb Rehman; Idris-Khodja, Noureddine; Li, Melissa W; Leibowitz, Avshalom; Paradis, Pierre; Rautureau, Yohann; Schiffrin, Ernesto L

2013-10-01

In human atherosclerosis, which is associated with elevated plasma and coronary endothelin (ET)-1 levels, ETA receptor antagonists improve coronary endothelial function. Mice overexpressing ET-1 specifically in the endothelium (eET-1) crossed with atherosclerosis-prone apolipoprotein E knockout mice (Apoe(-/-)) exhibit exaggerated high-fat diet (HFD)-induced atherosclerosis. Since endothelial dysfunction often precedes atherosclerosis development, we hypothesized that mice overexpressing endothelial ET-1 on a genetic background deficient in apolipoprotein E (eET-1/Apoe(-/-)) would have severe endothelial dysfunction. To test this hypothesis, we investigated endothelium-dependent relaxation (EDR) to acetylcholine in eET-1/Apoe(-/-) mice. EDR in mesenteric resistance arteries from 8- and 16-week-old mice fed a normal diet or HFD was improved in eET-1/Apoe(-/-) compared with Apoe(-/-) mice. Nitric oxide synthase (NOS) inhibition abolished EDR in Apoe(-/-). EDR in eET-1/Apoe(-/-) mice was resistant to NOS inhibition irrespective of age or diet. Inhibition of cyclooxygenase, the cytochrome P450 pathway, and endothelium-dependent hyperpolarization (EDH) resulted in little or no inhibition of EDR in eET-1/Apoe(-/-) compared with wild-type (WT) mice. In eET-1/Apoe(-/-) mice, blocking of EDH or soluble guanylate cyclase (sGC), in addition to NOS inhibition, decreased EDR by 36 and 30%, respectively. The activation of 4-aminopyridine-sensitive voltage-dependent potassium channels (Kv) during EDR was increased in eET-1/Apoe(-/-) compared with WT mice. We conclude that increasing eET-1 in mice that develop atherosclerosis results in decreased mutual dependence of endothelial signaling pathways responsible for EDR, and that NOS-independent activation of sGC and increased activation of Kv are responsible for enhanced EDR in this model of atherosclerosis associated with elevated endothelial and circulating ET-1.

Interaction between the Stress Phase Angle (SPA) and the Oscillatory Shear Index (OSI) Affects Endothelial Cell Gene Expression.

PubMed

Amaya, Ronny; Cancel, Limary M; Tarbell, John M

2016-01-01

Hemodynamic forces play an important role in the non-uniform distribution of atherosclerotic lesions. Endothelial cells are exposed simultaneously to fluid wall shear stress (WSS) and solid circumferential stress (CS). Due to variations in impedance (global factors) and geometric complexities (local factors) in the arterial circulation a time lag arises between these two forces that can be characterized by the temporal phase angle between CS and WSS (stress phase angle-SPA). Asynchronous flows (SPA close to -180°) that are most prominent in coronary arteries have been associated with localization of atherosclerosis. Reversing oscillatory flows characterized by an oscillatory shear index (OSI) that is great than zero are also associated with atherosclerosis localization. In this study we examined the relationship between asynchronous flows and reversing flows in altering the expression of 37 genes relevant to atherosclerosis development. In the case of reversing oscillatory flow, we observed that the asynchronous condition upregulated 8 genes compared to synchronous hemodynamics, most of them proatherogenic. Upregulation of the pro-inflammatory transcription factor NFκB p65 was confirmed by western blot, and nuclear translocation of NFκB p65 was confirmed by immunofluorescence staining. A comparative study between non-reversing flow and reversing flow found that in the case of synchronous hemodynamics, reversing flow altered the expression of 11 genes, while in the case of asynchronous hemodynamics, reversing flow altered the expression of 17 genes. Reversing flow significantly upregulated protein expression of NFκB p65 for both synchronous and asynchronous conditions. Nuclear translocation of NFκB p65 was confirmed for synchronous and asynchronous conditions in the presence of flow reversal. These data suggest that asynchronous hemodynamics and reversing flow can elicit proatherogenic responses in endothelial cells compared to synchronous hemodynamics without shear stress reversal, indicating that SPA as well as reversal flow (OSI) are important parameters characterizing arterial susceptibility to disease.

Interaction between the Stress Phase Angle (SPA) and the Oscillatory Shear Index (OSI) Affects Endothelial Cell Gene Expression

PubMed Central

Amaya, Ronny; Cancel, Limary M.; Tarbell, John M.

2016-01-01

Hemodynamic forces play an important role in the non-uniform distribution of atherosclerotic lesions. Endothelial cells are exposed simultaneously to fluid wall shear stress (WSS) and solid circumferential stress (CS). Due to variations in impedance (global factors) and geometric complexities (local factors) in the arterial circulation a time lag arises between these two forces that can be characterized by the temporal phase angle between CS and WSS (stress phase angle–SPA). Asynchronous flows (SPA close to -180°) that are most prominent in coronary arteries have been associated with localization of atherosclerosis. Reversing oscillatory flows characterized by an oscillatory shear index (OSI) that is great than zero are also associated with atherosclerosis localization. In this study we examined the relationship between asynchronous flows and reversing flows in altering the expression of 37 genes relevant to atherosclerosis development. In the case of reversing oscillatory flow, we observed that the asynchronous condition upregulated 8 genes compared to synchronous hemodynamics, most of them proatherogenic. Upregulation of the pro-inflammatory transcription factor NFκB p65 was confirmed by western blot, and nuclear translocation of NFκB p65 was confirmed by immunofluorescence staining. A comparative study between non-reversing flow and reversing flow found that in the case of synchronous hemodynamics, reversing flow altered the expression of 11 genes, while in the case of asynchronous hemodynamics, reversing flow altered the expression of 17 genes. Reversing flow significantly upregulated protein expression of NFκB p65 for both synchronous and asynchronous conditions. Nuclear translocation of NFκB p65 was confirmed for synchronous and asynchronous conditions in the presence of flow reversal. These data suggest that asynchronous hemodynamics and reversing flow can elicit proatherogenic responses in endothelial cells compared to synchronous hemodynamics without shear stress reversal, indicating that SPA as well as reversal flow (OSI) are important parameters characterizing arterial susceptibility to disease. PMID:27846267

Galpha13 regulates MEF2-dependent gene transcription in endothelial cells: role in angiogenesis.

PubMed

Liu, Guoquan; Han, Jingyan; Profirovic, Jasmina; Strekalova, Elena; Voyno-Yasenetskaya, Tatyana A

2009-01-01

The alpha subunit of heterotrimeric G13 protein is required for the embryonic angiogenesis (Offermanns et al., Science 275:533-536, 1997). However, the molecular mechanism of Galpha13-dependent angiogenesis is not understood. Here, we show that myocyte-specific enhancer factor-2 (MEF2) mediates Galpha13-dependent angiogenesis. Our data showed that constitutively activated Galpha13Q226L stimulated MEF2-dependent gene transcription. In addition, downregulation of endogenous Galpha13 inhibited thrombin-stimulated MEF2-dependent gene transcription in endothelial cells. Both Ca(2+)/calmodulin-dependent kinase IV (CaMKIV) and histone deacetylase 5 (HDAC5) were involved in Galpha13-mediated MEF2-dependent gene transcription. Galpha13Q226L also increased Ca(2+)/calmodulin-independent CaMKIV activity, while dominant negative mutant of CaMKIV inhibited MEF2-dependent gene transcription induced by Galpha13Q226L. Furthermore, Galpha13Q226L was able to derepress HDAC5-mediated repression of gene transcription and induce the translocation of HDAC5 from nucleus to cytoplasm. Finally, downregulation of endogenous Galpha13 and MEF2 proteins in endothelial cells reduced cell proliferation and capillary tube formation. Decrease of endothelial cell proliferation that was caused by the Galpha13 downregulation was partially restored by the constitutively active MEF2-VP16. Our studies suggest that MEF2 proteins are an important component in Galpha13-mediated angiogenesis.

Angiotensin II type 1 receptor blockers prevent tumor necrosis factor-alpha-mediated endothelial nitric oxide synthase reduction and superoxide production in human umbilical vein endothelial cells.

PubMed

Kataoka, Hiroki; Murakami, Ryuichiro; Numaguchi, Yasushi; Okumura, Kenji; Murohara, Toyoaki

2010-06-25

Decrease in endothelial nitric oxide synthase (eNOS) expression is one of the adverse outcomes of endothelial dysfunction. Tumor necrosis factor-alpha (TNF-alpha) is known to decrease eNOS expression and is an important mediator of endothelial dysfunction. We hypothesized that an angiotensin II type 1 (AT1) receptor blocker would improve endothelial function via not only inhibition of the angiotensin II signaling but also inhibition of the TNF-alpha-mediated signaling. Therefore we investigated whether an AT1 receptor blocker would restore the TNF-alpha-induced decrease in eNOS expression in cultured human umbilical vein endothelial cells (HUVEC). Pretreatment of HUVEC with an antioxidant (superoxide dismutase, alpha-tocopherol) or AT1 receptor blockers (olmesartan or candesartan) restored the TNF-alpha-dependent reduction of eNOS. The AT1 receptor blocker decreased the TNF-alpha-dependent increase of 8-isoprostane. The superoxide dismutase activities in HUVEC were stable during AT1 receptor blocker treatment, and the AT1 receptor blocker did not scavenge superoxide directly. The AT1 receptor blocker also decreased TNF-alpha-induced phosphorylation of I kappaB alpha and cell death. These results suggest that AT1 receptor blockers are able to ameliorate TNF-alpha-dependent eNOS reduction or cell injury by inhibiting superoxide production or nuclear factor-kappaB activation. (c) 2010 Elsevier B.V. All rights reserved.

Flavanol-rich cocoa ameliorates lipemia-induced endothelial dysfunction.

PubMed

Westphal, Sabine; Luley, Claus

2011-09-01

Consumption of flavanols improves chronic endothelial dysfunction. We investigated whether it can also improve acute lipemia-induced endothelial dysfunction. In this randomized, placebo-controlled, double-blind, crossover trial, 18 healthy subjects received a fatty meal with cocoa either rich in flavanols (918 mg) or flavanol-poor. Flow-mediated dilation (FMD), triglycerides, and free fatty acids were then determined over 6 h. After the flavanol-poor fat loading, the FMD deteriorated over 4 h. The consumption of flavanol-rich cocoa, in contrast, improved this deterioration in hours 2, 3, and 4 without abolishing it completely. Flavanols did not have any influence on triglycerides or on free fatty acids. Flavanol-rich cocoa can alleviate the lipemia-induced endothelial dysfunction, probably through an improvement in endothelial NO synthase.

Endothelial mineralocorticoid receptor activation mediates endothelial dysfunction in diet-induced obesity.

PubMed

Schäfer, Nicola; Lohmann, Christine; Winnik, Stephan; van Tits, Lambertus J; Miranda, Melroy X; Vergopoulos, Athanasios; Ruschitzka, Frank; Nussberger, Jürg; Berger, Stefan; Lüscher, Thomas F; Verrey, François; Matter, Christian M

2013-12-01

Aldosterone plays a crucial role in cardiovascular disease. 'Systemic' inhibition of its mineralocorticoid receptor (MR) decreases atherosclerosis by reducing inflammation and oxidative stress. Obesity, an important cardiovascular risk factor, is an inflammatory disease associated with increased plasma aldosterone levels. We have investigated the role of the 'endothelial' MR in obesity-induced endothelial dysfunction, the earliest stage in atherogenesis. C57BL/6 mice were exposed to a normal chow diet (ND) or a high-fat diet (HFD) alone or in combination with the MR antagonist eplerenone (200 mg/kg/day) for 14 weeks. Diet-induced obesity impaired endothelium-dependent relaxation in response to acetylcholine, whereas eplerenone treatment of obese mice prevented this. Expression analyses in aortic endothelial cells isolated from these mice revealed that eplerenone attenuated expression of pro-oxidative NADPH oxidase (subunits p22phox, p40phox) and increased expression of antioxidative genes (glutathione peroxidase-1, superoxide dismutase-1 and -3) in obesity. Eplerenone did not affect obesity-induced upregulation of cyclooxygenase (COX)-1 or prostacyclin synthase. Endothelial-specific MR deletion prevented endothelial dysfunction in obese (exhibiting high 'endogenous' aldosterone) and in 'exogenous' aldosterone-infused lean mice. Pre-incubation of aortic rings from aldosterone-treated animals with the COX-inhibitor indomethacin restored endothelial function. Exogenous aldosterone administration induced endothelial expression of p22phox in the presence, but not in the absence of the endothelial MR. Obesity-induced endothelial dysfunction depends on the 'endothelial' MR and is mediated by an imbalance of oxidative stress-modulating mechanisms. Therefore, MR antagonists may represent an attractive therapeutic strategy in the increasing population of obese patients to decrease vascular dysfunction and subsequent atherosclerotic complications.

Experimental study of laminar blood flow through an artery treated by a stent implantation: characterisation of intra-stent wall shear stress.

PubMed

Benard, Nicolas; Coisne, Damien; Donal, Erwan; Perrault, Robert

2003-07-01

The stimulation of endothelial cells by arterial wall shear stress (WSS) plays a central role in restenosis. The fluid-structure interaction between stent wire and blood flow alters the WSS, particularly between stent struts. We have designed an in vitro model of struts of an intra-vascular prosthesis to study blood flow through a 'stented' section. The experimental artery consisted of a transparent square section test vein, which reproduced the strut design (100x magnifying power). A programmable pump was used to maintain a steady blood flow. Particle image velocimetry method was used to measure the flow between and over the stent branches, and to quantify WSS. Several prosthesis patterns that were representative of the total stent strut geometry were studied in a greater detail. We obtained WSS values of between -1.5 and 1.5Pa in a weak SS area which provided a source of endothelial stimulation propitious to restenosis. We also compared two similar patterns located in two different flow areas (one at the entry of the stent and one further downstream). We only detected a slight difference between the weakest SS levels at these two sites. As the endothelial proliferation is greatly influenced by the SS, knowledge of the SS modification induced by the stent implantation could be of importance for intra-vascular prostheses design optimisation and thus can help to reduce the restenosis incidence rate.

Effect of Intermittent Energy Restriction on Flow Mediated Dilatation, a Measure of Endothelial Function: A Short Report.

PubMed

Headland, Michelle L; Clifton, Peter M; Keogh, Jennifer B

2018-06-04

Intermittent energy restriction is a popular alternative to daily energy restriction for weight loss; however, it is unknown if endothelial function, a risk factor for cardiovascular disease, is altered by periods of severe energy restriction. The objective of the study was to determine the impact of two consecutive very low energy intake days, which is the core component of the 5:2 intermittent energy restriction diet strategy, on endothelial function compared to consecutive ad libitum eating days. The secondary objective was to explore the effects of these dietary conditions on fasting glucose concentrations. This was a 4-week randomized, single-blinded, crossover study of 35 participants. Participants consumed a very low energy diet (500 calories for women, 600 calories for men) on two consecutive days per week and 5 days of habitual eating. In weeks 3 and 4 of the trial, participants had measurements of flow mediated dilatation (FMD) and blood samples taken following either 2 habitual eating days or 2 energy restricted days in a randomized order. FMD values were not different after the two eating states (8.6% vs. 8.3%, p = 0.7). All other outcome variables were unchanged. Endothelial function, as measured by flow mediated dilatation, was not altered by two consecutive very low energy intake days. Further investigations assessing the impact in specific population groups as well as different testing conditions would be beneficial.

Biology and flow cytometry of proangiogenic hematopoietic progenitors cells.

PubMed

Rose, Jonathan A; Erzurum, Serpil; Asosingh, Kewal

2015-01-01

During development, hematopoiesis and neovascularization are closely linked to each other via a common bipotent stem cell called the hemangioblast that gives rise to both hematopoietic cells and endothelial cells. In postnatal life, this functional connection between the vasculature and hematopoiesis is maintained by a subset of hematopoietic progenitor cells endowed with the capacity to differentiate into potent proangiogenic cells. These proangiogenic hematopoietic progenitors comprise a specific subset of bone marrow (BM)-derived cells that homes to sites of neovascularization and possess potent paracrine angiogenic activity. There is emerging evidence that this subpopulation of hematopoietic progenitors plays a critical role in vascular health and disease. Their angiogenic activity is distinct from putative "endothelial progenitor cells" that become structural cells of the endothelium by differentiation into endothelial cells. Proangiogenic hematopoietic progenitor cell research requires multidisciplinary expertise in flow cytometry, hematology, and vascular biology. This review provides a comprehensive overview of proangiogenic hematopoietic progenitor cell biology and flow cytometric methods to detect these cells in the peripheral blood circulation and BM. © 2014 International Society for Advancement of Cytometry.

Acetylcholine released by endothelial cells facilitates flow‐mediated dilatation

PubMed Central

Wilson, Calum; Lee, Matthew D.

2016-01-01

Key points The endothelium plays a pivotal role in the vascular response to chemical and mechanical stimuli.The endothelium is exquisitely sensitive to ACh, although the physiological significance of ACh‐induced activation of the endothelium is unknown.In the present study, we investigated the mechanisms of flow‐mediated endothelial calcium signalling.Our data establish that flow‐mediated endothelial calcium responses arise from the autocrine action of non‐neuronal ACh released by the endothelium. Abstract Circulating blood generates frictional forces (shear stress) on the walls of blood vessels. These frictional forces critically regulate vascular function. The endothelium senses these frictional forces and, in response, releases various vasodilators that relax smooth muscle cells in a process termed flow‐mediated dilatation. Although some elements of the signalling mechanisms have been identified, precisely how flow is sensed and transduced to cause the release of relaxing factors is poorly understood. By imaging signalling in large areas of the endothelium of intact arteries, we show that the endothelium responds to flow by releasing ACh. Once liberated, ACh acts to trigger calcium release from the internal store in endothelial cells, nitric oxide production and artery relaxation. Flow‐activated release of ACh from the endothelium is non‐vesicular and occurs via organic cation transporters. ACh is generated following mitochondrial production of acetylCoA. Thus, we show ACh is an autocrine signalling molecule released from endothelial cells, and identify a new role for the classical neurotransmitter in endothelial mechanotransduction. PMID:27730645

Vascular endothelial cells minimize the total force on their nuclei.

PubMed Central

Hazel, A L; Pedley, T J

2000-01-01

The vascular endothelium is a cellular monolayer that lines the arterial walls. It plays a vital role in the initiation and development of atherosclerosis, an occlusive arterial disease responsible for 50% of deaths in the Western world. The focal nature of the disease suggests that hemodynamic forces are an important factor in its pathogenesis. This has led to the investigation of the effects of mechanical forces on the endothelial cells themselves. It has been found that endothelial cells do respond to stresses induced by the flowing blood; in particular, they elongate and align with an imposed flow direction. In this paper, we calculate the distribution of force exerted on a three-dimensional hump, representing the raised cell nucleus, by a uniform shear flow. It is found that, for a nonaxisymmetric ellipsoidal hump, the least total force is experienced when the hump is aligned with the flow. Furthermore, for a hump of fixed volume, there is a specific aspect ratio combination that results in the least total force upon the hump, (0.38:2.2:1.0; height:length:width). This is approximately the same as the average aspect ratio taken up by the cell nuclei in vivo (0.27:2.23:1.0). It is possible, therefore, that the cells respond to the flow in such a way as to minimize the total force on their nuclei. PMID:10620272

Potential of Food and Natural Products to Promote Endothelial and Vascular Health.

PubMed

Auger, Cyril; Said, Amissi; Nguyen, Phuong Nga; Chabert, Philippe; Idris-Khodja, Noureddine; Schini-Kerth, Valérie B

2016-07-01

Endothelial dysfunction is now well established as a pivotal early event in the development of major cardiovascular diseases including hypertension, atherosclerosis, and diabetes. The alteration of the endothelial function is often triggered by an imbalance between the endothelial formation of vasoprotective factors including nitric oxide (NO) and endothelium-dependent hyperpolarization, and an increased level of oxidative stress involving several prooxidant enzymes such as NADPH oxidase and, often also, the appearance of cyclooxygenase-derived vasoconstrictors. Preclinical studies have indicated that polyphenol-rich food and food-derived products such as grape-derived products, black and red berries, green and black teas and cocoa, and omega-3 fatty acids can trigger activating pathways in endothelial cells promoting an increased formation of nitric oxide and endothelium-dependent hyperpolarization. Moreover, intake of such food-derived products has been associated with the prevention and/or the improvement of an established endothelial dysfunction in several experimental models of cardiovascular diseases and in humans with cardiovascular diseases. This review will discuss both experimental and clinical evidences indicating that different types of food and natural products are able to promote endothelial and vascular health, as well as the underlying mechanisms.

New assessment of endothelial function measured by short time flow-mediated vasodilation: Comparison with conventional flow-mediated vasodilation measurement.

PubMed

Matsui, Shogo; Kajikawa, Masato; Maruhashi, Tatsuya; Hashimoto, Haruki; Kihara, Yasuki; Chayama, Kazuaki; Goto, Chikara; Aibara, Yoshiki; Yusoff, Farina Mohamad; Kishimoto, Shinji; Nakashima, Ayumu; Noma, Kensuke; Kawaguchi, Tomohiro; Matsumoto, Takeo; Higashi, Yukihito

2018-05-04

Measurement of flow-mediated vasodilation (FMD) is an established method for assessing endothelial function. Measurement of FMD is useful for showing the relationship between atherosclerosis and endothelial function, mechanisms of endothelial dysfunction, and clinical implications including effects of interventions and cardiovascular events. To shorten and simplify the measurement of FMD, we have developed a novel technique named short time FMD (stFMD). We investigated the validity of stFMD for assessment of endothelial function compared with conventional FMD. We evaluated stFMD and conventional FMD in 82 subjects including patients with atherosclerotic risk factors and cardiovascular disease (66 men and 16 women, 57 ± 16 years). Both stFMD and conventional FMD were significantly correlated with age, systolic blood pressure, diastolic blood pressure and baseline brachial artery diameter. In addition, stFMD was significantly correlated with conventional FMD (r = 0.76, P < 0.001). Bland-Altman plot analysis showed good agreement between stFMD and conventional FMD. Moreover, stFMD in the at risk group and that in the cardiovascular disease group were significantly lower than that in the no risk group (4.6 ± 2.3% and 4.4 ± 2.2% vs. 7.3 ± 1.9%, P < 0.001, respectively). Optimal cutoff value of stFMD for diagnosing atherosclerosis was 7.0% (sensitivity of 71.0% and specificity of 85.0%). These findings suggest that measurement of stFMD, a novel and simple method, is useful for assessing endothelial function. Measurement of stFMD may be suitable for screening of atherosclerosis when repeated measurements of vascular function are required and when performing a clinical trial using a large population. URL for Clinical Trial: http://UMIN; Registration Number for Clinical Trial: UMIN000025458. Copyright © 2017 Elsevier B.V. All rights reserved.

Acute vascular effects of carbonated warm water lower leg immersion in healthy young adults.

PubMed

Ogoh, Shigehiko; Nagaoka, Ryohei; Mizuno, Takamasa; Kimura, Shohei; Shidahara, Yasuhiro; Ishii, Tomomi; Kudoh, Michinari; Iwamoto, Erika

2016-12-01

Endothelial dysfunction is associated with increased cardiovascular mortality and morbidity; however, this dysfunction may be ameliorated by several therapies. For example, it has been reported that heat-induced increases in blood flow and shear stress enhance endothelium-mediated vasodilator function. Under these backgrounds, we expect that carbon dioxide (CO 2 )-rich water-induced increase in skin blood flow improves endothelium-mediated vasodilation with less heat stress. To test our hypothesis, we measured flow-mediated dilation (FMD) before and after acute immersion of the lower legs and feet in mild warm (38°C) normal or CO 2 -rich tap water (1000 ppm) for 20 min in 12 subjects. Acute immersion of the lower legs and feet in mild warm CO 2 -rich water increased FMD (P < 0.01) despite the lack of change in this parameter upon mild warm normal water immersion. In addition, FMD was positively correlated with change in skin blood flow regardless of conditions (P < 0.01), indicating that an increase in skin blood flow improves endothelial-mediated vasodilator function. Importantly, the temperature of normal tap water must reach approximately 43°C to achieve the same skin blood flow level as that obtained during mild warm CO 2 -rich water immersion (38°C). These findings suggest that CO 2 -rich water-induced large increases in skin blood flow may improve endothelial-mediated vasodilator function while causing less heat stress. © 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Effects of venous needle turbulence during ex vivo hemodialysis on endothelial morphology and nitric oxide formation.

PubMed

Huynh, Thanh N; Chacko, Balu K; Teng, Xinjun; Brott, Brigitta C; Allon, Michael; Kelpke, Stacey S; Thompson, John A; Patel, Rakesh P; Anayiotos, Andreas S

2007-01-01

Arteriovenous grafts used for hemodialysis frequently develop intimal hyperplasia (IH), which ultimately leads to graft failure. Although the turbulent jet from the dialysis needle may contribute to vessel wall injury, its role in the pathogenesis of IH is relatively unexplored. In the current study, using bovine aortic endothelial cells (BAEC) cultured on the inner surface of a compliant tube, we evaluated the effects of simulated hemodialysis conditions on morphology and nitric oxide (NO) production. The flows via the graft and needle were 500 ml/min (Reynolds number=819) and 100ml/min (Reynolds number=954), respectively. In the presence of the needle jet for 6h, 19.3% (+/-1.53%) of BAEC were sheared off, whereas no loss of BAEC was observed in the presence of graft flow alone (P<0.05). In the presence of graft flow alone, assessment of cell orientation by the Saltykov method revealed that BAEC were oriented along the flow direction. This alignment, however, was lost in the presence of needle flow. Finally, NO production was also significantly decreased in the presence of the needle flow compared to the presence of graft flow alone (16+/-3.1 vs 34.7+/-1.9 nmol/10(6)cells/h, P<0.05). NO is a key player in vascular homeostasis mechanisms modulating vasomotor tone, inhibiting inflammation and smooth muscle cell proliferation. Thus, the loss of NO signaling and the loss of endothelial integrity caused by needle jet turbulence may contribute to the cascade of events leading to IH formation during hemodialysis.

Inter-Cellular Exchange of Cellular Components via VE-Cadherin-Dependent Trans-Endocytosis

PubMed Central

Sakurai, Takashi; Woolls, Melissa J.; Jin, Suk-Won

2014-01-01

Cell-cell communications typically involve receptor-mediated signaling initiated by soluble or cell-bound ligands. Here, we report a unique mode of endocytosis: proteins originating from cell-cell junctions and cytosolic cellular components from the neighboring cell are internalized, leading to direct exchange of cellular components between two adjacent endothelial cells. VE-cadherins form transcellular bridges between two endothelial cells that are the basis of adherence junctions. At such adherens junction sites, we observed the movement of the entire VE-cadherin molecule from one endothelial cell into the other with junctional and cytoplasmic components. This phenomenon, here termed trans-endocytosis, requires the establishment of a VE-cadherin homodimer in trans with internalization proceeding in a Rac1-, and actomyosin-dependent manner. Importantly, the trans-endocytosis is not dependent on any known endocytic pathway including clathrin-dependent endocytosis, macropinocytosis or phagocytosis. This novel form of cell-cell communications, leading to a direct exchange of cellular components, was observed in 2D and 3D-cultured endothelial cells as well as in the developing zebrafish vasculature. PMID:24603875

Assessing the Impact of Diabetes Mellitus on Donor Corneal Endothelial Cell Density.

PubMed

Liaboe, Chase A; Aldrich, Benjamin T; Carter, Pamela C; Skeie, Jessica M; Burckart, Kimberlee A; Schmidt, Gregory A; Reed, Cynthia R; Zimmerman, M Bridget; Greiner, Mark A

2017-05-01

To quantify changes in endothelial cell density (ECD) of donor corneal tissue in relation to the presence or absence of a medical history of diabetes mellitus diagnosis, treatment, and complications. A retrospective review was performed for all corneas collected at Iowa Lions Eye Bank between January 2012 and December 2015. For purposes of analysis, donor corneas were divided into 4 groups: nondiabetic, non-insulin-dependent diabetic, insulin-dependent diabetic without medical complications due to diabetes, and insulin-dependent diabetic with medical complications due to diabetes. ECD values (obtained through specular microscopy) and transplant suitability for endothelial transplantation (determined by the standard protocol of the eye bank) were compared among groups using linear mixed model analysis. In total, 4185 corneas from 2112 donors were included for analysis. Insulin-dependent diabetic samples with medical complications due to diabetes (N = 231 from 119 donors) showed lower ECD values compared with nondiabetic samples (-102 cells/mm, P = 0.049) and non-insulin-dependent diabetic samples (-117 cells/mm, P = 0.031). ECD values did not differ significantly among the remaining groups. The likelihood of suitability for endothelial transplantation did not differ among all 4 groups. Corneas from donors with insulin-dependent diabetes mellitus and medical complications resulting from the disease have lower mean ECD values compared with other donors. However, our analysis suggests that these corneas are equally likely to be included in the donor pool for corneal transplantation. Additional studies are needed to determine the mechanism(s) contributing to cell loss in donors with advanced diabetes and to assess associated endothelial cell functional impairment.

Gas6 Promotes Inflammatory (CCR2hiCX3CR1lo) Monocyte Recruitment in Venous Thrombosis.

PubMed

Laurance, Sandrine; Bertin, François-René; Ebrahimian, Talin; Kassim, Yusra; Rys, Ryan N; Lehoux, Stéphanie; Lemarié, Catherine A; Blostein, Mark D

2017-07-01

Coagulation and inflammation are inter-related. Gas6 (growth arrest-specific 6) promotes venous thrombosis and participates to inflammation through endothelial-innate immune cell interactions. Innate immune cells can provide the initiating stimulus for venous thrombus development. We hypothesize that Gas6 promotes monocyte recruitment during venous thrombosis. Deep venous thrombosis was induced in wild-type and Gas6-deficient (-/-) mice using 5% FeCl 3 and flow reduction in the inferior vena cava. Total monocyte depletion was achieved by injection of clodronate before deep venous thrombosis. Inflammatory monocytes were depleted using an anti-C-C chemokine receptor type 2 (CCR2) antibody. Similarly, injection of an anti-chemokine ligand 2 (CCL2) antibody induced CCL2 depletion. Flow cytometry and immunofluorescence were used to characterize the monocytes recruited to the thrombus. In vivo, absence of Gas6 was associated with a reduction of monocyte recruitment in both deep venous thrombosis models. Global monocyte depletion by clodronate leads to smaller thrombi in wild-type mice. Compared with wild type, the thrombi from Gas6 -/- mice contain less inflammatory (CCR2 hi CX 3 CR1 lo ) monocytes, consistent with a Gas6-dependent recruitment of this monocyte subset. Correspondingly, selective depletion of CCR2 hi CX 3 CR1 lo monocytes reduced the formation of venous thrombi in wild-type mice demonstrating a predominant role of the inflammatory monocytes in thrombosis. In vitro, the expression of both CCR2 and CCL2 were Gas6 dependent in monocytes and endothelial cells, respectively, impacting monocyte migration. Moreover, Gas6-dependent CCL2 expression and monocyte migration were mediated via JNK (c-Jun N-terminal kinase). This study demonstrates that Gas6 specifically promotes the recruitment of inflammatory CCR2 hi CX 3 CR1 lo monocytes through the regulation of both CCR2 and CCL2 during deep venous thrombosis. © 2017 American Heart Association, Inc.

Evidence for circulatory benefits of resveratrol in humans.

PubMed

Wong, Rachel H X; Coates, Alison M; Buckley, Jonathan D; Howe, Peter R C

2013-07-01

Impairments of endothelial function, which can be assessed noninvasively by flow-mediated dilation (FMD) of the brachial artery, contribute to the development of cardiovascular disease. Associations between FMD and cognition suggest a vascular component in the loss of cognitive function. Certain vasoactive nutrients that have been shown to improve FMD may also have the potential to enhance cerebral perfusion and cognition. Preclinical studies show that trans-resveratrol can enhance nitric oxide bioavailability, thereby increasing endothelium-dependent vasodilation. We have now shown that acute administration of resveratrol elicits dose-dependent increases of FMD with greater potency than other vasoactive nutrients and that this benefit is sustained following regular consumption. We describe the potential implications of this vasodilator benefit of resveratrol and its role in enhancing cerebrovascular and cognitive functions. © 2013 New York Academy of Sciences.

Study of endothelial cell apoptosis using fluorescence resonance energy transfer (FRET) biosensor cell line with hemodynamic microfluidic chip system.

PubMed

Yu, J Q; Liu, X F; Chin, L K; Liu, A Q; Luo, K Q

2013-07-21

To better understand how hyperglycemia induces endothelial cell dysfunction under the diabetic conditions, a hemodynamic microfluidic chip system was developed. The system combines a caspase-3-based fluorescence resonance energy transfer (FRET) biosensor cell line which can detect endothelial cell apoptosis in real-time, post-treatment effect and with a limited cell sample, by using a microfluidic chip which can mimic the physiological pulsatile flow profile in the blood vessel. The caspase-3-based FRET biosensor endothelial cell line (HUVEC-C3) can produce a FRET-based sensor protein capable of probing caspase-3 activation. When the endothelial cells undergo apoptosis, the color of the sensor cells changes from green to blue, thus sensing apoptosis. A double-labeling fluorescent technique (yo pro-1 and propidium iodide) was used to validate the findings revealed by the FRET-based caspase sensor. The results show high rates of apoptosis and necrosis of endothelial cells when high glucose concentration was applied in our hemodynamic microfluidic chip combined with an exhaustive pulsatile flow profile. The two apoptosis detection techniques (fluorescent method and FRET biosensor) are comparable; but FRET biosensor offers more advantages such as real-time observation and a convenient operating process to generate more accurate and reliable data. Furthermore, the activation of the FRET biosensor also confirms the endothelial cell apoptosis induced by the abnormal pulsatile shear stress and high glucose concentration is through caspase-3 pathway. A 12% apoptotic rate (nearly a 4-fold increase compared to the static condition) was observed when the endothelial cells were exposed to a high glucose concentration of 20 mM under 2 h exhaustive pulsatile shear stress of 30 dyne cm(-2) and followed with another 10 h normal pulsatile shear stress of 15 dyne cm(-2). Therefore, the most important finding of this study is to develop a novel endothelial cell apoptosis detection method, which combines the microfluidic chip system and FRET biosensor. This finding may provide new insight into how glucose causes endothelial cell dysfunction, which is the major cause of diabetes-derived complications.

