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Avaliação da expressão da PI3K e sua interação com a angiotensina II em núcleos centrais autonômicos de animais hipertensos. / Evaluation of PI3K expression and its interaction with angiotensin II on central autonomic nuclei of hypertensive animals.Leila Buttler 15 July 2011 (has links)
Uma das causas da hipertensão está relacionada a alterações de núcleos autonômicos responsáveis pelo controle dos reflexos cardiovasculares, destacando-se o PVN, o NTS e o RVLM. A ANG II, atuando nestes núcleos, participa do controle da PA. Estudos in vitro, demonstraram uma via de sinalização da ANG II que envolve a ativação da PI3K e da Akt. Neste estudo avaliamos as diferenças na atividade da PI3K, através da quantificação da subunidade p85 (PI3K) e da p-Akt por meio de immunoblotting desses núcleos centrais de animais SHR, WKY e Wistar, além da interação da PI3K com os efeitos cardiovasculares da ANG II injetada no PVN de Wistar acordados. A expressão da p85 não diferiu entre os núcleos estudados. No entanto, a p-Akt estava 1,5x mais expressa no PVN de SHR. Ainda, o aumento da PAM promovido pela injeção de ANG II no PVN foi reduzido 5 min após a injeção do inibidor da PI3K, com recuperação da resposta 30 min mais tarde, sugerindo que a via da PI3K está mais ativa no PVN de animais SHR e que a interação das vias ANG II-PI3K está relacionada com a hipertensão. / One cause of the hypertension is associated to changes in autonomic nuclei that control the cardiovascular reflexes. Among these nuclei are PVN, NST and RVLM. The ANG II, acting in these nuclei, contributes to the control of the AP. In vitro studies had demonstrated a signaling pathway of the ANG II that involves the activation of PI3K and Akt. In the present study we evaluated the PI3K activity via expression of p85 (PI3K) and p-Akt, through the immunoblotting of these central nuclei of SHR, WKY and Wistar. Moreover, we evaluated the interaction of PI3K to the cardiovascular effects of ANG II injected into the PVN of awake Wistar. The p85 expression was not different at all autonomic nuclei studied. However, the p-Akt expression was 1.5x higher in the PVN of SHR. Besides, the increase in the MAP elicited by the injection of ANG II into the PVN was reduced 5 min after the blockade of the PI3K, and restored 30 min later, suggesting that PI3K pathway is more activate at the PVN level of SHR and that the interaction between ANG II-PI3K is related to hypertension.
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Ação da angiotensina II no remodelamento da matriz extracelular perivascular em camundongos. / Action of angiotensin II in perivascular extracellular matrix remodeling in mice.Katia Aparecida da Silva Viegas 05 October 2012 (has links)
Neste estudo avaliou-se a ação da Angiotensina II (Ang II) e do bloqueio dos seus receptores AT1 e AT2 no remodelamento da matriz extracelular (MEC) e traçamos o perfil da sinalização intracelular envolvida no processo. O estudo foi feito in vivo em camundongos isogênicos C57Bl/6J submetidos a tratamento durante 7 e 14 dias com doses subpressoras de Ang II, bloqueador do receptor AT1 (Losartan) e uma combinação destes. Os animais foram sacrificados e procedeu-se a coleta de tecidos de artérias (aorta, carótida e femoral), coração, rins e pulmão para análise da síntese e degradação de componentes da MEC. Foram feitas avaliações hemodinâmicas, morfológicas em microscopia de luz, morfométricas, imunohistoquímicas para os componentes da matriz extracelular: colágeno (tipos I, III, IV e VI), fibronectina, tenascina-C, elastina, metaloproteinases (tipos 2 e 9), e quantificação de algumas proteínas ligadas à sinalização intracelular da via das Proteínas quinases ativadas por mitógenos (MAPK - Mitogen-activated protein kinases) usando-se Western Blotting. / In this study we evaluated the action of Angiotensin II (Ang II) and the blockade of AT1 and AT2 receptors in the remodeling of extracellular matrix (ECM) and outlines the process involved in intracellular signaling. The study was done in vivo in C57Bl/6J inbred mice undergoing treatment for 7 and 14 days subpressor doses of Ang II, AT1 receptor blocker (Losartan) and a combination thereof. The animals were sacrificed and proceeded to collect tissues of arteries (aorta, carotid and femoral arteries), heart, kidneys and lungs for analysis of the synthesis and degradation of ECM components. We assessed hemodynamic, morphological light microscopy, morphometry, immunohistochemistry for extracellular matrix components: collagen (types I, III, IV and VI), fibronectin, tenascin-C, elastin, metalloproteinases (types 2 and 9) and quantification of some proteins related to intracellular signaling pathways of the mitogen-activated protein kinase (MAPK) using Western Blotting.
