The Effect Of Fluid Shear Stress On The Barrier Function Of Endothelial Cells And An Investigation Of The Underlying Mechanism

ObjectiveAtherosclerotic plaques are mainly distributed in the low shear stress(LSS)region of arteries,but the molecular mechanism is still unclear.The maintenance of normal endothelial cell barrier function is an important condition to resist the formation of atherosclerosis.Studies have shown that fluid shear stress affects the autophagy of endothelial cells.The purpose of this study was to investigate the relationship between autophagy induced by fluid shear stress and endothelial barrier function,and its potential mechanism.MethodsHUVECs were exposed to low shear stress(LSS,5 dyne/cm~2)and high shear stress(HSS,30 dyne/cm~2)produced by a parallel plate chamber system.The LC3II/I ratio and the expression levels of P62 and Sphingosine 1 phosphate receptor-1(S1PR1)were determined by Western blotting.To investigate the relationship between S1PR1 and autophagy,we repeated the above assays after treating cells exposed to HSS with Baf-A1,an inhibitor of autophagic flux,and treating cells exposed to LSS with Rapamycin,an activator of autophagy.We examined the spatial localization and polymerization of F-actin by cytochemical staining to evaluate the changes in endothelial barrier function induced by shear stress.ResultsAn increase in the LC3II/I ratio was seen in cells exposed to LSS and HSS,however,the P62/GAPDH ratio was noticeably decreased in HSS group cells but increased in LSS group cells;P62 was increased in HSS group cells pretreated with Baf-A1 and decreased in LSS group cells pretreated with Rapamycin.The expression of S1PR1 was much higher on HUVECs exposed to HSS than on those exposed to LSS;pretreatment with Baf-A1 reduced S1PR1 expression on cells exposed to HSS;the expression of S1PR1 on cells exposed to LSS increased in the presence of Rapamycin.Dramatic enhancement of F-actin polymerization was seen in cells treated with Sphingosine 1 phosphate(S1P)and exposed to HSS;F-actin depolymerization occurred in cells treated with S1P and exposed to HSS in the presence of Baf-A1;after the addition of W146,stress fibres in cells were disordered;in cells exposed to LSS and treated with rapamycin and S1P,we found increased numbers of stress fibres and rough cellular outlines compared with those in non-pretreated cells exposed to LSS.ConclusionAutophagic flux in HUVECs is blocked under LSS conditions,which will down-regulate the expression of S1PR1 on HUVECs,this process is a critical signal pathway for the formation of atherosclerotic plaques in blood vessels promoted by LSS.Whereas the activation of autophagy by HSS up-regulates the expression of S1PR1 on HUVECs,maintains normal barrier function of HUVECs,which may be the molecular mechanism of HSS to resist the occurrence and development of atherosclerosis.