Chronic administration of the probiotic kefir improves the endothelial function in spontaneously hypertensive rats.

PubMed

Friques, Andreia G F; Arpini, Clarisse M; Kalil, Ieda C; Gava, Agata L; Leal, Marcos A; Porto, Marcella L; Nogueira, Breno V; Dias, Ananda T; Andrade, Tadeu U; Pereira, Thiago Melo C; Meyrelles, Silvana S; Campagnaro, Bianca P; Vasquez, Elisardo C

2015-12-30

The beverage obtained by fermentation of milk with kefir grains, a complex matrix containing acid bacteria and yeasts, has been shown to have beneficial effects in various diseases. However, its effects on hypertension and endothelial dysfunction are not yet clear. In this study, we evaluated the effects of kefir on endothelial cells and vascular responsiveness in spontaneously hypertensive rats (SHR). SHR were treated with kefir (0.3 mL/100 g body weight) for 7, 15, 30 and 60 days and compared with non-treated SHR and with normotensive Wistar-Kyoto rats. Vascular endothelial function was evaluated in aortic rings through the relaxation response to acetylcholine (ACh). The balance between reactive oxygen species (ROS) and nitric oxide (NO) synthase was evaluated through specific blockers in the ACh-induced responses and through flow cytometry in vascular tissue. Significant effects of kefir were observed only after treatment for 60 days. The high blood pressure and tachycardia exhibited by the SHR were attenuated by approximately 15 % in the SHR-kefir group. The impaired ACh-induced relaxation of the aortic rings observed in the SHR (37 ± 4 %, compared to the Wistar rats: 74 ± 5 %), was significantly attenuated in the SHR group chronically treated with kefir (52 ± 4 %). The difference in the area under the curve between before and after the NADPH oxidase blockade or NO synthase blockade of aortic rings from SHR were of approximately +90 and -60 %, respectively, when compared with Wistar rats. In the aortic rings from the SHR-kefir group, these values were reduced to +50 and -40 %, respectively. Flow cytometric analysis of aortic endothelial cells revealed increased ROS production and decreased NO bioavailability in the SHR, which were significantly attenuated by the treatment with kefir. Scanning electronic microscopy showed vascular endothelial surface injury in SHR, which was partially protected following administration of kefir for 60 days. In addition, the recruitment of endothelial progenitor cells was decreased in the non-treated SHR and partially restored by kefir treatment. Kefir treatment for 60 days was able to improve the endothelial function in SHR by partially restoring the ROS/NO imbalance and the endothelial architecture due to endothelial progenitor cells recruitment.

Vasculogenesis of decidua side population cells of first-trimester pregnancy.

PubMed

Wang, Qiushi; Shen, Licong; Huang, Wei; Song, Yong; Xiao, Li; Xu, Wenming; Liu, Ying

2013-05-07

Sufficient uterine blood supply is essential for the fetus to develop normally in the uterus. Several mechanisms are involved in the process of vessel development in deciduas and villus. We focus on whether first-trimester decidua side population (SP) cells contain cells capable of differentiating into endothelial cells. Eight decidua samples were collected from healthy women, 22- to 30-years old, undergoing elective terminations of early pregnancy (six to eight gestational weeks). The cell suspensions from human deciduas were stained by Hoechst 33342 and sorted by flow cytometry, further cultured under differentiation conditions and analyzed for specific markers. These cells were implanted into ischemic limbs of nude mice to test the capacity of angiogenesis in vivo by DiI tracers and immunohistochemistry. Decidua CD31(-)CD146(-) SP cells of first-trimester human pregnancy can differentiate into endothelial cells, express the corresponding specific markers of endothelial cells, such as CD31 and CD146, and form tube-like structures on Matrigel and part of newly formed vessels in the ischemic limbs of nude mice. Vascular endothelial growth factor was more effective in promoting proliferation of CD31(-)CD146(-)SP cells compared with other growth factors, and estrogen and progesterone at a final concentration of 10 μmol/L and 30 μmol/L, respectively, promoted the migration of CD31()-CD146(-)SP cells in a dose-dependent manner. CD31(-)CD146(-) SP cells may be involved in the formation of new vessels in the maternal aspect of the placenta in the first trimester.

[Effects of HOXB2 antisense oligodeoxynucleotides on the biological properties of primary human umbilical vein endothelial cells].

PubMed

Zhang, Xiaoqi; Liu, Xusheng

2002-07-01

To explore the effects of HOXB2 antisense oligodeoxynuc leotides (Asodn) on the biological properties of primary human umbilical vein endothelial cells (ECs). Fluorescent labelled Asodn was transfected into the endothelial cells of human unbilical vein mediated liposome and its distribution within endothelia was observed. (3)H-TdR incorporation test was employed to determine its effects on the DNA synthesis. Flow cytometry was applied to determine the change of the cell cycle. In the same time, RT-PCR was adopted to study the influence of Asodn on the expression of target genes. Fifteen minutes after the transfection, weak nucleic staining was observed. The fluorescent staining was the strongest 4 approximately 8 hours after the transfection and began to weaken in 16 hours. The proportion of cells in G1/0 phase in Asodn group was 53.4 +/- 3.1, significantly higher than that in control group (35.8 +/- 7.3, P < 0.01), and the proportion of cells in S phase in Asodn group was 42.2 +/- 3.5, significantly lower than that in control group (60.8 +/- 6.2, P < 0.01). The expression of HOXB2 mRNA was remarkably decreased during 24 to 48 hours. HOXB2 Asodn exerts inhibitory effects on EC proliferation dose-dependently, delays the conversion of G1 phase to S Phase, and inhibits the expression of HOXB2 mRNA. HOXB2 gene plays an important role in proliferation of endothelial cells and the mechanism is related to cell cycle.

Flavonoid-rich dark chocolate improves endothelial function and increases plasma epicatechin concentrations in healthy adults.

PubMed

Engler, Mary B; Engler, Marguerite M; Chen, Chung Y; Malloy, Mary J; Browne, Amanda; Chiu, Elisa Y; Kwak, Ho-Kyung; Milbury, Paul; Paul, Steven M; Blumberg, Jeffrey; Mietus-Snyder, Michele L

2004-06-01

Dark chocolate derived from the plant (Theobroma cacao) is a rich source of flavonoids. Cardioprotective effects including antioxidant properties, inhibition of platelet activity, and activation of endothelial nitric oxide synthase have been ascribed to the cocoa flavonoids. To investigate the effects of flavonoid-rich dark chocolate on endothelial function, measures of oxidative stress, blood lipids, and blood pressure in healthy adult subjects. The study was a randomized, double-blind, placebo-controlled design conducted over a 2 week period in 21 healthy adult subjects. Subjects were randomly assigned to daily intake of high-flavonoid (213 mg procyanidins, 46 mg epicatechin) or low-flavonoid dark chocolate bars (46 g, 1.6 oz). High-flavonoid chocolate consumption improved endothelium-dependent flow-mediated dilation (FMD) of the brachial artery (mean change = 1.3 +/- 0.7%) as compared to low-flavonoid chocolate consumption (mean change = -0.96 +/- 0.5%) (p = 0.024). No significant differences were noted in the resistance to LDL oxidation, total antioxidant capacity, 8-isoprostanes, blood pressure, lipid parameters, body weight or body mass index (BMI) between the two groups. Plasma epicatechin concentrations were markedly increased at 2 weeks in the high-flavonoid group (204.4 +/- 18.5 nmol/L, p < or = 0.001) but not in the low-flavonoid group (17.5 +/- 9 nmol/L, p = 0.99). Flavonoid-rich dark chocolate improves endothelial function and is associated with an increase in plasma epicatechin concentrations in healthy adults. No changes in oxidative stress measures, lipid profiles, blood pressure, body weight or BMI were seen.

Protective Effects of Hydrogen Sulfide in Hypoxic Human Umbilical Vein Endothelial Cells: A Possible Mitochondria-Dependent Pathway

PubMed Central

Shen, Yaqi; Guo, Wei; Wang, Zhijun; Zhang, Yuchen; Zhong, Liangjie; Zhu, Yizhun

2013-01-01

The aim of the study was to investigate the protective effects of sodium hydrosulfide (NaHS), a H2S donor, against hypoxia-induced injury in human umbilical vein endothelial cells (HUVECs) and also to look into the possible mechanisms by which H2S exerts this protective effect. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and scratch wound healing assay were chosen to measure the cell viability and migration-promoting effects. The fluorescent probe, DCFH-DA and 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1) were applied to detect the reactive oxygen species (ROS) level and mitochondrial membrane potential (ΔΨm). Furthermore, western blots were used to measure the expressions of the apoptosis-related proteins. Under hypoxic conditions, 300 μM and 600 μM of H2S could protect HUVECs against hypoxia-induced injury, as determined by MTT assay. Following the treatment of 60 μM NaHS for 18 h, scratch wound healing assays indicated that the scratch became much narrower than control group. After treatment with 60 μM, 120 μM, and 600 μM NaHS, and hypoxia for 30 min, flow cytometry demonstrated that the ROS concentrations decreased to 95.08% ± 5.52%, 73.14% ± 3.36%, and 73.51% ± 3.05%, respectively, compared with the control group. In addition, the JC-1 assay showed NaHS had a protective effect on mitochondria damage. Additionally, NaHS increased Bcl-2 expression and decreased the expression of Bax, Caspase-3 and Caspase-9 in a dose-dependent way. Our results suggest that H2S can protect endothelial cells and promote migration under hypoxic condition in HUVECs. These effects are partially associated with the preservation of mitochondrial function mediated by regulating the mitochondrial-dependent apoptotic pathway. PMID:23799362

Effects of sevuparin on rosette formation and cytoadherence of Plasmodium falciparum infected erythrocytes

PubMed Central

Saiwaew, Somporn; Sritabal, Juntima; Piaraksa, Nattaporn; Keayarsa, Srisuda; Ruengweerayut, Ronnatrai; Utaisin, Chirapong; Sila, Patima; Niramis, Rangsan; Udomsangpetch, Rachanee; Charunwatthana, Prakaykaew; Pongponratn, Emsri; Pukrittayakamee, Sasithon; Leitgeb, Anna M.; Wahlgren, Mats; Lee, Sue J.; Day, Nicholas P. J.; White, Nicholas J.; Dondorp, Arjen M.; Chotivanich, Kesinee

2017-01-01

In severe falciparum malaria cytoadherence of parasitised red blood cells (PRBCs) to vascular endothelium (causing sequestration) and to uninfected red cells (causing rosette formation) contribute to microcirculatory flow obstruction in vital organs. Heparin can reverse the underlying ligand-receptor interactions, but may increase the bleeding risks. As a heparin-derived polysaccharide, sevuparin has been designed to retain anti-adhesive properties, while the antithrombin-binding domains have been eliminated, substantially diminishing its anticoagulant activity. Sevuparin has been evaluated recently in patients with uncomplicated falciparum malaria, and is currently investigated in a clinical trial for sickle cell disease. The effects of sevuparin on rosette formation and cytoadherence of Plasmodium falciparum isolates from Thailand were investigated. Trophozoite stages of P. falciparum-infected RBCs (Pf-iRBCs) were cultured from 49 patients with malaria. Pf-iRBCs were treated with sevuparin at 37°C and assessed in rosetting and in cytoadhesion assays with human dermal microvascular endothelial cells (HDMECs) under static and flow conditions. The proportion of Pf-iRBCs forming rosettes ranged from 6.5% to 26.0% (median = 12.2%). Rosetting was dose dependently disrupted by sevuparin (50% disruption by 250 μg/mL). Overall 57% of P. falciparum isolates bound to HDMECs under static conditions; median (interquartile range) Pf-iRBC binding was 8.5 (3.0–38.0) Pf-iRBCs/1000 HDMECs. Sevuparin in concentrations ≥ 100 μg/mL inhibited cytoadherence. Sevuparin disrupts P. falciparum rosette formation in a dose dependent manner and inhibits cytoadherence to endothelial cells. The data support assessment of sevuparin as an adjunctive treatment to the standard therapy in severe falciparum malaria. PMID:28249043

Rho-kinase inhibition acutely augments blood flow in focal cerebral ischemia via endothelial mechanisms.

PubMed

Shin, Hwa Kyoung; Salomone, Salvatore; Potts, E Michelle; Lee, Sae-Won; Millican, Eric; Noma, Kensuke; Huang, Paul L; Boas, David A; Liao, James K; Moskowitz, Michael A; Ayata, Cenk

2007-05-01

Rho-kinase is a serine threonine kinase that increases vasomotor tone via its effects on both endothelium and smooth muscle. Rho-kinase inhibition reduces cerebral infarct size in wild type, but not endothelial nitric oxide synthase deficient (eNOS-/-) mice. The mechanism may be related to Rho-kinase activation under hypoxic/ischemic conditions and impaired vasodilation because of downregulation of eNOS activity. To further implicate Rho-kinase in impaired vascular relaxation during hypoxia/ischemia, we exposed isolated vessels from rat and mouse to 60 mins of hypoxia, and showed that hypoxia reversibly abolished acetylcholine-induced eNOS-dependent relaxation, and that Rho-kinase inhibitor hydroxyfasudil partially preserved this relaxation during hypoxia. We, therefore, hypothesized that if hypoxia-induced Rho-kinase activation acutely impairs vasodilation in ischemic cortex, in vivo, then Rho-kinase inhibitors would acutely augment cerebral blood flow (CBF) as a mechanism by which they reduce infarct size. To test this, we studied the acute cerebral hemodynamic effects of Rho-kinase inhibitors in ischemic core and penumbra during distal middle cerebral artery occlusion (dMCAO) in wild-type and eNOS-/- mice using laser speckle flowmetry. When administered 60 mins before or immediately after dMCAO, Rho-kinase inhibitors hydroxyfasudil and Y-27632 reduced the area of severely ischemic cortex. However, hydroxyfasudil did not reduce the area of CBF deficit in eNOS-/- mice, suggesting that its effect on CBF within the ischemic cortex is primarily endothelium-dependent, and not mediated by its direct vasodilator effect on vascular smooth muscle. Our results suggest that Rho-kinase negatively regulates eNOS activity in acutely ischemic brain, thereby worsening the CBF deficit. Therefore, rapid nontranscriptional upregulation of eNOS activity by small molecule inhibitors of Rho-kinase may be a viable therapeutic approach in acute stroke.

Effects of exercise training on cellular mechanisms of endothelial nitric oxide synthase regulation in coronary arteries after chronic occlusion

PubMed Central

Zhou, Minglong; Widmer, R. Jay; Xie, Wei; Jimmy Widmer, A.; Miller, Matthew W.; Schroeder, Friedhelm; Parker, Janet L.

2010-01-01

Exercise training enhances agonist-mediated relaxation in both control and collateral-dependent coronary arteries of hearts subjected to chronic occlusion, an enhancement that is mediated in part by nitric oxide. The purpose of the present study was to elucidate exercise training-induced adaptations in specific cellular mechanisms involved in the regulation of endothelial nitric oxide synthase (eNOS) in coronary arteries of ischemic hearts. Ameroid constrictors were surgically placed around the proximal left circumflex coronary artery (LCX) of adult female Yucatan miniature swine. Eight weeks postoperatively, animals were randomized into sedentary (pen-confined) or exercise training (treadmill run; 5 days/wk; 14 wk) protocols. Coronary artery segments (∼1.0 mm luminal diameter) were isolated from collateral-dependent (LCX) and control (nonoccluded left anterior descending) arteries 22 wk after ameroid placement. Endothelial cells were enzymatically dissociated, and intracellular Ca2+ responses (fura 2) to bradykinin stimulation were studied. Immunofluorescence and laser scanning confocal microscopy were used to quantify endothelial cell eNOS and caveolin-1 cellular distribution under basal and bradykinin-stimulated conditions. Immunoblot analysis was used to determine eNOS, phosphorylated (p)-eNOS, protein kinase B (Akt), pAkt, and caveolin-1 protein levels. Bradykinin-stimulated nitrite plus nitrate (NOx; nitric oxide metabolites) levels were assessed via HPLC. Exercise training resulted in significantly enhanced bradykinin-mediated increases in endothelial Ca2+ levels, NOx levels, and the distribution of eNOS-to-caveolin-1 ratio at the plasma membrane in endothelial cells of control and collateral-dependent arteries. Exercise training also significantly increased total eNOS and phosphorylated levels of eNOS (pSer1179) in collateral-dependent arteries. Total eNOS protein levels were also significantly increased in collateral-dependent arteries of sedentary animals. These data provide new insights into exercise training-induced adaptations in cellular mechanisms of nitric oxide regulation in collateral-dependent coronary arteries of chronically occluded hearts that contribute to enhanced nitric oxide production. PMID:20363881

Flow mediated dilation of the brachial artery: an investigation of methods requiring further standardization

PubMed Central

Peretz, Alon; Leotta, Daniel F; Sullivan, Jeffrey H; Trenga, Carol A; Sands, Fiona N; Aulet, Mary R; Paun, Marla; Gill, Edward A; Kaufman, Joel D

2007-01-01

Background In order to establish a consistent method for brachial artery reactivity assessment, we analyzed commonly used approaches to the test and their effects on the magnitude and time-course of flow mediated dilation (FMD), and on test variability and repeatability. As a popular and noninvasive assessment of endothelial function, several different approaches have been employed to measure brachial artery reactivity with B-mode ultrasound. Despite some efforts, there remains a lack of defined normal values and large variability in measurement technique. Methods Twenty-six healthy volunteers underwent repeated brachial artery diameter measurements by B-mode ultrasound. Following baseline diameter recordings we assessed endothelium-dependent flow mediated dilation by inflating a blood pressure cuff either on the upper arm (proximal) or on the forearm (distal). Results Thirty-seven measures were performed using proximal occlusion and 25 with distal occlusion. Following proximal occlusion relative to distal occlusion, FMD was larger (16.2 ± 1.2% vs. 7.3 ± 0.9%, p < 0.0001) and elongated (107.2 s vs. 67.8 s, p = 0.0001). Measurement of the test repeatability showed that differences between the repeated measures were greater on average when the measurements were done using the proximal method as compared to the distal method (2.4%; 95% CI 0.5–4.3; p = 0.013). Conclusion These findings suggest that forearm compression holds statistical advantages over upper arm compression. Added to documented physiological and practical reasons, we propose that future studies should use forearm compression in the assessment of endothelial function. PMID:17376239

Tumor Endothelial Cells

PubMed Central

Dudley, Andrew C.

2012-01-01

The vascular endothelium is a dynamic cellular “organ” that controls passage of nutrients into tissues, maintains the flow of blood, and regulates the trafficking of leukocytes. In tumors, factors such as hypoxia and chronic growth factor stimulation result in endothelial dysfunction. For example, tumor blood vessels have irregular diameters; they are fragile, leaky, and blood flow is abnormal. There is now good evidence that these abnormalities in the tumor endothelium contribute to tumor growth and metastasis. Thus, determining the biological basis underlying these abnormalities is critical for understanding the pathophysiology of tumor progression and facilitating the design and delivery of effective antiangiogenic therapies. PMID:22393533

The flow dependency of Tie2 expression in endotoxemia.

PubMed

Kurniati, Neng F; Jongman, Rianne M; vom Hagen, Franziska; Spokes, Katherine C; Moser, Jill; Regan, Erzsébet Ravasz; Krenning, Guido; Moonen, Jan-Renier A J; Harmsen, Martin C; Struys, Michel M R F; Hammes, Hans-Peter; Zijlstra, Jan G; Aird, William C; Heeringa, Peter; Molema, Grietje; van Meurs, Matijs

2013-07-01

Tie2 is predominantly expressed by endothelial cells and is involved in vascular integrity control during sepsis. Changes in Tie2 expression during sepsis development may contribute to microvascular dysfunction. Understanding the kinetics and molecular basis of these changes may assist in the development of therapeutic intervention to counteract microvascular dysfunction. To investigate the molecular mechanisms underlying the changes in Tie2 expression upon lipopolysaccharide (LPS) challenge. Studies were performed in LPS and pro-inflammatory cytokine challenged mice as well as in mice subjected to hemorrhagic shock, primary endothelial cells were used for in vitro experiments in static and flow conditions. Eight hours after LPS challenge, Tie2 mRNA loss was observed in all major organs, while loss of Tie2 protein was predominantly observed in lungs and kidneys, in the capillaries. A similar loss could be induced by secondary cytokines TNF-α and IL-1β. Ang2 protein administration did not affect Tie2 protein expression nor was Tie2 protein rescued in LPS-challenged Ang2-deficient mice, excluding a major role for Ang2 in Tie2 down regulation. In vitro, endothelial loss of Tie2 was observed upon lowering of shear stress, not upon LPS and TNF-α stimulation, suggesting that inflammation related haemodynamic changes play a major role in loss of Tie2 in vivo, as also hemorrhagic shock induced Tie2 mRNA loss. In vitro, this loss was partially counteracted by pre-incubation with a pharmacologically NF-кB inhibitor (BAY11-7082), an effect further substantiated in vivo by pre-treatment of mice with the NF-кB inhibitor prior to the inflammatory challenge. Microvascular bed specific loss of Tie2 mRNA and protein in vivo upon LPS, TNFα, IL-1β challenge, as well as in response to hemorrhagic shock, is likely an indirect effect caused by a change in endothelial shear stress. This loss of Tie2 mRNA, but not Tie2 protein, induced by TNFα exposure was shown to be controlled by NF-кB signaling. Drugs aiming at restoring vascular integrity in sepsis could focus on preventing the Tie2 loss.

A fish-based diet intervention improves endothelial function in postmenopausal women with type 2 diabetes mellitus: a randomized crossover trial.

PubMed

Kondo, Keiko; Morino, Katsutaro; Nishio, Yoshihiko; Kondo, Motoyuki; Nakao, Keiko; Nakagawa, Fumiyuki; Ishikado, Atsushi; Sekine, Osamu; Yoshizaki, Takeshi; Kashiwagi, Atsunori; Ugi, Satoshi; Maegawa, Hiroshi

2014-07-01

The beneficial effects of fish and n-3 polyunsaturated fatty acids (PUFAs) consumption on atherosclerosis have been reported in numerous epidemiological studies. However, to the best of our knowledge, the effects of a fish-based diet intervention on endothelial function have not been investigated. Therefore, we studied these effects in postmenopausal women with type 2 diabetes mellitus (T2DM). Twenty-three postmenopausal women with T2DM were assigned to two four-week periods of either a fish-based diet (n-3 PUFAs ≧ 3.0 g/day) or a control diet in a randomized crossover design. Endothelial function was measured with reactive hyperemia using strain-gauge plethysmography and compared with the serum levels of fatty acids and their metabolites. Endothelial function was determined with peak forearm blood flow (Peak), duration of reactive hyperemia (Duration) and flow debt repayment (FDR). A fish-based dietary intervention improved Peak by 63.7%, Duration by 27.9% and FDR by 70.7%, compared to the control diet. Serum n-3 PUFA levels increased after the fish-based diet period and decreased after the control diet, compared with the baseline (1.49 vs. 0.97 vs. 1.19 mmol/l, p < 0.0001). There was no correlation between serum n-3 PUFA levels and endothelial function. An increased ratio of epoxyeicosatrienoic acid/dihydroxyeicosatrienoic acid was observed after a fish-based diet intervention, possibly due to the inhibition of the activity of soluble epoxide hydrolase. A fish-based dietary intervention improves endothelial function in postmenopausal women with T2DM. Dissociation between the serum n-3 PUFA concentration and endothelial function suggests that the other factors may contribute to this phenomenon. Copyright © 2014 Elsevier Inc. All rights reserved.

Increased endothelial cell adhesion and elongation on micron-patterned nano-rough poly(dimethylsiloxane) films.

PubMed

Ranjan, Ashwini; Webster, Thomas J

2009-07-29

The success of synthetic vascular grafts is largely determined by their ability to promote vital endothelial cell functions such as adhesion, alignment, proliferation, and extracellular matrix (ECM) deposition. Developing such biomaterials requires the design and fabrication of materials that mimic select properties of native extracellular matrices. Furthermore, cells of the native endothelium have elongated and aligned morphology in the direction of blood flow, yet few materials promote this type of morphology initially, but rather rely on blood flow to orient endothelial cells. Therefore, the objective of this in vitro study was to design a biomaterial that mimics the conditions of the micro- and nano-environment of vascular intima tissue suitable for endothelial cell adhesion and elongation to improve the efficacy of small synthetic vascular grafts. Towards this end, patterned poly(dimethylsiloxane) (PDMS) films consisting of periodic arrays of nano-grooves (500 nm), with spacings ranging from 22 to 80 microm, and alternating nano- and micron roughness were fabricated using a novel electron beam physical vapor deposition method followed by polymer casting. By varying pattern spacing, the area of micron- and nano-rough surface was controlled. In vitro rat aortic endothelial cell adhesion and elongation studies indicated that endothelial cell function was enhanced on patterned PDMS surfaces with the widest spacing and greatest surface area of nano-roughness, as compared to more narrow pattern spacings and non-patterned PDMS surfaces. Specifically, endothelial cells adherent on PDMS patterned films of the widest spacing (greatest nano-rough area) displayed almost twice as much elongation as cells on non-patterned surfaces. For these reasons, the present study highlighted design criteria (the use of micron patterns of nano-features on PDMS) that may contribute to the intelligent design of new-generation vascular grafts.

Effect of specific surface microstructures on substrate endothelialisation and thrombogenicity: Importance for stent design.

PubMed

Lutter, Christoph; Nothhaft, Matthias; Rzany, Alexander; Garlichs, Christoph D; Cicha, Iwona

2015-01-01

In coronary artery disease, highly stenosed arteries are frequently treated by stent implantation, which thereafter necessitates a dual-antiplatelet therapy (DAPT) in order to prevent stent-thrombosis. We hypothesized that specific patterns of microstructures on stents can accelerate endothelialisation thereby reducing their thrombogenicity and the DAPT duration. Differently designed, 2-5 μm high elevations or hollows were lithographically etched on silicon plates, subsequently coated with silicon carbide. Smooth silicon plates and bare metal substrates were used as controls. To assess attachment and growth of human umbilical vein endothelial cells under static or flow conditions, actin cytoskeleton was visualised with green phalloidin. Endothelial migration was assessed in a modified barrier assay. To investigate surface thrombogenicity, platelets were incubated on the structured surfaces in static and flow conditions, and visualised with fluorescein-conjugated P-selectin antibody. Images were taken with incident-light fluorescent microscope for non-transparent objects. Compared to smooth surface, flat cubic elevations (5 μm edge length) improved endothelial cell attachment and growth under static and dynamic conditions, whereas smaller, spiky structures (2 μm edge length) had a negative influence on endothelialisation. Endothelial cell migration was fastest on flat cubic elevations, hollows, and smooth surfaces, whereas spiky structures and bare metal had a negative effect on endothelial migration. Thrombogenicity assays under static and flow conditions showed that platelet adhesion was reduced on the flat elevations and the smooth surface, as compared to the spiky structures, the hollow design and the bare metal substrates. Surface microstructures strongly influence endothelialisation of substrates. Designing stents with surface topography which accelerates endothelialisation and reduces thrombogenicity may be of clinical benefit by improving the safety profile of coronary interventions.

Treatment of denture-related stomatitis improves endothelial function assessed by flow-mediated vascular dilation.

PubMed

Osmenda, Grzegorz; Maciąg, Joanna; Wilk, Grzegorz; Maciąg, Anna; Nowakowski, Daniel; Loster, Jolanta; Dembowska, Elżbieta; Robertson, Douglas; Guzik, Tomasz; Cześnikiewicz-Guzik, Marta

2017-02-01

The presence of oral inflammation has recently been linked with the pathogenesis of cardiovascular diseases. While numerous studies have described links between periodontitis and endothelial dysfunction, little is known about the influence of denture-related stomatitis (DRS) on cardiovascular risk. Therefore, the aim of this study was to determine whether the treatment of DRS can lead to improvement of the clinical measures of vascular dysfunction. The DRS patients were treated with a local oral antifungal agent for 3 weeks. Blood pressure, flow-mediated dilatation (FMD) and nitroglycerine-mediated vascular dilatation (NMD) were measured during three study visits: before treatment, one day and two months after conclusion of antifungal therapy. Flow-mediated dilatation measurements showed significant improvement of endothelial function 2 months after treatment (FMD median 5%, 95 CI: 3-8.3 vs. 11%, 95% CI: 8.8-14.4; p < 0.01), while there was no difference in control, endothelium-independent vasorelaxations (NMD; median = 15.3%, 95% CI: 10.8-19.3 vs. 12.7%, 95% CI: 10.6-15; p = 0.3). Other cardiovascular parameters such as systolic (median = 125 mm Hg; 95% CI: 116-129 vs. 120 mm Hg, 95% CI: 116-126; p = 0.1) as well as diastolic blood pressure and heart rate (median = 65.5 bpm, 95% CI: 56.7-77.7 vs. 71 bpm, 95% CI: 66.7-75; p = 0.5) did not change during or after the treatment. Treatment of DRS is associated with improvement of endothelial function. Since endothelial dysfunction is known to precede the development of severe cardiovascular disorders such as atherosclerosis and hypertension, patients should be more carefully screened for DRS in general dental practice, and immediate DRS treatment should be advised.

Elevation in blood flow and shear rate prevents hyperglycemia-induced endothelial dysfunction in healthy subjects and those with type 2 diabetes.

PubMed

Greyling, Arno; Schreuder, Tim H A; Landman, Thijs; Draijer, Richard; Verheggen, Rebecca J H M; Hopman, Maria T E; Thijssen, Dick H J

2015-03-01

Hyperglycemia, commonly present after a meal, causes transient impairment in endothelial function. We examined whether increases in blood flow (BF) protect against the hyperglycemia-mediated decrease in endothelial function in healthy subjects and patients with type 2 diabetes mellitus (T2DM). Ten healthy subjects and 10 age- and sex-matched patients with T2DM underwent simultaneous bilateral assessment of brachial artery endothelial function by means of flow-mediated dilation (FMD) using high-resolution echo-Doppler. FMD was examined before and 60, 120, and 150 min after a 75-g oral glucose challenge. We unilaterally manipulated BF by heating one arm between minute 30 and minute 60. Oral glucose administration caused a statistically significant, transient increase in blood glucose in both groups (P < 0.001). Forearm skin temperature, brachial artery BF, and shear rate significantly increased in the heated arm (P < 0.001), and to a greater extent compared with the nonheated arm in both groups (interaction effect P < 0.001). The glucose load caused a transient decrease in FMD% (P < 0.05), whereas heating significantly prevented the decline (interaction effect P < 0.01). Also, when correcting for changes in diameter and shear rate, we found that the hyperglycemia-induced decrease in FMD can be prevented by local heating (P < 0.05). These effects on FMD were observed in both groups. Our data indicate that nonmetabolically driven elevation in BF and shear rate can similarly prevent the hyperglycemia-induced decline in conduit artery endothelial function in healthy volunteers and in patients with type 2 diabetes. Additional research is warranted to confirm that other interventions that increase BF and shear rate equally protect the endothelium when challenged by hyperglycemia. Copyright © 2015 the American Physiological Society.

Endothelial function across an oral contraceptive cycle in women using levonorgestrel and ethinyl estradiol.

PubMed

Torgrimson, Britta N; Meendering, Jessica R; Kaplan, Paul F; Minson, Christopher T

2007-06-01

Oral contraceptive pills (OCPs) are a popular contraception method. Currently, lower-dose ethinyl estradiol formulations are most commonly prescribed, although they have been linked to increased arterial vascular risk. The aim of this study was to investigate endothelial function in healthy young women using lower-dose ethinyl estradiol OCPs. We examined flow-mediated, endothelium-dependent and nitroglycerin-mediated, endothelium-independent vasodilation of the brachial artery, comparing two doses of ethinyl estradiol/levonorgestrel OCPs in 15 healthy young women on two study days: once during the active phase and once during the placebo phase of an OCP cycle. Group low dose (LD) (n=7) active pills contained 150 microg levonorgestrel/30 microg ethinyl estradiol versus Group very low dose (VLD) (n=8) with 100 microg levonorgestrel/20 microg ethinyl estradiol. Endothelium-dependent vasodilation was lower during the active phase in Group VLD (5.33 +/- 1.77% vs. 7.23 +/- 2.60%; P=0.024). This phase difference was not observed in Group LD (8.00 +/- 0.970% vs. 7.61 +/- 1.07%; P=0.647). Endothelium-independent vasodilation did not differ between phases in either group. Finally, we measured endothelium-dependent vasodilation in two additional women who received 10 microg of unopposed ethinyl estradiol. Endothelium-dependent vasodilation was increased by unopposed ethinyl estradiol compared with the placebo phase (10.88 +/- 2.34% vs. 6.97 +/- 1.83%). These results suggest that levonorgestrel may antagonize the activity of ethinyl estradiol. Thus both the progestin type and estradiol dose need to be considered when assessing arterial vascular risk of OCP use in women.

The procyanidin-induced pseudo laminar shear stress response: a new concept for the reversal of endothelial dysfunction.

