The SIRT1 activator SRT1720 reverses vascular endothelial dysfunction, excessive superoxide production, and inflammation with aging in mice

Abstract

Reductions in arterial SIRT1 expression and activity with aging are linked to vascular endothelial dysfunction. We tested the hypothesis that the specific SIRT1 activator SRT1720 improves endothelial function [endothelium-dependent dilation (EDD)] in old mice. Young (4-9 mo) and old (29-32 mo) male B6D2F1 mice treated with SRT1720 (100 mg/kg body wt) or vehicle for 4 wk were studied with a group of young controls. Compared with the young controls, aortic SIRT1 expression and activity were reduced (P < 0.05) and EDD was impaired (83 ± 2 vs. 96 ± 1%; P < 0.01) in old vehicle-treated animals. SRT1720 normalized SIRT1 expression/activity in old mice and restored EDD (95 ± 1%) by enhancing cyclooxygenase (COX)-2-mediated dilation and protein expression in the absence of changes in nitric oxide bioavailability. Aortic superoxide production and expression of NADPH oxidase 4 (NOX4) were increased in old vehicle mice (P < 0.05), and ex vivo administration of the superoxide scavenger TEMPOL restored EDD in that group. SRT1720 normalized aortic superoxide production in old mice, without altering NOX4 and abolished the improvement in EDD with TEMPOL, while selectively increasing aortic antioxidant enzymes. Aortic nuclear factor-κB (NF-κB) activity and tumor necrosis factor-α (TNF-α) were increased in old vehicle mice (P < 0.05), whereas SRT1720 normalized NF-κB activation and reduced TNF-α in old animals. SIRT1 activation with SRT1720 ameliorates vascular endothelial dysfunction with aging in mice by enhancing COX-2 signaling and reducing oxidative stress and inflammation. Specific activation of SIRT1 is a promising therapeutic strategy for age-related endothelial dysfunction in humans.

Keywords: COX-2 dilation; SIRT1; aging; superoxide; vascular inflammation.

Fig. 1.

Endothelium-dependent and endothelium-independent dilation. Dose responses to the endothelium-dependent dilator acetylcholine (ACh; A) and endothelium-independent dilator sodium nitroprusside (SNP; B) in young control (YC), young SRT1720-treated (YS), old vehicle-control (OV), and old SRT1720-treated (OS) mice. Dose responses to ACh (C) and SNP (D) in YC and young vehicle-treated (YV) mice. Values are means ± SE. ACh: YC, n = 38; OV, n = 17; YS, n = 24; OS, n = 28; YV, n = 11. SNP: YC, n = 23; OV, n = 11; YS, n = 13; OS, n = 17; YV, n = 6. Differences in dose-response curves were assessed with repeated-measures ANOVA. Least squares differences post hoc test was used to determine individual group differences to ACh. *P < 0.01 vs. YC; †P < 0.01 vs. OV.

Fig. 2.

Nitric oxide (NO)-, cyclooxygenase (COX)-, and COX-2-dependent modulation of endothelium-dependent dilation (EDD). EDD to ACh in the absence or presence of NO synthase inhibitor N^G-nitro-l-arginine methyl ester (l-NAME) in YC and YS mice (A) and OV and OS mice (B). C: NO-mediated ACh dilation (maximal dilation to ACh − maximal dilation to ACh + l-NAME) in YC, OV, YS, and OS mice. EDD to ACh in the absence or presence of l-NAME and l-NAME plus the COX inhibitor indomethacin (Indo) in OV and OS mice (D) and in YC and YS mice (E). F: maximal EDD to ACh in the absence or presence of Indo or the COX-2 specific inhibitor N-(2-cyclohexyloxy-4-nitrophenyl)-methanesulfon-amide (NS-398) in YC, OV, YS, and OS mice. Values are means ± SE. ACh + l-NAME: YC, n = 12; OV, n = 11; YS, n = 14; OS, n = 14. NO-mediated ACh dilation: YC, n = 12; OV, n = 10; YS, n = 12; OS, n = 12. ACh + l-NAME + Indo: YC, n = 8; OV, n = 7; YS, n = 8; OS, n = 7. ACh + Indo: YC, n = 11; OV, n = 6; YS, n = 8; OS, n = 9. ACh + NS-398: YC, n = 12; OV, n = 7; YS, n = 7; OS, n = 13. Differences in dose-response curves were assessed with repeated-measures ANOVA. Differences in NO-mediated ACh maximal dilation and in maximal dilation to ACh with or without Indo and NS-398 were determined via one-way ANOVA and least squares differences post hoc test. †P < 0.05 vs. YC ACh; ‡P < 0.05 YS ACh; §P < 0.05 vs. OV ACh; #P < 0.05 vs. OS ACh; ◇P < 0.05 vs. YC NO-mediated ACh dilation. *P < 0.05 vs. OS ACh + l-NAME.

Fig. 3.

