LKB1-AMPK–mTOR Pathway Involved In The Autophagy Induced By Oxidized Lp(a) In Human Umbilical Vascular Endothelial Cells

【Background and Objective】Recently, oxidized lipoprotein(a)(oxLp(a)) is considered to be a more potentarteriosclerotic factor than native Lp(a), although native Lp(a) can be a risk factor foratherosclerotic diseases. Lp(a) is easilier oxidized than LDL due to the unique apo(a). A studyreported that oxLp (a) wasdetected in endothelial cells, in human carotid and cerebral arteries,and the deposition of oxLp(a), but not native Lp(a), Another observation is that a weakercorrelation between the CIMT and the oxLp(a), in the multiple linear regression analysis. Agrowing body of evidence suggests that autophagy is very important in atheroscleroticdisease.there are reported that oxLDL could induced autophagy in HUVECs，and in this wayoxLDL is degradated, while the mechanisms is not clear really.Our lab had confirmed thatoxLp(a) could induced the ROS increasing in HUVECs, while ROS in the cells could induceoxidative stress,and oxidative stress is a well-known stimulus of autophagy to facilitate theremoval of damaged organelles though activation the LKB1-AMPK-mTOR signal pathway.Therefore,in this study,we investigate that wether oxLp(a) could induce HUVECsautophagy though upregulation the ROS level and activate the AMPK-mTOR pathway,todegradate oxLp(a) HUVECs uptake or the toxiferous protein in oxidative milieu, accordinglyautophagy has a protective effect on HUVECs under oxLp(a).【Methods】Here, we first provide evidence that autophagy is activated on the treatment of oxLp(a) atdifferent concentrations and time though LC3and beclin-1protein determination in HUVECs.The protein level was detected by Western Blot and RT-PCR was used for the test of beclin1 mRNA level under oxLp(a) in the dose-and time-dependent manner in HUVECs.Next, ROS production was detected by DCFH-DA staining with the dose-andtime-dependent manner, The protein level of LC3and beclin1were detected by Western Blotwhen HUVECs were treated with oxLp(a) and NAC(ROS scavenger).Thirdly,HUVECs were incubated with oxLp(a) at different time,3AB and Comp C wereadd into the oxLp(a),the protein expression of PAR protein and LC3protein,and thephosphorylation level of AMPK、p70S6K and4EBP1were monited by Western blot,MAP1-LC3immunofluorescence for the LC3level and MDC staining for the amountautophagic vacuole.【Results】1First, the cells were incubated for6h with increasing concentrations of oxLp(a) at25,50,100and200μg/ml,from the result,we could see that25–200μg/ml of oxLp(a) markedlyupregulated the level of LC3and beclin1proteins with the dose-dependent manner comparedto control, and the proteins reached one peaks at100μg/ml. Time control analysis of LC3ratioand beclin1proteins by treatment cells with100μg/ml of oxLp(a) indicated that theupregulation of the proteins reached peaks at6h and then decreased at12h compared tocontrol.While, the inhibitor of autophagy3MA and chloroquine incubate HUVECs with100μg/ml oxLp(a) for6h,LC3and beclin1protein had decreased strikingly and MAP1-LC3immunofluorescence and MDC staining had displayed weak fluorescence intensity.2To detect the oxLp(a)-induced intracellular ROS production in HUVECs,we choose H2O2(200μmol/L) as the positive control of ROS production and NAC as the ROS scavengers inROS production examination.The results showed that the intracellular ROS production wassignificantly increased by stimulation of oxLp(a)(100μg/ml). While,Pretreatment cells withNAC, There was down-ragulation of the oxLp(a)-induced ROS production in HUVECs.OxLp(a) could up-regulate cells ROS in a dose-and time-dependent manner compared withH2O2. The intracellular ROS induced by oxLp(a) and NAC on HUVECs was breakdown andthat NAC could strikingly suppress the autophagy protein expression induced by oxLp(a).Thishad suggest that oxLp(a) could increase the intracellular ROS in HUVECs and ROS hadinvoved in the process of autophagy induced by oxLp(a).3Treated cells with100μg/ml oxLp(a) there would be increasing expression of PAR and LC3proteins,while,after adding3AB(a specific PARP-1inhibitor) in oxLp(a),it showed thatthere was a reduction in the two proteins level.The cell immunofluorescence and MDCstaining suggested that3AB could obviously reverse effect of aotuphagy induced byoxLp(a),as well as the expression of PAR protein.Here we next examined the expression level of oxLp(a)on AMPK activation and mTORdownstream signaling molecule, S6K and4EBP1anf their phosphorylation.We treated cellswith100μg/ml for1020and30min,western blot analysis showed that oxLp(a) inducedsignificant increase of AMPK phosphorylation at Thr-172and decreased in phosphorylation ofp70S6K and4EBP1. Compound C(a specific AMPK inhibitor) was used to block AMPK tofuther proof that AMPK play a inportant role in oxLp(a)-induced autophagy. Compound Csignificantly suppressed oxLp(a)-induced activation of AMPK and restored thephosphorylation of p70S6K and4EBP1. Similar results were also observed in the test ofimmunofluorescence and MDC staining.At last, these results suggested that LKB1-AMPKactivation contributes to oxLp(a)-induced autophagy.【Conclusions】1OxLp(a) could induce autophagy in HUVECs and in the dose-and time-dependent mannerup-regulation the autophagy protein LC3and beclin1.2oxLp(a) could increase the intracellular ROS in HUVECs and ROS had invoved in theprocess of autophagy induced by oxLp(a).3LKB1-AMPK activation contributes to oxLp(a)-induced HUVECs autophagy and in a ROS-mediated manner.