Project 173709

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 whereby Kupffer cells modulate the formation and secretion of the components of the scar tissue is of great relevance for potential therapeutic intervention. We hypothesize that Kupffer cell-derived factors/reactive species play a critical role in the stellate cell fibrogenic response. We propose to: 1) 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; 2) determine if Kupffer cell-derived reactive species are the mediators for collagen I up-regulation in stellate cells measuring the concentration of oxidative and nitrosative species and incubating the co-cultures with antioxidants and inhibitors. 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 disect the contribution of each cell type to the effects observed in the co-culture; 3) 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; and 4) assess the contribution of chronic ethanol feeding to collagen I expression by stellate cells in co-culture with Kupffer cells and to determine whether increased liver calcification ocurs and this event leads to the fibrogenic response.