Functions and Mechanisms of Ref-1 in the Endothlium

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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.