A ativação do receptor para tromboxano-prostanóide (TP) está relacionada com o processo de desenvolvimento e manutenção da hipertensão arterial. O objetivo do presente trabalho foi investigar se a ativação do receptor TP promove a produção de fatores contráteis derivados do endotélio (EDCFs) e espécies reativas de oxigênio (ERO), contribuindo para a disfunção vascular observada na hipertensão renovascular. Aortas de ratos normotensos (2R) ou hipertensos (2R-1C) foram utilizadas para avaliar a expressão proteica da enzima cicloxigenase (COX-1, COX- 2), expressão gênica do receptor TP, expressão proteica da enzima NO-Sintase endotelial fosforilada (fosfo-eNOS) e produção de TXA2 ativada pelo U46619. Células endoteliais (CE) e do músculo liso vascular (CMLV) foram utilizadas para avaliar a produção de NO e ERO pelo estímulo com o U46619. Em aortas com (E+) ou sem endotélio (E-) de ratos 2R e 2R-1C foram realizadas curvas concentraçãoefeito cumulativas para o U46619 na ausência ou presença de Tiron, Catalase (Cat) e em aortas E+, na presença de L-NAME, Ibuprofeno (Ibu) ou L-NAME + Ibu. Em aortas E+ contraídas com U46619, foram realizadas curvas concentração-efeito para acetilcolina (ACh), na ausência ou presença de Tiron, Cat ou Ibu. Os resultados mostram que a expressão gênica para o receptor TP foi menor e a expressão proteica da COX-1 e COX-2 foi maior em aortas de ratos 2R-1C do que em 2R. Em aorta de ratos 2R e 2R-1C, o U46619 não promoveu a geração de TXA2 e não alterou a expressão proteica da fosfo-eNOS. O estímulo com U46619 promoveu o aumento de NO em CE e ERO em CE e CMLV. Em aortas E- a potência (pD2) do U46619 em induzir contração foi maior em 2R e 2R-1C em relação às preparações E+. Em aortas E- de ratos 2R, a contração ao U46619 foi menor com Cat ou Ibu do que no controle. Em preparações E+, Ibu ou Ibu+Tiron (mas não Ibu+L-NAME) reduziram a potência do U46619 em relação ao controle. Em aortas E+ de ratos 2R- 1C, a contração ao U46619 foi maior com Tiron e menor em presença de Tiron+Ibu em relação ao controle. Tiron+L-NAME ou Ibu não modificou a resposta contrátil ao U46619. Em aortas E-, Tiron ou Cat não modificaram a contração ao U46619. Quando contraídas com U46619 (10 nmol/L), o relaxamento à ACh foi menor em aorta de ratos 2R-1C em relação a 2R. Em aortas E+ de ratos 2R e 2R-1C, o relaxamento à ACh foi menor quando contraídas com U46619 (100 nmol/L) do que com U46619 (10 nmol/L). Tiron, Cat ou Ibu não modificaram o relaxamento à ACh em aorta de ratos 2R-1C comparadas a 2R. Em aorta de ratos 2R, o peróxido de hidrogênio regula a atividade das enzimas COX e eNOS. Por outro lado, em aorta de ratos 2R-1C, a regulação da atividade da COX pelas ERO depende do endotélio e modula negativamente a síntese de EDCFs. Palavras-chave: receptor TP, NO, ERO, COX, função vascular, hipertensão. / TP receptor activation is involved on the development and maintenance of hypertension. This study aimed to investigate if TP receptor activation induces endothelium-derived contractile factors (EDCFs) and reactive oxygen species (ROS) production, and if it contributes to the vascular dysfunction in renal hypertensive rats. Normotensive (2K) and renal hypertensive (2K-1C) rat aortas were used to evaluate COX-1 and -2 expression, genic expression of TP receptor, p-eNOS expression and TXA2 production induced by TP agonist, U46619. Endothelial cells (EC) and vascular smooth muscle cells (VSMC) were stimulated with U46619 to evaluate NO and ROS production. U46619- stimulated concentration-effect curves were performed in intact (E+) and denuded-endothelium (E-) aortas of 2K and 2K-1C rats, in the absence (Control) or presence of Tiron or Catalase (Cat). In E+ aortas, we used L-NAME, Ibuprofen or the combination of both. Concentration-effect curves were performed for acetylcholine (ACh) in