Mechanisms of Prostacyclin-Mediated Lung Endothelial Barrier Protection

描述（由申请方提供）：开发治疗急性肺损伤（ALI）和成人呼吸窘迫综合征（ARDS）的有效疗法仍然是一项具有挑战性的任务。许多用于测试新型保护剂的实验模型在ALI诱导期间利用预防性或同时治疗，而治疗后代表更临床相关的干预。与药物预处理阻断ALI发作相比，药物给药时机的这种差异可能对治疗效率和特定分子机制的激活产生显著影响，这些分子机制指导正在进行的损伤的消退。该提案将填补这一空白，并探索FDA批准的前列环素（PC）类似物伊洛前列素在体外和体内脓毒性ALI模型中的后治疗效果。炎症和内皮细胞（EC）通透性增加在ALI的病理生理学中起主要作用。在上一个周期的建议，我们的特点是第一次在无菌模型的呼吸机诱导的肺损伤PC介导的保护的分子机制。我们的初步研究表明，PC预处理对LPS诱导的肺部炎症和血管渗漏具有有效的保护作用。本提案将研究PC后处理在由革兰氏阳性热灭活金黄色葡萄球菌（HKSA）引起的脓毒性ALI的细胞培养和动物模型中的作用。我们推测Rap 1 GTdR信号通过促进EC屏障修复和抑制炎症性内皮细胞活化在PC诱导的ALI消退加速中起双重作用。Aim-1将评估PC后处理的效果，并确定Rap 1在加速HKSA挑战EC屏障恢复中的作用。目的-2将定义参与EC屏障恢复的Rap 1下游的分子机制。我们将研究Rap 1效应子KRIT 1和RIAM在增强EC粘附结构和重建EC屏障所必需的外周细胞骨架中的作用。目的-3将研究PC后处理对Rap 1、KRIT 1和RIAM刺激在下调HKSA诱导的肺EC激活中的作用。目的-4将阐明Rap 1，KRIT 1和Riam在PC促进的ALI恢复体内使用“功能丧失”和“功能获得”的分子方法和小鼠遗传模型的具体作用。这些研究将描述新的保护机制，并确定新的蛋白质靶点，用于预防与急性肺损伤相关的肺血管屏障功能障碍的未来治疗。 公共卫生相关性：成人呼吸窘迫综合征（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。目前，开发有效的治疗ALI/ARDS的疗法是一项重大挑战。本研究将探讨前列环素对肺血管内皮功能障碍的保护作用的分子机制，并首次描述革兰氏阳性脓毒性ALI模型中恢复的新机制。这些研究将扩大我们对导致ALI消退的分子机制的了解，并可能确定药物治疗的新靶点。

DESCRIPTION (provided by applicant): Development of effective therapies for treatment of acute lung injury (ALI) and adult respiratory distress syndrome (ARDS) remains a challenging task. Many experimental models for testing of novel protective agents utilize preventive or concurrent treatment during ALI induction, while post-treatment represents more clinically relevant intervention. Such differences in the timing of drug administration may have dramatic impact on the efficiency of treatment and activation of specific molecular mechanisms directing resolution of ongoing injury in contrast to blocking onset of ALI by drug pretreatment. This proposal will fill this void and explore effects of post-treatment with FDA-approved prostacyclin (PC) analog iloprost in the in vitro and in vivo septic ALI models. Inflammation and increased endothelial cell (EC) permeability play a major role in the pathophysiology of ALI. During the previous cycle of this proposal, we characterized for the first time the molecular mechanisms of PC-mediated protection in aseptic model of ventilator induced lung injury. Our preliminary studies suggest potent protective effects of PC pretreatment against LPS-induced lung inflammation and vascular leak. This proposal will investigate effects of PC post-treatment in cell culture and animal models of septic ALI caused by Gram-positive heat-inactivated Staphylococcus Aureus bacteria (HKSA). We hypothesize that signaling by Rap1 GTPase plays a dual role in PC-induced acceleration of ALI resolution via promotion of EC barrier repair and suppression of inflammatory endothelial activation. Aim-1 will evaluate effects of PC post-treatment and define a role of Rap1 in acceleration of barrier recovery in HKSA challenged EC. Aim-2 will define molecular mechanisms downstream of Rap1 involved in EC barrier recovery. We will study a role of Rap1 effectors KRIT1 and RIAM in enhancement of EC adhesive structures and peripheral cytoskeleton essential for re-establishment of EC barrier. Aim-3 will study a role of Rap1, KRIT1 and RIAM stimulation by PC post-treatment in downregulation of HKSA-induced pulmonary EC activation. Aim-4 will elucidate specific role of Rap1, KRIT1 and Riam in PC-facilitated ALI recovery in vivo using "loss of function" and "gain of function" molecular approaches and mouse genetic models. These studies will characterize novel protective mechanisms and identify new protein targets for future therapies aimed at prevention of the pulmonary vascular barrier dysfunction associated with acute lung injury. PUBLIC HEALTH RELEVANCE: Adult 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. Development of effective therapies for ALI/ARDS treatment currently represents major a challenge. This study will investigate molecular mechanisms underlying protective effects of prostacyclin against pulmonary vascular endothelial dysfunction and characterize for the first time novel mechanisms of recovery in the model of Gram-positive septic ALI. These studies will expand our knowledge about molecular mechanisms leading to ALI resolution and may identify new targets for drug therapies.