Existe uma relação entre o uso abrangente de inibidores da bomba de prótons (IBPs), como o omeprazol, e o aumento de risco cardiovascular. Essa relação está associada ao efeito dos IBPs de interferir na síntese e biodisponibilidade do óxido nítrico (NO), um fator importante na homeostase vascular. Também foi evidenciado que o omeprazol causa disfunção endotelial junto a um desequilíbrio redox em aortas, mediado pela ativação da enzima xantina oxidoredutase (XOR), responsável pelo catabolismo das purinas e geração de espécies reativas de oxigênio (ERO). As ERO decorrentes da atividade da XOR, podem aumentar a expressão e a atividade de metaloproteinases de matriz (MMPs), principalmente a MMP-2, que são promotoras de remodelamento tecidual. Assim, nosso objetivo foi analisar se o omeprazol causa remodelamento vascular em aorta de ratos, frente ao seu efeito de aumento do estresse oxidativo via XOR promovendo ativação de MMPs. Foram utilizados ratos wistar com peso entre 180-200 g (n=40), separados em 4 grupos de tratamento onde cada animal foi tratado com 0,5 mL de solução das drogas nas seguintes especificações: o grupo Controle (C) foi tratado com solução veículo tween 2% (vol./vol.) 1 vez ao dia por gavagem, o grupo Alopurinol (A) recebeu uma solução deste inibidor de XOR por gavagem (50 mg/kg/dia), o grupo Omeprazol (O) que recebeu uma solução de omeprazol diluída em tween 2% por via intraperitoneal (10 mg/kg/dia) e por fim, o grupo Omeprazol+Alopurinol (O+A) que recebeu as duas drogas concomitantemente. O protocolo experimental durou 4 semanas, durante as quais foram realizadas aferições da pressão arterial sistólica por pletismografia de cauda. Ao fim do tratamento, os animais foram submetidos à eutanásia, onde foi aferido o pH do lavado gástrico e foi coletada a aorta torácica para a análise de reatividade vascular, análise bioquímica de ERO, a análise morfométrica, e ensaio de atividade de MMPs. Não houve variação de pressão arterial em nenhum dos grupos. O tratamento com alopurinol não alterou nenhum dos parâmetros analisados em relação ao grupo controle neste estudo. O pH gástrico aumentou nos grupos tratados com omeprazol. Na reatividade vascular, observamos que o omeprazol diminuiu o efeito máximo da resposta vasodilatadora dos anéis de aorta à acetilcolina, mas que o tratamento associado ao alopurinol (O+A) preveniu essa diminuição. Em relação ao pD2, foi constatado que o tratamento com omeprazol resulta na diminuição da potência da acetilcolina em causar relaxamento vascular, e que a associação do tratamento com alopurinol, não foi capaz de prevenir essa diminuição. O grupo O também apresentou aumento de espécies reativas de oxigênio no leito vascular, observados no ensaio DHE e o tratamento com alopurinol preveniu este efeito. No ensaio de atividade gelatinolítica in situ observamos um aumento da atividade de MMPs no grupo O, e o tratamento com alopurinol também preveniu esse efeito. Na análise morfométrica observamos que o grupo O apresentou aumento dos parâmetros de remodelamento vascular, denotando um remodelamento hipertrófico, que foi prevenido pela associação com alopurinol. Com base nos resultados, é possível concluir que o tratamento com omeprazol causou remodelamento em aortas de ratos, e que esse efeito ocorreu paralelamente a outros prejuízos, como a diminuição da função vascular avaliada pela resposta à acetilcolina, aumento de espécies reativas de oxigênio e aumento de atividade de MMPs. Como todos esses efeitos resultantes do uso do omeprazol foram prevenidos pela associação do tratamento com alopurinol, é viável inferir que a XOR participe da via pela qual o omeprazol causa efeitos deletérios sobre a vasculatura / There is a relationship between the use of proton pump inhibitors (PPIs), such as omeprazole, and the increase of cardiovascular risk. This relation is associated with the effect of PPIs on nitric oxide synthesis and bioavailability, which is an important factor to vascular homeostasis. It also clear that omeprazole causes endothelial dysfunction by mechanisms involving xanthine oxidoreductase (XOR) mediated redox imbalance in aortas, which is responsible for purines catabolism, and generates reactive oxygen species (ROS). ROS derived from XOR activity, may increase matrix metalloproteinases expression and activity, mainly MMP-2 that are promoters of tissue