E+ aortas, contracted by U46619, in the absence or presence of Tiron, Catalase or Ibuprofen. In 2K-1C, the genic expression of TP receptor was lower and expression of COX-1 and -2 was higher than in 2K. The agonist did not have effect on TXA2 production or p-eNOS expression in both rat groups of aortas. U46619 induced production of NO in EC and ROS in EC and VSMC. The endothelium removal increased the potency of U46619 in 2K and 2K-1C aortas. In Eaortas of 2K, contraction induced by U46619 was impaired by Catalase. In E+ aortas, the potency was lower in the presence of Ibu ou Ibu+Tiron, but not Cat or Ibu+LNAME. In E+ aortas of 2K-1C, the contraction to U46619 was potentiated by Tiron but it was inhibited by Tiron+Ibu. Tiron and L-NAME or Ibuprofen had no effect on U46619-induced contraction. In E- aortas of 2K-1C, Tiron or Catalase had no effect on the contraction induced by U46619. ACh-induced relaxation was impaired in 2K and 2K-1C aortas contracted with 100 nmol/L U46619. Relaxation induced by ACh was lower in 2K-1C aortas contracted with 10 nmol/L U46619. Tiron, Catalase or Ibuprofen had no effect on the relaxation to ACh in 2K-1C rats aorta. Our results suggest that in 2K rat aortas, H2O2 regulates COX and eNOS activity. On the other hand, in 2K-1C rats aorta, ROS modulates COX activity and EDCFs production, which mechanisms are endothelium- dependent.

A ativação do receptor para tromboxano-prostanóide (TP) está relacionada com o processo de desenvolvimento e manutenção da hipertensão arterial. O objetivo do presente trabalho foi investigar se a ativação do receptor TP promove a produção de fatores contráteis derivados do endotélio (EDCFs) e espécies reativas de oxigênio (ERO), contribuindo para a disfunção vascular observada na hipertensão renovascular. Aortas de ratos normotensos (2R) ou hipertensos (2R-1C) foram utilizadas para avaliar a expressão proteica da enzima cicloxigenase (COX-1, COX- 2), expressão gênica do receptor TP, expressão proteica da enzima NO-Sintase endotelial fosforilada (fosfo-eNOS) e produção de TXA2 ativada pelo U46619. Células endoteliais (CE) e do músculo liso vascular (CMLV) foram utilizadas para avaliar a produção de NO e ERO pelo estímulo com o U46619. Em aortas com (E+) ou sem endotélio (E-) de ratos 2R e 2R-1C foram realizadas curvas concentraçãoefeito cumulativas para o U46619 na ausência ou presença de Tiron, Catalase (Cat) e em aortas E+, na presença de L-NAME, Ibuprofeno (Ibu) ou L-NAME + Ibu. Em aortas E+ contraídas com U46619, foram realizadas curvas concentração-efeito para acetilcolina (ACh), na ausência ou presença de Tiron, Cat ou Ibu. Os resultados mostram que a expressão gênica para o receptor TP foi menor e a expressão proteica da COX-1 e COX-2 foi maior em aortas de ratos 2R-1C do que em 2R. Em aorta de ratos 2R e 2R-1C, o U46619 não promoveu a geração de TXA2 e não alterou a expressão proteica da fosfo-eNOS. O estímulo com U46619 promoveu o aumento de NO em CE e ERO em CE e CMLV. Em aortas E- a potência (pD2) do U46619 em induzir contração foi maior em 2R e 2R-1C em relação às preparações E+. Em aortas E- de ratos 2R, a contração ao U46619 foi menor com Cat ou Ibu do que no controle. Em preparações E+, Ibu ou Ibu+Tiron (mas não Ibu+L-NAME) reduziram a potência do U46619 em relação ao controle. Em aortas E+ de ratos 2R- 1C, a contração ao U46619 foi maior com Tiron e menor em presença de Tiron+Ibu em relação ao controle. Tiron+L-NAME ou Ibu não modificou a resposta contrátil ao U46619. Em aortas E-, Tiron ou Cat não modificaram a contração ao U46619. Quando contraídas com U46619 (10 nmol/L), o relaxamento à ACh foi menor em aorta de ratos 2R-1C em relação a 2R. Em aortas E+ de ratos 2R e 2R-1C, o relaxamento à ACh foi menor quando contraídas com U46619 (100 nmol/L) do que com U46619 (10 nmol/L). Tiron, Cat ou