Vascular Endothelial Cells Senescence Impairs TRPV4-dependent Vasodilation Through ANXA2

Traditionally,most studies blame aging process on mitochondrial energy metabolism disorder,DNA damage or decreased immunity.As the largest system,circulatory system is the basis of maintaining the homeostasis of human body,it transports nutrients and oxygen and remove metabolic wastes to all body cell through blood vessels.Thus,the"vascular aging theory"came into being.The theory points out that vascular aging is the basis of body aging,dysfunction caused by vascular aging will accelerate the aging process of the body.Blood vessels are composed of endothelial cells and smooth muscle cells.In previous studies,endothelial TRPV4 induced Ca²⁺influx signal plays an important role in endothelium-dependent vasodilation.Studies have shown that in high-salt diet mouse models and high-fat diet mouse models,Endothelial TRPV4 function of resistance arteries is impaired and thus the regulation of blood pressure is weakened.However,there are no studies showing the effect of senescent endothelial cells on TRPV4.Here,we use Calcium sparklet,Whole-cell patch clamp and TRPV4-depandent vasodilation confirmed that TRPV4 activity is impaired in senescent endothelial cells.Western-blot and fluorescence imaging showed that lower expression of TRPV4 on cell membrane of senescent endothelial cells than normal endothelial cells.ANXA2,a part of Annexin family,ANXA2 are closely related to the construction of ion channels,include TRPV5 and TRPV6,TASK1 and Na_v1.8 channel.Through PLA experiment,we confirmed that ANXA2 and TRPV4 have interaction.immunoblotting experiment proved that after Knock down of ANXA2 in ECs with si ANXA2,the expression of TRPV4 in endothelial cells membrane decreased,finally affected the activity of TRPV4 in endothelial cellsIn this study,we examined the interaction between ANXA2 and TRPV4 and Effect of ANXA2 on TRPV4 expression on endothelial cell membrane.Experimental results show that accumulation of ROS caused by the increase of Nox2 expression in senescent endothelial cells will destroy the interaction between ANXA2 and TRPV4,leading to lower expression of TRPV4 on cell membrane.Ultimately,TRPV4 activity is impaired.Caveolin-1 is the main structural protein of caveolae,which can regulate many cellular signal transductions.In pulmonary arteries,endothelial Caveolin1 can promote TRPV4 activity through PKC（protein kinase C）.Interestingly,we found interaction between Caveolin1 and ANXA2 in our study,ANXA2 also has a membrane-driving effect on Caveolin1,resulting in the distancing of Caveolin1 and TRPV4 on cell membrane.The promoting effect of Caveolin1 to TRPV4 is weakened,and the activity of TRPV4 is inhibited indirectly.Here,we discover NOX2-ROS-ANXA2-TRPV4 pathway and NOX2-ROS-ANXA2/Caveolin1-TRPV4 pathway indicate how cellular senescence impairs TRPV4activity and TRPV4-dependent vasodialtion.Thus,inhibiting the production of ROS or activity of NOX2 in senescent endothelial cells may serve as an effective strategy for maintain that senescent vascular function.