Orientador: Edson Antunes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T10:48:45Z (GMT). No. of bitstreams: 1 Claudino_MarioAngelo_D.pdf: 1501824 bytes, checksum: bf8ff1de7d8df61303dfb69ed190a0b9 (MD5) Previous issue date: 2008 / Resumo: A disfunção erétil é definida como a incapacidade de alcançar ou manter ereção peniana adequada para a satisfação sexual, e ocorre em graus variáveis prejudicando a qualidade de vida do homem. A disfunção erétil está associada a fatores de risco como doenças cardiovasculares, hipertensão arterial, diabetes mellitus, hipercolesterolemia, envelhecimento, tabagismo e sedentarismo. O óxido nítrico (NO) é o principal mediador do relaxamento do músculo liso peniano. Evidências clínicas e experimentais indicam que a redução da disponibilidade de NO ou da reatividade ao NO endógeno leva à disfunção endotelial. Trabalhos recentes mostram que parte dos benefícios da atividade física regular no sistema cardiovascular é intensificar a produção de NO e/ou expressão da NOS pelas células endoteliais. Dessa forma, usando o modelo de inibição crônica da síntese de NO em ratos pelo L-NAME, investigamos os efeitos do treinamento físico regular na função erétil de ratos. Na primeira etapa, estudamos a influência do treinamento físico na função erétil de ratos saudáveis normotensos. Na segunda etapa, estudamos a influência do pré-condicionamento físico na função erétil de ratos submetidos a bloqueio crônico de NO. Na terceira etapa, avaliamos a eficácia terapêutica da atividade física regular na função erétil de ratos submetidos a bloqueio crônico de NO. Além das respostas funcionais (medidas de relaxamento de corpo cavernoso isolado e de pressão intracavernosa in vivo), realizamos a dosagem plasmática de nitrito/nitrato (NOx). A atividade plasmática da superóxido dismutase (SOD), a expressão protéica da NO síntase neuronal (nNOS), da nitrotirosina (3-NT) e da SOD, assim como a expressão gênica da gp91phox foram analisadas no corpo cavernoso. Em ratos sedentários, nossos resultados mostraram que o exercício físico por 8 semanas ampliou a resposta relaxante mediada pelo NO (NaNO2 acidificado), nitroprussiato de sódio (SNP) e estimulação elétrica (EFS), sem alterar a resposta contrátil em resposta à fenilefrina (PE) e endotelina-1 (ET-1). Nos ratos com deficiência crônica de NO, o pré-condicionamento preveniu o aumento da pressão arterial e amplificou o relaxamento do corpo cavernoso induzido pelo SNP, ACh e EFS; porém, não alterou a resposta relaxante ao BAY 41-2272 e sildenafil. Além disso, o pré-condicionamento normalizou a pressão intracavernosa e os níveis plasmáticos de NOx. Ao avaliarmos o efeito do treinamento físico após instalação do bloqueio crônico de NO (eficácia terapêutica), notamos que a atividade física atenuou a hipertensão arterial, potencializou o relaxamento induzido pelo SNP e reverteu a redução do relaxamento em resposta à ACh e EFS. Além disso, normalizou a pressão intracavernosa e atenuou os níveis de NOx. A expressão protéica da nNOS e da PnNOS não foi alterada pela atividade física; porém, o aumento da nitração protéica (3-NT) foi previnida pelo treinamento físico nos animais hipertensos. A expressão protéica da SOD não foi alterada pelo treinamento; entretanto, a atividade física regular atenuou a redução da atividade plasmática da SOD. Os níveis de expressão gênica da subunidade catalítica da NADPH oxidase, a gp91phox, foram reduzidos pela atividade física regular nos ratos deficientes crônicos de NO, e restaurados pelo pós-condicionamento. Portanto, concluimos que o treinamento físico (pré ou pós-condicionamento), é uma estratégia nãofarmacológica eficiente capaz de reduzir o estresse oxidativo, aumentando a biodisponibilidade de NO, que resulta na melhora da disfunção erétil / Abstract: Erectile dysfunction is defined as the consistent or recurrent inability of a man to attain and/or maintain a penile erection sufficient for sexual activity, and can occur in different levels leading to an impairment of men¿s quality of life. Risk factors such as cardiovascular diseases, arterial hypertension, diabetes mellitus, hypercholesterolemia, aging, smoking and life sedentary increase the prevalence of erectile dysfunction. Nitric oxide (NO) is the main mediator of relaxation of penile smooth muscle. Clinical and experimental evidences suggest that decreased NO biodisponibility or NO reactivity contribute to erectile dysfunction. Recent studies have shown that part of beneficial effects of regular physical activity in the cardiovascular system is due to an increase in NO production and/or NOS expression by endothelial cells. Therefore, the aim of this work was to study the influence of run training on the erectile function from rats under chronic NO blockade by long-term treatment with L-NAME. First, we investigated the influence of physical training in the erectile function of healthy, normotense rats. Second, we studied the influence of prior physical conditioning in the erectile function of chronic NO-defficient rats. Third, we evaluated the therapeutic efficacy of regular physical activity (pós-conditioning) in the erectile function in chronic NO-defficient rats. To achieve this, we evaluated the functional responses (measurements of cavernosal relaxation in vitro and intracavernosal pressure in vivo) and the plasma levels of nitrite/nitrate (NOx) and plasmatic superoxide dismutase (SOD) activity, protein expression of neuronal NOS (nNOS), nitrotyrosin (3-NT) and SOD, and gene expression of gp91phox in the corpus cavernosum. In healthy normotenses rats, physical training by 8 weeks improved the relaxant responses mediated by NO (added as acidified sodium nitrite solution; NaNO2), sodium nitricprusside (SNP) and electrical field stimulation (EFS), without affecting the contractile responses induced by phenilephrine (PE) and endothelin (ET-1). In NO-defficient rats, the prior