EXPRESSION OF BCL-2 IN LIVER GRAFTS AFTER ADENOVIRAL TRANSFER IMPROVES SURVIVAL FOLLOWING PROLONGED ISCHEMIA AND REPERFUSION IN RAT LIVER TRANSPLANTATION

Abstract

P50 Aims: Apoptosis represents a crucial mechanism of ischemia-reperfusion related graft injury after liver transplantation. Apoptosis may be inhibited by overexpression of the bcl-2 gene. Aim of the present study was to investigate the effects of adenoviral bcl-2 transfer into donor livers on ischemia and reperfusion injury and survival after rat liver transplantation. Methods: A non-replicative adenovirus, expressing bcl-2 under control of a tetracyclin-inducable promoter (adv TetOn bcl-2) was generated in 293 cells and hepatocytes in culture. Male Lewis rats (170-200g; n=11) were treated i.v. with 1x1010advTetOn bcl-2 in combination with a second adenovirus which transfers the TetOn repressor protein under control of a CMV promoter (advCMVRep) at a ratio of 4:1 at 48h prior to organ harvest. Induction of the virus was achieved by admixture of 0,286 mg/100g doxycycline to drinking water. Controls were pretreated with a control adenoviral construct (advCMV GFP) (n=12), or with solely 0,286 mg/100g doxycycline (n=12). Liver transplantations including arterial reconstruction were performed after cold graft storage in UW for 16h. Intrahepatic expression of bcl-2 was displayed by western blot and immunohistology. Survival was monitored for 7 days, which was considered as definite, tissue specimen were collected in a second set of experiments grafts at 24h and 7 days postreperfusion. Results: Following treatment with advTetOn bcl-2/adv CMVRep bcl-2 expression was evident in all grafts after 24h and 7 days in western blots and immunohistochemically, but was completely absent in control animals. Bcl-2 expression was detected in hepatocytes and to a high degree in hepatic endothelial cells. By means of TUNEL-assay a strikingly (p<0.05) reduced number of positive endothelial cells was evident after adenoviral bcl-2 transfer (0.1±0.3 cells/hpf, mean±SD) as compared to the control virus (4.8±2.3) and doxycyclin pretreated grafts (1.3±0.2). Survival after transplantation was 100% after bcl-2 treatment as compared to 50% in both control groups (p=0.035). Conclusions: Expression of bcl-2 following adenoviral transfer elevated survival from 50% to 100% after ischemia/reperfusion in rat liver transplantation. The protection of predominately endothelial cells may have protective effects on graft injury. The study show the feasibility of a transient, doxycyclin controlled adenoviral gene transfer in a transplantation model for solid organs and could thus represent a potential technique to prevent early graft injury following liver transplantation.