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Monoamine Oxydases Et Athérosclérose : Signalisation Mitogène Et Études in Vivo

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Monoamine Oxydases Et Athérosclérose : Signalisation Mitogène Et Études in Vivo UNIVERSITE TOULOUSE III - PAUL SABATIER Sciences THESE Pour obtenir le grade de DOCTEUR DE L’UNIVERSITE TOULOUSE III Discipline : Innovation Pharmacologique Présentée et soutenue par : Christelle Coatrieux le 08 octobre 2007 Monoamine oxydases et athérosclérose : signalisation mitogène et études in vivo Jury Monsieur Luc Rochette Rapporteur Professeur, Université de Bourgogne, Dijon Monsieur Ramaroson Andriantsitohaina Rapporteur Directeur de Recherche, INSERM, Angers Monsieur Philippe Valet Président Professeur, Université Paul Sabatier, Toulouse III Madame Nathalie Augé Examinateur Chargé de Recherche, INSERM Monsieur Angelo Parini Directeur de Thèse Professeur, Université Paul Sabatier, Toulouse III INSERM, U858, équipes 6/10, Institut Louis Bugnard, CHU Rangueil, Toulouse Résumé Les espèces réactives de l’oxygène (EROs) sont impliquées dans l’activation de nombreuses voies de signalisation cellulaires, conduisant à différentes réponses comme la prolifération. Les EROs, à cause du stress oxydant qu’elles génèrent, sont impliquées dans de nombreuses pathologies, notamment l’athérosclérose. Les monoamine oxydases (MAOs) sont deux flavoenzymes responsables de la dégradation des catécholamines et des amines biogènes comme la sérotonine ; elles sont une source importante d’EROs. Il a été montré qu’elles peuvent être impliquées dans la prolifération cellulaire ou l’apoptose du fait du stress oxydant qu’elles génèrent. Ce travail de thèse a montré que la MAO-A, en dégradant son substrat (sérotonine ou tyramine), active une voie de signalisation mitogène particulière : la voie métalloprotéase- 2/sphingolipides (MMP2/sphingolipides), et contribue à la prolifération de cellules musculaire lisses vasculaires induite par ces monoamines. De plus, une étude complémentaire a confirmé l’importance des EROs comme stimulus mitogène (utilisation de peroxyde d’hydrogène exogène), et a décrit plus spécifiquement les étapes en amont de l’activation de MMP2, ainsi que l’activation par la MMP2 de la sphingomyélinase neutre (première enzyme de la cascade des sphingolipides). Enfin, une étude a été menée in vivo sur un modèle de souris développant des plaques d’athérome (ApoE -/-), visant à étudier le rôle athéroprotecteur d’un inhibiteur de MAOs (IMAO), comparé à des piégeurs de carbonyle (ayant pour certains des propriétés IMAOs également). Cette étude montre un effet protecteur des piégeurs de carbonyles, indépendant de leurs propriétés IMAOs, et un faible effet de l’IMAO seul, dans la formation des lésions vasculaires primaires. En conclusion, ce travail a montré une implication du stress oxydant, et particulièrement des MAOs dans la prolifération des cellules musculaires lisses vasculaires. Il a permis également de mieux décrire l’interaction entre MMP2 et la sphingomyélinase neutre ainsi que la voie de signalisation activée en amont de MMP2. Enfin, bien que l’étude visant à montrer l’effet athéroprotecteur d’un IMAO sur lésion primaires n’ait pas montré d’efficacité aussi importante que celle notée pour des molécules (IMAOs ou non) piégeurs de carbonyles, ceci n’exclut pas un rôle des MAOs dans la formation des lésions avancées. Abstract Reactive oxygen species (ROS) are involved in the activation of many signaling pathways, leading to different responses, such as cell proliferation, inflammation, differentiation or apoptosis. The oxidative stress generated by ROS is involved in the initiation and evolution of many diseases including atherosclerosis. Monoamine oxidases (MAOs) are two flavoenzymes that catalyze the oxidative deamination of catecholamines and biogenic amines, such as serotonin and tyramine. The degradation of biogenic amines by MAOs generates a large amount of ROS that may play a role in the biological properties of these agents like cell proliferation or apoptosis. This study showed that ROS generated during the degradation of serotonin or tyramine by MAO-A, trigger the activation of a specific stress-induced mitogenic pathway, namely the metalloprotease2-neutral sphingomyelinase2 pathway (MMP2/nSMase2), which is involved in vascular smooth muscle cell proliferation. This ROS-induced MAO-A dependent signalling was mimicked by exogenous hydrogen peroxyde, which allowed to demonstrate that ROS- induced SMC proliferation requires the downstream activation of sphingosine kinase-1 (SK- 1), a key enzyme in survival and proliferation signalling via the generation of sphingosine 1- phosphate (S1P). Furthermore, we report preliminary data on the mechanisms involved upstream the activation of MMP2 and of nSMase2 (which implicate the proprotein convertase and MT1-MMP, the physiological activator of MMP2). Lastly, the protective effect of