title: MAPK mediates inflammatory response and cell death in rat pulmonary microvascular endothelial cells in an ischemia-reperfusion model of lung transplantation.
abstract: BACKGROUND
  Hypoxia-reoxygenation of cultured macrovascular endothelial cells is used to study ischemia-reperfusion (IR)-related cellular and molecular changes; however, these models do not accurately depict events in pulmonary microvascular endothelial cells (PMVECs) during conventional lung retrieval and transplantation. We used rat PMVECs in a new non-hypoxic cell-based lung transplantation model to assess these events.
  
  
  METHODS
  To simulate cold storage, rat PMVECs were preserved in 95% O2-5% CO2 at 4°C for 6 hours in low-potassium dextran solution. Dishes were warmed for 1 hour to room temperature for simulating implantation. Medium was added at 37°C in 50% O2-5% CO2-45% N2 to simulate reperfusion. Additional PMVECs were transfected with siRNA targeting mitogen-activated protein kinases (MAPKs) and then subjected to simulated IR.
  
  
  RESULTS
  MAPKs and NF-κB were activated during simulated reperfusion, and AP-1 was activated during ischemia and reperfusion. Increased malondialdehyde levels were found during cold ischemia, and apoptosis and production of IL-1β, IL-6, and TNF-α were observed during reperfusion. Silencing of MAPKs attenuated oxidative stress, inflammation and apoptosis. Silencing of JNK and p38 decreased NF-κB phosphorylation and increased inhibitor of NF-κB (IκB)α levels. Knockdown of ERK1/2 increased NF-κB phosphorylation but had no effect on IκBα expression. Silencing of JNK and ERK1/2, but not p38, decreased AP-1 phosphorylation.
  
  
  CONCLUSIONS
  Exposing rat PMVECs to simulated non-hypoxic IR caused lipid peroxidation, inflammation and apoptosis, which required MAPK-mediated NF-κB and AP-1 activation and distinct regulation of MAPKs by these 2 transcription factors. This model could be used to uncouple mechanisms of IR and evaluate potential therapeutics in alleviating IR injury.
authors.affiliation: Department of Anesthesiology (Hei Long Jiang Province Key Lab of Research on Anesthesiology and Critical Care Medicine), and the Second Affiliated Hospital, Harbin Medical University, Harbin, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Tan
authors.forename: Dongdong
authors.initials: D
authors.lastname: Liu
authors.forename: Xiangqi
authors.initials: X
authors.lastname: Lv
authors.forename: Linlin
authors.initials: L
authors.lastname: Wang
authors.forename: Can
authors.initials: C
authors.lastname: Zhao
authors.forename: Yuxiang
authors.initials: Y
authors.lastname: Che
authors.forename: Qing
authors.initials: Q
authors.lastname: Xie
authors.forename: Xiaoguang
authors.initials: X
authors.lastname: Cui
mesh_terms: D000818:Animals; D016923:Cell Death; D042783:Endothelial Cells; D004730:Endothelium, Vascular / Q000473:pathology*; D007249:Inflammation / Q000209:etiology*; D008168:Lung / Q000098:blood supply; D016040:Lung Transplantation; D008297:Male; D055806:Microvessels / Q000473:pathology*; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology*; D051381:Rats; D017208:Rats, Wistar; D015427:Reperfusion Injury / Q000150:complications*
chemical_list: D020928:Mitogen-Activated Protein Kinases
keywords: activator protein-1; ischemia-reperfusion; lung transplantation; microvascular endothelial cells; mitogen-activated protein kinases; nuclear factor-κB