Uma elastase-2 foi recentemente descrita como a principal enzima formadora de angiotensina (Ang) II no perfusato do leito arterial mesentérico (LAM) isolado de rato. Investigamos a interação dessa elastase-2 do perfusato do LAM isolado de rato (E-2LAMR) com alguns substratos e inibidores de elastases-2 e de quimases formadoras de Ang II. Os precursores de Ang II, [Pro11-D-Ala12]-Ang I e substrato tetradecapeptídeo de renina (TDP), foram convertidos em Ang II pela E-2LAMR com eficiências catalíticas de 8,6 min-1mM-1 e 5,1 min-1mM-1, respectivamente, enquanto os substratos cromogênicos N-succinil-Ala-Ala-Pro-Leu-p-nitroanilida e N-succinil-Ala-Ala-Pro-Phe-p-nitroanilida foram hidrolisados pela enzima com eficiências catalíticas de 10,6 min-1mM-1 e 7,6 min-1mM-1, respectivamente. O inibidor peptídico CH 5450 inibiu as atividades da E-2LAMR sobre os substratos Ang I (IC50=49 mM) e N-succinil-Ala-Ala-Pro-Phe-p-nitroanilida (IC50=4,8 mM), enquanto Acetil-Ala-Ala-Pro-Leu-clorometilcetona (Ac-AAPL-CK), um efetivo inibidor de elastases-2 pancreáticas, bloqueou eficientemente a atividade formadora de Ang II da E-2LAMR (IC50=4,5 mM). Em conjunto, esses dados confirmaram e estenderam as similaridades enzimológicas entre elastases-2 pancreáticas e a E-2LAMR. Além disso, a interação até então desconhecida da E-2LAMR com [Pro11-D-Ala12]-Ang I e CH 5450, ambos considerados como reagentes seletivos para quimases, sugere que as evidências para a formação de Ang II in vivo por quimases podem ter sido superestimadas em investigações prévias sobre vias geradoras de Ang II. Experimentos realizados com o LAM isolado de rato analisando o efeito vasoconstritor de Ang II, Ang I, TDP e [Pro11-D-Ala12]-Ang I mostraram a existência de uma via geradora de Ang II independente da ECA, a qual é sensível à quimostatina e Ac-AAPL-CK. Entre os possíveis candidatos para essa via alternativa à ECA aparece a E-2LAMR, uma enzima que não é inibida por captopril e que é sensível à quimostatina e Ac-AAPL-CK. Embora quimases, que também são sensíveis à quimostatina, também possam ser candidatos a essa via independente da ECA, com base nos fatos de que a quimase I de rato tem uma atividade predominante de degradação da Ang II e que não existem relatos na literatura de que quimases sejam sensíveis ao inibidor Ac-AAPL-CK, esses dados em conjunto sugerem um possível papel para a E-2LAMR, mas não quimases, como uma via alternativa à ECA para a geração de Ang II no LAM isolado de rato. A clonagem e o seqüenciamento do cDNA para a E-2LAMR foram alcançados pela combinação de transcrição reversa e reação da polimerase em cadeia. A seqüência do cDNA mostrou-se idêntica à do cDNA para a elastase-2 pancreática de rato; o cDNA tem 909 nucleotídeos mais uma cauda poli (A) e codifica uma preproenzima de 271 amino ácidos. A análise dos supostos amino ácidos no sítio de ligação da Ang I revelou características que poderiam explicar a atividade do tipo carboxidipeptidase necessária para a eficiente conversão de Ang I em Ang II. Adicionalmente, a seqüência revela características estruturais que poderiam contribuir para a ausência de atividade dessa enzima sobre a Ang II. O RNAm para a E-2LAMR foi expresso em LAM, pâncreas, pulmão, coração, rim, fígado e baço, mas não em aorta de rato. Células endoteliais do LAM em cultura expressaram o RNAm para a E-2LAMR e sintetizaram a enzima. A localização intravascular dessa enzima e sua habilidade em formar Ang II e não clivar esse peptídeo indicam que ela poderia ter uma participação significativa como um agente formador de Ang II no sistema cardiovascular. Esses resultados também podem indicar que a E-2LAMR é expressa em vasos de resistência, mas não em vasos de condutância. / An elastase-2 has been recently described as the major angiotensin (Ang) II-forming enzyme of the rat mesenteric arterial bed (MAB) perfusate. Here, we have investigated the interaction of affinity-purified rat MAB elastase-2 with some substrates and inhibitors of both pancreatic elastases-2 and Ang II-forming chymases. The Ang II precursors [Pro11-D-Ala12]-Ang I and renin substrate tetradecaptide (TDP) were converted into Ang II by the rat MAB elastase-2 with catalytic efficiencies of 8.6 min-1mM-1 and 5.1 min-1mM-1, respectively, and the chromogenic substrates N-succinyl-Ala-Ala-Pro-Leu-p-nitroanilide and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide were hydrolyzed by the enzyme with catalytic efficiencies of 10.6 min-1mM-1 and 7.6 min-1mM-1, respectively. The noncleavable peptide inhibitor CH 5450 inhibited the rat MAB elastase-2 activities toward the substrates Ang I (IC50=49 mM) and N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (IC50=4.8 mM), whereas N-acetyl-Ala-Ala-Pro-Leu-chloromethylketone (Ac-AAPL-CK), an effective active site-directed inhibitor of pancreatic elastases-2, efficiently blocked the Ang II-generating activity of the rat MAB enzyme (IC50=4.5 mM). Altogether, these data confirm and extend the enzymological similarities between pancreatic elastases-2 and their rat MAB counterpart. Moreover, the thus far unrealized interaction of rat MAB elastase-2 with [Pro11-D-Ala12]-Ang I and CH 5450, both regarded as selective for chymases, suggests that