Molecular Characterization of NoxA1 in VSMC NADPH Oxidase Activation

VSMC NADPH 氧化酶激活中 NoxA1 的分子表征

基本信息

批准号：
8845598
负责人：
MARSCHALL S. RUNGE
金额：
$ 15.11万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-07-20 至 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8845598
关键词：
Affect Agonist Angioplasty Animal Model Aorta Apolipoprotein E Arterial Fatty Streak Arterial Injury Arteries Atherosclerosis Attenuated Blood Pressure Blood Vessels Cardiac Cardiovascular Diseases Carotid Arteries Catalytic Domain Cell Communication Cell Culture Techniques Cell Line Complement Cytochromes b Data Disease Dissection Electrons Endothelium Enzymes Event Exhibits Family Functional disorder Gene Expression Generations Goals Growth Growth Factor Homologous Gene Human Hyperplasia Hypertension Infection Inflammatory K-562 Laboratories Lead Leukocytes Lipids Medial Membrane Methodology Molecular Mus Mutation NADP NADPH Oxidase Oxidases Oxidation-Reduction Oxidative Stress Oxygen Phosphorylation Phosphorylation Site Plasma Play Post-Translational Protein Processing Production Protein Kinase Reactive Oxygen Species Reagent Regulation Research Respiratory Burst Rodent Role Serine Signal Transduction Smooth Muscle Myocytes Stimulus Stroke Structure Superoxides Therapeutic Time Tunica Adventitia Vascular Diseases Vascular Proliferation Ventricular Function atherogenesis attenuation base deletion analysis genetic approach genetic regulatory protein human CYBA protein in vivo inhibitor/antagonist injured insight member neointima formation neutrophil cytosol factor 40K neutrophil cytosol factor 67K novel novel strategies overexpression protein protein interaction response restenosis small hairpin RNA small molecule libraries transcription factor vascular smooth muscle cell proliferation

项目摘要

DESCRIPTION (provided by applicant): Extensive evidence from animal models and human studies supports the hypothesis that NADPH oxidase derived oxidative stress is a major contributor to the pathophysiology of cardiovascular diseases. In particular, dysregulation of Nox1 NADPH oxidase has been implicated in many vascular diseases including atherosclerosis, aortic dissection, hypertension and stroke. Although molecular mechanisms activating the Nox2 (gp91phox) NADPH oxidase have been elucidated, the events that lead to dysregulation of Nox1 NADPH oxidase activity are not well understood. Our prior studies and preliminary data support the notion that NoxA1, the homologue of p67phox, is expressed in mouse and human vascular smooth muscle cells (VSMC) and is a key regulator of Nox1 NADPH oxidase activity. NoxA1-dependent reactive oxygen species (ROS) play an essential role in agonist-induced VSMC proliferation and growth-promoting, redox-sensitive protein kinase activation. NoxA1 is upregulated in aortas and atherosclerotic lesions of ApoE-/- mice and in intimal and medial VSMC of human early carotid atherosclerotic lesions. In this proposal we will investigate how NoxA1 and p47phox interaction regulates Nox1 NADPH oxidase activity in VSMC, the mechanisms that regulate NoxA1 expression and post translational modifications and their role in Nox1 NADPH oxidase activation. We will also determine the role of NoxA1-dependent ROS production in restenosis and atherosclerosis. We will screen a small molecule library to identify potential inhibitors of Nox1 NADPH oxidase activity to complement the genetic approaches. To accomplish these goals, we have assembled a unique set of reagents and methodologies including NoxA1-/-, ApoE-/-/NoxA1-/- and [SM22α-CreKI/+/NoxA1f/+] mice, Nox1y/-, p47phox-/- and NoxA1-/- mouse aortic VSMC cultures, adenoviral NoxA1 overexpression and shRNA constructs and a functional AlphaScreen and a 100,000 member library of small molecules for identifying potential Nox1 NADPH oxidase inhibitors. Our research strategy includes the following three specific aims. Aim 1: Define the molecular mechanisms of NoxA1-dependent NADPH oxidase activity in agonist-induced ROS generation in mouse and human aortic VSMC. Aim 2: Determine the effect of NoxA1 deficiency in restenosis and atherosclerosis. Aim 3: Identify novel and specific NoxA1 and p47phox interaction inhibitors as means to regulating Nox1 activity. Together, these studies will establis the role of VSMC Nox1 NADPH oxidase in vascular pathophysiology and potentially identify strategies/compounds for the regulation of this enzyme.

描述（由申请人提供）：来自动物模型和人类研究的大量证据支持以下假设：NADPH氧化酶衍生的氧化应激是导致心血管疾病病理生理学的主要因素。特别是，NOX1 NADPH氧化酶的失调与许多血管疾病有关，包括动脉粥样硬化，主动脉夹层，高血压和中风。尽管已经阐明了激活NOX2（GP91PHOX）NADPH氧化酶的分子机制，但导致NOX1 NADPH氧化酶活性失调的事件尚不清楚。我们先前的研究和初步数据支持以下观点：p67phox的同源物Noxa1在小鼠和人血管平滑肌细胞（VSMC）中表达，并且是NOX1 NADPH氧化酶活性的关键调节剂。 NOXA1依赖性活性氧（ROS）在激动剂诱导的VSMC增殖和生长促进，氧化还原敏感蛋白激酶激活中起着至关重要的作用。 NOXA1在APOE - / - 小鼠的动脉粥样硬化病变以及人类早期颈动脉粥样硬化病变的内膜和内侧VSMC中上调。在此提案中，我们将研究NOXA1和P47PHOX相互作用如何调节VSMC中NADPH氧化酶活性，该机制调节NOXA1表达和转化后修饰及其在NOX1 NADPH氧化酶激活中的作用。我们还将确定NOXA1依赖性ROS产生在再狭窄和动脉粥样硬化中的作用。我们将筛选一个小分子文库，以鉴定NOX1 NADPH氧化酶活性的潜在抑制剂，以补充遗传方法。 To accomplish these goals, we have assembled a unique set of reagents and methodologies including NoxA1-/-, ApoE-/-/NoxA1-/- and [SM22α-CreKI/+/NoxA1f/+] mice, Nox1y/-, p47phox-/- and NoxA1-/- mouse aortic VSMC cultures, adenoviral NoxA1 overexpression and shRNA构建体和功能性α和100,000个小分子的成员库，用于鉴定潜在的NADPH氧化酶抑制剂。我们的研究策略包括以下三个特定目标。 AIM 1：定义在小鼠和人主动脉VSMC中，定义了动力学诱导的ROS产生NOXA1依赖性NADPH氧化酶活性的分子机制。目标2：确定NOXA1缺乏对再狭窄和动脉粥样硬化的影响。 AIM 3：确定新颖和特定的NOXA1和P47PHOX相互作用抑制剂作为调节NOX1活性的手段。总之，这些研究将建立VSMC NOX1 NADPH氧化酶在血管病理生理学中的作用，并可能识别用于调节该酶的策略/化合物。