remodeling. Thus, our aim was to analyze if omeprazole entails vascular remodeling in rat\'s aorta, with its effect of causing oxidative stress via XOR, promoting MMPs activation. Male rats weighing between 180-200g (n=40) were assigned to 4 groups with different treatments, where each animal was treated with 0.5 mL of drug solution, following the specification per group: Control group (C) was treated with the vehicle tween 2% (vol./vol.) 1 time a day by gavage; Allopurinol group (A) that received a solution of this XOR inhibitor by gavage (50mg/kg/day), Omeprazole group (O) which was treated by intraperitoneal route with a solution of omeprazole diluted at tween 2% (10 mg/kg/day) and at last, the Omeprazole+Allopurinol group (O+A), that received both drugs concomitantly. The experimental protocol lasted 4 weeks, during which, were performed systolic blood pressure measurements by tail cuff plethysmography. By the end of treatments, the animals were submitted to euthanasia, then the pH of the gastric washing was measured, and the thoracic aorta was collected to study vascular reactivity, biochemical analysis of ROS, morphometric analysis and MMPs activity assay. There was no blood pressure variation in any of the treatment groups. Treatment with allopurinol did not alter any of the parameters that were analyzed in the present study, in comparison to control group. Gastric washing pH increased in groups treated with omeprazole. In vascular reactivity, it was noticed that omeprazole decreased the maximum effect of the aortic ring\'s vasodilator response to acetylcholine, while the omeprazole treatment associated with allopurinol (O+A) prevented this decrease. Regarding to pD2, it was observed that omeprazole treatment results in decreased acetylcholine potency to cause vascular relaxation, and the association to allopurinol treatment was not capable of preventing this decrease. The O group also presented increased reactive oxygen species levels in the vascular bed, according to DHE assay, and the treatment with allopurinol prevented this effect. With respect to in situ gelatinolytic activity assay, we noticed an increase in MMPs activity in the O group, and the treatment with allopurinol prevented that. The morphometric analysis showed the O group with increased vascular remodeling parameters, denoting a hypertrophic remodeling, which was prevented by the association with allopurinol. Based on these results, is possible to conclude that the treatment with omeprazole caused aortic remodeling in rats, and combined to this effect, some other were observed, such as the vascular function impairment evaluated by the response to acetylcholine, the increase of ROS and increase in MMPs activity. As the effects of omeprazole treatment were prevented by the association of treatment with allopurinol, it is reasonable to infer that XOR participates of the pathway by which omeprazole exerts its deleterious effects on the vasculature

Inibidores da Na+/K+ ATPase, como a ouabaína (OUA), estão associados à gênese/manutenção da hipertensão arterial (HA). Ratos tratados com OUA ficam hipertensos e suas artérias mesentéricas de resistência (AMR) apresentam disfunção vascular, estresse oxidativo e remodelamento estrutural e mecânico. Modelos de HA dependente de volume, como o DOCA-sal, têm aumento da concentração plasmática de OUA. Interagindo com a Na+/K+ ATPase, a OUA ativa vias de proliferação e fibrose, como a via MAPK, ativada quando da fosforilação da c-Src e do EGFR. A rostafuroxina, antagonista da OUA, reduz a pressão arterial (PA) e melhora a disfunção endotelial em AMR de ratos DOCA-sal. Dados do laboratório (não publicados) mostram que a rostafuroxina reverte o remodelamento mecânico e restaura parcialmente o remodelamento estrutural das AMR de ratos DOCA-sal. Assim, o objetivo deste trabalho foi avaliar os mecanismos de sinalização celular envolvidos nas mudanças estruturais e mecânicas das AMR de ratos DOCA-sal tratados com rostafuroxina. Ratos Wistar, uninefrectomizados, foram tratados com injeções subcutâneas de DOCA (em mg/kg/semana: 20 na primeira; 12 na segunda e terceira; 6 da quarta ao fim do experimento) e ingeriram água com NaCl (1%) e KCl (0,2%). Cinco semanas após o início do tratamento, um subgrupo passou a ser tratado, concomitantemente, com rostafuroxina (gavagem, 1mg/kg/dia) por 3 semanas. Ao final