Neprilysin and Pulmonary Vascular Remodeling: Cellular and Molecular Mechanisms

描述(由申请人提供)： 项目摘要慢性缺氧性肺动脉高压(PHTN)在普通人群和退伍军人中是一个主要的临床问题，并合并包括慢性阻塞性肺疾病(COPD)在内的大多数肺和心脏疾病。许多因素，包括香烟烟雾(CS)、氧化应激、炎症和最终的缺氧，导致了这种形式的慢性PHTN的血管功能障碍和重构。结构改变的显著特征是内/外膜交界处的肺动脉(PA)平滑肌细胞(SMC)的增殖和向更远端的迁移。奈普利辛(neprilysin，NEP)是一种具有细胞表面肽酶活性的跨膜蛋白，可降解特定的促炎和抗炎神经肽，并可能通过不依赖于肽酶的机制直接与细胞内蛋白偶联，参与信号转导。最近还描述了NEP的新型底物。越来越多的证据表明，慢性CS和低氧引起肺NEP活性和表达下降，其机制尚不清楚。我们现在有初步证据表明，NEP在晚期COPD患者的肺中也选择性地减少。NEP基因缺失的小鼠对慢性缺氧有夸大的肺血管重塑和PHTN反应，与野生型小鼠相比，常驻PA SMC的生长速度加快。人和小鼠NEP的下降在发生早期PA SMC增殖和迁移的远端血管中最为显著。我们还观察到，CS提取物(CSE)和低氧可降低人和小鼠PA-SMC中NEP的活性和表达。这些观察结果支持这样一种观点，即NEP可能对肺循环起到保护作用，并且可能是慢性PHTN的疾病修饰基因。为了扩展这些观察，我们将研究NEP在慢性CS和低氧诱导的PHTN中的作用，在互补的人类和小鼠组织和细胞准备中。我们将检验总体假设：在慢性肺疾病，如COPD中，NEP减少易导致肺血管重构增加。慢性CS(如低氧)降低NEP的活性和/或表达，最终导致驻留的PA SMC增殖增加、血管功能障碍、肺血管重构和PHTN。相反，增加的NEP保护肺血管系统免受慢性CS(和缺氧)所致的肺血管重塑和PHTN的发展，至少部分是通过减少常驻的PA SMC的生长来实现的。我们的目标是：1)确定NEP活性和表达在CS所致的人和小鼠肺血管重塑中的作用；2)确定CS减弱NEP活性和表达的关键机制(重点关注活性氧和氮物种[ROS/RNS]导致溶酶体和蛋白酶体降解蛋白质，以及低氧诱导转录因子HIF-1和HIF-2介导的基因表达下降)。该项目将利用人类肺组织和来自“对照”患者和严重COPD患者的PA SMCs、基因工程小鼠模型和独特的慢病毒载体来研究CS诱导的肺血管重构。我们将依次研究氧化应激如何失活NEP多肽酶活性，启动溶酶体和蛋白酶体中NEP蛋白的降解，然后通过诱导/激活HIF-1和HIF-2来降低NEP基因的表达。这些研究可能有助于我们加深对控制慢性CS和低氧诱导的PHTN易感性的机制的理解，也可以确定新的治疗策略(如直接或间接增加肺内NEP)来限制或逆转这一重要的临床问题。 公共卫生相关性： 项目简介：肺高压，或肺血管高压，通常是由于暴露在香烟烟雾和低氧水平(缺氧)中引起的；这是退伍军人的一个主要临床问题，使慢性阻塞性肺病和心力衰竭等肺部疾病复杂化。烟雾和低氧可能会导致肺动脉壁中的平滑肌细胞异常生长，导致血管直径缩小，增加右侧心脏的做功。一种名为Neprilysin的细胞表面蛋白可能在防止这种异常生长方面发挥重要作用。该项目将利用来自肺动脉高压患者的肺组织、人类血管细胞、基因工程小鼠模型和独特的试剂来确定Neprilysin对这种疾病过程的保护程度。这些研究可能有助于增加我们对控制吸烟和低氧所致肺血管疾病易感性的机制的理解，并最终可能导致对这一重要临床问题的新治疗策略的确定。

DESCRIPTION (provided by applicant): PROJECT ABSTRACT Chronic hypoxic pulmonary hypertension (PHTN) is a major clinical problem, in the general and Veteran populations, and complicates most lung and heart disorders, including chronic obstructive pulmonary disease (COPD). Many factors, including cigarette smoke (CS), oxidant stress, inflammation and eventually hypoxia, contribute to the observed vascular dysfunction and remodeling in this form of chronic PHTN. Proliferation of pulmonary artery (PA) smooth muscle cells (SMCs) at the medial/adventitial border and migration to more distal sites are prominent features of the structural change. Neprilysin (NEP) is a transmembrane protein with a cell surface peptidase activity that degrades select pro- and anti-inflammatory neuropeptides and may take part in signaling cascades by directly coupling to intracellular proteins via peptidase-independent mechanisms. Novel substrates of NEP have also recently been described. There is growing evidence that lung NEP activity and expression decrease in response to chronic CS and hypoxia by as yet unknown mechanisms. We now have preliminary evidence that NEP is also selectively reduced in lungs of patients with advanced COPD. NEP null mice have exaggerated pulmonary vascular remodeling and PHTN in response to chronic hypoxia, and resident PA SMCs have increased growth compared to their wild type counterparts. The decrease in human and mouse NEP is most striking in distal vessels where early PA SMC proliferation and migration occurs. We have also observed that CS extract (CSE) and hypoxia decrease NEP activity and expression in isolated human and mouse PA SMCs. These observations support