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Sirt6 deletion in bone marrow-derived cells increases atherosclerosis – Central role of macrophage scavenger receptor 1
Journal of Molecular and Cellular Cardiology 139 (2020) 24–32
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Sirt6 deletion in bone marrow-derived cells increases atherosclerosis – Central role of macrophage scavenger receptor 1
T
Tasneem Arsiwalaa, Jürgen Pahlaa, Lambertus J. van Titsa,b, Lavinia Biscegliec, Daniel S. Gaula,b, Sarah Costantinoa, Melroy X. Mirandaa,b, Kathrin Nussbaumd, Simona Stivalaa,e, Przemyslaw Blyszczuka,b, Julien Webera,b, Anne Tailleuxf, Sokrates Steina,b, Francesco Panenia,b, Jürg H. Beera,e, Melanie Greterd, Burkhard Becherd, Raul Mostoslavskyg, Urs Erikssona, ⁎ Bart Staelsf, Johan Auwerxh, Michael O. Hottigerc, Thomas F. Lüschera,i, Christian M. Mattera,b, a
Center for Molecular Cardiology, University of Zurich, Zurich, Switzerland Department of Cardiology, University Heart Center, Zurich University Hospital, Zurich, Switzerland c Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland d Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland e Internal Medicine Cantonal Hospital Baden, Baden, Switzerland f Univ. Lille - EGID; Inserm UMR1011; CHU Lille, Institut Pasteur de Lille, France g Massachusetts General Hospital, Cancer Center, Harvard Medical School, Boston, USA h Laboratory of Integrative & Systems Physiology, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland i Cardiology, Royal Brompton and Harefield Hospitals and Imperial College, London, United Kingdom b
ARTICLE INFO
ABSTRACT
Keywords: SIRT6 oxLDL MSR1 Atherosclerosis Foam cell
Aims: Sirtuin 6 (Sirt6) is a NAD+-dependent deacetylase that plays a key role in DNA repair, inflammation and lipid regulation. Sirt6-null mice show severe metabolic defects and accelerated aging. Macrophage-foam cell formation via scavenger receptors is a key step in atherogenesis. We determined the effects of bone marrowrestricted Sirt6 deletion on foam cell formation and atherogenesis using a mouse model. Methods and results: Sirt6 deletion in bone marrow-derived cells increased aortic plaques, lipid content and macrophage numbers in recipient Apoe−/− mice fed a high-cholesterol diet for 12 weeks (n = 12–14, p < .001). In RAW macrophages, Sirt6 overexpression reduced oxidized low-density lipoprotein (oxLDL) uptake, Sirt6 knockdown enhanced it and increased mRNA and protein levels of macrophage scavenger receptor 1 (Msr1), whereas levels of other oxLDL uptake and efflux transporters remained unchanged. Similarly, in human primary macrophages, Sirt6 knockdown increased MSR1 protein levels and oxLDL uptake. Double knockdown of Sirt6 and Msr1 abolished the increase in oxLDL uptake observed upon Sirt6 single knockdown. FACS analyses of macrophages from aortic plaques of Sirt6-deficient bone marrow-transplanted mice showed increased MSR1 protein expression. Double knockdown of Sirt6 and the transcription factor c-Myc in RAW cells abolished the increase in Msr1 mRNA and protein levels; c-Myc overexpression increased Msr1 mRNA and protein levels. Conclusions: Loss of Sirt6 in bone marrow-derived cells is proatherogenic; hereby macrophages play an important role given a c-Myc-dependent increase in MSR1 protein expression and an enhanced oxLDL uptake in human and murine macrophages. These findings assign endogenous SIRT6 in macrophages an important atheroprotective role.
1. Introduction Atherosclerosis is the leading cause of death in developed countries [1]. In atherogenesis, macrophages play a critical role, from initiation with fatty streaks up to plaque rupture. Atherosclerosis is enhanced
when an excess of oxidized low-density lipoproteins (oxLDL), that are formed by the oxidation of LDL particles, lead to foam cell formation within the subendothelial layer of the arterial wall. Foam cell formation is accelerated either by an increase in oxidized lipid uptake and/or by a decrease in cholesterol efflux. Scavenger receptors on macrophages
⁎ Corresponding author at: Department of Cardiology, University Heart Center, University Hospital Zurich and Center for Molecular Cardiology, University of Zurich, Raemistrasse 100, 8091 Zurich, Switzerland. E-mail address: [email protected] (C.M. Matter).
https://doi.org/10.1016/j.yjmcc.2020.01.002 Received 18 June 2019; Received in revised form 10 January 2020; Accepted 13 January 2020 Available online 21 January 2020 0022-2828/ © 2020 Elsevier Ltd. All rights reserved.
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Fig. 1. Hematopoietic homozygous Sirt6 deletion increases atherosclerosis. Bone marrow (BM) cells from Apoe−/− Sirt6+/+ or Apoe−/− Sirt6−/− mice were transplanted into recipient Apoe−/− mice that were fed a high-cholesterol diet for 12 weeks. A, Representative images of en face thoraco-abdominal aortae stained with oil-red O (ORO, left panel) and their corresponding plaque quantifications (Apoe−/− Sirt6+/+, n = 14; Apoe−/− Sirt6−/− n = 13, right panel). B, C, Representative images (left panel) and quantifications of aortic root cross sections (right panel) stained for lipids (ORO) and CD68 (n = 12 in each group). Scale bars in photomicrographs is 500 μm. D, Plasma levels of total cholesterol (Apoe−/− Sirt6+/+, n = 11; Apoe−/− Sirt6−/−, n = 9) and E, plasma triglycerides (TG; Apoe−/− Sirt6+/+, n = 11; Apoe−/− Sirt6−/−, n = 9). F, Plasma levels of MCP-1 (Apoe−/− Sirt6+/+, n = 11; Apoe−/− Sirt6−/−, n = 10) and G, IL-6 (Apoe−/− Sirt6+/+, n = 10; Apoe−/− Sirt6−/−, n = 9). Values are presented as mean ± SEM and analysed using unpaired Student's two-tailed t-test for fig. A, B, D and E data and Mann-Whitney U test for fig. C, F, G and H data. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
recognize and internalize the modified forms of LDL particles and trigger foam cell formation. Important scavenger receptors expressed on macrophages include scavenger receptor A, (in mice: macrophage scavenger receptor 1, MSR1), CD36 and oxidized low-density lipoprotein receptor 1 (LOX1). Sirtuins (SIRT1–7) comprise a family of nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases that are activated upon caloric restriction and prevent age-associated diseases [2]. Among the sirtuins, SIRT6 is a nuclear protein known to deacetylate histone H3 lysine 9 (H3K9) [3] and H3 lysine 56 (H3K56) [4,5]. It increases chromosomal stability by promoting repair of double strand breaks, a hallmark of genomic instability in aging tissues [6]. SIRT6 has gained attention for its role in human telomere and genome stabilization [3], gene expression and DNA repair [7], glucose and fat homeostasis [8–10], as well as inflammation [11,12]. Experiments with genetically engineered mouse models using Sirt6 loss-of-function or gain-of-function studies highlight the protective role of SIRT6 in aging, metabolism and inflammation: Homozygous Sirt6deficient mice demonstrate a progeria-like phenotype with severe hypoglycemia leading to death around 3 to 4 weeks of age [13]. In the myocardium, Sirt6 deficiency boosts insulin-like growth factor signaling and results in cardiac hypertrophy and heart failure [14]. In human
endothelial cells, SIRT6 depletion increases DNA damage, telomere dysfunction and senescence [15]. Hepatocyte-specific Sirt6 deficiency enhances Pcsk9 gene expression and elevates plasma LDL cholesterol levels [10]. Finally, cultured bone marrow-derived macrophages (BMDM) isolated from Sirt6-null mice reveal increased levels of Mcp-1 and Il-6, and hypersensitivity to lipopolysaccharide stimulation [16]. Sirt6 gain-of-function studies underline the above-mentioned beneficial effects of SIRT6: SIRT6 protects against cardiac hypertrophy, heart failure, myocardial and hypoxic damage [14,17,18] and improves lipid metabolism by decreasing plasma LDL cholesterol and Pcsk9 levels [10]. SIRT6 also functions as a co-repressor of c-MYC transcriptional activity [19]. Of note, c-MYC has been associated with the expression of scavenger receptors and foam cell formation [20]. Less is known about SIRT6 in atherogenesis and its particular role in macrophages: Recent studies have reported a putative protective role of endogenous SIRT6 in atherosclerosis using a partial loss-of-function approach: Constitutive heterozygous Sirt6 deletion enhanced atherosclerosis through an increase in inflammation [21,22]. Similarly, a systemic knockdown of Sirt6 using small hairpin RNA, increased plaque size and vulnerability features [23]. Finally, an in vitro study reported that SIRT6 reduced macrophage foam cell formation by inducing autophagy using a gain-of-function approach [24]. 25
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However, the role of bone marrow (BM)-specific Sirt6 deletion on atherosclerosis, its effects on foam cell formation and the key molecular mediators in this context remain unknown. Thus, we hypothesized that homozygous BM-specific Sirt6 deletion in apolipoprotein E knockout (Apoe−/−) mice enhances atherogenesis and increases macrophage foam cell formation by interacting with MSR1.
double knockdown of Msr1 and Sirt6. Msr1 and Sirt6 double knockdown abolished the increase in Dil-oxLDL uptake observed upon Sirt6 single knockdown (Fig. 2E). In contrast, adenovirus-mediated overexpression of Sirt6 (Supplemental Fig. 4B) reduced oxLDL uptake (Supplemental Fig. 4C) and reduced MSR1 protein expression compared with control adenovirus (Supplemental Fig. 4D). To translate our findings to human macrophages, we investigated whether SIRT6 also regulates oxLDL uptake in human primary macrophages via MSR1 expression. Similar to our findings in mice, SIRT6 knockdown in human macrophages increased uptake of Dil-oxLDL (Fig. 2F), which corresponded with increased MSR1 expression compared with scramble control (Fig. 2G).
