Heparan Research Articles

Antiviral efficacy of heparan sulfate and enoxaparin sodium against SARS‐CoV‐2

AbstractAs the world transitions from the acute phase of the COVID‐19 pandemic caused by SARS‐CoV‐2, the scientific community continues to explore various therapeutic avenues to control its spread and mitigate its ongoing effects. Among the promising candidates are heparan sulfate (HS) and enoxaparin (EX), which have emerged as potential virus inhibitors. HS, a type of glycosaminoglycan, plays a prominent role in the attachment of the virus to host cells. At the same time, EX, a low‐molecular‐weight heparin, is being investigated for its ability to disrupt the interaction between the spike protein of SARS‐CoV‐2 and the ACE2 receptor in human cells. Understanding the mechanisms through which these substances operate could lay the foundation for new strategies in the ongoing management of COVID‐19. This study aimed to examine the details of SARS‐CoV‐2's entry mechanisms and the role of HS in this process. Furthermore, it examines EX's mechanism of action, highlighting how it potentially inhibits SARS‐CoV‐2. The interactions between HS and the virus, alongside in‐vitro and in‐silico inhibition studies with HS and EX, are critically analyzed to assess their antiviral efficacy. Additionally, the antiviral activity of sulfated polysaccharides and the potential therapeutic applications of these findings are discussed.

Heparan sulphate binding controls in vivo half-life of the HpARI protein family

The parasitic nematode Heligmosomoides polygyrus bakeri secretes the HpARI family, which bind to IL-33, either suppressing (HpARI1 and HpARI2) or enhancing (HpARI3) responses to the cytokine. We previously showed that HpARI2 also bound to DNA via its first complement control protein (CCP1) domain. Here, we find that HpARI1 can also bind DNA, while HpARI3 cannot. Through the production of HpARI2/HpARI3 CCP1 domain-swapped chimeras, DNA-binding ability can be transferred, and correlates with in vivo half-life of administered proteins. We found that HpARI1 and HpARI2 (but not HpARI3) also binds to the extracellular matrix component heparan sulphate (HS), and structural modelling showed a basic charged patch in the CCP1 domain of HpARI1 and HpARI2 (but not HpARI3) which could facilitate these interactions. Finally, a mutant of HpARI2 was produced which lacked DNA and HS binding, and was also shown to have a short half-life in vivo. Therefore, we propose that during infection the suppressive HpARI1 and HpARI2 proteins have long-lasting effects at the site of deposition due to DNA and/or extracellular matrix interactions, while HpARI3 has a shorter half-life due to a lack of these interactions.

The role of heparan sulfate in enhancing the chemotherapeutic response in triple-negative breast cancer

BackgroundBreast cancer, one of the most common forms of cancer, is associated with the highest cancer-related mortality among women worldwide. In comparison to other types of breast cancer, patients diagnosed with the triple-negative breast cancer (TNBC) subtype have the worst outcome because current therapies do not produce long-lasting responses. Hence, innovative therapies that produce persisting responses are a critical need. We previously discovered that hyperactivating purinergic receptors (P2RXs) by increasing extracellular adenosine triphosphate (eATP) concentrations enhances TNBC cell lines’ response to chemotherapy. Heparan sulfate inhibits multiple extracellular ATPases, so it is a molecule of interest in this regard. In turn, heparanase degrades polysulfated polysaccharide heparan sulfate. Importantly, previous work suggests that breast cancer and other cancers express heparanase at high levels. Hence, as heparan sulfate can inhibit extracellular ATPases to facilitate eATP accumulation, it may intensify responses to chemotherapy. We postulated that heparanase inhibitors would exacerbate chemotherapy-induced decreases in TNBC cell viability by increasing heparan sulfate in the cellular microenvironment and hence, augmenting eATP.MethodsWe treated TNBC cell lines MDA-MB 231, Hs 578t, and MDA-MB 468 and non-tumorigenic immortal mammary epithelial MCF-10A cells with paclitaxel (cytotoxic chemotherapeutic) with or without the heparanase inhibitor OGT 2115 and/or supplemental heparan sulfate. We evaluated cell viability and the release of eATP. Also, we compared the expression of heparanase protein in cell lines and tissues by immunoblot and immunohistochemistry, respectively. In addition, we examined breast-cancer-initiating cell populations using tumorsphere formation efficiency assays on treated cells.ResultsWe found that combining heparanase inhibitor OGT 2115 with chemotherapy decreased TNBC cell viability and tumorsphere formation through increases in eATP and activation of purinergic receptors as compared to TNBC cells treated with single-agent paclitaxel.ConclusionOur data shows that by preventing heparan sulfate breakdown, heparanase inhibitors make TNBC cells more susceptible to chemotherapy by enhancing eATP concentrations.

