Macrophage Mitochondrial Energy Status Regulates Cholesterol Efflux and Is Enhanced by Anti-miR33 in Atherosclerosis

Abstract

Rationale: Therapeutically targeting macrophage reverse cholesterol transport is a promising approach to treat atherosclerosis. Macrophage energy metabolism can significantly influence macrophage phenotype, but how this is controlled in foam cells is not known. Bioinformatic pathway analysis predicts that miR-33 represses a cluster of genes controlling cellular energy metabolism that may be important in macrophage cholesterol efflux.

Objective: We hypothesized that cellular energy status can influence cholesterol efflux from macrophages, and that miR-33 reduces cholesterol efflux via repression of mitochondrial energy metabolism pathways.

Methods and results: In this study, we demonstrated that macrophage cholesterol efflux is regulated by mitochondrial ATP production, and that miR-33 controls a network of genes that synchronize mitochondrial function. Inhibition of mitochondrial ATP synthase markedly reduces macrophage cholesterol efflux capacity, and anti-miR33 required fully functional mitochondria to enhance ABCA1-mediated cholesterol efflux. Specifically, anti-miR33 derepressed the novel target genes PGC-1α, PDK4, and SLC25A25 and boosted mitochondrial respiration and production of ATP. Treatment of atherosclerotic Apoe(-/-) mice with anti-miR33 oligonucleotides reduced aortic sinus lesion area compared with controls, despite no changes in high-density lipoprotein cholesterol or other circulating lipids. Expression of miR-33a/b was markedly increased in human carotid atherosclerotic plaques compared with normal arteries, and there was a concomitant decrease in mitochondrial regulatory genes PGC-1α, SLC25A25, NRF1, and TFAM, suggesting these genes are associated with advanced atherosclerosis in humans.

Conclusions: This study demonstrates that anti-miR33 therapy derepresses genes that enhance mitochondrial respiration and ATP production, which in conjunction with increased ABCA1 expression, works to promote macrophage cholesterol efflux and reduce atherosclerosis.

Keywords: atherosclerosis; cholesterol; macrophages; microRNA-33, mouse; mitochondria.

Figure 1. Mitochondria are required for cholesterol efflux in macrophages and are predicted to be regulated by miR-33

(A) Human THP-1 macrophages transfected with control anti-miR or anti-miR33 were cholesterol-loaded for 24h before pre-treatment with oligomycin for 1h, and subsequently incubated with apoA1 for 6h. % cholesterol efflux is shown as a proportion of total radiolabeled cholesterol in the cell. (B) Peritoneal macrophages from wild-type C57BL6 or Pgc-1α^-/- mice were loaded with or without cholesterol for 24h, and cholesterol efflux to apoA1 was measured for 6h. % cholesterol efflux is shown as a proportion of total radiolabeled cholesterol in the cell. (C) Bioinformatic pathway analysis using the DAVID gene tool and Gene Set Enrichment Analysis predicted that miR-33 regulates multiple mitochondrial genes. Predicted miR-33 targets are depicted as yellow circles; interacting downstream genes are shown as violet circles; genes outlined in blue are previously confirmed miR-33 target genes, and dotted lines are novel miR-33 target genes confirmed in this study.

Figure 1. Mitochondria are required for cholesterol efflux in macrophages and are predicted to be regulated by miR-33

(A) Human THP-1 macrophages transfected with control anti-miR or anti-miR33 were cholesterol-loaded for 24h before pre-treatment with oligomycin for 1h, and subsequently incubated with apoA1 for 6h. % cholesterol efflux is shown as a proportion of total radiolabeled cholesterol in the cell. (B) Peritoneal macrophages from wild-type C57BL6 or Pgc-1α^-/- mice were loaded with or without cholesterol for 24h, and cholesterol efflux to apoA1 was measured for 6h. % cholesterol efflux is shown as a proportion of total radiolabeled cholesterol in the cell. (C) Bioinformatic pathway analysis using the DAVID gene tool and Gene Set Enrichment Analysis predicted that miR-33 regulates multiple mitochondrial genes. Predicted miR-33 targets are depicted as yellow circles; interacting downstream genes are shown as violet circles; genes outlined in blue are previously confirmed miR-33 target genes, and dotted lines are novel miR-33 target genes confirmed in this study.

Figure 2. Anti-miR33 treatment increases the expression of novel mitochondrial genes in macrophages

(A) 3′UTR assays for novel miR-33 target genes PGC-1α, PDK4 or SLC25A25. Cells were transfected with UTR constructs downstream of renilla luciferase in the presence of control miRNA or miR-33 mimics (80nM). Graphs depict luciferase activity relative to control miR after 24h. WT indicates the wild-type 3′UTR sequence, and mut indicates that miR-33 binding sites have been mutated by site-directed mutagenesis. (B-C). Peritoneal macrophages or THP-1 cells were transfected with 120nM control anti-miR or anti-miR33 for 48h. The relative mRNA (B) or protein (C) expression of select mitochondrial genes was determined. Data are representative of triplicates of at least n= 3 experiments and were analyzed using a t-test (* p≤0.05, ** p<0.001).

