Cleaves Heparan Sulfate Research Articles

DMD TREATMENT: ANIMAL MODELS: P.207Full-length but not truncated osteoprotegerin binds directly to muscle cells and increases rapidly dystrophic muscle force

Clinical studies have shown that osteoporosis and muscle degeneration can occur in tandem following neuromuscular diseases. The receptor-activator of nuclear factor κB (RANK), the receptor-activator of nuclear factor κB ligand (RANKL), and osteoprotegerin (OPG) triad, a key pathway for bone regulation, is involved in Duchenne muscular dystrophy physiopathology. OPG, a decoy receptor for RANKL, the truncated (TR-OPG-Fc) or full-length OPG (FL-OPG-Fc) fused with an Fc fragment of IgG1 can inhibit RANKL/RANK interactions in vivo or in vitro. TR-OPG-Fc contains only 4 cysteine-rich RANKL domains while FL-OPG-Fc contains 4 cysteine-rich RANKL domains, 2 TRAIL death domains and 1 heparin-binding domain. We have shown that a 10 day treatment with FL-OPG-Fc during the first acute phase of degeneration in dystrophin-deficient mdx mice completely restored force and resistance to eccentric contractions of fast-twitch muscle. Because FL-OPG-Fc was superior to muscle specific deletion of RANK or anti-RANKL, anti-TRAIL, TR-OPG-Fc treatments, we hypothesized that the heparin-binding domain found in FL-OPG-Fc interacts directly with muscle. WB and confocal microscopy demonstrated that FL-OPG-Fc, but not TR-OPG-Fc, binds to C2C12 myotube. We next showed that heparinase III, which cleaves heparan sulfate chains, reduces FL-OPG-Fc binding by 50%. To further support the importance of the heparin domain, FL-OPG-Fc was co-incubated with equal concentration of heparin, which abrogated by 85% its binding ability. Next, using ex vivo contractile properties of isolated fast-twitch EDL, we showed that a 24h FL-OPG-Fc treatment increased significantly the absolute and specific forces of dystrophic EDL. In conclusion, FL-OPG-Fc, through its heparin-binding domain, binds directly muscle cells and can rapidly increase absolute and specific maximal tetanic forces. Current investigations are directed towards a better understanding of the mechanisms by which FL-OPG-Fc improves dystrophic muscles.

Epithelial Heparan Sulfate Contributes to Alveolar Barrier Function and Is Shed during Lung Injury.

The lung epithelial glycocalyx is a carbohydrate-enriched layer lining the pulmonary epithelial surface. Although epithelial glycocalyx visualization has been reported, its composition and function remain unknown. Using immunofluorescence and mass spectrometry, we identified heparan sulfate (HS) and chondroitin sulfate within the lung epithelial glycocalyx. In vivo selective enzymatic degradation of epithelial HS, but not chondroitin sulfate, increased lung permeability. Using mass spectrometry and gel electrophoresis approaches to determine the fate of epithelial HS during lung injury, we detected shedding of 20 saccharide-long or greater HS into BAL fluid in intratracheal LPS-treated mice. Furthermore, airspace HS in clinical samples from patients with acute respiratory distress syndrome correlated with indices of alveolar permeability, reflecting the clinical relevance of these findings. The length of HS shed during intratracheal LPS-induced injury (≥20 saccharides) suggests cleavage of the proteoglycan anchoring HS to the epithelial surface, rather than cleavage of HS itself. We used pharmacologic and transgenic animal approaches to determine that matrix metalloproteinases partially mediate HS shedding during intratracheal LPS-induced lung injury. Although there was a trend toward decreased alveolar permeability after treatment with the matrix metalloproteinase inhibitor, doxycycline, this did not reach statistical significance. These studies suggest that epithelial HS contributes to the lung epithelial barrier and its degradation is sufficient to increase lung permeability. The partial reduction of HS shedding achieved with doxycycline is not sufficient to rescue epithelial barrier function during intratracheal LPS-induced lung injury; however, whether complete attenuation of HS shedding is sufficient to rescue epithelial barrier function remains unknown.

