A patogênese e o estadiamento do melanoma são clinicamente bem caracterizados, entretanto, muitas variações são observadas no que diz respeito à progressão e respostas terapêuticas. Mecanismos intrínsecos das células de melanoma contribuem para esse fenômeno, como a plasticidade fenotípica e a expressão de determinadas moléculas, como a galectina-3. Galectina-3 tem papel fundamental para o crescimento do tumor, metástase e montagem de uma rede vascular funcional. Dessa forma, o melanoma possui mecanismos alternativos de vascularização, onde células endoteliais e células tumorais formam a superfície luminal dos vasos e são expostas às forças fluídicas do sangue, o shear stress. Nós hipotetizamos que o shear promove a adaptação celular e células de melanoma se comunicam com (1) outras células tumorais para sustentar a progressão do tumor, ou (2) com células endoteliais para garantir a manutenção vascular. Para isso, comparamos dois modelos de geração do shear denominados pela agitação de placas concêntricas e rotação de um cone sobre placa, que é o modelo padrão dos estudos em células endoteliais. O modelo de placas concêntricas gerou resultados mais robustos, pois garantiu a viabilidade e a adesão celular em até 24h. Também investigamos o potencial angiogênico das células de melanoma após monitorar a ativação de células endoteliais em contato com o meio condicionado (MC) do shear. O shear aumentou significativamente a secreção de Angiopoietina-2, IL-8 e PLGF num contexto independente de galectina-3. Além disso, o shear diminuiu a taxa de crescimento das células que expressam galectina-3, o que não foi observado no tratamento com o MC. Já a capacidade de sobreviver em longo prazo foi maior quando as células tumorais são expostas ao MC derivado do shear, sem influência da expressão de galectina-3. Em relação ao efeito do MC liberado após shear para o remodelamento vascular, nós não observamos alteração na proliferação das células endoteliais. Entretanto, o MC do shear parece acelerar a formação de túbulos e favorecer a desorganização das fibras de actina, além de diminuir a fosforilação de ERK nas células endoteliais. Juntos, esses resultados mostram que o shear induz a secreção de moléculas pró-angiogênicas e pró-inflamatórias que promovem a sobrevivência e proliferação a longo prazo das células tumorais naive. Nas células endoteliais, o MC derivado do shear altera a sinalização de ERK e o rearranjo do citoesqueleto / Melanoma pathogenesis and staging are clinically well characterized, although a large variation is observed in progression and therapeutic responses. Intrinsic mechanisms of melanoma cells contribute to this phenomenon, such as phenotypic plasticity and the expression of certain molecules as galectin-3. Galectin-3 plays a prominent role in tumor growth, metastasis, and to build a functional vascular network. In this way, melanoma has alternative mechanisms of vascularization, in which both endothelial cells and tumor cells form the luminal surface of vessels and are in contact with flowing blood. Here, we postulated that shear stress promotes cell adaptation and melanoma cells communicate with (1) other tumor cells in order to support tumor progression; or (2) with endothelial cells to ensure vascular maintenance. We compared two methods for generating shear stress, namely revolution of concentric plates and stirring of a suspended cone on a plate, which is the gold standard method for studies on endothelial cells. The concentric plates model yielded more reliable results, since it ensured the greater viability and adhesion of cells up to 24h, as compared to the cone and plate system. We then investigated the angiogenic potential of melanoma cells by monitoring the activation of endothelial cells upon exposure to the conditioned medium (CM) from cells exposed to shear stress. Shear stress significantly increased the secretion of Angiopoietin-2, IL-8 and PLGF, in a galectin-3 independent manner. Also, shear stress decreased the growth rate of galectin-3 positive melanoma cells, but not when galectin-3 was down regulated. No such effect was observed with the shear-derived CM. Instead, we did observed long-term melanoma survival under low density upon treatment with the shear-derived CM. In this scenario, the expression of galectin-3 did not change the results. Regarding the effect of shear-derived CM to vascular remodeling, endothelial cells did not show any change in cell proliferation. However, the CM of melanoma cells upon shear accelerated the endothelial cell tube formation and disorganized the actin stress fibers. CM also decreased the phosphorylation of ERK. Taken together, these data indicate that the production of proangiogenic and proinflammatory molecules by melanoma cells under shear enables long-term survival and proliferation of naïve melanoma cells. To endothelial cells, shear-derived CM led to changes in ERK signaling and cytoskeletal rearrangement

