Rôle de la chimiokine RANTES/CCL5 et de ses ligands membranaires dans l angiogenèse (Application en biothérapie)

L angiogenese est un processus physiopathologique consistant en la formation de nouveaux vaisseaux. Parmi ies facteurs responsables de cette angiogenese les chimiokines sont des médiateurs de la formation des néo-vaisseaux. Le role angiogénique de la chimiokine RANTES/CCL5 est encore source de controverses. Nos travaux démontrent que RANTES/CCLS délivrée localement a l aide de biomatériau de nitrocellulose induit la formation de capillaires sanguins dans un modele de délivrance sous-cutané chez le rat. L effet angiogénique de RANTES/CCL5 a été caractérisé in vitro dans les cellules endothéliales HUV-EC-Cs. Nous demontrons que RANTES/CCL5 induit l étalement et la migration de cellules HUV-EC~Cs et participe a la formation de réseaux vasculaires par le biais d une sécrétion de VEGF. La liaison de RANTES/CCL5 a ses récepteurs protéiques, CCR1 et CCR5 ainsi qu aux protéoglycannes membranaires, CD44, syndécanne-1 et syndécanne-4 est indispensable a l induction de son effet pro-angiogénique. De plus, la liaison de RANTES/CCL5 aux chahes glycosaminoglycannes de type héparane sulfate est essentielle a ses effets biologiques in vitro. Ayant pour objectif de développer une stratégie thérapeutique permettant de lutter contre Vangiogenése, nous avons utilisé deux mutants de RANTESXCCLS caractérisés par une incapacité de lier les chaines lycosaminoglvcanniques. Ces mutants [44ANAA4I]-RANTES/CCL5 et [E66A]-RANTES/CCL5 sont moins efficaces que RANTES/CCL5 pour induire des effets cellulaires impliques dans l'angiogenese in vivo et in vitro. A Finverse, nous avons envisagé l utiIisation d un biomatériau permettant la déiivrance locale de RANTES/CCL5 dans un modele d ischémie de patte de souris, pathologie pour laquelle un traitement induisant Fangiogenese aurait un effet bénéfique pour la survie tissulaire.Angiogenesis is a physiopathological process defined by new blood vessel formation. Chemokines are described as possible mediators of angiogenesis. The angiogenic role of the chemokine RANTES/CCL5 is controversial. Our study demonstrated that the local delivery of RANTES/CCL5 by a nitrocellulose biomaterial in a rat subcutaneous model leads to the formation of vessels around the biomaterial. We described that the treatment with RANTES/CCL5 of the endothelial cells line (HUV-EC-Cs) induced the cell spreading, migration and the vascular network formation through VEGF secretion, in vitro. We performed the analysis of the role of the RANTES/CCL5 receptors, CCR1 or CCR5 and membrane proteogiycans, CD44, syndecan-1 or syndecan-4 in angiogenesis process induced by RANTES/CCL5. Moreover the binding of RANTES/CCL5 on glycosaminoglycan heparan sulfate chains was essential for these effects. The aim of this study was to develop therapeutics strategies in pathologies characterized by a deleterious angiogenesis such as cancers. The glycosaminoglycan-binding deficient mutants [MANAAQ]-RANTES/CCL5 and [E66A]- RANTES/CCL5 were less effective than RANTES/CCL5 to induce angiogenesis. In contrary, the association of RANTES/CCL5 with a polysaccharide biomaterial could constitute a strategy to local diffusion of this chemokine in order to induce a neovascularization in hypoxic tissue such as in the ischemic mice leg model.PARIS13-BU Sciences (930792102) / SudocSudocFranceF

Glycosaminoglycans and syndecan-4 are involved in SDF-1/CXCL12-mediated invasion of human epitheloid carcinoma HeLa cells.

International audienceBACKGROUND: In addition to their physiologic effects in inflammation and angiogenesis, chemokines are involved in cancer pathology. The CXC-chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12 mediates its biological activities through activation of G protein-coupled receptor CXCR4 and binds to glycosaminoglycans (GAGs). METHODS: Using Bio-coat cell migration chambers, specific antagonists, flow cytometry and RNA interference, we evaluate the involvement of heparan sulfate proteoglycans (HSPG) in the SDF-1/CXCL12-induced invasion of human cervix epitheloid carcinoma HeLa cells. RESULTS: The SDF-1/CXCL12-induced cell invasion is dependent on CXCR4. Furthermore, Protein Kinase C delta (PKC delta) and c-jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) are implicated in this event, but not extracellular signal-regulated kinase (ERK) 1/2. Moreover, the invasion of HeLa cells induced by SDF-1/CXCL12 was dependent on matrix metalloproteinase-9 (MMP-9). The pre-incubation of HeLa cells with heparin or with anti-heparan sulfate antibodies or with beta-d-xyloside inhibited SDF-1/CXCL12-mediated cell invasion. Furthermore, the down-regulation of syndecan-4, a heparan sulfate proteoglycan, decreased SDF-1/CXCL12-mediated HeLa cell invasion. CONCLUSION: GAGs, probably on syndecan-4, are involved in SDF-1/CXCL12-mediated cell chemotaxis. GENERAL SIGNIFICANCE: These data suggest that targeting the glycosaminoglycan/chemokine interaction could be a new therapeutic approach for carcinomas in which SDF-1/CXCL12 is involved

