Enzyme Heparanase Research Articles

Synthesis of Low-Molecular-Weight Fucoidan Analogue and Its Inhibitory Activities against Heparanase and SARS-CoV-2 Infection.

Heparan sulfate (HS) is ubiquitous on cell surfaces and is used as a receptor by many viruses including SARS-CoV-2. However, increased activity of the inflammatory enzyme heparanase (HPSE), which hydrolyses HS, in patients with COVID-19 not only increases the severity of symptoms but also may facilitate the spread of the virus by degrading HS on the cell surface. Therefore, synthetic HPSE blockades, which can bind to SARS-CoV-2 spike protein (SARS-CoV-2-S) and inhibit viral entry, have attracted much attention. This study investigated the development of a new dual-targeting antiviral agent against HPSE and SARS-CoV-2-S using fucoidan as a structural motif. It was found that all-sulfated fucoidan derivative 10, which exhibited the highest binding affinity to SARS-CoV-2-S among 13 derivatives, also showed the highest inhibitory activity against HPSE. Based on this, a newly designed and synthesized fucoidan analogue 16, in which the octyl group of 10 was changed to a cholestanyl group, was found to show higher activity than 10 but did not inhibit factor Xa associated with undesired anticoagulant effects. The binding affinity of 16 to SARS-CoV-2-S was significantly increased approximately 400-fold over that of 10.Furthermore, 16 effectively inhibited infection by the SARS-CoV-2 Wuhan strain and two Omicron subvariants.

Synthesis of Low‐Molecular‐Weight Fucoidan Analogue and Its Inhibitory Activities against Heparanase and SARS‐CoV‐2 Infection

Heparan sulfate (HS) is ubiquitous on cell surfaces and is used as a receptor by many viruses including SARS‐CoV‐2. However, increased activity of the inflammatory enzyme heparanase (HPSE), which hydrolyses HS, in patients with COVID‐19 not only increases the severity of symptoms but also may facilitate the spread of the virus by degrading HS on the cell surface. Therefore, synthetic HPSE blockades, which can bind to SARS‐CoV‐2 spike protein (SARS‐CoV‐2‐S) and inhibit viral entry, have attracted much attention. This study investigated the development of a new dual‐targeting antiviral agent against HPSE and SARS‐CoV‐2‐S using fucoidan as a structural motif. It was found that all‐sulfated fucoidan derivative 10, which exhibited the highest binding affinity to SARS‐CoV‐2‐S among 13 derivatives, also showed the highest inhibitory activity against HPSE. Based on this, a newly designed and synthesized fucoidan analogue 16, in which the octyl group of 10 was changed to a cholestanyl group, was found to show higher activity than 10 but did not inhibit factor Xa associated with undesired anticoagulant effects. The binding affinity of 16 to SARS‐CoV‐2‐S was significantly increased approximately 400‐fold over that of 10. Furthermore, 16 effectively inhibited infection by the SARS‐CoV‐2 Wuhan strain and two Omicron subvariants.

Abstract 2913: Co-expression of extracellular matrix enzymes heparanase or PH-20 augments the anti-tumor efficacy of folate receptor 1-targeting CAR T cells in an ovarian cancer model

