SIRT1 reduces endothelial activation without affecting vascular function in ApoE-/- mice.

Excessive production of reactive oxygen species (ROS) contributes to progression of atherosclerosis, at least in part by causing endothelial dysfunction and inflammatory activation. The class III histone deacetylase SIRT1 has been implicated in extension of lifespan. In the vasculature,SIRT1 gain-of-function using SIRT1 overexpression or activation has been shown to improve endothelial function in mice and rats via stimulation of endothelial nitric oxide (NO) synthase (eNOS). However, the effects of SIRT1 loss-of-function on the endothelium in atherosclerosis remain to be characterized. Thus, we have investigated the endothelial effects of decreased endogenous SIRT1 in hypercholesterolemic ApoE-/- mice. We observed no difference in endothelial relaxation and eNOS (Ser1177) phosphorylation between 20-week old male atherosclerotic ApoE-/- SIRT1+/- and ApoE-/- SIRT1+/+ mice. However, SIRT1 prevented endothelial superoxide production, inhibited NF-kappaB signaling, and diminished expression of adhesion molecules. Treatment of young hypercholesterolemic ApoE-/- SIRT1+/- mice with lipopolysaccharide to boost NF-kappaB signaling led to a more pronounced endothelial expression of ICAM-1 and VCAM-1 as compared to ApoE-/- SIRT1+/+ mice. In conclusion, endogenous SIRT1 diminishes endothelial activation in ApoE-/- mice, but does not affect endothelium-dependent vasodilatation.

Endogenous SIRT1 has been shown to decrease macrophage foam cell formation and atherogenesis in hypercholesterolemic ApoE -/-SIRT1 +/mice [17]. In non-atherosclerotic aortae of rats, dominant-negative SIRT1 transfection impairs endothelial function via eNOS inhibition ex vivo [18], and endothelial overexpression of human SIRT1 diminishes atherogenesis in ApoE -/mice and improves vascular function [19]. In addition, activation of SIRT1 prevents hyperglycemia-induced vascular cell senescence in mice with diabetes, thereby protecting from vascular dysfunction [20]. Nevertheless, the impact of a SIRT1 haploinsufficiency on endothelium-dependent vasomotion and endothelial cell activation in atherosclerotic mice remains to be determined.
In the present study, we therefore investigated the effects of a single SIRT1 allele on aortic relaxation and endothelial activation in 20-week-old atherosclerotic ApoE -/-SIRT1 +/+ and ApoE -/-SIRT1 +/mice.

Endogenous SIRT1 does not alter endothelial function in ApoE -/mice
Overexpression of human SIRT1 in mouse endothelial cells has been shown to diminish atherogenesis in ApoE -/mice. [19] However, the underlying mechanisms remain to be further characterized. To investigate the effect of endogenous SIRT1 on endotheliumdependent vasodilatation and endothelial inflammatory activation, we assessed endothelium-dependent function and inflammatory pathways in aortic rings from 20week-old atherosclerotic ApoE -/-SIRT1 +/+ or ApoE -/-SIRT1 +/mice. Interestingly, the acetylcholine-mediated relaxation of aortic rings after precontraction with norepinephrine did not differ between ApoE -/-SIRT1 +/+ and the haploinsufficient ApoE -/-SIRT1 +/mice ( Figure  1A). Vasoconstriction with norepinephrine and endothelium-independent vasodilatation with sodium nitroprusside were normal ( Figure 1B, C). eNOSderived NO plays an important role in vascular relaxation, and eNOS activity is mainly regulated by Akt-dependent Ser 1177 phosphorylation [21]. We observ-  Figure 1D). Our data indicate that endogenous SIRT1 in atherosclerotic ApoE -/mice does not affect endothelial function.

