Enzyme Heparanase Research Articles

Heparanase 2 (Hpa2) attenuates the growth of pancreatic carcinoma

While the pro-tumorigenic properties of the ECM-degrading heparanase enzyme are well documented, the role of its close homolog, heparanase 2 (Hpa2), in cancer is largely unknown. We examined the role of Hpa2 in pancreatic cancer, a malignancy characterized by a dense fibrotic ECM associated with poor response to treatment and bad prognosis. We show that pancreatic ductal adenocarcinoma (PDAC) patients that exhibit high levels of Hpa2 survive longer than patients with low levels of Hpa2. Strikingly, overexpression of Hpa2 in pancreatic carcinoma cells resulted in a most prominent decrease in the growth of tumors implanted orthotopically and intraperitoneally, whereas Hpa2 silencing resulted in bigger tumors. We further found that Hpa2 enhances endoplasmic reticulum (ER) stress response and renders cells more sensitive to external stress, associating with increased apoptosis. Interestingly, we observed that ER stress induces the expression of Hpa2, thus establishing a feedback loop by which Hpa2 enhances ER stress that, in turn, induces Hpa2 expression. This leads to increased apoptosis and attenuated tumor growth. Altogether, Hpa2 emerges as a powerful tumor suppressor in pancreatic cancer.

Alveolar epithelial glycocalyx shedding aggravates the epithelial barrier and disrupts epithelial tight junctions in acute respiratory distress syndrome

The main pathophysiological mechanism of acute respiratory distress syndrome (ARDS) invovles the increase in alveolar barrier permeability that is primarily caused by epithelial glycocalyx and tight junction (TJ) protein destruction. This study was performed to explore the effects of the alveolar epithelial glycocalyx on the epithelial barrier, specifically on TJ proteins, in ARDS. We used C57BL/6 mice and human lung epithelial cell models of lipopolysaccharide (LPS)-induced ARDS. Changes in alveolar permeability were evaluated via pulmonary histopathology analysis and by measuring the wet/dry weight ratio of the lungs. Degradation of heparan sulfate (HS), an important component of the epithelial glycocalyx, and alterations in levels of the epithelial TJ proteins (occludin, zonula occludens 1, and claudin 4) were assessed via ELISA, immunofluorescence analysis, and western blotting analysis. Real-time quantitative polymerase chain reaction was used to detect the mRNA of the TJ protein. Changes in glycocalyx and TJ ultrastructures in alveolar epithelial cells were evaluated through electron microscopy. In vivo and in vitro, LPS increased the alveolar permeability and led to HS degradation and TJ damage. After LPS stimulation, the expression of the HS-degrading enzyme heparanase (HPA) in the alveolar epithelial cells was increased. The HPA inhibitor N-desulfated/re-N-acetylated heparin alleviated LPS-induced HS degradation and reduced TJ damage. In vitro, recombinant HPA reduced the expression of the TJ protein zonula occludens-1 (ZO-1) and inhibited its mRNA expression in the alveolar epithelial cells. Taken together, our results demonstrate that shedding of the alveolar epithelial glycocalyx aggravates the epithelial barrier and damages epithelial TJ proteins in ARDS, with the underlying mechanism involving the effect of HPA on ZO-1.

Heparanase Inhibitors in Cancer Progression: Recent Advances.

An endo-β-glucuronidase enzyme, Heparanase (HPSE), degrades the side chains of polymeric heparan sulfate (HS), a glycosaminoglycan formed by alternate repetitive units of D-glucosamine and D-glucuronic acid/L-iduronic acid. HS is a major component of the extracellular matrix and basement membranes and has been implicated in processes of the tissue's integrity and functional state. The degradation of HS by HPSE enzyme leads to conditions like inflammation, angiogenesis, and metastasis. An elevated HPSE expression with a poor prognosis and its multiple roles in tumor growth and metastasis has attracted significant interest for its inhibition as a potential anti-neoplastic target. We reviewed the literature from journal publication websites and electronic databases such as Bentham, Science Direct, PubMed, Scopus, USFDA, etc., about HPSE, its structure, functions, and role in cancer. The present review is focused on Heparanase inhibitors (HPIns) that have been isolated from natural resources or chemically synthesized as new therapeutics for metastatic tumors and chronic inflammatory diseases in recent years. The recent developments made in the HPSE structure and function are also discussed, which can lead to the future design of HPIns with more potency and specificity for the target. HPIns can be a better target to be explored against various cancers.

