Growth factor- and heparin-dependent regulation of constitutive and agonist-mediated human endothelial barrier function

2006 ◽  
Vol 291 (5) ◽  
pp. H2126-H2135 ◽  
Author(s):  
Alan B. Moy ◽  
Ken Blackwell ◽  
Mack H. Wu ◽  
Harris J. Granger
Keyword(s):  
Growth Factors ◽  
Growth Factor ◽  
Numerical Model ◽  
Barrier Function ◽  
Umbilical Vein ◽  
Membrane Capacitance ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function ◽  
Barrier Dysfunction ◽  
Umbilical Vein Endothelial Cells

We report functional differences in constitutive and agonist-mediated endothelial barrier function between cultured primary and Clonetics human umbilical vein endothelial cells (pHUVEC and cHUVEC) grown in soluble growth factors and heparin. Basal transendothelial resistance (TER) was much lower in pHUVEC than in cHUVEC grown in medium supplemented with growth factors, such as basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and human epithelial growth factor (EGF), and heparin. On the basis of a numerical model of TER, the increased basal TER in cHUVEC was due to effects on cell-matrix adhesion and membrane capacitance. Heparin and bFGF increased constitutive TER in cultured pHUVEC, and heparin mediated additional increases in constitutive TER in pHUVEC supplemented with bFGF. EGF attenuated bFGF-mediated increases in TER. On the basis of the numerical model, in contrast to cHUVEC, heparin and bFGF augmented TER through effects on cell-cell adhesion and membrane capacitance in pHUVEC. Thrombin mediated quantitatively greater amplitude and a more sustained decline in TER in cultured cHUVEC than pHUVEC. Thrombin-mediated barrier dysfunction was attenuated in pHUVEC conditioned in EGF in the presence or absence of heparin. Thrombin-mediated barrier dysfunction was also attenuated when monolayers were exposed to low concentrations of heparin and further attenuated in the presence of bFGF. cAMP stimulation mediated differential attenuation of thrombin-mediated barrier dysfunction between pHUVEC and cHUVEC. VEGF displayed differential effects in TER in serum-free medium. Taken together, these data demonstrate marked differential regulation of constitutive and agonist-mediated endothelial barrier function in response to mitogens and heparin stimulation.

Augmentation of Vascular Endothelial Barrier Function by Heparin and Low Molecular Weight Heparin

1995 ◽  
Vol 73 (04) ◽  
pp. 706-712 ◽  
Author(s):  
P G Bannon ◽  
Mi-Jurng Kim ◽  
R T Dean ◽  
J Dawes
Keyword(s):  
Barrier Function ◽  
Umbilical Vein ◽  
Interleukin 1 ◽  
Endothelial Permeability ◽  
Endothelial Barrier ◽  
Vascular Endothelial ◽  
Transendothelial Electrical Resistance ◽  
Endothelial Barrier Function ◽  
Early Passage ◽  
Umbilical Vein Endothelial Cells

SummaryGlycosaminoglycans (GAGs) are an important component of endothelial barrier function. Early passage human umbilical vein endothelial cells were grown to confluence on transparent micropore filters and barrier function assessed as transendothelial electrical resistance (TEER) and permeability to albumin and sucrose. Unfractionated heparin and the LMW heparin Clexane decreased endothelial permeability to both sucrose and albumin and increased TEER. Chondroitin 6-sulphate also augmented barrier function, but other GAGs had no effect. Interleukin-1 increased permeability to albumin and sucrose and decreased TEER. Although heparin attenuated the effect of IL-1 on TEER and sucrose permeability, it could not restore the barrier to albumin transfer. Denuded endothelial matrix presented a negligible barrier, which was not enhanced by heparin. When sulphation of endogenous GAGs was inhibited by chlorate, barrier function was compromised and was not restored by exogenous heparin. Thus heparin enhances the barrier function of resting endothelium, but cannot completely overcome the increased permeability resulting from exposure to IL-1 or substitute for endogenous GAGs.

scholarly journals A collagen gel-coated, aligned nanofiber membrane for enhanced endothelial barrier function

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Dohui Kim ◽  
Seongsu Eom ◽  
Sang Min Park ◽  
Hyeonjun Hong ◽  
Dong Sung Kim
Keyword(s):  
Tight Junctions ◽  
Barrier Function ◽  
Synergistic Effects ◽  
Umbilical Vein ◽  
Collagen Gel ◽  
Electrospinning Process ◽  
Endothelial Barrier ◽  
Nanofiber Membrane ◽  
Endothelial Barrier Function ◽  
Barrier Dysfunction

