INTRODUÇÃO: O objetivo deste estudo é determinar as propriedades biomecânicas e histológicas de fragmentos da parede anterior de aneurismas da aorta abdominal. MÉTODOS: Dos pacientes submetidos à correção cirúrgica aberta de aneurisma da aorta abdominal, foram removidos fragmentos da parede anterior do saco aneurismático, divididos em dois espécimes. Um, destinado à análise histológica, para a quantificação de fibras colágenas, elásticas, musculares lisas e grau de atividade inflamatória e outro, pareado, submetido a teste destrutivo uniaxial, obtendo-se características biomecânicas, como, força, tensão e estresse de falência do fragmento. As médias das variáveis paramétricas foram avaliadas com teste t-Student ou análise de variância. Quando significante, utilizou-se teste de Tukey para discriminar as diferenças. As distribuições das variáveis não paramétricas foram avaliadas com teste Mann-Whitney ou análise de Kruskal-Wallis. Quando significante, utilizou-se teste de Dunn para discriminar as diferenças. Os valores de p<0,05 foram considerados estatisticamente significantes. RESULTADOS: Foram considerados os resultados das análises de fragmentos de 90 indivíduos. Os valores médios encontrados para as propriedades biomecânicas relacionadas à resistência do tecido aórtico (falência) foram força = 4,98±2,22 N, tensão = 13,18±5,98 N/cm e estresse = 103,14±47,09 N/cm2. A deformação média dos fragmentos até a falência foi de 0,39±0,12. Os fragmentos dos aneurismas de diâmetros transversos máximos maiores ou iguais a 5,5 cm apresentaram valores médios de força, tensão e estresse de falência (5,32±2,07 N, 13,83±5,58 N/cm e 103,02 N/cm2) maiores que os fragmentos de aneurismas de diâmetros menores que 5,5 cm (4,1±2,41 N, 10,82±6,48 N/cm, 77,03 N/cm2), com significância estatística para os três parâmetros de resistência do material. Não foram identificadas diferenças entre os valores médios de deformação de falência entre estes grupos (0,41±0,12 x 0,37±0,14 p = 0,260), bem como entre os valores médios de espessura dos fragmentos (1,58±0,41 x 1,53±0,42 mm p = 0,662). Os valores percentuais médios na composição dos fragmentos foram para as fibras colágenas (coloração de tricrômico de Masson) de 44,34±0,48%, para as fibras colágenas (coloração de picrosirius) de 61,85±10,14%, para as fibras musculares lisas (imuno histoquímica/alfa actina) de 3,46±2,23% e para as fibras elásticas (coloração de Verhoeff) inferior a 1% (traços). Não foram identificadas diferenças entre o percentual destes elementos na composição de fragmentos provenientes da parede anterior de aneurismas de diâmetro transverso máximo >= 5,5 cm e < 5,5 cm. Foi caracterizada uma atividade inflamatória mais intensa nos fragmentos provenientes de aneurismas de diâmetro transverso máximo >= 5,5 cm quando comparados aos fragmentos provenientes de aneurismas de diâmetro transverso máximo < 5,5 cm (grau 3 - 70% x 28,6% p = 0,011). Comparando-se os aneurismas sintomáticos versus os assintomáticos não foram identificadas diferenças significativas para as propriedades biomecânicas de falência dos fragmentos (força = 5,32±2,36 x 4,65±2,05 N, p = 0,155; tensão = 14,08±6,11 x 12,81±5,77 N/cm, p = 0,154; estresse = 103,02 x 84,76 N/cm2, p = 0,144 e deformidade = 0,38±0,12 x 0,41±0,13, p = 0,287), assim como para a espessura (1,56±0,41 x 1,57±0,41 mm p = 0,848) e composição histológica (fibras colágenas 44,67±11,17 x 44,02±13,79 % p = 0,808; fibras musculares lisas 2,52 x 2,35 %, p = 0,751; fibras elásticas inferior a 1%). CONCLUSÃO: Os fragmentos provenientes da parede anterior do saco aneurismático de aneurismas maiores mostraram-se mais resistentes, não se identificando diferenças entre os fragmentos quanto à espessura e conteúdo da matriz protéica. A maior resistência dos fragmentos de aneurismas maiores provavelmente está relacionada à adaptação da parede para suportar maior grau de sobrecarga hemodinâmica à medida que o diâmetro aumenta. Neste estudo esta adaptação não foi revelada pela análise histológica realizada e demonstra a limitação do estudo de fragmentos isolados de aneurismas para estimar o risco de ruptura dos mesmos / INTRODUCTION: The objective of this study was to determine the biomechanical and histological properties of fragments isolated from the anterior wall of abdominal aortic aneurysms. METHODS: Fragments of the anterior aneurysm wall were excised from the aneurysmatic sac of patients who underwent open surgery for repair of abdominal aortic aneurysm and divided into two specimens. One specimen was sent for histological analysis for quantification of collagen fibers, elastic fibers, smooth muscle cells and degree of inflammatory activity and the other, by uniaxial testing, was used to assess biomechanical properties, such as force, tension, and stress at the time of failure of the material. The means of parametric variables were evaluated with Student\'s t test or analysis of variance. When significant, we used the Tukey test to discriminate differences. The distributions of non-parametric variables were evaluated with Mann- Whitney or Kruskal-Wallis test. When significant, we used Dunn\'s test to discriminate differences. A p-value of less than 0.05 was considered statistically significant. RESULTS: Anterior-wall fragments from a total of 90 patients were considered. The average values of biomechanical parameters related to the resistance of the aorta (failure) were as follows: strength, 4.98±2.22 N; tension, 13.18±5.98 N/cm; and stress 103.14±47.09 N/cm2. The average deformation of the fragments at the time of the failure was 0.39±0.12. Fragments of aortic aneurysm with a maximum transverse diameter larger or equal to 5.5 cm showed average values for strength, tension, and stress at the time of the failure of the material (5.32±2.07 N, 13.83±5.58 N/cm, and 103.02 N/cm2, respectively), which were higher than those of fragments of aneurysms with diameters less than 5.5 cm (4.1±2.41 N, 10.82±6.48 N/cm, 77.03 N/cm2, respectively). The differences in the 3 parameters were statistically significant. However, no differences were observed between the groups in relation to average failure deformation (0.41±0.12 × 0.37±0.14; p = 0.260) and thickness of the analyzed fragments (1.58±0.41 × 1.53±0.42 mm; p = 0.662). The average values of fiber compositions of the fragments were as follows: collagen fibers, 44.34±0.48% and 61.85±10.14% (assessed using Masson trichrome staining and picrosirius red staining, respectively); smooth muscle cells, 3.46±2.23% (immunohistochemistry/alpha-actin); and elastic fibers, less than 