Cleaves Heparan Sulfate Research Articles

Trauma promotes heparan sulfate modifications and cleavage that disrupt homeostatic gene expression in microvascular endothelial cells.

Introduction: Heparan sulfate (HS) in the vascular endothelial glycocalyx (eGC) is a critical regulator of blood vessel homeostasis. Trauma results in HS shedding from the eGC, but the impact of trauma on HS structural modifications that could influence mechanisms of vascular injury and repair has not been evaluated. Moreover, the effect of eGC HS shedding on endothelial cell (EC) homeostasis has not been fully elucidated. The objectives of this work were to characterize the impact of trauma on HS sulfation and determine the effect of eGC HS shedding on the transcriptional landscape of vascular ECs. Methods: Plasma was collected from 25 controls and 49 adults admitted to a level 1 trauma center at arrival and 24h after hospitalization. Total levels of HS and angiopoietin-2, a marker of pathologic EC activation, were measured at each time point. Enzymatic activity of heparanase, the enzyme responsible for HS shedding, was determined in plasma from hospital arrival. Liquid chromatography-tandem mass spectrometry was used to characterize HS di-/tetrasaccharides in plasma. In vitro work was performed using flow conditioned primary human lung microvascular ECs treated with vehicle or heparinase III to simulate human heparanase activity. Bulk RNA sequencing was performed to determine differentially expressed gene-enriched pathways following heparinase III treatment. Results: We found that heparanase activity was increased in trauma plasma relative to controls, and HS levels at arrival were elevated in a manner proportional to injury severity. Di-/tetrasaccharide analysis revealed lower levels of 3-O-sulfated tetramers with a concomitant increase in ΔIIIS and ΔIIS disaccharides following trauma. Admission levels of total HS and specific HS sulfation motifs correlated with 24-h angiopoietin-2 levels, suggesting an association between HS shedding and persistent, pathological EC activation. In vitro pathway analysis demonstrated downregulation of genes that support cell junction integrity, EC polarity, and EC senescence while upregulating genes that promote cell differentiation and proliferation following HS shedding. Discussion: Taken together, our findings suggest that HS cleavage associated with eGC injury may disrupt homeostatic EC signaling and influence biosynthetic mechanisms governing eGC repair. These results require validation in larger, multicenter trauma populations coupled with in vivo EC-targeted transcriptomic and proteomic analyses.

Enzymatic Modulation of the Pulmonary Glycocalyx Enhances Susceptibility to Streptococcus pneumoniae.

The pulmonary epithelial glycocalyx is rich in glycosaminoglycans such as hyaluronan and heparan sulfate. Despite their presence, the importance of these glycosaminoglycans in bacterial lung infections remains elusive. To address this, we intranasally inoculated mice with Streptococcus pneumoniae in the presence or absence of enzymes targeting pulmonary hyaluronan and heparan sulfate, followed by characterization of subsequent disease pathology, pulmonary inflammation, and lung barrier dysfunction. Enzymatic degradation of hyaluronan and heparan sulfate exacerbated pneumonia in mice, as evidenced by increased disease scores and alveolar neutrophil recruitment. However, targeting epithelial hyaluronan in combination with Streptococcus pneumoniae infection further exacerbated systemic disease, indicated by elevated splenic bacterial load and plasma levels of pro-inflammatory cytokines. In contrast, enzymatic cleavage of heparan sulfate resulted in increased bronchoalveolar bacterial burden, lung damage and pulmonary inflammation in mice infected with Streptococcus pneumoniae. Accordingly, heparinase-treated mice also exhibited disrupted lung barrier integrity as evidenced by higher alveolar edema scores and vascular protein leakage into the airways. This finding was corroborated in a human alveolus-on-a-chip platform, confirming that heparinase treatment also disrupts the human lung barrier during Streptococcus pneumoniae infection. Notably, enzymatic pre-treatment with either hyaluronidase or heparinase also rendered human epithelial cells more sensitive to pneumococcal-induced barrier disruption, as determined by transepithelial electrical resistance measurements, consistent with our findings in murine pneumonia. Taken together, these findings demonstrate the importance of intact hyaluronan and heparan sulfate in limiting pneumococci-induced damage, pulmonary inflammation, and epithelial barrier function and integrity.

Design Principle of Heparanase Inhibitors: A Combined In Vitro and In Silico Study.

