Lipopolysaccharide-induced endothelial barrier breakdown is cyclic adenosine monophosphate dependent in vivo and in vitro.

PubMed

Schlegel, Nicolas; Baumer, Yvonne; Drenckhahn, Detlev; Waschke, Jens

2009-05-01

To determine whether cyclic adenosine monophosphate (cAMP) is critically involved in lipopolysaccharide (LPS)-induced breakdown of endothelial barrier functions in vivo and in vitro. Experimental laboratory research. Research laboratory. Wistar rats and cultured human microvascular endothelial cells. Permeability measurements in single postcapillary venules in vivo and permeability measurements and cell biology techniques in vitro. We demonstrate that within 120 minutes LPS increased endothelial permeability in rat mesenteric postcapillary venules in vivo and caused a barrier breakdown in human dermal microvascular endothelial cells in vitro. This was associated with the formation of large intercellular gaps and fragmentation of vascular endothelial cadherin immunostaining. Furthermore, claudin 5 immunostaining at cell borders was drastically reduced after LPS treatment. Interestingly, activity of the small GTPase Rho A, which has previously been suggested to mediate the LPS-induced endothelial barrier breakdown, was not increased after 2 hours. However, activity of Rac 1, which is known to be important for maintenance of endothelial barrier functions, was significantly reduced to 64 +/- 8% after 2 hours. All LPS-induced changes of endothelial cells were blocked by a forskolin-mediated or rolipram-mediated increase of cAMP. Consistently, enzyme-linked immunosorbent assay-based measurements demonstrated that LPS significantly decreased intracellular cAMP. In summary, our data demonstrate that LPS disrupts endothelial barrier properties by decreasing intracellular cAMP. This mechanism may involve inactivation of Rac 1 rather than activation of Rho A.

The Barrier Within: Endothelial Transport of Hormones

PubMed Central

Kolka, Cathryn M.; Bergman, Richard N.

2015-01-01

Hormones are involved in a plethora of processes including development and growth, metabolism, mood, and immune responses. These essential functions are dependent on the ability of the hormone to access its target tissue. In the case of endocrine hormones that are transported through the blood, this often means that the endothelium must be crossed. Many studies have shown that the concentrations of hormones and nutrients in blood can be very different from those surrounding the cells on the tissue side of the blood vessel endothelium, suggesting that transport across this barrier can be rate limiting for hormone action. This transport can be regulated by altering the surface area of the blood vessel available for diffusion through to the underlying tissue or by the permeability of the endothelium. Many hormones are known to directly or indirectly affect the endothelial barrier, thus affecting their own distribution to their target tissues. Dysfunction of the endothelial barrier is found in many diseases, particularly those associated with the metabolic syndrome. The interrelatedness of hormones may help to explain why the cluster of diseases in the metabolic syndrome occur together so frequently and suggests that treating the endothelium may ameliorate defects in more than one disease. Here, we review the structure and function of the endothelium, its contribution to the function of hormones, and its involvement in disease. PMID:22875454

The barrier within: endothelial transport of hormones.

PubMed

Kolka, Cathryn M; Bergman, Richard N

2012-08-01

Hormones are involved in a plethora of processes including development and growth, metabolism, mood, and immune responses. These essential functions are dependent on the ability of the hormone to access its target tissue. In the case of endocrine hormones that are transported through the blood, this often means that the endothelium must be crossed. Many studies have shown that the concentrations of hormones and nutrients in blood can be very different from those surrounding the cells on the tissue side of the blood vessel endothelium, suggesting that transport across this barrier can be rate limiting for hormone action. This transport can be regulated by altering the surface area of the blood vessel available for diffusion through to the underlying tissue or by the permeability of the endothelium. Many hormones are known to directly or indirectly affect the endothelial barrier, thus affecting their own distribution to their target tissues. Dysfunction of the endothelial barrier is found in many diseases, particularly those associated with the metabolic syndrome. The interrelatedness of hormones may help to explain why the cluster of diseases in the metabolic syndrome occur together so frequently and suggests that treating the endothelium may ameliorate defects in more than one disease. Here, we review the structure and function of the endothelium, its contribution to the function of hormones, and its involvement in disease.

Modelling the endothelial blood-CNS barriers: a method for the production of robust in vitro models of the rat blood-brain barrier and blood-spinal cord barrier

PubMed Central

2013-01-01

Background Modelling the blood-CNS barriers of the brain and spinal cord in vitro continues to provide a considerable challenge for research studying the passage of large and small molecules in and out of the central nervous system, both within the context of basic biology and for pharmaceutical drug discovery. Although there has been considerable success over the previous two decades in establishing useful in vitro primary endothelial cell cultures from the blood-CNS barriers, no model fully mimics the high electrical resistance, low paracellular permeability and selective influx/efflux characteristics of the in vivo situation. Furthermore, such primary-derived cultures are typically labour-intensive and generate low yields of cells, limiting scope for experimental work. We thus aimed to establish protocols for the high yield isolation and culture of endothelial cells from both rat brain and spinal cord. Our aim was to optimise in vitro conditions for inducing phenotypic characteristics in these cells that were reminiscent of the in vivo situation, such that they developed into tight endothelial barriers suitable for performing investigative biology and permeability studies. Methods Brain and spinal cord tissue was taken from the same rats and used to specifically isolate endothelial cells to reconstitute as in vitro blood-CNS barrier models. Isolated endothelial cells were cultured to expand the cellular yield and then passaged onto cell culture inserts for further investigation. Cell culture conditions were optimised using commercially available reagents and the resulting barrier-forming endothelial monolayers were characterised by functional permeability experiments and in vitro phenotyping by immunocytochemistry and western blotting. Results Using a combination of modified handling techniques and cell culture conditions, we have established and optimised a protocol for the in vitro culture of brain and, for the first time in rat, spinal cord endothelial cells

Modelling the endothelial blood-CNS barriers: a method for the production of robust in vitro models of the rat blood-brain barrier and blood-spinal cord barrier.

PubMed

Watson, P Marc D; Paterson, Judy C; Thom, George; Ginman, Ulrika; Lundquist, Stefan; Webster, Carl I

2013-06-18

Modelling the blood-CNS barriers of the brain and spinal cord in vitro continues to provide a considerable challenge for research studying the passage of large and small molecules in and out of the central nervous system, both within the context of basic biology and for pharmaceutical drug discovery. Although there has been considerable success over the previous two decades in establishing useful in vitro primary endothelial cell cultures from the blood-CNS barriers, no model fully mimics the high electrical resistance, low paracellular permeability and selective influx/efflux characteristics of the in vivo situation. Furthermore, such primary-derived cultures are typically labour-intensive and generate low yields of cells, limiting scope for experimental work. We thus aimed to establish protocols for the high yield isolation and culture of endothelial cells from both rat brain and spinal cord. Our aim was to optimise in vitro conditions for inducing phenotypic characteristics in these cells that were reminiscent of the in vivo situation, such that they developed into tight endothelial barriers suitable for performing investigative biology and permeability studies. Brain and spinal cord tissue was taken from the same rats and used to specifically isolate endothelial cells to reconstitute as in vitro blood-CNS barrier models. Isolated endothelial cells were cultured to expand the cellular yield and then passaged onto cell culture inserts for further investigation. Cell culture conditions were optimised using commercially available reagents and the resulting barrier-forming endothelial monolayers were characterised by functional permeability experiments and in vitro phenotyping by immunocytochemistry and western blotting. Using a combination of modified handling techniques and cell culture conditions, we have established and optimised a protocol for the in vitro culture of brain and, for the first time in rat, spinal cord endothelial cells. High yields of both CNS

Impedance analysis of GPCR-mediated changes in endothelial barrier function: overview, and fundamental considerations for stable and reproducible measurements

PubMed Central

Stolwijk, Judith A.; Matrougui, Khalid; Renken, Christian W.; Trebak, Mohamed

2014-01-01

The past 20 years have seen significant growth in using impedance-based assays to understand the molecular underpinning of endothelial and epithelial barrier function in response to physiological agonists, pharmacological and toxicological compounds. Most studies on barrier function use G protein coupled receptor (GPCR) agonists which couple to fast and transient changes in barrier properties. The power of impedance based techniques such as Electric Cell-Substrate Impedance Sensing (ECIS) reside in its ability to detect minute changes in cell layer integrity label-free and in real-time ranging from seconds to days. We provide a comprehensive overview of the biophysical principles, applications and recent developments in impedance-based methodologies. Despite extensive application of impedance analysis in endothelial barrier research little attention has been paid to data analysis and critical experimental variables, which are both essential for signal stability and reproducibility. We describe the rationale behind common ECIS data presentation and interpretation and illustrate practical guidelines to improve signal intensity by adapting technical parameters such as electrode layout, monitoring frequency or parameter (resistance versus impedance magnitude). Moreover, we discuss the impact of experimental parameters, including cell source, liquid handling and agonist preparation on signal intensity and kinetics. Our discussions are supported by experimental data obtained from human microvascular endothelial cells challenged with three GPCR agonists, thrombin, histamine and Sphingosine-1-Phosphate. PMID:25537398

Impedance analysis of GPCR-mediated changes in endothelial barrier function: overview and fundamental considerations for stable and reproducible measurements.

PubMed

Stolwijk, Judith A; Matrougui, Khalid; Renken, Christian W; Trebak, Mohamed

2015-10-01

The past 20 years has seen significant growth in using impedance-based assays to understand the molecular underpinning of endothelial and epithelial barrier function in response to physiological agonists and pharmacological and toxicological compounds. Most studies on barrier function use G protein-coupled receptor (GPCR) agonists which couple to fast and transient changes in barrier properties. The power of impedance-based techniques such as electric cell-substrate impedance sensing (ECIS) resides in its ability to detect minute changes in cell layer integrity label-free and in real-time ranging from seconds to days. We provide a comprehensive overview of the biophysical principles, applications, and recent developments in impedance-based methodologies. Despite extensive application of impedance analysis in endothelial barrier research, little attention has been paid to data analysis and critical experimental variables, which are both essential for signal stability and reproducibility. We describe the rationale behind common ECIS data presentation and interpretation and illustrate practical guidelines to improve signal intensity by adapting technical parameters such as electrode layout, monitoring frequency, or parameter (resistance versus impedance magnitude). Moreover, we discuss the impact of experimental parameters, including cell source, liquid handling, and agonist preparation on signal intensity and kinetics. Our discussions are supported by experimental data obtained from human microvascular endothelial cells challenged with three GPCR agonists, thrombin, histamine, and sphingosine-1-phosphate.

RhoB controls endothelial barrier recovery by inhibiting Rac1 trafficking to the cell border

PubMed Central

Marcos-Ramiro, Beatriz; García-Weber, Diego; Barroso, Susana; Feito, Jorge; Ortega, María C.; Cernuda-Morollón, Eva; Reglero-Real, Natalia; Fernández-Martín, Laura; Durán, Maria C.; Alonso, Miguel A.; Correas, Isabel; Cox, Susan; Ridley, Anne J.

2016-01-01

Endothelial barrier dysfunction underlies chronic inflammatory diseases. In searching for new proteins essential to the human endothelial inflammatory response, we have found that the endosomal GTPase RhoB is up-regulated in response to inflammatory cytokines and expressed in the endothelium of some chronically inflamed tissues. We show that although RhoB and the related RhoA and RhoC play additive and redundant roles in various aspects of endothelial barrier function, RhoB specifically inhibits barrier restoration after acute cell contraction by preventing plasma membrane extension. During barrier restoration, RhoB trafficking is induced between vesicles containing RhoB nanoclusters and plasma membrane protrusions. The Rho GTPase Rac1 controls membrane spreading and stabilizes endothelial barriers. We show that RhoB colocalizes with Rac1 in endosomes and inhibits Rac1 activity and trafficking to the cell border during barrier recovery. Inhibition of endosomal trafficking impairs barrier reformation, whereas induction of Rac1 translocation to the plasma membrane accelerates it. Therefore, RhoB-specific regulation of Rac1 trafficking controls endothelial barrier integrity during inflammation. PMID:27138256

Ascorbic Acid Prevents VEGF-induced Increases in Endothelial Barrier Permeability

PubMed Central

Ulker, Esad; Parker, William H.; Raj, Amita; Qu, Zhi-chao; May, James M.

2015-01-01

Vascular endothelial growth factor (VEGF) increases endothelial barrier permeability, an effect that may contribute to macular edema in diabetic retinopathy. Since vitamin C, or ascorbic acid, can tighten the endothelial permeability barrier, we examined whether it could prevent the increase in permeability due to VEGF in human umbilical vein endothelial cells (HUVECs). As previously observed, VEGF increased HUVEC permeability to radiolabeled inulin within 60 min in a concentration-dependent manner. Loading the cells with increasing concentrations of ascorbate progressively prevented the leakage caused by 100 ng/ml VEGF, with a significant inhibition at 13 μM and complete inhibition at 50 μM. Loading cells with 100 μM ascorbate also decreased basal generation of reactive oxygen species and prevented the increase caused by both 100 ng/ml VEGF. VEGF treatment decreased intracellular ascorbate by 25%, thus linking ascorbate oxidation to its prevention of VEGF-induced barrier leakage. The latter was blocked by treating the cells with 60 μM L-NAME (but not D-NAME) as well as by 30 μM sepiapterin, a precursor of tetrahydrobiopterin that is required for proper function of endothelial nitric oxide synthase (eNOS). These findings suggest that VEGF-induced barrier leakage uncouples eNOS. Ascorbate inhibition of the VEGF effect could thus be due either to scavenging superoxide or to peroxynitrite generated by the uncoupled eNOS, or more likely to its ability to recycle tetrahydrobiopterin, thus avoiding enzyme uncoupling in the first place. Ascorbate prevention of VEGF-induced increases in endothelial permeability opens the possibility that its repletion could benefit diabetic macular edema. PMID:26590088

Abl Tyrosine Kinase Phosphorylates Nonmuscle Myosin Light Chain Kinase to Regulate Endothelial Barrier Function

PubMed Central

Dudek, Steven M.; Chiang, Eddie T.; Camp, Sara M.; Guo, Yurong; Zhao, Jing; Brown, Mary E.; Singleton, Patrick A.; Wang, Lichun; Desai, Anjali; Arce, Fernando T.; Lal, Ratnesh; Van Eyk, Jennifer E.; Imam, Syed Z.

2010-01-01

Nonmuscle myosin light chain kinase (nmMLCK), a multi-functional cytoskeletal protein critical to vascular homeostasis, is highly regulated by tyrosine phosphorylation. We identified multiple novel c-Abl–mediated nmMLCK phosphorylation sites by mass spectroscopy analysis (including Y231, Y464, Y556, Y846) and examined their influence on nmMLCK function and human lung endothelial cell (EC) barrier regulation. Tyrosine phosphorylation of nmMLCK increased kinase activity, reversed nmMLCK-mediated inhibition of Arp2/3-mediated actin polymerization, and enhanced binding to the critical actin-binding phosphotyrosine protein, cortactin. EC challenge with sphingosine 1-phosphate (S1P), a potent barrier-enhancing agonist, resulted in c-Abl and phosphorylated nmMLCK recruitment into caveolin-enriched microdomains, rapid increases in Abl kinase activity, and spatial targeting of c-Abl to barrier-promoting cortical actin structures. Conversely, reduced c-Abl expression in EC (siRNA) markedly attenuated S1P-mediated cortical actin formation, reduced the EC modulus of elasticity (assessed by atomic force microscopy), reduced nmMLCK and cortactin tyrosine phosphorylation, and attenuated S1P-mediated barrier enhancement. These studies indicate an essential role for Abl kinase in vascular barrier regulation via posttranslational modification of nmMLCK and strongly support c-Abl-cortactin-nmMLCK interaction as a novel determinant of cortical actin-based cytoskeletal rearrangement critical to S1P-mediated EC barrier enhancement. PMID:20861316

Pericyte-derived sphingosine 1-phosphate induces the expression of adhesion proteins and modulates the retinal endothelial cell barrier.

PubMed

McGuire, Paul G; Rangasamy, Sampathkumar; Maestas, Joann; Das, Arup

2011-12-01

The mechanisms that regulate the physical interaction of pericytes and endothelial cells and the effects of these interactions on interendothelial cell junctions are not well understood. We determined the extent to which vascular pericytes could regulate pericyte-endothelial adhesion and the consequences that this disruption might have on the function of the endothelial barrier. Human retinal microvascular endothelial cells were cocultured with pericytes, and the effect on the monolayer resistance of endothelial cells and expression of the cell junction molecules N-cadherin and VE-cadherin were measured. The molecules responsible for the effect of pericytes or pericyte-conditioned media on the endothelial resistance and cell junction molecules were further analyzed. Our results indicate that pericytes increase the barrier properties of endothelial cell monolayers. This barrier function is maintained through the secretion of pericyte-derived sphingosine 1-phosphate. Sphingosine 1-phosphate aids in maintenance of microvascular stability by upregulating the expression of N-cadherin and VE-cadherin, and downregulating the expression of angiopoietin 2. Under normal circumstances, the retinal vascular pericytes maintain pericyte-endothelial contacts and vascular barrier function through the secretion of sphingosine 1-phosphate. Alteration of pericyte-derived sphingosine 1-phosphate production may be an important mechanism in the development of diseases characterized by vascular dysfunction and increased permeability.

Gliovascular and cytokine interactions modulate brain endothelial barrier in vitro.

PubMed

Chaitanya, Ganta V; Cromer, Walter E; Wells, Shannon R; Jennings, Merilyn H; Couraud, P Olivier; Romero, Ignacio A; Weksler, Babette; Erdreich-Epstein, Anat; Mathis, J Michael; Minagar, Alireza; Alexander, J Steven

2011-11-23

The glio-vascular unit (G-unit) plays a prominent role in maintaining homeostasis of the blood-brain barrier (BBB) and disturbances in cells forming this unit may seriously dysregulate BBB. The direct and indirect effects of cytokines on cellular components of the BBB are not yet unclear. The present study compares the effects of cytokines and cytokine-treated astrocytes on brain endothelial barrier. 3-dimensional transwell co-cultures of brain endothelium and related-barrier forming cells with astrocytes were used to investigate gliovascular barrier responses to cytokines during pathological stresses. Gliovascular barrier was measured using trans-endothelial electrical resistance (TEER), a sensitive index of in vitro barrier integrity. We found that neither TNF-α, IL-1β or IFN-γ directly reduced barrier in human or mouse brain endothelial cells or ECV-304 barrier (independent of cell viability/metabolism), but found that astrocyte exposure to cytokines in co-culture significantly reduced endothelial (and ECV-304) barrier. These results indicate that the barrier established by human and mouse brain endothelial cells (and other cells) may respond positively to cytokines alone, but that during pathological conditions, cytokines dysregulate the barrier forming cells indirectly through astrocyte activation involving reorganization of junctions, matrix, focal adhesion or release of barrier modulating factors (e.g. oxidants, MMPs). © 2011 Chaitanya et al; licensee BioMed Central Ltd.

Ascorbate protects endothelial barrier function during septic insult: Role of protein phosphatase type 2A.

PubMed

Han, Min; Pendem, Suresh; Teh, Suet Ling; Sukumaran, Dinesh K; Wu, Feng; Wilson, John X

2010-01-01

Endothelial barrier dysfunction contributes to morbidity in sepsis. We tested the hypothesis that raising the intracellular ascorbate concentration protects the endothelial barrier from septic insult by inhibiting protein phosphatase type 2A. Monolayer cultures of microvascular endothelial cells were incubated with ascorbate, dehydroascorbic acid (DHAA), the NADPH oxidase inhibitors apocynin and diphenyliodonium, or the PP2A inhibitor okadaic acid and then were exposed to septic insult (lipopolysaccharide and interferon-gamma). Under standard culture conditions that depleted intracellular ascorbate, septic insult stimulated oxidant production and PP2A activity, dephosphorylated phosphoserine and phosphothreonine residues in the tight junction-associated protein occludin, decreased the abundance of occludin at cell borders, and increased monolayer permeability to albumin. NADPH oxidase inhibitors prevented PP2A activation and monolayer leak, showing that these changes required reactive oxygen species. Okadaic acid, at a concentration that inhibited PP2A activity and monolayer leak, prevented occludin dephosphorylation and redistribution, implicating PP2A in the response of occludin to septic insult. Incubation with ascorbate or DHAA raised intracellular ascorbate concentrations and mitigated the effects of septic insult. In conclusion, ascorbate acts within microvascular endothelial cells to inhibit septic stimulation of oxidant production by NADPH oxidase and thereby prevents PP2A activation, PP2A-dependent dephosphorylation and redistribution of occludin, and disruption of the endothelial barrier. Copyright 2009 Elsevier Inc. All rights reserved.

Cancer cells remodel themselves and vasculature to overcome the endothelial barrier.

PubMed

Shenoy, Anitha K; Lu, Jianrong

2016-10-01

Metastasis refers to the spread of cancer cells from a primary tumor to distant organs mostly via the bloodstream. During the metastatic process, cancer cells invade blood vessels to enter circulation, and later exit the vasculature at a distant site. Endothelial cells that line blood vessels normally serve as a barrier to the movement of cells into or out of the blood. It is thus critical to understand how metastatic cancer cells overcome the endothelial barrier. Epithelial cancer cells acquire increased motility and invasiveness through epithelial-to-mesenchymal transition (EMT), which enables them to move toward vasculature. Cancer cells also express a variety of adhesion molecules that allow them to attach to vascular endothelium. Finally, cancer cells secrete or induce growth factors and cytokines to actively prompt vascular hyperpermeability that compromises endothelial barrier function and facilitates transmigration of cancer cells through the vascular wall. Elucidation of the mechanisms underlying metastatic dissemination may help develop new anti-metastasis therapeutics. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Edaravone Protects against Methylglyoxal-Induced Barrier Damage in Human Brain Endothelial Cells

PubMed Central

Tóth, Andrea E.; Walter, Fruzsina R.; Bocsik, Alexandra; Sántha, Petra; Veszelka, Szilvia; Nagy, Lajos; Puskás, László G.; Couraud, Pierre-Olivier; Takata, Fuyuko; Dohgu, Shinya; Kataoka, Yasufumi; Deli, Mária A.

2014-01-01

Background Elevated level of reactive carbonyl species, such as methylglyoxal, triggers carbonyl stress and activates a series of inflammatory responses leading to accelerated vascular damage. Edaravone is the active substance of a Japanese medicine, which aids neurological recovery following acute brain ischemia and subsequent cerebral infarction. Our aim was to test whether edaravone can exert a protective effect on the barrier properties of human brain endothelial cells (hCMEC/D3 cell line) treated with methylglyoxal. Methodology Cell viability was monitored in real-time by impedance-based cell electronic sensing. The barrier function of the monolayer was characterized by measurement of resistance and flux of permeability markers, and visualized by immunohistochemistry for claudin-5 and β-catenin. Cell morphology was also examined by holographic phase imaging. Principal Findings Methylglyoxal exerted a time- and dose-dependent toxicity on cultured human brain endothelial cells: a concentration of 600 µM resulted in about 50% toxicity, significantly reduced the integrity and increased the permeability of the barrier. The cell morphology also changed dramatically: the area of cells decreased, their optical height significantly increased. Edaravone (3 mM) provided a complete protection against the toxic effect of methylglyoxal. Co-administration of edaravone restored cell viability, barrier integrity and functions of brain endothelial cells. Similar protection was obtained by the well-known antiglycating molecule, aminoguanidine, our reference compound. Conclusion These results indicate for the first time that edaravone is protective in carbonyl stress induced barrier damage. Our data may contribute to the development of compounds to treat brain endothelial dysfunction in carbonyl stress related diseases. PMID:25033388

Pericyte Derived Sphinogosine 1-Phosphate Induces the Expression of Adhesion Proteins and Modulates the Retinal Endothelial Cell Barrier

PubMed Central

McGuire, P.G.; Rangasamy, S.; Maestas, J.; Das, A.

2011-01-01

Objective The mechanisms that regulate the physical interaction of pericytes and endothelial cells and the effects of these interactions on interendothelial cell junctions are not well understood. We determined the extent to which vascular pericytes could regulate pericyte-endothelial adhesion and the consequences that this disruption might have on the function of the endothelial barrier. Methods and Results Human retinal microvascular endothelial cells were co-cultured with pericytes, and the effect on the monolayer resistance of endothelial cells and expression of the cell junction molecules N-cadherin and VE-cadherin were measured. The molecules responsible for the effect of pericytes or pericyte conditioned media on the endothelial resistance and cell junction molecules were further analyzed. Our results indicate that pericytes increase the barrier properties of endothelial cell monolayers. This barrier function is maintained through the secretion of pericyte-derived sphingosine 1-phosphate (S1P). S1P aids in maintenance of microvascular stability by up-regulating the expression of N-cadherin and VE-cadherin, and down-regulating the expression of angiopoietin 2. Conclusion Under normal circumstances, the retinal vascular pericytes maintain pericyte-endothelial contacts and vascular barrier function through the secretion of S1P. Alteration of pericyte-derived S1P production may be an important mechanism in the development of diseases characterized by vascular dysfunction and increased permeability. PMID:21940944

Three-dimensional culture conditions differentially affect astrocyte modulation of brain endothelial barrier function in response to transforming growth factor β1.

PubMed

Hawkins, Brian T; Grego, Sonia; Sellgren, Katelyn L

2015-05-22

Blood-brain barrier (BBB) function is regulated by dynamic interactions among cell types within the neurovascular unit, including astrocytes and endothelial cells. Co-culture models of the BBB typically involve astrocytes seeded on two-dimensional (2D) surfaces, which recent studies indicate cause astrocytes to express a phenotype similar to that of reactive astrocytes in situ. We hypothesized that the culture conditions of astrocytes would differentially affect their ability to modulate BBB function in vitro. Brain endothelial cells were grown alone or in co-culture with astrocytes. Astrocytes were grown either as conventional (2D) monolayers, or in a collagen-based gel which allows them to grow in a three-dimensional (3D) construct. Astrocytes were viable in 3D conditions, and displayed a marked reduction in their expression of glial fibrillary acidic protein (GFAP), suggesting reduced activation. Stimulation of astrocytes with transforming growth factor (TGF)β1 decreased transendothelial electrical resistance (TEER) and reduced expression of claudin-5 in co-cultures, whereas treatment of endothelial cells in the absence of astrocytes was without effect. The effect of TGFβ1 on TEER was significantly more pronounced in endothelial cells cultured with 3D astrocytes compared to 2D astrocytes. These results demonstrate that astrocyte culture conditions differentially affect their ability to modulate brain endothelial barrier function, and suggest a direct relationship between reactive gliosis and BBB permeability. Moreover, these studies demonstrate the potential importance of physiologically relevant culture conditions to in vitro modeling of disease processes that affect the neurovascular unit. Copyright © 2015 Elsevier B.V. All rights reserved.

Contacting co-culture of human retinal microvascular endothelial cells alters barrier function of human embryonic stem cell derived retinal pigment epithelial cells.

PubMed

Skottman, H; Muranen, J; Lähdekorpi, H; Pajula, E; Mäkelä, K; Koivusalo, L; Koistinen, A; Uusitalo, H; Kaarniranta, K; Juuti-Uusitalo, K

2017-10-01

Here we evaluated the effects of human retinal microvascular endothelial cells (hREC) on mature human embryonic stem cell (hESC) derived retinal pigment epithelial (RPE) cells. The hESC-RPE cells (Regea08/017, Regea08/023 or Regea11/013) and hREC (ACBRI 181) were co-cultured on opposite sides of transparent membranes for up to six weeks. Thereafter barrier function, small molecule permeability, localization of RPE and endothelial cell marker proteins, cellular fine structure, and growth factor secretion of were evaluated. After co-culture, the RPE specific CRALBP and endothelial cell specific von Willebrand factor were appropriately localized. In addition, the general morphology, pigmentation, and fine structure of hESC-RPE cells were unaffected. Co-culture increased the barrier function of hESC-RPE cells, detected both with TEER measurements and cumulative permeability of FD4 - although the differences varied among the cell lines. Co-culturing significantly altered VEGF and PEDF secretion, but again the differences were cell line specific. The results of this study showed that co-culture with hREC affects hESC-RPE functionality. In addition, co-culture revealed drastic cell line specific differences, most notably in growth factor secretion. This model has the potential to be used as an in vitro outer blood-retinal barrier model for drug permeability testing. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Lipid rafts regulate PCB153-induced disruption of occludin and brain endothelial barrier function through protein phosphatase 2A and matrix metalloproteinase-2

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

Eum, Sung Yong, E-mail: seum@miami.edu; Jaraki, Dima; András, Ibolya E.

Occludin is an essential integral transmembrane protein regulating tight junction (TJ) integrity in brain endothelial cells. Phosphorylation of occludin is associated with its localization to TJ sites and incorporation into intact TJ assembly. The present study is focused on the role of lipid rafts in polychlorinated biphenyl (PCB)-induced disruption of occludin and endothelial barrier function. Exposure of human brain endothelial cells to 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153) induced dephosphorylation of threonine residues of occludin and displacement of occludin from detergent-resistant membrane (DRM)/lipid raft fractions within 1 h. Moreover, lipid rafts modulated the reduction of occludin level through activation of matrix metalloproteinase 2 (MMP-2)more » after 24 h PCB153 treatment. Inhibition of protein phosphatase 2A (PP2A) activity by okadaic acid or fostriecin markedly protected against PCB153-induced displacement of occludin and increased permeability of endothelial cells. The implication of lipid rafts and PP2A signaling in these processes was further defined by co-immunoprecipitation of occludin with PP2A and caveolin-1, a marker protein of lipid rafts. Indeed, a significant MMP-2 activity was observed in lipid rafts and was increased by exposure to PCB153. The pretreatment of MMP-2 inhibitors protected against PCB153-induced loss of occludin and disruption of lipid raft structure prevented the increase of endothelial permeability. Overall, these results indicate that lipid raft-associated processes, such as PP2A and MMP-2 activation, participate in PCB153-induced disruption of occludin function in brain endothelial barrier. This study contributes to a better understanding of the mechanisms leading to brain endothelial barrier dysfunction in response to exposure to environmental pollutants, such as ortho-substituted PCBs. - Highlights: • PCB153 disturbed human brain endothelial barrier through disruption of occludin. • Lipid raft

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

The endothelial glycocalyx

PubMed Central

Yang, Yimu; Schmidt, Eric P.

2013-01-01

Once thought to be a structure of small size and uncertain significance, the endothelial glycocalyx is now known to be an important regulator of endothelial function. Studies of the systemic vasculature have demonstrated that the glycocalyx forms a substantial in vivo endothelial surface layer (ESL) critical to inflammation, barrier function and mechanotransduction. The pulmonary ESL is significantly thicker than the systemic ESL, suggesting unique physiologic function. We have recently demonstrated that the pulmonary ESL regulates exposure of endothelial surface adhesion molecules, thereby serving as a barrier to neutrophil adhesion and extravasation. While the pulmonary ESL is not a critical structural component of the endothelial barrier to fluid and protein, it serves a major role in the mechanotransduction of vascular pressure, with impact on the active regulation of endothelial permeability. It is likely that the ESL serves numerous additional functions in vascular physiology, representing a fertile area for future investigation. PMID:24073386

microRNAs as Pharmacological Targets in Endothelial Cell Function and Dysfunction

PubMed Central

Chamorro-Jorganes, Aránzazu; Araldi, Elisa; Suárez, Yajaira

2013-01-01

Endothelial cell dysfunction is a term which implies the dysregulation of normal endothelial cell functions, including impairment of the barrier functions, control of vascular tone, disturbance of proliferative, migratory and morphogenic capacities of endothelial cells, as well as control of leukocyte trafficking. MicroRNAs (miRNAs) are short non-coding RNAs that have emerged as critical regulators of gene expression acting predominantly at the post-transcriptional level. This review summarizes the latest insights in the identification of endothelial-specific miRNAs and their targets, as well as their roles in controlling endothelial cell functions in both autocrine and paracrine manner. In addition, we discuss the therapeutic potential for the treatment of endothelial cell dysfunction and associated vascular pathophysiological conditions. PMID:23603154

Hypoxia/Aglycemia-Induced Endothelial Barrier Dysfunction and Tight Junction Protein Downregulation Can Be Ameliorated by Citicoline

PubMed Central

Pan, Qunwen; Zhao, Yuhui; Chen, Ji; Zhao, Bin; Chen, Yanfang

2013-01-01

This study explores the effect of citicoline on the permeability and expression of tight junction proteins (TJPs) in endothelial cells under hypoxia/aglycemia conditions. Hypoxia or oxygen and glucose deprivation (OGD) was utilized to induce endothelial barrier breakdown model on human umbilical vein endothelial cells (HUVECs) and mouse brain microvascular endothelial cells (bEnd.3s). The effect of citicoline on endothelial barrier breakdown models was determined at either low or high concentrations. FITC-Dextran flux was used to examine the endothelial permeability. The expression of TJPs was measured by immunofluorescence, Real-time PCR and Western Blot methods. Results showed that hypoxia or OGD increased the permeability of HUVECs accompanied with down-regulation of occludens-1 (ZO-1) and occludin at both mRNA and protein levels. Similarly in bEnd.3s, hypoxia increased the permeability and decreased the expression of ZO-1 and claudin-5. Citicoline treatment dose-dependently decreased the permeability in these two models, which paralleled with elevated expression of TJPs. The data demonstrate that citicoline restores the barrier function of endothelial cells compromised by hypoxia/aglycemia probably via up-regulating the expression of TJPs. PMID:24358213

Intracellular Ascorbate Prevents Endothelial Barrier Permeabilization by Thrombin.

PubMed

Parker, William H; Qu, Zhi-chao; May, James M

2015-08-28

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. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Crossing the endothelial barrier during metastasis.

PubMed

Reymond, Nicolas; d'Água, Bárbara Borda; Ridley, Anne J

2013-12-01

During metastasis, cancer cells disseminate to other parts of the body by entering the bloodstream in a process that is called intravasation. They then extravasate at metastatic sites by attaching to endothelial cells that line blood vessels and crossing the vessel walls of tissues or organs. This Review describes how cancer cells cross the endothelial barrier during extravasation and how different receptors, signalling pathways and circulating cells such as leukocytes and platelets contribute to this process. Identification of the mechanisms that underlie cancer cell extravasation could lead to the development of new therapies to reduce metastasis.

Mechanisms of pertussis toxin-induced barrier dysfunction in bovine pulmonary artery endothelial cell monolayers.

PubMed

Patterson, C E; Stasek, J E; Schaphorst, K L; Davis, H W; Garcia, J G

1995-06-01

We have previously characterized several G proteins in endothelial cells (EC) as substrates for the ADP-ribosyltransferase activity of both pertussis (PT) and cholera toxin and described the modulation of key EC physiological responses, including gap formation and barrier function, by these toxins. In this study, we investigated the mechanisms involved in PT-mediated regulation of bovine pulmonary artery endothelial cells barrier function. PT caused a dose-dependent increase in albumin transfer, dependent upon action of the holotoxin, since neither the heat-inactivated PT, the isolated oligomer, nor the protomer induced EC permeability. PT-induced gap formation and barrier dysfunction were additive to either thrombin- or thrombin receptor-activating peptide-induced permeability, suggesting that thrombin and PT utilize distinct mechanisms. PT did not result in Ca2+ mobilization or alter either basal or thrombin-induced myosin light chain phosphorylation. However, PT stimulated protein kinase C (PKC) activation, and both PKC downregulation and PKC inhibition attenuated PT-induced permeability, indicating that PKC activity is involved in PT-induced barrier dysfunction. Like thrombin-induced permeability, the PT effect was blocked by prior increases in adenosine 3',5'-cyclic monophosphate. Thus PT-catalyzed ADP-ribosylation of a G protein (possibly other than Gi) may regulate cytoskeletal protein interactions, leading to EC barrier dysfunction.

Immortalized endothelial cell lines for in vitro blood-brain barrier models: A systematic review.

PubMed

Rahman, Nurul Adhwa; Rasil, Alifah Nur'ain Haji Mat; Meyding-Lamade, Uta; Craemer, Eva Maria; Diah, Suwarni; Tuah, Ani Afiqah; Muharram, Siti Hanna

2016-07-01

Endothelial cells play the most important role in construction of the blood-brain barrier. Many studies have opted to use commercially available, easily transfected or immortalized endothelial cell lines as in vitro blood-brain barrier models. Numerous endothelial cell lines are available, but we do not currently have strong evidence for which cell lines are optimal for establishment of such models. This review aimed to investigate the application of immortalized endothelial cell lines as in vitro blood-brain barrier models. The databases used for this review were PubMed, OVID MEDLINE, ProQuest, ScienceDirect, and SpringerLink. A narrative systematic review was conducted and identified 155 studies. As a result, 36 immortalized endothelial cell lines of human, mouse, rat, porcine and bovine origins were found for the establishment of in vitro blood-brain barrier and brain endothelium models. This review provides a summary of immortalized endothelial cell lines as a guideline for future studies and improvements in the establishment of in vitro blood-brain barrier models. It is important to establish a good and reproducible model that has the potential for multiple applications, in particular a model of such a complex compartment such as the blood-brain barrier. Copyright © 2016 Elsevier B.V. All rights reserved.

Microcapsules functionalized with neuraminidase can enter vascular endothelial cells in vitro

PubMed Central

Liu, Weizhi; Wang, Xiaocong; Bai, Ke; Lin, Miao; Sukhorukov, Gleb; Wang, Wen

2014-01-01

Microcapsules made of polyelectrolyte multilayers exhibit no or low toxicity, appropriate mechanical stability, variable controllable degradation and can incorporate remote release mechanisms triggered by various stimuli, making them well suited for targeted drug delivery to live cells. This study investigates interactions between microcapsules made of synthetic (i.e. polystyrenesulfonate sodium salt/polyallylamine hydrochloride) or natural (i.e. dextran sulfate/poly-l-arginine) polyelectrolyte and human umbilical vein endothelial cells with particular focus on the effect of the glycocalyx layer on the intake of microcapsules by endothelial cells. Neuraminidase cleaves N-acetyl neuraminic acid residues of glycoproteins and targets the sialic acid component of the glycocalyx on the cell membrane. Three-dimensional confocal images reveal that microcapsules, functionalized with neuraminidase, can be internalized by endothelial cells. Capsules without neuraminidase are blocked by the glycocalyx layer. Uptake of the microcapsules is most significant in the first 2 h. Following their internalization by endothelial cells, biodegradable DS/PArg capsules rupture by day 5; however, there is no obvious change in the shape and integrity of PSS/PAH capsules within the period of observation. Results from the study support our hypothesis that the glycocalyx functions as an endothelial barrier to cross-membrane movement of microcapsules. Neuraminidase-loaded microcapsules can enter endothelial cells by localized cleavage of glycocalyx components with minimum disruption of the glycocalyx layer and therefore have high potential to act as drug delivery vehicles to reach tissues beyond the endothelial barrier of blood vessels. PMID:25339691

Astrocytes increase barrier properties and ZO-1 expression in retinal vascular endothelial cells.

PubMed

Gardner, T W; Lieth, E; Khin, S A; Barber, A J; Bonsall, D J; Lesher, T; Rice, K; Brennan, W A

1997-10-01

Diabetic retinopathy and other diseases associated with retinal edema are characterized by increased microvascular leakage. Astrocytes have been proposed to maintain endothelial function in the brain, suggesting that glial impairment may underlie the development of retinal edema. The purpose of this study was to test the effects of astrocytes on barrier properties in retinal microvascular endothelial cells. Bovine retinal microvascular endothelial cells were exposed to conditioned media from rat brain astrocytes. Transendothelial electrical resistance (TER) was determined on 24-mm Transwell (Cambridge, MA) polycarbonate filters with the End-Ohm device (World Precision Instruments, Sarasota, FL). ZO-1 protein content was quantified by microtiter enzyme-linked immunosorbent assay. Astrocyte-conditioned medium (ACM) significantly increased TER (P < 0.0001) and ZO-1 content (P < 0.01). Both serum-containing and serum-free N1B defined ACM increased ZO-1 expression, but heating abolished the effect. Serum-free ACM decreased cell proliferation by 16%. Astrocytes release soluble, heat-labile factors that increase barrier properties and tight junction protein content. These results suggest that astrocytes enhance blood-retinal barrier properties, at least in part by increasing tight junction protein expression. Our findings suggest that glial malfunction plays an important role in the pathogenesis of vasogenic retinal edema.

Inflammatory Responses and Barrier Function of Endothelial Cells Derived from Human Induced Pluripotent Stem Cells.

PubMed

Halaidych, Oleh V; Freund, Christian; van den Hil, Francijna; Salvatori, Daniela C F; Riminucci, Mara; Mummery, Christine L; Orlova, Valeria V

2018-05-08

Several studies have reported endothelial cell (EC) derivation from human induced pluripotent stem cells (hiPSCs). However, few have explored their functional properties in depth with respect to line-to-line and batch-to-batch variability and how they relate to primary ECs. We therefore carried out accurate characterization of hiPSC-derived ECs (hiPSC-ECs) from multiple (non-integrating) hiPSC lines and compared them with primary ECs in various functional assays, which included barrier function using real-time impedance spectroscopy with an integrated assay of electric wound healing, endothelia-leukocyte interaction under physiological flow to mimic inflammation and angiogenic responses in in vitro and in vivo assays. Overall, we found many similarities but also some important differences between hiPSC-derived and primary ECs. Assessment of vasculogenic responses in vivo showed little difference between primary ECs and hiPSC-ECs with regard to functional blood vessel formation, which may be important in future regenerative medicine applications requiring vascularization. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

Intracellular ascorbate tightens the endothelial permeability barrier through Epac1 and the tubulin cytoskeleton

PubMed Central

Parker, William H.; Rhea, Elizabeth Meredith; Qu, Zhi-Chao; Hecker, Morgan R.

2016-01-01

Vitamin C, or ascorbic acid, both tightens the endothelial permeability barrier in basal cells and also prevents barrier leak induced by inflammatory agents. Barrier tightening by ascorbate in basal endothelial cells requires nitric oxide derived from activation of nitric oxide synthase. Although ascorbate did not affect cyclic AMP levels in our previous study, there remains a question of whether it might activate downstream cyclic AMP-dependent pathways. In this work, we found in both primary and immortalized cultured endothelial cells that ascorbate tightened the endothelial permeability barrier by ∼30%. In human umbilical vein endothelial cells, this occurred at what are likely physiologic intracellular ascorbate concentrations. In so doing, ascorbate decreased measures of oxidative stress and also flattened the cells to increase cell-to-cell contact. Inhibition of downstream cyclic AMP-dependent proteins via protein kinase A did not prevent ascorbate from tightening the endothelial permeability barrier, whereas inhibition of Epac1 did block the ascorbate effect. Although Epac1 was required, its mediator Rap1 was not activated. Furthermore, ascorbate acutely stabilized microtubules during depolymerization induced by colchicine and nocodazole. Over several days in culture, ascorbate also increased the amount of stable acetylated α-tubulin. Microtubule stabilization was further suggested by the finding that ascorbate increased the amount of Epac1 bound to α-tubulin. These results suggest that physiologic ascorbate concentrations tighten the endothelial permeability barrier in unstimulated cells by stabilizing microtubules in a manner downstream of cyclic AMP that might be due both to increasing nitric oxide availability and to scavenging of reactive oxygen or nitrogen species. PMID:27605450

In vitro studies of the blood-brain barrier using isolated brain capillaries and cultured endothelial cells.

PubMed

Goldstein, G W; Betz, A L; Bowman, P D; Dorovini-Zis, K

1986-01-01

The endothelial cells in brain capillaries are the anatomic site of the blood-brain barrier. To learn more about the biology of these specialized cells, we developed methods to prepare suspensions of purified brain microvessels as well as primary cultures of endothelial cells in monolayer. These two preparations allow for direct investigation of the metabolism, transport properties, and receptor content of the brain capillary. We used isolated brain microvessels to study distribution of membrane carriers between the luminal and the abluminal plasma membrane of endothelial cells. We found that Na+K+-ATPase and the A-system amino-acid transport system are located predominantly on the abluminal surface of brain capillary endothelial cells. This distribution of transport carriers is consistent with the low permeability of potassium and small neutral amino acids in the blood-to-brain direction. It suggests, however, that both solutes can be actively transported across brain capillaries from the brain interstitial fluid to the blood. In tissue culture, the endothelial cells form continuous tight junctions with their neighbors. This results in a cellular layer impermeable to protein tracers. When exposed to hyperosmolar solutions, in an attempt to mimic the conditions that open the blood-brain barrier in vivo, we found a reversible separation of the tight junctions between contiguous endothelial cells. No indication of activation of pinocytosis was observed. In vitro systems provide a novel approach for studying the function of the blood-brain barrier and allow for observations not possible with intact animals.

HSP27 phosphorylation protects against endothelial barrier dysfunction under burn serum challenge.

PubMed

Sun, Huan-bo; Ren, Xi; Liu, Jie; Guo, Xiao-wei; Jiang, Xu-pin; Zhang, Dong-xia; Huang, Yue-sheng; Zhang, Jia-ping

2015-07-31

F-actin rearrangement is an early event in burn-induced endothelial barrier dysfunction. HSP27, a target of p38 MAPK/MK2 pathway, plays an important role in actin dynamics through phosphorylation. The question of whether HSP27 participates in burn-related endothelial barrier dysfunction has not been identified yet. Here, we showed that burn serum induced a temporal appearance of central F-actin stress fibers followed by a formation of irregular dense peripheral F-actin in pulmonary endothelial monolayer, concomitant with a transient increase of HSP27 phosphorylation that conflicted with the persistent activation of p38 MAPK/MK2 unexpectedly. The appearance of F-actin stress fibers and transient increase of HSP27 phosphorylation occurred prior to the burn serum-induced endothelial hyperpermeability. Overexpressing phospho-mimicking HSP27 (HSP27(Asp)) reversed the burn serum-induced peripheral F-actin rearrangement with the augmentation of central F-actin stress fibers, and more importantly, attenuated the burn serum-induced endothelial hyperpermeability; such effects were not observed by HSP27(Ala), a non-phosphorylated mutant of HSP27. HSP27(Asp) overexpression also rendered the monolayer more resistant to barrier disruption caused by Cytochalasin D, a chemical reagent that depolymerizes F-actin specifically. Further study showed that phosphatases and sumoylation-inhibited MK2 activity contributed to the blunting of HSP27 phosphorylation during the burn serum-induced endothelial hyperpermeability. Our study identifies HSP27 phosphorylation as a protective response against burn serum-induced endothelial barrier dysfunction, and suggests that targeting HSP27 wound be a promising therapeutic strategy in ameliorating burn-induced lung edema and shock development. Copyright © 2015 Elsevier Inc. All rights reserved.

Tight junction protein expression and barrier properties of immortalized mouse brain microvessel endothelial cells.

PubMed

Brown, Rachel C; Morris, Andrew P; O'Neil, Roger G

2007-01-26

Understanding the molecular and biochemical mechanisms regulating the blood-brain barrier is aided by in vitro model systems. Many studies have used primary cultures of brain microvessel endothelial cells for this purpose. However, primary cultures limit the generation of material for molecular and biochemical assays since cells grow slowly, are prone to contamination by other neurovascular unit cells, and lose blood-brain barrier characteristics when passaged. To address these issues, immortalized cell lines have been generated. In these studies, we assessed the suitability of the immortalized mouse brain endothelial cell line, bEnd3, as a blood-brain barrier model. RT-PCR and immunofluorescence indicated expression of multiple tight junction proteins. bEnd3 cells formed barriers to radiolabeled sucrose, and responded like primary cultures to disrupting stimuli. Exposing cells to serum-free media on their basolateral side significantly decreased paracellular permeability; astrocyte-conditioned media did not enhance barrier properties. The serum-free media-induced decrease in permeability was correlated with an increase in claudin-5 and zonula occludens-1 immunofluorescence at cell-cell contracts. We conclude that bEnd3 cells are an attractive candidate as a model of the blood-brain barrier due to their rapid growth, maintenance of blood-brain barrier characteristics over repeated passages, formation of functional barriers and amenability to numerous molecular interventions.

TIGHT JUNCTION PROTEIN EXPRESSION AND BARRIER PROPERTIES OF IMMORTALIZED MOUSE BRAIN MICROVESSEL ENDOTHELIAL CELLS

PubMed Central

Brown, Rachel C.; Morris, Andrew P.; O’Neil, Roger G.

2007-01-01

Understanding the molecular and biochemical mechanisms regulating the blood-brain barrier is aided by in vitro model systems. Many studies have used primary cultures of brain microvessel endothelial cells for this purpose. However, primary cultures limit the generation of material for molecular and biochemical assays since cells grow slowly, are prone to contamination by other neurovascular unit cells, and lose blood-brain barrier characteristics when passaged. To address these issues, immortalized cell lines have been generated. In these studies, we assessed the suitability of the immortalized mouse brain endothelial cell line, bEnd3, as a blood-brain barrier model. RT-PCR and immunofluorescence indicated expression of multiple tight junction proteins. bEnd3 cells formed barriers to radiolabeled sucrose, and responded like primary cultures to disrupting stimuli. Exposing cells to serum-free media on their basolateral side significantly decreased paracellular permeability; astrocyte-conditioned media did not enhance barrier properties. The serum-free media-induced decrease in permeability was correlated with an increase in claudin-5 and zonula occludens-1 immunofluorescence at cell-cell contracts. We conclude that bEnd3 cells are an attractive candidate as a model of the blood-brain barrier due to their rapid growth, maintenance of blood-brain barrier characteristics over repeated passages, formation of functional barriers and amenability to numerous molecular interventions. PMID:17169347

Expression of PKA inhibitor (PKI) gene abolishes cAMP-mediated protection to endothelial barrier dysfunction.

PubMed

Lum, H; Jaffe, H A; Schulz, I T; Masood, A; RayChaudhury, A; Green, R D

1999-09-01

We investigated the hypothesis that cAMP-dependent protein kinase (PKA) protects against endothelial barrier dysfunction in response to proinflammatory mediators. An E1-, E3-, replication-deficient adenovirus (Ad) vector was constructed containing the complete sequence of PKA inhibitor (PKI) gene (AdPKI). Infection of human microvascular endothelial cells (HMEC) with AdPKI resulted in overexpression of PKI. Treatment with 0.5 microM thrombin increased transendothelial albumin clearance rate (0.012 +/- 0.003 and 0.035 +/- 0.005 microl/min for control and thrombin, respectively); the increase was prevented with forskolin + 3-isobutyl-1-methylxanthine (F + I) treatment. Overexpression of PKI resulted in abrogation of the F + I-induced inhibition of the permeability increase. However, with HMEC infected with ultraviolet-inactivated AdPKI, the F + I-induced inhibition was present. Also, F + I treatment of HMEC transfected with reporter plasmid containing the cAMP response element-directed transcription of the luciferase gene resulted in an almost threefold increase in luciferase activity. Overexpression of PKI inhibited this induction of luciferase activity. The results show that Ad-mediated overexpression of PKI in endothelial cells abrogated the cAMP-mediated protection against increased endothelial permeability, providing direct evidence that cAMP-dependent protein kinase promotes endothelial barrier function.

Bicarbonate disruption of the pulmonary endothelial barrier via activation of endogenous soluble adenylyl cyclase, isoform 10

PubMed Central

Obiako, Boniface; Calchary, Wendy; Xu, Ningyong; Kunstadt, Ryan; Richardson, Bianca; Nix, Jessica

2013-01-01

It is becoming increasingly apparent that cAMP signals within the pulmonary endothelium are highly compartmentalized, and this compartmentalization is critical to maintaining endothelial barrier integrity. Studies demonstrate that the exogenous soluble bacterial toxin, ExoY, and heterologous expression of the forskolin-stimulated soluble mammalian adenylyl cyclase (AC) chimera, sACI/II, elevate cytosolic cAMP and disrupt the pulmonary microvascular endothelial barrier. The barrier-disruptive effects of cytosolic cAMP generated by exogenous soluble ACs are in contrast to the barrier-protective effects of subplasma membrane cAMP generated by transmembrane AC, which strengthens endothelial barrier integrity. Endogenous soluble AC isoform 10 (AC10 or commonly known as sAC) lacks transmembrane domains and localizes within the cytosolic compartment. AC10 is uniquely activated by bicarbonate to generate cytosolic cAMP, yet its role in regulation of endothelial barrier integrity has not been addressed. Here we demonstrate that, within the pulmonary circulation, AC10 is expressed in pulmonary microvascular endothelial cells (PMVECs) and pulmonary artery endothelial cells (PAECs), yet expression in PAECs is lower. Furthermore, pulmonary endothelial cells selectively express bicarbonate cotransporters. While extracellular bicarbonate generates a phosphodiesterase 4-sensitive cAMP pool in PMVECs, no such cAMP response is detected in PAECs. Finally, addition of extracellular bicarbonate decreases resistance across the PMVEC monolayer and increases the filtration coefficient in the isolated perfused lung above osmolality controls. Collectively, these findings suggest that PMVECs have a bicarbonate-sensitive cytosolic cAMP pool that disrupts endothelial barrier integrity. These studies could provide an alternative mechanism for the controversial effects of bicarbonate correction of acidosis of acute respiratory distress syndrome patients. PMID:23686854

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

Dengue Virus Infection Differentially Regulates Endothelial Barrier Function over Time through Type I Interferon Effects

PubMed Central

Liu, Ping; Woda, Marcia; Ennis, Francis A.; Libraty, Daniel H.

2013-01-01

Background The morbidity and mortality resulting from dengue hemorrhagic fever (DHF) are largely caused by endothelial barrier dysfunction and a unique vascular leakage syndrome. The mechanisms that lead to the location and timing of vascular leakage in DHF are poorly understood. We hypothesized that direct viral effects on endothelial responsiveness to inflammatory and angiogenesis mediators can explain the DHF vascular leakage syndrome. Methods We used an in vitro model of human endothelium to study the combined effects of dengue virus (DENV) type 2 (DENV2) infection and inflammatory mediators on paracellular macromolecule permeability over time. Results Over the initial 72 h after infection, DENV2 suppressed tumor necrosis factor (TNF)–α–mediated hyperpermeability in human umbilical vein endothelial cell (HUVEC) monolayers. This suppressive effect was mediated by type I interferon (IFN). By 1 week, TNF-α stimulation of DENV2-infected HUVECs synergistically increased cell cycling, angiogenic changes, and macromolecule permeability. This late effect could be prevented by the addition of exogenous type I IFN. Conclusions DENV infection of primary human endothelial cells differentially modulates TNF-α–driven angiogenesis and hyperpermeability over time. Type I IFN plays a central role in this process. Our findings suggest a rational model for the DHF vascular leakage syndrome. PMID:19530939

Lysophosphatidic acid receptor, LPA6, regulates endothelial blood-brain barrier function: Implication for hepatic encephalopathy.

PubMed

Masago, Kayo; Kihara, Yasuyuki; Yanagida, Keisuke; Hamano, Fumie; Nakagawa, Shinsuke; Niwa, Masami; Shimizu, Takao

2018-07-02

Cerebral edema is a life-threatening neurological condition characterized by brain swelling due to the accumulation of excess fluid both intracellularly and extracellularly. Fulminant hepatic failure (FHF) develops cerebral edema by disrupting blood-brain barrier (BBB). However, the mechanisms by which mediator induces brain edema in FHF remain to be elucidated. Here, we assessed a linkage between brain edema and lysophosphatidic acid (LPA) signaling by utilizing an animal model of FHF and in vitro BBB model. Azoxymethane-treated mice developed FHF and hepatic encephalopathy, associated with higher autotaxin (ATX) activities in serum than controls. Using in vitro BBB model, LPA disrupted the structural integrity of tight junction proteins including claudin-5, occludin, and ZO-1. Furthermore, LPA decreased transendothelial electrical resistances in in vitro BBB model, and induced cell contraction in brain endothelial monolayer cultures, both being inhibited by a Rho-associated protein kinase inhibitor, Y-27632. The brain capillary endothelial cells predominantly expressed LPA 6 mRNA, whose knockdown blocked the LPA-induced endothelial cell contraction. Taken together, the up-regulation of serum ATX in hepatic encephalopathy may activate the LPA-LPA 6 -G 12/13 -Rho pathway in brain capillary endothelial cells, leading to enhancement of BBB permeability and brain edema. Copyright © 2018 Elsevier Inc. All rights reserved.

Membrane organization determines barrier properties of endothelial cells and short-chain sphingolipid-facilitated doxorubicin influx.

PubMed

van Hell, A J; Klymchenko, A; Gueth, D M; van Blitterswijk, W J; Koning, G A; Verheij, M

2014-09-01

The endothelial lining and its outer lipid membrane are the first major barriers drug molecules encounter upon intravenous administration. Our previous work identified lipid analogs that counteract plasma membrane barrier function for a series of amphiphilic drugs. For example, short-chain sphingolipids (SCS), like N-octanoyl-glucosylceramide, effectively elevated doxorubicin accumulation in tumor cells, both in vitro and in vivo, and in endothelial cells, whereas other (normal) cells remained unaffected. We hypothesize here that local membrane lipid composition and the degree of lipid ordering define SCS efficacy in individual cells. To this end, we study the differential effect of SCS on bovine aortic endothelial cells (BAEC) in its confluent versus proliferative state, as a model system. While their (plasma membrane) lipidome stays remarkably unaltered when BAECs reach confluency, their lipids segregate to form apical and basolateral domains. Using probe NR12S, we reveal that lipids in the apical membrane are more condensed/liquid-ordered. SCS preferentially attenuate the barrier posed by these condensed membranes and facilitate doxorubicin influx in these particular membrane regions. We confirm these findings in MDCK cells and artificial membranes. In conclusion, SCS-facilitated drug traversal acts on condensed membrane domains, elicited by confluency in resting endothelium. Copyright © 2014 Elsevier B.V. All rights reserved.

Rap1 and Rap2 Antagonistically Control Endothelial Barrier Resistance

PubMed Central

Pannekoek, Willem-Jan; Linnemann, Jelena R.; Brouwer, Patricia M.; Bos, Johannes L.; Rehmann, Holger

2013-01-01

Rap1 and Rap2 are closely related proteins of the Ras family of small G-proteins. Rap1 is well known to regulate cell-cell adhesion. Here, we have analysed the effect of Rap-mediated signalling on endothelial permeability using electrical impedance measurements of HUVEC monolayers and subsequent determination of the barrier resistance, which is a measure for the ease with which ions can pass cell junctions. In line with its well-established effect on cell-cell junctions, depletion of Rap1 decreases, whereas activation of Rap1 increases barrier resistance. Despite its high sequence homology with Rap1, depletion of Rap2 has an opposite, enhancing, effect on barrier resistance. This effect can be mimicked by depletion of the Rap2 specific activator RasGEF1C and the Rap2 effector MAP4K4, establishing Rap2 signalling as an independent pathway controlling barrier resistance. As simultaneous depletion or activation of both Rap1 and Rap2 results in a barrier resistance comparable to control cells, Rap1 and Rap2 control barrier resistance in a reciprocal manner. This Rap1-antagonizing effect of Rap2 is established independent of junctional actin formation. These data establish that endothelial barrier resistance is determined by the combined antagonistic actions of Rap1 and Rap2. PMID:23469100

Carbachol inhibits TNF-α-induced endothelial barrier dysfunction through alpha 7 nicotinic receptors.

PubMed

Li, Yu-zhen; Liu, Xiu-hua; Rong, Fei; Hu, Sen; Sheng, Zhi-yong

2010-10-01

To test whether carbachol can influence endothelial barrier dysfunction induced by tumor necrosis factor (TNF)-α and whether the alpha 7 nicotinic receptor can mediate this process. Rat cardiac microvascular endothelial cells were exposed to carbachol followed by TNF-α treatment in the presence or the absence of α-bungarotoxin (an antagonist of the alpha 7 nicotinic receptor). Permeability of endothelial cells cultured on Transwell filters was assayed using FITC-albumin. F-actin was stained with FITC- phalloidin. Expression of vascular endothelial cadherin, intercellular adhesion molecule 1 (ICAM-1), phosphor-ERK1/2 and phosphor-JNK was detected using Western blot. Carbachol (2 μmol/L-2 mmol/L) prevented increase in endothelial cell permeability induced by TNF-α (500 ng/mL) in a dose-dependent manner. Further, it attenuated the down-regulation of vascular endothelial cadherin and the up-regulation of ICAM-1 induced by TNF-α. In addition, treatment of endothelial cells with carbachol decreased phosphor-ERK1/2 and phosphor-JNK. These effects of carbachol were blocked by α-bungarotoxin 3 μg/mL. These data suggest that the inhibitory effect of carbachol on TNF-α-induced endothelial barrier dysfunction mediated by the alpha 7 nicotinic receptor.