2. Results 2.1. Hematopoietic Sirt6 deletion increases atherosclerosis in Apoe−/− mice To test the effects of Sirt6 deletion in BM-derived cells on atherosclerotic plaque formation, BM isolated from Apoe−/-Sirt6+/+ or Apoe−/-Sirt6−/− donor mice were transplanted into sub-lethally irradiated Apoe−/− recipient mice (Supplemental Fig. 1). Of note, Apoe−/Sirt6−/− mice were not born at the expected Mendelian frequency (Apoe−/-Sirt6−/− 20 [5.76%], n = 347, approximately one pup every 15 litters). Even though Sirt6 knockout pups born on Apoe−/− background had no birth abnormalities, they showed early mortality similar to Sirt6 knockout mice. Following 6 weeks of recovery after BM reconstitution, mice were fed a HCD for 12 weeks. En face plaque staining of thoraco-abdominal aortae revealed a significant increase in atherosclerosis in Apoe−/-Sirt6−/− BM-recipient mice compared with Apoe−/Sirt6+/+ BM-recipient mice (Fig. 1A). Similarly, ORO staining of serial cross-sections of aortic roots showed an increase in lipid accumulation and atherosclerotic lesion size in Apoe−/-Sirt6−/− BM-recipient mice (Fig. 1B) compared with Apoe−/-Sirt6+/+ BM-recipient control mice. Furthermore, aortic root cross-sections from the Apoe−/-Sirt6−/− BMrecipient mice showed increased macrophage (CD68-positive staining) accumulation within atherosclerotic plaques (Fig. 1C). There were no significant differences in the weight course of these mice during the 12 weeks on HCD (data not shown). Plasma lipid and cytokine analyses showed that cholesterol levels were lower in the Apoe−/-Sirt6−/− BM-recipient mice, while triglycerides (TG), as well as MCP-1 and IL-6 levels remained unchanged (Fig. 1D-G). The cholesterol and triglyceride fractions are shown in Supplemental Fig. 2. Macrophages localized in the plaques of Apoe−/Sirt6−/− BM-recipient mice lacked SIRT6 expression compared with Apoe−/-Sirt6+/+ BM-recipient mice (Supplemental Fig. 3A), confirming that plaque-resident macrophages were derived from transplanted BM cells. Flow cytometric analyses proved the transplantation efficiency of CD45.1 BM cells into recipient mice (Supplemental Fig. 3B). Blood hematology counts between the two groups of mice were not changed (data not shown).
2.3. Sirt6−/− bone marrow transplantation increases MSR1 expression in mouse aortic plaque macrophages To assess whether SIRT6 regulates MSR1 in vivo, we analysed Msr1 expression in BM-recipient mice in the presence or absence of Sirt6, respectively. BM cells isolated from Apoe−/-Sirt6−/− BM-recipient mice, fed a HCD for 12 weeks, showed both increased mRNA and protein levels of Msr1, compared with BM cells from Apoe−/-Sirt6+/+ BM-transplanted mice (Fig. 3A, B). Furthermore, immunofluorescence analyses of aortic root cross sections suggested an increase in MSR1 staining in macrophages of Apoe−/Sirt6−/− BM-recipient mice, compared with Apoe−/-Sirt6+/+ BM-recipient mice (Fig. 3C). To quantify increased MSR1 expression in plaque-derived macrophages, plaques were isolated from the aortic arch. CD45+CD11b+ Ly6C+ F4/80+ macrophages were identified and MSR1 levels assessed by flow cytometry. Increased expression of MSR1 in aortic plaque macrophages of Apoe−/-Sirt6−/− BM-recipient mice was observed relative to Apoe−/− BM-recipient mice (Fig. 3D). Corresponding analyses in blood monocytes isolated from these mice showed no difference in MSR1 levels between both groups (Fig. 3E, F). Similar to aortic plaque macrophages, MSR1 levels were increased in BMDM from Apoe−/Sirt6−/− compared with Apoe−/-Sirt6+/+ mice (Fig. 3G) ex vivo. 2.4. Sirt6 knockdown increases Msr1 expression via c-MYC To identify the mechanism underlying the increase in Msr1 expression, we performed a ChIP of acetyl H3K9 and acetyl H3K56 in RAW 264.7 cells upon Sirt6 knockdown. There was no significant difference in enrichment for H3K9 and H3K56 acetylation at Msr1 promoter in the scramble and Sirt6 knockdown cells (Supplemental Fig. 5), suggesting that the increase in Msr1 expression upon Sirt6 knockdown does not reflect a Sirt6-mediated increase in acetylation of histone 3 at the Msr1 promoter. A recent study shows that SIRT6 co-represses c-MYC transcriptional activity and ribosomal gene expression [19]. Co-immunoprecipitation experiments were performed in RAW 264.7 cells to confirm the interaction between SIRT6 and c-MYC in our experimental setting (Supplemental Fig. 6). As c-MYC transcriptional activity is linked to oxLDL uptake [20] and the UCSC database shows that c-MYC binds to the enhancer region of Msr1, we investigated whether loss of c-MYC represses the increase in Msr1 levels observed upon Sirt6 knockdown. Indeed, double knockdown of c-Myc and Sirt6 prevented an increase in Msr1 expression in RAW 264.7 cells, both at the mRNA and protein level (Fig. 4A, B). In contrast, c-MYC overexpression was associated with Msr1 gene upregulation, thus confirming the role of c-MYC as a regulator of Msr1 transcription (Fig. 4C, Supplemental Fig. 7).
2.2. Sirt6 decreases oxLDL accumulation by suppressing Msr1 expression in murine and human macrophages To determine whether macrophage-specific deficiency of Sirt6 affects LDL uptake, the accumulation of oxLDL in RAW 264.7 cells was measured in vitro. Expression levels of SIRT6 were markedly reduced 48 h after Sirt6 silencing (Fig. 2A). Flow cytometric analyses revealed that Sirt6 silencing in macrophages increased oxLDL accumulation in vitro (Fig. 2B). Similarly, acLDL accumulation was increased upon Sirt6 knockdown in RAW 264.7 cells (Supplemental Fig. 4A). To address the mechanism underlying the increase in oxLDL uptake upon Sirt6 deficiency, we assessed the effects of Sirt6 silencing on the expression of genes involved in cellular cholesterol accumulation. Knockdown of Sirt6 increased scavenger receptor Msr1 mRNA and protein expression without altering mRNA expression of other scavenger receptors such as Cd36 or Lox1 (Fig. 2C, D). Furthermore, mRNA levels of cholesterol efflux transporters Abca1 and Abcg1 were not altered (Fig. 2C). To address the causal involvement of increased MSR1 expression in enhanced oxLDL accumulation, Dil-oxLDL uptake was measured upon
3. Discussion We demonstrate that BM-specific Sirt6 deletion is sufficient to increase modified LDL uptake in macrophages and enhance plaque 26
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Fig. 2. SIRT6 downregulates macrophage oxLDL uptake via MSR1 in mice and men. A, Western blot of SIRT6 protein levels upon scramble or Sirt6 knockdown from murine RAW 264.7 macrophages (n = 3 each). B, Quantification of Dil-oxLDL uptake upon scramble or Sirt6 knockdown (n = 6). C, Relative mRNA expression levels of genes involved in modified low-density lipoprotein uptake and efflux (n = 5); mRNA expression levels were normalized to Rps29 and Rps18. D, Sirt6 knockdown increases MSR1 protein compared to control levels in RAW 264.7 macrophages (n = 3). E, Quantification of DiloxLDL upon knockdown of Sirt6 and Msr1 (n = 3). Knockdown of SIRT6 in human primary macrophages F, enhances Dil-oxLDL uptake (n = 6) and G, increases MSR1 expression (n = 6). Values are represented as mean ± SEM and analysed using Mann-Whitney U test (B-D, F, G) and ordinary oneway ANOVA with Holm-Sidak's multiple comparison (E).
