NOX4-autophagy Pathway Mediates Monocytes-human Umbilical Vein Endothelial Cells Adhesion Enhanced By Apelin-13

Aim: Apelin, as the endogenous ligand of APJ receptor, has a variety of biological effects in the body. Our previous experiments research found that apelin-13 enhanced monocytes and human umbilical vein endothelial cells adhesion via PI3 K pathway. The objective of this study was to explore apelin-13 on ROS from NOX4 and autophagy in human umbilical vein endothelial cells, and discuss the mechanism of apelin-13 enhanced that monocytes adhere to endothelial cells. This will provide provide theoretical basis for atherosclerosis treatment.Methods:1. The expression of NOX4, Beclin1, LC3II/I, ICAM-1, β-Tublin and β-Actin were detected by Western Blot.2. Used DCFH-DA to label ROS, the generation of ROS was detected by fluorescence microscopy or multifunctional microplate.3. Adenovirus, which expressed mRFP-GFP-LC3 fusion protein, infected human umbilical vein endothelial cells, marking and tracing of intracellular LC3, then reflected intracellular autophagy flow by fluorescence microscopy.4. Used Calcein AM labeled THP-1 cells, fluorescent microscope and multifunctional microplate reader detected the cells adhesion.5. Used SiRNA to silence beclin-1 and LC3 in human umbilical vein endothelial cells, the effect of them on cell adhesion was observed.Results:1. Apelin-13 promotes the generation of ROS in human umbilical vein endothelial cells in dose(0-1.0μM) and time(0-24h) dependent manners.2. Apelin-13 promotes the expression of NOX4 in human umbilical vein endothelial cells in dose(0-1.0μM) and time(0-24h) dependent manners.3. NOX inhibitor DPI and PI3 K inhibitor LY294002 inhibited that apelin-13 promoted the generation of ROS in human umbilical vein endothelial cells.4. The Class III PI3 K inhibitor 3-MA inhibited that apelin-13 promoted the generation of ROS and the expression of NOX4 in human umbilical vein endothelial cells.5. Apelin-13 increased the expression of autophagy protein beclin1 and LC3-II/I in human umbilical vein endothelial cells in dose(0-1.0μM) and time(0-24h) dependent manners.6. Apelin-13 promoted autophagy flux in HUVECs. Used HCQ to treat HUVECs, the results also showed apelin-13 promoted autophagy flux.7. ROS scavenger NAC, H2O2 enzymes scavenger Catalase and NOX inhibitor DPI inhibited that the apelin-13 increased the expression of autophagy protein beclin1 and LC3-II/I in human umbilical vein endothelial cells.8. ROS scavenger NAC and H2O2 enzymes scavenger Catalase inhibited that the apelin-13 induced the increasing of autophagy flux in human umbilical vein endothelial cells.9. ROS scavenger NAC, H2O2 enzymes scavenger Catalase and NOX inhibitor DPI inhibited the adhesion of MCs-HUVECs enhanced by the apelin-13.10. Beclin1 siRNA and LC3 siRNA inhibited the adhesion of MCs-HUVECs enhanced by the apelin-13.11. Autophagy inducer Rapamycin enhanced MCs–HUVECs adhesion.12. ROS scavenger NAC, H2O2 enzymes scavenger Catalase NOX inhibitor DPI and Class PI3 K inhibitor 3Ⅲ-MA inhibited ICAM-1 expression upregulated by apelin-13 in HUVECs.13. Autophagy inducer Rapamycin upregulated ICAM-1 expression in HUVECs.Conclusion: Class III PI3K-NOX4-ROS-autophagy pathway mediates monocyte-human umbilical vein endothelial cells adhesion enhanced by apelin-13.