Oxidized Low Density Lipoprotein Induces Endothelial Cell Autophagy By Akt/mTOR/p70S6K Pathway

Objective Autophagy,which is an evolutionarily conserved process,plays an important role in cells growth,development and homeostasis.Oxidative stress is a well-known stimulus of autophagy that facilitates the removal of damaged material.In this study,we investigated the role of oxidized low-density lipoprotein(ox-LDL)on autophagy and Akt/m TOR/p70S6 K signaling in human umbilical endothelial vein cells(HUVECs).Methods HUVECs were exposed to ox-LDL(100 ?g/ml)and isopyknic PBS then collected after 6 h and 12 h.Ultrastructure changes of endothelial cells were examined by transmission electron microscope.The autophagy levels in different groups were measured by the m RNA expression levels of microtubule-associated protein 1 light chain 3(LC3)and sequestosome 1(SQSTM1/p62),which were assayed by quantitative RT-PCR.The protein expression levels of LC3,p62,P-Akt/Akt?P-m TOR/m TOR?P-p70S6 K /p70S6 K were investigated by Western blot.Results 1.Ultrastructure changes in cells were examined with transmission electron microscope at 6 h and 12 h post ox-LDL treatment.E M images showed normal cytoplasm,mitochondria,nucleus,and chromatin in control HUVECs,while few or no autophagosomes and lysosomes were observed.In contrast,the E M images from ox-LDL-treated HUVECs displayed many autophagosomes at various developmental stages which demonstrated that ox-LDL could induce autophagy in HUVECs.The number of autophagosomes at 6 h was considerably higher than those at 12 h.2.The detection of LC3-II is used as a marker of autophagy activation.Compared to the control group,the treatment group showed increased the m RNA and protein level of LC3-II(P<0.05)and decreased the level of p62(P<0.05).Additionally,p62 interacts with the autophagic effecter protein LC3 and is degraded through the autophagy–lysosomal pathway.3.Ox-LDL inhibited the phosphorylated protein expression levels within the Akt /m TOR/p70S6 K signaling pathway(P<0.01).However,the effect of ox-LDL on the Akt /m TOR /p70S6 K signaling pathway did not affect the total protein expression levels of Akt,m TOR and p70S6 K.Conclusion In the present study,we provided evidence suggesting that ox-LDL(100 ?g/ml)could induce autophagy in HUVECs.In particular,our study demonstrated that the number of autophagosomes was significantly increased after 6 h and 12 h in HUVECs treated with ox-LDL.In contrast to the control group,the m RNA and protein expression level of LC3-II was up regulated while the level of p62 was down regulated in the ox-LDL treatment group.This effect was more significant in the 6 h treatment group than in the 12 h group.Moreover,the phosphorylated protein expression levels within the Akt /m TOR/p70S6 K signaling pathway was down regulated.Based on this,we imply that ox-LDL induces autophagy in HUVECs by inhibiting the Akt/m TOR/p70S6 K signaling pathways.