The JMJD2A inhibitor, IOX1 [5-carboxy-8-hydroxyquinoline (5-carboxy-8-HQ)], was dissolved in dimethyl sulfoxide (DMSO) (both from Sigma, St. Louis, MO, USA) to yield a 250 µM stock solution and was subsequently diluted in Dulbecco's modified Eagle's medium (DMEM; HyClone, Logan, UT, USA) to the required concentration.

As previously described (9), VSMCs were isolated from the thoracic aortas of male Sprague-Dawley rats (weighing, 150–180 g). All animals used in our study were purchased from the Animal Center of Renmin Hospital of Wuhan University. The experimental procedures and animal care procedures were conducted in accordance with the Institutional Animal Care and Use Committee of Wuhan University. Briefly, rat thoracic aortas were harvested following euthanasia. After scraping off the adventitia and endothelium, thoracic aortas were cut into sections of 2×2 mm. The primary VSMCs were obtained using the tissue explants adherent method and cultured in DMEM (HyClone) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA) and 1% penicillin/streptomycin (PS; Beyotime Institute of Biotechnology, Bejing, China) in an atmosphere of 5% CO₂, at 37°C. The purity of the VMSCs was assessed by immunostaining with anti-smooth muscle α-actin antibody. VSMCs between the third and fifth passages were used for the experiments.

The cell proliferation assay was performed using a Cell Counting Kit-8 (CCK8; Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Briefly, approximately 8,000 VSMCs were seeded in 96-well plates in 200 µl of culture medium. Following synchronization with DMEM containing 0.1% FBS for 24 h, the VSMCs were pretreated with IOX1 at various concentrations (0, 50, 100 and 200 µM) for 2 h, and then stimulated with 1 µM Ang II (Sigma) for 24 h. CCK8 solution (20 µl) was then added and the OD value was measured at 450 nm using a microplate spectrophotometer (Infinite M200; Tecan Group Ltd., Männedorf, Switzerland).

A Transwell chamber assay (Corning Inc., Corning, NY, USA) was performed to evaluate the migration activity of the VMSCs. Synchronized (10⁵) VSMCs were seeded into the upper chamber in 200 µl of serum-free medium in the presence or absence of IOX1. Following inhibitor precondition for 2 h, 600 µl of DMEM supplemented with 1 µM Ang II were added to the lower chamber followed by incubation for 8 h. Cells remaining on the upper side of the membrane were gently wiped with a cotton swab. The migrated cells were fixed with methanol and stained with 0.5% crystal violet. The number of migrated cells was counted in 5 random fields under a microscope (Olympus Corporation, Tokyo, Japan) at a ×100 magnification.

Cell-cycle analysis was carried out by flow cytometry. The VSMCs were cultured in 6-cm culture plates (Corning Inc.) at a density of 5×10⁵ cells/plate. At 50% cell density, the VSMCs were synchronized for 24 h. Then VSMCs were then pre-treated with IOX1 (200 µM), followed by treatment with Ang II treatment for 24 h. The VMSCs were then trypsinized, harvested and washed twice with cold phosphate-buffered saline (PBS), and fixed with 70% cold ethanol and stored at 4°C overnight. After staining with prop-idium iodide (PI solution: 50 mg/ml PI, 100 mg/ml RNase A) at 37°C for 30 min, the cell cycle distribution was determined by flow cytometry (BD FACSCalibur; Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The percentage of cells in the G0/G1, S and G2-M phase of the cell cycle was determined using ModFitLT cell cycle analysis software (Verity Software House Inc., Topsham, ME, USA).

