9 research outputs found

    Investigation des mécanismes physiologiques menant à la libération du BDNF par les plaquettes et leur susceptibilité aux médicaments antiplaquettaires

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    Les plaquettes sont considĂ©rĂ©es comme l'un des rĂ©servoirs les plus importants non seulement des facteurs de croissance, mais aussi des facteurs neurotrophiques qui pourraient contribuer Ă  la rĂ©paration des lĂ©sions vasculaires et Ă  la prĂ©vention de la dĂ©tĂ©rioration neurologique. Parmi ces facteurs, le facteur neurotrophique dĂ©rivĂ© du cerveau (Brain-Derived Neurotrophic Factor ou BDNF) – une protĂ©ine appartenant Ă  la famille des neurotrophines– est largement exprimĂ©e Ă  la fois dans l'hippocampe et au niveau des plaquettes. Les plaquettes constituent un important rĂ©servoir de BDNF; cependant, on ne sait que peu de choses sur les facteurs modulant la libĂ©ration de ce dernier dans la circulation et si les mĂ©dicaments antiplaquettaires affectent cette sĂ©crĂ©tion. Dans le cadre de ce projet, nous avons Ă©mis l’hypothĂšse principale que les diffĂ©rentes voies d’activation plaquettaire peuvent mener Ă  une libĂ©ration de BDNF, oĂč celle-ci est affectĂ©e par les antiplaquettaires. A cette fin, les plaquettes ont Ă©tĂ© isolĂ©es Ă  partir d’échantillons sanguins de volontaires sains (Groupe 1), de patients souffrant de maladies cardiovasculaires stables requĂ©rant la prise de mĂ©dicaments antiplaquettaires [en prĂ©vention secondaire et en double thĂ©rapie Ă  l’acide acĂ©tylsalicylique (ASA ou Aspirine) en association avec un antagoniste du rĂ©cepteur P2Y12], (Groupe 2) ou en monothĂ©rapie Ă  l’ASA (Groupe 3), versus de patients atteints de maladies valvulaires ou de cardiomyopathies ne requĂ©rant pas la prise de mĂ©dicaments antiplaquettaires (Groupe 4). L’agrĂ©gation plaquettaire a Ă©tĂ© Ă©tudiĂ©e par agrĂ©gomĂ©trie optique en rĂ©ponse Ă  des agonistes spĂ©cifiques : adĂ©nosine diphosphate (ADP), acide arachidonique (AA), Ă©pinĂ©phrine, collagĂšne et Thrombin-receptor activated peptide 6 (TRAP-6 amide). Les antiplaquettaires testĂ©s sont dirigĂ©s contre la cyclo-oxygĂ©nase-1 ou COX-1 (ASA), contre le rĂ©cepteur de P2Y12 de l’ADP (AR-C) et contre le rĂ©cepteur αIIbÎČ3 du fibrinogĂšne (Abciximab). La libĂ©ration du BDNF a Ă©tĂ© quantifiĂ©e par ELISA. La prĂ©sence du BDNF et de son rĂ©cepteur Tropomyosin-Related Kinase Receptor type B (TrKB) a Ă©tĂ© dĂ©tectĂ©e par immunobuvardage. Nous avons montrĂ© que l’activation des plaquettes par les diffĂ©rents agonistes testĂ©s induit une agrĂ©gation plaquettaire de l’ordre de 80% et permet de libĂ©rer jusqu’à 5 fois plus de BDNF, passant de 2500 pg / 250 x 106 plaquettes Ă  l’état basal Ă  approximativement 13000 pg / 250 x 106 plaquettes Ă  l’état stimulĂ©. Tous les antiplaquettaires testĂ©s rĂ©duisent la libĂ©ration de BDNF par les plaquettes stimulĂ©es. Cependant, le niveau d’inhibition et sa significativitĂ© dĂ©pendent de la nature de l’agoniste; Ă  savoir que l’ASA rĂ©duit significativement la sĂ©crĂ©tion de BDNF en rĂ©ponse Ă  l’AA, Ă  l’épinĂ©phrine et au TRAP-6; alors que l’AR-C Ă©tait plus efficace en rĂ©ponse Ă  l’ADP, l’AA et l’épinĂ©phrine. L’Abciximab est un antagoniste qui inhibe la sĂ©crĂ©tion de BDNF en rĂ©ponse Ă  tous les agonistes, en inhibant aussi l’agrĂ©gation plaquettaire. Notons que la libĂ©ration de BDNF en rĂ©ponse au collagĂšne est inhibĂ©e par l’ASA et l’AR-C, alors que l’agrĂ©gation n’a pas Ă©tĂ© affectĂ©e. Ainsi, aucune corrĂ©lation positive et significative entre l’agrĂ©gation plaquettaire et la libĂ©ration de BDNF n’a pu ĂȘtre obtenue. La prĂ©sence des antiplaquettaires rĂ©duits Ă  diffĂ©rents degrĂ©s la libĂ©ration de BDNF chez les diffĂ©rents groupes des patients, malgrĂ© que son expression intraplaquettaire Ă©tait similaire entre les groupes. On remarque que les antiplaquettaires rĂ©duisent plus significativement la quantitĂ© du BDNF relĂąchĂ©e chez les patients sous mono ou double thĂ©rapie antiplaquettaire en comparaison avec les volontaires sains et les patients atteints de maladies valvulaires. Nous avons aussi dĂ©montrĂ© que le BDNF exogĂšne active les plaquettes isolĂ©es et lavĂ©es chez les volontaires sains, en induisant une forte agrĂ©gation stable et irrĂ©versible. Par contre, le BDNF exogĂšne n’arrive pas Ă  agrĂ©ger les plaquettes en plasma riche en plaquettes. De plus, nos rĂ©sultats indiquent que la forme tronquĂ©e du rĂ©cepteur BDNF, le TrKB, est exprimĂ©e au niveau des plaquettes de volontaires sains. L’inhibition de l’activitĂ© kinase du TrKB abolit l’agrĂ©gation induite par le BDNF. Ces rĂ©sultats suggĂšrent que l’action du BDNF dans les plaquettes lavĂ©es pourrait passer par l’intermĂ©diaire du TrKB. Cette Ă©tude nous permet de conclure que le BDNF est prĂ©sent dans les plaquettes et est libĂ©rĂ© suite Ă  l’activation plaquettaire et que cette libĂ©ration est rĂ©duite par les antiplaquettaires. Cependant, l’agrĂ©gation plaquettaire ne semble pas ĂȘtre associĂ©e directement Ă  la sĂ©crĂ©tion du BDNF, ce qui suggĂšre que d’autres mĂ©canismes sous-jacents pourraient intervenir dans le contrĂŽle de cette sĂ©crĂ©tion. Les