Le développement des anticorps monoclonaux thérapeutiques est en plein essor notamment à cause de leur bénéfice important pour le traitement des cancers. Cependant, à l’heure actuelle, aucun anticorps monoclonal sur le marché ou en phase III ne cible de RCPGs, en dépit de l’implication grandissante de ces récepteurs dans la carcinogenèse. Parmi les RCPGs les plus pertinents pour l’oncologie, souvent cités dans la littérature et dont certains inhibiteurs chimiques sont en phase clinique avancée, on trouve les deux sous-types de récepteurs des endothélines ETAR et ETBR. Dans ce contexte, mon projet de thèse a consisté à produire des anticorps monoclonaux capables de lier spécifiquement les récepteurs des endothélines, puis à les caractériser dans le but d’évaluer leur potentiel antitumoral. Grâce à une stratégie d’immunisation génique, un ensemble de 27 anticorps monoclonaux, tous spécifiques de la forme native d’ETBR, a été obtenu. Un de ces anticorps, nommé rendomab-B1, a fait l’objet d’une caractérisation précise et s’est révélé être un puissant inhibiteur allostérique d’ETBR. De plus, cette propriété antagoniste a permis de bloquer l’action autocrine antiapoptotique de l’ET-1 sur des cellules endothéliales vasculaires, suggérant ainsi que le rendomab-B1 pourrait être utilisé comme agent thérapeutique afin d’inhiber les effets tumorigènes liés à la suractivation de l’axe ET1/ETBR au niveau de l’endothélium vasculaire tumoral. Par ailleurs, le rendomab-B1 a également été testé sur des lignées de mélanomes humains ; l’absence de fixation de l’anticorps malgré la présence de récepteurs ETB fonctionnels à la surface de ces cellules suggère l’existence d’une forme moléculaire atypique du récepteur, potentiellement spécifique aux mélanomes. A la lumière de ces résultats, le rendomab-B1 apparaît comme un outil prometteur, à la fois pour l’étude structurale et fonctionnelle d’ETBR, mais aussi pour une éventuelle thérapie anticancéreuse. Enfin, les 26 autres anticorps monoclonaux anti-ETBR, actuellement en cours de caractérisation, constituent également des molécules potentiellement intéressantes pour un usage fondamental ou thérapeutique impliquant ETBR. Pour conclure, ces travaux ont démontré l’intérêt de la méthode d’immunisation génique pour la production d’anticorps monoclonaux anti-RCPGs à visée thérapeutique. / For a decade, monoclonal antibodies have become increasingly important for the biotherapeutic management of cancer. However, none of the monoclonal antibodies currently on the market or in late stage clinical trial do target a G-protein coupled receptor in spite of the emerging role of these receptors in tumor progression. Among the therapeutically relevant GPCRs for oncology, the endothelin receptors (ETAR and ETBR) are particularly attractive considering their overexpression in a wide range of tumors and their involvement in various stages of tumorigenesis. In this context, my PhD project consisted in producing and characterizing monoclonal antibodies directed against endothelin receptors with a view to use them as anti-tumor agents. Using an original DNA immunization strategy, we produced a panel of 27 monoclonal antibodies which selectively recognized ETBR expressed at the surface of transfected cells. One of these antibodies, named rendomab-B1, was extensively characterized and proved to be a potent allosteric antagonist of ETBR. Moreover, rendomab-B1 was able to disrupt the autocrine ET1-mediated survival loop on vascular endothelial cells, suggesting that this antibody could be used to prevent the pro-tumorigenic effect due to ET-1 and ETBR upregulation in the tumor-surrounding endothelium. Furthermore, rendomab-B1 binding onto ETBR was also assessed on melanoma cell lines and revealed that a tumor-specific form of ETBR may exist, as illustrated by the poor fixation of rendomab-B1 on these cells in spite of the presence of functional ETB receptors. Together, these results present rendomab-B1 as promising agent, not only for the structural and functional study of ETBR, but also for its therapeutic modulation in the case of cancer for instance. Finally, the other 26 monoclonal antibodies, whose characterization is still ongoing, also constitute potential tools for fundamental or therapeutic applications involving ETBR. To conclude, this work has highlighted the relevance of the DNA immunization approach to generate monoclonal antibodies against the native form of GPCRs.

