Coupling Of ?5 Integrins To Annexin A2 By Flow Drives Its Translocation To Membrane Lipid Rafts And Endothelial Activation

Atherosclerosis is a chronic and progressive inflammatory disease,which mainly occurs at the bifurcation and curvature of blood vessels in specific areas.The blood flow in this specific areas is mainly in the form of disturbed flow,and the wall shear stress is non-uniform and has an irregular distribution.Endothelial cells,which form an interface between the vessel wall and blood,can feel the stimulation of blood flow.When endothelial cells are stimulated by high shear force or laminar flow,the expression of related genes or proteins can play an anti-atherosclerosis role.In contrast,when stimulated by low shear forces or disturbed flow,mediate an inflammatory response that leads to atherosclerosis.Our previous studies have shown that integrin?5 in endothelial cells can translocate into lipid rafts and activate endothelial cells under the stimulation of disturbed flow,mediated inflammasome activation,increase the expression of endothelial cell inflammatory cytokines and adhesion factor,and genetic ablation of integrin?5 in LDLR-/-mice significantly reduced the aorta plaque area,further elucidating the important role of integrin?5 in the inflammatory response of Endothelial cells and the process of atherosclerosis occurs.However,the underlying mechanism has remained largely unknown.Therefore,the purpose of our study is to clarify the translocation mechanism of integrin?5 and reveal the molecular biology process of atherosclerosis disease.In order to find the disturbed flow promote integrin?5 to lipid rafts transfer mechanism and possible related proteins,Mass spectrometry studies revealed endothelial Annexin A2 as a potential carrier enable integrin?5 traffic in response to OSS.Immunofluorescence staining and immunoprecipitation assays found that OSS dramatically increased the binding between Annexin A2 and integrins?5 compared with static HUVECs.The translocation of integrin?5 from non-lipid raft to lipid raft fractions in response to OSS was dramatically reversed by si RNA-mediated silencing of Annexin A2 in HUVECs,and the expression of vascular cell adhesion molecule 1(VCAM-1)and intercellular adhesion molecule 1(ICAM-1)were strongly reduced.The immunoprecipitation of HEK295T cells suggesting that CTD of Annexin A2bound with integrin?5 even to a larger extent,suggesting that CTD of Annexin A2predominantly contributed to this interaction.BRET assays that OSS dephosphorylated Annexin A2 at Y24,changed its conformation and subsequently unmasked the active CTD of Annexin A2,which offer the chance to bind with integrin?5.In vivo,we used lenti-Anx A2^WT and lenti-Anx A2^Y24F to infect the endothelium of partially ligated carotid arteries in Apo E^-/-mice,at which disturbed flow occurs,and fed western-type diet immediately after the surgery.Compared to lenti-Anx A2^WTinfected mice,the enhanced protein levels of active form-?5(Act-?5)and VCAM-1in those with lenti-Anx A2^Y24F infection were observed by en face immunofluroscence staining and the carotid artery lesion areas showed a similar trend.Annexin A2 displays the classical property of calcium-dependent lipid binding at its convex side,HUVECs with EGTA failed to show the translocation of integrin?5-Annexin A2 complex to lipid raft,we also found that integrin?5 localized to lipid raft upon the stimulation with A23187 in HUVECs.The decrease of Annexin A2^{p Y24}level and the phosphorylation of FAK induced by OSS were partially reversed in PIEZO1 silenced HUVECs.Previous study reported that PTP1B was the only protein tyrosine phosphatase activated by Ca²⁺/calpain,both of the PTP1B inhibitor and silencing are able to abolish the dephosphorylation of Annexin A2^{p Y24} and FAK activation in response to OSS.In vivo,knock-down endothelial Ptp1B in Apo E^-/-mice and fed a western-type diet resulted in a dramatic reduction of activation of integrin?5,However,Annexin A2 deficiency fully reversed the phenotype of Ptp1B knock-down in Apo E^-/-mice.Our data elucidate that disturbed flow inhibits Tyr24 phosphorylation of Annexin A2,induces conformational changes in Annexin A2,promotes binding and translocation of Annexin A2 to integrin?5,and mediates the activation of integrin?5through the Piezo1-Ca²⁺-PTP1B pathway.This is a new endothelial mechanotransduction molecular mechanism,linking the atherogenic flow and integrins activation,thereby identifying a class of potential therapeutic targets for atherosclerosis.