Nox4 and Vascular Homeostasis

描述(申请人提供)：在正常情况下，内皮通过影响血管运动张力、血小板功能、白细胞运输、血管重塑和血管生成来调节血管动态平衡。这些功能的许多方面在一定程度上受到内皮细胞产生一氧化氮(NO7)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，生物活性不会受到损害，使这些患者容易发生包括心肌梗死和中风在内的血管事件。NO生物活性受损与血管产生过多的活性氧(ROS)有关，特别是超氧化物(7O2-)，它能迅速与NO7反应以抑制其生物活性。NADPH氧化酶(NOx)家族在限制NO7生物活性的病理性ROS产生中起着重要作用。然而，在这个应用中，我们提出了NADPH氧化酶亚型4(NOX4)是一种自相矛盾地促进正常NO7生物活性的内皮ROS来源的数据。我们的数据表明，细胞内ROS根据ROS产生的地点和产生的RO类型产生上下文反应。我们的发现将从根本上改变目前血管系统中涉及NO7和ROS的范式，并将具有超越血管疾病的广泛意义。因此，这一建议的中心假设是，基于有助于正常血管内稳态的相关ROS信号，NOX4是内皮细胞表型的重要决定因素。本研究的目的是确定内皮细胞中NOX4生理性信号的决定因素以及参与这一过程的潜在分子机制。为了实现这一目标，我们将首先确定调控内皮细胞中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，如EGF和VEGF是如何调节NOX4的催化活性和细胞内定位的。研究还将在内皮细胞中过度表达或缺乏NOX4的小鼠主动脉内皮细胞(MAECs)中进行。然后，我们将继续确定NOX4介导的eNOS活性调节的分子机制。对内皮细胞中NOX4水平的遗传操作将有助于我们确定对NO7生物活性和eNOS催化的影响。然后，我们将探索已知NOX4目标的参与，如Akt、PTP1B和SOD1。我们的数据表明NOX4与血管内皮生长因子信号转导有关，这促使我们定义了相关的确切机制。这些研究将被用来确定NOX4对细胞培养中内皮细胞表型的影响，如增殖、迁移和血管生成。最后，我们将利用内皮细胞过度表达或缺乏NOX4的小鼠，在体内确定NOX4对血管内皮细胞表型和血管疾病的影响。这些动物将被用来探测内皮细胞NOX4对血管7号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坚实的工作知识，了解NOX4如何有助于内皮表型的控制，以及如何在体内转化为内环境平衡反应。有了这些信息，我们应该有必要的洞察力来设计新的工具，以调节血管表型，着眼于血管疾病的治疗。 公共卫生相关性：内皮是血管的内层，其行为是血管的重要控制点。我们从经验中了解到，动脉粥样硬化高危人群的血管不能正常工作。事实上，那些血管功能最差的人心脏病发作的风险最高。目前的学说认为，在疾病的背景下，血管中自由基的产生是导致血管功能异常的原因。然而，在这项建议中，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NOX4，它能以一种有助于血管正常功能的方式产生自由基。这项提议旨在确定这种名为NOX4的酶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.