Heparanase Inhibitor Research Articles

PG545, a Heparan Sulfate Mimetic, Reduces Heparanase Expression In Vivo, Blocks Spontaneous Metastases and Enhances Overall Survival in the 4T1 Breast Carcinoma Model

PG545 is a clinically relevant heparan sulfate (HS) mimetic which, in addition to possessing anti-angiogenic properties, also acts as a heparanase inhibitor which may differentiate its mechanism(s) of action from approved angiogenesis inhibitors. The degradation of HS by heparanase has been strongly implicated in cell dissemination and the metastatic process. Thus, the anti-metastatic activity of PG545 has been linked to the enzymatic function of heparanase – the only endoglycosidase known to cleave HS, an important component of the extracellular matrix (ECM) which represents a potential avenue for therapeutic intervention for certain metastatic cancer indications. Recent concerns raised about the paucity of overall survival as an endpoint in mouse models of clinically relevant metastasis led us to examine the effect of PG545 on the progression of both primary tumor growth and the spontaneously metastasizing disease in the 4T1 syngeneic breast carcinoma model in a non-surgical and surgical (mastectomy) setting. PG545 significantly inhibited primary tumor growth but importantly also inhibited lung metastasis in treated mice, an effect not observed with the tyrosine kinase inhibitor sorafenib. Importantly, PG545 significantly enhanced overall survival compared to vehicle control and the sorafenib group, suggesting PG545’s inhibitory effect on heparanase is indeed a critical attribute to induce anti-metastatic activity. In addition to blocking a common angiogenic signalling pathway in tumor cells, the expression of heparanase in the primary tumor and lung was also significantly reduced by PG545 treatment. These results support the ongoing development of PG545 and highlight the potential utility in metastatic disease settings.

Heparanase Expression Correlates with Bone Destruction in the Patients with Multiple Myeloma

Abstract 1813Multiple myeloma is an incurable malignancy, and excessive bone destruction is a major cause of morbidity in myeloma patients. However, the biologic mechanisms involved in the pathogenesis of myeloma-induced bone disease are poorly understood. Heparanase, an enzyme that cleaves the heparan sulfate chains of proteoglycans, is upregulated in a variety of human tumors, including myeloma. In the present study, bone marrow biopsies from 40 myeloma patients were stained with antibodies raised against heparanase, RANKL (an osteoclastogenic cytokine), OPG (a decoy receptor for RANKL), TRAP (a marker of osteoclastogenesis) and osteocalcin (a marker of osteoblastogenesis). The radiologic studies for bone lesions of these patients were also recorded. We analyzed the correlations between heparanase expression in bone marrow myeloma cells with (1) the numbers of TRAP positive osteoclasts, (2) RANKL and OPG expression in myeloma cells and osteoblastic cells, (3) the numbers of osteocalcin positive osteoblasts in bone marrow, and (4) the presence/absence of lytic bone lesions. We found a positive correlation between heparanase expression and RANKL expression as well as the numbers of TRAP positive osteoclasts in myeloma and bone marrow cells, but no correlation was found between the expressions of heparanase and OPG in bone marrow cells (myeloma cells do not express OPG). In contrast, heparanase expression was negatively correlated with the numbers of osteocalcin positive osteoblasts. Taken together, these data suggest that heparanase expression by myeloma cells promotes osteoclastogenesis and at same time inhibits osteoblastogenesis. Clinical data show that 92% of patients with high level of heparanase had one or more lytic bone lesions, while only 63% of patients with median∼ low levels of heparanase had bone lesions (p<0.0001). In summary, enhanced heparanase expression in myeloma cells promotes bone resorption and inhibits bone formation; these events contribute to the uncontrolled bone destruction that is characteristic of myeloma. These data provide novel insight into the mechanisms driving myeloma bone disease and suggest that heparanase inhibitors are valid therapeutic targets for the treatment of multiple myeloma. Disclosures:No relevant conflicts of interest to declare.

