Characteristics of macroautophagy and molecular chaperone-mediated autophagy in oxidative stress injury of endothelial cells

Abstract

Objective To explore the characteristics of macroautophagy and molecular chaperone-mediated autophagy (CMA) in the oxidative stress injury of human umbilical vein endothelial cells (HUVECs). Methods After HUVECs were treated with oxidized low density lipoprotein (OX-LDL) at different concentrations(0、20、50、100、200 μg/mL), the production of reactive oxygen species (ROS) was measured to screen the optimal concentration to induce HUVECs oxidative stress. The cells were divided into 4 groups: control group (without any treatment), macroautophagy inhibition group (treated with 10 mmol/L 3-MA, specific inhibitor of macroautophagy), oxidative stress group (treated with 50 μg/mL OX-LDL), and macroautophagy inhibition-oxidative stress group (treated with 10 mmol/L 3-MA+50 μg/mL OX-LDL). The expression levels of proliferation marker protein PCNA, apoptosis marker Caspase-3, macroautophagy marker Beclin-1, and CMA markers Hsc70 and Lamp-2 were detected in each group with Western blotting. The proliferation, apoptosis, 2 kinds of autophagy under different treatments were evaluated respectively. After the cells of the oxidative stress group and macroautophagy inhibition-oxidative stress group were treated for different duration (1, 3, 6, 12 and 48 h), the proliferation and apoptosis of HUVECs were tested by CCK-8 assay and Annexin-V/PI double staining, respectively. Western blotting was adopted to determine the protein levels of Beclin-1, Hsc70 and Lamp-2. Finally, the time regulation characteristics of the 2 autophagy pathways before and after macroautophagy inhibition were analyzed. Results Under OX-LDL induced oxidative stress, the ROS level was elevated with the increase of OX-LDL concentration, and 50 μg/mL was finally selected as the appropriate concentration for subsequent experiments. As compared with the control group, the levels of Caspase-3, Beclin-1, Hsc70 and Lamp-2 were up-regulated, while that of PCNA was down-regulated in the oxidative stress group (P < 0.05). With the prolongation of treatment time, proliferation inhibition rate, overall apoptotic rate and Lamp-2 level were increased within 48 h, Hsc70 level was elevated after 6 h, and that of Beclin-1 was improved within 12 h in the oxidative stress group (P < 0.05). In the macroautophagy inhibition-oxidative stress group, the expression levels of Caspase-3, Hsc70 and Lamp-2 were also up-regulated, that of PCNA down-regulated (P < 0.05), those of Hsc70 and Lamp-2 were increased at the same time and that of Hsc70 was in an elevation after 3 h of treatment (P < 0.05). Conclusion Macroautophagy and CMA are activated successively during the process of oxidative stress, and CMA can be enhanced after macroautophagy inhibition. The early activation contributes to attenuate oxidative stress-induced damage to endothelial cells.

Keywords

• chaperone-mediated autophagy
• macroautophagy
• oxidative stress
• human umbilical vein endothelial cells