Glycosaminoglycan Mimetics Research Articles

Modulation of Glycosaminoglycans Affects PrPSc Metabolism but Does Not Block PrPSc Uptake.

Mammalian prions are unconventional infectious agents composed primarily of the misfolded aggregated host prion protein PrP, termed PrP(Sc). Prions propagate by the recruitment and conformational conversion of cellular prion protein into abnormal prion aggregates on the cell surface or along the endocytic pathway. Cellular glycosaminoglycans have been implicated as the first attachment sites for prions and cofactors for cellular prion replication. Glycosaminoglycan mimetics and obstruction of glycosaminoglycan sulfation affect prion replication, but the inhibitory effects on different strains and different stages of the cell infection have not been thoroughly addressed. We examined the effects of a glycosaminoglycan mimetic and undersulfation on cellular prion protein metabolism, prion uptake, and the establishment of productive infections in L929 cells by two mouse-adapted prion strains. Surprisingly, both treatments reduced endogenous sulfated glycosaminoglycans but had divergent effects on cellular PrP levels. Chemical or genetic manipulation of glycosaminoglycans did not prevent PrP(Sc) uptake, arguing against their roles as essential prion attachment sites. However, both treatments effectively antagonized de novo prion infection independently of the prion strain and reduced PrP(Sc) formation in chronically infected cells. Our results demonstrate that sulfated glycosaminoglycans are dispensable for prion internalization but play a pivotal role in persistently maintained PrP(Sc) formation independent of the prion strain. Recently, glycosaminoglycans (GAGs) became the focus of neurodegenerative disease research as general attachment sites for cell invasion by pathogenic protein aggregates. GAGs influence amyloid formation in vitro. GAGs are also found in intra- and extracellular amyloid deposits. In light of the essential role GAGs play in proteinopathies, understanding the effects of GAGs on protein aggregation and aggregate dissemination is crucial for therapeutic intervention. Here, we show that GAGs are dispensable for prion uptake but play essential roles in downstream infection processes. GAG mimetics also affect cellular GAG levels and localization and thus might affect prion propagation by depleting intracellular cofactor pools.

Allosteric inhibition of factor XIa. Sulfated non-saccharide glycosaminoglycan mimetics as promising anticoagulants

Recent development of sulfated non-saccharide glycosaminoglycan mimetics, especially sulfated pentagalloyl glucopyranoside (SPGG), as potent inhibitors of factor XIa (FXIa) (J. Med. Chem. 2013; 56:867-878 and J. Med. Chem. 2014; 57:4805-4818) has led to a strong possibility of developing a new line of factor XIa-based anticoagulants. In fact, SPGG represents the first synthetic, small molecule inhibitor that appears to bind in site remote from the active site. Considering that allosteric inhibition of FXIa is a new mechanism for developing a distinct line of anticoagulants, we have studied SPGG’s interaction with FXIa with a goal of evaluating its pre-clinical relevance. Comparative inhibition studies with several glycosaminoglycans revealed the importance of SPGG’s non-saccharide backbone. SPGG did not affect the activity of plasma kallikrein, activated protein C and factor XIIIa suggesting that SPGG-based anticoagulation is unlikely to affect other pathways connected with coagulation factors. SPGG’s effect on APTT of citrated human plasma was also not dependent on antithrombin or heparin cofactor II. Interestingly, SPGG’s anticoagulant potential was diminished by serum albumin as well as factor XI, while it could be reversed by protamine or polybrene, which implies possible avenues for developing antidote strategy. Studies with FXIa mutants indicated that SPGG engages Lys529, Arg530 and Arg532, but not Arg250, Lys252, Lys253 and Lys255. Finally, SPGG competes with unfractionated heparin, but not with polyphosphates and/or glycoprotein Ibα, for binding to FXIa. These studies enhance understanding on the first allosteric inhibitor of FXIa and highlight its value as a promising anticoagulant.

