Study population
From November 2018 to July 2019, 180 CAD patients and 210 healthy controls were recruited from Zhongnan Hospital of Wuhan University (Hubei, China) based on the previous inclusion and exclusion criteria [46]. Clinical characteristics, such as a history of type 2 diabetes mellitus (T2DM) and hypertension, fasting plasma glucose (FPG), lipid and Hcy levels, and leukocyte counts, were collected.
This study was approved by the Medical Ethics Committee of Zhongnan Hospital of Wuhan University (Approval number, 2018017, Wuhan, China) and was conducted in accordance with the Declaration of Helsinki. All participants signed informed consent forms.
Cell culture
THP-1 cells and human umbilical vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection (ATCC, Manassas, USA). THP-1 cells were cultured in RPMI 1640 medium (11875093, Gibco, CA, USA) supplemented with 10% (v/v) fetal bovine serum (FBS, 10099141, Gibco) and 1% (v/v) PS (100 IU/mL penicillin G & 100 IU/mL streptomycin, C0222, Beyotime, Shanghai, China). HUVECs were cultured in DMEM high glucose medium (11965092, Gibco) supplemented with 10% (v/v) FBS and 1% (v/v) PS. CD14 + primary monocytes were obtained from peripheral blood through magnetic bead sorting and were cultured in RPMI 1640 medium supplemented with 15% (v/v) FBS and 1% (v/v) PS. All cells were maintained in a humidified atmosphere containing 5% CO2 at 37 °C.
THP-1 cells (8 × 105 cells/mL) in the treatment group were exposed to 2 mL of medium containing 50 mg/L ox-LDL (YB-002, Yiyuan Biotech, Guangzhou, China); 50 μmol/L Hcy (H4628, Sigma-Aldrich, Darmstadt, Germany); 0.1, 0.5, or 1.0 μmol/L FA (F8758, Sigma-Aldrich); 50 mg/L ox-LDL plus 0.5 μmol/L FA; or 50 μmol/L Hcy plus 0.5 μmol/L FA for 72 h. (The medium and induction reagents were changed every 24 h.) CD14 + primary monocytes (6 × 105 cells/mL, 2 mL) were induced and cultured for 12 h using the same grouping method. Cells that were cultured in RPMI 1640 medium without induction reagents were used as a negative control.
ARID5B overexpression and knockout in THP-1 cells
The pGH125-ARID5B (NM_032199.3)-puro plasmid was constructed to induce overexpression. The ARID5B knockout plasmid was constructed using the CRISPR/Cas9 method. In brief, the unique single-guide RNA (gRNA) sequence (sgRNA sequence: 5’-gcagaccccaaaggtccttg-3’) of the ARID5B gene was designed and cloned into the lentiCRISPR v1 plasmid. To generate stable cell lines, HEK293 cells were cotransfected with pGH125-ARID5B or lentiCRISPR v1 plasmid and packaging plasmids using a transfection reagent (6366244001, Roche, USA) according to the manufacturer's instructions. After 72 h of culture, the viral supernatant was collected and used to infect THP-1 cells (1 × 105 cells/mL). After 72 h, THP-1 cells were selected using 0.3 μg/ml puromycin. Puromycin-resistant cells were then seeded into 96-well plates for clonal expansion and analyzed using qPCR and western blotting.
Animals, diets, and experimental procedures
Apolipoprotein E-deficient (ApoE−/−) mice have a homozygous deletion of the ApoE gene that was prepared by homologous recombination technology. This mouse model exhibits abnormal hyperlipidemia and can develop atherosclerosis under spontaneous or induced conditions. ApoE−/− mice have extensively been used to study the mechanisms underlying the initiation and progression of atherosclerosis. In this study, 8 wild-type and 48 ApoE−/− male C57BL/6 J mice aged 5 weeks were purchased from Charles River Laboratory Animal Technology Co., Ltd. (Beijing, China) in November 2019. All animals were housed in a pathogen-free facility with a temperature of 24 ± 1 °C, a relative humidity of 50 ± 1%, and a light/dark cycle of 12/12 h. All animal studies (including the mouse euthanasia procedure) were performed in compliance with the institutional animal care regulations and guidelines of Wuhan University and were conducted according to the AAALAC and IACUC guidelines (IACUC approval number, 2019174).
The wild-type control group was fed a normal diet (G1, WT + ND), and 48 ApoE−/− mice were randomly distributed into six groups (8 per group): (1) normal diet (G2, ApoE−/− + ND), (2) high-fat diet (G3, ApoE−/− + HFD), (3) high-fat diet + 1.6 μg/mL FA (F8758, Sigma-Aldrich) daily in drinking water (G4, ApoE−/− + HFD + FA), (4) normal diet + 1.8 g/L Hcy (H4628, Sigma-Aldrich) daily in drinking water (G5, ApoE−/− + ND + Hcy), (5) normal diet + 1.6 μg/mL FA & 1.8 g/L Hcy daily in drinking water (G6, ApoE−/− + ND + FA + Hcy), and (6) high-fat diet + 1.6 μg/mL FA & 1.8 g/L Hcy daily in drinking water (G7, ApoE−/− + HFD + FA + Hcy). Drinking water containing FA and Hcy was changed every 3 days.