Heparanase (HPSE) is an enzyme that cleaves heparan sulfate (HS) side chains from heparan sulfate proteoglycans (HSPGs). Overexpression of HPSE is associated with various types of cancer, inflammation, and immune disorders, making it a highly promising therapeutic target. Previously developed HPSE inhibitors that have advanced to clinical trials are polysaccharide-derived compounds or their mimetics; however, these molecules tend to suffer from poor bioavailability, side effects via targeting other saccharide binding proteins, and heterogeneity. Few small-molecule inhibitors have progressed to the preclinical or clinical stages, leaving a gap in HPSE drug discovery. In this study, a novel small molecule that can inhibit HPSE activity was discovered through high-throughput screening (HTS) using an ultrasensitive HPSE probe. Computational tools were employed to elucidate the mechanisms of inhibition. The essential structural features of the hit compound were summarized into a structure-activity relationship (SAR) theory, providing insights into the future design of HPSE small-molecule inhibitors.

Elevated Circulating Cell-Free Hemoglobin Drives Endothelial Glycocalyx Destruction and Inflammation in Sepsis

Rationale: Endothelial injury is a hallmark of sepsis-induced acute respiratory distress syndrome (ARDS). Endothelial barrier integrity is regulated by the endothelial glycocalyx, a matrix of proteoglycans and glycosaminoglycans (GAGs) that lines the vascular lumen. In sepsis, this glycocalyx is degraded by heparanase, which cleaves heparan sulfate, the primary glycocalyx GAG. Glycocalyx breakdown is linked to worse sepsis outcomes, but the mechanisms inducing heparanase activity in sepsis are unknown. We previously found that plasma levels of hemoglobin released from red blood cells (cell-free hemoglobin, CFH) are elevated in clinical sepsis and correlate with higher rates of ARDS and death. CFH also worsens endothelial injury and microvascular permeability in murine sepsis. We therefore hypothesized that CFH upregulates heparanase production to drive glycocalyx shedding and inflammation in sepsis. Methods: To test the association between CFH levels, heparanase expression, and glycocalyx degradation, circulating levels of heparanase, heparan sulfate, and total GAGs were measured in 77 sepsis patients and compared across CFH quartiles. In mice with intraabdominal polymicrobial sepsis with elevated CFH (CS [cecal slurry] + intravenous [IV] CFH), plasma heparanase, total GAGs, and syndecan-1 were quantified. Whole lung and plasma pro-inflammatory cytokines were also determined to investigate the impact of CFH on inflammation in sepsis. Results: In sepsis patients, higher circulating CFH levels were associated with increased plasma heparanase (p=0.0304), total GAGs (p=0.0178), and heparan sulfate (p=0.0112). CFH also exacerbated glycocalyx degradation in murine sepsis; circulating syndecan-1 (p=0.0152) and total GAGs (p=0.0094) were elevated in animals that received CS+IV CFH compared to CS alone. This effect may be mediated by upregulation of active heparanase, which was higher in the plasma of CS+IV CFH treated animals (p=0.1333). CFH also worsened illness severity, as CS+IV CFH animals had worse sepsis severity scores (p=0.0074). Whole lung tumor necrosis factor-alpha (TNF-a; p=0.0129) and interferon-gamma (IFN-g; p=0.0264) transcript levels were increased in the CS+IV CFH group, indicating greater lung inflammation. Systemic inflammation was also enhanced in the CS+IV CFH animals, demonstrated by higher plasma TNF-a (p<0.001) and IFN-g (p=0.0221) concentrations. Conclusions: In sepsis, CFH is associated with upregulation of heparanase and endothelial glycocalyx degradation, which may disrupt the endothelial barrier, causing downstream organ injury. Future studies to define the molecular pathways that underlie these findings and their effect on endothelial permeability will be an important next step towards identifying novel treatment targets to improve outcomes for patients with sepsis-associated ARDS. Funding F30HL170483, R01HL164937, R01HL158906, R01HL150783, R01HL125371. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Exploring Heparanase Levels in Tears: Insights From Herpes Simplex Virus-1 Keratitis Patients and Animal Studies.

