Mechanistic Study Of The Mechanism By Which Low Shear Stress Impairs Endothelial Function Via AT1R

Background: Reactive oxygen species(ROS)contributed to many aspects of physiological and pathological cardiovascular processes.However,the underlying mechanism of ROS induction by low shear stress(LSS)remains unclear.Accumulating evidence has shown that the angiotensin II type 1 receptor(AT1R)was involved in inflammation,apoptosis,and ROS production.Objective: Our aim was to explore the role of AT1 R in LSS-mediated ROS induction.Methods: We exposed human umbilical vein endothelial cells(HUVECs)to LSS(3dyne/cm2)for different periods of time(0,5,15,30,60,120 min).Western blotting was used to measure AT1 R,AT2R,AKT,ERK,eNOS protein and phosphorylation expression levels while q PCR to analysis AT1 R expression.HUVECs were cultured with a fluorescent probe,either DCFH,DHE or DAF,after being subjected to LSS.Flow cytometry also was used to further measure endothelial cell NO and ROS levels.Additionally,Cell Immunofluorescence was also used to examine the AT1 R expression.Furthermore,using immunohistochemistry we also detected AT1 R expression and ROS level in the inner curvature of the aortic arch and the descending aorta in C57BL/6 mice.The above molecular was assayed by corresponding methods under LSS 30 min after AT1 R was significantly inhibited by small interfering RNA and AT1 R antagonist Losartan(1 μM).Finally,we explore the AKT,ERK and eNOS phosphorylation at Ser633,1177 and Thr495 expression after human vein endothelial cells exposure to AKT and ERK inhibitor(LY29002 and PD98059,respectively).Results: Low shear stress significantly increased endothelial cell AT1 R and reactive oxygen species expression.eNOS phosphorylation at Ser1177 and Ser633 was markedly increased while eNOS phosphorylation at Thr495 was inhibited by Losartan(1 μM).NO was also upregulated and ROS was downregulated by Losartn.The dephosphorylation at Ser1177,Ser633 and phosphorylation at Thr495 was significantly reversed by si-AT1 R.The increase of eNOS phosphorylation at Ser1177 and Ser633 and decrease of eNOS phosphorylation at Thr495 was significantly reversed by AKT and ERK inhibitory.Moreover,the ROS level was significantly reduced by endogenous and exogenous NO donors(L-arginine,SNP)and increased by the eNOS inhibitor L-NAME.Conclusion: LSS induces ROS production via AT1R/eNOS/NO pathway.Background: Atherosclerosis was a chronic inflammation of vessel characterized by endothelial cell inflammation and vascular smooth muscle cells proliferation,resulting in ischemia of distal artery and causes cardiovascular events.Low shear stress(LSS)-induced endothelial inflammation was the basis for the development of atherosclerosis.However,the mechanism underlying LSS-induced inflammation is not well understood.The angiotensin II type 1 receptor(AT1R),a component of the renin-angiotensin system,participated in atherosclerotic plaque progression.Objective: Our aim was to investigate the role of AT1 R in LSS-induced endothelial activation.Methods: We exposed human umbilical vein endothelial cells(HUVECs)to LSS(3dyne/cm2)for different periods of time(0,30,60,120 min).Western blotting was used to measure AT1 R,ICAM1,VCAM1,NFκB、ERK、JNK、P38 protein and phosphorylation expression levels while q PCR to analysis AT1 R,ICAM1 and VCAM1 mRNA expression.Additionally,Cell Immunofluorescence was also used to examine the AT1 R,ICAM1,VCAM1 expression.Furthermore,using immunohistoch-emistry we also detected AT1 R,ICAM1,VCAM1 protein expression in the inner curvature of the aortic arch and the descending aorta in C57BL/6 mice.The above molecular was assayed by corresponding methods in respond to LSS after human vein endothelial cells preincubated with pharmacological inhibition and small interfering RNA.Finally,we detected the nuclear NFκB expression by nuclear extract after AT1 R and ERK were effectively inhibited.The effect of AT1 R antagonists on endothelial cell apoptosis was also measured by tunel staining and flow cytometry.Results: LSS significantly activated endothelial cell inflammatory markers ICAM1,VCAM1,AT1 R,and NFκB.Losartan(1 μM)and AT1 R siRNA significantly inhibited the expression of ICAM1,VCAM1,p-ERK,p-NFκB,and nuclear NFκB in endothelial cells,while ERK inhibitors and ERK siRNA significantly inhibited the significant inhibition of endothelial cells ICAM1,VCAM1,p-ERK,p-NFκB,and nuclear NFκB.In addition,Losartan also significantly reduced endothelial cell apoptosis.Conclusion: LSS induces endothelial inflammation via AT1R/ERK signaling and that Losartan has beneficial effects on endothelial inflammation.