Study On The Role And Mechanism Of Laminar Shear Stress-sensitive Gene EPHB2 In Atherosclerosis

Atherosclerosis is the main cause of cardiovascular and cerebrovascular diseases such as coronary heart disease and myocardial infarction.Despite years of hard work by scientific researchers and medical staff,the morbidity and mortality of cardiovascular and cerebrovascular diseases induced by atherosclerosis remain high.Atherosclerosis is caused by long-term excessive inflammation of the blood vessel wall,and the inflammatory response plays a vital role in the development of atherosclerosis at all stages.Therefore,targeting the inflammatory response of vascular endothelial cells to prevent and treat atherosclerosis has attracted much attention in recent years.Autophagy is a highly self-regulating process of cells,which plays an important role in many physiological and pathological processes of the body.Studies have shown that autophagy is involved in starvation adaptation,quality control of intracellular proteins and organelles,aging,tumor suppression,and oxidative stress.It plays an important role in the process of stimulation,prevention of neurodegenerative diseases and immunity,especially in the inflammatory response of cardiovascular diseases.Laminar shear stress（Lss）can regulate endothelial cell homeostasis by affecting gene expression in vascular endothelial cells,which is crucial in the development of atherosclerosis and the normal physiological function of blood vessels,and has anti-atherosclerotic properties.Therefore,using Lss-sensitive genes as an entry point to study cardiovascular diseases may be a promising strategy to combat the development of atherosclerosis.self-assembling peptide（SAP）can self-polymerize into a sponge-like fibrous network structure under physiological conditions,and this structure can make self-polypeptide have a controlled and sustained release effect of molecules,polypeptides,proteins,DNA and RNA.Studies have shown that SAP hydrogels with excellent biocompatibility and low immunogenicity can be used as carriers for sustained-release applications of therapeutic proteins,prolonging the action time of therapeutic proteins in the body to make them work more effectively.Therefore,SAP have great application prospects in the field of biomedicine.In this study,we found that laminar shear stress could inhibit tumor necrosis factor-α（TNF-α）induced increases in intercellular cell adhesion molecule-1（ICAM-1）,vascular cell adhesion molecule-1（VCAM-1）,cyclooxygenase-2（COX-2）,and matrix metalloproteinase-9（MMP-9）expression in human aortic endothelial cells（HAECs）and human umbilical vein endothelial cells（HUVECs）in an autophagy-dependent manner.In order to mine laminar shear stress-sensitive genes,HAECs used for whole-transcriptome sequencing were divided into three groups in this study:the static group（Static group）;The laminar shear stress of 12 dyn/cm² continued to act on HAECs for12 hours（Lss group）;And after 12 hours of laminar shear stress of 12 dyn/cm²,the HAECs returned to steady state culture for 24 hours（After Lss group）.Sequencing results showed that EPHB2 is a key gene in response to laminar shear stress.In addition,co-immunoprecipitation experiments showed that laminar shear stress can enhance EPHB2-mediated nuclear translocation of high mobility group protein 1（HMGB1）,which can interact with Beclin-1（BECN1）and finally induce autophagy.At the same time,this study identified a laminar shear stress-sensitive long non-coding RNA,lnc RNA LOC10798635,and successfully constructed a laminar shear stress-related lnc RNA LOC107986345/mi R-128-3p/EPHB2 regulatory axis.Further mechanistic studies showed that activation of the lnc RNA LOC107986345/mi R-128-3p/EPHB2axis can cause HMGB1 nuclear translocation,and the autophagy activation caused by this nuclear translocation plays an important role in the anti-inflammatory process of laminar shear stress.Further,this study intends to use the ligand ephrin B1 of EPHB2（Eph B2）to explore the feasibility of EPHB2 as a target for intervention in atherosclerosis.Preliminary studies have shown that simply injecting ephrin B1-Fc recombinant protein into mice will cause ephrin B1-Fc to be quickly lost in the blood.Therefore,we constructed SAP carried ephrin B1（ephrin B1-SAP）to interfere with the formation and development of atherosclerotic plaques in Apo E^-/-mice,and the results were gratifying.SAP can smoothly release ephrin B1-Fc into the blood of mice to prolong the time of ephrin B1-Fc in mice.In addition,HE staining results showed that ephrin B1-SAP reduced the plaque area and the proportion of inflammatory cells in the aorta of Apo E^-/-mice,and at the same time alleviated the degree of intima hyperplasia.Oil red O staining results showed that ephrin B1-SAP could effectively reduce the degree of lipid deposition in aortic plaques of Apo E^-/-mice.The results of immunohistochemistry and Western blot analysis experiments showed that ephrin B1-SAP inhibited the expression of inflammatory factors in vascular endothelial cells and promoted the expression of autophagy marker protein MAP1LC3B2.It is suggested that ephrin B1-SAP could activate autophagy and inhibit vascular inflammation in Apo E^-/-mice.This study proved the importance of EPHB2 as a target to investigate the occurrence and development of atherosclerosis,and opened up the application prospect of ephrin B1-SAP in inhibiting the formation of atherosclerotic plaques,providing a new strategy and means for effectively preventing and treating the occurrence and development of atherosclerosis.