Long-term goals of this research program are to elucidate cellular mechanisms underlying alcoholic liver fibrogenesis and to devise therapeutic modalities which are aimed at intervention of such mechanisms. During evolution of experimental alcoholic liver fibrosis in our rat model, Ito cells, perisinusoidal cells believed to be the primary source of extracellular matrices in the liver, undergo two distinct phases of activation characterized as the early proliferative and late fibrogenic responses. Our experimental evidence implicates two classes of mediators in initiation and acceleration of the Ito cell activation. They are cytokines and lipid peroxidation aldehydic products. Our findings to date support the paracrine and autocrine mechanisms of Ito cell activation which involve fibrogenic (TGFBeta) and mitogenic (PDGF, TGFalpha) cytokines. Both in vitro and in vivo evidence are also available to incriminate aldehydic products of lipid peroxidation such as malondialdehyde (MDA) and 4-hydroxynonenal (4HNE), as additional inducers of Ito cell collagen gene expression. To further delineate the specific roles, sources, and mechanisms of actions of these mediators in alcoholic liver fibrosis, we propose to test the following hypotheses: 1) proliferative and fibrogenic activation of Ito cells is initiated by enhanced paracrine stimulation with cytokines from Kupffer and sinusoidal endothelial cells, and perpetuated by induction of the autocrine mode of self-activation; 2) lipid peroxidation aldehydic products (MDA, 4HNE) their effects on cytosine gene expression by Kupffer and endothelial cells; 4) a high fat diet, a known nutritional factor for induction of alcoholic liver fibrosis, sensitizes Ito cells for mitogenic and fibrogenic effects of cytokines via alterations in expression of autocrine cytokines and their receptors; and 5) alcoholic liver fibrosis can be prevented if activities of mitogenic and fibrogenic cytokines and hepatic lipid peroxidation are suppressed. To test these hypotheses, we will examine expression of cytokines, their receptors, and aldehyde-protein adduct epitopes in Ito, Kupffer, and sinusoidal endothelial cell freshly isolated from the rat characterize the effects of MDA and 4HNE on Ito cell gene expression of matrix proteins and autocrine cytokines (TGFalpha, IFG-I and TGFBeta1), as well as gene expression of paracrine cytokines (TGFalpha, PDGF, TGFBeta, IL-6) by Kupffer, and endothelial cells. Lastly, we will test in our models anti- fibrotic modalities intended to block the actions of the proposed mediators by administration of: 1) anti-TGFBeta1 IgG or decorin to neutralize the fibrogenic TGFBeta activity; 2)interferon gamma to suppress the mitogenic and fibrogenic activation of Ito cells; and 3) glutathione monoester to replete a mitochondrial glutathione pool and to prevent hepatic lipid peroxidation and generation of MDA and 4HNE.
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