Epigenetic Regulation of Vascular Smooth Muscle Cells by Histone H3 Lysine 9 Dimethylation Attenuates Target Gene-Induction by Inflammatory Signaling

Supplemental Digital Content is available in the text.

Cultured VSMCs and ex vivo tissue from the medial layer of the aorta were washed in phosphate buffered saline (PBS) and fixed in 1% formaldehyde in PBS for 10 minutes at room temperature before quenching with 0.125 mM glycine for 5 minutes. Subsequently, the cells/tissue were washed three times in PBS. Ex vivo tissue was snap frozen and homogenised using a metal mortar. The cells/homogenised tissue were resuspended in nuclear lysis buffer (50 mM Tris-HCl pH 8.1, 10 mM EDTA pH 8.0, 1% SDS) supplemented with protease inhibitors (Roche) and incubated on ice for a minimum of 5 minutes. A Bioruptor Pico sonication device (Diagenode) set at high power, 30 seconds on and 30 seconds off for 15 to 20 cycles was used to fragment the chromatin to approximately 0.2-1 kb. The sonicated chromatin was centrifuged at 12,000 g at 4 ⁰C for 15 minutes to remove insoluble debris. The DNA concentration was determined using a Nanodrop ND-1000 spectrometer. Fragmentation of chromatin was confirmed by Bioanalyzer assessment (DNA High Sensitivity Analysis Kit Agilent Technologies) of a 0.5 μg aliquot after decrosslinking. To de-cross link, the 0.5 μg aliquots were incubated in elution buffer (20 mM Tris-HCl pH8.0, 5 mM EDTA pH 8.0, 50 mM NaCl, 1% SDS, 50 μg/ml Proteinase K, 100 μg/ml RNase A) at 37 ⁰C for an hour and then at 68 ⁰C overnight. Sonicated chromatin (2-20 μg) was incubated with the appropriate antibodies (Table III) or control IgG (Table IV) with BSA (100 μg/ml) and yeast tRNA (20 μg/ml), diluted 10x in RIPA-140 (10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 0.5 mM EGTA, 1% Triton X-100, 0.10% SDS, 0.10% sodium deoxycholate, 140 mM NaCl) supplemented with protease inhibitors (Roche), overnight at 4 ⁰C. A 10% aliquot of the chromatin used for each ChIP was directly de-crosslinked as described above. Protein A and protein G magnetic beads (600 μg, Life Technologies), washed twice in RIPA-140 buffer were added to the samples and incubated at 4 ⁰C on a rotating wheel for 2 hours. Samples were then washed twice in RIPA-140, twice in RIPA-500 (10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 0.5 mM EGTA, 1% Triton X-100, 0.10% SDS, 0.10% sodium deoxycholate, 500 mM NaCl), RIPA-LiCl (10 mM Tris-HCl pH 7.5, 1 mM EDTA pH 8.0, 0.5 mM EGTA, 1% Triton X-100, 0.10% SDS, 0.10% sodium deoxycholate, 250 mM LiCl) and twice in TE (10 mM Tris-HCl pH 8.1, 1 mM EDTA pH 8.0) before being resuspended in 200 μl of elution buffer. Samples were incubated in elution buffer at 37 ⁰C for an hour and then 68 ⁰C overnight. The gDNA samples were purified using a gel extraction kit (Qiagen) following the manufacturer's protocol and eluted in 50-100 μl of TE. The purified gDNA was analyzed by qPCR using primers listed in Tables V and VI. ChIP for NFkB-p65 and AP1-cJun Human VSMCs (3-4 million per ChIP) were washed in PBS and fixed with 1% formaldehyde and Cross-link gold (Diagenode) following the manufacturers protocol. Fixed cells were incubated at room temperature for 30 minutes, then quenched with 0.125 M glycine for 15 min at room temperature. The cells were then lysed using buffers from the iDeal ChIP-seq Kit for Transcription Factors (Diagenode), following the manufacturer's instructions. The chromatin was sheared using a Bioruptor Pico sonication device (Diagenode) set at high power, 30 seconds on and 30 seconds off for 20 cycles, to fragment the chromatin to approximately 0.2-1 kb. Fragmentation of chromatin was confirmed by Bioanalyzer assessment (DNA High Sensitivity Analysis Kit Agilent Technologies) of a 0.5 μg aliquot after de-crosslinking. To de-cross link, the 0.5 μg aliquots were incubated in elution buffer (20 mM Tris-HCl pH8.0, 5mM EDTA pH 8.0, 50 mM NaCl, 1% SDS, 50 μg/ml Proteinase K, 100 μg/ml RNase A) at 37 ⁰C for an hour and then at 68 ⁰C overnight. Chromatin immunoprecipitation was carried out using the iDeal ChIP-seq Kit for Transcription Factors using the appropriate antibodies or control IgG (Table III, IV), according to the manufacturers protocol. The gDNA samples were purified using iPure magnetic beads and eluted in 40ul of the elution buffer provided in the iDeal ChIP-seq Kit for Transcription Factors (Diagenode). The purified gDNA was analyzed by qRT-PCR using primers listed in Table VI.