Il est admis que l’axe endothéline (endothélines ET-1, -2 et -3 et leurs RCPG ETAR et ETBR), participe à la progression tumorale. Alors qu’ETAR est par exemple surexprimé dans le cancer de l’ovaire, ETBR l’est dans le mélanome. Cette surexpression, ainsi que l’implication d’ETA/BR dans la carcinogenèse, font de ces RCPG une cible tumorale pertinente. En raison de leurs forte spécificité, actions cytotoxiques variées, possibilités de couplage, les anticorps monoclonaux (AcM) sont des outils de choix en diagnostic et thérapie anti-cancéreuse. Cependant, on déplore actuellement l’absence d’AcM ciblant des RCPG sur le marché. Par une technique d’immunisation génique, 4 AcM anti-ETAR et 24 anti-ETBR ont été produits. Les résultats préliminaires obtenus avec les anti-ETAR sont prometteurs puisque ces AcM lient avec une haute affinité ETAR surexprimé dans des cellules CHO, l’un d’eux inhibant fortement la liaison du ligand. Mon travail de thèse s’est cependant concentré sur la caractérisation d’un anti-ETBR. Cet AcM reconnaît de façon spécifique et avec une forte affinité la conformation native d’ETBR surexprimé à la surface de cellules de mélanomes, suggérant l’existence d’une forme tumorale du récepteur. Suite à sa liaison aux cellules UACC-257 (lignée de mélanome), l’AcM se trouve internalisé. Dans ces cellules, malgré son incapacité à inhiber la liaison de l’ET, cet AcM inhibe l’activation de la voie PLC induite par le ligand et est également un fort inhibiteur de la migration due à l’activation de l’axe endothéline. Ces travaux soulignent l’intérêt de cet AcM comme outil diagnostique et thérapeutique dans le cas du mélanome. / It has been admitted that endothelin axis (endothelins ET-1, -2 and -3 and related GPCRs ETAR and ETBR) is involved in tumor progression. For instance, while ETAR is overexpressed in ovarian cancer, ETBR is in melanoma. This overexpression, as well as ETA/BR involvement in carcinogenesis, make these GPCRs a relevant tumor target. Because of their high specificity, various cytotoxic actions, possibilities of coupling, the monoclonal antibodies (mAbs) are useful tools in diagnosis and anti-cancer therapy. However, the absence of mAbs targeting GPCRs on the market is regrettable. Thanks to DNA immunization, 4 anti-ETAR mAbs and 24 anti-ETBR mAbs were produced. Preliminary results obtained with anti-ETAR are promising since these mAbs bind ETAR overexpressed in CHO cells with high affinity, one of them being a potent inhibitor of ligand binding. However, the aim of my PhD research works focused on the characterization of one anti-ETBR. This mAb specifically recognizes with high affinity the native conformation of ETBR overexpressed on the surface of melanoma cells, suggesting the existence of a tumor-specific receptor. Following its binding on UACC-257 cells (melanoma cell line), the mAb is internalized. In these cells, despite its inability to inhibit ET binding, this mAb is able to inhibit the ligand-induced activation of PLC pathway and display a potent inhibition of endothelin axis-induced migration. This work highlights the interest of this mAb as a tool for diagnosis and therapy in melanoma.

A hiperhomocisteinemia (HHcy) tem sido associada à disfunção endotelial, em decorrência do aumento de ânion superóxido (O2-) e redução da biodisponibilidade de óxido nítrico (NO), fatos estes que poderiam acarretar disfunção erétil. O objetivo deste trabalho foi estudar as consequências da HHcy sobre as respostas à endotelina-1 (ET-1) e fenilefrina (PhE) em corpos cavernosos de ratos, bem como os mecanismos envolvidos. Os animais foram divididos em dois grupos, os quais receberam água (controle) ou DL-homocisteina tiolactona (DL-HcyT, grupo HHcy), na dose de 1 g/Kg/dia, via oral por 15 dias. Análises morfológicas, de colágeno e expressão de -actina não revelaram macroalterações na estrutura de corpos cavernosos de ratos HHcy, sugerindo que alterações na funcionalidade destes tecidos não decorrem de modificações estruturais. A HHcy acarretou aumento dos níveis de O2- em corpos cavernosos de ratos, avaliados por microscopia confocal. A reatividade vascular foi avaliada para KCl, nitroprussiato de sódio (NPS), acetilcolina (ACh), ET-1, IRL-1620 e PhE. Não foram observadas alterações na reatividade vascular para KCl ou NPS. O relaxamento induzido por ACh foi reduzido em corpos cavernosos de ratos HHcy. A contração induzida por ET-1, via receptores ETA, mostrou-se aumentada em corpos cavernosos de ratos HHcy, sugerindo possível envolvimento de O2- basais em vias intracelulares, decorrentes da ativação de receptores ETA. Observou-se prejuízo do relaxamento induzido por ET-1 e IRL-1620 em corpos cavernosos de ratos HHcy, por ativação de receptores ETB. O prejuízo do relaxamento induzido por IRL-1620 foi decorrente da produção e/ou biodisponibilidade reduzida de NO. A expressão de RNAm para pré-pró-ET-1, enzima conversora de ET-1 e receptores ETA e ETB não foram alteradas em decorrência da HHcy. O Emax da PhE foi aumentado em corpos cavernosos de ratos HHcy, em decorrência de aumento nos níveis basais de O2- e redução de fatores moduladores negativos da contração, tal como peróxido de hidrogênio (H2O2), sugerindo possível prejuízo da enzima superóxido dismutase. A participação de metabólitos derivados da isoformas da enzima óxido nítrico sintase (NOS), eNOS, nNOS e iNOS que modulam negativamente a contração da PhE, mostraram-se importantes nesta resposta. Na HHcy, os metabólitos derivados principalmente da iNOS estão prejudicados, possivelmente por redução da atividade da NOS, processo de desacoplamento e/ou redução da biodisponibilidade de NO por interação com espécies reativas de oxigênio (ERO), formando peróxinitrito. A expressão de nitrotirosina, indicador da presença de peroxinitrito, não foi alterada em corpos cavernosos de ratos HHcy. As dosagens plasmáticas de nitrato mostraram redução