The Association Between Changes Of Blood Immune Indexes And Acute Coronary Syndrome

Part I The association between hematological indices of ACS and outcome 1.Background The deaths arising from Coronary atherosclerotic disease(CAD)have ranked second across the world with the development of economy and living standard in China.Advanced clinical symptoms during terminal stage of CAD have been found,including acute attack and dramatic deterioration.The outcome is not ideal without any medical intervention.Clinically,acute coronary syndrome(ACS)is defined as critical and terminal stage of CAD accompanying with unstable and dramatic deterioration.Definitely,ACS,as the worst type of CAD is divided into three subtypes,which challenge ability of physicians based on elevation of ST segment from ECG,ST-segment elevation myocardial infarction(STEMI),non-ST segment elevation myocardial infarction(NSTEMI)and unstable angina(UA).However,during clinical practice,the number of patients suffering ACS but not treated timely is still large,especially in China,therefore,the outcome of ACS is various.It is more difficult to perform risk classification of NSTEMI and UA.Currently,it is predominate to seek potential and optimized predictive indexes reflecting disease outcome to predict potential risk.It has been supposed that immune cells play an important role during development of ACS.More studies have focused on the effect of immune cells and inflammatory factors in evaluation of risk classification for ACS in spite of controversy.Recently,the diagnostic and predictive value of some hematological biomarkers has been assessed and confirmed in many studies,because they are more economical and available.2.Objective To explore potential indexes of ACS and predict development of disease,the biomarkers of patients suffering three subtypes of ACS were analyzed retrospectively including Neutrophil-to-lymphocyte ratio(NLR),Platelet-to-lymphocyte ratio(PLR),White blood cell count(WBC),Monocyte percentage(MO),Platelet width(PLW),myocardium injury,occlusion of coronary artery,stent implantation and 30-day mortality.3.Methods 3.1 Subjects A total of 309 patients suffering ACS admitted from Jun 2014 to Jun 2016 in 1st affiliated hospital of An Hui Medical University were enrolled and analyzed retrospectively.All patients were divided into three groups based on ST changes,50 in UA group,59 in STEMI group and 100 in NSTEMI group.ACS was diagnosed by history,CAG,EKG,echocardiography and biomarkers.3.2 Statistics Database was established by Excel 2007 and analysis was performed by SPSS 23.0.Mean ± standard deviation was used to describe quantitative data as normal distribution.Inter-group comparison was performed by t or chi-square test and non-parametric test.M(P25,P75)was used to describe quantitative data as skewed distribution.When significance of inter-group was found,pair comparisons among multi-groups were performed by Pairwise Comparisons;Pearson correlation coefficient was used to measure association among variables for quantitative with normal distribution,while,Spearman correlation coefficient was used to measure association among variables for quantitative with skewed distribution.Constituent ratio or frequency was used to describe qualitative data.Significant variables by univariate analysis(P<0.10)were set as independent variables and multivariate non-conditional logistic regression was used to screen risk factors of 30-day mortality.P<0.05 was significant.4.Results(1)It was shown by Spearman that there was positive association between NLR,PLR,WBC and c Tn I.The difference was significant(P<0.05).However,there was no association between MO and c Tn I.(2)It was shown by non-parametric test that there was no significance between NLR,PLR,PLW,MO,WBC and PCI treatment.(3)It was shown by non-parametric test that there was significance between NLR,PLR,MO,WBC and PCI treatment except for PLW.Meanwhile,compared with non-emergent PCI treatment,NLR,PLR and WBC of patients receiving emergent treatment were higher significantly,however,MO decreased significantly.Furthermore,In STEMI and NSTEMI group,it was shown that there was association between NLR,PLR and PCI treatment,compared with non-emergent PCI treatment,NLR and PLR of patients receiving emergent treatment were higher significantly,while,in UA group,compared with non-emergent PCI treatment,NLR,PLR and PLW of patients receiving emergent treatment were higher significantly,however,MO decreased significantly.(4)It was shown by non-parametric test that there was significance between NLR,PLR,MO,WBC and 30-day mortality except for PLW.Meanwhile,compared with non-mortality,NLR,PLR and WBC in 30-day mortality were significantly,however,MO decreased significantly.Furthermore,In STEMI and NSTEMI group,it was shown that there was association between NLR and 30-day mortality,compared with non-mortality,NLR of patients with 30-day mortality was higher significantly,while,in UA group,compared with with non-mortality,NLR,PLR and WBC of patients with 30-day mortality were higher significantly,however,MO decreased significantly.It was indicated NLR,PLR,MO and WBC could predict the mortality of ACS,especially for NLR.(5)SYNTAX scores were dividd into three arms based on international protocol,0-22 for low risk arm,23-32 for middle risk arm and ≥33 for high risk arm.It was shown by non-parametric test that there was no significance among NLR,PLR,PLW,MO and WBC(P>0.05).