Les Cellules Endothéliales Progénitrices ("Endothelial Progenitor Cells", EPCs) sont des précurseurs endothéliaux qui jouent un rôle émergeant en biologie vasculaire. Les EPCs ont été localisées dans le cordon ombilical, la moelle osseuse, le sang périphérique et dans certains tissus régénérateurs. Les interactions des EPCs avec les cellules sanguines et vasculaires peuvent largement influencer leurs propriétés biologiques et dicter leur fonctionnement pendant la réparation endothéliale. Plus spécifiquement, les interactions des EPCs avec les plaquettes circulantes induisent leur migration, leur recrutement et leur différentiation en cellules endothéliales aux sites de lésions vasculaires. Cependant, l’impact d’une telle interaction sur la fonction plaquettaire n’a pas été recherché. Le but de mon projet était de :1) générer des EPCs à partir des cellules mononucléaires du sang humain périphérique ("Peripheral Blood Mononuclear Cells", PBMCs); 2) étudier les interactions adhésives entre les EPCs et les plaquettes; 3) déterminer leur impact sur la fonction plaquettaire et la formation du thrombus et 4) décrire le mécanisme d’action des EPCs sur les plaquettes et le thrombus. Mises en culture sur une surface de fibronectine dans un milieu conditionné, les PBMCs fraîchement isolées possédaient une morphologie ronde et une petite taille. Après cinq jours, les PBMCs adhérentes donnaient naissance à des colonies, puis formaient une monocouche de cellules aplaties caractéristiques des EPCs après dix jours de culture. Les EPCs différenciées étaient positives pour l’Ulex-lectine et l’Acétyle des lipoprotéines de faible densité ("Acetylated Low Density Lipoprotein", Ac-LDL), exprimaient les marqueurs progéniteurs (CD34, P-sélectine, VEGFR2, vWF et VE-Cadhérine) tandis que les marqueurs leucocytaires (CD14, PSGL-1 et L-sélectine) étaient absents. Ces EPCs interagissaient avec les plaquettes activées par un mécanisme dépendant de la P-sélectine plaquettaire, inhibaient l’activation et l’agrégation plaquettaire et réduisaient significativement l’adhésion plaquettaire, principalement par l’action de prostacycline (PGI2). En fait, ceci était associé avec une augmentation de l’expression de la cyclooxygénase-2 (COX-2) et du monoxyde d’azote (NO) synthéthase inductible (iNOS). Toutefois, les effets inhibiteurs des EPCs sur la fonction plaquettaire ont été renversés par une inhibition de la COX et non pas du NO. Bien que les EPCs fussent en mesure de lier les plaquettes via la P-sélectine, leurs effets prédominants étaient médiés essentiellement par une sécrétion paracrine, impliquant la PGI2. Néanmoins, un rapprochement étroit ou un bref contact entre les EPCs et les plaquettes était requis pour que cette fonction soit complètement réalisée. D’ailleurs, cet aspect a été investigué chez des souris déficientes en P-sélectine (P-sel-/-) et chez leurs congénères de phénotype sauvage (Wild Type, WT). Chez les souris WT, les EPCs inhibaient l’agrégation plaquettaire dans le sang complet de manière concentration-dépendante alors que dans les souris P-sel-/-, l’action des EPCs n’avait pas d’effet significatif. De plus, en utilisant un modèle murin de thrombose artérielle, nous avons démontré que l’infusion systémique des EPCs altéraient la formation du thrombus et réduisaient significativement sa masse chez les souris WT, mais non pas chez les souris P-sel-/-. En outre, le nombre des EPCs incorporées au niveau du thrombus et de la paroi vasculaire était visiblement réduit chez les P-sel-/- par rapport aux souris WT. Dans cette étude, nous sommes parvenus à différentier adéquatement des EPCs à partir des PBMCs, nous avons étudié les interactions adhésives entre les EPCs et les plaquettes, et nous avons décrit leur impact sur la fonction plaquettaire et la formation du thrombus. De plus, nous avons identifié la PGI2 comme étant le principal facteur soluble sécrété par les EPCs en culture et responsable de leurs effets inhibiteurs sur l’activation, l’adhésion et l’agrégation plaquettaire in vitro. De surcroît, nous avons élucidé le mécanisme d’action des EPCs sur l’agrégation plaquettaire et la formation du thrombus, in vivo, et nous avons souligné le rôle de la P-sélectine plaquettaire dans ce processus. Ces résultats ajoutent de nouvelles connaissances sur la biologie des EPCs et définissent leur rôle potentiel dans la régulation de la fonction plaquettaire et la thrombogenèse. / Endothelial Progenitor Cells (EPCs) are believed to contribute to vascular biology and endothelial repair. EPCs have been isolated from umbilical cord, bone marrow, peripheral blood and in some regenerative tissues. Interactions of EPCs with vascular and blood cells can largely influence their functional properties and predict their destiny in the target tissues. More specifically, interactions of EPCs with circulating platelets provide the critical signal to ensure their migration and homing at the sites of vascular injury and their differentiation into endothelial cells. However, the functional consequences of such interactions on platelets remain unknown. Accordingly, this project was designed to investigate the impact of EPCs on platelet function and the specific objectives of this study were to: 1) generate EPCs from human peripheral blood mononuclear cells (PBMCs); 2) characterize the adhesive interactions between EPCs and platelets; 3) determine their impact on platelet function and thrombus formation and 4) elucidate the mechanistic action of EPCs on platelets and thrombus. Cultured on fibronectin in conditioned media, PBMCs differentiated, within ten days of culture, into EPCs, which were positive for Ulex-lectin and Ac-LDL (Acetylated Low Density Lipoprotein), and expressed progenitor markers (CD34, VEGFR2, vWF, and VE-Cadherin). These EPCs bind activated platelets through P-selectin-dependent mechanism, inhibited platelet activation, aggregation and adhesion, mainly via prostacyclin (PGI2) secretion. Indeed, this was associated with up-regulation of cyclooxygenase-2 (COX-2) and inducible nitric oxide (NO) synthase (iNOS). However, the effects on platelets were reversed by COX, but not by NO inhibition. Although EPCs bound platelets via platelet P-selectin, their predominant effects occurred via a paracrine secretion, implying PGI2. Nevertheless, a transitory link or brief contact between EPCs and platelets was required for this function to be fully realized. This was further depicted using a murine arterial thrombosis model in P-selectin deficient mice (P sel-/-) and their wild-type counterparts (WT). EPCs significantly impaired, in a concentration