The study was approved by the ethics committee of Ningxia Medical University (Yinchuan, China). Primary cultured VSMCs were obtained from human umbilical vein media (Department of Obstetrics, General Hospital of Ningxia Medical University, Yinchuan, China). Cells were cultured in Dulbecco’s Modified Eagle’s medium-Ham’s F12 media (Gibco-BRL, Gaithersburg, MD, USA) supplemented with 20% fetal calf serum (FCS; Gibco-BRL), 100 U/ml penicillin and streptomycin (Sigma-Aldrich Trading Co., Ltd, Shanghai, China) at 37°C in an incubator with 5% CO₂. The purity of the primary VSMC culture was confirmed by the characteristic ‘hill-and-valley’ growth pattern and immunocytochemistry of α-actin. VSMCs were used at between three and five passages for the experiment. Cells were seeded onto six-well plates and grown to 80% confluence. Cells were then serum-deprived for 24 h to reach synchrony, followed by the addition of 5% FCS for a further 24 h and the administration of Hcy (Sigma-Aldrich Trading Co., Ltd). Hcy was administered at the following concentrations: 0 (control), 50, 100, 200 and 500 μM and 500 μM plus folate (Sigma-Aldrich Trading Co., Ltd), respectively. Hcy was replenished every 8 h in compensation for its short half-life (total three times).

To assess cell proliferation, VSMCs were seeded in 96-well plates at 10³–10⁴ cells in 100 μl/well. The aforementioned concentrations of Hcy were then added and the cells were incubated for 72 h. Hcy was replenished every 8 h. A total of 20 μl MTT (5 mg/ml; Sigma-Aldrich Trading Co., Ltd) was added to each well and incubated at 37°C for 4 h. The supernatant was removed using a pipette, and 150 μl dimethyl sulfoxide was added to each well. After 10 min of incubation at room temperature, plates were read on a micro-enzyme-linked immunosorbent assay reader (Bio-Tek Instruments, Inc., Winooski, VT, USA) at 490 nm. Values were normalized using the control value.

Following treatment with various concentrations of Hcy, cells were analyzed using a FACStar™ Plus flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). In brief, cells were washed once with phosphate-buffered saline (PBS; 0.01 mol/l, pH 7.2) and fixed at −10°C for 5 min in paraformaldehyde-lysine-periodate fixation solution. Cells were then treated with 0.1% Triton X-100 and 0.5% RNase A, followed by the addition of propidium iodide. All samples were passed through 70-mm mesh prior to flow cytometric analysis; 30,000 nuclei were examined in each analysis. The percentage of cells was estimated using the Cell FIT analysis version 2.0 software (Becton, Dickinson and Company). Three separate experiments were performed with three different populations of cells.

Total RNA was isolated using TRIzol^® reagent (Invitrogen Life Technologies, Grand Island, NY, USA) and reverse transcribed using the RevertAid First Strand cDNA Synthesis kit (MBI Fermentas, Vilnius, Lithuania). GAPDH was used as an endogenous control, and the primer sequences were as follows: PDGF, 5′-TCTGCTGCTACCTGCGTCTGG-3′ (forward) and 5′-CACTGCACGTTGCGGTTGTT-3′ (reverse) and GAPDH, 5′-AGAAGGCTGGGGCTCATTTG-3′ (forward) and 5′-AGGGGCCATCCACAGTCTTC-3′ (reverse). qPCR analysis was performed using an FTC-3000 Real-Time PCR detection system (Funglyn Biotech Corp. Ltd., Toronto, ON, Canada). The RNA levels of each gene were calculated using the cycle threshold (Ct) value of the sample relative to that of GAPDH using the following formula: Ct=Ct_(GAPDH)−Ct_(sample). Final results, expressed as n-fold differences in target gene expression relative to the calibrator, termed N_target, were calculated using the following formula: N_target=2^{Ct(sample)−Ct(calibrator)}, where Ct values of the calibrator and sample were determined by subtracting the Ct value of the target gene from the Ct value.