The synchronized VSMCs were pretreated with IOX1 (200 µM) for 2 h and then stimulated with Ang II for 0, 3, 6, 12 and 24 h and lysed in RIPA lysis buffer (Beyotime Institute of Biotechnology) containing 1 mM PMSF and 10 mM phosphatase inhibitor (Roche, Mannheim, Germany) on ice for 15 min, and centrifuged for 5 min at 12,000 × g at 4°C. Total protein was quantified using a BCA protein assay kit (Beyotime Institute of Biotechnology) according to the manufacturer's instructions. Equal amounts of protein (35 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes. The PVDF membranes were blocked and incubated with antibodies against JMJD2A (SAB3500095; Sigma), H3K9me3 (ab8898; Abcam, Cambridge, MA, USA), cyclin D1 (2978T), cyclin E (4136S), p21 (2947T), p27 (3686T; all from Cell Signaling Technology, Inc., Danvers, MA, USA) and GAPDH (5632-1; Epitomics, Burlingame, CA, USA) overnight at 4°C. The washed membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (7074; Cell Signaling Technology, Inc.) for 2 h at room temperature, and subsequently detected using ECL reagent (Pierce, Rockford, IL, USA). The results of the western blot analysis were quantified using Quantity One software.

The mRNA expression levels of cyclin D1 and p21 were measured by RT-qPCR. The synchronized VSMCs were pre-treated with IOX1 (200 µM) for 2 h and then stimulated with Ang II for 12 h. Total RNA was extracted using the PicoPure RNA isolation kit (Applied Biosystems Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions and reverse transcribed into cDNA using a First-Strand Synthesis system (Invitrogen, Carlsbad, CA, USA). This was performed using the SYBR system on the ABI Prism 7500 Sequence Detection System (Applied Biosystems Life Technologies, Foster City, CA, USA). Each sample was run and analyzed in triplicate. Data were normalized to GAPDH expression and analyzed using the 2^−ΔΔCt method. The primer sequences used in the present study were as follows: cyclin D1 forward, 5′-TGTTCGTGGCCTCTAAGATGAAG-3′ and reverse, 5′-GGAAGTGTTCGATGAAATCGTG-3′; p21 forward, 5′-AGTATGCCGTCGTCTGTTCG-3′ and reverse, 5′-GAGTGCAAGACAGCGACAAG-3′; and GAPDH forward, 5′-GACATGCCGCCTGGAGAAAC-3′ and reverse, 5′-AGCCCAGGATGCCCTTTAGT-3′.

ChIP assay was performed using a ChIP assay kit (Pierce) according to the manfacturer's instructions. Briefly, the synchronized VSMCs were pre-treated with IOX1 (200 µM) for 2 h and then stimulated with Ang II for 12 h and fixed with 1% formaldehyde, washed and lysed. The cell lysates were sonicated to create chromatin fragments of 400–500 bp in length, diluted in ChIP dilution buffer and subjected to immunoprecipitation with anti-H3K9me3 antibodies (ab8898; Abcam) overnight at 4°C. The immune complexes were collected, washed and eluted with buffer. Protein-DNA cross-links were reversed overnight at 65°C and the DNA was extracted. ChIP-enriched DNA samples were analyzed by RT-qPCR using primers specific for cyclin D1 or p21. Data were analyzed using the 2^-ΔΔCt method and normalized with input samples. The results are expressed as a percentage (%) of the control values. The primer sequences were follows: cyclin D1 forward, CTCTGCCCGGCTTTGATCTCT-3′ and reverse, 5′-AGGCTGCAGGACTTTGCAACT-3′; and p21 forward, 5′-GTTCAGCCCTGGAACCGAAG-3′ and reverse, 5′-GTACCAAACACCCTTCACCTGGTAC-3′.

All results were analyzed using either a Student's t-test for between-group comparisons or the one-way ANOVA for multiple comparisons using SPSS 13 software. Data are presented as the means ± SEM of 3 or 6 experiments, as indicated in the figure legends. A value of p<0.05 was considered to indicate a statistically significant difference.

Subconfluent, quiescent VSMCs were exposed to Ang II for 0, 3, 6, 12 and 24 h. Western blot analysis was performed to assess the total protein levels of JMJD2A and its primary target, H3K9me3, in Ang II-stimulated VSMCs. The time course of JMJD2A and H3K9me3 protein expression is shown in Fig. 1. Ang II induced a slight increase in the JMJD2A protein levels at an early time point (3 h). The levels peaked at around 6 h and were maintained at these levels until the 12-h time point. Conversely, the protein expression of H3K9me3 decreased in a time-dependent manner in the Ang II-stimulated VSMCs.

