In vitro effects of hypoxia and reoxygenation on human umbilical endothelial cells

We investigated metabolic changes in human umbilical venous endothelial cells, when these were incubated under hypoxic followed by hyperoxic conditions, thus simulating hypoxia and reoxygenation. The human umbilical venous endothelial cells were incubated with a degassed buffer (oxygen content: 0-0.5%) for either 3 h or 24 h, followed by a 60 min incubation with oxygen-perfused buffer (oxygen content: 100%). Three hours of hypoxia led to a slight decrease in the ATP and creatine phosphate content (-16% +/- 5%), while a pronounced decrease of high energy phosphates (-54% +/- 4%) was observed after 24 h of hypoxia. Reoxygenating the cells after 3 h of hypoxia led to restoration of the content of high energy phosphates, while reoxygenation after 24 h resulted in a strong decrease (-66% +/- 4%). The prostaglandin I2 release during the first 3 h of hypoxia exceeded the release in the following 21 h. In all cases, reoxygenation increased the prostaglandin I2 release. Under normoxic conditions the ratio between oxidised glutathione and reduced glutathione shifted from 1:100 to 1:4.5 after 3 h of hypoxia. The content of lipid peroxidation products was almost unaffected during hypoxia, whereas reoxygenation resulted in a pronounced increase (+380% +/- 60%). The results of this in vitro study suggest that relatively long periods of hypoxia lead to a deficiency of high energy phosphates in the cell. Reoxygenation leads to the formation of oxygen-derived radicals, irrespectively of a prior hypoxia.