VE-cadherin In Endothelial Cells Research Articles

Proximity interactome of lymphatic VE-cadherin reveals mechanisms of junctional remodeling and reelin secretion

The adhesion receptor vascular endothelial (VE)-cadherin transduces an array of signals that modulate crucial lymphatic cell behaviors including permeability and cytoskeletal remodeling. Consequently, VE-cadherin must interact with a multitude of intracellular proteins to exert these functions. Yet, the full protein interactome of VE-cadherin in endothelial cells remains a mystery. Here, we use proximity proteomics to illuminate how the VE-cadherin interactome changes during junctional reorganization from dis-continuous to continuous junctions, triggered by the lymphangiogenic factor adrenomedullin. These analyses identified interactors that reveal roles for ADP ribosylation factor 6 (ARF6) and the exocyst complex in VE-cadherin trafficking and recycling. We also identify a requisite role for VE-cadherin in the in vitro and in vivo control of secretion of reelin—a lymphangiocrine glycoprotein with recently appreciated roles in governing heart development and injury repair. This VE-cadherin protein interactome shines light on mechanisms that control adherens junction remodeling and secretion from lymphatic endothelial cells.

Netrin‑4 promotes VE‑cadherin expression in endothelial cells through the NF‑κB signaling pathway.

Netrin-4 (NTN4), a secreted protein from the Netrin family, has been recognized for its role in vascular development, endothelial homeostasis and angiogenesis. Vascular endothelial (VE)-cadherin is a specialized adhesion protein located at the intercellular junctions of endothelial cells (ECs), and regulates migration, proliferation and permeability. To date, the relationship between NTN4 and VE-cadherin in ECs remains unclear. In the present study, human umbilical vein ECs (HUVECs) were transfected with NTN4 overexpression plasmid, resulting in NTN4 overexpression. Reverse transcription-quantitative PCR and western blotting were used to determine gene and protein expression. CCK8, wound healing, and Transwell assays were performed to evaluate cell proliferation, migration and permeability. NTN4 overexpression decreased HUVEC viability and migration. In addition, NTN4 overexpression increased the expression of VE-cadherin and decreased the permeability of HUVECs. Subsequent studies showed that NTN4 overexpression increased the NF-κB protein level and decreased IκB-α protein expression in HUVECs. In HUVECs treated with NF-κB inhibitor pyrrolidine dithiocarbamate, the expression of VE-cadherin failed to increase with NTN4 overexpression. Taken together, the results indicated that NTN4 overexpression increased VE-cadherin expression through the activation of the NF-κB signaling pathway in HUVECs. The present findings revealed a novel regulatory mechanism for VE-cadherin expression and suggested a novel avenue for future research on the role of NTN4 in endothelial barrier-related diseases.

Placental IL-1β inhibits PKM2 and leads to endothelial barrier injury in preeclampsia

Preeclampsia, a life-threatening hypertensive disorder of pregnancy, continues to be a major global health concern. Maternal endothelial dysfunction induced by components from the impaired placenta is a key hallmark of preeclampsia (PE). Increased levels of IL-1β have been observed in preeclamptic placentas, but its association with endothelial dysfunction has remained uncertain. Our hypothesis was that IL-1β released from the placenta links the placental abnormalities with maternal endothelial dysfunction in PE. Our investigation revealed elevated levels of IL-1β in preeclampsia placentas, which were negatively correlated with reduced VE-Cadherin expression in endothelial cells, indicating consistent endothelial dysfunction. Moreover, we identified the glycolytic enzyme pyruvate kinase 2 (PKM2) as a key regulator of glycolysis involved in the regulation of junctional VE-Cadherin in endothelial cells. Rescue assays further demonstrated that the overexpression of PKM2 was necessary to ameliorate endothelial barrier injury induced by IL-1β. Our study provides novel insights into the molecular mechanisms driving preeclampsia, highlighting the regulatory roles of IL-1β, PKM2, and VE-Cadherin in endothelial cells. These identified interactions may play a crucial role in the pathogenesis of preeclampsia, offering potential targets for therapeutic interventions to preserve endothelial integrity and alleviate the condition. This project was funded by Natural Science Foundation of Shandong (ZR2022MH195 and ZR2023MH211), and the Graduate Student Research Grant from Weifang Medical University. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Pseudomonas Aeruginosa Affects the Function of Pulmonary Vascular Endothelial Cells

