This proposal represents a continuation and expansion of our initial objective of attempting to understand the molecular mechanisms responsible for hepatic fibrogenesis. The final common pathway which occurs in all forms of liver fibrosis regardless of etiology is the increased deposition of extracellular matrix. We hypothesize that this process, which includes cell damage, recruitment and proliferation of inflammatory and mesenchymal cells, as well as extracellular matrix synthesis and degradation, is orchestrated by a variety of soluble peptides including cytokines.
The aims of the present proposal are to define the role of these modulatory cytokines in hepatic fibrosis and to investigate the mechanisms by which these effector proteins can be regulated.
Specific Aims : 1. To delineate the temporal expression of the cytokines involved in the pathogenesis of alcoholic liver disease. 2. To determine which cell types are synthesizing cytokines in alcoholic liver disease. 3. To ascertain how ethanol influences cytokine gene expression. 4. To determine the effects of prostaglandin E2 (PGE2) on matrix deposition, cytokine synthesis and transforming growth factor-beta (TGF-beta)receptors. 5. To investigate the effects of inhibiting cytokine activity in our rat model of alcoholic liver disease. Methods: The effects of the fibrogenic cytokines on extracellular matrix protein synthesis and deposition will be studied in primary cultures of hepatocytes, Ito cells, and Kupffer cells, and in the rat intragastric feeding model of alcohol-induced liver fibrosis. Cytokine and extracellular matrix synthesis will be evaluated by Northern hybridization analysis, nuclear run-on assays, morphological analysis, biochemical determinations, and RIAs. In situ hybridization analysis will be employed to localize cytokines in the animal model of alcoholic liver disease and in human liver specimens. The significance of PGE2 as an antifibrogenic agent will be evaluated in the same in vivo and in vitro model systems, while its effects on TGF-beta receptors will be investigated by affinity cross-linking studies. TGF-beta and interleukin-I activity will be inhibited in the in vivo model by the use of specific receptor antagonists. Health Relatedness: By accomplishing the aims of this proposal we shall formulate a general model for the pathogenesis of alcoholic liver disease and thereby aid in the formulation of rational therapy.
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