Neprilysin and Pulmonary Vascular Remodeling: Cellular and Molecular Mechanisms

脑啡肽酶和肺血管重塑：细胞和分子机制

• 批准号: 8245582
• 负责人: EDWARD CHARLES DEMPSEY
• 金额: --
• 依托单位: VA EASTERN COLORADO HEALTH CARE SYSTEM
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-01-01 至 2014-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8245582
• 关键词: Animal Model; Anti-Inflammatory Agents; Anti-inflammatory; Apoptosis; Arteries; Attenuated; Blood Circulation; Blood Vessels; Breathing; Caliber; Cell Proliferation; Cell Surface Proteins; Cell surface; Cells; Chronic; Chronic Obstructive Airway Disease; Clinical; Complex; Coupling; Development; Dipeptidyl-Peptidase IV; Disease; Distal; Employee Strikes; Etiology; Exercise; Exposure to; Functional disorder; Gene Expression; Gene Expression Regulation; Gene-Modified; Genetic Transcription; Genetically Engineered Mouse; Growth; HIF1A gene; Heart; Heart Diseases; Heart failure; Human; Hypoxia; Inflammation; Inflammatory; Injury; Integral Membrane Protein; Knockout Mice; Laboratories; Lead; Lentivirus Vector; Lung; Lung diseases; Lysosomes; Medial; Mediating; Mediator of activation protein; Membrane; Metalloproteases; Molecular; Mus; Neprilysin; Neuropeptides; Nitrogen; Oxidants; Oxygen; Pathogenesis; Patients; Peptide Hydrolases; Peptides; Peptidyl-Dipeptidase A; Play; Population; Predisposition; Prevention; Process; Property; Proteasome Inhibition; Proteins; Pulmonary Hypertension; Pulmonary artery structure; Pulmonary vessels; Reagent; Regulation; Reporting; Rest; Role; Side; Signal Transduction; Site; Smoke; Smooth Muscle; Smooth Muscle Myocytes; Stimulus; Structure; Structure of parenchyma of lung; Testing; Therapeutic Effect; Vascular Diseases; Vascular remodeling; Veterans; Work; cigarette smoke-induced; cigarette smoking; intercellular communication; interest; migration; mouse model; multicatalytic endopeptidase complex; muscle hypertrophy; novel; novel therapeutics; oxidant stress; pressure; prevent; protective effect; protein degradation; public health relevance; response; tissue/cell preparation; transcription factor

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.

描述（由申请人提供）： 慢性低氧性肺动脉高压（PHTN）是普通人群和退伍军人人群的主要临床问题，并使大多数肺和心脏疾病（包括慢性阻塞性肺病（COPD））复杂化。许多因素，包括香烟烟雾（CS），氧化应激，炎症和最终缺氧，有助于观察到的血管功能障碍和重塑这种形式的慢性PHTN。肺动脉（PA）平滑肌细胞（SMCs）在内/外膜边缘的增殖和迁移到更远的部位是结构变化的突出特征。脑啡肽酶（NEP）是具有细胞表面肽酶活性的跨膜蛋白，其降解选择的促炎和抗炎神经肽，并且可以通过经由肽酶非依赖性机制直接偶联至细胞内蛋白质而参与信号级联。最近也描述了NEP的新底物。有越来越多的证据表明，肺NEP活性和表达下降，以响应慢性CS和缺氧的机制尚未明确。我们现在有初步的证据表明，NEP在晚期COPD患者的肺部也有选择性地减少。NEP敲除小鼠对慢性缺氧有过度的肺血管重塑和PHTN，与野生型相比，常驻PA SMC的生长增加。人类和小鼠NEP的减少在远端血管中最为显著，在远端血管中发生早期PA SMC增殖和迁移。我们还观察到，CS提取物（CSE）和缺氧降低NEP活性和表达在分离的人和小鼠PA SMC。这些观察结果支持了NEP可能对肺循环产生保护作用的观点，并且可能是慢性形式的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和缺氧诱导的PHTN中的作用，在互补的人类和小鼠组织和细胞制剂中。我们将检验总体假设：NEP降低容易导致慢性肺部疾病（如COPD）中肺血管重塑增加。慢性CS（如缺氧）降低NEP活性和/或表达，最终导致驻留PA SMC增殖增加、血管功能障碍、肺血管重塑和PHTN。相比之下，增加的NEP至少部分地通过减少驻留PA SMC的生长来保护肺脉管系统免于响应于慢性CS（和缺氧）的肺血管重塑和PHTN的发展。我们的目标是：1）确定NEP活性和表达在由CS引起的人类和小鼠肺血管重塑中的参与; 2）确定CS对NEP活性和表达发挥减弱作用的关键机制（关注活性氧和氮物质[ROS/RNS]，其导致溶酶体和蛋白酶体的蛋白质降解以及由缺氧诱导的转录因子HIF-1和HIF-2介导的基因表达降低）。该项目将利用来自“对照”患者和严重COPD患者的人肺组织和PA SMC，基因工程小鼠模型和独特的慢病毒载体来研究CS诱导的肺血管重塑。我们将依次研究氧化应激如何灭活NEP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降低NEP基因表达。这些研究可能有助于增加我们对控制慢性CS和缺氧诱导的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中的NEP），以限制或逆转这一重要的临床问题。 公共卫生相关性： 项目叙述：肺动脉高压，或肺血管中的高压，通常是由暴露于香烟烟雾和低氧水平（缺氧）引起的;它是退伍军人群体中的主要临床问题，使肺部疾病如COPD和心力衰竭复杂化。肺动脉壁中的平滑肌细胞可能会因烟雾和缺氧而异常生长，导致血管直径减小，右侧心脏的工作增加。细胞表面蛋白Neprilysin可能在防止这种异常生长中发挥重要作用。该项目将利用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可以在多大程度上预防这种疾病。这些研究可能有助于增加我们对控制吸烟和缺氧诱导的肺血管疾病易感性的机制的理解，并可能最终导致识别这一重要临床问题的新治疗策略。