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Sample records for cardiac microvascular endothelial

  1. Expression and significance of fgl2 prothrombinase in cardiac microvascular endothelial cells of rats with type 2 diabetes.

    PubMed

    Ding, Yanping; Liu, Kun; Wang, Yan; Su, Guanhua; Deng, Heping; Zeng, Qiutang; Liao, Yuhua; Wang, Zhaohui

    2010-10-01

    Microthrombosis may be involved in the pathogenesis of cardiac microangiopathy due to diabetes. Recent studies have shown that fibrinogen-like protein 2 (fgl2) plays a pivotal role in microthrombosis in viral hepatitis, acute vascular xenograft rejection and cytokine-induced fetal loss syndrome. The current study was designed to examine the expression of fgl2 in microvascular endothelial cells and investigate the effects of microthrombi due to fgl2 on cardiac function and structure in rats with type 2 diabetes. Following induction of type 2 diabetes, 24 rats were observed dynamically. Fgl2 expression and related cardiac microthrombosis were examined. Local or circulating TNF-α was measured. Coronary flow (CF) per min was calculated as an index of cardiac microcirculation. Cardiac function and morphology were evaluated. It was found that Fgl2 was highly expressed in cardiac microvascular endothelial cells of rats with type 2 diabetes, which was promoted by local or circulating TNF-α. The Fgl2 expression was associated with cardiac hyaline microthrombosis. In parallel with the fgl2 expression, CF per min, cardiac diastolic or systolic function and cardiac morphology were aggravated to some extent. It was concluded that in rats with type 2 diabetes, microthrombosis due to fgl2 contributes to the impairment of cardiac diastolic or systolic function and morphological changes.

  2. Tongxinluo Reverses the Hypoxia-suppressed Claudin-9 in Cardiac Microvascular Endothelial Cells

    PubMed Central

    Liu, Kun; Wang, Xiu-Juan; Li, Yan-Ning; Li, Bin; Qi, Jin-Sheng; Zhang, Jing; Wang, Yu

    2016-01-01

    Background: Claudin-5, claudin-9, and claudin-11 are expressed in endothelial cells to constitute tight junctions, and their deficiency may lead to hyperpermeability, which is the initiating process and pathological basis of cardiovascular disease. Although tongxinluo (TXL) has satisfactory antianginal effects, whether and how it modulates claudin-5, claudin-9, and claudin-11 in hypoxia-stimulated human cardiac microvascular endothelial cells (HCMECs) have not been reported. Methods: In this study, HCMECs were stimulated with CoCl2 to mimic hypoxia and treated with TXL. First, the messenger RNA (mRNA) expression of claudin-5, claudin-9, and claudin-11 was confirmed. Then, the protein content and distribution of claudin-9, as well as cell morphological changes were evaluated after TXL treatment. Furthermore, the distribution and content histone H3K9 acetylation (H3K9ac) in the claudin-9 gene promoter, which guarantees transcriptional activation, were examined to explore the underlying mechanism, by which TXL up-regulates claudin-9 in hypoxia-stimulated HCMECs. Results: We found that hypoxia-suppressed claudin-9 gene expression in HCMECs (F = 7.244; P = 0.011) and the hypoxia-suppressed claudin-9 could be reversed by TXL (F = 61.911; P = 0.000), which was verified by its protein content changes (F = 29.142; P = 0.000). Moreover, high-dose TXL promoted the cytomembrane localization of claudin-9 in hypoxia-stimulated HCMECs, with attenuation of cell injury. Furthermore, high-dose TXL elevated the hypoxia-inhibited H3K9ac in the claudin-9 gene promoter (F = 37.766; P = 0.000), activating claudin-9 transcription. Conclusions: The results manifested that TXL reversed the hypoxia-suppressed claudin-9 by elevating H3K9ac in its gene promoter, playing protective roles in HCMECs. PMID:26879018

  3. Cardiotoxic drugs Herceptin and doxorubicin inhibit cardiac microvascular endothelial cell barrier formation resulting in increased drug permeability

    PubMed Central

    Wilkinson, Emma L.; Sidaway, James E.

    2016-01-01

    ABSTRACT Cardiotoxicity induced by anti-cancer therapeutics is a severe, and potentially fatal, adverse reaction of the heart in response to certain drugs. Current in vitro approaches to assess cardiotoxicity have focused on analysing cardiomyocytes. More recently it has become apparent that non-cardiomyocyte cells of the heart can potentially contribute to cardiotoxicity. Herceptin and doxorubicin are known to induce cardiotoxicity in the clinic. The effect of these drugs on the endothelial tight junction barrier was tested by analysing tight junction formation and zona occludens-1 (ZO-1) levels, revealing that Herceptin and doxorubicin are able to induce barrier perturbment and decrease barrier function in human cardiac microvascular endothelial cells (HCMECs) leading to increased permeability. Herceptin treatment had no effect on the tight junction barrier function in human dermal and human brain microvascular endothelial cells. HCMECs showed detectable levels of HER2 compared with the other endothelial cells suggesting that Herceptin binding to HER2 in these cells may interfere with tight junction formation. Our data suggests that doxorubicin and Herceptin can affect tight junction formation in the cardiac microvasculature leading to increased drug permeability and adverse effects on the cardiac myocytes. PMID:27543060

  4. [Knockdown of RUNX3 inhibits hypoxia-induced endothelial-to-mesenchymal transition of human cardiac microvascular endothelial cells].

    PubMed

    Liu, Yanhua; Li, Bingong; Wang, Yuqin; Wang, Delong; Zou, Jin; Ke, Xuan; Hao, Yanqin

    2016-12-01

    Objective To investigate the effects of Runt-related transcription factor 3 (RUNX3) knockdown on hypoxia-induced endothelial-to-mesenchymal transition (EndoMT) of human cardiac microvascular endothelial cells (HCMECs), and elucidate the underlying molecular mechanism. Methods HCMECs were cultured in hypoxic conditions and infected with RUNX3-RNAi lentivirus to knock-down the expression of RUNX3. Reverse transcription PCR was performed to detect the mRNA expressions of RUNX3 and EndoMT related genes such as CD31, vascular endothelial cadherin (VE-cadherin), α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP-1); Western blotting was used to determine the protein expressions of RUNX3, CD31, α-SMA and another molecules involved in EndoMT; and immunofluorescence cytochemistry was applied to observe the colocalization of CD31 and α-SMA. Results Hypoxia induced the transition of HCMECs to mesenchymal cells. Hypoxia up-regulated the expression of TGF-β2, Smad2/3, phosphorylation of Smad2/3 (p-Smad2/3), Notch-1, Hes1, and Hey1; knockdown of RUNX3 down-regulated the levels of Smad2/3, p-Smad2/3, Hes1, and Hey1 to different extents, and raised the levels of TGF-β2 and Notch-1. Conclusion Knockdown of RUNX3 in HCMECs attenuates hypoxia-induced EndoMT via partially inhibiting TGF-β and Notch signaling pathway.

  5. EGb 761 protects cardiac microvascular endothelial cells against hypoxia/reoxygenation injury and exerts inhibitory effect on ATM pathway.

    PubMed

    Zhang, Chao; Wang, Deng-Feng; Zhang, Zhuang; Han, Dong; Yang, Kan

    2016-12-14

    Ginkgo biloba extract (EGb 761) has been widely clinically used to reduce myocardial ischemia reperfusion injury (MIRI). Microvascular endothelial cells (MVECs) may be a proper cellular model in vitro for the effect and mechanism study against MIRI. However, the effect of EGb 761 on MVECs resisting hypoxia/reoxygenation (H/R) injury is little reported. In this study, H/R-injuried MVECs were treated with EGb 761, then cell viability, apoptosis, ROS production, SOD activity, caspase-3 activity, and the protein level of ATM, γ-H2AX, p53, Bax were measured. ATM siRNA was transfected to study the changes of protein in ATM pathway. EGb 761 presented protective effect on H/R-injuried MVECs with decreasing cell death, apoptosis and ROS, and elevated SOD activity. Next, EGb 761 could inhibit H/R-induced ATM, γ-H2AX, p53, Bax in a dose-dependent manner. Moreover, ATM siRNA also could inhibit H/R-induced ATM, γ-H2AX, p53, Bax. Overall, these findings verify EGb 761 protects cardiac MVECs from H/R injury, and for the first time, illustrate the influence on ATM pathway and apoptosis of EGb 761 via dampening ROS.

  6. Improvement of RNA fingerprinting efficiency for the analysis of differential gene expression in human cardiac macro- and microvascular endothelial cells.

    PubMed

    Bongrazio, M; Gräfe, M; Pries, A R; Gaehtgens, P; Zakrzewicz, A

    2001-06-01

    RNA fingerprinting by arbitrarily primed PCR (RAP-PCR) is a powerful tool to screen differential gene expression. However, PCR-based screening techniques show a high incidence of false positive results (40-90%). In order to increase the efficiency and feasibility of RAP-PCR, the original protocol was modified and applied to analyse differential gene expression in human coronary macro- (HCEC) and microvascular (HCMEC) endothelial cells. The major modifications introduced were: (i) the use of two primers for PCR amplification, instead of reverse-transcription primer alone; (ii) the use of three cycles at low stringency followed by further amplification at high stringency; (iii) optimization of amplification cycle number, template amount, and concentration of primers, dNTP, Mg(2+); (iv) detection of fingerprints by silver staining; and (v) direct sequencing using RAP-PCR primers. Analysis of untreated and TNF alpha -stimulated (100 U ml(-1)for 1, 4, and 24 h) HCEC and HCMEC displayed 11 differentially expressed products by 18 primer combinations. Confirmation of results by RT-PCR showed that the rate of false positives attributable to our screening method was less than 20%. Among detected RAP-PCR products, the expression of Mn-superoxide dismutase, A20 zinc finger protein, and three novel genes (A/a, 4/d, 7/c) was more strongly modulated by TNF in HCEC than HCMEC. A further novel gene (B/e) was strongly expressed in HCMEC while only barely detectable in HCEC. In conclusion, modification of RAP-PCR strongly reduced the incidence of false positives, eliminated a radioactive requirement, and allowed sequencing without prior cloning, supplying an improved technology able to identify new differentially expressed genes between macro- and microvascular endothelial cells.

  7. Glucagon-like peptide-1 preserves coronary microvascular endothelial function after cardiac arrest and resuscitation: potential antioxidant effects.

    PubMed

    Dokken, Betsy B; Piermarini, Charles V; Teachey, Mary K; Gura, Michael T; Dameff, Christian J; Heller, Brian D; Krate, Jonida; Ashgar, Aeen M; Querin, Lauren; Mitchell, Jennifer L; Hilwig, Ronald W; Kern, Karl B

    2013-02-15

    Glucagon-like peptide-1 (GLP-1) has protective effects in the heart. We hypothesized that GLP-1 would mitigate coronary microvascular and left ventricular (LV) dysfunction if administered after cardiac arrest and resuscitation (CAR). Eighteen swine were subjected to ventricular fibrillation followed by resuscitation. Swine surviving to return of spontaneous circulation (ROSC) were randomized to receive an intravenous infusion of either human rGLP-1 (10 pmol·kg(-1)·min(-1); n = 8) or 0.9% saline (n = 8) for 4 h, beginning 1 min after ROSC. CAR caused a decline in coronary flow reserve (CFR) in control animals (pre-arrest, 1.86 ± 0.20; 1 h post-ROSC, 1.3 ± 0.05; 4 h post-ROSC, 1.25 ± 0.06; P < 0.05). GLP-1 preserved CFR for up to 4 h after ROSC (pre-arrest, 1.31 ± 0.17; 1 h post-ROSC, 1.5 ± 0.01; 4 h post-ROSC, 1.55 ± 0.22). Although there was a trend toward improvement in LV relaxation in the GLP-1-treated animals, overall LV function was not consistently different between groups. 8-iso-PGF(2α), a measure of reactive oxygen species load, was decreased in post-ROSC GLP-1-treated animals [placebo, control (NS): 38.1 ± 1.54 pg/ml; GLP-1: 26.59 ± 1.56 pg/ml; P < 0.05]. Infusion of GLP-1 after CAR preserved coronary microvascular and LV diastolic function. These effects may be mediated through a reduction in oxidative stress.

  8. N-n-butyl haloperidol iodide ameliorates hypoxia/reoxygenation injury through modulating the LKB1/AMPK/ROS pathway in cardiac microvascular endothelial cells

    PubMed Central

    Lu, Binger; Wang, Bin; Zhong, Shuping; Zhang, Yanmei; Gao, Fenfei; Chen, Yicun; Zheng, Fuchun; Shi, Ganggang

    2016-01-01

    Endothelial cells are highly sensitive to hypoxia and contribute to myocardial ischemia/reperfusion injury. We have reported that N-n-butyl haloperidol iodide (F2) can attenuate hypoxia/reoxygenation (H/R) injury in cardiac microvascular endothelial cells (CMECs). However, the molecular mechanisms remain unclear. Neonatal rat CMECs were isolated and subjected to H/R. Pretreatment of F2 leads to a reduction in H/R injury, as evidenced by increased cell viability, decreased lactate dehydrogenase (LDH) leakage and apoptosis, together with enhanced AMP-activated protein kinase (AMPK) and liver kinase B1 (LKB1) phosphorylation in H/R ECs. Blockade of AMPK with compound C reversed F2-induced inhibition of H/R injury, as evidenced by decreased cell viability, increased LDH release and apoptosis. Moreover, compound C also blocked the ability of F2 to reduce H/R-induced reactive oxygen species (ROS) generation. Supplementation with the ROS scavenger N-acetyl-L-cysteine (NAC) reduced ROS levels, increased cell survival rate, and decreased both LDH release and apoptosis after H/R. In conclusion, our data indicate that F2 may mitigate H/R injury by stimulating LKB1/AMPK signaling pathway and subsequent suppression of ROS production in CMECs. PMID:27166184

  9. Qi-Shen-Yi-Qi Dripping Pills Promote Angiogenesis of Ischemic Cardiac Microvascular Endothelial Cells by Regulating MicroRNA-223-3p Expression

    PubMed Central

    Dai, Guo-Hua; Liu, Ning; Zhu, Jing-Wei; Yao, Jing; Yang, Chun; Ma, Pei-Ze; Song, Xian-Bo

    2016-01-01

    Traditional Chinese medicine (TCM) research shows that Qi-Shen-Yi-Qi Dripping Pills (QSYQ) can promote ischemic cardiac angiogenesis. Studies have shown that microRNAs (miRNAs) are the key component of gene regulation networks, which play a vital role in angiogenesis and cardiovascular disease. Mechanisms involving miRNA by which TCM promotes ischemic cardiac angiogenesis have not been reported. We found that microRNA-223-3p (mir-223-3p) was the core miRNA of angiogenesis of rats ischemic cardiac microvascular endothelial cells (CMECs) and inhibited angiogenesis by affecting RPS6KB1/HIF-1α signal pathway in previous study. Based on the results, we observed biological characteristics and optimal dosage for QSYQ intervening in rats ischemic CMECs angiogenesis and concluded that QSYQ low-dose group had the strongest ability to promote angiogenesis of ischemic myocardium. Using miRNA chip and real-time PCR techniques in this study, we identified mir-223-3p as the pivotal miRNA in QSYQ that regulated angiogenesis of ischemic CMECs. From real-time PCR and western blot analysis, research showed that gene and protein expression of factors located RPS6KB1/HIF-1α signaling pathway, including HIF-1α, VEGF, MAPK, PI3K, and AKT, were significantly upregulated by QSYQ to regulate angiogenesis of ischemic CMECs. This study showed that QSYQ promote ischemic cardiac angiogenesis by downregulating mir-223-3p expression in rats ischemic CMECs. PMID:27057198

  10. The CXCL10/CXCR3 axis promotes cardiac microvascular endothelial cell migration via the p38/FAK pathway in a proliferation-independent manner.

    PubMed

    Xia, Jing-Bo; Mao, Cheng-Zhou; Chen, Zhuo-Ying; Liu, Guang-Hui; Wu, Hai-Yan; Zhou, Deng-Cheng; Park, Kyu-Sang; Zhao, Hui; Kim, Soo-Ki; Cai, Dong-Qing; Qi, Xu-Feng

    2016-04-01

    CXCL10 is a chemokine with potent chemotactic activity for immune and non-immune cells expressing its receptor CXCR3. Previous studies have demonstrated that CXCL10 is involved in myocardial infarction. However, the role of CXCL10 in cardiac microvascular endothelial cell (CMEC) regulation and related mechanisms remains unclear. In this study, we investigated the effects of CXCL10 on the CMEC migration and explored its potential molecular mechanism by wound healing, cell proliferation and viability analysis. Furthermore, migration-related signaling pathways, including FAK, Erk, p38 and Smad, were examined by Western blotting. We found that CXCL10 significantly promotes CMEC migration under normal conditions and during hypoxia/ischemia. However, no significant differences in CMEC proliferation and viability were observed with or without CXCL10 treatment. CXCL10-mediated CMEC migration was greatly blocked by treatment with an anti-CXCR3 antibody. Although CXCL10 treatment promoted phosphorylation and activation of the FAK, Erk, and p38 pathways during hypoxia/ischemia, CXCL10-mediated CMEC migration was significantly blocked by p38 and FAK inhibitors, but not by an Erk inhibitor. Furthermore, CXCL10-mediated FAK activation was suppressed by the p38 inhibitor. These findings indicated that the CXCL10/CXCR3 pathway promotes the migration of CMECs under normal conditions and during hypoxia/ischemia in a proliferation-independent manner, at least in part, through regulation of the p38/FAK pathways.

  11. N-n-butyl Haloperidol Iodide Protects against Hypoxia/Reoxygenation Injury in Cardiac Microvascular Endothelial Cells by Regulating the ROS/MAPK/Egr-1 Pathway.

    PubMed

    Lu, Shishi; Zhang, Yanmei; Zhong, Shuping; Gao, Fenfei; Chen, Yicun; Li, Weiqiu; Zheng, Fuchun; Shi, Ganggang

    2016-01-01

    Endothelium dysfunction induced by reactive oxygen species (ROS) is an important initial event at the onset of myocardial ischemia/reperfusion in which the Egr-1 transcription factor often serves as a master switch for various damage pathways following reperfusion injury. We hypothesized that an intracellular ROS/MAPK/Egr-1 signaling pathway is activated in cardiac microvascular endothelial cells (CMECs) following hypoxia/reoxygenation (H/R). ROS generation, by either H/R or the ROS donor xanthine oxidase-hypoxanthine (XO/HX) activated all three MAPKs (ERK1/2, JNK, p38), and induced Egr-1 expression and Egr-1 DNA-binding activity in CMECs, whereas ROS scavengers (EDA and NAC) had the opposite effect following H/R. Inhibitors of all three MAPKs individually inhibited induction of Egr-1 expression by H/R in CMECs. Moreover, N-n-butyl haloperidol (F2), previously shown to protect cardiomyocytes subjected to I/R, dose-dependently downregulated H/R-induced ROS generation, MAPK activation, and Egr-1 expression and activity in CMECs, whereas XO/HX and MAPK activators (EGF, anisomycin) antagonized the effects of F2. Inhibition of the ROS/MAPK/Egr-1 signaling pathway, by either F2, NAC, or inhibition of MAPK, increased CMEC viability and the GSH/GSSG ratio, and decreased Egr-1 nuclear translocation. These results show that the ROS/MAPK/Egr-1 signaling pathway mediates H/R injury in CMECs, and F2 blocks this pathway to protect against H/R injury and further alleviate myocardial I/R injury.

  12. N-n-butyl Haloperidol Iodide Protects against Hypoxia/Reoxygenation Injury in Cardiac Microvascular Endothelial Cells by Regulating the ROS/MAPK/Egr-1 Pathway

    PubMed Central

    Lu, Shishi; Zhang, Yanmei; Zhong, Shuping; Gao, Fenfei; Chen, Yicun; Li, Weiqiu; Zheng, Fuchun; Shi, Ganggang

    2017-01-01

    Endothelium dysfunction induced by reactive oxygen species (ROS) is an important initial event at the onset of myocardial ischemia/reperfusion in which the Egr-1 transcription factor often serves as a master switch for various damage pathways following reperfusion injury. We hypothesized that an intracellular ROS/MAPK/Egr-1 signaling pathway is activated in cardiac microvascular endothelial cells (CMECs) following hypoxia/reoxygenation (H/R). ROS generation, by either H/R or the ROS donor xanthine oxidase-hypoxanthine (XO/HX) activated all three MAPKs (ERK1/2, JNK, p38), and induced Egr-1 expression and Egr-1 DNA-binding activity in CMECs, whereas ROS scavengers (EDA and NAC) had the opposite effect following H/R. Inhibitors of all three MAPKs individually inhibited induction of Egr-1 expression by H/R in CMECs. Moreover, N-n-butyl haloperidol (F2), previously shown to protect cardiomyocytes subjected to I/R, dose-dependently downregulated H/R-induced ROS generation, MAPK activation, and Egr-1 expression and activity in CMECs, whereas XO/HX and MAPK activators (EGF, anisomycin) antagonized the effects of F2. Inhibition of the ROS/MAPK/Egr-1 signaling pathway, by either F2, NAC, or inhibition of MAPK, increased CMEC viability and the GSH/GSSG ratio, and decreased Egr-1 nuclear translocation. These results show that the ROS/MAPK/Egr-1 signaling pathway mediates H/R injury in CMECs, and F2 blocks this pathway to protect against H/R injury and further alleviate myocardial I/R injury. PMID:28111550

  13. Statins suppress glucose-induced plasminogen activator inhibitor-1 expression by regulating RhoA and nuclear factor-κB activities in cardiac microvascular endothelial cells.

    PubMed

    Ni, Xiao-Qing; Zhu, Jian-Hua; Yao, Ning-Hua; Qian, Juan; Yang, Xiang-Jun

    2013-01-01

    The aim of this study was to investigate the possible proinflammatory signaling pathways involved in statin inhibition of glucose-induced plasminogen activator inhibitor-1 (PAI-1) expression in cardiac microvascular endothelial cells (CMECs). Primary rat CMECs were grown in the presence of 5.7 or 23 mmol/L glucose. PAI-1 mRNA and protein expression levels were measured by realtime polymerase chain reaction, Western blotting and enzyme-linked immunosorbent assay, respectively. A pull-down assay was performed to determine RhoA activity. IκBα protein expression was measured by Western blotting, nuclear factor (NF)-κB activation was detected by electrophoretic mobility shift assay and its transcription activity was determined by a dual luciferase reporter gene assay. PAI-1 mRNA and protein expression levels were both increased with high glucose concentrations, but they were significantly suppressed by simvastatin and atorvastatin treatment (P < 0.01) and the effects were reversed by mevalonate (100 μmol/L) and geranylgeranyl pyrophosphate (10 μmol/L) but not farnesyl pyrophosphate (10 μmol/L). Such effects were similar to those of a RhoA inhibitor, C3 exoenzyme (5 μg/mL), inhibitors of RhoA kinase (ROCK), Y-27632 (10 μmol/L) and hydroxyfasudil (10 μmol/L) and an NF-κB inhibitor, BAY 11-7082 (5 μmol/L). High glucose-induced RhoA and NF-κB activations in CMECs were both significantly inhibited by statins (P < 0.01). Simvastatin and atorvastatin equally suppress high glucose-induced PAI-1 expression. These effects of statins may occur partly by regulating the RhoA/ROCK-NF-κB pathway. The multifunctional roles of statins may be particularly beneficial for patients with metabolic syndrome.

  14. Rapid homogeneous endothelialization of high aspect ratio microvascular networks.

    PubMed

    Naik, Nisarga; Hanjaya-Putra, Donny; Haller, Carolyn A; Allen, Mark G; Chaikof, Elliot L

    2015-08-01

    Microvascularization of an engineered tissue construct is necessary to ensure the nourishment and viability of the hosted cells. Microvascular constructs can be created by seeding the luminal surfaces of microfluidic channel arrays with endothelial cells. However, in a conventional flow-based system, the uniformity of endothelialization of such an engineered microvascular network is constrained by mass transfer of the cells through high length-to-diameter (L/D) aspect ratio microchannels. Moreover, given the inherent limitations of the initial seeding process to generate a uniform cell coating, the large surface-area-to-volume ratio of microfluidic systems demands long culture periods for the formation of confluent cellular microconduits. In this report, we describe the design of polydimethylsiloxane (PDMS) and poly(glycerol sebacate) (PGS) microvascular constructs with reentrant microchannels that facilitates rapid, spatially homogeneous endothelial cell seeding of a high L/D (2 cm/35 μm; > 550:1) aspect ratio microchannels. MEMS technology was employed for the fabrication of a monolithic, elastomeric, reentrant microvascular construct. Isotropic etching and PDMS micromolding yielded a near-cylindrical microvascular channel array. A 'stretch - seed - seal' operation was implemented for uniform incorporation of endothelial cells along the entire microvascular area of the construct yielding endothelialized microvascular networks in less than 24 h. The feasibility of this endothelialization strategy and the uniformity of cellularization were established using confocal microscope imaging.

  15. Differentiation state determines neural effects on microvascular endothelial cells

    SciTech Connect

    Muffley, Lara A.; Pan, Shin-Chen; Smith, Andria N.; Ga, Maricar; Hocking, Anne M.; Gibran, Nicole S.

    2012-10-01

    Growing evidence indicates that nerves and capillaries interact paracrinely in uninjured skin and cutaneous wounds. Although mature neurons are the predominant neural cell in the skin, neural progenitor cells have also been detected in uninjured adult skin. The aim of this study was to characterize differential paracrine effects of neural progenitor cells and mature sensory neurons on dermal microvascular endothelial cells. Our results suggest that neural progenitor cells and mature sensory neurons have unique secretory profiles and distinct effects on dermal microvascular endothelial cell proliferation, migration, and nitric oxide production. Neural progenitor cells and dorsal root ganglion neurons secrete different proteins related to angiogenesis. Specific to neural progenitor cells were dipeptidyl peptidase-4, IGFBP-2, pentraxin-3, serpin f1, TIMP-1, TIMP-4 and VEGF. In contrast, endostatin, FGF-1, MCP-1 and thrombospondin-2 were specific to dorsal root ganglion neurons. Microvascular endothelial cell proliferation was inhibited by dorsal root ganglion neurons but unaffected by neural progenitor cells. In contrast, microvascular endothelial cell migration in a scratch wound assay was inhibited by neural progenitor cells and unaffected by dorsal root ganglion neurons. In addition, nitric oxide production by microvascular endothelial cells was increased by dorsal root ganglion neurons but unaffected by neural progenitor cells. -- Highlights: Black-Right-Pointing-Pointer Dorsal root ganglion neurons, not neural progenitor cells, regulate microvascular endothelial cell proliferation. Black-Right-Pointing-Pointer Neural progenitor cells, not dorsal root ganglion neurons, regulate microvascular endothelial cell migration. Black-Right-Pointing-Pointer Neural progenitor cells and dorsal root ganglion neurons do not effect microvascular endothelial tube formation. Black-Right-Pointing-Pointer Dorsal root ganglion neurons, not neural progenitor cells, regulate

  16. Targeting brain microvascular endothelial cells: a therapeutic approach to neuroprotection against stroke

    PubMed Central

    Yu, Qi-jin; Tao, Hong; Wang, Xin; Li, Ming-chang

    2015-01-01

    Brain microvascular endothelial cells form the interface between nervous tissue and circulating blood, and regulate central nervous system homeostasis. Brain microvascular endothelial cells differ from peripheral endothelial cells with regards expression of specific ion transporters and receptors, and contain fewer fenestrations and pinocytotic vesicles. Brain microvascular endothelial cells also synthesize several factors that influence blood vessel function. This review describes the morphological characteristics and functions of brain microvascular endothelial cells, and summarizes current knowledge regarding changes in brain microvascular endothelial cells during stroke progression and therapies. Future studies should focus on identifying mechanisms underlying such changes and developing possible neuroprotective therapeutic interventions. PMID:26807131

  17. Xiang-Qi-Tang and its active components exhibit anti-inflammatory and anticoagulant properties by inhibiting MAPK and NF-κB signaling pathways in LPS-treated rat cardiac microvascular endothelial cells.

    PubMed

    He, Chang-Liang; Yi, Peng-Fei; Fan, Qiao-Jia; Shen, Hai-Qing; Jiang, Xiao-Lin; Qin, Qian-Qian; Song, Zhou; Zhang, Cui; Wu, Shuai-Cheng; Wei, Xu-Bin; Li, Ying-Lun; Fu, Ben-Dong

    2013-04-01

    Xiang-Qi-Tang (XQT) is a Chinese herbal formula containing Cyperus rotundus, Astragalus membranaceus and Andrographis paniculata. Alpha-Cyperone (CYP), astragaloside IV (AS-IV) and andrographolide (AND) are the three major active components in this formula. XQT may modulate the inflammatory or coagulant responses. We therefore assessed the effects of XQT on lipopolysaccharide (LPS)-induced inflammatory model of rat cardiac microvascular endothelial cells (RCMECs). XQT, CYP, AS-IV and AND inhibited the production of tumor necrosis factor alpha (TNF-α), intercellular cell adhesion molecule-1 (ICAM-1) and plasminogen activator inhibitor-1 (PAI-1), and up-regulated the mRNA expression of Kruppel-like factor 2 (KLF2). XQT and CYP inhibited the secretion of tissue factor (TF). To further explore the mechanism, we found that XQT, or its active components CYP, AS-IV and AND significantly inhibited extracellular signal-regulated kinase (ERK), c-jun NH2-terminal kinase (JNK) and p38 phosphorylation protein expression as well as decreased the phosphorylation levels of nuclear factor κB (NF-κB) p65 proteins in LPS-stimulated RCMECs. These results suggested that XQT and its active components inhibited the expression of inflammatory and coagulant mediators via mitogen-activated protein kinase (MAPKs) and NF-κB signaling pathways. These findings may contribute to future research on the action mechanisms of this formula, as well as therapy for inflammation- or coagulation-related diseases.

  18. Endothelial Progenitor Cells in Diabetic Microvascular Complications: Friends or Foes?

    PubMed Central

    Yu, Cai-Guo; Zhang, Ning; Yuan, Sha-Sha; Ma, Yan; Yang, Long-Yan; Feng, Ying-Mei; Zhao, Dong

    2016-01-01

    Despite being featured as metabolic disorder, diabetic patients are largely affected by hyperglycemia-induced vascular abnormality. Accumulated evidence has confirmed the beneficial effect of endothelial progenitor cells (EPCs) in coronary heart disease. However, antivascular endothelial growth factor (anti-VEGF) treatment is the main therapy for diabetic retinopathy and nephropathy, indicating the uncertain role of EPCs in the pathogenesis of diabetic microvascular disease. In this review, we first illustrate how hyperglycemia induces metabolic and epigenetic changes in EPCs, which exerts deleterious impact on their number and function. We then discuss how abnormal angiogenesis develops in eyes and kidneys under diabetes condition, focusing on “VEGF uncoupling with nitric oxide” and “competitive angiopoietin 1/angiopoietin 2” mechanisms that are shared in both organs. Next, we dissect the nature of EPCs in diabetic microvascular complications. After we overview the current EPCs-related strategies, we point out new EPCs-associated options for future exploration. Ultimately, we hope that this review would uncover the mysterious nature of EPCs in diabetic microvascular disease for therapeutics. PMID:27313624

  19. The expression of ADAMTS13 in human microvascular endothelial cells.

    PubMed

    Wang, Anyou; Duan, Qiaohong; Wu, Jingsheng; Liu, Xin; Sun, Zimin

    2016-06-01

    ADAMTS13, as a specific von Willebrand factor (VWF)-cleaving protease, prevents microvascular thrombosis of VWF/platelet thrombi. It has been reported that human vascular endothelial cells could also synthesize and secrete ADAMTS13, and these reports were focused in human umbilical vascular endothelial cells. Considering the particularity of its huge quantity and structure of human microvascular endothelial cells (HMECs) in the body, whether ADAMTS13 is expressed in HMECs also needs to be confirmed. To investigate whether ADAMTS13 is expressed in HMECs. Real-time PCR (RT-PCR) amplification detected ADAMTS13 mRNA in HMEC-1 cell line. The expression and distribution of ADAMTS13 protein and VWF were detected by fluorescence immunoassay and western blot. We observed the expression and distribution of ADAMTS13 in HMECs. We confirmed the expression of ADAMTS13 mRNA in HMEC-1, and found that there were some partly common distributions of ADAMTS13 protein and VWF. This study provides the evidence that HMECs also express ADAMTS13. HMECs might also be a primary source for human plasma ADAMTS13. The overlap region for the distribution of ADAMTS13 and VWF suggests that ADAMTS13 might have a potential regulation role for VWF inside cells.

  20. Serum factors involved in human microvascular endothelial cell morphogenesis.

    PubMed

    Harvey, Kevin; Siddiqui, Rafat A; Sliva, Daniel; Garcia, Joe G N; English, Denis

    2002-09-01

    Our previous studies have demonstrated that lipid and protein angiogenic factors operate in tandem to induce optimal angiogenic responses in vivo. This study was undertaken to clarify the nature of the substances in human serum that are responsible for its remarkable ability to promote capillary morphogenesis in vitro. The ability of dilute (2%) human serum to promote the morphogenic differentiation of human dermal microvascular endothelial cells on Matrigel supports was depleted by more than 50% by treatment of the serum with activated charcoal, a procedure that effectively removes biologically active lipid growth factors. The remainder of the activity within serum was lost on heating to 60 degrees C for 60 minutes, indicating the involvement of a protein in the response. The ability of charcoal-treated serum to promote capillary morphogenesis was completely restored by the addition of sphingosine 1-phosphate (SPP, 500 nmol/L), but other lipids thought to be released into serum during clotting were ineffective. In addition, basic fibroblast growth factor (bFGF) effectively restored the ability of heat-treated serum to promote endothelial cell morphogenesis, but other protein growth factors, including vascular endothelial growth factor and platelet-derived growth factor, were ineffective. Together, SPP and bFGF were as effective as whole serum in promoting capillary morphogenesis. Responses to purified SPP were entirely sensitive to the effects of preexposure of the cells to pertussis toxin, whereas responses to bFGF were entirely pertussis toxin-resistant. Consistent with our hypothesis that two distinct factors in serum play a role in promoting capillary morphogenesis, responses induced by serum were inhibited approximately 50% by preexposure of endothelial cells to pertussis toxin. We conclude that platelet-released SPP acts in conjunction with circulating bFGF to promote capillary formation by microvascular endothelial cells. Lipid and protein growth factors

  1. Antiproliferative effect of elevated glucose in human microvascular endothelial cells

    NASA Technical Reports Server (NTRS)

    Kamal, K.; Du, W.; Mills, I.; Sumpio, B. E.

    1998-01-01

    Diabetic microangiopathy has been implicated as a fundamental feature of the pathological complications of diabetes including retinopathy, neuropathy, and diabetic foot ulceration. However, previous studies devoted to examining the deleterious effects of elevated glucose on the endothelium have been performed largely in primary cultured cells of macrovessel origin. Difficulty in the harvesting and maintenance of microvascular endothelial cells in culture have hindered the study of this relevant population. Therefore, the objective of this study was to characterize the effect of elevated glucose on the proliferation and involved signaling pathways of an immortalized human dermal microvascular endothelial cell line (HMEC-1) that possess similar characteristics to their in vivo counterparts. Human dermal microvascular endothelial cells (HMEC-1) were grown in the presence of normal (5 mM) or high D-glucose (20 mM) for 14 days. The proliferative response of HMEC-1 was compared under these conditions as well as the cAMP and PKC pathways by in vitro assays. Elevated glucose significantly inhibited (P < 0.05) HMEC-1 proliferation after 7, 10, and 14 days. This effect was not mimicked by 20 mM mannitol. The antiproliferative effect was more pronounced with longer exposure (1-14 days) to elevated glucose and was irreversible 4 days after a 10-day exposure. The antiproliferative effect was partially reversed in the presence of a PKA inhibitor, Rp-cAMP (10-50 microM), and/or a PKC inhibitor, Calphostin C (10 nM). HMEC-1 exposed to elevated glucose (20 mM) for 14 days caused an increase in cyclic AMP accumulation, PKA, and PKC activity but was not associated with the activation of downstream events such as CRE and AP-1 binding activity. These data support the hypothesis that HMEC-1 is a suitable model to study the deleterious effects of elevated glucose on microvascular endothelial cells. Continued studies with HMEC-1 may prove advantageous in delineation of the molecular

  2. Methodological issues in the assessment of skin microvascular endothelial function in humans.

    PubMed

    Cracowski, Jean-Luc; Minson, Christopher T; Salvat-Melis, Muriel; Halliwill, John R

    2006-09-01

    The study of microvascular function can be performed in humans using laser Doppler flowmetry of the skin. This technology lends itself to a wide range of applications for studying the endothelial function of skin blood vessels. We review the advantages and limitations of postocclusive hyperemia, local thermal hyperemia, acetylcholine iontophoresis, flowmotion and association with microdialysis as tools with which to investigate skin microvascular endothelial function in humans. Postocclusive hyperemia, thermal hyperemia and acetylcholine iontophoresis provide integrated indexes of microvascular function rather than specific endothelial markers. However, they are valuable tools and can be used as surrogate endpoints in clinical trials in which the assessment of microvascular function in humans is required.

  3. NAP reduces murine microvascular endothelial cells proliferation induced by hyperglycemia.

    PubMed

    D'Amico, Agata Grazia; Scuderi, Soraya; Maugeri, Grazia; Cavallaro, Sebastiano; Drago, Filippo; D'Agata, Velia

    2014-11-01

    Hyperglycemia has been identified as a risk factor responsible for micro- and macrovascular complications in diabetes. NAP (Davunetide) is a peptide whose neuroprotective actions are widely demonstrated, although its biological role on endothelial dysfunctions induced by hyperglycemia remains uninvestigated. In the present study we hypothesized that NAP could play a protective role on hyperglycemia-induced endothelial cell proliferation. To this end we investigated the effects of NAP on an in vitro model of murine microvascular endothelial cells grown in high glucose for 7 days. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay and cyclin D1 protein expression analysis revealed that NAP treatment significantly reduces viability and proliferation of the cells. Hyperglycemia induced the activation of mitogen-activated protein kinase/extracellular signal-regulated protein kinase and/or phosphatidylinositol-3 kinase/Akt pathways in a time-dependent manner. NAP treatment reduced the phosphorylation levels of ERK and AKT in cells grown in high glucose. These evidences suggest that NAP might be effective in the regulation of endothelial dysfunction induced by hyperglycemia.

  4. Syndecan-2 downregulation impairs angiogenesis in human microvascular endothelial cells

    SciTech Connect

    Noguer, Oriol Villena, Joan; Lorita, Jordi; Vilaro, Senen; Reina, Manuel

    2009-03-10

    The formation of new blood vessels, or angiogenesis, is a necessary process during development but also for tumour growth and other pathologies. It is promoted by different growth factors that stimulate endothelial cells to proliferate, migrate, and generate new tubular structures. Syndecans, transmembrane heparan sulphate proteoglycans, bind such growth factors through their glycosaminoglycan chains and could transduce the signal to the cytoskeleton, thus regulating cell behaviour. We demonstrated that syndecan-2, the major syndecan expressed by human microvascular endothelial cells, is regulated by growth factors and extracellular matrix proteins, in both bidimensional and tridimensional culture conditions. The role of syndecan-2 in 'in vitro' tumour angiogenesis was also examined by inhibiting its core protein expression with antisense phosphorothioate oligonucleotides. Downregulation of syndecan-2 reduces spreading and adhesion of endothelial cells, enhances their migration, but also impairs the formation of capillary-like structures. These results suggest that syndecan-2 has an important function in some of the necessary steps that make up the angiogenic process. We therefore propose a pivotal role of this heparan sulphate proteoglycan in the formation of new blood vessels.

  5. Brain microvascular endothelial cell transplantation ameliorates ischemic white matter damage.

    PubMed

    Puentes, Sandra; Kurachi, Masashi; Shibasaki, Koji; Naruse, Masae; Yoshimoto, Yuhei; Mikuni, Masahiko; Imai, Hideaki; Ishizaki, Yasuki

    2012-08-21

    Ischemic insults affecting the internal capsule result in sensory-motor disabilities which adversely affect the patient's life. Cerebral endothelial cells have been reported to exert a protective effect against brain damage, so the transplantation of healthy endothelial cells might have a beneficial effect on the outcome of ischemic brain damage. In this study, endothelin-1 (ET-1) was injected into the rat internal capsule to induce lacunar infarction. Seven days after ET-1 injection, microvascular endothelial cells (MVECs) were transplanted into the internal capsule. Meningeal cells or 0.2% bovine serum albumin-Hank's balanced salt solution were injected as controls. Two weeks later, the footprint test and histochemical analysis were performed. We found that MVEC transplantation improved the behavioral outcome based on recovery of hind-limb rotation angle (P<0.01) and induced remyelination (P<0.01) compared with the control groups. Also the inflammatory response was repressed by MVEC transplantation, judging from fewer ED-1-positive activated microglial cells in the MVEC-transplanted group than in the other groups. Elucidation of the mechanisms by which MVECs ameliorate ischemic damage of the white matter may provide important information for the development of effective therapies for white matter ischemia.

  6. Isolation of primary murine brain microvascular endothelial cells.

    PubMed

    Ruck, Tobias; Bittner, Stefan; Epping, Lisa; Herrmann, Alexander M; Meuth, Sven G

    2014-11-14

    The blood-brain-barrier is ultrastructurally assembled by a monolayer of brain microvascular endothelial cells (BMEC) interconnected by a junctional complex of tight and adherens junctions. Together with other cell-types such as astrocytes or pericytes, they form the neurovascular unit (NVU), which specifically regulates the interchange of fluids, molecules and cells between the peripheral blood and the CNS. Through this complex and dynamic system BMECs are involved in various processes maintaining the homeostasis of the CNS. A dysfunction of the BBB is observed as an essential step in the pathogenesis of many severe CNS diseases. However, specific and targeted therapies are very limited, as the underlying mechanisms are still far from being understood. Animal and in vitro models have been extensively used to gain in-depth understanding of complex physiological and pathophysiological processes. By reduction and simplification it is possible to focus the investigation on the subject of interest and to exclude a variety of confounding factors. However, comparability and transferability are also reduced in model systems, which have to be taken into account for evaluation. The most common animal models are based on mice, among other reasons, mainly due to the constantly increasing possibilities of methodology. In vitro studies of isolated murine BMECs might enable an in-depth analysis of their properties and of the blood-brain-barrier under physiological and pathophysiological conditions. Further insights into the complex mechanisms at the BBB potentially provide the basis for new therapeutic strategies. This protocol describes a method to isolate primary murine microvascular endothelial cells by a sequence of physical and chemical purification steps. Special considerations for purity and cultivation of MBMECs as well as quality control, potential applications and limitations are discussed.

  7. The role of intrinsic apoptotic signaling in hemorrhagic shock-induced microvascular endothelial cell barrier dysfunction.

    PubMed

    Sawant, Devendra A; Tharakan, Binu; Hunter, Felicia A; Childs, Ed W

    2014-11-01

    Hemorrhagic shock leads to endothelial cell barrier dysfunction resulting in microvascular hyperpermeability. Hemorrhagic shock-induced microvascular hyperpermeability is associated with worse clinical outcomes in patients with traumatic injuries. The results from our laboratory have illustrated a possible pathophysiological mechanism showing involvement of mitochondria-mediated "intrinsic" apoptotic signaling in regulating hemorrhagic shock-induced microvascular hyperpermeability. Hemorrhagic shock results in overexpression of Bcl-2 family of pro-apoptotic protein, BAK, in the microvascular endothelial cells. The increase in BAK initiates "intrinsic" apoptotic signaling cascade with the release of mitochondrial cytochrome c in the cytoplasm and activation of downstream effector caspase-3, leading to loss of endothelial cell barrier integrity. Thus, this review article offers a brief overview of important findings from our past and present research work along with new leads for future research. The summary of our research work will provide information leading to different avenues in developing novel strategies against microvascular hyperpermeability following hemorrhagic shock.

  8. Conditioned Media from Microvascular Endothelial Cells Cultured in Simulated Microgravity Inhibit Osteoblast Activity

    PubMed Central

    Cazzaniga, Alessandra; Castiglioni, Sara; Maier, Jeanette A. M.

    2014-01-01

    Background and Aims. Gravity contributes to the maintenance of bone integrity. Accordingly, weightlessness conditions during space flight accelerate bone loss and experimental models in real and simulated microgravity show decreased osteoblastic and increased osteoclastic activities. It is well known that the endothelium and bone cells cross-talk and this intercellular communication is vital to regulate bone homeostasis. Because microgravity promotes microvascular endothelial dysfunction, we anticipated that the molecular cross-talk between endothelial cells exposed to simulated microgravity and osteoblasts might be altered. Results. We cultured human microvascular endothelial cells in simulated microgravity using the rotating wall vessel device developed by NASA. Endothelial cells in microgravity show growth inhibition and release higher amounts of matrix metalloproteases type 2 and interleukin-6 than controls. Conditioned media collected from microvascular endothelial cells in simulated microgravity were used to culture human osteoblasts and were shown to retard osteoblast proliferation and inhibit their activity. Discussion. Microvascular endothelial cells in microgravity are growth retarded and release high amounts of matrix metalloproteases type 2 and interleukin-6, which might play a role in retarding the growth of osteoblasts and impairing their osteogenic activity. Conclusions. We demonstrate that since simulated microgravity modulates microvascular endothelial cell function, it indirectly impairs osteoblastic function. PMID:25210716

  9. Dobesilate enhances endothelial nitric oxide synthase-activity in macro- and microvascular endothelial cells

    PubMed Central

    Suschek, Christoph; Kolb, Hubert; Kolb-Bachofen, Victoria

    1997-01-01

    Dobesilate is used for normalizing vascular dysfunction in a number of diseases. In search for an effect on endothelial NO production, macrovascular endothelial cells from rat aorta, microvascular endothelial cells from rat exocrine pancreatic tissue, and capillary endothelial cells from rat islets, were cultured in the presence or absence of Mg-Dobesilate. The activity of constitutive nitric oxide synthase (ecNOS) in resident cells as well as of inducible nitric oxide synthase (iNOS) in cytokine-activated cells was measured indirectly by recording the citrulline concentrations in culture supernatants.In each of the different endothelial cells Mg-Dobesilate incubation (0.25–1 mM) for 24 h led to a significant and concentration-dependent increase in ecNOS-activities. With cytokine-activated endothelial cell cultures only moderate effects were seen with little or no concentration-dependency. Addition of the NOS-inhibitor NG-monomethyl-L-arginine led to a significant suppression of citrulline formation in all cultures as an evidence for the enzyme specificity of these effects.iNOS- and ecNOS-specific reverse transcription and semi-quantitative polymerase chain reaction (RT–PCR) with RNA from resident or cytokine-activated endothelial cells gave no evidence for an increase in NOS-specific mRNA after Mg-Dobesilate-treatment. Furthermore, Dobesilate-mediated enhancement of NO synthesis in resting endothelial cells was not due to iNOS induction in these cells, as no iNOS-specific signal was found by RT–PCR. PMID:9421302

  10. Host defenses to Rickettsia rickettsii infection contribute to increased microvascular permeability in human cerebral endothelial cells.

    PubMed

    Woods, Michael E; Olano, Juan P

    2008-03-01

    Rickettsiae are arthropod-borne intracellular bacterial pathogens that primarily infect the microvascular endothelium leading to systemic spread of the organisms and the major pathophysiological effect, increased microvascular permeability, and edema in vital organs such as the lung and brain. Much work has been done on mechanisms of immunity to rickettsiae, as well as the responses of endothelial cells to rickettsial invasion. However, to date, no one has described the mechanisms of increased microvascular permeability during acute rickettsiosis. We sought to establish an in vitro model of human endothelial-target rickettsial infection using the etiological agent of Rocky Mountain spotted fever, Rickettsia rickettsii, and human cerebral microvascular endothelial cells. Endothelial cells infected with R. rickettsii exhibited a dose-dependent decrease in trans-endothelial electrical resistance, which translates into increased monolayer permeability. Additionally, we showed that the addition of pro-inflammatory stimuli essential to rickettsial immunity dramatically enhanced this effect. This increase in permeability correlates with dissociation of adherens junctions between endothelial cells and is not dependent on the presence of nitric oxide. Taken together, these results demonstrate for the first time that increased microvascular permeability associated with rickettsial infection is partly attributable to intracellular rickettsiae and partly attributable to the immune defenses that have evolved to protect the host from rickettsial spread.

  11. Deeper Penetration of Erythrocytes into the Endothelial Glycocalyx Is Associated with Impaired Microvascular Perfusion

    PubMed Central

    Lee, Dae Hyun; Dane, Martijn J. C.; van den Berg, Bernard M.; Boels, Margien G. S.; van Teeffelen, Jurgen W.; de Mutsert, Renée; den Heijer, Martin; Rosendaal, Frits R.; van der Vlag, Johan; van Zonneveld, Anton Jan; Vink, Hans; Rabelink, Ton J.

    2014-01-01

    Changes in endothelial glycocalyx are one of the earliest changes in development of cardiovascular disease. The endothelial glycocalyx is both an important biological modifier of interactions between flowing blood and the vessel wall, and a determinant of organ perfusion. We hypothesize that deeper penetration of erythrocytes into the glycocalyx is associated with reduced microvascular perfusion. The population-based prospective cohort study (the Netherlands Epidemiology of Obesity [NEO] study) includes 6,673 middle-aged individuals (oversampling of overweight and obese individuals). Within this cohort, we have imaged the sublingual microvasculature of 915 participants using sidestream darkfield (SDF) imaging together with a recently developed automated acquisition and analysis approach. Presence of RBC (as a marker of microvascular perfusion) and perfused boundary region (PBR), a marker for endothelial glycocalyx barrier properties for RBC accessibility, were assessed in vessels between 5 and 25 µm RBC column width. A wide range of variability in PBR measurements, with a mean PBR of 2.14 µm (range: 1.43–2.86 µm), was observed. Linear regression analysis showed a marked association between PBR and microvascular perfusion, reflected by RBC filling percentage (regression coefficient β: −0.034; 95% confidence interval: −0.037 to −0.031). We conclude that microvascular beds with a thick (“healthy”) glycocalyx (low PBR), reflects efficient perfusion of the microvascular bed. In contrast, a thin (“risk”) glycocalyx (high PBR) is associated with a less efficient and defective microvascular perfusion. PMID:24816787

  12. Gene expression microarray data from human microvascular endothelial cells supplemented with a low concentration of niacin

    PubMed Central

    Hughes-Large, Jennifer M.; Borradaile, Nica M.

    2016-01-01

    The systemic lipid modifying drug, niacin, can directly improve human microvascular endothelial cell angiogenic function under lipotoxic conditions, possibly through activation of niacin receptors “Niacin receptor activation improves human microvascular endothelial cell angiogenic function during lipotoxicity” (Hughes-Large et al. 2014). Here we provide accompanying data collected using Affymetrix GeneChip microarrays to identify changes in gene expression in human microvascular endothelial cells treated with 10 μM niacin. Statistical analyses of robust multi-array average (RMA) values revealed that only 16 genes exhibited greater than 1.3-fold differential expression. Of these 16, only 5 were identified protein coding genes, while 3 of the remaining 11 genes appeared to be small nuclear/nucleolar RNAs. Altered expression of EFCAB4B, NAP1L2, and OR13C8 was confirmed by real time quantitative PCR. PMID:26937468

  13. Gene expression microarray data from human microvascular endothelial cells supplemented with a low concentration of niacin.

    PubMed

    Hughes-Large, Jennifer M; Borradaile, Nica M

    2016-03-01

    The systemic lipid modifying drug, niacin, can directly improve human microvascular endothelial cell angiogenic function under lipotoxic conditions, possibly through activation of niacin receptors "Niacin receptor activation improves human microvascular endothelial cell angiogenic function during lipotoxicity" (Hughes-Large et al. 2014). Here we provide accompanying data collected using Affymetrix GeneChip microarrays to identify changes in gene expression in human microvascular endothelial cells treated with 10 μM niacin. Statistical analyses of robust multi-array average (RMA) values revealed that only 16 genes exhibited greater than 1.3-fold differential expression. Of these 16, only 5 were identified protein coding genes, while 3 of the remaining 11 genes appeared to be small nuclear/nucleolar RNAs. Altered expression of EFCAB4B, NAP1L2, and OR13C8 was confirmed by real time quantitative PCR.

  14. Human microvascular endothelial cells express receptors for platelet-derived growth factor

    SciTech Connect

    Beitz, J.G.; Kim, Insoon; Calabresi, P.; Frackelton, A.R. Jr. )

    1991-03-01

    Endothelial cells have been widely thought to be unresponsive to platelet-derived growth factor (PDGF, a major growth factor released from stimulated platelets at the sites of vascular insults) and devoid of PDGF receptors. Nevertheless, in examining the growth-factor responses of microvascular endothelial cells isolated from human omental adipose tissue, the authors were surprised to detect PDGF-induced tyrosine phosphorylation of a 180-kDa glycoprotein, subsequently identified as the cellular receptor for PDGF by specific immunoprecipitation. Scatchard analysis of {sup 125}I-labeled PDGF binding to human microvascular endothelial cells revealed 30,000 PDGF receptors per cell with a K{sub d} of 0.14 nM. Normal cellular consequences of receptor activation were also observed, including tyrosine phosphorylation of a 42-kDa protein and serine phosphorylation of ribosomal protein S6. Furthermore, PDGF was mitogenic for these cells. Microvascular endothelial cells play a central role in neovascularization required for wound healing and solid tumor growth. Thus, the discovery of functional PFDG receptors on human microvascular endothelial cells suggests a direct role for PDGF in this process.

  15. Ascorbate inhibits NADPH oxidase subunit p47phox expression in microvascular endothelial cells.

    PubMed

    Wu, Feng; Schuster, David P; Tyml, Karel; Wilson, John X

    2007-01-01

    The production of reactive oxygen species (ROS) is central to the etiology of endothelial dysfunction in sepsis. Endothelial cells respond to infection by activating NADPH oxidases that are sources of intracellular ROS and potential targets for therapeutic administration of antioxidants. Ascorbate is an antioxidant that accumulates in these cells and improves capillary blood flow, vascular reactivity, arterial blood pressure, and survival in experimental sepsis. Therefore, the present study tested the hypothesis that ascorbate regulates NADPH oxidases in microvascular endothelial cells exposed to septic insult. We observed that incubation with Escherichia coli lipopolysaccharide (LPS) and interferon-gamma (IFNgamma) increased NADPH oxidase activity and expression of the enzyme subunit p47phox in mouse microvascular endothelial cells of skeletal muscle origin. Pretreatment of the cells with ascorbate prevented these increases. Polyethylene glycol-conjugated catalase and selective inhibitors of Jak2 also abrogated induction of p47phox. Exogenous hydrogen peroxide induced p47phox expression that was prevented by pretreatment of the cells with ascorbate. LPS+IFNgamma or hydrogen peroxide activated the Jak2/Stat1/IRF1 pathway and this effect was also inhibited by ascorbate. In conclusion, ascorbate blocks the stimulation by septic insult of redox-sensitive Jak2/Stat1/IRF1 signaling, p47phox expression, and NADPH oxidase activity in microvascular endothelial cells. Because endothelial NADPH oxidases produce ROS that can cause endothelial dysfunction, their inhibition by ascorbate may represent a new strategy for sepsis therapy.

  16. The effect of moesin overexpression on ageing of human dermal microvascular endothelial cells.

    PubMed

    Lee, Ju Hee; Hong, In Ae; Oh, Sang Ho; Kwon, Yeon Sook; Cho, Soo Hyun; Lee, Kwang Hoon

    2009-11-01

    Senescence of microvascular endothelial cells is known to play an important role in the pathophysiology of vascular diseases related to ageing, but the accurate mechanism or related genes are not known. Moesin, a cytoskeletal protein and the most potent candidate as an ageing-related protein, showed obvious changes in expression when compared before and after ageing. In this study, a lentivirus was used to overexpress moesin in endothelial cells. The expression of cell cycle mediators such as p16, cyclin D1 and cdk4, which can be the markers of ageing, was compared by RNA and was shown to be suppressed in moesin overexpressed endothelial cells. In conclusion, it can be said that the expression of moesin delays senescence of human dermal microvascular endothelial cells and this fundamental discovery can be used as a basis for understanding the mechanism of ageing and age-related diseases.

  17. Capsule independent uptake of the fungal pathogen Cryptococcus neoformans into brain microvascular endothelial cells.

    PubMed

    Sabiiti, Wilber; May, Robin C

    2012-01-01

    Cryptococcosis is a life-threatening fungal disease with a high rate of mortality among HIV/AIDS patients across the world. The ability to penetrate the blood-brain barrier (BBB) is central to the pathogenesis of cryptococcosis, but the way in which this occurs remains unclear. Here we use both mouse and human brain derived endothelial cells (bEnd3 and hCMEC/D3) to accurately quantify fungal uptake and survival within brain endothelial cells. Our data indicate that the adherence and internalisation of cryptococci by brain microvascular endothelial cells is an infrequent event involving small numbers of cryptococcal yeast cells. Interestingly, this process requires neither active signalling from the fungus nor the presence of the fungal capsule. Thus entry into brain microvascular endothelial cells is most likely a passive event that occurs following 'trapping' within capillary beds of the BBB.

  18. Usefulness of retinal microvascular endothelial dysfunction as a predictor of coronary artery disease.

    PubMed

    Al-Fiadh, Ali H; Wong, Tien Y; Kawasaki, Ryo; Clark, David J; Patel, Sheila K; Freeman, Melanie; Wilson, Andrew; Burrell, Louise M; Farouque, Omar

    2015-03-01

    Endothelial dysfunction is a key feature of atherosclerosis. Retinal microvascular endothelial function can be assessed using noninvasive dynamic vessel imaging techniques. Whether it is impaired in subjects with coronary artery disease (CAD) is unknown. The aim of this study was to examine the relation of retinal microvascular endothelial function with CAD. Vascular studies were performed in 197 prospectively recruited subjects divided into 2 groups: those without CAD but ≥2 cardiovascular risk factors (non-CAD controls; n = 119) and those with stable CAD (n = 78). Retinal microvascular endothelial dysfunction was assessed by measuring retinal arteriolar and venular dilatation to flicker light, a nitric oxide-dependent phenomenon, expressed as percentage increase over baseline diameter. Fingertip pulse-volume amplitude was measured to calculate reactive hyperaemia index and brachial artery flow-mediated dilatation assessed as measures of peripheral microvascular and conduit vessel endothelial function, respectively. Mean retinal arteriolar dilatation was attenuated in patients with CAD compared with non-CAD controls (1.51 ± 1.51% vs 2.37 ± 1.95%, p = 0.001). Retinal arteriolar dilatation was independently associated with CAD after adjustment for age, gender, cardiovascular risk factors, and medication use (odds ratio 1.60, 95% confidence interval 1.14 to 2.25, p = 0.007). Reactive hyperaemia index and flow-mediated dilatation were not different. In conclusion, the capacity of retinal arterioles to dilate in response to flicker light is an independent predictor of the presence of CAD and suggests that retinal microvascular endothelial dysfunction is a marker for underlying CAD.

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

    PubMed Central

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

    2015-01-01

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

  20. Dephosphorylation of Y685-VE-Cadherin Involved in Pulmonary Microvascular Endothelial Barrier Injury Induced by Angiotensin II.

    PubMed

    Wu, Zhiyong; Wang, Zhiwei; Dai, Feifeng; Liu, Huagang; Ren, Wei; Chang, Jinxing; Li, Bowen

    2016-01-01

    Angiotensin II (AngII) caused pulmonary microvascular endothelial barrier injury, which induced acute aortic dissection (AAD) combined with acute lung injury (ALI). However, the exact mechanism is unclear. We investigated the role of dephosphorylation of Y685-VE-cadherin in the AngII induced pulmonary microvascular endothelial barrier injury. Mice or pulmonary microvascular endothelial cells (PMVECs) were divided into control group, AngII group, AngII+PP2 (Src kinase inhibitor) group, and PP2 group. PP2 was used to inhibit the phosphorylation of Y685-VE-cadherin. Pathological changes, infiltration of macrophages and neutrophils, and pulmonary microvascular permeability were used to determine the pulmonary microvascular endothelial barrier function. Flow cytometry was used to determine the apoptosis of PMVECs, and immunofluorescence was used to determine the skeletal arrangement. Transendothelial resistance was used to detect the permeability of endothelial barrier. Phosphorylation of Y685-VE-cadherin was significantly reduced after AngII stimulation (P < 0.05), together with skeletal rearrangement, and elevation of endothelial permeability which finally induced endothelial barrier injury. After PP2 interference, the phosphorylation of Y685-VE-cadherin was further reduced and the endothelial permeability was further elevated. These data indicated that AngII could induce pulmonary injury by triggering endothelial barrier injury, and such process may be related to the dephosphorylation of Y685-VE-cadherin and the endothelial skeletal rearrangement.

  1. Dephosphorylation of Y685-VE-Cadherin Involved in Pulmonary Microvascular Endothelial Barrier Injury Induced by Angiotensin II

    PubMed Central

    Wang, Zhiwei; Dai, Feifeng; Liu, Huagang; Ren, Wei; Chang, Jinxing; Li, Bowen

    2016-01-01

    Angiotensin II (AngII) caused pulmonary microvascular endothelial barrier injury, which induced acute aortic dissection (AAD) combined with acute lung injury (ALI). However, the exact mechanism is unclear. We investigated the role of dephosphorylation of Y685-VE-cadherin in the AngII induced pulmonary microvascular endothelial barrier injury. Mice or pulmonary microvascular endothelial cells (PMVECs) were divided into control group, AngII group, AngII+PP2 (Src kinase inhibitor) group, and PP2 group. PP2 was used to inhibit the phosphorylation of Y685-VE-cadherin. Pathological changes, infiltration of macrophages and neutrophils, and pulmonary microvascular permeability were used to determine the pulmonary microvascular endothelial barrier function. Flow cytometry was used to determine the apoptosis of PMVECs, and immunofluorescence was used to determine the skeletal arrangement. Transendothelial resistance was used to detect the permeability of endothelial barrier. Phosphorylation of Y685-VE-cadherin was significantly reduced after AngII stimulation (P < 0.05), together with skeletal rearrangement, and elevation of endothelial permeability which finally induced endothelial barrier injury. After PP2 interference, the phosphorylation of Y685-VE-cadherin was further reduced and the endothelial permeability was further elevated. These data indicated that AngII could induce pulmonary injury by triggering endothelial barrier injury, and such process may be related to the dephosphorylation of Y685-VE-cadherin and the endothelial skeletal rearrangement. PMID:28119542

  2. Parasite biomass-related inflammation, endothelial activation, microvascular dysfunction and disease severity in vivax malaria.

    PubMed

    Barber, Bridget E; William, Timothy; Grigg, Matthew J; Parameswaran, Uma; Piera, Kim A; Price, Ric N; Yeo, Tsin W; Anstey, Nicholas M

    2015-01-01

    Plasmodium vivax can cause severe malaria, however its pathogenesis is poorly understood. In contrast to P. falciparum, circulating vivax parasitemia is low, with minimal apparent sequestration in endothelium-lined microvasculature, and pathogenesis thought unrelated to parasite biomass. However, the relationships between vivax disease-severity and total parasite biomass, endothelial autocrine activation and microvascular dysfunction are unknown. We measured circulating parasitemia and markers of total parasite biomass (plasma parasite lactate dehydrogenase [pLDH] and PvLDH) in adults with severe (n = 9) and non-severe (n = 53) vivax malaria, and examined relationships with disease-severity, endothelial activation, and microvascular function. Healthy controls and adults with non-severe and severe falciparum malaria were enrolled for comparison. Median peripheral parasitemia, PvLDH and pLDH were 2.4-fold, 3.7-fold and 6.9-fold higher in severe compared to non-severe vivax malaria (p = 0.02, p = 0.02 and p = 0.015, respectively), suggesting that, as in falciparum malaria, peripheral P. vivax parasitemia underestimates total parasite biomass, particularly in severe disease. P. vivax schizonts were under-represented in peripheral blood. Severe vivax malaria was associated with increased angiopoietin-2 and impaired microvascular reactivity. Peripheral vivax parasitemia correlated with endothelial activation (angiopoietin-2, von-Willebrand-Factor [VWF], E-selectin), whereas markers of total vivax biomass correlated only with systemic inflammation (IL-6, IL-10). Activity of the VWF-cleaving-protease, ADAMTS13, was deficient in proportion to endothelial activation, IL-6, thrombocytopenia and vivax disease-severity, and associated with impaired microvascular reactivity in severe disease. Impaired microvascular reactivity correlated with lactate in severe vivax malaria. Findings suggest that tissue accumulation of P. vivax may occur, with the hidden

  3. Parasite Biomass-Related Inflammation, Endothelial Activation, Microvascular Dysfunction and Disease Severity in Vivax Malaria

    PubMed Central

    Barber, Bridget E.; William, Timothy; Grigg, Matthew J.; Parameswaran, Uma; Piera, Kim A.; Price, Ric N.; Yeo, Tsin W.; Anstey, Nicholas M.

    2015-01-01

    Plasmodium vivax can cause severe malaria, however its pathogenesis is poorly understood. In contrast to P. falciparum, circulating vivax parasitemia is low, with minimal apparent sequestration in endothelium-lined microvasculature, and pathogenesis thought unrelated to parasite biomass. However, the relationships between vivax disease-severity and total parasite biomass, endothelial autocrine activation and microvascular dysfunction are unknown. We measured circulating parasitemia and markers of total parasite biomass (plasma parasite lactate dehydrogenase [pLDH] and PvLDH) in adults with severe (n = 9) and non-severe (n = 53) vivax malaria, and examined relationships with disease-severity, endothelial activation, and microvascular function. Healthy controls and adults with non-severe and severe falciparum malaria were enrolled for comparison. Median peripheral parasitemia, PvLDH and pLDH were 2.4-fold, 3.7-fold and 6.9-fold higher in severe compared to non-severe vivax malaria (p = 0.02, p = 0.02 and p = 0.015, respectively), suggesting that, as in falciparum malaria, peripheral P. vivax parasitemia underestimates total parasite biomass, particularly in severe disease. P. vivax schizonts were under-represented in peripheral blood. Severe vivax malaria was associated with increased angiopoietin-2 and impaired microvascular reactivity. Peripheral vivax parasitemia correlated with endothelial activation (angiopoietin-2, von-Willebrand-Factor [VWF], E-selectin), whereas markers of total vivax biomass correlated only with systemic inflammation (IL-6, IL-10). Activity of the VWF-cleaving-protease, ADAMTS13, was deficient in proportion to endothelial activation, IL-6, thrombocytopenia and vivax disease-severity, and associated with impaired microvascular reactivity in severe disease. Impaired microvascular reactivity correlated with lactate in severe vivax malaria. Findings suggest that tissue accumulation of P. vivax may occur, with the hidden

  4. Involvement of ROCK-mediated endothelial tension development in neutrophil-stimulated microvascular leakage

    PubMed Central

    Breslin, Jerome W.; Sun, Hengrui; Xu, Wenjuan; Rodarte, Charles; Moy, Alan B.; Wu, Mack H.; Yuan, Sarah Y.

    2009-01-01

    Neutrophil-induced coronary microvascular barrier dysfunction is an important pathophysiological event in heart disease. Currently, the precise cellular and molecular mechanisms of neutrophil-induced microvascular leakage are not clear. The aim of this study was to test the hypothesis that rho kinase (ROCK) increases coronary venular permeability in association with elevated endothelial tension. We assessed permeability to albumin (Pa) in isolated porcine coronary venules and in coronary venular endothelial cell (CVEC) monolayers. Endothelial barrier function was also evaluated by measuring transendothelial electrical resistance (TER) of CVEC monolayers. In parallel, we measured isometric tension of CVECs grown on collagen gels. Transference of constitutively active (ca)-ROCK protein into isolated coronary venules or CVEC monolayers caused a significant increase in Pa and decreased TER in CVECs. The ROCK inhibitor Y-27632 blocked the ca-ROCK-induced changes. C5a-activated neutrophils (106/ml) also significantly elevated venular Pa, which was dose-dependently inhibited by Y-27632 and a structurally distinct ROCK inhibitor, H-1152. In CVEC monolayers, activated neutrophils increased permeability with a concomitant elevation in isometric tension, both of which were inhibited by Y-27632 or H-1152. Treatment with ca-ROCK also significantly increased CVEC monolayer permeability and isometric tension, coupled with actin polymerization and elevated phosphorylation of myosin regulatory light chain on Thr18/Ser19. The data suggest that during neutrophil activation, ROCK promotes microvascular leakage in association with actin-myosin-mediated tension development in endothelial cells. PMID:16172166

  5. Angiopoietin-1 alters microvascular permeability coefficients in vivo via modification of endothelial glycocalyx

    PubMed Central

    Salmon, Andrew H.J.; Neal, Christopher R.; Sage, Leslie M.; Glass, Catherine A.; Harper, Steven J.; Bates, David O.

    2009-01-01

    Aims In this study, we wished to determine whether angiopoietin-1 (Ang1) modified the permeability coefficients of non-inflamed, intact continuous, and fenestrated microvessels in vivo and to elucidate the underlying cellular mechanisms. Methods and results Permeability coefficients were measured using the Landis–Michel technique (in frog and rat mesenteric microvessels) and an oncopressive permeability technique (in glomeruli). Ang1 decreased water permeability (LP: hydraulic conductivity) in continuous and fenestrated microvessels and increased the retention of albumin (σ: reflection coefficient) in continuous microvessels. Endothelial glycocalyx is common to these anatomically distinct microvascular beds, and contributes to the magnitude of both LP and σ. Ang1 treatment increased the depth of endothelial glycocalyx in intact microvessels and increased the content of glycosaminoglycan of cultured microvascular endothelial cell supernatant. Ang1 also prevented the pronase-induced increase in LP (attributable to selective removal of endothelial glycocalyx by pronase) by restoration of glycocalyx at the endothelial cell surface. The reduction in permeability was inhibited by a cell transport inhibitor, Brefeldin. Conclusion Ang1 modifies basal microvessel permeability coefficients, in keeping with previous reports demonstrating reduced solute flux in inflamed vessels. Anatomical, biochemical, and physiological evidence indicates that modification of endothelial glycocalyx is a novel mechanism of action of Ang1 that contributes to these effects. PMID:19297368

  6. Caveolae protect endothelial cells from membrane rupture during increased cardiac output

    PubMed Central

    Cheng, Jade P.X.; Mendoza-Topaz, Carolina; Howard, Gillian; Chadwick, Jessica; Shvets, Elena; Cowburn, Andrew S.; Dunmore, Benjamin J.; Crosby, Alexi; Morrell, Nicholas W.

    2015-01-01

    Caveolae are strikingly abundant in endothelial cells, yet the physiological functions of caveolae in endothelium and other tissues remain incompletely understood. Previous studies suggest a mechanoprotective role, but whether this is relevant under the mechanical forces experienced by endothelial cells in vivo is unclear. In this study we have sought to determine whether endothelial caveolae disassemble under increased hemodynamic forces, and whether caveolae help prevent acute rupture of the plasma membrane under these conditions. Experiments in cultured cells established biochemical assays for disassembly of caveolar protein complexes, and assays for acute loss of plasma membrane integrity. In vivo, we demonstrate that caveolae in endothelial cells of the lung and cardiac muscle disassemble in response to acute increases in cardiac output. Electron microscopy and two-photon imaging reveal that the plasma membrane of microvascular endothelial cells in caveolin 1−/− mice is much more susceptible to acute rupture when cardiac output is increased. These data imply that mechanoprotection through disassembly of caveolae is important for endothelial function in vivo. PMID:26459598

  7. Demonstration of actin filament stress fibers in microvascular endothelial cells in situ.

    PubMed

    Nehls, V; Drenckhahn, D

    1991-07-01

    We have developed a method for immunostaining the microvascular tree of rat mesenteric windows in situ. The procedure consists of three steps, i.e., mild fixation with formaldehyde, controlled proteolytic digestion of the mesothelial layer, and permeabilization with acetone. Discrimination between different microvascular segments was possible by double-fluorescent staining with antibodies to the smooth muscle isoform of alpha-actin and to nonmuscle myosin from platelets. Antibodies to nonmuscle myosin labeled numerous longitudinally oriented cables in endothelial cells of all microvascular segments (arterioles, metarterioles, pre-, mid-, and postcapillaries, small venules). Occasionally, the myosin-containing cables displayed the interrupted sarcomere-like staining pattern that is diagnostic for stress fibers. In contrast, staining of actin filaments with phalloidin-rhodamin resulted in a noninterrupted, continuous fluorescence of the stress fibers. A possible functional role of microvascular endothelial stress fibers is to serve as a tensile cytoskeletal scaffold that stabilizes the tubular, three-dimensional geometry of microvessels and, in addition, to help the endothelium resist the shear forces created by blood flow and by collision with red and white blood cells.

  8. Venlafaxine protects methylglyoxal-induced apoptosis in the cultured human brain microvascular endothelial cells.

    PubMed

    Lv, Qinghua; Gu, Chengyao; Chen, Caijing

    2014-05-21

    It was reported that venlafaxine protects microvascular endothelial cells injury in several models. But the mechanisms of venlafaxine protects cell injury still poor understanding. Here, we shows that in the cultured human brain microvascular endothelial cells (HBMEC), we found that venlafaxine protects methylglyoxal (MGO)-induced cell injury, and the venlafaxine significant reduction in the level of reactive oxygen species, down-regulated expression of pro-apoptotic activated caspase-3 and Bax, increased BDNF release and expression of anti-apoptotic Bcl-2 in the cultured HBMEC. Furthermore, we found that venlafaxine inhibits MGO-induced phosphorylation of JNK. Moreover, venlafaxine increased AKT phosphorylation and the protective effects of venlafaxine was inhibited by PI3K/AKT inhibitor. These findings suggest that venlafaxine protects MGO-induced HBMEC injury through PI3K/AKT and JNK pathway as the potential underlying mechanisms of HBMEC injury in diabetes.

  9. Emerging therapeutic strategies to prevent infection-related microvascular endothelial activation and dysfunction

    PubMed Central

    Darwish, Ilyse; Liles, W Conrad

    2013-01-01

    Recent evidence suggests that loss of endothelial barrier function and resulting microvascular leak play important mechanistic roles in the pathogenesis of infection-related end-organ dysfunction and failure. Several distinct therapeutic strategies, designed to prevent or limit infection-related microvascular endothelial activation and permeability, thereby mitigating end-organ injury/dysfunction, have recently been investigated in pre-clinical models. In this review, these potential therapeutic strategies, namely, VEGFR2/Src antagonists, sphingosine-1-phosphate agonists, fibrinopeptide Bβ15–42, slit2N, secinH3, angiopoietin-1/tie-2 agonists, angiopoietin-2 antagonists, statins, atrial natriuretic peptide, and mesenchymal stromal (stem) cells, are discussed in terms of their translational potential for the management of clinical infectious diseases. PMID:23863603

  10. Induction of Brain Microvascular Endothelial Cell Urokinase Expression by Cryptococcus neoformans Facilitates Blood-Brain Barrier Invasion

    PubMed Central

    Stie, Jamal; Fox, Deborah

    2012-01-01

    The invasive ability of the blood-borne fungal pathogen Cryptococcus neoformans can be enhanced through interactions with host plasma components, such as plasminogen. Previously we showed by in vitro studies that plasminogen coats the surface of C. neoformans and is converted to the active serine protease, plasmin, by host plasminogen activators. Viable, but not formaldehyde- or sodium azide-killed, cryptococcal strains undergo brain microvascular endothelial cell-dependent plasminogen-to-plasmin activation, which results in enhanced, plasmin-dependent cryptococcal invasion of primary bovine brain microvascular endothelial cells and fungal ability to degrade plasmin substrates. In the present work, brain microvascular endothelial cells cultured with viable, but not killed, cryptococcal strains led to significant increases in both urokinase mRNA transcription and cell-associated urokinase protein expression. Soluble urokinase was also detected in conditioned medium from brain microvascular endothelial cells cultured with viable, but not killed, C. neoformans. Exposure of plasminogen pre-coated viable C. neoformans to conditioned medium from strain-matched brain microvascular endothelial cell-fungal co-cultures resulted in plasminogen-to-plasmin activation and plasmin-dependent cryptococcal invasion. siRNA-mediated silencing of urokinase gene expression or the use of specific inhibitors of urokinase activity abrogated both plasminogen-to-plasmin activation on C. neoformans and cryptococcal-brain microvascular endothelial cell invasion. Our results suggest that pathogen exploitation of the host urokinase-plasmin(ogen) system may contribute to C. neoformans virulence during invasive cryptococcosis. PMID:23145170

  11. Characterizing nanoscale changes in the activity of VEGFR-2 on glioma microvascular endothelial cell membranes using atomic force microscopy

    PubMed Central

    Zhou, Dexiang; Zhan, Shengquan; Zhou, Dong; Wang, Peng; Chen, Guangzhong; Qin, Kun; Lin, Xiaofeng

    2017-01-01

    The aim of the current study was to demonstrate the distribution of VEGFR-2 on glioma microvascular endothelial cells on a nanoscale and investigate changes in VEGFR-2 activity following treatment with the VEGFR-2 inhibitor and agonist sorafenib and bradykinin, respectively. Three groups were evaluated in this study: Control glioma microvascular endothelial cells, sorafenib-treated glioma microvascular endothelial cells and bradykinin-treated glioma microvascular endothelial cells. Changes in the activity of VEGFR-2 on the glioma microvascular endothelial cell membranes following treatment with sorafenib and bradykinin were characterized by atomic force microscopy (AFM). Colloidal gold-labeled immune complexes and AFM were used to visualize the distribution of VEGFR-2 on the cell membranes. In the control group, VEGFR-2, which was observed as numerous globular structures, was evenly distributed on the cell surface membranes. The majority of the receptors were active. In the sorafenib group, only a few globular structures were observed on the cell membranes, with a density significantly lower than that in the control group (P<0.01). Furthermore, compared with the control group, fewer of the receptors were active. In the bradykinin group, numerous globular structures were densely distributed on the cell membranes, with a density significantly higher than that in the control group (P<0.01). The distribution and activity of VEGFR-2 on glioma microvascular endothelial cell membranes treated with sorafenib and bradykinin suggested that the activity of VEGFR-2 could be regulated by its inhibitor or agonist. PMID:28352319

  12. Thrombin stimulates albumin transcytosis in lung microvascular endothelial cells via activation of acid sphingomyelinase.

    PubMed

    Kuebler, Wolfgang M; Wittenberg, Claudia; Lee, Warren L; Reppien, Eike; Goldenberg, Neil M; Lindner, Karsten; Gao, Yizhuo; Winoto-Morbach, Supandi; Drab, Marek; Mühlfeld, Christian; Dombrowsky, Heike; Ochs, Matthias; Schütze, Stefan; Uhlig, Stefan

    2016-04-15

    Transcellular albumin transport occurs via caveolae that are abundant in lung microvascular endothelial cells. Stimulation of albumin transcytosis by proinflammatory mediators may contribute to alveolar protein leak in lung injury, yet the regulation of albumin transport and its underlying molecular mechanisms are so far incompletely understood. Here we tested the hypothesis that thrombin may stimulate transcellular albumin transport across lung microvascular endothelial cells in an acid-sphingomyelinase dependent manner. Thrombin increased the transport of fluorescently labeled albumin across confluent human lung microvascular endothelial cell (HMVEC-L) monolayers to an extent that markedly exceeds the rate of passive diffusion. Thrombin activated acid sphingomyelinase (ASM) and increased ceramide production in HMVEC-L, but not in bovine pulmonary artery cells, which showed little albumin transport in response to thrombin. Thrombin increased total caveolin-1 (cav-1) content in both whole cell lysates and lipid rafts from HMVEC-L, and this effect was blocked by inhibition of ASM or de novo protein biosynthesis. Thrombin-induced uptake of albumin into lung microvascular endothelial cells was confirmed in isolated-perfused lungs by real-time fluorescence imaging and electron microscopy of gold-labeled albumin. Inhibition of ASM attenuated thrombin-induced albumin transport both in confluent HMVEC-L and in intact lungs, whereas HMVEC-L treatment with exogenous ASM increased albumin transport and enriched lipid rafts in cav-1. Our findings indicate that thrombin stimulates transcellular albumin transport in an acid sphingomyelinase-dependent manner by inducing de novo synthesis of cav-1 and its recruitment to membrane lipid rafts.

  13. Early Activation of Primary Brain Microvascular Endothelial Cells by Nipah Virus Glycoprotein-Containing Particles

    PubMed Central

    Freitag, Tanja C.

    2015-01-01

    Nipah virus (NiV) is a highly pathogenic paramyxovirus that causes pronounced infection of brain endothelia and central nervous system (CNS) inflammation. Using primary porcine brain microvascular endothelial cells, we showed that upregulation of E-selectin precedes cytokine induction and is induced not only by infectious NiV but also by NiV-glycoprotein-containing virus-like particles. This demonstrates that very early events in NiV brain endothelial infection do not depend on NiV replication but can be triggered by the NiV glycoproteins alone. PMID:26676791

  14. Involvement of the cellular prion protein in the migration of brain microvascular endothelial cells.

    PubMed

    Watanabe, Takuya; Yasutaka, Yuki; Nishioku, Tsuyoshi; Kusakabe, Sae; Futagami, Koujiro; Yamauchi, Atsushi; Kataoka, Yasufumi

    2011-06-01

    The conversion of cellular prion protein (PrP(C)) to its protease-resistant isoform is involved in the pathogenesis of prion disease. Although PrP(C) is a ubiquitous glycoprotein that is present in various cell types, the physiological role of PrP(C) remains obscure. The present study aimed to determine whether PrP(C) mediates migration of brain microvascular endothelial cells. Small interfering RNAs (siRNAs) targeting PrP(C) were transfected into a mouse brain microvascular endothelial cell line (bEND.3 cells). siPrP1, selected among three siRNAs, reduced mRNA and protein levels of PrP(C) in bEND.3 cells. Cellular migration was evaluated with a scratch-wound assay. siPrP1 suppressed migration without significantly affecting cellular proliferation. This study provides the first evidence that PrP(C) may be necessary for brain microvascular endothelial cells to migrate into damaged regions in the brain. This function of PrP(C) in the brain endothelium may be a mechanism by which the neurovascular unit recovers from an injury such as an ischemic insult.

