Surfaces biomimétiques pour caractériser les interactions induites par les glycosaminoglycanes aux niveaux moléculaire, supramoléculaire et cellulaire

The oriented migration and controlled adhesion of cells is fundamental to many physiological and pathological processes. A family of linear polysaccharides, known as glycosaminoglycans (GAGs), help organizing and presenting signaling proteins, so-called chemokines, on the cell surface and in the extracellular matrix thus regulating cellular behavior. The objective of this PhD thesis was to develop biomimetic surfaces that are highly defined and tunable, for mechanistic studies of GAG-protein interactions on the molecular and supramolecular levels, and to probe cellular responses to defined biochemical and biophysical cues to better understand GAG-mediated cell-cell and cell-matrix communications.Applying oxime ligation, GAGs could be stably functionalized with biotin at the reducing end, and these features proved crucial for the reliable preparation of GAG-functionalized surfaces. A streptavidin monolayer served as a ‘molecular breadboard' to sequentially assemble biotinylated molecules with controlled orientation and surface densities. GAGs (heparan sulfate (HS) in particular), chemokines and other ECM components (e.g. integrin ligands promoting cell adhesion, RGD) were assembled into multifunctional surfaces that recapitulate selected aspects of the in vivo situation. Quartz crystal microbalance (QCM-D) and spectroscopic ellipsometry permitted us to characterize and control the supramolecular presentation of HS and RGD. These model surfaces were used to study the supramolecular interactions between HS and the selected chemokine stromal derived factor SDF-1α/CXCL12α and to analyze cellular responses to extracellular cues. Our data provide evidence that CXCL12α binding rigidifies HS assemblies, and that this effect is due to protein-mediated cross-linking of HS chains. The kinetics of chemokine binding to HS was quantified using surface plasmon resonance (SPR). We also demonstrate that the way in which the chemokine is presented, and in particular the presence of HS, is important for regulating myoblast behavior. Our data shows that the cell surface receptors CXCR4 (the CXCL12α receptor) and integrins (the RGD receptor) can act synergistically in controlling cellular adhesion and migration. These surfaces can generate novel insights in the field of glycobiology, e.g. in dissecting the function of GAGs in chemokine-mediated cellular migration.L'adhésion contrôlée et la migration orientée des cellules est fondamentale pour plusieurs processus physiologiques et pathologiques. Une famille de polysaccharides linéaires, connus sous le nom de glycosaminoglycanes (GAG) est impliquée dans l'organisation et la présentation des protéines de signalisation, les chimiokines, à la surface des cellules et dans la matrice extracellulaire (ECM). Les travaux concernent le développement de surfaces biomimétiques bien définies aux niveaux moléculaires et supramoléculaires pour l‘étude des mécanismes d'intéractions protéines-GAG et l'analyse de la réponse cellulaire à des signaux biochimiques et biophysiques spécifiques. L'objectif de cette étude est de mieux comprendre les communications cellule-cellule et cellule-matrice induites par les GAGs.En utilisant la ligation oxime, les GAGs peuvent être fonctionnalisés de manière stable par la biotine à leur extrémité réductrice, ce mode de couplage s'est avéré déterminant pour préparer des surfaces fonctionnalisées par les GAGs de manière stable. Une monocouche de streptavidine est utilisée comme plateforme modulable pour assembler séquentiellement les molécules biotinylées, avec une orientation et des densités de surface contrôlées. Des GAGs (les héparane sulfate (HS), en particulier), des chimiokines et d'autres composants de l'ECM (par exemple un ligand d'adhésion cellulaire, RGD) ont été assemblés reconstituant certains aspects des surfaces in vivo (cellules ou de l'ECM). La microbalance à quartz (QCM-D) et l'ellipsométrie spectroscopique nous ont permis de caractériser et de contrôler la présentation supramoléculaire du HS et du RGD. Ces surfaces modèles ont été utilisées pour étudier les interactions supramoléculaires entre le HS et la chimiokine SDF-1α/CXCL12α facteur d'origine stromale et pour analyser les réponses cellulaires aux signaux extracellulaires. Nos données apportent la preuve que la chimiokine, CXCL12α rigidifie les assemblages de HS, et que cet effet est dû à la réticulation des chaînes de HS induite par la protéine. La cinétique des interactions HS-chimiokine a été quantifiée en utilisant la résonance plasmonique de surface (SPR). Nous avons également démontré que le mode de présentation de la chimiokine sur la surface, en particulier la présence des HS, influence le comportement des myoblastes. Nos données montrent que les récepteurs cellulaires CXCR4 (récepteur de la CXCL12α) et l'intégrine (récepteur du RGD) peuvent agir en synergie pour contrôler l'adhésion et la migration cellulaire. Ces surfaces modèles fournissent des indications précieuses qui pourront être appliquées au domaine de la glycobiologie, par exemple, pour étudier le rôle des GAGs dans la migration cellulaire induite par les chimiokines

