Chen, Juan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 124-154). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Des altérations de fonctions physiologiques du réticulum endoplasmique (RE) induisent un processus appelé stress RE. Dans le domaine cardiovasculaire, plusieurs travaux ont montré que le stress RE contribue au développement de la majorité des pathologies cardiaques. En réponse au stress RE, la réponse UPR (Unfolded Protein Response) est activée afin de restaurer l’homéostasie du RE et de permettre la survie de la cellule. Néanmoins, dans le cas d’un stress RE excessif ou prolongé, les altérations ne pouvant plus être compensées, la cellule est éliminée par apoptose contribuant au développement de la pathologie cardiaque. Une thérapie prometteuse pour lutter contre ce type de pathologie consisterait donc à moduler la réponse au stress RE afin d’inhiber l’apoptose des cardiomyocytes. Au cours de ma thèse, je me suis intéressé aux modifications induites en réponse au stress RE dans le cœur et au rôle de la sirtuine 1 (SIRT1) dans la modulation de cette réponse. SIRT1 est une déacétylase activée par différents stress cardiaques et connue pour favoriser la survie cellulaire. D’une part, j’ai mis en évidence que le stress RE induit une modification importante de l’architecture des cardiomyocytes et en particulier une augmentation des contacts RE/mitochondries associée à une altération de la fonction mitochondriale. D’autre part, en utilisant une lignée cellulaire (H9c2), des cardiomyocytes de rat adulte et des souris invalidées pour SIRT1, j’ai démontré in vitro et in vivo (i) que SIRT1 est activée et joue un rôle cardioprotecteur en réponse au stress RE, (ii) que SIRT1 limite la réponse UPR en régulant spécifiquement la voie PERK, et (iii) que SIRT1 régule la voie PERK en déacétylant le facteur d’initiation de la traduction, eIF2 sur deux résidus lysine. Ces résultats montrent donc pour la première fois que SIRT1 est impliquée dans la régulation de la réponse apoptotique au stress RE des cardiomyocytes et suggèrent que cette déacétylase serait une cible thérapeutique intéressante pour prévenir l’apoptose dans les pathologies cardiaques liées au stress RE. / Impairment of physiological functions of the endoplasmic reticulum (ER) induces the so-called ER stress. ER stress has been implicated in many cardiovascular diseases including ischemic heart, hypertrophy and heart failure. To overcome the deleterious effect of ER stress, an evolutionarily conserved adaptive response known as Unfolded Protein Response (UPR) is activated in order to restore ER homeostasis and promote cell survival. Nevertheless, in the case of prolonged or severe ER stress, apoptotic cell death is ultimately activated to eliminate stressed cells, thus contributing to the development of the pathology. The modulation of ER stress response, in order to reduce cardiomyocyte apoptosis, thus appears as a promising therapeutic strategy for such pathologies. During my Ph.D thesis, I studied the modification that occur during ER stress response in the heart and the role of the sirtuine 1 (SIRT1) in the modulation of this response. SIRT1 is a deacetylase activated in response to many cardiac stresses to promote cell survival. First, we showed that ER stress induces important structural modifications of cardiomyocytes and in particular an increase in contact sites between ER and mitochondria associated with an alteration of the mitochondrial function. Secondly, using a cell line (H9c2), freshly isolated adult rat ventricular cardiomyocytes and SIRT1-KO mice, we demonstrated in vitro and in vivo (i) that SIRT1 is activated and plays a cardioprotective role in ER stress response, (ii) that SIRT1 attenuates the UPR by specifically regulating the PERK pathway, and (iii) that SIRT1 modulates PERK axis by deacetylating the translation initiation factor, eIF2on two lysine residues. Collectively, our results provide the first evidence that SIRT1 modulates ER stress-induced apoptosis in the heart and suggest that this deacetylase may represent a therapeutic target to prevent apoptosis in cardiac pathologies associated to ER stress.

