Articular cartilage plays a vital role in load-bearing joints, providing an almost frictionless surface to articulating bones. However, the avascular nature and low cell density of the tissue means that following injury, there is limited potential for regeneration and repair. With the ageing population, the prevalence and economic burden associated with osteoarthritis (OA) is increasing rapidly, but as of yet there are no fully effective ways to treat the condition. Research into novel therapies has therefore become a popular avenue of investigation, and human mesenchymal stem/stromal cells (hMSCs) have been highlighted as particularly promising targets. However, current, methods for inducing the chondrogenic differentiation of hMSCs, which typically employ the use of transforming growth factor beta 1 or 3 (TGFβ1/3), result in the production of hypertrophic rather than hyaline tissue, hampering translational progress. Growth differentiation factor 5 (GDF5) belongs to the TGFβ superfamily of proteins and is vital for skeletal formation, however its use in cartilage tissue engineering (TE) strategies has been somewhat neglected. Here we demonstrate that GDF5 significantly increases aggrecan gene expression (a marker of articular cartilage), without affecting collagen type X expression (a marker of chondrocyte hypertrophy), in chondrocyte pellet cultures derived from hMSCs, making it a promising target for the formation of permanent articular cartilage. The therapeutic application of growth factors is, at present, limited due to their expense, susceptibility to proteolytic degradation, and rapid clearance, leading to large quantities being required to get anywhere near the desired outcome. The highly sulfated glycosaminoglycan (GAG), heparin, is already extensively used in the clinic as an anticoagulant, and is also able to bind and potentiate the activity of a wide range of growth factors. As such, researchers are now using it to enhance stem cell expansion/ differentiation protocols, as well as to improve the delivery/ activity of growth factors in TE strategies. Here, we identify GDF5 as a novel heparin/heparan sulfate (HS)-binding protein, and show that endogenous HS proteoglycans (HSPGs) are vital for localizing GDF5 to the cell surface, but are not required for its signalling activity. Importantly, we report that clinically relevant doses of heparin (≥ 10 nM), but not equivalent concentrations of HS, inhibit GDF5’s biological activity, in both hMSC-derived chondrocyte pellet cultures, and in the skeletal cell line ATDC5. We demonstrate that these inhibitory effects are due to heparin (but not HS) inhibiting both GDF5 binding to endogenous HSPGs and GDF5-induced induction of Smad 1/5/8 signalling. This study may therefore explain the variable (and disappointing) results seen with heparin-loaded biomaterials for skeletal TE, and the adverse skeletal effects, such as osteoporosis, that have been reported in the clinic following long-term heparin treatment. Together, our results caution the use of heparin in the clinic and in TE applications, and prompt the transition to using more specific GAGs (e.g. HS derivatives or synthetics), with better-defined structures and fewer off-target effects, if optimal therapy is to be achieved. In the case of GDF5, we have used a variety of developed techniques to begin uncovering important structural and functional information regarding the HS-GDF5 interaction, which are hoped to ultimately pave the way towards achieving this aim. Although further analysis is necessary, our data indicate that relatively long HS sequences are required for binding, and that both ionic and non-ionic interactions play a role in the interaction. In addition we suggest that low- rather than high-affinity HS variants may be key to potentiating the activity of this growth factor.

The aim of the study was to investigate the expression NDST transcripts in a wide variety of tissues using RNA-sequencing experimental data from five published studies, using two common in silico tools: the Tophat-Cufflink pipeline and the HTSeq-DEXSeq pipeline. We show that to detect NDST alternative transcripts, paired-end sequencing should be used with replicates of samples or conditions together with 100 base read length to allow for reliable detection of the low expressed transcripts in the NDST family.  As a demonstration project, we also characterized HS synthesized by the adrenal carcinoma (ACC) cell line H295R and determined expression of NDSTs in the cells and in ACC tumor samples. We could show that roughly 65% of newly synthesized proteoglycans isolated after metabolic 35S-sulfate labeling of the cells are made up of heparan sulfate (HS) with an average chain length of 45 kDa. The HS chains show a high frequency of N-sulfation and a high total degree of sulfation. Interestingly, disaccharide analysis demonstrated a three-time higher amount of stored chondroitin sulfate (CS) compared to HS in the ACC cell line.

