The Role Of CD27-CD70 Signaling In Myocardial Infarction And Cardiac Remodeling

Part I Dynamic changes of serum sCD27 levels in AMI patients and correlation between the sCD27 level and progression of the disease.Objective: The purpose of our study is to explore the dynamic changes of serum sCD27 levels in acute myocardial infarction patients,and to investigate the correlation between the serum sCD27 and clinical indexes.Methods: From May 2017 to February 2018,175 subjects(43 negative control subjects,42 unstable angina patients,and 90 AMI patients)admitted to Wuhan Union Hospital were enrolled in the present study.Subjects with < 30% stenosis in a main coronary artery were enrolled into the negative control group.Patients in unstable angina(UA)group were considered when there is progressive increases in angina symptoms,angina at rest,and with ?50% stenosis in at least one of the major coronary arteries as confirmed by coronary arteriography.AMI patients were diagnosed according to the diagnostic criteria for acute myocardial infarction.The level of serum sCD27 was measured by enzyme-linkedimmunosorbent assay(Elisa),and the correlation between sCD27 level and clinical indexes was calculated by Pearson's test.Results: 1.Gensini score and the serum level of FBG in AMI group and UA group were significantly higher than those in negative control group.In addition,there were significant differences in smoking and HDL-C,c TNI,CK-MB between AMI group and negative control group.2.The serum sCD27 level increased significantly within 24 hours after the onset of acute myocardial infarction,and decreased to baseline on the 3rd day.The serum sCD27 level on the 7th day was significantly lower than that in the negative control group.3.There was a positive correlation between serum sCD27 level and c TNI,CK-MB,Gensini score in patients with acute myocardial infarction.Conclusion: The serum sCD27 level increased within 24 hours after the onset of AMI and was positively correlated with the c TNI,CK-MB and Gensini score,suggesting that sCD27 might be a new marker for early evaluation of the severity of acute myocardial infarction.Part II Effects of blocking CD27-CD70 pathway on cardiac function and ventricular RemodelingObjective: To explore the effects of CD27-CD70 pathway on collagen formation,myocardial fibrosis and extracellular matrix(ECM)formation after acute myocardial infarction.Try to find out the mechanism of ventricular remodeling after AMI.Methods: The AMI mouse model was induced by ligating the left anterior descending(LAD)branch of coronary artery.The AMI mice were randomly divided into three groups.One was injected with 500?g anti-CD70 in vivo antibody FR70(clone FR70;Bio X Cell,USA)on the day of operation,and 250?g on the 2nd,4th,6th,8th,10 th,12th day after operation,which was named AMI+FR70 group.One group was injected with 500?g isotype Ig G antibody(BE0090;Bio X cell,USA)on the day of operation,and 250?g on the 2nd,4th,6th,8th,10 th,12th day after operation,which was referred to as AMI+ Ig G group.The group without any additional treatment was called AMI group.In the sham group,we only opened the chest without ligate the left anterior descending branch of coronary artery.On the 3rd,7th and 14 th day after AMI,the mice were sacrificed.The cardiac function of the four groups was evaluated by echocardiogram on the 7th and 14 th day after the surgical operation.The indexes included LVFS,LVEF,LVESD,LVEDD.The mice were sacrificed after echocardiogram.The infarct size was evaluated by Masson's staining.Transferase-mediated deoxyuridine triphosphate-biotin nick end labeling(TUNEL)staining was used to evaluate the apoptosis of cardiac cells.Real-time PCR was used to evaluate the m RNA expression of collagen-I and collagen-III on the 7th and 14 th day after AMI.The protein level of collagen-I and matrix metalloproteinase-9 in the border zone was measured by western blot,in order to explore the effect of blocking CD27-CD70 pathway on the formation of ECM after AMI in mice.Results: The results of echocardiography on the 7th and 14 th day after AMI showed that the LVEF,LVFS in AMI group,AMI+ Ig G group and AMI+FR70 group were significantly lower than that in sham group,while the LVEDD,LVESD were significantly higher than that in sham group,indicating that the AMI model was successful established.Compared to the isotype-treated group,the LVEDD and LVESD were significantly increased and the EF and FS were significantly decreased in the FR70-treated group on day 7 and day 14.Results showed that FR70 treatment significantly increased the infarct size following MI at day 7 or day 14.Besides,there was no significant difference in area at risk(AAR)between the AMI groups at day 1.The m RNA level of collagen-I and collagen-III was significantly down-regulated in AMI+FR70 group than that of the AMI+Ig G group.The protein level of MMP-9 was increased.At the 14 th day of AMI,the collagen deposition in the infarct area of the FR70 treated group was significantly reduced compared with the AMI group and the Ig G treated group.The number of apoptotic cells was also increased.Conclusion: Blocking the CD27-CD70 pathway worsened the cardiac function of AMI mice,enlarged the infarct size and inhibited the ECM formation,resulted in aggravated ventricular remodeling and may increased the risk of cardiac rupture.Part III Effects of blocking CD27-CD70 pathway on immune response and angiogenesis after myocardial infarctionObjective: Excessive inflammatory response after acute myocardial infarction can lead to continuous myocardial damage which result in cardiac dysfunction and ventricular remodeling.After AMI,angiogenesis can increase the perfusion of ischemic myocardium,thus improve the symptoms of myocardial ischemia and hypoxia.It has been reported that blocking CD27-CD70 costimulation inhibits angiogenesis in hindlimb ischemia mouse model.The aim of this study was to further investigate the role of CD27-CD70 co-stimulation in inflammation and angiogenesis after myocardial ischemia.Methods: H&E staining was performed to observe the gross structure of infarct zone,border zone,remote zone and inflammatory cells infiltration.Flow cytometry was used to detect the changes of Tregs and Gr-1+CD11b+ neutrophils at day 3 after AMI.Immunofluorescence method was used to explore the number of F4/80 macrophages in the border zone in infarcted hearts.Real-time PCR method was used to evaluate the m RNA expression of inflammatory cytokines at day 7 after AMI.Theseinclude i NOS,TNF-?,IL-6,IL-1 ?,IL-10,Arginase-1 and TGF-?.At day 14 after AMI,the CD31 and ?-SMA positive cells in the infarcted area,peri-infarct area and remote area were evaluated by immunofluorescence.Results: Relative to sham controls,after surgery mice in the AMI model group exhibited increased separation of cardiac muscle fibers and necrosis with inflammatory cell infiltration,as detected in H&E-stained sections.Treatment with anti CD70 antibody aggravated this separation of cardiac muscle fibers,and was associated with increased inflammatory cell infiltration.FR70 treatment significantly decreased the number of CD4+CD25+Foxp3+Tregs in the infarcted heart at day 3 post-ischemia,while levels of CD11b+Gr1+ neutrophils were clearly up-regulated.The percentages of CD4+T cells and CD8+T cells did not change.Furthermore,real-time PCR assay showed that,FR70 treatment resulted in an increase in the cardiac expression of M1 macrophage markers i NOS,TNF-?,IL-6 and IL-1?,whereas expression of Arginase-1 which is associated with M2 macrophage polarization was down-regulated.FR70 treatment significantly reduced capillary density (expressed as the number of CD31-positive cells per field)in infarct area and peri-infarct area but not remote area.The ?-SMA positive arterioles were also decreased in the FR70-treated group as compared with the isotype treated group in the infarct and peri-infarct area.Conclusion: Blocking the CD27-CD70 pathway after AMI increased the infiltration of inflammatory cells and upregulated the expression level of inflammatory factors.The development of new capillaries and arterioles were inhibited,resulted in aggravated myocardial ischemia and hypoxia.