HDAC6 regulates cigarette smoke-induced endothelial barrier dysfunction and lung injury

香烟烟雾（CS）影响全球10亿人，并导致600万人过早死亡。 每年都有相关疾病。急性肺损伤（ALI）/急性呼吸窘迫综合征（ARDS）是一种生命- 死亡率高达40%新兴的流行病学研究表明， 肺部感染，并增加ALI/ ARDS的可能性。然而，不利影响的机制， ALI的CS未知。因此，迫切需要制定预防和治疗吸烟者ALI/ ARDS的策略。的 本申请的目的是使用人肺微血管内皮细胞（LMVEC）和临床前 动物模型，以确定CS损害肺内皮屏障功能的机制， 增加细菌感染后对ALI的脆弱性，并制定预防和恢复策略 易感人群中的内皮屏障功能和ALI。这项研究使用了最先进的技术， 全面的方法来解决CS增加内皮细胞通透性的中心假设 并通过一种机制使肺在感染后易患ALI，在该机制中，来自CS和线粒体的氧化剂 导致Akt氧化和随后的组蛋白去乙酰化酶6（HDAC 6）的GSK 3 β依赖性激活， 导致微管去乙酰化，导致动力蛋白相关蛋白（Drp）1易位到线粒体， 最终导致线粒体分裂和功能障碍，随后释放线粒体损伤- 相关分子模式（mtDAMP）。目的1：我们将首先确定HDAC 6在CS引发中的作用， 使用HDAC 6基因敲除小鼠检测铜绿假单胞菌感染后的ALI，内皮特异性 体内慢病毒HDAC 6 miRNA沉默，以及从HDAC 6敲除小鼠中分离的LMVEC;然后我们将 确定CS暴露导致HDAC 6激活的机制。目标2：我们将确定 HDAC 6在CS诱导的线粒体分裂和功能障碍中的作用以及它们在介导增加的 内皮通透性和铜绿假单胞菌感染后ALI的启动。我们将首先描述 CS暴露对Drp 1易位和线粒体分裂，融合，功能和mtDNA释放的影响， 体外和体内;然后我们将确定阻断HDAC 6或α-微管蛋白脱乙酰化对不良反应的影响。 CS在体外和体内对线粒体的影响;最后，我们将确定线粒体分裂的作用， CS诱导的体外内皮通透性增加和CS引发的线粒体ROS和mtDAMPs 体内ALI。本研究将为CS对肺内皮屏障功能的影响提供新的见解。 HDAC 6、线粒体分裂和mtDAMP之间的联系的建立可能导致新的治疗方法。 CS诱导的肺血管损伤和潜在的体循环血管损伤的方法。

Cigarette smoke (CS) affects 1 billion people worldwide and causes 6 million premature deaths from associated diseases each year. Acute lung injury (ALI)/ acute respiratory distress syndrome (ARDS) is a life- threatening condition with 40% mortality. Emerging epidemiology studies have shown that CS predisposes lungs to infections and increases the likelihood of ALI/ ARDS. However, the mechanism of adverse effects of CS on ALI is unknown. There is an urgent need for strategies to prevent and treat ALI/ ARDS in smokers. The objectives of this application is to use human lung microvascular endothelial cells (LMVEC) and preclinical animal models to identify mechanisms by which CS impairs pulmonary endothelial barrier function and increases vulnerability to ALI after bacterial infection and to develop strategies to prevent and restore endothelial barrier function and ALI in susceptible populations. This study uses state-of-art technologies and comprehensive approaches to address a central hypothesis that CS increases endothelial cell permeability and predisposes lungs to ALI after infection by a mechanism in which oxidants from CS and mitochondria cause Akt oxidation and subsequent GSK3β-dependent activation of histone deacetylase 6 (HDAC6), this results in microtubule deacetylation, leading to translocation of dynamin-related protein (Drp)1 to mitochondria, culminating in mitochondrial fission and dysfunction and subsequent release of mitochondrial damage- associated molecular patterns (mtDAMPs). Aim 1: We will first determine the role of HDAC6 in CS priming for ALI after Pseudomonas aeruginosa infection by using global HDAC6 knockout mice, endothelium-specific lentiviral HDAC6 miRNA silencing in vivo, and LMVEC isolated from HDAC6 knockout mice; we will then determine the mechanism by which CS exposure leads to HDAC6 activation. Aim 2: We will determine the role of HDAC6 in CS-induced mitochondrial fission and dysfunction and their roles in mediating increased endothelial permeability and priming for ALI after P. aeruginosa infection. We will first characterize the effects of CS exposure on Drp1 translocation and on mitochondrial fission, fusion, function, and release of mtDNA in vitro and in vivo; we will then determine the effect of blocking HDAC6 or α-tubulin deacetylation on the adverse effects of CS on mitochondria in vitro and in vivo; finally, we will determine the roles of mitochondrial fission, mitochondrial ROS and mtDAMPs in CS-induced increased endothelial permeability in vitro and CS priming for ALI in vivo. This study will provide innovative insights into the effect of CS on lung endothelial barrier function. Establishment of the links among HDAC6, mitochondrial fission and mtDAMPs may result in new therapeutic approaches to CS-induced vascular injury in the lung and potentially also in the systemic circulation.