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Although FOXP3+ regulatory T cells (Treg) depend on IL-2 produced by other cells for their survival and function, the levels of IL-2 in inflamed tissue are low, making it unclear how Treg access this critical resource. Here, we show that Treg use heparanase (HPSE) to access IL-2 sequestered by heparan sulfate (HS) within the extracellular matrix (ECM) of inflamed central nervous system tissue. HPSE expression distinguishes human and murine Treg from conventional T cells and is regulated by the availability of IL-2. HPSE-/- Treg have impaired stability and function in vivo, including in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Conversely, endowing monoclonal antibody-directed chimeric antigen receptor (mAbCAR) Treg with HPSE enhances their ability to access HS-sequestered IL-2 and their ability to suppress neuroinflammation in vivo. Together, these data identify a role for HPSE and the ECM in immune tolerance, providing new avenues for improving Treg-based therapy of autoimmunity. Regulatory T cell (Treg) maintenance and function require IL-2, yet this cytokine is only present in low levels in vivo. In this study, the authors demonstrate that that Treg use heparanase to access IL-2 bound to heparan sulfate proteoglycans in the extracellular matrix of inflamed brain tissue in mice. [ABSTRACT FROM AUTHOR]

Heparanase is the sole endoglucuronidase that degrades heparan sulfate in the cell surface and extracellular matrix (ECM). Several studies have reported the localization of heparanase in the cell nucleus, but the functional role of the nuclear enzyme is still obscure. Subjecting mouse embryonic fibroblasts (MEFs) derived from heparanase knockout (Hpse-KO) mice and applying transposase-accessible chromatin with sequencing (ATAC-seq), we revealed that heparanase is involved in the regulation of chromatin accessibility. Integrating with genome-wide analysis of chromatin states revealed an overall low activity in the enhancer and promoter regions of Hpse-KO MEFs compared with wild-type (WT) MEFs. Western blot analysis of MEFs and tissues derived from Hpse-KO vs. WT mice confirmed reduced expression of H3K27ac (acetylated lysine at N-terminal position 27 of the histone H3 protein). Our results offer a mechanistic explanation for the well-documented attenuation of inflammatory responses and tumor growth in Hpse-KO mice. [ABSTRACT FROM AUTHOR]

The kidney glomerular filtration barrier (GFB) is enriched with heparan sulfate (HS) proteoglycans, which contribute to its permselectivity. The endoglycosidase heparanase cleaves HS and hence appears to be involved in the pathogenesis of kidney injury and glomerulonephritis. We have recently reported, nonetheless, that heparanase overexpression preserved glomerular structure and kidney function in an experimental model of Adriamycin-induced nephropathy. To elucidate mechanisms underlying heparanase function in podocytes—key GFB cells, we utilized a human podocyte cell line and transgenic mice overexpressing heparanase. Notably, podocytes overexpressing heparanase (H) demonstrated significantly higher survival rates and viability after exposure to Adriamycin or hydrogen peroxide, compared with mock-infected (V) podocytes. Immunofluorescence staining of kidney cryo-sections and cultured H and V podocytes as well as immunoblotting of proteins extracted from cultured cells, revealed that exposure to toxic injury resulted in a significant increase in autophagic flux in H podocytes, which was reversed by the heparanase inhibitor, Roneparstat (SST0001). Heparanase overexpression was also associated with substantial transcriptional upregulation of autophagy genes BCN1, ATG5, and ATG12, following Adriamycin treatment. Moreover, cleaved caspase-3 was attenuated in H podocytes exposed to Adriamycin, indicating lower apoptotic cell death in H vs. V podocytes. Collectively, these findings suggest that in podocytes, elevated levels of heparanase promote cytoprotection. [ABSTRACT FROM AUTHOR]

Heparanase is an endo‐β‐glycosidase that cleaves heparan sulfate chains at the level of the glycosidic bond of selected glucuronic acid residues. Heparin fragments, that can compete with heparan sulfate for binding to heparanase, have been reported as weak heparanase inhibitors. To reinforce their inhibitory activity, a known β‐glucuronidase inhibitor has been introduced at their reducing end to directly interfere with the active site of the enzyme. We report here the synthesis of such oligosaccharides, displaying D‐glucaro‐δ‐lactam at their reducing end. Thanks to the trichloroacetimidate glycosylation method, we could devise experimental conditions allowing O‐glycosylation of the lactam ring, and we thus chemically prepared trisaccharides that were further elongated and sulfated using enzymatic methods. Heparanase inhibitory activity was not significantly improved by the presence of the D‐glucaro‐δ‐lactam ring. [ABSTRACT FROM AUTHOR]