Ibu não modificaram o relaxamento à ACh em aorta de ratos 2R-1C comparadas a 2R. Em aorta de ratos 2R, o peróxido de hidrogênio regula a atividade das enzimas COX e eNOS. Por outro lado, em aorta de ratos 2R-1C, a regulação da atividade da COX pelas ERO depende do endotélio e modula negativamente a síntese de EDCFs. Palavras-chave: receptor TP, NO, ERO, COX, função vascular, hipertensão. / TP receptor activation is involved on the development and maintenance of hypertension. This study aimed to investigate if TP receptor activation induces endothelium-derived contractile factors (EDCFs) and reactive oxygen species (ROS) production, and if it contributes to the vascular dysfunction in renal hypertensive rats. Normotensive (2K) and renal hypertensive (2K-1C) rat aortas were used to evaluate COX-1 and -2 expression, genic expression of TP receptor, p-eNOS expression and TXA2 production induced by TP agonist, U46619. Endothelial cells (EC) and vascular smooth muscle cells (VSMC) were stimulated with U46619 to evaluate NO and ROS production. U46619- stimulated concentration-effect curves were performed in intact (E+) and denuded-endothelium (E-) aortas of 2K and 2K-1C rats, in the absence (Control) or presence of Tiron or Catalase (Cat). In E+ aortas, we used L-NAME, Ibuprofen or the combination of both. Concentration-effect curves were performed for acetylcholine (ACh) in E+ aortas, contracted by U46619, in the absence or presence of Tiron, Catalase or Ibuprofen. In 2K-1C, the genic expression of TP receptor was lower and expression of COX-1 and -2 was higher than in 2K. The agonist did not have effect on TXA2 production or p-eNOS expression in both rat groups of aortas. U46619 induced production of NO in EC and ROS in EC and VSMC. The endothelium removal increased the potency of U46619 in 2K and 2K-1C aortas. In Eaortas of 2K, contraction induced by U46619 was impaired by Catalase. In E+ aortas, the potency was lower in the presence of Ibu ou Ibu+Tiron, but not Cat or Ibu+LNAME. In E+ aortas of 2K-1C, the contraction to U46619 was potentiated by Tiron but it was inhibited by Tiron+Ibu. Tiron and L-NAME or Ibuprofen had no effect on U46619-induced contraction. In E- aortas of 2K-1C, Tiron or Catalase had no effect on the contraction induced by U46619. ACh-induced relaxation was impaired in 2K and 2K-1C aortas contracted with 100 nmol/L U46619. Relaxation induced by ACh was lower in 2K-1C aortas contracted with 10 nmol/L U46619. Tiron, Catalase or Ibuprofen had no effect on the relaxation to ACh in 2K-1C rats aorta. Our results suggest that in 2K rat aortas, H2O2 regulates COX and eNOS activity. On the other hand, in 2K-1C rats aorta, ROS modulates COX activity and EDCFs production, which mechanisms are endothelium- dependent.

Although the etiology of diabetic nephropathy is still uncertain, proteinuria due to podocyte injury and loss (podocytopenia) are early features of the disease. Significant increases in thromboxane A2 (TXA2) production as well as expression of its receptor in animal models of diabetic nephropathy led to the hypothesis that TXA2 acting via its thromboxane-prostanoid (TP) receptor induces podocytopenia resulting in proteinuria. Systemic infusion of a TP antagonist demonstrated an important role of TXA2/TP signalling in our model of streptozotocin induced type-1 diabetic nephropathy by reducing kidney damage including proteinuria. Podocyte specific TP overexpressing mice did not demonstrate more pathologic or dynamic kidney damage than non-transgenic mice in STZ-induced diabetic nephropathy. Further assessment of the TP transgene functionality in this mice line is necessary to validate those results. Whereas the importance of TXA2/TP signalling is undeniable in diabetic nephropathy, it appears that podocyte TP receptors might not be directly targeted.

