Objective: Our previous studies found that 100 µg/ml oxidized low-density lipoprotein (ox-LDL) could up-regulate the autophagic level in human umbilical vein endothelial cells (HUVEC). The present study was conducted to observe the roles of oxidative stress and lectin-like oxidized low density lipoprotein-1 (LOX-1) in the ox-LDL-induced up-regulation of autophagy.
Methods: Prior to the ox-LDL exposure, LOX-1mAb, vitamin C and vitamin E were used to study the roles of LOX-1 and oxidative stress in the activation of autophagy. The contents of total-superoxide dismutase (T-SOD) and MDA (malondialdehyde) in the culture medium were detected with enzyme linked immunosorbent assay. Western blot was employed to detect the levels of autophagic marker microtubule-associated protein light chain 3 (MAP1-LC3)-II/LC3-I, beclin1 and lysosome associated membrane protein 2a (lamp2a).
Results: After the ox-LDL exposure, the down-regulated level of T-SOD [0.5 h (32.73 ± 1.09 vs 40.16 ± 1.28) U/ml, P < 0.01; 6 h (29.32 ± 1.56 vs 40.16 ± 1.28) U/ml, P < 0.01] and the up-regulated level of MDA [0.5 h (1.11 ± 0.04 vs 0.57 ± 0.05) nmol/ml, P < 0.01; 6 h (0.69 ± 0.03 vs 0.57 ± 0.05) nmol/ml, P < 0.05] in culture medium were also significant at 0.5 h and 6 h. The ox-LDL-induced increased ratio of LC3-II/LC3-I was reversed by the pretreatments of vitamin C and vitamin E (0.5 h, vitC: 3.11 ± 0.02 vs 4.31 ± 0.50, P < 0.05; vitE: 3.46 ± 0.19 vs 4.31 ± 0.50, P < 0.05; 6 h, vitC: 1.44 ± 0.05 vs 2.31 ± 0.16, P < 0.05), but not LOX-1mAb. LOX-1mAb decreased the ox-LDL-induced elevated level of lamp2a protein while vitamin C and vitamin E only inhibited the elevation of lamp2a at the timepoint of 6 h, but not 0.5 h.
Conclusion: Oxidative stress, rather than LOX-1, plays an important role in the ox-LDL-induced up-regulation of autophagy in HUVEC. The formation of autolysosomes is associated with the LOX-1-mediated endocytosis of ox-LDL. Oxidative stress only plays a minor role in the formation of autolysosomes induced by the engulfed ox-LDL.