evidence for the in vivo formation of Ang II by chymases may have been overestimated in previous investigations of Ang II-forming pathways. Experiments carried out in the isolated rat MAB analyzing the vasoconstrictor effect of Ang II, Ang I, TDP, and [Pro11-D-Ala12]-Ang I showed the existence of an ACE-independent pathway for Ang II generation, which is sensitive to chymostatin and Ac-AAPL-CK. Among the possible candidates for this ACE-independent pathway is rat MAB elastase-2, an enzyme that is not inhibited by captopril, and that is sensitive to chymostatin and Ac-AAPL-CK. Although chymases, which are also chymostatin-sensitive enzymes, might also be other possible candidates for this ACE-independent pathway, based on the fact that rat chymase I has a predominant Ang II-degrading activity, and because there are no reports in the literature that chymases are sensitive to Ac-AAPL-CK, altogether these data suggest a possible role for rat MAB elastase-2, but not chymases, as an alternative pathway to ACE for Ang II generation in the isolated rat MAB. The cloning and sequencing of the cDNA for the rat MAB elastase-2 was accomplished by reverse transcription-polymerase chain reaction. The sequence of this cDNA was found identical to the sequence of the rat pancreatic elastase-2; the cDNA is 909 nucleotides in length plus a poly (A) tail and encodes a preproenzyme of 271 amino acids. Analysis of the putative amino acids in the extended Ang I binding site of the rat MAB elastase-2 reveals features that could explain the dipeptidyl carboxypeptidase-like activity required for efficient Ang I to Ang II conversion. Additionally, the sequence reveals structural features that could contribute to the lack of activity of this enzyme toward Ang II. Rat MAB elastase-2 mRNA was expressed in rat mesenteric arteries, pancreas, lung, heart, kidney, liver, and spleen but not in aorta. Cultured mesenteric endothelial cells expressed the mRNA for rat MAB elastase-2 and synthesized the enzyme itself. The intravascular localization of this enzyme and its ability to generate Ang II and not destroy this peptide indicate that it might play a role in the rat cardiovascular system as an Ang II-forming agent. These results may also indicate that rat MAB elastase-2 is expressed in resistance vessels but not in conduit vessels.

Le Système Rénine Angiotensine tient une place prépondérante au sein de la circulation cérébrale. Les Antagonistes des Récepteurs AT1 à l'Angiotensine II (ARAII) ont prouvé leur efficacité dans la prévention de l'Accident Vasculaire Cérébral (AVC), indépendamment de leur effet anti-hypertenseur. Plusieurs mécanismes pourraient être impliqués dans cette cérébroprotection. D'une part, en bloquant les récepteurs AT1, les ARAII favorisent la stimulation des récepteurs AT2 à l'angiotensine II. Le caractère bénéfique lié à la stimulation des récepteurs AT2 s'oppose au caractère délétère lié à la stimulation des récepteurs AT1. Nous avons montré que cet équilibre AT1 - AT2 est modifié au niveau cérébrovasculaire suite à un régime hypersodé. En effet, la vasodilatation des artérioles cérébrales médiée par les récepteurs AT2 est abolie, ce qui pourrait constituer un élément délétère lors de la survenue d'un évènement ischémique. D'autre part, certains ARAII présentent une affinité pour les récepteurs PPAR-gamma. Cette activité, démontrée comme protectrice à différents niveaux vasculaires, pourrait également être bénéfique pour la circulation cérébrale. Nous avons en particulier montré que l'activation PPAR-gamma améliore les effets des ARAII au niveau de la circulation cérébrale (diamètre artériolaire, réactivité à l'angiotensine II). Les mécanismes en jeu semblent impliquer des modifications de la fonction des récepteurs AT1-AT2, indépendamment de leur expression. La stimulation des récepteurs AT2 et l'activation PPAR-gamma constituent donc deux propriétés particulières des ARAII. Ces propriétés pourraient participer au caractère cérébroprotecteur des ARAII, au-delà du seul blocage des récepteurs AT1. Le développement de molécules duales regroupant les activités antagoniste AT1 - agoniste PPAR-gamma pourrait constituer un avenir thérapeutique intéressant dans le traitement de l'hypertension en apportant une protection cérébrovasculaire supérieure aux traitements actuels / The Renin Angiotensin System plays a major role in cerebral circulation. AT1 receptor blockers (ARBs) afford protection against cerebrovascular complications that go beyond that to be expected from their blood pressure lowering action. Several mechanisms could explain such beneficial effects. Firstly, by blocking AT1 receptors, ARBs promote AT2 receptor stimulation by angiotensin II. The beneficial effect related to stimulation of AT2 receptors (vasodilation) counterbalances the deleterious actions of AT1 receptors stimulation. Changes in this ratio may then alter cerebral circulation. We demonstrated that the AT1- AT2 ratio is modified at the cerebrovascular level during high salt intake, which is a risk factor for stroke. The AT2-mediated vasodilation of pial arterioles is abolished. Secondly, some ARBs act as partial agonists of PPAR-gamma. Such an activity, which has been demonstrated to protect extracerebral vessels, could also be beneficial for cerebral circulation. Our results showed that PPAR-gamma activation improves ARB effects on cerebral circulation (arteriolar diameter, angiotensin II reactivity). The underlying mechanisms could imply functional regulation of AT1-AT2 receptors without any change in expression status. AT2 receptor stimulation and PPAR-gamma activity are two special properties of ARBs. These properties could contribute to the cerebroprotection induced by ARBs, beyond the AT1-receptor blockade. Development of new molecules with AT1-receptor blockade and PPAR-gamma activity could take part into the future therapeutic management of hypertension, providing a better cerebrovascular protection

