| Backgrounds and ObjectivesAtherosclerosis(AS)is an important cause of most cardiovascular diseases,strokes and peripheral vascular diseases.It is a focal vascular disease characterized by intimal thickening and plaque formation.At present,it is believed that atherosclerosis is not only related to metabolic disorders,but also a dynamic process closely related to inflammation,involving endothelial dysfunction,macrophage and vascular smooth muscle cell proliferation and apoptosis,and intimal calcification.Atherosclerosis is characterized by the infiltration of monocytes into the walls of large and middle arteries,forming atherosclerotic plaques.Monocytes differentiate into macrophages and foam cells,some of which undergo apoptosis and secondary necrosis,forming a necrotic core,and the plaque is easy to rupture to form clinical adverse events such as myocardial infarction and stroke.Monocytes/macrophages play a key role in this process by maintaining lipid homeostasis of vascular wall and regulating inflammatory responses,showing functional diversity at different stages of atherosclerosis.Plaque macrophages exhibit several different phenotypes.M1 macrophages are generally considered to be involved in inflammation and vulnerable plaques.M2 macrophages are considered to have thicker fibrous caps and less risk of rupture.Coronary atherosclerotic heart disease(CHD)is a pathological process characterized by the accumulation of atherosclerotic plaque in epicardial vessels.CHD is clinically divided into stable coronary artery disease(SCAD)and acute coronary syndrome(ACS).According to the definition in the Chinese Guidelines for the Diagnosis and Treatment of Stable Coronary Heart Disease 2018.SCAD include chronic stable exertional angina,ischemic cardiomyopathy,and the stable disease stage after ACS.SCAD accounts for the majority of CHD,and its epidemiology and morbidity are difficult to assess due to the different definition in different researches In general,it is more common in middle-aged women than men.but as age increases,the opposite characteristics appear,and it is more common in elderly men.The prognosis of SCAD patients is relatively better.The estimated annual mortality rate of the mixed population is 1.2%-2.4%,and the incidence of cardiac death is about 0.6%-1.4%.In general,patients with heart failure,reduced LVEF,a greater number of diseased blood vessels,more proximal coronary stenosis,a wider range of ischemia,more impaired multiple organ function,older age,and patients with significant depression have a worse prognosis.Prognosis assessment is an important part of SCAD patient management.Although SCAD is stable relative to the acute events of coronary artery disease,the underlying pathophysiological status is not always stable and may even become unstable at any time.How to be more economic,effective and quickly assess the prognosis in patients with SCAD,identify SCAD in the crowd "unstable" patients,screening high-risk patients,has been the urgent demand for clinicians.At present,the prognosis of patients with SCAD is mainly evaluated on the basis of risk factors,clinical symptomatology(especially the severity of angina pectoris)and the necessary non-invasive/invasive examinations(load electrocardiogram,single-photon emission computed tomography,cardiac MRI,coronary CTA,coronary angiography,etc.).Worse prognosis in SCAD patients is closely related to unstable plaques,but none of the above methods can directly identify unstable plaques.At present,the methods of identifying unstable plaques mainly include non-invasive/invasive imaging examination and inflammatory biomarker examination.However,atherosclerosis is a dynamic process.From the perspective of the patient’s entire coronary vascular bed,the occurrence,progression,plaque formation,rupture and repair of atherosclerotic plaques are ongoing.The information provided by either invasive or noninvasive imaging methods for predicting the development of atherosclerotic disease is limited Inflammatory markers have long been studied in patients with coronary heart disease,especially SCAD,and have shown good clinical diagnostic and prognostic value,providing important clinical reference value for primary and secondary prevention of coronary heart disease,screening of high-risk groups and risk stratification of prognosis.Mac-2 