Although the etiology of diabetic nephropathy is still uncertain, proteinuria due to podocyte injury and loss (podocytopenia) are early features of the disease. Significant increases in thromboxane A2 (TXA2) production as well as expression of its receptor in animal models of diabetic nephropathy led to the hypothesis that TXA2 acting via its thromboxane-prostanoid (TP) receptor induces podocytopenia resulting in proteinuria. Systemic infusion of a TP antagonist demonstrated an important role of TXA2/TP signalling in our model of streptozotocin induced type-1 diabetic nephropathy by reducing kidney damage including proteinuria. Podocyte specific TP overexpressing mice did not demonstrate more pathologic or dynamic kidney damage than non-transgenic mice in STZ-induced diabetic nephropathy. Further assessment of the TP transgene functionality in this mice line is necessary to validate those results. Whereas the importance of TXA2/TP signalling is undeniable in diabetic nephropathy, it appears that podocyte TP receptors might not be directly targeted.

Thromboxane A2 (TXA2) is an important mediator metabolized from arachidonic acid through the cyclooxygenase pathway, mainly in platelets and macrophages. It is a potent inducer of platelet aggregation and smooth muscle contraction. Its overproduction has been detected in pathologies such as stroke, asthma, myocardial infarction or atherosclerosis. The action of TXA2 is mediated by a specific G-protein coupled receptor (TP) of which two alternative spliced isoforms, TPalpha and TPbeta, have been described. The exact role of these two isoforms is not clearly understood. However, recent studies have described their implications in vascular physiology and pathology. The inhibition of the action of TXA2 on platelets and blood vessels would be interesting as original therapies against cardiovascular diseases. Consequently, the design of TP receptor antagonists remains of great interest in cardiovascular medicine. In the laboratory of medicinal chemistry (University of Liège, Belgium), several nitrobenzenesulfonylureas, derived from torasemide (a loop diuretic), have been previously described as TP receptor antagonists. Two compounds, BM573 and BM613 were among the most interesting molecules identified in that previous work. The present project is divided in two parts. First, we have determined the pharmacological properties of BM573 and BM613 as thromboxane synthase inhibitors and TP receptor antagonists, in vitro and in vivo. In our assays, these two compounds were proved to have high affinity for both TPalpha and TPbeta, to be potent antiplatelet agents, to inhibit thromboxane synthase and TP-mediated smooth muscle contraction. Additionally, they significantly reduced the size of the thrombus in a rat model of ferric chloride-induced arterial thrombosis. Consequently, we demonstrated that the TP receptor antagonists BM573 and BM613, belonging to the chemical family of nitrobenzenesulfonylureas, could be regarded as antiplatelet and antithrombotic agents potentially useful in thromboxane-related diseases such as stroke or myocardial infarction. Secondly, given the interesting pharmacological profile of BM573 and BM613, we have designed and synthesized several series of compounds derived from these two agents. We have evaluated the binding properties (affinity) of the first generation (+/- 35 original derivatives) of compounds on either TPalpha or TPbeta, transiently expressed in COS-7 cell lines. Additionally, we have measured the ability of our drugs to inhibit the intracellular calcium mobilization upon TPalpha or TPbeta stimulation. To confirm our results, we also assessed the antiplatelet properties of our drugs by means of determination of inhibition of human platelet aggregation. On the basis of the results obtained with these in vitro assays, we have synthesized and evaluated a second generation of derivatives (+/- 35 original compounds) and improved the selectivity of several original compounds for TP receptor isoforms. The originality of this work was to evaluate a large library of synthetic compounds on both TP receptor isoforms, using specific pharmacological tests. By means of structure-activity relationship studies, we were able to identify chemical groups implicated in selectivity and to propose lead compounds for development of highly specific TPalpha or TPbeta antagonists. Besides, we have identified an in vivo drug candidates for prevention of thrombosis