L'interferon-γ (IFNγ) est une cytokine immunomodulatrice puissante, également dotée d'une activité antivirale. Il possède deux ligands de haute affinité : un récepteur par lequel il transmet ses signaux et des polysaccharides complexes de la famille des héparanes sulfates (HS), tous deux situés à la surface cellulaire. In vivo, la liaison aux HS permet de concentrer localement la cytokine et de réguler son activité biologique par le biais d'une protection partielle du domaine C-terminal de la protéine. Ce domaine C-terminal, caractérisé par deux domaines basiques D1 et D2, est impliqué dans la reconnaissance du récepteur et des HS. Dans ce contexte, nos travaux se sont attachés à définir les aspects structuraux de l'interaction de l'IFNg, et plus précisément de son extrémité C-terminale, avec ses deux ligands. Pour cela, de divers mutants ponctuels, multiples et de délétion de l'IFNg ont été produites, purifiées et étudiées. Leur capacité à lier les HS et le récepteur de l'IFNg est déterminée par SPR puis leur influence sur l'activité antivirale de l'IFNg est déterminée. Les paramètres thermodynamiques de l'interaction IFNg:HS-oligosaccharides sont investigués. Par ailleurs, nous avons préparé une banque oligosaccharidique dérivée d'HS. Le criblage de cette banque, pour sa capacité à lier l'IFNγ, a permis de démontrer que l'IFNg reconnaissait des motifs de sulfatation particuliers. Finalement, nous avons tenté de cristallisé le complexe IFNg:HS-oligosaccharide, jusqu'à présent sans obtention de cristaux qui diffractent. Ces différentes approches visent à élucider le mécanisme de reconnaissance d'IFNg par des HS. Ceci afin de concevoir un mime de ce site d'interaction inhibant la signalisation de l'IFNg. Enfin, une compréhension plus détaillé de l'interaction de l'IFNg avec les HS et son récepteur reste à établir afin d'entièrement comprendre comment l'IFNg migre des HS vers l'IFNgR. / Interferon-γ (IFNγ) is a strong immunomodulating cytokine with some antiviral activity. It has two ligands for which it has high affinities: a receptor through which it transmits its signals, and complex polysaccharides of the heparan sulphate (HS) family. Both are situated on the cellular surface. In vivo, binding on the HS permits local concentration of the cytokine, and regulates its biological activity via a partial protection of the C-terminal region. This C-terminal region, characterised by two basic domains, D1 and D2, is implicated in the recognition of the receptor and the HS. In this context, we investigated the structural features for the interaction of IFNγ, and more specifically the importance of his C-terminus, with both of his cellular ligands. Therefore, we produced, purified and examined various mutants of IFNγ, including points, multiples and deletions mutants. There ability to bind to HS and the IFNγ receptor is examined by SPR and there influence on IFNγ's antiviral activity is determined. The thermodynamics complexation of IFNγ with the HS-oligosaccharides is examined. Moreover, we have prepared an oligosaccharidic library derived from HS. By screening this library for its capacity to bind IFNγ, we have demonstrated that the cytokine recognizes a particular sulphated pattern. Finely, we tried to crystallize the IFNγ:HS-oligosaccharide complex, without obtaining diffracting crystals yet. These studies contribute to clarify the mechanism of recognition of IFNγ by the HS. This would enable us to design a mimic of the interaction site for IFNγ on the HS, who inhibits inappropriate signaling of the cytokine. Finely, a detailed comprehension of the interaction of IFNγ with his receptor and with the HS needs to be established to fully understand how IFNγ migrates for the HS to its receptor.