binding protein(M2BP)is a binding protein of galactose-binding lectin secreted by activated macrophages.It is a secreted high glycosylated protein,a member of the superfamily of MSR rich cysteine domain,easy to be detected in plasma,and is currently recognized as a biomarker of viral infection and cancer.M2BP was found to be a measurable and soluble M1 macrophage marker in vitro.In clinical studies,it was found that the expression of M2BP was up regulated in subclinical carotid atherosclerosis and positively correlated with related biological markers,which could reflect occult atherosclerosis in the normal population.Studies on patients with coronary heart disease have shown that,compared with healthy people,M2BP levels in peripheral blood of patients with coronary heart disease were higher,and M2BP levels in patients with ACS were significantly higher than that in patients with SCAD,and the higher expression of M2BP indicates a worse prognosis.Although the above studies preliminarily revealed the correlation between M2BP and atherosclerosis,the mechanism of M2BP has not been further explored.Considering the importance of macrophages in atherogenesis and the lack of a complete understanding of the effects of M2BP on atherogenic monocyte/macrophage activation,we used both in vivo and in vitro models to elucidate the association between M2BP and atherogenesis.Also,there is a lack of clinical data of M2BP in risk stratification of patients with stable coronary heart disease,and there is no complete risk assessment system for SCAD patients.In view of this,we adopted a combination of basic and clinical experimental methods to explore the pathogenesis of M2BP in the process of atherosclerosis and plaque instability,to explore the predictive value of circulating M2BP level on the prognosis of SCAD patients,and to establish a risk prediction model for SCAD patients based on circulating M2BP level.MethodsPart Ⅰ The pathogenesis of M2BP in atherosclerosis.1.Effects of M2BP gene knockout on major metabolic indicators,atherosclerosis progression and plaque components of ApoE-/-mice.The experimental animals were ApoE-/-mice and M2BP-/-+ApoE-/-dual knockout mice,8-10 weeks age male mice.After 2 weeks of adaptive feeding,the transition to 12 weeks of high fat feeding.Blood serum was collected from two types of mice before and 12 weeks after the high-fat diet,and the body weight and lipid levels of the two groups were compared.RT-PCR was used to compare the M2BP mRNA levels in ApoE-/-mice before and after high-fat diet.The aorta segment from the ascending aorta to the abdominal aorta was cut and stored in a cryopreserved tube,frozen in liquid nitrogen,and stored in a refrigerator at-80℃.The gross aorta was stained with oil red O.The Image pro plus software was used to analyze and compare the atherosclerotic area between ApoE-/-mice and M2BP-/-+ApoE-/-mice.Separate the heart,aorta,and surrounding tissue and cut out the heart.Some samples were immersed in 4%paraformaldehyde solution for 24-48 hours,and then embedded in paraffin.The components of atherosclerotic plaques in the aortic root of the two groups of mice were compared by HE staining,Sirius red staining and immunohistochemistry.2.Effect of M2BP gene knockout on the migration activity of bone marrow mononuclear macrophage(BMDMs)and its adhesion capacity to vascular endothelial cells1)Western blot was used to compare the expression level of M2BP in undifferentiated THP-1 cells and activated macrophages.2)Transwell chemotactic experiment was used to compare the migration ability and activity of ApoE-/-mice and M2BP-/-+ ApoE-/-mice with and without MCP-1 as chemotactic agent.3)Cell adhesion experiment was conducted to compare the adhesion ability of mononuclear macrophages to human umbilical vein endothelial cells(HUVECs)between ApoE-/-mice and M2BP-/-+ApoE-/-mice.4)Two groups of living mice models of peritonitis were made with brewer’s thioacetate as an inducer(2 mL,concentration of 3%),and mononuclear cells in the circulation of living mice were induced to accumulate in a retroperitoneal direction to simulate the differentiation process of mononuclear-macrophage.After 3 days,the mice were killed,and the peritoneal lavage fluid was collected.Macrophage specific antibody CDllb was used to label macrophages and numbers of macrophages were