dos níveis de NO em ratos HHcy, sendo sugestivo de redução de sua biodisponibilidade. A HHcy não alterou a expressão de RNAm para eNOS, nNOS e iNOS em corpos cavernosos de ratos. Os metabólitos da enzima cicloxigenase-1 (COX-1) e COX-2 participam modulando negativamente a contração da PhE e a HHcy não alterou esta modulação. Concluindo, a HHcy intermediária, por sua capacidade de aumentar os níveis basais de O2-, pode afetar a função vasoativa, contração e relaxamento, do peptídeo ET-1, bem como aumentar a resposta de contração à PhE em decorrência de prejuízo de H2O2 e redução de metabólitos derivados da iNOS em corpos cavernosos de ratos. / Hyperhomocysteinemia (HHcy) has been associated with endothelial dysfunction, due to the increase in superoxide anion (O2-) and reduced bioavailability of nitric oxide (NO), these facts could result in erectile dysfunction. The objective of this work was to study the consequences of HHcy on the responses to endothelin-1 (ET-1) and phenylephrine (PhE) in rat corpus cavernosum, as well as the mechanisms involved. The animals were divided into two groups, which received water (control) or DL-homocysteine thiolactone (DL-HcyT; HHcy group) at a dose of 1 g/kg/day for 15 days orally. Morphological analysis and collagen and expression of -actin revealed no considerable alterations in the structure of the corpus cavernosum of HHcy rats, suggesting that alterations in the functionality of these tissues are not based on structural modifications. The HHcy resulted in increased levels of O2- in corpus cavernosum of rats, assessed by confocal microscopy. Vascular reactivity was assessed for substances KCl, sodium nitroprusside (SNP), acetylcholine (ACh), ET-1, IRL-1620 and PhE. There were no changes in vascular reactivity to KCl or NPS. The relaxation induced by ACh was reduced in HHcy rat corpus cavernosum. The contraction induced by ET-1, by ETA receptors, was increased in corpus cavernosum of HHcy rats, suggesting possible involvement of basal O2- in intracellular pathways by activation of ETA receptors. There was prejudice to the relaxation to ET-1 and IRL-1620 in HHcy rat corpus cavernosum by activation of ETB receptors. The decreased IRL-1620-induced relaxation was due to the reduced production and/or bioavailability of NO.The expression of mRNA for pre-pro-ET-1, endothelin converting enzyme and ETA and ETB receptors were not altered in HHcy. The Emax of PhE was increased in HHcy rat corpus cavernosum, due to increase in basal levels of O2- and reducing negative modulators factors of the contraction, such as hydrogen peroxide (H2O2), suggesting possible loss of the enzyme superoxide dismutase (SOD). The participation of metabolites derived from eNOS, nNOS and iNOS, that modulate negatively PhE-induced contraction appear to be important in this response. In HHcy, these metabolites, derived primary from iNOS, are damaged, possibly by reducing the activity of NOS, the process of decoupling and/or reduced bioavailability of NO by interaction with reactive oxidative species (ROS) to form peroxynitrite. The expression of nitrotyrosine, inidicador the presence of peroxynitrite, was not altered in HHcy rat corpus cavernosum. Plasma levels of nitrate showed reduced levels of NO in HHcy rats, being suggestive of reduced bioavailability. The HHcy did not alter the expression of mRNA for eNOS, iNOS and nNOS in rat corpus cavernosum. The metabolites of the enzyme cyclooxygenase-1 (COX-1) and COX-2 participate negatively modulating the contraction of PhE and HHcy does not seem to change this modulation. In conclusion, intermediate HHcy, by its ability to increase basal levels of O2-, may affect the vasoactive function, contraction and relaxation of ET-1 peptide, as well as increase the contraction induced by PhE due to decrease of H2O2 and reduced of the metabolites derived from iNOS in rat corpus cavernosum.

O rim é tanto um órgão-alvo como a principal fonte de produção de ET-1, peptídio que regula a excreção de Na+ e água por este órgão que expressa os seus respectivos receptores, ETA e ETB, além dos trocadores NHE1 e NHE3 que são essenciais para o equilíbrio ácido base e hidroeletrolítico das células. Assim, o objetivo deste estudo foi investigar, em células IRPTC, o papel de ET-1 na atividade dos trocadores NHE1 e NHE3. Nossos resultados indicam que o tratamento agudo com ET-1 (10-9 M) aumenta a velocidade de recuperação do pHi (dpHi/dt) nos dois primeiros minutos após o pulso ácido, sugerindo aumento na atividade dos trocadores NHE1 e NHE3, que ocorre via ativação dos receptores ETA e ETB e parece ser secundária à atividade da p90RSk e p38MAPK. O tratamento crônico com ET-1 (10-9 M) reduz a dpHi/dt nos dois primeiros minutos após o pulso ácido, o que sugere redução na atividade dos trocadores NHE1 e NHE3, que pode ser secundária à inibição da Na+, K+-ATPase por ET-1. / The kidney is both a target organ and the main source of ET-1 production, a peptide that regulates the excretion of Na+ and water by this organ that expresses its respective receptors, ETA and ETB, in addition to the NHE1 and NHE3 exchanger which are Essential for the basic acid and electrolyte balance of cells. Thus, the objective of this study was to investigate, in IRPTC cells, the role of ET-1 in the activity of the NHE1 and NHE3 exchanger. Our results indicate that the acute treatment with ET-1 (10-9 M) increases the rate of recovery of pHi (dpHi/dt) in the first two minutes after the acid pulse, suggesting an increase in the activity of the NHE1 and NHE3 exchanger, which occurs via activation Of ETA and ETB receptors and appears to be secondary to the activity of p90RSk and p38MAPK. Chronic treatment with ET-1 (10-9 M) reduces dpHi/dt in the first two minutes after the acid pulse, suggesting a reduction in NHE1 and NHE3 exchanger activity, which may be secondary to inhibition of Na+, K+- ATPase by ET-1.