formation in Apoe−/− mice through increased Msr1 expression in bone marrow cells and plaque macrophages. Corresponding in vitro experiments reveal that Sirt6 deficiency increases MSR1 levels both in mouse and human, likely via regulation of the transcription factor c-MYC. Thus, our findings identify MSR1 as a novel target for atheroprotective effects of SIRT6, whereby macrophages play a central role (Fig. 5). Our discoveries extend recent findings in which constitutive heterozygous deletion of Sirt6 enhanced atherosclerosis through an increase
in NKG2D ligand expression and inflammation [21,22]. Sirt6-induced atheroprotection mediated by BM-derived cells may involve all hematopoietic cells. We propose macrophages as the protagonist cells for the following reasons: Homozygous Sirt6 deficiency in BM-derived cells of Apoe−/− chimeric mice increased macrophage-specific MSR1 expression in aortic plaques. Moreover, our in vitro findings place MSR1 center stage for SIRT6-dependent foam cell formation. Ex vivo, BM-derived macrophages from Sirt6−/− mice have been reported to increase Mcp-1 27
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Fig. 3. Hematopoietic Sirt6 deficiency increases MSR1 expression in bone marrow cells and aortic plaque macrophages. A, Msr1 mRNA (n = 12) and B, MSR1 protein expression (n = 7) is increased in bone marrow (BM) cells isolated from Apoe−/-Sirt6−/− BM transplanted (BMT) mice compared to Apoe−/− Sirt6+/+ BMT mice. C, Immunofluorescence of MSR1 and CD68 suggesting more macrophage-associated MSR1 expression in cross sections of the aortic root of Apoe−/− Sirt6−/− BMT mice compared with Apoe−/-Sirt6+/+ BMT mice (scale bar = 25 μm). The qualitative immunofluorescence message provided in C is given in a quantitative fashion after sorting aortic arch plaques for macrophages: D, MSR1 protein levels are higher in F4/80+Ly6C+ aortic plaque macrophage population (n = 7) in Apoe−/− Sirt6−/− BMT mice compared with the Apoe−/− Sirt6+/+ BMT mice. E, F, MSR1 protein levels are unchanged in the Ly6Clow (Apoe−/− Sirt6+/+, n = 9; Apoe−/− Sirt6−/−, n = 10) and Ly6Chigh (Apoe−/− Sirt6+/+, n = 9; Apoe−/− Sirt6−/−, n = 10) blood monocyte populations of Apoe−/− Sirt6−/− and Apoe−/− Sirt6+/+ BMT mice. G, MSR1 protein expression after sorting for BM-derived macrophages (n = 4) obtained from Apoe−/− Sirt6+/+ and Apoe−/− Sirt6−/− mice. Values are presented as mean ± SEM and analysed using unpaired Student's two-tailed t-test for fig. A, E and F data and Mann-Whitney U test for fig. B, D and G.
and Il-6 expression levels [16]. In contrast, we could not detect any difference in plasma MCP-1 and IL-6 levels between both groups of BMrecipient mice, indicating that BM-restricted Sirt6 deletion does not mediate its pro-atherosclerotic effects by increasing systemic inflammation. This observation is in line with a previously published study showing that a myeloid specific deletion of Sirt6 leads to a reduced polarization of macrophages toward the inflammatory M2 type [25]. Moreover, our data show that cholesterol levels in mice lacking Sirt6 are decreased. Keeping in mind that MSR1 levels in our mouse model are increased, this is consistent with an earlier publication, which shows that a bone marrow-specific overexpression of MSR1 in Apoe−/− mice decreases cholesterol levels, possibly via increased cholesterol uptake of Kupffer cells (macrophage equivalent) in the liver [26]. Using complementary Sirt6 loss- and gain-of-function experiments,
our data show that SIRT6 decreases oxLDL uptake in RAW 264.7 macrophages, thereby diminishing foam cell formation. In addition, gene expression analyses upon Sirt6 knockdown revealed a specific increase in Msr1 levels with no difference in transcription of other scavenger receptors or cholesterol efflux genes. MSR1 expression in macrophages is known to increase the uptake of oxidized LDL particles in vitro [27]. Moreover, Msr1 loss-of-function studies using an Apoe−/− background have been reported to provide atheroprotection [27,28]. In parallel, polymorphisms in the MSR1 gene have been associated with human atherosclerosis [29]. These studies highlight the role of MSR1 in the uptake of modified LDL, foam cell formation and atherogenesis. However, the above-mentioned study, using bone marrow-specific MSR1 overexpression in an Apoe−/− background, did not find a difference in atherosclerosis between mice with and without MSR1 overexpression [26]. Of note, these mice were fed a normal chow diet. In contrast, we use a high cholesterol diet mimicking a western-type diet 28
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Fig. 4. Sirt6 knockdown increases MSR1 expression by enhancing c-MYC transcriptional activity. A, B, Knockdown of Sirt6 and c-Myc rescues the increase in Msr1 expression observed upon Sirt6 knockdown at both mRNA (n = 6) (A) and protein level (n = 6) (B). C, Msr1 gene expression following c-MYC overexpression. A control CRISPR Activation Plasmid was used as a negative control. Values are represented as mean ± SEM and analysed using ordinary one-way ANOVA with HolmSidak's multiple comparison; *:p < .05; **: p < .01; ***: p < .001; ****: p < .0001.
Fig. 5. Scheme illustrating the proposed molecular mechanism (left) and atherosclerosis phenotype (right panel) of Sirt6 loss-of-function in bone marrow-derived macrophages. A, Intact endogenous SIRT6 co-represses c-MYC and decreases Msr1 transcription and protein expression. These events diminish oxLDL uptake and foam cell formation in vitro and in vivo, thereby limiting progression of atherosclerosis. B, Sirt6 loss-of-function favors c-MYC transcriptional activity leading to enhanced Msr1 transcriptional activity and protein expression. The subsequent increase in oxLDL uptake is sufficient to enhance foam cell formation and atherosclerosis.
that is known to induce a more pronounced, macrophage-driven atherosclerosis phenotype. This increased cholesterol in mice with loss of protective SIRT6 is likely to increase levels of MSR1, enhance oxLDL uptake into macrophages eventually leading to increased
atherosclerosis. In line with these findings, double knockdown of Sirt6 and Msr1 prevented oxLDL uptake confirming that increased foam cell formation upon Sirt6 knockdown is Msr1-dependent. We translated these proof-of29
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principle in vitro findings to an ex vivo context: Using Apoe−/− mice, an environment reflecting markedly increased oxLDL, Sirt6 deletion in BMderived cells increased both mRNA and protein levels of the scavenger receptor Msr1. Increased MSR1 protein levels in Sirt6-deficient cells, in both BMDM obtained from donor BM cells before and after transplantation, suggests that MSR1 plays a central role for foam cell formation in vitro and in vivo. Interestingly, the ratio of upregulation of MSR1 upon Sirt6 deficiency is coherent with an increase in oxLDL uptake, strengthening the notion that indeed the increase in MSR1 is mediating these changes. To understand the interactions between SIRT6 and MSR1, we assessed whether SIRT6-dependent deacetylation at H3K9 and H3K56 modulated Msr1 at its promoter and found no difference. Since c-MYC binds to the Msr1 enhancer and SIRT6 is known to repress c-MYC activity [19], we investigated whether the observed increase in MSR1 levels was c-MYC-dependent. Indeed, double knockdown of c-Myc and Sirt6 prevented the SIRT6-dependent increase in MSR1 protein expression, suggesting that the SIRT6-dependent modulation of MSR1 expression is mediated by c-MYC. A recent study has shown that a rapid turnover of macrophages in atherosclerotic lesions is due to an increased Msr1 expression, which in turn promotes atherogenesis [30]. Indeed, we found an increase in MSR1 protein levels in macrophages isolated from aortic plaques of Apoe−/-Sirt6−/− BMT mice compared with Apoe−/-Sirt6+/+ BMT mice. Conversely, blood monocytes isolated from the same mice showed no difference in MSR1 protein levels. This suggests a specific effect of SIRT6 on Msr1 expression in macrophages. In line with our findings in mouse macrophages, SIRT6 knockdown in human primary macrophages also caused an increase in MSR1 expression and oxLDL uptake, thus translating our findings to humans and highlighting the importance of SIRT6 in foam cell formation and atherosclerosis.
