Human coronary artery samples
Atherosclerotic and control epicardial coronary artery segments were from human specimens with extensive atherosclerotic disease and healthy controls. The specimens were donated by the Shandong Red Cross Society.The experiment protocols were examined and approved by the review committee of Qilu Hospital, Jinan, China(ethics approval No. KYLL-2018(KS)-233).
ILF3 conditional transgenic and knockout mice (ILF3f/f mice) were generated with use of CRISPR-Cas9. The VSMC-specific ILF3 knockout (ILF3−/−/SMACre) and overexpression (ILF3over/SMACre) mice were bred fromILF3f/f mice crossed with Sm22a-creERT2+/− mice. Macrophage-specific ILF3 knockout (ILF3−/−/LyzCre) and ILF3-overexpressed (ILF3over/LyzCre) mice were bred from ILF3f/f mice crossed with Lyz2Cre+/− mice. ApoE−/− mice from Beijing Viewsolid Biotechnology (Beijing) were crossed with the offspring of ILF3−/−/SMACre, ILF3over/SMACre, ILF3−/−/LyzCre and ILF3over/LyzCre mice to generate ApoE−/−ILF3−/−/SMACre, ApoE−/−ILF3over/SMACre, ApoE−/−ILF3−/−/LyzCre and ApoE−/−ILF3over/LyzCre mice, respectively. The genotypes were verified by PCR. The study was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee of Shandong University (permit no.: 07006). Mice were fed a high-fat western diet (HFD) for 4 months and then sacrificed under general anesthesia with sodium pentobarbitone and with efforts to minimize suffering. Mice were weighed and organs were harvested and fixed in 4% paraformaldehyde.
Serum index levels
We collected 0.5 to 1.0 ml of blood from the left ventricle of mice at the time of tissue harvesting after 16 weeks feeding with the HFD. Serum levels of triglycerides (TG), total cholesterol (TC), blood glucose (BG), low density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), calcium and phosphorus were measured by standard enzymatic methods with commercial kits (Roche Diagnostics, Indianapolis, IN).
Immunohistochemistry (IHC) and immunofluorescence
For immunohistochemistry, paraffin-embedded sections were deparaffinized, rehydrated, underwent antigen retrieval and treated with hydrogen peroxide in sequence. Then, sections were blocked with 10% goat serum for 30 min at 37 °C, then incubated with specific primary antibodies including anti-ILF3 (Abcam, Cat# ab92355), anti-BMP2 (Novus, Cat# NBP1-19751), anti-Vimentin (Cell Signaling Technology, Cat#5741), anti-α-SMA (Sigma, Cat# A5228), anti-inducible nitric oxide synthase (iNOS; Abcam, Cat# ab3523), anti-arginase 1 (ARG-1;Abcam, Cat# ab239731), anti-OPN (Abcam, Cat# ab8448), anti-signal transducer and activator of transcription 1 (STAT1; Cell Signaling Technology, Cat#14994), anti-Runx2 (Abcam, Cat# ab192256) and anti-monocyte + macrophage antibody 2 (MOMA-2; Abcam, Cat# ab33451) overnight at 4 °C. After being washed 3 times, slides were exposed to streptavidin (horseradish peroxidase)-biotin labeled secondary antibody (ZSJQ-BIO, China) and reacted with diaminobenzidine. Finally, we used haematoxylin to counterstain the nucleus and a Nikon Eclipse 80imicroscope with a camera (DS-Ri1; Nikon) to acquire images.
For immunofluorescence, the same operational procedures were used as for immunohistochemistry for the first day. Then sections were incubated with primary antibodies including anti-iNOS (Abcam, Cat# ab3523), anti-ARG-1 (Abcam, Cat# ab239731), anti-OPN (Abcam, Cat# ab8448) and anti-α-SMA (Sigma, Cat# A5228) and reacted with fluorescent-labeled secondary antibodies and then 4', 6-diamidino-2-phenylindole (DAPI). Images were acquired by laser-scanning confocal microscopy (LSM710, Carl Zeiss).
