ADMA Acts As The New Risk Marker Of Coronary Heart Disease And The Relationship With Proinflammatory Factor MIF And AGEs

BackgroundCardiovascular disease, especially coronary heart disease (CHD) is a serious disease that threat human life and health.The pathogenesis of atherosclerosis (AS) is very complex. With in-depth research on the susceptible factors, inflammatory response plays an important role on the occurrence and process in the of coronary atherosclerosis.Current clinical practice, serum c-reactive protein (CRP) can be used as the most sensitive inflammatory indicators whice reflection of the development of atherosclerosis.But study found that CRP would rise under in all inflammatory symptoms, thus lacks specificity for the diagnosis of CHD.Epidemiological studies have shown that risk factors such as high cholesterol, high blood pressure, diabetes and smoking have close relationship with the occurrence and development of CHD. But about50%of patients with cardiovascular disease risk factors could not be used in the traditional sense. Early diagnosis for this kind of chronic and serious illness is important.Urgently need better biomarkers to predict the future risk of CHD, So that will can intervene at an early age, improve and even reverse the disease process. Asymmetry dimethyl arginine (ADMA) is a kind of endogenous nitric oxide synthase (NOS) inhibitor, it can lower the vascular active substances of nitric oxide (NO) production, decreased NO content and lead to endothelial dysfunction,this pathogenesis are closely related to many diseases.Many studies had proved that endothelial function plays an important role in the development and incidence of cardiovascular disease. In recent years, a lot of experimental research shown that high plasma ADMA levels is closely related to the disease of cardiovascular disease, ADMA may be a new risk factor of atherosclerosis.ADMA product by protein arginine methyl transferase (PRMT-1),and most of ADMA hydrolase by kidney dimethyl arginine a dimethylamine hydrolase (DDAH-2).Studies have shown that the rise of ADMA are related to the enzymes activity or content change of DDAH-2and PRMT-1.Macrophage migration inhibitory factor (MIF) is highly conserved in structure and a unique immune adjustment factor. MIF is a powerful pre-inflammatory cytokines. mainly from T cells and mononuclear macrophage. Researchs shown that in the endothelial cells and vascular smooth muscle cells that in the AS plaque are expressing high levels of MIF, it can promote the monocytes and macrophages gather to the lesion site, then activation, and release series of inflammatory factor, promotes plaque rupture and acute coronary syndrome (ACS). Some clinical studies have shown that MIF is associated with cardiovascular disease, such as arteriosclerosis, heart failure, etc.Advanced glycation end-products (AGEs) is a product that when protein through glycosylated modification. Studies have found that the abnormal rise in elderly patients, and the level was positively correlated with the incidence of atherosclerosis. Some research showed that it can increase the generation of ROS, and other studies showed that ROS can also affect DDAH-1/ADMA/PRMT-2pathways. So we may think that whether there are some relations betweenAGEs and ADMA.To explore the new biomarkers of coronary heart disease, this topic will be around the proinflammatory factor ADMA, MIF and AGEs and study their interaction relations.Part1Detection ADMA and MIFlevel in patients with CHD1.1ObjectionDetection ADMA andMIF content in patients with coronary heart disease, and then compared with healthy people, and analyzes the relationship between MIF and ADMA with disease. Discuss whether they can provide clinical potential roles that can be used as a predictor for coronary heart disease and a potential treatment rake.1.2Methods1.2.1Specimen collection and handlingIn July2011to December2011, collecting venous blood in EDTA anticoagulant tube in CAD patients(diagnosis via coronary artery angiography) on an empty stomach in the morning in cardiovascular institute of guangdong province people’s hospital,then the specimen through centrifugal (4℃,1500RPM/min) for20minutes, collected plasma in EP tubes, store at-70℃.1.2.2Detection MIF, ADMA level in patients with coronary heart disease(1) Detect the contents of the MIF in human plasma by ELISA. Detect MIF by quantitative enzyme-linked immunoassay (ELISA).(2) Detect the contents of ADMA in human plasma by ELISA. Detect ADMA by quantitative enzyme-linked immunoassay (ELISA).1.2.3Real-time fluorescent quantitative PCR detection of MIF, CD74mRNA expression level 1.2.4. ADMA and MIF correlation studiesStudy the correlation of ADMA and MIF by Pearson correlation analysis.1.3. Results1.3.1.The peripheral blood plasma content of MIF in the health group and CAD group is1103±508.0pg/ml(n=62),1422±697.2pg/ml(n-173) respectively (p<0.01),the difference was statistically significant. The duration of after accept PCI and MIF content had no significant relationship, but we can see after undergoing PCI, the content of MIF has a decrease trend over time.1.3.2.The peripheral blood plasma content of ADMA in the health group and CAD group is:170.7±19.93ng/ml,(n=24),253.8±12.88ng/ml(n=80) respectively,(p<0.01), the difference was statistically significant. ADMA and MIF shows correlation, p=0.035.1.3.3. The relatively expression level of MIF mRNA and CD74mRNAThe relatively expression level of MIF mRNA and CD74mRNA in Coronary heart disease has a tendency rise when comparison with healthy group, but no statistical difference.1.4ConclusionAccording to the results of thisstudy,plasma ADMA in CHD group was significantly increased, and increased with proinflammatory factor MIF. So indicated that ADMA may participate in the chronic inflammatory process, and can be used as aeffective marker of CHD that indicators the activity of the disease.Part2ADMA