physical conditioning attenuated the increased arterial pressure, and potentiated the relaxations of corpus cavernosum induced by SNP, ACh and EFS. However, no changes were found for BAY 41-2271 and sildenafil. In NO-defficient animals, the physical activity normalized the intracavernosal pressure and plasma NOx levels. The post-conditioning (therapeutic efficacy) also attenuated the L-NAME induced arterial hypertension, potentiated the SNP-induced relaxations and reverted the reductions of relaxing responses to ACh and EFS. Furthermore, post-conditioning nomalized the intracavernosal pressure and attenuated the plasma NOx levels. Protein expression of nNOS and PnNOS were not affected by post physical activity. However, the increase of protein nitration (3-NT) was reversed by postconditioning in the NO-defficient animals. The protein expression of SOD was not modified by exercise. Nevertheless, the physical activity attenuated the reduction of SOD plasma activity. The gp91phox expression was decreased in NO-defficient rats, and restored by physical training. In conclusion, we conclude that physical training (pre or post-conditioning) is an efficient nos-pharmcological approach able to reduce the oxidative stress, increasing the NO bioavailability that results in an improvement of erectile dysfunction / Doutorado / Doutor em Farmacologia

Objetivo: Avaliar a amplitude de pulso ocular (APO) em pacientes com hipertensão arterial sistêmica, comparando com o grupo controle, e eventuais influências da espessura central da córnea e do comprimento axial (COMPRaxial) do olho na APO. Método: Foram examinados 152 olhos de 76 pacientes com hipertensão arterial sistêmica e 136 olhos de 68 indivíduos que compuseram o grupo controle. Todas as medidas foram tomadas pelo mesmo examinador, no período das 7 às 10h da manhã, na seguinte ordem: pressão arterial sistêmica, tonometria de contorno dinâmico, COMPRaxial e paquimetria (PAQ). A análise estatística foi realizada utilizando-se o teste t para médias de dados pareados e, para as correlações, os coeficientes de Pearson ou o de Spearman. Considerou-se o nível de significância de 5%. Resultados: A APO média dos olhos direitos dos pacientes com hipertensão arterial sistêmica foi 2,10 (+/- 0.9 mmHg) e a dos olhos esquerdos foi 2,03 (+/-0,828 mmHg). A PAQ média dos olhos direitos foi 532.2 mµ (+/- 39 mµ), e a dos olhos esquerdos 532.1 mµ (+/- 36.5 mµ), não influenciando a APO. A variável COMPRaxial para o olho direito (OD) apresentou média de 23,44 mm (+/- 1.477 mm) e para o olho esquerdo (OE) foi de 23.343 em média (+/- 1,32 mm). Houve significância estatística quando estudada a influência do COMPRaxial sobre a APO, demonstrando correlação inversamente proporcional. A média da APO dos controles foi de 2,10 (+/- 0.9 mmHg) e para o OD e OE média de 2,03 (+/-0,828 mmHg). Quando comparados os valores médios da APO dos casos e controles, a diferença foi estatisticamente significante, sendo os valores dos controles maiores do que os encontrados nos pacientes com hipertensão arterial sistêmica (p <0 ,001). Constatou-se que existe diferença entre caso e controle, tanto para o OD quanto para o OE. Conclusões: O valor médio de APO foi menor nos pacientes com hipertensão arterial sistêmica do que nos controles; a APO não foi influenciada pela espessura central da córnea; e olhos com maior COMPRaxial apresentaram APO menor / Objective: The objectives of this study were to assess the ocular pulse amplitude (OPA) in patients with hypertension, compared with control group and to evaluate possible influences of central corneal thickness and axial length of eye in OPA. Method: We evaluated 152 eyes of 76 patients with hypertension and 136 eyes of 68 individuals who comprised the control group. All measurements were made by the same examiner in the period of 7 am and 10 am, in following order: blood pressure, dynamic contour tonometry, axial length and pachymetry. Statistical analysis was performed using the t test for paired data and averages for correlations, the Pearson correlation coefficients of Spearman. Considered the significance level of 5%. Results: The average OPA in right eyes of patients with hypertension was 2.10 (standard deviation (SD) 0.9 mmHg) and in left eyes was 2.03 (SD =0.828 mmHg). The average pachymetry of right eyes was 532.2 mµ (+/- 39 mµ); and for left eyes was 53.1 mµ ( +/-36.5 mµ). The variable diameter axial in right eye showed an average of 23.44 mm (+/- 1.477 mm); for the left eye 23,343 mm (+/- 1.32). There was statistical significance when studied the influence of the axial diameter of OPA, with inverse correlation. The average of controls OPA was presented mean 2.10 (+/- 0.9 mmHg) for the right eye and the left mean 2.03 (+/- 0.828 mmHg). When comparing the mean values of OPA in cases and controls the difference was statistically significant, the values of the controls are larger those found in patients with hypertension (p<0,001), so it was found difference between cases and controls, both for the right and left eye. Conclusions: The mean OPA was lower in patients with hypertension than in controls, the OPA was not influenced by central corneal thickness, and eyes with greater axial length showed lower OPA

Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-08-22T18:42:20Z No. of bitstreams: 1 tatianaresendecarvalho.pdf: 1506183 bytes, checksum: b3c7a483a60aa885fc2e59931d92b8b0 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-08-28T13:13:59Z (GMT) No. of bitstreams: 1 tatianaresendecarvalho.pdf: 1506183 bytes, checksum: b3c7a483a60aa885fc2e59931d92b8b0 (MD5) / Made available in DSpace on 2018-08-28T13:13:59Z (GMT). No. of bitstreams: 1 tatianaresendecarvalho.pdf: 1506183 bytes, checksum: b3c7a483a60aa885fc2e59931d92b8b0 (MD5) Previous issue date: 2018-03-21 / Estudo transversal realizado com 340 usuários hipertensos, acompanhados pela Estratégia Saúde da Família do município de São João del-Rei, selecionados a partir do registro das fichas de acompanhamento dos agentes comunitários de saúde, por amostragem aleatória simples. Os objetivos do estudo foram analisar a associação entre letramento funcional em saúde e adesão ao tratamento medicamentoso da HAS; descrever os níveis de letramento funcional em saúde da população de estudo; estimar a prevalência da não adesão ao tratamento medicamentoso da HAS e analisar os fatores associados à não adesão ao tratamento medicamentoso da HAS. A coleta de dados ocorreu entre os meses de junho à setembro de 2017 e foi realizada através de um formulário com questões estruturadas acerca dos fatores socioeconômicos, relacionados ao paciente, à doença, ao tratamento, à equipe e ao serviço de saúde. A avaliação da adesão ao tratamento medicamentoso foi feita através da Escala de Adesão Terapêutica de oito itens de Morisky (MMAS-8) e o nível de letramento funcional em saúde foi medido pelo B-TOFHLA. As análises estatísticas foram feitas utilizando-se o teste qui-quadrado e modelos de regressão de Poisson para estimar as razões de prevalência brutas e ajustadas, os respectivos intervalos de confiança (IC95%) e p-valor. A prevalência de não adesão ao tratamento farmacológico da HAS foi de 24,1% (IC 95%: 19,7 – 28,5). Os fatores associados à não adesão à terapia medicamentosa foram a credibilidade na importância dos medicamentos para o tratamento da HAS, a frequência na tomada dos medicamentos para a doença por dia, a compreensão das orientações e explicações dadas pelos profissionais de saúde e a dificuldade em conversar com os profissionais. Baixo letramento funcional em saúde foi encontrado em 80,2% dos hipertensos entrevistados. A proporção de usuários não aderentes ao tratamento com baixo letramento funcional em saúde foi 77% superior à proporção de usuários não aderentes à terapia medicamentosa com letramento funcional em saúde adequado (RP=1,77; IC95%:0,93 – 3,75). Os resultados mostram que mudanças baseadas na complexidade do regime terapêutico, uma adequada assistência à saúde, baseada nas particularidades de cada indivíduo, levando-se em conta a influência que o mesmo recebe do meio que o cerca podem contribuir para o aumento da adesão ao tratamento medicamentoso da HAS. É possível supor ainda que, identificando-se as limitações apresentadas pelos usuários em relação ao acesso e à compreensão das informações e orientações que lhe são repassadas, a equipe de saúde pode elaborar estratégias que favoreçam o processo de comunicação entre profissionais de saúde e usuários do sistema, compensando assim os baixos níveis de letramento funcional em saúde, fazendo com que as informações e orientações necessárias ao manejo e ao acompanhamento da doença sejam mais facilmente compreendidas pelos usuários. / This is a cross-sectional study performed with 340 hypertensive users who have been monitored by the Family Health Care Service from the city of São João del-Rei. They have been selected based on the follow-up records of the community health agents by simple random sampling. The aim of the study is to analyze the association between Functional Health Literacy and the adherence to the medical treatment for HAS (hypertension); to describe the levels of functional health literacy in the evaluated population; to estimate the predominant non-adherence to the drug treatment for hypertension and to analyze the factors that are connected to the non-adherence to the medical treatment for hypertension. Data collection has happened between June and September 2017 and it was carried out by using a questionnaire with questions about socioeconomic factors related to the patient, to their disease, to their treatment, the team who have assisted them and the health care service itself. The evaluation in order to measure the adherence to the drug treatment was carried out via the 8-item Morisky Medication Adherence Scale (MMAS-8) and the Functional Health Literacy level was measured by the B-TOFHLA. Statistical analyzes have been performed using the chi-square test and Poisson regression models in order to estimate crude and adjusted prevalence ratios, confidence intervals (IC95%), and p-value. The predominance of non-adherence to the pharmacological treatment for HAS was 24.1% (IC95%: 19.7-28.5). The main factors associated with the non-adherence to the drug therapy were: trustworthiness regarding the importance of drugs for the treatment of HAS, the frequency the user takes the prescribed medicine per day, the understanding of the instructions and explanations given by the health care professionals, and the struggle in talking to the professionals. Low functional health literacy has been found in 80.2% of the hypertensive patients who have been interviewed. The non-adherence ratio among patients with low functional health literacy is 77% higher than the proportion of non-adherence among patients with adequate functional health literacy (PR = 1.77, IC 95%: 0.93-3.75). The results have shown that changes based on the complexity of the therapeutic regime and an adequate health care provision based on the specificities of each individual, taking into account the influence that the patient gets from the environment he comes from can contribute to raise the levels of medication adherence for HAS. It is also possible to assume that, by identifying the patients limitations regarding the access and the overall comprehension of the information and instructions that are passed on to them, it allows the healthcare team to prepare and implement strategies that may favor the communication process between the healthcare professionals and users of the system, thus compensating for the low levels of functional health literacy and making the information and general guidelines that are necessary for the management and follow-up of the disease more easily understood by its users.

