1. Arterioscler Thromb Vasc Biol
Coenzyme Q10 Promotes Macrophage Cholesterol Efflux by Regulation of the Activator Protein-1/miR-378/ATP-Binding Cassette Transporter G1–Signaling PathwaySignificance
by Dongliang Wang, Xiao Yan, Min Xia, Yan Yang, Dan Li, Xinrui Li, Fenglin Song, and Wenhua Ling
Arterioscler Thromb Vasc Biol
Volume 34(9):
August 20, 2014
Copyright © American Heart Association, Inc. All rights reserved.

2. Coenzyme Q10 (CoQ10) modulates macrophage cholesterol homeostasis by enhancing cholesterol efflux.
Coenzyme Q10 (CoQ10) modulates macrophage cholesterol homeostasis by enhancing cholesterol efflux. J774.A1 (A) and THP-1 macrophages (B) were incubated with the vehicle dimethyl sulfoxide (DMSO; control), oxidized low-density lipoprotein (OxLDL; 50 μg/mL), CoQ10, or a combination of CoQ10 and OxLDL for 24 hours. After fixation by 4% paraformaldehyde, cells were stained with Oil Red O to detect lipid accumulation, and hematoxylin was used for counterstaining. The figure is representative of 3 independent experiments (magnification ×400). C and D, The density of lipid content was evaluated by alcohol extraction after staining. The absorbance at 540 nm was measured using a microplate reader. E and F, The intracellular levels of total cholesterol were analyzed by colorimetric assay kits. J774.A1 (G) and THP-1 macrophages (H) preloaded with 1.0 μCi/mL 3H-cholesterol and OxLDL were treated with CoQ10 or DMSO for 24 hours and subsequently incubated with apolipoprotein A-I (apoA-I; 15 μg/mL) or high-density lipoprotein (HDL; 100 μg/mL) for another 24 hours. Cholesterol efflux was measured as described in Materials and Methods in the online-only Data Supplement. C–H, The data are the mean±SEM (n=3). C–F, *P<0.05 vs (CoQ10 [−] and OxLDL [+]). G and H, *P<0.05 vs control. The data were analyzed with 1-way ANOVA and the Bonferroni–Dunn post hoc test.
Dongliang Wang et al. Arterioscler Thromb Vasc Biol. 2014;34:
Copyright © American Heart Association, Inc. All rights reserved.

3. Coenzyme Q10 (CoQ10) post-transcriptionally regulates ATP-binding cassette transporter G1 (ABCG1).
Coenzyme Q10 (CoQ10) post-transcriptionally regulates ATP-binding cassette transporter G1 (ABCG1). A–D, Oxidized low-density lipoprotein (OxLDL)–loaded J774.A1 and THP-1 macrophages were treated with CoQ10 (1, 10, 100 μmol/L) or the vehicle (dimethyl sulfoxide [DMSO]) for 24 hours. mRNA and protein expression of ATP-binding cassette transporter A1 (ABCA1), ABCG1, and scavenger receptor class B type I (SR-BI) were then determined by quantitative real-time polymerase chain reaction (qRT-PCR; A and C) and Western blotting (B and D), respectively. E and F, Cholesterol efflux to high-density lipoprotein (HDL) in OxLDL-loaded J774.A1 (E) and THP-1 macrophages (F) transfected with scrambled or ABCG1 small interfering RNA (siRNA) with or without CoQ10 (10 μmol/L). Inset, Western blotting for ABCG1. OxLDL-loaded J774.A1 (G) and THP-1 macrophages (I) were treated with CoQ10 (10 μmol/L) or the vehicle (DMSO) for 24 hours. Cells were then treated with 10 μg/mL of actinomycin D, and ABCG1 mRNA expression at indicated time points was quantified by qRT-PCR. OxLDL-loaded J774.A1 (H) and THP-1 macrophages (J) were cotransfected with mouse (m) and human (h) ABCG1 promoter and Renilla (for internal normalization) for 6 hours, respectively. Cells were then treated with CoQ10 (10 μmol/L) or DMSO for 12 hours, and the activity of the luciferase reporter was measured by the Dual-Luciferase Reporter Assay System. B, D, and inset of E and F are representative images of 3 independent assays. For the other panels, data are the mean±SEM (n=3–6). A and C, *P<0.05 vs control. The data were analyzed with 1-way ANOVA and the Bonferroni–Dunn post hoc test. E, F, H, and J, #P<0.05. The data were analyzed with Student t test.
Dongliang Wang et al. Arterioscler Thromb Vasc Biol. 2014;34:
Copyright © American Heart Association, Inc. All rights reserved.

