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Des modèles de dysfonction érectile (DE) ont été développés et étudiés chez le rat. Cependant, peu de choses sont connues dans certains modèles présentant des facteurs de risque de la DE, en l’occurrence le rat soumis à une diète riche en glucose, le rat avec une restriction de croissance intra-utérine (RCIU) et le rat avec l’infusion continue de l’angiotensine (Ang) II en ce qui concerne l’évaluation de la fonction érectile in vivo. C’est ainsi que nous nous sommes proposés cette étude. La fonction érectile a été mesurée par la stimulation électrique du nerf caverneux à des cohortes de rats mâles Sprague-Dawley de différents âges comme suit: 1) des rats jeunes de 12 semaines d’âge, adultes reproducteurs à la retraite d’environ 30 semaines d’âge et des rats de 27 et 29 semaines soumis à une diète riche en glucose par l’eau de breuvage (10%) à court (1 semaine), moyen (6 semaines) et long (12 ou 13 semaines) termes; 2) des rats jeunes âgés de 12 semaines recevant une infusion soit de l’Ang II (200ng/kg/min) soit de la saline par mini-pompes osmotiques sous-cutanées pendant 1 et 2 semaines; et 3) des rats adultes âgés de 36 semaines avec une RCIU. La fonction érectile a été représentée par des courbes de variations de pression intracaverneuse (PIC) et d’aire sous la courbe (ASC) normalisées avec la pression artérielle moyenne (PAM) en réponse à la stimulation nerveuse à différents volts. À la fin des expériences, les animaux ont été sacrifiés, le sang recueilli pour le dosage de la glycémie et de l’insulinémie; le pénis, l’aorte thoracique et le cœur prélevés à des fins d’analyses protéique et histologique. La diète riche en glucose à court terme a eu un effet néfaste sur la fonction érectile chez le jeune rat mais bénéfique chez le rat adulte reproducteur; de même que pour les études à long terme, la diète riche en glucose a amélioré ou renversé la DE associée au vieillissement. Les rats avec RCIU ont exhibé une baisse très significative de la fonction érectile. Ces détériorations ou améliorations de la fonction érectile avec le glucose ou dans la RCIU ont été principalement associées à des modifications de l’expression de l’Akt, en plus d’une augmentation de l’insuline sérique dans les groupes avec le glucose. L’Ang II a entraîné une baisse de la fonction érectile, statistiquement très significative après 1 semaine et maintenue après 2 semaines de traitement, sans causer des changements morphologiques au cœur et à l’aorte thoracique. En conclusion : l’atteinte du système érectile se fait précocement dans les complications cardiovasculaires liées à l’activation du SRA, l’implication de l’Akt dans la modulation de la fonction érectile dépend de type de vieillissement et de la diète, le RCIU est un facteur de risque pour la DE. / Rat is a most rodent used for erectile function studies, and many erectile dysfunction (ED) rat models have been developed and studied. However, according to our knowledge, erectile function has never been studied in high-glucose fed rat model, intrauterine growth restriction rat model (IUGR) and continuous angiotensin (Ang) II infusion rat model. In this context, we set up this study. We mesured simultaneously mean arterial pression (MAP), intracavernosal pression (ICP), aire under the curve (AUC); and erectile function was obtained by variations of the ratio ICP/MAP and AUC/MAP in response to electrical stimulation of cavernosal nerve (0.89 – 6.50 Volts) in male Sprague-Dawley rats in different protocols as follow: 1) young (12 week old), procreator adult (~30 week old) and adult (27 and 29 week old) rats randomized and fed with high-glucose diet in water (10%) or simple water for a short (1 week), middle (6 weeks) and long (12 and 13 weeks) terms; 2) adult (36 week old) IUGR rats; 3) young (12 week old) rats randomized and received a chronic infusion of AngII (200 ng/kg/min) or saline by osmotic minipumps for 1 and 2 weeks. At the end of procedures, blood for measuring serum glucose and insulin in rats with chronic high-glucose diet was obtained, the heart was excised and left ventricle was weighed. Aorta and penis were excised, cleaned and a section of each was formalin fixed for histological studies and the rest snap frozen for protein studies. The short term high-glucose diet impacted negatively in young rats but positively in procreator adult rats; also, the erectile function was improved or reversed by chronic high-glucose diet in adult rats. We also observed a diminished erectile function in RCIU. The improvement or deterioration of erectile function was associated with changes in Akt expression, besides an increased insulin serum in rats with high-glucose diet. A diminished erectile function was observed after 1 week continuous Ang II infusion and was maintained after 2 week infusion without detectable structural changes on heart and aorta. In conclusion: this study suggests that erectile system is a precoce cible of cardiovascular complications of renin-angiotensin system. High-glucose diet deteriores erectile function but maskes the ageing induced-ED, RCIU is associated with ED in later life. Akt involvement in erectile function modulation depends on aging type and the diet.

