Simulation of ischemic reperfusion in endothelial cell culture increases apoptosis.
The endothelial layer of the myocardial vasculature serves as an important protective barrier between blood and myocardium. Ischemic reperfusion (I/R) of the endothelium has been shown to initiate a series of events that leads to ischemic reperfusion injury in the heart. At the onset of ischemic reperfusion, endothelial cells initiate apoptosis, a process whereby the cells self-destruct. Ischemic reperfusion was simulated to study its effects on the induction of apoptosis in cultured human endothelial cells (ECV 304). In addition, the cells were treated with nitric oxide (NO) to test its effect on induction of apoptosis. To mimic hypoxia, four ECV 304 cultures were placed in a medium that had been bubbled with pure nitrogen gas for 24 hours. A continuous flow of nitrogen gas was applied to the culture flasks during the course of the 2-hour ischemic period. After 2 hours, the nitrogen was removed from the hypoxic cultures to simulate reperfusion. Exposure to NO was achieved through the NO-donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP) at 100 microM, Cell cultures were exposed to hypoxia only, hypoxia and SNAP, and SNAP only. One positive control was established by exposure to staurosporine. A second positive control was established by exposure to a 30-min heat treatment at 43 degrees C. Two cultures were left untreated to serve as negative controls. All cell cultures were incubated for 4 hours. Apoptosis was detected by the binding of annexin V-fluorescein isothiocyanate (annexin V-FITC). In addition, morphologic changes detected by electron microscopy were used. Apoptosis increased in all treated cultures, excluding SNAP only treated cells. It was concluded that I/R may lead to induction of apoptosis.
Holleyman
C
C
Circulatory Sciences Graduate Perfusion Program, Sarver Heart Center, University Medical Center, University of Arizona, Tucson 85724, USA.
Larson
D
D
Hunter
K
K
D017209:Apoptosis / Q000187:drug effects*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000473:pathology*; D006801:Humans; D066298:In Vitro Techniques; D017202:Myocardial Ischemia / Q000473:pathology*; D015428:Myocardial Reperfusion Injury / Q000473:pathology*; D009569:Nitric Oxide / Q000494:pharmacology; D010396:Penicillamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D014481:United States
C110881:S-nitro-N-acetylpenicillamine; D009569:Nitric Oxide; D010396:Penicillamine