The Mechanism Of EDIL3,an Endogenous Leukocyte Endothelial Adhesion Inhibitor,in Myocardial Infarction

BackgroundAcute myocardial infarction（MI）is irreversible myocardium injury caused by ischemia-induced cell death in the event of partial or complete coronary artery occlusion.MI is associated with many complications,including heart rupture,ventricular aneurysm formation,ventricular dilation,and heart failure.It is still one of the deadliest diseases with the highest mortality rate and disability rate worldwide.Infarcted myocardium activates a series of reparative responses during all three post-infarction stages.For example,during inflammatory phase,Danger-associated molecular patterns（DAMPs）recruits immune cells to the infarcted region to remove apoptotic cells and matrix fragments;during reparative phase,angiogenesis and collagen secretion are activated;during maturation phase,a mature collagen-based scar is formed.Throughout the entire course of post-infarction myocardial repair,a wide range of immune cells and inflammatory mediators associated with innate and adaptive immune responses are involved;over-or under-activation of inflammation responses in any reparative phase may lead to adverse myocardial remodeling.Therefore,it is of urgent importance to develop new therapies targeting adverse post-infarction inflammatory response.There is increasing evidence for the central role of leukocyte recruitment by healing tissue in post-infarction reparative immune response.The recruitment of leukocytes involves complicated adhesion cascades,including leukocytes’rolling,tight adhesion,and transmigration through endothelium and their infiltration into inflammatory tissues.During these processes,the interaction between Lymphocyte Function-associated Antigen 1（LFA-1）and intercellular adhesion molecule 1（ICAM1）is critical.Currently,there are three endogenous proteins that are known to regulate leukocyte recruitment:EGF-like repeat and discoidin I-like domain-containing protein 3（EDIL3）,Pentraxin-3（PTX3）and Growth differentiation factor-15（GDF-15）,Previous reports have already established PTX3 and GDF-15 as important regulatory molecules and potential biomarkers in MI.In contrast,there is not yet report on the role of EDIL3,an endogenous molecule that inhibits leukocyte-endothelium adhesion,in MI.Early studies claimed that EDIL3 is secreted by endothelial cells and promotes angiogenesis by binding to integrinα_vβ₃/α_vβ₅.Later research found that EDIL3 can also suppress neutrophils’infiltration into inflammatory regions;this suppression is exercised when EDIL3competitively binds to LFA-1,and thus interferes with binding between leukocyte LFA-1 and endothelial ICAM1.Furthermore,EDIL3 expressed by osteoclasts is shown to restrain further osteoclast genesis by interacting with integrinα_vβ₃.Via interaction with Phosphatidylserine from apoptotic cells,EDIL3 also mediates the endocytosis of these cells.Recently,it is established that myofibroblasts in infarcted myocardium express EDIL3 and its paralog Milk fat globule epidermal growth factor8（MFGE8）,both of which mediate endocytosis of dead myocardial cells and facilitate cardiac repair.Based on the known wide range of biological functions of EDIL3 as well as its distinct multi-domain architecture,we hypothesize that apart from its well-known role in leukocyte recruitment,EDIL3 may also be involved in other immune responses in post-infarction myocardial repair,including apoptotic cell clearance,angiogenesis,and collagen deposits.In this project,we seek to understand the role of EDIL3 in each phase of post-infarction cardiac repair,in an effort to provide clues for new therapeutic strategies.Research AimBy studying the role of EDIL3 after MI,we hope to further our understanding for the mechanism of post-infarction cardiac repair,and to provide new empirical and theoretical basis for therapeutic intervention against adverse post-infarction remodeling.MethodsSection one:Establishing the relationship between EDIL3 and MI severity1.Serum EDIL3 level in acute MI patients and its correlation with serum MI biomarkers and MI severity.Based on inclusion and exclusion criteria of acute myocardial infarction,patients are recruited,all of whom signed informed consent forms.Serum EDIL3 from these patients is quantified by Enzyme-linked immunosorbent assay（ELISA）.The median concentration of serum EDIL3 in