Flow cytometry
Carotid arteries were removed from animals culled by CO2 asphyxiation, dissected free from adipose and connective tissue and digested in 2.5 mg/ml of collagenase II (Invitrogen) and 2.5 U/ml of elastase (Worthington) in DMEM at 37 ⁰C, 5% CO2 (0.5 ml per carotid artery) for approximately 1 to 3 hours, mixing every 30 minutes, until a single cell suspension was reached. The dissociated cells were washed once in PBS, resuspended in 200 μl of FACS buffer (1% BSA in PBS), filtered through a 40 μm cell strainer and EYFP + VSMC were isolated on an Aria-fusion flow cytometry-assisted cell sorter (BD Bioscience).

Tissue processing for immunostaining
Carotid arteries were removed from animals culled by CO2 asphyxiation and dissected free from adipose and connective tissue, fixed with 4% (v/w) paraformaldehyde in PBS for 20 minutes at room temperature, washed for 5 minutes in PBS, incubated in 30% sucrose in PBS at 4 ⁰C overnight, embedded in TissueTek O.C.T. and snap frozen using liquid nitrogen. The arteries were cut transversely into 14 µm thick sections, mounted on SuperFrost Plus Adhesion microscope slides (Thermo Scientific) and stored at -80 ⁰C. Sections shown in Figure 1A are from the carotid arteries of animals fed a standard chow or atherosclerosis-incuding high fat diet.

Immunostaining of cryosections
Serial cryosections were rinsed in PBS, permeabilised in 0.5% Triton x-100 in PBS for 30 minutes at room temperature and blocked for 1 hour at room temperature in 1% BSA, 10% Normal Goat Serum (DAKO/Agilent), 0.1% Triton x-100 in PBS. Sections were then incubated with anti-H3K9me2 antibody conjugated to Alexa Fluor-647 (1:100, ab203851 Abcam) in blocking buffer overnight at 4 ⁰C. The sections were then washed twice for 5 minutes in PBS before staining the nuclei with 300 nM of DAPI in PBS for 10 minutes. The sections were then washed twice in PBS and mounted in RapiClear® 1.52 (SunJin Lab). Stained cryosections were imaged using confocal laser scanning microscopy (Leica SP5 or SP8) with laser lines and detectors set for maximal sensitivity without spectral overlap for DAPI (405 laser, 417-508 nm), CFP (458 laser, 454-502 nm), GFP (488 laser, 498-506 nm), YFP (514 laser, 525-560 nm), RFP (561 laser, 565-650 nm) and Alexa Fluor-647 (633 laser, 650-700 nm). Cryosections were imaged using a 20x oil objective, and data was acquired at an optical section resolution of 1024 x 1024. Image stacks (12 μm) were collected with 1 μm z-steps. Image J software was used for image processing and analysis. VSMCs were identified by expression of the fluorescent Myh11-lineage labeling reporter and their nuclei defined by DAPI staining. The mean pixel intensity of H3K9me2 staining in the nuclei of VSMCs was used to calculate H3K9me2 levels. Nuclei of individual Confetti+ cells included in the analysis were chosen without displaying the Alexa Fluor-647-H3K9me2 signal to avoid bias. A total of 20 ( Figure 1A, 4A) or 30 ( Figure 1C, Supplemental Figure 1B) Confetti+ VSMCs were analysed per animal (10 cells in each 12 μM image stacks per section, 2-3 sections per animal). Nuclear H3K9me2 levels for each individual VSMC was divided by the average VSMC nuclear H3K9me2 level of a control animal (standard diet, SD; no surgery left carotid artery or internal control right carotid artery) analysed in parallel.

Mouse VSMC Isolation
Whole aortas from C57BL/6J mice were dissected in PBS under an inverted Leica M80 microscope, applying aseptic technique. Fat and connective tissue were removed before the vessels were opened longitudinally to scrape off blood and endothelial cells. For primary mouse VSMC culture, each aorta was incubated in 1 ml of 1 mg/ml of collagenase II and 1 U/ml of elastase (Worthington) in DMEM at 37 ⁰C, 5% CO2 for 10 minutes to peel off and remove the adventitia. Aortas for RNA extraction were stored in RNAlater (ThermoFisher, AM7024) at -20 ⁰C. For RNA extraction or ChIP, the adventitia was removed manually in PBS without enzymatic digestion. The isolated medial layer of VSMCs was then washed in PBS before being snap frozen in liquid nitrogen or being further processed for culture.