Elastin-like peptides (ELPs) are stimulus-responsive protein-based polymers which are attractive material for biomedical research due to their biocompatibility and unique properties. The physical properties of ELPs are dependent on the chain length and the chosen amino acid at the guest residue position. This imparts unlimited flexibility in designing ELP based biomaterials with the desired physical properties. We have shown that in addition to their physical properties, ELPs have biological activities that are conducive to tissue regeneration. Specifically, we found that ELPs induce fibroblast proliferation via cell surface heparan sulfate proteoglycans (HSPG). Furthermore, our data suggests that ELP based materials with differential proliferative potential can be designed by controlling the interaction of ELPs with HSPGs by incorporating either hydrophobic or positively charged residues within the ELP sequence. Fibroblast proliferation is important for granulation tissue formation which is important in chronic wounds as well as in healing of other tissues. The customizable biological activity of ELPs coupled with their unique physical properties will enable us to design novel, sustainable and cost effective therapies for different tissue regeneration applications. ELPs can be genetically fused to biologically active peptides or proteins. These fusions can be expressed and readily purified since they maintain the phase transitioning property of the fused ELP domain. Moreover, depending on the ELP sequence chosen the chimeric fusion sequences can self-assemble into unique structures such as nanoparticles. These structures can then be applied to the injury site where they not only provide unique topographical cues or structural support but also act as delivery vehicles for the fused bioactive protein. We developed a multifunctional nanoparticle that is comprised of PMP-D2-ELP fusion protein and different functional peptide ELP fusion proteins to preserve the bioactivity of the functional group with the existence of elastase. These heterogeneous particles will be beneficial for the delivery of combination therapies to solve multiple problems that often existed in chronic wound healing or other tissue regeneration process. In summary, this study adds to our understanding of the biological activity of ELP and the interaction mechanism that allow the regulation of cellular behavior. Furthermore this work also investigated the potential therapeutic application of ELP as a delivery platform for chronic wound healing.

La production physiologique continue de cellules myélinisantes dans le système nerveux (SN) de mammifère offre de nouvelles perspectives thérapeutiques. Lors d’une atteinte de la myéline, une régénération endogène impliquant la génération d’oligodendrocytes s’engage. Ce processus repose sur la mobilisation de progéniteurs oligodendrocytaires parenchymateux et de progéniteurs de la zone sous-ventriculaire (SVZ). Cette réparation ne permet cependant pas une récupération fonctionnelle systématique. Nos travaux ont pour but d’identifier les facteurs qui contrôlent les différentes étapes de régénération. Ils révèlent une réexpression du CNTF et une surexpression des héparans sulfates protéoglycanes (HSPGs) suite à une démyélinisation du corps calleux. Ces changements de l’environnement péri-lésionnel régulent positivement le processus de remyélinisation. Nous avons montré un impact direct de l’expression post-lésionnelle du CNTF sur la mobilisation des deux sources cellulaires. Différents tests in vitro ont identifié le CNTF comme facteur chémoattractant pour ces cellules. Nos données montrent également que des modifications de sulfatation des héparans sulfates (HS) protéoglycanes contrôlées par la N-désacétylase-Sulfotransférase 1 des cellules du lignage oligodendrocytaire s’établissent en bordure de lésion et créent un microenvironnement favorable à la régénération. Divers test fonctionnels in vivo et in vitro révèlent le rôle clef des HSPGs dans la cinétique de démyélinisation et de remyélinisation en régulant la mobilisation des cellules du lignage oligodendrocytaire et l’activation microgliale. / In the mammal’s nervous system, the ongoing production of new myelinating cells on has open news therapeutic perspectives for demyelinating diseases. An endogenous regeneration process involving the generation of oligodendrocytes can occur following demyelination. This process relies on the mobilization of an endogenous reservoir of progenitor cells located in the adult brain: The parenchymal oligodendrocyte precursors and the subventricular zone derived neural progenitors. However, these endogenous repair attempts do not permit an efficient functional recovery. These failures are mainly due to mobilization, differentiation or to the generation of a hostile environment for the repair process. Our work is focusing on the identification of factors regulating those events. Our data show the reexpression of CNTF and overexpression of heparan sulphate proteoglycans (HSPGs) following a focal demyelination of the corpus callosum in adult mice. These environmental changes favor myelin repair. We show a direct impact of the post-lesional expression of CNTF on the mobilization of both cellular sources. Using various in vitro assays, we showed that CNTF is acting on the two cellular sources as a chemoattractant factor. Our data also show that sulfation modifications of HSPGs performed by the deacetylase-N-sulfotransferase 1 (Ndst1) on oligodendrocyte lineage cells occurred around the lesion and created a permissive microenvironment for the regenerative process. Various in vitro and in vivo functional assays demonstrated the key role of HSPGs in demyelination and remyelination dynamic by controlling mobilization of the oligodendrocyte lineage cells and microglial activation.