Angiogenic properties of the chemokine RANTES/CCL5

International audienc

The expanding roles of microRNAs in kidney pathophysiology

International audienc

Low molecular weight fucoidan prevents intimal hyperplasia in rat injured thoracic aorta through the modulation of matrix metalloproteinase-2 expression.

International audienceThe therapeutic potential of low molecular-weight fucoidan (LMWF), a sulfated polysaccharide extracted from brown seaweed was investigated on vascular smooth muscle cell (VSMC) and human vascular endothelial cell (HUV-EC-C) proliferation and migration in vitro and in a rat model of intimal hyperplasia. Sprague-Dawley rats were subjected to balloon injury in the thoracic aorta followed by two weeks' treatment with either LMWF (5mg/kg/day) or vehicle. Morphological analysis and proliferating cell nuclear antigen immunostaining at day 14 indicated that LMWF prevented intimal hyperplasia in rat thoracic aorta as compared with vehicle (neo-intima area, 3±0.50 versus 5±0.30mm, <0.01). In situ zymography showed that LMWF significantly decreased the activity of matrix metalloproteinase (MMP)-2 in the neo-intima compared to vehicle. The in vitro study demonstrated that 10μg/ml LMWF increased HUV-EC-C migration by 45±5% but reduced VSMC migration by 40±3%. LMWF also increased MMP-2 mRNA expression in HUV-EC-Cs and reduced it in VSMCs. MMP-2 level in the conditioned medium from cells incubated with 10μg/ml LMWF was 5.4-fold higher in HUV-EC-C, but 6-fold lower in VSMCs than in untreated control cells. Furthermore, decreasing MMP-2 expression in HUV-EC-Cs or VSMCs by RNA interference resulted in reduced LMWF-induced effects on cell migration

Sulfated oligosaccharides (heparin and fucoidan) binding and dimerization of stromal cell-derived factor-1 (SDF-1/CXCL 12) are coupled as evidenced by affinity CE-MS analysis

International audienceChemokine stromal cell-derived factor-1 (SDF-1) is a potent chemoattractant involved in leukocyte trafficking and metastasis. Heparan sulfate on the cell surface binds SDF-1 and may modulate its function as a coreceptor of this chemokine. A major effect of the glycosaminoglycan binding may be on the quaternary structure of SDF-1, which has been controversially reported as a monomer or a dimer. We have investigated the effect of sulfated oligosaccharides on the oligomerization of SDF-1 and of its mutated form SDF-1 (3/6), using affinity capillary electrophoresis (ACE) hyphenated to mass spectrometry (MS). Coupled to MS, ACE allowed the study for the first time of the effect of size-defined oligosaccharides on the quaternary organization of SDF-1 in muM range concentrations, i.e., lower values than the mM values previously reported in NMR, light scattering, and ultracentrifugation experiments. Our results showed that in the absence of sulfated oligosaccharides, SDF-1 is mostly monomeric in solution. However, dimer formation was observed upon interaction with heparin-sulfated oligosaccharides despite the mM Kd values for dimerization. A SDF-1/oligosaccharide 2/1 complex was detected, indicating that oligosaccharide binding promoted the dimerization of SDF-1. Heparin tetrasaccharide but not disaccharide promoted dimer formation, suggesting that the dimer required to be stabilized by a long enough bound oligosaccharide. The SDF-1/oligosaccharide 1/1 complex was only observed with heparin disaccharide and fucoidan pentasaccharide, pointing out the role of specific structural determinants in promoting dimer formation. These results underline the importance of dimerization induced by glycosaminoglycans for chemokine functionality

Manganese Superoxide Dismutase Dimorphism and Iron Overload, Hepatocellular Carcinoma, and Death in Hepatitis C Virus–Infected Patients

International audienc

Chemokine system polymorphisms, survival and hepatocellular carcinoma occurrence in patients with hepatitis C virus-related cirrhosis

AIM: To explore the influence of polymorphisms in genes encoding for the chemokines Stromal cell-Derived Factor-1 (SDF-1)/CXCL12 and Monocyte Chemotactic Protein-1 (MCP-1)/CCL2, or for the chemokine receptor CCR5 on the risks of liver-related death and hepatocellular carcinoma (HCC) occurrence in hepatitis C virus (HCV)-infected patients