Abstract Introduction: The tumor extracellular matrix (ECM) is a physical barrier which obstructs trafficking of CAR T cells to tumors. We hypothesize that co-expressing ECM remodeling/degrading enzymes heparanase (HPSE) or PH-20 with a CAR directed against folate receptor 1 (FolR1) will overcome the physical tumor microenvironment barrier and improve anti-tumor efficacy of anti-FolR1 CAR in a human ovarian cancer model. Methods: ECM CARs were constructed by combining anti-FolR1 ScFv with a CD8 hinge and transmembrane domain, 4-1BB, and CD3ζ endodomains, followed by a P2A element and full-length HPSE or GPI-anchored PH-20 sequence. CAR T cells were prepared from CD4+ and CD8+ T cells of healthy donors by lentiviral transduction. CAR expression was evaluated by flow cytometry; HPSE and PH-20 expression were measured via ELISA and Western blotting, respectively. CAR-mediated killing and cytokine release were observed in overnight co-culture assays with FolR1-expressing tumor cells. ECM CARs’ migration through CultrexTM and sodium hyaluronate matrices were assessed for HPSE and PH-20 function, respectively. ECM CARs’ efficacy was evaluated in vivo using a FolR1-expressing subcutaneous OV90 ovarian cancer xenograft model in NOD SCID Gamma (NSG) mice, and representative OV90 tumors were harvested at day 7 post-CAR T treatment for immunohistochemical staining. Results: FolR1 expression was confirmed in ovarian cancer cell lines and substrates targeted by HPSE (perlecan, glypican 3, CD138) and PH-20 (hyaluronan) were detected by immunofluorescence in human ovarian tumors’ sections. Lentiviral transduced primary T cells yielded robust expression of FolR1 CAR and ECM enzymes. Anti-FolR1 CAR and ECM CARs secreted pro-inflammatory cytokines IL-2, TNFα, IFNƳ and lysed OVCAR3 tumor cells with similar efficacy. ECM CARs with HPSE or PH-20 exhibited enhanced migration through ECM-rich matrices as compared to anti-FolR1 CAR alone. In vivo, all anti-FolR1 CAR T cells rejected OV90 tumors and extended survival compared to tumor alone or untransduced T cells (UTD). However, ECM CARs co-expressing HPSE or PH-20 accelerated tumor regression as compared to anti-FolR1 CAR alone. Tumor histology revealed CAR T cell infiltration (CD3+, CD8+) in mice treated with anti-FolR1 CARs but not UTD, indicating that anti-FolR1 CARs can effectively infiltrate OV90 tumors. Imaging studies are underway to evaluate the function of ECM CARs in the tumor microenvironment. Conclusions: The anti-FolR1 CARs boosted with ECM enzymes HPSE or PH-20 demonstrated specific lysis of FolR1-expressing cell lines, improved penetration through ECM-rich matrices in vitro, and accelerated tumor regression in an ovarian cancer model in vivo. Therefore, ECM CARs may help improve clinical outcomes in patients with ovarian tumors. Citation Format: Brittany L. Steimle, Peirong Hu, Ngoc Tran, Jia-Jye Lee, Kathleen Baysac, Kun Luo, Xu Wang, Genqing Liang, Saule Nurmukhambetova, Tri Tran, Pradyot Dash, Dina Schneider. Co-expression of extracellular matrix enzymes heparanase or PH-20 augments the anti-tumor efficacy of folate receptor 1-targeting CAR T cells in an ovarian cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2913.

Investigation of inhibitory effect of sulfated chitosan oligomer on human heparanase enzyme: in silico and in vitro studies

Deaths from cancer are widespread worldwide and the numbers continue to increase day by day. During the disease progression of cancer in cells, many of its metabolic activities change. Increased heparanase enzyme release is just one example. Following heparanase enzyme activity, many molecules interact with the remodeling of glycosaminoglycan structures, which triggers the release of different enzymes, cytokines, and growth factors, including fibroblast growth factors (FGF1 and FGF2), vascular endothelial growth factor (VEGF), hepatocyte growth factor, transforming growth factor β and platelet-derived growth factor. These are the most important factors in metastasis due to the formation of new vascular structures caused by those elements. To reduce tumor growth and metastasis, various drugs have been designed by modifying chitosan and its derivatives. In this study, we used chitosan oligomer (A), sulfated chitosan oligomer (ShCsO) (B), heparin (C), phosphate monomer (D1) of PI-88 and sulfate monomer (D2) of PI-88 as heparanase inhibitors. We modified the chitosan oligomer with chlorosulfonic acid to synthesize ShCsO to investigate its inhibitory effects on human serum heparanase. Also examined were molecular docking; molecular dynamics (MD); adsorption, distribution, metabolism, elimination and toxicity (ADMET); and target prediction. ShCsO decreased enzyme activity at a concentration of 0.0001 mg/mL. The docking scores of A, B and C from in silico studies were −6.254, −6.936 and −6.980 kcal/mol, respectively, and the scores for the two different PI-88 monomers were −5.741 and −5.824 kcal/mol. These results show that ShCsO may be a potential drug candidate for treating cancer. Communicated by Ramaswamy H. Sarma

