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First published online February 29, 2008
Journal of Experimental Biology 211, 911-920 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.012914

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Homocysteine-induced extracellular superoxide dismutase and its epigenetic mechanisms in monocytes

Yideng Jiang^1,*, Jianzhong Jiang², Jiantuan Xiong¹, Jun Cao¹, Nan Li¹, Guizhong Li and Shuren Wang³

¹ Department of Pathophysiology, Ningxia Medical College, Yinchuan, Ningxia 750004, China
² Department of Pathology, Ningxia Medical College, Yinchuan, Ningxia 750004, China
³ Department of Pathophysiology, West China College of Preclinical and Forensic Medical Sciences, Sichuan University, Chengdu, Sichuan 610041, China

^* Author for correspondence (e-mail: jwcjyd{at}163.com)

Accepted 9 January 2008

	Summary

TOP Summary INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION References

 
Although a modest homocysteine (Hcy) elevation is associated with an increased cardiovascular risk, the underlying mechanisms whereby Hcy triggers the accumulation of cholesterol and the roles of the extracellular superoxide dismutase (EC-SOD) in the development of foam cells have not yet been elucidated. In this study, we found both increased numbers of foam cells and an accumulation of cholesterol, and the H₂O₂ and oxidized low-density lipoprotein content also increased. Levels of EC-SOD were significantly suppressed by Hcy, however, while 5-azacytidine (AZC), a potent DNA methyltransferase (DNMT) inhibitor, increased the expression of EC-SOD. A quantitative real-time PCR of EC-SOD revealed that Hcy (100 µmol l^–1) accelerates DNA methylation of EC-SOD, but selectively increases the activity of DNA methyl transferase 1 (DNMT1). It showed that Hcy can reduce binding of methyl CpG and binding protein 2 (MeCP2) but has no effect on the activity of DNMT3. Moreover, chromatin immunoprecipitation assays demonstrated that Hcy increased the binding of acetylated histone H3 and H4 in monocytes. Based on the fact that the binding of MeCP2 with the EC-SOD was completely suppressed by AZC and trichostatin A [TSA, a histone deacetylase (HDAC) inhibitor], it is indicated that DNA methylation and HDAC mediate the binding of MeCP2 with EC-SOD gene. In conclusion, the study found that Hcy accelerates the development of foam cells by repressing EC-SOD transcription, and that Hcy exerts this function by upregulating DNA methylation via suppression of HDAC activity and increased DNMT1 activity.

Key words: EC-SOD, H₂O₂, ox-LDL, DNA methylation

	INTRODUCTION

TOP Summary INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION References

 
Epidemiologic and case control studies have consistently indicated that moderate and mild elevation of plasma homocysteine (Hcy) is an important and independent risk factor for arteriosclerosis and venous thrombosis disease in populations (Stampfer et al., 1992). The level of risk for cardiovascular disease attributable to homocysteine is equivalent to that associated with hypercholesterolemia, and elevated Hcy levels synergistically increase the risk associated with smoking and hypertension (Vilar et al., 2007). Recently, studies in animal models have revealed that hyperhomocysteinemia enhances the formation of atherosclerotic lesion and the neointimal hyperplasia followed by arterial injury (Hofmann et al., 2001). Although there is experimental evidence that Hcy causes endothelial dysfunction (Woo et al., 2000) and vascular smooth muscle cell proliferation (Majors et al., 1997), the fundamental biochemical mechanisms underlying these phenomena are not yet well understood.

Some biological effects of Hcy in vitro can be mimicked by hydrogen peroxide (H₂O₂) or other sulfhydryl-containing agents but inhibited by catalases (Starkebaum and Harlan, 1986). These results lend support to the hypothesis that increased oxidation mediated through the sulfhydryl group of Hcy is the major mechanism responsible for Hcy-induced vascular pathogenesis (Turhan et al., 2005). Moreover, another intermediary metabolite of methionine, cysteine, has similarities to the chemical structure and redox property of Hcy, but does not constitute a risk factor for cardiovascular disease. Why did homocysteine become a pro-oxidant and a well known risk factor for atherosclerosis (AS), while cysteine is usually anti-oxidative? Many plausible explanations have not probed this key mechanistic point. It is suggested that the above-mentioned characteristics alone are not sufficient to explain the deleterious effect of Hcy.

DNA methylation, i.e. the formation of 5-methylcytosine (5-mC) from a cytosine residue via methyltransferase, is an important factor regulating the development of gene expression at different stages (Maatouk et al., 2006; Yideng et al., 2007). Some reports have shown that DNA methylation might be important for atherogenesis because AS is at least partially regulated by DNA methylation. Investigations of ApoE knockout mice (Iwama et al., 1998) revealed that significant genomic hypomethylation is developed during the first replication of aortic smooth muscle cells (SMCs) in vivo, and that hypomethylation occurred in some specific genes, such as 15-lipoxygenase and ApoE, which are therefore indicated to be deeply involved in AS (Maatouk et al., 2006; Jiang et al., 2007a; Jiang et al., 2007b; Yi-Deng et al., 2007). This might result from a direct regulatory effect of hypomethylation on gene expression or a secondary effect via effects on DNA integrity and its function.

