Abstract 9971: Rock2 is the Predominant Rho Kinase Involved in Thrombin Receptor-Mediated Vascular Permeability

Abstract

Objective Endothelial barrier function is critically regulated by Rho kinase. Basal Rho kinase activity is essential for barrier maintenance, but its enhanced activity induced by vascular permeability factors such as thrombin causes barrier dysfunction involving acto-myosin contractility. Here, we investigated the isoform-specific roles of Rho kinase (ROCK1 and ROCK2) in regulating endothelial (hyper-)permeability. Methods 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). In the intact lungs of untreated and ROCK1/2 siRNA-transducing mice vascular permeability was induced by TFLLRN, an agonist of the thrombin receptor PAR1 and was assessed by lung Evans blue dye extravasation and wet-dry weight ratios of isolated lungs. Contractile forces were mapped by traction force microscopy. Results Downregulation of ROCK2, but not ROCK1 expression attenuated PAR1-mediated hyperpermeability of endothelial monolayers and the pulmonary microvasculature. Simultaneous downregulation of both Rho kinase isoforms further reduced the hyperpermeability. Surprisingly, inhibition of ROCK2 did not affect the net thrombin-induced contractile force enhancements and the formation of thrombin-induced F-actin stress fibers, but lowered basal traction forces, enforcing the basal barrier function of the endothelium. Conclusions These data show that ROCK2, but not ROCK1 is the critical Rho kinase for thrombin-induced endothelial barrier disruption. Rather than mediating the evoked contractile force enhancements through the formation of stress fibers, ROCK2 activity is of vital importance for the basal isometric tone of the endothelium. Thus, selective pharmacological inhibition of basal traction forces could provide a novel approach to stabilize the endothelial barrier, and limit vascular leak during derailed inflammatory episodes.