L'ostéoprotégérine, nouvel acteur dans l'angiogenèse (Rôle dans la formation de nouveaux vaisseaux et mécanisme d'action)

L Osteoprotégérine est une cytokine soluble qui joue un rôle clé dans le métabolisme osseux et est impliquée dans la réponse immunitaire et l hématopoïèse. Elle est associée à la dysfonction endothéliale et semble intervenir dans l angiogenèse. Cette cytokine constituerait en fait, un trait d union entre le tissu osseux et vasculaire. Son rôle dans la formation de la matrice osseuse est aujourd hui bien élucidé mais son implication dans la vascularisation reste à établir. L OPG est rapidement libérée par l endothélium dans des conditions inflammatoires et est donc en mesure d intervenir dans le processus de revascularisation initié par les cellules progénitrices endothéliales (PECs). Au cours de cette étude, nous avons tenté de comprendre le rôle joué par cette cytokines dans la néovascularisation induite in vitro, par une sous population de PECs appelées ECFCs (endothelial colony-forming cells), et sur la formation des néovaisseaux in vivo.Nous avons montré qu elle agit sur la souchitude des cellules CD34+, potentialise les propriétés proangiogènes des ECFCs in vitro, et participe au processus angiogénique in vivo. L OPG agit sur les ECFCs via le syndécanne-1, inhibe leur adhésion à la matrice extracellulaire, favorise leur migration et leur tubulogenèse via la voie SDF-1/CXCR4, et potentialise leur adhésion à l'endothélium activé. Les effets observés sont corrélés à la libération du SDF-1, une activation des voies de signalisation ERK1/2, Akt et mTOR et à une activation de l intégrine aVb3. Par ailleurs, nous avons montré que l'OPG potentialise l effet proangiogène du FGF-2 in vivo. Elle participe également au développement tumoral et à la dissémination des métastases, probablement via l'inhibition de l'apoptose des cellules tumorales, mais aussi par la promotion de l'angiogenèse tumorale.Osteoprotegerin is a key regulator of bone metabolism involved in the immune response, hematopoiesis, and endothelial dysfunction. It seems to be implicated in angiogenesis and may represent a link between bone and vascular system. Although its role in bone is well recognized, its involvement in vasculature remains to be established. In inflammatory conditions, OPG is constitutively released by endothelial cells and smooth muscle cells, and therefore is able to participate in blood vessels formation induced by endothelial progenitor cells (EPCs). In this study we attempted to determine, in vitro the precise role of OPG in angiogenesis process induced by a subpopulation of EPCs called endothelial colony-forming cells (ECFCs), and on neovessel formation in vivo.We found that OPG causes phenotype changes of ECFCs via the activation of different molecular pathways targeting cell clonogenicity, differentiation, proliferation, migration and adhesion. Our results suggest that OPG may interact with ECFCs through its binding to syndecan-1, to induce an anti-adhesive effect and thereby promoting ECFCs migration through a SDF-1/CXCR4 dependant pathway and the ERK1/2, Akt and the mTOR pathways activation. OPG can intervene on the autocrine effect of ECFCs by inducing their adhesion to activated endothelium and their tubulogenesis, and potentiate their paracrine effects by inducing SDF-1 release. Alternatively, it can promote ECFCs survival, probably, in a aVb3 integrin-dependent manner. In vivo, OPG potentiates FGF-2 proangiogenic effects and may participate in tumour growth, invasion and metastasis, possibly through inhibition of tumour cell apoptosis but also by promoting tumour angiogenesis.PARIS5-Bibliotheque electronique (751069902) / SudocSudocFranceF

In vitro and in vivo evaluation of a dextran-graft-polybutylmethacrylate copolymer coated on CoCr metallic stent