Silencing of SIRT1 enhances production of endothelial superoxide
Common risk factors predisposing to atherosclerosis, such as hypercholesterolemia or aging, are associated with oxidant stress at least in part due to an increased production of ROS [22]. We measured ROS production in human aortic endothelial cells (HAECs) treated with either scrambled-or SIRT1-siRNA. SIRT1 silencing elevated endothelial ROS levels upon TNFα stimulation, whereas under basal conditions there was no effect of SIRT1 silencing was observed ( Figure 2).

Enhanced expression of adhesion molecules in ApoE -/-SIRT1 +/plaques
Accumulating evidence suggests that chronic production of ROS may favor atherogenesis by inducing sustained endothelial inflammatory activation [2,5]. Expression of endothelial adhesion molecules play an important role in atherogenesis by promoting monocyte-derived macrophage recruitment and accumulation in the arterial intima [16]. Interestingly, expression of ICAM-1 and VCAM-1 was increased in atherosclerotic plaques of ApoE -/-SIRT1 +/compared with ApoE -/-SIRT1 +/+ mice ( Figure 3). These findings show that SIRT1 prevents adhesion molecule expression, an important step in endothelial cell activation.

SIRT1 regulates the expression of endothelial adhesion molecules via suppression of NF-κB signaling in vitro
NF-κB plays a central role in inflammatory processes and its signaling pathway is inhibited by SIRT1 via deacetylation [12,23]. NF-κB induces expression of adhesion molecules and inflammatory cytokines, and endothelial-specific inhibition of the NF-κB pathway protects mice from atherosclerosis [24]. SIRT1 has been shown to deacetylate the lysine residue K310 of RelA/p65 in human epithelial lung cells [12]. To test whether RelA/p65 signaling is suppressed by SIRT1 in HAECs, we quantified DNA-bound RelA/p65 in TNFαstimulated and unstimulated cells pretreated with the SIRT1 inhibitor splitomicin [25]. Binding of RelA/p65 to naked DNA was enhanced upon treatment with the SIRT1 inhibitor splitomicin after TNFα stimulation ( Figure 4A). To evaluate, if SIRT1 is also deacetylating K310 of RelA/p65 in HAECs, as previously reported  www.impactaging.com for HEK 293T cells [12], we stimulated SIRT1-or scrambled-siRNA-treated HAECs with TNFα and performed p65 immunoprecipitations. K310-p65 was increased in SIRT1-siRNA-treated HAECs ( Figure 4B). To further test if suppression of NF-κB signaling also affects the expression of adhesion molecules, we analyzed the expression of VCAM-1, a known NF-κB signaling target, in more detail. SIRT1-siRNA treatment enhanced expression of VCAM-1 in HAECs upon TNFα stimulation ( Figure 4C).

