The Effect Of DNA Methylation On The Gene Of Metal Metalloproteinases-9 And The Role Of It In The Atherosclerosis

Background Atherosclerosis is a chronic persistent inflammationdisease based on the dyslipidemia and characterized by the progressivedevelopment and sudden acceleration. A large body of evidence suggeststhat Metal metalloproteinases -9 (MMP-9) plays a important rolethroughout of the atherosclerosis and which was considered to havenegative effect on the pathogenesis in the past. But some recent studiesshow that the MMP-9 play the dual role in the atherosclerosisdevelopment. In one hand MMP-9 involved in plaque destabilization, disruption and thrombosis if its gene expresses abnormally high; but onthe other hand it involved in the normal development of tissues andorgans, repairing the impaired artery and the smooth muscles migrationwhich may stabilized the plaque. At the same time some studies showsthat the plasma MMP-9 concertration elevated in the old age crowd andwhich may explain why the atherosclerosis accelerated in the old, butthe mechanism of it is still unclear. Since the elevation of the plasmaMMP-9 concertration is persistent and happens with the age, the pastfinds that some external stimuli such as TNF-a, NF-κB, IL-18 et. alcould raises the expression of the gene of MMP-9 can not explain perfectly this phenomenon, there must be additional regulatorymechanism(s) is responsible for inappropriate expression, most notablythose resulting from mutations or polymorphism in the regulatory regionof the gene or modifications at the epigenetic level. DNA methylation inmammals plays a crucial role in development, X chromosomeinactivation, epigenetic silencing, aging, carcinogenesis, and certainhuman genetic disease. There are some clues indicated that modificationin DNA methylation induces the MMP-9 inappropriate expression insome carcinogenesis or immune disease. But whether this modificationhappens in the atherosclerosis has not been reported. The research aboutthat will open a new way for understand the relationship of the age, inflammation and the atherosclerosis.Objectives To investigate the difference of the MMP-9 gene DNAmethylation status between the normal and atherosclerosis patients, therelationship between the DNA methylation of MMP-9 gene, age and theMMP-9 plasma concertration. Analysis the effect of5-aza-2'-deoxycytidine and the ox-LDL on matrix metalloproteinaseexpression in the mononuclear macrophage cells in vivo.Methods: The 52 different types coronary heart disease patientsand 39 controls were included in this cross-sectional study. The DNAwere extracted from the white blood cell and the DNA methylationstatus of MMP-9 gene were studied using the MSP (methylation specific polymerase chain reaction), as well as the plasma MMP-9concentration were measured by ELISA; the monocytes which isolatedfrom peripheral blood of normal contral were incubated and wereintervened with 5-aza-2'-deoxycytidine and the ox-LDL, the MMP-9concentration and activity in the medium were measured, the mRNAlevel of MMP-9 were detected by reverse transcription polymerase chainreaction (RT-PCR). The dates were analysed using by SPSS software.Results: There is the lower level of the average density of the DNAmethylation of the MMP-9 gene promoter in the atherosclerosispatients were detect than normal contral, the plasma concentration ofMMP-9 are higher in the atherosclerosis patients. There is a markedinverse correlation between the age, the concentration of MMP-9 and theaverage density of the DNA methylation of MMP-9 in the stableatherosclerosis patients, but this marked inverse correlation were notfound in the normal and acute myocardial infarction patients. Themethylation inhibitors 5-aza-2'-deoxycytidine promote monocytesexpressing the MMP-9 singly slightly in vitro, the ox-LDL and the5-aza-2'-deoxycytidine combined intervention induced higher MMP-9expression level.Conclusions: The increasing of plasma level of MMP-9 in stableatherosclerosis patients has the relationship between the patients age.The age-related hypomethylation in the promoter of MMP-9 gene involved in the MMP-9 gene inappropriate expression in the agedatherosclerosis patients. The 5-aza-2'-deoxycytidine, a methylationinhibitors promote the monocytes expressing the MMP-9 highly with ornot with ox-LDL induced in vitro indicated that the hypomethylationinvolved in the dyslipidemia induced inflammation. Background: The matrix metalloproteinases-9 