Preliminary Study On The Relationship Between The Methylation Of Plasma Thrombomodulin Gene Promoter And CHD

BACKGROUND: Thrombomodulin(TM) is membrane glycoprotein that is located on the surface of endothelial cells discovered in recent years. As the receptor of thrombin, TM takes part in the activation of protein C(PC) and plays an important role in the process of anticoagulation. Besides, it can also degredate plasminogen activator inhibitor and promote fibrinolysis. Dysfunction of endothelium is the trigger of atherosclerosis, and changes in the anticoagulant function of endothelial cells could induce unbalance of angticoagulation/ fibrinolysis. TM could resist the promoting-atherosclerosis(AS) effect by damaging endothelial cells though binding to thombin. Therefore, as one of the factors that protects endothelial cells, and with its antithrombus function, it deserves great notice. Researches have indicated that plasma TM concentration of CHD patients is significantly higher than that of the control group, that is, the expression and the regulation of TM play an important role in the fuctions of the mechanism of pathophysiology of atherosclerosis.In recent years, epigenetical research on AS gradually becomes popular, and as a major epigenetical modification, methylation is one of the most common modifying ways in the process of postreplication and premodification. Large amount of researches demonstrated that AS is correlated to abnomal methylation of specific genes, and methylation status of AS-related TM gene is significantly high in pathological cases. Therefore, to investigate the methylation status and mechanism of expression and regulation of TM gene in coronary heart disease could devote to the further research about the pathogenesis of AS, and could provide theoretical bases for the prevention and treatment of CHD.AIM: (1) To investigate the relationship of the methylation of plasma thrombomodulin(TM) gene promoter between normal and coronary heart disease (CHD). (2) To investigate the relation between concentration of TM in plasma and methylation of TM gene promoter in normal and CHD. (3) To discuss the mechanisms of the atherosclerosis(AS) induced by TM gene.METHODS: (1)The sample population is consisted of 90 patients with CHD who were confirmed having stenosis or atheromatous plaque in their coronary artery by coronary angiography, and 75 cases without stenosis as the control group. (2) Methylation- specific PCR (MS-PCR) were employed to detect the TM gene promoter methylation state from the plasma of patients with CHD. (3) We investigate the concentration of TM in two groups by ELISA and did statistical analysis. RESULTS: (1)Methylation of TM gene promoter is detected in 41%(37/90) of the patients with CHD, and the rate of the control group is 20%(15/75).The methylation rate in CHD group is significantly higher than that of the control group(P<0.05). (2)Relationship between sex and the status of TM gene promoter: We detected TM gene promoter methylation in 43%(33/76) of the male patients with CHD, and the rate of male subjects in the control group is 19%(8/43). The rate in the case group is significantly higher than that in the control group(P<0.05). We detected TM gene promoter methylation in 29%(4/14) of the female patients with CHD, and the rate of the female subjects in the control group is 22%(7/32). There is no statistical difference between the two rates(P>0.05). (3)In the case group, methylation of TM gene promoter in 1-artery CHD group is detected in 44% of the patients (11/25) . The rate of 2-artery CHD group is 39%(9/23). And the rate of the over 3-artery CHD group is 40%(17/42). So there is no statistical significance among the methylation rate of 3 groups(P>0.05), and it was correlated with the pathological changes. (4) We divided all the subjects into four groups by age. We detected methylation rate in each group as follows: 19.35% in group below 49 years group; 29.17% in 50～59 years group; 33.33% in 60～69 years group; 42.86% in group above 70 years. With the increase of age, methylation rate of TM gene promoter had tendency to increase, but there is no statistical significance among the four group(P>0.05).(5) The mean plasma concentration of TM in the case group is 0.70±0.24 ng/ml, and the mean concentration in the control group is 0.52±0.18 ng/ml. The plasma concentration in case group is significantly higher than that in control group(P<0.05). (6) Relationship between sex and plasma TM concentration: The mean plasma TM concentration in the male subjects of the case group is 7.0±2.4ng/ml, and that in the control group is 5.2±1.8ng/ml. The plasma TM concentration of male subjects in the case group is significantly higher than that in the case group(P<0.05). The mean plasma TM concentration in female subjects of case group is 6.1±2.4ng/ml, and that in the control group is 5.6±2.2ng/ml. There is no statistical difference between the two concentrations(P>0.05).CONCLUSIONS: (1)Our results of 165 subjects indicated that, rate of methylation in promoter of TM gene in patients is significantly higher than the rate of control group indicates that menthylation of TM gene promoter in peripheral blood is closely related to the pathogenesis of CHD. It can inhibit or downregulate the expression of TM gene, and let it lose protection of resisting the promoting-atherosclerosis (AS) effect by damaging endothelial cells, and then cause AS.(2)The rate of methylation in promoter of TM gene in the male patients is significantly higher than the that of the male subjects in the control group, the non-existence of this phenomenon in female subjects suggests that high methylation status in TM in peripheral blood is closely related to male subjets who are liable to AS, and the mechanism need to be further proved.(3)In 90 patients with CHD, we did not observe that Methylation of plasma thrombomodulin gene promoter is related to the degree of disease, which indicates that methylation of TM gene in peripheral blood involed in beginning of AS, and the methylation is maintained.(4) Result that the plasma concentration in the case group is significantly higher than that in the control group suggested that TM produced by blood cells is not the main source of plasma TM. The increase of TM concentration in plasma caused by dysfunction of endothelial cells in CHD is much higher than the decrease which is caused by methylation of TM gene promoter in blood cells. (5) Result that the plasma TM concentration of male subjects in the case group is significantly higher than that in the case group which was not observed in female subjects indicated that TM is closely related to male subjects who are liable to AS, but the mechanism needs further research.