Surfaces biomimétiques pour caractériser les interactions induites par les glycosaminoglycanes aux niveaux moléculaire, supramoléculaire et cellulaire

The oriented migration and controlled adhesion of cells is fundamental to many physiological and pathological processes. A family of linear polysaccharides, known as glycosaminoglycans (GAGs), help organizing and presenting signaling proteins, so-called chemokines, on the cell surface and in the extracellular matrix thus regulating cellular behavior. The objective of this PhD thesis was to develop biomimetic surfaces that are highly defined and tunable, for mechanistic studies of GAG-protein interactions on the molecular and supramolecular levels, and to probe cellular responses to defined biochemical and biophysical cues to better understand GAG-mediated cell-cell and cell-matrix communications.Applying oxime ligation, GAGs could be stably functionalized with biotin at the reducing end, and these features proved crucial for the reliable preparation of GAG-functionalized surfaces. A streptavidin monolayer served as a ‘molecular breadboard' to sequentially assemble biotinylated molecules with controlled orientation and surface densities. GAGs (heparan sulfate (HS) in particular), chemokines and other ECM components (e.g. integrin ligands promoting cell adhesion, RGD) were assembled into multifunctional surfaces that recapitulate selected aspects of the in vivo situation. Quartz crystal microbalance (QCM-D) and spectroscopic ellipsometry permitted us to characterize and control the supramolecular presentation of HS and RGD. These model surfaces were used to study the supramolecular interactions between HS and the selected chemokine stromal derived factor SDF-1α/CXCL12α and to analyze cellular responses to extracellular cues. Our data provide evidence that CXCL12α binding rigidifies HS assemblies, and that this effect is due to protein-mediated cross-linking of HS chains. The kinetics of chemokine binding to HS was quantified using surface plasmon resonance (SPR). We also demonstrate that the way in which the chemokine is presented, and in particular the presence of HS, is important for regulating myoblast behavior. Our data shows that the cell surface receptors CXCR4 (the CXCL12α receptor) and integrins (the RGD receptor) can act synergistically in controlling cellular adhesion and migration. These surfaces can generate novel insights in the field of glycobiology, e.g. in dissecting the function of GAGs in chemokine-mediated cellular migration.L'adhésion contrôlée et la migration orientée des cellules est fondamentale pour plusieurs processus physiologiques et pathologiques. Une famille de polysaccharides linéaires, connus sous le nom de glycosaminoglycanes (GAG) est impliquée dans l'organisation et la présentation des protéines de signalisation, les chimiokines, à la surface des cellules et dans la matrice extracellulaire (ECM). Les travaux concernent le développement de surfaces biomimétiques bien définies aux niveaux moléculaires et supramoléculaires pour l‘étude des mécanismes d'intéractions protéines-GAG et l'analyse de la réponse cellulaire à des signaux biochimiques et biophysiques spécifiques. L'objectif de cette étude est de mieux comprendre les communications cellule-cellule et cellule-matrice induites par les GAGs.En utilisant la ligation oxime, les GAGs peuvent être fonctionnalisés de manière stable par la biotine à leur extrémité réductrice, ce mode de couplage s'est avéré déterminant pour préparer des surfaces fonctionnalisées par les GAGs de manière stable. Une monocouche de streptavidine est utilisée comme plateforme modulable pour assembler séquentiellement les molécules biotinylées, avec une orientation et des densités de surface contrôlées. Des GAGs (les héparane sulfate (HS), en particulier), des chimiokines et d'autres composants de l'ECM (par exemple un ligand d'adhésion cellulaire, RGD) ont été assemblés reconstituant certains aspects des surfaces in vivo (cellules ou de l'ECM). La microbalance à quartz (QCM-D) et l'ellipsométrie spectroscopique nous ont permis de caractériser et de contrôler la présentation supramoléculaire du HS et du RGD. Ces surfaces modèles ont été utilisées pour étudier les interactions supramoléculaires entre le HS et la chimiokine SDF-1α/CXCL12α facteur d'origine stromale et pour analyser les réponses cellulaires aux signaux extracellulaires. Nos données apportent la preuve que la chimiokine, CXCL12α rigidifie les assemblages de HS, et que cet effet est dû à la réticulation des chaînes de HS induite par la protéine. La cinétique des interactions HS-chimiokine a été quantifiée en utilisant la résonance plasmonique de surface (SPR). Nous avons également démontré que le mode de présentation de la chimiokine sur la surface, en particulier la présence des HS, influence le comportement des myoblastes. Nos données montrent que les récepteurs cellulaires CXCR4 (récepteur de la CXCL12α) et l'intégrine (récepteur du RGD) peuvent agir en synergie pour contrôler l'adhésion et la migration cellulaire. Ces surfaces modèles fournissent des indications précieuses qui pourront être appliquées au domaine de la glycobiologie, par exemple, pour étudier le rôle des GAGs dans la migration cellulaire induite par les chimiokines

