טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentTreibich Anna
SubjectInvolvement of Heparanase in Fibrotic Liver Regeneration
in the Murine Model
DepartmentDepartment of Medicine
Supervisor Professor Gad Spira
Full Thesis textFull thesis text - English Version


Abstract

Liver diseases associated with fibrosis require therapeutic intervention of which resection is the most common surgical treatment. Given the impaired regenerative capacity of fibrotic liver, radical resection as a curative means is far from ideal. The need to better understand the mechanisms of liver regeneration under fibrosis conditions is thus of immense importance.

Under conditions of liver injury, hepatic stellate cells are transactivated, changing their quiescent phenotype to proliferative myofibroblasts that synthesize and secrete extracellular matrix proteins, MMPs and TIMPs. HSC were also shown to express heparanase, an endo-β-D-glucoronidase capable of cleaving HS chains. Heparanase was shown to be involved in a wide range of normal and pathological processes including angiogenesis, tumor metastasis and wound healing response. Given the above, we postulated that heparanase may have a role during the development of liver fibrosis and liver regeneration.

In the present study we used CCL4 to induce fibrosis in C57BL/6 mice. Fibrosis was associated with elevated levels of heparanase, MMPs and their inhibitor TIMP-1. Such animals demonstrated impaired capacity to regenerate following 70% partial hepatectomy. Partial hepatectomy was associated with a decrease in heparanase mRNA levels 24 hours post surgery.

Consistent with its down-regulation during remodeling, liver regeneration following 70% PHx in heparanase knock-out mice was not impaired in comparison to their respective controls, and even better on day five post surgery. Of note is the fact that in heparanase knockout mice undergoing 70% PHx the levels of MMP-2, MMP-3 and VEGF significantly exceeded that of the control mice suggesting either a compensatory mechanism for the lack of heparanase, or defect in normal control of various genes expression.

Co-culture of HSC and primary hepatocytes allows mimicking in part the complex relationship between liver resident cells and examining the effect of cross-talk between them on the expression of various genes.

Under co-culture conditions the response of activated HSC to primary hepatocytes was analogous to inactivation of HSC in terms of expression of the examined genes. Activated HSC clone CFSC-8B, derived from CCl4-fibrotic rat liver, regain their quiescent phenotype and stop proliferating. The inactivation process was, as expected, accompanied by up-regulation of heparanase.

Using heparanase and VEGF over expression models we were able to demonstrate a link between expressions of the two genes, pointing to heparanase’s possible role in regulating VEGF expression.