1% (traces) (Verhoeff-van Gieson staining). No differences in fiber percentages were observed in the fragments from aneurysms with a maximum transverse diameter >= 5.5 cm and < 5.5 cm. A more intense inflammatory activity was assessed in fragments from aneurysms with maximum transverse diameter >= 5.5 cm than in fragments from aneurysms with maximum transverse diameter < 5.5 cm (grade 3 - 70% × 28.6%; p = 0.011). Compared to asymptomatic aneurysms, fragments from symptomatic aneurysms showed no significant differences in the biomechanical properties at the time of the failure (strength, 5.32±2.36 × 4.65±2.05 N, p = 0.155; tension, 14.08±6.11 × 12.81±5.77 N/cm, p = 0,154; stress, 103.02 × 84.76 N/cm2, p = 0.144; and deformity, 0.38±0.12 × 0.41±0.13, p = 0.287), thickness of the fragments (1.56±0.41 × 1.57±0.41 mm, p = 0.848) and histological composition (collagen fibers, 44.67±11.17 × 44.02±13.79%, p = 0.808; smooth muscle fibers, 2.52 × 2.35%, p = 0.751; elastic fibers, <1%). CONCLUSION: Fragments of the anterior wall removed from the aneurysmatic sac of large aneurysms appeared to be more resistant than those from small aneurysms. No differences between the aneurysm fragments were observed with respect to thickness and matrix protein content. The high resistance of fragments of larger aneurysms is probably attributable to the adaptation of the wall to support a high hemodynamic stress as the diameter of the aorta increases. In this study, this adaptation was not shown by histological analysis. This suggests a limitation of this study for assessing the risk of rupture based on isolated aneurysm fragments

Galectina-1 (Gal-1) pertence à família de lectinas que reconhecem -galactosídeos e pode ser expressa em vários tipos celulares, incluindo as células endoteliais e músculo liso. Esta lectina endógena possui propriedades imunomodulatórias e antiinflamatórias, dependentes de processos celulares, essenciais incluindo ativação, diferenciação, sobrevivência, fagocitose e adesão celular. Os eventos iniciais da lesão vascular são pouco conhecidos e, na literatura, são escassos os dados sobre a participação da Gal-1 nesses eventos. Portanto, neste trabalho, pretendeu-se avaliar a participação dessa lectina nos eventos iniciais da lesão vascular por hiperhomocisteinemia moderada induzida em camundongos. A dose padrão de 0,4g/Kg/dia de homocisteína-tiolactona foi administrada oralmente a camundongos selvagens (Gal-1+/+) e destituídos do gene da Gal-1 (Gal-1-/-), por diferentes tempos, causando uma elevação da concentração plasmática de homocisteína total, Este fato provocou alterações na reatividade vascular de contração na artéria aorta e de rolamento e adesão de leucócitos em vênulas mesentéricas. Foi detectada a presença da Gal-1 nas aortas de animais Gal-1+/+ em todos tempos de tratamento e no controle, observando, porém, um aumento dela no grupo tratados por 15 dias e, mais expressa em 24 horas (expressão protéica por Western blot e imunofluorescência e, gênica por Real Time PCR). Neste tempo, houve maiores alterações metabólicas significativas como triglicérides, LDL, colesterol total, glicose e de homocisteína. A análise da expressão das óxido nítrico sintases revelou não haver alterações para NOS induzida (iNOS) entre os grupos de animais Gal-1+/+ controle e tratados, nem mesmo em relação aos animais controles Gal-1-/-. Entretanto, o tratamento modulou a expressão da NOS endotelial (eNOS) nos animais Gal-1+/+, havendo uma redução da proteína para os tempos de 48 horas e 7dias (expressão protéica por imunohistoquímica - peroxidade e, gênica por RT-PCR). O tratamento não alterou a concentração plasmática de óxido nítrico (NO) em camundongos Gal-1+/+, mas foi detectada redução dos valores basais para animais Gal-1-/- e, uma redução com o tratamento por 15 dias. Portanto, foi verificado que a expressão de Gal-1 foi aumentada com o tratamento de Hcy-Taq nos tempos de 24 horas e 15 dias, onde se observam significativas e maiores alterações biológicas dos parâmetros de lesão vascular induzida pela hiperhomocisteinemia moderada analisadas: reatividade vascular, rolamento e adesão de leucócitos em vênulas mesentéricas, concentração de homocisteína plasmática, perfil lipídico e expressão da óxido nítrico sintase endotelial. / Galectin-1 (Gal-1) belongs to the lectin family of -galactosides-binding proteins and can be expressed by several cell types, including endothelial cells and vascular smooth cells. This endogenous lectin has immunological and anti-inflammatory properties dependent of essential cellular processes, including activation, differentiation, survival, phagocytosis and cell adhesion. There are few and inconclusive studies about the engagement of Gal-1 in vascular injury initial processes. Thus, this work was conducted to evaluate the engagement of Gal-1 in hyperhomocysteinemia-induced vascular injury. Wild type (Gal-1+/+) and Gal-1-knockout (Gal-1-/-) mice were fed with 0,4g/Kg/day with thiolactone-homocysteine (D,L Hcy-T) for different times, provoking increased plasmatic total homocysteine levels. That has indicated alterations in aortic vasomotor responses and mesenteric venial leukocyte rolling and adhesion. Gal-1 was detected in aortic frozen section of Gal-1+/+ mice for all times of treatment with D,L Hcy-T, but more detected for aorta of 15 days treated animal group and, more expressed, for 24 hours ones (protein expression by Western blot and immunofluorescence and, genetic by Real Time PCR). Besides, at 24 hours of treatment, many metabolic alterations were observed: increased LDL, triglycerides, cholesterol, glucose and homocysteine levels. Expressions for nitric oxide sintases (NOS) showed no alteration for inducible NOS (iNOS) for Gal-1+/+ mice (treated or not), even for Gal-1-/- untreat mice. However, the treatment was able to modulate endothelial NOS (eNOS) decreasing the expression at 48 hours and 7 days treated animals group (protein expression by imunoperoxidase and, genetic by RT-PCR). Gal-1-/- mice have less circulating constitutive nitric oxide (NO) than Gal-1+/+ ones, and, the treatment has reduced these levels only for Gal-1-/- 15 days treated mice but not for Gal-1+/+ ones . This work, therefore, has shown that Gal-1 is always expressed by aortas. However, increased expression of Gal-1 at 24 hours and 15 days of treatment has been often correlated to biological alterations in the in hyperhomocysteinemia-induced vascular injury: aortic vasomotor responses, leukocyte rolling and adhesion in mesenteric venues, plasmatic homocysteine, lipid metabolites and NOS expression.