The heparanase inhibitor PG545 is a potent anti-lymphoma drug: Mode of action

It is now well recognized that heparanase, an endo-β-D-glucuronidase capable of cleaving heparan sulfate (HS) side chains at a limited number of sites, promotes tumorigenesis by diverse mechanisms. Compelling evidence strongly implies that heparanase is a viable target for cancer therapy, thus encouraging the development of heparanase inhibitors as anti-cancer therapeutics. Here, we examined the efficacy and mode of action of PG545, an HS-mimetic heparanase inhibitor, in human lymphoma. We found that PG545 exhibits a strong anti-lymphoma effect, eliciting lymphoma cell apoptosis. Notably, this anti-lymphoma effect involves ER stress response that was accompanied by increased autophagy. The persistent ER stress evoked by PG545 is held responsible for cell apoptosis because apoptotic cell death was attenuated by an inhibitor of PERK, a molecular effector of ER stress. Importantly, PG545 had no such apoptotic effect on naïve splenocytes, further encouraging the development of this compound as anti-lymphoma drug. Surprisingly, we found that PG545 also elicits apoptosis in lymphoma cells that are devoid of heparanase activity (i.e., Raji), indicating that the drug also exerts heparanase-independent function(s) that together underlie the high potency of PG545 in preclinical cancer models.

PO-220 Defining the role of heparanase in breast cancer progression using the PyMT-MMTV mouse model

IntroductionHeparanase (HPSE) is a beta-d-endoglucuronidase and the only mammalian enzyme that cleaves heparan sulphate (HS), a major structural and regulatory component of the extracellular matrix (ECM) and the vascular basement membrane (BM). The expression of HPSE is tightly controlled and under physiological conditions is limited to immune cells, endothelial cells, placental trophoblasts and keratinocytes. However, during pathological conditions such as cancer, HPSE expression is dysregulated and high expression often correlates with poor patient survival. The cleavage of HS by HPSE expressing tumours degrades the ECM/BM and releases HS bound growth factors and cytokines, promoting cellular signalling in a positive feed-back mechanism. This in turn leads to enhanced primary tumour growth, metastasis, angiogenesis and inflammation. HPSE is therefore a critical promoter of the hallmarks of cancer and has generated significant interest as an anti-cancer drug target. However, despite decades of research, the precise mechanistic role of HPSE in the tumour microenvironment remains poorly defined. Breast cancer is the most prevalent malignancy in women worldwide. Clinical data reveals that the expression of HPSE in mammary tumours results in poor patient survival.Material and methodsWe recently generated a HPSE-deficient C57Bl/6 mouse strain (C57Bl/6xHPSE-/-) that were crossed with spontaneous mammary tumour developing PyMT-MMTV mice to generate the PyMT-MMTVxHPSE-/- strain, providing us with a valuable in vivo model to characterise the role of HPSE in early mammary tumour development, tumour progression and metastasis.Results and discussionsOur data indicate that although HPSE promoted tumour angiogenesis, overall tumour development and metastasis between PyMT-MMTV and PyMT-MMTVxHPSE-/- mice remained comparable. By examination of the tumour microenvironment, we also demonstrate that the tumour stroma positively contributes to mammary tumour HPSE activity, promoting tumour angiogenesis. In contrast, HPSE expressed in the tumour-bearing host appeared to play no significant role in tumour growth and metastasis. These data suggest that in the PyMT-MMTV model and indeed, in certain cancer settings such as breast cancer, HPSE may not play as significant a role to what has been proposed over the last two decades.ConclusionThese findings may have important implications for the ongoing development and application of HPSE inhibitors in the treatment of cancer.

