microRNA-33 Regulates Macrophage Autophagy in Atherosclerosis

Abstract

Objective: Defective autophagy in macrophages leads to pathological processes that contribute to atherosclerosis, including impaired cholesterol metabolism and defective efferocytosis. Autophagy promotes the degradation of cytoplasmic components in lysosomes and plays a key role in the catabolism of stored lipids to maintain cellular homeostasis. microRNA-33 (miR-33) is a post-transcriptional regulator of genes involved in cholesterol homeostasis, yet the complete mechanisms by which miR-33 controls lipid metabolism are unknown. We investigated whether miR-33 targeting of autophagy contributes to its regulation of cholesterol homeostasis and atherogenesis.

Approach and results: Using coherent anti-Stokes Raman scattering microscopy, we show that miR-33 drives lipid droplet accumulation in macrophages, suggesting decreased lipolysis. Inhibition of neutral and lysosomal hydrolysis pathways revealed that miR-33 reduced cholesterol mobilization by a lysosomal-dependent mechanism, implicating repression of autophagy. Indeed, we show that miR-33 targets key autophagy regulators and effectors in macrophages to reduce lipid droplet catabolism, an essential process to generate free cholesterol for efflux. Notably, miR-33 regulation of autophagy lies upstream of its known effects on ABCA1 (ATP-binding cassette transporter A1)-dependent cholesterol efflux, as miR-33 inhibitors fail to increase efflux upon genetic or chemical inhibition of autophagy. Furthermore, we find that miR-33 inhibits apoptotic cell clearance via an autophagy-dependent mechanism. Macrophages treated with anti-miR-33 show increased efferocytosis, lysosomal biogenesis, and degradation of apoptotic material. Finally, we show that treating atherosclerotic Ldlr^-/- mice with anti-miR-33 restores defective autophagy in macrophage foam cells and plaques and promotes apoptotic cell clearance to reduce plaque necrosis.

Conclusions: Collectively, these data provide insight into the mechanisms by which miR-33 regulates cellular cholesterol homeostasis and atherosclerosis.

Keywords: atherosclerosis; autophagy; hydrolysis; lipid droplets; macrophages.

Figure 1. miR-33 regulates lipid droplet catabolism

A) Coherent anti-Stokes Raman scattering (CARS) multiphoton imaging of cellular lipid droplets in peritoneal macrophages treated with miR-33 and control mimics and treated with acLDL for 24h. Quantification of lipid droplet volume by voxel analysis is shown at right. Scale bar = 10 μm. B) Cellular efflux of ³H-cholesterol to apolipoprotein A-I (apoA-I, 50μg/mL) in peritoneal macrophages treated with control or miR-33 mimics, in the presence or absence of the neutral lipolysis inhibtor paraoxon (Neutral) or the lysosomal inhibitor chloroquine (Lysosomal). C) Cellular efflux of ³H-cholesterol to apoA-I as in (B), in peritoneal macrophages treated with control or anti-miR-33 inhibitors.. *P < 0.05, **P < 0.01 compared to control treatment. NS, not significant.

Figure 2. Inhibition of endogenous miR-33 promotes autophagy in macrophage foam cells in vivo

A) Schematic diagram showing the experimental outline for antisense oligonucleotide treatment of male Apoe^−/− mice on a Western diet. B-C) qPCR of (B) miR-33 target genes or non-miR-33 targets, as well as (C) Foxo3, Tcfeb and their downstream transcriptional targets in macrophage foam cells isolated hyperlipidemic Apoe^−/− mice treated with anti-miR-33 or control anti-miR. D) Western blotting of lysates from peritoneal macrophages isolated from Apoe^−/− mice treated with anti-miR-33 or control anti-miR to detect LC3-I (cytosolic), LC3-II (autophagosomal) or the autophagy-chaperone protein p62. HSP90 is shown as an internal control. E) Fluorescence microscopy imaging of Adipophilin and neutral lipid (Nile Red-positive) droplets in peritoneal macrophages isolated from hyperlipidemic Apoe^−/− mice treated with anti-miR-33 or control anti-miR. Scale bar = 25μm. F) Fluorescence microscopy imaging of LC3 and neutral lipid (BODIPY-positive) droplets in peritoneal macrophages isolated from hyperlipidemic Apoe^−/− mice treated with anti-miR-33 or control anti-miR. Scale bar = 25μm. Data are the mean ± s.e.m. of 3 mice. *P<0.05, **P<0.005.

