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The endothelial dysfunction characterized by impaired nitric oxide (NO)-mediated and endothelium- dependent relaxation has been proved to play an essential role in initiation and progression of atherosclerosis. The another independent risk factor for atherosclerosis is hyperhomocysteinemia (HHcy),1 even moderate HHcy (plasma total homocysteine (Hcy) between 15 and 50 µmol/L) was found to be associated with an increased risk of cardiovascular disease.2 The increased level of Hcy was generally considered to impair the bioavailability of nitric oxide due to Hcy-induced oxidative stress. Hcy is an intermediate product in the transmethylation reactions including DNA methylation modification,3 which is deeply involved in the epigenetic regulation of gene expression. S-adenosylmethionine (SAM), which serves as the methyl donor for more than 100 different transmethylation reactions, is synthesized from methionine. A major product of SAM-dependent methyl transfer reactions is S-adenosylhomocysteine (SAH), which can undergo hydrolysis to form Hcy. Hcy can be remethylated to methionine. Therefore, an increased level of Hcy may interfere with DNA methylation modification and gene expression through this methionine cycle.
Recent studies have shown that aberrant DNA methylation was involved in the Hcy-related pathology.4-6 This mechanism may play a role in the dysfunction of endothelial NO system induced by Hcy in addition to oxidative stress. However, the survey on the promoter region of endothelial NO synthase (eNOS) gene shows that the promoter region of eNOS gene is lack of CpG island, which suggests that eNOS gene may not directly response to methylation modification. But an alternative approach in understanding the Hcy-related dysfunction of eNOS may be the DDAH/ADMA pathway. Asymmetric dimethylarginine (ADMA) is an endogenous NOS inhibitor. Elevation of circulating ADMA level has been shown to involve in the endothelial dysfunction in atherosclerosis, and the decreased activity of dimethylarginine dimethylaminohydrolase (DDAH, the key enzyme for removal of ADMA) is the major contributor for increase of ADMA.7-11 The promoter region of DDAH gene is found of high GC content, which was believed to be a structure feature prone to de novo methylation modification. Therefore, we postulated that HHcy-associated dysfunction of endothelial NO system may initiate from the aberrant methylation modification of DDAH gene. Two isoforms of DDAH have been identified: DDAH1 and DDAH2. DDAH1 is typically found in tissues of expressing neuronal NOS, whereas DDAH2 predominates in tissues of expressing eNOS.12 So we chose the DDAH2 as the target gene.
METHODS
Cell culture
The human umbilical cord was obtained from the Second Hospital Affiliated to West China University of Medical Sciences in Sichuan University. The umbilical vein was flushed with 30 ml of warm Dulbecco's phosphate buffered saline (PBS) and human umbilical vein endothelial cells (HUVECs) were isolated by enzymatic digestion with type Ⅱcollagenase (1 mg/ml). After 15 minutes incubation at 37°C, umbilical cord vein segments were perfused with 20 ml medium 199 (Gibco/Invitrogen, USA) containing L-glutamine (2 mmol/L), antibiotics, and 20% fetal bovine serum (FBS, Sijiqing, China). After the endothelial cell suspension was centrifuged at 1000 rpm for 6 minutes, the cell pellet was resuspended in 5 ml previous medium. This suspension was seeded into a 25-cm2 gelatin-coated culture flask and cultured at 37°C, 5% CO2 conditions. Confluent primary HUVECs were detached by 0.25% trypsin (Sigma, USA), and the third passages were used in experiment. Characterization of HUVECs was identified by von Willebrand factor staining. All experiments were performed on confluent monolayers of endothelial cells. HUVECs were serum-starved in M199 containing no FBS for 8 hours before treatment to avoid the confounding effects of serum. Cells were washed with Hank's balanced salt solution and cultured in medium containing 5% FBS in the absence or presence of Hcy (Sigma, USA) for 72 hours. Cells and medium were harvested to measure the activity and mRNA expression of DDAH and eNOS, the promoter CpG island methylation of DDAH2 gene, ADMA concentration, and NO content respectively. All measurements were performed in triplicate.
DDAH and NOS activity assay
The activity of DDAH in HUVECs was assayed by directly measuring the amount of ADMA metabolized by the enzyme.13 One unit of the enzyme was defined as the amount that catalyzed the formation of 1 mmol/L L-citrulline from ADMA per minute at 37ºC.