0024 : Angiogenesis potentialized by highly sulfated fucoidan: role of the chemokines and the proteoglycans

Fucoidans are natural sulfated polysaccharides used as glycosaminoglycans (GAG) mimetics. Both GAGs and chemokines are important to regulate angiogenesis and wound healing. Fucoidans interacts with pro-angiogenic chemokines, such as RANTES and SDF-1, leads to revascularization and increases endothelial cell migration. However this pro-angiogenic activity remains unclear and depends of the type of fucoidan. Innovative vascular therapies based on GAG mimetic should be developed. Upstream of developing a tissue engineering therapy, we propose to understand the beneficial effect of fucoidans on angiogenesis by a structure-function study. We hypothesize that the size and sulfation level could regulate the chemokines affinity and modulate its beneficial properties. We purified and characterized 5 fractions of fucoidans according to their sulfation rate. We tested their affinity to chemokines (Surface Plasmon Resonance), the effect on endothelial cells (HUVEC) migration (Boyden chamber) and their pro-angiogenic properties (Microvascular network formation). We also analyzed the effect of fucoidans on endogen proteoglycans and GAG expression (qRT-PCR, FACS). The structural analysis of fucoidans resulted in fractions (5-27kDa) composed of fucose, sulfate and uronic acid. The most sulfated fraction (5kDa with the ratio sulfate/fucose at 1.87) presented high affinity to biotinylated-SDF-1 and RANTES. Furthermore, this 5kDa fucoidan significantly increased HUVEC migration and microvascular network formation compared to other fractions. The 5 kDa fucoidan shows the highest pro-angiogenic effects on HUVEC. Sulfate-rich 5kDa fucoidan confirmed our hypothesis than small and highly sulfated fucoidan is attractive candidate to develop therapies based on revascularization. We now focus to develop regenerative cell therapy of ischemic heart based on the injection of progenitors cells coupled with pro-angiogenic fucoidan and chemokines.

Plasmin regulation through allosteric, sulfated, small molecules.

Plasmin, a key serine protease, plays a major role in clot lysis and extracellular matrix remodeling. Heparin, a natural polydisperse sulfated glycosaminoglycan, is known to allosterically modulate plasmin activity. No small allosteric inhibitor of plasmin has been discovered to date. We screened an in-house library of 55 sulfated, small glycosaminoglycan mimetics based on nine distinct scaffolds and varying number and positions of sulfate groups to discover several promising hits. Of these, a pentasulfated flavonoid-quinazolinone dimer 32 was found to be the most potent sulfated small inhibitor of plasmin (IC50 = 45 μM, efficacy = 100%). Michaelis-Menten kinetic studies revealed an allosteric inhibition of plasmin by these inhibitors. Studies also indicated that the most potent inhibitors are selective for plasmin over thrombin and factor Xa, two serine proteases in coagulation cascade. Interestingly, different inhibitors exhibited different levels of efficacy (40%–100%), an observation alluding to the unique advantage offered by an allosteric process. Overall, our work presents the first small, synthetic allosteric plasmin inhibitors for further rational design.

Synthetic, non-saccharide, glycosaminoglycan mimetics selectively target colon cancer stem cells.

Selective targeting of cancer stem-like cells (CSCs) is a paradigm-shifting approach. We hypothesized that CSCs can be targeted by interfering with functions of sulfated glycosaminoglycans, which play key roles in cancer cell growth, invasion and metastasis. We developed a tandem, dual screen strategy involving (1) assessing inhibition of monolayer versus spheroid growth and (2) assessing inhibition of primary versus secondary spheroid growth to identify G2.2, a unique sulfated nonsaccharide GAG mimetic (NSGM) from a focused library of 53 molecules, as a selective inhibitor of colon CSCs. The NSGM down-regulated several CSC markers through regulation of gene transcription, while closely related, inactive NSGMs G1.4 and G4.1 demonstrated no such changes. G2.2’s effects on CSCs were mediated, in part, through induction of apoptosis and inhibition of self-renewal factors. Overall, this work presents the proof-of-principle that CSCs can be selectively targeted through novel NSGMs, which are likely to advance fundamental understanding on CSCs while also aiding development of novel therapeutic agents.

395 Synthetic, Non-Saccharide Glycosaminoglycan Mimetics Selectively Target Colon Cancer Stem Cells

Selective targeting of cancer stem-like cells (CSCs) is a paradigm-shifting approach. We hypothesized that CSCs can be targeted by interfering with functions of sulfated glycosaminoglycans, which play key roles in cancer cell growth, invasion and metastasis. We developed a tandem, dual screen strategy involving (1) assessing inhibition of monolayer versus spheroid growth and (2) assessing inhibition of primary versus secondary spheroid growth to identify G2.2, a unique sulfated nonsaccharide GAG mimetic (NSGM) from a focused library of 53 molecules, as a selective inhibitor of colon CSCs. The NSGM down-regulated several CSC markers through regulation of gene transcription, while closely related, inactive NSGMs G1.4 and G4.1 demonstrated no such changes. G2.2’s effects on CSCs were mediated, in part, through induction of apoptosis and inhibition of self-renewal factors. Overall, this work presents the proof-of-principle that CSCs can be selectively targeted through novel NSGMs, which are likely to advance ...