The Western-type high-fat diet (20% fat, 1.25% cholesterol) was purchased from Peking Huafukang Laboratory Animal Center (Beijing, China). Mice without special treatment were given normal maintenance feed and normal drinking water. After 27 weeks, all mice were fasted overnight before ether anesthesia, after which blood and tissue samples were immediately collected and stored at −80 °C.
Macrophage polarization
To evaluate the regulatory effect of ARID5B on THP-1-derived macrophage polarization, the stable THP-1 cells (6 × 105 cells/mL, 2 mL) were induced to form M0 macrophages using 50 ng/mL PMA (P1585, Sigma-Aldrich) and cultured for 24 h for mRNA expression analysis or were further cultured for 48 h with new medium without PMA for apoptosis analysis.
DNA extraction, enzymatic digestion, and global 5-mC level detection
Genomic DNA was extracted using the phenol/chloroform method or a commercial DNA extraction kit (3001050, Simgen, Hangzhou, China) based on the sample size and was quantified by a NanoDrop 2000 (Thermo Scientific, Wilmington, USA). The enzymatic digestion of genomic DNA and the analysis of global 5-mC levels were performed according to our previously described ultrahigh-performance liquid chromatography–mass spectrometry/mass spectrometry (UPLC–MS/MS) method [24].
RNA extraction and RT–qPCR
Total RNA was extracted using TRIzol reagent (15596026, Invitrogen, CA, USA) or a commercial RNA extraction kit (5100050, Simgen) based on the sample size and was quantified by a NanoDrop 2000 (Thermo Scientific). Total RNA was reverse-transcribed into cDNA using a commercial reversal kit (FSQ-301, TOYOBO, Osaka, Japan). Real-time quantitative polymerase chain reaction (RT–qPCR) was performed using a Bio-Rad CFX96 real-time system (Bio-Rad, CA, USA). The relative expression levels of target genes were calculated using the comparative crossing threshold method of relative quantification (△Cq) and are expressed as fold change values. GAPDH was used as an internal reference gene. Each sample was analyzed at least three times, and the detailed primer information is shown in Additional file 2: Table S6.
Flow cytometry
Approximately 100 μL of human and mouse whole blood samples was used for monocyte subset analysis, which was performed according to our previously described method [19]. In brief, human blood samples were stained with a mixture of four mouse anti-human monoclonal fluorochrome-conjugated antibodies (anti-CD14-PE, 5 μL; anti-CD16-FITC, 10 μL; anti-CCR2-APC, 5 μL; anti-CD86-BV421, 2 μL) (55398, 555406, 558406, 562433, BD PharMingen, NJ, USA); mouse blood samples were stained with a mixture of three rat anti-mouse monoclonal fluorochrome-conjugated antibodies (anti-Ly6C-PE, 5 μL; anti-CD43-APC, 5 μL; anti-CD115-Alexa Fluor ® 488, 5 μL) (560592, 560663, 135512, BD PharMingen). Approximately 100 μL of cultured human CD14 + primary monocytes was stained with a mixture of three mouse anti-human monoclonal fluorochrome-conjugated antibodies (anti-CD14-PE, 5 μL; anti-CD16-FITC, 5 μL; anti-CCR2-APC, 5 μL) (BD PharMingen). The expression level of CCR2 was quantified by flow cytometry and was expressed as the median fluorescence intensity (MFI). Fluorescence was standardized using multiple peaks rainbow calibration beads (Spherotech, Chicago, USA) to ensure the reproducibility and comparability of the median fluorescence intensity (MFI) throughout the study period, as previously described [47]. Flow cytometry was performed using a Beckman CytoFLEX flow cytometer (Beckman Coulter, CA, USA). The gating strategy for human and mouse monocyte subsets is shown in Additional file 2: Fig. S4 and Fig. S5, respectively.
Monocyte subset sorting
Three monocyte subsets were sorted from 15 CAD patients and 8 healthy volunteers using a BD FACSAria III (BD PharMingen). In brief, 10 mL of EDTA-anticoagulated whole blood samples were collected, and red blood cells were lysed using 1 × RBC lysis buffer (555,899, BD PharMingen). White blood cells were stained with a mixture containing three mouse anti-human monoclonal fluorochrome-conjugated antibodies (anti-CD14-PE, 200 μL; anti-CD16-FITC, 400 μL; anti-CD86-APC, 50 μL) (555398, 555406, 555660, BD PharMingen). Monocytes were gated in an SSC/CD86 + dot plot, and then, based on CD14 and CD16 expression, CD86 + monocytes were divided into classical, intermediate, and nonclassical subsets. Based on the gating strategy, the three monocyte subsets were sorted separately. Flow cytometry was used to reanalyze the obtained monocytes, and the results indicated that the sorted monocyte subsets had good purity (Additional file 2: Fig. S6).