The novel HS-mimetic, Tet-29, regulates immune cell trafficking across barriers of the CNS during inflammation

BackgroundDisruption of the extracellular matrix at the blood–brain barrier (BBB) underpins neuroinflammation in multiple sclerosis (MS). The degradation of extracellular matrix components, such as heparan sulfate (HS) proteoglycans, can be prevented by treatment with HS-mimetics through their ability to inhibit the enzyme heparanase. The heparanase-inhibiting ability of our small dendrimer HS-mimetics has been investigated in various cancers but their efficacy in neuroinflammatory models has not been evaluated. This study investigates the use of a novel HS-mimetic, Tet-29, in an animal model of MS.MethodsNeuroinflammation was induced in mice by experimental autoimmune encephalomyelitis, a murine model of MS. In addition, the BBB and choroid plexus were modelled in vitro using transmigration assays, and migration of immune cells in vivo and in vitro was quantified by flow cytometry.ResultsWe found that Tet-29 significantly reduced lymphocyte accumulation in the central nervous system which, in turn, decreased disease severity in experimental autoimmune encephalomyelitis. The disease-modifying effect of Tet-29 was associated with a rescue of BBB integrity, as well as inhibition of activated lymphocyte migration across the BBB and choroid plexus in transwell models. In contrast, Tet-29 did not significantly impair in vivo or in vitro steady state-trafficking under homeostatic conditions.ConclusionsTogether these results suggest that Tet-29 modulates, rather than abolishes, trafficking across central nervous system barriers.

Interaction between mitochondrial homeostasis and barrier function in lipopolysaccharide-induced endothelial cell injury.

This study aimed to investigate the effects of mitochondrial homeostasis on lipopolysaccharide (LPS)-induced endothelial cell barrier function and the mechanisms that underlie these effects. Cells were treated with LPS or oligomycin (mitochondrial adenosine triphosphate synthase inhibitor) and the mitochondrial morphology, mitochondrial reactive oxygen species (mtROS), and mitochondrial membrane potential (ΔΨm) were evaluated. Moreover, the shedding of glycocalyx-heparan sulphate (HS), the levels of HS-specific degrading enzyme heparanase (HPA), and the expression of occludin and zonula occludens (ZO-1) of Tight Junctions (TJ)s, which are mediated by myosin light chain phosphorylation (p-MLC), were assessed. Examining the changes in mitochondrial homeostasis showed that adding heparinase III, which is an exogenous HPA, can destroy the integrity of glycocalyx. LPS simultaneously increased mitochondrial swelling, mtROS, and ΔΨm. Without oligomycin effects, HS, HPA levels, and p-MLC were found to be elevated, and the destruction of occludin and ZO-1 increased. Heparinase III not only damaged the glycocalyx by increasing HS shedding but also increased mitochondrial swelling and mtROS and decreased ΔΨm. Mitochondrial homeostasis is involved in LPS-induced endothelial cell barrier dysfunction by aggravating HPA and p-MLC levels. In turn, the integrated glycocalyx protects mitochondrial homeostasis.

