Control of Lung Permeability by Oxidized Phospholipids

描述(申请人提供)：血管通透性的改变是多种过程的特征，包括炎症、缺血/再灌注和呼吸机诱导的肺损伤，并可能增加肺泡泛滥、白细胞渗透和低氧血症，导致发病率和死亡率增加。伴随这些病理的氧化应激导致肺循环中氧化磷脂(OxPL)水平的增加。我们以前已经在体外和体内鉴定了一组屏障保护性Oxpls，并描述了基础肺内皮(EC)屏障功能的增强和炎性激动剂引起的通透性增加的减弱。OxPLs的屏障保护作用与增强外周肌动蛋白细胞骨架、增加小GTP酶Rac和CDC42介导的细胞-细胞和细胞-底物连接之间的相互作用以及Rac介导的Rho依赖的内皮高通透性途径的减弱有关。尽管内皮细胞骨架在内皮细胞通透性动态调节中的重要性已被广泛认识，但内皮细胞黏附复合体在内皮细胞屏障反应中的作用却知之甚少。我们的初步研究有力地表明，Oxpls在炎性激动剂诱导的肺内皮细胞屏障功能障碍模型中的屏障保护作用是由细胞-细胞黏附结构介导的，这种黏附结构受小GTP酶Rac和Rap1的调节。我们的研究表明，依赖于Rac和Rap1的信号也参与了OxPL介导的Rho途径的抑制。我们假设Oxpls的生物活性成分通过激活依赖于Rap1的黏附连接和紧密连接来促进肺血管屏障的完整性。我们还假设OxPL诱导的黏附连接的增强可能通过其负调控因子p190RhoGAP抑制Rho GTP酶，该负调控因子受黏附连接和脂筏的控制。特殊目的#1将测试一种新的上游机制，即OxPL通过参与脂筏和激活GRP78受体来诱导RAC激活。具体目标#2将评估Rap1及其细胞黏附效应因子afadin在Oxpls诱导的黏附连接和紧密连接增强中的参与。具体目标#3将探索p190RhoGAP在OxPL诱导的Rho抑制中RAC和RAP1依赖的机制中的作用。我们相信，这些研究将显著影响我们对氧化磷脂在EC屏障调节中的作用的理解，并将有助于识别新的蛋白质靶点，并为预防与急性肺炎症和损伤相关的肺血管屏障功能障碍提供新的治疗方法。 与公共卫生相关：肺损伤的急性期以内皮通透性增加和血气屏障受损为特征，这允许富含蛋白质的液体进入空气空间，导致肺水肿。然而，尽管最近在呼吸策略方面取得了进展，对ALI的病理生理学也有了更好的了解，但对于这种毁灭性的疾病，仍然没有多少有效的治疗方法。这项应用将研究氧化磷脂对由水肿性激动剂和与急性肺损伤相关的病理机械应变引起的肺血管内皮细胞渗漏的保护作用的分子机制，并将表征新型屏障保护性氧化磷脂化合物。

DESCRIPTION (provided by applicant): Alterations in vascular permeability are defining feature of diverse processes including inflammation, ischemia/reperfusion and ventilator-induced lung injury, and may increase alveolar flooding, leukocyte infiltration and hypoxemia leading to increased morbidity and mortality. Oxidative stress accompanying these pathologies results in increased levels of oxidized phospholipids (OxPL) in the pulmonary circulation. We have previously characterized a group of barrier-protective OxPLs in vitro and in vivo and described enhancement of basal pulmonary endothelial (EC) barrier function and attenuation of permeability increase caused by inflammatory agonists. Barrier protective effects of OxPLs have been linked to enhancement of peripheral actin cytoskeleton, increased interactions between cell-cell and cell-substrate junctions mediated by small GTPases Rac and Cdc42, and Rac-mediated attenuation of Rho-dependent pathways of endothelial hyper-permeability. Although importance of endothelial cytoskeleton in dynamic regulation of EC permeability is well recognized, the role of EC adhesive complexes in the EC barrier responses is less understood. Our preliminary studies strongly suggest that barrier protective effects of OxPLs in the models of lung EC barrier dysfunction induced by inflammatory agonists are mediated by cell-cell adhesive structures, which are regulated by small GTPases Rac and Rap1. Our studies indicate that Rac- and Rap1-dependent signaling is also involved in the OxPL- mediated inhibition of Rho pathway. We hypothesize that bioactive components of OxPLs promote the lung vascular barrier integrity via activation of Rap1-dependent engagement of adherens junctions and tight junctions. We also hypothesize that OxPL-induced enhancement of adherens junctions may inhibit Rho GTPase via its negative regulator p190RhoGAP controlled by adherens junctions and lipid rafts. Specific Aim #1 will test a novel upstream mechanism of OxPL-induced Rac activation via engagement of lipid rafts and activation of GRP78 receptor. Specific Aim #2 will evaluate involvement of Rap1 and its cell adhesion effector afadin in the OxPLs-induced adherens junction and tight junction enhancement. Specific Aim #3 will explore a role of p190RhoGAP in the Rac- and Rap1-dependent mechanisms of OxPL-induced Rho inhibition. We believe that these studies will significantly impact our understanding of the role of oxidized phospholipids in the EC barrier regulation and will help to identify novel protein targets and propose new therapies for prevention of pulmonary vascular barrier dysfunction associated with acute lung inflammation and injury. PUBLIC HEALTH RELEVANCE: The acute phase of lung injury is characterized by increased endothelial permeability and compromise of the blood-gas barrier, which allows an influx of protein-rich fluid into the air spaces, causing pulmonary edema. However, despite recent advances in ventilation strategies and a better understanding of the pathophysiology of ALI, there remain few effective treatments for this devastating illness. This application will investigate molecular mechanisms underlying protective effects of oxidized phospholipids against pulmonary vascular endothelial leak induced by edemagenic agonists and pathologic mechanical strain associated with acute lung injury and will characterize novel groups of barrier protective oxidized phospholipid compounds.