Carbachol inhibits TNF-α-induced endothelial barrier dysfunction through alpha 7 nicotinic receptors

PubMed Central

Li, Yu-zhen; Liu, Xiu-hua; Rong, Fei; Hu, Sen; Sheng, Zhi-yong

2010-01-01

Aim: To test whether carbachol can influence endothelial barrier dysfunction induced by tumor necrosis factor (TNF)-α and whether the alpha 7 nicotinic receptor can mediate this process. Methods: Rat cardiac microvascular endothelial cells were exposed to carbachol followed by TNF-α treatment in the presence or the absence of α-bungarotoxin (an antagonist of the alpha 7 nicotinic receptor). Permeability of endothelial cells cultured on Transwell filters was assayed using FITC-albumin. F-actin was stained with FITC- phalloidin. Expression of vascular endothelial cadherin, intercellular adhesion molecule 1 (ICAM-1), phosphor-ERK1/2 and phosphor-JNK was detected using Western blot. Results: Carbachol (2 μmol/L-2 mmol/L) prevented increase in endothelial cell permeability induced by TNF-α (500 ng/mL) in a dose-dependent manner. Further, it attenuated the down-regulation of vascular endothelial cadherin and the up-regulation of ICAM-1 induced by TNF-α. In addition, treatment of endothelial cells with carbachol decreased phosphor-ERK1/2 and phosphor-JNK. These effects of carbachol were blocked by α-bungarotoxin 3 μg/mL. Conclusion: These data suggest that the inhibitory effect of carbachol on TNF-α-induced endothelial barrier dysfunction mediated by the alpha 7 nicotinic receptor. PMID:20871620

Connexin channels provide a target to manipulate brain endothelial calcium dynamics and blood–brain barrier permeability

PubMed Central

De Bock, Marijke; Culot, Maxime; Wang, Nan; Bol, Mélissa; Decrock, Elke; De Vuyst, Elke; da Costa, Anaelle; Dauwe, Ine; Vinken, Mathieu; Simon, Alexander M; Rogiers, Vera; De Ley, Gaspard; Evans, William Howard; Bultynck, Geert; Dupont, Geneviève; Cecchelli, Romeo; Leybaert, Luc

2011-01-01

The cytoplasmic Ca2+ concentration ([Ca2+]i) is an important factor determining the functional state of blood–brain barrier (BBB) endothelial cells but little is known on the effect of dynamic [Ca2+]i changes on BBB function. We applied different agonists that trigger [Ca2+]i oscillations and determined the involvement of connexin channels and subsequent effects on endothelial permeability in immortalized and primary brain endothelial cells. The inflammatory peptide bradykinin (BK) triggered [Ca2+]i oscillations and increased endothelial permeability. The latter was prevented by buffering [Ca2+]i with BAPTA, indicating that [Ca2+]i oscillations are crucial in the permeability changes. Bradykinin-triggered [Ca2+]i oscillations were inhibited by interfering with connexin channels, making use of carbenoxolone, Gap27, a peptide blocker of connexin channels, and Cx37/43 knockdown. Gap27 inhibition of the oscillations was rapid (within minutes) and work with connexin hemichannel-permeable dyes indicated hemichannel opening and purinergic signaling in response to stimulation with BK. Moreover, Gap27 inhibited the BK-triggered endothelial permeability increase in in vitro and in vivo experiments. By contrast, [Ca2+]i oscillations provoked by exposure to adenosine 5′ triphosphate (ATP) were not affected by carbenoxolone or Gap27 and ATP did not disturb endothelial permeability. We conclude that interfering with endothelial connexin hemichannels is a novel approach to limiting BBB-permeability alterations. PMID:21654699

Saccharomyces cerevisiae show low levels of traversal across human endothelial barrier in vitro.

PubMed

Pérez-Torrado, Roberto; Querol, Amparo

2017-01-01

Background :   Saccharomyces cerevisiae is generally considered safe, and is involved in the production of many types of foods and dietary supplements. However, some isolates, which are genetically related to strains used in brewing and baking, have shown virulent traits, being able to produce infections in humans, mainly in immunodeficient patients. This can lead to systemic infections in humans. Methods : In this work, we studied S. cerevisiae isolates in an in vitro human endothelial barrier model, comparing their behaviour with that of several strains of the related pathogens Candida glabrata and Candida albicans . Results : The results showed that this food related yeast is able to cross the endothelial barrier in vitro . However, in contrast to C. glabrata and C. albicans , S. cerevisiae showed very low levels of traversal. Conclusions : We conclude that using an in vitro human endothelial barrier model with S. cerevisiae can be useful to evaluate the safety of S. cerevisiae strains isolated from foods.

Mitochondria and Endothelial Function

PubMed Central

Kluge, Matthew A.; Fetterman, Jessica L.; Vita, Joseph A.

2013-01-01

In contrast to their role in other cell types with higher energy demands, mitochondria in endothelial cells primarily function in signaling cellular responses to environmental cues. This article provides an overview of key aspects of mitochondrial biology in endothelial cells, including subcellular location, biogenesis, dynamics, autophagy, ROS production and signaling, calcium homeostasis, regulated cell death, and heme biosynthesis. In each section, we introduce key concepts and then review studies showing the importance of that mechanism to endothelial control of vasomotor tone, angiogenesis, and inflammatory activation. We particularly highlight the small number of clinical and translational studies that have investigated each mechanism in human subjects. Finally, we review interventions that target different aspects of mitochondrial function and their effects on endothelial function. The ultimate goal of such research is the identification of new approaches for therapy. The reviewed studies make it clear that mitochondria are important in endothelial physiology and pathophysiology. A great deal of work will be needed, however, before mitochondria-directed therapies are available for the prevention and treatment of cardiovascular disease. PMID:23580773

In vitro models of the blood–brain barrier: An overview of commonly used brain endothelial cell culture models and guidelines for their use

PubMed Central

Helms, Hans C; Abbott, N Joan; Burek, Malgorzata; Cecchelli, Romeo; Couraud, Pierre-Olivier; Deli, Maria A; Förster, Carola; Galla, Hans J; Romero, Ignacio A; Shusta, Eric V; Stebbins, Matthew J; Vandenhaute, Elodie; Weksler, Babette

2016-01-01

The endothelial cells lining the brain capillaries separate the blood from the brain parenchyma. The endothelial monolayer of the brain capillaries serves both as a crucial interface for exchange of nutrients, gases, and metabolites between blood and brain, and as a barrier for neurotoxic components of plasma and xenobiotics. This “blood-brain barrier” function is a major hindrance for drug uptake into the brain parenchyma. Cell culture models, based on either primary cells or immortalized brain endothelial cell lines, have been developed, in order to facilitate in vitro studies of drug transport to the brain and studies of endothelial cell biology and pathophysiology. In this review, we aim to give an overview of established in vitro blood–brain barrier models with a focus on their validation regarding a set of well-established blood–brain barrier characteristics. As an ideal cell culture model of the blood–brain barrier is yet to be developed, we also aim to give an overview of the advantages and drawbacks of the different models described. PMID:26868179

CORM-A1 prevents blood-brain barrier dysfunction caused by ionotropic glutamate receptor-mediated endothelial oxidative stress and apoptosis.

PubMed

Basuroy, Shyamali; Leffler, Charles W; Parfenova, Helena

2013-06-01

In cerebral microvascular endothelial cells (CMVEC) of newborn pigs, glutamate at excitotoxic concentrations (mM) causes apoptosis mediated by reactive oxygen species (ROS). Carbon monoxide (CO) produced by CMVEC or delivered by a CO-releasing molecule, CORM-A1, has antioxidant properties. We tested the hypothesis that CORM-A1 prevents cerebrovascular endothelial barrier dysfunction caused by glutamate excitotoxicity. First, we identified the glutamate receptors (GluRs) and enzymatic sources of ROS involved in the mechanism of endothelial apoptosis. In glutamate-exposed CMVEC, ROS formation and apoptosis were blocked by rotenone, 2-thenoyltrifluoroacetone (TTFA), and antimycin, indicating that mitochondrial complexes I, II, and III are the major sources of oxidative stress. Agonists of ionotropic GluRs (iGluRs) N-methyl-D-aspartate (NMDA), cis-ACPD, AMPA, and kainate increased ROS production and apoptosis, whereas iGluR antagonists exhibited antiapoptotic properties, suggesting that iGluRs mediate glutamate-induced endothelial apoptosis. The functional consequences of endothelial injury were tested in the model of blood-brain barrier (BBB) composed of CMVEC monolayer on semipermeable membranes. Glutamate and iGluR agonists reduced transendothelial electrical resistance and increased endothelial paracellular permeability to 3-kDa dextran. CORM-A1 exhibited potent antioxidant and antiapoptotic properties in CMVEC and completely prevented BBB dysfunction caused by glutamate and iGluR agonists. Overall, the endothelial component of the BBB is a cellular target for excitotoxic glutamate that, via a mechanism involving a iGluR-mediated activation of mitochondrial ROS production and apoptosis, leads to BBB opening that may be prevented by the antioxidant and antiapoptotic actions of CORMs. Antioxidant CORMs therapy may help preserve BBB functional integrity in neonatal cerebrovascular disease.

Shear stress and the endothelial transport barrier.

PubMed

Tarbell, John M

2010-07-15

The shear stress of flowing blood on the surfaces of endothelial cells that provide the barrier to transport of solutes and water between blood and the underlying tissue modulates the permeability to solutes and the hydraulic conductivity. This review begins with a discussion of transport pathways across the endothelium and then considers the experimental evidence from both in vivo and in vitro studies that shows an influence of shear stress on endothelial transport properties after both acute (minutes to hours) and chronic (hours to days) changes in shear stress. Next, the effects of shear stress on individual transport pathways (tight junctions, adherens junctions, vesicles and leaky junctions) are described, and this information is integrated with the transport experiments to suggest mechanisms controlling both acute and chronic responses of transport properties to shear stress. The review ends with a summary of future research challenges.

Anesthetic propofol overdose causes endothelial cytotoxicity in vitro and endothelial barrier dysfunction in vivo

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

Lin, Ming-Chung; Department of Anesthesiology, Chi Mei Medical Center, Liouying, Tainan, Taiwan; Chen, Chia-Ling

An overdose and a prolonged treatment of propofol may cause cellular cytotoxicity in multiple organs and tissues such as brain, heart, kidney, skeletal muscle, and immune cells; however, the underlying mechanism remains undocumented, particularly in vascular endothelial cells. Our previous studies showed that the activation of glycogen synthase kinase (GSK)-3 is pro-apoptotic in phagocytes during overdose of propofol treatment. Regarding the intravascular administration of propofol, we therefore hypothesized that propofol overdose also induces endothelial cytotoxicity via GSK-3. Propofol overdose (100 μg/ml) inhibited growth in human arterial and microvascular endothelial cells. After treatment, most of the endothelial cells experienced caspase-independent necrosis-likemore » cell death. The activation of cathepsin D following lysosomal membrane permeabilization (LMP) determined necrosis-like cell death. Furthermore, propofol overdose also induced caspase-dependent apoptosis, at least in part. Caspase-3 was activated and acted downstream of mitochondrial transmembrane potential (MTP) loss; however, lysosomal cathepsins were not required for endothelial cell apoptosis. Notably, activation of GSK-3 was essential for propofol overdose-induced mitochondrial damage and apoptosis, but not necrosis-like cell death. Intraperitoneal administration of a propofol overdose in BALB/c mice caused an increase in peritoneal vascular permeability. These results demonstrate the cytotoxic effects of propofol overdose, including cathepsin D-regulated necrosis-like cell death and GSK-3-regulated mitochondrial apoptosis, on endothelial cells in vitro and the endothelial barrier dysfunction by propofol in vivo. Highlights: ► Propofol overdose causes apoptosis and necrosis in endothelial cells. ► Propofol overdose triggers lysosomal dysfunction independent of autophagy. ► Glycogen synthase kinase-3 facilitates propofol overdose-induced apoptosis. ► Propofol overdose causes an

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

Compartmentalized, functional role of angiogenin during spotted fever group rickettsia-induced endothelial barrier dysfunction: evidence of possible mediation by host tRNA-derived small noncoding RNAs

PubMed Central

2013-01-01

Background Microvascular endothelial barrier dysfunction is the central enigma in spotted fever group (SFG) rickettsioses. Angiogenin (ANG) is one of the earliest identified angiogenic factors, of which some are relevant to the phosphorylation of VE-cadherins that serve as endothelial adherens proteins. Although exogenous ANG is known to translocate into the nucleus of growing endothelial cells (ECs) where it plays a functional role, nuclear ANG is not detected in quiescent ECs. Besides its nuclear role, ANG is thought to play a cytoplasmic role, owing to its RNase activity that cleaves tRNA to produce small RNAs. Recently, such tRNA-derived RNA fragments (tRFs) have been shown to be induced under stress conditions. All these observations raise an intriguing hypothesis about a novel cytoplasmic role of ANG, which is induced upon infection with Rickettsia and generates tRFs that may play roles in SFG rickettsioses. Methods C3H/HeN mice were infected intravenously with a sublethal dose of R. conorii. At days 1, 3, and 5 post infection (p.i.), liver, lung and brain were collected for immunofluorescence (IF) studies of R. conorii and angiogenin (ANG). Human umbilical vein endothelial cells (HUVECs) were infected with R. conorii for 24, 48, and 72 hrs before incubation with 1μg/ml recombinant human ANG (rANG) in normal medium for 2 hrs. HUVEC samples were subjected to IF, exogenous ANG translocation, endothelial permeability, and immunoprecipitation phosphorylation assays. To identify small non-coding RNAs (sncRNAs) upon rickettsial infection, RNAs from pulverized mouse lung tissues and HUVECs were subjected to library preparation and deep sequencing analysis using an Illumina 2000 instrument. Identified sncRNAs were confirmed by Northern hybridization, and their target mRNAs were predicted in silico using BLAST and RNA hybrid programs. Results In the present study, we have demonstrated endothelial up-regulation of ANG, co-localized with SFG rickettsial infection in

Shear stress and the endothelial transport barrier

PubMed Central

Tarbell, John M.

2010-01-01

The shear stress of flowing blood on the surfaces of endothelial cells that provide the barrier to transport of solutes and water between blood and the underlying tissue modulates the permeability to solutes and the hydraulic conductivity. This review begins with a discussion of transport pathways across the endothelium and then considers the experimental evidence from both in vivo and in vitro studies that shows an influence of shear stress on endothelial transport properties after both acute (minutes to hours) and chronic (hours to days) changes in shear stress. Next, the effects of shear stress on individual transport pathways (tight junctions, adherens junctions, vesicles and leaky junctions) are described, and this information is integrated with the transport experiments to suggest mechanisms controlling both acute and chronic responses of transport properties to shear stress. The review ends with a summary of future research challenges. PMID:20543206

Transient Intervals of Hyper-Gravity Enhance Endothelial Barrier Integrity: Impact of Mechanical and Gravitational Forces Measured Electrically

PubMed Central

Szulcek, Robert; van Bezu, Jan; Boonstra, Johannes; van Loon, Jack J. W. A.; van Nieuw Amerongen, Geerten P.

2015-01-01

Background Endothelial cells (EC) guard vascular functions by forming a dynamic barrier throughout the vascular system that sensitively adapts to ‘classical’ biomechanical forces, such as fluid shear stress and hydrostatic pressure. Alterations in gravitational forces might similarly affect EC integrity, but remain insufficiently studied. Methods In an unique approach, we utilized Electric Cell-substrate Impedance Sensing (ECIS) in the gravity-simulators at the European Space Agency (ESA) to study dynamic responses of human EC to simulated micro- and hyper-gravity as well as to classical forces. Results Short intervals of micro- or hyper-gravity evoked distinct endothelial responses. Stimulated micro-gravity led to decreased endothelial barrier integrity, whereas hyper-gravity caused sustained barrier enhancement by rapid improvement of cell-cell integrity, evidenced by a significant junctional accumulation of VE-cadherin (p = 0.011), significant enforcement of peripheral F-actin (p = 0.008) and accompanied by a slower enhancement of cell-matrix interactions. The hyper-gravity triggered EC responses were force dependent and nitric-oxide (NO) mediated showing a maximal resistance increase of 29.2±4.8 ohms at 2g and 60.9±6.2 ohms at 4g vs. baseline values that was significantly suppressed by NO blockage (p = 0.011). Conclusion In conclusion, short-term application of hyper-gravity caused a sustained improvement of endothelial barrier integrity, whereas simulated micro-gravity weakened the endothelium. In clear contrast, classical forces of shear stress and hydrostatic pressure induced either short-lived or no changes to the EC barrier. Here, ECIS has proven a powerful tool to characterize subtle and distinct EC gravity-responses due to its high temporal resolution, wherefore ECIS has a great potential for the study of gravity-responses such as in real space flights providing quantitative assessment of a variety of cell biological characteristics of any adherent

Neutrophil-endothelial cell interactions on endothelial monolayers grown on micropore filters.

PubMed Central

Taylor, R F; Price, T H; Schwartz, S M; Dale, D C

1981-01-01

We have developed a technique for growing endothelial monolayers on micropore filters. These monolayers demonstrate confluence by phase and electron microscopy and provide a functional barrier to passage of radiolabeled albumin. Neutrophils readily penetrate the monolayer in response to chemotaxin, whereas there is little movement in the absence of chemotaxin. This system offers unique advantages over available chemotaxis assays and may have wider applications in the study of endothelial function. Images PMID:7007441

Regulation and function of endothelial glycocalyx layer in vascular diseases.

PubMed

Sieve, Irina; Münster-Kühnel, Anja K; Hilfiker-Kleiner, Denise

2018-01-01

In the vascular system, the endothelial surface layer (ESL) as the inner surface of blood vessels affects mechanotransduction, vascular permeability, rheology, thrombogenesis, and leukocyte adhesion. It creates barriers between endothelial cells and blood and neighbouring cells. The glycocalyx, composed of glycoconjugates and proteoglycans, is an integral component of the ESL and a key element in inter- and intracellular communication and tissue homeostasis. In pathophysiological conditions (atherosclerosis, infection, ischemia/reperfusion injury, diabetes, trauma and acute lung injury) glycocalyx-degrading factors, i.e. reactive oxygen and nitrogen species, matrix metalloproteinases, heparanase and sialidases, damage the ESL, thereby impairing endothelial functions. This leads to increased capillary permeability, leucocyte-endothelium interactions, thrombosis and vascular inflammation, the latter further driving glycocalyx destruction. The present review highlights current knowledge on the vasculoprotective role of the ESL, with specific emphasis on its remodelling in inflammatory vascular diseases and discusses its potential as a novel therapeutic target to treat vascular pathologies. Copyright © 2017 Elsevier Inc. All rights reserved.

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

Regulation of human cerebro-microvascular endothelial baso-lateral adhesion and barrier function by S1P through dual involvement of S1P1 and S1P2 receptors.

PubMed

Wiltshire, Rachael; Nelson, Vicky; Kho, Dan Ting; Angel, Catherine E; O'Carroll, Simon J; Graham, E Scott

2016-01-27

Herein we show that S1P rapidly and acutely reduces the focal adhesion strength and barrier tightness of brain endothelial cells. xCELLigence biosensor technology was used to measure focal adhesion, which was reduced by S1P acutely and this response was mediated through both S1P1 and S1P2 receptors. S1P increased secretion of several pro-inflammatory mediators from brain endothelial cells. However, the magnitude of this response was small in comparison to that mediated by TNFα or IL-1β. Furthermore, S1P did not significantly increase cell-surface expression of any key cell adhesion molecules involved in leukocyte recruitment, included ICAM-1 and VCAM-1. Finally, we reveal that S1P acutely and dynamically regulates microvascular endothelial barrier tightness in a manner consistent with regulated rapid opening followed by closing and strengthening of the barrier. We hypothesise that the role of the S1P receptors in this process is not to cause barrier dysfunction, but is related to controlled opening of the endothelial junctions. This was revealed using real-time measurement of barrier integrity using ECIS ZΘ TEER technology and endothelial viability using xCELLigence technology. Finally, we show that these responses do not occur simply though the pharmacology of a single S1P receptor but involves coordinated action of S1P1 and S1P2 receptors.

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.

Wine and endothelial function.

PubMed

Caimi, G; Carollo, C; Lo Presti, R

2003-01-01

In recent years many studies have focused on the well-known relationship between wine consumption and cardiovascular risk. Wine exerts its protective effects through various changes in lipoprotein profile, coagulation and fibrinolytic cascades, platelet aggregation, oxidative mechanisms and endothelial function. The last has earned more attention for its implications in atherogenesis. Endothelium regulates vascular tone by a delicate balancing among vasorelaxing (nitric oxide [NO]) and vasoconstrincting (endothelins) factors produced by endothelium in response to various stimuli. In rat models, wine and other grape derivatives exerted an endothelium-dependent vasorelaxing capacity especially associated with the NO-stimulating activity of their polyphenol components. In experimental conditions, reservatrol (a stilbene polyphenol) protected hearts and kidneys from ischemia-reperfusion injury through antioxidant activity and upregulation of NO production. Wine polyphenols are also able to induce the expression of genes involved in the NO pathway within the arterial wall. The effects of wine on endothelial function in humans are not yet clearly understood. A favorable action of red wine or dealcoholized wine extract or purple grape juice on endothelial function has been observed by several authors, but discrimination between ethanol and polyphenol effects is controversial. It is, however likely that regular and prolonged moderate wine drinking positively affects endothelial function. The beneficial effects of wine on cardiovascular health are greater if wine is associated with a healthy diet. The most recent nutritional and epidemiologic studies show that the ideal diet closely resembles the Mediterranean diet.

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

An in vitro and in vivo study of peptide-functionalized nanoparticles for brain targeting: The importance of selective blood-brain barrier uptake.

PubMed

Bode, Gerard H; Coué, Gregory; Freese, Christian; Pickl, Karin E; Sanchez-Purrà, Maria; Albaiges, Berta; Borrós, Salvador; van Winden, Ewoud C; Tziveleka, Leto-Aikaterini; Sideratou, Zili; Engbersen, Johan F J; Singh, Smriti; Albrecht, Krystyna; Groll, Jürgen; Möller, Martin; Pötgens, Andy J G; Schmitz, Christoph; Fröhlich, Eleonore; Grandfils, Christian; Sinner, Frank M; Kirkpatrick, C James; Steinbusch, Harry W M; Frank, Hans-Georg; Unger, Ronald E; Martinez-Martinez, Pilar

2017-04-01

Targeted delivery of drugs across endothelial barriers remains a formidable challenge, especially in the case of the brain, where the blood-brain barrier severely limits entry of drugs into the central nervous system. Nanoparticle-mediated transport of peptide/protein-based drugs across endothelial barriers shows great potential as a therapeutic strategy in a wide variety of diseases. Functionalizing nanoparticles with peptides allows for more efficient targeting to specific organs. We have evaluated the hemocompatibilty, cytotoxicity, endothelial uptake, efficacy of delivery and safety of liposome, hyperbranched polyester, poly(glycidol) and acrylamide-based nanoparticles functionalized with peptides targeting brain endothelial receptors, in vitro and in vivo. We used an ELISA-based method for the detection of nanoparticles in biological fluids, investigating the blood clearance rate and in vivo biodistribution of labeled nanoparticles in the brain after intravenous injection in Wistar rats. Herein, we provide a detailed report of in vitro and in vivo observations. Copyright © 2016 Elsevier Inc. All rights reserved.

Glucocorticoid Induction of Occludin Expression and Endothelial Barrier Requires Transcription Factor p54 NONO

PubMed Central

Keil, Jason M.; Liu, Xuwen; Antonetti, David A.

2013-01-01

Purpose. Glucocorticoids (GCs) effectively reduce retinal edema and induce vascular barrier properties but possess unwanted side effects. Understanding GC induction of barrier properties may lead to more effective and specific therapies. Previous work identified the occludin enhancer element (OEE) as a GC-responsive cis-element in the promoters of multiple junctional genes, including occludin, claudin-5, and cadherin-9. Here, we identify two OEE-binding factors and determine their contribution to GC induction of tight junction (TJ) gene expression and endothelial barrier properties. Methods. OEE-binding factors were isolated from human retinal endothelial cells (HREC) using DNA affinity purification followed by MALDI-TOF MS/MS. Chromatin immunoprecipitation (ChIP) assays determined in situ binding. siRNA was used to evaluate the role of trans-acting factors in transcription of TJ genes in response to GC stimulation. Paracellular permeability was determined by quantifying flux through a cell monolayer, whereas transendothelial electrical resistance (TER) was measured using the ECIS system. Results. MS/MS analysis of HREC nuclear extracts identified the heterodimer of transcription factors p54/NONO (p54) and polypyrimidine tract-binding protein-associated splicing factor (PSF) as OEE-binding factors, which was confirmed by ChIP assay from GC-treated endothelial cells and rat retina. siRNA knockdown of p54 demonstrated that this factor is necessary for GC induction of occludin and claudin-5 expression. Further, p54 knockdown ablated the pro-barrier effects of GC treatment. Conclusions. p54 is essential for GC-mediated expression of occludin, claudin-5, and barrier induction, and the p54/PSF heterodimer may contribute to normal blood-retinal barrier (BRB) induction in vivo. Understanding the mechanism of GC induction of BRB properties may provide novel therapies for macular edema. PMID:23640037

Glucocorticoid induction of occludin expression and endothelial barrier requires transcription factor p54 NONO.

PubMed

Keil, Jason M; Liu, Xuwen; Antonetti, David A

2013-06-12

Glucocorticoids (GCs) effectively reduce retinal edema and induce vascular barrier properties but possess unwanted side effects. Understanding GC induction of barrier properties may lead to more effective and specific therapies. Previous work identified the occludin enhancer element (OEE) as a GC-responsive cis-element in the promoters of multiple junctional genes, including occludin, claudin-5, and cadherin-9. Here, we identify two OEE-binding factors and determine their contribution to GC induction of tight junction (TJ) gene expression and endothelial barrier properties. OEE-binding factors were isolated from human retinal endothelial cells (HREC) using DNA affinity purification followed by MALDI-TOF MS/MS. Chromatin immunoprecipitation (ChIP) assays determined in situ binding. siRNA was used to evaluate the role of trans-acting factors in transcription of TJ genes in response to GC stimulation. Paracellular permeability was determined by quantifying flux through a cell monolayer, whereas transendothelial electrical resistance (TER) was measured using the ECIS system. MS/MS analysis of HREC nuclear extracts identified the heterodimer of transcription factors p54/NONO (p54) and polypyrimidine tract-binding protein-associated splicing factor (PSF) as OEE-binding factors, which was confirmed by ChIP assay from GC-treated endothelial cells and rat retina. siRNA knockdown of p54 demonstrated that this factor is necessary for GC induction of occludin and claudin-5 expression. Further, p54 knockdown ablated the pro-barrier effects of GC treatment. p54 is essential for GC-mediated expression of occludin, claudin-5, and barrier induction, and the p54/PSF heterodimer may contribute to normal blood-retinal barrier (BRB) induction in vivo. Understanding the mechanism of GC induction of BRB properties may provide novel therapies for macular edema.

[Blood-nerve barrier: structure and function].

PubMed

Kanda, Takashi

2011-06-01

The blood-nerve barrier (BNB) is a dynamic interface between the endoneurial microenvironment and surrounding extracellular space or blood contents, and is localized the innermost layer of multilayered ensheathing perineurium and endoneurial microvessels. Since the BNB is a key structure controlling the internal milieu of the peripheral nerve parenchyma, adequate understanding of the BNB is crucial for developing treatment strategies for human peripheral nervous system disorders, including Guillain-Barré syndrome, chronic inflammatory demyelinating polyneuropathy, and diabetic and various metabolic/toxic neuropathies. However, fewer studies have been conducted on the BNB, if we compare against the number of studies on the blood-brain barrier. This is because of the lack of adequate human cell lines originating from the BNB. In our laboratory, human immortal cell lines from the BNB, namely, the endothelial cell line and pericyte cell line, have recently been established and vigorous investigations of their biological and physiological properties are now underway. Pericytes constituting the BNB were found to possess robust ability of controlling BNB integrity via secretion of various cytokines and growth factors including bFGF, VEGF, GDNF, BDNF, and angiopoietin-1. Unknown soluble factors secreted by pericytes also contribute to the upregulation of claudin-5 in endothelial cells in the BNB and thus, strengthen the barrier function of the BNB. In diabetic neuropathy, pericytes were shown to regulate the vascular basement membrane, while AGEs were shown to induce basement membrane hypertrophy and disrupt the BNB by increasing the autocrine secretion of VEGF and TGF-beta from pericytes. In this review article, we discuss the macroscopic and microscopic anatomy of the human BNB as well as the molecular mechanisms of mononuclear cell infiltration across the BNB.

Role of kinase-independent and -dependent functions of FAK in endothelial cell survival and barrier function during embryonic development.

PubMed

Zhao, Xiaofeng; Peng, Xu; Sun, Shaogang; Park, Ann Y J; Guan, Jun-Lin

2010-06-14

Focal adhesion kinase (FAK) is essential for vascular development as endothelial cell (EC)-specific knockout of FAK (conditional FAK knockout [CFKO] mice) leads to embryonic lethality. In this study, we report the differential kinase-independent and -dependent functions of FAK in vascular development by creating and analyzing an EC-specific FAK kinase-defective (KD) mutant knockin (conditional FAK knockin [CFKI]) mouse model. CFKI embryos showed apparently normal development through embryonic day (E) 13.5, whereas the majority of CFKO embryos died at the same stage. Expression of KD FAK reversed increased EC apoptosis observed with FAK deletion in embryos and in vitro through suppression of up-regulated p21. However, vessel dilation and defective angiogenesis of CFKO embryos were not rescued in CFKI embryos. ECs without FAK or expressing KD FAK showed increased permeability, abnormal distribution of vascular endothelial cadherin (VE-cadherin), and reduced VE-cadherin Y658 phosphorylation. Together, our data suggest that kinase-independent functions of FAK can support EC survival in vascular development through E13.5 but are insufficient for maintaining EC function to allow for completion of embryogenesis.

CD146 coordinates brain endothelial cell–pericyte communication for blood–brain barrier development

PubMed Central

Chen, Jianan; Luo, Yongting; Hui, Hui; Cai, Tanxi; Huang, Hongxin; Yang, Fuquan; Feng, Jing; Zhang, Jingjing; Yan, Xiyun

2017-01-01

The blood–brain barrier (BBB) establishes a protective interface between the central neuronal system and peripheral blood circulation and is crucial for homeostasis of the CNS. BBB formation starts when the endothelial cells (ECs) invade the CNS and pericytes are recruited to the nascent vessels during embryogenesis. Despite the essential function of pericyte–EC interaction during BBB development, the molecular mechanisms coordinating the pericyte–EC behavior and communication remain incompletely understood. Here, we report a single cell receptor, CD146, that presents dynamic expression patterns in the cerebrovasculature at the stages of BBB induction and maturation, coordinates the interplay of ECs and pericytes, and orchestrates BBB development spatiotemporally. In mouse brain, CD146 is first expressed in the cerebrovascular ECs of immature capillaries without pericyte coverage; with increased coverage of pericytes, CD146 could only be detected in pericytes, but not in cerebrovascular ECs. Specific deletion of Cd146 in mice ECs resulted in reduced brain endothelial claudin-5 expression and BBB breakdown. By analyzing mice with specific deletion of Cd146 in pericytes, which have defects in pericyte coverage and BBB integrity, we demonstrate that CD146 functions as a coreceptor of PDGF receptor-β to mediate pericyte recruitment to cerebrovascular ECs. Moreover, we found that the attached pericytes in turn down-regulate endothelial CD146 by secreting TGF-β1 to promote further BBB maturation. These results reveal that the dynamic expression of CD146 controls the behavior of ECs and pericytes, thereby coordinating the formation of a mature and stable BBB. PMID:28827364

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

The Drosophila blood-brain barrier: development and function of a glial endothelium.

PubMed

Limmer, Stefanie; Weiler, Astrid; Volkenhoff, Anne; Babatz, Felix; Klämbt, Christian

2014-01-01

The efficacy of neuronal function requires a well-balanced extracellular ion homeostasis and a steady supply with nutrients and metabolites. Therefore, all organisms equipped with a complex nervous system developed a so-called blood-brain barrier, protecting it from an uncontrolled entry of solutes, metabolites or pathogens. In higher vertebrates, this diffusion barrier is established by polarized endothelial cells that form extensive tight junctions, whereas in lower vertebrates and invertebrates the blood-brain barrier is exclusively formed by glial cells. Here, we review the development and function of the glial blood-brain barrier of Drosophila melanogaster. In the Drosophila nervous system, at least seven morphologically distinct glial cell classes can be distinguished. Two of these glial classes form the blood-brain barrier. Perineurial glial cells participate in nutrient uptake and establish a first diffusion barrier. The subperineurial glial (SPG) cells form septate junctions, which block paracellular diffusion and thus seal the nervous system from the hemolymph. We summarize the molecular basis of septate junction formation and address the different transport systems expressed by the blood-brain barrier forming glial cells.

A Cell Culture Approach to Optimized Human Corneal Endothelial Cell Function

PubMed Central

Bartakova, Alena; Kuzmenko, Olga; Alvarez-Delfin, Karen; Kunzevitzky, Noelia J.; Goldberg, Jeffrey L.

2018-01-01

Purpose Cell-based therapies to replace corneal endothelium depend on culture methods to optimize human corneal endothelial cell (HCEC) function and minimize endothelial-mesenchymal transition (EnMT). Here we explore contribution of low-mitogenic media on stabilization of phenotypes in vitro that mimic those of HCECs in vivo. Methods HCECs were isolated from cadaveric donor corneas and expanded in vitro, comparing continuous presence of exogenous growth factors (“proliferative media”) to media without those factors (“stabilizing media”). Identity based on canonical morphology and expression of surface marker CD56, and function based on formation of tight junction barriers measured by trans-endothelial electrical resistance assays (TEER) were assessed. Results Primary HCECs cultured in proliferative media underwent EnMT after three to four passages, becoming increasingly fibroblastic. Stabilizing the cells before each passage by switching them to a media low in mitogenic growth factors and serum preserved canonical morphology and yielded a higher number of cells. HCECs cultured in stabilizing media increased both expression of the identity marker CD56 and also tight junction monolayer integrity compared to cells cultured without stabilization. Conclusions HCECs isolated from donor corneas and expanded in vitro with a low-mitogenic media stabilizing step before each passage demonstrate more canonical structural and functional features and defer EnMT, increasing the number of passages and total canonical cell yield. This approach may facilitate development of HCEC-based cell therapies. PMID:29625488

Poly(I:C) Induces Human Lung Endothelial Barrier Dysfunction by Disrupting Tight Junction Expression of Claudin-5

DOE PAGES

Huang, Li -Yun; Stuart, Christine; Takeda, Kazuyo; ...

2016-08-09

Viral infections are often accompanied by pulmonary microvascular leakage and vascular endothelial dysfunction via mechanisms that are not completely defined. Here, we investigated the effect of the Toll-like receptor 3 (TLR3) ligand polyinosinic-polycytidylic acid [Poly(I:C)], a synthetic analog of viral double-stranded RNA (dsRNA) commonly used to simulate viral infections, on the barrier function and tight junction integrity of primary human lung microvascular endothelial cells. Poly(I:C) stimulated IL-6, IL-8, TNFα, and IFNβ production in conjunction with the activation of NF-κB and IRF3 confirming the Poly(I:C)-responsiveness of these cells. Poly(I:C) increased endothelialmonolayer permeability with a corresponding dose- and time-dependent decrease in themore » expression of claudin-5, a transmembrane tight junction protein and reduction of CLDN5 mRNA levels. Immunofluorescence experiments revealed disappearance of membrane-associated claudin-5 and co-localization of cytoplasmic claudin-5 with lysosomal-associated membrane protein 1. Chloroquine and Bay11-7082, inhibitors of TLR3 and NF-κB signaling, respectively, protected against the loss of claudin-5. Altogether, these findings provide new insight on how dsRNA-activated signaling pathways may disrupt vascular endothelial function and contribute to vascular leakage pathologies.« less

Antihypertensive therapy and endothelial function.

PubMed

Nadar, Sunil; Blann, Andrew D; Lip, Gregory Y H

2004-01-01

The benefits of treating hypertension in terms of reduction of morbidity and mortality are well established. However, it is debatable whether this benefit is derived entirely from the effects of a reduced blood pressure or whether these agents exert effects over and above blood pressure reduction on the endothelium. Hypertension is associated with adverse changes (whether damage or dysfunction) in the endothelium. Indeed, endothelial damage/dysfunction has have been demonstrated to be a reliable prognostic indicator of future cardiovascular events in hypertension. Of the various drug classes, calcium channel blockers and the ACE inhibitors have significant direct effects on the endothelium. This is in contrast to the beta blockers and alpha adrenergic blockers that appear to indirectly influence endothelial function solely as a result of lowered blood pressure. Antioxidants may have a beneficial effect on endothelial function as well, although their clinical use does not seem to translate into clinical benefit.

Bleeding complications in dengue are not associated with significant changes in the modulators of the endothelial barrier.

PubMed

Orsi, Fernanda Andrade; Angerami, Rodrigo Nogueira; Mazetto, Bruna Moraes; Quaino, Susan Kelly Picoli; De Paula, Erich Vinícius; Annichino-Bizzachi, Joyce Maria

2014-06-11

Bleeding complications in dengue may occur irrespective of the presence of plasma leakage. We compared plasma levels of modulators of the endothelial barrier among three dengue groups: bleedings without plasma leakage, dengue hemorrhagic fever, and non-complicated dengue. The aim was to evaluate whether the presence of subtle alterations in microvascular permeability could be detected in bleeding patients. Plasma levels of VEGF-A and its soluble receptors were not associated with the occurrence of bleeding in patients without plasma leakage. These results provide additional rationale for considering bleeding as a complication independent of endothelial barrier breakdown, as proposed by the 2009 WHO classification.

Vascular endothelial cells express isoforms of protein kinase A inhibitor.

PubMed

Lum, Hazel; Hao, Zengping; Gayle, Dave; Kumar, Priyadarsini; Patterson, Carolyn E; Uhler, Michael D

2002-01-01

The expression and function of the endogenous inhibitor of cAMP-dependent protein kinase (PKI) in endothelial cells are unknown. In this study, overexpression of rabbit muscle PKI gene into endothelial cells inhibited the cAMP-mediated increase and exacerbated thrombin-induced decrease in endothelial barrier function. We investigated PKI expression in human pulmonary artery (HPAECs), foreskin microvessel (HMECs), and brain microvessel endothelial cells (HBMECs). RT-PCR using specific primers for human PKI alpha, human PKI gamma, and mouse PKI beta sequences detected PKI alpha and PKI gamma mRNA in all three cell types. Sequencing and BLAST analysis indicated that forward and reverse DNA strands for PKI alpha and PKI gamma were of >96% identity with database sequences. RNase protection assays showed protection of the 542 nucleotides in HBMEC and HPAEC PKI alpha mRNA and 240 nucleotides in HBMEC, HPAEC, and HMEC PKI gamma mRNA. Western blot analysis indicated that PKI gamma protein was detected in all three cell types, whereas PKI alpha was found in HBMECs. In summary, endothelial cells from three different vascular beds express PKI alpha and PKI gamma, which may be physiologically important in endothelial barrier function.

Hantavirus-induced disruption of the endothelial barrier: neutrophils are on the payroll.

PubMed

Schönrich, Günther; Krüger, Detlev H; Raftery, Martin J

2015-01-01

Viral hemorrhagic fever caused by hantaviruses is an emerging infectious disease for which suitable treatments are not available. In order to improve this situation a better understanding of hantaviral pathogenesis is urgently required. Hantaviruses infect endothelial cell layers in vitro without causing any cytopathogenic effect and without increasing permeability. This implies that the mechanisms underlying vascular hyperpermeability in hantavirus-associated disease are more complex and that immune mechanisms play an important role. In this review we highlight the latest developments in hantavirus-induced immunopathogenesis. A possible contribution of neutrophils has been neglected so far. For this reason, we place special emphasis on the pathogenic role of neutrophils in disrupting the endothelial barrier.

Endothelial cells use dynamic actin to facilitate lymphocyte transendothelial migration and maintain the monolayer barrier

PubMed Central

Mooren, Olivia L.; Li, Jinmei; Nawas, Julie; Cooper, John A.

2014-01-01

The vascular endothelium is a highly dynamic structure, and the integrity of its barrier function is tightly regulated. Normally impenetrable to cells, the endothelium actively assists lymphocytes to exit the bloodstream during inflammation. The actin cytoskeleton of the endothelial cell (EC) is known to facilitate transmigration, but the cellular and molecular mechanisms are not well understood. Here we report that actin assembly in the EC, induced by Arp2/3 complex under control of WAVE2, is important for several steps in the process of transmigration. To begin transmigration, ECs deploy actin-based membrane protrusions that create a cup-shaped docking structure for the lymphocyte. We found that docking structure formation involves the localization and activation of Arp2/3 complex by WAVE2. The next step in transmigration is creation of a migratory pore, and we found that endothelial WAVE2 is needed for lymphocytes to follow a transcellular route through an EC. Later, ECs use actin-based protrusions to close the gap behind the lymphocyte, which we discovered is also driven by WAVE2. Finally, we found that ECs in resting endothelial monolayers use lamellipodial protrusions dependent on WAVE2 to form and maintain contacts and junctions between cells. PMID:25355948

Autophagy inhibitor 3-methyladenine protects against endothelial cell barrier dysfunction in acute lung injury.

PubMed

Slavin, Spencer A; Leonard, Antony; Grose, Valerie; Fazal, Fabeha; Rahman, Arshad

2018-03-01

Autophagy is an evolutionarily conserved cellular process that facilitates the continuous recycling of intracellular components (organelles and proteins) and provides an alternative source of energy when nutrients are scarce. Recent studies have implicated autophagy in many disorders, including pulmonary diseases. However, the role of autophagy in endothelial cell (EC) barrier dysfunction and its relevance in the context of acute lung injury (ALI) remain uncertain. Here, we provide evidence that autophagy is a critical component of EC barrier disruption in ALI. Using an aerosolized bacterial lipopolysaccharide (LPS) inhalation mouse model of ALI, we found that administration of the autophagy inhibitor 3-methyladenine (3-MA), either prophylactically or therapeutically, markedly reduced lung vascular leakage and tissue edema. 3-MA was also effective in reducing the levels of proinflammatory mediators and lung neutrophil sequestration induced by LPS. To test the possibility that autophagy in EC could contribute to lung vascular injury, we addressed its role in the mechanism of EC barrier disruption. Knockdown of ATG5, an essential regulator of autophagy, attenuated thrombin-induced EC barrier disruption, confirming the involvement of autophagy in the response. Similarly, exposure of cells to 3-MA, either before or after thrombin, protected against EC barrier dysfunction by inhibiting the cleavage and loss of vascular endothelial cadherin at adherens junctions, as well as formation of actin stress fibers. 3-MA also reversed LPS-induced EC barrier disruption. Together, these data imply a role of autophagy in lung vascular injury and reveal the protective and therapeutic utility of 3-MA against ALI.

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.

Role of Vascular and Lymphatic Endothelial Cells in Hantavirus Pulmonary Syndrome Suggests Targeted Therapeutic Approaches

PubMed Central

Gorbunova, Elena E.; Dalrymple, Nadine A.; Gavrilovskaya, Irina N.

2013-01-01

Abstract Background Hantaviruses in the Americas cause a highly lethal acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). Hantaviruses nonlytically infect microvascular and lymphatic endothelial cells and cause dramatic changes in barrier functions without disrupting the endothelium. Hantaviruses cause changes in the function of infected endothelial cells that normally regulate fluid barrier functions. The endothelium of arteries, veins, and lymphatic vessels are unique and central to the function of vast pulmonary capillary beds that regulate pulmonary fluid accumulation. Results We have found that HPS-causing hantaviruses alter vascular barrier functions of microvascular and lymphatic endothelial cells by altering receptor and signaling pathway responses that serve to permit fluid tissue influx and clear tissue edema. Infection of the endothelium provides several mechanisms for hantaviruses to cause acute pulmonary edema, as well as potential therapeutic targets for reducing the severity of HPS disease. Conclusions Here we discuss interactions of HPS-causing hantaviruses with the endothelium, roles for unique lymphatic endothelial responses in HPS, and therapeutic targeting of the endothelium as a means of reducing the severity of HPS disease. PMID:24024573

Role of vascular and lymphatic endothelial cells in hantavirus pulmonary syndrome suggests targeted therapeutic approaches.

PubMed

Mackow, Erich R; Gorbunova, Elena E; Dalrymple, Nadine A; Gavrilovskaya, Irina N

2013-09-01

Hantaviruses in the Americas cause a highly lethal acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). Hantaviruses nonlytically infect microvascular and lymphatic endothelial cells and cause dramatic changes in barrier functions without disrupting the endothelium. Hantaviruses cause changes in the function of infected endothelial cells that normally regulate fluid barrier functions. The endothelium of arteries, veins, and lymphatic vessels are unique and central to the function of vast pulmonary capillary beds that regulate pulmonary fluid accumulation. We have found that HPS-causing hantaviruses alter vascular barrier functions of microvascular and lymphatic endothelial cells by altering receptor and signaling pathway responses that serve to permit fluid tissue influx and clear tissue edema. Infection of the endothelium provides several mechanisms for hantaviruses to cause acute pulmonary edema, as well as potential therapeutic targets for reducing the severity of HPS disease. Here we discuss interactions of HPS-causing hantaviruses with the endothelium, roles for unique lymphatic endothelial responses in HPS, and therapeutic targeting of the endothelium as a means of reducing the severity of HPS disease.

Hydrophilic bile acids protect human blood-brain barrier endothelial cells from disruption by unconjugated bilirubin: an in vitro study

PubMed Central

Palmela, Inês; Correia, Leonor; Silva, Rui F. M.; Sasaki, Hiroyuki; Kim, Kwang S.; Brites, Dora; Brito, Maria A.

2015-01-01

Ursodeoxycholic acid and its main conjugate glycoursodeoxycholic acid are bile acids with neuroprotective properties. Our previous studies demonstrated their anti-apoptotic, anti-inflammatory, and antioxidant properties in neural cells exposed to elevated levels of unconjugated bilirubin (UCB) as in severe jaundice. In a simplified model of the blood-brain barrier, formed by confluent monolayers of a cell line of human brain microvascular endothelial cells, UCB has shown to induce caspase-3 activation and cell death, as well as interleukin-6 release and a loss of blood-brain barrier integrity. Here, we tested the preventive and restorative effects of these bile acids regarding the disruption of blood-brain barrier properties by UCB in in vitro conditions mimicking severe neonatal hyperbilirubinemia and using the same experimental blood-brain barrier model. Both bile acids reduced the apoptotic cell death induced by UCB, but only glycoursodeoxycholic acid significantly counteracted caspase-3 activation. Bile acids also prevented the upregulation of interleukin-6 mRNA, whereas only ursodeoxycholic acid abrogated cytokine release. Regarding barrier integrity, only ursodeoxycholic acid abrogated UCB-induced barrier permeability. Better protective effects were obtained by bile acid pre-treatment, but a strong efficacy was still observed by their addition after UCB treatment. Finally, both bile acids showed ability to cross confluent monolayers of human brain microvascular endothelial cells in a time-dependent manner. Collectively, data disclose a therapeutic time-window for preventive and restorative effects of ursodeoxycholic acid and glycoursodeoxycholic acid against UCB-induced blood-brain barrier disruption and damage to human brain microvascular endothelial cells. PMID:25821432

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.

Tight junctions and the modulation of barrier function in disease

PubMed Central

2008-01-01

Tight junctions create a paracellular barrier in epithelial and endothelial cells protecting them from the external environment. Two different classes of integral membrane proteins constitute the tight junction strands in epithelial cells and endothelial cells, occludin and members of the claudin protein family. In addition, cytoplasmic scaffolding molecules associated with these junctions regulate diverse physiological processes like proliferation, cell polarity and regulated diffusion. In many diseases, disruption of this regulated barrier occurs. This review will briefly describe the molecular composition of the tight junctions and then present evidence of the link between tight junction dysfunction and disease. PMID:18415116

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

The balance between Gαi-Cdc42/Rac and Gα12/13-RhoA pathways determines endothelial barrier regulation by sphingosine-1-phosphate

PubMed Central

Reinhard, Nathalie R.; Mastop, Marieke; Yin, Taofei; Wu, Yi; Bosma, Esmeralda K.; Gadella, Theodorus W. J.; Goedhart, Joachim; Hordijk, Peter L.

2017-01-01

The bioactive sphingosine-1-phosphatephosphate (S1P) is present in plasma, bound to carrier proteins, and involved in many physiological processes, including angiogenesis, inflammatory responses, and vascular stabilization. S1P can bind to several G-protein–coupled receptors (GPCRs) activating a number of different signaling networks. At present, the dynamics and relative importance of signaling events activated immediately downstream of GPCR activation are unclear. To examine these, we used a set of fluorescence resonance energy transfer–based biosensors for different RhoGTPases (Rac1, RhoA/B/C, and Cdc42) as well as for heterotrimeric G-proteins in a series of live-cell imaging experiments in primary human endothelial cells. These experiments were accompanied by biochemical GTPase activity assays and transendothelial resistance measurements. We show that S1P promotes cell spreading and endothelial barrier function through S1PR1-Gαi-Rac1 and S1PR1-Gαi-Cdc42 pathways. In parallel, a S1PR2-Gα12/13-RhoA pathway is activated that can induce cell contraction and loss of barrier function, but only if Gαi-mediated signaling is suppressed. Our results suggest that Gαq activity is not involved in S1P-mediated regulation of barrier integrity. Moreover, we show that early activation of RhoA by S1P inactivates Rac1 but not Cdc42, and vice versa. Together, our data show that the rapid S1P-induced increase in endothelial integrity is mediated by a S1PR1-Gαi-Cdc42 pathway. PMID:28954861

The adaptor CRADD/RAIDD controls activation of endothelial cells by proinflammatory stimuli.

PubMed

Qiao, Huan; Liu, Yan; Veach, Ruth A; Wylezinski, Lukasz; Hawiger, Jacek

2014-08-08

A hallmark of inflammation, increased vascular permeability, is induced in endothelial cells by multiple agonists through stimulus-coupled assembly of the CARMA3 signalosome, which contains the adaptor protein BCL10. Previously, we reported that BCL10 in immune cells is targeted by the "death" adaptor CRADD/RAIDD (CRADD), which negatively regulates nuclear factor κB (NFκB)-dependent cytokine and chemokine expression in T cells (Lin, Q., Liu, Y., Moore, D. J., Elizer, S. K., Veach, R. A., Hawiger, J., and Ruley, H. E. (2012) J. Immunol. 188, 2493-2497). This novel anti-inflammatory CRADD-BCL10 axis prompted us to analyze CRADD expression and its potential anti-inflammatory action in non-immune cells. We focused our study on microvascular endothelial cells because they play a key role in inflammation. We found that CRADD-deficient murine endothelial cells display heightened BCL10-mediated expression of the pleotropic proinflammatory cytokine IL-6 and chemokine monocyte chemoattractant protein-1 (MCP-1/CCL2) in response to LPS and thrombin. Moreover, these agonists also induce significantly increased permeability in cradd(-/-), as compared with cradd(+/+), primary murine endothelial cells. CRADD-deficient cells displayed more F-actin polymerization with concomitant disruption of adherens junctions. In turn, increasing intracellular CRADD by delivery of a novel recombinant cell-penetrating CRADD protein (CP-CRADD) restored endothelial barrier function and suppressed the induction of IL-6 and MCP-1 evoked by LPS and thrombin. Likewise, CP-CRADD enhanced barrier function in CRADD-sufficient endothelial cells. These results indicate that depletion of endogenous CRADD compromises endothelial barrier function in response to inflammatory signals. Thus, we define a novel function for CRADD in endothelial cells as an inducible suppressor of BCL10, a key mediator of responses to proinflammatory agonists. © 2014 by The American Society for Biochemistry and Molecular Biology

Pertussis toxin permeabilization enhances the traversal of Escherichia coli K1, macrophages, and monocytes in a cerebral endothelial barrier model in vitro.

PubMed

Seidel, Gabriela; Böcker, Kathrin; Schulte, Jessica; Wewer, Corinna; Greune, Lilo; Humberg, Verena; Schmidt, M Alexander

2011-03-01

The occasionally severe neurological complications following the human respiratory tract infection 'whooping cough' have been attributed to pertussis toxin (PT) expressed by the causative agent Bordetella pertussis. Disruption of the endothelial blood-brain barrier (BBB) by PT might facilitate the translocation of immune cells and of hematogenous microbial pathogens. To test this hypothesis, we investigated whether PT enhances the traversal of bacteria employing human brain microvascular endothelial cells (HBMEC) as an in vitro endothelial barrier model. PT incubation significantly increased the translocation of Escherichia coli K1 across the HBMEC barrier. Only intercellular E. coli K1 bacteria could be identified by electron microscopy suggesting paracellular translocation. In addition, the migration of differentiated HL60-derived macrophages and of human monocytic U937 cells through PT-treated HBMEC barriers was also enhanced. In comparison to E. coli C600, E. coli K1 showed prolonged survival in translocated HL60-derived and J774 macrophages as well as in U937 monocytes which suggested a contribution of the 'Trojan horse' mechanism. In summary, our findings demonstrate that the PT-induced permeabilization of endothelial barriers enhances the paracellular transmigration of microbes and immune cells. In vivo, this activity might lower the threshold of bacteremia facilitating secondary cerebral infections and the subsequent development of brain pathologies. Copyright © 2010 Elsevier GmbH. All rights reserved.

Pharmacological Treatment of Hypertension: Effects in Endothelial Function.

PubMed

Cobos-Segarra, Leonardo; Lopez-Jaramillo, Patricio; Ponte-Negretti Ci, Carlos; Villar, Raul; Penaherrera, Ernesto

2018-05-07

The vascular endothelium plays a crucial role to maintain the functional integrity of the cardiovascular system through the secretion of vasoactive substances such as prostacyclin and NO. Endothelial dysfunction participate in the genesis of HTA, but also hypertension produces endothelial damage. The mayor class of antihypertensive drugs have beneficial effects in the recuperation of the endothelial function, actions that are contributing to explain the impact of the adequate control of HTA in the reduction of CV events. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Plant-derived triterpene celastrol ameliorates oxygen glucose deprivation-induced disruption of endothelial barrier assembly via inducing tight junction proteins.

PubMed

Luo, Dan; Zhao, Jia; Rong, Jianhui

2016-12-01

The integrity and functions of blood-brain barrier (BBB) are regulated by the expression and organization of tight junction proteins. The present study was designed to explore whether plant-derived triterpenoid celastrol could regulate tight junction integrity in murine brain endothelial bEnd3 cells. We disrupted the tight junctions between endothelial bEnd3 cells by oxygen glucose deprivation (OGD). We investigated the effects of celastrol on the permeability of endothelial monolayers by measuring transepithelial electrical resistance (TEER). To clarify the tight junction composition, we analyzed the expression of tight junction proteins by RT-PCR and Western blotting techniques. We found that celastrol recovered OGD-induced TEER loss in a concentration-dependent manner. Celastrol induced occludin, claudin-5 and zonula occludens-1 (ZO-1) in endothelial cells. As a result, celastrol effectively maintained tight junction integrity and inhibited macrophage migration through endothelial monolayers against OGD challenge. Further mechanistic studies revealed that celastrol induced the expression of occludin and ZO-1) via activating MAPKs and PI3K/Akt/mTOR pathway. We also observed that celastrol regulated claudin-5 expression through different mechanisms. The present study demonstrated that celastrol effectively protected tight junction integrity against OGD-induced damage. Thus, celastrol could be a drug candidate for the treatment of BBB dysfunction in various diseases. Copyright © 2016 Elsevier GmbH. All rights reserved.

Omentin protects against LPS-induced ARDS through suppressing pulmonary inflammation and promoting endothelial barrier via an Akt/eNOS-dependent mechanism.

PubMed

Qi, Di; Tang, Xumao; He, Jing; Wang, Daoxin; Zhao, Yan; Deng, Wang; Deng, Xinyu; Zhou, Guoqi; Xia, Jing; Zhong, Xi; Pu, Shenglan

2016-09-08

Acute respiratory distress syndrome (ARDS) is characterized by increased pulmonary inflammation and endothelial barrier permeability. Omentin has been shown to benefit obesity-related systemic vascular diseases; however, its effects on ARDS are unknown. In the present study, the level of circulating omentin in patients with ARDS was assessed to appraise its clinical significance in ARDS. Mice were subjected to systemic administration of adenoviral vector expressing omentin (Ad-omentin) and one-shot treatment of recombinant human omentin (rh-omentin) to examine omentin's effects on lipopolysaccharide (LPS)-induced ARDS. Pulmonary endothelial cells (ECs) were treated with rh-omentin to further investigate its underlying mechanism. We found that a decreased level of circulating omentin negatively correlated with white blood cells and procalcitonin in patients with ARDS. Ad-omentin protected against LPS-induced ARDS by alleviating the pulmonary inflammatory response and endothelial barrier injury in mice, accompanied by Akt/eNOS pathway activation. Treatment of pulmonary ECs with rh-omentin attenuated inflammatory response and restored adherens junctions (AJs), and cytoskeleton organization promoted endothelial barrier after LPS insult. Moreover, the omentin-mediated enhancement of EC survival and differentiation was blocked by the Akt/eNOS pathway inactivation. Therapeutic rh-omentin treatment also effectively protected against LPS-induced ARDS via the Akt/eNOS pathway. Collectively, these data indicated that omentin protects against LPS-induced ARDS by suppressing inflammation and promoting the pulmonary endothelial barrier, at least partially, through an Akt/eNOS-dependent mechanism. Therapeutic strategies aiming to restore omentin levels may be valuable for the prevention or treatment of ARDS.

Multiple Rap1 effectors control Epac1-mediated tightening of endothelial junctions.

PubMed

Pannekoek, Willem-Jan; Vliem, Marjolein J; Bos, Johannes L

2018-02-17

Epac1 and Rap1 mediate cAMP-induced tightening of endothelial junctions. We have previously found that one of the mechanisms is the inhibition of Rho-mediated tension in radial stress fibers by recruiting the RhoGAP ArhGAP29 in a complex containing the Rap1 effectors Rasip1 and Radil. However, other mechanisms have been proposed as well, most notably the induction of tension in circumferential actin cables by Cdc42 and its GEF FGD5. Here, we have investigated how Rap1 controls FGD5/Cdc42 and how this interconnects with Radil/Rasip1/ArhGAP29. Using endothelial barrier measurements, we show that Rho inhibition is not sufficient to explain the barrier stimulating effect of Rap1. Indeed, Cdc42-mediated tension is induced at cell-cell contacts upon Rap1 activation and this is required for endothelial barrier function. Depletion of potential Rap1 effectors identifies AF6 to mediate Rap1 enhanced tension and concomitant Rho-independent barrier function. When overexpressed in HEK293T cells, AF6 is found in a complex with FGD5 and Radil. From these results we conclude that Rap1 utilizes multiple pathways to control tightening of endothelial junctions, possibly through a multiprotein effector complex, in which AF6 functions to induce tension in circumferential actin cables.

Endothelial microparticles: Sophisticated vesicles modulating vascular function

PubMed Central

Curtis, Anne M; Edelberg, Jay; Jonas, Rebecca; Rogers, Wade T; Moore, Jonni S; Syed, Wajihuddin; Mohler, Emile R

2015-01-01

Endothelial microparticles (EMPs) belong to a family of extracellular vesicles that are dynamic, mobile, biological effectors capable of mediating vascular physiology and function. The release of EMPs can impart autocrine and paracrine effects on target cells through surface interaction, cellular fusion, and, possibly, the delivery of intra-vesicular cargo. A greater understanding of the formation, composition, and function of EMPs will broaden our understanding of endothelial communication and may expose new pathways amenable for therapeutic manipulation. PMID:23892447

Serum from dengue virus-infected patients with and without plasma leakage differentially affects endothelial cells barrier function in vitro

PubMed Central

Baimukanova, Gyulnar; Lanteri, Marion Christine; Keating, Sheila Marie; Moraes Ferreira, Frederico; Heitman, John; Pannuti, Cláudio Sérgio; Pati, Shibani; Romano, Camila Malta; Cerdeira Sabino, Ester

2017-01-01

Background Although most of cases of dengue infections are asymptomatic or mild symptomatic some individuals present warning signs progressing to severe dengue in which plasma leakage is a hallmark. Methodology/Principal findings The present study used Electric Cell-substrate Impedance Sensing (ECIS®) which allows for electrical monitoring of cellular barrier function measuring changes in Transendothelial Electric Resistance (TEER) to investigate the parameters associated with dengue induced leakage. Three groups of individuals were tested: dengue-positives with plasma leakage (leakage), dengue-positives without plasma leakage (no leakage), and dengue-negatives (control). Data show that TEER values of human umbilical vein endothelial cells (HUVECs) was significantly lower after incubation with serum from subjects of the leakage group in comparison to the no leakage or control groups. The serum levels of CXCL1, EGF, eotaxin, IFN-γ, sCD40L, and platelets were significantly decreased in the leakage group, while IL-10, IL-6, and IP-10 levels were significantly increased. We also found a strong correlation between TEER values and augmented levels of IP-10, GM-CSF, IL-1α, and IL-8, as well as decreased levels of CXCL1 and platelets. Conclusions/Significance The present work shows that the magnitude of the immune response contributes to the adverse plasma leakage outcomes in patients and that serum components are important mediators of changes in endothelial homeostasis during dengue infections. In particular, the increased levels of IP-10 and the decreased levels of CXCL1 and platelets seem to play a significant role in the disruption of vascular endothelium associated with leakage outcomes after DENV infection. These findings may have important implications for both diagnostic and therapeutic approaches to predict and mitigate vascular permeabilization in those experiencing the most severe clinical disease outcomes after dengue infection. PMID:28586397

Specific Binding, Uptake, and Transport of ICAM-1-Targeted Nanocarriers Across Endothelial and Subendothelial Cell Components of the Blood-Brain Barrier

PubMed Central

Hsu, Janet; Rappaport, Jeff; Muro, Silvia

2014-01-01

Purpose The blood-brain barrier (BBB) represents a target for therapeutic intervention and an obstacle for brain drug delivery. Targeting endocytic receptors on brain endothelial cells (ECs) helps transporting drugs and carriers into and across this barrier. While most receptors tested are associated with clathrin-mediated pathways, clathrin-independent routes are rather unexplored. We have examined the potential for one of these pathways, cell adhesion molecule (CAM)-mediated endocytosis induced by targeting intercellular adhesion molecule 1 (ICAM-1), to transport drug carriers into and across BBB models. Methods Model polymer nanocarriers (NCs) coated with control IgG or antibodies against ICAM-1 (IgG NCs vs. anti-ICAM NCs; ~250-nm) were incubated with human brain ECs, astrocytes (ACs), or pericytes (PCs) grown as monocultures or bilayered (endothelial+subendothelial) co-cultures. Results ICAM-1 was present and overexpressed in disease-like conditions on ECs and, at a lesser extent, on ACs and PCs which are BBB subendothelial components. Specific targeting and CAM-mediated uptake of anti-ICAM NCs occurred in these cells, although this was greater for ECs. Anti-ICAM NCs were transported across endothelial monolayers and endothelial+subendothelial co-cultures modeling the BBB. Conclusions CAM-mediated transport induced by ICAM-1 targeting operates in endothelial and subendothelial cellular components of the BBB, which may provide an avenue to overcome this barrier. PMID:24558007

Specific binding, uptake, and transport of ICAM-1-targeted nanocarriers across endothelial and subendothelial cell components of the blood-brain barrier.

