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

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

Acidic preconditioning of endothelial colony-forming cells (ECFC) promote vasculogenesis under proinflammatory and high glucose conditions in vitro and in vivo

International audienceBACKGROUND:We have previously demonstrated that acidic preconditioning of human endothelial colony-forming cells (ECFC) increased proliferation, migration, and tubulogenesis in vitro, and increased their regenerative potential in a murine model of hind limb ischemia without baseline disease. We now analyze whether this strategy is also effective under adverse conditions for vasculogenesis, such as the presence of ischemia-related toxic molecules or diabetes, one of the main target diseases for cell therapy due to their well-known healing impairments.METHODS:Cord blood-derived CD34+ cells were seeded in endothelial growth culture medium (EGM2) and ECFC colonies were obtained after 14-21 days. ECFC were exposed at pH 6.6 (preconditioned) or pH 7.4 (nonpreconditioned) for 6 h, and then pH was restored at 7.4. A model of type 2 diabetes induced by a high-fat and high-sucrose diet was developed in nude mice and hind limb ischemia was induced in these animals by femoral artery ligation. A P value < 0.05 was considered statistically significant (by one-way analysis of variance).RESULTS:We found that acidic preconditioning increased ECFC adhesion and the release of pro-angiogenic molecules, and protected ECFC from the cytotoxic effects of monosodium urate crystals, histones, and tumor necrosis factor (TNF)α, which induced necrosis, pyroptosis, and apoptosis, respectively. Noncytotoxic concentrations of high glucose, TNFα, or their combination reduced ECFC proliferation, stromal cell-derived factor (SDF)1-driven migration, and tubule formation on a basement membrane matrix, whereas almost no inhibition was observed in preconditioned ECFC. In type 2 diabetic mice, intravenous administration of preconditioned ECFC significantly induced blood flow recovery at the ischemic limb as measured by Doppler, compared with the phosphate-buffered saline (PBS) and nonpreconditioned ECFC groups. Moreover, the histologic analysis of gastrocnemius muscles showed an increased vascular density and reduced signs of inflammation in the animals receiving preconditioned ECFC.CONCLUSIONS:Acidic preconditioning improved ECFC survival and angiogenic activity in the presence of proinflammatory and damage signals present in the ischemic milieu, even under high glucose conditions, and increased their therapeutic potential for postischemia tissue regeneration in a murine model of type 2 diabetes. Collectively, our data suggest that acidic preconditioning of ECFC is a simple and inexpensive strategy to improve the effectiveness of cell transplantation in diabetes, where tissue repair is highly compromised.Contexte: Nous avons précédemment démontré que le préconditionnement acide des ECFC humaines augmentait la prolifération, la migration et la tubulogenèse in vitro, ainsi que leur potentiel régénérateur dans un modèle murin d'ischémie des membres postérieurs. Nous analysons maintenant l'efficacité de cette stratégie dans des conditions défavorables à la vasculogenèse, telles que la présence de molécules toxiques associées à l'ischémie ou le diabète, l'une des principales maladies cibles de la thérapie cellulaire en raison de leurs altérations de guérison bien connues

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

Evaluation of commonly used tests to measure the effect of single-dose aspirin on mouse hemostasis

PG and CBL share equal senior authorship.International audienceDiscrepancies in preclinical studies of aspirin (ASA) antiplatelet activity in mouse models of bleeding and arterial thrombosis led us to evaluate commonly reported methods in order to propose a procedure for reliably measuring the effects of single dose ASA on mouse hemostasis. FVB and C57Bl6 mice received 100 mg/kg of ASA or vehicle orally 30 min or 3 h prior to investigate either hemostasis using the tail bleeding assay or carotid thrombosis induced by FeCl3, or to blood sampling for isolated platelet aggregation and TXB2 generation. Expected inhibition of COX1 by ASA was ascertained by a strong decrease in TXB2 production, and its effect on platelet function and hemostasis, by decreased collagen-induced aggregation and increased bleeding time, respectively. Strikingly, we determined that anti-hemostatic effects of ASA were more predictable 30 min after administration than 3 h later. Conversely, ASA did not alter time to arterial occlusion of the carotid upon FeCl3-induced thrombosis, suggesting ASA not to be used as reference inhibitor drug in this model of arterial thrombosis

