Inhibition of iduronic acid biosynthesis by ebselen reduces glycosaminoglycan accumulation in mucopolysaccharidosis type I fibroblasts.

Mucopolysaccharidosis type I (MPS-I) is a rare lysosomal storage disorder caused by deficiency of the enzyme alpha-L-iduronidase, which removes iduronic acid in both chondroitin/dermatan sulfate (CS/DS) and heparan sulfate (HS) and thereby contributes to the catabolism of glycosaminoglycans (GAGs). To ameliorate this genetic defect, the patients are currently treated by enzyme replacement and bone marrow transplantation, which have a number of drawbacks. This study was designed to develop an alternative treatment by inhibition of iduronic acid formation. By screening the Prestwick drug library, we identified ebselen as a potent inhibitor of enzymes that produce iduronic acid in CS/DS and HS. Ebselen efficiently inhibited iduronic acid formation during CS/DS synthesis in cultured fibroblasts. Treatment of MPS-I fibroblasts with ebselen not only reduced accumulation of CS/DS but also promoted GAG degradation. In early Xenopus embryos, this drug phenocopied the effect of downregulation of DS-epimerase 1, the main enzyme responsible for iduronic production in CS/DS, suggesting that ebselen inhibits iduronic acid production in vivo. However, ebselen failed to ameliorate the CS/DS and GAG burden in MPS-I mice. Nevertheless, the results propose a potential of iduronic acid substrate reduction therapy for MPS-I patients.

Role and Regulation of Mechanotransductive HIF-1α Stabilisation in Periodontal Ligament Fibroblasts.

Orthodontic tooth movement (OTM) creates compressive and tensile strain in the periodontal ligament, causing circulation disorders. Hypoxia-inducible factor 1α (HIF-1α) has been shown to be primarily stabilised by compression, but not hypoxia in periodontal ligament fibroblasts (PDLF) during mechanical strain, which are key regulators of OTM. This study aimed to elucidate the role of heparan sulfate integrin interaction and downstream kinase phosphorylation for HIF-1α stabilisation under compressive and tensile strain and to which extent downstream synthesis of VEGF and prostaglandins is HIF-1α-dependent in a model of simulated OTM in PDLF. PDLF were subjected to compressive or tensile strain for 48 h. In various setups HIF-1α was experimentally stabilised (DMOG) or destabilised (YC-1) and mechanotransduction was inhibited by surfen and genistein. We found that HIF-1α was not stabilised by tensile, but rather by compressive strain. HIF-1α stabilisation had an inductive effect on prostaglandin and VEGF synthesis. As expected, HIF-1α destabilisation reduced VEGF expression, whereas prostaglandin synthesis was increased. Inhibition of integrin mechanotransduction via surfen or genistein prevented stabilisation of HIF-1α. A decrease in VEGF expression was observed, but not in prostaglandin synthesis. Stabilisation of HIF-1α via integrin mechanotransduction and downstream phosphorylation of kinases seems to be essential for the induction of VEGF, but not prostaglandin synthesis by PDLF during compressive (but not tensile) orthodontic strain.

Surfen-mediated blockade of extratumoral chondroitin sulfate glycosaminoglycans inhibits glioblastoma invasion.