SIRT1, another nuclear sirtuin which shares atheroprotective effects with SIRT6 [35]. SIRT1 activators have been shown to decrease experimental atherosclerosis along pathways [36] that are similar to SIRT6 [10] and improve human endothelial dysfunction [37]. Human atherosclerosis is associated with an increased susceptibility to thrombosis, thereby leading to myocardial infarction and stroke. Along these lines, SIRT1 and SIRT3 have shown to confer protective effects in experimental thrombosis [38,39]. As pharmacological NAD+ boosters have become available as pansirtuin activators [2,40] with promising data at the experimental level [41–43], time is ready to explore unspecific sirtuin activators as a therapeutic approach in humans. Along these lines, NAD+ boosters may become particularly promising for treating or preventing atherothrombotic diseases such as coronary artery disease and myocardial infarction, cerebrovascular disease and stroke or peripheral arterial disease and acute limb ischemia [44]. 6. Methods 6.1. Animals All animal experiments were approved by the Cantonal Veterinary Office Zurich, Switzerland. All applicable international, national, and institutional guidelines for the care and use of animals were followed and all procedures performed involving animals were in accordance with the ethical standards of these guidelines. Apoe−/−Sirt6+/− mice (Sirt6+/− provided by Johan Auwerx, EPFL, Lausanne, Switzerland) and Apoe−/− mice (Charles River) were bred under pathogen–free conditions housed with a 12 h light – dark cycle. Apoe−/− Sirt6+/− mice were bred to yield Apoe−/− Sirt6−/− offspring. Since these mice show a strong metabolic phenotype with increased embryonic lethality, and the few surviving mice are usually dying within 3–4 weeks after birth, mice were sacrificed at the age of 3 weeks and the bone marrow isolated for transplantation [13]. To generate BM chimeras, we adapted a previous protocol [35] by using young donor mice: 6 week-old male Apoe−/− recipient mice were sub-lethally irradiated with two doses of 550 rads each (female mice were not tested). 3 week-old female and male donor mice were killed by CO2 inhalation and bones (hind limbs and hips) were flushed with sterile PBS to obtain bone marrow (BM) cells. Donor BM cells were injected intravenously (2.5 × 106 total cells per mouse). To prevent bacterial infection, 0.2% (vol/vol) Borgal (trimethoprim, sulfadoxin) was added to the drinking water for 1 week. Mice recovered for 6 weeks and then were fed a HCD, containing 1.25% cholesterol (D12108; Research Diets, New Brunswick, USA), for 12 weeks. To confirm BM chimerism, a control experiment with CD45.1 donor BM cells injected into CD45.2 positive Apoe−/− mice was performed: Cell mixtures were assessed by flow cytometry with monoclonal antibodies to CD45.1 (A20; BD Pharmingen) and CD45.2 (104; BD Pharmingen).
4. Limitations In our study we adopted bone marrow-restricted Sirt6 deletion to assess the role of hematopoietic cells in atherosclerosis. Additionally, we identified that SIRT6 regulates MSR1 by involving c-MYC. However, the exact molecular mechanism by which SIRT6 affects MSR1 remains to be determined. Furthermore, we cannot rule out that other bone marrow-derived cells that express MSR1 contribute to increased atherosclerosis in our model. Along these lines, deletion of Sirt6 may increase MSR1 expression in dendritic cells, which have been shown to express MSR1 [31]. Moreover, we cannot exclude a contribution of other potentially atherogenic bone marrow-derived cells such as neutrophils and T cells, independent of any MSR1 expression. In our study, we have only used male mice as bone marrow recipients to assess atherosclerosis. Further studies would be necessary to address whether a similar mechanism is operative in female mice. In fact, a sex-dependent phenotype has been reported using Sirt6 gain- or loss-of-function experiments regarding lifespan or insulin sensitivity, respectively [32,33].
6.2. Cell culture RAW 264.7 macrophages (Sigma-Aldrich) were cultured in a 5% CO2 incubator at 37 °C in DMEM-high glucose medium supplemented with 2 mM Glutamine and 10% fetal bovine serum (v/v). BM-derived macrophages (BMDM) were obtained by treating BM cells with 10 ng/ ml M-CSF for one week. To obtain human macrophages, buffy coats from healthy volunteers were used and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient centrifugation. Using CD14 Microbeads (Miltenyi Biotec), the monocyte population was isolated and plated with complete RPMI medium supplemented with 10% FBS and 10 ng/ml GM-CSF (Peprotech) for 8 days.
5. Conclusions and perspectives We demonstrate that Sirt6 loss-of-function in BM-derived cells is sufficient to increase atherosclerosis in Apoe−/− mice. Sirt6 deficiency upregulates Msr1 expression both in vivo and in vitro, in mice and human macrophages via c-MYC. Thus, our results reveal a novel, atheroprotective role of SIRT6. Hereby, macrophages play a key role as SIRT6 decreases foam cell formation by downregulating MSR1. In humans, SIRT6 expression levels are inversely associated with atherosclerosis: SIRT6 is decreased in human carotid plaques compared with normal arteries [21] and is lower in plaques from diabetic compared with non-diabetic patients [34]. There is translational potential for sirtuin activation related to
6.3. Immunohistochemistry, immunofluorescence and plaque analyses Aortic root tissues were embedded in OCT, followed by cryostat 30
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cutting into 5 μm thick sections. For immunohistochemistry, sections were fixed in acetone and incubated with the following primary antibodies: anti-CD68 (Bio-Rad), anti-SIRT6 (Cell Signaling), and antiMSR1 (Novus Biologicals). Subsequently, sections were incubated with Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) (MSR1, SIRT6) and Alexa Fluor 547 goat anti-rat IgG (eBioscience) (CD68) for 1 h. Aortic plaques were quantified en face (thoraco-abdominal) and in root cross sections using oil-red O (ORO) as described [35].
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Hilgendorf, G.F. Weber, I. Theurl, Y. Iwamoto, J.L. Figueiredo, et al., Local proliferation dominates lesional macrophage accumulation in atherosclerosis, Nat. Med. 19 (9) (2013) 1166–1172. [31] M. Becker, A. Cotena, S. Gordon, N. Platt, Expression of the class A macrophage scavenger receptor on specific subpopulations of murine dendritic cells limits their
6.4. Statistics Data are presented as mean ± SEM. Normal distribution was assessed with the D'Agostino-Pearson omnibus normality test using GraphPad Prism. For non-parametric data, the Mann – Whitney test was used. Three or more groups were compared using an ordinary one-way ANOVA with Holm-Sidak's multiple comparison. At least three independent experiments in triplicates were performed. Statistical significance was accepted at p ≤ .05. Analyses were performed using GraphPad Prism (version 5.0d, 2010). For multiple comparisons, significances are shown as stars: *:p < .05; **: p < .01; ***: p < .001; ****: p < .0001. Further Materials and Methods can be found in the supplements. Funding This work was supported by the Swiss National Science Foundation to JA (31003A-140780), JHB (310030–144152), TFL (310030–135815), CMM (310030–146923), CMM and JA (310030–165990), and to MOH (310030–157019/176177); by the National Institute of Health (USA) to JA (R01AG043930), Systems X (SysX.ch) to JA (2013–15), by Swiss Heart Foundation grants to CMM and JHB, the University Research Priority Program Integrative Human Physiology at the University of Zurich to CMM; the Hartmann-Müller Foundation and Novartis Foundation to CMM and LJvT, the Ecole Polytechnique Fédérale de Lausanne to JA, the University of Zurich to MOH, by the Kardio Foundation, Switzerland and the Budai Foundation, Lichtenstein to JHB – and the Zurich Heart House, Foundation for Cardiovascular Research, Zurich, Switzerland. Disclosures JA is a founder and scientific advisory board member of Mitobridge. The other authors do not declare a potential conflict of interest. Acknowledgements The graphical abstract was prepared using Servier Medical Art by Servier licensed under a Creative Commons Attribution 3.0 Unported License. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.yjmcc.2020.01.002. References [1] N. Townsend, L. Wilson, P. Bhatnagar, K. Wickramasinghe, M. Rayner, M. Nichols, Cardiovascular disease in Europe: epidemiological update 2016, Eur. Heart J. 37 (42) (2016) 3232–3245. [2] S. Winnik, J. Auwerx, D.A. Sinclair, C.M. Matter, Protective effects of sirtuins in cardiovascular diseases: from bench to bedside, Eur. Heart J. 36 (48) (2015) 3404–3412. [3] E. Michishita, R.A. McCord, E. Berber, M. Kioi, H. Padilla-Nash, M. Damian, et al., SIRT6 is a histone H3 lysine 9 deacetylase that modulates telomeric chromatin, Nature 452 (7186) (2008) 492–496. [4] E. Michishita, R.A. McCord, L.D. Boxer, M.F. Barber, T. Hong, O. Gozani, et al., Cell cycle-dependent deacetylation of telomeric histone H3 lysine K56 by human SIRT6,
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[38] A. Breitenstein, S. Stein, E.W. Holy, G.G. Camici, C. Lohmann, A. Akhmedov, et al., Sirt1 inhibition promotes in vivo arterial thrombosis and tissue factor expression in stimulated cells, Cardiovasc. Res. 89 (2) (2011) 464–472. [39] D.S. Gaul, J. Weber, L.J. Van Tits, S. Sluka, L. Pasterk, M.F. Reiner, et al., Loss of Sirt3 accelerates arterial thrombosis by increasing formation of neutrophil extracellular traps and plasma tissue factor activity, Cardiovasc. Res. 114 (8) (2018) 1178–1188. [40] S.A. Trammell, M.S. Schmidt, B.J. Weidemann, P. Redpath, F. Jaksch, R.W. Dellinger, et al., Nicotinamide riboside is uniquely and orally bioavailable in mice and humans, Nat. Commun. 7 (2016) 12948. [41] H. Zhang, D. Ryu, Y. Wu, K. Gariani, X. Wang, P. Luan, et al., NAD(+) repletion improves mitochondrial and stem cell function and enhances life span in mice, Science 352 (6292) (2016) 1436–1443. [42] A. Das, G.X. Huang, M.S. Bonkowski, A. Longchamp, C. Li, M.B. Schultz, et al., Impairment of an endothelial NAD(+)-H2S signaling network is a reversible cause of vascular aging, Cell 173 (1) (2018) 74–89 e20. [43] L. Rajman, K. Chwalek, D.A. Sinclair, Therapeutic potential of NAD-boosting molecules: the in vivo evidence, Cell Metab. 27 (3) (2018) 529–547. [44] M. Leslie, Whatever happened to, Science 353 (6305) (2016) 1198–1201.