4. miR-378 directly targets the 3′-untranslated region (3′-UTR) of ATP-binding cassette transporter G1 (ABCG1).
miR-378 directly targets the 3′-untranslated region (3′-UTR) of ATP-binding cassette transporter G1 (ABCG1). A–D, Oxidized low-density lipoprotein (OxLDL)–loaded J774.A1 and THP-1 macrophages were transfected with control miR (Con miR; 150 nmol/L) or the indicated concentrations of miR-378 for 48 hours. ABCG1 mRNA and protein expression were then determined by quantitative real-time polymerase chain reaction (qRT-PCR; A and C) and Western blotting (B and D). qRT-PCR (E and G) and Western blotting (F and H) analyses of mouse and human ABCG1 in OxLDL-loaded J774.A1 and THP-1 macrophages transfected with Con miR (150 nmol/L) or miR-378 (150 nmol/L) in the presence or absence of a control inhibitor (Con Inh,150 nmol/L) or anti–miR-378 (150 nmol/L). I and J, The miR-378 target sites in mouse and human ABCG1 are shown in the right panels of I and J. Mutants were generated in the mouse and human ABCG1 3′-UTR seed regions, as indicated. HEK293 cells were cotransfected with Con miR (40 nmol/L) or miR-378 (40 nmol/L), either 50 ng of pGL-3-wild-type (WT) or pGL3-mutant and 50 ng of pRL-TK for 48 hours, as described in Materials and Methods in the online-only Data Supplement. Luciferase reporter activities were then measured by the Dual-Luciferase Reporter Assay System. Cholesterol efflux to HDL from OxLDL-loaded J774.A1 (K and L) and THP-1 macrophages (M and N) transfected with the indicated concentrations of Con miR or miR-378, or Con Inh or anti–miR-378. B, D, F, and H, Representative images of 3 independent assays. A, C, E, and G, The data are expressed as the mean±SEM of fold of either Con miR (A and C) or Con miR and Con Inh (E and G; n=3). *P<0.05 vs Con miR or Con miR and Con Inh, 1-way ANOVA coupled with the Bonferroni–Dunn post hoc test. I and J, The data are expressed as the mean percentage of the 3′-UTR activity of Con miR±SEM (n=3). #P<0.05, Student t test. K–N, The data are the mean±SEM (n=3–6). *P<0.05 vs Con miR or Con Inh, 1-way ANOVA coupled with the Bonferroni–Dunn post hoc test.
Dongliang Wang et al. Arterioscler Thromb Vasc Biol. 2014;34:
Copyright © American Heart Association, Inc. All rights reserved.

5. miR-378 regulates coenzyme Q10 (CoQ10)–induced macrophage cholesterol efflux.
miR-378 regulates coenzyme Q10 (CoQ10)–induced macrophage cholesterol efflux. Oxidized low-density lipoprotein (OxLDL)–loaded J774.A1 and THP-1 macrophages were transfected with either a control miR (Con miR, 150 nmol/L) or miR-378 (40 or 150 nmol/L), or a control inhibitor (Con Inh, 150 nmol/L) or anti–miR-378 (150 nmol/L) for 24 hours. Cells were then treated with CoQ10 (10 μmol/L) or the vehicle (dimethyl sulfoxide [DMSO]) for another 24 hours. ATP-binding cassette transporter G1 (ABCG1) protein and mRNA expression were determined by Western blotting (A) and quantitative real-time polymerase chain reaction (B and D), respectively. The 3H-cholesterol–labeled and OxLDL-loaded J774.A1 and THP-1 macrophages were transfected with either Con miR or miR-378 (C), or Con Inh or anti–miR-378 (E) for 24 hours. Cells were then treated with CoQ10 or DMSO for another 24 hours, and cholesterol efflux to high-density lipoprotein (HDL) was determined. A, Representative images of 6 independent assays. B–E, The data are expressed as the mean±SEM of fold of either Con miR or Con Inh (n=6). *P<0.05 vs CoQ10, Student t test. NS indicates not significant.
Dongliang Wang et al. Arterioscler Thromb Vasc Biol. 2014;34:
Copyright © American Heart Association, Inc. All rights reserved.

