Apoptosis in chronic rejection of human cardiac allografts

Background. We investigated the role of apoptosis (programed cell death) in the pathogenesis of chronic rejection. Methods. Epicardial coronary arteries from cardiac allografts with chronic rejection were examined for apoptosis by the TUNEL assay. Double labeling was carried out using anti-CD3, anti-CD68, and anti-von Willenbrand factor (vWF) monoclonal antibodies. Additional immunostaining was carried using anti-Fas, anti-Fas-L, and anti-Bcl-2 monoclonal antibodies. Apoptosis-associated oligonucleosomal DNA degradation was assessed by DNA agarose gel electrophoresis. The transcription level of apoptosis-related caspase genes were determined using microarrays. Results. Apoptotic cells (TUNEL+) were detected within the arterial wall and in perivascular areas. Double labeling demonstrated that apoptotic cells included T cells (CD3+), monocyte/macrophages (CD68+), and vascular endothelial cells (VWF+). Numbers and densities of TUNEL+ cells did not correlate with the degree of arterial stenosis. Apoptosis-associated oligonucleosomal DNA degradation was assessed by agarose gel electrophoresis of DNA, which showed DNA fragments of approximately 180 bp and multimers thereof (DNA laddering gel), which are characteristic for DNA fragmentation in apoptotic cells. Microarray analysis demonstrated that the apoptosis related caspases 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, were all transcribed (caspases 8, 9, and 10 were highly up-regulated). These results are consistent with the involvement of apoptosis in chronic rejection. Immunoreactivity for Fas/Fas-L was present at the sites of apoptotic cells. Immunoreactivity for Bcl-2 was present in areas with very few apoptotic cells. Conclusions. Apoptotic cells include T cells, monocyte/macrophages, and endothelial cells. Apoptosis, likely through the Fas/Fas-L system, is involved in the pathogenesis of chronic rejection in cardiac allografts.