To investigate whether CD137 signaling can promote angiogenesis via regulating macrophage M1/M2 polarization. (1) The primary peritoneal macrophages in mice induced by 3% thiglycollate broth were divided into three groups: control group, CD137 signaling activated group and CD137 signaling inhibited group. Various specific markers of M1 and M2 macrophages were detected to observe the phenotype change of macrophages, and the macrophages protein expression of CD137, CD86 and CD206 was detected by flow cytometry (FCM). The protein and mRNA expression of induced nitric oxide synthase (iNOS), arginase Ⅰ(Arg-1) was determined by Western blot and RT-PCR, respectively. The secretion levels of IL-12 and IL-10 in culture supernatant of macrophages were detected by ELISA. (2) Macrophages were co-cultured with the endothelial cells (bEnd.3), and macrophages were implanted in the upper chamber, endothelial cells were implanted in stromal glue of the lower chamber. The experiment was divided into three groups: the control group, CD137 signaling activated group and peroxisome proliferator-activated receptor-γ (PPAR-γ) inhibited group, and tube formation ability of endothelial cells in each group was determined. (1) The purity of primary peritoneal macrophages in mice was (97.93±1.31)%. The expression of CD137 on the surface of macrophages was (97.40±2.70)%. (2) Compared with control group, the mRNA and protein expression levels of Arg-1 were significantly increased and the mRNA and protein expression of iNOS were significantly decreased in CD137 signaling activated group (all <0.05). Compared with CD137 signaling activated group, the mRNA and protein expression of Arg-1 were significantly lower and the mRNA and protein expression levels of iNOS were significantly higher in CD137 signaling inhibited group (all <0.05). FCM results showed that the average fluorescence intensity of CD206 was higher, while the average fluorescence intensity of CD86 was lower in CD137 signaling activated group than in control group (<0.05, <0.01, respectively); the expression of CD206 was significantly lower, while the expression of CD86 was higher, in the CD137 signaling inhibited group than in CD137 signaling activated group (<0.05, <0.01, respectively). ELISA results showed that the secretion of IL-10 was higher, and the secretion level of IL-12 was significantly lower in CD137 signaling activated group than in control group (both <0.01); the secretion of IL-10 was significantly lower and the secretion of IL-12 was significantly higher in CD137 signaling inhibited group than in CD137 signaling activated group (both <0.05). (3) Values of the formation of tube length and branch number were both longer in CD137 signaling activated group than control group (<0.05). The formation of the tube length and branch number were less in PPAR-γ inhibited group than in CD137 signaling activated group (<0.05). CD137 signaling can promote angiogenesis by regulating macrophage M1/M2 polarization.

References

PubMed