Nox4 and Vascular Homeostasis

Nox4 和血管稳态

• 批准号: 8759579
• 负责人: John Francis Keaney
• 金额: $ 47.38万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-15 至 2018-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8759579
• 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; public health relevance; research study; response; superoxide-generating NADPH oxidase; therapy design; tissue repair

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（由适用提供）：活性氧（ROS）包括超氧化物及其下游代谢产物。众所周知，这些物种在生理学和病理生理学中都起着多种作用。 ROS在体内的突出来源是人类中的NADPH氧化物（NOX）家族，该酶由7种同工型（NOX1-5，DUOX1，DUOX2）组成，具有不同的组织分布和调节机制。原型家庭成员（NOX2）是经典的“呼吸爆发氧化酶”，在严格的调节下产生高水平的ROS 对于东道国防御至关重要。 ROS在细胞信号传导中的重要性是在更谦虚的水平上，通常由其他NOX同工型产生，并且在其他细胞类型中进行了描述。在这方面，NOX4 NADPH氧化物同工型特别关注，因为它以过氧化氢（H2O2）形式产生ROS，并且主要在转录水平上进行调节。我们上前资金期的新兴数据表明，与`与O2产生的NADPH氧化物同工型相比，NOX4促进了物理血管适应和组织修复。在此应用中，我们提出了支持我们的中心假设的数据，即耐力运动的自适应血管效应需要内皮NOX4，包括增强的NO生物活性和耐药性。为了研究这一假设，我们将首先确定NOX4在耐力运动的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，然后评估为NOX4对耐耐力练习的反应中的抗氧化基因调节确定为ENOS/NO生物活性的血管适应，并上调NOX4的NOX4上调NRECROCTORAYEN nRECONTION，均以NRFF2-和PGC-pgc-cc-cc-cc-cc-cc-cc-cc-cc-cc-cc-cc-cc-cc-cc-cc-cc-cc-cc-cc-cc-cc-cc-cc-cc-cc-cc-cc-cc-cc-cc-cc-cc-cc-1-根据我们将执行有关全局（NRF2 - / - ，PGC-1¿-/ - ）和内皮特异性功能丧失模型（Eckonrf2，EckopGC-1）的锻炼方案，并评估AIM1中上面概述的途径。然后，我们将确定PGC-1是否足以通过内皮特异性PGC-1的动物模型模仿运动诱导的血管适应，我们创建的具有增强的无生物活性的功能。最后，我们将确定调查NOX4的机制及其对耐力运动的内皮反应的贡献。我们将使用已建立的颈动脉到朱古的分流系统，我们将建模增加流动模拟eNOS/NO的变化，抗血栓性活性和抗氧化活性的程度。然后，我们将测试该模型N NOX4 - / - 和Eckonox4小鼠，并确定上面列出的对NO生物活性以及抗血栓形成和抗氧化途径的影响。然后，我们将使用NOX4，AMP激酶，NRF2和PGC-1。的人类和鼠的内皮细胞模型来确定NOX4的分子机制，该分子机制指示NOX4决定对耐力运动的耐力运动，以避免生物活性，血栓耐受性，抗氧化和抗氧化。上面概述的实验应为我们提供有关NOX4如何对血管稳态贡献的扎实的工作知识。这些数据将是确定ROS如何在脉管系统中自适应的关键要素，重要的是，ROS如何积极地调节了脱离生物活性和血栓抗体。有了这些信息，我们应该有必要的见解来设计调节血管ROS的疗法，并更好地预测它们对正常血管生理学以及血管疾病的病理生理的影响。