Hyperoxia induces Egr-1 expression through activation of extracellular signal-regulated kinase 1/2 pathway

Early growth response gene (Egr-1) is a stress response gene activated by various forms of stress and growth factor signaling. We report that supraphysiologic concentrations of O(2) (hyperoxia) induced Egr-1 mRNA and protein expression in cultured alveolar epithelial cells, as well as in mouse lung in vivo. The contribution of the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK), p38 MAPK and PI3-kinase pathways to the activation of Egr-1 in response to hyperoxia was examined. Exposure to hyperoxia resulted in a rapid phosphorylation of ERK 1/2 kinases in mouse alveolar epithelial cells LA4. MEK inhibitor PD98059, but not inhibitors of p38 MAPK or PI3-kinase pathway, prevented Egr-1 induction by hyperoxia. The signaling cascade preceding Egr-1 activation was traced to epidermal growth factor receptor (EGFR) signaling. Hyperoxia is used as supplemental therapy in some diseases and typically results in elevated levels of reactive oxygen intermediates (ROI) in many lung cell types, the organ that receives highest O(2) exposure. Our results support a pathway for the hyperoxia response that involves EGF receptor, MEK/ERK pathway, and other unknown signaling components leading to Egr-1 induction. This forms a foundation for analysis of detailed mechanisms underlying Egr-1 activation during hyperoxia and understanding its consequences for regulating cell response to oxygen toxicity.     

Hyperoxia augments ER-stress-induced cell death independent of BiP loss

Cytotoxic reactive oxygen species are constantly formed as a by-product of aerobic respiration and are thought to contribute to aging and disease. Cells respond to oxidative stress by activating various pathways, whose balance is important for adaptation or induction of cell death. Our lab recently reported that BiP (GRP78), a proposed negative regulator of the unfolded protein response (UPR), declines during hyperoxia, a model of chronic oxidative stress. Here, we investigate whether exposure to hyperoxia, and consequent loss of BiP, activates the UPR or sensitizes cells to ER stress. Evidence is provided that hyperoxia does not activate the three ER stress receptors IRE1, PERK, and ATF6. Although hyperoxia alone did not activate the UPR, it sensitized cells to tunicamycin-induced cell death. Conversely, overexpression of BiP did not block hyperoxia-induced ROS production or increased sensitivity to tunicamycin. These findings demonstrate that hyperoxia and loss of BiP alone are insufficient to activate the UPR. However, hyperoxia can sensitize cells to toxicity from unfolded proteins, implying that chronic ROS, such as that seen throughout aging, could augment the UPR and, moreover, suggesting that the therapeutic use of hyperoxia may be detrimental for lung diseases associated with ER stress.     

Mitochondrial damage and metabolic compensatory mechanisms induced by hyperoxia in the U-937 cell line

Experimental hyperoxia represents a suitable in vitro model to study some pathogenic mechanisms related to oxidative stress. Moreover, it allows the investigation of the molecular pathophysiology underlying oxygen therapy and toxicity. In this study, a modified experimental set up was adopted to accomplish a model of moderate hyperoxia (50% O(2), 96 h culture) to induce oxidative stress in the human leukemia cell line, U-937. Spectrophotometric measurements of mitochondrial respiratory enzyme activities, NMR spectroscopy of culture media, determination of antioxidant enzyme activities, and cell proliferation and differentiation assays were performed. The data showed that moderate hyperoxia in this myeloid cell line causes: i) intriguing alterations in the mitochondrial activities at the levels of succinate dehydrogenase and succinate-cytochrome c reductase; ii) induction of metabolic compensatory adaptations, with significant shift to glycolysis; iii) induction of different antioxidant enzyme activities; iv) significant cell growth inhibition and v) no significant apoptosis. This work will permit better characterization the mitochondrial damage induced by hyperoxia. In particular, the data showed a large increase in the succinate cytochrome c reductase activity, which could be a fundamental pathogenic mechanism at the basis of oxygen toxicity.     

Hyperoxia-induced signal transduction pathways in pulmonary epithelial cells

Mechanical ventilation with hyperoxia is necessary to treat critically ill patients. However, prolonged exposure to hyperoxia leads to the generation of excessive reactive oxygen species (ROS), which can cause acute inflammatory lung injury. One of the major effects of hyperoxia is the injury and death of pulmonary epithelium, which is accompanied by increased levels of pulmonary proinflammatory cytokines and excessive leukocyte infiltration. A thorough understanding of the signaling pathways leading to pulmonary epithelial cell injury/death may provide some insights into the pathogenesis of hyperoxia-induced acute inflammatory lung injury. This review focuses on epithelial responses to hyperoxia and some of the major factors regulating pathways to epithelial cell injury/death, and proinflammatory responses on exposure to hyperoxia. We discuss in detail some of the most interesting players, such as NF-kappaB, that can modulate both proinflammatory responses and cell injury/death of lung epithelial cells. A better appreciation for the functions of these factors will no doubt help us to delineate the pathways to hyperoxic cell death and proinflammatory responses.     