The foam cells play a central role in the pathogenesis of atherosclerosis. The accumulation of cholesterol in foam cells is essentially a balance between cholesterol intake and cholesterol efflux. Extracellular superoxide dismutase (EC-SOD) is an anti-oxidative enzyme that catalyzes the dismutation of superoxide anion (O ^.–₂) to less reactive hydrogen peroxide (Reeves et al., 2002; Beckman, 1996; Crapo et al., 1992; Marklund, 1982), while the progression of AS and the change of EC-SOD are still unknown. How does Hcy induce foam cells and contribute to the accumulation of cholesterol? What roles does EC-SOD play? And what pathway does this interference go through? Based on these questions, the aim of the present study was to investigate the effect of Hcy on the activity of EC-SOD, as well as its relation with the methylation status of CpG sequences in DNA and the acetylation of histones H3 and H4. The aberration of DNA methylation and acetylation of histones H3 and H4 that was revealed, together with their pathways, could be potential targets for anti-atherosclerosis. Alteration of DNA methylation is potentially an important finding that could constitute the mechanism against AS featuring epigenetic gene silencing. The results suggested that methylation plays a role in the development of AS and had important consequences when it was exposed to Hcy. In addition, the results revealed that Hcy, being specifically converged on EC-SOD hypermethylation, contributed to the mechanisms leading to AS.

	MATERIALS AND METHODS

TOP Summary INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION References

 
Cell culture and cell treatment
Human blood from a healthy donor was drawn into heparinized syringes. Whole blood was separated into peripheral blood mononuclear cells and neutrophils using the density gradient from Nycoprep 1.077 (Axis-Shield PoC, Oslo, Norway). Monocytes were isolated from peripheral blood mononuclear cells by adherence to a serum-coated culture flask for 2 h. Adherent cells were detached and resuspended in RPMI-1640 medium containing 5% autologous plasma. Only cell preparations with a 95% viability or greater were used. The cells were then planted into six-well plates and grown to 80% confluence. The cells were then cultured with 0.5 µg ml^–1 phorbol myristate acetate (PMA) (Sigma-Aldrich, St Louis, USA) together with 100 µg ml^–1 oxidized low-density lipoprotein (ox-LDL) and different concentrations of Hcy.

Treatment with AZC and TSA
When the cells were in the exponential phase of growth, 30 mg l^–1 azacytidine (AZC; Sigma-Aldrich, St Louis, USA) and 50 ng ml^–1 trichostatin A (TSA; Sigma-Aldrich) were added to the monocytes and culture continued for 3 days, followed by three washes with phosphate buffered saline (PBS); culture using RPMI-1640 medium was continued for 2 days before experimentation.

Oil red O-stained foam cells and the analysis of the accumulation of cholesterol in cells
The cultured monocytes were washed with PBS three times, fixed in 2.5% glutaraldehyde for 3 h, dipped in 2.5% potassium dichromate for 16 h, and stained in 1% oil red O (Sigma-Aldrich) for 20 min to identify lipid droplets in cytoplasm. Cell nuclei were then stained in Hematoxylin for a few seconds. All products were washed with distilled H₂O and the monocyte-derived foam cells were observed and evaluated according to Wada et al.'s semi-quantitative method (Wada et al., 2002), i.e. the percentage of positive oil red O-staining cells.

The cultured human monocytes were resuspended in 0.5 ml sodium phosphate buffer solution (0.1 mol l^–1, pH 7.4). Each sample was sonified for 1 min with the microtip of a sonifier. Total cholesterol levels were determined as described (Rong et al., 1999). To determine free cholesterol (FC), cholesteryl esterhydrolase was omitted from the assay solution. Cholesteryl ester (CE) was determined by subtraction of the free cholesterol from total cholesterol (TC) levels.

Analysis of H₂O₂
H₂O₂ was analysed following the method of Peng et al. (Peng et al., 2006). Cultured monocytes were collected and diluted to 1.5x10⁷cells ml^–1 in PBS. After 1 h at 4°C, the cells were activated for 10 min at 37°C by addition of PMA (1 µg ml^–1). A 1 ml sample was removed from the cell suspension at 3 min intervals and filtered through a 0.22 µm syringe. Cell filtrates were collected at each time point during the reaction, and the H₂O₂ content of each filtrate was monitored as follows. 2.6 ml Tris-HCl buffer solution (pH 7.4, 0.1 mol l^–1), 0.1 ml 2.0 U ml^–1 horseradish peroxidase (HRP) solution and 0.1 ml cell filtrate were added to 0.2 ml p-hydroxyphenylacetic acid solution (pH 7.5). After 1 min, fluorescence was measured spectrofluorometrically (excitation wavelength, 325 nm; emission wavelength, 415 nm).