Objective To investigate the impact of Pseudomonas aeruginosa(PA) infection on the function of pulmonary vascular endothelial cells,and explore the mechanism of this bacterium in exacerbating lung inflammation in mice. Methods Two hours after human lung microvascular endothelial cell(HULEC-5a) were infected with the PA strain PAO1,the mRNA levels of autophagy-related gene 5(ATG5),6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3(PFKFB3),and calcium adhesion protein 5(CDH5) were determined by reverse transcription real-time fluorescent quantitative PCR(RT-qPCR).The protein levels of ATG5,PFKFB3,and vascular endothelial calcium adhesion protein(VE-cadherin) were detected by immunofluorescence.After the expression of ATG5 and PFKFB3 was respectively knocked down by small interfering RNA(siRNA),RT-qPCR was employed to measure the mRNA levels of ATG5,PFKFB3,and CDH5,and immunofluorescence to detect the protein levels of PFKFB3 and VE-cadherin.In addition,the lactate assay kit was used to determine the level of lactate in the cells.After mice were infected with PAO1,lung inflammation was assessed through histopathological section staining.Confocal microscopy was employed to capture and analyze fluorescence-labeled PFKFB3 and VE-cadherin in endothelial cells. Results Compared with the control group,the HULEC-5a cells infected with PAO1 showed up-regulated mRNA and protein levels of PFKFB3(all P<0.05),down-regulated mRNA level of CDH5(P=0.023),disrupted continuity and down-regulated protein level of VE-cadherin(P<0.001),and elevated lactate level(P=0.017).Compared with PAO1-infected HULEC-5a cells,knocking down PFKFB3 led to the up-regulated mRNA level of CDH5(P=0.043),lowered lactate level(P=0.047),and restored continuity of VE-cadherin;knocking down ATG5 led to up-regulated mRNA and protein levels of PFKFB3(P=0.013 and P=0.003),elevated lactate level(P=0.015),and down-regulated mRNA level of CDH5(P=0.020) and protein level of VE-cadherin(P=0.001).The HE staining results showed obvious red blood cell leakage,inflammatory cell infiltration,alveolar septal widening,and partial detachment of vascular endothelial cells in the alveoli of PA-infected mice.Immunofluorescence staining showed up-regulated expression of PFKFB3 and decreased fluorescence signal of VE-cadherin in endothelial cells of infected mice compared with normal mice. Conclusion PA may regulate the PFKFB3 pathway via AGT5 to disrupt the function of pulmonary vascular endothelial cells,thereby exacerbating the inflammation in the lungs of mice.

Placental exosomal miR-125b triggered endothelial barrier injury in preeclampsia

IntroductionPreeclampsia (PE) is an elusive life-threatening complication of pregnancy, and maternal endothelial dysfunction induced by components from the impaired placenta is a key hallmark of PE. Placenta-derived exosomes in maternal circulation have been correlated with risk of PE, however, the role of exosomes in PE remains to be determined. We hypothesized that placenta-released exosomes link the placental abnormalities with maternal endothelial dysfunction in PE. MethodsCirculating exosomes were collected from plasma samples of preeclamptic patients and normal pregnancies. Endothelial barrier function was examined by transendothelial electrical resistance (TEER) and cell permeability to FITC-dextran assays in human umbilical vein endothelial cells (HUVECs). miR-125b and VE-cadherin gene expression in exosomes and endothelial cells were assessed by qPCR and Western, and the possible post-transcriptional regulation of miR-125b on VE-cadherin was detected by luciferase assay. ResultsWe isolated placenta-derived exosomes in the maternal circulation and found that placenta-derived exosomes from preeclamptic patients (PE-exo) leads to endothelial barrier dysfunction. We then identified decreased expression of VE-cadherin in endothelial cells contribute to the breakdown of the endothelial barrier. Further investigations revealed increased exosomal miR-125b in PE-exo directly inhibited VE-cadherin in HUVECs, thereby mediating the adverse effect of PE-exo on endothelial barrier function. DiscussionPlacental exosomes link impaired placentation and endothelial dysfunction, thus providing new insight into the pathophysiology of preeclampsia. Exosomal miRNAs derived from placenta contribute to the endothelial dysfunction in PE and could be a promising therapeutic target for PE.