Les GAGs, en particulier les HS et les CS, sont des polysaccharides linéaires sulfatés situés à la surface des cellules et la matrice extracellulaire où ils influencent les fonctions des cellules. Les GAGs sont connus pour se lier et réguler l'activité d'un certain nombre de protéines différentes appelées «protéines de liaison héparine», y compris les chimiokines, facteurs de croissance, des enzymes et des molécules d'adhésion. Dans le cas du développement de la tumeur, la surexpression de l'héparanase a été observée. En conséquence, une variété d'oligosaccharides de HS et de CS est libérée. Néanmoins, leurs structures et leurs effets biologiques sont inconnus.De nombreux outils existent pour la caractérisation des GAG cependant, le développement de nouvelles technologies pour isoler des fragments du HS endogènes est nécessaire. Dans ce contexte, nous proposons d'utiliser la technologie d'empreinte moléculaire, ce qui permettrait d'obtenir des polymères avec des cavités capables de reconnaître certains types d'oligosaccharides mimétiques de HS, et par la suite d'étudier les HS endogènes.Les GAGs extraits de tumeurs xénogreffes et du sang, de 3 à 8 semaines au cours de la croissance tumorale, ont été quantifiés par dosage colorimétrique. Nous avons observé une diminution de la quantité des GAG tumoraux et une augmentation des GAG sanguin, au cours de la croissance de la tumeur. En outre, les GAGs tumoraux montrent une affinité croissante pour le FGF-2 au cours de la croissance tumorale.Nous avons étudié l'applicabilité de la «technique d'empreinte moléculaire» pour la production d'hydrogels imprimés capables de reconnaître spécifiquement le fondaparinux, un oligosaccharide analogue de l'héparine. Nous avons préparé une bibliothèque d'hydrogels imprimés afin d'optimiser leur synthèse et obtenir des matériaux qui reconnaissent spécifique et sélectivement cette molécule cible. Nos résultats montrent que, par un contrôle minutieux de la stœchiométrie et de la proportion de l'agent de réticulation utilisé lors de leur synthèse ainsi que la détermination des conditions de reconnaissance, les hydrogels imprimés reconnaissent spécifiquement les oligosaccharides mimétiques de HS.Ces travaux ouvrent des intéressantes perspectives d'application de la technologie d'impression moléculaire à l'analyse des séquences de GAGs extraits d'un milieu biologique. / GAGs, and particularly heparan sulfate (HS) and chondoitin sulfate (CS), are linear and sulfated polysaccharides located at the cell surface and extracellular matrix from where they influence the functions of cells. GAGs are known to bind and regulate the activity of a number of distinct proteins known as ‘heparin binding proteins' including chemokines, growth factors, enzymes and adhesion molecules. In the case of tumor development, heparanase over-expression has been observed. As a consequence, a variety of HS and CS oligosaccharides are released which structures and biological effects are unknown.Many tools exist for GAG characterization and a need to develop a new technology to isolate fragments of endogenous HS is required. In this context, we propose to use molecular imprinting technology that could allow to obtain polymers owing cavities able to recognize specific types of HS mimetic oligosaccharides and therefore the endogenous HS.GAGs extracted from xenografted tumors and blood, at 3 to 8 weeks during tumor growth, were quantified by a colorimetric assay. We observed a decrease in the amount of GAGs tumors and an increase of GAGs blood, during the tumor growth. Moreover, tumor GAGs were tested by competition toward growth factor with enzyme immunoassay showing increasing affinity for FGF-2 during tumor growth.We investigated the applicability of ‘Molecular Imprinting Technology' to the generation of imprinted hydrogels able of specifically recognize fondaparinux, an oligosaccharide analogue of heparin. We have prepared a library of imprinted hydrogels in order to optimize their synthesis and obtain materials that specifically and selectively recognize that oligosaccharide. Our results show that, by a careful control of the stoichiometry and crossliking choice for their synthesis and by adapting rebinding conditions, namely the temperature, imprinted hydrogels can readily be prepared to specifically recognize the HS mimetic used as model.This work opens an interesting outlook to analyze GAGs extracted from a biological medium by molecular imprinting technology