N‐Glycan and Heparan Sulphate Components of the Extracellular Matrix are Essential for Laminar Shear Stress Response of the Epithelial Sodium Channel (ENaC)

The epithelial sodium channel (ENaC), a mechanosensitive ion channel, is known to be activated by laminar shear stress (LSS) [1]. We hypothesise that the large extracellular loops of ENaC interact with the complex extracellular matrix (ECM) and that the application of LSS deforms both the extracellular matrix (ECM) and the interacting ENaC components resulting in a change in ENaC activity. This study investigated whether or not the N‐glycan and heparan sulphate components of the ECM are specifically involved in the LSS activation of ENaC.Human α, β and γ ENaC subunits were co‐expressed in oocytes surgically harvested from female Xenopus laevis. Two‐electrode voltage‐clamp was used to determine ENaC activity in response to LSS when different components of the ECM were degraded by neuraminidase and heparinase III enzymes. A significant LSS response was observed in ENaC expressing oocytes, compared to native (no ENaC) oocytes; normalized LSS current of 5.5 ± 1.1 compared to 0.06 ± 0.03 (p = 0.003, n =8–14). In ENaC expressing oocytes cleavage of N‐glycan from the ECM with neuraminidase (0.2U/mL for 2h at 21°C) significantly reduced the normalised LSS response from 4.5 ± 0.4 to 1.7 ± 0.4 (p=0.003, n=8). Destruction of heparan sulphate components of the ECM with heparinase III (0.5 U/mL for 30min at 21°C) significantly reduced the LSS response from 5.1 ± 1.2 to 1.2 ± 0.5 (p=0.009, n=12). Data indicates that both removal of N‐glycan and cleavage of heparan sulphate from the ECM reduce the activation of ENaC by LSS, indicating that they are crucial components of the ECM for mechanosensation by ENaC.Support or Funding InformationThis study was supported by the Royal Society Marsden fund.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Involvement of Heparanase in the Pathogenesis of Mesothelioma: Basic Aspects and Clinical Applications.

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. 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. 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). 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.

Overexpression of heparanase in mice promoted megakaryopoiesis

Heparanase, an endo-glucuronidase that specifically cleaves heparan sulfate (HS), is upregulated in several pathological conditions. In this study, we aimed to find a correlation of heparanase expression and platelets production. In the transgenic mice overexpressing human heparanase (Hpa-tg), hematological analysis of blood samples revealed a significantly higher number of platelets in comparison with wild-type (Ctr) mice, while no significant difference was found in leukocytes and red blood cell number between the two groups. Total number of thiazole orange positive platelets was increased in Hpa-tg vs. Ctr blood, reflecting a higher rate of platelets production. Concomitantly, megakaryocytes from Hpa-tg mice produced more and shorter HS fragments that were shed into the medium. Further, thrombopoietin (TPO) level was elevated in the liver and plasma of Hpa-tg mice. Together, the data indicate that heparanase expression promoted megakaryopoiesis, which may be through upregulated expression of TPO and direct effect of released HS fragments expressed in the megakaryocytes.

Arylamidonaphtalene sulfonate compounds as a novel class of heparanase inhibitors

The search for antimetastatic agents for cancer therapy may involve the ability of new compounds to maintain the tissue extracellular matrix integrity. Among known factors, heparanase, an endoglucuronidase responsible for heparan sulfate cleavage, is a promising target whose inhibition could represent a strong obstacle for metastatic cancerous mechanisms. The antimetastatic activity of some suramin derivatives reported in literature suggests a possible involvement of the heparanase enzyme. To confirm such hypothesis, we have investigated FCE27266, a molecule known for its antiangiogenic and antimetastatic properties. Other new derivatives were also synthesized and investigated. Our findings revealed that FCE27266 as well as some derivatives have a strong heparanase inhibition activity, together with no cytotoxic power. Moreover, a FCE27266 analogue (SST0546NA1; 17a) resulted also positive to lower gene expression of some proangiogenic factors.

Heparanase Upregulation Contributes to Porcine Reproductive and Respiratory Syndrome Virus Release.

