Mechanisms of Cigarette Smoke-Induced Acute Lung Injury

 描述（由申请人提供）： 吸烟是血管和肺部发病率和死亡率的一个重要原因，在退伍军人中增加。全身和肺内皮细胞（EC）损伤是香烟烟雾（CS）暴露的重要后果。CS与急性呼吸窘迫综合征（ARDS）的发病率增加相关，其特征是内皮通透性增加。我们实验室以前的研究表明，CS增加小鼠和培养的肺血管EC的肺血管通透性;这种作用涉及降低粘着斑激酶（FAK）和RhoA GT3激活。然而，参与这种损伤的CS成分尚不清楚。丙烯醛是高度反应性的α，β-不饱和醛， 存在于CS和吸烟者的呼出气冷凝物和血液中。我们的初步数据表明，丙烯醛，CS一样，增加肺血管EC渗透性在体内和体外;破坏肌动蛋白应力纤维，adherens连接，和黏着斑复合物;和减少RhoA和FAK激活。CS和丙烯醛诱导的肺内皮损伤是通过抑制醛应激在体内和体外。我们的总体目标是了解CS诱导肺内皮细胞功能障碍的机制。我们推测丙烯醛介导CS诱导的EC功能障碍，导致肺内皮细胞中RhoA和FAK通过羰基化增加通透性肺水肿。目标1.我们将确定：丙烯醛在体内和体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。B.丙烯醛对RhoA和FAK羰基化和失活的影响及其机制。C.如果抑制蛋白质羰基化减弱CS和丙烯醛诱导的血管通透性增加在体内和培养的肺EC。携带EC表面标记的循环细胞外囊泡（EV）已从吸烟者的血浆中分离出来，我们的初步数据表明，暴露于CS的小鼠血浆中的循环EV增加。我们的初步数据表明，从EC处理的丙烯醛分离的EV显着增加EC的渗透性，与EV分离的EC处理的车辆相比。因此，我们还假设CS中的丙烯醛通过EV降低肺内皮屏障功能。目标二。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：响应于CS和丙烯醛暴露的EV形成的表征。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EC（LMVEC）肺EC屏障功能的影响。C.从CS或丙烯醛暴露的LMVEC中分离的EV对未暴露的LMVEC和纯化蛋白质中RhoA和FAK的羰基化和活性的影响。D.如果抑制蛋白质羰基化可以防止CS或丙烯醛暴露小鼠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的影响及其在CS诱导的肺血管通透性中的作用。更好地了解CS诱导的肺EC损伤可能会增强对ARDS和可能涉及内皮损伤的慢性CS诱导疾病（如COPD）的理解。

 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.