| Myocardial infarction (MI) has become a serious threat to national healthy withmorbidity and mortality rising year by year. Acute heart failure is the major cause of deathin the acute phase of MI, but with the progress of medical treatment (drug thrombolysis,interventional stent and bypass surgery), the incidence of acute heart failure decreasedsignificantly. With the progression of the disease, it will enter into a chronicphase-ventricular remodeling. Ventricular remodeling following MI is an independent risk factor for many cardiovascular events, which is associated with an inflammatory reaction,followed by scar formation at the site of infarction as well as changes in the non-infarctedmyocardium, including interstitial fibrosis and vascular remodelling. Fibrous tissue thatforms at the site of cardiomyocyte loss preserves structural integrity and is integral to theheart’s recovery, whereas structural remodelling of viable myocardium impairs tissuebehaviour. Therefore, inappropriate ventricular remodeling will seriously affect cardiacfunction, eventually leading to chronic heart failure. Inhibiting inappropriate ventricularremodeling early may be an effective way to prevent or postpone heart failure for patientswith MI. Some studies show that macrophage polarization plays an important role in theregulation of fibrosis remodeling after MI. However, it is unclear that whether appropriatemacrophage polarization could prevente malignant ventricular fibrosis remodeling.Several signaling have been involved in macrophage polarization. TheRBP-JК-mediated Notch signaling is a very evolutionary conserved signaling，which playsan important regulatory role in cell growth, development, differentiation, apoptosis andtissue injury remodeling. In our laboratory, Dr. Wang Yaochun first found that Notchsignaling was closely related to macrophage polarization in mouse tumor model, blockingNotch signaling promoted M2type macrophage polarization. In the same year, Xiong et alreported that Notch1signaling-dependent macrophage M2b polarization might play apivotal role in the pathogenesis of systemic lupus erythematosus.In2012, Cao’s groupfound that blocking the Notch signaling by gamma secretase inhibitors might promoteTLR-induced inflammatory response and promote M1-type macrophages polarization. Inthe same year, one study reported that Notch signaling promotes M1-type polarization byupregulating IRF8that was published by Nature Immunology.Therefore, the Notchsignaling plays an important regulatory role in macrophage polarization. However, it isunclear about the mechanism of RBP-JК-mediated Notch signaling pathway on regulatingmacrophage polarization in fibrosis remodeling after chronic tissue injury is unclear,especially in the case of the MI.In this study, it is the the first time for us to perform comprehensive assessment onthe role of RBP-JК-mediated Notch signaling in regulating macrophage in fibrosis remodeling following chronic tissue injury and possible mechanism of RBP-JК-mediatedNotch signaling on regulating macrophage polarization in fibrosis remodeling followingMI. Our study may provide a novel treatment and prevention strategy on maladaptiveventricular fibrosis remodeling.AIMS1. To investigate whether the RBP-JК-mediated Notch signaling pathway involved inmacrophage regualtion on tissue remodeling following chronic tissue injury.2. To observe whether blocking Notch signaling pathway in macrophage after MI couldinfluence monocyte/macrophage within and outside the heart and infarct area.3. To explore the possible molecular mechanisms of the Notch signaling regulatingmacrophages in ventricular remodeling after MI.METHODS1. To establish conditional RBP-JКknockout mouse in the macrophage. According to theexperiment, the mice could be divided into the experimental group (RBP-JКknockoutmouse) and the control group (Wildtype mouse).2. To establish MI model(Gao Erhe) using12-week-old mice（10experimental mice;16control mice）. Comparative analyses of the heart function between experimental miceand control mice after4weeks by small animal ultrasound instrument.5. Comparative analyses of the heart weight ratio and lung weight ratio betweenexperimental mice and control mice after MI4weeks.6. Comparative analysis of infarct size and collagen content between experimental miceand control mice, which were conducted by light microscope (Masson’s trichromestain) and Stereomicroscope.7. To establish carbon tetrachloride-induced tissue injury model using8-week-old mice（experimental mice,12; control mice,12）. After10weeks,the livers, spleens, lungs,kidneys and hearts were isolated，which were used for comparative analyses of tissueinjury and fibrosis remodeling between experimental mice and control mice by lightmicroscope (Masson’s trichrome stain; Sirius red stain) and Stereomicroscope.8. Comparative analysis of the number and type of macrophage cells, apoptosis and the number of new blood vessels in MI area between experimental mice and control mice,which were conducted by laser scanning confocal microscope (immunofluorescence).9. Comparative analysis of HYP in non-infarcted area between experimental mice andcontrol mice by ELISA.10. Comparative analysis of transcription level of inflammatory cytokines,anti-inflammatory cytokines, and fibrosis-related factor in the infarcted area betweenexperimental mice and control mice by qRT-PCR.11. Comparative analysis of inflammatory cytokines (TNF-α, IL-6) and anti-inflammatorycytokines (IL-10) in serum between experimental mice and control mice by ELISA.12. After MI4weeks the blood, spleens and bone marrow were separated，which wereused for comparative analysis of the number and type of monocytes and macrophagesbetween experimental mice and control mice by flow cytometry.13. To induce primary cultured bone marrow-derived macrophages differentiating toinflammation-related M1–type macrophages under LPS plus INF-γ. Comparativeanalysis of the inflammatory cytokines and their associated NF-κB signaling betweenexperimental mice and control mice by Western blot、qRT-PCR and ELISARESULTS1. There was no significant difference on survival curves between knockout mice (KO)and wild-type mice (WT) after MI4weeks.2. After MI4weeks, the results showed by small animal ultrasound instrument: ejectionfraction (EF%)(54.04±7.293) in knockout group higher than that in the controlgroup (36.03±4.034%); left ventricular fractional shrinkage (FS%)(27.62±4.293)in knockout group higher than that in the control mice(17.31±2.180);left ventriculardiastolic anterior wall LVAWd (mm)(0.8613±0.07792), posterior wall LVPWd (mm)(0.6676±0.01436) and septal thickness LVIWd (mm)(3.367±0.2837) in knockoutmice were thinner than those in the control group (respectively,0.6465±0.03634;0.7284±0.06317;4.382±0.1748); left ventricular diastolic volume of the LVVol d (ul)(47.92±10.31) in knockout mice was less than that in the control group(87.87±8.209). 