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Journal of Molecular and Cellular Cardiology journal homepage: www.elsevier.com/locate/yjmcc
Sirt6 deletion in bone marrow-derived cells increases atherosclerosis – Central role of macrophage scavenger receptor 1
T
Tasneem Arsiwalaa, Jürgen Pahlaa, Lambertus J. van Titsa,b, Lavinia Biscegliec, Daniel S. Gaula,b, Sarah Costantinoa, Melroy X. Mirandaa,b, Kathrin Nussbaumd, Simona Stivalaa,e, Przemyslaw Blyszczuka,b, Julien Webera,b, Anne Tailleuxf, Sokrates Steina,b, Francesco Panenia,b, Jürg H. Beera,e, Melanie Greterd, Burkhard Becherd, Raul Mostoslavskyg, Urs Erikssona, ⁎ Bart Staelsf, Johan Auwerxh, Michael O. Hottigerc, Thomas F. Lüschera,i, Christian M. Mattera,b, a
Center for Molecular Cardiology, University of Zurich, Zurich, Switzerland Department of Cardiology, University Heart Center, Zurich University Hospital, Zurich, Switzerland c Department of Molecular Mechanisms of Disease, University of Zurich, Zurich, Switzerland d Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland e Internal Medicine Cantonal Hospital Baden, Baden, Switzerland f Univ. Lille - EGID; Inserm UMR1011; CHU Lille, Institut Pasteur de Lille, France g Massachusetts General Hospital, Cancer Center, Harvard Medical School, Boston, USA h Laboratory of Integrative & Systems Physiology, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland i Cardiology, Royal Brompton and Harefield Hospitals and Imperial College, London, United Kingdom b
ARTICLE INFO
ABSTRACT
Keywords: SIRT6 oxLDL MSR1 Atherosclerosis Foam cell
Aims: Sirtuin 6 (Sirt6) is a NAD+-dependent deacetylase that plays a key role in DNA repair, inflammation and lipid regulation. Sirt6-null mice show severe metabolic defects and accelerated aging. Macrophage-foam cell formation via scavenger receptors is a key step in atherogenesis. We determined the effects of bone marrowrestricted Sirt6 deletion on foam cell formation and atherogenesis using a mouse model. Methods and results: Sirt6 deletion in bone marrow-derived cells increased aortic plaques, lipid content and macrophage numbers in recipient Apoe−/− mice fed a high-cholesterol diet for 12 weeks (n = 12–14, p < .001). In RAW macrophages, Sirt6 overexpression reduced oxidized low-density lipoprotein (oxLDL) uptake, Sirt6 knockdown enhanced it and increased mRNA and protein levels of macrophage scavenger receptor 1 (Msr1), whereas levels of other oxLDL uptake and efflux transporters remained unchanged. Similarly, in human primary macrophages, Sirt6 knockdown increased MSR1 protein levels and oxLDL uptake. Double knockdown of Sirt6 and Msr1 abolished the increase in oxLDL uptake observed upon Sirt6 single knockdown. FACS analyses of macrophages from aortic plaques of Sirt6-deficient bone marrow-transplanted mice showed increased MSR1 protein expression. Double knockdown of Sirt6 and the transcription factor c-Myc in RAW cells abolished the increase in Msr1 mRNA and protein levels; c-Myc overexpression increased Msr1 mRNA and protein levels. Conclusions: Loss of Sirt6 in bone marrow-derived cells is proatherogenic; hereby macrophages play an important role given a c-Myc-dependent increase in MSR1 protein expression and an enhanced oxLDL uptake in human and murine macrophages. These findings assign endogenous SIRT6 in macrophages an important atheroprotective role.
1. Introduction Atherosclerosis is the leading cause of death in developed countries [1]. In atherogenesis, macrophages play a critical role, from initiation with fatty streaks up to plaque rupture. Atherosclerosis is enhanced
when an excess of oxidized low-density lipoproteins (oxLDL), that are formed by the oxidation of LDL particles, lead to foam cell formation within the subendothelial layer of the arterial wall. Foam cell formation is accelerated either by an increase in oxidized lipid uptake and/or by a decrease in cholesterol efflux. Scavenger receptors on macrophages
⁎ Corresponding author at: Department of Cardiology, University Heart Center, University Hospital Zurich and Center for Molecular Cardiology, University of Zurich, Raemistrasse 100, 8091 Zurich, Switzerland. E-mail address: [email protected] (C.M. Matter).
https://doi.org/10.1016/j.yjmcc.2020.01.002 Received 18 June 2019; Received in revised form 10 January 2020; Accepted 13 January 2020 Available online 21 January 2020 0022-2828/ © 2020 Elsevier Ltd. All rights reserved.
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Fig. 1. Hematopoietic homozygous Sirt6 deletion increases atherosclerosis. Bone marrow (BM) cells from Apoe−/− Sirt6+/+ or Apoe−/− Sirt6−/− mice were transplanted into recipient Apoe−/− mice that were fed a high-cholesterol diet for 12 weeks. A, Representative images of en face thoraco-abdominal aortae stained with oil-red O (ORO, left panel) and their corresponding plaque quantifications (Apoe−/− Sirt6+/+, n = 14; Apoe−/− Sirt6−/− n = 13, right panel). B, C, Representative images (left panel) and quantifications of aortic root cross sections (right panel) stained for lipids (ORO) and CD68 (n = 12 in each group). Scale bars in photomicrographs is 500 μm. D, Plasma levels of total cholesterol (Apoe−/− Sirt6+/+, n = 11; Apoe−/− Sirt6−/−, n = 9) and E, plasma triglycerides (TG; Apoe−/− Sirt6+/+, n = 11; Apoe−/− Sirt6−/−, n = 9). F, Plasma levels of MCP-1 (Apoe−/− Sirt6+/+, n = 11; Apoe−/− Sirt6−/−, n = 10) and G, IL-6 (Apoe−/− Sirt6+/+, n = 10; Apoe−/− Sirt6−/−, n = 9). Values are presented as mean ± SEM and analysed using unpaired Student's two-tailed t-test for fig. A, B, D and E data and Mann-Whitney U test for fig. C, F, G and H data. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
recognize and internalize the modified forms of LDL particles and trigger foam cell formation. Important scavenger receptors expressed on macrophages include scavenger receptor A, (in mice: macrophage scavenger receptor 1, MSR1), CD36 and oxidized low-density lipoprotein receptor 1 (LOX1). Sirtuins (SIRT1–7) comprise a family of nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases that are activated upon caloric restriction and prevent age-associated diseases [2]. Among the sirtuins, SIRT6 is a nuclear protein known to deacetylate histone H3 lysine 9 (H3K9) [3] and H3 lysine 56 (H3K56) [4,5]. It increases chromosomal stability by promoting repair of double strand breaks, a hallmark of genomic instability in aging tissues [6]. SIRT6 has gained attention for its role in human telomere and genome stabilization [3], gene expression and DNA repair [7], glucose and fat homeostasis [8–10], as well as inflammation [11,12]. Experiments with genetically engineered mouse models using Sirt6 loss-of-function or gain-of-function studies highlight the protective role of SIRT6 in aging, metabolism and inflammation: Homozygous Sirt6deficient mice demonstrate a progeria-like phenotype with severe hypoglycemia leading to death around 3 to 4 weeks of age [13]. In the myocardium, Sirt6 deficiency boosts insulin-like growth factor signaling and results in cardiac hypertrophy and heart failure [14]. In human
endothelial cells, SIRT6 depletion increases DNA damage, telomere dysfunction and senescence [15]. Hepatocyte-specific Sirt6 deficiency enhances Pcsk9 gene expression and elevates plasma LDL cholesterol levels [10]. Finally, cultured bone marrow-derived macrophages (BMDM) isolated from Sirt6-null mice reveal increased levels of Mcp-1 and Il-6, and hypersensitivity to lipopolysaccharide stimulation [16]. Sirt6 gain-of-function studies underline the above-mentioned beneficial effects of SIRT6: SIRT6 protects against cardiac hypertrophy, heart failure, myocardial and hypoxic damage [14,17,18] and improves lipid metabolism by decreasing plasma LDL cholesterol and Pcsk9 levels [10]. SIRT6 also functions as a co-repressor of c-MYC transcriptional activity [19]. Of note, c-MYC has been associated with the expression of scavenger receptors and foam cell formation [20]. Less is known about SIRT6 in atherogenesis and its particular role in macrophages: Recent studies have reported a putative protective role of endogenous SIRT6 in atherosclerosis using a partial loss-of-function approach: Constitutive heterozygous Sirt6 deletion enhanced atherosclerosis through an increase in inflammation [21,22]. Similarly, a systemic knockdown of Sirt6 using small hairpin RNA, increased plaque size and vulnerability features [23]. Finally, an in vitro study reported that SIRT6 reduced macrophage foam cell formation by inducing autophagy using a gain-of-function approach [24]. 25
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However, the role of bone marrow (BM)-specific Sirt6 deletion on atherosclerosis, its effects on foam cell formation and the key molecular mediators in this context remain unknown. Thus, we hypothesized that homozygous BM-specific Sirt6 deletion in apolipoprotein E knockout (Apoe−/−) mice enhances atherogenesis and increases macrophage foam cell formation by interacting with MSR1.