Increased Plasma Heparanase Activity in COVID-19 Patients.

Reports suggest a role of endothelial dysfunction and loss of endothelial barrier function in COVID-19. It is well established that the endothelial glycocalyx-degrading enzyme heparanase contributes to vascular leakage and inflammation. Low molecular weight heparins (LMWH) serve as an inhibitor of heparanase. We hypothesize that heparanase contributes to the pathogenesis of COVID-19, and that heparanase may be inhibited by LMWH. To test this hypothesis, heparanase activity and heparan sulfate levels were measured in plasma of healthy controls (n = 10) and COVID-19 patients (n = 48). Plasma heparanase activity and heparan sulfate levels were significantly elevated in COVID-19 patients. Heparanase activity was associated with disease severity including the need for intensive care, lactate dehydrogenase levels, and creatinine levels. Use of prophylactic LMWH in non-ICU patients was associated with a reduced heparanase activity. Since there is no other clinically applied heparanase inhibitor currently available, therapeutic treatment of COVID-19 patients with low molecular weight heparins should be explored.

Syndecan-1-Dependent Regulation of Heparanase Affects Invasiveness, Stem Cell Properties, and Therapeutic Resistance of Caco2 Colon Cancer Cells.

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.

Differential regulation of sFlt-1 splicing by U2AF65 and JMJD6 in placental-derived and endothelial cells.

Despite years of study, the gestational disorder preeclampsia (PE) remains poorly understood. One proposed mechanism of PE development is increased soluble VEGF receptor-1 (sFlt-1), ultimately causing angiogenic imbalance and endothelial dysfunction. The soluble protein is an alternative splice variant of FLT1, which also encodes for the full-length receptor Flt-1. The mechanism of the alternative splicing, and the reason for its inappropriate increase in preeclampsia, is not well understood. U2 auxiliary factor 65 (U2AF65) and jumonji C domain-containing protein 6 (JMJD6) have been implicated in the splicing of sFlt-1. Using siRNA knockdown and plasmid overexpression in immortalized placental trophoblasts (BeWo) and primary endothelial cells (HUVECs), we examined the role these proteins play in production of sFlt-1. Our results showed that U2AF65 has little, if any, effect on sFlt-1 splicing, and JMJD6 may enhance sFlt-1 splicing, but is not necessary for splicing to occur. Utilizing a hypoxic environment to mimic conditions of the preeclamptic placenta, as well as examining placentae in the reduced uterine perfusion pressure (RUPP) model of PE, which exhibits increased circulating sFlt-1, we found increased expression of JMJD6 in both hypoxic cells and placental tissue. Additionally, we observed a potential role for U2AF65 and JMJD6 to regulate the extracellular matrix enzyme heparanase, which may be involved in the release of sFlt-1 protein from the extracellular matrix. It will be important to study the role of these proteins in different tissues in the future, as changes in expression had differential effects on sFlt-1 splicing in the different cell types studied here.

The Good and Bad Sides of Heparanase-1 and Heparanase-2.