L'hypertension artérielle pulmonaire (HTAP) est caractérisée par un intense remodelage de la microcirculation pulmonaire affectant principalement les artérioles pulmonaires musculaires. Lorsqu'elle survient en l'absence de condition associée, l'HTAP est considérée comme idiopathique (HTAPi). L'HTAP représente une pan-vasculopathie au cours de laquelle chaque type cellulaire (cellules endothéliale, musculaire lisse, fibroblaste) constituant la paroi vasculaire joue un rôle spécifique dans la réponse à l'agression. Les buts de ce travail étaient d'explorer l'implication de différentes voies de signalisation dans l'initiation ou la progression de la maladie. Les différentes études ont été réalisées à partir de cultures de cellules musculaires lisses (CML) d'artère pulmonaire et de cellules endothéliales (CE) pulmonaires obtenues à partir de prélèvements pulmonaires humains obtenus lors de transplantation chez des patients souffrant d'HTAP réfractaire.Des études antérieures avaient souligné le rôle majeur de la sérotonine au cours de l'HTAP idiopathique. Dans une première étude, nous avons étudié le rôle respectif de la sérotonine (5-HT), de son transporteur (5-HTT) ou de ses récepteurs (5-HT1B, 5-HT2A et 5-HT2B) dans le remodelage vasculaire pulmonaire mis en évidence dans l'HTP associée à diverses conditions. Les résultats de cette première étude montraient qu'une surexpression du 5-HTT dans les CML d'artère pulmonaire est une voie physiopathologique commune impliquée dans le remodelage vasculaire pulmonaire observé dans l'HTAP idiopathique, la maladie veino-occlusive et l'HTAP associée à différentes pathologies.Des mécanismes inflammatoires jouent probablement un rôle important dans la physiopathologie du remodelage microvasculaire pulmonaire. En effet, des infiltrats composés de cellules inflammatoires mononucléées (macrophages, lymphocytes T et B et cellules dendritiques) sont fréquemment mis en évidence autour des lésions vasculaires pulmonaires de patients présentant une HTAP idiopathique. Les mécanismes impliqués dans le recrutement de ces cellules mononucléées demeurent mal compris et nous avons étudié le rôle d'une chimiokine, CC chemokine ligand 2 (CCL2). Les résultats de cette seconde étude montraient que CCL2 était surexprimée au cours de l'HTAP idiopathique. La source de cette surexpression semblait provenir des cellules endothéliales pulmonaires. CCL2 agissait non seulement sur le recrutement des monocytes mais également sur les cellules musculaires lisses vasculaires pulmonaires en stimulant leur prolifération et leur migration.Des mutations germinales de gènes codant pour des membres de la famille des récepteurs du TGF tels que BMPR2 (Bone Morphogenic Protein Receptor type 2) sont retrouvées dans près de 70% des cas d'HTAP familiale mais également chez 10 à 30 % des cas d'HTAPi apparemment non familiales. Ces patients sont regroupés sous le terme d'HTAP « héritable » (HTAPh). Nous avons, dans une troisième étude, évalué si la dysfonction des voies de signalisation secondaires aux mutations de BMPR2 pouvait avoir des conséquences sur la voie de l'endothéline 1 (ET-1) qui représente l'une des cibles thérapeutiques de choix au cours de l'HTAP. Les résultats de cette troisième étude montraient que l'ET-1 était surexprimée au cours de l'HTAP avec ou sans mutation de BMPR2. En revanche, une surexpression des récepteurs ET-A dans les CML était mise en évidence au cours de l'HTAPh et était associée à une augmentation de l'effet pro-proliférant de l'ET-1 sur les CML.Ces résultats révèlent que des facteurs vaso-actifs (ET-1, 5-HT) et inflammatoires jouent un rôle déterminant dans la physiopathologie de l'HTAP et pourraient représenter de nouvelles cibles thérapeutiques. / Pulmonary arterial hypertension (PAH) is characterized by intense pulmonary vascular remodelling affecting mainly the muscular pulmonary arteries and leading to increased pulmonary vascular resistance. When it occurs in the absence of associated conditions, PAH is regarded as idiopathic (iPAH). PAH represents a panvasculopathy in which each cell type constituting the vascular wall (endothelial cells, smooth muscle cells, fibroblast) plays a specific role. The aims of this work were to explore the implication of various pathways in the initiation or the progression of the disease. The various studies were carried out using pulmonary artery smooth muscle cells (PASMC) and pulmonary endothelial cells (PEC) obtained during lung transplantation from patients with refractory PAH.Former studies have emphasized the major role of serotonin (5-HT) in the process of pulmonary vascular remodelling in iPAH. In a first study, we studied the respective role of 5-HT, the 5-HT transporter (5-HTT) and several 5-HT receptors (5-HT1B, 5-HT2A and 5-HT2B) on PASMC proliferation in cells from patients with PH associated with various conditions. The results of this first study showed that 5-HTT overexpression in PASMC is a common pathogenic mechanism in various forms of PH.Inflammatory cytokines may affect pulmonary vascular remodelling in iPAH. Indeed, iPAH frequently reveals inflammatory infiltrates corresponding to macrophages, lymphocytes and dendritic cells in the range of plexiform lesions as well as in other vascular lesions. The mechanisms underlying pulmonary vessel infiltration by monocytes / macrophages are unclear and the role for inflammatory cells in pulmonary vascular remodeling remains to be elucidated. This second study showed that iPAH is associated with an overexpression of CCL2. PEC are a major source of CCL2, which behaves as chemoattractant for circulating inflammatory cells and as growth factor for PASMC.Germline mutations of bone morphogenetic protein (BMP) receptor type 2 (BMPR-2), a member of the transforming growth factor (TGF)-β receptor family, have been reported in nearly 70% of patients with the heritable form of the disease (hPAH), and in 10–30% of patients with sporadic iPAH. In a third study, we evaluated the functional consequences of BMPR-2 mutations on the endothelin 1 (ET-1) pathway which represents one of the therapeutic targets on PAH. The results of this third study showed that iPAH and hPAH were associated with a similar overexpression of ET-1. In contrast, ETA receptor mRNA levels which were increased in PASMC from patients with iPAH and hPAH compared to controls were much higher in hPAH than in iPAH cells. Consequently, the growth promoting effect of ET1 on PASMC was higher in PASMC from patients with iPAH, and was markedly elevated in PASMC from patients with hPAH. No changes in ETB receptor mRNA levels could be detected in PASMC from patients with iPAH or hPAH in comparison with controls.These results reveal that vasoactive factors (ET-1, 5-HT) and inflammatory factors play a determining role in the pathophysiology of PAH and could represent new therapeutic targets.

