La myosine est une protéine motrice impliquée dans la contraction du muscle lisse. Un de ces isoformes, appelé (+)insert lui procure une vélocité accrue. L’objectif de cette étude était d’évaluer la contribution de cet isoforme au bronchospasme observé chez les chevaux atteints de souffle, un modèle d’asthme humain. Nous avons procédé à l’analyse de l’expression génique de l’isoforme (+)insert dans les voies respiratoires de chevaux issus de 3 cohortes afin d’évaluer 1) la présence de cet isoforme chez les chevaux atteints de souffle ; 2) la réversibilité de son expression avec 2 traitements usuels (corticostéroïdes inhalés et retrait antigénique); et 3) la cinétique de réapparition de l’isoforme suite à une période de rémission de la maladie. Les résultats révèlent 1) une expression augmentée de l’isoforme (+)insert dans l’ensemble de l’arbre bronchique des chevaux atteints de souffle en exacerbation comparée aux contrôles et aux chevaux en rémission ; 2) les deux traitements conduisent à une réduction de l’expression de l’isoforme, mais l’administration de corticostéroïdes mène à une diminution plus rapide que celle induite par le retrait antigénique; et 3) une période de 30 jours ou plus d'exposition antigénique est nécessaire pour la réapparition de l’isoforme (+)insert dans l’arbre bronchique des chevaux atteints de souffle. Cette étude montre pour la première fois une modulation et une réversibilité de l’expression de l'isoforme (+)insert de la myosine en fonction du statut médical du sujet. Les données suggèrent que cet isoforme pourrait faire partie d’un mécanisme protecteur activé en réponse à une exposition antigénique prolongée. / Myosin is a motor protein implicated in smooth muscle contraction. The (+)insert isoform doubles smooth muscle velocity. The aim of the study was to evaluate the contribution of the fast contracting myosin isoform to bronchospasm in heaves-affected horses as model of human asthma. Gene expression analysis of the (+)insert myosin isoform in equine airways was performed in three different cohorts to evaluate 1) its presence and anatomical location in heaves-affected horses; 2) the reversibility of its expression with two common therapies (inhaled corticosteroids and antigen avoidance); and 3) its kinetics of reappearance following a chronic antigen exposure. Results showed 1) a significantly increased expression of the (+)insert isoform at all levels of the bronchial tree of horses with heaves in clinical exacerbation when compared to control horses and heaves-affected horses in clinical remission; 2) both treatments reduced the fast isoform expression in horses’ lower airways, but the administration of corticosteroids led to a faster response; and 3) (+)insert isoform does not increase in airways of all horses with heaves within 30 days of antigen exposure. The study shows for the first time a modulation of the fast contracting myosin isoform with the disease status. Data also suggest that (+)insert isoform is part of a protective mechanism enhanced in response to a prolonged antigen exposure.

Il a été démontré que les chevaux atteints du souffle présentent une augmentation de la masse de muscle lisse entourant les voies respiratoires comparativement à des chevaux sains (Herszberg, Ramos-Barbon et al. 2006, Leclere, Lavoie-Lamoureux et al. 2011). L’augmentation de la masse de muscle lisse ainsi observée résulte d’une hyperplasie, et possiblement, d’une hypertrophie des myocytes. Les traitements usuels du souffle ne sont que partiellement efficaces à diminuer cette augmentation. L’objectif de cette étude était d’explorer les mécanismes moléculaires impliqués dans ces changements affectant la cellule musculaire lisse dans la pathologie du souffle chez le cheval. Pour ce faire, nous avons examiné les effets d’une exposition antigénique sur l’expression du «Serum Response Factor» (SRF) dans le muscle lisse bronchique. Le SRF est un facteur de transcription localisé dans le noyau de la cellule musculaire lisse et régulant l’expression génique de celle-ci en favorisant un phénotype prolifératif ou contractile. Les résultats démontrent qu’avant exposition antigénique, les pourcentages de cellules exprimant le SRF sont faibles. Une augmentation significative du pourcentage de myocytes exprimant le SRF survient suite à une stimulation antigénique chez les chevaux atteints de souffle alors qu’aucune augmentation n’est observée chez les chevaux contrôles. Ces résultats suggèrent que le SRF pourrait contribuer au remodelage du muscle lisse péribronchique dans la pathologie du souffle. / Previous studies have shown that heaves-affected horses, when compared to age-matched control horses, have an increased smooth muscle mass surrounding their airways (Herszberg, Ramos-Barbon et al. 2006, Leclere, Lavoie-Lamoureux et al. 2011). Also, it is shown that hyperplasia, and possibly hypertrophy, contribute to this finding. Current therapies are only partially effective at reversing this finding. The goal of this study was to explore the molecular pathways involved in airway smooth muscle remodelling during heaves. We studied the Serum Response Factor (SRF), a nuclear transcriptional factor that controls gene expression in the smooth muscle cell and favour either a proliferative or a contractile phenotype. The results show that before antigenic exposition, the percentage of cells expressing SRF is low. A significant increase in the percentage of SRF-expressing myocytes occurs after antigenic stimulation in heaves-affected horses whereas no increase is observed in control subjects. These results suggest that the transcription factor SRF may contribute to airway smooth muscle remodeling in heaves-affected horses.

