טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentOhayon Olga
SubjectHeparanase Involvment in Hepatic Stellate Cell
Differentiation and Liver Remodeling
DepartmentDepartment of Medicine
Supervisor Professor Gad Spira
Full Thesis textFull thesis text - English Version


Abstract

Substantial evidence now exists to recognize hepatic stellate cells (HSC) as the main matrix-producing cells in the process of liver fibrosis.

Heparanase, an endo-β-D-glucoronidase, capable of cleaving heparan sulfate chains, was shown to have a role in a wide range of normal and pathological processes, such as angiogenesis, tumor metastasis and wound healing responses.

This research project had three main objectives. First, to explore the association between heparanase accumulation in the nucleus of HSC and HSC differentiation. Second to follow the expression of heparanase in thioacetamide induced fibrotic rats subjected to 70% PHx and the healing process. Finally, to explore the efficiency of a combined, antifibrotic and gene therapy on the capacity of fibrotic rats to regenerate following 70% PHx.

In the first part we utilized both primary HSC culture and HSC clones derived from a cirrhotic rat liver, CFSC-8B and CFSC-3H. Applying the pull-down assay to CFSC-8B cells we demonstrated a complex comprising heparanase, HSP-90 and β-actin. Treatment of CFSC-8B cells with either geldanamycin or cytochalasin-D, inhibitors of HSP-90 and β-actin respectively resulted in elevated levels of heparanase in the nucleus, expression of HSC differentiation markers and activation of the canonical and non canonical Wnt signaling pathways respectively.

In the second part of the study we followed the expression of heparanase in TAA induced. Our study showed that heparanase mRNA expression levels correlate with those of VEGF during the induction and recovery stages of liver fibrosis. We further demonstrated that treating fibrotic rat livers with halofuginone, a multipotent antifibrogenic drug, and subsequently subjecting them to HBT with human VEGF-165 resulted in elevated expression of heparanase mRNA. Moreover, these rats demonstrated an improved capacity to regenerate following 70% partial hepatectomy. In vitro, HF stimulated heparanase and VEGF mRNA expression in HSC.

Taken together, our results suggest that heparanase translocates from the cytoplasm to the nucleus and back, that the translocation of the protein is via HSP-90 and β-actin, and that the increase in heparanase content in the nucleus induces cell differentiation and activation of the Wnt signaling pathway. In addition, halofuginone enhances heparanase and VEGF expression and promotes liver regeneration.