| [Background and Objectives]Atherothrombosis plays a leading role in the pathogenesis of acute myocardial infarction(AMI),however,its mechanism is still not fully elucidated.This study aims to analyze the correlation between bactericidal/permeability-increasing protein(BPI)and NETosis firstly from the clinical perspective,through measuring the plasma levels of BPI,S100 calcium-binding protein A8/A9 complex(S100A8/A9)and myeloperoxidase-DNA complex(MPO-DNA)in different types of coronary heart disease(CHD),especially in AMI patients.Then,we try to verify the hypothesis that BPI promotes coronary atherothrombosis by activating platelets and enhancing NETosis in vitro and in vivo.Part Ⅰ Differential expression of plasma BPI associates with NETosis in AMI[Materials and Methods]1.This study enrolled a consecutive series of patients prospectively,who were admitted to the First Affiliated Hospital of Bengbu Medical College for coronary angiography(CAG)and/or percutaneous coronary intervention(PCI)between May 1 and August 31,2020.Individuals with ST-segment elevation myocardial infarction(STEMI),non-ST segment elevation myocardial infarction(NSTEMI),unstable angina pectoris(UAP),coronary atherosclerosis(CAS),and normal control(Control)were included according to inclusion criteria and exclusion criteria.Blood samples were both collected within 30 minutes after admission and at discharge.2.The plasma levels of BPI,high sensitivity C-reactive protein(hs-CRP),Interleukin-1β(IL-1β),MPO-DNA,and S100A8/A9 were measured by enzyme-linked immunosorbent assay(ELISA).3.The plasma levels of BPI,hs-CRP,IL-1β,MPO-DNA and S100A8/A9 in the AMI group(STEMI/NSTEMI)were compared with the control group.The severity of coronary atherosclerotic lesion was calculated based on the Gensini scoring system.Spearman’s correlation and unary linear regression were analyzed to reveal the correlations among the plasma BPI level with peripheral blood neutrophil counts,peripheral blood platelet counts,the plasma levels of hs-CRP,IL-1β,MPO-DNA,S100A8/A9,and the Gensini score in AMI patients.Receiver operating characteristic(ROC)curve was depicted to analyze the diagnostic efficacy for AMI of plasma BPI.4.The plasma levels of BPI,hs-CRP,IL-1β,MPO-DNA,and S100A8/A9 in different types of CHD were measured.Spearman’s correlation and unary linear regression were used to analyze the correlations among the plasma level of BPI with the plasma levels of hs-CRP,IL-1β,MPO-DNA,and S100A8/A9,and the Gensini score.Major adverse cardiovascular events(MACEs)were defined as recurrent myocardial infarction,hospitalization due to heart failure,recurrent angina,ischemic stroke,and all-cause death.All of patients including STEMI,NSTEMI,UAP,and CAS,were followed-up.Cox proportional hazards regression model was performed to detect the causality between plasma BPI and MACEs.The patients were divided into two groups:high-BPI group and low-BPI group,according to the median value of plasma BPI.Coagulation parameters including activated partial prothrombin time(APTT),D-dimer,fibrinogen,and international normalized ratio(INR)were compared between the high-BPI group and the low-BPI group.A Kaplan-Meier MACEs-free curve was used to compare MACEs episodes between two groups.Log-rank test was used to compare two MACEs-free survival curves.[Results]1.One hundred and twenty-two cases of AMI,134 cases of UAP,64 cases of CAS and 80 cases of Control were eventually included in this study.The patients with AMI were divided into STEMI(n=59)and NSTEMI(n=63).Compared with the control group,the plasma level of BPI in AMI group was higher[50.83(38.53,62.33)ng/mL vs 5.45(3.62,7.65)ng/mL,P<0.001].The STEMI group also had significantly higher plasma BPI level than the NSTEMI group(P<0.001).The plasma BPI level was positively correlated with peripheral blood neutrophil counts(rs=0.500,P<0.004),but not with peripheral blood platelet counts(rs=0.094,P=0.191)in AMI patients.The plasma BPI level was positively correlated with the plasma levels of hs-CRP,IL-1β,MPO-DNA,S100A8/A9(rs=0.834,P<0.001;rs=0.783,P<0.001;rs=0.740,P<0.001;rs=0.679,P<0.001),and with the Gensini score(rs=0.791,P<0.001)in AMI patients.2.The optimal cut-off value of plasma BPI was 35.17 ng/mL for AMI based on the ROC curve analysis.The ROC analysis also showed that BPI,CK-MB,and TnI all revealed diagnostic value for AMI.The values of area under the