the patient population is used to classify patients into high EDIL3 group and low EDIL3 group.We statistically analyze the difference in MI biomarkers,MI risk factors,and survival rate between these two groups.Spearman’s rank correlation is used to evaluate the correlation between EDIL3 and MI biomarkers in serum,including CK-MB,c Tn I and BNP.2.Investigation of the relationship between EDIL3 and MI in mice model.MI model of WT mice is established by surgical ligation of the left anterior descending artery.The m RNA level of EDIL3 is monitored by q PCR.Section two:Studying the effect of EDIL3 deficiency on post-infarction pathophysiology development.1.Confirmation of genotypes of EDIL3 knockout mice.PCR,q PCR and Western blot are used to confirm the genotypes of EDIL3 knockout mice at DNA,m RNA,and protein level,respectively.2.Analysis of the survival rate difference between WT and EDIL3 knockout mice after MI.MI model is separately constructed for WT and for EDIL3 deficient mice,followed by a continuous monitoring period of 28 days.Kaplan-Meier estimator is applied on the daily death record for the two groups of mice to estimate the survival function.Survival distribution from these two groups are compared by log-rank test.3.Analysis of the effect of EDIL3 deficiency on the post-MI cardiac function.Echocardiography is applied to compare cardiac function parameters,includingLeft ventricular ejection fraction（LVEF）,Fractional shortening（FS）,Left ventricular internal diameter at diastole（LVIDd）and Left ventricular internal diameter at systole（LVIDs）,between WT and EDIL3 knockout mice after MI.4.Investigation of the effect of EDIL3 deficiency on the infarcted size and collagen deposition.Masson trichrome staining is applied on sections obtained by slicing through cross section of infarcted mice heart.The infarcted area and collagen level in WT and in EDIL3 mice section is estimated by Image J,and the difference between the two groups are assessed by t-test.Section three:Analyzing the distribution of LFA-1,the receptor of EDIL3,in immunological cells.Flow cytometry is used to analyze the expression pattern of the EDIL3 receptor,LFA-1,in immunological cells,including lymphocytes,macrophages,and neutrophils.Section four:Quantifying the impact of EDIL3 deficiency on the recruitment and differentiation of immunological cells.1.Analysis of the effect of EDIL3 deficiency on the number,differentiation,and recruitment of neutrophils in peripheral blood and hearts.Flow cytometry is applied on single cell suspensions collected from blood and myocardium to compare the difference in the number of neutrophils between WT and EDIL3 mice after MI.Specifically,the number of total neutrophils,N1 neutrophils,and N2 neutrophils are measured.To further study the effect of EDIL3 deficiency on neutrophil recruitment,the Adoptive Cell Transfer（ACT）experiment is carried out:GFP labeled neutrophils induced ex vivo are injected to mice bloodstream,and myocardial tissues are collected three days after infarction in order to measure the level of exogenous neutrophils in myocardium.2.Analysis of the consequence of EDIL3 deficiency in terms of the number of monocytes in hearts.Flow cytometry is used on single cell suspensions collected from myocardium to compare the amount of myocardium monocytes between WT and EDIL3 knockout mice at multiple time points after MI.3.Analysis of the impact of EDIL3 deficiency on the number and differentiation of macrophages in hearts.The difference of the amounts of macrophages subtypes in myocardium between post-MI WT and EDIL3 knockout mice are compared by flow cytometry.Additionally,monocytes are isolated from bone marrow of WT and EDIL3knockout mice.These monocytes are induced ex vivo into M1 and M2a macrophages to study the effect of precense or absence of exogenous EDIL3 on differentiations of M1 and M2a macrophages.4.Analysis of the effect of EDIL3 deficiency on the recruitment and differentiation of lymphocytes in blood and in myocardium.Flow cytometry is utilized to compare the differences in the amounts of myocardium lymphocytes,including Th,Treg,andγδT-17 between WT and EDIL3knockout mice 7 days after infarction.Similarly,the differences in the amounts of B cells,T cells,Th,Tc,TEM,TCM,and Na?ve T cells in the peripheral bloods from WT and EDIL3 deficient mice 7 days after MI are compared by flow cytometry.Additionally,peripheral