Heparanase Blockade as a Novel Dual-Targeting Therapy for COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has caused over 5 million deaths worldwide. Pneumonia and systemic inflammation contribute to its high mortality. Many viruses use heparan sulfate proteoglycans as coreceptors for viral entry, and heparanase (HPSE) is a known regulator of both viral entry and inflammatory cytokines. We evaluated the heparanase inhibitor Roneparstat, a modified heparin with minimum anticoagulant activity, in pathophysiology and therapy for COVID-19. We found that Roneparstat significantly decreased the infectivity of SARS-CoV-2, SARS-CoV-1, and retroviruses (human T-lymphotropic virus 1 [HTLV-1] and HIV-1) in vitro. Single-cell RNA sequencing (scRNA-seq) analysis of cells from the bronchoalveolar lavage fluid of COVID-19 patients revealed a marked increase in HPSE gene expression in CD68+ macrophages compared to healthy controls. Elevated levels of HPSE expression in macrophages correlated with the severity of COVID-19 and the expression of inflammatory cytokine genes, including IL6, TNF, IL1B, and CCL2. In line with this finding, we found a marked induction of HPSE and numerous inflammatory cytokines in human macrophages challenged with SARS-CoV-2 S1 protein. Treatment with Roneparstat significantly attenuated SARS-CoV-2 S1 protein-mediated inflammatory cytokine release from human macrophages, through disruption of NF-κB signaling. HPSE knockdown in a macrophage cell line also showed diminished inflammatory cytokine production during S1 protein challenge. Taken together, this study provides a proof of concept that heparanase is a target for SARS-CoV-2-mediated pathogenesis and that Roneparstat may serve as a dual-targeted therapy to reduce viral infection and inflammation in COVID-19. IMPORTANCE The complex pathogenesis of COVID-19 consists of two major pathological phases: an initial infection phase elicited by SARS-CoV-2 entry and replication and an inflammation phase that could lead to tissue damage, which can evolve into acute respiratory failure or even death. While the development and deployment of vaccines are ongoing, effective therapy for COVID-19 is still urgently needed. In this study, we explored HPSE blockade with Roneparstat, a phase I clinically tested HPSE inhibitor, in the context of COVID-19 pathogenesis. Treatment with Roneparstat showed wide-spectrum anti-infection activities against SARS-CoV-2, HTLV-1, and HIV-1 in vitro. In addition, HPSE blockade with Roneparstat significantly attenuated SARS-CoV-2 S1 protein-induced inflammatory cytokine release from human macrophages through disruption of NF-κB signaling. Together, this study provides a proof of principle for the use of Roneparstat as a dual-targeting therapy for COVID-19 to decrease viral infection and dampen the proinflammatory immune response mediated by macrophages.

Heparin and Derivatives for Advanced Cell Therapies.

Heparin and its derivatives are saving thousands of human lives annually, by successfully preventing and treating thromboembolic events. Although the mode of action during anticoagulation is well studied, their influence on cell behavior is not fully understood as is the risk of bleeding and other side effects. New applications in regenerative medicine have evolved supporting production of cell-based therapeutics or as a substrate for creating functionalized matrices in biotechnology. The currently resurgent interest in heparins is related to the expected combined anti-inflammatory, anti-thrombotic and anti-viral action against COVID-19. Based on a concise summary of key biochemical and clinical data, this review summarizes the impact for manufacturing and application of cell therapeutics and highlights the need for discriminating the different heparins.

2-O, 3-O desulfated heparin (ODSH) increases bacterial clearance and attenuates lung injury in cystic fibrosis by restoring HMGB1-compromised macrophage function.

