YPEL2,a Novel Protein Mediating H₂O₂ Induces Endothelial Cellular Senescence Via P53/p21 Pathway

Background and objectiveAtherosclerosis（AS）is a chronic progressive disease characterized by disorders of lipid metabolism,which mainly affects large and medium-sized arteries and causes great harm to human health.The pathogenesis of AS is affected by many factors,and endothelial cellular senescence induced by oxidati ve stress may serve as a potential risk factor for deteriorating atherosclerotic plaque formation.Yippee-like protein 2（YPEL2）is a member of the YPEL protein family with highly homologous zinc finger domain and protein binding site.It may regulate the expression of target genes mediating with stimulus and function in multiple aspect to regulate disease occurrence and development.The regulatory pathway composed of p53 and p21 proteins is one of the pathways that affect cell proliferation and senescen ce,and is a key node in cell cycle regulation.Therefore,the purpose of our study is to investigate the possible roles and specific mechanisms of YPEL2 in regulating vascular endothelial cell senescence and AS plaque formation.Materials and methodsWe constructed the cellular senescence model by using human umbilical vein endothelial cells（HUVECs）treated with hydrogen peroxide（H₂O₂）,and collected 10 pairs of human carotid atherosclerotic plaques and normal carotid membrane for tissue validation.Additionally,18-month-old ApoE-/-mice were used as animal models for in vivo validation.Quantitative real-time polymerase chain reaction（qRT-PCR）and immunoblot experiments were performed to detect the expression levels of YPEL2 in senescent cells and plaques verse negative control.We constructed small interfering fragments of YPEL2 gene and plasmid overexpression vectors to transfect primary HUVECs.A serious of experiments including senescence-related β-galactosidase staining,CCK-8 proliferation detection,cell cycle PI staining,monocyte adhesion experiments,and Nitric oxide detection were used to investigate the effects of YPEL2 on H₂O₂ inducing senescence.Finally,qRT-PCR and immunoblot experiments were performed to investigate the specific mechanism of YPEL2 involving cellular senescence and whether it functions by activating the p53/p21 signaling pathway.Results1.H₂O₂ up-regulates the expression levels of YPEL2 in primary HUVECs in a concentration-dependent manner and promotes cell senescence;immunofluorescent staining shows that YPEL2 is mainly located in nucleus and its expression levels are up-regulated after H₂O₂ treatment;YPEL2 is significantly increased in human carotid atherosclerotic plaques and 18-month-old ApoE-/-mouse plaques in aortic valve.2.The results of senescence-related β-galactosidase staining,cell cycle PI staining and CCK-8 proliferation show that YPEL2 induces cellular senescence and promotes cycle arrest by mediating H₂O₂ in HUVECs;meanwhile,the results of monocytes adhesion and NO detections show that YPEL2 up-regulates the adhesion ability of HUVECs to monocytes and inhibits NO production.3.H₂O₂ regulates the expression levels of aging-related proteins including p53,p21,and LaminB1,additionally up-regulates the adhesion molecules including ICAM1 and VCAM1,while the effect of H₂O₂ is partially inhibited after interfering the expression of YPEL2.4.The overexpression of YPEL2 up-regulates p53 and its downstream target gene p21,and inhibits the levels of CyclinD1 and CDK4 and promotes pRb phosphorylation,while interfering p53 significantly inhibits the effect of YPEL2.Conclusions1.YPEL2 is up-regulated in human carotid atherosclerotic plaque and 18-monthold ApoE-/-mouse plaque in aortic valve.2.YPEL2 induces cellular senescence and promotes cycle arrest by mediating H₂O₂ in primary HUVECs,additionally up-regulates the adhesion ability of HUVECs to monocytes and inhibits NO synthesis.3.YPEL2 up-regulates p21 while inhibits the expression levels of CyclinD1 and CDK4 by interacting p53,and promotes pRb phosphorylation thereby inducing cellular senescence and cycle arrest.