(6)It was shown by multivariate logistic regression that there was association between c Tn I,NLR,PLR,MO and 30-day mortality,among which,c Tn I,PLR and NLR were risk factors of 30-day mortality.(7)It was shown by multivariate logistic regression that NLR was the most significant predictive factor of 30-day mortality,PLR and c Tnl were less and MO was at least,based on ROC curves of c Tn I,NLR,PLR and MO.5.Conclusion It is supposed that NLR is an economical and available predictive factor of ACS and it can be more effective when combined with PLR,PLW,MO and WBC.Part II The change of peripheral blood immune cells in acute coronary syndrome 1.Background It is well known that atherosclerosis is chronic inflammatory disease by innate immunity and adaptive immunity.Immune cells play an important role in etiology of disease.Acute coronary syndrome(ACS),as terminal stage of coronary atherosclerotic disease(CAD)is one of the most life-threatening cause in the world.The main feature of ACS is more unstable and fragile plaque compared with stable angina pectoris.Although most immune cells are effective for fragile plaques,however,the concrete mechanism is still unknown.Definitely,patients suffering ACS are the most ideal target when association between immune cells and fragile plaques is studied.Last decade,a series of immune cells including macrophages and different subsets of lymphocytes have been confirmed as agents involving chronic inflammatory reaction and promoting development of ACS.After part I,it has been concluded that hematological indexes can be qualified in predicting outcome of ACS,which means lymphocyte,monocytes,platelets are possible targets and may be critical during the process.Nevertheless,the association between different subsets of immune cells and three subtypes of ACS remains unclear,let alone the mechanism.2.Objective To explore the change of main immune cells from ACS including subsets of monocytes,T-cells and inhibitory MDSCs extracted from bone marrow and initially evaluate possible mechanism.3.Methods3.1 Subjects A total of 50 patients suffering ACS admitted in 1st affiliated hospital of An Hui Medical University from Jun 2016 to Oct 2016 and another 19 healthy subjects were enrolled.Patients were divided into two groups based on attack of acute infarction,AMI subgroup representing infarction including STEMI and NSTEMI,UA subgroup representing unstable angina.Thirdly,subgroup of healthy subjects was labeled as NG.ACS was diagnosed by history,CAG,EKG,echocardiography and biomarkers of myocardium injury.3.2 Methods Flow cytometry was used to assay subsets of monocytes,T-cells and inhibitory cells from bone marrow.CD14,CD16 and CD45 were used to label subset of monocytes.CD4,CD8 and CD3 were used to label subset of T-cells and CD14,HLA-DR and CD45 were used to label inhibitory MDSCs from bone marrow.Meanwhile,among monocytes,flow cytometry was also used to assay subsets of CD11b+CD206+,CD11b+CD68+ cells.3.3 Statistics Database was established by Excel 2007 and analysis was performed by SPSS 23.0.Mean ± standard deviation was used to describe quantitative data representing normal distribution.Chi-square test was used to compare the difference of inter-groups.If significance was found,SNK was used for pair comparison.M(P25,P75)was used to describe quantitative data representing skewed distribution.Non-parametric test was used to compare the difference of inter-groups,If significance was found,Pairwise Comparison was used for pair comparison.Pearson correlation coefficient was used to measure correlation among variates of quantitative data representing normal distribution,while,Spearman correlation coefficient was used to measure correlation among variates of quantitative data representing skewed distribution.P<0.05 was significant.4.Results 1)There was no significance among three groups in gender and age;2)It is indicated by univariate analysis that percentage of lymphocytes/karyocytes,CD3+CD4+T,percentage of mononuclear cells/karyocytes,CD14+CD16-,CD14+CD16+ and CD11b+CD68+ were significant in all three groups;3)General percentage of lymphocytes in three groups: NG>UA>Infarction.There was significance in pair comparison between AMI and UA as well as AMI and NG(P<0.05),however,there was no significance in pair comparison between UA and NG(P>0.05);4)The percentage of CD3+CD4+T/lymphocytes: Infarction>UA>NG.There was significance in pair comparison between AMI and NG(P<0.05),while,there was no significance in pair comparison between AMI and UA as well as UA and NG(P>0.05).However,there was no significance on CD3+T,CD3+CD8+T,CD4+/CD8+,CD4+CD8+ and CD4-CD8-in all three groups;5)The percentage of CD14+/karyocytes: NG>UA>Infarction.There was significance in pair comparison between AMI and NG(p<0.05).While,there was no significance between AMI and UA as well as UA and NG(P>0.05).The percentage of CD14+CD16-/mononuclear cells: NG>UA>Infarction.There was significance in pair comparison among AMI,UA and NG(P<0.05).The percentage of CD14+CD16+/mononuclear cells: AMI >UA>NG.There was significance in pair comparison among AMI,UA and NG(P<0.05);6)There was no significance for MDSCs in pair comparison among AMI,UA and NG(P>0.05);7)The percentage of CD11b+CD68+(as M1 with inflammation promotion): Infarction>UA>NG.There was significance in pair comparison between AMI and NG(P<0.05).While,there was no significance between AMI and UA as well as UA and NG(P>0.05).There was no