Binding of the chemokine CXCL12α to its natural extracellular matrix ligand heparan sulfate enables myoblast adhesion and facilitates cell motility

The chemokine CXCL12α is a potent chemoattractant that guides the migration of muscle precursor cells (myoblasts) during myogenesis and muscle regeneration. To study how the molecular presentation of chemokines influences myoblast adhesion and motility, we designed multifunctional biomimetic surfaces as a tuneable signalling platform that enabled the response of myoblasts to selected extracellular cues to be studied in a well-defined environment. Using this platform, we demonstrate that CXCL12α, when presented by its natural extracellular matrix ligand heparan sulfate (HS), enables the adhesion and spreading of myoblasts and facilitates their active migration. In contrast, myoblasts also adhered and spread on CXCL12α that was quasi-irreversibly surface-bound in the absence of HS, but were essentially immotile. Moreover, co-presentation of the cyclic RGD peptide as integrin ligand along with HS-bound CXCL12α led to enhanced spreading and motility, in a way that indicates cooperation between CXCR4 (the CXCL12α receptor) and integrins (the RGD receptors). Our findings reveal the critical role of HS in CXCL12α induced myoblast adhesion and migration. The biomimetic surfaces developed here hold promise for mechanistic studies of cellular responses to different presentations of biomolecules. They may be broadly applicable for dissecting the signalling pathways underlying receptor cross-talks, and thus may guide the development of novel biomaterials that promote highly specific cellular responses

Incorporation of Pentraxin 3 into Hyaluronan Matrices Is Tightly Regulated and Promotes Matrix Cross-linking

Mammalian oocytes are surrounded by a highly hydrated hyaluronan (HA)-rich extracellular matrix with embedded cumulus cells, forming the cumulus cell·oocyte complex (COC) matrix. The correct assembly, stability, and mechanical properties of this matrix, which are crucial for successful ovulation, transport of the COC to the oviduct, and its fertilization, depend on the interaction between HA and specific HA-organizing proteins. Although the proteins inter-α-inhibitor (IαI), pentraxin 3 (PTX3), and TNF-stimulated gene-6 (TSG-6) have been identified as being critical for COC matrix formation, its supramolecular organization and the molecular mechanism of COC matrix stabilization remain unknown. Here we used films of end-grafted HA as a model system to investigate the molecular interactions involved in the formation and stabilization of HA matrices containing TSG-6, IαI, and PTX3. We found that PTX3 binds neither to HA alone nor to HA films containing TSG-6. This long pentraxin also failed to bind to products of the interaction between IαI, TSG-6, and HA, among which are the covalent heavy chain (HC)·HA and HC·TSG-6 complexes, despite the fact that both IαI and TSG-6 are ligands of PTX3. Interestingly, prior encounter with IαI was required for effective incorporation of PTX3 into TSG-6-loaded HA films. Moreover, we demonstrated that this ternary protein mixture made of IαI, PTX3, and TSG-6 is sufficient to promote formation of a stable (i.e. cross-linked) yet highly hydrated HA matrix. We propose that this mechanism is essential for correct assembly of the COC matrix and may also have general implications in other inflammatory processes that are associated with HA cross-linking

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Well-defined biomimetic surfaces to characterize glycosaminoglycan-mediated interactions on the molecular, supramolecular and cellular levels