In this chapter, we will emphasize the importance of heparan sulfate proteoglycans (HSPG) in controlling various physiological and pathological molecular mechanisms and discuss how the heparanase enzyme can modulate the effects triggered by HSPG. Additionally, we will also navigate about the existing knowledge of the possible role of heparanase-2 in biological events. Heparan sulfate is widely distributed and evolutionarily conserved, evidencing its vital importance in cell development and functions such as cell proliferation, migration, adhesion, differentiation, and angiogenesis. During remodeling of the extracellular matrix, the breakdown of heparan sulfate by heparanase results in the release of molecules containing anchored glycosaminoglycan chains of great interest in heparanase-mediated cell signaling pathways in various physiological states, tumor development, inflammation, and other diseases. Taken together, it appears that heparanase plays a key role in the maintenance of the pathology of cancer and inflammatory diseases and is a potential target for anti-cancer therapies. Therefore, heparanase inhibitors are currently being examined in clinical trials as novel cancer therapeutics. Heparanase-2 has no enzymatic activity, displays higher affinity for heparan sulfate and the coding region alignment shows 40% identity with the heparanase gene. Heparanase-2 plays an important role in embryogenic development however its mode of action and biological function remain to be elucidated. Heparanase-2 functions as an inhibitor of the heparanase-1 enzyme and also inhibits neovascularization mediated by VEGF. The HPSE2 gene is repressed by the Polycomb complex, together suggesting a role as a tumor suppressor.

Regulation of Heparanase in Diabetes-Associated Pancreatic Carcinoma

While at least six types of cancer have been associated with diabetes, pancreatic ductal adenocarcinoma (PDAC) and diabetes exhibit a unique bidirectional relationship. Recent reports indicate that majority of PDAC patients display hyperglycemia, and ~50% have concurrent diabetes. In turn, hyperglycemic/diabetic state in PDAC patients fosters enhanced growth and dissemination of the tumor. Heparanase enzyme (the sole mammalian endoglycosidase degrading glycosaminoglycan heparan sulfate) is tightly implicated in PDAC progression, aggressiveness, and therapy resistance. Overexpression of heparanase is a characteristic feature of PDAC, correlating with poor prognosis. However, given the lack of heparanase expression in normal pancreatic tissue, the regulatory mechanisms responsible for induction of the enzyme in PDAC have remained largely unknown. Previously reported inducibility of heparanase gene by diabetic milieu components in several non-cancerous cell types prompted us to hypothesize that in the setting of diabetes-associated PDAC, hyperglycemic state may induce heparanase overexpression. Here, utilizing a mouse model of diet-induced metabolic syndrome/diabetes, we found accelerated PDAC progression in hyperglycemic mice, occurring along with induction of heparanase in PDAC. In vitro, we demonstrated that advanced glycation end-products (AGE), which are largely thought as oxidative derivatives resulting from chronic hyperglycemia, and the receptor for AGE (RAGE) are responsible for heparanase induction in PDAC cells. These findings underscore the new mechanism underlying preferential expression of heparanase in pancreatic cancer. Moreover, taken together with the well-established causal role of the enzyme in PDAC progression, our findings indicate that heparanase may sustain (at least in part) reciprocal causality between diabetes and pancreatic tumorigenesis.

VLA-4 phosphorylation during tumor and immune cell migration relies on its coupling to VEGFR2 and CXCR4 by syndecan-1.

When targeted by the tumor-promoting enzyme heparanase, cleaved and shed syndecan-1 (Sdc1)then couples VEGFR2 (also known as KDR) to VLA-4, activating VEGFR2 and the directed migration of myeloma cells. But how VEGFR2 activates VLA-4-mediated motility has remained unknown. We now report that VEGFR2 causes PKA-mediated phosphorylation of VLA-4 on S988, an event known to stimulate tumor metastasis while suppressing cytotoxic immune cells. A key partner in this mechanism is the chemokine receptor CXCR4, a well-known mediator of cell motility in response to gradients of the chemokine SDF-1 (also known as CXCL12). The entire machinery necessary to phosphorylate VLA-4, consisting of CXCR4, AC7 (also known as ADCY7) and PKA, is constitutively associated with VEGFR2 and is localized to the integrin by Sdc1. VEGFR2 carries out the novel phosphorylation of Y135 within the DRY microswitch of CXCR4, sequentially activating Gαiβγ, AC7 and PKA, which phosphorylates S988 on the integrin. This mechanism is blocked by a syndecan-mimetic peptide (SSTNVEGFR2), which, by preventing VEGFR2 linkage to VLA-4, arrests tumor cell migration that depends on VLA-4 phosphorylation and stimulates the LFA-1-mediated migration of cytotoxic leukocytes.