Flavivirus NS1 Triggers Tissue-Specific Disassembly of Intercellular Junctions Leading to Barrier Dysfunction and Vascular Leak in a GSK-3β-Dependent Manner.

The flavivirus nonstructural protein 1 (NS1) is secreted from infected cells and contributes to endothelial barrier dysfunction and vascular leak in a tissue-dependent manner. This phenomenon occurs in part via disruption of the endothelial glycocalyx layer (EGL) lining the endothelium. Additionally, we and others have shown that soluble DENV NS1 induces disassembly of intercellular junctions (IJCs), a group of cellular proteins critical for maintaining endothelial homeostasis and regulating vascular permeability; however, the specific mechanisms by which NS1 mediates IJC disruption remain unclear. Here, we investigated the relative contribution of five flavivirus NS1 proteins, from dengue (DENV), Zika (ZIKV), West Nile (WNV), Japanese encephalitis (JEV), and yellow fever (YFV) viruses, to the expression and localization of the intercellular junction proteins β-catenin and VE-cadherin in endothelial cells from human umbilical vein and brain tissues. We found that flavivirus NS1 induced the mislocalization of β-catenin and VE-cadherin in a tissue-dependent manner, reflecting flavivirus disease tropism. Mechanistically, we observed that NS1 treatment of cells triggered internalization of VE-cadherin, likely via clathrin-mediated endocytosis, and phosphorylation of β-catenin, part of a canonical IJC remodeling pathway during breakdown of endothelial barriers that activates glycogen synthase kinase-3β (GSK-3β). Supporting this model, we found that a chemical inhibitor of GSK-3β reduced both NS1-induced permeability of human umbilical vein and brain microvascular endothelial cell monolayers in vitro and vascular leakage in a mouse dorsal intradermal model. These findings provide insight into the molecular mechanisms regulating NS1-mediated endothelial dysfunction and identify GSK-3β as a potential therapeutic target for treatment of vascular leakage during severe dengue disease.

Placental IL‐1β impairs endothelial functions by reducing VE‐cadherin expression in PE

Preeclampsia (PE) is a pregnancy‐specific syndrome that affects 5~8% pregnancies and is a leading cause of maternal and neonatal morbidity and mortality, especially in low‐income and middle‐income countries. Widespread systemic inflammation and endothelial dysfunction are hallmarks of injury in PE, however, the molecular mechanisms linking inflammation and endothelial dysfunction remain largely unknown. We tested the hypothesis that placental IL‐1β impairs endothelial functions by inhibiting VE‐cadherin expression in PE. We found that NLRP3 inflammasome was overactivated in preeclamptic placenta, which induced more IL‐1β release. We also observed that IL‐1β significantly increased endothelial permeability of Human Umbilical Vein Endothelial Cells (HUVECs) in a dose‐response manner, and inhibition of IL‐1β receptor significantly attenuated the increase of endothelial permeability induced by IL‐1β. We further revealed that both mRNA and protein level of VE‐cadherin was reduced by IL‐1β in HUVECs. and overexpression of VE‐cadherin in endothelial cells reversed IL‐1β‐induced increase in endothelial permeability. Taken together, our preliminary data suggested that excessive placental IL‐1β significantly impaired endothelial functions by inhibiting VE‐cadherin expression.

Lactate induces vascular permeability via disruption of VE-cadherin in endothelial cells during sepsis.