Cell surface proteoglycans are important constituents of the glycocalyx and participate in cell–cell and cell–extracellular matrix (ECM) interactions, enzyme activation and inhibition, and multiple signaling routes, thereby regulating cell proliferation, survival, adhesion, migration, and differentiation. Heparanase, the sole mammalian heparan sulfate degrading endoglycosidase, acts as an "activator" of HS proteoglycans, thus regulating tissue hemostasis. Heparanase is a multifaceted enzyme that together with heparan sulfate, primarily syndecan-1, drives signal transduction, immune cell activation, exosome formation, autophagy, and gene transcription via enzymatic and nonenzymatic activities. An important feature is the ability of heparanase to stimulate syndecan-1 shedding, thereby impacting cell behavior both locally and distally from its cell of origin. Heparanase releases a myriad of HS-bound growth factors, cytokines, and chemokines that are sequestered by heparan sulfate in the glycocalyx and ECM. Collectively, the heparan sulfate–heparanase axis plays pivotal roles in creating a permissive environment for cell proliferation, differentiation, and function, often resulting in the pathogenesis of diseases such as cancer, inflammation, endotheliitis, kidney dysfunction, tissue fibrosis, and viral infection. [ABSTRACT FROM AUTHOR]

Unlike the intense research effort devoted to exploring the significance of heparanase in human diseases, very little attention was given to its close homolog, heparanase 2 (Hpa2). The emerging role of Hpa2 in a rare autosomal recessive congenital disease called urofacial syndrome (UFS), clearly indicates that Hpa2 is not a pseudogene but rather a gene coding for an important protein. Hpa2 lacks the heparan sulfate (HS)-degrading activity typical of heparanase, yet exhibits high affinity to HS, affinity that is 10-fold higher than that of heparanase. The consequences of this high-affinity interaction of Hpa2 with plasma membrane HSPG has not been explored yet. Here, we used highly purified Hpa2 protein to examine this aspect. We provide evidence that cells adhere to and spread on dishes coated with Hpa2. We also show that cell migration is attenuated markedly by exogenous addition of Hpa2 to primary and transformed cells, a function that agrees with the anti-cancer properties of Hpa2. Interestingly, we found that exogenous addition of Hpa2 also disrupts the morphology of cell colonies, resulting in cell scattering. This implies that under certain conditions and experimental settings, Hpa2 may exhibit pro-tumorigenic properties. We further developed a panel of anti-Hpa2 monoclonal antibodies (mAb) and show that these properties of Hpa2 are prevented by some of the newly-developed mAb, thus providing new molecular tools to better appreciate the significance of Hpa2 in health and disease. [ABSTRACT FROM AUTHOR]

The heparan sulfate proteoglycan Syndecan-1 binds cytokines, morphogens and extracellular matrix components, regulating cancer stem cell properties and invasiveness. Syndecan-1 is modulated by the heparan sulfate-degrading enzyme heparanase, but the underlying regulatory mechanisms are only poorly understood. In colon cancer pathogenesis, complex changes occur in the expression pattern of Syndecan-1 and heparanase during progression from well-differentiated to undifferentiated tumors. Loss of Syndecan-1 and increased expression of heparanase are associated with a change in phenotypic plasticity and an increase in invasiveness, metastasis and dedifferentiation. Here we investigated the regulatory and functional interplay of Syndecan-1 and heparanase employing siRNA-mediated silencing and plasmid-based overexpression approaches in the human colon cancer cell line Caco2. Heparanase expression and activity were upregulated in Syndecan-1 depleted cells. This increase was linked to an upregulation of the transcription factor Egr1, which regulates heparanase at the promoter level. Inhibitor experiments demonstrated an impact of focal adhesion kinase, Wnt and ROCK-dependent signaling on this process. siRNA-depletion of Syndecan-1, and upregulation of heparanase increased the colon cancer stem cell phenotype based on sphere formation assays and phenotypic marker analysis (Side-population, NANOG, KLF4, NOTCH, Wnt, and TCF4 expression). Syndecan-1 depletion increased invasiveness of Caco2 cells in vitro in a heparanase-dependent manner. Finally, upregulated expression of heparanase resulted in increased resistance to radiotherapy, whereas high expression of enzymatically inactive heparanase promoted chemoresistance to paclitaxel and cisplatin. Our findings provide a new avenue to target a stemness-associated signaling axis as a therapeutic strategy to reduce metastatic spread and cancer recurrence. [ABSTRACT FROM AUTHOR]