3. The left ventricular mass, and heart weight ratio and lung weight in the knockout micewere less than those in the wild type mice after MI4weeks.4. Masson staining of cardiac tissue sections showed: in heart of knockout mouse，theinfarct size decreased， myocardial viability increased in MI area， and fibrosisdeposition decreased in the infarct zone and non-infarct zone compared with theexperimental group.5. After carbon tetrachloride induced tissue injury10weeks, mouse livers, lungs, spleens,kidneys and hearts were isolated. We found that the injury in livers, lungs, spleens,kidneys and hearts in knockout mouse was slighter than that in the control group, whilethe extent of fibrosis remodeling of various organs was also slighter than that in thecontrol group by gross pictures, Masson Stain and Picro-Sirius Red Stain.6. After MI4weeks, the result showed that the number of macrophages andinflammation-related macrophages decreased in the infarct area of knockout micecompared with the control group by immunofluorescence staining and laser scanningconfocal microscope, while that there was no statistically significant difference betweenthe two groups in the number of anti-inflammatory-related macrophages. Tunel stainingshowed that cell apoptosis decreased in knockout mouse heart compared with theexperimental group. Immunofluorescence staining of cardiac tissue sections showedthat no statistically significant difference was observed between the two groups inneovascularization in infarction area, the junction of regional and non-infarcted area.7. After MI4weeks, no significant difference on the expression of inflammatorycytokines (TNF-α, IL-6) and anti-inflammatory cytokines (IL-10) in serum wasobserved between the two groups.8. After MI4weeks, in knockout mouse, the inflammatory response of spleens were notsignificantly increased through gross pictures and spleen weight ratio compared withthe experimental group. 9. Flow cytometry data showed that: in knockout mouse inflammation related (LY6CHighCD11b+) and anti-inflammatory related (LY6CLowCD11b+) monocytes in blood, bonemarrow and spleen decreased comparing with the experimental group after MI. Inaddition, in knockout mouse, monocyte-derived macrophages (CD11b+F4/80+) andinflammation-associated macrophages (F4/80+iNOS+) in spleen were less than thosein the experimental group.10. Confocal microscopy data showed in the primary cultured macrophage isolated fromknockout mouse the translocation of p65into the nucleus was inhibited and degradationof IκB was reduced compared with the control group after LPS plus INF-γ stimulationfor2hours. As while Flow cytometry data showed the expression ofinflammation-related factor TNF-α in macrophage was decreased in the knockoutmouse compared with the control group.11. Cell supernatant was collected from cultured macrophage and then performed ELISAassay. The results showed secretion of inflammation-related factor (TNF-α, IL-6, IL-12)decreased and anti-inflammatory-related factor (IL-10) increased in the knockout mousecompared with the control group after LPS plus INF-γ stimulation24hours:,. The dataof Immunofluorescence detection showed the expression of inflammation-related factorTNF-α and P65and P50were decreased and degradation of IκB was decreased in themacrophage of knockout mouse compared with the control group. The data of Westernblot showed in the knockout mouse macrophage, the expression of CYLD wasincreased, whereas the expression of P65and P50was decreased and the degradation ofIκB was decreased compared with the control group after LPS plus INF-γ stimulation24hours.CONCLUSIONS1. Using two different models of the mouse chronic injury, such as MI and carbontetrachloride induced organization injury, we verified RBP-JК–dependent Notch signaling in macrophages was closely related to tissue remodeling. To block RBP-JКinmacrophages can reduce chronic injury and suppress fibrosis remodeling.2. It was the first time that we found Notch signaling in macrophages was closelyassociated with the recovery of heart function after MI. It may provide a novel strategyto prevent or treat maladaptive ventricular fibrosis by blocking Notch signalingpathway in macrophages that can reduce cardiac injury and suppress fibrosisrestoration after MI and improve cardiac function.3. Analyzing the macrophage phenotype in the infarct area, we first found that blockingthe Notch signaling in macrophages could reduce the number of M1macrophages butnot affect the number of M2macrophages. Also it can affect the generation ofinflammation-related monocytes out of the MI area, such as blood, bone marrow andspleen. Furthermor it can reduce the number of inflammatory related macrophage.These studies may update the mechanism of the Notch signaling pathway regulationon macrophage polarization.4. Analyzing primary cultured macrophages, we demonstrated that the blockade of theNotch signaling in macrophages might inhibit the expression of P65and the P50andpromote upregulation of CYLD that can prohibit NF-κB signaling pathway bypreventing the translocation of p65into the nucleus and IκB degradation, and finallyreduce the secretion of inflammatory cytokines. Meanwhile, we also found thatblocking the Notch signaling in macrophages could promote the secretion ofanti-inflammatory-related cytokine IL-10. These data suggested blocking Notchsignaling in macrophages in vitro could inhibite M1-type macrophage polarization andpromote M2-type macrophage polarization.Take together, these studies may provide new therapy and prevention strategy onmaladaptive ventricular fibrosis remodeling. |
PDF Full Text Request