ANALYSIS OF THE CONTRIBUTION OF GENES HP, ACE, HPSE1, MTHFR AND HFE TO THE DEVELOPMENT OF HEART FAILURE

Objective: Heart Failure (HF) is a clinical syndrome characterised by typical symptoms, associated with a cardiac incapacity of pumping blood. Although there have been improvements in treating HF, this disease still has a high mortality rate. Genes HP, ACE, HPSE1, MTHFR, and HFE are involved in various events and regulatory mechanisms associated with the disease such as the functionality of Haptoglobin, the activity of ACE and Heparanase enzymes and the metabolisms of Folic Acid and Iron. The purpose of this study is to investigate the contribution of the variants Hp1/2 (HP), I/D (ACE), rs4693608 (HPSE1), C677T (MTHFR), H63D and C282Y (HFE) to the development of HF individually or in epistasis. Design and method: Genotype data previously obtained in 143 HF patients and 246 healthy individuals was used to carry out analysis. The analysis of the data was performed with SPSS software. The association between genotypes and alleles was tested by Chi-Square or Fisher tests. Odds Ratios were calculated as well as the corresponding confidence intervals. Results: We have observed a higher risk for developing HF in the presence of: 1) the T allele [OR (IC, 95) = 2,932] and CT genotype [OR (IC, 95) = 3,618] of the C677T polymorphism; 2) epistasis between II and YY genotypes of the I/D and C282Y polymorphisms [OR (IC, 95) = 5,191]; 3) epistasis between II and DD genotypes of the I/D and H63D polymorphisms [OR (IC, 95) = 6,626]; 4) epistasis between the 2 and C alleles of HP gene and C677T polymorphism [OR (IC, 95) = 4,351]; 5) epistasis between the C and A alleles of the C677T and rs4693608 polymorphisms [OR (IC, 95) = 6,804]. A protective effect was observed for the CC genotype of the C677T polymorphism [OR (IC, 95) = 0,341]; between the D allele of the I/D variation and the Y [OR (IC, 95) = 0,233] and D [OR (IC, 95) = 0,279] alleles of the C282Y and H63D polymorphisms, respectively. Conclusions: We have concluded that HP, ACE, HPSE1, MTHFR, and HFE genes contribute to the susceptibility of developing HF individually or in epistasis.

Abstract 2395: A bioluminescent probe for detection and imaging of heparanase enzymatic activity

Abstract Cancers of all types have been shown to overexpress the carbohydrate-processing enzyme heparanase during angiogenesis and invasion, and this enzymatic activity correlates positively with tumor stage and the presence of metastases. While detection of heparanase enzymatic activity holds potential for cancer diagnosis and staging, no probe has been achieved for in vivo systems. We have recently developed fluorogenic heparanase probes for single-step detection of in vitro heparanase enzymatic activity, drastically simplifying the detection of heparanase activity. The modular chemistry allows us to extend the utility further to improve the sensitivity of in vitro heparanase activity detection and enable in vivo detection. Thus, we have developed a bioluminescent heparanase probe, which upon activation by heparanase produces a substrate of firefly luciferase, a standard reporter enzyme for in vivo monitoring of tumor progression. This probe will be validated in multi-well assays of heparanase activity, and applied in real-time correlation of heparanase activity and tumor progression in, fLuc+ tumors in animal models. These studies hold promise in enabling highly sensitive detection of heparanase activity both in vitro and in vivo, and in allowing future research of real-time monitoring of heparanase activity with the tumor progression. Citation Format: Johnathan Somers, Kelton Schleyer, Jun Liu, Zhishen Wang, Arjun Kafle, Lina Cui. A bioluminescent probe for detection and imaging of heparanase enzymatic activity [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 2395.

Heparin, Heparan Sulphate and Sepsis: Potential New Options for Treatment.

Sepsis is a life-threatening hyperreaction to infection in which excessive inflammatory and immune responses cause damage to host tissues and organs. The glycosaminoglycan heparan sulphate (HS) is a major component of the cell surface glycocalyx. Cell surface HS modulates several of the mechanisms involved in sepsis such as pathogen interactions with the host cell and neutrophil recruitment and is a target for the pro-inflammatory enzyme heparanase. Heparin, a close structural relative of HS, is used in medicine as a powerful anticoagulant and antithrombotic. Many studies have shown that heparin can influence the course of sepsis-related processes as a result of its structural similarity to HS, including its strong negative charge. The anticoagulant activity of heparin, however, limits its potential in treatment of inflammatory conditions by introducing the risk of bleeding and other adverse side-effects. As the anticoagulant potency of heparin is largely determined by a single well-defined structural feature, it has been possible to develop heparin derivatives and mimetic compounds with reduced anticoagulant activity. Such heparin mimetics may have potential for use as therapeutic agents in the context of sepsis.