Des modèles de dysfonction érectile (DE) ont été développés et étudiés chez le rat. Cependant, peu de choses sont connues dans certains modèles présentant des facteurs de risque de la DE, en l’occurrence le rat soumis à une diète riche en glucose, le rat avec une restriction de croissance intra-utérine (RCIU) et le rat avec l’infusion continue de l’angiotensine (Ang) II en ce qui concerne l’évaluation de la fonction érectile in vivo. C’est ainsi que nous nous sommes proposés cette étude. La fonction érectile a été mesurée par la stimulation électrique du nerf caverneux à des cohortes de rats mâles Sprague-Dawley de différents âges comme suit: 1) des rats jeunes de 12 semaines d’âge, adultes reproducteurs à la retraite d’environ 30 semaines d’âge et des rats de 27 et 29 semaines soumis à une diète riche en glucose par l’eau de breuvage (10%) à court (1 semaine), moyen (6 semaines) et long (12 ou 13 semaines) termes; 2) des rats jeunes âgés de 12 semaines recevant une infusion soit de l’Ang II (200ng/kg/min) soit de la saline par mini-pompes osmotiques sous-cutanées pendant 1 et 2 semaines; et 3) des rats adultes âgés de 36 semaines avec une RCIU. La fonction érectile a été représentée par des courbes de variations de pression intracaverneuse (PIC) et d’aire sous la courbe (ASC) normalisées avec la pression artérielle moyenne (PAM) en réponse à la stimulation nerveuse à différents volts. À la fin des expériences, les animaux ont été sacrifiés, le sang recueilli pour le dosage de la glycémie et de l’insulinémie; le pénis, l’aorte thoracique et le cœur prélevés à des fins d’analyses protéique et histologique. La diète riche en glucose à court terme a eu un effet néfaste sur la fonction érectile chez le jeune rat mais bénéfique chez le rat adulte reproducteur; de même que pour les études à long terme, la diète riche en glucose a amélioré ou renversé la DE associée au vieillissement. Les rats avec RCIU ont exhibé une baisse très significative de la fonction érectile. Ces détériorations ou améliorations de la fonction érectile avec le glucose ou dans la RCIU ont été principalement associées à des modifications de l’expression de l’Akt, en plus d’une augmentation de l’insuline sérique dans les groupes avec le glucose. L’Ang II a entraîné une baisse de la fonction érectile, statistiquement très significative après 1 semaine et maintenue après 2 semaines de traitement, sans causer des changements morphologiques au cœur et à l’aorte thoracique. En conclusion : l’atteinte du système érectile se fait précocement dans les complications cardiovasculaires liées à l’activation du SRA, l’implication de l’Akt dans la modulation de la fonction érectile dépend de type de vieillissement et de la diète, le RCIU est un facteur de risque pour la DE. / Rat is a most rodent used for erectile function studies, and many erectile dysfunction (ED) rat models have been developed and studied. However, according to our knowledge, erectile function has never been studied in high-glucose fed rat model, intrauterine growth restriction rat model (IUGR) and continuous angiotensin (Ang) II infusion rat model. In this context, we set up this study. We mesured simultaneously mean arterial pression (MAP), intracavernosal pression (ICP), aire under the curve (AUC); and erectile function was obtained by variations of the ratio ICP/MAP and AUC/MAP in response to electrical stimulation of cavernosal nerve (0.89 – 6.50 Volts) in male Sprague-Dawley rats in different protocols as follow: 1) young (12 week old), procreator adult (~30 week old) and adult (27 and 29 week old) rats randomized and fed with high-glucose diet in water (10%) or simple water for a short (1 week), middle (6 weeks) and long (12 and 13 weeks) terms; 2) adult (36 week old) IUGR rats; 3) young (12 week old) rats randomized and received a chronic infusion of AngII (200 ng/kg/min) or saline by osmotic minipumps for 1 and 2 weeks. At the end of procedures, blood for measuring serum glucose and insulin in rats with chronic high-glucose diet was obtained, the heart was excised and left ventricle was weighed. Aorta and penis were excised, cleaned and a section of each was formalin fixed for histological studies and the rest snap frozen for protein studies. The short term high-glucose diet impacted negatively in young rats but positively in procreator adult rats; also, the erectile function was improved or reversed by chronic high-glucose diet in adult rats. We also observed a diminished erectile function in RCIU. The improvement or deterioration of erectile function was associated with changes in Akt expression, besides an increased insulin serum in rats with high-glucose diet. A diminished erectile function was observed after 1 week continuous Ang II infusion and was maintained after 2 week infusion without detectable structural changes on heart and aorta. In conclusion: this study suggests that erectile system is a precoce cible of cardiovascular complications of renin-angiotensin system. High-glucose diet deteriores erectile function but maskes the ageing induced-ED, RCIU is associated with ED in later life. Akt involvement in erectile function modulation depends on aging type and the diet.

