Agrin Regulates The Differentiation Of Epicardial Progenitor Cells Into Smooth Muscle Cells

Extracellular matrix(ECM)are extracellular molecules secreted by cells and provide a microenvironment for surrounding cells,which have structural support and signal transduction functions and play an active and critical role in cellular events.The spatiotemporal expression of cardiac ECM is essential for the development,regeneration and remodeling of the heart.Epicardial cells and fibroblasts are the main sources of cardiac ECM.The composition of cardiac ECM includes hyaluronic acid,fibronectin,periostin,tenascin-C,collagen and proteoglycan.ECM regulates cell proliferation,differentiation,migration and homeostasis by binding to cell surface receptors.The epicardium is a special epithelial tissue that wraps and protects the heart,which affects the heart development and angiogenesis.Epicardial progenitor cells(EPCs)have multi-differentiation potential,and specifically express transcription factors Tbx18,Wt1 and Tcf21 in heart.EPCs migrate into the heart and undergo epithelial-mesenchymal transition(EMT),which differentiate into smooth muscle cells forming a coronary skeleton structure.The differentiation of EPCs to smooth muscle cells is regulated by ECM.Agrin is a heparin sulfate proteoglycan,which is one of the components of cardiac ECM.It reported that agrin regulates cells' proliferation,migration and differentiation mainly by binding to dystroglycan(DAG)and muscle-specific receptor tyrosine kinase(MuSK).The latest research reported that agrin has the capacity to promote the of mouse cardiomyocytes proliferation after injury.The role of agrin in the differentiation and migration of EPCs is unclear.Understanding the role of agrin in the differentiation of EPCs into smooth muscle cells has clinical application value for coronary artery disease.Therefore,in this study we explored the spatiotemporal expression of agrin in the myocardium and the role of agrin in the differentiation of EPCs into smooth muscle cells.Part I: Spatiotemporal expression of Agrin in myocardium and epicardiumObjective: The cell-culture model of EPCs was established and the spatiotemporal expression level of agrin in myocardium was determined to further research of EPCs' differentiation.Methods: E11.5-day mouse embryonic ventricles were cultured and EPCs grew from the edge of the tissue.RT-PCR and immunofluorescence were used to determine the specific markers Tbx18 and Wt1.Agrin in mouse myocardium at different ages was detected by western blot,immunofluorescence,and RT-PCR.Results: The expanded cells with highly expression of transcription factors Tbx18 and WT1 and low expression of cTnT showed cobblestone-like morphology.The average level of Agrin in mouse myocardium increased from E11.5 to P0,and decreased from P0 to P28.Agrin is highly expressed in mouse epicardium and endocardium,and the expression level of agrin in EPCs was higher than that in the entire myocardium.Conclusion: The cultured primary cells are EPCs,and EPCs culture model successfully established.Agrin is expressed in mouse epicardium and EPCs,and the expression of agrin in mouse heart has the characteristics of spatiotemporal changes.Part II: Agrin binds to receptor ?-dystroglycan and inhibits the differentiation of epicardial progenitor cells into smooth muscle cellsObjective: To explore the role of agrin in the differentiation of epicardial progenitor cells into smooth muscle cells.Methods: EPCs were cultured in vitro and treated with different concentrations of agrin.RT-PCR and immunofluorescence were used to determine the expression of smooth muscle cell markers ?-SMA and MYH11,and the optimal concentration of agrin was selected for the cell culture.PCR and immunofluorescence were used to detect the receptors of agrin on the surface of EPCs.Results: The concentration of agrin at 200 ng / ml was used to culture EPCs for 5 days.Compared with the control group,the expression levels of ?-SMA and MYH11 were significantly reduced.The agrin receptor ?-dystryglycan(DAG)was expressed in EPCs and epicardium,and another receptor muscle-specific receptor tyrosine kinase(MuSK)was not determined.Proper concentration of agrin combined with DAG downregulated the expression of ?-SMA and MYH11 in EPCs.When DAG antibody was used to block the binding of agrin to the receptor DAG,the inhibitory effect of agrin on ?-SMA and MYH11 disappeared.Conclusion: The agrin receptor DAG is expressed on the surface of EPCs.The appropriate concentration of agrin inhibits the differentiation of EPCs into smooth muscle cells by binding with DAG.Part III: Agrin regulates the differentiation of epicardial progenitor cells into smooth muscle cells through Notch-1 signaling pathwayObjective: To observe the effect of agrin-mediated signaling on the differentiation of epicardial progenitor cells into smooth muscle cells.Methods: RT-PCR was used to detect the effect of agrin on Notch signaling pathway after epicardial progenitor cell intervention.The Notch-1 agonist Jagged-1 was used to activate the Notch-1 pathway of EPCs,and the effect of Notch-1 activation on the differentiation of EPCs was observed.RT-PCR and immunofluorescence were used to determine the key factor of Notch signaling and smooth muscle cell markers ?-SMA and MYH11 expression.With jagged-1 activating the Notch-1 pathway,agrin was used to interfere the EPCs with or without the DAG antibody to block the receptor.The expression of key factors of Notch-1 signaling pathway and smooth muscle cell markers ?-SMA and MYH11 were determined.With the treatment of agrin or / and Jagged-1,the cell shrinkage and migration ability were evaluated by gel shrinkage test and scratch test respectively.Results: Agrin reduced the expression of Notch-1,Hes-1 and Heyl in epicardial progenitor cells.The expression of Notch signaling pathway factors Notch-1,Hes-1 and Heyl and smooth muscle cell markers ?-SMA and MYH11 were increased with the treatment of 500 ng / ml concentration of Jagged-1.Under the notch-1 activation condition,the expression of Notch-1 and Hes-1 was suppressed and the ?-SMA and MYH11 was down-regulated in EPCs with agrin treatment.After blocking the receptor DAG,agrin's inhibitory effects on Notch-1 signaling and the expression of ?-SMA and MYH11 was disappeared.At the same time,Jagged-1 treatment increased gel contraction ability and decreased cell migration ability;while agrin treatment decreased the gel contraction ability and increased cell migration ability.Conclusion: Agrin inhibits the differentiation of epicardial progenitor cells into smooth muscle cells by down-regulating the Notch-1 signaling pathway.Agrin inhibits the differentiation of EPCs while promotes their migration ability.