PubMed

Hsu, Janet; Rappaport, Jeff; Muro, Silvia

2014-07-01

The blood-brain barrier (BBB) represents a target for therapeutic intervention and an obstacle for brain drug delivery. Targeting endocytic receptors on brain endothelial cells (ECs) helps transport drugs and carriers into and across this barrier. While most receptors tested are associated with clathrin-mediated pathways, clathrin-independent routes are rather unexplored. We have examined the potential for one of these pathways, cell adhesion molecule (CAM)-mediated endocytosis induced by targeting intercellular adhesion molecule -1 (ICAM-1), to transport drug carriers into and across BBB models. Model polymer nanocarriers (NCs) coated with control IgG or antibodies against ICAM-1 (IgG NCs vs. anti-ICAM NCs; ~250-nm) were incubated with human brain ECs, astrocytes (ACs), or pericytes (PCs) grown as monocultures or bilayered (endothelial+subendothelial) co-cultures. ICAM-1 was present and overexpressed in disease-like conditions on ECs and, at a lesser extent, on ACs and PCs which are BBB subendothelial components. Specific targeting and CAM-mediated uptake of anti-ICAM NCs occurred in these cells, although this was greater for ECs. Anti-ICAM NCs were transported across endothelial monolayers and endothelial+subendothelial co-cultures modeling the BBB. CAM-mediated transport induced by ICAM-1 targeting operates in endothelial and subendothelial cellular components of the BBB, which may provide an avenue to overcome this barrier.

KLF2 and KLF4 control endothelial identity and vascular integrity

PubMed Central

Sangwung, Panjamaporn; Zhou, Guangjin; Nayak, Lalitha; Chan, E. Ricky; Kang, Dong-Won; Zhang, Rongli; Lu, Yuan; Sugi, Keiki; Fujioka, Hisashi; Shi, Hong; Lapping, Stephanie D.; Ghosh, Chandra C.; Higgins, Sarah J.; Parikh, Samir M.; Jain, Mukesh K.

2017-01-01

Maintenance of vascular integrity in the adult animal is needed for survival, and it is critically dependent on the endothelial lining, which controls barrier function, blood fluidity, and flow dynamics. However, nodal regulators that coordinate endothelial identity and function in the adult animal remain poorly characterized. Here, we show that endothelial KLF2 and KLF4 control a large segment of the endothelial transcriptome, thereby affecting virtually all key endothelial functions. Inducible endothelial-specific deletion of Klf2 and/or Klf4 reveals that a single allele of either gene is sufficient for survival, but absence of both (EC-DKO) results in acute death from myocardial infarction, heart failure, and stroke. EC-DKO animals exhibit profound compromise in vascular integrity and profound dysregulation of the coagulation system. Collectively, these studies establish an absolute requirement for KLF2/4 for maintenance of endothelial and vascular integrity in the adult animal. PMID:28239661

Endothelial dysfunction in dengue virus pathology.

PubMed

Vervaeke, Peter; Vermeire, Kurt; Liekens, Sandra

2015-01-01

Dengue virus (DENV) is a leading cause of illness and death, mainly in the (sub)tropics, where it causes dengue fever and/or the more serious diseases dengue hemorrhagic fever and dengue shock syndrome that are associated with changes in vascular permeability. Despite extensive research, the pathogenesis of DENV is still poorly understood and, although endothelial cells represent the primary fluid barrier of the blood vessels, the extent to which these cells contribute to DENV pathology is still under debate. The primary target cells for DENV are dendritic cells and monocytes/macrophages that release various chemokines and cytokines upon infection, which can activate the endothelium and are thought to play a major role in DENV-induced vascular permeability. However, recent studies indicate that DENV also replicates in endothelial cells and that DENV-infected endothelial cells may directly contribute to viremia, immune activation, vascular permeability and immune targeting of the endothelium. Also, the viral non-structural protein-1 and antibodies directed against this secreted protein have been reported to be involved in endothelial cell dysfunction. This review provides an extensive overview of the effects of DENV infection on endothelial cell physiology and barrier function. Copyright © 2014 John Wiley & Sons, Ltd.

Endothelial Barrier and Metabolism: New Kids on the Block Regulating Bone Marrow Vascular Niches.

PubMed

Harjes, Ulrike; Verfaillie, Catherine; Carmeliet, Peter

2016-05-09

The vasculature of the bone marrow remains poorly characterized, yet crucial to maintain hematopoiesis and retain stem cells in a quiescent state. A recent study by Itkin et al. (2016) in Nature reports how vascular barrier integrity and endothelial cell metabolism regulate hematopoietic stem cell quiescence and leukocyte trafficking. Copyright © 2016 Elsevier Inc. All rights reserved.

Human astrocytes/astrocyte conditioned medium and shear stress enhance the barrier properties of human brain microvascular endothelial cells

PubMed Central

Siddharthan, Venkatraman; V. Kim, Yuri; Liu, Suyi; Kim, Kwang Sik

2009-01-01

The blood-brain barrier (BBB) is a structural and functional barrier that regulates the passage of molecules into and out of the brain to maintain the neural microenvironment. We have previously developed the in vitro BBB model with human brain microvascular endothelial cells (HBMEC). However, in vivo HBMEC are shown to interact with astrocytes and also exposed to shear stress through blood flow. In an attempt to develop the BBB model to mimic the in vivo condition we constructed the flow-based in vitro BBB model using HBMEC and human fetal astrocytes (HFA). We also examined the effect of astrocyte conditioned medium (ACM) in lieu of HFA to study the role of secreted factor(s) on the BBB properties. The tightness of HBMEC monolayer was assessed by the permeability of dextran and propidium iodide as well as by measuring the transendothelial electrical resistance (TEER). We showed that the HBMEC permeability was reduced and TEER was increased by non-contact, co-cultivation with HFA and ACM. The exposure of HBMEC to shear stress also exhibited decreased permeability. Moreover, HFA/ACM and shear flow exhibited additive effect of decreasing the permeability of HBMEC monolayer. In addition, we showed that the HBMEC expression of ZO-1 (tight junction protein) was increased by co-cultivation with ACM and in response to shear stress. These findings suggest that the non-contact co-cultivation with HFA helps maintain the barrier properties of HBMEC by secreting factor(s) into the medium. Our in vitro flow model system with the cells of human origin should be useful for studying the interactions between endothelial cells, glial cells, and secreted factor(s) as well as the role of shear stress in the barrier property of HBMEC. PMID:17368578

Wnt/β-catenin signaling controls development of the blood–brain barrier

PubMed Central

Liebner, Stefan; Corada, Monica; Bangsow, Thorsten; Babbage, Jane; Taddei, Andrea; Czupalla, Cathrin J.; Reis, Marco; Felici, Angelina; Wolburg, Hartwig; Fruttiger, Marcus; Taketo, Makoto M.; von Melchner, Harald; Plate, Karl Heinz; Gerhardt, Holger; Dejana, Elisabetta

2008-01-01

The blood–brain barrier (BBB) is confined to the endothelium of brain capillaries and is indispensable for fluid homeostasis and neuronal function. In this study, we show that endothelial Wnt/β-catenin (β-cat) signaling regulates induction and maintenance of BBB characteristics during embryonic and postnatal development. Endothelial specific stabilization of β-cat in vivo enhances barrier maturation, whereas inactivation of β-cat causes significant down-regulation of claudin3 (Cldn3), up-regulation of plamalemma vesicle-associated protein, and BBB breakdown. Stabilization of β-cat in primary brain endothelial cells (ECs) in vitro by N-terminal truncation or Wnt3a treatment increases Cldn3 expression, BBB-type tight junction formation, and a BBB characteristic gene signature. Loss of β-cat or inhibition of its signaling abrogates this effect. Furthermore, stabilization of β-cat also increased Cldn3 and barrier properties in nonbrain-derived ECs. These findings may open new therapeutic avenues to modulate endothelial barrier function and to limit the devastating effects of BBB breakdown. PMID:18955553

Loss of tight junction barrier function and its role in cancer metastasis.

PubMed

Martin, Tracey A; Jiang, Wen G

2009-04-01

As the most apical structure between epithelial and endothelial cells, tight junctions (TJ) are well known as functioning as a control for the paracellular diffusion of ions and certain molecules. It has however, become increasingly apparent that the TJ has a vital role in maintaining cell to cell integrity and that the loss of cohesion of the structure can lead to invasion and thus metastasis of cancer cells. This article will present data showing how modulation of expression of TJ molecules results in key changes in TJ barrier function leading to the successful metastasis of a number of different cancer types.

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.

Effect of extracorporeal cytokine removal on vascular barrier function in a septic shock patient.

PubMed

David, Sascha; Thamm, Kristina; Schmidt, Bernhard M W; Falk, Christine S; Kielstein, Jan T

2017-01-01

Sepsis and septic shock are major healthcare problems, affecting millions of individuals around the world each year. Pathophysiologically, septic multiple organ dysfunction (MOD) is a life-threatening condition caused by an overwhelming systemic inflammatory response of the host's organism to an infection. We experimentally tested if high circulating cytokine levels might increase vascular permeability-a critical hallmark of the disease-and if this phenomenon can be reversed by therapeutic cytokine removal (CytoSorb®) in an exemplary patient. A 32-year-old Caucasian female presented with septic shock and accompanying acute kidney injury (Sequential Organ Failure Assessment (SOFA) = 18) to our ICU. In spite of a broad anti-infective regimen, adequate fluid resuscitation, and high doses of inotropics and catecholamines, she remained refractory hypotensive. The extraordinary severity of septic shock suggested an immense overwhelming host response assumingly accompanied by a notable cytokine storm such as known from patients with toxic shock syndrome. Thus, a CytoSorb® filter was added to the dialysis circuit to remove excess shock-perpetuating cytokines. To analyze the endothelial phenotype in vitro before and after extracorporeal cytokine removal, we tested the septic shock patient's serum on human umbilical vein endothelial cells (HUVECs). The effect on endothelial integrity was assessed both on the morphological (fluorescent immunocytochemistry for VE-cadherin and F-actin) and functional (transendothelial electrical resistance (TER)) level that was recorded in real time with an "electric cell-substrate impedance sensing" (ECIS) system (ibidi). We found (1) severe alterations of cell-cell contacts and the cytoskeletal architecture and (2) profound functional permeability changes, the putative cellular correlate of the clinical vascular leakage syndrome. However, the endothelial barrier was protected from these profound adverse effects when HUVECs were challenged

Endothelial cells use dynamic actin to facilitate lymphocyte transendothelial migration and maintain the monolayer barrier.

PubMed

Mooren, Olivia L; Li, Jinmei; Nawas, Julie; Cooper, John A

2014-12-15

The vascular endothelium is a highly dynamic structure, and the integrity of its barrier function is tightly regulated. Normally impenetrable to cells, the endothelium actively assists lymphocytes to exit the bloodstream during inflammation. The actin cytoskeleton of the endothelial cell (EC) is known to facilitate transmigration, but the cellular and molecular mechanisms are not well understood. Here we report that actin assembly in the EC, induced by Arp2/3 complex under control of WAVE2, is important for several steps in the process of transmigration. To begin transmigration, ECs deploy actin-based membrane protrusions that create a cup-shaped docking structure for the lymphocyte. We found that docking structure formation involves the localization and activation of Arp2/3 complex by WAVE2. The next step in transmigration is creation of a migratory pore, and we found that endothelial WAVE2 is needed for lymphocytes to follow a transcellular route through an EC. Later, ECs use actin-based protrusions to close the gap behind the lymphocyte, which we discovered is also driven by WAVE2. Finally, we found that ECs in resting endothelial monolayers use lamellipodial protrusions dependent on WAVE2 to form and maintain contacts and junctions between cells. © 2014 Mooren et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Transplantation of in vitro cultured endothelial progenitor cells repairs the blood-brain barrier and improves cognitive function of APP/PS1 transgenic AD mice.

PubMed

Zhang, Shishuang; Zhi, Yongle; Li, Fei; Huang, Shan; Gao, Huabin; Han, Zhaoli; Ge, Xintong; Li, Dai; Chen, Fanglian; Kong, Xiaodong; Lei, Ping

2018-04-15

To date, the pathogenesis of Alzheimer's disease (AD) remains unclear. It is well-known that excessive deposition of Aβ in the brain is a crucial part of the pathogenesis of AD. In recent years, the AD neurovascular unit hypothesis has attracted much attention. Impairment of the blood-brain barrier (BBB) leads to abnormal amyloid-β (Aβ) transport, and chronic cerebral hypoperfusion causes Aβ deposition throughout the onset and progression of AD. Endothelial progenitor cells (EPCs) are the universal cells for repairing blood vessels. Our previous studies have shown that a reduced number of EPCs in the peripheral blood results in cerebral vascular repair disorder, cerebral hypoperfusion and neurodegeneration, which might be related to the cognitive dysfunction of AD patients. This study was designed to confirm whether EPCs transplantation could repair the blood-brain barrier, stimulate angiogenesis and reduce Aβ deposition in AD. The expression of ZO-1, Occludin and Claudin-5 was up-regulated in APP/PS1 transgenic mice after hippocampal transplantation of EPCs. Consistent with previous studies, EPC transplants also increased the microvessel density. We observed that Aβ senile plaque deposition was decreased and hippocampal cell apoptosis was reduced after EPCs transplantation. The Morris water maze test showed that spatial learning and memory functions were significantly improved in mice transplanted with EPCs. Consequently, EPCs could up-regulate the expression of tight junction proteins, repair BBB tight junction function, stimulate angiogenesis, promote Aβ clearance, and decrease neuronal loss, ultimately improve cognitive function. Taken together, these data demonstrate EPCs may play an important role in the therapeutic implications for vascular dysfunction in AD. Copyright © 2018 Elsevier B.V. All rights reserved.

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

Endothelial function is associated with myocardial diastolic function in women with systemic lupus erythematosus.

PubMed

Chin, Calvin W L; Chin, Chee-Yang; Ng, Marie X R; Le, Thu-Thao; Huang, Fei-Qiong; Fong, Kok-Yong; Thumboo, Julian; Tan, Ru-San

2014-09-01

Endothelial dysfunction is associated with traditional and systemic lupus erythematosus (SLE)-specific risk factors, and early data suggest reversibility of endothelial dysfunction with therapy. The clinical relevance of endothelial function assessment has been limited by the lack of studies, demonstrating its prognostic significance and impact on early myocardial function. Therefore, we aimed to determine the association between endothelial and myocardial diastolic function in SLE women. Women with SLE and no coronary artery disease were prospectively recruited and underwent radionuclide myocardial perfusion imaging (MPI) (Jetstream, Philips, the Netherlands) to exclude subclinical myocardial ischemia. Cardiac and vascular functions were assessed in all patients (Alpha 10, Aloka, Tokyo). Diastolic function was assessed using pulse wave early (E) and late mitral blood inflow and myocardial tissue Doppler (mean of medial and lateral annulus e') velocities. Endothelial function was measured using brachial artery flow-mediated vasodilatation (FMD%). Univariate and multivariate linear regressions were used to assess the association between FMD% and myocardial diastolic function, adjusting for potential confounders. Thirty-eight patients without detectable myocardial ischemia on MPI were studied (mean age 44 ± 10 years; mean disease duration 14 ± 6 years). About 61 % of patients had normal diastolic function (E/e' ≤ 8), and 5 % of patients had definite diastolic dysfunction with E/e' > 13 (mean 7.1 ± 2.9). FMD% was associated with E/e' (regression coefficient β = -0.35; 95 % CI -0.62 to -0.08; p = 0.01) independent of systolic blood pressure, age, and SLICC/ACR Damage Index.

Bile duct epithelial tight junctions and barrier function

PubMed Central

Rao, R.K.; Samak, G.

2013-01-01

Bile ducts play a crucial role in the formation and secretion of bile as well as excretion of circulating xenobiotic substances. In addition to its secretory and excretory functions, bile duct epithelium plays an important role in the formation of a barrier to the diffusion of toxic substances from bile into the hepatic interstitial tissue. Disruption of barrier function and toxic injury to liver cells appear to be involved in the pathogenesis of a variety of liver diseases such as primary sclerosing cholangitis, primary biliary cirrhosis and cholangiocarcinoma. Although the investigations into understanding the structure and regulation of tight junctions in gut, renal and endothelial tissues have expanded rapidly, very little is known about the structure and regulation of tight junctions in the bile duct epithelium. In this article we summarize the current understanding of physiology and pathophysiology of bile duct epithelium, the structure and regulation of tight junctions in canaliculi and bile duct epithelia and different mechanisms involved in the regulation of disruption and protection of bile duct epithelial tight junctions. This article will make a case for the need of future investigations toward our understanding of molecular organization and regulation of canalicular and bile duct epithelial tight junctions. PMID:24665411

Glycolysis-mediated control of blood-brain barrier development and function.

PubMed

Salmina, Alla B; Kuvacheva, Natalia V; Morgun, Andrey V; Komleva, Yulia K; Pozhilenkova, Elena A; Lopatina, Olga L; Gorina, Yana V; Taranushenko, Tatyana E; Petrova, Lyudmila L

2015-07-01

The blood-brain barrier (BBB) consists of differentiated cells integrating in one ensemble to control transport processes between the central nervous system (CNS) and peripheral blood. Molecular organization of BBB affects the extracellular content and cell metabolism in the CNS. Developmental aspects of BBB attract much attention in recent years, and barriergenesis is currently recognized as a very important and complex mechanism of CNS development and maturation. Metabolic control of angiogenesis/barriergenesis may be provided by glucose utilization within the neurovascular unit (NVU). The role of glycolysis in the brain has been reconsidered recently, and it is recognized now not only as a process active in hypoxic conditions, but also as a mechanism affecting signal transduction, synaptic activity, and brain development. There is growing evidence that glycolysis-derived metabolites, particularly, lactate, affect barriergenesis and functioning of BBB. In the brain, lactate produced in astrocytes or endothelial cells can be transported to the extracellular space via monocarboxylate transporters (MCTs), and may act on the adjoining cells via specific lactate receptors. Astrocytes are one of the major sources of lactate production in the brain and significantly contribute to the regulation of BBB development and functioning. Active glycolysis in astrocytes is required for effective support of neuronal activity and angiogenesis, while endothelial cells regulate bioavailability of lactate for brain cells adjusting its bidirectional transport through the BBB. In this article, we review the current knowledge with regard to energy production in endothelial and astroglial cells within the NVU. In addition, we describe lactate-driven mechanisms and action of alternative products of glucose metabolism affecting BBB structural and functional integrity in developing and mature brain. Copyright © 2015 Elsevier Ltd. All rights reserved.

Endothelial NOS activation induces the blood-brain barrier disruption via ER stress following status epilepticus.

PubMed

Ko, Ah-Reum; Kim, Ji Yang; Hyun, Hye-Won; Kim, Ji-Eun

2015-10-05

The blood-brain barrier (BBB) maintains the unique brain microenvironment, which is separated from the systemic circulating system. Since the endoplasmic reticulum (ER) is an important cell organelle that is responsible for protein synthesis, the correct folding and sorting of proteins contributing to cell survivals, ER stress is a potential cause of cell damage in various diseases. Therefore, it would be worthy to explore the the relationship between the ER stress and BBB disruption during vasogenic edema formation induced by epileptogenic insults. In the present study, we investigated the roles of ER stress in vasogenic edema and its related events in rat epilepsy models provoked by pilocarpine-induced status epilepticus (SE). SE-induced eNOS activation induces BBB breakdown via up-regulation of GRP78 expression and dysfunction of SMI-71 (an endothelial BBB marker) in the piriform cortex (PC). In addition, caveolin-1 peptide (an eNOS inhibitor) effectively attenuated GRP78 expression and down-regulation of SMI-71. Taken together, our findings suggest that eNOS-mediated ER stress may participate in SE-induced vasogenic edema formation. Therefore, the modulation of ER stress may be a considerable strategy for therapy in impairments of endothelial cell function. Copyright © 2015 Elsevier B.V. All rights reserved.

Ischemic preconditioning enhances integrity of coronary endothelial tight junctions

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

Li, Zhao; Jin, Zhu-Qiu, E-mail: zhu-qiu.jin@sdstate.edu

2012-08-31

Highlights: Black-Right-Pointing-Pointer Cardiac tight junctions are present between coronary endothelial cells. Black-Right-Pointing-Pointer Ischemic preconditioning preserves the structural and functional integrity of tight junctions. Black-Right-Pointing-Pointer Myocardial edema is prevented in hearts subjected to ischemic preconditioning. Black-Right-Pointing-Pointer Ischemic preconditioning enhances translocation of ZO-2 from cytosol to cytoskeleton. -- Abstract: Ischemic preconditioning (IPC) is one of the most effective procedures known to protect hearts against ischemia/reperfusion (IR) injury. Tight junction (TJ) barriers occur between coronary endothelial cells. TJs provide barrier function to maintain the homeostasis of the inner environment of tissues. However, the effect of IPC on the structure and function of cardiacmore » TJs remains unknown. We tested the hypothesis that myocardial IR injury ruptures the structure of TJs and impairs endothelial permeability whereas IPC preserves the structural and functional integrity of TJs in the blood-heart barrier. Langendorff hearts from C57BL/6J mice were prepared and perfused with Krebs-Henseleit buffer. Cardiac function, creatine kinase release, and myocardial edema were measured. Cardiac TJ function was evaluated by measuring Evans blue-conjugated albumin (EBA) content in the extravascular compartment of hearts. Expression and translocation of zonula occludens (ZO)-2 in IR and IPC hearts were detected with Western blot. A subset of hearts was processed for the observation of ultra-structure of cardiac TJs with transmission electron microscopy. There were clear TJs between coronary endothelial cells of mouse hearts. IR caused the collapse of TJs whereas IPC sustained the structure of TJs. IR increased extravascular EBA content in the heart and myocardial edema but decreased the expression of ZO-2 in the cytoskeleton. IPC maintained the structure of TJs. Cardiac EBA content and edema were reduced in IPC

Microvascular endothelial function and cognitive performance: The ELSA-Brasil cohort study.

PubMed

Brant, Luisa; Bos, Daniel; Araujo, Larissa Fortunato; Ikram, M Arfan; Ribeiro, Antonio Lp; Barreto, Sandhi M

2018-06-01

Impaired microvascular endothelial function may be implicated in the etiology of cognitive decline. Yet, current data on this association are inconsistent. Our objective is to investigate the relation of microvascular endothelial function to cognitive performance in the ELSA-Brasil cohort study. A total of 1521 participants from ELSA-Brasil free of dementia underwent peripheral arterial tonometry (PAT) to quantify microvascular endothelial function (PAT-ratio and mean baseline pulse amplitude (BPA)) and cognitive tests that covered the domains of memory, verbal fluency, and executive function at baseline. Cognitive tests in participants aged 55 years old and above were repeated during the second examination (mean follow-up: 3.5 (0.3) years). Linear regression and generalized linear models were used to evaluate the association between endothelial function, global cognitive performance, and performance on specific cognitive domains. In unadjusted cross-sectional analyses, we found that BPA and PAT-ratio were associated with worse global cognitive performance (mean difference for BPA: -0.07, 95% CI: -0.11; -0.03, p<0.01; mean difference for PAT-ratio: 0.11, 95% CI: 0.01; 0.20, p=0.02), worse performance on learning, recall, and word recognition tests (BPA: -0.87, 95% CI: -1.21; -0.52, p<0.01; PAT-ratio: 1.58, 95% CI: 0.80; 2.36, p<0.01), and only BPA was associated with worse performance in verbal fluency tests (-0.70, 95% CI: -1.19; -0.21, p<0.01). Adjustments for age, sex, and level of education rendered the associations statistically non-significant. Longitudinally, there was no association between microvascular endothelial and cognitive functions. The associations between microvascular endothelial function and cognition are explained by age, sex, and educational level. Measures of microvascular endothelial function may be of limited value with regard to preclinical cognitive deficits.

Adiponectin in Fresh Frozen Plasma Contributes to Restoration of Vascular Barrier Function after Hemorrhagic Shock

PubMed Central

Huby, Maria P.; Duan, Chaojun; Baer, Lisa; Peng, Zhanglong; Kozar, Rosemary A.; Doursout, Marie-Francoise; Holcomb, John B.; Wade, Charles E.; Ko, Tien C.

2015-01-01

Hemorrhagic shock is the leading cause of preventable deaths in civilian and military trauma. Use of fresh frozen plasma (FFP) in patients requiring massive transfusion is associated with improved outcomes. FFP contains significant amounts of adiponectin, which is known to have vascular protective function. We hypothesize that FFP improves vascular barrier function largely via adiponectin. Plasma adiponectin levels were measured in 19 severely injured patients in hemorrhagic shock (HS). Compared to normal individuals, plasma adiponectin levels decreased to 49% in HS patients prior to resuscitation (p<0.05) and increased to 64% post resuscitation (but not significant). In a HS mouse model, we demonstrated a similar decrease in plasma adiponectin to 54% but a significant increase to 79% by FFP resuscitation compared to baseline (p<0.05). HS disrupted lung vascular barrier function, leading to an increase in permeability. FFP resuscitation reversed these HS-induced effects. Immunodepletion of adiponectin from FFP abolished FFP's effects on blocking endothelial hyperpermeability in vitro, and on improving lung vascular barrier function in HS mice. Replenishment with adiponectin rescued FFP's effects. These findings suggest that adiponectin is an important component in FFP resuscitation contributing to the beneficial effects on vascular barrier function after HS. PMID:26263440

The endothelial glycocalyx: composition, functions, and visualization

PubMed Central

Reitsma, Sietze; Slaaf, Dick W.; Vink, Hans; van Zandvoort, Marc A. M. J.

2007-01-01

This review aims at presenting state-of-the-art knowledge on the composition and functions of the endothelial glycocalyx. The endothelial glycocalyx is a network of membrane-bound proteoglycans and glycoproteins, covering the endothelium luminally. Both endothelium- and plasma-derived soluble molecules integrate into this mesh. Over the past decade, insight has been gained into the role of the glycocalyx in vascular physiology and pathology, including mechanotransduction, hemostasis, signaling, and blood cell–vessel wall interactions. The contribution of the glycocalyx to diabetes, ischemia/reperfusion, and atherosclerosis is also reviewed. Experimental data from the micro- and macrocirculation alludes at a vasculoprotective role for the glycocalyx. Assessing this possible role of the endothelial glycocalyx requires reliable visualization of this delicate layer, which is a great challenge. An overview is given of the various ways in which the endothelial glycocalyx has been visualized up to now, including first data from two-photon microscopic imaging. PMID:17256154

PGC-1α dictates endothelial function through regulation of eNOS expression

PubMed Central

Craige, Siobhan M.; Kröller-Schön, Swenja; Li, Chunying; Kant, Shashi; Cai, Shenghe; Chen, Kai; Contractor, Mayur M.; Pei, Yongmei; Schulz, Eberhard; Keaney, John F.

2016-01-01

Endothelial dysfunction is a characteristic of many vascular related diseases such as hypertension. Peroxisome proliferator activated receptor gamma, coactivator 1α (PGC-1α) is a unique stress sensor that largely acts to promote adaptive responses. Therefore, we sought to define the role of endothelial PGC-1α in vascular function using mice with endothelial specific loss of function (PGC-1α EC KO) and endothelial specific gain of function (PGC-1α EC TG). Here we report that endothelial PGC-1α is suppressed in angiotensin-II (ATII)-induced hypertension. Deletion of endothelial PGC-1α sensitized mice to endothelial dysfunction and hypertension in response to ATII, whereas PGC-1α EC TG mice were protected. Mechanistically, PGC-1α promotes eNOS expression and activity, which is necessary for protection from ATII-induced dysfunction as mice either treated with an eNOS inhibitor (LNAME) or lacking eNOS were no longer responsive to transgenic endothelial PGC-1α expression. Finally, we determined that the orphan nuclear receptor, estrogen related receptor α (ERRα) is required to coordinate the PGC-1α -induced eNOS expression. In conclusion, endothelial PGC-1α expression protects from vascular dysfunction by promoting NO• bioactivity through ERRα induced expression of eNOS. PMID:27910955

Cathepsin D plays a role in endothelial-pericyte interactions during alteration of the blood-retinal barrier in diabetic retinopathy.

PubMed

Monickaraj, Finny; McGuire, Paul; Das, Arup

2018-05-01

Inflammation plays an important role in the pathogenesis of diabetic retinopathy. We have previously demonstrated the effect of cathepsin D (CD) on the mechanical disruption of retinal endothelial cell junctions and increased vasopermeability, as well as increased levels of CD in retinas of diabetic mice. Here, we have also examined the effect of CD on endothelial-pericyte interactions, as well as the effect of dipeptidyl peptidase-4 (DPP4) inhibitor on CD in endothelial-pericyte interactions in vitro and in vivo. Cocultured cells that were treated with pro-CD demonstrated a significant decrease in the expression of platelet-derived growth factor receptor-β, a tyrosine kinase receptor that is required for pericyte cell survival; N-cadherin, the key adherens junction protein between endothelium and pericytes; and increases in the vessel destabilizing agent, angiopoietin-2. The effect was reversed in cells that were treated with DPP4 inhibitor along with pro-CD. With pro-CD treatment, there was a significant increase in the phosphorylation of the downstream signaling protein, PKC-α, and Ca 2+ /calmodulin-dependent protein kinase II in endothelial cells and pericytes, which disrupts adherens junction structure and function, and this was significantly reduced with DPP4 inhibitor treatment. Increased CD levels, vasopermeability, and alteration in junctional-related proteins were observed in the retinas of diabetic rats, which were significantly changed with DPP4 inhibitor treatment. Thus, DPP4 inhibitors may be used as potential adjuvant therapeutic agents to treat increased vascular leakage observed in patients with diabetic macular edema.-Monickaraj, F., McGuire, P., Das, A. Cathepsin D plays a role in endothelial-pericyte interactions during alteration of the blood-retinal barrier in diabetic retinopathy.

Regulation of Human Brain Microvascular Endothelial Cell Adhesion and Barrier Functions by Memantine.

PubMed

Wang, Fei; Zou, Zhirong; Gong, Yi; Yuan, Dong; Chen, Xun; Sun, Tao

2017-05-01

Vascular risk factors have been linked to cognitive decline and dementia in the elderly. Microvascular inflammation, especially of the endothelium, may contribute to the progression of neurodegenerative events in Alzheimer's disease (AD). Memantine, an uncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist, is a licensed drug used for the treatment of moderate to severe AD. However, little information is available regarding its anti-inflammatory effects on the endothelium. In this study, we investigated the effects of memantine on human brain microvascular endothelial dysfunction induced by the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α). Our results show that memantine prevents the attachment of monocyte THP-1 cells to human brain microvascular endothelial cells (HBMVEs). An in vitro BBB model experiment displayed that memantine could rescue TNF-α-induced disruption of the in vitro BBB model. In addition, memantine also interferes with monocyte transmigration across the BBB model. Our results indicate that TNF-α significantly increased the expression of cell adhesion molecules, such as ICAM-1, VCAM-1, and E-selectin, which was prevented by pretreatment with memantine. Mechanistically, memantine reversed activation of the transcription factor NF-κB by preventing the phosphorylation and degradation of its inhibitor IκBα. Our data is the first to describe a novel anti-inflammatory mechanism driven by the endothelial cell-mediated neuroprotective effects of memantine.

Blood-nerve barrier: distribution of anionic sites on the endothelial plasma membrane and basal lamina of dorsal root ganglia.

PubMed

Bush, M S; Reid, A R; Allt, G

1991-09-01

Previous investigations of the blood-nerve barrier have correlated the greater permeability of ganglionic endoneurial vessels, compared to those of nerve trunks, with the presence of fenestrations and open intercellular junctions. Recent studies have demonstrated reduced endothelial cell surface charge in blood vessels showing greater permeability. To determine the distribution of anionic sites on the plasma membranes and basal laminae of endothelial cells in dorsal root ganglia, cationic colloidal gold and cationic ferritin were used. Electron microscopy revealed the existence of endothelial microdomains with differing labelling densities. Labelling indicated that caveolar and fenestral diaphragms and basal laminae are highly anionic at physiological pH, luminal plasma membranes and endothelial processes are moderately charged and abluminal plasma membranes are weakly anionic. Tracers did not occur in caveolae or cytoplasmic vesicles. In vitro tracer experiments at pH values of 7.3, 5.0, 3.5 and 2.0 indicated that the anionic charge on the various endothelial domains was contributed by chemical groups with differing pKa values. In summary, the labelling of ganglionic and sciatic nerve vessels was similar except for the heavy labelling of diaphragms in a minority of endoneurial vessels in ganglia. This difference is likely to account in part for the greater permeability of ganglionic endoneurial vessels. The results are discussed with regard to the blood-nerve and -brain barriers and vascular permeability in other tissues and a comparison made between the ultrastructure and anionic microdomains of epi-, peri- and endoneurial vessels of dorsal root ganglia and sciatic nerves.

Effects of Flavonoid-Containing Beverages and EGCG on Endothelial Function

PubMed Central

Shenouda, Sherene M.; Vita, Joseph A.

2009-01-01

Abnormalities of the vascular endothelium contribute to all stages of atherosclerosis from lesion development to clinical cardiovascular disease events. Recognized risk factors, including diabetes mellitus, hypertension, dyslipidemia, cigarette smoking, and sedentary lifestyle are associated with endothelial dysfunction. A variety of pharmacological and behavioral interventions have been shown to reverse endothelial dysfunction in patients with cardiovascular disease. A large number of epidemiological studies suggest that dietary factors, including increased intake of flavonoid-containing foods and beverages, reduce cardiovascular risk, and recent studies have shown that such beverages have favorable effects on endothelial function. These studies have engendered interest in the development of dietary supplements or drugs that would allow for more convenient and higher dose administration of flavonoids and might prove useful for prevention or treatment of cardiovascular disease. In this paper, we will review the contribution of endothelial dysfunction to the pathogenesis and clinical expression of atherosclerosis and recent data linking flavonoid and EGCG consumption to improved endothelial function and reduced cardiovascular risk. PMID:17906190

Atorvastatin affects negatively respiratory function of isolated endothelial mitochondria.

PubMed

Broniarek, Izabela; Jarmuszkiewicz, Wieslawa

2018-01-01

The purpose of this research was to elucidate the direct effects of two popular blood cholesterol-lowering drugs used to treat cardiovascular diseases, atorvastatin and pravastatin, on respiratory function, membrane potential, and reactive oxygen species formation in mitochondria isolated from human umbilical vein endothelial cells (EA.hy926 cell line). Hydrophilic pravastatin did not significantly affect endothelial mitochondria function. In contrast, hydrophobic calcium-containing atorvastatin induced a loss of outer mitochondrial membrane integrity, an increase in hydrogen peroxide formation, and reductions in maximal (phosphorylating or uncoupled) respiratory rate, membrane potential and oxidative phosphorylation efficiency. The atorvastatin-induced changes indicate an impairment of mitochondrial function at the level of ATP synthesis and at the level of the respiratory chain, likely at complex I and complex III. The atorvastatin action on endothelial mitochondria was highly dependent on calcium ions and led to a disturbance in mitochondrial calcium homeostasis. Uptake of calcium ions included in atorvastatin molecule induced mitochondrial uncoupling that enhanced the inhibition of the mitochondrial respiratory chain by atorvastatin. Our results indicate that hydrophobic calcium-containing atorvastatin, widely used as anti-atherosclerotic agent, has a direct negative action on isolated endothelial mitochondria. Copyright © 2017. Published by Elsevier Inc.

XOR inhibition with febuxostat accelerates pulmonary endothelial barrier recovery and improves survival in lipopolysaccharide-induced murine sepsis.

PubMed

Damarla, Mahendra; Johnston, Laura F; Liu, Gigi; Gao, Li; Wang, Lan; Varela, Lidenys; Kolb, Todd M; Kim, Bo S; Damico, Rachel L; Hassoun, Paul M

2017-08-01

Sepsis is a leading cause of death among patients in the intensive care unit, resulting from multi-organ failure. Activity of xanthine oxidoreductase (XOR), a reactive oxygen species (ROS) producing enzyme, is known to be elevated in nonsurvivors of sepsis compared to survivors. We have previously demonstrated that XOR is critical for ventilator-induced lung injury. Using febuxostat, a novel nonpurine inhibitor of XOR, we sought to determine the role of XOR inhibition in a murine model of sepsis-induced lung injury and mortality. C57BL/6J mice were subjected to intravenous (IV) lipopolysaccharide (LPS) for various time points, and lungs were harvested for analyses. Subsets of mice were treated with febuxostat, pre or post LPS exposure, or vehicle. Separate groups of mice were followed up for mortality after LPS exposure. After 24 hr of IV LPS , mice exhibited an increase in XOR activity in lung tissue and a significant increase in pulmonary endothelial barrier disruption. Pretreatment of animals with febuxostat before exposure to LPS, or treatment 4 h after LPS, resulted in complete abrogation of XOR activity. Inhibition of XOR with febuxostat did not prevent LPS-induced pulmonary vascular permeability at 24 h, however, it accelerated recovery of the pulmonary endothelial barrier integrity in response to LPS exposure. Furthermore, treatment with febuxostat resulted in significant reduction in mortality. Inhibition of XOR with febuxostat accelerates recovery of the pulmonary endothelial barrier and prevents LPS-induced mortality, whether given before or after exposure to LPS. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Cholesteryl ester transfer protein inhibition enhances endothelial repair and improves endothelial function in the rabbit.

PubMed

Wu, Ben J; Shrestha, Sudichhya; Ong, Kwok L; Johns, Douglas; Hou, Liming; Barter, Philip J; Rye, Kerry-Anne

2015-03-01

High-density lipoproteins (HDLs) can potentially protect against atherosclerosis by multiple mechanisms, including enhancement of endothelial repair and improvement of endothelial function. This study asks if increasing HDL levels by inhibiting cholesteryl ester transfer protein activity with the anacetrapib analog, des-fluoro-anacetrapib, enhances endothelial repair and improves endothelial function in New Zealand White rabbits with balloon injury of the abdominal aorta. New Zealand White rabbits received chow or chow supplemented with 0.07% or 0.14% (wt/wt) des-fluoro-anacetrapib for 8 weeks. Endothelial denudation of the abdominal aorta was carried out after 2 weeks. The animals were euthanized 6 weeks postinjury. Treatment with 0.07% and 0.14% des-fluoro-anacetrapib reduced cholesteryl ester transfer protein activity by 81±4.9% and 92±12%, increased plasma apolipoprotein A-I levels by 1.4±0.1-fold and 1.5±0.1-fold, increased plasma HDL-cholesterol levels by 1.8±0.2-fold and 1.9±0.1-fold, reduced intimal hyperplasia by 37±11% and 51±10%, and inhibited vascular cell proliferation by 25±6.1% and 35±6.7%, respectively. Re-endothelialization of the injured aorta increased from 43±6.7% (control) to 69±6.6% and 76±7.7% in the 0.07% and 0.14% des-fluoro-anacetrapib-treated animals, respectively. Aortic ring relaxation and guanosine 3',5'-cyclic monophosphate production in response to acetylcholine were also improved. Incubation of HDLs from the des-fluoro-anacetrapib-treated animals with human coronary artery endothelial cells increased cell proliferation and migration relative to control. These effects were abolished by knockdown of scavenger receptor-B1 and PDZ domain-containing protein 1 and by pharmacological inhibition of phosphatidylinositol-4,5-bisphosphate 3-kinase/Akt. Increasing HDL levels by inhibiting cholesteryl ester transfer protein reduces intimal thickening and regenerates functional endothelium in damaged New Zealand White rabbit aortas

Trauma exposure and endothelial function among midlife women.

PubMed

Thurston, Rebecca C; Barinas-Mitchell, Emma; von Känel, Roland; Chang, Yuefang; Koenen, Karestan C; Matthews, Karen A

2018-04-01

Trauma is a potent exposure that can have implications for health. However, little research has considered whether trauma exposure is related to endothelial function, a key process in the pathophysiology of cardiovascular disease (CVD). We tested whether exposure to traumatic experiences was related to poorer endothelial function among midlife women, independent of CVD risk factors, demographic factors, psychosocial factors, or a history of childhood abuse. In all, 272 nonsmoking perimenopausal and postmenopausal women aged 40 to 60 years without clinical CVD completed the Brief Trauma Questionnaire, the Child Trauma Questionnaire, physical measures, a blood draw, and a brachial ultrasound for assessment of brachial artery flow-mediated dilation (FMD). Relations between trauma and FMD were tested in linear regression models controlling for baseline vessel diameter, demographics, depression/anxiety, CVD risk factors, health behaviors, and, additionally, a history of childhood abuse. Over 60% of the sample had at least one traumatic exposure, and 18% had three or more exposures. A greater number of traumatic exposures was associated with lower FMD, indicating poorer endothelial function in multivariable models (beta, β [standard error, SE] -1.05 [0.40], P = 0.01). Relations between trauma exposure and FMD were particularly pronounced for three or more trauma exposures (b [SE] -1.90 [0.71], P = 0.008, relative to no exposures, multivariable). A greater number of traumatic exposures were associated with poorer endothelial function. Relations were not explained by demographics, CVD risk factors, mood/anxiety, or a by history of childhood abuse. Women with greater exposure to trauma over life maybe at elevated CVD risk.

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.

Uptake Mechanism of ApoE-Modified Nanoparticles on Brain Capillary Endothelial Cells as a Blood-Brain Barrier Model

PubMed Central

Wagner, Sylvia; Zensi, Anja; Wien, Sascha L.; Tschickardt, Sabrina E.; Maier, Wladislaw; Vogel, Tikva; Worek, Franz; Pietrzik, Claus U.; Kreuter, Jörg; von Briesen, Hagen

2012-01-01

Background The blood-brain barrier (BBB) represents an insurmountable obstacle for most drugs thus obstructing an effective treatment of many brain diseases. One solution for overcoming this barrier is a transport by binding of these drugs to surface-modified nanoparticles. Especially apolipoprotein E (ApoE) appears to play a major role in the nanoparticle-mediated drug transport across the BBB. However, at present the underlying mechanism is incompletely understood. Methodology/Principal Findings In this study, the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells was investigated to differentiate between active and passive uptake mechanism by flow cytometry and confocal laser scanning microscopy. Furthermore, different in vitro co-incubation experiments were performed with competing ligands of the respective receptor. Conclusions/Significance This study confirms an active endocytotic uptake mechanism and shows the involvement of low density lipoprotein receptor family members, notably the low density lipoprotein receptor related protein, on the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells. This knowledge of the uptake mechanism of ApoE-modified nanoparticles enables future developments to rationally create very specific and effective carriers to overcome the blood-brain barrier. PMID:22396775

Uptake mechanism of ApoE-modified nanoparticles on brain capillary endothelial cells as a blood-brain barrier model.

PubMed

Wagner, Sylvia; Zensi, Anja; Wien, Sascha L; Tschickardt, Sabrina E; Maier, Wladislaw; Vogel, Tikva; Worek, Franz; Pietrzik, Claus U; Kreuter, Jörg; von Briesen, Hagen

2012-01-01

The blood-brain barrier (BBB) represents an insurmountable obstacle for most drugs thus obstructing an effective treatment of many brain diseases. One solution for overcoming this barrier is a transport by binding of these drugs to surface-modified nanoparticles. Especially apolipoprotein E (ApoE) appears to play a major role in the nanoparticle-mediated drug transport across the BBB. However, at present the underlying mechanism is incompletely understood. In this study, the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells was investigated to differentiate between active and passive uptake mechanism by flow cytometry and confocal laser scanning microscopy. Furthermore, different in vitro co-incubation experiments were performed with competing ligands of the respective receptor. This study confirms an active endocytotic uptake mechanism and shows the involvement of low density lipoprotein receptor family members, notably the low density lipoprotein receptor related protein, on the uptake of the ApoE-modified nanoparticles into the brain capillary endothelial cells. This knowledge of the uptake mechanism of ApoE-modified nanoparticles enables future developments to rationally create very specific and effective carriers to overcome the blood-brain barrier.

Aging and vascular endothelial function in humans

PubMed Central

SEALS, Douglas R.; JABLONSKI, Kristen L.; DONATO, Anthony J.

2012-01-01

Advancing age is the major risk factor for the development of CVD (cardiovascular diseases). This is attributable, in part, to the development of vascular endothelial dysfunction, as indicated by reduced peripheral artery EDD (endothelium-dependent dilation) in response to chemical [typically ACh (acetylcholine)] or mechanical (intravascular shear) stimuli. Reduced bioavailability of the endothelium-synthesized dilating molecule NO (nitric oxide) as a result of oxidative stress is the key mechanism mediating reduced EDD with aging. Vascular oxidative stress increases with age as a consequence of greater production of reactive oxygen species (e.g. superoxide) without a compensatory increase in antioxidant defences. Sources of increased superoxide production include up-regulation of the oxidant enzyme NADPH oxidase, uncoupling of the normally NO-producing enzyme, eNOS (endothelial NO synthase) (due to reduced availability of the cofactor tetrahydrobiopterin) and increased mitochondrial synthesis during oxidative phosphorylation. Increased bioactivity of the potent endothelial-derived constricting factor ET-1 (endothelin-1), reduced endothelial production of/responsiveness to dilatory prostaglandins, the development of vascular inflammation, formation of AGEs (advanced glycation end-products), an increased rate of endothelial apoptosis and reduced expression of oestrogen receptor α (in postmenopausal females) also probably contribute to impaired EDD with aging. Several lifestyle and biological factors modulate vascular endothelial function with aging, including regular aerobic exercise, dietary factors (e.g. processed compared with non-processed foods), body weight/fatness, vitamin D status, menopause/oestrogen deficiency and a number of conventional and non-conventional risk factors for CVD. Given the number of older adults now and in the future, more information is needed on effective strategies for the prevention and treatment of vascular endothelial aging. PMID

How hormones influence composition and physiological function of the brain-blood barrier.

PubMed

Hampl, R; Bičíková, M; Sosvorová, L

2015-01-01

Hormones exert many actions in the brain. Their access and effects in the brain are regulated by the blood-brain barrier (BBB). Hormones as other substances may enter the brain and vice versa either by paracellular way requiring breaching tight junctions stitching the endothelial cells composing the BBB, or by passage through the cells (transcellular way). Hormones influence both ways through their receptors, both membrane and intracellular, present on/in the BBB. In the review the main examples are outlined how hormones influence the expression and function of proteins forming the tight junctions, as well as how they regulate expression and function of major protein transporters mediating transport of various substances including hormone themselves.

Evaluation of the Effects of Different Energy Drinks and Coffee on Endothelial Function.

PubMed

Molnar, Janos; Somberg, John C

2015-11-01

Endothelial function plays an important role in circulatory physiology. There has been differing reports on the effect of energy drink on endothelial function. We set out to evaluate the effect of 3 energy drinks and coffee on endothelial function. Endothelial function was evaluated in healthy volunteers using a device that uses digital peripheral arterial tonometry measuring endothelial function as the reactive hyperemia index (RHI). Six volunteers (25 ± 7 years) received energy drink in a random order at least 2 days apart. Drinks studied were 250 ml "Red Bull" containing 80 mg caffeine, 57 ml "5-hour Energy" containing 230 mg caffeine, and a can of 355 ml "NOS" energy drink containing 120 mg caffeine. Sixteen volunteers (25 ± 5 years) received a cup of 473 ml coffee containing 240 mg caffeine. Studies were performed before drink (baseline) at 1.5 and 4 hours after drink. Two of the energy drinks (Red Bull and 5-hour Energy) significantly improved endothelial function at 4 hours after drink, whereas 1 energy drink (NOS) and coffee did not change endothelial function significantly. RHI increased by 82 ± 129% (p = 0.028) and 63 ± 37% (p = 0.027) after 5-hour Energy and Red Bull, respectively. The RHI changed after NOS by 2 ± 30% (p = 1.000) and by 7 ± 30% (p = 1.000) after coffee. In conclusion, some energy drinks appear to significantly improve endothelial function. Caffeine does not appear to be the component responsible for these differences. Copyright © 2015 Elsevier Inc. All rights reserved.

Endothelial β-Catenin Signaling Is Required for Maintaining Adult Blood-Brain Barrier Integrity and Central Nervous System Homeostasis.

PubMed

Tran, Khiem A; Zhang, Xianming; Predescu, Dan; Huang, Xiaojia; Machado, Roberto F; Göthert, Joachim R; Malik, Asrar B; Valyi-Nagy, Tibor; Zhao, You-Yang

2016-01-12

The blood-brain barrier (BBB) formed by brain endothelial cells interconnected by tight junctions is essential for the homeostasis of the central nervous system. Although studies have shown the importance of various signaling molecules in BBB formation during development, little is known about the molecular basis regulating the integrity of the adult BBB. Using a mouse model with tamoxifen-inducible endothelial cell-restricted disruption of ctnnb1 (iCKO), we show here that endothelial β-catenin signaling is essential for maintaining BBB integrity and central nervous system homeostasis in adult mice. The iCKO mice developed severe seizures accompanied by neuronal injury, multiple brain petechial hemorrhages, and central nervous system inflammation, and all had postictal death. Disruption of endothelial β-catenin induced BBB breakdown and downregulation of the specific tight junction proteins claudin-1 and -3 in adult brain endothelial cells. The clinical relevance of the data is indicated by the observation of decreased expression of claudin-1 and nuclear β-catenin in brain endothelial cells of hemorrhagic lesions of hemorrhagic stroke patients. These results demonstrate the prerequisite role of endothelial β-catenin in maintaining the integrity of adult BBB. The results suggest that BBB dysfunction secondary to defective β-catenin transcription activity is a key pathogenic factor in hemorrhagic stroke, seizure activity, and central nervous system inflammation. © 2015 American Heart Association, Inc.

Involvement of specific macrophage-lineage cells surrounding arterioles in barrier and scavenger function in brain cortex.

PubMed Central

Mato, M; Ookawara, S; Sakamoto, A; Aikawa, E; Ogawa, T; Mitsuhashi, U; Masuzawa, T; Suzuki, H; Honda, M; Yazaki, Y; Watanabe, E; Luoma, J; Yla-Herttuala, S; Fraser, I; Gordon, S; Kodama, T

1996-01-01

The transport of solutes between blood and brain is regulated by a specific barrier. Capillary endothelial cells of brain are known to mediate barrier function and facilitate transport. Here we report that specific cells surrounding arterioles, known as Mato's fluorescent granular perithelial (FGP) cells or perivascular microglial cells, contribute to the barrier function. Immunohistochemical and in situ hybridization studies indicate that, in normal brain cortex, type I and type II macrophage scavenger receptors are expressed only in FGP/perivascular microglial cells, and surface markers of macrophage lineage are also detected on them. These cells mediate the uptake of macromolecules, including modified low density lipoprotein, horseradish peroxidase, and ferritin injected either into the blood or into the cerebral ventricles. Accumulation of scavenged materials with aging or after the administration of a high-fat diet results in the formation of honeycomb-like foam cells and the narrowing of the lumen of arterioles in the brain cortex. These results indicate involvement of FGP/perivascular microglial cells in the barrier and scavenger functions in the central nervous system. Images Fig. 1 Fig. 2 Fig. 4 Fig. 5 Fig. 6 PMID:8622926

Obstructive sleep apnoea syndrome, endothelial function and markers of endothelialization. Changes after CPAP.

PubMed

Muñoz-Hernandez, Rocio; Vallejo-Vaz, Antonio J; Sanchez Armengol, Angeles; Moreno-Luna, Rafael; Caballero-Eraso, Candela; Macher, Hada C; Villar, Jose; Merino, Ana M; Castell, Javier; Capote, Francisco; Stiefel, Pablo

2015-01-01

This study tries to assess the endothelial function in vivo using flow-mediated dilatation (FMD) and several biomarkers of endothelium formation/restoration and damage in patients with obstructive sleep apnoea (OSA) syndrome at baseline and after three months with CPAP therapy. Observational study, before and after CPAP therapy. We studied 30 patients with apnoea/hypopnoea index (AHI) >15/h that were compared with themselves after three months of CPAP therapy. FMD was assessed non-invasively in vivo using the Laser-Doppler flowmetry. Circulating cell-free DNA (cf-DNA) and microparticles (MPs) were measured as markers of endothelial damage and the vascular endothelial growth factor (VEGF) was determined as a marker of endothelial restoration process. After three month with CPAP, FMD significantly increased (1072.26 ± 483.21 vs. 1604.38 ± 915.69 PU, p< 0.005) cf-DNA and MPs significantly decreased (187.93 ± 115.81 vs. 121.28 ± 78.98 pg/ml, p<0.01, and 69.60 ± 62.60 vs. 39.82 ± 22.14 U/μL, p<0.05, respectively) and VEGF levels increased (585.02 ± 246.06 vs. 641.11 ± 212.69 pg/ml, p<0.05). These changes were higher in patients with more severe disease. There was a relationship between markers of damage (r = -0.53, p<0.005) but not between markers of damage and restoration, thus suggesting that both types of markers should be measured together. CPAP therapy improves FMD. This improvement may be related to an increase of endothelial restoration process and a decrease of endothelial damage.

Curcumin modulates endothelial permeability and monocyte transendothelial migration by affecting endothelial cell dynamics.

PubMed

Monfoulet, Laurent-Emmanuel; Mercier, Sylvie; Bayle, Dominique; Tamaian, Radu; Barber-Chamoux, Nicolas; Morand, Christine; Milenkovic, Dragan

2017-11-01

Curcumin is a phenolic compound that exhibits beneficial properties for cardiometabolic health. We previously showed that curcumin reduced the infiltration of immune cells into the vascular wall and prevented atherosclerosis development in mice. This study aimed to investigate the effect of curcumin on monocyte adhesion and transendothelial migration (TEM) and to decipher the underlying mechanisms of these actions. Human umbilical vein endothelial cells (HUVECs) were exposed to curcumin (0.5-1μM) for 3h prior to their activation by Tumor Necrosis Factor alpha (TNF-α). Endothelial permeability, monocyte adhesion and transendothelial migration assays were conducted under static condition and shear stress that mimics blood flow. We further investigated the impact of curcumin on signaling pathways and on the expression of genes using macroarrays. Pre-exposure of endothelial cells to curcumin reduced monocyte adhesion and their transendothelial migration in both static and shear stress conditions. Curcumin also prevented changes in both endothelial permeability and the area of HUVECs when induced by TNF-α. We showed that curcumin modulated the expression of 15 genes involved in the control of cytoskeleton and endothelial junction dynamic. Finally, we showed that curcumin inhibited NF-κB signaling likely through an antagonist interplay with several kinases as suggested by molecular docking analysis. Our findings demonstrate the ability of curcumin to reduce monocyte TEM through a multimodal regulation of the endothelial cell dynamics with a potential benefit on the vascular endothelial function barrier. Copyright © 2017 Elsevier Inc. All rights reserved.

Isoflavone supplementation and endothelial function in menopausal women.

PubMed

Hale, Georgina; Paul-Labrador, Maura; Dwyer, James H; Merz, C Noel Bairey

2002-06-01

Despite strong observational evidence for a beneficial role of oestrogen in cardiovascular disease, recent trial results suggest that hormone replacement therapy (HRT) may have adverse effects in menopausal women with established coronary heart disease. Isoflavones are oestrogen analogues found in plants with oestrogen-like properties and, because of a favourable side-effect profile, may be ideal alternatives to HRT with respect to cardiovascular benefits. Endothelial function is a marker of cardiovascular health. We aimed to determine the effect of isoflavones on endothelial function using the brachial artery reactivity test. Twenty-nine healthy menopausal women underwent entry and exit brachial artery reactivity testing following randomization to 2 weeks of an oral soy isoflavone concentrate containing 80 mg of soy isoflavones (Archer Daniel Midland Inc., IL, USA) or placebo. At study exit, there was no difference between placebo and isoflavone groups with respect to flow-mediated dilation (%FMD(max)), change (entry to exit) in %FMD(max) or response to nitroglycerine (%TNG). Subgroup analyses assessing lipid and oestrogen effects did not produce any significant results. These results suggest that short-term oral isoflavone supplements do not improve endothelial function in healthy menopausal women.

In smokers, Sonic hedgehog modulates pulmonary endothelial function through vascular endothelial growth factor.

PubMed

Henno, Priscilla; Grassin-Delyle, Stanislas; Belle, Emeline; Brollo, Marion; Naline, Emmanuel; Sage, Edouard; Devillier, Philippe; Israël-Biet, Dominique

2017-05-23

Tobacco-induced pulmonary vascular disease is partly driven by endothelial dysfunction. The Sonic hedgehog (SHH) pathway is involved in vascular physiology. We sought to establish whether the SHH pathway has a role in pulmonary endothelial dysfunction in smokers. The ex vivo endothelium-dependent relaxation of pulmonary artery rings in response to acetylcholine (Ach) was compared in 34 current or ex-smokers and 8 never-smokers. The results were expressed as a percentage of the contraction with phenylephrine. We tested the effects of SHH inhibitors (GANT61 and cyclopamine), an SHH activator (SAG) and recombinant VEGF on the Ach-induced relaxation. The level of VEGF protein in the pulmonary artery ring was measured in an ELISA. SHH pathway gene expression was quantified in reverse transcriptase-quantitative polymerase chain reactions. Ach-induced relaxation was much less intense in smokers than in never-smokers (respectively 24 ± 6% and 50 ± 7% with 10 -4 M Ach; p = 0.028). All SHH pathway genes were expressed in pulmonary artery rings from smokers. SHH inhibition by GANT61 reduced Ach-induced relaxation and VEGF gene expression in the pulmonary artery ring. Recombinant VEGF restored the ring's endothelial function. VEGF gene and protein expression levels in the pulmonary artery rings were positively correlated with the degree of Ach-induced relaxation and negatively correlated with the number of pack-years. SHH pathway genes and proteins are expressed in pulmonary artery rings from smokers, where they modulate endothelial function through VEGF.

Concerted regulation of retinal pigment epithelium basement membrane and barrier function by angiocrine factors.

PubMed

Benedicto, Ignacio; Lehmann, Guillermo L; Ginsberg, Michael; Nolan, Daniel J; Bareja, Rohan; Elemento, Olivier; Salfati, Zelda; Alam, Nazia M; Prusky, Glen T; Llanos, Pierre; Rabbany, Sina Y; Maminishkis, Arvydas; Miller, Sheldon S; Rafii, Shahin; Rodriguez-Boulan, Enrique

2017-05-19

The outer blood-retina barrier is established through the coordinated terminal maturation of the retinal pigment epithelium (RPE), fenestrated choroid endothelial cells (ECs) and Bruch's membrane, a highly organized basement membrane that lies between both cell types. Here we study the contribution of choroid ECs to this process by comparing their gene expression profile before (P5) and after (P30) the critical postnatal period when mice acquire mature visual function. Transcriptome analyses show that expression of extracellular matrix-related genes changes dramatically over this period. Co-culture experiments support the existence of a novel regulatory pathway: ECs secrete factors that remodel RPE basement membrane, and integrin receptors sense these changes triggering Rho GTPase signals that modulate RPE tight junctions and enhance RPE barrier function. We anticipate our results will spawn a search for additional roles of choroid ECs in RPE physiology and disease.

