SIRT1 regulates oxidant- and cigarette smoke-induced eNOS acetylation in endothelial cells: Role of resveratrol

Abstract

Endothelial nitric oxide synthase (eNOS) plays a crucial role in endothelial cell functions. SIRT1, a NAD(+)-dependent deacetylase, is shown to regulate endothelial function and hence any alteration in endothelial SIRT1 will affect normal vascular physiology. Cigarette smoke (CS)-mediated oxidative stress is implicated in endothelial dysfunction. However, the role of SIRT1 in regulation of eNOS by CS and oxidants are not known. We hypothesized that CS-mediated oxidative stress downregulates SIRT1 leading to acetylation of eNOS which results in reduced nitric oxide (NO)-mediated signaling and endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) exposed to cigarette smoke extract (CSE) and H(2)O(2) showed decreased SIRT1 levels, activity, but increased phosphorylation concomitant with increased eNOS acetylation. Pre-treatment of endothelial cells with resveratrol significantly attenuated the CSE- and oxidant-mediated SIRT1 levels and eNOS acetylation. These findings suggest that CS- and oxidant-mediated reduction of SIRT1 is associated with acetylation of eNOS which have implications in endothelial dysfunction.

Figure 1. Effect of CSE and H₂O₂ on SIRT1 levels and activity in HUVECs

SIRT1 levels: A) HUVECs were treated with CSE (0.5% and 1.0%) and H₂O₂ (25, 50 and 100 μM) for indicated time points. B) and C) Histograms represent the relative intensity of SIRT1 levels (bands) compared to respective controls (n=3). SIRT1 activity: D) HUVECs were treated with CSE (1.0%) and H₂O₂ (100 μM) for 1 hr, and at 4 hr alone or in combination with pretreatment of resveratrol (10 μM) and splitomicin (10 μM), and SIRT1 deacetylase activity was determined. Data are Mean ± SE (n=3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. controls.

Figure 1. Effect of CSE and H₂O₂ on SIRT1 levels and activity in HUVECs

SIRT1 levels: A) HUVECs were treated with CSE (0.5% and 1.0%) and H₂O₂ (25, 50 and 100 μM) for indicated time points. B) and C) Histograms represent the relative intensity of SIRT1 levels (bands) compared to respective controls (n=3). SIRT1 activity: D) HUVECs were treated with CSE (1.0%) and H₂O₂ (100 μM) for 1 hr, and at 4 hr alone or in combination with pretreatment of resveratrol (10 μM) and splitomicin (10 μM), and SIRT1 deacetylase activity was determined. Data are Mean ± SE (n=3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. controls.

Figure 2. Effect of CSE and H₂O₂ on SIRT1 phosphorylation in HUVECs

HUVECs were treated with CSE (0.5% and 1.0%) and H₂O₂ (25, 50 and 100 μM) for indicated time points. A) CSE and H₂O₂ increased the phosphorylation of SIRT1 (at Ser27 and Ser47). B) and C) Histograms represent relative intensity of phosphorylated SIRT1 levels compared to respective control treatments (n=3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. controls. p-SIRT1 (Ser27), phosphorylation of SIRT1 at Ser27; p- SIRT1 (Ser47), phosphorylation of SIRT1 at Ser 47.

Figure 3. Effect of proteasome inhibitor on CSE- and H₂O₂-induced degradation of SIRT1 degradation in HUVECs

A) HUVECs cells were pre-treated with or without proteasomal inhibitor N-Acetyl-Leu- Leu-Nle-CHO (ALLN, 5 μM) for 1 hr and then treated with CSE (1.0%) or H₂O₂ (100 μM) for 1 and 4 hr. B) and C) Histograms represent Mean ± SE of relative intensity of SIRT1 levels compared to respective controls (n=3). *P < 0.05, **P < 0.01, ***P < 0.001 vs. controls. ⁺P < 0.05, ⁺⁺P < 0.01 vs. CSE and H₂O₂ treatments alone.

Figure 4. Effect of CSE and H₂O₂ on eNOS acetylation and its regulation by SIRT1 in HUVECs

eNOS acetylation: A) HUVECs were treated with CSE (0.5% and 1.0%) and H₂O₂ (50 and 100 μM) for indicated time points. B) and C) Histograms represent the relative intensity of acetylated lysine levels of eNOS compared to respective controls (n=3). SIRT1 regulation of eNOS acetylation: D) HUVECs cells were pre-treated with or without SIRT1 activator (resveratrol, 10 μM) and inhibitor (splitomicin, 10 μM) for 1 hr and then treated with CSE (1.0%) for 4 hr. E) and F) Histograms represent the relative intensity of acetylated lysine levels of eNOS compared to respective controls (n=3). IgG was used as an isotype control. Data shown are Mean ± SE. **P < 0.01, ***P < 0.001 vs. control groups. ⁺⁺⁺P < 0.001 vs. CSE treatment alone.

Figure 4. Effect of CSE and H₂O₂ on eNOS acetylation and its regulation by SIRT1 in HUVECs

eNOS acetylation: A) HUVECs were treated with CSE (0.5% and 1.0%) and H₂O₂ (50 and 100 μM) for indicated time points. B) and C) Histograms represent the relative intensity of acetylated lysine levels of eNOS compared to respective controls (n=3). SIRT1 regulation of eNOS acetylation: D) HUVECs cells were pre-treated with or without SIRT1 activator (resveratrol, 10 μM) and inhibitor (splitomicin, 10 μM) for 1 hr and then treated with CSE (1.0%) for 4 hr. E) and F) Histograms represent the relative intensity of acetylated lysine levels of eNOS compared to respective controls (n=3). IgG was used as an isotype control. Data shown are Mean ± SE. **P < 0.01, ***P < 0.001 vs. control groups. ⁺⁺⁺P < 0.001 vs. CSE treatment alone.