Detection of acetylase, deacetylase and ox-LDL by ELISA
An enzyme-linked immunosorbent assay (ELISA) was carried out on all samples. Briefly, flat-bottomed 96-well microtiter plates were coated with 100 µl of 0.06 mol l^–1 sodium carbonate buffer (pH 9.6) containing 0.5 µg antigen ml^–1 at 4°C. Plates were blocked with 150 µl of PBS-Tween containing 1% BSA (Hangzhou Sijiqing Biological Engineering Material Co., Ltd, Huanzhou, China) at 20°C for 1 h and were washed five times with PBS-Tween. Based on the affinity titration curve for protein-G, a cell dilution of 1:600 was chosen. After diluting the samples in PBS-Tween, 100 µl of sample were added to the plates, which were incubated at 20°C for 1 h. Biotinylated acetylase (HAT), histone deacetylase (HDAC) and ox-LDL (Jackson Immunoresearch, West Grove, PA, USA) diluted 1:15000 in PBS-Tween were individually added (100 µl/well) and incubated at 20°C for 1 h. Streptavidin–peroxidase (Sigma-Aldrich) diluted 1:10000 in PBS-Tween was added and incubation continued at 20°C for 30 min. As the substrate, 50 µl of O-phenylenediamine (OPD) diluted in citric acid phosphate buffer (pH 5.0; 0.66 mg ml^–1) + 0.6 µl 30% H₂O₂ mg^–1 OPD was added and the mixture incubated in the dark at 20°C for 10 min. The enzyme reaction was stopped by adding 50 µl of 2 mol l^–1 H₂SO₄. The plates were read in a spectrophotometer at 492 nm.

Real-time reverse transcription-polymerase chain reaction
Total RNA was isolated using Trizol reagent (Invitrogen, Grand Island, NY, USA). The primer nucleotide sequences and probes are shown in Table 1. RNA was reverse transcribed by the Revert Aid first strand cDNA synthesis kit (MBI, Vilnius, Lithuania). Real-time PCR was performed using an FTC2000 real-time PCR detection system (Funglyn, Toronto, Canada) with the program as follows: 40 cycles at 95°C for 45 s, the annealing temperature (see Table 1) for 45 s and 60°C for 60 s. The melting curve analysis over the range 55° to 95° was performed by monitoring 6-FAM fluorescence as the temperature increased (in 0.5°C increments at 10 s intervals). The RNA level of each gene was acquired from the value of the threshold cycle (Ct) of the real-time PCR as related to that of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) through Eqn 1:

(1)

The final results, expressed as N-fold differences in the target gene expression and relative to the calibrator termed `N_target', were determined:

(2)

where Ct values of the calibrator and sample were determined by subtracting the Ct value of the target gene from the Ct value.

Western blot analysis
Electrophoresis was carried out on sodium dodecyl sulfate–polyacrylamide gels. Proteins were transferred to nitrocellulose membranes at 67 V for 2 h at room temperature with gentle agitation on a platform shaker, and were washed three times for 5 min in Tris-buffered saline plus Tween-20 (TBST). The membrane was incubated with a monoclonal anti-DNAmethyltransferase (DNMT)1, DNMT3, methyl-CpG (MeCP) binding domain (MBD), MeCP and extracellular superoxide dismutase (EC-SOD) antibody (1:250 dilution) (Jackson ImmunoResearch, West Grove, PA, USA) in 10 ml primary antibody dilution buffer with gentle agitation overnight at 4°C. The membrane was then washed three times with TBST and incubated with a second antibody (goat anti-rabbit horseradish peroxidase-conjugated immunoglobulin G; Jackson ImmunoResearch) in PSB at 1:2000 dilution containing 1% bovine serum albumin (New England Biolabs, Beverly, USA) for 1 h at room temperature. After washing again three times with TBST, the membrane was incubated with 10 ml LumiGLO (New England Biolabs, Beijing, China) with gentle agitation for 1 min at room temperature, then the excess developing solution was drained. The membrane could not be dried out, but instead was wrapped in a plastic wrap and exposed to X-ray film. The control value was taken as 100%. Values are reported relative to that of β-actin using the formula (relative value=experimental densitometry valuex100/β-actin value).

