SIRT-1 and vascular endothelial dysfunction with ageing in mice and humans

Abstract

We tested the hypothesis that reductions in the cellular deacetylase, sirtuin-1 (SIRT-1), contribute to vascular endothelial dysfunction with ageing via modulation of endothelial nitric oxide synthase (eNOS) acetylation/activation-associated nitric oxide (NO) production. In older (30 months, n = 14) vs. young (5-7 months, n = 16) B6D2F1 mice, aortic protein expression of SIRT-1 and eNOS phosphorylated at serine 1177 were lower (both P < 0.05), and acetylated eNOS was 6-fold higher (P < 0.05), whereas total eNOS did not differ (P = 0.65). Acetylcholine (ACh)-induced peak endothelium-dependent dilatation (EDD) was lower in isolated femoral arteries with ageing (P < 0.001). Incubation with sirtinol, a SIRT-1 inhibitor, reduced EDD in both young and older mice, abolishing age-related differences, whereas co-administration with l-NAME, an eNOS inhibitor, further reduced EDD similarly in both groups. Endothelium-independent dilatation to sodium nitroprusside (EID), was not altered by age or sirtinol treatment. In older (64 ± 1 years, n = 22) vs. young (25 ± 1 years, n = 16) healthy humans, ACh-induced forearm EDD was impaired (P = 0.01) and SIRT-1 protein expression was 37% lower in endothelial cells obtained from the brachial artery (P < 0.05), whereas EID did not differ. In the overall group, EDD was positively related to endothelial cell SIRT-1 protein expression (r = 0.44, P < 0.01). Reductions in SIRT-1 may play an important role in vascular endothelial dysfunction with ageing. SIRT-1 may be a key therapeutic target to treat arterial ageing.

Figure 1. Protein expression in aortas from young and older mice for eNOS, eNOS phosphorylated at serine 1177, ratio of peNOS to eNOS, SIRT-1 and acetylated eNOS

Protein expression is shown in aortas from young and older mice (n = 7–9 per group) for eNOS (A), eNOS phosphorylated at serine 1177 (peNOS) (B), ratio of peNOS to eNOS (C), SIRT-1 (D) and acetylated (Ac)-eNOS (E). Protein expression of eNOS, peNOS and SIRT-1 is expressed relative to GAPDH and immunopreciptated (IP) Ac-eNOS is expressed relative to no antibody IP control eNOS expression. Representative images are shown below the summary data. GAPDH images are from the same membrane after stripping and re-probing. Data are shown normalized to the young control mean values. Values are means ± SEM. *P < 0.05 vs. young.

Figure 2. Endothelium-dependent and -independent dilatation shown in untreated, sirtinol treated and sirtinol and l-NAME treated femoral arteries from young and older mice

Endothelium-dependent dilatation (acetylcholine, 1 × 10⁻⁹ to 1 × 10⁻⁴m; A) and endothelium-independent dilatation (sodium nitroprusside, 1 × 10⁻¹⁰ to 1 × 10⁻⁴m; B) are shown in untreated (n = 16, young, filled circles; n = 14, older, open circles), sirtinol treated (n = 14, young, filled squares; n = 13, older, open squares) and sirtinol and l-NAME (n = 15, young, filled diamonds; n = 13, older, open diamonds) treated femoral arteries from young and older mice. Values are means ± SEM. *P < 0.05 vs. young.

Figure 3. Endothelium-dependent and -independent dilatation shown in young and older healthy humans

Endothelium-dependent dilatation (forearm blood flow to intra-brachial artery infusion of acetylcholine; 1.0, 2.0, 4.0, and 8.0 μg 100⁻¹ ml forearm tissue; A) and endothelium-independent dilatation (forearm blood flow to intra-brachial artery infusion of sodium nitroprusside; 0.5, 1.0, 2.0 μg 100 ml⁻¹ forearm tissue; B) are shown in young (n = 16, filled circles) and older (n = 22, open circles) healthy humans. Values are means ± SEM. *P = 0.01 vs. young.

Figure 4. Arterial endothelial cell SIRT-1 protein expression and relation to endothelial dependent dilation

SIRT-1 protein expression is shown in endothelial cells obtained from brachial arteries of young (n = 14) and older (n = 18) healthy humans (A). Representative images of the immunofluorescence images of SIRT-1 from individual young and older subjects are shown below the group mean bars. The relation between acetylcholine-induced endothelium-dependent dilatation (area under the curve) and SIRT-1 endothelial cell protein expression is shown for all subjects (B: young, filled circles; older, open circles). Values are means ± SEM. *P < 0.05 vs. young.