Recent Advances on Plasmin Inhibitors for the Treatment of Fibrinolysis‐Related Disorders

Growing evidence suggests that plasmin is involved in a number of physiological processes in addition to its key role in fibrin cleavage. Plasmin inhibition is critical in preventing adverse consequences arising from plasmin overactivity, e.g., blood loss that may follow cardiac surgery. Aprotinin was widely used as an antifibrinolytic drug before its discontinuation in 2008. Tranexamic acid and ε-aminocaproic acid, two small molecule plasmin inhibitors, are currently used in the clinic. Several molecules have been designed utilizing covalent, but reversible, chemistry relying on reactive cyclohexanones, nitrile warheads, and reactive aldehyde peptidomimetics. Other major classes of plasmin inhibitors include the cyclic peptidomimetics and polypeptides of the Kunitz and Kazal-type. Allosteric inhibitors of plasmin have also been designed including small molecule lysine analogs that bind to plasmin's kringle domain(s) and sulfated glycosaminoglycan mimetics that bind to plasmin's catalytic domain. Plasmin inhibitors have also been explored for resolving other disease states including cell metastasis, cell proliferation, angiogenesis, and embryo implantation. This review highlights functional and structural aspects of plasmin inhibitors with the goal of advancing their design.

Glycosaminoglycan mimetic improves enrichment and cell functions of human endothelial progenitor cell colonies

Human circulating endothelial progenitor cells isolated from peripheral blood generate in culture cells with features of endothelial cells named late-outgrowth endothelial colony-forming cells (ECFC). In adult blood, ECFC display a constant quantitative and qualitative decline during life span. Even after expansion, it is difficult to reach the cell dose required for cell therapy of vascular diseases, thus limiting the clinical use of these cells. Glycosaminoglycans (GAG) are components from the extracellular matrix (ECM) that are able to interact and potentiate heparin binding growth factor (HBGF) activities. According to these relevant biological properties of GAG, we designed a GAG mimetic having the capacity to increase the yield of ECFC production from blood and to improve functionality of their endothelial outgrowth. We demonstrate that the addition of [OTR4131] mimetic during the isolation process of ECFC from Cord Blood induces a 3 fold increase in the number of colonies. Moreover, addition of [OTR4131] to cell culture media improves adhesion, proliferation, migration and self-renewal of ECFC. We provide evidence showing that GAG mimetics may have great interest for cell therapy applied to vascular regeneration therapy and represent an alternative to exogenous growth factor treatments to optimize potential therapeutic properties of ECFC.

Glycosaminoglycan Mimetic Associated to Human Mesenchymal Stem Cell-Based Scaffolds Inhibit Ectopic Bone Formation, but Induce Angiogenesis In Vivo

Tissue engineering approaches to stimulate bone formation currently combine bioactive scaffolds with osteocompetent human mesenchymal stem cells (hMSC). Moreover, osteogenic and angiogenic factors are required to promote differentiation and survival of hMSC through improved vascularization through the damaged extracellular matrix (ECM). Glycosaminoglycans (GAGs) are ECM compounds acting as modulators of heparin-binding protein activities during bone development and regenerative processes. GAG mimetics have been proposed as ECM stabilizers and were previously described for their positive effects on bone formation and angiogenesis after local treatment. Here, we developed a strategy associating the GAG mimetic [OTR4120] with bone substitutes to optimize stem cell-based therapeutic products. We showed that [OTR4120] was able to potentiate proliferation, migration, and osteogenic differentiation of hMSC in vitro. Its link to tricalcium phosphate/hydroxyapatite scaffolds improved their colonization by hMSC. Surprisingly, when these combinations were tested in an ectopic model of bone formation in immunodeficient mice, the GAG mimetics inhibit bone formation induced by hMSC and promoted an osteoclastic activity. Moreover, the inflammatory response was modulated, and the peri-implant vascularization stimulated. All together, these findings further support the ability of GAG mimetics to organize the local ECM to coordinate the host response toward the implanted biomaterial, and to inhibit the abnormal bone formation process on a subcutaneous ectopic site.