A hiperglicemia é um fator de risco na progressão da ND, que associada à atividade do SRA sistêmico e/ou intrarrenal e à síntese de ET1, resulta em perda da função renal. Objetivo: Investigar o papel da hiperglicemia e a relação com a síntese de Ang II e ET-1 no tecido renal no início da ND. Métodos: Ratos Wistar foram organizados em controle; diabéticos via STZ; tratados com losartan e outros com BQ123. Foram avaliados os parâmetros metabólicos e de função renal. Resultados: O losartan preveniu o efeito da STZ na expressão de renina medular, injúria tubular, expressão de desmina e de RNAm para TNFα e Nox4. O BQ123 não alterou o efeito da STZ em nenhum dos parâmetros estudados. Conclusão: A hiperglicemia tem predominância na injúria renal nos primeiros estágios da ND. Nessa condição, a Ang II sistêmica e/ou intrarrenal via receptor AT1 amplia os efeitos da hiperglicemia nos eventos iniciais da ND. / Hyperglycemia and a risk factor in the progression of ND, which associated with the RAS activity of systemic and/or intrarrenal and the synthesis of ET-1, results in loss of renal function. Aim: To investigate the role of hyperglycemia and a relationship with a synthesis of Ang II and ET-1 in the renal tissue at the beginning of DN. Methods: Wistar rats were organized in control; Diabetics via STZ; Treated with losartan and others with BQ123. Metabolic and renal function parameters were evaluated. Results: Losartan prevented the effect of STZ on the expression of medullary renin, tubular injury, desmin and mRNA expression for TNFα and Nox4. BQ123 did not alter the STZ effect in any of the parameters studied. Conclusion: Hyperglycemia has a predominance of renal injury in the early stages of ND. In this condition, Ang II systemic and/or intrarenal via the AT1 receptor amplifies the effects of hyperglycemia in the initial events of ND.

A hiperhomocisteinemia (HHcy) tem sido associada à disfunção endotelial, em decorrência do aumento de ânion superóxido (O2-) e redução da biodisponibilidade de óxido nítrico (NO), fatos estes que poderiam acarretar disfunção erétil. O objetivo deste trabalho foi estudar as consequências da HHcy sobre as respostas à endotelina-1 (ET-1) e fenilefrina (PhE) em corpos cavernosos de ratos, bem como os mecanismos envolvidos. Os animais foram divididos em dois grupos, os quais receberam água (controle) ou DL-homocisteina tiolactona (DL-HcyT, grupo HHcy), na dose de 1 g/Kg/dia, via oral por 15 dias. Análises morfológicas, de colágeno e expressão de -actina não revelaram macroalterações na estrutura de corpos cavernosos de ratos HHcy, sugerindo que alterações na funcionalidade destes tecidos não decorrem de modificações estruturais. A HHcy acarretou aumento dos níveis de O2- em corpos cavernosos de ratos, avaliados por microscopia confocal. A reatividade vascular foi avaliada para KCl, nitroprussiato de sódio (NPS), acetilcolina (ACh), ET-1, IRL-1620 e PhE. Não foram observadas alterações na reatividade vascular para KCl ou NPS. O relaxamento induzido por ACh foi reduzido em corpos cavernosos de ratos HHcy. A contração induzida por ET-1, via receptores ETA, mostrou-se aumentada em corpos cavernosos de ratos HHcy, sugerindo possível envolvimento de O2- basais em vias intracelulares, decorrentes da ativação de receptores ETA. Observou-se prejuízo do relaxamento induzido por ET-1 e IRL-1620 em corpos cavernosos de ratos HHcy, por ativação de receptores ETB. O prejuízo do relaxamento induzido por IRL-1620 foi decorrente da produção e/ou biodisponibilidade reduzida de NO. A expressão de RNAm para pré-pró-ET-1, enzima conversora de ET-1 e receptores ETA e ETB não foram alteradas em decorrência da HHcy. O Emax da PhE foi aumentado em corpos cavernosos de ratos HHcy, em decorrência de aumento nos níveis basais de O2- e redução de fatores moduladores negativos da contração, tal como peróxido de hidrogênio (H2O2), sugerindo possível prejuízo da enzima superóxido dismutase. A participação de metabólitos derivados da isoformas da enzima óxido nítrico sintase (NOS), eNOS, nNOS e iNOS que modulam negativamente a contração da PhE, mostraram-se importantes nesta resposta. Na HHcy, os metabólitos derivados principalmente da iNOS estão prejudicados, possivelmente por