Heparan sulfate proteoglycans interact with many extracellular matrix constituents, growth factors and enzymes. Degradation of heparan sulfate by endoglycosidic heparanase cleavage affects a variety of biological processes. We have purified a 50-kDa heparanase from human hepatoma and placenta, and now report cloning of the cDNA and gene encoding this enzyme. Expression of the cloned cDNA in insect and mammalian cells yielded 65-kDa and 50-kDa recombinant heparanase proteins. The 50-kDa enzyme represents an N-terminally processed enzyme, at least 100-fold more active than the 65-kDa form. The heparanase mRNA and protein are preferentially expressed in metastatic cell lines and specimens of human breast, colon and liver carcinomas. Low metastatic murine T-lymphoma and melanoma cells transfected with the heparanase cDNA acquired a highly metastatic phenotype in vivo, reflected by a massive liver and lung colonization. This represents the first cloned mammalian heparanase, to our knowledge, and provides direct evidence for its role in tumor metastasis. Cloning of the heparanase gene enables the development of specific molecular probes for early detection and treatment of cancer metastasis and autoimmune disorders.

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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-Dglucuronidase 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.Key words: heparan sulfate proteoglycans • extracellular matrix • endothelial cells • basic fibroblast growth factor • wound healing eparan sulfate proteoglycans (HSPGs) are most abundant in cell surfaces, extracellular matrix (ECM), and basement membranes (BMs) (1, 2). BMs represent specialized ECM structures that underlie endothelial cells (ECs) in the blood vessel wall, as well as epithelial cells in various tissues and organs. HSPGs, the major polysaccharide-containing component of BMs, play a key role in the self-assembly and integrity of the BM multimolecular architecture. This function is clearly ascribed to the heparan sulfate (HS) carbohydrate side chains (1, 2), interacting through specific attachment sites with the main protein components of H the ECM and BM, such as collagen IV, laminin, and fibronectin. HSPGs are also actively involved in orchestrating cellular responses in both normal and pathological conditions (1, 2), ranging from pregnancy and development to neovascularization and metastatic spread of malignant tumors.The importance of HS and especially its enzymatic degradation during angiogenesis and metastasis has attracted growing attention during the...

We have generated homozygous transgenic mice (hpa-tg) overexpressing human heparanase (endo-beta-D-glucuronidase) in all tissues and characterized the involvement of the enzyme in tissue morphogenesis, vascularization, and energy metabolism. Biochemical analysis of heparan sulfate (HS) isolated from newborn mice and adult tissues revealed 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 exhibited 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 function, reflected also by electron microscopy examination of the kidney tissue. The hpa-tg mice exhibited a reduced food consumption and body weight compared with control mice. The effect of heparanase on tissue remodeling and morphogenesis was best demonstrated 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 implantation, food consumption, tissue remodeling, and vascularization.

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