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

2016 ◽  
Vol 311 (2) ◽  
pp. L453-L466 ◽  
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.

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.

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 Effect of inflammation-mediated endothelial metabolic shift on endothelial barrier function

2021 ◽  
Vol 42 (Supplement_1) ◽  
Author(s):  
M Aslam ◽  
H Idrees ◽  
C W Hamm ◽  
Y Ladilov
Keyword(s):  
Endothelial Cells ◽  
Glucose Uptake ◽  
Barrier Function ◽  
Atp Synthesis ◽  
Fold Increase ◽  
Endothelial Barrier ◽  
Synthesis Rate ◽  
Endothelial Barrier Function ◽  
Metabolic Switch ◽  
Barrier Integrity

Abstract Background The integrity of the endothelial cell barrier of the microvasculature is compromised by inflammation. The increased vascular permeability leads to tissue injury and organ dysfunction. In recent years, considerable advances have been made in the understanding of signalling mechanisms regulating the endothelial barrier integrity. The role of endothelial metabolism as a modulator of endothelial barrier integrity is not yet well-studied. The aim of the present study was to investigate the effect of inflammation on endothelial metabolism and its role in the maintenance of endothelial barrier integrity. Methods The study was carried out on cultured human umbilical vein endothelial cells and rat coronary microvascular endothelial cells. Inflammatory condition was simulated by treating cells with low concentrations (1 ng/mL) of TNFα for 24h. Endothelial barrier function was analysed by measuring the flux of albumen through endothelial monolayers cultured on filter membranes. Gene expression was analysed by qPCR-based assays. The capacity of endothelial cells for maximal ATP synthesis rate was investigated by the real-time live-cell imaging using FRET-based ATP-biosensor (live cell FRET). Total cellular ATP concentration was measured using luminescence-based commercial kit (ATPLite, PerkinElmer). Mitochondrial mass was analysed by the ratio of mitochondrial DNA (mtDNA) to nuclear DNA (nDNA). The cellular glucose uptake was measured by fluorescent microscopy using a fluorescent analogue of glucose (2-NBDG). Results Treatment of human endothelial cells with TNFα resulted in significant suppression of mitochondrial and upregulation of glycolytic ATP synthesis rate, suggesting a metabolic switch. This was accompanied by a reduction in mitochondrial content (mtDNA/nDNA), reduction in total cellular ATP levels, an enhanced expression of glycolytic enzymes 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3) and phosphofructokinase 1 (PFK1), and enhanced glucose uptake by endothelial cells (n=5; p<0.05 for all parameters tested). Moreover, TNFα caused a 3-fold increase in endothelial permeability. Pharmacological inhibition of glycolysis either by partial replacement of glucose with 2-deoxy glucose (2DG) or an inhibition of PFKFB3 resulted in further worsening (a 5-fold increase in permeability) of TNFα-induced endothelial barrier failure. On the other hand pharmacological activation of AMPK, a potent inducer of mitochondrial biogenesis, could attenuate TNFα-induced but not 2DG-induced endothelial hyperpermeability. Conclusion The study demonstrates that TNFα induces metabolic switch towards glycolysis in endothelial cells. Moreover, the data suggest that upregulation of glycolysis may serve as an endogenous metabolic adaptation to the TNFα-induced suppression of mitochondrial ATP synthesis, which protects endothelial barrier integrity. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Justus-Liebig University GiessenDZHK (German Centre for Cardiovascular Research), partner site Rhein-Main, Bad Nauheim, Germany

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 Effects of Ebola Virus Glycoproteins on Endothelial Cell Activation and Barrier Function

2005 ◽  
Vol 79 (16) ◽  
pp. 10442-10450 ◽  
Author(s):  
Victoria M. Wahl-Jensen ◽  
Tatiana A. Afanasieva ◽  
Jochen Seebach ◽  
Ute Ströher ◽  
Heinz Feldmann ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Barrier Function ◽  
Cell Activation ◽  
Matrix Protein ◽  
Ebola Virus ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function ◽  
Endothelial Cell Activation ◽  
Tnf Α