L’hyperplasie et l’hypertrophie contribuent à l'augmentation de la masse de muscle lisse bronchique observée dans le souffle. Les cellules musculaires lisses (CML) présentent deux phénotypes; prolifératif ou contractile. Le serum response factor (SRF), un facteur de transcription impliqué dans l’activation de nombreux gènes, contribuerait à cette modulation phénotypique. Notamment, lorsqu'associé au cofacteur Elk-1, un phénotype prolifératif serait observé, alors qu'en présence de la myocardine (MYOCD) il y aurait induction d'un profil contractile. Récemment, il a été démontré que SRF est surexprimé dans les voies périphériques chez les chevaux atteints du souffle suite à une exposition antigénique. Cette étude vise à caractériser l'expression protéique et génique de SRF, Elk-1 et MYOCD dans les CML des voies respiratoires centrales et périphériques chez des chevaux atteints du souffle et des chevaux contrôles. L'évaluation de l’expression protéique de SRF, Elk-1 et MYOCD s’est effectuée par immunodétection sur des tissus provenant de biopsies thoracoscopiques ou endobronchiques, et ce, avant, à 1 et 30 jours du défi antigénique. L'expression génique a été étudiée par qPCR sur du muscle lisse disséqué de la trachée, et des bronches, ainsi que sur des voies respiratoires intermédiaires et périphériques. Les expressions génique et protéique de MYOCD sont augmentées uniquement dans les voies périphériques. L’expression génique de SRF et Elk-1 varient dans les voies centrales alors que le taux de protéines demeure stable. En conclusion, SRF et MYOCD pourraient être impliquées dans l’hypertrophie des voies respiratoires périphériques dans le souffle alors que l’hyperplasie ne semble pas être activée par Elk-1. / Airway smooth muscle (ASM) cells hyperplasia and hypertrophy contribute to the increased airway smooth muscle mass present in heaves. ASM cells express either a synthetic proliferative or a contractile phenotype. Serum response factor (SRF) is a transcription factor that has been shown to regulate myocyte differentiation in vitro in vascular and intestinal smooth muscles. When SRF is associated with Elk-1, it promotes ASM proliferation while myocardin (MYOCD) promotes the expression of contractile elements. Recently, SRF was shown to be overexpressed in the peripheral airways of heaves affected horses following an antigenic challenge. The objective of this study was to characterize the protein and gene expression of SRF, Elk-1 and MYOCD in ASM cells from central and peripheral airways of heaves affected horses and controls. Protein expression of Elk-1 and MYOCD was evaluated using immunohistochemistry while immunofluorescence was used for SRF detection in pulmonary peripheral and endobronchial biopsies before and at 1 and 30 days of antigenic exposure. Gene expression was investigated in ASM cells dissected from trachea and bronchi as well as from intermediate and peripheral airways using qPCR. MYOCD gene and protein expressions are increased only in peripheral airways. SRF and Elk-1 gene expression varied in the central airways while the positive cell percentage remains stable. In conclusion, the pulmonary peripheral airways hypertrophy observed in heaves seems to implicate SRF and MYOCD while the hyperplasia doesn’t seem to be activated by Elk-1.