L'adhésion contrôlée et la migration orientée des cellules est fondamentale pour plusieurs processus physiologiques et pathologiques. Une famille de polysaccharides linéaires, connus sous le nom de glycosaminoglycanes (GAG) est impliquée dans l'organisation et la présentation des protéines de signalisation, les chimiokines, à la surface des cellules et dans la matrice extracellulaire (ECM). Les travaux concernent le développement de surfaces biomimétiques bien définies aux niveaux moléculaires et supramoléculaires pour l‘étude des mécanismes d'intéractions protéines-GAG et l'analyse de la réponse cellulaire à des signaux biochimiques et biophysiques spécifiques. L'objectif de cette étude est de mieux comprendre les communications cellule-cellule et cellule-matrice induites par les GAGs.En utilisant la ligation oxime, les GAGs peuvent être fonctionnalisés de manière stable par la biotine à leur extrémité réductrice, ce mode de couplage s'est avéré déterminant pour préparer des surfaces fonctionnalisées par les GAGs de manière stable. Une monocouche de streptavidine est utilisée comme plateforme modulable pour assembler séquentiellement les molécules biotinylées, avec une orientation et des densités de surface contrôlées. Des GAGs (les héparane sulfate (HS), en particulier), des chimiokines et d'autres composants de l'ECM (par exemple un ligand d'adhésion cellulaire, RGD) ont été assemblés reconstituant certains aspects des surfaces in vivo (cellules ou de l'ECM). La microbalance à quartz (QCM-D) et l'ellipsométrie spectroscopique nous ont permis de caractériser et de contrôler la présentation supramoléculaire du HS et du RGD. Ces surfaces modèles ont été utilisées pour étudier les interactions supramoléculaires entre le HS et la chimiokine SDF-1α/CXCL12α facteur d'origine stromale et pour analyser les réponses cellulaires aux signaux extracellulaires. Nos données apportent la preuve que la chimiokine, CXCL12α rigidifie les assemblages de HS, et que cet effet est dû à la réticulation des chaînes de HS induite par la protéine. La cinétique des interactions HS-chimiokine a été quantifiée en utilisant la résonance plasmonique de surface (SPR). Nous avons également démontré que le mode de présentation de la chimiokine sur la surface, en particulier la présence des HS, influence le comportement des myoblastes. Nos données montrent que les récepteurs cellulaires CXCR4 (récepteur de la CXCL12α) et l'intégrine (récepteur du RGD) peuvent agir en synergie pour contrôler l'adhésion et la migration cellulaire. Ces surfaces modèles fournissent des indications précieuses qui pourront être appliquées au domaine de la glycobiologie, par exemple, pour étudier le rôle des GAGs dans la migration cellulaire induite par les chimiokines.The oriented migration and controlled adhesion of cells is fundamental to many physiological and pathological processes. A family of linear polysaccharides, known as glycosaminoglycans (GAGs), help organizing and presenting signaling proteins, so-called chemokines, on the cell surface and in the extracellular matrix thus regulating cellular behavior. The objective of this PhD thesis was to develop biomimetic surfaces that are highly defined and tunable, for mechanistic studies of GAG-protein interactions on the molecular and supramolecular levels, and to probe cellular responses to defined biochemical and biophysical cues to better understand GAG-mediated cell-cell and cell-matrix communications.Applying oxime ligation, GAGs could be stably functionalized with biotin at the reducing end, and these features proved crucial for the reliable preparation of GAG-functionalized surfaces. A streptavidin monolayer served as a ‘molecular breadboard' to sequentially assemble biotinylated molecules with controlled orientation and surface densities. GAGs (heparan sulfate (HS) in particular), chemokines and other ECM components (e.g. integrin ligands promoting cell adhesion, RGD) were assembled into multifunctional surfaces that recapitulate selected aspects of the in vivo situation. Quartz crystal microbalance (QCM-D) and spectroscopic ellipsometry permitted us to characterize and control the supramolecular presentation of HS and RGD. These model surfaces were used to study the supramolecular interactions between HS and the selected chemokine stromal derived factor SDF-1α/CXCL12α and to analyze cellular responses to extracellular cues. Our data provide evidence that CXCL12α binding rigidifies HS assemblies, and that this effect is due to protein-mediated cross-linking of HS chains. The kinetics of chemokine binding to HS was quantified using surface plasmon resonance (SPR). We also demonstrate that the way in which the chemokine is presented, and in particular the presence of HS, is important for regulating myoblast behavior. Our data shows that the cell surface receptors CXCR4 (the CXCL12α receptor) and integrins (the RGD receptor) can act synergistically in controlling cellular adhesion and migration. These surfaces can generate novel insights in the field of glycobiology, e.g. in dissecting the function of GAGs in chemokine-mediated cellular migration

Force-dependent breaching of the basement membrane.