PubMed

Corder, Roger; Warburton, Richard C; Khan, Noorafza Q; Brown, Ruth E; Wood, Elizabeth G; Lees, Delphine M

2004-11-01

Reduced endothelium-dependent vasodilator responses with increased synthesis of ET-1 (endothelin-1) are characteristics of endothelial dysfunction in heart failure and are predictive of mortality. Identification of treatments that correct these abnormalities may have particular benefit for patients who become refractory to current regimens. Hawthorn preparations have a long history in the treatment of heart failure. Therefore we tested their inhibitory effects on ET-1 synthesis by cultured endothelial cells. These actions were compared with that of GSE (grape seed extract), as the vasoactive components of both these herbal remedies are mainly oligomeric flavan-3-ols called procyanidins. This showed extracts of hawthorn and grape seed were equipotent as inhibitors of ET-1 synthesis. GSE also produced a potent endothelium-dependent vasodilator response on preparations of isolated aorta. Suppression of ET-1 synthesis at the same time as induction of endothelium-dependent vasodilation is a similar response to that triggered by laminar shear stress. Based on these results and previous findings, we hypothesize that through their pharmacological properties procyanidins stimulate a pseudo laminar shear stress response in endothelial cells, which helps restore endothelial function and underlies the benefit from treatment with hawthorn extract in heart failure.

E-Cigarette Aerosol Exposure Induces Reactive Oxygen Species, DNA Damage, and Cell Death in Vascular Endothelial Cells.

PubMed

Anderson, Chastain; Majeste, Andrew; Hanus, Jakub; Wang, Shusheng

2016-12-01

Cigarette smoking remains one of the leading causes of preventable death worldwide. Vascular cell death and dysfunction is a central or exacerbating component in the majority of cigarette smoking related pathologies. The recent development of the electronic nicotine delivery systems known as e-cigarettes provides an alternative to conventional cigarette smoking; however, the potential vascular health risks of e-cigarette use remain unclear. This study evaluates the effects of e-cigarette aerosol extract (EAE) and conventional cigarette smoke extract (CSE) on human umbilical vein endothelial cells (HUVECs). A laboratory apparatus was designed to produce extracts from e-cigarettes and conventional cigarettes according to established protocols for cigarette smoking. EAE or conventional CSE was applied to human vascular endothelial cells for 4-72 h, dependent on the assay. Treated cells were assayed for reactive oxygen species, DNA damage, cell viability, and markers of programmed cell death pathways. Additionally, the anti-oxidants α-tocopherol and n-acetyl-l-cysteine were used to attempt to rescue e-cigarette induced cell death. Our results indicate that e-cigarette aerosol is capable of inducing reactive oxygen species, causing DNA damage, and significantly reducing cell viability in a concentration dependent fashion. Immunofluorescent and flow cytometry analysis indicate that both the apoptosis and programmed necrosis pathways are triggered by e-cigarette aerosol treatment. Additionally, anti-oxidant treatment provides a partial rescue of the induced cell death, indicating that reactive oxygen species play a causal role in e-cigarette induced cytotoxicity. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Linagliptin reduces effects of ET-1 and TLR2-mediated cerebrovascular hyperreactivity in diabetes.

PubMed

Hardigan, Trevor; Abdul, Yasir; Ergul, Adviye

2016-08-15

The anti-hyperglycemic agent linagliptin, a dipeptidyl peptidase-4 inhibitor, has been shown to reduce inflammation and improve endothelial cell function. In this study, we hypothesized that DPP-IV inhibition with linagliptin would improve impaired cerebral blood flow in diabetic rats through improved insulin-induced cerebrovascular relaxation and reversal of pathological cerebrovascular remodeling that subsequently leads to improvement of cognitive function. Male type-2 diabetic Goto-Kakizaki (GK) and nondiabetic Wistar rats were treated with linagliptin, and ET-1 plasma levels and dose response curves to ET-1 (0.1-100nM) in basilar arteries were assessed. The impact of TLR2 antagonism on ET-1 mediated basilar contraction and endothelium-dependent relaxation to acetylcholine (ACh, 1nM-1M) in diabetic GK rats was examined with antibody directed against the TLR2 receptor (Santa Cruz, 5μg/mL). The expression of TLR2 in middle cerebral arteries (MCAs) from treated rats and in brain microvascular endothelial cells (BMVEC) treated with 100nM linagliptin was assessed. Linagliptin lowered plasma ET-1 levels in diabetes, and reduced ET-1-induced vascular contraction. TLR2 antagonism in diabetic basilar arteries reduced ET-1-mediated cerebrovascular dysfunction and improved endothelium-dependent vasorelaxation. Linagliptin treatment in the BMVEC was able to reduce TLR2 expression in cells from both diabetic and nondiabetic rats. These results suggest that inhibition of DPPIV using linagliptin improves the ET-1-mediated cerebrovascular dysfunction observed in diabetes through a reduction in ET-1 plasma levels and reduced cerebrovascular hyperreactivity. This effect is potentially a result of linagliptin causing a decrease in endothelial TLR2 expression and a subsequent increase in NO bioavailability. Copyright © 2016 Elsevier Inc. All rights reserved.

Cyclooxygenase inhibition improves endothelial vasomotor dysfunction of visceral adipose arterioles in human obesity

PubMed Central

Farb, Melissa G.; Tiwari, Stephanie; Karki, Shakun; Ngo, Doan TM; Carmine, Brian; Hess, Donald T.; Zuriaga, Maria A.; Walsh, Kenneth; Fetterman, Jessica L.; Hamburg, Naomi M.; Vita, Joseph A.; Apovian, Caroline M.; Gokce, Noyan

2013-01-01

Objective The purpose of this study was to determine whether cyclooxygenase inhibition improves vascular dysfunction of adipose microvessels from obese humans. Design and Methods In 20 obese subjects (age 37±12 yrs, BMI 47±8 kg/m2) we collected subcutaneous and visceral fat during bariatric surgery and characterized adipose depot-specific gene expression, endothelial cell phenotype, and microvascular function. Vasomotor function was assessed in response to endothelium-dependent agonists using videomicroscopy of small arterioles from fat. Results Arterioles from visceral fat exhibited impaired endothelium-dependent, acetylcholine-mediated vasodilation, compared to the subcutaneous depot (p<0.001). Expression of mRNA transcripts relevant to the cyclooxygenase pathway were upregulated in visceral compared to subcutaneous fat. Pharmacological inhibition of cyclooxygenase with indomethacin improved endothelium-dependent vasodilator function of arterioles from visceral fat by 2-fold (p=0.01), whereas indomethacin had no effect in the subcutaneous depot. Indomethacin increased activation via serine-1177 phosphorylation of endothelial nitric oxide synthase in response to acetylcholine in endothelial cells from visceral fat. Inhibition of endothelial nitric oxide synthase with Nω-nitro-L-arginine methyl ester abrogated the effects of cyclooxygenase-inhibition suggesting that vascular actions of indomethacin were related to increased nitric oxide bioavailability. Conclusions Our findings suggest that cyclooxygenase-mediated vasoconstrictor prostanoids partly contribute to endothelial dysfunction of visceral adipose arterioles in human obesity. PMID:23640904

Sialic acids regulate microvessel permeability, revealed by novel in vivo studies of endothelial glycocalyx structure and function

PubMed Central

Betteridge, Kai B.; Arkill, Kenton P.; Neal, Christopher R.; Harper, Steven J.; Foster, Rebecca R.; Satchell, Simon C.; Bates, David O.

2017-01-01

Key points We have developed novel techniques for paired, direct, real‐time in vivo quantification of endothelial glycocalyx structure and associated microvessel permeability.Commonly used imaging and analysis techniques yield measurements of endothelial glycocalyx depth that vary by over an order of magnitude within the same vessel.The anatomical distance between maximal glycocalyx label and maximal endothelial cell plasma membrane label provides the most sensitive and reliable measure of endothelial glycocalyx depth.Sialic acid residues of the endothelial glycocalyx regulate glycocalyx structure and microvessel permeability to both water and albumin. Abstract The endothelial glycocalyx forms a continuous coat over the luminal surface of all vessels, and regulates multiple vascular functions. The contribution of individual components of the endothelial glycocalyx to one critical vascular function, microvascular permeability, remains unclear. We developed novel, real‐time, paired methodologies to study the contribution of sialic acids within the endothelial glycocalyx to the structural and functional permeability properties of the same microvessel in vivo. Single perfused rat mesenteric microvessels were perfused with fluorescent endothelial cell membrane and glycocalyx labels, and imaged with confocal microscopy. A broad range of glycocalyx depth measurements (0.17–3.02 μm) were obtained with different labels, imaging techniques and analysis methods. The distance between peak cell membrane and peak glycocalyx label provided the most reliable measure of endothelial glycocalyx anatomy, correlating with paired, numerically smaller values of endothelial glycocalyx depth (0.078 ± 0.016 μm) from electron micrographs of the same portion of the same vessel. Disruption of sialic acid residues within the endothelial glycocalyx using neuraminidase perfusion decreased endothelial glycocalyx depth and increased apparent solute permeability to albumin in the same vessels in a time‐dependent manner, with changes in all three true vessel wall permeability coefficients (hydraulic conductivity, reflection coefficient and diffusive solute permeability). These novel technologies expand the range of techniques that permit direct studies of the structure of the endothelial glycocalyx and dependent microvascular functions in vivo, and demonstrate that sialic acid residues within the endothelial glycocalyx are critical regulators of microvascular permeability to both water and albumin. PMID:28524373

Effect of PEGylation on ligand-based targeting of drug carriers to the vascular wall in blood flow.

PubMed

Onyskiw, Peter J; Eniola-Adefeso, Omolola

2013-09-03

The blood vessel wall plays a prominent role in the development of many life-threatening diseases and as such is an attractive target for treatment. To target diseased tissue, particulate drug carriers often have their surfaces modified with antibodies or epitopes specific to vascular wall-expressed molecules, along with poly(ethylene glycol) (PEG) to improve carrier blood circulation time. However, little is known about the effect of poly(ethylene glycol) on carrier adhesion dynamics-specifically in blood flow. Here we examine the influence of different molecular weight PEG spacers on particle adhesion in blood flow. Anti-ICAM-1 or Sialyl Lewis(a) were grafted onto polystyrene 2 μm and 500 nm spheres via PEG spacers and perfused in blood over activated endothelial cells at physiological shear conditions. PEG spacers were shown to improve, reduce, or have no effect on the binding density of targeted-carriers depending on the PEG surface conformation, shear rate, and targeting moiety.

Bone Morphogenic Protein 4-Smad-Induced Upregulation of Platelet-Derived Growth Factor AA Impairs Endothelial Function.

PubMed

Hu, Weining; Zhang, Yang; Wang, Li; Lau, Chi Wai; Xu, Jian; Luo, Jiang-Yun; Gou, Lingshan; Yao, Xiaoqiang; Chen, Zhen-Yu; Ma, Ronald Ching Wan; Tian, Xiao Yu; Huang, Yu

2016-03-01

Bone morphogenic protein 4 (BMP4) is an important mediator of endothelial dysfunction in cardio-metabolic diseases, whereas platelet-derived growth factors (PDGFs) are major angiogenic and proinflammatory mediator, although the functional link between these 2 factors is unknown. The present study investigated whether PDGF mediates BMP4-induced endothelial dysfunction in diabetes mellitus. We generated Ad-Bmp4 to overexpress Bmp4 and Ad-Pdgfa-shRNA to knockdown Pdgfa in mice through tail intravenous injection. SMAD4-shRNA lentivirus, SMAD1-shRNA, and SMAD5 shRNA adenovirus were used for knockdown in human and mouse endothelial cells. We found that PDGF-AA impaired endothelium-dependent vasodilation in aortas and mesenteric resistance arteries. BMP4 upregulated PDGF-AA in human and mouse endothelial cells, which was abolished by BMP4 antagonist noggin or knockdown of SMAD1/5 or SMAD4. BMP4-impared relaxation in mouse aorta was also ameliorated by PDGF-AA neutralizing antibody. Tail injection of Ad-Pdgfa-shRNA ameliorates endothelial dysfunction induced by Bmp4 overexpression (Ad-Bmp4) in vivo. Serum PDGF-AA was elevated in both diabetic patients and diabetic db/db mice compared with nondiabetic controls. Pdgfa-shRNA or Bmp4-shRNA adenovirus reduced serum PDGF-AA concentration in db/db mice. PDGF-AA neutralizing antibody or tail injection with Pdgfa-shRNA adenovirus improved endothelial function in aortas and mesenteric resistance arteries from db/db mice. The effect of PDGF-AA on endothelial function in mouse aorta was also inhibited by Ad-Pdgfra-shRNA to inhibit PDGFRα. The present study provides novel evidences to show that PDGF-AA impairs endothelium-dependent vasodilation and PDGF-AA mediates BMP4-induced adverse effect on endothelial cell function through SMAD1/5- and SMAD4-dependent mechanisms. Inhibition of PGDF-AA ameliorates vascular dysfunction in diabetic mice. © 2016 American Heart Association, Inc.

Mechanisms of lung endothelial barrier disruption induced by cigarette smoke: role of oxidative stress and ceramides.

PubMed

Schweitzer, Kelly S; Hatoum, Hadi; Brown, Mary Beth; Gupta, Mehak; Justice, Matthew J; Beteck, Besem; Van Demark, Mary; Gu, Yuan; Presson, Robert G; Hubbard, Walter C; Petrache, Irina

2011-12-01

The epithelial and endothelial cells lining the alveolus form a barrier essential for the preservation of the lung respiratory function, which is, however, vulnerable to excessive oxidative, inflammatory, and apoptotic insults. Whereas profound breaches in this barrier function cause pulmonary edema, more subtle changes may contribute to inflammation. The mechanisms by which cigarette smoke (CS) exposure induce lung inflammation are not fully understood, but an early alteration in the epithelial barrier function has been documented. We sought to investigate the occurrence and mechanisms by which soluble components of mainstream CS disrupt the lung endothelial cell barrier function. Using cultured primary rat microvascular cell monolayers, we report that CS induces endothelial cell barrier disruption in a dose- and time-dependent manner of similar magnitude to that of the epithelial cell barrier. CS exposure triggered a mechanism of neutral sphingomyelinase-mediated ceramide upregulation and p38 MAPK and JNK activation that were oxidative stress dependent and that, along with Rho kinase activation, mediated the endothelial barrier dysfunction. The morphological changes in endothelial cell monolayers induced by CS included actin cytoskeletal rearrangement, junctional protein zonula occludens-1 loss, and intercellular gap formation, which were abolished by the glutathione modulator N-acetylcysteine and ameliorated by neutral sphingomyelinase inhibition. The direct application of ceramide recapitulated the effects of CS, by disrupting both endothelial and epithelial cells barrier, by a mechanism that was redox and apoptosis independent and required Rho kinase activation. Furthermore, ceramide induced dose-dependent alterations of alveolar microcirculatory barrier in vivo, measured by two-photon excitation microscopy in the intact rat. In conclusion, soluble components of CS have direct endothelial barrier-disruptive effects that could be ameliorated by glutathione modulators or by inhibitors of neutral sphingomyelinase, p38 MAPK, JNK, and Rho kinase. Amelioration of endothelial permeability may alleviate lung and systemic vascular dysfunction associated with smoking-related chronic obstructive lung diseases.

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry.

PubMed

Epshtein, Alona; Sakhneny, Lina; Landsman, Limor

2017-01-28

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, including endothelial, neuronal, and mesenchymal cells were shown to regulate cell differentiation and proliferation in the embryonic pancreas. In the adult, the function and mass of insulin-producing cells were shown to depend on cells in their microenvironment, including pericyte, immune, endothelial, and neuronal cells. Lastly, changes in the pancreas microenvironment were shown to regulate pancreas tumorigenesis. However, the cues underlying these processes are not fully defined. Therefore, characterizing the different cell types that comprise the pancreas microenvironment and profiling their gene expression are crucial to delineate the tissue development and function under normal and diseased states. Here, we describe a method that allows for the isolation of mesenchymal cells from the pancreas of embryonic, neonatal, and adult mice. This method utilizes the enzymatic digestion of mouse pancreatic tissue and the subsequent fluorescence-activated cell sorting (FACS) or flow-cytometric analysis of labeled cells. Cells can be labeled by either immunostaining for surface markers or by the expression of fluorescent proteins. Cell isolation can facilitate the characterization of genes and proteins expressed in cells of the pancreas mesenchyme. This protocol was successful in isolating and culturing highly enriched mesenchymal cell populations from the embryonic, neonatal, and adult mouse pancreas.

Evolution of the degradation mechanism of pure zinc stent in the one-year study of rabbit abdominal aorta model.

PubMed

Yang, Hongtao; Wang, Cong; Liu, Chaoqiang; Chen, Houwen; Wu, Yifan; Han, Jintao; Jia, Zichang; Lin, Wenjiao; Zhang, Deyuan; Li, Wenting; Yuan, Wei; Guo, Hui; Li, Huafang; Yang, Guangxin; Kong, Deling; Zhu, Donghui; Takashima, Kazuki; Ruan, Liqun; Nie, Jianfeng; Li, Xuan; Zheng, Yufeng

2017-11-01

In the present study, pure zinc stents were implanted into the abdominal aorta of rabbits for 12 months. Multiscale analysis including micro-CT, scanning electron microscopy (SEM), scanning transmission electron microscopy (STEM) and histological stainings was performed to reveal the fundamental degradation mechanism of the pure zinc stent and its biocompatibility. The pure zinc stent was able to maintain mechanical integrity for 6 months and degraded 41.75 ± 29.72% of stent volume after 12 months implantation. No severe inflammation, platelet aggregation, thrombosis formation or obvious intimal hyperplasia was observed at all time points after implantation. The degradation of the zinc stent played a beneficial role in the artery remodeling and healing process. The evolution of the degradation mechanism of pure zinc stents with time was revealed as follows: Before endothelialization, dynamic blood flow dominated the degradation of pure zinc stent, creating a uniform corrosion mode; After endothelialization, the degradation of pure zinc stent depended on the diffusion of water molecules, hydrophilic solutes and ions which led to localized corrosion. Zinc phosphate generated in blood flow transformed into zinc oxide and small amounts of calcium phosphate during the conversion of degradation microenvironment. The favorable physiological degradation behavior makes zinc a promising candidate for future stent applications. Copyright © 2017 Elsevier Ltd. All rights reserved.

Endomembrane H-Ras Controls Vascular Endothelial Growth Factor-induced Nitric-oxide Synthase-mediated Endothelial Cell Migration*

PubMed Central

Haeussler, Dagmar J.; Pimentel, David R.; Hou, Xiuyun; Burgoyne, Joseph R.; Cohen, Richard A.; Bachschmid, Markus M.

2013-01-01

We demonstrate for the first time that endomembrane-delimited H-Ras mediates VEGF-induced activation of endothelial nitric-oxide synthase (eNOS) and migratory response of human endothelial cells. Using thiol labeling strategies and immunofluorescent cell staining, we found that only 31% of total H-Ras is S-palmitoylated, tethering the small GTPase to the plasma membrane but leaving the function of the large majority of endomembrane-localized H-Ras unexplained. Knockdown of H-Ras blocked VEGF-induced PI3K-dependent Akt (Ser-473) and eNOS (Ser-1177) phosphorylation and nitric oxide-dependent cell migration, demonstrating the essential role of H-Ras. Activation of endogenous H-Ras led to recruitment and phosphorylation of eNOS at endomembranes. The loss of migratory response in cells lacking endogenous H-Ras was fully restored by modest overexpression of an endomembrane-delimited H-Ras palmitoylation mutant. These studies define a newly recognized role for endomembrane-localized H-Ras in mediating nitric oxide-dependent proangiogenic signaling. PMID:23548900

Activation of the endoplasmic reticulum stress response by the amyloid-beta 1-40 peptide in brain endothelial cells.

PubMed

Fonseca, Ana Catarina R G; Ferreiro, Elisabete; Oliveira, Catarina R; Cardoso, Sandra M; Pereira, Cláudia F

2013-12-01

Neurovascular dysfunction arising from endothelial cell damage is an early pathogenic event that contributes to the neurodegenerative process occurring in Alzheimer's disease (AD). Since the mechanisms underlying endothelial dysfunction are not fully elucidated, this study was aimed to explore the hypothesis that brain endothelial cell death is induced upon the sustained activation of the endoplasmic reticulum (ER) stress response by amyloid-beta (Aβ) peptide, which deposits in the cerebral vessels in many AD patients and transgenic mice. Incubation of rat brain endothelial cells (RBE4 cell line) with Aβ1-40 increased the levels of several markers of ER stress-induced unfolded protein response (UPR), in a time-dependent manner, and affected the Ca(2+) homeostasis due to the release of Ca(2+) from this intracellular store. Finally, Aβ1-40 was shown to activate both mitochondria-dependent and -independent apoptotic cell death pathways. Enhanced release of cytochrome c from mitochondria and activation of the downstream caspase-9 were observed in cells treated with Aβ1-40 concomitantly with caspase-12 activation. Furthermore, Aβ1-40 activated the apoptosis effectors' caspase-3 and promoted the translocation of apoptosis-inducing factor (AIF) to the nucleus demonstrating the involvement of caspase-dependent and -independent mechanisms during Aβ-induced endothelial cell death. In conclusion, our data demonstrate that ER stress plays a significant role in Aβ1-40-induced apoptotic cell death in brain endothelial cells suggesting that ER stress-targeted therapeutic strategies might be useful in AD to counteract vascular defects and ultimately neurodegeneration. © 2013.

Functional Definition of Progenitors Versus Mature Endothelial Cells Reveals Key SoxF-Dependent Differentiation Process.

PubMed

Patel, Jatin; Seppanen, Elke J; Rodero, Mathieu P; Wong, Ho Yi; Donovan, Prudence; Neufeld, Zoltan; Fisk, Nicholas M; Francois, Mathias; Khosrotehrani, Kiarash

2017-02-21

During adult life, blood vessel formation is thought to occur via angiogenic processes involving branching from existing vessels. An alternate proposal suggests that neovessels form from endothelial progenitors able to assemble the intimal layers. We here aimed to define vessel-resident endothelial progenitors in vivo in a variety of tissues in physiological and pathological situations such as normal aorta, lungs, and wound healing, tumors, and placenta, as well. Based on protein expression levels of common endothelial markers using flow cytometry, 3 subpopulations of endothelial cells could be identified among VE-Cadherin+ and CD45- cells. Lineage tracing by using Cdh5cre ERt2 /Rosa-YFP reporter strategy demonstrated that the CD31-/loVEGFR2lo/intracellular endothelial population was indeed an endovascular progenitor (EVP) of an intermediate CD31intVEGFR2lo/intracellular transit amplifying (TA) and a definitive differentiated (D) CD31hiVEGFR2hi/extracellular population. EVP cells arose from vascular-resident beds that could not be transferred by bone marrow transplantation. Furthermore, EVP displayed progenitor-like status with a high proportion of cells in a quiescent cell cycle phase as assessed in wounds, tumors, and aorta. Only EVP cells and not TA and D cells had self-renewal capacity as demonstrated by colony-forming capacity in limiting dilution and by transplantation in Matrigel plugs in recipient mice. RNA sequencing revealed prominent gene expression differences between EVP and D cells. In particular, EVP cells highly expressed genes related to progenitor function including Sox9 , Il33 , Egfr , and Pdfgrα. Conversely, D cells highly expressed genes related to differentiated endothelium including Ets1&2 , Gata2 , Cd31 , Vwf , and Notch . The RNA sequencing also pointed to an essential role of the Sox18 transcription factor. The role of SOX18 in the differentiation process was validated by using lineage-tracing experiments based on S ox18Cre ERt2 /Rosa-YFP mice. Besides, in the absence of functional SOX18/SOXF, EVP progenitors were still present, but TA and D populations were significantly reduced. Our findings support an entirely novel endothelial hierarchy, from EVP to TA to D, as defined by self-renewal, differentiation, and molecular profiling of an endothelial progenitor. This paradigm shift in our understanding of vascular-resident endothelial progenitors in tissue regeneration opens new avenues for better understanding of cardiovascular biology. © 2016 American Heart Association, Inc.

Flow cytometry for intracellular SPION quantification: specificity and sensitivity in comparison with spectroscopic methods

PubMed Central

Friedrich, Ralf P; Janko, Christina; Poettler, Marina; Tripal, Philipp; Zaloga, Jan; Cicha, Iwona; Dürr, Stephan; Nowak, Johannes; Odenbach, Stefan; Slabu, Ioana; Liebl, Maik; Trahms, Lutz; Stapf, Marcus; Hilger, Ingrid; Lyer, Stefan; Alexiou, Christoph

2015-01-01

Due to their special physicochemical properties, iron nanoparticles offer new promising possibilities for biomedical applications. For bench to bedside translation of super-paramagnetic iron oxide nanoparticles (SPIONs), safety issues have to be comprehensively clarified. To understand concentration-dependent nanoparticle-mediated toxicity, the exact quantification of intracellular SPIONs by reliable methods is of great importance. In the present study, we compared three different SPION quantification methods (ultraviolet spectrophotometry, magnetic particle spectroscopy, atomic adsorption spectroscopy) and discussed the shortcomings and advantages of each method. Moreover, we used those results to evaluate the possibility to use flow cytometric technique to determine the cellular SPION content. For this purpose, we correlated the side scatter data received from flow cytometry with the actual cellular SPION amount. We showed that flow cytometry provides a rapid and reliable method to assess the cellular SPION content. Our data also demonstrate that internalization of iron oxide nanoparticles in human umbilical vein endothelial cells is strongly dependent to the SPION type and results in a dose-dependent increase of toxicity. Thus, treatment with lauric acid-coated SPIONs (SEONLA) resulted in a significant increase in the intensity of side scatter and toxicity, whereas SEONLA with an additional protein corona formed by bovine serum albumin (SEONLA-BSA) and commercially available Rienso® particles showed only a minimal increase in both side scatter intensity and cellular toxicity. The increase in side scatter was in accordance with the measurements for SPION content by the atomic adsorption spectroscopy reference method. In summary, our data show that flow cytometry analysis can be used for estimation of uptake of SPIONs by mammalian cells and provides a fast tool for scientists to evaluate the safety of nanoparticle products. PMID:26170658

Elevated CXCL1 expression in gp130-deficient endothelial cells impairs neutrophil migration in mice

PubMed Central

Yao, Longbiao; Yago, Tadayuki; Shao, Bojing; Liu, Zhenghui; Silasi-Mansat, Robert; Setiadi, Hendra; Lupu, Florea

2013-01-01

Neutrophils emigrate from venules to sites of infection or injury in response to chemotactic gradients. How these gradients form is not well understood. Some IL-6 family cytokines stimulate endothelial cells to express adhesion molecules and chemokines that recruit leukocytes. Receptors for these cytokines share the signaling subunit gp130. We studied knockout mice lacking gp130 in endothelial cells. Unexpectedly, gp130-deficient endothelial cells constitutively expressed more CXCL1 in vivo and in vitro, and even more upon stimulation with tumor necrosis factor-α. Mobilization of this increased CXCL1 from intracellular stores to the venular surface triggered β2 integrin–dependent arrest of neutrophils rolling on selectins but impaired intraluminal crawling and transendothelial migration. Superfusing CXCL1 over venules promoted neutrophil migration only after intravenously injecting mAb to CXCL1 to diminish its intravascular function or heparinase to release CXCL1 from endothelial proteoglycans. Remarkably, mice lacking gp130 in endothelial cells had impaired histamine-induced venular permeability, which was restored by injecting anti–P-selectin mAb to prevent neutrophil rolling and arrest. Thus, excessive CXCL1 expression in gp130-deficient endothelial cells augments neutrophil adhesion but hinders migration, most likely by disrupting chemotactic gradients. Our data define a role for endothelial cell gp130 in regulating integrin-dependent adhesion and de-adhesion of neutrophils during inflammation. PMID:24081661

Endurance capacity is not correlated with endothelial function in male university students.

PubMed

Wang, Yan; Zeng, Xian-bo; Yao, Feng-juan; Wu, Fang; Su, Chen; Fan, Zhen-guo; Zhu, Zhu; Tao, Jun; Huang, Yi-jun

2014-01-01

Endurance capacity, assessed by 1000-meter (1000 m) run of male university students, is an indicator of cardiovascular fitness in Chinese students physical fitness surveillance. Although cardiovascular fitness is related to endothelial function closely in patients with cardiovascular diseases, it remains unclear whether endurance capacity correlates with endothelial function, especially with circulating endothelial microparticles (EMPs), a new sensitive marker of endothelial dysfunction in young students. The present study aimed to investigate the relationship between endurance capacity and endothelial function in male university students. Forty-seven healthy male university students (mean age, 20.1 ± 0.6 years; mean height, 172.4 ± 6.3 cm; and mean weight, 60.0 ± 8.2 kg) were recruited in this study. The measurement procedure of 1000 m run time was followed to Chinese national students Constitutional Health Criterion. Endothelium function was assessed by flow-mediated vasodilation (FMD) in the brachial artery measured by ultrasonic imaging, and the level of circulating EMPs was measured by flow cytometry. Cardiovascular fitness indicator--maximal oxygen uptake (VO2max)--was also measured on a cycle ergometer using a portable gas analyzer. 1000 m run time was correlated with VO2max (r  =  -0.399, p<0.05). However, there were no correlations between VO2max and FMD or levels of circulating CD31+/CD42- microparticles. Similarly, no correlations were found between 1000 m run time and FMD, and levels of circulating CD31+/CD42- microparticles in these male university students (p>0.05). The correlations between endurance capacity or cardiovascular fitness and endothelial function were not found in healthy Chinese male university students. These results suggest that endurance capacity may not reflect endothelial function in healthy young adults with well preserved FMD and low level of circulating CD31+/CD42-EMPs.

Endurance Capacity Is Not Correlated with Endothelial Function in Male University Students

PubMed Central

Wu, Fang; Su, Chen; Fan, Zhen-guo; Zhu, Zhu; Tao, Jun; Huang, Yi-jun

2014-01-01

Background Endurance capacity, assessed by 1000-meter (1000 m) run of male university students, is an indicator of cardiovascular fitness in Chinese students physical fitness surveillance. Although cardiovascular fitness is related to endothelial function closely in patients with cardiovascular diseases, it remains unclear whether endurance capacity correlates with endothelial function, especially with circulating endothelial microparticles (EMPs), a new sensitive marker of endothelial dysfunction in young students. The present study aimed to investigate the relationship between endurance capacity and endothelial function in male university students. Methods Forty-seven healthy male university students (mean age, 20.1±0.6 years; mean height, 172.4±6.3 cm; and mean weight, 60.0±8.2 kg) were recruited in this study. The measurement procedure of 1000 m run time was followed to Chinese national students Constitutional Health Criterion. Endothelium function was assessed by flow-mediated vasodilation (FMD) in the brachial artery measured by ultrasonic imaging, and the level of circulating EMPs was measured by flow cytometry. Cardiovascular fitness indicator - maximal oxygen uptake (VO2 max) - was also measured on a cycle ergometer using a portable gas analyzer. Results 1000 m run time was correlated with VO2max (r = −0.399, p<0.05). However, there were no correlations between VO2max and FMD or levels of circulating CD31+/CD42- microparticles. Similarly, no correlations were found between 1000 m run time and FMD, and levels of circulating CD31+/CD42- microparticles in these male university students (p>0.05). Conclusion The correlations between endurance capacity or cardiovascular fitness and endothelial function were not found in healthy Chinese male university students. These results suggest that endurance capacity may not reflect endothelial function in healthy young adults with well preserved FMD and low level of circulating CD31+/CD42-EMPs. PMID:25101975

Short-term exercise training improves flow-mediated dilation and circulating angiogenic cell number in older sedentary adults.

PubMed

Landers-Ramos, Rian Q; Corrigan, Kelsey J; Guth, Lisa M; Altom, Christine N; Spangenburg, Espen E; Prior, Steven J; Hagberg, James M

2016-08-01

Cardiovascular disease risk increases with age due, in part, to impaired endothelial function and decreased circulating angiogenic cell (CAC) number and function. We sought to determine if 10 days of aerobic exercise training improves endothelial function, CAC number, and intracellular redox balance in older sedentary adults. Eleven healthy subjects (4 men, 7 women), 61 ± 2 years of age participated in 60 min of aerobic exercise at 70% maximal oxygen consumption for 10 consecutive days while maintaining body weight. Before and after training, endothelial function was measured as flow-mediated dilation of the brachial artery and fasting blood was drawn to enumerate 3 CAC subtypes. Intracellular reactive oxygen species (ROS) and nitric oxide (NO) in CD34+ CACs were measured using fluorescent probes and reinforced via real-time quantitative polymerase chain reaction. Flow-mediated dilation improved significantly following training (10% ± 1.3% before vs. 16% ± 1.4% after training; P < 0.05). Likewise, CD34+/KDR+ number increased 104% and KDR+ number increased 151% (P < 0.05 for both), although CD34+ number was not significantly altered (P > 0.05). Intracellular NO and ROS levels in CD34+ CACs were not different after training (P > 0.05 for both). Messenger RNA expression of SOD1, endothelial nitric oxide synthase, and NADPH oxidase 2 and neutrophil cytosolic factor 1 in CD34+ CACs was not significantly altered with training (P > 0.05). In conclusion, 10 consecutive days of aerobic exercise increased flow-mediated dilation and CAC number in older, previously sedentary adults, but did not affect intracellular redox balance in CD34+ CACs. Overall, these data indicate that even short-term aerobic exercise training can have a significant impact on cardiovascular disease risk factors.

Relations of Arterial Stiffness and Brachial Flow-Mediated Dilation With New-Onset Atrial Fibrillation: The Framingham Heart Study.