6. Coenzyme Q10 (CoQ10) regulates miR-378 through activator protein-1 (AP-1).
Coenzyme Q10 (CoQ10) regulates miR-378 through activator protein-1 (AP-1). A and B, Left, Oxidized low-density lipoprotein (OxLDL)–loaded J774.A1 (A) and THP-1 macrophages (B) were treated with CoQ10 or the vehicle (dimethyl sulfoxide [DMSO]) for 24 hours. The protein expression of c-Jun and c-Fos was then determined by Western blotting. Right, OxLDL-loaded J774.A1 (A) and THP-1 macrophages (B) were treated with 12-O-tetra-decanoylphorbol-13-acetate (TPA; 50 nmol/L) or PBS control in the presence or absence of CoQ10 (10 μmol/L) for 24 hours. The miR-378 contents were then determined by quantitative real-time polymerase chain reaction. C and D, Western blotting analysis of ATP-binding cassette transporter G1 (ABCG1) expression in OxLDL-loaded J774.A1 (C) and THP-1 macrophages (D) transfected with scrambled or c-Jun small interfering RNA (siRNA) in the presence or absence of control miR (Con miR; 150 nmol/L) or miR-378 (150 nmol/L) for 48 hours. E and F, Top, mouse and human putative promoter elements of the mir-378 gene. The schematic diagram represents 2 potential binding sites. Bottom, Densitometry analysis of chromatin immunoprecipitation gels in OxLDL-loaded J774.A1 (E) and THP-1 macrophages (F) treated with TPA or PBS in the presence or absence of CoQ10 for 24 hours. Left, Luciferase reporter activities in J774.A1 (G) and THP-1 macrophages (H) transfected with a series of luciferase constructs containing full-length or truncated promoter elements of mouse or human mir-378 with wild-type putative AP-1–binding sites or mutant-binding sites. After transfection, cells were treated with TPA or PBS in the presence or absence of CoQ10 for 24 hours. Right, Luciferase activities in J774.A1 (G) and THP-1 macrophages (H) cotransfected with c-Jun siRNA and the luciferase reporter construct bearing the mouse or human mir-378 promoter. Left panels of A and B, as well as C and D, are representative images of 3 independent assays. Right panels of A and B, The data are the mean±SEM (n=3). #P<0.05, Student t test. Lower panels of E and F, and left panels of G and H, The data are the mean±SEM (n=3). *P<0.05 vs control, #P<0.05 vs (control+TPA), Student t test. Right panels of G and H, The data are the mean±SEM (n=3). *P<0.05 vs scramble siRNA, Student t test.
Dongliang Wang et al. Arterioscler Thromb Vasc Biol. 2014;34:
Copyright © American Heart Association, Inc. All rights reserved.

