MAPK mediates inflammatory response and cell death in rat pulmonary microvascular endothelial cells in an ischemia-reperfusion model of lung transplantation.

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% O-2-5% CO2 at 4 degrees 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 degrees C in 50% O-2-5% CO2-45% N-2 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-kappa B 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-1 beta, IL-6, and TNF-alpha were observed during reperfusion. Silencing of MAPKs attenuated oxidative stress, inflammation and apoptosis. Silencing of JNK and p38 decreased NF-kappa B phosphorylation and increased inhibitor of NF-kappa B (I kappa B)alpha levels. Knockdown of ERK1/2 increased NF-kappa B phosphorylation but had no effect on I kappa B alpha 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-kappa B 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. (c) 2013 International Society for Heart and Lung Transplantation. All rights reserved.