PubMed

Shaikh, Amir Y; Wang, Na; Yin, Xiaoyan; Larson, Martin G; Vasan, Ramachandran S; Hamburg, Naomi M; Magnani, Jared W; Ellinor, Patrick T; Lubitz, Steven A; Mitchell, Gary F; Benjamin, Emelia J; McManus, David D

2016-09-01

The relations of measures of arterial stiffness, pulsatile hemodynamic load, and endothelial dysfunction to atrial fibrillation (AF) remain poorly understood. To better understand the pathophysiology of AF, we examined associations between noninvasive measures of vascular function and new-onset AF. The study sample included participants aged ≥45 years from the Framingham Heart Study offspring and third-generation cohorts. Using Cox proportional hazards regression models, we examined relations between incident AF and tonometry measures of arterial stiffness (carotid-femoral pulse wave velocity), wave reflection (augmentation index), pressure pulsatility (central pulse pressure), endothelial function (flow-mediated dilation), resting brachial arterial diameter, and hyperemic flow. AF developed in 407/5797 participants in the tonometry sample and 270/3921 participants in the endothelial function sample during follow-up (median 7.1 years, maximum 10 years). Higher augmentation index (hazard ratio, 1.16; 95% confidence interval, 1.02-1.32; P=0.02), baseline brachial artery diameter (hazard ratio, 1.20; 95% confidence interval, 1.01-1.43; P=0.04), and lower flow-mediated dilation (hazard ratio, 0.79; 95% confidence interval, 0.63-0.99; P=0.04) were associated with increased risk of incident AF. Central pulse pressure, when adjusted for age, sex, and hypertension (hazard ratio, 1.14; 95% confidence interval, 1.02-1.28; P=0.02) was associated with incident AF. Higher pulsatile load assessed by central pulse pressure and greater apparent wave reflection measured by augmentation index were associated with increased risk of incident AF. Vascular endothelial dysfunction may precede development of AF. These measures may be additional risk factors or markers of subclinical cardiovascular disease associated with increased risk of incident AF. © 2016 American Heart Association, Inc.

Pulmonary endothelial pavement patterns.

PubMed Central

Kibria, G; Heath, D; Smith, P; Biggar, R

1980-01-01

The appearance of the endothelial pavement pattern was studied in the pulmonary trunk, pulmonary veins, aorta, and inferior vena cava of the rat by means of silver staining of the cell borders. The endothelial cell in each of the four blood vessels was found to have its own distinctive shape, fusiform and pointed in the direction of blood flow in the case of the aorta and larger and more rectangular in the pulmonary trunk and pulmonary veins. Detailed quantitation of the dimensions and surface area of the endothelial cells in each blood vessel was carried out by a photographic technique. Pulmonary hypertension was induced in one group of rats by feeding them on Crotalaria spectabilis seeds. The endothelial pavement pattern in their pulmonary trunks became disrupted with many of the cells assuming a fusiform shape reminiscent of aortic endothelium. Many small, new endothelial cells formed in the pulmonary trunk suggesting division of cells to line the enlarging blood vessels. In contrast the endothelial cells of the inferior vena cava merely increased in size to cope with the dilatation of this vein. Images PMID:7385090

Mathematical Modeling of Ischemia-Reperfusion Injury and Postconditioning Therapy.

PubMed

Fong, D; Cummings, L J

2017-11-01

Reperfusion (restoration of blood flow) after a period of ischemia (interruption of blood flow) can paradoxically place tissues at risk of further injury: so-called ischemia-reperfusion injury or IR injury. Recent studies have shown that postconditioning (intermittent periods of further ischemia applied during reperfusion) can reduce IR injury. We develop a mathematical model to describe the reperfusion and postconditioning process following an ischemic insult, treating the blood vessel as a two-dimensional channel, lined with a monolayer of endothelial cells that interact (respiration and mechanotransduction) with the blood flow. We investigate how postconditioning affects the total cell density within the endothelial layer, by varying the frequency of the pulsatile flow and the oxygen concentration at the inflow boundary. We find that, in the scenarios we consider, the pulsatile flow should be of high frequency to minimize cellular damage, while oxygen concentration at the inflow boundary should be held constant, or subject to only low-frequency variations, to maximize cell proliferation.

Defective fluid shear stress mechanotransduction mediates hereditary hemorrhagic telangiectasia

PubMed Central

Baeyens, Nicolas; Larrivée, Bruno; Ola, Roxana; Hayward-Piatkowskyi, Brielle; Dubrac, Alexandre; Huang, Billy; Ross, Tyler D.; Coon, Brian G.; Min, Elizabeth; Tsarfati, Maya; Tong, Haibin; Eichmann, Anne

2016-01-01

Morphogenesis of the vascular system is strongly modulated by mechanical forces from blood flow. Hereditary hemorrhagic telangiectasia (HHT) is an inherited autosomal-dominant disease in which arteriovenous malformations and telangiectasias accumulate with age. Most cases are linked to heterozygous mutations in Alk1 or Endoglin, receptors for bone morphogenetic proteins (BMPs) 9 and 10. Evidence suggests that a second hit results in clonal expansion of endothelial cells to form lesions with poor mural cell coverage that spontaneously rupture and bleed. We now report that fluid shear stress potentiates BMPs to activate Alk1 signaling, which correlates with enhanced association of Alk1 and endoglin. Alk1 is required for BMP9 and flow responses, whereas endoglin is only required for enhancement by flow. This pathway mediates both inhibition of endothelial proliferation and recruitment of mural cells; thus, its loss blocks flow-induced vascular stabilization. Identification of Alk1 signaling as a convergence point for flow and soluble ligands provides a molecular mechanism for development of HHT lesions. PMID:27646277

Gestational diabetes mellitus alters maternal and neonatal circulating endothelial progenitor cell subsets.

PubMed

Acosta, Juan C; Haas, David M; Saha, Chandan K; Dimeglio, Linda A; Ingram, David A; Haneline, Laura S

2011-03-01

The purpose of this study was to examine whether women with gestational diabetes mellitus (GDM) and their offspring have reduced endothelial progenitor cell subsets and vascular reactivity. Women with GDM, healthy control subjects, and their infants participated. Maternal blood and cord blood were assessed for colony-forming unit-endothelial cells and endothelial progenitor cell subsets with the use of polychromatic flow cytometry. Cord blood endothelial colony-forming cells were enumerated. Vascular reactivity was tested by laser Doppler imaging. Women with GDM had fewer CD34, CD133, CD45, and CD31 cells (circulating progenitor cells [CPCs]) at 24-32 weeks' gestation and 1-2 days after delivery, compared with control subjects. No differences were detected in colony-forming unit-endothelial cells or colony-forming unit-endothelial cells. In control subjects, CPCs were higher in the third trimester, compared with the postpartum period. Cord blood from GDM pregnancies had reduced CPCs. Vascular reactivity was not different between GDM and control subjects. The normal physiologic increase in CPCs during pregnancy is impaired in women with GDM, which may contribute to endothelial dysfunction and GDM-associated morbidities. Copyright © 2011 Mosby, Inc. All rights reserved.

Vascular effects of aldosterone: sorting out the receptors and the ligands.

PubMed

Feldman, Ross D; Gros, Robert

2013-12-01

Aldosterone has actions far beyond its role as a renal regulator of sodium reabsorption, and broader mechanisms of action than simply a transcriptional regulator. Aldosterone has a number of vascular effects, including regulation of vascular reactivity and vascular growth and/or development. Aldosterone-mediated effects on vascular reactivity reflect a balance between its endothelial-dependent vasodilator effects and its direct smooth muscle vasoconstrictor effects. The endothelial vasodilator effects of aldosterone are mediated by phosphatidylinositol 3-kinase-dependent activation of nitric oxide synthase. G-Protein oestrogen receptor (GPER) is a recently recognized G-protein coupled receptor (GPCR) that is activated by steroid hormones. It was first recognized as the GPCR mediating the rapid effects of oestrogens. Activation of GPER also mediates at least some of the vascular effects of aldosterone in smooth muscle and endothelial cells. In vascular endothelial cells, aldosterone activation of GPER mediates vasodilation. In contrast, activation of endothelial mineralocorticoid receptors has been linked to enhanced vasoconstrictor and/or impaired vasodilator responses. © 2013 Wiley Publishing Asia Pty Ltd.

Curcumin supplementation improves vascular endothelial function in healthy middle-aged and older adults by increasing nitric oxide bioavailability and reducing oxidative stress.

PubMed

Santos-Parker, Jessica R; Strahler, Talia R; Bassett, Candace J; Bispham, Nina Z; Chonchol, Michel B; Seals, Douglas R

2017-01-03

We hypothesized that curcumin would improve resistance and conduit artery endothelial function and large elastic artery stiffness in healthy middle-aged and older adults. Thirty-nine healthy men and postmenopausal women (45-74 yrs) were randomized to 12 weeks of curcumin (2000 mg/day Longvida®; n=20) or placebo (n=19) supplementation. Forearm blood flow response to acetylcholine infusions (FBF ACh ; resistance artery endothelial function) increased 37% following curcumin supplementation (107±13 vs. 84±11 AUC at baseline, P=0.03), but not placebo (P=0.2). Curcumin treatment augmented the acute reduction in FBF ACh induced by the nitric oxide synthase inhibitor NG monomethyl-L-arginine (L-NMMA; P=0.03), and reduced the acute increase in FBF ACh to the antioxidant vitamin C (P=0.02), whereas placebo had no effect (both P>0.6). Similarly, brachial artery flow-mediated dilation (conduit artery endothelial function) increased 36% in the curcumin group (5.7±0.4 vs. 4.4±0.4% at baseline, P=0.001), with no change in placebo (P=0.1). Neither curcumin nor placebo influenced large elastic artery stiffness (aortic pulse wave velocity or carotid artery compliance) or circulating biomarkers of oxidative stress and inflammation (all P>0.1). In healthy middle-aged and older adults, 12 weeks of curcumin supplementation improves resistance artery endothelial function by increasing vascular nitric oxide bioavailability and reducing oxidative stress, while also improving conduit artery endothelial function.

Curcumin supplementation improves vascular endothelial function in healthy middle-aged and older adults by increasing nitric oxide bioavailability and reducing oxidative stress

PubMed Central

Santos-Parker, Jessica R.; Strahler, Talia R.; Bassett, Candace J.; Bispham, Nina Z.; Chonchol, Michel B.; Seals, Douglas R.

2017-01-01

We hypothesized that curcumin would improve resistance and conduit artery endothelial function and large elastic artery stiffness in healthy middle-aged and older adults. Thirty-nine healthy men and postmenopausal women (45-74 yrs) were randomized to 12 weeks of curcumin (2000 mg/day Longvida®; n=20) or placebo (n=19) supplementation. Forearm blood flow response to acetylcholine infusions (FBFACh; resistance artery endothelial function) increased 37% following curcumin supplementation (107±13 vs. 84±11 AUC at baseline, P=0.03), but not placebo (P=0.2). Curcumin treatment augmented the acute reduction in FBFACh induced by the nitric oxide synthase inhibitor NG monomethyl-L-arginine (L-NMMA; P=0.03), and reduced the acute increase in FBFACh to the antioxidant vitamin C (P=0.02), whereas placebo had no effect (both P>0.6). Similarly, brachial artery flow-mediated dilation (conduit artery endothelial function) increased 36% in the curcumin group (5.7±0.4 vs. 4.4±0.4% at baseline, P=0.001), with no change in placebo (P=0.1). Neither curcumin nor placebo influenced large elastic artery stiffness (aortic pulse wave velocity or carotid artery compliance) or circulating biomarkers of oxidative stress and inflammation (all P>0.1). In healthy middle-aged and older adults, 12 weeks of curcumin supplementation improves resistance artery endothelial function by increasing vascular nitric oxide bioavailability and reducing oxidative stress, while also improving conduit artery endothelial function. PMID:28070018

An Integrative Review of Mechanotransduction in Endothelial, Epithelial (Renal) and Dendritic Cells (Osteocytes)

PubMed Central

Weinbaum, Sheldon; Duan, Yi; Thi, Mia M.; You, Lidan

2013-01-01

In this review we will examine from a biomechanical and ultrastructural viewpoint how the cytoskeletal specialization of three basic cell types, endothelial cells (ECs), epithelial cells (renal tubule) and dendritic cells (osteocytes), enables the mechano-sensing of fluid flow in both their native in vivo environment and in culture, and the downstream signaling that is initiated at the molecular level in response to fluid flow. These cellular responses will be discussed in terms of basic mysteries and paradoxes encountered by each cell type. In ECs fluid shear stress (FSS) is nearly entirely attenuated by the endothelial glycocalyx that covers their apical membrane and yet FSS is communicated to both intracellular and junctional molecular components in activating a wide variety of signaling pathways. The same is true in proximal tubule (PT) cells where a dense brush border of microvilli covers the apical surface and the flow at the apical membrane is negligible. A four decade old unexplained mystery is the ability of PT epithelia to reliably reabsorb 60% of the flow entering the tubule regardless of the glomerular filtration rate. In the cortical collecting duct (CCD) the flow rates are so low that a special sensing apparatus, a primary cilia is needed to detect very small variations in tubular flow. In bone it has been a century old mystery as to how osteocytes embedded in a stiff mineralized tissue are able to sense miniscule whole tissue strains that are far smaller than the cellular level strains required to activate osteocytes in vitro. PMID:23976901

Protective effects of flavanol-rich dark chocolate on endothelial function and wave reflection during acute hyperglycemia.

PubMed

Grassi, Davide; Desideri, Giovambattista; Necozione, Stefano; Ruggieri, Fabrizio; Blumberg, Jeffrey B; Stornello, Michele; Ferri, Claudio

2012-09-01

Nitric oxide plays a pivotal role in regulating vascular tone. Different studies show endothelial function is impaired during hyperglycemia. Dark chocolate increases flow-mediated dilation in healthy and hypertensive subjects with and without glucose intolerance; however, the effect of pretreatment with dark chocolate on endothelial function and other vascular responses to hyperglycemia has not been examined. Therefore, we aimed to investigate the effects of flavanol-rich dark chocolate administration on (1) flow-mediated dilation and wave reflections; (2) blood pressure, endothelin-1 and oxidative stress, before and after oral glucose tolerance test (OGTT). Twelve healthy volunteers (5 males, 28.2±2.7 years) randomly received either 100 g/d dark chocolate or flavanol-free white chocolate for 3 days. After 7 days washout period, volunteers were switched to the other treatment. Flow-mediated dilation, stiffness index, reflection index, peak-to-peak time, blood pressure, endothelin-1 and 8-iso-PGF(2α) were evaluated after each treatment phase and OGTT. Compared with white chocolate, dark chocolate ingestion improved flow-mediated dilation (P=0.03), wave reflections, endothelin-1 and 8-iso-PGF(2α) (P<0.05). After white chocolate ingestion, flow-mediated dilation was reduced after OGTT from 7.88±0.68 to 6.07±0.76 (P=0.027), 6.74±0.51 (P=0.046) at 1 and 2 h after the glucose load, respectively. Similarly, after white chocolate but not after dark chocolate, wave reflections, blood pressure, and endothelin-1 and 8-iso-PGF(2α) increased after OGTT. OGTT causes acute, transient impairment of endothelial function and oxidative stress, which is attenuated by flavanol-rich dark chocolate. These results suggest cocoa flavanols may contribute to vascular health by reducing the postprandial impairment of arterial function associated with the pathogenesis of atherosclerosis.

New stent surface materials: the impact of polymer-dependent interactions of human endothelial cells, smooth muscle cells, and platelets.

PubMed

Busch, Raila; Strohbach, Anne; Rethfeldt, Stefanie; Walz, Simon; Busch, Mathias; Petersen, Svea; Felix, Stephan; Sternberg, Katrin

2014-02-01

Despite the development of new coronary stent technologies, in-stent restenosis and stent thrombosis are still clinically relevant. Interactions of blood and tissue cells with the implanted material may represent an important cause of these side effects. We hypothesize material-dependent interaction of blood and tissue cells. The aim of this study is accordingly to investigate the impact of vascular endothelial cells, smooth muscle cells and platelets with various biodegradable polymers to identify a stent coating or platform material that demonstrates excellent endothelial-cell-supportive and non-thrombogenic properties. Human umbilical venous endothelial cells, human coronary arterial endothelial cells and human coronary arterial smooth muscle cells were cultivated on the surfaces of two established biostable polymers used for drug-eluting stents, namely poly(ethylene-co-vinylacetate) (PEVA) and poly(butyl methacrylate) (PBMA). We compared these polymers to new biodegradable polyesters poly(l-lactide) (PLLA), poly(3-hydroxybutyrate) (P(3HB)), poly(4-hydroxybutyrate) (P(4HB)) and a polymeric blend of PLLA/P(4HB) in a ratio of 78/22% (w/w). Biocompatibility tests were performed under static and dynamic conditions. Measurement of cell proliferation, viability, glycocalix width, eNOS and PECAM-1 mRNA expression revealed strong material dependency among the six polymer samples investigated. Only the polymeric blend of PLLA/P(4HB) achieved excellent endothelial markers of biocompatibility. Data show that PLLA and P(4HB) tend to a more thrombotic response, whereas the polymer blend is characterized by a lower thrombotic potential. These data demonstrate material-dependent endothelialization, smooth muscle cell growth and thrombogenicity. Although polymers such as PEVA and PBMA are already commonly used for vascular implants, they did not sufficiently meet the criteria for biocompatibility. The investigated biodegradable polymeric blend PLLA/P(4HB) evidently represents a promising material for vascular stents and stent coatings. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Involvement of the lysophosphatidic acid-generating enzyme autotaxin in lymphocyte-endothelial cell interactions.

PubMed

Nakasaki, Tae; Tanaka, Toshiyuki; Okudaira, Shinichi; Hirosawa, Michi; Umemoto, Eiji; Otani, Kazuhiro; Jin, Soojung; Bai, Zhongbin; Hayasaka, Haruko; Fukui, Yoshinori; Aozasa, Katsuyuki; Fujita, Naoya; Tsuruo, Takashi; Ozono, Keiichi; Aoki, Junken; Miyasaka, Masayuki

2008-11-01

Autotaxin (ATX) is a secreted protein with lysophospholipase D activity that generates lysophosphatidic acid (LPA) from lysophosphatidylcholine. Here we report that functional ATX is selectively expressed in high endothelial venules (HEVs) of both lymph nodes and Peyer's patches. ATX expression was developmentally regulated and coincided with lymphocyte recruitment to the lymph nodes. In adults, ATX expression was independent of HEV-expressed chemokines such as CCL21 and CXCL13, innate immunity signals including those via TLR4 or MyD88, and of the extent of lymphocyte trafficking across the HEVs. ATX expression was induced in venules at sites of chronic inflammation. Receptors for the ATX enzyme product LPA were constitutively expressed in HEV endothelial cells (ECs). In vitro, LPA induced strong morphological changes in HEV ECs. Forced ATX expression caused cultured ECs to respond to lysophosphatidylcholine, up-regulating lymphocyte binding to the ECs in a LPA receptor-dependent manner under both static and flow conditions. Although in vivo depletion of circulating ATX did not affect lymphocyte trafficking into the lymph nodes, we surmise, based on the above data, that ATX expressed by HEVs acts on HEVs in situ to facilitate lymphocyte binding to ECs and that ATX in the general circulation does not play a major role in this process. Tissue-specific inactivation of ATX will verify this hypothesis in future studies of its mechanism of action.

Antioxidant pretreatment and reduced arterial endothelial dysfunction after diving.

PubMed

Obad, Ante; Valic, Zoran; Palada, Ivan; Brubakk, Alf O; Modun, Darko; Dujić, Zeljko

2007-12-01

We have recently shown that a single air dive leads to acute arterial vasodilation and impairment of endothelium-dependent vasodilatation in humans. Additionally we have found that predive antioxidants at the upper recommended daily allowance partially prevented some of the negative effects of the dive. In this study we prospectively evaluated the effect of long-term antioxidants at a lower RDA dose on arterial endothelial function. Eight professional male divers performed an open sea air dive to 30 msw. Brachial artery flow-mediated dilation (FMD) was assessed before and after diving. The first dive, without antioxidants, caused significant brachial arterial diameter increase from 3.85 +/- 0.55 to 4.04 +/- 0.5 mm and a significant reduction of FMD from 7.6 +/- 2.7 to 2.8 +/- 2.1%. The second dive, with antioxidants, showed unchanged arterial diameter and significant reduction of FMD from 8.11 +/- 2.4 to 6.8 +/- 1.4%. The FMD reduction was significantly less with antioxidants. Vascular smooth muscle function, assessed by nitroglycerine (endothelium-independent dilation), was unaffected by diving. This study shows that long-term antioxidant treatment at a lower RDA dose ending 3-4 h before a dive reduces the endothelial dysfunction in divers. Since the scuba dive was of a similar depth and duration to those practiced by numerous recreational divers, this study raises the possibility of routine predive supplementation with antioxidants.

Dark chocolate consumption improves leukocyte adhesion factors and vascular function in overweight men.

PubMed

Esser, Diederik; Mars, Monica; Oosterink, Els; Stalmach, Angelique; Müller, Michael; Afman, Lydia A

2014-03-01

Flavanol-enriched chocolate consumption increases endothelium-dependent vasodilation. Most research so far has focused on flow-mediated dilation (FMD) only; the effects on other factors relevant to endothelial health, such as inflammation and leukocyte adhesion, have hardly been addressed. We investigated whether consumption of regular dark chocolate also affects other markers of endothelial health, and whether chocolate enrichment with flavanols has additional benefits. In a randomized double-blind crossover study, the effects of acute and of 4 wk daily consumption of high flavanol chocolate (HFC) and normal flavanol chocolate (NFC) on FMD, augmentation index (AIX), leukocyte count, plasma cytokines, and leukocyte cell surface molecules in overweight men (age 45-70 yr) were investigated. Sensory profiles and motivation scores to eat chocolate were also collected. Findings showed that a 4 wk chocolate intake increased FMD by 1%, which was paralleled by a decreased AIX of 1%, decreased leukocyte cell count, decreased plasma sICAM1 and sICAM3, and decreased leukocyte adhesion marker expression (P<0.05 for time effect), with no difference between HFC and NFC consumption. Flavanol enrichment did affect taste and negatively affected motivation to consume chocolate. This study provides new insights on how chocolate affects endothelial health by demonstrating that chocolate consumption, besides improving vascular function, also lowers the adherence capacity of leukocytes in the circulation.

Effect of acute moderate exercise on induced inflammation and arterial function in older adults.

PubMed

Ranadive, Sushant Mohan; Kappus, Rebecca Marie; Cook, Marc D; Yan, Huimin; Lane, Abbi Danielle; Woods, Jeffrey A; Wilund, Kenneth R; Iwamoto, Gary; Vanar, Vishwas; Tandon, Rudhir; Fernhall, Bo

2014-04-01

Acute inflammation reduces flow-mediated vasodilatation and increases arterial stiffness in young healthy individuals. However, this response has not been studied in older adults. The aim of this study, therefore, was to evaluate the effect of acute induced systemic inflammation on endothelial function and wave reflection in older adults. Furthermore, an acute bout of moderate-intensity aerobic exercise can be anti-inflammatory. Taken together, we tested the hypothesis that acute moderate-intensity endurance exercise, immediately preceding induced inflammation, would be protective against the negative effects of acute systemic inflammation on vascular function. Fifty-nine healthy volunteers between 55 and 75 years of age were randomized to an exercise or a control group. Both groups received a vaccine (induced inflammation) and sham (saline) injection in a counterbalanced crossover design. Inflammatory markers, endothelial function (flow-mediated vasodilatation) and measures of wave reflection and arterial stiffness were evaluated at baseline and at 24 and 48 h after injections. There were no significant differences in endothelial function and arterial stiffness between the exercise and control group after induced inflammation. The groups were then analysed together, and we found significant differences in the inflammatory markers 24 and 48 h after induction of acute inflammation compared with sham injection. However, flow-mediated vasodilatation, augmentation index normalized for heart rate (AIx75) and β-stiffness did not change significantly. Our results suggest that acute inflammation induced by influenza vaccination did not affect endothelial function in older adults.

Blood-Vessel Mimicking Structures by Stereolithographic Fabrication of Small Porous Tubes Using Cytocompatible Polyacrylate Elastomers, Biofunctionalization and Endothelialization

PubMed Central

Huber, Birgit; Engelhardt, Sascha; Meyer, Wolfdietrich; Krüger, Hartmut; Wenz, Annika; Schönhaar, Veronika; Tovar, Günter E. M.; Kluger, Petra J.; Borchers, Kirsten

2016-01-01

Blood vessel reconstruction is still an elusive goal for the development of in vitro models as well as artificial vascular grafts. In this study, we used a novel photo-curable cytocompatible polyacrylate material (PA) for freeform generation of synthetic vessels. We applied stereolithography for the fabrication of arbitrary 3D tubular structures with total dimensions in the centimeter range, 300 µm wall thickness, inner diameters of 1 to 2 mm and defined pores with a constant diameter of approximately 100 µm or 200 µm. We established a rinsing protocol to remove remaining cytotoxic substances from the photo-cured PA and applied thio-modified heparin and RGDC-peptides to functionalize the PA surface for enhanced endothelial cell adhesion. A rotating seeding procedure was introduced to ensure homogenous endothelial monolayer formation at the inner luminal tube wall. We showed that endothelial cells stayed viable and adherent and aligned along the medium flow under fluid-flow conditions comparable to native capillaries. The combined technology approach comprising of freeform additive manufacturing (AM), biomimetic design, cytocompatible materials which are applicable to AM, and biofunctionalization of AM constructs has been introduced as BioRap® technology by the authors. PMID:27104576

Modulating Vascular Hemodynamics With an Alpha Globin Mimetic Peptide (HbαX).

PubMed

Keller, T C Stevenson; Butcher, Joshua T; Broseghini-Filho, Gilson Brás; Marziano, Corina; DeLalio, Leon J; Rogers, Stephen; Ning, Bo; Martin, Jennifer N; Chechova, Sylvia; Cabot, Maya; Shu, Xiahong; Best, Angela K; Good, Miranda E; Simão Padilha, Alessandra; Purdy, Michael; Yeager, Mark; Peirce, Shayn M; Hu, Song; Doctor, Allan; Barrett, Eugene; Le, Thu H; Columbus, Linda; Isakson, Brant E

2016-12-01

The ability of hemoglobin to scavenge the potent vasodilator nitric oxide (NO) in the blood has been well established as a mechanism of vascular tone homeostasis. In endothelial cells, the alpha chain of hemoglobin (hereafter, alpha globin) and endothelial NO synthase form a macromolecular complex, providing a sink for NO directly adjacent to the production source. We have developed an alpha globin mimetic peptide (named HbαX) that displaces endogenous alpha globin and increases bioavailable NO for vasodilation. Here we show that, in vivo, HbαX administration increases capillary oxygenation and blood flow in arterioles acutely and produces a sustained decrease in systolic blood pressure in normal and angiotensin II-induced hypertensive states. HbαX acts with high specificity and affinity to endothelial NO synthase, without toxicity to liver and kidney and no effect on p50 of O 2 binding in red blood cells. In human vasculature, HbαX blunts vasoconstrictive response to cumulative doses of phenylephrine, a potent constricting agent. By binding to endothelial NO synthase and displacing endogenous alpha globin, HbαX modulates important metrics of vascular function, increasing vasodilation and flow in the resistance vasculature. © 2016 American Heart Association, Inc.

Self-reported racial discrimination and endothelial reactivity to acute stress in women.

PubMed

Wagner, Julie A; Tennen, Howard; Finan, Patrick H; Ghuman, Nimrta; Burg, Matthew M

2013-08-01

This study investigated the effect of self-reported racial discrimination on endothelial responses to acute laboratory mental stress among post-menopausal women. One-hundred thirteen women (n = 94 self-identified as White and n = 19 self-identified as racial/ethnic minority), 43% with type 2 diabetes, reported lifetime experiences of racial/ethnic discrimination. Repeated assessments of flow-mediated dilation were performed at baseline, immediately after 5 min of mental arithmetic and at 20-min recovery. Both White and racial/ethnic minority women reported lifetime discrimination, with rates significantly higher among minorities. Self-reported lifetime discrimination was associated with attenuated flow-mediated dilation at recovery. Confounding variables, including clinical characteristics, mood, personality traits, other life stressors and general distress, did not better account for the effect of racial discrimination. Neither race/ethnicity nor diabetes status moderated the effect. The perceived stressfulness of the mental arithmetic was not associated with the endothelial response. In conclusion, self-reported lifetime discrimination is associated with attenuated endothelial recovery from acute mental stress. Elucidating the effects of discrimination and the biological mechanisms through which it affects the vasculature may suggest interventions to improve health. Copyright © 2012 John Wiley & Sons, Ltd.

RAPAMYCIN INCREASES LENGTH AND MECHANOSENSORY FUNCTION OF PRIMARY CILIA IN RENAL EPITHELIAL AND VASCULAR ENDOTHELIAL CELLS.

PubMed

Sherpa, Rinzhin T; Atkinson, Kimberly F; Ferreira, Viviana P; Nauli, Surya M

2016-12-01

Primary cilia arebiophysically-sensitive organelles responsible for sensing fluid-flow and transducing this stimulus into intracellular responses. Previous studies have shown that the primary cilia mediate flow-induced calcium influx, and sensitivity of cilia function to flow is correlated to cilia length. Cells with abnormal cilia length or function can lead to a host of diseases that are collectively termed as ciliopathies. Rapamycin, a potent inhibitor of mTOR (mammalian target of rapamycin), has been demonstrated to be a potential pharmacological agent against the aberrant mTOR signaling seen in ciliopathies such as polycystic kidney disease (PKD) and tuberous sclerosis complex (TSC). Here we look at the effects of rapamycin on ciliary length and function for the first time. Compared to controls, primary cilia in rapamycin-treated porcine renal epithelial and mouse vascular endothelial cells showed a significant increase in length. Graded increases in fluid-shear stress further indicates that rapamycin enhances cilia sensitivity to fluid flow. Treatment with rapamycin led to G0 arrest in porcine epithelial cells while no significant change in cell cycle were observed in rapamycin-treated mouse epithelial or endothelial cells, indicating a species-specific effect of rapamycin. Given the previousin vitro and in vivo studies establishing rapamycin as a potential therapeutic agent for ciliopathies, such as PKD and TSC, our studies show that rapamycin enhances ciliary function and sensitivity to fluid flow. The results of our studies suggest a potential ciliotherapeutic effect of rapamycin.

Effects of 7-ketocholesterol on the activity of endothelial poly(ADP-ribose) polymerase and on endothelium-dependent relaxant function.

PubMed

Kiss, Levente; Chen, Min; Gero, Domokos; Módis, Katalin; Lacza, Zsombor; Szabó, Csaba

2006-12-01

Oxidative and nitrosative stress play an important role in the development of endothelial vascular dysfunction during early atherosclerosis. Oxidative stress activates the nuclear enzyme poly(ADP-ribose) polymerase (PARP) in endothelial cells. In patients with atherosclerosis the level of oxidized LDL in the plasma is elevated. In oxidized LDL various oxysterols have been identified, such as 7-ketocholesterol (7K). 7K has been shown to induce PARP activation in microglial cells. The aim of the current study was to clarify the effects of 7K on the activity of endothelial PARP and on the endothelium-dependent relaxant function of blood vessels. We treated human umbilical vein endothelial (HUVEC) cells with 2-16 microg/ml 7K as well as vascular rings harvested from BALB/c mouse thoracic aorta with 90 microg/ml 7K for 2 h. A group of mice was treated with 7K subcutaneously for 1 week (10 mg/kg/day). We also conducted in vitro and in vivo experiments using pretreatment with buthionine sulphoximine (BSO), a glutathione-lowering agent. The activity of PARP was calculated by measurement of tritiated NAD incorporation. The activity of PARP increased significantly in 7K-treated HUVEC cells. After BSO pretreatment, this increase was higher. Isolated vascular rings demonstrated no change in endothelium-dependent relaxant function after 2 h of incubation with 7K, even after BSO pretreatment. In vivo treatment with 7K for 1 week had no effect on the relaxant function. Our experimental results suggest that although 7-ketocholesterol can activate PARP enzyme in endothelial cells, it is not sufficient on its own to cause impairment in the endothelium-dependent vascular reactivity.

HZE ⁵⁶Fe-ion irradiation induces endothelial dysfunction in rat aorta: role of xanthine oxidase.

PubMed

Soucy, Kevin G; Lim, Hyun Kyo; Kim, Jae Hyung; Oh, Young; Attarzadeh, David O; Sevinc, Baris; Kuo, Maggie M; Shoukas, Artin A; Vazquez, Marcelo E; Berkowitz, Dan E

2011-10-01

Ionizing radiation has been implicated in the development of significant cardiovascular complications. Since radiation exposure is associated with space exploration, astronauts are potentially at increased risk of accelerated cardiovascular disease. This study investigated the effect of high atomic number, high-energy (HZE) iron-ion radiation on vascular and endothelial function as a model of space radiation. Rats were exposed to a single whole-body dose of iron-ion radiation at doses of 0, 0.5 or 1 Gy. In vivo aortic stiffness and ex vivo aortic tension responses were measured 6 and 8 months after exposure as indicators of chronic vascular injury. Rats exposed to 1 Gy iron ions demonstrated significantly increased aortic stiffness, as measured by pulse wave velocity. Aortic rings from irradiated rats exhibited impaired endothelial-dependent relaxation consistent with endothelial dysfunction. Acute xanthine oxidase (XO) inhibition or reactive oxygen species (ROS) scavenging restored endothelial-dependent responses to normal. In addition, XO activity was significantly elevated in rat aorta 4 months after whole-body irradiation. Furthermore, XO inhibition, initiated immediately after radiation exposure and continued until euthanasia, completely inhibited radiation-dependent XO activation. ROS production was elevated after 1 Gy irradiation while production of nitric oxide (NO) was significantly impaired. XO inhibition restored NO and ROS production. Finally, dietary XO inhibition preserved normal endothelial function and vascular stiffness after radiation exposure. These results demonstrate that radiation induced XO-dependent ROS production and nitroso-redox imbalance, leading to chronic vascular dysfunction. As a result, XO is a potential target for radioprotection. Enhancing the understanding of vascular radiation injury could lead to the development of effective methods to ameliorate radiation-induced vascular damage.

