Nox4 and Vascular Homeostasis

描述（由申请人提供）：在正常情况下，内皮通过其对血管舒缩张力、血小板功能、白细胞运输、血管重塑和血管生成的影响来调节血管稳态。这些功能的许多方面在一定程度上是由内皮细胞产生一氧化氮（NO7）调节的。在高血压和动脉粥样硬化等血管疾病患者中，一氧化氮生物活性受损，使这些患者易发生包括心肌梗死和中风在内的血管事件。一氧化氮生物活性受损与血管中活性氧（ROS）的过量产生有关，尤其是超氧化物（7O2-），它能与一氧化氮迅速反应，抑制其生物活性。NADPH氧化酶（Nox）家族酶在限制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，我们提供的数据表明，NADPH氧化酶异构体4 （Nox4）是内皮活性氧来源，矛盾的是促进正常的NO7生物活性。我们的数据表明，细胞内ROS产生的上下文反应基于ROS产生的位置和ROS产生的类型。我们的发现将从根本上改变目前关于NO7和ROS在血管系统中的研究范式，并将具有血管疾病以外的广泛意义。因此，该提案的中心假设是，Nox4是内皮细胞表型的重要决定因素，基于上下文ROS信号，有助于正常血管稳态。本提案的目的是确定内皮中生理性Nox4信号传导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，我们将首先确定内皮中Nox4催化活性调节的分子机制。这些研究将涉及内皮细胞和COS-7细胞，以确定Nox4的特定结构域，这些结构域决定了其细胞内定位和催化活性。然后，我们将研究某些受体配体，如EGF和VEGF如何调节Nox4的催化活性和细胞内定位。研究还将在内皮过度表达或缺乏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。内皮中Nox4水平的基因操作将帮助我们确定NO7生物活性和eNOS催化的意义。然后，我们将探讨已知的Nox4靶点如Akt、PTP1B和SOD1的参与。我们的数据暗示Nox4参与VEGF信号传导，促使我们确定其中的确切机制。这些研究将用于确定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，我们将确定Nox4对内皮细胞表型和血管疾病的影响，使用内皮细胞过度表达或缺乏Nox4的小鼠。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扎实的工作知识，了解Nox4如何促进内皮表型的控制，以及这如何转化为体内的稳态反应。有了这些信息，我们应该有必要的洞察力来设计新的工具来调节血管表型，并着眼于血管疾病的治疗。

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.