Interferon-induced Protein 35 (IFI35) Refgulates Endothelial Cell Function And Re-endothelialization Of Injured Arteries By Inhibiting The Nuclear Factor-kappa B (NF-?B) Pathway

Background and Aim:Endothelial cells(ECs)are the barrier and dynamic component of the cardiovascular system and play an essential role in the regulation of vascular function and the maintenance of homeostasis.After various causes of intimal injury(such as balloon dilatation or intracoronary stent implantation),endothelial cell proliferation,migration and repair of the damaged sites,or a process known as re-endothelialization,played an important role in the development of intimal hyperplasia and atherosclerosis.Therefore,it is of great significance to study the mechanisms of re-endothelialization and promote efficient endothelial recovery after vascular injury.Interferon-induced protein 35(IFI35)is an IFN-?-induced protein that plays important roles in the immune-inflammatory response.In this study,we tested whether IFI35 affects the proliferation,migration and re-endothelialization of endothelial cells.Methods:In our present study,primary human umbilical vein endothelial cells(HUVECs)were transfected with Ad-IFI35 or siRNA-IFI35 to evaluate its potential roles in cell proliferation and migration.Furthermore,through the IFI35 domain deletion assay and dual luciferase reporter assay,the effects of IFI35 on NF-?B/p65 signaling pathway and its potential mechanisms were explored.Finally,wire injury of the carotid artery was induced in C57BL/6 mice,which was followed by IFI35 or null adenovirus transduction.Evans blue and HE staining were performed to evaluate the re-endothelialization rate and neointima formation at different time points after overexpression of IFI35 in the injuried arteries.Results:As for the in vitro experiments,Edu staining,CCK8 and cell cycle analysis showed that IFI35 could significantly inhibit the proliferation of endothelial cells.In terms of cell migration,transwell assay and scratch-wound assay showed that IFI35 could inhibit the migration of endothelial cells.Conversely,after inhibiting the expression of IFI35,the proliferation and migration of endothelial cells were significantly enhanced,and the inhibitory effect of IFI35 on the proliferation and migration of endothelial cells could be inhibited by PDTC,an inhibitor of the NF-KB/p65 signaling pathway.In terms of mechanisms,IFI35 could reduce p65 translocation to the nucleus and the formation of cytoplasmic phosphorylated p65,thereby inhibiting NF-KB/p65 signaling pathway activation.Further studies showed that IFI35 could inhibit the activation of NF-?B/p65 signaling through the interaction of its NID1 domain and Nmi protein,and Nmi protein could significantly reduce the inhibition of IFI35 on the proliferation and migration of endothelial cells and the NF-KB/p65 signaling pathway.In vivo studies shows that in IFI35 adenovirus-transduced mice,the re-endothelialization rates at days 3 and day 7 were significantly reduced,compared to that in null adenovirus-transduced mice(5%and 35%,vs 20%and 50%,respectively).Meanwhile,subsequent neointimal hyperplasia of the injured arteries was obviously increased in IFI35 adenovirus-transduced mice.Conclusions:IFI35 could inhibit the proliferation and migration of endothelial cells,thereby delaying the re-endothelialization rate of the intima after the vascular wire injury,resulting in intimal hyperplasia.IFI35 could interact with Nmi through its NID1 domain,inhibit the activation of NF-?B/p65 signaling pathway,thereby weakening the proliferation and migration of endothelial cells.Therefore,IFI35 could be a new target for prevention and treatment of intimal hyperplasia after vascular injury.