A produção e liberação de substâncias vasodilatadoras pelas células endoteliais requer uma elevação sustentada na concentração intracelular de cálcio; essa elevação é consequente a um influxo de cálcio. Porém, a identidade do (s) canal (is) envolvido (s) nesse influxo ainda não foi (ram) determinada (s) conclusivamente. Existem evidências de que o gene TRPV4 (que codifica uma proteína permeável a cátions, inclusive ao cálcio) é expresso em células endoteliais. Porém, a falta de agentes que modulem especificamente a função dessa proteína não permitiu que o papel do TRPV4 no controle da função endotelial pudesse ser elucidado. Recentemente foram descritos dois novos compostos, o GSK1016790A (GSK) e o HC-067047 (HC), com ação ativadora e bloqueadora seletiva desse canal, respectivamente. Consequentemente, nesta dissertação descrevemos e interpretamos os resultados obtidos em experimentos concebidos para caracterizar o efeito do GSK1016790A (e com fins comparativos o efeito do 4PDD) em artérias isoladas de várias espécies. Para isso, empregamos anéis de artérias suspensos em cubas para órgão isolado para registro da tensão desenvolvida por esses anéis durante a contração isométrica provocada pela adição de fenilefrina; todos os experimentos foram realizados com solução de Krebs contendo diclofenaco (10 M). Inicialmente verificamos mediante imunohistoquímica a presença de imunorreatividade para o TRPV4 no endotélio da aorta torácica de rato. A adição de concentrações isoladas ou cumulativas de GSK produziu relaxamentos dependentes da concentração na aorta torácica de rato (CE50=0,5 nM; IC95%=0,35-0,72 nM; n=7); o 4PDD (1-10 µM), em concentrações isoladas, também produziu relaxamentos na aorta torácica de rato. Resultados semelhantes foram observados para o GSK na aorta torácica de coelho (CE50= 4,3 nM; IC95%=3,58-5,14 nM; n=5), de camundongo (CE50=1,4 nM; IC95%=0,85-2,24 nM; n=3) e de cobaia (CE50=0,2 nM; IC95%=0,12-0,22 nM; n=4). GSK relaxou também a aorta abdominal (CE50=6,5 nM; IC95%=3,71-11,3 nM; n=3) e a artéria femoral de coelho (CE50=17 nM; IC95%=16,8-18,7 nM; n=4); Os relaxamentos produzidos por ambas as drogas apareceram 1-2 min após a adição e atingiram o máximo em 5-8 min, foram reversíveis e não apresentaram taquifilaxia. Em todas as artérias os relaxamentos foram estritamente dependentes de endotélio e da presença de cálcio no meio extracelular. Na aorta torácica de rato, a pré-incubação com HC (5 minutos) aboliu o efeito do GSK sem afetar os relaxamentos produzidos pela acetilcolina. Em todas as artérias testadas os efeitos do GSK e do 4PDD foram revertidos completamente pelo HC (1-3 µM) ou pelo vermelho de rutênio (aorta torácica de rato e artérias de coelho, 1µM, VR). Esses resultados demonstram que os canais TRPV4 estão presentes na célula endotelial e que a sua ativação leva à produção de fatores relaxantes. Como corolário, esses resultados constituem indícios de que os canais TRPV4 podem participar da regulação da função das células endoteliais em situações fisiológicas e/ ou fisiopatológicas. / Production and release of vasodilator substances by endothelial cells require a sustained elevation of intracellular calcium which depends on calcium influx. The identity of the channels involved in this influx remains to be established. There is evidence that the TRPV4 gene (which encodes for a cation permeable channel including calcium) is expressed in endothelial cells; the lack of pharmacologic agents that selectively modulate the activity of TRPV4 channels has hindered the elucidation of its function in endothelial cells. Recently two new compounds, GSK1016790A (GSK) and HC-067047 (HC), which selectively activate or block TRPV4 channels, respectively, were described. This dissertation consists in the description and interpretation of results from experiments conceived to characterize the effect of GSK (and of 4PDD for comparison) in isolated arterial rings from several animal species. To this aim we used arterial rings mounted in isolated organ chambers; we recorded continuously the