Nox4 and Vascular Homeostasis

说明(申请人提供)：活性氧种(ROS)包括超氧化物及其下游代谢物。这些物种在生理学和病理生理学中都扮演着多种角色。体内ROS的一个重要来源是NADPH氧化酶(NOX)家族，在人类中由7种亚型(NOX1-5，DUOX1，DUOX2)组成，具有不同的组织分布和调节机制。典型的家族成员(NOX2)是典型的“呼吸爆发氧化酶”，在严格的调控下产生高水平的ROS 这对东道主的防守至关重要。在细胞信号转导中重要的ROS是在更适度的水平上产生的，通常是由其他NOx亚型产生的，并且已经在其他类型的细胞中描述过。在这方面，NOX4 NADPH氧化酶亚型特别令人感兴趣，因为它在成分上以过氧化氢(H_2O_2)的形式产生ROS，并且主要在转录水平上受到调节。我们上一个资助期的新数据表明，与产生�O2的NADPH氧化酶亚型不同，NOX4促进了生理血管适应和组织修复。在这一应用中，我们提供了支持我们的中心假设的数据，即内皮细胞NOX4是耐力运动的适应性血管效应所必需的，包括增强NO�生物活性和抗血栓形成。为了研究这一假说，我们将首先确定NOX4在耐力运动血管反应中的体内作用。在这些研究中，NOX4-/-和野生型小鼠将接受耐力运动，随后评估血管适应性，确定eNOS/NO�生物活性，并上调抗血栓(KLF2，血栓调节蛋白)和抗氧化(NRF2，PGC-1�)途径。为了确定内皮细胞NOX4的具体影响，我们还将在我们创建和表征的结构性和诱导性内皮特异性NOX4基因敲除(ECKONox4)模型中测试运动诱导的血管适应。然后，我们将确定抗氧化基因调节在NOX4对耐力运动的反应中的作用，因为我们的初步数据表明，NOX4上调了血管系统中依赖于NRF2和PGC-1的�途径。因此，我们将在全球(NRF2-/-，PGC-1�-/-)和内皮特异性功能丧失模型(ECKONRF2，ECKOPGC-1�)上执行我们的练习方案，并评估上面在AIM1中概述的通路。然后，我们将用我们创建的内皮特异性PGC-1�上调的动物模型来确定PGC-1�是否足以模拟运动诱导的血管适应，该动物模型的特点是增强了NO�的生物活性。最后，我们将确定NOX4的调节机制及其在耐力运动中对内皮细胞反应的贡献。使用已建立的颈动脉到颈静脉分流系统，我们将模拟血流增加在多大程度上模拟运动中eNOS/NO�、抗血栓活性和抗氧化活性的变化。然后，我们将在NOX4-/-和ECKONOX4小鼠身上测试该模型，并确定其对NO�生物活性以及上述抗血栓和抗氧化途径的影响。然后，我们将使用人和小鼠内皮细胞模型中的NOX4、AMP激酶、NRF2和PGC-1�操作来确定NOX4在NO�生物活性、血栓抵抗和抗氧化剂上调方面调控内皮细胞对耐力运动的反应的分子机制。上面概述的实验应该为我们提供关于NOX4如何有助于血管内稳态的坚实的工作知识。这些数据将是确定ROS如何在血管系统中适应以及重要的是ROS如何积极调节NO�生物活性和血栓抵抗的关键因素。有了这些信息，我们应该有必要的洞察力来设计调节血管ROS的治疗方法，并更好地预测它们对正常血管生理学和血管疾病的病理生理学的影响。

DESCRIPTION (provided by applicant): Reactive oxygen species (ROS) include superoxide and its downstream metabolites. These species are known to play multiple roles in both physiology and pathophysiology. A prominent source of ROS in vivo are the NADPH oxidase (Nox) family of enzymes that in humans consists of 7 isoforms (Nox1-5, Duox1, Duox2) with distinct tissue distribution and mechanisms of regulation. The prototypic family member (Nox2) is the classic "respiratory burst oxidase' that produces high levels of ROS under strict regulation that are critical for host defence. ROS important in cellular signaling are produced at more modest levels, often by other Nox isoforms, and have been described in other cell types. In this regard, the Nox4 NADPH oxidase isoform is of particular interest as it constitutively generates ROS in the form of hydrogen peroxide (H2O2) and is regulated principally at the transcriptional level. Emerging data from our previous funding period indicate that Nox4, in contrast to �O2- producing NADPH oxidase isoforms, promotes physiological vascular adaptation and tissue repair. In this application, we present data supporting our central hypothesis that endothelial Nox4 is required for the adaptive vascular effects of endurance exercise including enhanced NO� bioactivity and thrombosis resistance. To investigate this hypothesis, we will first determine the in vivo role of Nox4 in the vascular response to endurance exercise. For these studies, Nox4-/- and wild-type mice will undergo endurance exercise followed by assessment of vascular adaptation determined as eNOS/NO� bioactivity, and upregulation of antithrombotic (KLF2, thrombomodulin) and antioxidant (Nrf2, PGC-1�) pathways. To determine the specific impact of endothelial Nox4, we will also test exercise-induced vascular adaptation in constitutive and inducible endothelial- specific Nox4 knockout (ECKONox4) models we have created and characterized. We will then determine the role of antioxidant gene regulation in the Nox4 response to endurance exercise as our preliminary data indicate that Nox4 upregulates both Nrf2- and PGC-1�-dependent pathways in the vasculature. Accordingly we will perform our exercise protocol on global (Nrf2-/-, PGC-1�-/-) and endothelial-specific loss-of-function models (ECKONrf2, ECKOPGC-1�), and assess the pathways outlined above in Aim1. We will then determine if PGC-1� is sufficient to mimic exercise-induced vascular adaptation with an animal model of endothelial-specifc PGC-1� upregulation we have created that features enhanced NO� bioactivity. Finally, we will determine the mechanisms regulating Nox4 and its contribution to the endothelial response to endurance exercise. Using an established carotid-to-jugular shunt system, we will model the extent to which increased flow mimics the changes in eNOS/NO�, antithrombotic activity, and antioxidant activity seen with exercise. We will then test this model n Nox4-/- and ECKONox4 mice and determine the impact on NO� bioactivity and the antithrombotic and antioxidant pathways listed above. We will then use human and murine endothelial cell models of Nox4, AMP kinase, Nrf2, and PGC-1� manipulation to determine the molecular mechanisms whereby Nox4 dictates the endothelial response to endurance exercise with regards to NO� bioactivity, thrombosis resistance, and antioxidant upregulation. The experiments outlined above should provide us with a solid working knowledge of how Nox4 contributes to vascular homeostasis. These data will be a key element of determining how ROS can be adaptive in the vasculature and, importantly, how ROS positively regulate NO� bioactivity and thromboresistance. With this information in hand, we should have the requisite insight to design therapies that modulate vascular ROS and better predict their impact on normal vascular physiology and also the pathophysiology of vascular disease.