Les nouvelles technologies d'imagerie basées sur la tomodensitométrie en coupe permettent l'évaluation de très haute qualité de la structure 3D de l'aorte thoracique. La reconstruction virtuelle et les modèles géométriques de l'aorte sont indispensables à l'exploitation des images dont le temps de traitement manuel reste cependant considérable et les outils numériques insuffisants ou inadaptés pour mesurer correctement sa morphologie. L'aorte n'est pas un simple tube de conduction du sang mais un organe de régulation de la pulsatilité des ondes de pression provoquées par l'éjection cardiaque. Ses désordres biomécaniques peuvent accélérer la formation de calcifications dans sa paroi et entrainer des risques de graves complications, comme les anévrismes et les dissections. La réparation aortique basée sur l'implantation d'endoprothèses est en pleine évolution et requiert des renseignements morphologiques précis pour en améliorer le taux de succès. Notre objectif a été d'étudier la géométrie tridimensionnelle de l'aorte en développant des algorithmes appropriés. Une plateforme informatique a été conçue et testée pour étudier trois pathologies de l'aorte: l'athérosclérose calcifiée, l'anévrisme et la dissection. L'hypothèse du travail a été que la géométrie spécifique des artères de chaque individu joue un rôle complémentaire à celui des facteurs de risque traditionnels dans le développement de ces pathologies. Notre premier travail a montré que trois facteurs résument 80% de la variabilité géométrique de l'aorte thoracique: le volume aortique, le déroulement et la symétrie de l'arche aortique, avec des taux de variabilité respectifs de 46%, 22% et 12%. Dans deux travaux suivants, nous avons montré que les calcifications de l'aorte thoracique se concentrent principalement dans la crosse et dans le segment descendant proximal, et que cette distribution était associée à la morphologie de l'aorte indépendamment de l'âge, du sexe, de la surface corporelle et des facteurs de risque traditionnels. Le quatrième travail a montré que le score de dépôt calcique dans toute l'aorte thoracique incluant la crosse était plus étroitement associé aux complications non-cardiaques, vasculaires périphériques et cérébrales, que le score traditionnel de calcium coronaire. Il faut noter que la crosse aortique n'est pas visualisée dans les études de routine de calcium coronaire sans injection. Le cinquième travail décrit un modèle déformable capable de segmenter la lumière aortique dans un contexte pathologique. Il a été appliqué pour étudier de façon automatisée la taille d'un anévrisme abdominal avant et après la pose d'une endoprothèse. Dans le dernier travail, la méthode précédente a été adaptée pour étudier la géométrie aortique des patients atteints de dissection comparativement à un groupe témoin de patients qui en étaient indemnes. Trois variables géométriques ont été identifiées dans le modèle de prédiction du risque de dissection: le diamètre de la crosse, la longueur de l'aorte thoracique et l'âge. En conclusion, nos résultats montrent que les maladies aortiques sont étroitement associées à la géométrie de l'aorte indépendamment des facteurs de risque traditionnels. Les algorithmes que nous avons développés ouvrent la voie à l'automatisation et à une réduction de la variabilité des mesures. / New imaging technologies, including those associated with multislice computed tomography, allow to evaluate the structure of the thoracic aorta in 3D with an impressive resolution. Aortic virtual reconstruction and geometric modeling are essential for imaging evaluation because manual measurements are time-consuming, and the available tools still need to be adapted to complex aortic morphologies. The aorta is more than a simple tubular conduit vessel for blood. It also regulates the pulsatile pressure waves that are injected into the arterial system by the left ventricle. The biomechanical disorders produced by these waves can accelerate the formation of calcium deposits within the arterial wall. Furthermore, they are thought to be responsible for severe aortic complications, including aneurysms and dissections. Endovascular aortic repair is a modern technique based on the implantation of an endograft to restore the normal blood flow. Precise morphological measurements are required to improve this technique, for both surgery planning and patient follow up. Our objective was to develop original algorithms to study the aortic geometry in 3D. A computing platform was designed and tested to analyze three main aortic pathologies: calcified atherosclerosis, aneurysms and dissections. The hypothesis of our study was that the individual arterial geometry of a subject plays a complementary role in the development of vascular pathologies beyond traditional risk factors. Our first work revealed that 80% of the total geometric variability in the thoracic aorta might be explained using 3 factors: the aortic volume, the aortic arc unfolding and its asymmetry. Variability percentages accounted for 46%, 22% and 12%, respectively. The next 2 works, showed that calcifications in the thoracic aorta were concentrated in the aortic arch and in the proximal descending segment. This spatial distribution was associated with aortic morphology, independently of age, sex, body surface area and traditional risk factors. Our fourth article revealed that calcium deposits in the entire thoracic aorta (including the aortic arch) was associated with non-cardiac events, beyond the standard coronary artery calcium score. It is noteworthy that the aortic arch region is systematically excluded from standard scans. Our fifth manuscript described a novel deformable model applied to the aortic segmentation under pathological contexts. It was used to estimate the size and shape of abdominal aneurysms before and after endograft implantation. In the last work, this method was adapted to study the geometry of the thoracic aorta of patients with an aortic dissection with respect to a control group. Three anatomic variables were identified for the risk prediction model: the aortic arch diameter, the thoracic aortic length and the age of the patient. In conclusion, our results show that aortic diseases are closely associated with aortic geometry, independently from traditional risk factors. The developed algorithms improved the automation of measurements and reduced the variability of the estimations.

