ox-LDL-Induced Endothelial Progenitor Cell Oxidative Stress via p38/Keap1/Nrf2 Pathway

Nrf2 which was recently reported to regulate the antioxidant genes and cellular redox regulators was highly expressed in EPCs. However, its role in ox-LDL-induced EPC oxidative stress and apoptosis has not been fully illustrated.

EPCs isolated from human peripheral blood mononuclear cells were treated with different concentrations of ox-LDL, Keap1 siRNA, and a specific p38 MAPK inhibitor SB203580 and then used to assay the cytoplasmic Nrf2, nuclear Nrf2, NAD(P) H:quinone oxidoreductase 1 (NQO1) and Bax/Bcl-2 levels with Western blot, NQO1 mRNA levels with RT-PCR, ROS levels with H2DCF-DA, loss/disruption of mitochondrial membrane potential with JC-1, apoptosis with Annexin V and PI, migration with transwell chambers, and tube formation with Matrigel.

ox-LDL decreased the nuclear Nrf2/Histone H3 to cytoplasmic Nrf2/GAPDH ratio, NQO1 mRNA, and protein levels. ox-LDL enhanced ROS production, induced the loss of membrane potential, and increased the cell shrinkage, pyknotic nuclei, and apoptosis of EPCs. Keap1 siRNA increased Nrf2 nuclear translocation, NQO1 mRNA transcription, and protein expression and prevented ROS generation and formation of JC-1 monomers. ox-LDL increased the activation of p38. SB203580 significantly eliminated ox-LDL induced inhibition of Nrf2 nuclear translocation, depression of NQO1 mRNA transcription, generation of ROS, and formation of JC-1 monomers in EPCs. Keap1 siRNA decreased the Bax/Bcl-2 ratio which was increased by ox-LDL in EPCs. ox-LDL decreased EPC migration and tube formation. Keap1 siRNA preserved the migration and tube formation of EPCs.

ox-LDL activated EPCs p38/Keap1/Nrf2 pathway and induced oxidative stress, dysfunction, and apoptosis of EPCs.