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. 2014 Jun 16:12:170.
doi: 10.1186/1479-5876-12-170.

Hypermethylation of DDAH2 promoter contributes to the dysfunction of endothelial progenitor cells in coronary artery disease patients

Pan-Pan Niu, Yu Cao, Ting Gong, Jin-Hui Guo, Bi-Kui Zhang  1 , Su-Jie Jia
Affiliations

Hypermethylation of DDAH2 promoter contributes to the dysfunction of endothelial progenitor cells in coronary artery disease patients

Pan-Pan Niu et al. J Transl Med. .

Abstract

Background: Circulating endothelial progenitor cells (EPCs) may be a biomarker for vascular function and cardiovascular risk in patients with coronary artery disease (CAD). Dimethylarginine dimethylaminohydrolase 2 (DDAH2) regulates the function of EPCs. This study aimed to examine whether hypermethylation of DDAH2 promoter contributes to impaired function of EPCs in CAD patients.

Methods: Peripheral blood mono-nuclear cells from 25 CAD patients and 15 healthy volunteers were collected and differentiated into EPCs. EPCs were tested for their adhesive capability. DDAH2 mRNA expression was analyzed by real-time PCR, and the methylation of DDAH2 promoter was detected by bisulfite genomic sequencing.

Results: DDAH2 promoter in EPCs from CAD patients was hypermethylated and the methylation level was negatively correlated to DDAH2 mRNA level and adhesion function of EPCs. Homocysteine impaired the adhesion function of EPCs, accompanied by lower DDAH2 expression and higher methylation level of DDAH2 promoter, compared to controls. These effects of homocysteine were reversed by pretreatment with Aza, an inhibitor of DNA methyltransferase.

Conclusion: Hypermethylation in DDAH2 promoter is positively correlated to the dysfunction of EPCs in CAD patients. Homocysteine disrupts EPCs function via inducing the hypermethylation of DDAH2 promoter, suggesting a key role of epigenetic mechanism in the progression of atherosclerosis.

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Figures

Figure 1
Figure 1
Characterization of isolated EPCs. A. Representative images of cultured EPCs on 7th day, 14th day, and 20th day (magnification 200×). B. Double-color fluorescent imaging indicated Dil-acetylated LDL incorporation and FITC lectin binding by EPCs. The nuclei was stained by DAPI (magnification 200×). C. FACS showed the expression of stem cell marker CD45, VEGFR-2, CD34, and CD133 in EPCs at 14th day.
Figure 2
Figure 2
The adhesion ability of EPCs is lower in CAD patients than in controls. A. EPCs were isolated from CAD patients and controls and cultured for 14 days. A. Representative images of nuclear staining of adherent EPCs. B. Quantitative analysis of the number of adherent EPCs. *P < 0.05 vs. control group.
Figure 3
Figure 3
Hypermethylation of DDAH2 promoter and reduced DDAH2 expression in EPCs from CAD patients. A. The mean methylation status for each of the 12 CpG sites within DDAH2 promoter in EPCs from controls (n = 10). B. The mean methylation status for each of the 12 CpG sites within DDAH2 promoter in EPCs from CAD patients (n = 10). 1 represented fully methylated, 0 represented completely demethylated. C. The combined average methylation status of 7 CG pairs (positions +3144, +3162, +3170, +3200, +3229, +3261, and +3265) in DDAH2 intron 4 in EPCs was higher in CAD patients than in controls. *P < 0.05 vs. control group. D. The mRNA expression level of DDAH2 was lower in EPCs from CAD patients. **P < 0.01 vs. control group.
Figure 4
Figure 4
The adhesion ability of EPCs is negatively correlated with DDAH2 promoter methylation level in CAD patients (r = 0.730, P = 0.001).
Figure 5
Figure 5
Hcy induces the hypermethylation of DDAH2 promoter in EPCs. A. The mean methylation status for each of the 12 CpG sites within DDAH2 promoter in EPCs untreated. B. The mean methylation status for each of the 12 CpG sites within DDAH2 promoter in EPCs treated with 1 mM Hcy. C. The mean methylation status for each of the 12 CpG sites within DDAH2 promoter in EPCs treated with 1 mM Hcy and 5 μM 5-Aza. D. Relative mRNA expression of DDAH2 in EPCs of different groups. Values were expressed as means ± SD (n = 3). *P < 0.05, **P < 0.01 vs. Control; #P < 0.05 vs. 1 mM Hcy treated group.
Figure 6
Figure 6
Hcy decreases the adhesion ability of EPCs. A. Representative images of nuclear staining of adherent EPCs in control, 1 mM Hcy treated, and 1 mM Hcy plus 5 μM 5-Aza treated groups. B. Quantitative analysis of the number of adherent EPCs in each group. *P < 0.05 vs. control group; #P < 0.05 vs.1 mM Hcy treated group.
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