| Backgrounds and Objectives:Atherosclerosis(AS)is the major cause of cardiovascular diseases and also one of the research hotspots and urgent problems worldwide.AS is a chronic degenerative disease occurring in large and medium-sized artery,mainly characterized by vascular intimal injury or dysfunction,lipid accumulation,participation of a variety of inflammatory cells and cytokines,leading to the formation of atheromatous plaque and arterial wall hardening,finally causing luminal stenosis and target organ ischemic changes and corresponding symptoms.The mechanism of AS has not been fully elucidated by far due to its complexity.The process of AS is initiated by vascular endothelial injury or dysfunction,and proceeds and develops under the participation and contribution of adhesion molecules(ICAM-1、VCAM-1、E-selectin、P-selectin),chemokines(MCP-1、IL-8,etc.),a variety of inflammatory cells(monocytes-macrophages、T lymphocytes、mast cells、neutrophils,etc.),inflammatory cytokines(IL-1、IL6、TNF-aα、CRP、Lp-PLA2,etc.)and multiple matrix protease(MMPs、ASAMTSs、cathepsins,etc.).With the formation and expansion of lipid core,inflammatory reactions within plaque aggravate,giving rise to necrosis of macrophages and smooth muscle cells、neoangiogenesis and degradation of fibrous cap and extracellular matrix to accelerate the vulnerability and rupture of plaques.Thus it can be seen that inflammation is involved in the whole process of initiation,development,destabilization and eventual rupture of atherosclerotic plaques.Acute coronary syndrome(ACS)is a group of clinical syndromes caused by acute myocardial ischemia,consisting of unstable angina pectoris(UAP),non-ST elevation myocardial infarction(NSTEMI)and ST-elevation myocardial infarction(STEMI).ACS patients were the high-risk population in patients with coronary artery diseases(CAD).Although the total motality of ACS patients has been significantly reduced due to the optimal medication therapy and advancement in thrombolysis and coronary intervention,the in-hospital and outside incidence of recurrence of major adverse cardiovascular events(MACEs)is still high in short-term and long-term observation.Thus it is imperative to conduct early risk assessment and strengthen treatment and management of ACS patients in clinical practice.The role of GRACE score and TIMI score in risk stratification of ACS patients has been widely recognized and validated by multiple large clinical trials and recommended by guidelines as well.Nine indexes are applied in GRACE score system for risk assessment in ACS patients including age、resting heart rate、systolic blood pressure、initial serum creatinine、history of congestive heart failure、history of myocardial infarction、ST-segment depression、elevated cardiac enzymes and no in-hospital percutaneous coronary intervention.Recent researches demonstrated that GRACE score had a good performance in predicting in-hospital mortality and short-term and long-term prognosis after discharge in ACS patients.Rupture or erosion of intracoronary unstable plaque followed by thrombus formation is currently regarded as the main pathogenesis of ACS.Unstable plaque is often characterized by owing large necrotic core(30%of plaque)、thin fibrous cap(thickness usually<65 um)covering the necrotic core、macrophages infiltration、few smooth muscle cells、expansive remodelling preserving the lumen、neovascularization from vasa vasorum、plaque haemorrhage and thrombus formation.Vulnerability index was usually calculated and used for evaluating plaque instability in animal models of atherosclerosis and unstable plaque.Clinically,there are multiple imaging tests to detect the instability of plaques such as multi-slice spiral CT(MDCT),carotid ultrasound,optical coherence tomography(OCT)and intravascular ultrasound(IVUS).Coronary angiography is always regarded as the golden standard for diagnosis of CAD.The direct visual observation of the morphology of coronary artery plaque by coronary angiography may also help estimating the stability of coronary plaques roughly and effectively.Recent clinical studies showed that coronary complex lesions detected by coronary angiography could reflect plaque instability effectively and predict cardiovascular outcomes independently in CAD patients.Numerous clinical researches have explored the relationship between plasma proinflammatory cytokines and coronary complex lesions in order to search for new plasma biomarkers to reflect plaque stability.Mac-2 binding protein(M2BP)is a highly glycosylated secreted protein belonging to the macrophage scavenger receptor cysteine-rich domain superfamily.It is expressed by hematopoietic and