Antagonism of miR-33 in mice promotes reverse cholesterol transport and regression of atherosclerosis

Abstract

Plasma HDL levels have a protective role in atherosclerosis, yet clinical therapies to raise HDL levels have remained elusive. Recent advances in the understanding of lipid metabolism have revealed that miR-33, an intronic microRNA located within the SREBF2 gene, suppresses expression of the cholesterol transporter ABC transporter A1 (ABCA1) and lowers HDL levels. Conversely, mechanisms that inhibit miR-33 increase ABCA1 and circulating HDL levels, suggesting that antagonism of miR-33 may be atheroprotective. As the regression of atherosclerosis is clinically desirable, we assessed the impact of miR-33 inhibition in mice deficient for the LDL receptor (Ldlr-/- mice), with established atherosclerotic plaques. Mice treated with anti-miR33 for 4 weeks showed an increase in circulating HDL levels and enhanced reverse cholesterol transport to the plasma, liver, and feces. Consistent with this, anti-miR33-treated mice showed reductions in plaque size and lipid content, increased markers of plaque stability, and decreased inflammatory gene expression. Notably, in addition to raising ABCA1 levels in the liver, anti-miR33 oligonucleotides directly targeted the plaque macrophages, in which they enhanced ABCA1 expression and cholesterol removal. These studies establish that raising HDL levels by anti-miR33 oligonucleotide treatment promotes reverse cholesterol transport and atherosclerosis regression and suggest that it may be a promising strategy to treat atherosclerotic vascular disease.

Figure 1. Anti-miR33 treatment reduces miR-33 expression in the liver and causes derepression of its target genes.

Ldlr^–/– mice were fed a WD for 14 weeks and then subsequently treated for 4 weeks with PBS (no treatment) or control anti-miR or anti-miR33 2′F/MOE oligonucleotides. (A and B) Expression of (A) miR-33, (B) miR-33 target genes (Abca1, Abcg1, Hadhb, Crot, Cpt1a), and nontarget lipid metabolism genes (Insig1, Hmgcr, Srebf2) in the liver was quantified by RT-PCR. Con, control. (C) Western blot of hepatic ABCA1 and ABCG1 protein. The white line denotes lanes run noncontiguously. *P ≤ 0.05, compared with controls. (D) Plasma levels of ALT and AST.

Figure 2. Anti-miR33 increases circulating HDL in Ldlr^–/– mice.

(A) Plasma HDL levels after 4 weeks of the indicated treatment (n = 15 mice/group). *P ≤ 0.05, compared with controls. (B) FPLC lipoprotein profiles from pooled plasma (n = 4) of PBS-treated and control anti-miR and anti-miR33–treated mice. (C) Cholesterol ester (CE) and free cholesterol (FC) content of the HDL. *P ≤ 0.05, compared with controls. (D) Western blots of apoA1 and apoE in HDL lipoprotein fractions (fraction number 53–70 of FPLC profile).

Figure 3. RCT is increased in anti-miR33–treated mice.

After 4 weeks of the indicated, treatment Ldlr^–/– mice (n = 8/group) were injected subcutaneously with ³H-cholesterol–labeled, acLDL-loaded bone marrow–derived macrophages. Data are expressed as the percentage of the ³H-cholesterol tracer relative to that of total cpm tracer injected ± SD. (A) Time course of ³H-cholesterol distribution in plasma. (B) Hepatic ³H-cholesterol tracer levels after 48 hours. (C) Fecal ³H-cholesterol tracer levels. Feces were collected continuously from 0 to 48 hours after injection.*P ≤ 0.05, compared with controls.

Figure 4. Anti-miR33 treatment regresses atherosclerosis.

(A) Quantification of the lesion area of mice (n = 15/group) at baseline (14 weeks of WD) and after 4 weeks of the indicated treatment. Horizontal bars indicate the mean, and individual symbols indicate individual mice. PBS treatment is indicated by the dash. Data are the mean ± SEM. *P ≤ 0.05. (B) Representative hematoxylin- and eosin-stained aortic sinus sections from control anti-miR– and anti-miR33–treated mice. Original magnification: ×50 (top images); ×200 (bottom images).

Figure 5. Inhibition of miR-33 improves markers of atherosclerotic plaque stability.

Characterization of atherosclerotic lesion composition by (A) oil red O staining for neutral lipids, (B) immunohistochemical staining for the macrophage marker CD68, and (C) picrosirius red staining for collagen. Images show representative sections from control and anti-miR33–treated mice, and quantification (n = 10 mice/group) is shown in the accompanying bar graph. *P ≤ 0.05, compared with no treatment and control anti-miR. Original magnification: ×200 (A and C); ×100 (B).

Figure 6. Anti-miR oligonucleotides reach plaque macrophages and alter target gene expression.

(A) Immunohistochemical detection of the phosphorothioate backbone of the 2′F/MOE oligonucleotides (left panel) and CD68 (right panel) in serial sections of the aortic sinus. Original magnification: ×50 (top images); ×200 (lower images). (B) Expression of Abca1 mRNA in plaque CD68⁺ macrophages isolated by laser-capture microdissection and analyzed by qRT-PCR. PBS treatment is indicated by the dash. *P ≤ 0.05, compared with all other groups. (C) Gene expression profiling was performed on mRNA of lesional macrophages isolated by laser-capture microdissection, and a CDF analysis was performed. CDF analysis showed a significant enrichment in the expression of genes containing miR-33 binding sites in anti-miR33–treated mice (blue line) compared with that in control anti-miR–treated mice (pink line).

Figure 7. Macrophages from anti-miR33–treated plaques show reduced inflammatory gene expression.

Lesional CD68⁺ macrophages were laser captured from aortic sinus lesions of Ldlr^–/– mice receiving the indicated treatment, and mRNA was isolated. Expression of (A) inflammatory genes (Tnfa, Tlr6, and Tlr13) and (B) M1 and M2 markers (iNos, Arg1, and Il10) was analyzed by qRT-PCR. PBS treatment is indicated by the dash. Data are the mean expression levels from 4 mice per group ± SEM. *P < 0.05, compared with baseline.