Eyal Zcharia

Transgenic expression of mammalian heparanase uncovers physiological functions of heparan sulfate in tissue morphogenesis, vascularization, and feeding behavior

We have generated homozygous trans¬genic mice (hpa‐tg) 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)

Heparanase Enhances Syndecan-1 Shedding A NOVEL MECHANISM FOR STIMULATION OF TUMOR GROWTH AND METASTASIS

When shed from the cell surface, the heparan sulfate proteoglycan syndecan-1 can facilitate the growth, angiogenesis, and metastasis of tumors. Here we report that tumor cell expression of heparanase, an enzyme known to be a potent promoter of tumor progression and metastasis, regulates both the level and location of syndecan-1 within the tumor microenvironment by enhancing its synthesis and subsequent shedding from the tumor cell surface. Heparanase regulation of syndecan-1 is detected in both human myeloma and breast cancer cell lines. This regulation requires the presence of active enzyme, because mutated forms of heparanase lacking heparan sulfate-degrading activity failed to influence syndecan-1 expression or shedding. Removal of heparan sulfate from the cell surface using bacterial heparitinase dramatically accelerated syndecan-1 shedding, suggesting that the effects of heparanase on syndecan-1 expression by tumor cells may be due, at least in part, to enzymatic removal or reduction in the size of heparan sulfate chains. Animals bearing tumors formed from cells expressing high levels of heparanase or animals transgenic for heparanase expression exhibited elevated levels of serum syndecan-1 as compared with controls, indicating that heparanase regulation of syndecan-1 expression and shedding can occur in vivo and impact cancer progression and perhaps other pathological states. These results reveal a new mechanism by which heparanase promotes an aggressive tumor phenotype and suggests that heparanase and syndecan-1 act synergistically to fine tune the tumor microenvironment and ensure robust tumor growth.

Cell surface expression and secretion of heparanase markedly promote tumor angiogenesis and metastasis

The present study emphasizes the importance of cell surface expression and secretion of heparanase (endo-β-d-glucuronidase) in tumor angiogenesis and metastasis. For this purpose, nonmetastatic Eb mouse lymphoma cells were transfected with the predominantly intracellular human heparanase or with a readily secreted chimeric construct composed of the human enzyme and the chicken heparanase signal peptide. Eb cells overexpressing the secreted heparanase invaded a reconstituted basement membrane to a much higher extent than cells overexpressing the intracellular enzyme. Cell invasion was inhibited in the presence of laminaran sulfate, a potent inhibitor of heparanase activity and experimental metastasis. The increased invasiveness in vitro was reflected in vivo by rapid and massive liver colonization and accelerated mortality. In fact, mice inoculated with cells expressing the secreted enzyme succumb because of liver metastasis and dysfunction, as early as 10 days after s.c. inoculation of the cells, when their tumor burden did not exceed 1% of body weight. Cell surface localization and secretion of heparanase markedly stimulated tumor angiogenesis, as demonstrated by a 4–6-fold increase in vessel density and functionality evaluated by MRI of tumors produced by cells expressing the secreted vs. the nonsecreted heparanase, consistent with actual counting of blood vessels. Altogether, our results indicate that the potent proangoigenic and prometastatic properties of heparanase are tightly regulated by its cellular localization and secretion. The increased potency of the secreted enzyme makes it a promising target for anticancer drug development.

Heparanase mediates cell adhesion independent of its enzymatic activity

Heparanase is an endo‐β‐D‐glucuronidase that cleaves heparan sulfate and is implicated in diverse physiological and pathological processes. In this study we report on a novel direct involvement of heparanase in cell adhesion. We demonstrate that expression of heparanase in nonadherent lymphoma cells induces early stages of cell adhesion, provided that the enzyme is expressed on the cell surface. Heparanasemediated cell adhesion to extracellular matrix (ECM) results in integrin‐dependent cell spreading, tyrosine phosphorylation of paxillin, and reorganization of the actin cytoskeleton. The surface‐bound enzyme also augments cell invasion through a reconstituted basement membrane. Cell adhesion was augmented by cell surface heparanase regardless of whether the cells were transfected with active or point mutated inactive enzyme, indicating that heparanase functions as an adhesion molecule independent of its endoglycosidase activity. The combined feature of heparanase as an ECM‐degrading enzyme and a cell adhesion molecule emphasizes its significance in processes involving cell adhesion, migration, and invasion, including embryonic development, neovascularization, and cancer metastasis.— Goldshmidt, O., Zcharia, E., Cohen, M., Aingorn, H., Cohen, I., Nadav, L., Katz, B.‐Z., Geiger, B., Vlodavsky, I. Heparanase mediates cell adhesion independent of its enzymatic activity. FASEB J. 17, 1015–1025 (2003)