Made available in DSpace on 2015-05-14T13:00:08Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 7780446 bytes, checksum: c3d9b30a61c17cf2f7b0a011741a719d (MD5) Previous issue date: 2014-10-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Oxidative stress has been indicated as a central mechanism in Ang II-dependent hypertension. Furthermore, previous studies reported that oxidative stress is associated with the impairment of angiotensin converting enzyme 2 (ACE2) compensatory activity by the disintegrin and metalloprotease 17 (ADAM17) during hypertension. In this context, -lipoic acid (LA), a potent antioxidant, has been studied by its effects on cardiovascular system, however its effects on Ang II/ROS/ADAM17/ACE2 pathway have not been studied yet. Here we used two Ang II-dependent hypertensive models, the two-kidney-one-clip (2K1C) and deoxycorticosterone-sal (DOCA-salt) hypertension. Firstly, we analyzed the effects induced by chronic treatment with LA on blood pressure, heart rate and baroreflex sensitivity (sympathetic and parasympathetic components) in renovascular hypertensive rats. Male Wistar rats underwent 2K1C or sham surgery and were maintained untouched for four weeks to develop hypertension. Four weeks post-surgery, rats were orally treated with LA (60 mg/kg) or saline for 14 days. On the 15th day, mean arterial pressure (MAP) and heart rate (HR) were recorded. In addition, baroreflex sensitivity test using phenylephrine (8 μg/kg, i.v.) and sodium nitroprusside (25 μg/kg, i.v.) was performed. Chronic treatment with LA decreased blood pressure in hypertensive animals compared to 2K1C + saline group (133.5 ± 9.3 vs 176.5 ± 9.6 mmHg, p<0.05, respectively); however, no significant changes in baseline HR were observed. Regarding baroreflex, LA treatment increased the sensitivity of both the sympathetic and parasympathetic components (−2.80 ± 0.6 vs −2.61 ± 0.4 and −4.14 ± 0.6 vs. −3.85 ± 0.5 bpm.mm Hg−1, p < 0.05, n = 8, respectively). To investigate the relationship between ADAM17 and oxidative stress, Neuro2A cells were treated with Ang-II (100 nM) 24 h after vehicle or LA (500 μM). ADAM17 expression was increased by Ang-II (100.2 ± 0.8 vs 120.5 ± 9.1%, p<0.05) and decreased after LA (120.5 ± 9.1 vs 69.0 ± 0.3%, p<0.05). In other set of experiments, LA reduced ADAM17 (92.9 ± 5.3 vs control: 77.3 ± 3.3%, p<0.05) following its overexpression. Moreover, ADAM17 activity was reduced by LA in ADAM17-overexpressing cells (109.5 ± 19.8 vs 158.0 ± 20.0 FU/min/μg protein, p<0.05), in which ADAM17 overexpression increased oxidative stress (114.1 ± 2.5 vs 101.0 ± 1.0%, p<0.05). Conversely, LA-treated cells attenuated ADAM17 overexpression-induced oxidative stress (76.0 ± 9.1 vs 114.1 ± 2.5%, p<0.05). In DOCA-salt-hypertensive mice, a model in which ADAM17 expression and activity are increased, hypertension was blunted by pretreatment with LA (119.0 ± 2.4 vs 131.4 ± 2.2 mmHg, p<0.05). In addition, LA improved dysautonomia and baroreflex sensitivity. Furthermore, LA blunted the increase in the expression of NADPH oxidase subunits as well as the increase in ADAM17 and decrease in ACE2 activities in the hypothalamus of DOCA-salt hypertensive mice. Our data suggest that chronic treatment with LA promotes antihypertensive effect and improves baroreflex sensitivity and autonomic function in rats with renovascular hypertension as well as in DOCA-salt mice. Therefore, these data suggest that LA might preserve ACE2 compensatory activity by