  15. Platelets play an important role in TNF-induced microvascular endothelial cell pathology.

    PubMed Central

    Lou, J.; Donati, Y. R.; Juillard, P.; Giroud, C.; Vesin, C.; Mili, N.; Grau, G. E.

    1997-01-01

    Tumor necrosis factor-alpha (TNF) is known to be an important mediator in the pathogenesis of several inflammatory diseases. Vascular endothelial cells represent a major target of TNF effects. Platelet sequestration has been found in brain microvessels during experimental cerebral malaria and lung in experimental pulmonary fibrosis, implying that it may participate in TNF-dependent microvascular pathology. In this study, we investigated the mechanisms of platelet-endothelial interaction, using co-cultures between platelets and TNF-activated mouse brain microvascular endothelial cells (MVECs). Adhesion and fusion of platelets to MVECs was evidenced by electron microscopy, dye transfer, and flow cytometry. It was induced by TNF and interferon-gamma and depended on LFA-1 expressed on the platelet surface and ICAM-1 expressed on MVECs. The adhesion and fusion also led to the transfer of platelet markers on the MVEC surface, rendering these more adherent for leukocytes, and to an enhanced MVEC sensitivity to TNF-induced injury. These results suggest that platelets can participate in TNF-induced microvascular pathology. Images Figure 2 Figure 3 Figure 6 PMID:9358766

  16. Shear stress-induced Ets-1 modulates protease inhibitor expression in microvascular endothelial cells.

    PubMed

    Milkiewicz, Malgorzata; Uchida, Cassandra; Gee, Eric; Fudalewski, Tomasz; Haas, Tara L

    2008-11-01

    Elevated shear stress within the skeletal muscle microvasculature is implicated in the induction of a longitudinal splitting form of angiogenesis, which is characterized by the lack of basement membrane breakage. We investigated whether the transcriptional regulator, Ets-1, is responsive to changes in hemodynamic forces and if so, whether Ets-1 controls microvascular endothelial cell integrity by inducing the expression of inhibitors of matrix degrading proteases. Rats were treated with prazosin for 2, 4, and 7 days to increase in microvascular shear stress in hindlimb skeletal muscles. In complimentary in vitro experiments, rat microvascular skeletal muscle endothelial cells were exposed to laminar shear stress (15 dyne/cm(2)) for 0.5, 2, and 24 h. TaqMan PCR analysis of laser microdissected capillaries isolated from EDL muscles demonstrated transient (after 2 days) induction of Ets-1 gene expression. In cultured cells, a transient up-regulation of Ets-1 mRNA was observed after 2 h shear stimulation, accompanied by increased phosphorylation of Ets-1 and enhanced Ets-1 DNA binding activity. This response was modulated by ERK1/2 and p38 MAP kinases, but was not dependent on NOS or COX-2 activity. PAI-1, TIMP-1 and TIMP-3 mRNA were elevated significantly in prazosin treated EDL, and in response to shear stimulation in vitro. In cultured endothelial cells, Ets-1 RNA interference abolished the shear-induced increases in Ets-1, PAI-1, TIMP-1, and TIMP-3 mRNA expression. These results suggest that enhanced laminar shear stress may act to preserve the integrity of microvascular walls in part through Ets-1-dependent induction of protease inhibitors.

  17. Ghrelin stimulates angiogenesis in human microvascular endothelial cells: Implications beyond GH release

    SciTech Connect

    Li Aihua; Cheng Guangli; Zhu Genghui; Tarnawski, Andrzej S. . E-mail: atarnawski@yahoo.com

    2007-02-09

    Ghrelin, a peptide hormone isolated from the stomach, releases growth hormone and stimulates appetite. Ghrelin is also expressed in pancreas, kidneys, cardiovascular system and in endothelial cells. The precise role of ghrelin in endothelial cell functions remains unknown. We examined the expression of ghrelin and its receptor (GHSR1) mRNAs and proteins in human microvascular endothelial cells (HMVEC) and determined whether ghrelin affects in these cells proliferation, migration and in vitro angiogenesis; and whether MAPK/ERK2 signaling is important for the latter action. We found that ghrelin and GHSR1 are constitutively expressed in HMVEC. Treatment of HMVEC with exogenous ghrelin significantly increased in these cells proliferation, migration, in vitro angiogenesis and ERK2 phosphorylation. MEK/ERK2 inhibitor, PD 98059 abolished ghrelin-induced in vitro angiogenesis. This is First demonstration that ghrelin and its receptor are expressed in human microvascular endothelial cells and that ghrelin stimulates HMVEC proliferation, migration, and angiogenesis through activation of ERK2 signaling.

  18. The Proangiogenic Effect of Iroquois Homeobox Transcription Factor Irx3 in Human Microvascular Endothelial Cells*

    PubMed Central

    Scarlett, Kisha; Pattabiraman, Vaishnavi; Barnett, Petrina; Liu, Dong; Anderson, Leonard M.

    2015-01-01

    Angiogenesis is a dynamic process required for embryonic development. However, postnatal vascular growth is characteristic of multiple disease states. Despite insights into the multistep process in which adhesion molecules, extracellular matrix proteins, growth factors, and their receptors work in concert to form new vessels from the preexisting vasculature, there remains a lack of insight of the nuclear transcriptional mechanisms that occur within endothelial cells (ECs) in response to VEGF. Iroquois homeobox gene 3 (Irx3) is a transcription factor of the Iroquois family of homeobox genes. Irx homeodomain transcription factors are involved in the patterning and development of several tissues. Irx3 is known for its role during embryogenesis in multiple organisms. However, the expression and function of Irx3 in human postnatal vasculature remains to be investigated. Here we show that Irx3 is expressed in human microvascular endothelial cells, and expression is elevated by VEGF stimulation. Genetic Irx3 gain and loss of function studies in human microvascular endothelial cells resulted in the modulation of EC migration during wound healing, chemotaxis and invasion, and tubulogenesis. Additionally, we observed increased delta-like ligand 4 (Dll4) expression, which suggests an increase in EC tip cell population. Finally, siRNA screening studies revealed that transient knockdown of Hey1, a downstream Notch signaling mediator, resulted in increased Irx3 expression in response to VEGF treatment. Strategies to pharmacologically regulate Irx3 function in adult endothelial cells may provide new therapies for angiogenesis. PMID:25512384

  19. Hydrogen peroxide-induced calcium influx in lung microvascular endothelial cells involves TRPV4

    PubMed Central

    Suresh, Karthik; Servinsky, Laura; Reyes, Jose; Baksh, Syeda; Undem, Clark; Caterina, Michael; Pearse, David B.

    2015-01-01

    In acute respiratory distress syndrome, both reactive oxygen species (ROS) and increased intracellular calcium ([Ca2+]i) are thought to play important roles in promoting endothelial paracellular permeability, but the mechanisms linking ROS and [Ca2+]i in microvascular endothelial cells are not known. In this study, we assessed the effect of hydrogen peroxide (H2O2) on [Ca2+]i in mouse and human lung microvascular endothelial cells (MLMVEC and HLMVEC, respectively). We found that in both MLMVECs and HLMVECs, exogenously applied H2O2 increased [Ca2+]i through Ca2+ influx and that pharmacologic inhibition of the calcium channel transient receptor potential vanilloid 4 (TRPV4) attenuated the H2O2-induced Ca2+ influx. Additionally, knockdown of TRPV4 in HLMVEC also attenuated calcium influx following H2O2 challenge. Administration of H2O2 or TRPV4 agonists decreased transmembrane electrical resistance (TER), suggesting increased barrier permeability. To explore the regulatory mechanisms underlying TRPV4 activation by ROS, we examined H2O2-induced Ca2+ influx in MLMVECs and HLMVECs with either genetic deletion, silencing, or pharmacologic inhibition of Fyn, a Src family kinase. In both MLMVECs derived from mice deficient for Fyn and HLMVECs treated with either siRNA targeted to Fyn or the Src family kinase inhibitor SU-6656 for 24 or 48 h, the H2O2-induced Ca2+ influx was attenuated. Treatment with SU-6656 decreased the levels of phosphorylated, but not total, TRPV4 protein and had no effect on TRPV4 response to the external agonist, GSK1016790A. In conclusion, our data suggest that application of exogenous H2O2 increases [Ca2+]i and decreases TER in microvascular endothelial cells via activation of TRPV4 through a mechanism that requires the Src kinase Fyn. PMID:26453519

  20. Differential effects of Bartonella henselae on human and feline macro- and micro-vascular endothelial cells.

    PubMed

    Berrich, Moez; Kieda, Claudine; Grillon, Catherine; Monteil, Martine; Lamerant, Nathalie; Gavard, Julie; Boulouis, Henri Jean; Haddad, Nadia

    2011-01-01

    Bartonella henselae, a zoonotic agent, induces tumors of endothelial cells (ECs), namely bacillary angiomatosis and peliosis in immunosuppressed humans but not in cats. In vitro studies on ECs represent to date the only way to explore the interactions between Bartonella henselae and vascular endothelium. However, no comparative study of the interactions between Bartonella henselae and human (incidental host) ECs vs feline (reservoir host) ECs has been carried out because of the absence of any available feline endothelial cell lines.To this purpose, we have developed nine feline EC lines which allowed comparing the effects of Bartonella strains on human and feline micro-vascular ECs representative of the infection development sites such as skin, versus macro-vascular ECs, such as umbilical vein.Our model revealed intrinsic differences between human (Human Skin Microvascular ECs -HSkMEC and Human Umbilical Vein ECs - iHUVEC) and feline ECs susceptibility to Bartonella henselae infection.While no effect was observed on the feline ECs upon Bartonella henselae infection, the human ones displayed accelerated angiogenesis and wound healing.Noticeable differences were demonstrated between human micro- and macro-vasculature derived ECs both in terms of pseudo-tube formation and healing. Interestingly, Bartonella henselae effects on human ECs were also elicited by soluble factors.Neither Bartonella henselae-infected Human Skin Microvascular ECs clinically involved in bacillary angiomatosis, nor feline ECs increased cAMP production, as opposed to HUVEC.Bartonella henselae could stimulate the activation of Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) in homologous cellular systems and trigger VEGF production by HSkMECs only, but not iHUVEC or any feline ECs tested.These results may explain the decreased pathogenic potential of Bartonella henselae infection for cats as compared to humans and strongly suggest that an autocrine secretion of VEGF by human skin

  1. Platelet lysate gel and endothelial progenitors stimulate microvascular network formation in vitro: tissue engineering implications

    PubMed Central

    Fortunato, Tiago M.; Beltrami, Cristina; Emanueli, Costanza; De Bank, Paul A.; Pula, Giordano

    2016-01-01

    Revascularisation is a key step for tissue regeneration and complete organ engineering. We describe the generation of human platelet lysate gel (hPLG), an extracellular matrix preparation from human platelets able to support the proliferation of endothelial colony forming cells (ECFCs) in 2D cultures and the formation of a complete microvascular network in vitro in 3D cultures. Existing extracellular matrix preparations require addition of high concentrations of recombinant growth factors and allow only limited formation of capillary-like structures. Additional advantages of our approach over existing extracellular matrices are the absence of any animal product in the composition hPLG and the possibility of obtaining hPLG from patients to generate homologous scaffolds for re-implantation. This discovery has the potential to accelerate the development of regenerative medicine applications based on implantation of microvascular networks expanded ex vivo or the generation of fully vascularised organs. PMID:27141997

  2. Human microvascular lymphatic and blood endothelial cells produce fibrillin: deposition patterns and quantitative analysis.

    PubMed

    Rossi, Antonella; Gabbrielli, Erica; Villano, Marilisa; Messina, Mario; Ferrara, Francesco; Weber, Elisabetta

    2010-12-01

    Fibrillin microfibrils constitute a scaffold for elastin deposition in the wall of arteries and form the anchoring filaments that connect the lymphatic endothelium to surrounding elastic fibers. We previously reported that fibrillin is deposited in a honeycomb pattern in bovine arterial endothelial cells, which also deposit microfibril-associated glycoprotein (MAGP)-1, whereas thoracic duct endothelial cells form an irregular web. The present immunohistochemical study was designed to verify whether lymphatic and blood human dermal microvascular endothelial cells (HDMECs) isolated from human foreskin by the sequential use of a pan-endothelial marker, CD31, and the lymphatic specific marker, D2-40, deposit fibrillin and MAGP-1. In both cell types, fibrillin and MAGP-1 co-localized and were deposited with different patterns of increasing complexity co-existing in the same culture. Fibrillin microfibrils formed a wide-mesh honeycomb leaving fibrillin-free spaces that were gradually filled. This modality of fibrillin deposition, similar to that of bovine large artery endothelial cells, was basically the same in blood and lymphatic HDMECs. In some lymphatic HDMECs, fibrillin was initially deposited as uniformly scattered short fibrillin strands probably as a result of anchoring filaments carried over from the vessels of origin. Our findings show that blood and lymphatic endothelial cells participate in fibrillin deposition in human skin.

  3. A novel paradigm for heart failure with preserved ejection fraction: comorbidities drive myocardial dysfunction and remodeling through coronary microvascular endothelial inflammation.

    PubMed

    Paulus, Walter J; Tschöpe, Carsten

    2013-07-23

    Over the past decade, myocardial structure, cardiomyocyte function, and intramyocardial signaling were shown to be specifically altered in heart failure with preserved ejection fraction (HFPEF). A new paradigm for HFPEF development is therefore proposed, which identifies a systemic proinflammatory state induced by comorbidities as the cause of myocardial structural and functional alterations. The new paradigm presumes the following sequence of events in HFPEF: 1) a high prevalence of comorbidities such as overweight/obesity, diabetes mellitus, chronic obstructive pulmonary disease, and salt-sensitive hypertension induce a systemic proinflammatory state; 2) a systemic proinflammatory state causes coronary microvascular endothelial inflammation; 3) coronary microvascular endothelial inflammation reduces nitric oxide bioavailability, cyclic guanosine monophosphate content, and protein kinase G (PKG) activity in adjacent cardiomyocytes; 4) low PKG activity favors hypertrophy development and increases resting tension because of hypophosphorylation of titin; and 5) both stiff cardiomyocytes and interstitial fibrosis contribute to high diastolic left ventricular (LV) stiffness and heart failure development. The new HFPEF paradigm shifts emphasis from LV afterload excess to coronary microvascular inflammation. This shift is supported by a favorable Laplace relationship in concentric LV hypertrophy and by all cardiac chambers showing similar remodeling and dysfunction. Myocardial remodeling in HFPEF differs from heart failure with reduced ejection fraction, in which remodeling is driven by loss of cardiomyocytes. The new HFPEF paradigm proposes comorbidities, plasma markers of inflammation, or vascular hyperemic responses to be included in diagnostic algorithms and aims at restoring myocardial PKG activity.

  4. Differential susceptibility of equine and mouse brain microvascular endothelial cells to equine herpesvirus 1 infection.

    PubMed

    Hasebe, R; Kimura, T; Nakamura, K; Ochiai, K; Okazaki, K; Wada, R; Umemura, T

    2006-04-01

    Equine herpesvirus 1 (EHV-1) shows endotheliotropism in the central nervous system (CNS) of infected horses. However, infection of endothelial cells has not been observed in the CNS of infected mice. To explore the basis for this difference in endotheliotropism, we compared the susceptibility of equine brain microvascular endothelial cells (EBMECs) and mouse brain microvascular endothelial cells (MBMECs) to EHV-1 infection. The kinetics of viral growth in EBMECs was typical of a fully productive infection whereas viral infection in MBMECs seemed to be nonproductive. Immunofluorescence microscopy using anti-EHV-1 polyclonal antibody demonstrated viral antigen in infected EBMECs, but not infected MBMECs. EHV-1 immediate early (IE), early (ICP0), and late (gB, gD and gK) transcripts were expressed in infected EBMECs. However, none of these genes was detected in infected MBMECs by reverse transcription-polymerase chain reaction. Electron microscopic examination at the stage of viral entry showed that viral particles were present within uncoated vesicles in the cytoplasm of EBMECs, but absent from those of MBMECs. These results suggest that viral entry is an important determinant of the susceptibility of EBMECs and MBMECs to EHV-1 infection.

  5. Association of Microvascular Function and Endothelial Biomarkers With Clinical Outcome in Dengue: An Observational Study

    PubMed Central

    Yacoub, Sophie; Lam, Phung Khanh; Vu, Le Hoang Mai; Le, Thi Lien; Ha, Ngo Thanh; Toan, Tran Thi; Van, Nguyen Thu; Quyen, Nguyen Than Ha; Le Duyen, Huynh Thi; Van Kinh, Nguyen; Fox, Annette; Mongkolspaya, Juthathip; Wolbers, Marcel; Simmons, Cameron Paul; Screaton, Gavin Robert; Wertheim, Heiman; Wills, Bridget

    2016-01-01

    Background. The hallmark of severe dengue is increased microvascular permeability, but alterations in the microcirculation and their evolution over the course of dengue are unknown. Methods. We conducted a prospective observational study to evaluate the sublingual microcirculation using side-stream dark-field imaging in patients presenting early (<72 hours after fever onset) and patients hospitalized with warning signs or severe dengue in Vietnam. Clinical findings, microvascular function, global hemodynamics assessed with echocardiography, and serological markers of endothelial activation were determined at 4 time points. Results. A total of 165 patients were enrolled. No difference was found between the microcirculatory parameters comparing dengue with other febrile illnesses. The proportion of perfused vessels (PPV) and the mean flow index (MFI) were lower in patients with dengue with plasma than those without leakage (PPV, 88.1% vs 90.6% [P = .01]; MFI, 2.1 vs 2.4 [P = .007]), most markedly during the critical phase. PPV and MFI were correlated with the endothelial activation markers vascular cell adhesion molecule 1 (P < .001 for both) and angiopoietin 2 (P < .001 for both), negatively correlated. Conclusions. Modest microcirculatory alterations occur in dengue, are associated with plasma leakage, and are correlate with molecules of endothelial activation, angiopoietin 2 and vascular cell adhesion molecule 1. PMID:27230099

  6. Modulation of platelet-derived growth factor receptor expression in microvascular endothelial cells during in vitro angiogenesis.

    PubMed Central

    Marx, M; Perlmutter, R A; Madri, J A

    1994-01-01

    Microvascular endothelial cells in vivo exhibit a plastic phenotype, forming a nonproliferative, differentiated capillary network, while retaining their ability to respond to injury by proliferation, migration and neovascularization. The presence of PDGF receptors and PDGF responsiveness in microvascular endothelial cells and the significance of PDGF isoforms in the control of endothelial cell growth and differentiation remain controversial. Since culture of microvascular endothelial cells in a three-dimensional (3D) system induced cell differentiation and angiogenesis and inhibited proliferation, the present study investigates the role of different extracellular matrix environments in inducing different microvascular endothelial cell phenotypes on microvascular endothelial cell PDGF receptor expression and PDGF responsiveness. In conventional two-dimensional (2D) culture, microvascular endothelial cells expressed both PDGF receptor alpha and beta chains. Suramin treatment demonstrated continuous downregulation of the alpha receptor surface expression. PDGF BB and, to a lesser extent, PDGF AB were mitogenic in 2D-culture, PDGF AA failed to induce any proliferative response despite inducing receptor autophosphorylation. During in vitro angiogenesis induced by 3D-culture, both PDGF receptors were rapidly downregulated. Assessment of cell proliferation showed quiescent cells and PDGF unresponsiveness. We conclude that the induction of a differentiated phenotype during in vitro angiogenesis (tube formation) driven in part by the spatial organization of the surrounding matrix is associated with a downregulation of PDGF receptors. Identification of the molecular cell-matrix interactions involved in this receptor regulation may allow for targeted manipulation of cell growth in vivo and lead to novel therapeutic applications for PDGF. Images PMID:7506710

  7. Macro- and microvascular endothelial cells in vitro: maintenance of biochemical heterogeneity despite loss of ultrastructural characteristics

    SciTech Connect

    Stolz, D.B.; Jacobson, B.S. )

    1991-02-01

    Microvascular endothelial cells from bovine adrenal medulla and brain and macrovessel endothelial cells from bovine aorta were isolated and cultured under similar conditions in order to determine morphologic and biochemical heterogeneity in vitro. All three cell types exhibited nearly identical ultrastructural morphology and two-dimensional gel protein patterns of {sup 35}S-methionine-labeled whole cells. Two-dimensional gel analysis of {sup 35}S-methionine-labeled plasma membrane proteins however, revealed two-dimensional gel protein patterns unique to the tissue type from which the endothelial cells were isolated. This suggests that the functional significance of these specific endothelial cell types is manifested primarily in surface-associated proteins and that many of the differences are sustained in culture. To determine the potential of aorta, brain, and adrenal medulla endothelial cell (EC) cultures to respond to developmentally significant signals, morphology, growth pattern, and cell surface proteins were monitored in the presence and absence of growth factors. A 17 to 26% increase in cell density as well as an increase in the number of elongated and overlapping cells resulted when all three EC types were exposed to a mitogenic medium. Additionally, expression of specific glycoprotein profiles, as determined by Concanavalin A Western blotting of two-dimensional gels, was dependent on the presence or absence of growth factors in the medium. The ability to induce this morphologic and biochemical variation in the three endothelial cell types was maintained into later passage. Taken together, these data imply that endothelial cells isolated from different tissues exhibit and maintain biochemical heterogeneity and do not completely dedifferentiate into a common endothelial cell type in culture.

  8. Perfluorooctane sulfonate (PFOS) induces reactive oxygen species (ROS) production in human microvascular endothelial cells: role in endothelial permeability

    PubMed Central

    Qian, Yong; Ducatman, Alan; Ward, Rebecca; Leonard, Steve; Bukowski, Valerie; Guo, Nancy Lan; Shi, Xianglin; Vallyathan, Val; Castranova, Vincent

    2011-01-01

    Perfluorooctane sulfonate (PFOS) is a member of perfluoroalkyl acids (PFAA) containing an 8-carbon backbone. PFOS is a man-made chemical with carbon-fluorine bonds that are one of the strongest in organic chemistry and widely used in industry. Human occupational and environmental exposure to PFOS occurs globally. PFOS is non-biodegradable and persistent in the human body and environment. In this study, data demonstrated that exposure of human microvascular endothelial cells (HMVEC) to PFOS induced the production of reactive oxygen species (ROS) at both high and low concentrations. Morphologically, it was found that exposure to PFOS induced actin filament remodeling and endothelial permeability changes in HMVEC. Furthermore, data demonstrated the production of ROS plays a regulatory role in PFOS-induced actin filament remodeling and the increase in endothelial permeability. Our results indicate that the generation of ROS may play a role in PFOS-induced aberrations of the endothelial permeability barrier. The results generated from this study may provide a new insight into the potential adverse effects of PFOS exposure on humans at the cellular level. PMID:20391123

  9. Neuregulin1-β decreases IL-1β-induced neutrophil adhesion to human brain microvascular endothelial cells

    PubMed Central

    Wu, Limin; Walas, Samantha; Leung, Wendy; Sykes, David B.; Wu, Jiang; Lo, Eng H.; Lok, Josephine

    2014-01-01

    Neuroinflammation contributes to the pathophysiology of diverse diseases including stroke, traumatic brain injury, Alzheimer's Disease, Parkinson's Disease, and multiple sclerosis, resulting in neurodegeneration and loss of neurological function. The response of the microvascular endothelium often contributes to neuroinflammation. One such response is the up-regulation of endothelial adhesion molecules which facilitate neutrophil adhesion to the endothelium and their migration from blood to tissue. Neuregulin-1 (NRG1) is an endogenous growth factor which has been reported to have anti-inflammatory effects in experimental stroke models. We hypothesized that NRG1 would decrease the endothelial response to inflammation, and result in a decrease in neutrophil adhesion to endothelial cells. We tested this hypothesis in an in-vitro model of cytokine-induced endothelial injury, in which human brain microvascular endothelial cells (BMECs) were treated with IL-1β, along with co-incubation with vehicle or NRG1-β. Outcome measures included protein levels of endothelial ICAM-1, VCAM-1, and E-selectin; as well as the number of neutrophils that adhere to the endothelial monolayer. Our data show that NRG1-β decreased the levels of VCAM-1, E-selectin, and neutrophil adhesion to brain microvascular endothelial cells activated by IL1-β. These findings open new possibilities for investigating NRG1 in neuroprotective strategies in brain injury. PMID:24863743

  10. Magnetic particle spectroscopy allows precise quantification of nanoparticles after passage through human brain microvascular endothelial cells

    NASA Astrophysics Data System (ADS)

    Gräfe, C.; Slabu, I.; Wiekhorst, F.; Bergemann, C.; von Eggeling, F.; Hochhaus, A.; Trahms, L.; Clement, J. H.

    2016-06-01

    Crossing the blood-brain barrier is an urgent requirement for the treatment of brain disorders. Superparamagnetic iron oxide nanoparticles (SPIONs) are a promising tool as carriers for therapeutics because of their physical properties, biocompatibility, and their biodegradability. In order to investigate the interaction of nanoparticles with endothelial cell layers in detail, in vitro systems are of great importance. Human brain microvascular endothelial cells are a well-suited blood-brain barrier model. Apart from generating optimal conditions for the barrier-forming cell units, the accurate detection and quantification of SPIONs is a major challenge. For that purpose we use magnetic particle spectroscopy to sensitively and directly quantify the SPION-specific iron content. We could show that SPION concentration depends on incubation time, nanoparticle concentration and location. This model system allows for further investigations on particle uptake and transport at cellular barriers with regard to parameters including particles’ shape, material, size, and coating.

  11. Pericyte abundance affects sucrose permeability in cultures of rat brain microvascular endothelial cells.

    PubMed

    Parkinson, Fiona E; Hacking, Cindy

    2005-07-05

    The blood-brain barrier is a physical and metabolic barrier that restricts diffusion of blood-borne substances into brain. In vitro models of the blood-brain barrier are used to characterize this structure, examine mechanisms of damage and repair and measure permeability of test substances. The core component of in vitro models of the blood-brain barrier is brain microvascular endothelial cells. We cultured rat brain microvascular endothelial cells (RBMEC) from isolated rat cortex microvessels. After 2-14 days in vitro (DIV), immunohistochemistry of these cells showed strong labeling for zona occludens 1 (ZO-1), a tight junction protein expressed in endothelial cells. Pericytes were also present in these cultures, as determined by expression of alpha-actin. The present study was performed to test different cell isolation methods and to compare the resulting cell cultures for abundance of pericytes and for blood-brain barrier function, as assessed by 14C-sucrose flux. Two purification strategies were used. First, microvessels were preabsorbed onto uncoated plastic for 4 h, then unattached microvessels were transferred to coated culture ware. Second, microvessels were incubated with an antibody to platelet-endothelial cell adhesion molecule 1 (PECAM-1; CD31) precoupled to magnetic beads, and a magnetic separation procedure was performed. Our results indicate that immunopurification, but not preadsorption, was an effective method to purify microvessels and reduce pericyte abundance in the resulting cultures. This purification significantly reduced 14C-sucrose fluxes across cell monolayers. These data indicate that pericytes can interfere with the development of blood-brain barrier properties in in vitro models that utilize primary cultures of RBMECs.

  12. Mechanisms of modulation of brain microvascular endothelial cells function by thrombin.

    PubMed

    Brailoiu, Eugen; Shipsky, Megan M; Yan, Guang; Abood, Mary E; Brailoiu, G Cristina

    2017-02-15

    Brain microvascular endothelial cells are a critical component of the blood-brain barrier. They form a tight monolayer which is essential for maintaining the brain homeostasis. Blood-derived proteases such as thrombin may enter the brain during pathological conditions like trauma, stroke, and inflammation and further disrupts the permeability of the blood-brain barrier, via incompletely characterized mechanisms. We examined the underlying mechanisms evoked by thrombin in rat brain microvascular endothelial cells (RBMVEC). Our results indicate that thrombin, acting on protease-activated receptor 1 (PAR1) increases cytosolic Ca(2+) concentration in RBMVEC via Ca(2+) release from endoplasmic reticulum through inositol 1,4,5-trisphosphate receptors and Ca(2+) influx from extracellular space. Thrombin increases nitric oxide production; the effect is abolished by inhibition of the nitric oxide synthase or by antagonism of PAR1 receptors. In addition, thrombin increases mitochondrial and cytosolic reactive oxygen species production via PAR1-dependent mechanisms. Immunocytochemistry studies indicate that thrombin increases F-actin stress fibers, and disrupts the tight junctions. Thrombin increased the RBMVEC permeability assessed by a fluorescent flux assay. Taken together, our results indicate multiple mechanisms by which thrombin modulates the function of RBMVEC and may contribute to the blood-brain barrier dysfunction.

  13. The Effects of Copper on Brain Microvascular Endothelial Cells and Claudin Via Apoptosis and Oxidative Stress.

    PubMed

    Wang, Jian; Chen, Junquan; Tang, Zhaoxin; Li, Ying; Hu, Lianmei; Pan, Jiaqiang

    2016-11-01

    Many neurodegenerative diseases are related to copper although the effects on brain microvascular endothelial cells (BMECs) are poorly understood. In the present study, a primary BMEC culture model was established to evaluate the effects of copper on brain microvascular endothelial cells and whether claudin-1, claudin-3, claudin-5, and claudin-12 isoforms contribute to apoptosis and intrinsic antioxidant activity. Our results showed that copper ions had dual effects on BMECs by regulating intracellular reactive oxygen species (ROS) levels. Copper levels between 30 and 120 μM could enhance viability and promote proliferation. On the other hand, copper cytotoxicity was a result of apoptosis indicating a redox-independent manner of cell death. Expression levels of claudins were also regulated by copper in a concentration-dependent manner. We identified four claudin isoforms (1, 3, 5, and 12) and showed that their expression levels were regulated as a group by copper. Antioxidant activity of BMECs was also copper regulated, and superoxide dismutase and catalase were the main contributors to BMEC antioxidant functions. Together, our results indicated that copper had dual effects on BMEC growth and intrinsic antioxidant activities played a crucial role in BMEC survival and tight junction.

  14. EFFECT OF IRRADIATION ON MICROVASCULAR ENDOTHELIAL CELLS OF PAROTID GLANDS IN THE MINIATURE PIG

    PubMed Central

    Xu, Junji; Yan, Xing; Gao, Runtao; Mao, Lisha; Cotrim, Ana P.; Zheng, Changyu; Zhang, Chunmei; Baum, Bruce J.; Wang, Songlin

    2013-01-01

    Purpose To evaluate the effect of irradiation on microvascular endothelial cells in miniature pig parotid glands. Methods and Materials A single 25-Gy dose of irradiation (IR) was delivered to parotid glands of 6 miniature pigs. Three other animals served as non-IR controls. Local blood flow rate in glands was measured pre- and post-IR with an ultrasonic Doppler analyzer. Samples of parotid gland tissue were taken at 4 h, 24 h, 1 week, and 2 weeks after IR for microvascular density (MVD) analysis and sphingomyelinase (SMase) assay. Histopathology and immunohistochemical staining (anti-CD31 and anti-AQP1) were used to assess morphological changes. MVD was determined by calculating the number of CD31- or AQP1-stained cells per field. A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay was used to detect apoptotic cells. The activity of acid and neutral Mg2+-dependent SMase (ASMase and NSMase, respectively) was also assayed. Results Local parotid gland blood flow rate decreased rapidly at 4 h post-IR and remained below control levels throughout the 14-day observation period. Parotid MVD also declined from 4 to 24 hours and remained below control levels thereafter. The activity levels of ASMase and NSMase in parotid glands increased rapidly from 4 to 24 h post-IR and then declined gradually. The frequency of detecting apoptotic nuclei in the glands followed similar kinetics. Conclusions Single-dose IR led to a significant reduction of MVD and local blood flow rate, indicating marked damage to microvascular endothelial cells in miniature pig parotid glands. The significant and rapid increases of ASMase and NSMase activity levels may be important in this IR-induced damage. PMID:20832188

  15. Effect of Irradiation on Microvascular Endothelial Cells of Parotid Glands in the Miniature Pig

    SciTech Connect

    Xu Junji; Yan Xing; Gao Runtao; Mao Lisha; Cotrim, Ana P.; Zheng Changyu; Zhang Chunmei; Baum, Bruce J.; Wang Songlin

    2010-11-01

    Purpose: To evaluate the effect of irradiation on microvascular endothelial cells in miniature pig parotid glands. Methods and Materials: A single 25-Gy dose of irradiation (IR) was delivered to parotid glands of 6 miniature pigs. Three other animals served as non-IR controls. Local blood flow rate in glands was measured pre- and post-IR with an ultrasonic Doppler analyzer. Samples of parotid gland tissue were taken at 4 h, 24 h, 1 week, and 2 weeks after IR for microvascular density (MVD) analysis and sphingomyelinase (SMase) assay. Histopathology and immunohistochemical staining (anti-CD31 and anti-AQP1) were used to assess morphological changes. MVD was determined by calculating the number of CD31- or AQP1-stained cells per field. A terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) apoptosis assay was used to detect apoptotic cells. The activity of acid and neutral Mg{sup 2+}-dependent SMase (ASMase and NSMase, respectively) was also assayed. Results: Local parotid gland blood flow rate decreased rapidly at 4 h post-IR and remained below control levels throughout the 14-day observation period. Parotid MVD also declined from 4 to 24 hours and remained below control levels thereafter. The activity levels of ASMase and NSMase in parotid glands increased rapidly from 4 to 24 h post-IR and then declined gradually. The frequency of detecting apoptotic nuclei in the glands followed similar kinetics. Conclusions: Single-dose IR led to a significant reduction of MVD and local blood flow rate, indicating marked damage to microvascular endothelial cells in miniature pig parotid glands. The significant and rapid increases of ASMase and NSMase activity levels may be important in this IR-induced damage.

  16. 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.

  17. Inhibition of dipeptidyl peptidase 4 regulates microvascular endothelial growth induced by inflammatory cytokines

    SciTech Connect

    Takasawa, Wataru; Ohnuma, Kei; Hatano, Ryo; Endo, Yuko; Dang, Nam H.

    2010-10-08

    Research highlights: {yields} TNF-{alpha} or IL-1{beta} induces EC proliferation with reduction of CD26 expression. {yields} CD26 siRNA or DPP-4 inhibition enhances TNF-{alpha} or IL-1{beta}-induced EC proliferation. {yields} Loss of CD26/DPP-4 enhances aortic sprouting induced by TNF-{alpha} or IL-1{beta}. {yields} Capillary formation induced by TNF-{alpha} or IL-1{beta} is enahced in the CD26{sup -/-} mice. -- Abstract: CD26/DPP-4 is abundantly expressed on capillary of inflamed lesion as well as effector T cells. Recently, CD26/dipeptidyl peptidase 4 (DPP-4) inhibition has been used as a novel oral therapeutic approach for patients with type 2 diabetes. While accumulating data indicate that vascular inflammation is a key feature of both micro- and macro-vascular complications in diabetes, the direct role of CD26/DPP-4 in endothelial biology is to be elucidated. We herein showed that proinflammatory cytokines such as tumor necrosis factor or interleukin-1 reduce expression of CD26 on microvascular endothelial cells, and that genetical or pharmacological inhibition of CD26/DPP-4 enhances endothelial growth both in vitro and in vivo. With DPP-4 inhibitors being used widely in the treatment of type 2 diabetes, our data strongly suggest that DPP-4 inhibition plays a pivotal role in endothelial growth and may have a potential role in the recovery of local circulation following diabetic vascular complications.

  18. Increased susceptibility to amyloid-β toxicity in rat brain microvascular endothelial cells under hyperglycemic conditions.

    PubMed

    Carvalho, Cristina; Katz, Paige S; Dutta, Somhrita; Katakam, Prasad V G; Moreira, Paula I; Busija, David W

    2014-01-01

    We hypothesized that hyperglycemia-induced mitochondrial dysfunction and oxidative stress are closely associated with amyloid-β peptide (Aβ) toxicity in endothelial cells. Brain microvascular endothelial cells from rat (RBMEC) and mice (MBMEC) were isolated from adult Sprague-Dawley rats and homozygous db/db (Leprdb/Leprdb) and heterozygous (Dock7m/Leprdb) mice, and cultured under normo- and hyperglycemic conditions for 7 d followed by 24 h exposure to Aβ1-40. Some experiments were also performed with two mitochondrial superoxide (O2•-) scavengers, MitoTempo and Peg-SOD. Cell viability was measured by the Alamar blue assay and mitochondrial membrane potential (ΔΨm) by confocal microscopy. Mitochondrial O2•- and hydrogen peroxide (H2O2) production was assessed by fluorescence microscopy and H2O2 production was confirmed by microplate reader. Hyperglycemia or Aβ1-40 alone did not affect cell viability in RBMEC. However, the simultaneous presence of high glucose and Aβ1-40 reduced cell viability and ΔΨm, and enhanced mitochondrial O2•- and H2O2 production. MitoTempo and PEG-SOD prevented Aβ1-40 toxicity. Interestingly, MBMEC presented a similar pattern of alterations with db/db cultures presenting higher susceptibility to Aβ1-40. Overall, our results show that high glucose levels increase the susceptibility of brain microvascular endothelial cells to Aβ toxicity supporting the idea that hyperglycemia is a major risk factor for vascular injury associated with AD.

  19. Impaired microvascular reactivity and endothelial function in patients with Cushing's syndrome: influence of arterial hypertension.

    PubMed

    Prázný, M; Jezková, J; Horová, E; Lazárová, V; Hána, V; Kvasnicka, J; Pecen, L; Marek, J; Skrha, J; Krsek, M

    2008-01-01

    The aim of the study was to evaluate skin microvascular reactivity (MVR) and possible influencing factors (fibrinolysis, oxidative stress, and endothelial function) in patients with Cushing's syndrome. Twenty-nine patients with active Cushing's syndrome (ten of them also examined after a successful operation) and 16 control subjects were studied. Skin MVR was measured by laser Doppler flowmetry during post-occlusive (PORH) and thermal hyperemia (TH). Malondialdehyde and Cu,Zn-superoxide dismutase were used as markers of oxidative stress. Fibrinolysis was estimated by tissue plasminogen activator (tPA) and its inhibitor (PAI-1). N-acetyl-beta-glucosaminidase, E-selectin, P-selectin, and ICAM-1 were used as markers of endothelial function. Oxidative stress and endothelial dysfunction was present in patients with hypercortisolism, however, increased concentration of ICAM-1 was also found in patients after the operation as compared to controls (290.8+/-74.2 vs. 210.9+/-56.3 ng.ml(-1), p<0.05). Maximal perfusion was significantly lower in patients with arterial hypertension during PORH and TH (36.3+/-13.0 vs. 63.3+/-32.4 PU, p<0.01, and 90.4+/-36.6 vs. 159.2+/-95.3 PU, p<0.05, respectively) and similarly the velocity of perfusion increase during PORH and TH was lower (3.2+/-1.5 vs. 5.2+/-3.4 PU.s(-1), p<0.05, and 0.95+/-0.6 vs. 1.8+/-1.1 PU.s(-1), p<0.05, respectively). The most pronounced impairment of microvascular reactivity was present in patients with combination of arterial hypertension and diabetes mellitus.

  20. Hyperoxic sheep pulmonary microvascular endothelial cells generate free radicals via mitochondrial electron transport.

    PubMed Central

    Sanders, S P; Zweier, J L; Kuppusamy, P; Harrison, S J; Bassett, D J; Gabrielson, E W; Sylvester, J T

    1993-01-01

    Free radical generation by hyperoxic endothelial cells was studied using electron paramagnetic resonance (EPR) spectroscopy and the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Studies were performed to determine the radical species produced, whether mitochondrial electron transport was involved, and the effect of the radical generation on cell mortality. Sheep pulmonary microvascular endothelial cell suspensions exposed to 100% O2 for 30 min exhibited prominent DMPO-OH and, occasionally, additional smaller DMPO-R signals thought to arise from the trapping of superoxide anion (O2-.), hydroxyl (.OH), and alkyl (.R) radicals. Superoxide dismutase (SOD) quenched both signals suggesting that the observed radicals were derived from O2-.. Studies with deferoxamine suggested that the generation of .R occurred secondary to the formation of .OH from O2-. via an iron-mediated Fenton reaction. Blocking mitochondrial electron transport with rotenone (20 microM) markedly decreased radical generation. Cell mortality increased slightly in oxygen-exposed cells. This increase was not significantly altered by SOD or deferoxamine, nor was it different from the mortality observed in air-exposed cells. These results suggest that endothelial cells exposed to hyperoxia for 30 min produce free radicals via mitochondrial electron transport, but under the conditions of these experiments, this radical generation did not appear cause cell death. PMID:8380815

  1. Paracrine crosstalk between human hair follicle dermal papilla cells and microvascular endothelial cells.

    PubMed

    Bassino, Eleonora; Gasparri, Franco; Giannini, Valentina; Munaron, Luca

    2015-05-01

    Human follicle dermal papilla cells (FDPC) are a specialized population of mesenchymal cells located in the skin. They regulate hair follicle (HF) development and growth, and represent a reservoir of multipotent stem cells. Growing evidence supports the hypothesis that HF cycling is associated with vascular remodeling. Follicular keratinocytes release vascular endothelial growth factor (VEGF) that sustains perifollicular angiogenesis leading to an increase of follicle and hair size. Furthermore, several human diseases characterized by hair loss, including Androgenetic Alopecia, exhibit alterations of skin vasculature. However, the molecular mechanisms underlying HF vascularization remain largely unknown. In vitro coculture approaches can be successfully employed to greatly improve our knowledge and shed more light on this issue. Here we used Transwell-based co-cultures to show that FDPC promote survival, proliferation and tubulogenesis of human microvascular endothelial cells (HMVEC) more efficiently than fibroblasts. Accordingly, FDPC enhance the endothelial release of VEGF and IGF-1, two well-known proangiogenic growth factors. Collectively, our data suggest a key role of papilla cells in vascular remodeling of the hair follicle.

  2. GPR40/FFA1 and Neutral Sphingomyelinase Are Involved in Palmitate-Boosted Inflammatory Response of Microvascular Endothelial Cells to LPS

    PubMed Central

    Lu, Zhongyang; Li, Yanchun; Jin, Junfei; Zhang, Xiaoming; Hannun, Yusuf A.; Huang, Yan

    2015-01-01

    Objectives Increased levels of both saturated fatty acids (SFAs) and lipopolysaccharide (LPS) are associated with type 2 diabetes. However, it remains largely unknown how SFAs interact with LPS to regulate inflammatory responses in microvascular endothelial cells (MIC ECs) that are critically involved in atherosclerosis as a diabetic complication. In this study, we compared the effects of LPS, palmitic acid (PA), the most abundant saturated fatty acid, or the combination of LPS and PA on interleukin (IL)-6 expression by MIC ECs and explored the underlying mechanisms. Methods Human cardiac MIC ECs were treated with LPS, PA and LPS plus PA and the regulatory pathways including receptors, signal transduction, transcription and post-transcription, and sphingolipid metabolism for IL-6 expression were investigated. Results G protein-coupled receptor (GPR)40 or free fatty acid receptor 1 (FFA1), but not toll-like receptor 4, was involved in PA-stimulated IL-6 expression. PA not only stimulated IL-6 expression by itself, but also remarkably enhanced LPS-stimulated IL-6 expression via a cooperative stimulation on mitogen-activated protein kinase and nuclear factor kappa B signaling pathways, and both transcriptional and post-transcriptional activation. Furthermore, PA induced a robust neutral sphingomyelinase (nSMase)-mediated sphingomyelin hydrolysis that was involved in PA-augmented IL-6 upregulation. Conclusion PA boosted inflammatory response of microvascular endothelial cells to LPS via GPR40 and nSMase. PMID:25795558

  3. The Association of Serum Vascular Endothelial Growth Factor and Ferritin in Diabetic Microvascular Disease

    PubMed Central

    Guo, Li; Jiang, Fang; Tang, Yue-Ting; Si, Meng-Ya

    2014-01-01

    Abstract Background: Vascular endothelial growth factor (VEGF) is involved in the pathogenesis of diabetic microvascular disease. Most diabetes patients have higher serum levels of ferritin that may participate in diabetic vascular complications through high oxidative stress induced by iron. However, the mechanistic link between ferritin and VEGF is obscure. The study investigated the association of VEGF and ferritin in patients with diabetic microvascular disease. Patients and Methods: Sixty patients with type 2 diabetes mellitus (T2DM) and 26 healthy individuals were selected in this study. Serum ferritin, VEGF, hematological parameters, and clinical data were assessed in this cohort. The Spearman rank method was used to evaluate the associations among them. Results: Serum levels of VEGF and ferritin were significantly higher in diabetes patients compared with the controls; levels of both were elevated with development of the disease. There were positive correlations between VEGF and glucose levels and between VEGF and ferritin in diabetes groups, especially in patients with diabetic retinopathy. Positive correlations were also found between VEGF level and the parameters of age, hemoglobin, and albumin in patients with diabetes hypertension. Conclusions: Our data suggest that high ferritin levels in T2DM are closely related to the development of diabetic vascular complications through interaction with VEGF. PMID:24279470

  4. Lipopolysaccharide Induces Human Pulmonary Micro-Vascular Endothelial Apoptosis via the YAP Signaling Pathway

    PubMed Central

    Yi, Lei; Huang, Xiaoqin; Guo, Feng; Zhou, Zengding; Chang, Mengling; Tang, Jiajun; Huan, Jingning

    2016-01-01

    Gram-negative bacterial lipopolysaccharide (LPS) induces a pathologic increase in lung vascular leakage under septic conditions. LPS-induced human pulmonary micro-vascular endothelial cell (HPMEC) apoptosis launches and aggravates micro-vascular hyper-permeability and acute lung injury (ALI). Previous studies show that the activation of intrinsic apoptotic pathway is vital for LPS-induced EC apoptosis. Yes-associated protein (YAP) has been reported to positively regulate intrinsic apoptotic pathway in tumor cells apoptosis. However, the potential role of YAP protein in LPS-induced HPMEC apoptosis has not been determined. In this study, we found that LPS-induced activation and nuclear accumulation of YAP accelerated HPMECs apoptosis. LPS-induced YAP translocation from cytoplasm to nucleus by the increased phosphorylation on Y357 resulted in the interaction between YAP and transcription factor P73. Furthermore, inhibition of YAP by small interfering RNA (siRNA) not only suppressed the LPS-induced HPMEC apoptosis but also regulated P73-mediated up-regulation of BAX and down-regulation of BCL-2. Taken together, our results demonstrated that activation of the YAP/P73/(BAX and BCL-2)/caspase-3 signaling pathway played a critical role in LPS-induced HPMEC apoptosis. Inhibition of the YAP might be a potential therapeutic strategy for lung injury under sepsis. PMID:27807512

  5. Inhibition of Murine Pulmonary Microvascular Endothelial Cell Apoptosis Promotes Recovery of Barrier Function under Septic Conditions

    PubMed Central

    Wang, Lefeng; Mehta, Sanjay; Brock, Michael

    2017-01-01

    Sepsis is characterized by injury of the pulmonary microvasculature and the pulmonary microvascular endothelial cells (PMVEC), leading to barrier dysfunction and acute respiratory distress syndrome (ARDS). Our recent work identified a strong correlation between PMVEC apoptosis and microvascular leak in septic mice in vivo, but the specific role of apoptosis in septic PMVEC barrier dysfunction remains unclear. Thus, we hypothesize that PMVEC apoptosis is likely required for PMVEC barrier dysfunction under septic conditions in vitro. Septic stimulation (mixture of tumour necrosis factor α, interleukin 1β, and interferon γ [cytomix]) of isolated murine PMVEC resulted in a significant loss of barrier function as early as 4 h after stimulation, which persisted until 24 h. PMVEC apoptosis, as reflected by caspase activation, DNA fragmentation, and loss of membrane polarity, was first apparent at 8 h after cytomix. Pretreatment of PMVEC with the pan-caspase inhibitor Q-VD significantly decreased septic PMVEC apoptosis and was associated with reestablishment of PMVEC barrier function at 16 and 24 h after stimulation but had no effect on septic PMVEC barrier dysfunction over the first 8 h. Collectively, our data suggest that early septic murine PMVEC barrier dysfunction driven by proinflammatory cytokines is not mediated through apoptosis, but PMVEC apoptosis contributes to late septic PMVEC barrier dysfunction. PMID:28250575

  6. Impact of Calcium Signaling during Infection of Neisseria meningitidis to Human Brain Microvascular Endothelial Cells

    PubMed Central

    Asmat, Tauseef M.; Tenenbaum, Tobias; Jonsson, Ann-Beth

    2014-01-01

    The pili and outer membrane proteins of Neisseria meningitidis (meningococci) facilitate bacterial adhesion and invasion into host cells. In this context expression of meningococcal PilC1 protein has been reported to play a crucial role. Intracellular calcium mobilization has been implicated as an important signaling event during internalization of several bacterial pathogens. Here we employed time lapse calcium-imaging and demonstrated that PilC1 of meningococci triggered a significant increase in cytoplasmic calcium in human brain microvascular endothelial cells, whereas PilC1-deficient meningococci could not initiate this signaling process. The increase in cytosolic calcium in response to PilC1-expressing meningococci was due to efflux of calcium from host intracellular stores as demonstrated by using 2-APB, which inhibits the release of calcium from the endoplasmic reticulum. Moreover, pre-treatment of host cells with U73122 (phospholipase C inhibitor) abolished the cytosolic calcium increase caused by PilC1-expressing meningococci demonstrating that active phospholipase C (PLC) is required to induce calcium transients in host cells. Furthermore, the role of cytosolic calcium on meningococcal adherence and internalization was documented by gentamicin protection assay and double immunofluorescence (DIF) staining. Results indicated that chelation of intracellular calcium by using BAPTA-AM significantly impaired PilC1-mediated meningococcal adherence to and invasion into host endothelial cells. However, buffering of extracellular calcium by BAPTA or EGTA demonstrated no significant effect on meningococcal adherence to and invasion into host cells. Taken together, these results indicate that meningococci induce calcium release from intracellular stores of host endothelial cells via PilC1 and cytoplasmic calcium concentrations play a critical role during PilC1 mediated meningococcal adherence to and subsequent invasion into host endothelial cells. PMID:25464500

  7. A simple method for isolating and culturing the rat brain microvascular endothelial cells.

    PubMed

    Liu, Yang; Xue, Qiang; Tang, Qing; Hou, Min; Qi, Hongyi; Chen, Gang; Chen, Weihai; Zhang, Jifen; Chen, Yi; Xu, Xiaoyu

    2013-11-01

    Brain microvascular endothelial cells (BMECs), a main component of the blood-brain barrier, play a critical role in the pathogenesis of many brain diseases. The primary culture of BMECs has been used in various models for studying cerebrovascular diseases in vitro. However, there are still several problems existing in the isolation and cultivation of primary rat BMECs, such as low yield, contamination with other cell types, and requirement of a large number of animals and expensive growth factor. In this study, we describe a simple, economical (without any growth factor) and repeatable method to obtain endothelial cells with high purity (>99%) and yield (about 2.2×10(7) per rat) from cerebral cortexes of neonatal rat, mainly from gray matter. In vitro examinations determined that the isolated cells expressed typical phenotypic markers of differentiated brain endothelium such as multiple drug resistant protein, von Willebrand factor, platelet endothelial cell adhesion molecule 1 (PECAM-1/CD31), and intercellular adhesion molecule (ICAM). These cells also possessed morphological and ultra-structural characteristics that were observed by phase contrast microscope and electric microscope. Then GFAP and α-SMA were used, respectively, to identify astrocyte and pericyte which were potential to contaminate primary culturing of BMECs. And specific reaction of endothelial cells to external stimulation was tested by culture with TNF-α for 24h. All these results of our experiments supply that our protocol provides an effective and reliable method to obtain high purity and yield of rat BMECs and offers a useful tool for studying cellular physiology, cerebrovascular diseases, brain tumors, blood-brain barrier and neurovascular units, etc.

  8. Regulation of endothelial VCAM-1 expression in murine cardiac grafts. Roles for TNF and IL4.

    PubMed Central

    Bergese, S.; Pelletier, R.; Vallera, D.; Widmer, M.; Orosz, C.

    1995-01-01

    The in vivo mechanisms of vascular endothelial activation and VCAM-1 expression were studied in murine heterotopic cardiac grafts. Preliminary studies demonstrated that cardiac allograft endothelia develop reactivity with MECA-32 monoclonal antibody (MAb) and M/K-2 (anti-VCAM-1) MAb within 3 days of transplantation, whereas cardiac isografts develop MECA-32 reactivity but no M/K-2 reactivity. Additional studies demonstrated that a single treatment of cardiac isograft recipients with the anti-CD3 MAb 145-2C11 induces VCAM-1 expression on isograft microvascular endothelia within 24 hours. We have used this experimental system to identify the cytokines responsible for expression of VCAM-1 and MECA-32 MAb reactivity on graft vascular endothelia. We report that the expression of VCAM-1 on isograft endothelia that was induced with anti-CD3 MAb was blocked by simultaneous treatment with either pentoxifylline, soluble tumor necrosis factor (TNF) receptor (TNFR-Fc), anti-IL4 MAb, or soluble IL4R, but not by anti-IFN-gamma MAb. Alternatively, a similar pattern of isograft endothelial VCAM-1 expression was stimulated in the absence of anti-CD3 MAbs with a single injection of human recombinant TNF-alpha, or with recombinant murine IL4 provided as IL4/anti-IL4 MAb complexes. In addition, the IL4-induced VCAM-1 expression was completely blocked by a single intravenous treatment of the isograft recipients with TNFR:Fc. This suggests that high concentrations of TNF-alpha can stimulate endothelial VCAM-1 expression, but these concentrations are apparently not achieved in cardiac isografts. In the absence of an inducing agent such as anti-CD3 MAb, the stimulation of VCAM-1 expression with exogenous IL4 may reflect functional interaction between endogenous TNF and exogenous IL4, as suggested by the blocking experiments with TNFR:Fc. Although cardiac isograft endothelia normally develop reactivity with MECA-32 MAb within 3 days of transplantation, treatment of cardiac isograft

  9. Concise Review: Tissue-Specific Microvascular Endothelial Cells Derived from Human Pluripotent Stem Cells

    PubMed Central

    Wilson, Hannah K.; Canfield, Scott G.; Shusta, Eric V.; Palecek, Sean P.

    2014-01-01

    Accumulating evidence suggests that endothelial cells (ECs) display significant heterogeneity across tissue types, playing an important role in tissue regeneration and homeostasis. Recent work demonstrating the derivation of tissue-specific microvascular endothelial cells (TS-MVECs) from human pluripotent stem cells (hPSCs) has ignited the potential to generate tissue-specific models which may be applied to regenerative medicine and in vitro modeling applications. Here we review techniques by which hPSC-derived TS-MVECs have been made to date and discuss how current hPSC-EC differentiation protocols may be directed towards tissue-specific fates. We begin by discussing the nature of EC tissue specificity in vivo and review general hPSC-EC differentiation protocols generated over the last decade. Finally, we describe how specificity can be integrated into hPSC-EC protocols to generate hPSC-derived TS-MVECs in vitro, including EC and parenchymal cell co-culture, directed differentiation, and direct reprogramming strategies. PMID:25070152

  10. Microvascular endothelial cells exhibit optimal aspect ratio for minimizing flow resistance.

    PubMed

    Sumagin, Ronen; Brown, Carl W; Sarelius, Ingrid H; King, Michael R

    2008-04-01

    A recent analytical solution of the three-dimensional Stokes flow through a bumpy tube predicts that for a given bump area, there exists an optimal circumferential wavenumber which minimizes flow resistance. This study uses measurements of microvessel endothelial cell morphology to test whether this prediction holds in the microvasculature. Endothelial cell (EC) morphology was measured in blood perfused in situ microvessels in anesthetized mice using confocal intravital microscopy. EC borders were identified by immunofluorescently labeling the EC surface molecule ICAM-1 which is expressed on the surface but not in the EC border regions. Comparison of this theory with extensive in situ measurements of microvascular EC geometry in mouse cremaster muscle using intravital microscopy reveals that the spacing of EC nuclei in venules ranging from 27 to 106 microm in diameter indeed lies quite close to this predicted optimal configuration. Interestingly, arteriolar ECs are configured to minimize flow resistance not in the resting state, but at the dilated vessel diameter. These results raise the question of whether less organized circulatory systems, such as that found in newly formed solid tumors or in the developing embryo, may deviate from the optimal bump spacing predicted to minimize flow resistance.

  11. Transfer of functional microRNAs between glioblastoma and microvascular endothelial cells through gap junctions

    PubMed Central

    Thuringer, Dominique; Boucher, Jonathan; Jego, Gaetan; Pernet, Nicolas; Cronier, Laurent; Hammann, Arlette; Solary, Eric; Garrido, Carmen

    2016-01-01

    Extensive invasion and angiogenesis are hallmark features of malignant glioblastomas. Here, we co-cultured U87 human glioblastoma cells and human microvascular endothelial cells (HMEC) to demonstrate the exchange of microRNAs that initially involve the formation of gap junction communications between the two cell types. The functional inhibition of gap junctions by carbenoxolone blocks the transfer of the anti-tumor miR-145-5p from HMEC to U87, and the transfer of the pro-invasive miR-5096 from U87 to HMEC. These two microRNAs exert opposite effects on angiogenesis in vitro. MiR-5096 was observed to promote HMEC tubulogenesis, initially by increasing Cx43 expression and the formation of heterocellular gap junctions, and secondarily through a gap-junction independent pathway. Our results highlight the importance of microRNA exchanges between tumor and endothelial cells that in part involves the formation of functional gap junctions between the two cell types. PMID:27661112

  12. Transfer of functional microRNAs between glioblastoma and microvascular endothelial cells through gap junctions.

    PubMed

    Thuringer, Dominique; Boucher, Jonathan; Jego, Gaetan; Pernet, Nicolas; Cronier, Laurent; Hammann, Arlette; Solary, Eric; Garrido, Carmen

    2016-11-08

    Extensive invasion and angiogenesis are hallmark features of malignant glioblastomas. Here, we co-cultured U87 human glioblastoma cells and human microvascular endothelial cells (HMEC) to demonstrate the exchange of microRNAs that initially involve the formation of gap junction communications between the two cell types. The functional inhibition of gap junctions by carbenoxolone blocks the transfer of the anti-tumor miR-145-5p from HMEC to U87, and the transfer of the pro-invasive miR-5096 from U87 to HMEC. These two microRNAs exert opposite effects on angiogenesis in vitro. MiR-5096 was observed to promote HMEC tubulogenesis, initially by increasing Cx43 expression and the formation of heterocellular gap junctions, and secondarily through a gap-junction independent pathway. Our results highlight the importance of microRNA exchanges between tumor and endothelial cells that in part involves the formation of functional gap junctions between the two cell types.

  13. TGFB1 disrupts the angiogenic potential of microvascular endothelial cells of the corpus luteum.

    PubMed

    Maroni, Dulce; Davis, John S

    2011-07-15

    Cyclical formation and regression of the ovarian corpus luteum is required for reproduction. During luteal regression, the microvasculature of the corpus luteum is extensively disrupted. Prostaglandin F2α, a primary signal for luteal regression, induces the expression of transforming growth factor β1 (TGFB1) in the corpus luteum. This study determined the actions of TGFB1 on microvascular endothelial cells isolated from the bovine corpus luteum (CLENDO cells). We hypothesized that TGFB1 participates in the disruption of the microvasculature during luteal regression. TGFB1 activated the canonical SMAD signaling pathway in CLENDO cells. TGFB1 (1 ng/ml) significantly reduced both basal and fetal-calf-serum-stimulated DNA synthesis, without reducing cell viability. TGFB1 also significantly reduced CLENDO cell transwell migration and disrupted the formation of capillary-like structures when CLENDO cells were plated on Matrigel. By contrast, CLENDO cells plated on fibrillar collagen I gels did not form capillary-like structures and TGFB1 induced cell death. Additionally, TGFB1 caused loss of VE-cadherin from cellular junctions and loss of cell-cell contacts, and increased the permeability of confluent CLENDO cell monolayers. These studies demonstrate that TGFB1 acts directly on CLENDO cells to limit endothelial cell function and suggest that TGFB1 might act in the disassembly of capillaries observed during luteal regression.

  14. Role of JNK in network formation of human lung microvascular endothelial cells

    PubMed Central

    Medhora, Meetha; Dhanasekaran, Anuradha; Pratt, Phillip F.; Cook, Craig R.; Dunn, Laurel K.; Gruenloh, Stephanie K.; Jacobs, Elizabeth R.

    2010-01-01

    The signaling mechanisms in vasculogenesis and/or angiogenesis remain poorly understood, limiting the ability to regulate growth of new blood vessels in vitro and in vivo. Cultured human lung microvascular endothelial cells align into tubular networks in the three-dimensional matrix, Matrigel. Overexpression of MAPK phosphatase-1 (MKP-1), an enzyme that inactivates the ERK, JNK, and p38 pathways, inhibited network formation of these cells. Adenoviral-mediated overexpression of recombinant MKP-3 (a dual specificity phosphatase that specifically inactivates the ERK pathway) and dominant negative or constitutively active MEK did not attenuate network formation in Matrigel compared with negative controls. This result suggested that the ERK pathway may not be essential for tube assembly, a conclusion which was supported by the action of specific MEK inhibitor PD 184352, which also did not alter network formation. Inhibition of the JNK pathway using SP-600125 or L-stereoisomer (L-JNKI-1) blocked network formation, whereas the p38 MAPK blocker SB-203580 slightly enhanced it. Inhibition of JNK also attenuated the number of small vessel branches in the developing chick chorioallantoic membrane. Our results demonstrate a specific role for the JNK pathway in network formation of human lung endothelial cells in vitro while confirming that it is essential for the formation of new vessels in vivo. PMID:18263671

  15. Impaired endothelial function and microvascular asymmetrical dimethylarginine in angiotensin II-infused rats: effects of tempol.

    PubMed

    Wang, Dan; Luo, Zaiming; Wang, Xiaoyan; Jose, Pedro A; Falck, John R; Welch, William J; Aslam, Shakil; Teerlink, Tom; Wilcox, Christopher S

    2010-11-01

    Angiotensin (Ang) II causes endothelial dysfunction, which is associated with cardiovascular risk. We investigated the hypothesis that Ang II increases microvascular reactive oxygen species and asymmetrical dimethylarginine and switches endothelial function from vasodilator to vasoconstrictor pathways. Acetylcholine-induced endothelium-dependent responses of mesenteric resistance arterioles were assessed in a myograph and vascular NO and reactive oxygen species by fluorescent probes in groups (n=6) of male rats infused for 14 days with Ang II (200 ng/kg per minute) or given a sham infusion. Additional groups of Ang or sham-infused rats were given oral Tempol (2 mmol · L(-1)). Ang II infusion increased mean blood pressure (119±5 versus 89±7 mm Hg; P<0.005) and plasma malondialdehyde (0.57±0.02 versus 0.37±0.05 μmol · L(-1); P<0.035) and decreased maximal endothelium-dependent relaxation (18±5% versus 54±6%; P<0.005) and hyperpolarizing (19±3% versus 29±3%; P<0.05) responses and NO activity (0.9±0.1 versus 1.6±0.2 U; P<0.01) yet enhanced endothelium-dependent contraction responses (23±5% versus 5±5%; P<0.05) and reactive oxygen species production (0.82±0.05 versus 0.15±0.03 U; P<0.01). Ang II decreased the expression of dimethylarginine dimethylaminohydrolase 2 and increased asymmetrical dimethylarginine in vessels (450±50 versus 260±35 pmol/mg of protein; P<0.01) but not plasma. Tempol prevented any significant changes with Ang II. In conclusion, Ang redirected endothelial responses from relaxation to contraction, reduced vascular NO, and increased asymmetrical dimethylarginine. These effects were dependent on reactive oxygen species and could, therefore, be targeted with effective antioxidant therapy.

  16. Adherence to human lung microvascular endothelial cells (HMVEC-L) of Plasmodium vivax isolates from Colombia

    PubMed Central

    2013-01-01

    Background For years Plasmodium vivax has been considered the cause of benign malaria. Nevertheless, it has been observed that this parasite can produce a severe disease comparable to Plasmodium falciparum. It has been suggested that some physiopathogenic processes might be shared by these two species, such as cytoadherence. Recently, it has been demonstrated that P. vivax-infected erythrocytes (Pv-iEs) have the capacity to adhere to endothelial cells, in which intercellular adhesion molecule-1 (ICAM-1) seems to be involved in this process. Methods Adherence capacity of 21 Colombian isolates, from patients with P. vivax mono-infection to a microvascular line of human lung endothelium (HMVEC-L) was assessed in static conditions and binding was evaluated at basal levels or in tumor necrosis factor (TNF) stimulated cells. The adherence specificity for the ICAM-1 receptor was determined through inhibition with an anti-CD54 monoclonal antibody. Results The majority of P. vivax isolates, 13 out of 21 (61.9%), adhered to the HMVEC-L cells, but P. vivax adherence was at least seven times lower when compared to the four P. falciparum isolates. Moreover, HMVEC-L stimulation with TNF led to an increase of 1.6-fold in P. vivax cytoadhesion, similar to P. falciparum isolates (1.8-fold) at comparable conditions. Also, blockage of ICAM-1 receptor with specific antibodies showed a significant 50% adherence reduction. Conclusions Plasmodium vivax isolates found in Colombia are also capable of adhering specifically in vitro to lung endothelial cells, via ICAM-1 cell receptor, both at basal state and after cell stimulation with TNF. Collectively, these findings reinforce the concept of cytoadherence for P. vivax, but here, to a different endothelial cell line and using geographical distinct isolates, thus contributing to understanding P. vivax biology. PMID:24080027

  17. Trigemino-cardiac reflex during microvascular trigeminal decompression in cases of trigeminal neuralgia.

    PubMed

    Schaller, Bernhard

    2005-01-01

    The trigemino-cardiac reflex (TCR) is a well-recognized phenomenon consisting of bradycardia, arterial hypotension, apnea, and gastric hypermotility during ocular surgery or other manipulations in and around the orbit. Thus far, it could bee shown that central stimulation of the trigeminal nerve during transsphenoidal surgery and surgery for tumors in the cerebellopontine angle can lead to TCR. In cases of microvascular trigeminal decompression for trigeminal neuralgia, no data of the possible occurrence of TCR are available. TCR was defined as a drop in mean arterial blood pressure (MABP) and the heart rate (HR) of more than 20% to the baseline values before the stimulus and coinciding with the manipulation of the trigeminal nerve. Electronic anesthetic recorded perioperative HR and MABP values were reviewed retrospectively in 28 patients who received microvascular trigeminal decompression in cases of trigeminal neuralgia and were divided into two subgroups on the basis of occurrence of TCR during surgery. Of the 28 patients, 5 (18%) showed evidence of TCR during manipulation at the trigeminal radix by separation from microvascular structures. Their HR fell 46% and their MABP 57% during operative procedures near the trigeminal nerve as compared with levels immediately before the stimulus. After cessation of manipulation, HR and MABP returned (spontaneously) to levels before the stimulus. Risk factors of TCR were compared with results from the literature. In conclusion, the present results give evidence of TCR during manipulation of the central part of the trigeminal nerve during microvascular trigeminal decompression in cases of trigeminal neuralgia under a standardized anesthetic protocol.

  18. Microvascular permeability changes might explain cardiac tamponade after alcohol septal ablation for hypertrophic cardiomyopathy.

    PubMed

    Hsu, Jen-Te; Hsiao, Ju-Feng; Chang, Jung-Jung; Chung, Chang-Min; Chang, Shih-Tai; Pan, Kuo-Li

    2014-04-01

    Various sequelae of alcohol septal ablation for hypertrophic obstructive cardiomyopathy have been reported. Of note, some cases of cardiac tamponade after alcohol septal ablation cannot be well explained. We describe the case of a 78-year-old woman with hypertrophic obstructive cardiomyopathy in whom cardiac tamponade developed one hour after alcohol septal ablation, probably unrelated to mechanical trauma. At that time, we noted a substantial difference in the red blood cell-to-white blood cell ratio between the pericardial effusion (1,957.4) and the peripheral blood (728.3). In addition to presenting the patient's case, we speculate that a possible mechanism for acute tamponade--alcohol-induced changes in microvascular permeability--is a reasonable explanation for cases of alcohol septal ablation that are complicated by otherwise-unexplainable massive pericardial effusions.

  19. Hyperosmolarity attenuates TNFα–mediated pro-inflammatory activation of human pulmonary microvascular endothelial cells

    PubMed Central

    Banerjee, Anirban; Moore, Ernest E.; McLaughlin, Nathan J.; Lee, Luis; Jones, Wilbert L.; Johnson, Jeffrey L.; Nydam, Trevor L.; Silliman, Christopher C.

    2013-01-01

    Firm neutrophil (PMN)-endothelial (EC) adhesion is crucial to the PMN-mediated hyperinflammation observed in acute lung injury. Hypertonic saline (HTS) used for resuscitation of hemorrhagic shock has been associated with a decreased incidence of PMN-mediated lung injury/acute respiratory distress syndrome. We hypothesize that physiologically accessible hypertonic incubation (170mM vs. 140mM, osmolarity ranging from 360-300 mOsm/L) inhibits pro-inflammatory activation of human pulmonary microvascular endothelial cells (HMVECs). Pro-inflammatory activation of HMVECs was investigated in response to TNFα including IL-8 release, ICAM-1 surface expression, PMN adhesion, and signaling mechanisms under both isotonic (control) and hypertonic conditions. Hyperosmolarity alone had no effect on either basal IL-8 release or ICAM-1 surface expression, but did lead to concentration-dependent decreases in TNFα–induced IL-8 release, ICAM-1 surface expression, and PMN:HMVEC adhesion. Conversely, HTS activated p38 mitogen-activated protein kinase (MAPK) and enhanced TNFα activation of p38 MAPK. Despite this basal activation, hyperosmolar incubation attenuated TNFα stimulated IL-8 release and ICAM-1 surface expression and subsequent PMN adherence, while p38 MAPK inhibition did not further influence the effects of hyperosmolar conditions on ICAM-1 surface expression. In addition, TNFα induced NF-kB DNA binding, but HTS conditions attenuated this by 31% (p<0.01). In conclusion, HTS reduces PMN:HMVEC adhesion as well as TNFα-induced pro-inflammatory activation of primary HMVECs via attenuation of NF-kB signaling. PMID:23364439

  20. Bcl-2 silencing attenuates hypoxia-induced apoptosis resistance in pulmonary microvascular endothelial cells.

    PubMed

    Cao, Yongmei; Jiang, Zhen; Zeng, Zhen; Liu, Yujing; Gu, Yuchun; Ji, Yingying; Zhao, Yupeng; Li, Yingchuan

    2016-01-01

    Pulmonary arterial hypertension (PAH) is a life-threatening disorder that ultimately causes heart failure. While the underlying causes of this condition are not well understood, previous studies suggest that the anti-apoptotic nature of pulmonary microvascular endothelial cells (PMVECs) in hypoxic environments contributes to PAH pathogenesis. In this study, we focus on the contribution of Bcl-2 and hypoxia response element (HRE) to apoptosis-resistant endothelial cells and investigate the mechanism. PMVECs obtained from either normal rats or apoptosis-resistant PMVECs obtained from PAH rats were transduced with recombinant lentiviral vectors carrying either Bcl-2-shRNA or HRE combined Bcl-2-shRNA, and then cultured these cells for 24 h under hypoxic (5% O2) or normoxic (21% O2) conditions. In normal PMVECs, Bcl-2-shRNA or HRE combined with Bcl-2-shRNA transduction successfully decreased Bcl-2 expression, while increasing apoptosis as well as caspase-3 and P53 expression in a normoxic environment. In a hypoxic environment, the effects of Bcl-2-shRNA treatment on cell apoptosis, and on Bcl-2, caspase-3, P53 expression were significantly suppressed. Conversely, HRE activation combined with Bcl-2-shRNA transduction markedly enhanced cell apoptosis and upregulated caspase-3 and P53 expression, while decreasing Bcl-2 expression. Furthermore, in apoptosis-resistant PMVECs, HRE-mediated Bcl-2 silencing effectively enhanced cell apoptosis and caspase-3 activity. The apoptosis rate was significantly depressed when Lv-HRE-Bcl-2-shRNA was combined with Lv-P53-shRNA or Lv-caspase3-shRNA transduction in a hypoxic environment. These results suggest that HRE-mediated Bcl-2 inhibition can effectively attenuate hypoxia-induced apoptosis resistance in PMVECs by downregulating Bcl-2 expression and upregulating caspase-3 and P53 expression. This study therefore reveals critical insight into potential therapeutic targets for treating PAH.

  1. Hydroxysafflor yellow A protects methylglyoxal-induced injury in the cultured human brain microvascular endothelial cells.

    PubMed

    Li, Wenlu; Liu, Jie; He, Ping; Ni, Zhenzhen; Hu, Yangmin; Xu, Huimin; Dai, Haibin

    2013-08-09

    Individuals with diabetes have high concentration of methylglyoxal (MGO) and have advanced glycation end-products (AGEs) which play an important role in vascular complications, such as stroke. Our previous data demonstrated that hydroxysafflor yellow A (HSYA), a major active chemical component of the safflower yellow pigment, had antiglycation effect on the AGEs formation in vitro. It is not known whether HSYA can protect against MGO-induced injury in cultured human brain microvascular endothelial cells (HBMEC). Using cultured HBMEC, cell injury was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) formation, lactate dehydrogenase (LDH) release and AnnexinV/PI staining. Advanced glycogen end-products and caspase-3 formation were measured by Western blotting. Incubation of MGO for 24h concentration-dependently induced HBMEC injury, which was protected by HSYA from 10 to 100 μmol/l. Caspase-3 expression and AnnexinV/PI staining illustrated that the protection of HSYA was probably associated with inhibiting cell apoptosis. What's more, MGO promoted AGEs accumulation in the cultured HBMEC, which was also inhibited by 100 μmol/l HSYA. Thus, our results proved that HSYA could inhibit MGO-induced injury in the cultured HBMEC, which was associated with its antiglycation effect.

  2. Fermented Chinese Formula Shuan-Tong-Ling Protects Brain Microvascular Endothelial Cells against Oxidative Stress Injury

    PubMed Central

    Tan, Lingjing; Zhang, Xiang; Wang, Jinfeng; Li, Xiaoli; Huang, Weifeng; Yang, Songbai

    2016-01-01

    Fermented Chinese formula Shuan-Tong-Ling (STL), composed of fourteen medicinal herbs, was an experiential formula by Dr. Zhigang Mei for treating vascular encephalopathy, but the underlying mechanisms remained unknown. In this study, we aimed to investigate the protective effects of fermented STL on hydrogen peroxide- (H2O2-) induced injury in rat brain microvascular endothelial cells (BMECs) and the possible mechanisms. Cultured BMECs were treated with H2O2, STL, or nicotinamide (NAM, a SIRT1 inhibitor). Then, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was employed to detect cell proliferation and senescence-associated beta-galactosidase (SA-β-gal) was used to examine cell senescence. Cell nuclei were observed by 4′,6-diamidino-2-phenylindole. Additionally, changes in reactive oxygen species (ROS), superoxide dismutase (SOD), and glutathione (GSH) levels were measured. Expression of SIRT1, p21, and PGC-1α was determined by western blot. Cell proliferation significantly increased with STL treatment in a dose-dependent manner. H2O2 treatment could intensify cell senescence and nuclei splitting or pyknosis. With STL treatment, the reduced ROS level was accompanied by increased SOD and GSH activity. Further assays showed upregulation of SIRT1 and PGC-1α and downregulation of p21 after STL treatment. The results revealed that STL could protect BMECs against oxidative stress injury at least partially through the SIRT1 pathway. PMID:28096886

  3. Mycoplasmal cerebral vasculopathy in a lymphoma patient: presumptive evidence of Mycoplasma pneumoniae microvascular endothelial cell invasion in a brain biopsy.

    PubMed

    Rhodes, Roy H; Monastersky, Bruce T; Tyagi, Rachana; Coyne, Thomas

    2011-10-15

    A 73-year-old man had episodic encephalopathy, ataxia and neuropathy. Symptoms largely resolved but adenopathy later lead to the diagnosis of a low-grade follicular lymphoma. The neurological symptoms soon recurred with new pontine calcifications identified by computed tomography. Brain biopsy revealed microvascular endothelial cell nuclear changes. Electron microscopy identified small polymorphic bacteria without a cell wall and with terminal and attachment organelles within endothelial cells and clustered in some microvascular lumina. Immunostaining was positive for Mycoplasma pneumoniae and convalescent serum enzyme immunoassay was positive for M. pneumoniae IgG. The patient again recovered and he was neurologically stable 33 months after the initial episode. The ultrastructural findings of the bacterial cells are distinctive of some mycoplasmal species when compared to other small bacteria. Mycoplasma-like organisms are reported in four autopsied patients who had chronic encephalopathy, movement disorders, and some of the same light- and electron-microscopic findings in the brain as our patient. Direct neuroinvasion by Mycoplasma species has been suggested, while anatomic observations in our patient and in the four autopsy cases show microvascular invasion but not parenchymal invasion. Most mycoplasmal encephalitis may be immune-mediated. The frequency of neurovascular invasion is not known. It may be rare and it may persist.

  4. Cocaine Inhibits Store-Operated Ca2+ Entry in Brain Microvascular Endothelial Cells: Critical Role for Sigma-1 Receptors

    PubMed Central

    Brailoiu, G. Cristina; Deliu, Elena; Console-Bram, Linda M; Soboloff, Jonathan; Abood, Mary E; Unterwald, Ellen M; Brailoiu, Eugen

    2015-01-01

    Sigma-1 receptor (Sig-1R) is an intracellular chaperone protein with many ligands, located at the endoplasmic reticulum. Binding of cocaine to Sig-1R has previously been found to modulate endothelial functions. In the present study, we show that cocaine dramatically inhibits store-operated Ca2+ entry (SOCE), a Ca2+ influx mechanism promoted by depletion of intracellular Ca2+ stores, in rat brain microvascular endothelial cells. Using either Sig-1R shRNA or pharmacological inhibition with the unrelated Sig-1R antagonists BD-1063 and NE-100, we show that cocaine-induced SOCE inhibition is dependent on Sig-1R. In addition to revealing new insight into fundamental mechanisms of cocaine-induced changes in endothelial function, these studies provide an unprecedented role for Sig-1R as a SOCE inhibitor. PMID:26467159

  5. Cocaine inhibits store-operated Ca2+ entry in brain microvascular endothelial cells: critical role for sigma-1 receptors.

    PubMed

    Brailoiu, G Cristina; Deliu, Elena; Console-Bram, Linda M; Soboloff, Jonathan; Abood, Mary E; Unterwald, Ellen M; Brailoiu, Eugen

    2016-01-01

    Sigma-1 receptor (Sig-1R) is an intracellular chaperone protein with many ligands, located at the endoplasmic reticulum (ER). Binding of cocaine to Sig-1R has previously been found to modulate endothelial functions. In the present study, we show that cocaine dramatically inhibits store-operated Ca(2+) entry (SOCE), a Ca(2+) influx mechanism promoted by depletion of intracellular Ca(2+) stores, in rat brain microvascular endothelial cells (RBMVEC). Using either Sig-1R shRNA or pharmacological inhibition with the unrelated Sig-1R antagonists BD-1063 and NE-100, we show that cocaine-induced SOCE inhibition is dependent on Sig-1R. In addition to revealing new insight into fundamental mechanisms of cocaine-induced changes in endothelial function, these studies indicate an unprecedented role for Sig-1R as a SOCE inhibitor.

  6. High microvascular endothelial water permeability in mouse lung measured by a pleural surface fluorescence method.

    PubMed Central

    Carter, E P; Olveczky, B P; Matthay, M A; Verkman, A S

    1998-01-01

    Transport of water between the capillary and airspace compartments in lung encounters serial barriers: the alveolar epithelium, interstitium, and capillary endothelium. We previously reported a pleural surface fluorescence method to measure net capillary-to-airspace water transport. To measure the osmotic water permeability across the microvascular endothelial barrier in intact lung, the airspace was filled with a water-immiscible fluorocarbon. The capillaries were perfused via the pulmonary artery with solutions of specified osmolalites containing a high-molecular-weight fluorescent dextran. An increase in perfusate osmolality produced a prompt decrease in surface fluorescence due to dye dilution in the capillaries, followed by a slower return to initial fluorescence as capillary and lung interstitial osmolality equilibrate. A mathematical model was developed to determine the osmotic water permeability coefficient (Pf) of lung microvessels from the time course of pleural surface fluorescence. As predicted, the magnitude of the prompt change in surface fluorescence increased with decreased pulmonary artery perfusion rate and increased osmotic gradient size. With raffinose used to induce the osmotic gradient, Pf was 0.03 cm/s at 23 degrees C and was reduced 54% by 0.5 mM HgCl2. Temperature dependence measurements gave an Arrhenius activation energy (Ea) of 5.4 kcal/mol (12-37 degrees C). The apparent Pf induced by the smaller osmolytes mannitol and glycine was 0.021 and 0.011 cm/s (23 degrees C). Immunoblot analysis showed approximately 1.4 x 10(12) aquaporin-1 water channels/cm2 of capillary surface, which accounted quantitatively for the high Pf. These results establish a novel method for measuring osmotically driven water permeability across microvessels in intact lung. The high Pf, low Ea, and mercurial inhibition indicate the involvement of molecular water channels in water transport across the lung endothelium. PMID:9545071

  7. Metformin Represses Glucose Starvation Induced Autophagic Response in Microvascular Endothelial Cells and Promotes Cell Death.