antiplaquettaires rĂ©duisent la libĂ©ration de BDNF et il semble que l’action pro-agrĂ©gante du BDNF sur les plaquettes lavĂ©es passe par l’intermĂ©diaire du TrKB, sans exclure la possibilitĂ© que d’autres types de rĂ©cepteurs plaquettaires soient impliquĂ©s dans le signal dĂ©clenchĂ© par le BDNF. L’implication physiopathologique du BDNF libĂ©rĂ© suite Ă  l’activation plaquettaire ou sa biodisponibilitĂ© en prĂ©sence des antiplaquettaires au niveau cardiovasculaire reste Ă  ĂȘtre Ă©lucidĂ©e afin de rĂ©vĂ©ler son potentiel diagnostique ou thĂ©rapeutique.Platelets are considered one of the most important reservoirs not only of growth factors but also of neurotrophic factors that may contribute to the repair of vascular lesions and prevention of neurological deterioration. Among these factors, the Brain-Derived Neurotrophic Factor (BDNF), a protein belonging to the neurotrophin family, is largely expressed in both the hippocampus and platelets. In fact, platelets constitute an important reservoir of BDNF; however, little is known about the factors controlling its release into the circulation and whether antiplatelet drugs affect this secretion. Henceforth, the main hypothesis of this project is that platelet activation pathways lead to BDNF release which is affected by antiplatelet agents. For this purpose, platelets were isolated from the blood of four groups of human subjects following their consent. Group 1 consisted of healthy volunteers; Group 2 and Group 3 consisted of patients with stable cardiovascular disease on, respectively, dual antiplatelet therapy (aspirin + P2Y12 receptor antagonist) or monotherapy (aspirin) as secondary prevention; and Group 4 consisted of patients with valvular disease or cardiomyopathy who are not on antiplatelet therapy. Platelet aggregation was studied by optical aggregometry in response to the following agonists: adenosine diphosphate (ADP), arachidonic acid (AA), epinephrine, collagen, and thrombin-receptor activated peptide 6 (TRAP-6 amide). The antiplatelet agents that were tested antagonize cyclooxygenase-1 (COX-1) (acetylsalicylic acid (ASA) or aspirin), ADP P2Y12 receptor (AR-C), and fibrinogen receptor αIIbÎČ3 (Abciximab). BDNF release was quantified by ELISA. BDNF protein and its Tropomyosin-Related Kinase Receptor Type B (TrKB) receptor were detected by immunoblotting. Our results show that platelet activation in response to several agonists tested induced 80% platelet aggregation and augmented BDNF release by 5 folds, from 2500 pg / 250 x 106 platelets at baseline to approximately 13000 pg / 250 x 106 after stimulation. Moreover, all the tested antiplatelet agents reduced the release of BDNF by stimulated platelets. However, the level of reduction varied differentially between platelet antagonists depending on the platelet agonist used. Indeed, ASA significantly reduced BDNF secretion in response to AA, epinephrine, and TRAP-6, whereas AR-C was more effective in response to ADP, AA, and epinephrine. Abciximab inhibited BDNF secretion as well as platelet aggregation in response to all agonists. Noteworthy, the release of BDNF in response to collagen was inhibited by ASA and AR-C, while platelet aggregation was not affected. Accordingly, no significant correlation between platelet aggregation and BDNF release could be obtained. Although intra-platelet expression was similar in the different groups, the presence of antiplatelet agents reduced the release of BDNF to varying degrees between groups. As such, antiplatelet agents reduced BDNF release more significantly in patients on dual or mono antiplatelet therapy (Groups 2 and 3) as compared to healthy volunteers (Group 1) and valvular disease patients (Group 4). We have also shown that exogenous BDNF activated isolated/washed platelets from healthy volunteers, inducing strong, stable, and irreversible aggregation. In contrast, exogenous BDNF could not induce aggregation of platelets in platelet-rich plasma. In addition, our results indicate that the truncated form of the BDNF receptor, TrKB, is expressed in platelets of healthy volunteers. Hence, the inhibition of TrKB kinase activity abolished BDNF-induced aggregation. These results suggest that the action of BDNF in washed platelets might ensue through TrKB. We conclude from this study that BDNF is present in platelets and released following platelet activation, and its release is reduced by antiplatelet agents. However, platelet aggregation does not appear to be directly associated with BDNF secretion, suggesting that other underlying mechanisms may be involved in controlling its secretion. Antiplatelet agents reduce the release of BDNF, and it appears that the pro-aggregating action of BDNF on washed platelets ensues, non-exclusively, through TrKB, which means that other types of platelet receptors may also be involved in BDNF signaling. The pathophysiological implication of BDNF released following platelet activation or its bioavailability in the presence of antiplatelet agents in the cardiovascular system thus remain to be elucidated in order to reveal its diagnostic or therapeutic potential