Figure 3. Indirect regulation of mitochondrial gene expression by miR-33

(A-B) Nrf1 mRNA (A) and protein (B) expression in peritoneal macrophages treated with control anti-miR or anti-miR33. **p≤0.01 compared to control anti-miR. (C). Mitochondrial biogenesis in peritoneal macrophages from control and anti-miR33 treated cells. Mitochondrial DNA per ng of total cellular DNA was evaluated by quantitative PCR and is expressed relative to control. **p≤0.01 compared to control anti-miR (D) OXPHOS complexes I-V in macrophages transfected with control anti-miR or anti-miR33 by Western blot and quantified using Image J. *p≤0.05 compared to control anti-miR, as measured from duplicate lanes from at least 6 independent experiments.

Figure 4. Inhibition of miR-33 increases mitochondrial respiration and ATP production that contributes to macrophage cholesterol efflux potential

(A) Human macrophages were transfected with control anti-miR or anti-miR33 and the oxygen consumption rate was determined using the Seahorse XF24 Extracellular Flux Analyzer. Data depicted demonstrates mean ± SEM of n = 4 experiments. (B) Peritoneal macrophages were transfected with control miR, miR-33 mimics, control anti-miR or anti-miR33 for 48 h prior to the addition of apoA1 for 6h. Intracellular ATP levels were measured and are shown as either μM [ATP] per μg protein. (C) THP-1 macrophages transfected with control anti-miR or anti-miR33 for 24h were incubated with 1μCi/mL 3[H]-cholesterol and 37.5μg/mL acLDL for 24h prior to the addition of 25μg/mL apoA1 for 6h in the presence or absence of 10μM oligomycin. Percentage cholesterol efflux was determined relative to control and data shows mean ± SD of 6 replicates, and is representative of at least 3 independent experiments. (D) Peritoneal macrophages from either wild-type (WT) or Pgc-1α^-/- mice were transfected with cont anti-miR or anti-miR33 and intracellular ATP was measured as in (B) above. Data shown are % change relative to control ± SD from 3 replicates, and is representative from at least 3 independent experiments. (E) Peritoneal or BMDM macrophages from either WT, Pgc-1α^-/-, Pdk4^-/- or Slc25a25^-/- mice were transfected with anti-miRs and cholesterol efflux was measured as in (B). Data is shown as % increase in efflux to apoA1 by anti-miR33 compared to control anti-miR of 6 replicates, and is representative of n=3 independent experiments.

Figure 5. Anti-miR33 therapy decreases atherosclerosis in Apoe^-/- mice independently of HDL cholesterol

Apoe^-/- mice were simultaneously fed Western diet (0.2% cholesterol) and an administered anti-miR33 (or control anti-miR) oligonucleotides for 8 weeks prior to sacrifice (A) Quantification of atherosclerotic lesion areas in aortic sinus sections. Mean ± SEM for each treatment group is shown, *P<0.05, n=7-8 per group, student t-test. (B) Plasma HDL cholesterol levels at the end of study showed no difference between control and anti-miR33. (C) Coherent anti-Stokes Raman scattering (CARS), two-photon fluorescence (TPF) and second harmonic generation (SHG) microscopy was used to visualize lipid droplets (CARS, red), elastin (TPF, green) and collagen (SHG, blue) in atherosclerotic lesions of Apoe^-/- mice treated with control-anti-miR and anti-miR33. Dashed outlines depict lesion/lumen border. (D) Quantification of lipid content from CARS signal (total CARS area) in the aortic sinus from cont anti-miR and anti-miR33 treated Apoe^-/- mice. p≤0.01, n=3 per group. (E) Representative images of immunohistochemistry staining for PGC-1α and PDK4 in aortic sinus sections of Apoe^-/- mice treated with control-anti-miR or anti-miR33.

Figure 6. Novel miR-33 mitochondrial target gene expression is regulated in vivo

(A) Gene expression analysis of peritoneal macrophages isolated from western-fed Apoe^-/- mice treated with control anti-miR or anti-miR33 for 8 weeks (from Figure 5). (B). Relative expression of miR-33a (red) and miR-33b (blue) as well as PGC-1α (PPARGC1A), SLC25A25, SLC25A23, NRF1 and TFAM in healthy arteries (Normal) or carotids from patients with atherosclerosis (Plaque) from the Biobank of Karolinska Endarterectomy (BiKE).