Liver regeneration involves inflammatory cells and mediators which activate hepatic stellate cells (HSCs). HSCs release proteases that degrade components of the extracellular matrix (ECM) causing the release of growth factors such as hepatocyte growth factor (HGF). This initiates the proliferative phase (response of hepatocytes to HGF) then the resolution phase (ECM production and remodelling). The ECM components that bind the growth factors for regeneration are not known, however heparan sulfate proteoglycans (HSPGs) have been implicated. The aim of this project is to investigate the role of perlecan, an extracellular HSPG in liver development as a model of liver regeneration using wildtype (C57BL/6) and exon-3 deficient mice (HSPG2Δ3/Δ3).
Liver tissues were sectioned and probed for the presence of perlecan, HS and chondroitin sulfate (CS). Perlecan was localised around the periphery of both C57BL/6 and HSPG2Δ3/Δ3 mice livers however expression was remarkably reduced in HSPG2Δ3/Δ3 mice. Staining could be seen as patches surrounding hepatocytes, however no perlecan was detected in basement membrane structures. Neither HS (Ab10E4) nor CS (AbCS56) was detected in liver sections however staining for HS stub (Ab3G10) was seen in C57BL/6 mice livers. Comparisons were made to 20 week old human foetal liver samples. Perlecan, HS and CS were detected around basement membrane structures.
Liver tissues were homogenised and enriched for proteoglycans by anion exchange chromatography and then probed by ELISA. Proteoglycan-enriched fractions of C57BL/6 were found to contain perlecan, HS and CS. In contrast, perlecan, HS and CS showed reduced expression in HSPG2Δ3/Δ3 liver tissue.
Western blotting indicated that perlecan present in the livers of C57BL/6 mice was full length (~460 kDa) and decorated with HS while perlecan expression was reduced in HSPG2Δ3/Δ3 liver tissue and was not decorated with HS or CS.