Heparanase Regulates Response to Proteasomal Inhibition in Myeloma

Abstract 4902Multiple myeloma is an incurable hematologic malignancy. In patients, even after several rounds of high-dose chemotherapy, myeloma tumor cells survive, proliferate aggressively and re-establish the active disease. Understanding the molecular mechanisms that aid myeloma cells in evading chemotherapy is therefore critical for curing this deadly disease. Studies from our lab have established heparanase, an endoglycosidase expressed highly by myeloma tumor cells, as a master regulator of aggressive myeloma disease where it drives tumor growth, angiogenesis, invasion, and osteolysis. However, the effect of heparanase on response to chemotherapy is unknown. Addressing this question, we have made two striking discoveries: i) treatment with proteasomal inhibitors dramatically elevates heparanase expression and secretion in myeloma, and ii) high heparanase expression significantly reduces therapy-induced apoptosis and cell death in myeloma. Proteasomal inhibition by either bortezomib or MG132 dramatically elevated the expression of heparanase and its secretion into the conditioned media. When this conditioned media was added to tumor cells that had not been exposed to proteosome inhibitors, the heparanase was readily taken up by the cells. This is consistent with previous studies demonstrating that exogenous heparanase can be taken up by myeloma tumor cells resulting in activation of aggressive tumor behavior.Next, to test whether heparanase influences chemoresistance in myeloma, we evaluated the effect of bortezomib on inducing apoptosis of myeloma cells expressing either high (HPSE-high) or low (HPSE-low) levels of heparanase. Apoptotic cells were identified by staining for Annexin V and cell death was assessed by staining with propidium iodide. Results revealed that following treatment with bortezomib, HPSE-high cells had a significantly higher percentage of viable cells (Annexin V negative, PI negative) and a significantly smaller fraction of apoptotic cells (Annexin V positive) compared to HPSE-low cells. Similarly, even when myeloma cells were grown on fibronectin, cells expressing high levels of heparanase exhibited significantly decreased bortezomib-induced cell death. Consistent with the above findings, we found that several well established chemoresistant myeloma cell lines such as LR5, Dox 6 and Dox 40 all expressed high levels of heparanase. Together the above findings demonstrate that tumors respond to proteosome inhibitors by upregulating expression and secretion of heparanase and that tumor cells having high levels of heparanase resist proteosome inhibitor-induced apoptosis. These results indicate that heparanase may play an important role in chemoresistance of myeloma cells and that heparanase inhibitors used in tandem with other drugs may improve initial patient outcome and reduce incidence of relapse. Disclosures:No relevant conflicts of interest to declare.

Heapranase role in the treatment of avascular necrosis of femur head

BackgroundIdiopathic avascular necrosis (AVN) of bone causes significant morbidity in adults although the pathophysiology is unknown. The present treatment options include systemic biphosphonate therapy and local bone drilling decompression, ameliorating the healing process and their by render the weight bearing femur head less vulnerable to collapse. In the present study we demonstrate the involvement of heparanase in AVN and in the acceptable treatments. Methods56 female rats were studied. In 8 control rats AVN was induced by ligamentum teres ligation of the right femur while the left femur remained intact. In the rest of the rats, in addition to right femur AVN, treatment was added by subcutaneous biphosphonate therapy, femoral head drilling or combination of the treatments. All rats were scarified after 6weeks. Immunostaining of the femur heads were performed to heparanase, tissue factor pathway inhibitor (TFPI), tissue factor (TF) and hematoxylin-eosin. ResultsStaining of heparanase, TFPI and TF were most prominent in the bone-marrow tissue of the femur heads. Staining by hematoxylin-eosin revealed damaged femur heads with prominent heparanase and TFPI staining in the femur with AVN compared to the contra lateral side of the same rat. No difference was found in the TF staining between the two sides. In the drilling and / or biphosphonate therapy groups, in contrast to the control group, femur heads were preserved with no significant difference in heparanase and TFPI staining between the two sides. ConclusionsHeparanase and TFPI are locally elevated in the process of AVN and are normalized by the acceptable treatments. Inhibition of heparanase by heparins can potentially improve the nowadays therapy modalities.