Arterial SIRT1 expression and activity. Aortic protein expression of SIRT1 (A) and ratio of acetylated-p53 to total p53 (B) in YC, OV, YS, and OS mice. Data are expressed relative to GAPDH and normalized to YC mean value. Individual representative Western blot bands from a single image are at bottom. AU, arbitrary units. Values are means ± SE. SIRT1: YC, n = 15; OV, n = 11; YS, n = 14; OS, n = 14. Acetyl-p53/p53: YC, n = 7; OV, n = 8; YS, n = 5; OS, n = 5. Differences in protein expression were determined with one-way ANOVA and least squares differences post hoc test. *P < 0.05 vs. YC; †P < 0.05 vs. OV.

Fig. 4.

Aortic expression of COX enzymes. Aortic protein expression of COX-1 (A) and COX-2 (B) in YC, OV, YS, and OS mice. Data are expressed relative to GAPDH and normalized to YC mean value. Individual representative Western blot bands from a single image are at bottom. Values are means ± SE. COX-1: YC, n = 7; OV, n = 8; YS, n = 11; OS, n = 9. COX-2: YC, n = 6; OV, n = 6; YS, n = 8; OS, n = 6. Differences in protein expression were determined with one-way ANOVA. Least squares differences post hoc test was used to determine individual group differences to COX-2. *P = 0.05 vs. YC; †P < 0.05 vs. OV.

Fig. 5.

Arterial superoxide production, superoxide-dependent modulation of endothelium-dependent dilation, and oxidant enzyme expression. A: mean electron paramagnetic resonance (EPR) signal of aortic rings from YC, OV, YS, and OS mice. B: maximal dilation of carotid arteries to ACh and to ACh + TEMPOL, a superoxide scavenger, in YC, OV, YS, and OS mice. C: aortic protein expression of NADPH oxidase 4 (NOX4) in YC, OV, YS, and OS mice. Western blot data are expressed relative to GAPDH and normalized to YC mean value. Individual representative Western blot bands from a single image are at bottom. Values are means ± SE. EPR: YC, n = 16; OV, n = 5; YS, n = 7; OS, n = 7. ACh + TEMPOL: YC, n = 8; OV, n = 6; YS, n = 7; OS, n = 8. NOX4: YC, n = 9; OV, n = 8; YS, n = 7; OS, n = 7. Group n for ACh are listed in Fig. 1. Differences in EPR and maximal dilation in the presence or absence of TEMPOL were determined with one-way ANOVA and least squares differences post hoc test. Differences in NOX4 protein expression were determined with one-way ANOVA (P = 0.06) and two-way ANOVA (treatment × age). *P < 0.05 vs. YC; †P < 0.05 vs. OV; ‡P < 0.05 vs. YC ACh; §P < 0.05 vs. OV ACh.

Fig. 6.

Arterial antioxidant enzymes. Aortic protein expression of manganese superoxide dismutase (MnSOD; A), catalase (B), copper zinc superoxide dismutase (CuZnSOD; C), and extracellular superoxide dismutase (ecSOD; D) from YC, OV, YS, and OS mice. Data are expressed relative to GAPDH and normalized to YC mean value. Individual representative Western blot bands from a single image are at bottom. Values are means ± SE. MnSOD: YC, n = 16; OV, n = 13; YS, n = 15; OS, n = 15. Catalase: YC, n = 11; OV, n = 10; YS, n = 12; OS, n = 13. CuZnSOD: YC, n = 11; OV, n = 9; YS, n = 10; OS, n = 10. ecSOD: YC, n = 13; OV, n = 11; YS, n = 15; OS, n = 13. Differences in protein expression were determined with one-way ANOVA and least squares differences post hoc test. *P < 0.05 vs. YC; †P < 0.05 vs. OV.

Fig. 7.

Arterial inflammation. Aortic protein expression of ratio of nuclear factor-κB (NF-κB) subunit acetyl-p65 to total p65 (A), tumor necrosis factor-α (TNF-α; B), and interferon-γ (IFN-γ; C) in YC, OV, YS, and OS mice. Western data are expressed relative to GAPDH and normalized to YC mean value. Individual representative Western blot bands from a single image are at bottom. Values are mean ± SE. NF-κB acetyl-p65/p65: YC, n = 6; OV, n = 6; YS, n = 8; OS, n = 8. TNF-α: YC, n = 4; OV, n = 5; YS, n = 3; OS, n = 6. IFN-γ: YC, n = 4; OV, n = 5; YS, n = 3; OS, n = 6. Differences in protein expression and cytokine levels were determined with one-way ANOVA and least squares differences post hoc test. *P < 0.05 vs. YC; †P < 0.05 vs. OV; §P = 0.1 vs. OV.

Fig. 8.

Working hypothesis. In old mice, treatment with SRT1720 increases aortic SIRT1 activity and expression and this results in increased arterial COX-2 protein and enhanced COX-2 mediated vasodilation, as well as an upregulation of aortic antioxidant enzymes and reduced vascular oxidative stress, and a decrease in NF-κB acetyl-p65 levels and a reduction in arterial inflammation. These changes restore vascular homeostasis and ameliorate the age-related decline in endothelial function, which may reduce the risk of cardiovascular disease.