tension developed by them during isometric contractions elicited by phenylephrine (Phe); all the experiments were conducted using Krebs solution containing diclofenac (10 µM). Initially, we confirmed by immunohistochemistry the presence of anti-TRPV4 immunoreactivity in the endothelium of rat thoracic aorta. In rat thoracic aortic rings pre-constricted with Phe (0.1 µM) the addition of different concentrations of GSK (either single or cumulative concentrations) caused concentration-dependent relaxations (EC50=0.5 nM, 95%CI=0.35-0.72 nM, n=7); 4PDD (in single concentrations) also caused relaxations of rat thoracic aortic rings. Similar results were observed for GSK in thoracic aortic rings from rabbit (EC50=4.3 nM, 95%CI=3.58-5.14 nM, n=5), mouse (EC50=1.4 nM, 95%CI=0.85-2.24 nM, n=3) and guinea-pig (EC50=0.2 nM, 95%CI=0.12-0.22 nM, n=4). GSK also produced relaxations of rings from rabbit abdominal aorta (EC50=6.5 nM, 95%CI=3.71-11.3 nM, n=3) and femoral artery (EC50=17 nM, 95%CI=16.8-18.7 nM, n=4). Relaxations caused by both GSK and 4PDD started 1-2 min after their addition and reached a steady-state in 5-8min; they were reversible after washing-out and did not exhibit tachyphylaxis. In all the studied arteries GSK or 4PDD induced- relaxations were strictly endothelium- and extracellular calcium- dependent. Pre-incubation of rat thoracic aortic rings with HC (1 µM for 5min) abolished the effect of GSK but did not affect relaxations elicited by Ach (1 µM). In all the arterial rings HC (1-3 µM) also completely reverted the relaxations caused by GSK or 4PDD; in rabbit and rat thoracic aortic rings ruthenium red (1 µM) also completely reverted the relaxations caused by GSK or 4PDD. The present findings showing that TRPV4 channels are present in endothelial cells and that their activation results in the production and release of relaxing factors constitute an indication that TRPV4 channels could be involved in the regulation of endothelial cell functions under physiological or patho-physiological conditions.

A produção e liberação de substâncias vasodilatadoras pelas células endoteliais requer uma elevação sustentada na concentração intracelular de cálcio; essa elevação é consequente a um influxo de cálcio. Porém, a identidade do (s) canal (is) envolvido (s) nesse influxo ainda não foi (ram) determinada (s) conclusivamente. Existem evidências de que o gene TRPV4 (que codifica uma proteína permeável a cátions, inclusive ao cálcio) é expresso em células endoteliais. Porém, a falta de agentes que modulem especificamente a função dessa proteína não permitiu que o papel do TRPV4 no controle da função endotelial pudesse ser elucidado. Recentemente foram descritos dois novos compostos, o GSK1016790A (GSK) e o HC-067047 (HC), com ação ativadora e bloqueadora seletiva desse canal, respectivamente. Consequentemente, nesta dissertação descrevemos e interpretamos os resultados obtidos em experimentos concebidos para caracterizar o efeito do GSK1016790A (e com fins comparativos o efeito do 4PDD) em artérias isoladas de várias espécies. Para isso, empregamos anéis de artérias suspensos em cubas para órgão isolado para registro da tensão desenvolvida por esses anéis durante a contração isométrica provocada pela adição de fenilefrina; todos os experimentos foram realizados com solução de Krebs contendo diclofenaco (10 M). Inicialmente verificamos mediante imunohistoquímica a presença de imunorreatividade para o TRPV4 no endotélio da aorta torácica de rato. A adição de concentrações isoladas ou cumulativas de GSK produziu relaxamentos dependentes da concentração na aorta torácica de rato (CE50=0,5 nM; IC95%=0,35-0,72 nM; n=7); o 4PDD (1-10 µM), em concentrações isoladas, também produziu relaxamentos na aorta torácica de rato. Resultados semelhantes foram observados para o GSK na aorta torácica de coelho (CE50= 4,3 nM; IC95%=3,58-5,14 nM; n=5), de camundongo (CE50=1,4 nM; IC95%=0,85-2,24 nM; n=3) e de cobaia (CE50=0,2 nM; IC95%=0,12-0,22 nM; n=4). GSK relaxou também a aorta abdominal (CE50=6,5 nM; IC95%=3,71-11,3 nM; n=3) e