Extracellular vesicles from microglial cells activated by abnormal heparan sulfate oligosaccharides from Sanfilippo patients impair neuronal dendritic arborization

BackgroundIn mucopolysaccharidosis type III (MPS III, also known as Sanfilippo syndrome), a pediatric neurodegenerative disorder, accumulation of abnormal glycosaminoglycans (GAGs) induces severe neuroinflammation by triggering the microglial pro-inflammatory cytokines production via a TLR4-dependent pathway. But the extent of the microglia contribution to the MPS III neuropathology remains unclear. Extracellular vesicles (EVs) mediate intercellular communication and are known to participate in the pathogenesis of adult neurodegenerative diseases. However, characterization of the molecular profiles of EVs released by MPS III microglia and their effects on neuronal functions have not been described.MethodsHere, we isolated EVs secreted by the microglial cells after treatment with GAGs purified from urines of Sanfilippo patients (sfGAGs-EVs) or from age-matched healthy subjects (nGAGs-EVs) to explore the EVs’ proteins and small RNA profiles using LC–MS/MS and RNA sequencing. We next performed a functional assay by immunofluorescence following nGAGs- or sfGAGs-EVs uptake by WT primary cortical neurons and analyzed their extensions metrics after staining of βIII-tubulin and MAP2 by confocal microscopy.ResultsFunctional enrichment analysis for both proteomics and RNA sequencing data from sfGAGs-EVs revealed a specific content involved in neuroinflammation and neurodevelopment pathways. Treatment of cortical neurons with sfGAGs-EVs induced a disease-associated phenotype demonstrated by a lower total neurite surface area, an impaired somatodendritic compartment, and a higher number of immature dendritic spines.ConclusionsThis study shows, for the first time, that GAGs from patients with Sanfilippo syndrome can induce microglial secretion of EVs that deliver a specific molecular message to recipient naive neurons, while promoting the neuroinflammation, and depriving neurons of neurodevelopmental factors. This work provides a framework for further studies of biomarkers to evaluate efficiency of emerging therapies.Graphical

Enhancing intrinsic TGF-β signaling via heparan sulfate glycosaminoglycan regulation to promote mesenchymal stem cell capabilities and chondrogenesis for cartilage repair

Osteoarthritis burdens patients due to the limited regenerative capacity of chondrocytes. Traditional cartilage repair often falls short, necessitating innovative approaches. Mesenchymal stem cells (MSCs) show promise for regeneration. Heparan sulfate glycosaminoglycans (HS-GAGs) regulate cellular functions, making them a target for cartilage repair. This study highlights how Heparinase III (HepIII) cleaves intact HS-GAGs in bone marrow-derived MSCs (BM-MSCs), enhancing their capabilities and specifically promoting chondrogenesis. HepIII-treated BM-MSCs cultured in a hanging drop device for three days, significantly increased cell number and aggregation into a cell sphere with early chondrogenesis. HepIII promoted BM-MSCs toward chondrogenesis, increasing type II collagen, intact HS-GAGs, and sulfated GAG content, while upregulating chondrogenic and heparan sulfate proteoglycan genes. Treatment with the TGF-β inhibitor (SB-431542) in HepIII-treated BM-MSCs demonstrated enhanced intrinsic transforming growth factor-β (TGF-β) signaling and fibronectin expression. This approach also boosted BM-MSC self-renewal, immunosuppressive potential, and modified acetylated histone signatures, offering a cost-effective strategy for cartilage repair by addressing inflammation, metabolic changes, and the high costs of traditional TGF-β methods. From the results, HepIII-treated BM-MSCs show potential for use in combination with other biopolymers as injectable gels to improve cartilage repair in osteoarthritis patients in the near future.