blood samples collected from WT and EDIL3 knockout mice are sent to National Chengdu Center for Safety Evaluation of Drugs for complete blood count（CBC）.We statistically assess the difference between the two mice populations in terms of the amount of all leukocytes,lymphocytes,monocytes,neutrophils,as well as Neutrophil-lymphocyte ratio（NLR）and Platelet-lymphocyte ratio（PLR）.Section six:Analyzing the effect of EDIL3 deficiency on the expression of cytokines in post-MI hearts.1.Analysis of EDIL3 deficiency’s effect on the expression of chemokines and their receptors.The m RNA expression level of CCL2,CCL3,CCL5,CCR2,CX3CL1,and CX3CR1 are measured by q PCR for WT and EDIL3 knockout mice at multiple post-infarction time points.2.Analysis of EDIL3 deficiency’s effect on the expression of pro-and anti-inflammatory factorsWe used q PCR to quantify the m RNA expression of IL-1β,TNF-α,TGF-β,IL-17Aand IL-23p19 for WT and EDIL3 knockout mice at various time point after myocardial infarction.3.Analysis of EDIL3 deficiency’s impact on the expression of factors associated with collagen deposition.At various time point after myocardial infarction,q PCR is applied to detect the m RNA expression of MMP9,TIMP1,Col1a1 and Col3a1 for WT and EDIL3knockout mice.ResultsSection one:EDIL3 level in patient serum is negatively correlated with the severity of acute myocardial infarctionThe amount of MI biomarkers in patients,including creatine kinase-MB（CK-MB）,cardiac troponin I（c Tn I）,and myoglobin（MFB）,is statistically significantly different between the high EDIL3 level and low EDIL3 level groups,as classified by median serum EDIL3 level measured by ELISA.EDIL3 expression is negatively correlated with the level of CK-MB,c Tn I,and MGB.During a follow-up period of 40 months,we observed that patients in low EDIL3level group have higher mortality rate,implicating the potential role of EDIL3 in acute MI prognosis.Section two:EDIL3 deficiency is beneficial to post-infarction cardiac healing1.EDIL3 knockout mice have higher post-infarction survival rate and better cardiac function.In the established MI mouse model,EDIL3 knockout mice have higher survival rate within 28 days after infarction compared to WT mice.Via echocardiography monitor of WT and EDIL3 deficient mice at multiple time points after MI,we find that EDIL3 knockout mice have stronger LVEF and weaker LVIDd and LVIDs.These data suggest that EDIL3 deficiency is beneficial to cardiac repair and promotes post-infarction cardiac function.2.Infarcted size and collagen deposition are reduced in EDIL3 knockout mice.EDIL3 deficient mice have smaller infracted size and thicker ventricular wall compared to WT at 28 days after MI.This is in line with echocardiography evidences,which establish the benefits of EDIL3 deficiency in cardiac healing.Masson staining shows that in infarcted regions and their borders,EDIL3 knockout mice have lower level of collagen in left ventricular compared to WT mouse.This demonstrates the benefit of EDIL3 deficiency in reducing collagen deposition and preventing excessive myocardium fibrosis.Section three:LFA-1,the receptor of EDIL3,is mainly expressed by the neutrophils and macrophagesFlow cytometry shows that EDIL3’s receptor,LFA-1,is mainly synthesized by neutrophils and macrophages,while LFA-1 expression in lymphocyte is low.This suggests that EDIL3 is likely to affect post-infarction repair mainly through neutrophils and macrophages.Section four:EDIL3 deficiency facilitates neutrophils recruitment to infarcted regionAs demonstrated by flow cytometry,the level of both N1 and N2 neutrophils in the blood of EDIL3 knockout mice is lower than that in WT mice,no matter whether the mice are healthy or have suffered from MI.While less neutrophils are observed in healthy heart of EDIL3 deficient mice compared to WT,the myocardium from the EDIL3 deficient mice have both more N1 neutrophils and more N2 neutrophils three days after MI.As neutrophils are known to express EDIL3 receptors such as LFA-1,we hypothesize that EDIL3 deficiency facilitates neutrophils recruitment to infarcted heart.This is confirmed by the ACT experiment,where bloodstream neutrophils are more likely to be recruited to infarcted myocardium in EDIL3 deficient mice than in WT mice.Section five:EDIL3 deficiency affects the conversion of monocytesThe ratio of Ly6C^hi/Ly6C^lo monocytes in EDIL3 deficient