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Human umbilical vein endothelial cells submitted to hypoxia‐reoxygenation in vitro: Implication of free radicals, xanthine oxidase, and energy deficiency
Author(s) -
Michiels Carine,
Arnould Thierry,
Houbion Andrée,
Remacle José
Publication year - 1992
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.1041530109
Subject(s) - xanthine oxidase , umbilical vein , allopurinol , chemistry , antioxidant , biochemistry , glutathione peroxidase , glutathione , radical , pharmacology , xanthine , hypoxia (environmental) , in vivo , in vitro , enzyme , oxygen , biology , medicine , microbiology and biotechnology , organic chemistry
Ischemia‐reperfusion is observed in various diseases such as myocardium infarct. Different theories have been proposed to explain the reperfusion injury, among them that the free radical generation plays a crucial role. To study the mechanisms of the reperfusion injury, a hypoxia (H)‐reoxygenation (R) model upon human umbilical vein endothelial cells in culture was developed in order to mimic the in vivo situation. Different parameters were quantified and compared under H or H/R, and we found that oxygen readmission led to damage amplification after a short hypoxia period. To estimate the importance of various causes of toxicity, the effects of various protective molecules were compared. Different antioxidant molecules, iron‐chelating agent, xanthine oxidase inhibitors, and energy‐supplying molecules were very efficient protectors. Synergy could also be observed between the antioxidants and the energy‐supplying molecules or the xanthine oxidase inhibitors. The toxic effect of O 2 ·( − ) could be lowered by the presence of SOD or glutathione peroxidase in the culture medium, whereas glutathione peroxidase was the most efficient enzyme when injected into the cells. The production of O 2 ·( − ) and of H 2 O 2 by endothelial cells was directly estimated to be, respectively, of 0.17 and 0.035 μmol/min/mg prot during the R period. O 2 ·( − ) production was completely inhibited when allopurinol was added during H and R. In addition, a xanthine oxidase activity of 21.5 10 −6 U/mg prot could be observed by a direct assay in cells after H but not in control cells, thus confirming the previous conclusions of xanthine oxidase as a potent source of free radicals in these conditions. Thanks to the use of cultured human endothelial cells, a clear picture was obtained of the overall process leading to cell degenerescence during the reoxygenation process. We particularly could stress the importance of the low energetic state of these cells, which is a critical factor acting synergistically with the oxidant molecules to injure the cells. These results also open new possibilities for the development of new therapeutics for ischemia. © 1992 Wiley‐Liss, Inc.