Decellularized extracellular matrix from bovine ovarian tissue maintains the protein composition of the native matrisome

Recent approaches of regenerative reproductive medicine investigate the decellularized extracellular matrix to develop a transplantable engineered ovary (TEO). However, a full proteomic analysis is not usually performed after the decellularization process to evaluate the preservation of the extracellular matrix (ECM). In this study, the ECM of the bovine ovarian cortex was analyzed before and after decellularization using mass spectrometry and bioinformatics. A total of 155 matrisome proteins were identified in the native ECM of the bovine ovarian cortex, with 145 matrisome proteins detected in the decellularized ECM. After decellularization, only 10 matrisome proteins were lost, and notably, none belonged to the category of reproductive biological processes. Histology and histochemistry analyses were employed to assess the general morphology of both native and decellularized ECM, allowing for the identification of the most abundant ECM proteins. Moreover, our study highlighted collagen type VI alpha 3 and heparan sulfate proteoglycan 2 as the most abundant components in the bovine ovarian ECM, mirroring the composition observed in the human ovary. These findings enhance our understanding of the composition of both native and decellularized ECM, with the potential implications for the development of a TEO. SignificanceThe significance of the present study lies on the possibility of advancing towards developing a bioengineered ovary, which is the ultimate strategy to regain fertility in women. The results demonstrate that the decellularized extracellular matrix of the bovine ovary maintains the protein composition of the native matrisome, using a recently described decellularization protocol. The decellularized matrix may serve as scaffolding for seeding ovarian stromal cells and follicles to create a bioengineered ovary, and as closer its composition is to the native matrix the better. Also, comparing the bovine ovarian matrisome, which was described for the first time here, with the human ovarian matrisome, we could see a great similarity, suggesting that the bovine ovary decellularized matrix may serve as a model for developing a human bioengineered ovary.

Natural history of valve disease in patients with mucopolysaccharidosis II and the impact of enzyme replacement therapy.

Mucopolysaccharidosis II (MPS II, Hunter syndrome) is a rare, X-linked lysosomal storage disease caused by reduced activity of iduronate-2-sulfatase (I2S), with subsequent cellular accumulation of the glycosaminoglycans (GAGs), heparan sulfate, and dermatan sulfate (DS). DS is a major component of the extracellular matrix of heart valves, which can be affected in MPS II. We investigated the natural history of valve disease in MPS II and the impact of long-term intravenous enzyme replacement therapy (ERT) with recombinant I2S (idursulfase). In total, 604 cardiac examinations were assessed from serial follow-up of 80 male patients (49 neuronopathic). Valve disease was classified according to standard practice from hemodynamic features evident from echocardiography. The natural history group comprised 48 patients (up to 14.8 years of follow-up; median, 2.6 years; 24 patients started ERT during the study); 56 patients were treated (up to 14.2 years of follow-up; median, 6.2 years). Lifetime GAG burden (calculated from urinary GAG measurements) correlated significantly with the degree of valve disease. Onset of moderate-to-severe valve disease was significantly delayed in treated (median age at onset, 29.1 ± 2 [95% CI: 25.2-32.9] years; Kaplan-Meier estimation) versus untreated patients (17.6 ± 1 [95% Cl: 15.8-19.4] years; p < 0.0001). Cox regression modeling found that long-term ERT reduced the probability of developing severe valve disease (χ2, 32.736; significant after 5 years of ERT). Overall, this study found that valve disease severity in MPS II correlates with GAG burden and that progression is delayed by long-term ERT.

Identification of HSPG2 as a bladder pro-tumor protein through NID1/AKT signaling