Heparanase (HPSE) cleaves heparan sulfate proteoglycans during herpes simplex virus-1 (HSV-1) infection, aiding in viral egress and disease progression. Its action has been well established in in vitro and in vivo models, but its relevance in human patients remains unclear. This study aimed to specifically evaluate tear HPSE levels of patients with herpes simplex keratitis (HSK) and to correlate these findings with a commonly used murine model. Tear samples from patient and mice samples were collected at LV Prasad Eye Institute, Hyderabad, India, and at the University of Illinois, Chicago, IL, respectively. Tears were collected from HSV-1 patients, bacterial/fungal keratitis cases, and healthy individuals. For in vivo study, C57BL/6 mice were infected with HSV-1 (McKrae strain) followed by tear fluid collection at various time points (0-10 days). The HSV-1, bacterial keratitis, fungal keratitis, and healthy control groups each had 30 patients. There was a significant difference in HPSE expression in the HSV-1 infected eyes (1.55 ± 0.19 units/mL) compared to HSV-1 contralateral eyes (1.23 ± 0.13 units/mL; P = 0.82), bacterial keratitis eyes (0.87 ± 0.15 units/mL; P = 0.0078), fungal keratitis eyes (0.64 ± 0.09 units/mL; P < 0.00001), and normal controls (0.53 ± 0.06 units/mL; P < 0.00001). C57BL/6 mice tear HPSE expression in infected eyes was 0.66 to 5.57 ng heparan sulfate (HS) removed per minute when compared to non-infected eye (range, 0.70-3.67 ng HS removed per minute). To the best of our knowledge, this study is the first to report elevated HPSE levels in the tears of patients with different forms of HSV-1 keratitis, and it confirms similar findings in a murine model, providing a valuable basis for future in vivo and clinical research on HSV-1 ocular infection.

Analysis of Expression Pattern and Prognostic Value of the Heparanase in Breast Cancer Through CD274/CTLA-4 Immune Checkpoint Proteins.

Objectives: Heparanase (HPSE), an endoglycosidase that cleaves heparan sulfate, regulates various biological processes related to tumor progression. We explore the prognostic value of HPSE and its relationship with immunotherapy response in patients with breast cancer, to improve the effectiveness of immunotherapy and increase the survival outcomes. Methods: In the study, we explored the prognostic value of HPSE through the The Cancer Genome Atlas (TCGA) database. By using the single-sample gene set enrichment analysis (ssGSEA) method, we measured the infiltration levels of 24 immune cell types in the tumor microenvironment. Cancer Therapeutics Response Portal (CTRP) and PRISM datasets provide the area under the dose-response curve (AUC) to measure drug sensitivity. Using nomograms, we predicted overall survival ability. In vivo studies, we investigated the relationship between HPSE and immune checkpoint proteins and pro-inflammatory cytokines by immunohistochemistry of Triple-Negative Breast Cancer tumors in mice. Results: Our model demonstrated that the integrating of HPSE with the clinical stage effectively predicts patients' survival time, highlighting high HPSE expression as a prognostic risk factor for breast cancer. Then the Receiver Operating Characteristic (ROC) curve [AUC of 1 year = 0.747, AUC of 3 years = 0.731] and Decision Curve Analysis (DCA) curve illustrated the satisfactory discriminative capacity of our model, emphasizing its valuable clinical applicability. Immune-related results showed that HPSE correlates strongly with immune infiltrating cells, immune-related genes, and the anti-cancer immunity cycle. In vivo studies have demonstrated that HPSE in breast cancer is associated with increased expression of immune checkpoint proteins CD274 and cytotoxic T lymphocyte-associated protein 4 (CTLA-4) and is positively correlated with the pro-inflammatory cytokine TNF-α. Meanwhile, we analyzed the 11 types of drugs that are sensitive to the HPSE gene. Conclusion: Our results show that HPSE can serve as an effective biomarker to predict the prognosis of breast cancer patients and reflect the impact of immunotherapy.