Concerted regulation of retinal pigment epithelium basement membrane and barrier function by angiocrine factors

PubMed Central

Benedicto, Ignacio; Lehmann, Guillermo L.; Ginsberg, Michael; Nolan, Daniel J.; Bareja, Rohan; Elemento, Olivier; Salfati, Zelda; Alam, Nazia M.; Prusky, Glen T.; Llanos, Pierre; Rabbany, Sina Y.; Maminishkis, Arvydas; Miller, Sheldon S.; Rafii, Shahin; Rodriguez-Boulan, Enrique

2017-01-01

The outer blood-retina barrier is established through the coordinated terminal maturation of the retinal pigment epithelium (RPE), fenestrated choroid endothelial cells (ECs) and Bruch's membrane, a highly organized basement membrane that lies between both cell types. Here we study the contribution of choroid ECs to this process by comparing their gene expression profile before (P5) and after (P30) the critical postnatal period when mice acquire mature visual function. Transcriptome analyses show that expression of extracellular matrix-related genes changes dramatically over this period. Co-culture experiments support the existence of a novel regulatory pathway: ECs secrete factors that remodel RPE basement membrane, and integrin receptors sense these changes triggering Rho GTPase signals that modulate RPE tight junctions and enhance RPE barrier function. We anticipate our results will spawn a search for additional roles of choroid ECs in RPE physiology and disease. PMID:28524846

Alda-1 Protects Against Acrolein-Induced Acute Lung Injury and Endothelial Barrier Dysfunction.

PubMed

Lu, Qing; Mundy, Miles; Chambers, Eboni; Lange, Thilo; Newton, Julie; Borgas, Diana; Yao, Hongwei; Choudhary, Gaurav; Basak, Rajshekhar; Oldham, Mahogany; Rounds, Sharon

2017-12-01

Inhalation of acrolein, a highly reactive aldehyde, causes lung edema. The underlying mechanism is poorly understood and there is no effective treatment. In this study, we demonstrated that acrolein not only dose-dependently induced lung edema but also promoted LPS-induced acute lung injury. Importantly, acrolein-induced lung injury was prevented and rescued by Alda-1, an activator of mitochondrial aldehyde dehydrogenase 2. Acrolein also dose-dependently increased monolayer permeability, disrupted adherens junctions and focal adhesion complexes, and caused intercellular gap formation in primary cultured lung microvascular endothelial cells (LMVECs). These effects were attenuated by Alda-1 and the antioxidant N-acetylcysteine, but not by the NADPH inhibitor apocynin. Furthermore, acrolein inhibited AMP-activated protein kinase (AMPK) and increased mitochondrial reactive oxygen species levels in LMVECs-effects that were associated with impaired mitochondrial respiration. AMPK total protein levels were also reduced in lung tissue of mice and LMVECs exposed to acrolein. Activation of AMPK with 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside blunted an acrolein-induced increase in endothelial monolayer permeability, but not mitochondrial oxidative stress or inhibition of mitochondrial respiration. Our results suggest that acrolein-induced mitochondrial dysfunction may not contribute to endothelial barrier dysfunction. We speculate that detoxification of acrolein by Alda-1 and activation of AMPK may be novel approaches to prevent and treat acrolein-associated acute lung injury, which may occur after smoke inhalation.

Comparison of a Rat Primary Cell-Based Blood-Brain Barrier Model With Epithelial and Brain Endothelial Cell Lines: Gene Expression and Drug Transport.

PubMed

Veszelka, Szilvia; Tóth, András; Walter, Fruzsina R; Tóth, Andrea E; Gróf, Ilona; Mészáros, Mária; Bocsik, Alexandra; Hellinger, Éva; Vastag, Monika; Rákhely, Gábor; Deli, Mária A

2018-01-01

Cell culture-based blood-brain barrier (BBB) models are useful tools for screening of CNS drug candidates. Cell sources for BBB models include primary brain endothelial cells or immortalized brain endothelial cell lines. Despite their well-known differences, epithelial cell lines are also used as surrogate models for testing neuropharmaceuticals. The aim of the present study was to compare the expression of selected BBB related genes including tight junction proteins, solute carriers (SLC), ABC transporters, metabolic enzymes and to describe the paracellular properties of nine different culture models. To establish a primary BBB model rat brain capillary endothelial cells were co-cultured with rat pericytes and astrocytes (EPA). As other BBB and surrogate models four brain endothelial cells lines, rat GP8 and RBE4 cells, and human hCMEC/D3 cells with or without lithium treatment (D3 and D3L), and four epithelial cell lines, native human intestinal Caco-2 and high P-glycoprotein expressing vinblastine-selected VB-Caco-2 cells, native MDCK and MDR1 transfected MDCK canine kidney cells were used. To test transporter functionality, the permeability of 12 molecules, glucopyranose, valproate, baclofen, gabapentin, probenecid, salicylate, rosuvastatin, pravastatin, atorvastatin, tacrine, donepezil, was also measured in the EPA and epithelial models. Among the junctional protein genes, the expression level of occludin was high in all models except the GP8 and RBE4 cells, and each model expressed a unique claudin pattern. Major BBB efflux (P-glycoprotein or ABCB1) and influx transporters (GLUT-1, LAT-1) were present in all models at mRNA levels. The transcript of BCRP (ABCG2) was not expressed in MDCK, GP8 and RBE4 cells. The absence of gene expression of important BBB efflux and influx transporters BCRP, MRP6, -9, MCT6, -8, PHT2, OATPs in one or both types of epithelial models suggests that Caco-2 or MDCK models are not suitable to test drug candidates which are substrates

Importins α and β signaling mediates endothelial cell inflammation and barrier disruption.

PubMed

Leonard, Antony; Rahman, Arshad; Fazal, Fabeha

2018-04-01

Nucleocytoplasmic shuttling via importins is central to the function of eukaryotic cells and an integral part of the processes that lead to many human diseases. In this study, we addressed the role of α and β importins in the mechanism of endothelial cell (EC) inflammation and permeability, important pathogenic features of many inflammatory diseases such as acute lung injury and atherosclerosis. RNAi-mediated knockdown of importin α4 or α3 each inhibited NF-κB activation, proinflammatory gene (ICAM-1, VCAM-1, and IL-6) expression, and thereby endothelial adhesivity towards HL-60 cells, upon thrombin challenge. The inhibitory effect of α4 and α3 knockdown was associated with impaired nuclear import and consequently, DNA binding of RelA/p65 subunit of NF-κB and occurred independently of IκBα degradation. Intriguingly, knockdown of importins α4 and α3 also inhibited thrombin-induced RelA/p65 phosphorylation at Ser 536 , showing a novel role of α importins in regulating transcriptional activity of RelA/p65. Similarly, knockdown of importin β1, but not β2, blocked thrombin-induced activation of RelA/p65 and its target genes. In parallel studies, TNFα-mediated inflammatory responses in EC were refractory to knockdown of importins α4, α3 or β1, indicating a stimulus-specific regulation of RelA/p65 and EC inflammation by these importins. Importantly, α4, α3, or β1 knockdown also protected against thrombin-induced EC barrier disruption by inhibiting the loss of VE-cadherin at adherens junctions and by regulating actin cytoskeletal rearrangement. These results identify α4, α3 and β1 as critical mediators of EC inflammation and permeability associated with intravascular coagulation. Copyright © 2018 Elsevier Inc. All rights reserved.

Assessing endothelial function and providing calibrated UFMD data using a blood pressure cuff

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

Maltz, Jonathan S.

Methods and apparatus are provided for assessing endothelial function in a mammal. In certain embodiments the methods involve using a cuff to apply pressure to an artery in a subject to determine a plurality of baseline values for a parameter related to endothelial function as a function of applied pressure (P.sub.m); b) applying a stimulus to the subject; and applying external pressure P.sub.m to the artery to determine a plurality of stimulus-effected values for the parameter related to endothelial function as a function of applied pressure (P.sub.m); where the baseline values are determined from measurements made when said mammal ismore » not substantially effected by said stimulus and differences in said baseline values and said stimulus-effected values provide a measure of endothelial function in said mammal.« less

Evaluation of a static stretching intervention on vascular endothelial function and arterial stiffness.

PubMed

Shinno, Hiromi; Kurose, Satoshi; Yamanaka, Yutaka; Higurashi, Kyoko; Fukushima, Yaeko; Tsutsumi, Hiromi; Kimura, Yutaka

2017-06-01

Maintenance and enhancement of vascular endothelial function contribute to the prevention of cardiovascular disease and prolong a healthy life expectancy. Given the reversible nature of vascular endothelial function, interventions to improve this function might prevent arteriosclerosis. Accordingly, we studied the effects of a 6-month static stretching intervention on vascular endothelial function (reactive hyperaemia peripheral arterial tonometry index: RH-PAT index) and arterial stiffness (brachial-ankle pulse wave velocity: baPWV) and investigated the reversibility of these effects after a 6-month detraining period following intervention completion. The study evaluated 22 healthy, non-smoking, premenopausal women aged ≥40 years. Subjects were randomly assigned to the full-intervention (n = 11; mean age: 48.6 ± 2.8 years) or a half-intervention that included a control period (n = 11; mean age: 46.9 ± 3.6 years). Body flexibility and vascular endothelial function improved significantly after 3 months of static stretching. In addition to these improvements, arterial stiffness improved significantly after a 6-month intervention. However, after a 6-month detraining period, vascular endothelial function, flexibility, and arterial stiffness all returned to preintervention conditions, demonstrating the reversibility of the obtained effects. A 3-month static stretching intervention was found to improve vascular endothelial function, and an additional 3-month intervention also improved arterial stiffness. However, these effects were reversed by detraining.

Obstructive Sleep Apnoea Syndrome, Endothelial Function and Markers of Endothelialization. Changes after CPAP

PubMed Central

Sanchez Armengol, Angeles; Moreno-Luna, Rafael; Caballero-Eraso, Candela; Macher, Hada C.; Villar, Jose; Merino, Ana M; Castell, Javier; Capote, Francisco; Stiefel, Pablo

2015-01-01

Study objectives This study tries to assess the endothelial function in vivo using flow-mediated dilatation (FMD) and several biomarkers of endothelium formation/restoration and damage in patients with obstructive sleep apnoea (OSA) syndrome at baseline and after three months with CPAP therapy. Design Observational study, before and after CPAP therapy. Setting and Patients We studied 30 patients with apnoea/hypopnoea index (AHI) >15/h that were compared with themselves after three months of CPAP therapy. FMD was assessed non-invasively in vivo using the Laser-Doppler flowmetry. Circulating cell-free DNA (cf-DNA) and microparticles (MPs) were measured as markers of endothelial damage and the vascular endothelial growth factor (VEGF) was determined as a marker of endothelial restoration process. Measurements and results After three month with CPAP, FMD significantly increased (1072.26 ± 483.21 vs. 1604.38 ± 915.69 PU, p< 0.005) cf-DNA and MPs significantly decreased (187.93 ± 115.81 vs. 121.28 ± 78.98 pg/ml, p<0.01, and 69.60 ± 62.60 vs. 39.82 ± 22.14 U/μL, p<0.05, respectively) and VEGF levels increased (585.02 ± 246.06 vs. 641.11 ± 212.69 pg/ml, p<0.05). These changes were higher in patients with more severe disease. There was a relationship between markers of damage (r = -0.53, p<0.005) but not between markers of damage and restoration, thus suggesting that both types of markers should be measured together. Conclusions CPAP therapy improves FMD. This improvement may be related to an increase of endothelial restoration process and a decrease of endothelial damage. PMID:25815511

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

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

Permeability of Endothelial and Astrocyte Cocultures: In Vitro Blood–Brain Barrier Models for Drug Delivery Studies

PubMed Central

Li, Guanglei; Simon, Melissa J.; Cancel, Limary M.; Shi, Zhong-Dong; Ji, Xinying; Tarbell, John M.; Morrison, Barclay; Fu, Bingmei M.

2014-01-01

The blood–brain barrier (BBB) is a major obstacle for drug delivery to the brain. To seek for in vitro BBB models that are more accessible than animals for investigating drug transport across the BBB, we compared four in vitro cultured cell models: endothelial monoculture (bEnd3 cell line), coculture of bEnd3 and primary rat astrocytes (coculture), coculture with collagen type I and IV mixture, and coculture with Matrigel. The expression of the BBB tight junction proteins in these in vitro models was assessed using RT-PCR and immunofluorescence. We also quantified the hydraulic conductivity (Lp), transendothelial electrical resistance (TER) and diffusive solute permeability (P) of these models to three solutes: TAMRA, Dextran 10K and Dextran 70K. Our results show that Lp and P of the endothelial monoculture and coculture models are not different from each other. Compared with in vivo permeability data from rat pial microvessels, P of the endothelial monoculture and coculture models are not significantly different from in vivo data for Dextran 70K, but they are 2–4 times higher for TAMRA and Dextran 10K. This suggests that the endothelial monoculture and all of the coculture models are fairly good models for studying the transport of relatively large solutes across the BBB. PMID:20361260

Permeability of endothelial and astrocyte cocultures: in vitro blood-brain barrier models for drug delivery studies.

PubMed

Li, Guanglei; Simon, Melissa J; Cancel, Limary M; Shi, Zhong-Dong; Ji, Xinying; Tarbell, John M; Morrison, Barclay; Fu, Bingmei M

2010-08-01

The blood-brain barrier (BBB) is a major obstacle for drug delivery to the brain. To seek for in vitro BBB models that are more accessible than animals for investigating drug transport across the BBB, we compared four in vitro cultured cell models: endothelial monoculture (bEnd3 cell line), coculture of bEnd3 and primary rat astrocytes (coculture), coculture with collagen type I and IV mixture, and coculture with Matrigel. The expression of the BBB tight junction proteins in these in vitro models was assessed using RT-PCR and immunofluorescence. We also quantified the hydraulic conductivity (L (p)), transendothelial electrical resistance (TER) and diffusive solute permeability (P) of these models to three solutes: TAMRA, Dextran 10K and Dextran 70K. Our results show that L (p) and P of the endothelial monoculture and coculture models are not different from each other. Compared with in vivo permeability data from rat pial microvessels, P of the endothelial monoculture and coculture models are not significantly different from in vivo data for Dextran 70K, but they are 2-4 times higher for TAMRA and Dextran 10K. This suggests that the endothelial monoculture and all of the coculture models are fairly good models for studying the transport of relatively large solutes across the BBB.

Endothelial β-Catenin Signaling Is Required for Maintaining Adult Blood-Brain Barrier Integrity and CNS Homeostasis

PubMed Central

Tran, Khiem A.; Zhang, Xianming; Predescu, Dan; Huang, Xiaojia; Machado, Roberto F.; Göthert, Joachim R.; Malik, Asrar B.; Valyi-Nagy, Tibor; Zhao, You-Yang

2015-01-01

Background The blood-brain barrier (BBB) formed by brain endothelial cells (ECs) interconnected by tight junctions (TJs) is essential for the homeostasis of the central nervous system (CNS). Although studies have shown the importance of various signaling molecules in BBB formation during development, little is known about the molecular basis regulating the integrity of the adult BBB. Methods and Results Using a mouse model with tamoxifen-inducible EC-restricted disruption of ctnnb1 (iCKO), here we show that endothelial β-catenin signaling is essential for maintaining BBB integrity and CNS homeostasis in adult. The iCKO mice developed severe seizures accompanied by neuronal injury, multiple brain petechial hemorrhages, and CNS inflammation, and all died postictal. Disruption of endothelial β-catenin induced BBB breakdown and downregulation of specific TJ proteins Claudin-1 and -3 in adult brain ECs. The clinical relevance of the data is indicated by the observation of decreased expression of Claudin-1 and nuclear β-catenin in brain ECs of hemorrhagic lesions of hemorrhagic stroke patients. Conclusion These results demonstrate the prerequisite role of endothelial β-catenin in maintaining the integrity of adult BBB. The results suggest that BBB dysfunction secondary to defective β-catenin transcription activity is a key pathogenic factor in hemorrhagic stroke, seizure activity and CNS inflammation. PMID:26538583

Protective effects of dark chocolate on endothelial function and diabetes.

PubMed

Grassi, Davide; Desideri, Giovambattista; Ferri, Claudio

2013-11-01

Relationship between cocoa consumption and cardiovascular disease, particularly focusing on clinical implications resulting from the beneficial effects of cocoa consumption on endothelial function and insulin resistance. This could be of clinical relevance and may suggest the mechanistic explanation for the reduced risk of cardiovascular events reported in the different studies after cocoa intake. Increasing evidence supports a protective effect of cocoa consumption against cardiovascular disease. Cocoa and flavonoids from cocoa have been described to improve endothelial function and insulin resistance. A proposed mechanism could be considered in the improvement of the endothelium-derived vasodilator nitric oxide by enhancing nitric oxide synthesis or by decreasing nitric oxide breakdown. The endothelium plays a pivotal role in the arterial homeostasis, and insulin resistance is the most important pathophysiological feature in various prediabetic and diabetic states. Reduced nitric oxide bioavailability with endothelial dysfunction is considered the earliest step in the pathogenesis of atherosclerosis. Further, insulin resistance could account, at least in part, for the endothelial dysfunction. Endothelial dysfunction has been considered an important and independent predictor of future development of cardiovascular risk and events. Cocoa and flavonoids from cocoa might positively modulate these mechanisms with a putative role in cardiovascular protection.

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

Fatty Acid-Binding Protein 5 at the Blood-Brain Barrier Regulates Endogenous Brain Docosahexaenoic Acid Levels and Cognitive Function.

PubMed

Pan, Yijun; Short, Jennifer L; Choy, Kwok H C; Zeng, Annie X; Marriott, Philip J; Owada, Yuji; Scanlon, Martin J; Porter, Christopher J H; Nicolazzo, Joseph A

2016-11-16

Fatty acid-binding protein 5 (FABP5) at the blood-brain barrier contributes to the brain uptake of docosahexaenoic acid (DHA), a blood-derived polyunsaturated fatty acid essential for maintenance of cognitive function. Given the importance of DHA in cognition, the aim of this study was to investigate whether deletion of FABP5 results in cognitive dysfunction and whether this is associated with reduced brain endothelial cell uptake of exogenous DHA and subsequent attenuation in the brain levels of endogenous DHA. Cognitive function was assessed in male and female FABP5 +/+ and FABP5 -/- mice using a battery of memory paradigms. FABP5 -/- mice exhibited impaired working memory and short-term memory, and these cognitive deficits were associated with a 14.7 ± 5.7% reduction in endogenous brain DHA levels. The role of FABP5 in the blood-brain barrier transport of DHA was assessed by measuring 14 C-DHA uptake into brain endothelial cells and capillaries isolated from FABP5 +/+ and FABP5 -/- mice. In line with a crucial role of FABP5 in the brain uptake of DHA, 14 C-DHA uptake into brain endothelial cells and brain capillaries of FABP5 -/- mice was reduced by 48.4 ± 14.5% and 14.0 ± 4.2%, respectively, relative to those of FABP5 +/+ mice. These results strongly support the hypothesis that FABP5 is essential for maintaining brain endothelial cell uptake of DHA, and that cognitive deficits observed in FABP5 -/- mice are associated with reduced CNS access of DHA. Genetic deletion of fatty acid-binding protein 5 (FABP5) in mice reduces uptake of exogenous docosahexaenoic acid (DHA) into brain endothelial cells and brain capillaries and reduces brain parenchymal levels of endogenous DHA. Therefore, FABP5 in the brain endothelial cell is a crucial contributor to the brain levels of DHA. Critically, lowered brain DHA levels in FABP5 -/- mice occurred in tandem with cognitive deficits in a battery of memory paradigms. This study provides evidence of a critical role for FABP5

Endothelial cells are critical regulators of iron transport in a model of the human blood-brain barrier.

PubMed

Chiou, Brian; Neal, Emma H; Bowman, Aaron B; Lippmann, Ethan S; Simpson, Ian A; Connor, James R

2018-01-01

Iron delivery to the brain is essential for multiple neurological processes such as myelination, neurotransmitter synthesis, and energy production. Loss of brain iron homeostasis is a significant factor in multiple neurological disorders. Understanding the mechanism by which the transport of iron across the blood-brain barrier (BBB) is regulated is crucial to address the impact of iron deficiency on brain development and excessive accumulation of iron in neurodegenerative diseases. Using induced pluripotent stem cell (iPSC)-derived brain endothelial cells (huECs) as a human BBB model, we demonstrate the ability of transferrin, hepcidin, and DMT1 to impact iron transport and release. Our model reveals a new function for H-ferritin to transport iron across the BBB by binding to the T-cell immunoglobulin and mucin receptor 1. We show that huECs secrete both transferrin and H-ferritin, which can serve as iron sources for the brain. Based on our data, brain iron status can exert control of iron transport across the endothelial cells that constitute the BBB. These data address a number of pertinent questions such as how brain iron uptake is regulated at the regional level, the source of iron delivery to the brain, and the clinical strategies for attempting to treat brain iron deficiency.

Cytokine Signaling Modulates Blood-Brain Barrier Function

PubMed Central

Pan, Weihong; Stone, Kirsten P.; Hsuchou, Hung; Manda, Vamshi K.; Zhang, Yan; Kastin, Abba J.

2014-01-01

The blood-brain barrier (BBB) provides a vast interface for cytokines to affect CNS function. The BBB is a target for therapeutic intervention. It is essential, therefore, to understand how cytokines interact with each other at the level of the BBB and how secondary signals modulate CNS functions beyond the BBB. The interactions between cytokines and lipids, however, have not been fully addressed at the level of the BBB. Here, we summarize current understanding of the localization of cytokine receptors and transporters in specific membrane microdomains, particularly lipid rafts, on the luminal (apical) surface of the microvascular endothelial cells composing the BBB. We then illustrate the clinical context of cytokine effects on the BBB by neuroendocrine regulation and amplification of inflammatory signals. Two unusual aspects discussed are signaling crosstalk by different classes of cytokines and genetic regulation of drug efflux transporters. We also introduce a novel area of focus on how cytokines may act through nuclear hormone receptors to modulate efflux transporters and other targets. A specific example discussed is the ATP-binding cassette transporter-1 (ABCA-1) that regulates lipid metabolism. Overall, cytokine signaling at the level of the BBB is a crucial feature of the dynamic regulation that can rapidly change BBB function and affect brain health and disease. PMID:21834767

Regulation of human feto-placental endothelial barrier integrity by vascular endothelial growth factors: competitive interplay between VEGF-A165a, VEGF-A165b, PIGF and VE-cadherin.

PubMed

Pang, Vincent; Bates, David O; Leach, Lopa

2017-12-01

The human placenta nourishes and protects the developing foetus whilst influencing maternal physiology for fetal advantage. It expresses several members of the vascular endothelial growth factor (VEGF) family including the pro-angiogenic/pro-permeability VEGF-A 165 a isoform, the anti-angiogenic VEGF-A 165 b, placental growth factor (PIGF) and their receptors, VEGFR1 and VEGFR2. Alterations in the ratio of these factors during gestation and in complicated pregnancies have been reported; however, the impact of this on feto-placental endothelial barrier integrity is unknown. The present study investigated the interplay of these factors on junctional occupancy of VE-cadherin and macromolecular leakage in human endothelial monolayers and the perfused placental microvascular bed. Whilst VEGF-A 165 a (50 ng/ml) increased endothelial monolayer albumin permeability ( P <0.0001), equimolar concentrations of VEGF-A 165 b ( P >0.05) or PlGF ( P >0.05) did not. Moreover, VEGF-A 165 b (100 ng/ml; P <0.001) but not PlGF (100 ng/ml; P >0.05) inhibited VEGF-A 165 a-induced permeability when added singly. PlGF abolished the VEGF-A 165 b-induced reduction in VEGF-A 165 a-mediated permeability ( P >0.05); PlGF was found to compete with VEGF-A 165 b for binding to Flt-1 at equimolar affinity. Junctional occupancy of VE-cadherin matched alterations in permeability. In the perfused microvascular bed, VEGF-A 165 b did not induce microvascular leakage but inhibited and reversed VEGF-A 165 a-induced loss of junctional VE-cadherin and tracer leakage. These results indicate that the anti-angiogenic VEGF-A 165 b isoform does not increase permeability in human placental microvessels or HUVEC primary cells and can interrupt VEGF-A 165 a-induced permeability. Moreover, the interplay of these isoforms with PIGF (and s-flt1) suggests that the ratio of these three factors may be important in determining the placental and endothelial barrier in normal and complicated pregnancies. © 2017 The Author(s).

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.

SIRT1 reduces endothelial activation without affecting vascular function in ApoE-/- mice

PubMed Central

Stein, Sokrates; Schäfer, Nicola; Breitenstein, Alexander; Besler, Christian; Winnik, Stephan; Lohmann, Christine; Heinrich, Kathrin; Brokopp, Chad E.; Handschin, Christoph; Landmesser, Ulf; Tanner, Felix C.; Lüscher, Thomas F.; Matter, Christian M.

2010-01-01

Excessive production of reactive oxygen species (ROS) contributes to progression of atherosclerosis, at least in part by causing endothelial dysfunction and inflammatory activation. The class III histone deacetylase SIRT1 has been implicated in extension of lifespan. In the vasculature,SIRT1 gain-of-function using SIRT1 overexpression or activation has been shown to improve endothelial function in mice and rats via stimulation of endothelial nitric oxide (NO) synthase (eNOS). However, the effects of SIRT1 loss-of-function on the endothelium in atherosclerosis remain to be characterized. Thus, we have investigated the endothelial effects of decreased endogenous SIRT1 in hypercholesterolemic ApoE-/- mice. We observed no difference in endothelial relaxation and eNOS (Ser1177) phosphorylation between 20-week old male atherosclerotic ApoE-/- SIRT1+/- and ApoE-/- SIRT1+/+ mice. However, SIRT1 prevented endothelial superoxide production, inhibited NF-κB signaling, and diminished expression of adhesion molecules. Treatment of young hypercholesterolemic ApoE-/- SIRT1+/- mice with lipopolysaccharide to boost NF-κB signaling led to a more pronounced endothelial expression of ICAM-1 and VCAM-1 as compared to ApoE-/- SIRT1+/+ mice. In conclusion, endogenous SIRT1 diminishes endothelial activation in ApoE-/- mice, but does not affect endothelium-dependent vasodilatation. PMID:20606253

Sleep Restriction Impairs Blood–Brain Barrier Function

PubMed Central

He, Junyun; Hsuchou, Hung; He, Yi; Kastin, Abba J.; Wang, Yuping

2014-01-01

The blood–brain barrier (BBB) is a large regulatory and exchange interface between the brain and peripheral circulation. We propose that changes of the BBB contribute to many pathophysiological processes in the brain of subjects with chronic sleep restriction (CSR). To achieve CSR that mimics a common pattern of human sleep loss, we quantified a new procedure of sleep disruption in mice by a week of consecutive sleep recording. We then tested the hypothesis that CSR compromises microvascular function. CSR not only diminished endothelial and inducible nitric oxide synthase, endothelin1, and glucose transporter expression in cerebral microvessels of the BBB, but it also decreased 2-deoxy-glucose uptake by the brain. The expression of several tight junction proteins also was decreased, whereas the level of cyclooxygenase-2 increased. This coincided with an increase of paracellular permeability of the BBB to the small tracers sodium fluorescein and biotin. CSR for 6 d was sufficient to impair BBB structure and function, although the increase of paracellular permeability returned to baseline after 24 h of recovery sleep. This merits attention not only in neuroscience research but also in public health policy and clinical practice. PMID:25355222

Comparison of Endothelial Function in Asian Indians Versus Caucasians.

PubMed

Pusalavidyasagar, Snigdha; Sert Kuniyoshi, Fatima H; Shamsuzzaman, Abu S M; Singh, Prachi; Maharaj, Shantal; Leinveber, Pavel; Nykodym, Jiri; Somers, Virend K

2016-09-01

Asian Indians have markedly increased mortality due to coronary artery disease (CAD). Impaired endothelial function has been linked to an increased risk of acute cardiovascular events. We tested the hypothesis that endothelial function was attenuated in Asian Indians and Caucasians. We studied 14 Asian Indians [mean age: 30 ± 6 years; mean body mass index (BMI): 25 ± 3 kg/m(2)] and 11 Caucasians (mean age: 30 ± 5 years; mean BMI: 26 ± 2 kg/m(2)). All 25 subjects were healthy men and nonsmokers without any history of CAD or diabetes and were not taking medications. Endothelial function was evaluated by ultrasound measures of flow-mediated dilatation (FMD) and endothelium-independent nonflow mediated vasodilatation (NFMD) of the brachial artery, in the morning immediately after awakening (6 a.m.) in a fasting state. Mean age, BMI, apnea-hypopnea index, heart rate, and blood pressure were similar in both groups (P = >0.05). When correcting for body surface area, brachial artery diameter was not different between the two groups (2.1% ± 0.3% vs. 2.2% ± 0.4%; P = 0.29). FMD and NFMD were similar in Asian Indians and Caucasians (5.9% ± 4.1% vs. 5.7% ± 2.6%, P = 0.70; 16.4% ± 8% vs. 14.8% ± 4.1%, P = 0.58, respectively). Endothelial function in Asian Indian men is not attenuated in comparison to Caucasian men.

Tumour necrosis factor-α-mediated disruption of cerebrovascular endothelial barrier integrity in vitro involves the production of proinflammatory interleukin-6.

PubMed

Rochfort, Keith D; Collins, Laura E; McLoughlin, Alisha; Cummins, Philip M

2016-02-01

The co-involvement of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) during blood-brain barrier (BBB) injury has been reported in various models of neuroinflammation, although the precise functional interplay between these archetypal proinflammatory cytokines remains largely undefined within this context. In the current paper, we tested the hypothesis that TNF-α-mediated BBB disruption is measurably attributable in-part to induction of microvascular endothelial IL-6 production. In initial experiments, we observed that treatment of human brain microvascular endothelial cells (HBMvECs) with TNF-α (0-100 ng/mL, 0-24 h) robustly elicited both time- and dose-dependent induction of IL-6 expression and release, as well as expression of the IL-6 family receptor, GP130. Further experiments demonstrated that the TNF-α-dependent generation of reactive oxygen species, down-regulation of adherens/tight junction proteins, and concomitant elevation of HBMvEC permeability, were all significantly attenuated by blockade of IL-6 signalling using either an anti-IL-6 neutralizing antibody or an IL-6 siRNA. Based on these observations, we conclude that TNF-α treatment of HBMvECs in vitro activates IL-6 production and signalling, events that were shown to synergize with TNF-α actions to elicit HBMvEC permeabilization. These novel findings offer a constructive insight into the specific contribution of downstream cytokine induction to the injurious actions of TNF-α at the BBB microvascular endothelium interface. The co-involvement of tumour necrosis factor-α (TNF-α) and interleukin-6 (IL-6) during blood-brain barrier (BBB) injury has been widely reported. Using human brain microvascular endothelial cells (HBMvEC), we show that TNF-α-mediated BBB disruption is measurably attributable in-part to induction of endothelial IL-6 production and signalling. We demonstrate that the TNF-α-dependent generation of reactive oxygen species (ROS), down-regulation of

Microvascular endothelial function and severity of primary open angle glaucoma.

PubMed

Bukhari, S M I; Kiu, K Y; Thambiraja, R; Sulong, S; Rasool, A H G; Liza-Sharmini, A T

2016-12-01

PurposeThe role of microvascular endothelial dysfunction on severity of primary open angle glaucoma (POAG) was investigated in this study.Patients and methodsA prospective cohort study was conducted. One hundred and fourteen ethnically Malay patients (114 eyes) with POAG treated at the eye clinic of Hospital University Sains Malaysia between April 2012 and December 2014 were recruited. Patients aged between 40 and 80 years with two consecutive reliable and reproducible Humphrey visual field 24-2 analyses were selected. Patients who were diagnosed with any other type of glaucoma, previous glaucoma-filtering surgery, or other surgeries except uncomplicated cataract and pterygium surgery were excluded. Humphrey visual field analysis 24-2 was used to stratify the severity of glaucoma using Advanced Glaucoma Intervention Study (AGIS) score at the time of recruitment. Microvascular endothelial function was assessed using Laser Doppler fluximetry and iontophoresis. Iontophoresis process with acetylcholine (ACh) and sodium nitroprusside (SNP) was used to measure microvascular endothelium-dependent and -independent vasodilatation, respectively.ResultsBased on the AGIS score, 55 patients showed mild glaucoma, with 29 moderate and 30 severe. There was statistically significant difference in microvascular endothelial function (ACh% and ACh max ) between mild and moderate POAG cases (P=0.023) and between mild and severe POAG cases (P<0.001). There was negative correlation between microvascular endothelial function and severity of POAG (r=-0.457, P<0.001).ConclusionMicrovascular endothelial dysfunction may have a role in influencing the severity of POAG in Malay patients.

The effect on endothelial function of vitamin C during methionine induced hyperhomocysteinaemia.

PubMed

Hanratty, C G; McGrath, L T; McAuley, D F; Young, I S; Johnston, D G

2001-01-01

Manipulation of total homocysteine concentration with oral methionine is associated with impairment of endothelial-dependent vasodilation. This may be caused by increased oxidative stress. Vitamin C is an aqueous phase antioxidant vitamin and free radical scavenger. We hypothesised that if the impairment of endothelial function related to experimental hyperhomocysteinaemia was free radically mediated then co-administration of vitamin C should prevent this. Ten healthy adults took part in this crossover study. Endothelial function was determined by measuring forearm blood flow (FBF) in response to intra-arterial infusion of acetylcholine (endothelial-dependent) and sodium nitroprusside (endothelial-independent). Subjects received methionine (100 mg/Kg) plus placebo tablets, methionine plus vitamin C (2 g orally) or placebo drink plus placebo tablets. Study drugs were administered at 9 am on each study date, a minimum of two weeks passed between each study. Homocysteine (tHcy) concentration was determined at baseline and after 4 hours. Endothelial function was determined at 4 hours. Responses to the vasoactive substances are expressed as the area under the curve of change in FBF from baseline. Data are mean plus 95% Confidence Intervals. Following oral methionine tHcy concentration increased significantly versus placebo. At this time endothelial-dependent responses were significantly reduced compared to placebo (31.2 units [22.1-40.3] vs. 46.4 units [42.0-50.8], p < 0.05 vs. Placebo). Endothelial-independent responses were unchanged. Co-administration of vitamin C did not alter the increase in homocysteine or prevent the impairment of endothelial-dependent responses (31.4 [19.5-43.3] vs. 46.4 units [42.0-50.8], p < 0.05 vs. Placebo) This study demonstrates that methionine increased tHcy with impairment of the endothelial-dependent vasomotor responses. Administration of vitamin C did not prevent this impairment and our results do not support the hypothesis that the

Direct visualization of the arterial wall water permeability barrier using CARS microscopy

PubMed Central

Lucotte, Bertrand M.; Powell, Chloe; Knutson, Jay R.; Combs, Christian A.; Malide, Daniela; Yu, Zu-Xi; Knepper, Mark; Patel, Keval D.; Pielach, Anna; Johnson, Errin; Borysova, Lyudmyla; Balaban, Robert S.

2017-01-01

The artery wall is equipped with a water permeation barrier that allows blood to flow at high pressure without significant water leak. The precise location of this barrier is unknown despite its importance in vascular function and its contribution to many vascular complications when it is compromised. Herein we map the water permeability in intact arteries, using coherent anti-Stokes Raman scattering (CARS) microscopy and isotopic perfusion experiments. Generation of the CARS signal is optimized for water imaging with broadband excitation. We identify the water permeation barrier as the endothelial basolateral membrane and show that the apical membrane is highly permeable. This is confirmed by the distribution of the AQP1 water channel within endothelial membranes. These results indicate that arterial pressure equilibrates within the endothelium and is transmitted to the supporting basement membrane and internal elastic lamina macromolecules with minimal deformation of the sensitive endothelial cell. Disruption of this pressure transmission could contribute to endothelial cell dysfunction in various pathologies. PMID:28373558

Functional characterization of human pluripotent stem cell-derived arterial endothelial cells.

PubMed

Zhang, Jue; Chu, Li-Fang; Hou, Zhonggang; Schwartz, Michael P; Hacker, Timothy; Vickerman, Vernella; Swanson, Scott; Leng, Ning; Nguyen, Bao Kim; Elwell, Angela; Bolin, Jennifer; Brown, Matthew E; Stewart, Ron; Burlingham, William J; Murphy, William L; Thomson, James A

2017-07-25

Here, we report the derivation of arterial endothelial cells from human pluripotent stem cells that exhibit arterial-specific functions in vitro and in vivo. We combine single-cell RNA sequencing of embryonic mouse endothelial cells with an EFNB2-tdTomato/EPHB4-EGFP dual reporter human embryonic stem cell line to identify factors that regulate arterial endothelial cell specification. The resulting xeno-free protocol produces cells with gene expression profiles, oxygen consumption rates, nitric oxide production levels, shear stress responses, and TNFα-induced leukocyte adhesion rates characteristic of arterial endothelial cells. Arterial endothelial cells were robustly generated from multiple human embryonic and induced pluripotent stem cell lines and have potential applications for both disease modeling and regenerative medicine.

Arterial endothelial function measurement method and apparatus

DOEpatents

Maltz, Jonathan S; Budinger, Thomas F

2014-03-04

A "relaxoscope" (100) detects the degree of arterial endothelial function. Impairment of arterial endothelial function is an early event in atherosclerosis and correlates with the major risk factors for cardiovascular disease. An artery (115), such as the brachial artery (BA) is measured for diameter before and after several minutes of either vasoconstriction or vasorelaxation. The change in arterial diameter is a measure of flow-mediated vasomodification (FMVM). The relaxoscope induces an artificial pulse (128) at a superficial radial artery (115) via a linear actuator (120). An ultrasonic Doppler stethoscope (130) detects this pulse 10-20 cm proximal to the point of pulse induction (125). The delay between pulse application and detection provides the pulse transit time (PTT). By measuring PTT before (160) and after arterial diameter change (170), FMVM may be measured based on the changes in PTT caused by changes in vessel caliber, smooth muscle tone and wall thickness.

Loss of Endothelial Barrier in Marfan Mice (mgR/mgR) Results in Severe Inflammation after Adenoviral Gene Therapy

PubMed Central

Weymann, Alexander; Arif, Rawa; Weber, Antje; Zaradzki, Marcin; Richter, Karsten; Ensminger, Stephan; Robinson, Peter Nicholas; Wagner, Andreas H.; Karck, Matthias; Kallenbach, Klaus

2016-01-01

Objectives Marfan syndrome is an autosomal dominant inherited disorder of connective tissue. The vascular complications of Marfan syndrome have the biggest impact on life expectancy. The aorta of Marfan patients reveals degradation of elastin layers caused by increased proteolytic activity of matrix metalloproteinases (MMPs). In this study we performed adenoviral gene transfer of human tissue inhibitor of matrix metalloproteinases-1 (hTIMP-1) in aortic grafts of fibrillin-1 deficient Marfan mice (mgR/mgR) in order to reduce elastolysis. Methods We performed heterotopic infrarenal transplantation of the thoracic aorta in female mice (n = 7 per group). Before implantation, mgR/mgR and wild-type aortas (WT, C57BL/6) were transduced ex vivo with an adenoviral vector coding for human TIMP-1 (Ad.hTIMP-1) or β-galactosidase (Ad.β-Gal). As control mgR/mgR and wild-type aortas received no gene therapy. Thirty days after surgery, overexpression of the transgene was assessed by immunohistochemistry (IHC) and collagen in situ zymography. Histologic staining was performed to investigate inflammation, the neointimal index (NI), and elastin breaks. Endothelial barrier function of native not virus-exposed aortas was evaluated by perfusion of fluorescent albumin and examinations of virus-exposed tissue were performed by transmission electron microscopy (TEM). Results IHC and ISZ revealed sufficient expression of the transgene. Severe cellular inflammation and intima hyperplasia were seen only in adenovirus treated mgR/mgR aortas (Ad.β-Gal, Ad.hTIMP-1 NI: 0.23; 0.43), but not in native and Ad.hTIMP-1 treated WT (NI: 0.01; 0.00). Compared to native mgR/mgR and Ad.hTIMP-1 treated WT aorta, the NI is highly significant greater in Ad.hTIMP-1 transduced mgR/mgR aorta (p = 0.001; p = 0.001). As expected, untreated Marfan grafts showed significant more elastolysis compared to WT (p = 0.001). However, elastolysis in Marfan aortas was not reduced by adenoviral overexpression of hTIMP-1

Comparison of Endothelial Function in Asian Indians Versus Caucasians

PubMed Central

Sert Kuniyoshi, Fatima H.; Shamsuzzaman, Abu S.M.; Singh, Prachi; Maharaj, Shantal; Leinveber, Pavel; Nykodym, Jiri; Somers, Virend K.

2016-01-01

Abstract Background: Asian Indians have markedly increased mortality due to coronary artery disease (CAD). Impaired endothelial function has been linked to an increased risk of acute cardiovascular events. We tested the hypothesis that endothelial function was attenuated in Asian Indians and Caucasians. Methods: We studied 14 Asian Indians [mean age: 30 ± 6 years; mean body mass index (BMI): 25 ± 3 kg/m2] and 11 Caucasians (mean age: 30 ± 5 years; mean BMI: 26 ± 2 kg/m2). All 25 subjects were healthy men and nonsmokers without any history of CAD or diabetes and were not taking medications. Endothelial function was evaluated by ultrasound measures of flow-mediated dilatation (FMD) and endothelium-independent nonflow mediated vasodilatation (NFMD) of the brachial artery, in the morning immediately after awakening (6 a.m.) in a fasting state. Results: Mean age, BMI, apnea–hypopnea index, heart rate, and blood pressure were similar in both groups (P = >0.05). When correcting for body surface area, brachial artery diameter was not different between the two groups (2.1% ± 0.3% vs. 2.2% ± 0.4%; P = 0.29). FMD and NFMD were similar in Asian Indians and Caucasians (5.9% ± 4.1% vs. 5.7% ± 2.6%, P = 0.70; 16.4% ± 8% vs. 14.8% ± 4.1%, P = 0.58, respectively). Conclusion: Endothelial function in Asian Indian men is not attenuated in comparison to Caucasian men. PMID:27172431

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.

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

A systematic review of vascular and endothelial function: effects of fruit, vegetable and potassium intake.

PubMed

Blanch, N; Clifton, P M; Keogh, J B

2015-03-01

To review the relationships between: 1) Potassium and endothelial function; 2) Fruits and vegetables and endothelial function; 3) Potassium and other measures of vascular function; 4) Fruits and vegetables and other measures of vascular function. An electronic search for intervention trials investigating the effect of potassium, fruits and vegetables on vascular function was performed in MEDLINE, EMBASE and the Cochrane Library. Potassium appears to improve endothelial function with a dose of >40 mmol/d, however the mechanisms for this effect remain unclear. Potassium may improve measures of vascular function however this effect may be dependent on the effect of potassium on blood pressure. The effect of fruit and vegetables on endothelial function independent of confounding variables is less clear. Increased fruit and vegetable intake may improve vascular function only in high risk populations. Increasing dietary potassium appears to improve vascular function but the effect of increasing fruit and vegetable intake per se on vascular function is less clear. Copyright © 2014 Elsevier B.V. All rights reserved.

Ionophore and Biometal Modulation of P-glycoprotein Expression and Function in Human Brain Microvascular Endothelial Cells.

PubMed

McInerney, Mitchell P; Volitakis, Irene; Bush, Ashley I; Banks, William A; Short, Jennifer L; Nicolazzo, Joseph A

2018-03-05

Biometals such as zinc and copper have been shown to affect tight junction expression and subsequently blood-brain barrier (BBB) integrity. Whether these biometals also influence the expression and function of BBB transporters such as P-glycoprotein (P-gp) however is currently unknown. Using the immortalised human cerebral microvascular endothelial (hCMEC/D3) cell line, an in-cell western assay (alongside western blotting) assessed relative P-gp expression after treatment with the metal ionophore clioquinol and biometals zinc and copper. The fluorescent P-gp substrate rhodamine-123 was employed to observe functional modulation, and inductively coupled plasma mass spectrometry (ICP-MS) provided information on biometal trafficking. A 24-h treatment with clioquinol, zinc and copper (0.5, 0.5 and 0.1 μM) induced a significant upregulation of P-gp (1.7-fold) assessed by in-cell western and this was confirmed with western blotting (1.8-fold increase). This same treatment resulted in a 23% decrease in rhodamine-123 accumulation over a 1 h incubation. ICP-MS demonstrated that while t8his combination treatment had no effect on intracellular zinc concentrations, the treatment significantly enhanced bioavailable copper (4.6-fold). Enhanced delivery of copper to human brain microvascular endothelial cells is associated with enhanced expression and function of the important efflux pump P-gp, which may provide therapeutic opportunities for P-gp modulation.

Sleep restriction impairs blood-brain barrier function.

PubMed

He, Junyun; Hsuchou, Hung; He, Yi; Kastin, Abba J; Wang, Yuping; Pan, Weihong

2014-10-29

The blood-brain barrier (BBB) is a large regulatory and exchange interface between the brain and peripheral circulation. We propose that changes of the BBB contribute to many pathophysiological processes in the brain of subjects with chronic sleep restriction (CSR). To achieve CSR that mimics a common pattern of human sleep loss, we quantified a new procedure of sleep disruption in mice by a week of consecutive sleep recording. We then tested the hypothesis that CSR compromises microvascular function. CSR not only diminished endothelial and inducible nitric oxide synthase, endothelin1, and glucose transporter expression in cerebral microvessels of the BBB, but it also decreased 2-deoxy-glucose uptake by the brain. The expression of several tight junction proteins also was decreased, whereas the level of cyclooxygenase-2 increased. This coincided with an increase of paracellular permeability of the BBB to the small tracers sodium fluorescein and biotin. CSR for 6 d was sufficient to impair BBB structure and function, although the increase of paracellular permeability returned to baseline after 24 h of recovery sleep. This merits attention not only in neuroscience research but also in public health policy and clinical practice. Copyright © 2014 the authors 0270-6474/14/3414697-10$15.00/0.

Differential permissivity of human cerebrovascular endothelial cells to enterovirus infection and specificities of serotype EV-A71 in crossing an in vitro model of the human blood-brain barrier.

PubMed

Volle, Romain; Archimbaud, Christine; Couraud, Pierre-Olivier; Romero, Ignacio A; Weksler, Babette; Mirand, Audrey; Pereira, Bruno; Henquell, Cécile; Peigue-Lafeuille, Hélène; Bailly, Jean-Luc

2015-07-01

Human cerebral microvascular endothelial cells (hCMEC/D3 cell line) form a steady polarized barrier when cultured in vitro on a permeable membrane. Their susceptibility to enterovirus (EV) strains was analysed to investigate how these viruses may cross the blood-brain barrier. A sample of 88 virus strains was selected on phylogenetic features amongst 43 epidemiologically relevant types of the four EV species A-D. The EV-A71 genome was replicated at substantial rates, whilst the infectious virus was released at extremely low but sustained rates at both barrier sides for at least 4 days. EV-A71 antigens were detected in a limited number of cells. The properties of the endothelial barrier (structure and permeability) remained intact throughout infection. The chronic EV-A71 infection was in sharp contrast to the productive infection of cytolytic EVs (e.g. echoviruses E-6 and E-30). The hCMEC/D3 barriers infected with the latter EVs exhibited elevated proportions of apoptotic and necrotic cells, which resulted in major injuries to the endothelial barriers with a dramatic increase of paracellular permeability and virus crossing to the abluminal side. The following intracellular rearrangements were also seen: early destruction of the actin cytoskeleton, remodelling of intracellular membranes and reorganization of the mitochondrion network in a small cluster near the perinuclear space.

[Blood-nerve barrier and peripheral nerve regeneration].

PubMed

Kanda, Takashi

2013-01-01

Blood-nerve barrier (BNB) restricts the movement of soluble mediators and leukocytes from the blood contents to the peripheral nervous system (PNS) parenchyma and thus maintains the endoneurial homeostasis. However, it interferes the supply of various neurotrophic factors from the blood constituents and stops the drainage of toxic substances out of the PNS parenchyma, resulting in the inhibition of peripheral nerve regeneration. If the manipulation of BNB function is possible, regeneration of peripheral nerve may be facilitated via the alteration of peripheral nerve microenvironment and ample supply of neurotrophic substances. A possible method to manipulate the BNB for therapeutic purposes is to modify the endothelial function using siRNAs, oligonucleotides and virus vectors. Another possible method is to modify BNB pericytes: small hydrophobic substances that can reach the pericyte membrane through the endothelial monolayer and strengthen the pericytic activity, including the release of various cytokines/chemokines that influence endothelial function, may also be useful as drug candidates to control the BNB function.

A Triple Culture Model of the Blood-Brain Barrier Using Porcine Brain Endothelial cells, Astrocytes and Pericytes.

PubMed

Thomsen, Louiza Bohn; Burkhart, Annette; Moos, Torben

2015-01-01

In vitro blood-brain barrier (BBB) models based on primary brain endothelial cells (BECs) cultured as monoculture or in co-culture with primary astrocytes and pericytes are useful for studying many properties of the BBB. The BECs retain their expression of tight junction proteins and efflux transporters leading to high trans-endothelial electric resistance (TEER) and low passive paracellular permeability. The BECs, astrocytes and pericytes are often isolated from small rodents. Larger species as cows and pigs however, reveal a higher yield, are readily available and have a closer resemblance to humans, which make them favorable high-throughput sources for cellular isolation. The aim of the present study has been to determine if the preferable combination of purely porcine cells isolated from the 6 months old domestic pigs, i.e. porcine brain endothelial cells (PBECs) in co-culture with porcine astrocytes and pericytes, would compare with PBECs co-cultured with astrocytes and pericytes isolated from newborn rats with respect to TEER value and low passive permeability. The astrocytes and pericytes were grown both as contact and non-contact co-cultures as well as in triple culture to examine their effects on the PBECs for barrier formation as revealed by TEER, passive permeability, and expression patterns of tight junction proteins, efflux transporters and the transferrin receptor. This syngenic porcine in vitro BBB model is comparable to triple cultures using PBECs, rat astrocytes and rat pericytes with respect to TEER formation, low passive permeability, and expression of hallmark proteins signifying the brain endothelium (tight junction proteins claudin 5 and occludin, the efflux transporters P-glycoprotein (PgP) and breast cancer related protein (BCRP), and the transferrin receptor).

Loss of Endothelial Barrier Antigen Immunoreactivity in Rat Retinal Microvessels is Correlated with Clostridium perfringens Type D Epsilon Toxin-induced Damage to the Blood-Retinal Barrier.

PubMed

Mander, K A; Finnie, J W

2018-01-01

Clostridium perfringens type D epsilon toxin (ETX) is a potent neurotoxin producing a severe, and often fatal, neurological disorder in ruminant livestock. Microvascular damage appears to be the fundamental action of ETX in the brain and, recently, similar vascular injury, with subsequent severe vasogenic oedema, has been reported in the retina of rats given ETX. Endothelial barrier antigen (EBA) is a useful marker of an intact blood-brain barrier in rats and it has been shown that loss of EBA immunoreactivity is correlated with ETX-induced cerebral microvascular damage in this species. This paper reports, for the first time, that loss of EBA immunoexpression also occurs in rat retinal microvessels exposed to ETX, the marked reduction in EBA immunopositivity acting as a useful marker for blood-retinal barrier breakdown produced by this neurotoxin. Copyright © 2017 Elsevier Ltd. All rights reserved.

Endothelial function in highly endurance-trained and sedentary, healthy young women.

PubMed

Moe, Ingvild T; Hoven, Heidi; Hetland, Eva V; Rognmo, Oivind; Slørdahl, Stig A

2005-05-01

Endothelial function is reduced by age, chronic heart failure, coronary artery disease, hypertension or type 2 diabetes, and it is shown that aerobic exercise may reverse this trend. The effect of a high aerobic training status on endothelial function in young, healthy subjects is however less clear. The present study was designed to determine whether endothelial function is improved in highly endurance-trained young women compared to sedentary, healthy controls. Brachial artery diameter was measured in 16 endurance-trained (age: 23.7 +/- 2.5 years, maximal oxygen uptake (VO2max): 60.6 +/- 4.5 ml/kg per min) and 14 sedentary females (age: 23.7 +/- 2.1 years, VO2max: 40.5 +/- 5.6 ml/kg per min) at rest, during flow-mediated dilation (FMD) and after sublingual glycerol trinitrate administration, using high-resolution ultrasound. FMD did not differ between the endurance-trained and the sedentary females (14.8% vs 16.4%, p = NS), despite a substantial difference in VO2max of 50% (p < 0.001). The endurance-trained group possessed however, a 9% larger resting brachial artery diameter when adjusted for body surface area. The results of the present study suggest that endothelial function is well preserved in young, healthy women, and that a high aerobic training status due to long term aerobic training does not improve the dilating capacity any further.

Regulation of Endothelial Permeability by Glutathione S-Transferase Pi Against Actin Polymerization.

PubMed

Yang, Yang; Yin, Fangyuan; Hang, Qiyun; Dong, Xiaoliang; Chen, Jiao; Li, Ling; Cao, Peng; Yin, Zhimin; Luo, Lan

2018-01-01

Inflammation-induced injury of the endothelial barrier occurs in several pathological conditions, including atherosclerosis, ischemia, and sepsis. Endothelial cytoskeleton rearrangement is an important pathological mechanism by which inflammatory stimulation triggers an increase of vascular endothelial permeability. However, the mechanism maintaining endothelial cell barrier function against inflammatory stress is not fully understood. Glutathione S-transferase pi (GSTpi) exists in various types of cells and protects them against different stresses. In our previous study, GSTpi was found to act as a negative regulator of inflammatory responses. We used a Transwell permeability assay to test the influence of GSTpi and its transferase activity on the increase of endothelial permeability induced by tumor necrosis factor alpha (TNF-α). TNF-α-induced actin remodeling and the influence of GSTpi were observed by using laser confocal microscopy. Western blotting was used to test the influence of GSTpi on TNF-α-activated p38 mitogen-activated protein kinase (MAPK)/MK2/heat shock protein 27 (HSP27). GSTpi reduced TNF-α-induced stress fiber formation and endothelial permeability increase by restraining actin cytoskeleton rearrangement, and this reduction was unrelated to its transferase activity. We found that GSTpi inhibited p38MAPK phosphorylation by directly binding p38 and influenced downstream substrate HSP27-induced actin remodeling. GSTpi inhibited TNF-α-induced actin remodeling, stress fiber formation and endothelial permeability increase by inhibiting the p38MAPK/HSP27 signaling pathway. © 2018 The Author(s). Published by S. Karger AG, Basel.

The Blood–Brain Barrier

PubMed Central

Daneman, Richard; Prat, Alexandre

2015-01-01

Blood vessels are critical to deliver oxygen and nutrients to all of the tissues and organs throughout the body. The blood vessels that vascularize the central nervous system (CNS) possess unique properties, termed the blood–brain barrier, which allow these vessels to tightly regulate the movement of ions, molecules, and cells between the blood and the brain. This precise control of CNS homeostasis allows for proper neuronal function and also protects the neural tissue from toxins and pathogens, and alterations of these barrier properties are an important component of pathology and progression of different neurological diseases. The physiological barrier is coordinated by a series of physical, transport, and metabolic properties possessed by the endothelial cells (ECs) that form the walls of the blood vessels, and these properties are regulated by interactions with different vascular, immune, and neural cells. Understanding how these different cell populations interact to regulate the barrier properties is essential for understanding how the brain functions during health and disease. PMID:25561720

Smad4 deletion in blood vessel endothelial cells promotes ovarian cancer metastasis.

PubMed

Yang, Jie; Wang, Ya; Zeng, Zhen; Qiao, Long; Zhuang, Liang; Gao, Qinglei; Ma, Ding; Huang, Xiaoyuan

2017-05-01

SMAD4 is a critical co-smad in signal transduction pathways activated in response to transforming growth factor-β (TGF-β)-related ligands, regulating cell growth and differentiation. The roles played by SMAD4 inactivation in tumors highlighted it as a tumor-suppressor gene. Herein, we report that loss of SMAD4 expression in vascular endothelial cells promotes ovarian cancer invasion. SiRNA transfer of this gene in the HUVEC reduced SMAD4 protein expression and function. Although it reduced the vessel endothelial cell tubule formation in vitro and in vivo, it did not affect the tumor growth significantly in vivo. However, it weakened the barrier integrity in endothelial cells and increased vessel permeability and the ovarian cancer liver metastasis. We documented reduced angiogenesis and increased invasion histologically and by intravital microscopy, and gained mechanistic insight at the messenger and gene level. Finally, we found a negative reciprocal regulation between SMAD4 and FYN. FYN is one of the Src family kinases (SFK), activation of which can cause dissociation of cell-cell junctions and adhesion, resulting in paracellular hypermeability. Upon SMAD4 deletion, we detected high expression levels of FYN in vessel endothelial cells, suggesting the mechanism of the ovarian tumor cells cross the endothelial barrier and transform to an invasive phenotype.

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.

Heterozygous Null Bone Morphogenetic Protein Receptor Type 2 Mutations Promote SRC Kinase-dependent Caveolar Trafficking Defects and Endothelial Dysfunction in Pulmonary Arterial Hypertension*

PubMed Central

Prewitt, Allison R.; Ghose, Sampa; Frump, Andrea L.; Datta, Arumima; Austin, Eric D.; Kenworthy, Anne K.; de Caestecker, Mark P.

2015-01-01

Hereditary pulmonary arterial hypertension (HPAH) is a rare, fatal disease of the pulmonary vasculature. The majority of HPAH patients inherit mutations in the bone morphogenetic protein type 2 receptor gene (BMPR2), but how these promote pulmonary vascular disease is unclear. HPAH patients have features of pulmonary endothelial cell (PEC) dysfunction including increased vascular permeability and perivascular inflammation associated with decreased PEC barrier function. Recently, frameshift mutations in the caveolar structural protein gene Caveolin-1 (CAV-1) were identified in two patients with non-BMPR2-associated HPAH. Because caveolae regulate endothelial function and vascular permeability, we hypothesized that defects in caveolar function might be a common mechanism by which BMPR2 mutations promote pulmonary vascular disease. To explore this, we isolated PECs from mice carrying heterozygous null Bmpr2 mutations (Bmpr2+/−) similar to those found in the majority of HPAH patients. We show that Bmpr2+/− PECs have increased numbers and intracellular localization of caveolae and caveolar structural proteins CAV-1 and Cavin-1 and that these defects are reversed after blocking endocytosis with dynasore. SRC kinase is also constitutively activated in Bmpr2+/− PECs, and localization of CAV-1 to the plasma membrane is restored after treating Bmpr2+/− PECs with the SRC kinase inhibitor 3-(4-chlorophenyl)-1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (PP2). Late outgrowth endothelial progenitor cells isolated from HPAH patients show similar increased activation of SRC kinase. Moreover, Bmpr2+/− PECs have impaired endothelial barrier function, and barrier function is restored after treatment with PP2. These data suggest that heterozygous null BMPR2 mutations promote SRC-dependent caveolar trafficking defects in PECs and that this may contribute to pulmonary endothelial barrier dysfunction in HPAH patients. PMID:25411245

Could an endoneurial endothelial crosstalk between Wnt/β-catenin and Sonic Hedgehog pathways underlie the early disruption of the infra-orbital blood-nerve barrier following chronic constriction injury?

PubMed

Moreau, Nathan; Mauborgne, Annie; Couraud, Pierre-Olivier; Romero, Ignacio A; Weksler, Babette B; Villanueva, Luis; Pohl, Michel; Boucher, Yves

2017-01-01

Blood–nerve barrier disruption is pivotal in the development of neuroinflammation, peripheral sensitization, and neuropathic pain after peripheral nerve injury. Activation of toll-like receptor 4 and inactivation of Sonic Hedgehog signaling pathways within the endoneurial endothelial cells are key events, resulting in the infiltration of harmful molecules and immunocytes within the nerve parenchyma. However, we showed in a previous study that preemptive inactivation of toll-like receptor 4 signaling or sustained activation of Sonic Hedgehog signaling did not prevent the local alterations observed following peripheral nerve injury, suggesting the implication of another signaling pathway. Using a classical neuropathic pain model, the infraorbital nerve chronic constriction injury (IoN-CCI), we investigated the role of the Wnt/β-catenin pathway in chronic constriction injury-mediated blood–nerve barrier disruption and in its interactions with the toll-like receptor 4 and Sonic Hedgehog pathways. In the IoN-CCI model versus control, mRNA expression levels and/or immunochemical detection of major Wnt/Sonic Hedgehog pathway (Frizzled-7, vascular endothelial-cadherin, Patched-1 and Gli-1) and/or tight junction proteins (Claudin-1, Claudin-5, and Occludin) readouts were assessed. Vascular permeability was assessed by sodium fluorescein extravasation. IoN-CCI induced early alterations in the vascular endothelial-cadherin/β-catenin/Frizzled-7 complex, shown to participate in local blood–nerve barrier disruption via a β-catenin-dependent tight junction protein downregulation. Wnt pathway also mediated a crosstalk between toll-like receptor 4 and Sonic Hedgehog signaling within endoneurial endothelial cells. Nevertheless, preemptive inhibition of Wnt/β-catenin signaling before IoN-CCI could not prevent the downregulation of key Sonic Hedgehog pathway readouts or the disruption of the infraorbital blood–nerve barrier, suggesting that Sonic Hedgehog pathway

Trafficking of adeno-associated virus vectors across a model of the blood-brain barrier; a comparative study of transcytosis and transduction using primary human brain endothelial cells.