Tie2 Activation Promotes Protection and Reconstitution of the Endothelial Glycocalyx in Human Sepsis.

The endothelial glycocalyx (eGC), a carbohydrate-rich layer lining the luminal surface of the endothelium, provides a first vasoprotective barrier against vascular leakage in sepsis. We hypothesized that angiopoietin-2 (Angpt-2), antagonist of the endothelium-stabilizing receptor Tie2, induces a rapid loss of the eGC in human sepsis. Using intravital microscopy, we measured the perfused boundary region (PBR), an inverse parameter of eGC dimensions in sublingual microvessels, in patients with sepsis and age-matched nonseptic subjects. Median PBR values were significantly higher in patients compared with controls and correlated with serum Angpt-2 levels. To transfer and further explore these findings in a cell culture system, we exposed endothelial cells (ECs) to serum (5%) from a subgroup of septic patients and nonseptic controls. Confocal and atomic force microscopy revealed that sepsis serum, but not control serum, induced thinning of the eGC on human ECs in vitro, which correlated with paired PBR values obtained in vivo (r = 0.96, p < 0.01). Inhibition of Angpt-2 or Tie2 activation completely abolished eGC damage. Mechanistically, sepsis-induced eGC breakdown required the loss of its main constituent heparan sulfate; a result of heparan sulfate-specific enzyme heparanase, which was suppressed by Tie2 activation. Finally, Tie2 activation, but not Angpt-2 inhibition, initiated after septic or enzymatic damage provoked rapid refurbishment of the eGC. Our data indicate that eGC breakdown in human sepsis is mediated via Tie2 deactivation by Angpt-2. Activation of Tie2 seems to accelerate recovery of the eGC and might hold promise as a therapeutic target in human sepsis.

Conditional Deletion of Phd2 in Spongiotrophoblasts Mimics Early Onset Preeclampsia

Preeclampsia is a common pregnancy disorder which is characterized by new onset hypertension and endothelial dysfunction. Despite efforts to determine the causal factors of this disease, little progress has been made in discerning the etiology. The hypoxic and ischemic placenta, however, is generally accepted as the source for secreted factors in the maternal circulation, such as sFLT-1, which drive the maternal syndrome.Using BeWo placental trophoblast cells, we measured the role of hypoxia on sFLT-1 mRNA as well as protein production. We also exposed the cells to treatment with heparin and heparanase inhibitor OGT-2115.We found that under hypoxic conditions mRNA levels of sFLT-1 were unchanged compared to normoxic controls. Although the message level did not differ under hypoxic conditions, the sFLT-1 release into the media was significantly greater in hypoxia. Additionally, we found that sFLT-1 is able to bind heparan strands in the extracellular matrix with its heparin binding site. These heparan strands can be cleaved by the extracellular enzyme heparanase. We found that heparanase expression was significantly increased in hypoxia, and inhibiting the actions of heparanase attenuated the release of sFLT-1 into the media.While the placenta remains a source of sFLT-1, the mechanism of increased circulating sFLT-1 may differ than simple upregulation of the protein. These data demonstrate the potential importance of the role heparanase may play in releasing previously made sFLT-1 into the maternal circulation.

Sexual Dimorphism in Regulation of Trophoblast Respiration and Metabolic Reprogramming with Obesity and Gestational Diabetes

Preeclampsia is a common pregnancy disorder which is characterized by new onset hypertension and endothelial dysfunction. Despite efforts to determine the causal factors of this disease, little progress has been made in discerning the etiology. The hypoxic and ischemic placenta, however, is generally accepted as the source for secreted factors in the maternal circulation, such as sFLT-1, which drive the maternal syndrome.Using BeWo placental trophoblast cells, we measured the role of hypoxia on sFLT-1 mRNA as well as protein production. We also exposed the cells to treatment with heparin and heparanase inhibitor OGT-2115.We found that under hypoxic conditions mRNA levels of sFLT-1 were unchanged compared to normoxic controls. Although the message level did not differ under hypoxic conditions, the sFLT-1 release into the media was significantly greater in hypoxia. Additionally, we found that sFLT-1 is able to bind heparan strands in the extracellular matrix with its heparin binding site. These heparan strands can be cleaved by the extracellular enzyme heparanase. We found that heparanase expression was significantly increased in hypoxia, and inhibiting the actions of heparanase attenuated the release of sFLT-1 into the media.While the placenta remains a source of sFLT-1, the mechanism of increased circulating sFLT-1 may differ than simple upregulation of the protein. These data demonstrate the potential importance of the role heparanase may play in releasing previously made sFLT-1 into the maternal circulation.