    PubMed

    Samuel, Samson Mathews; Ghosh, Suparna; Majeed, Yasser; Arunachalam, Gnanapragasam; Emara, Mohamed M; Ding, Hong; Triggle, Chris R

    2017-03-05

    Metformin, the most frequently administered drug for the treatment of type 2 diabetes, is being investigated for its potential in the treatment of various types of cancer; however, the cellular basis for this putative anti-cancer action remains controversial. In the current study we examined the effect of metformin on endoplasmic reticulum (ER) stress and autophagy in glucose-starved micro-vascular endothelial cells (MECs). The rationale for our experimental protocol is that in a growing tumor MECs are subjected to hypoxia and nutrient/glucose starvation that results from the reduced supply and relatively high consumption of glucose. Mouse MECs (MMECs) were glucose-starved for up to 48h in the absence or presence of metformin (50μM and 2mM) and the status of ER stress, autophagic, cell survival and apoptotic markers were assessed. Activation of ER stress and autophagy was observed in glucose starved MECs as evidenced by the significant increase in the levels of ER stress and autophagic markers while accumulation of LC3B stained punctae in the MECs confirmed autophagic activation. Treatment with 2mM metformin, independent of AMPK, significantly reversed glucose starvation induced ER stress and autophagy in MECs, but, at 24h, did not decrease cell viability; however, at 48h, persistent ER stress and metformin associated inhibition of autophagy decreased cell viability, caused cell cycle arrest in G2/M and increased the number of cells in the sub-G0/G1 phase of cell cycle. Treatment with metformin reduced phosphorylation of Akt and mTOR and inhibited downstream targets of mTOR. Our findings support the argument that treatment with metformin when used in combination with glycolytic inhibitors will inhibit pro-survival autophagy and promote cell death and potentially prove to be the basis for an effective anti-cancer strategy.

  8. Galantamine and carbon monoxide protect brain microvascular endothelial cells by heme oxygenase-1 induction

    SciTech Connect

    Nakao, Atsunori; Kaczorowski, David J.; Zuckerbraun, Brian S.; Lei Jing; Faleo, Gaetano; Deguchi, Kentaro; McCurry, Kenneth R.; Billiar, Timothy R.; Kanno, Shinichi

    2008-03-14

    Galantamine, a reversible inhibitor of acetylcholine esterase (AChE), is a novel drug treatment for mild to moderate Alzheimer's disease and vascular dementia. Interestingly, it has been suggested that galantamine treatment is associated with more clinical benefit in patients with mild-to-moderate Alzheimer disease compared to other AChE inhibitors. We hypothesized that the protective effects of galantamine would involve induction of the protective gene, heme oxygenase-1 (HO-1), in addition to enhancement of the cholinergic system. Brain microvascular endothelial cells (mvECs) were isolated from spontaneous hypertensive rats. Galantamine significantly reduced H{sub 2}O{sub 2}-induced cell death of mvECs in association with HO-1 induction. These protective effects were completely reversed by nuclear factor-{kappa}B (NF-{kappa}B) inhibition or HO inhibition. Furthermore, galantamine failed to induce HO-1 in mvECs which lack inducible nitric oxide synthase (iNOS), supplementation of a nitric oxide (NO) donor or iNOS gene transfection on iNOS-deficient mvECs resulted in HO-1 induction with galantamine. These data suggest that the protective effects of galantamine require NF-{kappa}B activation and iNOS expression, in addition to HO-1. Likewise, carbon monoxide (CO), one of the byproducts of HO, up-regulated HO-1 and protected mvECs from oxidative stress in a similar manner. Our data demonstrate that galantamine mediates cytoprotective effects on mvECs through induction HO-1. This pharmacological action of galantamine may, at least in part, account for the superior clinical efficacy of galantamine in vascular dementia and Alzheimer disease.

  9. Ocimum sanctum Linn. stimulate the expression of choline acetyltransferase on the human cerebral microvascular endothelial cells

    PubMed Central

    Kusindarta, Dwi Liliek; Wihadmadyatami, Hevi; Haryanto, Aris

    2016-01-01

    Aim: This research was conducted to identify the expression of choline acetyltransferase (ChAT) in human cerebral microvascular endothelial cells (HCMECs) and to clarify the capability of Ocimum sanctum Linn. ethanolic extract to stimulate the presence of ChAT in the aging HCMECs. Materials and Methods: In this study, we perform an in vitro analysis some in the presence of an ethanolic extract of O. sanctum Linn. as a stimulator for the ChAT expression. HCMECs are divided become two groups, the first is in low passage cells as a model of young aged and the second is in a high passage as a model of aging. Furthermore to analysis the expression of ChAT without and with extract treatments, immunocytochemistry and flow cytometry analysis were performed. In addition, ChAT sandwich enzyme-linked immunosorbent assay is developed to detect the increasing activity of the ChAT under normal, and aging HCMECs on the condition treated and untreated cells. Results: In our in vitro models using HCMECs, we found that ChAT is expressed throughout intracytoplasmic areas. On the status of aging, the ethanolic extract from O. sanctum Linn. is capable to stimulate and restore the expression of ChAT. The increasing of ChAT expression is in line with the increasing activity of this enzyme on the aging treated HCMECs. Conclusions: Our observation indicates that HCMECs is one of the noncholinergic cells which is produced ChAT. The administrated of O. sanctum Linn. ethanolic extract may stimulate and restore the expression of ChAT on the deteriorating cells of HCMECs, thus its may give nerve protection and help the production of acetylcholine. PMID:28096604

  10. Cinnamaldehyde reduces IL-1beta-induced cyclooxygenase-2 activity in rat cerebral microvascular endothelial cells.

    PubMed

    Guo, Jian-You; Huo, Hai-Ru; Zhao, Bao-Sheng; Liu, Hong-Bin; Li, Lan-Fang; Ma, Yue-Ying; Guo, Shu-Ying; Jiang, Ting-Liang

    2006-05-10

    Cinnamaldehyde is a principle compound isolated from Guizhi-Tang, which is a famous traditional Chinese medical formula used to treat influenza, common cold and other pyretic conditions. The aim of the present study was to investigate the effects of cinnamaldehyde on expression and activity of cyclooxygenase (COX) and prostaglandin E(2) (PGE(2)) in rat cerebral microvascular endothelial cells (RCMEC). RCMEC were cultured, and identified by immunohistochemistry for von Willebrand factor in cytoplasm of the cells. Then cells were incubated in M199 medium containing interleukin (IL)-1beta in the presence or absence of cinnamaldehyde. After incubation, the medium was collected and the amount of PGE(2) was measured by enzyme-linked immunosorbent assay (ELISA). The cells were harvested, mRNA expression and activity of COX were analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR) with SYBR Green dye and ELISA respectively. Positive immunostaining for von Willebrand factor was present diffusely in the cytoplasm of >95% RCMEC. IL-1beta increased the mRNA expression and activity of COX-2, and production of PGE(2) in a dose- and time-dependent manner in RCMEC, while mRNA and activity of COX-1 were not significantly altered. Cinnamaldehyde significantly decreased IL-1beta-induced COX-2 activity and PGE(2) production in a dose-dependent manner, while it showed no inhibitory effect on IL-1beta-induced COX-2 mRNA expression in cultured RCMEC. In conclusion, cinnamaldehyde reduces IL-1beta-induced COX-2 activity, but not IL-1beta-induced COX-2 mRNA expression, and consequently inhibits production of PGE(2) in cultured RCMEC.

  11. BMPRII influences the response of pulmonary microvascular endothelial cells to inflammatory mediators.

    PubMed

    Vengethasamy, Leanda; Hautefort, Aurélie; Tielemans, Birger; Belge, Catharina; Perros, Frédéric; Verleden, Stijn; Fadel, Elie; Van Raemdonck, Dirk; Delcroix, Marion; Quarck, Rozenn

    2016-11-01

    Mutations in the bone morphogenetic protein receptor (BMPR2) gene have been observed in 70 % of patients with heritable pulmonary arterial hypertension (HPAH) and in 11-40 % with idiopathic PAH (IPAH). However, carriers of a BMPR2 mutation have only 20 % risk of developing PAH. Since inflammatory mediators are increased and predict survival in PAH, they could act as a second hit inducing the development of pulmonary hypertension in BMPR2 mutation carriers. Our specific aim was to determine whether inflammatory mediators could contribute to pulmonary vascular cell dysfunction in PAH patients with and without a BMPR2 mutation. Pulmonary microvascular endothelial cells (PMEC) and arterial smooth muscle cells (PASMC) were isolated from lung parenchyma of transplanted PAH patients, carriers of a BMPR2 mutation or not, and from lobectomy patients or lung donors. The effects of CRP and TNFα on mitogenic activity, adhesiveness capacity, and expression of adhesion molecules were investigated in PMECs and PASMCs. PMECs from BMPR2 mutation carriers induced an increase in PASMC mitogenic activity; moreover, endothelin-1 secretion by PMECs from carriers was higher than by PMECs from non-carriers. Recruitment of monocytes by PMECs isolated from carriers was higher compared to PMECs from non-carriers and from controls, with an elevated ICAM-1 expression. CRP increased adhesion of monocytes to PMECs in carriers and non-carriers, and TNFα only in carriers. PMEC from BMPR2 mutation carriers have enhanced adhesiveness for monocytes in response to inflammatory mediators, suggesting that BMPR2 mutation could generate susceptibility to an inflammatory insult in PAH.

  12. Phloretin ameliorates 2-chlorohexadecanal-mediated brain microvascular endothelial cell dysfunction in vitro

    PubMed Central

    Üllen, Andreas; Fauler, Günter; Bernhart, Eva; Nusshold, Christoph; Reicher, Helga; Leis, Hans-Jörg; Malle, Ernst; Sattler, Wolfgang

    2012-01-01

    2-Chlorohexadecanal (2-ClHDA), a chlorinated fatty aldehyde, is formed via attack on ether-phospholipids by hypochlorous acid (HOCl) that is generated by the myeloperoxidase–hydrogen peroxide–chloride system of activated leukocytes. 2-ClHDA levels are elevated in atherosclerotic lesions, myocardial infarction, and neuroinflammation. Neuroinflammatory conditions are accompanied by accumulation of neutrophils (an ample source of myeloperoxidase) in the brain. Microvessel damage by inflammatory mediators and/or reactive oxidants can induce blood–brain barrier (BBB) dysfunction, a pathological condition leading to cerebral edema, brain hemorrhage, and neuronal death. In this in vitro study we investigated the impact of 2-ClHDA on brain microvascular endothelial cells (BMVEC), which constitute the morphological basis of the BBB. We show that exogenously added 2-ClHDA is subject to rapid uptake and metabolism by BMVEC. Using C16 structural analogues of 2-ClHDA we found that the cytotoxic potential decreases in the following order: 2-ClHDA>hexadecanal>palmitic acid>2-ClHDA-dimethylacetal. 2-ClHDA induces loss of barrier function, mitochondrial dysfunction, apoptosis via activation of caspase 3, and altered intracellular redox balance. Finally we investigated potential protective effects of several natural polyphenols on in vitro BBB function. Of the compounds tested, phloretin almost completely abrogated 2-ClHDA-induced BMVEC barrier dysfunction and cell death. These data suggest that 2-ClHDA has the potential to induce BBB breakdown under inflammatory conditions and that phloretin confers protection in this experimental setting. PMID:22982051

  13. Modulation of transforming growth factor beta receptor levels on microvascular endothelial cells during in vitro angiogenesis.

    PubMed Central

    Sankar, S; Mahooti-Brooks, N; Bensen, L; McCarthy, T L; Centrella, M; Madri, J A

    1996-01-01

    Microvascular endothelial cells (RFCs) cultured in two-dimensional (2D) cultures proliferate rapidly and exhibit an undifferentiated phenotype. Addition of transforming growth factor beta1 (TGFbeta1) increases fibronectin expression and inhibits proliferation. RFCs cultured in three-dimensional (3D) type I collagen gels proliferate slowly and are refractory to the anti-proliferative effects of TGF beta1. TGF beta1 promotes tube formation in 3D cultures. TGF beta1 increases fibronectin expression and urokinase plasminogen activator (uPA) activity and plasminogen activator inhibitor-1 (PAI-1) levels in 3D cultures. Since the TGF beta type I and II receptors have been reported to regulate different activities induced by TGF beta1, we compared the TGF beta receptor profiles on cells in 2D and 3D cultures. RFCs in 3D cultures exhibited a significant loss of cell surface type II receptor compared with cells in 2D cultures. The inhibitory effect of TGF beta1 on proliferation is suppressed in transfected 2D cultures expressing a truncated form of the type II receptor, while its stimulatory effect on fibronectin production is reduced in both 2D and 3D transfected cultures expressing a truncated form of the type I receptor. These data suggest that the type II receptor mediates the antiproliferative effect of TGF beta1 while the type I receptor mediates the matrix response of RFCs to TGF beta1 and demonstrate that changes in the matrix environment can modulate the surface expression of TGF beta receptors, altering the responsiveness of RFCs to TGF beta1. PMID:8617876

  14. 2,3,7,8-TCDD exposure, endothelial dysfunction and impaired microvascular reactivity.

    PubMed

    Pelclová, Daniela; Prázny, Martin; Skrha, Jan; Fenclová, Zdenka; Kalousová, Marta; Urban, Pavel; Navrátil, Tomás; Senholdová, Zdenka; Smerhovsky, Zdenek

    2007-09-01

    Vascular function was examined in subjects with long-term high level of serum 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) during their follow-up visits. Their earlier mean peak TCDD level at the time of exposure in 1965-1968 was estimated in the range of 3300-74 000 pg/g lipids. Ten former pesticide production workers heavily exposed to TCDD (age 57 +/- 2 years, TCDD about 170 pg/g lipids) were examined in 2001. Extended group of 15 TCDD-exposed men (age 59 +/- 3 years, TCDD about 130 pg/g lipids) underwent the same examination in 2004. Findings were compared with a control group of 14 healthy men (age 54 +/- 2 years). Skin microvascular reactivity (MVR) was measured by laser Doppler perfusion monitoring in the forearm during post-occlusive reactive hyperemia (PORH) and thermal hyperemia (TH). Several parameters of MVR in men exposed to TCDD were significantly impaired, compared with the control group and further progression of the impairment of MVR has been observed between years 2001 and 2004. Serum concentration of E-selectin and inhibitor of tissue plasminogen activator 1 (PAI-1) was significantly higher in exposed subjects (56.0 +/- 18.4 ng/mL versus 40.0 +/- 12.0 ng/mL, P = 0.022 and 90.9 +/- 33.3 ng/mL versus 45.0 +/- 18.0, P = 0.002, respectively). In addition, PORH in the forearm was significantly negatively associated with SOD activity (r = -0.77, P = 0.009) as well as the velocity of perfusion increase during TH (r = -0.68, P = 0.03) and TH% (r = -0.78, P = 0.008). Our data document the presence of endothelial dysfunction in TCDD-exposed men.

  15. [Effects of transient exposure to high glucose on biological behaviors of human dermal microvascular endothelial cells].

    PubMed

    Qiao, L; Yang, H Z; Li, X C; Huang, X Q; Yuan, B; Zhou, Z D

    2017-02-20

    Objective: To observe the effects of transient exposure to high glucose on biological behaviors of human dermal microvascular endothelial cells cultured in vitro. Methods: The dividing method and treatment of cells for the detection of all indexes in this study were as follows. Human dermal microvascular endothelial cells of the 4th passage were divided into 3 groups according to the random number table, with 12 wells in each group. Cells in control group (C) were cultured with complete culture solution containing 5 mmol/L D-glucose for 7 d. Cells in transient high glucose group (THG) were cultured with complete culture solution containing 30 mmol/L D-glucose for 2 d and complete culture solution containing 5 mmol/L D-glucose for 5 d. Cells in prolonged high glucose group (PHG) were cultured with complete culture solution containing 30 mmol/L D-glucose for 7 d. (1) The cell morphology in groups C and PHG on culture day 7 and that in group THG on culture day 2 and 7 was observed by inverted optical microscope. (2) On culture day 0, 2, 4, and 7, cell proliferation rate was determined by cell viability analyzing counter. (3) After culture day 2, the scratch experiment was performed, and the cells were further cultured. At post scratch hour (PSH) 0, 24, 48, 72, 96, and 120, the scratch area was measured, and the cell migration rates of the latter 5 time points were calculated. (4) On culture day 0, 2, 4, and 7, the cell apoptosis rate was determined by cell analyzer. (5) Cells were seeded into Matrigel to culture for 24 h after culture day 7. The formation of vessel-like structure was observed by inverted optical microscope. The length and number of branch point of vessel-like structure were calculated. (6) On culture day 2, 4, and 7, mRNA expression of vascularization-related gene tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) was determined with real-time fluorescent quantitative reverse transcription polymerase chain reaction. Data were processed with

  16. Galectin-1 suppresses methamphetamine induced neuroinflammation in human brain microvascular endothelial cells: Neuroprotective role in maintaining blood brain barrier integrity.

    PubMed

    Parikh, Neil U; Aalinkeel, R; Reynolds, J L; Nair, B B; Sykes, D E; Mammen, M J; Schwartz, S A; Mahajan, S D

    2015-10-22

    Methamphetamine (Meth) abuse can lead to the breakdown of the blood-brain barrier (BBB) integrity leading to compromised CNS function. The role of Galectins in the angiogenesis process in tumor-associated endothelial cells (EC) is well established; however no data are available on the expression of Galectins in normal human brain microvascular endothelial cells and their potential role in maintaining BBB integrity. We evaluated the basal gene/protein expression levels of Galectin-1, -3 and -9 in normal primary human brain microvascular endothelial cells (BMVEC) that constitute the BBB and examined whether Meth altered Galectin expression in these cells, and if Galectin-1 treatment impacted the integrity of an in-vitro BBB. Our results showed that BMVEC expressed significantly higher levels of Galectin-1 as compared to Galectin-3 and -9. Meth treatment increased Galectin-1 expression in BMVEC. Meth induced decrease in TJ proteins ZO-1, Claudin-3 and adhesion molecule ICAM-1 was reversed by Galectin-1. Our data suggests that Galectin-1 is involved in BBB remodeling and can increase levels of TJ proteins ZO-1 and Claudin-3 and adhesion molecule ICAM-1 which helps maintain BBB tightness thus playing a neuroprotective role. Galectin-1 is thus an important regulator of immune balance from neurodegeneration to neuroprotection, which makes it an important therapeutic agent/target in the treatment of drug addiction and other neurological conditions.

  17. Collagen-nanofiber hydrogel composites promote contact guidance of human lymphatic microvascular endothelial cells and directed capillary tube formation.

    PubMed

    Laco, Filip; Grant, M Helen; Black, Richard A

    2013-06-01

    Collagen and fibronectin matrices are known to stimulate migration of microvascular endothelial cells and the process of tubulogenesis, but the physical, chemical, and topographical cues for directed vessel formation have yet to be determined. In this study, growth, migration, elongation, and tube formation of human lymphatic microvascular endothelial cells (LECs) were investigated on electrospun poly(D,L-lactic-co-glycolic acid) (PLGA) and poly(L-lactic-co-D-lactic acid) (PLDL) nanofiber-coated substrates, and correlated with fiber density and diameter. Directed migration of LECs was observed in the presence of aligned nanofibers, whereas random fiber alignment slowed down migration and growth of LECs. Cell guidance was significantly enhanced in the presence of more hydrophobic PLDL polymer nanofibers compared to PLGA (10:90). Subsequent experiments with tube-forming assays reveal the ability of resorbable hydrophobic nanofibers >300 nm in diameter to promote cell guidance in collagen gels without direct cell-fiber contact, in contrast to the previously reported contact-guidance phenomena. Our results show that endothelial cell guidance is possible within nanofiber/collagen-gel constructs that mimic the native extracellular matrix in terms of size and orientation of fibrillar components.

  18. Chemokine-Like Factor 1-Derived C-Terminal Peptides Induce the Proliferation of Dermal Microvascular Endothelial Cells in Psoriasis

    PubMed Central

    Tan, Yaqi; Wang, Yixuan; Li, Li; Xia, Jinyu; Peng, Shiguang; He, Yanling

    2015-01-01

    Psoriasis is an inflammatory disease characterized by the abnormal proliferation of skin cells, including dermal microvascular endothelial cells. Recently, chemokine-like factor 1 (CKLF1) was found to participate in the local inflammation and cell proliferation. To explore its role in the pathogenesis of psoriasis, the expression of both CKLF1 and its receptor (CCR4) was determined in the psoriatic lesions. Also, the effect of the C-terminal peptides (C19 and C27) of CKLF1 on the proliferation of human umbilical vein endothelial cells was studied in vitro. By immunohistochemistry and immunofluorescence, the expression of both CKLF1 and CCR4 was determined in the psoriatic lesions. The effect of C-terminal peptides on human umbilical vein endothelial cells (HUVECs) was studied in vitro by the evaluation of cell proliferation and apoptosis. The in vivo assessment was performed accordingly through the subcutaneous injection peptides on BALB/c mice. The results showed that, by immunohistochemistry, both CKLF1 and CCR4 were increasingly expressed in psoriatic lesions as compared to normal skins. Moreover, the primary umbilical vein endothelial cells exhibited higher proliferation ratio under the C19 or C27 stimulation, which was even enhanced by the addition of psoriatic sera or TNF-α. Furthermore, the enhancement of peptide simulation was accompanied with the activation of ERK1/2-MAPKs pathway. In addition, such effect of C19 and C27 was mirrored by the hyperproliferation of cutaneous microvessels in BALB/c mice that were subcutaneously injected with the two peptides. Therefore, we concluded that CKLF1 plays a role in the pathogenesis of psoriasis by promoting the proliferation of microvascular endothelial cells that possibly correlates with ERK1/2-MAPKs activation. PMID:25915746

  19. Involvement of the transcription factor NF-kappaB in tubular morphogenesis of human microvascular endothelial cells by oxidative stress.

    PubMed Central

    Shono, T; Ono, M; Izumi, H; Jimi, S I; Matsushima, K; Okamoto, T; Kohno, K; Kuwano, M

    1996-01-01

    Oxygen radicals are induced under various pathologic conditions associated with neovascularization. Oxygen radicals modulate angiogenesis in cultured human microvascular endothelial cells by an unknown mechanism. Treatment of human microvascular endothelial cells for 15 min with 0.1 to 0.5 mM hydrogen peroxide (H2O2) or 100 U of tumor necrosis factor alpha per ml induced tubular morphogenesis in type I collagen gels. Gel shift assays with nuclear extracts demonstrated that H2O2 increases the binding activities of two transcription factors, NF-kappaB and AP-1, but not of Spl. Tumor necrosis factor alpha increased the binding activities of all three factors. A supershift assay with specific antibodies against JunB, JunD, and c-Jun (Jun family) showed that the antibody against c-Jun supershifted the AP-1 complex after H2O2 treatment. Coadministration of the antisense sequence of NF-kappaB inhibited H2O2-dependent tubular morphogenesis, and the antisense c-Jun oligonucleotide caused partial inhibition. The angiogenic factor responsible for H2O2-induced tubular morphogenesis was examined. Cellular mRNA levels of vascular endothelial growth factor and interleukin-8 (IL-8), but not those of transforming growth factor alpha, were increased after treatment with 0.5 mM H2O2. Coadministration of anti-IL-8 antibody inhibited tubular morphogenesis enhanced by H2O2, and IL-8 itself also enhanced the formation of tube-like structures. Treatment with antisense NF-kappaB oligonucleotide completely blocked H2O2-dependent IL-8 production by endothelial cells. The tubular morphogenesis of vascular endothelial cells after treatment with oxidative stimuli and its possible association with NF-kappaB and IL-8, is examined. PMID:8754823

  20. Ca2+ homeostasis in microvascular endothelial cells from an insulin-dependent diabetic model: role of endosomes/lysosomes

    NASA Astrophysics Data System (ADS)

    Sanka, Shankar C.; Bennett, David C.; Rojas, Jose D.; Tasby, Geraldine B.; Meininger, Cynthia J.; Wu, Guoyao; Wesson, Donald E.; Pfarr, Curtis M.; Martinez-Zaguilan, Raul

    2000-04-01

    Cytosolic Ca2+ ([Ca2+]cyt) regulates several cellular functions, e.g. cell growth, contraction, secretion, etc. In many cell types, ion homeostasis appears to be coupled with glucose metabolism. In certain cell types, a strict coupling between glycolysis and the activity of Sarcoplasmic/Endoplasmic Reticulum Ca2+-ATPases (SERCA) has been suggested. Glucose metabolism is altered in diabetes. We hypothesize that: (1) Ca2+ homeostasis is altered in microvascular endothelial cells from diabetic animals due to the dysfunction of glycolysis coupling the activity of SERCA; (2) endosomal/lysosomal compartments expressing SERCA are involved in the dysfunction associated with diabetes.

  1. Involvement of RhoA/ROCK1 signaling pathway in hyperglycemia-induced microvascular endothelial dysfunction in diabetic retinopathy

    PubMed Central

    Lu, Qian-Yi; Chen, Wei; Lu, Li; Zheng, Zhi; Xu, Xun

    2014-01-01

    Diabetic retinopathy (DR) is a well-known serious complication of diabetes mellitus (DM), and can eventually advance to end-stage blindness. In the early stage of DR, endothelial cell barrier disorganized primarily and tight junction (TJ) protein composition transformed subsequently. The small GTPase RhoA and its downstream effector Rho-associated coiled-coil containing protein kinase 1 (ROCK1) regulate a mass of cellular processes, including cell adherence, proliferation, permeability and apoptosis. Although RhoA inhibitors have provided substantial clinical benefit as hypertonicity therapeutics, their use is limited by complex microenvironment as DR. While ample evidence indicates that TJ can be influenced by the RhoA/ROCK1 signaling, the underlying mechanisms remain incompletely understood. Here, we have uncovered a significant signaling network involved in diabetic retinal microvascular endothelial dysfunction (RMVED). Our results indicated that the activation of RhoA/ROCK1 pathway due to high glucose played a key role in microvascular endothelial cell dysfunction (MVED) by way of directly inducing TJ proteins over-expression during DR. We demonstrated that inhibition of RhoA/ROCK1 may attenuate the hypertonicity of endothelial cell caused by high glucose microenvironment meanwhile. Besides, chemical and pharmacological inhibitors of RhoA/ROCK1 pathway may partly block inflammation due to DR. Simultaneously, the apoptosis aroused by high glucose was also prevented considerably by fasudil, a kind of pharmacological inhibitor of RhoA/ROCK1 pathway. These findings indicate that RhoA/ROCK1 signaling directly modulates MVED, suggesting a novel therapeutic target for DR. PMID:25400825

  2. Exosomal signaling during hypoxia mediates microvascular endothelial cell migration and vasculogenesis.

    PubMed

    Salomon, Carlos; Ryan, Jennifer; Sobrevia, Luis; Kobayashi, Miharu; Ashman, Keith; Mitchell, Murray; Rice, Gregory E

    2013-01-01

    Vasculogenesis and angiogenesis are critical processes in fetal circulation and placental vasculature development. Placental mesenchymal stem cells (pMSC) are known to release paracrine factors (some of which are contained within exosomes) that promote angiogenesis and cell migration. The aims of this study were: to determine the effects of oxygen tension on the release of exosomes from pMSC; and to establish the effects of pMSC-derived exosomes on the migration and angiogenic tube formation of placental microvascular endothelial cells (hPMEC). pMSC were isolated from placental villi (8-12 weeks of gestation, n = 6) and cultured under an atmosphere of 1%, 3% or 8% O2. Cell-conditioned media were collected and exosomes (exo-pMSC) isolated by differential and buoyant density centrifugation. The dose effect (5-20 µg exosomal protein/ml) of pMSC-derived exosomes on hPMEC migration and tube formation were established using a real-time, live-cell imaging system (Incucyte™). The exosome pellet was resuspended in PBS and protein content was established by mass spectrometry (MS). Protein function and canonical pathways were identified using the PANTHER program and Ingenuity Pathway Analysis, respectively. Exo-pMSC were identified, by electron microscopy, as spherical vesicles, with a typical cup-shape and diameters around of 100 nm and positive for exosome markers: CD63, CD9 and CD81. Under hypoxic conditions (1% and 3% O2) exo-pMSC released increased by 3.3 and 6.7 folds, respectively, when compared to the controls (8% O2; p<0.01). Exo-pMSC increased hPMEC migration by 1.6 fold compared to the control (p<0.05) and increased hPMEC tube formation by 7.2 fold (p<0.05). MS analysis identified 390 different proteins involved in cytoskeleton organization, development, immunomodulatory, and cell-to-cell communication. The data obtained support the hypothesis that pMSC-derived exosomes may contribute to placental vascular adaptation to low oxygen tension under both

  3. Effect of angiopoietin-like protein 4 on rat pulmonary microvascular endothelial cells exposed to LPS

    PubMed Central

    WANG, YUXI; CHEN, HAILONG; LI, HAILONG; ZHANG, JINGWEN; GAO, YANYAN

    2013-01-01

    Pulmonary microvascular endothelial cells (PMVECs) possess highly proliferative and angiogenic capacities and are localized at the critical interface between the blood and microvessel wall; they are the primary targets of inflammatory cytokines during lung inflammation. Angiopoietin-like protein 4 (Angptl4) is a circulating protein that has recently been implicated in the regulation of angiogenesis and metastasis. This study aimed to investigate the effect of Angptl4 on rat PMVECs (RPMVECs) exposed to lipopolysaccharide (LPS). The cell culture was stimulated with LPS. Total Angptl4 cDNA was obtained from Source BioScience. The PCR product was cloned into the pcDNA3.1-eGFP or the pcDNA3.1-eGFP-Angptl4 vector, which were then transfected into the RPMVECs using SuperFect transfection reagent. The Angptl4 mRNA levels, protein levels and cell morphology of the RPMVECs in the experimental groups were detected using real time-PCR, western blot analysis, MTT assay, ELISA and confocal microscopy methods, respectively. The Angptl4 expression vector, pcDNA3.1-eGFP-Angptl4, was successfully constructed. The Angptl4 mRNA level in the LPS-pcDNA3.1-eGFP-transfected group (blank control) was slightly increased and was significantly higher in the experimental group compared with the empty vector and blank control group with significant differences. Pro-apoptotic caspase-8, -9 and Bax protein were inhibited, while p-AKT/AKT and p-MEK1/2 protein expression was also decreased. The rosiglitazone group had significantly decreased levels of the inflammatory cytokine, tumor necrosis factor (TNF)-α (P<0.01). The overexpression of Angptl4 inhibited the LPS-induced increase in the permeability of the RPMVECs, which was associated with the depolymerization of central F-actin in the RPMVECs. In conclusion, our study demonstrates that the overexpression of Angptl4 exerts protective, anti-inflammatory and anti-angiogenic effects. It represents a novel therapeutic target gene for the treatment

  4. Palmitate-induced inflammatory pathways in human adipose microvascular endothelial cells promote monocyte adhesion and impair insulin transcytosis.

    PubMed

    Pillon, Nicolas J; Azizi, Paymon M; Li, Yujin E; Liu, Jun; Wang, Changsen; Chan, Kenny L; Hopperton, Kathryn E; Bazinet, Richard P; Heit, Bryan; Bilan, Philip J; Lee, Warren L; Klip, Amira

    2015-07-01

    Obesity is associated with inflammation and immune cell recruitment to adipose tissue, muscle and intima of atherosclerotic blood vessels. Obesity and hyperlipidemia are also associated with tissue insulin resistance and can compromise insulin delivery to muscle. The muscle/fat microvascular endothelium mediates insulin delivery and facilitates monocyte transmigration, yet its contribution to the consequences of hyperlipidemia is poorly understood. Using primary endothelial cells from human adipose tissue microvasculature (HAMEC), we investigated the effects of physiological levels of fatty acids on endothelial inflammation and function. Expression of cytokines and adhesion molecules was measured by RT-qPCR. Signaling pathways were evaluated by pharmacological manipulation and immunoblotting. Surface expression of adhesion molecules was determined by immunohistochemistry. THP1 monocyte interaction with HAMEC was measured by cell adhesion and migration across transwells. Insulin transcytosis was measured by total internal reflection fluorescence microscopy. Palmitate, but not palmitoleate, elevated the expression of IL-6, IL-8, TLR2 (Toll-like receptor 2), and intercellular adhesion molecule 1 (ICAM-1). HAMEC had markedly low fatty acid uptake and oxidation, and CD36 inhibition did not reverse the palmitate-induced expression of adhesion molecules, suggesting that inflammation did not arise from palmitate uptake/metabolism. Instead, inhibition of TLR4 to NF-κB signaling blunted palmitate-induced ICAM-1 expression. Importantly, palmitate-induced surface expression of ICAM-1 promoted monocyte binding and transmigration. Conversely, palmitate reduced insulin transcytosis, an effect reversed by TLR4 inhibition. In summary, palmitate activates inflammatory pathways in primary microvascular endothelial cells, impairing insulin transport and increasing monocyte transmigration. This behavior may contribute in vivo to reduced tissue insulin action and enhanced tissue

  5. Anti inflammatory and anti angiogenic effect of black raspberry extract on human esophageal and intestinal microvascular endothelial cells

    PubMed Central

    Medda, Rituparna; Lyros, Orestis; Schmidt, Jamie L.; Jovanovic, Nebojsa; Nie, Linghui; Link, Benjamin J.; Otterson, Mary F.; Stoner, Gary D.; Shaker, Reza; Rafiee, Parvaneh

    2014-01-01

    Polyphenolic compounds (anthocyanins, flavonoid glycosides) in berries prevent the initiation, promotion, and progression of carcinogenesis in rat’s digestive tract and esophagus, in part, via anti-inflammatory pathways. Angiogenesis has been implicated in the pathogenesis of chronic inflammation and tumorigenesis. In this study, we investigated the anti-inflammatory and anti-angiogenic effects of black raspberry extract (BRE) on two organ specific primary human intestinal microvascular endothelial cells, (HIMEC) and human esophageal microvascular endothelial cells (HEMEC), isolated from surgically resected human intestinal and donor discarded esophagus, respectively. HEMEC and HIMEC were stimulated with TNF-α/IL-1β with or without BRE. The anti-inflammatory effects of BRE were assessed based upon COX-2, ICAM-1 and VCAM-1 gene and protein expression, PGE2 production, NFκB p65 subunit nuclear translocation as well as endothelial-leukocyte adhesion. The anti-angiogenic effects of BRE were assessed on cell migration, proliferation and tube formation following VEGF stimulation as well as on activation of Akt, MAPK and JNK signaling pathways. BRE inhibited TNF-α/IL-1β-induced NFκB p65 nuclear translocation, PGE2 production, up-regulation of COX-2, ICAM-1 and VCAM-1 gene and protein expression and leukocyte binding in HEMEC but not in HIMEC. BRE attenuated VEGF-induced cell migration, proliferation and tube formation in both HEMEC and HIMEC. The anti-angiogenic effect of BRE is mediated by inhibition of Akt, MAPK and JNK phosphorylations. BRE exerted differential anti-inflammatory effects between HEMEC and HIMEC following TNF-α/IL-1β activation whereas demonstrated similar anti-angiogenic effects following VEGF stimulation in both cell lines. These findings may provide more insight into the anti-tumorigenic capacities of BRE in human disease and cancer. PMID:25446010

  6. Anti inflammatory and anti angiogenic effect of black raspberry extract on human esophageal and intestinal microvascular endothelial cells.

    PubMed

    Medda, Rituparna; Lyros, Orestis; Schmidt, Jamie L; Jovanovic, Nebojsa; Nie, Linghui; Link, Benjamin J; Otterson, Mary F; Stoner, Gary D; Shaker, Reza; Rafiee, Parvaneh

    2015-01-01

    Polyphenolic compounds (anthocyanins, flavonoid glycosides) in berries prevent the initiation, promotion, and progression of carcinogenesis in rat's digestive tract and esophagus, in part, via anti-inflammatory pathways. Angiogenesis has been implicated in the pathogenesis of chronic inflammation and tumorigenesis. In this study, we investigated the anti-inflammatory and anti-angiogenic effects of black raspberry extract (BRE) on two organ specific primary human intestinal microvascular endothelial cells, (HIMEC) and human esophageal microvascular endothelial cells (HEMEC), isolated from surgically resected human intestinal and donor discarded esophagus, respectively. HEMEC and HIMEC were stimulated with TNF-α/IL-1β with or without BRE. The anti-inflammatory effects of BRE were assessed based upon COX-2, ICAM-1 and VCAM-1 gene and protein expression, PGE2 production, NFκB p65 subunit nuclear translocation as well as endothelial cell-leukocyte adhesion. The anti-angiogenic effects of BRE were assessed on cell migration, proliferation and tube formation following VEGF stimulation as well as on activation of Akt, MAPK and JNK signaling pathways. BRE inhibited TNF-α/IL-1β-induced NFκB p65 nuclear translocation, PGE2 production, up-regulation of COX-2, ICAM-1 and VCAM-1 gene and protein expression and leukocyte binding in HEMEC but not in HIMEC. BRE attenuated VEGF-induced cell migration, proliferation and tube formation in both HEMEC and HIMEC. The anti-angiogenic effect of BRE is mediated by inhibition of Akt, MAPK and JNK phosphorylations. BRE exerted differential anti-inflammatory effects between HEMEC and HIMEC following TNF-α/IL-1β activation whereas demonstrated similar anti-angiogenic effects following VEGF stimulation in both cell lines. These findings may provide more insight into the anti-tumorigenic capacities of BRE in human disease and cancer.

  7. Rickettsia rickettsii infection of human macrovascular and microvascular endothelial cells reveals activation of both common and cell type-specific host response mechanisms.

    PubMed

    Rydkina, Elena; Turpin, Loel C; Sahni, Sanjeev K

    2010-06-01

    Although inflammation and altered barrier functions of the vasculature, due predominantly to the infection of endothelial cell lining of small and medium-sized blood vessels, represent salient pathological features of human rickettsioses, the interactions between pathogenic rickettsiae and microvascular endothelial cells remain poorly understood. We have investigated the activation of nuclear transcription factor-kappa B (NF-kappaB) and p38 mitogen-activated protein (MAP) kinase, expression of heme oxygenase 1 (HO-1) and cyclooxygenase 2 (COX-2), and secretion of chemokines and prostaglandins after Rickettsia rickettsii infection of human cerebral, dermal, and pulmonary microvascular endothelial cells in comparison with pulmonary artery cells of macrovascular origin. NF-kappaB and p38 kinase activation and increased HO-1 mRNA expression were clearly evident in all cell types, along with relatively similar susceptibility to R. rickettsii infection in vitro but considerable variations in the intensities/kinetics of the aforementioned host responses. As expected, the overall activation profiles of macrovascular endothelial cells derived from human pulmonary artery and umbilical vein were nearly identical. Interestingly, cerebral endothelial cells displayed a marked refractoriness in chemokine production and secretion, while all other cell types secreted various levels of interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) in response to infection. A unique feature of all microvascular endothelial cells was the lack of induced COX-2 expression and resultant inability to secrete prostaglandin E(2) after R. rickettsii infection. Comparative evaluation thus yields the first experimental evidence for the activation of both common and unique cell type-specific host response mechanisms in macrovascular and microvascular endothelial cells infected with R. rickettsii, a prototypical species known to cause Rocky Mountain spotted fever in humans.

  8. An ultrastructural analysis of endothelial change paralleling platelet aggregation in a light/dye model of microvascular insult.

    PubMed Central

    Povlishock, J. T.; Rosenblum, W. I.; Sholley, M. M.; Wei, E. P.

    1983-01-01

    Those microvascular endothelial events that parallel the evolution of platelet aggregation were evaluated in a well-controlled animal model. Cat pial microvessels were observed through a cranial window while local platelet aggregation was produced by intravenous injection of sodium fluorescein and simultaneous exposure of the pial vessels to light from a filtered mercury lamp that excited the fluorescein. The vessels were fixed in situ when the in vivo observations of a preselected vessel indicated early, intermediate, or advanced aggregation in that vessel. The preselected vessel was then harvested for ultrastructural study together with adjacent vessels from the illuminated field. These vessels and appropriate controls were compared in semiserial thin sections. The onset of platelet aggregation in both venules and arterioles was accompanied by focal endothelial lucency, vacuole formation, luminal membrane rupture, and swelling of the nuclear envelope. These changes were not found in control material. With intermediate aggregation these changes were more common, while with advanced aggregation these abnormalities occurred together with focal endothelial denudation. Thus, in this model denudation occurred only with advanced aggregation and was not a prerequisite for aggregation. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 PMID:6824062

  9. Expression of VEGF-related proteins in cultured human brain microvascular endothelial cells and pericytes after exposure to methylmercury.

    PubMed

    Hirooka, Takashi; Yamamoto, Chika; Yasutake, Akira; Eto, Komyo; Kaji, Toshiyuki

    2013-01-01

    The localization of neuropathological lesions along deep sulci and fissures is one of the characteristics of a cerebrum damaged by methylmercury. Edematous changes in white matter have been proposed as the cause of the localization of lesions; however, the molecular mechanisms underlying methylmercury-induced edema remain unclear. Since the vascular endothelial growth factor (VEGF) system regulates vascular permeability and can be involved in the progression of edematous changes, we examined the effect of methylmercury on the expression of VEGF-related proteins in cultured human brain microvascular endothelial cells and pericytes. After methylmercury exposure, mRNA and protein levels of VEGF-A in pericytes and placenta growth factor (PlGF) and VEGF-receptor-1/-2 in endothelial cells were elevated. The induction of pericyte VEGF-A expression was independent of hypoxia-inducible factor-α and hypoxia-response element signaling. Taken together, these results suggest that methylmercury activates the VEGF system in brain microvessels in a paracrine fashion. When the activation occurs in narrow areas such as along the deep sulci in the cerebrum, hyperpermeability and subsequent edematous changes would cause a circulatory disturbance and result in neural cell damage. We propose this as a reason for the localization of the neuropathological lesions along the deep sulci and fissures in the cerebral cortex, such as the calcarine fissure, in patients with Minamata disease.

  10. Activation of sonic hedgehog signaling attenuates oxidized low-density lipoprotein-stimulated brain microvascular endothelial cells dysfunction in vitro.

    PubMed

    Jiang, Xiu-Long; Chen, Ting; Zhang, Xu

    2015-01-01

    The study was performed to investigate the role of sonic hedgehog (SHH) in the oxidized low-density lipoprotein (oxLDL)-induced blood-brain barrier (BBB) disruption. The primary mouse brain microvascular endothelial cells (MBMECs) were exposed to oxLDL. The results indicated that treatment of MBMECs with oxLDL decreased the cell viability, and oxidative stress was involved in oxLDL-induce MBMECs dysfunction with increasing intracellular ROS and MDA formation as well as decreasing NO release and eNOS mRNA expression. In addition, SHH signaling components, such as SHH, Smo and Gli1, mRNA and protein levels were significantly decreased after incubation with increasing concentrations of oxLDL. Treatment with oxLDL alone or SHH loss-of-function significantly increased the permeability of MBMECs, and overexpression of SHH attenuated oxLDL-induced elevation of permeability in MBMECs. Furthermore, SHH gain-of-function could reverse oxLDL-induced apoptosis through inhibition caspase3 and caspase8 levels in MBMECs. Taken together, these results demonstrated that the suppression of SHH in MBMECs might contribute to the oxLDL-induced disruption of endothelial barrier. However, the overexpression of SHH could reverse oxLDL-induced endothelial cells dysfunction in vitro.

  11. Metoprolol Improves Endothelial Function in Patients with Cardiac Syndrome X

    PubMed Central

    Majidinia, Maryam; Rasmi, Yousef; Khadem Ansari, Mohammad Hassan; Seyed-Mohammadzad, MirHossein; Saboory, Ehsan; Shirpoor, Alireza

    2016-01-01

    Endothelial dysfunction which is manifested by the loss of nitric oxide bioavailability, is an increasingly recognized cause of cardiac syndrome X (CSX) and beta blockers are used for the treatment of this syndrome. Thus, the aim of this study was to investigate effects of metoprolol, as a beta blocker, on endothelial function in CSX patients. The study included 25 CSX patients (20 female/ 5 male, mean age: 55.36±10.31 years) who received metoprolol (50 mg BID) for one month. In addition, 25 healthy controls (20 female/ 5 male, mean age: 54.32 ±9.27 years) were enrolled. Levels of endothelin-1, E-selectin, and vascular cell adhesion molecule-1 (VCAM-1) in controls and CSX patients were measured, both at the baseline and after the treatment, by the enzyme-linked immunosorbent assay. In CSX patients, at the baseline, levels of E-selectin and VCAM-1 were significantly higher than those of the controls. In addition, levels of these biomarkers in CSX patients after the treatment significantly decreased compared to the baseline. In spite of similar tendency, these differences were not significant for endothelin-1. In conclusion, metoprolol therapy improves endothelial function. Thus, it may be a suggested choice for CSX treatment. However, further studies are needed to confirm the clinical significance of metoprolol therapy for CSX patients. PMID:27980592

  12. Anti-TNFα therapy transiently improves high density lipoprotein cholesterol levels and microvascular endothelial function in patients with rheumatoid arthritis: a Pilot Study

    PubMed Central

    2012-01-01

    Background Rheumatoid arthritis (RA) is associated with increased morbidity and mortality from cardiovascular disease (CVD). This can be only partially attributed to traditional CVD risk factors such as dyslipidaemia and their downstream effects on endothelial function. The most common lipid abnormality in RA is reduced levels of high-density lipoprotein (HDL) cholesterol, probably due to active inflammation. In this longitudinal study we hypothesised that anti-tumor necrosis factor-α (anti-TNFα) therapy in patients with active RA improves HDL cholesterol, microvascular and macrovascular endothelial function. Methods Twenty-three RA patients starting on anti-TNFα treatment were assessed for HDL cholesterol level, and endothelial-dependent and -independent function of microvessels and macrovessels at baseline, 2-weeks and 3 months of treatment. Results Disease activity (CRP, fibrinogen, DAS28) significantly decreased during the follow-up period. There was an increase in HDL cholesterol levels at 2 weeks (p < 0.05) which was paralleled by a significant increase in microvascular endothelial-dependent function (p < 0.05). However, both parameters returned towards baseline at 12 weeks. Conclusion Anti-TNFα therapy in RA patients appears to be accompanied by transient but significant improvements in HDL cholesterol levels, which coexists with an improvement in microvascular endothelial-dependent function. PMID:22824166

  13. Functional expression of choline transporter like-protein 1 (CTL1) and CTL2 in human brain microvascular endothelial cells.

    PubMed

    Iwao, Beniko; Yara, Miki; Hara, Naomi; Kawai, Yuiko; Yamanaka, Tsuyoshi; Nishihara, Hiroshi; Inoue, Takeshi; Inazu, Masato

    2016-02-01

    In this study, we examined the molecular and functional characterization of choline transporter in human brain microvascular endothelial cells (hBMECs). Choline uptake into hBMECs was a saturable process that was mediated by a Na(+)-independent, membrane potential and pH-dependent transport system. The cells have two different [(3)H]choline transport systems with Km values of 35.0 ± 4.9 μM and 54.1 ± 8.1 μM, respectively. Choline uptake was inhibited by choline, acetylcholine (ACh) and the choline analog hemicholinium-3 (HC-3). Various organic cations also interacted with the choline transport system. Choline transporter-like protein 1 (CTL1) and CTL2 mRNA were highly expressed, while mRNA for high-affinity choline transporter 1 (CHT1) and organic cation transporters (OCTs) were not expressed in hBMECs. CTL1 and CTL2 proteins were localized to brain microvascular endothelial cells in human brain cortical sections. Both CTL1 and CTL2 proteins were expressed on the plasma membrane and mitochondria. CTL1 and CTL2 proteins are mainly expressed in plasma membrane and mitochondria, respectively. We conclude that choline is mainly transported via an intermediate-affinity choline transport system, CTL1 and CTL2, in hBMECs. These transporters are responsible for the uptake of extracellular choline and organic cations. CTL2 participate in choline transport mainly in mitochondria, and may be the major site for the control of choline oxidation.

  14. Cytotoxic effects of aflatoxin B1 on human brain microvascular endothelial cells of the blood-brain barrier.

    PubMed

    Qureshi, Humaira; Hamid, Saeed S; Ali, Syed Shayan; Anwar, Javeria; Siddiqui, Anwar Ali; Khan, Naveed Ahmed

    2015-05-01

    Aflatoxins are mycotoxins produced by Aspergillus spp. Although AFB1 is implicated as a carcinogen in hepatocellular carcinoma, brain autopsies in affected areas have revealed its presence in 81% of cases. Given its haematogenous spread, here we determined the cytotoxic effects of AFB1 on primary human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier, human umbilical vein endothelial cells (HUVEC) as well as immortalized epithelial cells of human hepatocellular carcinoma (Huh7). The cell types were exposed to AFB1 (3-32 nM) for 24 h and release of lactate dehydrogenase was measured as cell cytotoxicity marker. Furthermore, DNA was collected from both cell types and DNA adduct formation was determined by immunoblot using anti-AFB1-DNA adduct antibody. At 32 nM, AFB1 killed >85% HBMEC, while controls showed minimal effects (P < .05). Similar concentrations of AFB1 showed 22% cell death of HUVEC, while the same concentration did not kill Huh7. At low concentrations, in other words, 3.2 nM, AFB1 produced DNA adduct formation in HBMEC, while high concentration (32 nM) did not form DNA adducts. For HUVEC, 16 nM and 32 nM exhibited DNA adduct formation. For Huh7, 3.2 nM did not form DNA adducts, while 32 nM exhibited DNA adduct formation. For the first time, we report that AFB1 affected the viability of primary endothelial cells but not immortalized Huh7 cells. Cytotoxicity of brain endothelial cells suggests extra-hepatic complications post-AFB1 exposure.

  15. Human microvascular endothelial cellular interaction with atomic N-doped DLC compared with Si-doped DLC thin films.

    PubMed

    Okpalugo, T I T; Murphy, H; Ogwu, A A; Abbas, G; Ray, S C; Maguire, P D; McLaughlin, J; McCullough, R W

    2006-08-01

    This article reports results of endothelial cell interaction with atom beam source N-doped a-C:H (diamond-like carbon, DLC) as it compares with that of Si-doped DLC thin films. The RF plasma source exhibits up to 40% N-dissociation and N-atomic fluxes of approximately 0.85 x 10(18) atoms/s, which ensures better atomic nitrogen incorporation. Two different types of nitrogen species (with and without the use of sweep plates to remove charged ions) were employed for nitrogen doping. The number of attached endothelial cells is highest on Si-DLC, followed by the N-DLC (where the sweep plates were used to remove ions), the N-DLC (without the use of sweep plates), undoped DLC, and finally the uncoated sample. The contact angle values for these films suggest that water contact angle is higher in the atomic nitrogen neutral films and Si-DLC films compared to the ionized-nitrogen specie doped films and undoped DLC thin films, suggesting that the more hydrophobic films, semiconducting films, and film with relieved stress have better interaction with human microvascular endothelial cells. It seems evident that N-doping increases the Raman I(D)/I(G) ratios, whereas N-neutral doping decreases it slightly and Si-doping decreases it even further. In this study, lower Raman I(D)/I(G) ratios are associated with increased sp(3)/sp(2) ratio, an increased H concentration, photoluminescence intensity, and a higher endothelial cellular adhesion. These investigations could be relevant to biocompatibility assessment of nanostructured biomaterials and tissue engineering.

  16. Mst1 inhibits CMECs autophagy and participates in the development of diabetic coronary microvascular dysfunction

    PubMed Central

    Lin, Jie; Zhang, Lei; Zhang, Mingming; Hu, Jianqiang; Wang, Tingting; Duan, Yu; Man, Wanrong; Wu, Bin; Feng, Jiaxu; Sun, Lei; Li, Congye; Zhang, Rongqing; Wang, Haichang; Sun, Dongdong

    2016-01-01

    Cardiovascular complications account for a substantial proportion of morbidity and mortality in diabetic patients. Abnormalities of cardiac microvascular endothelial cells (CMECs) lead to impaired cardiac microvascular vessel integrity and subsequent cardiac dysfunction, underlining the importance of coronary microvascular dysfunction. In this study, experimental diabetes models were constructed using Mst1 transgenic, Mst1 knockout and sirt1 knockout mice. Diabetic Mst1 transgenic mice exhibited impaired cardiac microvessel integrity and decreased cardiac function. Mst1 overexpression deceased CMECs autophagy as evidenced by decreased LC3 expression and enhanced protein aggregation when subjected to high glucose culture. Mst1 knockout improved cardiac microvessel integrity and enhanced cardiac functions in diabetic mice. Mst1 knockdown up-regulated autophagy as indicated by more typical autophagosomes and increased LC3 expression in CMECs subjected to high glucose cultures. Mst1 knockdown also promoted autophagic flux in the presence of bafilomycin A1. Mst1 overexpression increased CMECs apoptosis, whereas Mst1 knockout decreased CMECs apoptosis. Sirt1 knockout abolished the effects of Mst1 overexpression in cardiac microvascular injury and cardiac dysfunction. In conclusion, Mst1 knockout preserved cardiac microvessel integrity and improved cardiac functions in diabetic mice. Mst1 decreased sirt1 activity, inhibited autophagy and enhanced apoptosis in CMECs, thus participating in the pathogenesis of diabetic coronary microvascular dysfunction. PMID:27680548

  17. Hsp90 inhibition suppresses NF-κB transcriptional activation via Sirt-2 in human lung microvascular endothelial cells.

    PubMed

    Thangjam, Gagan S; Birmpas, Charalampos; Barabutis, Nektarios; Gregory, Betsy W; Clemens, Mary Ann; Newton, Joseph R; Fulton, David; Catravas, John D

    2016-05-15

    The ability of anti-heat shock protein 90 (Hsp90) drugs to attenuate NF-κB-mediated transcription is the major basis for their anti-inflammatory properties. While the molecular mechanisms underlying this effect are not clear, they appear to be distinct in human endothelial cells. We now show for the first time that type 2 sirtuin (Sirt-2) histone deacetylase binds human NF-κB target gene promoter and prevents the recruitment of NF-κB proteins and subsequent assembly of RNA polymerase II complex in human lung microvascular endothelial cells. Hsp90 inhibitors stabilize the Sirt-2/promoter interaction and impose a "transcriptional block," which is reversed by either inhibition or downregulation of Sirt-2 protein expression. Furthermore, this process is independent of NF-κB (p65) Lysine 310 deacetylation, suggesting that it is distinct from known Sirt-2-dependent mechanisms. We demonstrate that Sirt-2 is recruited to NF-κB target gene promoter via interaction with core histones. Upon inflammatory challenge, chromatin remodeling and core histone H3 displacement from the promoter region removes Sirt-2 and allows NF-κB/coactivator recruitment essential for RNA Pol II-dependent mRNA induction. This novel mechanism may have important implications in pulmonary inflammation.

  18. Fibrin and collagen differentially but synergistically regulate sprout angiogenesis of human dermal microvascular endothelial cells in 3-dimensional matrix.

    PubMed

    Feng, Xiaodong; Tonnesen, Marcia G; Mousa, Shaker A; Clark, Richard A F

    2013-01-01

    Angiogenesis is a highly regulated event involving complex, dynamic interactions between microvascular endothelial cells and extracellular matrix (ECM) proteins. Alteration of ECM composition and architecture is a hallmark feature of wound clot and tumor stroma. We previously reported that during angiogenesis, endothelial cell responses to growth factors are modulated by the compositional and mechanical properties of a surrounding three-dimensional (3D) extracellular matrix (ECM) that is dominated by either cross-linked fibrin or type I collagen. However, the role of 3D ECM in the regulation of angiogenesis associated with wound healing and tumor growth is not well defined. This study investigates the correlation of sprout angiogenesis and ECM microenvironment using in vivo and in vitro 3D angiogenesis models. It demonstrates that fibrin and type I collagen 3D matrices differentially but synergistically regulate sprout angiogenesis. Thus blocking both integrin alpha v beta 3 and integrin alpha 2 beta 1 might be a novel strategy to synergistically block sprout angiogenesis in solid tumors.

  19. Pitfalls in assessing microvascular endothelial barrier function: impedance-based devices versus the classic macromolecular tracer assay

    PubMed Central

    Bischoff, Iris; Hornburger, Michael C.; Mayer, Bettina A.; Beyerle, Andrea; Wegener, Joachim; Fürst, Robert

    2016-01-01

    The most frequently used parameters to describe the barrier properties of endothelial cells (ECs) in vitro are (i) the macromolecular permeability, indicating the flux of a macromolecular tracer across the endothelium, and (ii) electrical impedance of ECs grown on gold-film electrodes reporting on the cell layer’s tightness for ion flow. Due to the experimental differences between these approaches, inconsistent observations have been described. Here, we present the first direct comparison of these assays applied to one single cell type (human microvascular ECs) under the same experimental conditions. The impact of different pharmacological tools (histamine, forskolin, Y-27632, blebbistatin, TRAP) on endothelial barrier function was analyzed by Transwell® tracer assays and two commercial impedance devices (xCELLigence®, ECIS®). The two impedance techniques provided very similar results for all compounds, whereas macromolecular permeability readings were found to be partly inconsistent with impedance. Possible reasons for these discrepancies are discussed. We conclude that the complementary combination of both approaches is highly recommended to overcome the restrictions of each assay. Since the nature of the growth support may contribute to the observed differences, structure-function relationships should be based on cells that are consistently grown on either permeable or impermeable growth supports in all experiments. PMID:27025965

  20. Molecular and Cellular Characterization of Space Flight Effects on Microvascular Endothelial Cell Function - PreparatoryWork for the SFEF Project

    NASA Astrophysics Data System (ADS)

    Balsamo, Michele; Barravecchia, Ivana; Mariotti, Sara; Merenda, Alessandra; De Cesari, Chiara; Vukich, Marco; Angeloni, Debora

    2014-12-01

    Exposure to microgravity during space flight (SF) of variable length induces suffering of the endothelium (the cells lining all blood vessels), mostly responsible for health problems found in astronauts and animals returning from space. Of interest to pre-nosological medicine, the effects of microgravity on astronauts are strikingly similar to the consequences of sedentary life, senescence and degenerative diseases on Earth, although SF effects are accelerated and reversible. Thus, microgravity is a significant novel model for better understanding of common pathologies. A comprehensive cell and molecular biology study is needed in order to explain pathophysiological findings after SFs. This project will study the effects of microgravity and cosmic radiation on endothelial cells (ECs) cultured on the International Space Station through analysis of 1) cell transcriptome, 2) DNA methylome, 3) DNA damage and cell senescence, 4) variations in cell cycle and cell morphology. This project has been selected by the European Space Agency and the Italian Space Agency and is presently in preparation. The ground study presented here was performed to determine the biological and engineering requirements that will allow us to retrieve suitable samples after culturing, fixing and storing ECs in space. We expect to identify molecular pathways activated by space microgravity in microvascular ECs, which may shed light on pathogenic molecular mechanisms responsible for endothelial suffering shared by astronauts and individuals affected with aging, degenerative and sedentary life-associated pathologies on Earth.

  1. Quantitative in vitro assay to measure neutrophil adhesion to activated primary human microvascular endothelial cells under static conditions.

    PubMed

    Wilhelmsen, Kevin; Farrar, Katherine; Hellman, Judith

    2013-08-23

    The vascular endothelium plays an integral part in the inflammatory response. During the acute phase of inflammation, endothelial cells (ECs) are activated by host mediators or directly by conserved microbial components or host-derived danger molecules. Activated ECs express cytokines, chemokines and adhesion molecules that mobilize, activate and retain leukocytes at the site of infection or injury. Neutrophils are the first leukocytes to arrive, and adhere to the endothelium through a variety of adhesion molecules present on the surfaces of both cells. The main functions of neutrophils are to directly eliminate microbial threats, promote the recruitment of other leukocytes through the release of additional factors, and initiate wound repair. Therefore, their recruitment and attachment to the endothelium is a critical step in the initiation of the inflammatory response. In this report, we describe an in vitro neutrophil adhesion assay using calcein AM-labeled primary human neutrophils to quantitate the extent of microvascular endothelial cell activation under static conditions. This method has the additional advantage that the same samples quantitated by fluorescence spectrophotometry can also be visualized directly using fluorescence microscopy for a more qualitative assessment of neutrophil binding.

  2. Evaluation of multiwalled carbon nanotube cytotoxicity in cultures of human brain microvascular endothelial cells grown on plastic or basement membrane.

    PubMed

    Eldridge, Brittany N; Xing, Fei; Fahrenholtz, Cale D; Singh, Ravi N

    2017-03-09

    There is a growing interest in the use of multiwalled carbon nanotubes (MWCNTs) to treat diseases of the brain. Little is known about the effects of MWCNTs on human brain microvascular endothelial cells (HBMECs), which make up the blood vessels in the brain. In our studies, we evaluate the cytotoxicity of MWCNTs and acid oxidized MWNCTs, with or without a phospholipid-polyethylene glycol coating. We determined the cytotoxic effects of MWCNTs on both tissue-mimicking cultures of HBMECs grown on basement membrane and on monolayer cultures of HBMECs grown on plastic. We also evaluated the effects of MWCNT exposure on the capacity of HBMECs to form rings after plating on basement membrane, a commonly used assay to evaluate angiogenesis. We show that tissue-mimicking cultures of HBMECs are less sensitive to all types of MWCNTs than monolayer cultures of HBMECs. Furthermore, we found that MWCNTs have little impact on the capacity of HBMECs to form rings. Our results indicate that relative cytotoxicity of MWCNTs is significantly affected by the type of cell culture model used for testing, and supports further research into the use of tissue-mimicking endothelial cell culture models to help bridge the gap between in vitro and in vivo toxicology.

  3. The multifaceted responses of primary human astrocytes and brain microvascular endothelial cells to the Lyme disease spirochete, Borrelia burgdorferi.

    PubMed

    Brissette, Catherine A; Kees, Eric D; Burke, Margaret M; Gaultney, Robert A; Floden, Angela M; Watt, John A

    2013-08-16

    The vector-borne pathogen, Borrelia burgdorferi, causes a multi-system disorder including neurological complications. These neurological disorders, collectively termed neuroborreliosis, can occur in up to 15% of untreated patients. The neurological symptoms are probably a result of a glial-driven, host inflammatory response to the bacterium. However, the specific contributions of individual glial and other support cell types to the pathogenesis of neuroborreliosis are relatively unexplored. The goal of this project was to characterize specific astrocyte and endothelial cell responses to B. burgdorferi. Primary human astrocytes and primary HBMEC (human brain microvascular endothelial cells) were incubated with B. burgdorferi over a 72-h period and the transcriptional responses to the bacterium were analyzed by real-time PCR arrays. There was a robust increase in several surveyed chemokine and related genes, including IL (interleukin)-8, for both primary astrocytes and HBMEC. Array results were confirmed with individual sets of PCR primers. The production of specific chemokines by both astrocytes and HBMEC in response to B. burgdorferi, including IL-8, CXCL-1, and CXCL-10, were confirmed by ELISA. These results demonstrate that primary astrocytes and HBMEC respond to virulent B. burgdorferi by producing a number of chemokines. These data suggest that infiltrating phagocytic cells, particularly neutrophils, attracted by chemokines expressed at the BBB (blood-brain barrier) may be important contributors to the early inflammatory events associated with neuroborreliosis.

  4. Sex-Specific Factors in Microvascular Angina

    PubMed Central

    Humphries, Karin H.; Bairey Merz, C. Noel

    2014-01-01

    Among women presenting for evaluation of suspected ischemic symptoms, a diagnosis of normal coronary arteries is five times more common, as compared to men. These women are often labeled as cardiac syndrome X (CSX), a subset of which have microvascular angina (MA) due to microvascular coronary dysfunction (MCD). MCD is not benign and is associated with an annual 2.5% cardiac event rate. Non-invasive testing for MCD remains insensitive although newer imaging modalities such as adenosine cardiac magnetic resonance imaging (CMRI) appear promising. The gold standard for diagnosis of MCD is coronary reactivity testing (CRT), an invasive technique which is not available in many countries. With regard to treatment, large scale trials are lacking. While research is ongoing, the current platform of therapy consists of anti-anginal, anti-platelet and endothelial modifying agents (primarily angiotensin converting enzyme inhibitors and statins). PMID:24582724

  5. 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

  6. Influence of curvature on the morphology of brain microvascular endothelial cells

    NASA Astrophysics Data System (ADS)

    Ye, Mao; Yang, Zhen; Wong, Andrew; Searson, Peter; Searson Group Team

    2013-03-01

    There are hundreds or thousands of endothelial cells around the perimeter of a single artery or vein, and hence an individual cell experiences little curvature. In contrast, a single endothelial cell may wrap around itself to form the lumen of a brain capillary. Curvature plays a key role in many biological, chemical and physical processes, however, its role in dictating the morphology and polarization of brain capillary endothelial cells has not been investigated. We hypothesize that curvature and shear flow play a key role in determining the structure and function of the blood-brain barrier (BBB). We have developed the ``rod'' assay to study the influence of curvature on the morphology of confluent monolayers of endothelial cells. In this assay cells are plated onto glass rods pulled down to the desired diameter in the range from 5 - 500 μm and coated with collagen. We show that curvature has a significant influence on the morphology of endothelial cells and may have an important role in blood-brain barrier function.

  7. Gene expression analysis reveals marked differences in the transcriptome of infantile hemangioma endothelial cells compared to normal dermal microvascular endothelial cells

    PubMed Central

    2013-01-01

    Background Infantile hemangiomas are benign vascular tumors primarily found on the skin in 10% of the pediatric population. The etiology of this disease is largely unknown and while large scale genomic studies have examined the transcriptomes of infantile hemangioma tumors as a whole, no study to date has compared the global gene expression profiles of pure infantile hemangioma endothelial cells (HEMECs) to that of normal human dermal microvascular endothelial cells (HDMVECs). Methods To shed light on the molecular differences between these normal and aberrant dermal endothelial cell types, we performed whole genome microarray analysis on purified cultures of HEMECs and HDMVECs. We then utilized qPCR and immunohistochemistry to confirm our microarray results. Results Our array analysis identified 125 genes whose expression was upregulated and 104 genes whose expression was downregulated by greater than two fold in HEMECs compared to HDMVECs. Bioinformatics analysis revealed three major classifications of gene functions that were altered in HEMECs including cell adhesion, cell cycle, and arachidonic acid production. Several of these genes have been reported to be critical regulators and/or mutated in cancer, vascular tumors, and vascular malformations. We confirmed the expression of a subset of these differentially expressed genes (ANGPT2, ANTXR1, SMARCE1, RGS5, CTAG2, LTBP2, CLDN11, and KISS1) using qPCR and utilized immunohistochemistry on a panel of paraffin embedded infantile hemangioma tumor tissues to demonstrate that the cancer/testis antigen CTAG2 is highly abundant in vessel-dense proliferating infantile hemangiomas and with significantly reduced levels during tumor involution as vascular density decreases. Conclusion Our data reveal that the transcriptome of HEMECs is reflective of a pro-proliferative cell type with altered adhesive characteristics. Moveover, HEMECs show altered expression of many genes that are important in the progression and prognosis

  8. Caveolin-1 mediates tissue plasminogen activator-induced MMP-9 up-regulation in cultured brain microvascular endothelial cells.

    PubMed

    Jin, Xinchun; Sun, Yanyun; Xu, Ji; Liu, Wenlan

    2015-03-01

    Thrombolysis with tissue plasminogen activator (tPA) increases matrix metalloproteinase-9 (MMP-9) activity in the ischemic brain, which exacerbates blood-brain barrier injury and increases the risk of symptomatic cerebral hemorrhage. The mechanism through which tPA enhances MMP-9 activity is not well understood. Here we report an important role of caveolin-1 in mediating tPA-induced MMP-9 synthesis. Brain microvascular endothelial cell line bEnd3 cells were incubated with 5 or 20 μg/ml tPA for 24 hrs before analyzing MMP-9 levels in the conditioned media and cellular extracts by gelatin zymography. tPA at a dose of 20 μg/mL tPA, but not 5 μg/mL, significantly increased MMP-9 level in cultured media while decreasing it in cellular extracts. Concurrently, tPA treatment induced a 2.3-fold increase of caveolin-1 protein levels in endothelial cells. Interestingly, knockdown of Cav-1 with siRNA inhibited tPA-induced MMP-9 mRNA up-regulation and MMP-9 increase in the conditioned media, but did not affect MMP-9 decrease in cellular extracts. These results suggest that caveolin-1 critically contributes to tPA-mediated MMP-9 up-regulation, but may not facilitate MMP-9 secretion in endothelial cells. Thrombolysis with tissue plasminogen activator (tPA) increases matrix metalloproteinase-9 (MMP-9) activity in the ischemic brain, which exacerbates ischemic blood brain barrier (BBB) injury and increases the risk of symptomatic cerebral hemorrhage. Our results suggest a novel mechanism underlying this tPA-MMP 9 axis. In response to tPA treatment, caveolin-1 protein levels increased in endothelial cells, which mediate MMP-9 mRNA up-regulation and its secretion into extracellular space. Caveolin-1 may, however, not facilitate MMP-9 secretion in endothelial cells. Our data suggest caveolin-1 as a novel therapeutic target for protecting the BBB against ischemic damage. The schematic outlines tPA-induced MMP-9 upreguation.

  9. Carnosine protects brain microvascular endothelial cells against rotenone-induced oxidative stress injury through histamine H₁ and H₂ receptors in vitro.

    PubMed

    Zhang, Luyi; Yao, Ke; Fan, Yanying; He, Ping; Wang, Xiaofen; Hu, Weiwei; Chen, Zhong

    2012-12-01

    Although it is believed that carnosine has protective effects on various cell types, its effect on microvascular endothelial cells has not been well defined. In the present study, we investigated the protective effects of carnosine in microvascular endothelial cells using an in vitro rotenone-induced oxidative stress model. Mouse brain microvascular endothelial cells were exposed to 1 μmol/L rotenone for 18 h. In some experiments, carnosine (100 nmol/L-1 mmol/L) was added 30 min prior to rotenone exposure. When used, histamine receptor antagonists (100 nmol/L-10 μmol/L) were added 15 min before carnosine treatment. After rotenone exposure, apoptosis of microvascular cells was analysed by Hoechst 33342 staining, whereas mitochondrial membrane potential was assessed by JC-1 staining. Intracellular carnosine and histamine levels were determined using HPLC or ultra-HPLC. Over the range 1 μmol/L-1 mmol/L, carnosine concentration-dependently decreased the number of apoptotic cells after 18 h exposure to rotenone. This effect was reversed by the histamine H1 receptor antagonists pyrilamine and diphenhydramine (1 and 10 μmol/L) and the H2 receptor antagonists cimetidine (100 nmol/L-10 μmol/L) and zolatidine (10 μmol/L). α-Fluoromethylhistidine (100 μmol/L), a selective and irreversible inhibitor of histidine decarboxylase, also significantly inhibited the protective effects of carnosine. At 0.1 mmol/L, carnosine restored the decrease in mitochondrial membrane potential after 6 h exposure to 1 μmol/L rotenone and this effect was also reversed by the H1 and H2 receptor antagonists. Moreover, intracellular carnosine levels increased as early as 1 h after carnosine treatment, whereas intracellular histamine levels increased 18 h after carnosine treatment. The results of the present study indicate that carnosine protects brain microvascular endothelial cells against rotenone-induced oxidative stress injury via histamine H1 and H2 receptors. The

  10. Endothelial progenitor cells, microvascular obstruction, and left ventricular remodeling in patients with ST elevation myocardial infarction undergoing primary percutaneous coronary intervention.

    PubMed

    Porto, Italo; De Maria, Giovanni Luigi; Leone, Antonio Maria; Dato, Ilaria; D'Amario, Domenico; Burzotta, Francesco; Niccoli, Giampaolo; Trani, Carlo; Biasucci, Luigi Marzio; Bolognese, Leonardo; Crea, Filippo

    2013-09-15

    Endothelial progenitor cells (EPCs) are released from the bone marrow during cardiac ischemic events, potentially influencing vascular and myocardial repair. We assessed the clinical and angiographic correlates of EPC mobilization at the time of primary percutaneous coronary intervention in 78 patients with ST elevation myocardial infarction and the impact of both baseline and follow-up EPC levels on left ventricular (LV) remodeling. Blood samples were drawn from the aorta and the culprit coronary artery for cytofluorimetric EPC detection (CD34+CD45dimKDR+ cells, in percentage of cytofluorimetric counts). Area at risk was assessed by Bypass Angioplasty Revascularization Investigation myocardial jeopardy index, thrombotic burden as thrombus score and microvascular obstruction (MVO) as a combination of ST segment resolution and myocardial blush grade. Echocardiographic evaluation of LV remodeling was performed at 1-year follow-up in 54 patients, whereas peripheral EPC levels were reassessed in 40 patients. EPC levels during primary percutaneous coronary intervention were significantly higher in intracoronary than in aortic blood (0.043% vs 0.0006%, p <0.001). Both intracoronary and aortic EPC were related to area at risk extent, to intracoronary thrombus score (p <0.001), and inversely to MVO (p = 0.001). Peripheral EPC levels at 1-year follow-up were lower in patients with LV remodeling than in those without (0.001% [0.001 to 0.002] vs 0.003% [0.002 to 0.010]; p = 0.01) and independently predicted absence of remodeling at multivariate analysis. In conclusion, a rapid intracoronary EPC recruitment takes place in the early phases of ST elevation myocardial infarction, possibly reflecting an attempted reparative response. The extent of this mobilization seems to be correlated to the area at risk and to the amount of MVO. Persistently low levels of EPC are associated to LV remodeling.

  11. Involvement of PI3K and ROCK signaling pathways in migration of bone marrow-derived mesenchymal stem cells through human brain microvascular endothelial cell monolayers.

    PubMed

    Lin, Mei-Na; Shang, De-Shu; Sun, Wei; Li, Bo; Xu, Xin; Fang, Wen-Gang; Zhao, Wei-Dong; Cao, Liu; Chen, Yu-Hua

    2013-06-04

    Bone marrow-derived mesenchymal stem cells (MSC) represent an important and easily available source of stem cells for potential therapeutic use in neurological diseases. The entry of circulating cells into the central nervous system by intravenous administration requires, firstly, the passage of the cells across the blood-brain barrier (BBB). However, little is known of the details of MSC transmigration across the BBB. In the present study, we employed an in vitro BBB model constructed using a human brain microvascular endothelial cell monolayer to study the mechanism underlying MSC transendothelial migration. Transmigration assays, transendothelial electrical resistance (TEER) and horseradish peroxidase (HRP) flux assays showed that MSC could transmigrate through human brain microvascular endothelial cell monolayers by a paracellular pathway. Cell fractionation and immunofluorescence assays confirmed the disruption of tight junctions. Inhibition assays showed that a Rho-kinase (ROCK) inhibitor (Y27632) effectively promoted MSC transendothelial migration; conversely, a PI3K inhibitor (LY294002) blocked MSC transendothelial migration. Interestingly, adenovirus-mediated interference with ROCK in MSC significantly increased MSC transendothelial migration, and overexpression of a PI3K dominant negative mutant in MSC cells could block transendothelial migration. Our findings provide clear evidence that the PI3K and ROCK pathways are involved in MSC migration through human brain microvascular endothelial cell monolayers. The information yielded by this study may be helpful in constructing gene-modified mesenchymal stem cells that are able to penetrate the BBB effectively for cell therapy.

  12. Dexamethasone suppresses JMJD3 gene activation via a putative negative glucocorticoid response element and maintains integrity of tight junctions in brain microvascular endothelial cells.

    PubMed

    Na, Wonho; Shin, Jee Y; Lee, Jee Y; Jeong, Sangyun; Kim, Won-Sun; Yune, Tae Y; Ju, Bong-Gun

    2017-01-01

    The blood-brain barrier (BBB) exhibits a highly selective permeability to support the homeostasis of the central nervous system (CNS). The tight junctions in the BBB microvascular endothelial cells seal the paracellular space to prevent diffusion. Thus, disruption of tight junctions results in harmful effects in CNS diseases and injuries. It has recently been demonstrated that glucocorticoids have beneficial effects on maintaining tight junctions in both in vitro cell and in vivo animal models. In the present study, we found that dexamethasone suppresses the expression of JMJD3, a histone H3K27 demethylase, via the recruitment of glucocorticoid receptor α (GRα) and nuclear receptor co-repressor (N-CoR) to the negative glucocorticoid response element (nGRE) in the upstream region of JMJD3 gene in brain microvascular endothelial cells subjected to TNFα treatment. The decreased JMJD3 gene expression resulted in the suppression of MMP-2, MMP-3, and MMP-9 gene activation. Dexamethasone also activated the expression of the claudin 5 and occludin genes. Collectively, dexamethasone attenuated the disruption of the tight junctions in the brain microvascular endothelial cells subjected to TNFα treatment. Therefore, glucocorticoids may help to preserve the integrity of the tight junctions in the BBB via transcriptional and post-translational regulation following CNS diseases and injuries.

  13. 26S Proteasome regulation of Ankrd1/CARP in adult rat ventricular myocytes and human microvascular endothelial cells

    SciTech Connect

    Samaras, Susan E.; Chen, Billy; Koch, Stephen R.; Sawyer, Douglas B.; Lim, Chee Chew; Davidson, Jeffrey M.

    2012-09-07

    Highlights: Black-Right-Pointing-Pointer The 26S proteasome regulates Ankrd1 levels in cardiomyocytes and endothelial cells. Black-Right-Pointing-Pointer Ankrd1 protein degrades 60-fold faster in endothelial cells than cardiomyocytes. Black-Right-Pointing-Pointer Differential degradation appears related to nuclear vs. sarcolemmal localization. Black-Right-Pointing-Pointer Endothelial cell density shows uncoupling of Ankrd1 mRNA and protein levels. -- Abstract: Ankyrin repeat domain 1 protein (Ankrd1), also known as cardiac ankyrin repeat protein (CARP), increases dramatically after tissue injury, and its overexpression improves aspects of wound healing. Reports that Ankrd1/CARP protein stability may affect cardiovascular organization, together with our findings that the protein is crucial to stability of the cardiomyocyte sarcomere and increased in wound healing, led us to compare the contribution of Ankrd1/CARP stability to its abundance. We found that the 26S proteasome is the dominant regulator of Ankrd1/CARP degradation, and that Ankrd1/CARP half-life is significantly longer in cardiomyocytes (h) than endothelial cells (min). In addition, higher endothelial cell density decreased the abundance of the protein without affecting steady state mRNA levels. Taken together, our data and that of others indicate that Ankrd1/CARP is highly regulated at multiple levels of its expression. The striking difference in protein half-life between a muscle and a non-muscle cell type suggests that post-translational proteolysis is correlated with the predominantly structural versus regulatory role of the protein in the two cell types.

  14. Endogenous microRNAs in human microvascular endothelial cells regulate mRNAs encoded by hypertension-related genes.

    PubMed

    Kriegel, Alison J; Baker, Maria Angeles; Liu, Yong; Liu, Pengyuan; Cowley, Allen W; Liang, Mingyu

    2015-10-01

    The goal of this study was to systematically identify endogenous microRNAs (miRNAs) in endothelial cells that regulate mRNAs encoded by genes relevant to hypertension. Small RNA deep sequencing was performed in cultured human microvascular endothelial cells. Of the 50 most abundant miRNAs identified, 30 had predicted target mRNAs encoded by genes with known involvement in hypertension or blood pressure regulation. The cells were transfected with anti-miR oligonucleotides to inhibit each of the 30 miRNAs and the mRNA abundance of predicted targets was examined. Of 95 miRNA-target pairs examined, the target mRNAs were significantly upregulated in 35 pairs and paradoxically downregulated in 8 pairs. The result indicated significant suppression of the abundance of mRNA encoded by ADM by endogenous miR-181a-5p, ATP2B1 by the miR-27 family, FURIN by miR-125a-5p, FGF5 by the let-7 family, GOSR2 by miR-27a-3p, JAG1 by miR-21-5p, SH2B3 by miR-30a-5p, miR-98, miR-181a-5p, and the miR-125 family, TBX3 by the miR-92 family, ADRA1B by miR-22-3p, ADRA2A by miR-30a-5p and miR-30e-5p, ADRA2B by miR-30e-5p, ADRB1 by the let-7 family and miR-98, EDNRB by the miR-92 family, and NOX4 by the miR-92 family, miR-100-5p, and miR-99b-5p (n=3-9; P<0.05 versus scrambled anti-miR). Treatment with anti-miR-21 decreased blood pressure in mice fed a 4% NaCl diet. Inhibition of the miRNAs targeting NOX4 mRNA increased H2O2 release from endothelial cells. The findings indicate widespread, tonic control of mRNAs encoded by genes relevant to blood pressure regulation by endothelial miRNAs and provide a novel and uniquely informative basis for studying the role of miRNAs in hypertension.

  15. Glucose starvation is required for insulin stimulation of glucose uptake and metabolism in cultured microvascular endothelial cells

    SciTech Connect

    Gerritsen, M.E.; Burke, T.M.; Allen, L.A.

