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 w
as carried out using anti-CD3, anti-CD68, and anti-von Willenbrand factor (
vWF) monoclonal antibodies. Additional immunostaining was carried using ant
i-Fas, anti-Fas-L, and anti-Bcl-2 monoclonal antibodies. Apoptosis-associat
ed oligonucleosomal DNA degradation was assessed by DNA agarose gel electro
phoresis. The transcription level of apoptosis-related caspase genes were d
etermined using microarrays.
Results. Apoptotic cells (TUNEL+) were detected within the arterial wall an
d in perivascular areas. Double labeling demonstrated that apoptotic cells
included T cells (CD3+), monocyte/macrophages (CD68+), and vascular endothe
lial 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 lad
dering gel), which are characteristic for DNA fragmentation in apoptotic ce
lls. 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 w
ere highly up-regulated). These results are consistent with the involvement
of apoptosis in chronic rejection. Immunoreactivity for Fas/Fas-L was pres
ent 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 end
othelial cells. Apoptosis, likely through the Fas/Fas-L system, is involved
in the pathogenesis of chronic rejection in cardiac allografts.