CD137-CD137L Signaling Affects Angiogenesis by Mediating Phenotypic Conversion of Macrophages

Background: Angiogenesis in atherosclerotic plaque is an important factor causing plaque hemorrhage, vulnerability, and rupture, and different phenotypes of macrophages have different effects on angiogenesis. Our previous study has demonstrated CD137-CD137L signaling, a pair of inflammatory costimulatory molecules, can promote angiogenesis in atherosclerotic plaque. Therefore, we aimed to investigate whether this signaling could affect angiogenesis by regulating phenotypic transition of macrophages. Methods: Male mouse primary peritoneal macrophages were extracted by intraperitoneal injection of thioglycollate, and then flow cytometry was used to detect the expression of CD137. Flow cytometry, Western blotting, quantitative real-time PCR, and enzyme-linked immunosorbent assay (ELISA) were used to assess the phenotypic changes of macrophages after different treatment. Mouse brain microvascular endothelial cells (bEnd.3) were cocultured with macrophages, and tube formation was assessed to examine angiogenesis. Results: We found that the number of junctions and branches of bEnd.3 were increased when CD137-CD137L signaling was activated, while such number was further increased when bEnd.3 were cocultured with macrophages. Flow cytometry showed that CD137 was expressed on almost all primary peritoneal macrophages. The expression of CD86 was decreased in the agonist CD137L group and increased in the agonist CD137L + inhibitory anti-CD137 antibody group after adding the CD137 inhibitor. The expression of CD206 in each group exhibited opposite trend compared with CD86. Moreover, the expression of inducible nitric oxide synthase at the mRNA, and protein levels were decreased after stimulating CD137-CD137L signaling, and such downward trend was reversed when CD137-CD137L signaling was inhibited. Furthermore, the expression of arginase-1 was opposite to that of inducible nitric oxide synthase. Enzyme-linked immunosorbent assay indicated that the content of interleukin-12 (IL-12) in the supernatant of macrophages in the agonist CD137L group was lower than that in the control group, and its content in the inhibited group was higher than that in the activated group. The change of interleukin-10 (IL-10) content in macrophage supernatant was opposite to that of IL-12. When AKT serine/threonine kinase 1 (Akt1) inhibitor was used to inhibit the phenotypic transformation of macrophages induced by CD137-CD137L, the number of junctions and branches formed by bEnd.3 was decreased compared with the coculture group. Conclusions: These results indicated that CD137-CD137L signaling could promote angiogenesis by regulating phenotypic transition of macrophages of male mice.