PurposeHeparan sulfate proteoglycans (HSPGs) are complex molecules found on the cell membrane and within the extracellular matrix, increasingly recognized for their role in tumor progression. This study aimed to investigate the involvement of Heparan sulfate proteoglycan 2 (HSPG2) in the progression of bladder cancer.MethodsWe identified HSPG2 as a promoter of bladder tumor progression using single-cell RNA sequencing and transcriptome analysis of sequencing data from seven patient samples obtained from the Gene Expression Omnibus (GEO) database (GSE135337). Transcript profiles of 28 normal tissues and 407 bladder tumor tissues were analyzed for HSPG2 expression and survival outcomes using the Sanger tools and cBioPortal databases. HSPG2-overexpressing T24 and Biu-87 cell lines were generated, and cell proliferation and migration were assessed using CCK-8 and Transwell assays. Western blotting and immunostaining were performed to evaluate the activation of Nidogen-1 (NID1)/protein kinase B (AKT) signaling. Mouse models with patient-derived tumor organoids (HSPG2high and HSPG2low) were established to assess anticancer effects.ResultsOur results demonstrated a marked upregulation of HSPG2 in malignant bladder tumors, which correlated significantly with poor patient prognosis. HSPG2 overexpression consistently enhanced bladder tumor cell proliferation and conferred chemotherapy resistance, as shown in both in vitro and in vivo experiments. Mechanistically, HSPG2 upregulated NID1 expression, leading to the activation of the AKT pro-survival signaling pathway and promoting sustained tumor growth in bladder cancer.ConclusionThis study highlights the critical pro-tumor role of HSPG2/NID1/AKT signaling in bladder cancer and suggests its potential as a therapeutic target in clinical treatment.

Micro scale chromatography of human plasma proteins for nano LC-ESI-MS/MS

Organic precipitation of proteins with acetonitrile demonstrated complete protein recovery and improved chromatography of human plasma proteins. The separation of 25 μL of human plasma into 22 fractions on a QA SAX resin facilitated more effective protein discovery despite the limited sample size. Micro chromatography of plasma proteins over quaternary amine (QA) strong anion exchange (SAX) resins performed best, followed by diethylaminoethyl (DEAE), heparin (HEP), carboxymethyl cellulose (CMC), and propyl sulfate (PS) resins. Two independent statistical methods, Monte Carlo comparison with random MS/MS spectra and the rigorous X!TANDEM goodness of fit algorithm protein p-values corrected to false discovery rate q-values (q ≤ 0.01) agreed on at least 12,000 plasma proteins, each represented by at least three fully tryptic corrected peptide observations. There was qualitative agreement on 9393 protein/gene symbols between the linear quadrupole versus orbital ion trap but also quantitative agreement with a highly significant linear regression relationship between log observation frequency (F value 4,173, p-value 2.2e-16). The use of a QA resin showed nearly perfect replication of all the proteins that were also found using DEAE-, HEP-, CMC-, and PS-based chromatographic methods combined and together estimated the size of the size of the plasma proteome as ≥12,000 gene symbols.

Effect of Biopolymers and Functionalized by Them Vaterite Microparticles on Platelet Aggregation

Vaterite microparticles, metastable form of calcium carbonate, are promising forms of delivery of medicinal compounds. For more efficient delivery of target molecules (increased incorporation and retention), vaterite microparticles must be functionalized with biopolymers. In this article the effect of polysaccharides, mucin and vaterite microparticles, as well as hybrid vaterite microparticles with the above-mentioned biopolymers was studied on platelet aggregation. It was found that fucoidan, heparin and dextran sulfate (when added to platelet-rich plasma) and mucin (when added to isolated platelets) initiated cell aggregation. Pectin and chondroitin sulfate inhibited ADP- and thrombin-induced aggregation in a dose-dependent manner, mucin suppressed ADP-induced, and dextran sulfate suppressed thrombin-induced platelet aggregation. Vaterite microparticles at a concentration of 100–1000 μg/ml did not affect the aggregation of isolated platelets, but caused 10–15% cell aggregation in plasma; at the same time, at a concentration of 1000 μg/ml vaterite microparticles prevented agonist-induced cell aggregation by ~30%. It has been established that hybrid vaterite microparticles with fucoidan or heparin, when added both to platelet-rich plasma and to isolated cells, are capable to initiate platelet aggregation. Vaterite microparticles functionalized with pectin or chondroitin sulfate had no effect on spontaneous cell aggregation, and did not affect (with chondroitin sulfate) or inhibit (with pectin) agonist-induced platelet aggregation. Thus, the use of hybrid vaterite microparticles with pectin or fucoidan/heparin may be promising for the delivery of drugs aimed at modulating (inhibition with pectin or activation with fucoidan/heparin) the platelet component of hemostasis.

