Organ and limb revascularization procedures are performed more than 500,000 times annually in the United States. Under emergent conditions, successful arterial reconstructions continue to result in loss of limb and limb function. Mechanisms thought to contribute to limb loss include intracellular generation of free radical/peroxide species and extracellular accumulation of neutrophils, which may induce membrane injury. In vivo models of ischemia and reperfusion demonstrate increased vascular permeability, implicating endothelial injury. This evidence has prompted some investigators to postulate that the endothelium is an """"""""ischemic shock tissue """""""". Alterations in basic endothelial cell functions are believed to exacerbate tissue injury as a consequence of loss of endothelial cell integrity, increased capillary thrombosis and subsequent irreversible end-organ damage. Specific and important endothelial cell functions include the production of prostaglandins and nitric oxide(an endothelial cell derived relaxant factor-EDRF) thought to be important in thrombosis and hemostasis. Considerable in vivo historical data exists which suggests that prostacyclin is released during ischemia, causing vasodilation and inhibiting platelet aggregation.In addition, in vitro studies suggest that nitric oxide, prostacyclin and prostacyclin analogues may detoxify the effects of neutrophil activation by preventing degranulation. In vivo studies have documented that administration of prostacyclin can decrease tissue infarction in both the myocardium and skeletal muscle during severe ischemia.In vivo data also exists which suggests that inhibition of EDRF synthesis salvages ischemic myocardium from reperfusion injury. The cytotoxic or cytoprotective characteristics of nitric oxide are being hotly investigated. Using a novel model of bovine aortic endothelial cells grown on microcarrier beads, we propose to assess the quantitative and temporal aspects of eicosanoids and nitric oxide biosynthesis during conditions of hypoxia and reoxygenation. This model allows alteration of conditions to result in ischemia (i.e. depletion of energy substrates) as well as hypoxia. In addition we believe endothelial cells from pulmonary artery may have a different pattern of response to the release of eicosanoids and nitric oxide during hypoxic/reoxygenation conditions. In both these cell lines, we propose to: 1) determine whether hypoxia and/or reoxygenation alters the temporal profile of prostaglandin/NO release, 2) to determine whether depletion of energy substrates (i.e. glucose) during hypoxia exacerbates alterations in endothelial eicosanoid/Nitric Oxide release.3) To determine if eicosanoid/nitric oxide release returns to baseline levels during reoxygenation following pure hypoxia or ischemia. 4) To determine whether free-radical scavengers potentiate or diminish alterations in eicosanoid/nitric oxide release resulting from conditions of hypoxia and/or ischemia. These data will shed information on changes in endothelial cell function long after the ischemic insult.