Invasive spread of glioblastoma (GBM) is linked to changes in chondroitin sulfate (CS) proteoglycan (CSPG)-associated sulfated glycosaminoglycans (GAGs) that are selectively up-regulated in the tumor microenvironment (TME). We hypothesized that inhibiting CS-GAG signaling in the TME would stem GBM invasion. Rat F98 GBM cells demonstrated enhanced preferential cell invasion into oversulfated 3-dimensional composite of CS-A and CS-E [4- and 4,6-sulfated CS-GAG (COMP)] matrices compared with monosulfated (4-sulfated) and unsulfated hyaluronic acid matrices in microfluidics-based choice assays, which is likely influenced by differential GAG receptor binding specificities. Both F98 and human patient-derived glioma stem cells (GSCs) demonstrated a high degree of colocalization of the GSC marker CD133 and CSPGs. The small molecule sulfated GAG antagonist bis-2-methyl-4-amino-quinolyl-6-carbamide (surfen) reduced invasion and focal adhesions in F98 cells encapsulated in COMP matrices and blocked CD133 and antichondroitin sulfate antibody (CS-56) detection of respective antigens in F98 cells and human GSCs. Surfen-treated F98 cells down-regulated CSPG-binding receptor transcripts and protein, as well as total and activated ERK and protein kinase B. Lastly, rats induced with frontal lobe tumors and treated with a single intratumoral dose of surfen demonstrated reduced tumor burden and spread compared with untreated controls. These results present a first demonstration of surfen as an inhibitor of sulfated GAG signaling to stem GBM invasion.-Logun, M. T., Wynens, K. E., Simchick, G., Zhao, W., Mao, L., Zhao, Q., Mukherjee, S., Brat, D. J., Karumbaiah, L. Surfen-mediated blockade of extratumoral chondroitin sulfate glycosaminoglycans inhibits glioblastoma invasion.

Widespread use of Lactobacillus OppA, a surface located protein, as an adhesin that recognises epithelial cell surface glycosaminoglycans.

Specific adherence is the first requisite that a microorganism has to fulfil to become established onto a mucosal surface. It was previously shown that the OppA surface protein of Lactobacillus salivarius Lv72 bound HeLa cell cultures through interaction with glycosaminoglycans (GAGs). To determine whether this is a peculiarity of that strain or whether it can be extended to other lactobacilli, 12 strains, belonging to six species, were confronted with HeLa-cell cultures in the presence of soluble GAGs. Interference was observed to six of them, heparan sulphate and chondroitin sulphate C being more interfering than chondroitin sulphate A or chondroitin sulphate B. Furthermore, inhibition of the biosynthesis of GAGs or their elimination from the cell surface with specific enzymes also resulted in reduced adherence. Analysis of the surface proteome of Lactobacillus crispatus Lv25 and of Lactobacillus reuteri RC14 revealed single proteins that immunoreacted with antibodies raised against OppA, the main adhesin of L. salivarius Lv72. Upon MALDI-TOF-TOF analysis, they were identified as OppA-like proteins, thus indicating that these proteins participate as adhesins in attachment of diverse lactobacilli to the surface of human epithelial cells.

The Role of Dimethyl Sulfoxide (DMSO) in Gene Expression Modulation and Glycosaminoglycan Metabolism in Lysosomal Storage Disorders on an Example of Mucopolysaccharidosis.

Obstacles to effective therapies for mucopolysaccharidoses (MPSs) determine the need for continuous studies in order to enhance therapeutic strategies. Dimethyl sulfoxide (DMSO) is frequently utilised as a solvent in biological studies, and as a vehicle for drug therapy and the in vivo administration of water-insoluble substances. In the light of the uncertainty on the mechanisms of DMSO impact on metabolism of glycosaminoglycans (GAGs) pathologically accumulated in MPSs, in this work, we made an attempt to investigate and resolve the question of the nature of GAG level modulation by DMSO, the isoflavone genistein solvent employed previously by our group in MPS treatment. In this work, we first found the cytotoxic effect of DMSO on human fibroblasts at concentrations above 3%. Also, our results displayed the potential role of DMSO in the regulation of biological processes at the transcriptional level, then demonstrated a moderate impact of the solvent on GAG synthesis. Interestingly, alterations of lysosomal ultrastructure upon DMSO treatment were visible. As there is growing evidence in the literature that DMSO can affect cellular pathways leading to numerous changes, it is important to expand our knowledge concerning this issue.

The inhibition of glycosaminoglycan incorporation influences the cell proliferation and cytodifferentiation in cultured embryonic mouse molars.

