Project 2: Mechanisms of Increased Lung Disease Secondary to Polymorphisms in the ACE gene

目前慢性阻塞性肺病（COPD）的治疗试图改善症状， 防止恶化。研究已经确定了在一个内含子中的插入（I）/缺失（D）多态性， 血管紧张素转换酶基因。D/D纯合子（约占人群的25%）表现出更严重的 慢性阻塞性肺疾病，但机制尚不清楚。我们的主要目标是研究和靶向ACE在 COPD的发展。COPD患者受到慢性炎症的困扰，导致肺 合规肺泡巨噬细胞（AM）是COPD局部炎症反应的关键。ACE是 在AM表面表达，其丰度调节血管紧张素II（ANGII）的水平， 结合AM上的ANGII I型（AT 1）受体。COPD中的炎症引起支气管痉挛，这可能导致 在缺氧中。缺氧，反过来，导致增加血管紧张素Ⅱ，一个关键的炎症介质，创造一个恶性循环。 周期我们的初步数据显示，AM产生对缺氧的炎症反应， AT 1受体阻滞剂氯沙坦。我们还发现肺血管紧张素转换酶和血管紧张素Ⅱ的增加， ACE D/D基因型。我们的数据表明，内含子序列编码microRNA（miRNAs），小RNA 可以调节蛋白质丰度。我们的总体假设是ACE D/D基因型增加 ACE蛋白的翻译，导致缺氧期间ANGII反应过度， 关于COPD目的1：验证ACE基因D/D多态性与ACE升高相关的假设 与I/I受试者相比，活动和肺部炎症。同时，我们将确定这些水平是否预示着 肺顺应性的差异，如通过磁共振弹性成像（MRE）量化的。在目标2中，我们将测试 缺氧导致ACE和ANGII呈剂量依赖性增加的假设在AM中被夸大 D/D和I/I受试者的数据。我们的初步数据表明ACE特异性miRNAs在血管内皮细胞中的表达， 内含子序列目的3将检验D/D受试者中这些miRNAs表达减少的假设， 与I/I受试者相比，导致ACE翻译增加。这将有助于发现以前的 未识别的靶点可能降低肺中的ACE活性。iTarget COBRE将促进这些 通过为职业发展提供强有力的指导和多学科的环境， 科学成长。可视化分子相互作用核心将促进我们的MRE成像分析，而 Molecular Tools Core将提供用于调节miRNA的细胞水平的构建体。我们的蛋白质翻译 研究将受益于与凯滕巴赫实验室加强科学互动。麦克莱伦医生和 Grigoryan将提供蛋白质设计和蛋白质生物化学方面的专业知识。这些研究将确定新的 针对COPD的抗炎策略，包括AT 1受体阻滞剂的潜在再利用 氯沙坦。我们的研究将同时发现调节ACE的其他靶点，从而导致开发 炎症性肺病的新疗法。

Current treatments for chronic obstructive pulmonary disease (COPD) attempt to ameliorate symptoms and prevent exacerbations. Studies have identified an insertion (I)/deletion (D) polymorphism in an intron of the angiotensin converting enzyme (ACE) gene. D/D homozygotes (~25% of the population) exhibit more severe COPD but the mechanism is unclear. Our major goal is to investigate and target the role of ACE in the development of COPD. Patients with COPD are plagued by chronic inflammation, leading to increased lung compliance. Alveolar macrophages (AMs) are critical to the local inflammatory response in COPD. ACE is expressed on the surface of AMs and its abundance regulates levels of angiotensin II (ANGII), which in turn binds the ANGII type I (AT1) receptor on AMs. Inflammation in COPD causes bronchospasm, which can result in hypoxia. Hypoxia, in turn, causes an increase in ANGII, a key inflammatory mediator, creating a vicious cycle. Our preliminary data show that AMs generate an inflammatory response to hypoxia that is blocked by the AT1 receptor blocker, losartan. We also found increased pulmonary ACE and ANGII in subjects with the ACE D/D genotype. Our data suggest that the intronic sequence encodes microRNAs (miRNAs), small RNAs that can regulate protein abundance. Our overarching hypothesis is that the ACE D/D genotype increases translation of ACE protein, leading to an exaggerated ANGII response during hypoxia, increasing the severity of COPD. Aim 1 will test the hypothesis that the ACE D/D polymorphism is associated with elevated ACE activity and lung inflammation compared to I/I subjects. In parallel, we will determine if these levels predict differences in lung compliance, as quantified by magnetic resonance elastography (MRE). In Aim 2, we will test the hypothesis that hypoxia causes a dose-dependent increase in ACE and ANGII that is exaggerated in AMs from D/D versus I/I subjects. Our preliminary data suggest the expression of ACE-specific miRNAs from the intronic sequence. Aim 3 will test the hypothesis that reduced expression of these miRNAs in D/D subjects causes an increase in ACE translation compared to I/I subjects. This will foster discovery of previously unrecognized targets that could decrease ACE activity in the lung. The iTarget COBRE will facilitate these studies by providing strong mentorship for career development and a multidisciplinary environment to promote scientific growth. The Visualizing Molecular Interactions Core will facilitate our MRE imaging analysis, while the Molecular Tools Core will provide constructs for modulating cellular levels of miRNAs. Our protein translation studies will benefit from enhanced scientific interactions with the Kettenbach Laboratory. Drs. McLellan and Grigoryan will offer expertise in protein design and protein biochemistry. These studies will determine novel anti-inflammatory strategies to target COPD, including the potential repurposing of the AT1 receptor blocker losartan. Our studies will simultaneously uncover additional targets regulating ACE, leading to the development of novel therapeutics for inflammatory lung diseases.