Abstract
The endothelial adherens junction is formed by complexes of transmembrane adhesive proteins, of which beta-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 junction-cytoskeleton interaction. In this study, we investigated the effect of neutrophil activation on endothelial monolayer integrity and on beta-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 beta-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 beta-catenin in neutrophil-exposed monolayers. Correlatively, there was an increase in actin stress fiber formation in treated cells. Finally, beta-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 beta-catenin and VE-cadherin.
Highlights
The wall of exchange vessels consists of a layer of endothelial cells that connect to each other with closely opposed intercellular junctions
The results showed that tyrosine phosphorylation of vascular endothelial (VE)-cadherin and -catenin was increased when cultured coronary venular endothelial cells (CVECs) were exposed to activated polymorphonuclear leukocytes (PMNs). -Catenin and VE-cadherin staining revealed a marked decrease in the amount of these proteins at the cell periphery upon stimulation by PMNs
Our preliminary studies revealed that coating the polycarbonate membranes with gelatin was superior to fibronectin, and the gelatin imposed no major restriction to albumin clearance
Summary
The wall of exchange vessels consists of a layer of endothelial cells that connect to each other with closely opposed intercellular junctions. Upon PMN adhesion, VE-cadherin proteolysis is accompanied by the disappearance of both VE-cadherin and catenins from AJ [14, 16, 17] Another hyperpermeability agonist, vascular endothelial growth factor, has been shown to increase the transendothelial flux of albumin concomitant with a loss of VE-cadherin [18]. Vascular endothelial growth factor, has been shown to increase the transendothelial flux of albumin concomitant with a loss of VE-cadherin [18] These studies have provided a possible linkage between PMN activation and VE-cadherin disorganization in the regulation of endothelial barrier function. This paper is available on line at http://www.jbc.org tional proteins in response to PMNs in microvascular endothelial cells
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