Isolation of primary monocytes
Approximately 50 mL of fresh peripheral blood was collected from healthy volunteers, and sodium citrate was used for anticoagulation. First, peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation using 1.077 g/mL Ficoll/Hypaque (P4350, Solarbio, Beijing, China). Then, monocytes were purified from PBMCs using the positive selection method with a magnetic-activated cell sorter (MACS) system (130-042-201, Miltenyi, Bergisch Gladbach, Germany) and CD14 microbeads (130-050-201, Miltenyi) according to the manufacturer’s instructions. Flow cytometry was used to identify the purity of the obtained monocytes, which were labelled with CD14 monoclonal fluorochrome-conjugated antibodies (555398, BD PharMingen). Monocyte purity was at least 90% (Additional file 2: Fig. S7).
MDRE–qPCR assay
A methylation-dependent restriction enzyme digestion-based qPCR (MDRE–qPCR) assay was used to examine the methylation level of the ARID5B gene cg25953130 CpG site. DNA samples (including sample DNA and positive and negative methylation controls) were first diluted to 40 ng/μL with ultrapure water. The diluted DNA samples were digested using a 10 μL mixture of 5 μL of DNA, 0.4 μL of FspEI (R0662S, NEB, Beijing, China), 1 μL of buffer, 0.2 μL of activator, and 3.4 μL of ddH2O. Another 10 μL mixture (5 μL of DNA, 1 μL of buffer, 0.2 μL of activator, and 3.8 μL of ddH2O) without enzymatic digestion was used as a negative control. The reaction mixtures were incubated at 37 °C for 3 h and then incubated at 80 °C for 20 min for enzyme inactivation. The cg25953130 methylation level was measured using a 10 μL mixture including 1 μL of enzyme-digested product (or negative control), 5 μL of 2 × T5 Fast qPCR Mix (TSE202, Tsingke, Beijing, China), 1 μL of forward primer (5’-GAATTGGAATAGCGCCAGGT-3’), 1 μL of reverse primer (5’-AAGGAAATATGAATGTG CTCACG-3′), and 3.2 μL of ddH2O. The reaction mixtures were incubated at 95 °C for 1 min, followed by 40 cycles (denaturation, 95 °C for 10 s; annealing, 58 °C for 5 s; extension, 72 °C for 15 s). The efficiency of the restriction digestion was evaluated as follows: The methylation level of the unmethylated negative control should approach 0%, and the methylated positive control should approach 100%. Otherwise, the samples were redigested. The formula for calculating the methylation level was as follows: cg25953130 methylation level (%) = 100% × [1 − 2△Cq (control − digestion) group].
HUVEC adhesion assay
One day prior to the adhesion assay, 1 mL of HUVECs was seeded into 12-well plates at a density of 5 × 105 cells/mL and cultured in 5% CO2 at 37 °C until the cells reached 90% confluence. In the meantime, 1 mL of THP-1 cells (1 × 106 cells/mL) was stained with 1 μL of the fluorescent probe BCECF-AM (5 mM) (Beyotime, Shanghai, China) in the dark for 30 min. Then, the cells were washed twice using phosphate-buffered saline (PBS, pH = 7.4, P1022, Solarbio). After being resuspended, 1 mL (5 × 105 cells/mL) of the stained THP-1 cells was added to the monolayer formed by the HUVECs. After the cells were incubated at 37 °C for 60 min, the nonadherent cells were washed away using PBS. Under an inverted fluorescence microscope, five fields per well were randomly selected and photographed at 100 × magnification to calculate the number of adherent THP-1 cells. Image-Pro Plus (v 6.0) software was used for image analysis.
Transwell assay
THP-1 chemotaxis in response to MCP-1 was evaluated with a Transwell chamber with a pore size of 8 μm (CLS3422, Corning, Toledo, USA). Approximately 0.6 mL of RPMI 1640 medium containing 200 ng/mL MCP-1 (SRP3109, Sigma-Aldrich) and 5% (v/v) FBS was first added to the well of a 24-well plate. Then, 0.2 mL of THP-1 cells (1 × 106 cells/mL) was added to the Transwell chamber. The chamber was placed into the well and incubated at 37 °C for 60 min. PBS (pH = 7.4) was used to wash away the medium, and the cells on the upper side of the filters were washed twice. The cells on the underside of the filters were fixed with 1.0 mL of 4% paraformaldehyde (BL539A, Biosharp, Hefei, China) for 10 min and then stained with 0.6 mL of 0.1% crystal violet staining solution (94,448, Sigma-Aldrich) for 10 min. Under an inverted microscope, five fields per chamber were randomly selected and photographed at 100 × magnification to calculate the number of migrated THP-1 cells. Image-Pro Plus (v 6.0) software was used for image analysis.