International audienceIntroduction: The major complications of stent implantation are restenosis and late stent thrombosis. PBMA polymers are used for stent coating because of their mechanical properties. We previously synthesized and characterized Dextrangraft-polybutylmethacrylate copolymer (Dex-PBMA) as a potential stent coating. In this study, we evaluated the haemocompatibility and biocompatibility properties of Dex-PBMA in vitro and in vivo.Methods: Here, we investigated: (1) the effectiveness of polymer coating under physiological conditions and its ability to release Tacrolimus®, (2) the capacity of Dex-PBMA to inhibit Staphylococcus aureus adhesion, (3) the thrombin generation and the human platelet adhesion in static and dynamic conditions, (4) thebiocompatibility properties in vitro on human endothelial colony forming cells (ECFC) and on mesenchymal stem cells (MSC) and in vivo in rat models, and (5) we implanted Dex-PBMA and Dex-PBMA TAC coated stents in neointimal hyperplasia restenosis rabbit model. Results: Dex-PBMA coating efficiently prevented bacterial adhesion and release Tacrolimus®. Dex-PBMA exhibit haemocompatibility properties under flow and ECFC and MSC compatibility. In vivo, no pathological foreign body reaction was observed neither after intramuscular nor intravascular aortic implantation. After Dex-PBMA and Dex-PBMATAC coated stents 30 days implantation in a restenosis rabbit model, an endothelial cell coverage was observed and the lumenpatency was preserved.Conclusion: Based on our findings, Dex-PBMA exhibited vascular compatibility and can potentially be used as a coating for metallic coronary stents

A motif within the N-terminal domain of TSP-1 specifically promotes the proangiogenic activity of endothelial colony-forming cells

Thrombospondin-1 (TSP-1) gives rise to fragments that have both pro- and anti-angiogenic effects in vitro and in vivo. the TSP-HepI peptide (2.3 kDa), located in the N-terminal domain of TSP-1, has proangiogenic effects on endothelial cells. We have previously shown that TSP-1 itself exhibits a dual effect on endothelial colony-forming cells (ECFC) by enhancing their adhesion through its TSP-HepI fragment while reducing their proliferation and differentiation into vascular tubes (tubulogenesis) in vitro. This effect is likely mediated through CD47 binding to the TSP-1 C-terminal domain. Here we investigated the effect of TSP-HepI peptide on the angiogenic properties of ECFC in vitro and in vivo. TSP-HepI peptide potentiated FGF-2-induced neovascularisation by enhancing ECFC chemotaxis and tubulogenesis in a Matrigel plug assay. ECFC exposure to 20 mu g/mL of TSP-HepI peptide for 18 h enhanced cell migration (p < 0.001 versus VEGF exposure), upregulated alpha 6-integrin expression, and enhanced their cell adhesion to activated endothelium under physiological shear stress conditions at levels comparable to those of SDF-1 alpha. the adhesion enhancement appeared to be mediated by the heparan sulfate proteoglycan (HSPG) syndecan-4, as ECFC adhesion was significantly reduced by a syndecan-4-neutralising antibody. ECFC migration and tubulogenesis were stimulated neither by a TSP-HepI peptide with a modified heparin-binding site (S/TSP-HepI) nor when the glycosaminoglycans (GAGS) moieties were removed from the ECFC surface by enzymatic treatment. Ex vivo TSP-HepI priming could potentially serve to enhance the effectiveness of therapeutic neovascularisation with ECFC. (C) 2012 Elsevier Inc. All rights reserved.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Groupe d'Etude et de Recherches sur l'Hemostase (GEHT)Region Ile-de-France (CORDDIM)Leducq TransAtlantic Network of ExcellenceUniv Estado Rio de Janeiro, Dept Biol Celular, Lab Biol Celula Endotelial & Angiogenese LabAngio, Inst Biol Roberto Alcantara Gomes, BR-20550011 Rio de Janeiro, RJ, BrazilINSERM, U765, Paris, FranceUniv Paris 05, Paris, FranceUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, São Paulo, BrazilUniv Fed Rio de Janeiro, Inst Ciencias Biomed, Rio de Janeiro, RJ, BrazilHop Europeen Georges Pompidou, AP HP, Dept Haematol, Paris, FranceINSERM, Paris Cardiovasc Res Ctr, U970, Paris, FranceUniversidade Federal de São Paulo, Escola Paulista Med, Dept Biofis, São Paulo, BrazilLeducq TransAtlantic Network of Excellence: 04CVD01-LENALeducq TransAtlantic Network of Excellence: 04CVD02 -LINATCNPq: E-26/110.780/2010CAPES: 629/09Web of Scienc

