Nox4 and Vascular Homeostasis

Nox4 和血管稳态

• 批准号: 8109965
• 负责人: John Francis Keaney
• 金额: $ 48.97万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-15 至 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8109965
• 关键词: Animals; Arginine; Atherosclerosis; Behavior; Biological Assay; Blood Platelets; Blood Vessels; COS-7 Cell; Catalysis; Cell Culture Techniques; Chimera organism; Citrulline; Cyclic GMP; Data; Disease; EGF gene; Endothelial Cells; Endothelium; Enzymes; Event; Eye; Family; Free Radicals; Hand; Homeostasis; Human; Hypertension; Individual; Knowledge; Leukocyte Trafficking; Ligands; Link; Mediating; Molecular; Mus; Myocardial Infarction; NADPH Oxidase; Nitric Oxide; PTPN1 gene; Pathologic; Patients; Phenotype; Physiological; Play; Process; Production; Protein Isoforms; Reactive Oxygen Species; Regulation; Risk; Role; Signal Transduction; Site; Solid; Source; Staging; Stroke; Superoxides; Translating; Vascular Diseases; Vascular Endothelial Growth Factors; Vascular remodeling; Vasomotor; Work; angiogenesis; base; cell behavior; design; experience; genetic manipulation; high risk; in vivo; insight; migration; overexpression; public health relevance; receptor; research study; response; tool

DESCRIPTION (provided by applicant): Under normal circumstances, the endothelium regulates vascular homeostasis via its influence on vasomotor tone, platelet function, leukocyte trafficking, vascular remodeling, and angiogenesis. Many aspects of these functions are regulated, in part, by the endothelial production of nitric oxide (NO7). In patients with vascular diseases such as hypertension and atherosclerosis, NO bioactivity is impaired predisposing these patients to vascular events including myocardial infarction and stroke. Impaired NO bioactivity has been linked to excess vascular production of reactive oxygen species (ROS), particularly superoxide (7O2-), that rapidly reacts with NO7 to quench its bioactivity. The NADPH oxidase (Nox) family of enzymes plays a prominent role in pathologic ROS production that limits NO7 bioactivity. In this application, however, we present data that NADPH oxidase isoform 4 (Nox4) is an endothelial ROS source that paradoxically promotes normal NO7 bioactivity. Our data indicate that intracellular ROS produce contextual responses based upon the site of ROS production and the type of ROS produced. Our findings will radically change current paradigms involving NO7 and ROS in the vasculature and will have broad implications beyond vascular disease. The central hypothesis of this proposal, therefore, is that Nox4 is an important determinant of endothelial cell phenotype based upon contextual ROS signaling that contributes to normal vascular homeostasis. The objective of this proposal is to identify determinants of physiologic Nox4 signaling in the endothelium and the underlying molecular mechanisms involved in this process. In order to achieve this objective, we will first determine the molecular mechanisms for regulation of Nox4 catalytic activity in the endothelium. These studies will involve endothelial and COS-7 cells to determine the specific domains of Nox4 that dictate its intracellular localization and catalytic activity. Then we will examine how certain receptor ligands, such as EGF, and VEGF modulate Nox4 catalytic activity and intracellular localization. Studies will also be performed in mouse aortic endothelial cells (MAECs) from mice that either overexpress or lack Nox4 in the endothelium. We will then move on to determine the molecular mechanisms responsible for Nox4-mediated modulation of eNOS activity. Genetic manipulation of Nox4 levels in the endothelium will help us determine the implications for NO7 bioactivity and eNOS catalysis. We will then probe the involvement of known Nox4 targets such as Akt, PTP1B, and SOD1. Our data implicate Nox4 in VEGF signaling, prompting us to define the precise mechanisms involved. These studies will be used to set the stage for determining the implications of Nox4 on endothelial cell phenotype in cell culture such as proliferation, migration, and angiogenesis. Finally, we will determine the implications of Nox4 on endothelial cell phenotype and vascular disease in vivo using mice that either overexpress or lack Nox4 in the endothelium. These animals will be used to probe endothelial Nox4 on vascular NO7 bioactivity and angiogenesis. We expect these experiments to provide us with a solid working knowledge of how Nox4 contributes to the control of endothelial phenotype and how this translates into homeostatic responses in vivo. With this information in hand, we should have the requisite insight to design new tools directed at modulating vascular phenotype with an eye toward the treatment of vascular disease. PUBLIC HEALTH RELEVANCE: The endothelium is the lining of blood vessels and its behavior is an important control point for blood vessels. We know from experience that blood vessels in people at risk for atherosclerosis do not work normally. In fact, those individuals with the worst function in their blood vessels are at the highest risk for heart attack. Current dogma suggests that the production of free radicals in blood vessels is responsible for some of the abnormal function of blood vessels in the setting of disease. However, in this proposal, we provide evidence that free radicals produced in the blood vessels are actually part of normal function. We have found a particular enzyme, known as Nox4 that produces radicals in a manner that helps the normal function of blood vessels. This proposal is designed to determine how this enzyme, Nox4, produces more normal function.