Human skin fibroblast response is differentially regulated by galactofucan and low molecular weight galactofucan

Abstract Wound healing is a sum of mechanisms that have to be finely modulated to reconstitute a tissue similar to the original. Several pathologies induce a decrease of healing rate and patients have to be treated to help the closure of wound. Glycosaminoglycan (GAG) mimetics have proven to be valuable in stimulating wound healing. In this study, galactofucan (fucoidan) was extracted from the brown seaweed Saccharina longicruris during two harvest periods (M05 and N05 fractions). Crude galactofucan M05 and N05 were subjected to radical depolymerization (RDP) for 4 h to obtain 2 RDP fractions (M05 RDP and N05 RDP fractions). Each fraction was added on cultured dermal fibroblasts and growth rate, collagen and MMP secretion were analyzed after treatment. Crude and RDP galactofucan influenced fibroblast growth, apoptosis and the synthesis of matrix metalloproteinase (MMP) and collagen according to the molecular weight of the polymer. Both crude galactofucan extracts reduced cell growth and increased MMP secretion, whereas only M05 stimulated apoptosis. By contrast, RDP fractions significantly increased collagen-I secretion and have no negative effect on fibroblast proliferation. Thus, crude and RDP galactofucan exhibit different metabolic activities on fibroblasts, which may be of interest in the area of wound healing.

Self‐evolving oxidative stress with identifiable pre‐ and postmitochondrial phases in PC12 cells

During the neurodegenerative process in several brain diseases, oxidative stress is known to play important roles in disease severity and evolution. Although early events of stress, such as increased lipid peroxidation and decreased superoxide dismutase, are known to characterize early onsets of these diseases, little is known about the events that participate in maintaining the chronic evolving phase influencing the disease progression in neurons. Here, we used differentiated PC12 cells to identify premitochondrial and postmitochondrial events occurring during the oxidative stress cascade leading to apoptosis. Our data indicate that an acute and strong oxidative impulse (500 μM H(2)O(2), 30 min) can induce, in this model, a 24-hr self-evolving stress, which advances from a premitochondrial phase characterized by lysosomes and cathepsin B and D translocations to cytosol and early mitochondrial membrane hyperpolarization. This phase lasts for about 5 hr and is followed by a postmitochondrial phase distinguished by mitochondrial membrane depolarization, reactive oxygen species increase, caspase-9 and caspase-3 activations, and apoptosis. Inhibition of cathepsins B and D suggests that cells can be protected at the premitochondrial phase of stress evolution and that new cathepsins regulators, such as glycosaminoglycans mimetics, can be considered as new therapeutic prototypes for neurodegeneration. Insofar as early oxidative stress markers have been related to the early onset of neurodegeneration, strategies protecting cells at the premitochondrial phase of oxidative stress may have important therapeutic applications.

Heparin-induced Leukocytosis Requires 6-O-Sulfation and Is Caused by Blockade of Selectin- and CXCL12 Protein-mediated Leukocyte Trafficking in Mice

Leukocytosis refers to an increase in leukocyte count above the normal range in the blood and is a common laboratory finding in patients. In many cases, the mechanisms underlying leukocytosis are not known. In this study, we examined the effects, the structural determinants, and the underlying mechanisms of heparin-induced leukocytosis, a side effect occurring in 0.44% of patients receiving heparin. We observed that heparin induced both lymphocytosis and neutrophilia, and the effects required heparin to be 6-O-sulfated but did not require its anticoagulant activity. Cell mobilization studies revealed that the lymphocytosis was attributable to a combination of blockage of lymphocyte homing and the release of thymocytes from the thymus, whereas the neutrophilia was caused primarily by neutrophil release from the bone marrow and demargination in the vasculature. Mechanistic studies revealed that heparin inhibits L- and P-selectin, as well as the chemokine CXCL12, leading to leukocytosis. Heparin is known to require 6-O-sulfate to inhibit L- and P-selectin function, and in this study we observed that 6-O-sulfate is required for its interaction with CXCL12. We conclude that heparin-induced leukocytosis requires glucosamine 6-O-sulfation and is caused by blockade of L-selectin-, P-selectin-, and CXCL12-mediated leukocyte trafficking.