Muscle mechanical behavior potentially plays an important role in some of the most common bladder disorders. These include overactive bladder, which can involve involuntary contractions during bladder filling, and impaired contractility or underactive bladder, which may involve weak or incomplete contractions during voiding. Actin-myosin cross-bridges in detrusor smooth muscle (DSM) are responsible for contracting and emptying the bladder. The total tension produced by muscle is the sum of its preload and active tensions. Studies suggest that actin-myosin cross-links are involved in adjustable preload stiffness (APS), which is characterized by a preload tension curve that can be shifted along the length axis as a function of strain history and activation history. DSM also exhibits length adaptation in which the active tension curve can exhibit a similar shift. Actin-myosin cross-bridges are also responsible for myogenic contractions in response to quick stretch of DSM strips and spontaneous rhythmic contractions (SRC) that may occur during bladder filling. Studies show that SRC may participate in the mechanical regulation of both APS and length adaptation. However, the mechanical mechanisms by which actin-myosin interactions enable this interrelated combination of behaviors remain to be determined and were the primary focus of this dissertation. The objectives of this study were to: 1) provide evidence to support the hypothesis that a common mechanism is responsible for SRC and myogenic contraction, 2) develop a sensor-based mechanical model to demonstrate that SRC in one cell is sufficient to trigger stretch-induced myogenic contraction in surrounding cells and propagate the contraction, and 3) develop a conceptual model with actin-myosin cross-bridges and cross-links that produces the coupled mechanical behaviors of APS, SRC, and length adaptation in DSM. Improved understanding of bladder biomechanics may enable the identification of specific targets for the development of new treatments for overactive and underactive bladder.

Les tumeurs stromales gastro-intestinales (GIST) sont les tumeurs mésenchymateuses les plus fréquentes du système digestif. Elles ont pour origine les cellules interstitielles de Cajal (ICC) ou les cellules précurseurs mésenchymateuses communes aux ICCs et aux cellules musculaires lisses (SMC). Les GISTs sont des tumeurs qui sont chimiorésistantes et radiorésistantes. L'identification de mutations activatrices des gènes KIT (75-80%) ou/et PDGFRA (5-10%) a ouvert la voie à un traitement systémique chez les patients GIST sous forme d'Imatinib, un inhibiteur de tyrosine-kinase. Si ce traitement aboutit à une réponse clinique d'amélioration, un certain nombre d'effet secondaire sont néanmoins observés, comme les résistances au traitement. Afin d'améliorer le traitement initial, la physiopathologie du GIST doit progresser. La musculature de l'appareil digestif est une structure complexe composée de SMCs, de neurones entériques, de fibroblastes et d'ICCs. Au cours du développement, le mésoderme splanchnique donnera lieu au moins à deux types de cellules, les SMCs et les ICCs. Récemment, notre laboratoire a montré que la protéine de liaison à l'ARN RBPMS2 (pour RNA Binding Protein with Multiple Splicing 2) est impliquée dans le développement et le remodelage des SMCs digestives. Les travaux que j'ai réalisés au cours de ma thèse avaient pour objectifs d'étudier l'expression et la fonction de RBPMS2 dans les tumeurs GISTs humains. Nous avons analysé l'expression de RBPMS2 dans les GIST humains et nous avons démontré que RBPMS2 était fortement exprimé dans les tumeurs GISTs de manière indépendante de l'activité KIT. Nous avons également analysé la fonction de RBPMS2 en culture et avons montré que l'expression ectopique de RBPMS2 dans les SMCs humaines adultes et différenciées culture conduisaient à l'augmentation de leur taux de prolifération et altèreraient leur différenciation. Ces résultats suggèrent que RBPMS2 et les voies de signalisation qu´il contrôle pourraient être des cibles thérapeutiques potentielles dans la thérapie des tumeurs GISTs. / Gastrointestinal stromal tumors (GIST) are the most common mesenchymal neoplasm of the GI tract. They are supposed to arise from the interstitial cells of Cajal (ICCs) or from a mesenchymal precursor cell, common of ICCs and smooth muscle cells (SMCs). GISTs are highly resistant to conventional chemotherapy and radiotherapy. However, a targeted therapy is now proposed. These tumors have activating mutations in two closely related genes, the KIT (75-80%) or/and the PDGFRA (5-10%). Targeting these mutated activated proteins with Imatinib mesylate, a small-molecule tyrosine kinase inhibitor, has proven efficient in GIST treatment. However, resistance to Imatinib finally develops and new-targeted therapies are necessary. The musculature of the gastrointestinal (GI) tract is a highly complex structure composed of visceral SMCs, enteric neurons, fibroblast-like cells and ICCs. During the development, the splanchnic mesoderm will give rise at least to two cell types, ICCs and SMCs. Recently our laboratory showed that the RNA Binding Protein with Multiple Splicing 2 (RBPMS2) is involved into the development and remodeling of SMC.My PhD works investigate the expression and function of RBPMS2 in human GISTs. We analyzed the expression of RBPMS2 in human GISTs and we found that RBPMS2 was abnormally highly expressed in the tumoral cells of GISTs. We also analyzed the function of RBPMS2 into human adult SMC cell culture and demonstrated that ectopic expression of RBPMS2 in mature and differentiated SMC cultures increases their proliferation rate and alters their differentiation. These findings suggest that RBPMS2 could be a potential target for cancer therapy.