Primary mouse VSMC Culture
After isolating the medial layer of VSMCs, the tissue was further digested in 1 ml of 2.5 mg/ml of collagenase II (Invitrogen) and 2.5 U/ml of elastase (Worthington) in DMEM at 37 ⁰C, 5% CO2 for approximately 1 to 3 hours, mixing every 20 minutes, until a single cell suspension was reached. The dissociated VSMCs were plated in VSMC growth medium (DMEM supplemented with 10% (v/v) of FBS, 100 U/ml of penicillin, 100 mg/ml of streptomycin and 2 mM of glutamine) into one well of a 12-well plate (Corning) per aorta. VSMCs were maintained at 37 ⁰C, 5% CO2 in growth medium and split 1:2 every 3-5 days when approximately 90% confluent. For passaging, the cells were washed in PBS and incubated in trypsin at 37 ⁰C, 5% CO2 for approximately 5 minutes. Experiments were carried out using passage 4 VSMCs.

Human VSMC Culture
Human aortic VSMCs were isolated from patients undergoing aortic valve replacement surgery with ethics committee approval. The gender and age of donors is provided in the Major Resources Tables. Adventitia and endothelial cells were manually removed from 2-3 mm² sections of aorta. The medial VSMC layer was then placed in a 6 well plate with a cover slip placed on top and cultured in growth medium (DMEM supplemented with 10% (v/v) of FBS, 100 U/ml of penicillin, 100 mg/ml of streptomycin and 2 mM of glutamine). VSMCs were cultured to approximately 90% confluence before passaging. For passaging, the cells were washed in PBS and incubated in trypsin at 37 ⁰C, 5% CO2 for 3-5 minutes. Experiments were carried out using passage 6-15 VSMCs.

Treatment of VSMC cultures
Mouse and human VSMCs were seeded in 12-well plates in growth medium at a ratio of 1:2. When the cells became 70% confluent, the media was changed to chemically defined medium (CDM; 1:1 mixture of Iscove's modified Dulbecco's medium plus Ham's F-12 medium, both supplemented with GlutaMAX (Gibco, Life technologies), 5 mg/ml bovine serum albumin, 450 μM monothioglycerol, 15 mg/ml Transferrin, 7 mg/ml Insulin, 1 x lipids (100 x chemically defined lipid concentrate (Gibco, Life Technologies), 1 x penicillin/streptomycin (Gibco, Life Technologies). After 24 hours, the media was replaced with CDM with 1 μM UNC0638 (UNC, Tocris) and/or 10 μM SP600125 (Abcam) or vehicle control (DMSO) and the medium was refreshed a further 24 hours later. After 48 hours of treatment, 2 ng/ml of human recombinant IL-1a (Peprotech) or 90 ng/ml of human recombinant TNF-a (Peprotech) was added to the appropriate wells. After 6 hours of IL-1a or TNF-a stimulation, unless otherwise indicated in the figure legends, the cells were washed twice with PBS, harvested by trypsinisation, washed twice with PBS and cell pellets stored at -80 ⁰C.

G9A SiRNA knock down
Cultured human VSMCs (300,000 at passage 7-10) were plated per well of a 6 well plate (35 mm diameter) in VSMC growth medium. Eight hours after plating, the media was changed to CDM without penicillin/streptomycin and 16 hours later the cells were transfected with either a 100 nM pool of siRNA targeted against G9A (sc-43777, Santa Cruz) or a 100 nM pool of control scrambled siRNA (sc-37007, Santa Cruz) using HiPerfect transfection reagent (Qiagen), following the manufacturers protocol (12 μl of HiPerfect was used per transfection). 48 hours after transfection the cells were treated with or without 2 ng/ml of human recombinant IL-1a (Peprotech) for 6 hours, or as specified, before harvest.

Dye Quenched-gelatin Extra Cellular Matrix (ECM) degradation assay
Immediately after UNC0638 and/or IL-1a treatment of primary mouse VSMCs, 1 ml of 50 μg/ml of DQ-gelatin (Thermofisher Scientific), dissolved in CDM, was added per 9.5 cm 2 well. After 2 hours of DQ-gelatin treatment, the cells were washed twice in CDM before incubating with 300 nM of DAPI in PBS for 2 minutes. The cells were then washed once with PBS and mounted in VECTASHIELD Antifade mounting medium (Vector Laboratories). Degradation-induced fluorescence was used to measure MMP activity. The DQ-gelatin-treated VSMCs were imaged using a 20x objective with a fluorescence microscope (ZEISS Axio Vert A.1). The corrected total fluorescence (CTF) was quantified using ImageJ software as follows; CTF = [(area x mean pixel intensity) -(area x mean pixel intensity of 5 background readings)]/ cell number. Cell numbers were determined by counting DAPI stained nuclei. The average CTF of 5 fields of view was used to quantify the MMP activity of each sample.

