| Background and objective:Cerebrovascular disease(CVD)is the first leading cause of death and most frequent cause of long-term disability in urban and rural population in China.And cerebral infarction(CI)is the most common subtype,which contributes to 60-80%of CVD.It is a complex disease which involves interactions between environmental and genetic factors.Epigenetics is the bridge connecting environmental and genetic factors.DNA methylation is considered to be a major type of epigenetic regulation that generally silences gene expression and affects disease.Elevation of plasma homocysteine level is an independent risk factor for cerebrovascular disease and has also been reported to induce modulation of gene expression through alteration of the methylation status by metabolic interfering with the level of S-adenosyl methionine(SAM,donor of methyl group).Thrombomodulin(TM)is a kind of membrane glycoprotein expressed on the surface of vein endothelial cells.It functions as a kind of receptor of thrombin and plays an important role in anti-coagulation,anti-inflammation and promoting fibrinolysis.Thus,TM is important in preventing the occurrence of Cl.It has been reported that the hypermethylation of plasma TM gene promoter in patients with coronary heart disease(CHD)may correlate with the genesis and development of CHD.Till now,no articles have shown whether the methylation of circulating TM gene promoter correlates with the genesis and development of CI.This study was designed to investigate the alteration of methylation status of plasma TM gene promoter in CI patients,and to discuss the relationship among plasma Hcy,the methylation status and the expression of mRNA of circulating TM gene,as well as finding the epigenetic mechanisms of hyperhomocysteinemia-induced CI,so as to provide a new theoretical basis for the prevention and treatment of CI.Methods:120 CI patients and 80 controls were enrolled in this case-control study.All of CI patients enrolled from the First Affiliated Hospital of Baotou Medical College in China during the period of May 2013 to November 2014,who accorded with cerebrovascular disease diagnosis standard of the 4th cerebrovascular disease meeting in 1995,were confirmed as new cerebral infarction by MRI or CT.All of control individuals were recruited from healthy persons and patients of benign paroxysmal positional vertigo and migraine in the same period,with no evidence of cerebrovascular disease in history and physical examination,and/or with the results of head CT/MRI normal.The clinical data,risk factors and the severity of neurological impairment assessed by National Institutes of Health Stroke Scale(NIHSS)were recorded within 24 hours of admission.All of CI patients received head magnetic resonance imaging and the size of infarction was recorded.Cubital vein samples of all participants were taken for extracting DNA and RNA in peripheral blood mononuclear cells.The methylation status of circulating TM gene promoter were analyzed by nested-methylation-specific polymerase chain reaction(nMSP).The mRNA expressions of circulating TM gene were detected by real-time PCR(RT-PCR).Plasma concentrations of soluble TM(sTM)were quantified using a Human TM ELISA kit of Chinese Diagnostica.The plasma Hcy concentration was determined on an automatic biochemical analyzer.The data were analyzed with the SPSS software package(version 17.0 for Windows).Results:(1)The cases and controls were selected to be age-and sex-comparable,while these two factors were no longer significantly different.There were significant differences in cholesterol,the prevalence of hypertension,diabetes,smoking and drinking between CI patients and controls.There were no significant differences in triglyceride between two groups.(2)There were three forms in TM gene methylation status:unmethylation,hemimethylation and full methylation.Methylation was detected in 89(74.2%)of the 120 CI specimens,and detected in 38(47.5%)of the 80 control samples.The results demonstrated that cases had a higher TM methylation level than controls(X~2 =14.724,p= 0.00).According to the size of infarction,CI patients were divided into three subgroups:massive infarction group,middle infarction group and lacunar infarction group,and the methylation frequencies of three subgroups were 90.3%,76.2%,and 61.7%,respectively.The methylation frequencies tended to increase with the size of infarction,but there were significant differences only between massive infarction group and lacunar infarction group(X~2=7.777,p=0.005),whereas no significant differences were found between lacunar infarction group and middle infarction group,or between massive infarction group and middle infarction group.The NIHSS scores of CI patients were 6.55±4.25,which were positively associated with the degree of TM gene methylation(r=0.462,p=0.00).(3)The plasma Hcy level in the cerebral infarction groups was significantly higher than that of the controls(14.31 ± 4.61 mmol/L vs 10.25 ± 2.9 mmol/L;t=7.566,p=0.00).The correlation analysis showed that plasma Hcy levels were positively correlated with the degree of TM gene methylation(r=0.701,p=0.00).(4)Bivariate logistic regression analysis was used to explore the association of the methylation status of TM gene promoter with age,sex,smoking,drinking,low density lipoprotein(LDL)and blood glucose in CI patients.The results showed that age and smoking,instead of sex,drinking,LDL and blood glucose,were influencing factors of the methylation status of TM gene Promoter in CI patients.(5)The expression of TM significantly attenuated in case groups compared to control groups.TM expression of the fully methylated,hemimethylated and unmethylated in cases decreased to 12%,25%and 64%of controls,respectively,which was negatively related with the degree of TM gene methylation(r =-0.711,p=0.00).(6)50 Cl patients with HHcy were given oral folic acid,mecobalamin and vitamine B6 for 12 weeks,and the plasma Hcy level was reduced significantly compared with that of pretreatment(12.36±2.80?mol/L vs 19.02±2.44?mol/L;t=16.314,p=0.00).Full methylation frequencies were reduced compared with that of pretreatment(42%vs 54%),but no statistical differences were found between before and after treatment(X~2 =1.442,p=0.230).The expression of TM mRNA was increased significantly compared with that of pretreatment(0.2514±0.1295 vs 0.2149±0.1170;t=3.205,p=0.02).(7)The plasma sTM level in cases was significantly higher than in controls(t=17.268,p=0.00),with the mean ±SD levels of 4.33 ± 0.84 ng/ml and 2.81 ± 0.38 ng/ml,respectively.The correlation analysis showed that the plasma sTM levels were positively associated with the size of infarction and the NIHSS scores in CI patients,with the correlation coefficient of 0.611 and 0.544,respectively.Conclusions:(1)CI patients had a higher TM methylation levels than controls.The TM methylation levels of CI patients were closely linked with Hcy levels,positively associated with the size of infarction and the NIHSS scores,and negatively related with the expression of TM mRNA,which revealed that hyperhomocysteine may induce hypermethylation of TM and be associated with dysregulation of TM expression,and thus this pathogenic effect may play a key role in occurrence and development of CI.(2)Age and smoking both are influencing factors of the methylation status of TM gene promoter in CI patients,while sex,drinking,LDL and blood glucose are not influencing factors of the methylation status of TM gene promoter in CI patients.(3)After oral administration of folic acid,mecobalamin and vitamine B6 for 12 weeks,the plasma Hcy levels were significantly decreased in CI patients with HHcy,but it may take longer time to reverse the hypermethylated status of TM gene promoter.(4)The higher levels of plasma sTM in cases were positively associated with the size of infarction and the NIHSS scores,which can be an indicator for vascular endothelial injury to some extent,but it is not due to downregulated TM gene expression. |
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