Role of mitochondria in chemically-induced hypoxie injury of EAhy926 endothelial cells

Complete or partial occlusion of blood vessels results in anoxic or hypoxic injury to the affected tissues. Endothelial cells, which line the blood vessels, and their mitochondria are probably the first to be affected by the oxygen loss. This is due to the proximity of the cells to oxygen and the dependence of mitochondria on oxygen to produce energy. To ascertain the role of mitochondria in the hypoxic injury process we have used a membrane potential sensitive dye. We have observed that the mitochondria typically go from a high membrane potential state to one significantly lower within 1 hour of chemically-induced hypoxia. This loss of membrane potential is accompanied by a more than 80% drop in ATP content as determined by the luciferin /luciferase assay. The cells start to die with the first hour as determined by the decrease of calcein (a vital dye) fluorescence. However, complete cell death does not occur until 6-7 hours later as determined by propidium iodide incorporation. Following the decrease in Δψ, a decrease in mitochondrial calcein fluorescence intensity occurs indicative of the mitochondrial permeability transition. These data suggest that mitochondria are primary targets of hypxoic injury in endothelial cells.