and MIF interaction research 2.1ObjectionAnalyzes the relationship between ADMA and MIF using human endothelial cells, discuss the possible pathophysiological mechanisms of MIF promote atherosclerosis.2.2Method2.2.1The cultivation of Eahy926cellsUsing DMEM F12medium,10%FBS, when cell fusion up to70%, wash twice with PBS, then add in the culture medium containing1%FBS, after night,add the finalconcentration MIF with different concentration (0,2,5,20ng/ml), and culture with different time(24h), respectively.2.2.2Extraction of total cell RNAAfter processing cells according with the2.2.1method, using Trizol method to extract the cell RNA.2.2.3RT-qPCR detection of associated protein’s mRNA relatively expression level.Firstly, reverse the transcription for cDNA, and then the Q-RCR detection tissue factor (TF), matrix protease (MMP-1), endothelin (ET-1), and DDAH-2, PRMT-1, and endothelial nitric oxide synthese (eNOS) in mRNA level.2.2.4Detect the cell supernatant NO level use nitrate nitrous acid enzymatic method.After processing cells according to the2.2.1method, collecting cells culture medium supernatant fluid for test NO.2.2.5Detection ROS level Eahy926cells use fluorescence microscopeAfter processing cells according to the2.2.1method,adding suitable amount of DCFH-DA solution, to the final concentration of DCFH-DA up to10um in the solution, put the culture plate in the37℃incubator, and incubated for15min. After 15min,add Hoechst33342in proportion of1to1000, continuing incubation15min in37℃incubator. Then remove cells supernatant, repeat wash2-3times with PBS, then add each hole with1mL DMEM F12medium. Use fluorescence microscope to analyze signal changes of cell fluorescence and taken fluorescence photographs.2.3Results2.3.1Related proteins’ mRNA relatively expression level in total cell RNAWhen using20ng/ml MIF stimulus Eahy926cells, ET-1, TF, MMP-1mRNA expression levels were significantly increased (p<0.05); at the same time, eNOS, DDAH2mRNA level that relevant to NO production decreased (p<0.05), the change of RPMT-1has no statistical significance.(Detailed results as shown in figure2-1)2.3.2The NO level in cell supernatantNO average level of NO is about14.4μM in20ng/ml MIF group, and Control group was27.5μM (p<0.05), the difference was statistically significant.2.3.3ROS in endothelial cellsAdd20ng/ml MIF group’s ROS level p has significantly increased compared to control group, other concentration group’s ROS production did not obvious.(Detailed results as shown in figure2-2)2.3.4. MIF expression in endothelial cells when treatment with ADMAIn the20and50μM ADMA group, MIF expression increased markedly.(Detailed results as shown in figure2-3and2-4)2.4ConclusionMIF can influence the expression of inflammatory cytokines and the generation of ROS, thus affects ADMA metabolism related enzymes. At the same time, ADMA can raise MIF expression in endothelial cells.Part3ADMA and AGEs interaction research3.1. ObjectionAnalyze whether AGEs can affect DDAH-2/ADMA/PRMT-1pathways, lead to affect the generation of NO, and result in endothelial dysfunction, and lead to atherosclerosis.3.2. Methods3.2.1. Analyze the effects of AGEs to ADMA related metabolism enzyme of DDAH-2and PRMT-1through the level of gene expressionAdd different concentrations of AGEs (0,20,50,100μg/ml) to intervention Eahy926cells, and add the same concentration without glycosylation globulin as control, cultivate with different time(24、48h). After treatment, use Trozol method to extract total RNA, real-time fluorescent quantitative PCR to detection DDAH-2and PRMT-1gene expression levels.3.2.2. Analyze the effects of AGEs to ADMA related metabolism enzyme of DDAH-2and PRMT-1through the level of protein expressionAfter processing cells according to the3.2.1method, extraction of total protein, using Western Blot to detect DDAH-2and PRMT-1protein expression level.3.2.3Use fluorescence microscope to detect the ROS whice the endothelial cells producted after intervention by AGEs.After processing cells according to the3.2.1method, and then detection ROS according to the2.2.5method. 3.3Results3.3.1. The effects of AGEs to ADMA related metabolism enzyme of DDAH-2and PRMT-1through the level of gene expressionIn the50μg/ml and100μg/ml group, PRMT-1mRNA expression level increased significantly compared with control group, the DDAH-2mRNA decreased significantly compared with control subjects (p<0.05), at the same time, there was no statistically significant difference between the two groups. And the25μg/ml group only small changes compared with the control group.(Detailed results as shown in figure3-1)3.3.2The effects of AGEs to ADMA related metabolism enzyme of DDAH-2and PRMT-1through the level of protein expressionIn the100μg/ml group, PRMT-1protein expression increased obviously, and DDAH-2decreased, but25μg/m and group changed little.(Detailed results as shown in figure3-2and3-3)3.3.3The ROS whice the endothelial cells producted after intervention by AGEs.Compared with control group, the25μg/ml and50μg/ml,1OOμg/ml group, the expression of ROS have increased significantly, while100μg/ml group increased significantly.(Detailed results as shown in figure3-4)3.4ConclusionAGEs can affect the DDAH-2/ADMA/PRMT-1pathway.SummaryAccording to the results of thisstudy,plasma ADMA in CHD group was significantly increased, and increased with proinflammatory factor MIF. So indicated that ADMA may participate in the chronic inflammatory process, and can be used as aeffective marker of CHD that indicators the activity of the disease.MIF can influence the expression of inflammatory cytokines and the generation of ROS,thus affects ADMA metabolism related enzymes. At the same time, ADMA can raise MIF expression in endothelial cells, thus promotes the formation of the AS, verified the proinflammatory role of ADMA.AGEs can induction the generation of ROS, thus affect the DDAH-2/ADMA/PRMT-1pathway.