breaking the feed-forward cycle between ADAM17 and oxidative stress, resulting in reduction in hypertension. / O estresse oxidativo tem sido apontado como um mecanismo fundamental na hipertensão arterial dependente de Ang II. Além disso, o estresse oxidativo está associado com a regulação negativa da enzima conversora de angiotensina tipo 2 (ECA2) por meio da ação da desintegrina e metaloprotease 17 (ADAM17) na hipertensão. Neste contexto, o ácido lipóico (AL), um potente antioxidante, vem sendo estudado por suas ações sobre o sistema cardiovascular e foi selecionado para o estudo na modulação da via Ang II/ROS/ADAM17/ECA2. Neste trabalho foram utilizados dois modelos de hipertensão dependentes de Angiotensina II (Ang II): dois-rins-um-clipe (2R1C) e o modelo desoxicorticosterona-sal (DOCA-sal). Primeiramente, ratos Wistar foram submetidos à cirurgia 2R1C ou Sham, para avaliação do tratamento crônico do AL sobre os parâmetros cardiovasculares. Quatro semanas após a indução da hipertensão renovascular, os ratos foram tratados com AL (60 mg/kg) ou solução salina durante 14 dias, por via oral. No 15º dia, a pressão arterial média (PAM) e freqüência cardíaca (FC) foram registradas. Além disso, o teste da sensibilidade do barorreflexo, utilizando fenilefrina (8 μg/kg, i.v) e nitroprussiato de sódio (25 μg/kg, i.v) foi realizado. O tratamento crônico com AL reduziu a pressão arterial em animais hipertensos, quando comparado ao grupo 2R1C + salina (133,5 ± 9,3 vs 176,5 ± 9,6 mmHg, p<0,05, respectivamente); no entanto, não foram observadas alterações significativas na FC. O tratamento com AL aumentou a sensibilidade de ambos os componentes simpático e parassimpático do barorreflexo (-2,80 ± 0,6 vs -2,61 ± 0,4 e - 4,14 ± 0,6 vs -3,85 ± 0,5 bpm.mm Hg-1, p<0,05, respectivamente). Para investigar a relação entre ADAM17 e o estresse oxidativo, células Neuro2A foram tratadas com Ang II (100 nM) 24h após veículo ou AL (500 μM). Ang II aumentou a expressão da ADAM17 (100,2 ± 0,8 vs 120,5 ± 9,1%, p<0,05) e foi reduzida pelo tratamento com o AL (120,5 ± 9,1 vs 69,0 ± 0,3%, p<0,05). Em outro experimento, AL reduziu a expressão da ADAM17 (92,9 ± 5,3 vs controle: 77,3 ± 3,3%, p<0,05) em células que superexpressaram o ADAM17, bem como sua atividade foi atenuada após o tratamento com AL (109,5 ± 19,8 vs 158,0 ± 20,0 FU/min/mg de proteína, p<0,05). A produção de espécies reativas de oxigênio (ROS) mostrou-se aumentada em células com superexpressão da ADAM17 (114,1 ± 2,5 vs 101,0 ± 1,0%, p <0,05), porém em células tratadas com o AL, a formação de ROS foi atenuada (76,0 ± 9,1 vs 114,1 ± 2,5%, p<0,05). Em camundongos DOCA-sal, modelo em que a expressão e a atividade de ADAM17 encontram-se elevadas, a hipertensão foi diminuída pelo tratamento com AL (119,0 ± 2,4 vs 131,4 ± 2,2 mmHg, p<0,05). Além disso, o AL melhorou a disfunção autonômica e a sensibilidade do barorreflexo. O AL aboliu o aumento da expressão da NADPH-oxidase, bem como o aumento da atividade da ADAM17 e promoveu uma redução na atividade da ECA2 no hipotálamo dos animais DOCA-sal. Esses dados sugerem que o tratamento crônico com AL promove um efeito anti-hipertensivo, com melhora da sensibilidade do barorreflexo e função autonômica em ratos com hipertensão renovascular, bem como no modelo DOCA-sal. Portanto, podemos sugerir que o AL preserva a atividade compensatória da ECA2 por romper o ciclo de retroalimentação positiva entre a ADAM17 e o estress oxidativo, resultando na redução da hipertensão arterial.