Circulating lactate levels are a critical biomarker for sepsis and are positively correlated with sepsis-associated mortality. We investigated whether lactate plays a biological role in causing endothelial barrier dysfunction in sepsis. We showed that lactate causes vascular permeability and worsens organ dysfunction in CLP sepsis. Mechanistically, lactate induces ERK-dependent activation of calpain1/2 for VE-cadherin proteolytic cleavage, leading to the enhanced endocytosis of VE-cadherin in endothelial cells. In addition, we found that ERK2 interacts with VE-cadherin and stabilizes VE-cadherin complex in resting endothelial cells. Lactate-induced ERK2 phosphorylation promotes ERK2 disassociation from VE-cadherin. In vivo suppression of lactate production or genetic depletion of lactate receptor GPR81 mitigates vascular permeability and multiple organ injury and improves survival outcome in polymicrobial sepsis. Our study reveals that metabolic cross-talk between glycolysis-derived lactate and the endothelium plays a critical role in the pathophysiology of sepsis.

Rivaroxaban protects from the oxysterol-induced damage and inflammatory activation of the vascular endothelium

ABSTRACT Background Rivaroxaban is one of the direct factor Xa inhibitors. Its function in the inactivated coagulation cascade is unclear. The aim of the study was to assess the effect of rivaroxaban on the endothelial integrity and inflammatory properties of endothelial cells stimulated by 25-hydroxycholesterol (25-OHC). Methods HUVECs were stimulated with 25-OHC, rivaroxaban and 25-OHC+ rivaroxaban. HUVEC integrity and permeability were measured using the xCELLigence system and paracellular flux assay. The mRNA expression of tissue factor, ICAM-1, VEGF, IL-33, MCP-1, TNF-α was analyzed in the real-time PCR. Apoptosis and viability were measured by flow cytometry. The VEGF protein concentration was assessed by ELISA. The confocal microscope was used to evaluate the expression of VE-cadherin in endothelial cells. Results 25-OHC decreased endothelial cell integrity and increased the mRNA expression of IL-33, tissue factor, ICAM-1, MCP-1, VEGF, TNF-α as compared to unstimulated controls. Following the stimulation with rivaroxaban, HUVEC restored integrity disrupted by 25-OHC (p < .01). In HUVECs pre-stimulated with oxysterol, rivaroxaban decreased mRNA expression of IL-33, TNF-α, chemokines MCP-1, ICAM-1, VEGF and tissue factor (p < .01). Rivaroxaban 100 mg/ml+25-OHC increased the VE-cadherin expression in endothelium as compared to 25-OHC (p < .05). Conclusion Our finding suggests that rivaroxaban may restore the endothelial barrier and inhibit the inflammatory activation caused by oxysterol in vitro.

The Role of MRPL23 Antisense RNA 1 (MRPL23-AS1) in the Pre-Metastatic Microenvironment of Malignancy During the Process of Epithelial-Mesenchymal Transition

We aimed to explore MRPL23-AS1’s role in the pre-metastatic microenvironment of malignancy during epithelial-mesenchymal transition (EMT). Identification and verification of lncRNA-interacting proteins in salivary adenoid cystic carcinoma (SACC) cells were conducted via RNA-pulldown, silver staining, and Western blotting. RIP and RIP-seq were sequentially administered to verify the binding partners of lncRNA. CHIRP was performed to detect the promoter DNA in the downstream of lncRNA-protein complex. Ultimately CHIP-qPCR detected the effects of lncRNA on the binding degree of its interacting protein to the promoter DNA in the downstream genes and the methyla-tion level of histones in the promoter region. The exosomes secreted by different SACC cells were extracted from culture supernatant to measure lncRNA expression via qPCR. MRPL23-AS1 interacted with EZH2 protein and promoted EZH2 binding to E-cadherin gene promoter region along with the H3K27 methylation. MRPL23-AS1 could promote EMT of SACC cells and increase pulmonary vascular endothelial cells permeability via exosomes secretion. MRPL23-AS1 up-regulated VEGFA, while down-regulated E-cadherin and VE-cadherin in endothelial cells. Exosomes rich in MRPL23-AS1 could boost lung metastasis in vivo. MRPL23-AS1 inhibits E-cadherin level and promotes EMT of SACC cells, suggesting that it might be a biomarker and therapeutic target for lung cancer.