double knockdown of Msr1 and Sirt6. Msr1 and Sirt6 double knockdown abolished the increase in Dil-oxLDL uptake observed upon Sirt6 single knockdown (Fig. 2E). In contrast, adenovirus-mediated overexpression of Sirt6 (Supplemental Fig. 4B) reduced oxLDL uptake (Supplemental Fig. 4C) and reduced MSR1 protein expression compared with control adenovirus (Supplemental Fig. 4D). To translate our findings to human macrophages, we investigated whether SIRT6 also regulates oxLDL uptake in human primary macrophages via MSR1 expression. Similar to our findings in mice, SIRT6 knockdown in human macrophages increased uptake of Dil-oxLDL (Fig. 2F), which corresponded with increased MSR1 expression compared with scramble control (Fig. 2G).
2. Results 2.1. Hematopoietic Sirt6 deletion increases atherosclerosis in Apoe−/− mice To test the effects of Sirt6 deletion in BM-derived cells on atherosclerotic plaque formation, BM isolated from Apoe−/-Sirt6+/+ or Apoe−/-Sirt6−/− donor mice were transplanted into sub-lethally irradiated Apoe−/− recipient mice (Supplemental Fig. 1). Of note, Apoe−/Sirt6−/− mice were not born at the expected Mendelian frequency (Apoe−/-Sirt6−/− 20 [5.76%], n = 347, approximately one pup every 15 litters). Even though Sirt6 knockout pups born on Apoe−/− background had no birth abnormalities, they showed early mortality similar to Sirt6 knockout mice. Following 6 weeks of recovery after BM reconstitution, mice were fed a HCD for 12 weeks. En face plaque staining of thoraco-abdominal aortae revealed a significant increase in atherosclerosis in Apoe−/-Sirt6−/− BM-recipient mice compared with Apoe−/Sirt6+/+ BM-recipient mice (Fig. 1A). Similarly, ORO staining of serial cross-sections of aortic roots showed an increase in lipid accumulation and atherosclerotic lesion size in Apoe−/-Sirt6−/− BM-recipient mice (Fig. 1B) compared with Apoe−/-Sirt6+/+ BM-recipient control mice. Furthermore, aortic root cross-sections from the Apoe−/-Sirt6−/− BMrecipient mice showed increased macrophage (CD68-positive staining) accumulation within atherosclerotic plaques (Fig. 1C). There were no significant differences in the weight course of these mice during the 12 weeks on HCD (data not shown). Plasma lipid and cytokine analyses showed that cholesterol levels were lower in the Apoe−/-Sirt6−/− BM-recipient mice, while triglycerides (TG), as well as MCP-1 and IL-6 levels remained unchanged (Fig. 1D-G). The cholesterol and triglyceride fractions are shown in Supplemental Fig. 2. Macrophages localized in the plaques of Apoe−/Sirt6−/− BM-recipient mice lacked SIRT6 expression compared with Apoe−/-Sirt6+/+ BM-recipient mice (Supplemental Fig. 3A), confirming that plaque-resident macrophages were derived from transplanted BM cells. Flow cytometric analyses proved the transplantation efficiency of CD45.1 BM cells into recipient mice (Supplemental Fig. 3B). Blood hematology counts between the two groups of mice were not changed (data not shown).
2.3. Sirt6−/− bone marrow transplantation increases MSR1 expression in mouse aortic plaque macrophages To assess whether SIRT6 regulates MSR1 in vivo, we analysed Msr1 expression in BM-recipient mice in the presence or absence of Sirt6, respectively. BM cells isolated from Apoe−/-Sirt6−/− BM-recipient mice, fed a HCD for 12 weeks, showed both increased mRNA and protein levels of Msr1, compared with BM cells from Apoe−/-Sirt6+/+ BM-transplanted mice (Fig. 3A, B). Furthermore, immunofluorescence analyses of aortic root cross sections suggested an increase in MSR1 staining in macrophages of Apoe−/Sirt6−/− BM-recipient mice, compared with Apoe−/-Sirt6+/+ BM-recipient mice (Fig. 3C). To quantify increased MSR1 expression in plaque-derived macrophages, plaques were isolated from the aortic arch. CD45+CD11b+ Ly6C+ F4/80+ macrophages were identified and MSR1 levels assessed by flow cytometry. Increased expression of MSR1 in aortic plaque macrophages of Apoe−/-Sirt6−/− BM-recipient mice was observed relative to Apoe−/− BM-recipient mice (Fig. 3D). Corresponding analyses in blood monocytes isolated from these mice showed no difference in MSR1 levels between both groups (Fig. 3E, F). Similar to aortic plaque macrophages, MSR1 levels were increased in BMDM from Apoe−/Sirt6−/− compared with Apoe−/-Sirt6+/+ mice (Fig. 3G) ex vivo. 2.4. Sirt6 knockdown increases Msr1 expression via c-MYC To identify the mechanism underlying the increase in Msr1 expression, we performed a ChIP of acetyl H3K9 and acetyl H3K56 in RAW 264.7 cells upon Sirt6 knockdown. There was no significant difference in enrichment for H3K9 and H3K56 acetylation at Msr1 promoter in the scramble and Sirt6 knockdown cells (Supplemental Fig. 5), suggesting that the increase in Msr1 expression upon Sirt6 knockdown does not reflect a Sirt6-mediated increase in acetylation of histone 3 at the Msr1 promoter. A recent study shows that SIRT6 co-represses c-MYC transcriptional activity and ribosomal gene expression [19]. Co-immunoprecipitation experiments were performed in RAW 264.7 cells to confirm the interaction between SIRT6 and c-MYC in our experimental setting (Supplemental Fig. 6). As c-MYC transcriptional activity is linked to oxLDL uptake [20] and the UCSC database shows that c-MYC binds to the enhancer region of Msr1, we investigated whether loss of c-MYC represses the increase in Msr1 levels observed upon Sirt6 knockdown. Indeed, double knockdown of c-Myc and Sirt6 prevented an increase in Msr1 expression in RAW 264.7 cells, both at the mRNA and protein level (Fig. 4A, B). In contrast, c-MYC overexpression was associated with Msr1 gene upregulation, thus confirming the role of c-MYC as a regulator of Msr1 transcription (Fig. 4C, Supplemental Fig. 7).
2.2. Sirt6 decreases oxLDL accumulation by suppressing Msr1 expression in murine and human macrophages To determine whether macrophage-specific deficiency of Sirt6 affects LDL uptake, the accumulation of oxLDL in RAW 264.7 cells was measured in vitro. Expression levels of SIRT6 were markedly reduced 48 h after Sirt6 silencing (Fig. 2A). Flow cytometric analyses revealed that Sirt6 silencing in macrophages increased oxLDL accumulation in vitro (Fig. 2B). Similarly, acLDL accumulation was increased upon Sirt6 knockdown in RAW 264.7 cells (Supplemental Fig. 4A). To address the mechanism underlying the increase in oxLDL uptake upon Sirt6 deficiency, we assessed the effects of Sirt6 silencing on the expression of genes involved in cellular cholesterol accumulation. Knockdown of Sirt6 increased scavenger receptor Msr1 mRNA and protein expression without altering mRNA expression of other scavenger receptors such as Cd36 or Lox1 (Fig. 2C, D). Furthermore, mRNA levels of cholesterol efflux transporters Abca1 and Abcg1 were not altered (Fig. 2C). To address the causal involvement of increased MSR1 expression in enhanced oxLDL accumulation, Dil-oxLDL uptake was measured upon
3. Discussion We demonstrate that BM-specific Sirt6 deletion is sufficient to increase modified LDL uptake in macrophages and enhance plaque 26
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Fig. 2. SIRT6 downregulates macrophage oxLDL uptake via MSR1 in mice and men. A, Western blot of SIRT6 protein levels upon scramble or Sirt6 knockdown from murine RAW 264.7 macrophages (n = 3 each). B, Quantification of Dil-oxLDL uptake upon scramble or Sirt6 knockdown (n = 6). C, Relative mRNA expression levels of genes involved in modified low-density lipoprotein uptake and efflux (n = 5); mRNA expression levels were normalized to Rps29 and Rps18. D, Sirt6 knockdown increases MSR1 protein compared to control levels in RAW 264.7 macrophages (n = 3). E, Quantification of DiloxLDL upon knockdown of Sirt6 and Msr1 (n = 3). Knockdown of SIRT6 in human primary macrophages F, enhances Dil-oxLDL uptake (n = 6) and G, increases MSR1 expression (n = 6). Values are represented as mean ± SEM and analysed using Mann-Whitney U test (B-D, F, G) and ordinary oneway ANOVA with Holm-Sidak's multiple comparison (E).