PubMed

Merkel, Steven F; Andrews, Allison M; Lutton, Evan M; Mu, Dakai; Hudry, Eloise; Hyman, Bradley T; Maguire, Casey A; Ramirez, Servio H

2017-01-01

Developing therapies for central nervous system (CNS) diseases is exceedingly difficult because of the blood-brain barrier (BBB). Notably, emerging technologies may provide promising new options for the treatment of CNS disorders. Adeno-associated virus serotype 9 (AAV9) has been shown to transduce cells in the CNS following intravascular administration in rodents, cats, pigs, and non-human primates. These results suggest that AAV9 is capable of crossing the BBB. However, mechanisms that govern AAV9 transendothelial trafficking at the BBB remain unknown. Furthermore, possibilities that AAV9 may transduce brain endothelial cells or affect BBB integrity still require investigation. Using primary human brain microvascular endothelial cells as a model of the human BBB, we performed transduction and transendothelial trafficking assays comparing AAV9 to AAV2, a serotype that does not cross the BBB or transduce endothelial cells effectively in vivo. Results of our in vitro studies indicate that AAV9 penetrates brain microvascular endothelial cells barriers more effectively than AAV2, but has reduced transduction efficiency. In addition, our data suggest that (i) AAV9 penetrates endothelial barriers through an active, cell-mediated process, and (ii) AAV9 fails to disrupt indicators of BBB integrity such as transendothelial electrical resistance, tight junction protein expression/localization, and inflammatory activation status. Overall, this report shows how human brain endothelial cells configured in BBB models can be utilized for evaluating transendothelial movement and transduction kinetics of various AAV capsids. Importantly, the use of a human in vitro BBB model can provide import insight into the possible effects that candidate AVV gene therapy vectors may have on the status of BBB integrity. Read the Editorial Highlight for this article on page 192. © 2016 International Society for Neurochemistry.

Brain endothelial TAK1 and NEMO safeguard the neurovascular unit

PubMed Central

Ridder, Dirk A.; Wenzel, Jan; Müller, Kristin; Töllner, Kathrin; Tong, Xin-Kang; Assmann, Julian C.; Stroobants, Stijn; Weber, Tobias; Niturad, Cristina; Fischer, Lisanne; Lembrich, Beate; Wolburg, Hartwig; Grand’Maison, Marilyn; Papadopoulos, Panayiota; Korpos, Eva; Truchetet, Francois; Rades, Dirk; Sorokin, Lydia M.; Schmidt-Supprian, Marc; Bedell, Barry J.; Pasparakis, Manolis; Balschun, Detlef; D’Hooge, Rudi; Löscher, Wolfgang; Hamel, Edith

2015-01-01

Inactivating mutations of the NF-κB essential modulator (NEMO), a key component of NF-κB signaling, cause the genetic disease incontinentia pigmenti (IP). This leads to severe neurological symptoms, but the mechanisms underlying brain involvement were unclear. Here, we show that selectively deleting Nemo or the upstream kinase Tak1 in brain endothelial cells resulted in death of endothelial cells, a rarefaction of brain microvessels, cerebral hypoperfusion, a disrupted blood–brain barrier (BBB), and epileptic seizures. TAK1 and NEMO protected the BBB by activating the transcription factor NF-κB and stabilizing the tight junction protein occludin. They also prevented brain endothelial cell death in a NF-κB–independent manner by reducing oxidative damage. Our data identify crucial functions of inflammatory TAK1–NEMO signaling in protecting the brain endothelium and maintaining normal brain function, thus explaining the neurological symptoms associated with IP. PMID:26347470

The impact of physical training on endothelial function in myocardial infarction survivors: pilot study.

PubMed

Peller, Michał; Balsam, Paweł; Główczyńska, Renata; Ossoliński, Krzysztof; Gilarowska, Anna; Kołtowski, Łukasz; Grabowski, Marcin; Filipiak, Krzysztof J; Opolski, Grzegorz

Endothelial dysfunction (ED) may indirectly influence the outcome of patients with coronary artery disease. To assess the influence of cardiac rehabilitation (CR) on endothelial function in patients after ST-segment elevation myocardial infarction (STEMI). Twenty-nine patients scheduled for CR were included in the study. CR began at least four weeks after STEMI and consisted of 12 or 24 training sessions. Endothelial function assessment was performed before and after CR, using reactive hyperaemia peripheral arterial tonometry. Before the CR, ED was diagnosed in 16 of 29 (55.2%) patients. A total of 25 patients had two assessments of endothelial function: before and after CR. In univariate analysis the factors of negative response of endothelial function to CR were: higher baseline hyperaemia index (lnRHI) (odds ratio [OR] for positive response to CR 0.01; 95% confidence interval [CI] 0.00-0.33; p = 0.01) and higher peak serum troponin I level during index hospitalisation (OR 0.97; 95% CI 0.94-1.00; p = 0.04). The independent, negative predictor of response to CR was lnRHI (OR 0.01; 95% CI 0.01-0.16; p = 0.03). Patients training for 24 sessions (n = 16) had similar lnRHI changes to those of patients training for 12 sessions (n = 9); [0.16 (-0.06)-0.30 vs. 0.10 (0.05-0.15); p = 0.44, respectively]. ED is a frequent abnormality in STEMI survivors. Despite the lack of statistically significant improvement of endothelial function after CR in the analysed group of patients, some factors can influence the efficacy of this type of physical activity. The best effect of CR on endothelial function was observed in patients with baseline ED.

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

Resistance-based interval exercise acutely improves endothelial function in type 2 diabetes.

PubMed

Francois, Monique E; Durrer, Cody; Pistawka, Kevin J; Halperin, Frank A; Little, Jonathan P

2016-11-01

Different modes of exercise, disease, and training status can modify endothelial shear stress and result in distinct effects on endothelial function. To date, no study has examined the influence of type 2 diabetes (T2D) and training status on the acute endothelial response to different modes of interval exercise (INT). We examined the effect of a single session of resistance- and cardio-based INT compared with a time-matched control on endothelial function in 12 age-matched T2D participants, 12 untrained, and 11 trained adults (aged 56 ± 7 yr). Flow-mediated dilation (%FMD) of the brachial artery was assessed at baseline and immediately, 1, and 2 h after an acute bout of cardio interval (C-INT), resistance interval (R-INT), and seated control (CTL); these interventions were randomized and separated by >2 days. C-INT involved seven 1-min cycling intervals at 85% of peak power with 1-min recovery between. R-INT involved the same pattern of seven 1-min intervals using leg resistance exercises. Endothelial function (%FMD) was improved after R-INT in all groups (Condition × Time interaction, P < 0.01), an effect that was most robust in T2D where %FMD was higher immediately (+4.0 ± 2.8%), 1 h (+2.5 ± 2.5%), and 2 h (+1.9 ± 1.9%) after R-INT compared with CTL (P < 0.01 for all). C-INT improved %FMD in T2D at 1-h postexercise (+1.6 ± 2.2%, P = 0.03) compared with CTL. In conclusion, R-INT acutely improves endothelial function throughout the 2-h postexercise period in T2D patients. The long-term impact of resistance exercise performed in an interval pattern is warranted. Copyright © 2016 the American Physiological Society.

Resistance-based interval exercise acutely improves endothelial function in type 2 diabetes

PubMed Central

Francois, Monique E.; Durrer, Cody; Pistawka, Kevin J.; Halperin, Frank A.

2016-01-01

Different modes of exercise, disease, and training status can modify endothelial shear stress and result in distinct effects on endothelial function. To date, no study has examined the influence of type 2 diabetes (T2D) and training status on the acute endothelial response to different modes of interval exercise (INT). We examined the effect of a single session of resistance- and cardio-based INT compared with a time-matched control on endothelial function in 12 age-matched T2D participants, 12 untrained, and 11 trained adults (aged 56 ± 7 yr). Flow-mediated dilation (%FMD) of the brachial artery was assessed at baseline and immediately, 1, and 2 h after an acute bout of cardio interval (C-INT), resistance interval (R-INT), and seated control (CTL); these interventions were randomized and separated by >2 days. C-INT involved seven 1-min cycling intervals at 85% of peak power with 1-min recovery between. R-INT involved the same pattern of seven 1-min intervals using leg resistance exercises. Endothelial function (%FMD) was improved after R-INT in all groups (Condition × Time interaction, P < 0.01), an effect that was most robust in T2D where %FMD was higher immediately (+4.0 ± 2.8%), 1 h (+2.5 ± 2.5%), and 2 h (+1.9 ± 1.9%) after R-INT compared with CTL (P < 0.01 for all). C-INT improved %FMD in T2D at 1-h postexercise (+1.6 ± 2.2%, P = 0.03) compared with CTL. In conclusion, R-INT acutely improves endothelial function throughout the 2-h postexercise period in T2D patients. The long-term impact of resistance exercise performed in an interval pattern is warranted. PMID:27638878

Effect of Saxagliptin on Circulating Endothelial Progenitor Cells and Endothelial Function in Newly Diagnosed Type 2 Diabetic Patients.

PubMed

Li, Fang; Chen, Jiachao; Leng, Fei; Lu, Zhiqiang; Ling, Yan

2017-06-01

Endothelial dysfunction is associated with the risk of cardiovascular complications in diabetic patients. Endothelial progenitor cells (EPCs) and flow-mediated dilation (FMD) are common markers of endothelial function. In this study, we aim to investigate whether the DPP-4 inhibitor saxagliptin modulate EPCs number and FMD in newly diagnosed, treatment-naive type 2 diabetic patients. This was a controlled, randomized, open-label clinical trial. Saxagliptin group and metformin group consumed either saxagliptin 5 mg per day or metformin 1 500 mg per day respectively for 12 weeks. Changes of FMD and EPCs number after 12-week intervention were the primary endpoints. 31 patients were initially enrolled and randomized to saxagliptin group (n=16) and metformin group (n=15). 27 patients completed the trial (saxagliptin group n=14 and metformin group n=13), and 4 patients dropped out during the study. FMD and EPCs number increased significantly in both saxagliptin group and metformin group, and there was no significant difference between groups. 2-h postprandial plasma glucose, HbA1c and diastolic blood pressure improved significantly in both groups, and there was no significant difference between groups. Saxagliptin and metformin had comparable beneficial effects on endothelial function. © Georg Thieme Verlag KG Stuttgart · New York.

Nebivolol: impact on cardiac and endothelial function and clinical utility.

PubMed

Toblli, Jorge Eduardo; DiGennaro, Federico; Giani, Jorge Fernando; Dominici, Fernando Pablo

2012-01-01

Endothelial dysfunction is a systemic pathological state of the endothelium characterized by a reduction in the bioavailability of vasodilators, essentially nitric oxide, leading to impaired endothelium-dependent vasodilation, as well as disarrangement in vascular wall metabolism and function. One of the key factors in endothelial dysfunction is overproduction of reactive oxygen species which participate in the development of hypertension, atherosclerosis, diabetes, cardiac hypertrophy, heart failure, ischemia-reperfusion injury, and stroke. Because impaired endothelial activity is believed to have a major causal role in the pathophysiology of vascular disease, hypertension, and heart failure, therapeutic agents which modify this condition are of clinical interest. Nebivolol is a third-generation β-blocker with high selectivity for β1-adrenergic receptors and causes vasodilation by interaction with the endothelial L-arginine/ nitric oxide pathway. This dual mechanism of action underscores several hemodynamic qualities of nebivolol, which include reductions in heart rate and blood pressure and improvements in systolic and diastolic function. Although nebivolol reduces blood pressure to a degree similar to that of conventional β-blockers and other types of antihypertensive drugs, it may have advantages in populations with difficult-to-treat hypertension, such as patients with heart failure along with other comorbidities, like diabetes and obesity, and elderly patients in whom nitric oxide-mediated endothelial dysfunction may be more pronounced. Furthermore, recent data indicate that nebivolol appears to be a cost-effective treatment for elderly patients with heart failure compared with standard care. Thus, nebivolol is an effective and well tolerated agent with benefits above those of traditional β-blockers due to its influence on nitric oxide release, which give it singular hemodynamic effects, cardioprotective activity, and a good tolerability profile. This

Anti–IL-6 neutralizing antibody modulates blood-brain barrier function in the ovine fetus

PubMed Central

Zhang, Jiyong; Sadowska, Grazyna B.; Chen, Xiaodi; Park, Seon Yeong; Kim, Jeong-Eun; Bodge, Courtney A.; Cummings, Erin; Lim, Yow-Pin; Makeyev, Oleksandr; Besio, Walter G.; Gaitanis, John; Banks, William A.; Stonestreet, Barbara S.

2015-01-01

Impaired blood-brain barrier function represents an important component of hypoxic-ischemic brain injury in the perinatal period. Proinflammatory cytokines could contribute to ischemia-related blood-brain barrier dysfunction. IL-6 increases vascular endothelial cell monolayer permeability in vitro. However, contributions of IL-6 to blood-brain barrier abnormalities have not been examined in the immature brain in vivo. We generated pharmacologic quantities of ovine-specific neutralizing anti-IL-6 mAbs and systemically infused mAbs into fetal sheep at 126 days of gestation after exposure to brain ischemia. Anti–IL-6 mAbs were measured by ELISA in fetal plasma, cerebral cortex, and cerebrospinal fluid, blood-brain barrier permeability was quantified using the blood-to-brain transfer constant in brain regions, and IL-6, tight junction proteins, and plasmalemma vesicle protein (PLVAP) were detected by Western immunoblot. Anti–IL-6 mAb infusions resulted in increases in mAb (P < 0.05) in plasma, brain parenchyma, and cerebrospinal fluid and decreases in brain IL-6 protein. Twenty-four hours after ischemia, anti–IL-6 mAb infusions attenuated ischemia-related increases in blood-brain barrier permeability and modulated tight junction and PLVAP protein expression in fetal brain. We conclude that inhibiting the effects of IL-6 protein with systemic infusions of neutralizing antibodies attenuates ischemia-related increases in blood-brain barrier permeability by inhibiting IL-6 and modulates tight junction proteins after ischemia.—Zhang, J., Sadowska, G. B., Chen, X., Park, S. Y., Kim, J.-E., Bodge, C. A., Cummings, E., Lim, Y.-P., Makeyev, O., Besio, W. G., Gaitanis, J., Banks, W. A., Stonestreet, B. S. Anti–IL-6 neutralizing antibody modulates blood-brain barrier function in the ovine fetus. PMID:25609424

The effects of anti-obesity intervention with orlistat and sibutramine on microvascular endothelial function.

PubMed

Al-Tahami, Belqes Abdullah Mohammad; Ismail, Ab Aziz Al-Safi; Bee, Yvonne Tee Get; Awang, Siti Azima; Salha Wan Abdul Rani, Wan Rimei; Sanip, Zulkefli; Rasool, Aida Hanum Ghulam

2015-01-01

Obesity is associated with impaired microvascular endothelial function. We aimed to determine the effects of orlistat and sibutramine treatment on microvascular endothelial function, anthropometric and lipid profile, blood pressure (BP), and heart rate (HR). 76 subjects were recruited and randomized to receive orlistat 120 mg three times daily or sibutramine 10 mg daily for 9 months. Baseline weight, BMI, BP, HR and lipid profile were taken. Microvascular endothelial function was assessed using laser Doppler fluximetry and iontophoresis process. Maximum change (max), percent change (% change) and peak flux (peak) in perfusion to acetylcholine (ACh) and sodium nitroprusside (SNP) iontophoresis were used to quantify endothelium dependent and independent vasodilatations. 24 subjects in both groups completed the trial. After treatment, weight and BMI were decreased for both groups. AChmax, ACh % change and ACh peak were increased in orlistat-treated group but no difference was observed for sibutramine-treated group. BP and total cholesterol (TC) were reduced for orlistat-treated group. HR was reduced for orlistat-treated group but was increased in sibutramine-treated group. 9 months treatment with orlistat significantly improved microvascular endothelial function. This was associated with reductions in weight, BMI, BP, HR, TC and low density lipoprotein cholesterol. No effect was seen in microvascular endothelial function with sibutramine.

Arginase Inhibition Improves Microvascular Endothelial Function in Patients With Type 2 Diabetes Mellitus.

PubMed

Kövamees, Oskar; Shemyakin, Alexey; Checa, Antonio; Wheelock, Craig E; Lundberg, Jon O; Östenson, Claes-Göran; Pernow, John

2016-11-01

The development of microvascular complications in diabetes is a complex process in which endothelial dysfunction is important. Emerging evidence suggests that arginase is a key mediator of endothelial dysfunction in type 2 diabetes mellitus by reciprocally regulating nitric oxide bioavailability. The aim of this prospective intervention study was to test the hypothesis that arginase activity is increased and that arginase inhibition improves microvascular endothelial function in patients with type 2 diabetes and microvascular dysfunction. Microvascular endothelium-dependent and -independent dilatation was determined in patients with type 2 diabetes (n = 12) and healthy age-matched control subjects (n = 12) with laser Doppler flowmetry during iontophoretic application of acetylcholine and sodium nitroprusside, respectively, before and after administration of the arginase inhibitor N ω -hydroxy-nor-L-arginine (120 min). Plasma ratios of amino acids involved in arginase and nitric oxide synthase activities were determined. The laser Doppler flowmetry data were the primary outcome variable. Microvascular endothelium-dependent dilatation was impaired in subjects with type 2 diabetes (P < .05). After administration of N ω -hydroxy-nor-L-arginine, microvascular endothelial function improved significantly in patients with type 2 diabetes to the level observed in healthy controls. Endothelium-independent vasodilatation did not change significantly. Subjects with type 2 diabetes had higher levels of ornithine and higher ratios of ornithine/citrulline and ornithine/arginine (P < .05), suggesting increased arginase activity. Arginase inhibition improves microvascular endothelial function in patients with type 2 diabetes and microvascular dysfunction. Arginase inhibition may represent a novel therapeutic strategy to improve microvascular endothelial function in patients with type 2 diabetes.

Single photon emission computed tomography and positron emission tomography imaging of multi-drug resistant P-glycoprotein--monitoring a transport activity important in cancer, blood-brain barrier function and Alzheimer's disease.

PubMed

Piwnica-Worms, David; Kesarwala, Aparna H; Pichler, Andrea; Prior, Julie L; Sharma, Vijay

2006-11-01

Overexpression of multi-drug resistant P-glycoprotein (Pgp) remains an important barrier to successful chemotherapy in cancer patients and impacts the pharmacokinetics of many important drugs. Pgp is also expressed on the luminal surface of brain capillary endothelial cells wherein Pgp functionally comprises a major component of the blood-brain barrier by limiting central nervous system penetration of various therapeutic agents. In addition, Pgp in brain capillary endothelial cells removes amyloid-beta from the brain. Several single photon emission computed tomography and positron emission tomography radiopharmaceutical have been shown to be transported by Pgp, thereby enabling the noninvasive interrogation of Pgp-mediated transport activity in vivo. Therefore, molecular imaging of Pgp activity may enable noninvasive dynamic monitoring of multi-drug resistance in cancer, guide therapeutic choices in cancer chemotherapy, and identify transporter deficiencies of the blood-brain barrier in Alzheimer's disease.

Select Rab GTPases Regulate the Pulmonary Endothelium via Endosomal Trafficking of Vascular Endothelial-Cadherin.

PubMed

Chichger, Havovi; Braza, Julie; Duong, Huetran; Boni, Geraldine; Harrington, Elizabeth O

2016-06-01

Pulmonary edema occurs in settings of acute lung injury, in diseases, such as pneumonia, and in acute respiratory distress syndrome. The lung interendothelial junctions are maintained in part by vascular endothelial (VE)-cadherin, an adherens junction protein, and its surface expression is regulated by endocytic trafficking. The Rab family of small GTPases are regulators of endocytic trafficking. The key trafficking pathways are regulated by Rab4, -7, and -9. Rab4 regulates the recycling of endosomes to the cell surface through a rapid-shuttle process, whereas Rab7 and -9 regulate trafficking to the late endosome/lysosome for degradation or from the trans-Golgi network to the late endosome, respectively. We recently demonstrated a role for the endosomal adaptor protein, p18, in regulation of the pulmonary endothelium through enhanced recycling of VE-cadherin to adherens junction. Thus, we hypothesized that Rab4, -7, and -9 regulate pulmonary endothelial barrier function through modulating trafficking of VE-cadherin-positive endosomes. We used Rab mutants with varying activities and associations to the endosome to study endothelial barrier function in vitro and in vivo. Our study demonstrates a key role for Rab4 activation and Rab9 inhibition in regulation of vascular permeability through enhanced VE-cadherin expression at the interendothelial junction. We further showed that endothelial barrier function mediated through Rab4 is dependent on extracellular signal-regulated kinase phosphorylation and activity. Thus, we demonstrate that Rab4 and -9 regulate VE-cadherin levels at the cell surface to modulate the pulmonary endothelium through extracellular signal-regulated kinase-dependent and -independent pathways, respectively. We propose that regulating select Rab GTPases represents novel therapeutic strategies for patients suffering with acute respiratory distress syndrome.

Endothelial progenitor cells bind and inhibit platelet function and thrombus formation.

PubMed

Abou-Saleh, Haissam; Yacoub, Daniel; Théorêt, Jean-François; Gillis, Marc-Antoine; Neagoe, Paul-Eduard; Labarthe, Benoit; Théroux, Pierre; Sirois, Martin G; Tabrizian, Maryam; Thorin, Eric; Merhi, Yahye

2009-12-01

Interactions of endothelial progenitor cells (EPCs) with vascular and blood cells contribute to vascular homeostasis. Although platelets promote the homing of EPCs to sites of vascular injury and their differentiation into endothelial cells, the functional consequences of such interactions on platelets remain unknown. Herein, we addressed the interactions between EPCs and platelets and their impact on platelet function and thrombus formation. Cultured on fibronectin in conditioned media, human peripheral blood mononuclear cells differentiated, within 10 days of culture, into EPCs, which uptake acetylated low-density lipoprotein, bind ulex-lectin, lack monocyte/leukocyte markers (CD14, P-selectin glycoprotein ligand-1, L-selectin), express progenitor/endothelial markers (CD34, vascular endothelial growth factor receptor-2, von Willebrand factor, and vascular endothelial cadherin), and proliferate in culture. These EPCs bound activated platelets via CD62P and inhibited its translocation, glycoprotein IIb/IIIa activation, aggregation, and adhesion to collagen, mainly via prostacyclin secretion. Indeed, this was associated with upregulation of cyclooxygenase-2 and inducible nitric oxide synthase. However, the effects on platelets in vitro were reversed by cyclooxygenase and cyclooxygenase-2 inhibition but not by nitric oxide or inducible nitric oxide synthase inhibition. Moreover, in a ferric chloride-induced murine arterial thrombosis model, injection of EPCs led to their incorporation into sites of injury and impaired thrombus formation, leading to an incomplete occlusion with 50% residual flow. Peripheral blood mononuclear cell-derived EPCs bind platelets via CD62P and inhibit platelet activation, aggregation, adhesion to collagen, and thrombus formation, predominantly via upregulation of cyclooxygenase-2 and secretion of prostacyclin. These findings add new insights into the biology of EPCs and define their potential roles in regulating platelet function and

Endothelial glycocalyx dysfunction in disease: albuminuria and increased microvascular permeability.

PubMed

Salmon, Andrew H J; Satchell, Simon C

2012-03-01

Appreciation of the glomerular microcirculation as a specialized microcirculatory bed, rather than as an entirely separate entity, affords important insights into both glomerular and systemic microvascular pathophysiology. In this review we compare regulation of permeability in systemic and glomerular microcirculations, focusing particularly on the role of the endothelial glycocalyx, and consider the implications for disease processes. The luminal surface of vascular endothelium throughout the body is covered with endothelial glycocalyx, comprising surface-anchored proteoglycans, supplemented with adsorbed soluble proteoglycans, glycosaminoglycans and plasma constituents. In both continuous and fenestrated microvessels, this endothelial glycocalyx provides resistance to the transcapillary escape of water and macromolecules, acting as an integral component of the multilayered barrier provided by the walls of these microvessels (ie acting in concert with clefts or fenestrae across endothelial cell layers, basement membranes and pericytes). Dysfunction of any of these capillary wall components, including the endothelial glycocalyx, can disrupt normal microvascular permeability. Because of its ubiquitous nature, damage to the endothelial glycocalyx alters the permeability of multiple capillary beds: in the glomerulus this is clinically apparent as albuminuria. Generalized damage to the endothelial glycocalyx can therefore manifest as both albuminuria and increased systemic microvascular permeability. This triad of altered endothelial glycocalyx, albuminuria and increased systemic microvascular permeability occurs in a number of important diseases, such as diabetes, with accumulating evidence for a similar phenomenon in ischaemia-reperfusion injury and infectious disease. The detection of albuminuria therefore has implications for the function of the microcirculation as a whole. The importance of the endothelial glycocalyx for other aspects of vascular function

Endothelial Activation and Blood-Brain Barrier Disruption in Neurotoxicity after Adoptive Immunotherapy with CD19 CAR-T Cells.

PubMed

Gust, Juliane; Hay, Kevin A; Hanafi, Laïla-Aïcha; Li, Daniel; Myerson, David; Gonzalez-Cuyar, Luis F; Yeung, Cecilia; Liles, W Conrad; Wurfel, Mark; Lopez, Jose A; Chen, Junmei; Chung, Dominic; Harju-Baker, Susanna; Özpolat, Tahsin; Fink, Kathleen R; Riddell, Stanley R; Maloney, David G; Turtle, Cameron J

2017-12-01

Lymphodepletion chemotherapy followed by infusion of CD19-targeted chimeric antigen receptor-modified T (CAR-T) cells can be complicated by neurologic adverse events (AE) in patients with refractory B-cell malignancies. In 133 adults treated with CD19 CAR-T cells, we found that acute lymphoblastic leukemia, high CD19 + cells in bone marrow, high CAR-T cell dose, cytokine release syndrome, and preexisting neurologic comorbidities were associated with increased risk of neurologic AEs. Patients with severe neurotoxicity demonstrated evidence of endothelial activation, including disseminated intravascular coagulation, capillary leak, and increased blood-brain barrier (BBB) permeability. The permeable BBB failed to protect the cerebrospinal fluid from high concentrations of systemic cytokines, including IFNγ, which induced brain vascular pericyte stress and their secretion of endothelium-activating cytokines. Endothelial activation and multifocal vascular disruption were found in the brain of a patient with fatal neurotoxicity. Biomarkers of endothelial activation were higher before treatment in patients who subsequently developed grade ≥4 neurotoxicity. Significance: We provide a detailed clinical, radiologic, and pathologic characterization of neurotoxicity after CD19 CAR-T cells, and identify risk factors for neurotoxicity. We show endothelial dysfunction and increased BBB permeability in neurotoxicity and find that patients with evidence of endothelial activation before lymphodepletion may be at increased risk of neurotoxicity. Cancer Discov; 7(12); 1404-19. ©2017 AACR. See related commentary by Mackall and Miklos, p. 1371 This article is highlighted in the In This Issue feature, p. 1355 . ©2017 American Association for Cancer Research.

Abnormal Barrier Function in Gastrointestinal Disorders.

PubMed

Farré, Ricard; Vicario, María

2017-01-01

There is increasing concern in identifying the mechanisms underlying the intimate control of the intestinal barrier, as deregulation of its function is strongly associated with digestive (organic and functional) and a number of non-digestive (schizophrenia, diabetes, sepsis, among others) disorders. The intestinal barrier is a complex and effective defensive functional system that operates to limit luminal antigen access to the internal milieu while maintaining nutrient and electrolyte absorption. Intestinal permeability to substances is mainly determined by the physicochemical properties of the barrier, with the epithelium, mucosal immunity, and neural activity playing a major role. In functional gastrointestinal disorders (FGIDs), the absence of structural or biochemical abnormalities that explain chronic symptoms is probably close to its end, as recent research is providing evidence of structural gut alterations, at least in certain subsets, mainly in functional dyspepsia (FD) and irritable bowel syndrome (IBS). These alterations are associated with increased permeability, which seems to reflect mucosal inflammation and neural activation. The participation of each anatomical and functional component of barrier function in homeostasis and intestinal dysfunction is described, with a special focus on FGIDs.

A novel approach to prevent endothelial hyperpermeability: the Crataegus extract WS® 1442 targets the cAMP/Rap1 pathway.

PubMed

Bubik, Martin F; Willer, Elisabeth A; Bihari, Peter; Jürgenliemk, Guido; Ammer, Hermann; Krombach, Fritz; Zahler, Stefan; Vollmar, Angelika M; Fürst, Robert

2012-01-01

Endothelial hyperpermeability followed by edema formation is a hallmark of many severe disorders. Effective drugs directly targeting endothelial barrier function are widely lacking. We hypothesized that the hawthorn (Crataegus spp.) extract WS® 1442, a proven multi-component drug against moderate forms of heart failure, would prevent vascular leakage by affecting endothelial barrier-regulating systems. In vivo, WS® 1442 inhibited the histamine-evoked extravasation of FITC-dextran from mouse cremaster muscle venules. In cultured human endothelial cells, WS® 1442 blocked the thrombin-induced FITC-dextran permeability. By applying biochemical and microscopic techniques, we revealed that WS® 1442 abrogates detrimental effects of thrombin on adherens junctions (vascular endothelial-cadherin), the F-actin cytoskeleton, and the contractile apparatus (myosin light chain). Mechanistically, WS® 1442 inhibited the thrombin-induced rise of intracellular calcium (ratiometric measurement), followed by an inactivation of PKC and RhoA (pulldown assay). Moreover, WS® 1442 increased endothelial cAMP levels (ELISA), which consequently activated PKA and Rap1 (pulldown assay). Utilizing pharmacological inhibitors or siRNA, we found that PKA is not involved in barrier protection, whereas Epac1, Rap1, and Rac1 play a crucial role in the WS® 1442-induced activation of cortactin, which triggers a strong cortical actin rearrangement. In summary, WS® 1442 effectively protects against endothelial barrier dysfunction in vitro and in vivo. It specifically interacts with endothelial permeability-regulating systems by blocking the Ca(2+)/PKC/RhoA and activating the cAMP/Epac1/Rap1 pathway. As a proven safe herbal drug, WS® 1442 opens a novel pharmacological approach to treat hyperpermeability-associated diseases. This in-depth mechanistic work contributes to a better acceptance of this herbal remedy. Copyright © 2011 Elsevier Ltd. All rights reserved.

Functional Polarity of Microvascular Brain Endothelial Cells Supported by Neurovascular Unit Computational Model of Large Neutral Amino Acid Homeostasis

PubMed Central

Taslimifar, Mehdi; Buoso, Stefano; Verrey, Francois; Kurtcuoglu, Vartan

2018-01-01

The homeostatic regulation of large neutral amino acid (LNAA) concentration in the brain interstitial fluid (ISF) is essential for proper brain function. LNAA passage into the brain is primarily mediated by the complex and dynamic interactions between various solute carrier (SLC) transporters expressed in the neurovascular unit (NVU), among which SLC7A5/LAT1 is considered to be the major contributor in microvascular brain endothelial cells (MBEC). The LAT1-mediated trans-endothelial transport of LNAAs, however, could not be characterized precisely by available in vitro and in vivo standard methods so far. To circumvent these limitations, we have incorporated published in vivo data of rat brain into a robust computational model of NVU-LNAA homeostasis, allowing us to evaluate hypotheses concerning LAT1-mediated trans-endothelial transport of LNAAs across the blood brain barrier (BBB). We show that accounting for functional polarity of MBECs with either asymmetric LAT1 distribution between membranes and/or intrinsic LAT1 asymmetry with low intraendothelial binding affinity is required to reproduce the experimentally measured brain ISF response to intraperitoneal (IP) L-tyrosine and L-phenylalanine injection. On the basis of these findings, we have also investigated the effect of IP administrated L-tyrosine and L-phenylalanine on the dynamics of LNAAs in MBECs, astrocytes and neurons. Finally, the computational model was shown to explain the trans-stimulation of LNAA uptake across the BBB observed upon ISF perfusion with a competitive LAT1 inhibitor. PMID:29593549

Anti-IL-6 neutralizing antibody modulates blood-brain barrier function in the ovine fetus.

PubMed

Zhang, Jiyong; Sadowska, Grazyna B; Chen, Xiaodi; Park, Seon Yeong; Kim, Jeong-Eun; Bodge, Courtney A; Cummings, Erin; Lim, Yow-Pin; Makeyev, Oleksandr; Besio, Walter G; Gaitanis, John; Banks, William A; Stonestreet, Barbara S

2015-05-01

Impaired blood-brain barrier function represents an important component of hypoxic-ischemic brain injury in the perinatal period. Proinflammatory cytokines could contribute to ischemia-related blood-brain barrier dysfunction. IL-6 increases vascular endothelial cell monolayer permeability in vitro. However, contributions of IL-6 to blood-brain barrier abnormalities have not been examined in the immature brain in vivo. We generated pharmacologic quantities of ovine-specific neutralizing anti-IL-6 mAbs and systemically infused mAbs into fetal sheep at 126 days of gestation after exposure to brain ischemia. Anti-IL-6 mAbs were measured by ELISA in fetal plasma, cerebral cortex, and cerebrospinal fluid, blood-brain barrier permeability was quantified using the blood-to-brain transfer constant in brain regions, and IL-6, tight junction proteins, and plasmalemma vesicle protein (PLVAP) were detected by Western immunoblot. Anti-IL-6 mAb infusions resulted in increases in mAb (P < 0.05) in plasma, brain parenchyma, and cerebrospinal fluid and decreases in brain IL-6 protein. Twenty-four hours after ischemia, anti-IL-6 mAb infusions attenuated ischemia-related increases in blood-brain barrier permeability and modulated tight junction and PLVAP protein expression in fetal brain. We conclude that inhibiting the effects of IL-6 protein with systemic infusions of neutralizing antibodies attenuates ischemia-related increases in blood-brain barrier permeability by inhibiting IL-6 and modulates tight junction proteins after ischemia. © FASEB.

Endothelial Function and Weight Loss: Comparison of Low-Carbohydrate and Low-Fat Diets

PubMed Central

Mohler, Emile R.; Sibley, Alexandra A.; Stein, Richard; Davila-Roman, Victor; Wyatt, Holly; Badellino, Karen; Rader, Daniel J.; Klein, Samuel; Foster, Gary D.

2012-01-01

The effect of weight loss on obesity-associated endothelial dysfunction is not clear because of conflicting data, demonstrating both improvement and no change in endothelial function after weight loss in obese subjects. A two-year prospective study (n=121) was conducted to examine: 1) the effect of obesity and weight loss (either a low-carbohydrate or and low-fat diet) on flow mediated vasodilatation (FMD), a measure of endothelial function. Participants reduced body weight by 7.1±4.4%, 8.7±6.8% 7.1±7.8% and 4.1±7.7% at 3, 6, 12 and 24 months, respectively with no significant differences between the low-fat and low-carbohydrate groups. Endothelial function was inversely correlated with waist circumference, triglyceride level, and directly correlated with leptin in obese persons prior to weight loss. These weight losses did not confer any improvements in FMD. There were no differences between the low-fat and low-carbohydrate diets in FMD at any time point. At 6 months (r = 0.26, p = 0.04) and one year (r = 0.28, p = 0.03), there were positive correlations between change in FMD and change in leptin but not at two years. There was no significant improvement in endothelial function after 7.1±7.8% weight loss at one year and 4.1±7.7% at two years, achieved by either a low carbohydrate or a low fat diet. PMID:23404949

Sildenafil restores endothelial function in the apolipoprotein E knockout mouse

PubMed Central

2013-01-01

Background Atherosclerosis is an inflammatory process of the arterial walls and is initiated by endothelial dysfunction accompanied by an imbalance in the production of reactive oxygen species (ROS) and nitric oxide (NO). Sildenafil, a selective phosphodiesterase-5 (PDE5) inhibitor used for erectile dysfunction, exerts its cardiovascular effects by enhancing the effects of NO. The aim of this study was to investigate the influence of sildenafil on endothelial function and atherosclerosis progression in apolipoprotein E knockout (apoE−/−) mice. Methods ApoE−/− mice treated with sildenafil (Viagra®, 40 mg/kg/day, for 3 weeks, by oral gavage) were compared to the untreated apoE−/− and the wild-type (WT) mice. Aortic rings were used to evaluate the relaxation responses to acetylcholine (ACh) in all of the groups. In a separate set of experiments, the roles of NO and ROS in the relaxation response to ACh were evaluated by incubating the aortic rings with L-NAME (NO synthase inhibitor) or apocynin (NADPH oxidase inhibitor). In addition, the atherosclerotic lesions were quantified and superoxide production was assessed. Results Sildenafil restored the vasodilator response to acetylcholine (ACh) in the aortic rings of the apoE−/− mice. Treatment with L-NAME abolished the vasodilator responses to ACh in all three groups of mice and revealed an augmented participation of NO in the endothelium-dependent vasodilation in the sildenafil-treated animals. The normalized endothelial function in sildenafil-treated apoE−/− mice was unaffected by apocynin highlighting the low levels of ROS production in these animals. Moreover, morphological analysis showed that sildenafil treatment caused approximately a 40% decrease in plaque deposition in the aorta. Conclusion This is the first study demonstrating the beneficial effects of chronic treatment with sildenafil on endothelial dysfunction and atherosclerosis in a model of spontaneous hypercholesterolemia. These data

Nitric-oxide synthase trafficking inducer is a pleiotropic regulator of endothelial cell function and signaling

PubMed Central

2017-01-01

Endothelial nitric-oxide synthase (eNOS) and its bioactive product, nitric oxide (NO), mediate many endothelial cell functions, including angiogenesis and vascular permeability. For example, vascular endothelial growth factor (VEGF)-mediated angiogenesis is inhibited upon reduction of NO bioactivity both in vitro and in vivo. Moreover, genetic disruption or pharmacological inhibition of eNOS attenuates angiogenesis during tissue repair, resulting in delayed wound closure. These observations emphasize that eNOS-derived NO can promote angiogenesis. Intriguingly, eNOS activity is regulated by nitric-oxide synthase trafficking inducer (NOSTRIN), which sequesters eNOS, thereby attenuating NO production. This has prompted significant interest in NOSTRIN's function in endothelial cells. We show here that NOSTRIN affects the functional transcriptome of endothelial cells by down-regulating several genes important for invasion and angiogenesis. Interestingly, the effects of NOSTRIN on endothelial gene expression were independent of eNOS activity. NOSTRIN also affected the expression of secreted cytokines involved in inflammatory responses, and ectopic NOSTRIN overexpression functionally restricted endothelial cell proliferation, invasion, adhesion, and VEGF-induced capillary tube formation. Furthermore, NOSTRIN interacted directly with TNF receptor-associated factor 6 (TRAF6), leading to the suppression of NFκB activity and inhibition of AKT activation via phosphorylation. Interestingly, TNF-α-induced NFκB pathway activation was reversed by NOSTRIN. We found that the SH3 domain of NOSTRIN is involved in the NOSTRIN-TRAF6 interaction and is required for NOSTRIN-induced down-regulation of endothelial cell proteins. These results have broad biological implications, as aberrant NOSTRIN expression leading to deactivation of the NFκB pathway, in turn triggering an anti-angiogenic cascade, might inhibit tumorigenesis and cancer progression. PMID:28235804

Association of Abacavir and Impaired Endothelial Function in Treated and Suppressed HIV-Infected Patients

PubMed Central

Hsue, Priscilla Y.; Hunt, Peter W.; Wu, Yuaner; Schnell, Amanda; Ho, Jennifer E.; Hatano, Hiroyu; Xie, Yu; Martin, Jeffrey N.; Ganz, Peter; Deeks, Steven G.

2009-01-01

Background HIV-infected patients have accelerated atherosclerosis. Abacavir has been associated with increased risk of cardiovascular events, for reasons that remain to be elucidated. As endothelial dysfunction is central to the pathogenesis of atherosclerosis, we tested the hypothesis that current treatment with abacavir is associated with impaired endothelial function. Methods We studied a cohort of 61 antiretroviral-treated patients who had undetectable plasma HIV RNA levels. Endothelial function was assessed by measuring flow-mediated vasodilation (FMD) of the brachial artery. We compared FMD in patients treated with or without abacavir, while adjusting for traditional risk factors and HIV-specific characteristics. Results The median age was 50 years (IQR 45–57). The median duration of HIV infection was 18 years, and the median CD4 cell count was 369 cells/mm3. Thirty subjects (49%) were receiving abacavir. Overall, the median FMD in the HIV-infected patients was low (3.5%; IQR 2.3–5.6%). The FMD was lower in the abacavir-treated patients than those not on abacavir (2.8% vs. 4.9%, p=0.01). After adjustment for traditional risk factors, HIV specific factors, and baseline brachial artery diameter, current abacavir use was independently associated with lower FMD (p=0.017). Duration of therapy and CD4 count were not associated with reduced FMD. Conclusions Endothelial function, a central mechanism in atherosclerosis and a marker of cardiovascular risk, is impaired among antiretroviral-treated patients with undetectable viral loads. Current use of abacavir was independently associated with impaired endothelial function. This finding suggests that abnormal endothelial function may underlie the clinically observed increased risk in myocardial infarction among abacavir-treated patients. PMID:19542863

Endothelial permeability is controlled by spatially defined cytoskeletal mechanics: atomic force microscopy force mapping of pulmonary endothelial monolayer.

PubMed

Birukova, Anna A; Arce, Fernando T; Moldobaeva, Nurgul; Dudek, Steven M; Garcia, Joe G N; Lal, Ratnesh; Birukov, Konstantin G

2009-03-01

Actomyosin contraction directly regulates endothelial cell (EC) permeability, but intracellular redistribution of cytoskeletal tension associated with EC permeability is poorly understood. We used atomic force microscopy (AFM), EC permeability assays, and fluorescence microscopy to link barrier regulation, cell remodeling, and cytoskeletal mechanical properties in EC treated with barrier-protective as well as barrier-disruptive agonists. Thrombin, vascular endothelial growth factor, and hydrogen peroxide increased EC permeability, disrupted cell junctions, and induced stress fiber formation. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine, hepatocyte growth factor, and iloprost tightened EC barriers, enhanced peripheral actin cytoskeleton and adherens junctions, and abolished thrombin-induced permeability and EC remodeling. AFM force mapping and imaging showed differential distribution of cell stiffness: barrier-disruptive agonists increased stiffness in the central region, and barrier-protective agents decreased stiffness in the center and increased it at the periphery. Attenuation of thrombin-induced permeability correlates well with stiffness changes from the cell center to periphery. These results directly link for the first time the patterns of cell stiffness with specific EC permeability responses.

High-intensity Interval training enhances mobilization/functionality of endothelial progenitor cells and depressed shedding of vascular endothelial cells undergoing hypoxia.

PubMed

Tsai, Hsing-Hua; Lin, Chin-Pu; Lin, Yi-Hui; Hsu, Chih-Chin; Wang, Jong-Shyan

2016-12-01

Exercise training improves endothelium-dependent vasodilation, whereas hypoxic stress causes vascular endothelial dysfunction. Monocyte-derived endothelial progenitor cells (Mon-EPCs) contribute to vascular repair process by differentiating into endothelial cells. This study investigates how high-intensity interval (HIT) and moderate-intensity continuous (MCT) exercise training affect circulating Mon-EPC levels and EPC functionality under hypoxic condition. Sixty healthy sedentary males were randomized to engage in either HIT (3-min intervals at 40 and 80 % VO 2max for five repetitions, n = 20) or MCT (sustained 60 % VO 2max , n = 20) for 30 min/day, 5 days/week for 6 weeks, or to a control group (CTL) that did not received exercise intervention (n = 20). Mon-EPC characteristics and EPC functionality under hypoxic exercise (HE, 100 W under 12 % O 2 ) were determined before and after HIT, MCT, and CTL. The results demonstrated that after the intervention, the HIT group exhibited larger improvements in VO 2peak , estimated peak cardiac output (Q C ), and estimated peak perfusions of frontal cerebral lobe (Q FC ) and vastus lateralis (Q VL ) than the MCT group. Furthermore, HIT (a) increased circulating CD14 ++ /CD16 - /CD34 + /KDR + (Mon-1 EPC) and CD14 ++ /CD16 + /CD34 + /KDR + (Mon-2 EPC) cell counts, (b) promoted the migration and tube formation of EPCs, (c) diminished the shedding of endothelial (CD34 - /KDR + /phosphatidylserine + ) cells, and (d) elevated plasma nitrite plus nitrate, stromal cell-derived factor-1, matrix metalloproteinase-9, and vascular endothelial growth factor-A concentrations at rest or following HE, compared to those of MCT. In addition, Mon-1 and -2 EPC counts were directly related to VO 2peak and estimated peak Q C , Q FC , and Q VL . HIT is superior to MCT for improving hemodynamic adaptation and Mon-EPC production. Moreover, HIT effectively enhances EPC functionality and suppresses endothelial injury undergoing hypoxia.

Assessment of macrovascular endothelial function using pulse wave analysis and its association with microvascular reactivity in healthy subjects.

PubMed

Ibrahim, N N I N; Rasool, A H G

2017-08-01

Pulse wave analysis (PWA) and laser Doppler fluximetry (LDF) are non-invasive methods of assessing macrovascular endothelial function and microvascular reactivity respectively. The aim of this study was to assess the correlation between macrovascular endothelial function assessed by PWA and microvascular reactivity assessed by LDF. 297 healthy and non-smoking subjects (159 females, mean age (±SD) 23.56 ± 4.54 years) underwent microvascular reactivity assessment using LDF followed by macrovascular endothelial function assessments using PWA. Pearson's correlation showed no correlation between macrovascular endothelial function and microvascular reactivity (r = -0.10, P = 0.12). There was no significant correlation between macrovascular endothelial function assessed by PWA and microvascular reactivity assessed by LDF in healthy subjects. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Endothelial Progenitor Cells Bind and Inhibit Platelet Function and Thrombus Formation

PubMed Central

Abou-Saleh, Haissam; Yacoub, Daniel; Théorêt, Jean-François; Gillis, Marc-Antoine; Neagoe, Paul-Eduard; Labarthe, Benoit; Théroux, Pierre; Sirois, Martin G.; Tabrizian, Maryam; Thorin, Eric; Merhi, Yahye

2013-01-01

Background Interactions of endothelial progenitor cells (EPCs) with vascular and blood cells contribute to vascular homeostasis. Although platelets promote the homing of EPCs to sites of vascular injury and their differentiation into endothelial cells, the functional consequences of such interactions on platelets remain unknown. Herein, we addressed the interactions between EPCs and platelets and their impact on platelet function and thrombus formation. Methods and Results Cultured on fibronectin in conditioned media, human peripheral blood mononuclear cells differentiated, within 10 days of culture, into EPCs, which uptake acetylated low-density lipoprotein, bind ulex-lectin, lack monocyte/leukocyte markers (CD14, P-selectin glycoprotein ligand-1, L-selectin), express progenitor/endothelial markers (CD34, vascular endothelial growth factor receptor-2, von Willebrand factor, and vascular endothelial cadherin), and proliferate in culture. These EPCs bound activated platelets via CD62P and inhibited its translocation, glycoprotein IIb/IIIa activation, aggregation, and adhesion to collagen, mainly via prostacyclin secretion. Indeed, this was associated with upregulation of cyclooxygenase-2 and inducible nitric oxide synthase. However, the effects on platelets in vitro were reversed by cyclooxygenase and cyclooxygenase-2 inhibition but not by nitric oxide or inducible nitric oxide synthase inhibition. Moreover, in a ferric chloride–induced murine arterial thrombosis model, injection of EPCs led to their incorporation into sites of injury and impaired thrombus formation, leading to an incomplete occlusion with 50% residual flow. Conclusions Peripheral blood mononuclear cell– derived EPCs bind platelets via CD62P and inhibit platelet activation, aggregation, adhesion to collagen, and thrombus formation, predominantly via upregulation of cyclooxygenase-2 and secretion of prostacyclin. These findings add new insights into the biology of EPCs and define their potential

Induced Pluripotent Stem Cell-Derived Endothelial Cells in Insulin Resistance and Metabolic Syndrome.

PubMed

Carcamo-Orive, Ivan; Huang, Ngan F; Quertermous, Thomas; Knowles, Joshua W

2017-11-01

Insulin resistance leads to a number of metabolic and cellular abnormalities including endothelial dysfunction that increase the risk of vascular disease. Although it has been particularly challenging to study the genetic determinants that predispose to abnormal function of the endothelium in insulin-resistant states, the possibility of deriving endothelial cells from induced pluripotent stem cells generated from individuals with detailed clinical phenotyping, including accurate measurements of insulin resistance accompanied by multilevel omic data (eg, genetic and genomic characterization), has opened new avenues to study this relationship. Unfortunately, several technical barriers have hampered these efforts. In the present review, we summarize the current status of induced pluripotent stem cell-derived endothelial cells for modeling endothelial dysfunction associated with insulin resistance and discuss the challenges to overcoming these limitations. © 2017 American Heart Association, Inc.

Effect of amplitude and duration of impulsive pressure on endothelial permeability in in vitro fluid percussion trauma.

PubMed

Nakadate, Hiromichi; Inuzuka, Koji; Akanuma, Suguru; Kakuta, Akira; Aomura, Shigeru

2014-04-16

Intracranial pressure changes during head impact cause brain injuries such as vasogenic edema and cerebral contusion. However, the influence of impulsive pressure on endothelial function has not yet been fully studied in vitro. In this study, we developed a pressure loading device that produced positive and negative pressures by modifying an in vitro fluid percussion model and examined the effects of the amplitude and duration of the pressures on endothelial permeability. Human umbilical vein endothelial cells were subjected to three types of positive pressure (average amplitude/average duration of 352 kPa/23 ms, 73 kPa/27 ms, and 70 kPa/44 ms) and three types of negative pressure (-72 kPa/41 ms, -67 kPa/104 ms, and -91 kPa/108 ms), and the transendothelial electrical resistance (TEER) was measured between 15 min and 24 h after pressure loading for quantifying the formation of an integral monolayer of endothelial cells. After loading, vascular endothelial- (VE-) cadherin, an endothelium-specific cell-cell adhesion molecule involved in endothelial barrier function, was stained and observed using fluorescence microscopy. The pressure loading device could produce positive pressure pulses with amplitudes of 53-1348 kPa and durations of 9-29.1 ms and negative pressure pulses with amplitudes of -52 - -93 kPa and durations of 42.9-179.5 ms. The impulsive pressure reduced the TEER associated with the change in VE-cadherin localization. Additionally, TEER decreased considerably at 15 min and 6 h post-loading, with these changes being significant in positive pressure with larger amplitude and shorter duration and in all types of negative pressures compared to pre-loading. The changes in intracranial pressure during head impact impair endothelial barrier function by the disruption of the integrity of endothelial cell-cell junctions, and the degree of increase in endothelial permeability depends on the amplitude, duration, and direction (compressive and tensile) of the

Low Immunogenic Endothelial Cells Maintain Morphological and Functional Properties Required for Vascular Tissue Engineering.

PubMed

Lau, Skadi; Eicke, Dorothee; Carvalho Oliveira, Marco; Wiegmann, Bettina; Schrimpf, Claudia; Haverich, Axel; Blasczyk, Rainer; Wilhelmi, Mathias; Figueiredo, Constança; Böer, Ulrike

2018-03-01

The limited availability of native vessels suitable for the application as hemodialysis shunts or bypass material demands new strategies in cardiovascular surgery. Tissue-engineered vascular grafts containing autologous cells are considered ideal vessel replacements due to the low risk of rejection. However, endothelial cells (EC), which are central components of natural blood vessels, are difficult to obtain from elderly patients of poor health. Umbilical cord blood represents a promising alternative source for EC, but their allogeneic origin corresponds with the risk of rejection after allotransplantation. To reduce this risk, the human leukocyte antigen class I (HLA I) complex was stably silenced by lentiviral vector-mediated RNA interference (RNAi) in EC from peripheral blood and umbilical cord blood and vein. EC from all three sources were transduced by 93.1% ± 4.8% and effectively, HLA I-silenced by up to 67% compared to nontransduced (NT) cells or transduced with a nonspecific short hairpin RNA, respectively. Silenced EC remained capable to express characteristic endothelial surface markers such as CD31 and vascular endothelial cadherin important for constructing a tight barrier, as well as von Willebrand factor and endothelial nitric oxide synthase important for blood coagulation and vessel tone regulation. Moreover, HLA I-silenced EC were still able to align under unidirectional flow, to take up acetylated low-density lipoprotein, and to form capillary-like tube structures in three-dimensional fibrin gels similar to NT cells. In particular, addition of adipose tissue-derived mesenchymal stem cells significantly improved tube formation capability of HLA I-silenced EC toward long and widely branched vascular networks necessary for prevascularizing vascular grafts. Thus, silencing HLA I by RNAi represents a promising technique to reduce the immunogenic potential of EC from three different sources without interfering with EC-specific morphological and

Effect of angiotensin-converting enzyme inhibitors on vascular endothelial function in hypertensive patients after intensive periodontal treatment.

PubMed

Rubio, María C; Lewin, Pablo G; De la Cruz, Griselda; Sarudiansky, Andrea N; Nieto, Mauricio; Costa, Osvaldo R; Nicolosi, Liliana N

2016-04-01

There is a relation between vascular endothelial function, atherosclerotic disease, and inflammation. Deterioration of endothelial function has been observed twenty-four hours after intensive periodontal treatment. This effect may be counteracted by the action of angiotensin-converting enzyme inhibitors, which improve endothelial function. The aim of the present study was to evaluate vascular endothelial function after intensive periodontal treatment, in hypertensive patients treated with angiotensinconverting enzyme inhibitors. A prospective, longitudinal, comparative study involving repeated measurements was conducted. Fifty-two consecutive patients with severe periodontal disease were divided into two groups, one comprising hypertensive patients treated with converting enzyme inhibitors and the other comprising patients with no clinical signs of pathology and not receiving angiotensin-converting enzyme inhibitors. Endothelial function was assessed by measuring postischemic dilation of the humeral artery (baseline echocardiography Doppler), and intensive periodontal treatment was performed 24h later. Endothelial function was re-assessed 24h and 15 days after periodontal treatment. Results were analyzed using the SPSS 20 statistical software package. Student's t test and MANOVA were calculated and linear regression analysis with 95% confidence intervals and α<0.05 was performed. Arterial dilation at 24 hours was lower compared to baseline in both groups; values corresponding to the groups receiving angiotensin-converting enzyme inhibitors were 11.89 ± 4.87 vs. 7.30 ± 2.90% (p<0.01) and those corresponding to the group not receiving ACE inhibitors were 12.72 ± 4.62 vs. 3.56 ± 2.39 (p<0.001). The differences between groups were statistically significant (p<0.001). The increase in endothelial dysfunction after intensive periodontal treatment was significantly lower in hypertensive patients treated with angiotensin-converting enzyme inhibitors. Endothelial

YAP and TAZ regulate adherens junction dynamics and endothelial cell distribution during vascular development

PubMed Central

Neto, Filipa; Klaus-Bergmann, Alexandra; Ong, Yu Ting; Alt, Silvanus; Vion, Anne-Clémence; Szymborska, Anna; Carvalho, Joana R; Hollfinger, Irene; Bartels-Klein, Eireen; Franco, Claudio A

2018-01-01

Formation of blood vessel networks by sprouting angiogenesis is critical for tissue growth, homeostasis and regeneration. How endothelial cells arise in adequate numbers and arrange suitably to shape functional vascular networks is poorly understood. Here we show that YAP/TAZ promote stretch-induced proliferation and rearrangements of endothelial cells whilst preventing bleeding in developing vessels. Mechanistically, YAP/TAZ increase the turnover of VE-Cadherin and the formation of junction associated intermediate lamellipodia, promoting both cell migration and barrier function maintenance. This is achieved in part by lowering BMP signalling. Consequently, the loss of YAP/TAZ in the mouse leads to stunted sprouting with local aggregation as well as scarcity of endothelial cells, branching irregularities and junction defects. Forced nuclear activity of TAZ instead drives hypersprouting and vascular hyperplasia. We propose a new model in which YAP/TAZ integrate mechanical signals with BMP signaling to maintain junctional compliance and integrity whilst balancing endothelial cell rearrangements in angiogenic vessels. PMID:29400648

Flow-mediated changes in pulse wave velocity: a new clinical measure of endothelial function.

PubMed

Naka, Katerina K; Tweddel, Ann C; Doshi, Sagar N; Goodfellow, Jonathan; Henderson, Andrew H

2006-02-01

To test whether measuring hyperaemic changes in pulse wave velocity (PWV) could be used as a new method of assessing endothelial function for use in clinical practice. Flow-mediated changes in vascular tone may be used to assess endothelial function and may be induced by distal hyperaemia, while endothelium-mediated changes in vascular tone can influence PWV. These three known principles were combined to provide and test a novel method of measuring endothelial function by the acute effects of distal hyperaemia on upper and lower limb PWV (measured by a recently developed method). Flow-mediated changes in upper and lower limb PWV were compared in 17 healthy subjects and seven patients with stable chronic heart failure (CHF), as a condition where endothelial function is impaired but endothelium-independent dilator responses are retained. Corroborative measurements of PWV and brachial artery diameter responses to endothelium-dependent and -independent pharmacological stimuli were performed in a further eight healthy subjects. Flow-mediated reduction of PWV (by 14% with no change in blood pressure) was found in normal subjects but was almost abolished in patients with CHF. PWV responses appear to be inversely related to and relatively greater than brachial artery diameter responses. The method may offer potential advantages of practical use and sensitivity over conduit artery diameter responses to measure endothelial dysfunction.

[Recent studies on corneal epithelial barrier function].

PubMed

Liu, F F; Li, W; Liu, Z G; Chen, W S

2016-08-01

Corneal epithelium, the outermost layer of eyeball, is the main route for foreign materials to enter the eye. Under physiological conditions, the corneal epithelial superficial cells form a functionally selective permeability barrier. Integral corneal epithelial barrier function not only ensures the enrolling of nutrients which is required for regular metabolism, but also prevents foreign bodies, or disease-causing microorganism invasion. Recently, a large number of clinical and experimental studies have shown that abnormal corneal epithelial barrier function is the pathological basis for many ocular diseases. In addition, some study found that corneal epithelial barrier constitutes a variety of proteins involved in cell proliferation, differentiation, apoptosis, and a series of physiological and pathological processes. This paper reviewed recent studies specifically on the corneal epithelial barrier, highlights of its structure, function and influence factors. (Chin J Ophthalmol, 2016, 52: 631-635).

The role of the intestinal microvasculature in inflammatory bowel disease: studies with a modified Caco-2 model including endothelial cells resembling the intestinal barrier in vitro.

PubMed

Kasper, Jennifer Y; Hermanns, Maria Iris; Cavelius, Christian; Kraegeloh, Annette; Jung, Thomas; Danzebrink, Rolf; Unger, Ronald E; Kirkpatrick, Charles James

The microvascular endothelium of the gut barrier plays a crucial role during inflammation in inflammatory bowel disease. We have modified a commonly used intestinal cell model based on the Caco-2 cells by adding microvascular endothelial cells (ISO-HAS-1). Transwell filters were used with intestinal barrier-forming Caco-2 cells on top and the ISO-HAS-1 on the bottom of the filter. The goal was to determine whether this coculture mimics the in vivo situation more closely, and whether the model is suitable to evaluate interactions of, for example, prospective nanosized drug vehicles or contrast agents with this coculture in a physiological and inflamed state as it would occur in inflammatory bowel disease. We monitored the inflammatory responsiveness of the cells (release of IL-8, soluble intercellular adhesion molecule 1, and soluble E-selectin) after exposure to inflammatory stimuli (lipopolysaccharide, TNF-α, INF-γ, IL1-β) and a nanoparticle (Ba/Gd: coprecipitated BaSO 4 and Gd(OH) 3 ), generally used as contrast agents. The barrier integrity of the coculture was evaluated via the determination of transepithelial electrical resistance and the apparent permeability coefficient (P app ) of NaFITC. The behavior of the coculture Caco-1/ISO-HAS-1 was compared to the respective monocultures Caco-2 and ISO-HAS-1. Based on transepithelial electrical resistance, the epithelial barrier integrity of the coculture remained stable during incubation with all stimuli, whereas the P app decreased after exposure to the cytokine mixture (TNF-α, INF-γ, IL1-β, and Ba/Gd). Both the endothelial and epithelial monocultures showed a high inflammatory response in both the upper and lower transwell-compartments. However, in the coculture, inflammatory mediators were only detected on the epithelial side and not on the endothelial side. Thus in the coculture, based on the P app , the epithelial barrier appears to prevent a potential inflammatory overreaction in the underlying

Exercise training improves endothelial function in young prehypertensives

PubMed Central

Beck, Darren T; Casey, Darren P; Martin, Jeffrey S; Emerson, Blaze D; Braith, Randy W

2015-01-01

Prehypertensives exhibit marked endothelial dysfunction, a risk factor for future cardiovascular morbidity and mortality. However, the ability of exercise to ameliorate endothelial dysfunction in prehypertensives is grossly underinvestigated. This prospective randomized and controlled study examined the separate effects of resistance and endurance training on conduit artery endothelial function in young prehypertensives. Forty-three unmedicated prehypertensive (systolic blood pressure [SBP]=120–139 mmHg; diastolic blood pressure [DBP]=80–89 mmHg) but otherwise healthy men and women and 15 normotensive matched time-controls (NMTC); n = 15) between 18 and 35 y of age met screening requirements and participated in the study. Prehypertensive subjects were randomly assigned to either a resistance exercise training (PHRT; n = 15), endurance exercise training (PHET; n = 13) or time-control group (PHTC; n = 15). The treatment groups performed exercise training three days per week for eight weeks. The control groups did not initiate exercise programs throughout the study. Flow mediated dilation (FMD) of the brachial artery, biomarkers of enodothelial function and peripheral blood pressure were evaluated before and after exercise intervention or time-matched control. PHRT and PHET reduced resting SBP (9.6 ± 3.6 and 11.9 ± 3.4 mmHg, respectively; P < 0.05) and DBP (8.0 ± 5.1 and 7.2 ± 3.4 mmHg, respectively; P < 0.05). Exercise training improved brachial artery FMD absolute diameter, percent dilation and normalized percent dilation by 30%, 34% and 19% for PHRT, P < 0.05; and by 54%, 63% and 75% for PHET, P < 0.05; respectively. PHRT and PHET increased plasma concentrations of 6-keto prostaglandin F1α (19% and 22%, respectively; P < 0.05), NOx (19% and 23%, respectively; P < 0.05), and reduced endothelin-1 by (16% and 24%, respectively; P < 0.01). This study provides novel evidence that resistance and endurance exercise separately have beneficial effects on resting

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

Endothelial function in patients with migraine during the interictal period.