ABSTRACT Ebola virus causes severe hemorrhagic fever with high mortality rates in humans and nonhuman primates. Vascular instability and dysregulation are disease-decisive symptoms during severe infection. While the transmembrane glycoprotein GP1,2 has been shown to cause endothelial cell destruction, the role of the soluble glycoproteins in pathogenesis is largely unknown; however, they are hypothesized to be of biological relevance in terms of target cell activation and/or increase of endothelial permeability. Here we show that virus-like particles (VLPs) consisting of the Ebola virus matrix protein VP40 and GP1,2 were able to activate endothelial cells and induce a decrease in barrier function as determined by impedance spectroscopy and hydraulic conductivity measurements. In contrast, the soluble glycoproteins sGP and Δ-peptide did not activate endothelial cells or change the endothelial barrier function. The VLP-induced decrease in barrier function was further enhanced by the cytokine tumor necrosis factor alpha (TNF-α), which is known to induce a long-lasting decrease in endothelial cell barrier function and is hypothesized to play a key role in Ebola virus pathogenesis. Surprisingly, sGP, but not Δ-peptide, induced a recovery of endothelial barrier function following treatment with TNF-α. Our results demonstrate that Ebola virus GP1,2 in its particle-associated form mediates endothelial cell activation and a decrease in endothelial cell barrier function. Furthermore, sGP, the major soluble glycoprotein of Ebola virus, seems to possess an anti-inflammatory role by protecting the endothelial cell barrier function.

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.

scholarly journals SARS-CoV-2 causes severe epithelial inflammation and barrier dysfunction

2021 ◽  
Author(s):  
Stefanie Deinhardt-Emmer ◽  
Sarah Böttcher ◽  
Clio Häring ◽  
Liane Giebeler ◽  
Andreas Henke ◽  
...  
Keyword(s):  
Cell Culture ◽  
Endothelial Cells ◽  
Epithelial Cells ◽  
Barrier Function ◽  
Endothelial Barrier ◽  
Endothelial Barrier Function ◽  
Endothelial Layer ◽  
Culture Systems ◽  
Viral Spread ◽  
Cell Culture Systems

Infections with SARS-CoV-2 can be asymptomatic, but they can also be accompanied by a variety of symptoms that result in mild to severe coronavirus disease-19 (COVID-19) and are sometimes associated with systemic symptoms. Although the viral infection originates in the respiratory system, it is unclear how the virus can overcome the alveolar barrier, which is observed in severe COVID-19 disease courses. To elucidate the viral effects on the barrier integrity and immune reactions, we used mono-cell culture systems and a complex human chip model composed of epithelial, endothelial, and mononuclear cells. Our data show that SARS-CoV-2 efficiently infected epithelial cells with high viral loads and inflammatory response, including interferon expression. By contrast, the adjacent endothelial layer was neither infected nor did it show productive virus replication or interferon release. With prolonged infection, both cell types were damaged, and the barrier function was deteriorated, allowing the viral particles to overbear. In our study, we demonstrate that although SARS-CoV-2 is dependent on the epithelium for efficient replication, the neighboring endothelial cells are affected, e.g., by the epithelial cytokines or components induced during infection, which further results in the damage of the epithelial/endothelial barrier function and viral dissemination. IMPORTANCE SARS-CoV-2 challenges healthcare systems and societies worldwide in unprecedented ways. Although numerous new studies have been conducted, research to better understand the molecular pathogen-host interactions are urgently needed. For this, experimental models have to be developed and adapted. In the present study we used mono cell-culture systems and we established a complex chip model, where epithelial and endothelial cells are cultured in close proximity. We demonstrate that epithelial cells can be infected with SARS-CoV-2, while the endothelium did not show any infection signs. Since SARS-CoV-2 is able to establish viremia, the link to thromboembolic events in severe COVID-19 courses is evident. However, whether the endothelial layer is damaged by the viral pathogens or whether other endothelial-independent homeostatic factors are induced by the virus is essential for understanding the disease development. Therefore, our study is important as it demonstrates that the endothelial layer could not be infected by SARS-CoV-2 in our in vitro experiments, but we were able to show the destruction of the epithelial-endothelial barrier in our chip model. From our experiments we can assume that virus-induced host factors disturbed the epithelial-endothelial barrier function and thereby promote viral spread.

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.

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