Apoptosis assay
A commercial Annexin V-FITC/PI Apoptosis Kit (BB-4101, BestBio, Shanghai, China) was used to evaluate the early and late apoptosis rates according to the manufacturer's instructions. In brief, 0.5 mL of cultured cells (1 × 106 cells/mL) was first stained with 5 μL of Annexin V-FITC in the dark for 15 min and then stained with 5 μL of PI for 5 min. The apoptosis rate was immediately analyzed using flow cytometry.
Western blotting
Cells were lysed in ice-cold RIPA lysis buffer (R0020, Solarbio), and protein levels were quantified using a BCA protein assay kit (P0012S, Beyotime). The extracted cellular proteins were mixed with loading buffer (v/v = 4:1) (P1040, Solarbio) and boiled for 10 min. Protein samples (30 μg per well) were separated by 8% SDS–PAGE gels and transferred to PVDF membranes. The membranes were blocked at room temperature with 5% BSA in 1 × TBST (0.2% Tween-20, pH = 7.6) buffer for 2 h and then incubated with primary antibodies against GAPDH (1:100,000, A19056, ABclonal, Wuhan, China) and ARID5B (1:2000, NBP1-83622, Novus, Shanghai, China) at 4 °C overnight. After being washed with 1 × TBST buffer four times (10 min each time), the membranes were then incubated with HRP-conjugated mouse anti-rabbit (1:5000, AS061, ABclonal) and goat anti-mouse (1:5000, AS003, ABclonal) secondary antibodies at room temperature for 1 h. Protein bands were visualized by chemiluminescence using an ECL reagent (PE0010, Solarbio) and photographed by a Tanon-5200 Chemiluminescent Imaging System (Tanon, Shanghai, China). Image-Pro Plus (v 6.0) software was used to perform the densitometric semiquantitative analysis of protein bands. The protein expression was normalized against GAPDH.
Assessment of atherosclerotic plaques
Atherosclerotic plaque formation in the mouse aortic sinus was assessed using oil red O staining (O0625, Sigma-Aldrich). Heart samples were embedded in OCT (4583, Sakura, USA) compound and frozen in liquid nitrogen. The frozen tissue was thawed at − 20 °C for 30 min, after which the aortic sinus was sectioned into consecutive 6-μm-thick sections at − 20 °C. Aortic sinus sections with three complete aortic valves were selected for oil red O staining and photographed using a microscope (Nikon, Tokyo, Japan) at 40 × magnification. The degree of atherosclerotic lesions was evaluated by determining the ratio of atherosclerotic plaque areas to vessel lumen areas, which were quantified using Image-Pro Plus (v 6.0) software.
Immunofluorescence staining
Frozen aortic sinus sections were dried and fixed in cold acetone for 10 min and were repaired using sodium citrate–EDTA. Aortic sinus sections were then washed with PBS buffer (pH = 7.4) three times and blocked with 2% BSA for 1 h at 37 °C. Then, the sections were incubated with rabbit anti-mouse MOMA (Monocyte & Macrophage) (1:50, ab33451, Abcam, Shanghai, China) primary antibodies at 4 °C overnight and with Alexa Fluor® 488-conjugated goat anti-rabbit (1:200, ab150077, Abcam) secondary antibodies for 50 min at 25 °C. After being washed with PBS buffer three times, the sections were incubated with DAPI (62248, Thermo Scientific). After the sections were washed and slightly dried, they were mounted with an anti-fluorescence quenching mounting medium. A Nikon upright fluorescence microscope (Nikon) was used to observe and obtain fluorescence images. The positive areas of the sections were quantified using Image-Pro Plus (v 6.0) software.
Statistical analysis
Based on their distributions, continuous variables are presented as the mean ± SD or as the median (interquartile range). Comparisons between continuous variables were performed using Student’s t test or the Mann–Whitney U test.
Categorical variables are presented as frequencies (n, %), and the chi-square test was used to evaluate the significance of any differences between groups. Spearman or Pearson bivariate correlation analyses were used to analyze the correlations in the present study. Multivariate regression analysis was used to evaluate the effects of CAD risk factors on the methylation level of cg25953130. SPSS software version 17.0 (SPSS, Chicago, USA) and GraphPad Prism 8.0 (GraphPad, San Diego, USA) were used for statistical analyses. A p < 0.05 (two-tailed) was considered to be statistically significant.
