Ischemia-reperfusion-induced apoptotic endothelial cells isolated from rat skeletal muscle.
BACKGROUND
The purpose of the present study was to investigate ischemia-reperfusion-induced apoptosis and necrosis in endothelial cells isolated from skeletal muscle.


METHODS
A vascular pedicle isolated rat gracilis muscle model was used. After surgical preparation, clamps were applied to the vascular pedicle to create 4 hours of ischemia and released for reperfusion (ischemia-reperfusion group, n = 9). Clamping was omitted in sham ischemia-reperfusion rats (sham ischemia-reperfusion group, n = 9). The muscle samples were harvested after 20 hours of reperfusion for the process of cell isolation. One hundred thousand cells from each sample were stained by monoclonal anti-CD146-fluorescein (a principal marker for mature endothelial cells), Annexin V-PE, or 7-aminoactinomycin D to detect and quantify apoptotic and necrotic cells. Twenty thousand cells from each sample were scanned and analyzed by flow cytometry.


RESULTS
The average +/- SEM of CD146-fluorescein-positive cells was 20.0 +/- 2.9 percent, suggesting that these cells might be endothelial cells from the muscle microvasculature. In the population of gated CD146-fluorescein-positive cells, the average percentage of apoptotic cells (stained by Annexin V-PE) was 15.9 +/- 2.2 percent in the sham ischemia-reperfusion group and 33.5 +/- 5.3 percent in the ischemia-reperfusion group (p < 0.01), the average percentage of necrotic/apoptotic cells (stained by both 7-aminoactinomycin D and Annexin V-PE) was 17.8 +/- 4.1 percent in the sham ischemia-reperfusion group and 39.2 +/- 3.1 percent in the ischemia-reperfusion group (p < 0.01).


CONCLUSIONS
Given the results of the present study, the authors hypothesize that the endothelial cells lining microscopic blood vessels are among the major contributors to ischemia-reperfusion-induced cell apoptosis and necrosis detected from rat skeletal muscle.
Wang
Wei Z
WZ
Las Vegas, Nev. From the Department of Surgery, Division of Plastic Surgery, University of Nevada School of Medicine.
Fang
Xin-Hua
XH
Stephenson
Linda L
LL
Khiabani
Kayvan T
KT
Zamboni
William A
WA
D000818:Animals; D017209:Apoptosis; D004195:Disease Models, Animal; D042783:Endothelial Cells / Q000473:pathology / Q000502:physiology*; D007511:Ischemia / Q000473:pathology / Q000503:physiopathology*; D008297:Male; D008833:Microcirculation; D018482:Muscle, Skeletal / Q000098:blood supply / Q000473:pathology / Q000503:physiopathology*; D009336:Necrosis; D051381:Rats; D017207:Rats, Sprague-Dawley; D015427:Reperfusion Injury / Q000473:pathology / Q000503:physiopathology*