Chez les patients présentant un syndrome métabolique, le développement des comorbidités cardiaques et rénales associées au diabète de type 2 sont liées à des altérations au niveau vasculaire. Afin d’évaluer l’effet protecteur rénal et cardiaque de nouvelles molécules, le modèle de rat Zucker fatty/Spontaneously hypertensive heart failure F1 hybrid (ZSF1) semblait approprié. Cependant, son développement lent et les impacts rénaux et cardiaques modestes en limitaient son utilisation. Notre but fut d’exacerber ces altérations par deux méthodes. Nous avons d’abord effectué une néphrectomie unilatérale chez le rat ZSF1 et évalué l’évolution des fonctions cardiaque et rénale en fonction de l’âge. Seule une exacerbation de la dysfonction rénale a été mise en évidence. Néanmoins nous avons pu démontrer l’effet protecteur rénal de l’inhibiteur de l’enzyme de conversion de l’angiotensine lisinopril ainsi que d’un composé antioxydant, le bardoxolone. Notre seconde stratégie a consisté à infuser de l’angiotensine II (AngII) à des rats ZSF1. L’hypertension déjà existante dans ce modèle a été fortement accrue, et le niveau d’aldostérone circulante a été significativement augmenté. Dans ce contexte, les fonctions cardiaque et rénale ont été dégradées de manière importante. Enfin nous avons montré que dans ce modèle, un inhibiteur de l’aldostérone synthase induisait une meilleure protection rénale que l’antagoniste des récepteurs à l’aldostérone éplérénone. Nous avons donc mis en évidence que le rat ZSF1-AngII est un modèle de dysfonction cardio-rénale permettant d’évaluer l’effet protecteur de composés sur les fonctions rénale et cardiaque, dans un contexte de syndrome métabolique / In the context of metabolic syndrome, development of Type 2 Diabetes is associated with (and influenced by) cardiac and renal comorbidities linked to micro- and macro-vasculature alterations. To assess the efficacy of new compounds on targeted organs in the context of metabolic syndrome, the Zucker fatty/Spontaneously hypertensive heart failure F1 hybrid (ZSF1) rat model could be suitable assuming cardiorenal alterations would develop in a short timeframe. Actually, the ZSF1 rat model recapitulates features of human metabolic syndrome, but develops relatively late (1year-time) and moderate cardiac and renal dysfunctions. The aim of our work was to exacerbate cardiorenal impairments in terms of onset and extent. Two options were explored. On one hand, unilateral nephrectomy was performed in ZSF1 rats, and cardiac and renal functions were longitudinally assessed. This surgical insult only significantly deteriorated renal function, which was prevented by standard of care, lisinopril and new renal protective agent, bardoxolone. On the other hand, subcutaneous infusion of angiotensin II (AngII) was used in the aim to induce hemodynamic and hormonal stress and thus to enhance cardiorenal impairments. AngII-infused ZSF1 rats displayed significant hypertension and increased levels of circulating aldosterone. Moreover, renal and cardiac functions dropped, concomitantly. We showed in this model that an aldosterone synthase inhibitor induced overall better renal protection than the mineralocorticoid receptor antagonist eplerenone. Our data showed that ZSF1-AngII rat is a suitable model to evaluate the cardio and renoprotective effects of drugs in the context of metabolic syndrome

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

Les accidents vasculaires cérébraux sont la deuxième cause de mortalité dans le monde, le développement de nouvelles thérapeutiques est donc urgent. Deux acteurs jouent un rôle majeur dans la régulation de la circulation cérébrale : le monoxyde d’azote (NO) et le système rénine angiotensine (SRA). Le chapitre 1 de ce manuscrit s’intéresse tout d’abord au NO, son rôle physiologique et ses voies de signalisation. Nous présentons les donneurs de NO disponibles sur le marché ainsi que ceux en développement. La bucillamine dinitrosée, développée dans notre laboratoire, fait l’objet d’une évaluation in vitro et in vivo. La deuxième partie de l’introduction s’intéresse au SRA, en rappelant son rôle prépondérant dans le maintien de la pression artérielle et de la régulation cérébrovasculaire. Nous présentons les récepteurs de l’angiotensine II (AngII), AT1 et AT2, responsables respectivement d’une vasoconstriction et d’une vasodilatation des artères cérébrales. Enfin la dernière partie présente la régulation des