mice is higher than WT.Section six:EDIL3 affects the differentiation of macrophages1.Deficiency of EDIL3 increases the amount of CD11b^hi pro-inflammatory macrophages after infarction.1,4 and 7 days after MI,more CD11b^hi pro-inflammatory macrophages areconsistently detected in EDIL3 deficient mice hearts compared to those in WT mice.2.EDIL3 deficiency promotes the differentiation of pro-inflammatory macrophages after infarction.More Mertk^-MHC II^lo pro-inflammatory macrophages can be observed in EDIL3knockout mice compared to WT mice are observed 4 days after MI.This suggests that EDIL3 deficiency alters microenvironment for macrophage differentiation after infarction.3.Absence of EDIL3 elevates the amounts of monocyte-derived macrophages.4 days after MI,the myocardium of EDIL3 knockout mice possesses more monocyte-derived Ly6C⁺macrophages compared to WT.Section seven:EDIL3 deficiency influence the post-MI proliferation and differentiation of lymphocytes1.Deficiency of EDIL3 reduces NLR and PLR in circulatory system.Complete blood count tests for post-infarction mice show that EDIL3 knockout mice have less neutrophils and more lymphocytes in the blood compared to WT mice.Furthermore,compared to WT,post-infarction EDIL3 knockout mice have lower NLR and lower PLR,both of which are clinical indication of better prognosis.These data also suggest that the mechanism of protective effect of EDIL3 deficiency might involve neutrophils and lymphocytes.2.Absence of EDIL3 promotes T cell proliferation in peripheral blood.Compared to that in WT mice,the bloodstream of EDIL3 knockout mice have more CD4⁺T cells 7 days after MI.A closer look reveals that these additional CD4⁺T cell are mainly effector memory T cells.3.EDIL3 deficiency decreased the number of Th1 and Tc1 in infarcted hearts.In post-MI myocardium,no significant difference is observed between WT andEDIL3 deficient mice in terms of the number of Th17,γδT-17 and Treg cells.While EDIL3 deficient mice have decreased the number of Th and Tc cells in infarcted hearts.Section eight:EDIL3 regulates the expression of cytokines associated with cardiac repair1.EDIL3 deficiency up-regulates the expression of chemokines related to neutrophils.While there is no significant difference in post-infarction CCL2,CCL3,CCR2 and CX3CR1 expression between WT and EDIL3 deficient mice heart,CCL5 is more highly expressed in EDIL3 deficient mice.This shows that while EDIL3 deficient is not correlated with the expression of chemokines and chemokine receptors associated with monocytes,EDIL3 absence promotes neutrophil-related chemokine expression.2.EDIL3 deficiency affects the expression of pro-inflammatory and anti-inflammatory factors,including IL-1βand TNF-α.EDIL3 deficiency does not affect the expression of IL-17A,IL-23p19 or TGF-β after MI,but does elevate the expression level of IL-1βand TNF-α,which implicates the role of EDIL3 in modulating local immune microenvironment in infarcted myocardium.3.EDIL3 deficiency affects the expression of factors related to collagen deposition.In infarcted hearts,the expression of collagen related proteins,including Col1a1and Col3a1,are lower in EDIL3 knockout mice,while their MMP9 and TIMP1expression is slightly elevated.This implies that EDIL3 deficiency can prevent over-fibrosis of myocardium by inhibiting collagen deposition.ConclusionClinical evidences demonstrated that serum EDIL3 level was negatively correlated with the level of myocardium infarction biomarkers,such as CK-MB,c Tn I,and MGB.Similarly,patient follow-up data also confirms that lower EDIL3 level was associated with worse prognosis.These all confirm that EDIL3 was negatively correlated with the severity of MI.In the myocardium infarction mouse model,EDIL3 knockout mice have higher survival rate after infarction surgery compared to wild type mice,with less collagen deposition and better cardiac function.In the infarcted hearts of EDIL3knockout mice,there were more neutrophils and pro-inflammatory macrophages;the expression levels of IL-1βand TNF-α,two factors inhibiting collagen secretion by myofibroblasts,were higher,while expressions of factors responsible for collagen synthesis were down-regulated.Therefore,EDIL3 deficiency might improve cardiac healing after myocardial infarction by promoting the recruitment of neutrophils to infarcted region and regulating the polarization of macrophages.