Involvement of local lamellipodia in endothelial barrier function.

Jerome W Breslin,Xun E Zhang,Rebecca A Worthylake,Flavia M Souza-Smith,Yulia Komarova

doi:10.1371/journal.pone.0117970

Abstract

Recently we observed that endothelial cells cultured in tightly confluent monolayers display frequent local lamellipodia, and that thrombin, an agent that increases endothelial permeability, reduces lamellipodia protrusions. This led us to test the hypothesis that local lamellipodia contribute to endothelial barrier function. Movements of subcellular structures containing GFP-actin or VE-cadherin-GFP expressed in endothelial cells were recorded using time-lapse microscopy. Transendothelial electrical resistance (TER) served as an index of endothelial barrier function. Changes in both lamellipodia dynamics and TER were assessed during baseline and after cells were treated with either the barrier-disrupting agent thrombin, or the barrier-stabilizing agent sphingosine-1-phosphate (S1P). The myosin II inhibitor blebbistatin was used to selectively block lamellipodia formation, and was used to test their role in the barrier function of endothelial cell monolayers and isolated, perfused rat mesenteric venules. Myosin light chain (MLC) phosphorylation was assessed by immunofluorescence microscopy. Rac1 and RhoA activation were evaluated using G-LISA assays. The role of Rac1 was tested with the specific inhibitor NSC23766 or by expressing wild-type or dominant negative GFP-Rac1. The results show that thrombin rapidly decreased both TER and the lamellipodia protrusion frequency. S1P rapidly increased TER in association with increased protrusion frequency. Blebbistatin nearly abolished local lamellipodia protrusions while cortical actin fibers and stress fibers remained intact. Blebbistatin also significantly decreased TER of cultured endothelial cells and increased permeability of isolated rat mesenteric venules. Both thrombin and S1P increased MLC phosphorylation and activation of RhoA. However, thrombin and S1P had differential impacts on Rac1, correlating with the changes in TER and lamellipodia protrusion frequency. Overexpression of Rac1 elevated, while NSC23766 and dominant negative Rac1 reduced barrier function and lamellipodia activity. Combined, these data suggest that local lamellipodia, driven by myosin II and Rac1, are important for dynamic changes in endothelial barrier integrity.

Highlights

The endothelium of capillary and postcapillary venules is a semi-permeable barrier critical for normal blood-tissue exchange of fluids and solutes
The importance of junctional protein complexes, such as those composed of VE-cadherin and its associated catenins have been well established in the control of microvascular permeability [1,2]
A more detailed understanding of the time course of the cytoskeletal and junctional mechanisms elicited by agents that alter endothelial barrier function requires the ability to more precisely view changes in these subcellular structures in living endothelial cells

Summary

Introduction

The endothelium of capillary and postcapillary venules is a semi-permeable barrier critical for normal blood-tissue exchange of fluids and solutes. During inflammation, this barrier becomes compromised, allowing increased transport of plasma proteins into the surrounding tissues. During barrier compromise due to inflammatory mediators, current theory suggests that centripetal tension can put stress on the junctions and limit their strength, delaying recovery of normal barrier integrity [3,4,5]. Various inflammatory stimuli promote development of actin stress fibers, which are thought to increase centripetal tension [6,7]. Agents that reduce permeability, such as the bioactive lipid sphingosine-1-phosphate (S1P), have been reported to increase the number of cortical actin fibers, stabilizing the cell periphery and strengthening junctions between endothelial cells [8,9]

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