7. Coenzyme Q10 (CoQ10) promotes macrophage reverse cholesterol transport (RCT) in vivo.
Coenzyme Q10 (CoQ10) promotes macrophage reverse cholesterol transport (RCT) in vivo. A–G, Thirty-week-old male apolipoprotein E–deficient (apoE−/−) mice were orally gavaged with CoQ10 (600 mg/kg BW) or the vehicle (normal saline) once daily for 14 days. The blood samples were collected 4 hours after CoQ10 treatment at day 14. Serum CoQ10 concentrations were then determined by high-performance liquid chromatography with electrochemical detection (A). c-Jun and c-Fos protein expression (B), miR-378 content (C), and protein expression of ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1), and scavenger receptor class B type I (SR-BI; D) in aortas isolated from CoQ10- or the vehicle-treated apoE−/− mice were determined by Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR). The aortas were pooled with 4 mice in each group. Thioglycollate-elicited mouse peritoneal macrophages (MPMs) were obtained for the qRT-PCR and Western blotting analyses of cellular c-Jun and c-Fos protein (E) expression, miR-378 contents (F), ABCA1, ABCG1, and SR-BI protein expression levels (G), and cholesterol efflux to apolipoprotein A-I (apoA-I) and high-density lipoprotein (HDL; H). Isolated MPMs were pooled with 4 mice in each group. The method of injection of thioglycollate to obtain MPMs is described in Materials and Methods in the online-only Data Supplement. I, Thirty-week-old male apoE−/− mice were orally gavaged with CoQ10 (600 mg/kg BW) or the vehicle once daily for 14 days. On day 12, 3H-cholesterol–labeled and oxidized low-density lipoprotein (OxLDL)–loaded MPMs (typically 4.8×106 cells at 5.2×106 cpm per mouse) were intraperitoneally injected into the mice. After cell injection, blood at 6, 24, and 48 hours, liver at 48 hours (after the animals were euthanized), and feces within the 48-hour experimental period were collected for the analysis of macrophage RCT. 3H-cholesterol recovery in the feces, liver, and plasma were measured. J, Four-hour fasted apoE−/− mice that were intraperitoneally injected with thioglycollate were orally treated with CoQ10 (600 mg/kg BW) for 0 to 24 hours. Serum CoQ10 concentrations, cellular miR-378, and ABCG1 mRNA expression levels in isolated MPMs were quantified (n=6). The results of serum CoQ10 levels are the mean±SEM. The results for miR-378 contents and ABCG1 mRNA levels are the mean±SEM of fold of the control (untreated mice served as controls), which was set to 1. B, D, E, and G, Representative images of 3 independent assays. A, The data are the mean±SEM (n=12). *P<0.05 vs control, Student t test. C, F, and H, The data are the mean±SEM (n=3). *P<0.05 vs control, #P<0.05, Student t test. I, The data are the mean±SEM (n=12). #P<0.05, Student t test.
Dongliang Wang et al. Arterioscler Thromb Vasc Biol. 2014;34:
Copyright © American Heart Association, Inc. All rights reserved.

8. Coenzyme Q10 (CoQ10) inhibits endogenous foam cell formation and the progression of atherosclerosis in vivo.
Coenzyme Q10 (CoQ10) inhibits endogenous foam cell formation and the progression of atherosclerosis in vivo. Male 30-week-old apolipoprotein E–deficient (apoE−/−) mice were euthanized (baseline) or subjected to oral gavage of CoQ10 (600 mg/kg BW), T (10 mg/kg BW), or the vehicle (normal saline; control) once daily for 4 weeks. Four days before euthanasia, mice (n=12 per group) were intraperitoneally injected with thioglycollate to obtain mouse peritoneal macrophages (MPMs) for Oil Red O staining (A), analyses of total lipid content (B), and total cholesterol content (C). D and E, Representative Oil Red O staining of aortic sinus lesion (D) and quantification of the aortic sinus lesion area (E). F, Total cholesterol content in the thoracic and abdominal aorta. G, Western blotting analyses of ATP-binding cassette transporter G1 (ABCG1) protein expression in aortas and MPMs isolated from baseline, the vehicle-, CoQ10-, or T treated apoE−/− mice. The aortas and MPMs were pooled with 4 mice in each group, respectively. H, A unified paradigm depicting the role of CoQ10 in promoting macrophage cholesterol efflux in vitro and in vivo. A and G, Representative images of 3 independent assays. D, Representative images of 12 independent experiments. B, C, and F, The data are the mean±SEM (n=3). E, The data are the mean±SEM (n=12). *P<0.05 vs control, #P<0.05 vs baseline, 1-way ANOVA coupled with the Bonferroni–Dunn post hoc test. NS indicates not significant.
Dongliang Wang et al. Arterioscler Thromb Vasc Biol. 2014;34:
Copyright © American Heart Association, Inc. All rights reserved.