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

Estudos na fase crônica da hipertensão comprovam a eficácia do treinamento aeróbio (T) em reduzir parcialmente a pressão arterial (PA), no qual correlacionavam-se com o remodelamento vascular. Pouco se sabe sobre o potencial efeito benéfico do T iniciado em fase pré-hipertensiva. Os objetivos foram analisar em SHR jovens (4 semanas) e seus controles WKY os efeitos sequenciais do T sobre níveis de PA, alterações vasculares, conteúdo de colágeno total, associando-as à modulação neural. Animais em fase pré-hipertensiva realizaram T de baixa intensidade (50-60%) ou sedentarismo por 8 semanas. Foram avaliados os parâmetros funcionais e seus componentes espectrais, alterações estruturais ocorridas em artérias (femoral e renal), arteríolas (músculo sóleo e temporal) e com conteúdo de colágeno. O T reduziu a PAM no SHR na 4ª e 8ª semana, provocou bradicardia de repouso em ambos os grupos na 8ª, melhorou a variabilidade da FC (WKY e SHR) e impediu o aumento da variabilidade da PAS nos SHR. Hipertensão provocou aumento da razão parede/luz e conteúdo de colágeno em ambas as artérias, T impediu esse aumento na artéria femoral, efeito não foi observado na artéria renal. T impediu a queda da razão parede/luz em arteríolas do músculo sóleo sem efeitos no músculo sóleo. / Studies in the chronic phase of hypertension prove the effectiveness of aerobic training (T) in partially reduce blood pressure (BP), which correlated with vascular remodeling. Little is known about the potential beneficial effect of T started in pre-hypertensive stage. The objectives were to analyze in young SHR (4 weeks) and their WKY controls the sequential effects of T on BP levels, vascular changes, total collagen content, linking them to the neural modulation. Animals in the pre-hypertension stage T made of low intensity (50-60%) or inactivity for 8 weeks. We evaluated the functional parameters and their spectral components, structural changes in arteries (femoral and renal), arterioles (soleus muscle and temporal) and collagen content. OT reduced MAP in SHR at 4 and 8 weeks, caused bradycardia standby in both groups in 8th improved HR variability (WKY and SHR) and prevent the increase of SBP variability in SHR. Hypertension caused ratio increase / wall light and collagen content in both arteries, t prevented this increase in femoral artery, an effect was not observed in the renal artery. T prevented the fall of reason / wall light in arterioles of the soleus muscle without effects in the soleus muscle.

A patogênese e o estadiamento do melanoma são clinicamente bem caracterizados, entretanto, muitas variações são observadas no que diz respeito à progressão e respostas terapêuticas. Mecanismos intrínsecos das células de melanoma contribuem para esse fenômeno, como a plasticidade fenotípica e a expressão de determinadas moléculas, como a galectina-3. Galectina-3 tem papel fundamental para o crescimento do tumor, metástase e montagem de uma rede vascular funcional. Dessa forma, o melanoma possui mecanismos alternativos de vascularização, onde células endoteliais e células tumorais formam a superfície luminal dos vasos e são expostas às forças fluídicas do sangue, o shear stress. Nós hipotetizamos que o shear promove a adaptação celular e células de melanoma se comunicam com (1) outras células tumorais para sustentar a progressão do tumor, ou (2) com células endoteliais para garantir a manutenção vascular. Para isso, comparamos dois modelos de geração do shear denominados pela agitação de placas concêntricas e rotação de um cone sobre placa, que é o modelo padrão dos estudos em células endoteliais. O modelo de placas concêntricas gerou resultados mais robustos, pois garantiu a viabilidade e a adesão celular em até 24h. Também investigamos o potencial angiogênico das células de melanoma após monitorar a ativação de células endoteliais em contato com o meio condicionado (MC) do shear. O shear aumentou significativamente a secreção de Angiopoietina-2, IL-8 e PLGF num contexto independente de galectina-3. Além disso, o shear diminuiu a taxa de crescimento das células que