Lesão do DNA mitocondrial (mtDNA) promove disfunção desta organela, contribuindo para a gênese do envelhecimento e fisiopatologia de doenças como aterosclerose e diabetes. A mitocôndria é a principal fonte quantitativa de espécies reativas de oxigênio (ROS) em células, e o complexo NAD(P)H oxidase a principal fonte de ROS envolvidas na sinalização celular. A possível inter-relação entre estas duas importantes vias produtoras de ROS não está definida. O objetivo deste estudo foi investigar o perfil de alterações na expressão e atividade da NAD(P)H oxidase de células musculares lisas vasculares (VSMC) em resposta a perturbações mínimas da função mitocondrial análogas às esperadas em doenças crônico-degenerativas vasculares. Inicialmente, validamos modelo in vitro de disfunção mitocondrial induzida por incubação de VSMC com brometo de etídio (24 - 72 h). Lesões mínimas do mtDNA foram documentadas por alterações nos produtos de amplificação (PCR) da região repetitiva da D-loop e redução da taxa de consumo de oxigênio total em ~15% vs. basal (p<0,05). Este grau de lesão não foi suficiente para induzir alterações morfológicas evidentes ou apoptose, e foi associado ao retardo de 25 - 30% no aumento de população celular induzido por soro fetal bovino. Nestas condições, não se detectou aumento da produção basal de superóxido ou mudanças nos níveis de glutationa, óxidos de nitrogênio, ou da atividade superóxido dismutase. A produção basal de peróxido de hidrogênio aumentou ~15%. Após disfunção mitocondrial, houve significativo aumento (30 - 45%) na atividade basal do complexo NAD(P)H oxidase em fração de membrana de VSMC. Entretanto, a ativação da oxidase pela AII, conhecido agonista da oxidase vascular, foi essencialmente abolida, indicando dependência funcional da ativação da oxidase com a integridade da mitocôndria. Em sintonia com esses dados, na condição basal, ocorreu aumento de expressão da isoforma Nox4 da oxidase, enquanto o aumento do mRNA da Nox1 normalmente visto após AII foi minimizado. Por outro lado, o aumento da atividade da NADPH oxidase causado pelo estressor do RE tunicamicina (indutor de Nox4) foi também abolido pela disfunção mitocondrial, entretanto, ocorreu aumento do mRNA da Nox4, indicando que as alterações funcionais da oxidase nesta situação não decorrem apenas de mudanças da expressão. Dissociação semelhante entre expressão e atividade ocorreu após exposição de 72 horas ao EtBr (i.e., durante adaptação). Nesta, ocorreu maior expressão do mRNA de Nox1 e Nox4 com AII, sem aumento da atividade da oxidase em membranas. Incubação do EtBr por 24 horas não induziu per se aumento consistente nos índices de estresse do RE e induziu inversão do padrão do tráfego subcelular da dissulfeto isomerase protéica (PDI), uma chaperona redox descrita recentemente como reguladora da NADPH oxidase. Após 72 horas de incubação com EtBr, a expressão de chaperonas marcadoras de estresse do RE foi bastante diminuída e o tráfego da PDI teve o padrão restaurado. Demonstramos por microscopia confocal evidências preliminares de possível co-localização entre Nox1 e mitocôndria. Estes dados sugerem uma relevante inter-relação funcional entre mitocôndria e complexo NAD(P)H oxidase, associada pelo menos a alterações de expressão e/ou tráfego subcelular de subunidades catalíticas e reguladoras desse complexo. / Mitochondrial DNA (mtDNA) damage induces dysfunction of this organelle, contributing to the genesis of aging and to the pathophysiology of diseases such as atherosclerosis and diabetes. Mitochondria are the main quantitative source of reactive oxygen species (ROS) in cells, while NAD(P)H oxidase complex is a major source of cell signaling-associated ROS. The possible crosstalk between these two relevant sources of ROS is unclear. The aim of this study was to investigate changes in activity and/or expression of vascular smooth muscle cell (VSMC) NAD(P)H oxidase in response to minor perturbations of mitochondrial function similar to those expected to occur in chronic degenerative vascular diseases. Initially, we validated an in vitro model of mitochondrial dysfunction in VSMC, through incubation with ethidium bromide (24 - 72 h). Minimal mtDNA damage after EtBr was shown by distinct amplification patterns (at PCR) of D-loop repetitive region and by ~ 15% oxygen consumption decrease vs. basal (p<0.05). Such mtDNA damage was not sufficient to induce morphologic changes