counted.3.Effect of M2BP gene knockout on activation of MAPK signal transduction pathway1)Western blot experiments were conducted to compare the activation of MAPK signal transduction pathway in ApoE-/-mice and M2BP-/-+ ApoE-/-mice.2)Cell adhesion experiments were conducted to compare the effects of different concentrations of rhM2BP(0,0.5,1,5 ug/ml)stimulation on BMDMs adhesion of ApoE-/-mice in vitro.3)Transwell chemotaxis experiments was conducted to compare the effects of different concentrations of rhM2BP(0,0.5,1,5 ug/ml)stimulation on BMDMs migration of ApoE-/-mice in vitro.4)Western blot experiments were conducted to compare phosphorylation activation of p38-MAPK in BMDMs of ApoE-/-mice stimulated by different concentrations of rhM2BP(0,0.5,1,5 ug/ml)in vitro.5)Establishment of ApoE-/-+Lenti-p38 mouse model:ApoE-/-mice were fed with high fat for 8 weeks,and 200ul of p38mapk-shrna lentivirus containing 107TU were injected into tail vein.After that,high-fat feeding continued for 4 weeks.BMDMs were isolated and cultured.6)Western blot experiments were conducted to compare the phosphorylation and activation of p38-mapk in BMDMs of ApoE-/-mice,M2BP-/-+ApoE-/-mice and ApoE-/-+Lenti-p38 mice.7)Transwell chemotaxis experiments were conducted to compare BMDMs migration ability of ApoE-/-mice,M2BP-/-+ApoE-/-mice and ApoE-/-+Lenti-p38 mice.8)Cell adhesion experiments were conducted to compare the adhesion of BMDMs to HUVECs in ApoE-/-mice,M2BP-/-+ApoE-/-mice and ApoE-/-+Lenti-p38 mice.9)In vivo peritonitis models of ApoE-/-mice,M2BP-/-+ApoE-/-mice and ApoE-/-+Lenti-p38 mice were prepared by using brewer’s thioglycolate as the inducer,and the migration capacity of mononuclear macrophages in vivo of the three groups of mice was compared.Part Ⅱ The establishment of a stable coronary artery disease risk prediction model based on circulating M2BP level.A total of 333 SCAD patients admitted to the department of cardiology of our hospital from January 2016 to December 2016 were enrolled in this study.After admission,the medical history was routinely collected and the concentration of M2BP was determined by ELISA.The study end points were 1)recurrent angina requiring hospitalization,2)non-fatal acute myocardial infarction,3)cardiogenic death,and 4)death from any other causes.Recurrent angina pectoris,non-fatal acute myocardial infarction and cardiogenic death requiring hospitalization were defined as Major Adverse Cardiovascular Events(MACE).Patients with SCAD were divided into low M2BP group(1st),medium M2BP group(2nd)and high M2BP group(3rd)according to the three-digit plasma M2BP level.Kaplan-Meier method was used to plot survival curves for the three groups,and log-rank test was used to compare the statistical differences in the incidence of abnormal outcomes among the three groups.Cox risk ratio model was used to determine the independent risk factors affecting the prognosis of patients with SCAD in this study cohort.Here,we adopted two modeling methods:1)the univariate Cox regression model was used to screen the factors with statistical correlation to the outcome;for the variables with p<0.1 and the factors related to the prognosis of SCAD in clinical consideration were continued to screen in the multivariate Cox regression model,and the variables with statistical significance were selected as the risk factors.2)By using the method of "input" different clinical variables were selected and added to Cox multi-factor model to construct multiple Cox models,and their HR values were compared to analyze whether M2BP had independent prediction value for the occurrence of endpoint events.Nomogram was plotted for visually present.Bootstrap self-sampling method was used to verify the prediction effect of the model constructed in method 1).The consistency index(C-index)was calculated to evaluate the performance of the constructed prediction model.Decision Curve Analysis(DCA)was used to explore clinical practicability.ResultsPartⅠ The pathogenesis of M2BP in atherosclerosis.1.M2BP gene knockout had no significant effect on the major metabolic indicators of ApoE-/-mice,such as body weight,total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C)and triglyceride(TG)levels.2.M2BP mRNA level of ApoE-/-mice increased by more than 5 times after high-fat diet(p<0.01).3.Gross aortic oil red O staining