Tryptophan depletion under conditions that imitate insulin resistance enhances fatty acid oxidation and induces endothelial dysfunction through reactive oxygen species-dependent and independent pathways.

PubMed

Eleftheriadis, Theodoros; Pissas, Georgios; Sounidaki, Maria; Antoniadi, Georgia; Rountas, Christos; Liakopoulos, Vassilios; Stefanidis, Loannis

2017-04-01

In atherosclerosis-associated pathologic entities characterized by malnutrition and inflammation, L-tryptophan (TRP) levels are low. Insulin resistance is an independent cardiovascular risk factor and induces endothelial dysfunction by increasing fatty acid oxidation. It is also associated with inflammation and low TRP levels. Low TRP levels have been related to worse cardiovascular outcome. This study evaluated the effect of TRP depletion on endothelial dysfunction under conditions that imitate insulin resistance. Fatty acid oxidation, harmful pathways due to increased fatty acid oxidation, and endothelial dysfunction were assessed in primary human aortic endothelial cells cultured under normal glucose, low insulin conditions in the presence or absence of TRP. TRP depletion activated general control non-derepressible 2 kinase and inhibited aryl hydrocarbon receptor. It increased fatty acid oxidation by increasing expression and activity of carnitine palmitoyltransferase 1. Elevated fatty acid oxidation increased the formation of reactive oxygen species (ROS) triggering the polyol and hexosamine pathways, and enhancing protein kinase C activity and methylglyoxal production. TRP absence inhibited nitric oxide synthase activity in a ROS-dependent way, whereas it increased the expression of ICAM-1 and VCAM-1 in a ROS independent and possibly p53-dependent manner. Thus, TRP depletion, an amino acid whose low levels have been related to worse cardiovascular outcome and to inflammatory atherosclerosis-associated pathologic entities, under conditions that imitate insulin resistance enhances fatty acid oxidation and induces endothelial dysfunction through ROS-dependent and independent pathways. These findings may offer new insights at the molecular mechanisms involved in accelerated atherosclerosis that frequently accompanies malnutrition and inflammation.

Oxidative stress-induced iron signaling is responsible for peroxide-dependent oxidation of dichlorodihydrofluorescein in endothelial cells: role of transferrin receptor-dependent iron uptake in apoptosis.

PubMed

Tampo, Yoshiko; Kotamraju, Srigiridhar; Chitambar, Christopher R; Kalivendi, Shasi V; Keszler, Agnes; Joseph, Joy; Kalyanaraman, B

2003-01-10

Dichlorodihydrofluorescein (DCFH) is one of the most frequently used probes for detecting intracellular oxidative stress. In this study, we report that H2O2-dependent intracellular oxidation of DCFH to a green fluorescent product, 2',7'-dichlorofluorescein (DCF), required the uptake of extracellular iron transported through a transferrin receptor (TfR) in endothelial cells. H2O2-induced DCF fluorescence was inhibited by the monoclonal IgA-class anti-TfR antibody (42/6) that blocked TfR endocytosis and the iron uptake. H2O2-mediated inactivation of cytosolic aconitase was responsible for activation of iron regulatory protein-1 and increased expression of TfR, resulting in an increased iron uptake into endothelial cells. H2O2-mediated caspase-3 proteolytic activation was inhibited by anti-TfR antibody. Similar results were obtained in the presence of a lipid hydroperoxide. We conclude that hydroperoxide-induced DCFH oxidation and endothelial cell apoptosis required the uptake of extracellular iron by the TfR-dependent iron transport mechanism and that the peroxide-induced iron signaling, in general, has broader implications in oxidative vascular biology.

Effects of antioxidants on endothelial function in human saphenous vein in an ex vivo model.

PubMed

Sharif, Muhammed Anees; Bayraktutan, Ulvi; Arya, Nityanand; Badger, Stephen A; O'Donnell, Mark E; Young, Ian S; Soong, Chee V

2009-01-01

This ex vivo study is aimed at determining the beneficial effects of antioxidant agents on human saphenous vein endothelial function. Vein rings harvested during infrainguinal bypass surgery were assessed in an organ bath for endothelium-dependent relaxation, initially without and then with the addition of 10 microM manganese tetrakis benzoic acid porphyrin (MnTBAP), 0.01% N-acetylcysteine (NAC), 0.02% NAC, 10 microM vitamin C, and 100 microM vitamin C. Fifty-five vein rings from 22 patients were analyzed. MnTBAP improved the endothelium-dependent relaxation when compared with control (57.0% vs 37.8%, P < .01). Addition of 0.01% or 0.02% NAC did not improve the endothelium-dependent vasorelaxation (28.2% vs 18.6%, P = ns and 37.8% vs 29.8%, P = ns, respectively). Although 10-microM vitamin C failed to improve endothelial function (50.6% vs 37.2%, P = ns), 100-microM vitamin C significantly enhanced endothelium-dependent relaxation (66.5% vs 38.3%, P < .001). These results suggest that the addition of MnTBAP and high-dose vitamin C can improve the endothelial function of harvested saphenous vein segments in an ex vivo model.

Inhibition of nitric oxide synthesis in forearm vasculature of insulin-dependent diabetic patients: blunted vasoconstriction in patients with microalbuminuria.

PubMed

Elliott, T G; Cockcroft, J R; Groop, P H; Viberti, G C; Ritter, J M

1993-12-01

1. Microalbuminuria is a risk factor for cardiovascular disease in patients with insulin-dependent diabetes mellitus, and may be a marker of microvascular dysfunction including endothelial damage. The purpose of this study was to determine whether vasoconstrictor responses to NG-monomethyl-L-arginine, an inhibitor of endothelium-derived relaxing factor/nitric oxide biosynthesis, differ between healthy subjects and insulin-dependent patients with or without microalbuminuria. 2. Twenty-eight insulin-dependent diabetic patients (14 with normal albumin excretion, 14 with microalbuminuria) were studied under euglycaemic conditions, together with 14 healthy control subjects. Forearm vascular responses to brachial artery infusions of NG-monomethyl-L-arginine, sodium nitroprusside (an endothelium-independent nitrovasodilator) and carbachol (an endothelium-dependent vasodilator) were determined by strain gauge plethysmography. 3. Basal blood flow and vasodilator responses were similar in each group. NG-Monomethyl-L-arginine reduced blood flow by 41.3 +/- 2.3% (mean +/- SEM) in healthy control subjects, 34.0 +/- 3.4% in diabetic patients without microalbuminuria and 29.2 +/- 2.0% in diabetic patients with microalbuminuria. Diabetic patients differed from healthy subjects (P = 0.005), due to a difference between control subjects and microalbuminuric diabetic patients (P < 0.001). NG-Monomethyl-L-arginine did not influence nitroprusside responses but reduced carbachol responses in control subjects and normoalbuminuric diabetic patients but not in microalbuminuric diabetic patients. 4. These results provide evidence of abnormal endothelium-derived relaxing factor/nitric oxide biosynthesis in insulin-dependent diabetic patients with microalbuminuria.

Endothelial disruptive proinflammatory effects of nicotine and e-cigarette vapor exposures.

PubMed

Schweitzer, Kelly S; Chen, Steven X; Law, Sarah; Van Demark, Mary; Poirier, Christophe; Justice, Matthew J; Hubbard, Walter C; Kim, Elena S; Lai, Xianyin; Wang, Mu; Kranz, William D; Carroll, Clinton J; Ray, Bruce D; Bittman, Robert; Goodpaster, John; Petrache, Irina

2015-07-15

The increased use of inhaled nicotine via e-cigarettes has unknown risks to lung health. Having previously shown that cigarette smoke (CS) extract disrupts the lung microvasculature barrier function by endothelial cell activation and cytoskeletal rearrangement, we investigated the contribution of nicotine in CS or e-cigarettes (e-Cig) to lung endothelial injury. Primary lung microvascular endothelial cells were exposed to nicotine, e-Cig solution, or condensed e-Cig vapor (1-20 mM nicotine) or to nicotine-free CS extract or e-Cig solutions. Compared with nicotine-containing extract, nicotine free-CS extract (10-20%) caused significantly less endothelial permeability as measured with electric cell-substrate impedance sensing. Nicotine exposures triggered dose-dependent loss of endothelial barrier in cultured cell monolayers and rapidly increased lung inflammation and oxidative stress in mice. The endothelial barrier disruptive effects were associated with increased intracellular ceramides, p38 MAPK activation, and myosin light chain (MLC) phosphorylation, and was critically mediated by Rho-activated kinase via inhibition of MLC-phosphatase unit MYPT1. Although nicotine at sufficient concentrations to cause endothelial barrier loss did not trigger cell necrosis, it markedly inhibited cell proliferation. Augmentation of sphingosine-1-phosphate (S1P) signaling via S1P1 improved both endothelial cell proliferation and barrier function during nicotine exposures. Nicotine-independent effects of e-Cig solutions were noted, which may be attributable to acrolein, detected along with propylene glycol, glycerol, and nicotine by NMR, mass spectrometry, and gas chromatography, in both e-Cig solutions and vapor. These results suggest that soluble components of e-Cig, including nicotine, cause dose-dependent loss of lung endothelial barrier function, which is associated with oxidative stress and brisk inflammation.

Homocysteine impaired endothelial function through compromised vascular endothelial growth factor/Akt/endothelial nitric oxide synthase signalling.

PubMed

Yan, Ting-Ting; Li, Qian; Zhang, Xuan-Hong; Wu, Wei-Kang; Sun, Juan; Li, Lin; Zhang, Quan; Tan, Hong-Mei

2010-11-01

1. Hyperhomocysteinaemia (HHcy) is associated with endothelial dysfunction and has been recognized as a risk factor of cardiovascular disease. The present study aimed to investigate the effect of homocysteine (Hcy) on endothelial function in vivo and in vitro, and the underlying signalling pathways. 2. The HHcy animal model was established by intragastric administration with l-methionine in rats. Plasma Hcy and nitric oxide (NO) concentration were measured by fluorescence immunoassay or nitrate reductase method, respectively. Vasorelaxation in response to acetylcholine and sodium nitroprusside were carried out on aortic rings. Human umbilical vein endothelial cells (HUVEC) were treated with indicated concentrations of Hcy in the in vitro experiments. Intracellular NO level and NO concentration in culture medium were assayed. The alterations of possible signalling proteins were detected by western blot analysis. 3. l-methionine administration induced a significant increase in plasma Hcy and decrease in plasma NO. Endothelium-dependent relaxation of aortic rings in response to acetylcholine was impaired in l-methionine-administrated rats. The in vitro study showed that Hcy reduced both intracellular and culture medium NO levels. Furthermore, Hcy decreased phosphorylation of endothelial nitric oxide synthase (eNOS) at serine-1177 and phosphorylation of Akt at serine-473. Hcy-induced dephosphorylation of eNOS at Ser-1177 was partially reversed by insulin (Akt activator) and GF109203X (PKC inhibitor). Furthermore, Hcy reduced vascular endothelial growth factor (VEGF) expression in a dose-dependent manner. 4. In conclusion, Hcy impaired endothelial function through compromised VEGF/Akt/endothelial nitric oxide synthase signalling. These findings will be beneficial for further understanding the role of Hcy in cardiovascular disease. © 2010 Blackwell Publishing Asia Pty Ltd.

Angiotensin-converting enzyme 2 activation improves endothelial function.

PubMed

Fraga-Silva, Rodrigo A; Costa-Fraga, Fabiana P; Murça, Tatiane M; Moraes, Patrícia L; Martins Lima, Augusto; Lautner, Roberto Q; Castro, Carlos H; Soares, Célia Maria A; Borges, Clayton L; Nadu, Ana Paula; Oliveira, Marilene L; Shenoy, Vinayak; Katovich, Michael J; Santos, Robson A S; Raizada, Mohan K; Ferreira, Anderson J

2013-06-01

Diminished release and function of endothelium-derived nitric oxide coupled with increases in reactive oxygen species production is critical in endothelial dysfunction. Recent evidences have shown that activation of the protective axis of the renin-angiotensin system composed by angiotensin-converting enzyme 2, angiotensin-(1-7), and Mas receptor promotes many beneficial vascular effects. This has led us to postulate that activation of intrinsic angiotensin-converting enzyme 2 would improve endothelial function by decreasing the reactive oxygen species production. In the present study, we tested 1-[[2-(dimetilamino)etil]amino]-4-(hidroximetil)-7-[[(4-metilfenil)sulfonil]oxi]-9H-xantona-9 (XNT), a small molecule angiotensin-converting enzyme 2 activator, on endothelial function to validate this hypothesis. In vivo treatment with XNT (1 mg/kg per day for 4 weeks) improved the endothelial function of spontaneously hypertensive rats and of streptozotocin-induced diabetic rats when evaluated through the vasorelaxant responses to acetylcholine/sodium nitroprusside. Acute in vitro incubation with XNT caused endothelial-dependent vasorelaxation in aortic rings of rats. This vasorelaxation effect was attenuated by the Mas antagonist D-pro7-Ang-(1-7), and it was reduced in Mas knockout mice. These effects were associated with reduction in reactive oxygen species production. In addition, Ang II-induced reactive oxygen species production in human aortic endothelial cells was attenuated by preincubation with XNT. These results showed that chronic XNT administration improves the endothelial function of hypertensive and diabetic rat vessels by attenuation of the oxidative stress. Moreover, XNT elicits an endothelial-dependent vasorelaxation response, which was mediated by Mas. Thus, this study indicated that angiotensin-converting enzyme 2 activation promotes beneficial effects on the endothelial function and it is a potential target for treating cardiovascular disease.

ANGIOTENSIN-CONVERTING ENZYME 2 ACTIVATION IMPROVES ENDOTHELIAL FUNCTION

PubMed Central

Fraga-Silva, Rodrigo A.; Costa-Fraga, Fabiana P.; Murça, Tatiane M.; Moraes, Patrícia L.; Lima, Augusto Martins; Lautner, Roberto Q.; Castro, Carlos H.; Soares, Célia Maria A.; Borges, Clayton L.; Nadu, Ana Paula; Oliveira, Marilene L.; Shenoy, Vinayak; Katovich, Michael J.; Santos, Robson A.S.; Raizada, Mohan K.; Ferreira, Anderson J.

2013-01-01

Diminished release and function of endothelium-derived nitric oxide (NO) coupled with increases in reactive oxygen species (ROS) production is critical in endothelial dysfunction. Recent evidences have shown that activation of the protective axis of the renin-angiotensin system composed by angiotensin-converting enzyme2 (ACE2), Angiotensin-(1-7) [Ang-(1-7)] and Mas receptor promotes many beneficial vascular effects. This has led us to postulate that activation of intrinsic ACE2 would improve endothelial function by decreasing the ROS production. In the present study, we tested 1-[[2-(dimetilamino)etil]amino]-4-(hidroximetil)-7-[[(4-metilfenil)sulfonil]oxi]-9H-xantona-9 (XNT), a small molecule ACE2 activator, on endothelial function to validate this hypothesis. In vivo treatment with XNT (1mg/kg/day for 4 weeks) improved the endothelial function of spontaneously hypertensive rats and of streptozotocin-induced diabetic rats when evaluated through the vasorelaxant responses to acetylcholine/sodium nitroprusside. Acute in vitro incubation with XNT caused endothelial-dependent vasorelaxation in aortic rings of rats. This vasorelaxation effect was attenuated by the Mas antagonist D-pro7-Ang-(1-7) and it was reduced in Mas knockout mice. These effects were associated with reduction in ROS production. In addition, Ang II-induced ROS production in human aortic endothelial cells was attenuated by pre-incubation with XNT. These results showed that chronic XNT administration improves the endothelial function of hypertensive and diabetic rat vessels by attenuation of the oxidative stress. Moreover, XNT elicits an endothelial-dependent vasorelaxation response, which was mediated by Mas. Thus, this study indicated that ACE2 activation promotes beneficial effects on the endothelial function and it is a potential target for treating cardiovascular disease. PMID:23608648

Endothelial mineralocorticoid receptor activation mediates endothelial dysfunction in diet-induced obesity

PubMed Central

Schäfer, Nicola; Lohmann, Christine; Winnik, Stephan; van Tits, Lambertus J.; Miranda, Melroy X.; Vergopoulos, Athanasios; Ruschitzka, Frank; Nussberger, Jürg; Berger, Stefan; Lüscher, Thomas F.; Verrey, François; Matter, Christian M.

2013-01-01

Received 22 July 2012; revised 29 January 2013; accepted 4 March 2013 Aims Aldosterone plays a crucial role in cardiovascular disease. ‘Systemic’ inhibition of its mineralocorticoid receptor (MR) decreases atherosclerosis by reducing inflammation and oxidative stress. Obesity, an important cardiovascular risk factor, is an inflammatory disease associated with increased plasma aldosterone levels. We have investigated the role of the ‘endothelial’ MR in obesity-induced endothelial dysfunction, the earliest stage in atherogenesis. Methods and results C57BL/6 mice were exposed to a normal chow diet (ND) or a high-fat diet (HFD) alone or in combination with the MR antagonist eplerenone (200 mg/kg/day) for 14 weeks. Diet-induced obesity impaired endothelium-dependent relaxation in response to acetylcholine, whereas eplerenone treatment of obese mice prevented this. Expression analyses in aortic endothelial cells isolated from these mice revealed that eplerenone attenuated expression of pro-oxidative NADPH oxidase (subunits p22phox, p40phox) and increased expression of antioxidative genes (glutathione peroxidase-1, superoxide dismutase-1 and -3) in obesity. Eplerenone did not affect obesity-induced upregulation of cyclooxygenase (COX)-1 or prostacyclin synthase. Endothelial-specific MR deletion prevented endothelial dysfunction in obese (exhibiting high ‘endogenous’ aldosterone) and in ‘exogenous’ aldosterone-infused lean mice. Pre-incubation of aortic rings from aldosterone-treated animals with the COX-inhibitor indomethacin restored endothelial function. Exogenous aldosterone administration induced endothelial expression of p22phox in the presence, but not in the absence of the endothelial MR. Conclusion Obesity-induced endothelial dysfunction depends on the ‘endothelial’ MR and is mediated by an imbalance of oxidative stress-modulating mechanisms. Therefore, MR antagonists may represent an attractive therapeutic strategy in the increasing population of obese patients to decrease vascular dysfunction and subsequent atherosclerotic complications. PMID:23594590

Flow Perturbation Mediates Neutrophil Recruitment and Potentiates Endothelial Injury via TLR2 in Mice – Implications for Superficial Erosion

PubMed Central

Franck, Grégory; Mawson, Thomas; Sausen, Grasiele; Salinas, Manuel; Masson, Gustavo Santos; Cole, Andrew; Beltrami-Moreira, Marina; Chatzizisis, Yiannis; Quillard, Thibault; Tesmenitsky, Yevgenia; Shvartz, Eugenia; Sukhova, Galina K.; Swirski, Filip K.; Nahrendorf, Matthias; Aikawa, Elena; Croce, Kevin J.; Libby, Peter

2017-01-01

Rationale Superficial erosion currently causes up to a third of acute coronary syndromes (ACS), yet we lack understanding of its mechanisms. Thrombi due to superficial intimal erosion characteristically complicate matrix-rich atheromata in regions of flow perturbation. Objective This study tested in vivo the involvement of disturbed flow, and of neutrophils, hyaluronan, and TLR2 ligation in superficial intimal injury, a process implicated in superficial erosion. Methods and Results : In mouse carotid arteries with established intimal lesions tailored to resemble the substrate of human eroded plaques, acute flow perturbation promoted downstream endothelial cell (EC) activation, neutrophil accumulation, EC death and desquamation, and mural thrombosis. Neutrophil loss-of-function limited these findings. TLR2 agonism activated luminal ECs, and deficiency of this innate immune receptor decreased intimal neutrophil adherence in regions of local flow disturbance, reducing EC injury and local thrombosis (p<0.05). Conclusions These results implicate flow disturbance, neutrophils, and TLR2 signaling as mechanisms that contribute to superficial erosion, a cause of ACS of likely growing importance in the statin era. PMID:28428204

Two ways to feel the pressure: an endothelial Ca(2+) entry channel with dual mechanosensitivity.

PubMed

Groschner, Klaus

2002-01-01

One impressive function of the vascular endothelium is its ability to adjust the release of vasoactive mediators such as NO and PGI(2) almost instantaneously to changes in blood flow or blood pressure. Besides this fast feedback response to hemodynamic alterations, the endothelium is subject to long-term adaptations that are crucial for prevention of pathological processes such as atherogenesis. Among the various signals that are sensed by endothelial cells, mechanical forces which arise from pulsatile blood flow are probably most important for fast as well as long-term control of blood vessel function by the endothelium.

Alterations in NO- and PGI2- dependent function in aorta in the orthotopic murine model of metastatic 4T1 breast cancer: relationship with pulmonary endothelial dysfunction and systemic inflammation.

PubMed

Buczek, E; Denslow, A; Mateuszuk, L; Proniewski, B; Wojcik, T; Sitek, B; Fedorowicz, A; Jasztal, A; Kus, E; Chmura-Skirlinska, A; Gurbiel, R; Wietrzyk, J; Chlopicki, S

2018-05-22

Patients with cancer develop endothelial dysfunction and subsequently display a higher risk of cardiovascular events. The aim of the present work was to examine changes in nitric oxide (NO)- and prostacyclin (PGI 2 )-dependent endothelial function in the systemic conduit artery (aorta), in relation to the formation of lung metastases and to local and systemic inflammation in a murine orthotopic model of metastatic breast cancer. BALB/c female mice were orthotopically inoculated with 4T1 breast cancer cells. Development of lung metastases, lung inflammation, changes in blood count, systemic inflammatory response (e.g. SAA, SAP and IL-6), as well as changes in NO- and PGI 2 -dependent endothelial function in the aorta, were examined 2, 4, 5 and 6 weeks following cancer cell transplantation. As early as 2 weeks following transplantation of breast cancer cells, in the early metastatic stage, lungs displayed histopathological signs of inflammation, NO production was impaired and nitrosylhemoglobin concentration in plasma was decreased. After 4 to 6 weeks, along with metastatic development, progressive leukocytosis and systemic inflammation (as seen through increased SAA, SAP, haptoglobin and IL-6 plasma concentrations) were observed. Six weeks following cancer cell inoculation, but not earlier, endothelial dysfunction in aorta was detected; this involved a decrease in basal NO production and a decrease in NO-dependent vasodilatation, that was associated with a compensatory increase in cyclooxygenase-2 (COX-2)- derived PGI 2 production. In 4 T1 metastatic breast cancer in mice early pulmonary metastasis was correlated with lung inflammation, with an early decrease in pulmonary as well as systemic NO availability. Late metastasis was associated with robust, cancer-related, systemic inflammation and impairment of NO-dependent endothelial function in the aorta that was associated with compensatory upregulation of the COX-2-derived PGI 2 pathway.

Caspase-1 Inflammasome Activation Mediates Homocysteine-Induced Pyrop-Apoptosis in Endothelial Cells

PubMed Central

Xi, Hang; Zhang, Yuling; Xu, Yanjie; Yang, William Y; Jiang, Xiaohua; Sha, Xiaojin; Cheng, Xiaoshu; Wang, Jingfeng; Qin, Xuebin; Yu, Jun; Ji, Yong; Yang, Xiaofeng; Wang, Hong

2016-01-01

Rationale Endothelial injury is an initial mechanism mediating cardiovascular disease. Objective Here, we investigated the effect of hyperhomocysteinemia (HHcy) on programed cell death in endothelial cells (EC). Methods and Results We established a novel flow-cytometric gating method to define pyrotosis (Annexin V−/Propidium iodide+). In cultured human EC, we found that: 1). Hcy and Lipopolysaccharide (LPS) individually and synergistically induced inflammatory pyroptotic and non-inflammatory apoptotic cell death. 2). Hcy/LPS induced caspase-1 activation prior to caspase-8, -9, -3 activations. 3). Caspase-1/3 inhibitors rescued Hcy/LPS-induced pyroptosis/apoptosis, but caspase-8/9 inhibitors had differential rescue effect. 4). Hcy/LPS induced NLRP3 protein, caused NLRP3-containing inflammasome assembly, caspase-1 activation and IL-1β cleavage/activation. 5). Hcy/LPS elevated intracellular reactive oxidative species (ROS). 6). Intracellular oxidative gradient determined cell death destiny as intermediate intracellular ROS levels are associated with pyroptosis, whereas, high ROS corresponded to apoptosis. 7). Hcy/LPS induced mitochondrial membrane potential collapse and cytochrome-c release, and increased Bax/Bcl-2 ratio which were attenuated by antioxidants and caspase-1 inhibitor. 8). Antioxidants extracellular superoxide dismutase and catalase prevented Hcy/LPS-induced caspase-1 activation, mitochondrial dysfunction and pyroptosis/apoptosis. In cystathionine β-synthase deficient (Cbs−/−) mice, severe HHcy induced caspase-1 activation in isolated lung EC and caspase-1 expression in aortic endothelium, and elevated aortic caspase-1,9 protein/activity and Bax/Bcl-2 ratio in Cbs−/− aorta and HUVEC. Finally, Hcy-induced DNA fragmentation was reversed in caspase-1−/− EC. HHcy-induced aortic endothelial dysfunction was rescued in caspase-1−/− and NLRP3−/− mice. Conclusion HHcy preferentially induces EC pyroptosis via caspase-1-dependent inflammasome activation leading to endothelial dysfunction. We termed caspase-1 responsive pyroptosis and apoptosis as pyrop-apoptosis. PMID:27006445

Circulating endothelial progenitor cells, Th1/Th2/Th17-related cytokines, and endothelial dysfunction in resistant hypertension.

PubMed

Magen, Eli; Feldman, Arie; Cohen, Ziona; Alon, Dora Ben; Minz, Evegeny; Chernyavsky, Alexey; Linov, Lina; Mishal, Joseph; Schlezinger, Menacham; Sthoeger, Zev

2010-02-01

A possible link between chronic vascular inflammation and arterial hypertension is now an object of intensive studies. To compare Th1/Th2/Th17 cells-related cytokines, circulating endothelial progenitor cells (EPC), and endothelial function in subjects with resistant arterial hypertension (RAH) and controlled arterial hypertension (CAH). Blood pressure was measured by electronic sphygmomanometer. EPC were identified as CD34+/CD133+/kinase insert domain receptor (KDR)+ cells by flow cytometry. Th1/Th2/Th17 cells-related cytokines were identified using the Human Th1/Th2/Th17 Cytokines MultiAnalyte ELISArray Kit. Endothelium-dependent (FMD) vasodilatation of brachial artery was measured by Doppler ultrasound scanning. RAH group (n = 20) and CAH group (n = 20) and 17 healthy individuals (control group) were recruited. In the RAH group, lower blood levels of EPC number (42.4 +/- 16.7 cells/mL) and EPC% (0.19 +/- 0.08%) were observed than in the CAH group (93.1 +/- 88.7 cells/mL; P = 0.017; 0.27 +/- 0.17; P = 0.036) and control group (68.5 +/- 63.6 cells/mL; P < 0.001; 0.28 +/- 0.17%; P = 0.003), respectively. Plasma transforming growth factor-beta1 levels were significantly higher in the RAH group (1767 +/- 364 pg/mL) than in the CAH group (1292 +/- 349; P < 0.001) and in control group (1203 +/- 419 pg/mL; P < 0.001). In the RAH group, statistically significant negative correlation was observed between systolic blood pressure and EPC% (r = -0.72, P < 0.01). FMD in the RAH group was significantly lower (5.5 +/- 0.8%) than in the CAH group (9.2 +/- 1.4; P < 0.001) and in healthy controls (10.1 +/- 1.1%; P < 0.001). RAH is characterized by reduced circulating EPC, substantial endothelial dysfunction, and increased plasma transforming growth factor-beta1 levels.

Relationship between biomarkers of inflammation, oxidative stress and endothelial/microcirculatory function in successful aging versus healthy youth: a transversal study.

PubMed

Bottino, Daniel Alexandre; Lopes, Flávia Gomes; de Oliveira, Francisco José; Mecenas, Anete de Souza; Clapauch, Ruth; Bouskela, Eliete

2015-04-08

There is a functional decline of endothelial- dependent vasodilatation in the aging process. The aims of this study were to investigate if various microcirculatory parameters could correlate to anthropometrical variables, oxidative stress and inflammatory biomarkers in successful aging and compare the results to young healthy controls. Healthy elderly women (HE, 74.0 ± 8.7 years, n = 11) and young controls (YC, 23.1 ± 3.6 years, n = 24) were evaluated through nailfold videocapillaroscopy (NVC), venous occlusion plethysmography (VOP) and laboratorial analysis. Functional capillary density (FCD) and diameters, maximum red blood cell velocity (RBCVmax) during the reactive hyperemia response/RBCVbaseline after 1 min arterial occlusion at the finger base, time to reach RBCVmax were determined by NVC, peak increment of forearm blood flow (FBF) during the reactive hyperemia response (%Hyper) and after 0.4 mg sublingual nitroglycerin (%Nitro) by VOP and lipidogram, fibrinogen, fasting and postload glucose, oxidized LDL-cholesterol (oxLDL), sICAM, sVCAM, sE-Selectin, interleukines 1 and 6 and TNF-α by laboratorial analysis. Correlations and linear multiple regression (LMR) between %Hyper, %Nitro, microcirculatory parameters, oxidative stress and inflammatory biomarkers were investigated. sVCAM, sE-Selectin and oxLDL were higher and RBCVmax/RBCVbaseline and %Hyper lower in HE, while %Nitro and FCD remained unchanged. Fibrinogen, LDL-cholesterol, oxLDL correlated negatively to %Hyper while sVCAM correlated negatively to %Hyper and RBCVmax/RBCVbaseline. Healthy aged women presented dilated capillaries with sustained perfusion and endothelial dysfunction with preserved vascular smooth muscle reactivity. Fibrinogen, LDL-cholesterol, oxidized-LDL and sVCAM correlated negatively to endothelial function but not to microcirculatory parameters. Oxidized-LDL and sVCAM could determine %Hyper through LMR. Oxidized-LDL and sVCAM might be used as endothelial dysfunction biomarkers for elderly with normal cardiovascular risk factors.

Hypothyroidism Is Associated With Coronary Endothelial Dysfunction in Women.

PubMed

Sara, Jaskanwal D; Zhang, Ming; Gharib, Hossein; Lerman, Lilach O; Lerman, Amir

2015-07-29

Hypothyroidism is associated with an increased risk of coronary artery disease, beyond that which can be explained by its association with conventional cardiovascular risk factors. Coronary endothelial dysfunction precedes atherosclerosis, has been linked to adverse cardiovascular events, and may account for some of the increased risk in patients with hypothyroidism. The aim of this study was to determine whether there is an association between epicardial and microvascular coronary endothelial dysfunction and hypothyroidism. In 1388 patients (mean age 50.5 [12.3] years, 34% male) presenting with stable chest pain to Mayo Clinic, Rochester, MN for diagnostic coronary angiography, and who were found to have nonobstructive coronary artery disease (<40% stenosis), we invasively assessed coronary artery endothelial-dependent microvascular and epicardial function by evaluating changes in coronary blood flow (% Δ CBF Ach) and diameter (% Δ CAD Ach), respectively, in response to intracoronary infusions of acetylcholine. Patients were divided into 2 groups: hypothyroidism, defined as a documented history of hypothyroidism or a thyroid-stimulating hormone (TSH) >10.0 mU/mL, n=188, and euthyroidism, defined as an absence of a history of hypothyroidism in the clinical record and/or 0.3

L-arginine and arginine analogues: effects on isolated blood vessels and cultured endothelial cells.

PubMed Central

Schmidt, H. H.; Baeblich, S. E.; Zernikow, B. C.; Klein, M. M.; Böhme, E.

1990-01-01

1. The present study examined effects of arginine (Arg) and various Arg analogues on the vascular tone of rabbit and rat aortic rings, the release of nitrite from cultured bovine aortic endothelial cells and the metabolism of L-Arg in bovine and porcine endothelial cell homogenates. The respective D-enantiomers or N-alpha-benzoyl-L-arginine ethyl ester did not substitute for L-Arg. 2. In bovine aortic endothelial cells, the release of nitrite was only observed in the presence of L-Arg or L-Arg methyl ester in the cell culture medium. 3. In dialyzed homogenates of porcine and bovine aortic endothelial cells, L-Arg was metabolized independently of NADPH and Ca2+ to yield L-ornithine (L-Orn) and L-citrulline (L-Cit). No concomitant nitrite formation was detected. 4. Pretreatment of rabbit and rat aortic rings with L-canavanine (L-Can) or NG-monomethyl-L-Arg (L-NMMA) inhibited ATP- and acetylcholine-induced relaxations (endothelium-dependent) but not glyceryltrinitrate-induced relaxations (endothelium-independent). 5. In rabbit aortic rings, Arg and monomeric Arg analogues induced endothelium-independent relaxations. L-Arg methyl ester induced an endothelium-independent contraction, and L-NMMA induced a relaxation in the absence of endothelium and a contraction in the presence of endothelium. Polymeric basic amino acids such as poly L-Arg induced endothelium-dependent relaxations (inhibited by L-Can), a subsequent refractoriness to endothelium-dependent vasodilators (not prevented by L-Can) and endothelial cell death.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2282457

The presence of African American race predicts improvement in coronary endothelial function after supplementary L-arginine.