Interactions biospecifiques entre polystyrenes fonctionnalises et proteines plasmatiques

SIGLEINIST TD 19867 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Fucoidan Stimulates Monocyte Migration via ERK/p38 Signaling Pathways and MMP9 Secretion

Critical limb ischemia (CLI) induces the secretion of paracrine signals, leading to monocyte recruitment and thereby contributing to the initiation of angiogenesis and tissue healing. We have previously demonstrated that fucoidan, an antithrombotic polysaccharide, promotes the formation of new blood vessels in a mouse model of hindlimb ischemia. We examined the effect of fucoidan on the capacity of peripheral blood monocytes to adhere and migrate. Monocytes negatively isolated with magnetic beads from peripheral blood of healthy donors were treated with fucoidan. Fucoidan induced a 1.5-fold increase in monocyte adhesion to gelatin (p &lt; 0.05) and a five-fold increase in chemotaxis in Boyden chambers (p &lt; 0.05). Fucoidan also enhanced migration 2.5-fold in a transmigration assay (p &lt; 0.05). MMP9 activity in monocyte supernatants was significantly enhanced by fucoidan (p &lt; 0.05). Finally, Western blot analysis of fucoidan-treated monocytes showed upregulation of ERK/p38 phosphorylation. Inhibition of ERK/p38 phosphorylation abrogated fucoidan enhancement of migration (p &lt; 0.01). Fucoidan displays striking biological effects, notably promoting monocyte adhesion and migration. These effects involve the ERK and p38 pathways, and increased MMP9 activity. Fucoidan could improve critical limb ischemia by promoting monocyte recruitment

Effet d'un peptide mimant le domaine N-terminal de la thrombospondine-1 sur les propriétés fonctionnelles des progéniteurs endothéliaux in vitro et in vivo (proposition d'un produit de thérapie cellulaire)