Sirt1 (Sirtuine 1) est une protéine histone déacétylase dépendante de NAD+ qui stimule la néoglucogénèse et inhibe la glycolyse dans le foie, et qui augmente l’oxydation des acides gras dans le muscle strié squelettique. Le but de ce travail de thèse a été de définir les fonctions métaboliques de Sirt1 dans le muscle strié squelettique. Nous avons tout d’abord montré, à l’aide d’un modèle de souris déficientes pour le gène Sirt1, que Sirt1 régulait l’expression de l’hexokinase II et de SREBP-1c, protéine régulatrice de l’expression de l’hexokinase. De plus, un modèle d’électrotransfert de gènes permettait de mettre en évidence que Sirt1 régulait l’expression de SREBP-1c de façon LXR dépendante. Enfin, l’inhibition de Sirt1 par l’EX527 aboutissait à une diminution de la consommation de glucose chez des myotubes C2C12. Prises ensemble, ces données suggèrent un rôle important de Sirt1 dans la régulation du métabolisme du glucose dans le muscle strié squelettique. Dans un second temps, nous avons déterminé le rôle potentiel de Sirt1 lors d’un jeûne chez des myotubes C2C12. Un jeûne entraînait une augmentation de l’activité cathepsine B + L et une déphosphorylation des protéines AktS473, GSK3S21/S9, p70S6KT412 et S6 S235/S236 qui précédait une amyotrophie des myotubes. La renutrition aboutissait à une rephosphorylation de ces protéines et à un retour à la normale de la taille des myotubes. L’activité cathepsine B + L restait cependant élevée. Enfin, le niveau en ARNm de Sirt1 était augmenté de façon transitoire lors de la renutrition. D’autres mesures de marqueurs des voies protéolytiques et de l’activité de Sirt1 sont à envisager. Nos données ainsi que celles de la littérature suggèrent que Sirt1 pourrait avoir un rôle dans la régulation de l’autophagie lors du jeûne. Pour conclure, ce travail de thèse met en évidence un rôle pour Sirt1 dans la régulation du métabolisme du glucose dans le muscle strié squelettique et apporte de nouvelles perspectives dans l’étude de la régulation de ce métabolisme en conditions pathologiques / Sirt1 (Sirtuin 1) is a NAD+-dependent histone deacetylase, which stimulates gluconeogenesis and inhibits glycolysis in the liver, and which increases fatty acid oxidation in skeletal muscle. The aim of this thesis was to define the metabolic functions of Sirt1 in skeletal muscle. We first showed, using a mouse model lacking the Sirt1 gene, that Sirt1 regulated expression of hexokinase II and SREBP-1c, a protein that regulates hexokinase expression. In addition, a model of gene electrotransfer allowed us to show that Sirt1 regulated expression of SREBP-1c in a LXR-dependent manner. Finally, inhibition of Sirt1 by EX527 resulted in a decrease of glucose consumption in C2C12 myotubes. Taken together, these data suggest an important role of Sirt1 in the regulation of glucose metabolism in skeletal muscle. Secondly, we determined the potential role of Sirt1 during fasting in C2C12 myotubes. Fasting resulted in an increase in cathepsin B + L activity and a dephosphorylation of AktS473, GSK3S21/S9, p70S6KT412 and S6 S235/S236 preceding a myotubes atrophy. Refeeding led to a rephosphorylation of these proteins and a return to normal size of myotubes. However, cathepsin B + L activity remained elevated. Finally, the level of Sirt1 mRNA was transiently increased during refeeding. Other measures of proteolytic pathways and Sirt1 activity markers will be determined. Our data and those of the literature suggest that Sirt1 could play a role in autophagyregulation during fasting. To conclude, this thesis highlights a role for Sirt1 in the regulation of glucose metabolism in skeletal muscle and provides new perspectives in the study of regulation of this metabolism in pathological conditions

