Recent work from this laboratory and others has shown that alcoholic liver disease is associated with oxidative stress via mechanisms most likely involving Kupffer cells, yet effective therapeutic modalities are still lacking. This led us to hypothesize now that delivery of antioxidant genes would prevent early hepatic damage due to alcohol. The first goal of these studies is to prevent oxidative stress by gene delivery using superoxide dismutase(SOD) and catalase to small Wistar rats required for the Tsukamoto-French model. Initially, recombinant adeno associated virus (rAAV) vectors containing transgenes for the jellyfish (Aequorea victoria) green fluorescent protein (GFP) will be injected into small male and female Wistar rats to determine the time course of gene transduction and protein expression after vector injection. Gene transduction and protein expression will be quantitated using in situ PCR and fluorescence image-analysis, respectively. Next, similar experiments will be performed with rAAV containing SOD and catalase transgenes at various dose levels. The protective effect of delivery of SOD/CAT genes against oxidative stress will be assessed in a model of hypoxia/reoxygenation in perfused liver. We predict that hepatic expression of SOD/CAT will protect the liver from oxidative stress. Our second goal is to determine whether Kupffer or parenchymal cells predominate in mechanisms of oxidative stress. To do this, Kupffer and parenchymal cells will be selectively targeted with rAAV containing SOD/CAT. Initially, to determine optimal doses and times for maximal gene incorporation selectively into isolated Kupffer cells, rAAV containing GFP transgenes will be given to isolated cultured Kupffer cells for various times and gene transduction and protein expression monitored. Seeding of liver by injection of transduced Kupffer cells after elimination of native Kupffer cells with gadolinium chloride (GGdCl3) will be monitored via real-time video microscopy of GFP fluorescence in living animals using techniques developed by our team. Parenchymal cells will be selectively transduced in vivo in GdCl3-treated rats. Protection from oxidative stress will be assessed in the perfused liver with SOD/CAT exclusively in Kupffer or parenchymal cells. We expect that selectively targeting Kupffer and parenchymal cells with SOC/CAT will determine which cell-type predominates in oxidative stress associated with early liver injury due to alcohol. Our third goal will be to prevent alcoholic liver injury in vivo with gene delivery using a clinically relevant enteral alcohol administration model, the so-called gold standard. Accordingly, male and female Wistar rats will be injected with rAAV containing SOC/CAT. Animals will be placed on the Tsukamoto-French enteral alcohol delivery protocol for at least two months, and livery injury will be assessed from enzyme release and histology. Hypoxia and free radicals will be measured using a 2- nitroimidazole hypoxia marker and the spin trapping technique with electron spin resonance, respectively. We expect that oxidative stress will explain gender differences in alcoholic liver injury and that delivery of SOD/CAT genes in a rAAV vector targeting Kupffer cells will prevent this disease completely.
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