Les progéniteurs endothéliaux circulants (EPC) sont recrutées par des sites de néovascularisation active chez l adulte et contribuent à la réparation endothéliale et à la biologie vasculaire, dans un processus appelé vasculogenèse post-natale. Ainsi, les EPC représentent une source importante pour la thérapie de néovascularisation dans la maladie artérielle périphérique, l ischémie du myocarde et l accident vasculaire cérébral. Considérant que les EPC potentiellement fonctionnels sont limités dans la circulation périphérique, plusieurs molécules pro-angiogènes ont été étudiées comme agents pharmacologiques dans le but d'améliorer le recrutement des progéniteurs dans la moelle osseuse vers la circulation et la capacité d'adhérence à l'endothélium afin d obtenir une transplantation plus efficace. La thrombospondine-1 (TSP-1) est une glycoprotéine sécrétée lors de situations ischémiques qui joue un double rôle : elle est capable d inhiber et de stimuler l'angiogenèse. Cette double activité biologique est attribuée aux interactions de ses multiples fragments avec différents récepteurs de la surface cellulaire ainsi qu avec des molécules solubles ou protéines ancrées à la matrice extracellulaire. L'effet pro-angiogène de la TSP-1 est assigné au domaine N-terminal qui a une forte affinité à l'héparine et aux protéoglycanes. Les recherches antérieures de notre laboratoire ont indiqué deux séquences peptidiques situées dans le domaine N-terminal de la TSP-1 (TSP-HepI, acides aminés 17-35 et TSP-HepII, acides aminés 78-94), capables d'induire la différenciation des cellules endothéliales matures (HUVEC) dans les structures vasculaires in vitro en interagissant avec le syndécanne-4, un protéoglycane héparin-sulfate membranaire impliqué dans l'adhésion focale. L objectif de ce travail était d'étudier le potentiel de la TSP-1 comme molécule stratégique pour promouvoir une augmentation de la néovascularisation dans les tissus ischémiés, induite par des EPC en essais in vitro et in vivo. Dans un premier temps, nous avons démontré que les EPC du sang périphérique, isolées et accrues avec un milieu de culture modifié dans notre laboratoire (EGM-2/ENR), présentaient les caractéristiques phénotypiques et fonctionnelles des cellules formant des colonies endothéliales (ECFC) avec un haut potentiel prolifératif. Par ailleurs, ces progéniteurs endothéliaux nommées PB-ECFC ont sécrèté trois fois plus la quantité de fibronectine que les HUVEC et ont montré un réarrangement des filaments d'actine compatible avec un phénotype migratoire. Ensuite, nous avons démontré que les peptides TSP-HepI et TSP-HepII stimulaient les étapes-clés de l'angiogenèse in vitro (le chimiotactisme, l'adhésion cellulaire et la différenciation en structures capillary-like), induites par des PB-ECFC et des ECFC isolées du sang de cordon ombilical. En plus de cela, le TSP-HepI potentialise la néovascularisation in vivo induit par le FGF-2 dans les essais de matrigel plug chez la souris. Le pré-conditionnement ex vivo d ECFC isolées du sang de cordon ombilical avec la TSP-HepI a stimulé leur migration, l'augmentation de l'expression de l'intégrine-a6 et a augmenté leur adhésion à l'endothélium activé sous condition physiologique de flux. Ce dernier effet semble être influé par le syndécanne-4, on constate que l'adhésion des ECFC est considérablement réduite en présence d un anticorps anti-syndécan-4. La migration et la tubulogénèse ont été réduites en utilisant un peptide TSP-HepI modifié dans les sites de liaison à l héparine (S/TSP-HepI), ou lorsque HSPG a subi une dégradation enzymatique. Ces résultats suggèrent ainsi que le domaine N-terminal de la TSP-1 pourrait être utilisé comme molécule pharmacologique pour augmenter le potentiel biologique des ECFC dans la thérapie cellulaire de néovascularisation.Pas de résumé anglaisPARIS5-Bibliotheque electronique (751069902) / SudocSudocFranceF

Type 2 diabetes alters mesenchymal stem cell secretome composition and angiogenic properties

International audienceThis study aimed at characterizing the impact of type 2 diabetes mellitus (T2DM) on the bone marrow mesenchymal stem cell (BMMSC) secre-tome and angiogenic properties. BMMSCs from Zucker diabetic fatty rats (ZDF) (a T2DM model) and Zucker LEAN littermates (control) were cultured. The supernatant conditioned media (CM) from BMMSCs of diabetic and control rats were collected and analysed. Compared to results obtained using CM from LEAN-BMMSCs, the bioactive content of ZDF-BMMSC CM (i) differently affects endothelial cell (HUVEC) functions in vitro by inducing increased (3.5-fold; P < 0.01) formation of tubule-like structures and migration of these cells (3-fold; P < 0.001), as well as promotes improved vascular formation in vivo, and (ii) contains different levels of angiogenic factors (e.g. IGF1) and mediators, such as OSTP, CATD, FMOD LTBP1 and LTBP2, which are involved in angiogenesis and/or extracellular matrix composition. Addition of neutralizing antibodies against IGF-1, LTBP1 or LTBP2 in the CM of BMMSCs from diabetic rats decreased its stimulatory effect on HUVEC migration by approximately 60%, 40% or 40%, respectively. These results demonstrate that BMMSCs from T2DM rats have a unique secretome with distinct angiogenic properties and provide new insights into the role of BMMSCs in aberrant angiogenesis in the diabetic milieu

Ceramide 1 phosphate is a potent chemoattractant factor of endothelial colony forming cells and improve post ischemia tissue regeneration