Background: Pulmonary arterial hypertension (PAH) is a devastating disease characterized by increased pulmonary artery pressure, leading to right ventricle hypertrophy and ultimately heart failure and death. Sirtuin 1 (SIRT1) is an NAD+ dependent protein deacetylase that has been strongly implicated as a crucial link between longevity, stress response and maintenance of vascular health. In this thesis, we investigated the role of SIRT1 in the pulmonary vascular hypoxic response and the pathogenesis of pulmonary hypertension (PH) working under the hypothesis that SIRT1 plays a protective role in the pulmonary vasculature and that lack of SIRT1 would lead to worsening of PH in a model of chronic hypoxia (CH). Results: We determined that global SIRT1 knockout or SIRT1 catalytic inactivation resulted in a marked increase in right ventricle pressure and remodeling compared to wildtype mice in CH. Furthermore, hypoxia-induced erythrocytosis and pulmonary vascular remodeling were profoundly increased in both SIRT1 mouse lines. Subsequent molecular assessment revealed that SIRT1 knockout, but not inactivation, led to a significant increase in mRNA levels of hypoxia inducible factor (HIF)-1α and significantly higher activity in hypoxia, leading to elevated lactate dehydrogenase A (LDHA) and BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) in the lungs. Interestingly, both knockout and inactivation of SIRT1 enhanced the activity of HIF2α in the hypoxic lungs and kidneys, leading to increased erythropoietin (EPO) and plasminogen activator inhibitor-1 (PAI-1). Moreover, SIRT1 knockout or inactivation was associated with a trend towards hypoxic-independent increases in HIF3α mRNA in the lungs. Prevention of glycolytic shift using dichloroacetate (DCA) did not result in improvement in this model, yet resveratrol (RSV), a SIRT1 activator/mimic, partially prevented PH only in absence of SIRT1 activity. Finally, selective endothelial cell SIRT1 deletion was sufficient to cause worse PH in the CH model. Conclusions: SIRT1 plays a protective role in the hypoxic response through transcriptional and non-transcriptional control of the hypoxia inducible factors, thus protecting against worse hypoxia-induced PH. SIRT1 could be a novel target for future therapies in PAH.

BACKGROUND: Sirtuin-1 (SirT1) is a NAD+-dependent deacetylase essential for maintaining the structure and function of the vasculature. Reduced SirT1 expression and activity has been correlated with the development of vascular diseases, mainly attributed to loss of SirT1’s anti-oxidant and anti-inflammatory beneficial effects. We previously found that deletion of vascular smooth muscle (VSM) SirT1 in mice is associated with increased matrix metalloproteinases (MMPs) and the subsequent development of aortic dissections or ruptures in response to the hypertensive peptide angiotensin II. Based on these previous findings, we hypothesize that loss of SirT1 activity is involved in the pathogenesis of AA. SirT1 is a stress response gene, its deacetylase activity can be impaired by excessive oxidative stress. We postulate that mutating three cysteine residues in SirT1’s catalytic domain can prevent its inactivation by oxidative insults and protect against AA and other vascular diseases. OBJECTIVES: assess the role of SirT1 in a genetic mouse model of Marfan Syndrome that develops AA; (2) Determine design and optimize an enzyme-based colorimetric ELISA to determine SirT1 activity in mouse VSM cells and aortas; (3) Produce an adeno-associated virus (AAV) expressing an oxidant-resistant triple mutant SirT1 in VSM cells that has the potential to mitigate the downstream outcomes derived from alterations in SirT1 activity, such as MMPs activation and development of AA in mgR-/- mice. METHODS: mgR-/- and littermate mgR+/+ (WT) mice aortas and VSM cells were cultured in conditioned medium and the activity of released MMPs was determined by in-gel zymography. For the development of the SirT1 activity assay, we designed a multi-step sandwich ELISA that captures a biotin- and FLAG-tagged acetylated p53 peptide, used as SirT1 deacetylase substrate. Amounts of acetylated and total p53 peptide were sequentially detected with antibodies and colorimetric substrates as index of SirT1 deacetylase activity. AAVs expressing a control or triple mutant SirT1 (3M) were produced in HEK293T cells; VSM cells were then infected with control or 3M AAV and SirT1 protein expression levels were measured by Western Blot. RESULTS: MMPs activity is increased in aortas and VSMC of mgR-/- mice; the first stage of optimization of the SirT1 activity assay successfully defined the assay conditions and experimental design, and it is ready to be optimized with mgR-/- cell and tissue samples; our novel control and SirT1 triple mutant AAVs were produced and successfully overexpressed in VSM cells. / 2024-03-14T00:00:00Z

