CD137 Signaling Pathway Mediates Vascular Remodeling Via Regulating Endothelial Cell Function

Background Vascular intimal hyperplasia is involved in the initiation and development of Atherosclerosis(AS),restenosis after angioplasty and other vascular remodeling diseases.The excessive proliferation and migration of vascular smooth muscle causes intimal hyperplasia,which is an central link in pathological process of vascular remodeling.Neovascularization in plaque plays a critical role in plaque rupture and malignant vascular remodeling.The inflammatory costimulatory molecule CD137,ligation with CD137L(CD137 signaling)mediates various pathological processes and affects the development of atherosclerosis and plaque instability.However,the mechanisms that CD137 signaling mediated effects of endothelial cell function changes on angiogenesis and intimal hyperplasia remain unclear.Objectives To investigate the mechanisms of CD137 signaling mediated vascular remodeling(angiogenesis and intimal hyperplasia)via regulating endothelial cell function.Methods 1.CD137 signaling regulates angiogenesis by activating VEGFR2 pathway and promoting VEGFA secretion in endothelial cells(1)To clarify the role of CD137 molecule in angiogenesis In vivo: The number of neovessels in Apo E^-/- and Apo E^-/- CD137-/-mice atherosclerotic plaques were observed by immunohistochemical staining.Aortic ring angiogenesis assay was used to observe the aortic ring sprouting of CD137-/-and C57BL/6J mice.In vitro: CD137 si RNA transfection was conducted to observe the effect of CD137 silencing on human umbilical vein endothelial cells(HUVEC)proliferation,migration and tube formation ability.(2)CD137 signaling affects angiogenesis through VEGFR2/Akt/e NOS pathway The effect of activating the CD137 signaling on VEGFR2/Akt/e NOS pathway was observed by q RT-PCR and Western blot.Western blot,cell proliferation,cell migration,HUVEC tube formation,and aortic ring assay were applied to observe the effects of inhibiting the VEGFR2/Akt/e NOS pathway on the expression of angiogenesis related molecular expression,endothelial cell proliferation,migration,tube formation and aortic ring sprouting mediated by CD137 signaling when treated VEGFR2 inhibitors(Cabozantinib),Akt inhibitors(LY294002)or e NOS inhibitors(L-NAME)alone or combined.(3)CD137 signaling mediates VEGFR2 activation through promoting VEGFA secretion of endothelial cells Western blot was used to detected the expression of VEGFA in endothelial cells induced by CD137 signaling.Western blot and enzyme-linked immunosorbent assay(ELISA)were used to detect the secretion of VEGFA in endothelial cells mediated by CD137 signaling.2.CD137 signaling mediates intimal hyperplasia via reduced endothelial TET2 protein shift(1)CD137 signaling affects endothelial cell function to regulate phenotypic switch of vascular smooth muscle cells Tissue explants adherent method was applied to harvest primary mouse VSMC.?-SMA immunofluorescence staining was applied to identify the primary VSMC.Western blot was used to detect the expression of VSMC phenotypic proteins in EC(mouse brain endothelial cell line Bend.3)and VSMC co-culture system.Grouping: Control group: Upper chamber: EC,Lower chamber: VSMC;PDGF-BB group: Upper chamber: EC,Lower chamber: PDGF-BB treated VSMC and CD137 agonistic group: Upper chamber: CD137 L treated EC,Lower chamber: PDGF-BB treated VSMC.(2)CD137 signaling reduces endothelial exosomes mediated TET2 protein shift and affects vascular smooth muscle cell phenotypic switch,biological function in vitro and intimal hyperplasia.In vitro: Western blot,nanoparticle tracking analysis(NTA)and transmission electron microscopy(TEM)was used to characterize endothelial derived exosomes.CM-Dil was used to label exosomes in order to observe the exosome internalization.Western blot was used to detect the expression of VSMC phenotype proteins and TET2 protein.The VSMC proliferation and migration ability were detected via Edu-555 assay and Transwell assay.Grouping: Control group,PDGF-BB group,EC-CD137L-exos group and PDGF-BB+ECCD137L-exos group. In vivo: Construction and identification of the mice carotid artery wire injury model.CD31 immunofluorescence staining of the mouse injured carotid artery showed no obvious green fluorescence was seen.Hematoxylin-eosin(HE)staining was used to observe the intimal hyperplasia after carotid artery injury.The expression of TET2 protein and myosin heavy chain 11 protein were detected by immunofluorescence staining.Grouping: Sham group,Control(model)group,EC-CD137L-exos group and EC-CD137L-exos group.Exosomes were injected via the tail vein once a week for four weeks.