or apoptosis, whereas serum-stimulated increase in cell number was prevented by 25-30%. Under those conditions, baseline superoxide production, as well as levels of glutathione or nitrogen oxides or superoxide dismutase activity were unchanged. Baseline hydrogen peroxide production increased ~15%. VSMC membrane fraction NADPH oxidase activity was increased by 30-45% after mitochondrial dysfunction. However, oxidase activation due to AII (100 nM, 4h) was markedly abrogated, indicating that A-II-driven oxidase activation requires integrity of mitochondrial function. Accordingly, there were increases in baseline mRNA expression of Nox4 oxidase isoform, while the expected increase in Nox1 by AII was minimized. On the other hand, the NADPH oxidase activity induced by the endoplasmic reticulum stressor tunicamycin (Nox4 inducer) after mitochondrial dysfunction was abrogated, however simultaneously with increased Nox4 mRNA, thus indicating that the observed functional alterations in the oxidase complex in these conditions cannot be associated only to mRNA expression changes. After VSMC EtBr incubation for 72 h, similar dissociation between expression and activity was observed, with increase in Nox 1 and Nox4 mRNA by AII, without parallel increase in membrane fraction oxidase activity. Although there was little change in ER stress markers after 24h EtBr, protein disulfide isomerase (PDI), a redox chaperone recently described by us as a novel NAD(P)H oxidase regulator, exhibited a reversal of its subcellular traffic pattern. After 72 h EtBr, the expression of ER markers was strongly decreased and normal PDI traffic was restored. Confocal microscopy suggested possible co-localization between Nox1 and mitochondria. These results suggest a functionally relevant crosstalk between mitochondria and NADPH oxidase complex associated at least to changes in expression and/or subcellular traffic of catalytic or regulatory subunits of this complex.

INTRODUÇÃO: Os pacientes com asma crônica podem compartilhar similaridades clínicas e fisiológicas com pacientes com doença pulmonar obstrutiva crônica, tal como reversibilidade parcial ao broncodilatador ou pouca obstrução persistente do fluxo expiratório. Entretanto, não existem estudos comparando a patologia destas duas doenças em pacientes com idade similares e mesma gravidade da doença. MÉTODOS: Nós comparamos as dimensões das grandes e pequenas vias aéreas de 12 pacientes adultos (média±erro padrão, 32±3 anos) e 15 pacientes idosos e pré-idosos idosos (65±1 ano) não tabagista que foram a óbito por asma fatal com 14 pacientes tabagistas crônicos que foram a óbito por DPOC grave (71± 1 ano) e 19 pacientes-controle (56±1 ano). Usando a coloração de Movat e H&E, e a técnica de análise de imagens, nós quantificamos a espessura da membrana basal (MB) (valores expressos em ?m) a área de glândula submucosa nas grandes vias aéreas. Nas grandes e pequenas vias aéreas quantificamos a área de camada interna, a área de músculo liso e a área de camada externa. As áreas foram normalizadas pelo perímetro da MB (?m/?m2). RESULTADOS: os pacientes asmáticos adultos apresentaram a MB, área de músculo liso e a área da camada externa nas grandes e pequenas vias aéreas mais espessas, quando comparadas com os controles com idade similar com DPOC grave. Nos pacientes idosos e pré-idosos com asma, houve uma sobreposição na espessura da MB e na área da glândula submucosa, enquanto que nas pequenas e grandes vias aéreas a área de músculo liso foi mais espessa quando comparados com os controles com idade similar com pacientes com DPOC grave. Os pacientes com DPOC apresentaram nas pequenas e grandes vias aéreas as áreas de músculo liso menor quando comparada aos controles com idade similar. Os asmáticos adultos apresentaram a área de músculo liso maior quando comparada aos asmáticos idosos. CONCLUSÃO: Nossos dados fornecem novas informações sobre as mudanças patológicas que podem nos ajudar a entender melhor as similaridades e diferenças patológicas no pacientes adultos e idosos com asma comparados ao DPOC / Background: In some patients with chronic asthma, clinical and physiological similarities with chronic obstructive pulmonary disease may co-exist, such as partial reversibility to bronchodilators despite persistent expiratory airflow obstruction. However, pathologic analyses comparing both diseases in patients of similar age and disease severity are