Protective role of ANP in the models of acute lung injury

描述(申请人提供)：急性肺损伤、败血症、肺部炎症和呼吸机诱导的肺损伤是与肺血管屏障功能障碍相关的危及生命的疾病，可能导致肺水肿。在这些病理中，肺循环中心钠素(ANP)水平的升高表明其在调节肺损伤过程中具有潜在的作用。尽管ANP对血管张力、血浆容量和肾功能的生理作用已为人所知，但最近的研究发现了新的ANP生物学活性，包括对内皮屏障功能的影响，以及ANP对肺内皮细胞保护作用的分子机制仍有待阐明。我们以前的研究表明，小GTP酶Rac和Rho在调节内皮通透性和肌动蛋白细胞骨架和细胞接触的特异性重塑方面存在相互作用。我们的初步研究表明，ANP对炎症激动剂诱导的内皮细胞和急性肺损伤动物模型的肺内皮细胞屏障功能障碍具有屏障保护作用，并与ANP通过cAMP依赖的蛋白激酶(PKA)下调Rho信号和细胞骨架重构以及新的EPAC-Rap1-Tiam1/Vav2-RAC机制有关。我们推测，ANP可能通过抑制Rho依赖的内皮高通透性途径来减轻与急性肺损伤相关的急性肺内皮细胞功能障碍。我们还假设，ANP可能通过触发PKA和新的EPAC/Rap介导的信号通路，激活依赖RAC的EC屏障保护通路，从而增强受损肺的血管屏障功能。具体目标1将研究EPAC/Rap和PKA介导的机制在激活与ANP屏障保护作用相关的RAC依赖的信号转导中的作用。具体目标2将集中在ANP通过依赖RAC/PAK的微管稳定和调节微管相关的Rho特异性鸟嘌呤核苷酸交换因子gef-H1而诱导Rho下调的分子机制。在败血症和无菌肺损伤的动物模型中，将使用siRNA转基因、ANP基因敲除小鼠和抢救方法来描述ANP在体内减轻急性肺损伤中的潜在作用。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%。肺损伤的急性期以肺泡-毛细血管屏障通透性增加为特征，这允许富含蛋白质的液体进入空气空间，导致肺水肿。利用多学科的生化、细胞生物学和分子方法，这项应用将研究涉及心钠素诱导的肺内皮屏障增强的新的信号通路。由于急性肺损伤的分子基础尚不清楚，目前尚无特异的药物治疗方法，这些研究将加深我们对肺内皮细胞屏障功能调节的了解，并将支持心钠素在急性肺损伤治疗中的潜在治疗意义。

DESCRIPTION (provided by applicant): Acute lung injury, sepsis, lung inflammation, and ventilator-induced lung injury are life-threatening conditions associated with lung vascular barrier dysfunction, which may lead to pulmonary edema. Increased levels of atrial natriuretic peptide (ANP) in lung circulation reported in these pathologies suggest its potential role in modulation of lung injury process. Although physiological effects of ANP on the vascular tone, plasma volume, and renal function are well known, recent studies discovered novel ANP biological activities including effects on endothelial barrier function, and molecular mechanisms of ANP protective effects on pulmonary EC remain to be elucidated. Our previous studies demonstrated reciprocal relations between small GTPases Rac and Rho in the regulation of endothelial permeability and specific remodeling of actin cytoskeleton and cell contacts. Our preliminary studies strongly suggest the barrier protective effects of ANP against lung EC barrier dysfunction induced by inflammatory agonists in endothelial cell models and animal models of acute lung injury and link them to the ANP-induced downregulation of Rho signaling and cytoskeletal remodeling mediated via cAMP-dependent protein kinase (PKA) and novel Epac-Rap1-Tiam1/Vav2- Rac mechanism. We hypothesize that ANP may attenuate acute pulmonary endothelial dysfunction associated with acute lung injury via inhibition of Rho-dependent pathways of endothelial hyper-permeability. We also hypothesize that ANP may enhance the vascular barrier function in the injured lung by triggering PKA and novel Epac/Rap-mediated signaling leading to activation of Rac-dependent pathways of EC barrier protection. Specific Aim 1 will investigate a role of Epac/Rap and PKA-mediated mechanisms in the activation of Rac-dependent signaling associated with ANP barrier protective effects. Specific Aim 2 will focus on molecular mechanisms of ANP-induced Rho downregulation via Rac/PAK-dependent microtubule stabilization and regulation of microtubule-associated Rho-specific guanine nucleotide exchange factor GEF-H1. Specific Aim 3 will use siRNA transfections, ANP knockout mice and rescue approaches in the animal models of septic and aseptic lung injury to delineate potential role of ANP in the alleviation of acute lung injury in vivo. 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 permeability of the alveolar-capillary barrier, which allows an influx of protein-rich fluid into the air spaces, causing pulmonary edema. Using multidisciplinary biochemical, cell biology and molecular approaches, this application will examine novel signaling pathways involved in atrial natriudetic peptide-induced lung endothelial barrier enhancement. As molecular basis of acute lung injury is poorly understood, and no specific pharmacologic therapies are currently available, these studies will enhance our understanding of the regulation of lung endothelial barrier function, and will support potential therapeutic significance of atrial natriuretic peptide in the management of acute lung injury.