Nox4 and Vascular Homeostasis

描述（由申请人提供）：活性氧（ROS）包括超氧化物及其下游代谢产物。已知这些物种在生理学和病理生理学中发挥多种作用。体内ROS的主要来源是NADPH氧化酶（Nox）家族，在人体内由7种亚型（Nox 1 -5、Duox 1、Duox 2）组成，具有不同的组织分布和调节机制。原型家族成员（Nox 2）是经典的“呼吸爆发氧化酶”，在严格的调控下产生高水平的ROS 对宿主防御至关重要在细胞信号传导中重要的ROS通常由其他Nox同种型以更适度的水平产生，并且已经在其他细胞类型中描述。在这方面，Nox 4 NADPH氧化酶同工型是特别感兴趣的，因为它组成性地产生过氧化氢（H2 O2）形式的ROS，并且主要在转录水平上调节。我们上一个资助期的新数据表明，与产生<$O2的NADPH氧化酶亚型相比，Nox 4促进了生理血管适应和组织修复。在本申请中，我们提供的数据支持我们的中心假设，即内皮Nox 4是耐力运动的适应性血管效应所必需的，包括增强的NO生物活性和抗血栓形成。为了研究这一假设，我们将首先确定Nox 4在耐力运动血管反应中的体内作用。对于这些研究，Nox 4-/-和野生型小鼠将进行耐力运动，然后评估血管适应性，确定为eNOS/NO？生物活性，以及抗血栓形成（KLF 2，血栓调节蛋白）和抗氧化剂（Nrf 2，PGC-1？）途径的上调。为了确定内皮Nox 4的具体影响，我们还将在我们创建和表征的组成型和诱导型内皮特异性Nox 4敲除（ECKONox 4）模型中测试运动诱导的血管适应。然后，我们将确定抗氧化剂基因调控在Nox 4对耐力运动的反应中的作用，因为我们的初步数据表明，Nox 4上调血管系统中的Nrf 2和PGC-1？依赖性通路。因此，我们将在整体（Nrf 2-/-，PGC-1-/-）和内皮特异性功能丧失模型（ECKONrf 2，ECKOPGC-1）上执行我们的运动方案，并评估上述Aim 1中概述的途径。然后，我们将确定PGC-1 <$$>是否足以模拟运动诱导的血管适应与内皮特异性PGC-1 <$$>上调的动物模型，我们已经创建了增强NO <$生物活性的功能。最后，我们将确定调节Nox 4的机制及其对耐力运动内皮反应的贡献。使用一个已建立的颈动脉-颈静脉分流系统，我们将模拟增加的血流模拟eNOS/NO <$、抗血栓活性和运动中观察到的抗氧化活性变化的程度。然后，我们将在Nox 4-/-和ECKONOX 4小鼠中测试该模型，并确定对NO生物活性以及上述抗血栓形成和抗氧化途径的影响。然后，我们将使用Nox 4、AMP激酶、Nrf 2和PGC-1？操纵的人和鼠内皮细胞模型来确定Nox 4决定内皮细胞对耐力运动的反应的分子机制，包括NO？生物活性、抗血栓形成和抗氧化剂上调。上面概述的实验应该为我们提供了一个坚实的工作知识，Nox 4如何有助于血管内稳态。这些数据将是确定ROS如何在血管系统中自适应的关键因素，重要的是，ROS如何积极调节NO生物活性和血栓阻力。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.