Since the expression of JMJD2A was upregulated in the Ang II-stimulated VSMCs, we intended to block JMJD2A with the specific inhibitor, IOX1, and to examine the effect of JMJD2A inhibition on Ang II-stimulated VSMCs. A significant increse in proliferation was observed in the subconfluent, quiescent VSMCs following stimulation with Ang II in the absence of serum (Fig. 2). The VSMCs pre-treated with IOX1 (0, 50, 100 and 200 µM) exhibited a decrease in proliferation in a dose-dependent manner. The most prominent suppressive effect on VSMC proliferation was observed following treatment with IOX1 at 200 µM (decreased in proliferation of 44.73%). We then wished to examine whether IOX1 suppresses the migration of Ang II-stimulated VSMCs. Pretreated VSMCs pre-treated with 200 µM IOX1 were stimulated with Ang II. The enhanced migration of the Ang II-stimulated VSMCs was indeed suppressed by pretreatment with IOX1 (Fig. 3). These data indicated that IOX1 inhibited the proliferation and migration of VSMCs stimulated with Ang II.

We then examined the effects of IOX1 on the cell cycle of VSMCs. Following absolute synchronization, the VSMCs were stimulated with Ang II for 24 h in the presence or absence of IOX1. Cell cycle analysis using flow cytometry revealed that there was a marked increase in the percentage of Ang II-stimulated cells in the S phase compared with the quiescent (unstimulated) cells. This increase in the S phase cell population was decreased by IOX1 treatment (Fig. 4). The percentage of cells in the G0/G1 phase were increased in the cells pre-treated with IOX1 (Fig. 4), indicating that IOX1 significantly inhibited the proliferation of the VSMCs by slowing down the progression of the cell cycle from the G0/G1 to the S phase.

Considering that IOX1 suppressed the proliferation of VSMCs and blocked cell cycle progression, particularly at the G1/S phase transition, we measured the expression levels of various cell cycle-related proteins in the Ang II-stimulated VSMCs in the presence or absence of IOX1 (50, 100 and 200 µM). Stimulation with Ang II increased the expression of cyclin D1 and cyclin E (Fig. 5). This increase in cyclin D1 expression was attenuated by IOX1 in a concentration-dependent manner. However, IOX1 had a marginal effect on cyclin E expression. In addition, we measured the protein expression levels of the CDKIs, p21 and p27. The levels of p27 were not influenced by Ang II, whereas the p21 levels were decreased in the Ang II-stimulated VSMCs. When the cells were pre-treated with to IOX1, p21 expression was upregulated in the Ang II-stimulated VSMCs. To determine whether the changes in the protein expression levels of cyclin D1 and p21 are regulated by IOX1 at the transcriptional level, RT-qPCR was performed to measure their mRNA expression levels. The mRNA expression levels were similar to the protein levels. IOX1 decreased cyclin D1 mRNA expression and upregulated p21 mRNA expression (Fig. 6). These data suggested that IOX1 inhibited the Ang II-induced VSMC proliferation through selective regulation of the cell cycle-related proteins, cyclin D1 and p21.

To determine whether IOX1 epigenetically mediates the expression of cyclin D1 and p21 in Ang II-stimulated VSMCs, we first examined the global changes in H3K9me3, the main substrate of JMJD2A. IOX1 enhanced the total protein levels of H3K9me3 which were reduced in the Ang II-stimulated VSMCs (Fig. 7A). To investigate the mechanisms through which IOX1 regulates cyclin D1 and p21 expression, ChIP assays were performed us ing anti-H3K9me3 to examine the effects of IOX1 on the cyclin D1 and p21 promoters in the Ang II-stimulated VSMCs. The results revealed that the levels of H3K9me3 in the cyclin D1 and p21 promoter regions were decreased in the Ang II-stimulated cells compared to to the untreated control cells. Pre-treatment with 200 µM of IOX1 increased the levels of H3K9me3 in the promoters of these genes (Fig. 7B).