PubMed

Silva, Federico A; Rueda-Clausen, Christian F; Silva, Sandra Y; Zarruk, Juan G; Guzmán, Juan C; Morillo, Carlos A; Vesga, Boris; Pradilla, Gustavo; Flórez, Mildred; López-Jaramillo, Patricio

2007-01-01

The aim of this study is to evaluate endothelial function in migraineurs subjects during the asymptomatic period. Migraine has been proposed as a risk factor for cerebrovascular events. The underlying mechanisms that relate these 2 pathologies are unknown. Nitric oxide (NO) has been proposed as the final causative molecule of migraine. Increased NO metabolites concentrations have been reported in migraineurs subjects during acute migraine attacks, but there is no evidence indicating alterations in endothelial NO release during the symptom free period in theses subjects. Fifty migraineurs subjects and 25 healthy subjects matched by gender and age were included. Every subject underwent a complete examination that included medical history, physical examination, resting electrocardiogram, forearm flow-mediated vasodilation (FMD), blood determinations of fasting nitrates and nitrites (NO(2) (-)+ NO(3) (-)), glucose, lipid profile, creatinine, C-reactive protein, and blood cell count. No differences in FMD or NO(2) (-)+ NO(3) (-) were detected among groups. The only difference between migraineurs and control subjects was a higher mean blood pressure 92.1 (8.8) mmHg versus 86.7 (8.2) mmHg P= .01. The endothelial function is not altered during the interictal period in migraineurs subjects.

Regulation of vascular endothelial function by procyanidin-rich foods and beverages.

PubMed

Caton, Paul W; Pothecary, Mark R; Lees, Delphine M; Khan, Noorafza Q; Wood, Elizabeth G; Shoji, Toshihiko; Kanda, Tomomasa; Rull, Gurvinder; Corder, Roger

2010-04-14

Flavonoid-rich diets are associated with a lower mortality from cardiovascular disease. This has been linked to improvements in endothelial function. However, the specific flavonoids, or biologically active metabolites, conferring these beneficial effects have yet to be fully defined. In this experimental study of the effect of flavonoids on endothelial function cultured endothelial cells have been used as a bioassay with endothelin-1 (ET-1) synthesis being measured an index of the response. Evaluation of the relative effects of extracts of cranberry juice compared to apple, cocoa, red wine, and green tea showed inhibition of ET-1 synthesis was dependent primarily on their oligomeric procyanidin content. Procyanidin-rich extracts of cranberry juice triggered morphological changes in endothelial cells with reorganization of the actin cytoskeleton and increased immunostaining for phosphotyrosine residues. These actions were independent of antioxidant activity. Comparison of the effects of apple procyanidin monomers through heptamer showed a clear structure-activity relationship. Although monomer, dimer, and trimer had little effect on ET-1 synthesis, procyanidin tetramer, pentamer, hexamer, and heptamer produced concentration-dependent decreases with IC(50) values of 5.4, 1.6, 0.9, and 0.7 microM, respectively. Levels of ET-1 mRNA showed a similar pattern of decreases, which were inversely correlated with increased expression of Kruppel-like factor 2 (KLF2), a key endothelial transcription factor with a broad range of antiatherosclerotic actions including suppression of ET-1 synthesis. Future investigations of procyanidin-rich products should assess the role KLF2 induction plays in the beneficial vascular effects of high flavonoid consumption.

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.

SH2 domain-containing protein tyrosine phosphatase 2 and focal adhesion kinase protein interactions regulate pulmonary endothelium barrier function.

PubMed

Chichger, Havovi; Braza, Julie; Duong, Huetran; Harrington, Elizabeth O

2015-06-01

Enhanced protein tyrosine phosphorylation is associated with changes in vascular permeability through formation and dissolution of adherens junctions and regulation of stress fiber formation. Inhibition of the protein tyrosine phosphorylase SH2 domain-containing protein tyrosine phosphatase 2 (SHP2) increases tyrosine phosphorylation of vascular endothelial cadherin and β-catenin, resulting in disruption of the endothelial monolayer and edema formation in the pulmonary endothelium. Vascular permeability is a hallmark of acute lung injury (ALI); thus, enhanced SHP2 activity offers potential therapeutic value for the pulmonary vasculature in diseases such as ALI, but this has not been characterized. To assess whether SHP2 activity mediates protection against edema in the endothelium, we assessed the effect of molecular activation of SHP2 on lung endothelial barrier function in response to the edemagenic agents LPS and thrombin. Both LPS and thrombin reduced SHP2 activity, correlated with decreased focal adhesion kinase (FAK) phosphorylation (Y(397) and Y(925)) and diminished SHP2 protein-protein associations with FAK. Overexpression of constitutively active SHP2 (SHP2(D61A)) enhanced baseline endothelial monolayer resistance and completely blocked LPS- and thrombin-induced permeability in vitro and significantly blunted pulmonary edema formation induced by either endotoxin (LPS) or Pseudomonas aeruginosa exposure in vivo. Chemical inhibition of FAK decreased SHP2 protein-protein interactions with FAK concomitant with increased permeability; however, overexpression of SHP2(D61A) rescued the endothelium and maintained FAK activity and FAK-SHP2 protein interactions. Our data suggest that SHP2 activation offers the pulmonary endothelium protection against barrier permeability mediators downstream of the FAK signaling pathway. We postulate that further studies into the promotion of SHP2 activation in the pulmonary endothelium may offer a therapeutic approach for patients

Weight loss improves biomarkers endothelial function and systemic inflammation in obese postmenopausal Saudi women.

PubMed

Abd El-Kader, Shehab Mahmoud; Saiem Al-Dahr, Mohammed H

2016-06-01

Although postmenopausal associated disorders are important public health problems worldwide, to date limited studies evaluated the endothelial function and systemic inflammation response to weight loss in obese postmenopausal women. This study was done to evaluate the endothelial function and systemic inflammation response to weight loss in obese postmenopausal Saudi women. Eighty postmenopausal obese Saudi women (mean age 52.64±6.13 year) participated in two groups: Group (A) received aerobic exercise on treadmill and diet whereas, group (B) received no intervention. Markers of inflammation and endothelial function were measured before and after 3 months at the end of the study. The values of body mass index(BMI), interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), C-reactive protein (CRP), inter-cellular adhesion molecule (ICAM-1), vascular cell adhesion molecule (VCAM-1) and plasminogen activator inhibitor-1 activity (PAI-1:Ac) were significantly decreased in group (A), while changes were not significant in group (B). Also, there were significant differences between mean levels of the investigated parameters in group (A) and group (B) after treatment. Weight loss ameliorates inflammatory cytokines and markers of endothelial function in obese postmenopausal Saudi women.

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

Mice with targeted inactivation of ppap2b in endothelial and hematopoietic cells display enhanced vascular inflammation and permeability.

PubMed

Panchatcharam, Manikandan; Salous, Abdel K; Brandon, Jason; Miriyala, Sumitra; Wheeler, Jessica; Patil, Pooja; Sunkara, Manjula; Morris, Andrew J; Escalante-Alcalde, Diana; Smyth, Susan S

2014-04-01

Lipid phosphate phosphatase 3 (LPP3), encoded by the PPAP2B gene, is an integral membrane enzyme that dephosphorylates, and thereby terminates, the G-protein-coupled receptor-mediated signaling actions of lysophosphatidic acid (LPA) and sphingosine-1-phosphate. LPP3 is essential for normal vascular development in mice, and a common PPAP2B polymorphism is associated with increased risk of coronary artery disease in humans. Herein, we investigate the function of endothelial LPP3 to understand its role in the development and human disease. We developed mouse models with selective LPP3 deficiency in endothelial and hematopoietic cells. Tyrosine kinase Tek promoter-mediated inactivation of Ppap2b resulted in embryonic lethality because of vascular defects. LPP3 deficiency in adult mice, achieved using a tamoxifen-inducible Cre transgene under the control of the Tyrosine kinase Tek promoter, enhanced local and systemic inflammatory responses. Endothelial, but not hematopoietic, cell LPP3 deficiency led to significant increases in vascular permeability at baseline and enhanced sensitivity to inflammation-induced vascular leak. Endothelial barrier function was restored by pharmacological or genetic inhibition of either LPA production by the circulating lysophospholipase D autotaxin or of G-protein-coupled receptor-dependent LPA signaling. Our results identify a role for the autotaxin/LPA-signaling nexus as a mediator of endothelial permeability in inflammation and demonstrate that LPP3 limits these effects. These findings have implications for therapeutic targets to maintain vascular barrier function in inflammatory states.

Functional and Biochemical Endothelial Profiling In Vivo in a Murine Model of Endothelial Dysfunction; Comparison of Effects of 1-Methylnicotinamide and Angiotensin-converting Enzyme Inhibitor

PubMed Central

Bar, Anna; Olkowicz, Mariola; Tyrankiewicz, Urszula; Kus, Edyta; Jasinski, Krzysztof; Smolenski, Ryszard T.; Skorka, Tomasz; Chlopicki, Stefan

2017-01-01

Although it is known that 1-methylnicotinamide (MNA) displays vasoprotective activity in mice, as yet the effect of MNA on endothelial function has not been demonstrated in vivo. Here, using magnetic resonance imaging (MRI) we profile the effects of MNA on endothelial phenotype in mice with atherosclerosis (ApoE/LDLR-/-) in vivo, in comparison to angiotensin (Ang) -converting enzyme (ACE) inhibitor (perindopril), with known vasoprotective activity. On a biochemical level, we analyzed whether MNA- or perindopril-induced improvement in endothelial function results in changes in ACE/Ang II-ACE2/Ang-(1–7) balance, and L-arginine/asymmetric dimethylarginine (ADMA) ratio. Endothelial function and permeability were evaluated in the brachiocephalic artery (BCA) in 4-month-old ApoE/LDLR-/- mice that were non-treated or treated for 1 month or 2 months with either MNA (100 mg/kg/day) or perindopril (10 mg/kg/day). The 3D IntraGate®FLASH sequence was used for evaluation of BCA volume changes following acetylcholine (Ach) administration, and for relaxation time (T1) mapping around BCA to assess endothelial permeability using an intravascular contrast agent. Activity of ACE/Ang II and ACE2/Ang-(1–7) pathways as well as metabolites of L-arginine/ADMA pathway were measured using liquid chromatography/mass spectrometry-based methods. In non-treated 6-month-old ApoE/LDLR-/- mice, Ach induced a vasoconstriction in BCA that amounted to –7.2%. 2-month treatment with either MNA or perindopril resulted in the reversal of impaired Ach-induced response to vasodilatation (4.5 and 5.5%, respectively) and a decrease in endothelial permeability (by about 60% for MNA-, as well as perindopril-treated mice). Improvement of endothelial function by MNA and perindopril was in both cases associated with the activation of ACE2/Ang-(1–7) and the inhibition of ACE/Ang II axes as evidenced by an approximately twofold increase in Ang-(1–9) and Ang-(1–7) and a proportional decrease in Ang II

Early outgrowth cells versus endothelial colony forming cells functions in platelet aggregation.

PubMed

Bou Khzam, Lara; Bouchereau, Olivier; Boulahya, Rahma; Hachem, Ahmed; Zaid, Younes; Abou-Saleh, Haissam; Merhi, Yahye

2015-11-09

Endothelial progenitor cells (EPCs) have been implicated in neoangiogenesis, endothelial repair and cell-based therapies for cardiovascular diseases. We have previously shown that the recruitment of EPCs to sites of vascular lesions is facilitated by platelets where EPCs, in turn, modulate platelet function and thrombosis. However, EPCs encompass a heterogeneous population of progenitor cells that may exert different effects on platelet function. Recent evidence suggests the existence of two EPC subtypes: early outgrowth cells (EOCs) and endothelial colony-forming cells (ECFCs). We aimed at characterizing these two EPC subtypes and at identifying their role in platelet aggregation. EOCs and ECFCs were generated from human peripheral blood mononuclear cells (PBMCs) seeded in conditioned media on fibronectin and collagen, respectively. The morphological, phenotypical and functional characteristics of EOCs and ECFCs were assessed by optical and confocal laser scanning microscopes, cell surface markers expression, and Matrigel tube formation. The impact of EOCs and ECFCs on platelet aggregation was monitored in collagen-induced optical aggregometry and compared with PBMCs and human umbilical vein endothelial cells (HUVECs). The levels of the anti-platelet agents' nitric oxide (NO) and prostacyclin (PGI2) released from cultured cells as well as the expression of their respective producing enzymes NO synthases (NOS) and cyclooxygenases (COX) were also assessed. We showed that EOCs display a monocytic-like phenotype whereas ECFCs have an endothelial-like phenotype. We demonstrated that both EOCs and ECFCs and their supernatants inhibited platelet aggregation; however ECFCs were more efficient than EOCs. This could be related to the release of significantly higher amounts of NO and PGI2 from ECFCs, in comparison to EOCs. Indeed, ECFCs, like HUVECs, constitutively express the endothelial (eNOS)-and inducible (iNOS)-NOS isoforms, and COX-1 and weakly express COX-2, whereas

[Blood-brain barrier part III: therapeutic approaches to cross the blood-brain barrier and target the brain].

PubMed

Weiss, N; Miller, F; Cazaubon, S; Couraud, P-O

2010-03-01

Over the last few years, the blood-brain barrier has come to be considered as the main limitation for the treatment of neurological diseases caused by inflammatory, tumor or neurodegenerative disorders. In the blood-brain barrier, the close intercellular contact between cerebral endothelial cells due to tight junctions prevents the passive diffusion of hydrophilic components from the bloodstream into the brain. Several specific transport systems (via transporters expressed on cerebral endothelial cells) are implicated in the delivery of nutriments, ions and vitamins to the brain; other transporters expressed on cerebral endothelial cells extrude endogenous substances or xenobiotics, which have crossed the cerebral endothelium, out of the brain and into the bloodstream. Recently, several strategies have been proposed to target the brain, (i) by by-passing the blood-brain barrier by central drug administration, (ii) by increasing permeability of the blood-brain barrier, (iii) by modulating the expression and/or the activity of efflux transporters, (iv) by using the physiological receptor-dependent blood-brain barrier transport, and (v) by creating new viral or chemical vectors to cross the blood-brain barrier. This review focuses on the illustration of these different approaches. Copyright (c) 2009 Elsevier Masson SAS. All rights reserved.

Anger, depression and anxiety associated with endothelial function in childhood and adolescence.

PubMed

Osika, W; Montgomery, S M; Dangardt, F; Währborg, P; Gan, L M; Tideman, E; Friberg, P

2011-01-01

Psychosocial adversity is a risk factor for cardiovascular disease (CVD) in adults. The authors assessed associations of reactive hyperaemia peripheral arterial tonometry (RH-PAT), a measure of endothelial function predictive of CVD, with self-assessed psychological health among school children. A total of 248 healthy school children (mean (SD) age 14.0 (1.0); 136 girls and 112 boys) underwent RH-PAT testing. They completed the Beck Youth Inventories (BYI) of emotional and social impairment scales, which is used to screen for depression, anxiety, anger and disruptive behaviour. No sex differences were observed for the RH-PAT score. Statistically significant differences were observed for the BYI scores; girls had higher scores for depression, anger and anxiety. Among the girls, there were statistically significant associations between lower RH-PAT scores and higher scores for anger (B coefficient=-0.100, p=0.040), depression (-0.108, p=0.009) and anxiety (-0.138, p=0.039) after adjustment for age. Among the boys, disruptive behaviour was associated with higher RH-PAT scores (0.09, p=0.006). The girls have higher levels of self-assessed anger; depression and anxiety compared with the boys, and these characteristics are associated with lower RH-PAT scores, indicating attenuated endothelial function. Among the boys, disruptive behaviour was associated with better endothelial function. Although psychological ill-health is associated with impaired endothelial function and CVD among adults, such processes may also be relevant to children. Psychosocial adversity in childhood might be a risk factor for subsequent CVD.

Deterioration of endothelial function of micro- and macrocirculation in patients with diabetes type 1 and 2.

PubMed

Besic, Hana; Jeraj, Luka; Spirkoska, Ana; Jezovnik, Mateja K; Poredoš, Pavel

2017-08-01

Vascular complications are an important cause of morbidity in patients with diabetes mellitus (DM). Endothelial dysfunction is an early marker of atherosclerosis and has already been shown in macrocirculation of diabetic patients; however, data on endothelial function of microcirculation is scarce. Our aim was to evaluate endothelial function in macro- and microcirculation and their interrelationship in patients with type 1 and 2 DM. The study included 30 patients with type 1 DM, 30 patients with type 2 DM and 25 healthy controls. The endothelial function of large arteries was studied measuring flow-mediated dilation (FMD). Peripheral arterial tonometry was used for investigation of the endothelial function of microcirculation, measuring Reactive Hyperemia Index (RHI) and Augmentation Index (AI). In comparison to controls, both DM groups had decreased FMD: type 1 (4.0±5.0% vs. 10.0±7.8%, P=0.005) and type 2 (5.0±0.6% vs. 10.0±7.8%, P=0.007). However, only type 2 DM group had a lower RHI (1.71±0.44 vs. 2.05±0.54, P=0.017) in comparison to controls. Patients with type 1 and 2 DM had deteriorated functional capability of macrocirculation. However, endothelial dysfunction of microcirculation was present only in type 2 DM patients. Type 2 DM patients were also at higher risk for atherosclerosis because of the more frequent presence of risk factors.

Impaired endothelial function in lone atrial fibrillation.

PubMed

Polovina, Marija; Potpara, Tatjana; Giga, Vojislav; Stepanović, Jelena; Ostojić, Miodrag

2013-10-01

Impaired endothelial function has been previously documented in patients with atrial fibrillation (AF) and underlying comorbidities or older patients with idiopathic AF. The aim of this study was to evaluate systemic endothelial function in younger AF patients (less than < 60 years old) with lone AF (that is, without associated cardiopulmonary comorbidities, including arterial hypertension), by comparing brachial artery flow-mediated dilation (FMD) in lone AF patients with FMD of healthy subjects in sinus rhythm. Two groups of participants were prospectively enrolled. The first group comprised of 38 AF patients (the mean age 45 +/- 11 years, 68% male) with persistent (> 7 days) lone AF. The second group comprised of 28 healthy controls in sinus rhythm (the mean age 43 +/- 13, 53% male), matched by age, gender and atherosclerotic risk factors. All the participants underwent physical examination, laboratory analysis [including determination of C-reactive protein (CRP)], standard echocardiography and exercise-stress testing. Brachial artery FMD and endothelium independent dilation (NMD) were assessed with a high-resolution ultrasound probe and arterial diameters taken from 5 consecutive cardiac cycles were averaged for each measurement to accommodate to beat-to-beat flow variations in AF. There were no differences between the 2 groups regarding age, gender and most clinical, laboratory and echocardiographic characteristics (all p > 0.05), apart from the increased heart rate (p = 0.018), body mass index (p = 0.027), CRP levels (p = 0.007) and left atrial anteroposterior dimension (p < 0.001) in AF patients. FMD of AF patients [median value 5.0%, interquartile range (IQR) 2.87%-7.50%] was significantly lower (p < 0.001) than FMD of healthy controls (median value 8.85%, IQR 5.80%-12.50%), whereas there were no differences in median NMD values (p > 0.05). In the multivariate analysis, the independent FMD determinants in our study population were the presence of AF

Wnt activation of immortalized brain endothelial cells as a tool for generating a standardized model of the blood brain barrier in vitro.

PubMed

Paolinelli, Roberta; Corada, Monica; Ferrarini, Luca; Devraj, Kavi; Artus, Cédric; Czupalla, Cathrin J; Rudini, Noemi; Maddaluno, Luigi; Papa, Eleanna; Engelhardt, Britta; Couraud, Pierre Olivier; Liebner, Stefan; Dejana, Elisabetta

2013-01-01

Reproducing the characteristics and the functional responses of the blood-brain barrier (BBB) in vitro represents an important task for the research community, and would be a critical biotechnological breakthrough. Pharmaceutical and biotechnology industries provide strong demand for inexpensive and easy-to-handle in vitro BBB models to screen novel drug candidates. Recently, it was shown that canonical Wnt signaling is responsible for the induction of the BBB properties in the neonatal brain microvasculature in vivo. In the present study, following on from earlier observations, we have developed a novel model of the BBB in vitro that may be suitable for large scale screening assays. This model is based on immortalized endothelial cell lines derived from murine and human brain, with no need for co-culture with astrocytes. To maintain the BBB endothelial cell properties, the cell lines are cultured in the presence of Wnt3a or drugs that stabilize β-catenin, or they are infected with a transcriptionally active form of β-catenin. Upon these treatments, the cell lines maintain expression of BBB-specific markers, which results in elevated transendothelial electrical resistance and reduced cell permeability. Importantly, these properties are retained for several passages in culture, and they can be reproduced and maintained in different laboratories over time. We conclude that the brain-derived endothelial cell lines that we have investigated gain their specialized characteristics upon activation of the canonical Wnt pathway. This model may be thus suitable to test the BBB permeability to chemicals or large molecular weight proteins, transmigration of inflammatory cells, treatments with cytokines, and genetic manipulation.

Effect of shear stress on iPSC-derived human brain microvascular endothelial cells (dhBMECs).

PubMed

DeStefano, Jackson G; Xu, Zinnia S; Williams, Ashley J; Yimam, Nahom; Searson, Peter C

2017-08-04

The endothelial cells that form the lumen of capillaries and microvessels are an important component of the blood-brain barrier. Cell phenotype is regulated by transducing a range of biomechanical and biochemical signals in the local microenvironment. Here we report on the role of shear stress in modulating the morphology, motility, proliferation, apoptosis, and protein and gene expression, of confluent monolayers of human brain microvascular endothelial cells derived from induced pluripotent stem cells. To assess the response of derived human brain microvascular endothelial cells (dhBMECs) to shear stress, confluent monolayers were formed in a microfluidic device. Monolayers were subjected to a shear stress of 4 or 12 dyne cm -2 for 40 h. Static conditions were used as the control. Live cell imaging was used to assess cell morphology, cell speed, persistence, and the rates of proliferation and apoptosis as a function of time. In addition, immunofluorescence imaging and protein and gene expression analysis of key markers of the blood-brain barrier were performed. Human brain microvascular endothelial cells exhibit a unique phenotype in response to shear stress compared to static conditions: (1) they do not elongate and align, (2) the rates of proliferation and apoptosis decrease significantly, (3) the mean displacement of individual cells within the monolayer over time is significantly decreased, (4) there is no cytoskeletal reorganization or formation of stress fibers within the cell, and (5) there is no change in expression levels of key blood-brain barrier markers. The characteristic response of dhBMECs to shear stress is significantly different from human and animal-derived endothelial cells from other tissues, suggesting that this unique phenotype that may be important in maintenance of the blood-brain barrier. The implications of this work are that: (1) in confluent monolayers of dhBMECs, tight junctions are formed under static conditions, (2) the formation

Concurrent generation of functional smooth muscle and endothelial cells via a vascular progenitor.

PubMed

Marchand, Melanie; Anderson, Erica K; Phadnis, Smruti M; Longaker, Michael T; Cooke, John P; Chen, Bertha; Reijo Pera, Renee A

2014-01-01

Smooth muscle cells (SMCs) and endothelial cells (ECs) are typically derived separately, with low efficiencies, from human pluripotent stem cells (hPSCs). The concurrent generation of these cell types might lead to potential applications in regenerative medicine to model, elucidate, and eventually treat vascular diseases. Here we report a robust two-step protocol that can be used to simultaneously generate large numbers of functional SMCs and ECs from a common proliferative vascular progenitor population via a two-dimensional culture system. We show here that coculturing hPSCs with OP9 cells in media supplemented with vascular endothelial growth factor, basic fibroblast growth factor, and bone morphogenetic protein 4 yields a higher percentage of CD31(+)CD34(+) cells on day 8 of differentiation. Upon exposure to endothelial differentiation media and SM differentiation media, these vascular progenitors were able to differentiate and mature into functional endothelial cells and smooth muscle cells, respectively. Furthermore, we were able to expand the intermediate population more than a billion fold to generate sufficient numbers of ECs and SMCs in parallel for potential therapeutic transplantations.

Inhibition of c-Src protects paraquat induced microvascular endothelial injury by modulating caveolin-1 phosphorylation and caveolae mediated transcellular permeability.

PubMed

Huang, Yu; He, Qing

2017-06-01

The mechanisms underlying paraquat induced acute lung injury (ALI) is still not clear. C-Src plays an important role in the regulation of microvascular endothelial barrier function and the pathogenesis of ALI. In the present study, we found that paraquat induced cell toxicity and an increase of reactive oxygen species (ROS) in endothelium. Paraquat exposure also induced significant increase of caveolin-1 phosphorylation, caveolae trafficking and albumin permeability in endothelial monolayers. C-Src depletion by siRNA significantly attenuate paraquat induced cell toxicity, caveolin-1 phosphorylation, caveolae formation and endothelial hyperpermeability. N-acetylcysteine (NAC) failed to protect endothelial monolayers against paraquat induced toxicity. Thus, our findings suggest that paraquat exposure increases paracellular endothelial permeability by increasing caveolin-1 phosphorylation in a c-Src dependant manner. The depletion of c-Src might protect microvascular endothelial function by regulating caveolin-1 phosphorylation and caveolae trafficking during paraquat exposure, and might have potential therapeutic effects on paraquat induced ALI. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Blood-brain barrier hyperpermeability precedes demyelination in the cuprizone model.

PubMed

Berghoff, Stefan A; Düking, Tim; Spieth, Lena; Winchenbach, Jan; Stumpf, Sina K; Gerndt, Nina; Kusch, Kathrin; Ruhwedel, Torben; Möbius, Wiebke; Saher, Gesine

2017-12-01

In neuroinflammatory disorders such as multiple sclerosis, the physiological function of the blood-brain barrier (BBB) is perturbed, particularly in demyelinating lesions and supposedly secondary to acute demyelinating pathology. Using the toxic non-inflammatory cuprizone model of demyelination, we demonstrate, however, that the onset of persistent BBB impairment precedes demyelination. In addition to a direct effect of cuprizone on endothelial cells, a plethora of inflammatory mediators, which are mainly of astroglial origin during the initial disease phase, likely contribute to the destabilization of endothelial barrier function in vivo. Our study reveals that, at different time points of pathology and in different CNS regions, the level of gliosis correlates with the extent of BBB hyperpermeability and edema. Furthermore, in mutant mice with abolished type 3 CXC chemokine receptor (CXCR3) signaling, inflammatory responses are dampened and BBB dysfunction ameliorated. Together, these data have implications for understanding the role of BBB permeability in the pathogenesis of demyelinating disease.

High glucose, glucose fluctuation and carbonyl stress enhance brain microvascular endothelial barrier dysfunction: Implications for diabetic cerebral microvasculature.

PubMed

Li, Wei; Maloney, Ronald E; Aw, Tak Yee

2015-08-01

We previously demonstrated that in normal glucose (5mM), methylglyoxal (MG, a model of carbonyl stress) induced brain microvascular endothelial cell (IHEC) dysfunction that was associated with occludin glycation and prevented by N-acetylcysteine (NAC). Herein, we investigated the impact of high glucose and low GSH, conditions that mimicked the diabetic state, on MG-induced IHEC dysfunction. MG-induced loss of transendothelial electrical resistance (TEER) was potentiated in IHECs cultured for 7 or 12 days in 25 mM glucose (hyperglycemia); moreover, barrier function remained disrupted 6h after cell transfer to normal glucose media (acute glycemic fluctuation). Notably, basal occludin glycation was elevated under these glycemic states. TEER loss was exaggerated by inhibition of glutathione (GSH) synthesis and abrogated by NAC, which corresponded to GSH decreases and increases, respectively. Significantly, glyoxalase II activity was attenuated in hyperglycemic cells. Moreover, hyperglycemia and GSH inhibition increased MG accumulation, consistent with a compromised capacity for MG elimination. α-Oxoaldehydes (MG plus glyoxal) levels were elevated in streptozotocin-induced diabetic rat plasma. Immunohistochemistry revealed a prevalence of MG-positive, but fewer occludin-positive microvessels in the diabetic brain in vivo, and Western analysis confirmed an increase in MG-occludin adducts. These results provide the first evidence that hyperglycemia and acute glucose fluctuation promote MG-occludin formation and exacerbate brain microvascular endothelial dysfunction. Low occludin expression and high glycated-occludin contents in diabetic brain in vivo are factors that would contribute to the dysfunction of the cerebral microvasculature during diabetes. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

High glucose, glucose fluctuation and carbonyl stress enhance brain microvascular endothelial barrier dysfunction: Implications for diabetic cerebral microvasculature

PubMed Central

Li, Wei; Maloney, Ronald E.; Aw, Tak Yee

2015-01-01

We previously demonstrated that in normal glucose (5 mM), methylglyoxal (MG, a model of carbonyl stress) induced brain microvascular endothelial cell (IHEC) dysfunction that was associated with occludin glycation and prevented by N-acetylcysteine (NAC). Herein, we investigated the impact of high glucose and low GSH, conditions that mimicked the diabetic state, on MG-induced IHEC dysfunction. MG-induced loss of transendothelial electrical resistance (TEER) was potentiated in IHECs cultured for 7 or 12 days in 25 mM glucose (hyperglycemia); moreover, barrier function remained disrupted 6 h after cell transfer to normal glucose media (acute glycemic fluctuation). Notably, basal occludin glycation was elevated under these glycemic states. TEER loss was exaggerated by inhibition of glutathione (GSH) synthesis and abrogated by NAC, which corresponded to GSH decreases and increases, respectively. Significantly, glyoxalase II activity was attenuated in hyperglycemic cells. Moreover, hyperglycemia and GSH inhibition increased MG accumulation, consistent with a compromised capacity for MG elimination. α-Oxoaldehydes (MG plus glyoxal) levels were elevated in streptozotocin-induced diabetic rat plasma. Immunohistochemistry revealed a prevalence of MG-positive, but fewer occludin-positive microvessels in the diabetic brain in vivo, and Western analysis confirmed an increase in MG–occludin adducts. These results provide the first evidence that hyperglycemia and acute glucose fluctuation promote MG–occludin formation and exacerbate brain microvascular endothelial dysfunction. Low occludin expression and high glycated-occludin contents in diabetic brain in vivo are factors that would contribute to the dysfunction of the cerebral microvasculature during diabetes. PMID:25867911

An isogenic blood-brain barrier model comprising brain endothelial cells, astrocytes, and neurons derived from human induced pluripotent stem cells.

PubMed

Canfield, Scott G; Stebbins, Matthew J; Morales, Bethsymarie Soto; Asai, Shusaku W; Vatine, Gad D; Svendsen, Clive N; Palecek, Sean P; Shusta, Eric V

2017-03-01

The blood-brain barrier (BBB) is critical in maintaining a physical and metabolic barrier between the blood and the brain. The BBB consists of brain microvascular endothelial cells (BMECs) that line the brain vasculature and combine with astrocytes, neurons and pericytes to form the neurovascular unit. We hypothesized that astrocytes and neurons generated from human-induced pluripotent stem cells (iPSCs) could induce BBB phenotypes in iPSC-derived BMECs, creating a robust multicellular human BBB model. To this end, iPSCs were used to form neural progenitor-like EZ-spheres, which were in turn differentiated to neurons and astrocytes, enabling facile neural cell generation. The iPSC-derived astrocytes and neurons induced barrier tightening in primary rat BMECs indicating their BBB inductive capacity. When co-cultured with human iPSC-derived BMECs, the iPSC-derived neurons and astrocytes significantly elevated trans-endothelial electrical resistance, reduced passive permeability, and improved tight junction continuity in the BMEC cell population, while p-glycoprotein efflux transporter activity was unchanged. A physiologically relevant neural cell mixture of one neuron: three astrocytes yielded optimal BMEC induction properties. Finally, an isogenic multicellular BBB model was successfully demonstrated employing BMECs, astrocytes, and neurons from the same donor iPSC source. It is anticipated that such an isogenic facsimile of the human BBB could have applications in furthering understanding the cellular interplay of the neurovascular unit in both healthy and diseased humans. Read the Editorial Highlight for this article on page 843. © 2016 International Society for Neurochemistry.

Effects of humidified and dry air on corneal endothelial cells during vitreal fluid-air exchange.

PubMed

Cekiç, Osman; Ohji, Masahito; Hayashi, Atsushi; Fang, Xiao Y; Kusaka, Shunji; Tano, Yasuo

2002-07-01

To report the immediate anatomic and functional alterations in corneal endothelial cells following use of humidified air and dry air during vitreal fluid-air exchange in rabbits. Experimental study. Rabbits undergoing pars plana vitrectomy and lensectomy were perfused with either dry or humidified air during fluid-air exchange for designated durations. Three different experiments were performed. First, control and experimental corneas were examined by scanning electron microscopy (SEM). Second, corneas were stained with Phalloidin-FITC and examined by fluorescein microscopy. Finally, third, transendothelial permeability for carboxyfluorescein was determined using a diffusion chamber. While different from the corneal endothelial cells, those cells exposed to humidified air were less stressed than cells exposed to dry air by SEM. Actin cytoskeleton was found highly disorganized with dry air exposure. Humidified air maintained the normal actin cytoskeleton throughout the 20 minutes of fluid-air exchange. Paracellular carboxyfluorescein leakage was significantly higher in dry air insufflated eyes compared with that of the humidified air after 5, 10, and 20 minutes of fluid-air exchange (P =.002, P =.004, and P =.002, respectively). Dry air stress during fluid-air exchange causes significant immediate alterations in monolayer appearance, actin cytoskeleton, and barrier function of corneal endothelium in aphakic rabbit eyes. Use of humidified air largely prevents the alterations in monolayer appearance, actin cytoskeleton, and barrier function of corneal endothelial cells.

Lysosomal Trapping Is Present in Retinal Capillary Endothelial Cells: Insight into Its Influence on Cationic Drug Transport at the Inner Blood-Retinal Barrier.

PubMed

Kubo, Yoshiyuki; Seko, Narumi; Usui, Takuya; Akanuma, Shin-Ichi; Hosoya, Ken-Ichi

2016-01-01

Lysosomal trapping was investigated in the retinal capillary endothelial cells that are responsible for the inner blood-retinal barrier (BRB) using LysoTracker(®) Red (LTR). Using confocal microscopy on TR-iBRB2 cells, an in vitro model of the inner BRB, the presence of lysosomal trapping in retinal capillary endothelial cells was suggested since TR-iBRB2 cells exhibited punctuate intracellular localization of LTR that was attenuated by NH4Cl treatment. The study confirmed that LTR uptake by retinal capillary endothelial cells took place in a time- and temperature-dependent manner, and exhibited the 1.58-fold greater uptake at pH 8.4 than that at pH 7.4 while there was no change in uptake between pH 6.4 and pH 7.4, suggesting that passive diffusion is not enough to explain LTR uptake. The inhibition study showed the possible influence of lysosomal trapping on cationic drug transport by retinal capillary endothelial cells since LTR uptake was significantly inhibited by cationic amphiphilic drugs. Inhibition profiling and the estimation of IC50 suggested the influence of lysosomal trapping on propranolol and low-affinity pyrilamine transport while lysosomal trapping had only a partial effect on verapamil, clonidine, nicotine and high-affinity pyrilamine transport in retinal capillary endothelial cells.

Role of folic acid in nitric oxide bioavailability and vascular endothelial function

PubMed Central

Kenney, W. Larry

2017-01-01

Folic acid is a member of the B-vitamin family and is essential for amino acid metabolism. Adequate intake of folic acid is vital for metabolism, cellular homeostasis, and DNA synthesis. Since the initial discovery of folic acid in the 1940s, folate deficiency has been implicated in numerous disease states, primarily those associated with neural tube defects in utero and neurological degeneration later in life. However, in the past decade, epidemiological studies have identified an inverse relation between both folic acid intake and blood folate concentration and cardiovascular health. This association inspired a number of clinical studies that suggested that folic acid supplementation could reverse endothelial dysfunction in patients with cardiovascular disease (CVD). Recently, in vitro and in vivo studies have begun to elucidate the mechanism(s) through which folic acid improves vascular endothelial function. These studies, which are the focus of this review, suggest that folic acid and its active metabolite 5-methyl tetrahydrofolate improve nitric oxide (NO) bioavailability by increasing endothelial NO synthase coupling and NO production as well as by directly scavenging superoxide radicals. By improving NO bioavailability, folic acid may protect or improve endothelial function, thereby preventing or reversing the progression of CVD in those with overt disease or elevated CVD risk. PMID:27974600

Sulfoglucuronosyl paragloboside promotes endothelial cell apoptosis in inflammation: Elucidation of a novel glycosphingolipid-signaling pathway

PubMed Central

Dasgupta, Somsankar; Wang, Guanghu; Yu, Robert K.

2011-01-01

Sulfoglucuronosyl paragloboside (SGPG), a minor glycosphingolipid (GSL) of endothelial cells, is a ligand for L-selectin and has been implicated in neuro-inflammatory diseases, such as Guillian-Barré syndrome. Inflammatory cytokines, such as TNFα and IL-1β, up-regulate SGPG expression by stimulating gene expression for glucuronosyltransferases, both P and S forms (GlcATp and GlcATs), and the HNK-1 sulfotransferase (HNK-1 ST). Transfection of a human cerebromicrovascular endothelial cell (SV-HCEC) line with HNK-1 ST siRNA down-regulated SGPG expression, inhibited cytokine-stimulated T cell adhesion, and offered protection against apoptosis. However, the precise mechanisms of SGPG elevation in endothelial cell death (apoptosis) and the maintenance of blood-brain or blood-nerve barrier (BBB or BNB) integrity in inflammation have not been elucidated. Blocking SGPG expression inhibited cytokine-mediated stimulation of NF-κB activity but stimulated MAP kinase (ERK) activity. Furthermore, elevation of SGPG by over-expression of GlcATp and GlcATs triggered endothelial cell apoptosis, with GlcATs being more potent than GlcATp. While SGPG-mediated endothelial cell apoptosis was preceded by inhibiting the intracellular NF-κB activity, interfering with Akt and ERK activation and stimulating caspase 3 in SV-HCECs, HNK-1ST siRNA transfection also interfered with IKB phosphorylation but stimulated ERK activation. Our data indicate that SGPG is a critical regulatory molecule for maintaining endothelial cell survival and BBB/BNB barrier function. PMID:21916893

Role of lipid phosphate phosphatase 3 in human aortic endothelial cell function

PubMed Central

Touat-Hamici, Zahia; Weidmann, Henri; Blum, Yuna; Proust, Carole; Durand, Hervé; Iannacci, Francesca; Codoni, Veronica; Gaignard, Pauline; Thérond, Patrice; Civelek, Mete; Karabina, Sonia A.; Lusis, Aldons J.; Cambien, François; Ninio, Ewa

2016-01-01

Aims Lipid phosphate phosphatase 3; type 2 phosphatidic acid phosphatase β (LPP3; PPAP2B) is a transmembrane protein dephosphorylating and thereby terminating signalling of lipid substrates including lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P). Human LPP3 possesses a cell adhesion motif that allows interaction with integrins. A polymorphism (rs17114036) in PPAP2B is associated with coronary artery disease, which prompted us to investigate the possible role of LPP3 in human endothelial dysfunction, a condition promoting atherosclerosis. Methods and results To study the role of LPP3 in endothelial cells we used human primary aortic endothelial cells (HAECs) in which LPP3 was silenced or overexpressed using either wild type or mutated cDNA constructs. LPP3 silencing in HAECs enhanced secretion of inflammatory cytokines, leucocyte adhesion, cell survival, and migration and impaired angiogenesis, whereas wild-type LPP3 overexpression reversed these effects and induced apoptosis. We also demonstrated that LPP3 expression was negatively correlated with vascular endothelial growth factor expression. Mutations in either the catalytic or the arginine-glycine-aspartate (RGD) domains impaired endothelial cell function and pharmacological inhibition of S1P or LPA restored it. LPA was not secreted in HAECs under silencing or overexpressing LPP3. However, the intra- and extra-cellular levels of S1P tended to be correlated with LPP3 expression, indicating that S1P is probably degraded by LPP3. Conclusions We demonstrated that LPP3 is a negative regulator of inflammatory cytokines, leucocyte adhesion, cell survival, and migration in HAECs, suggesting a protective role of LPP3 against endothelial dysfunction in humans. Both the catalytic and the RGD functional domains were involved and S1P, but not LPA, might be the endogenous substrate of LPP3. PMID:27694435

Effect of amplitude and duration of impulsive pressure on endothelial permeability in in vitro fluid percussion trauma

PubMed Central

2014-01-01

Background Intracranial pressure changes during head impact cause brain injuries such as vasogenic edema and cerebral contusion. However, the influence of impulsive pressure on endothelial function has not yet been fully studied in vitro. In this study, we developed a pressure loading device that produced positive and negative pressures by modifying an in vitro fluid percussion model and examined the effects of the amplitude and duration of the pressures on endothelial permeability. Methods Human umbilical vein endothelial cells were subjected to three types of positive pressure (average amplitude/average duration of 352 kPa/23 ms, 73 kPa/27 ms, and 70 kPa/44 ms) and three types of negative pressure (−72 kPa/41 ms, −67 kPa/104 ms, and −91 kPa/108 ms), and the transendothelial electrical resistance (TEER) was measured between 15 min and 24 h after pressure loading for quantifying the formation of an integral monolayer of endothelial cells. After loading, vascular endothelial- (VE-) cadherin, an endothelium-specific cell-cell adhesion molecule involved in endothelial barrier function, was stained and observed using fluorescence microscopy. Results The pressure loading device could produce positive pressure pulses with amplitudes of 53–1348 kPa and durations of 9–29.1 ms and negative pressure pulses with amplitudes of −52–−93 kPa and durations of 42.9–179.5 ms. The impulsive pressure reduced the TEER associated with the change in VE-cadherin localization. Additionally, TEER decreased considerably at 15 min and 6 h post-loading, with these changes being significant in positive pressure with larger amplitude and shorter duration and in all types of negative pressures compared to pre-loading. Conclusions The changes in intracranial pressure during head impact impair endothelial barrier function by the disruption of the integrity of endothelial cell-cell junctions, and the degree of increase in endothelial permeability depends on the

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

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.

Helminths and intestinal barrier function

PubMed Central

McKay, Derek M.; Shute, Adam; Lopes, Fernando

2017-01-01

ABSTRACT Approximately one-sixth of the worlds' population is infected with helminths and this class of parasite takes a major toll on domestic livestock. The majority of species of parasitic helminth that infect mammals live in the gut (the only niche for tapeworms) where they contact the hosts' epithelial cells. Here, the helminth-intestinal epithelial interface is reviewed in terms of the impact on, and regulation of epithelial barrier function, both intrinsic (epithelial permeability) and extrinsic (mucin, bacterial peptides, commensal bacteria) elements of the barrier. The data available on direct effects of helminths on epithelial permeability are scant, fragmentary and pales in comparison with knowledge of mobilization of immune reactions and effector cells in response to helminth parasites and how these impact intestinal barrier function. The interaction of helminth-host and helminth-host-bacteria is an important determinant of gut form and function and precisely defining these interactions will radically alter our understanding of normal gut physiology and pathophysiological reactions, revealing new approaches to infection with parasitic helminths, bacterial pathogens and idiopathic auto-inflammatory disease. PMID:28452686

Vascular endothelial growth factor-D is a key molecule that enhances lymphatic metastasis of soft tissue sarcomas

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

Yanagawa, Takashi, E-mail: tyanagaw@med.gunma-u.ac.jp; Shinozaki, Tetsuya; Watanabe, Hideomi

2012-04-15

Studies on lymph node metastasis of soft tissue sarcomas are insufficient because of its rarity. In this study, we examined the expressions of vascular endothelial growth factor (VEGF)-C and VEGF-D in soft tissue sarcomas metastasized to lymph nodes. In addition, the effects of the two molecules on the barrier function of a lymphatic endothelial cell monolayer against sarcoma cells were analyzed. We examined 7 patients who had soft tissue sarcomas with lymph node metastases and who had undergone neither chemotherapy nor radiotherapy before lymphadenectomy. Immunohistochemistry revealed that 2 of 7 sarcomas that metastasized to lymph nodes expressed VEGF-C both inmore » primary and metastatic lesions. On the other hand, VEGF-D expression was detected in 4 of 7 primary and 7 of 7 metastatic lesions, respectively. Interestingly, 3 cases that showed no VEGF-D expression at primary sites expressed VEGF-D in metastatic lesions. Recombinant VEGF-C at 10{sup -8} and VEGF-D at 10{sup -7}and 10{sup -8} g/ml significantly increased the random motility of lymphatic endothelial cells compared with controls. VEGF-D significantly increased the migration of sarcoma cells through lymphatic endothelial monolayers. The fact that VEGF-D induced the migration of fibrosarcomas through the lymphatic endothelial monolayer is the probable reason for the strong relationship between VEGF-D expression and lymph node metastasis in soft tissue sarcomas. The important propensities of this molecule for the increase of lymph node metastases are not only lymphangiogenesis but also down-regulation of the barrier function of lymphatic endothelial monolayers, which facilitates sarcoma cells entering the lymphatic circulation.« less

Astrocytic TYMP and VEGFA drive blood–brain barrier opening in inflammatory central nervous system lesions

PubMed Central

Chapouly, Candice; Tadesse Argaw, Azeb; Horng, Sam; Castro, Kamilah; Zhang, Jingya; Asp, Linnea; Loo, Hannah; Laitman, Benjamin M.; Mariani, John N.; Straus Farber, Rebecca; Zaslavsky, Elena; Nudelman, German; Raine, Cedric S.

2015-01-01

In inflammatory central nervous system conditions such as multiple sclerosis, breakdown of the blood–brain barrier is a key event in lesion pathogenesis, predisposing to oedema, excitotoxicity, and ingress of plasma proteins and inflammatory cells. Recently, we showed that reactive astrocytes drive blood–brain barrier opening, via production of vascular endothelial growth factor A (VEGFA). Here, we now identify thymidine phosphorylase (TYMP; previously known as endothelial cell growth factor 1, ECGF1) as a second key astrocyte-derived permeability factor, which interacts with VEGFA to induce blood–brain barrier disruption. The two are co-induced NFκB1-dependently in human astrocytes by the cytokine interleukin 1 beta (IL1B), and inactivation of Vegfa in vivo potentiates TYMP induction. In human central nervous system microvascular endothelial cells, VEGFA and the TYMP product 2-deoxy-d-ribose cooperatively repress tight junction proteins, driving permeability. Notably, this response represents part of a wider pattern of endothelial plasticity: 2-deoxy-d-ribose and VEGFA produce transcriptional programs encompassing angiogenic and permeability genes, and together regulate a third unique cohort. Functionally, each promotes proliferation and viability, and they cooperatively drive motility and angiogenesis. Importantly, introduction of either into mouse cortex promotes blood–brain barrier breakdown, and together they induce severe barrier disruption. In the multiple sclerosis model experimental autoimmune encephalitis, TYMP and VEGFA co-localize to reactive astrocytes, and correlate with blood–brain barrier permeability. Critically, blockade of either reduces neurologic deficit, blood–brain barrier disruption and pathology, and inhibiting both in combination enhances tissue preservation. Suggesting importance in human disease, TYMP and VEGFA both localize to reactive astrocytes in multiple sclerosis lesion samples. Collectively, these data identify TYMP

Ethinyl estradiol-to-desogestrel ratio impacts endothelial function in young women✩

PubMed Central

Meendering, Jessica R.; Torgrimson, Britta N.; Miller, Nicole P.; Kaplan, Paul F.; Minson, Christopher T.

2010-01-01

Background Ethinyl estradiol (EE) and progestins have the ability to alter endothelial function. The type of progestin and the ratio of EE to progestin used in oral contraceptive pills (OCPs) may determine how they affect the arterial vasculature. Study Design In this study, we investigated endothelial function across a cycle in very low dose (VLD) and low dose (LD) combination EE and desogestrel (DSG) OCP users during two phases: active (VLD=20 mcg EE/150 mcg DSG; LD=30 mcg EE/150 mcg DSG) and pill-free. Endothelial function was also measured during an EE-only hormone phase (10 mcg EE) in group VLD. Results Endothelium-dependent vasodilation was greater during the active phase compared to the pill-free phase in group LD (9.02±0.72% vs. 7.33±0.84%; p=.029). This phase difference was not observed in group VLD (5.86±0.63% vs. 6.56±0.70%; p=.108). However, endothelium-dependent vasodilation was higher during the EE-only phase, compared to the active and pill-free phases (8.92±0.47% vs. 5.86±0.63%, and 6.56±0.70%; pb.001) in group VLD. Conclusions These data suggest DSG may antagonize the vasodilatory activity of EE and that this effect is further modulated by the EE-toDSG ratio. PMID:19041440

Effect of breaking up sedentary time with callisthenics on endothelial function.

PubMed

Carter, Sophie E; Gladwell, Valerie F

2017-08-01

Periods of prolonged sitting impairs endothelial function in lower limb conduit arteries, which is attenuated with physical activity breaks. The effect of activity breaks on upper limb arteries has not been examined. This study assessed changes in brachial artery endothelial function following either a prolonged sitting period or breaking up this sedentary time by performing sets of callisthenics exercises. Ten healthy participants (6 men) completed 2 conditions in a counterbalanced order: (a) 1-h 26-min sitting, or (b) breaking up this period every 20 min by performing a set of 5 callisthenics exercises. Brachial artery endothelial function was assessed via ultrasound using the flow-mediated dilation (FMD) technique prior to and following each condition, while brachial shear rate (SR) was acquired after each set of callisthenics. There was no significant change in FMD over time (P = 0.09) or between conditions (P = 0.12). Compared to sitting, brachial SR increased following each set of callisthenics, with a significant difference after the third break (Sit: 33.94 ± 12.79 s -1 ; Callisthenics: 57.16 ± 30.48 s -1 , P = 0.02). Alterations in SR in the upper limbs suggest callisthenics may be an effective intervention to break up sedentary time and attenuate the potentially deleterious effects of prolonged sitting on cardiovascular health.

Gestational diabetes, preeclampsia and cytokine release: similarities and differences in endothelial cell function.

PubMed

Rao, Rashmi; Sen, Suvajit; Han, Bing; Ramadoss, Sivakumar; Chaudhuri, Gautam

2014-01-01

Gestational diabetes, pre-eclampsia as well as intra-uterine infection during pregnancy affects the function of the endothelium both in the mother and the fetus leading to endothelial dysfunction. Gestational diabetes is also associated with an increased incidence of pre-eclampsia and it is likely that both the hyperglycemia as well as the release of cytokines especially TNFα during hyperglycemia may play an important role in the pathogenesis of endothelial dysfunction leading to preeclampsia. Similarly, some but not all studies have suggested that infection of the mother under certain circumstances can also lead to preeclampsia as women with either a bacterial or viral infection were at a higher risk of developing preeclampsia, compared to women without infection and infection also leads to a release in TNFα. Endothelial cells exposed to either high glucose or TNFα leads to an increase in the production of H2O2 and to a decrease in endothelial cell proliferation. The cellular and molecular mechanisms involved in this phenomenon are discussed.Gestational diabetes, pre-eclampsia as well as intra-uterine infection during pregnancy has profound effects on the fetus and long term effects on the neonate. All three conditions affect the function of the endothelium both in the mother and the fetus leading to endothelial dysfunction. Gestational diabetes is also associated with an increased incidence of pre-eclampsia and it is likely that both the hyperglycemia as well as the release of cytokines especially TNFα during hyperglycemia may play an important role in the pathogenesis of endothelial dysfunction leading to preeclampsia. It has also been suggested although not universally accepted that under certain circumstances maternal infection may also predispose to pre-eclampsia. Pre-eclampsia is also associated with the release of TNFα and endothelial dysfunction. However, the cellular and molecular mechanism(s) leading to the endothelial dysfunction by either

Non-invasive endothelial function assessment in patients with neurofibromatosis type 1: a cross-sectional study

PubMed Central

2013-01-01

Background Neurofibromatosis type 1 (NF1) is a multi-systemic disease caused by neurofibromin deficiency. The reduced life expectancy of patients with NF1 has been attributed to NF1-associated malignant neoplasms. However, an analysis of death certificates in the USA suggests that vascular disease could be an important cause of early death among these patients. Endothelial dysfunction (ED) is related to vasculopathy and is an early marker of subclinical atherosclerosis. Since neurofibromin has already been demonstrated to affect endothelial cell function, ED may be associated with NF1. The purpose of this study was to assess endothelial function in patients with NF1 using a non-invasive method. Methods NF1 patients and healthy control subjects, aged 18 to 35 years, were included. Subjects were excluded if they had any risk factor for vascular disease or any other condition known to affect endothelial function. Endothelial function was assessed using reactive hyperemia-peripheral arterial tone (RH-PAT) technology. ED was defined as a reactive hyperemia index (RHI) lower than 1.35. Results Four of the 29 (13.8%) NF1 patients and 1 of the 30 (3.3%) healthy volunteers had ED (p = 0.153). RHI medians and interquartile intervals were 1.8 (1.58-2.43) for the NF1 group and 2.02 (1.74 – 2.49) for the control group (p = 0.361). Conclusion The prevalence of ED was similar in NF1 patients and healthy controls. PMID:23497412

The effects of Ramadan fasting on endothelial function in patients with cardiovascular diseases.

PubMed

Yousefi, B; Faghfoori, Z; Samadi, N; Karami, H; Ahmadi, Y; Badalzadeh, R; Shafiei-Irannejad, V; Majidinia, M; Ghavimi, H; Jabbarpour, M

2014-07-01

Endothelial dysfunction, which can be manifested by loss of nitric oxide bioavailability, is an increasingly recognized cause of cardiovascular diseases. Previous studies showed that diets affect endothelial function and modify cardiovascular risks. This study aimed to assess the effects of Ramadan fasting, as a diet intervention, on endothelial function. The study population consisted of 21 male patients (mean age: 52±9 years) with cardiovascular risks (coronary artery disease, cerebrovascular or peripheral arterial diseases). The biochemical variables in serum of patients were measured 2 days before and after Ramadan fasting. The levels of asymmetric dimethylarginine (ADMA) and vascular endothelial growth factor (VEGF) were evaluated using the enzyme-linked immunosorbent assay. Nitric oxide (NO) and Malondialdehyde (MDA) levels were measured by the Griess and thiobarbituric acid reaction substances assay, respectively. NO levels in patients after Ramadan fasting were significantly higher compared with the baseline value (85.1±11.54 vs 75.8±10.7 μmol/l) (P<0.05). Post-Ramadan levels of ADMA decreased significantly in comparison with pre-Ramadan levels (802.6±60.9 vs 837.6±51.0 nmol/l) (P<0.05). In addition, the levels of VEGF and MDA changed during Ramadan fasting, but these changes were not statistically significant (228.1±27.1 vs 222.7±22.9 pg/ml and 3.2±0.7 vs 3.6±1.1 μmol/l, respectively). Ramadan fasting may have beneficial effects on endothelial function and can modulate cardiovascular risks. Further studies are needed to confirm the clinical significance of Ramadan fasting on cardiovascular health.

Enhanced Re-Endothelialization of Decellularized Rat Lungs

PubMed Central

Stabler, Collin T.; Caires, Luiz C.; Mondrinos, Mark J.; Marcinkiewicz, Cezary; Lazarovici, Philip; Wolfson, Marla R.

2016-01-01

Decellularized lung tissue has been recognized as a potential platform to engineer whole lung organs suitable for transplantation or for modeling a variety of lung diseases. However, many technical hurdles remain before this potential may be fully realized. Inability to efficiently re-endothelialize the pulmonary vasculature with a functional endothelium appears to be the primary cause of failure of recellularized lung scaffolds in early transplant studies. Here, we present an optimized approach for enhanced re-endothelialization of decellularized rodent lung scaffolds with rat lung microvascular endothelial cells (ECs). This was achieved by adjusting the posture of the lung to a supine position during cell seeding through the pulmonary artery. The supine position allowed for significantly more homogeneous seeding and better cell retention in the apex regions of all lobes than the traditional upright position, especially in the right upper and left lobes. Additionally, the supine position allowed for greater cell retention within large diameter vessels (proximal 100–5000 μm) than the upright position, with little to no difference in the small diameter distal vessels. EC adhesion in the proximal regions of the pulmonary vasculature in the decellularized lung was dependent on the binding of EC integrins, specifically α1β1, α2β1, and α5β1 integrins to, respectively, collagen type-I, type-IV, and fibronectin in the residual extracellular matrix. Following in vitro maturation of the seeded constructs under perfusion culture, the seeded ECs spread along the vascular wall, leading to a partial reestablishment of endothelial barrier function as inferred from a custom-designed leakage assay. Our results suggest that attention to cellular distribution within the whole organ is of paramount importance for restoring proper vascular function. PMID:26935764

Endothelial actions of atrial and B-type natriuretic peptides.

PubMed

Kuhn, Michaela

2012-05-01

The cardiac hormone atrial natriuretic peptide (ANP) is critically involved in the maintenance of arterial blood pressure and intravascular volume homeostasis. Its cGMP-producing GC-A receptor is densely expressed in the microvascular endothelium of the lung and systemic circulation, but the functional relevance is controversial. Some studies reported that ANP stimulates endothelial cell permeability, whereas others described that the peptide attenuates endothelial barrier dysfunction provoked by inflammatory agents such as thrombin or histamine. Many studies in vitro addressed the effects of ANP on endothelial proliferation and migration. Again, both pro- and anti-angiogenic properties were described. To unravel the role of the endothelial actions of ANP in vivo, we inactivated the murine GC-A gene selectively in endothelial cells by homologous loxP/Cre-mediated recombination. Our studies in these mice indicate that ANP, via endothelial GC-A, increases endothelial albumin permeability in the microcirculation of the skin and skeletal muscle. This effect is critically involved in the endocrine hypovolaemic, hypotensive actions of the cardiac hormone. On the other hand the homologous GC-A-activating B-type NP (BNP), which is produced by cardiac myocytes and many other cell types in response to stressors such as hypoxia, possibly exerts more paracrine than endocrine actions. For instance, within the ischaemic skeletal muscle BNP released from activated satellite cells can improve the regeneration of neighbouring endothelia. This review will focus on recent advancements in our understanding of endothelial NP/GC-A signalling in the pulmonary versus systemic circulation. It will discuss possible mechanisms accounting for the discrepant observations made for the endothelial actions of this hormone-receptor system and distinguish between (patho)physiological and pharmacological actions. Lastly it will emphasize the potential therapeutical implications derived from the

Diesel Exhaust Particle Exposure Causes Redistribution of Endothelial Tube VE-Cadherin

PubMed Central

Chao, Ming-Wei; Kozlosky, John; Po, Iris P.; Strickland, Pamela Ohman; Svoboda, Kathy K. H.; Cooper, Keith; Laumbach, Robert; Gordon, Marion K.

2010-01-01

Whether diesel exhaust particles (DEPs) potentially have a direct effect on capillary endothelia was examined by following the adherens junction component, vascular endothelial cell cadherin (VE-cadherin). This molecule is incorporated into endothelial adherens junctions at the cell surface, where it forms homodimeric associations with adjacent cells and contributes to the barrier function of the vasculature (Dejana et al., 2008; Venkiteswaran et al., 2002; Villasante et al., 2007). Human umbilical vein endothelial cells (HUVECs) that were pre-formed into capillary-like tube networks in vitro were exposed to DEPs for 24 hr. After exposure, the integrity of VE-cadherin in adherens junctions was assessed by immunofluorescence analysis, and demonstrated that increasing concentrations of DEPs caused increasing redistribution of VE-cadherin away from the cell-cell junctions toward intracellular locations. Since HUVEC tube networks are three-dimensional structures, whether particles entered the endothelial cells or tubular lumens was also examined. The data indicate that translocation of the particles does occur. The results, obtained in a setting that removes the confounding effects of inflammatory cells or blood components, suggest that if DEPs encounter alveolar capillaries in vivo, they may be able to directly affect the endothelial cell-cell junctions. PMID:20887764

Growth factor-induced morphological, physiological and molecular characteristics in cerebral endothelial cells.

