The Expression Characteristics Of Leukocyte Microparticles In CAS Population And The Mechanism Of Induction Of Macrophage Polarization

BackgroundMacrophages are the most important inflammatory cells in atherosclerotic plaques.The imbalance of macrophage subtypes of classical activated type(pro-inflammatory M1 type)and alternative activated type(anti-inflammatory M2 type)is closely related to the development and outcome of atherosclerotic plaques.Microparticles(MPs),tiny vesicles generated by activated or apoptotic cells,are not only involved in the pathophysiological process of the body,but also have potential value in predicting and diagnosing diseases.Previous study found that leukocyte-derived microparticles(LMPs)were significantly increased in early carotid atherosclerosis(CAS)patients and were strongly close to the carotid artery intimal-media thickness(CIMT)and plaque index.However,are LMPs promoting the development of AS related to its regulation of macrophage polarization?What is the mechanism of macrophage polarization regulating by LMPs?These have not been reported yet.Syndrome element is the basic element of TCM syndrome.Whether there is any difference of LMPs level among different syndromes elements in CAS patients remains to be explored.In this study,the clinical trials and cell experiments were performed to explore the clinical value of LMPs in patients with CAS and the mechanism of LMPs regulating macrophage phenotype,to provide a new research idea for the prevention and treatment of AS by integrated chinese and western medicine.Aims1.Clinical trials were conducted to analyze the expression characteristics of LMPs in the peripheral circulation of CAS patients and its correlation with carotid atherosclerotic plaque,the level of monocytes-macrophages subtypes,and the difference of LMPs,et al.among different syndromes,to evaluate the clinical significance and value of LMPs in early CAS patients.2.Cell experiments were performed to study the molecular mechanism of macrophage polarization inducing by LMPs which produced by macrophages/foam cells,providing new molecular targets for prevention and treatment of AS.Methods1.The methodological investigation of MPs:Peripheral venous blood from 5 healthy subjects was collected,and platelet-free plasma was collected by gradient centrifugation method.Blank controls,isotype controls(ICs),CD3,CD11a,Fc-block,Fc+ICs,Fc+CD3 and Fc+CD11a were tested by flow cytometry(Cytoflex LX flow cytometry).The mean fluorescence intensity(MFI)was analyzed to compare the effect of ICs with Fc-block in reducing non-specific binding and background signal in detecting MPs,so as to provide basis for the next research.2.Collection,detection and analysis of clinical LMPs:According to inclusion and exclusion criteria,12 patients with early CAS and 13 healthy subjects were selected from Dongzhimen hospital of Beijing university of Chinese medicine.Carotid ultrasound was used to measure CIMT,plaque thickness at the crosscut and plaque index of the subjects.Peripheral venous blood was collected and platelet-free plasma was obtained by gradient centrifugation.LMPs,including CD14+monocyte microparticles(mMPs),CD66b+neutrophil microparticles(NMPs),CD3+ lymphocyte microparticles(LyMPs)and CD142+ tissue factor microparticles(TF-MPs),were detected by Cytoflex LX flow cytometry.To observe the expression characteristics of circulating LMPs in subjects and analyze the correlation between LMPs and CIMT,plaque thickness at the crosscut and plaque index.3.Monocyte-Macrophage collection,subtype detection and analysis:The subjects belong to the same group as method 2.CD 14+ CD163-and CD 14+CD163+were used to tag M1 and M2 monocyte-macrophage respectively in peripheral blood.The level of monocytemacrophage subtype was test by FC500 flow cytometry and correlation between M1,M2 monocyte-macrophage subtypes and LMPs,plaque lesion was analyzed.4.Literature research about syndromes distribution of CAS:Taking "carotid artery","carotid atherosclerosis","carotid artery plaque","carotid atherosclerosis plaque","carotid artery atheromatous plaque" and "carotid atherosclerotic plaques","the doctor of traditional Chinese medicine","Chinese medicine" and "Chinese medicine","syndrome","syndrome element","dialectic","disease","pathogenesis" and "treatment" as subject terms to search CAS relevant literatures in CNKI,the data resources of WANFANG.The literatures were screened according to inclusion and exclusion criteria,and the TCM syndrome elements in the included literatures were extracted and analyzed to obtain the TCM syndrome distribution rule of CAS.5.Comparative analysis of LMPs level in patients with qi deficiency、blood stasis and phlegm turbidity:The CAS group sample size was expanded to 20 patients based on the former inclusion and exclusion