epithelial cells,but is also present in many other tissues including colon,duodenum,stomach,and lung.In addition,M2BP is detectable in many body fluids like semen,saliva,urine,tears,human milk,and plasma.As a proinflammatory protein,M2BP was highly expressed in condition of tumor and virus infection and has been proven to be associated with metastasis and poor prognosis of cancer.Recently,several researches indicated that M2BP could activate human monocytes and upregulate the synthesis and secretion of many inflammatory cytokines to facilitate human host immune response.Moreover,plasma levels of M2BP were found to be significantly elevated in patients with CAD than in healthy controls.Furthermore,plasma levels of M2BP were revealed to be correlated with traditional risk factors、metabolism and oxidative stress.Recent evidence showed that M2BP was highly expressed and secreted by proinflammatory M1-polarized macrophage in vitro and colocalized with CD68 + plaque macrophages in vivo.Besides,results from a retrospective research showed that plasma M2BP levels was independently predictive of long-term mortality in 149 CAD patients diagnosed by Coronary computed tomography angiography(CCTA).Given the proinflammatory characteristics of M2BP and mounting evidence showing its potential association with AS and CAD,we hypothesized that M2BP was involved in the process of AS and plaque destabilization.Thus our research sought to investigate the role and related mechanisms of M2BP in development of AS and plaque destabilization by means of combination of basic research and cl ini cal observation,thus providing new theoretical basis and therapeutic target for the pathogenesis of coronary heart disease.Content:1.We measured and compared plasma M2BP levels in patients with SAP、ACS and control group.Meanwhile,we analyzed the association of plasma M2BP levels and coronary complex lesions and its prognostic value in ACS patients.2.We analyzed and compared the mRNA expression levels of M2BP in peripheral blood mononuclear cells(PBMCs)in patients with SAP、ACS and healthy controls.The relationship between M2BP expression and other inflammatory cytokines and the predictive value of M2BP expression on coronary complex lesions were also explored.3.Fifty-one patients receiving carotid endarterectomy were consecutively recruited to analyze the expression levels of M2BP in symptomatic patients and unstable carotid plaques in combination with their symptoms and carotid artery ultrasound.4.To study the effects of lentivirus-induced silencing of M2BP in vivo on plaque formation and instability in ApoE knockout mice.5.To investigate the influence of M2BP stimulation on expression of inflammatory cytokines in THP-1 and HUVEC and related mechanisms.Methods:1.Plasma levels of M2BP in CAD patients and its prognostic value in ACS patientsA total of 348 patients undergoing coronary angiography in our hospital from January 2015 to December 2015 were consecutively recruited in our study.Patients were divided into three groups:216 patients with ACS,82 patients with SAP,and 50 controls.Medical history of each patient was collected regularly and peripheral blood samples were obtained upon admission for measurement of M2BP by Elisa.Evaluation of plaque morphology and quantitative assessment of coronary artery stenosis were performed by two cardiologists blinded to the study protocol and patient information.Coronary lesions were defined as complex or simple according to Ambrose’s methods.Among SAP patients,30 of them were randomly selected for serial detection of plasma M2BP levels before and at 1、2、6 and 24h after PCI.GRACE scoring system was applied for risk stratification in ACS patients and they were followed up for one year.The primary outcome was the composite end point of major adverse cardiovascular outcomes(MACEs),defined as cardiac death,nonfatal myocardial infarction,and recurrence of unstable angina that required re-hospitalization.The Cox proportional hazard model was performed to identify independent prognostic predictors.2.The mRNA expression in PBMC of CAD patients and its predictive value on coronary complex lesionsA total of 60 patients undergoing coronary angiography in our hospital from January 2016 to March 2016 were randomly selected in our study,including 40 ACS patients、20 SAP patients and 10 healthy controls.Medical history of each patient was collected regularly and peripheral blood samples were collected upon admission for measurement of inflammatory factors(hs-CRP、IL-6、TNF-α)by Elisa.Ficoll density gradient centrifugation was used for isolation of PBMC from blood sample.The total RNA was extracted