BACKGROUND: High mobility group box 1 protein (HMGB1) is an alarmin following its release by immune cells upon cellular activation or stress. High levels of extracellular HMGB1 play a critical role in impairing the clearance of invading pulmonary pathogens and dying neutrophils in the injured lungs of cystic fibrosis (CF) and acute respiratory distress syndrome (ARDS). A heparin derivative, 2-O, 3-O desulfated heparin (ODSH), has been shown to inhibit HMGB1 release from a macrophage cell line and is efficacious in increasing bacterial clearance in a mouse model of pneumonia. Thus, we hypothesized that ODSH can attenuate the bacterial burden and inflammatory lung injury in CF and we conducted experiments to determine the underlying mechanisms. METHODS: We determined the effects of ODSH on lung injury produced by Pseudomonas aeruginosa (PA) infection in CF mice with the transmembrane conductance regulator gene knockout (CFTR-/-). Mice were given ODSH or normal saline intraperitoneally, followed by the determination of the bacterial load and lung injury in the airways and lung tissues. ODSH binding to HMGB1 was determined using surface plasmon resonance and in silico docking analysis of the interaction of the pentasaccharide form of ODSH with HMGB1. RESULTS: CF mice given 25 mg/kg i.p. of ODSH had significantly lower PA-induced lung injury compared to mice given vehicle alone. The CF mice infected with PA had decreased levels of nitric oxide (NO), increased levels of airway HMGB1 and HMGB1-impaired macrophage phagocytic function. ODSH partially attenuated the PA-induced alteration in the levels of NO and airway HMGB1 in CF mice. In addition, ODSH reversed HMGB1-impaired macrophage phagocytic function. These effects of ODSH subsequently decreased the bacterial burden in the CF lungs. In a surface plasmon resonance assay, ODSH interacted with HMGB1 with high affinity (KD = 3.89 × 10-8 M) and induced conformational changes that may decrease HMGB1's binding to its membrane receptors, thus attenuating HMGB1-induced macrophage dysfunction. CONCLUSIONS: The results suggest that ODSH can significantly decrease bacterial infection-induced lung injury in CF mice by decreasing both HMGB1-mediated impairment of macrophage function and the interaction of HMGB1 with membrane receptors. Thus, ODSH could represent a novel approach for treating CF and ARDS patients that have HMGB1-mediated lung injury.

Heparan sulfate analogues regulate tumor-derived exosome formation that attenuates exosome functions in tumor processes.

Heparan sulfate (HS) is involved in many biological activities, including the biogenesis and uptake of exosomes, which are related to the occurrence and development of tumors. This study investigated the role of HS analogues (heparin, low molecular weight heparin, and 6-O-desulfated heparin) in modulating exosome secretion, composition and functions. Exosomes derived from B16F10 cells exposed to different HS analogues were isolated and characterized by TEM, western blotting and Nanosight analyses. The number, size and protein cargo of exosomes secreted by HS analogues-induced B16F10 cells were detected. The findings indicated the reduced tumor-derived exosome secretion and protein cargo as reflected by lower levels of CD63, TSG101, heparinase and IL-6 in exosomes derived from heparin-induced B16F10 cells as compared with 6-O-desulfated heparin-induced tumor cells. Further functional assays demonstrated that exosomes from tumor cells exposed to heparin weakened tumor proliferation, migration and invasion most significantly among various exosomes derived from B16F10 cells treated with different HS analogues. Moreover, the sulfate group at 6-O position of heparan sulfate has been proved to play an important role in tumor-derived exosome formation and functions. This study suggested a vital view to develop more specific and efficient HS-based strategies in cancer treatment for targeting tumor-derived exosomes.

Structure, Immunogenicity, and Conformation-Dependent Receptor Binding of the Postfusion Human Metapneumovirus F Protein.