Abstract Herein, a collagen gel-coated and aligned nanofiber membrane named Col-ANM is developed, which remarkably improves endothelial barrier function by providing biochemical and topographical cues simultaneously. Col-ANM is fabricated by collagen gel coating process on an aligned polycaprolactone (PCL) nanofiber membrane, which is obtained by a simple electrospinning process adopting a parallel electrode collector. Human umbilical vein endothelial cells (HUVECs) cultured on Col-ANM exhibit remarkably enhanced endothelial barrier function with high expression levels of intercellular junction proteins of ZO-1 and VE-cadherin, a high TEER, and a cellular permeability compared with the artificial porous membranes in commercial cell culture well inserts. The enhanced endothelial barrier function is conjectured to be attributed to the synergistic effects of topographical and biochemical cues provided by the aligned PCL nanofibers and collagen gel in the Col-ANM, respectively. Finally, the reactive oxygen species is applied to the HUVEC monolayer formed on the Col-ANM to destroy the tight junctions between HUVECs. The destruction of the tight junctions is demonstrated by the decreased TEER value over time. Results indicate the potential of Col-ANM in modeling endothelial barrier dysfunction-related diseases.

scholarly journals Regulation of the Actin Cytoskeleton by Thrombin in Human Endothelial Cells: Role of Rho Proteins in Endothelial Barrier Function

1998 ◽  
Vol 9 (9) ◽  
pp. 2639-2653 ◽  
Author(s):  
Valérie Vouret-Craviari ◽  
Patrice Boquet ◽  
Jacques Pouysségur ◽  
Ellen Van Obberghen-Schilling
Keyword(s):  
Endothelial Cells ◽  
Barrier Function ◽  
Umbilical Vein ◽  
Stress Fiber ◽  
Fiber Formation ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function ◽  
Cell Rounding ◽  
Stress Fiber Formation ◽  
Umbilical Vein Endothelial Cells

Endothelial barrier function is regulated at the cellular level by cytoskeletal-dependent anchoring and retracting forces. In the present study we have examined the signal transduction pathways underlying agonist-stimulated reorganization of the actin cytoskeleton in human umbilical vein endothelial cells. Receptor activation by thrombin, or the thrombin receptor (proteinase-activated receptor 1) agonist peptide, leads to an early increase in stress fiber formation followed by cortical actin accumulation and cell rounding. Selective inhibition of thrombin-stimulated signaling systems, including Gi/o (pertussis toxin sensitive), p42/p44, and p38 MAP kinase cascades, Src family kinases, PI-3 kinase, or S6 kinase pathways had no effect on the thrombin response. In contrast, staurosporine and KT5926, an inhibitor of myosin light chain kinase, effectively blocked thrombin-induced cell rounding and retraction. The contribution of Rho to these effects was analyzed by using bacterial toxins that either activate or inhibit the GTPase. Escherichia coli cytotoxic necrotizing factor 1, an activator of Rho, induced the appearance of dense actin cables across cells without perturbing monolayer integrity. Accordingly, lysophosphatidic acid, an activator of Rho-dependent stress fiber formation in fibroblasts, led to reorganization of polymerized actin into stress fibers but failed to induce cell rounding. Inhibition of Rho with Clostridium botulinum exoenzyme C3 fused to the B fragment of diphtheria toxin caused loss of stress fibers with only partial attenuation of thrombin-induced cell rounding. The implication of Rac and Cdc42 was analyzed in transient transfection experiments using either constitutively active (V12) or dominant-interfering (N17) mutants. Expression of RacV12 mimicked the effect of thrombin on cell rounding, and RacN17 blocked the response to thrombin, whereas Cdc42 mutants were without effect. These observations suggest that Rho is involved in the maintenance of endothelial barrier function and Rac participates in cytoskeletal remodeling by thrombin in human umbilical vein endothelial cells.

Effect of p18 on Endothelial Barrier Function by Mediating Vascular Endothelial Rab11a-VE-cadherin Recycling

2021 ◽  
Author(s):  
Bo-Wen Xu ◽  
Zhi-Qiang Cheng ◽  
Xu-Ting Zhi ◽  
Xiao-Mei Yang ◽  
Zhi-Bo Yan
Keyword(s):  
Barrier Function ◽  
Adherens Junctions ◽  
Cecal Ligation ◽  
Underlying Mechanism ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function ◽  
Barrier Dysfunction ◽  
Recycling Endosome

Abstract Endothelial barrier integrity requires recycling of VE-cadherin to adherens junctions. Both p18 and Rab11a play significant roles in VE-cadherin recycling. However, the underlying mechanism and the role of p18 in activating Rab11a have yet to be elucidated. Performing in vitro and in vivo experiments, we showed that p18 protein bound to VE-cadherin before Rab11a through its VE-cadherin-binding domain (aa 1–39). Transendothelial resistance showed that overexpression of p18 promoted the circulation of VE-cadherin to adherens junctions and the recovery of the endothelial barrier. Silencing of p18 caused endothelial barrier dysfunction and prevented Rab11a-positive recycling endosome accumulation in the perinuclear recycling compartments. Furthermore, p18 knockdown in pulmonary microvessels markedly increased vascular leakage in mice challenged with lipopolysaccharide and cecal ligation puncture. This study showed that p18 regulated the pulmonary endothelial barrier function in vitro and in vivo by regulating the binding of Rab11a to VE-cadherin and the activation of Rab11a.