Structure-based lead optimization to improve potency and selectivity of a novel tetrahydroimidazo[1,2-a]pyridine-5-carboxylic acid series of heparanase-1 inhibitor

Heparanase-1 (HPSE1) is an endo-β-d-glucuronidase that is the only mammalian enzyme known to cleave heparan sulfate (HS) of heparan sulfate proteoglycans (HSPG), a key component of the glycocalyx layer of the vascular endothelium matrix. Inhibition of HPSE1 has therapeutic potential for cancer and proteinuric kidney diseases. We previously reported that 2 showed a moderate potency as an HPSE1 inhibitor and an issue of selectivity against exo-β-d-glucuronidase (GUSβ) and glucocerebrosidase (GBA) remained. A structure-based lead optimization of 2 using X-ray co-crystal structure analysis and fragment molecular orbital calculation resulted in 4e, which showed a more than 7-fold increase in HPSE1 inhibitory activity. The subsequent introduction of a methyl group into the 6-hydroxy group of 4e resulted in 18 with reduced inhibitory activities against GUSβ and GBA while maintaining the inhibitory activity against HPSE1. The inhibitory activities of 18 against serum HPSE1 in mice were significant and lasted for 4 h at doses of 3, 30, and 100 mg/kg. Compound 18 could be a novel lead compound for HPSE1 inhibitors with improved inhibitory activity against HPSE1 and increased HPSE1 selectivity over GUSβ and GBA.

Abstract 3105: Design principle of heparanase inhibitors: A combined in vitro and in silico study

Abstract Heparanase (HPSE) is an endo-β-D-glucuronidase and the only known enzyme responsible for the cleavage of heparan sulfate (HS) side chains from heparan sulfate proteoglycans (HSPGs). The enzymatic activity of HPSE mediates ECM remodeling, regulates growth factors, and modulates related signaling pathways, thus HPSE has emerged as a novel therapeutic target for various diseases, including most types of cancers. In the last two decades, a number of HPSE inhibitors have been reported by several labs worldwide, with most of them belonging to the heparan sulfate mimics category. So far, few of the small molecule HPSE inhibitors have progressed into clinical trials and none has gained approval by regulatory agencies, leaving a blank in HPSE drug discovery. Here we present the discovery of a novel HPSE small molecule inhibitor by high-throughput screening using an ultrasensitive HPSE enzymatic detecting probe developed in our lab and provide the mechanisms of action behind the small molecule’s HPSE inhibitory activity. By doing a series of molecular dynamics (MD) simulations, we discovered the binding profiles on the derivatives of the lead compound. We summarized the essential structural features of the lead compound to provide insights into the design of future HPSE small molecule inhibitors. Citation Format: Yuzhao Zhang, Meijun Xiong, Zixin Chen, Gustavo Seabra, Jun Liu, Chenglong Li, Lina Cui. Design principle of heparanase inhibitors: A combined in vitro and in silico study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3105.

Abstract 4977: Development of small molecule inhibitors of protein interaction with glycosaminoglycans

Abstract Glycosaminoglycans (GAGs), such as heparan sulfate and chondroitin sulfate, are an important component of the tumor microenvironment (TME). These carbohydrates are attached to proteoglycans at the surface of tumor cells and play a vital role in malignant transformation. Heparan sulfate (HS) glycosaminoglycans have been demonstrated to play a pleiotropic role in regulating the activity of important cellular receptors and downstream signaling pathways, and are implicated in cancer cell proliferation, angiogenesis, invasion, and metastasis. Heparinase, an endoglycosidase that specifically cleaves heparan sulfate side chains of heparan sulfate proteoglycans is expressed in several sarcoma subtypes. We measured heparinase activity in several cell lines and found that A673, CHP100, CHLA9 and RD-ES Ewing’s sarcoma cell lines have significantly higher levels of heparinase activity compared to the heparinase-deficient CHO-K1 psgD-677 cell line. Interference with heparinase or heparin sulfate function(s) may represent a therapeutic approach in sarcoma. To this end, we tested these cell lines by measuring cell growth using a cell viability assay, in the presence or absence of GTC3300P, a small molecule inhibitor of protein interaction with glycosaminoglycan. We demonstrate that the Ewing’s sarcoma cell lines exhibit potent and significant inhibition in cell growth in vitro, in the presence of drug. Western blot analysis shows induction of cleaved Poly-ADP Ribose Polymerase (PARP), yH2AX, as well as cleaved caspase-3, known markers of apoptosis and down regulation of mTORC1 targets such as phospho-S6 (p-S6), in Ewing’s sarcoma. Apoptosis was confirmed by the induction of sub-G1 population in Ewing’s sarcoma by cell cycle analysis. Previous reports have indicated that heparinase activity is strongly implicated in cell migration and invasion and is associated with tumor metastasis, angiogenesis and inflammation. This was confirmed by LC-MS/MS proteomics and bioinformatics (gene ontology and gene set enrichment) analysis of these multiple cell lines, which identified alterations in numerous biomarkers in these pathways, including multiple ribosomal protein subunits, spliceosomes components, and complement coagulation cascade. We further conducted a cell migration assay and demonstrate that GTC3300 significantly inhibits cell migration in Ewing’s sarcoma cell lines, but not in heparinase-deficient psg677 cells. Taken together our data strongly suggests that targeting protein interaction with glycosaminoglycans including heparan sulfate in Ewing’s sarcoma represents a novel approach and merits further investigation Citation Format: Tahir N. Sheikh, Elgilda Musi, Alex J. Rai, Gary K. Schwartz. Development of small molecule inhibitors of protein interaction with glycosaminoglycans. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4977.