Porcine reproductive and respiratory syndrome virus (PRRSV) continues to cause substantial economic losses to the pig industry worldwide. Heparan sulfate (HS) is used by PRRSV for initial attachment to target cells. However, the role of HS in the late phase of PRRSV infection and the mechanism of virus release from host cells remain largely unknown. In this study, we showed that PRRSV infection caused a decrease in HS expression and upregulated heparanase, the only known enzyme capable of degrading HS. We subsequently demonstrated that the NF-κB signaling pathway and cathepsin L protease were involved in regulation of PRRSV infection-induced heparanase. In addition, we found that ablation of heparanase expression using small interfering RNA duplexes increased cell surface expression of HS and suppressed PRRSV replication and release, whereas overexpression of heparanase reduced HS surface expression and enhanced PRRSV replication and release. These data suggest that PRRSV activates NF-κB and cathepsin L to upregulate and process heparanase, and then the active heparanase cleaves HS, resulting in viral release. Our findings provide new insight into the molecular mechanism of PRRSV egress from host cells, which might help us to further understand PRRSV pathogenesis.IMPORTANCE Porcine reproductive and respiratory syndrome virus (PRRSV) causes great economic losses each year to the pig industry worldwide. The molecular mechanism of PRRSV release from host cells largely remains a mystery. In this study, we demonstrate that PRRSV activates NF-κB and cathepsin L to upregulate and process heparanase, and then the active heparanase is released to the extracellular space and exerts enzymatic activity to cleave heparan sulfate, resulting in viral release. Our findings provide new insight into the molecular mechanism of PRRSV egress from host cells, which might help us to further understand PRRSV pathogenesis.

Abstract 894: Heparanase-induced shedding of syndecan-1 promotes cancer cell invasion : prevention by inhibitory synstatin peptide

Abstract Syndecan-1 (Sdc1, CD138) is highly expressed in multiple myeloma (MM). Heparanase (HPSE), an enzyme that cleaves heparan sulfate (HS) chains on Sdc1, induces shedding of the Sdc1 ectodomain; this is associated with poor prognosis in this disease. However, the link between Sdc1 shedding and poor prognosis remains unclear. We now show that HPSE-induced shed Sdc1 (sSdc1) engages α4β1 integrin (VLA-4) and vascular endothelial cell growth factor receptor-2 (VEGFR2) to form a ternary receptor complex on the cell surface. This coupling of VEGFR2 to clusters of VLA-4 via sSdc1 leads to autoactivation of VEGFR2 and the subsequent activation of protein kinase A (PKA) bound to the VEGFR2 cytoplasmic domain. PKA activation proceeds via VEGFR2-mediated stimulation of the G-protein coupled cytokine receptor CXCR4 and the subsequent generation of cAMP by its activation of adenylate cyclase 7. This mechanism is prevented by SSTNVEGFR2, a peptide that competes for the VEGFR2 docking site in the Sdc1 ectodomain, thereby disrupting VEGFR2 binding to sSdc1. VLA-4 has been shown to mediate the polarized invasion of a variety of cells due to α4 integrin phosphorylation on Ser988 by PKA, followed by Rac GTPase activation, a process that is restricted to the leading edge of the cell. But how this is confined at the leading edge is wholly unknown. We find that HPSE-mediated shedding of Sdc1 promotes an invasive phenotype by re-localizing VLA-4 and VEGFR2 to the leading edge of migrating myeloma cells as well as human T-acute lymphoblastic leukemia, melanoma, and endothelial cells where VEGFR2-dependent PKA activation causes α4 integrin phosphorylation. These results reveal a novel mechanism in which the expression of HPSE, acting by causing Sdc1 shedding, potentially regulates angiogenesis and the extravasation and invasion of cancer cells and identifies SSTNVEGFR2 as a promising cancer therapeutic. Citation Format: Oisun Jung, Alan Rapraeger. Heparanase-induced shedding of syndecan-1 promotes cancer cell invasion : prevention by inhibitory synstatin peptide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 894. doi:10.1158/1538-7445.AM2017-894

Evaluation of the C-domain of heparanase during AGE-induced macrophage inflammatory response.

