Fibrosis of the hepatic subendothelial (Disse) space is a prominent component of the inflammation elicited by ethanol, and appears to correlate with clinically significant liver disease. The lesion consists of a collagen-rich new-matrix that replaces the low- density matrix normally present in this location. In recent and on-going studies from this laboratory, the lipocyte (fat-storing or Ito cell) has been examined in pure primary culture and found to be a major source of several matrix proteins including collagen, laminin and proteoglycans. In vivo, cells referred to as 'myofibroblasts' have been identified following chronic alcohol consumption and localized specifically to areas of fibrogenesis. Morphologic studies suggest that lipocytes and myofibriblasts represent the same cell in various stages of transition. The working hypothesis for the present studies is that ethanol 'activates' lipocytes, causing their conversion from resting, vitamin A-storing cells to matrix-secreting myofibroblasts. The pathway of lipocyte activation is the principal focus of the proposed research. Several possibilities will be explored, including stimulation by acetaldehyde or by soluble mediators of inflammation released as by-products of ethanol metabolism. The first series of experiments will establish wheter lipocyte activation occurs as a direct result of ethanol metabolism. This will be accomplished using a novel cell culture approach, in which hepatocytes and lipocytes are co-cultured as 'microorganoids' and incubated with varying concentrations of ethanol. Additional studies will examine whether lipocyte activation is indirect, involving compounds produced by neutrophils of Kupffer cells previously exposed to conditioned medium from ethanol-treated hepatocytes. Lipocyte activation will be judged with studies of ultrastructure as well as quantitative measurements of matrix protein secretion, matrix gene expression, and cell proliferation. Recently developed methods for evaluating matrix gene expression and for culture of individual parenchymal and non- parenchymal cell populations allow a direct approach to the pathogenesis of this common and important condition.
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