    1988-03-01

    In the present study we determined the uptake and disposition of glucose in serum-deprived rabbit coronary microvessel endothelial (RCME) cells. RCME cells exhibited stereospecific hexose uptake inhibited by cytochalasin B. Pretreatment of the RCME cells with potassium cyanide or 2,4-dinitrophenol inhibited 2-deoxyglucose uptake but not 3-O-methylglucose transport. A major proportion (30-60%) of the 2-deoxyglucose present in the RCME cells was not phosphorylated. These two observations suggested that the rate-limiting step in the uptake of 2-deoxyglucose was not transport but rather the phosphorylation of 2-deoxyglucose to 2-deoxyglucose 6-phosphate. When glucose-deprived cells were incubated 2 hr with (U-14C)glucose the disposition of the label was as follows: glycogen 60%, acid-soluble fraction 30%, and lipid less than 5%. In contrast glucose-fed cells exhibited lower overall glucose incorporation, and a slightly different disposition: glycogen 45%, acid-soluble fraction 50%, and lipid 5%. Glucose-deprived RCME cells also exhibited greater basal levels of 2-deoxyglucose uptake compared to glucose-fed cells. RCME cells incubated in the absence of glucose and serum for 16 hr exhibited dose-dependent insulin stimulation of hexose uptake and subsequent metabolism to macromolecules (i.e., glycogen and the acid-soluble fraction). Significant effects of insulin were observed with concentrations as low as 2 x 10(-10) M, well within the physiological range. In contrast, cells preincubated in serum-free culture medium containing 5.5 mM glucose did not exhibit insulin-enhanced hexose uptake or glucose metabolism (even at doses as high as 10(-7) M). These studies indicate that the effects of insulin on rabbit coronary microvascular endothelial cell glucose uptake and metabolism require both serum and glucose deprivation.

  16. Focal adhesion kinase is involved in type III group B streptococcal invasion of human brain microvascular endothelial cells.

    PubMed

    Shin, Sooan; Paul-Satyaseela, Maneesh; Maneesh, Paul-Satyaseela; Lee, Jong-Seok; Romer, Lewis H; Kim, Kwang Sik

    2006-01-01

    Group B streptococcus (GBS), the leading cause of neonatal meningitis, has been shown to invade human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier. GBS invasion of HBMEC has been shown to require the host cell actin cytoskeleton rearrangements. The present study examined the mechanisms underlying actin cytoskeleton rearrangements that are involved in type III GBS invasion of HBMEC. We showed that type III GBS invasion was inhibited by genistein, a general tyrosine kinase inhibitor (mean 54% invasion decrease at 100 microM), and LY294002, a phosphatidylinositol 3 (PI3) kinase inhibitor (mean 70% invasion decrease at 50 microM), but not by PP2, an inhibitor of the Src family tyrosine kinases. We subsequently showed that the focal adhesion kinase (FAK) was the one of the host proteins tyrosine phosphorylated by type III GBS. Over-expression of a dominant negative form of the FAK C-terminal domain significantly decreased type III GBS invasion of HBMEC (mean 51% invasion decrease). In addition, we showed that FAK phosphorylation correlated with its association of paxillin, an adapter protein of actin filament, and PI3-kinase subunit p85. This is the first demonstration that FAK phosphorylation and its association with paxillin and PI3 kinase play a key role in type III GBS invasion of HBMEC.

  17. Proteomic differences between microvascular endothelial cells and the EA.hy926 cell line forming three-dimensional structures.

    PubMed

    Ma, Xiao; Sickmann, Albert; Pietsch, Jessica; Wildgruber, Robert; Weber, Gerhard; Infanger, Manfred; Bauer, Johann; Grimm, Daniela

    2014-03-01

    Proteomic changes of two types of human endothelial cells (ECs) were determined and compared to morphological alterations occurring during the scaffold-free in vitro formation of 3D structures resembling vascular intimas. The EA.hy926 cell line and human microvascular ECs (HMVECs) were cultured on a random positioning machine or static on ground (normal gravity) for 5 and 7 days, before their morphology was examined and their protein content was analysed by MS after free-flow electrophoretic separation. A total of 1175 types of proteins were found in EA.hy926 cells and 846 in HMVEC forming 3D structures faster than the EA.hy926 cells. Five hundred and eighty-four of these kinds of proteins were present in both types of cells. They included a number of metabolic enzymes, of structure-related and stress proteins. Comparing proteins of EA.hy926 cells growing either adherently on ground or in 3D aggregates on the random positioning machine revealed that ribosomal proteins were enhanced, while tubes are formed and various components of 26S proteasomes remained prevalent in static normal gravity control cells only. The fast developing tube-like 3D structures of HMVEC suggested a transient augmentation of ribosomal proteins during the 3D assembling of ECs.

  18. Proteomic analysis of human brain microvascular endothelial cells reveals differential protein expression in response to enterovirus 71 infection.

    PubMed

    Luo, Wenying; Zhong, Jiayu; Zhao, Wei; Liu, Jianjun; Zhang, Renli; Peng, Liang; Hong, Wenxu; Huang, Sheng He; Cao, Hong

    2015-01-01

    2D DIGE technology was employed on proteins prepared from human brain microvascular endothelial cells (HBMEC), to study the differentially expressed proteins in cells at 0 h, 1 h, 16 h, and 24 h after infection. Proteins found to be differentially expressed were identified with matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDITOF/TOF MS) analysis. We identified 43 spots showing changes of at least 2.5 fold up- or downregulated expressions in EV71-infected cells at different time when comparing to control, and 28 proteins could be successfully identified by MALDI TOF/TOF mass spectrometry analysis. 4 proteins were significantly upregulated, and 6 proteins were downregulated, another 18 proteins were different expression at different incubation time. We identified changes in the expression of 12 cellular metabolism-related proteins, 5 molecules involved in cytoskeleton, 3 molecules involved in energy metabolism, 2 molecules involved in signal transduction, 1 molecule involved in the ubiquitin-proteasome pathway, 1 molecule involved in cell cycle, 1 molecule involved in apoptosis-related protein, 1 molecular chaperone, and 2 unknown proteins. These findings build up a comprehensive profile of the HBMEC proteome and provide a useful basis for further analysis of the pathogenic mechanism that underlies EV71 infections to induce severe neural complications.

  19. Exosomes contribute to the transmission of anti-HIV activity from TLR3-activated brain microvascular endothelial cells to macrophages

    PubMed Central

    Sun, Li; Wang, Xu; Zhou, Yu; Zhou, Run-Hong; Ho, Wen-Zhe; Li, Jie-Liang

    2017-01-01

    Human brain microvascular endothelial cells (HBMECs), the major cell type in the blood-brain barrier (BBB), play a key role in maintaining brain homeostasis. However, their role in the BBB innate immunity against HIV invasion of the central nervous system (CNS) remains to be determined. Our early work showed that TLR3 signaling of HBMECs could produce the antiviral factors that inhibit HIV replication in macrophages. The present study examined whether exosomes from TLR3-activated HBMECs mediate the intercellular transfer of antiviral factors to macrophages. Primary human macrophages could take up exosomes from TLR3-activated HBMECs. HBMECs-derived exosomes contained multiple antiviral factors, including several key IFN-stimulated genes (ISGs; ISG15, ISG56, and Mx2) at mRNA and protein levels. The depletion of exosomes from TLR3-activated HBMECs culture supernatant diminished HBMECs-mediated anti-HIV activity in macrophages. In conclusion, we demonstrate that exosomes shed by HBMECs are able to transport the antiviral molecules to macrophages. This finding suggests the possibility that HIV nonpermissive BBB cells (HBMECs) can help to restore the antiviral state in HIV-infected macrophages, which may be a defense mechanism against HIV neuroinvasion. PMID:27496004

  20. Ornithine decarboxylase and extracellular polyamines regulate microvascular sprouting and actin cytoskeleton dynamics in endothelial cells

    SciTech Connect

    Kucharzewska, Paulina; Welch, Johanna E.; Svensson, Katrin J.; Belting, Mattias

    2010-10-01

    The polyamines are essential for cancer cell proliferation during tumorigenesis. Targeted inhibition of ornithine decarboxylase (ODC), i.e. a key enzyme of polyamine biosynthesis, by {alpha}-difluoromethylornithine (DFMO) has shown anti-neoplastic activity in various experimental models. This activity has mainly been attributed to the anti-proliferative effect of DFMO in cancer cells. Here, we provide evidence that unperturbed ODC activity is a requirement for proper microvessel sprouting ex vivo as well as the migration of primary human endothelial cells. DFMO-mediated ODC inhibition was reversed by extracellular polyamine supplementation, showing that anti-angiogenic effects of DFMO were specifically related to polyamine levels. ODC inhibition was associated with an abnormal morphology of the actin cytoskeleton during cell spreading and migration. Moreover, our data suggest that de-regulated actin cytoskeleton dynamics in DFMO treated endothelial cells may be related to constitutive activation of the small GTPase CDC42, i.e. a well-known regulator of cell motility and actin cytoskeleton remodeling. These insights into the potential role of polyamines in angiogenesis should stimulate further studies testing the combined anti-tumor effect of polyamine inhibition and established anti-angiogenic therapies in vivo.

  1. VE-cadherin involved in the pulmonary microvascular endothelial cell barrier injury induced by angiotensin II through modulating the cellular apoptosis and skeletal rearrangement

    PubMed Central

    Wu, Zhiyong; Liu, Huagang; Ren, Wei; Dai, Feifeng; Chang, Jinxing; Li, Bowen

    2016-01-01

    Objective: Angiotensin II (AngII) involved in the pathogenesis of pulmonary injury through impairing the integrity of pulmonary microvascular endothelial barrier, but the mechanism is still not clear. We aim to determine the roles of VE-cadherin, playing crucial roles in the adhesion of the vascular endothelial barrier and the barrier function, in the pulmonary microvascular endothelial cell (PMVEC) barrier injury mediated by AngII. Methods: Mice acute lung injury (ALI) model was induced through pumping of AngII. The infiltration of macrophages and neutrophils as well as the PMVEC permeability were determined in order to determine the barrier injury in vivo and in vitro. Knockdown of VE-cadherin was established using siRNA technique, and its roles in the apoptosis and skeletal rearrangement in the PMVECs were evaluated. Results: After AngII interference, the expression of VE-cadherin in the PMVECs and pulmonary tissues in mice was down-regulated. Upon VE-cadherin knockdown through siRNA technique, AngII induced susceptibility of PMVECs to apoptosis. Knockdown of VE-cadherin contributed to the skeletal rearrangement in the endothelial cells, together with increase of permeability. Conclusions: VE-cadherin expression is closely related to the apoptosis and skeletal rearrangement of PMVECs induced by AngII. PMID:27830014

  2. Recent Insights in the Paracrine Modulation of Cardiomyocyte Contractility by Cardiac Endothelial Cells

    PubMed Central

    Andriantsitohaina, Ramaroson

    2014-01-01

    The cardiac endothelium is formed by a continuous monolayer of cells that line the cavity of the heart (endocardial endothelial cells (EECs)) and the luminal surface of the myocardial blood vessels (intramyocardial capillary endothelial cells (IMCEs)). EECs and IMCEs can exercise substantial control over the contractility of cardiomyocytes by releasing various factors such as nitric oxide (NO) via a constitutive endothelial NO-synthase (eNOS), endothelin-1, prostaglandins, angiotensin II, peptide growth factors, and neuregulin-1. The purpose of the present paper is actually to shortly review recent new information concerning cardiomyocytes as effectors of endothelium paracrine signaling, focusing particularly on contractile function. The modes of action and the regulatory paracrine role of the main mediators delivered by cardiac endothelial cells upon cardiac contractility identified in cardiomyocytes are complex and not fully described. Thus, careful evaluation of new therapeutic approaches is required targeting important physiological signaling pathways, some of which have been until recently considered as deleterious, like reactive oxygen species. Future works in the field of cardiac endothelial cells and cardiac function will help to better understand the implication of these mediators in cardiac physiopathology. PMID:24745027

  3. Measures of endothelial dysfunction predict response to cardiac resynchronisation therapy

    PubMed Central

    Warriner, David R; Lawford, Patricia; Sheridan, Paul J

    2016-01-01

    Objectives Cardiac resynchronisation therapy (CRT) improves morbidity and mortality in heart failure (HF). Impaired endothelial function, as measured by flow-mediated dilation (FMD) is associated with increased morbidity and mortality in HF and may help to differentiate responders from non-responders. Methods 19 patients were recruited, comprising 94% men, mean age 69±8 years, New York Heart Association functional classes II–IV, QRSd 161±21 ms and mean left ventricular ejection fraction 26±8%. Markers of response and FMD were measured at baseline, 6 and 12 months following CRT. Results 14 patients were responders to CRT. Responders had significant improvements in VO2 (12.6±1.7 to 14.7±1.5 mL/kg/min, p<0.05), quality of life score (44.4±22.9–24.1±21.3, p<0.01), left ventricular end diastolic volume (201.5±72.5 mL–121.3±72.0 mL, p<0.01) and 6-min walk distance (374.0±112.8 m at baseline to 418.1±105.3 m, p<0.05). Baseline FMD in responders was 2.9±1.9% and 7.4±3.73% in non-responders (p<0.05). Conclusions Response to CRT at 6 and 12 months is predicted by baseline FMD. This study confirms that FMD identifies responders to CRT, due to endothelium-dependent mechanisms alone. PMID:27335654

  4. Brain microvascular endothelial cell association and distribution of a 5 nm ceria engineered nanomaterial

    PubMed Central

    Dan, Mo; Tseng, Michael T; Wu, Peng; Unrine, Jason M; Grulke, Eric A; Yokel, Robert A

    2012-01-01

    Purpose: Ceria engineered nanomaterials (ENMs) have current commercial applications and both neuroprotective and toxic effects. Our hypothesis is that ceria ENMs can associate with brain capillary cells and/or cross the blood–brain barrier. Methods: An aqueous dispersion of ∼5 nm ceria ENM was synthesized and characterized in house. Its uptake space in the Sprague Dawley rat brain was determined using the in situ brain perfusion technique at 15 and 20 mL/minute flow rates; 30, 100, and 500 μg/mL ceria perfused for 120 seconds at 20 mL/minute; and 30 μg/mL perfused for 20, 60, and 120 seconds at 20 mL/minute. The capillary depletion method and light and electron microscopy were used to determine its capillary cell and brain parenchymal association and localization. Results: The vascular space was not significantly affected by brain perfusion flow rate or ENM, demonstrating that this ceria ENM did not influence blood–brain barrier integrity. Cerium concentrations, determined by inductively coupled plasma mass spectrometry, were significantly higher in the choroid plexus than in eight brain regions in the 100 and 500 μg/mL ceria perfusion groups. Ceria uptake into the eight brain regions was similar after 120-second perfusion of 30, 100, and 500 μg ceria/mL. Ceria uptake space significantly increased in the eight brain regions and choroid plexus after 60 versus 20 seconds, and it was similar after 60 and 120 seconds. The capillary depletion method showed 99.4% ± 1.1% of the ceria ENM associated with the capillary fraction. Electron microscopy showed the ceria ENM located on the endothelial cell luminal surface. Conclusion: Ceria ENM association with brain capillary endothelial cells saturated between 20 and 60 seconds and ceria ENM brain uptake was not diffusion-mediated. During the 120-second ceria ENM perfusion, ceria ENM predominately associated with the surface of the brain capillary cells, providing the opportunity for its cell uptake or redistribution

  5. Edaravone protected human brain microvascular endothelial cells from methylglyoxal-induced injury by inhibiting AGEs/RAGE/oxidative stress.

    PubMed

    Li, Wenlu; Xu, Hongjiao; Hu, Yangmin; He, Ping; Ni, Zhenzhen; Xu, Huimin; Zhang, Zhongmiao; Dai, Haibin

    2013-01-01

    Subjects with diabetes experience an increased risk of cerebrovascular disease and stroke compared with nondiabetic age-matched individuals. Increased formation of reactive physiological dicarbonyl compound methylglyoxal (MGO) seems to be implicated in the development of diabetic vascular complication due to its protein glycation and oxidative stress effect. Edaravone, a novel radical scavenger, has been reported to display the advantageous effects on ischemic stroke both in animals and clinical trials; however, little is known about whether edaravone has protective effects on diabetic cerebrovascular injury. Using cultured human brain microvascular endothelial cells (HBMEC), protective effects of edaravone on MGO and MGO enhancing oxygen-glucose deprivation (OGD) induced injury were investigated. Cell injury was measured by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) formation, cell account, lactate dehydrogenase (LDH) release and Rhodamine 123 staining. Advanced glycation end-products (AGEs) formation and receptor for advanced glycation end-products (RAGE) expression were measured by western blotting. Cellular oxidative stress was measured by reactive oxygen species (ROS) release. Treatment of MGO for 24 h significantly induced HBMEC injury, which was inhibited by pretreatment of edaravone from 10-100 µmol/l. What's more, treatment of MGO enhanced AGEs accumulation, RAGE expression and ROS release in the cultured HBMEC, which were inhibited by 100 µmol/l edaravone. Finally, treatment of MGO for 24 h and then followed by 3 h OGD insult significantly enhanced cell injury when compared with OGD insult only, which was also protected by 100 µmol/l edaravone. Thus, edaravone protected HBMEC from MGO and MGO enhancing OGD-induced injury by inhibiting AGEs/RAGE/oxidative stress.

  6. Insulin-like growth factor-1 secreted by brain microvascular endothelial cells attenuates neuron injury upon ischemia.

    PubMed

    Wang, Jun; Tang, Yibo; Zhang, Wei; Zhao, Haiping; Wang, Runjun; Yan, Yangyang; Xu, Liwei; Li, Pengtao

    2013-08-01

    Insulin-like growth factor (IGF)-1 is essential for the development of the nervous system, and is present in many cell types. Relatively little is known about IGF-1 expression in brain microvascular endothelial cells (BMECs). For in vivo studies, we examined the expression of IGF-1 and insulin-like growth factor-binding protein (IGFBP)-2 after focal cerebral ischemia for 12 h, 24 h, 3 days and 7 days, utilizing a permanent middle cerebral artery occlusion (MCAO) model in rats. For in vitro studies, we examined the levels of IGF-1 and IGFBP-2 in the culture medium or primary culture of BMECs injured by oxygen-glucose deprivation (OGD). Then, we elucidated the protective effects of IGF-1 on cortical neurons injured by OGD and the possible mechanism. In addition, we investigated the effect of BMEC-conditioned medium on IGF-1 receptor expression in neurons. The results showed that IGF-1 expression increased in serum and brain tissue, whereas IGFBP-2 expression decreased in brain tissue of MCAO-injured rats. In primary culture of BMECs, the expression levels of IGF-1 and IGFBP-2 were significantly higher under OGD conditions in culture. IGF-1 administration improved neuron viability upon normoxia or OGD, and upregulated p-Akt expression. This effect was reversed by LY294002, a specific inhibitor of the phosphoinositide 3-kinase-Akt signaling pathway. Furthermore, conditioned medium from OGD-treated BMECs substantially suppressed neuron viability and the expression of IGF-1 receptor simultaneously. These data demonstrate that therapeutic strategies that prioritize the early recovery of the IGF-1 system in BMECs might be promising in ischemic injury.

  7. Microvascular density and endothelial area correlate with Ki-67 proliferative index in surgically-treated pancreatic ductal adenocarcinoma patients

    PubMed Central

    AMMENDOLA, MICHELE; SACCO, ROSARIO; MARECH, ILARIA; SAMMARCO, GIUSEPPE; ZUCCALÀ, VALERIA; LUPOSELLA, MARIA; PATRUNO, ROSA; GIORDANO, MARCELLA; RUGGIERI, EUSTACHIO; ZIZZO, NICOLA; GADALETA, COSMO DAMIANO; RANIERI, GIROLAMO

    2015-01-01

    Previous experimental and clinical data have indicated that tumour cell proliferation is associated with angiogenesis; in addition, an increased microvascular density (MVD) of tumours has been associated with poor prognosis in solid and haematological malignancies. However, limited data exists regarding the association between tumour cell proliferation and angiogenesis in primary tumour tissue from pancreatic ductal adenocarcinoma (PDAC) patients; therefore, the present study aimed to investigate this association. A series of 31 PDAC patients with stage Tumour (T)2–3 Node (N)0–1 Metastasis (M)0 were recruited into the present study and subsequently underwent surgery. PDAC tissue and adjacent normal tissue (ANT), resected during surgery, were evaluated using immunohistochemistry and image analysis methods to determine MVD, endothelial area (EA) and Ki-67 expression, which is an indicator of cell proliferation rate. The results demonstrated a correlation between the above parameters with each other as well as the main clinico-pathological features of PDAC. Significant differences were identified in MVD, EA and Ki-67 proliferation index between PDAC and ANT. It was demonstrated that MVD, EA and Ki-67 proliferation index were significantly correlated with each other in tumour tissue (r=0.69–0.81; P=0.001–0.003). However, no other significant correlations were identified. These data therefore suggested that angiogenesis and cell proliferation rate were significantly increased in PDAC compared with ANT, which provides a biological basis for the potential use of novel combinations of angiogenesis inhibitors and anti-proliferative chemotherapeutic drugs in the treatment of PDAC. PMID:26622606

  8. Protective effects of total flavonoids in Caragana against hypoxia/reoxygenation-induced injury in human brain microvascular endothelial cells.

    PubMed

    He, Qian-Song; Zhang, Li; Fan, Zi-Yuan; Feng, Guo; Wang, Fu-Jiang; Liu, Zheng-Qi; Tang, Ting; Kuang, Shi-Xiang

    2017-02-22

    This study aimed to explore the protective effect of total flavonoids in Caragana against hypoxia/reoxygenation (H/R)-induced injury in human brain microvascular endothelial cells (BMECs). Human BMECs were selected and assigned into control, H/R, H/R+NMP, H/R+Low dose, H/R+Moderate dose, H/R+High dose groups. MTT and Transwell assays were used to detect cell viability and migration, respectively. Cell adhesion rate and tube formation were also detected. Real-time polymerase chain reaction (RT-PCR) and Western blotting were performed to test HIF-1α, VEGF and Notch1 mRNA and protein expressions. Compared with the H/R group, the cell viability rates in the H/R+NMP, H/R+Moderate dose and H/R+High dose groups were increased. The cell adhesion rates in the H/R+NMP, H/R+Moderate dose and H/R+High dose groups were significantly different from those in the H/R group. As compared to the H/R group, the cell migration abilities in the H/R+NMP, H/R+Moderate dose and H/R+High dose groups were enhanced. Compared with the H/R group, the number and length of tubes of BMECs in the H/R+NMP, H/R+High dose and H/R+Moderate dose groups were increased. HIF-1α, VEGF and Notch1 mRNA and protein expressions were higher in the H/R+Low dose, H/R+Moderate dose and H/R+High dose groups than in the H/R group. These findings revealed that total flavonoids in Caragana can protect BMECs from H/R-induced injury in a dose-dependent manner and it also may promote angiogenesis in BMECs by activating HIF- 1α-VEGF-Notch 1 signaling pathway.

  9. AM966, an Antagonist of Lysophosphatidic Acid Receptor 1, Increases Lung Microvascular Endothelial Permeability through Activation of Rho Signaling Pathway and Phosphorylation of VE-Cadherin

    PubMed Central

    Cai, Junting; Suber, Tomeka

    2017-01-01

    Maintenance of pulmonary endothelial barrier integrity is important for reducing severity of lung injury. Lysophosphatidic acid (LPA) regulates cell motility, cytoskeletal rearrangement, and cell growth. Knockdown of LPA receptor 1 (LPA1) has been shown to mitigate lung injury and pulmonary fibrosis. AM966, an LPA1 antagonist exhibiting an antifibrotic property, has been considered to be a future antifibrotic medicine. Here, we report an unexpected effect of AM966, which increases lung endothelial barrier permeability. An electric cell-substrate sensing (ECIS) system was used to measure permeability in human lung microvascular endothelial cells (HLMVECs). AM966 decreased the transendothelial electrical resistance (TEER) value immediately in a dose-dependent manner. VE-cadherin and f-actin double immunostaining reveals that AM966 increases stress fibers and gap formation between endothelial cells. AM966 induced phosphorylation of myosin light chain (MLC) through activation of RhoA/Rho kinase pathway. Unlike LPA treatment, AM966 had no effect on phosphorylation of extracellular signal-regulated kinases (Erk). Further, in LPA1 silencing cells, we observed that AM966-increased lung endothelial permeability as well as phosphorylation of VE-cadherin and focal adhesion kinase (FAK) were attenuated. This study reveals that AM966 induces lung endothelial barrier dysfunction, which is regulated by LPA1-mediated activation of RhoA/MLC and phosphorylation of VE-cadherin. PMID:28348461

  10. PJ-34 inhibits PARP-1 expression and ERK phosphorylation in glioma-conditioned brain microvascular endothelial cells.

    PubMed

    Motta, Carla; D'Angeli, Floriana; Scalia, Marina; Satriano, Cristina; Barbagallo, Davide; Naletova, Irina; Anfuso, Carmelina Daniela; Lupo, Gabriella; Spina-Purrello, Vittoria

    2015-08-15

    Inhibitors of PARP-1(Poly(ADP-ribose) polymerase-1) act by competing with NAD(+), the enzyme physiological substrate, which play a protective role in many pathological conditions characterized by PARP-1 overactivation. It has been shown that PARP-1 also promotes tumor growth and progression through its DNA repair activity. Since angiogenesis is an essential requirement for these activities, we sought to determine whether PARP inhibition might affect rat brain microvascular endothelial cells (GP8.3) migration, stimulated by C6-glioma conditioned medium (CM). Through wound-healing experiments and MTT analysis, we demonstrated that PARP-1 inhibitor PJ-34 [N-(6-Oxo-5,6-dihydrophenanthridin-2-yl)-N,N-dimethylacetamide] abolishes the migratory response of GP8.3 cells and reduces their viability. PARP-1 also acts in a DNA independent way within the Extracellular-Regulated-Kinase (ERK) signaling cascade, which regulates cell proliferation and differentiation. By western analysis and confocal laser scanning microscopy (LSM), we analyzed the effects of PJ-34 on PARP-1 expression, phospho-ERK and phospho-Elk-1 activation. The effect of MEK (mitogen-activated-protein-kinase-kinase) inhibitor PD98059 (2-(2-Amino-3-methoxyphenyl)-4 H-1-benzopyran-4-one) on PARP-1 expression in unstimulated and in CM-stimulated GP8.3 cells was analyzed by RT-PCR. PARP-1 expression and phospho-ERK activation were significantly reduced by treatment of GP8.3 cells with PJ-34 or PD98059. By LSM, we further demonstrated that PARP-1 and phospho-ERK are coexpressed and share the same subcellular localization in GP8.3 cells, in the cytoplasm as well as in nucleoplasm. Based on these data, we propose that PARP-1 and phospho-ERK interact in the cytosol and then translocate to the nucleus, where they trigger a proliferative response. We also propose that PARP-1 inhibition blocks CM-induced endothelial migration by interfering with ERK signal-transduction pathway.

  11. Systemic sclerosis sera affect fibrillin-1 deposition by dermal blood microvascular endothelial cells: therapeutic implications of cyclophosphamide

    PubMed Central

    2013-01-01

    Introduction Systemic sclerosis (SSc) is a connective tissue disorder characterized by endothelial cell injury, autoimmunity and fibrosis. The following three fibrillin-1 alterations have been reported in SSc. (1) Fibrillin-1 microfibrils are disorganized in SSc dermis. (2) Fibrillin-1 microfibrils produced by SSc fibroblasts are unstable. (3) Mutations in the FBN1 gene and anti-fibrillin-1 autoantibodies have been reported in SSc. Fibrillin-1 microfibrils, which are abundantly produced by blood and lymphatic microvascular endothelial cells (B-MVECs and Ly-MVECs, respectively), sequester in the extracellular matrix the latent form of the potent profibrotic cytokine transforming growth factor β (TGF-β). In the present study, we evaluated the effects of SSc sera on the deposition of fibrillin-1 and microfibril-associated glycoprotein 1 (MAGP-1) and the expression of focal adhesion molecules by dermal B-MVECs and Ly-MVECs. Methods Dermal B-MVECs and Ly-MVECs were challenged with sera from SSc patients who were treatment-naïve or under cyclophosphamide (CYC) treatment and with sera from healthy controls. Fibrillin-1/MAGP-1 synthesis and deposition and the expression of αvβ3 integrin/phosphorylated focal adhesion kinase and vinculin/actin were evaluated by immunofluorescence and quantified by morphometric analysis. Results Fibrillin-1 and MAGP-1 colocalized in all experimental conditions, forming a honeycomb pattern in B-MVECs and a dense mesh of short segments in Ly-MVECs. In B-MVECs, fibrillin-1/MAGP-1 production and αvβ3 integrin expression significantly decreased upon challenge with sera from naïve SSc patients compared with healthy controls. Upon challenge of B-MVECs with sera from CYC-treated SSc patients, fibrillin-1/MAGP-1 and αvβ3 integrin levels were comparable to those of cells treated with healthy sera. Ly-MVECs challenged with SSc sera did not differ from those treated with healthy control sera in the expression of any of the molecules assayed

  12. Anti-platelet factor 4/heparin antibodies from patients with heparin-induced thrombocytopenia provoke direct activation of microvascular endothelial cells.

    PubMed

    Blank, Miri; Shoenfeld, Yehuda; Tavor, Sigal; Praprotnik, Sonja; Boffa, Marie Claire; Weksler, Babette; Walenga, M Jeanine; Amiral, Jean; Eldor, Amiram

    2002-02-01

    Heparin-induced thrombocytopenia (HIT) is a serious complication that occurs in approximately 1-5% of patients treated with heparin and may be associated with severe thrombotic events. HIT is mediated by antibodies directed mostly to epitope(s) formed by complexes between heparin or other anionic mucopolysaccharides and platelet factor 4 (PF4). Anti-PF4/heparin IgG antibodies from six patients with HIT were affinity purified and assessed for interaction with human microvascular and macrovascular endothelial cells (EC). The antibodies directly activated primary cultures of human bone marrow microvascular EC (HBMEC) and SV40 immortalized HBMEC (TrHBMEC) only in the presence of PF4, but did not activate macrovascular human umbilical vein EC (HUVEC) under the same conditions. These antibodies were found to bind to TrHBMEC through the F(ab)(2) portion of the anti-PF4/heparin IgG. TrHBMEC activation was characterized by an augmented release of IL-6, von Willebrand factor, soluble thrombomodulin, and by an elevated expression of the adhesion molecules P-selectin, E-selectin and vascular cellular endothelial molecule-I to different degrees. Enhanced monocyte adhesion to PF4/heparin antibody-treated TrHBMEC (33-72% adhesion) was also observed. None of these effects occurred with unstimulated HUVEC. However, pre-treatment of HUVEC with tumor necrosis factor-alpha resulted in the same changes observed with microvascular EC exposed to the HIT antibodies. Our findings indicate that anti-PF4/heparin antibodies directly activate microvascular EC while interaction with macrovascular EC requires pre-activation. These results may explain some of the specific clinical manifestations in HIT.

  13. NEU1 and NEU3 sialidase activity expressed in human lung microvascular endothelia: NEU1 restrains endothelial cell migration, whereas NEU3 does not.

    PubMed

    Cross, Alan S; Hyun, Sang Won; Miranda-Ribera, Alba; Feng, Chiguang; Liu, Anguo; Nguyen, Chinh; Zhang, Lei; Luzina, Irina G; Atamas, Sergei P; Twaddell, William S; Guang, Wei; Lillehoj, Erik P; Puché, Adam C; Huang, Wei; Wang, Lai-Xi; Passaniti, Antonino; Goldblum, Simeon E

    2012-05-04

    The microvascular endothelial surface expresses multiple molecules whose sialylation state regulates multiple aspects of endothelial function. To better regulate these sialoproteins, we asked whether endothelial cells (ECs) might express one or more catalytically active sialidases. Human lung microvascular EC lysates contained heat-labile sialidase activity for a fluorogenic substrate, 2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid (4-MU-NANA), that was dose-dependently inhibited by the competitive sialidase inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid but not its negative control. The EC lysates also contained sialidase activity for a ganglioside mixture. Using real time RT-PCR to detect mRNAs for the four known mammalian sialidases, NEU1, -2, -3, and -4, NEU1 mRNA was expressed at levels 2700-fold higher that those found for NEU2, -3, or -4. Western analyses indicated NEU1 and -3 protein expression. Using confocal microscopy and flow cytometry, NEU1 was immunolocalized to both the plasma membrane and the perinuclear region. NEU3 was detected both in the cytosol and nucleus. Prior siRNA-mediated knockdown of NEU1 and NEU3 each decreased EC sialidase activity for 4-MU-NANA by >65 and >17%, respectively, and for the ganglioside mixture by 0 and 40%, respectively. NEU1 overexpression in ECs reduced their migration into a wound by >40%, whereas NEU3 overexpression did not. Immunohistochemical studies of normal human tissues immunolocalized NEU1 and NEU3 proteins to both pulmonary and extrapulmonary vascular endothelia. These combined data indicate that human lung microvascular ECs as well as other endothelia express catalytically active NEU1 and NEU3. NEU1 restrains EC migration, whereas NEU3 does not.

  14. Metformin induces up-regulation of blood-brain barrier functions by activating AMP-activated protein kinase in rat brain microvascular endothelial cells.

    PubMed

    Takata, Fuyuko; Dohgu, Shinya; Matsumoto, Junichi; Machida, Takashi; Kaneshima, Shuji; Matsuo, Mai; Sakaguchi, Shinya; Takeshige, Yuki; Yamauchi, Atsushi; Kataoka, Yasufumi

    2013-04-19

    Blood-brain barrier (BBB) disruption occurs frequently in CNS diseases and injuries. Few drugs have been developed as therapeutic candidates for facilitating BBB functions. Here, we examined whether metformin up-regulates BBB functions using rat brain microvascular endothelial cells (RBECs). Metformin, concentration- and time-dependently increased transendothelial electrical resistance of RBEC monolayers, and decreased RBEC permeability to sodium fluorescein and Evans blue albumin. These effects of metformin were blocked by compound C, an inhibitor of AMP-activated protein kinase (AMPK). AMPK stimulation with an AMPK activator, AICAR, enhanced BBB functions. These findings indicate that metformin induces up-regulation of BBB functions via AMPK activation.

  15. Quantitative assessment of brain microvascular and tissue oxygenation during cardiac arrest and resuscitation in pigs.

    PubMed

    Yu, J; Ramadeen, A; Tsui, A K Y; Hu, X; Zou, L; Wilson, D F; Esipova, T V; Vinogradov, S A; Leong-Poi, H; Zamiri, N; Mazer, C D; Dorian, P; Hare, G M T

    2013-07-01

    Cardiac arrest is associated with a very high rate of mortality, in part due to inadequate tissue perfusion during attempts at resuscitation. Parameters such as mean arterial pressure and end-tidal carbon dioxide may not accurately reflect adequacy of tissue perfusion during cardiac resuscitation. We hypothesised that quantitative measurements of tissue oxygen tension would more accurately reflect adequacy of tissue perfusion during experimental cardiac arrest. Using oxygen-dependent quenching of phosphorescence, we made measurements of oxygen in the microcirculation and in the interstitial space of the brain and muscle in a porcine model of ventricular fibrillation and cardiopulmonary resuscitation. Measurements were performed at baseline, during untreated ventricular fibrillation, during resuscitation and after return of spontaneous circulation. After achieving stable baseline brain tissue oxygen tension, as measured using an Oxyphor G4-based phosphorescent microsensor, ventricular fibrillation resulted in an immediate reduction in all measured parameters. During cardiopulmonary resuscitation, brain oxygen tension remained unchanged. After the return of spontaneous circulation, all measured parameters including brain oxygen tension recovered to baseline levels. Muscle tissue oxygen tension followed a similar trend as the brain, but with slower response times. We conclude that measurements of brain tissue oxygen tension, which more accurately reflect adequacy of tissue perfusion during cardiac arrest and resuscitation, may contribute to the development of new strategies to optimise perfusion during cardiac resuscitation and improve patient outcomes after cardiac arrest.

  16. Endothelial to mesenchymal transition contributes to arsenic-trioxide-induced cardiac fibrosis

    PubMed Central

    Zhang, Yong; Wu, Xianxian; Li, Yang; Zhang, Haiying; Li, Zhange; Zhang, Ying; Zhang, Longyin; Ju, Jiaming; Liu, Xin; Chen, Xiaohui; Glybochko, Peter V.; Nikolenko, Vladimir; Kopylov, Philipp; Xu, Chaoqian; Yang, Baofeng

    2016-01-01

    Emerging evidence has suggested the critical role of endothelial to mesenchymal transition (EndMT) in fibrotic diseases. The present study was designed to examine whether EndMT is involved in arsenic trioxide (As2O3)-induced cardiac fibrosis and to explore the underlying mechanisms. Cardiac dysfunction was observed in rats after exposure to As2O3 for 15 days using echocardiography, and the deposition of collagen was detected by Masson’s trichrome staining and electron microscope. EndMT was indicated by the loss of endothelial cell markers (VE-cadherin and CD31) and the acquisition of mesenchymal cell markers (α-SMA and FSP1) determined by RT-PCR at the mRNA level and Western blot and immunofluorescence analysis at the protein level. In the in-vitro experiments, endothelial cells acquired a spindle-shaped morphology accompanying downregulation of the endothelial cell markers and upregulation of the mesenchymal cell markers when exposed to As2O3. As2O3 activated the AKT/GSK-3β/Snail signaling pathway, and blocking this pathway with PI3K inhibitor (LY294002) abolished EndMT in As2O3-treated endothelial cells. Our results highlight that As2O3 is an EndMT-promoting factor during cardiac fibrosis, suggesting that targeting EndMT is beneficial for preventing As2O3-induced cardiac toxicity. PMID:27671604

  17. MiR-466b-1-3p regulates P-glycoprotein expression in rat cerebral microvascular endothelial cells.

    PubMed

    Yang, Xiaobo; Ren, Weimin; Shao, Yiye; Chen, Yinghui

    2017-04-03

    Epilepsy is one of the most common neurological disorders, and approximately one-third of epilepsy cases are resistant to treatment with anti-epileptic drug (AED). P-glycoprotein (P-gp) is a multi-drug transporter that is thought to play a pivotal role in multiple drug resistance (MDR) in epilepsy. The regulatory mechanism of P-gp remains largely unknown; however, recent studies have demonstrated that microRNAs (miRNAs) may regulate the chemo-resistance mediated by P-gp. This study investigated the effect of specific miRNAs that regulate P-gp expression in rat cerebral microvascular endothelial cells (RCMECs). Primary cultures of RCMECs were treated with phenobarbital (PB) at various concentrations to induce P-gp overexpression. MiRNA microarrays were used to investigate the expression profiles of miRNAs in the resistant RCMECs induced by PB and corresponding non-resistant cells. Our data demonstrated decreased miR-466b-1-3p expression in the resistant cells compared with the non-resistant cells. Moreover, the recombinant RNA of 466b-1-3p (mimic) and the artificial antisense RNA of miR-466b-1-3p (inhibitor) were constructed and transfected into resistant RCMECs. The expression and function of P-gp were measured by Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR) and flow cytometry using rhodamine efflux. The mRNA and protein levels of P-gp increased as the concentration of PB increased, whereas miR-466b-1-3p levels decreased with increasing PB concentrations (P<0.05). The miR-466b-1-3p mimic down-regulated P-gp expression, whereas the miR-466b-1-3p inhibitor up-regulated P-gp expression (P<0.05). These findings demonstrate that miR-466b-1-3p may regulate PB-induced P-gp expression in RCMECs.

  18. Human Pulmonary Microvascular Endothelial Cells Support Productive Replication of Highly Pathogenic Avian Influenza Viruses: Possible Involvement in the Pathogenesis of Human H5N1 Virus Infection

    PubMed Central

    Zeng, Hui; Pappas, Claudia; Belser, Jessica A.; Houser, Katherine V.; Zhong, Weiming; Wadford, Debra A.; Stevens, Troy; Balczon, Ron; Katz, Jacqueline M.

    2012-01-01

    Highly pathogenic avian influenza (HPAI) H5N1 viruses continue to cause sporadic human infections with a high fatality rate. Respiratory failure due to acute respiratory distress syndrome (ARDS) is a complication among hospitalized patients. Since progressive pulmonary endothelial damage is the hallmark of ARDS, we investigated host responses following HPAI virus infection of human pulmonary microvascular endothelial cells. Evaluation of these cells for the presence of receptors preferred by influenza virus demonstrated that avian-like (α2-3-linked) receptors were more abundant than human-like (α2-6-linked) receptors. To test the permissiveness of pulmonary endothelial cells to virus infection, we compared the replication of selected seasonal, pandemic (2009 H1N1 and 1918), and potentially pandemic (H5N1) influenza virus strains. We observed that these cells support productive replication only of HPAI H5N1 viruses, which preferentially enter through and are released from the apical surface of polarized human endothelial monolayers. Furthermore, A/Thailand/16/2004 and A/Vietnam/1203/2004 (VN/1203) H5N1 viruses, which exhibit heightened virulence in mammalian models, replicated to higher titers than less virulent H5N1 strains. VN/1203 infection caused a significant decrease in endothelial cell proliferation compared to other subtype viruses. VN/1203 virus was also found to be a potent inducer of cytokines and adhesion molecules known to regulate inflammation during acute lung injury. Deletion of the H5 hemagglutinin (HA) multibasic cleavage site did not affect virus infectivity but resulted in decreased virus replication in endothelial cells. Our results highlight remarkable tropism and infectivity of the H5N1 viruses for human pulmonary endothelial cells, resulting in the potent induction of host inflammatory responses. PMID:22072765

  19. The microvascular effects of insulin resistance and diabetes on cardiac structure, function, and perfusion: a cardiovascular magnetic resonance study

    PubMed Central

    Larghat, Abdulghani M.; Swoboda, Peter P.; Biglands, John D.; Kearney, Mark T.; Greenwood, John P.; Plein, Sven

    2014-01-01

    Aims Type 2 diabetes mellitus is an independent risk factor for the development of heart failure. To better understand the mechanism by which this occurs, we investigated cardiac structure, function, and perfusion in patients with and without diabetes. Methods and results Sixty-five patients with no stenosis >30% on invasive coronary angiography were categorized into diabetes (19) and non-diabetes (46) which was further categorized into prediabetes (30) and controls (16) according to the American Diabetes Association guidelines. Each patient underwent comprehensive cardiovascular magnetic resonance assessment. Left-ventricular (LV) mass, relative wall mass (RWM), Lagrangian circumferential strain, LV torsion, and myocardial perfusion reserve (MPR) were calculated. LV mass was higher in diabetics than non-diabetics (112.8 ± 39.7 vs. 91.5 ± 21.3 g, P = 0.01) and in diabetics than prediabetics (112.8 ± 39.7 vs. 90.3 ± 18.7 g, P = 0.02). LV torsion angle was higher in diabetics than non-diabetics (9.65 ± 1.90 vs. 8.59 ± 1.91°, P = 0.047), and MPR was lower in diabetics than non-diabetics (2.10 ± 0.76 vs. 2.84 ± 1.25 mL/g/min, P = 0.01). There was significant correlation between MPR and early diastolic strain rate (r = −0.310, P = 0.01) and LV torsion (r = −0.306, P = 0.01). In multivariable linear regression analysis, non-diabetics waist–hip ratio, but not body mass index, had a significant association with RWM (Beta = 0.34, P = 0.02). Conclusion Patients with diabetes have increased LV mass, LV torsion, and decreased MPR. There is a significant association between decreased MPR and increased LV torsion suggesting a possible mechanistic link between microvascular disease and cardiac dysfunction in diabetes. PMID:25117473

  20. Uncaria tomentosa alkaloidal fraction reduces paracellular permeability, IL-8 and NS1 production on human microvascular endothelial cells infected with dengue virus.

    PubMed

    Lima-Junior, Raimundo Sousa; Mello, Cintia da Silva; Siani, Antonio Carlos; Valente, Ligia M Marino; Kubelka, Claire Fernandes

    2013-11-01

    Dengue is the major Arbovirus in the world, annually causing morbidity and death. Severe dengue is associated with changes in the endothelial barrier function due to the production of inflammatory mediators by immune cells and by the endothelium. Dengue virus (DENV) replicates efficiently in human endothelial cells in vitro and elicits immune responses resulting in endothelial permeability. Uncaria tomentosa (Willd.) DC.(Rubiaceae), known as cat's claw, has been used in folk medicine for the treatment of a wide-array of symptoms, and several scientific studies reported its antiviral, immunomodulatory, anti-inflammatory and antioxidant properties. Here we infected a human lineage of dermal microvascular endothelial cells (HMEC-1) with DENV-2 and treated it with an alkaloidal fraction from U. tomentosa bark (AFUT). We showed antiviral and immunomodulatory activities of U. tomentosa by determining the NS1 antigen and IL-8 in supernatant of DENV-2 infected HMEC-1. Furthermore, by measurement of transendothelial electrical resistance (TEER) we demonstrated, for the first time, that a plant derivative contributed to the reduction of paracellular permeability in DENV-2 infected HMEC-1. We also showed that IL-8 contributed significantly to the induction of permeability. Although further investigations should be conducted before a new drug can be suggested, our in vitro data support evidence that AFUT could be potentially useful in developing a treatment for severe dengue.

  1. Modulatory effect of curcumin on survival of irradiated human intestinal microvascular endothelial cells: role of Akt/mTOR and NF-κB

    PubMed Central

    Binion, David G.; Wellner, Michael; Behmaram, Behnaz; Floer, Martin; Mitton, Elizabeth; Nie, Linghui; Zhang, Zhihong; Otterson, Mary F.

    2010-01-01

    Radiation therapy is an essential modality in the treatment of colorectal cancers. Radiation exerts an antiangiogenic effect on tumors, inhibiting endothelial proliferation and survival in the tumor microvasculature. However, damage from low levels of irradiation can induce a paradoxical effect, stimulating survival in endothelial cells. We used human intestinal microvascular endothelial cells (HIMEC) to define effects of radiation on these gut-specific endothelial cells. Low-level irradiation (1–5 Gy) activates NF-κB and the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, which is involved in cell cycle reentry and cell survival in HIMEC. A downstream target of PI3K/Akt is mammalian target of rapamycin (mTOR), which contributes to endothelial proliferation and angiogenesis. The aim of this study was to investigate the signaling molecules involved in the radiosensitizing effects of curcumin on HIMEC subjected to low levels of irradiation. We have demonstrated that exposure of HIMEC to low levels of irradiation induced Akt and mTOR phosphorylation, which was attenuated by curcumin, rapamycin, LY294002, and mTOR small interference RNA (siRNA). Activation of NF-κB by low levels of irradiation was inhibited by curcumin, SN-50, and mTOR siRNA. Curcumin also induced apoptosis by induction of caspase-3 cleavage in irradiated HIMEC. In conclusion, curcumin significantly inhibited NF-κB and attenuated the effect of irradiation-induced prosurvival signaling through the PI3K/Akt/mTOR and NF-κB pathways in these gut-specific endothelial cells. Curcumin may be a potential radiosensitizing agent for enhanced antiangiogenic effect in colorectal cancer radiation therapy. PMID:20299603

  2. Peoniflorin suppresses tumor necrosis factor-α induced chemokine production in human dermal microvascular endothelial cells by blocking nuclear factor-κB and ERK pathway.

    PubMed

    Chen, Tao; Guo, Zai-Pei; Jiao, Xiao-Yan; Jia, Rui-Zhen; Zhang, Yu-Hong; Li, Jing-Yi; Huang, Xu-Lei; Liu, Hong-Jie

    2011-07-01

    Peoniflorin (PF) extracted from the root of Paeonia lactiflora pall displays anti-inflammation and antioxidant properties in several animal models. Chemokines are vital for directing the movement of circulating leukocytes to the sites of inflammation and are involved in the pathogenesis of various inflammatory skin diseases. Herein, we investigated the effects and potential mechanisms of PF on tumor necrosis factor-α (TNF-α) induced chemokine production in human dermal microvascular endothelial cells. Human dermal microvascular endothelial cell line (HMEC-1) was treated by TNF-α with or without PF. PF markedly attenuated TNF-α-induced chemokines (including CCL2, CCL5, CCL20, CXCL8, CXCL16 and CX3CL1) mRNA expression in HMEC-1. PF also reduced the secretion of these chemokines in culture supernatants. In addition, endothelial activation in the presence of PF markedly blocked the chemotactic activities of TNF-α-stimulated HMEC-1 supernatant on promyelocytic leukemia cell line (HL-60) or the acute mature monocytic leukemia cell line (THP-1) cell migration. Furthermore, Western blot data revealed TNF-α upregulated phosphorylation of inhibitor of κB-α (IκBα) and phosphorylation of extracellular signal-regulated kinase (ERK)1/2, which was almost completely reversed by PF. Finally, PF inhibited nuclear factor-κB (NF-κB) nuclear translocation to the nucleus. Taken together, our data provide the first evidence that PF has an anti-inflammatory ability against TNF-α-induced chemokine production and leukocyte migration, which may be at least partly related to the inhibition of NF-κB and ERK pathway. PF may be a candidate medicine for the treatment of inflammatory skin diseases.

  3. Overexpression of actin-depolymerizing factor blocks oxidized low-density lipoprotein-induced mouse brain microvascular endothelial cell barrier dysfunction.

    PubMed

    Wang, Jun; Sun, Lu; Si, Yan-Fang; Li, Bao-Min

    2012-12-01

    The aim of present work was to elucidate the role of actin-depolymerizing factor (ADF), an important regulator of actin cytoskeleton, in the oxidized low-density lipoprotein (ox-LDL)-induced blood-brain barrier (BBB) disruption. The primary mouse brain microvascular endothelial cells (MBMECs) were exposed to ox-LDL. Treatment with LDL served as control. It was found that ADF mRNA level and protein expression were decreased when exposed to ox-LDL in MBMECs. Then, we investigated the influence of ADF overexpression on ox-LDL-treated MBMECs. Structurally, overexpression of ADF inhibited ox-LDL-induced F-actin formation. Functionally, overexpression of ADF attenuated ox-LDL-induced disruption of endothelial barrier marked by restoration of transendothelial electrical resistance, permeability of Evans Blue and expression of tight junction-associated proteins including ZO-1 and occludin, and blocked ox-LDL-induced oxidative stress marked by inhibition of reactive oxygen species (ROS) formation and activity of NADPH oxidase and Nox2 expression. However, overexpression of ADF in control cells had no significant effect on endothelial permeability and ROS formation. In conclusion, overexpression of ADF blocks ox-LDL-induced disruption of endothelial barrier. In addition, siRNA-mediated downregulation of ADF expression aggravated ox-LDL-induced disruption of endothelial barrier and ROS formation. These findings identify ADF as a key signaling molecule in the regulation of BBB integrity and suggest that ADF might be used as a target to modulate diseases accompanied by ox-LDL-induced BBB compromise.

  4. Cerebral Microvascular Endothelial Cell Apoptosis after Ischemia: Role of Enolase-Phosphatase 1 Activation and Aci-Reductone Dioxygenase 1 Translocation.

    PubMed

    Zhang, Yuan; Wang, Ting; Yang, Ke; Xu, Ji; Ren, Lijie; Li, Weiping; Liu, Wenlan

    2016-01-01

    Enolase-phosphatase 1 (ENOPH1), a newly discovered enzyme of the methionine salvage pathway, is emerging as an important molecule regulating stress responses. In this study, we investigated the role of ENOPH1 in blood brain barrier (BBB) injury under ischemic conditions. Focal cerebral ischemia induced ENOPH1 mRNA and protein expression in ischemic hemispheric microvessels in rats. Exposure of cultured brain microvascular endothelial cells (bEND3 cells) to oxygen-glucose deprivation (OGD) also induced ENOPH1 upregulation, which was accompanied by increased cell death and apoptosis reflected by increased 3-(4, 5-Dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide formation, lactate dehydrogenase release and TUNEL staining. Knockdown of ENOPH1 expression with siRNA or overexpressing ENOPH1 with CRISPR-activated plasmids attenuated or potentiated OGD-induced endothelial cell death, respectively. Moreover, ENOPH1 knockdown or overexpression resulted in a significant reduction or augmentation of reactive oxygen species (ROS) generation, apoptosis-associated proteins (caspase-3, PARP, Bcl-2 and Bax) and Endoplasmic reticulum (ER) stress proteins (Ire-1, Calnexin, GRP78 and PERK) in OGD-treated endothelial cells. OGD upregulated the expression of ENOPH1's downstream protein aci-reductone dioxygenase 1 (ADI1) and enhanced its interaction with ENOPH1. Interestingly, knockdown of ENOPH1 had no effect on OGD-induced ADI1 upregulation, while it potentiated OGD-induced ADI1 translocation from the nucleus to the cytoplasm. Lastly, knockdown of ENOPH1 significantly reduced OGD-induced endothelial monolayer permeability increase. In conclusion, our data demonstrate that ENOPH1 activation may contribute to OGD-induced endothelial cell death and BBB disruption through promoting ROS generation and the activation of apoptosis associated proteins, thus representing a new therapeutic target for ischemic stroke.

  5. Cerebral Microvascular Endothelial Cell Apoptosis after Ischemia: Role of Enolase-Phosphatase 1 Activation and Aci-Reductone Dioxygenase 1 Translocation

    PubMed Central

    Zhang, Yuan; Wang, Ting; Yang, Ke; Xu, Ji; Ren, Lijie; Li, Weiping; Liu, Wenlan

    2016-01-01

    Enolase-phosphatase 1 (ENOPH1), a newly discovered enzyme of the methionine salvage pathway, is emerging as an important molecule regulating stress responses. In this study, we investigated the role of ENOPH1 in blood brain barrier (BBB) injury under ischemic conditions. Focal cerebral ischemia induced ENOPH1 mRNA and protein expression in ischemic hemispheric microvessels in rats. Exposure of cultured brain microvascular endothelial cells (bEND3 cells) to oxygen-glucose deprivation (OGD) also induced ENOPH1 upregulation, which was accompanied by increased cell death and apoptosis reflected by increased 3-(4, 5-Dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide formation, lactate dehydrogenase release and TUNEL staining. Knockdown of ENOPH1 expression with siRNA or overexpressing ENOPH1 with CRISPR-activated plasmids attenuated or potentiated OGD-induced endothelial cell death, respectively. Moreover, ENOPH1 knockdown or overexpression resulted in a significant reduction or augmentation of reactive oxygen species (ROS) generation, apoptosis-associated proteins (caspase-3, PARP, Bcl-2 and Bax) and Endoplasmic reticulum (ER) stress proteins (Ire-1, Calnexin, GRP78 and PERK) in OGD-treated endothelial cells. OGD upregulated the expression of ENOPH1’s downstream protein aci-reductone dioxygenase 1 (ADI1) and enhanced its interaction with ENOPH1. Interestingly, knockdown of ENOPH1 had no effect on OGD-induced ADI1 upregulation, while it potentiated OGD-induced ADI1 translocation from the nucleus to the cytoplasm. Lastly, knockdown of ENOPH1 significantly reduced OGD-induced endothelial monolayer permeability increase. In conclusion, our data demonstrate that ENOPH1 activation may contribute to OGD-induced endothelial cell death and BBB disruption through promoting ROS generation and the activation of apoptosis associated proteins, thus representing a new therapeutic target for ischemic stroke. PMID:27630541

  6. Endothelial Jarid2/Jumonji is required for normal cardiac development and proper Notch1 expression.

    PubMed

    Mysliwiec, Matthew R; Bresnick, Emery H; Lee, Youngsook

    2011-05-13

    Jarid2/Jumonji critically regulates developmental processes including cardiovascular development. Jarid2 knock-out mice exhibit cardiac defects including hypertrabeculation with noncompaction of the ventricular wall. However, molecular mechanisms underlying Jarid2-mediated cardiac development remain unknown. To determine the cardiac lineage-specific roles of Jarid2, we generated myocardial, epicardial, cardiac neural crest, or endothelial conditional Jarid2 knock-out mice using Cre-loxP technology. Only mice with an endothelial deletion of Jarid2 recapitulate phenotypic defects observed in whole body mutants including hypertrabeculation and noncompaction of the ventricle. To identify potential targets of Jarid2, combinatorial approaches using microarray and candidate gene analyses were employed on Jarid2 knock-out embryonic hearts. Whole body or endothelial deletion of Jarid2 leads to increased endocardial Notch1 expression in the developing ventricle, resulting in increased Notch1-dependent signaling to the adjacent myocardium. Using quantitative chromatin immunoprecipitation analysis, Jarid2 was found to occupy a specific region on the endogenous Notch1 locus. We propose that failure to properly regulate Notch signaling in Jarid2 mutants likely leads to the defects in the developing ventricular chamber. The identification of Jarid2 as a potential regulator of Notch1 signaling has broad implications for many cellular processes including development, stem cell maintenance, and tumor formation.

  7. Generation of Brain Microvascular Endothelial-Like Cells from Human Induced Pluripotent Stem Cells by Co-Culture with C6 Glioma Cells

    PubMed Central

    Minami, Haruka; Tashiro, Katsuhisa; Okada, Atsumasa; Hirata, Nobue; Yamaguchi, Tomoko; Takayama, Kazuo; Mizuguchi, Hiroyuki; Kawabata, Kenji

    2015-01-01

    The blood brain barrier (BBB) is formed by brain microvascular endothelial cells (BMECs) and tightly regulates the transport of molecules from blood to neural tissues. In vitro BBB models from human pluripotent stem cell (PSCs)-derived BMECs would be useful not only for the research on the BBB development and function but also for drug-screening for neurological diseases. However, little is known about the differentiation of human PSCs to BMECs. In the present study, human induced PSCs (iPSCs) were differentiated into endothelial cells (ECs), and further maturated to BMECs. Interestingly, C6 rat glioma cell-conditioned medium (C6CM), in addition to C6 co-culture, induced the differentiation of human iPSC-derived ECs (iPS-ECs) to BMEC-like cells, increase in the trans-endothelial electrical resistance, decreased in the dextran transport and up-regulation of gene expression of tight junction molecules in human iPS-ECs. Moreover, Wnt inhibitors attenuated the effects of C6CM. In summary, we have established a simple protocol of the generation of BMEC-like cells from human iPSCs, and have demonstrated that differentiation of iPS-ECs to BMEC-like cells is induced by C6CM-derived signals, including canonical Wnt signals. PMID:26061227

  8. Effect of caveolin-1 on the expression of tight junction-associated proteins in rat glioma-derived microvascular endothelial cells

    PubMed Central

    Li, Yao; Liu, Li-Bo; Ma, Teng; Wang, Ping; Xue, Yi-Xue

    2015-01-01

    Caveolin-1 affects the permeability of blood-tumor barrier (BTB) by regulating the expression of tight junction-associated proteins. However, the effect is still controversial. In the present work, we studied the regulative effect of caveolin-1 on the expression of tight junction-associated proteins and BTB via directly silencing and overexpressing of caveolin-1 by recombinant adenovirus transduction of glioma-derived microvascular endothelial cells in rat brain. The results show that the caveolin-1 downregulation resulted in decreased expression of tight junction-associated proteins, opening of tight junctions, and increasing the permeability of BTB, whereas the overexpression of caveolin-1 presented the opposite effects. Therefore, we conclude that caveolin-1 regulates the expression of tight junction-associated proteins in a positive manner, which further plays a role in the regulation of BTB permeability. This finding provides a novel therapeutic target for selectively opening of BTB. PMID:26722502

  9. Cardiac tissue development for delivery of embryonic stem cell-derived endothelial and cardiac cells in natural matrices.

    PubMed

    Turner, William S; Wang, Xiaoling; Johnson, Scott; Medberry, Christopher; Mendez, Jose; Badylak, Stephen F; McCord, Marian G; McCloskey, Kara E

    2012-11-01

    The packaging and delivery of cells for cardiac regeneration has been explored using a variety biomaterials and delivery methods, but these studies often ignore one or more important design factors critical for rebuilding cardiac tissue. These include the biomaterial architecture, strength and stiffness, cell alignment, and/or incorporation of multiple cell types. In this article, we explore the combinatorial use of decellularized tissues, moldable hydrogels, patterned cell-seeding, and cell-sheet engineering and find that a combination of these methods is optimal in the recreation of transplantable cardiac-like tissue in vivo. We show that decellularized urinary bladder matrix (UBM), that is compliant and suturable, supports the survival of cell cultures but does not allow maintenance of cell-to-cell contacts of transferred cell-sheets (presumably, due to its rough surface). Moreover, the UBM material must be filled with hyaluronan (HA) hydrogels for smoothing rough surfaces and allowing the delivery of greater cell numbers. We additionally incorporated our previously developed "wrinkled" microchip for inducing alignment of cardiac cells with a laser-etched mask for co-seeding patterned "channels" of cells. This article also introduces a novel method of plasma coating for cell-sheet engineering that compares well with electron bean irradiation methods and may be combined with our "wrinkled" surfaces to facilitate the alignment of cardiac cells into sheets. Our data shows that an optimal design for generating cardiac tissue would include (1) decellularized matrix seeded with endothelial cells in a HA layered with (2) prealigned cardiac cell-sheets fabricated using our "wrinkled" microchips and thermo-responsive polymer [poly(N-isopropylacrylamide)] cell sheet transfer system.

  10. Autophagy protects human brain microvascular endothelial cells against methylglyoxal-induced injuries, reproducible in a cerebral ischemic model in diabetic rats.

    PubMed

    Fang, Lili; Li, Xue; Zhong, Yinbo; Yu, Jing; Yu, Lina; Dai, Haibin; Yan, Min

    2015-10-01

    Cerebral microvascular endothelial cells (ECs) are crucial for brain vascular repair and maintenance, but their physiological function may be impaired during ischemic stroke and diabetes. Methylglyoxal (MGO), a reactive dicarbonyl produced during glucose metabolism, could exacerbate ischemia-induced EC injury and dysfunction. We investigated the protective effect of autophagy on cultured human brain microvascular endothelial cells (HBMEC) that underwent MGO treatment. A further study was conducted to explore the underlying mechanisms of the protective effect. Autophagic activity was assessed by evaluating protein levels, using western blot. 3-methyladenine (3-MA), bafilomycin A1, ammonium chloride (AC), Beclin 1 siRNA, and chloroquine (CQ) were used to cause autophagy inhibition. Alarmar blue assay and lactate dehydrogenase release assay were used to evaluate cell viability. Streptozotocin was administered to induce type I diabetes in rats and post-permanent middle cerebral artery occlusion was performed to elicit cerebral ischemia. Blood-brain barrier permeability was also assessed. Our study found that MGO reduced HBMEC cell viability in a concentration- and time-dependent manner, and triggered the responsive autophagy activation. Autophagy inhibitors bafilomycin A1, AC, 3-MA, and BECN1 siRNA exacerbated MGO-induced HBMEC injury. FAK phosphorylation inhibitor PF573228 inhibited MGO-triggered autophagy and enhanced lactate dehydrogenase release. Meanwhile, similar autophagy activation in brain vascular ECs was observed during permanent middle cerebral artery occlusion-induced cerebral ischemia in diabetic rats, while chloroquine-induced autophagy inhibition enhanced blood-brain barrier permeability. Taken together, our study indicates that autophagy triggered by MGO defends HBMEC against injuries.

  11. West Nile virus infection modulates human brain microvascular endothelial cells tight junction proteins and cell adhesion molecules: Transmigration across the in vitro blood-brain barrier

    PubMed Central

    Verma, Saguna; Lo, Yeung; Chapagain, Moti; Lum, Stephanie; Kumar, Mukesh; Gurjav, Ulziijargal; Luo, Haiyan; Nakatsuka, Austin; Nerurkar, Vivek R.

    2009-01-01

    Neurological complications such as inflammation, failure of the blood-brain barrier (BBB), and neuronal death contribute to the mortality and morbidity associated with WNV-induced meningitis. Compromised BBB indicates the ability of the virus to gain entry into the CNS via the BBB, however, the underlying mechanisms, and the specific cell types associated with WNV-CNS trafficking are not well understood. Brain microvascular endothelial cells, main component of the BBB, represent a barrier to virus dissemination into the CNS and could play key role in WNV spread via hematogenous route. To investigate WNV entry into the CNS, we infected primary human brain microvascular endothelial (HBMVE) cells with the neurovirulent strain of WNV (NY99) and examined WNV replication kinetics together with the changes in the expressions of key tight junction proteins (TJP) and cell adhesion molecules (CAM). WNV infection of HBMVE cells was productive as analyzed by plaque assay and qRT-PCR, and did not induce cytopathic effect. Increased mRNA and protein expressions of TJP (claudin-1) and CAM (vascular cell adhesion molecule and E-selectin) were observed at days 2 and 3 after infection, respectively, which coincided with the peak in WNV replication. Further, using an in vitro BBB model comprised of HBMVE cells, we demonstrate that cell-free WNV can cross the BBB, without compromising the BBB integrity. These data suggest that infection of HBMVE cells can facilitate entry of cell-free virus into the CNS without disturbing the BBB, and increased CAM may assist in the trafficking of WNV-infected immune cells into the CNS, via ‘Trojan horse’ mechanism, thereby contributing to WNV dissemination in the CNS and associated pathology. PMID:19135695

  12. Key genes and proteins involved in CTCM-reducing microvascular endothelial cell permeability induced by SLT-IIv using gene chips and DIGE.

    PubMed

    Yi, Pengfei; Guo, Yang; Wang, Xin; Mu, Xiang; Wei, Xubin

    2010-01-01

    An Affymetrix mouse genome array and differential in-gel electrophoresis (DIGE) techniques were used to investigate the pharmacological mechanisms of a mixture of herbs, designated CTCM, a compound of traditional Chinese medicine, for the treatment of increased permeability in mouse intestinal microvascular endothelial cells (MIMECs) induced by the Shiga-like toxin type II variant (SLT-IIv). MIMECs were challenged with 10microg/ml SLT-IIv for 12h and then treated with CTCM at a concentration of 200microg/ml for 12h. Total RNA and proteins from each treatment group were extracted from cultured MIMECs for analysis by the Affymetrix GeneChip Mouse Genome 430 2.0 microarray and DIGE. The results obtained demonstrated that there were one genes downregulated and one genes upregulated, one protein downregulated and four proteins upregulated in the SLT-IIv group compared to the control group. In the CTCM group, four genes were upregulated, three genes were downregulated, a single protein was downregulated and a single protein was upregulated when compared to the control group. When the CTCM-treated group was compared to the SLT-IIv group, expression of one gene was found to be increased, and all other genes were decreased, with five proteins downregulated. Analysis of the data suggested that CTCM specifically and effectively reduced microvascular endothelial cell permeability to SLT-IIv in the treatment of pig edema disease. In the CTCM-treated group, hspa9 expression was increased in both gene chip and DIGE analysis, so it may be a key protein in reducing cell permeability and utilized in medical treatments.

  13. The Src family tyrosine kinases src and yes have differential effects on inflammation-induced apoptosis in human pulmonary microvascular endothelial cells.

    PubMed

    Nelin, Leif D; White, Hilary A; Jin, Yi; Trittmann, Jennifer K; Chen, Bernadette; Liu, Yusen

    2016-05-01

    Endothelial cells are essential for normal lung function: they sense and respond to circulating factors and hemodynamic alterations. In inflammatory lung diseases such as acute respiratory distress syndrome, endothelial cell apoptosis is an inciting event in pathogenesis and a prominent pathological feature. Endothelial cell apoptosis is mediated by circulating inflammatory factors, which bind to receptors on the cell surface, activating signal transduction pathways, leading to caspase-3-mediated apoptosis. We hypothesized that yes and src have differential effects on caspase-3 activation in human pulmonary microvascular endothelial cells (hPMVEC) due to differential downstream signaling effects. To test this hypothesis, hPMVEC were treated with siRNA against src (siRNAsrc), siRNA against yes (siRNAyes), or their respective scramble controls. After recovery, the hPMVEC were treated with cytomix (LPS, IL-1β, TNF-α, and IFN-γ). Treatment with cytomix induced activation of the extracellular signal-regulated kinase (ERK) pathway and caspase-3-mediated apoptosis. Treatment with siRNAsrc blunted cytomix-induced ERK activation and enhanced cleaved caspase-3 levels, while treatment with siRNAyes enhanced cytomix-induced ERK activation and attenuated levels of cleaved caspase-3. Inhibition of the ERK pathway using U0126 enhanced cytomix-induced caspase-3 activity. Treatment of hPMVEC with cytomix induced Akt activation, which was inhibited by siRNAsrc. Inhibition of the phosphatidylinositol 3-kinase/Akt pathway using LY294002 prevented cytomix-induced ERK activation and augmented cytomix-induced caspase-3 cleavage. Together, our data demonstrate that, in hPMVEC, yes activation blunts the ERK cascade in response to cytomix, resulting in greater apoptosis, while cytomix-induced src activation induces the phosphatidylinositol 3-kinase pathway, which leads to activation of Akt and ERK and attenuation of apoptosis.

  14. In vivo demonstration of red cell-endothelial interaction, sickling and altered microvascular response to oxygen in the sickle transgenic mouse.

    PubMed Central

    Kaul, D K; Fabry, M E; Costantini, F; Rubin, E M; Nagel, R L

    1995-01-01

    Intravascular sickling, red cell-endothelium interaction, and altered microvascular responses have been suggested to contribute to the pathophysiology of human sickle cell disease, but have never been demonstrated under in vivo flow. To address this issue, we have examined a transgenic mouse line, alphaHbetaSbetaS-Antilles [betaMDD] which has a combined high (78%) expression of beta S and beta S-Antilles globins. In vivo microcirculatory studies using the cremaster muscle preparation showed adhesion of red cells, restricted to postcapillary venules, in transgenic mice but not in control mice. Electron microscopy revealed distinct contacts between the red cell membrane and the endothelium surface. Some red cells exhibiting sickling were regularly observed in the venular flow. Infusion of transgenic mouse red cells into the ex vivo mesocecum vasculature also showed adhesion of mouse red cells exclusively in venules. Under resting conditions (pO2, 15-20 mmHg), there were no differences in the cremaster microvascular diameters of control and transgenic mice; however, transgenic mice showed a drastic reduction in microvascular red cell velocities (Vrbc) with maximal Vrbc decrease (> 60%) occurring in venules, the sites of red cell adhesion and sickling. Local, transient hyperoxia (pO2, 150 mmHg) resulted in striking differences between control and transgenic mice. In controls, oxygen caused a 69% arteriolar constriction, accompanied by 75% reduction in Vrbc. In contrast, in transgenic mice, hyperoxia resulted in only 8% decrease in the arteriolar diameter and in 68% increase in VrBC; the latter is probably due to an improved flow behavior of red cells as a consequence of unsickling. In summary, the high expression of human sickle hemoglobin in the mouse results not only in intravascular sickling but also red cell-endothelium interaction. The altered microvascular response to oxygen could be secondary to blood rheological changes, although possible intrinsic differences

  15. Endothelial Mineralocorticoid Receptor Deletion Prevents Diet-Induced Cardiac Diastolic Dysfunction in Females.

    PubMed

    Jia, Guanghong; Habibi, Javad; DeMarco, Vincent G; Martinez-Lemus, Luis A; Ma, Lixin; Whaley-Connell, Adam T; Aroor, Annayya R; Domeier, Timothy L; Zhu, Yi; Meininger, Gerald A; Barrett Mueller, Katelee; Jaffe, Iris Z; Sowers, James R

    2015-12-01

    Overnutrition and insulin resistance are especially prominent risk factors for the development of cardiac diastolic dysfunction in females. We recently reported that consumption of a Western diet (WD) containing excess fat (46%), sucrose (17.5%), and high fructose corn syrup (17.5%) for 16 weeks resulted in cardiac diastolic dysfunction and aortic stiffening in young female mice and that these abnormalities were prevented by mineralocorticoid receptor blockade. Herein, we extend those studies by testing whether WD-induced diastolic dysfunction and factors contributing to diastolic impairment, such as cardiac fibrosis, hypertrophy, inflammation, and impaired insulin signaling, are modulated by excess endothelial cell mineralocorticoid receptor signaling. Four-week-old female endothelial cell mineralocorticoid receptor knockout and wild-type mice were fed mouse chow or WD for 4 months. WD feeding resulted in prolonged relaxation time, impaired diastolic septal wall motion, and increased left ventricular filling pressure indicative of diastolic dysfunction. This occurred in concert with myocardial interstitial fibrosis and cardiomyocyte hypertrophy that were associated with enhanced profibrotic (transforming growth factor β1/Smad) and progrowth (S6 kinase-1) signaling, as well as myocardial oxidative stress and a proinflammatory immune response. WD also induced cardiomyocyte stiffening, assessed ex vivo using atomic force microscopy. Conversely, endothelial cell mineralocorticoid receptor deficiency prevented WD-induced diastolic dysfunction, profibrotic, and progrowth signaling, in conjunction with reductions in macrophage proinflammatory polarization and improvements in insulin metabolic signaling. Therefore, our findings indicate that increased endothelial cell mineralocorticoid receptor signaling associated with consumption of a WD plays a key role in the activation of cardiac profibrotic, inflammatory, and growth pathways that lead to diastolic dysfunction in

  16. Endothelial cell dysfunction and cardiac hypertrophy in the STOX1 model of preeclampsia

    PubMed Central

    Ducat, Aurélien; Doridot, Ludivine; Calicchio, Rosamaria; Méhats, Celine; Vilotte, Jean-Luc; Castille, Johann; Barbaux, Sandrine; Couderc, Betty; Jacques, Sébastien; Letourneur, Franck; Buffat, Christophe; Le Grand, Fabien; Laissue, Paul; Miralles, Francisco; Vaiman, Daniel

    2016-01-01

    Preeclampsia is a disease of pregnancy involving systemic endothelial dysfunction. However, cardiovascular consequences of preeclampsia are difficult to analyze in humans. The objective of the present study is to evaluate the cardiovascular dysfunction induced by preeclampsia by examining the endothelium of mice suffering of severe preeclampsia induced by STOX1 overexpression. Using Next Generation Sequencing on endothelial cells of mice carrying either transgenic or control embryos, we discovered significant alterations of gene networks involved in inflammation, cell cycle, and cardiac hypertrophy. In addition, the heart of the preeclamptic mice revealed cardiac hypertrophy associated with histological anomalies. Bioinformatics comparison of the networks of modified genes in the endothelial cells of the preeclamptic mice and HUVECs exposed to plasma from preeclamptic women identified striking similarities. The cardiovascular alterations in the pregnant mice are comparable to those endured by the cardiovascular system of preeclamptic women. The STOX1 mice could help to better understand the endothelial dysfunction in the context of preeclampsia, and guide the search for efficient therapies able to protect the maternal endothelium during the disease and its aftermath. PMID:26758611

  17. Talin1 is required for cardiac Z-disk stabilization and endothelial integrity in zebrafish

    PubMed Central

    Wu, Qing; Zhang, Jiaojiao; Koh, Wonshill; Yu, Qingming; Zhu, Xiaojun; Amsterdam, Adam; Davis, George E.; Arnaout, M. Amin; Xiong, Jing-Wei

    2015-01-01

    Talin (tln) binds and activates integrins to couple extracellular matrix–bound integrins to the cytoskeleton; however, its role in heart development is not well characterized. We identified the defective gene and the resulting cardiovascular phenotypes in zebrafish tln1fl02k mutants. The ethylnitrosourea-induced fl02k mutant showed heart failure, brain hemorrhage, and diminished cardiac and vessel lumens at 52 h post fertilization. Positional cloning revealed a nonsense mutation of tln1 in this mutant. tln1, but neither tln2 nor -2a, was dominantly expressed in the heart and vessels. Unlike tln1 and -2 in the mouse heart, the unique tln1 expression in the heart enabled us, for the first time, to determine the critical roles of Tln1 in the maintenance of cardiac sarcomeric Z-disks and endothelial/endocardial cell integrity, partly through regulating F-actin networks in zebrafish. The similar expression profiles of tln1 and integrin β1b (itgb1b) and synergistic function of the 2 genes revealed that itgb1b is a potential partner for tln1 in the stabilization of cardiac Z-disks and vessel lumens. Taken together, the results of this work suggest that Tln1-mediated Itgβ1b plays a crucial role in maintaining cardiac sarcomeric Z-disks and endothelial/endocardial cell integrity in zebrafish and may also help to gain molecular insights into congenital heart diseases.—Wu, Q., Zhang, J., Koh, W., Yu, Q., Zhu, X., Amsterdam, A., Davis, G. E., Arnaout, M. A., Xiong, J.-W. Talin1 is required for cardiac Z-disk stabilization and endothelial integrity in zebrafish. PMID:26310270

  18. Antiangiogenic Effect of (±)-Haloperidol Metabolite II Valproate Ester [(±)-MRJF22] in Human Microvascular Retinal Endothelial Cells.

    PubMed

    Olivieri, Melania; Amata, Emanuele; Vinciguerra, Shila; Fiorito, Jole; Giurdanella, Giovanni; Drago, Filippo; Caporarello, Nunzia; Prezzavento, Orazio; Arena, Emanuela; Salerno, Loredana; Rescifina, Antonio; Lupo, Gabriella; Anfuso, Carmelina Daniela; Marrazzo, Agostino

    2016-11-10

    (±)-MRJF22 [(±)-2], a novel prodrug of haloperidol metabolite II (sigma-1 receptor antagonist/sigma-2 receptor agonist ligand) obtained by conjugation to valproic acid (histone deacetylase inhibitor) via an ester bond, exhibits antiangiogenic activity, being able to reduce human retinal endothelial cell (HREC) viability in a comparable manner to bevacizumab. Moreover, (±)-2 was able to significantly reduce viable cells count, endothelial cell migration, and tube formation in vascular endothelial growth factor A (VEGF-A) stimulated HREC cultures.

  19. Particulate matter from indoor environments of classroom induced higher cytotoxicity and leakiness in human microvascular endothelial cells in comparison with those collected from corridor.

    PubMed

    Chua, M L; Setyawati, M I; Li, H; Fang, C H Y; Gurusamy, S; Teoh, F T L; Leong, D T; George, S

    2016-09-23

    We investigated the physicochemical properties (size, shape, elemental composition, and endotoxin) of size resolved particulate matter (PM) collected from the indoor and corridor environments of classrooms. A comparative hazard profiling of these PM was conducted using human microvascular endothelial cells (HMVEC). Oxidative stress-dependent cytotoxicity responses were assessed using quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and high content screening (HCS), and disruption of monolayer cell integrity was assessed using fluorescence microscopy and transwell assay. Scanning electron microscopy (SEM) coupled with energy-dispersive X-ray spectroscopy (EDX) analysis showed differences in the morphology and elemental composition of PM of different sizes and origins. While the total mass of PM collected from indoor environment was lower in comparison with those collected from the corridor, the endotoxin content was substantially higher in indoor PM (e.g., ninefold higher endotoxin level in indoor PM8.1-20 ). The ability to induce oxidative stress-mediated cytotoxicity and leakiness in cell monolayer were higher for indoor PM compared to those collected from the corridor. In conclusion, this comparative analysis suggested that indoor PM is relatively more hazardous to the endothelial system possibly because of higher endotoxin content.

  20. Hexachlorobenzene promotes angiogenesis in vivo, in a breast cancer model and neovasculogenesis in vitro, in the human microvascular endothelial cell line HMEC-1.

    PubMed

    Pontillo, Carolina; Español, Alejandro; Chiappini, Florencia; Miret, Noelia; Cocca, Claudia; Alvarez, Laura; Kleiman de Pisarev, Diana; Sales, María Elena; Randi, Andrea Silvana

    2015-11-19

    Exposure to environmental pollutants may alter proangiogenic ability and promotes tumor growth. Hexachlorobenzene (HCB) is an organochlorine pesticide found in maternal milk and in lipid foods, and a weak ligand of the aryl hydrocarbon receptor (AhR). HCB induces migration and invasion in human breast cancer cells, as well as tumor growth and metastasis in vivo. In this study, we examined HCB action on angiogenesis in mammary carcinogenesis. HCB stimulates angiogenesis and increases vascular endothelial growth factor (VEGF) expression in a xenograft model with the human breast cancer cell line MDA-MB-231. Human microvascular endothelial cells HMEC-1 exposed to HCB (0.005, 0.05, 0.5 and 5μM) showed an increase in cyclooxygenase-2 (COX-2) and VEGF protein expression involving AhR. In addition, we found that HCB enhances VEGF-Receptor 2 (VEGFR2) expression, and activates its downstream pathways p38 and ERK1/2. HCB induces cell migration and neovasculogenesis in a dose-dependent manner. Cells pretreatment with AhR, COX-2 and VEGFR2 selective inhibitors, suppressed these effects. In conclusion, our results show that HCB promotes angiogenesis in vivo and in vitro. HCB-induced cell migration and tubulogenesis are mediated by AhR, COX-2 and VEGFR2 in HMEC-1. These findings may help to understand the association among HCB exposure, angiogenesis and mammary carcinogenesis.

  1. Effect of oxygen and glucose deprivation on VEGF and its receptors in microvascular endothelial cells co-cultured with mast cells.

    PubMed

    Wang, Zhihua; Tao, Jianping; Zhang, Qingyong; Wei, Meng

    2015-09-01

    The aim of this study was to determine the correlation between angiogenesis and the differential expression of vascular endothelial growth factor (VEGF) and its receptors in myocardial microvascular endothelial cells (MMVECs) co-cultured with mast cells (MCs) or mast cell granules (MCGs) under oxygen and glucose deprivation (OGD). MMVECs and MCs were isolated from Wistar rats. MCs spontaneously degranulated in OGD. The expression of VEGF peaked at 8 h and decreased from 16 h in OGD. However, the expression of its receptor, fms-like tyrosine kinase-1 (Flt-1), and fetal liver kinase-1 (Flk-1), decreased significantly, and angiogenic potential of MMVECs decreased in OGD. Expression of VEGF, Flt-1, and Flk-1 increased significantly when MMVECs were co-cultured with MCGs or active MCs, but MCs had only a limited ability to induce angiogenesis in OGD. The angiogenic potential of MMVECs cultured in OGD (even with MCGs) was inferior to that of MMVECs cultured under normoxic conditions. OGD have a profound effect on angiogenesis, which is more pronounced than the effect of MCs on angiogenesis.