PubMed

Houghton, Jan L; Philbin, Edward F; Strogatz, David S; Torosoff, Mikhail T; Fein, Steven A; Kuhner, Patricia A; Smith, Vivienne E; Carr, Albert A

2002-04-17

The purpose of our study was to determine if the presence of African American ethnicity modulates improvement in coronary vascular endothelial function after supplementary L-arginine. Endothelial dysfunction is an early stage in the development of coronary atherosclerosis and has been implicated in the pathogenesis of hypertension and cardiomyopathy. Amelioration of endothelial dysfunction has been demonstrated in patients with established coronary atherosclerosis or with risk factors in response to infusion of L-arginine, the precursor of nitric oxide. Racial and gender patterns in L-arginine responsiveness have not, heretofore, been studied. Invasive testing of coronary artery and microvascular reactivity in response to graded intracoronary infusions of acetylcholine (ACh) +/- L-arginine was carried out in 33 matched pairs of African American and white subjects with no angiographic coronary artery disease. Pairs were matched for age, gender, indexed left ventricular mass, body mass index and low-density lipoprotein cholesterol. In addition to the matching parameters, there were no significant differences in peak coronary blood flow (CBF) response to intracoronary adenosine or in the peak CBF response to ACh before L-arginine infusion. However, absolute percentile improvement in CBF response to ACh infusion after L-arginine, as compared with before, was significantly greater among African Americans as a group (45 +/- 10% vs. 4 +/- 6%, p = 0.0016) and after partitioning by gender. The mechanism of this increase was mediated through further reduction in coronary microvascular resistance. L-arginine infusion also resulted in greater epicardial dilator response after ACh among African Americans. We conclude that intracoronary infusion of L-arginine provides significantly greater augmentation of endothelium-dependent vascular relaxation in those of African American ethnicity when compared with matched white subjects drawn from a cohort electively referred for coronary angiography. Our findings suggest that there are target populations in which supplementary L-arginine may be of therapeutic benefit in the amelioration of microvascular endothelial dysfunction. In view of the excess prevalence of cardiomyopathy among African Americans, pharmacologic correction of microcirculatory endothelial dysfunction in this group is an important area of further investigation and may ultimately prove to be clinically indicated.

AGE/RAGE-Induced EMP Release via the NOX-Derived ROS Pathway

PubMed Central

Chen, Ying-Hua; Chen, Zhang-Wei; Li, Hong-Mei

2018-01-01

Objective Diabetes is associated with accelerated formation of advanced glycation end products (AGEs) that are extensively found in circulating endothelial microparticles (EMPs). This study aimed to investigate whether AGEs have a direct effect on EMP formation and the possible underlying mechanism. Methods In vitro, cultured human umbilical vein endothelial cells (HUVECs) were incubated with AGEs (200 and 400 μg/ml) for 24 hours with or without pretreatment with anti-RAGE antibody, NOX inhibitor, or ROS scavenger. The number of CD31-positive EMPs was assessed by flow cytometry. Results The number of EMPs was significantly increased in HUVECs stimulated by AGEs in a dose-dependent manner. In addition, receptors for AGEs (RAGE), NAD(P)H oxidase (NOX), and reactive oxygen species (ROS) were increased by AGEs as compared to the control group. These changes could be reversed when HUVECs were pretreated with anti-RAGE antibody. Moreover, inhibition of NOX as well as antioxidant treatment reduced the release of EMPs induced by AGEs. Conclusion Our study suggested that AGEs increased EMP generation, which was mediated by RAGE signaling through NOX-derived ROS. PMID:29744367

AGE/RAGE-Induced EMP Release via the NOX-Derived ROS Pathway.

PubMed

Chen, Ying-Hua; Chen, Zhang-Wei; Li, Hong-Mei; Yan, Xin-Feng; Feng, Bo

2018-01-01

Diabetes is associated with accelerated formation of advanced glycation end products (AGEs) that are extensively found in circulating endothelial microparticles (EMPs). This study aimed to investigate whether AGEs have a direct effect on EMP formation and the possible underlying mechanism. In vitro, cultured human umbilical vein endothelial cells (HUVECs) were incubated with AGEs (200 and 400  μ g/ml) for 24 hours with or without pretreatment with anti-RAGE antibody, NOX inhibitor, or ROS scavenger. The number of CD31-positive EMPs was assessed by flow cytometry. The number of EMPs was significantly increased in HUVECs stimulated by AGEs in a dose-dependent manner. In addition, receptors for AGEs (RAGE), NAD(P)H oxidase (NOX), and reactive oxygen species (ROS) were increased by AGEs as compared to the control group. These changes could be reversed when HUVECs were pretreated with anti-RAGE antibody. Moreover, inhibition of NOX as well as antioxidant treatment reduced the release of EMPs induced by AGEs. Our study suggested that AGEs increased EMP generation, which was mediated by RAGE signaling through NOX-derived ROS.

Taspine isolated from Radix et Rhizoma Leonticis inhibits growth of human umbilical vein endothelial cell (HUVEC) by inducing its apoptosis.

PubMed

Zhang, Yanmin; He, Langchong; Zhou, Yali

2008-01-01

The present study was to evaluate the effects of taspine isolated from Radix et Rhizoma Leonticsi on the growth and apoptosis of human umbilical vein endothelial cell (HUVEC) line by MTT and flow cytometer, respectively. At the same time, a series of changes were observed in HUVEC treated by taspine, including microstructure, protein expression of bax, bcl-2 and VEGF. The change of microstructure was observed by transmission electron microscope (TEM). The protein expression of bax and bcl-2 was detected by immunohistochemistry (IHC), and VEGF protein secreted was determined by enzyme-linked immunosorbent assay (ELISA). The results showed taspine could inhibit growth and induce apoptosis of HUVEC in a dose-dependent manner. Cell cycle was significantly stopped at the S phase. Under electronic microscope, the morphology of HUVEC treated with taspine showed nuclear karyopycnosis, chromatin agglutination and typical apoptotic body. Bcl-2 and VEGF expressions were decreased and bax expression was increased. All these results demonstrate that taspine has an inhibitory effect on growth of HUVEC and can induce its apoptosis.

A Parallel-Plate Flow Chamber for Mechanical Characterization of Endothelial Cells Exposed to Laminar Shear Stress

PubMed Central

Wong, Andrew K.; LLanos, Pierre; Boroda, Nickolas; Rosenberg, Seth R.; Rabbany, Sina Y.

2017-01-01

Shear stresses induced by laminar fluid flow are essential to properly recapitulate the physiological microenvironment experienced by endothelial cells (ECs). ECs respond to these stresses via mechanotransduction by modulating their phenotype and biomechanical characteristics, which can be characterized by Atomic Force Microscopy (AFM). Parallel Plate Flow Chambers (PPFCs) apply unidirectional laminar fluid flow to EC monolayers in vitro. Since ECs in sealed PPFCs are inaccessible to AFM probes, cone-and-plate viscometers (CPs) are commonly used to apply shear stress. This paper presents a comparison of the efficacies of both methods. Computational Fluid Dynamic simulation and validation testing using EC responses as a metric have indicated limitations in the use of CPs to apply laminar shear stress. Monolayers subjected to laminar fluid flow in a PPFC respond by increasing cortical stiffness, elongating, and aligning filamentous actin in the direction of fluid flow to a greater extent than CP devices. Limitations using CP devices to provide laminar flow across an EC monolayer suggest they are better suited when studying EC response for disturbed flow conditions. PPFC platforms allow for exposure of ECs to laminar fluid flow conditions, recapitulating cellular biomechanical behaviors, whereas CP platforms allow for mechanical characterization of ECs under secondary flow. PMID:28989541

Dendritic Cell Transmigration through Brain Microvessel Endothelium Is Regulated by MIP-1α Chemokine and Matrix Metalloproteinases1

PubMed Central

Zozulya, Alla L.; Reinke, Emily; Baiu, Dana C.; Karman, Jozsef; Sandor, Matyas; Fabry, Zsuzsanna

2007-01-01

Dendritic cells (DCs) accumulate in the CNS during inflammatory diseases, but the exact mechanism regulating their traffic into the CNS remains to be defined. We now report that MIP-1α increases the transmigration of bone marrow-derived, GFP-labeled DCs across brain microvessel endothelial cell monolayers. Furthermore, occludin, an important element of endothelial tight junctions, is reorganized when DCs migrate across brain capillary endothelial cell monolayers without causing significant changes in the barrier integrity as measured by transendothelial electrical resistance. We show that DCs produce matrix metalloproteinases (MMP) -2 and -9 and GM6001, an MMP inhibitor, decreases both baseline and MIP-1α -induced DC transmigration. These observations suggest that DC transmigration across brain endothelial cell monolayers is partly MMP dependent. The migrated DCs express higher levels of CD40, CD80, and CD86 costimulatory molecules and induce T cell proliferation, indicating that the transmigration of DCs across brain endothelial cell monolayers contributes to the maintenance of DC Ag-presenting function. The MMP dependence of DC migration across brain endothelial cell monolayers raises the possibility that MMP blockers may decrease the initiation of T cell recruitment and neuroinflammation in the CNS. PMID:17182592

Akt/FOXO3a signaling modulates the endothelial stress response through regulation of heat shock protein 70 expression.

PubMed

Kim, Hyo-Soo; Skurk, Carsten; Maatz, Henrike; Shiojima, Ichiro; Ivashchenko, Yuri; Yoon, Suk-Won; Park, Young-Bae; Walsh, Kenneth

2005-06-01

To identify new antiapoptotic targets of the PI3K-Akt signaling pathway in endothelial cells, adenovirus-mediated Akt1 gene transfer and oligonucleotide microarrays were used to examine Akt-regulated transcripts. DNA microarray analysis revealed that HSP70 expression underwent the greatest fold activation of 12,532 transcripts examined in human umbilical vein endothelial cells (HUVEC) transduced with constitutively active Akt1. Akt1 gene transfer increased HSP70 transcript expression by 24.8-fold as determined by quantitative PCR and promoted a dose-dependent up-regulation of HSP70 protein as determined by Western immunoblot analysis. Gene transfer of FOXO3a, a downstream target of Akt in endothelial cells, significantly suppressed both basal and stress-induced HSP70 protein expression. FOXO3a induced caspase-9-dependent apoptosis in HUVEC, and cotransduction with Ad-HSP70 rescued endothelial cells from FOXO3a-induced apoptosis under basal and stress conditions. Our results identify HSP70 as a new antiapoptotic target of Akt-FOXO3a signaling in endothelial cells that controls viability through modulation of the stress-induced intrinsic cell death pathway.

Clathrin-dependent entry and vesicle-mediated exocytosis define insulin transcytosis across microvascular endothelial cells

PubMed Central

Azizi, Paymon M.; Zyla, Roman E.; Guan, Sha; Wang, Changsen; Liu, Jun; Bolz, Steffen-Sebastian; Heit, Bryan; Klip, Amira; Lee, Warren L.

2015-01-01

Transport of insulin across the microvasculature is necessary to reach its target organs (e.g., adipose and muscle tissues) and is rate limiting in insulin action. Morphological evidence suggests that insulin enters endothelial cells of the microvasculature, and studies with large vessel–derived endothelial cells show insulin uptake; however, little is known about the actual transcytosis of insulin and how this occurs in the relevant microvascular endothelial cells. We report an approach to study insulin transcytosis across individual, primary human adipose microvascular endothelial cells (HAMECs), involving insulin uptake followed by vesicle-mediated exocytosis visualized by total internal reflection fluorescence microscopy. In this setting, fluorophore-conjugated insulin exocytosis depended on its initial binding and uptake, which was saturable and much greater than in muscle cells. Unlike its degradation within muscle cells, insulin was stable within HAMECs and escaped lysosomal colocalization. Insulin transcytosis required dynamin but was unaffected by caveolin-1 knockdown or cholesterol depletion. Instead, insulin transcytosis was significantly inhibited by the clathrin-mediated endocytosis inhibitor Pitstop 2 or siRNA-mediated clathrin depletion. Accordingly, insulin internalized for 1 min in HAMECs colocalized with clathrin far more than with caveolin-1. This study constitutes the first evidence of vesicle-mediated insulin transcytosis and highlights that its initial uptake is clathrin dependent and caveolae independent. PMID:25540431

Estradiol induces endothelial cell migration and proliferation through estrogen receptor-enhanced RhoA/ROCK pathway.

PubMed

Oviedo, Pilar J; Sobrino, Agua; Laguna-Fernandez, Andrés; Novella, Susana; Tarín, Juan J; García-Pérez, Miguel-Angel; Sanchís, Juan; Cano, Antonio; Hermenegildo, Carlos

2011-03-30

Migration and proliferation of endothelial cells are involved in re-endothelialization and angiogenesis, two important cardiovascular processes that are increased in response to estrogens. RhoA, a small GTPase which controls multiple cellular processes, is involved in the control of cell migration and proliferation. Our aim was to study the role of RhoA on estradiol-induced migration and proliferation and its dependence on estrogen receptors activity. Human umbilical vein endothelial cells were stimulated with estradiol, in the presence or absence of ICI 182780 (estrogen receptors antagonist) and Y-27632 (Rho kinase inhibitor). Estradiol increased Rho GEF-1 gene expression and RhoA (gene and protein expression and activity) in an estrogen receptor-dependent manner. Cell migration, stress fiber formation and cell proliferation were increased in response to estradiol and were also dependent on the estrogen receptors and RhoA activation. Estradiol decreased p27 levels, and significantly raised the expression of cyclins and CDK. These effects were counteracted by the use of either ICI 182780 or Y-27632. In conclusion, estradiol enhances the RhoA/ROCK pathway and increases cell cycle-related protein expression by acting through estrogen receptors. This results in an enhanced migration and proliferation of endothelial cells. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Adult males with haemophilia have a different macrovascular and microvascular endothelial function profile compared with healthy controls.

PubMed

Sun, H; Yang, M; Fung, M; Chan, S; Jawi, M; Anderson, T; Poon, M-C; Jackson, S

2017-09-01

Endothelial function has been identified as an independent predictor of cardiovascular risk in the general population. It is unclear if the haemophilia population has a different endothelial function profile compared to the healthy population. This prospective study aims to assess if there is a difference in endothelial function between haemophilia patients and healthy controls, and the impact of endothelial function on vascular outcomes in the haemophilia population. Baseline cardiovascular risk factors and endothelial function were presented. Adult males with haemophilia A or B recruited from the British Columbia and Southern Alberta haemophilia treatment centres were matched to healthy male controls by age and cardiovascular risk factors. Macrovascular endothelial function was assessed by brachial artery flow-mediated dilation (FMD) and nitroglycerin-mediated dilation (NMD), and microvascular endothelial function was assessed by hyperaemic velocity time integral (VTI). Multivariable linear regression was used to assess the association between haemophilia and endothelial function. A total of 81 patients with haemophilia and 243 controls were included. Patients with haemophilia had a similar FMD and NMD compared to controls, although haemophilia was associated with higher FMD on multivariable analysis. Haemophilia was associated with significantly lower VTI on univariate and multivariable analyses, regardless of haemophilia type and severity. Adult males with haemophilia appear to have lower microvascular endothelial function compared to healthy controls. Future studies to assess the impact of endothelial dysfunction on cardiovascular events in the haemophilia population are needed. © 2017 John Wiley & Sons Ltd.

Activation of PPARδ prevents endothelial dysfunction induced by overexpression of amyloid-β precursor protein

PubMed Central

d'Uscio, Livius V.; Das, Pritam; Santhanam, Anantha V.R.; He, Tongrong; Younkin, Steven G.; Katusic, Zvonimir S.

2012-01-01

Aims Existing evidence suggests that amyloid-β precursor protein (APP) causes endothelial dysfunction and contributes to pathogenesis of atherosclerosis. In the present study, experiments were designed to: (1) determine the mechanisms underlying endothelial dysfunction and (2) define the effects of peroxisome proliferator-activated receptor delta (PPARδ) ligand on endothelial function in transgenic Tg2576 mice overexpressing mutated human APP. Methods and results Confocal microscopy and western blot analyses of wild-type mice aortas provided evidence that APP protein is mainly present in endothelial cells. Overexpression of APP significantly impaired endothelium-dependent relaxations to acetylcholine and phosphorylation of endothelial nitric oxide synthase at Ser1177 in aortas. HPLC analysis revealed that tetrahydrobiopterin (BH4) levels were reduced in Tg2576 mice aortas. This was caused by increased oxidation of BH4 and reduced expression and activity of GTP-cyclohydrolase I. Furthermore, gp91phox protein expression and superoxide anion production were increased in aortas of Tg2576 mice. This augmented superoxide formation was completely prevented by the NADPH oxidase inhibitor VAS2870. Expression of copper-/zinc-superoxide dismutase (Cu/ZnSOD) and extracellular SOD was downregulated. Treatment with PPARδ ligand GW501516 (2 mg/kg/day) for 14 days significantly increased BH4 bioavailability and improved endothelium-dependent relaxations in Tg2576 mice aortas. GW501516 also normalized protein expression of gp91phox and SODs, thereby reducing production of superoxide anion in the aortas. Conclusion Our results suggest that in APP transgenic mice loss of nitric oxide and increased oxidative stress are the major causes of endothelial dysfunction. The vascular protective effects of GW501516 in Tg2576 mice appear to be critically dependent on prevention of superoxide anion production. PMID:22886847

Activation of PPARδ prevents endothelial dysfunction induced by overexpression of amyloid-β precursor protein.

PubMed

d'Uscio, Livius V; Das, Pritam; Santhanam, Anantha V R; He, Tongrong; Younkin, Steven G; Katusic, Zvonimir S

2012-12-01

Existing evidence suggests that amyloid-β precursor protein (APP) causes endothelial dysfunction and contributes to pathogenesis of atherosclerosis. In the present study, experiments were designed to: (1) determine the mechanisms underlying endothelial dysfunction and (2) define the effects of peroxisome proliferator-activated receptor delta (PPARδ) ligand on endothelial function in transgenic Tg2576 mice overexpressing mutated human APP. Confocal microscopy and western blot analyses of wild-type mice aortas provided evidence that APP protein is mainly present in endothelial cells. Overexpression of APP significantly impaired endothelium-dependent relaxations to acetylcholine and phosphorylation of endothelial nitric oxide synthase at Ser(1177) in aortas. HPLC analysis revealed that tetrahydrobiopterin (BH(4)) levels were reduced in Tg2576 mice aortas. This was caused by increased oxidation of BH(4) and reduced expression and activity of GTP-cyclohydrolase I. Furthermore, gp91phox protein expression and superoxide anion production were increased in aortas of Tg2576 mice. This augmented superoxide formation was completely prevented by the NADPH oxidase inhibitor VAS2870. Expression of copper-/zinc-superoxide dismutase (Cu/ZnSOD) and extracellular SOD was downregulated. Treatment with PPARδ ligand GW501516 (2 mg/kg/day) for 14 days significantly increased BH(4) bioavailability and improved endothelium-dependent relaxations in Tg2576 mice aortas. GW501516 also normalized protein expression of gp91(phox) and SODs, thereby reducing production of superoxide anion in the aortas. Our results suggest that in APP transgenic mice loss of nitric oxide and increased oxidative stress are the major causes of endothelial dysfunction. The vascular protective effects of GW501516 in Tg2576 mice appear to be critically dependent on prevention of superoxide anion production.

High glucose condition increases NADPH oxidase activity in endothelial microparticles that promote vascular inflammation.

PubMed

Jansen, Felix; Yang, Xiaoyan; Franklin, Bernardo S; Hoelscher, Marion; Schmitz, Theresa; Bedorf, Jörg; Nickenig, Georg; Werner, Nikos

2013-04-01

Diabetes is a major risk factor for cardiovascular diseases. Circulating endothelial microparticles (EMP) are increased in diabetic patients, but their potential contribution in atherogenesis is unclear. We sought to determine the role of EMP derived under high glucose conditions in the development of atherosclerosis. EMP were generated from human coronary endothelial cells (HCAEC) exposed to high glucose concentrations in order to mimic diabetic conditions. These EMP were defined as 'injured' EMP (iEMP) and their effects were compared with EMP generated from 'healthy' untreated HCAEC. iEMP injection significantly impaired endothelial function in ApoE(-/-) mice compared with EMP and vehicle treatment. Immunofluorescent experiments showed increased macrophage infiltration and adhesion protein expression in atherosclerotic lesions of iEMP-treated ApoE(-/-) mice compared with controls. To further investigate the underlying mechanism of iEMP-induced vascular inflammation, additional in vitro experiments were performed. iEMP, but not EMP, induced activation of HCAEC in a time- and dose-dependent manner and increased monocyte adhesion. Further experiments demonstrated that iEMP induced activation of HCAEC by phosphorylation of p38 into its biologically active form phospho-p38. Inhibition of p38 activation abrogated iEMP-dependent induction of adhesion proteins and monocyte adhesion on HCAEC. Moreover, we could demonstrate that iEMP show increased NADPH oxidase activity and contain significantly higher level of reactive oxygen species (ROS) than EMP. iEMP triggered ROS production in HCAEC and thereby activate p38 in an ROS-dependent manner. High glucose condition increases NADPH oxidase activity in endothelial microparticles that amplify endothelial inflammation and impair endothelial function by promoting activation of the endothelium. These findings provide new insights into the pathogenesis of diabetes-associated atherosclerosis.

SIRT1 inhibits NADPH oxidase activation and protects endothelial function in the rat aorta: implications for vascular aging.

PubMed

Zarzuelo, María José; López-Sepúlveda, Rocío; Sánchez, Manuel; Romero, Miguel; Gómez-Guzmán, Manuel; Ungvary, Zoltan; Pérez-Vizcaíno, Francisco; Jiménez, Rosario; Duarte, Juan

2013-05-01

Vascular aging is characterized by up-regulation of NADPH oxidase, oxidative stress and endothelial dysfunction. Previous studies demonstrate that the activity of the evolutionarily conserved NAD(+)-dependent deacetylase SIRT1 declines with age and that pharmacological activators of SIRT1 confer significant anti-aging cardiovascular effects. To determine whether dysregulation of SIRT1 promotes NADPH oxidase-dependent production of reactive oxygen species (ROS) and impairs endothelial function we assessed the effects of three structurally different inhibitors of SIRT1 (nicotinamide, sirtinol, EX527) in aorta segments isolated from young Wistar rats. Inhibition of SIRT1 induced endothelial dysfunction, as shown by the significantly reduced relaxation to the endothelium-dependent vasodilators acetylcholine and the calcium ionophore A23187. Endothelial dysfunction induced by SIRT1 inhibition was prevented by treatment of the vessels with the NADPH oxidase inhibitor apocynin or superoxide dismutase. Inhibition of SIRT1 significantly increased vascular superoxide production, enhanced NADPH oxidase activity, and mRNA expression of its subunits p22(phox) and NOX4, which were prevented by resveratrol. Peroxisome proliferator-activated receptor-α (PPARα) activation mimicked the effects of resveratrol while PPARα inhibition prevented the effects of this SIRT1 activator. SIRT1 co-precipitated with PPARα and nicotinamide increased the acetylation of the PPARα coactivator PGC-1α, which was suppressed by resveratrol. In conclusion, impaired activity of SIRT1 induces endothelial dysfunction and up-regulates NADPH oxidase-derived ROS production in the vascular wall, mimicking the vascular aging phenotype. Moreover, a new mechanism for controlling endothelial function after SIRT1 activation involves a decreased PGC-1α acetylation and the subsequent PPARα activation, resulting in both decreased NADPH oxidase-driven ROS production and NO inactivation. Copyright © 2013 Elsevier Inc. All rights reserved.

Salicylate Treatment Improves Age-Associated Vascular Endothelial Dysfunction: Potential Role of Nuclear Factor κB and Forkhead Box O Phosphorylation

PubMed Central

Durrant, Jessica R.; Connell, Melanie L.; Folian, Brian J.; Donato, Anthony J.; Seals, Douglas R.

2011-01-01

We hypothesized that I kappa B kinase (IKK)-mediated nuclear factor kappa B and forkhead BoxO3a phosphorylation will be associated with age-related endothelial dysfunction. Endothelium-dependent dilation and aortic protein expression/phosphorylation were determined in young and old male B6D2F1 mice and old mice treated with the IKK inhibitor, salicylate. IKK activation was greater in old mice and was associated with greater nitrotyrosine and cytokines. Endothelium-dependent dilation, nitric oxide (NO), and endothelial NO synthase phosphorylation were lower in old mice. Endothelium-dependent dilation and NO bioavailability were restored by a superoxide dismutase mimetic. Nuclear factor kappa B and forkhead BoxO3a phosphorylation were greater in old and were associated with increased expression/activity of nicotinamide adenine dinucleotide phosphate oxidase and lower manganese superoxide dismutase expression. Salicylate lowered IKK phosphorylation and reversed age-associated changes in nitrotyrosine, endothelium-dependent dilation, NO bioavailability, endothelial NO synthase, nuclear factor kappa B and forkhead BoxO3a phosphorylation, nicotinamide adenine dinucleotide phosphate oxidase, and manganese superoxide dismutase. Increased activation of IKK with advancing age stimulates nuclear factor kappa B and inactivates forkhead BoxO3a. This altered transcription factor activation contributes to a pro-inflammatory/pro-oxidative arterial phenotype that is characterized by increased cytokines and nicotinamide adenine dinucleotide phosphate oxidase and decreased manganese superoxide dismutase leading to oxidative stress-mediated endothelial dysfunction. PMID:21303813

A new clinical method for the assessment of penile endothelial function using the flow mediated dilation with plethysmography technique.

PubMed

Dayan, Lior; Greunwald, Illan; Vardi, Yoram; Jacob, Giris

2005-04-01

Penile endothelial function (EnF) is 1 of the major factors involved in the pathophysiology of erectile dysfunction. EnF assessment could offer crucial information on the etiology and degree of severity of cavernosal vascular pathology. In the present study we propose a new technique for the evaluation of penile EnF and test its applicability using strain gauge plethysmography. A total of 23 healthy subjects (13 younger and 10 older than 40 years) with no history of erectile dysfunction were studied. The flow mediated dilation technique was applied to the arm and penis in both age groups for the assessment of EnF. Baseline blood flow and the sequential flow recordings after rapid cessation of 5 minutes of ischemia were obtained in both organs. Baseline flow in the penis was significantly higher (approximately 3-fold) than that in the forearm and was not affected by age in either organ. Both measures of penile EnF, ie area under the flow-time curve (AUC) and maximal flow obtained after ischemia were significantly lower in the older group compared to the younger group (p <0.01 and p <0.02, respectively). Individual penile AUC and maximal flow were significantly correlated with age (r = 0.55, p <0.01 and r = 0.50, p <0.02, respectively). Finally a positive, significant correlation existed between penile and forearm AUC (p <0.05, r = 0.48). The implementation of the flow mediated dilation technique using mercury strain gauge plethysmography is simple and applicable for the assessment of penile EnF. Endothelial function parameters in the penis were found to correlate with those in the forearm, thus support for the validity of the technique is given. Further strength for the validity of this procedure in the penis comes from the comparison between the forearm and penis, and the relation to subject age.

Effect of ascorbic acid on endothelial dysfunction of epicardial coronary arteries in chronic smokers assessed by cold pressor testing.

PubMed

Schindler, T H; Magosaki, N; Jeserich, M; Olschewski, M; Nitzsche, E; Holubarsch, C; Solzbach, U; Just, H

2000-01-01

In chronic smokers there is evidence for increased formation of oxygen-derived free radicals within the vessel wall impairing endothelial function. It has been suggested that the inactivation of endothelium-derived nitric oxide by oxygen free radicals contributes to endothelial dysfunction. Hence, we tested the hypothesis that in chronic smokers the antioxidant ascorbic acid could improve abnormal endothelial function of epicardial coronary arteries. Thirty-one patients (mean age 57 +/- 9 years) referred for routine diagnostic catheterization for evaluation of chest pain and without angiographically significant coronary artery stenoses were randomly assigned to one of the study groups to assess vasomotor response of epicardial coronary arteries due to cold pressor testing (CPT) before and after intravenous infusion of 3 g of ascorbic acid or 100 ml x 0.9% saline infusion. In 6 controls (mean age 55 +/- 3 years) CPT led to a similar increase in luminal area before and after ascorbic acid administration (26.5 +/- 15.0 vs. 28.4 +/- 17.7%, p = NS). In 15 chronic smokers (mean age 55 +/- 9 years), CPT induced a decrease in the luminal area of -18.5 +/- 6.3%. This flow-dependent vasoconstriction was significantly reversed to 7.7 +/- 6.2% (p < or = 0.03) vasodilation after intravenous ascorbic acid administration. In 10 chronic smokers (mean age 57 +/- 11 years) saline infusion (placebo) did not have a significant effect on CPT-induced vasoconstriction (-12.7 +/- 5.1 vs. -13.1 +/- 5.1%, p = NS). The CPT-induced increase in luminal area in chronic smokers after ascorbic acid infusion was significant compared to controls and placebo (each p < or = 0.05). Our assessment of endothelium-independent responses to nitroglycerin revealed no significant differences between the single study groups (p = NS). In chronic smokers acute intravenous administration of ascorbic acid significantly improves CPT-induced coronary endothelium-dependent dysfunction. According to the current understanding, this effect is due to improved cellular redox imbalance and prevention of nitric oxide inactivation in the endothelium and subendothelial space.

Endothelium as a transducing surface.

PubMed

Ryan, U S

1989-02-01

Endothelial cells responses to a variety of agonists include release of endothelium dependent vasodilators, such as endothelium dependent relaxing factor (EDRF) and prostacyclin (PGI2). These substances act on vascular smooth muscle to cause relaxation and also have potent anti-aggregatory effects on platelets. A study of the mechanisms of signal transduction involved in these processes was undertaken. An investigation of intracellular calcium using FURA-2 and INDO-1 loaded endothelial cells shows transient elevation in response to vasodilator agonists. The calcium content of endothelial cells calculated using 45Ca flux techniques is increased in response to bradykinin and thrombin. Receptor activation leads to increased phosphoinositide turnover in endothelial cells and activates protein kinase C, the latter may be involved in feedback regulation. Patch clamp studies have demonstrated receptor-operated ionic channels in the endothelial cell membrane. Thus, intracellular calcium concentration is elevated in response to receptor activation, both as a result of liberation of calcium from intracellular stores and calcium entry from extracellular sources. Endothelial cells also respond to particulate stimuli. They can selectively bind and phagocytize bacteria. Phagocytosis leads to generation of superoxide aionin, a process which also seems to be controlled by elevation of intracellular calcium and activation of protein kinase C. In addition phagocytosis activates endothelial cells resulting in increased migration, division and further phagocytosis. All in all, the plethora of different endothelial responses to a variety of stimuli suggests a complex and multipotent cell type.(ABSTRACT TRUNCATED AT 250 WORDS)

Stable knock-down of the sphingosine 1-phosphate receptor S1P1 influences multiple functions of human endothelial cells.

PubMed

Krump-Konvalinkova, Vera; Yasuda, Satoshi; Rubic, Tina; Makarova, Natalia; Mages, Jörg; Erl, Wolfgang; Vosseler, Claudia; Kirkpatrick, C James; Tigyi, Gabor; Siess, Wolfgang

2005-03-01

Sphingosine 1-phosphate (S1P) is a bioactive phospholipid acting both as a ligand for the G protein-coupled receptors S1P1-5 and as a second messenger. Because S1P1 knockout is lethal in the transgenic mouse, an alternative approach to study the function of S1P1 in endothelial cells is needed. All human endothelial cells analyzed expressed abundant S1P1 transcripts. We permanently silenced (by RNA interference) the expression of S1P1 in the human endothelial cell lines AS-M.5 and ISO-HAS.1. The S1P1 knock-down cells manifested a distinct morphology and showed neither actin ruffles in response to S1P nor an angiogenic reaction. In addition, these cells were more sensitive to oxidant stress-mediated injury. New S1P1-dependent gene targets were identified in human endothelial cells. S1P1 silencing decreased the expression of platelet-endothelial cell adhesion molecule-1 and VE-cadherin and abolished the induction of E-selectin after cell stimulation with lipopolysaccharide or tumor necrosis factor-alpha. Microarray analysis revealed downregulation of further endothelial specific transcripts after S1P1 silencing. Long-term silencing of S1P1 enabled us for the first time to demonstrate the involvement of S1P1 in key functions of endothelial cells and to identify new S1P1-dependent gene targets.

Endosome-to-Plasma Membrane Recycling of VEGFR2 Receptor Tyrosine Kinase Regulates Endothelial Function and Blood Vessel Formation

PubMed Central

Jopling, Helen M.; Odell, Adam F.; Pellet-Many, Caroline; Latham, Antony M.; Frankel, Paul; Sivaprasadarao, Asipu; Walker, John H.; Zachary, Ian C.; Ponnambalam, Sreenivasan

2014-01-01

Rab GTPases are implicated in endosome-to-plasma membrane recycling, but how such membrane traffic regulators control vascular endothelial growth factor receptor 2 (VEGFR2/KDR) dynamics and function are not well understood. Here, we evaluated two different recycling Rab GTPases, Rab4a and Rab11a, in regulating endothelial VEGFR2 trafficking and signalling with implications for endothelial cell migration, proliferation and angiogenesis. In primary endothelial cells, VEGFR2 displays co-localisation with Rab4a, but not Rab11a GTPase, on early endosomes. Expression of a guanosine diphosphate (GDP)-bound Rab4a S22N mutant caused increased VEGFR2 accumulation in endosomes. TfR and VEGFR2 exhibited differences in endosome-to-plasma membrane recycling in the presence of chloroquine. Depletion of Rab4a, but not Rab11a, levels stimulated VEGF-A-dependent intracellular signalling. However, depletion of either Rab4a or Rab11a levels inhibited VEGF-A-stimulated endothelial cell migration. Interestingly, depletion of Rab4a levels stimulated VEGF-A-regulated endothelial cell proliferation. Rab4a and Rab11a were also both required for endothelial tubulogenesis. Evaluation of a transgenic zebrafish model showed that both Rab4 and Rab11a are functionally required for blood vessel formation and animal viability. Rab-dependent endosome-to-plasma membrane recycling of VEGFR2 is important for intracellular signalling, cell migration and proliferation during angiogenesis. PMID:24785348

Effect of repeated sprints on postprandial endothelial function and triacylglycerol concentrations in adolescent boys.