Heparanase accelerates wound angiogenesis and wound healing in mouse and rat models

Orchestration of the rapid formation and reorganization of new tissue observed in wound healing involves not only cells and polypeptides but also the extracellular matrix (ECM) microenvironment. The ability of heparan sulfate (HS) to interact with major components of the ECM suggests a key role for HS in maintaining the structural integrity of the ECM. Heparanase, an endoglycosidase‐degrading HS in the ECM and cell surface, is involved in the enzymatic machinery that enables cellular invasion and release of HS‐bound polypeptides residing in the ECM. Bioavailabilty and activation of multitude mediators capable of promoting cell migration, proliferation, and neovascularization are of particular importance in the complex setting of wound healing. We provide evidence that heparanase is normally expressed in skin and in the wound granulation tissue. Heparanase stimulated keratinocyte cell migration and wound closure in vitro. Topical application of recombinant heparanase significantly accelerated wound healing in a flap/punch model and markedly improved flap survival. These heparanase effects were associated with enhanced wound epithelialization and blood vessel maturation. Similarly, a marked elevation in wound angiogenesis, evaluated by MRI analysis and histological analyses, was observed in heparanase‐overexpressing transgenic mice. This effect was blocked by a novel, newly developed, heparanase‐inhibiting glycol‐split fragment of heparin. These results clearly indicate that elevation of heparanase levels in healing wounds markedly accelerates tissue repair and skin survival that are mediated primarily by an enhanced angiogenic response.—Zcharia, E., Zilka, R., Yaar, A., Yacoby‐Zeevi, O., Zetser, A., Metzger, S., Sarid, R., Naggi, A., Casu, B., Ilan, N., Vlodavsky, I., Abramovitch, R. Heparanase accelerates wound angiogenesis and wound healing in mouse and rat models. FASEB J. 19, 211–221 (2005)

Newly Generated Heparanase Knock-Out Mice Unravel Co-Regulation of Heparanase and Matrix Metalloproteinases

Background Heparanase, a mammalian endo-β-D-glucuronidase, specifically degrades heparan sulfate proteoglycans ubiquitously associated with the cell surface and extracellular matrix. This single gene encoded enzyme is over-expressed in most human cancers, promoting tumor metastasis and angiogenesis. Principal Findings We report that targeted disruption of the murine heparanase gene eliminated heparanase enzymatic activity, resulting in accumulation of long heparan sulfate chains. Unexpectedly, the heparanase knockout (Hpse-KO) mice were fertile, exhibited a normal life span and did not show prominent pathological alterations. The lack of major abnormalities is attributed to a marked elevation in the expression of matrix metalloproteinases, for example, MMP2 and MMP14 in the Hpse-KO liver and kidney. Co-regulation of heparanase and MMPs was also noted by a marked decrease in MMP (primarily MMP-2,-9 and 14) expression following transfection and over-expression of the heparanase gene in cultured human mammary carcinoma (MDA-MB-231) cells. Immunostaining (kidney tissue) and chromatin immunoprecipitation (ChIP) analysis (Hpse-KO mouse embryonic fibroblasts) suggest that the newly discovered co-regulation of heparanase and MMPs is mediated by stabilization and transcriptional activity of β-catenin. Conclusions/Significance The lack of heparanase expression and activity was accompanied by alterations in the expression level of MMP family members, primarily MMP-2 and MMP-14. It is conceivable that MMP-2 and MMP-14, which exert some of the effects elicited by heparanase (i.e., over branching of mammary glands, enhanced angiogenic response) can compensate for its absence, in spite of their different enzymatic substrate. Generation of viable Hpse-KO mice lacking significant abnormalities may provide a promising indication for the use of heparanase as a target for drug development.

Heparanase powers a chronic inflammatory circuit that promotes colitis-associated tumorigenesis in mice.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease that is closely associated with colon cancer. Expression of the enzyme heparanase is clearly linked to colon carcinoma progression, but its role in UC is unknown. Here we demonstrate for what we believe to be the first time the importance of heparanase in sustaining the immune-epithelial crosstalk underlying colitis-associated tumorigenesis. Using histological specimens from UC patients and a mouse model of dextran sodium sulfate-induced colitis, we found that heparanase was constantly overexpressed and activated throughout the disease. We demonstrate, using heparanase-overexpressing transgenic mice, that heparanase overexpression markedly increased the incidence and severity of colitis-associated colonic tumors. We found that highly coordinated interactions between the epithelial compartment (contributing heparanase) and mucosal macrophages preserved chronic inflammatory conditions and created a tumor-promoting microenvironment characterized by enhanced NF-κB signaling and induction of STAT3. Our results indicate that heparanase generates a vicious cycle that powers colitis and the associated tumorigenesis: heparanase, acting synergistically with the intestinal flora, stimulates macrophage activation, while macrophages induce production (via TNF-α-dependent mechanisms) and activation (via secretion of cathepsin L) of heparanase contributed by the colon epithelium. Thus, disruption of the heparanase-driven chronic inflammatory circuit is highly relevant to the design of therapeutic interventions in colitis and the associated cancer.

