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Heparanase is an endo-B-D-glucuronidase involved in cleavage of heparan sulfate moieties and hence participates in extracellular matrix (ECM) degradation and remodeling. Traditionally, heparanase activity was correlated with the metastatic potential of a variety of tumor-derived cell types. Cloning of the heparanase gene indicated that heparanase expression is up-regulated in a variety of primary human tumors. In some cases, heparanase up-regulation correlated with increased tumor vascularity, an angiogenic feature that could be recapitulated in a number of in vitro and in vivo models. The mechanism by which heparanase enhances angiogenic responses is not entirely clear but is thought to be mediated primarily by release of ECM-resident angiogenic growth factors such as basic fibroblast growth factor and vascular endothelial growth factor (VEGF). Here, we examined the possibility that heparanase directly participates in VEGF gene regulation. We provide evidence that heparanase overexpression in human embryonic kidney 293, MDA-MB-435 human breast carcinoma, and rat C6 glioma cells resulted in a 3-to 6-fold increase in VEGF protein and mRNA levels, which correlated with elevation of p38 phosphorylation. Moreover, heparanase down-regulation in B16 mouse melanoma cells by a specific siRNA vector was accompanied by a decrease in VEGF and p38 phosphorylation levels, suggesting that VEGF gene expression is regulated by endogenous heparanase. Interestingly, a specific p38 inhibitor did not attenuate VEGF up-regulation by heparanase whereas Src inhibitors completely abrogated this effect. These results indicate, for the first time, that heparanase is actively involved in the regulation of VEGF gene expression, mediated by activation of Src family

Heparanase is a heparan sulfate degrading endoglycosidase participating in extracellular matrix degradation and remodeling. Heparanase is synthesized as a 65 kDa non-active precursor that subsequently undergoes proteolytic cleavage, yielding 8 kDa and 50 kDa protein subunits that heterodimerize to form an active enzyme. The protease responsible for heparanase processing is currently unknown, as is the sub-cellular processing site. In this study, we characterize an antibody (733) that preferentially recognizes the active 50 kDa heparanase form as compared to the non-active 65 kDa heparanase precursor. We have utilized this and other anti-heparanase antibodies to study the cellular localization of the latent 65 kDa and active 50 kDa heparanase forms during uptake and processing of exogenously added heparanase. Interestingly, not only the processed 50 kDa, but also the 65 kDa heparanase precursor was localized to perinuclear vesicles, suggesting that heparanase processing occurs in lysosomes. Indeed, heparanase processing was completely inhibited by chloroquine and bafilomycin A1, inhibitors of lysosome proteases. Similarly, processing of membrane-targeted heparanase was also chloroquine-sensitive, further ruling out the plasma membrane as the heparanase processing site. Finally, we provide evidence that antibody 733 partially neutralizes the enzymatic activity of heparanase, suggesting that the N-terminal region of the molecule is involved in assuming an active conformation. Monoclonal antibodies directed to this region are likely to provide specific heparanase inhibitors and hence assist in resolving heparanase functions under normal and pathological conditions.

Heparanase is a mammalian endoglycosidase that degrades heparan sulfate (HS) at specific intra-chain sites. Blood-borne neutrophils, macrophages, mast cells, and platelets exhibit heparanase activity that is thought to be stored in specific granules. The degranulated heparanase is implicated in extravasation of metastatic tumor cells and activated cells of the immune system. Degranulation and heparanase release in response to an inflammatory stimulus or platelet activation would facilitate cellular extravasation directly, by altering the composition and structural integrity of the extracellular matrix, or indirectly, by releasing HS-bound proinflammatory cytokines and chemokines. We hypothesized that in addition to such indirect effect, the released heparanase may also locally affect and activate neighboring cells such as endothelial cells. Here, we provide evidence that addition of the 65-kDa latent heparanase to endothelial cells enhances Akt signaling. 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 of heparanase stimulated phosphatidylinositol 3-kinasedependent endothelial cell migration and invasion. 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.Heparanase is an endo-␤-D-glucuronidase capable of cleaving heparan sulfate (HS) 1 side chains at a limited number of sites, yielding HS fragments of still appreciable size (ϳ5-7 kDa) (1-3). Participating in extracellular matrix (ECM) degradation and remodeling, heparanase activity has been traditionally correlated with the metastatic potential of tumor-derived cells (4 -7). Similarly, heparanase has been shown to facilitate cell invasion associated with angiogenesis, autoimmunity, and inflammation (6 -9). Among the few cell types that express heparanase under normal physiological conditions, platelets possess a high heparanase activity and were used as a source for heparanase purification (2, 10). In fact, serum heparanase is mainly derived from activated platelets (11). Heparanase was localized to tertiary granules of neutrophils (12, 13) and mast cells (7) and was released upon tumor necrosis factor-␣ and calcium ionophore treatments, respectively. Heparanase release by degranulation has been implicated in diapedesis and extravasation of a number of immune cells, including neutrophils, macrophages, and lymphocytes (8, 14, 15), while heparanase inhibitors exhibited an anti-inflammatory activity (15). Cleavage of HS side chains by degranulated heparanase during inflammation may facilitate the passage of blood-borne normal and malignant cells into tissues by altering the composition and structural integrity of the subendothelial ECM (1,8,14). In addition, heparanase may facilitate the release of a multitude of HS-bound growth factors, cytokines and chemokines that would, in tu...