a artéria femoral de coelho (CE50=17 nM; IC95%=16,8-18,7 nM; n=4); Os relaxamentos produzidos por ambas as drogas apareceram 1-2 min após a adição e atingiram o máximo em 5-8 min, foram reversíveis e não apresentaram taquifilaxia. Em todas as artérias os relaxamentos foram estritamente dependentes de endotélio e da presença de cálcio no meio extracelular. Na aorta torácica de rato, a pré-incubação com HC (5 minutos) aboliu o efeito do GSK sem afetar os relaxamentos produzidos pela acetilcolina. Em todas as artérias testadas os efeitos do GSK e do 4PDD foram revertidos completamente pelo HC (1-3 µM) ou pelo vermelho de rutênio (aorta torácica de rato e artérias de coelho, 1µM, VR). Esses resultados demonstram que os canais TRPV4 estão presentes na célula endotelial e que a sua ativação leva à produção de fatores relaxantes. Como corolário, esses resultados constituem indícios de que os canais TRPV4 podem participar da regulação da função das células endoteliais em situações fisiológicas e/ ou fisiopatológicas. / Production and release of vasodilator substances by endothelial cells require a sustained elevation of intracellular calcium which depends on calcium influx. The identity of the channels involved in this influx remains to be established. There is evidence that the TRPV4 gene (which encodes for a cation permeable channel including calcium) is expressed in endothelial cells; the lack of pharmacologic agents that selectively modulate the activity of TRPV4 channels has hindered the elucidation of its function in endothelial cells. Recently two new compounds, GSK1016790A (GSK) and HC-067047 (HC), which selectively activate or block TRPV4 channels, respectively, were described. This dissertation consists in the description and interpretation of results from experiments conceived to characterize the effect of GSK (and of 4PDD for comparison) in isolated arterial rings from several animal species. To this aim we used arterial rings mounted in isolated organ chambers; we recorded continuously the tension developed by them during isometric contractions elicited by phenylephrine (Phe); all the experiments were conducted using Krebs solution containing diclofenac (10 µM). Initially, we confirmed by immunohistochemistry the presence of anti-TRPV4 immunoreactivity in the endothelium of rat thoracic aorta. In rat thoracic aortic rings pre-constricted with Phe (0.1 µM) the addition of different concentrations of GSK (either single or cumulative concentrations) caused concentration-dependent relaxations (EC50=0.5 nM, 95%CI=0.35-0.72 nM, n=7); 4PDD (in single concentrations) also caused relaxations of rat thoracic aortic rings. Similar results were observed for GSK in thoracic aortic rings from rabbit (EC50=4.3 nM, 95%CI=3.58-5.14 nM, n=5), mouse (EC50=1.4 nM, 95%CI=0.85-2.24 nM, n=3) and guinea-pig (EC50=0.2 nM, 95%CI=0.12-0.22 nM, n=4). GSK also produced relaxations of rings from rabbit abdominal aorta (EC50=6.5 nM, 95%CI=3.71-11.3 nM, n=3) and femoral artery (EC50=17 nM, 95%CI=16.8-18.7 nM, n=4). Relaxations caused by both GSK and 4PDD started 1-2 min after their addition and reached a steady-state in 5-8min; they were reversible after washing-out and did not exhibit tachyphylaxis. In all the studied arteries GSK or 4PDD induced- relaxations were strictly endothelium- and extracellular calcium- dependent. Pre-incubation of rat thoracic aortic rings with HC (1 µM for 5min) abolished the effect of GSK but did not affect relaxations elicited by Ach (1 µM). In all the arterial rings HC (1-3 µM) also completely reverted the relaxations caused by GSK or 4PDD; in rabbit and rat thoracic aortic rings ruthenium red (1 µM) also completely reverted the relaxations caused by GSK or 4PDD. The present findings showing that TRPV4 channels are present in endothelial cells and that their activation results in the production and release of relaxing factors constitute an indication that TRPV4 channels could be involved in the regulation of endothelial cell functions under physiological or patho-physiological conditions.

