Low Shear Stress Induces ENOS Uncoupling Via Impairing Autophagy Flux

Background : Uncoupled endothelial nitric oxide synthase(eNOS)produces O2.-instead of nitric oxide(NO).Earlier,we reported rapamycin,an autophagy inducer and inhibitor of cellular proliferation,attenuated low shear stress(SS)induced O2.-production.Nevertheless,it is unclear whether autophagy palys a critical role in the regulation of eNOS uncoupling.Objective:This study aimed to investigate the modulation of autophagy on eNOS uncoupling induced by low SS exposure.Methods:Human umbilical vein endothelial cells(HUVECs)were subjected to the parallel flow chamber,which applied SS of 2 dyne/cm2 in vitro.The carotid arteries ligation of C57BL6 mice were performed in vivo.eNOS uncoupling changing the amount of superoxide and nitric oxide(NO)induced by low shear stress were detected by chemiluminescence staining.we used autophagy inhibitors 、autophagy agonists and Atg5 lentivirus,which modulalte the expression of related protein to study the effect of autophagy on low shear stress induced eNOS uncoupling,Results:It’s demonstrated that low SS induced eNOS uncoupling We found that low SS induced endothelial O2.-burst,which was accompanied by reduced NO release.Furthermore,inhibition of eNOS by L-NAME conspicuously attenuated low SS-induced O2.-releasing,indicating eNOS uncoupling.Low SS switched on endothelial autophagy while conferring endothelial autophagic flux blockade.Autophagy markers such as LC3 II/I ratio,amount of Beclin1,as well as ULK1/Atg1 were increased during low SS exposure,whereas autophagic degradation of p62/SQSTM1 was markedly reduced,implying impaired autophagic flux.eNOS uncoupling induced by low SS is caused by autophagic flux blockade while independent of initial O2.-generation.we found low SS-induced NO reduction could be reversed by rapamycin or WYE-354,but not by N-acetylcysteine or apocynin,via restoration of autophagic flux.Low SS-induced autophagic flux impairment regulates eNOS phosphorylation.we found phosphorylation of eNOS Thr495 by low SS or PMA stimulation was regulated by autophagy.Notably,eNOS Ser1177 phosphorylation boosted by rapamycin,was in favor of the eNOS recoupling through restoration of autophagic flux.Autophagic flux inhibtion by 3-MA,Baf A1,CQ and low SS did not alter acetylated state of eNOS Conclusion:These results together suggest a novel mechanism for regulation of eNOS uncoupling by low SS via autophagy-mediated eNOS phosphorylation,which is implicated in geometrical nature of atherogenesis.