scholarly journals FBXW7 regulates endothelial barrier function by suppression of the cholesterol synthesis pathway and prenylation of RhoB

2019 ◽  
Vol 30 (5) ◽  
pp. 607-621 ◽  
Author(s):  
Manon C. A. Pronk ◽  
Jisca Majolée ◽  
Anke Loregger ◽  
Jan S. M. van Bezu ◽  
Noam Zelcer ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Barrier Function ◽  
Rho Gtpases ◽  
Umbilical Vein ◽  
Cholesterol Biosynthesis ◽  
Endothelial Barrier ◽  
Biosynthesis Pathway ◽  
Endothelial Barrier Function ◽  
Cholesterol Biosynthesis Pathway

Rho GTPases control both the actin cytoskeleton and adherens junction stability and are recognized as essential regulators of endothelial barrier function. They act as molecular switches and are primarily regulated by the exchange of GDP and GTP. However, posttranslational modifications such as phosphorylation, prenylation, and ubiquitination can additionally alter their localization, stability, and activity. F-box proteins are involved in the recognition of substrate proteins predestined for ubiquitination and subsequent degradation. Given the importance of ubiquitination, we studied the effect of the loss of 62 members of the F-box protein family on endothelial barrier function in human umbilical vein endothelial cells. Endothelial barrier function was quantified by electrical cell impedance sensing and macromolecule passage assay. Our RNA interference–based screen identified FBXW7 as a key regulator of endothelial barrier function. Mechanistically, loss of FBXW7 induced the accumulation of the RhoB GTPase in endothelial cells, resulting in their increased contractility and permeability. FBXW7 knockdown induced activation of the cholesterol biosynthesis pathway and changed the prenylation of RhoB. This effect was reversed by farnesyl transferase inhibitors and by the addition of geranylgeranyl pyrophosphate. In summary, this study identifies FBXW7 as a novel regulator of endothelial barrier function in vitro. Loss of FBXW7 indirectly modulates RhoB activity via alteration of the cholesterol biosynthesis pathway and, consequently, of the prenylation status and activity of RhoB, resulting in increased contractility and disruption of the endothelial barrier.

scholarly journals Driving Rho GTPase activity in endothelial cells regulates barrier integrity

2010 ◽  
Vol 103 (01) ◽  
pp. 40-55 ◽  
Author(s):  
Cora Beckers ◽  
Victor van Hinsbergh ◽  
Geerten van Nieuw Amerongen
Keyword(s):  
Barrier Function ◽  
Rho Gtpases ◽  
Rho Gtpase ◽  
Three Dimensional ◽  
Regulatory Proteins ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function ◽  
Barrier Dysfunction ◽  
Gtpase Activation ◽  
The Individual

SummaryIn the past decade understanding of the role of the Rho GTPases RhoA, Rac1 and Cdc42 has been developed from regulatory proteins that regulate specific actin cytoskeletal structures – stress fibers, lamellipodia and filopodia – to complex integrators of cytoskeletal structures that can exert multiple functions depending on the cellular context. Fundamental to these functions are three-dimensional complexes between the individual Rho GTPases, their specific activators (GEFs) and inhibitors (GDIs and GAPs), which greatly outnumber the Rho GTPases themselves, and additional regulatory proteins. By this complexity of regulation different vasoactive mediators can induce various cytoskeletal structures that enable the endothelial cell (EC) to respond adequately. In this review we have focused on this complexity and the consequences of Rho GTPase regulation for endothelial barrier function. The permeability inducers thrombin and VEGF are presented as examples of G-protein coupled receptor- and tyrosine kinase receptormediated Rho GTPase activation, respectively. These mediators induce complex but markedly different networks of activators, inhibitors and effectors of Rho GTPases, which alter the endothelial barrier function. An interesting feature in this regulation is that Rho GTPases often have both barrier-protecting and barrier-disturbing functions. While Rac1 enforces the endothelial junctions, it becomes part of a barrier-disturbing mechanism as activator of reactive oxygen species generating NADPH oxidase. Similarly RhoA is protective under basal conditions, but becomes involved in barrier dysfunction after activation of ECs by thrombin. The challenge and promise lies in unfolding this complex regulation, as this will provide leads for new therapeutic opportunities.

scholarly journals Chronic ingestion of alcohol modulates expression of ubiquitin editing enzyme A20 in lung macrophages