Human metapneumovirus (hMPV) is an important cause of acute viral respiratory infection. As the only target of neutralizing antibodies, the hMPV fusion (F) protein has been a major focus for vaccine development and targeting by drugs and monoclonal antibodies (MAbs). While X-ray structures of trimeric prefusion and postfusion hMPV F proteins from genotype A, and monomeric prefusion hMPV F protein from genotype B have been determined, structural data for the postfusion conformation for genotype B is lacking. We determined the crystal structure of this protein and compared the structural differences of postfusion hMPV F between hMPV A and B genotypes. We also assessed the receptor binding properties of the hMPV F protein to heparin and heparan sulfate (HS). A library of HS oligomers was used to verify the HS binding activity of hMPV F, and several compounds showed binding to predominantly prefusion hMPV F, but had limited binding to postfusion hMPV F. Furthermore, MAbs to antigenic sites III and the 66-87 intratrimeric epitope block heparin binding. In addition, we evaluated the efficacy of postfusion hMPV B2 F protein as a vaccine candidate in BALB/c mice. Mice immunized with hMPV B2 postfusion F protein showed a balanced Th1/Th2 immune response and generated neutralizing antibodies against both subgroup A2 and B2 hMPV strains, which protected the mice from hMPV challenge. Antibody competition analysis revealed the antibodies generated by immunization target two known antigenic sites (III and IV) on the hMPV F protein. Overall, this study provides new characteristics of the hMPV F protein, which may be informative for vaccine and therapy development. IMPORTANCE Human metapneumovirus (hMPV) is an important cause of viral respiratory disease. In this paper, we report the X-ray crystal structure of the hMPV fusion (F) protein in the postfusion conformation from genotype B. We also assessed binding of the hMPV F protein to heparin and heparan sulfate, a previously reported receptor for the hMPV F protein. Furthermore, we determined the immunogenicity and protective efficacy of postfusion hMPV B2 F protein, which is the first study using a homogenous conformation of the protein. Antibodies generated in response to vaccination give a balanced Th1/Th2 response and target two previously discovered neutralizing epitopes.

Clinical Features and Treatment Response in Immune-Mediated Small Fiber Neuropathy with Trisulfated Heparin Disaccharide or Fibroblast Growth Factor Receptor 3 Antibodies.

OBJECTIVE: Novel antibodies to trisulfated heparin disaccharide (TS-HDS) and fibroblast growth factor receptor 3 (FGFR-3) have been recently described in otherwise cryptogenic small fiber neuropathy (SFN) cases. Our goal was to further describe clinical features in such cases and to analyze treatment responses. METHODS: In a retrospective analysis, 40 cases of cryptogenic SFN in a university neuropathy clinic were identified. Of these, TS-HDS and FGFR-3 cases were identified, and clinical features and treatment responses were analyzed. RESULTS: In this cohort, 95% were women, and 55% had either TS-HDS or FGFR-3 antibodies (77% of these had TS-HDS). Of the seropositive group, 41% had a nonlength dependent epidermal nerve fiber density on skin punch biopsy (OR = 1.80). In the seropositive group, 82% had neuropathic pain as their primary symptom (OR = 1.73). Also 32% of seropositive patients reported widespread pain (OR = 1.63). 63% of seropositive cases presented acutely (OR = 11.0). In the seropositive group, 23% had an initial erroneous diagnosis (OR = 1.47). Eight seropositive patients improved on intravenous immunoglobulin treatment, with a 42% reduction in pain scores (P = 0.02), a 44% reduction in the Utah Neuropathy Score, and improved epidermal nerve fiber density post-treatment. CONCLUSIONS: TS-HDS and FGFR-3 antibodies may be present in a high proportion of cryptogenic SFN cases with acute onset, nonlength dependent pathology, and primary neuropathic and widespread pain. They are often misdiagnosed as other conditions including fibromyalgia. These cases may be responsive to immune treatment, especially with intravenous immunoglobulin.

Low anticoagulant heparin-iron complex targeting inhibition of hepcidin ameliorates anemia of chronic disease in rodents.

Hepcidin is the only known hormone negatively regulates systemic iron availability, its excess contributes to anemia of chronic disease (ACD).Heparin has been shown to be an efficient hepcidin inhibitor both in vitro and in vivo, but its powerful anticoagulant activity limits this therapeutic application. To this end, heparin-iron complex was prepared by electrostatic interaction and/or coordination between heparin and dihydroxy iron solution ([Fe(OH)2]+) under the condition of ultrasonic assisted. We assessed the anticoagulant activity of heparin-iron in vitro and vivo by sheep plasma, chromogenic substrate method and tail-bleeding in mice, respectively. Anti-hepcidin effect of heparin-iron was detected in HepG2 cell and LPS induced acute inflammation mice by qRT-PCR and ELISA. Turpentine-induced anemia mice were established to evaluate the effect of heparin-iron in ACD. Mice were treated with heparin-iron for 4 weeks. The results indicated that heparin-iron has significantly reduced anticoagulant activity in vitro and in vivo, strongly decreases hepcidin mRNA and IL-6 induced high level of secreted hepcidin in HepG2 cell. Heparin-iron was also found to cause a reduction on hepcidin expression through BMP/SMAD and JAK/STAT3 pathways in LPS induced acute inflammation model in mice. In ACD mice, heparin-iron could lower elevated serum hepcidin and improve anemia. These findings demonstrated low anticoagulant heparin-iron has potential applications for the treatment of ACD with high hepcidin levels.