(3)Overexpression of TET2 inhibits endothelial exosomes mediated intimal hyperplasia after endothelial CD137 signaling activation.In vitro: overexpressed TET2 lentivirus was used to transfect endothelial cells and the transfection efficiency was detected by q RT-PCR and Western blot.The effects of endothelial derived overexpression of TET2 exosomes on VSMC phenotypic protein expression,proliferation and migration function were detected.Grouping: PDGF-BB group,PDGF-BB+Lv-TET2-exos group,PDGF-BB+CD137L-exos group and PDGF-BB+(LvTET2+CD137L)-exos group.In vivo: To observe the effect of the above endothelial derived exosomes on intimal hyperplasia.Grouping: Sham group,Control group,Lv-TET2-exos group,CD137L-exos group and(Lv-TET+CD137L)-exos group.Results 1.Endothelial CD137 is involved in the process of angiogenesis CD137 knockout in vivo reduced microvessel number in atherosclerotic plaque.Silencing CD137 in vitro reduced the aortic ring sprouting number,endothelial cell proliferation,migration and tube formation.2.CD137 signaling activates the VEGFR2/ Akt/e NOS pathway in endothelial cells Activation of the endothelial CD137 signaling promotes VEGFR2 transcription and translation and thus up-regulates the expression of p-VEGFR2,p-Akt and p-e NOS.3.CD137 signaling promotes angiogenesis via activating the VEGFR2/ Akt/e NOS pathway Compared with the control group,blocking the VEGFR2/Akt/e NOS pathway attenuated the CD137 signaling mediated endothelial proliferation,migration,tube formation and the aortic ring sprouting.4.CD137 signaling activates VEGFR2/Akt/e NOS pathway via promoting VEGFA secretion of endothelial cells Compared with the control group,the VEGFA content from EC and its supernatant increased in the CD137 agonist group,while the content of VEGFA in exosomes decreased.Compared with the CD137 agonist group,VEGFA content from EC and its supernatant decreased and VEGFA content in exosomes increased in the Anti-CD137 agonist group.Compared with the control group,the levels of VEGFA increased in EC and it's supernatant in the CD137 agonist group,while decreased significantly in the anti-CD137 group compared with the CD137 agonist group.In addition,the results of exosome detection in the endothelial supernatant showed that the level of VEGFA increased in the CD137 agonist group compared with the control group,while the level of VEGFA decreased in the antiCD137 group compared with the CD137 agonist group.5.Activation of CD137 signaling in endothelial cells promotes the phenotypic switch of VSMC induced by PDGF-BB during EC-VSMC co-culture.Compared with the PDGF-BB treated group,the expression of PDGF-BB-induced synthetic protein Vimentin and the contractile phenotypic markers ?-SMA,Calponin and MYH11 were obviously up-regulated after activation of the CD137 signaling.6.Activation of CD137 signaling regulates the transfer of endothelial cell derived exosomes to VSMC and promotes the proliferation,migration and neointima formation In vitro: compared with the PDGF-BB group,endothelial-derived exosomes(EC-CTRLexos)treated VMSC reduced the pro-phenotypic switch,pro-proliferation and promigration effects of VSMC.Compared with the EC-CTRL-exos group,activation of endothelial CD137 signaling(EC-CD137L-exos)attenuated the inhibition effect of endothelial-derived exosomes on VSMC phenotypic switch,proliferation and migration.In vivo: compared with the sham group,the neointima formation was obviously observed in injured carotid artery(CTRL)group.Compared with the CTRL group,the EC-CTRLexos group significantly inhibited neointima formation.Compared with the EC-CTRLexos group,the EC-CD137L-exos group significantly increased the neointima formation. 7.CD137 signaling affects the expression of endothelial TET2 and promotes the phenotypic switch of vascular smooth muscle cells In vitro: compared with the EC group,the expression of TET2 protein in EC and its derived exosomes significantly decreased after activation of the CD137 signaling.Compared with the EC group,overexpressed TET2 protein exosomes(PDGF-BB+Lv-TET2-exos)significantly inhibited the phenotypic switch,proliferation and migration of VMSC.However,compared with the CD137 signaling activated(PDGF-BB+CD137L-exos)group,overexpressed TET2 protein exosomes [PDGF-BB+(Lv-TET2+CD137L)-exos] group obviously inhibited the phenotypic switch,proliferation and migration of VMSC.In vivo: compared with the CTRL group,Lv-TET2-exos group significantly reduced the neointima formation.The(Lv-TET2+CD137L)-exos group significantly reduced neointima formation compared with the CD137L-exos group.Conclusions 1.CD137 signaling promotes angiogenesis via activation of VEGFA/VEGFR2/Akt/e NOS pathway.2.CD137 signaling promotes intimal hyperplasia through down-regulation of endothelial TET2 protein expression and shift to vascular smooth muscle cell.