Nox4 and Vascular Homeostasis

描述（由申请方提供）：在正常情况下，内皮通过影响血管张力、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地由内皮细胞产生的一氧化氮（NO 7）调节。在患有血管疾病如高血压和动脉粥样硬化的患者中，NO生物活性受损，使这些患者易患血管事件，包括心肌梗死和中风。受损的NO生物活性与血管中活性氧（ROS）的过量产生有关，特别是超氧化物（7 O2-），其迅速与NO 7反应以淬灭其生物活性。NADPH氧化酶（Nox）家族的酶在限制NO 7生物活性的病理性ROS产生中起着重要作用。然而，在本申请中，我们提供的数据表明，NADPH氧化酶亚型4（Nox 4）是一种内皮ROS源，矛盾地促进正常的NO 7生物活性。我们的数据表明，细胞内ROS产生上下文响应的基础上的网站的ROS生产和类型的ROS生产。我们的研究结果将从根本上改变目前的范式，涉及NO 7和ROS在血管系统中，并将有广泛的影响超出血管疾病。因此，该提议的中心假设是，Nox 4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细胞中生理性Nox 4信号传导的决定因素以及参与该过程的潜在分子机制。为了实现这一目标，我们将首先确定调节内皮细胞中Nox 4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Nox 4的特定结构域。然后，我们将研究某些受体配体，如EGF和VEGF如何调节Nox 4催化活性和细胞内定位。还将在来自内皮中过表达或缺乏Nox 4的小鼠的小鼠主动脉内皮细胞（MAEC）中进行研究。然后，我们将继续确定负责Nox 4介导的eNOS活性调节的分子机制。内皮细胞中Nox 4水平的遗传操作将有助于我们确定NO 7生物活性和eNOS催化的影响。然后，我们将探索已知的Nox 4靶点，如Akt，PTP 1B和SOD 1的参与。我们的数据暗示Nox 4参与VEGF信号传导，促使我们确定所涉及的确切机制。这些研究将用于确定Nox 4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。最后，我们将使用内皮细胞中过表达或缺乏Nox 4的小鼠来确定Nox 4对内皮细胞表型和体内血管疾病的影响。这些动物将用于探测内皮Nox 4对血管NO 7生物活性和血管生成的影响。我们希望这些实验能为我们提供一个坚实的工作知识，Nox 4如何有助于控制内皮细胞表型，以及这如何转化为体内稳态反应。有了这些信息，我们应该有必要的洞察力来设计新的工具，直接调节血管表型，着眼于血管疾病的治疗。 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，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。目前的教条认为，血管中自由基的产生是疾病背景下血管功能异常的原因。然而，在这项提案中，我们提供了证据，证明血管中产生的自由基实际上是正常功能的一部分。我们发现了一种特殊的酶，称为Nox 4，它以一种有助于血管正常功能的方式产生自由基。这项提案旨在确定这种酶Nox 4如何产生更正常的功能。

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.