International audienceCeramide 1 phosphate (C1P) is a well-known chemotaxis inductor in macrophages and murine progenitor cells. In endothelial colony forming cells (ECFC), we have shown that C1P improved proliferation and tubule formation, although C1P chemotactic effects remain unknown. Considering that, C1P levels are elevated in ischemic tissues and that ECFC migration is a key step post-ischemia tissue revascularization, we here aimed to study whether C1P is an ECFC chemoattractant factor and improve their revascularization abilities in vivo. Human cord blood-derived CD34+ cells were cultured in EGM2 and, after 14-18 days, ECFC colonies were obtained. ECFC were treated with C1P short chain analog C8-C1P. N=3-6, p<0.05, one-way ANOVA. We found that C8-C1P is a potent chemoattractant factor for ECFC not only per se, but also combined with SDF1 (transwells, Figure 1A). C8-C1P-induced chemotaxis was completely suppressed by pharmacological inhibitors of ERK1/2 and AKT pathways (Figure 1). In vivo, we observed that C8-C1P not only has a potent vasculogenic effect by itself, but also potentiated plug vascularization mediated by ECFC (Geltrex plug implants, Figure 2). Moreover, in a murine model of hind limb ischemia, intramuscular injection of C8-C1P enhanced blood perfusion in the ischemic limb and slightly increased the revascularization mediated by untreated ECFC transplantation. Furthermore, administration of C1P-pretreated ECFC together with intramuscular C1P resulted in a significant improvement of leg reperfusion compared to each condition alone (Figure 2). In conclusion, C8-C1P induce ECFC chemotaxis in vitro, through AKT and ERK1/2 activation, and in vivo in a hind limb ischemia model, where C8-C1P not only attract ECFC to the ischemic muscle, but also augmented ECFC revascularization abilities. Our results highlight the therapeutic potential of C1P to improve post ischemia tissue regeneration

la tension d'oxygène régule les fonctions paracrines des cellules souches mésenchymateuses

International audienceMesenchymal stem cells (MSCs) have captured the attention and research endeavors of the scientific world because of their differentiation potential. However, there is accumulating evidence suggesting that the beneficial effects of MSCs are predominantly due to the multitude of bioactive mediators secreted by these cells. Because the paracrine potential of MSCs is closely related to their microenvironment, the present study investigated and characterized select aspects of the human MSC (hMSC) secretome and assessed its in vitro and in vivo bioactivity as a function of oxygen tension, specifically near anoxia (0.1% O2) and hypoxia (5% O2), conditions that reflect the environment to which MSCs are exposed during MSC-based therapies in vivo. In contrast to supernatant conditioned media (CM) obtained from hMSCs cultured at either 5% or 21% of O2, CM from hMSCs cultured under near anoxia exhibited significantly (p < .05) enhanced chemotactic and proangiogenic properties and a significant (p < .05) decrease in the inflammatory mediator content. An analysis of the hMSC secretome revealed a specific profile under near anoxia: hMSCs increase their paracrine expression of the angiogenic mediators vascular endothelial growth factor (VEGF)-A, VEGF-C, interleukin-8, RANTES, and monocyte chemoattractant protein 1 but significantly decrease expression of several inflammatory/immunomodulatory mediators. These findings provide new evidence that elucidates aspects of great importance for the use of MSCs in regenerative medicine and could contribute to improving the efficacy of such therapies.SIGNIFICANCE:The present study investigated and characterized select aspects of the human mesenchymal stem cell (hMSC) secretome and assessed its in vitro and in vivo biological bioactivity as a function of oxygen tension, specifically near anoxia (0.1% O2) and hypoxia (5% O2), conditions that reflect the environment to which MSCs are exposed during MSC-based therapies in vivo. The present study provided the first evidence of a shift of the hMSC cytokine signature induced by oxygen tension, particularly near anoxia (0.1% O2). Conditioned media obtained from hMSCs cultured under near anoxia exhibited significantly enhanced chemotactic and proangiogenic properties and a significant decrease in the inflammatory mediator content. These findings provide new evidence that elucidates aspects of great importance for the use of MSCs in regenerative medicine, could contribute to improving the efficacy of such therapies, and most importantly highlighted the interest in using conditioned media in therapeutic modalities.Les cellules souches mésenchymateuses (CSM) sont très attractives pour la thérapie cellulaire en raison de leur potentiel de différenciation. Cependant, de nombreuses preuves suggèrent que les effets bénéfiques des CSM sont principalement dus à la multitude de médiateurs bioactifs sécrétés par ces cellules. Parce que le potentiel paracrine des CSM est étroitement lié à leur microenvironnement, la présente étude a examiné et caractérisé certains aspects du sécrétome du CSM humain (CSMh) et évalué sa bioactivité in vitro et in vivo en fonction de la tension en oxygène, en particulier près de l'anoxie. (0,1% O 2) et l’hypoxie (5% O 2), conditions qui reflètent l’environnement auquel les MSC sont exposés au cours de traitements in vivo à base de MSC. Contrairement aux milieux conditionnés (MC) obtenus à partir de CSM cultivées à 5% ou à 21% d'oxygène, les MC provenant de CSM cultivées presque en anoxie présentaient des propriétés chimiotactiques et proangiogéniques renforcées de manière significative (p <0,05) et une diminution significative du contenu en médiateur inflammatoire p<0,05). Une analyse du sécrétome des CSM a révélé un profil spécifique dans l'anoxie proche: les CSM accroissent leur expression paracrine du facteur de croissance endothélial vasculaire (VEGF)-A, VEGF-C, l'interleukine-8, RANTES et la protéine 1 de monocyte, un agent chimioattractant monocytaire mais diminue de façon significative l'expression de plusieurs médiateurs inflammatoires / immomodulateurs. Ces résultats fournissent de nouvelles preuves qui élucident des aspects d'une grande importance pour l'utilisation des CSM en médecine régénérative et pourraient contribuer à améliorer l'efficacité de tels traitements

