Mechanisms of Prostacyclin-Mediated Lung Endothelial Barrier Protection

描述（由申请人提供）：前列环素（PGI 2）是环氧合酶的产物，参与血管功能、伤口修复、炎症过程和急性肺损伤的调节。雾化的PGI 2诱导对高氧肺损伤或由缺血/再灌注引起的肺损伤的显著保护，并且增加的PGI 2稳定代谢物水平与较轻的呼吸窘迫相关。然而，PGI 2对肺内皮细胞（EC）的保护作用的分子机制还不清楚。我们已发表的数据表明，小GTP酶Rac和Rho在通过肌动蛋白细胞骨架和细胞接触的特异性重塑调节内皮通透性中的相互作用。我们的初步研究表明，PGI 2对人肺EC的屏障保护作用，并将其与Rac依赖的肌动蛋白细胞骨架重塑和增强粘附连接（AJ）介导的cAMP依赖的蛋白激酶（PKA）和新的Epac-Rap 1-Tiam 1/Vav 2-Rac机制。我们的研究还表明PGI 2对凝血酶诱导的肺EC屏障功能障碍具有有效的保护作用。我们假设PGI 2通过激活PKA和Epac介导的信号传导对肺EC发挥屏障保护作用，从而通过增强外周肌动蛋白细胞骨架和粘附连接激活EC屏障保护的Rac依赖性途径。我们还推测，PGI 2可能通过抑制内皮细胞高通透性的Rho依赖性途径，减轻与呼吸机诱导的肺损伤（VILI）相关的急性肺EC屏障功能障碍。具体目标#1将研究PGI 2诱导的肺EC细胞骨架重塑和屏障保护的PKA-和cAMP/-Epac-Rap 1-Tiam/Vav 2-Rac依赖性机制。具体目标#2将研究与PGI 2诱导的屏障保护相关的PGI 2诱导的AJ重塑的机制，并研究VE-钙粘蛋白介导的Rac调节（通过Epac 1、Tiam 1、Vav 2、Rap 1和Rac的局部募集）。具体目标#3将探索PGI 2诱导的Rho信号传导的调节，这是PGI 2在凝血酶诱导的肺EC屏障功能障碍的细胞培养模型和呼吸机诱导的肺损伤的动物模型中的保护作用的基础。我们相信，这些研究可能会发现新的蛋白质靶点，并提出新的治疗方法，用于预防与急性肺部炎症和损伤相关的肺血管屏障功能障碍。 公共卫生相关性：急性呼吸窘迫综合征（ARDS）是导致发病和死亡的主要原因，总死亡率为30- 40%。肺损伤的急性期的特征在于内皮渗透性增加和血气屏障受损，这允许富含蛋白质的流体流入空气空间，引起肺水肿。然而，尽管最近在通气策略方面取得了进展，并且对ALI的病理生理学有了更好的理解，但对于这种毁灭性疾病仍然很少有有效的治疗方法。本申请将研究前列环素对水肿激动剂诱导的肺血管内皮渗漏和与呼吸机诱导的肺损伤相关的病理性机械应变的保护作用的分子机制。

DESCRIPTION (provided by applicant): Prostacyclin (PGI2), a product of cyclooxygenase, has been implicated in the regulation of vascular function, wound repair, inflammatory processes, and acute lung injury. Aerosolized PGI2 induces marked protection against hyperoxic lung injury or lung damage caused by ischemia/reperfusion, and increased levels of PGI2 stable metabolites have been associated with less severe respiratory distress. However molecular mechanisms of PGI2 protective effects on pulmonary endothelial cells (EC) are not well understood. Our published data have shown a reciprocal role for small GTPases Rac and Rho in regulation of endothelial permeability via specific remodeling of actin cytoskeleton and cell contacts. Our preliminary studies strongly suggest barrier protective effects of PGI2 on human pulmonary EC and link them to the Rac-dependent actin cytoskeletal remodeling and enhancement of adherens junctions (AJ) mediated via cAMP-dependent protein kinase (PKA) and novel Epac-Rap1-Tiam1/Vav2-Rac mechanism. Our studies also indicate potent protective effects of PGI2 against thrombin-induced lung EC barrier dysfunction. We hypothesize that PGI2 exerts barrier protective effects on lung EC via activation of PKA- and Epac-mediated signaling leading to activation of Rac- dependent pathways of EC barrier protection via enhancement of peripheral actin cytoskeletal and adherens junctions. We also hypothesize that PGI2 may attenuate acute lung EC barrier dysfunction associated with ventilator induced lung injury (VILI) via inhibition of Rho-dependent pathways of endothelial hyperpermeability. Specific Aim #1 will study PKA- and cAMP/-Epac-Rap1-Tiam/Vav2-Rac-dependent mechanisms underlying PGI2-induced pulmonary EC cytoskeletal remodeling and barrier protection. Specific Aim #2 will study mechanisms of PGI2-induced AJ remodeling associated with PGI2-induced barrier protection and investigate VE-cadherin-mediated regulation of Rac via local recruitment of Epac1, Tiam1, Vav2, Rap1 and Rac. Specific Aim #3 will explore PGI2-induced modulation of Rho signaling underlying protective effects of PGI2 in cell culture model of thrombin-induced pulmonary EC barrier dysfunction and in animal model of ventilator-induced lung injury. We believe that these studies may 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: Acute respiratory distress syndrome (ARDS) remains a major cause of morbidity and mortality with an overall mortality rate of 30-40%. 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 prostacyclin against pulmonary vascular endothelial leak induced by edemagenic agonists and pathologic mechanical strain associated with ventilator induced lung injury.