Activity of heparanase, responsible for cleavage of heparan sulfate (HS), is strongly implicated in tumor metastasis. This is due primarily to remodeling of the extracellular matrix (ECM) that becomes more prone to invasion by metastatic tumor cells. In addition, heparanase promotes the development of blood and lymph vessels that mobilize disseminated cells to distant organs. Here, we provide evidence for an additional mechanism by which heparanase affects cell motility, namely the destruction of E-cadherin based adherent junctions (AJ). We found that overexpression of heparanase or its exogenous addition results in reduced E-cadherin levels in the cell membrane. This was associated with a substantial increase in the phosphorylation levels of E-cadherin, β-catenin, and p120-catenin, the latter recognized as a substrate of Src. Indeed, we found that Src phosphorylation is increased in heparanase overexpressing cells, associating with a marked decrease in the interaction of E-cadherin with β-catenin, which is instrumental for AJ integrity and cell-cell adhesion. Notably, the association of E-cadherin with β-catenin in heparanase overexpressing cells was restored by Src inhibitor, along with reduced cell migration. These results imply that heparanase promotes tumor metastasis by virtue of its enzymatic activity responsible for remodeling of the ECM, and by signaling aspects that result in Src-mediated phosphorylation of E-cadherin/catenins and loosening of cell-cell contacts that are required for maintaining the integrity of epithelial sheets. [ABSTRACT FROM AUTHOR]

Heparanase is an endo‐β‐d‐glucuronidase that cleaves heparan sulfate (HS) side chains of heparan sulfate proteoglycans. Compelling evidence tie heparanase levels with all steps of tumor formation including tumor initiation, growth, metastasis and chemo‐resistance, likely involving augmentation of signaling pathways and gene transcription. In order to reveal the molecular mechanism(s) underlying the protumorigenic properties of heparanase, we established an inducible (Tet‐on) system in U87 human glioma cells and applied gene array methodology in order to identify genes associated with heparanase induction. We found that CD24, a mucin‐like cell adhesion protein, is consistently upregulated by heparanase and by heparanase splice variant devoid of enzymatic activity, whereas heparanase gene silencing was associated with decreased CD24 expression. This finding was further substantiated by a similar pattern of heparanase and CD24 immunostaining in glioma patients (Pearson's correlation; R = 0.66, p = 0.00001). Noteworthy, overexpression of CD24 stimulated glioma cell migration, invasion, colony formation in soft agar and tumor growth in mice suggesting that CD24 functions promote tumor growth. Likewise, anti‐CD24 neutralizing monoclonal antibody attenuated glioma tumor growth, and a similar inhibition was observed in mice treated with a neutralizing mAb directed against L1 cell adhesion molecule (L1CAM), a ligand for CD24. Importantly, significant shorter patient survival was found in heparanase‐high/CD24‐high tumors vs. heparanase‐high/CD24‐low tumors for both high‐grade and low‐grade glioma (p = 0.02). Our results thus uncover a novel heparanase–CD24–L1CAM axis that plays a significant role in glioma tumorigenesis. What's new? The heparanase enzyme has been associated with carcinogenesis in many ways but how it affects glioma development remains unknown. Here, the authors find that heparanase overexpression enhances the expression of the mucin‐like cell adhesion protein CD24. CD24 promotes glioma growth while anti‐CD24 antibodies attenuate the growth of glioma xenografts, providing a molecular mechanism underlying the protumorigenic properties of heparanase in glioma. [ABSTRACT FROM AUTHOR]