Pequenas alterações da função renal durante a internação hospitalar e mais especificamente no período pós-operatório determinam significativo aumento na morbi-mortalidade hospitalar. A fim de determinar a incidência de insuficiência renal aguda (IRA) e os fatores de risco para seu desenvolvimento no pós-operatório de cirurgia para correção de aneurismas da aorta tóraco-abdominal e abdominal, foi realizado estudo retrospectivo das operações eletivas no ano de 2000. Foi encontrada alta incidência de IRA, tanto IRA leve, descrita como elevação de 25% da creatinina basal, em 57% dos pacientes, como uma forma mais grave, descrita como elevação de 50% e encontrada em 33% dos pacientes. Os fatores de risco independentes para IRA leve foram a presença de clampeamento acima do óstio das artérias renais (OR 6,9, IC 95% 1,32-36,12) e o tempo total de anestesia maior que 339 min (OR 1,00 para cada min acima de 339min, IC 95% 1,003-1,016). Para lesão mais grave, a instabilidade hemodinâmica, representada pela necessidade do uso de droga vasoativa no pós-operatório, também foi fator independente para o desenvolvimento de IRA (OR 7,4, IC 95% 1,84-30,16). A mortalidade hospitalar encontrada no estudo foi de 42,8% nos pacientes que tiveram clampeamento supra-renal, e 21,4% nos pacientes com clampeamento apenas infra-renal. Neste estudo, os fatores de risco independentes para óbito foram a filtração glomerular (FG) menor que 49 mL/min (OR 17,07, IC 95% 2,00-145,23), a necessidade de clampeamento acima das artérias renais (OR 9,6, IC 95% 1,37-67,88), desenvolvimento de IRA considerada como aumento da creatinina de 50% em relação a basal (OR 8,8, IC 95% 1,31-59,39) e a hiperglicemia no pós-operatório (OR 19,99, IC 2,32-172,28). Conhecendo a incidência de IRA, os fatores de risco para o seu desenvolvimento e a morbi-mortalidade associada ao seu desenvolvimento, foi testada N-acetilcisteína (NAC) como medida protetora, através de estudo randomizado, placebo-controlado e duplo-cego com os pacientes elegíveis para correção cirúrgica eletiva de aneurismas da aorta tóraco-abdominal e abdominal. NAC foi administrado por via oral 1200mg duas vezes ao dia 24 horas antes da operação, e após esta por 48 horas de forma endovenosa, 600mg de 12 em 12horas. Quarenta e dois pacientes foram incluídos no estudo, 18 no grupo NAC e 24 no grupo controle. A creatinina e FG basal foram semelhantes entre os grupos (1,19±0,33 vs 1,37±0,49 mg/dL; e 64,6±26,22 vs 65,7±28,32 ml/min, NAC vs controle, respectivamente, p=0,17 e p=0,90). A incidência de IRA leve, definida como aumento de 25% da creatinina basal, foi de 36% (13/36), mas não foi estatisticamente diferente entre os dois grupos (7/14, 50% no grupo NAC e 6/22, 27,3% no grupo controle, p=0,16). A mortalidade hospitalar foi de 23% (10/42) e também não foi diferente (p=0,209) entre os grupos, 33,3% no NAC e 16,7% no controle, o mesmo ocorrendo com o tempo de internação na UTI (2,93±1,53 vs 2,52±1,36 dias, p=0,40). Dessa forma, esse estudo sugere que embora possa haver algum efeito biológico da NAC, o seu uso na prevenção da IRA associada à operação eletiva de aneurisma da aorta abdominal não é justificável / Small alterations of the renal function during hospital stay and more specifically in the postoperative period determine significant increase in hospital mortality. In order to determine the incidence of AKI (acute kidney injury) and the risk factors for its development in the postoperative period of elective surgical open repair of thoracoabdominal and abdominal aortic aneurisms, it was carried out a retrospective study through the year of 2000. It was found high incidence of AKI, defined as a 25% increase in baseline serum creatinine (SCr), which occurred in 57% of the patients, and in a more strict definition, 50% increase in SCr, which occurred in 33%. The independent risk factors for AKI 25% was the presence of supra-renal clamping (OR 6,9, IC 95% 1,32-36,12) and the total anesthesia duration greater than 339 min (OR 1,00 for each min above 339 min, IC 95% 1,003-1,016). For more serious AKI (50%), the hemodynamic instability, represented for the need of vasoactive drugs in the postoperative period, also was an independent risk factor (OR 7,4, IC 95% 1,84-30,16). The mortality rate found in the study was 42,8% in patients who had supra-renal aortic clamping, and 21,4% in those with only infra-renal aortic clamping. In this study, the independent risk factors for death was baseline glomerular filtration rate (GFR) less than 49mL/min (OR 17,07, IC 95% 2,00-145,23), the need of supra-renal aortic clamping (OR 9,6, IC 95% 1,37-67,88), AKI defined as an increase of 50% in baseline SCr (OR 8,8, IC 95% 1.31-59,39) and the hyperglycemia in the postoperative period (OR 19,99, IC 2,32-172,28). Thereafter, we tested the protective effect of NAC through a randomized, placebo-controlled, double-blind study with patients eligible for elective open surgical repair of thoracoabdominal e abdominal aortic aneurisms. NAC was give po 1200mg twice daily 24 hours before the operation, and for 48 hours after surgery 600mg twice daily iv. Forty-two patients were enrolled in the study, 18 in group NAC and 24 in the control group. The baseline SCr and baseline GFR did not differ between groups (1,19±0,33 vs 1,37±0,49 mg/dL; and 64,6±26,22 vs 65,7±28,32 ml/min), NAC vs control, respectively, p=0,17 e p=0,90. The incidence of AKI defined as 25% increase in baseline SCr was 36% (13/36), but it was not statistically different between groups (7/14, 50% in NAC and 6/22, 27.3% in the control group, p=0,16). Hospital mortality was 23% (10/42) and was not different (p=0,209) in NAC group (33,3%) when compared with control (16,7% ). Likewise, the length of ICU stay did not differ (2,93±1,53 vs 2,52±1,36 days, p=0,40). This study suggests that although a biological effect of NAC cannot be excluded, its use in the prevention of AKI associated with elective aortic aneurysms cannot be justified

Este estudo avaliou a função vascular em anéis de aorta e no leito vascular mesentérico (LVM) de camundongos com deleção dos receptores <font face=\"symbol\">a2A e <font face=\"symbol\">a2Cadrenérgicos (KO) com 3, 5 e 7 meses, os quais apresentam uma hiperatividade simpática acompanhada de cardiomiopatia. Os KO apresentaram um aumento da freqüência cardíaca em todos os grupos avaliados, e hipertrofia ventricular esquerda aos 5 e 7 meses. Na aorta, o relaxamento dependente (acetilcolina) e independente (nitroprussiato de sódio) do endotélio e da via font face=\"symbol\">a-adrenérgica (isoproterenol), assim como a contração (fenilefrina e serotonina) e a mobilização de Ca2+ não foram alterados nos KO aos 3, 5 e 7 meses. Nos KO aos 3 meses, o relaxamento mediado pelos receptores ?2-adrenérgicos (clonidina) foi reduzido. Tanto a contração (noradrenalina) como o relaxamento (acetilcolina) no LVM dos KO aos 7 meses não foi alterado. Assim, sugere-se que os vasos arteriais parecem ser menos sensíveis do que o coração aos efeitos crônicos da hiperatividade simpática nos camundongos com deleção dos receptores <font face=\"symbol\">a2A e <font face=\"symbol\">a2C adrenérgicos. / This study assed the vascular function in aortic rings and in mesenteric vascular bed (MVB) from mice with disruption of <font face=\"symbol\">a2A and <font face=\"symbol\">a2Cadrenoceptors (KO) with 3, 5 and 7 months of age, that present sympathetic hyperactivity associated with cardiomyopathy. Heart rate was increased in all KO groups, and left ventricular hypertrophy was observed only in 5 and 7 month-old KO. There are no changes in the relaxation induced by acetylcholine (ACh), sodium nitroprusside and isoproterenol in aortic rings from all groups. In addition, the contraction induced by phenylephrine and serotonin, and Ca2+ handling did not change. However, in aorta from 3 month-old KO the relaxation induced by clonidine (<font face=\"symbol\">a2-adrenergic agonist) was reduced. In MVB from 7 month-old KO, neither the contraction (noradrenaline) nor relaxation (ACh) was modified. The results suggest that arterial vessel has been more resistant than heart to chronic effects induced by sympathetic hyperactivity observed in mice with disruption o<font face=\"symbol\">a2A and <font face=\"symbol\">a2C-adrenoceptors.