Additional file 2: of Acidic preconditioning of endothelial colony-forming cells (ECFC) promote vasculogenesis under proinflammatory and high glucose conditions in vitro and in vivo

ECFC adhesion under proinflammatory and high glucose conditions. Nonpreconditioned or preconditioned ECFC (npECFC or pECFC, respectively) were incubated with high glucose, TNFα, or their combination, and seeded onto fibronectin, collagen, or TNFα-activated HUVEC. After (A) 30 min or (B) 2 h, the number of adherent cells was counted. Results are expressed as percentage of nonpreconditioned ECFC (n = 5). *p < 0.05 vs untreated npECFC. #p < 0.05 vs npECFC with the same treatment. (DOCX 478 kb

Additional file 1: of Acidic preconditioning of endothelial colony-forming cells (ECFC) promote vasculogenesis under proinflammatory and high glucose conditions in vitro and in vivo

ECFC cell death induced by histones or MSU is not affected under high glucose conditions. Nonpreconditioned ECFC (npECFC) were incubated with histones (A) or MSU crystals (B) at the indicated concentrations, in the presence or absence of high glucose, and cell death was analyzed after 24 h (n = 3–5). (DOCX 135 kb

Additional file 3: of Acidic preconditioning of endothelial colony-forming cells (ECFC) promote vasculogenesis under proinflammatory and high glucose conditions in vitro and in vivo

Histologic analysis of gastrocnemius muscles. PBS, nonpreconditioned, or preconditioned ECFC (npECFC or pECFC, respectively) were infused intravenously in normoglycemic and type 2 diabetic (T2D) mice 5 h after ischemia-inducing surgery. Histologic analysis of gastrocnemius muscles, stained with hematoxylin and eosin (H/E) or Masson’s trichrome, was performed after 14 days postischemia in normoglycemic and type 2 diabetic (T2D) mice (n = 6 per group). Original magnification, 100×. Scale bar = 20 μm. (DOCX 1573 kb

Ceramide 1-Phosphate Protects Endothelial Colony Forming Cells From Apoptosis and Increases Vasculogenesis In Vitro and In Vivo

Objective: Ceramide 1-phosphate (C1P) is a bioactive sphingolipid highly augmented in damaged tissues. Because of its abilities to stimulate migration of murine bone marrow–derived progenitor cells, it has been suggested that C1P might be involved in tissue regeneration. In the present study, we aimed to investigate whether C1P regulates survival and angiogenic activity of human progenitor cells with great therapeutic potential in regenerative medicine such as endothelial colony–orming cells (ECFCs). Approach and Results: C1P protected ECFC from TNFα (tumor necrosis factor-α)-induced and monosodium urate crystal–induced death and acted as a potent chemoattractant factor through the activation of ERK1/2 (extracellular signal-regulated kinases 1 and 2) and AKT pathways. C1P treatment enhanced ECFC adhesion to collagen type I, an effect that was prevented by β1 integrin blockade, and to mature endothelial cells, which was mediated by the E-selectin/CD44 axis. ECFC proliferation and cord-like structure formation were also increased by C1P, as well as vascularization of gel plug implants loaded or not with ECFC. In a murine model of hindlimb ischemia, local administration of C1P alone promoted blood perfusion and reduced necrosis in the ischemic muscle. Additionally, the beneficial effects of ECFC infusion after ischemia were amplified by C1P pretreatment, resulting in a further and significant enhancement of leg reperfusion and muscle repair. Conclusions: Our findings suggest that C1P may have therapeutic relevance in ischemic disorders, improving tissue repair by itself, or priming ECFC angiogenic responses such as chemotaxis, adhesion, proliferation, and tubule formation, which result in a better outcome of ECFC-based therapy.Fil: Mena, Hebe Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Zubiry, Paula Romina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Dizier, Blandine. Inserm; Francia. Universite de Paris; FranciaFil: Mignon, Virginie. Inserm; Francia. Universite de Paris; FranciaFil: Parborell, Maria Fernanda Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Schattner, Mirta Ana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Boisson Vidal, Catherine. Inserm; Francia. Universite de Paris; FranciaFil: Negrotto, Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; Argentin