ADENOVIRUS-MEDIATED INDUCIBLE NITRIC-OXIDE SYNTHASE GENE-TRANSFER INHIBITS HEPATOCYTE APOPTOSIS

Background. Apoptosis limits hepatocyte viability in bioartificial liv ers in vitro and may contribute to liver dysfunction in vivo. Nitric o xide (NO) inhibits hepatocyte apoptosis; however, methods to deliver N O in a sustained manner to hepatocytes are limited. Here, we tested th e feasibility of inducible NO synthase (iNOS) gene transfer as an appr oach to deliver an intracellular source of NO to inhibit spontaneous a nd tumor necrosis factor-alpha (TNI-alpha)-induced apoptosis in cultur ed hepatocytes. Methods. An adenoviral vector carrying the human iNOS gene (AdiNOS) was used to overexpress iNOS in cultured rat hepatocytes . Spontaneous apoptosis was induced by prolonged culture (4 days), and stimulated apoptosis was induced by exposure to TNF-alpha + actinomyc in D (TNF-alpha+ActD). Nitrite (NO2-), cell viability and cellular cas pase-3-like protease activity were measured. Results, AdiNOS gene tran sfer resulted in sustained NO production and protected hepatocytes fro m spontaneous and TNF-alpha+ActD-induced apoptosis. Apoptosis was asso ciated with increases in caspase-3-like protease activity which was su ppressed by iNOS gene transfer in an NO-dependent manner Dithiothreito l partially reversed the NO-induced suppression of caspase-3-like acti vity, which is consistent with S-nitrosylation of caspase-3. Conclusio ns. Adenovirus-mediated iNOS gene transfer effectively blocks spontane ous and TNF-alpha+ActD-induced cell killing in hepatocytes. iNOS gene transfer could be used to suppress apoptotic hepatocyte death in vitro and possibly in vivo.