PubMed

Sedgwick, Matthew J; Morris, John G; Nevill, Mary E; Barrett, Laura A

2015-01-01

This study investigated whether repeated, very short duration sprints influenced endothelial function (indicated by flow-mediated dilation) and triacylglycerol concentrations following the ingestion of high-fat meals in adolescent boys. Nine adolescent boys completed two, 2-day main trials (control and exercise), in a counter-balanced, cross-over design. Participants were inactive on day 1 of the control trial but completed 40 × 6 s maximal cycle sprints on day 1 of the exercise trial. On day 2, capillary blood samples were collected and flow-mediated dilation measured prior to, and following, ingestion of a high-fat breakfast and lunch. Fasting flow-mediated dilation and plasma triacylglycerol concentration were similar in the control and exercise trial (P > 0.05). In the control trial, flow-mediated dilation was reduced by 20% and 27% following the high-fat breakfast and lunch; following exercise these reductions were negated (main effect trial, P < 0.05; interaction effect trial × time, P < 0.05). The total area under the plasma triacylglycerol concentration versus time curve was 13% lower on day 2 in the exercise trial compared to the control trial (8.65 (0.97) vs. 9.92 (1.16) mmol · l(-1) · 6.5 h, P < 0.05). These results demonstrate that repeated 6 s maximal cycle sprints can have beneficial effects on postprandial endothelial function and triacylglycerol concentrations in adolescent boys.

Endothelial mechanotransduction proteins and vascular function are altered by dietary sucrose supplementation in healthy young male subjects.

PubMed

Gliemann, Lasse; Rytter, Nicolai; Lindskrog, Mads; Slingsby, Martina H Lundberg; Åkerström, Thorbjörn; Sylow, Lykke; Richter, Erik A; Hellsten, Ylva

2017-08-15

Mechanotransduction in endothelial cells is a central mechanism in the regulation of vascular tone and vascular remodelling Mechanotransduction and vascular function may be affected by high sugar levels in plasma because of a resulting increase in oxidative stress and increased levels of advanced glycation end-products (AGE). In healthy young subjects, 2 weeks of daily supplementation with 3 × 75 g of sucrose was found to reduce blood flow in response to passive lower leg movement and in response to 12 W of knee extensor exercise. This vascular impairment was paralleled by up-regulation of platelet endothelial cell adhesion molecule (PECAM)-1, endothelial nitric oxide synthase, NADPH oxidase and Rho family GTPase Rac1 protein expression, an increased basal phosphorylation status of vascular endothelial growth factor receptor 2 and a reduced phosphorylation status of PECAM-1. There were no measurable changes in AGE levels. The findings of the present study demonstrate that daily high sucrose intake markedly affects mechanotransduction proteins and has a detrimental effect on vascular function. Endothelial mechanotransduction is important for vascular function but alterations and activation of vascular mechanosensory proteins have not been investigated in humans. In endothelial cell culture, simple sugars effectively impair mechanosensor proteins. To study mechanosensor- and vascular function in humans, 12 young healthy male subjects supplemented their diet with 3 × 75 g sucrose day -1 for 14 days in a randomized cross-over design. Before and after the intervention period, the hyperaemic response to passive lower leg movement and active knee extensor exercise was determined by ultrasound doppler. A muscle biopsy was obtained from the thigh muscle before and after acute passive leg movement to allow assessment of protein amounts and the phosphorylation status of mechanosensory proteins and NADPH oxidase. The sucrose intervention led to a reduced flow response to passive movement (by 17 ± 2%) and to 12 W of active exercise (by 9 ± 1%), indicating impaired vascular function. A reduced flow response to passive and active exercise was paralleled by a significant up-regulation of platelet endothelial cell adhesion molecule (PECAM-1), endothelial nitric oxide synthase, NADPH oxidase and the Rho family GTPase Rac1 protein expression in the muscle tissue, as well as an increased basal phosphorylation status of vascular endothelial growth factor receptor 2 and a reduced phosphorylation status of PECAM-1. The phosphorylation status was not acutely altered with passive leg movement. These findings indicate that a regular intake of high levels of sucrose can impair vascular mechanotransduction and increase the oxidative stress potential, and suggest that dietary excessive sugar intake may contribute to the development of vascular disease. © 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.

Aquaporin-1 shifts the critical transmural pressure to compress the aortic intima and change transmural flow: theory and implications.

PubMed

Joshi, Shripad; Jan, Kung-Ming; Rumschitzki, David S

2015-12-01

Transmural-pressure (ΔP)-driven plasma advection carries macromolecules into the vessel wall, the earliest prelesion atherosclerotic event. The wall's hydraulic conductivity, LP, the water flux-to-ΔP ratio, is high at low pressures, rapidly decreases, and remains flat to high pressures (Baldwin AL, Wilson LM. Am J Physiol Heart Circ Physiol 264: H26-H32, 1993; Nguyen T, Toussaint, Xue JD, Raval Y, Cancel CB, Russell LM, Shou S, Sedes Y, Sun O, Yakobov Y, Tarbell JM, Jan KM, Rumschitzki DS. Am J Physiol Heart Circ Physiol 308: H1051-H1064, 2015; Tedgui A, Lever MJ. Am J Physiol Heart Circ Physiol. 247: H784-H791, 1984. Shou Y, Jan KM, Rumschitzki DS. Am J Physiol Heart Circ Physiol 291: H2758-H2771, 2006) due to pressure-induced subendothelial intima (SI) compression that causes endothelial cells to partially block internal elastic laminar fenestrae. Nguyen et al. showed that rat and bovine aortic endothelial cells express the membrane protein aquaporin-1 (AQP1) and transmural water transport is both transcellular and paracellular. They found that LP lowering by AQP1 blocking was perplexingly ΔP dependent. We hypothesize that AQP1 blocking lowers average SI pressure; therefore, a lower ΔP achieves the critical force/area on the endothelium to partially block fenestrae. To test this hypothesis, we improve the approximate model of Huang et al. (Huang Y, Rumschitzki D, Chien S, Weinbaum SS. Am J Physiol Heart Circ Physiol 272: H2023-H2039, 1997) and extend it by including transcellular AQP1 water flow. Results confirm the observation by Nguyen et al.: wall LP and water transport decrease with AQP1 disabling. The model predicts 1) low-pressure LP experiments correctly; 2) AQP1s contribute 30-40% to both the phenomenological endothelial + SI and intrinsic endothelial LP; 3) the force on the endothelium for partial SI decompression with functioning AQP1s at 60 mmHg equals that on the endothelium at ∼43 mmHg with inactive AQP1s; and 4) increasing endothelial AQP1 expression increases wall LP and shifts the ΔP regime where LP drops to significantly higher ΔP than in Huang et al. Thus AQP1 upregulation (elevated wall LP) might dilute and slow low-density lipoprotein binding to SI extracellular matrix, which may be beneficial for early atherogenesis. Copyright © 2015 the American Physiological Society.

Endothelial function is unaffected by changing between carvedilol and metoprolol in patients with heart failure-a randomized study

PubMed Central

2011-01-01

Background Carvedilol has been shown to be superior to metoprolol tartrate to improve clinical outcomes in patients with heart failure (HF), yet the mechanisms responsible for these differences remain unclear. We examined if there were differences in endothelial function, insulin stimulated endothelial function, 24 hour ambulatory blood pressure and heart rate during treatment with carvedilol, metoprolol tartrate and metoprolol succinate in patients with HF. Methods Twenty-seven patients with mild HF, all initially treated with carvedilol, were randomized to a two-month treatment with carvedilol, metoprolol tartrate or metoprolol succinate. Venous occlusion plethysmography, 24-hour blood pressure and heart rate measurements were done before and after a two-month treatment period. Results Endothelium-dependent vasodilatation was not affected by changing from carvedilol to either metoprolol tartrate or metoprolol succinate. The relative forearm blood flow at the highest dose of serotonin was 2.42 ± 0.33 in the carvedilol group at baseline and 2.14 ± 0.24 after two months continuation of carvedilol (P = 0.34); 2.57 ± 0.33 before metoprolol tartrate treatment and 2.42 ± 0.55 after treatment (p = 0.74) and in the metoprolol succinate group 1.82 ± 0.29 and 2.10 ± 0.37 before and after treatment, respectively (p = 0.27). Diurnal blood pressures as well as heart rate were also unchanged by changing from carvedilol to metoprolol tartrate or metoprolol succinate. Conclusion Endothelial function remained unchanged when switching the beta blocker treatment from carvedilol to either metoprolol tartrate or metoprolol succinate in this study, where blood pressure and heart rate also remained unchanged in patients with mild HF. Trial registration Current Controlled Trials NCT00497003 PMID:21999413

Endothelial targeting of high-affinity multivalent polymer nanocarriers directed to intercellular adhesion molecule 1.

PubMed

Muro, Silvia; Dziubla, Thomas; Qiu, Weining; Leferovich, John; Cui, Xiumin; Berk, Erik; Muzykantov, Vladimir R

2006-06-01

Targeting of diagnostic and therapeutic agents to endothelial cells (ECs) provides an avenue to improve treatment of many maladies. For example, intercellular adhesion molecule 1 (ICAM-1), a constitutive endothelial cell adhesion molecule up-regulated in many diseases, is a good determinant for endothelial targeting of therapeutic enzymes and polymer nanocarriers (PNCs) conjugated with anti-ICAM (anti-ICAM/PNCs). However, intrinsic and extrinsic factors that control targeting of anti-ICAM/PNCs to ECs (e.g., anti-ICAM affinity and PNC valency and flow) have not been defined. In this study we tested in vitro and in vivo parameters of targeting to ECs of anti-ICAM/PNCs consisting of either prototype polystyrene or biodegradable poly(lactic-coglycolic) acid polymers (approximately 200 nm diameter spheres carrying approximately 200 anti-ICAM molecules). Anti-ICAM/PNCs, but not control IgG/PNCs 1) rapidly (t1/2 approximately 5 min) and specifically bound to tumor necrosis factor-activated ECs in a dose-dependent manner (Bmax approximately 350 PNC/cell) at both static and physiological shear stress conditions and 2) bound to ECs and accumulated in the pulmonary vasculature after i.v. injection in mice. Anti-ICAM/PNCs displayed markedly higher EC affinity versus naked anti-ICAM (Kd approximately 80 pM versus approximately 8 nM) in cell culture and, probably because of this factor, higher value (185.3 +/- 24.2 versus 50.5 +/- 1.5% injected dose/g) and selectivity (lung/blood ratio 81.0 +/- 10.9 versus 2.1 +/- 0.02, in part due to faster blood clearance) of pulmonary targeting. These results 1) show that reformatting monomolecular anti-ICAM into high-affinity multivalent PNCs boosts their vascular immuno-targeting, which withstands physiological hydrodynamics and 2) support potential anti-ICAM/PNCs utility for medical applications.

Leptin receptors are expressed in coronary arteries, and hyperleptinemia causes significant coronary endothelial dysfunction.

PubMed

Knudson, Jarrod D; Dincer, U Deniz; Zhang, Cuihua; Swafford, Albert N; Koshida, Ryoji; Picchi, Andrea; Focardi, Marta; Dick, Gregory M; Tune, Johnathan D

2005-07-01

Obesity is associated with marked increases in plasma leptin concentration, and hyperleptinemia is an independent risk factor for coronary artery disease. As a result, the purpose of this investigation was to test the following hypotheses: 1) leptin receptors are expressed in coronary endothelial cells; and 2) hyperleptinemia induces coronary endothelial dysfunction. RT-PCR analysis revealed that the leptin receptor gene is expressed in canine coronary arteries and human coronary endothelium. Furthermore, immunocytochemistry demonstrated that the long-form leptin receptor protein (ObRb) is present in human coronary endothelium. The functional effects of leptin were determined using pressurized coronary arterioles (<130 microm) isolated from Wistar rats, Zucker rats, and mongrel dogs. Leptin induced pharmacological vasodilation that was abolished by denudation and the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine methyl ester and was absent in obese Zucker rats. Intracoronary leptin dose-response experiments were conducted in anesthetized dogs. Normal and obese concentrations of leptin (0.1-3.0 microg/min ic) did not significantly change coronary blood flow or myocardial oxygen consumption; however, obese concentrations of leptin significantly attenuated the dilation to graded intracoronary doses of acetylcholine (0.3-30.0 microg/min). Additional experiments were performed in canine coronary rings, and relaxation to acetylcholine (6.25 nmol/l-6.25 micromol/l) was significantly attenuated by obese concentrations of leptin (625 pmol/l) but not by physiological concentrations of leptin (250 pmol/l). The major findings of this investigation were as follows: 1) the ObRb is present in coronary arteries and coupled to pharmacological, nitric oxide-dependent vasodilation; and 2) hyperleptinemia produces significant coronary endothelial dysfunction.

Effect of metabolic abnormalities on endothelial dysfunction in normotensive offspring of subject with hypertension.

PubMed

Žižek, B; Žižek, D; Bedenčič, K; Jerin, A; Poredoš, P

2013-08-01

Essential hypertension (EH) is often accompanied by hyperinsulinemia/insulin resistance (IR) and deranged adiponectin secretion. IR may in turn be associated with endothelial dysfunction and increased levels of asymmetric dimethylarginine (ADMA). Therefore, we aimed to determine metabolic abnormalities in normotensive offspring of subjects with essential hypertension (familial trait-FT) and to examine their relations to endothelium-dependent vasodilation of the brachial artery (BA). We included 77 subjects, 38 were normotensive individuals with FT aged 28-39 (mean 33) years and 39 age-matched Controls without FT. Insulin, adiponectin and ADMA plasma levels were determined by radioimmunoassay. Using high-resolution ultrasound, BA diameters at rest and during reactive hyperemia (flow-mediated dilation-FMD) were measured. Subjects with FT had higher insulin and lower adiponectin levels than controls (13.65±6.70 vs. 7.09±2.20 mE/L; P<0.001 and 13.60±5.98 vs. 17.27±7.17 mg/L respectively; P<0.05). Insulin and adiponectin levels were negatively interrelated (r=-0.33, P=0.003). ADMA levels were comparable in both groups. The study group had worse FMD than Controls (6.11±3.28 vs. 10.20±2.07%; P<0.001). IR was independently associated with FMD (partial R2=0.23, P<0.001). Increased insulin and decreased adiponectin levels along with endothelial dysfunction are present in normotensive subjects with FT. IR and hypoadiponectinemia are interrelated, but only hyperinsulinemia has an independent adverse influence on endothelial function. Results of our study did not confirm the role of ADMA in pathogenesis of evolving hypertension.

[Co-adaptation of enzymatic systems of cells and blood supply in smooth muscle tumors of the corpus uteri].

PubMed

Lazaarev, A F; Avbalian, A M; Bobrov, I P; Klimachev, V V; Mischenko, E V

2008-01-01

We investigated co-adaptation of enzymatic systems of cells using data on activity of NAD(Ph)-dependent enzymes and AgNOR proteins of vascular endothelium vis-a-vis angiogenesis in benign and malignant smooth muscle tumors of the corpus uteri. Overall metabolic activity (NAD-H2 diaphorase) was found to directly correlate with angiogenesis and endothelial vessel proliferation (r = 0.76 and 0.84, respectively). SDH-regulated oxidation in the main metabolic succession of a tricarbonic acid cycle depended on blood supply and endothelial vessel proliferation (r = 0.84 and 0.92, respectively; p = 0.04). A similar relationship was shown for anaerobic glycolysis of SDH (LDH content), on the one hand, and blood supply and endothelial vessel proliferation(r = 0.57 and 0.70, respectively; p = 0.02), on the other. Hence, metabolic profile varied in unaltered myometrium and tumor with variable cellular density and peculiar extracellular matrix. The highest levels of metabolic activity with NAD(Ph)-dependent enzyme co-adaptation was observed in sarcomas which were also characterized by the highest vascular density for endothelial proliferation.

Prevention of neutrophil extravasation by α2-adrenoceptor-mediated endothelial stabilization.

PubMed

Herrera-García, Ada María; Domínguez-Luis, María Jesús; Arce-Franco, María; Armas-González, Estefanía; Álvarez de La Rosa, Diego; Machado, José David; Pec, Martina K; Feria, Manuel; Barreiro, Olga; Sánchez-Madrid, Francisco; Díaz-González, Federico

2014-09-15

Adrenergic receptors are expressed on the surface of inflammation-mediating cells, but their potential role in the regulation of the inflammatory response is still poorly understood. The objectives of this work were to study the effects of α2-adrenergic agonists on the inflammatory response in vivo and to determine their mechanism of action. In two mouse models of inflammation, zymosan air pouch and thioglycolate-induced peritonitis models, the i.m. treatment with xylazine or UK14304, two α2-adrenergic agonists, reduced neutrophil migration by 60%. The α2-adrenergic antagonist RX821002 abrogated this effect. In flow cytometry experiments, the basal surface expression of L-selectin and CD11b was modified neither in murine nor in human neutrophils upon α2-agonist treatment. Similar experiments in HUVEC showed that UK14304 prevented the activation-dependent upregulation of ICAM-1. In contrast, UK14304 augmented electrical resistance and reduced macromolecular transport through a confluent HUVEC monolayer. In flow chamber experiments, under postcapillary venule-like flow conditions, the pretreatment of HUVECs, but not neutrophils, with α2-agonists decreased transendothelial migration, without affecting neutrophil rolling. Interestingly, α2-agonists prevented the TNF-α-mediated decrease in expression of the adherens junctional molecules, VE-cadherin, β-catenin, and plakoglobin, and reduced the ICAM-1-mediated phosphorylation of VE-cadherin by immunofluorescence and confocal analysis and Western blot analysis, respectively. These findings indicate that α2-adrenoceptors trigger signals that protect the integrity of endothelial adherens junctions during the inflammatory response, thus pointing at the vascular endothelium as a therapeutic target for the management of inflammatory processes in humans. Copyright © 2014 by The American Association of Immunologists, Inc.

Diabetes enhances vulnerability to particulate air pollution-associated impairment in vascular reactivity and endothelial function.

PubMed

O'Neill, Marie S; Veves, Aristidis; Zanobetti, Antonella; Sarnat, Jeremy A; Gold, Diane R; Economides, Panayiotis A; Horton, Edward S; Schwartz, Joel

2005-06-07

Epidemiological studies suggest that people with diabetes are vulnerable to cardiovascular health effects associated with exposure to particle air pollution. Endothelial and vascular function is impaired in diabetes and may be related to increased cardiovascular risk. We examined whether endothelium-dependent and -independent vascular reactivity was associated with particle exposure in individuals with and without diabetes. Study subjects were 270 greater-Boston residents. We measured 24-hour average ambient levels of air pollution (fine particles [PM2.5], particle number, black carbon, and sulfates [SO4(2-)]) approximately 500 m from the patient examination site. Pollutant concentrations were evaluated for associations with vascular reactivity. Linear regressions were fit to the percent change in brachial artery diameter (flow mediated and nitroglycerin mediated), with the particulate pollutant index, apparent temperature, season, age, race, sex, smoking history, and body mass index as predictors. Models were fit to all subjects and then stratified by diagnosed diabetes versus at risk for diabetes. Six-day moving averages of all 4 particle metrics were associated with decreased vascular reactivity among patients with diabetes but not those at risk. Interquartile range increases in SO4(2-) were associated with decreased flow-mediated (-10.7%; 95% CI, -17.3 to -3.5) and nitroglycerin-mediated (-5.4%; 95% CI, -10.5 to -0.1) vascular reactivity among those with diabetes. Black carbon increases were associated with decreased flow-mediated vascular reactivity (-12.6%; 95% CI, -21.7 to -2.4), and PM2.5 was associated with nitroglycerin-mediated reactivity (-7.6%; 95% CI, -12.8 to -2.1). Effects were stronger in type II than type I diabetes. Diabetes confers vulnerability to particles associated with coal-burning power plants and traffic.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu Junji; Yan Xing; Gao Runtao

Purpose: To evaluate the effect of irradiation on microvascular endothelial cells in miniature pig parotid glands. Methods and Materials: A single 25-Gy dose of irradiation (IR) was delivered to parotid glands of 6 miniature pigs. Three other animals served as non-IR controls. Local blood flow rate in glands was measured pre- and post-IR with an ultrasonic Doppler analyzer. Samples of parotid gland tissue were taken at 4 h, 24 h, 1 week, and 2 weeks after IR for microvascular density (MVD) analysis and sphingomyelinase (SMase) assay. Histopathology and immunohistochemical staining (anti-CD31 and anti-AQP1) were used to assess morphological changes. MVDmore » was determined by calculating the number of CD31- or AQP1-stained cells per field. A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay was used to detect apoptotic cells. The activity of acid and neutral Mg{sup 2+}-dependent SMase (ASMase and NSMase, respectively) was also assayed. Results: Local parotid gland blood flow rate decreased rapidly at 4 h post-IR and remained below control levels throughout the 14-day observation period. Parotid MVD also declined from 4 to 24 hours and remained below control levels thereafter. The activity levels of ASMase and NSMase in parotid glands increased rapidly from 4 to 24 h post-IR and then declined gradually. The frequency of detecting apoptotic nuclei in the glands followed similar kinetics. Conclusions: Single-dose IR led to a significant reduction of MVD and local blood flow rate, indicating marked damage to microvascular endothelial cells in miniature pig parotid glands. The significant and rapid increases of ASMase and NSMase activity levels may be important in this IR-induced damage.« less

Inducible nitric oxide synthase inhibits oxygen consumption in collateral-dependent myocardium.

PubMed

Chen, Yingjie; Zhang, Ping; Li, Jingxin; Xu, Xin; Bache, Robert J

2014-02-01

Following coronary artery occlusion growth of collateral vessels can provide an effective blood supply to the dependent myocardium. The ischemia, which results in growth of collateral vessels, recruits an inflammatory response with expression of cytokines and growth factors, upregulation of endothelial nitric oxide (NO) synthase (eNOS) in vascular endothelial cells, and expression of inducible nitric oxide synthase (iNOS) in both vessels and cardiac myocytes. Because NO is a potent collateral vessel dilator, this study examined whether NO derived from iNOS or constitutive NOS regulates myocardial blood flow (MBF) in the collateral region. Nonselective NOS inhibition with N(G)-nitro-l-arginine (LNA) caused vasoconstriction with a significant decrease in MBF to the collateral region during exercise. In contrast, the highly selective iNOS inhibitor 1400W caused a 21 ± 5% increase of MBF in the collateral region. This increase in MBF following selective iNOS blockade was proportionate to an increase in myocardial O2 consumption (MVo2). The results suggest that NO produced by iNOS inhibits MVo2 in the collateralized region, so that the increase in MBF following iNOS blockade was the result of metabolic vasodilation secondary to an increase in MVo2. Thus the coordinated expression of iNOS to restrain MVo2 and eNOS to maintain collateral vasodilation act to optimize the O2 supply-demand relationship and protect the collateralized myocardium from ischemia.

Endothelial function varies according to insulin resistance disease type.

PubMed

Beckman, Joshua A; Goldfine, Allison B; Dunaif, Andrea; Gerhard-Herman, Marie; Creager, Mark A

2007-05-01

We examined the relationship between insulin resistance and vascular function in three insulin-resistant states (type 2 diabetes, non-HIV lipodystrophic diabetes, and nondiabetic polycystic ovary syndrome [PCOS]) and in healthy control subjects. The population included 12 women with type 2 diabetes, 6 with lipodystrophic diabetes, 10 with PCOS, and 19 healthy female subjects. Metabolic measures included insulin sensitivity by the homeostasis model assessment, lipids, free fatty acids, and adiponectin. High-resolution B-mode ultrasound was used to determine endothelium-dependent and -independent vasodilation. Type 2 diabetic, liposdystrophic, and PCOS subjects were insulin resistant compared with control subjects (P = 0.001). Flow-mediated vasodilation was reduced in diabetic (3.4 +/- 1.3%) compared with control (7.3 +/- 1.1%) subjects but not in lipodystrophic (7.7 +/- 1.2%) or PCOS (9.9 +/- 0.7%) subjects (P = 0.005). Nitroglycerin-mediated vasodilation was attenuated in both diabetic (15.2 +/- 2.0%) and lipodystrophic (16.7 +/- 3.6%) subjects compared with healthy control (24.6 +/- 2.4%) and PCOS (23.2 +/- 1.8%) subjects (P = 0.019). Insulin resistance, free fatty acids, adiponectin, or C-reactive protein did not associate with vascular dysfunction. Among these different types of patients with insulin resistance, we found abnormal endothelium-dependent vasodilation only in the patients with type 2 diabetes. We postulate that variations in the mechanism of insulin resistance may affect endothelial function differently than glucose homeostasis.

Impaired endothelium-dependent vasodilatation in women with previous gestational diabetes.

PubMed

Anastasiou, E; Lekakis, J P; Alevizaki, M; Papamichael, C M; Megas, J; Souvatzoglou, A; Stamatelopoulos, S F

1998-12-01

To assess whether otherwise healthy women with a history of gestational diabetes mellitus (GDM) may have abnormalities in endothelial function at a very early stage, before glucose intolerance occurs. A total of 33 women with previous GDM (17 nonobese [BMI < 27] and 16 obese [BMI > or = 27]) and 19 healthy nonobese women were examined. A 75-g oral glucose tolerance test was performed, and insulin levels and biochemical parameters were also measured. Using high-resolution ultrasound, we measured vasodilatory responses of the brachial artery during reactive hyperemia (endothelium-dependent vasodilatation), and after nitroglycerin administration, an endothelium-independent vasodilator. Flow-mediated dilatation (FMD) was significantly and equally decreased in both groups of women with previous GDM, compared with control subjects (1.6 +/- 3.7% in the nonobese GDM group and 1.6 +/- 2.5% in the obese GDM group vs. 10.3 +/- 4.4% in control subjects, P < 0.001). FMD correlated inversely with serum uric acid levels, BMI, serum total cholesterol, and basal insulin resistance (homeostasis model assessment). Nitrate-induced dilatation was significantly decreased only in the obese GDM group compared with control subjects, (21.4 +/- 5.1 vs. 27.9 +/- 9.5, P < 0.05). Endothelial dysfunction, which is considered as a very early index of atherogenesis, is already present in both obese and nonobese women with a history of GDM, even when they have normal glucose tolerance.

Platelet and not erythrocyte microparticles are procoagulant in transfused thalassaemia major patients.

PubMed

Agouti, Imane; Cointe, Sylvie; Robert, Stéphane; Judicone, Coralie; Loundou, Anderson; Driss, Fathi; Brisson, Alain; Steschenko, Dominique; Rose, Christian; Pondarré, Corinne; Bernit, Emmanuelle; Badens, Catherine; Dignat-George, Françoise; Lacroix, Romaric; Thuret, Isabelle

2015-11-01

The level of circulating platelet-, erythrocyte-, leucocyte- and endothelial-derived microparticles detected by high-sensitivity flow cytometry was investigated in 37 β-thalassaemia major patients receiving a regular transfusion regimen. The phospholipid procoagulant potential of the circulating microparticles and the microparticle-dependent tissue factor activity were evaluated. A high level of circulating erythrocyte- and platelet-microparticles was found. In contrast, the number of endothelial microparticles was within the normal range. Platelet microparticles were significantly higher in splenectomized than in non-splenectomized patients, independent of platelet count (P < 0·001). Multivariate analysis indicated that phospholipid-dependent procoagulant activity was influenced by both splenectomy (P = 0·001) and platelet microparticle level (P < 0·001). Erythrocyte microparticles were not related to splenectomy, appear to be devoid of proper procoagulant activity and no relationship between their production and haemolysis, dyserythropoiesis or oxidative stress markers could be established. Intra-microparticle labelling with anti-HbF antibodies showed that they originate only partially (median of 28%) from thalassaemic erythropoiesis. In conclusion, when β-thalassaemia major patients are intensively transfused, the procoagulant activity associated with thalassaemic erythrocyte microparticles is probably diluted by transfusions. In contrast, platelet microparticles, being both more elevated and more procoagulant, especially after splenectomy, may contribute to the residual thrombotic risk reported in splenectomized multi-transfused β-thalassaemia major patients. © 2015 John Wiley & Sons Ltd.

Endothelium dependent and independent responses in coronary artery disease measured at angioplasty.

PubMed Central

Holdright, D R; Clarke, D; Poole-Wilson, P A; Fox, K; Collins, P

1993-01-01

OBJECTIVE--To investigate the effects of substance P and papaverine, two drugs that increase coronary blood flow by different mechanisms, on vasomotion in stenotic coronary arteries at percutaneous transluminal coronary angioplasty (PTCA). DESIGN--Coronary blood flow responses to substance P and papaverine were measured in stenotic coronary arteries at the time of PTCA with quantitative angiography and a Doppler flow probe. SETTING--A cardiothoracic referral centre. PATIENTS--15 patients undergoing elective PTCA of a discrete epicardial coronary artery stenosis. INTERVENTIONS--Pharmacological coronary flow reserve was determined with papaverine 5-10 minutes before and after successful PTCA. Endothelium dependent responses to 2 minute infusions of substance P (10-15 pmol.min-1) were assessed immediately before PTCA. MAIN OUTCOME MEASURES--Coronary blood flow responses and changes in epicardial coronary artery area at stenotic, proximal, and distal sites with papaverine and substance P. RESULTS--Stenotic sites dilated with papaverine before PTCA (17.7%(6.9%) (mean (SEM)) area increase, p < 0.05 v baseline). Substance P dilated stenotic sites (16.8%(5.7%) area increase, p < 0.05) and proximal (14.3%(5.4%), p < 0.05) and distal sites (41.7%(9.3%), p < 0.005). Coronary flow reserve increased but did not reach normal values after PTCA (2.3(0.4) before PTCA v 3.0(0.4) after PTCA, p < 0.05) and was associated with an increase in peak flow with papaverine. Angioplasty did not alter baseline flow. After PTCA papaverine caused significant vasoconstriction at the stenotic site (-13.6%(4.3%) area decrease, p < 0.05). There was a negative correlation (r = -0.68, p < 0.05) between the dilator response with papaverine before PTCA and the constrictor response after PTCA. CONCLUSIONS--Substance P causes endothelium dependent dilatation in atheromatous coronary arteries, even at sites of overt atheroma. The cause of the paradoxical constrictor response to papaverine after PTCA is uncertain, but unopposed flow mediated vasoconstriction (the myogenic response) after balloon induced endothelial denudation may be one of several contributory factors. PMID:7518687

Targeting Microvascular Pericytes in Angiogenic Vessels of Prostate Cancer

DTIC Science & Technology

2006-04-01

Schlingemann RO. 2004. In vivo angiogenic phenotype of endothelial cells and pericytes induced by vascular endothelial growth factor -a. J Histochem Cytochem...R, McDonald DM. Age-related changes in vascular endothelial growth factor dependency and angiopoietin-1-induced plasti- city of adult blood vessels...hematopoietic progenitor cells and their progeny in vivo . We used the basic fibroblast growth factor (bFGF)- induced mouse corneal neovascularization

Optimization of human corneal endothelial cell culture: density dependency of successful cultures in vitro.

PubMed

Peh, Gary S L; Toh, Kah-Peng; Ang, Heng-Pei; Seah, Xin-Yi; George, Benjamin L; Mehta, Jodhbir S

2013-05-03

Global shortage of donor corneas greatly restricts the numbers of corneal transplantations performed yearly. Limited ex vivo expansion of primary human corneal endothelial cells is possible, and a considerable clinical interest exists for development of tissue-engineered constructs using cultivated corneal endothelial cells. The objective of this study was to investigate the density-dependent growth of human corneal endothelial cells isolated from paired donor corneas and to elucidate an optimal seeding density for their extended expansion in vitro whilst maintaining their unique cellular morphology. Established primary human corneal endothelial cells were propagated to the second passage (P2) before they were utilized for this study. Confluent P2 cells were dissociated and seeded at four seeding densities: 2,500 cells per cm2 ('LOW'); 5,000 cells per cm2 ('MID'); 10,000 cells per cm2 ('HIGH'); and 20,000 cells per cm2 ('HIGH(×2)'), and subsequently analyzed for their propensity to proliferate. They were also subjected to morphometric analyses comparing cell sizes, coefficient of variance, as well as cell circularity when each culture became confluent. At the two lower densities, proliferation rates were higher than cells seeded at higher densities, though not statistically significant. However, corneal endothelial cells seeded at lower densities were significantly larger in size, heterogeneous in shape and less circular (fibroblastic-like), and remained hypertrophic after one month in culture. Comparatively, cells seeded at higher densities were significantly homogeneous, compact and circular at confluence. Potentially, at an optimal seeding density of 10,000 cells per cm2, it is possible to obtain between 10 million to 25 million cells at the third passage. More importantly, these expanded human corneal endothelial cells retained their unique cellular morphology. Our results demonstrated a density dependency in the culture of primary human corneal endothelial cells. Sub-optimal seeding density results in a decrease in cell saturation density, as well as a loss in their proliferative potential. As such, we propose a seeding density of not less than 10,000 cells per cm2 for regular passage of primary human corneal endothelial cells.

Epigallocatechin 3-gallate inhibits 7-ketocholesterol-induced monocyte-endothelial cell adhesion.