Diabetic vasculopathy is intensified by macrophage inflammation caused by advanced glycation end products (AGEs). Heparanase (HPA) is a unique endoglycosidase, which cleaves heparan sulfate proteoglycans (HSPGs) including syndecan-1 (Syn-1) to further stimulate macrophage cell migration and inflammation. The present study was planned to evaluated the role of C-domain (if any) of HPA in AGE inflammatory response in macrophages. Cell viability was assessed using MTT assay, migration assay, ELISA for tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) levels, mRNA expression by RT-PCR and heparan degrading enzyme assay for HPA activity. In the present study, we found that pretreatment with anti-HPA antibody, which recognizes the C-domain of HPA inhibited macrophage migration, secretion of IL-1β and TNF-α as well as decreased HPA enzymatic activity and increased Syn-1 protein expression in AGE-induced macrophages. Compared with anti-HPA antibody pretreatment, co-pretreatment with anti-HPA plus Syn-1 antibodies promoted macrophage migration, and secretion of IL-1β and TNF-α significantly in AGE-induced macrophages. In addition, pretreatment with anti-HPA or anti-HPA plus Syn-1 antibodies did not markedly change the mRNA levels of IL-1β and TNF-α concentration AGE-treated macrophages. The results showed that C-domain of HPA mediates AGE-induced macrophage migration and inflammatory cytokine release via Syn-1 protein expression. Furthermore, C-domain of HPA may have a key role in diabetic vascular complication-associated inflammatory response.

Upregulated Expression of Heparanase and Heparanase 2 in the Brains of Alzheimer's Disease.

Heparan sulfate proteoglycans (HSPGs) promote amyloid-β peptide and tau fibrillization in Alzheimer's disease (AD) and provide resistance against proteolytic breakdown. Heparanase (HPSE) is the only enzyme that cleaves heparan sulfate (HS). Heparanase 2 (HPSE2) lacks HS-degrading activity, although it is able to interact with HS with high affinity. To analyze HPSE and HPSE2 expressions at different stages of AD. RT-PCR was used to analyze transcription levels of both heparanases at different stages of AD, and immunohistochemistry was performed to localize each one in different parts of the brain. Both proteins appeared overexpressed at different stages of AD. Immunohistochemistry indicated that the presence of the heparanases was related to AD pathology, with intracellular deposits found in degenerated neurons. At the extracellular level, HPSE was observed only in neuritic plaques with a fragmented core, while HPSE2 appeared in those with compact cores as well. Given the involvement of HSPGs in AD pathology, there would seem to be a relationship between the regulation of heparanase expression, the features of the disease, and a possible therapeutic alternative.

Gemcitabine-induced heparanase promotes aggressiveness of pancreatic cancer cells via activating EGFR signaling.

Pancreatic cancer (PC), characterized by aggressive local invasion and metastasis, is one of the most malignant cancers. Gemcitabine is currently used as the standard drug for the treatment of advanced and metastatic PC, but with limited efficacy. In this study, we demonstrated that gemcitabine increased the expression of heparanase (HPA1), the only known mammalian endoglycosidase capable of cleaving heparan sulfate, both in vitro and in vivo. Furthermore, overexpression of HPA1 in PC cell lines enhanced proliferation and invasion, accompanied with elevated phosphorylation of EGFR. In addition, we showed that the NF-κB pathway mediated the gemcitabine-induced HPA1 expression. Importantly, we found that an HPA1 inhibitor attenuated gemcitabine-induced invasion of PC cells. Finally, we showed that HPA1 was of negative prognostic value for PC patients. Taken together, our results demonstrated that gemcitabine-induced HPA1 promotes proliferation and invasion of PC cells through activating EGFR, implying that HPA1 may serve as promising therapeutic target in the treatment of PC.

The Role of Heparanase in the Pathogenesis of Acute Pancreatitis: A Potential Therapeutic Target

Acute pancreatitis (AP) is one of the most common diseases in gastroenterology. However, neither the etiology nor the pathophysiology of the disease is fully understood and no specific or effective treatment has been developed. Heparanase is an endoglycosidase that cleaves heparan sulfate (HS) side chains of HS sulfate proteoglycans into shorter oligosaccharides, activity that is highly implicated in cellular invasion associated with cancer metastasis and inflammation. Given that AP involves a strong inflammatory aspect, we examined whether heparanase plays a role in AP. Here, we provide evidence that pancreatic heparanase expression and activity are significantly increased following cerulein treatment. Moreover, pancreas edema and inflammation, as well as the induction of cytokines and signaling molecules following cerulein treatment were attenuated markedly by heparanase inhibitors, implying that heparanase plays a significant role in AP. Notably, all the above features appear even more pronounced in transgenic mice over expressing heparanase, suggesting that these mice can be utilized as a sensitive model system to reveal the molecular mechanism by which heparanase functions in AP. Heparanase, therefore, emerges as a potential new target in AP, and heparanase inhibitors, now in phase I/II clinical trials in cancer patients, are hoped to prove beneficial also in AP.