scarce. Methods: We compared the large and small airway dimensions in 12 younger (mean±SD, age 32 yr±3 yr) and 15 older (65 yr±1 yr) non-smoking fatal asthmatics with 14 chronic smokers with severe, fatal COPD (71 yr±1 yr) and 19 control patients (56 yr±1 yr). Using H&E, Movat\'s pentachrome staining and image analysis, we quantified large airway basement membrane (BM) thickness (?m); submucosal gland area; and large and small airway inner wall, smooth muscle and outer wall areas. Areas were normalized by BM perimeter (?m2/?m). Results: Younger adult fatal asthmatics had thicker BM, smooth muscle, and outer wall areas in both small and large airways when compared to agematched controls and fatal COPD patients. In older asthmatics, there was an overlap in BM thickness and submucosal gland area, whereas both large and small airway smooth muscle areas were thicker compared to age-matched controls and fatal COPD patients. COPD patients had thinner large and small airway smooth muscle areas compared to age-matched controls. Younger asthmatics had thicker small airway smooth muscle area compared to older asthmatics. Conclusion: Our data provide novel pathological substrate changes that may help us better understand physiological similarities and differences in younger and older patients with asthma compared to COPD.

La « semicarbazide-sensitive amine oxidase » (SSAO) catalyse la déamination oxydative des amines primaires en aldéhyde, peroxyde d’hydrogène et ammoniac. Elle participe à la différenciation cellulaires, l’inflammation et la transmigration leucocytaire à travers l’endothélium lymphatique. Nos objectifs ont été d’étudier le rôle de la SSAO (i) dans la différenciation chondrocytaire hypertrophique, en relation avec le développement de l’arthrose en utilisant des chondrocytes de rat en culture primaire et des genoux arthrosiques de patients (ii) dans le développement de l’athérosclérose en invalidant des souris ApoE-/- qui développent naturellement l’athérosclérose pour le gène de la SSAO. Au niveau articulaire, la SSAO a été détectée dans le cartilage de rat et humain. In vitro, la SSAO (activité et expression) augmentent au cours de la différenciation terminale de chondrocytes de rat. Son inhibition par le LJP1586 entraîne un retard de différenciation chondrocytaire. La SSAO augmente également dans les zones arthrosiques du cartilage humain parallèlement à l’augmentation de l’hypertrophie. La SSAO jouerait donc un rôle dans la différenciation terminale des chondrocytes (hypertrophie) possiblement via le transport de glucose et dans le développement de la maladie. Au niveau vasculaire, les souris femelles ApoE-/-SSAO-/- de 25 semaines présentent une augmentation de la surface des plaques d’athérome par rapport aux ApoE-/-. Ceci est associée à une diminution de l’expression d’α-actine dans le média sous les plaques et de smMHC dans l’aorte abdominale (AA) sans modification ni de l’infiltration des lymphocytes T; ni des monocytes/ macrophages dans la paroi artérielle, ni du profil cytokinique pro-/anti-inflammatoire dans la rate. A 15 semaines, les souris femelles ApoE-/-SSAO-/-, sm-MHC a diminué dans les AA de ces souris par rapport aux ApoE-/- ainsi qu’une réorientation du trafic des cellules immunitaires vers la paroi aortique sans modification significative de la surface des plaques a été détecté. La SSAO jouerait donc un rôle précoce dans le développement de l’athérosclérose via une modification du trafic des cellules immunitaires et du phénotype des CML dans la paroi / The semicarbazide-sensitive amine oxidase (SSAO) catalyzes the oxidative deamination of primary amines into aldehydes, hydrogen peroxide and ammonia. The SSAO was implicated in cellular differentiation, inflammation and transmigration of leukocyte through the lymphatic. The objectives of this work were to study the role of SSAO (i) in chondrocyte differentiation and in the development of osteoarthritis using rat chondrocyte primary cell culture and osteroarthritic samples from patients. (ii) in the development of atherosclerosis using ApoE-/- mice, which develop naturally atherosclerosis, invalidated for the SSAO gene. Concerning the articulation, the SSAO (expression and activity) was detected in the rat and human cartilage. In vitro, SSAO increases during chondrocyte terminal differentiation (hypertrophy) and the inhibition of its activity by LJP1586, decreases the level of