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Regulation of cardiac responses to increased load:role of endothelin-1, angiotensin II and collagen XVPiuhola, J. (Jarkko) 14 July 2002 (has links)
Abstract
Chronic overload of the heart is the major cause of left ventricular hypertrophy (LVH) and eventually heart failure. It is generally accepted that autocrine/paracrine factors, such as angiotensin II (Ang II) and endothelin-1 (ET-1) contribute to the development of LVH. Cardiac hypertrophy and failure are characterized by attenuated responsiveness to β- adrenergic stimulation and accumulation of collagenous material to the left ventricular wall. The present study aimed to characterize the roles of ET-1 and Ang II in the regulation of cardiac function. The role of the plasmamembrane Ca2+-ATPase (PMCA) in ET-1 induced cardiac responses and the role of type XV collagen in cardiac function were also studied.
Both ET-1 infusion and mechanical loading were able to induce positive inotropic effect and induction of early response genes in isolated perfused hearts. ET-1 also induced strong vasoconstriction. Cardiomyocyte-specific PMCA overexpression inhibited the ET-1 induced hypertrophic response, while inotropic response remained unaltered. ET-1 was found to induce release of adrenomedullin (AM), a potent vasorelaxing and inotropic peptide. Infusion of AM antagonized the vasoconstrictive effect of ET-1 independently of nitric oxide. In hypertrophied rat hearts ET-1 was found to contribute significantly to the Frank-Starling response, a fundamental mechanism regulating contractile performance of the heart. In mice hearts, ET-1 was found to play a dual role in load induced elevation of contractile strength: ETA receptors mediated an increase, while ETB receptors mediated an inhibitory effect on contrcatile force. Ang II was not contributing to the contractile response to load in either rat or mice hearts. Blunted response to β-adrenergic stimulus and increased vulnerability as a result of exercise was observed in mice lacking collagen XV.
In conclusion, the present results underscore the importance of the local factors, especially ET-1, in regulation of cardiac function, not only in terms of hypertrophic but also in terms of contractile response to load. The results also suggest a role for PMCA in regulation of cardiac function. Lack of type XV collagen was found to result in cardiac dysfunction with many features similar to those of early heart failure.
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Angiotensina II e sua associação com fator-I de crescimento semelhante à insulina, insulina e células foliculares na maturação de oócitos bovinos e conseqüente desenvolvimento embrionário / Angiotensin II and its association with insulin-like growth factor-I, insulin and follicular cells on bovine oocyte maturation and consequent embryo developmentStefanello, Jerônimo Rubert 06 May 2005 (has links)
The aim of the present study was to evaluate the action of angiotensin II (Ang II), insulin-like growth factor-I (IGF-I) and insulin (Ins) on bovine oocyte nuclear and cytoplasmic maturation with follicular cells. Cumulus-oocyte complexes (COCs) were cultured for 22 h with follicular cells and Ang II, IGF-I or Ins. In this experiment, two control groups were used, where the oocytes were cultured with (control with cells) or without (control without cells) follicular cells. Two experiments were performed with these five groups, in the presence or absence of LH and FSH. Only the oocytes cultured in the Ang II group (88.2±1.8 and 90.7±4.3%), respectively without and with LH/FSH) had resumption of meiosis in similar rates than those cultured in the absence of follicular cells (89.7±0.3 and 92.6±2.6; P<0.01). In a second experiment, the action of Ang II and its association with IGF-I or insulin on oocyte meiotic maturation was observed for 7 (germinal vesicle breakdown - GVBD), 12 (metaphase I - MI) and 22 (metaphase II - MII) hours, in a similar experimental design of the previous experiment. The Ang II, Ang II + IGF-I and Ang II + Ins, independent of gonadotrophins, were able to overturns the inhibition of meiotic maturation caused by follicular cells, in the same rates of control group without cells, in the three evaluated period (P<0.01). One last study was performed to investigate the effect of follicular cells, Ang II, IGF-I, Ins and its associations during oocyte maturation on the rate of subsequent embryo development. The oocytes were submitted to maturation similarly to the second experiment for 1 h (1+23 h), 12 h (12+12 h) or 24 h in the presence of follicular cells and its respective treatments + the period to complete 24 h in maturation media. In 1 + 23 h, the cleavage, blastocyst and hatching rates were not different among these five groups. In 12 + 12 h, the oocytes matured in the Ang II + IGF-I group developed into blastocyst (43.8±0.6) and hatching/total oocytes (17.1±1.2) in a higher rates than the others treatments (P<0.05). In 24 h, the association of Ang II + IGF-I improved blastocyst rate in comparison to the others treatments with follicular cells (P<0.05). Also, only in this group hatched blastocysts were obtained after 24 h in the presence of follicular cells. In conclusion, Ang II overturned the inhibitory effect on bovine oocyte nuclear maturation caused by follicular cells, independent of the presence of gonadotrophins, IGF-I and insulin. However, the oocyte