Heparanase and Vascular Endothelial Growth Factor Expression Is Increased in Hypoxia-Induced Retinal Neovascularization

Heparanase and VEGF are related closely to angiogenesis in cancer. The purpose of our study was to evaluate the expression and correlation of heparanase and VEGF in hypoxia-induced retinal neovascularization. C57BL/6 oxygen-induced retinopathy (OIR) mice and human retinal microvascular endothelial cells (HRECs) were treated with the hypoxia mimetic agent cobalt chloride (CoCl₂), and in the presence of the heparanase inhibitor phosphomannopentaose sulfate (Muparfostat, PI-88). Heparanase activity was assayed in HRECs, and the expression of heparanase, VEGF protein and mRNA were evaluated by immunofluorescence, ELISA, Western blot, and real-time PCR while retinal flat mounts were used to evaluate the area of neovascularization of mice retina. HREC heparanase activity was increased by treatment with CoCl₂, but was decreased by PI-88. Immunofluorescence showed that heparanase and VEGF staining was intense in hypoxia-treated HRECs and OIR mice retina, while VEGF staining was faint in the normoxia and PI-88-treated ones. Western blot and real-time PCR results indicated that the expression of heparanase and VEGF was increased under hypoxic conditions, and the increase of VEGF was inhibited by PI-88. Retinal flat mounts showed that the area of new vessels in retina of OIR mice was increased compared to the normoxic mice, and this effect was inhibited by PI-88. Heparanase is upregulated and associated with the VEGF expression in hypoxia-induced retinal diseases. Heparanase is involved in hypoxia-induced neovascularization through promoting VEGF expression and may be a new therapeutic target for hypoxia-induced neovascularization retinal diseases.

The pulmonary endothelial glycocalyx regulates neutrophil adhesion and lung injury during experimental sepsis

Sepsis, a systemic inflammatory response to infection, commonly progresses to acute lung injury (ALI), an inflammatory lung disease with high morbidity. We postulated that sepsis-associated ALI is initiated by degradation of the pulmonary endothelial glycocalyx, leading to neutrophil adherence and inflammation. Using intravital microscopy, we found that endotoxemia in mice rapidly induced pulmonary microvascular glycocalyx degradation via tumor necrosis factor-α (TNF-α)-dependent mechanisms. Glycocalyx degradation involved the specific loss of heparan sulfate and coincided with activation of endothelial heparanase, a TNF-α-responsive, heparan sulfate-specific glucuronidase. Glycocalyx degradation increased the availability of endothelial surface adhesion molecules to circulating microspheres and contributed to neutrophil adhesion. Heparanase inhibition prevented endotoxemia-associated glycocalyx loss and neutrophil adhesion and, accordingly, attenuated sepsis-induced ALI and mortality in mice. These findings are potentially relevant to human disease, as sepsis-associated respiratory failure in humans was associated with higher plasma heparan sulfate degradation activity; moreover, heparanase content was higher in human lung biopsies showing diffuse alveolar damage than in normal human lung tissue.

Use of PG545, a heparanase inhibitor, to inhibit pancreatic cancer tumor cell proliferation and migration in vitro and in vivo.

234 Background: Pancreatic Ductal Adenocarcinoma (PDAC) is highly resistant to conventional chemotherapy, as result there is an ongoing search to find novel effective strategies. Resistance is due in part to the high proportion of stromal tissue within the primary tumor. This intricate ECM (extracellular matrix) includes heparan-sulfate glycosaminoglycans which participate in tumor progression, angiogenesis and metastasis. PG545 is a heparanase inhibitor developed to target these pathways. Methods: In vitro cell viability assays were performed using WST-1 reagent and migration was evaluated using T- scratch assay. Animal survival experiments were performed by intraperitoneal injection of AsPC-1 (0.75 x 10^6) cells. In vivo tumor growth experiments were performed by orthotopic injection of PanO2-HY (5x10^5) cells. Results: PG545 significantly inhibited proliferation of tumor cells (AsPC-1 and PanO2) and fibroblasts (WI-38). PG545 caused only a modest inhibition in endothelial cell (HUVECs) proliferation. Migration was significantly inhibited by 1 µM PG545 in AsPC-1 and PanO2 after 12 hours. In a metastatic model of pancreatic cancer, treatment with PG545 (10 mg/kg 1st week, 5 mg/kg 2nd week) improved survival (35 days) compared to saline (22 days) and gemcitabine (28 days). In an immunocompetent orthotopic model, mice treated with PG545 (5 mg/kg twice weekly) had significantly decreased tumor weights after 3 weeks of therapy (p=0.002). Total metastatic events were also reduced in PG545 compared to gemcitabine and control treatment in the PanO2 model. Conclusions: PG545 inhibits tumor cell proliferation and migration in vitro and prolongs survival and inhibits tumor growth in vivo. Additionally it inhibits metastasis in vivo. Further studies are underway to elucidate the mechanism of inhibition and changes to pancreatic tumor microenvironment.

