Issue |
J Extra Corpor Technol
Volume 33, Number 3, September 2001
|
|
---|---|---|
Page(s) | 175 - 180 | |
DOI | https://doi.org/10.1051/ject/2001333175 | |
Published online | 14 August 2023 |
Simulation of Ischemic Reperfusion in Endothelial Cell Culture Increases Apoptosis
Circulatory Sciences Graduate Perfusion Program, Sarver Heart Center, University Medical Center, University of Arizona, Tucson, Arizona
* Address correspondence and reprint requests to: Douglas F. Larson, PhD, CCP, Room 4402, Department of Surgery, University of Arizona, Tucson, AZ 85724.
Received:
15
April
2000
Accepted:
22
May
2001
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 NOdonor (±)-S-nitroso-N-acetylpenicillamine (SNAP) at 100 µM. 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°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.
Key words: apoptosis / endothelial cell / annexin V / reperfusion injury / NFkB / nitric oxide
© 2001 AMSECT
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