cytoplasmic maturation evaluated through embryo development was improved when the Ang II and IGF-I were presents in maturation media with follicular cells for 12 + 12 hours. / O objetivo do presente estudo foi de avaliar a ação da angiotensina II (Ang II), fator-I de crescimento semelhante à insulina (IGF-I) e insulina (Ins) na maturação nuclear e citoplasmática de oócitos bovinos na presença de células foliculares. Os complexos cumulus-oócitos (CCOs) foram cultivados por 22 h na presença de células foliculares e Ang II, IGF-I ou Ins. Nesse experimento, foram utilizados dois grupos controles, onde os oócitos foram cultivados na presença (controle com células) ou na ausência (controle sem células) de células foliculares. Dois experimentos foram realizados com esses cinco grupos, sendo um com e outro sem adição de LH e FSH. Somente os oócitos cultivados no grupo Ang II (88,2±1,8 e 90,7±4,3%, respectivamente sem e com LH/FSH) reiniciaram a meiose em índices similares àqueles cultivados na ausência de células foliculares (89,7±0,3 e 92,6±2,6; P<0,01). Em um segundo experimento, foi avaliada a ação da Ang II e sua associação ao IGF-I ou insulina na maturação de oócitos por 7 (rompimento da vesícula germinativa - RVG), 12 (metáfase I - MI) e 22 (metáfase II - MII) horas, em um delineamento similar ao do experimento anterior. A Ang II, Ang II + IGF-I e Ang II + Ins, independente de gonadotrofinas, foram capazes de reverter a inibição da maturação causada pelas células foliculares, nos mesmos níveis do grupo controle sem células (P<0,01), nos três horários estudados. Um último experimento foi realizado para avaliar o efeito das células foliculares, Ang II, IGF-I, Ins e suas associações durante a maturação de oócitos sobre o índice de desenvolvimento embrionário subseqüente. Os oócitos foram maturados em grupos similares aos do segundo experimento por 1 h (1+23 h), 12 h (12+12 h) ou 24 h na presença de células foliculares e seus respectivos tratamentos + o período para completar 24 h em meio de maturação. Em 1 + 23 h, não houve diferença nos resultados de clivagem, blastocistos e eclosão entre os cinco grupos. No sistema 12 + 12 h, os oócitos no grupo Ang II + IGF-I atingiram índices de blastocistos (43,8±0,6) e eclosão/total de oócitos (17,1±1,2) superiores aos outros tratamentos (P<0,05). Em 24 h, a associação de Ang II + IGF-I obteve maiores índices de blastocistos em comparação aos demais tratamentos contendo células foliculares (P<0,05). Somente nesse grupo foram obtidos blastocistos eclodidos após 24 h na presença de células foliculares. Em conclusão, a Ang II reverte o efeito inibitório da maturação nuclear de oócitos bovinos causado pelas células foliculares, independente da presença de gonadotrofinas, IGF-I e insulina. No entanto, a capacitação oocitária, medida pelo desenvolvimento embrionário, é superior quando a Ang II e IGF-I estão presentes em meios contendo células foliculares em cultivos de 12 + 12 horas.
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Caractérisation de modèles murins du syndrome d'Ehlers-Danlos vasculaire / Characterization of vascular Ehlers-Danlos syndrome mouse modelsFaugeroux, Julie 14 November 2013 (has links)
Le Syndrome d’Ehlers-Danlos vasculaire (SEDv) est une pathologie génétique rare à transmission autosomique dominante. Les patients sont prédisposés à la survenue de ruptures vasculaires, digestives et utérines induisant une létalité précoce. Cette pathologie est due à des mutations du gène COL3A1 (codant le collagène de type III), majoritairement de type faux-sens, agissant via un mécanisme dominant négatif. Plus rarement, de larges délétions ou des mutations non-sens induisent une haplo-insuffisance. In fine, les conséquences de ces mutations portent essentiellement sur la synthèse du collagène de type III, aboutissant à une fragilité artérielle importante. A ce jour, aucun traitement curatif n’est disponible. L’inactivation du gène Col3a1 chez la souris entraine une mortalité in utero et néonatale quasi-systématique en cas d’homozygotie, mais les souris Col3a1+/- sont exemptes de phénotype vasculaire évident. Afin d’obtenir un modèle de dissection artérielle liée à un défaut de collagène de type III, une perfusion chronique d’Angiotensine II (Ang II) à dose pressive a été utilisée. Nos résultats montrent que l’haplo-insuffisance en collagène de type III confère une sensibilité artérielle majeure à l’Ang II. Cette fragilité est caractérisée par le développement de dissections/ruptures de l’aorte ascendante et induit des décès prématurés de ces animaux. Ce phénotype est lié non seulement à l’élévation de pression artérielle mais aussi à l’activation des voies de signalisation de l’Ang II. Enfin, nous montrons qu’un traitement associant un bêtabloquant (propranolol) et un vasodilatateur artériel (hydralazine) permet de réduire la mortalité induite par l’Ang II. Ces résultats suggèrent l’intérêt de l’ajout d’un traitement préventif par inhibiteur de l’Ang II au traitement bêtabloquant (Celiprolol) recommandé dans la pathologie humaine.Parallèlement, nous avons généré un modèle murin knock-in génétiquement plus proche des patients SEDv, par l’introduction d’une mutation ponctuelle faux-sens (Gly183Ser) observée chez un patient. L’analyse préliminaire de ce modèle montre que les souris Col3a1+/G183S décèdent spontanément, dès 4 semaines, de ruptures/dissections de l’aorte ascendante. Cependant, ces souris ne présentent de modification ni