Sodium bisulfite-sequencing assay
A standard curve was generated by preparing different target sequence ratios of methylated versus unmethylated alleles, as described previously (Pang et al., 2000). The bisulfite modification of gDNA has been described previously (Reeves et al., 2002). The genomic DNA was isolated from the cultured cells by using the E.Z.N.A Tissue DNA Kit (Omega Bio-tek, Guanzhou, China). The bisulfite modification was performed with the EZ DNA Methylation Kit (Zymo Research, Orange, CA, USA) following the manufacturer's instructions. Afterwards, DNA was precipitated with glycogen as carrier and resuspended in 20 µl of water.

A novel quantitative analysis of methylated alleles, which is essentially a major improvement over a previous method based on real-time PCR (MethyLight), was used (Peng et al., 2006). We used a VIC-labeled probe that specifically hybridizes to the sequence derived from the methylated allele, together with a FAM-labeled probe that binds to the sequence generated from the unmethylated allele. The amount of the fluorescent dye released during PCR is measured by a real-time PCR system and is directly proportional to the amount of PCR product. Binding sites of the probes covered three differently methylated CpG dinucleotides. The improved sequence specificity facilitates the relative quantification of the methylated and the unmethylated alleles that were simultaneously amplified in a single tube.

PCR primers were designed to amplify the bisulfite-converted antisense strand of the EC-SOD. The PCR primers, probes and stategy for designing the MethyLight reaction are shown in Fig. 1. PCR was carried out by using a 96-well optical tray with caps at a final reaction volume of 20 µl containing 10 µl TaqMan universal PCR master mix, 2 µl bisulfite-treated DNA, 2.5 µmol l^–1 of each primer of EC-SOD, 150 nmol l^–1 of each of the fluorescently labeled probes, EC-SODmet and EC-SODunmet. The initial denaturation at 95°C for 5 min to activate the AmpliTaq Gold DNA polymerase was followed by 40 cycles of the denaturation at 95°C for 15 s and the annealing and extension at 60°C for 1 min (Zeschnigk et al., 2004).

View larger version (23K): [in this window] [in a new window]   Fig. 1. Sequences of EC-SOD primers and probes. The probes for modified EC-SOD are indicated by shaded boxes, and the polymerase chain reaction (PCR) primers for the EC-SOD sequence are indicated by arrows and bold, italic type. The sequence of EC-SOD after sodium bisulfite modification is also shown. The solid line arrows indicate the extension direction. Methylated and unmethylated PCR have the same primers. Fluorescence detection was carried out by two passages. When PCR was carried out with methylation-modified gDNA, the fluorescence of the methylated probe was detected by the one passage, similar to PCR with unmethylation-modified gDNA. The other kind of fluorescence of unmethylated probes was detected by the other passages. Upper row, original sequence; lower row, bisulfite modified sequence (for display, assume all CpG sites are methylated); +, CpG site; non CpG `C' converted to `T'; >>>, left primer; <<<, right primer.

(3)

where FAM (495 nm) is arboxyfluoroscein, succinimidyl ester and HEX (535 nm) is 5-hexachloro-fluoroscein. To account for the differential efficacy of the PCR (methylated/unmethylated) and the activity of the probes, we restate the model as:

(4)

where a and b represent the additional effects. The following equation was deduced from the results generated by the standard curve, where (a=2, b=0.68):

(5)

Each MethyLight reaction was performed 3–6 times, and data are reported as the mean percent of the methylated or the unmethylated reference value within the three measurements.

Chromatin immunoprecipitation
Chromatin immunoprecipitation (ChIP) was performed according to published protocol (Umlauf et al., 2004) with modifications. In brief, the foam cells were collected and washed twice with Hank's solution. The cells were rinsed with ice-cold Hank's solution and incubated on ice for 10 min in 4 ml buffer. Nuclei were pelleted by centrifugation at 14 000 g for 15 min at 4°C, resuspended in 1% SDS, 50 mmol l^–1 Tris-HCl and 10 mmol l^–1 EDTA with the fresh protease inhibitor cocktail, and incubated on ice for 10 min. Chromatin was sonicated (Sonic Dismembrator 550, Fisher Scientific Microtip, Pittsburgh, PA, USA) to an average length of 500 bp. To preclear the chromatin, the sonicated cell suspension was diluted tenfold with a buffer containing 0.01% SDS, 1.1% Triton X-100, 1.2 mmol l^–1 EDTA, 16.7 mmol l^–1 Tris-HCl, pH 8.1, and 167 mmol l^–1 NaCl, and incubated with 80 µl salmon sperm DNA or protein A Agarose and 50% Slurry (Upstate Biotechnology Inc, Charlottesville, VA, USA) for 3 h with rotation at 4°C. One-third of the precleared chromatin was incubated with 2 µg of one of the following polyclonal antibodies overnight: anti-H3Ac, anti-H4Ac (Upstate Biotechnology Inc, Charlottesville, VA, USA), and control antibodies (Santa Cruz Biotechnology, Inc. Santa Cruz, CA, USA). One-tenth of the precleared chromatin was saved as the spare for the following experiment. Each immunoprecipitation was recovered, washed and eluted from the beads. ChIP DNA pellets were resuspended in 60 µl of Triton-EDTA (TE) and analyzed by SDS-PAGE.