Heparanase 1 involvement in prostate physiopathology.

The prostate is a compound exocrine gland of the male reproductive tract universally present in mammals. It is highly responsive to androgen and can be committed by a variety of pathological complications as prostatitis, benign, and malignant proliferative changes, which may be intensified by aging. Prostate intensively turnover its extracellular matrix (ECM) either at homeostasis or disease which includes a dynamically change of glycosaminoglycan composition during the life of an individual. Among the different enzymes playing a role in such changes, heparanase-1 is responsible for cleaving heparan sulfate (HS) at a limited number of sites, clearly involved in tissue remodeling. Its activity has been strongly implicated in cell invasion associated with cancer metastasis, a consequence of the structural modification that loosens the ECM barrier. In the present review we focuses in some aspects of the prostate physiology and diseases, particular prostate cancer, evidencing how the HPSE-1 activity encompasses the relationship of both processes.

Identification of Novel Class of Triazolo-Thiadiazoles as Potent Inhibitors of Human Heparanase and their Anticancer Activity

BackgroundExpression 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.MethodsIn 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.ResultsAmong 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.ConclusionsOverall, we identified biologically active heparanase inhibitor which could serve as a lead structure in developing compounds that target heparanase in cancer.

Involvement of heparanase in the pathogenesis of acute kidney injury: nephroprotective effect of PG545.

Despite the high prevalence of acute kidney injury (AKI) and its association with increased morbidity and mortality, therapeutic approaches for AKI are disappointing. This is largely attributed to poor understanding of the pathogenesis of AKI. Heparanase, an endoglycosidase that cleaves heparan sulfate, is involved in extracellular matrix turnover, inflammation, kidney dysfunction, diabetes, fibrosis, angiogenesis and cancer progression. The current study examined the involvement of heparanase in the pathogenesis of ischemic reperfusion (I/R) AKI in a mouse model and the protective effect of PG545, a potent heparanase inhibitor. I/R induced tubular damage and elevation in serum creatinine and blood urea nitrogen to a higher extent in heparanase over-expressing transgenic mice vs. wild type mice. Moreover, TGF-β, vimentin, fibronectin and α-smooth muscle actin, biomarkers of fibrosis, and TNFα, IL6 and endothelin-1, biomarkers of inflammation, were upregulated in I/R induced AKI, primarily in heparanase transgenic mice, suggesting an adverse role of heparanase in the pathogenesis of AKI. Remarkably, pretreatment of mice with PG545 abolished kidney dysfunction and the up-regulation of heparanase, pro-inflammatory (i.e., IL-6) and pro-fibrotic (i.e., TGF-β) genes induced by I/R. The present study provides new insights into the involvement of heparanase in the pathogenesis of ischemic AKI. Our results demonstrate that heparanase plays a deleterious role in the development of renal injury and kidney dysfunction, attesting heparanase inhibition as a promising therapeutic approach for AKI.

The Functions of Heparanase in Human Diseases.

The study of the heparanase has long been paid wide attention. Heparanase, an endo-β-D-glucuronidase, is capable of specifically degrading heparan sulfate (HS), one of the excellular matrix (ECM) components. It exerts its enzymatic activity catalyzing the cleavage of the β (1,4)-glycosidic bond between glucuronic acid and glucosamine residue. HS cleavage results in remodelling of the extracellular matrix as well as in regulating the release of many HS-linked molecules such as growth factors, cytokines and enzymes involved in inflammation, wound healing and tumour invasion. Varieties of experiments indicated that heparanase mRNA is overexpressed in human tumors, including breast cancer, gastrointestinal tumors, and esophageal carcinomas. A pro-metastatic and pro-angiogenic role for heparanase has been widely verified and high levels of heparanase correlate with reduced survival of cancer patients. Except protumor function, heparanase also plays a role in inflammation, angiogenesis, placentas and procoagulant activities. Heparanase is found to have many other functions in recent years, since many experiments have been carried out to identify this significant enzyme's new features. These newly found functions are related to the cellular activities such as autophagy and epithelial to mesenchymal transition (EMT). And together with other heparanase functions, autophagy and EMT are verified to be involved in several clinical disorders, for example, renal diseases. Considering that, once inactivated, there are no other enzymes capable of performing the same function, it is apparent that heparanase can be an effective and promising therapy target. This short review aims to establish the currently known function of this enzyme and provide evidence for heparanase targeted therapy.