T cell dysfunction has a crucial role in establishing and maintaining viral persistence. We have previously shown a decline in miR-181a, which regulates CD4+ T cell responses via DUSP6 overexpression, in individuals with hepatitis C virus (HCV) infection. Here, we describe accelerated T cell senescence in HCV-infected individuals compared with age-and sex-matched healthy subjects. Mechanistic studies revealed that up-regulation of transcription factor ∆Np63 led to the decline of miR-181a expression, resulting in an overexpression of the antiaging protein Sirt1, in CD4+ T cells from HCV-infected individuals. Either reconstituting miR-181a or silencing ∆Np63 or Sirt1 expression in CD4+ T cells led to accelerated T cell senescence, as evidenced by an increased senescence-associated b-galactosidase (SA-β-gal) expression, shortened telomere length, and decreased EdU incorporation; this suggests that HCV-induced T cell senescence is counterregulated by the ∆Np63-miR-181a-Sirt1 pathway. An increase of IL-2 production was observed in these senescent CD4+ T cells and was driven by a markedly reduced frequency of Foxp3+ regulatory T (Treg) cells and increased number of Foxp3- effector T (Teff) cells upon manipulating the ∆Np63-miR-181a-Sirt1 pathway. In conclusion, these findings provide novel mechanistic insights into how HCV uses cellular senescent pathways to regulate T cell functions, revealing new targets for rejuvenating impaired T cell responses during chronic viral infection.

Sirtuin-1 (SirT1) is a nicotinamide adenine dinucleotide (NAD+)-dependent lysine deacetylase expressed ubiquitously in the body. In the vasculature, SirT1 is present in endothelial and vascular smooth muscle cells (VSMCs), where it has been shown to promote anti-inflammatory and anti-oxidant effects. As a result, SirT1 is known to play a protective role in the vasculature wall from pathologies such as atherosclerosis, arterial stiffness, and aortic aneurysm. Hence, SirT1 is considered an attractive therapeutic target for vascular diseases and potentially, aging-related and metabolic diseases. However, currently available SirT1 activators have failed to reach the clinic. Thus, novel approaches to activate SirT1 are needed. In this study, we first sought to optimize a novel fluorescence-based SirT1 activity assay, with which to reliably assess intracellular SirT1 activity and the efficacy of SirT1 activators and inhibitors. We next sought to use the SirT1 activity assay to screen novel compounds identified by an in silico docking analysis and hypothesized to activate SirT1. Lastly, we generated adeno-associated viruses (AAV) overexpressing wildtype (WT) or a redox-resistant (3M) SirT1 to analyze the effects of overexpressing SirT1 in VSMCs, in normal and oxidative stress conditions. For the activity assay, our results showed that an optimal standard curve range was between 0 ng and 12 ng of substrate (acetylated-p53 peptide). After testing different commercially available human recombinant SirT1s, the Anaspec SirT1 of the highest concentration showed a decrease in measured fluorescence for acetylated-p53 peptide with higher SirT1 (ng), indicating the enzyme and the assay were functional. However, when novel small molecules (A4, B4, and G3) hypothesized to activate SirT1 were added to reactions, the total p53 peptide fluorescence values increased compared to the control, suggesting some interference of the molecules with the assay detection. After AAV infection in VSMCs, SirT1 expression, measured by HA-tag, increased for AAV WT (n=3, p=0.04) and similarly for AAV 3M SirT1, indicating that the AAVs efficiently infect VSMCs. SirT1 activity, measured by Western Blot as decreased acetylated-histone (H3), also appeared to increase for both AAV WT and AAV 3M. A similar trend was shown for VSMCs under oxidant stress conditions (n=2). In conclusion, we successfully established a standard curve range for a novel SirT1 activity assay. Further trials are needed to ensure activity assay reproducibility before testing the efficacy of SirT1 activators and inhibitors. Infection of AAV WT and 3M SirT1 led to an increase in the expression and activity of SirT1 in VSMCs. The expression of SirT1 by AAV may be a promising therapeutic option for in vivo prevention and treatment of vascular diseases. / 2026-03-01T00:00:00Z