INTRODUCTION : Les greffons de veine saphène interne (GVS) sont encore régulièrement utilisés comme conduits en chirurgie de pontage aorto-coronarien (PAC). Les dommages subis par les segments de veine saphène pendant le prélèvement et le stockage favorisent une dysfonction endothéliale qui se manifeste par une diminution de la production d'oxyde nitrique et/ou par une augmentation du niveau de stress oxydant pouvant entraîner une défaillance du greffon veineux se traduisant par une occlusion. La solution saline héparinée est la solution de préservation de référence malgré plusieurs études démontrant ses effets néfastes sur les GVS. GALA est une solution de préservation de greffons autologues vasculaires spécialement développée pour préserver l'intégrité structurale et fonctionnelle de la couche endothéliale des greffons utilisés en chirurgie de pontages aorto-coronariens. OBJECTIF : Comparer la préservation de l'intégrité des fonctions endothéliales des greffons de veine saphène après le stockage dans la solution GALA versus dans la solution saline héparinée dans le cadre d’une étude contrôlée et randomisée en étudiant la réactivité vasculaire en chambres d’organes. RÉSULTATS : Les segments de GVS d'un total de quinze patients ont été obtenus et divisés en anneaux de 3 mm de largeur. Il n'y avait pas de différences significatives dans les niveaux de contraction en réponse au chlorure de potassium, à la phényléphrine, ni dans les concentrations de phényléphrine nécessaires pour atteindre le niveau de contraction cible entre les anneaux du groupe GALA versus le groupe de saline héparinée. Les courbes dose-réponse du groupe solution GALA ont démontré une amélioration significative des relaxations dépendantes de l'endothélium par rapport au groupe solution saline héparinée. Les contractions et relaxations indépendantes de l'endothélium induites respectivement par la phényléphrine et le nitroprussiate de sodium étaient similaires dans les anneaux de GVS des deux groupes. CONCLUSION : L’utilisation intra-opératoire d'une solution développée spécifiquement pour la préservation de l’intégrité endothéliales présente un potentiel d’avantages cliniques chez les patients qui subissent une chirurgie de PAC. Les observations précédentes suggèrent que la solution GALA pourrait réduire la dysfonction endothéliale associée à la défaillance des greffons veineux et incite des évaluations à long terme plus approfondies dans le cadre d’essais cliniques. / INTRODUCTION: Saphenous vein grafts (SVGs) are still commonly used as conduits for coronary artery bypass grafting (CABG). Injury to SVGs during harvesting and storage promotes endothelial dysfunction, which is attributed to a decrease in production of nitric oxide and/or increased level of oxidative stress that can lead to vein graft failure (VGF). Heparinized saline is still the standard of care intraoperative preservation solution despite several studies demonstrating its detrimental effects on SVGs. GALA is an innovative one-time intraoperative graft storage solution developed to preserve endothelial integrity. OBJECTIVE: To investigate, in a randomized controlled study, endothelial functional integrity of saphenous vein grafts following storage in GALA vs heparinized saline using ex vivo vascular reactivity studies in organ chamber experiments. RESULTS: Segments of saphenous vein grafts from a total of fifteen patients were obtained and divided into 3 mm wide rings for evaluation. There were no significant differences in the levels of contraction in response to potassium chloride and to phenylephrine between groups, nor in the concentrations of phenylephrine needed to achieve the target level of contraction in saphenous vein graft rings. Concentration-response curves of the GALA group demonstrated a significant improvement in endothelium-dependent relaxations compared to the heparinized saline group. Endothelium-independent contractions and relaxations induced by phenylephrine and sodium nitroprusside, respectively, were not altered in saphenous vein graft rings from both groups. CONCLUSIONS: Intraoperative application of a solution developed for graft preservation demonstrated a potential benefit to protect endothelial and vascular functional integrity in saphenous vein grafts of patients undergoing CABG. These data suggest that the GALA solution may reduce endothelial dysfunction associated with vein graft failure and warrant further long-term evaluation in clinical trials.