Heparanase is an endoglycosidase which cleaves heparan sulfate (HS) and hence participates in degradation and remodeling of the extracellular matrix (ECM). Heparanase is preferentially expressed in human tumors and its over-expression in tumor cells confers an invasive phenotype in experimental animals. The enzyme also releases angiogenic factors from the ECM and thereby induces an angiogenic response in vivo. Heparanase upregulation correlates with increased tumor vascularity and poor postoperative survival of cancer patients. Heparanase is synthesized as a 65 kDa inactive precursor that undergoes proteolytic cleavage, yielding 8 kDa and 50 kDa protein subunits that heterodimerize to form an active enzyme. Heparanase exhibits also non-enzymatic activities, independent of its involvement in ECM degradation. Among these, are the enhancement of Akt signaling, stimulation of PI3K- and p38-dependent endothelial cell migration, and up regulation of VEGF, all contributing to its potent pro-angiogenic activity. Studies on relationships between structure and heparanase inhibition activity of nonanticogulant heparins systematically differing in their O-sulfation patterns, degrees of N-acetylation, and glycol-splitting of both pre-existing nonsulfated uronic acid residues (prevalently D-glucuronic) and/or those (Liduronic acid/L-galacturonic acid) generated by graded 2-O-desulfation, have permitted to select effective inhibitors of the enzymatic activity of heparanase. N-acetylated, glycol-split heparins emerged as especially strong inhibitors of heparanase, exerting little or no release of growth factors from ECM. N-acetylated glycol-split species of heparin, as well as heparanase gene silencing inhibit tumor metastasis, angiogenesis and inflammation in experimental animal models. These observations and the unexpected identification of a single functional heparanase, suggest that the enzyme is a promising target for anti-cancer and anti-inflammatory drug development.

Extracellular matrix (ECM) and basement membranes (BMs) present a physical barrier that requires enzymatic degradation during endothelial cell (EC) sprouting at early stages of angiogenesis. These multimolecular structures also serve as a storage depot for heparin‐binding angiogenic factors. Heparan sulfate proteoglycans (HSPGs) are responsible for the self‐assembly and integrity of the ECM and BM structure, as well as for sequestration of growth and differentiation factors. Recently, we reported the cloning of heparanase, an endo‐β‐ d ‐glucuronidase degrading heparan sulfate (HS), and provided direct evidence for its role in tumor metastasis. We now demonstrate that heparanase is intimately involved in angiogenesis and elucidate its mode of action. Apart from its direct involvement in ECM degradation and EC migration (vascular sprouting), heparanase releases active basic fibroblast growth factor (bFGF) from the subendothelial ECM, as well as bFGF‐stimulating HS degradation fragments from the EC surface. Interestingly, ECM‐derived HS fragments induced little or no potentiation of the growth‐promoting activity of bFGF. The angiogenic effect of heparanase was demonstrated in vivo (via the Matrigel plug assay) by showing a three‐ to fourfold increase in neovascularization induced by murine T‐lymphoma cells after stable transfection with the heparanase gene. Increased tissue vascularity was also observed in a mouse wound‐healing model in response to topical administration of recombinant heparanase. Immunohistochemical staining of human colon carcinoma tissue revealed a high expression of the heparanase protein in the endothelium of sprouting capillaries and small vessels, but not of mature quiescent blood vessels. The ability of heparanase to promote tumor angiogenesis and its involvement in tumor metastasis make it a promising target for cancer therapy.

We have generated homozygous trans¬genic mice ( hpa‐ t g ) overexpressing human hepara¬nase (endo‐β‐ D ‐glucuronidase) in all tissues and char¬acterized the involvement of the enzyme in tissue morphogenesis, vascularization, and energy metabo¬lism. Biochemical analysis of heparan sulfate (HS) isolated from newborn mice and adult tissues re¬vealed a profound decrease in the size of HS chains derived from hpa ‐tg vs. control mice. Despite this, the mice appeared normal, were fertile, and exhib¬ited a normal life span. A significant increase in the number of implanted embryos was noted in the hpa ‐tg vs. control mice. Overexpression of heparanase resulted in increased levels of urinary protein and creatinine, suggesting an effect on kidney func¬tion, reflected also by electron microscopy examina¬tion of the kidney tissue. The hpa ‐tg mice exhibited a reduced food consumption and body weight com¬pared with control mice. The effect of heparanase on tissue remodeling and morphogenesis was best dem¬onstrated by the phenotype of the hpa ‐tg mammary glands, showing excess branching and widening of ducts associated with enhanced neovascularization and disruption of the epithelial basement membrane. The hpa ‐tg mice exhibited an accelerated rate of hair growth, correlated with high expression of heparanase in hair follicle keratinocytes and increased vascularization. Altogether, characterization of the hpa ‐tg mice emphasizes the involvement of heparanase and HS in processes such as embryonic implan¬tation, food consumption, tissue remodeling, and vascularization.—Zcharia, E., Metzger, S., ChajekShaul, T., Aingorn, H., Elkin, M., Friedmann, Y., Weinstein, T., Li, J.‐P., Lindahl, U., Vlodavsky, I. Transgenic expression of mammalian heparanase uncovers physiological functions of heparan sulfate in tissue morphogenesis, vascularization, and feeding behavior. FASEB J. 18, 252–263 (2004)