The extracellular matrix (ECM) contains a variety of complex macromolecules including proteoglycans (PGs) and glycosaminoglycans (GAGs). PG consists of a protein core with covalently attached carbohydrate side chains called GAGs. Several PGs, including versican, biglycan, decorin and syndecan are involved in odontogenesis while the role of GAGs in those PGs in this process remains unclarified. The purpose of this study was to investigate the influence of GAGs on tooth development. The mandibular first molars at early bell stage were cultivated with or without 4-methylumbelliferyl-ß-D-xyloside (Xyl-MU). The cultured tooth germs were metabolically labelled with [35S] Na2SO4, then PGs in tooth germs and cultured medium were extracted separately and analyzed by gel filtration. Morphological changes were evaluated on days 2, 4, 6, and histological changes were examined by hematoxylin-eosin (HE) staining and transmission electron microscope (TEM). Related proteins and genes of cytodifferentiation were further examined by immunohistochemistry (IHC) and quantitive real-time PCR (qPCR) respectively. Meanwhile, BrdU incorporation assay was used to explore the effect of Xyl-MU on the cell proliferation of cultured tooth germs. The results demonstrated that the incorporation of GAGs to PGs in cultured tooth germs was heavily inhibited by Xyl-MU. Accompanied by the inhibition of GAGs incorporation, Xyl-MU altered tooth morphogenesis and delayed the differentiation of ameloblasts and odontoblasts. Proliferation of inner enamel epithelium (IEE) was also inhibited. Therefore, we draw a conclusion that the inhibition of GAGs incorporation influences the cell proliferation and cytodifferentiation in cultured embryonic mouse molars.

Glycosaminoglycan Neutralization in Coagulation Control.

The glycosaminoglycans (GAGs) heparan sulfate, dermatan sulfate, and heparin are important anticoagulants that inhibit clot formation through interactions with antithrombin and heparin cofactor II. Unfractionated heparin, low-molecular-weight heparin, and heparin-derived drugs are often the main treatments used clinically to handle coagulatory disorders. A wide range of proteins have been reported to bind and neutralize these GAGs to promote clot formation. Such neutralizing proteins are involved in a variety of other physiological processes, including inflammation, transport, and signaling. It is clear that these interactions are important for the control of normal coagulation and influence the efficacy of heparin and heparin-based therapeutics. In addition to neutralization, the anticoagulant activities of GAGs may also be regulated through reduced synthesis or by degradation. In this review, we describe GAG neutralization, the proteins involved, and the molecular processes that contribute to the regulation of anticoagulant GAG activity.

Comparative Quantification Method for Glycosylated Products Elongated on ß-Xylosides Using a Stable Isotope-Labeled Saccharide Primer.

The structures and amounts of glycosaminoglycan (GAG) produced by cells have attracted much interest because GAG biosynthesis activity can change in cellular processes such as disease and differentiation. ß-Xylosides, also called saccharide primers, have been used as artificial acceptors not only to generate GAG oligosaccharides in cells and tissues but also to investigate their biosynthetic pathways. Various analytical methods have been applied to confirm the structure and amounts of GAG oligosaccharides elongated using saccharide primers, yet sample preparation processes such as solid-phase extraction in analysis can cause experimental error and disrupt accurate comparative quantification of glycosylated products. In this study, we developed a new quantification method using a deuterium-labeled saccharide primer. The "heavy" and "light" primers were chemically synthesized, and priming abilities were confirmed by liquid chromatography-tandem mass spectrometry. Relative peak areas of light/heavy products showed good linearity and were well correlated with the theoretical amounts of glycosylated products. Then, as a validation study, we carried out a biosynthesis inhibition assay using known GAG biosynthesis inhibitors. According to the relative quantification using saccharide primers, differences in the mode-of-action among the four GAG biosynthesis inhibitors were dependent on the GAG biosynthetic pathway. Our results indicate that the method will likely forge a new path for comparative glycosaminoglycomics using cultured cells and tissues.

Targeting heparin and heparan sulfate protein interactions.

