Proteoglycan Turnover in Hepatic Stellate Cell Activation

Background: Chronic liver disease is a leading cause of death and morbidity worldwide. In liver injury, much is known about the changes in expression of collagens and matrix metalloproteinases, which occurs concomitant with decreased fibroblast apoptosis.  However, little is known about changes in expression of proteoglycans and the newly identified ADAMTS (A Disintegrin And Metalloproteinases with Thrombospondin Motifs) enzymes which cleave these proteoglycans. In other tissues, proteoglycans such as versican are key components of fibrotic extracellular matrix that have a structural role and modulate cellular functions; however none have been extensively studied in the liver.  Isolated hepatic stellate cells have been shown to activate in culture, expressing alpha-smooth muscle actin which may mimic their pathological activation in fibrogenesis, and has previously been shown to induce expression of ECM constituents. Methods: This project investigates changes in expression of versican and other candidate extracellular matrix proteins, as well as ADAMTS enzymes, in cultured hepatic stellate cells at 0-21 day time points.  Expression was analyzed using both quantitative and qualitative PCR and agarose gel electrophoresis.    Stellate cells were isolated from the livers of C57BL16 mice.  RNA was extracted and analyzed for purity using spectrophotometry and integrity by agarose gel electrophoresis.  Both RT-PCR and qPCR analysis were peformed using normalized concentrations of RNA starting material and validated primer sets. Results: Immunofluorescence analysis of isolated stellate cells showed alpha-smooth muscle actin expression supportive of fibroblast activation as well as desmin staining confirming stellate cell purity.  Candidate proteoglycans and ADAMTS enzymes were expressed at all time point of cultured stellate cells.  Expression of versican was found to increase as activation progressed, first modestly, then up to 5 fold by day 14.  Expression of associated processing enzymes ADAMTS 1 and 4 was suppressed during early activation and returned to day 1 levels by day 10. Conclusion: In other tissues and fibrotic tumor stroma, proteoglycans appear to play both a structural and regulatory role in the wound healing response through a diverse range of mechanisms.  The suppression of processing enzymes during early fibroblast expression may contribute to the formation of a versican-rich matrix which supports the proliferation and remodeling of liver extracellular matrix seen in liver disease.