Regulation of endothelial permeability via rhoA/ROCK

描述（由申请人提供）：本提案的目的是表征小GTPase rhoA及其下游效应物rho激酶（ROCK）在中性粒细胞诱导的内皮通透性变化中的作用。冠状动脉微血管异常过度渗漏是心脏病的早期事件，主要归因于炎症介质和多形核白细胞（PMN）的激活，主要是中性粒细胞。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-基质粘附，但rhoA和ROCK在内皮屏障调节中的作用尚不清楚。我们的前期研究表明，pmn诱导内皮rhoA活化，ROCK抑制可减轻pmn诱导的高通透性和肌动蛋白组织的变化，并且局灶黏附激酶（FAK）在通透性调节中起重要作用。我打算研究rhoA/ROCK在pmn诱导的高通透性中的作用，重点研究rhoA和ROCK介导的肌动蛋白细胞骨架和细胞-基质粘附的变化。我们将比较pmn诱导的rhoA活化、内皮通透性、FAK和paxiltin酪氨酸磷酸化、F/G-actin比值变化和内皮细胞张力的时间过程。我们还将研究rhoA/ROCK抑制如何影响这些pmn刺激的变化。这项研究将大大增加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.