de leurs paramètres hémodynamiques, ni de leurs diamètres aortiques. En revanche, environ 20 % des souris Col3a1+/G183S présentent des plaies au niveau du dos et des pattes. Ce nouveau modèle de souris est actuellement le seul à récapituler aussi fidèlement le phénotype des patients SEDv. Il devrait donc permettre de tester différentes approches thérapeutiques. / Vascular Ehlers-Danlos (vEDS) syndrome is a rare, inherited, autosomal dominant disease that results from mutations in the COL3A1 gene, encoding type III collagen. Patients are mostly affected by missense mutations probably acting through a dominant negative mechanism. A few patients present large deletions or nonsense mutations leading to a haploinsufficient mechanism. These mutations are supposed to lead to a defect in the synthesis and secretion of collagen type III, resulting in arterial wall fragility. Consequently, vEDS is mostly characterized by ruptures/dissections in arteries at a young age, which ultimately lead to premature death. While there is currently no surgical or therapeutic treatments available, a recent study reported the beneficial effect of the beta-blocker celiprolol, which prevents vascular complications in patients.To investigate the vascular phenotype of vEDS, a mouse model of this disease has been generated by the complete and ubiquitous inactivation of the COL3A1 gene. Col3a1-/- mice exhibit severe perinatal mortality and die prematurely from spontaneous vascular rupture. However, Col3a1+/- mice are viable and exhibit no obvious vascular phenotype. To determine the susceptibility of Col3a1+/- mice to develop vascular rupture/dissection, an experimental model of aneurysm induction was used, through the chronic infusion of Angiotensin II (Ang II). Our results showed that Ang II infusion led to severe premature mortality in Col3a1+/- compared to wild type. This fragility was characterized by the development of rupture/dissection in the ascending aorta. These lesions could be caused by the elevation of blood pressure and/or the activation of Ang II signaling pathways. We showed that treatment with a beta-blocker (propranolol) and an arterial vasodilator (hydralazine) reduced the mortality induced by Ang II in Col3a1+/- mice. These results suggest the beneficial effect of adding a preventive treatment inhibitor of Ang II to the beta-blocker treatment recommended in human pathology.Meanwhile, given that a majority of human vEDS cases is caused by missense mutations in the COL3A1 gene, we established a knock-in mouse model bearing a point mutation (Gly183Ser) found in vEDS patients. The preliminary characterization of this model showed that Col3a1+/G183S mice die spontaneously as early as 4 weeks of age from a dissection or rupture of the ascending aorta. However, these mice do not showed any changes of their hemodynamic parameters or aortic diameter. Furthermore, about 20 % of mouse Col3a1+/G183S display wounds in the back and legs. This new mouse model is currently the only that mimic more closely the human disease and could therefore be used to test different therapeutic strategies.
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Agtr1, Wnk1, Cul3 : nouveaux acteurs dans la signalisation et la régulation de la pression artérielleLatreche, Sabrina 28 November 2014 (has links)
L’hypertension artérielle est une maladie induite par de multiples facteurs génétiques et environnementaux. De nombreuses pathologies y sont associées. A travers ce travail, j’ai abordé trois aspects de la régulation de la pression artérielle in vivo et in vitro. Dans une première partie, j’ai étudié le rôle de l’activation du récepteur AT1 de l’angiotensine II dans le développement de la fibrose, indépendamment de l’hypertension artérielle. Un modèle animal exprimant un récepteur constitutivement actif et des modèles cellulaires (MEF, HEK293, H295) exprimant le récepteur constitutivement actif de façon inductible ont été utilisés. Contrairement aux souris sur fond mixte, les souris mutées sur fond pur C57Bl6 ne développent pas de fibrose cardiaque et rénale et ont une hypertension modérée, qui est difficile à réduire par les anti-hypertenseurs. De plus, les cellules fibroblastiques MEF ne sont pas un bon modèle pour étudier la fibrose induite par l’angiotensine II. Seule l’ostéopontine est un marqueur induit par l’expression du récepteur AT1 contitutivement actif. Ces différents modèles, étudiés extensivement, ne sont donc pas adaptés pour répondre aux questions posées. Dans la seconde partie de ma thèse, un travail collaboratif a permis de mettre en évidence le rôle majeur de Wnk1 au cours de l’hypertension et du remodelage cardiovasculaire induits par une infusion chronique d’angiotensine II. En effet, les souris Wnk1+/- (haplo-insuffisantes pour le gène Wnk1) présentent une résistance transitoire à l’hypertension induite par l’angiotensine II, particulièrement au cours de la première semaine d’infusion. Cette résistance est associée à une altération du remodelage hypertrophique cardiovasculaire mais la fonction rénale et la sécrétion d’aldostérone sont préservées. Au niveau mécanistique, nos résultats ont identifié Wnk1 comme un activateur important de la phosphorylation de Mypt1, un marqueur connu de l’activité de la voie Rho-kinase. Les aortes de souris Wnk1+/- présentent une diminution transitoire de la phosphorylation de Mypt1 après une semaine d’infusion d’angiotensine II. De façon importante, nous montrons