Capillarization precedes fibrosis in alcoholic liver disease. In capillarization, sinusoidal endothelial cells (SEC) lose fenestrae and form a basement membrane, a loss of phenotype that has not been examined previously. We propose to examine determinants of SEC phenotype in a series of in vitro and in vivo experiments. In vitro, isolated cells will be examined in homotypic culture and in co-culture with other liver cells to determine whether SEC phenotype is regulated by paracrine regulation or by heterotypic contact. The in vitro studies will utilize cells isolated from normal rats and from liver disease models, notably models of steatosis, alcoholic liver disease and fibrosis using thioacetamide. Studies in SEC from liver disease models will examine changes in response to determinants of normal SEC phenotype by heterotypic cells. Studies in hepatocytes and stellate cells from liver disease models will look for changes in factors that determine SEC phenotype. A proteomic approach will be used to identify and examine proteins involved in paracrine regulation. In vivo studies will provide confirmation of the relevance of the soluble factors implicated as determinants of SEC phenotype using continuous intraportal infusions of the soluble factors or their inhibitors in normal rats or rats from the above mentioned liver disease models by monitoring changes in liver histology. Differences in gene expression between SEC from normal and liver disease model rats will be characterized to understand what regulates SEC phenotype. These studies will be used to identify markers of SEC that are altered in the models, to confirm the findings of the proteomic studies and to identify common pathways that regulate expression of genes that are expressed in normal SEC, but not in SEC isolated from liver disease models. ? ?
