Mechanochemical regulation of endothelial permeability

描述（由申请人提供）：肺内皮是一种关键的、半选择性的细胞屏障，它可以防止水、溶质、大分子和细胞从脉管系统中泄漏。高潮气量机械通气导致的肺内皮细胞（EC）屏障完整性受损与EC所经历的循环拉伸模式改变有关，并导致通透性增加、肺泡充血、白细胞浸润、低氧血症以及发病率和死亡率增加。我们之前已经确定了屏障保护（鞘氨醇1-磷酸）和屏障破坏（凝血酶）引起的EC渗透性变化的细胞骨架机制，并描述了小GTPases Rac和Rho在与EC屏障调节相关的细胞局灶粘连和粘附连接的重塑中的关键作用。我们发表的结果和初步数据强烈表明，机械刺激对Rac和Rho活性的调节显著影响激动剂诱导的EC通透性调节。我们最初的假设是，循环拉伸（CS）对Rac和Rho gtpase的大小依赖性调节可能决定了高潮气量机械通气期间肺EC屏障功能障碍的严重程度和屏障恢复率。我们还假设细胞骨架（肌动蛋白，皮质蛋白），局灶粘附（paxillin, PKL/GIT2）和粘附连接（VE-cadherin, β -catenin）效应小gtpase的相互作用可能在机械力的肺屏障调节中起关键作用。特异性目标#1将结合体外和体内方法确定肺EC所经历的CS的生理和病理相关水平，并将探索大小依赖性Rac和Rho调节与EC细胞骨架变化之间的联系。特异性目标#2将研究生理和病理CS水平在激动剂诱导的EC细胞骨架、细胞接触和单层完整性调节中的作用。Specific Aim #3将确定Rac和rho特异性gef （Tiam1, Vav2, betaPIX， GEF-H1和p115-RhoGEF）对小GTPase活性的机械化学调节的分子机制。我们推测，本研究获得的结果将显著影响我们对小gtpase通过涉及细胞骨架和细胞粘附重塑的特定机制（SA#1和SA#2）在EC适应机械化学环境变化中的作用的理解，发现gef介导的机械化学刺激调节Rac和Rho的新机制（SA#3）。并使我们对呼吸机诱导的肺损伤中内皮功能障碍的细胞机制有了新的认识。

DESCRIPTION (provided by applicant): Pulmonary endothelium is a critical, semi-selective cellular barrier, which prevents the leakage of water, solutes, macromolecules and cells from the vasculature. Compromised pulmonary endothelial cell (EC) barrier integrity caused by mechanical ventilation at high tidal volumes is associated with altered patterns of cyclic stretch experienced by EC, and leads to increased permeability, alveolar flooding, leukocyte infiltration, hypoxemia, and increased morbidity and mortality. We have previously identified cytoskeletal mechanisms of EC permeability changes caused by barrier-protective (sphingosine 1-phosphate) and barrier-disruptive (thrombin) agents and described a critical involvement of small GTPases Rac and Rho in remodeling of cell focal adhesions and adherens junctions associated with EC barrier regulation. Our published results and preliminary data strongly suggest, that modulation of Rac and Rho activities by mechanical stimuli significantly impacts agonist-induced regulation of EC permeability. Our original hypothesis is that magnitude-dependent regulation of Rac and Rho GTPases by cyclic stretch (CS) may determine severity of the pulmonary EC barrier dysfunction and rates of barrier restoration during mechanical ventilation at high tidal volume. We also hypothesize that interactions of cytoskeletal (actin, cortactin), focal adhesion (paxillin, PKL/GIT2), and adherens junction (VE-cadherin, beta-catenin) effectors of small GTPases may be critically involved in the lung barrier regulation by mechanical forces. Specific Aim #1 will determine physiological and pathologically relevant levels of CS experienced by pulmonary EC using combination of in vitro and in vivo approaches and will explore a link between magnitude-dependent Rac and Rho regulation and EC cytoskeletal changes. Specific Aim #2 will investigate a role of physiological and pathological CS levels in the agonist-induced regulation of EC cytoskeleton, cell contacts, and monolayer integrity. Specific Aim #3 will identify molecular mechanisms of mechanochemical regulation of small GTPase activities by Rac- and Rho-specific GEFs (Tiam1, Vav2, betaPIX, GEF-H1 and p115-RhoGEF). We speculate that the results obtained in this study will significantly impact our understanding of the role of small GTPases in the EC adaptation to alterations of mechanochemical environment via specific mechanisms involving cytoskeletal and cell adhesion remodeling (SA#1 and SA#2), discover novel mechanisms of GEF-mediated regulation of Rac and Rho by mechanochemical stimuli (SA#3), and allow us to develop new insights into cellular mechanisms underlying endothelial dysfunction in ventilator-induced lung injury.