描述（由申请人提供）：在正常情况下，内皮通过其对血管舒张张张力，血小板功能，白细胞运输，血管重塑和血管生成的影响来调节血管稳态。这些功能的许多方面部分通过一氧化氮的内皮产生（NO7）来调节。在血管疾病（如高血压和动脉粥样硬化）的患者中，没有生物活性受到损害，使这些患者遭受了包括心肌梗塞和中风在内的血管事件。没有生物活性障碍与活性氧（ROS）的血管产生过多有关，尤其是超氧化物（7o2-），该物种与NO7迅速反应以淬灭其生物活性。 NADPH氧化酶（NOX）酶家族在限制No7生物活性的病理ROS产生中起着重要作用。但是，在此应用中，我们介绍了NADPH氧化酶同工型4（NOX4）是一种矛盾地促进正常NO7生物活性的内皮ROS源。我们的数据表明，基于ROS产生的位点和产生的ROS的类型，细胞内ROS产生上下文响应。我们的发现将在脉管系统中从根本上改变涉及No7和ROS的当前范式，并将具有血管疾病以外的广泛影响。因此，该提案的中心假设是NOX4是基于有助于正常血管稳态的上下文ROS信号传导的内皮细胞表型的重要决定因素。该建议的目的是确定内皮中生理NOX4信号的决定因素以及该过程中涉及的基本分子机制。为了实现这一目标，我们将首先确定内皮中NOX4催化活性调节的分子机制。这些研究将涉及内皮和COS-7细胞，以确定NOX4的特定结构域决定其细胞内定位和催化活性。然后，我们将研究某些受体配体，例如EGF和VEGF如何调节NOX4催化活性和细胞内定位。研究还将在小鼠的小鼠主动脉内皮细胞（MAEC）中进行，这些小鼠过表达或在内皮中缺乏NOX4。然后，我们将继续进行确定负责NOX4介导的ENOS活性调节的分子机制。内皮中NOX4水平的遗传操纵将有助于我们确定对No7生物活性和eNOS催化的影响。然后，我们将探测已知的NOX4靶标（例如AKT，PTP1B和SOD1）的参与。我们的数据暗示NOX4在VEGF信号传导中，促使我们定义了所涉及的精确机制。这些研究将用于确定NOX4对内皮细胞表型在细胞培养（例如增殖，迁移和血管生成）中的影响奠定基础。最后，我们将使用过表达或在内皮中缺乏NOX4的小鼠在体内确定NOX4对内皮细胞表型和血管疾病的含义。这些动物将用于探测内皮NOX4在血管NO7生物活性和血管生成上。我们希望这些实验能为我们提供有关NOX4如何促进内皮表型的控制以及如何转化为体内稳态反应的扎实的工作知识。借助此信息，我们应该有必要的见解来设计针对调节血管表型的新工具，以注重治疗血管疾病。 公共卫生相关性：内皮是血管的衬里，其行为是血管的重要控制点。我们从经验中知道，处于动脉粥样硬化风险的人的血管正常起作用。实际上，那些在血管中功能最差的人患心脏病的风险最高。目前的教条表明，血管中自由基的产生是疾病环境中血管的某些异常功能的原因。但是，在此提案中，我们提供了证据，表明血管中产生的自由基实际上是正常功能的一部分。我们发现了一种特定的酶，称为NOX4，该酶以帮助血管正常功能的方式产生自由基。该建议旨在确定该酶NOX4如何产生更正常的功能。