The heparan sulfate mimetic Muparfostat aggravates steatohepatitis in obese mice due to its binding affinity to lipoprotein lipase.

Heparanase is the only confirmed endoglycosidase that cleaves heparan sulfate (HS), a ubiquitous glycosaminoglycan with various essential roles in multiple pathological processes. Thus, the development of heparanase inhibitors has become an attractive strategy for drug discovery, especially in tumour therapy, in which HS mimetics are the most promising compounds. The various biological effects of heparanase also suggest a role for HS mimetics in many non-cancer indications, such as type 1 diabetes. However, the potential benefits of HS mimetics in obesity-related type 2 diabetes have not been elucidated. In this study, we investigated muparfostat (PI-88), a developed HS mimetic currently enrolled in Phase III clinical trials, in obese mouse models and in vitro cultured murine hepatocytes. Daily administration of muparfostat for 4 weeks caused hyperlipidaemia and aggravated hepatic steatosis in obese mice models, but not in lean animals. In cultured hepatocytes, muparfostat did not alter lipid accumulation. Acute tests suggested that muparfostat binds to lipoprotein lipase in competition with HS on vascular endothelial cell surfaces, thereby reducing the degradation of circulating triglycerides by lipoprotein lipase and subsequent uptake of fatty acids into vascular endothelial cells and causing hyperlipidaemia. This hyperlipidaemia aggravates hepatic steatosis and causes liver injury in muparfostat-treated obese mice. The binding activity of HS mimetics to lipoprotein lipase should be investigated as an additional pharmacological effect during heparanase inhibitor drug discovery. This study also provides novel evidence for an increased risk of drug-induced liver injury in obese individuals.

Heparanase-1: From Cancer Biology to a Future Antiviral Target.

Heparan sulfate proteoglycans (HSPGs) are a major constituent of the extracellular matrix (ECM) and are found to be implicated in viral infections, where they play a role in both cell entry and release for many viruses. The enzyme heparanase-1 is the only known endo-beta-D-glucuronidase capable of degrading heparan sulphate (HS) chains of HSPGs and is thus important for regulating ECM homeostasis. Heparanase-1 expression is tightly regulated as the uncontrolled cleavage of HS may result in abnormal cell activation and significant tissue damage. The overexpression of heparanase-1 correlates with pathological scenarios and is observed in different human malignancies, such as lymphoma, breast, colon, lung, and hepatocellular carcinomas. Interestingly, heparanase-1 has also been documented to be involved in numerous viral infections, e.g., HSV-1, HPV, DENV. Moreover, very recent reports have demonstrated a role of heparanase-1 in HCV and SARS-CoV-2 infections. Due to the undenied pro-carcinogenic role of heparanase-1, multiple inhibitors have been developed, some reaching phase II and III in clinical studies. However, the use of heparanase inhibitors as antivirals has not yet been proposed. If it can be assumed that heparanase-1 is implicated in numerous viral life cycles, its inhibition by specific heparanase-acting compounds should result in a blockage of viral infection. This review addresses the perspectives of using heparanase inhibitors, not only for cancer treatment, but also as antivirals. Eventually, the development of a novel class antivirals targeting a cellular protein could help to alleviate the resistance problems seen with some current antiretroviral therapies.