Extracellular vesicles released during hypoxia transport heparanase and enhance macrophage migration, endothelial tube formation and cancer cell stemness

Heparanase is upregulated during the progression of most cancers and via its enzyme activity promotes extracellular matrix degradation, angiogenesis and cell migration. Heparanase expression is often associated with enhanced tumor aggressiveness and chemoresistance. We previously demonstrated that increased heparanase expression in tumor cells enhances secretion and alters the composition of tumor-released exosomes. In the present study, we discovered that extracellular vesicles (EVs) secreted by human multiple myeloma cells growing in hypoxic conditions exhibited elevated levels of heparanase cargo compared to EVs from cells growing in normoxic conditions. When macrophages (RAW 264.7 monocyte/macrophage-like cells) were exposed to EVs released by tumor cells growing in either hypoxic or normoxic conditions, macrophage migration and invasion was elevated by EVs from hypoxic conditions. The elevated invasion of macrophages was blocked by a monoclonal antibody that inhibits heparanase enzyme activity. Moreover, the heparanase-bearing EVs from hypoxic cells greatly enhanced endothelial cell tube formation consistent with the known role of heparanase in promoting angiogenesis. EVs from hypoxic tumor cells when compared with EVs from normoxic cells also enhanced cancer stemness properties of both CAG and RPMI 8226 human myeloma cells. Together these data indicate that under hypoxic conditions, tumor cells secrete EVs having an elevated level of heparanase as cargo. These EVs can act on both tumor and non-tumor cells, enhancing tumor progression and tumor cell stemness that likely supports chemoresistance and relapse of tumor.

Evaluation of Immunohistochemical Expression of Heparanase in Helicobacter pylori-Associated Chronic Gastritis.

Chronic gastritis (CG) is a very common disease. More than half of the worldwide population suffers from symptoms of CG. This disease has received great attention since the discovery of H. pylori as the most important cause of CG. Symptoms experienced by patients with CG are attributed to H. pylori-induced inflammatory reactions. Heparanase (HPSE) is a mammalian β-endoglucoronidase. In inflammation; HPSE degrades and remodels the extracellular matrix's heparan sulfate polysaccharide chains liberating heparan sulfate-bound cytokines and chemokines, HPSE also facilitates movement of inflammatory cells. This study aimed to detect the function of HPSE in CG by correlating levels of HPSE expression with histopathological features of CG, including H. pylori infection, acute and chronic inflammatory cells, mucosal atrophic and/or metaplastic features. Ninety-five upper endoscopic-guided gastric punch biopsies were enrolled in this study. From each specimen, formalin-fixed and paraffin-embedded tissue blocks were prepared. Tissue sections were stained by Hematoxylin and eosin, Giemsa, and anti-heparanase antibody. HPSE expression was statistically associated with H. pylori infection (P-value < .000), and intensity of chronic lymphocytic inflammatory infiltrate in the gastric mucosal tissues (P = .004). High levels of HPSE expression were also related to the presence of neutrophils in the gastric surface epithelium and lamina propria (P-value < .009). HPSE expression was upregulated in H. pylori-associated chronic gastritis. Thus, future therapeutic agents that could specifically inhibit HPSE enzyme activity, may aid in the reduction of sequelae of H. pylori infection.

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.

Heparin-Superparamagnetic Iron Oxide Nanoparticles for Theranostic Applications.