expressam galectina-3, o que não foi observado no tratamento com o MC. Já a capacidade de sobreviver em longo prazo foi maior quando as células tumorais são expostas ao MC derivado do shear, sem influência da expressão de galectina-3. Em relação ao efeito do MC liberado após shear para o remodelamento vascular, nós não observamos alteração na proliferação das células endoteliais. Entretanto, o MC do shear parece acelerar a formação de túbulos e favorecer a desorganização das fibras de actina, além de diminuir a fosforilação de ERK nas células endoteliais. Juntos, esses resultados mostram que o shear induz a secreção de moléculas pró-angiogênicas e pró-inflamatórias que promovem a sobrevivência e proliferação a longo prazo das células tumorais naive. Nas células endoteliais, o MC derivado do shear altera a sinalização de ERK e o rearranjo do citoesqueleto / Melanoma pathogenesis and staging are clinically well characterized, although a large variation is observed in progression and therapeutic responses. Intrinsic mechanisms of melanoma cells contribute to this phenomenon, such as phenotypic plasticity and the expression of certain molecules as galectin-3. Galectin-3 plays a prominent role in tumor growth, metastasis, and to build a functional vascular network. In this way, melanoma has alternative mechanisms of vascularization, in which both endothelial cells and tumor cells form the luminal surface of vessels and are in contact with flowing blood. Here, we postulated that shear stress promotes cell adaptation and melanoma cells communicate with (1) other tumor cells in order to support tumor progression; or (2) with endothelial cells to ensure vascular maintenance. We compared two methods for generating shear stress, namely revolution of concentric plates and stirring of a suspended cone on a plate, which is the gold standard method for studies on endothelial cells. The concentric plates model yielded more reliable results, since it ensured the greater viability and adhesion of cells up to 24h, as compared to the cone and plate system. We then investigated the angiogenic potential of melanoma cells by monitoring the activation of endothelial cells upon exposure to the conditioned medium (CM) from cells exposed to shear stress. Shear stress significantly increased the secretion of Angiopoietin-2, IL-8 and PLGF, in a galectin-3 independent manner. Also, shear stress decreased the growth rate of galectin-3 positive melanoma cells, but not when galectin-3 was down regulated. No such effect was observed with the shear-derived CM. Instead, we did observed long-term melanoma survival under low density upon treatment with the shear-derived CM. In this scenario, the expression of galectin-3 did not change the results. Regarding the effect of shear-derived CM to vascular remodeling, endothelial cells did not show any change in cell proliferation. However, the CM of melanoma cells upon shear accelerated the endothelial cell tube formation and disorganized the actin stress fibers. CM also decreased the phosphorylation of ERK. Taken together, these data indicate that the production of proangiogenic and proinflammatory molecules by melanoma cells under shear enables long-term survival and proliferation of naïve melanoma cells. To endothelial cells, shear-derived CM led to changes in ERK signaling and cytoskeletal rearrangement

Pulmonary arterial hypertension (PAH) is a group of diseases characterized by avascular obstruction leading to a progressive increase of the resistances in the pulmonary blood flow. The recent progress in the understanding of mechanisms at the origin of this disease underlines the role of extrapulmonary cells, such as circulating stem cells and bone marrow-derived progenitor cells in vascular remodeling and in PAH development. In this thesis we have shown implication of the progenitor cells and chemotactic axis in the vascular remodeling in human and experimental PAH. This work could help to develop new therapies allowing more specific and more effective treatments leading to improved survival of PAH patients. / L’hypertension artérielle pulmonaire (HTAP) est un groupe de maladies qui se caractérise par une obstruction vasculaire conduisant à une augmentation progressive des résistances à l’écoulement sanguin. Le remodelage vasculaire qui implique toutes les couches de la paroi du vaisseau est considéré comme un élément clé dans la pathogenèse de l'HTAP. Les progrès récents dans la compréhension des mécanismes à l'origine de cette maladie soulignent le rôle de cellules