Abstract
Background 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. Methods 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. Results 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. Conclusion ox-LDL activated EPCs p38/Keap1/Nrf2 pathway and induced oxidative stress, dysfunction, and apoptosis of EPCs.
Highlights
Coronary and cerebral artery occlusion induced by atherosclerotic plaque formation and disruption is one of the major causes of the mortality and morbidity within the developed and modern societies
In endothelial progenitor cells (EPCs) challenged with 20 μg/mL oxidized-low density lipoprotein (ox-LDL) for 24 hours, the NAD(P) H:quinone oxidoreductase 1 (NQO1) protein level was markedly decreased, and pretreatment of EPCs with Keap1 siRNA transfection 24 hours before ox-LDL treatment significantly promoted NQO1 levels (Figures 2(j) and 2(k)). These results indicated that ox-LDL reduced NQO1 levels at the transcription and protein level in a time- and concentration-dependent manner in EPCs, which might have been implicated to Kelch-like ECH-associated protein-1- (Keap1-)dependent regulation of Nuclear factor erythroid 2-related factor 2 (Nrf2)
We found that ox-LDL significantly activated p38 and the inhibition of p38 mitogenactivated protein kinase (MAPK) reversed the depression of Nrf2 nuclear translocation and NQO1 mRNA transcription by ox-LDL in EPCs
Summary
Nrf which was recently reported to regulate the antioxidant genes and cellular redox regulators was highly expressed in EPCs. EPCs isolated from human peripheral blood mononuclear cells were treated with different concentrations of ox-LDL, Keap siRNA, and a specific p38 MAPK inhibitor SB203580 and used to assay the cytoplasmic Nrf, nuclear Nrf, 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 enhanced ROS production, induced the loss of membrane potential, and increased the cell shrinkage, pyknotic nuclei, and apoptosis of EPCs. Keap siRNA increased Nrf nuclear translocation, NQO1 mRNA transcription, and protein expression and prevented ROS generation and formation of JC-1 monomers. SB203580 significantly eliminated ox-LDL induced inhibition of Nrf nuclear translocation, depression of NQO1 mRNA transcription, generation of ROS, and formation of JC-1 monomers in EPCs. Keap siRNA decreased the Bax/Bcl-2 ratio which was increased by ox-LDL in EPCs. ox-LDL decreased EPC migration and tube formation. Conclusion. ox-LDL activated EPCs p38/Keap1/Nrf pathway and induced oxidative stress, dysfunction, and apoptosis of EPCs
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