The vascular endothelium is a significant source of reactive oxygen species and a critical target for tissue injury due to overproduction of oxygen free radicals. Excessive production of reactive oxygen species including superoxide (O2-), hydrogen peroxide (H2O2) and hydroxyl radical (OH-) occurs secondary to diverse phenomena including trauma, acute inflammation sepsis, tissue ischemia-reperfusion, oxygen toxicity and exposure to xenobiotics capable of redox cycling. Oxidant-induced alteration of endothelial cell structure, function or barrier properties can result in modification of endothelial vasoactive properties, increased vascular permeability leading to edema and initiation of other pathologic processes, including platelet aggregation, atherogenesis and activation of neutrophils. Fulfillment of the Specific Aims of the proposal will illuminate mechanisms of oxygen radical injury to metabolically and functionally unique large vessel and microvascular-derived indothelial cells isolated from thoracic aorta and brain capillaries. cells will be examined from both a biochemical and functional perspective. Free radical metabolism studies will be performed in concert with pharmacologic interventions for oxidant damage, which include conjugation of antioxidant enzymes to high molecular weight surface-active molecules and entrapment of antioxidant enzymes in liposomes (which serve as vectors for intracellular enzyme entry). Radioisotopic techniques and absorption and fluorescence spectroscopy will be applied for both identification and quantitation of reactive oxygen species in specific cellular compartments and after release from and intact cells. Controlled manipulation of antioxidant defenses via conjugated or liposome- entrapped antioxidant enzymes will also yield mechanistic information aboun generation and reactions of specific oxygen metabolities in endothelial cells. Measurement of free radical injury to cells and changes in vascular metabolic and barrier functions after oxidant stress will provide a practical means to test the capacity of antioxidant agents in preventing oxidant- mediated vascular injury. These proposed studies represent a combination of cell biology, new drug delivery technologies and sophisticated approaches to measuring oxidant production and reactions in intact cell systems. From this, we will better understand mechanisms of vascular free radical production and injury. Achieving this, we also gain insight from both biochemical and cellular perspective into the rational use of antioxidant agents for therapeutic intervention in tissue pathology secondary to overproduction of reactive oxygen metabolites.
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