has beenconsidered as a important inflammation mediators which could promotethe development of artherosclerosis. But the new evidence show that theMMP-9 may play the dural role in the artherosclerosis included theprotective and destructive effect. At the same time there are someevidences shows that the concentrations of MMP-9 increase with age.The reason about that is still not clear. There are some clues indicatedthat modification in DNA methylation induces the MMP-9 inappropriateexpression in some carcinogenesis or immune disease. But whether thismodification happens in the atherosclerosis has not been reported.Objectives: To investigate the difference of the MMP-9 gene DNAmethylation status between the normal and atherosclerosis patients, therelationship between the DNA methylation of MMP-9 gene, age and theMMP-9 plasma concertration.Methods: The 52 different types coronary heart disease patientsand 39 controls were included in this cross-sectional study. The DNAwere extracted from the white blood cell and the DNA methylation status of MMP-9 gene were studied using the MSP (methylationspecific polymerase chain reaction) and sequence, as well as the plasmaMMP-9 concentration were measured by ELISA. The dates wereanalysis using the SPSS 11.0 soft to find the relationship between theage, the MMP-9 concentration and the methylation levels.Results: The MMP-9 concertration and the average methylationdensity of the promoter of the MMP-9 gene between the young and oldage groups in normal 0.41±0.4vs0.2±0.1, P＞0.05); the averagemethylation density of average methylation density of the promoter ofthe MMP-9 gene is lowed in the stable angina group and the acutemyocardial infarction group than in the normal (6.1±2.5%vs 42.3±5.2%, P＜0.05; 25.6±4.7% vs42.3±5.2%, P＜0.05); Corresponding withthe MMP-9 higher in both patient groups than in contral. A markedpositive correlation is detected between the ages and the concentrationof MMP-9(r=0.41, P＜0.05)and a inverse correlation is detectedbetween the average density of the DNA methylation of MMP-9 and age(r=-0.78, P＜0.05)in the stable atherosclerosis patients, but this markedcorrelation were not found in the normal and acute myocardial infarctionpatients groups.Conclusion: In the stable artherosclerosis patients, there exist a hypomethylation in the promoter of MMP-9 gene, which may results inthe MMP-9 concertration increasing with age. Background: The ox-LDL has been reported to induce the MMP-9expression in human mononuclear macrophage cell but there were someargument in recent study. At the same time there were some evidenceshows that the DNA methylation involved in the regulatory mechanismof MMP-9 gene expression. In order to make sure that the effect of theDNA methylation and the ox-LDL on the MMP-9 gene expression, wetest these hypotheses by using the ox-LDL and the DNA methylationinhibitor to interfere in the cultured mononuclear macrophage cells.Objective: to observe the effect of the DNA methylation inhibitor5-aza-2'-deoxycytidine on the MMP-9 expression in the mononuclearmacrophage cells, and the effect of ox-LDL combined with DNAmethylation inhibitor 5-aza-2'-deoxycytidine on the expression of theMMP-9.Methods: the monocytes which isolated from peripheral blood ofnormal donors were incubated and were intervened with5-aza-2'-deoxycytidine or combined with the different concentrationox-LDL, the MMP-9 concentration and activity in the medium weremeasured, the mRNA level of MMP-9 were measured by reverse transcription polymerase chain reaction (RT-PCR). The difference ofMMP-9 contration and mRNA levels between the blank and theintervention group cells were analysed using by SPSS software.Results: The methylation inhibitors 5-aza-2'-deoxycytidinepromote monocytes expressing the MMP-9 singly slightly in vitro(0.5μmol/L: 0.82±0.06vs0.12±0.05, P＜0.05; 0.75μmol/L: 0.88±0.08vs0.12±0.05, P＜0.05), the ox-LDL and the 5-aza-2'-deoxycytidinecombined intervention induced higher MMP-9 expression level(0.52±0.06vs0.42±0.04, P＜0.05).Conclusions: Intervention with 5-aza-2'-deoxycytidine induced themononuclear macrophage cells highly expressing MMP-9 in vitro, thiseffect were enhanced when the intervention were combined with theox-LDL. This result refer to that the DNA methylation involved in theregulatory mechanism of the MMP-9 and the argument of the effect ofox-LDL on the expression of macrophage cells may lies in the differentmethylation status cells were used in individual experiments.