A deterioração da qualidade do ar ameaça a qualidade de vida dos habitantes nos grandes centros urbanos. O material particulado é constituído por partículas sólidas, liquidas ou ambas, suspensas no ar. Os idosos são o grupo mais susceptível aos efeitos negativos da poluição atmosférica e essa população vem crescendo exponencialmente no mundo inteiro, afetando quase todos os países do mundo, inclusive o Brasil, atualmente representado por um contingente populacional correspondente a 12% da população. Há fortes evidências científicas que a família das sirtuínas tem grande influência no processo do envelhecimento e longevidade em mamíferos. Alguns estudos apontam uma função protetora de Sirt1 contra danos oxidativos e inflamatórios. O objetivo desse estudo é avaliar o impacto das partículas de exaustão do diesel (DEP) sobre as alterações pulmonares decorrentes do processo do envelhecimento e a possível influência das sirtuínas nesse processo. Camundongos na idade de 02 meses e 15 meses foram expostos às DEPs ou ar filtrado por um período de 30 dias. Os parâmetros funcionais foram mensurados pelo FlexiVent. Após as exposições os pulmões foram coletados e o perfil inflamatório avaliado no lavado broncoalveolar e no soro sanguíneo, por meio das mensurações das citocinas inflamatórias. O estresse oxidativo foi avaliado pela técnica espectrofotometria e as expressões gênicas das sirtuínas foram mensuradas pela técnica de RT-PCR no homogenato do tecido pulmonar. De forma geral, além das diferenças fisiológicas relacionadas com a idade observadas entre os grupos, às exposições as DEPs afetaram o perfil inflamatório e as enzimas antioxidantes nos animais jovens e idosos. Além disso, o grupo idoso apresentou comprometeram no ganho de peso e aumento na deposição de colágeno no tecido pulmonar. As exposições diminuíram os níveis de expressão gênica de Sirt6, enquanto Sirt1 foi diminuída com a idade. Em conjunto, os dados obtidos enfatizam que as exposições ambientais aceleraram as alterações associadas ao envelhecimento pulmonar / The deterioration of the air quality is a threat to the habitants\' quality of life of the urban areas. The particulate matter is constituted of solid, liquid or both particles suspended in the air. The elderly is the most susceptible group to the harmful effects of the air pollution and this population is growing exponentially all over the world, affecting almost all the countries, including Brazil, representing 12.1% of the population. There are strong scientific evidences that the Sirtuins family has great influence on the aging and longevity process in mammals. Some studies points out a Sirt1 protective function against oxidative and inflammatory damage. This study aims to evaluate the impact of the diesel exhaust particulate (DEP) on the pulmonary changes resulting from the aging process and the possible influence of the sirtuins in this process. Two and fifteen months old mice were exposed to DEP for a period of 30 days. Functional assessment of these animals was measured by FlexiVent. After the exposures period, the lungs were collected and the inflammatory profile was evaluated in the bronchoalveolar lavage fluid and blood serum, by means of the measurements of the inflammatory cytokines. Oxidative stress was evaluated by the spectrophotometric technique and the sirtuin gene expression was measured by the RT-PCR technique in the pulmonary tissue homogenate. Our data showed that, besides the age-related physiological differences observed between groups; exposure to DEPs affected the inflammatory profile and antioxidant enzymes activity in young and old animals. In addition, the elderly group presented compromised in weight gain and increased collagen deposition in lung tissue. The exposures decreased gene expression levels of Sirt6, while Sirt1 was decreased due to age. Taken together, the data obtained emphasize that environmental exposures accelerated changes associated with pulmonary aging

