Abstract 364: ROCK2 Regulates Endothelial Hyperpermeability Responses Through Regulation of Basal Junctional Forces

Abstract

Rationale Our previous studies suggested distinct roles for the Rho kinase isoforms ROCK1 and ROCK2 in regulating vascular permeability in vivo. Although being the dominant isoform, it remained unclear how ROCK2 regulates endothelial permeability. Objective To investigate the mechanism of ROCK2-mediated endothelial hyperpermeability responses through selective targeting of either Rho kinase isoform. Methods and Results Endothelial hyperpermeability was induced in vitro by thrombin and was assessed by the passage of a tracer or by Electrical Cell-substrate Impedance Sensing (ECIS). Endothelial contractile forces were mapped by traction force microscopy. Silencing of ROCK2, but not ROCK1 attenuated thrombin-induced hyperpermeability of culured human endothelial monolayers. Simultaneous downregulation of both Rho kinase isoforms further reduced the hyperpermeability. Surprisingly, ROCK2 inhibition did affect neither the thrombin-induced formation of contractile F-actin stress fibers nor the thrombin-induced contractile force enhancements, but rather protected the endothelium from barrier disruption by lowering basal tension in endothelial junctions. Conclusions These data identify a novel mechanism for regulating endothelial hyperpermeability: ROCK2 activity is essential for the development of a critical basal isometric cellular tone (the so-called prestress), which presence is necessary for opening of the endothelial barrier in response to inflammatory mediators. GPvNA is supported by the Dutch Heart Foundation (grant 2011T072)