Inhibiting DNA Methylation by 5-Aza-2'-deoxycytidine ameliorates atherosclerosis through suppressing macrophage inflammation - PubMed (original) (raw)

. 2014 Dec;155(12):4925-38.

doi: 10.1210/en.2014-1595. Epub 2014 Sep 24.

Xianfeng Wang, Lin Jia, Ashis K Mondal, Abdoulaye Diallo, Gregory A Hawkins, Swapan K Das, John S Parks, Liqing Yu, Huidong Shi, Hang Shi, Bingzhong Xue

Affiliations

Inhibiting DNA Methylation by 5-Aza-2'-deoxycytidine ameliorates atherosclerosis through suppressing macrophage inflammation

Qiang Cao et al. Endocrinology. 2014 Dec.

Abstract

Inflammation marks all stages of atherogenesis. DNA hypermethylation in the whole genome or specific genes is associated with inflammation and cardiovascular diseases. Therefore, we aimed to study whether inhibiting DNA methylation by DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) ameliorates atherosclerosis in low-density lipoprotein receptor knockout (Ldlr(-/-)) mice. Ldlr(-/-) mice were fed an atherogenic diet and adminisered saline or 5-aza-dC (0.25 mg/kg) for up to 30 weeks. 5-aza-dC treatment markedly decreased atherosclerosis development in Ldlr(-/-) mice without changes in body weight, plasma lipid profile, macrophage cholesterol levels and plaque lipid content. Instead, this effect was associated with decreased macrophage inflammation. Macrophages with 5-aza-dC treatment had downregulated expression of genes involved in inflammation (TNF-α, IL-6, IL-1β, and inducible nitric oxidase) and chemotaxis (CD62/L-selectin, chemokine [C-C motif] ligand 2/MCP-1 [CCL2/MCP-1], CCL5, CCL9, and CCL2 receptor CCR2). This resulted in attenuated macrophage migration and adhesion to endothelial cells and reduced macrophage infiltration into atherosclerotic plaques. 5-aza-dC also suppressed macrophage endoplasmic reticulum stress, a key upstream signal that activates macrophage inflammation and apoptotic pathways. Finally, 5-aza-dC demethylated liver X receptor α (LXRα) and peroxisome proliferator-activated receptor γ1 (PPARγ1) promoters, which are both enriched with CpG sites. This led to overexpression of LXRα and PPARγ, which may be responsible for 5-aza-dC's anti-inflammatory and atheroprotective effect. Our findings provide strong evidence that DNA methylation may play a significant role in cardiovascular diseases and serve as a therapeutic target for prevention and treatment of atherosclerosis.

PubMed Disclaimer

Figures

Figure 1.

Figure 1.

5-Aza-dC ameliorates atherosclerosis development in _Ldlr_−/− mice. A, Representative cross-sectional image of aortic sinus stained with Oil Red O. B, Quantification of aortic lesion area. C, Representative images of aortic surface lesions. D, Aortic surface lesion area normalized to total aortic surface area. Eight-week-old male _Ldlr_−/− mice (n = 6 per group) were fed an atherogenic diet and were ip injected with either saline or 5-aza-dC (0.25 mg/kg body weight) 3 times a week. Quantification of atherosclerosis was conducted as described in Materials and Methods. For B and D, data are expressed as mean ± SEM; n = 6 per group. *, P < .05; **, P < .01.

Figure 2.

Figure 2.

5-aza-dC reduces macrophage content and inflammation in intima of artery wall in _Ldlr_−/− mice. A, Representative cross-sectional image of aortic sinus stained with CD68 antibody. B, Quantification of CD68-positive lesion area (square millimeters) and percentage in total lesion area. C, 5-Aza-dC decreases the percentage of F4/80+ cells in live aortic cells. Eight-week-old male _Ldlr_−/− mice (n = 6 per group) were treated with 5-aza-dC as described in Materials and Methods. For A and 2, the upper one-third of the heart was dissected and embedded in Optimum cutting temperature (OCT) medium, cryosectioned at 8-μm intervals, and stained with anti-CD68 antibodies. For C, aortas were digested to release aortic cells. The cells were stained with APC-conjugated anti-F4/80 antibodies and were analyzed using BD FACSCanto II as described in Materials and Methods. D, Expression of inflammatory genes in aortic macrophages. Aortic macrophages were isolated using a magnetic-activated cell sorting system as described in Materials and Methods. The expression of inflammatory genes was measured by real-time RT-PCR and normalized to cyclophilin; n = 8 per group. Data are expressed as mean ± SEM. *, P < .05; **, P < .01.

Figure 3.

Figure 3.

5-aza-dC suppresses macrophage inflammation, migration, and adhesion. A–G, 5-Aza-dC inhibits LPS-stimulated (A–F) and stearate-stimulated (G) expression of proinflammatory genes in macrophages. Raw264.7 macrophages were pretreated with 5-aza-dC (0.5μM) for 4 days and then treated with LPS (100 ng/mL) (A–F) or stearate (C18:0, 250μM) (G) for 4 hours. Gene expression was measured by real-time RT-PCR and normalized to cyclophilin; n = 4 per group. H–I, 5-Aza-dC reduces macrophage adhesion to endothelial cells (H) and migration (I). BMDMs were pretreated with 0.5μM 5-Aza-dC for 4 days, and BMDM-endothelium adhesion or migration assays were performed as described in Materials and Methods; n = 5 per group. All data are expressed as mean ± SEM. *, P < .05; **, P < .01.

Figure 4.

Figure 4.