from monocytes with the Trizol reagent and RT-PCR was performed to detect gene expression of M2BP and the other inflammatory cytokines.Plaques were divided into complex lesions or simple lesions according to morphology evaluation as previously described.PBMCs were also isolated for detection of gene expression of M2BP during 1-month outpatient follow-up for comparison.Logistic multivariate model and ROC curve were conducted for evaluate the predictive value of M2BP gene expression on coronary complex lesions.3.The expression of M2BP in human carotid artery plaques and its association with plaque destabilizationA total of 51 patients undergoing carotid endarterectomy were consecutively enrolled in our study.Patients with cerebrovascular symptoms(TIA、stroke and amaurosis fugax)within the 6 months before surgery were classified as symptomatic(n=36),the other 15 patients without neurological symptoms were defined as asymptomatic.Carotid plaques were also divided into echogenic、heterogeneous and echolucent plaques according to manifestations of carotid artery ultrasound.Medical history of each patient was collected regularly and peripheral blood samples were collected upon admission for measurement of M2BP by Elisa.Carotid plaque samples were partly collected for RT-PCR analysis of M2BP gene expression,the other parts were used for immunohistochemical staining and Masson staining.Positive staining area was measured and analyzed by Image pro plus.The relationship between the expression of M2BP and carotid ultrasound,symptoms and the components of the plaques was compared at different levels:peripheral plasma level,gene expression in plaque and positive staining area in plaque.4.The influence of M2BP on plaque development and stability and mechanisms of its inflammatory regulation4.1 In vivo study of the impact of M2BP silencing on plaque development and stabilityA total of 60 8-week-old male ApoE-/-mice were fed a high-fat diet for 8 weeks after 2-week adaptive feeding and then divided into 3 groups:(1)PBS control group(intravenous injected with 200ul PBS)、(2)Lenti-null group(intravenous injected with 200ul null lentivirus)、(3)Lenti-M2BP group(intravenous injected with 200ul lentivirus containing M2BP).All mice were fed for another 4 weeks before sacrifice.Blood was obtained and centrifuged to separate serum for detection of lipids and M2BP by Elisa.The mouse hearts were dissected and fixed in 4%paraformaldehyde overnight.Part of them were embedded in OCT compound to make cryosections for oil red staining.The other parts were embedded in paraffine to make paraffin sections for hematoxylin eosin staining、Sirius red staining and immunohistochemical staining(α-SMC、CD68、IL-6、MCP-1、ICAM-1、Lp-PLA2、MMP2、MMP9).The aortic trees(from aortic arch to abdominal aorta)were dissected from the heart and stained with Oil Red O for en face morphometric analysis of the atherosclerotic lesions.Frozen arteries were analyzed for protein expression by Western blot and for gene expression by RT-PCR.Image pro plus was used for quantitative analysis and comparison of positive staining area of different cytokines.The vulnerability index was calculated accordingly and compared across groups.4.2 In vitro study of the mechanisms of inflammatory regulation of M2BP1)Different concentrations of ox-LDL(0、10、20、40、80μg/ml)were used to stimulate THP-1 cells for different time points(6h and 24h).Conditioned media were collected for Elisa detection of M2BP.2)Different inflammatory cytokines(ox-LDL 20μg/ml、IL-1β 10ng/ml、TNF-α1 Ong/ml)were incubated with THP-1 cells alone or combinatorially for different time points(6h and 24h).Conditioned media were collected for Elisa detection of M2BP.3)Different inflammatory cytokines(ox-LDL20μg/ml、IL-1β10ng/ml、TNF-α10ng/ml)were incubated with HUVEC alone or combinatorially for 24h.Supernatants were collected for Elisa detection of M2BP.4)Different concentrations of rhM2BP(0、0.1、0.5、1、5μg/ml)were used to stimulate THP-1 cells for different time points(6h and 24h).Conditioned media were collected for Elisa detection of multiple inflammatory cytokines(IL-6,IL-8,TNF-a,MMP-9,Lp-PLA2).5)Different concentrations of rhM2BP(0、0.1、0.5、1、5μg/ml)were used to stimulate HUVEC for 24h.Supernatants were collected for Elisa detection of chemokines(IL-8 and MCP-1).6)Different concentrations of rhM2BP(0、0.1、0.5、1、5μg/ml)were used to stimulate HUVEC for 6h.Protein was extracted for detection of protein expression levels of adhesion molecules(ICAM-1、VCAM-1 and E-selectin).7)We performed cell adhesion assay to explore the effect of different concentrations of rhM2BP(0、0.1、0.5、1、5μg/ml)on the adhesion between