récepteurs de l’AngII par le NO, en particulier via la S-nitrosation du récepteur, la liaison d’un groupement NO sur une fonction thiol d’un résidu cystéine. Nous présentons les travaux de Leclerc qui montrent que l’exposition de cellules surexprimant le récepteur AT1 à un donneur de NO entraine une diminution d’affinité de l’AngII pour AT1 (Leclerc et al., 2006). Le chapitre 2 est consacré aux études expérimentales. L’objectif des travaux présentés dans cette thèse est d’étudier l’importance de la S-nitrosation des récepteurs de l’AngII au niveau cérébrovasculaire. Tout d’abord nous abordons la problématique actuelle concernant l’aspécificité des anticorps anti-AT1. Nous montrons que le nouvel anticorps monoclonal anti-AT1, censé être plus spécifique, ne reconnaît pas AT1 en western blot et en immunofluorescence, rendant donc son utilisation impossible. Nous faisons ensuite la démonstration pharmacologique des effets de la S-nitrosation sur les récepteurs de l’AngII. Nous montrons que l’exposition à un donneur de NO (S-nitrosoglutathion ou nitroprussiate de sodium) abolit spécifiquement la vasoconstriction médiée par AT1 comparé à d’autres vasoconstricteurs partageant ou non sa voie de signalisation. De plus cette exposition abolit aussi le tonus myogénique AT1-dépendant indépendant de la stimulation par l’AngII suggérant que l’altération survient sur le récepteur lui-même. Nous montrons par ailleurs que cet effet (i) ne dépend pas du NO endogène, (ii) se fait par une S-nitrosation plutôt que par la voie de la GMPc/GCs. Enfin nous étudions l’internalisation du récepteur par cytométrie en flux, sur un modèle hétérologue d’expression AT1. Nos résultats montrent que le GSNO ne modifie pas la localisation d’AT1 à la membrane et n’empêche pas son internalisation, indiquant que la voie ß-arrestine n’est pas impactée par la nitrosation.L’ensemble de ces résultats permet d’établir que la S-nitrosation d’AT1 constitue une cible thérapeutique potentiellement intéressante dans les AVC, où l’augmentation de la vasoconstriction médiée par AT1 est délétère / Stroke is the second leading cause of death worldwide, the development of new therapeutics is thus urgent. Two actors play a major role in the regulation of cerebral circulation: nitric oxide (NO) and the renin-angiotensin system (RAS). The first chapter of this manuscript focuses on NO, its role and its signaling pathways. We present the available NO donors as well as those in development. Dinitrosobucillamine, a new NO donor developed in our team, is evaluated in vitro and in vivo. The second part of the introduction focuses on RAS and its preponderant role in blood pressure maintenance and cerebrovascular regulation. We present the angiotensin II (AngII) receptors, AT1 and AT2 responsible for vasoconstriction and vasodilation of cerebral arteries, respectively. Finally, the last part presents the regulation of AngII receptors by NO, in particular through S-nitrosation of the receptors, the covalent bound between NO and cysteine residues. We present the work of Leclerc, showing that exposure of cells overexpressing AT1 to NO causes a decrease in AngII affinity for AT1 (Leclerc et al., 2006). The second chapter is devoted to the experimental studies. The objective of this work is to study the importance of AngII receptor S-nitrosation at the cerebrovascular level. First, we address the current problematic concerning the nonspecificity of anti-AT1 antibodies. We show here that the new monoclonal anti-AT1 antibody, which is supposed to be more specific, does not recognize AT1 in western blot and immunofluorescence, making its use impossible. We then make a pharmacological demonstration of S-nitrosation effects on AngII receptors. We show that exposure to NO donors (S-nitrosoglutathione or sodium nitroprusside) specifically abolishes AT1-mediated vasoconstriction compared to other vasoconstrictors sharing or not its signaling pathway. Moreover, this exposure also abolishes AT1-mediated AngII-independent myogenic tone, suggesting an alteration on the receptor itself. We also show that this effect (i) does not depend on endogenous NO, (ii) is mediated by S-nitrosation rather than by the cGMP/sGC pathway. Finally, we study AT1 internalization by flow cytometry on a heterologous model of AT1 expression. Our results show that GSNO does not alter AT1 cell surface localization and does not prevent its internalization, indicating that the ß-arrestin pathway is not impacted by nitrosation

