Moderate hyperoxia induces inflammation, apoptosis and necrosis in human umbilical vein endothelial cells: An in-vitro study.

BACKGROUND: Perioperative oxygen (O2) therapy can cause hyperoxia. Extreme hyperoxia can injure the cardiovascular system and remote organs. OBJECTIVE: Our primary objective was to test the hypothesis that exposure to moderate hyperoxia will induce injury to human umbilical vein endothelial cells (HUVECs), a model for studying the vascular endothelium under controlled conditions. DESIGN: In-vitro cell culture study. SETTING: Department of Anaesthesia, General Intensive Care and Pain Management, Medical University of Vienna, Austria. Study period from the beginning of October 2013 to the end of July 2014. CELLS: HUVECs were isolated from fresh umbilical cords. INTERVENTIONS: HUVECs were exposed to constant hyperoxia (40% O2), cyclic hyperoxia/anoxia (40%/0% O2, average 20% O2), constant normoxia (21% O2) and constant anoxia (0% O2) using a cell culture bioreactor. MAIN OUTCOME MEASURES: Cell growth, viability and release of IL-6, IL-8 and macrophage migration inhibitory factor were assessed at baseline and after 6, 12, 24 and 48 h of treatment. A phosphokinase array was performed after 60 min of treatment to identify activated cellular signalling pathways. RESULTS: Constant hyperoxia and cyclic hyperoxia/anoxia impeded cell growth, reduced viability, triggered a proinflammatory response, proven by IL-6, IL-8 and migration inhibitory factor release, and induced apoptosis and necrosis. The inflammatory and cytotoxicity responses were highest in the constant hyperoxia group. Phosphokinase arrays revealed that different O2 concentrations activated distinct sets of cytoprotective and cell death-associated kinases, including mitogen-activated protein kinases, Src kinases, p53, Akt, mitogen-activated and stress-activated kinase, Lyn, Lck, p70S6, signal transducers and activators of transcription 5b and 6, glycogen synthase kinase 3a/b and 5' AMP-activated protein kinases 1/2. CONCLUSION: Continuous moderate hyperoxia and cyclic moderate hyperoxia/anoxia-induced endothelial inflammation, apoptosis and necrosis. Given the large surface area of the vascular endothelium, moderately elevated O2 levels may contribute to cardiovascular inflammation and injury. TRIAL REGISTRATION: This in-vitro study was not registered in a database.

Argon Preconditioning Protects Airway Epithelial Cells against Hydrogen Peroxide-Induced Oxidative Stress.

BACKGROUND: Oxidative stress is the predominant pathogenic mechanism of ischaemia-reperfusion (IR) injury. The noble gas argon has been shown to alleviate oxidative stress-related myocardial and cerebral injury. The risk of lung IR injury is increased in some major surgeries, reducing clinical outcome. However, no study has examined the lung-protective efficacy of argon preconditioning. The present study investigated the protective effects of argon preconditioning on airway epithelial cells exposed to hydrogen peroxide (H2O2) to induce oxidative stress. METHODS: A549 airway epithelial cells were treated with a cytotoxic concentration of H2O2 after exposure to standard air or 30 or 50% argon/21% oxygen/5% carbon dioxide/rest nitrogen for 30, 45 or 180 min. Cells were stained with annexin V/propidium iodide, and apoptosis was evaluated by fluorescence-activated cell sorting. Protective signalling pathways activated by argon exposure were identified by Western blot analysis for phosphorylated candidate molecules of the mitogen-activated protein kinase and protein kinase B (Akt) pathways. RESULTS: Preconditioning with 50% argon for 30, 45 and 180 min and 30% argon for 180 min caused significant protection of A549 cells against H2O2-induced apoptosis, with increases in cellular viability of 5-47% (p < 0.0001). A small adverse effect was also observed, which presented as a 12-15% increase in cellular necrosis in argon-treated groups. Argon exposure resulted in early activation of c-Jun N-terminal kinase (JNK) and p38, peaking 10- 30 min after the start of preconditioning, and delayed activation of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, peaking after 60-90 min. CONCLUSIONS: Argon preconditioning protects airway epithelial cells from H2O2-induced apoptotic cell death. Argon activates the JNK, p38, and ERK1/2 pathways, but not the Akt pathway. The cytoprotective properties of argon suggest possible prophylactic applications in surgery-related IR injury of the lungs.

Pretreatment With Argon Protects Human Cardiac Myocyte-Like Progenitor Cells from Oxygen Glucose Deprivation-Induced Cell Death by Activation of AKT and Differential Regulation of Mapkinases.

BACKGROUND: The noble gas argon induces cardioprotection in a rabbit model of myocardial ischemia and reperfusion. However, no studies in human primary cells or subjects have been performed so far. We used human cardiac myocyte-like progenitor cells (HCMs) to investigate the protective effect on the cellular level. METHODS: HCMs were pretreated with 30% or 50% argon before oxygen-glucose deprivation (OGD) and reperfusion. We evaluated apoptotic states by flow cytometry and the activation of mitogen-activated protein kinase (MAPKs) members extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK), p38 MAPkinase, and protein kinase B (Akt) by Westernblot analysis and by activity assays of downstream transcription factors. Specific inhibitors were used to proof a significant participation of these pathways in the protection by argon. Beneficial effects were further assessed by TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay, lactate dehydrogenase (LDH), mitochondrial deoxyribonucleic acid (mtDNA), and cytokine release. RESULTS: Pretreatment with 30% or 50% argon for 90 min before OGD resulted in a significant protection of HCMs against apoptosis. This effect was reversed by the application of MAPK and Akt inhibitors during argon exposure. Argon 30% reduced the release of LDH by 33% and mtDNA by 45%. The release of interleukin 1ß was reduced by 44% after OGD and more than 90% during reperfusion. CONCLUSIONS: Pretreatment with argon protects HCMs from apoptosis under ischemic conditions via activation of Akt, Erk, and biphasic regulation of JNK. Argon gas is cheap and easily administrable, and might be a novel therapy to reduce myocardial ischemia-reperfusion injury.