PubMed

Krizbai, I A; Bauer, H; Amberger, A; Hennig, B; Szabó, H; Fuchs, R; Bauer, H C

2000-09-01

The capacity of vascular endothelial cells to modulate their phenotype in response to changes in environmental conditions is one of the most important characteristics of this cell type. Since different growth factors may play an important signalling role in this adaptive process we have investigated the effect of endothelial cell growth factor (ECGF) on morphological, physiological and molecular characteristics of cerebral endothelial cells (CECs). CECs grown in the presence of ECGF and its cofactor heparin exhibit an epithelial-like morphology (type I CECs). Upon removal of growth factors, CECs develop an elongated spindle-like shape (type II CECs) which is accompanied by the reorganization of actin filaments and the induction of alpha-actin expression. Since one of the most important functions of CECs is the creation of a selective diffusion barrier between the blood and the central nervous system (CNS), we have studied the expression of junction-related proteins in both cell types. We have found that removal of growth factors from endothelial cultures leads to the downregulation of cadherin and occludin protein levels. The loss of junctional proteins was accompanied by a significant increase in the migratory activity and an altered protease activity profile of the cells. TGF-beta1 suppressed endothelial migration in all experiments. Our data provide evidence to suggest that particular endothelial functions are largely controlled by the presence of growth factors. The differences in adhesiveness and migration may play a role in important physiological and pathological processes of endothelial cells such as vasculogenesis or tumor progression.

The effects of vitamin E and omega-3 PUFAs on endothelial function among adolescents with metabolic syndrome.

PubMed

Ahmadi, Alireza; Gharipour, Mojgan; Arabzadeh, Gholamreza; Moin, Payam; Hashemipour, Mahin; Kelishadi, Roya

2014-01-01

The present study aims to explore the effects of vitamin E and omega-3 on endothelial function indicators among adolescents with metabolic syndrome. In a randomized, double blind, and placebo-controlled trial, 90 young individuals, aged 10 to 18 years, with metabolic syndrome were randomly assigned to receive either vitamin E tablets (400 IU/day) or omega-3 tablets (2.4 gr/day) or placebo. For assessing endothelial functional state, the serum level of vascular endothelial growth factor (VEGF) was measured by ELISA test. The use of omega-3 supplementation for eight weeks led to significant increase in serum HDL level compared with the group treated with vitamin E or placebo group. In this regard, no significant correlations were found between the change in VEGF and baseline levels of other markers including anthropometric indices and serum lipids. Omega-3 could significantly reduce VEGF with the presence of other baseline variables (Beta = -12.55; P = 0.012). The administration of omega-3 can effectively improve endothelial function in adolescents with metabolic syndrome by reducing the level of serum VEGF, as a major index for atherosclerosis progression and endothelial destabilization. Omega-3 can be proposed as a VEGF antagonist for improving endothelial function in metabolic syndrome. The clinical implications of our findings should be assessed in future studies.

Phospholipase C-ε signaling mediates endothelial cell inflammation and barrier disruption in acute lung injury

PubMed Central

Bijli, Kaiser M.; Fazal, Fabeha; Slavin, Spencer A.; Leonard, Antony; Grose, Valerie; Alexander, William B.; Smrcka, Alan V.

2016-01-01

Phospholipase C-ε (PLC-ε) is a unique PLC isoform that can be regulated by multiple signaling inputs from both Ras family GTPases and heterotrimeric G proteins and has primary sites of expression in the heart and lung. Whereas the role of PLC-ε in cardiac function and pathology has been documented, its relevance in acute lung injury (ALI) is unclear. We used PLC-ε−/− mice to address the role of PLC-ε in regulating lung vascular inflammation and injury in an aerosolized bacterial LPS inhalation mouse model of ALI. PLC-ε−/− mice showed a marked decrease in LPS-induced proinflammatory mediators (ICAM-1, VCAM-1, TNF-α, IL-1β, IL-6, macrophage inflammatory protein 2, keratinocyte-derived cytokine, monocyte chemoattractant protein 1, and granulocyte-macrophage colony-stimulating factor), lung neutrophil infiltration and microvascular leakage, and loss of VE-cadherin compared with PLC-ε+/+ mice. These data identify PLC-ε as a critical determinant of proinflammatory and leaky phenotype of the lung. To test the possibility that PLC-ε activity in endothelial cells (EC) could contribute to ALI, we determined its role in EC inflammation and barrier disruption. RNAi knockdown of PLC-ε inhibited NF-κB activity in response to diverse proinflammatory stimuli, thrombin, LPS, TNF-α, and the nonreceptor agonist phorbol 13-myristate 12-acetate (phorbol esters) in EC. Depletion of PLC-ε also inhibited thrombin-induced expression of NF-κB target gene, VCAM-1. Importantly, PLC-ε knockdown also protected against thrombin-induced EC barrier disruption by inhibiting the loss of VE-cadherin at adherens junctions and formation of actin stress fibers. These data identify PLC-ε as a novel regulator of EC inflammation and permeability and show a hitherto unknown role of PLC-ε in the pathogenesis of ALI. PMID:27371732

Phospholipase C-ε signaling mediates endothelial cell inflammation and barrier disruption in acute lung injury.

PubMed

Bijli, Kaiser M; Fazal, Fabeha; Slavin, Spencer A; Leonard, Antony; Grose, Valerie; Alexander, William B; Smrcka, Alan V; Rahman, Arshad

2016-08-01

Phospholipase C-ε (PLC-ε) is a unique PLC isoform that can be regulated by multiple signaling inputs from both Ras family GTPases and heterotrimeric G proteins and has primary sites of expression in the heart and lung. Whereas the role of PLC-ε in cardiac function and pathology has been documented, its relevance in acute lung injury (ALI) is unclear. We used PLC-ε(-/-) mice to address the role of PLC-ε in regulating lung vascular inflammation and injury in an aerosolized bacterial LPS inhalation mouse model of ALI. PLC-ε(-/-) mice showed a marked decrease in LPS-induced proinflammatory mediators (ICAM-1, VCAM-1, TNF-α, IL-1β, IL-6, macrophage inflammatory protein 2, keratinocyte-derived cytokine, monocyte chemoattractant protein 1, and granulocyte-macrophage colony-stimulating factor), lung neutrophil infiltration and microvascular leakage, and loss of VE-cadherin compared with PLC-ε(+/+) mice. These data identify PLC-ε as a critical determinant of proinflammatory and leaky phenotype of the lung. To test the possibility that PLC-ε activity in endothelial cells (EC) could contribute to ALI, we determined its role in EC inflammation and barrier disruption. RNAi knockdown of PLC-ε inhibited NF-κB activity in response to diverse proinflammatory stimuli, thrombin, LPS, TNF-α, and the nonreceptor agonist phorbol 13-myristate 12-acetate (phorbol esters) in EC. Depletion of PLC-ε also inhibited thrombin-induced expression of NF-κB target gene, VCAM-1. Importantly, PLC-ε knockdown also protected against thrombin-induced EC barrier disruption by inhibiting the loss of VE-cadherin at adherens junctions and formation of actin stress fibers. These data identify PLC-ε as a novel regulator of EC inflammation and permeability and show a hitherto unknown role of PLC-ε in the pathogenesis of ALI. Copyright © 2016 the American Physiological Society.

Effects of dark chocolate on endothelial function in patients with non-alcoholic steatohepatitis.

PubMed

Loffredo, L; Baratta, F; Ludovica, P; Battaglia, S; Carnevale, R; Nocella, C; Novo, M; Pannitteri, G; Ceci, F; Angelico, F; Violi, F; Del Ben, M

2018-02-01

Oxidative stress plays a pivotal role in inducing endothelial dysfunction and progression from simple fatty liver steatosis (FLD) to non-alcoholic steatohepatitis (NASH). Polyphenols could reduce oxidative stress and restore endothelial function by inhibiting the nicotinamide-adenine-dinucleotide-phosphate (NADPH) oxidase isoform Nox2. The aim of this study was to assess endothelial function and oxidative stress in a population affected by simple FLD and NASH. Furthermore, we analysed the effect of high vs low content of cocoa polyphenols on endothelial function and oxidative stress in patients with NASH. In a cross-sectional study we analysed endothelial function, as assessed by flow-mediated dilation (FMD), and oxidative stress, as assessed by Nox2 activation, serum isoprostanes and nitric oxide bioavailability (NOx), in patients with NASH (n = 19), FLD (n = 19) and controls (n = 19). Then, we performed a randomized, cross-over study in 19 subjects with NASH comparing the effect of 14-days administration of 40 g of chocolate at high (dark chocolate, cocoa >85%) versus low content (milk chocolate, cocoa <35%) of polyphenols on FMD and oxidative stress. Compared to controls, NASH and FLD patients had higher Nox2 activity and isoprostanes levels and lower FMD and NOx, with a significant gradient between FLD and NASH. The interventional study showed that, compared to baseline, FMD and NOx increased (from 2.9 ± 2.4 to 7.2 ± 3.0% p < 0.001 and from 15.9 ± 3.6 to 20.6 ± 4.9 μM, p < 0.001, respectively) in subjects given dark but not in those given milk chocolate. A simple linear regression analysis showed that Δ (expressed by difference of values between before and after 14 days of chocolate assumption) of FMD was associated with Δ of Nox2 activity (Rs = -0.323; p = 0.04), serum isoprostanes (Rs: -0.553; p < 0.001) and NOx (Rs: 0.557; p < 0.001). Cocoa polyphenols improve endothelial function via Nox2 down-regulation in NASH patients

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

Sex Differences Influencing Micro- and Macrovascular Endothelial Phenotype In Vitro.

PubMed

Huxley, Virginia H; Kemp, Scott S; Schramm, Christine; Sieveking, Steve; Bingaman, Susan; Yu, Yang; Zaniletti, Isabella; Stockard, Kevin; Wang, Jianjie

2018-06-09

Endothelial dysfunction is an early hallmark of multiple disease states that also display sex differences with respect to age of onset, frequency, and severity. Results of in vivo studies of basal and stimulated microvascular barrier function revealed sex differences difficult to ascribe to specific cells or environmental factors. The present study evaluated endothelial cells (EC) isolated from macro- and/or microvessels of reproductively mature rats under the controlled conditions of low-passage culture to test the assumption that EC phenotype would be sex-independent. The primary finding was that EC, regardless of where they are derived, retain a sex-bias in low-passage culture, independent of varying levels of reproductive hormones. Implications of the work include the fallacy of expecting a universal set of mechanisms derived from study of EC from one sex and/or one vascular origin to apply uniformly to all EC under unstimulated conditions no less in the disease state. Vascular endothelial cells (EC) are heterogeneous with respect to phenotype reflecting at least organ of origin, location within the vascular network, and physical forces. Sex, as an independent influence on EC functions in health or etiology, susceptibility, and progression of dysfunction in numerous disease states, has been largely ignored. The current study focussed on EC isolated from aorta (macrovascular) and skeletal muscle vessels (microvascular) of age-matched male and female rats under identical conditions of short term (passage 4) culture. We tested the hypothesis that genomic sex would not influence endothelial growth, wound healing, morphology, lactate production, or messenger RNA and protein expression of key proteins (sex hormone receptors for androgen (AR) and oestrogen (ERα and ERβ); PECAM-1 and VE-CAD mediating barrier function; α v β 3 and N-Cadherin influencing matrix interactions; ICAM-1 and VCAM-1 mediating EC/white cell adhesion). The hypothesis was rejected as EC origin

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.

Alterations in blood-brain barrier function following acute hypertension: comparison of the blood-to-brain transfer of horseradish peroxidase with that of alpha-aminisobutyric acid

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

Ellison, M.D.B.

The blood-brain barrier (BBB) selectively restricts the blood-to-brain passage of many solutes owing to unique properties of cerebrovascular endothelial cell membranes. To date, experimental study of the BBB has been accomplished primarily through the use of two different methodological approaches. Morphological studies have mostly employed large molecular weight (MW) tracers to detect morphological alterations underlying increased permeability. Physiological studies, employing smaller, more physiologic tracers have successfully described, quantitatively, certain functional aspects of blood-to-brain transfer. The current work attempts to merge these two approaches and to consider barrier function/dysfunction from both a morphological and a functional perspective. Specifically, the study comparesmore » in rats, following acute hypertension, the cerebrovascular passage of /sup 14/C-alpha-aminoisobutyric acid (AIB) and that of horseradish peroxidase (HRP). The blood-to-brain passage of AIB and HRP were compared following acute hypertension, with regard to both the distributions of the tracer extravasation patterns and the magnitude of tracer extravasation. The results of this study suggest that traditional morphological barrier studies alone do not reveal all aspects of altered barrier status and that multiple mechanisms underlying increased BBB permeability may operate simultaneously during BBB dysfunction.« less

Hydroquinone stimulates inflammatory functions in microvascular endothelial cells via NF-κB nuclear activation.

PubMed

Hebeda, Cristina Bichels; Pinedo, Fernanda Júdice; Vinolo, Marco Aurélio Ramirez; Curi, Rui; Farsky, Sandra Helena Poliselli

2011-11-01

Hydroquinone impairs several leucocyte cell functions, which alter the immune response. Although endothelial cell functions are important for the development of immune responses, hydroquinone actions on endothelial cell have not been shown. Therefore, the effect of hydroquinone exposure (10 or 100 μM for 2 hr) on primary culture of microvascular endothelial cells (PMECs) obtained from the cremaster muscle of Wistar rats incubated in the presence or absence of lipopolysaccharide (LPS, 2 μg/mL) was investigated. Hydroquinone treatment induced the membrane expression of cell adhesion molecules (CAMs) from the immunoglobulin superfamilies ICAM-1 (intercellular), VCAM-1(vascular) and PECAM-1 (platelet endothelial) and induced the secretion of cytokines interleukin-1β (IL-1β) and tumour necrosis factor-α (TNF-α). The effects were dependent on transcriptional modifications because enhanced CAM mRNA expression as well as both cytokines and nuclear factor κB (NF-κB) nuclear activation was found. These effects may be due to the direct action of hydroquinone rather than its quinone metabolites, because endothelial cells do not present myeloperoxidase enzyme and hydroquinone incubation did not induce the expression of cytochrome P450 2E1 (CYP2E1) or prostaglandin H synthase 1. In addition, the incubation of endothelial cells with benzoquinone (10 μM, 2 hr) impaired PECAM-1 expression and did not modify NF-κB nuclear activation. Taken together, the data herein presented reveal that hydroquinone evokes pro-inflammatory properties in endothelial cells that are triggered by the enhancement of NF-κB nuclear translocation-dependent gene transcription. © 2011 The Authors. Basic & Clinical Pharmacology & Toxicology © 2011 Nordic Pharmacological Society.

Standards for the Protection of Skin Barrier Function.

PubMed

Giménez-Arnau, Ana

2016-01-01

The skin is a vital organ, and through our skin we are in close contact with the entire environment. If we lose our skin we lose our life. The barrier function of the skin is mainly driven by the sophisticated epidermis in close relationship with the dermis. The epidermal epithelium is a mechanically, chemically, biologically and immunologically active barrier submitted to continuous turnover. The barrier function of the skin needs to be protected and restored. Its own physiology allows its recovery, but many times this is not sufficient. This chapter is focused on the standards to restore, treat and prevent barrier function disruption. These standards were developed from a scientific, academic and clinical point of view. There is a lack of standardized administrative recommendations. Still, there is a walk to do that will help to reduce the social and economic burden of diseases characterized by an abnormal skin barrier function. © 2016 S. Karger AG, Basel.

Platelet Activating Factor-Induced Ceramide Micro-Domains Drive Endothelial NOS Activation and Contribute to Barrier Dysfunction

PubMed Central

Predescu, Sanda; Knezevic, Ivana; Bardita, Cristina; Neamu, Radu Florin; Brovcovych, Viktor; Predescu, Dan

2013-01-01

The spatial and functional relationship between platelet activating factor-receptor (PAF-R) and nitric oxide synthase (eNOS) in the lateral plane of the endothelial plasma membrane is poorly characterized. In this study, we used intact mouse pulmonary endothelial cells (ECs) as well as endothelial plasma membrane patches and subcellular fractions to define a new microdomain of plasmalemma proper where the two proteins colocalize and to demonstrate how PAF-mediated nitric oxide (NO) production fine-tunes ECs function as gatekeepers of vascular permeability. Using fluorescence microscopy and immunogold labeling electron microscopy (EM) on membrane patches we demonstrate that PAF-R is organized as clusters and colocalizes with a subcellular pool of eNOS, outside recognizable vesicular profiles. Moreover, PAF-induced acid sphingomyelinase activation generates a ceramide-based microdomain on the external leaflet of plasma membrane, inside of which a signalosome containing eNOS shapes PAF-stimulated NO production. Real-time measurements of NO after PAF-R ligation indicated a rapid (5 to 15 min) increase in NO production followed by a > 45 min period of reduction to basal levels. Moreover, at the level of this new microdomain, PAF induces a dynamic phosphorylation/dephosphorylation of Ser, Thr and Tyr residues of eNOS that correlates with NO production. Altogether, our findings establish the existence of a functional partnership PAF-R/eNOS on EC plasma membrane, at the level of PAF-induced ceramide plasma membrane microdomains, outside recognized vesicular profiles. PMID:24086643

Human Herpesvirus-8-Transformed Endothelial Cells Have Functionally Activated Vascular Endothelial Growth Factor/Vascular Endothelial Growth Factor Receptor

PubMed Central

Masood, Rizwan; Cesarman, Ethel; Smith, D. Lynne; Gill, Parkash S.; Flore, Ornella

2002-01-01

Kaposi’s sarcoma is a vascular tumor commonly associated with human immunodeficiency virus (HIV)-1 and human herpesvirus (HHV-8) also known as Kaposi’s sarcoma-associated herpesvirus. The principal features of this tumor are abnormal proliferation of vascular structures lined with spindle-shaped endothelial cells. HHV-8 may transform a subpopulation of endothelial cells in vitro via viral and cellular gene expression. We hypothesized that among the cellular genes, vascular endothelial growth factors (VEGFs) and their cognate receptors may be involved in viral-mediated transformation. We have shown that HHV-8-transformed endothelial cells (EC-HHV-8) express higher levels of VEGF, VEGF-C, VEGF-D, and PlGF in addition to VEGF receptors-1, -2, and -3. Furthermore, antibodies to VEGF receptor-2 inhibited cell proliferation and viability. Similarly, inhibition of VEGF gene expression with antisense oligonucleotides inhibited EC-HHV-8 cell proliferation/viability. The growth and viability of primary endothelial cells and a fibroblast cell line however were unaffected by either the VEGF receptor-2 antibody or the VEGF antisense oligodeoxynucleotides. VEGF and VEGF receptors are thus induced in EC-HHV-8 and participate in the transformation. Inhibitors of VEGF may thus modulate the disease process during development and progression. PMID:11786394

Sulfoglucuronosyl paragloboside promotes endothelial cell apoptosis in inflammation: elucidation of a novel glycosphingolipid-signaling pathway.

PubMed

Dasgupta, Somsankar; Wang, Guanghu; Yu, Robert K

2011-11-01

Sulfoglucuronosyl paragloboside (SGPG), a minor glycosphingolipid of endothelial cells, is a ligand for L-selectin and has been implicated in neuro-inflammatory diseases, such as Guillian-Barré syndrome. Inflammatory cytokines, such as TNFα and IL-1β, up-regulate SGPG expression by stimulating gene expression for glucuronosyltransferases, both P and S forms (GlcATp and GlcATs), and the human natural killer antigen (HNK-1) sulfotransferase (HNK-1 ST). Transfection of a human cerebromicrovascular endothelial cell (SV-HCEC) line with HNK-1 ST siRNA down-regulated SGPG expression, inhibited cytokine-stimulated T-cell adhesion, and offered protection against apoptosis. However, the precise mechanisms of SGPG elevation in endothelial cell apoptosis and the maintenance of blood-brain or blood-nerve barrier integrity in inflammation have not been elucidated. Blocking SGPG expression inhibited cytokine-mediated stimulation of NF-κB activity but stimulated MAP kinase activity. Furthermore, elevation of SGPG by over-expression of GlcATp and GlcATs triggered endothelial cell apoptosis, with GlcATs being more potent than GlcATp. Although SGPG-mediated endothelial cell apoptosis was preceded by inhibiting the intracellular NF-κB activity, interfering with Akt and ERK activation and stimulating caspase 3 in SV-HCECs, HNK-1ST siRNA transfection also interfered with IκB phosphorylation but stimulated ERK activation. Our data indicate that SGPG is a critical regulatory molecule for maintaining endothelial cell survival and blood-brain or blood-nerve barrier function. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.

Endothelial Response to Glucocorticoids in Inflammatory Diseases

PubMed Central

Zielińska, Karolina A.; Van Moortel, Laura; Opdenakker, Ghislain; De Bosscher, Karolien; Van den Steen, Philippe E.

2016-01-01

The endothelium plays a crucial role in inflammation. A balanced control of inflammation requires the action of glucocorticoids (GCs), steroidal hormones with potent cell-specific anti-inflammatory properties. Besides the classic anti-inflammatory effects of GCs on leukocytes, recent studies confirm that endothelial cells also represent an important target for GCs. GCs regulate different aspects of endothelial physiology including expression of adhesion molecules, production of pro-inflammatory cytokines and chemokines, and maintenance of endothelial barrier integrity. However, the regulation of endothelial GC sensitivity remains incompletely understood. In this review, we specifically examine the endothelial response to GCs in various inflammatory diseases ranging from multiple sclerosis, stroke, sepsis, and vasculitis to atherosclerosis. Shedding more light on the cross talk between GCs and endothelium will help to improve existing therapeutic strategies and develop new therapies better tailored to the needs of patients. PMID:28018358

You're Only as Old as Your Arteries: Translational Strategies for Preserving Vascular Endothelial Function with Aging

PubMed Central

Kaplon, Rachelle E.; Gioscia-Ryan, Rachel A.; LaRocca, Thomas J.

2014-01-01

Endothelial dysfunction develops with age and increases the risk of age-associated vascular disorders. Nitric oxide insufficiency, oxidative stress, and chronic low-grade inflammation, induced by upregulation of adverse cellular signaling processes and imbalances in stress resistance pathways, mediate endothelial dysfunction with aging. Healthy lifestyle behaviors preserve endothelial function with aging by inhibiting these mechanisms, and novel nutraceutical compounds that favorably modulate these pathways hold promise as a complementary approach for preserving endothelial health. PMID:24985329

SHBG and endothelial function in older subjects.

PubMed

Maggio, Marcello; Cattabiani, Chiara; Lauretani, Fulvio; Mantovani, Marco; Buttò, Valeria; De Vita, Francesca; Volpi, Riccardo; Artoni, Andrea; Giallauria, Francesco; Zuliani, Giovanni; Aloe, Rosalia; Lippi, Giuseppe; Ceresini, Graziano; Cederholm, Tommy; Ceda, Gian Paolo; Lind, Lars

2013-10-03

Endothelial dysfunction is predictor of cardiovascular diseases that have different prevalence in men and women before menopause. Sex hormones and sex hormone binding globulin (SHBG), novel risk factors for diabetes and cardiovascular diseases even in older individuals, might explain this difference. However, the relationship between these hormones and endothelial function has never been addressed in the elderly. 430 men and,424 women 70 years older of Prospective Study of the Vasculature in Uppsala Seniors study, with complete data on SHBG, testosterone(T), estradiol(E2), endothelium-independent vasodilation (EIDV), endothelium-dependent vasodilation(EDV), flow-mediated vasodilation (FMD) and the pulse wave analysis (reflection index, RI) were evaluated. Multivariate regression analysis adjusted for confounders was used to assess the relationship between T, E2, SHBG and endothelial function. In men we found a positive relationship between SHBG and EDV (β ± SE 3.60 ± 0.83, p<0.0001), EIDV (2.42 ± 0.58, p<0.0001) but not with FMD. The relationship between SHBG and EDV and EIDV was maintained after adjustment for sex (1.64 ± 0.47, p<0.001 and 1.79 ± 0.35, p<0.0006, respectively). After adjustment for confounders, the relationship between SHBG and EDV and EIDV was still statistically significant (2.63 ± 0.90 and 1.86 ± 0.63, p = 0.004 for both). In women SHBG and EIDV were positively associated (1.58 ± 0.46; p = 0.0007), and this relationship was independent of sex (1.79 ± 0.35; p<0.001). No significant interaction SHBG * SEX was found for EIDV (p = 0.72). In a combined analysis in two sexes, SHBG and EIDV were positively associated (1.13 ± 0.45; p = 0.01). SHBG was not associated with EDV, FMD and RI. No significant relationship was found between T or E2 and EDV, EIDV, FMD or RI in both sexes. In older men SHBG, but not T and E2, is positively and independently associated with EDV in resistance arteries. In both sexes, SHBG was positively and independently

Eicosapentaenoic acid improves endothelial function and nitric oxide bioavailability in a manner that is enhanced in combination with a statin.

PubMed

Mason, R Preston; Dawoud, Hazem; Jacob, Robert F; Sherratt, Samuel C R; Malinski, Tadeusz

2018-07-01

The endothelium exerts many vasoprotective effects that are largely mediated by release of nitric oxide (NO). Endothelial dysfunction represents an early but reversible step in atherosclerosis and is characterized by a reduction in the bioavailability of NO. Previous studies have shown that eicosapentaenoic acid (EPA), an omega-3 fatty acid (O3FA), and statins individually improve endothelial cell function, but their effects in combination have not been tested. Through a series of in vitro experiments, this study evaluated the effects of a combined treatment of EPA and the active metabolite of atorvastatin (ATM) on endothelial cell function under conditions of oxidative stress. Specifically, the comparative and time-dependent effects of these agents on endothelial dysfunction were examined by measuring the levels of NO and peroxynitrite (ONOO - ) released from human umbilical vein endothelial cells (HUVECs). The data suggest that combined treatment with EPA and ATM is beneficial to endothelial function and was unique to EPA and ATM since similar improvements could not be recapitulated by substituting another O3FA docosahexaenoic acid (DHA) or other TG-lowering agents such as fenofibrate, niacin, or gemfibrozil. Comparable beneficial effects were observed when HUVECs were pretreated with EPA and ATM before exposure to oxidative stress. Interestingly, the kinetics of EPA-based protection of endothelial function in response to oxidation were found to be significantly different than those of DHA. Lastly, the beneficial effects on endothelial function generated by combined treatment of EPA and ATM were reproduced when this study was expanded to an ex vivo model utilizing rat glomerular endothelial cells. Taken together, these findings suggest that a combined treatment of EPA and ATM can inhibit endothelial dysfunction that occurs in response to conditions such as hyperglycemia, oxidative stress, and dyslipidemia. Copyright © 2018 The Authors. Published by Elsevier

Improvement of endothelial function by pitavastatin: a meta-analysis.

PubMed

Katsiki, Niki; Reiner, Željko; Tedeschi Reiner, Eugenia; Al-Rasadi, Khalid; Pirro, Matteo; Mikhailidis, Dimitri P; Sahebkar, Amirhossein

2018-02-01

Dyslipidemia is commonly associated with endothelial dysfunction and increased cardiovascular risk. Pitavastatin has been shown to reduce total and low-density lipoprotein cholesterol, to increase high-density lipoprotein (HDL)-cholesterol and improve HDL function. Furthermore, several trials explored its effects on flow-mediated dilation (FMD), as an index of endothelial function. The authors evaluated the effect of pitavastatin therapy on FMD. The authors performed a systematic review and meta-analysis of all clinical trials exploring the impact of pitavastatin on FMD. The search included PubMed-Medline, Scopus, ISI Web of Knowledge and Google Scholar databases. Quantitative data synthesis was performed using a random-effects model, with weighted mean difference (WMD) and 95% confidence interval (CI) as summary statistics. Six eligible studies comprising 7 treatment arms were selected for this meta-analysis. Overall, WMD was significant for the effect of pitavastatin on FMD (2.45%, 95% CI: 1.31, 3.60, p < 0.001) and the effect size was robust in the leave-one-out sensitivity analysis. This meta-analysis of all available clinical trials revealed a significant increase of FMD induced by pitavastatin.

Targeting vascular (endothelial) dysfunction

PubMed Central

Steven, Sebastian; Weber, Alina; Shuvaev, Vladimir V.; Muzykantov, Vladimir R.; Laher, Ismail; Li, Huige; Lamas, Santiago

2016-01-01

Abstract Cardiovascular diseases are major contributors to global deaths and disability‐adjusted life years, with hypertension a significant risk factor for all causes of death. The endothelium that lines the inner wall of the vasculature regulates essential haemostatic functions, such as vascular tone, circulation of blood cells, inflammation and platelet activity. Endothelial dysfunction is an early predictor of atherosclerosis and future cardiovascular events. We review the prognostic value of obtaining measurements of endothelial function, the clinical techniques for its determination, the mechanisms leading to endothelial dysfunction and the therapeutic treatment of endothelial dysfunction. Since vascular oxidative stress and inflammation are major determinants of endothelial function, we have also addressed current antioxidant and anti‐inflammatory therapies. In the light of recent data that dispute the prognostic value of endothelial function in healthy human cohorts, we also discuss alternative diagnostic parameters such as vascular stiffness index and intima/media thickness ratio. We also suggest that assessing vascular function, including that of smooth muscle and even perivascular adipose tissue, may be an appropriate parameter for clinical investigations. Linked Articles This article is part of a themed section on Redox Biology and Oxidative Stress in Health and Disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.12/issuetoc PMID:27187006

Role of folic acid in nitric oxide bioavailability and vascular endothelial function.

PubMed

Stanhewicz, Anna E; Kenney, W Larry

2017-01-01

Folic acid is a member of the B-vitamin family and is essential for amino acid metabolism. Adequate intake of folic acid is vital for metabolism, cellular homeostasis, and DNA synthesis. Since the initial discovery of folic acid in the 1940s, folate deficiency has been implicated in numerous disease states, primarily those associated with neural tube defects in utero and neurological degeneration later in life. However, in the past decade, epidemiological studies have identified an inverse relation between both folic acid intake and blood folate concentration and cardiovascular health. This association inspired a number of clinical studies that suggested that folic acid supplementation could reverse endothelial dysfunction in patients with cardiovascular disease (CVD). Recently, in vitro and in vivo studies have begun to elucidate the mechanism(s) through which folic acid improves vascular endothelial function. These studies, which are the focus of this review, suggest that folic acid and its active metabolite 5-methyl tetrahydrofolate improve nitric oxide (NO) bioavailability by increasing endothelial NO synthase coupling and NO production as well as by directly scavenging superoxide radicals. By improving NO bioavailability, folic acid may protect or improve endothelial function, thereby preventing or reversing the progression of CVD in those with overt disease or elevated CVD risk. © The Author(s) 2016. Published by Oxford University Press on behalf of the International Life Sciences Institute. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Diverse Functional Outcomes of Plasmodium falciparum Ligation of EPCR: Potential Implications for Malarial Pathogenesis

PubMed Central

Gillrie, Mark R.; Avril, Marion; Brazier, Andrew J.; Davis, Shevaun P.; Stins, Monique F.; Smith, Joseph D.; Ho, May

2015-01-01

Summary P. falciparum-infected erythrocytes (IRBC) expressing the domain cassettes (DC) 8 and 13 of the cytoadherent ligand PfEMP1 adhere to the endothelial protein C receptor (EPCR). By interfering with EPCR anti-coagulant and pro-endothelial barrier functions, IRBC adhesion could promote coagulation and vascular permeability that contribute to the pathogenesis of cerebral malaria. In this study, we examined adhesion of DC8- and DC13-expressing parasite lines to endothelial cells from different microvasculature, and the consequences of EPCR engagement on endothelial cell function. We found that IRBC from IT4var19 (DC8) and IT4var07 (DC13) parasite lines adhered to human brain, lung, and dermal endothelial cells under shear stress. However, the relative contribution of EPCR to parasite cytoadherence on the different types of endothelial cell varied. We also observed divergent functional outcomes for DC8 CIDRα1.1 and DC13 CIDRα1.4 domains. IT4var07 CIDRα1.4 inhibited generation of activated protein C (APC) on lung and dermal endothelial cells and blocked the APC-EPCR binding interaction on brain endothelial cells. IT4var19 CIDRα1.1 inhibited thrombin-induced endothelial barrier dysfunction in lung endothelial cells, while IT4var07 CIDRα1.4- inhibited the protective effect of APC on thrombin-induced permeability. Overall, these findings reveal a much greater complexity of how CIDRα1-expressing parasites may modulate malaria pathogenesis through EPCR adhesion. PMID:26119044

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

Hypokalemia correlated with arterial stiffness but not microvascular endothelial function in patients with primary aldosteronism.

PubMed

Chang, Yi-Yao; Chen, Aaron; Chen, Ying-Hsien; Hung, Chi-Sheng; Wu, Vin-Cent; Wu, Xue-Ming; Lin, Yen-Hung; Ho, Yi-Lwun; Wu, Kwan-Dun

2015-06-01

Hypokalemia in primary aldosteronism (PA) patients correlates with higher levels of cardiovascular events and altered left ventricular geometry. However, the influence of aldosterone on microvascular endothelial function and the effect of hypokalemia on the vascular structure still remain unclear. We investigated the peripheral arterial functions, including the endothelial function of microvasculature and arterial stiffness in PA and essential hypertension (EH) patients, and the correlation between hypokalemia and peripheral arterial function among PA patients. Twenty patients diagnosed as EH and 37 patients with PA were enrolled in this study. Reactive hyperemia index (RHI) and the augmentation index (AI) were obtained by non-invasive peripheral arterial tonometry. Twenty EH patients and a total of 37 PA patients, including 21 patients with normokalemia and 16 patients with hypokalemia, were enrolled and divided into groups 1, 2 and 3 respectively. PA patients had significantly higher AI (p=0.024) but not RHI than EH patients. RHI showed no difference between groups 1, 2 and 3. Group 3 had higher AI than either group 1 or group 2. In the whole study population, serum potassium level, after multivariate regression analysis testing, was the only factor associated with AI (ß= -0.102; p=0.002). In PA patients, serum potassium level was the only significant factor correlated with AI. (r= -0.458; p=0.004) CONCLUSIONS: PA patients had higher arterial stiffness but comparable microvascular endothelial function to EH patients. Hypokalemia correlated with arterial stiffness but not microvascular endothelial function in PA patients. © The Author(s) 2014.

Exercise training improves endothelial function in resistance arteries of young prehypertensives.

PubMed

Beck, D T; Martin, J S; Casey, D P; Braith, R W

2014-05-01

Prehypertension is associated with reduced conduit artery endothelial function and perturbation of oxidant/antioxidant status. It is unknown whether endothelial dysfunction persists to resistance arteries and whether exercise training affects oxidant/antioxidant balance in young prehypertensives. We examined resistance artery function using venous occlusion plethysmography measurement of forearm (FBF) and calf blood flow (CBF) at rest and during reactive hyperaemia (RH), as well as lipid peroxidation (8-iso-PGF2α) and antioxidant capacity (Trolox-equivalent antioxidant capacity; TEAC) before and after exercise intervention or time control. Forty-three unmedicated prehypertensive and 15 matched normotensive time controls met screening requirements and participated in the study (age: 21.1±0.8 years). Prehypertensive subjects were randomly assigned to resistance exercise training (PHRT; n=15), endurance exercise training (PHET; n=13) or time-control groups (PHTC; n=15). Treatment groups exercised 3 days per week for 8 weeks. Peak and total FBF were lower in prehypertensives than normotensives (12.7±1.2 ml min(-1) per100 ml tissue and 89.1±7.7 ml min(-1) per 100 ml tissue vs 16.3±1.0 ml min(-1) per 100 ml tissue and 123.3±6.4 ml min(-1) per 100 ml tissue, respectively; P<0.05). Peak and total CBF were lower in prehypertensives than normotensives (15.3±1.2 ml min(-1) per 100 ml tissue and 74±8.3 ml min(-1) per 100 ml tissue vs 20.9±1.4 ml min(-1) per 100 ml tissue and 107±9.2 ml min(-1) per 100 ml tissue, respectively; P<0.05). PHRT and PHET improved humoral measures of TEAC (+24 and +30%) and 8-iso-PGF2α (-43 and -40%, respectively; P < or = 0.05). This study provides evidence that young prehypertensives exhibit reduced resistance artery endothelial function and that short-term (8 weeks) resistance or endurance training are effective in improving resistance artery endothelial function and oxidant

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

Consumption of High-Polyphenol Dark Chocolate Improves Endothelial Function in Individuals with Stage 1 Hypertension and Excess Body Weight

PubMed Central

Nogueira, Lívia de Paula; Knibel, Marcela Paranhos; Torres, Márcia Regina Simas Gonçalves; Nogueira Neto, José Firmino; Sanjuliani, Antonio Felipe

2012-01-01

Background. Hypertension and excess body weight are important risk factors for endothelial dysfunction. Recent evidence suggests that high-polyphenol dark chocolate improves endothelial function and lowers blood pressure. This study aimed to evaluate the association of chocolate 70% cocoa intake with metabolic profile, oxidative stress, inflammation, blood pressure, and endothelial function in stage 1 hypertensives with excess body weight. Methods. Intervention clinical trial includes 22 stage 1 hypertensives without previous antihypertensive treatment, aged 18 to 60 years and presents a body mass index between 25.0 and 34.9 kg/m2. All participants were instructed to consume 50 g of chocolate 70% cocoa/day (2135 mg polyphenols) for 4 weeks. Endothelial function was evaluated by peripheral artery tonometry using Endo-PAT 2000 (Itamar Medical). Results. Twenty participants (10 men) completed the study. Comparison of pre-post intervention revealed that (1) there were no significant changes in anthropometric parameters, percentage body fat, glucose metabolism, lipid profile, biomarkers of inflammation, adhesion molecules, oxidized LDL, and blood pressure; (2) the assessment of endothelial function through the reactive hyperemia index showed a significant increase: 1.94 ± 0.18 to 2.22 ± 0.08, P = 0.01. Conclusion.In individuals with stage 1 hypertension and excess body weight, high-polyphenol dark chocolate improves endothelial function. PMID:23209885

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.

The Staphylococcus aureus Alpha-Toxin Perturbs the Barrier Function in Caco-2 Epithelial Cell Monolayers by Altering Junctional Integrity

PubMed Central

Vikström, Elena; Magnusson, Karl-Eric; Vécsey-Semjén, Beatrix; Colque-Navarro, Patricia; Möllby, Roland

2012-01-01

Increased microvascular permeability is a hallmark of sepsis and septic shock. Intestinal mucosal dysfunction may allow translocation of bacteria and their products, thereby promoting sepsis and inflammation. Although Staphylococcus aureus alpha-toxin significantly contributes to sepsis and perturbs the endothelial barrier function, little is known about possible effects of S. aureus alpha-toxin on human epithelial barrier functions. We hypothesize that S. aureus alpha-toxin in the blood can impair the intestinal epithelial barrier and thereby facilitate the translocation of luminal bacteria into the blood, which may in turn aggravate a septic condition. Here, we showed that staphylococcal alpha-toxin disrupts the barrier integrity of human intestinal epithelial Caco-2 cells as evidenced by decreased transepithelial electrical resistance (TER) and reduced cellular levels of junctional proteins, such as ZO-1, ZO-3, and E-cadherin. The Caco-2 cells also responded to alpha-toxin with an elevated cytosolic calcium ion concentration ([Ca2+]i), elicited primarily by calcium influx from the extracellular environment, as well as with a significant reduction in TER, which was modulated by intracellular calcium chelation. Moreover, a significantly larger reduction in TER and amounts of the junctional proteins, viz., ZO-3 and occludin, was achieved by basolateral than by apical application of the alpha-toxin. These experimental findings thus support the hypothesis that free staphylococcal alpha-toxin in the bloodstream may cause intestinal epithelial barrier dysfunction and further aggravate the septic condition by promoting the release of intestinal bacteria into the underlying tissues and the blood. PMID:22354024

Interleukin 2 Activates Brain Microvascular Endothelial Cells Resulting in Destabilization of Adherens Junctions.

PubMed

Wylezinski, Lukasz S; Hawiger, Jacek

2016-10-28

The pleiotropic cytokine interleukin 2 (IL2) disrupts the blood-brain barrier and alters brain microcirculation, underlying vascular leak syndrome that complicates cancer immunotherapy with IL2. The microvascular effects of IL2 also play a role in the development of multiple sclerosis and other chronic neurological disorders. The mechanism of IL2-induced disruption of brain microcirculation has not been determined previously. We found that both human and murine brain microvascular endothelial cells express constituents of the IL2 receptor complex. Then we established that signaling through this receptor complex leads to activation of the transcription factor, nuclear factor κB, resulting in expression of proinflammatory interleukin 6 and monocyte chemoattractant protein 1. We also discovered that IL2 induces disruption of adherens junctions, concomitant with cytoskeletal reorganization, ultimately leading to increased endothelial cell permeability. IL2-induced phosphorylation of vascular endothelial cadherin (VE-cadherin), a constituent of adherens junctions, leads to dissociation of its stabilizing adaptor partners, p120-catenin and β-catenin. Increased phosphorylation of VE-cadherin was also accompanied by a reduction of Src homology 2 domain-containing protein-tyrosine phosphatase 2, known to maintain vascular barrier function. These results unravel the mechanism of deleterious effects induced by IL2 on brain microvascular endothelial cells and may inform the development of new measures to improve IL2 cancer immunotherapy, as well as treatments for autoimmune diseases affecting the central nervous system. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The research on endothelial function in women and men at risk for cardiovascular disease (REWARD) study: methodology

PubMed Central

2011-01-01

Background Endothelial function has been shown to be a highly sensitive marker for the overall cardiovascular risk of an individual. Furthermore, there is evidence of important sex differences in endothelial function that may underlie the differential presentation of cardiovascular disease (CVD) in women relative to men. As such, measuring endothelial function may have sex-specific prognostic value for the prediction of CVD events, thus improving risk stratification for the overall prediction of CVD in both men and women. The primary objective of this study is to assess the clinical utility of the forearm hyperaemic reactivity (FHR) test (a proxy measure of endothelial function) for the prediction of CVD events in men vs. women using a novel, noninvasive nuclear medicine -based approach. It is hypothesised that: 1) endothelial dysfunction will be a significant predictor of 5-year CVD events independent of baseline stress test results, clinical, demographic, and psychological variables in both men and women; and 2) endothelial dysfunction will be a better predictor of 5-year CVD events in women compared to men. Methods/Design A total of 1972 patients (812 men and 1160 women) undergoing a dipyridamole stress testing were recruited. Medical history, CVD risk factors, health behaviours, psychological status, and gender identity were assessed via structured interview or self-report questionnaires at baseline. In addition, FHR was assessed, as well as levels of sex hormones via blood draw. Patients will be followed for 5 years to assess major CVD events (cardiac mortality, non-fatal MI, revascularization procedures, and cerebrovascular events). Discussion This is the first study to determine the extent and nature of any sex differences in the ability of endothelial function to predict CVD events. We believe the results of this study will provide data that will better inform the choice of diagnostic tests in men and women and bring the quality of risk stratification in

Acute effects of coffee on endothelial function in healthy subjects.

PubMed

Buscemi, S; Verga, S; Batsis, J A; Donatelli, M; Tranchina, M R; Belmonte, S; Mattina, A; Re, A; Cerasola, G

2010-05-01

Coffee is the most widely consumed beverage in the world, but its effect on the cardiovascular system has not been fully understood. Coffee contains caffeine and antioxidants, which may influence endothelial function, both of which have not yet been investigated. The objective of this study was to investigate the acute effects of coffee on endothelial function measured by brachial artery flow-mediated dilation (FMD). A total of 20 (10 males and 10 females) healthy non-obese subjects underwent a double-blind, crossover study. Subjects ingested one cup of caffeinated (CC) and one cup of decaffeinated (DC) Italian espresso coffee in random order at 5- to 7-day intervals. Following CC ingestion, FMD decreased progressively and significantly (mean+/-s.e.m.: 0 min, 7.7+/-0.6; 30 min, 6.3+/-0.7; 60 min, 6.0+/-0.8%; ANOVA (analysis of variance), P<0.05), but it did not significantly increase after DC ingestion (0 min, 6.9+/-0.6; 30 min, 8.1+/-0.9; 60 min, 8.5+/-0.9%; P=0.115). Similarly, CC significantly increased both systolic and diastolic blood pressure; this effect was not observed after DC ingestion. Blood glucose concentrations remained unchanged after ingestion of both CC and DC, but insulin (0 min, 15.8+/-0.9; 60 min, 15.0+/-0.8 muU/ml; P<0.05) and C-peptide (0 min, 1.25+/-0.09; 60 min, 1.18+/-0.09 ng/ml; P<0.01) blood concentrations decreased significantly only after CC ingestion. CC acutely induced unfavorable cardiovascular effects, especially on endothelial function. In the fasting state, insulin secretion is also likely reduced after CC ingestion. Future studies will determine whether CC has detrimental clinically relevant effects, especially in unhealthy subjects.

Endothelial-specific inhibition of NF-κB enhances functional haematopoiesis.

PubMed

Poulos, Michael G; Ramalingam, Pradeep; Gutkin, Michael C; Kleppe, Maria; Ginsberg, Michael; Crowley, Michael J P; Elemento, Olivier; Levine, Ross L; Rafii, Shahin; Kitajewski, Jan; Greenblatt, Matthew B; Shim, Jae-Hyuck; Butler, Jason M

2016-12-21

Haematopoietic stem cells (HSCs) reside in distinct niches within the bone marrow (BM) microenvironment, comprised of endothelial cells (ECs) and tightly associated perivascular constituents that regulate haematopoiesis through the expression of paracrine factors. Here we report that the canonical NF-κB pathway in the BM vascular niche is a critical signalling axis that regulates HSC function at steady state and following myelosuppressive insult, in which inhibition of EC NF-κB promotes improved HSC function and pan-haematopoietic recovery. Mice expressing an endothelial-specific dominant negative IκBα cassette under the Tie2 promoter display a marked increase in HSC activity and self-renewal, while promoting the accelerated recovery of haematopoiesis following myelosuppression, in part through protection of the BM microenvironment following radiation and chemotherapeutic-induced insult. Moreover, transplantation of NF-κB-inhibited BM ECs enhanced haematopoietic recovery and protected mice from pancytopenia-induced death. These findings pave the way for development of niche-specific cellular approaches for the treatment of haematological disorders requiring myelosuppressive regimens.

Endothelial-specific inhibition of NF-κB enhances functional haematopoiesis

PubMed Central

Poulos, Michael G.; Ramalingam, Pradeep; Gutkin, Michael C.; Kleppe, Maria; Ginsberg, Michael; Crowley, Michael J. P.; Elemento, Olivier; Levine, Ross L.; Rafii, Shahin; Kitajewski, Jan; Greenblatt, Matthew B.; Shim, Jae-Hyuck; Butler, Jason M.

2016-01-01

Haematopoietic stem cells (HSCs) reside in distinct niches within the bone marrow (BM) microenvironment, comprised of endothelial cells (ECs) and tightly associated perivascular constituents that regulate haematopoiesis through the expression of paracrine factors. Here we report that the canonical NF-κB pathway in the BM vascular niche is a critical signalling axis that regulates HSC function at steady state and following myelosuppressive insult, in which inhibition of EC NF-κB promotes improved HSC function and pan-haematopoietic recovery. Mice expressing an endothelial-specific dominant negative IκBα cassette under the Tie2 promoter display a marked increase in HSC activity and self-renewal, while promoting the accelerated recovery of haematopoiesis following myelosuppression, in part through protection of the BM microenvironment following radiation and chemotherapeutic-induced insult. Moreover, transplantation of NF-κB-inhibited BM ECs enhanced haematopoietic recovery and protected mice from pancytopenia-induced death. These findings pave the way for development of niche-specific cellular approaches for the treatment of haematological disorders requiring myelosuppressive regimens. PMID:28000664

A critical role for phosphatidylinositol (3,4,5)-trisphosphate-dependent Rac exchanger 1 in endothelial junction disruption and vascular hyperpermeability

PubMed Central

Naikawadi, Ram P.; Cheng, Ni; Vogel, Stephen M.; Qian, Feng; Wu, Dianqing; Malik, Asrar B.; Ye, Richard D.

2013-01-01

Rationale The small GTPase Rac is critical to vascular endothelial functions, yet its regulation in endothelial cells remains unclear. Understanding the upstream pathway may delineate Rac activation mechanisms and its role in maintaining vascular endothelial barrier integrity. Objective By investigating P-Rex1, one of the Rac-specific guanine nucleotide exchange factors (GEFs) previously known for G protein-coupled receptor (GPCR) signaling, we sought to determine whether Rac-GEF is a nodal for signal integration and potential target for drug intervention. Methods and Results Using gene deletion and siRNA silencing approach, we investigated the role of P-Rex1 in lung microvascular endothelial cells (HLMVECs). TNF-α exposure led to disruption of endothelial junctions, and silencing P-Rex1 protected junction integrity. TNF-α stimulated Rac activation and ROS production in a P-Rex1-dependent manner. Removal of P-Rex1 significantly reduced ICAM-1 expression, PMN transendothelial migration and leukocyte sequestration in TNF-α challenged mouse lungs. The P-Rex1 knockout mice were also refractory to lung vascular hyper-permeability and edema in a LPS-induced sepsis model. Conclusions These results demonstrate for the first time that P-Rex1 expressed in endothelial cells is activated downstream of TNF-α, which is not a GPCR agonist. Our data identify P-Rex1 as a critical mediator of vascular barrier disruption. Targeting P-Rex1 may effectively protect against TNF-α and LPS-induced endothelial junction disruption and vascular hyper-permeability. PMID:22965143

Optimizing donor heart outcome after prolonged storage with endothelial function analysis and continuous perfusion.

PubMed

Poston, Robert S; Gu, Junyan; Prastein, Deyanira; Gage, Fred; Hoffman, John W; Kwon, Michael; Azimzadeh, Agnes; Pierson, Richard N; Griffith, Bartley P

2004-10-01

By minimizing tissue ischemia, continuous perfusion (CP) during organ transport may increase the safety of "marginal donors." My colleagues and I investigated whether an analysis of donor heart viability predicts recovery of grafts challenged with a 24-hour preservation interval. Dog hearts underwent cold static storage (CS) for 8 hours (n = 8) or 24 hours (n = 2) or CP for 24 hours with cold asanguinous, oxygenated solution (n = 8). Myocardial systolic and diastolic function and oxygen and lactate consumption were assessed at base line, during CP, and after Langendorff blood reperfusion. Base line endothelial function was evaluated by the percentage transcoronary change ([coronary sinus - aorta]/aorta) in myeloperoxidase and by platelet function and coronary flow reserve after 20 seconds of coronary artery occlusion. During CP, the endothelium was assessed by transcoronary protein release and coronary resistance. Edema was assessed by weight gain and histology. Base line systolic and metabolic functions showed no relation to post-Langendorff function. Compared with CS, CP resulted in a greater recovery in systolic function (87% +/- 35% vs 65% +/- 15% of baseline; p = 0.05) and a shorter interval required for lactate consumption to exceed production (7.0 +/- 6.8 minutes vs 15.0 +/- 8.9 minutes; p = 0.06). Endothelial function was heterogeneous: coronary flow reserve, 2.7 +/- 0.7; percentage change in myeloperoxidase, -8.4% +/- 6.8%; and change in platelet function, 4.3% +/- 3.5%, as determined by thromboelastography angle at base line. Protein release during CP for 24 hours was 8.3 +/- 7.1 g. Two factors predicted more than 75% systolic pressure generation recovery: use of CP and normal endothelial function (p = 0.05; Fisher's exact test). However, CP led to edema according to histology, weight gain (72 +/- 29 g), and impaired diastolic function versus CS (end-diastolic pressure-volume relationship, 1.4 +/- 0.4 mm Hg/mL vs 0.8 +/- 0.3 mm Hg/mL; p = 0.08). Better

Mangiferin protects mitochondrial function by preserving mitochondrial hexokinase-II in vessel endothelial cells.

PubMed

Song, Junna; Li, Yi; Song, Junmei; Hou, Fangjie; Liu, Baolin; Li, Aiying

2017-07-01

Hexokinase-II (HK-II) confers protection against cell death and this study was designed to investigate the effect of mangiferin on the regulation of mitochondrial HK-II. In vessel endothelial cells, saturated fatty acid palmitate (PA) stimulation induced HK-II detachment from mitochondria due to cellular acidification. Mangiferin reduced lactate accumulation by improving pyruvate dehydrogenase activity, promoted Akt translocation to HK-II and prevented HK-II detachment from mitochondria. Knockdown of Akt2 diminished the protective effect of mangiferin on mitochondrial HK-II, confirming the role of Akt in the regulation of HK-II. Mangiferin prevented mitochondrial permeability transition pore opening, restored mitochondrial membrane potential and thereby protected cell from apoptosis. In high-fat diet fed mice, oral administration of mangiferin induced Akt phosphorylation, increased HK-II binding to mitochondria and resultantly protected vessel endothelial function, demonstrating its protective effect on endothelial integrity in vivo. This finding provided a novel strategy for the protection of mitochondrial function in the endothelium. Copyright © 2017 Elsevier B.V. All rights reserved.

Astrocytic TYMP and VEGFA drive blood-brain barrier opening in inflammatory central nervous system lesions.

PubMed

Chapouly, Candice; Tadesse Argaw, Azeb; Horng, Sam; Castro, Kamilah; Zhang, Jingya; Asp, Linnea; Loo, Hannah; Laitman, Benjamin M; Mariani, John N; Straus Farber, Rebecca; Zaslavsky, Elena; Nudelman, German; Raine, Cedric S; John, Gareth R

2015-06-01

In inflammatory central nervous system conditions such as multiple sclerosis, breakdown of the blood-brain barrier is a key event in lesion pathogenesis, predisposing to oedema, excitotoxicity, and ingress of plasma proteins and inflammatory cells. Recently, we showed that reactive astrocytes drive blood-brain barrier opening, via production of vascular endothelial growth factor A (VEGFA). Here, we now identify thymidine phosphorylase (TYMP; previously known as endothelial cell growth factor 1, ECGF1) as a second key astrocyte-derived permeability factor, which interacts with VEGFA to induce blood-brain barrier disruption. The two are co-induced NFκB1-dependently in human astrocytes by the cytokine interleukin 1 beta (IL1B), and inactivation of Vegfa in vivo potentiates TYMP induction. In human central nervous system microvascular endothelial cells, VEGFA and the TYMP product 2-deoxy-d-ribose cooperatively repress tight junction proteins, driving permeability. Notably, this response represents part of a wider pattern of endothelial plasticity: 2-deoxy-d-ribose and VEGFA produce transcriptional programs encompassing angiogenic and permeability genes, and together regulate a third unique cohort. Functionally, each promotes proliferation and viability, and they cooperatively drive motility and angiogenesis. Importantly, introduction of either into mouse cortex promotes blood-brain barrier breakdown, and together they induce severe barrier disruption. In the multiple sclerosis model experimental autoimmune encephalitis, TYMP and VEGFA co-localize to reactive astrocytes, and correlate with blood-brain barrier permeability. Critically, blockade of either reduces neurologic deficit, blood-brain barrier disruption and pathology, and inhibiting both in combination enhances tissue preservation. Suggesting importance in human disease, TYMP and VEGFA both localize to reactive astrocytes in multiple sclerosis lesion samples. Collectively, these data identify TYMP as an

Engineering of Surface Functionality onto Polystyrene Microcarriers for the Attachment and Growth of Human Endothelial Cells

NASA Astrophysics Data System (ADS)

Xiong, Gordon M.; Foord, John S.; Griffiths, Jon-Paul; Parker, Emily M.; Moloney, Mark G.; Choong, Cleo

2014-08-01

This work reports the effects of introducing diverse chemical functionalities onto the surface of polystyrene microcarrier beads on their ability to function as injectable cell carriers. Cellular adhesion and proliferation, as well as cellular outgrowths from microcarrier surfaces, using human umbilical vein endothelial cells (HUVECs), were examined in detail. It was observed that initial cell adhesion appeared to be most significantly decreased by hydrophobicity, whilst cell proliferation appeared to be improved in most chemical functional groups over unmodified polystyrene. Overall, our study highlights the importance of surface chemistry in directing the growth and function of human endothelial cells.

An EMMPRIN-γ-catenin-Nm23 complex drives ATP production and actomyosin contractility at endothelial junctions.

PubMed

Moreno, Vanessa; Gonzalo, Pilar; Gómez-Escudero, Jesús; Pollán, Ángela; Acín-Pérez, Rebeca; Breckenridge, Mark; Yáñez-Mó, María; Barreiro, Olga; Orsenigo, Fabrizio; Kadomatsu, Kenji; Chen, Christopher S; Enríquez, José A; Dejana, Elisabetta; Sánchez-Madrid, Francisco; Arroyo, Alicia G

2014-09-01

Cell-cell adhesions are important sites through which cells experience and resist forces. In endothelial cells, these forces regulate junction dynamics and determine endothelial barrier strength. We identify the Ig superfamily member EMMPRIN (also known as basigin) as a coordinator of forces at endothelial junctions. EMMPRIN localization at junctions correlates with endothelial junction strength in different mouse vascular beds. Accordingly, EMMPRIN-deficient mice show altered junctions and increased junction permeability. Lack of EMMPRIN alters the localization and function of VE-cadherin (also known as cadherin-5) by decreasing both actomyosin contractility and tugging forces at endothelial cell junctions. EMMPRIN ensures proper actomyosin-driven maturation of competent endothelial junctions by forming a molecular complex with γ-catenin (also known as junction plakoglobin) and Nm23 (also known as NME1), a nucleoside diphosphate kinase, thereby locally providing ATP to fuel the actomyosin machinery. These results provide a novel mechanism for the regulation of actomyosin contractility at endothelial junctions and might have broader implications in biological contexts such as angiogenesis, collective migration and tissue morphogenesis by coupling compartmentalized energy production to junction assembly. © 2014. Published by The Company of Biologists Ltd.

An EMMPRIN–γ-catenin–Nm23 complex drives ATP production and actomyosin contractility at endothelial junctions

PubMed Central

Moreno, Vanessa; Gonzalo, Pilar; Gómez-Escudero, Jesús; Pollán, Ángela; Acín-Pérez, Rebeca; Breckenridge, Mark; Yáñez-Mó, María; Barreiro, Olga; Orsenigo, Fabrizio; Kadomatsu, Kenji; Chen, Christopher S.; Enríquez, José A.; Dejana, Elisabetta; Sánchez-Madrid, Francisco; Arroyo, Alicia G.

2014-01-01

ABSTRACT Cell–cell adhesions are important sites through which cells experience and resist forces. In endothelial cells, these forces regulate junction dynamics and determine endothelial barrier strength. We identify the Ig superfamily member EMMPRIN (also known as basigin) as a coordinator of forces at endothelial junctions. EMMPRIN localization at junctions correlates with endothelial junction strength in different mouse vascular beds. Accordingly, EMMPRIN-deficient mice show altered junctions and increased junction permeability. Lack of EMMPRIN alters the localization and function of VE-cadherin (also known as cadherin-5) by decreasing both actomyosin contractility and tugging forces at endothelial cell junctions. EMMPRIN ensures proper actomyosin-driven maturation of competent endothelial junctions by forming a molecular complex with γ-catenin (also known as junction plakoglobin) and Nm23 (also known as NME1), a nucleoside diphosphate kinase, thereby locally providing ATP to fuel the actomyosin machinery. These results provide a novel mechanism for the regulation of actomyosin contractility at endothelial junctions and might have broader implications in biological contexts such as angiogenesis, collective migration and tissue morphogenesis by coupling compartmentalized energy production to junction assembly. PMID:24994937

Human brain microvascular endothelial cells resist elongation due to shear stress.

PubMed

Reinitz, Adam; DeStefano, Jackson; Ye, Mao; Wong, Andrew D; Searson, Peter C

2015-05-01

Endothelial cells in straight sections of vessels are known to elongate and align in the direction of flow. This phenotype has been replicated in confluent monolayers of bovine aortic endothelial cells and human umbilical vein endothelial cells (HUVECs) in cell culture under physiological shear stress. Here we report on the morphological response of human brain microvascular endothelial cells (HBMECs) in confluent monolayers in response to shear stress. Using a microfluidic platform we image confluent monolayers of HBMECs and HUVECs under shear stresses up to 16 dyne cm(-2). From live-cell imaging we quantitatively analyze the cell morphology and cell speed as a function of time. We show that HBMECs do not undergo a classical transition from cobblestone to spindle-like morphology in response to shear stress. We further show that under shear stress, actin fibers are randomly oriented in the cells indicating that there is no cytoskeletal remodeling. These results suggest that HBMECs are programmed to resist elongation and alignment under shear stress, a phenotype that may be associated with the unique properties of the blood-brain barrier. Copyright © 2015 Elsevier Inc. All rights reserved.