Heparanase regulation of sFLT-1 release in trophoblasts in vitro

IntroductionPreeclampsia is a common pregnancy disorder which is characterized by new onset hypertension and endothelial dysfunction. Despite efforts to determine the causal factors of this disease, little progress has been made in discerning the etiology. The hypoxic and ischemic placenta, however, is generally accepted as the source for secreted factors in the maternal circulation, such as sFLT-1, which drive the maternal syndrome. MethodsUsing BeWo placental trophoblast cells, we measured the role of hypoxia on sFLT-1 mRNA as well as protein production. We also exposed the cells to treatment with heparin and heparanase inhibitor OGT-2115. ResultsWe found that under hypoxic conditions mRNA levels of sFLT-1 were unchanged compared to normoxic controls. Although the message level did not differ under hypoxic conditions, the sFLT-1 release into the media was significantly greater in hypoxia. Additionally, we found that sFLT-1 is able to bind heparan strands in the extracellular matrix with its heparin binding site. These heparan strands can be cleaved by the extracellular enzyme heparanase. We found that heparanase expression was significantly increased in hypoxia, and inhibiting the actions of heparanase attenuated the release of sFLT-1 into the media. DiscussionWhile the placenta remains a source of sFLT-1, the mechanism of increased circulating sFLT-1 may differ than simple upregulation of the protein. These data demonstrate the potential importance of the role heparanase may play in releasing previously made sFLT-1 into the maternal circulation.

Specificity and action pattern of heparanase Bp, a β-glucuronidase from Burkholderia pseudomallei.

The specificity and action pattern of a β-glucuronidase derived from the pathogenic bacteria Burkholderia pseudomallei and expressed in Escherichia coli as a recombinant protein has been evaluated. While this enzyme shows activity on a number of glycosaminoglycans, our study has focused on its action on heparin, heparan sulfate and their biosynthetic intermediates as well as chemoenzymatically synthesized, structurally defined heparan sulfate oligosaccharides. These heparin/heparan sulfate (HP/HS) substrates examined varied in size and structure, but all contained an uronic acid (UA) residue β-(1→4) linked to a glucosamine residue. On the substrates tested, this enzyme (heparanase Bp) acted only on a glucuronic acid residue β-(1→4) linked to an N-acetylglucosamine, N-sulfoglucosamine or N-acetyl-6-O-sulfoglucosamine residue. A substrate was required to have a length of pentasaccharide or longer and heparanase Bp acted with a random endolytic action pattern on HP/HS. The specificity and glycohydrolase mechanism of action of heparanase Bp resembles mammalian heparanase and is complementary to the bacterial heparin lyases, which act through an eliminase mechanism on a glucosamine residue (1→4) linked to a UA residue, suggesting its utility as a tool for the structural determination of HP/HS as well as representing a possible model for the medically relevant mammalian heparanase. The utility heparanase Bp was demonstrated by the oligosaccharide mapping of heparin, which afforded resistant intact highly sulfated domains ranging from tetrasaccharide to >28-mer with a molecular weight >9000.

The Development of Assays for Heparanase Enzymatic Activity: Towards a Gold Standard.