dependent-manner, collagen-induced whole blood platelet aggregation in WT mice; whereas in P-sel-/- mice, EPCs had no significant effect. Moreover, in murin model of arterial thrombosis, infusion of EPCs altered thrombus formation and significantly reduced the mass of thrombi generated in WT, but not in P-sel-/- mice. Furthermore, the number of EPCs recruited within the thrombi and along the vascular wall was visually reduced in P-sel-/- mice as compared to WT mice. In this project, we succeeded in adequately differentiating EPCs from PBMCs, we characterized the adhesive interaction between EPCs and platelets, and we addressed the impact of EPCs on platelet function and thrombus formation. Moreover, we identified PGI2 as the principal soluble factor secreted by cultured EPCs and responsible of their inhibitory effects on platelet function in vitro. In addition, using a murine model of arterial carotid injury in WT and P-sel-/- mice, we elucidate the mechanistic action of EPCs on platelet aggregation and thrombus formation, in vivo, and we highlighted the role of platelet P-selectin in this process. These findings add new insights into the biology of EPCs and reveal a potential role for EPCs in regulating platelet function, which in turn may limit thrombogenesis and maintain hemostasis at the sites of vascular injury.

A hipertensão arterial pulmonar é uma síndrome clínica e hemodinâmica, caracterizada pelo aumento de resistência vascular na microcirculação. A vasoconstrição presente na doença ocorre principalmente devido à disfunção endotelial, induzindo a um estado pró-trombótico onde a participação das plaquetas é inequívoca. A trombina, principal agonista da ativação plaquetária, exerce seus efeitos nas células por meio de receptores ativados por proteases. Através da citometria de fluxo, este trabalho teve por objetivo analisar (1) a expressão do receptor ativado por protease do tipo 1 (PAR-1) na membrana de plaquetas, em seu estado íntegro ou clivado, (2) a atividade, mediante a formação de agregados entre plaquetas e leucócitos e, plaquetas e monócitos após estímulo de receptores ativados por proteases, além (3) da expressão de selectina-P na membrana plaquetária após estimulação. Foram estudados 30 pacientes portadores de hipertensão arterial pulmonar sob tratamento ambulatorial no Instituto do Coração da Faculdade de Medicina da Universidade de São Paulo. A idade destes pacientes variou de 11 a 78 anos, e a média da pressão sistólica da artéria pulmonar foi de 89+29 mmHg. Em 30% dos pacientes, o número de plaquetas esteve abaixo de 150x103/l, e em 40%, o nível do hematócrito apresentou-se acima de 50%. No presente estudo, não foi observada diferença significante na expressão do PAR-1 íntegro em plaquetas de pacientes comparativamente aos controles (p = 0,2). Em contrapartida, as plaquetas dos pacientes apresentaram menor quantidade de receptor clivado (p = 0,01), sugerindo internalização destes receptores. Com relação à avaliação da atividade de PAR-1 através da formação de agregados entre plaquetas e leucócitos e, plaquetas e monócitos após estimulação por agentes capazes de atuar sobre o receptor de trombina, não foram observadas diferenças entre pacientes e controles, ou seja, em ambos os casos houve a formação de agregados (p = 0,2 e p = 0,4, respectivamente). Em relação aos leucócitos, o SFLLRN, estimulou o receptor independentemente do seu estado, íntegro ou clivado, nos pacientes (p < 0,05), enquanto que nos indivíduos normais, a resposta só foi observada quando o receptor íntegro foi estimulado por trombina (p < 0,05). Tanto nos pacientes estudados, como nos controles, a estimulação do PAR-1 plaquetário, promoveu aumento da expressão de selectina-P na superfície plaquetária (p < 0,0001), embora não houvesse diferença entre os grupos (p = 0,9). Estes resultados demonstram que as plaquetas dos pacientes não são refratárias à liberação de seu conteúdo granular, e encontram-se aptas a responder aos estímulos tanto quanto as plaquetas dos controles. Este estudo reforça a importância da terapia antiplaquetária em pacientes com hipertensão arterial pulmonar. / Pulmonary hypertension is a clinical and hemodynamic syndrome, characterized by the increase of vascular resistance in lungs, generally through various mechanisms, involving vasoconstriction and remodeling of the arterial wall. Endothelial dysfunction in pulmonary arterial hypertension leads to a prothrombotic status, in which platelet participation seems to be unequivocal. Thrombin is the most potent platelet activator and exerts its effects on cells, through protease-activated receptors. Flow cytometry procedure was employed to assess (1) platelet protease-activated receptor 1 (PAR-1) expression, in both uncleaved and cleaved forms, (2) PAR-1 activity, through platelet-leukocyte and platelet-monocyte aggregates formation in response to the thrombin receptor stimulus, and finally (3) platelet P-selectin expression after stimulus. Thirty patients with pulmonary arterial hypertension (age 11 to 78 years) under treatment at the Heart Institute, University of São Paulo were enrolled. The systolic pulmonary arterial pressure was 89+29 mmHg. The platelet count was < 150x103/l in 30% of the patients, and the hematocrit level was > 50% in 40% of the patients. In the present study, there was no relevant difference in the level of intact platelet protease-activated receptor 1 expression in patients and controls (p = 0.2). On the other hand, the expression of cleaved receptors was decreased in patients (p = 0.01) platelets, what suggests internalization. There was no difference on platelet-leukocyte, and platelet-monocyte aggregates in response to the thrombin receptor stimulus between patients and controls, in other words, in both cases there was the aggregates formation (p = 0.2). However, aggregates formation in patients appeared to occur predominately in response to an agent (SFLLRN) capable to stimulate the receptor, independent of its state, intact or cleaved (p < 0.05). Otherwise, in healthy individuals, the response occurred especially when the intact receptor was stimulated by thrombin (p < 0.05). In these patients, similarly to the controls, platelet protease-activated receptor 1 stimulation induced membrane P-selectin expression (p < 0.001), although there was no difference between the groups (p = 0.9). These findings suggest that platelets from patients are not refractory to its granular content secretion and are capable to respond to stimulus as the controls platelets. This study re-enforce the importance of anti platelet therapy in pulmonary arterial hypertension patients.