Statistics
Results are expressed as mean ± s.e.m. The data were analyzed with one-way ANOVA and additional analysis was carried out with the Student–Newman–Keuls' test for multiple comparisons within treatment groups, or t-test between two groups. P<0.05 was considered significant.

	RESULTS

TOP Summary INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION References

 
The effect of homocysteine on cholesteryl ester accumulation in monocytes
We investigated the molecular mechanisms underlying the effect of Hcy on the accumulation of cholesteryl ester during the development of foam cells. To evaluate any possible cellular damage caused by Hcy, we first determined the percentage of foam cells. When the monocytes were grown to subconfluence and then co-cultured with ox-LDL and PMA along with 50, 100, 200 or 500 µmol l^–1 Hcy for 72 h, a great number of foam cells were found by oil red O staining (Fig. 2) and the percentage of foam cells in the positive group (cultured with ox-LDL and PMA but not Hcy) (46.20±5.80%) was much higher than the control group (without ox-LDL, PMA and Hcy) (4.80±1.50%) (P<0.05). The percentage of foam cells in 50, 100, 200 or 500 µmol l^–1 Hcy groups were much higher than that in the positive group, i.e. increased by about 1.25, 1.92, 1.52 or 1.42-fold, respectively (P<0.05 or P<0.01), thus suggesting that Hcy induced the formation of foam cells. The maximum level was at 100 µmol l^–1 Hcy, which is similar to the plasma level observed in hyperhomocysteinemia (Fig. 2A). When Hcy was cultured in control medium for an additional 24, 48 or 72 h, the percentage of foam cells increased to 66.2%, 80.2% and 92.5%, respectively, in comparison with the control group (Fig. 2B). The inhibitory effects of folate, AZC and TSA resulted in the percentage of foam cells decreasing to about 72.4%, 54.65% and 51.95% of the positive control group, respectively (Fig. 2C).

View larger version (22K): [in this window] [in a new window]   Fig. 2. Percentage of positive oil red O staining cells in groups of different concentrations of Hcy (50, 100, 200 and 500 µmol l–1; A), time of incubation (24, 48 and 72 h; B) and antagonist (C) group (values are means ± s.e.m., N=6). The percentage of positive oil red O-staining cells was evaluated by the semi-quantitative analysis; P<0.05, compared with control group; *P<0.05, **P<0.01, compared with the positive group.

The effect of Hcy on H₂O₂ and Ox-LDL in foam cells
It has been reported that oxidative damage of lipids and the consequent formation of foam cells is a key step in the onset and the development of atherosclerosis. H₂O₂ and ox-LDL were involved in the formation of foam cells (Peng et al., 2006). From the above description, we can see that the maximum effect was observed at 100 µmol l^–1 Hcy (Fig. 2A) so we added 100 µmol l^–1 Hcy to the monocyte culture with PMA and ox-LDL. We found that the quantities of H₂O₂ and ox-LDL in foam cells were significantly increased in the presence of 100 µmol l^–1 Hcy. After 72 h the increased levels reached 11.59- and 3.04-fold of the control group, respectively, and the quantity of H₂O₂ and ox-LDL increased in a time-dependent manner. We also examined the effect of folate, AZC and TSA on H₂O₂ and ox-LDL production in Hcy-treated monocytes. After incubation with folate, AZC and TSA, H₂O₂ and ox-LDL production was substantially inhibited (Fig. 3), suggesting that there are underlying relationships between H₂O₂, ox-LDL and the accumulation of cholesterol.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Levels of (A) H2O2 and (B) ox-LDL produced by activated monocytes. H2O2 was measured by fluorescence spectrofluorophotometry and the content of ox-LDL was detected by ELISA. See Materials and methods for details.

View larger version (19K): [in this window] [in a new window]   Fig. 4. Relative EC-SOD protein (A) and mRNA (B) levels. Monocytes were treated with 100 µmol l–1 Hcy in control medium for 24, 48 or 72 h. Total RNA was isolated and real-time PCR was carried out to detect mRNA levels. Protein levels of EC-SOD (A) were examined by western blotting (B). Values are means ± s.e.m. from 3 separate experiments with triplicated samples (N=6).*P<0.05, compared with positive group; P<0.05, compared with 100 µmol l–1 Hcy group (72 h).