do protocolo, os animais foram anestesiados, mortos, as AMR dissecadas e fixadas em paraformaldeído (4%) para as análises histológicas ou armazenadas para Western Blot. Análise estatística aplicada: ANOVA uma ou duas vias. Avaliada por meio da plestismografia de cauda, o tratamento com DOCA-sal aumentou a PA; a rostafuroxina reduziu, mas não normalizou a mesma aos valores do grupo SHAM. A razão de colágeno I/III e a expressão proteica dos mediadores fibróticos TGFβ1 e CTGF foi maior nas AMR dos DOCA, alterações restauradas após tratamento com rostafuroxina. Além do mais, houve aumento da espessura da lâmina elástica interna das AMR dos DOCA, o que foi revertido pela rostafuroxina. A expressão de endotelina-1 foi maior nas AMR tanto dos DOCA tratados ou não com rostafuroxina; a expressão do receptor ETΑ foi reduzida nas AMR de ambos os grupos. Já a expressão do receptor ETΒ foi maior nas AMR dos DOCA quando comparados aos SHAM, sendo normalizada pela rostafuroxina. A expressão de c-SrcTyr418 e EGFR, bem como das enzimas c-RAF, ERK 1/2, p-ERK 1/2 e p38MAPK foi maior nas AMR dos DOCA, sendo restauradas com a rostafuroxina. Os dados sugerem que o remodelamento das AMR do grupo DOCA pode ser atribuído à ação direta da OUA sobre a via fibrótica e sobre a elastina, interferindo na rigidez e no diâmetro interno, e também à sua ação indireta, mediada pela endotelina-1 que, agindo em receptores ETB e transativando o EGFR, ativa a via da MAPK, a qual está associada aos eventos de proliferação e fibrose celular. / Na+/K+ ATPase inhibitors, such as ouabain (OUA), are associated with genesis/maintenance of hypertension. Rats treated with OUA are hypertensive and their mesenteric resistance arteries (MRA) exhibit vascular dysfunction, oxidative stress and structural and mechanical remodeling. Volume-dependent hypertensive models, as DOCA-salt, have increased plasmatic OUA levels. Interacting with Na+/K+ ATPase, OUA activates proliferation and fibrosis pathways, such as MAPK pathway, which is activated from c-Src and EGFR phosphorylation. Rostafuroxin, an OUA antagonist, reduces arterial pressure (AP) and improves endothelial dysfunction in MRA of DOCA-salt rats. Unpublished data of our laboratory have shown that rostafuroxin reverses the mechanical and partially restores the structural remodeling of MRA of DOCA-salt rats. Thus, the aim of this study was to evaluate the cellular signaling mechanisms involved in the structural and mechanical changes in MRA of DOCA-salt rats treated with rostafuroxin. Male Wistar rats, uninephrectomized, were treated with subcutaneous injections of DOCA (mg/kg/week: 20 in the 1st; 12 in the 2nd and 3rd; 6 from 4th to the end of the experiment) and drunk water with NaCl (1%) and KCl (0,2%). After 5 weeks of treatment, a subgroup started being treated, concomitantly, with rostafuroxin (gavage, 1mg/kg/day) for 3 weeks. At the end of the protocol, animals were anesthetized, killed, the MRA dissected out and fixed in paraformaldehyde (4%) to histological analyses or stored to Western Blot. Statistical analysis applied: ANOVA one- or two-way. Evaluated by pletismography, the DOCA-salt treatment raised the AP levels; rostafuroxin reduced it, but did not normalized to the values of SHAM group. Collagen I/III ratio and protein expression of fibrosis mediators TGFβ1 and CTGF was higher in MRA of DOCA and rostafuroxin restored these parameters. In addition, an increment in the thickness of inner elastic lamina was observed in MRA of DOCA, which was reversed by rostafuroxin treatment. Endothelin-1 expression was higher in MRA of both DOCA and rostafuroxin treated rats; ETΑ receptor was less expressed in both groups. On the other hand, ETΒ was more expressed in MRA from DOCA and normalized to SHAM levels by rostafuroxin treatment. The expression of c-SrcTyr418 and EGFR, as well as of the enzymes c-RAF, ERK 1/2, p-ERK 1/2 and p38MAPK was higher in MRA of DOCA when compared to SHAM, being restores with rostafuroxin. Taken together, the present data suggest that MRA remodeling in DOCA group can be attributed to direct action of OUA on elastin and fibrotic pathway, interfering in stiffness and inner diameter, as well as to its indirect action, mediated by endothelin-1, which acting in ETB receptors and activating EGFR, activates MAPK pathway, which is associated to proliferation and fibroses events.

