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, and this constitutes the major goal of this application. We hypothesize that Kupffer cell-derived factors/reactive species play a critical role in the stellate cell fibrogenic response. In this application we propose: 1) to explore 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. We will analyze whether the effect on collagen I expression involves transcriptional regulation by carrying out nuclear in vitro transcription run-on assays, promoter studies, mRNA stability, and Northern blot analysis. To study if protein synthesis is affected, the rate of collagen I synthesis and degradation will be calculated; 2) to determine if Kupffer cell-derived reactive species are the mediators for collagen I up-regulation in stellate cells, concentration of prooxidant and nitrosative species will be measured and the effect of incubating the co-cultures with antioxidants and inhibitors will be studied. Studies to identify the source of reactive species in the co-culture (e.g. NADPH oxidase, xanthine oxidase, mitochondria, cytochrome P450 2E1, and inducible nitric oxide synthase) will involve addition of inhibitors or chemical inducers. The use of conditioned medium from Kupffer cells added to stellate cells, and transfection with siRNA to selectively silence the sources of reactive species in one cell type or the other will help to dissect the contribution of each cell type to the effects observed in the co-culture; 3) to characterize the contribution of arachidonic acid, as a representative polyunsaturated fatty acid, on collagen I expression in the co-cultures and to compare it to that of other fatty acids. Mechanistic approaches will include evaluating the contribution of lipid peroxidation reactions and the arachidonic acid metabolic pathways; and 4) to assess the contribution of chronic ethanol feeding to collagen I expression by stellate cells in co-culture with Kupffer cells. The basal expression of collagen I in control stellate cells will be compared to that of stellate cells from ethanol-treated rats, and the contribution of reactive species will be assessed. Mechanistic studies will follow to understand how Kupffer cells modulate the fibrogenic response in stellate cells from ethanol-treated rats. We hope that this co-culture model 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.
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