showed that after 12 weeks of high-fat diet,the aortic atherosclerosis area of M2BP-/-+ApoE-/-mice decreased by about 27%(12.3±0.34%vs 16.9±0.29,p<0.01)compared with ApoE-/-mice.4.HE staining results showed that compared with ApoE-/-mice,M2BP-/-+ApoE-/-mice had a lower degree of atherosclerosis(11.9±0.25%vs 15.9±0.39,p<0.01).Collagen staining(Sirius staining),macrophage specificity marker CD68,and vascular smooth muscle cell specificity marker a-SMA immunohistochemical staining suggested that M2BP-/-+ApoE-/-mice had lower collagen composition,less smooth muscle cells,and less macrophage aggregation than ApoE-/-mice.5.Western blot results showed that,compared with undifferentiated THP-1 mononuclear cells,the expression level of M2BP increased by 230%after activation and differentiation of THP-1 mononuclear cells into macrophages.6.Compared with ApoE-/-mice,BMDMs derived from M2BP-/-+ApoE-/-mice showed weaker migration and activity without MCP-1 as a chemoattractant,which was not improved or reversed with the addition of MCP-1(50ng/ml).Similarly,BMDMs derived from M2BP-/-+ApoE-/-mice had a weak ability to adhere to HUVECs,and this situation did not improve over time.7.In the live mouse model of peritonitis,compared with ApoE-/-mice,the number of macrophages in the peritoneal lavage fluid of M2BP-/-+ApoE-/-mice was significantly reduced(4.12*106 vs 7.96*106,p=0.035).8.Western blot showed that,compared with ApoE-/-mice,M2BP-/-+ApoE-/-mice had significantly decreased p38-MAPK activity,while ERK-MAPK and JNK-MAPK activities had no significant changes.9.The effects of stimulation of rhM2BP at different concentrations on BMDMs migration and adhesion were analyzed in vitro,and the phosphorylation activation of p38-MAPK was detected by Western blot.The results showed that M2BP concentration dependence promoted BMDMs migration and adhesion to HUVECs,which was accompanied by M2BP concentration dependence enhancement of p38-MAPK activity.10.ApoE-/-mice were transfected with adenovirus containing p38 interfering RNA.Western blot confirmed that ApoE-/-mice had the most significant p38-MAPK activation degree,followed by M2BP-/-+ApoE-/-mice,and the lowest p38 RNAi mice.At the same time,BMDMs migration,adhesion and the trend of the collection of retroperitoneal macrophages showed the same trend.Part Ⅱ The establishment of a stable coronary artery disease risk prediction model based on circulating M2BP level.1.Subjects were divided into three groups according to the trine of M2BP concentration.There were no significant differences among the three groups in gender,age,history of hypertension,history of diabetes,history of dyslipidemia and current smoking.The incidence of old myocardial infarction was higher in the group with higher M2BP concentration(2nd,3rd)than in the group with lower M2BP concentration(1st)(6.35%vs 30.63%vs 30.91%,p<0.001).2.M2BP level of patients with MACE was significantly higher than that of patients without MACE(15.32(12.45-21.92)vs 11.42(8.36-15.07),μg/ml,p<0.001).3.Survival analysis showed that with the increase of M2BP level,the event-free survival rates of recurrent angina requiring hospitalization(p=0.017),non-fatal acute myocardial infarction(p=0.062),all-cause death(p=0.046)and MACE(p=0.0001)all decreased,among which the p value of non-fatal acute myocardial infarction did not reach statistical significance,but still showed the trend.4.The variables of the multivariate Cox risk proportion model were M2BP(HR=1.1528,95%ci=1.1028-1.2051,p<0.001),previous history of myocardial infarction(HR=3.3598,95%ci=1.7855-6.3221,p<0.001),hypersensitive troponin T(HR=1.3145,95%ci=1.1531-1.4985,p<0.001).The calculated c-index of the final model is 0.852.Conclusions1.Under the action of various atherosclerotic pathogenic factors,M2BP participates in the process of atherosclerosis.2.M2BP participates in the pathophysiological processes of monocyte-macrophage activation,directed aggregation and adhesion at least through the p38-MAPK intracellular signal transduction pathway.3.The circulating M2BP level has a good independent predictive value for the prognosis of patients with SCAD.4.Based on the circulating M2BP level,the SCAD patient risk prediction model is established,which has high predictive value,strong validity and reliability. |
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