Les Cellules Endothéliales Progénitrices ("Endothelial Progenitor Cells", EPCs) sont des précurseurs endothéliaux qui jouent un rôle émergeant en biologie vasculaire. Les EPCs ont été localisées dans le cordon ombilical, la moelle osseuse, le sang périphérique et dans certains tissus régénérateurs. Les interactions des EPCs avec les cellules sanguines et vasculaires peuvent largement influencer leurs propriétés biologiques et dicter leur fonctionnement pendant la réparation endothéliale. Plus spécifiquement, les interactions des EPCs avec les plaquettes circulantes induisent leur migration, leur recrutement et leur différentiation en cellules endothéliales aux sites de lésions vasculaires. Cependant, l’impact d’une telle interaction sur la fonction plaquettaire n’a pas été recherché. Le but de mon projet était de :1) générer des EPCs à partir des cellules mononucléaires du sang humain périphérique ("Peripheral Blood Mononuclear Cells", PBMCs); 2) étudier les interactions adhésives entre les EPCs et les plaquettes; 3) déterminer leur impact sur la fonction plaquettaire et la formation du thrombus et 4) décrire le mécanisme d’action des EPCs sur les plaquettes et le thrombus. Mises en culture sur une surface de fibronectine dans un milieu conditionné, les PBMCs fraîchement isolées possédaient une morphologie ronde et une petite taille. Après cinq jours, les PBMCs adhérentes donnaient naissance à des colonies, puis formaient une monocouche de cellules aplaties caractéristiques des EPCs après dix jours de culture. Les EPCs différenciées étaient positives pour l’Ulex-lectine et l’Acétyle des lipoprotéines de faible densité ("Acetylated Low Density Lipoprotein", Ac-LDL), exprimaient les marqueurs progéniteurs (CD34, P-sélectine, VEGFR2, vWF et VE-Cadhérine) tandis que les marqueurs leucocytaires (CD14, PSGL-1 et L-sélectine) étaient absents. Ces EPCs interagissaient avec les plaquettes activées par un mécanisme dépendant de la P-sélectine plaquettaire, inhibaient l’activation et l’agrégation plaquettaire et réduisaient significativement l’adhésion plaquettaire, principalement par l’action de prostacycline (PGI2). En fait, ceci était associé avec une augmentation de l’expression de la cyclooxygénase-2 (COX-2) et du monoxyde d’azote (NO) synthéthase inductible (iNOS). Toutefois, les effets inhibiteurs des EPCs sur la fonction plaquettaire ont été renversés par une inhibition de la COX et non pas du NO. Bien que les EPCs fussent en mesure de lier les plaquettes via la P-sélectine, leurs effets prédominants étaient médiés essentiellement par une sécrétion paracrine, impliquant la PGI2. Néanmoins, un rapprochement étroit ou un bref contact entre les EPCs et les plaquettes était requis pour que cette fonction soit complètement réalisée. D’ailleurs, cet aspect a été investigué chez des souris déficientes en P-sélectine (P-sel-/-) et chez leurs congénères de phénotype sauvage (Wild Type, WT). Chez les souris WT, les EPCs inhibaient l’agrégation plaquettaire dans le sang complet de manière concentration-dépendante alors que dans les souris P-sel-/-, l’action des EPCs n’avait pas d’effet significatif. De plus, en utilisant un modèle murin de thrombose artérielle, nous avons démontré que l’infusion systémique des EPCs altéraient la formation du thrombus et réduisaient significativement sa masse chez les souris WT, mais non pas chez les souris P-sel-/-. En outre, le nombre des EPCs incorporées au niveau du thrombus et de la paroi vasculaire était visiblement réduit chez les P-sel-/- par rapport aux souris WT. Dans cette étude, nous sommes parvenus à différentier adéquatement des EPCs à partir des PBMCs, nous avons étudié les interactions adhésives entre les EPCs et les plaquettes, et nous avons décrit leur impact sur la fonction plaquettaire et la formation du thrombus. De plus, nous avons identifié la PGI2 comme étant le principal facteur soluble sécrété par les EPCs en culture et responsable de leurs effets inhibiteurs sur l’activation, l’adhésion et l’agrégation plaquettaire in vitro. De surcroît, nous avons élucidé le mécanisme d’action des EPCs sur l’agrégation plaquettaire et la formation du thrombus, in vivo, et nous avons souligné le rôle de la P-sélectine plaquettaire dans ce processus. Ces résultats ajoutent de nouvelles connaissances sur la biologie des EPCs et définissent leur rôle potentiel dans la régulation de la fonction plaquettaire et la thrombogenèse. / Endothelial Progenitor Cells (EPCs) are believed to contribute to vascular biology and endothelial repair. EPCs have been isolated from umbilical cord, bone marrow, peripheral blood and in some regenerative tissues. Interactions of EPCs with vascular and blood cells can largely influence their functional properties and predict their destiny in the target tissues. More specifically, interactions of EPCs with circulating platelets provide the critical signal to ensure their migration and homing at the sites of vascular injury and their differentiation into endothelial cells. However, the functional consequences of such interactions on platelets remain unknown. Accordingly, this project was designed to investigate the impact of EPCs on platelet function and the specific objectives of this study were to: 1) generate EPCs from human peripheral blood mononuclear cells (PBMCs); 2) characterize the adhesive interactions between EPCs and platelets; 3) determine their impact on platelet function and thrombus formation and 4) elucidate the mechanistic action of EPCs on platelets and thrombus. Cultured on fibronectin in conditioned media, PBMCs differentiated, within ten days of culture, into EPCs, which were positive for Ulex-lectin and Ac-LDL (Acetylated Low Density Lipoprotein), and expressed progenitor