5-Aza-dC inhibits macrophage ER stress. A and B, 5-Aza-dC inhibits ERN1/IRE-1α (A) and Chop (B) mRNA expression in macrophages. Raw264.7 macrophages were treated with 5-aza-dC (0.5μM) for up to 6 days. ERN1/IRE-1α (A) and Chop (B) mRNA expression was measured by real-time RT-PCR and normalized to cyclophilin; n = 4 per group. C–E, 5-Aza-dC inhibits phosphorylated (p-) and total ERN1/IRE-1α in macrophages. Raw264.7 macrophages were pretreated with 5-aza-dC (0.5μM) for 2 days and then were stimulated with stearate (C18:0, 250μM) for 1 day. Phosphorylated and total ERN1/IRE-1α was measured by immunoblotting. A representative blot is shown in C. The blots were quantitated for total (D) and phosphorylated (E) ERN1/IRE-1α expression with a Li-COR Odyssey infrared imager system and normalized to that of β-actin; n = 3 per group. All data are expressed as mean ± SEM. **, P < .01 (A and B); for D and E, groups labeled with different superscripts are statistically different from each other, P < .05.

Figure 5.

Figure 5.

5-Aza-dC reduces ER stress and apoptosis in atherosclerotic plaques of _Ldlr_−/− mice. A and B, Representative cross-sectional image of aortic sinus stained with phospho-ERN1/IRE-1α (A) or Chop (B) antibodies. C and D, Quantification of phospho-ERN1/IRE-1α (C) or Chop (D) positive areas. E and F, 5-Aza-dC reduces apoptosis in atherosclerotic plaques of _Ldlr_−/− mice: E, representative images of apoptotic cells in aortic sinus; F, quantification of apoptotic cell numbers per section. All data are expressed as mean ± SEM; n = 6 per group. *, P < .05; **, P < .01. Abbreviation: TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Figure 6.

Figure 6.

5-Aza-dC induces LXRα and PPARγ1 expression via regulating their promoter DNA methylation. A and B, 5-Aza-dC stimulates LXRα mRNA in Raw26.47 macrophages (A) and aortic macrophages (B). C and D, 5-Aza-dC stimulates PPARγ mRNA in Raw264.7 macrophages (C) and aortic macrophages (D). In A and C, Raw264.7 macrophages were treated with 5-aza-dC (0.5μM) for up to 6 days; n = 6 per group. In B and D, aortic macrophages were isolated from saline- or 5-aza-dC-treated _Ldlr_−/− mice; n = 6 per group. LXRα and PPARγ mRNA were measured by real-time RT-PCR and normalized to cyclophilin. E and F, Promoter activities of LXRα (E) and PPARγ1 (F) were regulated by DNA methylation. LXRα and PPARγ1 promoters (Supplemental Figure 5, A and B) were cloned into pGL3-luciferase expression vector. Either unmethylated or fully methylated reporter constructs were transfected into Raw264.7 cells, and luciferase activity was measured; n = 3 per group. G and H, 5-Aza-dC demethylates LXRα promoter. Raw264.7 macrophages were treated with 5-aza-dC (0.5μM) for up to 6 days. CpG methylation was measured by pyrosequencing; n = 3 per group. In G, the average DNA methylation level at the LXRα promoter from CpG sites 9 to 25 (according to Supplemental Figure 6A) was calculated and expressed as a function of treatment time. H, DNA methylation levels at individual CpG sites on LXRα promoter (according to Supplemental Figure 5A) in Raw264.7 macrophages treated with 5-aza-dC for 6 days. I, DNA methylation levels at PPARγ1 promoter (according to Supplemental Figure 5B) in Raw264.7 macrophages treated with 5-aza-dC for 4 days; n = 3 per group. All data are expressed as mean ± SEM. For E and F, groups labeled with different superscripts are statistically different from each other, P < .05; for the rest of the graphs: *, P < .05; **, P < .01.

Figure 7.

Figure 7.

PPARγ and LXRα knockdowns substantially reverse the inhibitory effects of 5-aza-dC on inflammation and ER stress. A–F, PPARγ and LXRα knockdowns additively reverse the inhibitory effects of 5-aza-dC on TNF-α (A), IL-6 (B), CCL2 (C), CCL5 (D), IRE-1α (E), and Chop (F) mRNA expression in Raw264.7 macrophages. Raw264.7 macrophages were reversely transfected with PPARγ1 and/or LXRα siRNAs. Cells were then pretreated with 5-aza-dC (0.5μM) for 4 days. In A–D, cells were further stimulated with LPS (100 ng/mL) for 4 hours. Gene expression was measured by real-time RT-PCR and normalized to cyclophilin. All data are expressed as mean ± SE; n = 6 per group. Groups labeled with different letters are statistically significantly different, P < .05.

References

    1. Galkina E, Ley K. Immune and inflammatory mechanisms of atherosclerosis (*). Annu Rev Immunol. 2009;27:165–197. - PMC - PubMed
    1. Libby P, Ridker PM, Hansson GK. Inflammation in atherosclerosis: from pathophysiology to practice. J Am Coll Cardiol. 2009;54:2129–2138. - PMC - PubMed
    1. Erbay E, Babaev VR, Mayers JR, et al. Reducing endoplasmic reticulum stress through a macrophage lipid chaperone alleviates atherosclerosis. Nat Med. 2009;15:1383–1391. - PMC - PubMed
    1. Tabas I. The role of endoplasmic reticulum stress in the progression of atherosclerosis. Circ Res. 2010;107:839–850. - PMC - PubMed
    1. Luczak MW, Jagodzinski PP. The role of DNA methylation in cancer development. Folia Histochem Cytobiol. 2006;44:143–154. - PubMed

Publication types

MeSH terms

Substances

Grants and funding

LinkOut - more resources