A hipertensão arterial sistêmica (HAS) apresenta alterações vasculares significativas como o remodelamento vascular e o aumento da atividade das metaloproteinases de matriz (MMPs), principalmente a MMP-2, responsáveis pela proteólise da matriz extracelular. No remodelamento vascular, ocorre uma mudança no fenótipo das células musculares lisas vasculares (CMLV) de contrátil para sintético, com redução de proteínas da maquinaria contrátil como a calponina-1. Essa alteração fenotípica confere às CMLV a capacidade de migração e proliferação, contribuindo ao remodelamento arterial durante a HAS. A calponina-1 foi degradada pela MMP-2 em aorta de ratos endotoxêmicos e este efeito contribuiu para a menor contração vascular da sepse. Sua redução foi associada ao aumento de atividade de MMP-2 e proliferação de CMLV em aortas de ratos hipertensos. A hipótese do presente trabalho é de que a MMP-2 contribui para as alterações morfofuncionais induzidas pela hipertensão em artérias de condutância e de resistência pela regulação de calponina-1. Para testar tal hipótese, ratos Wistar foram submetidos ao modelo de hipertensão dois rins-um clipe (2R-1C) ou apenas laparatomia e tratados por via oral com doxiciclina (inibidor da atividade de MMPs na dose de 30 mg/kg/dia) ou água durante sete dias. A pressão arterial sistólica (PAS) foi aferida diariamente por pletismografia de cauda. Aorta e artérias mesentéricas foram removidas para a execução de zimografia in situ e em gel, imunofluorescência para calponina-1, imunohistoquímica para Ki-67 e análise morfológica. Artérias mesentéricas e aortas também foram utilizadas para curva concentração-efeito com fenilefrina. Observou-se que a PAS aumentou em ratos 2R-1C e o tratamento com doxiciclina não a reduziu. A análise morfológica da aorta mostrou que a razão média por lúmen e a área de secção transversal aumentaram nos animais hipertensos em relação aos grupos Sham. Não houve alteração nestes parâmetros nas artérias mesentéricas. Houve aumento de proliferação das CMLV em aortas de ratos 2R-1C e a doxiciclina reverteu essa alteração. Nas artérias mesentéricas não foi observada alteração na proliferação celular. A atividade gelatinolítica de MMP-2 e sua expressão estão aumentadas nos dois leitos arteriais de ratos 2R-1C e o tratamento com doxiciclina as reduziu. A expressão de calponina-1 está reduzida nas aortas e aumentada nas artérias de resistência de animais 2R-1C e o tratamento reverteu estes efeitos. A contração à fenilefrina das aortas e artérias mesentéricas está aumentada nos animais 2R-1C e o tratamento a reduziu. Os resultados indicam que a MMP-2 tem ações diferentes na regulação de calponina-1 nos dois leitos arteriais. Nas artérias de condutância, a redução de calponina-1 pela MMP-2 pode ser o gatilho para a mudança de fenótipo das células musculares lisas vasculares, o que conduz ao remodelamento hipertrófico. Nas artérias de resistência, a MMP-2 pode contribuir para a função contrátil do leito arterial aumentando os níveis de calponina-1. / Hypertension is a global public health problem that lead to significant vascular changes. Among them, the chronic remodeling and increased activity of matrix metalloproteinase (MMP)-2, which is responsible for extracellular matrix proteolysis, are the main mediators of the vascular maladaptation. During vascular remodeling, there is a change in the phenotype of vascular smooth muscle cells (VSMC) of contractile to synthetic form, with a reduction of contractile proteins such as calponin-1. This phenotype switch provides to the VSMC the ability of migration and proliferation, thus contributing to hypertension-induced arterial remodeling. Calponin-1 was degraded by MMP-2 in rat endotoxemic aortas and this effect contributed to the vascular hypocontractility. In addition, its reduction was associated with increased activity of MMP-2 and proliferation of VSMC in aortas of rats submitted to renovascular hypertension. Therefore, the hypothesis of this study is that MMP-2 contributes to hypertension-induced morphological and functional changes in conductance and resistance arteries by regulating calponin-1. To test such hypothesis, male Wistar rats were submitted to the two kidney- one clipe (2K-1C) hypertension model and were treated with doxycycline (inhibitor of MMPs activity at 30 mg/kg/day) or water for one week. Systolic blood pressure (SBP) was daily checked by tail-cuff plethysmography. Aortas and mesenteric arteries were removed to perform in situ and gel zymography, immunofluorescence for calponin-1, immunohistochemistry for Ki-67 and morphological analysis. Mesenteric arteries and aortas were also used for concentration-effect curve for phenylephrine. It was observed that SBP has increased in 2K-1C rats and doxycycline did not reduce it. Morphological analysis of the aorta showed that both media per lumen ratio and arterial cross sectional area increased in hypertensive animals compared to Sham. There was no changes in this two parameters in mesenteric arteries. Furthermore, there was an increase of VSMC proliferation in rat aortas and doxycycline was able to revert this alteration. In the mesenteric arteries, no changes were observed in VSMC proliferation. Gelatinolytic activity of MMP-2 and its expression were increased in both arterial beds of 2K-1C rats and treatment with doxycycline reduced it. Calponin-1 expression was reduced in aortas and increased in resistance arteries from 2K-1C animals and the treatment was able to improve both scenarios. The contractile function of aortas and mesenteric arteries was increased in 2K-1C rats and treatment has reduced it. So, what follows is that MMP-2 has different actions in the regulation of calponin-1 in both arterial beds. In conductance arteries, reduction of calponin-1 by MMP-2 may trigger the VSMC phenotype switch, which leads to the hypertrophic remodeling. In resistance arteries, MMP-2 contributes to the hypercontractility of the arterial bed by increasing calponin-1. This effect may be particularly related to extracellular actions of MMP-2.