extrapulmonaires, telles que des cellules souches circulantes et des progéniteurs dérivés de la moelle osseuse, dans le remodelage vasculaire et dans le développement de l’HTAP. Les thérapeutiques ciblées sur la dysfonction endothéliale ne permettent pas à l'heure actuelle de guérir cette maladie, il est donc nécessaire d’identifier d'autres mécanismes physiopathologiques permettant de développer de nouvelles stratégies thérapeutiques. C’est pourquoi nous avons exploré l’implication des cellules progénitrices et des signaux chimiotactiques dans le remodelage vasculaire au cours de l’HTAP humaine et expérimentale. Nous avons mis en évidence un recrutement de cellules c-kit+ (incluant des progéniteurs et des mastocytes) ainsi que l’expansion de vasa vasorum dans les poumons des patients atteints d’HTAP. Grâce à l’utilisation du modèle expérimentale d’HTAP induit par l’hypoxie chez la souris, nous avons montré le rôle central de la chimiokine CXCL12 et de ses deux récepteurs CXCR4 et CXCR7 dans le recrutement des progéniteurs c-kit+. Le traitement combiné par les antagonistes AMD3100 et CCX771, grâce à leurs actions synergiques, inhibe le remodelage vasculaire pulmonaire, l’hypertrophie cardiaque droite, ainsi que le recrutement des progéniteurs c-kit+, induits par l’hypoxie. Par ailleurs, le blocage de c-kit par l’imatinib améliore également les paramètres d’hémodynamique et diminue le recrutement périvasculaire des cellules c-kit+, probablement en inhibant leurs expansion dans la moelle osseuse. Nous avons également mis en évidence une altération d’une population de progéniteurs mésenchymateux d’origine hématopoïétique, appelés fibrocytes, dans le sang des patients souffrant d’HTAP.Ce travail pourrait contribuer à développer des actions thérapeutiques ciblées permettant la mise en place de traitements à la fois plus spécifiques et plus efficaces susceptibles d’améliorer la survie des patients HTAP.

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.

A degradação da matriz extracelular e de proteínas da parede aórtica associada à inflamação é uma das principais características dos aneurismas da aorta abdominal (AAA). O objetivo deste trabalho foi investigar a participação das metaloproteinases 2 e 9 na formação de AAAs, através de um modelo experimental inédito de indução de AAA em ratos Wistar, desencadeado por duas potenciais causas de secreção e ativação de MMP-2 e 9: alteração do fluxo sanguíneo e lesão vascular externa na aorta. A formação de aneurismas foi observada em 60%-70% dos animais, apresentando diâmetro de 7 a 8 vezes maior que o diâmetro normal da aorta. Histologicamente observou-se remodelamento, intensa resposta inflamatória, destruição maciça de fibras elásticas e aumento da síntese de colágeno na parede aórtica. A expressão de ambas as formas de MMP-2 foram observadas tanto nos AAAs como nos grupos controles, neste com menor atividade, entretanto a expressão da pró e da MMP-9 ativa foram encontradas apenas nos AAAs. Conclusão: Nossos resultados sugerem que tanto as MMP-2 quanto as MMP-9 apresentam importante papel no desenvolvimento de AAA e este novo modelo de indução de AAA, pode ajudar a elucidar os mecanismos que desencadeiam a secreção e ativação das MMP-2 e MMP-9 na formação de aneurismas. / Degradation of extracellular matrix and proteins associated with inflammation of the aortic wall is the main characteristics of the abdominal aortic aneurysms (AAA). The aim of this study was investigate the participation of Metalloproteinase 2 and 9 in AAA formation in Wistar rats. A novel experimental model of AAA was developed, providing two potential causes of MMPs secretion and activation, turbulent flow (caused by surgically induced extrinsic stenosis) and outside vascular injury is detailed described. The days analyzed were the 3rd and the 7th post surgery. Aneurysms were observed to occur in 60-70% of the Group AAA, exhibiting a major transversal diameter to 7 from 8 times larger than controls and sham groups. Histologically, the aneurysms wall showed extensive structural remodeling, intense inflammatory response, massive elastic fibers destruction and abundant collagen deposition. Increased pro- and active MMP-2 was demonstrated in the AAA and controls groups, whereas pro- and active MMP-9 were found to be expressed only in the AAA group. Conclusions: MMP-2 and MMP-9 may have a pivotal role in the development of experimental AAA. This model can help to elucidate the mechanisms which trigger off MMP-2 and MMP-9 secretion and activation causing aneurysms.