A Novel Peptide from VP1 of EV-D68 Exhibits Broad-Spectrum Antiviral Activity Against Human Enteroviruses

Enteroviruses have been a historical concern since the identification of polioviruses in humans. Wild polioviruses have almost been eliminated, while multiple species of non-polio enteroviruses and their variants co-circulate annually. To date, at least 116 types have been found in humans and are grouped into the species Enterovirus A–D and Rhinovirus A–C. However, there are few available antiviral drugs, especially with a universal pharmaceutical effect. Here, we demonstrate that peptide P25 from EV-D68 has broad antiviral activity against Ev A–D enteroviruses in vitro. P25, derived from the HI loop and β-I sheet of VP1, operates through a conserved hydrophilic motif -R---K-K--K- and the hydrophobic F near the N-terminus. It could prevent viral infection of EV-A71 by competing for the heparan sulfate (HS) receptor, and binding and stabilizing virions by suppressing the release of the viral genome. P25 also inhibited the generation of infectious viral particles by reducing viral protein synthesis. The molecular docking revealed that P25 might bind to the pocket opening area, a potential target for broad-spectrum antivirals. Our findings implicate the multiple antiviral effects of peptide P25, including blocking viral binding to the HS receptor, impeding viral genome release, and reducing progeny particles, which could be a novel universal anti-enterovirus drug candidate.

Comparative Bindings of Glycosaminoglycans with CXCL8 Monomer and Dimer: Insights from Conformational Dynamics and Kinetics of Hydrogen Bonds.

GAGs bind to both the monomeric and dimeric forms of CXCL8, helping to form a concentration gradient of the chemokine that facilitates the recruitment of neutrophils to an injury site and supports other biological functions. In this study, atomistic molecular dynamics simulations were conducted to investigate the binding behavior of two hexameric GAGs sulfated at two different positions, chondroitin sulfate (CS) and heparan sulfate (HS), with the monomer (SIL8) and dimer (DIL8) forms of the CXCL8 protein. The results support that the conformational diversity of CS and HS appeared to be more when binding with monomer SIL8 than dimer DIL8. CS gained more configurational entropy from glycosidic linkage flexibility when bound to SIL8 than DIL8, with a higher energy barrier, whereas HS exhibited a lower energy barrier for configurational entropy when bound to SIL8 and DIL8. The monomer SIL8 exhibited more favorable and preferential binding with GAGs compared to DIL8. Formation of hydrogen bonds with the basic amino acids of SIL8 and GAG was more rigid and required higher activation energy to break than the other identified hydrogen bondings. Water molecules involved in hydrogen bonding with GAGs, excluding those with basic amino acids of DIL8, showed longer lifetimes and slower relaxation compared to SIL8. This suggests that water-mediated interactions also favor binding of DIL8 with GAGs. Despite having more basic amino acids, DIL8 did not display stronger binding than SIL8, indicating the significant role of basic residues in stabilizing the GAG-protein interactions in the monomers. The reason could be that the greater number of nonbasic amino acids in dimeric CXCL8 stabilizes the complex by forming water-mediated hydrogen bonds, reducing the conformational preferences for binding with GAGs. In contrast, the monomeric form of CXCL8 exhibits a higher conformational preference for protein-GAG interactions.

The accomplices: Heparan sulfates and N-glycans foster SARS-CoV-2 spike:ACE2 receptor binding and virus priming

Although it is well established that the SARS-CoV-2 spike glycoprotein binds to the host cell ACE2 receptor to initiate infection, far less is known about the tissue tropism and host cell susceptibility to the virus. Differential expression across different cell types of heparan sulfate (HS) proteoglycans, with variably sulfated glycosaminoglycans (GAGs), and their synergistic interactions with host and viral N-glycans may contribute to tissue tropism and host cell susceptibility. Nevertheless, their contribution remains unclear since HS and N-glycans evade experimental characterization. We, therefore, carried out microsecond-long all-atom molecular dynamics simulations, followed by random acceleration molecular dynamics simulations, of the fully glycosylated spike:ACE2 complex with and without highly sulfated GAG chains bound. By considering the model GAGs as surrogates for the highly sulfated HS expressed in lung cells, we identified key cell entry mechanisms of spike SARS-CoV-2. We find that HS promotes structural and energetic stabilization of the active conformation of the spike receptor-binding domain (RBD) and reorientation of ACE2 toward the N-terminal domain in the same spike subunit as the RBD. Spike and ACE2 N-glycans exert synergistic effects, promoting better packing, strengthening the protein:protein interaction, and prolonging the residence time of the complex. ACE2 and HS binding trigger rearrangement of the S2' functional protease cleavage site through allosteric interdomain communication. These results thus show that HS has a multifaceted role in facilitating SARS-CoV-2 infection, and they provide a mechanistic basis for the development of GAG derivatives with anti-SARS-CoV-2 potential.