Les pathologies vasculaires provoquent un remodelage de la paroi artérielle pouvant entraîner une modification de sa rigidité et de son comportement mécanique. L’objectif de cette thèse est de proposer des méthodes de caractérisation mécanique permettant d’identifier les changements de propriétés mécaniques artérielles dans le cadre de deux situations pathologiques : l’anévrysme de l’aorte abdominale (AAA) et l’hypertension artérielle (HTA). La première étude a consisté à évaluer in vitro les modifications de fonctionnalité de l’artère dans le cas d’un AAA obtenu par le modèle de xénogreffe chez le rat qui permet de reproduire certains aspects de la pathologie humaine et qui est utilisé pour la mise au point de thérapies cellulaires. Une analyse des variations régionales des propriétés mécaniques du tissu anévrysmal a d’abord été menée en effectuant des tests de traction sur anneaux d’AAA. Des tests d’extension-inflation ont ensuite été réalisés sur la structure vasculaire pour des conditions de chargement reproduisant celles observées in vivo. Dans chacun des cas, une méthode inverse couplée à un modèle numérique par éléments finis a été développée afin d’identifier les paramètres matériaux du tissu vasculaire. Dans la deuxième étude, la rigidité artérielle a été mesurée in vivo sur une population de patients atteints d’HTA et de sujets sains en utilisant deux méthodes non-invasives qui ont été développées et optimisées : l’imagerie ultrarapide de l’onde de pouls et l’élastographie par ondes de cisaillement de la paroi artérielle. Ces deux méthodes s’appuient sur un échographe ultrarapide. La vitesse de l’onde de pouls locale sur un segment de la carotide a ainsi pu être évaluée, ainsi que la vitesse de propagation d’ondes de cisaillement générées dans la paroi à plusieurs instants du cycle cardiaque. Les deux approches in vitro et in vivo ont ainsi permis d’évaluer certains changements de propriétés mécaniques de la paroi artérielle dans des cas pathologiques. Bien que tous les mécanismes biologiques de l’AAA et de l’HTA soient complexes, ce travail permet de contribuer à une meilleure compréhension des pathologies vasculaires pouvant ainsi aider au choix ou au développement de traitements adaptés, tant d’un point de vue pharmacologique que dans le cadre de nouvelles thérapies cellulaires. / Vascular pathologies are generally accompanied by a remodeling of the arterial wall that may lead to modifications of its stiffness and mechanical behavior. The goal of this thesis is to propose methods of mechanical characterization allowing to detect the changes in arterial mechanical properties in the case of two pathologies: abdominal aortic aneurysm (AAA) and arterial hypertension (HTA). The first study consisted in evaluating in vitro the arterial functional modifications in the case of an AAA obtained by the rat xenograft model which reproduces several biological features of the human pathology and is used to develop cell therapies. First, the assessment of regional variations in mechanical properties of aneurysmal tissue was conducted by carrying out traction tests on rings from AAAs. Then, extension-inflation tests were conducted on the vascular structure for loading conditions replicating those observed in vivo. In each case, an inverse method associated with a numerical finite element model was developed to identify the material parameters of vascular tissue. In the second study, arterial stiffness was measured in vivo for a population of hypertensive patients and healthy subjects using two non-invasive methods which were developed and optimized: ultrafast imaging of the pulse wave and shear wave elastography of the arterial wall. These two methods are based on an ultrafast ultrasound scanner. Thus the local pulse wave velocity on a segment of the carotid artery was assessed, as well as the propagation speed of shear waves created in the arterial wall at several moments during the cardiac cycle. Both in vitro and in vivo approaches enabled to evaluate some changes in mechanical properties of the arterial wall in pathological cases. Although all the biological mechanisms of AAA and HTA are complex, this work provides a contribution to a better understanding of vascular pathologies and can thereby assist in the choice or development of adapted treatments, from both a pharmacological point of view, and within the context of new cell therapies.

La signalisation purinergique participe à de nombreux processus physiopathologiques dans le système cardiovasculaire. Alors que les nucléotides extracellulaires sont considérés comme des « signaux de danger » ; la NTPDase1(CD39), ectonucléotidase à l’origine de leur hydrolyse, permet de maintenir l’homéostasie vasculaire par ses actions anti-thrombotiques etimmuno-modulatrices. Le rôle de CD39 dans la fonction vasculaire liée à l’hypertension artérielle(HTA) reste méconnu. L’HTA, facteur de risque majeur de complications cardiovasculaires, est caractérisée par un remodelage structurel(hypertrophie, fibrose) et fonctionnel (hypercontractilité, dysfonction endothéliale) des vaisseaux, causées notamment par un stress oxydatif et une inflammation périvasculaire. L’objectif de notre projet a consisté à étudier l’évolution de CD39 ainsi que son rôle potentiel dans la condition vasculaire pathologique de l’HTA. Nous mettons en évidence une diminution de l’expression et de l’activité du CD39 vasculaire dans l’HTA. Une diminution de l’activité ADPase du CD39 soluble a également été observée au niveau circulant. L’étude des éléments à l’origine de cette diminution montre une sensibilité du transcrit vasculaire de CD39 à certaines cytokinespro- et anti-inflammatoires, mais également à une tension mécanique. Une étude in vivo du potentiel rôle de CD39 (souris déficientes pour le gène de CD39 (Entpd1) et traitement à l’apyrase) dans un modèle d’HTA à l’Angiotensine-II a également été réalisée. L’ensemble de ces données suggère qu’une diminution du CD39 vasculaire et circulant pourrait contribuer à majorer les altérations vasculaires contemporaines de l’HTA. / Purinergic signaling is involved in numerous physiopathological processes in cardiovascular system. While extracellular nucleotides are considered as « danger signals » ; the NTPDase1(CD39), ectonucleotidase responsible for their hydrolysis, preserves vascular homeostasis by itsanti-thrombotic and immunomodulatory actions.The role of CD39 in vascular function related to arterial hypertension remains unknown. Hypertension, the major risk factor of cardiovascular complications, is characterized by structural remodeling (hypertrophy, fibrosis) and functional (hypercontractility, endothelial dysfunction) of vessels caused in particular by perivascular oxidative stress and inflammation.Our aim was to investigate the evolution of CD39 expression / function and its potential contribution in the pathological vascular condition of hypertension. We highlighted a decrease invascular CD39 expression and activity in the context of hypertension. A decrease in soluble ADPase activity specific to CD39 was also observed in blood circulation. Investigation of elements responsible for this decrease reveals asensitivity of vascular CD39 transcription to several pro- and anti-inflammatory cytokines and to mechanical tension. In vivo study of potential role of CD39 (mice deficient for CD39 gene (Entpd1) and treatment with apyrase) in Angiotensin-II model of hypertension was also carried out. All these data suggest that a decrease in circulating and vascular CD39 may contribute to vascular changes associated with hypertension.