Figure 3. miR-33 regulation of cholesterol efflux requires autophagy

A, B) Cellular efflux of ³H-cholesterol to apoA-I in wild type and Atg5^−/− macrophages treated with miR-33 and control (A) mimics or inhibitors (B). C) Immunofluorescence imaging of Bodipy-stained lipid droplets in Ctrl anti-miR or anti-miR-33 treated wild type and Atg5^−/− macrophages loaded with acetylated LDL. Cells were imaged prior to (control) or after incubation with apoA-I for 4h to promote cholesterol efflux. Scale bar, 25μm. D) Quantification of neutral lipid content of cells shown in (C). E) qRT-PCR of Abca1 mRNA in cells shown in (C). *P<0.05, **P<0.005. NS, not significant.

Figure 4. miR-33 inhibition restores defective autophagy in atherosclerosis

A) Schematic diagram showing the treatment regimen of male Ldlr^−/− mice on a Western diet. B) Western blotting of the autophagy chaperone protein p62 in aortic lysates of Ldlr^−/− mice (n=4) that were fed a western diet for 14 weeks and then treated for 4 weeks with anti-miR-33 or control anti-miR. C) Representative immunofluorescence staining of CD68 (red) and p62 (green) and their colocalization (yellow) in atherosclerotic plaques of Ldlr^−/− mice described in (A). At left, quantification of p62 fluorescence intensity in plaques is shown (n=7 mice / group). D) Confocal microscopy of p62 (green) and CD68 (red), and their colocalization (merge), in aortic sinus atherosclerotic plaques of Ldlr^−/− mice undergoing atherosclerosis regression. Dashed lines indicate plaque borders. L= lumen. Scale bar = 100μm.

Figure 5. miR-33 inhibition increases LC3 expression in atherosclerotic plaques

A) Representative immunofluorescence staining of CD68 (red) and LC3 (green) and their colocalization (yellow) in atherosclerotic plaques of Ldlr^−/− mice described in (A). At right, quantification of LC3 fluorescence intensity in plaques is shown (n=7 mice / group). B) Confocal microscopy of LC3 (green) and CD68 (red), and their colocalization (merge), in aortic sinus atherosclerotic plaques of Ldlr^−/− mice undergoing atherosclerosis regression. Dashed lines indicate plaque borders. L= lumen. Scale bar = 100μm.

Figure 6. miR-33 regulates efferocytosis in atherosclerosis

A) Quantification of necrotic area (depicted by arrows) in the aortic roots of ctrl anti-miR or anti-miR-33 treated mice, with representative images of the aortic root lesions stained with H&E presented at left of graphs. n=6 mice/group. Original magnification, ×10. B-D and F) Apoptotic Jurkat cells were added onto peritoneal macrophages treated with ctrl anti-miR or anti-miR-33, and efferocytosis was assayed and expressed as the percent of cells having efferocytosed (B), the number of ingested apoptotic cells per macrophage (mΦ) (C) or the number of cells containing cellular debris (F). In (D), the ability of miR-33 inhibition to regulate efferocytosis in autophagy-deficient Atg5^−/− macrophages was measured as compared to wild-type. E) Fluorescence microscopy of lysosomal-associated membrane protein 1 (LAMP-1) in macrophages treated with anti-miR-33 or control anti-miR. (B, C, E, F) Scale bar = 50μm. *P < 0.05, **P < 0.01 compared to control treatment. NS, not significant.