Functions and Mechanisms of Ref-1 in the Endothlium

描述（由申请人提供）：由活性氧（ROS）和内皮性一氧化氮（NO）的产生和消除决定的内皮还原氧化（氧化还原）状态的扰动有助于许多血管疾病的发病机制，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，本研究提出了一个新概念，即ref-i调节内皮细胞ROS和NO的产生，因此是内皮细胞氧化还原状态的关键决定因素。拟开展的实验将测试ref-1在调节内皮细胞ROS和NO生成中的作用和机制。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，以及其影响内皮细胞NO和ROS生成的可能机制将被探讨。在调节这些介质的活动/表达中重要的ref-1结构域将被表征。除了确定ref-1在培养内皮细胞中的功能外，在全血管中的腺病毒基因转移实验也将阐明其在调节内皮源性生物可利用NO和内皮依赖性血管张力中的作用。最后，将明确enos衍生的NO和由rac1调控的NAD (P) H氧化酶衍生的ROS在调节内皮细胞中ref-1的功能、表达和亚细胞定位中的作用。Ref-1作为转录和DNA修复酶的主要调控因子，参与细胞增殖、凋亡和分化等基本过程。ref-1在心血管系统中的重要性才刚刚开始得到重视。通过研究ref-1在内皮中的调控作用及其在内皮生理和病理生理中的作用，并通过研究ref-1的转录和新的非转录作用机制，本研究有望推进我们目前对其在血管生物学和疾病中的功能的理解。

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.