PubMed

Yamagata, Kazuo; Tanaka, Noriko; Suzuki, Koichi

2013-07-01

7-Ketocholesterol (7KC) induces monocytic adhesion to endothelial cells, and induces arteriosclerosis while high-density lipoprotein (HDL) inhibits monocytic adhesion to the endothelium. Epigallocatechin 3-gallate (EGCG) was found to have a protective effect against arteriosclerosis. Therefore, the purpose of this study was to examine the possible HDL-like mechanisms of EGCG in endothelial cells by investigating whether EGCG inhibits 7KC-induced monocyte-endothelial cell adhesion by activating HDL-dependent signal transduction pathways. 7KC and/or EGCG were added to human endothelial cells (ISO-HAS), and the adhesion of pro-monocytic U937 cells was examined. The expression of genes associated with HDL effects such as Ca(2+)/calmodulin-dependent kinase II (CaMKKII), liver kinase B (LKD1), PSD-95/Dlg/ZO-1 kinase 1 (PDZK1), phosphatidylinositol 3-kinase (PI3K), intercellular adhesion molecule-1 (ICAM-1), monocyte chemotactic protein-1 (MCP-1), and endothelial nitric oxide synthase (eNOS) was examined by RT-PCR, and ICAM-1 protein expression was evaluated by western blot (WB). Production of reactive oxygen species (ROS) was examined with H2DCFDA. 7KC significantly induced adhesion of U937 cells to human endothelial cells while significantly increasing gene expressions of ICAM-1 and MCP-1 and decreasing eNOS and CaMKKII gene expressions. EGCG inhibited 7KC-induced monocytic adhesion to endothelial cells, and induced expression of eNOS and several genes involved in the CaMKKII pathway. Stimulation of endothelial cells with EGCG produced intracellular ROS, whereas treatment with N-acetylcysteine (NAC) blocked EGCG-induced expression of eNOS and CaMKKII. These results suggest that inhibition of monocyte-endothelial cell adhesion by EGCG is associated with CaMKKII pathway activation by ROS. Inhibition of 7KC-induced monocyte-endothelial cell adhesion induced by EGCG may function similarly to HDL. Copyright © 2013 Elsevier Inc. All rights reserved.

Metabolic and structural integrity of magnetic nanoparticle-loaded primary endothelial cells for targeted cell therapy.

PubMed

Orynbayeva, Zulfiya; Sensenig, Richard; Polyak, Boris

2015-05-01

To successfully translate magnetically mediated cell targeting from bench to bedside, there is a need to systematically assess the potential adverse effects of magnetic nanoparticles (MNPs) interacting with 'therapeutic' cells. Here, we examined in detail the effects of internalized polymeric MNPs on primary rat endothelial cells' structural intactness, metabolic integrity and proliferation potential. The intactness of cytoskeleton and organelles was studied by fluorescent confocal microscopy, flow cytometry and high-resolution respirometry. MNP-loaded primary endothelial cells preserve intact cytoskeleton and organelles, maintain normal rate of proliferation, calcium signaling and mitochondria energy metabolism. This study provides supportive evidence that MNPs at doses necessary for targeting did not induce significant adverse effects on structural integrity and functionality of primary endothelial cells - potential cell therapy vectors.

Exposure to ultrafine particles, intracellular production of reactive oxygen species in leukocytes and altered levels of endothelial progenitor cells.

PubMed

Jantzen, Kim; Møller, Peter; Karottki, Dorina Gabriela; Olsen, Yulia; Bekö, Gabriel; Clausen, Geo; Hersoug, Lars-Georg; Loft, Steffen

2016-06-01

Exposure to particles in the fine and ultrafine size range has been linked to induction of low-grade systemic inflammation, oxidative stress and development of cardiovascular diseases. Declining levels of endothelial progenitor cells within systemic circulation have likewise been linked to progression of cardiovascular diseases. The objective was to determine if exposure to fine and ultrafine particles from indoor and outdoor sources, assessed by personal and residential indoor monitoring, is associated with altered levels of endothelial progenitor cells, and whether such effects are related to leukocyte-mediated oxidative stress. The study utilized a cross sectional design performed in 58 study participants from a larger cohort. Levels of circulating endothelial progenitor cells, defined as either late (CD34(+)KDR(+) cells) or early (CD34(+)CD133(+)KDR(+) cells) subsets were measured using polychromatic flow cytometry. We additionally measured production of reactive oxygen species in leukocyte subsets (lymphocytes, monocytes and granulocytes) by flow cytometry using intracellular 2',7'-dichlorofluoroscein. The measurements encompassed both basal levels of reactive oxygen species production and capacity for reactive oxygen species production for each leukocyte subset. We found that the late endothelial progenitor subset was negatively associated with levels of ultrafine particles measured within the participant residences and with reactive oxygen species production capacity in lymphocytes. Additionally, the early endothelial progenitor cell levels were positively associated with a personalised measure of ultrafine particle exposure and negatively associated with both basal and capacity for reactive oxygen species production in lymphocytes and granulocytes, respectively. Our results indicate that exposure to fine and ultrafine particles derived from indoor sources may have adverse effects on human vascular health. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

Exercise training improves function of circulating angiogenic cells in patients with chronic heart failure.

PubMed

Van Craenenbroeck, Emeline M; Hoymans, Vicky Y; Beckers, Paul J; Possemiers, Nadine M; Wuyts, Kurt; Paelinck, Bernard P; Vrints, Christiaan J; Conraads, Viviane M

2010-09-01

Alterations in circulating angiogenic cells (CAC) and endothelial progenitor cells (EPC), known to contribute to endothelial repair, could explain the reversal of endothelial function in response to exercise training. Moreover, training-induced vascular remodeling might affect the acute response of EPC and CAC following a single exercise bout. We studied the impact of exercise training on CAC function and numbers of CD34(+)/KDR(+) EPC in patients with chronic heart failure (CHF) and we assessed the effect of acute exercise on CAC and EPC in sedentary and trained patients. Twenty-one sedentary CHF patients underwent 6-month exercise training and were compared to a non-trained control group (n = 17) and 10 healthy age-matched subjects. At baseline and follow-up, flow-mediated dilation was assessed and graded exercise testing (GXT) was performed. Before and immediately after GXT, CAC migratory capacity was assessed in vitro and circulating CD34(+)/KDR(+) EPC were quantified using flow cytometry. At baseline, CAC migration was significantly impaired in sedentary CHF patients but normalized acutely after GXT. Training corrected endothelial dysfunction, which coincided with a 77% increase in CAC migration (P = 0.0001). Moreover, the GXT-induced improvement detected at baseline was no longer observed after training. Numbers of CD34(+)/KDR(+) EPC increased following 6-month exercise training (P = 0.021), but were not affected by GXT, either prior or post-training. In conclusion, the present findings demonstrate for the first time that exercise training in CHF reverses CAC dysfunction and increases numbers of CD34(+)/KDR(+) EPC, which is accompanied by improvement of peripheral endothelial function. The acute exercise-induced changes in CAC function wane with exercise training, suggesting that repetitive exercise bouts progressively lead to functional endothelial repair.

Microvascular Endothelial Dysfunction in Sedentary, Obese Humans is mediated by NADPH Oxidase; Influence of Exercise Training

PubMed Central

La Favor, Justin D.; Dubis, Gabriel S.; Yan, Huimin; White, Joseph D.; Nelson, Margaret A.M.; Anderson, Ethan J.; Hickner, Robert C.

2016-01-01

Objective The objectives of this study were to determine the impact of in vivo reactive oxygen species (ROS) on microvascular endothelial function in obese human subjects and to determine the efficacy of an aerobic exercise intervention on alleviating obesity-associated dysfunctionality. Approach and Results Young, sedentary men and women were divided into lean (BMI 18–25; n=14), intermediate (BMI 28–32.5; n=13), and obese (BMI 33–40; n=15) groups. A novel microdialysis technique was utilized to detect elevated interstitial hydrogen peroxide (H2O2) and superoxide levels in the vastus lateralis of obese compared to both lean and intermediate subjects. Nutritive blood flow was monitored in the vastus lateralis via the microdialysis-ethanol technique. A decrement in acetylcholine-stimulated blood flow revealed impaired microvascular endothelial function in the obese subjects. Perfusion of apocynin, an NADPH oxidase (Nox) inhibitor, lowered (normalized) H2O2 and superoxide levels and reversed microvascular endothelial dysfunction in obese subjects. Following 8-weeks of exercise, H2O2 levels were decreased in the obese subjects and microvascular endothelial function in these subjects was restored to levels similar to lean subjects. Skeletal muscle protein expression of the Nox subunits p22phox, p47phox, and p67phox were increased in obese relative to lean subjects, where p22phox and p67phox expression was attenuated by exercise training in obese subjects. Conclusions This study implicates Nox as a source of excessive ROS production in skeletal muscle of obese individuals, and links excessive Nox derived ROS to microvascular endothelial dysfunction in obesity. Furthermore, aerobic exercise training proved to be an effective strategy for alleviating these maladies. PMID:27765769

Circulating platelet aggregates damage endothelial cells in culture.

PubMed

Aluganti Narasimhulu, Chandrakala; Nandave, Mukesh; Bonilla, Diana; Singaravelu, Janani; Sai-Sudhakar, Chittoor B; Parthasarathy, Sampath

2017-06-01

Presence of circulating endothelial cells (CECs) in systemic circulation may be an indicator of endothelial damage and/or denudation, and the body's response to repair and revascularization. Thus, we hypothesized that aggregated platelets (AgPlts) can disrupt/denude the endothelium and contribute to the presence of CEC and EC-derived particles (ECDP). Endothelial cells were grown in glass tubes and tagged with/without 0.5 μm fluorescent beads. These glass tubes were connected to a mini-pump variable-flow system to study the effect of circulating AgPlts on the endothelium. ECs in glass tube were exposed to medium alone, nonaggregated platelets (NAgPlts), AgPlts, and 90 micron polystyrene beads at a flow rate of 20 mL/min for various intervals. Collected effluents were cultured for 72 h to analyze the growth potential of dislodged but intact ECs. Endothelial damage was assessed by real time polymerase chain reaction (RT-PCR) for inflammatory genes and Western blot analysis for von Willebrand factor. No ECs and ECDP were observed in effluents collected after injecting medium alone and NAgPlts, whereas AgPlts and Polybeads drastically dislodged ECs, releasing ECs and ECDP in effluents as the time increased. Effluents collected when endothelial cell damage was seen showed increased presence of von Willebrand factor as compared to control effluents. Furthermore, we analyzed the presence of ECs and ECDPs in heart failure subjects, as well as animal plasma samples. Our study demonstrates that circulating AgPlts denude the endothelium and release ECs and ECDP. Direct mechanical disruption and shear stress caused by circulating AgPlts could be the underlying mechanism of the observed endothelium damage. Copyright © 2017 Elsevier Inc. All rights reserved.

Acute effects of high-fat meals enriched with walnuts or olive oil on postprandial endothelial function.

PubMed

Cortés, Berenice; Núñez, Isabel; Cofán, Montserrat; Gilabert, Rosa; Pérez-Heras, Ana; Casals, Elena; Deulofeu, Ramón; Ros, Emilio

2006-10-17

We sought to investigate whether the addition of walnuts or olive oil to a fatty meal have differential effects on postprandial vasoactivity, lipoproteins, markers of oxidation and endothelial activation, and plasma asymmetric dimethylarginine (ADMA). Compared with a Mediterranean diet, a walnut diet has been shown to improve endothelial function in hypercholesterolemic patients. We hypothesized that walnuts would reverse postprandial endothelial dysfunction associated with consumption of a fatty meal. We randomized in a crossover design 12 healthy subjects and 12 patients with hypercholesterolemia to 2 high-fat meal sequences to which 25 g olive oil or 40 g walnuts had been added. Both test meals contained 80 g fat and 35% saturated fatty acids, and consumption of each meal was separated by 1 week. Venipunctures and ultrasound measurements of brachial artery endothelial function were performed after fasting and 4 h after test meals. In both study groups, flow-mediated dilation (FMD) was worse after the olive oil meal than after the walnut meal (p = 0.006, time-period interaction). Fasting, but not postprandial, triglyceride concentrations correlated inversely with FMD (r = -0.324; p = 0.024). Flow-independent dilation and plasma ADMA concentrations were unchanged, and the concentration of oxidized low-density lipoproteins decreased (p = 0.051) after either meal. The plasma concentrations of soluble inflammatory cytokines and adhesion molecules decreased (p < 0.01) independently of meal type, except for E-selectin, which decreased more (p = 0.033) after the walnut meal. Adding walnuts to a high-fat meal acutely improves FMD independently of changes in oxidation, inflammation, or ADMA. Both walnuts and olive oil preserve the protective phenotype of endothelial cells.

Acute EGCG supplementation reverses endothelial dysfunction in patients with coronary artery disease.

PubMed

Widlansky, Michael E; Hamburg, Naomi M; Anter, Elad; Holbrook, Monika; Kahn, David F; Elliott, James G; Keaney, John F; Vita, Joseph A

2007-04-01

Epidemiological studies demonstrate an inverse relation between dietary flavonoid intake and cardiovascular risk. Recent studies with flavonoid-containing beverages suggest that the benefits of these nutrients may relate, in part, to improved endothelial function. We hypothesized that dietary supplementation with epigallocatechin gallate (EGCG), a major catechin in tea, would improve endothelial function in humans. We examined the effects of EGCG on endothelial function in a double blind, placebo-controlled, crossover design study. We measured brachial artery flow-mediated dilation by vascular ultrasound at six time points: prior to treatment with EGCG or placebo, two hours after an initial dose of EGCG (300 mg) or placebo, and after two weeks of treatment with EGCG (150 mg twice daily) or placebo. The order of treatments (EGCG or placebo) was randomized and there was a one-week washout period between treatments. A total of 42 subjects completed the study, and brachial artery flow-mediated dilation improved from 7.1 +/- 4.1 to 8.6 +/- 4.7% two hours after the first dose of 300 mg of EGCG (P = 0.01), but was similar to baseline (7.8 +/- 4.2%, P = 0.12) after two weeks of treatment with the final measurements made approximately 14 hours after the last dose. Placebo treatment had no significant effect, and there were no changes in reactive hyperemia or the response to sublingual nitroglycerin. The changes in vascular function paralleled plasma EGCG concentrations, which increased from 2.6 +/- 10.9 to 92.8 +/- 78.7 ng/ml after acute EGCG (P < 0.001), but were unchanged from baseline after two weeks of treatment (3.4 +/- 13.1 ng/ml). EGCG acutely improves endothelial function in humans with coronary artery disease, and may account for a portion of the beneficial effects of flavonoid-rich food on endothelial function.

Quantifying effects of cyclic stretch on cell-collagen substrate adhesiveness of vascular endothelial cells.

PubMed

Omidvar, Ramin; Tafazzoli-Shadpour, Mohammad; Mahmoodi-Nobar, Farbod; Azadi, Shohreh; Khani, Mohammad-Mehdi

2018-05-01

Vascular endothelium is continuously subjected to mechanical stimulation in the form of shear forces due to blood flow as well as tensile forces as a consequence of blood pressure. Such stimuli influence endothelial behavior and regulate cell-tissue interaction for an optimized functionality. This study aimed to quantify influence of cyclic stretch on the adhesive property and stiffness of endothelial cells. The 10% cyclic stretch with frequency of 1 Hz was applied to a layer of endothelial cells cultured on a polydimethylsiloxane substrate. Cell-substrate adhesion of endothelial cells was examined by the novel approach of atomic force microscope-based single-cell force spectroscopy and cell stiffness was measured by atomic force microscopy. Furthermore, the adhesive molecular bonds were evaluated using modified Hertz contact theory. Our results show that overall adhesion of endothelial cells with substrate decreased after cyclic stretch while they became stiffer. Based on the experimental results and theoretical modeling, the decrease in the number of molecular bonds after cyclic stretch was quantified. In conclusion, in vitro cyclic stretch caused alterations in both adhesive capacity and elastic modulus of endothelial cells through mechanotransductive pathways as two major determinants of the function of these cells within the cardiovascular system.

Flow-mediated vasodilation is augmented in a corkscrew collateral artery compared with that in a native artery in patients with thromboangiitis obliterans (Buerger disease).

PubMed

Fujii, Yuichi; Fujimura, Noritaka; Mikami, Shinsuke; Maruhashi, Tatsuya; Kihara, Yasuki; Chayama, Kazuaki; Noma, Kensuke; Higashi, Yukihito

2011-12-01

A healthy endothelium maintains vascular tone and structure. The purpose of this study was to evaluate endothelial function in corkscrew collateral arteries in Buerger disease. We measured flow-mediated vasodilation (FMD) in corkscrew arteries in 26 patients with Buerger disease, in control arteries in 26 healthy subjects, and in native arteries in 16 patients with Buerger disease. Hyperemic flow was lower in corkscrew arteries than in native arteries in patients with Buerger disease and in control arteries in healthy subjects. There was no significant difference between hyperemic flow in patients with Buerger disease in whom measurements were performed in native arteries and that in healthy subjects. FMD was lower in corkscrew arteries and native arteries in patients with Buerger disease than in control arteries in healthy subjects. There was no significant difference between FMD in corkscrew arteries in patients with Buerger disease and in that in native arteries. The ratio of FMD to hyperemic flow was significantly smaller in native arteries in patients with Buerger disease than in corkscrew arteries and in control arteries in healthy subjects (5.5 ± 6.2 vs 8.8 ± 8.9 and 9.6 ± 7.6 mL/min, P < .001, respectively). There was no significant difference in the ratio of FMD to hyperemic flow between corkscrew arteries in Buerger disease and control arteries in healthy subjects. Nitroglycerin-induced vasodilation was similar in all leg arteries. Endothelial function of a corkscrew collateral artery in patients with Buerger disease is maintained, while endothelial function is impaired in a native artery in Buerger disease. Copyright © 2011 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.

Hyperglycemia-induced PATZ1 negatively modulates endothelial vasculogenesis via repression of FABP4 signaling.

PubMed

Chen, Ren-An; Sun, Xiao-Mian; Yan, Chang-You; Liu, Li; Hao, Miao-Wang; Liu, Qiang; Jiao, Xi-Ying; Liang, Ying-Min

2016-09-02

Vascular endothelial dysfunction, a central hallmark of diabetes, predisposes diabetic patients to numerous cardiovascular complications. The POZ/BTB and AT-hook-containing zinc finger protein 1 (PATZ1), is an important transcriptional regulatory factor and regulates divergent pathways depending on the cellular context, but its role in endothelial cells remains poorly understood. Herein, we report for the first time that endothelial PATZ1 expression was abnormally upregulated in diabetic endothelial cells (ECs) regardless of diabetes classification. This stimulatory effect was further confirmed in the high glucose-treated human umbilical vein endothelial cells (HUVECs). From a functional standpoint, transgenic overexpression of PATZ1 in endothelial colony forming cells (ECFCs) blunted angiogenesis in vivo and rendered endothelial cells unresponsive to established angiogenic factors. Mechanistically, PATZ1 acted as a potent transcriptional corepressor of fatty acid-binding protein 4 (FABP4), an essential convergence point for angiogenic and metabolic signaling pathways in ECs. Taken together, endothelial PATZ1 thus potently inhibits endothelial function and angiogenesis via inhibition of FABP4 expression, and abnormal induction of endothelial PATZ1 may contribute to multiple aspects of vascular dysfunction in diabetes. Copyright © 2016. Published by Elsevier Inc.

Endothelial Ca2+ oscillations reflect VEGFR signaling-regulated angiogenic capacity in vivo

PubMed Central

Yokota, Yasuhiro; Nakajima, Hiroyuki; Wakayama, Yuki; Muto, Akira; Kawakami, Koichi; Fukuhara, Shigetomo; Mochizuki, Naoki

2015-01-01

Sprouting angiogenesis is a well-coordinated process controlled by multiple extracellular inputs, including vascular endothelial growth factor (VEGF). However, little is known about when and how individual endothelial cell (EC) responds to angiogenic inputs in vivo. Here, we visualized endothelial Ca2+ dynamics in zebrafish and found that intracellular Ca2+ oscillations occurred in ECs exhibiting angiogenic behavior. Ca2+ oscillations depended upon VEGF receptor-2 (Vegfr2) and Vegfr3 in ECs budding from the dorsal aorta (DA) and posterior cardinal vein, respectively. Thus, visualizing Ca2+ oscillations allowed us to monitor EC responses to angiogenic cues. Vegfr-dependent Ca2+ oscillations occurred in migrating tip cells as well as stalk cells budding from the DA. We investigated how Dll4/Notch signaling regulates endothelial Ca2+ oscillations and found that it was required for the selection of single stalk cell as well as tip cell. Thus, we captured spatio-temporal Ca2+ dynamics during sprouting angiogenesis, as a result of cellular responses to angiogenic inputs. DOI: http://dx.doi.org/10.7554/eLife.08817.001 PMID:26588168

Involvement of myosin light-chain kinase in endothelial cell retraction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wysolmerski, R.B.; Lagunoff, D.

Permeabilized bovine pulmonary artery endothelial cell monolayers were used to investigate the mechanism of endothelial cell retraction. Postconfluent endothelial cells permeabilized with saponin retracted upon exposure to ATP and Ca{sup 2+}. Retraction was accompanied by thiophosphorylation of 19,000-Da myosin light chains when adenosine 5'-(gamma-({sup 35}S)thio)triphosphate was included in the medium. Both retraction and thiophosphorylation of myosin light chains exhibited a graded quantitative dependence on Ca{sup 2+}. When permeabilized monolayers were extracted in buffer D containing 100 mM KCl and 30 mM MgCl2 for 30 min, the cells failed to retract upon exposure to ATP and Ca{sup 2+}, and no thiophosphorylationmore » of myosin light chains occurred. The ability both to retract and to thiophosphorylate myosin light chains was restored by the addition to the permeabilized, extracted cells of myosin light-chain kinase and calmodulin together but not by either alone. These studies indicate that endothelial cell retraction, as does smooth muscle contraction, depends on myosin light-chain kinase phosphorylation of myosin light chains.« less

Activation of protein kinase C and disruption of endothelial monolayer integrity by sodium arsenite-Potential mechanism in the development of atherosclerosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pereira, Flavia E.; Coffin, J. Douglas; Beall, Howard D.

2007-04-15

Arsenic exposure has been shown to exacerbate atherosclerosis, beginning with activation of the endothelium that lines the vessel wall. Endothelial barrier integrity is maintained by proteins of the adherens junction (AJ) such as vascular endothelial cadherin (VE-cadherin) and {beta}-catenin and their association with the actin cytoskeleton. In the present study, human aortic endothelial cells (HAECs) were exposed to 1, 5 and 10 {mu}M sodium arsenite [As(III)] for 1, 6, 12 and 24 h, and the effects on endothelial barrier integrity were determined. Immunofluorescence studies revealed formation of actin stress fibers and non-uniform VE-cadherin and {beta}-catenin staining at cell-cell junctions thatmore » were concentration- and time-dependent. Intercellular gaps were observed with a measured increase in endothelial permeability. In addition, concentration-dependent increases in tyrosine phosphorylation (PY) of {beta}-catenin and activation of protein kinase C{alpha} (PKC{alpha}) were observed. Inhibition of PKC{alpha} restored VE-cadherin and {beta}-catenin staining at cell-cell junctions and abolished the As(III)-induced formation of actin stress fibers and intercellular gaps. Endothelial permeability and PY of {beta}-catenin were also reduced to basal levels. These results demonstrate that As(III) induces activation of PKC{alpha}, which leads to increased PY of {beta}-catenin downstream of PKC{alpha} activation. Phosphorylation of {beta}-catenin plausibly severs the association of VE-cadherin and {beta}-catenin, which along with formation of actin stress fibers, results in intercellular gap formation and increased endothelial permeability. To the best of our knowledge, this is the first report demonstrating that As(III) causes a loss of endothelial monolayer integrity, which potentially could contribute to the development of atherosclerosis.« less

Biomarkers of coronary endothelial health: correlation with invasive measures of collateral function, flow and resistance in chronically occluded coronary arteries and the effect of recanalization.

PubMed

Ladwiniec, Andrew; Ettelaie, Camille; Cunnington, Michael S; Rossington, Jennifer; Thackray, Simon; Alamgir, Farquad; Hoye, Angela

2016-06-01

In the presence of a chronically occluded coronary artery, the collateral circulation matures by a process of arteriogenesis; however, there is considerable variation between individuals in the functional capacity of that collateral network. This could be explained by differences in endothelial health and function. We aimed to examine the relationship between the functional extent of collateralization and levels of biomarkers that have been shown to relate to endothelial health. We measured four potential biomarkers of endothelial health in 34 patients with mature collateral networks who underwent a successful percutaneous coronary intervention (PCI) for a chronic total coronary occlusion (CTO) before PCI and 6-8 weeks after PCI, and examined the relationship of biomarker levels with physiological measures of collateralization. We did not find a significant change in the systemic levels of sICAM-1, sE-selectin, microparticles or tissue factor 6-8 weeks after PCI. We did find an association between estimated retrograde collateral flow before CTO recanalization and lower levels of sICAM-1 (r=0.39, P=0.026), sE-selectin (r=0.48, P=0.005) and microparticles (r=0.38, P=0.03). Recanalization of a CTO and resultant regression of a mature collateral circulation do not alter systemic levels of sICAM-1, sE-selectin, microparticles or tissue factor. The identified relationship of retrograde collateral flow with sICAM-1, sE-selectin and microparticles is likely to represent an association with an ability to develop collaterals rather than their presence and extent.

Targeted overexpression of endothelial nitric oxide synthase in endothelial cells improves cerebrovascular reactivity in Ins2Akita-type-1 diabetic mice.

PubMed

Chandra, Saurav B; Mohan, Sumathy; Ford, Bridget M; Huang, Lei; Janardhanan, Preethi; Deo, Kaiwalya S; Cong, Linlin; Muir, Eric R; Duong, Timothy Q

2016-06-01

Reduced bioavailability of nitric oxide due to impaired endothelial nitric oxide synthase (eNOS) activity is a leading cause of endothelial dysfunction in diabetes. Enhancing eNOS activity in diabetes is a potential therapeutic target. This study investigated basal cerebral blood flow and cerebrovascular reactivity in wild-type mice, diabetic mice (Ins2(Akita+/-)), nondiabetic eNOS-overexpressing mice (TgeNOS), and the cross of two transgenic mice (TgeNOS-Ins2(Akita+/-)) at six months of age. The cross was aimed at improving eNOS expression in diabetic mice. The major findings were: (i) Body weights of Ins2(Akita+/-) and TgeNOS-Ins2(Akita+/-) were significantly different from wild-type and TgeNOS mice. Blood pressure of TgeNOS mice was lower than wild-type. (ii) Basal cerebral blood flow of the TgeNOS group was significantly higher than cerebral blood flow of the other three groups. (iii) The cerebrovascular reactivity in the Ins2(Akita+/-) mice was significantly lower compared with wild-type, whereas that in the TgeNOS-Ins2(Akita+/-) was significantly higher compared with the Ins2(Akita+/-) and TgeNOS groups. Overexpression of eNOS rescued cerebrovascular dysfunction in diabetic animals, resulting in improved cerebrovascular reactivity. These results underscore the possible role of eNOS in vascular dysfunction in the brain of diabetic mice and support the notion that enhancing eNOS activity in diabetes is a potential therapeutic target. © The Author(s) 2015.

C-reactive protein induces release of both endothelial microparticles and circulating endothelial cells in vitro and in vivo: further evidence of endothelial dysfunction.

PubMed

Devaraj, Sridevi; Kumaresan, Pappanaicken R; Jialal, Ishwarlal

2011-12-01

Inflammation is pivotal in atherosclerosis. A key early event in atherosclerosis is endothelial dysfunction. C-reactive protein (CRP), the prototypic marker of inflammation in humans, is a risk marker for cardiovascular disease, and there is mounting evidence to support its role in atherothrombosis. CRP has been shown to promote endothelial dysfunction both in vitro and in vivo. Emerging biomarkers of endothelial dysfunction include circulating endothelial cells (CECs) and endothelial microparticles (EMPs). However, there is a paucity of data examining the effect of CRP on CEC and EMP production in vitro and in vivo. In this report, we treated human aortic endothelial cells (HAECs) with increasing concentrations of CRP (0-50 μg/mL) or boiled CRP. We counted CECs and EMPs by flow cytometry. Although CRP treatment resulted in a significant increase in release of both CECs and EMPs, boiled CRP failed to have an effect. Pretreatment of HAECs with sepiapterin or diethylenetriamine NONOate, both of which preserve nitric oxide (NO), resulted in attenuation of CRP's effects on CECs and EMPs. CD32 and CD64 blocking antibodies but not CD16 antibody or lectin-like oxidized LDL receptor 1 small interfering RNA (LOX-1 siRNA) prevented CRP-induced production of CECs and EMPs. Furthermore, delivery of human CRP to Wistar rats compared with human serum albumin resulted in significantly increased CECs and EMPs, corroborating the in vitro findings. We provide novel data that CRP, via NO deficiency, promotes endothelial dysfunction by inducing release of CECs and EMPs, which are biomarkers of endothelial dysfunction.

Endothelial cell-derived microparticles induce plasmacytoid dendritic cell maturation: potential implications in inflammatory diseases.

PubMed

Angelot, Fanny; Seillès, Estelle; Biichlé, Sabeha; Berda, Yael; Gaugler, Béatrice; Plumas, Joel; Chaperot, Laurence; Dignat-George, Françoise; Tiberghien, Pierre; Saas, Philippe; Garnache-Ottou, Francine

2009-11-01

Increased circulating endothelial microparticles, resulting from vascular endothelium dysfunction, and plasmacytoid dendritic cell activation are both encountered in common inflammatory disorders. The aim of our study was to determine whether interactions between endothelial microparticles and plasmacytoid dendritic cells could contribute to such pathologies. Microparticles generated from endothelial cell lines, platelets or activated T cells were incubated with human plasmacytoid dendritic cells sorted from healthy donor blood or with monocyte-derived dendritic cells. Dendritic cell maturation was evaluated by flow cytometry, cytokine secretion as well as naive T-cell activation and polarization. Labeled microparticles were also used to study cellular interactions. Endothelial microparticles induced plasmacytoid dendritic cell maturation. In contrast, conventional dendritic cells were resistant to endothelial microparticle-induced maturation. In addition to upregulation of co-stimulatory molecules, endothelial microparticle-matured plasmacytoid dendritic cells secreted inflammatory cytokines (interleukins 6 and 8, but no interferon-alpha) and also induced allogeneic naive CD4(+) T cells to proliferate and to produce type 1 cytokines such as interferon-gamma and tumor necrosis factor-alpha. Endothelial microparticle endocytosis by plasmacytoid dendritic cells appeared to be required for plasmacytoid dendritic cell maturation. Importantly, the ability of endothelial microparticles to induce plasmacytoid dendritic cells to mature was specific as microparticles derived from activated T cells or platelets (the major source of circulating microparticules in healthy subjects) did not induce such plasmacytoid dendritic cell maturation. Our data show that endothelial microparticles specifically induce plasmacytoid dendritic cell maturation and production of inflammatory cytokines. This novel activation pathway may be implicated in various inflammatory disorders and endothelial microparticles could be an important immunmodulatory therapeutic target.

Reduction of obesity, as induced by leptin, reverses endothelial dysfunction in obese (Lep(ob)) mice

NASA Technical Reports Server (NTRS)

Winters, B.; Mo, Z.; Brooks-Asplund, E.; Kim, S.; Shoukas, A.; Li, D.; Nyhan, D.; Berkowitz, D. E.

2000-01-01

Obesity is a major health care problem and is associated with significant cardiovascular morbidity. Leptin, a neuroendocrine hormone released by adipose tissue, is important in modulating obesity by signaling satiety and increasing metabolism. Moreover, leptin receptors are expressed on vascular endothelial cells (ECs) and mediate angiogenesis. We hypothesized that leptin may also play an important role in vasoregulation. We investigated vasoregulatory mechanisms in the leptin-deficient obese (ob/ob) mouse model and determined the influence of leptin replacement on endothelial-dependent vasorelaxant responses. The direct effect of leptin on EC nitric oxide (NO) production was also tested by using 4, 5-diaminofluorescein-2 diacetate staining and measurement of nitrate and nitrite concentrations. Vasoconstrictor responses to phenylephrine, norepinephrine, and U-46619 were markedly enhanced in aortic rings from ob/ob mice and were modulated by NO synthase inhibition. Vasorelaxant responses to ACh were markedly attenuated in mesenteric microvessels from ob/ob mice. Leptin replacement resulted in significant weight loss and reversal of the impaired endothelial-dependent vasorelaxant responses observed in ob/ob mice. Preincubation of ECs with leptin enhanced the release of NO production. Thus leptin-deficient ob/ob mice demonstrate marked abnormalities in vasoregulation, including impaired endothelial-dependent vasodilation, which is reversed by leptin replacement. These findings may be partially explained by the direct effect of leptin on endothelial NO production. These vascular abnormalities are similar to those observed in obese, diabetic, leptin-resistant humans. The ob/ob mouse may, therefore, be an excellent new model for the study of the cardiovascular effects of obesity.

Intracellular Ascorbate Prevents Endothelial Barrier Permeabilization by Thrombin*

PubMed Central

Parker, William H.; Qu, Zhi-chao; May, James M.

2015-01-01

Intracellular ascorbate (vitamin C) has previously been shown to tighten the endothelial barrier and maintain barrier integrity during acute inflammation in vitro. However, the downstream effectors of ascorbate in the regulation of endothelial permeability remain unclear. In this study, we evaluated ascorbate as a mediator of thrombin-induced barrier permeabilization in human umbilical vein endothelial cells and their immortalized hybridoma line, EA.hy926. We found that the vitamin fully prevented increased permeability to the polysaccharide inulin by thrombin in a dose-dependent manner, and it took effect both before and after subjection to thrombin. Thrombin exposure consumed intracellular ascorbate but not the endogenous antioxidant GSH. Likewise, the antioxidants dithiothreitol and tempol did not reverse permeabilization. We identified a novel role for ascorbate in preserving cAMP during thrombin stimulation, resulting in two downstream effects. First, ascorbate maintained the cortical actin cytoskeleton in a Rap1- and Rac1-dependent manner, thus preserving stable adherens junctions between adjacent cells. Second, ascorbate prevented actin polymerization and formation of stress fibers by reducing the activation of RhoA and phosphorylation of myosin light chain. Although ascorbate and thrombin both required calcium for their respective effects, ascorbate did not prevent thrombin permeabilization by obstructing calcium influx. However, preservation of cAMP by ascorbate was found to depend on both the production of nitric oxide by endothelial nitric-oxide synthase, which ascorbate is known to activate, and the subsequent generation cGMP by guanylate cyclase. Together, these data implicate ascorbate in the prevention of inflammatory endothelial barrier permeabilization and explain the underlying signaling mechanism. PMID:26152729