  2. Differential transcription profiles of long non-coding RNAs in primary human brain microvascular endothelial cells in response to meningitic Escherichia coli

    PubMed Central

    Yang, Ruicheng; Huang, Fei; Fu, Jiyang; Dou, Beibei; Xu, Bojie; Miao, Ling; Liu, Wentong; Yang, Xiaopei; Tan, Chen; Chen, Huanchun; Wang, Xiangru

    2016-01-01

    Accumulating studies have indicated the influence of long non-coding RNAs (lncRNAs) on various biological processes as well as disease development and progression. However, the lncRNAs involved in bacterial meningitis and their regulatory effects are largely unknown. By RNA-sequencing, the transcriptional profiles of host lncRNAs in primary human brain microvascular endothelial cells (hBMECs) in response to meningitic Escherichia coli were demonstrated. Here, 25,257 lncRNAs were identified, including 24,645 annotated lncRNAs and 612 newly found ones. A total of 895 lncRNAs exhibited significant differences upon infection, among which 382 were upregulated and 513 were downregulated (≥2-fold, p < 0.05). Via bioinformatic analysis, the features of these lncRNAs, their possible functions, and the potential regulatory relationships between lncRNAs and mRNAs were predicted. Moreover, we compared the transcriptional specificity of these differential lncRNAs among hBMECs, human astrocyte cell U251, and human umbilical vein endothelial cells, and demonstrated the novel regulatory effects of proinflammatory cytokines on these differential lncRNAs. To our knowledge, this is the first time the transcriptional profiles of host lncRNAs involved in E. coli-induced meningitis have been reported, which shall provide novel insight into the regulatory mechanisms behind bacterial meningitis involving lncRNAs, and contribute to better prevention and therapy of CNS infection. PMID:27958323

  3. Gap junction-mediated transfer of miR-145-5p from microvascular endothelial cells to colon cancer cells inhibits angiogenesis.

    PubMed

    Thuringer, Dominique; Jego, Gaetan; Berthenet, Kevin; Hammann, Arlette; Solary, Eric; Garrido, Carmen

    2016-05-10

    Gap junctional communication between cancer cells and blood capillary cells is crucial to tumor growth and invasion. Gap junctions may transfer microRNAs (miRs) among cells. Here, we explore the impact of such a transfer in co-culture assays, using the antitumor miR-145 as an example. The SW480 colon carcinoma cells form functional gap junction composed of connexin-43 (Cx43) with human microvascular endothelial cells (HMEC). When HMEC are loaded with miR-145-5p mimics, the miR-145 level drastically increases in SW480. The functional inhibition of gap junctions, using either a gap channel blocker or siRNA targeting Cx43, prevents this increase. The transfer of miR-145 also occurs from SW480 to HMEC but not in non-contact co-cultures, excluding the involvement of soluble exosomes. The miR-145 transfer to SW480 up-regulates their Cx43 expression and inhibits their ability to promote angiogenesis. Our results indicate that the gap junctional communication can inhibit tumor growth by transferring miRs from one endothelial cell to neighboring tumor cells. This "bystander" effect could find application in cancer therapy.

  4. Upregulation of COX-2/PGE2 by ET-1 mediated through Ca2+-dependent signals in mouse brain microvascular endothelial cells.

    PubMed

    Lin, Chih-Chung; Hsieh, Hsi-Lung; Chi, Pei-Ling; Yang, Chien-Chung; Hsiao, Li-Der; Yang, Chuen-Mao

    2014-06-01

    Endothelin-1 (ET-1), a proinflammatory mediator, is elevated in the regions of several brain inflammatory disorders, implying that ET-1 may contribute to inflammatory responses. The deleterious effects of ET-1 on brain endothelial cells may aggravate brain inflammation mediated through the upregulation of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) system. However, the signaling mechanisms underlying ET-1-induced COX-2 expression in mouse brain microvascular endothelial cells (bEnd.3 cells) remain unclear. Herein, we investigated the effects of Ca2+-dependent protein kinases on ET-1-induced COX-2 expression and PGE2 release in bEnd.3 cells. The data obtained with Western blotting, reverse transcription PCR, and intracellular Ca2+ analyses showed that ET-1-induced COX-2 expression was mediated through phosphatidylinositol-phospholipase C (PI-PLC) and phosphatidylcholine-phospholipase C (PC-PLC)/Ca2+-dependent activation of protein kinase C-alpha (PKC-α) and calmodulin kinase II (CaMKII) cascades. Next, we demonstrated that ET-1 stimulated intracellular Ca2+ increase, phoshorylation of PKC-α, CaMKII, and mitogen-activated protein kinases (MAPKs) (ERK1/2, p38 MAPK, and JNK1/2) and then activated the activating transcription factor 2 (ATF2)/activator protein 1 (AP-1) via Gq/i protein-coupled ETB receptors. Moreover, the data of chromatin immunoprecipitation and promoter reporter assay demonstrated that the activated ATF2/AP-1 and p300 bound to its corresponding binding sites within COX-2 promoter, thereby turning on COX-2 gene transcription. Finally, upregulation of COX-2 by ET-1 promoted PGE2 biosynthesis and release in these cells. Taken together, these results demonstrate that in bEnd.3 cells, Ca2+-dependent PKC-α and CaMKII linking to MAPKs, ATF2/AP-1, and p300 cascade is essential for ET-1-induced COX-2 upregulation. Understanding the mechanisms of COX-2/PGE2 system upregulated by ET-1 on brain microvascular endothelial cells may provide rational

  5. The protective role of isorhamnetin on human brain microvascular endothelial cells from cytotoxicity induced by methylglyoxal and oxygen-glucose deprivation.

    PubMed

    Li, Wenlu; Chen, Zhigang; Yan, Min; He, Ping; Chen, Zhong; Dai, Haibin

    2016-02-01

    As the first target of stroke, cerebral endothelial cells play a key role in brain vascular repair and maintenance, and their function is impeded in diabetes. Methylglyoxal (MGO), a reactive dicarbonyl produced during glucose metabolism, accumulates in diabetic patients. MGO and MGO-induced advanced glycation end-products (AGEs) could ameliorate stroke-induced brain vascular damage, closely related with ECs dysfunction. Using MGO plus oxygen-glucose deprivation (OGD) to mimic diabetic stroke, we reported the protective effect of isorhamnetin on OGD-induced cytotoxicity after MGO treatment on primary human brain microvascular endothelial cells (HBMEC) and explored the underlying mechanisms. Treatment of MGO for 24 h significantly enhanced 3-h OGD-induced HBMEC toxic effect, which was inhibited by pretreatment of isorhamnetin (100 μmol/L). Moreover, the protective effect of isorhamnetin is multiple function dependent, which includes anti-inflammation, anti-oxidative stress and anti-apoptosis effects. Besides its well-known inhibition on the mitochondria-dependent or intrinsic apoptotic pathway, isorhamnetin also reduced activation of the extrinsic apoptotic pathway, as characterized by the decreased expression and activity of caspase 3 and caspase 8. Furthermore, pretreatment with isorhamnetin specifically inhibited FAS/FASL expression and suppressed nuclear factor-kappa B nuclear translocation. Taken together, our results indicated that isorhamnetin protected against OGD-induced cytotoxicity after MGO treatment in cultured HBMEC due to its multiple protective effects and could inhibit Fas-mediated extrinsic apoptosis. Therefore, isorhamnetin is a promising reagent for the treatment of hyperglycemia and ischemia-induced cerebral vascular degeneration. A proposed model of the potential protective mechanism of isorhamnetin, a metabolite of quercetin, on methylglyoxal (MGO) treatment plus oxygen-glucose deprivation (OGD) exposure-induced cytotoxicity in cultured human

  6. The novel mitochondria-targeted hydrogen sulfide (H2S) donors AP123 and AP39 protect against hyperglycemic injury in microvascular endothelial cells in vitro.

    PubMed

    Gerő, Domokos; Torregrossa, Roberta; Perry, Alexis; Waters, Alicia; Le-Trionnaire, Sophie; Whatmore, Jacqueline L; Wood, Mark; Whiteman, Matthew

    2016-11-01

    The development of diabetic vascular complications is initiated, at least in part, by mitochondrial reactive oxygen species (ROS) production in endothelial cells. Hyperglycemia induces superoxide production in the mitochondria and initiates changes in the mitochondrial membrane potential that leads to mitochondrial dysfunction. Hydrogen sulfide (H2S) supplementation has been shown to reduce the mitochondrial oxidant production and shows efficacy against diabetic vascular damage in vivo. However, the half-life of H2S is very short and it is not specific for the mitochondria. We have therefore evaluated two novel mitochondria-targeted anethole dithiolethione and hydroxythiobenzamide H2S donors (AP39 and AP123 respectively) at preventing hyperglycemia-induced oxidative stress and metabolic changes in microvascular endothelial cells in vitro. Hyperglycemia (HG) induced significant increase in the activity of the citric acid cycle and led to elevated mitochondrial membrane potential. Mitochondrial oxidant production was increased and the mitochondrial electron transport decreased in hyperglycemic cells. AP39 and AP123 (30-300nM) decreased HG-induced hyperpolarisation of the mitochondrial membrane and inhibited the mitochondrial oxidant production. Both H2S donors (30-300nM) increased the electron transport at respiratory complex III and improved the cellular metabolism. Targeting H2S to mitochondria retained the cytoprotective effect of H2S against glucose-induced damage in endothelial cells suggesting that the molecular target of H2S action is within the mitochondria. Mitochondrial targeting of H2S also induced >1000-fold increase in the potency of H2S against hyperglycemia-induced injury. The high potency and long-lasting effect elicited by these H2S donors strongly suggests that these compounds could be useful against diabetic vascular complications.

  7. Evidence for a discrete UTP receptor in cardiac endothelial cells.

    PubMed Central

    Yang, S.; Buxton, I. L.; Probert, C. B.; Talbot, J. N.; Bradley, M. E.

    1996-01-01

    1. We have examined the effects of various purine and pyrimidine nucleotides upon cells cultured from guinea-pig cardiac endothelium (CEC), and find the P2Y-agonist 2-methylthioadenosine triphosphate (2MeSATP) to be a potent (EC50 = 85 +/- 10.2 nM) stimulator of increase in intracellular calcium concentrations, while uridine 5'-triphosphate (UTP) and adenosine 5'-triphosphate (ATP) are less potent but equipotent with one another (EC50s = 2.1 +/- 0.3 and 1.8 +/- 0.2 microM, respectively). 2. While the P2Y receptor exhibited rapid homologous desensitization, this had no effect upon subsequent responsiveness of CEC to either ATP or UTP. Effects of maximal concentrations of ATP and UTP were not only additive, but did not cross-desensitize. Responses to UTP (but not to ATP or 2MeSATP) were blocked by treatment with pertussis toxin (PTX); all three nucleotides appeared to liberate calcium from an intracellular pool. 3. Suramin (30 microM) significantly (P < 0.05) increased the EC50 for ATP-dependent increases in intracellular calcium (5.3 +/- 2.2 microM vs. 2.0 +/- 0.9 microM in the absence of suramin), while it completely blocked the response to 2MeSATP. Suramin had no effect upon responses to UTP at concentrations of 100 microM. 4. We conclude that in addition to the P2Y and P2U subtypes of the ATP receptor, an additional receptor responsive to UTP but exhibiting no affinity for purine nucleotides is present in CEC; this "pyrimidine receptor' liberates intracellular calcium via a G-protein, and may partly mediate the contractile response to UTP in the coronary vasculature. PMID:8730756

  8. Immunoglobulin M-enriched human intravenous immunoglobulins reduce leukocyte-endothelial cell interactions and attenuate microvascular perfusion failure in normotensive endotoxemia.

    PubMed

    Hoffman, Johannes N; Fertmann, Jan M; Vollmar, Brigitte; Laschke, Matthias W; Jauch, Karl W; Menger, Michael D

    2008-01-01

    Clinical studies indicate potential differences in the efficacy of immunoglobulin (Ig) preparations in patients with sepsis. A recent meta-analysis showed improved survival rates with IgM-enriched Igs. It was the objective of the present study to characterize microcirculatory actions of different clinically used Ig preparations in a rodent endotoxin model by intravital microscopy. Male Syrian golden hamsters 6 to 8 weeks old with a body weight of 60 to 80 g were investigated by intravital fluorescence microscopy. Endotoxemia was induced by administration of 2 mg/kg (i.v.) endotoxin (LPS, Escherichia coli). Two different Ig preparations containing IgM, IgA, and IgG (intravenous IgM group; n = 6; 5 mL Pentaglobin/kg body weight, i.v.) or exclusively IgG (intravenous IgG group; n = 5; 5 mL Flebogamma/kg body weight, i.v.) were applied 5 min before LPS. Saline-treated endotoxemic animals served as controls (control; n = 8). In controls, LPS induced massive leukocyte-endothelial cell interactions, pronounced microvascular leakage, a decrease of systemic platelet count, and distinct capillary perfusion failure (P < 0.05). Both intravenous IgM and IgG reduced venular leakage (P< 0.05) and ameliorated the decrease in platelet count (P < 0.05). Of interest, intravenous IgM was capable of significantly (P< 0.05) reducing leukocyte adhesion in venules. This was associated with normalization of capillary perfusion at 24 h of endotoxemia, whereas intravenous IgG could not prevent LPS-mediated microvascular perfusion failure. We demonstrate that IgM-enriched Igs are superior to IgG alone in attenuating LPS-induced leukocytic inflammation and microcirculatory dysfunction. Our findings can explain better efficacy of IgM-enriched Igs in patients with severe sepsis.

  9. Endothelial p53 Deletion Improves Angiogenesis and Prevents Cardiac Fibrosis and Heart Failure Induced by Pressure Overload in Mice

    PubMed Central

    Gogiraju, Rajinikanth; Xu, Xingbo; Bochenek, Magdalena L.; Steinbrecher, Julia H.; Lehnart, Stephan E.; Wenzel, Philip; Kessel, Michael; Zeisberg, Elisabeth M.; Dobbelstein, Matthias; Schäfer, Katrin

    2015-01-01

    Background Cardiac dysfunction developing in response to chronic pressure overload is associated with apoptotic cell death and myocardial vessel rarefaction. We examined whether deletion of tumor suppressor p53 in endothelial cells may prevent the transition from cardiac hypertrophy to heart failure. Methods and Results Mice with endothelial‐specific deletion of p53 (End.p53‐KO) were generated by crossing p53fl/fl mice with mice expressing Cre recombinase under control of an inducible Tie2 promoter. Cardiac hypertrophy was induced by transverse aortic constriction. Serial echocardiography measurements revealed improved cardiac function in End.p53‐KO mice that also exhibited better survival. Cardiac hypertrophy was associated with increased p53 levels in End.p53‐WT controls, whereas banded hearts of End.p53‐KO mice exhibited lower numbers of apoptotic endothelial and non‐endothelial cells and altered mRNA levels of genes regulating cell cycle progression (p21), apoptosis (Puma), or proliferation (Pcna). A higher cardiac capillary density and improved myocardial perfusion was observed, and pharmacological inhibition or genetic deletion of p53 also promoted endothelial sprouting in vitro and new vessel formation following hindlimb ischemia in vivo. Hearts of End.p53‐KO mice exhibited markedly less fibrosis compared with End.p53‐WT controls, and lower mRNA levels of p53‐regulated genes involved in extracellular matrix production and turnover (eg, Bmp‐7, Ctgf, or Pai‐1), or of transcription factors involved in controlling mesenchymal differentiation were observed. Conclusions Our analyses reveal that accumulation of p53 in endothelial cells contributes to blood vessel rarefaction and fibrosis during chronic cardiac pressure overload and suggest that endothelial cells may be a therapeutic target for preserving cardiac function during hypertrophy. PMID:25713289

  10. Thymosin Beta4 Regulates Cardiac Valve Formation Via Endothelial-Mesenchymal Transformation in Zebrafish Embryos

    PubMed Central

    Shin, Sun-Hye; Lee, Sangkyu; Bae, Jong-Sup; Jee, Jun-Goo; Cha, Hee-Jae; Lee, You Mie

    2014-01-01

    Thymosin beta4 (TB4) has multiple functions in cellular response in processes as diverse as embryonic organ development and the pathogeneses of disease, especially those associated with cardiac coronary vessels. However, the specific roles played by TB4 during heart valve development in vertebrates are largely unknown. Here, we identified a novel function of TB4 in endothelialmesenchymal transformation (EMT) in cardiac valve endocardial cushions in zebrafish. The expressions of thymosin family members in developing zebrafish embryos were determined by whole mount in situ hybridization. Of the thymosin family members only zTB4 was expressed in the developing heart region. Cardiac valve development at 48 h post fertilization was defected in zebrafish TB4 (zTB4) morpholino-injected embryos (morphants). In zTB4 morphants, abnormal linear heart tube development was observed. The expressions of bone morphogenetic protein (BMP) 4, notch1b, and hyaluronic acid synthase (HAS) 2 genes were also markedly reduced in atrio-ventricular canal (AVC). Endocardial cells in the AVC region were stained with anti-Zn5 antibody reactive against Dm-grasp (an EMT marker) to observe EMT in developing cardiac valves in zTB4 morphants. EMT marker expression in valve endothelial cells was confirmed after transfection with TB4 siRNA in the presence of transforming growth factor β (TGFβ) by RT-PCR and immunofluorescent assay. Zn5-positive endocardial AVC cells were not observed in zTB4 morphants, and knockdown of TB4 suppressed TGF-β-induced EMT in ovine valve endothelial cells. Taken together, our results demonstrate that TB4 plays a pivotal role in cardiac valve formation by increasing EMT. PMID:24732964

  11. The TGFβ type II receptor plays a critical role in the endothelial cells during cardiac development.

    PubMed

    Robson, Andrew; Allinson, Kathleen R; Anderson, Robert H; Henderson, Deborah J; Arthur, Helen M

    2010-09-01

    TGFβ signalling is required for normal cardiac development. To investigate which cell types are involved, we used mice carrying a floxed Type II TGFβ receptor (Tgfbr2fl) allele and Cre-lox genetics to deplete this receptor in different regions of the heart. The three target tissues and corresponding Cre transgenic lines were atrioventricular myocardium (using cGata6-Cre), ventricular myocardium (using Mlc2v-Cre), and vascular endothelium (using tamoxifen-activated Cdh5(PAC)-CreERT2). Spatio-temporal Cre activity in each case was tracked via lacZ activation from the Rosa26R locus. Atrioventricular-myocardial-specific Tgfbr2 knockout (KO) embryos had short septal leaflets of the tricuspid valve, whereas ventricular myocardial-specific KO embryos mainly exhibited a normal cardiac phenotype. Inactivation of Tgfbr2 in endothelial cells from E11.5 resulted in deficient ventricular septation, accompanied by haemorrhage from cerebral blood vessels. We conclude that TGFβ signalling through the Tgfbr2 receptor, in endothelial cells, plays an important role in cardiac development, and is essential for cerebral vascular integrity.

  12. Fibronectin biosynthesis and cell-surface expression by cardiac and non-cardiac endothelial cells.

    PubMed Central

    Johnson, C. M.; Helgeson, S. C.

    1993-01-01

    We examined the biosynthesis and surface expression of fibronectin, an adhesive glycoprotein, in several types of cultured porcine endothelial cells: pulmonary artery, thoracic aorta, coronary artery, aortic valve, and mitral valve. We used immunocytochemical staining to compare the levels of fibronectin present in these same tissues in vivo. Using endogenous radiolabeling, we found that all cell types except aortic valve endothelial cells synthesized and released into the culture media substantial quantities of fibronectin. Using radioiodination of intact cells, we found that, whereas both thoracic aorta and pulmonary artery cells had measurable fibronectin on the surface, aortic valve, mitral valve, and coronary artery cells had little cell-surface fibronectin present. Immunocytochemical staining showed that all endothelial regions except aortic valve had substantial quantities of immunoreactive fibronectin in vivo. These data suggest that the aortic valve endothelium may be distinct from other endothelia. Such differences could be important for the pathogenesis of valvular disease. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:8494044

  13. Exosomes from iPSCs Delivering siRNA Attenuate Intracellular Adhesion Molecule-1 Expression and Neutrophils Adhesion in Pulmonary Microvascular Endothelial Cells.

    PubMed

    Ju, Zhihai; Ma, Jinhui; Wang, Chen; Yu, Jie; Qiao, Yeru; Hei, Feilong

    2017-04-01

    The pro-inflammatory activation of pulmonary microvascular endothelial cells resulting in continuous expression of cellular adhesion molecules, and subsequently recruiting primed neutrophils to form a firm neutrophils-endothelium (PMN-EC) adhesion, has been examined and found to play a vital role in acute lung injury (ALI). RNA interference (RNAi) is a cellular process through harnessing a natural pathway silencing target gene based on recognition and subsequent degradation of specific mRNA sequences. It opens a promising approach for precision medicine. However, this application was hampered by many obstacles, such as immunogenicity, instability, toxicity problems, and difficulty in across the biological membrane. In this study, we reprogrammed urine exfoliated renal epithelial cells into human induced pluripotent stem cells (huiPSCs) and purified the exosomes (Exo) from huiPSCs as RNAi delivery system. Through choosing the episomal system to deliver transcription factors, we obtained a non-integrating huiPSCs. Experiments in both vitro and vivo demonstrated that these huiPSCs possess the pluripotent properties. The exosomes of huiPSCs isolated by differential centrifugation were visualized by transmission electron microscopy (TEM) showing a typical exosomal appearance with an average diameter of 122 nm. Immunoblotting confirmed the presence of the typical exosomal markers, including CD63, TSG 101, and Alix. Co-cultured PKH26-labeled exosomes with human primary pulmonary microvascular endothelial cells (HMVECs) confirmed that they could be internalized by recipient cells at a time-dependent manner. Then, electroporation was used to introduce siRNA against intercellular adhesion molecule-1 (ICAM-1) into exosomes to form an Exo/siRNA compound. The Exo/siRNA compound efficiently delivered the target siRNA into HMVECs causing selective gene silencing, inhibiting the ICAM-1 protein expression, and PMN-EC adhesion induced by lipopolysaccharide (LPS). These data suggest

  14. Perforin Mediates Endothelial Cell Death and Resultant Transplant Vascular Disease in Cardiac Allografts

    PubMed Central

    Choy, Jonathan C.; Kerjner, Alexandra; Wong, Brian W.; McManus, Bruce M.; Granville, David J.

    2004-01-01

    T cell-induced endothelial injury is an important event in the development of transplant vascular disease (TVD), the leading expression of chronic rejection of vascularized organ transplants. However, the precise contribution of perforin to vascular damage in allografts and resultant TVD has not been addressed in vivo. Minor histocompatability antigen mismatched mouse heterotopic cardiac transplants were performed from 129J donors into C57Bl/6 (wild-type (WT)) or perforin knockout (PKO) recipients. Perforin was abundant in immune infiltrates in the myocardium and vasculature of transplanted hearts in WT mice. Allograft coronary arteries in both WT and PKO mice had considerable vasculitis. There was also marked endothelial disruption, as well as TUNEL-positivity in the endothelial region, in coronary arteries of hearts transplanted into WT mice that was not evident in PKO recipients (P = 0.05). At 30 days post-transplantation, intimal thickening was assessed on elastic Van Gieson-stained ventricular sections. There was an average of 54.2 ± 6.7% luminal narrowing of coronary arteries in allografts from WT mice as compared to 13.4 ± 5.1% luminal narrowing in PKO counterparts (P < 0.00002). In summary, perforin plays a primary role in endothelial damage and the resultant onset and progression of TVD. PMID:15215168

  15. Activation of melatonin receptor (MT1/2) promotes P-gp transporter in methamphetamine-induced toxicity on primary rat brain microvascular endothelial cells.

    PubMed

    Jumnongprakhon, Pichaya; Sivasinprasasn, Sivanan; Govitrapong, Piyarat; Tocharus, Chainarong; Tocharus, Jiraporn

    2017-02-20

    Melatonin has been known as a neuroprotective agent for the central nervous system (CNS) and the blood-brain barrier (BBB), which is the primary structure that comes into contact with several neurotoxins including methamphetamine (METH). Previous studies have reported that the activation of melatonin receptors (MT1/2) by melatonin could protect against METH-induced toxicity in brain endothelial cells via several mechanisms. However, its effects on the P-glycoprotein (P-gp) transporter, the active efflux pump involved in cell homeostasis, are still unclear. Thus, this study investigated the role of melatonin and its receptors on the METH-impaired P-gp transporter in primary rat brain microvascular endothelial cells (BMVECs). The results showed that METH impaired the function of the P-gp transporter, significantly decreasing the efflux of Rho123 and P-gp expression, which caused a significant increase in the intracellular accumulation of Rho123, and these responses were reversed by the interaction of melatonin with its receptors. Blockade of the P-gp transporter by verapamil caused oxidative stress, apoptosis, and cell integrity impairment after METH treatment, and these effects could be reversed by melatonin. Our results, together with previous findings, suggest that the interaction of melatonin with its receptors protects against the effects of the METH-impaired P-gp transporter and that the protective role in METH-induced toxicity was at least partially mediated by the regulation of the P-gp transporter. Thus, melatonin and its receptors (MT1/2) are essential for protecting against BBB impairment caused by METH.

  16. Shear stress and 17beta-estradiol modulate cerebral microvascular endothelial Na-K-Cl cotransporter and Na/H exchanger protein levels.

    PubMed

    Chang, Elaine; O'Donnell, Martha E; Barakat, Abdul I

    2008-01-01

    Ion transporters of blood-brain barrier (BBB) endothelial cells play an important role in regulating the movement of ions between the blood and brain. During ischemic stroke, reduction in cerebral blood flow is accompanied by transport of Na and Cl from the blood into the brain, with consequent brain edema formation. We have shown previously that a BBB Na-K-Cl cotransporter (NKCC) participates in ischemia-induced brain Na and water uptake and that a BBB Na/H exchanger (NHE) may also participate. While the abrupt reduction of blood flow is a prominent component of ischemia, the effects of flow on BBB NKCC and NHE are not known. In the present study, we examined the effects of changes in shear stress on NKCC and NHE protein levels in cerebral microvascular endothelial cells (CMECs). We have shown previously that estradiol attenuates both ischemia-induced cerebral edema and CMEC NKCC activity. Thus, in the present study, we also examined the effects of estradiol on NKCC and NHE protein levels in CMECs. Exposing CMECs to steady shear stress (19 dyn/cm(2)) increased the abundance of both NKCC and NHE. Estradiol abolished the shear stress-induced increase in NHE but not NKCC. Abrupt reduction of shear stress did not alter NKCC or NHE abundance in the absence of estradiol, but it decreased NKCC abundance in estradiol-treated cells. Our results indicate that changes in shear stress modulate BBB NKCC and NHE protein levels. They also support the hypothesis that estradiol attenuates edema formation in ischemic stroke in part by reducing the abundance of BBB NKCC protein.

  17. Human Brain Microvascular Endothelial Cells Derived from the BC1 iPS Cell Line Exhibit a Blood-Brain Barrier Phenotype.

    PubMed

    Katt, Moriah E; Xu, Zinnia S; Gerecht, Sharon; Searson, Peter C

    2016-01-01

    The endothelial cells that form capillaries in the brain are highly specialized, with tight junctions that minimize paracellular transport and an array of broad-spectrum efflux pumps that make drug delivery to the brain extremely challenging. One of the major limitations in blood-brain barrier research and the development of drugs to treat central nervous system diseases is the lack of appropriate cell lines. Recent reports indicate that the derivation of human brain microvascular endothelial cells (hBMECs) from human induced pluripotent stem cells (iPSCs) may provide a solution to this problem. Here we demonstrate the derivation of hBMECs extended to two new human iPSC lines: BC1 and GFP-labeled BC1. These hBMECs highly express adherens and tight junction proteins VE-cadherin, ZO-1, occludin, and claudin-5. The addition of retinoic acid upregulates VE-cadherin expression, and results in a significant increase in transendothelial electrical resistance to physiological values. The permeabilities of tacrine, rhodamine 123, and Lucifer yellow are similar to values obtained for MDCK cells. The efflux ratio for rhodamine 123 across hBMECs is in the range 2-4 indicating polarization of efflux transporters. Using the rod assay to assess cell organization in small vessels and capillaries, we show that hBMECs resist elongation with decreasing diameter but show progressive axial alignment. The derivation of hBMECs with a blood-brain barrier phenotype from the BC1 cell line highlights that the protocol is robust. The expression of GFP in hBMECs derived from the BC1-GFP cell line provides an important new resource for BBB research.

  18. Beta-Adrenoceptor Activation Reduces Both Dermal Microvascular Endothelial Cell Migration via a cAMP-Dependent Mechanism and Wound Angiogenesis

    PubMed Central

    O'Leary, Andrew P; Fox, James M; Pullar, Christine E

    2015-01-01

    Angiogenesis is an essential process during tissue regeneration; however, the amount of angiogenesis directly correlates with the level of wound scarring. Angiogenesis is lower in scar-free foetal wounds while angiogenesis is raised and abnormal in pathophysiological scarring such as hypertrophic scars and keloids. Delineating the mechanisms that modulate angiogenesis and could reduce scarring would be clinically useful. Beta-adrenoceptors (β-AR) are G protein-coupled receptors (GPCRs) expressed on all skin cell-types. They play a role in wound repair but their specific role in angiogenesis is unknown. In this study, a range of in vitro assays (single cell migration, scratch wound healing, ELISAs for angiogenic growth factors and tubule formation) were performed with human dermal microvascular endothelial cells (HDMEC) to investigate and dissect mechanisms underpinning β-AR-mediated modulation of angiogenesis in chick chorioallantoic membranes (CAM) and murine excisional skin wounds. β-AR activation reduced HDMEC migration via cyclic adenosine monophosphate (cAMP)-dependent and protein kinase A (PKA)-independent mechanisms as demonstrated through use of an EPAC agonist that auto-inhibited the cAMP-mediated β-AR transduced reduction in HDMEC motility; a PKA inhibitor was, conversely, ineffective. ELISA studies demonstrated that β-AR activation reduced pro-angiogenic growth factor secretion from HDMECs (fibroblast growth factor 2) and keratinocytes (vascular endothelial growth factor A) revealing possible β-AR-mediated autocrine and paracrine anti-angiogenic mechanisms. In more complex environments, β-AR activation delayed HDMEC tubule formation and decreased angiogenesis both in the CAM assay and in murine excisional skin wounds in vivo. β-AR activation reduced HDMEC function in vitro and angiogenesis in vivo; therefore, β-AR agonists could be promising anti-angiogenic modulators in skin. J. Cell. Physiol. 230: 356–365, 2015. © 2014 The Authors. Journal

  19. Sevoflurane prevents lipopolysaccharide-induced barrier dysfunction in human lung microvascular endothelial cells: Rho-mediated alterations of VE-cadherin.

    PubMed

    Huang, Yiran; Tan, Qindong; Chen, Rui; Cao, Biao; Li, Wenhong

    Acute lung injury (ALI) mainly occurs as increased permeability of lung tissue and pleural effusion. Inhaled anesthetic sevoflurane has been demonstrated to alleviate lung permeability by upregulating junction proteins after ischemia-reperfusion. However, the exact mechanisms of its protective effect on reperfusion injury remain elusive. The aim of this study was to assess possible preconditioning with sevoflurane in an in vitro model of lipopolysaccharide (LPS)-induced barrier dysfunction in human lung microvascular endothelial cells (HMVEC-Ls). In this study, HMVEC-Ls were exposed to minimum alveolar concentration of sevoflurane for 2 h. LPS significantly increased the permeability of HMVEC-L. Moreover, the distribution of junction protein, vascular endothelial (VE)-cadherin, in cell-cell junction area and the total expression in HMVEC-Ls were significantly decreased by LPS treatment. However, the abnormal distribution and decreased expression of VE-cadherin and hyperpermeability of HMVEC-Ls were significantly reversed by pretreatment with sevoflurane. Furthermore, LPS-induced activation of the RhoA/ROCK signaling pathway was significantly inhibited with sevoflurane. Such activation, abnormal distribution and decreased expression of VE-cadherin and hyperpermeability of HMVEC-Ls were significantly inhibited with sevoflurane pretreatment or knockdown of RhoA or ROCK-2. In conclusion, sevoflurane prevented LPS-induced rupture of HMVEC-L monolayers by suppressing the RhoA/ROCK-mediated VE-cadherin signaling pathway. Our results may explain, at least in part, some beneficial effects of sevoflurane on pulmonary dysfunction such as ischemia-reperfusion injury.

  20. Hyperglycaemia promotes human brain microvascular endothelial cell apoptosis via induction of protein kinase C-ßI and prooxidant enzyme NADPH oxidase

    PubMed Central

    Shao, Beili; Bayraktutan, Ulvi

    2014-01-01

    Blood–brain barrier disruption represents a key feature in hyperglycaemia-aggravated cerebral damage after an ischaemic stroke. Although the underlying mechanisms remain largely unknown, activation of protein kinase C (PKC) is thought to play a critical role. This study examined whether apoptosis of human brain microvascular endothelial cells (HBMEC) might contribute to hyperglycaemia-evoked barrier damage and assessed the specific role of PKC in this phenomenon. Treatments with hyperglycaemia (25 mM) or phorbol myristate acetate (PMA, a protein kinase C activator, 100 nM) significantly increased NADPH oxidase activity, O2•- generation, proapoptotic protein Bax expression, TUNEL-positive staining and caspase-3/7 activities. Pharmacological inhibition of NADPH oxidase, PKC-a, PKC-ß or PKC-ßI via their specific inhibitors and neutralisation of O2•- by a cell-permeable superoxide dismutase mimetic, MnTBAP normalised all the aforementioned increases induced by hyperglycaemia. Suppression of these PKC isoforms also negated the stimulatory effects of hyperglycaemia on the protein expression of NADPH oxidase membrane-bound components, Nox2 and p22-phox which determine the overall enzymatic activity. Silencing of PKC-ßI gene through use of specific siRNAs abolished the effects of both hyperglycaemia and PMA on endothelial cell NADPH oxidase activity, O2•- production and apoptosis and consequently improved the integrity and function of an in vitro model of human cerebral barrier comprising HBMEC, astrocytes and pericytes. Hyperglycaemia-mediated apoptosis of HBMEC contributes to cerebral barrier dysfunction and is modulated by sequential activations of PKC-ßI and NADPH oxidase. PMID:24936444

  1. Human Brain Microvascular Endothelial Cells Derived from the BC1 iPS Cell Line Exhibit a Blood-Brain Barrier Phenotype

    PubMed Central

    Gerecht, Sharon; Searson, Peter C.

    2016-01-01

    The endothelial cells that form capillaries in the brain are highly specialized, with tight junctions that minimize paracellular transport and an array of broad-spectrum efflux pumps that make drug delivery to the brain extremely challenging. One of the major limitations in blood-brain barrier research and the development of drugs to treat central nervous system diseases is the lack of appropriate cell lines. Recent reports indicate that the derivation of human brain microvascular endothelial cells (hBMECs) from human induced pluripotent stem cells (iPSCs) may provide a solution to this problem. Here we demonstrate the derivation of hBMECs extended to two new human iPSC lines: BC1 and GFP-labeled BC1. These hBMECs highly express adherens and tight junction proteins VE-cadherin, ZO-1, occludin, and claudin-5. The addition of retinoic acid upregulates VE-cadherin expression, and results in a significant increase in transendothelial electrical resistance to physiological values. The permeabilities of tacrine, rhodamine 123, and Lucifer yellow are similar to values obtained for MDCK cells. The efflux ratio for rhodamine 123 across hBMECs is in the range 2–4 indicating polarization of efflux transporters. Using the rod assay to assess cell organization in small vessels and capillaries, we show that hBMECs resist elongation with decreasing diameter but show progressive axial alignment. The derivation of hBMECs with a blood-brain barrier phenotype from the BC1 cell line highlights that the protocol is robust. The expression of GFP in hBMECs derived from the BC1-GFP cell line provides an important new resource for BBB research. PMID:27070801

  2. Human blood-brain barrier receptors for Alzheimer's amyloid-beta 1- 40. Asymmetrical binding, endocytosis, and transcytosis at the apical side of brain microvascular endothelial cell monolayer.

    PubMed Central

    Mackic, J B; Stins, M; McComb, J G; Calero, M; Ghiso, J; Kim, K S; Yan, S D; Stern, D; Schmidt, A M; Frangione, B; Zlokovic, B V

    1998-01-01

    A soluble monomeric form of Alzheimer's amyloid-beta (1-40) peptide (sAbeta1-40) is present in the circulation and could contribute to neurotoxicity if it crosses the brain capillary endothelium, which comprises the blood-brain barrier (BBB) in vivo. This study characterizes endothelial binding and transcytosis of a synthetic peptide homologous to human sAbeta1-40 using an in vitro model of human BBB. 125I-sAbeta1-40 binding to the brain microvascular endothelial cell monolayer was time dependent, polarized to the apical side, and saturable with high- and low-affinity dissociation constants of 7.8+/-1.2 and 52.8+/-6.2 nM, respectively. Binding of 125I-sAbeta1-40 was inhibited by anti-RAGE (receptor for advanced glycation end products) antibody (63%) and by acetylated low density lipoproteins (33%). Consistent with these data, transfected cultured cells overexpressing RAGE or macrophage scavenger receptor (SR), type A, displayed binding and internalization of 125I-sAbeta1-40. The internalized peptide remains intact > 94%. Transcytosis of 125I-sAbeta1-40 was time and temperature dependent, asymmetrical from the apical to basolateral side, saturable with a Michaelis constant of 45+/-9 nM, and partially sensitive to RAGE blockade (36%) but not to SR blockade. We conclude that RAGE and SR mediate binding of sAbeta1-40 at the apical side of human BBB, and that RAGE is also involved in sAbeta1-40 transcytosis. PMID:9710442

  3. Modulation of human dermal microvascular endothelial cell and human gingival fibroblast behavior by micropatterned silica coating surfaces for zirconia dental implant applications.

    PubMed

    Laranjeira, Marta S; Carvalho, Ângela; Pelaez-Vargas, Alejandro; Hansford, Derek; Ferraz, Maria Pia; Coimbra, Susana; Costa, Elísio; Santos-Silva, Alice; Fernandes, Maria Helena; Monteiro, Fernando Jorge

    2014-04-01

    Dental ceramic implants have shown superior esthetic behavior and the absence of induced allergic disorders when compared to titanium implants. Zirconia may become a potential candidate to be used as an alternative to titanium dental implants if surface modifications are introduced. In this work, bioactive micropatterned silica coatings were produced on zirconia substrates, using a combined methodology of sol-gel processing and soft lithography. The aim of the work was to compare the in vitro behavior of human gingival fibroblasts (HGFs) and human dermal microvascular endothelial cells (HDMECs) on three types of silica-coated zirconia surfaces: flat and micropatterned (with pillars and with parallel grooves). Our results showed that cells had a higher metabolic activity (HGF, HDMEC) and increased gene expression levels of fibroblast-specific protein-1 (FSP-1) and collagen type I (COL I) on surfaces with pillars. Nevertheless, parallel grooved surfaces were able to guide cell growth. Even capillary tube-like networks of HDMEC were oriented according to the surface geometry. Zirconia and silica with different topographies have shown to be blood compatible and silica coating reduced bacteria adhesion. All together, the results indicated that microstructured bioactive coating seems to be an efficient strategy to improve soft tissue integration on zirconia implants, protecting implants from peri-implant inflammation and improving long-term implant stabilization. This new approach of micropatterned silica coating on zirconia substrates can generate promising novel dental implants, with surfaces that provide physical cues to guide cells and enhance their behavior.

  4. Photodynamic efficacy of liposome-delivered hypocrellin B in microvascular endothelial cells in vitro and chicken combs in vivo: a potential photosensitizer for port wine stain

    NASA Astrophysics Data System (ADS)

    Chen, H. X.; Yang, Z. F.; Zou, X. B.; Zhu, J. G.; Deng, H.; Zhao, J. Q.; Gu, Y.

    2013-02-01

    Photodynamic therapy (PDT) has been proved a successful method for port wine stain (PWS), but the prolonged skin photosensitivity induced by the photosensitizers used currently seriously limits the clinical application of PDT. In this study, we investigate the feasibility of hypocrellin B (HB), a promising second-generation photosensitizer for the treatment of PWS. The photodynamic effect of liposome-delivered HB was evaluated in vitro with microvascular endothelial cells (MEC) and in vivo with chicken combs. The dark cytotoxicity and photocytotoxicity of liposomal HB in MEC were evaluated using the MTT assay. Gross and histological examinations were performed to investigate the selective occlusion of the superficial dermal microvasculature in the chicken comb. The result showed that photocytotoxicity of liposomal HB was dependent on both light dose and drug concentration. PDT with HB (0.5-1 mg kg-1) and a light dose of 120 J cm-2 showed selective destruction of the superficial dermal microvasculature of the chicken comb, leaving the overlying epidermis intact. This is the first study to investigate the potential efficacy of HB-PDT as a novel modality for the treatment of PWS. These findings suggest that liposomal HB is a safe and effective photosensitizer for PWS.

  5. Modulation of human dermal microvascular endothelial cell and human gingival fibroblast behavior by micropatterned silica coating surfaces for zirconia dental implant applications

    PubMed Central

    Laranjeira, Marta S; Carvalho, Ângela; Pelaez-Vargas, Alejandro; Hansford, Derek; Ferraz, Maria Pia; Coimbra, Susana; Costa, Elísio; Santos-Silva, Alice; Fernandes, Maria Helena; Monteiro, Fernando Jorge

    2014-01-01

    Dental ceramic implants have shown superior esthetic behavior and the absence of induced allergic disorders when compared to titanium implants. Zirconia may become a potential candidate to be used as an alternative to titanium dental implants if surface modifications are introduced. In this work, bioactive micropatterned silica coatings were produced on zirconia substrates, using a combined methodology of sol–gel processing and soft lithography. The aim of the work was to compare the in vitro behavior of human gingival fibroblasts (HGFs) and human dermal microvascular endothelial cells (HDMECs) on three types of silica-coated zirconia surfaces: flat and micropatterned (with pillars and with parallel grooves). Our results showed that cells had a higher metabolic activity (HGF, HDMEC) and increased gene expression levels of fibroblast-specific protein-1 (FSP-1) and collagen type I (COL I) on surfaces with pillars. Nevertheless, parallel grooved surfaces were able to guide cell growth. Even capillary tube-like networks of HDMEC were oriented according to the surface geometry. Zirconia and silica with different topographies have shown to be blood compatible and silica coating reduced bacteria adhesion. All together, the results indicated that microstructured bioactive coating seems to be an efficient strategy to improve soft tissue integration on zirconia implants, protecting implants from peri-implant inflammation and improving long-term implant stabilization. This new approach of micropatterned silica coating on zirconia substrates can generate promising novel dental implants, with surfaces that provide physical cues to guide cells and enhance their behavior. PMID:27877662

  6. Post-mortem culture of Balamuthia mandrillaris from the brain and cerebrospinal fluid of a case of granulomatous amoebic meningoencephalitis, using human brain microvascular endothelial cells.

    PubMed

    Jayasekera, Samantha; Sissons, James; Tucker, Julie; Rogers, Claire; Nolder, Debbie; Warhurst, David; Alsam, Selwa; White, Jonathan M L; Higgins, E M; Khan, Naveed Ahmed

    2004-10-01

    The first isolation in the UK of Balamuthia mandrillaris amoebae from a fatal case of granulomatous amoebic meningoencephalitis is reported. Using primary cultures of human brain microvascular endothelial cells (HBMECs), amoebae were isolated from the brain and cerebrospinal fluid (CSF). The cultures showed a cytopathic effect at 20-28 days, but morphologically identifiable B. mandrillaris amoebae were seen in cleared plaques in subcultures at 45 days. The identification of the organism was later confirmed using PCR on Chelex-treated extracts. Serum taken while the patient was still alive reacted strongly with slide antigen prepared from cultures of the post-mortem isolate, and also with those from a baboon B. mandrillaris strain at 1:10,000 in indirect immunofluorescence, but with Acanthamoeba castellanii (Neff) at 1:160, supporting B. mandrillaris to be the causative agent. If the presence of amoebae in the post-mortem CSF reflects the condition in life, PCR studies on CSF and on biopsies of cutaneous lesions may also be a valuable tool. The role of HBMECs in understanding the interactions of B. mandrillaris with the blood-brain barrier is discussed.

  7. Effects of cinnamaldehyde on PGE2 release and TRPV4 expression in mouse cerebral microvascular endothelial cells induced by interleukin-1beta.

    PubMed

    Ma, Yue-Ying; Huo, Hai-Ru; Li, Cang-Hai; Zhao, Bao-Sheng; Li, Lan-Fang; Sui, Feng; Guo, Shu-Ying; Jiang, Ting-Liang

    2008-03-01

    Cinnamaldehyde is a principle compound isolated from Guizhi-Tang (GZT), which is a famous traditional Chinese medical formula used to treat influenza, common cold and other pyretic conditions. Transient receptor potential vanilloid subtype 4 (TRPV4) is expressed in the anterior hypothalamus and may act as thermosensor. The purpose of the present study was to investigate the effects of cinnamaldehyde on the production of prostaglandin E2 (PGE2) and the expression of TRPV4 in mouse cerebral microvascular endothelial cell strain (b.End3). In the research work, the b.End3 cells were cultured in DMEM medium containing interleukin-1beta (IL-1beta) in the presence or absence of ruthenium red (RR), a kind of known TRPV4 inhibitor, or different concentrations of cinnamaldehyde. The results suggested that IL-1beta significantly increase production of PGE2 and cinnamaldehyde evidently decrease IL-1beta-induced PGE2 production, while RR showed no inhibitory effect on PGE2 production. Moreover, it was identified that TRPV4 was expressed at the mRNA and protein levels in b.End3 cells. IL-1beta could up-regulate the expression of TRPV4, RR and cinnamaldehyde could down-regulate the high expression of mRNA and protein of TRPV4 by IL-1beta induced in b.End3 cells. In conclusion, cinnamaldehyde decreased the production of PGE2 and the expression of TRPV4 in b.End3 cells induced by IL-1beta.

  8. Oxidative Stress Induced by Cigarette Smoke Extracts in Human Brain Cells (T98G) and Human Brain Microvascular Endothelial Cells (HBMEC) in Mono- and Co-Culture.

    PubMed

    Kim, Ju-Hyeong; Cho, Myung-Haing; Choi, Kyung-Chul; Lee, Kyuhong; Kim, Kwang-Sik; Shim, Soon-Mi

    2015-01-01

    The objective of the current study was to examine oxidative stress induced by cigarette smoke extract (CSE) or cigarette smoke condensate (CSC) in human brain cells (T98G) and human brain microvascular endothelial cells (HBMEC) in mono- and co-culture systems. Cell viability of T98G cells exposed to CSC (0.05-4 mg/ml) was significantly decreased compared to CSE (0.025-20%). There were no marked differences between quantities of reactive oxygen species (ROS) generation by either CSE (2, 4, and 10%) or CSC (0.2, 0.4, and 0.8 mg/ml) treatment compared to control. However, a significant effect was noted in ROS generation following CSC incubation at 4mg/ml. Cellular integrity of HBMEC decreased to 74 and 64% within 120 h of exposure at the IC50 value of CSE and CSC, respectively. This study suggests that chronic exposure to cigarette smoking might initiate damage to the blood-brain barrier.

  9. Soluble mediators produced by the crosstalk between microvascular endothelial cells and dengue-infected primary dermal fibroblasts inhibit dengue virus replication and increase leukocyte transmigration.

    PubMed

    Bustos-Arriaga, José; Mita-Mendoza, Neida K; Lopez-Gonzalez, Moises; García-Cordero, Julio; Juárez-Delgado, Francisco J; Gromowski, Gregory D; Méndez-Cruz, René A; Fairhurst, Rick M; Whitehead, Stephen S; Cedillo-Barrón, Leticia

    2016-04-01

    When dengue virus (DENV)-infected mosquitoes use their proboscis to probe into human skin during blood feeding, both saliva and virus are released. During this process, cells from the epidermis and dermis layers of the skin, along with small blood vessels, may get exposed to or infected with DENV. In these microenvironments of the skin, the presence of DENV initiates a complex interplay among the DENV-infected and non-infected neighboring cells at the initial bite site. Previous studies suggested that DENV-infected human dermal fibroblasts (HDFs) participate in the immune response against DENV by secreting soluble mediators of innate immunity. In the present study, we investigated whether DENV-infected HDFs activate human dermal microvascular endothelial cells (HDMECs) in co-cultures. Our results suggest that co-cultures of DENV-infected HDFs and HDMECs elicit soluble mediators that are sufficient to reduce viral replication, activate HDMECs, and induce leukocyte migration through HDMEC monolayers. These effects were partly dependent on HDF donor and DENV serotype, which may provide novel insights into the natural variation in host susceptibility to DENV disease.

  10. In vitro model of cerebral ischemia by using brain microvascular endothelial cells derived from human induced pluripotent stem cells.

    PubMed

    Kokubu, Yasuhiro; Yamaguchi, Tomoko; Kawabata, Kenji

    2017-04-29

    Brain-derived microvascular endothelial cells (BMECs), which play a central role in blood brain barrier (BBB), can be used for the evaluation of drug transport into the brain. Although human BMEC cell lines have already been reported, they lack original properties such as barrier integrity. Pluripotent stem cells (PSCs) can be used for various applications such as regenerative therapy, drug screening, and pathological study. In the recent study, an induction method of BMECs from PSCs has been established, making it possible to more precisely study the in vitro human BBB function. Here, using induced pluripotent stem (iPS) cell-derived BMECs, we examined the effects of oxygen-glucose deprivation (OGD) and OGD/reoxygenation (OGD/R) on BBB permeability. OGD disrupted the barrier function, and the dysfunction was rapidly restored by re-supply of the oxygen and glucose. Interestingly, TNF-α, which is known to be secreted from astrocytes and microglia in the cerebral ischemia, prevented the restoration of OGD-induced barrier dysfunction in an apoptosis-independent manner. Thus, we could establish the in vitro BBB disease model that mimics the cerebral ischemia by using iPS cell-derived BMECs.

  11. Nitric oxide from brain microvascular endothelial cells may initiate the compensatory response to mild hypoxia of astrocytes in a hypoxia-inducible factor-1α dependent manner

    PubMed Central

    Shi, Qinghai; Liu, Xin; Wang, Ning; Zheng, Xinchuan; Fu, Jianfeng; Zheng, Jiang

    2016-01-01

    The physiological level of nitric oxide (NO) released by brain microvascular endothelial cells (BMECs) at normoxia can block the degradation of hypoxia-inducible factor-1α (HIF-1α) in astrocytes and initiate the compensatory response to hypoxia. However, it is unclear whether this occurs at mild hypoxia. This study was to investigate the expression of HIF-1α, VEGF and LDHA and the lactic acid production in astrocytes with or without co-culture with BMECs after mild hypoxia exposure. During mild hypoxia (5% O2), exogenous NO blocked the degradation of HIF-1α in astrocytes but up-regulated the transcription of VEGF and LDHA, accompanied by elevated expression of VEGF protein and increased production of lactic acid. This was further confirmed by silencing of HIF-1α expression in astrocytes. In astrocytes co-cultured with primary rat BMEC under mild hypoxia, NO was released by the BMECs and prevented the degradation of HIF-1α in astrocytes, leading to the up-regulated mRNA expression of VEGF and LDHA, elevated VEGF protein expression and increased production of lactic acid. In BMECs, NO was derived from intracellular eNOS. Based on these findings, we hypothesize that, under mild hypoxia, even though astrocytes do not respond to hypoxia, NO produced by BMECs may transmit a hypoxia signal to astrocytes, triggering their adaptive response via HIF-1α. PMID:27904676

  12. Differentially expressed genes of human microvascular endothelial cells in response to anti-dengue virus NS1 antibodies by suppression subtractive hybridization.

    PubMed

    Yin, Yue; Jiang, Lan; Fang, Danyun; Jiang, Lifang; Zhou, Junmei

    2013-06-01

    It has been previously shown that anti-dengue virus (DENV) nonstructural protein NS1 antibodies could act as autoantibodies that direct against one or more of the host's own proteins, which has potential implications for dengue hemorrhagic fever pathogenesis. In the present study, we have employed suppression subtractive hybridization (SSH) to identify the differentially expressed genes from human microvascular endothelial cells (HMEC-1) in response to anti-dengue virus type 2 NS1 antibodies (anti-DENV2 NS1 Abs). A total of 35 clones from the SSH cDNA library were randomly selected for further analysis using bioinformatics tools after vector screening. After searching for sequence homology in NCBI GenBank database with BLASTN and BLASTX programs, 23 obtained sequences with significant matches (E-values <1×10(-4)) in the SSH library. The predicted genes in the subtracted library include immune response molecules (CD59 antigen preproprotein preproprotein, MURR1), signal transduction molecules (Nuclear casein kinase and cyclin-dependent kinase substrate 1), calcium-binding proteins (S100A6, Annexin A2 isoform 1/2), and cell-membrane component (Yip1 domain family). From these clones, 5 upregulated genes were selected for differential expression profiling by real-time RT-PCR to confirm their upregulated status. The results confirmed their differential upregulation, and thus verified the success of SSHs and the likely involvement of these genes in dengue pathogenesis.

  13. Modulation of human dermal microvascular endothelial cell and human gingival fibroblast behavior by micropatterned silica coating surfaces for zirconia dental implant applications

    NASA Astrophysics Data System (ADS)

    Laranjeira, Marta S.; Carvalho, Ângela; Pelaez-Vargas, Alejandro; Hansford, Derek; Ferraz, Maria Pia; Coimbra, Susana; Costa, Elísio; Santos-Silva, Alice; Fernandes, Maria Helena; Monteiro, Fernando Jorge

    2014-04-01

    Dental ceramic implants have shown superior esthetic behavior and the absence of induced allergic disorders when compared to titanium implants. Zirconia may become a potential candidate to be used as an alternative to titanium dental implants if surface modifications are introduced. In this work, bioactive micropatterned silica coatings were produced on zirconia substrates, using a combined methodology of sol-gel processing and soft lithography. The aim of the work was to compare the in vitro behavior of human gingival fibroblasts (HGFs) and human dermal microvascular endothelial cells (HDMECs) on three types of silica-coated zirconia surfaces: flat and micropatterned (with pillars and with parallel grooves). Our results showed that cells had a higher metabolic activity (HGF, HDMEC) and increased gene expression levels of fibroblast-specific protein-1 (FSP-1) and collagen type I (COL I) on surfaces with pillars. Nevertheless, parallel grooved surfaces were able to guide cell growth. Even capillary tube-like networks of HDMEC were oriented according to the surface geometry. Zirconia and silica with different topographies have shown to be blood compatible and silica coating reduced bacteria adhesion. All together, the results indicated that microstructured bioactive coating seems to be an efficient strategy to improve soft tissue integration on zirconia implants, protecting implants from peri-implant inflammation and improving long-term implant stabilization. This new approach of micropatterned silica coating on zirconia substrates can generate promising novel dental implants, with surfaces that provide physical cues to guide cells and enhance their behavior.

  14. Three-dimensional cardiac microtissues composed of cardiomyocytes and endothelial cells co-differentiated from human pluripotent stem cells

    PubMed Central

    van Meer, Berend J.; Tertoolen, Leon G. J.

    2017-01-01

    ABSTRACT Cardiomyocytes and endothelial cells in the heart are in close proximity and in constant dialogue. Endothelium regulates the size of the heart, supplies oxygen to the myocardium and secretes factors that support cardiomyocyte function. Robust and predictive cardiac disease models that faithfully recapitulate native human physiology in vitro would therefore ideally incorporate this cardiomyocyte-endothelium crosstalk. Here, we have generated and characterized human cardiac microtissues in vitro that integrate both cell types in complex 3D structures. We established conditions for simultaneous differentiation of cardiomyocytes and endothelial cells from human pluripotent stem cells following initial cardiac mesoderm induction. The endothelial cells expressed cardiac markers that were also present in primary cardiac microvasculature, suggesting cardiac endothelium identity. These cell populations were further enriched based on surface markers expression, then recombined allowing development of beating 3D structures termed cardiac microtissues. This in vitro model was robustly reproducible in both embryonic and induced pluripotent stem cells. It thus represents an advanced human stem cell-based platform for cardiovascular disease modelling and testing of relevant drugs. PMID:28279973

  15. The NRF2 knockout rat: a new animal model to study endothelial dysfunction, oxidant stress, and microvascular rarefaction

    PubMed Central

    Priestley, Jessica R. C.; Kautenburg, Katie E.; Casati, Marc C.; Endres, Bradley T.; Geurts, Aron M.

    2015-01-01

    Nuclear factor (erythroid-derived 2)-like-2 (NRF2) is a master antioxidant and cell protective transcription factor that upregulates antioxidant defenses. In this study we developed a strain of Nrf2 null mutant rats to evaluate the role of reduced NRF2-regulated antioxidant defenses in contributing to endothelial dysfunction and impaired angiogenic responses during salt-induced ANG II suppression. Nrf2−/− mutant rats were developed using transcription activator-like effector nuclease technology in the Sprague-Dawley genetic background, and exhibited a 41-bp deletion that included the start codon for Nrf2 and an absence of immunohistochemically detectable NRF2 protein. Expression of mRNA for the NRF2-regulated indicator enzymes heme oxygenase-1, catalase, superoxide dismutase 1, superoxide dismutase 2, and glutathione reductase was significantly lower in livers of Nrf2−/− mutant rats fed high salt (HS; 4% NaCl) for 2 wk compared with wild-type controls. Endothelium-dependent dilation to acetylcholine was similar in isolated middle cerebral arteries (MCA) of Nrf2−/− mutant rats and wild-type littermates fed low-salt (0.4% NaCl) diet, and was eliminated by short-term (3 days) HS diet in both strains. Low-dose ANG II infusion (100 ng/kg sc) reversed salt-induced endothelial dysfunction in MCA and prevented microvessel rarefaction in wild-type rats fed HS diet, but not in Nrf2−/− mutant rats. The results of this study indicate that suppression of NRF2 antioxidant defenses plays an essential role in the development of salt-induced oxidant stress, endothelial dysfunction, and microvessel rarefaction in normotensive rats and emphasize the potential therapeutic benefits of directly upregulating NRF2-mediated antioxidant defenses to ameliorate vascular oxidant stress in humans. PMID:26637559

  16. Morphology of microvascular changes and endothelial regeneration in experimental ozone-induced parathyroiditis. III. Some pathologic considerations

    SciTech Connect

    Atwal, O.S.; Pemsingh, R.S.

    1981-03-01

    Proliferation of the vascular endothelium of the parathyroid glands of dogs exposed to ozone inhalation was studied. Direct observation of mitoses in the endothelial cells were made. Focal hemorrhages in the form of the presence of erythrocytes, clotting, and fibrin deposition in the extravascular spaces were seen. Ultrastructural analysis of transverse section of blood vessels showed intravascular platelet aggregation. Lymphoid cells were observed in the lumen of the blood vessels as well as in the perivascular areas as infiltrates. The possibility is discussed that present vascular reactions signify the morphologic equivalent of an immunologic response.

  17. Endothelial Nogo-B regulates sphingolipid biosynthesis to promote pathological cardiac hypertrophy during chronic pressure overload

    PubMed Central

    Huang, Yan; Azevedo, Paula S.; Siragusa, Mauro; Bielawski, Jacek; Giordano, Frank J.

    2016-01-01

    We recently discovered that endothelial Nogo-B, a membrane protein of the ER, regulates vascular function by inhibiting the rate-limiting enzyme, serine palmitoyltransferase (SPT), in de novo sphingolipid biosynthesis. Here, we show that endothelium-derived sphingolipids, particularly sphingosine-1-phosphate (S1P), protect the heart from inflammation, fibrosis, and dysfunction following pressure overload and that Nogo-B regulates this paracrine process. SPT activity is upregulated in banded hearts in vivo as well as in TNF-α–activated endothelium in vitro, and loss of Nogo removes the brake on SPT, increasing local S1P production. Hence, mice lacking Nogo-B, systemically or specifically in the endothelium, are resistant to the onset of pathological cardiac hypertrophy. Furthermore, pharmacological inhibition of SPT with myriocin restores permeability, inflammation, and heart dysfunction in Nogo-A/B–deficient mice to WT levels, whereas SEW2871, an S1P1 receptor agonist, prevents myocardial permeability, inflammation, and dysfunction in WT banded mice. Our study identifies a critical role of endothelial sphingolipid biosynthesis and its regulation by Nogo-B in the development of pathological cardiac hypertrophy and proposes a potential therapeutic target for the attenuation or reversal of this clinical condition. PMID:27158676

  18. LDL-Lipids from patients with hypercholesterolaemia and Alzheimer’s disease are inflammatory to microvascular endothelial cells: Mitigation by statin intervention

    PubMed Central

    Dias, H. K. I.; Brown, C. L. R.; Polidori, M. C.; Lip, G.Y.H.

    2016-01-01

    Elevated LDL concentration in mid-life increases the risk of developing Alzheimer’s disease (AD) in later life. Increased oxidative modification (oxLDL) and nitration is observed during dementia and hypercholesterolemia. We investigated the hypothesis that statin intervention in mid-life mitigates the inflammatory effects of oxLDL on the microvasculature. Human microvascular endothelial cells (HMVEC) were maintained on transwells to mimic the microvasculature and exposed to patient and control LDL. Blood was obtained from statin-naïve, normo- and hyperlipidaemic subjects, AD with vascular dementia (AD-plus) and AD subjects (n=10/group) at baseline. Only hyperlipidaemic subjects with normal cognitive function received 40mg simvastatin intervention/day for three months. Blood was re-analysed from normo- and hyper-lipidaemic subjects after three months. LDL isolated from statin-naïve hyperlipidaemic, AD and AD-plus subjects was more oxidised (agarose gel electrophoretic mobility, protein carbonyl content and 8-isoprostane F2α) compared to control subjects. Statin intervention decreased protein carbonyls (2.5±0.4 Vs 3.95±0.2nmol/mg; P<0.001) and 8-isoprostane F2α (30.4±4.0 pg/ml Vs 43.5±8.42 pg/ml; P<0.05). HMVEC treatment with LDL-lipids from hyperlipidaemic, AD and AD-plus subjects impaired endothelial tight junction expression and decreased total glutathione levels (AD; 18.61±1.3, AD-plus; 16.5±0.7nmol/mg protein) compared to untreated cells (23.8±1.2 vs nmol/mg protein). Basolateral IL-6 secretion was increased by LDL-lipids from hyperlipidaemic (78.4±1.9 pg/ml), AD (63.2±5.9 pg/ml) and AD-plus (80.8±0.9 pg/ml) groups compared to healthy subject lipids (18.6±3.6 pg/ml). LDL-Lipids isolated after statin intervention did not affect endothelial function. In summary, LDL-lipids from hypercholesterolaemic, AD and AD-plus patients are inflammatory to HMVEC. In vivo intervention with statins reduces the damaging effects of LDL-lipids on HMVEC. PMID

  19. p130Cas Scaffolds the Signalosome To Direct Adaptor-Effector Cross Talk during Kaposi's Sarcoma-Associated Herpesvirus Trafficking in Human Microvascular Dermal Endothelial Cells

    PubMed Central

    Bandyopadhyay, Chirosree; Veettil, Mohanan Valiya; Dutta, Sujoy

    2014-01-01

    ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) interacts with cell surface receptors, such as heparan sulfate, integrins (α3β1, αVβ3, and αVβ5), and EphrinA2 (EphA2), and activates focal adhesion kinase (FAK), Src, phosphoinositol 3-kinase (PI3-K), c-Cbl, and RhoA GTPase signal molecules early during lipid raft (LR)-dependent productive macropinocytic entry into human dermal microvascular endothelial cells. Our recent studies have identified CIB1 as a signal amplifier facilitating EphA2 phosphorylation and subsequent cytoskeletal cross talk during KSHV macropinocytosis. Although CIB1 lacks an enzymatic activity and traditional adaptor domain or known interacting sequence, it associated with the KSHV entry signal complex and the CIB1-KSHV association was sustained over 30 min postinfection. To identify factors scaffolding the EphA2-CIB1 signal axis, the role of major cellular scaffold protein p130Cas (Crk-associated substrate of Src) was investigated. Inhibitor and small interfering RNA (siRNA) studies demonstrated that KSHV induced p130Cas in an EphA2-, CIB1-, and Src-dependent manner. p130Cas and Crk were associated with KSHV, LRs, EphA2, and CIB1 early during infection. Live-cell microscopy and biochemical studies demonstrated that p130Cas knockdown did not affect KSHV entry but significantly reduced productive nuclear trafficking of viral DNA and routed KSHV to lysosomal degradation. p130Cas aided in scaffolding adaptor Crk to downstream guanine nucleotide exchange factor phospho-C3G possibly to coordinate GTPase signaling during KSHV trafficking. Collectively, these studies demonstrate that p130Cas acts as a bridging molecule between the KSHV-induced entry signal complex and the downstream trafficking signalosome in endothelial cells and suggest that simultaneous targeting of KSHV entry receptors with p130Cas would be an attractive potential avenue for therapeutic intervention in KSHV infection. IMPORTANCE Eukaryotic cell adaptor molecules

  20. Endothelial Cell Death, Angiogenesis, and Microvascular Function after Castration in an Androgen-Dependent Tumor: Role of Vascular Endothelial Growth Factor

    NASA Astrophysics Data System (ADS)

    Jain, Rakesh K.; Safabakhsh, Nina; Sckell, Axel; Chen, Yi; Jiang, Ping; Benjamin, Laura; Yuan, Fan; Keshet, Eli

    1998-09-01

    The sequence of events that leads to tumor vessel regression and the functional characteristics of these vessels during hormone--ablation therapy are not known. This is because of the lack of an appropriate animal model and monitoring technology. By using in vivo microscopy and in situ molecular analysis of the androgen-dependent Shionogi carcinoma grown in severe combined immunodeficient mice, we show that castration of these mice leads to tumor regression and a concomitant decrease in vascular endothelial growth factor (VEGF) expression. Androgen withdrawal is known to induce apoptosis in Shionogi tumor cells. Surprisingly, tumor endothelial cells begin to undergo apoptosis before neoplastic cells, and rarefaction of tumor vessels precedes the decrease in tumor size. The regressing vessels begin to exhibit normal phenotype, i.e., lower diameter, tortuosity, vascular permeability, and leukocyte adhesion. Two weeks after castration, a second wave of angiogenesis and tumor growth begins with a concomitant increase in VEGF expression. Because human tumors often relapse following hormone--ablation therapy, our data suggest that these patients may benefit from combined anti-VEGF therapy.

  1. Circulating Endothelial Cells and Endothelial Function predict Major Adverse Cardiac Events and Early Adverse Left Ventricular Remodeling in Patients with ST-Segment Elevation Myocardial Infarction

    PubMed Central

    Magdy, Abdel Hamid; Bakhoum, Sameh; Sharaf, Yasser; Sabry, Dina; El-Gengehe, Ahmed T; Abdel-Latif, Ahmed

    2016-01-01

    Endothelial progenitor cells (EPCs) and circulating endothelial cells (CECs) are mobilized from the bone marrow and increase in the early phase after ST-elevation myocardial infarction (STEMI). The aim of this study was to assess the prognostic significance of CECs and indices of endothelial dysfunction in patients with STEMI. In 78 patients with acute STEMI, characterization of CD34+/VEGFR2+ CECs, and indices of endothelial damage/dysfunction such as brachial artery flow mediated dilatation (FMD) were determined. Blood samples for CECs assessment and quantification were obtained within 24 hours of admission and FMD was assessed during the index hospitalization. At 30 days follow up, the primary composite end point of major cardiac adverse events (MACE) consisting of all-cause mortality, recurrent non-fatal MI, or heart failure and the secondary endpoint of early adverse left ventricular (LV) remodeling were analyzed. The 17 patients (22%) who developed MACE had significantly higher CEC level (P = 0.004), vWF level (P =0.028), and significantly lower FMD (P = 0.006) compared to the remaining patients. Logistic regression analysis showed that CECs level and LV ejection fraction were independent predictors of MACE. The areas under the receiver operating characteristic curves (ROC) for CEC level, FMD, and the logistic model with both markers were 0.73, 0.75, and 0.82 respectively for prediction of the MACE. The 16 patients who developed the secondary endpoint had significantly higher CEC level compared to remaining patients (p =0.038). In conclusion, increased circulating endothelial cells and endothelial dysfunction predicted the occurrence of major adverse cardiac events and adverse cardiac remodeling in patients with STEMI. PMID:26864952

  2. Hcp family proteins secreted via the type VI secretion system coordinately regulate Escherichia coli K1 interaction with human brain microvascular endothelial cells.

    PubMed

    Zhou, Yan; Tao, Jing; Yu, Hao; Ni, Jinjing; Zeng, Lingbing; Teng, Qihui; Kim, Kwang Sik; Zhao, Guo-Ping; Guo, Xiaokui; Yao, Yufeng

    2012-03-01

    Type VI secretion systems (T6SSs) are involved in the pathogenicity of several gram-negative bacteria. Based on sequence analysis, we found that a cluster of Escherichia coli virulence factors (EVF) encoding a putative T6SS exists in the genome of the meningitis-causing E. coli K1 strain RS218. The T6SS-associated deletion mutants exhibited significant defects in binding to and invasion of human brain microvascular endothelial cells (HBMEC) compared with the parent strain. Hcp family proteins (the hallmark of T6SS), including Hcp1 and Hcp2, were localized in the bacterial outer membrane, but the involvements of Hcp1 and Hcp2 have been shown to differ in E. coli-HBMEC interaction. The deletion mutant of hcp2 showed defects in the bacterial binding to and invasion of HBMEC, while Hcp1 was secreted in a T6SS-dependent manner and induced actin cytoskeleton rearrangement, apoptosis, and the release of interleukin-6 (IL-6) and IL-8 in HBMEC. These findings demonstrate that the T6SS is functional in E. coli K1, and two Hcp family proteins participate in different steps of E. coli interaction with HBMEC in a coordinate manner, e.g., binding to and invasion of HBMEC, the cytokine and chemokine release followed by cytoskeleton rearrangement, and apoptosis in HBMEC. This is the first demonstration of the role of T6SS in meningitis-causing E. coli K1, and T6SS-associated Hcp family proteins are likely to contribute to the pathogenesis of E. coli meningitis.

  3. Development and Validation of an In-Cell Western for Quantifying P-Glycoprotein Expression in Human Brain Microvascular Endothelial (hCMEC/D3) Cells.

    PubMed

    McInerney, Mitchell P; Pan, Yijun; Short, Jennifer L; Nicolazzo, Joseph A

    2017-01-05

    An in-cell western (ICW) protocol detecting the relative expression of P-glycoprotein (P-gp) in human cerebro-microvascular endothelial cells (hCMEC/D3) was developed and optimized, with the intention of improving throughput relative to western blotting (WB). For validation of the ICW protocol, hCMEC/D3 cells were incubated with known P-gp upregulators (10 μM rifampicin and 5 μM SR12813) and treated with siRNA targeted against MDR1, before measuring changes in P-gp expression, using both ICW and WB in parallel. To confirm a relationship between the detected P-gp expression and function, the uptake of the P-gp substrate rhodamine-123 was assessed following SR12813 treatment. Rifampicin and SR12813 significantly upregulated P-gp expression (1.5-fold and 1.9-fold, respectively) compared to control, as assessed by the ICW protocol. WB analysis of the same treatments revealed 1.4-fold and 1.5-fold upregulations. MDR1 siRNA reduced P-gp abundance by 20% and 35% when assessed by ICW and WB, respectively. SR12813 treatment reduced rhodamine-123 uptake by 18%, indicating that the observed changes in P-gp expression by ICW were associated with comparable functional changes. The correlation of P-gp upregulation by WB, rhodamine-123 uptake, and the ICW protocol provide validation of a new ICW method as an alternative method for quantification of P-gp in hCMEC/D3 cells.

  4. Mast cells positive to tryptase, endothelial cells positive to protease-activated receptor-2, and microvascular density correlate among themselves in hepatocellular carcinoma patients who have undergone surgery

    PubMed Central

    Ammendola, Michele; Sacco, Rosario; Sammarco, Giuseppe; Piardi, Tullio; Zuccalà, Valeria; Patruno, Rosa; Zullo, Alessandra; Zizzo, Nicola; Nardo, Bruno; Marech, Ilaria; Crovace, Alberto; Gadaleta, Cosmo Damiano; Pessaux, Patrick; Ranieri, Girolamo

    2016-01-01

    Background Mast cells (MCs) can stimulate angiogenesis, releasing several proangiogenic cytokines stored in their cytoplasm. In particular MCs can release tryptase, a potent in vivo and in vitro proangiogenic factor via proteinase-activated receptor-2 (PAR-2) activation and mitogen-activated protein kinase phosphorylation. Nevertheless, no data are available concerning the relationship between MC density positive to tryptase (MCDPT), endothelial cells positive to PAR-2 forming microvascular density (PAR-2-MVD), and classical MVD (C-MVD) in hepatocellular carcinoma (HCC) angiogenesis. This study analyzed the correlation between MCDPT, PAR-2-MVD, and C-MVD, each correlated to the others and to the main clinicopathological features, in early HCC patients who underwent surgery. Methods A series of 53 HCC patients with early stage (stage 0 according to the Barcelona Clinic Liver Cancer Staging Classification) were selected and then underwent surgery. Tumor tissue samples were evaluated by means of immunohistochemistry and image analysis methods in terms of number of MCDPT, PAR-2-MVD, and C-MVD. Results A significant correlation between MCDPT, PAR-2-MVD, and C-MVD groups, each correlated to the others, was found by Pearson t-test analysis (r ranged from 0.67 to 0.81; P-value ranged from 0.01 to 0.03). No other significant correlation was found. Conclusion Our in vivo pilot data suggest that MCDPT and PAR-2-MVD may play a role in HCC angiogenesis and could be further evaluated as a target of antiangiogenic therapy. PMID:27499640

  5. Aryl hydrocarbon Receptor is Necessary to Protect Fetal Human Pulmonary Microvascular Endothelial Cells against Hyperoxic Injury: Mechanistic Roles of Antioxidant Enzymes and RelB

    PubMed Central

    Zhang, Shaojie; Patel, Ananddeep; Chu, Chun; Jiang, Weiwu; Wang, Lihua; Welty, Stephen E.; Moorthy, Bhagavatula; Shivanna, Binoy

    2015-01-01

    Hyperoxia contributes to the development of bronchopulmonary dysplasia (BPD) in premature infants. Activation of the aryl hydrocarbon receptor (AhR) protects adult and newborn mice against hyperoxic lung injury by mediating increases in the expression of phase I (cytochrome P450 (CYP) 1A) and phase II (NADP(H) quinone oxidoreductase (NQO1)) antioxidant enzymes (AOE). AhR positively regulates the expression of RelB, a component of the nuclear factor-kappaB (NF-κB) protein that contributes to anti-inflammatory processes in adult animals. Whether AhR regulates the expression of AOE and RelB, and protects fetal primary human lung cells against hyperoxic injury is unknown. Therefore, we tested the hypothesis that AhR-deficient fetal human pulmonary microvascular endothelial cells (HPMEC) will have decreased RelB activation and AOE, which will in turn predispose them to increased oxidative stress, inflammation, and cell death compared to AhR-sufficient HPMEC upon exposure to hyperoxia. AhR-deficient HPMEC showed increased hyperoxia-induced reactive oxygen species (ROS) generation, cleavage of poly (ADP-ribose) polymerase (PARP), and cell death compared to AhR-sufficient HPMEC. Additionally, AhR-deficient cell culture supernatants displayed increased macrophage inflammatory protein 1α and 1β, indicating a heightened inflammatory state. Interestingly, loss of AhR was associated with a significantly attenuated CYP1A1, NQO1, superoxide dismutase 1(SOD1), and nuclear RelB protein expression. These findings support the hypothesis that decreased RelB activation and AOE in AhR-deficient cells is associated with increased hyperoxic injury compared to AhR-sufficient cells. PMID:25831079

  6. Brain Invasion by Mouse Hepatitis Virus Depends on Impairment of Tight Junctions and Beta Interferon Production in Brain Microvascular Endothelial Cells

    PubMed Central

    Bleau, Christian; Filliol, Aveline; Samson, Michel

    2015-01-01

    ABSTRACT Coronaviruses (CoVs) have shown neuroinvasive properties in humans and animals secondary to replication in peripheral organs, but the mechanism of neuroinvasion is unknown. The major aim of our work was to evaluate the ability of CoVs to enter the central nervous system (CNS) through the blood-brain barrier (BBB). Using the highly hepatotropic mouse hepatitis virus type 3 (MHV3), its attenuated variant, 51.6-MHV3, which shows low tropism for endothelial cells, and the weakly hepatotropic MHV-A59 strain from the murine coronavirus group, we investigated the virus-induced dysfunctions of BBB in vivo and in brain microvascular endothelial cells (BMECs) in vitro. We report here a MHV strain-specific ability to cross the BBB during acute infection according to their virulence for liver. Brain invasion was observed only in MHV3-infected mice and correlated with enhanced BBB permeability associated with decreased expression of zona occludens protein 1 (ZO-1), VE-cadherin, and occludin, but not claudin-5, in the brain or in cultured BMECs. BBB breakdown in MHV3 infection was not related to production of barrier-dysregulating inflammatory cytokines or chemokines by infected BMECs but rather to a downregulation of barrier protective beta interferon (IFN-β) production. Our findings highlight the importance of IFN-β production by infected BMECs in preserving BBB function and preventing access of blood-borne infectious viruses to the brain. IMPORTANCE Coronaviruses (CoVs) infect several mammals, including humans, and are associated with respiratory, gastrointestinal, and/or neurological diseases. There is some evidence that suggest that human respiratory CoVs may show neuroinvasive properties. Indeed, the severe acute respiratory syndrome coronavirus (SARS-CoV), causing severe acute respiratory syndrome, and the CoVs OC43 and 229E were found in the brains of SARS patients and multiple sclerosis patients, respectively. These findings suggest that hematogenously spread

  7. The Interaction between Circulating Complement Proteins and Cutaneous Microvascular Endothelial Cells in the Development of Childhood Henoch-Schönlein Purpura

    PubMed Central

    Yang, Yao-Hsu; Tsai, I-Jung; Chang, Chun-Jung; Chuang, Ya-Hui; Hsu, Hui-Yao; Chiang, Bor-Luen

    2015-01-01

    Objective In addition to IgA, the deposition of complement (C)3 in dermal vessels is commonly found in Henoch-Schönlein purpura (HSP). The aim of this study is to elucidate the role of circulating complement proteins in the pathogenesis of childhood HSP. Methods Plasma levels of C3a, C4a, C5a, and Bb in 30 HSP patients and 30 healthy controls were detected by enzyme-linked immunosorbent assay (ELISA). The expression of C3a receptor (C3aR), C5a receptor (CD88), E-selectin, intercellular adhesion molecule 1 (ICAM-1), C3, C5, interleukin (IL)-8, monocyte chemotactic protein (MCP)-1, and RANTES by human dermal microvascular endothelial cells (HMVEC-d) was evaluated either by flow cytometry or by ELISA. Results At the acute stage, HSP patients had higher plasma levels of C3a (359.5 ± 115.3 vs. 183.3 ± 94.1 ng/ml, p < 0.0001), C5a (181.4 ± 86.1 vs. 33.7 ± 26.3 ng/ml, p < 0.0001), and Bb (3.7 ± 2.6 vs. 1.0 ± 0.6 μg/ml, p < 0.0001), but not C4a than healthy controls. Although HSP patient-derived acute phase plasma did not alter the presentation of C3aR and CD88 on HMVEC-d, it enhanced the production of endothelial C3 and C5. Moreover, C5a was shown in vitro to up-regulate the expression of IL-8, MCP-1, E-selectin, and ICAM-1 by HMVEC-d with a dose-dependent manner. Conclusion In HSP, the activation of the complement system in part through the alternative pathway may have resulted in increased plasma levels of C3a and C5a, which, especially C5a, may play a role in the disease pathogenesis by activating endothelium of cutaneous small vessels. PMID:25760949

  8. Cold Pressor Stress Cardiac Magnetic Resonance Myocardial Flow Reserve Is Not Useful for Detection of Coronary Endothelial Dysfunction in Women with Signs and Symptoms of Ischemia and No Obstructive CAD

    PubMed Central

    Landes, Sofy; Dela Cruz, Sherwin; Wei, Janet; AlBadri, Ahmed; Shufelt, Chrisandra; Mehta, Puja; Thomson, Louise E.; Diniz, Marcio A.; Zhang, Xiao; Petersen, John W.; Anderson, R. David; Pepine, Carl J.; Berman, Daniel S.; Bairey Merz, C. Noel

    2017-01-01

    Background Coronary endothelial function testing using acetylcholine is not routinely available, while non-pharmacological cold pressor testing (CPT) is considered an endothelial stressor. Noninvasive cardiac magnetic resonance imaging (CMRI) myocardial perfusion reserve index (MPRI) can detect coronary microvascular dysfunction (CMD). We evaluated if CPT stress CMRI MPRI could detect invasive coronary endothelial dysfunction. Methods Coronary reactivity testing was performed in 189 women with symptoms and signs of ischemic but no obstructive coronary artery disease as previously described plus CPT stress. Subjects also underwent pharmacologic and CPT stress during CMRI (1.5 T). Statistical analysis comparing CPT MPRI between groups was performed by Welch`s t-test and Mann-Whitney where appropriate. Anderson-Darling test and Levene test were considered to verify the normality and homogeneity of variances assumptions. Correlation analyses between CPT MPRI and both invasive and noninvasive measures of CMD were performed using Spearman correlation. Results While CPT MPRI correlated with pharmacological stress MPRI, it did not correlate with invasive measures of CMD including invasively measured responses to intracoronary (IC) adenosine, IC acetylcholine, CPT, or IC nitroglycerin. Additionally CPT MPRI was not significantly different between subjects with normal compared to abnormal pharm stress MPRI or normal compared to abnormal invasive CMD parameters. Conclusion Despite correlation with pharmacological stress MPRI, non-invasive CPT MPRI does not appear to be useful for detecting CMD in symptomatic women. PMID:28081214

  9. Genome-wide transcription profile of endothelial cells after cardiac transplantation in the rat.

    PubMed

    Mikalsen, B; Fosby, B; Wang, J; Hammarström, C; Bjaerke, H; Lundström, M; Kasprzycka, M; Scott, H; Line, P-D; Haraldsen, G

    2010-07-01

    Transcriptome analyses of organ transplants have until now usually focused on whole tissue samples containing activation profiles from different cell populations. Here, we enriched endothelial cells from rat cardiac allografts and isografts, establishing their activation profile at baseline and on days 2, 3 and 4 after transplantation. Modulated transcripts were assigned to three categories based on their regulation profile in allografts and isografts. Categories A and B contained the majority of transcripts and showed similar regulation in both graft types, appearing to represent responses to surgical trauma. By contrast, category C contained transcripts that were partly allograft-specific and to a large extent associated with interferon-gamma-responsiveness. Several transcripts were verified by immunohistochemical analysis of graft lesions, among them the matricellular protein periostin, which was one of the most highly upregulated transcripts but has not been associated with transplantation previously. In conclusion, the majority of the differentially expressed genes in graft endothelial cells are affected by the transplantation procedure whereas relatively few are associated with allograft rejection.