Correlation between heparanase and vascular endothelial growth factor in human retinal microvascular endothelial cells induced by hypoxia

Objective To investigate the effects of heparanase and vascular endothelial growth factor (VEGF) and their correlation in CoCl2-induced human retinal microvascular endothelial cells (HRECs) in an hypoxia model.Methods Human eyes were selected to establish CoCl2-induced HRECs hypoxia model in this study.Four experimental groups were studied:normal control group,hypoxia group (CoCl2 100 μmol/L,48 hours),PI-88 group (specific competitive inhibitor of heparanase: phosphomannopentaose sulfate,PI-88,5 μg/ml,combined with CoCl2 100 μmol/L,48 hours) and PBS control group.Heparanase,VEGF and Pol Ⅱ expression in HRECs of normal and hypoxia group were analyzed with immunofluorescence.Western blot was used to evaluate the expression of heparanase and VEGF in HRECs of normal,hypoxia,PI-88 and PBS control groups. Results Immunofluorescence studies showed that the expression of heparanase and VEGF in cytoplasm was intense in hypoxia HRECs,but faint in normal group.Heparanase was also observed in the nucleus of hypoxia HRECs.Western blot results showed that the expression of Hpa and VEGF protein was increased significantly in hypoxia group compared with normal group (Hpa:F=- 4.005,P ＜ 0.05 ; VEGF:F =- 4.063,P ＜ 0.05),and VEGF was decreased in HRECs treated with PI-88 (F=5.963,P＜0.05).Conclusions Heparanase is up-regulated that resulted in increase of VEGF expression,therefore enhanced angiogenesis in CoCl2-induced hypoxia HRECs. Key words: Retinal neovascularization/pathophysiology; Vascular endothelial growth factors; Anoxia; Retinal vessels; Endothelial cells

Adjuvant therapy options following curative treatment of hepatocellular carcinoma: A systematic review of randomized trials

AimsNumerous postoperative therapies for preventing recurrence of hepatocellular carcinoma (HCC) have been reported, but their efficacy remains controversial and knowledge about adverse effects is limited. A systematic review of randomized controlled trials (RCTs) was performed to gain a comprehensive picture of the efficacy and risks of these therapies. MethodsMEDLINE, EMBASE and the Cochrane Library were systematically searched through July 2011. Risk ratios (RRs) and 95% confidence intervals (CIs) were calculated. ResultsA total of 2989 patients from 28 RCTs involving 10 postoperative therapies were included. For interferon therapy, the estimated RR for the 2-year recurrence rate was 0.84 (95% CI 0.73–0.97, P = 0.02) and the overall survival (OS) was 1.15 (95% CI 1.07–1.22, P < 0.001). Postoperative therapy with the vitamin K2 analog did not lead to a significant reduction in the 1-year recurrence rate, with a pooled RR of 0.60 (95% CI 0.28–1.27, P = 0.18). However, it did slightly improve the 1-year OS, with a pooled RR of 1.03 (95% CI 1.00–1.05, P = 0.03). Transarterial chemotherapy with or without embolization, adoptive immunotherapy and heparanase inhibitor PI-88 therapy may delay tumor recurrence. The effects of acyclic retinoid, lipiodol-iodine-131 and tumor vaccine treatment were promising but require further study. All postoperative therapies except interferon administered intramuscularly were well tolerated by the majority of patients. ConclusionsUse of adjuvant interferon is definitely associated with an increase in OS. Postoperative therapies involving acyclic retinoid, lipidol-iodine-131, or tumor vaccine may improve the OS of patients with HCC after curative treatment.