Heparin glycosaminoglycans mediate a range of biological events, including anticoagulation as well as a diversity of cell proliferation and differentiation processes. Heparin saccharides have been shown to act as inhibitors against angiogenesis and metastasis of tumour cells. This thesis describes work developing chemistry towards varying length oligosaccharide sequences with potential to offer variable sulfation patterns. The main synthetic components to this work were contribution to developing scalable syntheses of an orthogonally protected L-Iduronic acid unit and a differentially protected D-glucosamine unit. The synthetic work also evaluated a recently reported diazo transfer reagent, which allowed for earlier placement of azide protection over that of previously developed routes within the group. This provided a cheaper, more atom efficient route towards protected D-glucosamine building blocks. Glycosylation of the developed D-GlcN donor units with the L-Ido acceptor allowed the production of key disaccharides which facilitated an efficient iterative glycosylation strategy towards longer oligosaccharides, ultimately providing a differentially protected pentasaccharide. The project evaluated methods towards generating various dimeric heparin type systems through forming new O4 ether linkages between GlcN residues across various short linker fragments. The most successful of these dimerisations used a methallyl dichloride core which allowed for further derivatisation towards dihydroxylated species, the analysis of which highlighted some interesting proton NMR data. The final aspect of this project began development of chemistry towards non-reducing end-labelled oligosaccharide sequences by implementation of a masked aldehyde unit on the C4 hydroxyl of GlcN synthesised from the allylated GlcN precursor via dihydroxylation chemistry. Incorporation of this moiety (protected as a 1,2-dibenzyl glycol) within both a trisaccharide and a pentasaccharide was achieved. Further development of this chemistry should allow for late step oxidative cleavage to reveal the reactive aldehyde, potentially allowing for attachment of various amine functionalised fluorophores via reductive amination. Radiolabelling of such a species should also be possible through sodium borotritide reduction for example.

L’Héparane sulfate (HS) est un polysaccharide linéaire et sulfaté présent à la surface des cellules ou dans le milieu extracellulaire des tissus animaux. Le long des chaines d'HS, des régions présentant une densité de charge négative élevée (domaines S) alternent avec des régions plus faiblement chargées (domaines A). Différents motifs SAS sont ainsi exposés à la surface des cellules et permettent des interactions spécifiques avec de nombreuses protéines comme l’interféron gamma (INF-γ). Cette cytokine interagit avec haute affinité avec les chaines d'HS, ce qui module son activité in vivo (accumulation et localisation tissulaire, clairance sanguine). Pour moduler l’activité de l’INF-γ en inhibant ses interactions avec les chaines d'HS de la surface des cellules, nous avons entrepris la synthèse de mimes de motifs SAS, dans lesquels des fragments synthétiques de domaines S sont liés par un espaceur de longueur modulable. Pour effectuer cette conjugation, nous avons choisi d'utiliser deux types de chimie click la "CuAAC" et la "ligation oxime". Cette stratégie a nécessité de mettre au point des fonctionnalisations orthogonales des extrémités réductrices et non réductrices d'oligosaccharides synthétiques. Nous avons mis au point les réactions sur un disaccharide modèle dérivé du cellobiose, puis les avons transférées à la modification d'un tetrasaccharide synthétique d'HS. Dans