Background: Mammalian cells express a single functional heparanase, an endoglycosidase that cleaves heparan sulfate and thereby promotes tumor metastasis, angiogenesis, and inflammation. Malignant mesothelioma is highly aggressive and has a poor prognosis because of the lack of markers for early diagnosis and resistance to conventional therapies. The purpose of this study was to elucidate the mode of action and biological significance of heparanase in mesothelioma and test the efficacy of heparanase inhibitors in the treatment of this malignancy.Methods: The involvement of heparanase in mesothelioma was investigated by applying mouse models of mesothelioma and testing the effect of heparanase gene silencing (n = 18 mice per experiment; two different models) and heparanase inhibitors (ie, PG545, defibrotide; n = 18 per experiment; six different models). Synchronous pleural effusion and plasma samples from patients with mesothelioma (n = 35), other malignancies (12 non-small cell lung cancer, two small cell lung carcinoma, four breast cancer, three gastrointestinal cancers, two lymphomas), and benign effusions (five patients) were collected and analyzed for heparanase content (enzyme-linked immunosorbent assay). Eighty-one mesothelioma biopsies were analyzed by H-Score for the prognostic impact of heparanase using immunohistochemistry. All statistical tests were two-sided.Results: Mesothelioma tumor growth, measured by bioluminescence or tumor weight at termination, was markedly attenuated by heparanase gene silencing (P = .02) and by heparanase inhibitors (PG545 and defibrotide; P < .001 and P = .01, respectively). A marked increase in survival of the mesothelioma-bearing mice (P < .001) was recorded. Heparanase inhibitors were more potent in vivo than conventional chemotherapy. Clinically, heparanase levels in patients' pleural effusions could distinguish between malignant and benign effusions, and a heparanase H-score above 90 was associated with reduced patient survival (hazard ratio = 1.89, 95% confidence interval = 1.09 to 3.27, P = .03).Conclusions: Our results imply that heparanase is clinically relevant in mesothelioma development. Given these preclinical and clinical data, heparanase appears to be an important mediator of mesothelioma, and heparanase inhibitors are worthy of investigation as a new therapeutic modality in mesothelioma clinical trials. [ABSTRACT FROM AUTHOR]

Heparanase, the sole heparan sulfate (HS)-degrading endoglycosidase, regulates multiple biological activities that enhance tumor growth, metastasis, angiogenesis, and inflammation. Heparanase accomplishes this by degrading HS and thereby regulating the bioavailability of heparin-binding proteins; priming the tumor microenvironment; mediating tumor–host crosstalk; and inducing gene transcription, signaling pathways, exosome formation, and autophagy that together promote tumor cell performance and chemoresistance. By contrast, heparanase-2, a close homolog of heparanase, lacks enzymatic activity, inhibits heparanase activity, and regulates selected genes that promote normal differentiation, endoplasmic reticulum stress, tumor fibrosis, and apoptosis, together resulting in tumor suppression. The emerging premise is that heparanase is a master regulator of the aggressive phenotype of cancer, while heparanase-2 functions as a tumor suppressor. [ABSTRACT FROM AUTHOR]

Background: Expression and activity of heparanase, an endoglycosidase that cleaves heparan sulfate (HS) side chains of proteoglycans, is associated with progression and poor prognosis of many cancers which makes it an attractive drug target in cancer therapeutics.Methods: In the present work, we report the in vitro screening of a library of 150 small molecules with the scaffold bearing quinolones, oxazines, benzoxazines, isoxazoli(di)nes, pyrimidinones, quinolines, benzoxazines, and 4-thiazolidinones, thiadiazolo[3,2-a]pyrimidin-5-one, 1,2,4-triazolo-1,3,4-thiadiazoles, and azaspiranes against the enzymatic activity of human heparanase. The identified lead compounds were evaluated for their heparanase-inhibiting activity using sulfate [35S] labeled extracellular matrix (ECM) deposited by cultured endothelial cells. Further, anti-invasive efficacy of lead compound was evaluated against hepatocellular carcinoma (HepG2) and Lewis lung carcinoma (LLC) cells.Results: Among the 150 compounds screened, we identified 1,2,4-triazolo-1,3,4-thiadiazoles bearing compounds to possess human heparanase inhibitory activity. Further analysis revealed 2,4-Diiodo-6-(3-phenyl-[1, 2, 4]triazolo[3,4-b][1, 3, 4]thiadiazol-6yl)phenol (DTP) as the most potent inhibitor of heparanase enzymatic activity among the tested compounds. The inhibitory efficacy was demonstrated by a colorimetric assay and further validated by measuring the release of radioactive heparan sulfate degradation fragments from [35S] labeled extracellular matrix. Additionally, lead compound significantly suppressed migration and invasion of LLC and HepG2 cells with IC50 value of ~5 μM. Furthermore, molecular docking analysis revealed a favourable interaction of triazolo-thiadiazole backbone with Asn-224 and Asp-62 of the enzyme.Conclusions: Overall, we identified biologically active heparanase inhibitor which could serve as a lead structure in developing compounds that target heparanase in cancer. [ABSTRACT FROM AUTHOR]