PG545, A Heparanase Inhibitor, Inhibits Pancreatic Cancer Tumor Cell Proliferation in Vitro and in Vivo

PG545, A Heparanase Inhibitor, Inhibits Pancreatic Cancer Tumor Cell Proliferation in Vitro and in Vivo

Heparan sulfate and heparanase play key roles in mouse β cell survival and autoimmune diabetes.

The autoimmune type 1 diabetes (T1D) that arises spontaneously in NOD mice is considered to be a model of T1D in humans. It is characterized by the invasion of pancreatic islets by mononuclear cells (MNCs), which ultimately leads to destruction of insulin-producing β cells. Although T cell dependent, the molecular mechanisms triggering β cell death have not been fully elucidated. Here, we report that a glycosaminoglycan, heparan sulfate (HS), is expressed at extraordinarily high levels within mouse islets and is essential for β cell survival. In vitro, β cells rapidly lost their HS and died. β Cell death was prevented by HS replacement, a treatment that also rendered the β cells resistant to damage from ROS. In vivo, autoimmune destruction of islets in NOD mice was associated with production of catalytically active heparanase, an HS-degrading enzyme, by islet-infiltrating MNCs and loss of islet HS. Furthermore, in vivo treatment with the heparanase inhibitor PI-88 preserved intraislet HS and protected NOD mice from T1D. Our results identified HS as a critical molecular requirement for islet β cell survival and HS degradation as a mechanism for β cell destruction. Our findings suggest that preservation of islet HS could be a therapeutic strategy for preventing T1D.

Heparanase Is Essential for the Development of Diabetic Nephropathy in Mice

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

Effect of heparanase inhibitor on proliferation of umbilical vein vascular endothelial cell and expression of heparanase

Background Experimental study showed that heparanase (HPA)is overexpressed in choroidal neovascularization,suggesting that it may play a role in the pathogenesis of angiogenesis.To certify HPA inhibitor suppress the formation and development of new blood vessel has an important significance for the treatment of choroidal neovascularization.Objective The aim of this study was to investigate the effect of HPA inhibitor on the proliferation of human umbilical vein vascular endothelial cell (UVEC) and the expression of HPA.Methods Hunan UVEC was primarily cultured and passaged and the third generation cells were used in the experiment.Phosphomannopentaose sulfate (PI-88) solution,a HPA inhibitor,was prepared with endothelial cell medium and the end concentrations were 400.00,200.00,100.00,50.00,25.00,12.50,6.25 mg/L respectively.The cells were treated with PI-88 solutions for 24,48 and 72 hours.The growth and proliferation of human UVEC were analyzed using MTT colorimetric assay at absorbance 570 nm.The expression of HPA in the cells was detected by immunochemistry in 48 hours after addition of PI-88.Results Cultured human UVEC showed the fusiform and polygon in shape.The A570 values of human CVEC were significantly different among various concentrations of PI-88 groups (F=2.721,P=0.053 ) and different action time (F=9.656,P =0.002).When PI-88 was administered for 24 hours,the A570 values of human UVEC were insignificantly altered in comparison with the one without PI-88 culture group (P＞0.05 ).However,in 72 hours after experiment,the A570 values were significantly declined as the PI-88 concentration was ＞50.00 mg/L ( P＜0.05 ).When PI-88 was administered for 48 and 72 hours,the A570 values of human UVEC were significantly higher than those of 24 hours in ＜50.00 mg/L groups (P＜0.01 ),but no statistical differences were seen in ＞100.00 mg/L groups among various time points (P＞0.05 ).HPA was intently expressed in the cytoplasm of human UVEC.However,at 48 hours after addition of ＞25.00 mg/L PI-88,the HPA expression was obviously weaker.Conclusions PI-88 can suppress the growth and proliferation of human UVEC at the dose-and time-dependent manner by downregulating the expression of HPA in the cells. Key words: Phosphomannopentaose sulfate; Heparanase; Umbilical vein vascular endothelial cell