aussi que l’infusion chronique d’angiotensine II induit une activation de l’expression du gène Wnk1 au niveau aortique, et la surexpression de Wnk1 in vitro active de façon importante et reproductible la phosphorylation de Mypt1, indépendamment de l’activation de Spak (substrat bien caractérisé de Wnk1). En conclusion, ce travail a permis d’identifier Wnk1 comme un nouveau gène cible de l’angiotensine II au niveau vasculaire et a révélé un nouveau mécanisme mis en jeu au cours de l’hypertension et du remodelage cardiovasculaire qui lui est associé. Cette étude fait l’objet d’un article que je signe en premier auteur et qui est actuellement soumis pour publication. Dans une dernière partie, j’ai étudié le rôle de la culline3 dans la régulationde la voie RhoKinase. Les mutations du gène Cul3 ont très récemment été identifiées comme responsables du syndrome de Gordon. Ce gène code une protéine d’échafaudage d’un complexe d’ubiquitination important et ubiquitaire (CRL3) conduisant à la dégradation protéique. La voie des Rho-kinases joue un rôle majeur dans le tonus vasculaire et sa régulation par les agents relaxants ou constricteurs. Des travauxrécents suggèrent que la dégradation de RhoA implique le complexe culline3-ring-ligase (CRL3). Nous avons voulu établir les liens structuraux et fonctionnels entre ce complexe d’ubiquitination et la voie Rho-kinase dans des modèles cellulaires, pour ainsi expliquer tout ou partie du mécanisme moléculaire conduisant des mutations constitutionnelles du gène Cul3 à produire une hypertension artérielle. Les interactions protéiques entre deux adaptateurs différents et la culline3 nous ont permis de montrer que la culline3 mutée entraine une modification d’affinité spécifique selon ses partenaires. Les interactions entre RhoA et le CRL3 n’ont pas pu être démontré. (...) / Hypertension is a disease due to multiple genetic and environmental factors. Many cardiovascular diseases are associated. During my PhD thesis, I addressed three aspects of the regulation of blood pressure in vivo and in vitro. In the first part, I studied the role of angiotensin II AT1 receptor activation in the development of fibrosis, independently of hypertension. I used animal and cellular models (MEF, HEK293, H295) expressing a constitutively active receptor. The results show that the mutant mice did not develop cardiac or renal fibrosis in a pure C57Bl6 strain. Furthermore, their moderate hypertension has not been normalized with two antihypertensives. The pure C57BL6 genetic background seems to be the cause of this moderate phenotype. Furthermore, MEF cells are not a good model to study fibrosis induced by angiotensin II. Only osteopontin is a marker induced by expression of the mutant receptor. As the mouse models and despite of their originality, these cellular models appear to be inappropriate to study AngII-dependent fibrosis. These limitations together with the weakness of the AT1 mutant phenotype lead to us to stop this project. In the second part of my thesis, a collaborative study allows us to show that Wnk1-haploinsufficiency in mice is responsible for a strong and transitory resistance to angiotensin II (AngII)-induced hypertension associated with a significant reduction of cardiovascular remodeling and a preservation of renal function and aldosterone release. Mechanistically, we unravel a critical role for Wnk1 in the activation of the phosphorylation of Mypt1, a known marker of the Rho-kinase pathway activity. Wnk1-haploinsufficient mice display a significant and transitory decrease of AngII-induced phospho- Mypt1 in the aorta, concomitant to the hypertension-resistance. Importantly, we further evidence that, in the vasculature, AngII chronic infusion induces a significant upregulation in Wnk1 gene expression which causes in vitro a significant increase in Mypt1 phosphorylation independently of spak activation. Our results provide new insight into the downstream vascular signaling pathway of AngII and unravel a previously unsuspected mechanism linking Wnk1 to hypertension and vascular remodeling. In the last part, I studied the role of vascular Cullin3 (Cul3) in the development of hypertension. Mutations in this gene have been recently identified as responsible for a familial hypertension with hyperkalaemia, FHHt. The Cul3 gene encodes an important and ubiquitous ubiquitin scaffold protein participating to a protein degradation complex (CRL3). The Rho-kinase pathway plays a major role in vascular tone and its regulation by relaxing or vasoconstricting agents. Recent studies suggest that the degradation of RhoA involves the CRL3 complex. I started to analyze the structural and functional links between this ubiquitination complex and the Rho kinase pathway in cellular models to explain all or part of the molecular mechanisms leading constitutional mutations of Cul3 gene to produce hypertension. I have shown that the Cul3Δ9 mutant presents an increased neddylation compared to the wild form and mofications of its affinity for some adaptors. However, in this preliminary work, its interactions with and its role in the degradation of RhoA have not been demonstrated yet. This PhD thesis has helped to address several aspects of the pathophysiology of the vessels and the role of angiotensin II in these regulations using modern tools, original mouse and cell line models. This has particularly highlighted a new target of angiotensin II and a new WNK1 vascular role.