Rationnel : L’artérite à cellules géantes (ACG) est une vascularite primitive des gros vaisseaux dont le diagnostic repose sur la mise en évidence d’un infiltrat inflammatoire et de cellules géantes à la biopsie d’artère temporale (BAT). On note également un remodelage de la paroi vasculaire lié à une prolifération des cellules musculaires lisses vasculaires (CMLV) pouvant aboutir à une occlusion artérielle. Objectif : Caractériser les auto-anticorps dirigés contre les cellules endothéliales (CE) et les CMLV au cours de l’ACG et préciser le rôle des CMLV dans le remodelage pariétal. Méthodes : La recherche d’auto-anticorps a reposé sur un immunoblot 2D couplé à la spectrométrie de masse. Les protéomes des CMLV d’artère ombilicale, d’artère pulmonaire et d’aorte humaines normales a été comparés par protéomique différentielle (2D-DIGE). Nous avons utilisé la 2D-DIGE et des puces d’expression pan-génomiques pour comparer les CMLV issues de BAT de patients suspects d’ACG (avec un diagnostic final d’ACG ou non), afin d’identifier les mécanismes contribuant à la prolifération des CMLV. Résultats : Chez 15 patients atteints d’ACG, nous avons notamment identifié la lamine, la vinculine et l’annexine A5 comme cible des auto-anticorps anti-CMLV. Les antigènes cibles identifiés sont liés à Grb2, une protéine adaptatrice impliquée dans la prolifération des CMLV. Nous avons mis en évidence des protéomes différents au sein des CMLV humaines normales selon leur origine vasculaire et avons principalement identifié des protéines du cytosquelette et du métabolisme énergétique.A partir des CMLV isolées des BAT et à l’aide d’Ingenuity®, nous avons identifié l’endothéline 1 (ET-1) et la paxilline comme des molécules impliquées dans le remodelage vasculaire. En immunohistochimie et par qPCR, nous avons confirmé l’expression de l’ET-1 et de ses récepteurs ETAR et ETBR au sein des artères temporales de patients atteints d’ACG. Enfin, nous avons inhibé la prolifération des CMLV avec du macitentan, un inhibiteur d’ETAR et en particulier avec son métabolite actif, mais pas avec d’autres inhibiteurs des récepteurs de l’ET-1. Conclusion : Nous avons identifié chez les patients atteints d’ACG des anticorps anti-CMLV dont le rôle pathogéne potentiel reste à définir. Les différences protéiques observées à partir des CMLV humaines normales pourraient correspondre à des phénotypes différents. A partir d’un matériel biologique unique, nous avons pu montrer que la prolifération excessive des CMLV au cours de l’ACG pouvait être inhibée par le macitentan ce qui permet d’envisager un usage thérapeutique de cette molécule. / Background : Giant cell arteritis (GCA) is a large vessel vasculitis and its diagnosis usually relies on the identification of an inflammatory infiltrate made of mononuclear cells and giant cells upon temporal artery biopsy. There is also a remodeling process in the arterial wall due to an excessive proliferation of vascular smooth muscle cells (VSMC) which can sometimes lead to arterial occlusion. Purpose: Identify auto-antibodies targeting either endothelial cells (EC) and/or VSMC during GCA and better understand the role of VSMC in the remodeling process. Methods : Auto-antibodies were detected by a 2-dimensionnal immunoblot and their target antigens were identified by mass spectrometry. Proteoms of umbilical artery, pulmonary artery and aorta VSMC were compared by 2 dimension differential in gel electrophoresis (2D-DIGE). In order to identify mechanisms involved in VSMC proliferation in GCA, we used both 2D-DIGE and pan genomic chips in order to compare VSMC isolated at the time of temporal artery biopsy (TAB) from patients with a final diagnosis of GCA or another diagnosis. Results : In 15 patients with GCA, we identified lamin, vinculin and Annexin A5 as target antigens of anti-VSMC antibodies. Target antigens were linked with Grb2, an adaptator protein involved in VSMC proliferation. Normal VSMC originating from different vascular beds have differ in protein contents with differential expression of cytoskeleton and energy metabolism proteins. We compared VSMC from TAB with Ingenuity software and identified endothelin-1 (ET-1) and paxillin as proteins involved in vessel remodeling. We confirmed by immunohistichemistry and qPCR that ET-1 and its receptor ETAR and ETBR were expressed in temporal arteries from patients with GCA. Last, we reduced VSMC proliferation with Macitentan, an ETAR and ETBR antagonist and significantly inhibited VSMC proliferation with its active metabolite whereas other ET-1 inhibitors had no effect. Conclusion : We identified anti-VSMC auto-antibodies in patients with GCA. Their pathogenic role remains to be determined. Normal VSMC from different vascular locations differ in protein conten which might reflect different phenotypes and different properties. The escessive proliferation of VSMC from patients with GCA was inhibited by Macitentan. This drug might constitute a future therapeutic option.