Heparanase Activation of the Coagulation System in a Mice Sepsis Model

Abstract 378 Background:Heparanase that is abundant in platelets and neutrophils is implicated in cell invasion, tumor metastasis and angiogenesis. Recently, we demonstrated that heparanase is directly involved in the regulation of the hemostatic system. Heparanase was shown to form a complex and enhance tissue factor (TF) activity, resulting in increased factor Xa production (Nadir et al, Haematologica, 2010). In the present study, the effect of heparanase on sepsis was investigated. Methods: ICR mice, heparanase knock-out mice and heparanase over-expression mice were studied. Sepsis was induced by intra-peritoneal injection of lipopolysaccharides (LPS). Four hours after injection, blood was obtained via temple plexus puncture followed by mice sacrificing. Levels of thrombin-antithrombin (TAT), D-Dimer and IL-6 were tested using ELISA. Plasma factor VII levels were evaluated using the Western blot analysis and fibrinogen levels in the lung, kidney and spleen were estimated by immunostaining. Results: Intra-peritoneal injection of heparanase (0.5μg/mg) compared to PBS injection, increased the TAT (p<0.05) and D-Dimer (p<0.05) levels, which were similar to the ones induced by LPS (5μg/mg), although the IL-6 levels did not rise and mice did not show signs of illness. Moreover, heparanase injection half an hour following LPS administration resulted in reduced levels of TAT, D-Dimer, fibrinogen and of factor VII (p<0.001), increased levels of IL-1 (p<0.001) and profuse blood leak from puncture, compatible with severe disseminated intravascular coagulation (DIC). Similarly, injection of LPS to heparanase knock-out mice led to lower levels of TAT (p<0.001), D-Dimer (p<0.001) and IL-6 (p<0.001) compared to control mice, while injection of LPS to heparanase over-expression mice resulted in DIC. Conclusions: Heparanase is found to induce activation of the coagulation system without inflammatory response. It also contributes to laboratory enhancement of sepsis resulting in DIC. Inhibitors of heparanase may potentially attenuate morbidity and mortality in sepsis. Disclosures:No relevant conflicts of interest to declare.

Heparanase enhances nerve-growth-factor-induced PC12 cell neuritogenesis via the p38 MAPK pathway

Heparanase is involved in the cleavage of the HS (heparan sulfate) chain of HSPGs (HS proteoglycans) and hence participates in remodelling of the ECM (extracellular matrix) and BM (basement membrane). In the present study we have shown that NGF (nerve growth factor) promoted nuclear enrichment of EGR1 (early growth response 1), a transcription factor for heparanase, and markedly induced heparanase expression in rat adrenal pheochromocytoma (PC12) cells. K252a, an antagonist of the NGF receptor TrkA (tyrosine kinase receptor A), decreased heparanase protein expression induced by NGF in PC12 cells. Suramin, a heparanase inhibitor, decreased heparanase in PC12 cells and blocked NGF-induced PC12 neuritogenesis. Stable overexpression of heparanase activated p38 MAPK (mitogen-activated protein kinase) by phosphorylation and enhanced the neurite outgrowth induced by NGF, whereas knock down of heparanase impaired this process. However, overexpression of latent pro-heparanase with a Y156A mutation still led to enhanced NGF-induced neurite outgrowth and increased p38 MAPK phosphorylation. Inhibition of p38 MAPK by SB203580 suppressed the promotion of NGF-induced neuritogenesis by the wild-type and mutant heparanase. The impaired differentiation by knock down of heparanase could be restored by transfection of wild-type or mutant heparanase in PC12 cells. The results of the present study suggest that heparanase, at least in the non-enzymatic form, may promote NGF-induced neuritogenesis via the p38 MAPK pathway.