Mucopolysaccharidosis type I: founder effect of the p.P533R mutation in North Africa

BackgroundMucopolysaccharidosis type I is a lysosomal storage disease resulting from a deficiency in alpha-L-iduronidase (IDUA), which causes the accumulation of partially degraded dermatan sulfate and heparan sulfate. This retrospective study, spanning eight years, analyzed data from 45 MPSI patients. The report aimed to explore the potential origin of the p.P533R mutation in the Maghrebin population by constructing a single-nucleotide polymorphism haplotype around the IDUA gene, in order to propose a molecular proof of a founder effect of the MPSI/p.P533R allele.Patients and methodsAll of the studied patients were from Libya (2), Mauritania (1) Morocco (21) and Tunisia (21) with first cousins being the most frequent union. The diagnosis of MPSI patients often involves the combination of urinary screening, leukocyte IDUA activity determination, and DNA molecular analysis. In our study, to identify the common p.P533R mutation, we performed both DNA sequencing and tetra-primer ARMS PCR assay. Additionally, Haploview was used to determine the specific haplotype that cosegregates with the p.P533R mutation. Controls were genotyped to ensure that all the SNPs were in Hardy–Weinberg equilibrium.ResultsIn the present report we confirmed the very strong impact of consanguinity on the incidence of MPSI disease. Furthermore, studied families of mixed ancestry shared common and specific haplotype, which was observed in studied populations, suggesting the presence of a founder effect in the North Africa.ConclusionThe p.P533R missense mutation was identified in each patient originated from Libya, Mauritania, Morocco and Tunisia. Furthermore, these MPSI patients exhibited the same IDUA haplotype. The occurrence of a shared AAGGGTG haplotype, among North African populations may be attributed to substantial historical gene exchange between these groups, likely stemming from migration, inter-ethnic marriage, or other forms of interaction throughout history.

Heparin treatment is associated with a delayed diagnosis of Alzheimer’s dementia in electronic health records from two large United States health systems

AbstractRecent studies suggest that heparan sulfate proteoglycans (HSPG) contribute to the predisposition to, protection from, and potential treatment and prevention of Alzheimer’s disease (AD). Here, we used electronic health records (EHR) from two different health systems to examine whether heparin therapy was associated with a delayed diagnosis of AD dementia. Longitudinal EHR data from 15,183 patients from the Mount Sinai Health System (MSHS) and 6207 patients from Columbia University Medical Center (CUMC) were used in separate survival analyses to compare those who did or did not receive heparin therapy, had a least 5 years of observation, were at least 65 years old by their last visit, and had subsequent diagnostic code or drug treatment evidence of possible AD dementia. Analyses controlled for age, sex, comorbidities, follow-up duration and number of inpatient visits. Heparin therapy was associated with significant delays in age of clinical diagnosis of AD dementia, including +1.0 years in the MSMS cohort (P &lt; 0.001) and +1.0 years in the CUMC cohort (P &lt; 0.001). While additional studies are needed, this study supports the potential roles of heparin-like drugs and HSPGs in the protection from and prevention of AD dementia.

