Mechanism Of Endothelial Barrier Dysfunction Induced By Low Shear Stress Mediated By Autophagic Flux/S1P(R) Signaling Pathway

Objective:The low shear stress(LSS)area of artery is the high incidence area of atherosclerosis(AS).Endothelial cell barrier dysfunction was considered to be one of the important pathophysiological processes in AS.Studies have shown that the blocked autophagic flux is involved in the occurrence and development of AS caused by LSS,but its molecular mechanism has not been fully clarified.S1PR1 is the most abundant subtype of S1 PR 1-5receptor in endothelial cells,which plays an important role in maintaining the functional integrity of endothelial barrier.In this study,we investigated the changes of autophagy state on S1PR1 expression and endothelial cell barrier function under different shear forces by intervening ATG8,a key molecule in the formation of autophagosome membrane during autophagy initiation,and the role of ATG8 during LSS induced AS.Methods:(1)Western blot was used to measure the expression levels of autophagy marker protein LC3 and autophagy substrate protein p62 in human umbilical vein endothelial cells(HUVECs)treated with high shear stress(HSS)(30dyne / cm2)and low shear stress(LSS)(5dyne / cm2)for 1h.(2)To investigated the changes of intercellular junction,HUVECs were HSS and LSS for 1h,then stained fibrous actin(F-actin)with phalloidin and stained β-Catenin with Immunofluorescence staining.Also measure the transcription and protein level of S1PR1.(3)HUVECs were transfected with siRNA targeting ATG8 in vitro to construct a model of lower autophagy response under LSS and autophagy initiated but blocked autophagic flux under HSS.(4)After transfection,HSS and LSS were applied for 1 hour,the expression levels of p62,LC3 and S1PR1 were measured,and stained F-actin and β-Catenin to observe the arrangement and morphology of cells.Results:(1)Shear stress induced autophagy activation of endothelial cells,and autophagic flux was unobstructed during HSS and was blocked during LSS.(2)S1PR1 transcription and protein expression increased,and cells were closely connected with clear cell contour and boundary during HSS.During LSS,the transcription level of S1PR1 increased significantly,but the protein expression level did not change significantly compared with the control group.At the same time,vacuolate-like changes occurred at the cell junction,the gap increased,a large number of stress fibers appeared,and the cell contour was unclear.(3)After transfecting siRNA which targeted ATG8 into HUVECs,the protein level of S1PR1 protein during HSS and LSS was lower than that in the non-transfected group.During HSS,the intercellular connection was not tight,and the cytoskeleton protein staining showed increased stress fibers and unclear cell contour,indicating that the intercellular connection was damaged.The cell morphology and connection during LSS were the same as those in the non-transfected group.Conclusion:The blocked of autophagic flux induced by LSS can damage the barrier function of endothelial cells by regulating the expression of S1PR1,which maybe is the molecular mechanism of LSS induced AS.