Proteins were extracted from the cells using cell lysis buffer and separated using 12% SDS-PAGE. The proteins and the prestained marker (MBI Fermentas, Amherst, NY, USA) were then transferred onto a polyvinylidene fluoride Immobilon^®-P Transfer Membrane (Millipore, Billerica, MA, USA) with a pore size of 0.45 mm using a Trans-Blot^® Semi-Dry Transfer Cell model 755 (Bio-Rad, Hercules, CA, USA) for 90 min. The membrane was incubated at room temperature in PBS-Tween 20 (PBS-T) buffer containing 5% non-fat milk for 4 h. Membranes were then cut as required and placed in a hybridization bag with 1 ml anti-β-actin or -PDGF primary antibodies (Sigma-Aldrich, St. Louis, MO, USA) and incubated at 4°C overnight. The membranes were subsequently washed three times using PBS-T buffer and the secondary antibody was added for 2 h at room temperature. The membranes were washed a further three times with Tris buffered saline with Tween 20 and incubated with horseradish peroxidase substrate (BeyoECL Plus A/B; Beyotime Institute of Biotechnology, Shanghai, China) for 1 min. Blots were developed using X-film (Kodak, Tokyo, Japan).

Genomic DNA was isolated from VSMCs using the Wizard^® Genomic DNA purification kit (Promega Corp., Madison, WI, USA) and nMS-PCR was performed for the detection of PDGF gene methylation. Following standard sodium bisulfite DNA modification using the EZ DNA Methylation-Gold™ kit (Zymo Research, Irvine, CA, USA), two-step PCR amplifications were performed. nMS-PCR initially uses an outer primer pair that does not contain any CpG sites, followed by second step PCR using conventional PCR primers. CpG islands were identified in the PDGF promoter region using a CpG Island Search engine (http://www.uscnorris.com/cpgislands2/cpg.aspx; http://www.cbs.dtu.dk/services/promoter/). The primers used in the nMS-PCR assay were as follows: PDGF-outer primer, 5′-TTTTTTTGTTTTGAAATTTTGGTTAA-3′ (forward) and 5′-CAAAATCCCAACAAAAAAAATCTCC-3′ (reverse); PDGF-methylated primer, 5′-TTTGGAAATTAATGATAAGTTAGGC-3′ (forward) and 5′-AAGCATCATAAAAAACAAACGCATC-3′ (reverse); PDGF-unmethylated primer, 5′-TTGGAAATTAATGATAAGTTAGGTGA-3′ (forward) and 5′-AAACATCATAAAAAACAAACACATCA-3′ (reverse). To reduce mispriming and to increase efficiency, touchdown PCR was used for the amplification. The PCR products were separated using electrophoresis on a 2% agarose gel containing ethidium bromide. DNA bands were visualized using ultraviolet light and methylation was calculated using the following formula: Methylation % = methylation/(methylation + unmethylation) × 100.

The concentrations of SAM and SAH were determined using HPLC. Cells were centrifuged, washed twice with cold PBS and maintained on ice. The cell pellets were subsequently homogenized in four volumes of 0.4 μM HClO₄. A 200 μl aliquot of the acid extract was loaded into a C18 column (Shimadzu, Kyoto, Japan), run by a Hitachi L2000 HPLC system (Hitachi, Tokyo, Japan). The absorption of the eluted compounds was monitored at an excitation wavelength of 254 nm. Elution of SAM and SAH was achieved at a flow rate of 1.0 ml/min using mobile-phase ammonium formate solution. Chromatograms were recorded using a D-2000 Elite integrator. SAM and SAH standards were used to identify the elution peaks, and the SAM and SAH tissue values were calculated using the standard curve.