Changes in Lipoprotein Lipase in the Heart Following Diabetes Onset

Due to its constant pumping and contraction, the heart requires a substantial amount of energy, with fatty acids (FAs) providing a major part of its adenosine triphosphate (ATP). However, the heart is incapable of making this substrate and attains its FAs from multiple sources, including the action of lipoprotein lipase (LPL). LPL is produced in cardiomyocytes and subsequently secreted to its heparan sulfate proteoglycan (HSPG) binding sites on the plasma membrane. To then move LPL to the endothelial cell (EC) lumen, glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) attaches to interstitial LPL and transfers it to the vascular lumen, where the LPL is ready to perform its function of breaking down circulating triglycerides (TG) into FAs. The endo-β-glucuronidase heparanase (Hpa) is unique in that it is the only known mammalian enzyme to cleave heparan sulfate (HS), thereby promoting the abovementioned release of LPL from the cardiomyocyte HSPG. In diabetes, it has been suggested that changes in how the heart generates energy are responsible for the development of diabetic cardiomyopathy (DCM). Following moderate diabetes, with the reduction in glucose utilization, the heart increases its LPL activity at the vascular lumen due to an increase in Hpa action. Although this adaptation might be beneficial to compensate for the underutilization of glucose by the heart, it is toxic over the long term, as harmful lipid metabolite accumulation, along with augmented FA oxidation and thus oxidative stress, leads to cell death. This coincides with the loss of a cardioprotective growth factor—namely, vascular endothelial growth factor B (VEGFB). This review discusses interconnections between Hpa, LPL, and VEGFB and their potential implications in DCM. Given that mechanism-based therapeutic care for DCM is unavailable, understanding the pathology of this cardiomyopathy, along with the contribution of LPL, will help us advance its clinical management.

Lead identification of novel tetrahydroimidazo[1,2-a]pyridine-5-carboxylic acid derivative as a potent heparanase-1 inhibitor

Heparanase-1 (HPSE1) is an endo-β-d-glucuronidase that cleaves heparan sulfate proteoglycans into short-chain heparan sulfates (HS). The inhibition of HPSE1 has therapeutic potential for proteinuric diseases such as nephrotic syndrome because increased HPSE1 expression is associated with the loss of HS in the glomerular basement membrane, leading to the development of proteinuria. The present study examined the generation of a lead compound focusing on chemical structures with a sugar moiety, such as glycosides and sugar analogs, taking their physical properties into consideration. Compound 10, an exo-β-d-glucuronidase (GUSβ) inhibitor, was found to have a weak inhibitory activity against endo-β-d-glucuronidase HPSE1. A structure–activity relationship study using the X-ray co-crystal structure of 10 and HPSE1 resulted in 12a, which showed a more than 14-fold increase in HPSE1 inhibitory activity compared with that of 10. Compound 12a could be a novel lead compound for the development of a potent HPSE1 inhibitor.

Targeting of Heparanase in the Murine Model Protects against Radiation-Induced Nephropathy

<h3>Purpose/Objective(s)</h3> Adverse effects are common in virtually all types of treatment modalities in cancer including that of radiation therapy. Several agents are clinically used to aid in the reduction of side effects of anti-cancer therapy, however these agents <i>per se</i> are typically not endowed with tumor-suppressing properties. Herein we report that the extracellular matrix (ECM)-degrading enzyme heparanase (a valid target for anti-cancer drug development) is also a promising candidate for interventions aimed at preventing development of radiation nephropathy - a significant side effect of radiation therapy in the treatment of abdominal/pelvic malignancies. Heparanase is the sole mammalian endoglucuronidase that cleaves heparan sulfate in the ECM. The enzymatic action of heparanase has not only been found to promote tumor development and progression at various anatomic sites but is upregulated in essentially all tumor types found in the abdominal/pelvic cavity, whose radiation treatment may result in the development of radiation nephropathy. <h3>Materials/Methods</h3> Eleven-week-old female C3H/HeNHsd mice were administered 10 Gy of radiation using brachytherapy to induce radiation nephropathy. Subcutaneous injections of Roneparstat, a specific heparanase inhibitor, were administered to the mice and urine samples were collected over 24h. Urinary albumin was measured using an ELISA kit. Prior to and following irradiation, heparanase and early growth response (Egr1) transcription factor mRNA levels were determined by qRT-PCR in both HK-2 human proximal tubule epithelial cells and HEK-293 human embryonic kidney cells. <h3>Results</h3> In this study, we evaluated the potential of targeting heparanase to diminish radiation therapy-induced kidney damage. We demonstrated that clinically relevant doses of ionizing radiation upregulate heparanase expression in renal cells (most likely through a transcription factor Egr 1 –dependent mechanism). Moreover, utilizing a murine model of abdominal radiation therapy, we found that the specific heparanase inhibitor, Roneparstat, abolished radiation-induced albuminurea –the hallmark of radiation nephropathy. <h3>Conclusion</h3> Given the well-documented anti-cancer effects of heparanase inhibition, our present findings attest this enzyme as a unique target in the landscape of cancer therapy due to its dual action. Indeed, targeting of heparanase exerts not only direct anti-tumor effects but also protects from kidney damage induced by radiation - the backbone of cancer therapy across a broad range of abdominal/pelvic malignancies.