    Head‐to‐Head comparison of consensus‐recommended platelet function tests to assess P2Y12 Inhibition : insights for multi‐center trials

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    The vasodilator‐associated stimulated phosphoprotein (VASP) phosphorylation level is a highly specific method to assess P2Y12 receptor inhibition. Traditionally, VASP phosphorylation is analyzed by flow cytometry, which is laborious and restricted to specialized laboratories. Recently, a simple ELISA kit has been commercialized. The primary objective of this study was to compare the performance of VASP assessment by ELISA and flow cytometry in relation to functional platelet aggregation testing by MultiplateÂź whole‐blood aggregometry. Blood from 24 healthy volunteers was incubated with increasing concentration of a P2Y12 receptor inhibitor (AR‐C 66096). Platelet function testing was carried out simultaneously by MultiplateÂź aggregometry and by VASP assessment through ELISA and flow cytometry. As expected, increasing concentrations of the P2Y12 receptor inhibitor induced a proportional inhibition of platelet aggregation and P2Y12 receptor activation across the modalities. Platelet reactivity index values of both ELISA‐ and flow cytometry‐ based VASP assessment methods correlated strongly (r = 0.87, p < 0.0001) and showed minimal bias (1.05%). Correlation with MultiplateÂź was slightly higher for the flow cytometry‐based VASP assay (r = 0.79, p < 0.0001) than for the ELISA‐based assay (r = 0.69, p < 0.0001). Intraclass correlation (ICC) was moderate for all the assays tested (ICC between 0.62 and 0.84). However, categorization into low, optimal, or high platelet reactivity based on these assays was strongly concordant (Îș between 0.86 and 0.92). In conclusion, the consensus‐recommended assays with their standardized cut‐offs should not be used interchangeably in multi‐center clinical studies but, rather, they should be standardized throughout sites

    Soluble CD40 ligand stimulates the pro-angiogenic function of peripheral blood angiogenic outgrowth cells via increased release of matrix metalloproteinase-9.