Cytochrome P450 dependent metabolism of fluindione in vitro in rat and human microsomes and in vivo in rat.

International audienceFluindione (2-(4’fluoro-phenyl)-phenylindan1,3-dione) is an anti-vitamin K (AVK) marketed in 1967 in France and in Luxembourg (1-3). It is still the leading AVK in France in 2016 (around 800.000 patients, 82% of the market). However very little is known on the metabolism of this drug. In order to better understand the pharmacokinetics of this drug (4,5), we started to study the in vitro and in vivo metabolism of fluindione. Thus the incubation of fluindione with rat liver microsomes showed a major metabolite X, having a similar UV spectrum as fluindione, but slighly less polar and a high resolution mass spectrum showing addition of an oxygen atom. Its formation was dependent on NADPH and O2 and was inhibited by benzyl-imidazole, a general cytochrome P450 inhibitor. Semi-preparative incubations of fluindione allowed to get an 1H NMR spectrum showing the presence of 8 non exchangeable protons corresponding to an asymmetric aromatic A ring and an unchanged fluorophenyl. Incubation with human liver microsomes showed the formation of this metabolite X. Use of recombinant P450 showed that CYP1A2, 2B6, 2C9, 2D6, 3A4 and 3A5 could produce this metabolite. CYP2C9 and CYP1A2 had good affinities (less than 30 µM).The chemical synthesis of several candidate metabolites was performed: 2-hydroxy-fluindione, 2-(4’-fluorophenyl)-4-hydroxy-coumarin, 2-(4’-fluorophenyl)-4-hydroxy-isocoumarin (new). X corresponded to none of these candidates.An equimolar mixture of fluindione and 13C labeled fluindione on the aromatic A ring was injected IP in rats (20 mg/kg). Analysis of plasma showed the formation of metabolite X and of two minor metabolites. The first minor one Y as identified to (2-(4’hydroxy-phenyl)-phenyl)-indan1,3-dione formed by defluorination and hydroxylation of the fluorophenyl group (a known metabolic reaction). A second minor metabolite Z in very low amount could be the equivalent of metabolite X with fluorine replaced by hydroxyl. In rat urine the sulfate of X and the sulfate of Z and a trace of Y could be found.In human (10 mg per os) fluindione and metabolite X were detected.Further experiments are ongoing in order to finally identify metabolite X