A hipertensão arterial sistêmica (HAS) apresenta alterações vasculares significativas como o remodelamento vascular e o aumento da atividade das metaloproteinases de matriz (MMPs), principalmente a MMP-2, responsáveis pela proteólise da matriz extracelular. No remodelamento vascular, ocorre uma mudança no fenótipo das células musculares lisas vasculares (CMLV) de contrátil para sintético, com redução de proteínas da maquinaria contrátil como a calponina-1. Essa alteração fenotípica confere às CMLV a capacidade de migração e proliferação, contribuindo ao remodelamento arterial durante a HAS. A calponina-1 foi degradada pela MMP-2 em aorta de ratos endotoxêmicos e este efeito contribuiu para a menor contração vascular da sepse. Sua redução foi associada ao aumento de atividade de MMP-2 e proliferação de CMLV em aortas de ratos hipertensos. A hipótese do presente trabalho é de que a MMP-2 contribui para as alterações morfofuncionais induzidas pela hipertensão em artérias de condutância e de resistência pela regulação de calponina-1. Para testar tal hipótese, ratos Wistar foram submetidos ao modelo de hipertensão dois rins-um clipe (2R-1C) ou apenas laparatomia e tratados por via oral com doxiciclina (inibidor da atividade de MMPs na dose de 30 mg/kg/dia) ou água durante sete dias. A pressão arterial sistólica (PAS) foi aferida diariamente por pletismografia de cauda. Aorta e artérias mesentéricas foram removidas para a execução de zimografia in situ e em gel, imunofluorescência para calponina-1, imunohistoquímica para Ki-67 e análise morfológica. Artérias mesentéricas e aortas também foram utilizadas para curva concentração-efeito com fenilefrina. Observou-se que a PAS aumentou em ratos 2R-1C e o tratamento com doxiciclina não a reduziu. A análise morfológica da aorta mostrou que a razão média por lúmen e a área de secção transversal aumentaram nos animais hipertensos em relação aos grupos Sham. Não houve alteração nestes parâmetros nas artérias mesentéricas. Houve aumento de proliferação das CMLV em aortas de ratos 2R-1C e a doxiciclina reverteu essa alteração. Nas artérias mesentéricas não foi observada alteração na proliferação celular. A atividade gelatinolítica de MMP-2 e sua expressão estão aumentadas nos dois leitos arteriais de ratos 2R-1C e o tratamento com doxiciclina as reduziu. A expressão de calponina-1 está reduzida nas aortas e aumentada nas artérias de resistência de animais 2R-1C e o tratamento reverteu estes efeitos. A contração à fenilefrina das aortas e artérias mesentéricas está aumentada nos animais 2R-1C e o tratamento a reduziu. Os resultados indicam que a MMP-2 tem ações diferentes na regulação de calponina-1 nos dois leitos arteriais. Nas artérias de condutância, a redução de calponina-1 pela MMP-2 pode ser o gatilho para a mudança de fenótipo das células musculares lisas vasculares, o que conduz ao remodelamento hipertrófico. Nas artérias de resistência, a MMP-2 pode contribuir para a função contrátil do leito arterial aumentando os níveis de calponina-1. / Hypertension is a global public health problem that lead to significant vascular changes. Among them, the chronic remodeling and increased activity of matrix metalloproteinase (MMP)-2, which is responsible for extracellular matrix proteolysis, are the main mediators of the vascular maladaptation. During vascular remodeling, there is a change in the phenotype of vascular smooth muscle cells (VSMC) of contractile to synthetic form, with a reduction of contractile proteins such as calponin-1. This phenotype switch provides to the VSMC the ability of migration and proliferation, thus contributing to hypertension-induced arterial remodeling. Calponin-1 was degraded by MMP-2 in rat endotoxemic aortas and this effect contributed to the vascular hypocontractility. In addition, its reduction was associated with increased activity of MMP-2 and proliferation of VSMC in aortas of rats submitted to renovascular hypertension. Therefore, the hypothesis of this study is that MMP-2 contributes to hypertension-induced morphological and functional changes in conductance and resistance arteries by regulating calponin-1. To test such hypothesis, male Wistar rats were submitted to the two kidney- one clipe (2K-1C) hypertension model and were treated with doxycycline (inhibitor of MMPs activity at 30 mg/kg/day) or water for