Mechanisms of Prostacyclin-Mediated Lung Endothelial Barrier Protection

描述(申请人提供)：前列环素(PGI2)是环氧合酶的产物，参与调节血管功能、伤口修复、炎症过程和急性肺损伤。雾化吸入PGI2对高氧性肺损伤或缺血/再灌流所致的肺损伤具有明显的保护作用，且PGI2稳定代谢产物水平的升高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GTP酶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起着相互作用。我们的初步研究表明，PGI2对人肺内皮细胞具有屏障保护作用，并与Rac依赖的肌动蛋白细胞骨架重塑和通过cAMP依赖的蛋白激酶(PKA)和新的EPAC-Rap1-Tiam1/Vav2-Rac机制介导的黏附连接(AJ)增强有关。我们的研究还表明，PGI2对凝血酶诱导的肺内皮细胞屏障功能障碍具有有效的保护作用。我们推测，PGI2通过激活PKA和EPAC介导的信号通路对肺内皮细胞发挥屏障保护作用，从而激活RAC依赖的EC屏障保护通路，增强外周肌动蛋白、细胞骨架和黏附连接。我们还假设PGI2可能通过抑制Rho依赖的内皮高通透性通路来减轻与呼吸机诱导的肺损伤(VILI)相关的急性肺内皮细胞屏障功能障碍。具体目标#1将研究依赖PKA和cAMP/-EPAC-Rap1-Tiam/Vav2-Rac的机制，这些机制是PGI2诱导的肺内皮细胞骨架重塑和屏障保护的基础。具体目的#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，并研究VE-钙粘附素通过局部募集Epac1、Tiam1、Vav2、Rap1和Rac对RAC的调节。具体目的#3将在凝血酶诱导的肺内皮细胞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，探讨PGI2对Rho信号的调节作用。我们相信，这些研究可能确定新的蛋白质靶点，并提出预防与急性肺炎症和损伤相关的肺血管屏障功能障碍的新疗法。 公共卫生相关性：急性呼吸窘迫综合征(ARDS)仍然是发病率和死亡率的主要原因，总死亡率为30%-40%。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。前列环素对水肿性激动剂引起的肺血管内皮细胞渗漏和呼吸机所致肺损伤相关的病理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.