The cells were sonicated after resuspending in 1 ml sodium phosphate buffer (0.1 mmol/L, pH 6.5). The lysates were used to assay NOS activity by the conversion of L-arginine to NO with a commercial kit according to the manufacturer's instruction (Jiancheng, Nanjing, China).
Determination of ADMA concentration
Cell medium of 1 ml was pipetted into a tube containing 5-sulfosalicylic acid, and the mixture stored at 4°C for 10 minutes. The precipitated protein was removed by centrifugation at 2500 g for 15 minutes (4°C) and the supernatant was used for measurement of ADMA concentration by high-performance liquid chromatography (HPLC).14
Measurement of NO content
Levels of nitrite/nitrate, the stable end products of NO, were measured using the commercial kit (Jiancheng). Briefly, nitrate was converted to nitrite with nitrite reductase, and the total nitrite was measured with the Griess reagent. The absorbance was determined at 550 nm with a spectrophotometer.
Determinations of eNOS and DDAH2 mRNA expression
Total RNA of HUVECs was isolated using Trizol isolation reagent (Invitrogen Gaithersburg, USA) after cells incubated with Hcy for 72 hours. Values of A260 and A280 were measured and A260/A280 ratio of all samples was over 1.8. one µg total RNA was reversely transcribed into cDNA respectively. That is, 1 µg total RNA was added oligo (dT) and M-MLV (Promega Shanghai, China), and the mixture was incubated at 37°C for 1 hour followed by 15 minutes incubation at 70°C. The total reaction volume was 25 µl for PCR reaction. The oligonucleotide primers were designed on the basis of the nucleotide sequence in GenBank (L26914 for eNOS and BC001435 for DDAH2). Primer sequences and PCR reaction conditions are shown in Table 1. The PCR-amplified products were electrophoresed on 1.0% agarose gel and visualized with ethidium bromide staining. The mRNA expression of eNOS and DDAH2 were standardized with the corresponding β-actin (GenBank NM 001101) as an internal control respectively. Relative intensity of band was analyzed by BIO-RAD software.
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Table 1. Primer sequences and PCR reaction conditions |
Nested methylation-specific PCR analysis
Total genomic DNA was extracted by DNA isolation kit (TianGen Beijing, China) according to the manufacturer's instruction. The final preparations had a ratio of A260: A280 between 1.6 and 1.8. Two µg DNA from each sample was treated with sodium bisulfite (Sigma, USA) at 50°C for 16 hours. Nested methylation-specific PCR (nMSP) was used for the detection of methylation pattern in promoter region CpG island of DDAH2 gene (GenBank AF129756). This method is based on the fact that treatment of denatured DNA with sodium bisulfite converts all cytosine residues to uracil, which are then amplified as thymines in the PCR reactions. In contrast, 5-methylcytosine is resistant to bisulfite deamination under the reaction conditions and is amplified as cytosine. nMSP consists of two-step PCR amplifications. The first step of nMSP uses an outer primer pair set that does not contain any CpG. The second step PCR was carried out with the conventional PCR primers. Primer sequences and nMSP reaction conditions are shown in Table 2.
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Table 2. Primers sequences and nMSP reaction conditions |
To reduce misprinting and to increase efficiency, touchdown (TD) PCR was used in the amplification. Following hot start, samples were subjected to 20 cycles in a TD program (94ºC for 45 seconds, annealing temperature for 45 seconds, and 72ºC for 45 seconds for 20 cycles, followed by a 1ºC decrease of the annealing temperature every second cycle). After completion of the TD program, 20 cycles were subsequently run (94ºC for 45 seconds, 45ºC for 45 seconds, and 72ºC for 45 seconds), ending with a 5 minutes extension at 72ºC. The PCR products from the second PCR reaction were electrophoresed on 1.0% agarose gel and visualized with ethidium bromide staining.
Statistical analysis
Results are expressed as mean ± standard deviation (SD). Data were analyzed using SPSS 11.0 for Windows (SPSS Inc, Chicago, USA). Comparisons between 2 groups were evaluated using the Student t test. Multiple comparisons were performed using ANOVA. A value of P<0.05 was considered statistically significant.
RESULTS
Effect of homocysteine on DDAH activity
The results showed the dose-dependent inhibitive effect of Hcy on DDAH activity. Ten µmol/L Hcy had no significant effect on DDAH enzyme activity in HUVECs, while 30, 100, and 300 µmol/L Hcy obviously inhibited DDAH activity compared with control group (0.65±0.09, 0.57±0.09, 0.30±0.05, 0.11±0.07 vs 0.69±0.08 respectively; Fig. 1).