In this study, superparamagnetic iron oxide nanoparticles (SPIONs) were engineered with an organic coating composed of low molecular weight heparin (LMWH) and bovine serum albumin (BSA), providing heparin-based nanoparticle systems (LMWH@SPIONs). The purpose was to merge the properties of the heparin skeleton and an inorganic core to build up a targeted theranostic nanosystem, which was eventually enhanced by loading a chemotherapeutic agent. Iron oxide cores were prepared via the co-precipitation of iron salts in an alkaline environment and oleic acid (OA) capping. Dopamine (DA) was covalently linked to BSA and LMWH by amide linkages via carbodiimide coupling. The following ligand exchange reaction between the DA-BSA/DA-LMWH and OA was conducted in a biphasic system composed of water and hexane, affording LMWH@SPIONs stabilized in water by polystyrene sulfonate (PSS). Their size and morphology were investigated via dynamic light scattering (DLS) and transmission electron microscopy (TEM), respectively. The LMWH@SPIONs’ cytotoxicity was tested, showing marginal or no toxicity for samples prepared with PSS at concentrations of 50 µg/mL. Their inhibitory activity on the heparanase enzyme was measured, showing an effective inhibition at concentrations comparable to G4000 (N-desulfo-N-acetyl heparin, a non-anticoagulant and antiheparanase heparin derivative; Roneparstat). The LMWH@SPION encapsulation of paclitaxel (PTX) enhanced the antitumor effect of this chemotherapeutic on breast cancer cells, likely due to an improved internalization of the nanoformulated drug with respect to the free molecule. Lastly, time-domain NMR (TD-NMR) experiments were conducted on LMWH@SPIONs obtaining relaxivity values within the same order of magnitude as currently used commercial contrast agents.

Heparanase Is a Putative Mediator of Endothelial Glycocalyx Damage in COVID-19 - A Proof-of-Concept Study.

Coronavirus disease 2019 (COVID-19) is a systemic disease associated with injury (thinning) of the endothelial glycocalyx (eGC), a protective layer on the vascular endothelium. The aim of this translational study was to investigate the role of the eGC-degrading enzyme heparanase (HPSE), which is known to play a central role in the destruction of the eGC in bacterial sepsis. Excess activity of HPSE in plasma from COVID-19 patients correlated with several markers of eGC damage and perfused boundary region (PBR, an inverse estimate of glycocalyx dimensions of vessels with a diameter 4-25 µm). In a series of translational experiments, we demonstrate that the changes in eGC thickness of cultured cells exposed to COVID-19 serum correlated closely with HPSE activity in concordant plasma samples (R = 0.82, P = 0.003). Inhibition of HPSE by a nonanticoagulant heparin fragment prevented eGC injury in response to COVID-19 serum, as shown by atomic force microscopy and immunofluorescence imaging. Our results suggest that the protective effect of heparin in COVID-19 may be due to an eGC-protective off-target effect.

CREB3 Plays an Important Role in HPSE-Facilitated HSV-1 Release in Human Corneal Epithelial Cells

Herpes simplex virus type-1 (HSV-1) exploits several host factors to enhance its replication and release from infected cells. It induces the production of host enzyme heparanase (HPSE) to aid in egress. While the mechanism by which HPSE assists in viral release is well-characterized, other host factors that are recruited along with HPSE for viral release are less well understood. In this study, we identify cyclic-AMP-responsive element-binding protein3 (CREB3) as a key player in HPSE-facilitated HSV-1 egress. When CREB3 is transiently upregulated in human corneal epithelial cells, HSV-1 release from the infected cells is correspondingly enhanced. This activity is linked to HPSE expression such that HPSE-transfected corneal epithelial (HCE) cells more highly express CREB3 than wild-type cells while the cells knocked out for HPSE show very little CREB3 expression. CREB3-transfected HCE cells showed significantly higher export of HPSE upon infection than wild-type cells. Our data suggests that coat protein complex II (COPII), which mediates HPSE trafficking, is also upregulated via a CREB3-dependent pathway during HSV-1 infection. Finally, the co-transfection of CREB3 and HPSE in HCE cells shows the highest viral release compared to either treatment alone, establishing CREB3 as a key player in HPSE-facilitated HSV-1 egress.