Clinically, non-invasive carcinomas are confined to the epithelial side of the basement membrane and are classified as benign, whereas invasive cancers invade through the basement membrane and thereby acquire the potential to metastasize. Recent findings suggest that, in addition to protease-mediated degradation and chemotaxis-stimulated migration, basement membrane invasion by malignant cells is significantly influenced by the stiffness of the associated interstitial extracellular matrix and the contractility of the tumor cells that is dictated in part by their oncogenic genotype. In this review, we highlight recent findings that illustrate unifying molecular mechanisms whereby these physical cues contribute to tissue fibrosis and malignancy in three epithelial organs: breast, pancreas, and liver. We also discuss the clinical implications of these findings and the biological properties and clinical challenges linked to the unique biology of each of these organs

Differential structural remodelling of heparan sulfate by chemokines: the role of chemokine oligomerization.

Chemokines control the migration of cells in normal physiological processes and in the context of disease such as inflammation, autoimmunity and cancer. Two major interactions are involved: (i) binding of chemokines to chemokine receptors, which activates the cellular machinery required for movement; and (ii) binding of chemokines to glycosaminoglycans (GAGs), which facilitates the organization of chemokines into haptotactic gradients that direct cell movement. Chemokines can bind and activate their receptors as monomers; however, the ability to oligomerize is critical for the function of many chemokines in vivo Chemokine oligomerization is thought to enhance their affinity for GAGs, and here we show that it significantly affects the ability of chemokines to accumulate on and be retained by heparan sulfate (HS). We also demonstrate that several chemokines differentially rigidify and cross-link HS, thereby affecting HS rigidity and mobility, and that HS cross-linking is significantly enhanced by chemokine oligomerization. These findings suggest that chemokine-GAG interactions may play more diverse biological roles than the traditional paradigms of physical immobilization and establishment of chemokine gradients; we hypothesize that they may promote receptor-independent events such as physical re-organization of the endothelial glycocalyx and extracellular matrix, as well as signalling through proteoglycans to facilitate leukocyte adhesion and transmigration

Redox-Driven Host-Guest Interactions Allow the Controlled Release of Captured Cells on RGD-Functionalized Surfaces

International audienc

Well-defined biomimetic surfaces to characterize glycosaminoglycan-mediated interactions on the molecular, supramolecular and cellular levels.

International audienceGlycosaminoglycans (GAGs) are ubiquitously present at the cell surface and in extracellular matrix, and crucial for matrix assembly, cell-cell and cell-matrix interactions. The supramolecular presentation of GAG chains, along with other matrix components, is likely to be functionally important but remains challenging to control and to characterize, both in vivo and in vitro. We present a method to create well-defined biomimetic surfaces that display GAGs, either alone or together with other cell ligands, in a background that suppresses non-specific binding. Through the design of the immobilization platform - a streptavidin monolayer serves as a molecular breadboard for the attachment of various biotinylated ligands - and a set of surface-sensitive in situ analysis techniques (including quartz crystal microbalance and spectroscopic ellipsometry), the biomimetic surfaces are tailor made with tight control on biomolecular orientation, surface density and lateral mobility. Analysing the interactions between a selected GAG (heparan sulphate, HS) and the HS-binding chemokine CXCL12α (also called SDF-1α), we demonstrate that these surfaces are versatile for biomolecular and cellular interaction studies. T-lymphocytes are found to adhere specifically to surfaces presenting CXCL12α, both when reversibly bound through HS and when irreversibly immobilized on the inert surface, even in the absence of any bona fide cell adhesion ligand. Moreover, surfaces which present both HS-bound CXCL12α and the intercellular adhesion molecule 1 (ICAM-1) synergistically promote cell adhesion. Our surface biofunctionalization strategy should be broadly applicable for functional studies that require a well-defined supramolecular presentation of GAGs along with other matrix or cell-surface components