curve(AUC)of BPI,CK-MB,and TnI were 0.938(0.893～0.983),0.709(0.610～0.807),and 0.824(0.747～0.901)respectively.There was a significant difference between BPI and CK-MB in the diagnostic efficacy for AMI(BPI vs CK-MB:Z=3.673,P=0.0001).3.The plasma levels BPI and hs-CRP,IL-1β,MPO-DNA,S100A8/A9 in different types of CHD were significantly higher than that in the control group.The plasma BPI level was positively correlated with the plasma levels of hs-CRP,IL-1β,MPO-DNA,S100A8/A9,and with the Gensini score in patients with varied degrees of coronary atherosclerotic lesion.4.According to the median value of plasma BPI concentration 41.10 ng/mL,the patients with varied degrees of coronary atherosclerotic lesion were divided into two groups:high-BPI group(BPI>41.10 ng/mL)and low-BPI group(BPI ≤41.10 ng/mL).Compared with the low-BPI group,APTT and INR were lower in the high-BPI group.Meanwhile,the plasma levels of D-dimer and fibrinogen elevated significantly in the high-BPI group(all P<0.05).After following-up 22.18 months,62 patients(62/320,19.38%)developed MACEs,including 42 patients(42/160,26.25%)in the high-BPI group and 20 patients(20/160,12.5%)in the low-BPI group.In the high-BPI group,MACEs were increased significantly,compared with that in the low BPI group(log-rank test χ2=7.803,P=0.021).Cox analysis showed that BPI was an independent risk factor for MACEs in patients with varied degrees of coronary atherosclerotic lesion.Part Ⅱ BPI promotes NETosis by activating platelets[Materials and Methods]1.Fifteen cases of 30 mL blood samples were collected randomly in the AMI group and the control group,to prepare platelet-rich plasma(PRP).Platelets were labeled with anti-CD41-FITC and anti-CD62p-PE antibodies,and the proportions of CD41+/CD62p+activated platelets in the control group,low-concentration BPI group,(40 ng/mL),high-concentration BPI group(400 ng/mL),AMI group,and AMI+BPI antibody group(5μg)were analyzed by flow cytometry.Human peripheral blood neutrophils were isolated and identified by flow cytometry with CD177 antibody.The NETs structure was observed by confocal microscopy after MPO/HistoneH3 double immunofluorescence staining in the AMI group and the control group.2.Thrombus tissue was aspirated from infarction-related artery of STEMI patients during PCI.Platelet membrane glycoprotein(GP)Ⅱb(CD41)was stained with anti-CD41 antibody.The co-expression of CD41 and BPI in coronary atherosclerotic thrombus tissues was observed by laser confocal microscope.Triple immunofluorescence staining(MPO+Histone H3+S100A8/9)was used to observe the structural components of NETs and the co-expression of S100A8/9 protein in coronary atherosclerotic thrombosis.3.Fresh anticoagulant whole blood was collected from the celiac artery of wild-type C57BL/6 mice at 8～10 weeks to prepare PRP.Platelets were labeled with anti-CD41-FITC and anti-CD62p-PE antibodies,and the proportions of CD41+/CD62p+activated platelets in the control group and BPI-pretreated group(40 ng/mL)group were analyzed by flow cytometry.Platelet aggregation function in mice was observed by spreading experiment.The expression of p-NF-κB and p-selectin in BPI-activated platelets was tested by Western Blot.4.Mice peripheral blood neutrophils were isolated and identified by flow cytometry with Ly6C antibody.Mice platelets with and without BPI pre-treatment were co-cultured with mice neutrophils.Flow cytometry was employed to detect the reactive oxygen species(ROS)production in neutrophils.The NETs structure was observed by confocal microscopy after MPO/HistoneH3 double immunofluorescence staining.NETosis and the release of S100A8/A9 were viewed by confocal microscopy after MPO/HistoneH3/S 100A8/9 triple inununofluorescence staining.The concentration of S100A8/A9 protein in the cell co-culture supernatant was measured by ELISA.5.Wild-type C57BL/6 mice were randomly grouped:sham control group,model group and BPI pre-treatment group.Ferric chloride(FeCl3)induced thrombosis in the common carotid artery of mice.The mice in BPI pre-treatment group were injected with mouse BPI recombinant protein(200 μL,0.4 mg/mL)through the tail vein one day before the model establishment.The time and length of common carotid artery thrombosis were observed.Immunofluorescence staining(CD41+Fibrin/MPO+Histone H3)was used to observe the co-expression of activated platelets and Fibrin,and the NETs structure in the thrombus tissue.Western Blot was used to test the expression of CD41 and Fibrinogen protein in thrombus tissue.