Heparin and heparan sulfate glycosaminoglycans are long, linear polysaccharides that are made up of alternating dissacharide sequences of sulfated uronic acid and amino sugars. Unlike heparin, which is only found in mast cells, heparan sulfate is ubiquitously expressed on the cell surface and in the extracellular matrix of all animal cells. These negatively-charged glycans play essential roles in important cellular functions such as cell growth, adhesion, angiogenesis, and blood coagulation. These biomolecules are also involved in pathophysiological conditions such as pathogen infection and human disease. This review discusses past and current methods for targeting these complex biomolecules as a novel therapeutic strategy to treating disorders such as cancer, neurodegenerative diseases, and infection.

Nonsteroidal anti-inflammatory drugs modulate cellular glycosaminoglycan synthesis by affecting EGFR and PI3K signaling pathways.

In this report, selected non-steroidal anti-inflammatory drugs (NSAIDs), indomethacin and nimesulide, and analgesics acetaminophen, alone, as well as in combination with isoflavone genistein as potential glycosaminoglycan (GAG) metabolism modulators were considered for the treatment of mucopolysaccharidoses (MPSs) with neurological symptoms due to the effective blood-brain barrier (BBB) penetration properties of these compounds. We found that indomethacin and nimesulide, but not acetaminophen, inhibited GAG synthesis in fibroblasts significantly, while the most pronounced impairment of glycosaminoglycan production was observed after exposure to the mixture of nimesulide and genistein. Phosphorylation of the EGF receptor (EGFR) was inhibited even more effective in the presence of indomethacin and nimesulide than in the presence of genistein. When examined the activity of phosphatidylinositol-3-kinase (PI3K) production, we observed its most significant decrease in the case of fibroblast exposition to nimesulide, and afterwards to indomethacin and genistein mix, rather than indomethacin used alone. Some effects on expression of individual GAG metabolism-related and lysosomal function genes, and significant activity modulation of a number of genes involved in intracellular signal transduction pathways and metabolism of DNA and proteins were detected. This study documents that NSAIDs, and their mixtures with genistein modulate cellular glycosaminoglycan synthesis by affecting EGFR and PI3K signaling pathways.

Drugs affecting glycosaminoglycan metabolism.

Glycosaminoglycans (GAGs) are charged polysaccharides ubiquitously present at the cell surface and in the extracellular matrix. GAGs are crucial for cellular homeostasis, and their metabolism is altered during pathological processes. However, little consideration has been given to the regulation of the GAG milieu through pharmacological interventions. In this review, we provide a classification of small molecules affecting GAG metabolism based on their mechanism of action. Furthermore, we present evidence to show that clinically approved drugs affect GAG metabolism and that this could contribute to their therapeutic benefit.

Arresting progressive atherosclerosis by immunization with an anti-glycosaminoglycan monoclonal antibody in apolipoprotein E-deficient mice.

Atherogenesis is associated with the early retention of low-density lipoproteins (LDL) in the arterial intima by interaction with glycosaminoglycan (GAG)-side chains of proteoglycans. Retained LDL undergo reactive oxygen species-mediated oxidation. Oxidized LDL trigger oxidative stress (OS) and inflammation, contributing to atherosclerosis development. Recently, we reported the preventive anti-atherogenic properties of the chimeric mouse/human monoclonal antibody (mAb) chP3R99-LALA, which were related to the induction of anti-chondroitin sulfate antibody response able to inhibit chondroitin sulfate dependent LDL-enhanced oxidation. In the present work, we aimed at further investigating the impact of chP3R99-LALA mAb vaccination on progressive atherosclerosis in apolipoprotein E-deficient mice (apoE(-/-)) fed with a high-fat high-cholesterol diet receiving 5 doses (50 µg) of the antibody subcutaneously, when ~5% of the aortic area was covered by lesions. Therapeutic immunization with chP3R99-LALA mAb halted atherosclerotic lesions progression. In addition, aortic OS was modulated, as shown by a significant (p<0.05) reduction of lipid and protein oxidation, preservation of antioxidant enzymes activity and reduced glutathione, together with a decrease of nitric oxide levels. chP3R99-LALA mAb immunization also regulated aortic NF-κB activation, diminishing the proinflammatory IL1-ß and TNF-α gene expression as well as the infiltration of macrophages into the arterial wall. The therapeutic immunization of apoE(-/-) with progressive atheromas and persistent hypercholesterolemia using chP3R99-LALA mAb arrested further development of lesions, accompanied by a decrease of aortic OS and NF-κB-regulated pro-inflammatory cytokine gene expression. These results contribute to broaden the potential use of this anti-GAG antibody-based immunotherapy as a novel approach to target atherosclerosis at different phases of progression.

