The Effect Of CD137-CD137L Signaling On The Phenotypic Transition Of Macrophages And Angiogenesis

ObjectiveTo investigate the effect of CD137-CD137L signaling on phenotypic transition of macrophages and angiogenesis.Methods1.Obtain Bone marrow-derived macrophages(BMDM).BMDM were obtained by differentiation of bone marrow cells(BMCs)in the presence of macrophage colony-stimulating factor(M-CSF).The morphology changes,structure,phagocytic function and the expression of markers(F4/80,CD86,CD206)of the cells were detected under inverted phase-contrast microscopy,Wright staining,ink phagocytosis assay and flow cytometry respectively.2.After interfering with CD137-CD137L signal of BMDM[Control group(sterile PBS),CD137-CD137L signaling activation group(recombinant CD137L protein),CD137-CD137L signaling inhibition group(anti-CD137 antibody+recombinant CD137L protein)],the morphological changes of cells in each group were observed under inverted phase contrast microscope.Western Blot was carried out to detect the expression levels of the characteristic markers(αSMA,Collagen I,Vimentin)of myofibroblasts in each group.3.After interfering with CD137-CD137L signal of BMDM,the expression levels and the distribution of CD206 andαSMA in cells of each group were detected by immunofluorescence technique.4.After interfering with CD137-CD137L signal of BMDM,the supernatant of each group was extracted to culture endothelial cells,and the migration and tube formation ability of the endothelial cells were detected by wound healing test and endothelial cells tube formation assay respectively.5.After interfering with CD137-CD137L signal of BMDM,the supernatant of each group was extracted to culture the aortic ring in vitro.Aortic ring angiogenesis was performed to observe the effect of CD137-CD137L signal on angiogenesis.Results1.The results of wright staining,ink phagocytosis test and flow cytometry showed that BMDM with high phagocytic activity and high purity was induced by M-CSF.2.Compared with the control group,the expression level ofαSMA,Vimentin and Collagen I of cells in the CD137-CD137L signal activation group were significantly increased.While the expression level ofαSMA,Vimentin and Collagen I in CD137-CD137L signal inhibition group were significantly down-regulated compared with the activation group,3.Compared with the control group,the green fluorescence intensity of cells and the percentage of CD206~+αSMA~+cells in the CD137-CD137L signal activation group were increased significantly.Compared with the activation group,the green fluorescence intensity of cells and the percentage of CD206~+αSMA~+cells were significantly down-regulated in CD137-CD137L signal inhibition group.4.Compared with the control group,the percentage of the wound healing area of endothelial cells cultured with supernatant of the CD137-CD137L signal activation group was significantly increased.Compared with the activation group,the percentage of wound healing area of endothelial cells cultured with supernatant of CD137-CD137L signal inhibition group was decreased significantly.5.The aortic rings cultured in supernatant of CD137-CD137L signal activation groups was observed vascular sprouting,while the aortic rings cultured in the supernatant of control group and CD137 signal inhibition group had no vascular sprouting.Conclusions1.CD137-CD137L signaling regulates the transition of bone marrow-derived macrophages into myofibroblast(MMT).2.CD137-CD137L signaling enhance the migration and tube formation ability of endothelial cells through regulating MMT.