  10. 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.

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

    PubMed Central

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

    2016-01-01

    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. PMID:26813587

  12. Listeriolysin O mediates cytotoxicity against human brain microvascular

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Penetration of the brain microvascular endothelial layer is one of the routes L. monocytogenes use to breach the blood-brain barrier. Because host factors in the blood severely limit direct invasion of human brain microvascular endothelial cells (HBMECs) by L. monocytogenes, alternative mechanisms m...

  13. Aryl hydrocarbon receptor is necessary to protect fetal human pulmonary microvascular endothelial cells against hyperoxic injury: Mechanistic roles of antioxidant enzymes and RelB

    SciTech Connect

    Zhang, Shaojie; Patel, Ananddeep; Chu, Chun; Jiang, Weiwu; Wang, Lihua; Welty, Stephen E.; Moorthy, Bhagavatula; Shivanna, Binoy

    2015-07-15

    Hyperoxia contributes to the development of bronchopulmonary dysplasia (BPD) in premature infants. Activation of the aryl hydrocarbon receptor (AhR) protects adult and newborn mice against hyperoxic lung injury by mediating increases in the expression of phase I (cytochrome P450 (CYP) 1A) and phase II (NADP(H) quinone oxidoreductase (NQO1)) antioxidant enzymes (AOE). AhR positively regulates the expression of RelB, a component of the nuclear factor-kappaB (NF-κB) protein that contributes to anti-inflammatory processes in adult animals. Whether AhR regulates the expression of AOE and RelB, and protects fetal primary human lung cells against hyperoxic injury is unknown. Therefore, we tested the hypothesis that AhR-deficient fetal human pulmonary microvascular endothelial cells (HPMEC) will have decreased RelB activation and AOE, which will in turn predispose them to increased oxidative stress, inflammation, and cell death compared to AhR-sufficient HPMEC upon exposure to hyperoxia. AhR-deficient HPMEC showed increased hyperoxia-induced reactive oxygen species (ROS) generation, cleavage of poly(ADP-ribose) polymerase (PARP), and cell death compared to AhR-sufficient HPMEC. Additionally, AhR-deficient cell culture supernatants displayed increased macrophage inflammatory protein 1α and 1β, indicating a heightened inflammatory state. Interestingly, loss of AhR was associated with a significantly attenuated CYP1A1, NQO1, superoxide dismutase 1(SOD1), and nuclear RelB protein expression. These findings support the hypothesis that decreased RelB activation and AOE in AhR-deficient cells is associated with increased hyperoxic injury compared to AhR-sufficient cells. - Highlights: • AhR deficiency potentiates oxygen toxicity in human fetal lung cells. • Deficient AhR signaling increases hyperoxia-induced cell death. • AhR deficiency increases hyperoxia-induced ROS generation and inflammation. • Anti-oxidant enzyme levels are attenuated in AhR-deficient lung cells

  14. Evidence of myocardial scarring and microvascular obstruction on cardiac magnetic resonance imaging in a series of patients presenting with myocardial infarction without obstructed coronary arteries.

    PubMed

    Hermens, Jeannine A J M; van Es, Jan; von Birgelen, Clemens; Op den Akker, Jeroen W; Wagenaar, Lodewijk J

    2014-08-01

    Patients with acute chest pain, electrocardiographic ST-elevation and significant elevation of cardiac troponin but without obstructive coronary artery disease represent a diagnostic and therapeutic dilemma. Cardiac magnetic resonance imaging (CMR) can elucidate underlying alternative causes of troponin elevation including detection of (minor) myocardial infarction (MI) by identifying myocardial scarring as delayed enhancement. Of 77 patients, who were admitted between March 2009 and December 2012 with electrocardiographic (ECG) and biochemical evidence of acute MI without obstructive coronary artery disease, 45 patients underwent CMR that showed in 11/77 (14%) late gadolinium enhancement (LGE), compatible with myocardial scarring. We analyzed clinical, echocardiographic, and CMR data of these patients. Elevated troponin I levels were observed in all patients (median 1.3 ng/l, IQR 0.44-187) with median peak creatinine phosphokinase of 485 U/l (IQR 234-618). Echocardiographic wall motion abnormalities were detected in 8/11 (73%) patients; in 75% of these segments, ECG abnormalities were observed in corresponding leads. CMR detected LGE in the inferior (4/11), the inferolateral (5/11), the inferoseptal (2/11), the anterior (3/11), apical (3/11) and in the lateral segments (2/11). In addition, in all but two patients, these segments matched ECG abnormalities in corresponding leads. CMR identified microvascular obstruction in 4/11 (36%) patients. Patients with clinical, ECG, and biochemical signs of acute MI but unobstructed coronary arteries may have CMR-detectable myocardial scars. Information on myocardial scarring may help to make the diagnosis and draw therapeutic consequences. This case series underlines the value of contrast-enhanced CMR for myocardial tissue characterization.

  15. Serum from Diesel Exhaust-Exposed Rats with Cardiac Dysfunction Alters Aortic Endothelial Cell Function In Vitro: Circulating Mediators as Causative Factors?

    EPA Science Inventory

    Although circulating inflammatory mediators are strongly associated with adverse cardiovascular outcomes triggered by inhaled air pollution, direct cause-effect linkage has not been established. Given that endothelial toxicity often precedes and precipitates cardiac dysfunction, ...

  16. Endothelial, cardiac muscle and skeletal muscle exhibit different viscous and elastic properties as determined by atomic force microscopy

    NASA Technical Reports Server (NTRS)

    Mathur, A. B.; Collinsworth, A. M.; Reichert, W. M.; Kraus, W. E.; Truskey, G. A.

    2001-01-01

    This study evaluated the hypothesis that, due to functional and structural differences, the apparent elastic modulus and viscous behavior of cardiac and skeletal muscle and vascular endothelium would differ. To accurately determine the elastic modulus, the contribution of probe velocity, indentation depth, and the assumed shape of the probe were examined. Hysteresis was observed at high indentation velocities arising from viscous effects. Irreversible deformation was not observed for endothelial cells and hysteresis was negligible below 1 microm/s. For skeletal muscle and cardiac muscle cells, hysteresis was negligible below 0.25 microm/s. Viscous dissipation for endothelial and cardiac muscle cells was higher than for skeletal muscle cells. The calculated elastic modulus was most sensitive to the assumed probe geometry for the first 60 nm of indentation for the three cell types. Modeling the probe as a blunt cone-spherical cap resulted in variation in elastic modulus with indentation depth that was less than that calculated by treating the probe as a conical tip. Substrate contributions were negligible since the elastic modulus reached a steady value for indentations above 60 nm and the probe never indented more than 10% of the cell thickness. Cardiac cells were the stiffest (100.3+/-10.7 kPa), the skeletal muscle cells were intermediate (24.7+/-3.5 kPa), and the endothelial cells were the softest with a range of elastic moduli (1.4+/-0.1 to 6.8+/-0.4 kPa) depending on the location of the cell surface tested. Cardiac and skeletal muscle exhibited nonlinear elastic behavior. These passive mechanical properties are generally consistent with the function of these different cell types.

  17. Rosetting Plasmodium falciparum-infected erythrocytes bind to human brain microvascular endothelial cells in vitro, demonstrating a dual adhesion phenotype mediated by distinct P. falciparum erythrocyte membrane protein 1 domains.

    PubMed

    Adams, Yvonne; Kuhnrae, Pongsak; Higgins, Matthew K; Ghumra, Ashfaq; Rowe, J Alexandra

    2014-03-01

    Adhesion interactions between Plasmodium falciparum-infected erythrocytes (IE) and human cells underlie the pathology of severe malaria. IE cytoadhere to microvascular endothelium or form rosettes with uninfected erythrocytes to survive in vivo by sequestering IE in the microvasculature and avoiding splenic clearance mechanisms. Both rosetting and cytoadherence are mediated by the parasite-derived IE surface protein family Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). Rosetting and cytoadherence have been widely studied as separate entities; however, the ability of rosetting P. falciparum strains to cytoadhere has received little attention. Here, we show that IE of the IT/R29 strain expressing a rosette-mediating PfEMP1 variant (IT4var09) cytoadhere in vitro to a human brain microvascular endothelial cell line (HBEC-5i). Cytoadherence was inhibited by heparin and by treatment of HBEC-5i with heparinase III, suggesting that the endothelial receptors for IE binding are heparan sulfate proteoglycans. Antibodies to the N-terminal regions of the IT4var09 PfEMP1 variant (NTS-DBL1α and DBL2γ domains) specifically inhibited and reversed cytoadherence down to low concentrations (<10 μg/ml of total IgG). Surface plasmon resonance experiments showed that the NTS-DBLα and DBL2γ domains bind strongly to heparin, with half-maximal binding at a concentration of ∼0.5 μM in both cases. Therefore, cytoadherence of IT/R29 IE is distinct from rosetting, which is primarily mediated by NTS-DBL1α interactions with complement receptor 1. These data show that IT4var09-expressing parasites are capable of dual interactions with both endothelial cells and uninfected erythrocytes via distinct receptor-ligand interactions.

  18. Quantitative evaluation of endothelial progenitors and cardiac valve endothelial cells: proliferation and differentiation on poly-glycolic acid/poly-4-hydroxybutyrate scaffold in response to vascular endothelial growth factor and transforming growth factor beta1.

    PubMed

    Dvorin, Evan L; Wylie-Sears, Jill; Kaushal, Sunjay; Martin, David P; Bischoff, Joyce

    2003-06-01

    Three-dimensional scaffolds made of bioabsorbable polymeric constituents are currently being tested for use in tissue engineering of various tissues. A composite scaffold of poly-glycolic acid (PGA) non-woven mesh dip-coated in a 1% solution of poly-4-hydroxybutyrate (P4HB) was shown to be suitable as a scaffold for creation of tissue-engineered trileaflet pulmonic valve replacements in sheep [Hoerstrup, S.P., et al., Circulation 102(Suppl. 3), III44, 2000]. However, little is known about how cells seeded on PGA/P4HB respond in vitro to soluble factors supplied in the culture medium. To optimize tissue development in vitro, before implantation, we set out to develop quantitative biochemical assays to measure how cells seeded on PGA/P4HB respond to growth and differentiation factors. Herein we show that ovine aortic valvular endothelial cells and circulating endothelial progenitor cells (EPCs) seeded onto PGA/P4HB proliferate in response to vascular endothelial growth factor and transdifferentiate to a mesenchymal phenotype in response to transforming growth factor beta(1). Transdifferentiation from an endothelial to mesenchymal phenotype is a critical step during embryonic development of cardiac valves. Our results demonstrate that valvular endothelial cells and EPCs isolated from peripheral blood can recapitulate critical developmental steps on PGA/P4HB. These results demonstrate that PGA/P4HB provides a conducive environment for cellular proliferation, differentiation, and tissue development.

  19. Simultaneous Non-Invasive Assessment of Systemic and Coronary Endothelial Function Iantorno et al: Cardiac MRI and Endothelial Function

    PubMed Central

    Schär, Michael; Krishnaswamy, Rupa; Soleimanifard, Sahar; Steinberg, Angela; Stuber, Matthias; Gerstenblith, Gary; Weiss, Robert G.

    2016-01-01

    Background Normal endothelial function is a measure of vascular health and dysfunction a predictor of coronary events. Nitric Oxide (NO)-mediated coronary artery endothelial function (CEF), as assessed by vasomotor reactivity during isometric handgrip exercise (IHE), was recently quantified noninvasively with MRI. Because the internal mammary artery (IMA) is often visualized during coronary MRI we propose the strategy of simultaneously assessing systemic and coronary endothelial function noninvasively by MRI during IHE. Methods and Results Changes in cross-sectional area (CSA) and blood flow (BF) in the right coronary artery (RCA) and the IMA in 25 CAD patients and 26 healthy subjects during IHE were assessed using 3T MRI. In 8 healthy subjects a NO synthase inhibitor was infused to evaluate the role of NO in the IMA-IHE response. Inter-observer IMA-IHE reproducibility was good for CSA (R=0.91) and BF (R=0.91). In healthy subjects, CSA and BF of the IMA increased during IHE and these responses were significantly attenuated by L-NMMA (p<0.01 vs. placebo). In CAD patients, the RCA did not dilate with IHE and dilation of the IMA was less than that of the healthy subjects (p=0.01). The BF responses of both the RCA and IMA to IHE were also significantly reduced in CAD patients. Conclusions MRI-detected IMA responses to IHE primarily reflect NO-dependent endothelial function, are reproducible and reduced in CAD patients. Endothelial function in both coronary and systemic (IMA) arteries can now be measured noninvasively with the same imaging technique and promises novel insights into systemic and local factors affecting vascular health. PMID:26919997

  20. Phospholipase Cε Modulates Rap1 Activity and the Endothelial Barrier

    PubMed Central

    DiStefano, Peter V.; Smrcka, Alan V.; Glading, Angela J.

    2016-01-01

    The phosphoinositide-specific phospholipase C, PLCε, is a unique signaling protein with known roles in regulating cardiac myocyte growth, astrocyte inflammatory signaling, and tumor formation. PLCε is also expressed in endothelial cells, however its role in endothelial regulation is not fully established. We show that endothelial cells of multiple origins, including human pulmonary artery (HPAEC), human umbilical vein (HUVEC), and immortalized brain microvascular (hCMEC/D3) endothelial cells, express PLCε. Knockdown of PLCε in arterial endothelial monolayers decreased the effectiveness of the endothelial barrier. Concomitantly, RhoA activity and stress fiber formation were increased. PLCε-deficient arterial endothelial cells also exhibited decreased Rap1-GTP levels, which could be restored by activation of the Rap1 GEF, Epac, to rescue the increase in monolayer leak. Reintroduction of PLCε rescued monolayer leak with both the CDC25 GEF domain and the lipase domain of PLCε required to fully activate Rap1 and to rescue endothelial barrier function. Finally, we demonstrate that the barrier promoting effects PLCε are dependent on Rap1 signaling through the Rap1 effector, KRIT1, which we have previously shown is vital for maintaining endothelial barrier stability. Thus we have described a novel role for PLCε PIP2 hydrolytic and Rap GEF activities in arterial endothelial cells, where PLCε-dependent activation of Rap1/KRIT1 signaling promotes endothelial barrier stability. PMID:27612188

  1. Phospholipase Cε Modulates Rap1 Activity and the Endothelial Barrier.

    PubMed

    DiStefano, Peter V; Smrcka, Alan V; Glading, Angela J

    2016-01-01

    The phosphoinositide-specific phospholipase C, PLCε, is a unique signaling protein with known roles in regulating cardiac myocyte growth, astrocyte inflammatory signaling, and tumor formation. PLCε is also expressed in endothelial cells, however its role in endothelial regulation is not fully established. We show that endothelial cells of multiple origins, including human pulmonary artery (HPAEC), human umbilical vein (HUVEC), and immortalized brain microvascular (hCMEC/D3) endothelial cells, express PLCε. Knockdown of PLCε in arterial endothelial monolayers decreased the effectiveness of the endothelial barrier. Concomitantly, RhoA activity and stress fiber formation were increased. PLCε-deficient arterial endothelial cells also exhibited decreased Rap1-GTP levels, which could be restored by activation of the Rap1 GEF, Epac, to rescue the increase in monolayer leak. Reintroduction of PLCε rescued monolayer leak with both the CDC25 GEF domain and the lipase domain of PLCε required to fully activate Rap1 and to rescue endothelial barrier function. Finally, we demonstrate that the barrier promoting effects PLCε are dependent on Rap1 signaling through the Rap1 effector, KRIT1, which we have previously shown is vital for maintaining endothelial barrier stability. Thus we have described a novel role for PLCε PIP2 hydrolytic and Rap GEF activities in arterial endothelial cells, where PLCε-dependent activation of Rap1/KRIT1 signaling promotes endothelial barrier stability.

  2. Fumaric Acid Esters Do Not Reduce Inflammatory NF-κB/p65 Nuclear Translocation, ICAM-1 Expression and T-Cell Adhesiveness of Human Brain Microvascular Endothelial Cells.

    PubMed

    Haarmann, Axel; Nehen, Mathias; Deiß, Annika; Buttmann, Mathias

    2015-08-13

    Dimethyl fumarate (DMF) is approved for disease-modifying treatment of patients with relapsing-remitting multiple sclerosis. Animal experiments suggested that part of its therapeutic effect is due to a reduction of T-cell infiltration of the central nervous system (CNS) by uncertain mechanisms. Here we evaluated whether DMF and its primary metabolite monomethyl fumarate (MMF) modulate pro-inflammatory intracellular signaling and T-cell adhesiveness of nonimmortalized single donor human brain microvascular endothelial cells at low passages. Neither DMF nor MMF at concentrations of 10 or 50 µM blocked the IL-1β-induced nuclear translocation of NF-κB/p65, whereas the higher concentration of DMF inhibited the nuclear entry of p65 in human umbilical vein endothelium cultured in parallel. DMF and MMF also did not alter the IL-1β-stimulated activation of p38 MAPK in brain endothelium. Furthermore, neither DMF nor MMF reduced the basal or IL-1β-inducible expression of ICAM-1. In accordance, both fumaric acid esters did not reduce the adhesion of activated Jurkat T cells to brain endothelium under basal or inflammatory conditions. Therefore, brain endothelial cells probably do not directly mediate a potential blocking effect of fumaric acid esters on the inflammatory infiltration of the CNS by T cells.

  3. Salvianolic acid B improves the disruption of high glucose-mediated brain microvascular endothelial cells via the ROS/HIF-1α/VEGF and miR-200b/VEGF signaling pathways.

    PubMed

    Yang, Ming-Chao; You, Fu-Li; Wang, Zhe; Liu, Xiang-Nan; Wang, Yan-Feng

    2016-09-06

    The study investigated the roles and mechanisms of Salvianolic acid B (Sal B) on permeability of rat brain microvascular endothelial cells (RBMECs) exposed to high glucose. The results demonstrated that Sal B greatly up-regulated the expression of tight junction (TJ) proteins and decreased the permeability of RBMECs compared with the control group. And the increase of reactive oxidative species (ROS) production, the upregulation of hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) protein induced by high glucose were antagonized by Sal B. In addition, a great decrease of microRNA-200b (miR-200b) was observed in the RBMECs under high-glucose condition, which was significantly increased by Sal B pretreatment. And overexpression of miR-200b markedly attenuated the RBMECs permeability and inhibited the expression of VEGF protein by targeting with 3'-UTR of its mRNA. This led to the conclusion that Sal B-mediated improvement of blood-brain barrier dysfunction induced by high-glucose is related to the ROS/HIF-1α/VEGF and miR-200b/VEGF signaling pathways.

  4. Circulating factors induced by caloric restriction in the nonhuman primate Macaca mulatta activate angiogenic processes in endothelial cells.

    PubMed

    Csiszar, Anna; Sosnowska, Danuta; Tucsek, Zsuzsanna; Gautam, Tripti; Toth, Peter; Losonczy, Gyorgy; Colman, Ricki J; Weindruch, Richard; Anderson, Rozalyn M; Sonntag, William E; Ungvari, Zoltan

    2013-03-01

    Moderate caloric restriction (CR) without malnutrition increases healthspan in virtually every species studied, including nonhuman primates. In mice, CR exerts significant microvascular protective effects resulting in increased microvascular density in the heart and the brain, which likely contribute to enhanced tolerance to ischemia and improved cardiac performance and cognitive function. Yet, the underlying mechanisms by which CR confer microvascular protection remain elusive. To test the hypothesis that circulating factors triggered by CR regulate endothelial angiogenic capacity, we treated cultured human endothelial cells with sera derived from Macaca mulatta on long-term (over 10 years) CR. Cells treated with sera derived from ad-libitum-fed control monkeys served as controls. We found that factors present in CR sera upregulate vascular endothelial growth factor (VEGF) signaling and stimulate angiogenic processes, including endothelial cell proliferation and formation of capillary-like structures. Treatment with CR sera also tended to increase cellular migration (measured by a wound-healing assay using electric cell-substrate impedance sensing [ECIS] technology) and adhesion to collagen. Collectively, we find that circulating factors induced by CR promote endothelial angiogenic processes, suggesting that increased angiogenesis may be a potential mechanism by which CR improves cardiac function and prevents vascular cognitive impairment.

  5. West Nile virus-induced cell adhesion molecules on human brain microvascular endothelial cells regulate leukocyte adhesion and modulate permeability of the in vitro blood-brain barrier model.

    PubMed

    Roe, Kelsey; Orillo, Beverly; Verma, Saguna

    2014-01-01

    Characterizing the mechanisms by which West Nile virus (WNV) causes blood-brain barrier (BBB) disruption, leukocyte infiltration into the brain and neuroinflammation is important to understand the pathogenesis of WNV encephalitis. Here, we examined the role of endothelial cell adhesion molecules (CAMs) in mediating the adhesion and transendothelial migration of leukocytes across human brain microvascular endothelial cells (HBMVE). Infection with WNV (NY99 strain) significantly induced ICAM-1, VCAM-1, and E-selectin in human endothelial cells and infected mice brain, although the levels of their ligands on leukocytes (VLA-4, LFA-1and MAC-1) did not alter. The permeability of the in vitro BBB model increased dramatically following the transmigration of monocytes and lymphocytes across the models infected with WNV, which was reversed in the presence of a cocktail of blocking antibodies against ICAM-1, VCAM-1, and E-selectin. Further, WNV infection of HBMVE significantly increased leukocyte adhesion to the HBMVE monolayer and transmigration across the infected BBB model. The blockade of these CAMs reduced the adhesion and transmigration of leukocytes across the infected BBB model. Further, comparison of infection with highly neuroinvasive NY99 and non-lethal (Eg101) strain of WNV demonstrated similar level of virus replication and fold-increase of CAMs in HBMVE cells suggesting that the non-neuropathogenic response of Eg101 is not because of its inability to infect HBMVE cells. Collectively, these results suggest that increased expression of specific CAMs is a pathological event associated with WNV infection and may contribute to leukocyte infiltration and BBB disruption in vivo. Our data further implicate that strategies to block CAMs to reduce BBB disruption may limit neuroinflammation and virus-CNS entry via 'Trojan horse' route, and improve WNV disease outcome.

  6. Cardiovascular Action of Insulin in Health and Disease: Endothelial L-Arginine Transport and Cardiac Voltage-Dependent Potassium Channels

    PubMed Central

    Dubó, Sebastián; Gallegos, David; Cabrera, Lissette; Sobrevia, Luis; Zúñiga, Leandro; González, Marcelo

    2016-01-01

    Impairment of insulin signaling on diabetes mellitus has been related to cardiovascular dysfunction, heart failure, and sudden death. In human endothelium, cationic amino acid transporter 1 (hCAT-1) is related to the synthesis of nitric oxide (NO) and insulin has a vascular effect in endothelial cells through a signaling pathway that involves increases in hCAT-1 expression and L-arginine transport. This mechanism is disrupted in diabetes, a phenomenon potentiated by excessive accumulation of reactive oxygen species (ROS), which contribute to lower availability of NO and endothelial dysfunction. On the other hand, electrical remodeling in cardiomyocytes is considered a key factor in heart failure progression associated to diabetes mellitus. This generates a challenge to understand the specific role of insulin and the pathways involved in cardiac function. Studies on isolated mammalian cardiomyocytes have shown prolongated action potential in ventricular repolarization phase that produces a long QT interval, which is well explained by attenuation in the repolarizing potassium currents in cardiac ventricles. Impaired insulin signaling causes specific changes in these currents, such a decrease amplitude of the transient outward K+ (Ito) and the ultra-rapid delayed rectifier (IKur) currents where, together, a reduction of mRNA and protein expression levels of α-subunits (Ito, fast; Kv 4.2 and IKs; Kv 1.5) or β-subunits (KChIP2 and MiRP) of K+ channels involved in these currents in a MAPK mediated pathway process have been described. These results support the hypothesis that lack of insulin signaling can produce an abnormal repolarization in cardiomyocytes. Furthermore, the arrhythmogenic potential due to reduced Ito current can contribute to an increase in the incidence of sudden death in heart failure. This review aims to show, based on pathophysiological models, the regulatory function that would have insulin in vascular system and in cardiac electrophysiology. PMID

  7. Endothelial actions of atrial and B-type natriuretic peptides

    PubMed Central

    Kuhn, Michaela

    2012-01-01

    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

  8. 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

  9. Vascular endothelial growth factor blockade promotes the transition from compensatory cardiac hypertrophy to failure in response to pressure overload.

    PubMed

    Izumiya, Yasuhiro; Shiojima, Ichiro; Sato, Kaori; Sawyer, Douglas B; Colucci, Wilson S; Walsh, Kenneth

    2006-05-01

    Cardiac hypertrophy is associated with upregulation of vascular endothelial growth factor (VEGF) in the myocardium. Here, we evaluated the effects of a decoy VEGF receptor on heart morphology and function to a murine model of pressure overload hypertrophy. Mice were administered adenoviral vector encoding a decoy VEGF receptor (Ad-Flk), and their hearts were subjected to pressure overload by transverse aortic constriction (TAC). Treatment with Ad-Flk led to a net reduction in capillary density in hearts subjected to TAC. Ad-Flk also led to a reduction in TAC-induced cardiac hypertrophy and promoted left ventricle dilatation and a loss in contractile function. Treatment with Ad-Flk markedly increased myocardial fibrosis and collagen gene upregulation. In contrast, Ad-Flk had no effect on any of these parameters in sham-treated mice. Administration of a VEGF trap reagent diminished pressure overload cardiac hypertrophy and promoted the progression to heart failure but had no effect on sham-treated animals. These findings suggest that VEGF is required to maintain myocardial capillary density and that reductions in the vascular bed are associated with the transition from compensatory hypertrophy to failure.

  10. Low intensity exercise prevents disturbances in rat cardiac insulin signaling and endothelial nitric oxide synthase induced by high fructose diet.

    PubMed

    Stanišić, Jelena; Korićanac, Goran; Ćulafić, Tijana; Romić, Snježana; Stojiljković, Mojca; Kostić, Milan; Pantelić, Marija; Tepavčević, Snežana

    2016-01-15

    Increase in fructose consumption together with decrease in physical activity contributes to the development of metabolic syndrome and consequently cardiovascular diseases. The current study examined the preventive role of exercise on defects in cardiac insulin signaling and function of endothelial nitric oxide synthase (eNOS) in fructose fed rats. Male Wistar rats were divided into control, sedentary fructose (received 10% fructose for 9 weeks) and exercise fructose (additionally exposed to low intensity exercise) groups. Concentration of triglycerides, glucose, insulin and visceral adipose tissue weight were determined to estimate metabolic syndrome development. Expression and/or phosphorylation of cardiac insulin receptor (IR), insulin receptor substrate 1 (IRS1), tyrosine-specific protein phosphatase 1B (PTP1B), Akt, extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) and eNOS were evaluated. Fructose overload increased visceral adipose tissue, insulin concentration and homeostasis model assessment index. Exercise managed to decrease visceral adiposity and insulin level and to increase insulin sensitivity. Fructose diet increased level of cardiac PTP1B and pIRS1 (Ser307), while levels of IR and ERK1/2, as well as pIRS1 (Tyr 632), pAkt (Ser473, Thr308) and pERK1/2 were decreased. These disturbances were accompanied by reduced phosphorylation of eNOS at Ser1177. Exercise managed to prevent most of the disturbances in insulin signaling caused by fructose diet (except phosphorylation of IRS1 at Tyr 632 and phosphorylation and protein expression of ERK1/2) and consequently restored function of eNOS. Low intensity exercise could be considered as efficient treatment of cardiac insulin resistance induced by fructose diet.

  11. GATA6 Promotes Angiogenic Function and Survival in Endothelial Cells by Suppression of Autocrine Transforming Growth Factor β/Activin Receptor-like Kinase 5 Signaling*

    PubMed Central

    Froese, Natali; Kattih, Badder; Breitbart, Astrid; Grund, Andrea; Geffers, Robert; Molkentin, Jeffery D.; Kispert, Andreas; Wollert, Kai C.; Drexler, Helmut; Heineke, Joerg

    2011-01-01

    Understanding the transcriptional regulation of angiogenesis could lead to the identification of novel therapeutic targets. We showed here that the transcription factor GATA6 is expressed in different human primary endothelial cells as well as in vascular endothelial cells of mice in vivo. Activation of endothelial cells was associated with GATA6 nuclear translocation, chromatin binding, and enhanced GATA6-dependent transcriptional activation. siRNA-mediated down-regulation of GATA6 after growth factor stimulation led to a dramatically reduced capacity of macro- and microvascular endothelial cells to proliferate, migrate, or form capillary-like structures on Matrigel. Adenoviral overexpression of GATA6 in turn enhanced angiogenic function, especially in cardiac endothelial microvascular cells. Furthermore, GATA6 protected endothelial cells from undergoing apoptosis during growth factor deprivation. Mechanistically, down-regulation of GATA6 in endothelial cells led to increased expression of transforming growth factor (TGF) β1 and TGFβ2, whereas enhanced GATA6 expression, accordingly, suppressed Tgfb1 promoter activity. High TGFβ1/β2 expression in GATA6-depleted endothelial cells increased the activation of the activin receptor-like kinase 5 (ALK5) and SMAD2, and suppression of this signaling axis by TGFβ neutralizing antibody or ALK5 inhibition restored angiogenic function and survival in endothelial cells with reduced GATA6 expression. Together, these findings indicate that GATA6 plays a crucial role for endothelial cell function and survival, at least in part, by suppressing autocrine TGFβ expression and ALK5-dependent signaling. PMID:21127043

  12. Calyculin A Reveals Serine/Threonine Phosphatase Protein Phosphatase 1 as a Regulatory Nodal Point in Canonical Signal Transducer and Activator of Transcription 3 Signaling of Human Microvascular Endothelial Cells

    PubMed Central

    Zgheib, Carlos; Zouein, Fouad A.; Chidiac, Rony; Kurdi, Mazen

    2012-01-01

    Vascular inflammation is initiated by stimuli acting on endothelial cells. A clinical feature of vascular inflammation is increased circulating interleukin 6 (IL-6) type cytokines such as leukemia inhibitory factor (LIF), but their role in vascular inflammation is not fully defined. IL-6 type cytokines activate transcription factor signal transducer and activator of transcription 3 (STAT3), which has a key role in inflammation and the innate immune response. Canonical STAT3 gene induction is due to phosphorylation of (1) Y705, leading to STAT3 dimerization and DNA binding and (2) S727, enhancing homodimerization and DNA binding by recruiting p300/CBP. We asked whether enhancing S727 STAT3 phosphorylation using the protein phosphatase 1 (PP1) inhibitor, calyculin A, would enhance LIF-induced gene expression in human microvascular endothelial cells (HMEC-1). Cotreatment with calyculin A and LIF markedly increased STAT3 S727 phosphorylation, without affecting the increase in the nuclear fraction of STAT3 phosphorylated on Y705. PP2A inhibitors, okadaic acid and fostriecin, did not enhance STAT3 S727 phosphorylation. Surprisingly, calyculin A eliminated LIF-induced gene expression: (1) calyculin A reduced binding of nuclear extracts to a STAT3 consensus site, thereby reducing the overall level of binding observed with LIF; and (2) calyculin A caused p300/CBP phosphorylation, thus resulting in reduced acetylation activity and degradation. Together, these findings reveal a pivotal role of a protein serine/threonine phosphatases that is likely PP1 in HMEC in controlling STAT3 transcriptional activity. PMID:22142222

  13. Donor antibodies to HNA-3a implicated in TRALI reactions prime neutrophils and cause PMN-mediated damage to human pulmonary microvascular endothelial cells in a two-event in vitro model.

    PubMed

    Silliman, Christopher C; Curtis, Brian R; Kopko, Patricia M; Khan, Samina Y; Kelher, Marguerite R; Schuller, Randy M; Sannoh, Baindu; Ambruso, Daniel R

    2007-02-15

    Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related mortality. Antibodies to HNA-3a are commonly implicated in TRALI. We hypothesized that HNA-3a antibodies prime neutrophils (PMNs) and cause PMN-mediated cytotoxicity through a two-event pathogenesis. Isolated HNA-3a+ or HNA-3a- PMNs were incubated with plasma containing HNA-3a antibodies implicated in TRALI, and their ability to prime the oxidase was measured. Human pulmonary microvascular endothelial cells (HMVECs) were activated with endotoxin or buffer, HNA-3a+ or HNA-3a- PMNs were added, and the coculture was incubated with plasma+/-antibodies to HNA-3a. PMN-mediated damage was measured by counting viable HMVECs/mm2. Plasma containing HNA-3a antibodies primed the fMLP-activated respiratory burst of HNA-3a+, but not HNA-3a-, PMNs and elicited PMN-mediated damage of LPS-activated HMVECs when HNA-3a+, but not HNA-3a-, PMNs were used. Thus, antibodies to HNA-3a primed PMNs and caused PMN-mediated HMVEC cytotoxicity in a two-event model identical to biologic response modifiers implicated in TRALI.

  14. Endothelial cells are progenitors of cardiac pericytes and vascular smooth muscle cells

    PubMed Central

    Chen, Qi; Zhang, Hui; Liu, Yang; Adams, Susanne; Eilken, Hanna; Stehling, Martin; Corada, Monica; Dejana, Elisabetta; Zhou, Bin; Adams, Ralf H.

    2016-01-01

    Mural cells of the vessel wall, namely pericytes and vascular smooth muscle cells, are essential for vascular integrity. The developmental sources of these cells and molecular mechanisms controlling their progenitors in the heart are only partially understood. Here we show that endocardial endothelial cells are progenitors of pericytes and vascular smooth muscle cells in the murine embryonic heart. Endocardial cells undergo endothelial–mesenchymal transition and convert into primitive mesenchymal progenitors expressing the platelet-derived growth factor receptors, PDGFRα and PDGFRβ. These progenitors migrate into the myocardium, differentiate and assemble the wall of coronary vessels, which requires canonical Wnt signalling involving Frizzled4, β-catenin and endothelial cell-derived Wnt ligands. Our findings identify a novel and unexpected population of progenitors for coronary mural cells with potential relevance for heart function and disease conditions. PMID:27516371

  15. Neisseria meningitidis causes cell cycle arrest of human brain microvascular endothelial cells at S phase via p21 and cyclin G2.

    PubMed

    Oosthuysen, Wilhelm F; Mueller, Tobias; Dittrich, Marcus T; Schubert-Unkmeir, Alexandra

    2016-01-01

    Microbial pathogens have developed several mechanisms to modulate and interfere with host cell cycle progression. In this study, we analysed the effect of the human pathogen Neisseria meningitidis on cell cycle in a brain endothelial cell line as well as in primary brain endothelial cells. We found that N.  Meningitidis causes an accumulation of cells in the S phase early at 3 and at 24 h post-infection that was paralleled by a decrease of cells in G2/M phase. Importantly, the outer membrane proteins of the colony opacity-associated (Opa) protein family as well as the Opc protein proved to trigger the accumulation of cells in the S phase. A focused cell cycle reverse transcription quantitative polymerase chain reaction-based array and integrated network analysis revealed changes in the abundance of several cell cycle regulatory mRNAs, including the cell cycle inhibitors p21(WAF1/CIP1) and cyclin G2. These alterations were reflected in changes in protein expression levels and/or relocalization in N. meningitidis-infected cells. Moreover, an increase in p21(WAF1/CIP1) expression was found to be p53 independent. Genetic ablation of p21(WAF1/CIP1) and cyclin G2 abrogated N. meningitidis-induced S phase accumulation. Finally, by measuring the levels of the biomarker 8-hydroxydeoxyguanosine and phosphorylation of the histone variant H2AX, we provide evidence that N. meningitidis induces oxidative DNA damage in infected cells.

  16. Roles of LOX-1 in microvascular dysfunction.

    PubMed

    Lubrano, Valter; Balzan, Silvana

    2016-05-01

    Studies from human and animal models with metabolic disease and hypertension highlight atrophic remodeling, reduced lumen size and thinner vascular walls of microvessels with profound density reduction. This impaired vascular response limits the perfusion of peripheral tissues inducing organ damage. These conditions are strongly associated with oxidative stress and in particular with the up-regulation of lectin-like oxidized low density lipoprotein receptor-1 (LOX-1). Several factors such as cytokines, shear stress, and advanced glycation end-products, especially oxLDL, can up-regulate LOX-1. The activation of this receptor induces the production of adhesion molecules, cytokines and the release of reactive oxygen species via NADPH oxidase. LOX-1 is considered a potent mediator of endothelial dysfunction and it is significantly associated with reduced microvascular endothelium NO-dependent vasodilation in hypercholesterolemia and hypertension. Microvascular endothelial cells increased the expression of IL-6 in association with the increased concentration of LDL and its degree of oxidation. Moreover, increased IL-6 levels are associated with up-regulation of LOX-1 in a dose-dependent manner. Another consequence of microvascular inflammation is the generation of small amounts of ROS, similar to those induced by low concentration of oxLDL (<5 μg/mL) which induces capillary tube formation of endothelial cells, through LOX-1 up-regulation. In light of its central role, LOX-1 represents an attractive therapeutic target for the treatment of human atherosclerotic diseases and microvascular disorders.

  17. Hypoxia augments the calcium-activated chloride current carried by anoctamin-1 in cardiac vascular endothelial cells of neonatal mice

    PubMed Central

    Wu, Ming-Ming; Lou, Jie; Song, Bin-Lin; Gong, Yuan-Feng; Li, Yan-Chao; Yu, Chang-Jiang; Wang, Qiu-Shi; Ma, Tian-Xing; Ma, Ke; Hartzell, H Criss; Duan, Dayue Darrel; Zhao, Dan; Zhang, Zhi-Ren

    2014-01-01

    BACKGROUND AND PURPOSE The molecular identity of calcium-activated chloride channels (CaCCs) in vascular endothelial cells remains unknown. This study sought to identify whether anoctamin-1 (Ano1, also known as TMEM16A) functions as a CaCC and whether hypoxia alters the biophysical properties of Ano1 in mouse cardiac vascular endothelial cells (CVECs). EXPERIMENTAL APPROACH Western blot, quantitative real-time PCR, confocal imaging analysis and patch-clamp analysis combined with pharmacological approaches were used to determine whether Ano1 was expressed and functioned as CaCC in CVECs. KEY RESULTS Ano1 was expressed in CVECs. The biophysical properties of the current generated in the CVECs, including the Ca2+ and voltage dependence, outward rectification, anion selectivity and the pharmacological profile, are similar to those described for CaCCs. The density of ICl(Ca) detected in CVECs was significantly inhibited by T16Ainh-A01, an Ano1 inhibitor, and a pore-targeting, specific anti-Ano1 antibody, and was markedly decreased in Ano1 gene knockdown CVECs. The density of ICl(Ca) was significantly potentiated in CVECs exposed to hypoxia, and this hypoxia-induced increase in the density of ICl(Ca) was inhibited by T16Ainh-A01 or anti-Ano1 antibody. Hypoxia also increased the current density of ICl(Ca) in Ano1 gene knockdown CVECs. CONCLUSIONS AND IMPLICATIONS Ano1 formed CaCC in CVECs of neonatal mice. Hypoxia enhances Ano1-mediated ICl(Ca) density via increasing its expression, altering the ratio of its splicing variants, sensitivity to membrane voltage and to Ca2+. Ano1 may play a role in the pathophysiological processes during ischaemia in heart, and therefore, Ano1 might be a potential therapeutic target to prevent ischaemic damage. PMID:24758567

  18. TNF-α stimulates endothelial palmitic acid transcytosis and promotes insulin resistance

    PubMed Central

    Li, Wenjing; Yang, Xiaoyan; Zheng, Tao; Xing, Shasha; Wu, Yaogong; Bian, Fang; Wu, Guangjie; Li, Ye; Li, Juyi; Bai, Xiangli; Wu, Dan; Jia, Xiong; Wang, Ling; Zhu, Lin; Jin, Si

    2017-01-01

    Persistent elevation of plasma TNF-α is a marker of low grade systemic inflammation. Palmitic acid (PA) is the most abundant type of saturated fatty acid in human body. PA is bound with albumin in plasma and could not pass through endothelial barrier freely. Albumin-bound PA has to be transported across monolayer endothelial cells through intracellular transcytosis, but not intercellular diffusion. In the present study, we discovered that TNF-α might stimulate PA transcytosis across cardiac microvascular endothelial cells, which further impaired the insulin-stimulated glucose uptake by cardiomyocytes and promoted insulin resistance. In this process, TNF-α-stimulated endothelial autophagy and NF-κB signaling crosstalk with each other and orchestrate the whole event, ultimately result in increased expression of fatty acid transporter protein 4 (FATP4) in endothelial cells and mediate the increased PA transcytosis across microvascular endothelial cells. Hopefully the present study discovered a novel missing link between low grade systemic inflammation and insulin resistance. PMID:28304381

  19. miR-146a Attenuates Inflammatory Pathways Mediated by TLR4/NF-κB and TNFα to Protect Primary Human Retinal Microvascular Endothelial Cells Grown in High Glucose

    PubMed Central

    Ye, Eun-Ah; Steinle, Jena J.

    2016-01-01

    Pathological mechanisms underlying diabetic retinopathy are still not completely understood. Increased understanding of potential cellular pathways responsive to hyperglycemia is essential to develop novel therapeutic strategies for diabetic retinopathy. A growing body of evidence shows that microRNA (miRNA) play important roles in pathological mechanisms involved in diabetic retinopathy, as well as possessing potential as novel therapeutic targets. The hypothesis of this study was that miR-146a plays a key role in attenuating hyperglycemia-induced inflammatory pathways through reduced TLR4/NF-κB and TNFα signaling in primary human retinal microvascular endothelial cells (REC). We cultured human REC in normal (5 mM) glucose or transferred to high glucose medium (25 mM) for 3 days. Transfection was performed on REC with miRNA mimic (hsa-miR-146a-5p). Our results demonstrate that miR-146a expression was decreased in human REC cultured in high glucose. Overexpression of miR-146a using mimics reduced the levels of TLR4/NF-κB and TNFα in REC cultured in high glucose. Both MyD88-dependent and -independent signaling were decreased by miR-146a overexpression in REC in high glucose conditions. The results suggest that miR-146a is a potential therapeutic target for reducing inflammation in REC through inhibition of TLR4/NF-κB and TNFα. Our study will contribute to understanding of diabetic retinal pathology, as well as providing important clues to develop therapeutics for clinical applications. PMID:26997759

  20. Vascular Endothelial Growth Factor-Delivery Systems for Cardiac Repair: An Overview

    PubMed Central

    Simón-Yarza, Teresa; Formiga, Fabio R.; Tamayo, Esther; Pelacho, Beatriz; Prosper, Felipe; Blanco-Prieto, María J.

    2012-01-01

    Since the discovery of the Vascular Endothelial Growth Factor (VEGF) and its leading role in the angiogenic process, this has been seen as a promising molecule for promoting neovascularization in the infarcted heart. However, even though several clinical trials were initiated, no therapeutic effects were observed, due in part to the short half life of this factor when administered directly to the tissue. In this context, drug delivery systems appear to offer a promising strategy to overcome limitations in clinical trials of VEGF. The aim of this paper is to review the principal drug delivery systems that have been developed to administer VEGF in cardiovascular disease. Studies published in the last 5 years are reviewed and the main features of these systems are explained. The tissue engineering concept is introduced as a therapeutic alternative that holds promise for the near future. PMID:22737191

  1. Multiple Functions of Glutamate Uptake via Meningococcal GltT-GltM l-Glutamate ABC Transporter in Neisseria meningitidis Internalization into Human Brain Microvascular Endothelial Cells

    PubMed Central

    Yanagisawa, Tatsuo; Kim, Kwang Sik; Yokoyama, Shigeyuki; Ohnishi, Makoto

    2015-01-01

    We previously reported that Neisseria meningitidis internalization into human brain microvasocular endothelial cells (HBMEC) was triggered by the influx of extracellular l-glutamate via the GltT-GltM l-glutamate ABC transporter, but the underlying mechanism remained unclear. We found that the ΔgltT ΔgltM invasion defect in assay medium (AM) was alleviated in AM without 10% fetal bovine serum (FBS) [AM(−S)]. The alleviation disappeared again in AM(−S) supplemented with 500 μM glutamate. Glutamate uptake by the ΔgltT ΔgltM mutant was less efficient than that by the wild-type strain, but only upon HBMEC infection. We also observed that both GltT-GltM-dependent invasion and accumulation of ezrin, a key membrane-cytoskeleton linker, were more pronounced when N. meningitidis formed larger colonies on HBMEC under physiological glutamate conditions. These results suggested that GltT-GltM-dependent meningococcal internalization into HBMEC might be induced by the reduced environmental glutamate concentration upon infection. Furthermore, we found that the amount of glutathione within the ΔgltT ΔgltM mutant was much lower than that within the wild-type N. meningitidis strain only upon HBMEC infection and was correlated with intracellular survival. Considering that the l-glutamate obtained via GltT-GltM is utilized as a nutrient in host cells, l-glutamate uptake via GltT-GltM plays multiple roles in N. meningitidis internalization into HBMEC. PMID:26099588

  2. Loss of microvascular negative charges accompanied by interstitial edema in septic rats' heart.

    PubMed

    Gotloib, L; Shostak, A; Galdi, P; Jaichenko, J; Fudin, R

    1992-01-01

    We studied the effect of Gram-negative sepsis on negative charges of heart capillaries and myocardial cells. We used a rat model of multiorgan failure, with ruthenium red (RR) and polyethyleneimine (PEI) as cationic binding tracers. Twenty-four hours after induction of sepsis, negative charges had decreased in glycocalyx and basement membrane of myocardial capillary endothelial cells. There were substantial amounts of interstitial edema. Density of anionic charges in the sarcolemmal glycocalyx complex of cardiac cells was markedly reduced. Myocardial cells' mitochondria consistently showed morphologic changes, whose severity ranged between stages II and IV C of Trump. Thirteen days after induction of sepsis, capillary endothelial and myocardial cells had recovered almost completely and showed no intracellular edema. Gram-negative sepsis caused a significant reduction in negative charges normally present in the microvascular wall as well as on myocardial cells. Consequently, several membranes limiting the various compartments of heart tissue lost their structural integrity. This morphometric data could explain the development of protein-rich interstitial edema and defective cell volume regulation observed in cardiac muscle of endotoxin-shocked animals. This myocardial edema may be at the origin of the cardiac dysfunction observed in both experimental and human septic shock.

  3. Tongxinluo (TXL), a Traditional Chinese Medicinal Compound, Improves Endothelial Function After Chronic Hypoxia Both In Vivo and In Vitro

    PubMed Central

    Zheng, Cui-Ying; Song, Li-Li; Wen, Jin-Kun; Li, Li-Min; Guo, Zong-Wei; Zhou, Pei-Pei; Wang, Chang; Li, Yong-Hui; Ma, Dong

    2015-01-01

    Abstract: Vascular injury after chronic hypoxia leads to endothelial injury and structural damage to tight junctions (TJs), thereby resulting in a variety of cardiovascular diseases. Thus, attenuating hypoxia-induced damage has great significance for the prevention and treatment of cardiovascular disease. The aim of this study was to investigate whether the endothelial protection conferred by tongxinluo (TXL), a traditional Chinese medicinal compound, is related to its regulation of TJ protein expression. In vivo, we found that TXL could promote hypoxia-induced angiogenesis in lung and liver tissue. In vitro, we found that CoCl2 treatment significantly reduced the expression of the TJ proteins occludin, claudin-1, VE-cadherin, and beta-catenin in cultured human cardiac microvascular endothelial cells. TXL pretreatment abrogated the CoCl2-induced downregulation of these TJ proteins. Conversely, overexpression of Krüppel-like factor 4 (KLF4) inhibited the expression of TJ proteins in human cardiac microvascular endothelial cells, an effect that was reversed by TXL pretreatment. Further experiments showed that TXL could promote endothelial cell proliferation by increasing KLF4 phosphorylation, thereby reversing the effect of KLF4 on the expression of TJ proteins. These findings provide a new molecular mechanism for the TXL-induced increase in TJ protein expression. PMID:26065642

  4. Heterogeneous ageing of skeletal muscle microvascular function.

    PubMed

    Muller-Delp, Judy M

    2016-04-15

    The distribution of blood flow to skeletal muscle during exercise is altered with advancing age. Changes in arteriolar function that are muscle specific underlie age-induced changes in blood flow distribution. With advancing age, functional adaptations that occur in resistance arterioles from oxidative muscles differ from those that occur in glycolytic muscles. Age-related adaptations of morphology, as well as changes in both endothelial and vascular smooth muscle signalling, differ in muscle of diverse fibre type. Age-induced endothelial dysfunction has been reported in most skeletal muscle arterioles; however, unique alterations in signalling contribute to the dysfunction in arterioles from oxidative muscles as compared with those from glycolytic muscles. In resistance arterioles from oxidative muscle, loss of nitric oxide signalling contributes significantly to endothelial dysfunction, whereas in resistance arterioles from glycolytic muscle, alterations in both nitric oxide and prostanoid signalling underlie endothelial dysfunction. Similarly, adaptations of the vascular smooth muscle that occur with advancing age are heterogeneous between arterioles from oxidative and glycolytic muscles. In both oxidative and glycolytic muscle, late-life exercise training reverses age-related microvascular dysfunction, and exercise training appears to be particularly effective in reversing endothelial dysfunction. Patterns of microvascular ageing that develop among muscles of diverse fibre type and function may be attributable to changing patterns of physical activity with ageing. Importantly, aerobic exercise training, initiated even at an advanced age, restores muscle blood flow distribution patterns and vascular function in old animals to those seen in their young counterparts.

  5. Cardiac Magnetic Resonance Myocardial Perfusion Reserve Index Is Reduced in Women With Coronary Microvascular Dysfunction: A National Heart, Lung and Blood Institute-Sponsored Study From the Women's Ischemia Syndrome Evaluation (WISE)

    PubMed Central

    Thomson, Louise E.J.; Wei, Janet; Agarwal, Megha; Haft-Baradaran, Afsaneh; Shufelt, Chrisandra; Mehta, Puja K.; Gill, Edward; Johnson, B. Delia; Kenkre, Tanya; Handberg, Eileen; Li, Debiao; Sharif, Behzad; Berman, Daniel S.; Petersen, John; Pepine, Carl J.; Bairey Merz, C. Noel

    2015-01-01

    Background Women with signs and symptoms of ischemia and no obstructive coronary artery disease often have coronary microvascular dysfunction (CMD), diagnosed by invasive coronary reactivity testing (CRT). While traditional noninvasive stress imaging is often normal in CMD, cardiac magnetic resonance imaging (CMRI) may be able to detect CMD in this population. Methods and Results Vasodilator stress CMRI was performed in 118 women with suspected CMD who had undergone CRT and 21 asymptomatic reference subjects. Semi quantitative evaluation of the first-pass perfusion images was completed to determine myocardial perfusion reserve index (MPRI). The relationship between CRT findings and MPRI was examined by Pearson correlations, logistic regression and sensitivity/specificity. Symptomatic women had lower mean pharmacologic stress MPRI compared to reference subjects (1.71±0.43 vs. 2.23±0.37, p<0.0001). Lower MPRI was predictive of one or more abnormal CRT variables (OR = 0.78 [0.70, 0.88], p<0.0001, c-statistic 0.78 [0.68, 0.88]). An MPRI threshold of 1.84 predicted CRT abnormality with sensitivity 73% and specificity 74%. Conclusions Noninvasive CMRI MPRI can detect CMD defined by invasive CRT. Further work is aimed to optimize the non-invasive identification and management of CMD patients. PMID:25801710

  6. The Association between Circulating MicroRNA Levels and Coronary Endothelial Function

    PubMed Central

    Widmer, R. Jay; Chung, Woo-Young; Herrmann, Joerg; Jordan, Kyra L.; Lerman, Lilach O.; Lerman, Amir

    2014-01-01

    Human microRNAs (miRs) have been implicated in human diseases presumably through the downregulation and silencing of targeted genes via post-translational modifications. However, their role in the early stage of coronary atherosclerosis is not known. The aim of this study was to test the hypothesis that patients with early atherosclerosis and coronary endothelial dysfunction (CED) have alterations in transcoronary miR gradients. Patients underwent coronary angiography and endothelial function testing in the cardiac catheterization laboratory. Patients were divided into abnormal (n = 26) and normal (n = 22) microvascular coronary endothelial function based on intracoronary response to infused acetylcholine measured as a percent change in coronary blood flow (CBF) and arterial diameter. Blood samples were obtained simultaneously from the aorta and coronary sinus at the time of catheterization for RNA isolation, and miR subsequently assessed. Baseline characteristics were similar in both groups. Patients with microvascular CED displayed transcoronary gradients significantly elevated in miR-92a and miR-133 normalized to C-elegans-39 miR. Percent change in CBF and the transcoronary gradient of miR-133 displayed a significant inverse correlation (r2 = 0.11, p = 0.03). Thus, we present novel data whereupon selected miRs demonstrate elevated transcoronary gradients in patients with microvascular CED. The current findings support further studies on the mechanistic role of miRs in coronary atherosclerosis and in humans. PMID:25310838

  7. Regulation of human aortic endothelial cell-derived mesenchymal growth factors by allogeneic lymphocytes in vitro. A potential mechanism for cardiac allograft vasculopathy.

    PubMed Central

    Wagner, C R; Morris, T E; Shipley, G D; Hosenpud, J D

    1993-01-01

    Cardiac allograft vasculopathy is thought to be triggered by an alloreactive response to the donor coronary vasculature, resulting in smooth muscle cell proliferation and ultimate occlusion of the donor coronary arteries. To determine whether allogeneic lymphocytes are capable of regulating endothelial-derived smooth muscle cell (SMC) growth factors, human aortic endothelial cells (HAECs) were exposed to allogeneic lymphocytes. The HAEC-lymphocyte co-cultures were assessed for (a) lymphocyte proliferation in response to the allogeneic HAECs; (b) release of soluble factors that stimulate human aortic SMC proliferation; and (c) alteration of HAEC mRNA levels for a panel of known SMC growth factors. Co-culture conditioned medium increased SMC proliferation, compared to medium conditioned by HAECs alone. HAECs exposed to allogeneic lymphocytes increased their expression of mRNA for basic fibroblast growth factor, transforming growth factors alpha and beta, and platelet derived growth factor A and B chains. These results demonstrate that allogeneic lymphocytes are capable of inducing HAECs to increase mRNA levels for several mesenchymal growth factors and to release bioactive products capable of stimulating SMC cell proliferation in vitro. Additionally, the data support the hypothesis that alloreactive lymphocytes can stimulate allogeneic donor endothelial cells to produce growth factors that may contribute to the intimal thickening seen in cardiac allograft vasculopathy. Images PMID:8376585

  8. Skin microvascular reactivity in trained adolescents.

    PubMed

    Roche, Denise M; Rowland, T W; Garrard, M; Marwood, S; Unnithan, V B

    2010-04-01

    Whilst endothelial dysfunction is associated with a sedentary lifestyle, enhanced endothelial function has been documented in the skin of trained individuals. The purpose of this study was to investigate whether highly trained adolescent males possess enhanced skin microvascular endothelial function compared to their untrained peers. Seventeen highly and predominantly soccer trained boys (V(O)(2)(peak): 55 +/- 6 mL kg(-1) min(-1)) and nine age- and maturation-matched untrained controls (V(O)(2)(peak): 43 +/- 5 mL kg(-1) min(-1)) aged 13-15 years had skin microvascular endothelial function assessed using laser Doppler flowmetry. Baseline and maximal thermally stimulated skin blood flow (SkBF) responses were higher in forearms of trained subjects compared to untrained participants [baseline SkBF: 11 +/- 4 vs. 9 +/- 3 perfusion units (PU), p < 0.05; SkBF(max): 282 +/- 120 vs. 204 +/- 68 PU, p < 0.05]. Similarly, cutaneous vascular conductance (CVC) during local heating was superior in the forearm skin of trained versus untrained individuals (CVC(max): 3 +/- 1 vs. 2 +/- 1 PU mmHg(-1), p < 0.05). Peak hyperaemia following arterial occlusion and area under the reactive hyperaemia curve were also greater in forearm skin of the trained group (peak hyperaemia: 51 +/- 21 vs. 35 +/- 15 PU, p < 0.05; area under curve: 1596 +/- 739 vs. 962 +/- 796 PUs, p < 0.05). These results suggest that chronic exercise training in adolescents is associated with enhanced microvascular endothelial vasodilation in non-glabrous skin.

  9. Microvascular Targets for Anti-Fibrotic Therapeutics

    PubMed Central

    Pu, Kai-Ming T.; Sava, Parid; Gonzalez, Anjelica L.

    2013-01-01

    Fibrosis is characterized by excessive extracellular matrix deposition and is the pathological outcome of repetitive tissue injury in many disorders. The accumulation of matrix disrupts the structure and function of the native tissue and can affect multiple organs including the lungs, heart, liver, and skin. Unfortunately, current therapies against the deadliest and most common fibrosis are ineffective. The pathogenesis of fibrosis is the result of aberrant wound healing, therefore, the microvasculature plays an important role, contributing through regulation of leukocyte recruitment, inflammation, and angiogenesis. Further exacerbating the condition, microvascular endothelial cells and pericytes can transdifferentiate into matrix depositing myofibroblasts. The contribution of the microvasculature to fibrotic progression makes its cellular components and acellular products attractive therapeutic targets. In this review, we examine many of the cytokine, matrix, and cellular microvascular components involved in fibrosis and discuss their potential as targets for fibrotic therapies with a particular focus on developing nanotechnologies. PMID:24348218

  10. A comparative study of the cell cycle status and primitive cell adhesion molecule profile of human CD34+ cells cultured in stroma-free versus porcine microvascular endothelial cell cultures.

    PubMed

    Chute, J P; Saini, A A; Kampen, R L; Wells, M R; Davis, T A

    1999-02-01

    Porcine microvascular endothelial cells (PMVECs) plus cytokines support a rapid proliferation and expansion of human CD34+CD38- cells that are capable of multilineage engraftment within the bone marrow of a secondary host. CD34+CD38- cells contain the self-renewing, long-term culture-initiating cells (LTC-IC) that are ideal targets for retroviral gene transfer experiments. Previous experiments attempting retroviral infection of CD34+CD38- cells have failed partly because these cells do not enter cell cycle in response to cytokine combinations. In this study, we determined the cell cycle status and the cell adhesion molecule profile on purified CD34+ cells and the CD34+CD38- subset before and after ex vivo expansion on PMVECs. Purified human CD34+ cells were cocultured with PMVECs for 7 days in the presence of optimal concentrations of granulocyte/macrophage-colony-stimulating factor (GM-CSF) + interleukin (IL)-3 + IL-6 + stem cell factor (SCF) + Flt-3 ligand. The total CD34+ population and the CD34+CD38- subset increased 8.4- and 67-fold, respectively, with absolute increases in the number of colony-forming unit-granulocyte macrophage (CFU-GM) (28.2-fold), CFU-Mix (8.7 fold), and burst-forming unit-erythroid (BFU-E) (4.0-fold) progenitor cells. After 7 days of coculture with PMVECs, 44% of the CD34+CD38+ subset were found to be in G1, and 51% were in G2/S/M phase of the cell cycle. More remarkably, 53% of the CD34+CD38- subset were in G1, and 17% were in G2/S/M phase after 7 days of PMVEC coculture. In contrast, only 22% of the CD34+CD38- subset remaining after 7 days of stroma-free culture were in G1, and 6% were in G2/S/M phase. Despite the high level of cellular activation and proliferation induced by PMVEC coculture, the surface expression of adhesion molecules CD11a (LFA-1), CD11b, CD15s (sialyl-Lewis x), CD43, and CD44 (HCAM) on the total CD34+ population was maintained, and the surface expression of CD49d (VLA-4), CD54 (ICAM), CD58, and CD62L (L selectin

  11. Role of mitochondrial dysfunction in hyperglycaemia-induced coronary microvascular dysfunction: Protective role of resveratrol.

    PubMed

    Joshi, Mandar S; Williams, David; Horlock, Duncan; Samarasinghe, Thilini; Andrews, Karen L; Jefferis, Ann-Maree; Berger, Philip J; Chin-Dusting, Jaye P; Kaye, David M

    2015-05-01

    Microvascular complications are now recognized to play a major role in diabetic complications, and understanding the mechanisms is critical. Endothelial dysfunction occurs early in the course of the development of complications; the precise mechanisms remain poorly understood. Mitochondrial dysfunction may occur in a diabetic rat heart and may act as a source of the oxidative stress. However, the role of endothelial cell-specific mitochondrial dysfunction in diabetic vascular complications is poorly studied. Here, we studied the role of diabetes-induced abnormal endothelial mitochondrial function and the resultant endothelial dysfunction. Understanding the role of endothelial mitochondrial dysfunction in diabetic vasculature is critical in order to develop new therapies. We demonstrate that hyperglycaemia leads to mitochondrial dysfunction in microvascular endothelial cells, and that mitochondrial inhibition induces endothelial dysfunction. Additionally, we show that resveratrol acts as a protective agent; resveratrol-mediated mitochondrial protection may be used to prevent long-term diabetic cardiovascular complications.

  12. Candida glabrata binds to glycosylated and lectinic receptors on the coronary endothelial luminal membrane and inhibits flow sense and cardiac responses to agonists.

    PubMed

    Torres-Tirado, David; Knabb, Maureen; Castaño, Irene; Patrón-Soberano, Araceli; De Las Peñas, Alejandro; Rubio, Rafael

    2016-01-01

    Candida glabrata (CG) is an opportunistic fungal pathogen that initiates infection by binding to host cells via specific lectin-like adhesin proteins. We have previously shown the importance of lectin-oligosaccharide binding in cardiac responses to flow and agonists. Because of the lectinic-oligosaccharide nature of CG binding, we tested the ability of CG to alter the agonist- and flow-induced changes in cardiac function in isolated perfused guinea pig hearts. Both transmission and scanning electron microscopy showed strong attachment of CG to the coronary endothelium, even after extensive washing. CG shifted the coronary flow vs. auricular-ventricular (AV) delay relationship upward, indicating that greater flow was required to achieve the same AV delay. This effect was completely reversed with mannose, partially reversed with galactose and N-acetylgalactosamine, but hyaluronan had no effect. Western blot analysis was used to determine binding of CG to isolated coronary endothelial luminal membrane (CELM) receptors, and the results indicate that flow-sensitive CELM receptors, ANG II type I, α-adrenergic 1A receptor, endothelin-2, and VCAM-1 bind to CG. In addition, CG inhibited agonist-induced effects of bradykinin, angiotensin, and phenylephrine on AV delay, coronary perfusion pressure, and left ventricular pressure. Mannose reversed the inhibitory effects of CG on the agonist responses. These results suggest that CG directly binds to flow-sensitive CELM receptors via lectinic-oligosaccharide interactions with mannose and disrupts the lectin-oligosaccharide binding necessary for flow-induced cardiac responses.

  13. What Causes Coronary Microvascular Disease?

    MedlinePlus

    ... Living With Clinical Trials Links Related Topics Angina Atherosclerosis Coronary Heart Disease Coronary Heart Disease Risk Factors ... Microvascular Disease? The same risk factors that cause atherosclerosis may cause coronary microvascular disease. Atherosclerosis is a ...

  14. Fibrinogen Induces Alterations of Endothelial Cell Tight Junction Proteins

    PubMed Central

    PATIBANDLA, PHANI K.; TYAGI, NEETU; DEAN, WILLIAM L.; TYAGI, SURESH C.; ROBERTS, ANDREW M.; LOMINADZE, DAVID

    2009-01-01

    We previously showed that an elevated content of fibrinogen (Fg) increased formation of filamentous actin and enhanced endothelial layer permeability. In the present work we tested the hypothesis that Fg binding to endothelial cells (ECs) alters expression of actin-associated endothelial tight junction proteins (TJP). Rat cardiac microvascular ECs were grown in gold plated chambers of an electrical cell-substrate impedance system, 8-well chambered, or in 12-well plates. Confluent ECs were treated with Fg (2 or 4 mg/ml), Fg (4 mg/ml) with mitogen-activated protein kinase (MEK) kinase inhibitors (PD98059 or U0126), Fg (4 mg/ml) with anti-ICAM-1 antibody or BQ788 (endothelin type B receptor blocker), endothelin-1, endothelin-1 with BQ788, or medium alone for 24 h. Fg induced a dose-dependent decrease in EC junction integrity as determined by transendothelial electrical resistance (TEER). Western blot analysis and RT-PCR data showed that the higher dose of Fg decreased the contents of TJPs, occludin, zona occluden-1 (ZO-1), and zona occluden-2 (ZO-2) in ECs. Fg-induced decreases in contents of the TJPs were blocked by PD98059, U0126, or anti-ICAM-1 antibody. While BQ788 inhibited endothelin-1-induced decrease in TEER, it did not affect Fg-induced decrease in TEER. These data suggest that Fg increases EC layer permeability via the MEK kinase signaling pathway by affecting occludin, ZO-1, and ZO-2, TJPs, which are bound to actin filaments. Therefore, increased binding of Fg to its major EC receptor, ICAM-1, during cardiovascular diseases may increase microvascular permeability by altering the content and possibly subcellular localization of endothelial TJPs. PMID:19507189

  15. Cardiac shockwave therapy improves myocardial function in patients with refractory coronary artery disease by promoting VEGF and IL-8 secretion to mediate the proliferation of endothelial progenitor cells

    PubMed Central

    CAI, HONG-YAN; LI, LIN; GUO, TAO; WANG, YU; MA, TIE-KUN; XIAO, JIAN-MING; ZHAO, LING; FANG, YIN; YANG, PING; ZHAO, HU

    2015-01-01

    Cardiac shockwave therapy (CSWT) is a potential and effective remedy to promote revascularization in the ischemic myocardium of patients with refractory coronary heart disease (CHD). The technique is both safe and non-invasive; however, the underlying molecular mechanism remains unclear. The aim of this study was to evaluate the efficacy of CSWT in treating CHD patients and investigate a potential mechanism. A total of 26 patients with CHD were enrolled in the study, and CSWT was performed over a 3-month period. The efficacy of CSWT was assessed using several clinical parameters. Peripheral blood (PB) was collected prior to and following treatment. The number of circulating endothelial progenitor cells (EPCs) in the PB was counted using a flow cytometer, and the levels of vascular endothelial growth factor (VEGF), interleukin-8 (IL-8), stromal cell-derived factor 1 and matrix metalloproteinase 9 in the PB were analyzed. Mononuclear cells were isolated from the PB and cultured in vitro. The EPCs and EPC-colony forming units (EPC-CFUs) in the PB mononuclear cell culture were counted using an inverted phase contrast microscope. Following CSWT, the tested clinical parameters were significantly improved. The levels of circulating EPCs, VEGF and IL-8 in the PB were significantly increased, as were the EPCs and EPC-CFUs from the PB mononuclear cell culture. We suggest that EPC proliferation, mediated by VEGF and IL-8 secretion, may be among the potential mechanisms associated with CSWT. PMID:26668649

  16. Macrophage migration inhibitory factor promotes cardiac stem cell proliferation and endothelial differentiation through the activation of the PI3K/Akt/mTOR and AMPK pathways

    PubMed Central

    CUI, JINJIN; ZHANG, FENGYUN; WANG, YONGSHUN; LIU, JINGJIN; MING, XING; HOU, JINGBO; LV, BO; FANG, SHAOHONG; YU, BO

    2016-01-01

    Macrophage migration inhibitory factor (MIF) has pleiotropic immune functions in a number of inflammatory diseases. Recent evidence from expression and functional studies has indicated that MIF is involved in various aspects of cardiovascular disease. In this study, we aimed to determine whether MIF supports in vitro c-kit+CD45− cardiac stem cell (CSC) survival, proliferation and differentiation into endothelial cells, as well as the possible mechanisms involved. We observed MIF receptor (CD74) expression in mouse CSCs (mCSCs) using PCR and immunofluorescence staining, and MIF secretion by mCSCs using PCR and ELISA in vitro. Increasing amounts of exogenous MIF did not affect CD74 expression, but promoted mCSC survival, proliferation and endothelial differentiation. By contrast, treatment with an MIF inhibitor (ISO-1) or siRNA targeting CD74 (CD74-siRNA) suppressed the biological changes induced by MIF in the mCSCs. Increasing amounts of MIF increased the phosphorylation of Akt and mammalian target of rapamycin (mTOR), which are known to support cell survival, proliferation and differentiation. These effects of MIF on the mCSCs were abolished by LY294002 [a phosphoinositide 3-kinase (PI3K) inhibitor] and MK-2206 (an Akt inhibitor). Moreover, adenosine monophosphate-activated protein kinase (AMPK) phosphorylation increased following treatment with MIF. The AMPK inhibitor, compound C, partly blocked the pro-proliferative effects of MIF on the mCSCs. In conclusion, our results suggest that MIF promotes mCSC survival, proliferation and endothelial differentiation through the activation of the PI3K/Akt/mTOR and AMPK signaling pathways. Thus, MIF may prove to be a potential therapeutic factor in the treatment of heart failure and myocardial infarction by activating CSCs. PMID:27035848

  17. Cardiac fibroblasts support endothelial cell proliferation and sprout formation but not the development of multicellular sprouts in a fibrin gel co-culture model.

    PubMed

    Twardowski, Rachel L; Black, Lauren D

    2014-05-01

    A primary impediment to cardiac tissue engineering lies in the inability to adequately vascularize the constructs to optimize survival upon implantation. During normal angiogenesis, endothelial cells (ECs) require a support cell to form mature patent lumens and it has been demonstrated that pericytes, vascular smooth muscle cells and mesenchymal stem cells (MSCs) are all able to support the formation of mature vessels. In the heart, cardiac fibroblasts (CFs) provide important electrical and mechanical functions, but to date have not been sufficiently studied for their role in angiogenesis. To study CFs role in angiogenesis, we co-cultured different concentrations of various cell types in fibrin hemispheres with appropriate combinations of their specific media, to determine the optimal conditions for EC growth and sprout formation through DNA analysis, flow cytometry and immunohistology. ECs proliferated best when co-cultured with CFs and analysis of immunohistological images demonstrated that ECs formed the longest and most numerous sprouts with CFs as compared to MSCs. However, ECs were able to produce more multicellular sprouts when in culture with the MSCs. Moreover, these effects were dependent on the ratio of support cell to EC in co-culture. Overall, CFs provide a good support system for EC proliferation and sprout formation; however, MSCs allow for more multicellular sprouts, which is more indicative of the in vivo process.

  18. Skin microvascular reactivity in patients with hypothyroidism.

    PubMed

    Mihor, Ana; Gergar, Maša; Gaberšček, Simona; Lenasi, Helena

    2016-11-04

    Hypothyroidism is associated with impaired vascular function; however, little is known about its impact on microcirculation. We aimed to determine skin microvascular reactivity in hypothyroidism focusing on endothelial function and the sympathetic response. We measured skin laser Doppler (LD) flux (LDF) on the volar forearm and the finger pulp using LD flowmetry in hypothyroid patients (N = 13) and healthy controls (N = 15). Skin microvascular reactivity was assessed by a three-minute occlusion of the brachial artery, inducing postocclusive reactive hyperaemia (PRH), and by a four-minute local cooling of the hand. An electrocardiogram (ECG), digital artery blood pressure and skin temperature at the measuring sites were recorded. Baseline LDF, the digital artery blood pressure and the heart rate were comparable between patients and controls. On the other hand, patients exhibited significantly longer PRH duration, significantly higher blood pressure during cooling (unpaired t-test, p <0.05) and lower, albeit not significant, LDF in the ipsilateral finger pulp during cooling compared to controls. Unexpectedly, the results of the present study point to an increased vasodilator capacity of skin microcirculation and an apparent increase in sympathetic reactivity after local cooling in hypothyroid patients. Hypothyroidism induces subtle changes of some haemodynamic parameters in skin microcirculation implying altered endothelial function and altered sympathetic reactivity.

  19. Transcriptional Regulation of Cystathionine-γ-Lyase in Endothelial Cells by NADPH Oxidase 4-Dependent Signaling.

    PubMed

    Mistry, Rajesh K; Murray, Thomas V A; Prysyazhna, Oleksandra; Martin, Daniel; Burgoyne, Joseph R; Santos, Celio; Eaton, Philip; Shah, Ajay M; Brewer, Alison C

    2016-01-22

    The gasotransmitter, hydrogen sulfide (H2S) is recognized as an important mediator of endothelial cell homeostasis and function that impacts upon vascular tone and blood pressure. Cystathionine-γ-lyase (CSE) is the predominant endothelial generator of H2S, and recent evidence suggests that its transcriptional expression is regulated by the reactive oxygen species, H2O2. However, the cellular source of H2O2 and the redox-dependent molecular signaling pathway that modulates this is not known. We aimed to investigate the role of Nox4, an endothelial generator of H2O2, in the regulation of CSE in endothelial cells. Both gain- and loss-of-function experiments in human endothelial cells in vitro demonstrated Nox4 to be a positive regulator of CSE transcription and protein expression. We demonstrate that this is dependent upon a heme-regulated inhibitor kinase/eIF2α/activating transcription factor 4 (ATF4) signaling module. ATF4 was further demonstrated to bind directly to cis-regulatory sequences within the first intron of CSE to activate transcription. Furthermore, CSE expression was also increased in cardiac microvascular endothelial cells, isolated from endothelial-specific Nox4 transgenic mice, compared with wild-type littermate controls. Using wire myography we demonstrate that endothelial-specific Nox4 transgenic mice exhibit a hypo-contractile phenotype in response to phenylephrine that was abolished when vessels were incubated with a CSE inhibitor, propargylglycine. We, therefore, conclude that Nox4 is a positive transcriptional regulator of CSE in endothelial cells and propose that it may in turn contribute to the regulation of vascular tone via the modulation of H2S production.

  20. Transcriptional Regulation of Cystathionine-γ-Lyase in Endothelial Cells by NADPH Oxidase 4-Dependent Signaling*

    PubMed Central

    Mistry, Rajesh K.; Murray, Thomas V. A.; Prysyazhna, Oleksandra; Martin, Daniel; Burgoyne, Joseph R.; Santos, Celio; Eaton, Philip; Shah, Ajay M.; Brewer, Alison C.

    2016-01-01

    The gasotransmitter, hydrogen sulfide (H2S) is recognized as an important mediator of endothelial cell homeostasis and function that impacts upon vascular tone and blood pressure. Cystathionine-γ-lyase (CSE) is the predominant endothelial generator of H2S, and recent evidence suggests that its transcriptional expression is regulated by the reactive oxygen species, H2O2. However, the cellular source of H2O2 and the redox-dependent molecular signaling pathway that modulates this is not known. We aimed to investigate the role of Nox4, an endothelial generator of H2O2, in the regulation of CSE in endothelial cells. Both gain- and loss-of-function experiments in human endothelial cells in vitro demonstrated Nox4 to be a positive regulator of CSE transcription and protein expression. We demonstrate that this is dependent upon a heme-regulated inhibitor kinase/eIF2α/activating transcription factor 4 (ATF4) signaling module. ATF4 was further demonstrated to bind directly to cis-regulatory sequences within the first intron of CSE to activate transcription. Furthermore, CSE expression was also increased in cardiac microvascular endothelial cells, isolated from endothelial-specific Nox4 transgenic mice, compared with wild-type littermate controls. Using wire myography we demonstrate that endothelial-specific Nox4 transgenic mice exhibit a hypo-contractile phenotype in response to phenylephrine that was abolished when vessels were incubated with a CSE inhibitor, propargylglycine. We, therefore, conclude that Nox4 is a positive transcriptional regulator of CSE in endothelial cells and propose that it may in turn contribute to the regulation of vascular tone via the modulation of H2S production. PMID:26620565

  1. Microvascular Autonomic Composites

    DTIC Science & Technology

    2012-01-06

    characterization of carbon nanotube yarns, 3-D braids, and their composites. SAMPE Journal 43: 6-19. Bogdanovich A and Mohamed MH. 2009. Three-Dimensional... carbon in red and bromine in yellow. The fracture surfaces were analyzed by SEM to show film was indistinguishable from the matrix, but by using the...nature, the mother of composite materials, applying microvascular technology to create skin, cartilages, tendons, bones and teeth. Cellulose fiber

  2. Sepsis-induced elevation in plasma serotonin facilitates endothelial hyperpermeability

    PubMed Central

    Li, Yicong; Hadden, Coedy; Cooper, Anthonya; Ahmed, Asli; Wu, Hong; Lupashin, Vladimir V.; Mayeux, Philip R.; Kilic, Fusun

    2016-01-01

    Hyperpermeability of the endothelial barrier and resulting microvascular leakage are a hallmark of sepsis. Our studies describe the mechanism by which serotonin (5-HT) regulates the microvascular permeability during sepsis. The plasma 5-HT levels are significantly elevated in mice made septic by cecal ligation and puncture (CLP). 5-HT-induced permeability of endothelial cells was associated with the phosphorylation of p21 activating kinase (PAK1), PAK1-dependent phosphorylation of vimentin (P-vimentin) filaments, and a strong association between P-vimentin and ve-cadherin. These findings were in good agreement with the findings with the endothelial cells incubated in serum from CLP mice. In vivo, reducing the 5-HT uptake rates with the 5-HT transporter (SERT) inhibitor, paroxetine blocked renal microvascular leakage and the decline in microvascular perfusion. Importantly, mice that lack SERT showed significantly less microvascular dysfunction after CLP. Based on these data, we propose that the increased endothelial 5-HT uptake together with 5-HT signaling disrupts the endothelial barrier function in sepsis. Therefore, regulating intracellular 5-HT levels in endothelial cells represents a novel approach in improving sepsis-associated microvascular dysfunction and leakage. These new findings advance our understanding of the mechanisms underlying cellular responses to intracellular/extracellular 5-HT ratio in sepsis and refine current views of these signaling processes during sepsis. PMID:26956613

  3. Sepsis-induced elevation in plasma serotonin facilitates endothelial hyperpermeability.

    PubMed

    Li, Yicong; Hadden, Coedy; Cooper, Anthonya; Ahmed, Asli; Wu, Hong; Lupashin, Vladimir V; Mayeux, Philip R; Kilic, Fusun

    2016-03-09

    Hyperpermeability of the endothelial barrier and resulting microvascular leakage are a hallmark of sepsis. Our studies describe the mechanism by which serotonin (5-HT) regulates the microvascular permeability during sepsis. The plasma 5-HT levels are significantly elevated in mice made septic by cecal ligation and puncture (CLP). 5-HT-induced permeability of endothelial cells was associated with the phosphorylation of p21 activating kinase (PAK1), PAK1-dependent phosphorylation of vimentin (P-vimentin) filaments, and a strong association between P-vimentin and ve-cadherin. These findings were in good agreement with the findings with the endothelial cells incubated in serum from CLP mice. In vivo, reducing the 5-HT uptake rates with the 5-HT transporter (SERT) inhibitor, paroxetine blocked renal microvascular leakage and the decline in microvascular perfusion. Importantly, mice that lack SERT showed significantly less microvascular dysfunction after CLP. Based on these data, we propose that the increased endothelial 5-HT uptake together with 5-HT signaling disrupts the endothelial barrier function in sepsis. Therefore, regulating intracellular 5-HT levels in endothelial cells represents a novel approach in improving sepsis-associated microvascular dysfunction and leakage. These new findings advance our understanding of the mechanisms underlying cellular responses to intracellular/extracellular 5-HT ratio in sepsis and refine current views of these signaling processes during sepsis.