A common sugar-nucleotide-mediated mechanism of inhibition of (glycosamino)glycan biosynthesis, as evidenced by 6F-GalNAc (Ac3).

Glycosaminoglycan (GAG) polysaccharides have been implicated in a variety of cellular processes, and alterations in their amount and structure have been associated with diseases such as cancer. In this study, we probed 11 sugar analogs for their capacity to interfere with GAG biosynthesis. One analog, with a modification not directly involved in the glycosidic bond formation, 6F-N-acetyl-d-galactosamine (GalNAc) (Ac3), was selected for further study on its metabolic and biologic effect. Treatment of human ovarian carcinoma cells with 50 µM 6F-GalNAc (Ac3) inhibited biosynthesis of GAGs (chondroitin/dermatan sulfate by ∼50-60%, heparan sulfate by ∼35%), N-acetyl-d-glucosamine (GlcNAc)/GalNAc containing glycans recognized by the lectins Datura stramonium and peanut agglutinin (by ∼74 and ∼43%, respectively), and O-GlcNAc protein modification. With respect to function, 6F-GalNAc (Ac3) treatment inhibited growth factor signaling and reduced in vivo angiogenesis by ∼33%. Although the analog was readily transformed in cells into the uridine 5'-diphosphate (UDP)-activated form, it was not incorporated into GAGs. Rather, it strongly reduced cellular UDP-GalNAc and UDP-GlcNAc pools. Together with data from the literature, these findings indicate that nucleotide sugar depletion without incorporation is a common mechanism of sugar analogs for inhibiting GAG/glycan biosynthesis.

Modulation of expression of genes involved in glycosaminoglycan metabolism and lysosome biogenesis by flavonoids.

Flavonoids were found previously to modulate efficiency of synthesis of glycosaminoglycans (GAGs), compounds which are accumulated in cells of patients suffering from mucopolysaccharidoses (MPSs). The aim of this work was to determine effects of different flavonoids (genistein, kaempferol, daidzein) used alone or in combinations, on expression of genes coding for proteins involved in GAG metabolism. Analyses with DNA microarray, followed by real-time qRT-PCR revealed that genistein, kaempferol and combination of these two compounds induced dose- and time-dependent remarkable alterations in transcript profiles of GAG metabolism genes in cultures of wild-type human dermal fibroblasts (HDFa). Interestingly, effects of the mixture of genistein and kaempferol were stronger than those revealed by any of these compounds used alone. Similarly, the most effective reduction in levels of GAG production, in both HDFa and MPS II cells, was observed in the presence of genistein, keampferol and combination of these compounds. Forty five genes were chosen for further verification not only in HDFa, but also in MPS II fibroblasts by using real-time qRT-PCR. Despite effects on GAG metabolism-related genes, we found that genistein, kaempferol and mixture of these compounds significantly stimulated expression of TFEB. Additionally, a decrease in MTOR transcript level was observed at these conditions.

Enhanced delivery of daidzein into fibroblasts and neuronal cells with cationic derivatives of gamma-cyclodextrin for the control of cellular glycosaminoglycans.

