The ability to sense and respond to decreases in environmental oxygen is crucial for mammalian survival. Among the numerous cell types in which appropriate sensing of and adaptation to hypoxia are critical are vascular endothelial cells (EC). Given their unique location, EC are frequently and directly exposed to decreases in blood oxygen tension. Moreover, as a result of their location, EC are important mediators of vascular tone, coagulation, immune response, vascular permeability and angiogenesis. Thus, the ability of EC to synthesize and release various mitogenic, pro-coagulant, vasoactive and immunologic substances in response to hypoxia, and the adaptive strategies these cells have developed to tolerate wide variations in environmental oxygen availability, are critical to maintaining homeostasis while, in certain instances, contributing to the pathogenesis of disease. We have previously described upregulation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) specifically in EC during exposure to hypoxia. We have also described a novel DNA enhancer element in the GAPDH promoter region, adjacent to an hypoxia-inducible factor 1-binding site, that is necessary for GAPDH upregulation by hypoxia. In related studies, we have determined that the mechanism of GAPDH upregulation by hypoxia in EC is different from that of other hypoxia-regulated genes in other cell types, in that upregulation is not abolished by carbon monoxide, inhibitors of heme synthesis or specific inhibitors of protein phosphorylation. This suggests that the upregulation of GAPDH by hypoxia in EC is mechanistically distinct and may be EC specific. The specific upregulation of GAPDH by hypoxia in EC may be related to their remarkable hypoxia tolerance and/or their specific functions within the vascular wall. The long-term objectives of this proposal are to characterize EC-specific hypoxia sensing mechanisms that regulate gene expression. The specific aims of this proposal are to: (1) characterize the cis- and trans-acting factors required for GAPDH regulation by hypoxia in EC, (2) define the EC specificity of GAPDH gene upregulation by hypoxia, (3) characterize signal transduction mechanisms involved in EC upregulation of GAPDH during hypoxia.