Study On Protection And Mechanisms Of Pigment Epithelium-derived Factor Regulating Macrophage Polarization In Oxygen-induced Retinopathy

Purpose:Pigment epithelium derived factor(PEDF),as a multifunctional factor,has been extensively studied,and increasing researches have shown that neovascularization(NV)is closely related to inflammation and immune cells.The purpose of this study is to investigate the mechanism and the role of PEDF played in macrophage activation and polarization shift as well as angiogenesis during the process of ocular neovascularization(NV)both in vivo and in vitro.Methods:1.C57BL/6J(Postnatal day 7,P7)mice were prepared and randomly divided into normal control group,oxygen induced retinopathy(OIR)model group,recombinant PEDF treated group and phosphate buffered saline(PBS)treated control group.Retina whole mount staining was performed to analyze the NV area and macrophage recruitment.Inflammation and cytokines associated with macrophage phenotype in retinas were evaluated by realtime RT–PCR.Macrophages were sorted by CD11b microbeads and the polarization pattern of CD11b~+macrophages was analyzed by flow cytometry at different time points.2.Multiple macrophages were cultivated in hypoxia with different concentrations of PEDF(0,1,2,5,10 ng/ml).The cytokines assoctiated with macrophage polarization were evaluated by realtime RT-PCR and western blots.Human umbilical vascular endothelial cells(HUVECs)were cultured with supernatants of polarized macrophages.Cell proliferation and tube formation assay were performed to study the effect of polarized macrophages on endothelial cells.At P12,OIR mice received intravitreal injection of EGFP-labeled M0-,M1-and M2-like macrophages,retina whole mount staining was performed to analyze the influence of different polarized macrophages on NV formation.3.C57BL/6J newborn mice were divided into control and OIR model groups randomly.Mouse macrophage cell line RAW264.7 cells and bone marrow derived macrophages(BMDMs)were cultured under normoxia or hypoxia.Immunofluorescence staining and western blots were performed to detect the expression of PEDF receptor(PEDF-R)in macrophages.BMDMs were cultivated in hypoxia with different inhibitors of PEDF-R.Realtime RT-PCR was applied to examine the expression of cytokines associated with macrophage polarization.4.C57BL/6J mice were randomly divided into normal control group,OIR model group,recombinant PEDF treated group and PBS treated control group.At P17,Mf were isolated from different groups.BMDMs were cultivated under hypoxia with 10 ng/ml PEDF and inhibitors of mitogen-activated protein kinase(MAPK)signal pathway and Notch1protein.Western blots were performed to detect the activation of MAPK and Notch1signal pathway.Realtime RT-PCR was applied to examine the expression of cytokines associated with macrophage polarization.Results:Recombinant PEDF significantly supressed the process of macrophage recruitment and activation as well as RNV.M1-and M2-like macrophages significantly increased NV formation and its supernatants elevated HUVECs proliferation and tube formation,.PEDF significantly inhibited hypoxic-induced macrophage polarization both in vivo and in vitro.PEDF regulated macrophage polarization in hypoxia via Adipose triglyceride lipase(ATGL)mediated MAPK signal pathway and Notch1 protein.Conclusions:Recombinant PEDF could suppressed neovasculation by regulating macrophage recruitment and polarization,indicating a paracrine role of PEDF in endothelial cells and macrophage cross talk in angiogenesis.