Cationic colloidal silica membrane perturbation as a means of examining changes at the sinusoidal surface during liver regeneration

By employing the cationic colloidal silica membrane density perturbation te chnique, we examined growth factor receptor and extracellular matrix (ECM) changes at the sinusoidal surface during rat liver regeneration 72 hours af ter 70% partial hepatectomy (PHx). At this time after PHx, hepatocyte divis ion has mostly subsided, white sinusoidal endothelial cell (SEC) proliferat ion is initiating, resulting in avascular hepatocyte islands. Because of th e discontinuous nature of the surface of liver SEC, ECM proteins underlying the SEC, as web as SEC luminal membrane proteins, are available to absorpt ion to the charged silica beads when the liver is perfused with the colloid . Subsequent liver homogenization and density centrifugation yield two sepa rate fractions, enriched in SECs as well as hepatocyte basolateral membrane -specific proteins up to 50-fold over whole Liver lysates. This technique f acilitates examination of changes in protein composition that influence or occur as a result of SEC mitogenesis and migration during regeneration of t he liver. When ECM and receptor proteins from SEC-enriched fractions were e xamined by Western immunoblotting, urokinase plasminogen activator receptor , fibronectin, and plasmin increased at the SEC surface 72 hours after PHx. Epidermal growth factor receptor, plasminogen, SPARC (secreted protein, ac idic and rich in cysteine, also called osteonectin or BM40), and collagen I V decreased, and fibrinogen subunits and c-Met expression remained constant 72 hours after PHx when compared to control liver. These results display t he usefulness of the cationic colloidal silica membrane isolation protocol. They also show considerable modulation of surface components that may regu late angiogenic processes at the end stage of Liver regeneration during the reformation of sinusoids.