A deterioração da qualidade do ar ameaça a qualidade de vida dos habitantes nos grandes centros urbanos. O material particulado é constituído por partículas sólidas, liquidas ou ambas, suspensas no ar. Os idosos são o grupo mais susceptível aos efeitos negativos da poluição atmosférica e essa população vem crescendo exponencialmente no mundo inteiro, afetando quase todos os países do mundo, inclusive o Brasil, atualmente representado por um contingente populacional correspondente a 12% da população. Há fortes evidências científicas que a família das sirtuínas tem grande influência no processo do envelhecimento e longevidade em mamíferos. Alguns estudos apontam uma função protetora de Sirt1 contra danos oxidativos e inflamatórios. O objetivo desse estudo é avaliar o impacto das partículas de exaustão do diesel (DEP) sobre as alterações pulmonares decorrentes do processo do envelhecimento e a possível influência das sirtuínas nesse processo. Camundongos na idade de 02 meses e 15 meses foram expostos às DEPs ou ar filtrado por um período de 30 dias. Os parâmetros funcionais foram mensurados pelo FlexiVent. Após as exposições os pulmões foram coletados e o perfil inflamatório avaliado no lavado broncoalveolar e no soro sanguíneo, por meio das mensurações das citocinas inflamatórias. O estresse oxidativo foi avaliado pela técnica espectrofotometria e as expressões gênicas das sirtuínas foram mensuradas pela técnica de RT-PCR no homogenato do tecido pulmonar. De forma geral, além das diferenças fisiológicas relacionadas com a idade observadas entre os grupos, às exposições as DEPs afetaram o perfil inflamatório e as enzimas antioxidantes nos animais jovens e idosos. Além disso, o grupo idoso apresentou comprometeram no ganho de peso e aumento na deposição de colágeno no tecido pulmonar. As exposições diminuíram os níveis de expressão gênica de Sirt6, enquanto Sirt1 foi diminuída com a idade. Em conjunto, os dados obtidos enfatizam que as exposições ambientais aceleraram as alterações associadas ao envelhecimento pulmonar / The deterioration of the air quality is a threat to the habitants\' quality of life of the urban areas. The particulate matter is constituted of solid, liquid or both particles suspended in the air. The elderly is the most susceptible group to the harmful effects of the air pollution and this population is growing exponentially all over the world, affecting almost all the countries, including Brazil, representing 12.1% of the population. There are strong scientific evidences that the Sirtuins family has great influence on the aging and longevity process in mammals. Some studies points out a Sirt1 protective function against oxidative and inflammatory damage. This study aims to evaluate the impact of the diesel exhaust particulate (DEP) on the pulmonary changes resulting from the aging process and the possible influence of the sirtuins in this process. Two and fifteen months old mice were exposed to DEP for a period of 30 days. Functional assessment of these animals was measured by FlexiVent. After the exposures period, the lungs were collected and the inflammatory profile was evaluated in the bronchoalveolar lavage fluid and blood serum, by means of the measurements of the inflammatory cytokines. Oxidative stress was evaluated by the spectrophotometric technique and the sirtuin gene expression was measured by the RT-PCR technique in the pulmonary tissue homogenate. Our data showed that, besides the age-related physiological differences observed between groups; exposure to DEPs affected the inflammatory profile and antioxidant enzymes activity in young and old animals. In addition, the elderly group presented compromised in weight gain and increased collagen deposition in lung tissue. The exposures decreased gene expression levels of Sirt6, while Sirt1 was decreased due to age. Taken together, the data obtained emphasize that environmental exposures accelerated changes associated with pulmonary aging