Heparanase Is Essential for the Development of Diabetic Nephropathy in Mice

Diabetic nephropathy (DN) is the major life-threatening complication of diabetes. Abnormal permselectivity of glomerular basement membrane (GBM) plays an important role in DN pathogenesis. Heparanase is the predominant enzyme that degrades heparan sulfate (HS), the main polysaccharide of the GBM. Loss of GBM HS in diabetic kidney was associated with increased glomerular expression of heparanase; however, the causal involvement of heparanase in the pathogenesis of DN has not been demonstrated. We report for the first time the essential involvement of heparanase in DN. With the use of Hpse-KO mice, we found that deletion of the heparanase gene protects diabetic mice from DN. Furthermore, by investigating the molecular mechanism underlying induction of the enzyme in DN, we found that transcription factor early growth response 1 (Egr1) is responsible for activation of heparanase promoter under diabetic conditions. The specific heparanase inhibitor SST0001 markedly decreased the extent of albuminuria and renal damage in mouse models of DN. Our results collectively underscore the crucial role of heparanase in the pathogenesis of DN and its potential as a highly relevant target for therapeutic interventions in patients with DN.

Heparanase induces tissue factor expression in vascular endothelial and cancer cells.

Summary.  Background: Over‐expression of tissue factor (TF) and activation of the coagulation system are common in cancer patients. Heparanase is an endo‐β‐d‐glucuronidase that cleaves heparan sulfate chains on cell surfaces and in the extracellular matrix, activity that closely correlates with cell invasion, angiogenesis and tumor metastasis. The study was undertaken to investigate the involvement of heparanase in TF expression. Methods: Tumor‐derived cell lines were transfected with heparanase cDNA and TF expression was examined. The effect of exogenous addition of active and inactive heparanase on TF expression and activity was studied in tumor cell lines and primary human umbilical vein endothelial cells. TF expression was also explored in heparanase over‐expressing transgenic (Tg) mice. Blast cells were collected from acute leukemia patients and TF and heparanase expression levels were analyzed. Results: Over‐expression of heparanase in tumor‐derived cell lines resulted in a 2‐fold increase in TF expression levels, and a similar trend was observed in heparanase Tg mice in vivo. Likewise, exogenous addition of heparanase to endothelial or tumor‐derived cells resulted in enhanced TF expression and activity. Interestingly, TF expression was also induced in response to enzymatically inactive heparanase, suggesting that this effect was independent of heparanase enzymatic activity. The regulatory effect of heparanase on TF expression involved activation of the p38 signaling pathway. A positive correlation between TF expression levels and heparanase activity was found in blasts collected from 22 acute leukemia patients. Conclusions: Our results indicate that in addition to its well‐known function as an enzyme paving a way for invading cells, heparanase also participates in the regulation of TF gene expression and its related coagulation pathways.

Role of promoter methylation in regulation of the mammalian heparanase gene.

Mammalian heparanase (endo-β-glucuronidase) degrades heparan sulfate proteoglycans and is an important modulator of the extracellular matrix and associated factors. The enzyme is preferentially expressed in neoplastic tissues and contributes to tumour metastasis and angiogenesis. To investigate the epigenetic regulation of the heparanase locus, methylation-specific and bisulfite PCR were performed on a panel of 22 human cancer cell lines. Cytosine methylation of the heparanase promoter was associated with inactivation of the affected allele. Despite lack of sequence homology, extensively methylated CpG islands were found both in human choriocarcinoma (JAR) and rat glioma (C-6) cells which lack heparanase activity. Treatment of these cells with demethylating agents (5-azacytidine, 5-aza-2′-deoxycytidine) resulted in stable dose- and time-dependant promoter hypomethylation accompanied by reappearance of heparanase mRNA, protein and enzymatic activity. An inhibitor of histone deacetylase, Trichostatin A, failed to induce either of these effects. Upregulation of heparanase expression and activity by demethylating drugs was associated with a marked increase in lung colonization by pretreated C-6 rat glioma cells. The increased metastatic potential in vivo was inhibited in mice treated with laminaran sulfate, a potent inhibitor of heparanase activity. We propose a model wherein expression of mammalian heparanase gene is modulated by the interplay between trans-activating genetic and cis-inhibitory epigenetic elements in its promoter.