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

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.

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

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab172 ◽

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.

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

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.37.suppl_1.45 ◽

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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Intermittent hypoxia-induced endothelial barrier dysfunction requires ROS-dependent MAP kinase activation

AJP Cell Physiology ◽

10.1152/ajpcell.00313.2013 ◽

2014 ◽

Vol 306 (8) ◽

pp. C745-C752 ◽

Cited By ~ 33

Author(s):

Vladislav V. Makarenko ◽

Peter V. Usatyuk ◽

Guoxiang Yuan ◽

May M. Lee ◽

Jayasri Nanduri ◽

...

Keyword(s):

Barrier Function ◽

Intermittent Hypoxia ◽

Map Kinases ◽

Fiber Formation ◽

Endothelial Barrier ◽

Endothelial Barrier Function ◽

Barrier Dysfunction ◽

Endothelial Barrier Dysfunction ◽

The Impact ◽

Junction Proteins

The objective of the present study was to determine the impact of simulated apnea with intermittent hypoxia (IH) on endothelial barrier function and assess the underlying mechanism(s). Experiments were performed on human lung microvascular endothelial cells exposed to IH-consisting alternating cycles of 1.5% O2 for 30s followed by 20% O2 for 5 min. IH decreased transendothelial electrical resistance (TEER) suggesting attenuated endothelial barrier function. The effect of IH on TEER was stimulus dependent and reversible after reoxygenation. IH-exposed cells exhibited stress fiber formation and redistribution of cortactin, vascular endothelial-cadherins, and zona occludens-1 junction proteins along with increased intercellular gaps at cell-cell boundaries. Extracellular signal-regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK) were phosphorylated in IH-exposed cells. Inhibiting either ERK or JNK prevented the IH-induced decrease in TEER and the reorganization of the cytoskeleton and junction proteins. IH increased reactive oxygen species (ROS) levels, and manganese (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachloride, a membrane-permeable antioxidant, prevented ERK and JNK phosphorylation as well as IH-induced changes in endothelial barrier function. These results demonstrate that IH via ROS-dependent activation of MAP kinases leads to reorganization of cytoskeleton and junction proteins resulting in endothelial barrier dysfunction.

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Chlorogenic acid alleviates sepsis-induced endothelial barrier dysfunction and the underlying mechanism

10.21203/rs.3.rs-23474/v1 ◽

2020 ◽

Author(s):

Zisen Zhang ◽

Yue Wu ◽

Yu Zhu ◽

Xiao-yong Peng ◽

Ming-ying Xue ◽

...

Keyword(s):

Septic Shock ◽

Underlying Mechanism ◽

Shock Group ◽

Endothelial Barrier ◽

Vascular Endothelial ◽

Endothelial Barrier Function ◽

Barrier Dysfunction ◽

Endothelial Barrier Dysfunction ◽

Liver And Kidney ◽

Kidney Blood Flow

Abstract Background Chlorogenic acid (CGA) has been shown to improve inflammatory cytokines in patients with ulcerative colitis. Vascular endothelial barrier dysfunction is closely related to sepsis. We hypothesize that CGA plays an important role in treating sepsis by protecting the mesenteric endothelial barrier. Methods Severe sepsis was established by cecal ligation and puncture (CLP) in SD rats. Immediately after the induction of sepsis, CGA (20 mg/kg) was administered intravenously, and the effects of CGA on mesenteric vascular leakage, hemodynamics, liver and kidney blood flow, and the ultrastructure of the mesenteric endothelial cells were determined. The transendothelial electrical resistance and the Transwell permeability assays were used for observing endothelial barrier function in vascular endothelial cells (VECs). ZO-1 and VE-cadherin expression were observed by immunohistochemistry and western blotting. Results FITC-BSA leakage from mesenteric micro-vessels was significantly increased after septic shock, which was significantly improved by CGA. Liver and kidney blood flow were increased by 41.2% and 38.4%, respectively, after CGA administration compared with the septic shock group. Hemodynamic status was significantly decreased after septic shock, and significantly improved by CGA. The 72-h survival rate of septic shock rats in the CGA group (50%) was significantly higher than the septic shock group (6.25%). CGA improved the tight junction opening after septic shock and also significantly up-regulated the expression of ZO-1 and VE-cadherin. CGA also protected endothelial hyperpermeability against lipopolysaccharide-stimulated VEC injury by increasing the expression of ZO-1 and VE-cadherin in vitro. Conclusion CGA was beneficial for endothelial barrier function in rats with septic shock, which is the major contribution to CGA with respecting to improving hemodynamic status and organ perfusion. The underlying mechanism involved CGA up-regulation of ZO-1 and VE-cadherin.