Aromatase inhibitor anastrozole modifies cellular functions in gingival fibroblasts and endothelial cells: possible periodontal complications of aromatase inhibitor treatment.

Recent studies have shown that treatment with aromatase inhibitors contributes to an increased prevalence of periodontitis. In this study, we assessed effects of the aromatase inhibitor anastrozole on cellular function of human gingival fibroblasts (HGFs) and endothelial cells. Expression levels of collagen, extracellular matrix (ECM) proteins, matrix metalloproteinases (MMPs), and tissue inhibitors of metalloproteinases (TIMPs) were examined in HGFs exposed to anastrozole. Furthermore, inflammatory responses in HGFs cultured with anastrozole were evaluated in the presence of Porphyromonas gingivalis lipopolysaccharide. We also evaluated the vascular permeability and vascular endothelial (VE)-cadherin expression of endothelial cells exposed to anastrozole. Anastrozole enhanced expression levels of collagen, ECM proteins, TIMPs, and inflammatory cytokines in HGFs, as well as vascular permeability of endothelial cells. In addition, anastrozole reduced expression levels of MMPs in HGFs and VE-cadherin in endothelial cells. These results suggest that anastrozole modulates various cellular functions in HGFs and endothelial cells.

566-P: Sulodexide Suppresses NLRP3 Inflammasome Activation via NOX4/ROS Signaling in the Retinal Microvasculature

Aims: Sulodexide (SDX) protects endothelial cells via restoring endothelial glycocalyx, but the effect of SDX on endothelial inflammation remains unknown. We showed that SDX potently inhibited the activation of NLRP3 inflammasome in diabetic retinas, but the underlying mechanism was not clear. As reactive oxygen species (ROS) activates NLRP3 inflammasome, and NADPH oxidase 4 (NOX4) is a key generator of ROS in endothelial cells, this study thus investigated if NOX4/ROS signaling mediates the effect of SDX on NLRP3 inflammasome, hoping to provide novel insights into SDX in treating diabetic retinopathy (DR). Methods: Diabetes was induced in mice by high fat diet and streptozotocin, followed by i.p. injection of SDX (10mg/kg) every other day for 3 months. Human retinal microvascular endothelial cells were exposed to high glucose, accompanied with overexpression or knocking down of NOX4 and a serial concentration of SDX. The retinal levels of NOX4, NLRP3 inflammasome, inflammatory factors, junction components, vascular leakage and ROS levels were determined. Results: SDX treatment dramatically suppressed the hyperglycemia-induced overexpression of NOX4 and activation of NLRP3 inflammasome in retinas compared to controls. Moreover, SDX preserved the levels of ZO-1, VE-cadherin and ameliorated the retinal vascular permeability. In addition, SDX significantly ameliorated high glucose-induced NOX4 up-regulation, ROS production and NLRP3 inflammasome activation, and restored the reduction of ZO-1 and VE-cadherin in endothelial cells. Conversely, overexpression of NOX4 exacerbated endothelial inflammation through over production of ROS and subsequent activation of NLRP3 inflammasome, which was rescued by SDX treatment. Conclusion: Our study suggests that SDX protects the retinal microvasculature by inhibiting NOX4/ROS-induced NLRP3 inflammasome activation, thus provides novel evidence of utilizing SDX for the treatment of DR. Disclosure T. Li: None. X. He: None. R. Peng: None. X. Fu: None. G. Shi: None. Y. Chen: None. Funding Research and Development Plans in Key Areas of Guangdong Province (2019B020227003); National Natural Science Foundation of China (81770826); National Key Research and Development Program of China (2017YFA0105803)