markers (CD34, VEGFR2, vWF, and VE-Cadherin). These EPCs bind activated platelets through P-selectin-dependent mechanism, inhibited platelet activation, aggregation and adhesion, mainly via prostacyclin (PGI2) secretion. Indeed, this was associated with up-regulation of cyclooxygenase-2 (COX-2) and inducible nitric oxide (NO) synthase (iNOS). However, the effects on platelets were reversed by COX, but not by NO inhibition. Although EPCs bound platelets via platelet P-selectin, their predominant effects occurred via a paracrine secretion, implying PGI2. Nevertheless, a transitory link or brief contact between EPCs and platelets was required for this function to be fully realized. This was further depicted using a murine arterial thrombosis model in P-selectin deficient mice (P sel-/-) and their wild-type counterparts (WT). EPCs significantly impaired, in a concentration dependent-manner, collagen-induced whole blood platelet aggregation in WT mice; whereas in P-sel-/- mice, EPCs had no significant effect. Moreover, in murin model of arterial thrombosis, infusion of EPCs altered thrombus formation and significantly reduced the mass of thrombi generated in WT, but not in P-sel-/- mice. Furthermore, the number of EPCs recruited within the thrombi and along the vascular wall was visually reduced in P-sel-/- mice as compared to WT mice. In this project, we succeeded in adequately differentiating EPCs from PBMCs, we characterized the adhesive interaction between EPCs and platelets, and we addressed the impact of EPCs on platelet function and thrombus formation. Moreover, we identified PGI2 as the principal soluble factor secreted by cultured EPCs and responsible of their inhibitory effects on platelet function in vitro. In addition, using a murine model of arterial carotid injury in WT and P-sel-/- mice, we elucidate the mechanistic action of EPCs on platelet aggregation and thrombus formation, in vivo, and we highlighted the role of platelet P-selectin in this process. These findings add new insights into the biology of EPCs and reveal a potential role for EPCs in regulating platelet function, which in turn may limit thrombogenesis and maintain hemostasis at the sites of vascular injury.

A hipertensão arterial pulmonar é uma síndrome clínica e hemodinâmica, caracterizada pelo aumento de resistência vascular na microcirculação. A vasoconstrição presente na doença ocorre principalmente devido à disfunção endotelial, induzindo a um estado pró-trombótico onde a participação das plaquetas é inequívoca. A trombina, principal agonista da ativação plaquetária, exerce seus efeitos nas células por meio de receptores ativados por proteases. Através da citometria de fluxo, este trabalho teve por objetivo analisar (1) a expressão do receptor ativado por protease do tipo 1 (PAR-1) na membrana de plaquetas, em seu estado íntegro ou clivado, (2) a atividade, mediante a formação de agregados entre plaquetas e leucócitos e, plaquetas e monócitos após estímulo de receptores ativados por proteases, além (3) da expressão de selectina-P na membrana plaquetária após estimulação. Foram estudados 30 pacientes portadores de hipertensão arterial pulmonar sob tratamento ambulatorial no Instituto do Coração da Faculdade de Medicina da Universidade de São Paulo. A idade destes pacientes variou de 11 a 78 anos, e a média da pressão sistólica da artéria pulmonar foi de 89+29 mmHg. Em 30% dos pacientes, o número de plaquetas esteve abaixo de 150x103/l, e em 40%, o nível do hematócrito apresentou-se acima de 50%. No presente estudo, não foi observada diferença significante na expressão do PAR-1 íntegro em plaquetas de pacientes comparativamente aos controles (p = 0,2). Em contrapartida, as plaquetas dos pacientes apresentaram menor quantidade de receptor clivado (p = 0,01), sugerindo internalização destes receptores. Com relação à avaliação da atividade de PAR-1 através da formação de agregados entre plaquetas e leucócitos e, plaquetas e monócitos após estimulação por agentes capazes de atuar sobre o receptor de trombina, não foram observadas diferenças entre pacientes e controles, ou seja, em ambos os casos houve a formação de agregados (p = 0,2 e p = 0,4, respectivamente). Em relação aos leucócitos, o SFLLRN, estimulou o receptor independentemente do seu estado, íntegro ou clivado, nos pacientes (p < 0,05), enquanto que nos indivíduos normais, a resposta só foi observada quando o receptor íntegro foi estimulado por trombina (p < 0,05). Tanto nos pacientes estudados, como nos controles, a estimulação do PAR-1 plaquetário, promoveu aumento da expressão de selectina-P na superfície plaquetária (p < 0,0001), embora não houvesse diferença entre os grupos (p = 0,9). Estes resultados demonstram que as plaquetas dos pacientes não são refratárias à liberação de seu conteúdo granular, e encontram-se aptas a responder aos estímulos tanto quanto as plaquetas dos controles. Este estudo reforça a importância da terapia antiplaquetária em pacientes com hipertensão arterial pulmonar. / Pulmonary hypertension is a clinical and hemodynamic syndrome, characterized by the increase of vascular resistance in lungs, generally through various mechanisms, involving vasoconstriction and remodeling of the arterial wall. Endothelial dysfunction in pulmonary arterial hypertension leads to a prothrombotic status, in which platelet participation seems to be unequivocal. Thrombin is the most potent platelet activator and exerts its effects on cells, through protease-activated receptors. Flow cytometry procedure was employed to assess (1) platelet protease-activated receptor 1 (PAR-1) expression, in both uncleaved and cleaved forms, (2) PAR-1 activity, through platelet-leukocyte and platelet-monocyte aggregates formation