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

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

Osteoprotegerin induces CD34 + differentiation in Endothelial Progenitor Cells

International audienceEndothelial progenitor cells (EPCs) are the main hypothetical cells that could give rise to vessels and in particular one subtype isolated from peripheral or cord bloods: endothelial colony forming cells (ECFCs). These ECFCs are clonogenic precursors committed to endothelial lineage and have robust vasculogenic properties. However, their low number and poor expansion properties when isolated from human adult bloods, currently limit their use as an autologous cell therapy product. We previously reported that osteoprotegerin (OPG), a well-characterized regulator of bone metabolism, contributes to ischemic tissue revascularization, tumor growth in vivo, and potentiates ECFCs proangiogenic properties through the secretion of SDF-1. The current study investigated the role of OPG in ECFCs differentiation and expansion from cord blood CD34+ cells. OPG increased the number of ECFCs after endothelial differentiation of CD34+ cells, enhancing the time of EPCs colonies initial appearance and the growth kinetic of endothelial cell progeny. OPG-exposed ECFCs expressed higher levels of CD34+ compared to control ECFCs. In conclusion, our findings provide novel insights into OPG in regulation of CD34+ progenitor cells. These results give new opportunities for ex vivo expansion of human ECFCs using OPG as a cell culture component for future ECFC product manufacture according to GMP

Osteoprotegerin regulates cancer cell migration through SDF-1/CXCR4 axis and promotes tumour development by increasing neovascularization

International audienceABSTRACTWe previously reported that OPG is involved in ischemic tissue neovascularization through the secretion of SDF-1 by pretreated-OPG endothelial colony-forming cells (ECFCs). As the vascularization is one of the key factor influencing the tumour growth and cancer cell dissemination, we investigated whether OPG was able to modulate the invasion of human MNNG-HOS osteosarcoma and DU145 prostate cancer cell lines in vitro and in vivo. Cell motility was analysed in vitro by using Boyden chambers. Human GFP-labelled MMNG-HOS cells were inoculated in immunodeficient mice and the tumour nodules formed were then injected with OPG and/or FGF-2, AMD3100 or 0.9% NaCl (control group). Tumour growth was manually followed and angiogenesis was assessed by immunohistochemistry. In vitro, SDF-1 released by OPG-pretreated ECFCs markedly attracted both MNNG-HOS and DU145 cells and induced spontaneous migration of cancer cells. In vivo, tumour volumes were significantly increased in OPG-treated group compared to the control group and OPG potentiated the effect of FGF-2. Concomitantly, OPG alone or combined with FGF-2 increased the number of new vasculature compared to the control group. Interestingly AMD3100, an inhibitor of SDF-1, prevented the in vivo effects of OPG induced by SDF-1 This study provides experimental evidence that OPG promotes tumour development trough SDF-1/CXCR4 axis

Low-Molecular-Weight Fucoidan Induces Endothelial Cell Migration via the PI3K/AKT Pathway and Modulates the Transcription of Genes Involved in Angiogenesis

Low-molecular-weight fucoidan (LMWF) is a sulfated polysaccharide extracted from brown seaweed that presents antithrombotic and pro-angiogenic properties. However, its mechanism of action is not well-characterized. Here, we studied the effects of LMWF on cell signaling and whole genome expression in human umbilical vein endothelial cells and endothelial colony forming cells. We observed that LMWF and vascular endothelial growth factor had synergistic effects on cell signaling, and more interestingly that LMWF by itself, in the absence of other growth factors, was able to trigger the activation of the PI3K/AKT pathway, which plays a crucial role in angiogenesis and vasculogenesis. We also observed that the effects of LMWF on cell migration were PI3K/AKT-dependent and that LMWF modulated the expression of genes involved at different levels of the neovessel formation process, such as cell migration and cytoskeleton organization, cell mobilization and homing. This provides a better understanding of LMWF's mechanism of action and confirms that it could be an interesting therapeutic approach for vascular repair

Endoglin Is an Endothelial Housekeeper against Inflammation: Insight in ECFC-Related Permeability through LIMK/Cofilin Pathway

International audienceEndoglin (Eng) is an endothelial cell (EC) transmembrane glycoprotein involved in adhesion and angiogenesis. Eng mutations result in vessel abnormalities as observed in hereditary hemorrhagic telangiectasia of type 1. The role of Eng was investigated in endothelial functions and permeability under inflammatory conditions, focusing on the actin dynamic signaling pathway. Endothelial Colony-Forming Cells (ECFC) from human cord blood and mouse lung/aortic EC (MLEC, MAEC) from Eng+/+ and Eng+/− mice were used. ECFC silenced for Eng with Eng-siRNA and ctr-siRNA were used to test tubulogenesis and permeability +/− TNFα and +/− LIM kinase inhibitors (LIMKi). In silico modeling of TNFα–Eng interactions was carried out from PDB IDs 5HZW and 5HZV. Calcium ions (Ca2+) flux was studied by Oregon Green 488 in epifluorescence microscopy. Levels of cofilin phosphorylation and tubulin post-translational modifications were evaluated by Western blot. F-actin and actin–tubulin distribution/co-localization were evaluated in cells by confocal microscopy. Eng silencing in ECFCs resulted in a decrease of cell sprouting by 50 ± 15% (p < 0.05) and an increase in pseudo-tube width (41 ± 4.5%; p < 0.001) compared to control. Upon TNFα stimulation, ECFC Eng–siRNA displayed a significant higher permeability compared to ctr-siRNA (p < 0.01), which is associated to a higher Ca2+ mobilization (p < 0.01). Computational analysis suggested that Eng mitigated TNFα activity. F-actin polymerization was significantly increased in ECFC Eng-siRNA, MAEC+/−, and MLEC+/− compared to controls (p < 0.001, p < 0.01, and p < 0.01, respectively) as well as actin/tubulin distribution (p < 0.01). Furthermore, the inactive form of cofilin (P-cofilin at Ser3) was significantly decreased by 36.7 ± 4.8% in ECFC Eng-siRNA compared to ctr-siRNA (p < 0.001). Interestingly, LIMKi reproduced the absence of Eng on TNFα-induced ECFC-increased permeability. Our data suggest that Eng plays a critical role in the homeostasis regulation of endothelial cells under inflammatory conditions (TNFα), and loss of Eng influences ECFC-related permeability through the LIMK/cofilin/actin rearrangement-signaling pathway