La formation de podosomes dans les cellules endothéliales (Ces) aortiques en réponse au TGFβ est bien décrite in vitro (dans des boîtes de culture) et ex vivo (dans des segments de vaisseaux aortiques vivants). Le projet de cette thèse était de franchir l’étape suivante en démontrant la présence de podosomes in vivo en réponse à du TGFβ d’origine endogène. Pour ce faire, nous avons choisi d’utiliser un modèle de souris génétiquement modifiées présentant spontanément des niveaux de TGFβ élevés dans la paroi aortique, raisonnant que cet environnement devrait être propice à l’apparition des structures. La souris Marfan (FbnC1039G/+) constitue le modèle murin de la maladie humaine appelée « syndrome de Marfan ». Chez la souris comme chez l’homme, une mutation dans le gène codant pour la fibrilline-1 conduit à une augmentation des niveaux de TGFβ dans la paroi aortique et dans le sang circulant. Nous avons utilisé ce modèle pour rechercher la présence de podosomes dans l'endothélium aortique. La visualisation « en face » de l'endothélium marqué pour l’actine fibrillaire, la cortactine, la protéine adaptatrice Tks5 et la metalloprotéase MT1-MMP, a révélé des rosettes de podosomes semblables à celles détectées ex vivo en réponse à du TGFβ d’origine exogène. Ces rosettes peuvent être trouvées dans l'endothélium de l'aorte ascendante ou descendante. L’analyse du tissu sous-jacent révèle une détérioration de la lame basale qui apparait parsemée de zones dépourvues d’un de ses constituants majeurs, le collagène IV. Nous émettons l'hypothèse que les rosettes de podosomes sont impliquées dans la formation de ces zones de dégradation du collagène IV. Pour examiner les conséquences du déficit en fibrilline-1 pour les CEs et confirmer les données in vivo concernant la souris Marfan, nous avons utilisé deux approches in vitro. D’abord, nous avons mis au point un protocole pour isoler les CEs aortiques à partir des souris Marfan. Ensuite, nous avons, par la stratégie des siRNA, procédé à la déplétion en fibrilline-1 de CEs aortiques d’origine bovine (BAEc). Les cellules endothéliales aortiques isolées des souris Marfan conservent in vitro le phénotype « TGFβ » et forment podosomes capables de dégrader la matrice extracellulaire sans aucune stimulation exogène. Dans le modèle des cellules BAE, le dosage du TGFβ indique que, in vitro, la déplétion de ces cellules en fibrilline-1 provoque une augmentation de TGFβ biologiquement actif associé à la fraction cellulaire. Ce TGFβ conduit alors à la formation de podosomes dans les CEs. Nous avons observé d’autres conséquences du déficit en fibriline-1 dans l’endothélium in situ. L’analyse topologique de la surface de l’endothélium aortique de la souris Marfan révèle des phénomènes de « blebbing », des filopodes, des anomalies des jonctions cellules-cellules. L’analyse ultrastructurale en microscopie électronique à transmission révèle que l’endothélium de la souris Marfan a une apparence radicalement distincte de celui observé chez les témoins sauvages. La lame élastique, fragilisée par le déficit en fibrilline-1, disparaît comme digérée, par endroits. On observe une sécrétion anormale de matrice extracellulaire. Les cellules présentent un phénotype activé ou des signes d’apoptose. Ces études fournissent la première démonstration de l'apparition de podosomes endothéliaux in vivo et suggèrent leur implication en physiopathologie vasculaire. Elles fournissent également la première démonstration que l’endothelium aortique est profondément modifié dans le modèle murin de la maladie de Marfan. / Podosome formation in aortic endothelial cells exposed to TGFβ has been well described in vitro (in tissue culture dishes) and ex vivo (in living aortic vessel segments). The aim of the project was to go to the next step and demonstrate the occurrence of podosomes in vivo in response to endogenous TGFβ. For this purpose, we chose to use a genetically engineered mouse model, which spontaneously presents high TGFβ levels in the aorta, reasoning that this would be a favorable environment for these structures to appear. Marfan mice represent the murine model of the human disease Marfan syndrome. Similar to the human disease, a mutation in the fibrillin-1 gene (C1039G/+) leads to enhanced TGFβ levels in the aortic wall as well as in circulating blood. We therefore used the Marfan mouse model in search of podosomes in the aortic endothelium. “En face” viewing of the endothelium stained for filamentous actin, cortactin, Tks5 adaptator protein and MT1-MMP metalloprotease detected podosome rosettes with features similar to those detected in the ex vivo situation. Podosome rosettes were found in both descending and ascending aorta. Analysis of the underlying tissue with collagen IV staining revealed a basement membrane scattered with staining-free patches, most likely corresponding to collagen IV degradation. We propose that podosome rosettes are involved in basement membrane degradation in this mouse. To examine the consequences of fibrillin-1 deficiency in endothelial cells and confirm the data obtained in vivo in Marfan mouse aorta, we used two in vitro approaches. First, we set up a protocol to isolate aortic endothelial cells from Marfan aortas, second, we depleted fibrillin-1 from BAE cells by siRNA silencing. Isolated Marfan aortic endothelial cells retained in vitro the TGFβ activated phenotype and formed functional podosomes without any exogenous stimulation. TGFβ levels measurements in fibrillin-1 depleted aortic endothelial cells confirmed that fibrillin-1 deficiency triggers an increase in active, cell associated TGFβ, which in turns, leads to podosome formation in endothelial cells. Finally we studied other alterations caused by the fibrillin-1 defect at the endothelial cell level in situ. Topological analysis of the Marfan mouse aortic endothelium monolayer revealed cell blebbing, numerous filopodia and showed altered cell-cell junctions. At the ultrastructural level, transmission electronic microscopy revealed that the Marfan mouse endothelium had an appearance dramatically distinct from that observed in control littermates. The elastic lamina, weakened by fibrillin-1 deficit, disappeared in some places. The vessel wall also showed abundant extracellular matrix proteins. Endothelial cells presented an activated or apoptotic phenotype. These studies provide the first demonstration for the occurrence of endothelial podosomes in vivo and suggest their involvement in vascular physiopathology. In addition, they provide evidence that the aortic endothelium is profoundly altered in the murine model of Marfan syndrome.