[Results]1.The proportions of CD41+/CD62p+activated platelets in the control group,low-concentration BPI group(40 ng/mL),high-concentration BPI group(400 ng/mL),AMI group,and AMI+BPI antibody group(5μg)were 13.9%,28.8%,38.3%,38.1%and 22.1%,respectively.The proportion of activated platelets in the control group increased after being pretreated with BPI(P<0.001;vs Ctrl).Compared with the control group,the proportion of activated platelets in AMI group was higher(P<0.01;vs Ctrl),and correspondingly declined after being pretreated with BPI antibody.The proportion of NETosis in neutrophils discharging the structure of NETs(MPO/Histone H3 double immunofluorescence staining)was higher in peripheral blood of patients with AMI,compared with the control group,observed by laser confocal microscopy.The co-expression of BPI,CD41,NETs structural components and S100A8/9 protein was found in coronary atherosclerotic thrombus tissue from infarction-related artery of STEMI patients,observed by laser confocal microscopy.2.The proportion of CD41+/CD62p+activated platelets in mice increased after being pretreated with BPI(P<0.01;Ctrl vs BPI:16.4%vs 34.7%).In mice,BPI-activated platelets spread more significantly observed with a Leica SPE microscope,from round to filamentous or irregular,and increased in volume.In mice,BPI-activated platelets expressed a higher quantity of p-selectin and p-NF-κB protein than the control group.3.In mice,neutrophils have higher ROS production after co-incubation with BPI-pretreated platelets compared with the untreated platelets and the control group detected by flow cytometry.The proportion of neutrophils presented with NETs structure was higher compared with the untreated platelets and the control group,observed by laser confocal microscopy,and the secretion of S100A8/A9 protein by neutrophils was up-regulated.4.In the mice model of FeCl3-induced common carotid artery thrombosis,the time of common carotid artery thrombosis was shorter in BPI-pretreated group(BPI-pretreated group:54.33 ± 6.41 sec vs Model group:70.50 ± 7.58 sec,P<0.005),and length was longer compared with the model group(BPI-pretreated group:4.12 ± 0.68 mm vs Model group:3.12± 0.36 mm,P<0.01),The expression of CD41 and fibrin in thrombus tissues from BPI-pretreated group was significantly higher,and the area of CD41-fibrin co-localization was larger compared with the model group.Meanwhile,the expression of MPO and Histone H3 in thrombus tissues from BPI-pretreated group was significantly higher,and the area of MPO-Histone H3 co-localization(NETs structure)was larger compared with the model group.The expression of CD41 and fibrinogen proteins in thrombus tissues was higher in BPI-pretreated group compared with the model group.These results indicated that platelet GPⅡb/Ⅲa receptors were activated,whose binding ability to fibrinogen was enhanced,and NETosis was facilitated.[Conclusions]1.In patients with different types of CHD,the plasma BPI level was up-regulated and positively correlated with the severity of inflammation and coronary atherosclerotic lesion(Gensini score).At the same time,the elevated plasma BPI level was associated with an increased propensity for thrombosis and increased incidence of MACEs.In AMI patients especially,the plasma BPI level was up-regulated and positively correlated with NETosis biomarkers.These results suggest that there may be some possible mechanisms underlying how BPI promotes NETosis and coronary atherothrombosis.2.In vitro,BPI could activate platelets,enhance platelets aggregation and spreading,and up-regulate the expression of p-selectin and p-NF-κB in platelets.BPI-activated platelets increase ROS production in neutrophils,enhance NETosis and the release of S100A8/A9 protein,facilitate atherothrombosis through platelet-neutrophil interaction.In vivo,BPI could activate platelets effectively,increase the binding between platelets and fibrinogen,enhance NETosis,and promote arterial thrombosis in the mice model of FeCl3-induced common carotid artery thrombosis.3.Clinically,BPI may be a promising biomarker for AMI,and may provide a reliable reference for the early diagnosis of AMI,disease evaluation,new drug development and personalized precision therapy. |
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