AMPKα2调控肺动脉平滑肌细胞表型转化在吸烟肺血管重构中的作用和机制研究

Cigarette smoke-induced oxidative stress is the main cause of pulmonary arterial hypertension(PAH) associated with chronic obstructive pulmonary disease(COPD). Phenotypic modulation of pulmonary artery smooth muscle cell(PASMC) accompanied by pulmonary vascular remodeling plays a pivotal role in the pathogenesis of PAH. Recent studies suggest that the AMP-activated protein kinase (AMPK) is a potential target for suppressing oxidative stress and controlling inflammation, albeit through different functions of two isoforms of the AMPK (α1 and α2). Based on our preliminary study, we hypothesize that AMPKα2 (but not α1) specifically regulates cigarette smoke-induced modulation of PASMC phenotype and their proliferation. In this study, AMPKα1 and α2 knockout mice will be analyzed, as compared with the wild-type mice, for their susceptibility to cigarette smoke-induced pulmonary vascular remodeling in the context of a whole animal. In conjugation with the use of AMPK agonist and inhibitor, by assessing the severity of vessel wall oxidative stress and vascular remodeling, we will establish the role of AMPKα2 in cigarette smoke-induced pulmonary vascular remodeling, and further evaluate the effectiveness of using AMPK activator to reverse this pathological process. Finally, using a panel of in vitro assays based on primarily cultured PASMC isolated from the AMPK isoform knockout mice exposed to cigarette smoke extract(CSE) and nicotine respectively, we will demonstrate molecular mechanisms, at levels of redox signaling, and gene transcription, through which AMPKα2 regulates phenotypic modulation of PASMC under oxidative stress. Finally, our results will lead to the development of novel therapeutic strategy that targets specific AMPK isoform to reverse or prevent early-stage pulmonary hypertension associated with COPD.

尽管已公认香烟暴露致高氧化应激状态直接导致COPD肺血管重构及肺动脉高压形成，但其确切机制并未阐明。肺动脉平滑肌细胞（PASMC）表型转化是肺血管重构的关键始动环节，我们前期工作发现抗氧化应激靶点AMPK亚型AMPKα2激活后抑制香烟提取物诱导的PASMC表型转化和过度增殖，在此基础上，本项目拟选取吸烟COPD患者肺组织标本，利用野生型、分别敲除AMPKα1和AMPKα2基因小鼠制备香烟熏制肺血管重构模型，从整体水平揭示AMPKα2活性水平与氧化应激状态、肺血管重构及肺动脉高压严重程度的相关性；评价活化AMPK或特异性激活AMPKα2对香烟致肺血管重构和肺动脉高压的干预作用。并从上述模型中分离PASMC给予香烟提取物/尼古丁处理，从氧化还原信号通路、转录水平探讨AMPKα2抑制PASMC表型转化参与肺血管重构的分子机制，旨在揭示COPD肺动脉高压的发病机制，为确立有效的治疗手段提供新思路。

肺动脉高压是慢性阻塞性肺病（COPD）的重要并发症之一，直接影响患者的预后，然而目前对于COPD相关肺动脉高压尚缺乏疗效可靠的防治手段。吸烟是COPD的主要危险因素，动物模型证实香烟暴露可直接导致肺血管重构和肺动脉高压形成。本项目选取吸烟人肺组织标本，制备香烟致肺血管重构动物模型，从调控肺动脉平滑肌细胞（PASMCs）表型转化入手，探讨AMPK信号通路参与香烟暴露致肺血管重构的机制，明确利用AMPK信号通路活化药物二甲双胍（Metformin）是否逆转吸烟肺血管重构，为吸烟COPD患者肺血管重构及肺动脉高压的发病机制的阐明及探索防治策略提供新思路。本项目采用回顾性病例对照分析，证实吸烟、血清炎症因子水平增高是COPD患者肺动脉高压发生的独立危险因素。动物实验证实大鼠腹腔注射香烟提取物（CSE）引起肺小动脉重构，Metformin能显著减轻CSE诱导肺组织和肺小血管周围的炎症反应，抑制肺血管重构。体外研究发现5%CSE诱导PASMCs从收缩型向合成型（表型转化）和过度增殖，伴有AMPK磷酸化水平下降，mTOR磷酸化水平增加，Metformin可显著逆转上述过程；慢病毒载体介导的siRNA技术分别下调PASMCs中AMPKα1和α2表达，发现AMPKα2下调后抵消Metformin抑制CSE诱导PASMCs的表型转化；而AMPKα1和α2下调均部分抵消Metformin抑制CSE诱导PASMCs的增殖，提示AMPKα1和α2不同亚型在CSE诱导PASMCs表型转化和增殖中扮演不同的角色；Metformin抑制PASMCs表型转化的机制依赖于AMPKα2的活化，mTOR磷酸化抑制。本项目从动物和细胞水平获得Metformin激活AMPK或激活AMPKα2能够阻止/逆转吸烟致肺血管重构的证据，为Metformin用于临床早期干预COPD相关肺动脉高压提供理论依据。