Ionizing radiation increases the endothelial permeability and the transendothelial migration of tumor cells through ADAM10-activation and subsequent degradation of VE-cadherin

BackgroundWe analyzed the changes in permeability of endothelial cell layers after photon irradiation, with a focus on the metalloproteases ADAM10 and ADAM17, and on VE-cadherin, components crucial for the integrity of endothelial intercellular junctions, and their roles in the transmigration of cancer cells through endothelial cell monolayers.MethodsPrimary HUVEC were irradiated with 2 or 4 Gy photons at a dose rate of 5 Gy/min. The permeability of an irradiated endothelial monolayer for macromolecules and tumor cells was analyzed in the presence or absence of the ADAM10/17 inhibitors GI254023X and GW280264X. Expression of ADAM10, ADAM17 and VE-Cadherin in endothelial cells was quantified by immunoblotting and qRT. VE-Cadherin was additionally analyzed by immunofluorescence microscopy and ELISA.ResultsIonizing radiation increased the permeability of endothelial monolayers and the transendothelial migration of tumor cells. This was effectively blocked by a selective inhibition (GI254023X) of ADAM10. Irradiation increased both, the expression and activity of ADAM10, which led to increased degradation of VE-cadherin, but also led to higher rates of VE-cadherin internalization. Increased degradation of VE-cadherin was also observed when endothelial monolayers were exposed to tumor-cell conditioned medium, similar to when exposed to recombinant VEGF.ConclusionsOur results suggest a mechanism of irradiation-induced increased permeability and transendothelial migration of tumor cells based on the activation of ADAM10 and the subsequent change of endothelial permeability through the degradation and internalization of VE-cadherin.

Lysophosphatidic Acid Receptor 4 Activation Augments Drug Delivery in Tumors by Tightening Endothelial Cell-Cell Contact

Vascular normalization in tumors may improve drug delivery and anti-tumor immunity. Angiogenesis inhibitors induce hypoxia, which may facilitate malignant progression; therefore, we investigated other methods to promote vascular maturation. Here, we show that lysophosphatidic acid (LPA) enhances blood flow by promoting fine vascular networks, thereby improving vascular permeability and suppressing tumor growth when combined with anti-cancer drug treatment. Six different G protein-coupled receptors have been identified as LPA receptors (LPA1-6). In studies using mutant mice, we found that LPA4 is involved in vascular network formation. LPA4 activation induces circumferentialactin bundling beneath the cell membrane and enhances linear adherens junction formation by VE-cadherin in endothelial cells. Therefore, we conclude that activation of LPA4 is a promising approach for vascular regulation.

The Structure and Function of VE‐cadherin in Endothelial Cells

Endothelial cells, cells that line the blood vessels, are responsible for transporting the flow of blood to tissues for the purpose of oxygenating and supplying nutrients. These cells are connected, side‐to‐side, by adherens junctions (AJs). The proteins responsible for keeping AJs together are the adhesion molecules called VE‐cadherins. The York Community High School MSOE Center for BioMolecular Modeling SMART Team used 3‐D modeling and printing technology to examine structure‐function relationships of these proteins. Each VE‐cadherin is composed of five extracellular domains, a transmembrane domain, and a cytoplasmic domain. Each domain is made up of a seven stranded beta barrel fold. One such strand is called the A strand, and this strand, in the EC1 domain, allows for two VE‐cadherins from two adjacent endothelial cells to connect together. This formation of cell‐cell adhesion between the two VE‐cadherins is due to heterophilic interaction. Disruption of VE‐cadherin‐mediated cell‐cell adhesion results in increased permeability of the endothelial cells which can cause edema and hemorrhages. There are several factors that can disrupt this VE‐cadherin‐VE‐cadherin cell adhesion event, such as bacterial infection‐induced sepsis. In the presence of an optimal concentration of calcium and magnesium, however, VE‐cadherin molecules are able to promote a type of paracellular pathway that mediates the passage of hormones, oxygen, and nutrients. VE‐cadherin is an integral protein that functions as an adhesion zipper for endothelial cells; therefore, without these adhesion molecules, the endothelium will not be able to work as a selective barrier.Support or Funding InformationCenter for BioMolecular Modeling, Milwaukee School of Engineering

Overexpressing IRS1 in Endothelial Cells Enhances Angioblast Differentiation and Wound Healing in Diabetes and Insulin Resistance.

