Molecular markers of apoptosis during acute rejection after heart transplantation

Acute rejection is an important cause of morbidity and occasional mortality after heart transplantation. The role of apoptosis in rejection has not been adequately defined. To clarify this issue we investigated the changes in several molecular markers of apoptosis and proteins involved in the regulation of the apoptotic pathway. Fas, bax, bak, bclxs and CPP32 act as pro- apoptotic regulators whereas bcl-2 acts as an anti-apoptotic regulator. Ten patients were prospectively followed for 3 months after heart transplantation. Biopsies were performed at 1,2,3,4,5,6,8,10 and 12 weeks and on clinical indication of rejection. 108 biopsies were obtained. Internationa Society of Heart & Lung Transplantation (ISHLT) grades of rejection were no rejection (60), Grade 1A (26), Grade 1B (6), Grade 3A (12), Grade 3B (2) and Grade 4 (2). All biopsies were examined for expression of bax, bcl-2, bclxs, bak, Fas and CPP32 by immunocytochemistry. The C-terminal deoxynucleotydyl transferase-mediated dUTP-fluoroscein nick end labelling (TUNEL) technique was applied to biopsies from patients before and during episodes of moderate/severe acute rejection. The TUNEL technique detected occasional foci of apoptotic cells in biopsies before rejection and an increase in the number of apoptotic cells during rejection. Fas, bax, bclxs and bak were strongly expressed in cardiac myocytes both prior to and during episodes of rejection, expression not appearing to change with rejection. CPP32 (pro-apoptotic) and bcl-2 (anti-apoptotic) were not expressed during no rejection, but were weakly expressed in cardiac myocytes during rejection episodes. Strong CPP32 and bcl-2 staining was seen in inflammatory cells during moderate/severe rejection. In conclusion although an increase in apoptotic cells was detected during rejection using the TUNEL technique, no change in the molecular markers were seen.