the idea that NEP may exert a protective effect on the lung circulation and could be a disease modifying gene for chronic forms of PHTN. To extend these observations, we will investigate the role of NEP in chronic CS- and hypoxia-induced PHTN, in complementary human and mouse tissue and cell preparations. We will test the Overall Hypothesis that: decreased NEP predisposes to the development of increased pulmonary vascular remodeling in chronic lung disorders, like COPD. Chronic CS (like hypoxia) reduces NEP activity and/or expression, ultimately leading to increased proliferation of resident PA SMCs, vascular dysfunction, pulmonary vascular remodeling, and PHTN. In contrast, increased NEP protects the lung vasculature from the development of pulmonary vascular remodeling and PHTN in response to chronic CS (and hypoxia) at least in part, by decreasing the growth of resident PA SMCs. Our Aims are: 1) determine the involvement of NEP activity and expression in human and mouse pulmonary vascular remodeling due to CS; 2) determine key mechanisms by which CS exerts an attenuating effect on NEP activity and expression (focus on reactive oxygen and nitrogen species [ROS/RNS] leading to protein degradation by lysosomes and proteasomes and decreased gene expression mediated by the hypoxia-induced transcription factors HIF-1 and HIF-2). This project will draw on human lung tissue and PA SMCs from 'control' patients and those with severe COPD, genetically engineered mouse models, and unique lentiviral vectors to study CS-induced pulmonary vascular remodeling. We will sequentially examine how oxidant stress inactivates NEP peptidase activity, initiates degradation of NEP protein in the lysosome and proteasome, and later decreases NEP gene expression by induction/activation of HIF-1 and HIF-2. These studies may help increase our understanding of mechanisms that control susceptibility to chronic CS- and hypoxia-induced PHTN and could also identify new therapeutic strategies (like directly or indirectly increasing NEP in the lung) to limit or reverse this important clinical problem. PUBLIC HEALTH RELEVANCE: Project Narrative: Pulmonary hypertension, or high pressure in the lung blood vessels, is often caused by exposure to cigarette smoke and low oxygen levels (hypoxia); it is a major clinical problem in the Veteran population, complicating lung disorders like COPD and heart failure. Smooth muscle cells in the walls of lung arteries may grow abnormally in response to smoke and hypoxia, leading to a reduction in vessel diameter and increased work for the right side of the heart. A cell surface protein, Neprilysin, may play an important role in preventing this abnormal growth. This project will draw on lung tissue from patients with pulmonary hypertension, human vascular cells, genetically engineered mouse models, and unique reagents to determine the extent to which Neprilysin can protect against this disease process. These studies may help to increase our understanding of mechanisms that control susceptibility to smoke and hypoxia-induced pulmonary vascular disease and may ultimately lead to the identification of new therapeutic strategies for this important clinical problem.