Nox4 and Vascular Homeostasis

Nox4 和血管稳态

基本信息

批准号：
8900326
负责人：
John Francis Keaney
金额：
$ 46.67万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-07-15 至 2018-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8900326
关键词：
Animal Model Animals Antioxidants Arteries Blood Vessels Blood coagulation Bone Marrow Transplantation Cell Culture Techniques Cell model Chemicals Data Drug or chemical Tissue Distribution Elements Endothelial Cells Enzymes Exercise Exercise stress test Family Family member Functional disorder Funding Gene Expression Regulation Genetic Models Hand Health Hematopoietic Homeostasis Host Defense Human Hydrogen Peroxide Injury Invaded Knock-out Knowledge Modeling Molecular Mus NADPH Oxidase Normal Cell Organism Pathway interactions Physiological Physiology Play Protein Isoforms Protocols documentation Reactive Oxygen Species Regulation Research Resistance Role Shunt Device Signal Pathway Signal Transduction Skeletal Muscle Smooth Muscle Solid Source Stress Superoxides System Testing Thrombomodulin Thrombosis Tissues Tube Up-Regulation Vascular Diseases Wild Type Mouse Work Wound Healing adenylate kinase cell killing cell type improved functioning in vivo insight interest killings loss of function prevent research study response superoxide-generating NADPH oxidase therapy design tissue repair

项目摘要

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.

描述（由申请人提供）：活性氧（ROS）包括超氧化物及其下游代谢物，已知这些物质在生理学和病理生理学中发挥多种作用，体内 ROS 的一个重要来源是 NADPH 氧化酶（Nox）家族。人类中由 7 种同工型（Nox1-5、Duox1、Duox2）组成的酶，具有不同的组织分布和调节机制。 (Nox2)是经典的“呼吸爆发氧化酶”，在严格调控下产生高水平的ROS 对宿主防御至关重要的 ROS 通常由其他 Nox 亚型产生，并且已在其他细胞类型中得到描述。在这方面，Nox4 NADPH 氧化酶亚型特别令人感兴趣，因为它是组成型。与此相反，Nox4 以过氧化氢 (H2O2) 的形式产生 ROS，并且主要在转录水平上受到调节。产生 O2 的 NADPH 氧化酶异构体可促进生理性血管适应和组织修复，在此应用中，我们提供的数据支持我们的中心假设，即内皮 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能力。为了研究这一假设，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-/- 和野生型小鼠将进行耐力运动，然后进行评估。血管适应被确定为 eNOS/NO� 生物活性，以及抗血栓（KLF2、血栓调节蛋白）和抗氧化剂（Nrf2、PGC-1�）途径的上调。为了确定内皮 Nox4 的具体影响，我们还将测试运动诱导的血管适应。我们创建并表征了本构型和诱导型内皮特异性 Nox4 敲除 (ECKONox4) 模型，然后我们将确定抗氧化基因调控在该模型中的作用。 Nox4 对运动的反应，因为我们的初步耐力数据表明 Nox4 上调脉管系统中 Nrf2 和 PGC-1� 依赖性通路，因此我们将在全局 (Nrf2-/-、PGC-1�-/-) 上执行我们的运动方案。）和内皮特异性功能丧失模型（ECKONrf2、ECKOPGC-1�），并评估上述 Aim1 中概述的途径，然后我们将确定是否。 PGC-1� 足以通过我们创建的内皮特异性 PGC-1� 上调动物模型来模拟运动引起的血管适应，该模型具有增强的 NO� 生物活性。最后，我们将确定调节 Nox4 的机制及其对血管适应的贡献。使用已建立的颈动脉至颈静脉分流系统，我们将模拟 eNOS/NO�、抗血栓活性和抗氧化活性变化的程度。然后，我们将在 Nox4-/- 和 ECKONox4 小鼠中测试该模型，并确定对上面列出的 NOx 生物活性以及抗血栓和抗氧化途径的影响，然后我们将使用 Nox4、AMP 激酶的人类和小鼠内皮细胞模型。、Nrf2 和 PGC-1� 操作，以确定 Nox4 在 NO� 生物活性、血栓形成抵抗和抗氧化上调方面决定内皮对耐力运动的反应的分子机制。上述实验应该为我们提供关于 Nox4 如何促进血管稳态的扎实工作知识。这些数据将是确定 ROS 如何在脉管系统中适应的关键要素，更重要的是，确定 ROS 如何积极调节 NOx 生物活性和抗血栓性。有了这些信息，我们应该有必要的洞察力来设计调节血管活性氧的疗法，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。