ce travail, nous avons optimisé deux réactions clef : une alkylation anomérique dans l’eau et une allylation de fonction alcool dans des conditions neutres / Heparan sulfate (HS) is a linear polysaccharide found in animal tissues at the cell surface or in the extracellular matrix. HS chains display alternating highly negatively charged regions (S) and less charged ones (A). SAS domains with different topologies can thus be exposed at the cell surface with the aim of interacting specifically with different proteins. Gamma interferon (INF-γ) is a cytokine that binds tightly to HS chains. This interaction allows controlling numerous bioactivities of the cytokine (accumulation and location in tissues as well as blood clearance). The discovery of HS fragment able to modulate the activity of IFN-γ could open the way to new innovative therapeutics. To this aim we launched a program aiming at synthesizing mimetic of the SAS motifs found in HS. We devised a strategy allowing linking two synthetic S fragments of HS through a spacer. To this aim we selected two click chemistry reactions: the "CuAAC" triazole formation and "oxime ligation". To implement this strategy, we optimized, on a disaccharide model derived from cellobiose, a methodology allowing the functionalization of the reducing and non-reducing end of synthetic oligosaccharides by to orthogonal reactive functions. Then we extended the methodology to a HS tetrasaccharide fragment. In this work, we optimized two key reactions: an anomeric alkylation in water and a hydroxyl allylation in neutral condition

京都大学 / 0048 / 新制・論文博士 / 博士(医科学) / 乙第12872号 / 論医科博第1号 / 新制||医科||4(附属図書館) / 31590 / (主査)教授 野田 亮, 教授 楠見 明弘, 教授 瀬原 淳子 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

L'Héparane Sulfate (HS), un polysaccharide linéaire, module les activités biologiques de nombreuses protéines. Afin d'élucider les interactions entre l'HS et les protéines, la synthèse chimique d'HS est un outil précieux, mais elle peut être difficile. Notre équipe a montré que des mélanges combinatoires obtenus par auto-assemblage de différentes combinaisons de dérivés disaccharidiques (lactose et lactose persulfaté) sur surfaces planes d'or peuvent reconnaître spécifiquement certaines protéines se liant à l'HS, telles que les isoformes de la chimiokine CXCL12 ou IFNγ. Avec ces dérivés, nous avons réalisé un auto-assemblage sur des nanoparticules d'or. Mais à cause de la toxicité des nanoparticules d'or, nous avons aussi adapté cette méthode à des nanoparticules lipidiques. En utilisant les conditions qui ont déjà été améliorées pendant la synthèse des dérivés lactose et lactose persulfaté, nous avons préparé deux autres dérivés disaccharidiques plus proches de la structure réelle d'HS. Ces nouveaux dérivés sont utilisés pour réaliser des nanoparticules d'or et nanoparticules lipidiques afin de comparer les propriétés avec les lactose et lactose persulfaté. Les tests d'affinité avec différentes protéines sont en cours de réalisation. / Héparan Sulfate (HS) is a linear polysaccharide that modulates the biological activities of numerous proteins. In order to elucidate the interaction between HS and proteins, the synthesis of HS is an invaluable tool, but the synthesis is sometimes difficult. Our group has demonstrated that the combinatorial mixtures obtained by self-assembly of different combinations of disaccharide derivatives (lactose and persulfated lactose) on gold plan surfaces could recognize specifically some HS binding proteins, such as the isoforms of the chemokine CXCL12 or IFNγ. Because of the toxicity of gold nanoparticles, we have also adapted this method to lipid nanoparticles. Using the conditions that have already improved during the synthesis of lactose and persulfated lactose derivatives, we have synthesized two other disaccharide derivatives, which were closer to the real structure of HS. These new derivatives were used to prepare the gold and lipid nanoparticles at the aim of comparing the properties with lactose and persulfated lactose. The tests of affinities with different proteins are in progress.