The enzyme heparanase, an endo-β-glucuronidase, degrades heparan sulfate (HS) chains on the cell surface and in the extracellular matrix. Heparanase regulates numerous biological processes that drive tumour growth, metastasis and angiogenesis. In addition to its key role in cancer progression, it has also been implicated in an ever-growing number of other diseases, particularly those associated with inflammation. The importance of heparanase in biology has led to numerous efforts over the years to develop assays to monitor its activity and to screen for new inhibitors as potential drug candidates. Despite these efforts and the commercialization of a few kits, most heparanase assays are still complex, labour intensive, costly or have limited application. Herein we review the various methods for assaying heparanase enzymatic activity, focusing on recent developments towards new assays that hold the promise of accelerating research into this important enzyme.

Host Enzymes Heparanase and Cathepsin L Promote Herpes Simplex Virus 2 Release from Cells.

Herpes simplex virus 2 (HSV-2) can productively infect many different cell types of human and nonhuman origin. Here we demonstrate interconnected roles for two host enzymes, heparanase (HPSE) and cathepsin L, in HSV-2 release from cells. In vaginal epithelial cells, HSV-2 causes heparan sulfate shedding and upregulation in HPSE levels during the productive phase of infection. We also noted increased levels of cathepsin L and show that regulation of HPSE by cathepsin L via cleavage of HPSE proenzyme is important for infection. Furthermore, inhibition of HPSE by a specific inhibitor, OGT 2115, dramatically reduces HSV-2 release from vaginal epithelial cells. Likewise, we show evidence that the inhibition of cathepsin L is detrimental to the infection. The HPSE increase after infection is mediated by an increased NF-κB nuclear localization and a resultant activation of HPSE transcription. Together these mechanisms contribute to the removal of heparan sulfate from the cell surface and thus facilitate virus release from cells.IMPORTANCE Genital infections by HSV-2 represent one of the most common sexually transmitted viral infections. The virus causes painful lesions and sores around the genitals or rectum. Intermittent release of the virus from infected tissues during sexual activities is the most common cause of transmission. At the molecular level, cell surface heparan sulfate (HS) is known to provide attachment sites for HSV-2. While the removal of HS during HSV-1 release has been shown, not much is known about the host factors and their regulators that contribute to HSV-2 release from natural target cell types. Here we suggest a role for the host enzyme heparanase in HSV-2 release. Our work reveals that in addition to the regulation of transcription by NF-κB, HPSE is also regulated posttranslationally by cathepsin L and that inhibition of heparanase activity directly affects HSV-2 release. We provide unique insights into the host mechanisms controlling HSV-2 egress and spread.

Heparan sulfate is a plasma biomarker of acute cellular allograft rejection.

Despite advances in management of immunosuppression, graft rejection remains a significant clinical problem in solid organ transplantation. Non-invasive biomarkers of graft rejection can facilitate earlier diagnosis and treatment of acute rejection. The purpose of this study was to investigate the potential role of heparan sulfate as a novel biomarker for acute cellular rejection. Heparan sulfate is released from the extracellular matrix during T-cell infiltration of graft tissue via the action of the enzyme heparanase. In a murine heart transplant model, serum heparan sulfate is significantly elevated during rejection of cardiac allografts. Moreover, expression of the enzyme heparanase is significantly increased in activated T-cells. In human studies, plasma heparan sulfate is significantly elevated in kidney transplant recipients with biopsy-proven acute cellular rejection compared to healthy controls, recipients with stable graft function, and recipients without acute cellular rejection on biopsy. Taken together, these findings support further investigation of heparan sulfate as a novel biomarker of acute cellular rejection in solid organ transplantation.

Substitution-Inert Polynuclear Platinum Complexes as Metalloshielding Agents for Heparan Sulfate.

Cleavage of heparan sulfate proteoglycans (HSPGs) by the enzyme heparanase modulates tumour-related events including angiogenesis, cell invasion, and metastasis. Metalloshielding of heparan sulfate (HS) by positively charged polynuclear platinum complexes (PPCs) effectively inhibits physiologically critical HS functions. Studies using bacterial P. heparinus heparinase II showed that a library of Pt complexes varying in charge and nuclearity and the presence or absence of a dangling amine inhibits the cleavage activity of the enzyme on the synthetic pentasaccharide, Fondaparinux (FPX). Charge-dependent affinity of PPC for FPX was seen in competition assays with methylene blue and ethidium bromide. The dissociation constant (Kd ) of TriplatinNC for FPX was directly measured by isothermal titration calorimetry (ITC). The trend in DFT calculated interaction energies with heparin fragments is consistent with the spectroscopic studies. Competitive inhibition of TAMRA-R9 internalization in human carcinoma (HCT116) cells along with studies in HCT116, wildtype CHO and mutant CHO-pgsA745 (lacking HS/CS) cells confirm that HSPG-mediated interactions play an important role in the cellular accumulation of PPCs.

