Activated Neutrophils Induce Hyperpermeability and Phosphorylation of Adherens Junction Proteins in Coronary Venular Endothelial Cells

Abstract

The endothelial adherens junction is formed by complexes of transmembrane adhesive proteins, of which ␤-catenin is known to connect the junctional protein vascular endothelial (VE)-cadherin to the cytoskeleton and to play a signaling role in the regulation of junctioncytoskeleton interaction. In this study, we investigated the effect of neutrophil activation on endothelial monolayer integrity and on ␤-catenin and VE-cadherin modification. Treatment of cultured bovine coronary endothelial monolayers with C5a-activated neutrophils resulted in an increase in permeability as measured by albumin clearance across the monolayer. Furthermore, large scale intercellular gap formation was observed in coincidence with the hyperpermeability response. Immunofluorescence analysis showed that ␤-catenin and VE-cadherin staining changed from a uniform distribution along the membrane of control cells to a diffuse pattern for both proteins and finger-like projections for ␤-catenin in neutrophil-exposed monolayers. Correlatively, there was an increase in actin stress fiber formation in treated cells. Finally, ␤-catenin and VE-cadherin from neutrophil-treated endothelial cells showed a significant increase in tyrosine phosphorylation. Our results are the first to link neutrophil-mediated changes in adherens junctions with intercellular gap formation and hyperpermeability in microvascular endothelial cells. These data suggest that neutrophils may regulate endothelial barrier function through a process conferring conformational changes to ␤-catenin and VE-cadherin.The wall of exchange vessels consists of a layer of endothelial cells that connect to each other with closely opposed intercellular junctions. A major function of the junctional connection is to maintain the semi-permeable property of the endothelial barrier and to control the transvascular passage of solutes, fluid, and blood cells. Four types of junctions associated with endothelial cells have been identified: adherens junctions (AJ), 1 tight junctions, gap junctions, and complexus adherentes (1, 2). AJ, formed by transmembrane adhesive proteins called cadherins, appear to be the main complex regulating macromolecular permeability in microvascular endothelium. Cadherins, specifically vascular endothelial (VE)-cadherin, are associated with the actin cytoskeleton through a family of proteins called catenins, including ␣-catenin, ␤-catenin, and plakoglobin (3, 4). The endothelial permeability is affected by many agonists including ␣-thrombin, histamine, and phorbol esters (5-9) as well as by a group of inflammatory cells, namely polymorphonuclear leukocytes (PMNs) (10 -14). At the site of injury or inflammation, circulating PMNs often adhere to and subsequently migrate through the endothelium and enter surrounding tissues (15). It has long been documented that the process of PMN adherence and migration is associated with an increase in endothelial permeability (10, 11). Although much work has been dedicated to identify PMN-derived hyperpermeability factors (11-14), little is k...

Regulation of transendothelial migration of colon cancer cells by E-selectin-mediated activation of p38 and ERK MAP kinases

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ICAM-1-Mediated, Src- and Pyk2-Dependent Vascular Endothelial Cadherin Tyrosine Phosphorylation Is Required for Leukocyte Transendothelial Migration

“…Previously, it was reported that coincubation of EC and C5a-activated neutrophils causes increased tyrosine phosphorylation of AJ proteins; however, the specific mechanisms of leukocyte-induced modification of AJ proteins remain unclear (26,27). To examine specifically the consequences of leukocyte binding to EC on the phosphorylation of VE-cadherin, we treated EC with TNF-␣, which has been shown to increase expression of cell adhesion molecules such as ICAM-1 and VCAM-1 (28).…”

The role of cytoskeleton in the regulation of vascular endothelial barrier function

“…An additional binding partner, p120, is thought to be involved in the control over membrane-localized cadherin turnover and stability [Komarova et al, 2007]. The adhesiveness of adherence junctions seems to be strongly regulated by tyrosine phosphorylation [Tinsley et al, 1999;Weis et al, 2004;Vestweber, 2007]; established vascular barrier disruptors histamine, VEGF and TNFα increase tyrosine phosphorylation of one or more proteins within adherence junctions [Angelini et al, 2006;Andriopoulou et al, 1999;Esser et al, 1998]. The phosphorylation was attributed to several tyrosine protein kinases, including Src, Fyn, Yes and Pyk2 [Lambeng et al, 2005;van Buul et al, 2005;Gong et al, 2008].…”