Hcy selectively reduces DNMT1, DNMT3, MBD, MeCP2 mRNA and protein expression
We have previously reported that Hcy has an effect on the activity of DNA methyltransferases and CpG binding proteins in monocytes, and DNA methyltransferases and CpG binding proteins involved in DNA methylation (Jiang et al., 2007a; Jiang et al., 2007b). DNMT1, DNMT3, MBD and MeCP2 are key genes regulating the gene transition. To understand how Hcy affects the regulation of foam cells, we examined the mRNA and the protein expression of DNMT1, DNMT3, MBD and MeCP2 in monocytes cultured with folate, AZC and TSA. To characterize the time course of this effect, RNA and protein were harvested from foam cells treated with Hcy for 24, 48 and 72 h and analyzed by real time RT-PCR together with western blotting analysis (Fig. 5). Both the reduction in MeCP2 and the increase of DNMT1 started as early as 24 h. MeCP2 mRNA decreased to 20.9%, 43.87% and 41.23% of the control group levels after 24, 48 and 72 h. However, DNMT1 mRNA increased to 27.23%, 38.17% and 59.3% of the control group levels after 24, 48 and 72 h, respectively (Fig. 6C). Because DNMT3 and MBD remained unchanged, we treated monocytes with 100 µmol l^–1 Hcy and folate, AZC as well as TSA for 72 h, and then measured the mRNA expression of DNMT1, DNMT3, MBD and MeCP2. While folate, AZC and TSA affected the mRNA expression of DNMT1 and MeCP2, it had only minor effects on the mRNA level of DNMT3 and MBD (Fig. 5B).

View larger version (21K): [in this window] [in a new window]   Fig. 5. The relative mRNA levels of DNMT1, DNMT3, MBD2, MeCP2 in monocytes cultured for different times (A) and with different antagonists (B). Fluorescence was versus the PCR cycle number for both reactions and each sample dilubition is indicated. Each gene RNA level was acquired from the value of the threshold cycle (Ct) of the real-time PCR relative to that of GAPDH using Eqn 1. The final results, expressed as N-fold differences in target gene expression relative to the calibrator, termed `Ntarget', are determined as following: N=2Ct(sample)–Ct(calibrator) (Eqn 2), where Ct values of the calibrator and sample were determined by subtracting the Ct value of the target gene from the Ct value. Values are means ± s.e.m., representative of three separate experiments.

View larger version (38K): [in this window] [in a new window]   Fig. 6. Effects of Hcy on DNMT1, DNMT3, MBD2, MeCP2 protein levels in human monocytes incubated for different times (A) and with different antagonists (B). The monocytes were incubated in the absence or presence of Hcy at various concentrations and immunoblots (top) analyzed by densitometry (below). The results were expressed as percentage of control (100%) ± s.e.m., each performed in duplicate. Relative value was related to that of β-actin through the formula (relative value=experiment densitometry valuex100/β-actin value).

The effect of Hcy on the protein level of DNMT1, DNMT3, MBD2 and MeCP2 was measured by western blot (Fig. 6), with similar results to the mRNA expression of DNMT1, DNMT3, MBD2 and MeCP2. The lowest or highest protein expression was also in the 100 µmol l^–1 Hcy group, and there was a significant difference among DNMT1, DNMT3, MBD2 and MeCP2 protein levels after various incubation times with Hcy. Thus, Hcy selectively decreases DNMT1 and MeCP2 mRNA levels in monocytes in a time-dependent manner. Both the reduction in the expression of MeCP2 mediated by Hcy and the increase of DNMT1 were pathophysiologically relevant and independent. The effects of folate, AZC and TSA on the protein expression of DNMT1, MECP2, DNMT3 and MBD were consistent with the mRNA expression.

Change of homocysteine-induced DNA methylation of EC-SOD
To analyze the possible role of Hcy on levels of EC-SOD DNA methylation, we used a quantitative TaqMan-based real-time PCR. It is already known that AZC and TSA can induce general hypomethylation, so we cultured the primary monocytes in the presence of Hcy, folate, AZC and TSA. It was found that the homocysteine after 24, 48 and 72 h incubation led to significant increases in EC-SOD DNA methylation, by 85.55%, 135.28% and 145.9%, respectively (P<0.05). In the control group and the 100 µmol l^–1 Hcy group, when cultured with folate, AZC and TSA, the levels of DNA methylation of EC-SOD decreased by 27.9%, 28.8% and 39.5%, respectively (P<0.01) (Fig. 7). These findings suggested that there is a specificity of Hcy on DNA methylation in human monocytes.

View larger version (8K): [in this window] [in a new window]   Fig. 7. EC-SOD real-time PCR methylated levels (see Materials and methods) *P<0.05, **P<0.001, compared with positive group; P<0.05 compared with 100 µmol l–1 Hcy group (72 h).

