Mechanisms of Cigarette Smoke-Induced Acute Lung Injury

 描述(由申请人提供)： 吸烟是导致血管和肺部疾病和死亡的一个重要原因，在退伍军人中这种情况有所增加。全身和肺内皮细胞(EC)损伤都是吸烟(CS)暴露的重要后果。CS与急性呼吸窘迫综合征(ARDS)的发病率增加有关，其特征是内皮通透性增加。我们实验室以前的研究表明，CS增加了小鼠和培养的肺血管内皮细胞的肺血管通透性；这种作用涉及降低粘着斑激酶(FAK)和RhoA GTP酶的激活。然而，参与这一损伤的CS的成分尚不清楚。丙烯醛是一种活性很强的α-不饱和醛， 存在于CS以及吸烟者呼出的呼气冷凝物和血液中。我们的初步数据表明，与CS一样，丙烯醛在体内和体外都能增加肺血管内皮细胞的通透性；破坏肌动蛋白应激纤维、黏附连接和焦点黏附复合体；并降低RhoA和FAK的激活。在体内和体外，通过抑制乙醛应激可以预防CS和丙烯醛引起的肺内皮细胞损伤。我们的总体目标是了解CS诱导的肺内皮细胞功能障碍的机制。我们推测，丙烯醛通过RhoA和FAK在肺内皮细胞中的羰化作用，介导CS诱导的EC功能障碍，导致通透性肺水肿增加。目的1.研究丙烯醛在体内外对肺微血管屏障功能的影响，包括：a.丙烯醛对小鼠肺微血管通透性的影响。2.丙烯醛对RhoA和FAK的羰化和失活的影响及其机制(S)。在体内和培养的肺内皮细胞中，如果抑制蛋白质的羰化可以减轻CS和丙烯醛诱导的血管通透性增加。从吸烟者的血浆中分离到带有EC表面标志的循环细胞外小泡(EV)，我们的初步数据表明，CS暴露的小鼠血浆中循环EV增加。我们的初步数据显示，与用赋形剂处理的EC分离的EV相比，经丙烯醛处理的EC分离的EV显著增加了EC的通透性。因此，我们还假设CS中的丙烯醛通过EV降低了肺内皮细胞屏障功能。目的2.我们将确定循环和肺EV在CS和丙烯醛诱导的肺损伤中的作用，包括：a.CS和丙烯醛暴露所致EV形成的特征。B.CS或丙烯醛暴露小鼠的循环和肺EV对健康小鼠和培养的小鼠肺微血管内皮细胞(LMVEC)肺内皮细胞屏障功能的影响。C.从CS或丙烯醛暴露的LMVEC中分离出的EV对未暴露的LMVEC和纯化的蛋白质中的RhoA和FAK活性以及羰化作用的影响。D.如果抑制蛋白质羰化，可以防止CS或丙烯醛暴露的小鼠EV引起的肺血管损伤。这些研究将全面评估丙烯醛对肺EC和EV的影响，以及它们在CS诱导的肺血管通透性中的作用。更好地了解CS诱导的肺内皮细胞损伤可能会提高对ARDS和更多可能涉及内皮损伤的慢性CS诱导的疾病(如COPD)的了解。

 DESCRIPTION (provided by applicant): Cigarette smoking is an important cause of vascular and pulmonary morbidity and mortality and is increased among veterans. Both systemic and pulmonary endothelial cell (EC) injuries are important consequences of Cigarette Smoke (CS) exposure. CS is associated with an increased incidence of Acute Respiratory Distress Syndrome (ARDS), with the hallmark of increased endothelial permeability. Previous studies from our laboratory indicate that CS increases lung vascular permeability in mice and in cultured lung vascular EC; an effect involving decreased focal adhesion kinase (FAK) and RhoA GTPase activation. However, the components of CS involved in this injury are unknown. Acrolein is a highly reactive a, ß- unsaturated aldehyde that is present in CS and in exhaled breath condensates and blood of smokers. Our preliminary data indicate that acrolein, like CS, increases lung vascular EC permeability in vivo and in vitro; disrupts actin stress fibers, adherens junctions, and focal adhesion complexes; and decreases RhoA and FAK activation. CS- and acrolein-induced lung endothelial injury is prevented by inhibition of aldehyde stress in vivo and in vitro. Our Overall Objective is to understand the mechanism of CS-induced lung endothelial cell dysfunction. We hypothesize that acrolein mediates CS-induced EC dysfunction resulting in increased permeability pulmonary edema via carbonylation of RhoA and FAK in lung endothelial cells. Aim 1. We will determine: The effects of acrolein on lung microvascular barrier function in vivo and in vitro, including: a. The effects f acrolein on mouse lung microvascular permeability. b. The effects of acrolein on RhoA and FAK carbonylation and inactivation and the mechanism(s) of these changes. c. If inhibition of protein carbonylation attenuates CS- and acrolein-induced increased vascular permeability in vivo and in cultured lung EC. Circulating extracellular vesicles (EV) bearing EC surface markers have been isolated from plasma of smokers and our preliminary data indicate that there are increased circulating EV in plasma of mice exposed to CS. Our preliminary data show that EV isolated from EC treated with acrolein significantly increased EC permeability, compared to EV isolated from EC treated with vehicle. Therefore, we also hypothesize that acrolein in CS decreases lung endothelial barrier function via EV. Aim 2. We will determine the role of circulating and lung EV in CS- and acrolein-induced lung injury, including: a. Characterization of formation of EV in response to CS and acrolein exposure. b. The effects of circulating and lung EV from CS- or acrolein-exposed mice on lung EC barrier function in healthy mice and in cultured mouse lung microvascular EC (LMVEC). c. The effect of EV isolated from CS- or acrolein-exposed LMVEC on carbonylation and activities of RhoA and FAK in unexposed LMVEC and in purified proteins. d. If inhibition of protein carbonylation prevents lung vascular injury caused by EV from CS- or acrolein-exposed mice. These studies will comprehensively assess the effects of acrolein on lung EC and EV and their roles in CS-induced lung vascular permeability. Better understanding of CS-induced lung EC injury is likely to enhance understanding of both ARDS and more chronic CS-induced diseases that may involve endothelial injury, such as COPD.