ABSTRACT
Hepatic stellate cells (HSC) and liver myofibroblasts (MFB) are two cell populations most likely responsible for the synthesis of most connective tissue components in fibrotic liver. They differ in their origin and location, and possibly in patterns of gene expression. Normal and carbon tetrachloride-cirrhotic livers from rats were used to isolate HSC. Liver was perfused with pronase and collagenase solutions, followed by centrifugation of the cell suspension on a density gradient. HSC were quiescent 2 days after plating on plastic but they became activated after another 5 days in culture. When the culture was passaged 5 times, its character changed profoundly as HSC were replaced by MFB. Microarray analysis was used to determine gene expression in quiescent HSC, activated HSC and MFB. The expression of 49 genes coding for connective tissue proteins, proteoglycans, metalloproteinases and their inhibitors, growth factors and cellular markers was determined. The pattern of gene expression changed during HSC activation and there were distinct differences between HSC and MFB. Little difference between normal cells and cells isolated from cirrhotic liver was found.
Subject(s)
Connective Tissue/metabolism , Fibroblasts/metabolism , Liver/metabolism , Myocytes, Smooth Muscle/metabolism , RNA, Messenger/biosynthesis , Animals , Carbon Tetrachloride/toxicity , Cells, Cultured , Connective Tissue/drug effects , Extracellular Matrix/chemistry , Fibroblasts/cytology , Fibroblasts/drug effects , Gene Expression , Immunohistochemistry , Liver/cytology , Liver/drug effects , Liver Cirrhosis, Experimental/metabolism , Metalloproteases/genetics , Metalloproteases/metabolism , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/drug effects , Oligonucleotide Array Sequence Analysis , Proteoglycans/genetics , Proteoglycans/metabolism , RNA, Messenger/drug effects , Rats , Reverse Transcriptase Polymerase Chain Reaction , Tissue Inhibitor of Metalloproteinases/genetics , Tissue Inhibitor of Metalloproteinases/metabolismABSTRACT
Hepatic stellate cells (HSC) are located in Disse spaces of normal rat liver. In their quiescent state they serve as a storage site for vitamin A. In fibrotic liver they become activated, proliferate and they undergo transdifferentiation into myofibroblast-like cells. Changes in the cell phenotype are accompanied by changes in the cellular cytoskeleton. We have studied the expression of alpha-smooth muscle actin and intermediate filament proteins vimentin, desmin and glial fibrillary acidic protein (GFAP) by immunocytochemistry in HSC cultured for 2 or 7 days after isolation. Normal or cirrhotic rat liver was perfused with solutions of pronase and collagenase and HSC were isolated by density gradient centrifugation of the resulting cell suspension. Liver cirrhosis was produced in rats by repeated carbon tetrachloride administration. Vimentin was detected in all cells from normal and cirrhotic liver. The concentration of desmin in the cells from cirrhotic liver was slightly higher than that in normal cells and it increased with time in culture. GFAP could be detected only in normal cells 2 days after their isolation. In contrast, alpha smooth muscle actin (alpha-SMA) was absent from normal cells at this time but its expression was pronouced later. In most cells from cirrhotic liver this antigen was already present on the second day of culture and its expression further increased.