A modification of the assay developed by Hattori et al (18) was used to determine the activities of DNA methyltransferases. VSMCs (1×10⁶) were homogenized using a glass pestle containing 500 μl lysis buffer (100 mmol/l NaCl, 10 mmol/l Tris-HCl, pH 8.0, 25 mmol/l EDTA, 0.5% SDS and proteinase K 0.2 g/l). The suspension was freeze-thaw cycled between −70 and 37°C three times. VSMC protein exacts were stored at −70°C prior to analysis. The reaction contained cell homogenates (5 μg protein), 0.25 μg poly(deoxyinosinic-deoxycytidylic) acid and 11.1×10¹⁰% Bq [methyl-³H] SAM in a total volume of 20 μl, and was incubated at 37°C for 2 h. RNA was removed by adding 20 μl RNase A (2 g/l) and incubating at room temperature for 5 min. DNA was purified using the E.Z.N.A.^® Cycle-Pure kit (Omega Bio-Tek, Inc., Norcross, GA, USA), and the purified genomic DNA was spotted onto a Whatman GF/C filter disc and dried at 80°C for 5 min, prior to counting in a Packard 1600 TR Liquid Scintillation Counter (Packard Instrument Co., Meriden, CT, USA) for determination of C-5 MT-ase activity. Each reaction was performed in triplicate and the assay was repeated three times to blind the source of the samples. In order to exclude background protein, all samples were initially assayed with the control containing the whole cell lysate, but without poly(deoxyinosinic-deoxycytidylic) acid. C-5 MT-ase activity was expressed as the quantity of incorporated [methyl-³H] groups into the poly(deoxyinosinic-deoxycytidylic) acid (cpm/1 μg protein).

Each experiment was repeated three times. Results are expressed as the mean ± standard deviation. Statistical comparisons of single parameters between two groups were performed using the paired Student’s t-test. Kruskal-Wallis one-way analysis of variance was used to compare the means of multiple groups, followed by the Dunn test. A value of P≤0.05 was considered to indicate a statistically significant difference.

The primary culture of confluent VSMCs exhibited a typical elongated ribbon or spindle-shaped appearance with a characteristic ‘hill and valley’ pattern (Fig. 1A), and immunocytochemistry revealed 98% positive α-actin staining (Fig. 1B). MTT assay showed that VSMC viability significantly increased by 1.2-, 2.2-, 3.6- and 4.3-fold subsequent to treatment with 50, 100, 200 and 500 μM Hcy, respectively, compared with the control group (P<0.01). Furthermore, folate treatment had an antagonistic effect against Hcy-induced VSMC proliferation, with a 63% decrease in VSMC viability observed in the folate plus 500 μM Hcy group compared with the 500 μM Hcy group (P<0.01) (Fig. 1C).

Flow cytometry revealed that, as Hcy concentration increased, the proportion of the cell population in S phase increased, and that in G₀/G₁ phase decreased. Folate showed an inhibitory effect against Hcy on the VSMC cell cycle (Fig. 2).

qPCR and western blot analyses revealed a dose-dependent increase in PDGF mRNA and protein expression with Hcy treatment, in parallel with the increase in VSMC proliferation observed under Hcy treatment. PDGF has a key role in VSMC proliferation; therefore, this parallel alteration in VSMC PDGF expression and proliferation suggests a causative role for PDGF in Hcy-induced VSMC proliferation (Fig. 3).

DNA methylation occurs almost exclusively at CpG dinucleotides, and CpG methylation is often associated with gene silencing and vice versa. The DNA methylation level of the CpG islands in the PDGF gene promoter was analyzed using nMS-PCR. The PDGF gene promoter was found to be significantly hypomethylated in a dose-dependent manner upon Hcy treatment (Fig. 4). These data indicate that Hcy treatment inhibits the methylation of the PDGF gene, and this demethylation effect may be involved in the upregulation of PDGF expression and VSMC proliferation in the pathogenesis of AS.

SAM and SAH concentrations are important factors in the transmethylation process. In the present study, SAM and SAH levels were assessed using HPLC. In the Hcy-treated groups, the intracellular levels of SAH were significantly higher than those in the control group, showing up to a three-fold increase. However, upon Hcy treatment the concentration of SAM was observed to decrease, leading to a significant decrease in the ratio of SAM/SAH. SAM is a primary methyl-donor while SAH is a potent inhibitor of methyltransferase activity; therefore, the SAM/SAH ratio may be critical in the modification of DNA methylation. However, the changes in SAM and SAH levels did not exhibit the dose-effect trend with Hcy concentration (Fig. 5).

C-5 MT-ase activity is another key regulator of the transmethylation reaction. Hcy was found to significantly upregulate the activity of C-5 MT-ase compared with the control cells (Fig. 6); however, no significant dose-dependent increase was observed.