INTRODUÇÃO: A exposição contínua desde o nascimento à poluição atmosférica de grandes centros urbanos de origem predominante veicular, contribui para eventos cardiopulmonares em adultos. A inflamação e remodelamento induzidos pelos poluentes podem modular a resposta vascular a mediadores e alterar a sua patência e reatividade contribuindo para eventos isquêmicos. MÉTODOS: Verificar alterações inflamatórias e estruturais nos pulmões e corações de camundongos Swiss machos cronicamente expostos, desde o nascimento até a idade adulta, às câmaras seletivas com filtros (câmara limpa) e sem filtros (câmara poluída) para particulados e gases tóxicos, situadas a 20m de um cruzamento de tráfego intenso da cidade de São Paulo, 24 h/7dias/semana/4 meses. Realizaram-se medidas internas e externas dos poluentes: PM2,5; NO2 e Black Carbon (BC) e análises morfométricas nos pulmões e corações. RESULTADOS: A filtragem da câmara limpa clareou 100% de BC, 52% de PM2,5 e 32% de NO2. Nos pulmões, as alterações predominaram na região peribronquiolar. Os animais submetidos à câmara poluída apresentaram inflamação peribronquiolar caracterizada por aumento do infiltrado perivascular (p=0,022), hiperplasia do epitélio bronquiolar (p=0,037), escore inflamatório total (p=0,015), e diminuição do infiltrado polimorfonuclear na região do septo alveolar distal (p=0.001). A parede dos vasos peribronquiolares não demonstrou alteração da razão lúmen/parede (L/P) entre os grupos (0,094), porém, os animais submetidos à câmara poluída, apresentaram remodelamento estrutural com diminuição de fibras elásticas na adventícia (p= 0,006) e na túnica média (p= 0,008) e das fibras colágenas apenas na adventícia (p= 0,016). No miocárdio, verificamos aumento do número de núcleos por área do VE, sem alteração de peso dos corações entre os grupos (p=0,005). As coronárias dos camundongos expostos à câmara poluída também não demonstraram diferenças na razão L/P, porém a poluição induziu remodelamento através de aumento de fibras colágenas e elásticas na camada adventícia e média dos vasos (p<0.001; p=0,03 respectivamente), mesmo quando categorizados os vasos por seu calibre e por ventrículo separadamente (p<0,001). CONCLUSÃO: A poluição atmosférica da cidade de São Paulo, Brasil, induziu alterações inflamatórias e estruturais nos pulmões e corações dos animais normais expostos desde o nascimento até a idade adulta. A resposta pulmonar caracterizou-se por inflamação e diminuição das fibras da matriz extracelular dos vasos, enquanto as coronárias apresentaram fibrose e elastogênese em toda parede vascular. Esses achados reforçam a hipótese de que a poluição atmosférica de grandes centros urbanos induz alterações silenciosas porém significativas, órgão-específicas, modulando a resposta vascular, podendo contribuir para alteração da patência e reatividade dos vasos e eventos isquêmicos na idade adulta / INTRODUCTION: The continuous exposition since birth to atmospheric pollution of great urban centers originated by vehicles predominantly, contributes to cardiopulmonary events in adults. The remodeling and inflammation induced by airborne pollutants may modulate the vascular response to mediators and alter the vascular patency and reactivity which may contribute to ischemic events. METHODS: To verify the inflammatory and structural alterations of the lungs and heart of Swiss male mice chronically and continuously exposed since birth to adulthood, to selective chambers with (clean) or without (polluted) filter devices for particles and toxic gases, 7days/24h/4 months, located 20m far from a cross-road with heavy traffic in Sao Paulo city downtown. Measurements of PM2,5; NO2 and Black Carbon (BC) inside the chambers and in the external environment and morphometric analyses of the lung and heart were made. RESULTS: Filters cleared 100% of BC, 47% of PM2,5 and 68% of NO2 in the clean chamber. In the lungs, pollutants affected predominantly the peribronchiolar area. Animals submitted to the polluted chamber presented peribronchiolar inflammation characterized by increased perivascular influx (p=0.022), bronchiolar epithelial hyperplasia (p=0.037), total inflammatory scores (p=0.015), and decrement of polymorphonuclear cells infiltration in the distal septa (p=0.001). There was no difference of the lumen/wall ratio (L/W) of peribronchiolar vessels (p=0.094) between groups. However, mice from polluted chamber presented structural remodeling characterized by decrement of elastic fibers in both adventitia (p=0.006) and media layers (p= 0.008) and collagen content