The goal of this proposal is to characterize the role of the small GTPase rhoA and its downstream effectors, rho kinase (ROCK), in neutrophil-induced changes in endothelial permeability. Abnormally excessive coronary microvascular leakage is an early event in heart disease, and is largely attributed to inflammatory mediators and the activation of polymorphonuclear leukocytes (PMN), predominantly neutrophil. Regulation of the endothelial barrier is a complex process involving the actin cytoskeleton, intercellular adhesions, and cell-matrix adhesion. Studies indicate that rhoA and ROCK regulate the actin cytoskeleton and cell-matrix adhesion, however, the role of rhoA and ROCK in endothelial barrier regulation is unclear. Our pilot studies demonstrate PMN-induced endothelial rhoA activation, that ROCK inhibition attenuates PMN-induced hyperpermeability and changes in actin organization, and that focal adhesion kinase (FAK) plays an important role in permeability regulation. I intend to investigate the role of rhoA/ROCK in PMN-induced hyperpermeability, with emphasis on rhoA and ROCK-mediated changes in the actin cytoskeleton and cell-matrix adhesion. We will compare time-courses of PMN-induced rhoA activation, endothelial permeability, tyrosine phosphorylation of FAK and paxiltin, F/G-actin ratio changes, and endothelial cell tension. We will also investigate how rhoA/ROCK inhibition affects these PMN-stimulated changes. This study will greatly increase our understanding of endothelial barrier regulation. ? ?
