Nox4 and Vascular Homeostasis

Nox4 和血管稳态

• 批准号: 8292092
• 负责人: John Francis Keaney
• 金额: $ 48.8万
• 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/8292092
• 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（申请人提供）：在正常情况下，内皮通过影响血管舒缩张力、血小板功能、白细胞运输、血管重塑和血管生成来调节血管稳态。这些功能的许多方面部分受到内皮细胞一氧化氮 (NO7) 生成的调节。在患有高血压和动脉粥样硬化等血管疾病的患者中，NO 生物活性受损，导致这些患者容易发生血管事件，包括心肌梗塞和中风。 NO 生物活性受损与血管产生过多的活性氧 (ROS)，特别是超氧化物 (7O2-) 相关，超氧化物 (7O2-) 会快速与 NO7 反应以淬灭其生物活性。 NADPH 氧化酶 (Nox) 家族在限制 NO7 生物活性的病理性 ROS 产生中发挥着重要作用。然而，在本申请中，我们提供的数据表明，NADPH 氧化酶同工型 4 (Nox4) 是一种内皮 ROS 来源，但它却相反地促进正常的 NO7 生物活性。我们的数据表明，细胞内 ROS 会根据 ROS 产生的位点和产生的 ROS 类型产生上下文响应。我们的研究结果将从根本上改变当前涉及脉管系统中 NO7 和 ROS 的范式，并将对血管疾病之外产生广泛的影响。因此，该提议的中心假设是，Nox4 是内皮细胞表型的重要决定因素，其基于有助于正常血管稳态的背景 ROS 信号传导。该提案的目的是确定内皮细胞中生理性 Nox4 信号传导的决定因素以及该过程中涉及的潜在分子机制。为了实现这一目标，我们将首先确定调节内皮细胞中Nox4催化活性的分子机制。这些研究将涉及内皮细胞和 COS-7 细胞，以确定决定其细胞内定位和催化活性的 Nox4 的特定结构域。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。还将对内皮细胞中过度表达或缺乏 Nox4 的小鼠主动脉内皮细胞 (MAEC) 进行研究。然后我们将继续确定 Nox4 介导的 eNOS 活性调节的分子机制。内皮细胞中 Nox4 水平的基因操作将帮助我们确定对 NO7 生物活性和 eNOS 催化的影响。然后我们将探讨已知的 Nox4 靶标（例如 Akt、PTP1B 和 SOD1）的参与情况。我们的数据表明 Nox4 参与 VEGF 信号传导，促使我们定义所涉及的精确机制。这些研究将用于确定 Nox4 对细胞培养中内皮细胞表型（如增殖、迁移和血管生成）的影响奠定基础。最后，我们将使用内皮细胞中过度表达或缺乏 Nox4 的小鼠来确定 Nox4 对内皮细胞表型和体内血管疾病的影响。这些动物将被用来探测内皮Nox4对血管NO7生物活性和血管生成的影响。我们希望这些实验能够为我们提供关于 Nox4 如何有助于控制内皮表型以及如何转化为体内稳态反应的扎实的工作知识。掌握了这些信息，我们应该有必要的洞察力来设计旨在调节血管表型的新工具，着眼于血管疾病的治疗。 公共卫生相关性：内皮是血管的内层，其行为是血管的重要控制点。根据经验我们知道，有动脉粥样硬化风险的人的血管不能正常工作。事实上，那些血管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造成疾病时血管功能异常的原因。然而，在这项提案中，我们提供的证据表明，血管中产生的自由基实际上是正常功能的一部分。我们发现了一种称为 Nox4 的特殊酶，它能产生自由基，从而有助于血管的正常功能。该提案旨在确定这种酶 Nox4 如何产生更正常的功能。