Synthesis and Cell Growth Inhibitory Activity of Six Non-glycosaminoglycan-Type Heparin-Analogue Trisaccharides.

The design and synthesis of heparin mimetics with high anticancer activity but no anticoagulant activity is an important task in medicinal chemistry. Herein, we present the efficient synthesis of five Glc-GlcA-Glc-sequenced and one Glc-IdoA-Glc-sequenced non-glycosaminoglycan, heparin-related trisaccharides with various sulfation/sulfonylation and methylation patterns. The cell growth inhibitory effects of the compounds were tested against four cancerous human cell lines and two non-cancerous cell lines. Two d-glucuronate-containing tetra-O-sulfated, partially methylated trisaccharides displayed remarkable and selective inhibitory effects on the growth of ovary carcinoma (A2780) and melanoma (WM35) cells. Methyl substituents on the glucuronide unit proved to be detrimental, whereas acetyl substituents were beneficial to the cytostatic activity of the sulfated derivatives.

Heparin fragments induce cervical inflammation by recruiting immune cells through Toll-like receptor 4 in nonpregnant mice.

Inflammation is a hallmark in the human cervix remodelling. A possible candidate inducing the inflammatory driven ripening of the cervix is the matrix component heparan sulphate, which has been shown to be elevated in late pregnancy in the cervix and uterus. Heparin and a glycol-split low molecular weight heparin (gsHep) with low anticoagulant potency has been shown to enhance myometrial contraction and interleukin (IL)-8 production by cervical fibroblasts. The aim of this study was to investigate the mechanism by which heparin promotes cervical inflammation. Wild-type, Toll-like receptor 4 (TLR4), Myeloid differentiation primary response gene 88 (MyD88) and Interferon regulatory factor 3 (IRF3)-deficient mice were treated by deposition of gsHep into the vaginas of nonpregnant mice. To identify which cells that responded to the heparin fragments, a rhodamine fluorescent construct of gsHep was used, which initially did bind to the epithelial cells and were at later time points located in the sub-mucosa. The heparin fragments induced a strong local inflammatory response in wild-type mice shown by a rapid infiltration of neutrophils and to a lesser extent macrophages into the epithelium and the underlying extracellular matrix of the cervix. Further, a marked migration into the cervical and vaginal lumen was seen by both neutrophils and macrophages. The induced mucosal inflammation was strongly reduced in TLR4- and IRF3-deficient mice. In conclusion, our findings suggest that a TLR4/IRF3-mediated innate immune response in the cervical mucosa is induced by gsHep. This low anticoagulant heparin version, a novel TLR4 agonist, could contribute to human cervical ripening during the initiation of labour.

The Anticoagulant and Nonanticoagulant Properties of Heparin.

Heparins represent one of the most frequently used pharmacotherapeutics. Discovered around 1926, routine clinical anticoagulant use of heparin was initiated only after the publication of several seminal papers in the early 1970s by the group of Kakkar. It was shown that heparin prevents venous thromboembolism and mortality from pulmonary embolism in patients after surgery. With the subsequent development of low-molecular-weight heparins and synthetic heparin derivatives, a family of related drugs was created that continues to prove its clinical value in thromboprophylaxis and in prevention of clotting in extracorporeal devices. Fundamental and applied research has revealed a complex pharmacodynamic profile of heparins that goes beyond its anticoagulant use. Recognition of the complex multifaceted beneficial effects of heparin underscores its therapeutic potential in various clinical situations. In this review we focus on the anticoagulant and nonanticoagulant activities of heparin and, where possible, discuss the underlying molecular mechanisms that explain the diversity of heparin's biological actions.

The use of heparin and heparin-like molecules in cancer treatment: a review.