The Heparanase Inhibitor PG545 Attenuates Colon Cancer Initiation and Growth, Associating with Increased p21 Expression

Heparanase activity is highly implicated in cellular invasion and tumor metastasis, a consequence of cleavage of heparan sulfate and remodeling of the extracellular matrix underlying epithelial and endothelial cells. Heparanase expression is rare in normal epithelia, but is often induced in tumors, associated with increased tumor metastasis and poor prognosis. In addition, heparanase induction promotes tumor growth, but the molecular mechanism that underlines tumor expansion by heparanase is still incompletely understood. Here, we provide evidence that heparanase down regulates the expression of p21 (WAF1/CIP1), a cyclin-dependent kinase inhibitor that attenuates the cell cycle. Notably, a reciprocal effect was noted for PG545, a potent heparanase inhibitor. This compound efficiently reduced cell proliferation, colony formation, and tumor xenograft growth, associating with a marked increase in p21 expression. Utilizing the APC Min+/− mouse model, we show that heparanase expression and activity are increased in small bowel polyps, whereas polyp initiation and growth were significantly inhibited by PG545, again accompanied by a prominent induction of p21 levels. Down-regulation of p21 expression adds a novel feature for the emerging pro-tumorigenic properties of heparanase, while the potent p21 induction and anti-tumor effect of PG545 lends optimism that it would prove an efficacious therapeutic in colon carcinoma patients.

Specific heparanase inhibition reverses glucose-induced mesothelial-to-mesenchymal transition.

Epithelial-to-mesenchymal transition (EMT) of peritoneal mesothelial cells induced by high glucose (HG) levels is a major biological mechanism leading to myofibroblast accumulation in the omentum of patients on peritoneal dialysis (PD). Heparanase (HPSE), an endoglycosidase that cleaves heparan sulfate chains, is involved in the EMT of several cell lines, and may have a major role in this pro-fibrotic process potentially responsible for the failure of dialysis. Its specific inhibition may therefore plausibly minimize this pathological condition. An in vitro study employing several biomolecular strategies was conducted to assess the role of HPSE in the HG-induced mesothelial EMT process, and to measure the effects of its specific inhibition by SST0001, a N-acetylated glycol-split heparin with a strong anti-HPSE activity. Rat mesothelial cells were grown for 6 days in HG (200 mM) culture medium with or without SST0001. Then EMT markers (VIM, α-SMA, TGF-β) and vascular endothelial growth factor (VEGF) (a factor involved in neoangiogenesis) were measured by real-time PCR and immunofluorescence/western blotting. As a functional analysis, trans-epithelial resistance (TER) and permeability to albumin were also measured in our in vitro model using a Millicell-ERS ohmmeter and a spectrophotometer, respectively. Our results showed that 200 mM of glucose induced a significant gene and protein up-regulation of VEGF and all EMT markers after 6 days of culture. Intriguingly, adding SST0001 on day 3 reversed these biological and cellular effects. HPSE inhibition also restored the normal TER and permeability lost during the HG treatment. Taken together, our data confirm that HG can induce EMT of mesothelial cells, and that HPSE plays a central part in this process. Our findings also suggest that pharmacological HPSE inhibition could prove a valuable therapeutic tool for minimizing fibrosis and avoiding a rapid decline in the efficacy of dialysis in patients on PD, though clinical studies and/or trials would be needed to confirm the clinical utility of this treatment.