one week. Systolic blood pressure (SBP) was daily checked by tail-cuff plethysmography. Aortas and mesenteric arteries were removed to perform in situ and gel zymography, immunofluorescence for calponin-1, immunohistochemistry for Ki-67 and morphological analysis. Mesenteric arteries and aortas were also used for concentration-effect curve for phenylephrine. It was observed that SBP has increased in 2K-1C rats and doxycycline did not reduce it. Morphological analysis of the aorta showed that both media per lumen ratio and arterial cross sectional area increased in hypertensive animals compared to Sham. There was no changes in this two parameters in mesenteric arteries. Furthermore, there was an increase of VSMC proliferation in rat aortas and doxycycline was able to revert this alteration. In the mesenteric arteries, no changes were observed in VSMC proliferation. Gelatinolytic activity of MMP-2 and its expression were increased in both arterial beds of 2K-1C rats and treatment with doxycycline reduced it. Calponin-1 expression was reduced in aortas and increased in resistance arteries from 2K-1C animals and the treatment was able to improve both scenarios. The contractile function of aortas and mesenteric arteries was increased in 2K-1C rats and treatment has reduced it. So, what follows is that MMP-2 has different actions in the regulation of calponin-1 in both arterial beds. In conductance arteries, reduction of calponin-1 by MMP-2 may trigger the VSMC phenotype switch, which leads to the hypertrophic remodeling. In resistance arteries, MMP-2 contributes to the hypercontractility of the arterial bed by increasing calponin-1. This effect may be particularly related to extracellular actions of MMP-2.

L’hypertension artérielle pulmonaire (HTAP) correspond à un groupe de maladies qui se caractérise par une obstruction vasculaire suite à une vasoconstriction excessive, une prolifération cellulaire et la création de thromboses in situ, conduisant à une augmentation progressive des résistances vasculaires pulmonaires puis au décès. De nombreuses avancées dans la prise en charge de l’HTAP ont été réalisées ces dernières années avec la mise à disposition de médicaments principalement vasodilatateurs. Cependant, aucun de ces médicaments n’est curatif de la maladie témoignant de la nécessité à obtenir de nouvelles thérapeutiques. Des molécules axant leur effet sur la lutte contre la prolifération cellulaire liée à l’activation des récepteurs à tyrosine-kinases (RTK) ou le stress oxydant (SO) paraissent aujourd’hui comme de potentielles innovations thérapeutiques dans l’HTAP. Cependant, à l’heure actuelle, les données sur le SO dans l’HTAP sont trop peu détaillées pour cibler correctement cette voie physiopathologique. De même, les inhibiteurs de tyrosine-kinases ont montré un bénéfice dans la prise en charge de l’HTAP mais associé à des effets indésirables graves tels qu’une toxicité cardiaque. Dans ce travail, nous avons approfondi le mécanisme d’action du SO dans la physiopathologie de l’HTAP et complété l’identification des RTK dans le remodelage vasculaire pulmonaire afin de permettre la mise au point de thérapeutiques efficaces avec un rapport bénéfice risque favorable pour le patient. / Pulmonary arterial hypertension (PAH) corresponds to a group of diseases characterized by a vascular obstruction due to vasoconstriction, cellular proliferation and in situ thrombosis, leading to a progressive increase in pulmonary vascular resistances. New knowledge in the PAH management were performed in the last few years, specifically for vasodilators. However, none of those treatments cure the disease and new drugs are still needed. Molecules targeting cellular proliferation induced by tyrosine kinases receptors (TKR) activation or oxidative stress (OS) seem to be potential therapeutic innovations. However, knowledge on OS in PAH is not enough accomplished in PAH to target accurately this pathophysiologic pathway. Similarly, tyrosine kinase inhibitors have shown efficacy in PAH management but associated with severe adverse events as cardiac toxicity. In this study, mechanism of action of OS in pathophysiology of PAH was detailed and identification of TKR involved in vascular remodeling was completed in order to find efficient therapeutics with a favorable risk benefit ratio for PAH patient.