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Evaluating Angiotensin II Type 1 Receptor Changes in Post- Renal Insufficiency and in Left Anterior Descending Artery Ligation Animal Models Using [11C]Methyl-CandesartanMackasey, Kumiko January 2012 (has links)
Non invasive in vivo imaging will lead to better understanding of Angiotensin II Type 1 Receptor’s (AT1R) role in disease progression and may guide therapy in cardiovascular patients. Two models were used in this project: 5/6 nephrectomy and transient left anterior descending (LAD) ligation. Rats were scanned with [13N]ammonia and [11C]methyl-candesartan, both of which are Positron Emission Tomography (PET) tracers, at 8 weeks (nephrectomy) and 2 weeks (LAD ligation) after surgery. Western blot analysis was used to corroborate PET data. Nephrectomy: Renal AT1R image analysis displayed a 40% decrease in kidney AT1R in nephrectomized animals compared to sham (p<0.05) which was confirmed with Western blot and biodistribution. LAD ligation: Left Ventricle AT1R Western blot analysis exhibited a 60% increase in 20min ligation (p<0.05) with maintained myocardial blood flow. In conclusion, changes in renal AT1R were successfully imaged using [11C]methyl-candesartan in nephrectomized animals, and 20min LAD ligation/reperfusion is an appropriate model to image an increase in cardiac AT1R following ischemic injury.
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Characterization of [18F]FPyKYNE-Losartan as a Novel PET Tracer for Imaging AT1 ReceptorsHachem, Maryam January 2015 (has links)
The Angiotensin II Type I Receptor (AT1R) is the main receptor that produces most of the physiological actions of the Renin Angiotensin System (RAS). Alterations of AT1R expression in renal and cardiovascular diseases make this receptor an attractive target for developing an imaging agent to monitor its expression in disease states. [18F]FPyKYNE-Losartan has been developed as a derivative of the clinically used AT1R blocker Losartan. The aim of this work was to characterize this tracer and evaluate its potential as an imaging agent for AT1Rs, thereby, progressing towards human imaging of the AT1R. MicroPET imaging in rats and PET imaging in pigs displayed specific AT1R binding, high kidney-to-blood and image contrast, and slow clearance from kidneys. [18F]FPyKYNE-Losartan was shown to have 2 types of labeled metabolites in rat plasma and kidneys: hydrophilic and hydrophobic, whereas, only hydrophilic metabolite(s) in pig plasma. Plasma protein binding of [18F]FPyKYNE-Losartan was determined, by an in vitro ultrafiltration method, to be 97% which is very similar to that of Losartan (98%). FPyKYNE-Losartan displayed full antagonism of Ang II pressor effect in rats in vivo, with an ED50 of 25.5 mg/Kg and 4-times (25%) less potency than Losartan. In vitro binding studies confirmed the binding selectivity of [18F]FPyKYNE-Losartan. Bmax and Kd parameters were determined to be 348 ± 112 fmol/mm2 and 49.4 nM, respectively. Rat dosimetry studies exhibited that the sex averaged effective doses of [18F]FPyKYNE-Losartan according to ICRP 60 and 103 protocols are 2.97E-02 (mSv/MBq) and 3.06E-02 (mSv/MBq), respectively, which are within an acceptable range compared with other F-18 labeled tracers and within the safety limits of the FDA. In conclusion, [18F]FPyKYNE-Losartan has an excellent potential for translation towards human imaging to monitor AT1R expression and guide therapy.