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Driving Rho GTPase activity in endothelial cells regulates barrier integrity

Thrombosis and Haemostasis ◽

10.1160/th09-06-0403 ◽

2010 ◽

Vol 103 (01) ◽

pp. 40-55 ◽

Cited By ~ 130

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.

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

Scientific Reports ◽

10.1038/s41598-019-51560-8 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 7

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.

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

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00185.2006 ◽

2006 ◽

Vol 291 (5) ◽

pp. H2126-H2135 ◽

Cited By ~ 3

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.

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Using cultured endothelial cells to study endothelial barrier dysfunction: Challenges and opportunities

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00393.2015 ◽

2016 ◽

Vol 311 (2) ◽

pp. L453-L466 ◽

Cited By ~ 30

Author(s):

Jurjan Aman ◽

Ester M. Weijers ◽

Geerten P. van Nieuw Amerongen ◽

Asrar B. Malik ◽

Victor W. M. van Hinsbergh

Keyword(s):

Endothelial Cells ◽

Barrier Function ◽

Fluid Shear Stress ◽

Alternative Methods ◽

Endothelial Barrier ◽

Endothelial Barrier Function ◽

Barrier Dysfunction ◽

Vascular Leak ◽

Cell Based Therapy ◽

Depth Analysis

Despite considerable progress in the understanding of endothelial barrier regulation and the identification of approaches that have the potential to improve endothelial barrier function, no drug- or stem cell-based therapy is presently available to reverse the widespread vascular leak that is observed in acute respiratory distress syndrome (ARDS) and sepsis. The translational gap suggests a need to develop experimental approaches and tools that better mimic the complex environment of the microcirculation in which the vascular leak develops. Recent studies have identified several elements of this microenvironment. Among these are composition and stiffness of the extracellular matrix, fluid shear stress, interaction of endothelial cells (ECs) with pericytes, oxygen tension, and the combination of toxic and mechanic injurious stimuli. Development of novel cell culture techniques that integrate these elements would allow in-depth analysis of EC biology that closely approaches the (patho)physiological conditions in situ. In parallel, techniques to isolate organ-specific ECs, to define EC heterogeneity in its full complexity, and to culture patient-derived ECs from inducible pluripotent stem cells or endothelial progenitor cells are likely to advance the understanding of ARDS and lead to development of therapeutics. This review 1) summarizes the advantages and pitfalls of EC cultures to study vascular leak in ARDS, 2) provides an overview of elements of the microvascular environment that can directly affect endothelial barrier function, and 3) discusses alternative methods to bridge the gap between basic research and clinical application with the intent of improving the translational value of present EC culture approaches.

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S-1-Propenylcysteine Improves TNF-α-Induced Vascular Endothelial Barrier Dysfunction by Suppressing the GEF-H1/RhoA/Rac Pathway

10.21203/rs.3.rs-62924/v1 ◽

2020 ◽

Author(s):

Kayo Kunimura ◽

Satomi Miki ◽

Miyuki Takashima ◽

Jun-ichiro Suzuki

Keyword(s):