The levels of acetylated histones H3 and H4 analysis and the effect of Hcy on HAT and HDAC
There is mounting evidence that mutant transcription factors, often resulting from chromosome translocations, contribute to the pathogenesis of corepressors and histone deacetylases (HDACs) and thereby result in altered chromatin architecture and modified gene expression. In this case, HDACs play a key role in the control of gene expression through chromatin modification.

We chose a strategy whereby histones were isolated from the nuclei of foam cells by acid extraction and detected by ChIP. As shown in Fig. 8, acetylated histones H3 and H4 in monocytes were treated with the 100 µmol l^–1 Hcy for varied time points. The levels of acetylated histone H3 and H4 were significantly elevated after incubation compared with the control group. The maximum level of acetylated histone H3 and H4 production was observed after 72 h incubation with 100 µmol l^–1 Hcy. These observations indicate that the relatively low concentration of Hcy, within the pathophysiological range, can directly trigger the increase of acetylated histones H3 and H4 in cultured monocytes. Treatment of cells with 100 µmol l^–1 Hcy induced a time-dependent accumulation of acetylated histones H3 and H4. But when treated with folate, AZC and TSA, the amount of acetylated histone H3 and H4 was decreased compared with levels in the 100 µmol l^–1 Hcy group (72 h).

View larger version (26K): [in this window] [in a new window]   Fig. 8. Relative levels of histone H3 (A) and H4 (B) determined by ChIP assay. The monocytes were treated with 100 µmol l–1 Hcy, folate, AZC and TSA for 24, 48 and 72 h. Nuclear extracts were immunoprecipitated with antibody against AcH3 and AcH4, and relative levels of histones H3 and H4 were analyzed by SDS–PAGE. *P<0.05, compared with positive group; P<0.05, compared with 100 µmol l–1 Hcy group (72 h).

HAT and HDAC are enzymes that catalyze the deacetylation and the acetylation of lysine residues located in the NH₂-terminal tails of histones and non-histone proteins. To determine whether Hcy is able to induce the secretion of HAT and HDAC in monocytes, we next examined whether the homocysteine-induced upregulation of HAT and HDAC expression resulted in an increase of acetylated histone H3 and H4. The amounts of HAT and HDAC secreted from the cultured monocytes were determined by ELISA, and HDAC was significantly decreased in foam cells pretreated with homocysteine in both a time- and dose-dependent manner (Fig. 9). However, HAT levels did not increase on treatment with folate, AZC and TSA, but the HDAC expression was consistent with the results of acetylated histone H3 and H4 (Fig. 9). These results suggested that homocysteine could sensitize acetylated histone H3 and H4 through the suppression of HDAC activity.

View larger version (7K): [in this window] [in a new window]   Fig. 9. Effect of Hcy on homocysteine (HAT) and histone deacetylase (HDAC) levels in cultured monocytes. Levels of HAT and HDAC in supernatant of cultured monocytes were measured by ELISA assays after incubation with 100 µmol l–1 Hcy for the indicated times. Data are expressed as mean ± s.e.m. and were representative of at least 6 independent experiments. *P<0.05 compared with corresponding untreated groups.

	DISCUSSION

TOP Summary INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION References

 
Hyperhomocysteinemia has been recognized as an independent risk factor that predicts adverse cardiovascular aspects of patients such as ischemic heart disease and stroke (Clarke et al., 1991). However, the underlying mechanisms of these unfavorable effects of homocysteine have not been elucidated. Why does Hcy become a pro-oxidant and an important risk factor for atherosclerosis? Since then many plausible explanations have not covered this key point, and the results of the present study demonstrate for the first time that Hcy not only inhibits the secretion of EC-SOD and accelerates the accumulation of cholesterol but also induces the gene promotor hypermethylation of EC-SOD and the acetylation of histone H3 and H4 in cultured human monocytes.

First, we tested whether H₂O₂ and ox-LDL act as mediators in the Hcy-induced expression of EC-SOD and found that Hcy elevated H₂O₂ and ox-LDL secretions, suggesting that H₂O₂ and ox-LDL, as strong oxidants, are important risk factors for atherosclerosis (Ross et al., 1999; Wentworth et al., 2003; Liu et al., 1996). Recently it has been reported that reactive cholesterol ozonized products trigger the formation of foam cells in tissue macrophages (Zelko and Folz, 2003). Our present study showed that PMA-activated monocytes, when cultured with ox-LDL, were obviously transformed into foam cells. But when monocytes were cultured with Hcy, the levels of H₂O₂ and ox-LDL were significantly increased, indicating that Hcy is involved in the formation of foam cells and the accumulation of cholesterol. In addition, there is evidence that in a mild homocysteine elevation, so-called hyperhomocysteinemia, H₂O₂ and ox-LDL internalize excessive amounts of lipids and become foam cells. Our study also showed that AZC and TSA could partially block the formation of Hcy-induced H₂O₂ and ox-LDL.

