[Cyclosporin A inhibits the adhesion of neutrophil with ECV-304 induced by hypoxia/reoxygenation via ROS-Cyclophilin A-ERK1/2 pathway].
To investigate the inhibition of cyclosporin A (CsA) on neutrophil adhesion to human umbilical vein endothelial cells (HUVECs, ECV-304) induced by hypoxia/reoxygenation and further explore its mechanism, a 1 h hypoxia/4 h reoxygenation model was reproduced using ECV-304. The adhesion rate of neutrophils to ECV-304 was determined by measuring the activity of endogenous hexosaminidase. The expression of endothelial cell adhesion molecules of E-selectin and ICAM-1 was measured by flow cytometry. The expression of cyclophilin A (CyPA) and the activation of ERK1/2 was compared among experimental groups by Western blot. The content of reactive oxygen species (ROS) was measured by Fenton reaction. After being stimulated with 1 h hypoxia/4 h reoxygenation, ECV-304 showed an enhanced neutrophil adhensiveness in association with an increased surface expression of E-selectin and ICAM-1. In parallel, the content of ROS was also increased. These effects were significantly suppressed by the addition of CsA. Most importantly, the expression of CyPA was significantly increased following 1 h hypoxia/4 h reoxygenation, which was accompanied with an increased activation of ERK1/2. Treatment with CyPA inhibitor CsA and CyPA antisense oligonucleotides significantly inhibited the activation of ERK1/2 and decreased the adhesion of neutrophils to ECV-304. The specific ERK1/2 inhibitor PD98059 caused an inhibition of neutrophil adhesion to hypoxia/reoxygenation-stimulated ECV-304. Our data confirm that CsA inhibits neutrophil adhesion to hypoxia/reoxygenation stimulated ECV-304 by a mechanism involving inhibition of the signal transduction of ROS, CyPA and ERK1/2.
Zhou
Si-Gui
SG
Institute of Pharmacy and Pharmacology, Nanhua University, Hengyang, Hunan 421001, China.
Xu
Li-Peng
LP
Liao
Duan-Fang
DF
Lei
Xiao-Yong
XY
Yan
Feng-Xiang
FX
Zhu
Bing-Yang
BY
D002448:Cell Adhesion; D015687:Cell Hypoxia; D002478:Cells, Cultured; D021983:Cyclophilins / Q000096:biosynthesis* / Q000235:genetics; D016572:Cyclosporine / Q000494:pharmacology*; D004730:Endothelium, Vascular / Q000166:cytology*; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis / Q000235:genetics; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D009504:Neutrophils / Q000166:cytology*; D017382:Reactive Oxygen Species / Q000378:metabolism; D015427:Reperfusion Injury / Q000503:physiopathology; D015398:Signal Transduction; D014471:Umbilical Veins / Q000166:cytology
D017382:Reactive Oxygen Species; D018799:Intercellular Adhesion Molecule-1; D016572:Cyclosporine; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D021983:Cyclophilins