Oxygen radicals generated during anoxia followed by reoxygenation reduce the synthesis of tissue-type plasminogen activator and plasminogen activator inhibitor-1 in human endothelial cell culture.
The effect of anoxia and reoxygenation on the synthesis and secretion of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) was studied in primary cultures of human umbilical vein endothelial cells. Sublethal anoxia, determined by trypan blue dye exclusion and lactate dehydrogenase release, was produced by cell culture under a 95% N2, 5% CO2 atmosphere for 2-24 h and was followed by reoxygenation with 95% air, 5% CO2 for 24 or 48 h. Anoxia did not alter the levels of mRNA for t-PA or PAI-1 in the cells or the secretion of t-PA or PAI-1 into the medium. At 24 h, t-PA secreted into conditioned medium was 7.0 +/- 1.4 ng/2 x 10(6) cells (n = 9) and PAI-1 was 300 +/- 13 IU/2 x 10(6) cells (n = 9), whereas the content of t-PA mRNA was 2.2 pg/micrograms of RNA and PAI-1 mRNA was 180 pg/micrograms of RNA. During reoxygenation, however, t-PA antigen and PAI-1 activity as well as mRNA for PAI-1 decreased proportionally to the duration of anoxia, to reach 27 +/- 1.0, 49 +/- 2.0, and 47 +/- 14% of control values, respectively, within 24 h of anoxia. t-PA mRNA also decreased significantly during reoxygenation following anoxia, but the extent could not be accurately quantitated. Addition, during anoxia, of a 200 micrograms/ml concentration of the superoxide anion radical scavenger superoxide dismutase or of a 5 mM concentration of the iron chelator deferoxamine mesylate prevented the subsequent decrease of t-PA antigen during reoxygenation; addition of these compounds during reoxygenation had no effect. Superoxide dismutase, but not deferoxamine mesylate, when added during anoxia prevented the subsequent decrease in PAI-1 activity. These studies suggest that the marked alteration of endothelial cell fibrinolysis during anoxia followed by reoxygenation is most likely mediated by a mechanism dependent on oxygen radicals. Impaired endothelial cell fibrinolysis may contribute to the pathophysiology of ischemia/reperfusion injury.
Shatos
M A
MA
Department of Surgery, University of Vermont College of Medicine, Burlington 05405.
Doherty
J M
JM
Stump
D C
DC
Thompson
E A
EA
Collen
D
D
D000332:Aerobiosis; D000693:Anaerobiosis; D002478:Cells, Cultured; D003676:Deferoxamine / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000378:metabolism*; D016166:Free Radical Scavengers; D005609:Free Radicals; D006801:Humans; D000860:Hypoxia / Q000378:metabolism; D007700:Kinetics; D015849:Plasminogen Inactivators / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000494:pharmacology; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis*
D016166:Free Radical Scavengers; D005609:Free Radicals; D015849:Plasminogen Inactivators; D013482:Superoxide Dismutase; D010959:Tissue Plasminogen Activator; D003676:Deferoxamine