Introdução: A correção do aneurisma pela técnica endovascular ganha mais espaço com o aprimoramento do material utilizado, buscando-se um sistema ideal. Os estudos com esses dispositivos necessitam de modelos experimentais que devem apresentar características anatômicas e fisiopatológicas similares aos aneurismas em humanos. Apresentamos um novo modelo com bolsa de pericárdio bovino em suínos, com abordagem cirúrgica por via retroperitonial. Materiais e métodos: Estudo prospectivo, de coorte, avaliado pelo comitê de bioética, utilizando 11 suínos de raça Large White e do sexo feminino, com idade de 12 semanas, dividido em duas fases. A fase I consistiu na confecção do aneurisma, abordando a aorta retroperitonial com anestesia geral; na fase II, após 15 dias, realizou-se a arteriografia, eutanásia e retirada da peça para estudo anatomopatológico. Resultados: Em todos os animais, observou-se formação de trombos murais, como ocorre em aneurismas verdadeiros que comprometem de 30 a 90% da luz. À microscopia foram observadas calcificações na região do trombo em 82% dos animais. Havia infiltrado linfoplasmocitário no enxerto e região perienxerto, com fibrose em nove animais. Em três suínos havia espessamento miointimal considerável, e em oito, microcalcificações. A mortalidade foi nula, sem complicações cirúrgicas locais, isquêmicas e de ruptura. Conclusão: O modelo corresponde ao objetivo inicial de manter a aorta, seus ramos colaterais e terminais pérvios, com achados estruturais semelhantes aos de aneurismas encontrados em humanos: reação inflamatória parietal, periadvencial, trombo e calcificações da parede. As técnicas anestésica e operatória desenvolvidas foram satisfatórias e de morbimortalidade nula no período avaliado. / Background: The correction of an aneurysm using endovascular techniques has gained importance with the improvement of materials. The study of endovascular prostheses requires experimental models that reproduce anatomic and physiopathologic characteristics of human aneurysms. This study describes a new swine model that used bovine pericardium and a retroperitoneal surgical approach. Material and methods: This prospective cohort study used eleven Large White female pigs with a mean age of 12 weeks in the two study phases. In phase I, the aneurysm was produced and surgery using a retroperitoneal approach to the aorta under general anesthesia was performed; and in phase II, 15 days later, the animals underwent arteriography and were later killed, and specimens were removed for histological analysis Results: The formation of parietal thrombus, which is also found in true aneurysms that involve 30 to 90% of the lumen, was observed in all animals. Microscopic analysis showed calcifications in the area of thrombus in 82% of the animals. There was lymphoplasmocytic infiltration in the graft and adjacent area, with fibrosis in nine animals. Three pigs had substantial myointimal thickening, and eight had microcalcifications. Mortality was zero, and there were no ruptures, ischemia or surgery site. Conclusion: The aorta and its collateral and terminal branches remained healthy, and structural findings were similar to those observed in human aneurysms: wall inflammation, periadventitial thrombus and wall calcification. The anesthetic and operatory techniques were satisfactory, and there was no morbidity or mortality during the study.

Les acides gras polyinsaturés oméga-3 à longue chaîne (AGPIω3-LC), principalement représentés par l'acide eicosapentaénoïque (EPA) et l'acide docosahexaénoïque (DHA), présentent des effets bénéfiques vis-à-vis de l'athérosclérose. Leur action au niveau vasculaire est suggérée. Les mécanismes d'action sont mal compris du fait de la complexité d'action des AGPIω3-LC au niveau cellulaire. Les AGPIω3-LC affectent de nombreuses protéines y compris les facteurs de transcription, ce qui engendre la modulation de l'expression de nombreux gènes. La complexité s'accroît si l'on considère l'ensemble des métabolites oxygénés issus des AGPIω3-LC. Les métabolites issus de la peroxydation lipidique peuvent être produits en abondance au cours de l'athérogenèse mais leur bioactivité est quasiment inconnue. Des points majeurs restent à éclaircir comme l'identification des cibles cellulaires et moléculaires au niveau vasculaire et la bioactivité des métabolites peroxydés. Objectifs : étudier l'impact du DHA au niveau vasculaire et évaluer la bioactivité de certains métabolites issus de la peroxydation lipidique du DHA. Les travaux visant à étudier les propriétés athéro-protectrices du DHA n'ont jusqu'à présent pas permis d'obtenir une vision globale de son spectre d'action. La nutrigénomique a paru être une approche pertinente pour étudier les effets du DHA au niveau vasculaire. Une étude in vivo a été réalisée sur des souris athérosclérotiques LDLR-/- recevant ou non une supplémentation en DHA (2% de l'apport énergétique journalier). Le rôle athéro-protecteur du DHA a été confirmé dans une étude précédente rapportant une diminution de 35% de l'étendue des lésions au niveau aortique dans le groupe DHA. Au niveau protéique, nous avons montré une altération significative de l'expression de protéines intervenant principalement au niveau des métabolismes glucidique et lipidique mais aussi dans les défenses anti-oxydantes. Au niveau génique, la composante inflammatoire s'est avérée être une cible majeure du DHA au niveau vasculaire. La supplémentation en DHA a réduit l'expression de gènes impliqués dans l'adhésion cellulaire, la chimiotaxie et la présentation de l'antigène. Plusieurs gènes se sont avérés être des marqueurs phénotypiques des macrophages et l'analyse des régulateurs transcriptionnels montrait une implication probable de PPARγ, IFNγ et NFκB dans les modulations d'expression génique observées. Nos analyses suggèrent une orientation préférentielle des macrophages vers un phénotype réparateur de type M2 chez les souris recevant la supplémentation en DHA. Une analyse immunohistochimique au niveau aortique a révélé une plus grande abondance d'arginase I. Une approche plus ciblée alliant une étude in vitro sur un modèle de macrophages humains et l'utilisation de métabolites peroxydés spécifiques du DHA, les neuroprostanes, nous a permis de conforter notre hypothèse selon laquelle le DHA pourrait agir au moins en partie via ses métabolites peroxydés. L'exposition des macrophages à deux types de neuroprostanes (les 4-F4t et les 14-A4-Neuroprostanes) a permis de réduire significativement l'expression et la sécrétion de plusieurs médiateurs pro-inflammatoires. Des résultats préliminaires suggèrent que ces effets anti-inflammatoires des neuroprostanes seraient indépendants de PPARγ mais liés à une inhibition de la voie NFκB. L'étude in vivo et l'utilisation de la nutrigénomique nous ont permis d'explorer de façon ouverte et non biaisée l'impact du DHA au niveau vasculaire. Les résultats sont que le DHA active le métabolisme énergétique et les défenses anti-oxydantes et diminue l'état inflammatoire avec une implication probable du DHA dans