Oil-red O staining andSirius red staining
Briefly, frozen sections were hydrated and washed, then immersed in 0.3% Oil-red O or Sirius red stain. Then sections were counterstained with haematoxylin and visualized under a Nikon Eclipse 80 imicroscope with a camera (DS-Ri1; Nikon). Oil-red O stain was used to detect lipids and Sirius red to examine collagen in aortic root plaques. Atherosclerotic plaque instability index was computed by using the formula (Oil-red O area + MOMA-2 area)/(α-SMA area + collagen I area).
Human aortic vascular smooth muscle cells (HAVSMCs) were obtained from the ScienCell (Carlsbad, CA, US) and cultured in SMC medium (SMCM) including 2% fetal bovine serum (FBS), 1% SMC growth supplement (SMCGS) and 1% penicillin/streptomycin solution. Peritoneal macrophages were collected from ApoE−/−, ILF3−/−/LyzCre and ILF3over/LyzCre mice as described8. After being injected intraperitoneally with 6% starch for 3 days, mice were euthanized by spinal dislocation. Macrophages were collected from the peritoneal cavity by perfusion with cold phosphate-buffered saline (PBS, GibcoBRL Life Technologies). Then macrophages were grown in Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% FBS (Gibco). All cells were incubated at 37 °C in a humidified atmosphere and used after passages 3–5. Cells were starved for 24 h before each experiment. For in vitro experiments, we used osteogenic medium containing 10 mM β-glycerophosphate (β-GP, Sigma) to stimulate cell calcification for 3–14 days. Cells were treated with oxidized LDL (oxLDL; 50 µg/ml) to simulate a high lipid condition.
Lentivirus (Lv) and siRNA transfection
Lentiviruses and siRNA duplex were supplied by GenePharma (Suzhou, China). Lentiviruses encoding overexpressed ILF3 (Lv-ILF3) were incubated with VSMCs or macrophages at multiplicity of infection (MOI) of 10. After 24 h, the medium containing lentiviruses was removed and replaced with fresh medium. The siRNA duplex encoding ILF3 knockout cDNA (si-ILF3) was transfected into VSMCs or macrophages by using Lipofectamine 3000 Reagent Protocol (Invitrogen, NY, USA) in Opti-medium (Gibco) for 6 h and then replaced with fresh medium. The Homo sapiens siRNA sequence was 5'-CCUGUGUGAGAAAUCCAUU-3'. The lentivirus NCBI reference sequence is NO. NM_001137673.
After HAVSMCs were transfected with ILF3 siRNA, RNAs were extracted with TRIzol reagent. We used the Qubit2.0 Fluorometer (Life Technologies, USA) and Nanodrop One spectrophotometer (Thermo Fisher Scientific, USA) to check RNA concentration and quality. The total RNA integrity was determined by using an Agilent 2100 Bioanalyzer (Agilent Technologies, USA) to ensure RNA sample integrity number (RIN) > 7. The VAHTS Total RNA-seq (H/M/R) Library Prep Kit (Vazyme, China) was used to construct RNA-seq strand-specific libraries according to the manufacturer’s instructions. The quantity, insert size and concentration of purified cDNA libraries were validated by using the Qubit2.0 Fluorometer and Nanodrop One spectrophotometer. Library clusters were produced by cBot and sequencing involved the Illumina NovaSeq 6000 platform (Illumina, USA). The library generation and sequencing were performed by Genechem (Shanghai). According to the results of RNA-seq, Gene Ontology (GO) analysis and KEGG pathway analysis were used to identify cardiovascular systemic diseases and calcification-related signaling pathways.
After treatment, cells were fixed in 4% paraformaldehyde for 10 min and washed with PBS for 3 times. Then, cells were punched with 0.1% Triton X100 and blocked with 10% goat serum for 40 min at 37 °C. Cells were incubated with the primary antibodies anti-STAT1 (Cell Signaling Technology, Cat#14994) and anti-Runx2 (Abcam, Cat# ab76956) overnight at 4 °C. The second day, cells were reacted with fluorescent-labeled secondary antibodies and then 4', 6-diamidino-2-phenylindole (DAPI). Images were obtained by laser-scanning confocal microscopy (LSM710, Carl Zeiss).