differentiation. In human arthritic cartilage, SSAO was higher that in healthy cartilage, in association with an increase in hypertrophic markers. The SSAO plays a role in the terminal differentiation of chondrocytes and might be involved in the development of osteoarthritis. At the vascular level, 25 week-old female ApoE-/-SSAO-/- mice presented a 50% increase in plaque surface associated with an 80% decrease in α-actin expression in the media of aortic sinus and a decrease in sm-MCH in abdominal aortas (AA) compared to ApoE-/- mice. These results were not due neither to a modification of monocytes/ macrophages, Tcell infiltration in the plaque nor in a pro- or anti-inflammatory cytokine change in spleen. In 15 week-old ApoE-/-SSAO-/- mice, even if no modification of plaque surface was found, a decrease in sm-MHC was noticed in the AA from ApoE-/-SSAO-/- compare to ApoE-/- mice. More over, the immune cell trafficking was increased in the aortic wall of ApoE-/-SSAO-/- compared to ApoE-/- mice. Thus, SSAO is involved in the early development of atherosclerosis in changing the immune cell trafficking and the VSMC phenotype

La rigidité artérielle et la fibrose ont une valeur prédictive dans le développement des maladies cardiovasculaires (CV). Ces 2 phénotypes impliquent les cellules musculaires lisses vasculaires (CMLVs) notamment des récepteurs membranaires et les protéines du cytosquelette. Les objectifs ont été d’étudier : (i) l’influence du récepteur minéralocorticoïde (MR) sur la réactivité vasculaire, (ii) le rôle de l’intégrine αvβ3 dans le développement de la rigidité artérielle et la fibrose vasculaire, et (iii) l’impact de la vimentine et la synémine sur la structure et la fonction artérielle. Ces trois études ont utilisées des souris avec invalidation génétiques des protéines d’intérêt. Résultats : l’absence du MR diminue la réactivité vasculaire en altérant le couplage contraction/relaxation des CMLVs via des mécanismes Ca2+- et NO-dépendants (une diminution de la vasoconstriction en réponse au Ca2+ extracellulaire et une altération de la vasorelaxation endothélium-dépendante en réponse à l’acétylcholine). L’invalidation de la sous-unité αv prévient la fibrose en réponse à l’administration d’angiotensine II. L’absence de la vimentine et non celle de la synémine augmente la rigidité artérielle via des changements des adhésions focales des CMLVs mais aussi des cellules endothéliales. En conclusion, les récepteurs membranaires et protéines intracellulaires étudiées influencent la fonction et la structure des artères grâce à des actions spécifiques sur le tonus musculaire, la mécanotransduction et l’organisation ultra-structurale des CMLVs. Ces études montrent au niveau cellulaire et moléculaire le déterminisme plurifactoriel des phénotypes de rigidité-fibrose de la paroi artérielle. Ces résultats nécessitent des travaux plus mécanistiques pour affirmer l’implication de ces protéines dans les maladies CV liées au vieillissement / Arterial stiffness and fibrosis have a predictive value in the development of cardiovascular diseases (CV). These two phenotypes involve vascular smooth muscle cells (VSMCs) including membrane receptors and cytoskeletal proteins. The objectives were to examine: (i) the influence of the mineralocorticoid receptor (MR) on vascular reactivity, (ii) the role of avb3 integrin in the development of arterial stiffness and vascular fibrosis, and (iii) the impact of vimentin and synemin on arterial structure and function. The mice with genetic invalidation of the proteins of interest were used in these three studies. Results: the absence of MR decreased vascular reactivity by altering the contraction/relaxation coupling of VSMC through Ca2+- and NO-dependent mechanisms (a decrease of vasoconstriction in response to extracellular Ca2+ and impaired endothelium-dependent vasorelaxation in response to acetylcholine). The invalidation of the αv subunit prevented fibrosis in response to the administration of angiotensin II. The absence of vimentin, and not that of the synemin, increased arterial stiffness via changes in focal adhesions of VSMCs as well as endothelial cells. In conclusion, the studied membrane receptors and intracellular proteins that influenced the structure and function of arteries through specific actions on muscle tone, the mechanotransduction and the ultra-structural organization of VSMCs. These studies show the multifactorial dependency of the stiffness-fibrosis phenotypes of the arterial wall at the cellular and molecular levels. These results require more mechanistic work to determine the role of these proteins in CV diseases related to aging