  4. Spliced stromal cell-derived factor-1α analog stimulates endothelial progenitor cell migration and improves cardiac function in a dose-dependent manner after myocardial infarction

    PubMed Central

    Hiesinger, William; Frederick, John R.; Atluri, Pavan; McCormick, Ryan C.; Marotta, Nicole; Muenzer, Jeffrey R.; Woo, Y. Joseph

    2011-01-01

    Objectives Stromal cell-derived factor (SDF)-1α is a potent endogenous endothelial progenitor cell (EPC) chemokine and key angiogenic precursor. Recombinant SDF-1α has been demonstrated to improve neovasculogenesis and cardiac function after myocardial infarction (MI) but SDF-1α is a bulky protein with a short half-life. Small peptide analogs might provide translational advantages, including ease of synthesis, low manufacturing costs, and the potential to control delivery within tissues using engineered biomaterials. We hypothesized that a minimized peptide analog of SDF-1α, designed by splicing the N-terminus (activation and binding) and C-terminus (extracellular stabilization) with a truncated amino acid linker, would induce EPC migration and preserve ventricular function after MI. Methods EPC migration was first determined in vitro using a Boyden chamber assay. For in vivo analysis, male rats (n=48) underwent left anterior descending coronary artery ligation. At infarction, the rats were randomized into 4 groups and received peri-infarct intramyocardial injections of saline, 3 μg/kg of SDF-1α, 3 μg/kg of spliced SDF analog, or 6 μg/kg spliced SDF analog. After 4 weeks, the rats underwent closed chest pressure volume conductance catheter analysis. Results EPCs showed significantly increased migration when placed in both a recombinant SDF-1α and spliced SDF analog gradient. The rats treated with spliced SDF analog at MI demonstrated a significant dose-dependent improvement in end-diastolic pressure, stroke volume, ejection fraction, cardiac output, and stroke work compared with the control rats. Conclusions A spliced peptide analog of SDF-1α containing both the N- and C- termini of the native protein induced EPC migration, improved ventricular function after acute MI, and provided translational advantages compared with recombinant human SDF-1α. PMID:20951261

  5. Soluble epoxide hydrolase inhibition improves coronary endothelial function and prevents the development of cardiac alterations in obese insulin-resistant mice

    PubMed Central

    Roche, Clothilde; Besnier, Marie; Cassel, Roméo; Harouki, Najah; Coquerel, David; Guerrot, Dominique; Nicol, Lionel; Loizon, Emmanuelle; Remy-Jouet, Isabelle; Morisseau, Christophe; Mulder, Paul; Ouvrard-Pascaud, Antoine; Madec, Anne-Marie; Richard, Vincent

    2015-01-01

    This study addressed the hypothesis that inhibiting the soluble epoxide hydrolase (sEH)-mediated degradation of epoxy-fatty acids, notably epoxyeicosatrienoic acids, has an additional impact against cardiovascular damage in insulin resistance, beyond its previously demonstrated beneficial effect on glucose homeostasis. The cardiovascular and metabolic effects of the sEH inhibitor trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB; 10 mg/l in drinking water) were compared with those of the sulfonylurea glibenclamide (80 mg/l), both administered for 8 wk in FVB mice subjected to a high-fat diet (HFD; 60% fat) for 16 wk. Mice on control chow diet (10% fat) and nontreated HFD mice served as controls. Glibenclamide and t-AUCB similarly prevented the increased fasting glycemia in HFD mice, but only t-AUCB improved glucose tolerance and decreased gluconeogenesis, without modifying weight gain. Moreover, t-AUCB reduced adipose tissue inflammation, plasma free fatty acids, and LDL cholesterol and prevented hepatic steatosis. Furthermore, only the sEH inhibitor improved endothelium-dependent relaxations to acetylcholine, assessed by myography in isolated coronary arteries. This improvement was related to a restoration of epoxyeicosatrienoic acid and nitric oxide pathways, as shown by the increased inhibitory effects of the nitric oxide synthase and cytochrome P-450 epoxygenase inhibitors l-NA and MSPPOH on these relaxations. Moreover, t-AUCB decreased cardiac hypertrophy, fibrosis, and inflammation and improved diastolic function, as demonstrated by the increased E/A ratio (echocardiography) and decreased slope of the end-diastolic pressure-volume relation (invasive hemodynamics). These results demonstrate that sEH inhibition improves coronary endothelial function and prevents cardiac remodeling and diastolic dysfunction in obese insulin-resistant mice. PMID:25724490

  6. Tissue kallikrein-modified human endothelial progenitor cell implantation improves cardiac function via enhanced activation of akt and increased angiogenesis.

    PubMed

    Yao, Yuyu; Sheng, Zulong; Li, YeFei; Fu, Cong; Ma, Genshan; Liu, Naifeng; Chao, Julie; Chao, Lee

    2013-05-01

    Endothelial progenitor cells (EPCs) have been shown to enhance angiogenesis not only by incorporating into the vasculature but also by secreting cytokines, thereby serving as an ideal vehicle for gene transfer. As tissue kallikrein (TK) has pleiotropic effects in inhibiting apoptosis and oxidative stress, and promoting angiogenesis, we evaluated the salutary potential of kallikrein-modified human EPCs (hEPCs; Ad.hTK-hEPCs) after acute myocardial infarction (MI). We genetically modified hEPCs with a TK gene and evaluated cell survival, engraftment, revascularization, and functional improvement in a nude mouse left anterior descending ligation model. hEPCs were manipulated to overexpress the TK gene. In vitro, the antiapoptotic and paracrine effects were assessed under oxidative stress. TK protects hEPCs from oxidative stress-induced apoptosis via inhibition of activation of caspase-3 and -9, induction of Akt phosphorylation, and secretion of vascular endothelial growth factor. In vivo, the Ad.hTK-hEPCs were transplanted after MI via intracardiac injection. The surviving cells were tracked after transplantation using near-infrared optical imaging. Left ventricular (LV) function was evaluated by transthoracic echocardiography. Capillary density was quantified using immunohistochemical staining. Engrafted Ad.hTK-hEPCs exhibited advanced protection against ischemia by increasing LV ejection fraction. Compared with Ad.Null-hEPCs, transplantation with Ad.hTK-hEPCs significantly decreased cardiomyocyte apoptosis in association with increased retention of transplanted EPCs in the myocardium. Capillary density and arteriolar density in the infarct border zone was significantly higher in Ad.hTK-hEPC-transplanted mice than in Ad.Null-hEPC-treated mice. Transplanted hEPCs were clearly incorporated into CD31(+) capillaries. These results indicate that implantation of kallikrein-modified EPCs in the heart provides advanced benefits in protection against ischemia-induced MI by

  7. Monitoring in microvascular surgery.

    PubMed

    Furnas, H; Rosen, J M

    1991-03-01

    The importance of monitoring in microvascular surgery is underscored by the high reported salvage rates of failing free flaps and replants. In this overview, we begin by defining the physiology of ischemic tissue with emphasis given to the no-reflow phenomenon and the secondary critical ischemia times. Based on the physiological changes accompanying ischemia, several variables are defined that can be monitored to reflect the vascular state of a free flap or replant. Multifarious monitoring systems are then reviewed, including clinical observation, temperature, isotope clearance, ultrasonic Doppler, laser Doppler, transcutaneous oxygen tension, reflection plethysmography, dermofluorometry, pH, electromagnetic flowmetry, serial hematocrits, interstitial fluid pressure, and magnetic resonance imaging.

  8. In Vitro Endothelialization Test of Biomaterials Using Immortalized Endothelial Cells

    PubMed Central

    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. PMID:27348615

  9. Connecting heart failure with preserved ejection fraction and renal dysfunction: the role of endothelial dysfunction and inflammation.

    PubMed

    Ter Maaten, Jozine M; Damman, Kevin; Verhaar, Marianne C; Paulus, Walter J; Duncker, Dirk J; Cheng, Caroline; van Heerebeek, Loek; Hillege, Hans L; Lam, Carolyn S P; Navis, Gerjan; Voors, Adriaan A

    2016-06-01

    Renal dysfunction in heart failure with preserved ejection fraction (HFpEF) is common and is associated with increased mortality. Impaired renal function is also a risk factor for developing HFpEF. A new paradigm for HFpEF, proposing a sequence of events leading to myocardial remodelling and dysfunction in HFpEF, was recently introduced, involving inflammatory, microvascular, and cardiac components. The kidney might play a key role in this systemic process. Renal impairment causes metabolic and systemic derangements in circulating factors, causing an activated systemic inflammatory state and endothelial dysfunction, which may lead to cardiomyocyte stiffening, hypertrophy, and interstitial fibrosis via cross-talk between the endothelium and cardiomyocyte compartments. Here, we review the role of endothelial dysfunction and inflammation to explain the link between renal dysfunction and HFpEF, which allows for identification of new early risk markers, prognostic factors, and unique targets for intervention.

  10. Intracranial microvascular free flaps.

    PubMed

    Levine, Steven; Garfein, Evan S; Weiner, Howard; Yaremchuk, Michael J; Saadeh, Pierre B; Gurtner, Geoffrey; Levine, Jamie P; Warren, Stephen M

    2009-02-01

    Large acquired intracranial defects can result from trauma or surgery. When reoperation is required because of infection or tumor recurrence, management of the intracranial dead space can be challenging. By providing well-vascularized bulky tissue, intracranial microvascular free flaps offer potential solutions to these life-threatening complications. A multi-institutional retrospective chart and radiographic review was performed of all patients who underwent microvascular free-flap surgery for salvage treatment of postoperative intracranial infections between 1998 and 2006. A total of six patients were identified with large intracranial defects and postoperative intracranial infections. Four patients had parenchymal resections for tumor or seizure and two patients had posttraumatic encephalomalacia. All patients underwent operative debridement and intracranial free-flap reconstruction using the latissimus dorsi muscle (N=2), rectus abdominis muscle (N=2), or omentum (N=2). All patients had titanium (N=4) or Medpor (N=2) cranioplasties. We concluded that surgery or trauma can result in significant intracranial dead space. Treatment of postoperative intracranial infection can be challenging. Vascularized free tissue transfer not only fills the void, but also provides a delivery system for immune cells, antibodies, and systemically administered antibiotics. The early use of this technique when intracranial dead space and infection coexist is beneficial.

  11. Cardiac Non-myocyte Cells Show Enhanced Pharmacological Function Suggestive of Contractile Maturity in Stem Cell Derived Cardiomyocyte Microtissues

    PubMed Central

    Ravenscroft, Stephanie M.; Pointon, Amy; Williams, Awel W.; Cross, Michael J.; Sidaway, James E.

    2016-01-01

    The immature phenotype of stem cell derived cardiomyocytes is a significant barrier to their use in translational medicine and pre-clinical in vitro drug toxicity and pharmacological analysis. Here we have assessed the contribution of non-myocyte cells on the contractile function of co-cultured human embryonic stem cell derived cardiomyocytes (hESC-CMs) in spheroid microtissue format. Microtissues were formed using a scaffold free 96-well cell suspension method from hESC-CM cultured alone (CM microtissues) or in combination with human primary cardiac microvascular endothelial cells and cardiac fibroblasts (CMEF microtissues). Contractility was characterized with fluorescence and video-based edge detection. CMEF microtissues displayed greater Ca2+ transient amplitudes, enhanced spontaneous contraction rate and remarkably enhanced contractile function in response to both positive and negative inotropic drugs, suggesting a more mature contractile phenotype than CM microtissues. In addition, for several drugs the enhanced contractile response was not apparent when endothelial cell or fibroblasts from a non-cardiac tissue were used as the ancillary cells. Further evidence of maturity for CMEF microtissues was shown with increased expression of genes that encode proteins critical in cardiac Ca2+ handling (S100A1), sarcomere assembly (telethonin/TCAP) and β-adrenergic receptor signalling. Our data shows that compared with single cell-type cardiomyocyte in vitro models, CMEF microtissues are superior at predicting the inotropic effects of drugs, demonstrating the critical contribution of cardiac non-myocyte cells in mediating functional cardiotoxicity. PMID:27125969

  12. Ionizing radiation promotes the acquisition of a senescence-associated secretory phenotype and impairs angiogenic capacity in cerebromicrovascular endothelial cells: role of increased DNA damage and decreased DNA repair capacity in microvascular radiosensitivity.

    PubMed

    Ungvari, Zoltan; Podlutsky, Andrej; Sosnowska, Danuta; Tucsek, Zsuzsanna; Toth, Peter; Deak, Ferenc; Gautam, Tripti; Csiszar, Anna; Sonntag, William E

    2013-12-01

    Cerebromicrovascular rarefaction is believed to play a central role in cognitive impairment in patients receiving whole-brain irradiation therapy. To elucidate the mechanism underlying the deleterious effects of γ-irradiation on the cerebral microcirculation, rat primary cerebromicrovascular endothelial cells (CMVECs) were irradiated in vitro. We found that in CMVECs, γ-irradiation (2-8 Gy) elicited increased DNA damage, which was repaired less efficiently in CMVECs compared with neurons, microglia, and astrocytes. Increased genomic injury in CMVECs associated with increased apoptotic cell death. In the surviving cells, γ-irradiation promotes premature senescence (indicated by SA-β-galactosidase positivity and upregulation of p16 (INK4a) ), which was associated with impaired angiogenic capacity (decreased proliferation and tube-forming capacity). γ-Irradiated CMVECs acquired a senescence-associated secretory phenotype, characterized by upregulation of proinflammatory cytokines and chemokines (including IL-6, IL-1α, and MCP-1). Collectively, increased vulnerability of γ-irradiated CMVECs and their impaired angiogenic capacity likely contribute to cerebromicrovascular rarefaction and prevent regeneration of the microvasculature postirradiation. The acquisition of a senescence-associated secretory phenotype in irradiated CMVECs is biologically highly significant as changes in the cytokine microenvironment in the hippocampus may affect diverse biological processes relevant for normal neuronal function (including regulation of neurogenesis and the maintenance of the blood brain barrier).

  13. Imaging Microvascular Dysfunction and Mechanisms for Female-Male Differences in CAD.

    PubMed

    Patel, Monica B; Bui, Linh P; Kirkeeide, Richard L; Gould, K Lance

    2016-04-01

    Microvascular dysfunction or disease is most commonly associated with diffuse epicardial coronary atherosclerosis and endothelial dysfunction, whereas it is less common as a distinct, separate, isolated pathophysiology. The different manifestations of coronary artery disease in women relate in part to their smaller coronary arteries, higher coronary blood flow, and higher endothelial shear stress, which have profound effects on endothelial function and development or resistance to atherosclerosis, its symptomatic presentation, outcomes, and treatment. The complex interactions of focal stenosis, diffuse epicardial atherosclerotic coronary narrowing, and microvascular dysfunction make definitive diagnosis and management difficult by use of standard noninvasive and invasive physiological and anatomic technologies. However, quantitative rest-stress myocardial perfusion, best documented by positron emission tomography, combined with clinical circumstances usually provides a definitive diagnosis to guide management, including vigorous risk factor management and revascularization for patients with physiologically severe epicardial stenosis by quantitative positron emission tomography.

  14. Systemic activation of the transient receptor potential vanilloid subtype 4 channel causes endothelial failure and circulatory collapse: Part 2.

    PubMed

    Willette, Robert N; Bao, Weike; Nerurkar, Sandhya; Yue, Tian-Li; Doe, Chris P; Stankus, Gerald; Turner, Gregory H; Ju, Haisong; Thomas, Heath; Fishman, Cindy E; Sulpizio, Anthony; Behm, David J; Hoffman, Sandra; Lin, Zuojun; Lozinskaya, Irina; Casillas, Linda N; Lin, Min; Trout, Robert E Lee; Votta, Bartholomew J; Thorneloe, Kevin; Lashinger, Erin S R; Figueroa, David J; Marquis, Robert; Xu, Xiaoping

    2008-08-01

    The transient receptor potential (TRP) vanilloid subtype 4 (V4) is a nonselective cation channel that exhibits polymodal activation and is expressed in the endothelium, where it contributes to intracellular Ca2+ homeostasis and regulation of cell volume. The purpose of the present study was to evaluate the systemic cardiovascular effects of GSK1016790A, a novel TRPV4 activator, and to examine its mechanism of action. In three species (mouse, rat, and dog), the i.v. administration of GSK1016790A induced a dose-dependent reduction in blood pressure, followed by profound circulatory collapse. In contrast, GSK1016790A had no acute cardiovascular effects in the TRPV4-/- null mouse. Hemodynamic analyses in the dog and rat demonstrate a profound reduction in cardiac output. However, GSK1016790A had no effect on rate or contractility in the isolated, buffer-perfused rat heart, and it produced potent endothelial-dependent relaxation of rodent-isolated vascular ring segments that were abolished by nitric-oxide synthase (NOS) inhibition (N-nitro-L-arginine methyl ester; L-NAME), ruthenium red, and endothelial NOS (eNOS) gene deletion. However, the in vivo circulatory collapse was not altered by NOS inhibition (L-NAME) or eNOS gene deletion but was associated with (concentration and time appropriate) profound vascular leakage and tissue hemorrhage in the lung, intestine, and kidney. TRPV4 immunoreactivity was localized in the endothelium and epithelium in the affected organs. GSK1016790A potently induced rapid electrophysiological and morphological changes (retraction/condensation) in cultured endothelial cells. In summary, inappropriate activation of TRPV4 produces acute circulatory collapse associated with endothelial activation/injury and failure of the pulmonary microvascular permeability barrier. It will be important to determine the role of TRPV4 in disorders associated with edema and microvascular congestion.

  15. Cardiac catheterization

    MedlinePlus

    Catheterization - cardiac; Heart catheterization; Angina - cardiac catheterization; CAD - cardiac catheterization; Coronary artery disease - cardiac catheterization; Heart valve - cardiac catheterization; Heart failure - ...

  16. Retinal microvascular plasticity in a premature neonate.

    PubMed

    Kandasamy, Y; Hartley, L; Smith, R

    2017-01-31

    Dilation and abnormal tortuosity of retinal vessels are the hallmarks of severe retinopathy of prematurity (ROP) in premature infants. The stages of ROP are defined by vessel appearance at the interface between the vascular and avascular retinal areas. Deregulated signaling pathways involving hypoxia-inducible factors such as vascular endothelial growth factor (VEGF) are involved in the pathogenesis of ROP. VEGF-antagonists are increasingly being used as 'off-label medication' to treat this condition, with some success. We present Baby SM (female), who was born prematurely at 24 weeks gestation in a tertiary neonatal intensive care unit, and with a birth weight of 640 g. On screening at 35 weeks postmenstrual age (PMA), she was noted to have ROP, which became severe by 37 weeks PMA. She received one dose of intravitreal VEGF antagonist (Bevacizumab), resulting in a decrease in vessel tortuosity and dilation. However, repeat imaging at 4 weeks showed a re-emergence of vessel tortuosity. We believe the observed changes demonstrate an inherent retinal microvascular plasticity in premature neonates. With improved survival of extremely premature neonates and the availability of retinal imaging technology, we are now able to observe this plasticity.

  17. Sphingosine-1-Phosphate Signaling in Endothelial Disorders.

    PubMed

    Sanchez, Teresa

    2016-06-01

    Numerous preclinical studies indicate that sustained endothelial activation significantly contributes to tissue edema, perpetuates the inflammatory response, and exacerbates tissue injury ultimately resulting in organ failure. However, no specific therapies aimed at restoring endothelial function are available as yet. Sphingosine-1-phosphate (S1P) is emerging as a potent modulator of endothelial function and endothelial responses to injury. Recent studies indicate that S1PR are attractive targets to treat not only disorders of the arterial endothelium but also microvascular dysfunction caused by ischemic or inflammatory injury. In this article, we will review the current knowledge of the role of S1P and its receptors in endothelial function in health and disease, and we will discuss the therapeutic potential of targeting S1PR not only for disorders of the arterial endothelium but also the microvasculature. The therapeutic targeting of S1PR in the endothelium could help to bridge the gap between biomedical research in vascular biology and clinical practice.

  18. Transcriptional network analysis for the regulation of left ventricular hypertrophy and microvascular remodeling.

    PubMed

    Moreno-Moral, Aida; Mancini, Massimiliano; D'Amati, Giulia; Camici, Paolo; Petretto, Enrico

    2013-12-01

    Hypertension and cardiomyopathies share maladaptive changes of cardiac morphology, eventually leading to heart failure. These include left ventricular hypertrophy (LVH), myocardial fibrosis, and structural remodeling of coronary microcirculation, which is the morphologic hallmark of coronary microvascular dysfunction. To pinpoint the complex molecular mechanisms and pathways underlying LVH-associated cardiac remodeling independent of blood pressure effects, we employed gene network approaches to the rat heart. We used the Spontaneously Hypertensive Rat model showing many features of human hypertensive cardiomyopathy, for which we collected histological and histomorphometric data of the heart and coronary vasculature, and genome-wide cardiac gene expression. Here, we provide a large catalogue of gene co-expression networks in the heart that are significantly associated with quantitative variation in LVH, microvascular remodeling, and fibrosis-related traits. Many of these networks were significantly conserved to human idiopathic and/or ischemic cardiomyopathy patients, suggesting a potential role for these co-expressed genes in human heart disease.

  19. Brain endothelial dysfunction in cerebral adrenoleukodystrophy.

    PubMed

    Musolino, Patricia L; Gong, Yi; Snyder, Juliet M T; Jimenez, Sandra; Lok, Josephine; Lo, Eng H; Moser, Ann B; Grabowski, Eric F; Frosch, Matthew P; Eichler, Florian S

    2015-11-01

    See Aubourg (doi:10.1093/awv271) for a scientific commentary on this article.X-linked adrenoleukodystrophy is caused by mutations in the ABCD1 gene leading to accumulation of very long chain fatty acids. Its most severe neurological manifestation is cerebral adrenoleukodystrophy. Here we demonstrate that progressive inflammatory demyelination in cerebral adrenoleukodystrophy coincides with blood-brain barrier dysfunction, increased MMP9 expression, and changes in endothelial tight junction proteins as well as adhesion molecules. ABCD1, but not its closest homologue ABCD2, is highly expressed in human brain microvascular endothelial cells, far exceeding its expression in the systemic vasculature. Silencing of ABCD1 in human brain microvascular endothelial cells causes accumulation of very long chain fatty acids, but much later than the immediate upregulation of adhesion molecules and decrease in tight junction proteins. This results in greater adhesion and transmigration of monocytes across the endothelium. PCR-array screening of human brain microvascular endothelial cells after ABCD1 silencing revealed downregulation of both mRNA and protein levels of the transcription factor c-MYC (encoded by MYC). Interestingly, MYC silencing mimicked the effects of ABCD1 silencing on CLDN5 and ICAM1 without decreasing the levels of ABCD1 protein itself. Together, these data demonstrate that ABCD1 deficiency induces significant alterations in brain endothelium via c-MYC and may thereby contribute to the increased trafficking of leucocytes across the blood-brain barrier as seen in cerebral adrenouleukodystrophy.

  20. Inflammation-induced microvascular insulin resistance is an early event in diet-induced obesity.

    PubMed

    Zhao, Lina; Fu, Zhuo; Wu, Jing; Aylor, Kevin W; Barrett, Eugene J; Cao, Wenhong; Liu, Zhenqi

    2015-12-01

    Endothelial dysfunction and vascular insulin resistance usually coexist and chronic inflammation engenders both. In the present study, we investigate the temporal relationship between vascular insulin resistance and metabolic insulin resistance. We assessed insulin responses in all arterial segments, including aorta, distal saphenous artery and the microvasculature, as well as the metabolic insulin responses in muscle in rats fed on a high-fat diet (HFD) for various durations ranging from 3 days to 4 weeks with or without sodium salicylate treatment. Compared with controls, HFD feeding significantly blunted insulin-mediated Akt (protein kinase B) and eNOS [endothelial nitric oxide (NO) synthase] phosphorylation in aorta in 1 week, blunted vasodilatory response in small resistance vessel in 4 weeks and microvascular recruitment in as early as 3 days. Insulin-stimulated whole body glucose disposal did not begin to progressively decrease until after 1 week. Salicylate treatment fully inhibited vascular inflammation, prevented microvascular insulin resistance and significantly improved muscle metabolic responses to insulin. We conclude that microvascular insulin resistance is an early event in diet-induced obesity and insulin resistance and inflammation plays an essential role in this process. Our data suggest microvascular insulin resistance contributes to the development of metabolic insulin resistance in muscle and muscle microvasculature is a potential therapeutic target in the prevention and treatment of diabetes and its related complications.

  1. A Novel Human Tissue-Engineered 3-D Functional Vascularized Cardiac Muscle Construct

    PubMed Central

    Valarmathi, Mani T.; Fuseler, John W.; Davis, Jeffrey M.; Price, Robert L.

    2017-01-01

    Organ tissue engineering, including cardiovascular tissues, has been an area of intense investigation. The major challenge to these approaches has been the inability to vascularize and perfuse the in vitro engineered tissue constructs. Attempts to provide oxygen and nutrients to the cells contained in the biomaterial constructs have had varying degrees of success. The aim of this current study is to develop a three-dimensional (3-D) model of vascularized cardiac tissue to examine the concurrent temporal and spatial regulation of cardiomyogenesis in the context of postnatal de novo vasculogenesis during stem cell cardiac regeneration. In order to achieve the above aim, we have developed an in vitro 3-D functional vascularized cardiac muscle construct using human induced pluripotent stem cell-derived embryonic cardiac myocytes (hiPSC-ECMs) and human mesenchymal stem cells (hMSCs). First, to generate the prevascularized scaffold, human cardiac microvascular endothelial cells (hCMVECs) and hMSCs were co-cultured onto a 3-D collagen cell carrier (CCC) for 7 days under vasculogenic culture conditions. In this milieu, hCMVECs/hMSCs underwent maturation, differentiation, and morphogenesis characteristic of microvessels, and formed extensive plexuses of vascular networks. Next, the hiPSC-ECMs and hMSCs were co-cultured onto this generated prevascularized CCCs for further 7 or 14 days in myogenic culture conditions. Finally, the vascular and cardiac phenotypic inductions were analyzed at the morphological, immunological, biochemical, molecular, and functional levels. Expression and functional analyses of the differentiated cells revealed neo-angiogenesis and neo-cardiomyogenesis. Thus, our unique 3-D co-culture system provided us the apt in vitro functional vascularized 3-D cardiac patch that can be utilized for cellular cardiomyoplasty. PMID:28194397

  2. A Novel Human Tissue-Engineered 3-D Functional Vascularized Cardiac Muscle Construct.

    PubMed

    Valarmathi, Mani T; Fuseler, John W; Davis, Jeffrey M; Price, Robert L

    2017-01-01

    Organ tissue engineering, including cardiovascular tissues, has been an area of intense investigation. The major challenge to these approaches has been the inability to vascularize and perfuse the in vitro engineered tissue constructs. Attempts to provide oxygen and nutrients to the cells contained in the biomaterial constructs have had varying degrees of success. The aim of this current study is to develop a three-dimensional (3-D) model of vascularized cardiac tissue to examine the concurrent temporal and spatial regulation of cardiomyogenesis in the context of postnatal de novo vasculogenesis during stem cell cardiac regeneration. In order to achieve the above aim, we have developed an in vitro 3-D functional vascularized cardiac muscle construct using human induced pluripotent stem cell-derived embryonic cardiac myocytes (hiPSC-ECMs) and human mesenchymal stem cells (hMSCs). First, to generate the prevascularized scaffold, human cardiac microvascular endothelial cells (hCMVECs) and hMSCs were co-cultured onto a 3-D collagen cell carrier (CCC) for 7 days under vasculogenic culture conditions. In this milieu, hCMVECs/hMSCs underwent maturation, differentiation, and morphogenesis characteristic of microvessels, and formed extensive plexuses of vascular networks. Next, the hiPSC-ECMs and hMSCs were co-cultured onto this generated prevascularized CCCs for further 7 or 14 days in myogenic culture conditions. Finally, the vascular and cardiac phenotypic inductions were analyzed at the morphological, immunological, biochemical, molecular, and functional levels. Expression and functional analyses of the differentiated cells revealed neo-angiogenesis and neo-cardiomyogenesis. Thus, our unique 3-D co-culture system provided us the apt in vitro functional vascularized 3-D cardiac patch that can be utilized for cellular cardiomyoplasty.

  3. Skin microvascular reactivity in children and adolescents with type 1 diabetes in relation to levels of physical activity and aerobic fitness.

    PubMed

    Roche, Denise M; Edmunds, Sarah; Cable, Tim; Didi, Mo; Stratton, Gareth

    2008-11-01

    No studies to date have evaluated the relationship between exercise and microvascular function in youth with type 1 diabetes mellitus (T1DM). Twenty-nine complication free children and adolescents with T1DM were assessed for skin microvascular reactivity, aerobic fitness (VO2peak) and physical activity. VO2peak but not physical activity was significantly and independently associated with maximal hyperemia of the skin microcirculation (p < .01). No significant associations were found between venoarteriolar reflex (VAR) vasoconstriction and VO2peak or physical activity. Aerobic fitness may be an important indicator or mediator of effective microvascular endothelial function in youth with T1DM.

  4. Endurance, interval sprint, and resistance exercise training: impact on microvascular dysfunction in type 2 diabetes.

    PubMed

    Olver, T Dylan; Laughlin, M Harold

    2016-02-01

    Type 2 diabetes (T2D) alters capillary hemodynamics, causes capillary rarefaction in skeletal muscle, and alters endothelial and vascular smooth muscle cell phenotype, resulting in impaired vasodilatory responses. These changes contribute to altered blood flow responses to physiological stimuli, such as exercise and insulin secretion. T2D-induced microvascular dysfunction impairs glucose and insulin delivery to skeletal muscle (and other tissues such as skin and nervous), thereby reducing glucose uptake and perpetuating hyperglycemia and hyperinsulinemia. In patients with T2D, exercise training (EX) improves microvascular vasodilator and insulin signaling and attenuates capillary rarefaction in skeletal muscle. EX-induced changes subsequently augment glucose and insulin delivery as well as glucose uptake. If these adaptions occur in a sufficient amount of tissue, and skeletal muscle in particular, chronic exposure to hyperglycemia and hyperinsulinemia and the risk of microvascular complications in all vascular beds will decrease. We postulate that EX programs that engage as much skeletal muscle mass as possible and recruit as many muscle fibers within each muscle as possible will generate the greatest improvements in microvascular function, providing that the duration of the stimulus is sufficient. Primary improvements in microvascular function occur in tissues (skeletal muscle primarily) engaged during exercise, and secondary improvements in microvascular function throughout the body may result from improved blood glucose control. We propose that the added benefit of combined resistance and aerobic EX programs and of vigorous intensity EX programs is not simply "more is better." Rather, we believe the additional benefit is the result of EX-induced adaptations in and around more muscle fibers, resulting in more muscle mass and the associated microvasculature being changed. Thus, to acquire primary and secondary improvements in microvascular function and improved

  5. Nupr1/Chop signal axis is involved in mitochondrion-related endothelial cell apoptosis induced by methamphetamine.

    PubMed

    Cai, D; Huang, E; Luo, B; Yang, Y; Zhang, F; Liu, C; Lin, Z; Xie, W-B; Wang, H

    2016-03-31

    Methamphetamine (METH) abuse has been a serious global public health problem for decades. Previous studies have shown that METH causes detrimental effects on the nervous and cardiovascular systems. METH-induced cardiovascular toxicity has been, in part, attributed to its destructive effect on vascular endothelial cells. However, the underlying mechanism of METH-caused endothelium disruption has not been investigated systematically. In this study, we identified a novel pathway involved in endothelial cell apoptosis induced by METH. We demonstrated that exposure to METH caused mitochondrial apoptosis in human umbilical vein endothelial cells and rat cardiac microvascular endothelial cells in vitro as well as in rat cardiac endothelial cells in vivo. We found that METH mediated endothelial cell apoptosis through Nupr1-Chop/P53-PUMA/Beclin1 signaling pathway. Specifically, METH exposure increased the expression of Nupr1, Chop, P53 and PUMA. Elevated p53 expression raised up PUMA expression, which initiated mitochondrial apoptosis by downregulating antiapoptotic Bcl-2, followed by upregulation of proapoptotic Bax, resulting in translocation of cytochrome c (cyto c), an apoptogenic factor, from the mitochondria to cytoplasm and activation of caspase-dependent pathways. Interestingly, increased Beclin1, upregulated by Chop, formed a ternary complex with Bcl-2, thereby decreasing the dissociative Bcl-2. As a result, the ratio of dissociative Bcl-2 to Bax was also significantly decreased, which led to translocation of cyto c and initiated more drastic apoptosis. These findings were supported by data showing METH-induced apoptosis was significantly inhibited by silencing Nupr1, Chop or P53, or by PUMA or Beclin1 knockdown. Based on the present data, a novel mechanistic model of METH-induced endothelial cell toxicity is proposed. Collectively, these results highlight that the Nupr1-Chop/P53-PUMA/Beclin1 pathway is essential for mitochondrion-related METH-induced endothelial

  6. Hypoxia inducible factor-1: regulation by nitric oxide in posthypoxic microvascular endothelium.

    PubMed

    Natarajan, Ramesh; Jones, Drew G; Fisher, Bernard J; Wallace, Timothy J; Ghosh, Shobha; Fowler, Alpha A

    2005-10-01

    Microvascular endothelial cells provide a critical regulatory interface between blood constituents and tissue. Hypoxia inducible factor-1 (HIF-1) is a key transcription factor required for expression of hypoxia-dependent genes. We employed a model of hypoxia and reoxygenation (H/R) using the dermal microvascular endothelial cell line HMEC-1 to examine the effects of altered oxygen concentrations on microvascular HIF-1 expression and nitric oxide (NO) formation. Hypoxia increased inducible NO synthase (iNOS) mRNA in a time-dependent manner in HMEC-1. However, endothelial NO synthase mRNA progressively declined during hypoxia. H/R promoted significant increases in cellular nitrite levels that were significantly abrogated by the specific iNOS inhibitor N6-(1-iminoethyl)-L-lysine, di hy drochloride. Exogenous NO promoted stabilization of the alpha subunit of HIF-1 and produced functional DNA binding. Exposure of HMEC-1 to H/R resulted in previously unrecognized biphasic HIF-1alpha stabilization during reoxygenation. When the iNOS gene was silenced through the use of iNOS-specific small interfering RNA, HIF-1alpha stabilization and HIF-1 activation were dramatically diminished, suggesting that inducible NOS-derived NO is a key factor sustaining HIF-1 activation during both hypoxia and reoxygenation.

  7. CTC-Endothelial Cell Interactions during Metastasis

    DTIC Science & Technology

    2014-06-01

    study of CTC-Endothelial interactions, as it introduced cell aggregation in the chamber, likely because of the presence of contaminating RBCs in PBMC...interactions, as it introduced cell aggregation in the chamber, likely because of the presence of contaminating RBCs in PBMC preparations, which disturbed the...microvascular endothelium via E- selectin/E-selectin ligand interactions under shear flow theoretically promote extravasation and contribute to the

  8. Binding of human endothelium to Ulex europaeus I-coated Dynabeads: application to the isolation of microvascular endothelium.

    PubMed

    Jackson, C J; Garbett, P K; Nissen, B; Schrieber, L

    1990-06-01

    A major problem encountered when isolating human microvascular endothelium is the presence of contaminating cells such as fibroblasts that rapidly over-grow the endothelial cells. We describe here a simple, rapid technique for purifying endothelial cells derived from the microvasculature of neonatal foreskin and osteoarthritic and rheumatoid arthritic synovium. This technique is based on the selective binding of the lectin Ulex europaeus I (UEA I) to the endothelial cell surface via fucose residues. Initially UEA I was covalently bound to tosyl-activated super-paramagnetic polystyrene beads (Dynabeads) by incubation for 24 h at room temperature. Cells were isolated by extracting microvascular segments from enzyme-treated (trypsin and Pronase) cubes of tissue. The mixed population of cells obtained were purified by incubating them at 4 degrees C for 10 min with the UEA I-coated Dynabeads. Endothelium bound to the beads whilst contaminating cells were removed by five washes with HBSS using a magnetic particle concentrator. The endothelial cells thus obtained grew to confluence as a cobblestone-like monolayer and expressed von Willebrand factor antigen. The cells were released from the Dynabeads by the competitive binding of fucose (10 min at 4 degrees C). This new method is simple and reproducible and allows pure human microvascular endothelial cells to be cultured within 2 h of obtaining a specimen.

  9. Assessment of endothelial and neurovascular function in human skin microcirculation.

    PubMed

    Roustit, Matthieu; Cracowski, Jean-Luc

    2013-07-01

    Peripheral microvascular dysfunction has been described in many physiological and pathological conditions. Owing to its accessibility, the cutaneous microcirculation provides a unique index of microvascular function. Skin microvascular function has therefore been proposed as a prognostic marker or for evaluating the effect of drugs on the microcirculation. Various reactivity tests, coupled with techniques measuring skin blood flux, are used to non-invasively explore both endothelial and neurovascular microvascular functioning in humans. We review the advantages and limitations of the main reactivity tests, including post-occlusive reactive hyperemia, local thermal hyperemia, pressure-induced vasodilation, and iontophoresis of vasodilators, combined with measurement techniques such as laser Doppler and laser speckle contrast imaging. Recent advances in our comprehension of the physiological pathways underlying these reactivity tests, as well as technological developments in microcirculation imaging, have provided reliable and reproducible tools for studying the microcirculation.

  10. Rab5-mediated VE-cadherin internalization regulates the barrier function of the lung microvascular endothelium

    PubMed Central

    Yang, Junjun; Yao, Wei; Qian, Guisheng; Wei, Zhenghua

    2016-01-01

    The small GTPase Rab5 has been well defined to control the vesicle-mediated plasma membrane protein transport to the endosomal compartment. However, its function in the internalization of vascular endothelial (VE)-cadherin, an important component of adherens junctions, and as a result regulating the endothelial cell polarity and barrier function remain unknown. Here, we demonstrated that lipopolysaccharide (LPS) simulation markedly enhanced the activation and expression of Rab5 in human pulmonary microvascular endothelial cells (HPMECs), which is accompanied by VE-cadherin internalization. In parallel, LPS challenge also induced abnormal cell polarity and dysfunction of the endothelial barrier in HPMECs. LPS stimulation promoted the translocation of VE-cadherin from the plasma membrane to intracellular compartments, and intracellularly expressed VE-cadherin was extensively colocalized with Rab5. Small interfering RNA (siRNA)-mediated depletion of Rab5a expression attenuated the disruption of LPS-induced internalization of VE-cadherin and the disorder of cell polarity. Furthermore, knockdown of Rab5 inhibited the vascular endothelial hyperpermeability and protected endothelial barrier function from LPS injury, both in vitro and in vivo. These results suggest that Rab5 is a critical mediator of LPS-induced endothelial barrier dysfunction, which is likely mediated through regulating VE-cadherin internalization. These findings provide evidence, implicating that Rab5a is a potential therapeutic target for preventing endothelial barrier disruption and vascular inflammation. PMID:26112597

  11. Rab5-mediated VE-cadherin internalization regulates the barrier function of the lung microvascular endothelium.

    PubMed

    Yang, Junjun; Yao, Wei; Qian, Guisheng; Wei, Zhenghua; Wu, Guangyu; Wang, Guansong

    2015-12-01

    The small GTPase Rab5 has been well defined to control the vesicle-mediated plasma membrane protein transport to the endosomal compartment. However, its function in the internalization of vascular endothelial (VE)-cadherin, an important component of adherens junctions, and as a result regulating the endothelial cell polarity and barrier function remain unknown. Here, we demonstrated that lipopolysaccharide (LPS) simulation markedly enhanced the activation and expression of Rab5 in human pulmonary microvascular endothelial cells (HPMECs), which is accompanied by VE-cadherin internalization. In parallel, LPS challenge also induced abnormal cell polarity and dysfunction of the endothelial barrier in HPMECs. LPS stimulation promoted the translocation of VE-cadherin from the plasma membrane to intracellular compartments, and intracellularly expressed VE-cadherin was extensively colocalized with Rab5. Small interfering RNA (siRNA)-mediated depletion of Rab5a expression attenuated the disruption of LPS-induced internalization of VE-cadherin and the disorder of cell polarity. Furthermore, knockdown of Rab5 inhibited the vascular endothelial hyperpermeability and protected endothelial barrier function from LPS injury, both in vitro and in vivo. These results suggest that Rab5 is a critical mediator of LPS-induced endothelial barrier dysfunction, which is likely mediated through regulating VE-cadherin internalization. These findings provide evidence, implicating that Rab5a is a potential therapeutic target for preventing endothelial barrier disruption and vascular inflammation.

  12. Hyperosmolality-mediated peritoneal microvascular vasodilation is linked to aquaporin function.

    PubMed

    Zakaria, El Rasheid; Althani, Asma; Fawzi, Ashraf A; Fituri, Omar M

    2014-01-01

    Glucose-based peritoneal dialysis (PD) solutions dilate the parietal and visceral peritoneal microvasculature by endothelium-dependent mechanisms that primarily involve hyperosmolality. This PD-mediated dilation occurs by active intracellular glucose uptake and adenosine Al receptor activation, and by hyperosmolality-stimulated glibenclamide-sensitive potassium channels. Both pathways invoke NO as a second messenger for vasodilation. We hypothesized that during crystalloid-induced osmosis, the osmotic water flux through the transendothelial water-exclusive aquaporin 1 (AQP1) channels is the primary mechanism whereby the endothelium is being stimulated to instigate hyperosmolality-driven vasodilation. Four microvascular levels (diameters in the range 6 - 100 microm) were visualized by intravital videomicroscopy of the terminal ileum in anesthetized rats. Microvascular diameters and flow were measured after topical exposure to a 5% hypertonic mannitol or 2.5% glucose-based PD solution, at baseline and after brief tissue pre-treatment (with 0.1% glutaraldehyde for 10 seconds) or after combined tissue pre-treatment and pharmacologic blockade of AQP1 with HgCl2 (100 micromol/L). Vascular endothelial integrity was verified by the response to acetylcholine (10(-4) mol/L) and sodium nitroprusside (10(-4) mol/L). The hyperosmolar solutions both caused rapid and sustained vasodilation at all microvascular levels, which was not altered by tissue pre-treatment. Inhibition of AQP1 completely abolished the mannitol-induced vasodilation and markedly attenuated the PD fluid-mediated vasodilation. Neither glutaraldehyde pre-treatment nor HgCl2 affected tissue integrity or endothelial cell function. We conclude that the peritoneal microvascular vasodilation caused by hyperosmolar PD fluid is instigated by the osmotic water flux through AQP1. Clinical PD solutions have components other than hyperosmolality that can induce endothelium-dependent peritoneal microvascular vasodilation

  13. Vascular endothelial overexpression of human CYP2J2 (Tie2-CYP2J2 Tr) modulates cardiac oxylipin profiles and enhances coronary reactive hyperemia in mice

    PubMed Central

    Hanif, Ahmad; Edin, Matthew L.; Zeldin, Darryl C.; Morisseau, Christophe; Falck, John R.

    2017-01-01

    Arachidonic acid is metabolized to epoxyeicosatrienoic acids (EETs) by cytochrome (CYP) P450 epoxygenases, and to ω-terminal hydroxyeicosatetraenoic acids (HETEs) by ω-hydroxylases. EETs and HETEs often have opposite biologic effects; EETs are vasodilatory and protect against ischemia/reperfusion injury, while ω-terminal HETEs are vasoconstrictive and cause vascular dysfunction. Other oxylipins, such as epoxyoctadecaenoic acids (EpOMEs), hydroxyoctadecadienoic acids (HODEs), and prostanoids also have varied vascular effects. Post-ischemic vasodilation in the heart, known as coronary reactive hyperemia (CRH), protects against potential damage to the heart muscle caused by ischemia. The relationship among CRH response to ischemia, in mice with altered levels of CYP2J epoxygenases has not yet been investigated. Therefore, we evaluated the effect of endothelial overexpression of the human cytochrome P450 epoxygenase CYP2J2 in mice (Tie2-CYP2J2 Tr) on oxylipin profiles and CRH. Additionally, we evaluated the effect of pharmacologic inhibition of CYP-epoxygenases and inhibition of ω-hydroxylases on CRH. We hypothesized that CRH would be enhanced in isolated mouse hearts with vascular endothelial overexpression of human CYP2J2 through modulation of oxylipin profiles. Similarly, we expected that inhibition of CYP-epoxygenases would reduce CRH, whereas inhibition of ω-hydroxylases would enhance CRH. Compared to WT mice, Tie2-CYP2J2 Tr mice had enhanced CRH, including repayment volume, repayment duration, and repayment/debt ratio (P < 0.05). Similarly, inhibition of ω-hydroxylases increased repayment volume and repayment duration, in Tie2-CYP2J2 Tr compared to WT mice (P < 0.05). Endothelial overexpression of CYP2J2 significantly changed oxylipin profiles, including increased EETs (P < 0.05), increased EpOMEs (P < 0.05), and decreased 8-iso-PGF2α (P < 0.05). Inhibition of CYP epoxygenases with MS-PPOH attenuated CRH (P < 0.05). Ischemia caused a decrease in mid

  14. Effects of hyperoxia on microvascular cells in vitro

    SciTech Connect

    D'Amore, P.A.; Sweet, E.

    1987-02-01

    Microvascular cells are most vulnerable to direct oxygen damage. Using an in vitro model system we have investigated the effect of elevated oxygen on the proliferation, morphology, and integrity of microvascular endothelial cells (EC) and pericytes. Cultivation of these cells at oxygen concentrations of 40% for 1 wk resulted in the inhibition of EC proliferation but had no effect on the growth of the pericytes. Similarly, hyperoxia induced a dramatic change in the shape of the EC, increasing their spread area by close to six-fold. Under the same conditions, the spread area of the pericytes was unaffected. To understand the effect of the hyperoxic treatment on the cells, the integrity of various membrane systems was assessed. /sup 51/Cr release was used to monitor plasma membrane integrity. There was no difference in chromium release by EC and pericytes over the 7 d of growth under normoxic and hyperoxic conditions. Mitochondrial integrity was examined by staining the cells with Rhodamine 123, which is selectively accumulated by the mitochondria. The staining pattern of the mitochondria of both EC and pericytes was altered by growth in the elevated oxygen. Finally, the lysosomes were visualized using acridine orange. The acridine orange staining pattern revealed enlarged and perinuclear lysosomes in the EC but no change in the pericyte lysosomal staining pattern. Thus, the cells of the microvasculature seem to be differentially affected by hyperoxia, a fact that may be significant in the etiology of reperfusion injury, ischemic disease, and pathologies associated with prematurity.

  15. Diabetic microvascular complications: possible targets for improved macrovascular outcomes

    PubMed Central

    D’Elia, John A; Bayliss, George; Roshan, Bijan; Maski, Manish; Gleason, Ray E; Weinrauch, Larry A

    2011-01-01

    The results of recent outcome trials challenge hypotheses that tight control of both glycohemoglobin and blood pressure diminishes macrovascular events and survival among type 2 diabetic patients. Relevant questions exist regarding the adequacy of glycohemoglobin alone as a measure of diabetes control. Are we ignoring mechanisms of vasculotoxicity (profibrosis, altered angiogenesis, hypertrophy, hyperplasia, and endothelial injury) inherent in current antihyperglycemic medications? Is the polypharmacy for lowering cholesterol, triglyceride, glucose, and systolic blood pressure producing drug interactions that are too complex to be clinically identified? We review angiotensin–aldosterone mechanisms of tissue injury that magnify microvascular damage caused by hyperglycemia and hypertension. Many studies describe interruption of these mechanisms, without hemodynamic consequence, in the preservation of function in type 1 diabetes. Possible interactions between the renin–angiotensin–aldosterone system and physiologic glycemic control (through pulsatile insulin release) suggest opportunities for further clinical investigation. PMID:21694944

  16. Microvascular Function in Aging Among Women Living with HIV.

    PubMed

    Monsuez, Jean-Jacques; Belin, Catherine; Bouchaud, Olivier

    2016-12-01

    Combined antiretroviral therapy (CART) has turned HIV-infection to a treatable chronic disease during which many patients survive to middle and older age. However, they prematurely develop non-AIDS comorbidities such as cardiovascular disease, metabolic syndrome, diabetes, and HIV-associated neurocognitive disorders (HAND). Microcirculatory changes and endothelial dysfunction occur early both in HIV-infected and in aging patients, in whom they usually precede cardiovascular and neurocognitive impairments. Also, mild cognitive involvement has been reported in women during the menopausal transition. Disruption of the blood-brain barrier, as well as microvascular and cerebral blood flow changes, has been reported in HIV patients with HAND, including postmenopausal women. However, most studies addressing this issue included women aged less than 50 years. Whether HIV-infected women growing older with CART would be subsequently exposed to an increased progression of cognitive impairment overtime remains unknown.

  17. Adult cutaneous hemangiomas are composed of nonreplicating endothelial cells.

    PubMed

    Tuder, R M; Young, R; Karasek, M; Bensch, K

    1987-12-01

    Thirty-four human "cherry" dermal hemangiomas were studied by electron microscopy, immunohistochemistry, and cell culture to assess the neoplastic nature of these lesions. Electron microscopy of nine hemangiomas revealed a pronounced thickening of the basement membrane (0.6 to 14 micron) in 93% of the total 158 vascular structures examined within the lesions. This increase was caused mainly by multiple layers of basal lamina, which were irregular in outline and frequently associated with pericytes. Basement membrane changes were present both in the periphery of the hemangiomas, as well as in the center of the lesions. Immature vessels could not be identified and mitoses were absent in all endothelial cells. Using an immunohistochemical marker (Ki67) specific for proliferating cells in G2 and S phases, positive staining was not found in the endothelial cells lining the hemangiomatous vessels, whereas basal epidermal keratinocytes in the same preparations and cultured microvascular endothelial cells expressed the antigen. Endothelial cells of nine hemangiomas did not stain with an activation-related antibody (E12) specific for endothelial cells. When endothelial cells from 14 hemangiomas were isolated and cultured under conditions that support the growth of normal human skin microvascular endothelial cells, the cells of hemangiomatous origin failed to grow. We conclude that the adult hemangiomas may not be true neoplasms, but a tissue overgrowth composed of mature vessels resembling dermal venules, lined by endothelial cells with virtually no turnover.

  18. Effect of high altitude and exercise on microvascular parameters in acclimatized subjects.

    PubMed

    Bauer, Andreas; Demetz, Florian; Bruegger, Dirk; Schmoelz, Martin; Schroepfer, Sebastian; Martignoni, André; Baschnegger, Heiko; Hoelzl, Josef; Thiel, Manfred; Choukér, Alexander; Peter, Klaus; Gamble, John; Christ, Frank

    2006-02-01

    The role of microvascular fluid shifts in the adaptation to hypobaric hypoxia and its contribution to the pathophysiology of AMS (acute mountain sickness) is unresolved. In a systematic prospective study, we investigated the effects of hypobaric hypoxia and physical exercise alone, and in combination, on microvascular fluid exchange and related factors. We used computer-assisted VCP (venous congestion plethysmography) on the calves of ten altitude-acclimatized volunteers. We investigated the effects of: (i) actively climbing to an altitude of 3196 m, (ii) airlifting these subjects to the same altitude, and (iii) exercise at low altitude. CFC (capillary filtration capacity), Pvi (isovolumetric venous pressure) and Qa (calf blood flow) were assessed before and after each procedure and then repeated after an overnight rest. Measurements of CFC showed no evidence of increased microvascular permeability after any of the procedures. Pvi was significantly decreased (P<0.001) from 20.3+/-4.4 to 8.9+/-4.3 mmHg after active ascent, and was still significantly lower (P=0.009) after overnight rest at high altitude (13.6+/-5.9 mmHg). No such changes were observed after the passive ascent (16.7+/-4.0 mmHg at baseline; 17.3+/-4.5 mmHg after passive ascent; and 19.9+/-5.3 mmHg after overnight rest) or after exercise at low altitude. After the active ascent, Qa was significantly increased. We also found a significant correlation between Qa, Pvi and the number of circulating white blood cells. In conclusion, we found evidence to support the hypothesis that increased microvascular permeability associated with AMS does not occur in acclimatized subjects. We also observed that the microvascular equilibrium pressure (Pvi) fell in inverse relation to the increase in Qa, especially in hypoxic exercise. We hypothesize that this inverse relationship reflects the haemodynamic changes at the microvascular interface, possibly attributable to the flow-induced increases in endothelial surface

  19. Long-term high-fat diet induces hippocampal microvascular insulin resistance and cognitive dysfunction.

    PubMed

    Fu, Zhuo; Wu, Jing; Nesil, Tanseli; Li, Ming D; Aylor, Kevin W; Liu, Zhenqi

    2017-02-01

    Insulin action on hippocampus improves cognitive function, and obesity and type 2 diabetes are associated with decreased cognitive function. Cerebral microvasculature plays a critical role in maintaining cerebral vitality and function by supplying nutrients, oxygen, and hormones such as insulin to cerebral parenchyma, including hippocampus. In skeletal muscle, insulin actively regulates microvascular opening and closure, and this action is impaired in the insulin-resistant states. To examine insulin's action on hippocampal microvasculature and parenchyma and the impact of diet-induced obesity, we determined cognitive function and microvascular insulin responses, parenchyma insulin responses, and capillary density in the hippocampus in 2- and 8-mo-old rats on chow diet and 8-mo-old rats on a long-term high-fat diet (6 mo). Insulin infusion increased hippocampal microvascular perfusion in rats on chow diet by ~80-90%. High-fat diet feeding completely abolished insulin-mediated microvascular responses and protein kinase B phosphorylation but did not alter the capillary density in the hippocampus. This was associated with a significantly decreased cognitive function assessed using both the two-trial spontaneous alternation behavior test and the novel object recognition test. As the microvasculature provides the needed endothelial surface area for delivery of nutrients, oxygen, and insulin to hippocampal parenchyma, we conclude that hippocampal microvascular insulin resistance may play a critical role in the development of cognitive impairment seen in obesity and diabetes. Our results suggest that improvement in hippocampal microvascular insulin sensitivity might help improve or reverse cognitive function in the insulin-resistant states.

  20. Endothelial Progenitor Cells Physiology and Metabolic Plasticity in Brain Angiogenesis and Blood-Brain Barrier Modeling

    PubMed Central

    Malinovskaya, Natalia A.; Komleva, Yulia K.; Salmin, Vladimir V.; Morgun, Andrey V.; Shuvaev, Anton N.; Panina, Yulia A.; Boitsova, Elizaveta B.; Salmina, Alla B.

    2016-01-01

    Currently, there is a considerable interest to the assessment of blood-brain barrier (BBB) development as a part of cerebral angiogenesis developmental program. Embryonic and adult angiogenesis in the brain is governed by the coordinated activity of endothelial progenitor cells, brain microvascular endothelial cells, and non-endothelial cells contributing to the establishment of the BBB (pericytes, astrocytes, neurons). Metabolic and functional plasticity of endothelial progenitor cells controls their timely recruitment, precise homing to the brain microvessels, and efficient support of brain angiogenesis. Deciphering endothelial progenitor cells physiology would provide novel engineering approaches to establish adequate microfluidically-supported BBB models and brain microphysiological systems for translational studies. PMID:27990124

  1. Coronary Microvascular Dysfunction and Microvascular Angina: A Systematic Review of Therapies

    PubMed Central

    Marinescu, Mark A; Löffler, Adrián I.; Ouellette, Michelle; Smith, Lavone; Kramer, Christopher M.; Bourque, Jamieson

    2015-01-01

    Angina without coronary artery disease (CAD) has substantial morbidity and is present in 10–30% of patients undergoing angiography. Coronary microvascular dysfunction (CMD) is present in 50–65% of these patients. The optimal treatment of this cohort is undefined. We performed a systematic review to evaluate treatment strategies for objectively defined CMD in the absence of CAD. We included studies assessing therapy in human subjects with angina and coronary flow reserve (CFR) or myocardial perfusion reserve (MPR) <2.5 by positron emission tomography (PET), cardiac magnetic resonance imaging (CMR), dilution methods, or intracoronary Doppler in the absence of coronary artery stenosis ≥50% or structural heart disease. Only 8 articles met strict inclusion criteria. The articles were heterogeneous, using different treatments, end-points, and definitions of CMD. Small sample sizes severely limit the power of these studies, with an average of 11 patients per analysis. Studies evaluating, sildenafil, quinapril, estrogen, and transcutaneous electrical nerve stimulation (TENS) application demonstrated benefits in their respective endpoints. No benefit was found with L-arginine, doxazosin, pravastatin, and diltiazem. Our systematic review highlights that there is little data to support therapies for CMD. We assess the data meeting rigorous inclusion criteria and review the related but excluded literature. We additionally describe the next steps needed to address this research gap, including a standardized definition of CMD, routine assessment of CMD in studies of chest pain without obstructive CAD, and specific therapy assessment in the population with confirmed CMD. PMID:25677893

  2. Endothelial progenitor cells in chronic obstructive pulmonary disease and emphysema.

    PubMed

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

    2017-01-01

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

  3. Endothelial progenitor cells in chronic obstructive pulmonary disease and emphysema

    PubMed Central

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

    2017-01-01

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

  4. Bubble-Induced Endothelial Microparticles Promote Endothelial Dysfunction

    PubMed Central

    Huang, Guoyang; Zhang, Kun; Qing, Long; Liu, Wenwu; Xu, Weigang

    2017-01-01

    Decompression sickness is a systemic pathophysiological process caused by bubbles and endothelial microparticles (EMPs) are established markers reflecting competency of endothelial function and vascular biology. Here, we investigated the effects of bubble-induced EMPs on endothelial cells in vitro and vivo. Rat pulmonary microvascular endothelial cells (PMVECs) were isolated and stimulated by bubbles and bubble-induced EMPs were collected and incubated with normal PMVECs in vitro. Cell viability and apoptosis were detected using Cell Counting Kit-8 assay and Annexin V FITC/PI double staining, respectively. Cell permeability and pro-inflammatory cytokines were determined by electric cell substrate impedance sensing and enzyme-linked immunosorbent assay, respectively. Intracellular nitric oxide and reactive oxygen species production were analyzed microscopically. In vivo study, bubble-induced EMPs were intravenously injected to the rats and soluble thrombomodulin, intercellular adhesion molecule 1, and vascullar adhesion molecule 1 were involved in evaluating endothelial dysfunction. In our study, bubble stimulus resulted in a significant increase of EMPs release by 3 fold. Bubble-induced EMPs significantly decreased cell viability and increased cell apoptosis. Moreover, bubble-induced EMPs induced abnormal increase of cell permeability and over-expression of pro-inflammatory cytokines. Intracellular ROS production increased while NO production decreased. These negative effects caused by bubble-induced EMPs were remarkably suppressed when EMPs pretreated with surfactant FSN-100. Finally, intravenous injection of bubble-induced EMPs caused elevations of soluble thrombomodulin and pro-inflammatory cytokines in the circulation. Altogether, our results demonstrated that bubble-induced EMPs can mediate endothelial dysfunction in vitro and vivo, which can be attenuated by EMPs abatement strategy. These data expanded our horizon of the detrimental effects of bubble

  5. Informational dynamics of vasomotion in microvascular networks: a review.

    PubMed

    Pradhan, R K; Chakravarthy, V S

    2011-02-01

    Vasomotion refers to spontaneous oscillation of small vessels observed in many microvascular beds. It is an intrinsic phenomenon unrelated to cardiac rhythm or neural and hormonal regulation. Vasomotion is found to be particularly prominent under conditions of metabolic stress. In spite of a significant existent literature on vasomotion, its physiological and pathophysiological roles are not clear. It is thought that modulation of vasomotion by vasoactive substances released by metabolizing tissue plays a role in ensuring optimal delivery of nutrients to the tissue. Vasomotion rhythms exhibit a great variety of temporal patterns from regular oscillations to chaos. The nature of vasomotion rhythm is believed to be significant to its function, with chaotic vasomotion offering several physiological advantages over regular, periodic vasomotion. In this article, we emphasize that vasomotion is best understood as a network phenomenon. When there is a local metabolic demand in tissue, an ideal vascular response should extend beyond local microvasculature, with coordinated changes over multiple vascular segments. Mechanisms of information transfer over a vessel network have been discussed in the literature. The microvascular system may be regarded as a network of dynamic elements, interacting, either over the vascular anatomical network via gap junctions, or physiologically by exchange of vasoactive substances. Drawing analogies with spatiotemporal patterns in neuronal networks of central nervous system, we ask if properties like synchronization/desynchronization of vasomotors have special significance to microcirculation. Thus the contemporary literature throws up a novel view of microcirculation as a network that exhibits complex, spatiotemporal and informational dynamics.

  6. Focally regulated endothelial proliferation and cell death in human synovium.

    PubMed Central

    Walsh, D. A.; Wade, M.; Mapp, P. I.; Blake, D. R.

    1998-01-01

    Angiogenesis and vascular insufficiency each may support the chronic synovial inflammation of rheumatoid arthritis. We have shown by quantitative immunohistochemistry and terminal uridyl deoxynucleotide nick end labeling that endothelial proliferation and cell death indices were each increased in synovia from patients with rheumatoid arthritis compared with osteoarthritic and noninflamed controls, whereas endothelial fractional areas did not differ significantly among disease groups. Markers of proliferation were associated with foci immunoreactive for vascular endothelial growth factor and integrin alpha(v)beta3, whereas cell death was observed in foci in which immunoreactivities for these factors were weak or absent. No association was found with thrombospondin immunoreactivity. The balance between angiogenesis and vascular regression in rheumatoid synovitis may be determined by the focal expression of angiogenic and endothelial survival factors. Increased endothelial cell turnover may contribute to microvascular dysfunction and thereby facilitate persistent synovitis. Images Figure 1 Figure 3 Figure 4 PMID:9502411

  7. Globular adiponectin ameliorates metabolic insulin resistance via AMPK-mediated restoration of microvascular insulin responses.

    PubMed

    Zhao, Lina; Fu, Zhuo; Wu, Jing; Aylor, Kevin W; Barrett, Eugene J; Cao, Wenhong; Liu, Zhenqi

    2015-09-01

    Adiponectin is an adipokine with anti-inflammatory and anti-diabetic properties. Hypoadiponectinaemia is closely associated with endothelial dysfunction and insulin resistance in obesity and diabetes. Insulin resistance is present in muscle microvasculature and this may contribute to decreased insulin delivery to, and action in, muscle. In this study we examined whether adiponectin ameliorates metabolic insulin resistance by affecting muscle microvascular recruitment. We demonstrated that a high-fat diet induces vascular adiponectin and insulin resistance but globular adiponectin administration can restore vascular insulin responses and improve insulin's metabolic action via an AMPK- and nitric oxide-dependent mechanism. This suggests that globular adiponectin might have a therapeutic potential for improving insulin resistance and preventing cardiovascular complications in patients with diabetes via modulation of microvascular insulin responses. Hypoadiponectinaemia is closely associated with endothelial dysfunction and insulin resistance, and microvasculature plays a critical role in the regulation of insulin action in muscle. Here we tested whether adiponectin replenishment could improve metabolic insulin sensitivity in male rats fed a high-fat diet (HFD) via the modulation of microvascular insulin responses. Male Sprague-Dawley rats were fed either a HFD or low-fat diet (LFD) for 4 weeks. Small resistance artery myograph changes in tension, muscle microvascular recruitment and metabolic response to insulin were determined. Compared with rats fed a LFD, HFD feeding abolished the vasodilatory actions of globular adiponectin (gAd) and insulin on pre-constricted distal saphenous arteries. Pretreatment with gAd improved insulin responses in arterioles isolated from HFD rats, which was blocked by AMP-activated protein kinase (AMPK) inhibition. Similarly, HFD abolished microvascular responses to either gAd or insulin and decreased insulin-stimulated glucose disposal by

  8. Insulin resistance and vessel endothelial function.

    PubMed Central

    van Oostrom, A J H H M; Cabezas, M Castro; Rabelink, T J

    2002-01-01

    IRS is a complex disease consisting of a clustering of metabolic disorders, of which hyperglycaemia, hyper-insulinaemia and dyslipidaemia are the most important. Endothelial dysfunction plays an important role in the pathogenesis of atherosclerosis. The effects of hyperinsulinaemia seem to depend on lipidaemia and glycaemia. Hyperglycaemia and hyperlipidaemia have detrimental effects on endothelial function in the fasting as well as the postprandial states. In both situations, the generation of ROS and vasoactive molecules plays a major role in interfering with the atheroprotective endothelium-dependent NO system. Treatment of IRS in regard to endothelial function should be focused initially on lifestyle improvement, such as stopping smoking and eating a balanced diet containing antioxidant vitamins, folic-acid, L-arginine and long-chain omega-3 unsaturated FA. Strict glucose control has shown to improve endothelial function and decrease microvascular complications. However, macrovascular complications, in line with endothelial functional improvement, have so far been reduced only when treatment was focused on other characteristics of the IRS syndrome, in particular dyslipidaemia. Other relevant treatments include ACE inhibitors and thiazolidinediones, and probably tetrahydrobiopterin and folic acid supplementation. Future studies should address the effects of therapeutic neovascularization on endothelial dysfunction. PMID:12216328

  9. The Role of Cardiac Side Population Cells in Cardiac Regeneration

    PubMed Central

    Yellamilli, Amritha; van Berlo, Jop H.

    2016-01-01

    The heart has a limited ability to regenerate. It is important to identify therapeutic strategies that enhance cardiac regeneration in order to replace cardiomyocytes lost during the progression of heart failure. Cardiac progenitor cells are interesting targets for new regenerative therapies because they are self-renewing, multipotent cells located in the heart. Cardiac side population cells (cSPCs), the first cardiac progenitor cells identified in the adult heart, have the ability to differentiate into cardiomyocytes, endothelial cells, smooth muscle cells, and fibroblasts. They become activated in response to cardiac injury and transplantation of cSPCs into the injured heart improves cardiac function. In this review, we will discuss the current literature on the progenitor cell properties and therapeutic potential of cSPCs. This body of work demonstrates the great promise cSPCs hold as targets for new regenerative strategies. PMID:27679798

  10. NEU1 and NEU3 Sialidase Activity Expressed in Human Lung Microvascular Endothelia

    PubMed Central

    Cross, Alan S.; Hyun, Sang Won; Miranda-Ribera, Alba; Feng, Chiguang; Liu, Anguo; Nguyen, Chinh; Zhang, Lei; Luzina, Irina G.; Atamas, Sergei P.; Twaddell, William S.; Guang, Wei; Lillehoj, Erik P.; Puché, Adam C.; Huang, Wei; Wang, Lai-Xi; Passaniti, Antonino; Goldblum, Simeon E.

    2012-01-01

    The microvascular endothelial surface expresses multiple molecules whose sialylation state regulates multiple aspects of endothelial function. To better regulate these sialoproteins, we asked whether endothelial cells (ECs) might express one or more catalytically active sialidases. Human lung microvascular EC lysates contained heat-labile sialidase activity for a fluorogenic substrate, 2′-(4-methylumbelliferyl)-α-d-N-acetylneuraminic acid (4-MU-NANA), that was dose-dependently inhibited by the competitive sialidase inhibitor, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid but not its negative control. The EC lysates also contained sialidase activity for a ganglioside mixture. Using real time RT-PCR to detect mRNAs for the four known mammalian sialidases, NEU1, -2, -3, and -4, NEU1 mRNA was expressed at levels 2700-fold higher that those found for NEU2, -3, or -4. Western analyses indicated NEU1 and -3 protein expression. Using confocal microscopy and flow cytometry, NEU1 was immunolocalized to both the plasma membrane and the perinuclear region. NEU3 was detected both in the cytosol and nucleus. Prior siRNA-mediated knockdown of NEU1 and NEU3 each decreased EC sialidase activity for 4-MU-NANA by >65 and >17%, respectively, and for the ganglioside mixture by 0 and 40%, respectively. NEU1 overexpression in ECs reduced their migration into a wound by >40%, whereas NEU3 overexpression did not. Immunohistochemical studies of normal human tissues immunolocalized NEU1 and NEU3 proteins to both pulmonary and extrapulmonary vascular endothelia. These combined data indicate that human lung microvascular ECs as well as other endothelia express catalytically active NEU1 and NEU3. NEU1 restrains EC migration, whereas NEU3 does not. PMID:22403397

  11. Endothelial Gata5 transcription factor regulates blood pressure

    PubMed Central

    Messaoudi, Smail; He, Ying; Gutsol, Alex; Wight, Andrew; Hébert, Richard L.; Vilmundarson, Ragnar O.; Makrigiannis, Andrew P.; Chalmers, John; Hamet, Pavel; Tremblay, Johanne; McPherson, Ruth; Stewart, Alexandre F. R.; Touyz, Rhian M.; Nemer, Mona

    2015-01-01

    Despite its high prevalence and economic burden, the aetiology of human hypertension remains incompletely understood. Here we identify the transcription factor GATA5, as a new regulator of blood pressure (BP). GATA5 is expressed in microvascular endothelial cells and its genetic inactivation in mice (Gata5-null) leads to vascular endothelial dysfunction and hypertension. Endothelial-specific inactivation of Gata5 mimics the hypertensive phenotype of the Gata5-null mice, suggestive of an important role for GATA5 in endothelial homeostasis. Transcriptomic analysis of human microvascular endothelial cells with GATA5 knockdown reveals that GATA5 affects several genes and pathways critical for proper endothelial function, such as PKA and nitric oxide pathways. Consistent with a role in human hypertension, we report genetic association of variants at the GATA5 locus with hypertension traits in two large independent cohorts. Our results unveil an unsuspected link between GATA5 and a prominent human condition, and provide a new animal model for hypertension. PMID:26617239

  12. Endurance, interval sprint, and resistance exercise training: impact on microvascular dysfunction in type 2 diabetes

    PubMed Central

    Laughlin, M. Harold

    2015-01-01

    Type 2 diabetes (T2D) alters capillary hemodynamics, causes capillary rarefaction in skeletal muscle, and alters endothelial and vascular smooth muscle cell phenotype, resulting in impaired vasodilatory responses. These changes contribute to altered blood flow responses to physiological stimuli, such as exercise and insulin secretion. T2D-induced microvascular dysfunction impairs glucose and insulin delivery to skeletal muscle (and other tissues such as skin and nervous), thereby reducing glucose uptake and perpetuating hyperglycemia and hyperinsulinemia. In patients with T2D, exercise training (EX) improves microvascular vasodilator and insulin signaling and attenuates capillary rarefaction in skeletal muscle. EX-induced changes subsequently augment glucose and insulin delivery as well as glucose uptake. If these adaptions occur in a sufficient amount of tissue, and skeletal muscle in particular, chronic exposure to hyperglycemia and hyperinsulinemia and the risk of microvascular complications in all vascular beds will decrease. We postulate that EX programs that engage as much skeletal muscle mass as possible and recruit as many muscle fibers within each muscle as possible will generate the greatest improvements in microvascular function, providing that the duration of the stimulus is sufficient. Primary improvements in microvascular function occur in tissues (skeletal muscle primarily) engaged during exercise, and secondary improvements in microvascular function throughout the body may result from improved blood glucose control. We propose that the added benefit of combined resistance and aerobic EX programs and of vigorous intensity EX programs is not simply “more is better.” Rather, we believe the additional benefit is the result of EX-induced adaptations in and around more muscle fibers, resulting in more muscle mass and the associated microvasculature being changed. Thus, to acquire primary and secondary improvements in microvascular function and

  13. Early Systemic Microvascular Damage in Pigs with Atherogenic Diabetes Mellitus Coincides with Renal Angiopoietin Dysbalance

    PubMed Central

    Khairoun, Meriem; van den Heuvel, Mieke; van den Berg, Bernard M.; Sorop, Oana; de Boer, Rients; van Ditzhuijzen, Nienke S.; Bajema, Ingeborg M.; Baelde, Hans J.; Zandbergen, Malu; Duncker, Dirk J.; Rabelink, Ton J.; Reinders, Marlies E. J.; Rotmans, Joris I.

    2015-01-01

    Background Diabetes mellitus (DM) is associated with a range of microvascular complications including diabetic nephropathy (DN). Microvascular abnormalities in the kidneys are common histopathologic findings in DN, which represent one manifestation of ongoing systemic microvascular damage. Recently, sidestream dark-field (SDF) imaging has emerged as a noninvasive tool that enables one to visualize the microcirculation. In this study, we investigated whether changes in the systemic microvasculature induced by DM and an atherogenic diet correlated spatiotemporally with renal damage. Methods Atherosclerotic lesion development was triggered in streptozotocin-induced DM pigs (140 mg/kg body weight) by administering an atherogenic diet for approximately 11 months. Fifteen months following induction of DM, microvascular morphology was visualized in control pigs (n = 7), non-diabetic pigs fed an atherogenic diet (ATH, n = 5), and DM pigs fed an atherogenic diet (DM+ATH, n = 5) using SDF imaging of oral mucosal tissue. Subsequently, kidneys were harvested from anethesized pigs and the expression levels of well-established markers for microvascular integrity, such as Angiopoietin-1 (Angpt1) and Angiopoietin-2 (Angpt2) were determined immunohistochemically, while endothelial cell (EC) abundance was determined by immunostaining for von Willebrand factor (vWF). Results Our study revealed an increase in the capillary tortuosity index in DM+ATH pigs (2.31±0.17) as compared to the control groups (Controls 0.89±0.08 and ATH 1.55±0.11; p<0.05). Kidney biopsies showed marked glomerular lesions consisting of mesangial expansion and podocyte lesions. Furthermore, we observed a disturbed Angpt2/ Angpt1balance in the cortex of the kidney, as evidenced by increased expression of Angpt2 in DM+ATH pigs as compared to Control pigs (p<0.05). Conclusion In the setting of DM, atherogenesis leads to the augmentation of mucosal capillary tortuosity, indicative of systemic microvascular damage

  14. Reduced Microvascular Density in Omental Biopsies of Children with Chronic Kidney Disease

    PubMed Central

    Grabe, Niels; Lahrmann, Bernd; Nasser, Hamoud; Freise, Christian; Schneider, Axel; Lingnau, Anja; Degenhardt, Petra; Ranchin, Bruno; Sallay, Peter; Cerkauskiene, Rimante; Malina, Michal; Ariceta, Gema; Schmitt, Claus Peter; Querfeld, Uwe

    2016-01-01

    Background Endothelial dysfunction is an early manifestation of cardiovascular disease (CVD) and consistently observed in patients with chronic kidney disease (CKD). We hypothesized that CKD is associated with systemic damage to the microcirculation, preceding macrovascular pathology. To assess the degree of “uremic microangiopathy”, we have measured microvascular density in biopsies of the omentum of children with CKD. Patients and Methods Omental tissue was collected from 32 healthy children (0–18 years) undergoing elective abdominal surgery and from 23 age-matched cases with stage 5 CKD at the time of catheter insertion for initiation of peritoneal dialysis. Biopsies were analyzed by independent observers using either a manual or an automated imaging system for the assessment of microvascular density. Quantitative immunohistochemistry was performed for markers of autophagy and apoptosis, and for the abundance of the angiogenesis-regulating proteins VEGF-A, VEGF-R2, Angpt1 and Angpt2. Results Microvascular density was significantly reduced in uremic children compared to healthy controls, both by manual imaging with a digital microscope (median surface area 0.61% vs. 0.95%, p<0.0021 and by automated quantification (total microvascular surface area 0.89% vs. 1.17% p = 0.01). Density measured by manual imaging was significantly associated with age, height, weight and body surface area in CKD patients and healthy controls. In multivariate analysis, age and serum creatinine level were the only independent, significant predictors of microvascular density (r2 = 0.73). There was no immunohistochemical evidence for apoptosis or autophagy. Quantitative staining showed similar expression levels of the angiogenesis regulators VEGF-A, VEGF-receptor 2 and Angpt1 (p = 0.11), but Angpt2 was significantly lower in CKD children (p = 0.01). Conclusions Microvascular density is profoundly reduced in omental biopsies of children with stage 5 CKD and associated with diminished

  15. Ultrasound fails to induce proliferation of human brain and mouse endothelial cell lines

    NASA Astrophysics Data System (ADS)

    Rodemer, Claus; Jenne, Jürgen; Fatar, Marc; Hennerici, Michael G.; Meairs, Stephen

    2012-11-01

    Both in vitro and in vivo studies suggest that ultrasound (US) is capable of inducing angiogenesis. There is no information, however, on whether ultrasound can induce proliferation of brain endothelial cells. We therefore explored the angiogenic potential of ultrasound on a novel immortalised human brain endothelial cell line (hCMEC/D3) and on mouse brain microvascular endothelial cells (bEND3). Ultrasound failed to enhance cell proliferation in both cell lines at all acoustic pressures studied. Endothelial cell damage occurred at 0.24 MPa with significantly slower proliferation. Cells growing in Opticell{trade mark, serif} dishes did not show damage or reduced proliferation at these pressures.

  16. Two-Photon Imaging within the Murine Thorax without Respiratory and Cardiac Motion Artifact

    PubMed Central

    Presson, Robert G.; Brown, Mary Beth; Fisher, Amanda J.; Sandoval, Ruben M.; Dunn, Kenneth W.; Lorenz, Kevin S.; Delp, Edward J.; Salama, Paul; Molitoris, Bruce A.; Petrache, Irina

    2011-01-01

    Intravital microscopy has been recognized for its ability to make physiological measurements at cellular and subcellular levels while maintaining the complex natural microenvironment. Two-photon microscopy (TPM), using longer wavelengths than single-photon excitation, has extended intravital imaging deeper into tissues, with minimal phototoxicity. However, due to a relatively slow acquisition rate, TPM is especially sensitive to motion artifact, which presents a challenge when imaging tissues subject to respiratory and cardiac movement. Thoracoabdominal organs that cannot be exteriorized or immobilized during TPM have generally required the use of isolated, pump-perfused preparations. However, this approach entails significant alteration of normal physiology, such as a lack of neural inputs, increased vascular resistance, and leukocyte activation. We adapted techniques of intravital microscopy that permitted TPM of organs maintained within the thoracoabdominal cavity of living, breathing rats or mice. We obtained extended intravital TPM imaging of the intact lung, arguably the organ most susceptible to both respiratory and cardiac motion. Intravital TPM detected the development of lung microvascular endothelial activation manifested as increased leukocyte adhesion and plasma extravasation in response to oxidative stress inducers PMA or soluble cigarette smoke extract. The pulmonary microvasculature and alveoli in the intact animal were imaged with comparable detail and fidelity to those in pump-perfused animals, opening the possibility for TPM of other thoracoabdominal organs under physiological and pathophysiological conditions. PMID:21703395

  17. Revisiting Cardiac Cellular Composition

    PubMed Central

    Pinto, Alexander R.; Ilinykh, Alexei; Ivey, Malina J.; Kuwabara, Jill T.; D'Antoni, Michelle L.; Debuque, Ryan; Chandran, Anjana; Wang, Lina; Arora, Komal; Rosenthal, Nadia; Tallquist, Michelle D.

    2015-01-01

    Rationale Accurate knowledge of the cellular composition of the heart is essential to fully understand the changes that occur during pathogenesis and to devise strategies for tissue engineering and regeneration. Objective To examine the relative frequency of cardiac endothelial cells, hematopoietic-derived cells and fibroblasts in the mouse and human heart. Methods and Results Using a combination of genetic tools and cellular markers, we examined the occurrence of the most prominent cell types in the adult mouse heart. Immunohistochemistry revealed that endothelial cells constitute over 60%, hematopoietic-derived cells 5–10%, and fibroblasts under 20% of the non-myocytes in the heart. A refined cell isolation protocol and an improved flow cytometry approach provided an independent means of determining the relative abundance of non-myocytes. High dimensional analysis and unsupervised clustering of cell populations confirmed that endothelial cells are the most abundant cell population. Interestingly, fibroblast numbers are smaller than previously estimated, and two commonly assigned fibroblast markers, Sca-1 and CD90, underrepresent fibroblast numbers. We also describe an alternative fibroblast surface marker that more accurately identifies the resident cardiac fibroblast population. Conclusions This new perspective on the abundance of different cell types in the heart demonstrates that fibroblasts comprise a relatively minor population. By contrast, endothelial cells constitute the majority of non-cardiomyocytes and are likely to play a greater role in physiologic function and response to injury than previously appreciated. PMID:26635390

  18. Role of genetic polymorphisms of ion channels in the pathophysiology of coronary microvascular dysfunction and ischemic heart disease.

    PubMed

    Fedele, Francesco; Mancone, Massimo; Chilian, William M; Severino, Paolo; Canali, Emanuele; Logan, Suzanna; De Marchis, Maria Laura; Volterrani, Maurizio; Palmirotta, Raffaele; Guadagni, Fiorella

    2013-11-01

    Conventionally, ischemic heart disease (IHD) is equated with large vessel coronary disease. However, recent evidence has suggested a role of compromised microvascular regulation in the etiology of IHD. Because regulation of coronary blood flow likely involves activity of specific ion channels, and key factors involved in endothelium-dependent dilation, we proposed that genetic anomalies of ion channels or specific endothelial regulators may underlie coronary microvascular disease. We aimed to evaluate the clinical impact of single-nucleotide polymorphisms in genes encoding for ion channels expressed in the coronary vasculature and the possible correlation with IHD resulting from microvascular dysfunction. 242 consecutive patients who were candidates for coronary angiography were enrolled. A prospective, observational, single-center study was conducted, analyzing genetic polymorphisms relative to (1) NOS3 encoding for endothelial nitric oxide synthase (eNOS); (2) ATP2A2 encoding for the Ca²⁺/H⁺-ATPase pump (SERCA); (3) SCN5A encoding for the voltage-dependent Na⁺ channel (Nav1.5); (4) KCNJ8 and KCNJ11 encoding for the Kir6.1 and Kir6.2 subunits of K-ATP channels, respectively; and (5) KCN5A encoding for the voltage-gated K⁺ channel (Kv1.5). No significant associations between clinical IHD manifestations and polymorphisms for SERCA, Kir6.1, and Kv1.5 were observed (p > 0.05), whereas specific polymorphisms detected in eNOS, as well as in Kir6.2 and Nav1.5 were found to be correlated with IHD and microvascular dysfunction. Interestingly, genetic polymorphisms for ion channels seem to have an important clinical impact influencing the susceptibility for microvascular dysfunction and IHD, independent of the presence of classic cardiovascular risk factors.