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    The role of endothelial progenitor cells in vascular repair is related to their incorporation at sites of vascular lesions, differentiation into endothelial cells, and release of various angiogenic factors specifically by a subset of early outgrowth endothelial progenitor cells (EOCs). It has been shown that patients suffering from cardiovascular disease exhibit increased levels of circulating and soluble CD40 ligand (sCD40L), which may influence the function of EOCs. We have previously shown that the inflammatory receptor CD40 is expressed on EOCs and its ligation with sCD40L impairs the anti-platelet function of EOCs. In the present study, we aimed at investigating the effect of sCD40L on the function of EOCs in endothelial repair. Human peripheral blood mononuclear cell-derived EOCs express CD40 and its adaptor proteins, the tumor necrosis factor receptor-associated factors; TRAF1, TRAF2 and TRAF3. Stimulation of EOCs with sCD40L increased the expression of TRAF1, binding of TRAF2 to CD40 and phosphorylation of p38 mitogen activated protein kinase (MAPK). In an in vitro wound healing assay, stimulation of EOCs with sCD40L increased the release of matrix metalloproteinase 9 (MMP-9) in a concentration-dependent manner and significantly enhanced the angiogenic potential of cultured human umbilical vein endothelial cells (HUVECs). Inhibition of p38 MAPK reversed sCD40L-induced MMP-9 release by EOCs, whereas inhibition of MMP-9 reversed their pro-angiogenic effect on HUVECs. This study reveals the existence of a CD40L/CD40/TRAF axis in EOCs and shows that sCD40L increases the pro-angiogenic function of EOCs on cultured HUVECs by inducing a significant increase in MMP-9 release via, at least, the p38 MAPK signaling pathway

    sCD40L increases MMP-9 release by EOCs.

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    <p>A) Representative gelatin zymography gel of MMP-2 and MMP-9 release by HUVECs and EOCs. Histogram represents the mean of data ± SEM of optical density. n ≄ 4, **P<0.01 <i>vs</i>. HUVECs. B) Representative gelatin zymography gel showing the release of MMP-9 by control EOCs and the effects of increasing concentration of sCD40L (0.1, 0.5 and 1.0 ÎŒg/mL) following 24 hours stimulation of EOCs. Histogram represents the mean of data ± SEM of optical density ratio. n ≄ 3,*P<0.05 <i>vs</i>. control. </p

    CD40 and TRAF expression and binding to CD40 in EOCs.

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    <p>A) Comparative CD40 and TRAF expression profiles in PBMCs and EOCs, as determined by Western Blots of cell lysates analyzed for CD40, TRAF1, 2, 3, 5 and 6 expressions; the loading control ÎČ-actin is representative of the proteins loaded for each TRAF (blots are representative of n ≄ 4). B) Representative blots showing the effect of sCD40L on TRAF association with CD40. EOCs were stimulated with sCD40L (1 ÎŒg/mL) for 15 or 30 minutes or left untreated (baseline) and CD40 was immunoprecipitated from lysates using an anti-CD40 monoclonal antibody. Immunoprecipitates were then assayed for TRAF1, 2 and 3 by SDS-PAGE. Histogram represents the mean of data ± SEM of fold increase in optical density over baseline. n ≄ 4, **P<0.01 <i>vs</i>. baseline. C) Representative blots showing the effect of sCD40L on TRAF expression in EOCs following 24 hour stimulation with sCD40L (1 ÎŒg/mL); the loading control ÎČ-actin is representative of the proteins loaded for each TRAF. Histogram represents the mean of data ± SEM of fold increase in optical density over control. n=3, *P<0.05. </p

    Enhancement of MMP-9 release in sCD40L-stimulated EOCs is p38 MAPK-dependent.

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    <p>Representative gelatin zymography gel of supernatants of EOCs and sCD40L (1 ÎŒg/mL)-, p38 inhibitor SB203580 (10 ÎŒM)-, and double sCD40L- and SB203580-treated EOCs. Histogram represents the mean of data ± SEM of fold increase in optical density over control EOCs. n ≄ 3, *P<0.05 <i>vs</i>. other groups. </p
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