BACKGROUND: Heparin and heparin-like molecules have shown some promise in the treatment of several cancers. These molecules have roles in angiogenesis, cell proliferation, immune system modulation, cell migration, and cellular invasion. The pathways and mechanisms used by these molecules to inhibit the proliferation of cancer cells aid in understanding the utilization of these molecules in potential treatments. Our aim is to review the use of heparin and heparin-like molecules in cancer treatment, explore the results, and discuss their potential downfalls. METHODS: Publications on heparin and heparin-like molecules and compounds were collected from the PubMed and EMBASE databases. Boolean operators and MeSH terms related to heparin, heparin-like molecules, and cancer were used to conduct this search. The articles were reviewed by the authors. RESULTS: Several heparin mimetics are showing promise in cancer treatment. Various studies using mimetics alone or in combination with chemotherapy have been conducted and have yielded mixed results. They work on multiple target molecules, mostly receptors such as fibroblast growth factor and endothelial growth factor. The main types of cancers targeted by these drugs are multiple myeloma, pancreatic cancer, hepatocellular carcinoma (HCC), and other solid tumors. CONCLUSION: Although limited clinical evidence of efficacy and potential pitfalls are present, heparin and heparin-like molecules have shown potential in the management of cancer patients. Additional research is required to fully understand the biological mechanisms utilized by these molecules in cancer treatment.

Hepatitis B Virus Entry into Cells.

Hepatitis B virus (HBV), an enveloped partially double-stranded DNA virus, is a widespread human pathogen responsible for more than 250 million chronic infections worldwide. Current therapeutic strategies cannot eradicate HBV due to the persistence of the viral genome in a special DNA structure (covalently closed circular DNA, cccDNA). The identification of sodium taurocholate co-transporting polypeptide (NTCP) as an entry receptor for both HBV and its satellite virus hepatitis delta virus (HDV) has led to great advances in our understanding of the life cycle of HBV, including the early steps of infection in particular. However, the mechanisms of HBV internalization and the host factors involved in this uptake remain unclear. Improvements in our understanding of HBV entry would facilitate the design of new therapeutic approaches targeting this stage and preventing the de novo infection of naïve hepatocytes. In this review, we provide an overview of current knowledge about the process of HBV internalization into cells.

Studies on the Mechanisms of Anti-Inflammatory Activity of Heparin- and Hyaluronan-Containing Multilayer Coatings-Targeting NF-κB Signalling Pathway.

The use of implants can be hampered by chronic inflammatory reactions, which may result in failure of the implanted device. To prevent such an outcome, the present study examines the anti-inflammatory properties of surface coatings made of either hyaluronic acid (HA) or heparin (Hep) in combination with chitosan (Chi) prepared as multilayers through the layer-by-layer (LbL) technique. The properties of glycosaminoglycan (GAG)-modified surfaces were characterized in terms of surface topography, thickness and wettability. Results showed a higher thickness and hydrophilicity after multilayer formation compared to poly (ethylene imine) control samples. Moreover, multilayers containing either HA or Hep dampened the inflammatory response visible by reduced adhesion, formation of multinucleated giant cells (MNGCs) and IL-1ß release, which was studied using THP-1 derived macrophages. Furthermore, investigations regarding the mechanism of anti-inflammatory activity of GAG were focused on nuclear transcription factor-кB (NF-κB)-related signal transduction. Immunofluorescence staining of the p65 subunit of NF-κB and immunoblotting were performed that showed a significant decrease in NF-κB level in macrophages on GAG-based multilayers. Additionally, the association of FITC-labelled GAG was evaluated by confocal laser scanning microscopy and flow cytometry showing that macrophages were able to associate with and take up HA and Hep. Overall, the Hep-based multilayers demonstrated the most suppressive effect making this system most promising to control macrophage activation after implantation of medical devices. The results provide an insight on the anti-inflammatory effects of GAG not only based on their physicochemical properties, but also related to their mechanism of action toward NF-κB signal transduction.

Heparin and non-anticoagulant heparin attenuate histone-induced inflammatory responses in whole blood.