DISCUSSION
Enhanced atherogenesis in ApoE -/-SIRT1 +/mice is causally linked to increased expression of adhesion molecules in aortae. Indeed, we provide in vitro and in vivo evidence that underlines this concept by demonstrating that ApoE -/-SIRT1 +/mice exhibit increased endothelial expression of ICAM-1 and VCAM-1 upon LPS injection. Importantly, upregulation of these adhesion molecules promotes recruitment of monocytes and T cells to luminal endothelial cells [27].
In concert with increased levels of IL-1β, TNFα, and P-Sel in the activated arterial wall, these molecular events are sufficient to recruit circulating leukocytes to athero-sclerotic lesions, especially monocyte-derived macrophages and T cells.
At the molecular level, the inhibitory effects of SIRT1 on adhesion molecule expression may be mediated via RelA/p65 signaling. Our data show that SIRT1 suppresses binding of RelA/p65 to naked DNA, therefore interfering with a crucial step in the transcriptional activation of NF-κB. These findings are in line with previous reports showing that SIRT1 deacetylases RelA/p65 at the lysine residue K310 in human epithelial lung cells [12]. In agreement with these reports, we demonstrate that this mechanism of RelA/p65 signaling suppression is present in HAECs. www.impactaging.com Surprisingly, we observe no endothelial dysfunction in ApoE -/-SIRT1 +/mice. In contrast, Pearson et al. showed improved endothelial function in mice kept on a diet with a very high resveratrol content (2400 mg/kg/food) that could be mediated by SIRT1 activation [28]. However, such effects may also be related to activation of AMPK by resveratrol or via other targets of this compound [29,30]. Furthermore, adenovirus-mediated inhibition of endothelial SIRT1 diminishes endothelium-dependent vasodilatation in rat aortic rings and decreases bioavailable NO levels [18]. Others reported improved relaxation in ApoE -/mice with endothelial SIRT1 overexpression that were kept on a high-fat diet [19]. However, in this study WT aortic rings showed also marked endothelial dysfunction by relaxing only up to 50%, thereby casting doubts on the endothelial integrity of the preparations [19]. In contrast, we observed no change in endothelial function or aortic eNOS activity between hypercholesterolemic ApoE -/-SIRT1 +/and ApoE -/-SIRT1 +/+ mice, suggesting that the endothelial-protective effects of SIRT1 include factors other than eNOS-dependent NO production. Indeed, we detected a profound increase in ROS-production after silencing of SIRT1 in TNFalpha-stimulated endothelial cells, indicating that endogenous SIRT1 inhibits agonist-induced ROS production in endothelial cells. Of note, an excessive production of ROS has been implicated in endothelial inflammatory activation and the pathogenesis of atherosclerosis [31]. Therefore, inhibition of excessive endothelial ROS production likely represents an important endothelial-protective action of endogenous SIRT1.
Taken together, our results show that SIRT1 does not influence endothelium-dependent vascular function in ApoE -/mice, but it prevents superoxide production in endothelial cells and reduces the expression of inflammatory adhesion molecules by suppressing NF-κB signaling. Although the specificity of available SIRT1 activators has been questioned recently [32], it is likely that SIRT1 activators may prevent atherosclerosis and other inflammatory diseases by hindering prooxidative and inflammatory processes.

MATERIALS AND METHODS
Animals. ApoE -/-SIRT1 +/and ApoE -/-SIRT1 +/+ mice were described previously [17]. Male mice were fed a high-cholesterol diet (1.25% total cholesterol, Research Diets) for 12 weeks starting at the age of 8 weeks. All animal procedures were approved by the local animal committee and performed in accordance with our institutional guidelines.
Immunohistochemistry and immunofluorescence. Serial cryosections from the aortic sinus were stained with rabbit anti-von Willebrand Factor (Dako), rat anti-CD31, rat anti-VCAM-1 (BD Biosciences), rat anti-ICAM-1 (Serotec). Fluorescence was analyzed on a Leica TCS SP2 confocal microscope and means were taken from n=6 different mice evaluating 6 serial cryosections/tissue from each mouse.
RNA and protein analysis. Total RNA isolated from proximal aortae and HAEC was extracted with TRIZOL (Invitrogen), reverse transcribed, and the cDNA quantified by SYBR green qPCR using specific primers. For protein analysis, aortic tissue lysates were blotted and incubated with rabbit anti-SIRT1, rabbit anti-eNOS (Santa Cruz Biotechnology), and rabbit anti-Phospho-eNOS (Ser 1177 ) (Cell Signaling Technology).
Quantification of DNA-bound RelA/p65. HAEC were pretreated with splitomicin for one hour and stimulated with 10 ng/ml TNFα for 30 minutes. Nuclear extracts of aortic tissue samples were obtained with the Nuclear Extract kit (ActiveMotif) using a Dounce pestle, and a RelA/p65 transcription factor assay was performed using the TransAM kit (ActiveMotif) according to the manufacturer's instructions.
Statistical analyses. Data are presented as mean ± SEM. Statistical significance of differences was calculated using an ANOVA with post hoc Tukey's test or Student's unpaired t test. Significance was accepted at p<0.05.