criteria.according to the syndrome elements of CAS obtained from literatures research three major syndrome elements "qi deficiency","blood stasis" and "phlegm turbidity" were selected to observe their distribution in the enrolled population.The level of LMPs,monocyte-macrophage,et al.among the syndromes was also compared and analyzed.6.Induction of foam cells and identification of LMPs in vitro:THP-1 were used to induce macrophages for 24h with 100ng/mL Phorbol 12-myristate 13-Acetate(PMA),followed by 80ug/mL oxidized low-density lipoprotein(ox-LDL)for 48h to establish the foam cell model.Then cholesterol ester measurement and oil red O staining were performed to identify the foam cells.LMPs derived from foam cells were extracted and identified by flow cytometry and transmission electron microscopy.7.Detection of macrophage polarization and related signaling pathways by LMPs:The LMPs derived from foam cells were labeled by fluorescence and co-cultured with macrophages.It contained two different concentrations groups,1 ×106 LMPs/mL(106 LMPs),1 ×107 LMPs/mL(107 LMPs)and control group with no LMPs.The relationship between LMPs and macrophages was observed by laser confocal,and the phenotypes of M1 and M2 macrophages were detected by flow cytometry.The immunofluorescence was used to observe the effect of LMPs on the fluorescence intensity and nuclear translocation of macrophage NF-κB p65.The change of phospho-IKKα/β(p-IKKα/β)was detected by western blotting to elucidate the mechanism of macrophage polarization inducing by LMPs.Results1 The methodological investigation of MPs:The MFI of ICs group,CD3 group and CD11a group was significantly higher than that of blank group(P<0.01),and the MFI of the ICs group was lower than that of CD3 group but significantly higher than that of CD11a group.If ICs were used as a gate,positive results would appear in CD3 group and false negative results would present in CD11a group.After applying Fc-block,MFI in CD3 group did not change significantly while MFI in the Fc+CD11a group was significantly lower than that in CD11a group and higher than blank group(P<0.01).It indicated that Fc-block could block the non-specific binding of CD11a antibody whereas had no effect on CD3.2 Expression characteristics of LMPs in early CAS patients and their correlation with the parameters of carotid ultrasound:The results found that the level of LMPs(including CD14+mMPs,CD66b+NMPs,CD3+LyMPs)and CD142+MPs in peripheral blood increased in CAS patients.There was statistically significant difference in the level of CD14+mMPs(P<0.01).Correlation analysis showed that CD14+mMPs and CD66b+NMPs were positively correlated with CIMT(P=0.026,r=0.485;P=0.025,r=0.488,respectively).In addition,CD3+LyMPs and CD66b+NMPs were significantly linked to the plaque thickness at the crosscut(P=0.005,r=0.874;P=0.011,r=0.826,respectively).CD142+MPs were not only highly correlated with the plaque thickness at the crosscut(P=0.001,r=0.922),but also correlated with the plaque index(P=0.094,r=0.504).3 Correlation between LMPs and monocyte-macrophage subtypes in early CAS patients:Compared with healthy group,the tendency of CD14+CD163-M1 and the ratio of M1/M2 monocyte-macrophage increased in CAS patients(P=0.053 and P=0.09).The level of CD 14+CD163+M2 monocyte-macrophage in healthy group was slightly increased compared with CAS group but the difference was not statistically significant(P>0.05).Correlation analysis showed that M2 monocyte-macrophage was weakly correlated with CD142+MPs(P=0.099,r=-0.352).Additionally,the number and the ratio of M1/M2 monocyte-macrophage had no correlation with MPs(P>0.05).4 The Literatures research on the distribution of TCM syndrome elements in CAS patients:According to inclusion and exclusion criteria,25 literatures were screened,involving 5782 patients.A total of 13 syndrome elements "phlegm turbidity","blood stasis","qi deficiency","yin deficiency","heat evil","qi stagnation","yang-preponderance","blood deficiency","yang deficiency","endogenous wind","dampness evil","kidney essence deficiency" and "poison evil" were obtained.The syndrome elements of CAS patients were mainly phlegm turbidity,blood stasis and qi deficiency,accounting for 49.20%、45.19%and 24.28%respectively.Yin deficiency(9.60%)accounted for the highest proportion of stable plaques while phlegm turbidity(30.43%)had the highest frequency of occurrence in unstable plaques.Blood stasis was the most common factors in mild,moderate and severe carotid stenosis.In addition,statistical analysis of plaque location and syndrome distribution frequency showed that no matter the plaque was located in the common carotid artery,internal carotid artery,carotid dilatation or bifurcation of the carotid artery,phlegm turbidity occurred most frequently,followed by blood stasis or qi