Activation of stellate cells, a key issue in the pathogenesis of hepatic fibrosis, is mediated by factors released from damaged hepatocytes and activated Kupffer cells. Understanding the mechanisms by which Kupffer cells modulate the formation and secretion of the components of the scar tissue is of great relevance for potential therapeutic intervention. Our hypothesis is that Kupffer cell-derived factors/reactive species play a critical role in the stellate cell fibrogenic response. Our lab is interested in:

1. Exploring the impact of Kupffer cells on stellate cell collagen I production using an in vitro co-culture model of primary rat Kupffer cells and primary stellate cells.

2. Determining if Kupffer cell-derived reactive oxygen/nitrogen species are the mediators for collagen I up-regulation in stellate cells.

3. Studying the contribution of arachidonic acid, as a representative polyunsaturated fatty acid, to collagen I expression in the co-cultures.

4. Assessing the effects of chronic ethanol feeding to collagen I expression by stellate cells in co-culture with Kupffer cells and the potential "two-hit phenomenon". Mechanistic studies are under way to understand how Kupffer cells modulate the scarring process and extracellular matrix remodeling in stellate cells from ethanol-fed rats. We are confident that this approach will help us to define the effects of Kupffer cells on the stellate cell fibrogenic response and to dissect potential therapeutic targets for preventing liver disease.

Alcohol-induced liver injury involves significant mitochondrial damage and up-regulation of inducible nitric oxide synthase (NOS2) leading to excessive generation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) affecting cell survival. Understanding the molecular mechanisms of pathological nitric oxide (NO) overproduction by NOS2 is of great relevance for efficient pharmacotherapy to prevent alcohol hepatotoxicity.

Our laboratory has used a combination of a cutting edge proteomics technique along with a Systems Biology approach to elucidate mitochondrial proteins involved in ALD that could impact NO synthesis. Preliminary results in support of this Application identified argininosuccinate synthase (ASS) as up-regulated by chronic alcohol feeding. Furthermore, livers from patients with alcoholic liver disease (ALD) or with hepatocellular carcinoma also showed increased ASS suggesting a potential link between ASS and ALD. ASS is an enzyme from the urea and the L-citrulline/NO cycles which could have a rate-limiting role for high-output NO synthesis via NOS2.

Virtually nothing is known on how alcohol modulates ASS expression and how the L-arginine "recycling" pathway may impact NO generation and liver injury. We hypothesize that up-regulation of ASS by alcohol-derived species may increase the availability of intracellular substrate for NO synthesis by NOS2 contributing to the pathophysiology of ALD. We will test this hypothesis and study the mechanistic aspects involved in ASS induction, and the biological relevance along the following Specific Aims:

1. To dissect whether the alcohol-mediated up-regulation of ASS plays a role in increased NO synthesis by NOS2 in hepatocytes; the following possibilities will be addressed: Does the up-regulation of ASS by alcohol increase intracellular L-arginine availability for NO synthesis by NOS2 in hepatocytes? Could alcohol increase L-arginine influx playing a role in NO synthesis?

2. To identify the mechanism by which alcohol induces ASS, key questions considered are: Do increased ROS act as sensors leading to up-regulation of ASS by alcohol? Does ASS undergo S-nitrosylation to regulate NO production? If it occurs, this will indicate a novel feedback mechanism triggered by alcohol whereby NO limits its own synthesis by governing substrate regeneration for high-output NO synthesis.

3. To assess the biological relevance of the alcohol-mediated induction of ASS for NO synthesis in vivo, wild-type mice, Ass+/-, and mice injected with either EGFP.AAV8 or ASS.EGFP.AAV8 will be fed the control or the alcohol Lieber-DeCarli diets and liver function and biochemical measures of oxidative stress and nitro-cellular stress will be evaluated as a read-out for the contribution of ASS to liver injury.

Understanding the mechanisms by which reactive oxygen species modulate both the expression and splicing of the transcription factor KLF6 which plays a crucial role in the development of alcoholic liver disease, is of great relevance for potential therapeutic intervention. We believe that reactive oxygen species in general, and reactive oxygen species derived from cytochrome P450 2E1 metabolism in particular, may play a critical role in regulating KLF6 splicing and its biological actions.

We are currently exploring the significance of CYP2E1-derived reactive oxygen species modulation of KLF6 expression and splicing in CYP2E1-expressing cells and in primary hepatocytes isolated from chronic alcohol fed rats. Experiments are under way to evaluate the role of stress-activated kinases in modulating the up-regulation and splicing of KLF6 under reactive oxygen species. We plan to analyze the effects of prooxidants and of glutathione depletion and to evaluate the contribution of each KLF6 splice isoform to the effects mediated by prooxidants in selected KLF6 down-stream targets. In addition, we are establishing the relevance in vivo of the spliced isoforms KLF6_V1 and KLF6_V2 in alcoholic liver disease using animal models.