redução da atividade da NOS, processo de desacoplamento e/ou redução da biodisponibilidade de NO por interação com espécies reativas de oxigênio (ERO), formando peróxinitrito. A expressão de nitrotirosina, indicador da presença de peroxinitrito, não foi alterada em corpos cavernosos de ratos HHcy. As dosagens plasmáticas de nitrato mostraram redução dos níveis de NO em ratos HHcy, sendo sugestivo de redução de sua biodisponibilidade. A HHcy não alterou a expressão de RNAm para eNOS, nNOS e iNOS em corpos cavernosos de ratos. Os metabólitos da enzima cicloxigenase-1 (COX-1) e COX-2 participam modulando negativamente a contração da PhE e a HHcy não alterou esta modulação. Concluindo, a HHcy intermediária, por sua capacidade de aumentar os níveis basais de O2-, pode afetar a função vasoativa, contração e relaxamento, do peptídeo ET-1, bem como aumentar a resposta de contração à PhE em decorrência de prejuízo de H2O2 e redução de metabólitos derivados da iNOS em corpos cavernosos de ratos. / Hyperhomocysteinemia (HHcy) has been associated with endothelial dysfunction, due to the increase in superoxide anion (O2-) and reduced bioavailability of nitric oxide (NO), these facts could result in erectile dysfunction. The objective of this work was to study the consequences of HHcy on the responses to endothelin-1 (ET-1) and phenylephrine (PhE) in rat corpus cavernosum, as well as the mechanisms involved. The animals were divided into two groups, which received water (control) or DL-homocysteine thiolactone (DL-HcyT; HHcy group) at a dose of 1 g/kg/day for 15 days orally. Morphological analysis and collagen and expression of -actin revealed no considerable alterations in the structure of the corpus cavernosum of HHcy rats, suggesting that alterations in the functionality of these tissues are not based on structural modifications. The HHcy resulted in increased levels of O2- in corpus cavernosum of rats, assessed by confocal microscopy. Vascular reactivity was assessed for substances KCl, sodium nitroprusside (SNP), acetylcholine (ACh), ET-1, IRL-1620 and PhE. There were no changes in vascular reactivity to KCl or NPS. The relaxation induced by ACh was reduced in HHcy rat corpus cavernosum. The contraction induced by ET-1, by ETA receptors, was increased in corpus cavernosum of HHcy rats, suggesting possible involvement of basal O2- in intracellular pathways by activation of ETA receptors. There was prejudice to the relaxation to ET-1 and IRL-1620 in HHcy rat corpus cavernosum by activation of ETB receptors. The decreased IRL-1620-induced relaxation was due to the reduced production and/or bioavailability of NO.The expression of mRNA for pre-pro-ET-1, endothelin converting enzyme and ETA and ETB receptors were not altered in HHcy. The Emax of PhE was increased in HHcy rat corpus cavernosum, due to increase in basal levels of O2- and reducing negative modulators factors of the contraction, such as hydrogen peroxide (H2O2), suggesting possible loss of the enzyme superoxide dismutase (SOD). The participation of metabolites derived from eNOS, nNOS and iNOS, that modulate negatively PhE-induced contraction appear to be important in this response. In HHcy, these metabolites, derived primary from iNOS, are damaged, possibly by reducing the activity of NOS, the process of decoupling and/or reduced bioavailability of NO by interaction with reactive oxidative species (ROS) to form peroxynitrite. The expression of nitrotyrosine, inidicador the presence of peroxynitrite, was not altered in HHcy rat corpus cavernosum. Plasma levels of nitrate showed reduced levels of NO in HHcy rats, being suggestive of reduced bioavailability. The HHcy did not alter the expression of mRNA for eNOS, iNOS and nNOS in rat corpus cavernosum. The metabolites of the enzyme cyclooxygenase-1 (COX-1) and COX-2 participate negatively modulating the contraction of PhE and HHcy does not seem to change this modulation. In conclusion, intermediate HHcy, by its ability to increase basal levels of O2-, may affect the vasoactive function, contraction and relaxation of ET-1 peptide, as well as increase the contraction induced by PhE due to decrease of H2O2 and reduced of the metabolites derived from iNOS in rat corpus cavernosum.

