Mechanisms of Prostacyclin-Mediated Lung Endothelial Barrier Protection

描述（由申请人提供）：Prostacyclin （PGI2）是环加氧酶的产物，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PGI2对高氧性肺损伤或缺血/再灌注引起的肺损伤具有显著的保护作用，PGI2稳定代谢物水平的增加与较轻的呼吸窘迫有关。然而，PGI2对肺内皮细胞（EC）保护作用的分子机制尚不清楚。我们发表的数据表明，小gtpase Rac和Rho通过肌动蛋白细胞骨架和细胞接触的特异性重塑，在调节内皮通透性方面发挥了相互作用。我们的初步研究强烈提示PGI2对人肺EC具有屏障保护作用，并将其与camp依赖性蛋白激酶（PKA）和新的Epac-Rap1-Tiam1/Vav2-Rac机制介导的rac依赖性肌动蛋白细胞骨架重塑和粘附连接增强（AJ）联系起来。我们的研究还表明，PGI2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PGI2通过激活PKA-和epac介导的信号通路，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依赖通路，从而对肺EC发挥屏障保护作用。我们还假设PGI2可能通过抑制内皮细胞高通透性的rho依赖通路来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。Specific Aim #1将研究PKA-和cAMP/- epac - rap1 - tiam / vav2 - rac依赖pgi2诱导肺EC细胞骨架重塑和屏障保护的机制。Specific Aim #2将研究pgi2诱导的AJ重塑与pgi2诱导的屏障保护相关的机制，并通过Epac1、Tiam1、Vav2、Rap1和Rac的局部募集来研究ve -cadherin介导的Rac调控。特异性目标#3将探讨PGI2诱导的Rho信号调节在凝血酶诱导的肺EC屏障功能障碍细胞培养模型和呼吸机诱导的肺损伤动物模型中的保护作用。我们相信这些研究可能会发现新的蛋白靶点，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。

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.