Glycosaminoglycans mimetics potentiate the clonogenicity, proliferation, migration and differentiation properties of rat mesenchymal stem cells

Successful use of stem cell-based therapeutic products is conditioned by transplantation of optimized cells in permissive microenvironment. Mesenchymal stem cell (MSC) fates are tightly regulated by humoral factors, cellular interactions and extracellular matrix (ECM) components, such as glycosaminoglycans (GAG), which are complex polysaccharides with structural heterogeneity. During osteogenesis, a temporally controlled expression of particular GAG species is required to interact with specific growth promoting and differentiating factors to regulate their biological activities. As a comparative tool to study natural GAG, we used structurally and functionally related synthetic GAG mimetics. One of these compounds [OTR 4120] was previously shown to stimulate bone repair in rat models. Here, we demonstrate that structurally distinct GAG mimetics stimulate differentially clonogenicity, proliferation, migration and osteogenic phenotype of MSC in vitro, according to their specific chemical signature, underlying the role of sulfate and acetyl groups in specific interactions with heparin binding factors (HBF). These effects are dependent on FGF-2 interactions since they are inhibited by a FGF receptor 1 signaling pathway blocker. These data suggest that the in vivo [OTR 4120] bone regenerative effect could be due to its ability to induce MSC migration and osteogenic differentiation. To conclude, we provide evidences showing that GAG mimetics may have great interest for bone regeneration therapy and represent an alternative to exogenous growth factor treatments to optimize potential therapeutic properties of MSC.

Computational analyses of the catalytic and heparin-binding sites and their interactions with glycosaminoglycans in glycoside hydrolase family 79 endo-β-d-glucuronidase (heparanase)

Mammalian heparanase is an endo-β-glucuronidase associated with cell invasion in cancer metastasis, angiogenesis and inflammation. Heparanase cleaves heparan sulfate proteoglycans in the extracellular matrix and basement membrane, releasing heparin/heparan sulfate oligosaccharides of appreciable size. This in turn causes the release of growth factors, which accelerate tumor growth and metastasis. Heparanase has two glycosaminoglycan-binding domains; however, no three-dimensional structure information is available for human heparanase that can provide insights into how the two domains interact to degrade heparin fragments. We have constructed a new homology model of heparanase that takes into account the most recent structural and bioinformatics data available. Heparin analogs and glycosaminoglycan mimetics were computationally docked into the active site with energetically stable ring conformations and their interaction energies were compared. The resulting docked structures were used to propose a model for substrates and conformer selectivity based on the dimensions of the active site. The docking of substrates and inhibitors indicates the existence of a large binding site extending at least two saccharide units beyond the cleavage site (toward the nonreducing end) and at least three saccharides toward the reducing end (toward heparin-binding site 2). The docking of substrates suggests that heparanase recognizes the N-sulfated and O-sulfated glucosamines at subsite +1 and glucuronic acid at the cleavage site, whereas in the absence of 6-O-sulfation in glucosamine, glucuronic acid is docked at subsite +2. These findings will help us to focus on the rational design of heparanase-inhibiting molecules for anticancer drug development by targeting the two heparin/heparan sulfate recognition domains.

Heparin Binds 8 kDa Gelsolin Cross-β-Sheet Oligomers and Accelerates Amyloidogenesis by Hastening Fibril Extension

Glycosaminoglycans (GAGs) are highly sulfated linear polysaccharides prevalent in the extracellular matrix, and they associate with virtually all amyloid deposits in vivo. GAGs accelerate the aggregation of many amyloidogenic peptides in vitro, but little mechanistic evidence is available to explain why. Herein, spectroscopic methods demonstrate that GAGs do not affect the secondary structure of the monomeric 8 kDa amyloidogenic fragment of human plasma gelsolin. Moreover, monomerized 8 kDa gelsolin does not bind to heparin under physiological conditions. In contrast, 8 kDa gelsolin cross-β-sheet oligomers and amyloid fibrils bind strongly to heparin, apparently because of electrostatic interactions between the negatively charged polysaccharide and a positively charged region of the 8 kDa gelsolin assemblies. Our observations are consistent with a scaffolding mechanism whereby cross-β-sheet oligomers, upon formation, bind to GAGs, accelerating the fibril extension phase of amyloidogenesis, possibly by concentrating and orienting the oligomers to more efficiently form amyloid fibrils. Notably, heparin decreases the 8 kDa gelsolin concentration necessary for amyloid fibril formation, likely a consequence of fibril stabilization through heparin binding. Because GAG overexpression, which is common in amyloidosis, may represent a strategy for minimizing cross-β-sheet oligomer toxicity by transforming them into amyloid fibrils, the mechanism described herein for GAG-mediated acceleration of 8 kDa gelsolin amyloidogenesis provides a starting point for therapeutic strategy development. The addition of GAG mimetics, small molecule sulfonates shown to reduce the amyloid load in animal models of amyloidosis, to a heparin-accelerated 8 kDa gelsolin aggregation reaction mixture neither significantly alters the rate of amyloidogenesis nor prevents oligomers from binding to GAGs, calling into question their commonly accepted mechanism.