in response to the thrombin receptor stimulus, and finally (3) platelet P-selectin expression after stimulus. Thirty patients with pulmonary arterial hypertension (age 11 to 78 years) under treatment at the Heart Institute, University of São Paulo were enrolled. The systolic pulmonary arterial pressure was 89+29 mmHg. The platelet count was < 150x103/l in 30% of the patients, and the hematocrit level was > 50% in 40% of the patients. In the present study, there was no relevant difference in the level of intact platelet protease-activated receptor 1 expression in patients and controls (p = 0.2). On the other hand, the expression of cleaved receptors was decreased in patients (p = 0.01) platelets, what suggests internalization. There was no difference on platelet-leukocyte, and platelet-monocyte aggregates in response to the thrombin receptor stimulus between patients and controls, in other words, in both cases there was the aggregates formation (p = 0.2). However, aggregates formation in patients appeared to occur predominately in response to an agent (SFLLRN) capable to stimulate the receptor, independent of its state, intact or cleaved (p < 0.05). Otherwise, in healthy individuals, the response occurred especially when the intact receptor was stimulated by thrombin (p < 0.05). In these patients, similarly to the controls, platelet protease-activated receptor 1 stimulation induced membrane P-selectin expression (p < 0.001), although there was no difference between the groups (p = 0.9). These findings suggest that platelets from patients are not refractory to its granular content secretion and are capable to respond to stimulus as the controls platelets. This study re-enforce the importance of anti platelet therapy in pulmonary arterial hypertension patients.

No description available.

No description available.

A disfunção microvascular, envolvendo células endoteliais, plaquetas e leucócitos, está presente na hipertensão arterial pulmonar (HAP), associando-se a risco aumentado de trombose e menor sobrevida. Estudos sobre disfunção microvascular são escassos em outras formas da doença que não a idiopática. Os objetivos do estudo foram: caracterizar a disfunção microvascular em diferentes formas de HAP através da dosagem de marcadores bioquímicos, avaliando possíveis correlações com índices de gravidade; investigar os efeitos da administração de rosuvastatina em níveis circulantes de marcadores de disfunção microvascular nesses pacientes; e investigar possível associação entre o nível plasmático dos marcadores e prognóstico. Foram incluídos sessenta pacientes: 14 com HAP idiopática ou hereditária, e 46 com HAP associada a cardiopatia congênita (HAPCCg) sem hipoxemia (N=18) ou com hipoxemia (N=28), com idades entre 13 e 60 anos. Foram dosados os níveis plasmáticos circulantes do antígeno do fator de von Willebrand (vWF:Ag), ativador tecidual do plasminogênio (t-PA); inibidor do ativador do plasminogênio (PAI-1), fator de necrose tumoral (TNF-), proteína C reativa (PCR), selectina-P; interleucina-6 (IL-6); e interleucina-10 (IL -10), na condição basal e após 30, 60 e 180 dias de tratamento, por método imunoenzimático. Após randomização, administrouse placebo (N=30) ou dose única oral diária (10mg) de rosuvastatina (N=30), por seis meses. Dados demográficos e funcionais como idade, distância caminhada em seis minutos, saturação periférica de oxigênio em repouso e após esforço, bem como hematócrito, também foram registrados. Pacientes com HAPCCg foram acompanhados por um período de 0,7 a 4,0 anos (mediana de 3,6 anos). Na condição basal, excetuando-se TNF- e PCR, todas as proteínas apresentaram-se significantemente elevadas em relação aos controles (p<0,001), havendo correlação com índices de gravidade clínica. No estudo com rosuvastatina, houve redução significante nos níveis de selectina-P em relação ao placebo (p=0,037), ao longo do tratamento. Houve melhora na saturação periférica de oxigênio após seis minutos de caminhada, no grupo estatina, em pacientes com HAPCCg com hipoxemia, em relação ao placebo. Considerando-se o período de acompanhamento, em portadores de HAPCCg, níveis plasmáticos persistentemente elevados do vWF:Ag (média de quatro determinações), acima do nível correspondente ao percentil 95 dos controles (139 U/d/L) associaram-se maior risco de morte (razão de risco 6,56, IC 95% 1,46 a 29,4, p=0.014), sem alteração após ajustamento para variáveis demográficas, funcionais e de tratamento, à análise multivariada. Assim, a disfunção microvascular está presente em indivíduos com HAP idiopática, hereditária ou associada a cardiopatias congênitas. Na HAP, o uso crônico de rosuvastatina em dose baixa associase à redução do nível circulante de selectina-P, e propicia aumento na saturação periférica de oxigênio ao final do exercício, em indivíduos com HAPCCg e hipoxemia. Em indivíduos portadores de HAPCCg, níveis plasmáticos persistentemente elevados do vWF:Ag são indicativo de pior prognóstico / Microvascular dysfunction, involving endothelial cells, platelets and leukocytes, is present in pulmonary arterial hypertension (PAH), and is associated to higher risk to thrombotic complications and mortality. Most data about microvascular dysfunction in PAH do not include other forms of the disease beyond idiopathic PAH. The present study was planned to measure plasma levels microvascular dysfunction markers in two different forms of PAH, and investigate possible correlations with indices of severity of the disease; to investigate the effects of chronic rosuvastatin administration versus placebo on the circulating levels of these markers; and to investigate possible associations between levels of these parameters and prognosis. Sixty patients (aged 13 to 60 years) were included, 14 with idiopathic or hereditary PAH, and 46 with congenital heart disease-associated PAH (CHDPAH), in the absence (N=18) or presence (N=28) of hypoxemia. Plasma levels of von Willebrand factor antigen (vWF:Ag), tissue-plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), tumor necrosis factor alpha (TNF-), reactive C protein (RCP), P-selectin, interleukin-6 (IL- 6), and interleukin-10 (IL-10) were measured before treatment and 30, 90, and 180 days on treatment using high-sensitivity enzyme-linked immunosorbent assay kits. Patients were randomly assigned to placebo (N=30) or a single oral dose of rosuvastatin (N=30), 10mg/day, for six months. Demographic and functional data such as age, six-minute walk distance, peripheral oxygen saturation at rest and at the end of the six-minute walk, as well as the hematocrit, were recorded. Patients with CHDPAH were followed-up for 0.7 to 4.0 years (median 3.6 years). At baseline, levels of all proteins (except TNF- and RCP) were significantly increased in patients versus controls (p<0,001), and correlated significantly with indices of severity of the disease. P-selectin level was lower in the rosuvastatin group compared with placebo throughout the treatment (p = 0.037). In hypoxemic CHDPAH patients, the peripheral oxygen saturation, at the end of the six-minute walk, was higher in the rosuvastatin group, compared with placebo. During the follow-up of patients with CHDPAH, an average vWF:Ag (mean of four determinations) above the level corresponding to the 95th percentile of controls (139 U/dL) was associated with a high risk of death (hazard ratio 6.56, 95% CI 1.46 to 29.4, p=0.014). This was not modified after adjustment for demographic, functional and treatment-related variables in multivariate analysis. In conclusion, microvascular dysfunction is present in individuals with idiopathic, hereditary and the congenital heart disease-associated PAH. The chronic use of low-dose rosuvastatin is associated to reduction of circulating levels of P-selectin. In patients with CHDPAH with hypoxemia, rosuvastatin also increases peripheral oxygen saturation during exercise. In CHDPAH patients, a sustained increase in plasma vWF:Ag is indicative of poor prognosis

La tomographie par émission de positons (TEP) est une technique d’imagerie moléculaire avec de meilleures performances que la tomographie par émission monophotonique. Son utilisation contribue à l’amélioration des prises en charge des patients. Dans les centres dépourvus de cyclotrons, le 68Ga disponible à partir d’un générateur constitue une alternative pour le développement de traceurs TEP. Pour pouvoir développer des 68Ga-traceurs, un travail de caractérisation de la qualité des éluats a été effectué. Des méthodes de marquage adaptées ont été mises en place et validées. Nous nous sommes intéressés à trois cibles moléculaires particulièrement intéressantes dans les pathologies cardiovasculaires: les récepteurs de la somatostatine (SSTR) surexprimés dans les tumeurs neuroendocrines (TNE) mais constituant aussi une cible d’intérêt dans les pathologies cardiovasculaires à composante inflammatoire ; la phosphatidylsérine (PS), un marqueur de l’apoptose cellulaire et de l’activation plaquettaire ; la P-sélectine, un marqueur des activations plaquettaire et endothéliale. Les traceurs suivants ont été développés: 1) Analogues de la somatostatine ciblant les SSTR: a)68Ga-DOTANOC validé pour l’imagerie des TNE-Gastroentéropancréatiques dans le cadre d’un essai clinique multicentrique. b) 68Ga-NODAGANOC testé in vitro sur des cellules d’adénocarcinome pancréatique. Cette validation initiale dans l’application la plus fréquente(oncologie) a pour objectif de faciliter le passage vers des applications cardiovasculaires futures (athérosclérose, myocardite...) ; 2) Un peptide ciblant la PS : le 68Ga-P04087 ; 3) Un polysaccharide ciblant la P-sélectine: 68Ga-NODAGA-Asphy. Les deux derniers traceurs ont été testés sur un modèle d’endocardite infectieuse chez le rat. / The Positron emission tomography (PET) is a molecular imaging technique with usually better performances than Single-Photon Emission Computed Tomography. Consequently, the use of PET and appropriate tracers could enable clinicians to make a better therapeutic decision, thus improving the management of patients. In centers without cyclotrons, 68Ga available from a generator is an alternative for the development of PET tracers. In order to develop 68Ga labeled-molecules, a characterization of the quality of the eluates was performed. Radiolabeling techniques adapted to the quality of the starting material were developed and validated. In this thesis we focused on three particularly interesting molecular targets in cardiovascular pathologies: somatostatin receptors (SSTR), overexpressed in neuroendocrine tumors (NETs) but also constituting a target of interest in cardiovascular diseases with an inflammatory component; phosphatidylserine (PS), a marker of cell apoptosis and platelet activation; P-selectin, a marker of platelet and endothelial activation.The following tracers have been developed: 1) Somatostatin analogues which target SSTR: a) 68Ga-DOTANOC validated for Gastroenteropancreatic-NETs imaging and used in a multicenter clinical trial. b) 68Ga-NODAGANOC tested in vitro on pancreatic adenocarcinoma cells. This initial validation in the most common application (oncology) aims to facilitate the transition to future cardiovascular applications (atherosclerosis, myocarditis ...) 2) A peptide for PS targeting: 68Ga-P04087; 3) A polysaccharide for P-selectin targeting: 68Ga-NODAGA-Asphy. The last two radiolabeled molecules were tested in a rat model of infective endocarditis.

