Ph.D Thesis

Ph.D StudentGingis-Velitski Svetlana
SubjectRegulation of Heparanase Cellular Uptake and
DepartmentDepartment of Medicine
Supervisors PROF. Israel Vlodavsky
Full Thesis textFull thesis text - English Version


Heparanase is a mammalian endoglycosidase that degrades heparan sulfate (HS) at specific intrachain sites, an activity that is strongly implicated in cell dissemination associated with tumor metastasis, inflammation, and angiogenesis. Thus, heparanase bioavailability, accessibility, and activity should be kept tightly regulated. We provide evidence that HS is not only a substrate for, but also a regulator of heparanase. Co-localization studies and cell fractionation following heparanase addition have identified syndecan family members as molecules responsible for heparanase uptake. We hypothesized that in addition to such indirect effect, the released heparanase may also locally affect and activate neighboring cells such as endothelial cells. We provide evidence that addition of the latent heparanase to endothelial cells enhances Akt phosphprylation. Heparanase-mediated Akt phosphorylation was independent of its enzymatic activity or the presence of cell membrane HS proteoglycans and was augmented by heparin. Moreover, addition and overexpression of heparanase stimulated endothelial cell migration, invasion, survival and neovascularization. These results suggest, for the first time, that heparanase activates endothelial cells and elicits angiogenic responses directly. This effect appears to be mediated by as yet unidentified heparanase receptor. We examined the possible function of the mannose 6-phosphate receptor and low density lipoprotein-receptor related protein, recently implicated in cellular uptake of heparanase, as heparanase receptors mediating Akt phosphorylation. We found that addition of heparanase to MPR- or LRP-deficient fibroblasts elicited Akt activation indistinguishable from control fibroblasts. In contrast, disruption of lipid rafts abrogated Akt/PKB phosphorylation following heparanase addition. These results suggest that lipid raft resident receptor(s) mediate heparanase signaling. The clinical significance of heparanase in tumor progression and metastasis, as well as the fact that only one heparan sulfate degrading enzyme was identified, makes heparanase an attractive target for the development of anti heparanase drugs. We screened a panel of monoclonal antibodies in an attempt to identify a monoclonal antibody that will inhibit heparanase enzymatic activity. None of the antibodies tested was capable of heparanase inhibition. Interestingly, one hybridoma, 6F8, exhibited the opposite effect and significantly enhanced heparanase activity. The antibody facilitated invasion of tumor-derived cells in vitro, enhanced the migration of human keratinocytes and improved wound healing in vivo, in a mouse punch model. These results further support a role of heparanase in the course of wound healing and, moreover, suggest that monoclonal antibodies can be applied clinically for the enhancement, rather than inhibition, of selected enzymes.