The effect of enhancing insulin’s actions in endothelial cells (ECs) to improve angiogenesis and wound healing was studied in obesity and diabetes. Insulin receptor substrate 1 (IRS1) was overexpressed in ECs using the VE-cadherin promoter to create ECIRS1 TG mice, which elevated pAkt activation and expressions of vascular endothelial growth factor (VEGF), Flk1, and VE-cadherin in ECs and granulation tissues (GTs) of full-thickness wounds. Open wound and epithelialization rates and angiogenesis significantly improved in normal mice and high fat (HF) diet–induced diabetic mice with hyperinsulinemia in ECIRS1 TG versus wild type (WT), but not in insulin-deficient diabetic mice. Increased angioblasts and EC numbers in GT of ECIRS1 mice were due to proliferation in situ rather than uptake. GT in HF-fed diabetic mice exhibited parallel decreases in insulin and VEGF-induced pAkt and EC numbers by >50% without changes in angioblasts versus WT mice, which were improved in ECIRS1 TG mice on normal chow or HF diet. Thus, HF-induced diabetes impaired angiogenesis by inhibiting insulin signaling in GT to decrease the differentiation of angioblasts to EC, which was normalized by enhancing insulin’s action targeted to EC, a potential target to improve wound healing in diabetes and obesity.

Abstract 8: RNA-binding Protein Quaking Maintains Endothelial Barrier Function Through β-catenin and VE-cadherin

Endothelial barrier function plays a major role in the onset of atherosclerosis. This barrier is determined largely by adherens junctions. Remarkably little is known about their regulation at the post[[Unable to Display Character: &amp;#8208;]]transcriptional level. We find that the RNA-binding protein Quaking (QKI), known for its function in embryonic blood vessel formation, is highly expressed in quiescent adult endothelial cells (EC) in vivo. In vitro, EC displayed increased levels of QKI when cultured under laminar atheroprotective flow. Using KLF2 overexpression and a human QKI promoter reporter gene, we found that KLF2 mediates this increase in QKI expression. Subsequently we aimed to investigate the role of QKI in EC vascular integrity. Silencing of QKI markedly impaired (0.65 fold ±0.13; p&lt;0.05) the capacity to form a high resistance endothelial monolayer, as measured using Electric cell-substrate impedance sensing. To confirm a role for QKI in maintaining EC barrier function in vivo, we measured Bradykinin-induced vascular leakage in QKI viable mice (QKIv), which express decreased levels of the QKI protein. Indeed, QKIv mice displayed a 20% (p&lt;0.05) increase in extravascular accumulation of Evans blue-labeled albumin compared to wild type littermates. Interestingly, the mRNA of both β-catenin and VE-cadherin, the prime adhesion proteins in EC adherens junctions, contain conserved QKI-binding sites. Moreover the targeted reduction of QKI resulted in a reduction of β-catenin and VE-cadherin protein expression. Importantly, we identified a direct role for QKI in regulating mRNA biology of β-catenin and VE-cadherin, as RNA immunoprecipitation and luciferase-reporter assays revealed that QKI can directly bind to the mRNA and induces transcript translation, respectively. This effects was perturbed upon reduced QKI expression. In conclusion, we show that QKI functions as a critical regulator of β-catenin and VE-cadherin in endothelial cells, and the modulation of QKI expression affects endothelial monolayer integrity, in vitro and in vivo. These studies provide novel insight into the importance of post-transcriptional regulation of components of the endothelial adherens junction, and may have wide ranging implications for the preservation of vascular integrity in disease.