Barrier Function ◽

Vascular Diseases ◽

Endothelial Barrier ◽

Vascular Endothelial ◽

Barrier Dysfunction ◽

Endothelial Barrier Dysfunction ◽

Junctional Proteins ◽

Tnf Α ◽

Gene And Protein Expression ◽

Sulfur Containing

Abstract Background: Vascular endothelial barrier function is maintained by cell-to-cell junctional proteins and contributes to vascular homeostasis. Various risk factors such as inflammation disrupt barrier function through down-regulation of these proteins and promote vascular diseases such as atherosclerosis. Previous studies have demonstrated that aged garlic extract (AGE) and its sulfur-containing constituents exert the protective effects against several vascular diseases such as atherosclerosis. In this study, we examined whether AGE and its sulfur-containing constituents improve the endothelial barrier dysfunction elicited by a pro-inflammatory cytokine, Tumor-necrosis factor-α (TNF-α), and explored their mode of action on TNF-α signaling pathway.Methods: Human umbilical vein endothelial cells (HUVECs) were treated with test substances in the presence of TNF-α for various time periods. The endothelial permeability was measured by using a transwell permeability assay. The localization of cell-to-cell junctional proteins and actin cytoskeletons were visualized by immunostaining. RhoA and Rac activities were assessed by using GTP-binding protein pulldown assay. Gene and protein expression levels of signaling molecules were analyzed by real-time PCR and western blotting, respectively. Results: We found that AGE and its major sulfur-containing constituent, S-1-propenylcysteine (S1PC), reduced hyperpermeability elicited by TNF-α in HUVECs. In addition, S1PC inhibited TNF-α-induced production of myosin light chain (MLC) kinase and inactivation of MLC phosphatase through the suppression of the Rac and RhoA signaling pathways, respectively, which resulted in the dephosphorylation of MLC2, a key factor of actin remodeling. Moreover, S1PC inhibited the phosphorylation and activation of guanine nucleotide exchange factor-H1 (GEF-H1), a common upstream key molecule and activator of Rac and RhoA. These effects of S1PC were accompanied by its ability to protect the disruption of junctional proteins on the cell-cell contact regions and the increase of actin stress fibers induced by TNF-α. Conclusions: The present study suggested that AGE and S1PC improve endothelial barrier disruption through the protection of junctional proteins on plasma membrane.

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S-1-propenylcysteine improves TNF-α-induced vascular endothelial barrier dysfunction by suppressing the GEF-H1/RhoA/Rac pathway

Cell Communication and Signaling ◽

10.1186/s12964-020-00692-w ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Kayo Kunimura ◽

Satomi Miki ◽

Miyuki Takashima ◽

Jun-ichiro Suzuki

Keyword(s):

Barrier Function ◽

Vascular Diseases ◽

Endothelial Barrier ◽

Major Constituent ◽

Vascular Endothelial ◽

Barrier Dysfunction ◽

Endothelial Barrier Dysfunction ◽

Junctional Proteins ◽

Tnf Α ◽

Sulfur Containing

Abstract Background Vascular endothelial barrier function is maintained by cell-to-cell junctional proteins and contributes to vascular homeostasis. Various risk factors such as inflammation disrupt barrier function through down-regulation of these proteins and promote vascular diseases such as atherosclerosis. Previous studies have demonstrated that aged garlic extract (AGE) and its sulfur-containing constituents exert the protective effects against several vascular diseases such as atherosclerosis. In this study, we examined whether AGE and its sulfur-containing constituents improve the endothelial barrier dysfunction elicited by a pro-inflammatory cytokine, Tumor-necrosis factor-α (TNF-α), and explored their mode of action on TNF-α signaling pathway. Methods Human umbilical vein endothelial cells (HUVECs) were treated with test substances in the presence of TNF-α for various time periods. The endothelial permeability was measured by using a transwell permeability assay. The localization of cell-to-cell junctional proteins and actin cytoskeletons were visualized by immunostaining. RhoA and Rac activities were assessed by using GTP-binding protein pulldown assay. Gene and protein expression levels of signaling molecules were analyzed by real-time PCR and western blotting, respectively. Results We found that AGE and its major sulfur-containing constituent, S-1-propenylcysteine (S1PC), reduced hyperpermeability elicited by TNF-α in HUVECs. In addition, S1PC inhibited TNF-α-induced production of myosin light chain (MLC) kinase and inactivation of MLC phosphatase through the suppression of the Rac and RhoA signaling pathways, respectively, which resulted in the dephosphorylation of MLC2, a key factor of actin remodeling. Moreover, S1PC inhibited the phosphorylation and activation of guanine nucleotide exchange factor-H1 (GEF-H1), a common upstream key molecule and activator of Rac and RhoA. These effects of S1PC were accompanied by its ability to prevent the disruption of junctional proteins on the cell–cell contact regions and the increase of actin stress fibers induced by TNF-α. Conclusions The present study suggested that AGE and its major constituent, S1PC, improve endothelial barrier disruption through the protection of junctional proteins on plasma membrane.

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