Heparanase Inhibition Prevents Liver Steatosis in E0 Mice.

Background: Non-alcoholic fatty liver disease affects up to 30% of adults in the USA, and is associated with a higher incidence of chronic liver morbidity and mortality. Several molecular pathways are involved in the pathology of liver steatosis, including lipid uptake, lipogenesis, lipolysis, and beta-oxidation. The enzyme heparanase has been implicated in liver steatosis. Herein, we investigated the effect of heparanase inhibition on liver steatosis in E0 mice. Methods: In vivo experiments: Male wild-type mice fed with either chow diet (n = 4) or high-fat diet (n = 6), and male E0 mice fed with chow diet (n = 8) or high-fat diet (n = 33) were included. Mice on a high-fat diet were treated for 12 weeks with PG545 at low dose (6.4 mg/kg/week, ip, n = 6) or high dose (13.3 mg/kg/week, ip, n = 7), SST0001 (1.2 mg/mouse/day, ip, n = 6), or normal saline (control, n = 14). Animals were sacrificed two days after inducing peritonitis. Serum was analyzed for biochemical parameters. Mouse peritoneal macrophages (MPMs) were harvested and analyzed for lipid content. Livers were harvested for histopathological analysis of steatosis, lipid content, and the expression of steatosis-related factors at the mRNA level. In vitro experiments: MPMs were isolated from untreated E0 mice aged 8–10 weeks and were cultured and treated with either PG545 or SST0001, both at 50 µg/mL for 24 h, followed by assessment of mRNA expression of steatosis related factors. Results: Heparanase inhibition significantly attenuated the development of liver steatosis, as was evident by liver histology and lipid content. Serum analysis indicated lowering of cholesterol and triglycerides levels in mice treated with heparanase inhibitors. In liver tissue, assessment of mRNA expression of key factors in lipid uptake, lipolysis, lipogenesis, and beta-oxidation exhibited significant downregulation following PG545 treatment and to a lesser extent when SST0001 was applied. However, in vitro treatment of MPMs with PG545, but not SST0001, resulted in increased lipid content in these cells, which is opposed to their effect on MPMs of treated mice. This may indicate distinct regulatory pathways in the system or isolated macrophages following heparanase inhibition. Conclusion: Heparanase inhibition significantly attenuates the development of liver steatosis by decreasing tissue lipid content and by affecting the mRNA expression of key lipid metabolism regulators.

On the Biology of Werner's Complex

AbstractWerner's Complex, as a cationic coordination complex (CCC), has hitherto unappreciated biological properties derived from its binding affinity to highly anionic biomolecules such as glycosaminoglycans (GAGs) and nucleic acids. Competitive inhibitor and spectroscopic assays confirm the high affinity to GAGs heparin, heparan sulfate (HS), and its pentasaccharide mimetic Fondaparinux (FPX). Functional consequences of this affinity include inhibition of FPX cleavage by bacterial heparinase and mammalian heparanase enzymes with inhibition of cellular invasion and migration. Werner's Complex is a very efficient condensing agent for DNA and tRNA. In proof‐of‐principle for translational implications, it is demonstrated to display antiviral activity against human cytomegalovirus (HCMV) at micromolar concentrations with promising selectivity. Exploitation of non‐covalent hydrogen‐bonding and electrostatic interactions has motivated the unprecedented discovery of these properties, opening new avenues of research for this iconic compound.

On the Biology of Werner's Complex.