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Propriedades conformacionais de hormônios peptídicos ligantes de receptores acoplados a proteínas G em solução e em presença de membranas modelo / Conformational properties of peptide hormones binding to G protein coupled receptors in solution and in the presence of model membranesNélida Simona Marín Huachaca 31 May 2007 (has links)
Os hormônios peptídicos Angiotensina II (Ang II) e bradicinina (BK) ativam transdução de sinal através da ligação a Receptores Acoplados a Proteínas G (GPCR). Este trabalho propõe o estudo de propriedades conformacionais, através de espectroscopia de fluorescência da Ang II e BK e de seus análogos contendo o marcador de spin ácido 2,2,6,6-tetrametilpiperidina-1-oxil-4-amino-4-carboxílico, TOAC (TOAC1-Ang II, TOAC3-Ang II, TOAC0-BK, TOAC3-BK). Os peptídeos foram estudados em solução (efeito do pH) e também na presença de membranas modelo, micelas e bicamadas, formadas por anfifílicos zwitteriônicos ou aniônicos. Foi monitorada a fluorescência intrínseca dos resíduos aromáticos (Tyr4 na Ang II e Phe5 e Phe8 na BK). O efeito de supressão da fluorescência pelo TOAC foi utilizado para obter informação sobre a proximidade desse resíduo aos grupos fluoróforos. Foi observada dependência da fluorescência com o pH e regiões de pKs dos grupamentos ionizáveis. Os espectros evidenciaram também a interação peptídeo-membrana modelo. Interações mais fortes ocorreram entre os peptídeos e membranas com carga superficial negativa, evidenciando a importância de interações eletrostáticas para a ligação. Porém, interações hidrofóbicas também estão envolvidas, como verificado pela ligação dos peptídeos a membranas zwitteriônicas. Estudos com variação de pH também mostraram o papel dessa variável na interação peptídeo-membrana e a alteração de pKs de resíduos ionizáveis decorrentes da interação. A titulação com concentrações crescentes de membranas permitiu o cálculo das constantes de associação. A ligação a membranas é função da conformação dos peptídeos. Em particular, a presença de TOAC na posição 3 parece diminuir a afinidade desses análogos por membranas. Supressão de fluorescência foi efetuada empregando três diferentes abordagens: 1) supressão pela molécula aquossolúvel acrilamida, 2) supressão da fluorescência de fosfolipídeos contendo o fluoróforo NBD em diferentes posições da molécula pelos análogos marcados com TOAC, 3) supressão da fluorescência dos peptídeos por ésteres metílicos do ácido esteárico contendo o grupamento nitróxido em diferentes posições da cadeia. Esses estudos permitiram determinar a localização dos peptídeos na interface água-membrana. Medidas de anisotropia de fluorescência também evidenciaram a ligação dos peptídeos a membranas, revelando maior imobilidade dos mesmos nessas condições. Foi ainda estudado um peptídeo que contém os resíduos 92-100 (fEL1) do receptor AT1 de Ang II humano. Predições baseadas na estrutura cristalina da rodopsina estimam que essa seqüência localiza-se na primeira alça extra-celular do receptor. A seqüência contém a Tyr92, considerada um resíduo importante para a ligação hormônio-receptor. Resultados preliminares sugeriram que fEL1 interage com Ang II e TOAC1-Ang II, mas não com TOAC3-Ang II. Este último resultado provavelmente deve-se à dobra causada por TOAC que restringe a liberdade de movimento do esqueleto peptídico. Essa característica provavelmente determina a falta de atividade biológica de TOAC3-Ang II e TOAC3-BK, enquanto os análogos marcados no N-terminal retém atividade parcial (Nakaie et al., 2002). Tem sido proposto que peptídeos ligantes de GPCR se ligariam à bicamada lipídica e atingiriam seu receptor através da difusão pela bicamada. Em solução aquosa essas moléculas são flexíveis, existindo um equilíbrio dinâmico entre várias conformações. A ligação à bicamada lipídica estabilizaria uma ou algumas conformações, entre elas aquela que o ligante adota ao ligar-se ao receptor. O presente estudo contribui para a compreensão, a nível molecular, do processo de interação entre os hormônios peptídicos e membranas lipídicas. / The peptide hormones Angiotensin II (Ang II) and bradykinin (BK) trigger signal transduction by binding to G Protein Coupled Receptors (GPCR). This work proposes the study of conformational properties of Ang II and BK, as well as their analogues containing the spin label 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid, TOAC (TOAC1-Ang II, TOAC3-Ang II, TOAC0-BK, TOAC3-BK) making use of fluorescence spectroscopy. Studies were performed in solution (effect of pH) and also of the interaction between the peptides and model membranes - micelles and bilayers - formed by amphiphiles, either zwitterionic or negatively charged. The intrinsic fluorescence of aromatic residues (Tyr4 in Ang II and Phe5 and Phe8 in BK) was monitored. Fluorescence quenching by the TOAC-carrying analogues provided information about the proximity between TOAC and the fluorophores. The fluorescence was pH-dependent and evinced regions corresponding to pKs of ionizable groups. Peptide-model membrane interactions were also examined. Stronger interactions were detected between the peptides and membranes formed by negatively charged amphiphiles, pointing to the importance of electrostatic interactions for binding. However, hydrophobic interactions were also involved, as suggested by the fact that the peptides also bound to zwitterionic membranes. Variable pH studies showed the effect of this parameter on peptide-membrane interaction. The peptide-membrane interactions promoted changes in the pKs of ionizable residues. Titrations with increasing membrane concentrations allowed calculation of binding constants. Binding to membranes is a function of peptide conformation. In particular, TOAC at position 3 seems to decrease the affinity of both Ang II and BK for membranes. Fluorescence quenching studies made use of three different approaches: 1) quenching by water soluble acrylamide, 2) quenching of the fluorescence of phospholipids carrying the fluorescent group NBD in different positions by the spin-labeled TOAC-bearing analogues, 3) quenching of the peptides fluorescence by methyl esters of stearic acid containing the nitroxide moiety at different positions in the acyl chain. These studies indicated that the peptides are located at the water-membrane interface. Measurements of fluorescence anisotropy also evinced binding of the peptides to the membranes and showed that the peptides undergo more restricted motion under these conditions. A peptide containing residues 92-100 (fEL1) of the Ang II AT1 human receptor was also studied. Predictions based on rhodopsin crystalline structure estimate that this sequence is located in the receptor´s first extra-cellular loop. Preliminary results suggest that fEL1 interacts with Ang II and TOAC1-Ang II, but not TOAC3-Ang II. This latter result is probably due to the TOAC-induced bend that restricts the freedom of motion of the peptide backbone. This feature is probably the cause of lack of biological activity of TOAC3-Ang II and TOAC3-BK, while the N-terminally labeled analogues retain partial activity (Nakaie et al., 2002). GPCR-binding peptides have been proposed to bind to the lipid bilayer and reach their receptors by diffusion in the bilayer. In aqueous solution these molecules exist as a dynamic equilibrium between various flexible conformations, among them, the receptor-bound conformation. The present study provides contributions for the understanding, at the molecular level, of the interaction between the peptide hormones and lipid membranes.