significance for CD11b+CD206+(as M2 with inflammation inhibition)in pair comparison among AMI,UA and NG(P>0.05).5.Conclusion 1)The subset of monocytes is closely associated with ACS,of which,CD14+CD16-represents negative association,while,CD14+CD16+ represents positive association;2)A special set of cells during transition from monocytes to macrophages may be in peripheral blood,of which,M1 with inflammation promotion represents positive association with acute ACS,whose function and classification should be explored further;3)In this study,there was on significance between MDSCs and ACS;4)Adaptive immunity should be associated with unstable plaques of ACS and CD3+CD4+T may play a role in early stage of ACS.Part III The association between serum nicotinamide phosphoribosyl transferase(NAMPT)and mononuclear cells/subtypes of macrophages among patients suffering ACS 1.Background Atherosclerosis is chronic and inflammatory disease of vessel wall,which is a main leading death cause.Acute coronary syndrome(ACS)is terminal stage of coronary atherosclerotic heart disease(CAD).Accumulation of Lipid and immune cells within vessel wall is feature of atherosclerosis and etiology of ACS is associated with unstable atherosclerosis plaque and plaque-related infarction or occlusion.Currently,inflammatory reaction has been found during course of atherosclerosis and mononuclear macrophage plays an important role in participating and adjusting atherosclerosis-related inflammatory reaction.Macrophages of different types representing corresponding phenotypes manage special function during inflammatory reaction and are divided into two subtypes,M1 promoting inflammatory reaction and M2 inhibiting inflammatory reaction.Both M1 and M2 are found within plaque and the interaction between M1 and M2 deciding trend of plaque.Polarization of mononuclear macrophages within plaque is influenced by many factors,therefore,phenotypes of mononuclear macrophages center the development of atherosclerosis.However,the mechanism of polarization still remains unknown although its importance has been confirmed.Recently,the association between nicotinamide phosphoribosyl transferase(NAMPT)and cardiac disease has been found.Intracellular NAMPT(i NAMPT)is rate-limiting enzyme of NAD synthesis,while extracellular(e NAMPT)features as cytokines/adipocytokines,which means NAMPT is the link between metabolism and inflammatory.Although elevated serum NAMPT is considered as an independent influential factor of ACS and NAMPT is possibly associated with formation and rupture of plaque,it is still unknown whether NAMPT can adjust polarization of serum mononuclear macrophages.2.Objective(1)To explore the association between NAMPT and mononuclear cells/subtypes of macrophages by comparison between expressions of serum NAMPT and peripheral blood mononuclear cells(PBMC)/subtypes of macrophages from patients suffering ACS and healthy subjects;(2)To explore the association between i NAMPT or e NAMPT and peripheral blood mononuclear cells(PBMC)/subtypes of macrophages.3.Methods(1)Subjects There were two groups in the study.A total of 61 patients suffering ACS admitted 1st affiliated hospital of An Hui Medical University from Jun 2015 to Dec 2016 were enrolled as ACS group.Another 20 matched healthy subjects without any cardiac disease were enrolled as control group.(2)Sampling and isolation of PBMC Samples from patients and healthy subjects were collected and tested by molecule biomarkers including Tn I,flow cytometry.PBMC was isolated.(3)Cultivation of primary human macrophages Samples were divided into two parts.One part was isolated to obtain PBMC for inducing polarization of M1 and M2.Meanwhile,r NAMPT,FK866 inhibiting NAMPT and neutralizing antibody of NAMPT were added into cells in another part.The expression of cytokines and polarization-related biomarkers of macrophages at different points was assayed.(4)RNA extraction and PCR Pure RNA was extracted and target genes were tested quantitatively by commercial PCR primers.(5)Assay of e NAMPT and cytokines The level of serum e NAMPT was tested by ELISA.The level of IL-1ra,IL-6,IL-10 and TNF-α was also tested.3.6 Flow cytometry Ratio of M1/M2 was calculated by multi-parameters flow cytometry.3.7 Western Blot Proteins within supernate were collected after pyrolysis of fresh cells.Concentration of proteins was tested following electrophoresis and transmembrane.Photos were made by chemiluminescence after label coloration with primary and secondary antibodies.3.8 Statistics Obtained data were handled based on objective.P<0.05 was significant.4.Results(1)There was significance on hypertension,troponin and creatine kinase in UA,NSTEMI,STEMI and UG based on clinical data;(2)The expression of e NAMPT and i NAMPT in patients suffering ACS increased;(3)The polarization of M1 from peripheral blood mononuclear cells(PBMC)/subtypes of macrophages was found during acute stage of ACS;(4)The level of NAMPT RNA and e NAMPT within M1 mononuclear cells/macrophages was higher than that within M2 mononuclear cells/macrophages;(5)Inhibition of e NAMPT and i NAMPT did not influence polarization of M2 but decreased that of M1.Elevated NAMPT did not influence polarization of M2 but promoted that of M1.5.ConclusionNAMPT is active in etiology of ACS by partly adjusting function of mononuclear cells/subtypes of macrophages.Elevated NAMPT promotes inflammation possibly through adjusting polarization of M1 during ACS.