deficiency.5 Comparison of LMPs level and other parameters of CAS patients with qi deficiency、phlegm turbidity and blood stasis:Three syndrome elements "phlegm turbidity" "blood stasis" and "qi deficiency" were selected to study the distribution of syndrome elements in the included subjects.Among the 20 CAS patients,blood stasis was the predominant syndrome elements(100%),followed by phlegm turbidity(70%)and qi deficiency(40%).14 patients presented two syndrome elements,including 10 patients(50%)with "blood stasis" and "phlegm turbidity",4 patients(20%)with "qi deficiency" and "blood stasis".4 cases(20%)with "blood stasis","qi deficiency" and "phlegm turbidity".However,statistical analysis found that there was no difference in the parameters of LMPs,et al.among the three syndromes of "blood stasis","qi deficiency" and "phlegm turbidity"(P>0.05).6 Induction of foam cells and identification of LMPs6.1 Induction and identification of foam cells:After culturing macrophages with 80μg/mL ox-LDL for 48 hours.The content of cholesterol esters was accounted for 50.2%,56.7%,and 48.64%of the total cholesterol,which could reach 50%basically.Oil red O staining showed a large number of red lipid particles in the cells,indicating that the experimental conditions could fundamentally transform macrophages to foam cells.6.2 Identification of LMPs:Extracting vesicles derived from foam cells and the results of flow cytometry analysis showed that annexin-V positive expression of MPs marker was abundant in 100nm-1000nm gate.Furthermore,circular vesicles with size over 100nm could be seen under transmission electron microscope.The above results indicated that the vesicles extracted in this experiment were LMPs.7 Effect of LMPs on macrophage polarization and changes in related signaling pathway proteins7.1 Effect of LMPs on macrophage subtypes:LMPs derived from foam cells were cocultured with macrophages,and laser confocal microscopy showed that LMPs were in close contact with macrophages.Under the microscope,different concentrations LMPs had no obvious effect on the morphology of macrophages.They were all irregular adherent cells such as disk-shaped and fusiform cells,with pseudopodia stretching out and agglomerating.Flow cytometry showed that,compared with the control group(48.87%±5.29%),the proportion of CD11b+CD11c+CD206-M1 macrophage in the 106LMPs group and 107 LMPs group were significantly increased(69.49%±3.91%,67.4%±1.9%),and the difference was statistically significant(P<0.01).There was no difference in the level of M1 macrophage induced by 106LMPs and 107LMPs(P>0.05).The ratio of M1/M2 macrophage was also increased in 106LMPs and 107LMPs group than that of control group,and there was a difference between 107LMPs and control group(P<0.05).The level of CD11b+CD11cCD206+M2 macrophage elevated in control group,which higher than that in 106LMPs and 107LMPs groups(P=0.096).7.2 Effect of LMPs on the polarization related signaling pathways of macrophages:The immunofluorescence showed that the fluorescence intensity of NF-κB p65 enhanced with the increase of LMPs concentration.According to the quantitative analysis of MFI in each group by Image-Pro Plus,compared with the control group,MFI in 106LMPs group showed an increasing trend(P=0.086)and significantly increased in 107LMPs group(P<0.01).Compared with the 106LMPs group,the MFI of the 107LMPs group statistically significant rose(P<0.05).The level of p-IKKα/β protein increased in 106LMPs group(P=0.094)and 107LMPs group(P<0.01)by western blot while the protein level of 107LMPs group was higher than that of 106LMPs group(P=0.05).Conclusions1.Both ICs and Fc-block have limitations for detecting MPs.In the research,ICs or Fc-block should be selected according to the sample type and antigen type through pre-trial.2.The level of LMPs and CD142+MPs increased in early CAS patients,and have closely correlation with plaque lesion.3.The level of M1 and M2 monocyte-macrophage are unbalanced in early CAS patients.Moreover,MPs are correlated with monocyte-macrophage subtypes,and may be involved in regulating the subtypes of monocyte-macrophage.4."Phlegm turbidity","blood stasis",and "qi deficiency" are the main syndrome elements of AS,which is consistent with the understanding of the pathogenesis of AS.The included CAS patients in this trial are mostly characterized by "blood stasis and phlegm turbidity",and there is no difference in the parameters of LMPs,et al.among different syndromes.5.The foam cell-derived LMPs can promote the polarization of macrophages toward M1 subtypes,and inhibit macrophage polarization toward M2.The mechanism of foam cellderived LMPs promote the polarization of M1 macrophages may be related to the activation of IKKα/β/NF-κB pathway,suggesting that LMPs are able to promote the inflammatory response of AS.