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Fig. 1. Effect of homocysteine (Hcy) on DDAH activity in HUVECs. HUVECs were cultured in medium containing Hcy (0, 10, 30, 100, and 300 µmol/L) for 72 hours. DDAH enzyme activity (U/mgprot) in cell lysates was determined by measurements of L-citrulline formation. *P<0.05, **P<0.01 vs no Hcy. |
Effect of Hcy on ADMA concentration
Since DDAH is the key enzyme for metabolism of endogenous NOS inhibitor ADMA, the inhibition of DDAH activity may result in an increased accumulation of ADMA. Our study showed that Hcy dose-dependently increased ADMA levels in the culture medium of HUVECs compared with control group (0.63 ± 0.09, 0.82 ± 0.09, 1.24 ± 0.10 and 1.48 ± 0.07 vs 0.55 ± 0.08 respectively; Fig. 2).
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Fig. 2. Effect of homocysteine (Hcy) on ADMA accumulation in HUVECs. HUVECs were exposed to Hcy (0, 10, 30, 100, and 300 µmol/L) for 72 hours. ADMA concentration in the medium was measured by HPLC. Values are expressed as µmol/L ADMA per 106 cells. *P<0.05, **P<0.01 vs no Hcy. |
Effects of Hcy on eNOS activity and NO content
Treatment of HUVECs with Hcy for 72 hours inhibited the eNOS activity and the production of NO (Table 3), which showed a similar dose-dependent changing pattern with DDAH and ADMA.
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Table 3. Effect of homocysteine (Hcy) on NOS activity and NO content in HUVECs |
Effects of Hcy on DDAH2 and eNOS mRNA expression
After 72 hours treatment of HUVECs with Hcy, RT-PCR showed a decreased DDAH mRNA expression, which was statistically significant in 300 µmol/L Hcy group compared with no Hcy (P<0.05, Fig. 3).
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Fig. 3. The mRNA expression of DDAH2 in HUVECs cultured with homocysteine (Hcy) for 72 hours. Hcy of 10 and 30 µmol/L slightly increased DDAH mRNA expression, whereas 100 and 300 µmol/L Hcy obviously decreased DDAH mRNA expression. lane M: DL2000; lane 1: 0; lane 2: 10 μmol/L; Lane 3: 30 μmol/L; lane 4: 100 μmol/L; lane 5: 300 μmol/L. *P<0.05 compared with no Hcy. |
RT-PCR also showed a decreased eNOS mRNA expression by the treatment of Hcy, which was statistically significant in 100 and 300 µmol/L Hcy groups compared with no Hcy (P<0.05, Fig. 4).
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Fig. 4. The mRNA expression of eNOS in HUVECs cultured with homocysteine (Hcy) for 72 hours. Hcy decreased eNOS mRNA expression in HUVECs and was obvious in 100 and 300μmol/L groups. Lane M: DL2000; lane 1: 0; lane 2: 10 μmol/L; lane 3: 30 μmol/L; lane 4: 100 μmol/L; lane 5: 300 μmol/L. *P<0.05 compared with no Hcy. |
Effect of Hcy on methylation pattern in promoter sequence of DDAH2 gene
The CpG dinucleotides in promoter sequence of DDAH2 gene were partial methylated and partial unmethylated under normal condition in cultured HUVECs. Treatment of Hcy altered this methylation pattern. Low concentration of Hcy (10 and 30 µmol/L) induced a demethylation modification, which showed a hypomethylation pattern compared with no Hcy treatment, while high concentration of Hcy (100 and 300 µmol/L) induced de novo methylation, i.e. hypermethylation in the promoter CpG dinucleotides of DDAH2 gene (Fig. 5). Since the hypermethylation in the promoter of gene was believed to be gene-silencing, this result therefore strongly suggested a causative mechanism of Hcy-induced hypermethylation of DDAH2 gene for the inhibition of DDAH2 mRNA transcription shown in Fig. 4.
