Regulation of endothelial permeability via rhoA/ROCK

描述(由申请人提供)：本提案的目标是表征小GTP酶RhoA及其下游效应因子Rho Kinase(ROCK)在中性粒细胞诱导的内皮通透性变化中的作用。异常过多的冠状动脉微血管渗漏是心脏病的早期事件，主要归因于炎症介质和以中性粒细胞为主的多形核白细胞(PMN)的激活。内皮屏障的调节是一个复杂的过程，涉及肌动蛋白细胞骨架、细胞间黏附和细胞-基质黏附。研究表明，RhoA和ROCK调节肌动蛋白细胞骨架和细胞与基质的黏附，但RhoA和ROCK在内皮屏障调节中的作用尚不清楚。我们的初步研究表明，PMN诱导内皮细胞RhoA激活，ROCK抑制减轻PMN诱导的高通透性和肌动蛋白组织的变化，并且粘着斑激酶(FAK)在通透性调节中发挥重要作用。我打算研究RhoA/ROCK在PMN诱导的高通透性中的作用，重点是RhoA和ROCK介导的肌动蛋白细胞骨架和细胞-基质黏附的变化。我们将比较PMN诱导的RhoA激活、内皮通透性、FAK和paxiltin的酪氨酸磷酸化、F/G-肌动蛋白比率变化和内皮细胞张力的时间进程。我们还将研究RhoA/ROCK抑制如何影响PMN刺激的这些变化。这项研究将极大地提高我们对内皮屏障调节的理解。

DESCRIPTION (provided by applicant): 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.