Apoptosis of endothelial cells precedes myocyte cell apoptosis in ischemia/reperfusion injury

Background-Apoptosis contributes to cell loss after ischemia/reperfusion in jury in the heart. This study describes the time course and level of apopto sis in different cell types in the intact heart during ischemia/reperfusion injury. Methods and Results-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 s tained by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labe ling (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 isch emia alone. Early in reperfusion, TUNEL staining was colocalized with endot helial cells from small coronary vessels. Endothelial apoptosis peaked at 1 hour of reperfusion and, at this time, there was clear perivascular locali zation of apoptotic cardiac myocytes, whose number was inversely proportion al 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 ischemi a alone, but it colocalized with TUNEL staining over the 3 time points of r eperfusion, Immunocytochemical findings were confirmed by electron microsco py and Western blotting. Conclusions-In the very early stages of reperfusion, apoptosis is first see n in the endothelial cells from small coronary vessels. The radial spread o f apoptosis to surrounding cardiac myocytes suggests that reperfusion induc es the release of soluble pro-apoptotic mediators from endothelial cells th at promote myocyte apoptosis.