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Investigating the Mechanisms involved in Traffic-Generated Air Pollution: Mediated Disruption of the Blood-Brain Barrier in a Wild Type Mouse Model using a Pharmaceutical Intervention ApproachSuwannasual, Usa 08 1900 (has links)
This study investigated whether oxLDL and/or angiotensin (Ang) II signaling pathways mediate traffic-generated air pollution- exposure induced alterations in blood-brain barrier (BBB) integrity and permeability in a healthy wild type (C57Bl/6) mouse model; additionally, whether these outcomes are exacerbated by a high fat-diet investigated. An environmentally relevant concentration of a mixture of vehicle engine exhaust (MVE) was used. To investigate the hypotheses, 12 wk old male C57Bl/6 mice on either a high fat (HF) or low fat (LF) diet were randomly assigned to inhalational exposure of either filtered-air (FA) or 30 µg PM/m3 diesel exhaust + 70 µg PM/m3 gasoline exhaust (MVE) for 6 hr/day for 30 days. Additionally, we examined mechanisms involved in MVE-mediated alterations BBB integrity using a novel BBB co-culture in vitro model, consisting of mouse primary cerebral vascular endothelial cells on an apical transwell and astrocytes in the basal compartment, which was treated with plasma from the mice on our exposure study. Our in vivo exposure study results showed that MVE inhalation resulted in increased circulating plasma oxLDL and Ang II, compared to FA controls. Additionally, we observed increased cerebral microvascular expression of oxLDL receptors, LOX-1 and CD-36, and Ang II receptor subtype 1 (AT1) in MVE-exposed C57Bl/6 mice, which was further exacerbated with consumption of an HF diet. Increased signaling of both Ang II and oxLDL was associated with decreased BBB integrity, as evidenced by the concurrent reduction in expression of tight junction (TJ) protein claudin-5 and increased permeability of sodium fluorescein (Na-F) from the blood into the cerebral parenchyma. Our results suggest that possible mechanisms involved in oxLDL and/or Ang II-mediated alterations in BBB integrity include oxidative stress and upregulated expression and activity of matrix metalloproteinase (MMP)-9, which is associated with degradation of TJ proteins in the BBB. Our in vitro BBB co-culture results confirm our in vivo findings, as we observe increased BBB permeability (TEER) and decreased integrity (decreased expression of TJ proteins) in the endothelial (apical) layer when treated with plasma from MVE-exposed mice, which was further exacerbated when treated with plasma from MVE-exposed mice on an HF diet. Pre-treatment of the endothelial cells with the AT1 receptor antagonist, Losartan, prior to applying plasma, resulted in attenuation of the alterations observed in endothelial integrity in the BBB co-culture treated with plasma from either MVE+LF or MVE+HF animals. These results suggest Ang II – AT1 signaling mediate, at least in part, the alterations in the BBB integrity observed after exposure to MVE. Moreover, we observed that treatment of the endothelial (apical) layer with plasma from MVE-exposed animals resulted in increased production of inflammatory mediators interleukin-6 (IL-6) and transforming growth factor-β in the astrocyte media (basal compartment). Additionally, these same astrocytes also displayed increased production of angiotensin-converting enzyme (ACE) and also AT1 receptor mRNA expression, while showing decreased expression of the aryl hydrocarbon receptor (AhR) and glutathione peroxidase (GPx). Collectively, these results suggest that exposure to the ubiquitous environmental air pollutant, vehicle engine emissions, results in increased oxLDL and Ang II signaling in the cerebral microvasculature, which is associated with decreased vessel integrity and increased oxidative stress and inflammatory signaling in the CNS. The observed detrimental outcomes are even further exacerbated when coupled with the consumption of an HF diet.
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