MAO inhibitors has been evaluated in vivo on an animal model for atherosclerosis, the apoE-/- mice. This study indicated that hydrazinic MAO inhibitors were very efficient in inhibiting the development of atherosclerotic lesions in apoE -/- mice, but this was due to the carbonyl scavenger properties of hydrazine and not to their IMAO effect, because non hydrazinic IMAO did not exhibited any protective effect. These data indicate that MAO-dependent oxidative stress is not involved in the formation of early atherosclerotic lesions, which doesn’t exclude an involvement in more advanced states, plaque rupture and remodelling. In conclusion, this work allowed to demonstrate a role of MAO-dependent oxidative stress in SMC proliferation, via a new stress-induced signalling mechanism, the MMP2/nSMase2/SK-1 pathway, and suggests a role for ROS in the formation and the stability/fragility of fibrous cap in atherosclerosis. Principales abréviations utilisées AGE : Advanced Glycoxydation MAPK: Mitogen-Activated Protein Kinase Endproduct MDA: Malondialdehyde ALE : Advanced Lipoxydation Endproduct MMP: Matrix MetalloProtease ApoE -/- : Knockout ApolipoprotéineE MT-MMP: Membrane-type CE : Cellules Endothéliales MetalloProtease CML : Cellules Musculaires Lisses NAD(P)H oxydase : Nicotinamide adénine dinucléotide phosphate oxydase ERK : Extracellular-signal Regulated Kinase NF-κB : Nuclear Factor -κB ERO : Espèce Réactive de l’Oxygène PDGF : Platelet-derived Growth Factor FAD : Flavin Adenin Dinucleotid SERT : Transporteur à la sérotonine H2O2 : Peroxyde d’hydrogène Skase : Sphingosine kinase HDL : High Density Lipoprotein S1P : Sphingosine 1-phosphate 4-HNE : 4-Hydroxynonenal SM : Sphingomyéline 5-HT : 5-hydroxytryptamine (sérotonine) nSMase : Sphingomyélinase euter IMAO : Inhibiteur de monoamine oxydase TIMP: Tissue inhibitor of MMP LDL: Low-Density Lipoprotein TNF alpha (ou TNFα):Tumor Necrosis Factor alpha LDLox: Low-Density Lipoprotein oxydées Trp : Tryptophane MAO: Monoamine oxydase SOMMAIRE I. Les Monoamine Oxydases : généralités..................................................................................................... 1 Isoformes............................................................................................................................................................ 1 Structure............................................................................................................................................................. 2 Localisation........................................................................................................................................................ 2 Fonction des MAOs............................................................................................................................................ 5 a. Réaction enzymatique catalysée .......................................................................................................... 5 b. Rôle au niveau du système nerveux central ......................................................................................... 5 c. Rôle au niveau des organes périphériques ........................................................................................... 7 d. Nouveau rôle lié à la production d’EROs ............................................................................................ 7 Substrats et inhibiteurs....................................................................................................................................... 8 a. Substrats............................................................................................................................................... 9 b. Inhibiteurs .......................................................................................................................................... 10 Pathologies associées....................................................................................................................................... 11 a. Les maladies neurodégénératives et psychiatriques........................................................................... 11 b. Le diabète........................................................................................................................................... 12 II. Monoamine oxydases et stress oxydant ................................................................................................... 14 Apoptose........................................................................................................................................................... 14 Hypertrophie (cardiomyocytes)........................................................................................................................ 15 Prolifération....................................................................................................................................................
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