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Sirt1 inhibition induces aortic endothelial dysfunction. Role of oxidative stress and PPARα. Sirtuins are a family of redox-sensitive NAD+-dependent deacetylases. It has been previously described that mammalian homolog SIRT1 deacetylates endothelial nitric oxide synthase (eNOS) and thus increases eNOS catalytic activity (Mattagajasingh et al., 2007). Hypothesizing that the beneficial effects of resveratrol (one of the main polyphenols in red wine) on vascular function is mediated, in part, by its role as a SIRT1 activator (Howitz et al., 2003), we investigated whether the inhibition of SIRT1 induces endothelial dysfunction and if resveratrol is able to prevent it. Experiments were conducted in aortic rings from male Wistar rats. Rings were incubated in Krebs solution for 5 h in a cell culture incubator in the absence or presence of nicotinamide (NAM) (1 mM) or sirtinol (30 µM), both of them SIRT1 inhibitors, and in the presence of resveratrol (0.1 mM), the NADPH oxidase inhibitor apocynin (100 μM), the peroxisome proliferator-activated receptor-α (PPARα) agonist clofibrate (1 μM), and resveratrol plus the PPARα antagonist GW6471 (1 μM). After equilibration, arteries were stimulated with phenylephrine (1 µM) and a concentration-response curve was constructed by cumulative addition of acetylcholine (ACh). In the case of NAM after stimulation with phenylephrine (1 µM) a concentration-response curve was also constructed by cumulative addition of calcium ionophore (A23187). The protocol using ACh was repeated adding superoxide dismutase (SOD, 100 U/ml) to the organ chamber 30 min before the addition of phenylephrine. NADPH oxidase activity was measured by lucigenin enhanced chemiluminiscence. Aortic superoxide anion (O2 -) production by dihydroethidium (DHE) fluorescence and gene and protein expression by RT-PCR and western blots, respectively. Statistically analysis for multiple comparisons was carried out by one-way ANOVA analysis followed by Bonferroni post hoc test of 6-9 separate experiments. Incubation of the aortic rings for 5 h in the absence of NAM or Sirtinol produced no significant changes in the contractile response to phenylephrine or in the relaxant response to ACh or A23187. Incubating NAM or Sirtinol for 5 h led to the development of endothelial dysfunction as indicated by the reduction in the maximal relaxant effect of ACh (45.2 ± 7.4% and 67.5 ± 3.2%, respectively, and control 84.9 ± 3.5%, p<0.01) or A23187 (57.4 ± 8.3% NAM vs control 78.8 ± 4.6%, p<0.05), while coincubation with resveratrol prevented endothelial dysfunction induced by NAM or Sirtinol (Maximal effect ACh=85.2 ± 9.5% and 86.6 ± 3.8%, respectively). The impaired relaxant response to ACh induced by NAM was completely restored by SOD and apocynin and partially restored by SOD in the case of Sirtinol. Acetyl-histone-3 protein expression was increased by both NAM and sirtinol and inhibited when the rings were coincubated by resveratrol. Ethidium fluorescence was increased by NAM (by ~63%) which was abolished by apocynin. The NADPH oxidase activity and the mRNA expression of its catalytic subunit NOX4 was also increased by NAM (~2.7 and 8.0 times, respectively) and suppressed by coincubation with resveratrol. The improvement in relaxant response to ACh and the reduction in NOX4 expression induced by resveratrol in NAM incubated rings were suppressed by coincubation with the PPARα antagonist GW6471. Moreover, clofibrate exibited similar protective effects than resveratrol in NAM induced impairment of ACh relaxation and increased NOX4 expression. In conclusion, SIRT1 inhibition induced endothelial dysfunction, which was associated to increased NADPH oxidase-driven O2 - production which was prevented by resveratrol, as a result of SIRT-1 activation and the subsequent PPARα activation to downregulate NOX4 expression. Mattagajasingh I et al., (2007). PNAS 104:14855-60. Howitz KT et al., (2003). Nature 425:191-6. Supported by SAF2010-22066.
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