l'orientation phénotypique des macrophages vers un phénotype réparateur de type M2. Les résultats obtenus in vitro sur macrophages primaires humains confirment que les métabolites peroxydés du DHA contribuent à ses propriétés anti-inflammatoires. / Numerous studies have reported beneficial effects of long chain omega-3 polyunsaturated fatty acids (LC-ω3PUFAs) on atherosclerosis and associated cardiovascular events. These fatty acids are mainly represented by eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). LC-ω3PUFAs exert their athero-protective action mainly by a reduction of triglyceridemia, increased endothelial relaxation, and reduced inflammation. Their specific action at the vascular level is suggested. Their mechanisms of action are still only partially understood because of the complexity of action of LC-ω3PUFAs at the cellular level. LC-ω3PUFAs affect several membrane or cytosolic proteins, including transcription factors, which modulate numerous gene expression. The complexity is further increased when considering a wide range of oxygenated metabolites derived from LC-ω3PUFAs. Among them, the metabolites from lipid peroxidation may be produced in abundance during atherogenesis but their bioactivity is almost unknown. There are still major issues to be clarified, such as the identification of cellular and molecular targets at the vascular level ; the bioactivity of peroxidized metabolites that could play a key role in the prevention of atherosclerosis. The objective of this work was to study the impact of DHA at the vascular level and to evaluate the bioactivity of selected metabolites from DHA lipid peroxidation. Studies on anti-atherogenic properties of DHA have so far been carried out only in a targeted manner that does not provide a comprehensive and integrated view of its spectrum of action. Nutrigenomics appears to be a relevant approach to study the effects of DHA at the vascular level. In vivo study was performed on LDLR-/- atherosclerotic mice supplemented or not with DHA. The athero-protective effect of DHA has been confirmed in the previous study performed by our team reporting a 35% decrease of the atherosclerotic lesion in the DHA group. Aorta proteome study demonstrated a significant alteration of the expression of proteins involved mainly in the carbohydrate and lipid metabolism and also in antioxidant defenses. At the transcriptome level, the inflammatory component of atherosclerosis appears to be a major target of DHA at the vascular level. More precisely, DHA supplementation reduced the expression of genes involved in cell adhesion, chemotaxis and antigen presentation. Several genes were found to be phenotypic markers of macrophages and analysis of transcriptional regulators showed a possible contribution of PPARγ, IFNγ and NFκB to the observed modulation of gene expression. Our results suggest a preferential orientation of macrophages to a M2 type repair phenotype in mice receiving DHA supplementation. This was confirmed by immunohistochemical analysis at the aorta which revealed a greater abundance of arginase I. We chose a focused in vitro study of the action of specific DHA peroxidized metabolites: the neuroprostanes on a human macrophages. This study allowed us to confirm our hypothesis that DHA may act at least in part via its peroxidized metabolites. The macrophages exposition to 2 types of neuroprostanes (4-F4t and 14-A4-Neuroprostanes) has significantly reduced the expression and secretion of several pro-inflammatory mediators. Preliminary results suggest that these anti-inflammatory effects are PPARγ-independent but related to inhibition of NFκB pathway. In vivo study, and nutrigenomics approach, allowed us to explore the impact of DHA at the vascular level by an open and unbiased way. The results are that DHA activates energy metabolism and antioxidant defenses and reduces the inflammatory component of atherosclerosis with a possible involvement of DHA in the phenotypic direction of macrophages to a M2 type repair phenotype. The results obtained in vitro on primary human macrophages confirmed that DHA peroxidized metabolites contribute to its anti-inflammatory properties.

Orientador: Cristina Antoniali Silva / Banca: Fernando Silva Carneiro / Banca: Carlos Alan Candido Dias Junior / Banca: Graziela Scalianti Ceravolo / Banca: Angela de Castro Resende / Resumo: A redução da reatividade vascular à fenilefrina (PE) em aorta de ratas espontaneamente hipertensas (SHR) ao final da prenhez é dependente de maior produção e/ou maior biodisponibilidade de óxido nítrico (NO), consequente do aumento da fosforilação da enzima óxido nítrico sintase endotelial (eNOS) via PI3K/Akt. A glicosilação do tipo N-acetil-glucosamina (O-GlcNAc) é uma modificação pós-traducional que compete com a fosforilação pelos mesmos sítios de ligação nas proteínas. A O-GlcNAcilação da eNOS em serina1177 leva a redução da sua atividade enquanto a fosforilação leva a sua ativação. Além destes mecanismos, a interação da eNOS com outras proteínas é capaz de regular positiva ou negativamente a sua atividade. O objetivo deste trabalho foi analisar possíveis alterações nos mecanismos de modificação pós-traducional que controlam a ativação da eNOS os quais poderiam contribuir para maior ativação e maior biodisponibilidade de NO observada em artérias de ratas prenhes. Foram avaliados o conteúdo proteico O-GlcNAc e também expressão das enzimas que participam desta modificação, O-GlcNAc transferase (OGT) e O-GlcNAcase (OGA) por Western Blotting e a atividade da OGA por ensaio bioquímico em aorta e em artéria mesentérica (2º ou 3º ramo) de ratas não prenhes (NP) e prenhes (P), normotensas (Wistar) e SHR. Ensaios de Western Blotting foram realizados também para análise da expressão das seguintes proteínas: Cav-1, p-Cav-1, CaM e Hsp90. Realizamos a contagem do número de cavéolas en... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Reduction of vascular reactivity to phenylephrine (PE) in aortaof spontaneously hypertensive rats (SHR) at the end of pregnancy is dependent on higherproduction and/or higerbioavailability of nitric oxide (NO), as a consequence of increased endothelial nitric oxide synthase enzyme (eNOS) phosphorylation,by PI3K/Akt.Glycosylation with O-linked N-acetylglucosamine (O-GlcNAc)is a post-translational modification that competes with phosphorylation by the same binding sites in proteins. O-GlcNAcylation of eNOSon serine siteleads to a reduction in its activity while eNOS phosphorylation leads to its activation. In addition to these mechanisms, the interaction of eNOS with other proteins is able to regulate positively or negatively its activity. The objective of this studywas to analyze possible changes in the mechanisms of post-translational modification that control the eNOS activation, which could contribute to its the greater activation and greater bioavailability of NO observed in arteriesof pregnant rats. The O-GlcNAc-protein content and also the enzymesexpressionthat participate in this modification, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) was assessed by Western Blotting, and OGA activity were evaluated by biochemical assay in the aorta and in the artery mesenteric (2ndor 3rdbranch) of non-pregnant (NP) and pregnant (P), normotensiverats(Wistar) and SHR.Western Blotting assays were also performed for expression analysis of the following proteins: Cav-1, p-Cav-1, CaM and Hsp90. We performed the counting of the number of endothelial caveolaein the aorta and the mesenteric artery in the presence or absence of methyl-β-cyclodextrin (dextrin, 10 mmol/L) by electronicmicroscopy.In functional studies, we evaluated the participation of the OGA enzyme, by inhibition with PugNAc (100 μmol/L) and of the caveolae, using a caveolae disassembler, (Complete abstract electronic access below) / Doutor