L’hypertension artérielle (HTA) est un problème de santé publique. Largement répandue dans le monde, elle touchait 40% des adultes âgés de plus de 25 ans et causait 9.4 millions de décès en 2008. Cette pathologie est la cause la plus commune de décès dans les pays développés et constitue un facteur de risque majeur d’atteintes cardiaques, rénales et cérébrales. Bien qu'étudiés depuis maintenant plus d'un demi-siècle, les mécanismes des atteintes systémiques liées à l'hypertension restent encore peu connus. L'augmentation de la pression artérielle est multifactorielle et découle aussi du dérèglement de certains systèmes tels le système rénine-angiotensine et le système endothéline. De récentes études ont suggéré un rôle de l’immunité innée dans le développement de l’HTA et des lésions qui y sont associées. L’endothéline-1 est un puissant vasoconstricteur. Sa production est déclenchée par différents stimuli dont l’AngII et des cytokines pro-inflammatoires. L’ET-1 agit via deux récepteurs : ETAR et ETBR. Pour ce projet, nous avons généré des souris ne possédant pas le récepteur ETB myéloïde (LysM-Cre Ednrb lox/lox). Ces souris ont été soumises à une perfusion chronique d’AngII associée à un régime hyper-sodé. Nous avons observé que les deux groupes de souris développent la même hypertension et les mêmes atteintes cardiaques. En revanche les atteintes des organes cibles sont moins importantes chez les souris LysM-Cre Ednrb lox/lox. La fonction rénale de ces dernières est préservée et, histologiquement, moins de lésions sont observées. Cette protection semble être due à l’incapacité des cellules myéloïdes à infiltrer les reins. En effet, la chimioattraction des cellules myéloïdes vers ET-1 est dépendante du récepteur ETB myéloïde. De plus, les cellules inflammatoires qui sont observées dans les reins des souris LysM-Cre Ednrb lox/lox présentent un phénotype anti-inflammatoire contrairement à leur contrôles. / Arterial hypertension is a major risk factor for atherosclerosis, coronary artery disease, stroke, and chronic kidney disease (CKD) and is one of the most prominent contributors to death worldwide. However, despite the frequency of hypertension, its cause in the majority of adults is unknown. Hypertension is complex, with no single mechanism entirely explaining the blood pressure (BP) rise in any given model. The past 50 years have seen growing evidence implicating the immune system. Recent data suggest that macrophages (M)/monocytes contribute to, and protect from, hypertension and its associated end organ injury. Endothelin-1 (ET-1) is the most potent endogenous vasoconstrictor. Its production is triggered by multiple stimuli including Ang II and pro-inflammatory cytokines. ET-1 acts by binding to two distinct receptors, the endothelin-A (ETA) and the endothelin-B (ETB) receptors. Interestingly, ET antagonism can blunt BP elevation in an Ang II model suggesting that ET-1 largely mediates the effects of Ang II. Here, we have generated mice specifically lacking ETB receptors on myeloid cells. We have shown that the development of hypertension associated with Ang II infusion is not dependent on these cells. Similarly, the cardiac dysfunction seen after 6 weeks of Ang II was similar between knockout and control mice. Interestingly, mice deficient of ETB receptors on myeloid cells alone were protected from Ang II induced vascular dysfunction and kidney injury. This protection appeared to relate to an inability for ETB receptor deficient Mto infiltrate the kidneys due to impaired chemokinesis towards ET-1. Furthermore, the Minfiltrating he kidney in response to Ang II in myeloid ETB receptor deficient mice overwhelmingly possessed an anti-inflammatory phenotype.

A hipótese do estudo é a de que a Angiotensina II (AngII) é capaz de desencadear processos inflamatórios iniciais que compõem parte dos mecanismos envolvidos na lesão vascular ou no seu progresso. Os objetivos foram avaliar a expressão de marcadores iniciais de inflamação em resposta à ação da AngII e por qual receptor (AT1 ou AT2) esta levaria à expressão destes marcadores inflamatórios. O estudo foi realizado em camundongos machos C57Bl/6J submetidos ao tratamento com doses subpressoras de AngII, bloqueadores dos receptores AT1 e AT2 e uma combinação destes. Os tempos de tratamento foram determinados através de uma curva de tempo-resposta feita com injeções de AngII. Após definição da curva temporal foram preparados 6 grupos de animais: controle, tratados AngII, Losartan, AngII+Losartan, PD123319 e AngII+PD123319. Foram feitas avaliações dos marcadores inflamatórios nos corações por imunohistoquímica para citocinas IL-1<font face=\"Symbol\">b e IL-6, TNF<font face=\"Symbol\">a, TGF-<font face=\"Symbol\">b e MCP-1, a molécula de adesão ICAM-1, e foram quantificados a IL-6 e o TNF-<font face=\"Symbol\">a séricos pela técnica ELISA. / The study hypothesis is that the angiotensin II (Ang II) is capable of triggering inflammatory processes that comprise the initial part of the mechanisms involved in vascular injury or in progress. The objectives were to evaluate the expression of early markers of inflammation in response to the action of Ang II and which receptor (AT1 and AT2) this would lead to the expression of these inflammatory markers. The study was conducted in male C57Bl/6J mice undergoing treatment with subpressor doses of AngII, blockers of AT1 and AT2 receptors and a combination thereof. Treatment times were determined using a time-response curve performed with injections of AngII. After definition of time curve were prepared six animal groups: control, treated Ang II, losartan, Ang II + losartan, PD123319 and Ang II + PD123319. Evaluations were made in the hearts of inflammatory markers by immunohistochemistry for IL-1<font face=\"Symbol\">b and IL-6, TNF<font face=\"Symbol\">a, TGF-<font face=\"Symbol\">b and MCP-1, the adhesion TNF-<font face=\"Symbol\">a serum by ELISA.