2019 ◽  
Vol 6 ◽  
Author(s):  
Quan-Yong Huang ◽  
Yu-Chuan Chen ◽  
Shui-Ping Liu
Keyword(s):  
Evans Blue ◽  
Barrier Function ◽  
Underlying Mechanism ◽  
Endothelial Barrier ◽  
Lung Edema ◽  
Chronic Alcohol ◽  
Endothelial Barrier Function ◽  
Barrier Dysfunction ◽  
Endothelial Barrier Dysfunction ◽  
Editing Enzyme

Background: Alcohol abuse is involved in the pathogenesis of multiple organ disorders; the underlying mechanism is incompletely understood. The ubiquitin editing enzyme A20 is involved in regulating activities in the cell. Suppression of A20 is suggested as one factor in the initiation of  inflammation. This study investigates the mechanism by which chronic alcohol consumption modulates the levels of ubiquitin editing enzyme A20 in macrophages and further contributes to induce endothelial barrier dysfunction in the lung. Methods: Mice were gavage-fed with 40% alcohol daily for 0- 3 weeks. Airway macrophages were collected by lung lavage. Expression of ubiquitin editing enzyme A20 in isolated macrophages was assessed at both mRNA and protein levels. The endothelial barrier function of the lung was evaluated by the Evans blue method. Results: Mice treated with alcohol for 3 weeks showed an increase in cell infiltration in the lung in response to exposure to peptidoglycan; over 80% of the infiltrated cells were macrophages. Furthermore, we observed that A20 level was suppressed in macrophages of mice treated with alcohol; the levels of tumor necrosis factor, interleukin-6 and nuclear factor kappa B in macrophage were increased. In addition, the endothelial barrier function of the lung was compromised, showing excessive infiltration of Evans blue in the lung indicating lung edema. Pretreatment with synthesized A20 inhibited alcohol-induced lung endothelial barrier dysfunction. Conclusions: We conclude that chronic alcohol ingestion disturbs the endothelial barrier function in the lung by modulating macrophage properties. Increase in A20 in the cell may have potential for the treatment of inflammatory disorders.

Abstract 45: Endothelial Aryl Hydrocarbon Receptor Nucleatranslator (Arnt) is a Novel Regulator of Cardiac Endothelial Barrier Integrity in Diabetes

2017 ◽  
Vol 37 (suppl_1) ◽  
Author(s):  
Maura Knapp ◽  
Mei Zheng ◽  
Nikola Sladojevic ◽  
Qiong Zhao ◽  
Konstaintin G Birukov ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Barrier Function ◽  
Endothelial Permeability ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function ◽  
Barrier Dysfunction ◽  
Endothelial Barrier Dysfunction ◽  
Aryl Hydrocarbon ◽  
Underlying Mechanisms

Background: Diabetes leads to endothelial barrier dysfunction and altered endothelial permeability, which results in increased cardiovascular risk. ARNT, also known as HIF-1β, a transcription factor that functions as a master regulator of glucose homeostasis, has been implicated in diabetes. Endothelial-specific ARNT deletion (ArntΔEC) in mice is embryonically lethal, with hemorrhage occurring in the heart during the embryonic stage. However, the particular role of endothelial ARNT(ecARNT) in diabetes is largely unknown. We have found a significant decrease in ARNT expression in both diabetic rodent endothelial cells and diabetic human hearts. We hypothesize that a loss of ecARNT mediates endothelial barrier dysfunction during diabetes. Methods and Results: We generated inducible endothelial specific ARNT knockout mice (ecARNT-/-) by crossing mice with loxP sequences flanking exon 6 of ARNT with Cre ERT2 mice under the VE-cadherin promoter. A 90% deletion of ecARNT was achieved following two weeks of oral tamoxifen administration. ecARNT-/- mice exhibit severe blood vessel leakage, which is restricted to the heart, suggesting a distinct function for ecARNT in different tissues. Cardiomyopathy is evident 6 months after ARNT deletion. In vitro , trans-endothelial electrical resistance (TER) and transwell assays have confirmed endothelial barrier disruption in cardiac microvascular endothelial cells (CMEC) isolated from both ecARNT-/- hearts and diabetic (DB/DB) mouse hearts. To determine the underlying mechanisms by which ARNT may regulate endothelial barrier function, we performed DNA sequencing on CMEC isolated from control, ecARNT-/-, and DB/DB mice. Data suggest a significant increase in TNFa signaling, including ELAM-1 and ICAM-1 in CMEC isolated from ecARNT-/- CMEC and diabetic CMEC. Moreover, use of anti-TNFa antibody rescues endothelial barrier dysfunction in CMEC isolated from ecARNT-/- mice. Taken together, these results suggest that a reduction in ecARNT during diabetes may mediate endothelial barrier dysfunction through a TNFa signaling pathway. Conclusion: ecARNT is a critical mediator of endothelial barrier function and could potentially serve as a therapeutic target for diabetic cardiovascular diseases.

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