Girdin/GIV regulates transendothelial permeability by controlling VE-cadherin trafficking through the small GTPase, R-Ras

Vascular permeability is regulated by intercellular junction organization of endothelial cells, the dysfunction of which is implicated in numerous pathological conditions. Molecular mechanisms of how endothelial cells regulate intercellular junction in response to extracellular signals, however, have so far remained elusive. This study identified that Girdin (also termed GIV), an Akt substrate functioning in post natal angiogenesis, was expressed in a mature endothelial monolayer, where it regulated VE-cadherin trafficking to maintain vascular integrity. Girdin depletion abrogated VEGF-induced VE-cadherin endocytosis and the disassembly of adherens junctions in a monolayer of endothelial cells, thus leading to a significant decrease in the permeability. We also showed that activated R-Ras, a member of the Ras family GTPase, known to be a master regulator of transendothelial permeability, interacts with Girdin, and facilitates the complex formation between Girdin and VE-cadherin in endothelial cells. However, the increased permeability mediated by the loss of R-Ras was rescued by Girdin depletion, thus suggesting that the interaction of Girdin with R-Ras functions in VE-cadherin trafficking pathways distinct from endocytosis. The recycling of VE-cadherin was promoted by the exogenous expression of the active mutant of R-Ras, which was attenuated in the Girdin-depleted endothelial cells. These results show that Girdin regulates transendothelial permeability in synergy with R-Ras and VE-cadherin in an endothelial monolayer.

Effect of proinflammatory activation on F-actin distribution in cultured human endothelial cells under conditions of experimental microgravity.

We compared the state of actin cytoskeleton, morphology, and expression of VE-cadherin in endothelial cells of human umbilical cord vein under conditions of TNF-α-mediated activation and microgravity modeling and found that 3D-clinorotation for 24 h impaired the integrity of endothelial monolayer, altered cell morphology, induced cytoskeleton reorganization, and reduced the expression of VE-cadherin. The combination of experimental microgravity and proinflammatory activation led to more pronounced clearing of the perinuclear space from microfilaments and accumulation of depolymerized actin, which confirms additive effect of the studied factors on actin cytoskeleton of endothelial cells.

Neural Wiskott-Aldrich Syndrome Protein (N-WASP)-mediated p120-Catenin Interaction with Arp2-Actin Complex Stabilizes Endothelial Adherens Junctions

Stable adherens junctions (AJs) are required for formation of restrictive endothelial barrier. Vascular endothelial cadherin from contiguous endothelial cells forms AJs, which are stabilized intracellularly by binding of p120-catenin and cortical actin. Mechanisms inducing cortical actin formation and enabling its linkage with p120-catenin remain enigmatic. We altered the function of neural Wiskott-Aldrich syndrome protein (N-WASP), which induces actin polymerization through actin-related protein 2/3 complex (Arp2/3), to address the role of N-WASP in regulating AJ stability and thereby endothelial permeability. We show that depletion of N-WASP in endothelial cells impaired AJ adhesion and favored the organization of actin from cortical actin to stress fibers, resulting thereby in formation of leaky endothelial barrier. Exposure of the N-WASP-depleted endothelial cell monolayer to the permeability-increasing mediator, thrombin, exaggerated AJ disruption and stress fiber formation, leading to an irreversible increase in endothelial permeability. We show that N-WASP binds p120-catenin through its verprolin cofilin acid (VCA) domain, induces cortical actin formation through Arp2, and links p120-catenin with cortical actin. The interaction of N-WASP with p120-catenin, actin, and Arp2 requires phosphorylation of N-WASP at the Tyr-256 residue by focal adhesion kinase. Expression of the VCA domain of N-WASP or phosphomimicking (Y256D)-N-WASP mutant in endothelial cells stabilizes AJs and facilitates barrier recovery after thrombin stimulation. Our study demonstrates that N-WASP, by mediating p120-catenin interaction with actin-polymerizing machinery, maintains AJs and mitigates disruption of endothelial barrier function by edemagenic agents, therefore representing a novel target for preventing leaky endothelial barrier syndrome.