Em concentrações elevadas no plasma ou no tecido renal a Angiotensina II (Ang II) induz, alterações na hemodinâmica renal, injúria glomerular, aumento da síntese de componentes da matriz extracelular glomerular, estresse oxidativo e apoptose de células glomerulares, incluindo os podócitos. Os podócitos possuem um sistema reninaangiotensina (SRA) próprio e expressam os receptores AT1 e AT2 para o peptídeo, além do trocador Na+/H+ isoforma 1 (NHE1). O NHE1 está envolvido com a resistência e indução de apoptose, controle do volume celular e manutenção do fenótipo celular. Assim, o objetivo deste estudo foi investigar em podócitos, o papel da Ang II na indução de apoptose, e os eventos intracelulares associados à atividade do NHE1 nesta condição. Nossos resultados indicam que o tratamento com Ang II em alta concentração por 24 horas promove apoptose em podócitos. Nesta condição o NHE1, promove ativação da via de sinalização intracelular p38 MAPK e aumenta a atividade do NHE1 levando a alcalose, ativação da Bax e apoptose nos podócitos. / It has been observed that high plasma, or kidney tissue concentrations of angiotensin II (Ang II) leads to changes in renal hemodynamics, severe glomerular injury, increased synthesis of glomerular extracellular matrix components, oxidative stress and apoptosis in glomerular cells, including podocyte and mesangial cells. Podocytes a local renin-angiotensin system (RAS), expresses the AT1 and AT2 receptors for Ang II and the Na + / H + exchanger (NHE1). The NHE1 is involved with resistance and induction of apoptosis, cell volume control and maintenance of cell phenotype. Thus, the goal of this study was to investigate in podocytes the role of Ang II in the induction of apoptosis, and intracellular events linked to the NHE1 activity in this condition. Our results indicate that the treatment with Ang II, in a high dose, for 24 hours induces apoptosis in podocytes, and promotes oxidative stress. However, the activation of NADPH oxidase subunits Nox4 and p22 (phox) and pro- apoptotic pprotein Bax, came before the late apoptosis observed in 24 hours of treatment with Ang II. Under physiological conditions, the NHE1 activity contributes to cell survival by preventing cytosolic acidification. Moreover, Ang II via the AT1 receptor, activates intracellular signaling pathway p38 MAPK and increases the NHE1 activiy leading to alkalosis, Bax activation and apoptosis in podocytes.

Demonstramos na fase crônica da hipertensão por coarctação, depressão acentuada do controle reflexo da freqüência cardíaca (Michelini et al, Hypertension, 1992; 19:II159-II163) e que o tratamento crônico com losartan, embora não reverta os elevados níveis de pressão, normaliza o controle reflexo da freqüência cardíaca (Santos et al, Am. J. Physiol, 1995; 269:H812-H818). No presente estudo investigamos em ratos hipertensos e seus controles normotensos os efeitos do bloqueio crônico dos receptores AT1 sobre a atividade aferente do nervo depressor aórtico (NDA, protocolo I) e sobre a atividade simpática esplâncnica (ASE, protocolo II) na situação basal e após estimulação por hipotensões e hipertensões transitórias. Ratos Wistar adultos machos (180-300g) foram tratados cronicamente com veículo (VEH=água destilada, 0,1ml/100g/dia, po.) ou losartan (LOS= 10mg/kg/dia, po.) por 8 dias. No 4º dia após a determinação da pressão de cauda, foram submetidos à coarctação subdiafragmática da aorta (CH) ou à cirurgia fictícia (SHAM) e ao final do 7º dia, instrumentados com cânula arterial para registro direto da pressão arterial, realizada no 8º dia, com os animais acordados. Foram a seguir anestesiados para isolamento do nervo depressor aórtico ou do simpático esplâncnico, que foram registrados simultaneamente à pressão arterial em duas situações distintas: a) durante a situação basal (registro da atividade espontânea por 10-15 minutos), b) durante estimulação dos pressorreceptores por variações transitórias da pressão arterial (infusões/retiradas de sangue e injeções/infusões de fenilefrina e nitroprussiato de sódio iv.). Os registros da atividade neural foram retificados, integrados e aquisitados simultaneamente aos registros de pressão arterial em computador (programa de aquisição de dados Codas - Windaq DI-200, Ohio, USA). Tratamento com LOS determinou queda significante da pressão de cauda (104+-3 vs. 117+-3 mmHg nos grupos VEH). Em ambos os grupos VEH e LOS, CH determinou aumento significativo e similar da pressão arterial (em média 29%) que foi acompanhada, nos grupos tratados com VEH, por significativo aumento da variabilidade associada à depressão no ganho da correlação da atividade do NDA com a pressão arterial (CHVEH=1,04+-0,11 vs. SHAMVEH=1,63+-0,14 %/ciclo/mmHg, p<0,05, protocolo I) e por facilitação da resposta da ASE (CHVEH=-10,36+-1,05 vs. SHAMVEH=-5,81+-0,60 %/ciclo/mmHg, p<0,05, protocolo II). Nos grupos tratados com LOS, o estabelecimento da hipertensão foi acompanhado por redução da variabilidade e significativa melhora no ganho do NDA, uma vez que na faixa fisiológica de variações da pressão a depressão da atividade do NDA foi significativamente menor (CHLOS=3,30+-0,33 vs. CHVEH=2,18+-0,37 %/ciclo/mmHg, p<0,05, uma recuperação de 40% quando comparado aos respectivos controles ~5,01+-0,33 %/ciclo/mmHg). Houve ainda nos CHLOS normalização do ganho de resposta de ASE (CHLOS=-6,58+-0,62 %/ciclo/mmHg que não diferia dos SHAMLOS e SHAMVEH). O tratamento crônico com LOS determinou ainda correção parcial da atividade basal do NDA, mas não alterou a descarga espontânea da ASE. As alterações de atividade do NDA e ASE observadas nos CHLOS ocorreram simultaneamente, mas foram independentes da redução da pressão arterial pelo LOS. Nossos dados sugerem que a Ang II, ativada durante o desenvolvimento da CH, deprime a sinalização aferente pelo NDA, sendo responsável pela facilitação da resposta simpática durante alterações transitórias da pressão arterial. Nossos dados permitem ainda discriminar entre o efeito pressor e o efeito modulatório da Ang II, indicando restauração parcial do ganho da NDA e normalização da resposta simpática após o bloqueio crônico dos receptores AT1, mesmo na presença da hipertensão. / In the chronic phase of coarctation hypertension (CH) we have shown both marked depression of baroreceptor reflex control of heart rate (Michelini et al, Hypertension, 1992, 19: II159-II163) and normalization of the depressed reflex control even with the persistence of hypertension in losartan-treated animals (Santos et al, Am. J. Physiol, 1995, 269: H812-H818). In the present study we analyzed the effects of chronic AT1tors blockade on the both the afferent aortic nerve activity (AON, protocol I) and splanchnic sympathetic nerve activity (SSNA, protocol II) of CH and sham operated controls in two conditions: spontaneous activity (basal condition) and stimulated discharge during transient increases/decreases in arterial pressure. Male Wistar rats (180-300g aged 2-3 months) were chronically treated with vehicle (VEH=distilled water, 1ml/kg/day, po.) or losartan (LOS=10mg/kg/day, po.) during 8 days. Tail pressure was measured at the beginning and on the 4th day, before subdiaphragmatic aortic coarctation (CH) or sham surgery (SHAM). On day 7 the rats were instrumented with a catheter to record arterial pressure in conscious freely moving rats on day 8. Rats were then anesthetized to record AON or SSNA simultaneously with pressure during 10-15 min (basal or spontaneous activity) and during baroreceptor loading/unloading (infusion/withdrawal of blood or injections/infusions of phenylephrine and sodium nitroprusside iv.). Nerve activity was rectified and integrated and acquired simultaneously with pressure in a computer (Windaq DI-200, Ohio, USA). Losartan-treatment caused a significant decrease in tail pressure (104+-3 vs. 117+-3 mmHg in VEH groups). In both LOS- and VEH-treated groups, CH caused significant and similar increases in arterial pressure (mean of 29%) that was accompanied in the VEH groups by both significant increase in variability and depression of the AON/activity/pressure relationship (CHVEH=1,04+-0,11 vs. SHAMVEH=1,63+-0,14 %/cycle/mmHg, p<0,05, protocol I) and by potentiation of SSNA responses (CHVEH=-10,36+-1,05 vs. SHAMVEH=-5,81+-0,60 %/cycle/mmHg, p<0,05, protocol II). In the LOS-treated groups, establishment of CH was accompanied by smaller variability and marked improvement in AON gain: in the physiological range of pressure changes, depression of AON activity was significantly smaller (CHLOS=3,30+-0,33 vs. CHVEH=2,18+-0,37 %/cycle/mmHg, p<0,05, a recovery of 40% when compared to respective controls of ~5,01+-0,33 %/cycle/mmHg). CHLOS showed also normalization of SSNA responses: gain in the CHLOS=-6,58+-0,62 %/cycle/mmHg, that was not different from SHAMLOS and SHAMVEH groups. In addition chronic treatment with LOS caused partial correction of spontaneous AON activity but did not change spontaneous SSNA discharge. Both AON and SSNA responses observed in CHLOS occurred simultaneously but were independent from pressure reductions observed in these groups. The date suggested that Ang II, activated during development of hypertension, depresses the afferent signaling by aortic receptors and is factor responsible for the facilitation of SSNA during pressure changes. The date permits to discriminate between pressor and a modulatory effects of Ang II and indicates that after chronic AT1 receptors blockade hypertension persists, but there is a partial restoration of AON gain accompanied by normalization of the sympathetic response.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2019-01-31T10:46:56Z No. of bitstreams: 2 Tese - Allancer Divino de Carvalho Nunes - 2018.pdf: 10933019 bytes, checksum: ec8f5a33649eddf1e5ff660120efcca9 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2019-01-31T10:49:09Z (GMT) No. of bitstreams: 2 Tese - Allancer Divino de Carvalho Nunes - 2018.pdf: 10933019 bytes, checksum: ec8f5a33649eddf1e5ff660120efcca9 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2019-01-31T10:49:09Z (GMT). No. of bitstreams: 2 Tese - Allancer Divino de Carvalho Nunes - 2018.pdf: 10933019 bytes, checksum: ec8f5a33649eddf1e5ff660120efcca9 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-04-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Despite Angiotensin-(1-7) [Ang-(1-7)] and Angiotensin II (Ang II) be considered important regulators of the cardiovascular system, its direct effects on cardiac contractility remain unclear and even controversial. Several studies, using different preparations, have showed that Ang-(1-7) or Ang II produce positive, negative or no inotropic effects. Thus, the aim of this study was to increase the knowledge about the