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 metastasis and inflammation. In addition to its structural role in extracellular matrix assembly and integrity, HS sequesters a multitude of polypeptides that reside in the extracellular matrix as a reservoir. A variety of growth factors, cytokines, chemokines, and enzymes can be released by heparanase activity and profoundly affect cell and tissue function. 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. Addition of heparin or xylosides to cell cultures resulted in a pronounced accumulation of, heparanase in the culture medium, whereas sodium chlorate had no such effect. Moreover, cellular uptake of heparanase was markedly reduced in HS-deficient CHO-745 mutant cells, heparan sulfate proteoglycan-deficient HT-29 colon cancer cells, and heparinasetreated cells. We also studied the heparanase biosynthetic route and found that the half-life of the active enzyme is ϳ30 h. This and previous localization studies suggest that heparanase resides in the endosomal/lysosomal compartment for a relatively long period of time and is likely to play a role in the normal turnover of HS. Co-localization studies and cell fractionation following heparanase addition have identified syndecan family members as candidate molecules responsible for heparanase uptake, providing an efficient mechanism that limits extracellular accumulation and function of heparanase.

Summary. Background: Over-expression of tissue factor (TF) and activation of the coagulation system are common in cancer patients. Heparanase is an endo-b-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.

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.

Heparanase is an endoglycosidase that specifically cleaves heparan sulfate side chains, a class of glycosaminoglycans abundantly present in the extracellular matrix and on the cell surface. Heparanase activity is strongly implicated in tumor angiogenesis and metastasis attributed to remodeling of the subepithelial and subendothelial basement membranes. We hypothesized that similar to its proangiogenic capacity, heparanase is also engaged in lymphangiogenesis and utilized the D2-40 monoclonal antibody to study lymphangiogenesis in tumor specimens obtained from 65 head and neck carcinoma patients. Lymphatic density was analyzed for association with clinical parameters and heparanase staining. We provide evidence that lymphatic vessel density (LVD) correlates with head and neck lymph node metastasis (N-stage, p 5 0.007) and inversely correlates with tumor cell differentiation (p 5 0.007). Notably, heparanase staining correlated with LVD (p 5 0.04) and, moreover, with VEGF C levels (p 5 0.01). We further demonstrate that heparanase overexpression by epidermoid, breast, melanoma and prostate carcinoma cells induces a 3-to 5-fold elevation in VEGF C expression in vitro and facilitates tumor xenograft lymphangiogenesis in vivo, whereas heparanase gene silencing was associated with decreased VEGF C levels. These findings suggest that heparanase plays a unique dual role in tumor metastasis, facilitating tumor cell invasiveness and inducing VEGF C expression, thereby increasing the density of lymphatic vessels that mobilize metastatic cells. ' 2008 Wiley-Liss, Inc.Key words: heparanase; head and neck carcinoma; lymphatic vessels; VEGF C; D2-40Heparanase is an endo-b-glucuronidase that cleaves heparan sulfate (HS) side chains of HS proteoglycans (HSPG). Heparanase activity has long been correlated with cell invasion associated with cancer metastasis, a consequence of structural modification that loosens the extracellular matrix (ECM) barrier. 1,2 This notion gained further support by employing siRNA and ribozyme technologies, 3,4 clearly depicting heparanase-mediated HS cleavage and ECM remodeling as critical requisites for inflammation, tumor angiogenesis and tumor metastasis. More recently, heparanase upregulation was documented in an increasing number of human carcinomas and hematological malignancies. [5][6][7] In many cases, heparanase induction correlated with increased tumor metastasis, vascular density and shorter postoperative survival rate, thus providing a strong clinical support for the prometastatic function of the enzyme. 5,6 These studies depict compelling evidence for the clinical relevance of the enzyme, making it an attractive target for the development of anticancer drugs. 5,[8][9][10][11][12] The role that heparanase plays in the primary tumor is less well understood, but likely involves angiogenic aspects. Elevation of microvessel density correlated with heparanase induction in solid 13-17 and hematological 18 malignancies and was also evident in tumor xenografts produced by cells overexpressing hepar...