in the adventitia (p= 0.016). In the heart, there was an increase of nucleus of myocytes/left ventricle area, but no alteration of the myocardial weight between groups (p=0.005). Although the coronary arterioles from mice exposed to polluted ambient did not show differences of L/W ratio when compared to those from clean ambient, pollutants induced increment of collagen and elastic fibers in the adventitia and media vascular layer (p<0.001; p=0.03 respectively), even when vessels were categorized by caliber and per ventricle chamber separately (p<0.001). CONCLUSION: The continuous exposure to atmospheric pollution of São Paulo city, Brazil, since birth to adulthood, induced inflammatory and structural alterations in the lungs and heart. The lung response was characterized by inflammation and decrement of extracellular matrix fibers while in the heart, there was fibrosis and elastin increment in the coronary arteriolar wall. These results reinforce the hypothesis that urban airborne pollution predominantly generated by traffic, induces silent but significant organ-specific alterations, which may impair vascular patency and reactivity and may contribute to ischemic cardiac events in the adulthood

Neste estudo, os objetivos propostos foram: 1) verificar o efeito da sobrecarga salina 1% sobre a pressão arterial e variáveis relacionadas ao controle hídrico em camundongos machos e fêmeas; 2) comparar os efeitos da sobrecarga salina sobre o remodelamento vascular de artérias elásticas e de resistência em machos e fêmeas. Para tal, foram usados camundongos C57Bl/6 machos e fêmeas tratados por 2 ou 12 semanas com solução de NaCl 1% para beber ou água fresca (grupo controle). Os animais foram pesados semanalmente e acompanhados em gaiola metabólica ao final do estudo (coleta de água e urina). A pressão arterial (PA) e frequência cardíaca (FC) foram medidas por pletismografia de cauda. Os animais foram eutanasiados, tiveram peso e comprimento naso-anal medidos, sendo coletados coração (análise de artérias cardíacas) e aorta para análise morfométrica, avaliando-se fibras colágenas e lamelas elásticas. Também foi avaliada a relação percentual peso cardíaco/peso corporal. Urina e sangue foram coletados para quantificação de proteínas totais, creatinina, sódio e cloreto. Ao final dos tempos de sobrecarga salina, não foram observadas diferenças significantes de peso corporal, pressão arterial e frequência cardíaca, hematócrito e medida naso-anal entre os grupos. Verificamos aumento de peso relativo do coração em períodos mais curtos de sobrecarga em machos (2 semanas) e mais longos em fêmeas (12 semanas). Os valores urinários de sódio, proteína total e creatinina aumentaram com a sobrecarga salina, tanto em machos como em fêmeas. A avaliação morfométrica das artérias elásticas apontou aumento do número de lamelas em machos, enquanto a avaliação das artérias musculares apontou aumento da deposição colágena em fêmeas. / In this study, the objectives were: 1) determine the effect of 1% salt overload on blood pressure and related variables controlling water in male and female mice, 2) to compare the effects of salt overload on vascular remodeling of elastic arteries and strength in both males and females. To this end, we used C57BL / 6 males and females treated for 2 or 12 weeks with 1% NaCl solution for drinking or fresh water (control group). The animals were weighed weekly and monitored in metabolic cage at the end of the study (collecting water and urine). Blood pressure (BP) and heart rate (HR) were measured by tail plethysmography. The animals were euthanized, had weight and naso-anal length measured heart being collected (analysis of heart arteries) and aorta for morphometric analysis, evaluating elastic lamellae and collagen fibers. We also evaluated the percentage ratio heart weight / body weight. Blood and urine were collected for measurement of total protein, creatinine, sodium and chloride. At the end of the times of saline overload, no significant differences were observed in body weight, blood pressure and heart rate, and hematocrit measured naso-anal between groups. We observed increase in relative heart weight in shorter periods of overload in males (2 weeks) and longer in females (12 weeks). The values of urinary sodium, total protein and creatinine increased with saline overload in both males and females. The morphometric analysis of the elastic arteries showed an increase in the number of lamellae in males, while the evaluation of muscular arteries showed increased collagen deposition in females.