Hyperpigmentation in human solar lentigo is promoted by heparanase-induced loss of heparan sulfate chains at the dermal–epidermal junction

Skin pigmentation induced by ultraviolet B radiation is caused in part by inflammation mediated by cytokines secreted from keratinocytes and fibroblasts in the irradiated area. Heparanase is also activated in the irradiated skin, and this leads to loss of heparan sulfate at the dermal-epidermal junction (DEJ), resulting in uncontrolled diffusion of heparan sulfate-binding cytokines through the DEJ. However, it is not clear whether heparanase-induced loss of heparan sulfate at the DEJ is involved in the pigmentation process in sun-exposed skin. We examined the role of heparan sulfate in the pigmentation process of human pigmented skin and in pigmented skin-equivalent model. Heparan sulfate and blood vessels in human pigmented skin, solar lentigo, and non-pigmented skin were evaluated by means of immunohistochemistry. Pigmented skin equivalent models were cultured with or without heparanase inhibitor and the pigmentation levels were compared. In solar lentigo, heparan sulfate was hardly observed, presumably due to the increase of heparanase at the DEJ, in spite of the deposition of core protein of perlecan (also known as heparan sulfate proteoglycan). The number of blood vessels was significantly increased in solar lentigo. In the pigmented skin equivalent model, heparanase inhibitor increased the staining intensity of heparan sulfate at the DEJ and markedly reduced melanogenesis in the epidermis. Our results indicate that heparanase-induced loss of heparan sulfate at the DEJ is involved in the pigmentation process of human skin. Consequently, heparanase inhibitors can be expected to exert a protective effect against ultraviolet exposure-induced skin pigmentation.

Heparanase-mediated Loss of Nuclear Syndecan-1 Enhances Histone Acetyltransferase (HAT) Activity to Promote Expression of Genes That Drive an Aggressive Tumor Phenotype

Heparanase acts as a master regulator of the aggressive tumor phenotype in part by enhancing expression of proteins known to drive tumor progression (e.g. VEGF, MMP-9, hepatocyte growth factor (HGF), and RANKL). However, the mechanism whereby this enzyme regulates gene expression remains unknown. We previously reported that elevation of heparanase levels in myeloma cells causes a dramatic reduction in the amount of syndecan-1 in the nucleus. Because syndecan-1 has heparan sulfate chains and because exogenous heparan sulfate has been shown to inhibit the activity of histone acetyltransferase (HAT) enzymes in vitro, we hypothesized that the reduction in nuclear syndecan-1 in cells expressing high levels of heparanase would result in increased HAT activity leading to stimulation of protein transcription. We found that myeloma cells or tumors expressing high levels of heparanase and low levels of nuclear syndecan-1 had significantly higher levels of HAT activity when compared with cells or tumors expressing low levels of heparanase. High levels of HAT activity in heparanase-high cells were blocked by SST0001, an inhibitor of heparanase. Restoration of high syndecan-1 levels in heparanase-high cells diminished nuclear HAT activity, establishing syndecan-1 as a potent inhibitor of HAT. Exposure of heparanase-high cells to anacardic acid, an inhibitor of HAT activity, significantly suppressed their expression of VEGF and MMP-9, two genes known to be up-regulated following elevation of heparanase. These results reveal a novel mechanistic pathway driven by heparanase expression, which leads to decreased nuclear syndecan-1, increased HAT activity, and up-regulation of transcription of multiple genes that drive an aggressive tumor phenotype.

Pre-clinical and clinical significance of heparanase in Ewing’s sarcoma

Heparanase is an endoglycosidase that specifically cleaves heparan sulphate side chains of heparan sulphate proteoglycans, activity that is strongly implicated in cell migration and invasion associated with tumour metastasis, angiogenesis and inflammation. Heparanase up-regulation was documented in an increasing number of human carcinomas, correlating with reduced post-operative survival rate and enhanced tumour angiogenesis. Expression and significance of heparanase in human sarcomas has not been so far reported. Here, we applied the Ewing’s sarcoma cell line TC71 and demonstrated a potent inhibition of cell invasion in vitro and tumour xenograft growth in vivo upon treatment with a specific inhibitor of heparanase enzymatic activity (compound SST0001, non-anticoagulant N-acetylated, glycol split heparin). Next, we examined heparanase expression and cellular localization by immunostaining of a cohort of 69 patients diagnosed with Ewing’s sarcoma. Heparanase staining was noted in all patients. Notably, heparanase staining intensity correlated with increased tumour size (P = 0.04) and with patients’ age (P = 0.03), two prognostic factors associated with a worse outcome. Our study indicates that heparanase expression is induced in Ewing’s sarcoma and associates with poor prognosis. Moreover, it encourages the inclusion of heparanase inhibitors (i.e. SST0001) in newly developed therapeutic modalities directed against Ewing’s sarcoma and likely other malignancies.

