Mechanochemical regulation of endothelial permeability

描述(申请人提供)：肺内皮细胞是一种关键的、半选择性的细胞屏障，它防止水、溶质、大分子和细胞从血管系统渗漏。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经历的周期性拉伸模式改变有关，并导致通透性增加、肺泡泛滥、白细胞渗透、低氧血症以及增加发病率和死亡率。我们以前已经确定了屏障保护性(1-磷酸鞘氨醇)和屏障干扰性(凝血酶)药物引起EC通透性变化的细胞骨架机制，并描述了小GTP酶Rac和Rho在与EC屏障调节相关的细胞局灶性黏附和黏附连接重塑中的关键参与。我们发表的结果和初步数据有力地表明，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，循环牵张(CS)对RAC和Rho GTP酶的大小依赖调节可能决定了高潮气量机械通气时肺内皮细胞屏障功能障碍的严重程度和屏障恢复的速度。我们还推测，小分子GTP酶的细胞骨架(肌动蛋白，Cortactin)、焦点黏附(Paxlin，PKL/GIT2)和黏附连接(VE-cadherin，β-catenin)效应器之间的相互作用可能在机械力调节肺屏障中起关键作用。具体目标#1将采用体外和体内相结合的方法，确定肺内皮细胞经历的生理和病理相关的CS水平，并探索大小依赖的RAC和Rho调节与EC细胞骨架变化之间的联系。具体目标#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。具体目标#3将确定RAC和Rho特异的GEF(Tiam1、Vav2、betaPIX、gef-h1和p115-Rhogef)对小GTP酶活性进行机械力化学调节的分子机制。我们推测，这项研究的结果将显著影响我们对小GTP酶通过涉及细胞骨架和细胞黏附重塑的特定机制(SA#1和SA#2)在EC适应机械力化学环境变化中的作用的理解，发现环境基金通过机械力化学刺激(SA#3)调节RAC和Rho的新机制，并使我们能够对呼吸机诱导的肺损伤中内皮功能障碍的细胞机制产生新的见解。

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.