La grossesse est un état physiologique particulier où de nombreux changements fonctionnels et structuraux surviennent. Chez la rate, pour répondre aux besoins grandissants du fœtus, l’artère utérine se développe pour atteindre le double de son diamètre original avant parturition. Par conséquent, le débit sanguin utérin augmente d’environ vingt fois. Pour ce faire, les vaisseaux utérins sont l’objet d’un remodelage caractérisé par une hypertrophie et une hyperplasie des différentes composantes de la paroi. De plus, ce remodelage est complètement réversible après la parturition, par opposition au remodelage vasculaire « pathologique » qui affecte les artères systémiques, dans l’hypertension chronique, par exemple. La grossesse s’accompagne aussi de modifications hormonales importantes, comme les œstrogènes dont la concentration s’accroît progressivement au cours de cette période. Elle atteindra une concentration trois cents fois plus élevée avant terme que chez une femme non gravide. Cette hormone possède de multiples fonctions, ainsi qu’un mode d’action à la fois génomique et non génomique. Considérant l’ensemble de ces éléments, nous avons formulé l’hypothèse que l’œstradiol serait responsable de modifier la circulation utérine durant la grossesse, par son action vasorelaxante, mais aussi en influençant le remodelage de la vasculature utérine. Nous avons montré que le 17β-Estradiol (17β-E2) produit une relaxation due à un effet non génomique des artères utérines en agissant directement sur le muscle lisse par un mécanisme indépendant du monoxyde d’azote et des récepteurs classiques aux œstrogènes (ERα, ERβ). De plus, la relaxation induite par le 17β-E2 dans l’artère utérine durant la gestation est réduite par rapport à celle des artères des rates non gestantes. Ceci serait attribuable à une diminution de monoxyde d’azote provenant de la synthase de NO neuronale dans les muscles lisses des artères utérines. Nos résultats démontrent que le récepteur à l’œstrogène couplé aux protéines G (GPER), la protéine kinase A (PKA) et la protéine kinase G (PKG) ne sont pas impliqués dans la signalisation intracellulaire associée à l’effet vasorelaxant induit par le 17β-E2. Cependant, nous avons montré une implication probable des canaux potassiques sensibles au voltage, ainsi qu’un rôle possible des canaux potassiques de grande conductance activés par le potentiel et le calcium (BKCa). En effet, le penitrem A, un antagoniste présumé des canaux potassiques à grande conductance, réduit la réponse vasoralaxante du 17β-E2. Toutefois, une autre action du penitrem A n’est pas exclue, car l’ibériotoxine, reconnue pour inhiber les mêmes canaux, n’a pas d’effet sur cette relaxation. Quoi qu’il en soit, d’autres études sont nécessaires pour obtenir une meilleure compréhension des mécanismes impliqués dans la relaxation non génomique sur le muscle lisse des artères utérines. Quant à l’implication de l’œstrogène sur le remodelage des artères utérines durant la gestation, nous avons tenté d’inhiber la synthèse d’œstrogènes durant la gestation en utilisant un inhibiteur de l’aromatase. Plusieurs paramètres ont été évalués (paramètres sanguins, réactivité vasculaire, pression artérielle) sans changements significatifs entre le groupe contrôle et celui traité avec l’inhibiteur. Le même constat a été fait pour le dosage plasmatique de l’œstradiol, ce qui suggère l’inefficacité du blocage de l’aromatase dans ces expériences. Ainsi, notre protocole expérimental n’a pas réussi à inhiber la synthèse d’œstrogène durant la grossesse chez le rat et, ce faisant, nous n’avons pas pu vérifier notre hypothèse. En conclusion, nous avons démontré que le 17β-E2 agit de façon non génomique sur les muscles lisses des artères utérines qui implique une action sur les canaux potassiques de