Une augmentation de la concentration de l’angiotensine II (Ang II) contribue à la prolifération, la migration et l’hypertrophie des cellules musculaires lisses vasculaires (CMLVs) par l’activation des voies des mitogen-activated protein kinases (MAPK) et de la phosphoinositide 3-kinase (PI3K)/protéine kinase B (PKB/Akt). L’Ang II induit l’activation du facteur de transcription early growth response protein-1 (Egr-1) et sa suractivation est remarquée dans les lésions athérosclérotiques et les modèles animaux de lésions vasculaires. La régulation des facteurs de transcription est effectuée par des histones désacétylases (HDACs) qui désacétylent les lysines des histones et protéines non-histones. L’Ang II induit la phosphorylation et l’export nucléaire de la classe IIa des HDACs, particulièrement les HDAC5, et une augmentation de celles-ci est observée dans les maladies vasculaires. L’Ang II est un puissant activateur des voies des MAPK et de la PI3K/Akt, toutefois l’implication de ces voies dans la phosphorylation des HDAC5 et l’expression de l’Egr-1 dans les CMLVs reste inexplorée. Dans cette étude, l’Ang II a induit la phosphorylation des HDAC5 sur la sérine 498 dans les A10 CMLVs. Un blocage pharmacologique de l’extracellular signal-regulated kinase 1/2 (ERK1/2) par U0126 n’a montré aucun effet significatif sur la phosphorylation et l’exclusion nucléaire des HDAC5 induite par l’Ang II. Par contre, l’inhibition de la voie PI3K par wortmannin, de l’Akt par SC66 ou le knockdown de l’Akt par des petits ARN interférents (siRNA) a atténué la phosphorylation et l’export nucléaire des HDAC5 induits par l’Ang II. Par ailleurs, l’inhibition de l’Akt ou le knockdown de cette kinase a diminué l’expression de l’Egr-1 induite dans les CMLVs stimulées par l’Ang II. L’inhibition des HDACs de la classe IIa par MC1568 ou TMP-195 ou bien le knockdown des HDAC5 a diminué l’expression de l’Egr-1 induite par l’Ang II. De plus, le blocage de l’export nucléaire des HDAC5 par la leptomycine B ou la KPT-330 a empêché la localisation cytoplasmique des HDAC5 et a atténué l’expression de l’Egr-1 en réponse à une stimulation de l’Ang II. L’hypertrophie vasculaire induite par l’Ang II a pu être inhibée par la suppression de l’HDAC5 et l’Egr-1. En conclusion, l’Ang II induit la phosphorylation et l’exclusion nucléaire des HDAC5 par la voie PI3K/Akt et non celle de ERK1/2; de plus, l’Ang II induit l’expression de l’Egr-1 à l’aide des HDAC5 via la voie Akt contribuant ainsi à l’hypertrophie des CMLVs. / Elevated concentration of angiotensin II (Ang II) contributes to vascular smooth muscle cells (VSMCs) proliferation, migration and hypertrophy by the activation of the mitogen-activated protein kinases (MAPK) and phosphoinositide 3-kinase (PI3K)/protein kinase B (PKB/Akt) pathways. Ang II induced the expression of early growth response protein-1 (Egr-1), which is a transcription factor that is upregulated in atherosclerosis lesions and in animal models of vascular injuries. The activation or derepression of gene transcription is mediated by histone deacetylases (HDACs), which deacetylate lysine residues from histone and non-histones proteins. Ang II-induced the phosphorylation and nuclear export of class IIa HDACs, notably HDAC5, and its elevated activation is observed in vascular pathologies. Ang II is a potent activator of the MAPK and PI3K/Akt pathways, however their implication in the phosphorylation of HDAC5 and Egr-1 expression in VSMCs remain unexplored. In this study, Ang II-induced HDAC5 phosphorylation at serine 498 in A10 VSMCs and pharmacological blockade of the extracellular signal-regulated kinase 1/2 (ERK1/2) by U0126 did not affect the phosphorylation and nuclear exclusion of HDAC5 in response to Ang II. Whereas, pharmacological inhibition of the PI3K by wortmannin, Akt by SC66 or small interfering RNA (siRNA)-induced silencing of Akt attenuated Ang II-induced HDAC5 phosphorylation and its nuclear export. Furthermore, inhibition or knockdown of Akt suppressed Ang II-induced Egr-1 expression. In addition, the inhibition of class IIa HDAC5 