and pathological platelet aggregation./Le thromboxane A2 (TXA2) est un métabolite de la cascade de lacide arachidonique (AA) par la voie des cyclooxygénases et de la thromboxane synthase, principalement formé dans les plaquettes et les macrophages. Le TXA2 est un puissant inducteur de lagrégation plaquettaire et de la contraction des muscles lisses vasculaires et bronchiques. Par ailleurs, une augmentation des taux en TXA2 a été constatée dans différentes pathologies : l'infarctus du myocarde, l'atherosclérose, les accidents vasculaires cérébraux, ou encore l'asthme. Laction du TXA2 sur les tissus résulte de la stimulation dun récepteur appartenant à la famille des récepteurs couplés aux protéines G. Ce récepteur au TXA2 (TP) présente deux isoformes générées par épissage alternatif, TPalpha et TPbeta. Le rôle physiologique exact de ces deux isoformes n'est pas encore connu. Cependant, de récents travaux ont mis en évidence leur importance, notamment dans la physiologie vasculaire et dans certaines pathologies. Linhibition de laction du TXA2 au niveau des plaquettes et des vaisseaux sanguins pourrait donc être une stratégie thérapeutique innovante pour traiter et prévenir les maladies cardiovasculaires. En conséquence, le développement dantagonistes des récepteurs TP reste dun grand intérêt en médecine cardiovasculaire. Des études de pharmacomodulation avaient permis au Laboratoire de Chimie Pharmaceutique (Université de Liège, Belgique) d'identifier des nitrobenzènesulfonylurées, dérivées du torasémide (un diurétique de lanse), présentant un puissant antagonisme des récepteurs TP. Parmi ceux-ci, deux composés, le BM573 et le BM613, faisaient parties des molécules les plus intéressantes identifiées au cours de ces précédentes recherches. Ce projet est divisé en deux parties. Premièrement, nous avons déterminé les propriétés pharmacologiques du BM573 et du BM613 en tant quinhibiteurs de la thromboxane synthase et antagonistes des récepteurs TP, in vitro et in vivo. Au cours de nos expériences, ces deux composés se sont révélés posséder une grande affinité pour TPalpha et TPbeta, être de puissants agents antiplaquettaires, des inhibiteurs de la thromboxane synthase et de la contraction des muscles lisses induite par le TXA2. En outre, lutilisation de ces produits dans un modèle de thrombose artérielle induite par le chlorure ferrique chez le rat a provoqué une réduction significative du thrombus formé. En conséquence, nous avons démontré que le BM573 et le BM613, appartenant à la famille chimique des nitrobenzenesulfonylurées, pouvaient être considérés comme des agents antiplaquettaires et antithrombotiques, potentiellement utiles en tant quagents thérapeutiques dans des pathologies associées au TXA2 telles que linfarctus du myocarde ou laccident vasculaire cérébral. Ensuite, nous nous sommes concentrés sur l'activité de cette famille de composés (les nitrobenzènesulfonylurées) vis-à-vis des deux isoformes du récepteur au thromboxane. Pour ce faire, nous avons conçu et synthétisé de nombreuses séries de composés dérivés du BM573 et du BM613. Nous avons tout dabord évalué laffinité de la première génération de composés (+/- 35 dérivés) sur des lignées cellulaires (COS-7) exprimant sélectivement soit TPalpha soit TPbeta. De plus, nous avons mesuré la capacité de ces composés à inhiber la mobilisation de calcium intracellulaire ([Ca2+]i) induite par la stimulation des deux isoformes TPalpha et TPbeta séparément. Nos résultats ont été confirmés sur agrégation plaquettaire humaine. Sur la base des résultats obtenus avec cette première génération de produits, nous avons synthétisé une seconde génération (+/- 35 dérivés) de composés, et avons réussi à augmenter la sélectivité en faveur de TPbeta pour certains produits. Loriginalité de ce travail réside dans le fait que nous avons évalué un nombre de produits importants sur TPalpha et TPbeta, au moyen de tests pharmacologiques spécifiques. Grâce à des études de relation structure-activité, nous avons identifié des groupements chimiques impliqués dans la sélectivité entre les deux isoformes. Nous pouvons donc proposer des structures "chef de file" pouvant être utiles pour le développement de composés hautement sélectifs, soit pour TPalpha, soit pour TPbeta. Par ailleurs, nous avons identifié in vivo des candidats pour le développement dagents thérapeutiques pour la prévention des thromboses et des autres pathologies provoquées par une activation plaquettaires excessive.