Effects Of YAP On Vascular Endothelial Senescence In Rats And Underlying Mechanism

Background:Vascular senescence based on physiological aging and influenced by various risk factors,is characterized by structural and functional change,mainly involving endothelial cells,smooth muscle cells and fiber cells,which is the common pathological basis of many cardiovascular and cerebrovascular diseases.Vascular endothelial senescence,accompanied by the increases of inflammatory factors and reactive oxygen species and the decreases of nitric oxide,is a key site for the treatment of vascular aging,which causes to vascular dysfunction and a series of vascular diseases including atherosclerosis,hypertension,diabetes vascular diseases,etc.Yes-associated protein(YAP),a major effector of the Hippo signaling pathway plays an important role in vascular inflammation,the hallmark of senescence.Recently,it has been found that YAP is demonstrated to control autophagy by associating with TBK1 in the cytoplasm or regulating of Armus and other members of this family.Autophagy,a normal physiological activity,can be activated by hunger or other emergency to degrade the dysfunctional large molecules including proteins,nucleic acids,lipids,and organelles substances by autophagy-lysosome.When autophagy function dysfunction,the mechanism of cellular catabolism collapse causing mitochondrial dysfunction,ATP in the cell decreased and reactive oxygen species increased and leading cell senescence.In addition,mTOR,an important signaling pathways in regulating autophagy,has a mutual regulating effect with the YAP signaling pathway.Therefore,we hypothesized that YAP could promote vascular endothelial senescence through mTOR-mediating autophagy.Objectives:The purpose of this study was to investigate the expression level of YAP during rat vascular senescence and to explore the roles of YAP in rat vascular endothelial aging and underlying mechanism.Methods and results:1)The vascular tissues were obtained from young and old SD rats and assessed by analysis of the SA-β-gal staining and semi-quantification of senescence markers P1 6 and P53 and YAP.We found that YAP was highly expressed in vascular tissues of old rat and its level was correlated with the upregulation of senescence markers P21,P16 and P53.2)To ascertain the roles of YAP on rat vascular endothelial senescence.In vitro,we firstly treated human umbilical vein endothelial cells or vascular tissues in rats with 0.5mg/mL of LPS to establish an aging model.Then,the silencing or overexpression of YAP respectively were performed in HUVECs and rat vascular tissues.Vascular endothelial senescence was assessed by analysis of the SA-β-gal staining and semi-quantification of senescence markers P16,P21,P53,TERT and TRF1 expression.In vivo,4 weeks old SD mice were randomly assigned to the four groups(n=4 for each group):0 PFU group,6.32×104 PFU group,6.32×105 PFU group,and 12.64×105 PFU group.The corresponding dose of overexpressed adenovirus was injected into the tail vein,and the samples were collected and stained with SA-β-gal after 4 weeks.The results showed that YAP silencing reduces rat vascular endothelial senescence,while YAP overexpression promotes rat vascular endothelial senescence.3)to investigate whether YAP affects rat vascular endothelial senescence through mTOR-mediating autophagy.Human umbilical vein endothelial cells or rat vascular tissues were treated with si YAP、Ad-YAP and rapamycin and then the results were assessed by SA-β-gal staining and Western Blot to detect the expressions of p-mTOR,mTOR,Beclin1,LC3Ⅰ,LC3Ⅱ,P62,YAP,P16,P21,P53,TERT and TRF1.The results showed that YAP activates the mTOR signaling pathway and inhibits autophagy during YAP-promoted rat vascular endothelial senescence.When rapamycin was used to inhibit the mTOR signaling pathway,the effects of YAP on senescence and autophagy was relieved.Conclusions:This study showed that YAP can promote rat vascular endothelial senescence,and the mechanism of YAP promoting rat vascular endothelial senescence could be by mTOR-mediating autophagy,which may provide certain reference for the molecular target treatment of vascular endothelial senescence related diseases.