Second, we have distinguished the potential enzymatic sources of EC-SOD. From our results, we found that decreased EC-SOD levels relative to elevated homocysteine levels could be a response to homocysteine-induced oxidative damage and could thus constitute a protective mechanism, with the effect of countering oxidative stress. All of these results are consistent with the increased circulating EC-SOD levels associated with reduced cardiovascular risk (Ross, 1993). Based on these findings, the increased EC-SOD should be helpful in diminishing the risk of vascular damages.

Furthermore, although the molecular mechanisms underlying Hcy-induced atherosclerosis have been the subject of large-scale research, most previous studies have only focused on the influence of Hcy on endothelial cells (Benditt and Benditt, 1973). It is still not known whether DNA methylation of EC-SOD contributes to lesion formation or whether it occurs as a consequence of the pathological process. However, as it has been reported earlier (Turhan et al., 2005), our results indicate a causal relationship between the methylation and the EC-SOD expression. It is conceivable that hypermethylation has at least partly developed through the decreased cell proliferation, which is a typical feature in atherogenesis (Campbell et al., 1981). Similar to cancer cells, the EC-SOD methylation level leading to the formation of foam cells was high, so it is also possible that hypermethylation of genes is involved in the inhibition of cell growth. Preliminary results indicate that similar hypomethylation can be found in smooth muscle cells isolated from the intima of balloon-denudated arteries (Schwartz et al., 1995). These findings also support the hypothesis that DNA methylation could reflect a difference in gene expression and be the potential target of gene regulation (Willems et al., 1993).

Just as expected, the results of our study showed that the activity of DNMT1 increased and the expression levels of MeCP2 decreased in monocytes after incubation with Hcy. A potential plausible explanation was the compensatory reaction of the methylation mechanisms against Hcy-induced hypomethylation. It has been reported that methyltransferase activity in cancer tissue actually increased despite genome-wide hypomethylation (Haaf, 1995). Methyltransferase activity can be seen as a compensatory mechanism to maintain genomic methylation patterns, as only two rounds of replication are required for genomic hypomethylation if the activity of maintenance methylation provided by DNMT or other methyltransferases is not effective. The decline of S-adenosylmethionine could result from the excessive consumption due to the increased activity of DNMT (Lee et al., 1996).

Last but not least, it is now generally accepted that precise regulation of gene expression by epigenetic mechanisms is required to maintain the normal development of mammals. The epigenetic code is most likely to consist of both DNA methylation and histone modifications. But there still remains controversy on the role of the epigenetic code in the tissue-specific gene expression during the normal development of mammals, and the DNA methylation code has a critical effect that CpG methylation, which correlates with the suppression of cell transcription. In contrast, a variety of histone modifications, including acetylation and methylation at various lysine residues and other modifications, constitute the more complex histone code (Bonaldi et al., 2004). In general, acetylation of histone H3 and H4 correlates with gene activation, while deacetylation correlates with gene silencing (Zhang and Tang, 2003). Based on the results of our study, Hcy might operate with mechanisms that are involved in the upregulation of histone H3 and H4 in foam cells. Elucidation of the role of Hcy in the expression of EC-SOD in vivo, especially in the development of atherosclerotic plaque in patients with vascular disorder, could provide new insight into our understanding of hyperhomocysteine.

View larger version (4K): [in this window] [in a new window]   Fig. 10. Proposed mechanism of homocysteine-induced accumulation of cholesterol and the formation of foam cells.

LIST OF ABBREVIATIONS

5-mC

5-methylcytosine

AS

artherosclerosis

AZC

azacytidine

C-5MT-ase

C-5 DNA rnethyltransferase

CE

cholesteryl ester

ChIP

chromatin immunoprecipitation

Ct

threshold cycle

DNMT

DNA methyltransferase

EC-SOD

extracellular superoxide dismutase

ELISA

enzyme-linked immunosorbent assay

FC

free cholesterol

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

HAT

acetylase

Hcy

homocysteine

HDAC

histone deacetylase

HRP

horseradish peroxidase

MBD

methyl-CpG binding domain

MeCP2

binding of methyl CpG and the binding protein 2

OPD

O-phenylene diamine

ox-LDL

oxide-low-density lymphocyte

PCR

polymerase chain reaction

PMA

phorbol myristate acetate

SMC

smooth muscle cell

TBST

Tris-buffered saline plus Tween-20

TC

total cholesterol

TE

Triton-EDTA

TSA

trichostatin A

VIC (450nm)

victoria

	Acknowledgments

 
This project was supported by the project of science and technology research of Ningxia Higher Education (Registration No. 2007.292) and the project of focal point scientific research of Health Department of Ningxia Hui autonomous region in China (Registration No. W200720).

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