Cytotoxic and pro-inflammatory histones are present in neutrophil extracellular traps (NETs) and are elevated in blood in several inflammatory conditions, sepsis being a major example. Compounds which can attenuate activities of histones are therefore of interest, with heparin being one such material that has previously been shown to bind to histones. Heparin, a successful anticoagulant for nearly a century, has been shown experimentally to bind to histones and exhibit a protective effect in inflammatory conditions. In the present study carried out in whole blood, heparin and selectively desulfated heparin reduced histone induced inflammatory markers such as interleukin 6 (IL 6), interleukin 8 (IL 8) and tissue factor and C3a, a complement component. The selectively desulfated heparins, with reduced anticoagulant activities, retained a high degree of effectiveness as an anti-histone agent, whereas fully desulfated heparin was found to be ineffective. The results from this study indicate that the presence of sulfate and other specific structural features are required for heparin to attenuate the inflammatory action of histones in whole blood.

Roneparstat: Development, Preclinical and Clinical Studies.

A growing interest around heparanase and its role in cancer, inflammation and other diseases prompted the identification of specific inhibitors of this enzyme and the exploration of their potential therapeutic role. Roneparstat, a 15-25 kDa N-acetylated and glycol split heparin, is one of the most potent and widely studied heparanase inhibitors. These studies generated a large body of data, which allowed to characterize Roneparstat properties and to endorse its potential therapeutic role. Multiple Myeloma represents the indication that most of the studies, including the phase I clinical trial, addressed. However, Roneparstat antitumor activity activity has been documented in other cancers, and in non-oncological conditions.In addition, assessing Roneparstat activity in different experimental models contributed to understanding heparanase role and the biological factors that may be affected by heparanase inhibition in more detail. Finally, some studies elucidated the molecular mechanisms regulating the enzyme-inhibitor kinetics, thus providing important data for the identification and design of new inhibitors.The objective of this chapter is to provide a comprehensive overview of the most significant studies involving Roneparstat and discuss its potential role in therapy.

Non-Anticoagulant Heparins as Heparanase Inhibitors.

The chapter will review early and more recent seminal contributions to the discovery and characterization of heparanase and non-anticoagulant heparins inhibiting its peculiar enzymatic activity. Indeed, heparanase displays a unique versatility in degrading heparan sulfate chains of several proteoglycans expressed in all mammalian cells. This endo-ß-D-glucuronidase is overexpressed in cancer, inflammation, diabetes, atherosclerosis, nephropathies and other pathologies. Starting from known low- or non-anticoagulant heparins, the search for heparanase inhibitors evolved focusing on structure-activity relationship studies and taking advantage of new chemical-physical analytical methods which have allowed characterization and sequencing of polysaccharide chains. New methods to screen heparanase inhibitors and to evaluate their mechanism of action and in vivo activity in experimental models prompted their development. New non-anticoagulant heparin derivatives endowed with anti-heparanase activity are reported. Some leads are under clinical evaluation in the oncology field (e.g., acute myeloid leukemia, multiple myeloma, pancreatic carcinoma) and in other pathological conditions (e.g., sickle cell disease, malaria, labor arrest).

Novel pH-triggered biocompatible polymeric micelles based on heparin-α-tocopherol conjugate for intracellular delivery of docetaxel in breast cancer.

In this study, pH-triggered polymeric micelle comprising α-tocopherol (TOC) and heparin (HEP) was developed and loaded with docetaxel (DTX). The amphiphilic copolymer was synthesized by grafting TOC onto HEP backbone by a pH-cleavable bond. DTX-loaded micelles were characterized in terms of critical micelle concentration (CMC), particle size, zeta potential, entrapment efficiency (EE), pH-responsive behavior, and drug release. In vitro cytotoxicity of the micelles against breast cancer cells was investigated by MTT assay. The cellular uptake of coumarin-loaded micelles was also evaluated. Furthermore, the pharmacokinetics of DTX-loaded micelles was evaluated and compared with that of Taxotere®.HEP-CA-TOC copolymers showed low CMC values and high EE. At pH 7.4, the micelles remained stable in size and shape, whereas considerable changes in particle size and morphology were observed at pH 5.5. DTX-loaded micelles showed pH-dependent drug release profiles. Coumarin-loaded micelles showed higher cellular uptake than free coumarin. Therefore, the DTX-loaded micelles showed more toxicity against breast cancer cells than free DTX. A significant increase in T1/2 ß, AUC0-∞ and MRT was observed in DTX-loaded micelle treated group as compared to the group treated with Taxotere®.The results suggest that the pH-sensitive HEP-modified micelles could be promising for enhanced intracellular drug delivery of DTX for cancer treatment.