L’endothélium joue un rôle primordial dans le développement et le maintien des multiples fonctions vasculaires. Il est ainsi largement impliqué dans des situations pathologiques comme les maladies cardio-vasculaires. La description de marqueurs endothéliaux circulants a permis une exploration non invasive de l'endothélium. Notre équipe s’est intéressée principalement aux cellules endothéliales circulantes (CEC), dont le taux reflète la lésion ou l’activation de l’endothélium, et aux progéniteurs endothéliaux circulants (PEC), marqueurs de régénération endothéliale. La découverte en 1997 par Asahara de la présence chez l’adulte de ces PEC, participant à la formation de nouveaux vaisseaux par vasculogenèse, a ouvert de nouvelles perspectives, notamment pour la thérapie cellulaire des pathologies ischémiques. Ce travail a consisté à développer les méthodes d’étude de ces cellules dans plusieurs contextes. Tout d’abord, nous avons exploré l’utilité de ces marqueurs dans la physiopathologie de l’hypertension artérielle pulmonaire (HTAP). Puis nous avons analysé le potentiel de mobilisation des progéniteurs endothéliaux à partir de la paroi vasculaire lors d’une ischémie locale chez des volontaires sains dans le cadre du développement d’un produit de thérapie cellulaire autologue. Une partie de ce projet a été de mettre en place et d’optimiser les techniques d’étude de ces marqueurs. Les CEC ont été quantifiées par immunoséparation magnétique (IMS), technique mise au point en 1992 (Dignat-George 1992) et transférée dans notre laboratoire. La quantification des PEC a été réalisée par cytométrie en flux et par culture cellulaire. En culture, deux types de PEC sont décrits : les PEC précoces, dont l’origine est monocytaire et pour lesquels la culture est déjà standardisée, et les « Endothelial Colony Forming Cells » (ECFC), seules cellules présentant des caractéristiques de cellules endothéliales progénitrices et pouvant être proposées comme produit de thérapie cellulaire. Nous avons optimisé la quantification des ECFC en culture en étudiant l’effet de diverses matrices et de la densité d’ensemencement des cellules mononucléées issues du sang total sur l’obtention de ces cellules et leurs propriétés angiogènes. La dysfonction endothéliale a été décrite comme un élément central dans le développement de l’HTAP dont le diagnostic repose sur la mesure de la pression artérielle pulmonaire par cathétérisme cardiaque droit. En l’absence de marqueur biologique non invasif dans cette maladie, nous avons quantifié les CEC et les progéniteurs circulants dans deux études. Une étude réalisée chez des patients adultes a montré une augmentation spécifique des CEC dans l’HTAP et non dans l’hypertension pulmonaire thromboembolique chronique. Ainsi les CEC semblent être le reflet des lésions endothéliales pulmonaires et non de la sévérité clinique des patients. L’autre étude a montré l’intérêt de la quantification des CEC dans la prise en charge thérapeutique des enfants souffrant d’HTAP secondaire à une cardiopathie congénitale, dont les formes irréversibles présentaient des taux élevés de CEC. Nous avons ainsi défini un nouveau marqueur non invasif à utilité diagnostique et pronostique. Les PEC sont des cellules rares dans le sang circulant, difficiles à expandre, et dont les essais de mobilisation médullaire se sont révélés insuffisants. L’hypothèse récente d’une réserve vasculaire des progéniteurs endothéliaux nous a conduits à étudier l’effet d’un processus d’ischémie locale sur la mobilisation de ces cellules chez des volontaires sains. Deux groupes d'âge ont été inclus afin d'évaluer l'impact du vieillissement sur la méthode de mobilisation étudiée. Malgré un effet de cette ischémie sur la dilatation endothéliale cette méthode n’a pas permis de mobiliser significativement les PEC issus de la paroi endothéliale, quel que soit l'âge des sujets. A l’inverse, l’hypoxie a eu un effet délétère sur les capacités angiogènes des ECFC. / The endothelium plays a key role in the development and the homeostasis of vascular functions. It is also well involved in pathological situations like cardiovascular diseases. Thanks to the description of circulating endothelial markers, non invasive study of the endothelium is now possible. Our group was particularly interested in circulating endothelial cells (CECs), the level of which reflects an endothelial activation or lesion, and to circulating endothelial progenitors cells (EPCs), markers of endothelial repair. EPC description by Asahara in 1997 in adult blood, involved in new blood vessel formation by vasculogenesis, offered new perspectives, specially for cell therapy in ischemic diseases. This work consisted in the development of methods to study these markers in different contexts. First, we explored the interest of these markers in the physiopathology of pulmonary arterial hypertension (PAH). Then we evaluated endothelial progenitors mobilization from the vascular wall by a local ischemia process in healthy volunteers, in the perspective of an autologous cell therapy product development. One part of this project was the implementation and optimization of the methods to study CEC and EPC. CEC were quantified by magnetic immunoseparation. This technique was developped in 1992 by F. Dignat-George's group and transferred in our laboratory. EPC were quantified by flow cytometry and cell culture. Two types of EPC are described in culture: the early EPC, which originate from monocyte lineage and which culture is standardized, and the « Endothelial Colony Forming Cells » (ECFC), the only cells presenting endothelial progenitor cell properties and which use as a cell therapy product can be considered. ECFC quantification by culture was optimized by assessment of the impact of diverse matrices and seeding concentrations of mononuclear cells isolated from whole blood, on ECFC commitment and their angiogenic properties. Endothelial dysfunction was described as a central element in the development of PAH, which diagnosis is based on the use of right heart catheterization. Due to the lack of noninvasive marker for this disease, CEC and circulating progenitors were quantified in two studies. One of them realized in adult patients showed a specific increase of CEC in PAH and not in post-embolic PH. CEC would then reflect the presence of specific endothelial lesions and not the clinical state of the patients. The other study demonstrated the interest of CEC quantification in the therapeutic care of children with PAH secondary to congenital heart disease, for whom patients in irreversible state had a higher level of CEC. We then defined a new noninvasive biomarker.that can be used for the diagnosis and prognosis of PAH. EPC are rare events in whole blood, difficult to expand and for which, mobilization protocols revealed insufficient. The recent hypothesis of a vascular reservoir for endothelial progenitor led us to study the effect of a local ischemia procedure on the mobilization of these cells in healthy volunteers. Two age groups were included to assess the impact of aging on this procedure. Despite a significant endothelial dilation with the local ischemia, no EPC were mobilized, whatever the age group. Ischemia even altered ECFC angiogenic properties.