by MC1568, TMP-195 or HDAC5 knockdown by siRNA reduced Ang II-induced Egr-1 expression. The blockade of the nuclear export of HDAC5 by leptomycin B or KPT-330 prevented the cytoplasmic localization of HDAC5 and attenuated the expression of Egr-1 by Ang II in VSMCs. Moreover, HDAC5 or Egr-1 depletion prevented Ang II-induced cell hypertrophy. In summary, Ang II-induced HDAC5 phosphorylation and its nuclear export is mediated by the PI3K/Akt and not the ERK1/2 pathway, in addition, Ang II-induced Egr-1 expression involves the implication of HDAC5 via the Akt pathway which subsequently leads to VSMC hypertrophy.

<p>This study addresses the issue of how to convert peptides into drug-like non- peptides with retained biological activities at peptide receptors. Angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, Ang II) was used as a model peptide. </p><p>Knowledge of the bioactive conformations of endogenous peptides is invaluable for the conversion of peptides into less peptidic analogues. Effectively constrained cyclic analogues, with retained pharmacological activities, may provide valuable information about the bioactive conformations of the peptide in question. </p><p>This thesis describes the development of synthesis for a number of protected, aldehyde-functionalized building blocks for standard solid phase peptide synthesis, and their use for the preparation of cyclic peptide analogues. The effect of variations in the side-chain lengths of the building blocks, on the outcome of the cyclizations was studied. Incorporation of a building block derived from L-aspartic acid afforded bicyclization towards the C-terininal end of the peptide, while for the corresponding L-glutamic acid derived building block, N-terminal directed bicyclization was achieved. A building block derived from L-2-aminoadipic acid was exploited for monocyclization furnishing <i>cis-</i> and <i>trans-</i> vinyl sulfide bridged peptide analogues. </p><p>The described cyclization methods have been applied to the synthesis of a number of conformationally constrained Ang II analogues, for which the pharmacological properties have been evaluated. Two of the Ang II analogues synthesized displayed high affinities and full agonist activities at the AT₁ angiotensin receptor, and have proven to be useful tools in the search for the bioactive conformation of Ang II.</p>

This study addresses the issue of how to convert peptides into drug-like non- peptides with retained biological activities at peptide receptors. Angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, Ang II) was used as a model peptide. Knowledge of the bioactive conformations of endogenous peptides is invaluable for the conversion of peptides into less peptidic analogues. Effectively constrained cyclic analogues, with retained pharmacological activities, may provide valuable information about the bioactive conformations of the peptide in question. This thesis describes the development of synthesis for a number of protected, aldehyde-functionalized building blocks for standard solid phase peptide synthesis, and their use for the preparation of cyclic peptide analogues. The effect of variations in the side-chain lengths of the building blocks, on the outcome of the cyclizations was studied. Incorporation of a building block derived from L-aspartic acid afforded bicyclization towards the C-terininal end of the peptide, while for the corresponding L-glutamic acid derived building block, N-terminal directed bicyclization was achieved. A building block derived from L-2-aminoadipic acid was exploited for monocyclization furnishing cis- and trans- vinyl sulfide bridged peptide analogues. The described cyclization methods have been applied to the synthesis of a number of conformationally constrained Ang II analogues, for which the pharmacological properties have been evaluated. Two of the Ang II analogues synthesized displayed high affinities and full agonist activities at the AT1 angiotensin receptor, and have proven to be useful tools in the search for the bioactive conformation of Ang II.

