CD137 Signaling Promotes Endothelial Apoptosis by Inhibiting Nrf2 Pathway, and Upregulating NF-κB Pathway.

BACKGROUND: Endothelial dysfunction and apoptosis resulting from oxidative stress can lead to the development of atherosclerosis. Our group has previously showed that CD137 signaling contributes to the progression of atherosclerosis and the vulnerability of plaques. The aim of this study is to investigate the effects of CD137 signaling in atherosclerosis on endothelial cells (ECs) apoptosis and to explore the underlying mechanisms. METHODS: Serum samples were collected from 11 patients with acute myocardial infarction and 4 controls. Peritoneal injection of agonist-CD137 recombinant protein in ApoE-/- mice was used to determine whether CD137 signaling can promote apoptosis in vivo, and human umbilical vein endothelial cells treated with agonist-CD137 recombinant protein, M5580 (a Nrf2 pathway agonist) and CAPE (a NF-κB pathway inhibitor) were used to explore the effect of Nrf2 and NF-κB pathway in CD137 signaling-induced ECs apoptosis in vitro. RESULTS: ELISA showed that Bcl-2 in the serum of AMI patients was lower than that of the control group, while TNF-α and sCD137 were higher than that of the control group. Confocal microscopy and Western blot analysis showed that the nuclear translocation of Nrf2 in the agonist-CD137 group was significantly inhibited, and the expression of its downstream antioxidant enzymes was also decreased when compared with control. Immunofluorescence and Western blot results showed that the nuclear translocation of NF-κB in the agonist-CD137 group was enhanced, and ELISA results showed that the secretion of proinflammatory cytokines in the agonist-CD137 group was increased. Immunofluorescence results revealed that ROS production in the agonist-CD137 group was higher than that in control, M5580 (a Nrf2 pathway agonist) and CAPE (a NF-κB pathway inhibitor) groups. In vitro studies using HUVECs and in vivo studies using high-fat-fed ApoE-/- mice showed that the number of apoptotic endothelial cells was the highest in the agonist-CD137 group. By contrast, both M5580 and CAPE treatments were able to reduce CD137 induced ECs apoptosis. CONCLUSIONS: Our results showed that CD137 signaling promotes ECs apoptosis through prooxidative and proinflammatory mechanisms, mediated by Nrf2 and NF-κB pathways, respectively.

CD137 signaling induces macrophage M2 polarization in atherosclerosis through STAT6/PPARδ pathway.

CD137 signaling plays an important role in the formation and development of atherosclerotic plaques. The purpose of the present study was to investigate the effects of CD137 signaling on macrophage polarization during atherosclerosis and to explore the underlying mechanisms. The effect of CD137 signaling on macrophage phenotype in atherosclerotic plaques was determined by intraperitoneal injection of agonist-CD137 recombinant protein in apolipoprotein E-deficient (ApoE-/-) mice, an established in vivo model of atherosclerosis. Murine peritoneal macrophages and RAW 264.7 cells were treated with AS1517499 and siPPARδ (peroxisome proliferator-activated receptor δ) to study the role of STAT6 (signal transducers and activators of transcription 6)/PPARδ signaling in CD137-induced M2 macrophage polarization in vitro. Results from both in vivo and in vitro experiments showed that CD137 signaling can transform macrophages into the M2 phenotype during the process of atherosclerotic plaque formation and regulate the angiogenic features of M2 macrophages. Furthermore, activation of the CD137 signaling pathway induces phosphorylation of STAT6 and enhances the expression of PPARδ. We further found that macrophage M2 polarization is reduced when the STAT6/PPARδ pathway is inhibited. Together, these data show a role for the STAT6/PPARδ signaling pathway in the CD137 signaling-induced M2 macrophage polarization pathway.

Exosome derived from CD137-modified endothelial cells regulates the Th17 responses in atherosclerosis.

The role of exosomes derived from endothelial cells (ECs) in the progression of atherosclerosis (AS) and inflammation remains largely unexplored. We aimed to investigate whether exosome derived from CD137-modified ECs (CD137-Exo) played a major role in AS and to elucidate the potential mechanism underlying the inflammatory effect. Exosomes derived from mouse brain microvascular ECs treated with agonist anti-CD137 antibody were used to explore the effect of CD137 signalling in AS and inflammation in vitro and vivo. CD137-Exo efficiently induced the progression of AS in ApoE-/- mice. CD137-Exo increased the proportion of Th17 cells both in vitro and vivo. The IL-6 contained in CD137-Exo which is regulated by Akt and NF-КB pathway was verified to activate Th17 cell differentiation. IL-17 increased apoptosis, inhibited cell viability and improved lactate dehydrogenase (LDH) release in ECs subjected to inflammation induced by lipopolysaccharide (LPS). The expression of soluble intercellular adhesion molecule1 (sICAM-1), monocyte chemoattractant protein-1 (MCP-1) and E-selectin in the supernatants of ECs after IL-17 treatment was dramatically increased. CD137-Exo promoted the progression of AS and Th17 cell differentiation via NF-КB pathway mediated IL-6 expression. This finding provided a potential method to prevent local and peripheral inflammation in AS.

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.