ALK1 est un récepteur de la famille du TGF-β, principalement exprimé dans les cellules endothéliales. Le ligand physiologique et circulant d'ALK1, BMP9, a été découvert par notre laboratoire en 2007, ce qui a ouvert des possibilités d'étude de la fonction d'ALK1. La première partie de ma thèse a été consacrée à l'analyse fonctionnelle de mutants d'ALK1, retrouvés sur des patients atteints de la maladie de Rendu-Osler de type 2, en réponse à BMP9. Cette étude a permis de : 1) proposer l'haploinsuffisance fonctionnelle comme modèle de la maladie ; 2) développer un test diagnostique pour discriminer les mutations pathogènes des polymorphismes rares, basé sur leur réponse à BMP9 ; 3) d'avoir une meilleure connaissance des acides aminés d'ALK1 importants dans la réponse à BMP9. Un second travail a consisté en la production de la forme mature de BMP9 et du domaine extracellulaire d'ALK1 en vue de l'étude de la structure cristallographique du complexe. L'expression des protéines et leur purification sont en phase d'optimisation. Enfin, un troisième projet consistait en l'analyse du rôle de BMP9 dans l'angiogenèse in vivo. La neutralisation de BMP9 par deux stratégies distinctes induit une augmentation de la densité vasculaire dans la rétine de la souris. Le mécanisme est en cours d'investigation. / ALK1 is a TGF-β family receptor, mainly expressed on endothelial cells. The physiologic and circulating ligand of ALK1, BMP9, was discovered by our laboratory in 2007, which opened opportunities for studying the function of ALK1. The first part of my thesis was on the functional analysis of ALK1 mutants from HHT-2 patients in response to BMP9. This study allowed us to: 1) propose functional haploinsufficiency as a model for HHT-2; 2) develop a diagnostic tool to discriminate pathogenic mutations from rare polymorphisms, based on their BMP9 response; 3) increase our knowledge of important amino acids in ALK1 for the BMP9 response. A second work was on the production of the mature form of BMP9 and of the extracellular domain of ALK1 in order to study the crystallographic structure of the complex. The expression of these proteins and their purification are in optimization phase. Lastly, a third project was on the analysis of the role of BMP9 in angiogenesis in vivo. Neutralization of BMP9 using two strategies induces an increase of the vascular density of the retina in mouse. Mechanism of action is under investigation.

In this study, we investigated the role of Ang II in pathological vascular remodeling. We sought to determine whether the humoral or the mechanical effects of Ang II are the dominant factor driving the remodeling process. The following experimental groups were used: control group (untreated mice), mice treated with an angiotensin receptor blocker (Candesartan, 0.5 mg/kg/day,SQ), an ACE inhibitor (Captopril, 6 mg/kg/day), and a calcium-channel blocker (Amlodipine, 7.5 mg/kg/day). All mice (n=6 per experimental group) were from the C57Bl/6 background. The carotid ligation model was implemented to study the differences in vascular remodeling. Additionally, multiple time points (7-, 14-, and 21-days post-surgery) were used to track the progression of remodeling. In Day-7 analysis, all three treatment groups yielded similar remodeling patterns as evidenced by a significant reduction in neointimal area, medial thickening and hypertrophy compared with the control group. Histomorphometric analysis of carotid sections collected 1mm below the ligation demonstrated that the Amlodipine group had 26% reduction in total vessel area, Candesartan a 36% reduction, and Captopril a 28% reduction (p less than 0.05 in all groups compared with Control), as well as a parallel 38-40% drop in medial thickness. In Day-14 analysis, no significant differences between the Controls and treatment groups were observed, although differences were emerging between the treatment groups. Candesartan was found to reduce the extent of negative remodeling observed between the 7- and 14-day Control data, whereas the Captopril group did not exhibit this trend. All treatment groups exhibited less neointimal formation than Controls, similar to Day-7. By the 21-day time point, the Captopril group underwent positive remodeling, resembling the Candesartan and Amlodipine groups. Although total vessel area was analogous among all groups, neointimal areas were significantly decreased in the treatment groups. Blood pressure plays a pivotal role in the modulation of vascular remodeling in response to mechanical injury. Although intermediate timepoint analysis suggests that humoral aspects of ACE inhibition or angiotensin-receptor blockade yielded unique effects on the overall vessel caliber, upon reaching the late, 21-day time point, the mechanical factors became predominant. These data support the importance of blood pressure control in the attenuation of pathological vascular remodeling.

