Scarabelli T, Stephanou A, Rayment N, Pasini E, Comini L, Curello S, Ferrari R, Knight R, Latchman D
Medical Molecular Biology Unit, Institute of Child Health, University College London, UK.
Circulation. 2001 Jul 17;104(3):253-6. doi: 10.1161/01.cir.104.3.253.
Apoptosis contributes to cell loss after ischemia/reperfusion injury in the heart. This study describes the time course and level of apoptosis in different cell types in the intact heart during ischemia/reperfusion injury.
Isolated Langendorff-perfused rat hearts were subjected to perfusion alone (control) or to 35 minutes of regional ischemia, either alone or followed by 5, 60, or 120 minutes of reperfusion. Sections were stained by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and propidium iodide and with anti-von Willebrand factor, anti-desmin, or anti-active caspase 3 antibodies; they were then visualized by confocal microscopy. Sections were also examined by electron microscopy. No TUNEL-positive cells were seen in control hearts or hearts exposed to ischemia alone. Early in reperfusion, TUNEL staining was colocalized with endothelial cells from small coronary vessels. Endothelial apoptosis peaked at 1 hour of reperfusion and, at this time, there was clear perivascular localization of apoptotic cardiac myocytes, whose number was inversely proportional to their distance from a positive vessel. After 2 hours of reperfusion, apoptotic cardiac myocytes assumed a more homogeneous distribution. Active caspase 3 labeling was seen independent of DNA fragmentation during ischemia alone, but it colocalized with TUNEL staining over the 3 time points of reperfusion. Immunocytochemical findings were confirmed by electron microscopy and Western blotting.
In the very early stages of reperfusion, apoptosis is first seen in the endothelial cells from small coronary vessels. The radial spread of apoptosis to surrounding cardiac myocytes suggests that reperfusion induces the release of soluble pro-apoptotic mediators from endothelial cells that promote myocyte apoptosis.
细胞凋亡参与心脏缺血/再灌注损伤后的细胞丢失。本研究描述了在缺血/再灌注损伤期间完整心脏中不同细胞类型凋亡的时间进程和水平。
将离体的Langendorff灌注大鼠心脏单独进行灌注（对照），或进行35分钟的局部缺血，缺血后单独再灌注5、60或120分钟。切片用末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法（TUNEL）、碘化丙啶染色，并使用抗血管性血友病因子、抗结蛋白或抗活性半胱天冬酶3抗体进行染色；然后通过共聚焦显微镜观察。切片也通过电子显微镜检查。在对照心脏或仅接受缺血的心脏中未观察到TUNEL阳性细胞。在再灌注早期，TUNEL染色与小冠状动脉的内皮细胞共定位。内皮细胞凋亡在再灌注1小时达到峰值，此时凋亡的心肌细胞在血管周围有明显定位，其数量与它们距阳性血管的距离成反比。再灌注2小时后，凋亡的心肌细胞呈现更均匀的分布。在单独缺血期间可见活性半胱天冬酶3标记独立于DNA片段化，但在再灌注的3个时间点上它与TUNEL染色共定位。免疫细胞化学结果通过电子显微镜和蛋白质印迹法得到证实。
在再灌注的极早期，凋亡首先出现在小冠状动脉的内皮细胞中。凋亡向周围心肌细胞的径向扩散表明，再灌注诱导内皮细胞释放可溶性促凋亡介质，从而促进心肌细胞凋亡。