Two cationic derivatives of γ-cyclodextrin (GCD) were synthesized by functionalization with glycidyltrimethylammonium chloride (GTMAC) and ethylenediamine (EDA). Both these derivatives (GCD-GTMAC and GCD-EDA) have been shown to interact strongly with anionic biopolymers, unfractionated heparin (UFH) and mucin, the latter showing their mucoadhesive properties. They form inclusion complexes with daidzein (DAI), an isoflavone displaying a multitude of physiological effects, much more efficiently than the unmodified GCD. It was also shown that the complexes of these GCD derivatives with DAI and Nile Red penetrate human fibroblasts and murine hippocampal neuronal cells indicating that cationic GCD derivatives can be considered as potential delivery systems for isoflavones and other poorly water soluble compounds. Moreover, it was found that DAI delivered in cationic GCD complexes decreased the level of the cellular glycosaminoglycans (GAGs) in normal fibroblasts suggesting their possible application in the control of GAGs in mucopolysaccharidoses, lysosomal storage diseases caused by pathological accumulation of GAGs in the cells.

Role of glycosaminoglycans in infectious disease.

Glycosaminoglycans (GAGs) have been shown to bind to a wide variety of microbial pathogens, including viruses, bacteria, parasites, and fungi in vitro. GAGs are thought to promote pathogenesis by facilitating pathogen attachment, invasion, or evasion of host defense mechanisms. However, the role of GAGs in infectious disease has not been extensively studied in vivo and therefore their pathophysiological significance and functions are largely unknown. Here we describe methods to directly investigate the role of GAGs in infections in vivo using mouse models of bacterial lung and corneal infection. The overall experimental strategy is to establish the importance and specificity of GAGs, define the essential structural features of GAGs, and identify a biological activity of GAGs that promotes pathogenesis.

Targeting arterial wall sulfated glycosaminoglycans in rabbit atherosclerosis with a mouse/human chimeric antibody.

The progression of atherosclerosis is favored by increasing amounts of chondroitin sulfate proteoglycans in the artery wall. We previously reported the reactivity of chP3R99 monoclonal antibody (mAb) with sulfated glycosaminoglycans and its association with the anti-atherogenic properties displayed. Now, we evaluated the accumulation of this mAb in atherosclerotic lesions and its potential use as a probe for specific in vivo detection of the disease. Atherosclerosis was induced in NZW rabbits (n = 14) by the administration of Lipofundin 20% using PBS-receiving animals as control (n = 8). Accumulation of chP3R99 mAb in atherosclerotic lesions was assessed either by immunofluorescence detection of human IgG in fresh-frozen sections of aorta, or by immunoscintigraphy followed by biodistribution of the radiotracer upon administration of (99m)Tc-chP3R99 mAb. Immunofluorescence studies revealed the presence of chP3R99 mAb in atherosclerotic lesions 24 h after intravenous administration, whereas planar images showed an evident accumulation of (99m)Tc-chP3R99 mAb in atherosclerotic rabbit carotids. Accordingly, (99m)Tc-chP3R99 mAb uptake by lesioned aortic arch and thoracic segment was increased 5.6-fold over controls and it was 3.9-folds higher in carotids, in agreement with immunoscintigrams. Moreover, the deposition of (99m)Tc-chP3R99 mAb in the artery wall was associated both with the presence and size of the lesions in the different portions of evaluated arteries and was greater than in non-targeted organs. In conclusion, chP3R99 mAb preferentially accumulates in arterial atherosclerotic lesions supporting the potential use of this anti-glycosaminoglycans antibody for diagnosis and treatment of atherosclerosis.

Sugars in the microenvironment: the sticky problem of HA turnover in tumors.

The properties and behavior of tumor cells are closely regulated by their microenvironment. Accordingly, stromal cells and extracellular matrix components can have a pronounced effect on cancer initiation, growth, and progression. The linear glycosaminoglycan hyaluronan (HA) is a major component of the extracellular matrix. Altered synthesis and degradation of HA in the tumor context has been implicated in many aspects of tumor biology. In particular, the accumulation of small HA oligosaccharides (sHA) in the tumor interstitial space may play a decisive role, due to the ability of sHA to activate a number of biological processes that are not modulated by high molecular weight (HMW)-HA. In this article, we review the normal physiological role and metabolism of HA and then survey the evidence implicating HA in tumor growth and progression, focusing in particular on the potential contribution of sHA to these processes.