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Apoptosis as well as necrosis may play an important role in hepatic ischemia/reperfusion (I/R) injury. Akt, a serine-threonine protein kinase, is known to promote cell survival. We investigated whether gene transfer of constitutively active or dominant negative Akt could affect hepatic I/R injury.Hepatic I/R injury was induced in rats by Pringle's maneuver for 20 min followed by reperfusion. Adenoviruses encoding a constitutively active form of Akt (myrAkt), a dominant negative form of Akt (dnAkt), or beta-galactosidase (LacZ) were injected through the tail vein 72 h before hepatic I/R.Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining demonstrated a significant increase in the positive cells 240 min after reperfusion. Immunoblotting with phospho-Akt antibody showed phosphorylation of Akt from 90 to 180 min after reperfusion. The expression of myrAkt reduced the number of TUNEL-positive cells and hepatic necrosis around the central veins in the liver after reperfusion. This expression also significantly inhibited the increase in serum alanine aminotransferase (297 +/- 131 IU/L, P0.05) 120 min after I/R, compared with increases in uninfected (1761 +/- 671 IU/L), LacZ adenovirus (1528 +/- 671 IU/L)-, and dnAkt adenovirus (1342 +/- 485 IU/L)-infected rats. MyrAkt expression phosphorylated Bad and inhibited the release of cytochrome-c after reperfusion. No difference in nuclear translocation of nuclear factor (NF)-kappaB, p65 was seen among the three groups of rats, however.Adenoviral gene transfer of myrAkt could inhibit apoptotic cell death and subsequent hepatic I/R injury in the rat, through Bad, not NF-kappaB.

Background. Proliferative cholangitis (PC) associated with hepatolithiasis results in stricture of the main bile ducts and is a major cause of residual and/or recurrent stones after repeated treatment for hepatolithiasis. The transcription factor E2F controls the expression of several genes involved in cell proliferation. The aim of this study was to inhibit PC using cytostatic gene therapy by transferring fusigenic anionic liposome–hemagglutinating virus of Japan (HVJ–anionic liposome) complexes containing a synthetic double-stranded oligodeoxynucleotide with high affinity for E2F (E2F decoy). Materials and methods. PC was induced by introducing a fine nylon thread into the bile duct in a rat model. HVJ–anionic liposomes containing the E2F decoy were administered directly into the biliary tract. HVJ–anionic liposomes containing a missense oligodeoxynucleotide (scramble decoy) were also given as a control. The count of peribiliary glands in the bile duct, 5′-bromodeoxyuridine (BrdU) labeling index, and immunohistochemical staining for proliferating cell nuclear antigen (PCNA) in the bile duct were compared among untransfected, scramble decoy-transfected, and E2F decoy-transfected rats. Results. E2F decoy-transfected bile ducts showed inhibition of the papillary proliferation of the biliary epithelium and peribiliary gland hyperplasia. BrdU incorporation and PCNA expression in the bile ducts were inhibited in E2F decoy-transfected rats. Conclusion. Our cytostatic gene therapy approach using direct E2F decoy transfer into the biliary tract suppressed PC in a rat model and may offer an effective therapeutic option for reducing recurrence following treatment for hepatolithiasis.

The effects of organ preservation on mitochondrial oxidative phosphorylation activity, adenylate hepatic energy charge, cytochrome content, and redox state of NAD+/NADH couple in rat liver transplantation were compared between a nonpreservation group and a preservation group with grafts preserved for 12 hr in Euro-Collin's solution. At 3 hr after transplantation, the energy charge in the preservation group decreased to 0.60 +/- 0.02 from the control value of 0.86 +/- 0.01, accompanied by a reduction of intramitochondrial redox state of NAD+/NADH couple. In contrast, in the nonpreservation group, the decrease in energy charge was minimally decreased to 0.79 +/- 0.04 due to the compensatory enhancement of mitochondrial oxidative phosphorylation activity. These results suggest that an enhanced mitochondrial ATP synthesis and a reduced intramitochondrial redox state are important factors affecting survival following rat liver transplantation.

Porcine livers preserved for 72 hr using continuous hypothermic perfusion (CHP) were studied in order to compare the effects of CHP on energy metabolism with those of simple cold storage (SCS). The livers of the CHP group were perfused in situ for 72 hr at 7 degrees C with University of Wisconsin (UW) solution and FC-43 as an oxygen carrier, while those of the S1 group were stored ex situ for 48 hr at 4 degrees C in UW solution, and those of the S2 group for 72 hr. They were then recirculated with human blood at 37 degrees C for 2 hr for the evaluation of their viability. At the end of preservation, significantly higher levels of total adenine nucleotides (TAN) and energy charge (EC) were observed in the CHP group compared with the S1 and S2 groups. At 2 hr recirculation, the level of TAN was significantly higher in the CHP group than in the S1 and S2 groups. The EC level was also higher in the CHP group than in the S2 group. During recirculation, the ketone body ratio (acetoacetate/beta-hydroxybutyrate) was higher in the CHP group than in the S2 group. The values in the CHP group were above 1.0 after 45 min recirculation. There were no significant differences in the pyruvate/lactate ratio and lactate level between the CHP and S1 groups. However, these values were significantly different from those in the S2 group. The present findings demonstrate that CHP using UW solution and the oxygen carrier was better able to preserve the energy metabolism of the porcine liver for 72 hr than 48-hr SCS in UW solution.