  19. Flow motion dynamics of microvascular blood flow and oxygenation: Evidence of adaptive changes in obesity and type 2 diabetes mellitus/insulin resistance.

    PubMed

    Clough, Geraldine F; Kuliga, Katarzyna Z; Chipperfield, Andrew J

    2017-02-01

    An altered spatial heterogeneity and temporal stability of network perfusion can give rise to a limited adaptive ability to meet metabolic demands. Derangement of local flow motion activity is associated with reduced microvascular blood flow and tissue oxygenation, and it has been suggested that changes in flow motion activity may provide an early indicator of declining, endothelial, neurogenic, and myogenic regulatory mechanisms and signal the onset and progression of microvascular pathophysiology. This short conference review article explores some of the evidence for altered flow motion dynamics of blood flux signals acquired using laser Doppler fluximetry in the skin in individuals at risk of developing or with cardiometabolic disease.

  20. Activation of endothelial β-catenin signaling induces heart failure

    PubMed Central

    Nakagawa, Akito; Naito, Atsuhiko T.; Sumida, Tomokazu; Nomura, Seitaro; Shibamoto, Masato; Higo, Tomoaki; Okada, Katsuki; Sakai, Taku; Hashimoto, Akihito; Kuramoto, Yuki; Oka, Toru; Lee, Jong-Kook; Harada, Mutsuo; Ueda, Kazutaka; Shiojima, Ichiro; Limbourg, Florian P.; Adams, Ralf H.; Noda, Tetsuo; Sakata, Yasushi; Akazawa, Hiroshi; Komuro, Issei

    2016-01-01

    Activation of β-catenin-dependent canonical Wnt signaling in endothelial cells plays a key role in angiogenesis during development and ischemic diseases, however, other roles of Wnt/β-catenin signaling in endothelial cells remain poorly understood. Here, we report that sustained activation of β-catenin signaling in endothelial cells causes cardiac dysfunction through suppressing neuregulin-ErbB pathway in the heart. Conditional gain-of-function mutation of β-catenin, which activates Wnt/β-catenin signaling in Bmx-positive arterial endothelial cells (Bmx/CA mice) led to progressive cardiac dysfunction and 100% mortality at 40 weeks after tamoxifen treatment. Electron microscopic analysis revealed dilatation of T-tubules and degeneration of mitochondria in cardiomyocytes of Bmx/CA mice, which are similar to the changes observed in mice with decreased neuregulin-ErbB signaling. Endothelial expression of Nrg1 and cardiac ErbB signaling were suppressed in Bmx/CA mice. The cardiac dysfunction of Bmx/CA mice was ameliorated by administration of recombinant neuregulin protein. These results collectively suggest that sustained activation of Wnt/β-catenin signaling in endothelial cells might be a cause of heart failure through suppressing neuregulin-ErbB signaling, and that the Wnt/β-catenin/NRG axis in cardiac endothelial cells might become a therapeutic target for heart failure. PMID:27146149

  1. A comparison of the antigen-presenting capabilities of class II MHC-expressing human lung epithelial and endothelial cells.

    PubMed Central

    Cunningham, A C; Zhang, J G; Moy, J V; Ali, S; Kirby, J A

    1997-01-01

    Human lung alveolar epithelial cells constitutively express class II major histocompatibility complex (MHC). Human lung microvascular endothelial and small airway epithelial cells can be induced to express class II MHC by stimulation with the pro-inflammatory cytokine interferon-gamma. The levels of class II MHC on lung epithelial and endothelial cells were comparable to those seen on an Epstein-Barr virus (EBV)-transformed B-cell line. However, the costimulatory molecules B7-1 and B7-2 were not expressed. The ability of the class II MHC expressing human lung parenchymal cells to present alloantigen to CD4+ T lymphocytes was investigated. Freshly isolated human alveolar epithelial cells (type II pneumocytes) and monolayers of interferon-gamma-stimulated small airway epithelial and lung microvascular endothelial cells were co-cultured with allogeneic CD4+ T lymphocytes and proliferation determined by [3H]thymidine incorporation. A clear difference was observed between effects of the epithelial and endothelial cells on CD4+ T-lymphocyte activation. Alveolar and small airway epithelial cells failed to stimulate the proliferation of allogeneic CD4+ T lymphocytes whereas lung microvascular endothelial cells did stimulate proliferation. This difference could not be explained by the levels of class II MHC or the lack of B7-1 and B7-2 solely. Microvascular endothelial cells, and not alveolar or small airway epithelial cells, possess B7-independent costimulatory pathways. PMID:9301537

  2. Radiation Effects on the Cytoskeleton of Endothelial Cells and Endothelial Monolayer Permeability

    SciTech Connect

    Gabrys, Dorota; Greco, Olga; Patel, Gaurang; Prise, Kevin M.; Tozer, Gillian M.; Kanthou, Chryso

    2007-12-01

    Purpose: To investigate the effects of radiation on the endothelial cytoskeleton and endothelial monolayer permeability and to evaluate associated signaling pathways, which could reveal potential mechanisms of known vascular effects of radiation. Methods and Materials: Cultured endothelial cells were X-ray irradiated, and actin filaments, microtubules, intermediate filaments, and vascular endothelial (VE)-cadherin junctions were examined by immunofluorescence. Permeability was determined by the passage of fluorescent dextran through cell monolayers. Signal transduction pathways were analyzed using RhoA, Rho kinase, and stress-activated protein kinase-p38 (SAPK2/p38) inhibitors by guanosine triphosphate-RhoA activation assay and transfection with RhoAT19N. The levels of junction protein expression and phosphorylation of myosin light chain and SAPK2/p38 were assessed by Western blotting. The radiation effects on cell death were verified by clonogenic assays. Results: Radiation induced rapid and persistent actin stress fiber formation and redistribution of VE-cadherin junctions in microvascular, but not umbilical vein endothelial cells, and microtubules and intermediate filaments remained unaffected. Radiation also caused a rapid and persistent increase in microvascular permeability. RhoA-guanosine triphosphatase and Rho kinase were activated by radiation and caused phosphorylation of downstream myosin light chain and the observed cytoskeletal and permeability changes. SAPK2/p38 was activated by radiation but did not influence either the cytoskeleton or permeability. Conclusion: This study is the first to show rapid activation of the RhoA/Rho kinase by radiation in endothelial cells and has demonstrated a link between this pathway and cytoskeletal remodeling and permeability. The results also suggest that the RhoA pathway might be a useful target for modulating the permeability and other effects of radiation for therapeutic gain.

  3. Promotion of airway anastomotic microvascular regeneration and alleviation of airway ischemia by deferoxamine nanoparticles

    PubMed Central

    Tian, Wen; Sung, Yon K.; Sun, Wenchao; Hsu, Joe L.; Manickam, Sathish; Wagh, Dhananjay; Joubert, Lydia-Marie; Semenza, Gregg L.; Rajadas, Jayakumar; Nicolls, Mark R.

    2014-01-01

    Airway tissue ischemia and hypoxia in human lung transplantation is a consequence of the sacrifice of the bronchial circulation during the surgical procedure and is a major risk factor for the development of airway anastomotic complications. Augmented expression of hypoxia-inducible factor (HIF)-1α promotes microvascular repair and alleviates allograft ischemia and hypoxia. Deferoxamine mesylate (DFO) is an FDA-approved iron chelator which has been shown to upregulate cellular HIF-1α. Here, we developed a nanoparticle formulation of DFO that can be topically applied to airway transplants at the time of surgery. In a mouse orthotopic tracheal transplant (OTT) model, the DFO nanoparticle was highly effective in enhancing airway microvascular perfusion following transplantation through the production of the angiogenic factors, placental growth factor (PLGF) and stromal cell-derived factor (SDF)-1. The endothelial cells in DFO treated airways displayed higher levels of p-eNOS and Ki67, less apoptosis, and decreased production of perivascular reactive oxygen species (ROS) compared to vehicle-treated airways. In summary, a DFO formulation topically-applied at the time of surgery successfully augmented airway anastomotic microvascular regeneration and the repair of alloimmune-injured microvasculature. This approach may be an effective topical transplant-conditioning therapy for preventing airway complications following clinical lung transplantation. PMID:24161166

  4. Effect of caffeine contained in a cup of coffee on microvascular function in healthy subjects.

    PubMed

    Noguchi, Katsuhiko; Matsuzaki, Toshihiro; Sakanashi, Mayuko; Hamadate, Naobumi; Uchida, Taro; Kina-Tanada, Mika; Kubota, Haruaki; Nakasone, Junko; Sakanashi, Matao; Ueda, Shinichiro; Masuzaki, Hiroaki; Ishiuchi, Shogo; Ohya, Yusuke; Tsutsui, Masato

    2015-02-01

    Recent epidemiological studies have demonstrated that coffee drinking is associated with reduced mortality of cardiovascular disease. However, its precise mechanisms remain to be clarified. In this study, we examined whether single ingestion of caffeine contained in a cup of coffee improves microvascular function in healthy subjects. A double-blind, placebo-controlled, crossover study was performed in 27 healthy volunteers. A cup of either caffeinated or decaffeinated coffee was drunk by the subjects, and reactive hyperemia of finger blood flow was assessed by laser Doppler flowmetry. In an interval of more than 2 days, the same experimental protocol was repeated with another coffee in a crossover manner. Caffeinated coffee intake slightly but significantly elevated blood pressure and decreased finger blood flow as compared with decaffeinated coffee intake. There was no significant difference in heart rate between caffeinated and decaffeinated coffee intake. Importantly, caffeinated coffee intake significantly enhanced post-occlusive reactive hyperemia of finger blood flow, an index of microvascular endothelial function, compared with decaffeinated coffee intake. These results provide the first evidence that caffeine contained in a cup of coffee enhances microvascular function in healthy individuals.

  5. Components of the interleukin-33/ST2 system are differentially expressed and regulated in human cardiac cells and in cells of the cardiac vasculature.

    PubMed

    Demyanets, Svitlana; Kaun, Christoph; Pentz, Richard; Krychtiuk, Konstantin A; Rauscher, Sabine; Pfaffenberger, Stefan; Zuckermann, Andreas; Aliabadi, Arezu; Gröger, Marion; Maurer, Gerald; Huber, Kurt; Wojta, Johann

    2013-07-01

    Interleukin-33 (IL-33) is a recently described member of the IL-1 family of cytokines, which was identified as a ligand for the ST2 receptor. Components of the IL-33/ST2 system were shown to be expressed in normal and pressure overloaded human myocardium, and soluble ST2 (sST2) has emerged as a prognostic biomarker in myocardial infarction and heart failure. However, expression and regulation of IL-33 in human adult cardiac myocytes and fibroblasts was not tested before. In this study we found that primary human adult cardiac fibroblasts (HACF) and human adult cardiac myocytes (HACM) constitutively express nuclear IL-33 that is released during cell necrosis. Tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-1β significantly increased both IL-33 protein and IL-33 mRNA expression in HACF and HACM as well as in human coronary artery smooth muscle cells (HCASMC). The nuclear factor-κB (NF-κB) inhibitor dimethylfumarate inhibited TNF-α- and IL-1β-induced IL-33 production as well as nuclear translocation of p50 and p65 NF-κB subunits in these cells. Mitogen-activated protein/extracellular signal-regulated kinase inhibitor U0126 abrogated TNF-α-, IFN-γ-, and IL-1β-induced and Janus-activated kinase inhibitor I reduced IFN-γ-induced IL-33 production. We detected IL-33 mRNA in human myocardial tissue from patients undergoing heart transplantation (n=27) where IL-33 mRNA levels statistically significant correlated with IFN-γ (r=0.591, p=0.001) and TNF-α (r=0.408, p=0.035) mRNA expression. Endothelial cells in human heart expressed IL-33 as well as ST2 protein. We also reveal that human cardiac and vascular cells have different distribution patterns of ST2 isoforms (sST2 and transmembrane ST2L) mRNA expression and produce different amounts of sST2 protein. Both human macrovascular (aortic and coronary artery) and heart microvascular endothelial cells express specific mRNA for both ST2 isoforms (ST2L and sST2) and are a source for sST2 protein, whereas

  6. Altered long non-coding RNA transcriptomic profiles in brain microvascular endothelium after cerebral ischemia.

    PubMed

    Zhang, J; Yuan, L; Zhang, X; Hamblin, M H; Zhu, T; Meng, F; Li, Y; Chen, Y E; Yin, K J

    2016-03-01

    The brain endothelium is an important therapeutic target for the inhibition of cerebrovascular dysfunction in ischemic stroke. Previously, we documented the important regulatory roles of microRNAs in the cerebral vasculature, in particular the cerebral vascular endothelium. However, the functional significance and molecular mechanisms of other classes of non-coding RNAs in the regulation of cerebrovascular endothelial pathophysiology after stroke are completely unknown. Using RNA sequencing (RNA-seq) technology, we profiled long non-coding RNA (lncRNA) expressional signatures in primary brain microvascular endothelial cells (BMECs) after oxygen-glucose deprivation (OGD), an in vitro mimic of ischemic stroke conditions. After 16h of OGD exposure, the expression levels for 362 of the 10,677 lncRNAs analyzed changed significantly, including a total of 147 lncRNAs increased and 70 lncRNAs decreased by more than 2-fold. Among them, the most highly upregulated lncRNAs include Snhg12, Malat1, and lnc-OGD 1006, whereas the most highly downregulated lncRNAs include 281008D09Rik, Peg13, and lnc-OGD 3916. Alteration of the most highly upregulated/downregulated ODG-responsive lncRNAs was further confirmed in cultured BMECs after OGD as well as isolated cerebral microvessels in mice following transient middle cerebral artery occlusion (MCAO) and 24h reperfusion by the quantitative real-time PCR approach. Moreover, promoter analysis of altered ODG-responsive endothelial lncRNA genes by bioinformatics showed substantial transcription factor binding sites on lncRNAs, implying potential transcriptional regulation of those lncRNAs. These findings are the first to identify OGD-responsive brain endothelial lncRNAs, which suggest potential pathological roles for these lncRNAs in mediating endothelial responses to ischemic stimuli. Endothelial-selective lncRNAs may function as a class of novel master regulators in cerebrovascular endothelial pathologies after ischemic stroke.

  7. Early gene response of human brain endothelial cells to Listeria monocytogenes

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The gene expression of human brain microvascular endothelial cells (HBMEC) to Listeria monocytogenes at 4 hour infection was analyzed. Four hours after infection, the expression of 456 genes of HBMEC had changed (p<0.05). We noted that many active genes were involved in the formyl-methionylleucylph...

  8. Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Kemper, Björn; Bauwens, Andreas; Vollmer, Angelika; Ketelhut, Steffi; Langehanenberg, Patrik; Müthing, Johannes; Karch, Helge; von Bally, Gert

    2010-05-01

    Digital holographic microscopy (DHM) enables quantitative multifocus phase contrast imaging for nondestructive technical inspection and live cell analysis. Time-lapse investigations on human brain microvascular endothelial cells demonstrate the use of DHM for label-free dynamic quantitative monitoring of cell division of mother cells into daughter cells. Cytokinetic DHM analysis provides future applications in toxicology and cancer research.

  9. The human brain intracerebral microvascular system: development and structure

    PubMed Central

    Marín-Padilla, Miguel

    2012-01-01

    The capillary from the meningeal inner pial lamella play a crucial role in the development and structural organization of the cerebral cortex extrinsic and intrinsic microvascular compartments. Only pial capillaries are capable of perforating through the cortex external glial limiting membrane (EGLM) to enter into the nervous tissue, although incapable of perforating the membrane to exit the brain. Circulatory dynamics and functional demands determine which capillaries become arterial and which capillaries become venous. The perforation of the cortex EGLM by pial capillaries is a complex process characterized by three fundamental stages: (1) pial capillary contact with the EGLM with fusion of vascular and glial basal laminae at the contact site, (2) endothelial cell filopodium penetration through the fussed laminae with the formation of a funnel between them that accompanies it into the nervous tissue while remaining open to the meningeal interstitium and, (3) penetration of the whole capillary carrying the open funnel with it and establishing an extravascular Virchow-Robin Compartment (V-RC) that maintains the perforating vessel extrinsic (outside) the nervous tissue through its entire length. The V-RC is walled internally by the vascular basal lamina and externally by the basal lamina of joined glial cells endfeet. The VRC outer glial wall appear as an extension of the cortex superficial EGLM. All the perforating vessels within the V-RCs constitute the cerebral cortex extrinsic microvascular compartment. These perforating vessels are the only one capable of responding to inflammatory insults. The V-RC remains open (for life) to the meningeal interstitium permitting the exchanges of fluid and of cells between brain and meninges. The V-RC function as the brain sole drainage (prelymphatic) system in both physiological as well as pathological situations. During cortical development, capillaries emerge from the perforating vessels, by endothelial cells growing sprouts

  10. Direct vasorelaxation by a novel phytoestrogen tanshinone IIA is mediated by nongenomic action of estrogen receptor through endothelial nitric oxide synthase activation and calcium mobilization.

    PubMed

    Fan, Guanwei; Zhu, Yan; Guo, Hao; Wang, Xiaoying; Wang, Hong; Gao, Xiumei

    2011-03-01

    Salvia miltiorrhiza (Danshen) has been widely used in China and other Asian countries for treating various cardiovascular diseases resulting from its ability to improve coronary microcirculation and increase coronary blood flow. Tanshinone IIA (Tan IIA), the major active lipophilic ingredient responsible for the beneficial actions of Salvia miltiorrhiza, has been shown to induce vasodilation in coronary arteries. Because our recent study identified Tan IIA as a new member of the phytoestrogens, we hypothesized that its action might be mediated by estrogen receptor (ER) in vascular endothelial cells. The aim of the present study was to assess whether cardiovascular protection exerted by Tan IIA is mediated by the ER signal pathway and whether the genomic or nongenomic action of ER is involved within arteries and vascular endothelial cells. The effect of Tan IIA on blood vessels was investigated by vascular ring assay using endothelium-intact and endothelium-denuded rat aortas. Similar to estrogen, Tan IIA caused an nitric oxide- and endothelium-dependent relaxation, which was blocked by ER antagonist ICI 182,780. Primary cardiac microvascular endothelial cells were used as a model to study the cellular and molecular mechanisms of Tan IIA-induced vasorelaxation. We demonstrate that Tan IIA is capable of activating the estrogen receptor signal pathway, leading to increased endothelial nitric oxide synthase gene expression, nitric oxide production, ERK1/2 phosphorylation, and Ca mobilization. Collectively, these effects contribute to Tan IIA's vasodilative activity effects of y ER antagonist Cnt of cardiovascular diseases. Our findings support a continued effort in discovering and developing novel phytoestrogens as an alternative hormone replacement therapy for safer and more effective treatment of cardiovascular diseases.

  11. Endothelial colony forming cells ameliorate endothelial dysfunction via secreted factors following ischemia-reperfusion injury.

    PubMed

    Collett, Jason A; Mehrotra, Purvi; Crone, Allison; Shelley, W Christopher; Yoder, Mervin C; Basile, David P

    2017-02-22

    Damage to endothelial cells contributes to acute kidney injury (AKI) by leading to impaired perfusion. Endothelial colony-forming cells (ECFCs) are endothelial precursor cells with high proliferative capacity, pro-angiogenic activity, and in vivo vessel forming potential. We hypothesized that ECFCs may ameliorate the degree of AKI and/or promote repair of the renal vasculature following ischemia/reperfusion (I/R). Rat pulmonary microvascular ECs (PMVEC) with high proliferative potential were compared with pulmonary artery ECs (PAEC) with low proliferative potential in rats subjected to renal I/R. PMVEC administration reduced renal injury and hastened recovery as indicated by serum creatinine and tubular injury scores, while PAEC did not. Vehicle-treated control animals showed consistent reductions in renal medullary blood flow (MBF) within 2 hours of reperfusion, while PMVEC protected against loss in MBF as measured by laser Doppler. Interestingly, PMVEC mediated protection occurred in the absence of homing to the kidney. Conditioned medium (CM) from human cultured cord blood ECFC also conveyed beneficial effects against I/R injury and loss of MBF. Moreover, ECFC-CM significantly reduced the expression of adhesion molecules such as ICAM-1 and p-selectin, and decreased the number of differentiated lymphocytes typically recruited into the kidney following renal ischemia. Taken together, these data suggest that ECFC secrete factors that preserve renal function post ischemia, in part, by preserving microvascular function.

  12. Clinical and pathological assessment of different suture techniques for microvascular anastomosis in rat femoral artery

    PubMed Central

    El-Shazly, Mohamed

    2007-01-01

    This study examined the clinical and pathological features after a microvascular anastomosis of a rat femoral artery using four different suture techniques. Sixty Sprage-Dawely rats were divided randomly into 4 groups. Fifteen bisected arteries (one from each animal) in Group I, II, III and IV were sutured with the simple interrupted suture, continuous suture, sleeve suture and cuff suture, respectively. The anastomosis times in Group I, II, III and IV were 28.67, 14.67, 15.47 and 15.93 min, respectively. Immediate bleeding that stopped without intervention (grade I) was observed in 67%, 73% and 60% of the anastomosed vessels in Groups II, III and IV, respectively, while 60% of the vessels in Group I showed light bleeding that was inhibited by gentile pressure (grade II). All vessels examined appeared to be patent at 5 and 15 min after the anastomosis. On the 7th day postoperatively, the vessels of Group I showed the highest patency rate (93%) compared with Groups II (67%), III (73%) and IV (87%). Moreover, there were more pronounced pathological changes in Group I than in the other groups. These changes included endothelial loss, endothelial proliferation, degeneration and necrosis of the tunica media. Suture materials surrounded by an inflammatory reaction were also observed. In conclusion, the simple interrupted suture is preferable for microvascular anastomosis due to its highest patency rate. The other techniques investigated can be good alternatives because of their short anastomotic time and moderate pathological changes. PMID:17679774

  13. Non-invasive evaluation of vasomotor and metabolic functions of microvascular endothelium in human skin.

    PubMed

    Fedorovich, Andrey A

    2012-07-01

    Correlation between metabolic and microhemodynamic processes in skin was assessed through acute pharmacological test with metabolically active Actovegin in 28 healthy volunteers. Laser Doppler flowmetry in combination with wavelet analysis of blood flow oscillations was used to identify functional state of arteriolar-venular areas of microvascular bed in the right forearm skin; capillary blood flow parameters were assessed through computer capillaroscopy in the nail bed of the right hand on the 4th finger. The metabolic effect (improved oxygen uptake and glucose disposal by tissues) was accompanied by significant increase in endothelial rhythm amplitude by 98% (p<0.00006), neurogenic rhythm amplitude by 50% (p<0.003) and myogenic rhythm amplitude by 54% (p<0.03), with capillary blood flow rate increasing by 90μm/s (p<0.04), pericapillary zone reducing by 15μm (p<0.0001) and diastolic blood pressure dropping by 4mm Hg (p<0.02). These results show close correlation between metabolic and microhemodynamic processes, which suggests that the amplitude activity within the range of endothelial rhythm (0.0095-0.021Hz) during laser Doppler flowmetry reflects not only solely vasomotor function but also metabolic function of microvascular endothelium.

  14. Specific albumin binding to microvascular endothelium in culture

    SciTech Connect

    Schnitzer, J.E.; Carley, W.W.; Palade, G.E. )

    1988-03-01

    The specific binding of rat serum albumin (RSA) to confluent microvascular endothelial cells in culture derived from the vasculature of the rat epididymal fat pad was studied at 4{degree}C by radioassay and immunocytochemistry. Radioiodinated RSA ({sup 125}I-RSA) binding to the cells reached equilibrium at {approximately} 20 min incubation. Albumin binding was a slowly saturating function over concentrations ranging from 0.01 to 50 mg/ml. Specific RSA binding with a moderate apparent affinity constant of 1.0 mg/ml and with a maximum binding concentration of 90 ng/cm{sup 2} was immunolocalized with anti-RSA antibody to the outer (free) side of the enothelium. Scatchard analysis of the binding yielded a nonlinear binding curve with a concave-upward shape. Dissociation rate analysis supports negative cooperativity of albumin binding, but multiple binding sites may also be present. Albumin binding fulfilled many requirements for ligand specificity including saturability, reversibility, competibility, and dependence on both cell type and cell number. The results are discussed in terms of past in situ investigations on the localization of albumin binding to vascular endothelium and its effect on transendothelial molecular transport.

  15. Evidence of Microvascular Changes in the Retina following Kawasaki Disease

    PubMed Central

    Chen, Katherine Y. H.; Burgner, David P.; Wong, Tien Y.; Saw, Seang Mei; Quek, Swee Chye; Pang, Audrey Y. C.; Leo, Seo Wei; Wong, Inez B.; Zannino, Diana; Curtis, Nigel; Cheung, Michael; Cheung, Carol Y.; Lim, Terence C. W.

    2017-01-01

    It is unclear whether all children with Kawasaki disease (KD) have increased later cardiovascular risk. The retinal microvasculature reflects changes in the microcirculation and is associated with traditional cardiovascular risk factors and events. The aim of this study was to investigate retinal microvascular parameters in two populations of patients with previous KD and control participants. We performed case-control studies of 116 (57 patients and 59 control participants) Australian and 156 (78 patients and 78 control participants) Singaporean individuals, at least two years since their acute illness. Standardised retinal photographs were graded by trained technicians using a semi-automated software, which quantifies the retinal microvasculature (calibre, branching angle, fractal dimensions, and tortuosity). Retinal venules of Singaporean KD patients were 9.67 μm (95% CI 4.87 to 14.51, p < 0.001) larger than control participants following correction for traditional cardiovascular risk factors. An incremental increase in the size of retinal venules in those with coronary artery abnormalities was observed. There was limited evidence that retinal venules were larger in Australian KD patients with coronary artery abnormalities compared to control participants (7.34 μm, 95% CI 1.30 to 15.99, p = 0.10). Differences in retinal microvasculature were particularly evident in Singaporean KD patients. Larger retinal venules may reflect chronic inflammation and endothelial dysfunction, and are associated with coronary artery disease in adults. PMID:28094311

  16. Microvascular network topology of the human retinal vessels.

    PubMed

    Schröder, S; Brab, M; Schmid-Schönbein, G W; Reim, M; Schmid-Schönbein, H

    1990-01-01

    A quantitative analysis of blood flow in the human retinal vessels requires a detailed picture of the microvascular network topology. In order to lay the foundation for a quantitative microcirculatory network analysis of the human retina, a novel technique for tissue preparation and network characterization was developed. After injection of hydrogen peroxide into the human bulb, the microvasculature was filled with oxygen produced by endothelial catalase and visualized after embedding in a mixture of cedar oil and gum damar. The vessel topology was documented in the form of photomicrographs, which permitted complete reconstruction of the microvasculature on transparent overlays. By considering the complete capillary system it was possible to divide the retinal network into dichotomous, asymmetric arteriolar and venular trees. The Strahler ordering method, which considers both dichotomous and side branching configurations, was selected and applied to analyze the retinal vascular trees, using the capillaries as the zero order reference vessels. The number of vessel segments was found to be an approximate logarithmic function of the order number, in accordance with Horton's law. Vessel lengths within each order were found to be log-normal distributed, and median lengths for different orders could be approximated by a 2nd degree polynomial curve. Diameters within each order could be approximated by a Gaussian distribution, and the mean values for different orders could be expressed by an exponential curve. These data provide the basis for conductance, pressure and flow computations within the retinal microvessels.

  17. Prognosis of invasive breast cancer after adjuvant therapy evaluated with VEGF microvessel density and microvascular imaging.

    PubMed

    Li, Ying; Wei, Xi; Zhang, Sheng; Zhang, Jin

    2015-11-01

    The aim of this study was to investigate the role of ultrasonographic microvascular imaging in the evaluation of prognosis of patients with invasive breast cancer treated by adjuvant therapies. A total of 121 patients with invasive breast cancer underwent ultrasonographic contrast-enhanced imaging, vascular endothelial growth factor (VEGF) staining, and microvessel density (MVD) counts. The parameters of microvascular imaging and the expression of VEGF and MVD in primary breast cancer were calculated. The correlation between these factors and the overall and progression-free survival rate were analyzed using the Kaplan-Meier method. Among 121 cases, the positive VEGF cases were 75 and negative ones were 46. The cut point of 52.3 was calculated by the regressive curve for MVD counts. The data showed the mean intensity (MI) was positively associated with both the MVD counts (r = .51, p < .001) and VEGF expression (r = .35, p < .001). For the prognosis of patients, high VEGF expression and MVD counts were associated with reduced progressive and survival times (PFS, p = .032 and p = .034; OS, p = .041 and p = .038, respectively). The correlation between parameters of microvascular imaging, VEGF expressive status, and the MVD counts were established. The cut point of mean intensity (MI = 40) was used to investigate as an independent predictor for PFS (p = .021) and OS (p = .025), respectively, due to a strong correlation between MVD counts and VEGF expression in patients with invasive breast cancer. The microvascular imaging could be a visual and helpful tool to predict the prognosis of patients with invasive breast cancer treated by adjuvant therapies.

  18. Roles of limbal microvascular net and limbal stroma in regulating maintenance of limbal epithelial stem cells.

    PubMed

    Huang, Minghai; Wang, Bowen; Wan, Pengxia; Liang, Xuanwei; Wang, Xiaoran; Liu, Ying; Zhou, Qiang; Wang, Zhichong

    2015-02-01

    Knowledge of the microenvironment (niche) of stem cells is helpful for stem-cell-based regenerative medicine. In the eye, limbal epithelial stem cells (corneal epithelial stem cells) provide the self-renewal capacity of the corneal epithelium and are essential for maintaining corneal transparency and vision. Limbal epithelial stem cell deficiency results in significant visual deterioration. Successful treatment of this type of blinding disease requires studies of the limbal epithelial stem cells and their microenvironment. We investigate the function of the limbal microvascular net and the limbal stroma in the maintenace of the limbal epithelial stem cell niche in vivo and examine the regulation of limbal epithelial stem cell survival, proliferation and differentiation in vivo. We assess the temporal and spatial changes in the expression patterns of the following markers during a six-month follow-up of various rabbit limbal autograft transplantation models: vascular endothelial cell marker CD31, corneal epithelium differentiation marker K3, limbal epithelial stem-cell-associated markers P63 and ABCG2 and proliferating cell nuclear marker Ki67. Our results suggest that limbal epithelial stem cells cannot maintain their stemness or proliferation without the support of the limbal microvascular net microenvironment. Thus, both the limbal microvascular net and the limbal stroma play important roles as components of the limbal epithelial stem cell niche maintaining limbal epithelial stem cell survival and proliferation and the avoidance of differentiation. The limbal stroma constitutes the structural basis of the limbal epithelial stem cell niche and the limbal microvascular net is a requirement for this niche. These new insights should aid the eventual construction of tissue-engineered cornea for corneal blind patients in the future.

  19. Can C-peptide mediated anti-inflammatory effects retard the development of microvascular complications of type 1 diabetes?

    PubMed

    Luppi, Patrizia; Kallas, Åsa; Wahren, John

    2013-07-01

    Hyperglycemia is considered to be the major cause of microvascular complications of diabetes. Growing evidence highlights the importance of hyperglycemia-mediated inflammation in the initiation and progression of microvascular complications in type 1 diabetes. We hypothesize that lack of proinsulin C-peptide and lack of its anti-inflammatory properties contribute to the development of microvascular complications. Evidence gathered over the past 20 years shows that C-peptide is a biologically active peptide in its own right. It has been shown to reduce formation of reactive oxygen species and nuclear factor-κB activation induced by hyperglycemia, resulting in inhibition of cytokine, chemokine and cell adhesion molecule formation as well as reduced apoptotic activity. In addition, C-peptide stimulates and induces the expression of both Na⁺, K⁺-ATPase and endothelial nitric oxide synthase. Animal studies and small-scale clinical trials in type 1 diabetes patients suggest that C-peptide replacement combined with regular insulin therapy exerts beneficial effects on kidney and nerve dysfunction. Further clinical trials in patients with microvascular complications including measurements of inflammatory markers are warranted to explore the clinical significance of the aforementioned, previously unrecognized, C-peptide effects.

  20. Sickle erythrocytes inhibit human endothelial cell DNA synthesis

    SciTech Connect

    Weinstein, R.; Zhou, M.A.; Bartlett-Pandite, A.; Wenc, K. )

    1990-11-15

    Patients with sickle cell anemia experience severe vascular occlusive phenomena including acute pain crisis and cerebral infarction. Obstruction occurs at both the microvascular and the arterial level, and the clinical presentation of vascular events is heterogeneous, suggesting a complex etiology. Interaction between sickle erythrocytes and the endothelium may contribute to vascular occlusion due to alteration of endothelial function. To investigate this hypothesis, human vascular endothelial cells were overlaid with sickle or normal erythrocytes and stimulated to synthesize DNA. The erythrocytes were sedimented onto replicate monolayers by centrifugation for 10 minutes at 17 g to insure contact with the endothelial cells. Incorporation of 3H-thymidine into endothelial cell DNA was markedly inhibited during contact with sickle erythrocytes. This inhibitory effect was enhanced more than twofold when autologous sickle plasma was present during endothelial cell labeling. Normal erythrocytes, with or without autologous plasma, had a modest effect on endothelial cell DNA synthesis. When sickle erythrocytes in autologous sickle plasma were applied to endothelial monolayers for 1 minute, 10 minutes, or 1 hour and then removed, subsequent DNA synthesis by the endothelial cells was inhibited by 30% to 40%. Although adherence of sickle erythrocytes to the endothelial monolayers was observed under these experimental conditions, the effect of sickle erythrocytes on endothelial DNA synthesis occurred in the absence of significant adherence. Hence, human endothelial cell DNA synthesis is partially inhibited by contact with sickle erythrocytes. The inhibitory effect of sickle erythrocytes occurs during a brief (1 minute) contact with the endothelial monolayers, and persists for at least 6 hours of 3H-thymidine labeling.

  1. Usefulness of cardiac MRI in the prognosis and follow-up of ischemic heart disease.

    PubMed

    Hidalgo, A; Pons-Lladó, G

    2015-01-01

    Cardiac magnetic resonance imaging (MRI) is an important tool that makes it possible to evaluate patients with cardiovascular disease; in addition to infarction and alterations in myocardial perfusion, cardiac MRI is useful for evaluating other phenomena such as microvascular obstruction and ischemia. The main prognostic factors in cardiac MRI are ventricular dysfunction, necrosis in late enhancement sequences, and ischemia in stress sequences. In acute myocardial infarction, cardiac MRI can evaluate the peri-infarct zone and quantify the size of the infarct. Furthermore, cardiac MRI's ability to detect and evaluate microvascular obstruction makes it a fundamental tool for establishing the prognosis of ischemic heart disease. In patients with chronic ischemic heart disease, cardiac MRI can detect ischemia induced by pharmacological stress and can diagnose infarcts that can be missed on other techniques.

  2. Angina pectoris in patients without flow-limiting coronary artery disease (cardiac syndrome X). A forest of a variety of trees.

    PubMed

    Cocco, Giuseppe; Jerie, Paul

    2015-01-01

    Coronary heart disease (CHD) represents an important problem worldwide. At present, more women than men are evaluated for CHD and it has been recognized that the prevalence of this pathology in women is at least the same as in men. We have learned that cardiac syndrome X (CSX) is frequent because worldwide each year millions of people (mostly women) with angina pectoris without flow-limiting epicardial pathology are identified. Data from large myocardial infarction registries suggest a 5% to 25% prevalence of cases without flow-limiting coronary pathology. It must, however, be considered that these people are said to have normal coronary arteries by visual analysis of biplane coronarography. On the other hand, as demonstrated from autopsy, and in vivo by ultrasound intravascular studies, it would be more appropriate to say that in the majority of these cases no obstructive or flow-limiting coronary pathology was detected by coronarography. In CSX, endothelial dysfunction and microvascular dysfunction, sometimes with coronary microvascular spasm and epicardial coronary artery spasm, have been recognized as pathophysiologic mechanisms. In CSX, symptoms and pathologic signs are the same in patients with flow-limiting coronary pathology. The difference lies in the fact that the mechanisms of myocardial ischemia are microvascular and flow-limiting epicardial coronary pathology is absent. By interplay, the pathologic entities at work in CSX are linked with poor long-term outcome. The prevalence of these outcomes is probably smaller than in patients with flow-limiting coronary pathology but we lack precise values. Nonetheless, severe cardiovascular complications are frequent in CSX and it is thus the pathology is not benign. Drugs used in coronary ischemic disease are empirically prescribed to treat CSX, but we lack data from specific trials. It seems that statins and ranolazine might exert positive effects. However, specific research to target interventions in CSX would

  3. Re-endothelialization of rat lung scaffolds through passive, gravity-driven seeding of segment-specific pulmonary endothelial cells.

    PubMed

    Scarritt, Michelle E; Pashos, Nicholas C; Motherwell, Jessica M; Eagle, Zachary R; Burkett, Brian J; Gregory, Ashley N; Mostany, Ricardo; Weiss, Daniel J; Alvarez, Diego F; Bunnell, Bruce A

    2016-12-12

    Effective re-endothelialization is critical for the use of decellularized scaffolds for ex vivo lung engineering. Current approaches yield insufficiently re-endothelialized scaffolds that hemorrhage and become thrombogenic upon implantation. Herein, gravity-driven seeding coupled with bioreactor culture facilitated widespread distribution and engraftment of endothelial cells throughout rat lung scaffolds. Initially, human umbilical vein endothelial cells (HUVECs) were seeded into the pulmonary artery by either gravity-driven, variable flow perfusion seeding or pump-driven, pulsatile flow perfusion seeding. Gravity seeding evenly distributed cells and supported cell survival and re-lining of the vascular walls while perfusion pump-driven seeding led to increased cell fragmentation and death. Using gravity seeding, rat pulmonary artery endothelial cells (PAECs) and rat pulmonary vein endothelial cells (PVECs) attached in intermediate and large vessels, while rat pulmonary microvascular endothelial cells (MVECs) deposited mostly in microvessels. Combination seeding of PAECs, PVECs, and MVECs led to positive VE-cadherin staining. In addition, combination seeding improved barrier function as assessed by serum albumin extravasation; however, leakage was observed in the distal portions of the re-endothelialized tissue suggesting that recellularization of the alveoli is necessary to complete barrier function of the capillary-alveolar network. Overall, these data indicate that vascular recellularization of rat lung scaffolds is achieved through gravity seeding. This article is protected by copyright. All rights reserved.

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

    SciTech Connect

    Lin, Ming-Chung; Chen, Chia-Ling; Yang, Tsan-Tzu; Choi, Pui-Ching; Hsing, Chung-Hsi; Lin, Chiou-Feng

    2012-12-01

    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-like 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 increase

  5. Treatment of hemimasticatory spasm with microvascular decompression.

    PubMed

    Wang, Yong-Nan; Dou, Ning-Ning; Zhou, Qiu-Meng; Jiao, Wei; Zhu, Jin; Zhong, Jun; Li, Shi-Ting

    2013-01-01

    Hemimasticatory spasm is a rare disorder characterized by paroxysmal involuntary contraction of the jaw-closing muscles. As the ideology and pathogenesis of the disease are still unclear, there has been no treatment that could give rise to a good outcome so far. Herein, we tried to use surgical management to cure the disease. Six patients with the disease were included in this study. These patients underwent microvascular decompression of the motor fibers of the trigeminal root. After the operation, all faces of the patients felt relaxed at varied degrees, except for 1 patient. Our study showed that microvascular decompression of the trigeminal nerve could lead to a better outcome. However, a control study with a large sample is needed before this technique is widely used.

  6. EFFECTS OF DOBUTAMINE ON INTESTINAL MICROVASCULAR BLOOD FLOW HETEROGENEITY AND OXYGEN EXTRACTION DURING SEPTIC SHOCK.

    PubMed

    Ospina-Tascón, Gustavo Adolfo; García Marín, Alberto Federico; Echeverri, Gabriel J; Bermúdez, William Fernando; Madriñán Navia, Humberto José; Valencia, Juan David; Quiñones, Edgardo; Rodríguez, Fernando; Marulanda, Angela; Arango Davila, César Augusto; Bruhn, Alejandro; Hernández, Glenn; De Backer, Daniel

    2017-03-23

    Derangements of microvascular blood flow distribution might contribute to disturbing oxygen extraction by peripheral tissues. We evaluated the dynamic relationships between the mesenteric oxygen extraction ratio (mes-ERO2) and the heterogeneity of microvascular blood flow at the gut and sublingual mucosa, during the development and resuscitation of septic shock in a swine model of fecal peritonitis. Jejunal-villi and sublingual microcirculation were evaluated using a portable intravital-microscopy technique. Simultaneously, we obtained arterial, mixed-venous and mesenteric blood gases, and jejunal-tonometric measurements. During resuscitation, pigs were randomly allocated to fixed-dose of dobutamine (5 µgr/kg/min) or placebo, while three sham models with identical monitoring served as controls. At the time-of-shock, we observed a significant decreased proportion of perfused intestinal-villi (villi-PPV) and sublingual percentage of perfused small-vessels (SL-PPV), paralleling an increase in mes-ERO2 in both dobutamine and placebo groups. After starting resuscitation, villi-PPV and SL-PPV significantly increased in the dobutamine group with subsequent improvement of functional capillary density, while mes-ERO2 exhibited a corresponding significant decrease (repeated-measures ANOVA, p=0.02 and p=0.04 for time*group-interactions and inter-group differences for villi-PPV and mes-ERO2, respectively). Variations in villi-PPV were paralleled by variations in mes-ERO2 (R(2)=0.88, p<0.001) and these, in turn, by mesenteric lactate changes (R(2)=0.86, p<0.001). There were no significant differences in cardiac output and systemic oxygen delivery throughout the experiment. In conclusion, dynamic changes in microvascular blood flow heterogeneity at jejunal mucosa are closely related to the mesenteric oxygen extraction ratio, suggesting a crucial role for microvascular blood flow distribution on oxygen uptake during development and resuscitation from septic shock.

  7. Vitamin D and retinal microvascular damage

    PubMed Central

    Mutlu, Unal; Ikram, M Arfan; Hofman, Albert; de Jong, Paulus T V M; Uitterlinden, Andre G; Klaver, Caroline C W; Ikram, M Kamran

    2016-01-01

    Abstract Vitamin D has been linked to various cardiovascular risk factors including indices of large-vessel disease. However, it remains unclear whether vitamin D is also associated with microvascular damage. In a community-dwelling population, we studied associations between vitamin D serum levels and retinal microvascular damage defined as retinopathy signs, narrower arterioles, and wider venules. From the population-based Rotterdam Study, we included 5675 participants (age ≥45 years) with vitamin D data and gradable retinal photographs. Serum levels of vitamin D were measured using an antibody-based assay. Retinal exudates, microaneurysms, cotton wool spots, and dot/blot hemorrhages were graded on fundus photographs by experienced graders in the whole sample; retinal vascular calibers, that is, arteriolar and venular diameters, were semiautomatically measured in a subsample (n = 2973). We examined the cross-sectional association between vitamin D and retinal microvascular damage using logistic and linear regression models, adjusting for age, sex, and cardiovascular risk factors. We found that persons with lower vitamin D levels were more likely to have retinopathy (adjusted odds ratio per standard deviation (SD) decrease of vitamin D = 1.30; 95% confidence interval (CI): = 1.12–1.49). Furthermore, lower vitamin D levels were associated with wider venular calibers (adjusted mean difference per SD decrease in vitamin D = 1.35; 95% CI = 0.64–2.06). This association was strongest among men (P for interaction = 0.023). Lower levels of vitamin D are associated with retinal microvascular damage, suggesting that the link with cardiovascular risk may partly run through changes in the microvasculature. PMID:27930528

  8. Hyperglycemia and Diabetes Downregulate the Functional Expression of TRPV4 Channels in Retinal Microvascular Endothelium

    PubMed Central

    Monaghan, Kevin; McNaughten, Jennifer; McGahon, Mary K.; Kelly, Catriona; Kyle, Daniel; Yong, Phaik Har

    2015-01-01

    Retinal endothelial cell dysfunction is believed to play a key role in the etiology and pathogenesis of diabetic retinopathy. Numerous studies have shown that TRPV4 channels are critically involved in maintaining normal endothelial cell function. In the current paper, we demonstrate that TRPV4 is functionally expressed in the endothelium of the retinal microcirculation and that both channel expression and activity is downregulated by hyperglycaemia. Quantitative PCR and immunostaining demonstrated molecular expression of TRPV4 in cultured bovine retinal microvascular endothelial cells (RMECs). Functional TRPV4 activity was assessed in cultured RMECs from endothelial Ca2+-responses recorded using fura-2 microfluorimetry and electrophysiological recordings of membrane currents. The TRPV4 agonist 4α-phorbol 12,13-didecanoate (4-αPDD) increased [Ca2+]i in RMECs and this response was largely abolished using siRNA targeted against TRPV4. These Ca2+-signals were completely inhibited by removal of extracellular Ca2+, confirming their dependence on influx of extracellular Ca2+. The 4-αPDD Ca2+-response recorded in the presence of cyclopiazonic acid (CPA), which depletes the intracellular stores preventing any signal amplification through store release, was used as a measure of Ca2+-influx across the cell membrane. This response was blocked by HC067047, a TRPV4 antagonist. Under voltage clamp conditions, the TRPV4 agonist GSK1016790A stimulated a membrane current, which was again inhibited by HC067047. Following incubation with 25mM D-glucose TRPV4 expression was reduced in comparison with RMECs cultured under control conditions, as were 4αPDD-induced Ca2+-responses in the presence of CPA and ion currents evoked by GSK1016790A. Molecular expression of TRPV4 in the retinal vascular endothelium of 3 months’ streptozotocin-induced diabetic rats was also reduced in comparison with that in age-matched controls. We conclude that hyperglycaemia and diabetes reduce the

  9. Fabrication, characterization, and modeling of microvascular composites

    NASA Astrophysics Data System (ADS)

    Ryan, Thomas J.

    Composite laminates of glass fiber and epoxy pre-preg were fabricated with microvascular channels. The channels were created using polylactic acid (PLA) filament that evaporates at a temperature of 392 °F (200 °C) above the resin cure temperature of 250 °F (121 °C). After the composite is cured, the panel was removed from the oven and allowed to cool to room temperature. The panel is then reheated to 392 °F to vaporize the filament, leaving a cylindrical channel. A microvascular channel can be used for withdrawing heat, damage detection and self-healing. However, increasing the temperatures of the laminate above the cure temperature of the resin causes excess cross linking, potentially decreasing the mechanical properties. Tensile and flexural mechanical tests were performed on composite specimens and tensile tests were performed on neat resin specimens. A three-dimensional finite element model (FEM) was developed to study the progressive deformation and damage mechanics under tensile loading. The load carrying capacity of the microvascular composite was shown to decrease by 40% from a standard composite material. This paper will present the details of the fabrication, characterization and modeling techniques that were used in this study.

  10. Transcript Analysis Reveals a Specific HOX Signature Associated with Positional Identity of Human Endothelial Cells

    PubMed Central

    Toshner, Mark; Dunmore, Benjamin J.; McKinney, Eoin F.; Southwood, Mark; Caruso, Paola; Upton, Paul D.; Waters, John P.; Ormiston, Mark L.; Skepper, Jeremy N.; Nash, Gerard; Rana, Amer A.; Morrell, Nicholas W.

    2014-01-01

    The endothelial cell has a remarkable ability for sub-specialisation, adapted to the needs of a variety of vascular beds. The role of developmental programming versus the tissue contextual environment for this specialization is not well understood. Here we describe a hierarchy of expression of HOX genes associated with endothelial cell origin and location. In initial microarray studies, differential gene expression was examined in two endothelial cell lines: blood derived outgrowth endothelial cells (BOECs) and pulmonary artery endothelial cells. This suggested shared and differential patterns of HOX gene expression between the two endothelial lines. For example, this included a cluster on chromosome 2 of HOXD1, HOXD3, HOXD4, HOXD8 and HOXD9 that was expressed at a higher level in BOECs. Quantative PCR confirmed the higher expression of these HOXs in BOECs, a pattern that was shared by a variety of microvascular endothelial cell lines. Subsequently, we analysed publically available microarrays from a variety of adult cell and tissue types using the whole “HOX transcriptome” of all 39 HOX genes. Using hierarchical clustering analysis the HOX transcriptome was able to discriminate endothelial cells from 61 diverse human cell lines of various origins. In a separate publically available microarray dataset of 53 human endothelial cell lines, the HOX transcriptome additionally organized endothelial cells related to their organ or tissue of origin. Human tissue staining for HOXD8 and HOXD9 confirmed endothelial expression and also supported increased microvascular expression of these HOXs. Together these observations suggest a significant involvement of HOX genes in endothelial cell positional identity. PMID:24651450

  11. Transcript analysis reveals a specific HOX signature associated with positional identity of human endothelial cells.

    PubMed

    Toshner, Mark; Dunmore, Benjamin J; McKinney, Eoin F; Southwood, Mark; Caruso, Paola; Upton, Paul D; Waters, John P; Ormiston, Mark L; Skepper, Jeremy N; Nash, Gerard; Rana, Amer A; Morrell, Nicholas W

    2014-01-01

    The endothelial cell has a remarkable ability for sub-specialisation, adapted to the needs of a variety of vascular beds. The role of developmental programming versus the tissue contextual environment for this specialization is not well understood. Here we describe a hierarchy of expression of HOX genes associated with endothelial cell origin and location. In initial microarray studies, differential gene expression was examined in two endothelial cell lines: blood derived outgrowth endothelial cells (BOECs) and pulmonary artery endothelial cells. This suggested shared and differential patterns of HOX gene expression between the two endothelial lines. For example, this included a cluster on chromosome 2 of HOXD1, HOXD3, HOXD4, HOXD8 and HOXD9 that was expressed at a higher level in BOECs. Quantative PCR confirmed the higher expression of these HOXs in BOECs, a pattern that was shared by a variety of microvascular endothelial cell lines. Subsequently, we analysed publically available microarrays from a variety of adult cell and tissue types using the whole "HOX transcriptome" of all 39 HOX genes. Using hierarchical clustering analysis the HOX transcriptome was able to discriminate endothelial cells from 61 diverse human cell lines of various origins. In a separate publically available microarray dataset of 53 human endothelial cell lines, the HOX transcriptome additionally organized endothelial cells related to their organ or tissue of origin. Human tissue staining for HOXD8 and HOXD9 confirmed endothelial expression and also supported increased microvascular expression of these HOXs. Together these observations suggest a significant involvement of HOX genes in endothelial cell positional identity.

  12. Selective capture of endothelial and perivascular cells from brain microvessels using laser capture microdissection.

    PubMed

    Kinnecom, Katie; Pachter, Joel S

    2005-12-01

    Laser capture microdissection (LCM) of the major cell types comprising brain microvessels offers a powerful technology to explore the molecular basis of the blood-brain barrier in health and disease. However, the ability to selectively retrieve endothelial or perivascular cells, without cross-contamination from the other, has proven difficult. Additionally, histochemical methods previously described for use with LCM have not allowed for identification of all the different size branches of the microvascular tree. Here, we describe a double immunostaining method, combining bright-field and fluorescence microscopy, and using an extensive dehydration with xylene, to clearly identify and spatially resolve endothelial from perivascular cells within all size microvascular branches in frozen brain sections. LCM of these sections, coupled with RNA analysis by reverse-transcription polymerase chain reaction, revealed that captured endothelial cells show endothelial markers but no detectable markers for astrocytes or smooth muscle cells/pericytes. Conversely, captured astrocytes or smooth muscle cells/pericytes demonstrate their respective markers, but not those of endothelial cells. This approach has applicability to microarray analysis, thereby enabling global gene profiling of the different cell types along the entirety of the brain microvascular tree.

  13. Endothelial Cell Growth and Differentiation on Collagen-Immobilized Polycaprolactone Nanowire Surfaces.

    PubMed

    Leszczak, Victoria; Baskett, Dominique A; Popat, Ketul C

    2015-06-01

    The success of cardiovascular implants is associated with the development of an endothelium on material surface, critical to the prevention of intimal hyperplasia, calcification and thrombosis. A thorough understanding of the interaction between vascular endothelial cells and the biomaterial involved is essential in order to have a successful application which promotes healing and regeneration through integration with native tissue. In this study, we have developed collagen immobilized nanostructured surfaces with controlled arrays of high aspect ratio nanowires for the growth and maintenance of human microvascular endothelial cells (HMVECs). The nanowire surfaces were fabricated from polycaprolactone using a novel nanotemplating technique, and were immobilized with collagen utilizing an aminolysis method. The collagen immobilized nanowire surfaces were characterized using contact angle measurements, scanning electron microscopy and X-ray photoelectron spectroscopy. Human microvascular endothelial cells were used to evaluate the efficacy of the collagen immobilized nanowire surfaces to promote cell adhesion, proliferation, viability and differentiation. The results presented here indicate significantly higher cellular adhesion, proliferation and viability on nanowire and collagen immobilized surfaces as compared to the control surface. Further, HMVECs have a more elongated body and low shape factor on nanostructured surfaces. The differentiation potential of collagen immobilized nanowire surfaces was also evaluated by immunostaining and western blotting for key endothelial cell markers that are expressed when human microvascular endothelial cells are differentiated. Results indicate that expression of VE-cadherin is increased on collagen immobilized surfaces while the expression of von Willebrand factor is statistically similar on all surfaces.

  14. Cardiac catheterization - discharge

    MedlinePlus

    Catheterization - cardiac - discharge; Heart catheterization - discharge: Catheterization - cardiac; Heart catheterization; Angina - cardiac catheterization discharge; CAD - cardiac catheterization discharge; Coronary artery disease - cardiac catheterization ...

  15. Endothelial activation and dysfunction in the pathogenesis of influenza A virus infection

    PubMed Central

    Armstrong, Susan M; Darwish, Ilyse; Lee, Warren L

    2013-01-01

    The development of severe influenza has been attributed, in part, to a heightened innate immune response. Recent evidence suggests that endothelial activation, loss of barrier function, and consequent microvascular leak may also serve important mechanistic roles in the pathogenesis of severe influenza. The aim of this review is to summarize the current evidence in support of endothelial activation and dysfunction as a central feature preceding the development of severe influenza. We also discuss the effect of influenza on platelet–endothelial interactions. PMID:23863601

  16. Effects of Aggregatibacter actinomycetemcomitans leukotoxin on endothelial cells.

    PubMed

    Dietmann, Anelia; Millonig, Alban; Combes, Valery; Couraud, Pierre-Olivier; Kachlany, Scott C; Grau, Georges E

    2013-01-01

    Aggregatibacter actinomycetemcomitans is a human pathogen that produces leukotoxin (LtxA) as a major virulence factor. In this study the effect of LtxA on microvascular endothelial cell viability and phenotype was studied. High doses of single LtxA treatment (500 ng/ml to 5 μg/ml) significantly and irreversibly decreased cell proliferation and induced apoptosis, as assessed by tetrazolium salt and annexin V assay, respectively. Apoptosis was partially inhibited by the pan-caspase inhibitor, z-VAD-fmk. LtxA caused a cell cycle arrest in the G2/M phase after 72 h. Between 500 ng/ml and 5 μg/ml, after long- or short-term stimulation LtxA increased the expression of ICAM-1 and VCAM-1, as well as the percentages of endothelial cells expressing these adhesion molecules. Thus, A. actinomycetemcomitans LtxA has substantial pro-inflammatory effects on human brain endothelial cells by upregulation of ICAM-1 and VCAM-1. Furthermore, LtxA in higher concentration was found to decrease proliferation and induces apoptosis in microvascular endothelial cells.

  17. GPCR signaling and cardiac function.

    PubMed

    Capote, Leany A; Mendez Perez, Roberto; Lymperopoulos, Anastasios

    2015-09-15

    G protein-coupled receptors (GPCRs), such as β-adrenergic and angiotensin II receptors, located in the membranes of all three major cardiac cell types, i.e. myocytes, fibroblasts and endothelial cells, play crucial roles in regulating cardiac function and morphology. Their importance in cardiac physiology and disease is reflected by the fact that, collectively, they represent the direct targets of over a third of the currently approved cardiovascular drugs used in clinical practice. Over the past few decades, advances in elucidation of their structure, function and the signaling pathways they elicit, specifically in the heart, have led to identification of an increasing number of new molecular targets for heart disease therapy. Here, we review these signaling modalities employed by GPCRs known to be expressed in the cardiac myocyte membranes and to directly modulate cardiac contractility. We also highlight drugs and drug classes that directly target these GPCRs to modulate cardiac function, as well as molecules involved in cardiac GPCR signaling that have the potential of becoming novel drug targets for modulation of cardiac function in the future.

  18. Hypoxia-Induced Reactive Oxygen Species Cause Chromosomal Abnormalities in Endothelial Cells in the Tumor Microenvironment

    PubMed Central

    Hida, Yasuhiro; Maishi, Nako; Towfik, Alam Mohammad; Inoue, Nobuo; Shindoh, Masanobu; Hida, Kyoko

    2013-01-01

    There is much evidence that hypoxia in the tumor microenvironment enhances tumor progression. In an earlier study, we reported abnormal phenotypes of tumor-associated endothelial cells such as those resistant to chemotherapy and chromosomal instability. Here we investigated the role of hypoxia in the acquisition of chromosomal abnormalities in endothelial cells. Tumor-associated endothelial cells isolated from human tumor xenografts showed chromosomal abnormalities, >30% of which were aneuploidy. Aneuploidy of the tumor-associated endothelial cells was also shown by simultaneous in-situ hybridization for chromosome 17 and by immunohistochemistry with anti-CD31 antibody for endothelial staining. The aneuploid cells were surrounded by a pimonidazole-positive area, indicating hypoxia. Human microvascular endothelial cells expressed hypoxia-inducible factor 1 and vascular endothelial growth factor A in response to either hypoxia or hypoxia-reoxygenation, and in these conditions, they acquired aneuploidy in 7 days. Induction of aneuploidy was inhibited by either inhibition of vascular endothelial growth factor signaling with vascular endothelial growth factor receptor 2 inhibitor or by inhibition of reactive oxygen species by N-acetyl-L-cysteine. These results indicate that hypoxia induces chromosomal abnormalities in endothelial cells through the induction of reactive oxygen species and excess signaling of vascular endothelial growth factor in the tumor microenvironment. PMID:24260373

  19. Aged garlic extract improves homocysteine-induced endothelial dysfunction in macro- and microcirculation.

    PubMed

    Weiss, Norbert; Ide, Nagatoshi; Abahji, Thomas; Nill, Lars; Keller, Christiane; Hoffmann, Ulrich

    2006-03-01

    Endothelial dysfunction caused by increases in vascular oxidant stress that decrease bioavailable nitric oxide (NO) plays a critical role in the vascular pathobiology of hyperhomocysteinemia. Boosting cellular glutathione levels or increasing the activity of cellular glutathione peroxidase can compensate for homocysteine's effects on endothelial function. Aged garlic extract (AGE) contains water- and oil-soluble sulfur compounds that modify the intracellular thiol and redox state, minimize intracellular oxidant stress, and stimulate NO generation in endothelial cells and animals. We performed a placebo-controlled, blinded, crossover trial to examine whether AGE reduces macro- and microvascular endothelial dysfunction during acute hyperhomocysteinemia induced by an oral methionine challenge in healthy subjects. Acute hyperhomocysteinemia leads to a significant decrease in flow-mediated vasodilation of the brachial artery as determined by vascular ultrasound, indicative of macrovascular endothelial dysfunction. In addition, acute hyperhomocysteinemia leads to a decrease in acetylcholine-stimulated skin perfusion as measured by laser-Doppler flowmetry. This indicates microvascular endothelial dysfunction, which is presumably a result of impairment of the endothelium-derived hyperpolarizing factor pathway. Pretreatment with AGE for 6 wk significantly diminished the adverse effects of acute hyperhomocysteinemia in both vascular territories. We conclude that AGE may at least partly prevent a decrease in bioavailable NO and endothelium-derived hyperpolarizing factor during acute hyperhomocysteinemia. This pilot study warrants further investigations on the effects of AGE on endothelial dysfunction in patients with other cardiovascular risk factors or established vascular disease and on the clinical outcome of patients with cardiovascular disease.

  20. Stromal Cells in Dense Collagen Promote Cardiomyocyte and Microvascular Patterning in Engineered Human Heart Tissue.

    PubMed

    Roberts, Meredith A; Tran, Dominic; Coulombe, Kareen L K; Razumova, Maria; Regnier, Michael; Murry, Charles E; Zheng, Ying

    2016-04-01

    Cardiac tissue engineering is a strategy to replace damaged contractile tissue and model cardiac diseases to discover therapies. Current cardiac and vascular engineering approaches independently create aligned contractile tissue or perfusable vasculature, but a combined vascularized cardiac tissue remains to be achieved. Here, we sought to incorporate a patterned microvasculature into engineered heart tissue, which balances the competing demands from cardiomyocytes to contract the matrix versus the vascular lumens that need structural support. Low-density collagen hydrogels (1.25 mg/mL) permit human embryonic stem cell-derived cardiomyocytes (hESC-CMs) to form a dense contractile tissue but cannot support a patterned microvasculature. Conversely, high collagen concentrations (density ≥6 mg/mL) support a patterned microvasculature, but the hESC-CMs lack cell-cell contact, limiting their electrical communication, structural maturation, and tissue-level contractile function. When cocultured with matrix remodeling stromal cells, however, hESC-CMs structurally mature and form anisotropic constructs in high-density collagen. Remodeling requires the stromal cells to be in proximity with hESC-CMs. In addition, cocultured cardiac constructs in dense collagen generate measurable active contractions (on the order of 0.1 mN/mm(2)) and can be paced up to 2 Hz. Patterned microvascular networks in these high-density cocultured cardiac constructs remain patent through 2 weeks of culture, and hESC-CMs show electrical synchronization. The ability to maintain microstructural control within engineered heart tissue enables generation of more complex features, such as cellular alignment and a vasculature. Successful incorporation of these features paves the way for the use of large scale engineered tissues for myocardial regeneration and cardiac disease modeling.

  1. Low dose radiation-induced endothelial cell retraction.

    PubMed

    Kantak, S S; Diglio, C A; Onoda, J M

    1993-09-01

    We characterized in vitro the effects of gamma-radiation (12.5-100 cGy) on pulmonary microvascular endothelial cell (PMEC) morphology and F-actin organization. Cellular retraction was documented by phase-contrast microscopy and the organization of actin microfilaments was determined by immunofluorescence. Characterization included radiation dose effects, their temporal duration and reversibility of the effects. A dose-dependent relationship between the level of exposure (12.5-100 cGy) and the rate and extent of endothelial retraction was observed. Moreover, analysis of radiation-induced depolymerization of F-actin microfilament stress fibres correlated positively with the changes in PMEC morphology. The depolymerization of the stress fibre bundles was dependent on radiation dose and time. Cells recovered from exposure to reform contact inhibited monolayers > or = 24 h post-irradiation. Concomitantly, the depolymerized microfilaments reorganized to their preirradiated state as microfilament stress fibres arrayed parallel to the boundaries of adjacent contact-inhibited cells. The data presented here are representative of a series of studies designed to characterize low-dose radiation effects on pulmonary microvascular endothelium. Our data suggest that post-irradiation lung injuries (e.g. oedema) may be induced with only a single fraction of therapeutic radiation, and thus microscopic oedema may initiate prior to the lethal effects of radiation on the microvascular endothelium, and much earlier than would be suggested by the time course for clinically-detectable oedema.

  2. Association of Maternal Antiangiogenic Profile at Birth With Early Postnatal Loss of Microvascular Density in Offspring of Hypertensive Pregnancies

    PubMed Central

    Yu, Grace Z.; Aye, Christina Y.L.; Lewandowski, Adam J.; Davis, Esther F.; Khoo, Cheen P.; Newton, Laura; Yang, Cheng T.; Al Haj Zen, Ayman; Simpson, Lisa J.; O’Brien, Kathryn; Cook, David A.; Granne, Ingrid; Kyriakou, Theodosios; Channon, Keith M.; Watt, Suzanne M.

    2016-01-01

    Offspring of hypertensive pregnancies are more likely to have microvascular rarefaction and increased blood pressure in later life. We tested the hypothesis that maternal angiogenic profile during a hypertensive pregnancy is associated with fetal vasculogenic capacity and abnormal postnatal microvascular remodeling. Infants (n=255) born after either hypertensive or normotensive pregnancies were recruited for quantification of postnatal dermal microvascular structure at birth and 3 months of age. Vasculogenic cell potential was assessed in umbilical vein endothelial cells from 55 offspring based on in vitro microvessel tube formation and proliferation assays. Maternal angiogenic profile (soluble fms-like tyrosine kinase-1, soluble endoglin, vascular endothelial growth factor, and placental growth factor) was measured from postpartum plasma samples to characterize severity of pregnancy disorder. At birth, offspring born after hypertensive pregnancy had similar microvessel density to those born after a normotensive pregnancy, but during the first 3 postnatal months, they had an almost 2-fold greater reduction in total vessel density (−17.7±16.4% versus −9.9±18.7%; P=0.002). This postnatal loss varied according to the vasculogenic capacity of the endothelial cells of the infant at birth (r=0.49; P=0.02). The degree of reduction in both in vitro and postnatal in vivo vascular development was proportional to levels of antiangiogenic factors in the maternal circulation. In conclusion, our data indicate that offspring born to hypertensive pregnancies have reduced vasculogenic capacity at birth that predicts microvessel density loss over the first 3 postnatal months. Degree of postnatal microvessel reduction is proportional to levels of antiangiogenic factors in the maternal circulation at birth. PMID:27456522

  3. Longitudinal assessment of endothelial function in the microvasculature of mice in-vivo.

    PubMed

    Belch, Jill J F; Akbar, Naveed; Alapati, Venkateswara; Petrie, John; Arthur, Simon; Khan, Faisel

    2013-01-01

    Endothelial dysfunction is associated with early development of cardiovascular disease, making longitudinal measurements desirable. We devised a protocol using laser Doppler imaging (LDI) and iontophoresis of acetylcholine (ACh) and sodium nitroprusside (SNP) to assess the skin microcirculation longitudinally in mice every 4 weeks for 24 weeks in two groups of C57BL/6 mice, chow versus high-cholesterol diet(known to induce endothelial dysfunction). LDI measurements were compared with vascular function (isometric tension) measured using wire myography in the tail artery in response to ACh and SNP. Microvascular responses to ACh were significantly reduced in cholesterol-fed versus chow-fed mice from week 4 onwards (P<0.005, ANOVA). Pre-treatment with N(G)-nitro-L-arginine methyl-ester-hydrochloride (L-NAME) showed a significant reduction in ACh response compared with vehicle-treated animals (P<0.05) at baseline and at 12 weeks. In cholesterol-fed mice, ACh responses were 226 ± 21 and 180 ± 21 AU (P=0.03) before and after L-NAME, respectively. A reduction in ex-vivo ACh response was detected in the tail artery in cholesterol-fed mice, and a significant correlation found between peak microvascular ACh response and maximum ACh response in the tail artery (r=0.699, P=0.017). No changes were found in SNP responses in the microvasculature or tail artery. Using this protocol, we have shown longitudinal decreases in microvascular endothelial function to cholesterol feeding. L-NAME studies confirm that the reduced vasodilatation to ACh in cholesterol-fed mice was mediated partly through reduced NO bioavailability. Wire myography of tail arteries confirmed that in-vivo measurements of microvascular function reflect ex-vivo vascular function in other beds. Longitudinal assessments of skin microvascular function in mice could provide a useful translatable model for assessing early endothelial dysfunction.

  4. Direct ink writing of microvascular networks

    NASA Astrophysics Data System (ADS)

    Wu, Willie

    Nature is replete with examples of embedded microvascular systems that enable efficient fluid flow and distribution for autonomic healing, cooling, and energy harvesting. The ability to incorporate microvascular networks in functional materials systems is therefore both scientifically and technologically important. In this PhD thesis, the direct-write assembly of planar and 3D biomimetic microvascular networks within polymer and hydrogel matrices is demonstrated. In addition, the influence of network design of fluid transport efficiency is characterized. Planar microvascular networks composed of periodic lattices of uniformal microchannels and hierarchical, branching architectures are constructed by direct-write assembly of a fugitive organic ink. Several advancements are required to facilitate their patterning, including pressure valving, dual ink printing, and dynamic pressure variation to allow tunable control of ink deposition. The hydraulic conductance is measured using a high pressure flow meter as a function of network design. For a constant vascular volume and areal coverage, 2- and 4-generation branched architectures that obey Murray's Law exhibited the highest hydraulic conductivity. These experimental observations are in good agreement with predictions made by analytic models. 3D microvascular networks are fabricated by omnidirectional printing a fugitive organic ink into a photopolymerizable hydrogel matrix that is capped with fluid filler of nearly identical composition. Using this approach, 3D networks of arbitrary design can be patterned. After ink deposition is complete, the matrix and fluid filler are chemically cross-linked via UV irradiation, and the ink is removed by liquefication. Aqueous solutions composed of a triblock copolymer of polyethylene oxide (PEO)-polypropylene oxide (PPO)-PEO constitute the materials system of choice due to their thermal- and concentration-dependent phase behavior. Specifically, the fugitive ink consists of a 23 w

  5. Expression of endothelial nitric oxide synthase and vascular endothelial growth factor in association with neovascularization in human primary astrocytoma*

    PubMed Central

    Pan, Jian-wei; Zhan, Ren-ya; Tong, Ying; Zhou, Yong-qing; Zhang, Ming

    2005-01-01

    Objective: To investigate the relationship between the expression of endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF) and angiogenesis in primary astrocytoma. Methods: Thirty-seven primary astrocytomas and 4 astrocytic hyperplasia samples were collected and divided into three groups according to histological grade. The expression of eNOS, VEGF and factor VIII related antigen (FVIIIRAg) were assayed by immunohistochemistry. Microvascular density was assessed by FVIIIRAg immunoreactivity. The intensity of immunoreactivity was graded according to the percentage of positive tumor cells. Results: No eNOS and VEGF were expressed in the astrocytes and vascular endothelium in astrocytic hyperplasia. The expression of eNOS or VEGF was light in low-grade astrocytoma and strong in glioblastoma. eNOS expression in astrocytoma was very positively correlated with VEGF. eNOS and VEGF expression in anaplastic astrocytoma was median in contrast to the low grade astrocytoma and glioblastoma. Lower microvascular density was found in low grade astrocytoma than that in higher grade malignant ones. The expressions of eNOS and VEGF were correlated with microvascular density and tumor malignancy. Conclusion: This finding suggests that eNOS and VEGF may have cooperative effect in tumor angiogenesis and play an important role in the pathogenesis of primary astrocytoma. PMID:15973775

  6. Human Myocardial Pericytes: Multipotent Mesodermal Precursors Exhibiting Cardiac Specificity

    PubMed Central

    Chen, William C.W.; Baily, James E.; Corselli, Mirko; Diaz, Mary; Sun, Bin; Xiang, Guosheng; Gray, Gillian A.; Huard, Johnny; Péault, Bruno

    2015-01-01

    Perivascular mesenchymal precursor cells (i.e. pericytes) reside in skeletal muscle where they contribute to myofiber regeneration; however, the existence of similar microvessel-associated regenerative precursor cells in cardiac muscle has not yet been documented. We tested whether microvascular pericytes within human myocardium exhibit phenotypes and multipotency similar to their anatomically and developmentally distinct counterparts. Fetal and adult human heart pericytes (hHPs) express canonical pericyte markers in situ, including CD146, NG2, PDGFRβ, PDGFRα, αSMA, and SM-MHC, but not CD117, CD133 and desmin, nor endothelial cell (EC) markers. hHPs were prospectively purified to homogeneity from ventricular myocardium by flow cytometry, based on a combination of positive- (CD146) and negative-selection (CD34, CD45, CD56, and CD117) cell lineage markers. Purified hHPs expanded in vitro were phenotypically similar to human skeletal muscle-derived pericytes (hSkMPs). hHPs express MSC markers in situ and exhibited osteo- chondro-, and adipogenic potentials but, importantly, no ability for skeletal myogenesis, diverging from pericytes of all other origins. hHPs supported network formation with/without ECs in Matrigel cultures; hHPs further stimulated angiogenic responses under hypoxia, markedly different from hSkMPs. The cardiomyogenic potential of hHPs was examined following 5-azacytidine treatment and neonatal cardiomyocyte co-culture in vitro, and intramyocardial transplantation in vivo. Results indicated cardiomyocytic differentiation in a small fraction of hHPs. In conclusion, human myocardial pericytes share certain phenotypic and developmental similarities with their skeletal muscle homologs, yet exhibit different antigenic, myogenic, and angiogenic properties. This is the first example of an anatomical restriction in the developmental potential of pericytes as native mesenchymal stem cells. PMID:25336400

  7. Kaposi's sarcoma-associated herpesvirus infection of blood endothelial cells induces lymphatic differentiation.

    PubMed

    Carroll, Patrick A; Brazeau, Elizabeth; Lagunoff, Michael

    2004-10-10

    Kaposi's sarcoma-associated herpesvirus (KSHV) is necessary for KS, a highly vascularized tumor predominated by endothelial-derived spindle cells that express markers of lymphatic endothelium. Following KSHV infection of TIME cells, an immortalized human dermal microvascular endothelial cell (DMVEC) line, expression of many genes specific to lymphatic endothelium, including VEGFR3, podoplanin, LYVE-1, and Prox-1, is significantly increased. Increases in VEGFR3 and podoplanin protein are also demonstrated following latent infection. Examination of cytokine secretion showed that KSHV infection significantly induces hIL-6 while strongly inhibiting secretion of IL-8, a gene product that is decreased by differentiation of blood to lymphatic endothelial cells. These studies support the hypotheses that latent KSHV infection of blood endothelial cells drives their differentiation to lymphatic endothelial cells.

  8. Increased circulating levels of tissue kallikrein in systemic sclerosis correlate with microvascular involvement

    PubMed Central

    Del Rosso, A; Distler, O; Milia, A; Emanueli, C; Ibba-Manneschi, L; Guiducci, S; Conforti, M; Generini, S; Pignone, A; Gay, S; Madeddu, P; Matucci-Cerinic, M

    2005-01-01

    Background: In systemic sclerosis (SSc) the lack of an angiogenic response to hypoxia may be due to inappropriate synthesis of angiogenic and angiostatic factors. Tissue kallikrein (t-kallikrein), regulating the kallikrein-kinin system and acting on the microcirculation, is a potent angiogenic agent, and kallistatin is its natural inhibitor. Objective: To evaluate, in patients with SSc, t-kallikrein and kallistatin levels and their correlation with clinical features and measures of microvascular involvement. Patients and methods: Serum levels of t-kallikrein and kallistatin (ELISA) and t-kallikrein skin expression (immunohistochemistry) were studied in patients with SSc, and evaluated for subset (dSSc or lSSc), clinical and immunological features, and microvascular involvement (ulcers, telangiectasias, nailfold videocapillaroscopy). Results: Circulating levels of t-kallikrein were higher in SSc than in controls (p<0.001). T-kallikrein did not differ between lSSc and dSSc, although it was higher in lSSc than in controls (p<0.001).T-kallikrein levels were higher in patients with early and active capillaroscopic pattern than in those with late pattern (p = 0.019 and 0.023). Patients with giant capillaries and capillary microhaemorrhages had higher t-kallikrein concentrations than patients with architectural derangement (p = 0.04). No differences in kallistatin levels were detected between patients with SSc and controls, or between lSSc and dSSc. In early SSc skin, the presence of t-kallikrein was found in endothelial and in perivascular inflammatory cells, while no staining in skin of advanced SSc was detected. Conclusion: T-kallikrein levels are increased in patients with SSc, particularly in lSSc, and are associated with early and active capillaroscopic patterns. T-kallikrein may play a part in SSc microvascular changes. PMID:15708892

  9. Zingiber officinale attenuates retinal microvascular changes in diabetic rats via anti-inflammatory and antiangiogenic mechanisms

    PubMed Central

    Dongare, Shirish; Mathur, Rajani; Saxena, Rohit; Mathur, Sandeep; Agarwal, Renu; Nag, Tapas C.; Srivastava, Sushma; Kumar, Pankaj

    2016-01-01

    Purpose Diabetic retinopathy is a common microvascular complication of long-standing diabetes. Several complex interconnecting biochemical pathways are activated in response to hyperglycemia. These pathways culminate into proinflammatory and angiogenic effects that bring about structural and functional damage to the retinal vasculature. Since Zingiber officinale (ginger) is known for its anti-inflammatory and antiangiogenic properties, we investigated the effects of its extract standardized to 5% 6-gingerol, the major active constituent of ginger, in attenuating retinal microvascular changes in rats with streptozotocin-induced diabetes. Methods Diabetic rats were treated orally with the vehicle or the ginger extract (75 mg/kg/day) over a period of 24 weeks along with regular monitoring of bodyweight and blood glucose and weekly fundus photography. At the end of the 24-week treatment, the retinas were isolated for histopathological examination under a light microscope, transmission electron microscopy, and determination of the retinal tumor necrosis factor-α (TNF-α), nuclear factor-kappa B (NF-κB), and vascular endothelial growth factor (VEGF) levels. Results Oral administration of the ginger extract resulted in significant reduction of hyperglycemia, the diameter of the retinal vessels, and vascular basement membrane thickness. Improvement in the architecture of the retinal vasculature was associated with significantly reduced expression of NF-κB and reduced activity of TNF-α and VEGF in the retinal tissue in the ginger extract–treated group compared to the vehicle-treated group. Conclusions The current study showed that ginger extract containing 5% of 6-gingerol attenuates the retinal microvascular changes in rats with streptozotocin-induced diabetes through anti-inflammatory and antiangiogenic actions. Although precise molecular targets remain to be determined, 6-gingerol seems to be a potential candidate for further investigation. PMID:27293376

  10. Myocardial perfusion echocardiography and coronary microvascular dysfunction

    PubMed Central

    Barletta, Giuseppe; Del Bene, Maria Riccarda

    2015-01-01

    Our understanding of coronary syndromes has evolved in the last two decades out of the obstructive atherosclerosis of epicardial coronary arteries paradigm to include anatomo-functional abnormalities of coronary microcirculation. No current diagnostic technique allows direct visualization of coronary microcirculation, but functional assessments of this circulation are possible. This represents a challenge in cardiology. Myocardial contrast echocardiography (MCE) was a breakthrough in echocardiography several years ago that claimed the capability to detect myocardial perfusion abnormalities and quantify coronary blood flow. Research demonstrated that the integration of quantitative MCE and fractional flow reserve improved the definition of ischemic burden and the relative contribution of collaterals in non-critical coronary stenosis. MCE identified no-reflow and low-flow within and around myocardial infarction, respectively, and predicted the potential functional recovery of stunned myocardium using appropriate interventions. MCE exhibited diagnostic performances that were comparable to positron emission tomography in microvascular reserve and microvascular dysfunction in angina patients. Overall, MCE improved echocardiographic evaluations of ischemic heart disease in daily clinical practice, but the approval of regulatory authorities is lacking. PMID:26730291

  11. Disruption of in vitro endothelial barrier integrity by Japanese encephalitis virus-Infected astrocytes.

    PubMed

    Chang, Cheng-Yi; Li, Jian-Ri; Chen, Wen-Ying; Ou, Yen-Chuan; Lai, Ching-Yi; Hu, Yu-Hui; Wu, Chih-Cheng; Chang, Chen-Jung; Chen, Chun-Jung

    2015-05-08

    Blood-brain barrier (BBB) characteristics are induced and maintained by crosstalk between brain microvascular endothelial cells and neighboring cells. Using in vitro cell models, we previously found that a bystander effect was a cause for Japanese encephalitis-associated endothelial barrier disruption. Brain astrocytes, which neighbor BBB endothelial cells, play roles in the maintenance of BBB integrity. By extending the scope of relevant studies, a potential mechanism has been shown that the activation of neighboring astrocytes could be a cause of disruption of endothelial barrier integrity during the course of Japanese encephalitis viral (JEV) infection. JEV-infected astrocytes were found to release biologically active molecules that activated ubiquitin proteasome, degraded zonula occludens-1 (ZO-1) and claudin-5, and disrupted endothelial barrier integrity in cultured brain microvascular endothelial cells. JEV infection caused astrocytes to release vascular endothelial growth factor (VEGF), interleukin-6 (IL-6), and matrix metalloproteinases (MMP-2/MMP-9). Our data demonstrated that VEGF and IL-6 released by JEV-infected astrocytes were critical for the proteasomal degradation of ZO-1 and the accompanying disruption of endothelial barrier integrity through the activation of Janus kinase-2 (Jak2)/signal transducer and activator of transcription-3 (STAT3) signaling as well as the induction of ubiquitin-protein ligase E3 component, n-recognin-1 (Ubr 1) in endothelial cells. MMP-induced endothelial barrier disruption was accompanied by MMP-mediated proteolytic degradation of claudin-5 and ubiquitin proteasome-mediated degradation of ZO-1 via extracellular VEGF release. Collectively, these data suggest that JEV infection could activate astrocytes and cause release of VEGF, IL-6, and MMP-2/MMP-9, thereby contributing, in a concerted action, to the induction of Japanese encephalitis-associated BBB breakdown. GLIA 2015.

  12. Prevascularization of self-organizing engineered heart tissue by human umbilical vein endothelial cells abrogates contractile performance.

    PubMed

    Sondergaard, Claus Svane; Witt, Russell; Mathews, Grant; Najibi, Skender; Le, Lisa; Clift, Tracy; Si, Ming-Sing

    2012-12-01

    Establishing vascularization is a critical obstacle to the generation of engineered heart tissue (EHT) of substantial thickness. Addition of endothelial cells to the formative stages of EHT has been demonstrated to result in prevascularization, or the formation of capillary-like structures. The detailed study of the effects of prevascularization on EHT co