Sirt1 inhibits resistin expression in aortic stenosis

Abstract

The development of human calcified aortic stenosis (AS) includes age-dependent processes that have been involved in atherosclerosis, such as infiltration of macrophages in aortic valves, which then promote production of many pro-inflammatory cytokines, including resistin. However, the molecular mechanisms contributing to these processes are not established. Since Sirt1 has been shown to modulate macrophage biology and inflammation, we examined its levels in human AS and tested its impact on resistin expression. Sirt1 mRNA (p = 0.01) and protein (p<0.05) levels were reduced in explanted valves from AS patients (n = 51) compared to those from control (n = 11) patients. Sirt1 mRNA levels were negatively associated with resistin mRNA levels quantified in AS valves (p = 0.02). Stimulation of Sirt1 by resveratrol or virus-driven overexpression robustly diminished resistin mRNA and protein expression in macrophages, whereas down-regulation of Sirt1 triggered a large increase in resistin expression. These effects were direct, as chromatin immunoprecipitation assays showed that Sirt1 physically interacted with the resistin promoter region at an AP-1 response element. Moreover, Sirt1 blocked c-jun-induced resistin transactivation in gene reporter assays. These findings demonstrate that, in calcified AS, levels of Sirt1 are reduced whereas those of resistin are increased within aortic valve leaflets. Our results also suggest that this loss of Sirt1 expression alleviates its inhibition of resistin transcription in macrophages. Although the overall contribution of this process to the underlying mechanisms for AS disease development remains unresolved, these observations suggest that modification of Sirt1 expression and/or activity could represent a novel approach against inflammation in AS.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Low Sirt1 levels in aortic stenosis are inversely associated with resistin expression.

A, Levels of Sirt1 and resistin mRNA levels in aortic valves from 11 control and 51 patients with AS. Gene mRNA was normalized to the housekeeping gene L27. B, Linear regression between relative resistin and Sirt1 mRNA levels. Each point represents a single patient. C, Sirt1 protein levels in aortic valves from control and AS patients. Bar graph depicts the signal intensity relative to that of a non-specific band. D, Sirt1 protein levels in aortic valve tissue from control and AS patients as visualized by immunofluorescence (IF, top panels) and immunohistochemistry (lower panels). In IF, FITC (green) signals were against Sirt1, whereas Hoescht (blue) signals strain nuclei. Representative pictures are shown. In all panels, * indicates a significant difference between groups.

Figure 2. Activation of Sirt1 inhibits resistin expression in macrophages.

A, Sirt1 and resistin mRNA levels in U937 macrophages treated for 24 h with LPS (1 µg/mL) and resveratrol (50 µM) or their respective vehicle. B, Secreted levels of resistin in U937 macrophages treated as in A. Bars are representative of 3 experiments done in triplicate. In all panels, * indicates a significant difference between groups.

Figure 3. Downregulation of Sirt1 increases resistin expression in macrophages.

Sirt1 (A) and resistin (B) mRNA levels in RAW264 macrophages infected with viruses for Sirt1 overexpression (pBabe-Sirt1) or downregulation (pSuper-Sirt1 RNAi) or their respective control vectors. Scr: scrambled sequence. (C) Resistin mRNA levels in pBabe- or pBabe-Sirt1-infected RAW264 macrophages treated for 24 h with or without LPS (1 µg/mL). Bars are representative of 3 experiments done in triplicate. In all panels, * indicates a significant difference between groups.

Figure 4. Repression of the human resistin promoter by Sirt1.

A, Gene reporter assays using 3.0 kb of the human resistin promoter in 293 T cells transfected with c-Jun, Sirt1 or empty control plasmids and treated for 24 h with resveratrol (20 µM) or vehicle before quantification of the luminescent signal. Bars are representative of 3 experiments done in triplicate. * indicates a significant difference between groups. B-C, ChIP assay of the AP-1 response element on the resistin promoter using antibodies against Sirt1, IgG, or c-jun on U-937 cells treated with 1 µg/ml LPS or vehicle for 24 h.