la membrane cellulaire. Toutefois, notre protocole expérimental n’a pas été en mesure d’évaluer les effets génomiques associés au remodelage vasculaire utérin durant la gestation et d’autres études devront être effectuées. / Pregnancy is a peculiar physiological state in which many structural and functional changes occur. In rats, to meet the growing needs of the fetus, uterine artery expands to reach twice its original diameter before parturition. Therefore, uterine blood flow increases by about twenty times. To do this, the uterine vessels undergo substantial remodelling characterized by hypertrophy and hyperplasia of the various components of the wall. Furthermore, this remodelling is completely reversible after parturition, as opposed to “pathological vascular remodelling” that affects systemic arteries in chronic hypertension, for instance. Pregnancy is also accompanied by significant hormonal changes, such as estrogens whose concentration in the blood increases progressively during this period. It reaches concentrations three hundred times higher before term than in non-pregnant women. This hormone has multiple functions and mediates its effects by genomic and non-genomic pathways. Considering these elements, we hypothesized that estradiol would be responsible for remodeling the uterine circulation during pregnancy, by its vasorelaxant action, but also by influencing the remodeling of the uterine vasculature. We have shown that 17β-Estradiol (17β-E2) produced non-genomic relaxation of uterine arteries by acting directly on smooth muscle using a mechanism independent of nitric oxide and classical estrogen receptors (ERα, ERβ). Moreover, the relaxation induced by 17β-E2 in the uterine artery during pregnancy is reduced compared to that of arteries from non-pregnant animals. This is the result of decrease of nitric oxide derived from neuronal NO synthase in smooth muscle of uterine arteries. Our results also show that the estrogen receptor coupled to G proteins (GPER), protein kinase A (PKA) and protein kinase G (PKG) are NOT involved in intracellular signaling associated with the vasorelaxant effect induced by the 17β-E2. Moreover, we have shown a possible involvement of potassium channels sensitive to voltage and a possible involvement of large conductance calcium-activated potassium channels (BKCa). Indeed, the penitrem A, a suspected antagonist of BKCa, reduced the vasoralaxant responses of 17β-E2. However, some other actions of penitrem A are not excluded because iberiotoxin, known to inhibit the same channels, had no effect on this relaxation. However, further studies are needed to obtain a better understanding of the mechanisms involved in the relaxation non-genomics on the smooth muscle of uterine arteries. As for the involvement of estrogen on the remodeling of uterine arteries during pregnancy, we attempted to inhibit the synthesis of estrogen during pregnancy using an aromatase inhibitor. Several parameters were evaluated (plasma values, vascular reactivity, blood pressure) without significant changes between the control group and those treated with the inhibitor. The same observation was made for the determination of plasma estradiol, suggesting the uneffectiveness of aromatase blocking in these experiments. Thus, our experimental protocol failed to inhibit the synthesis of estrogen in rats during pregnancy and, in so doing, we cannot confirm or refute our hypothesis. In conclusion, we demonstrated that 17β-E2 acts on smooth muscle of the uterine arteries by a non-genomic pathway by apparently acting on potassium channels. However, our experimental protocol was not able to assess the genomic effects associated with uterine vascular remodeling during pregnancy and further studies are required to ascertain this aspect of our work.