La calcification vasculaire est l’accumulation de cristaux de calcium dans les vaisseaux sanguins à travers un processus pathologique qui ressemble à la formation de l’os ou du cartilage. Elle apparaît notamment chez les patients diabétiques ou atteints d’une insuffisance rénale chronique. La conséquence principale de la calcification vasculaire est la perte de l’élasticité qui est indispensable pour la fonction des larges artères, elle est de plus associée à la mortalité des patients hémodialysés. Les traitements contre la calcification vasculaire sont généralement limités à ceux qui corrigent les facteurs causatifs des problèmes de santé mais aucune intervention efficace, spécifique et ciblée n’est disponible. Par conséquence, une compréhension profonde des mécanismes moléculaires impliqués dans la calcification vasculaire est nécessaire dans le but de trouver de nouvelles cibles thérapeutiques. La phospholipase D catalyse l’hydrolyse des phospholipides en acide phosphatidique et une tête polaire, elle est aussi impliquée dans différentes fonctions cellulaires et maladies. Il a été démontré qu’elle peut être activée par des facteurs impliqués dans l’ostéogenèse et par d’autres impliqués dans la calcification vasculaire. Ainsi, nous avons étudié le rôle de la phospholipase D dans la calcification vasculaire dans 3 modèles différents. Le premier est un modèle in-vitro de cellules musculaires lisses murines (lignée cellulaire MOVAS), elles sont cultivées en présence d’acide ascorbique et de β-glycérophosphate. Le deuxième est un modèle ex-vivo d’explants d’aortes cultivés en présence de fortes concentrations de phosphate et le troisième est un modèle in-vivo d’insuffisance rénale chronique produite chez des rats. Dans ce dernier modèle, la calcification vasculaire est induite par un régime riche en phosphore et en calcium et par des injections de vitamine D active. La calcification dans ces trois modèles a été suivie par l’analyse de la minéralisation en dosant les dépôts de calcium, de l’activité phosphatase alcaline, et de l’expression de différents marqueurs ostéo-chondrocytaires. Une augmentation de l’expression génique de Pld1 a été observée dans les trois modèles, en particulier au cours des premières étapes de la calcification, et a été accompagnée d'une activité accrue de la phospholipase D dans les modèles in vitro et ex-vivo. L’inhibition de l’activité phospholipase D dans ces deux modèles ou de la phospholipase D1 dans le modèle MOVAS a bloqué complètement la calcification. Par contre, l’inhibition spécifique de la phospholipase D2 n’a pas montré des effets significatifs. Deux voies par lesquelles la phospholipase D peut être activée ont été testées, la voie de la protéine kinase C et la voie de la sphingosine-1-phosphate. Ces deux voies métaboliques se sont révélées être impliquées dans le processus de calcification mais pas forcément dans l’activation de la phospholipase D au cours de ce processus. Des résultats préliminaires ont montré que la phospholipase D pourrait agir après activation de la sphingosine kinase 2 dont l’activité s’est avérée nécessaire pour la calcification dans le modèle MOVAS. Des études supplémentaires sont nécessaires pour comprendre par quels mécanismes la phospholipase D est activée et comment elle agit. La phospholipase D pourrait être une nouvelle cible thérapeutique pour le traitement de la calcification vasculaire vu que son inhibition ne semble pas avoir des effets secondaires chez les patients / Vascular calcification is the accumulation of calcium phosphate crystals in blood vessels via a pathological process that resembles physiological bone or cartilage formation. Calcification in the medial layer is mainly seen in diabetic and chronic kidney disease patients. Its main consequence is the loss of elasticity which is indispensable for the function of large arteries. Accordingly, vascular medial calcification was significantly associated with mortality in hemodialysis patients. Vascular calcification treatments are limited to those that correct its causative health problems, but no efficient, specific and targeted interventions are available. Therefore, a deep understanding of its molecular mechanisms is needed to find novel therapeutic targets. Phospholipase D catalyses the hydrolysis of phospholipids into phosphatidic acid and a head group. It is implicated in different cellular functions and diseases. It was found to be activated by factors involved in osteogenesis and others involved in vascular calcification. Thus, we investigated its role in vascular calcification in 3 models: an in-vitro model of murine smooth muscle cell line MOVAS cultured with ascorbic acid and β-glycerophosphate, an ex-vivo model of rat aortas cultured in high phosphate medium, and an in-vivo model of adenine-induced kidney disease in rats in which vascular calcification is induced by further administration of high phosphorus/calcium diet and active vitamin D injections. Calcification was detected in these models using different approaches including alkaline phosphatase activity, calcium dosage, and/or evaluation of osteo-chondrocytic markers expression. Pld1 expression was seen upregulated in all the three models, especially during early stages of calcification, and was accompanied with increased phospholipase D activity in the in-vitro and ex-vivo model. The inhibition of total phospholipase D activity in these two models, or that of phospholipase D1 in case of MOVAS model, abolished calcification. Phospholipase D2-specific inhibition did not induce significant effects. Two pathways by which phospholipase D can be activated were tested, protein kinase C and sphingosine 1-phosphate pathways, but they were found to be involved in calcification but not necessary for phospholipase D activation during this process. Alternatively, the preliminary results showed that PLD may be acting by activation of sphingosine kinase 2 whose activity was found necessary for calcification in the MOVAS model. Further investigations are needed to understand the mechanisms by which phospholipase D is activated and by which it is acting. Phospholipase D could be a novel target for vascular calcification especially that its inhibition in patients did not induce adverse health effects