Key role of heparan sulfate chains in assembly of anchoring complex at the dermal-epidermal junction

Epidermal basement membrane forms anchoring complex composed of hemidesmosomes, anchoring filaments, lamina densa and anchoring fibrils to link epidermis to dermis. However, the anchoring complex is rarely formed in skin equivalent models, probably because of degradation of extracellular matrix (ECM) proteins and heparan sulfate chains by matrix metalloproteinases (MMPs) and heparanase, respectively. To explore the roles of ECM proteins and heparan sulfate in anchoring complex assembly, we used specific inhibitors of MMPs and heparanase, and the formation of anchoring complex was analysed in terms of polarized deposition of collagen VII, BP180 and β4 integrin at the dermal-epidermal junction (DEJ) by means of immunohistochemistry and transmission electron microscopy (TEM). The deposition of collagen VII was polarized to the basal side by the addition of MMP inhibitor, and the staining intensity was increased by combined treatment with MMP inhibitor and heparanase inhibitor, which enhanced anchoring fibril formation as observed by TEM. BP180 was polarized to the basal side by heparanase inhibitor, which protects HS chains, but not by MMP inhibitor. MMP inhibitor improved the polarization of β4 integrin. Hemidesmosomes were formed in the presence of each inhibitor, as observed by TEM, and formation was greatly enhanced by the combined treatment. These findings suggest that heparan sulfate chains, in addition to ECM proteins at the DEJ, play an important role in the assembly of anchoring complex, especially hemidesmosomes and anchoring fibrils.

Significance of heparanase in cancer and inflammation.

Heparan sulfate proteoglycans (HSPGs) are primary components at the interface between virtually every eukaryotic cell and its extracellular matrix. HSPGs not only provide a storage depot for heparin-binding molecules in the cell microenvironment, but also decisively regulate their accessibility, function and mode of action. As such, they are intimately involved in modulating cell invasion and signaling loops that are critical for tumor growth, inflammation and kidney function. In a series of studies performed since the cloning of the human heparanase gene, we and others have demonstrated that heparanase, the sole heparan sulfate degrading endoglycosidase, is causally involved in cancer progression, inflammation and diabetic nephropathy and hence is a valid target for drug development. Heparanase is causally involved in inflammation and accelerates colon tumorigenesis associated with inflammatory bowel disease. Notably, heparanase stimulates macrophage activation, while macrophages induce production and activation of latent heparanase contributed by the colon epithelium, together generating a vicious cycle that powers colitis and the associated tumorigenesis. Heparanase also plays a decisive role in the pathogenesis of diabetic nephropathy, degrading heparan sulfate in the glomerular basement membrane and ultimately leading to proteinuria and kidney dysfunction. Notably, clinically relevant doses of ionizing radiation (IR) upregulate heparanase expression and thereby augment the metastatic potential of pancreatic carcinoma. Thus, combining radiotherapy with heparanase inhibition is an effective strategy to prevent tumor resistance and dissemination in IR-treated pancreatic cancer patients. Also, accumulating evidence indicate that peptides derived from human heparanase elicit a potent anti-tumor immune response, suggesting that heparanase represents a promising target antigen for immunotherapeutic approaches against a broad variety of tumours. Oligosaccharide-based compounds that inhibit heparanase enzymatic activity were developed, aiming primarily at halting tumor growth, metastasis and angiogenesis. Some of these compounds are being evaluated in clinical trials, targeting both the tumor and tumor microenvironment.