Endothelial cell-derived
oxygen free radicals are important mediators of postischemic injury; however, the mechanisms that trigger this radical generation are not known, and it is not known if this process can occur in human cells and tissues. The
enzyme xanthine oxidase can be an important source of radical generation; however, it has been reported that this
enzyme may not be present in human endothelium. To determine the presence and mechanisms of radical generation in human vascular endothelial cells subjected to
anoxia and reoxygenation, electron paramagnetic resonance measurements were performed on cultured human aortic endothelial cells using the spin trap 5,5-dimethyl-1-pyrroline N-
oxide (DMPO). These measurements were correlated with cellular injury,
xanthine oxidase activity, and alterations in cellular
nucleotides. Upon reoxygenation after 60 min of
anoxia, large
DMPO-OH (aN = aH = 14.9 G) and smaller DMPO-R (aN = 15.8 G, aH = 22.8 G) signals were seen.
Superoxide dismutase totally quenched this radical generation. The ferric
iron chelator deferoxamine prevented cell death and totally quenched the DMPO-R signal with a 40% decrease in the
DMPO-OH signal.
Xanthine oxidase was shown to be present in these cells and to be the primary source of
free radicals. While the concentration of this
enzyme did not change after
anoxia, the concentration of its substrate,
hypoxanthine, markedly increased, resulting in increased
free radical generation upon reoxygenation. Thus, reoxygenated human vascular endothelial cells generate
superoxide free radicals, which further react with
iron to form the reactive
hydroxyl radical, which in turn causes cell death.
Xanthine oxidase was the primary source of radical generation with this process triggered by the breakdown of
ATP to the substrate
hypoxanthine during
anoxia.