effect of the low concentration of Ang-(1-7) and Ang II on cardiac contractility, coronary vascular function and the possible receptors and mechanisms of action involved in these effects. The in vivo effects of Ang-(1-7) and Ang II on cardiac contractility were evaluated in anesthetized rats. The Ang-(1-7) 4 nmol/L and Ang II 40 nmol/L caused negative inotropism in anesthetized rats. None of the peptides were able to change heart rate or arterial blood pressure in anesthetized rats. The direct effects of the Ang-(1-7) and Ang II on cardiac contractility were evaluated in isolated perfused rat hearts. After a basal period (30-40 minutes), the hearts were perfused for an additional 15 min with Ang-(1-7) and Ang II (20 pmol/L) in presence or absence of Mas receptor antagonist A-779 (2 nmol/L), AT2 receptor antagonist PD1233190 (2 nmol/L), AT1 receptor antagonist Losartan (1μmol/L), MrgD receptor antagonist D-PRO (2 nmol/L), ACE2 inhibitor DX600 (2nmol/L), Nitric Oxide synthase (NOS) inhibitor L-Name (10 nmol/L), Guanylyl cyclase (GC) inhibitor ODQ (200 nmol/L) and Adenylyl cyclase (AC) inhibitor MDL 12,330A (1μmol/L). Low concentrations of Ang-(1-7) and Ang II reduced the left ventricular end-systolic pressure (LVESP). The A-779 did not blocked the effect of Ang-(1-7) or Ang II. PD123319 and Losartan inhibited the Ang-(1-7) but not Ang II-induced negative inotropic effects. On the other hand, D-PRO was able to block the negative inotropic effect of the Ang II and Ang-(1-7). Furthermore, L-Name, ODQ and MDL 12,330A inhibited the Ang-(1-7) but not Ang II-induced negative inotropic effects. Similarly, low concentrations of Ang- (1-7) and Ang II-induced coronary vasodilatation. The A-779, D-PRO, L-Name, MDL 12,330A blocked the effect of Ang-(1-7) or Ang II. The DX600 blocked the vasodilatation induced by Ang II. PD123319, Losartan and ODQ inhibited the Ang-(1-7) but not Ang II-induced coronary vasodilatation. In addiction, the pharmacological blocked and gene silencing of AT1 receptor in endothelial human cells stimulated with Ang-(1-7) decreased AKT phosphorilation induced by Ang-(1-7). These data demonstrate that Ang-(1-7) and Ang II, at picomolar concentrations, induce significant and similar negative inotropic and coronary vasodilatation effects involving complex interaction mechanisms between many different receptors, altering intracellular signaling and their constitutive activity. / Embora a Angiotensina-(1-7) e Angiotensina II sejam considerados importantes peptídeos para regulação do sistema cardiovascular, seus efeitos sobre a contratilidade cardíaca ainda não estão totalmente elucidados. Diversos estudos, utilizando diferentes preparações, têm demonstrado que a Ang II e a Ang-(1-7) promovem efeito inotrópico positivo, negativo ou nenhum efeito inotrópico. Diante disso, o objetivo deste estudo foi ampliar os conhecimentos sobre os efeitos da Ang II e Ang-(1-7) no controle da contratilidade ventricular e função vascular coronariana. Além disso, avaliaremos o envolvimento dos receptores envolvidos nestes efeitos e os possíveis mecanismos de ação. Os efeitos in vivo da Ang-(1-7) e Ang II na contratilidade cardíaca foram avaliados em ratos anestesiados com uretana através da canulação intraventricular esquerda. A infusão de Ang-(1-7) 4 nmol/L e Ang II 40 nmol/L promoveram inotropismo negativo em corações de ratos anestesiados. Nenhum dos peptídeos foram capazes de alterar a pressão arterial e frequência cardíaca em ratos anestesiados. Os efeitos diretos da Ang- (1-7) e Ang II na contratilidade cardíaca foram avaliados em corações isolados de ratos. Após o período basal (30-40 minutos), os corações foram perfundidos durante 15 minutos com Ang-(1-7) ou Ang II na concentração de 20 pmol/L na presença ou ausência de antagonista do receptor Mas A-779 (2 nmol/L), antagonista do receptor AT2 PD123319 (2 nmol/L), antagonista do receptor AT1 Losartan (1μmol/L), antagonista do receptor MrgD D-PRO (2 nmol/L),inibidor da enzima ECA2 DX600 (2nmol/L), inibidor de Óxido nítrico sintase (NOS) L-Name (10 nmol/L), inibidor de Guanilato Ciclase (GC) ODQ (200 nmol/L) e inibidor de Adenilato Ciclase (AC) MDL 12,330A (1μmol/L). Concentrações picomolares de Ang-(1-7) e Ang II reduziram a pressão intraventricular sistólica (PIS). O A-779 não bloqueou os efeitos da Ang-(1-7) ou Ang II. PD123319 e Losartan inibiram somente o efeito inotrópico da Ang-(1-7). Por outro lado, D-PRO foi capaz de bloquear o efeito inotrópico de ambos os peptídeos angiotensinérgicos. Além disso, L-Name, ODQ e MDL 12,330A bloquearam apenas o efeito inotrópico da Ang-(1-7). Semelhantemente, baixas concentrações de Ang-(1-7) e Ang II induziram vasodilatação coronariana. O A- 779, D-PRO, L-Name, MDL 12,330A bloquearam a vasodilatação induzida por Ang-(1-7) e Ang II. O DX600 bloqueou a vasodilatação coronariana induzida por Ang II. PD 123319, Losartan e ODQ inibiram apenas a vasodilatação coronariana induzida por Ang-(1-7). Além disso, o bloqueio farmacológico e o silenciamento gênico do receptor AT1 em células endoteliais humanas estimuladas com Ang-(1-7) reduziram a fosforilação de AKT promovida por este peptídeo. Esses resultados demonstram que Ang-(1-7) e Ang II, em concentrações picomolares induzem significante e similar efeito inotrópico negativo e vasodilatação coronariana, envolvendo complexos mecanismos de interação entre os receptores.