Il a été démontré que la sirtuine 1 (Sirt-1), une histone désacétylase de classe III, est surexprimée dans le coeur des rats spontanément hypertendus (SHR). Nous avons récemment montré que les cellules musculaires lisses vasculaires (CMLV) des SHR présentent une expression accrue de Sirt-1 par rapport aux rats Wistar Kyoto (WKY) de même âge qui contribue à l’augmentation de la régulation de la protéine Giα impliquée dans la pathogenèse de l'hypertension. La présente étude a été effectuée pour étudier le rôle de l'augmentation de l'expression de la Sirt-1 dans la pathogenèse de l'hypertension chez les SHR et pour explorer les mécanismes moléculaires impliqués dans cette réponse. Dans cette étude, un inhibiteur sélectif de la Sirt-1, EX-527 (5 mg/kg de poids corporel), a été injecté par voie intrapéritonéale chez des rats SHR adultes de 8 semaines et des rats WKY de même âge, deux fois par semaine pendant 3 semaines. La pression artérielle (PA) et la fréquence cardiaque ont été mesurées deux fois par semaine par la méthode non invasive du brassard autour de la queue. Le traitement avec l’inhibiteur spécifique de la Sirt-1, l'EX-527, a atténué les augmentations de PA (de 76 mmHg) et de fréquence cardiaque chez les rats SHR. La surexpression de Sirt-1 et des protéines Giα dans le coeur, les CMLV et l'aorte a été atténuée au niveau des contrôles par l'inhibiteur de la Sirt-1. L'inhibition de la Sirt-1 a également atténué les niveaux accrus des anions superoxydes, l’activité de la NADPH oxydase et la surexpression des sous-unités de la NADPH oxydase ; les protéines Nox2, Nox4 et P47phox dans les CMLV isolées des SHR traités par l’EX-527. De plus, les niveaux réduits du monoxyde d'azote synthase endothélial (eNOS) et du monoxyde d'azote (NO) et les niveaux accrus de la peroxynitrite (ONOO-) dans les CMLV des SHR ont également été rétablis à des niveaux contrôles par l'inhibiteur de la Sirt-1. Ces résultats suggèrent que l'inhibition de la surexpression de la Sirt-1, en diminuant les niveaux accrus des protéines Giα et du stress nitro-oxydant, atténue la PA élevée chez les rats SHR. Il est donc possible de suggérer que les inhibiteurs de la Sirt-1 puissent être utilisés comme des agents thérapeutiques dans le traitement des complications cardiovasculaires associées à l'hypertension. / Sirtuin-1 (Sirt-1), class III histone deacetylase, has been shown to be overexpressed in hearts from spontaneously hypertensive rats (SHR). We recently showed that vascular smooth muscle cells (VSMC) from SHR exhibit enhanced expression of Sirt-1 as compared to age-matched Wistar Kyoto (WKY) rats, which contributes to the upregulation of Giα protein implicated in the pathogenesis of hypertension. The present study was undertaken to investigate the role of upregulated Sirt-1 expression in the pathogenesis of hypertension in SHR and to explore the underlying molecular mechanisms involved in this response. For this study, a selective inhibitor of Sirt-1, EX-527 (5mg/kg of body weight), was injected intraperitoneally into 8-week-old adult SHR and age-matched WKY rats twice per week for 3 weeks. The blood pressure (BP) and heart rate was measured twice a week by the CODA™ non-invasive tail cuff method. Treatment of SHR with Sirt-1-specific inhibitor, EX-527, attenuated high BP by 76 mmHg and inhibited the augmented heart rate. The overexpression of Sirt-1 and Giα proteins in heart, VSMC and aorta was attenuated to the control levels by Sirt-1 inhibitor. Inhibition of Sirt-1 also attenuated the enhanced levels of superoxide anion, NADPH oxidase activity and the overexpression of NADPH oxidase subunits; Nox2, Nox4 and P47phox proteins in VSMC isolated from EX-527-treated SHR. Furthermore, the decreased levels of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) and increased levels of peroxynitrite (ONOO-) in VSMC from SHR were also restored to control levels by Sirt-1 inhibitor. These results suggest that the inhibition of overexpression of Sirt-1 through decreasing the enhanced levels of Giα proteins and nitro-oxidative stress attenuates the high BP in SHR. It may thus be suggested that inhibitors of Sirt-1 may have the potential to be used as therapeutic agents in the treatment of cardiovascular complications associated with hypertension.