Objective: To investigate the aberrant DNA methylation in promoter region of estrogen receptor alpha (ERalpha) in atherosclerosis (As) and the possible involvement of homocysteine (Hcy) in its pathogenesis.
Methods: The blood samples were collected from 54 patients with As approved by carotid colorized ultrasound examination and 28 healthy control subjects. The methylation status of CpG islands in ER-alpha gene promoter region of genome DNA was analyzed by nested-methylation-specific PCR (nMSP). tHcy was examined by fluorescent-biochemical method. Spearman rank correlation was used to analyse the relationship between the degree of methylation in ER-alpha gene and the level of tHcy. Cultured smooth muscle cells of Homo sapiens were treated by Hcy with different concentrations and different treating time, again the DNA methylation status was assayed by nMSP, and the proliferation of SMC was assayed by MTT.
Results: Hypermethylation of ER-alpha gene promoter region was found in 38 cases of atherosclerosis patients, and the methylation frequency was 70.4%. While in healthy controls, just 8 of 28 samples hypermethylation was found, only 28.6% methylation frequency was detected, much lower than the one in atherosclerosis group (p<0.05). Meanwhile, the level of tHcy in atherosclerosis group was significantly higher than that in control group (p<0.05). The spearman rank correlation analysis explored an obvious correlation between the degree of methylation in ER-alpha gene and the level of tHcy (r=0.809, p<0.05), and the severity of atherosclerotic lesion was also heightened along with the increment of plasma level of tHcy. The cultured SMCs treated by Hcy resulted in de novo methylation in promoter region of ERalpha gene with a concentration and treating time-dependent manner, and a dose-dependent promoting effect on SMC proliferation. The in vivo and in vitro data coincidently showed that the Hcy could promote the hypermethylation of ERalpha gene, which may be an important mechanism for the pathogenesis of As.
Conclusion: Hypermethylation of CpG islands in ER-alpha gene promoter region was found in much higher frequency in atherosclerosis patients, which is positively correlated with the increased level of plasma tHcy and the severity of atherosclerotic lesion, and the in vitro experimental results further extended above clinical data that HHcy can lead to the hypermethylation of ER-alpha gene, and hence to promote the occurrence and development of As.