Impaired Autophagy Induced by oxLDL/β2GPI/anti-β2GPI Complex through PI3K/AKT/mTOR and eNOS Signaling Pathways Contributes to Endothelial Cell Dysfunction

Endothelial cell dysfunction plays a fundamental role in the pathogenesis of atherosclerosis (AS), and endothelial autophagy has protective effects on the development of AS. Our previous study had shown that oxidized low-density lipoprotein/β2-glycoprotein I/anti-β2-glycoprotein I antibody (oxLDL/β2GPI/anti-β2GPI) complex could promote the expressions of inflammatory cytokines and enhance the adhesion of leukocytes to endothelial cells. In the present study, we aimed to assess the effects of oxLDL/β2GPI/anti-β2GPI complex on endothelial autophagy and explore the associated potential mechanisms. Human umbilical vein endothelial cells (HUVECs) and mouse brain endothelial cell line (bEnd.3) were used as models of the vascular endothelial cells. Autophagy was evaluated by examining the expressions of autophagic proteins using western blotting analysis, autophagosome accumulation using transmission electron microscopy, and RFP-GFP-LC3 adenoviral transfection and autophagic flux using lysosome inhibitor chloroquine. The expressions of phospho-PI3K, phospho-AKT, phospho-mTOR, and phospho-eNOS were determined by western blotting analysis. 3-Methyladenine (3-MA) and rapamycin were used to determine the role of autophagy in oxLDL/β2GPI/anti-β2GPI complex-induced endothelial cell dysfunction. We showed that oxLDL/β2GPI/anti-β2GPI complex suppressed the autophagy, evidenced by an increase in p62 protein, a decrease in LC3-II and Beclin1, and a reduction of autophagosome generation in endothelial cells. Moreover, inhibition of autophagy was associated with PI3K/AKT/mTOR and eNOS signaling pathways. Rapamycin attenuated oxLDL/β2GPI/anti-β2GPI complex-induced endothelial inflammation, oxidative stress, and apoptosis, whereas 3-MA alone induced the endothelial injury. Our results suggested that oxLDL/β2GPI/anti-β2GPI complex inhibited endothelial autophagy via PI3K/AKT/mTOR and eNOS signaling pathways and further contributed to endothelial cell dysfunction. Collectively, our findings provided a novel mechanism for vascular endothelial injury in AS patients with an antiphospholipid syndrome (APS) background.