Role of syndecan-1 and exogenous heparin in hepatoma sphere formation.

Glycosaminoglycan-modified proteoglycans play important roles in many cell activities, including cell differentiation and stem cell development. Tumor sphere formation ability is one of properties in cancer stem cells (CSCs). The correlation between CSC markers and proteoglycan remains to be clarified. Upon hepatoma sphere formation, expression of CSC markers CD13, CD90, CD133, and CD44, as well the syndecan family protein syndecan-1 (SDC1), increased as analyzed by PCR. Further examination by suppression of CD13 expression showed downregulation of SDC1 and CD44 gene expression, whereas suppression of SDC1 gene expression downregulated CD13 and CD44 gene expression. Suppression of SDC1 gene expression also suppressed sphere development, as analyzed by a novel sphereocrit assay to quantify the level of sphere formation. The heparin disaccharide components, but not those of chondroitin disaccharide, changed with hepatoma sphere development, revealing the increased levels of N-sulfation and 2-O-sulfation. These explained the inhibition of hepatoma sphere formation by exogenous heparin. In conclusion, we found that SDC1 affected CSC marker CD13 and CD44 expression. SDC1 proteoglycan and heparin components changed and affected hepatoma sphere development. Application of heparin mimics in reduction of hepatoma stem cells might be possible.

Interaction with the heparin-derived binding inhibitors destabilizes galectin-3 protein structure.

The ß-galactoside-binding protein, galectin-3, is extensively involved in cancer development, progression and metastasis through multiple mechanisms. Inhibition of the galectin-3-mediated actions is increasingly considered as a promising therapeutic approach for cancer treatment. Our early studies have identified several novel galectin-3 binding inhibitors from chemical modification of the anticoagulant drug heparin. These heparin-derived galectin-3 binding inhibitors, which show no anticoagulant activity and bind to the galectin-3 canonical carbohydrate-binding site, induce galectin-3 conformational changes and inhibit galectin-3-mediated cancer cell adhesion, invasion and angiogenesis in vitro and reduce metastasis in mice. In this study, we determined the binding affinities of these heparin-derived ligands to galectin-3 using an isothermal titration calorimetry (ITC) ligand displacement approach. Such ITC experiments showed that the 2-de-O-sulphated, N-acetylated (compound E) and 6-de-O-sulphated, N-acetylated (F) heparin-derived ligands and their ultra-low molecular weight sub-fractions (E3 and F3) bind to galectin-3 with KD ranging from 0.96 to 1.32 mM.Differential scanning fluorimetry analysis revealed that, in contrast to the disaccharide ligand, N-acetyl-lactosamine, which binds to the fully folded form of galectin-3 and promotes galectin-3 thermal stability, the heparin-derived ligands preferentially bind to the unfolded state of galectin-3 and cause destabilization of the galectin-3 protein structure. These results provide molecular insights into the interaction of galectin-3 with the heparin-derived ligands and explain the previously demonstrated in vitro and in vivo effects of these binding inhibitors on galectin-3-mediated cancer cell behaviours.

Current Diagnosis and Treatment of Painful Small Fiber Neuropathy.

PURPOSE OF REVIEW: Small fiber neuropathy (SFN) could cause significant morbidity due to neuropathic pain and autonomic dysfunction. SFN is underdiagnosed and the knowledge on the condition is limited among general public and health care professionals. This review is intended to enhance the understanding of SFN symptoms, causes, diagnostic tools, and therapeutic options. RECENT FINDINGS: There is evidence of SFN in up to 40% patients with fibromyalgia. The causes of SFN are glucose metabolism defect, dysimmune, gluten sensitivity and celiac disease, monoclonal gammopathy, vitamin deficiencies, toxic agents, cancer, and unknown etiology. Auto-antibodies targeting neuronal antigens trisulfated heparin disaccharide (TS-HDS) and fibroblast growth factor 3 (FGFR3) are found in up to 20% of patients with SFN. Treatment of SFN includes treating the etiology and managing symptoms. SFN should be considered in patients with wide-spread body pain. The search for known causes of SFN is a crucial step in disease management.