formation in Apoe−/− mice through increased Msr1 expression in bone marrow cells and plaque macrophages. Corresponding in vitro experiments reveal that Sirt6 deficiency increases MSR1 levels both in mouse and human, likely via regulation of the transcription factor c-MYC. Thus, our findings identify MSR1 as a novel target for atheroprotective effects of SIRT6, whereby macrophages play a central role (Fig. 5). Our discoveries extend recent findings in which constitutive heterozygous deletion of Sirt6 enhanced atherosclerosis through an increase
in NKG2D ligand expression and inflammation [21,22]. Sirt6-induced atheroprotection mediated by BM-derived cells may involve all hematopoietic cells. We propose macrophages as the protagonist cells for the following reasons: Homozygous Sirt6 deficiency in BM-derived cells of Apoe−/− chimeric mice increased macrophage-specific MSR1 expression in aortic plaques. Moreover, our in vitro findings place MSR1 center stage for SIRT6-dependent foam cell formation. Ex vivo, BM-derived macrophages from Sirt6−/− mice have been reported to increase Mcp-1 27
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Fig. 3. Hematopoietic Sirt6 deficiency increases MSR1 expression in bone marrow cells and aortic plaque macrophages. A, Msr1 mRNA (n = 12) and B, MSR1 protein expression (n = 7) is increased in bone marrow (BM) cells isolated from Apoe−/-Sirt6−/− BM transplanted (BMT) mice compared to Apoe−/− Sirt6+/+ BMT mice. C, Immunofluorescence of MSR1 and CD68 suggesting more macrophage-associated MSR1 expression in cross sections of the aortic root of Apoe−/− Sirt6−/− BMT mice compared with Apoe−/-Sirt6+/+ BMT mice (scale bar = 25 μm). The qualitative immunofluorescence message provided in C is given in a quantitative fashion after sorting aortic arch plaques for macrophages: D, MSR1 protein levels are higher in F4/80+Ly6C+ aortic plaque macrophage population (n = 7) in Apoe−/− Sirt6−/− BMT mice compared with the Apoe−/− Sirt6+/+ BMT mice. E, F, MSR1 protein levels are unchanged in the Ly6Clow (Apoe−/− Sirt6+/+, n = 9; Apoe−/− Sirt6−/−, n = 10) and Ly6Chigh (Apoe−/− Sirt6+/+, n = 9; Apoe−/− Sirt6−/−, n = 10) blood monocyte populations of Apoe−/− Sirt6−/− and Apoe−/− Sirt6+/+ BMT mice. G, MSR1 protein expression after sorting for BM-derived macrophages (n = 4) obtained from Apoe−/− Sirt6+/+ and Apoe−/− Sirt6−/− mice. Values are presented as mean ± SEM and analysed using unpaired Student's two-tailed t-test for fig. A, E and F data and Mann-Whitney U test for fig. B, D and G.
and Il-6 expression levels [16]. In contrast, we could not detect any difference in plasma MCP-1 and IL-6 levels between both groups of BMrecipient mice, indicating that BM-restricted Sirt6 deletion does not mediate its pro-atherosclerotic effects by increasing systemic inflammation. This observation is in line with a previously published study showing that a myeloid specific deletion of Sirt6 leads to a reduced polarization of macrophages toward the inflammatory M2 type [25]. Moreover, our data show that cholesterol levels in mice lacking Sirt6 are decreased. Keeping in mind that MSR1 levels in our mouse model are increased, this is consistent with an earlier publication, which shows that a bone marrow-specific overexpression of MSR1 in Apoe−/− mice decreases cholesterol levels, possibly via increased cholesterol uptake of Kupffer cells (macrophage equivalent) in the liver [26]. Using complementary Sirt6 loss- and gain-of-function experiments,
our data show that SIRT6 decreases oxLDL uptake in RAW 264.7 macrophages, thereby diminishing foam cell formation. In addition, gene expression analyses upon Sirt6 knockdown revealed a specific increase in Msr1 levels with no difference in transcription of other scavenger receptors or cholesterol efflux genes. MSR1 expression in macrophages is known to increase the uptake of oxidized LDL particles in vitro [27]. Moreover, Msr1 loss-of-function studies using an Apoe−/− background have been reported to provide atheroprotection [27,28]. In parallel, polymorphisms in the MSR1 gene have been associated with human atherosclerosis [29]. These studies highlight the role of MSR1 in the uptake of modified LDL, foam cell formation and atherogenesis. However, the above-mentioned study, using bone marrow-specific MSR1 overexpression in an Apoe−/− background, did not find a difference in atherosclerosis between mice with and without MSR1 overexpression [26]. Of note, these mice were fed a normal chow diet. In contrast, we use a high cholesterol diet mimicking a western-type diet 28
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Fig. 4. Sirt6 knockdown increases MSR1 expression by enhancing c-MYC transcriptional activity. A, B, Knockdown of Sirt6 and c-Myc rescues the increase in Msr1 expression observed upon Sirt6 knockdown at both mRNA (n = 6) (A) and protein level (n = 6) (B). C, Msr1 gene expression following c-MYC overexpression. A control CRISPR Activation Plasmid was used as a negative control. Values are represented as mean ± SEM and analysed using ordinary one-way ANOVA with HolmSidak's multiple comparison; *:p < .05; **: p < .01; ***: p < .001; ****: p < .0001.
Fig. 5. Scheme illustrating the proposed molecular mechanism (left) and atherosclerosis phenotype (right panel) of Sirt6 loss-of-function in bone marrow-derived macrophages. A, Intact endogenous SIRT6 co-represses c-MYC and decreases Msr1 transcription and protein expression. These events diminish oxLDL uptake and foam cell formation in vitro and in vivo, thereby limiting progression of atherosclerosis. B, Sirt6 loss-of-function favors c-MYC transcriptional activity leading to enhanced Msr1 transcriptional activity and protein expression. The subsequent increase in oxLDL uptake is sufficient to enhance foam cell formation and atherosclerosis.
that is known to induce a more pronounced, macrophage-driven atherosclerosis phenotype. This increased cholesterol in mice with loss of protective SIRT6 is likely to increase levels of MSR1, enhance oxLDL uptake into macrophages eventually leading to increased
atherosclerosis. In line with these findings, double knockdown of Sirt6 and Msr1 prevented oxLDL uptake confirming that increased foam cell formation upon Sirt6 knockdown is Msr1-dependent. We translated these proof-of29
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principle in vitro findings to an ex vivo context: Using Apoe−/− mice, an environment reflecting markedly increased oxLDL, Sirt6 deletion in BMderived cells increased both mRNA and protein levels of the scavenger receptor Msr1. Increased MSR1 protein levels in Sirt6-deficient cells, in both BMDM obtained from donor BM cells before and after transplantation, suggests that MSR1 plays a central role for foam cell formation in vitro and in vivo. Interestingly, the ratio of upregulation of MSR1 upon Sirt6 deficiency is coherent with an increase in oxLDL uptake, strengthening the notion that indeed the increase in MSR1 is mediating these changes. To understand the interactions between SIRT6 and MSR1, we assessed whether SIRT6-dependent deacetylation at H3K9 and H3K56 modulated Msr1 at its promoter and found no difference. Since c-MYC binds to the Msr1 enhancer and SIRT6 is known to repress c-MYC activity [19], we investigated whether the observed increase in MSR1 levels was c-MYC-dependent. Indeed, double knockdown of c-Myc and Sirt6 prevented the SIRT6-dependent increase in MSR1 protein expression, suggesting that the SIRT6-dependent modulation of MSR1 expression is mediated by c-MYC. A recent study has shown that a rapid turnover of macrophages in atherosclerotic lesions is due to an increased Msr1 expression, which in turn promotes atherogenesis [30]. Indeed, we found an increase in MSR1 protein levels in macrophages isolated from aortic plaques of Apoe−/-Sirt6−/− BMT mice compared with Apoe−/-Sirt6+/+ BMT mice. Conversely, blood monocytes isolated from the same mice showed no difference in MSR1 protein levels. This suggests a specific effect of SIRT6 on Msr1 expression in macrophages. In line with our findings in mouse macrophages, SIRT6 knockdown in human primary macrophages also caused an increase in MSR1 expression and oxLDL uptake, thus translating our findings to humans and highlighting the importance of SIRT6 in foam cell formation and atherosclerosis.