A disfunção microvascular, envolvendo células endoteliais, plaquetas e leucócitos, está presente na hipertensão arterial pulmonar (HAP), associando-se a risco aumentado de trombose e menor sobrevida. Estudos sobre disfunção microvascular são escassos em outras formas da doença que não a idiopática. Os objetivos do estudo foram: caracterizar a disfunção microvascular em diferentes formas de HAP através da dosagem de marcadores bioquímicos, avaliando possíveis correlações com índices de gravidade; investigar os efeitos da administração de rosuvastatina em níveis circulantes de marcadores de disfunção microvascular nesses pacientes; e investigar possível associação entre o nível plasmático dos marcadores e prognóstico. Foram incluídos sessenta pacientes: 14 com HAP idiopática ou hereditária, e 46 com HAP associada a cardiopatia congênita (HAPCCg) sem hipoxemia (N=18) ou com hipoxemia (N=28), com idades entre 13 e 60 anos. Foram dosados os níveis plasmáticos circulantes do antígeno do fator de von Willebrand (vWF:Ag), ativador tecidual do plasminogênio (t-PA); inibidor do ativador do plasminogênio (PAI-1), fator de necrose tumoral (TNF-), proteína C reativa (PCR), selectina-P; interleucina-6 (IL-6); e interleucina-10 (IL -10), na condição basal e após 30, 60 e 180 dias de tratamento, por método imunoenzimático. Após randomização, administrouse placebo (N=30) ou dose única oral diária (10mg) de rosuvastatina (N=30), por seis meses. Dados demográficos e funcionais como idade, distância caminhada em seis minutos, saturação periférica de oxigênio em repouso e após esforço, bem como hematócrito, também foram registrados. Pacientes com HAPCCg foram acompanhados por um período de 0,7 a 4,0 anos (mediana de 3,6 anos). Na condição basal, excetuando-se TNF- e PCR, todas as proteínas apresentaram-se significantemente elevadas em relação aos controles (p<0,001), havendo correlação com índices de gravidade clínica. No estudo com rosuvastatina, houve redução significante nos níveis de selectina-P em relação ao placebo (p=0,037), ao longo do tratamento. Houve melhora na saturação periférica de oxigênio após seis minutos de caminhada, no grupo estatina, em pacientes com HAPCCg com hipoxemia, em relação ao placebo. Considerando-se o período de acompanhamento, em portadores de HAPCCg, níveis plasmáticos persistentemente elevados do vWF:Ag (média de quatro determinações), acima do nível correspondente ao percentil 95 dos controles (139 U/d/L) associaram-se maior risco de morte (razão de risco 6,56, IC 95% 1,46 a 29,4, p=0.014), sem alteração após ajustamento para variáveis demográficas, funcionais e de tratamento, à análise multivariada. Assim, a disfunção microvascular está presente em indivíduos com HAP idiopática, hereditária ou associada a cardiopatias congênitas. Na HAP, o uso crônico de rosuvastatina em dose baixa associase à redução do nível circulante de selectina-P, e propicia aumento na saturação periférica de oxigênio ao final do exercício, em indivíduos com HAPCCg e hipoxemia. Em indivíduos portadores de HAPCCg, níveis plasmáticos persistentemente elevados do vWF:Ag são indicativo de pior prognóstico / Microvascular dysfunction, involving endothelial cells, platelets and leukocytes, is present in pulmonary arterial hypertension (PAH), and is associated to higher risk to thrombotic complications and mortality. Most data about microvascular dysfunction in PAH do not include other forms of the disease beyond idiopathic PAH. The present study was planned to measure plasma levels microvascular dysfunction markers in two different forms of PAH, and investigate possible correlations with indices of severity of the disease; to investigate the effects of chronic rosuvastatin administration versus placebo on the circulating levels of these markers; and to investigate possible associations between levels of these parameters and prognosis. Sixty patients (aged 13 to 60 years) were included, 14 with idiopathic or hereditary PAH, and 46 with congenital heart disease-associated PAH (CHDPAH), in the absence (N=18) or presence (N=28) of hypoxemia. Plasma levels of von Willebrand factor antigen (vWF:Ag), tissue-plasminogen activator (t-PA), plasminogen activator inhibitor-1 (PAI-1), tumor necrosis factor alpha (TNF-), reactive C protein (RCP), P-selectin, interleukin-6 (IL- 6), and interleukin-10 (IL-10) were measured before treatment and 30, 90, and 180 days on treatment using high-sensitivity enzyme-linked immunosorbent assay kits. Patients were randomly assigned to placebo (N=30) or a single oral dose of rosuvastatin (N=30), 10mg/day, for six months. Demographic and functional data such as age, six-minute walk distance, peripheral oxygen saturation at rest and at the end of the six-minute walk, as well as the hematocrit, were recorded. Patients with CHDPAH were followed-up for 0.7 to 4.0 years (median 3.6 years). At baseline, levels of all proteins (except TNF- and RCP) were significantly increased in patients versus controls (p<0,001), and correlated