Because of its impact on multiple biological pathways, heparanase has emerged as a major regulator of cancer, inflammation and other disease processes. Heparanase accomplishes this by degrading heparan sulfate which regulates the abundance and location of heparin-binding growth factors thereby influencing multiple signaling pathways that control gene expression, syndecan shedding and cell behavior. In addition, heparanase can act via nonenzymatic mechanisms that directly activate signaling at the cell surface. Clinical trials testing heparanase inhibitors as anticancer therapeutics are showing early signs of efficacy in patients further emphasizing the biological importance of this enzyme. This review focuses on recent developments in the field of heparanase regulation of cancer and inflammation, including the impact of heparanase on exosomes and autophagy, and novel mechanisms whereby heparanase regulates tumor metastasis, angiogenesis and chemoresistance. In addition, the ongoing development of heparanase inhibitors and their potential for treating cancer and inflammation are discussed. [ABSTRACT FROM AUTHOR]

The pulmonary vasculature constitutively expresses the integrin lymphocyte function-associated antigen-1 ligands intercellular adhesion molecule (ICAM)-1 and -2. In this study, effector T cells were temporarily entrapped by the lung vasculature on their way to inflamed lymph nodes, and this entrapment was strongly reduced in ICAM-1 and -2 double-deficient mice (79 and 86% reduction for CD8+ and CD4+ effectors, respectively, compared with wild-type mice). Although the pulmonary vasculature has been suggested to be masked by the heparan sulfate-containing glycocalyx, which is susceptible to heparanase-mediated shedding, lung and lymphocyte heparanase have been found to be unnecessary for this entrapment. Systemic LPS induced rapid neutrophil entrapment in the lung vasculature, but in contrast to T-cell entrapment, this sequestration was ICAM-1, ICAM-2, and heparanase independent. Furthermore, neutrophil migration into the bronchoalveolar space induced by LPS inhalation and LPS-induced leakage of red blood cells into this space were not dependent on lung ICAMs or heparanase activity. Nevertheless, heparanase was critical for neutrophil accumulation in smoke-exposed lungs. Our results indicate that, whereas T cells use ICAM-1 and -2 for temporary pulmonary entrapment, neutrophils get sequestered and extravasate into inflamed lungs independent of ICAMs. This is the first demonstration that the pulmonary vasculature is differentially recognized by T cells and neutrophils. [ABSTRACT FROM AUTHOR]

Heparanase, the exclusive mammalian heparan sulfate-degrading enzyme, has been suggested to be utilized by leukocytes to penetrate through the dense basement membranes surrounding blood venules. Despite its established role in tumor cell invasion, heparanase function in leukocyte extravasation has never been demonstrated. We found that TH1/TC1-type effector T cells are highly enriched for this enzyme, with a 3.6-fold higher heparanase mRNA expression compared with naive lymphocytes. Using adoptive transfer of wild-type and heparanase-deficient effector T cells into inflamed mice, we show that T-cell heparanase was not required for extravasation inside inflamed lymph nodes or skin. Leukocyte extravasation through acute inflamed skin vessels was also heparanase independent. Furthermore, neutrophils emigrated to the inflamed peritoneal cavity independently of heparanase expression on either the leukocytes or on the endothelial and mesothelial barriers, and overexpression of the enzyme on neutrophils did not facilitate their emigration. However, heparanase absence significantly reduced monocyte emigration into the inflamed peritoneal cavity. These results collectively suggest that neither leukocyte nor endothelial heparanase is required for T-cell and neutrophil extravasation through inflamed vascular barriers, whereas this enzyme is required for optimal monocyte recruitment to inflamed peritoneum. [ABSTRACT FROM AUTHOR]