A endotelina-1 (ET-1) é um peptídeo vasoconstritor que exerce um papel importante no controle do tônus do corpo cavernoso. No entanto, tem sido demonstrado que esse peptídeo também está envolvido na disfunção erétil (DE) associada ao diabetes mellitus e hipertensão. O consumo de etanol aumenta os níveis plasmáticos de ET-1 e a resposta contrátil a esse peptídeo em tecidos vasculares. Os objetivos deste trabalho foram o de estudar as consequências funcionais e celulares do consumo crônico de etanol sobre o sistema endotelinérgico no corpo cavernoso e identificar os mediadores envolvidos nessa resposta. Ratos Wistar foram divididos em dois grupos, os quais receberam água (controle) ou solução de etanol a 20% (vol./vol.) por seis semanas. Nossos resultados mostram que em tiras de tecido cavernoso, não houve alteração da resposta de relaxamento induzida pela adrenomedulina e nitroprussiato de sódio após tratamento com etanol. Com relação à acetilcolina, o consumo crônico de etanol reduziu o relaxamento induzido pelo referido agonista. Além disso, observou-se redução dos níveis plasmáticos e teciduais de nitrato no grupo etanol. Em conjunto, esses resultados sugerem que o tratamento crônico com etanol reduz a síntese/liberação do NO tecidual sem prejuízo em sua via de sinalização. O tratamento com etanol aumentou os níveis plasmáticos de ET-1 e a resposta contrátil induzida por esse peptídeo em corpo cavernoso de ratos. A contração induzida pela fenilefrina ou KCl 120 mmol/L não foi afetada pelo tratamento com etanol, sugerindo que os efeitos do tratamento sobre a reatividade do corpo cavernoso não são inespecíficos. O antagonista dos receptores ETB, o BQ788, não alterou a resposta de contração induzida pela ET-1 em corpo cavernoso de animais do grupo controle ou etanol. Não houve alteração da resposta de relaxamento induzida pelo IRL1620, um agonista seletivo dos receptores ETB. O tratamento com etanol não alterou os níveis de RNAm assim como a expressão protéica dos receptores ETB. Esses resultados mostram que o aumento da contração induzida pela ET-1 após tratamento com etanol não está relacionado à redução do relaxamento mediado pelos receptores ETB. Em nosso estudo o BQ123, antagonista seletivo dos receptores ETA, deslocou a curva cumulativa para ET-1 para direita em músculo cavernoso de ratos do grupo controle com consequente redução do valor de pD2. O mesmo não foi observado no tecido de animais do grupo etanol, indicando que a resposta mediada pelos receptores ETA está favorecida após o tratamento. O consumo de etanol não afetou os níveis de RNAm dos componentes do sistema endotelinérgico (ET-1, ECE-1, receptores ETA e ETB) e das isoformas da enzima óxido nítrico sintase (NOS) (eNOS, nNOS e iNOS), porém aumentou a expressão protéica do receptor ETA, da ET-1 e da iNOS no músculo cavernoso. O tratamento com etanol induziu aumento do estresse oxidativo sistêmico assim como dos níveis de ânions superóxido (O2-) no corpo cavernoso. As espécies reativas de oxigênio (ERO), os metabólitos derivados da NOS e da ciclooxigenase (COX) modulam negativamente a contração induzida por ET-1 e mostraram-se importantes no aumento da contração à ET-1 observada no corpo cavernoso de animais tratados com etanol. O Y27632, um inibidor da Rho-cinase, reduziu a resposta contrátil da ET-1 em corpo cavernoso de animais de ambos os grupos. Portanto, os resultados mostram que o tratamento com etanol aumenta a resposta contrátil da ET-1 por mecanismos que envolvem o aumento da expressão dos receptores ETA e das ERO e a via da Rho-cinase. / Endothelin-1 (ET-1) is a vasoconstrictor peptide that plays an important role in controlling the tone of the cavernosal smooth muscle (CSM). ET-1 is also involved in erectile dysfunction (ED) associated with diabetes mellitus and hypertension. Ethanol consumption increases plasma levels of ET-1 and the contractile response to this peptide in vascular tissues. This study aimed to investigate the cellular and functional consequences of chronic ethanol consumption on the endothelinergic system in CSM as well as the mediators involved in this response. Male Wistar rats were treated with ethanol 20% (vol./vol.) for 6 weeks. Reactivity experiments were performed on isolated CSM. Our findings show that adrenomedullin and sodium nitroprusside-induced relaxation was not altered after treatment with ethanol. On the other hand, acetylcholine-induced relaxation was reduced in CSM from ethanol-treated rats. Moreover, chronic ethanol consumption reduced plasma and CSM nitrate levels. These observations suggest that chronic ethanol consumption reduces NO synthesis/release but does not alter NO signaling pathway. Ethanol consumption increases plasma levels of ET-1 and the contractile response to this peptide in isolated CSM. Chronic ethanol consumption did not alter the contraction induced by phenylephrine or KCl 120mmol/L in isolated CSM strips. These observations suggest that the effects of chronic ethanol consumption on the CSM reactivity are nonspecific. BQ788, a selective ETB receptor antagonist, did not alter ET-1-induced contraction in CSM from both control and ethanol-treated rats. The relaxation induced by IRL1620, a selective ETB receptors agonist, was not affected by ethanol consumption. mRNA levels and protein expression for ETB receptor were not affected by ethanol consumption. We concluded that CSM hyper-reactivity to ET-1 is not related to reduction of ETB receptor-mediated relaxation. BQ123, a selective ETA receptor antagonist, shifted the concentration-response curve for ET-1 to the right in CSM from control rats. However, this response was not observed in CSM from ethanol group, indicating that the response mediated by the ETA receptor is favored after ethanol treatment. It was found that chronic ethanol consumption did not alter mRNA levels for the components of the endothelinergic system (ET-1, ECE-1, ETA and ETB receptors) and the isoforms of nitric oxide synthase (NOS) (eNOS, nNOS and iNOS), but increased protein expression for ETA receptor, ET-1 and iNOS. Ethanol induced systemic and cavernosal oxidative stress. Reactive oxygen species (ROS), metabolites derived from cyclooxygenase (COX) and NOS, modulate negatively ET-1-induced contraction and appear to be important mediators of ethanol-induced ET-1 hyper-reactivity in the isolated CSM. Y27632, a Rho-kinase inhibitor, reduced ET-1-induced contraction in CSM from both control and ethanol-treated rats. Our results show that chronic ethanol consumption increases ET-1 induced contraction in isolated CSM and that this response is mediated by the Rho-kinase pathway and an increase in ROS generation and ETA receptor expression.