Western blot analysis
Western blot analysis was conducted as previously described 8. Membranes were incubated with primary antibodies including anti-ILF3 (Abcam, Cat# ab92355), anti-β-actin (Sigma, Cat# SAB2100037), anti-STAT1 (Cell Signaling Technology, Cat# 14994), anti-BMP2 (Abcam, Cat# ab14933), anti-Runx2 (Cell Signaling Technology, Cat# 12556), anti-Vimentin (Cell Signaling Technology, Cat# 5741), anti-iNOS (Abcam, Cat# ab178945), anti-α-SMA (Sigma, Cat# A5228), anti-OPN (Abcam, Cat# ab8448), anti-ARG-1 (Abcam, Cat# ab239731), anti-p-smad1/5 (Cell Signaling Technology, Cat# 9516) and anti-Smad1 (Cell Signaling Technology, Cat#6944). Secondary horseradish peroxidase-coupled antibodies were prepared and incubated with membranes. Images were obtained by using chemiluminescence (Millipore, Billerica, MA).
Total RNA was extracted from cells with TRIzol reagent and reverse-transcribed to cDNA by using HiScriptIIIRT SuperMix for qPCR (Vazyme, Nanjing, China). Quantitative RT-PCR involved using the SYBR Green Master mix kit (Roche, USA). The average cycle threshold (Ct) method was used to determine mRNA expression. The 2-ΔΔCT method was used to calculate the relative change of mRNA. The primer sequences for ILF3, BMP2, STAT1 and β-actin are in supplemental Table S1.
Alizarin-red and von Kossa staining
For Alizarin-red staining, after fixing in 4% paraformaldehyde, cells were immersed in 1% Alizarin-red solution (Solarbio, China) for 15 min. The sections of aortic root were deparaffinized and dyed with Alizarin-red for 5 min. Von Kossa staining involved using a kit (Solarbio). The cells were exposed to 5% silver nitrate solution and exposed to ultravioletray for 1 h.
Alkaline phosphatase (ALP) activity and calcium content detection
ALP activity was detected by using an ALP assay kit (Beyotime, China) and normalized to total protein concentration by the BCA Protein Assay Kit (Beyotime, China). Calcium content was determined with the Calcium Assay Kit (Nanjing Jiancheng Bioengineering Institute, China) and normalized to protein content.
Luciferase activity assay
The promoter regions of BMP2 (from the 5' region − 1360 to + 597 bp) and STAT1 (from the 5' region − 526 to + 246 bp) were ligated into the pGL3-basic vector (Cat#: E1751, Promega, WI, USA), then pGL3-BMP2-Luc and pGL3-STAT1-Luc were synthesized by digesting the plasmid with KpnI and XhoI and subcloning into the luciferase reporter vector. To further explore the binding site of ILF3 on the promoters of BMP2 and STAT1, we constructed several pGL3-BMP2-Luc reporter plasmids with progressively deleted 5'-flanking regions from the − 1360- to + 597-bp region and pGL3-STAT1-Luc reporter plasmids with progressively deleted 5'-flanking regions from the − 526- to + 246-bp region. Various lengths of the promoter region of BMP2 sequence including − 1160 to + 597, -960 to + 597, -760 to + 597, -560 to + 597, -360 to + 597, -160 to + 597, +41 to + 597, +241 to + 597 and + 425 to + 597 bp regions and STAT1 sequences including − 330 to + 246, -140 to + 246 and + 51 to + 246 bp regions were constructed. HEK293T cells were grown in 24-well plates and co-transfected with luciferase reporter plasmids and a Renilla reporter plasmid (pRL-TK, Promega) by using Lipofectamine 3000 Reagent Protocolin Opti-medium. After 24 h, firefly and renilla luciferase signals were determined by using the Dual-Luciferase reporter Assay Kit (Promega).
Chromatin immunoprecipitation assay (ChIP)
ChIP assay involved using a ChIP Assay kit (CST, USA). Nucleoprotein complexes were extracted from HAVSMCs. For immunoprecipitation, anti-ILF3 antibody (Abcam, Cat# ab131004), normal IgG antibody (CST, USA) and Histone H3 antibody (CST, USA) were used. Specific primers targeting different DNA sites in the − 160 to + 40 bp fragment of BMP2 and the − 140 to + 50 bp fragment of STAT1 are in supplemental Table 1.
Data are presented as mean ± SEM from three replicate experiments. Student’s t test and one-way ANOVA were used to evaluate differences between 2 groups and multiple groups, respectively, by using SPSS 18.0. Differences were considered significant at P < 0.05.