Background: Heparanase, an endoglycosidase that cleaves heparan sulfate (HS), is involved in various biologic processes. Recently, an association between heparanase and glomerular injury was suggested. The present study examines the involvement of heparanase in the pathogenesis of Adriamycin-induced nephrotic syndrome (ADR-NS) in a mouse model. Methods: BALB/c wild-type (wt) mice and heparanase overexpressing transgenic mice (hpa-TG) were tail-vein injected with either Adriamycin (ADR, 10 mg/kg) or vehicle. Albuminuria was investigated at days 0, 7, and 14 thereafter. Mice were sacrificed at day 15, and kidneys were harvested for various analyses: structure and ultrastructure alterations, podocyte proteins expression, and heparanase enzymatic activity. Results: ADR-injected wt mice developed severe albuminuria, while ADR-hpa-TG mice showed only a mild elevation in urinary albumin excretion. In parallel, light microscopy of stained cross sections of kidneys from ADR-injected wt mice, but not hpa-TG mice, showed mild to severe glomerular and tubular damage. Western blot and immunofluorescence analyses revealed significant reduction in nephrin and podocin protein expression in ADR-wt mice, but not in ADR-hpa-TG mice. These results were substantiated by electron-microscopy findings showing massive foot process effacement in injected ADR-wt mice, in contrast to largely preserved integrity of podocyte architecture in ADR-hpa-TG mice. Conclusions: Our results suggest that heparanase may play a nephroprotective role in ADR-NS, most likely independently of HS degradation. Moreover, hpa-TG mice comprise an invaluable in vivo platform to investigate the interplay between heparanase and glomerular injury. [ABSTRACT FROM AUTHOR]

Abstract: Heparanase, the sole mammalian endoglycosidase degrading heparan sulfate, is causally involved in cancer metastasis, angiogenesis, inflammation and kidney dysfunction. Despite the wide occurrence and impact of heparan sulfate proteoglycans in vascular biology, the significance of heparanase in vessel wall disorders is underestimated. Blood vessels are highly active structures whose morphology rapidly adapts to maintain vascular function under altered systemic and local conditions. In some pathologies (restenosis, thrombosis, atherosclerosis) this normally beneficial adaptation may be detrimental to overall function. Enzymatic dependent and independent effects of heparanase on arterial structure mechanics and repair closely regulate arterial compliance and neointimal proliferation following endovascular stenting. Additionally, heparanase promotes thrombosis after vascular injury and contributes to a pro-coagulant state in human carotid atherosclerosis. Importantly, heparanase is closely associated with development and progression of atherosclerotic plaques, including stable to unstable plaque transition. Consequently, heparanase levels are markedly increased in the plasma of patients with acute myocardial infarction. Noteworthy, heparanase activates macrophages, resulting in marked induction of cytokine expression associated with plaque progression towards vulnerability. Together, heparanase emerges as a regulator of vulnerable lesion development and potential target for therapeutic intervention in atherosclerosis and related vessel wall complications. [Copyright &y& Elsevier]

Heparanase is an endoglucuronidase that cleaves heparan sulfate chains of proteoglycans. In many malignancies, high heparanase expression and activity correlate with an aggressive tumour phenotype. A major consequence of heparanase action in cancer is a robust up-regulation of growth factor expression and increased shedding of syndecan-1 (a transmembrane heparan sulfate proteoglycan). Substantial evidence indicates that heparanase and syndecan-1 work together to drive growth factor signalling and regulate cell behaviours that enhance tumour growth, dissemination, angiogenesis and osteolysis. Preclinical and clinical studies have demonstrated that therapies targeting the heparanase/syndecan-1 axis hold promise for blocking the aggressive behaviour of cancer. [ABSTRACT FROM AUTHOR]

Heparanase is an endo-β-glucuronidase that cleaves heparan sulfate side chains, leading to structural modifications that loosen the extracellular matrix barrier and associated with tumor metastasis, inflammation and angiogenesis. In addition, the highly sulfated heparan sulfate proteoglycans are important constituents of the glomerular basement membrane and its permselective properties. Recent studies suggest a role for heparanase in several experimental and human glomerular diseases associated with proteinuria such as diabetes, minimal change disease, and membranous nephropathy. Here, we quantified blood and urine heparanase levels in renal transplant recipients and patients with chronic kidney disease (CKD), and assessed whether alterations in heparanase levels correlate with proteinuria and renal function. We report that in transplanted patients, urinary heparanase was markedly elevated, inversely associated with estimated glomerular filtration rate (eGFR), suggesting a relationship between heparanase and graft function. In CKD patients, urinary heparanase was markedly elevated and associated with proteinuria, but not with eGFR. In addition, urinary heparanase correlated significantly with plasma heparanase in transplanted patients. Such a systemic spread of heparanase may lead to damage of cells and tissues alongside the kidney. The newly described association between heparanase, proteinuria and decreased renal function is expected to pave the way for new therapeutic options aimed at attenuating chronic renal allograft nephropathy, leading to improved graft survival and patient outcome. [ABSTRACT FROM AUTHOR]