Heparanase: A Novel Therapeutic Target for the Treatment of Atherosclerosis.

Cardiovascular disease (CVD) is the leading cause of death and disability worldwide, and its management places a huge burden on healthcare systems through hospitalisation and treatment. Atherosclerosis is a chronic inflammatory disease of the arterial wall resulting in the formation of lipid-rich, fibrotic plaques under the subendothelium and is a key contributor to the development of CVD. As such, a detailed understanding of the mechanisms involved in the development of atherosclerosis is urgently required for more effective disease treatment and prevention strategies. Heparanase is the only mammalian enzyme known to cleave heparan sulfate of heparan sulfate proteoglycans, which is a key component of the extracellular matrix and basement membrane. By cleaving heparan sulfate, heparanase contributes to the regulation of numerous physiological and pathological processes such as wound healing, inflammation, tumour angiogenesis, and cell migration. Recent evidence suggests a multifactorial role for heparanase in atherosclerosis by promoting underlying inflammatory processes giving rise to plaque formation, as well as regulating lesion stability. This review provides an up-to-date overview of the role of heparanase in physiological and pathological processes with a focus on the emerging role of the enzyme in atherosclerosis.

Role of Heparanase and Syndecan-1 in HSV-1 Release from Infected Cells.

Herpes Simplex Virus 1 (HSV-1) is a neurotropic human virus that belongs to the Alphaherpesvirinae subfamily of Herpesviridae. Establishment of its productive infection and progression of disease pathologies depend largely on successful release of virions from the virus-producing cells. HSV-1 is known to exploit many host factors for its release. Recent studies have shown that heparanase (HPSE) is one such host enzyme that is recruited for this purpose. It is an endoglycosidase that cleaves heparan sulfate (HS) from the surface of infected cells. HS is a virus attachment coreceptor that is commonly found on cell surfaces as HS proteoglycans e.g., syndecan-1 (SDC-1). The current model suggests that HSV-1 during the late stage of infection upregulates HPSE, which in turn enhances viral release by removing the virus-trapping HS moieties. In addition to its role in directly enabling viral release, HPSE accelerates the shedding of HS-containing ectodomains of SDC-1, which enhances HSV-1 release via a similar mechanism by upregulating CREB3 and COPII proteins. This review outlines the role of HPSE and SDC-1 as newly assigned host factors that facilitate HSV-1 release during a lytic infection cycle.

Non-enzymatic heparanase enhances gastric tumor proliferation via TFEB-dependent autophagy

Heparanase (HPA) is the predominant enzyme that cleaves heparan sulfate and plays a critical role in a variety of pathophysiological processes. HPA activity has been traditionally correlated with tumor metastasis due to participation in the cleavage and remodeling of the extracellular matrix (ECM). Apart from its well-characterized catalytic properties, HPA was noticed to exert biological functions not rely on its enzymatic activity. This feature is supported by studies showing induction of signaling events, such as Src and AKT, by nonenzymatic HPA mutant. We provide evidence here that active HPA and inactive HPA mutant proteins enhance gastric cancer cell growth, possibly attributed to TFEB-mediated autophagy. Similarly, HPA gene silencing resulted in decreased gastric cancer cell proliferation and autophagy. Besides, TFEB inhibition reduced cell growth and autophagy induced by nonenzymatic HPA. Notably, HPA and TFEB were significantly elevated in gastric carcinomas compared with the adjacent gastric tissue. Moreover, the elevation of HPA gene expression and upregulation of TFEB levels have been associated with advanced clinical stage and poor prognosis of gastric cancer, providing strong clinical support for a connection between TFEB and HPA. Thus, neutralizing the nonenzymatic function of HPA and the related TFEB-driven autophagy may profoundly impact gastric cancer progression.