Molecular Dynamics Simulations of CXCL-8 and Its Interactions with a Receptor Peptide, Heparin Fragments, and Sulfated Linked Cyclitols

CXCL-8 (Interleukin 8) is a CXC chemokine with a central role in the human immune response. We have undertaken extensive in silico analyses to elucidate the interactions of CXCL-8 with its various binding partners, which are crucial for its biological function. Sequence and structure analyses showed that residues in the thirdq β-sheet and basic residues in the heparin binding site are highly variable, while residues in the second β-sheet are highly conserved. Molecular dynamics simulations in aqueous solution of dimeric CXCL-8 have been performed with starting geometries from both X-ray and NMR structures showed shearing movements between the two antiparallel C-terminal helices. Dynamic conservation analyses of these simulations agreed with experimental data indicating that structural differences between the two structures at quaternary level arise from changes in the secondary structure of the N-terminal loop, the 3(10)-helix, the 30s, 40s, and 50s loops and the third β-sheet, resulting in a different interhelical separation. Nevertheless, the observation of these different states indicates that CXCL-8 has the potential to undergo conformational changes, and it seems likely that this feature is relevant to the mode of binding of glycosaminoglycan (GAG) mimetics such as cyclitols. Simulations of the receptor peptide fragment-CXCL-8 complex identified several specific interactions of the receptor peptide with CXCL-8 that could be exploited in the structure-based design of competitive peptides and nonpeptidic molecules targeting CXCL-8 for combating inflammatory diseases. Simulations of the CXCL-8 dimer complexed with a 24-mer heparin fragment and of the CXCL-8-receptor peptide complex revealed that Arg60, Lys64, and Arg68 in the dimer bind to cyclitols in a horseshoe pattern, defining a region which is spatially distinct from the receptor binding site. There appears to be an optimum number of sulfates and an optimum length of alkyl spacers required for the interaction of cyclitol inhibitors with the dimeric form of CXCL-8. Calculation of the binding affinities of cyclitol inhibitors reflected satisfactorily the ranking of experimentally determined inhibitory potencies. The findings of these molecular modeling studies will help in the search for inhibitors which can modulate various CXCL-8 biological activities and serve as an excellent model system to study CXC-inhibitor interactions.

Glycosaminoglycan mimetics trigger IP3-dependent intracellular calcium release in myoblasts

Glycosaminoglycans (GAG) are sulfated polysaccharides that play an important role in regulating cell functions. GAG mimetics called RGTAs (for ReGeneraTing Agents) have been shown to stimulate tissue repair. In particular they accelerate myogenesis, in part via their heparin-mimetic property towards growth factors. RGTAs also increase activity of calcium-dependent intracellular protease suggesting an effect on calcium cellular homeostasis. This effect was presently investigated on myoblasts in vitro using one member of the RGTA family molecule named OTR4120. We have shown that OTR4120 or heparin induced transient increases of intracellular calcium concentration ([Ca2+]i) in pre-fusing myoblasts from both mouse SolD7 cell line and rat skeletal muscle satellite cells grown in primary culture by mobilising sarcoplasmic reticulum store. This [Ca2+]i was not mediated by ryanodine receptors but instead resulted from stimulation of the Inositol-3 phosphate-phospholipase C activation pathway. OTR4120-induced calcium transient was not mediated through an ATP, nor a tyrosine kinase, nor an acetylcholine receptor but principally through serotonin 5-HT2A receptor. This original finding shows that the GAG mimetic can induce calcium signal through serotonin receptors and the IP3 pathway may be relevant to its ability to favour myoblast differentiation. It supports a novel and unexpected function of GAGs in the regulation of calcium homeostasis.

