ENDOTHELIAL CELL CYCLE ANTIOXIDANTS AND RADIATION

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Vascular endothelial cells (EC) are primary targets of free radical/oxidant stress in many diseases including radiation toxicity. When endothelium is injured, the EC divide to repair the vascular barrier. This proliferative response probably occurs in a milieu of sustained free radical/oxidant stress. We hypothesize that proliferating EC have different susceptibilities to freee radical/oxidant stress depending on their position in the DNA synthetic or cell cycle. We hypothesize that these different susceptibilities are due partly to variations in antioxidant/free radical defense mechanisms. EC will be cultured from bovine aorta and irradiated (0-10 Gy, 137 Cs, gamma rays). Cewll cycle distribution will be quantified by flow cytometry. The EC will be enriched/synchronized in cell cycle by centrifugal elutriation, fluorescence activated cell sorting and (3H) thymidine suicide. Cytotoxicity, acute and proliferative cell death, will be assessed by the number of adherent cells, release of lactate dehydrogenase and survival parameters derived from colony counts. Superoxide dismutase, and catalase activities plus components of the glutathione redox cycle (reduced (GSH) and oxidized glutathione, and the enzymatic activities of glutathione peroidase, glutathione reductase, glutathione synthetases, glucose-6- phosphodehydrogenase and 6-phosphogluconate dehydrogenase) will be measured. Innate levels of the antioxidant defenses will be related to cell cycle position and cytotoxicity both before and after irradiation. The defenses then will be blocked (DDTC, ATZ, selenium-deficient medium, BCNU, BSO and CDNB). Next, in the presence and absence of the blockers, the enzyme activities will be raised by treatments with unaltered, polyethylene glycol conjugated or liposome encapsulated enzymes. The effects of these alterations on cytotoxicity and cell cycle progression (monitored by flow cytometry and autoradiography) will be measured. These experiments will clarify mechanisms of EC destruction and proliferation and should help with the development of specific treatments for cardiopulmonary and vascular disease.