Anhand von immunhistochemischen Färbemethoden wurde die Expression von CD 31 (immunhistochemischer Marker für Endothelzellen) und -Aktin (immunhistochemischer Marker für glatte Muskelzellen) auf Zellen der intramyokardialen Blutgefäße herztransplantierter Patienten histomorphometrisch ausgewertet. In die Bewertung der myokardialen Strukturen ging auch die Untersuchung der jeweiligen zugehörigen HE-Färbungen mit ein. Ziel dieser Untersuchungen war die Beurteilung von qualitativen und quantitativen Unterschieden dieser Marker während eines Untersuchungszeitraumes von 14 Monaten nach Herztransplantation (HTx) bei Patienten mit einer angiographisch diagnostizierten Transplantatvaskulopathie (TVP) mit Patienten ohne einer prä- bzw. postmortem diagnostizierten TVP. Der Einsatz dieser Immunomarker richtete sich hierbei auf die selektive Darstellung der terminalen Strombahn mit der Fragestellung, ob ein intramyokardiales morphologisches Korrelat zu der TVP der epikardialen Blutgefäße existiert und ob die histomorphometrische Auswertung der bewerteten Strukturen eine mögliche Frühdiagnose der TVP nach HTx zuläßt. Des weiteren sollte eine mögliche Assoziation der TVP mit den demographischen Charakteristika sowie der Anzahl und dem Schweregrad der Rejektionsepisoden der untersuchten Patienten überprüft werden. Als Basis dienten dabei in Paraffin eingebettete rechtsventrikuläre Rejektionskontrollbiopsien, die im Rahmen der routinemäßig durchgeführten Abstoßungsdiagnostik entnommenen wurden. Nach morphologischer Bewertung dieser Schnitte im Hinblick auf die Existenz und den Schweregrad einer Abstoßungsreaktion wurden diese immunhistochemisch aufbereitet und anschließend quantitativausgewertet. Im Rahmen dieser Arbeit weist die quantitativ- histomorphometrische Erfassung intramyokardialer Blutgefäße in rechtsventrikulären Rejektionskontrollbiopsien auf die mögliche Frühdiagnose einer TVP nach HTx hin. Dieses Verfahren könnte die zur Zeit verwendeten diagnostischen Methoden ergänzen. Jedoch sind noch weitere prospektive klinische Studien für die Validierung dieser Ergebnisse notwendig. / In this study, immunohistochemical and histomorphometric investigations were performed in order to investigate the expression of CD 31 (immunohistochemical marker for endothelial cells) and -Aktin (immunohistochemical marker for smooth muscle cells) on cells of intramyocardial blood vessels in heart transplant (HTx) patients. The evaluation of myocardial structures also implicated the investigation of the corresponding histological H & E stainings. The aim of this study was to ascertain whether HTx patients with angiographic evidence of graft vessel disease (GVD) showed different qualitative and quantitative expression of the above mentioned immunomarkers than HTx patients with no angiopraphic or postmortem signs of this phenomenon. The investigation time included the first 14 months after HTx. The use of these immunomarkers made possible the selective representation of the terminal vascular system to answer the question, as to whether there existed an intramyocardial morphological correlate to GVD of epicardial coronary arteries and whether histomorphometric evaluation of small vessels permits early diagnosis of GVD after HTx. In addition, demographic data, as well the number and grade of rejection episodes of the studied patients, were reviewed in order to ascertain whether there might be an association of these characteristics with GVD. The material studied consisted of paraffin-embedded right ventricular rejection control samples from routine postoperative diagnostic management, which were used to reveal acute rejection episodes. After morphological evaluation of histological slices with regard to the appearence and severity of acute rejection, immunohistochemical staining was performed and finally a quantitative investigation was done. According to the results of this study, quantitative histomorphometric investigations of intramyocardial blood vessels in right ventricular rejection control samples permit the early diagnosis of GVD after HTx and completes the present diagnostic tools. But further prospective clinical studies are necessary to confirm these results.