THP-lcells and HUVEC.8)The migration of THP-1 cells were measured by Transwell migration assay after stimulation by different concentrations of rhM2BP(0、0.1、0.5、1、5μg/ml).9)We explored the expression and extracellular activity of MMP9 and MMP2 in THP-1 cells after rhM2BP stimulation.Furthermore,we investigated whether NF-kB and MAPK signal pathway were involved in this process via using specific inhibitors of NF-kB and MAPK signal pathway.Result:1.Plasma levels of M2BP in CAD patients and its prognostic value in ACS patientsPatients in SAP and ACS group were observed to own more risk factors(smoking、diabetes mellitus、hyperlipemia)than control group(p<0.001).Moreover,patients with CAD had a higher plasma M2BP level than control group(p<0.001).Likewise,plasma M2BP level was significantly elevated in patients with UAP than those in SAP and control group(p<0.001).However,in patients with ACS,no significant differences were found in patients with NSTEMI、STEMI and UAP patients regarding plasma M2BP level.There was no significant correlation between plasma M2BP and hs-TnT level via spearman correlation analysis(r=0.081,p=0.240).In patients with ACS,peripheral plasma M2BP level had a positive correlation with GRACE score(r=0.218,p=0.001)and risk stratification assessed by GRACE system.For randomly selected 30 SAP patients,plasma M2BP level increased significantly 1h after PCl,then decrease gradually to baseline at 24h.In addition,we found that M2BP plasma level had no significant association with the number of coronary artery lesion.Nevertheless,in ACS patients,plasma M2BP level was significantly correlated to the presence and degree of coronary complex lesions(p<0.001).During 1-year(11.7±2.7 months)follow-up of 216 ACS patients,a total of 45(20.8%)cases of MACEs occurred.Patients in MACE group were observed to have a significantly higher level of plasma M2BP than patients without MACE(p<0.001).Cox multivariate analysis revealed that plasma M2BP level was still an independent predictor of poor prognosis in ACS patients after adjustment for other clinical confounders.Furthermore,plasma M2BP combined with GRACE score can improve the diagnostic value of MACE in patients with ACS.2.The mRNA expression of M2BP in PBMC of CAD patients and its predictive value on coronary complex lesionsACS patients were observed to have an elevated mRNA expression level of M2BP in PBMCs than SAP group and healthy controls(p<0.001).Moreover,gene expression of M2BP in PBMCs tended to be higher in acute phase than chronic stable phase(p<0.001).Spearman correlation analysis demonstrated that mRNA expression of M2BP in PBMC was positively correlated with gene expression of inflammatory cytokines in PBMC and plasma level of proinflammatory mediators.The expression of M2BP in PBMCs was higher in patients with complex lesions(n=48)than simple lesions(n=12)(p=0.004).And there was a positive correlation between M2BP expression with numbers of coronary complex lesions.ROC analysis indicated that gene expression of M2BP in PBMCs had a better performance than plasma hs-CRP in diagnosis of coronary complex lesions(AUC:0.696 vs 0.588,p=0.109)in our cohort.Logistic multivariate analysis revealed that gene expression of M2BP in PBMCs was still independently predictive of coronary complex lesions after multivariate adjustment of clinical risk factors(OR=2.586,95%CI=1.124-5.948,p=0.025).3.The expression of M2BP in human carotid artery plaques and its association with plaque destabilizationOur study showed that M2BP was widely expressed in the nucleus、cytoplasm and extracellular space of carotid plaques,and was mainly distributed in extracellular space as a secretory factor.Immuohistochemical staining of CD68、α-SMC and M2BP demonstrated that M2BP was mainly colocalized with macrophages but was seldom expressed by SMC.Moreover,the expression of M2BP in ruptured type Ⅲ plaque and type II plaque with large lipid core was significantly higher than in type Ⅰ fibrous plaque.We also observed that M2BP was highly expressed in vulnerable sites including shoulder area、necrotic core and ruptured fibrous cap but weakly distributed in stable areas such as intact fibrous cap and fibrous matrix tissue within plaques.In addition,symptomatic patients tended to have an elevated expression of M2BP than asymptomatic ones especially those with ischemic symptoms within 1 month.Besides,we found that expression of M2BP was significantly higher in patients with TIA and stroke than patients with amaurosis fugax.In addition,M2BP expression levels showed a decreased trend with increased time since latest symptoms onset.It was also obvious that expression of