Homoclinic orbits and critical points of barrier functions

NASA Astrophysics Data System (ADS)

Cannarsa, Piermarco; Cheng, Wei

2015-06-01

We interpret the close link between the critical points of Mather's barrier functions and minimal homoclinic orbits with respect to the Aubry sets on {{T}}n . We also prove a critical point theorem for barrier functions and the existence of such homoclinic orbits on {{T}}2 as an application.

Acute and subacute effects of EV iron sucrose on endothelial functions in hemodialysis patients.

PubMed

Ozkurt, Sultan; Ozenc, Fatma; Degirmenci, Nevbahar Akcar; Temiz, Gokhan; Musmul, Ahmet; Sahin, Garip; Yalcin, Ahmet Ugur

2012-01-01

Iron support is an important component of treatment of anemia in hemodialysis (HD) patients. However, there are concerns about endovenous (EV) iron therapy that may cause endothelial dysfunction (ED) by increasing oxidative stress (OS) and lead to cardiovascular events. In this study, we aimed to evaluate the effects of high and repeated doses of EV iron sucrose on endothelial functions in acute and subacute phases. We included 15 HD patients to our study. There were 16 patients with iron deficiency but normal kidney functions in control group. We also evaluated endothelium-dependent vasodilatation (EDV) and nitroglycerin-induced vasodilatation (NIV) from the brachial artery by ultrasonography at the beginning of the study, and then 200 mg EV iron sucrose was given initially to both groups for 1 h in 250 cc 0.9% saline and 4 h after the end of the infusion (acute phase) sonographic vasodilatation parameters were measured from brachial artery. These measurements and laboratory tests were repeated 1 week after the end of a total 1000 mg EV iron sucrose replacement (200 mg/week). There was a statistically significant increase in hemoglobin and ferritin levels after the EV iron sucrose therapy in both control and patient groups. EDV values in the HD group were significantly lower than that in the control group before therapy (6.25% vs. 10.53%, p < 0.05). EV iron sucrose therapy did not alter EDV and NIV values at the 4th hour and 6th week in both control and patient groups. According to our study, compared with the control group with normal kidney functions, HD patients had impaired endothelial functions. However, in HD patients, high and repeated doses of EV iron sucrose do not have deleterious effects on endothelial functions at acute and subacute phases and can be used safely in that patient group.

Telmisartan enhances mitochondrial activity and alters cellular functions in human coronary artery endothelial cells via AMP-activated protein kinase pathway.

PubMed

Kurokawa, Hirofumi; Sugiyama, Seigo; Nozaki, Toshimitsu; Sugamura, Koichi; Toyama, Kensuke; Matsubara, Junichi; Fujisue, Koichiro; Ohba, Keisuke; Maeda, Hirofumi; Konishi, Masaaki; Akiyama, Eiichi; Sumida, Hitoshi; Izumiya, Yasuhiro; Yasuda, Osamu; Kim-Mitsuyama, Shokei; Ogawa, Hisao

2015-04-01

Mitochondrial dysfunction plays an important role in cellular senescence and impaired function of vascular endothelium, resulted in cardiovascular diseases. Telmisartan is a unique angiotensin II type I receptor blocker that has been shown to prevent cardiovascular events in high risk patients. AMP-activated protein kinase (AMPK) plays a critical role in mitochondrial biogenesis and endothelial function. This study assessed whether telmisartan enhances mitochondrial function and alters cellular functions via AMPK in human coronary artery endothelial cells (HCAECs). In cultured HCAECs, telmisartan significantly enhanced mitochondrial activity assessed by mitochondrial reductase activity and intracellular ATP production and increased the expression of mitochondria related genes. Telmisartan prevented cellular senescence and exhibited the anti-apoptotic and pro-angiogenic properties. The expression of genes related anti-oxidant and pro-angiogenic properties were increased by telmisartan. Telmisartan increased endothelial NO synthase and AMPK phosphorylation. Peroxisome proliferator-activated receptor gamma signaling was not involved in telmisartan-induced improvement of mitochondrial function. All of these effects were abolished by inhibition of AMPK. Telmisartan enhanced mitochondrial activity and exhibited anti-senescence effects and improving endothelial function through AMPK in HCAECs. Telmisartan could provide beneficial effects on vascular diseases via enhancement of mitochondrial activity and modulating endothelial function through AMPK activation. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Shiftwork and Decline in Endothelial Function Among Police Officers

PubMed Central

Charles, Luenda E.; Zhao, Songzhu; Fekedulegn, Desta; Violanti, John M.; Andrew, Michael E.; Burchfiel, Cecil M.

2016-01-01

Background Our objective was to assess the influence of shiftwork on change in endothelial function. Methods This longitudinal study was conducted in 188 police officers (78.2% men). Shiftwork status (day, afternoon, night) was assessed objectively using daily Buffalo, NY payroll work history records. Brachial artery flow-mediated dilation (FMD) was assessed using ultrasound. Mean change in FMD% between 2004–2009 and 2010–2015 was compared across shiftwork using analysis of variance/covariance. Results Overall, mean FMD% decreased from 5.74 ± 2.83 to 3.88 ± 2.11 over an average of 7 years among all officers; P < 0.0001. Effect modification by gender was significant. Among men (but not women), those who worked day shifts had a smaller mean (±SE) decrease in FMD% (−0.89 ± 0.35) compared with those who worked the afternoon (−2.69 ± 0.39; P = 0.001) or night shifts (−2.31 ± 0.45; P = 0.020) after risk factor adjustment. Conclusions Larger declines in endothelial function were observed among men who worked afternoon or night shifts. Further investigation is warranted. Am. J. Ind. Med. Published 2016. This article is a U.S. Government work and is in the public domain in the USA PMID:27245641

Mesenchymal-endothelial-transition contributes to cardiac neovascularization

PubMed Central

Ubil, Eric; Duan, Jinzhu; Pillai, Indulekha C.L.; Rosa-Garrido, Manuel; Wu, Yong; Bargiacchi, Francesca; Lu, Yan; Stanbouly, Seta; Huang, Jie; Rojas, Mauricio; Vondriska, Thomas M.; Stefani, Enrico; Deb, Arjun

2014-01-01

Endothelial cells contribute to a subset of cardiac fibroblasts by undergoing endothelial-to-mesenchymal-transition, but whether cardiac fibroblasts can adopt an endothelial cell fate and directly contribute to neovascularization after cardiac injury is not known. Here, using genetic fate map techniques, we demonstrate that cardiac fibroblasts rapidly adopt an endothelial cell like phenotype after acute ischemic cardiac injury. Fibroblast derived endothelial cells exhibit anatomical and functional characteristics of native endothelial cells. We show that the transcription factor p53 regulates such a switch in cardiac fibroblast fate. Loss of p53 in cardiac fibroblasts severely decreases the formation of fibroblast derived endothelial cells, reduces post infarct vascular density and worsens cardiac function. Conversely, stimulation of the p53 pathway in cardiac fibroblasts augments mesenchymal to endothelial transition, enhances vascularity and improves cardiac function. These observations demonstrate that mesenchymal-to-endothelial-transition contributes to neovascularization of the injured heart and represents a potential therapeutic target for enhancing cardiac repair. PMID:25317562

FOXO3 Modulates Endothelial Gene Expression and Function by Classical and Alternative Mechanisms*

PubMed Central

Czymai, Tobias; Viemann, Dorothee; Sticht, Carsten; Molema, Grietje; Goebeler, Matthias; Schmidt, Marc

2010-01-01

FOXO transcription factors represent targets of the phosphatidylinositol 3-kinase/protein kinase B survival pathway controlling important biological processes, such as cell cycle progression, apoptosis, vascular remodeling, stress responses, and metabolism. Recent studies suggested the existence of alternative mechanisms of FOXO-dependent gene expression beyond classical binding to a FOXO-responsive DNA-binding element (FRE). Here we analyzed the relative contribution of those mechanisms to vascular function by comparing the transcriptional and cellular responses to conditional activation of FOXO3 and a corresponding FRE-binding mutant in human primary endothelial cells. We demonstrate that FOXO3 controls expression of vascular remodeling genes in an FRE-dependent manner. In contrast, FOXO3-induced cell cycle arrest and apoptosis occurs independently of FRE binding, albeit FRE-dependent gene expression augments the proapoptotic response. These findings are supported by bioinformatical analysis, which revealed a statistical overrepresentation of cell cycle regulators and apoptosis-related genes in the group of co-regulated genes. Molecular analysis of FOXO3-induced endothelial apoptosis excluded modulators of the extrinsic death receptor pathway and demonstrated important roles for the BCL-2 family members BIM and NOXA in this process. Although NOXA essentially contributed to FRE-dependent apoptosis, BIM was effectively induced in the absence of FRE-binding, and small interfering RNA-mediated BIM depletion could rescue apoptosis induced by both FOXO3 mutants. These data suggest BIM as a critical cell type-specific mediator of FOXO3-induced endothelial apoptosis, whereas NOXA functions as an amplifying factor. Our study provides the first comprehensive analysis of alternatively regulated FOXO3 targets in relevant primary cells and underscores the importance of such genes for endothelial function and integrity. PMID:20123982

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

Drugs of abuse and blood-brain barrier endothelial dysfunction: A focus on the role of oxidative stress

PubMed Central

Sajja, Ravi K; Rahman, Shafiqur

2015-01-01

Psychostimulants and nicotine are the most widely abused drugs with a detrimental impact on public health globally. While the long-term neurobehavioral deficits and synaptic perturbations are well documented with chronic use of methamphetamine, cocaine, and nicotine, emerging human and experimental studies also suggest an increasing incidence of neurovascular complications associated with drug abuse. Short- or long-term administration of psychostimulants or nicotine is known to disrupt blood-brain barrier (BBB) integrity/function, thus leading to an increased risk of brain edema and neuroinflammation. Various pathophysiological mechanisms have been proposed to underlie drug abuse-induced BBB dysfunction suggesting a central and unifying role for oxidative stress in BBB endothelium and perivascular cells. This review discusses drug-specific effects of methamphetamine, cocaine, and tobacco smoking on brain microvascular crisis and provides critical assessment of oxidative stress-dependent molecular pathways focal to the global compromise of BBB. Additionally, given the increased risk of human immunodeficiency virus (HIV) encephalitis in drug abusers, we have summarized the synergistic pathological impact of psychostimulants and HIV infection on BBB integrity with an emphasis on unifying role of endothelial oxidative stress. This mechanistic framework would guide further investigations on specific molecular pathways to accelerate therapeutic approaches for the prevention of neurovascular deficits by drugs of abuse. PMID:26661236

Cyclic adenosine monophosphate levels and the function of skin microvascular endothelial cells.

PubMed

Tuder, R M; Karasek, M A; Bensch, K G

1990-02-01

The maintenance of the normal epithelioid morphology of human dermal microvascular endothelial cells (MEC) grown in vitro depends strongly on the presence of factors that increase intracellular levels of cyclic AMP. Complete removal of dibutyryl cAMP and isobutylmethylxanthine (IMX) from the growth medium results in a progressive transition from an epithelioid to a spindle-shaped cell line. This transition cannot be reversed by the readdition of dibutyryl cAMP and IMX to the growth medium or by addition of agonists that increase cAMP levels. Spindle-shaped MEC lose the ability to express Factor VIII rAG and DR antigens and to bind peripheral blood mononuclear leukocyte (PBML). Ultrastructural analyses of transitional cells and spindle-shaped cells show decreased numbers of Weibel-Palade bodies in transitional cells and their complete absence in spindle-shaped cells. Interferon-gamma alters several functional properties of both epithelioid and spindle-shaped cells. In the absence of dibutyryl cAMP it accelerates the transition from epithelial to spindle-shaped cells, whereas in the presence of cyclic AMP interferon-gamma increases the binding of PBMLs to both epithelioid and spindle-shaped MEC and the endocytic activity of the endothelial cells. These results suggest that cyclic AMP is an important second messenger in the maintenance of several key functions of microvascular endothelial cells. Factors that influence the levels of this messenger in vivo can be expected to influence the angiogenic and immunologic functions of the microvasculature.

Angiocrine functions of organ-specific endothelial cells

PubMed Central

Rafii, Shahin; Butler, Jason M; Ding, Bi-Sen

2016-01-01

Preface Endothelial cells lining blood vessel capillaries are not just passive conduits for delivering blood. Tissue-specific endothelium establish specialized vascular niches that deploy specific sets of growth factors, known as angiocrine factors, which actively participate in inducing, specifying, patterning, and guiding organ regeneration and maintaining homeostasis and metabolism. Angiocrine factors upregulated in response to injury orchestrates self-renewal and differentiation of tissue-specific repopulating resident stem and progenitor cells into functional organs. Uncovering the precise mechanisms whereby physiological-levels of angiocrine factors are spatially and temporally produced, and distributed by organotypic endothelium to repopulating cells, will lay the foundation for driving organ repair without scarring. PMID:26791722

The vascular barrier-protecting hawthorn extract WS® 1442 raises endothelial calcium levels by inhibition of SERCA and activation of the IP3 pathway.

PubMed

Willer, Elisabeth A; Malli, Roland; Bondarenko, Alexander I; Zahler, Stefan; Vollmar, Angelika M; Graier, Wolfgang F; Fürst, Robert

2012-10-01

WS® 1442 has been proven as an effective and safe therapeutical to treat mild forms of congestive heart failure. Beyond this action, we have recently shown that WS® 1442 protects against thrombin-induced vascular barrier dysfunction and the subsequent edema formation by affecting endothelial calcium signaling. The aim of the study was to analyze the influence of WS® 1442 on intracellular calcium concentrations [Ca(2+)](i) in the human endothelium and to investigate the underlying mechanisms. Using ratiometric calcium measurements and a FRET sensor, we found that WS® 1442 concentration-dependently increased basal [Ca(2+)](i) by depletion of the endoplasmic reticulum (ER) and inhibited a subsequent histamine-triggered rise of [Ca(2+)](i). Interestingly, the augmented [Ca(2+)](i) did neither trigger an activation of the contractile machinery nor led to a barrier breakdown (macromolecular permeability). It also did not impair endothelial cell viability. As assessed by patch clamp recordings, WS® 1442 did only slightly affect endothelial Na(+)/K(+)-ATPase, but increased [Ca(2+)](i) by inhibiting the sarcoplasmic/endoplasmic reticulum Ca(2+) ATPase (SERCA) and by activating the inositol 1,4,5-trisphosphate (IP(3)) pathway. Most importantly, WS® 1442 did not induce store-operated calcium entry (SOCE), but even irreversibly prevented histamine-induced SOCE. Taken together, WS® 1442 prevented the deleterious hyperpermeability-associated rise of [Ca(2+)](i) by a preceding, non-toxic release of Ca(2+) from the ER. WS® 1442 interfered with SERCA and the IP(3) pathway without inducing SOCE. The elucidation of this intriguing mechanism helps to understand the complex pharmacology of the cardiovascular drug WS® 1442. Copyright © 2012 Elsevier Ltd. All rights reserved.

Maspin impairs the function of endothelial cells: an implying pathway of preeclampsia.

PubMed

Zhang, Ying; Liu, Hao; Shi, Xinwei; Qiao, Fuyuan; Zeng, Wanjiang; Feng, Ling; Deng, Dongrui; Liu, Haiyi; Wu, Yuanyuan

2017-09-29

Widespread endothelial injury contributes to the occurrence of preeclampsia. Maspin, first identified as a tumor suppressor, plays a critical role in cell invasion and angiogenesis. Our previous studies found that the expression of maspin was increased in preeclampsic placenta. In this research, we studied the function of human umbilical vein endothelial cells (HUVECs) to explore the role and possible mechanism of maspin gene in the pathogenesis of preeclampsia. HUVECs were treated with different concentration of recombinant human maspin protein (r-maspin) during normoxia and hypoxia, we detected the proliferation, apoptosis, migration and tube formation of HUVECs. We also assessed nitride oxide (NO) synthesis and the expression of matrix metalloproteinase 2 (MMP2) to further explore the underlying molecular mechanism. There was only slight maspin expression at mRNA level in HUVECs. Treated HUVECs with r-maspin, the proliferation of HUVECs was significantly promoted both under normoxia and hypoxia. The tubes formed by HUVECs were significantly inhibited and NO synthesis was significantly reduced by r-maspin. Meantime, r-maspin also inhibited MMP2 expression and activity in HUVECs. However, there was no significant change in the migration and apoptosis of HUVECs. Maspin may be an important participant for mediating endothelial function and ultimately leads to the occurence of preeclamsia.

Glucocorticoids induce transactivation of tight junction genes occludin and claudin-5 in retinal endothelial cells via a novel cis-element.

PubMed

Felinski, Edward A; Cox, Amy E; Phillips, Brett E; Antonetti, David A

2008-06-01

Tight junctions between vascular endothelial cells help to create the blood-brain and blood-retinal barriers. Breakdown of the retinal tight junction complex is problematic in several disease states including diabetic retinopathy. Glucocorticoids can restore and/or preserve the endothelial barrier to paracellular permeability, although the mechanism remains unclear. We show that glucocorticoid treatment of primary retinal endothelial cells increases content of the tight junction proteins occludin and claudin-5, co-incident with an increase in barrier properties of endothelial monolayers. The glucocorticoid receptor antagonist RU486 reverses both the glucocorticoid-stimulated increase in occludin content and the increase in barrier properties. Transcriptional activity from the human occludin and claudin-5 promoters increases in retinal endothelial cells upon glucocorticoid treatment, and is dependent on the glucocorticoid receptor (GR) as demonstrated by siRNA. Deletion analysis of the occludin promoter reveals a 205bp sequence responsible for the glucocorticoid response. However, this region does not possess a canonical glucocorticoid response element and does not bind to the GR in a chromatin immunoprecipitation (ChIP) assay. Mutational analysis of this region revealed a novel 40bp occludin enhancer element (OEE), containing two highly conserved regions of 10 and 13 base pairs, that is both necessary and sufficient for glucocorticoid-induced gene expression in retinal endothelial cells. These data suggest a novel mechanism for glucocorticoid induction of vascular endothelial barrier properties through increased occludin and claudin-5 gene expression.

GLUCOCORTICOIDS INDUCE TRANSACTIVATION OF TIGHT JUNCTION GENES OCCLUDIN AND CLAUDIN-5 IN RETINAL ENDOTHELIAL CELLS VIA A NOVEL CIS-ELEMENT

PubMed Central

Felinski, Edward A.; Cox, Amy E.; Phillips, Brett E.; Antonetti, David A.

2008-01-01

Tight junctions between vascular endothelial cells help to create the blood-brain and blood-retinal barriers. Breakdown of the retinal tight junction complex is problematic in several disease states including diabetic retinopathy. Glucocorticoids can restore and/or preserve the endothelial barrier to paracellular permeability, although the mechanism remains unclear. We show that glucocorticoid treatment of primary retinal endothelial cells increases content of the tight junction proteins occludin and claudin-5, co-incident with an increase in barrier properties of endothelial monolayers. The glucocorticoid receptor antagonist RU486 reverses both the glucocorticoid-stimulated increase in occludin content and the increase in barrier properties. Transcriptional activity from the human occludin and claudin-5 promoters increases in retinal endothelial cells upon glucocorticoid treatment, and is dependent on the glucocorticoid receptor (GR) as demonstrated by siRNA. Deletion analysis of the occludin promoter reveals a 205 bp sequence responsible for the glucocorticoid response. However, this region does not posses a canonical glucocorticoid response element and does not bind to the GR in a chromatin immunoprecipitation (ChIP) assay. Mutational analysis of this region revealed a novel 40 bp occludin enhancer element (OEE), containing two highly-conserved regions of 10 and 13 base pairs, that is both necessary and sufficient for glucocorticoid-induced gene expression in retinal endothelial cells. These data suggest a novel mechanism for glucocorticoid induction of vascular endothelial barrier properties through increased occludin and claudin-5 gene expression. PMID:18501346

Critical role of TRPP2 and TRPC1 channels in stretch-induced injury of blood-brain barrier endothelial cells.

PubMed

Berrout, Jonathan; Jin, Min; O'Neil, Roger G

2012-02-03

The microvessels of the brain are very sensitive to mechanical stresses such as observed in traumatic brain injury (TBI). Such stresses can quickly lead to dysfunction of the microvessel endothelial cells, including disruption of blood-brain barrier (BBB). It is now evident that elevation of cytosolic calcium levels ([Ca2+]i) can compromise the BBB integrity, however the mechanism by which mechanical injury can produce a [Ca2+]i increase in brain endothelial cells is unclear. To assess the effects of mechanical/stretch injury on [Ca2+]i signaling, mouse brain microvessel endothelial cells (bEnd3) were grown to confluency on elasticized membranes and [Ca2+]i monitored using fura 2 fluorescence imaging. Application of an injury, using a pressure/stretch pulse of 50 ms, induced a rapid transient increase in [Ca2+]i. In the absence of extracellular Ca2+, the injury-induced [Ca2+]i transient was greatly reduced, but not fully eliminated, while unloading of Ca2+ stores by thapsigargin treatment in the absence of extracellular Ca2+ abolished the injury transient. Application of LOE-908 and amiloride, TRPC and TRPP2 channel blockers, respectively, both reduced the transient [Ca2+]i increase. Further, siRNA knockdown assays directed at TRPC1 and TRPP2 expression also resulted in a reduction of the injury-induced [Ca2+]i response. In addition, stretch injury induced increases of NO production and actin stress fiber formation, both of which were markedly reduced upon treatment with LOE908 and/or amiloride. We conclude that mechanical injury of brain endothelial cells induces a rapid influx of calcium, mediated by TRPC1 and TRPP2 channels, which leads to NO synthesis and actin cytoskeletal rearrangement. Copyright © 2011. Published by Elsevier B.V.

The expression of VE-cadherin in breast cancer cells modulates cell dynamics as a function of tumor differentiation and promotes tumor-endothelial cell interactions.

PubMed

Rezaei, Maryam; Cao, Jiahui; Friedrich, Katrin; Kemper, Björn; Brendel, Oliver; Grosser, Marianne; Adrian, Manuela; Baretton, Gustavo; Breier, Georg; Schnittler, Hans-Joachim

2018-01-01

The cadherin switch has profound consequences on cancer invasion and metastasis. The endothelial-specific vascular endothelial cadherin (VE-cadherin) has been demonstrated in diverse cancer types including breast cancer and is supposed to modulate tumor progression and metastasis, but underlying mechanisms need to be better understood. First, we evaluated VE-cadherin expression by tissue microarray in 392 cases of breast cancer tumors and found a diverse expression and distribution of VE-cadherin. Experimental expression of fluorescence-tagged VE-cadherin (VE-EGFP) in undifferentiated, fibroblastoid and E-cadherin-negative MDA-231 (MDA-VE-EGFP) as well as in differentiated E-cadherin-positive MCF-7 human breast cancer cell lines (MCF-VE-EGFP), respectively, displayed differentiation-dependent functional differences. VE-EGFP expression reversed the fibroblastoid MDA-231 cells to an epithelial-like phenotype accompanied by increased β-catenin expression, actin and vimentin remodeling, increased cell spreading and barrier function and a reduced migration ability due to formation of VE-cadherin-mediated cell junctions. The effects were largely absent in both MDA-VE-EGFP and in control MCF-EGFP cell lines. However, MCF-7 cells displayed a VE-cadherin-independent planar cell polarity and directed cell migration that both developed in MDA-231 only after VE-EGFP expression. Furthermore, VE-cadherin expression had no effect on tumor cell proliferation in monocultures while co-culturing with endothelial cells enhanced tumor cell proliferation due to integration of the tumor cells into monolayer where they form VE-cadherin-mediated cell contacts with the endothelium. We propose an interactive VE-cadherin-based crosstalk that might activate proliferation-promoting signals. Together, our study shows a VE-cadherin-mediated cell dynamics and an endothelial-dependent proliferation in a differentiation-dependent manner.

Carbohydrate restriction with postmeal walking effectively mitigates postprandial hyperglycemia and improves endothelial function in type 2 diabetes.

PubMed

Francois, Monique E; Myette-Cote, Etienne; Bammert, Tyler D; Durrer, Cody; Neudorf, Helena; DeSouza, Christopher A; Little, Jonathan P

2018-01-01

Postprandial hyperglycemia has deleterious effects on endothelial function. Restricting carbohydrate intake and postmeal walking have each been shown to reduce postprandial hyperglycemia, but their combination and subsequent effects on endothelial function have not been investigated. Here, we sought to examine the effect of blunting postprandial hyperglycemia by following a low-carbohydrate diet, with or without postmeal walking exercise, on markers of vascular health in type 2 diabetes (T2D). In a randomized crossover design, individuals with T2D ( n = 11) completed three 4-day controlled diet interventions consisting of 1) low-carbohydrate diet alone (LC), 2) low-carbohydrate diet with 15-min postmeal walks (LC + Ex), and 3) low-fat control diet (CON). Fasting blood samples and brachial artery flow-mediated dilation (%FMD) were measured before and after each intervention. Total circulating microparticles (MPs), endothelial MPs, platelet MPs, monocyte-platelet aggregates, and adhesion molecules were assessed as biomarkers of vascular health. There was a significant condition × time interaction for %FMD ( P = 0.01), with post hoc tests revealing improved %FMD after LC + Ex (+0.8 ± 1.0%, P = 0.02), with no change after LC or CON. Endothelial MPs were significantly reduced with the LC diet by ~45% (from 99 ± 60 to 44 ± 31 MPs/μl, P = 0.02), with no change after LC + Ex or CON (interaction: P = 0.04). Total MPs were lower (main effect time: P = 0.02), whereas monocyte-platelet aggregates were higher (main effect time: P < 0.01) after all interventions. Plasma adhesion molecules and C-reactive protein were unaltered. Attenuating postprandial hyperglycemic excursions using a low-carbohydrate diet combined with postmeal walking appears to be an effective strategy to improve endothelial function in individuals with T2D. NEW & NOTEWORTHY Carbohydrate restriction and postmeal walking lower postprandial hyperglycemia in individuals with type 2 diabetes. Here, we show

Fingolimod promotes blood-nerve barrier properties in vitro.

PubMed

Nishihara, Hideaki; Maeda, Toshihiko; Sano, Yasuteru; Ueno, Maho; Okamoto, Nana; Takeshita, Yukio; Shimizu, Fumitaka; Koga, Michiaki; Kanda, Takashi

2018-04-01

The main effect of fingolimod is thought to be functional antagonism of lymphocytic S1P1 receptors and the prevention of lymphocyte egress from lymphoid tissues, thereby reducing lymphocyte infiltration into the nervous system. However, a growing number of reports suggest that fingolimod also has a direct effect on several cell types in the nervous system. Although we previously reported that fingolimod enhances blood-brain barrier (BBB) functions, there have been no investigations regarding the blood-nerve barrier (BNB). In this study, we examine how fingolimod affects the BNB. An immortalized human peripheral nerve microvascular endothelial cell line (HPnMEC) was used to evaluate BNB barrier properties. We examined tight junction proteins and barrier functions of HPnMECs in conditioned medium with or without fingolimod-phosphate and blood sera from patients with typical chronic inflammatory demyelinating polyneuropathy (CIDP). Incubation with fingolimod-phosphate increased levels of claudin-5 mRNA and protein as well as TEER values in HPnMECs. Conversely, typical CIDP sera decreased claudin-5 mRNA/protein levels and TEER values in HPnMECs; however, pretreatment with fingolimod-phosphate inhibited the effects of the typical CIDP sera. Fingolimod-phosphate directly modifies the BNB and enhances barrier properties. This mechanism may be a viable therapeutic target for CIDP, and fingolimod may be useful in patients with typical CIDP who have severe barrier disruption.

Exposure to Lipopolysaccharide and/or Unconjugated Bilirubin Impair the Integrity and Function of Brain Microvascular Endothelial Cells

PubMed Central

Cardoso, Filipa L.; Kittel, Ágnes; Veszelka, Szilvia; Palmela, Inês; Tóth, Andrea; Brites, Dora; Deli, Mária A.; Brito, Maria A.

2012-01-01

Background Sepsis and jaundice are common conditions in newborns that can lead to brain damage. Though lipopolysaccharide (LPS) is known to alter the integrity of the blood-brain barrier (BBB), little is known on the effects of unconjugated bilirubin (UCB) and even less on the joint effects of UCB and LPS on brain microvascular endothelial cells (BMEC). Methodology/Principal Findings Monolayers of primary rat BMEC were treated with 1 µg/ml LPS and/or 50 µM UCB, in the presence of 100 µM human serum albumin, for 4 or 24 h. Co-cultures of BMEC with astroglial cells, a more complex BBB model, were used in selected experiments. LPS led to apoptosis and UCB induced both apoptotic and necrotic-like cell death. LPS and UCB led to inhibition of P-glycoprotein and activation of matrix metalloproteinases-2 and -9 in mono-cultures. Transmission electron microscopy evidenced apoptotic bodies, as well as damaged mitochondria and rough endoplasmic reticulum in BMEC by either insult. Shorter cell contacts and increased caveolae-like invaginations were noticeable in LPS-treated cells and loss of intercellular junctions was observed upon treatment with UCB. Both compounds triggered impairment of endothelial permeability and transendothelial electrical resistance both in mono- and co-cultures. The functional changes were confirmed by alterations in immunostaining for junctional proteins β-catenin, ZO-1 and claudin-5. Enlargement of intercellular spaces, and redistribution of junctional proteins were found in BMEC after exposure to LPS and UCB. Conclusions LPS and/or UCB exert direct toxic effects on BMEC, with distinct temporal profiles and mechanisms of action. Therefore, the impairment of brain endothelial integrity upon exposure to these neurotoxins may favor their access to the brain, thus increasing the risk of injury and requiring adequate clinical management of sepsis and jaundice in the neonatal period. PMID:22586454

Long-term effects of bariatric surgery on peripheral endothelial function and coronary microvascular function.

PubMed

Tarzia, Pierpaolo; Lanza, Gaetano A; Sestito, Alfonso; Villano, Angelo; Russo, Giulio; Figliozzi, Stefano; Lamendola, Priscilla; De Vita, Antonio; Crea, Filippo

We previously demonstrated that bariatric surgery (BS) leads to a short-term significant improvement of endothelial function and coronary microvascular function. In this study we assessed whether BS maintains its beneficial effect at long-term follow up. We studied 19 morbidly obese patients (age 43±9years, 12 women) without any evidence of cardiovascular disease who underwent BS. Patients were studied before BS, at 3 months and at 4.0±1.5years follow up. Peripheral vascular function was assessed by flow-mediated dilation (FMD) and nitrate-mediated dilation (NMD), i.e., brachial artery diameter changes in response to post-ischemic forearm hyperhaemia and to nitroglycerin administration, respectively. Coronary microvascular function was assessed by measuring coronary blood flow (CBF) response to intravenous adenosine and to cold pressor test (CPT) in the left anterior descending coronary artery. Together with improvement of anthropometric and metabolic profile, at long-term follow-up patients showed a significant improvement of FMD (6.43±2.88 vs. 8.21±1.73%, p=0.018), and CBF response to both adenosine (1.73±0.48 vs. 2.58±0.54; p<0.01) and CPT (1.43±0.30 vs. 2.23±0.48; p<0.01), compared to basal values. No differences in vascular end-points were shown at 3-month and 4-year follow-up after BS. Our data show that, in morbidly obese patients, BS exerts beneficial and long lasting effects on peripheral endothelial function and on coronary microvascular dilator function. Copyright © 2016 Asia Oceania Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.

Quercetin, Hyperin, and Chlorogenic Acid Improve Endothelial Function by Antioxidant, Antiinflammatory, and ACE Inhibitory Effects.

PubMed

Huang, Wu-Yang; Fu, Lin; Li, Chun-Yang; Xu, Li-Ping; Zhang, Li-Xia; Zhang, Wei-Min

2017-05-01

In recent years, the blueberry cultivation and processing industry developed quickly because blueberries are super-fruit with healthy function. Blueberry leaves are byproducts of the blueberry industry, which are rich in bioactive phenolics, such as quercetin (Q), hyperin (H), and chlorogenic acid (C). This study investigated protective effects of 3 phenolics (Q, H, and C) from leaves of rabbiteye blueberry Vaccinium ashei on human umbilical vein endothelial cells. The results showed that all these 3 phenolics could improve endothelial function by inhibiting oxidative damage and proinflammatory cytokines caused by tumor necrosis factor-α (TNF-α). The cell vitalities of endothelial cells pretreated with Q, H, and C were higher than those stimulated with TNF-α only. These phenolics could decrease reactive oxygen species and xanthine oxidase-1 levels and increase superoxide dismutase and heme oxygenase-1 levels in endothelial cells. They also could decrease the protein expressions of intercellular adhesion molecule-1, vascular cell adhesion molecule-1, and monocyte chemotactic protein-1 induced by TNF-α. In addition, Q, H, and C also exhibited vasodilatory effect by reducing the angiotensin I-converting enzyme (ACE) protein levels in endothelial cells. Mostly 3 phenolics exhibited bioactivities as a function of concentration, but the effects not always depended on the concentration. The antioxidant and antiinflammatory effects of Q seemed to be more pronounced than H; however, H exhibited higher cell vitalities. The results indicated that phenolics from rabbiteye blueberry leaves could be potential antioxidants, inflammation and ACE inhibitors, and rabbiteye blueberry leaves provide a new resources of phytochemicals beneficial for cardiovascular health. © 2017 Institute of Food Technologists®.

Magnetic forces enable controlled drug delivery by disrupting endothelial cell-cell junctions

NASA Astrophysics Data System (ADS)

Qiu, Yongzhi; Tong, Sheng; Zhang, Linlin; Sakurai, Yumiko; Myers, David R.; Hong, Lin; Lam, Wilbur A.; Bao, Gang

2017-06-01

The vascular endothelium presents a major transport barrier to drug delivery by only allowing selective extravasation of solutes and small molecules. Therefore, enhancing drug transport across the endothelial barrier has to rely on leaky vessels arising from disease states such as pathological angiogenesis and inflammatory response. Here we show that the permeability of vascular endothelium can be increased using an external magnetic field to temporarily disrupt endothelial adherens junctions through internalized iron oxide nanoparticles, activating the paracellular transport pathway and facilitating the local extravasation of circulating substances. This approach provides a physically controlled drug delivery method harnessing the biology of endothelial adherens junction and opens a new avenue for drug delivery in a broad range of biomedical research and therapeutic applications.

Blueberries improve endothelial function, but not blood pressure, in adults with metabolic syndrome: a randomized, double-blind, placebo-controlled clinical trial.

PubMed

Stull, April J; Cash, Katherine C; Champagne, Catherine M; Gupta, Alok K; Boston, Raymond; Beyl, Robbie A; Johnson, William D; Cefalu, William T

2015-05-27

Blueberry consumption has been shown to have various health benefits in humans. However, little is known about the effect of blueberry consumption on blood pressure, endothelial function and insulin sensitivity in humans. The present study investigated the role of blueberry consumption on modifying blood pressure in subjects with metabolic syndrome. In addition, endothelial function and insulin sensitivity (secondary measurements) were also assessed. A double-blind and placebo-controlled study was conducted in 44 adults (blueberry, n = 23; and placebo, n = 21). They were randomized to receive a blueberry or placebo smoothie twice daily for six weeks. Twenty-four-hour ambulatory blood pressure, endothelial function and insulin sensitivity were assessed pre- and post-intervention. The blood pressure and insulin sensitivity did not differ between the blueberry and placebo groups. However, the mean change in resting endothelial function, expressed as reactive hyperemia index (RHI), was improved significantly more in the group consuming the blueberries versus the placebo group (p = 0.024). Even after adjusting for confounding factors, i.e., the percent body fat and gender, the blueberry group still had a greater improvement in endothelial function when compared to their counterpart (RHI; 0.32 ± 0.13 versus -0.33 ± 0.14; p = 0.0023). In conclusion, daily dietary consumption of blueberries did not improve blood pressure, but improved (i.e., increased) endothelial function over six weeks in subjects with metabolic syndrome.

Nanomechanics and Sodium Permeability of Endothelial Surface Layer Modulated by Hawthorn Extract WS 1442

PubMed Central

Peters, Wladimir; Drueppel, Verena; Kusche-Vihrog, Kristina; Schubert, Carola; Oberleithner, Hans

2012-01-01

The endothelial glycocalyx (eGC) plays a pivotal role in the physiology of the vasculature. By binding plasma proteins, the eGC forms the endothelial surface layer (ESL) which acts as an interface between bloodstream and endothelial cell surface. The functions of the eGC include mechanosensing of blood flow induced shear stress and thus flow dependent vasodilation. There are indications that levels of plasma sodium concentrations in the upper range of normal and beyond impair flow dependent regulation of blood pressure and may therefore increase the risk for hypertension. Substances, therefore, that prevent sodium induced endothelial dysfunction may be attractive for the treatment of cardiovascular disease. By means of combined atomic force - epifluorescence microscopy we studied the impact of the hawthorn (Crataegus spp.) extract WS 1442, a herbal therapeutic with unknown mechanism of action, on the mechanics of the ESL of ex vivo murine aortae. Furthermore, we measured the impact of WS 1442 on the sodium permeability of endothelial EA.hy 926 cell monolayer. The data show that (i) the ESL contributes by about 11% to the total endothelial barrier resistance for sodium and (ii) WS 1442 strengthens the ESL resistance for sodium up to about 45%. This mechanism may explain some of the vasoprotective actions of this herbal therapeutic. PMID:22253842

Nanomechanics and sodium permeability of endothelial surface layer modulated by hawthorn extract WS 1442.

PubMed

Peters, Wladimir; Drüppel, Verena; Drueppel, Verena; Kusche-Vihrog, Kristina; Schubert, Carola; Oberleithner, Hans

2012-01-01

The endothelial glycocalyx (eGC) plays a pivotal role in the physiology of the vasculature. By binding plasma proteins, the eGC forms the endothelial surface layer (ESL) which acts as an interface between bloodstream and endothelial cell surface. The functions of the eGC include mechanosensing of blood flow induced shear stress and thus flow dependent vasodilation. There are indications that levels of plasma sodium concentrations in the upper range of normal and beyond impair flow dependent regulation of blood pressure and may therefore increase the risk for hypertension. Substances, therefore, that prevent sodium induced endothelial dysfunction may be attractive for the treatment of cardiovascular disease. By means of combined atomic force-epifluorescence microscopy we studied the impact of the hawthorn (Crataegus spp.) extract WS 1442, a herbal therapeutic with unknown mechanism of action, on the mechanics of the ESL of ex vivo murine aortae. Furthermore, we measured the impact of WS 1442 on the sodium permeability of endothelial EA.hy 926 cell monolayer. The data show that (i) the ESL contributes by about 11% to the total endothelial barrier resistance for sodium and (ii) WS 1442 strengthens the ESL resistance for sodium up to about 45%. This mechanism may explain some of the vasoprotective actions of this herbal therapeutic.

Role of the Retinal Vascular Endothelial Cell in Ocular Disease

PubMed Central

Bharadwaj, Arpita S.; Appukuttan, Binoy; Wilmarth, Phillip A.; Pan, Yuzhen; Stempel, Andrew J.; Chipps, Timothy J.; Benedetti, Eric E.; Zamora, David O.; Choi, Dongseok; David, Larry L.; Smith, Justine R.

2012-01-01

Retinal endothelial cells line the arborizing microvasculature that supplies and drains the neural retina. The anatomical and physiological characteristics of these endothelial cells are consistent with nutritional requirements and protection of a tissue critical to vision. On the one hand, the endothelium must ensure the supply of oxygen and other nutrients to the metabolically active retina, and allow access to circulating cells that maintain the vasculature or survey the retina for the presence of potential pathogens. On the other hand, the endothelium contributes to the blood-retinal barrier that protects the retina by excluding circulating molecular toxins, microorganisms, and pro-inflammatory leukocytes. Features required to fulfill these functions may also predispose to disease processes, such as retinal vascular leakage and neovascularization, and trafficking of microbes and inflammatory cells. Thus, the retinal endothelial cell is a key participant in retinal ischemic vasculopathies that include diabetic retinopathy and retinopathy of prematurity, and retinal inflammation or infection, as occurs in posterior uveitis. Using gene expression and proteomic profiling, it has been possible to explore the molecular phenotype of the human retinal endothelial cell and contribute to understanding of the pathogenesis of these diseases. In addition to providing support for the involvement of well-characterized endothelial molecules, profiling has the power to identify new players in retinal pathologies. Findings may have implications for the design of new biological therapies. Additional progress in this field is anticipated as other technologies, including epigenetic profiling methods, whole transcriptome shotgun sequencing, and metabolomics, are used to study the human retinal endothelial cell. PMID:22982179

An In Vitro Model of the Blood-Brain Barrier: Naegleria fowleri Affects the Tight Junction Proteins and Activates the Microvascular Endothelial Cells.

PubMed

Coronado-Velázquez, Daniel; Betanzos, Abigail; Serrano-Luna, Jesús; Shibayama, Mineko

2018-04-14

Naegleria fowleri causes a fatal disease known as primary amoebic meningoencephalitis. This condition is characterized by an acute inflammation that originates from the free passage of peripheral blood cells to the central nervous system through the alteration of the blood-brain barrier. In this work, we established models of the infection in rats and in a primary culture of endothelial cells from rat brains with the aim of evaluating the activation and the alterations of these cells by N. fowleri. We proved that the rat develops the infection similar to the mouse model. We also found that amoebic cysteine proteases produced by the trophozoites and the conditioned medium induced cytopathic effect in the endothelial cells. In addition, N. fowleri can decrease the transendothelial electrical resistance by triggering the destabilization of the tight junction proteins claudin-5, occludin, and ZO-1 in a time-dependent manner. Furthermore, N. fowleri induced the expression of VCAM-1 and ICAM-1 and the production of IL-8, IL-1β, TNF-α, and IL-6 as well as nitric oxide. We conclude that N. fowleri damaged the blood-brain barrier model by disrupting the intercellular junctions and induced the presence of inflammatory mediators by allowing the access of inflammatory cells to the olfactory bulbs. © 2018 The Author(s) Journal of Eukaryotic Microbiology © 2018 International Society of Protistologists.

Relation of endothelial function to cardiovascular risk in women with sedentary occupations and without known cardiovascular disease.

PubMed

Lippincott, Margaret F; Carlow, Andrea; Desai, Aditi; Blum, Arnon; Rodrigo, Maria; Patibandla, Sushmitha; Zalos, Gloria; Smith, Kevin; Schenke, William H; Csako, Gyorgy; Waclawiw, Myron A; Cannon, Richard O

2008-08-01

Our purpose was to determine predictors of endothelial function and potential association with cardiovascular risk in women with sedentary occupations, in whom obesity-associated risk factors may contribute to excess morbidity and mortality. Ninety consecutive women (age range 22 to 63 years, 22 overweight (body mass index [BMI] > or =25 to 29.9 kg/m(2)) and 42 obese (BMI > or = 30 kg/m(2)), had vital signs, lipids, insulin, glucose, high-sensitivity C-reactive protein, and sex hormones measured. Endothelial function was determined using brachial artery flow-mediated dilation after 5 minutes of forearm ischemia. Treadmill stress testing was performed with gas exchange analysis at peak exercise (peak oxygen consumption [Vo(2)]) to assess cardiorespiratory fitness. Brachial artery reactivity was negatively associated with Framingham risk score (r = -0.3542, p = 0.0007). Univariate predictors of endothelial function included peak Vo(2) (r = 0.4483, p <0.0001), age (r = -0.3420, p = 0.0010), BMI (r = -0.3065, p = 0.0035), and high-sensitivity C-reactive protein (r = -0.2220, p = 0.0400). Using multiple linear regression analysis with stepwise modeling, peak Vo(2) (p = 0.0003) was the best independent predictor of brachial artery reactivity, with age as the only other variable reaching statistical significance (p = 0.0436) in this model. In conclusion, endothelial function was significantly associated with cardiovascular risk in women with sedentary occupations, who were commonly overweight or obese. Even in the absence of routine exercise, cardiorespiratory fitness, rather than conventional risk factors or body mass, is the dominant predictor of endothelial function and suggests a modifiable approach to risk.

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

A Two-Tier Golgi-Based Control of Organelle Size Underpins the Functional Plasticity of Endothelial Cells

PubMed Central

Ferraro, Francesco; Kriston-Vizi, Janos; Metcalf, Daniel J.; Martin-Martin, Belen; Freeman, Jamie; Burden, Jemima J.; Westmoreland, David; Dyer, Clare E.; Knight, Alex E.; Ketteler, Robin; Cutler, Daniel F.

2014-01-01

Summary Weibel-Palade bodies (WPBs), endothelial-specific secretory granules that are central to primary hemostasis and inflammation, occur in dimensions ranging between 0.5 and 5 μm. How their size is determined and whether it has a functional relevance are at present unknown. Here, we provide evidence for a dual role of the Golgi apparatus in controlling the size of these secretory carriers. At the ministack level, cisternae constrain the size of nanostructures (“quanta”) of von Willebrand factor (vWF), the main WPB cargo. The ribbon architecture of the Golgi then allows copackaging of a variable number of vWF quanta within the continuous lumen of the trans-Golgi network, thereby generating organelles of different sizes. Reducing the WPB size abates endothelial cell hemostatic function by drastically diminishing platelet recruitment, but, strikingly, the inflammatory response (the endothelial capacity to engage leukocytes) is unaltered. Size can thus confer functional plasticity to an organelle by differentially affecting its activities. PMID:24794632

Force control of endothelium permeability in mechanically stressed pulmonary micro-vascular endothelial cells.

PubMed

Wang, Bin; Caluch, Adam; Fodil, Redouane; Féréol, Sophie; Zadigue, Patricia; Pelle, Gabriel; Louis, Bruno; Isabey, Daniel

2012-01-01

Mechanical factors play a key role in the pathogenesis of Acute Respiratory Distress Syndrome (ARDS) and Ventilator-Induced Lung Injury (VILI) as contributing to alveolo-capillary barrier dysfunction. This study aims at elucidating the role of the cytoskeleton (CSK) and cell-matrix adhesion system in the stressed endothelium and more precisely in the loss of integrity of the endothelial barrier. We purposely develop a cellular model made of a monolayer of confluent Human Pulmonary Microvascular Endothelial Cells (HPMVECs) whose cytoskeleton (CSK) is directly exposed to sustained cyclic mechanical stress for 1 and 2 h. We used RGD-coated ferromagnetic beads and measured permeability before and after stress application. We find that endothelial permeability increases in the stressed endothelium, hence reflecting a loss of integrity. Structural and mechanical results suggest that this endothelial barrier alteration would be due to physically-founded discrepancies in latero-basal reinforcement of adhesion sites in response to the global increase in CSK stiffness or centripetal intracellular forces. Basal reinforcement of adhesion is presently evidenced by the marked redistribution of αvβ3 integrin with cluster formation in the stressed endothelium.

Endothelial Function and Serum Concentration of Toxic Metals in Frequent Consumers of Fish

PubMed Central

Buscemi, Silvio; Vasto, Sonya; Di Gaudio, Francesca; Grosso, Giuseppe; Bergante, Sonia; Galvano, Fabio; Massenti, Fatima Maria; Amodio, Emanuele; Rosafio, Giuseppe; Verga, Salvatore

2014-01-01

Background Endothelial dysfunction is involved in the pathogenesis of atherosclerosis. Consumption of fish is associated with reduced cardiovascular risk, but there is paucity of data concerning its effect on endothelial function. Furthermore, investigation of the effects of fish consumption on health must take into account the ingestion of contaminants, including transition metals and some metalloids, which may have unfavorable effects on health, including those on the cardiovascular system. We investigated the association between fish consumption, endothelial function (flow mediated dilation of the brachial artery), and serum concentration of some toxic metals in apparently healthy people. Methods Twenty-nine high fish consumers (at least 3 portions a week) were compared with 25 low fish consumers (less than 1 portion a week). All participants were free of diabetes, cardiovascular or other systemic diseases. Serum metal (antimonium, arsenic, mercury, lead, cobalt, copper, zinc, selenium, strontium) concentrations were measured in subgroups of 24 high fish consumers and 19 low fish consumers. Results Both groups exhibited similar habitual dietary patterns, age and anthropometric characteristics. The high fish consumers had higher flow mediated dilation (9.7±1.8 vs. 7.3±1.9%; P<0.001), but also higher serum concentrations of mercury (5.87±2.69 vs. 1.65±1.10 mcg/L; P<0.001) and arsenic (6.04±3.25 vs. 2.30±1.58 mcg/L; P<0.001). The fasting plasma glucose concentrations were significantly correlated with both mercury (r = 0.39; P = 0.01) and arsenic concentrations (r = 0.55; P<0.001). Conclusions Habitual consumption of high amounts of fish is associated with better endothelial function despite higher serum concentrations of mercury and arsenic. PMID:25401695

Endothelial Cells Derived from the Blood-Brain Barrier and Islets of Langerhans Differ in their Response to the Effects of Bilirubin on Oxidative Stress Under Hyperglycemic Conditions.

PubMed

Kapitulnik, Jaime; Benaim, Clara; Sasson, Shlomo

2012-01-01

Unconjugated bilirubin (UCB) is a neurotoxic degradation product of heme. Its toxic effects include induction of apoptosis, and ultimately neuronal cell death. However, at low concentrations, UCB is a potent antioxidant that may protect cells and tissues against oxidative stress by neutralizing toxic metabolites such as reactive oxygen species (ROS). High glucose levels (hyperglycemia) generate reactive metabolites. Endothelial cell dysfunction, an early vascular complication in diabetes, has been associated with hyperglycemia-induced oxidative stress. Both glucose and UCB are substrates for transport proteins in microvascular endothelial cells of the blood-brain barrier (BBB). In the current study we show that UCB (1-40 μM) induces apoptosis and reduces survival of bEnd3 cells, a mouse brain endothelial cell line which serves as an in vitro model of the BBB. These deleterious effects of UCB were enhanced in the presence of high glucose (25 mM) levels. Interestingly, the bEnd3 cells exhibited an increased sensitivity to the apoptotic effects of UCB when compared to the MS1 microcapillary endothelial cell line. MS1 cells originate from murine pancreatic islets of Langerhans, and are devoid of the barrier characteristics of BBB-derived endothelial cells. ROS production was increased in both bEnd3 and MS1 cells exposed to high glucose, as compared with cells exposed to normal (5.5 mM) glucose levels. While UCB (0.1-40 μM) did not alter ROS production in cells exposed to normal glucose, relatively low ("physiological") UCB concentrations (0.1-5 μM) attenuated ROS generation in both cell lines exposed to high glucose levels. Most strikingly, higher UCB concentrations (20-40 μM) increased ROS generation in bEnd3 cells exposed to high glucose, but not in similarly treated MS1 cells. These results may be of critical importance for understanding the vulnerability of the BBB endothelium upon exposure to increasing UCB levels under hyperglycemic conditions.

Endothelial Cells Derived from the Blood-Brain Barrier and Islets of Langerhans Differ in their Response to the Effects of Bilirubin on Oxidative Stress Under Hyperglycemic Conditions

PubMed Central

Kapitulnik, Jaime; Benaim, Clara; Sasson, Shlomo

2012-01-01

Unconjugated bilirubin (UCB) is a neurotoxic degradation product of heme. Its toxic effects include induction of apoptosis, and ultimately neuronal cell death. However, at low concentrations, UCB is a potent antioxidant that may protect cells and tissues against oxidative stress by neutralizing toxic metabolites such as reactive oxygen species (ROS). High glucose levels (hyperglycemia) generate reactive metabolites. Endothelial cell dysfunction, an early vascular complication in diabetes, has been associated with hyperglycemia-induced oxidative stress. Both glucose and UCB are substrates for transport proteins in microvascular endothelial cells of the blood-brain barrier (BBB). In the current study we show that UCB (1–40 μM) induces apoptosis and reduces survival of bEnd3 cells, a mouse brain endothelial cell line which serves as an in vitro model of the BBB. These deleterious effects of UCB were enhanced in the presence of high glucose (25 mM) levels. Interestingly, the bEnd3 cells exhibited an increased sensitivity to the apoptotic effects of UCB when compared to the MS1 microcapillary endothelial cell line. MS1 cells originate from murine pancreatic islets of Langerhans, and are devoid of the barrier characteristics of BBB-derived endothelial cells. ROS production was increased in both bEnd3 and MS1 cells exposed to high glucose, as compared with cells exposed to normal (5.5 mM) glucose levels. While UCB (0.1–40 μM) did not alter ROS production in cells exposed to normal glucose, relatively low (“physiological”) UCB concentrations (0.1–5 μM) attenuated ROS generation in both cell lines exposed to high glucose levels. Most strikingly, higher UCB concentrations (20–40 μM) increased ROS generation in bEnd3 cells exposed to high glucose, but not in similarly treated MS1 cells. These results may be of critical importance for understanding the vulnerability of the BBB endothelium upon exposure to increasing UCB levels under hyperglycemic

Acute effect of sidestream cigarette smoke extract on vascular endothelial function.

PubMed

Argacha, J F; Fontaine, D; Adamopoulos, D; Ajose, A; van de Borne, P; Fontaine, J; Berkenboom, G

2008-09-01

Acute exposure to passive smoking adversely affects vascular function by promoting oxidative stress and endothelial dysfunction. However, it is not known whether tobacco sidestream (SS) smoke has a greater deleterious effect on the endothelium than non-tobacco SS smoke and whether these effects are related to nicotinic endothelial stimulation. To test these hypotheses, endothelial-dependent relaxation and superoxide anion production were assessed in isolated rat aortas incubated with tobacco SS smoke, non-tobacco SS smoke, or pure nicotine. Tobacco SS smoke decreased the maximal relaxation to acetylcholine (Ach) from 79 +/- 6% to 57 +/- 7.3% (% inhibition of phenylephrine-induced plateau, P < 0.001) and increased superoxide anion production from 31 +/- 9.7 to 116 +/- 24 count/10 sec/mg (P < 0.01, lucigenin-enhanced chemiluminescence technique). The non-tobacco SS smoke extract had no significant effect on the response to Ach but increased superoxide anion production in the aortic wall to 133 +/- 2 count/10 sec/mg (P < 0.001). Furthermore, concentration-response curves to Ach and superoxide production remained unaltered with nicotine (0.001, 0.01, or 0.1 mM). In conclusion, despite similar increases in vascular wall superoxide production with tobacco and non-tobacco SS smoke, only the tobacco SS smoke extracts affected endothelium-dependent vasorelaxation. Nicotine alone does not reproduce the effects seen with tobacco SS smoke, suggesting that the acute endothelial toxicity of passive smoking cannot simply be ascribed to a nicotine-dependent mechanism.

Coniferyl Aldehyde Attenuates Radiation Enteropathy by Inhibiting Cell Death and Promoting Endothelial Cell Function

PubMed Central

Son, Yeonghoon; Jang, Jun-Ho; Lee, Yoon-Jin; Kim, Sung-Ho; Ko, Young-Gyo; Lee, Yun-Sil; Lee, Hae-June

2015-01-01

Radiation enteropathy is a common complication in cancer patients. The aim of this study was to investigate whether radiation-induced intestinal injury could be alleviated by coniferyl aldehyde (CA), an HSF1-inducing agent that increases cellular HSP70 expression. We systemically administered CA to mice with radiation enteropathy following abdominal irradiation (IR) to demonstrate the protective effects of CA against radiation-induced gastrointestinal injury. CA clearly alleviated acute radiation-induced intestinal damage, as reflected by the histopathological data and it also attenuated sub-acute enteritis. CA prevented intestinal crypt cell death and protected the microvasculature in the lamina propria during the acute and sub-acute phases of damage. CA induced HSF1 and HSP70 expression in both intestinal epithelial cells and endothelial cells in vitro. Additionally, CA protected against not only the apoptotic cell death of both endothelial and epithelial cells but also the loss of endothelial cell function following IR, indicating that CA has beneficial effects on the intestine. Our results provide novel insight into the effects of CA and suggest its role as a therapeutic candidate for radiation-induced enteropathy due to its ability to promote rapid re-proliferation of the intestinal epithelium by the synergic effects of the inhibition of cell death and the promotion of endothelial cell function. PMID:26029925

In Vitro Endothelialization Test of Biomaterials Using Immortalized Endothelial Cells.

PubMed

Kono, Ken; Hiruma, Hitomi; Kobayashi, Shingo; Sato, Yoji; Tanaka, Masaru; Sawada, Rumi; Niimi, Shingo

2016-01-01

Functionalizing biomaterials with peptides or polymers that enhance recruitment of endothelial cells (ECs) can reduce blood coagulation and thrombosis. To assess endothelialization of materials in vitro, primary ECs are generally used, although the characteristics of these cells vary among the donors and change with time in culture. Recently, primary cell lines immortalized by transduction of simian vacuolating virus 40 large T antigen or human telomerase reverse transcriptase have been developed. To determine whether immortalized ECs can substitute for primary ECs in material testing, we investigated endothelialization on biocompatible polymers using three lots of primary human umbilical vein endothelial cells (HUVEC) and immortalized microvascular ECs, TIME-GFP. Attachment to and growth on polymer surfaces were comparable between cell types, but results were more consistent with TIME-GFP. Our findings indicate that TIME-GFP is more suitable for in vitro endothelialization testing of biomaterials.

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

Gpr124 is essential for blood-brain barrier integrity in central nervous system disease.

PubMed

Chang, Junlei; Mancuso, Michael R; Maier, Carolina; Liang, Xibin; Yuki, Kanako; Yang, Lu; Kwong, Jeffrey W; Wang, Jing; Rao, Varsha; Vallon, Mario; Kosinski, Cynthia; Zhang, J J Haijing; Mah, Amanda T; Xu, Lijun; Li, Le; Gholamin, Sharareh; Reyes, Teresa F; Li, Rui; Kuhnert, Frank; Han, Xiaoyuan; Yuan, Jenny; Chiou, Shin-Heng; Brettman, Ari D; Daly, Lauren; Corney, David C; Cheshier, Samuel H; Shortliffe, Linda D; Wu, Xiwei; Snyder, Michael; Chan, Pak; Giffard, Rona G; Chang, Howard Y; Andreasson, Katrin; Kuo, Calvin J

2017-04-01

Although blood-brain barrier (BBB) compromise is central to the etiology of diverse central nervous system (CNS) disorders, endothelial receptor proteins that control BBB function are poorly defined. The endothelial G-protein-coupled receptor (GPCR) Gpr124 has been reported to be required for normal forebrain angiogenesis and BBB function in mouse embryos, but the role of this receptor in adult animals is unknown. Here Gpr124 conditional knockout (CKO) in the endothelia of adult mice did not affect homeostatic BBB integrity, but resulted in BBB disruption and microvascular hemorrhage in mouse models of both ischemic stroke and glioblastoma, accompanied by reduced cerebrovascular canonical Wnt-β-catenin signaling. Constitutive activation of Wnt-β-catenin signaling fully corrected the BBB disruption and hemorrhage defects of Gpr124-CKO mice, with rescue of the endothelial gene tight junction, pericyte coverage and extracellular-matrix deficits. We thus identify Gpr124 as an endothelial GPCR specifically required for endothelial Wnt signaling and BBB integrity under pathological conditions in adult mice. This finding implicates Gpr124 as a potential therapeutic target for human CNS disorders characterized by BBB disruption.

Gene expression profiles of brain endothelial cells during embryonic development at bulk and single-cell levels.

PubMed

Hupe, Mike; Li, Minerva Xueting; Kneitz, Susanne; Davydova, Daria; Yokota, Chika; Kele-Olovsson, Julianna; Hot, Belma; Stenman, Jan M; Gessler, Manfred

2017-07-11

The blood-brain barrier is a dynamic interface that separates the brain from the circulatory system, and it is formed by highly specialized endothelial cells. To explore the molecular mechanisms defining the unique nature of vascular development and differentiation in the brain, we generated high-resolution gene expression profiles of mouse embryonic brain endothelial cells using translating ribosome affinity purification and single-cell RNA sequencing. We compared the brain vascular translatome with the vascular translatomes of other organs and analyzed the vascular translatomes of the brain at different time points during embryonic development. Because canonical Wnt signaling is implicated in the formation of the blood-brain barrier, we also compared the brain endothelial translatome of wild-type mice with that of mice lacking the transcriptional cofactor β-catenin ( Ctnnb1 ). Our analysis revealed extensive molecular changes during the embryonic development of the brain endothelium. We identified genes encoding brain endothelium-specific transcription factors ( Foxf2 , Foxl2 , Foxq1 , Lef1 , Ppard , Zfp551 , and Zic3 ) that are associated with maturation of the blood-brain barrier and act downstream of the Wnt-β-catenin signaling pathway. Profiling of individual brain endothelial cells revealed substantial heterogeneity in the population. Nevertheless, the high abundance of Foxf2 , Foxq1 , Ppard , or Zic3 transcripts correlated with the increased expression of genes encoding markers of brain endothelial cell differentiation. Expression of Foxf2 and Zic3 in human umbilical vein endothelial cells induced the production of blood-brain barrier differentiation markers. This comprehensive data set may help to improve the engineering of in vitro blood-brain barrier models. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

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.