L’athérosclérose est une pathologie cardiovasculaire chronique impliquant de nombreux acteurs. Les cellules du muscle lisse vasculaire (CMLV) jouent un important rôle dans la pathogénicité. Lors de la formation des plaques athérosclérotiques, ces cellules entraînent l’augmentation de la taille de l’athérome, accentuent la formation du chapeau fibreux et à long terme, contribuent à l’instabilité de la plaque. Dans cette étude, nous nous sommes intéressés à l’impact d’ARF6 sur les cellules du muscle lisse vasculaire et ses implications pathologiques dans l’athérosclérose. Les ARF sont des GTPases agissant comme interrupteurs moléculaires dans divers processus physiologiques tels que le trafic vésiculaire intracellulaire et le remodelage des lipides membranaires. ARF6 est importante pour la prolifération et la migration cellulaire des CMLV, deux phénomènes importants dans le développement de l’athérosclérose. Nous émettons donc l’hypothèse que la GTPase ARF6 est impliquée dans la progression de l’athérosclérose. En premier lieu, nous avons étudié l’effet de la GTPase dans le phénomène de l’invasion cellulaire. Dans l’athérosclérose, plusieurs facteurs environnementaux influencent l’invasion des CMLV. Nous avons voulu vérifier l’effet d’ARF6 sur l’invasion des CMLV médiée par le facteur de croissance dérivé des plaquettes (PDGF-BB) et l’angiotensine II (Ang II). Dans un modèle humain, l’invasion était diminuée en l’absence d’ARF6. Nous avons démontré que ce mécanisme résultait d’un effet d’ARF6 sur l’activité de la métalloprotéinase matricielle MMP14. En second lieu, nous avons voulu évaluer l’effet d’ARF6 dans un modèle in vivo d’athérosclérose. En utilisant un modèle accéléré d’athérosclérose inductible, nous avons inhibé ARF6 dans les cellules du muscle lisse. Après dix semaines de diète riche en gras, nous avons observé une diminution de la taille des lésions athérosclérotiques dans les souris ARF6 KO, accompagnée d’une réduction de l’expression des facteurs pro-inflammatoires tels qu’IL-6. Dans un modèle in vitro, l’absence d’ARF6 réduisait l’absorption lipidique en agissant sur l’expression des transporteurs. De plus, ARF6 régulait des voies de signalisation impliquées dans l’inflammation. En somme, nous avons démontré l’importance d’ARF6 dans la modulation pathologique des CMLV dans l’athérosclérose. Ainsi, ARF6 contribue à la pathogénicité des CMLV en modulant leur invasion cellulaire tout en jouant un rôle pro-inflammatoire. / Atherosclerosis is a chronic cardiovascular disease characterized by an accumulation of lipids, followed by the infiltration of macrophages and vascular smooth muscle cells (VSMC). VSMC are responsible for the increase of lesion size, the formation of a fibrous cap, and eventually contributing to the plaque instability. In this study, we were interested in the role of ARF6 in the vascular smooth muscle cells and its pathological implications in atherosclerosis. ARF are a family of GTPases that act as molecular switches and are involved in diverse physiological mechanisms, such as vesicular traffic and membrane lipid transformation. In VSMC, ARF6 is important for cell proliferation and migration, two processes involved in atherosclerosis. We therefore hypothesize that the GTPase ARF6 is involved in the development of atherosclerosis through its impact on VSMC. First, we studied the role of ARF6 in the mechanism of cell invasion. In atherosclerosis, multiple environmental factors affect VSMC invasion. We verified the impact of ARF6 on platelet-derived growth factor (PDGF-BB) and angiotensin II (Ang II)-mediated invasion. Using a human model, we observed a reduction of invasion in the absence of ARF6. We have demonstrated that this mechanism is due to the effect of ARF6 on the activity of the matrix metalloproteinase MMP14. Second, we wanted to verify the role of ARF6 in atherosclerosis in an in vivo model. Using an accelerated inducible atherosclerosis model, we inhibited ARF6 in smooth muscle cells. After ten weeks of high-fat diet, we observed a reduction in the size of atherosclerotic lesions in ARF6 KO mice. This reduction was accompanied by a decrease in the expression of proinflammatory factors. In our in vitro model, ARF6 depletion reduced lipid uptake by downregulating the lipidic transporter expression. Also, ARF6 was responsible to activate inflammation signaling pathways. In summary, we have demonstrated the impact of ARF6 in the pathological modulation of VSMC in atherosclerosis. Indeed, ARF6 contributes to the pathogenicity of VSMC through its ability to modulate cell invasion and induce proinflammatory actions.