Glycosaminoglycan mimetics inhibit SDF-1/CXCL12-mediated migration and invasion of human hepatoma cells

We have recently reported that the CXC-chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12 induces proliferation, migration, and invasion of the Huh7 human hepatoma cells through its G-protein-coupled receptor CXCR4 and that glycosaminoglycans (GAGs) are involved in these events. Here, we demonstrate by surface plasmon resonance that the chemokine binds to GAG mimetics obtained by grafting carboxylate, sulfate or acetate groups onto a dextran backbone. We also demonstrate that chemically modified dextrans inhibit SDF-1/CXCL12-mediated in vitro chemotaxis and anchorage-independent cell growth in a dose-dependent manner. The binding of GAG mimetics to the chemokine and their effects in modulating the SDF-1/CXCL12 biological activities are mainly related to the presence of sulfate groups. Furthermore, the mRNA expression of enzymes involved in heparan sulfate biosynthesis, such as exostosin-1 and -2 or N-deacetylase N-sulfotransferases remained unchanged, but heparanase mRNA and protein expressions in Huh7 cells were decreased upon GAG mimetic treatment. Moreover, decreasing heparanase-1 mRNA levels by RNA interference significantly reduced SDF-1/CXCL12-induced extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation. Therefore, we suggest that GAG mimetic effects on SDF-1/CXCL12-mediated hepatoma cell chemotaxis may rely on decreased heparanase expression, which impairs SDF-1/CXCL12's signaling. Altogether, these data suggest that GAG mimetics may compete with cellular heparan sulfate chains for the binding to SDF-1/CXCL12 and may affect heparanase expression, leading to reduced SDF-1/CXCL12 mediated in vitro chemotaxis and growth of hepatoma cells.

Glycosaminoglycan mimetics–induced mobilization of hematopoietic progenitors and stem cells into mouse peripheral blood: Structure/function insights

Glycosaminoglycans (GAG) are major components of bone marrow extracellular matrix because they have the property to interact with cells and growth factors in hematopoietic niches. In this study, we investigated the effect of two different chemically defined GAG mimetics on mobilization of hematopoietic stem and progenitor cells (HSPCs) in mice peripheral blood. Mobilization was achieved by intraperitoneal injection of GAG mimetics. Mobilized cells were characterized phenotypically by reverse transcription polymerase chain reaction and fluorescence-activated cell sorting analysis and functionally by colony-forming cell, cobblestone area-forming cell and long-term culture-initiating cell assays in vitro. Radioprotection assays were performed to confirm the functionality of primitive hematopoietic cells in vivo. Involvement of stromal-derived factor-1 (SDF-1) and matrix metalloproteinase-9 (MMP-9) were investigated. GAG mimetics treatment induces hyperleukocytosis and mobilization of HSPC. They synergize with the effects of granulocyte colony-stimulating factor or AMD3100 on hematopoietic progenitors mobilization. Reconstitution of lethally irradiated recipient mice with peripheral blood mononuclear cells from GAG mimetic-treated donor mice improves engraftment and survival. BiAcore studies indicate that the mimetics interact directly with SDF-1. In addition, GAG mimetics-induced mobilization is associated with increased levels of pro- and active MMP-9 from bone marrow cells and increased level of SDF-1 in peripheral blood. Finally, mobilization is partially inhibited by co-injection with anti-SDF-1 antibody. This study demonstrates that GAG mimetics induce efficient mobilization of HSPCs, associated with an activation of pro-MMP-9 and a modification in the SDF-1 concentration gradient between bone marrow and peripheral blood. We suggest that structural features of GAGs can modify the nature of mobilized cells.

The Structure of Glycosaminoglycans and their Interactions with Proteins

Glycosaminoglycans (GAGs) are important complex carbohydrates that participate in many biological processes through the regulation of their various protein partners. Biochemical, structural biology and molecular modelling approaches have assisted in understanding the molecular basis of such interactions, creating an opportunity to capitalize on the large structural diversity of GAGs in the discovery of new drugs. The complexity of GAG-protein interactions is in part due to the conformational flexibility and underlying sulphation patterns of GAGs, the role of metal ions and the effect of pH on the affinity of binding. Current understanding of the structure of GAGs and their interactions with proteins is here reviewed: the basic structures and functions of GAGs and their proteoglycans, their clinical significance, the three-dimensional features of GAGs, their interactions with proteins and the molecular modelling of heparin binding sites and GAG-protein interactions. This review focuses on some key aspects of GAG structure-function relationships using classical examples that illustrate the specificity of GAG-protein interactions, such as growth factors, anti-thrombin, cytokines and cell adhesion molecules. New approaches to the development of GAG mimetics as possible new glycotherapeutics are also briefly covered.