Interferon Causes Endothelial Injury in Humans

Therapy with exogenous interferon and human conditions that feature endogenous interferon upregulation may be associated with endothelial damage that primarily involves small blood vessels. Endothelial injury associated with interferon may display different clinical expression, including thrombotic microangiopathy, Raynaud’s phenomenon, vasculopathy of dermatomyositis and atrophic papulosis, interferon-associated skin angiopathy, systemic capillary leak syndrome, collapsing glomerulopathy, interstitial lung disease, pulmonary hypertension, and retinopathy. Interferon- induced endothelial damage involves complement-mediated injury, although pathogenic mechanisms by which interferon promote abnormal complement activation on endothelial cells are not fully understood. Human interferon-γ (type II interferon) binds to heparan sulfate on the endothelial surface, suggesting that overproduction of interferon-γ may hinder factor H attachment to the same location. Absence of factor H on self surfaces promotes activation of the alternative pathway of complement and complement-mediated endothelial damage. Type I interferon typically induces the generation of antibodies. Type I interferon upregulation may elicit the formation of autoantibodies against factor H. These autoantibodies block factor H binding to endothelial surfaces, abolishing the protective effect of factor H on complement-mediated damage. In addition, interferon induces insulin resistance which is associated with reduced heparan sulfate in the extracellular matrix, including the endothelial surface. Decreased amount of heparan sulfate suppresses factor H attachment, promoting activation of the alternative pathway of complement. Complement blockade with eculizumab (a monoclonal antibody against C5) improves endothelial damage in patients with thrombotic microangiopathy and other situations associated with interferon upregulation and interferon-induced endothelial injury, suggesting that complement-mediated injury is clinically relevant under conditions that feature interferon overproduction.

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.

Immunogenic Sulfated l-Idose Homo Oligosaccharides Elicit Neutralizing Antibody against Native Heparan Sulfate with Biomarker and Therapeutic Possibilities.

Heparan sulfate (HS) is a non-immunogenic antigen, and developing antibodies against specific sulfated patterns in HS poses significant challenges. Herein, we employed an innovative immunization strategy that exploits the molecular mimicry of HS to generate antibodies against HS sequences. Mice were immunized with synthetic sulfated oligo-l-idose (ID49) that mimics optimum 67% of the conserved structure of HS ligands. This immunization of ID49@CRM197 with alum and Freund's adjuvant resulted in the production of robust IgG antibody responses targeting ID49 and cross-reactivity with the N-sulfated HS ligands compared to N-unsubstituted and N-acetate domain synthetic HS ligands. Such a pharmacological agent exhibited distinct staining of tissue sections and cell lines and induced complement-dependent cell cytotoxicity against SK-BR-3 cancer cells. Moreover, these antibodies inhibited heparin-mediated anticoagulation activity similar to that of protamine. These findings highlight the biomarker and possible therapeutic capability of the antibodies.

Myeloid deficiency of heparan sulfate 6-O-endosulfatases impairs bone marrow hematopoiesis

The heparan sulfate (HS) 6-O-endosulfatases or the Sulfs (Sulf1 and Sulf2) are the only known enzymes that can modify HS sulfation status extracellularly and have been shown to regulate diverse biological processes. The role of the Sulfs in bone marrow (BM) hematopoiesis is not known. In this study, we generated a novel mouse line with myeloid-specific deletion of the Sulfs by crossing Sulf1/2 double floxed mice with the LysM-cre line. The LysM-Sulf knockout (KO) male mice exhibited age-dependent expansion of hematopoietic stem cells and the granulocyte-monocyte lineages in the BM, whereas common lymphoid progenitors and B lymphocyte populations were significantly reduced. Although megakaryocytic and erythroid progenitors were not reduced in the BM, the LysM-Sulf KO males suffered age-dependent reduction of red blood cells (RBCs) and platelets in the peripheral blood, suggesting that the production of RBCs and platelets was arrested at later stages. In addition, LysM-Sulf KO males displayed progressive splenomegaly with extramedullary hematopoiesis. Compared to males, LysM-Sulf KO females exhibited a much-reduced phenotype, and ovariectomy had little effect. Mechanistically, reduced TGF-β/Smad2 but enhanced p53/p21 signaling were observed in male but not female LysM-Sulf KO mice. Finally, HS disaccharide analysis via LC-MS/MS revealed increased HS 6-O-sulfation in the BM from both male and female LysM-Sulf KO mice, however, the distribution of 6-O-sulfated motifs were different between the sexes with compensatory increase in Sulf1 expression observed only in LysM-Sulf KO females. In conclusion, our study reveals that myeloid deficiency of the Sulfs leads to multilineage abnormalities in BM hematopoiesis in an age- and sex-dependent manner.