significantly with indices of severity of the disease. P-selectin level was lower in the rosuvastatin group compared with placebo throughout the treatment (p = 0.037). In hypoxemic CHDPAH patients, the peripheral oxygen saturation, at the end of the six-minute walk, was higher in the rosuvastatin group, compared with placebo. During the follow-up of patients with CHDPAH, an average vWF:Ag (mean of four determinations) above the level corresponding to the 95th percentile of controls (139 U/dL) was associated with a high risk of death (hazard ratio 6.56, 95% CI 1.46 to 29.4, p=0.014). This was not modified after adjustment for demographic, functional and treatment-related variables in multivariate analysis. In conclusion, microvascular dysfunction is present in individuals with idiopathic, hereditary and the congenital heart disease-associated PAH. The chronic use of low-dose rosuvastatin is associated to reduction of circulating levels of P-selectin. In patients with CHDPAH with hypoxemia, rosuvastatin also increases peripheral oxygen saturation during exercise. In CHDPAH patients, a sustained increase in plasma vWF:Ag is indicative of poor prognosis

Résumé Responsable de près de 7 millions de décès prématurés par an à travers le monde, la pollution atmosphérique représente un problème sanitaire mondial majeur. L’exposition humaine à la pollution particulaire dont les émissions de diesel représentent la principale source, est responsable d’une élévation de la mortalité et de l’incidence d’événements cardiovasculaires. Les mécanismes physiopathologiques sous-tendant cette toxicité aigue restent à ce jour largement méconnus. A l’aide d’une méthodologie d’exposition standardisée aux émissions de diesel, nous avons exploré les effets vasculaires périphériques et pulmonaires ainsi que les effets hémostatiques et plaquettaires secondaires à l’inhalation particulaire chez des sujets sains. L’étude dynamique de la microcirculation périphérique par Laser Doppler Imager nous a permis de démontrer une altération de la vasomotricité endothélium-dépendante induite par l’exposition aux émissions de diesel. La dysfonction observée est associée à une diminution de la biodisponibilité locale du NO ainsi qu’à une production radicalaire accrue au sein de la cellule endothéliale. Au cours d’une épreuve dynamique d’élévation du débit cardiaque par infusion de dobutamine, l’exposition aiguë aux émissions de diesel entraîne une élévation de la résistance vasculaire pulmonaire et une diminution de la distensibilité vasculaire pulmonaire mesurées par échocardiographie. Enfin, l’expression de surface des marqueurs d’activation plaquettaire CD62p (P-selectine) et CD63 est augmentée après exposition aux émissions de diesel, en corrélation avec la quantité de particules inhalées et constitue ainsi un état d’activation plaquettaire accru sans toutefois de modification de l’agrégation plaquettaire. A travers ces effets vasculaires systémiques et pulmonaires ainsi que ces effets hémostatiques, l’exposition aux émissions de diesel entraîne une toxicité cardiovasculaire aiguë, agissant en synergie, capable de déclencher la survenue d’événements cardiovasculaires.Abstract Responsible for up to 7 million deaths/year, air pollution is a major worldwide health burden. Particulate human exposure mainly originates from diesel exhaust and is associated with an increased cardiovascular mortality and an increased onset of cardiovascular events. Physiopathologic mechanisms underlying this acute toxicity remain largely unknown. We used a standardized diesel exhaust exposure protocol to explore not only the peripheral and pulmonary vascular effects, but also the hemostatic and platelet modifications in healthy subjects after particulate inhalation. Diesel exhaust exposure impairs the endothelium-dependant vasodilation during a dynamic evaluation of the peripheral microcirculation assessed by Laser Doppler Imager, whilst the endothelial independent vasodilatation remains unaffected. Vascular dysfunction is associated to a decreased local NO bioavailability and an increased endothelial reactive oxygen species production. During a dynamic cardiac stress test using continuous dobutamine infusion, acute diesel exhaust exposure induces an increased pulmonary vascular resistance associated to a decreased pulmonary vascular distensibility, both evaluated by echocardiography. Finally, platelet activation is modified after diesel exhaust exposure with an increased CD62p (P-selectin) and CD63 platelet surface expression, correlated with the total amount of inhaled particles. We observed a state of platelet activation without change in platelet aggregation. Through these multiples effects, combining systemic and pulmonary vascular impairment with platelet prothrombotic modifications, diesel exhaust exposure induces an acute cardiovascular toxicity, which can synergistically trigger acute events. / Doctorat en Sciences médicales (Médecine) / info:eu-repo/semantics/nonPublished