Introdução: A disfunção erétil (DE) é definida como a incapacidade de alcançar ou manter a ereção do pênis para um desempenho sexual satisfatório, contribuindo significativamente para a baixa qualidade de vida e morbidade psicossocial masculina. A endotelina-1 (ET-1), um potente peptídeo vasoconstritor que promove contração lenta e sustentada em células de músculo liso vascular, possui grande importância na fisiopatologia da DE. Diversos estudos mostram que o aumento da expressão de mediadores inflamatórios está intimamente ligado ao desenvolvimento da DE. O inflamassoma é um complexo multiprotéico do sistema imune inato que atua através da ativação da caspase-1 e resulta na maturação de citocinas pró- inflamatórias, tais como interleucina- IL (IL-l?). O receptor NLRP3 faz parte do inflamassoma e sua ativação leva a clivagem de caspase-1 e consequente secreção de IL-1?. A ET-1, também possui papel importante na inflamação crônica vascular, mediando a liberação de citocinas pró-inflamatórias. No entanto, ainda é desconhecido se a ação pró- inflamatória da ET-1 em células de músculo liso é mediada pela ativação da via do inflamassoma. Hipótese: A ET-1 ativa o NLRP3 em células do músculo liso do corpo cavernoso (CMLCC), promovendo alterações na reatividade do corpo cavernoso (CC). Objetivo: Avaliar o papel da endotelina-1 na ativação do NLRP3 em CMLCC de camundongos. Métodos: CMLCC de camundongos C578BL/6 (WT) e NLRP3-/- foram cultivadas em meio de cultura DMEM acrescido de soro fetal bovino (SFB), 10%, foram pré- incubadas com endotelina-1 nas concentrações de 10-9, 10-8 e 10-7 M, em presença de LPS ou veículo. Avaliamos o efeito da deleção do NLRP3 sobre a reatividade do CC (contratilidade e relaxamento mediante estímulos por campo elétrico e/ou farmacológico). Após, avaliamos o efeito da ET-1 na ativação do NLRP3, nas alterações sobre a reatividade do CC de camundongos WT, e se estas persistiriam nos camundongos NLRP3-/- e caspase1/11-/- . Resultados: As células apresentaram-se fluorescentes para marcação para ?-actina e não para Von Willebrand, caracterizando assim que não houve contaminação com células endoteliais. A incubação com a ET-1 10-7 M por 24 h na presença de LPS ou veículo aumentou a atividade da caspase-1 em CMLCC de camundongos WT e este efeito não ocorreu nas CMLCC de camundongos NLRP3-/-. Não se observou diferença com relação à massa corporal ou massa dos órgãos entre os animais WT e NLRP3-/-. O CC de animais NLRP3-/- apresenta prejuízo para o relaxamento mediado por nitroprussiato de sódio (NPS) quando comparado com as tiras de CC de camundongos WT. A incubação com ET-1 10-7 M por 4 horas promove aumento na contração para fenilefrina (PE) e prejuízo no relaxamento induzido por nitroprussiato de sódio (NPS), e o mesmo efeito não é observado nas tiras de CC de camundongos NLRP3-/- e caspase1/11-/-. Conclusão: O NLRP3 contribui para o aumento na contração e prejuízo no relaxamento produzido pela ET-1 em CC de camundongos, possivelmente através da ativação da caspase-1 / Introduction: Erectile dysfunction (ED) is defined as the inability to achieve or maintain penile erection to perform sexual intercourse, it contributes significantly to the low quality of life and male psychosocial morbidity. Endothelin-1 (ET-1), a potent vasoconstrictor peptide that promotes slow and sustained contraction of vascular smooth muscle cells, has great importance in the pathophysiology of ED. Several studies show that increased expression of inflammatory mediators is closely linked to the development of ED. The inflammasome is a multiproteic complex of the innate immune system that acts through activation of caspase-1, which leads to maturation of pro-inflammatory cytokines such as interleukin-1 beta (IL-l?). The activation of NLRP3 receptor, part of the inflammasome, leads to caspase-1 cleavage and subsequent secretion of IL-1?. ET-1 also plays an important role in chronic vascular inflammation by mediating the release of pro-inflammatory cytokines. However, it is still unknown whether pro-inflammatory actions of ET-1 on smooth muscle cells is mediated by the activation of the inflammasome. Hypothesis: ET-1 activates NLRP3 in smooth muscle cells of the corpora cavernosa (SMCCC), promoting changes in corpus cavernosum (CC) reactivity. Objective: To evaluate the role of endothelin-1 in the activation of the NLRP3 in SMCCC of mice. Methods: SMCCC of C57BL/6 (WT) and NLRP3-/- mice were grown in DMEM culture medium supplemented with bovine fetal serum (FBS) 10%, pre-incubated with endothelin-1 at concentrations of 10-9, 10- 8 and 10-7M, in the presence of LPS or vehicle. We evaluated the effect of the NLRP3 deletion on the reactivity of the CC (contractility and relaxation by electric field and/or pharmacological stimulation). After that, we evaluated the ET-1 effect on activation NLRP3, changes on the reactivity of the CC of WT, and if these alterations would persist NLRP3-/- and caspase1/11-/- mice. Results: The cells presented fluorescent labeling to ?