The hepatocyte proteome in organotypic rat liver models and the influence of the local microenvironment

Lucas T. Vu, Sophia M. Orbach, W. Keith Ray, Margaret E. Cassin, Padmavathy Rajagopalan, Richard F. Helm

Liver models that closely mimic the in vivo microenvironment are useful for understanding liver functions, capabilities, and intercellular communication processes. Three-dimensional (3D) liver models assembled using hepatocytes and liver sinusoidal endothelial cells (LSECs) separated by a polyelectrolyte multilayer (PEM) provide a functional system while also permitting isolation of individual cell types for proteomic analyses. To better understand the mechanisms and processes that underlie liver model function, hepatocytes were maintained as monolayers and 3D PEM-based formats in the presence or absence of primary LSECs. The resulting hepatocyte proteomes, the proteins in the PEM, and extracellular levels of urea, albumin and glucose after three days of culture were compared. All systems were ketogenic and found to release glucose. The presence of the PEM led to increases in proteins associated with both mitochondrial and peroxisomal-based β-oxidation. The PEMs also limited production of structural and migratory proteins associated with dedifferentiation. The presence of LSECs increased levels of Phase I and Phase II biotransformation enzymes as well as several proteins associated with the endoplasmic reticulum and extracellular matrix remodeling. The proteomic analysis of the PEMs indicated that there was no significant change after three days of culture. These results are discussed in relation to liver model function. Heterotypic cell-cell and cell-ECM interactions exert different effects on hepatocyte functions and phenotypes.