FIRST award that is directed to the mechanisms involving regulation of human endothelial cell xanthine oxidase-xanthine-dehydrogenase (XO-XDH). Xanthine oxidase is an efficient oxygen radical-producing enzyme which may impact greatly upon human vascular and inflammatory diseases. Despite its importance, nothing is known of the genetic and environmental factors which govern XO expression in humans, as these factors appear to differ from other species. In addition, appropriate experimental human systems to study the specific effects of XO on endothelial cell physiology and biochemistry are lacking. It is hypothesized that hypoxia, a characteristic of ischemic vascular disease, increases the transcription of XO-XDH and also leads to sulfuration and activation of the molybdenum active site. Preliminary data are provided which demonstrate 1) that oxygen tension exerts a significant and consistent effect of XO and XDH activity in human umbilical vein endothelial cells (HUVEC) and other cells 2) oxygen effects XO-XDH at both pre- and post-translational levels 3) reoxygenation alters XO-related oxygen metabolite production and other relevant physiologic parameters in EC 4) antibodies have been developed and cDNAs of human XO-XDH have and are being cloned; and 5) transfection strategies using different techniques and promoters have been optimized for HUVEC. The objectives of this proposal are 1) to investigate the mechanism of regulation of human endothelial cell XO-XDH by determining the effect of oxygen tension on XO-XDH transcription, protein levels, activity and molybdenum site integrity 2) to determine the specific contribution of XO to hypoxia induced proinflammatory changes of human endothelial cells by creating XO-XDH under and over expressing HUVEC transfectants containing the human XO-XDH gene in the antisense or sense orientations. The overall goal is to provide a critical understanding of the factors which determine expression of human endothelial cells XO-XDH, and identify physiologic perturbations which accompany selective changes in cellular XO-XDH levels.
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