Les cardiopathies congénitales (CC) représentent la première cause d’anomalie malformative à la naissance. Les progrès considérables des années 80 (CEC néonatale, diagnostic prénatal) en ont modifié l’épidémiologie, avec un transfert de la mortalité de la pédiatrie à l’âge adulte. Dans ce contexte, l’évaluation de la qualité de vie liée à la santé (QdV) des enfants et adultes porteurs de CC devient un critère de jugement important, en recherche clinique comme dans les soins. Nous avons mené 4 études prospectives de QdV chez des patients avec CC: une étude chez 282 enfants de 8 à 18 ans avec CC comparés à 180 enfants contrôles; une étude sur 202 enfants avec CC corrélant QdV et VO2; une étude de QdV sur 208 adolescents et adultes porteurs d'HTAP sur CC; et une étude sur l’évolution de la QdV de 111 enfants sous AVK participant à un programme d’éducation thérapeutique. Les patients avec CC simple ont manifesté une QdV similaire à celle de la population générale. Ceux avec une cardiopathie complexe ont été préférentiellement impactés sur leur bien-être physique mais ont développé aussi des mécanismes de coping. En pédiatrie, l’évaluation de la QdV par les parents était plus péjorative mais parfois plus pertinente que celle des enfants. Nous avons mis en évidence le lien entre QdV et VO2 chez l’enfant cardiaque. Les résultats de nos travaux devraient permettre d’aider les cardiologues, cardiopédiatres et chirurgiens cardiaques dans leurs annonces diagnostiques, en particulier lors des moments cruciaux de notre sur-spécialité médico-chirurgicale: diagnostic prénatal, réanimation, transition vers l’âge adulte, prise en charge palliative d’une cardiopathie sévère. / Congenital heart diseases (CHD) are the leading cause of birth malformations. The tremendous progress since the 80’s (neonatal bypass, prenatal diagnosis) have changed the epidemiology, transferring mortality from pediatrics to adulthood. Therefore assessing the health-related quality of life (QoL) of children and adults suffering from CHD has become an important issue, in both clinical research and patients’ follow-up. We carried out 4 prospective QoL studies in patients with CHD: a study in 282 CHD children aged 8 to 18 compared with 180 controls; a study among 202 CHD children correlating their QoL scores to VO2; a QoL study among 208 adolescents and adults with PAH-CHD; and a study among 111 children in a therapeutic anticoagulation education program aiming to measure the evolution of their QoL. Patients with simple CHD showed a similar QoL to that of the control population. Those with complex heart diseases were preferentially affected in their physical well-being but also developed mechanisms of coping in other dimensions. In pediatrics, the evaluation of the QoL by parents is essential, sometimes more accurate than that of children themselves. As in previous studies in adults with heart failure, we found a significant relationship between QoL and physical performance during exercise in CHD children. The results of our work should help cardiologists, cardiac surgeons and pediatric cardiologists in their diagnostic announcement, especially during crucial moments of this medical and surgical subspecialty: prenatal diagnosis, intensive care, transition of care from adolescence to adulthood, palliative treatment of a complex CHD.

Pulmonary arterial hypertension (PAH) is a lethal disease, characterized by functional or structural abnormalities involving distal pulmonary arterioles that result in increased pulmonary vascular resistance (PVR) and ultimately right heart failure. Our objective is to establish a conditional transgenic system in mice, to test the hypothesis that lung EC apoptosis at the level of distal pulmonary arterioles is necessary and sufficient to cause a PAH phenotype. In a pilot study, the Fas-Induced Apoptosis (FIA) construct was expressed under the control of endothelial-specific Tie2 promoter in transgenic mice (i.e. EFIA mice). Administration of a small molecule dimerizing agent, AP20187, resulted in lung modest dose-dependent PAH, which was associated with proliferative vascular lesions localized to distal lung arterioles in a small proportion of mice. Due to the low level of transgene expression in preliminary EFIA lines, we re-designed the transgenic vector by incorporating a more robust endothelial promoter (superTie2). The new construct was transfected into HUVEC and BAEC and analyzed by monitoring immunofluorescence (DsRed). Data from the EFIA model suggests that EC apoptosis may be sufficient to induce a PAH phenotype with the characteristic lung vascular lesions. The EFIA model will allow us to better explore the mechanism that links distal lung EC apoptosis with reactive vascular cell proliferation in the pathogenesis of this devastating disease.