Elevation of blood pressure leads to structural and functional alterations in vasculature, resulting in increased arterial stiffness, which in turn is a predictor of future hypertension and cardiovascular risks. Angiotensin II (Ang II) plays a crucial role in blood pressure regulation. In addition to its hemodynamic effects, Ang II activates both innate and adaptive immunity. The objective of this study is to define the roles of CD4⁺ T lymphocyte subsets in the progression of vascular remodeling and stiffening induced by Ang II. A mouse model of Ang II infusion was used to induce hypertension and vascular diseases. In the WT mice, Ang II infusion led to an increased aortic stiffness within 7 days of the treatment as well as an increase in aortic remodeling within 14 days of the treatment. Interestingly, RAG1(-/-) mice, lacking functional T and B lymphocytes were prevented from the vascular stiffening and remodeling caused by Ang II. Characterization of T cell subsets in the perivascular aortic infiltrates showed that there was a sequential activation of peri-arotic Th1 and Th17 during the time course of Ang II treatment, which was associated with the initial increased aortic stiffness and the subsequent remodeling, respectively. To extend the concept, roles of suppressive regulatory T cells (Tregs) were further examined. Proliferation of Tregs was successfully induced in vivo using a cytokine complex of IL-2 and anti-IL-2 mAb clone JES6-1. Ang II-infused mice that received the IL-2/anti-IL-2 complex exhibited a reduced vascular remodeling and stiffening caused by Ang II. Stimulation of Tregs with the IL-2/anti-IL-2 complex also suppressed the Th1 and Th17 responses and reduced immune cells infiltrates in the aortas. Since hypertension is closely related to the kidney and renal homeostasis is also tightly regulated by Ang II, the kidney function was determined in this Ang II-hypertensive model. In the wild type mice, two weeks infusion of Ang II resulted in an increased glomerular filtration rate (GFR) whereas immunodeficient RAG1(-/-) mice exhibited a marked decrease in GFR. Subsequent experiments showed that Th17 was crucial in renal hemodynamic response to Ang II, partly by regulating secretion of vasodilatory prostaglandin E₂.

Chronic neonatal pulmonary hypertension (PHT) frequently presents with rightventricular (RV) dysfunction. In neonatal rats exposed to chronic hypoxia, RV dysfunction is reversed by sustained rescue treatment with a Rho-kinase (ROCK) inhibitor – the caveat being systemic hypotension. We therefore examined the reversing effects of pulmonary-selective ROCK inhibition. Rat pups were exposed to air or hypoxia from birth for 21 days and received sustained rescue treatment with aerosolized Fasudil (81 mg/ml t.i.d for 15 min) or i.p. Y27632 (15 mg/kg b.i.d) from days 14-21. Inhaled Fasudil normalized pulmonary vascular resistance, and reversed pulmonary vascular remodeling but did not improve RV systolic function. Systemic, but not pulmonary-selective, ROCK inhibition attenuated increased RV ROCK activity. Our findings indicate that RV dysfunction in chronic hypoxic PHT is not merely a result of increased afterload, but rather may be due to increased activity of ROCK in the right ventricle.

Chronic neonatal pulmonary hypertension (PHT) frequently presents with rightventricular (RV) dysfunction. In neonatal rats exposed to chronic hypoxia, RV dysfunction is reversed by sustained rescue treatment with a Rho-kinase (ROCK) inhibitor – the caveat being systemic hypotension. We therefore examined the reversing effects of pulmonary-selective ROCK inhibition. Rat pups were exposed to air or hypoxia from birth for 21 days and received sustained rescue treatment with aerosolized Fasudil (81 mg/ml t.i.d for 15 min) or i.p. Y27632 (15 mg/kg b.i.d) from days 14-21. Inhaled Fasudil normalized pulmonary vascular resistance, and reversed pulmonary vascular remodeling but did not improve RV systolic function. Systemic, but not pulmonary-selective, ROCK inhibition attenuated increased RV ROCK activity. Our findings indicate that RV dysfunction in chronic hypoxic PHT is not merely a result of increased afterload, but rather may be due to increased activity of ROCK in the right ventricle.