-actin, but not for Von Willebrand factor, characterizing absence of endothelial cells contamination. The incubation with 10-7 M ET-1 for 24 h in the presence of LPS or vehicle increased caspase-1 activity in SMCCC from WT, but not from NLRP3-/- mice. No difference was observed in body mass or weight of the organs between WT and NLRP3-/- animals. The CC from NLRP3-/- animals displayed impaired relaxation mediated by sodium nitroprusside (SNP) when compared to WT CC. The incubation with ET-1 10-7 M for 4 hours promoted an increase in the contraction to phenylephrine (PE) and reduced relaxation induced by sodium nitroprusside (SNP). The same effect was not observed in CC strips from NLRP3-/- and caspase1/11-/- mice. Conclusion: NLRP3 contributes to the increase in contraction and impaired relaxation produced by ET-1 in mice CC, possibly by activation of caspase-1

Background: Air pollution is associated with negative health effects. Exposure to combustion-derived particulate matter (PM) air pollution has been related to increased incidence of cardiovascular and respiratory morbidity and mortality, specifically in susceptible populations. Ambient particles, with a diameter of less than 2.5 mm, have been suggested to be the strongest contributor to these health effects. Diesel exhaust (DE) is a major source of small combustion-derived PM air pollution world wide.  In healthy volunteers, exposure to DE, has been associated with airway inflammation and impaired vasomotor function and endogenous fibrinolysis. The aims of this thesis were to further elucidate the underlying mechanisms to the reported cardiovascular effects following exposure to DE, with specific focus on endothelin-1 (ET-1). Additionally, the vascular effects of the major gaseous component of DE, nitrogen dioxide (NO2), were assessed together with the impact of an exhaust particle trap to reduce the observed negative vascular effects after DE exposure. Methods: In all studies healthy, non-smoking male volunteers were included and exposed for one hour during intermittent exercise in a randomised double-blind crossover fashion. In studies I-III, subjects were exposed to DE at a particulate matter concentration of approximately 300 μg/m3 and filtered air, on two different occasions. In study V an additional exposure was employed, during which DE was filtered through an exhaust particle trap. In study IV subjects were exposed to nitrogen dioxide (NO2) at 4 ppm or filtered air. In study I, thrombus formation and platelet activation were assessed using the Badimon ex vivo perfusion chamber and flow cytometry. Study II comprised the determination of arterial stiffness including pulse wave analysis and velocity. In studies III-V, vascular assessment was performed using venous occlusion plethysmography. In studies IV and V, the vascular responses to intra-arterially infused endothelial-dependent and endothelial-independent vasodilatators were registered. In study III, vascular responses to intra-arterial infusion of Endothelin-1 (ET-1) and ET-1-receptor antagonists were assessed. Venous occlusion phlethysmography was in all cases performed 4-6 hours following exposures. Blood samples for markers of inflammation, coagulation and platelet activation were collected before and throughout the study periods in studies III and V. Results: Exposure to DE increased ex vivo thrombus formation and arterial stiffness, in terms of augmentation index. DE inhalation impaired vasomotor function and endogenous fibrinolysis. The exhaust particle trap reduced the particle concentration by 98% and abolished the effects on vasomotor function, endogenous fibrinolysis and ex vivo thrombus formation. Plasma concentrations of ET-1 and its precursor big-ET-1 were unchanged following exposure. Dual endothelial receptor antagonism caused similar vasodilatation after both exposures, although vasodilatation to the endothelin-A receptor alone was blunted after DE exposure. ET-1 infusion induced vasoconstriction only following DE exposure. Exposure to nitrogen dioxide did not affect vascular function. Conclusion: Inhalation of diesel exhaust in young healthy men impaired important and complementary aspects of vascular function in humans; regulation of vascular tone and endogenous fibrinolysis as well as increased ex vivo thrombus formation. The use of an exhaust particle trap significantly reduced particle emissions and abolished the DE-induced vascular and prothrombotic effects. The adverse vascular effects following DE exposure do not appear to be directly mediated through the endothelin system. Neither is NO2 suggested to be a major arbiter of the DE-induced cardiovascular responses. Arterial stiffness is a non-invasive and easily accessible method and could thus be employed to address vascular function in larger field studies. Taken together, this thesis has given further knowledge about the mechanisms underlying the DE-induced vascular effects.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / "August 2000." Typescript. Vita. Includes bibliographical references (leaves 186-210). Also available on the Internet.