SIRT1, another nuclear sirtuin which shares atheroprotective effects with SIRT6 [35]. SIRT1 activators have been shown to decrease experimental atherosclerosis along pathways [36] that are similar to SIRT6 [10] and improve human endothelial dysfunction [37]. Human atherosclerosis is associated with an increased susceptibility to thrombosis, thereby leading to myocardial infarction and stroke. Along these lines, SIRT1 and SIRT3 have shown to confer protective effects in experimental thrombosis [38,39]. As pharmacological NAD+ boosters have become available as pansirtuin activators [2,40] with promising data at the experimental level [41–43], time is ready to explore unspecific sirtuin activators as a therapeutic approach in humans. Along these lines, NAD+ boosters may become particularly promising for treating or preventing atherothrombotic diseases such as coronary artery disease and myocardial infarction, cerebrovascular disease and stroke or peripheral arterial disease and acute limb ischemia [44]. 6. Methods 6.1. Animals All animal experiments were approved by the Cantonal Veterinary Office Zurich, Switzerland. All applicable international, national, and institutional guidelines for the care and use of animals were followed and all procedures performed involving animals were in accordance with the ethical standards of these guidelines. Apoe−/−Sirt6+/− mice (Sirt6+/− provided by Johan Auwerx, EPFL, Lausanne, Switzerland) and Apoe−/− mice (Charles River) were bred under pathogen–free conditions housed with a 12 h light – dark cycle. Apoe−/− Sirt6+/− mice were bred to yield Apoe−/− Sirt6−/− offspring. Since these mice show a strong metabolic phenotype with increased embryonic lethality, and the few surviving mice are usually dying within 3–4 weeks after birth, mice were sacrificed at the age of 3 weeks and the bone marrow isolated for transplantation [13]. To generate BM chimeras, we adapted a previous protocol [35] by using young donor mice: 6 week-old male Apoe−/− recipient mice were sub-lethally irradiated with two doses of 550 rads each (female mice were not tested). 3 week-old female and male donor mice were killed by CO2 inhalation and bones (hind limbs and hips) were flushed with sterile PBS to obtain bone marrow (BM) cells. Donor BM cells were injected intravenously (2.5 × 106 total cells per mouse). To prevent bacterial infection, 0.2% (vol/vol) Borgal (trimethoprim, sulfadoxin) was added to the drinking water for 1 week. Mice recovered for 6 weeks and then were fed a HCD, containing 1.25% cholesterol (D12108; Research Diets, New Brunswick, USA), for 12 weeks. To confirm BM chimerism, a control experiment with CD45.1 donor BM cells injected into CD45.2 positive Apoe−/− mice was performed: Cell mixtures were assessed by flow cytometry with monoclonal antibodies to CD45.1 (A20; BD Pharmingen) and CD45.2 (104; BD Pharmingen).
4. Limitations In our study we adopted bone marrow-restricted Sirt6 deletion to assess the role of hematopoietic cells in atherosclerosis. Additionally, we identified that SIRT6 regulates MSR1 by involving c-MYC. However, the exact molecular mechanism by which SIRT6 affects MSR1 remains to be determined. Furthermore, we cannot rule out that other bone marrow-derived cells that express MSR1 contribute to increased atherosclerosis in our model. Along these lines, deletion of Sirt6 may increase MSR1 expression in dendritic cells, which have been shown to express MSR1 [31]. Moreover, we cannot exclude a contribution of other potentially atherogenic bone marrow-derived cells such as neutrophils and T cells, independent of any MSR1 expression. In our study, we have only used male mice as bone marrow recipients to assess atherosclerosis. Further studies would be necessary to address whether a similar mechanism is operative in female mice. In fact, a sex-dependent phenotype has been reported using Sirt6 gain- or loss-of-function experiments regarding lifespan or insulin sensitivity, respectively [32,33].
6.2. Cell culture RAW 264.7 macrophages (Sigma-Aldrich) were cultured in a 5% CO2 incubator at 37 °C in DMEM-high glucose medium supplemented with 2 mM Glutamine and 10% fetal bovine serum (v/v). BM-derived macrophages (BMDM) were obtained by treating BM cells with 10 ng/ ml M-CSF for one week. To obtain human macrophages, buffy coats from healthy volunteers were used and peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient centrifugation. Using CD14 Microbeads (Miltenyi Biotec), the monocyte population was isolated and plated with complete RPMI medium supplemented with 10% FBS and 10 ng/ml GM-CSF (Peprotech) for 8 days.
5. Conclusions and perspectives We demonstrate that Sirt6 loss-of-function in BM-derived cells is sufficient to increase atherosclerosis in Apoe−/− mice. Sirt6 deficiency upregulates Msr1 expression both in vivo and in vitro, in mice and human macrophages via c-MYC. Thus, our results reveal a novel, atheroprotective role of SIRT6. Hereby, macrophages play a key role as SIRT6 decreases foam cell formation by downregulating MSR1. In humans, SIRT6 expression levels are inversely associated with atherosclerosis: SIRT6 is decreased in human carotid plaques compared with normal arteries [21] and is lower in plaques from diabetic compared with non-diabetic patients [34]. There is translational potential for sirtuin activation related to
6.3. Immunohistochemistry, immunofluorescence and plaque analyses Aortic root tissues were embedded in OCT, followed by cryostat 30
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cutting into 5 μm thick sections. For immunohistochemistry, sections were fixed in acetone and incubated with the following primary antibodies: anti-CD68 (Bio-Rad), anti-SIRT6 (Cell Signaling), and antiMSR1 (Novus Biologicals). Subsequently, sections were incubated with Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen) (MSR1, SIRT6) and Alexa Fluor 547 goat anti-rat IgG (eBioscience) (CD68) for 1 h. Aortic plaques were quantified en face (thoraco-abdominal) and in root cross sections using oil-red O (ORO) as described [35].
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6.4. Statistics Data are presented as mean ± SEM. Normal distribution was assessed with the D'Agostino-Pearson omnibus normality test using GraphPad Prism. For non-parametric data, the Mann – Whitney test was used. Three or more groups were compared using an ordinary one-way ANOVA with Holm-Sidak's multiple comparison. At least three independent experiments in triplicates were performed. Statistical significance was accepted at p ≤ .05. Analyses were performed using GraphPad Prism (version 5.0d, 2010). For multiple comparisons, significances are shown as stars: *:p < .05; **: p < .01; ***: p < .001; ****: p < .0001. Further Materials and Methods can be found in the supplements. Funding This work was supported by the Swiss National Science Foundation to JA (31003A-140780), JHB (310030–144152), TFL (310030–135815), CMM (310030–146923), CMM and JA (310030–165990), and to MOH (310030–157019/176177); by the National Institute of Health (USA) to JA (R01AG043930), Systems X (SysX.ch) to JA (2013–15), by Swiss Heart Foundation grants to CMM and JHB, the University Research Priority Program Integrative Human Physiology at the University of Zurich to CMM; the Hartmann-Müller Foundation and Novartis Foundation to CMM and LJvT, the Ecole Polytechnique Fédérale de Lausanne to JA, the University of Zurich to MOH, by the Kardio Foundation, Switzerland and the Budai Foundation, Lichtenstein to JHB – and the Zurich Heart House, Foundation for Cardiovascular Research, Zurich, Switzerland. Disclosures JA is a founder and scientific advisory board member of Mitobridge. The other authors do not declare a potential conflict of interest. Acknowledgements The graphical abstract was prepared using Servier Medical Art by Servier licensed under a Creative Commons Attribution 3.0 Unported License. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.yjmcc.2020.01.002. References [1] N. Townsend, L. Wilson, P. Bhatnagar, K. Wickramasinghe, M. Rayner, M. Nichols, Cardiovascular disease in Europe: epidemiological update 2016, Eur. Heart J. 37 (42) (2016) 3232–3245. [2] S. Winnik, J. Auwerx, D.A. Sinclair, C.M. Matter, Protective effects of sirtuins in cardiovascular diseases: from bench to bedside, Eur. Heart J. 36 (48) (2015) 3404–3412. [3] E. Michishita, R.A. McCord, E. Berber, M. Kioi, H. Padilla-Nash, M. Damian, et al., SIRT6 is a histone H3 lysine 9 deacetylase that modulates telomeric chromatin, Nature 452 (7186) (2008) 492–496. [4] E. Michishita, R.A. McCord, L.D. Boxer, M.F. Barber, T. Hong, O. Gozani, et al., Cell cycle-dependent deacetylation of telomeric histone H3 lysine K56 by human SIRT6,
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