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Fig. 5. The methylation status of the promoter CpG island of the HUVECs DDAH2 gene was determined by nMSP. Homocysteine (Hcy) treatment for 72 hours altered the methylation pattern of the promoter CpG island of DDAH gene. Ten and 30 µmol/L Hcy induced a partial demethylation (hypomethylation), while 100 and 300 µmol/L Hcy induced de novo methylation (hypermethylation) in the promoter CpG island of DDAH2 (M: methylated; U: unmethylated). |
DISCUSSION
Accumulating evidences over the past decades have shown that HHcy as an independent risk factor for atherosclerosis could impair NO production and bioavailability in endothelial cells,15-17 but the explanation for the injurious mechanism is biologically plausible. Hcy could promote oxidant injury to vascular cells, particularly to the endothelium and eNOS through the auto-oxidative reaction of Hcy, which has been suggested as an essential mechanism for Hcy-induced dysfunction and impairment of endothelium. But cysteine (similar chemical structure with just one less methyl residue) level in normal plasma exceeds that of Hcy by 20–30 fold, and it undergoes similar oxidative chemistry, cysteine is usually not considered as a risk factor for cardiovascular disease.18 Until now the Hcy-cysteine puzzle has not been convincingly and evidentially explained.
Hcy is an important metabolic byproduct in the methionine cycle19 and involves in the DNA methylation modification, but cysteine does not. This difference may be an intrinsic trait for their distinct effect on pathogenesis of atherosclerosis. Hcy can alter DNA methylation pattern and gene expression, but cysteine cannot. So we investigate the possible pathway of NO system impairment in endothelial cells by Hcy-induced aberrant DNA methylation modification. Since the promoter region of eNOS gene is lack of CpG dinucleotides pairs, which suggests that Hcy may not be able to directly alter the transcription of eNOS gene through the methylation modification in its promoter region. But the impairment of DDAH/ADMA/NOS/NO pathway is an important known mechanism for dysfunction of NO system, which may be an alternative approach for impairment of NO system through the methylation modification.
Our results demonstrated that Hcy in low concentration (10 and 30 µmol/L) induced hypomethylation, whereas high concentration of Hcy (100 and 300 µmol/L) induced hypermethylation in the promoter region CpG island of DDAH2 gene. The transcription of DDAH2 mRNA was correspondingly upregulated in low concentration of Hcy that induced hypomethylation, and downregulated in high concentration of Hcy that induced hypermethylation. DDAH2 activity in HUVECs decreased along with the step up of Hcy concentration, and relevant elevation of ADMA level was observed. Meanwhile, eNOS activity and NO contents showed corresponding decrease. The alterations in DDAH/ADMA/NOS/NO pathway showed highly correlative, which indicated that the pathogenic process of dysfunction of endothelial NO system induced by HHcy could partially originate from Hcy-caused aberrant methylation modification in DDAH2 gene.
DNA methylation has emerged as an important epigenetic modification that regulates the expression and function of the genes without altering the primary sequence of the DNA in the mammalian genome. In general, DNA methylation represses transcription, and loss of methylation is associated with gene activation. Increasing evidence indicates that human diseases, including cancer20 and atherosclerosis, are either caused or impacted by aberrant methylation. That is, DNA methylation may represent an important process whereby atherosclerosis risk factors affect the function of the genome, and induce atherosclerotic impairment. Recent studies have demonstrated atherosclerosis-related methylation changes of specific genes, including estrogen receptor-α (ERα),21 15-Lipoxygenase (15-LO),22 p53,23 apolipoprotein B,24 etc.25,26 To further explore the mechanisms responsible for dysfunction of eNOS induced by HHcy, we added a novel gene involved in the aberrant methylation modification induced by Hcy- DDAH2, which may be an essential mechanism for understanding the Hcy-cysteine puzzle. In addition, our results also demonstrated the DDAH/ADMA/NOS/NO pathway for Hcy-related dysfunction of NO system.
In summary, the present study demonstrated that the dysfunction of NO system in HHcy may partially originate from the aberrant methylation modification of DDAH2 gene besides oxidative stress injury, and the impairment of DDAH2 resulted in an elevation of ADMA, which competitively inhibited eNOS activity and decreased the production of NO. This process may be an important pathway for dysfunction of endothelial NO system and pathogenesis of atherosclerosis. Our results gained further insight into the mechanism underlying the HHcy-induced endothelial dysfunction, and unveiled a reasonable explanation for Hcy-cysteine puzzle. That is, HHcy may interfere with the methylation modification of DDAH2 gene, and indirectly impaired the function of NO system through the DDAH/ADMA/NOS/NO pathway.
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