Functions and Mechanisms of Ref-1 in the Endothlium

描述（由申请人提供）：内皮氧化还原 (redox) 状态的扰动由活性氧 (ROS) 和内皮一氧化氮 (NO) 的产生和消除决定，导致许多血管疾病的发病机制，例如动脉粥样硬化、再狭窄和高血压。氧化还原因子-1 (ref-1) 是一种普遍存在的 DNA 修复酶/转录调节因子。 ref-1 是否以及如何影响内皮氧化还原状态和功能尚不清楚。基于初步证据，该提案提出了 ref-i 调节内皮细胞 ROS 和 NO 产生的新概念，因此是内皮氧化还原状态的关键决定因素。拟议的实验将测试 ref-1 在调节内皮 ROS 和 NO 产生中的作用和机制。将探讨 ref-1 对 eNOS、Akt 激酶和 NAD(P)H 氧化酶活性以及热休克蛋白表达的影响，作为其影响内皮 NO 和 ROS 生成的可能机制。对调节这些介质的活性/表达很重要的 ref-1 域将被表征。除了确定 ref-1 在培养的内皮细胞中的功能外，全血管中的腺病毒基因转移实验还将阐明其在调节内皮衍生的生物可利用的 NO 和内皮依赖性血管张力中的作用。最后，将定义eNOS衍生的NO和源自rac1调节的NAD（P）H氧化酶的ROS在调节内皮中ref-1的功能、表达和亚细胞定位中的作用。Ref-1作为转录的主调节因子和DNA修复酶，参与增殖、凋亡和分化等基本细胞过程。 ref-1 在心血管系统中的重要性才刚刚开始被认识到。通过研究 ref-1 在内皮细胞中的调节、其在内皮生理学和病理生理学中的作用，并通过检查 ref-1 的转录和新型非转录作用机制，该提案有望增进我们目前对其在血管生物学和疾病中功能的理解。

DESCRIPTION (provided by the applicant): Perturbations in the reduction-oxidation (redox) status of the endothelium, determined by the production and elimination of reactive oxygen species (ROS) and endothelial nitric oxide (NO), contributes to the pathogenesis of many vascular disorders such as atherosclerosis, restenosis, and hypertension.Redox factor-1 (ref-1) is a ubiquitous DNA repair enzyme/transcriptional regulator. If and how ref-1 affects endothelial redox status and function is not known. Based on preliminary evidence, this proposal advances the novel concept that ref-i regulates endothelial production of ROS and NO, and therefore is a crucial determinant of endothelial redox state. Proposed experiments will test the role and mechanisms of ref-1 in regulating the production of endothelial ROS and NO. The effect of ref-1 on eNOS, Akt kinase, and NAD(P)H oxidase activities, and heat shock protein expression, as possible mechanisms for its effects on endothelial NO and ROS generation will be explored. The domains of ref-1 that are important in regulating the activities/expression of these mediators will be characterized. In addition to defining the function(s) of ref-1 in cultured endothelial cells, adenoviral gene transfer experiments in whole vessels will also elucidate its role in regulating endothelium-derived bioavailable NO, and endothelium-dependent vascular tone. Finally, the roles of eNOS-derived NO, and ROS derived from the rac1 -regulated NAD (P) H oxidase in regulating the function, expression, and sub-cellular localization of ref-1 in the endothelium will be defined.Ref-1, as a master regulator of transcription and a DNA repair enzyme, participates in fundamental cellular processes such as proliferation, apoptosis, and differentiation. The importance of ref-1 in the cardiovascular system is only beginning to be appreciated. By investigating the regulation of ref-1 in the endothelium, its role in endothelial physiology and pathophysiology, and by examining both transcriptional and novel non-transcriptional mechanisms of action of ref-1, this proposal promises to advance our current understanding of its functions in vascular biology and disease.