The schematic diagram for the role of EC-derived exosomes and CD137 signaling on PDGF-BB induced VSMCs phenotype switch by regulating the expression of TET2 protein. In vitro, CD137 L as CD137 ligand was used to activate CD137 signaling of ECs. After activating CD137 signaling, collected exosomes released by EC, purification of these exosomes and added to VSMCs which pretreatment with PDGF-BB then measure phenotypic proteins, MYH11, Calponin, α-SMA and Vimentin. In vivo, 0.36 mm diameter guide wire was used to injure common carotid artery intima. Administration of EC-exosomes from different treatments into C57BL/6 J by tail vein on day1, 7, 14, 21 and sacrificed on day 28 to get carotid arteries for neointimal formation detection.

Activated endothelial cells (ECs) play an important role in the development of atherosclerosis (AS) because they form neointima resulting from abnormal vascular smooth muscle cell (VSMC) hyperplasia. TET2 was recently reported as a major regulator of the phenotypic switch of VSMCs, and it also protects ECs from noxious stimuli and represses inflammation in AS. The purpose of the present study is to determine whether activation of CD137 signaling can regulate the function of VSMCs by altering endothelial TET2 expression and to explore the potential mechanisms. VSMCs were isolated from C57BL/6 J mice, and the influence of ECs on VSMCs was analyzed using a co-culture system. Western blotting was used to detect the protein expression levels of TET2 and CD9, CD81, and CD63 as well as VSMC phenotypic markers. RT-PCR was performed to detect the TET2 mRNA levels. EdU proliferation assay and transwell migration assay were employed to assess the functions of VSMCs. EXO-spin kits were used to extract and purify exosomes. Transmission electron microscopy and nanoparticle tracking analysis were applied to characterize exosomes. ECs transduced with the TET2-overexpression lentivirus showed that the activation of endothelial CD137 signaling decreased TET2 expression and the TET2 content in exosomes. EC-derived exosomes were internalized by VSMCs, which inhibited phenotypic switching in vitro and neointimal formation in vivo. However, exosomes derived from CD137-activated ECs exhibited weakened protective effects on VSMCs. In conclusion, activation of endothelial CD137 signaling attenuated the repressive effects of EC-derived exosomes on platelet-derived growth factor (PDGF)-BB-induced phenotypic switching of VSMCs and neointimal formation after carotid injury.