Heparanase is an endo-β-d-glucuronidase capable of cleaving heparan sulfate side chains at a limited number of sites, yielding heparan sulfate fragments of still appreciable size. Importantly, heparanase activity correlates with the metastatic potential of tumor-derived cells, attributed to enhanced cell dissemination as a consequence of heparan sulfate cleavage and remodeling of the extracellular matrix and basement membrane underlying epithelial and endothelial cells. Similarly, heparanase activity is implicated in neovascularization, inflammation and autoimmunity, involving the migration of vascular endothelial cells and activated cells of the immune system. The cloning of a single human heparanase cDNA 10 years ago enabled researchers to critically approve the notion that heparan sulfate cleavage by heparanase is required for structural remodeling of the extracellular matrix, thereby facilitating cell invasion. Progress in the field has expanded the scope of heparanase function and its significance in tumor progression and other pathologies. Notably, although heparanase inhibitors attenuated tumor progression and metastasis in several experimental systems, other studies revealed that heparanase also functions in an enzymatic activity-independent manner. Thus, inactive heparanase was noted to facilitate adhesion and migration of primary endothelial cells and to promote phosphorylation of signaling molecules such as Akt and Src, facilitating gene transcription (i.e. vascular endothelial growth factor) and phosphorylation of selected Src substrates (i.e. endothelial growth factor receptor). The concept of enzymatic activity-independent function of heparanase gained substantial support by the recent identification of the heparanase C-terminus domain as the molecular determinant behind its signaling capacity. Identification and characterization of a human heparanase splice variant (T5) devoid of enzymatic activity and endowed with protumorigenic characteristics, elucidation of cross-talk between heparanase and other extracellular matrix-degrading enzymes, and identification of single nucleotide polymorphism associated with heparanase expression and increased risk of graft versus host disease add other layers of complexity to heparanase function in health and disease. [ABSTRACT FROM AUTHOR]

Mammalian heparanase, an endoglycosidase-degrading heparan sulfate, is synthesized as a latent 65 kDa precursor that undergoes proteolytic processing, primarily by cathepsin-L, yielding 8 kDa and 50 kDa subunits that heterodimerize to form a highly active enzyme. Enhanced heparanase expression in human tumors correlates with metastatic potential, tumor vascularity, and reduced postoperative survival of cancer patients, attributed to enzymatic and nonenzymatic activities of the heparanase protein. Urinary and plasma levels of heparanase are elevated in cancer patients and suppressed in response to effective anticancer treatments. These observations and the anticancerous effect of heparanase gene silencing and of heparanase-inhibiting molecules suggest that the enzyme is a promising target for anticancer drug development. [ABSTRACT FROM AUTHOR]

Patients on chronic hemodialysis (HD) are at high risk for developing atherosclerosis and cardiovascular complications. Heparanase, an endoglycosidase that cleaves heparan sulfate (HS) side chains of proteoglycans, is involved in extracellular matrix degradation and, as such, may be involved in the atherosclerotic lesion progression. We hypothesize that heparanase is elevated in HD patients, partly due to its release from primed circulating polymorphonuclear leukocytes (PMNLs), undergoing degranulation. Priming of PMNLs was assessed by levels of CD11b and the rate of superoxide release. Heparanase mRNA expression in PMNLs was determined by RT-PCR. PMNL and plasma levels of heparanase were determined by immunoblotting, immunofluorescence, and flow cytometry analyses. The levels of soluble HS in plasma were measured by a competition ELISA. This study shows that PMNLs isolated from HD patients have higher mRNA and protein levels of heparanase compared with normal control (NC) subjects and that heparanase levels correlate positively with PMNL priming. Plasma levels of heparanase were higher in HD patients than in NC subjects and were further elevated after the dialysis session. In addition, heparanase expression inversely correlates with plasma HS levels. A pronounced expression of heparanase was found in human atherosclerotic lesions. The increased heparanase activity in the blood of HD patients results at least in part from the degranulation of primed PMNLs and may contribute to the acceleration of the atherosclerotic process. Our findings highlight primed PMNLs as a possible source for the increased heparanase in HD patients, posing heparanase as a new risk factor for cardiovascular complications and atherosclerosis. [ABSTRACT FROM AUTHOR]