Immunostaining of cultured VSMCs
After treatment of primary human VSMCs with UNC0638 (48h) and IL-1a or TNF-a (1h), the cells were washed twice with PBS and fixed with 4% paraformaldehyde for 10 minutes at room temperature. Cells were subsequently incubated with 0.5% Triton-X100 (Sigma) for 20 minutes at room temperature before being washed three times with PBS and blocked with 10% normal goat serum (Dako) in PBS for 1 hour at room temperature. Cells were then incubated with NFkB p65 antibody in blocking buffer (1 μg/ml, Santa Cruz sc-372) for 2 hours at room temperature and washed three times with blocking buffer. After 1 hour of incubation with Alexa Fluor 488conjugated goat anti-rabbit IgG (2 μg/ml, Abcam ab150077) at room temperature, the cells were washed twice with PBS before incubating with 300 nM of DAPI in PBS for 2 minutes. Secondary only control stainings were done in parallel. Finally, the cells were then washed once with PBS and mounted in VECTASHIELD Antifade mounting medium (Vector laboratories). The cells were imaged using a 10x objective with a fluorescent microscope (ZEISS Axio Vert A.1). The nuclear/cytoplasmic ratio of p65 was measured using ImageJ software (http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Intensity_Ratio_Nuclei_Cytoplasm_Tool). The nuclear/cytoplasmic ratio measures the mean pixel intensity of nuclear area defined by DAPI divided by the mean grey value of the cytoplasmic area. The average nuclear/cytoplasmic p65 ratio of 3 fields of view was used to calculate the nuclear/cytoplasmic ratio of p65 of each sample.

Western blotting
Cell pellets were lysed directly in 2X Laemmli sample buffer (LSB, 20% glycerol, 4% SDS, 100 mM Tris HCl pH 6.8, 200 mM DTT), incubated at 98 ⁰C for 5 minutes and sonicated for 1 minute at medium intensity using a standard Bioruptor (Diagenode). For ex vivo tissue samples the adventitia was removed as described above. For the comparison of H3K9me2 levels in animals after HFD ( Figure 1B) the tissue (medial layer, plaque, intima) were crushed using a pestle in 2 x LSB, incubated at 98 degrees and sonicated. For the analysis of A366-treated and control tissue ( Figure 4C), the endothelial cell layer was manually removed by gentle scraping before processing. Total protein (10-30 μg) was resolved with SDS-PAGE and transferred to PVDF membranes. The membranes were incubated in blocking buffer (5% w/v nonfat dry milk in TBS (50 mM Tris-HCl, pH 7.5, 150 mM NaCl)) for one hour at room temperature. The membranes were then probed with primary antibodies (Table III) overnight at 4 ⁰C followed by secondary antibodies conjugated to horseradish peroxidase (HRP , Table IV) for 30-60 minutes at room temperature. All antibody incubation steps were done in blocking buffer. Protein was detected using the ECL system (GE Healthcare).

Reverse transcription -quantitative Real Time-Polymerase Chain Reaction (RT-qPCR)
Total RNA was extracted with TRIzol (Life Technologies) and 0.05-0.5 μg was reverse transcribed using a QuantiTect Reverse Transcription kit (Qiagen), according to the manufacturer's directions. For quantification, cDNA (diluted 1:2-1:10) was amplified, in technical duplicates, with a QuantiTect SYBR Green PCR kit (Qiagen) and primer pairs listed in Tables VII and VIII, using a Corbett Life Science Rotor Gene 6000 PCR system. A standard curve was used to calculate relative transcript levels, which were normalized to the average of two housekeeping genes (indicated in figure  legends).

Statistical analysis
The data were analysed using Kruskal-Wallis one-way analysis of variance with Dunn's test to compare specific sample pairs, Mann-Whitney U test, Wilcoxon rank-sum test or a linear model (described below) as detailed in figure legends.
The H3K9me2 immunostaining data shown in Figures 1A, 1C, 4B and Supplemental Figure IB, were analysed for statistical significance using a linear model (fitted in R). The log-transformed H3K9me2/DAPI scores for each cell were expressed as a linear function of the batch (experimental day) and "treatment" groups (e.g. "no surgery" vs. "control" in Figure 1C). In all cases, the model itself was significant (P<2.2x10 -16 ) and P-values report where treatment groups were significantly different from controls: HFD vs. SD in Figure 1A (P<2.2x10 -16 ), Post injury vs. Control in Figure 1C  To test whether the observed reduction of H3K9me2 in animals fed a high fat versus a standard diet (western blow shown in Figure 1B), we performed one-sided exact Wilcoxon rank-sum tests. We tested against the alternative that H3K9me2 levels were lesser for the high-fat diet than the standard diet respectively (P=0.05). Similar analysis revealed P=0.05 for p-p65 being higher in high fat diet samples compared to controls.

Supplemental Tables
Supplemental Table I