M2BP was higher in echolucent and heterogeneous plaques than echogenic plaques.Spearman correlation analysis disclosed that M2BP positive staining area had a positive correlation with CD68+ macrophage positive staining area(p=0.374,r=0.007)and TUNEL positive staining area(r=0.329,p=0.018)but had a negative correlation with SMC positive staining area(r=-0.309,p=0.027)and collagen area(r=-0.304,p=0.030).4.The influence of M2BP on plaque development and stability and mechanisms of its inflammatory regulation4.1 In vivo study of the impact of M2BP silencing on plaque development and stabilityNo significant difference was found regarding body weight and peripheral blood lipid level(TC,LDL-c,HDL-c and TG)among the three groups of mice in our study.Compared with lenti-null group and PBS group,M2BP lentivirus transfection significantly reduced the gene and protein expression of M2BP in atherosclerotic plaques and systemically in peripheral blood(p<0.05),indicating that transfection was effective in silencing M2BP in vivo.The en face area analysis of the whole aorta stained by oil red showed that AS spectrum was significantly lower in lenti-M2BP group than in lenti-null group and PBS group.The same trend was observed in comparison of cross-sectional lesion area of aortic sinus.Immunohistochemical staining and special staining revealed that silencing M2BP in vivo significantly decreased the macrophage and lipid content in plaques.In contrast,the SMC and collagen staining area was increased accordingly.Thus M2BP knockdown deceased the vulnerability index of atherosclerotic plaques.Furthermore,immunohistochemical staining and RT-PCR analysis uncovered that the expression levels of adhesion molecule(ICAM-1)、proinflammatory cytokines(IL-6、MCP-1、Lp-PLA2)、matrix metalloproteinase(MMP9 and MMP2)and neovascularization(CD31、VEGFA、Flt-1)were significantly reduced after lentivirus transfection of M2BP(p<0.05).4.2 In vitro study of the mechanisms of inflammatory regulation of M2BPOx-LDL was able to upregulate M2BP expression in THP-1 cells in a dose-dependent and time-dependent manner.Multiple inflammatory factors(ox-LDL 20μg/ml、IL-1β10ng/ml、TNF-α10ng/ml)could increase the expression of M2BP in THP-1 cells and HUVEC dose-dependently and/or time-dependently,with a particularly enhancing effect when these stimuli were combined.Likewise,recombinant protein rhM2BP stimulation was capable of upregulating the synthesis and secretion of inflammatory cytokines(IL-6,IL-8,TNF-α,MMP-9,Lp-PLA2)in THP-1 monocytes.Besides,rhM2BP increased the secretion of chemokines(IL-8 and MCP-1)and protein expression of adhesion molecules(ICAM-1、VCAM-1 and E-selectin)in HUVEC dose-dependently.Furthermore,rhM2BP could enhance the chemotaxis of THP-1 monocytes and adhesion between HUVEC with THP-1 monocytes.We also found that rhM2BP could upregulate the expression and extracellular activity of MMP9 and MMP2 in THP-1 cells in a dose-dependent and time-dependent manner.Stimulation with 2ug/ml rhM2BP for 1h was ’able to induce significant phosphorylation and activation of NF-kB and MA PK(ERK、p38 and JNK)in THP-1 monocytes.Furthermore,inhibition of NF-kB and MAPK signaling pathways with their specific inhibitors could significantly downregulate the expression and activity of MMP9 and MMP2.Conclusion:(1)M2BP was highly expressed in peripheral blood of ACS patients and was associated with coronary complex lesions.The plasma M2BP level had an independent prognostic value in ACS patients.Besides,plasma M2BP level combined with GRACE score can improve the prognostic value of ACS.(2)PBMC is an important cytological source of peripheral blood M2BP.The mRNA expression of M2BP in the PBMC of ACS patients was significantly increased and it was proven to own a good diagnostic value and independent predictive value for coronary complex lesions.(3)M2BP is highly expressed in unstable plaques and vulnerable regions and significantly correlated with clinical manifestations of carotid plaques,indicating that M2 BP was involved in the pathogenesis of plaque destabilization.(4)M2BP could promote the development and destabilization of atherosclerotic plaques via upregulating the expression of proinflammatory cytokines、chemokines and adhesion molecules and enhancing adhesion between monocytes and vascular endothelium、chemotaxis of monocytes and neovascularization within plaques.(5)M2BP increased the expression of MMP2 and MMP9 in monocytes and macrophages by activating intracellular NF-kB p65 and MAPK(P38,ERK and JNK)signaling pathway to facilitate collagen degradation,thereby promoting the vulnerability of atherosclerotic plaques. |
PDF Full Text Request