Measurement and characterization of free radical generation in reoxygenated human endothelial cells

The endothelial cell is thought to be an important site of free radical generation in ischemic tissues. It has been demonstrated that endothelial cells from several species generate a burst of free radical generation upon reoxygenation; however, it has been suggested that human endothelial cells are not similarly capable of generating free radicals on reoxygenation. In view of the central importance of revascularization with accompanying reoxygenation in the clinical treatment of tissue ischemia/infarction, we have performed studies to determine the presence, mechanism, and kinetics of free radical generation in human endothelial cells. Therefore, we subjected cultured human umbilical vein endothelial cells to anoxia followed by reoxygenation. Cell suspensions of 10⁷ cells/ml were subjected to varying periods of anoxia and reoxygenation. On reoxygenation with addition of a 50 mM concentration of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), after 90 min of anoxia an electron paramagnetic resonance (EPR) signal was observed consisting of 2 components: a quartet 1:2:2:1 DMPO-OH signal, a(N) = A(H) = 14.9 G, and a six-peaked DMPO-R signal, a(N) = 15.6 G a(H) = 22.9 G, whereas cells in air gave no signal. The observed signal was quenched by superoxide dismutase (SOD) or catalase. Deferoxamine decreased the measured radical signals by 40%. Cyclooxygenase blockers did not decrease radical generation, but the xanthine oxidase blocker oxypurinol did decrease radical generation by 60%. Both the magnitude of radical generation and the cellular xanthine oxidase activity were measured to be 4- to 5-fold lower than that seen in identical preparations of bovine aortic endothelial cells, further suggesting that the presence and concentration of xanthine oxidase has a major influence on the magnitude of the radical generation. Thus human endothelial cells can generate oxygen free radicals when subjected to anoxia and reoxygenation. Both xanthine oxidase and redox cycling of chelatable iron are important mechanisms of this free radical generation.