Glycocalyx heparan sulfate cleavage promotes endothelial cell angiopoietin-2 expression by impairing shear stress-related AMPK/FoxO1 signaling.

Angiopoietin-2 (Ang-2) is a key mediator of vascular disease during sepsis, and elevated plasma levels of Ang-2 are associated with organ injury scores and poor clinical outcomes. We have previously observed that biomarkers of endothelial glycocalyx (EG) damage correlate with plasma Ang-2 levels, suggesting a potential mechanistic linkage between EG injury and Ang-2 expression during states of systemic inflammation. However, the cell signaling mechanisms regulating Ang-2 expression following EG damage are unknown. In the current study, we determined the temporal associations between plasma heparan sulfate (HS) levels as a marker of EG erosion and plasma Ang-2 levels in children with sepsis and in mouse models of sepsis. Second, we evaluated the role of shear stress–mediated 5′-adenosine monophosphate–activated protein kinase (AMPK) signaling in Ang-2 expression following enzymatic HS cleavage from the surface of human primary lung microvascular endothelial cells (HLMVECs). We found that plasma HS levels peaked before plasma Ang-2 levels in children and mice with sepsis. Further, we discovered that impaired AMPK signaling contributed to increased Ang-2 expression following HS cleavage from flow-conditioned HLMVECs, establishing a paradigm by which Ang-2 may be upregulated during sepsis.

Heparanase is a prognostic biomarker independent of tumor purity and hypoxia based on bioinformatics and immunohistochemistry analysis of esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell carcinoma (ESCC) is a common malignant tumor of the digestive tract with a poor prognosis. The tumor microenvironment (TME) is mainly composed of tumor cells, stromal cells, and immune cells and plays an important role in ESCC development. There are substantial differences in tumor purity among different parts of ESCC tissues, consisting of distinct immune and stromal cells and variations in the status of hypoxia. Thus, prognostic models of ESCC based on bioinformatic analysis of tumor tissues are unreliable.MethodDifferentially expressed genes (DEGs) independent of tumor purity and hypoxia were screened by Spearman correlation analysis of public ESCC cohorts. Subsequently, the DEGs were subjected to Cox regression analysis. Then, we constructed a protein–protein interaction (PPI) network of the DEGs using Cytoscape. Intersection analysis of the univariate Cox and PPI results indicated that heparanase (HPSE), an endo-β-D-glucuronidase capable of cleaving heparan sulfate side chains, was a predictive factor. Gene set enrichment analysis (GSEA) was used to reveal the potential function of HPSE, and single-cell sequencing data were analyzed to evaluate the distribution of HPSE in immune cells. Furthermore, a human ESCC tissue microarray was used to validate the expression and prognostic value of HPSE.ResultWe found that HPSE was downregulated in ESCC tissues and was not correlated with tumor purity or hypoxia status. HPSE is involved in multiple biological processes. ESCC patients with low HPSE expression in cancerous tissues exhibited poor prognosis.ConclusionsThese results indicate that low HPSE expression in cancerous tissues correlates with poor prognosis in patients with ESCC. HPSE is a novel prognostic biomarker independent of tumor purity and hypoxia status in ESCC.

Abstract 1419: Single molecule detection of human heparanase

Abstract Heparanase, a mammalian endo-β-D-glucuronidase, is the only known enzyme responsible for the cleavage of heparan sulfate (HS) side chains of heparan sulfate proteoglycans (HSPGs). Its activity is implicated in various disease conditions such as tumor growth, angiogenesis, metastasis, chronic inflammation and diabetic nephropathy. Detecting the activity of heparanase thus is a promising approach for the discovery of diagnostics and therapeutics for these diseases. We have developed an activity-based disaccharide probe for heparanase, which offers over 700-fold of fluorescence increase in the presence of human heparanase. In this project, we aim to build up an ultrasensitive single heparanase detection system by coupling a sensitive fluorogenic probe with a microwell-based device. The microwell-based device is designed to achieve single enzyme separation and significantly increase the sensitivity of detection. This system is being validated using recombinant human heparanase and will be used to analyze clinical samples from breast cancer patients. Citation Format: Zhishen Wang. Single molecule detection of human heparanase [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1419.