Werner's Complex, as a cationic coordination complex (CCC), has hitherto unappreciated biological properties derived from its binding affinity to highly anionic biomolecules such as glycosaminoglycans (GAGs) and nucleic acids. Competitive inhibitor and spectroscopic assays confirm the high affinity to GAGs heparin, heparan sulfate (HS), and its pentasaccharide mimetic Fondaparinux (FPX). Functional consequences of this affinity include inhibition of FPX cleavage by bacterial heparinase and mammalian heparanase enzymes with inhibition of cellular invasion and migration. Werner's Complex is a very efficient condensing agent for DNA and tRNA. In proof-of-principle for translational implications, it is demonstrated to display antiviral activity against human cytomegalovirus (HCMV) at micromolar concentrations with promising selectivity. Exploitation of non-covalent hydrogen-bonding and electrostatic interactions has motivated the unprecedented discovery of these properties, opening new avenues of research for this iconic compound.

The glycocalyx, a novel key in understanding of mechanism of diabetic nephropathy: a commentary.

Diabetes is a chronic and progressive disease that usually causes disrupts the function of the body's organs and can eventually lead to cardiomyopathy, nephropathy, retinopathy, and neuropathy. Diabetic nephropathy (DN) is the most common cause of chronic kidney disease and causes chronic structural changes in different parts of the affected kidney. Glycocalyx layer is one of the most important components of the vascular base found in the endothelium throughout the body's arteries and it has been shown that glycocalyx is also damaged during diabetic nephropathy. Our goal is to conduct this systematic review study is to find the cause-and-effect relationship between glycocalyx and diabetic nephropathy and also to clarify the role of the endothelial renal glycocalyx in understanding of mechanism of the course of diabetic nephropathy, and to provide an accurate background for further important studies. All databases included MEDLINE (PubMed), Science Direct, Scopus, Ovid and Google Scholar were systematically searched for related published articles. In all databases, the following search strategy was implemented and these key words (in the title/abstract) were used: "diabetes" AND "glycocalyx" OR "diabetic nephropathy" AND "glycocalyx". A total of 19 articles were retrieved from all databases using search strategy. After screening based on the title and abstract, number of 17 of them selected for full text assessment. Finally, after extracting the key points and making connections between the articles, we came up with new points to consider. It can be said that diabetes with the action of reactive oxygen species through oxidative stress, increases ICAM-1 and TNF-α and decreases heparanase enzyme, it affects the glomerular endothelium and eventually leads to albuminuria and destruction of the Glx layer. Diabetes causes super-structural changes in the kidney nephrons at the glomerular level. The glomerular filter barrier, which includes the epithelial cell called the podocyte, endothelial pore cells, and basal membrane of the glomerulus, plays a major role in stabilizing the selective glomerular function in healthy individuals. Diabetic nephropathy also causes changes in endothelial glycocalyx.

Heparan Sulfate Proteoglycans in Diabetes.

Diabetes is a complex disorder responsible for the mortality and morbidity of millions of individuals worldwide. Although many approaches have been used to understand and treat diabetes, the role of proteoglycans, in particular heparan sulfate proteoglycans (HSPGs), has only recently received attention. The HSPGs are heterogeneous, highly negatively charged, and are found in all cells primarily attached to the plasma membrane or present in the extracellular matrix (ECM). HSPGs are involved in development, cell migration, signal transduction, hemostasis, inflammation, and antiviral activity, and regulate cytokines, chemokines, growth factors, and enzymes. Hyperglycemia, accompanying diabetes, increases reactive oxygen species and upregulates the enzyme heparanase that degrades HSPGs or affects the synthesis of the HSPGs altering their structure. The modified HSPGs in the endothelium and ECM in the blood vessel wall contribute to the nephropathy, cardiovascular disease, and retinopathy seen in diabetes. Besides the blood vessel, other cells and tissues in the heart, kidney, and eye are affected by diabetes. Although not well understood, the adipose tissue, intestine, and brain also reveal HSPG changes associated with diabetes. Further, HSPGs are significantly involved in protecting the β cells of the pancreas from autoimmune destruction and could be a focus of prevention of type I diabetes. In some circumstances, HSPGs may contribute to the pathology of the disease. Understanding the role of HSPGs and how they are modified by diabetes may lead to new treatments as well as preventative measures to reduce the morbidity and mortality associated with this complex condition.