The heparan sulfate degrading enzyme heparanase is up‐regulated by the EWS‐Fli1 fusion protein in Ewing sarcoma

Ewing sarcoma is an aggressive form of bone and soft tissue cancer that predominantly affects adolescents. It is commonly caused by the partial translocation of the Ewing sarcoma protein (EWS) gene to the gene encoding the Ets family transcription factor Fli1, resulting in the formation of the EWS‐Fli1 fusion protein. Ewing sarcoma is often highly metastatic, resulting in a poor survival rate of approximately 20%. Expression of the endoglycosidase heparanase (HPSE), a heparan sulfate‐degrading enzyme that contributes to extracellular matrix remodelling, is up‐regulated in approximately 90% of all cancers, including Ewing sarcoma, and possesses pro‐metastatic and ‐angiogenic functions. It is therefore not surprising that it is also a marker of poor prognosis for cancer patients. While increased expression of HPSE in Ewing sarcoma has been reported to correlate with increased tumour size, disease severity and a poor patient outcome, the mechanism by which HPSE expression is increased in Ewing sarcoma has not been defined. We have identified a novel link between the EWS‐Fli1 fusion protein and the expression of HPSE. Using a combination of patient‐derived samples and cell lines, we have been able to show that an increase in expression of EWS‐Fli1 correlates with increased HPSE expression and activity. We also demonstrated that increased EWS‐Fli1 expression is responsible for increased HPSE expression and enzymatic activity by binding to the HPSE gene promoter and driving its expression. These findings highlight a possible role for HPSE in the progression and prognosis of Ewing sarcoma that may contribute to the aggressive nature of the disease.Support or Funding InformationAustralian National Health and Medical Research CouncilThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Prophylactic Antiheparanase Activity by PG545 Is Antiviral In Vitro and Protects against Ross River Virus Disease in Mice

Recently we reported on the efficacy of pentosan polysulfate (PPS), a heparan sulfate mimetic, to reduce the recruitment of inflammatory infiltrates and protect the cartilage matrix from degradation in Ross River virus (RRV)-infected PPS-treated mice. Here, we describe both prophylactic and therapeutic treatment with PG545, a low-molecular-weight heparan sulfate mimetic, for arthritogenic alphaviral infection. We first assessed antiviral activity in vitro through a 50% plaque reduction assay. Increasing concentrations of PG545 inhibited plaque formation prior to viral adsorption in viral strains RRV T48, Barmah Forest virus 2193, East/Central/South African chikungunya virus (CHIKV), and Asian CHIKV, suggesting a strong antiviral mode of action. The viral particle-compound dissociation constant was then evaluated through isothermal titration calorimetry. Furthermore, prophylactic RRV-infected PG545-treated mice had reduced viral titers in target organs corresponding to lower clinical scores of limb weakness and immune infiltrate recruitment. At peak disease, PG545-treated RRV-infected mice had lower concentrations of the matrix-degrading enzyme heparanase in conjunction with a protective effect on tissue morphology, as seen in the histopathology of skeletal muscle. Enzyme-linked immunosorbent assay quantification of cartilage oligomeric matrix protein and cross-linked C-telopeptides of type II collagen as well as knee histopathology showed increased matrix protein degradation and cartilage erosion in RRV-infected phosphate-buffered saline-treated mice compared to their PG545-treated RRV-infected counterparts. Taken together, these findings suggest that PG545 has a direct antiviral effect on arthritogenic alphaviral infection and curtails RRV-induced inflammatory disease when administered as a prophylaxis.