Heparanase is a mammalian endo-β-D-glucuronidase capable of cleaving HS side chains at a limited number of sites, activity that is strongly implicated in tumor metastasis, neovascularization, inflammation, and autoimmunity. Clinically, up-regulation of heparanase mRNA and protein expression has been documented in a variety of primary human tumors, correlating with reduced postoperative survival and increased lymph node and distant metastasis, thus providing strong clinical support for the prometastatic feature of the enzyme and making it an attractive target for the development of anticancer and anti-inflammatory drugs. Screening a panel of monoclonal antibodies for their ability to inhibit heparanase enzymatic activity, we noted that one hybridoma, 6F8, exhibited the opposite effect and significantly enhanced heparanase activity. Here, we provide evidence that antibody 6F8 enhances the activity of recombinant and cellular heparanase, facilitates invasion of tumor-derived cells in vitro, and improves wound healing in a mouse punch model in vivo. These results 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 certain enzymes. [ABSTRACT FROM AUTHOR]

Heparan sulfate (HS) side chains of HS proteoglycans bind to and assemble extracellular matrix proteins and play important roles in cell-cell and cell-extracellular matrix interactions. HS chains bind a multitude of bioactive molecules and thereby function in the control of multiple normal and pathological processes. Enzymatic degradation of HS by heparanase, a mammalian endoglycosidase, affects the integrity and functional state of tissues and is involved in, among other processes, inflammation, angiogenesis, and cancer metastasis. Here, we report the cloning of heparanase from four Israeli species of the blind subterranean mole rat (Spalax ehrenbergi superspecies), 85% homologous to the human enzyme. Unlike its limited expression in human tissues, heparanase is highly expressed in diverse Spalax tissues. Moreover, we have identified a unique splice variant of the Spalax enzyme lacking 16 aa encoded by exon 7. This deletion resulted in a major defect in trafficking and processing of the heparanase protein, leading to a loss of its enzymatic activity. Interspecies variation was noted in the sequence and in the expression of the splice variant of the heparanase gene in blind mole rats living under different ecogeographical stresses, indicating a possible role in adaptation to stress in Spalax evolution. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

We have generated homozygous transgenic mice (hpa-tg) overexpressing human heparanase (endo-β-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. [ABSTRACT FROM AUTHOR]

Heparan sulfate proteoglycans are essential for biological processes regulated by fibroblast growth factors (FGFs). Heparan sulfate (HS) regulates the activity of FGFs by acting as a coreceptor at the cell surface, enhancing FGF-FGFR affinity, and being a storage reservoir for FGFs in the extracellular matrix (ECM). Here we demonstrate a critical role for heparanase during mouse submandibular gland (SMG) branching morphogenesis. Heparanase, an endoglycosidase, colocalized with perlecan in the basement membrane and in epithelial clefts of SMGs. Inhibition of heparanase activity in organ culture decreased branching morphogenesis, and this inhibition was rescued specifically by FGF10 and not by other FGFs. By contrast, exogenous heparanase increased SMG branching and MAPK signaling and, surprisingly, when isolated epithelia were cultured in a three-dimensional ECM with FGF10, it increased the number of lateral branches and end buds. In a solid-phase binding assay, an FGF10-FGFR2b complex was released from the ECM by heparanase. In addition, surface plasmon resonance (SPR) analysis showed that FGF10 and the FGF10-FGFR2b complex bound to purified perlecan HS and could be released by heparanase. We used the FGF10-FGFR2b complex as a probe for HS in SMGs, and it colocalized with perlecan in the basement membrane and partly colocalized with syndecan 1 in the epithelium, and binding was reduced by treatment with heparanase. In summary, our results show heparanase releases FGF10 from perlecan HS in the basement membrane, increasing MAPK signaling, epithelial clefting, and lateral branch formation, which results in increased branching morphogenesis. [ABSTRACT FROM AUTHOR]