Stromelysin-1 and hepatic stellate cells

Vyas, Samir Kumar (1996) Stromelysin-1 and hepatic stellate cells. University of Southampton, Doctoral Thesis.

Abstract

This thesis examines the synthesis and secretion of stromelysin-1 in a culture model of rat HSC activation, its role in HSC proliferation and its binding to the HSC surface membrane through a high affinity binding site.

Highly pure (>98% characterised by Vitamin A autofluorescence) HSC were prepared by pronase/collagenase perfusion, differential centrifugation and centrifugal elutriation. By Northern analysis, stromelysin-1 mRNA was undetectable in freshly isolated HSC, peaked after 3 days and declined below the detection limit at 7 days (n=6) relative to ribosomal S14 mRNA. Released stromelysin-1 activity was characterised by zymography of serum-free HSC conditioned media (HSCCM) in the presence of enzyme inhibitors and by Western blotting. Intracellular stromelysin was demonstrated in cultured desmin-positive HSC by dual indirect immunofluorescence. Interestingly, the intensity of stromelysin expression was vividly heterogeneous with large clumps of strongly positive, rounded cells containing pleomorphic nuclei. Continued stromelysin-1 activity was seen by zymography of HSCCM harvested after 3, 7, 14 and 21 days in culture but net quantitative ¹⁴C-β-casein degradation (per μg DNA) was reduced sevenfold in prolonged culture. Analysis of cell conditioned media by zymography showed that stromelysin release by HSC was downregulated by culture on a model basement membrane and was not a feature of cultured Kupffer cells, sinusoidal endothelial cells or hepatocytes.

Confocal autofluorescence subtraction microscopy displayed cell surface immunofluoresence for endogenously secreted stromelysin by freshly cultured HSC.

These data show that HSC synthesise and secrete stromelysin in early primary culture. I addition, expression of stromelysin-1 by cultured HSC may be important in permitting or mediation cellular proliferation in the presence of serum. Current evidence indicates this is an extracellular effect mediated by the proteinase activity of stromelysin and that hepatic stellate cells possess an as yet uncharacterised high affinity cell surface binding site for stromelysin-1.

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