Extracellular ATP-mediated signaling for survival in hyperoxia-induced oxidative stress

Respiratory failure is a serious consequence of lung cell injury caused by treatment with high inhaled oxygen concentrations. Human lung microvascular endothelial cells (HLMVEC) are a principal target of hyperoxic injury (hyperoxia). Cell stress can cause release of ATP, and this extracellular nucleotide can activate purinoreceptors and mediate responses essential for survival. In this investigation, exposure of endothelial cells to an oxidative stress, hyperoxia, caused rapid but transient ATP release (20.03 +/- 2.00 nm/10(6) cells in 95% O(2) versus 0.08 +/- 0.01 nm/10(6) cells in 21% O2 at 30 min) into the extracellular milieu without a concomitant change in intracellular ATP. Endogenously produced extracellular ATP-enhanced mTOR-dependent uptake of glucose (3467 +/- 102 cpm/mg protein in 95% oxygen versus 2100 +/- 112 cpm/mg protein in control). Extracellular addition of ATP-activated important cell survival proteins like PI 3-kinase and extracellular-regulated kinase (ERK-1/2). These events were mediated primarily by P2Y receptors, specifically the P2Y2 and/or P2Y6 subclass of receptors. Extracellular ATP was required for the survival of HLMVEC in hyperoxia (55 +/- 10% surviving cells with extracellular ATP scavengers [apyrase + adenosine deaminase] versus 95 +/- 12% surviving cells without ATP scavengers at 4 d of hyperoxia). Incubation with ATP scavengers abolished ATP-dependent ERK phosphorylation stimulated by hyperoxia. Further, ERK activation also was found to be important for cell survival in hyperoxia, as treatment with PD98059 enhanced hyperoxia-mediated cell death. These findings demonstrate that ATP release and subsequent ATP-mediated signaling events are vital for survival of HLMVEC in hyperoxia.     

Effects of continuous and episodic hyperoxia on stress and hepatic glutathione levels in one-summer-old rainbow trout (Oncorhynchus mykiss)

One-summer-old rainbow trout ( Oncorhynchus mykiss ) were exposed to continuous hyperoxia (173 ± 24%) and three hyperoxic/normoxic treatments for 14 days. Hepatic glutathione status as the indicator of oxidative stress, as well as classical stress indicators such as hemoglobin, hematocrit and plasma cortisol levels, were measured during normoxic, constantly hyperoxic and the following episodically hyperoxic oxygen treatment regimes: 12 h hyperoxia:12 h normoxia (12 HYP:12 NOR), 24 HYP:24 NOR and 48 HYP:24 NOR. Constant hyperoxia tended to shrink erythrocytes, but the 12 HYP:12 NOR treatment increased the number of erythrocytes and thus enhanced the oxygen carrying capacity of blood. Similarly, a trend toward an elevation in plasma cortisol concentrations was detected in 12 HYP:12 NOR treatment group. The finding that elevated hepatic glutathione (GSH) levels (P < 0.01), indicative of enhanced potential of the liver tissue to resist oxidative stress, coincided with elevated cortisol levels might suggest that in the 12 HYP:12 NOR treatment physiological processes were recruited to increase oxygen carrying capacity in blood and to elevate protection against oxyradicals. However, none of the episodic hyperoxia treatments or continuous hyperoxia caused mortality or resulted in better growth. These data indicate that continuous hyperoxia (173 ± 24%) and hyperoxic-normoxic treatments may be applied in intensive culture of rainbow trout provided that fish have at least 24 h in normoxia prior to the next bout of hyperoxia. Shorter recovery periods, like in a 12 HYP:12 NOR treatment, may result in the increased need of oxygen in tissues followed by an activation of glutathione dependent defence system against an increased oxygen load.     

FLIP inhibits endothelial cell apoptosis during hyperoxia by suppressing Bax

High oxygen tension (hyperoxia) causes pulmonary cell death, involving apoptosis, necrosis, or mixed death phenotypes, though the underlying mechanisms remain unclear. In mouse lung endothelial cells (MLEC) hyperoxia activates both extrinsic (Fas-dependent) and intrinsic (mitochondria-dependent) apoptotic pathways. We examined the hypothesis that FLIP, an inhibitor of caspase-8, can protect endothelial cells against the lethal effects of hyperoxia. Hyperoxia caused the time-dependent downregulation of FLIP in MLEC. Overexpression of FLIP attenuated intracellular reactive oxygen species generation during hyperoxia exposure, by downregulating extracellular-regulated kinase-1/2 activation and p47(phox) expression. FLIP prevented hyperoxia-induced trafficking of the death-inducing signal complex from the Golgi apparatus to the plasma membrane. Furthermore, FLIP blocked the activations of caspase-8/Bid, caspases -3/-9, and inhibited the mitochondrial translocation and activation of Bax, resulting in protection against hyperoxia-induced cell death. Under normoxic conditions, FLIP expression increased the phosphorylation of p38 mitogen-activated protein kinase leading to increased phosphorylation of Bax during hyperoxic stress. Furthermore, FLIP expression markedly inhibited protein kinase C activation and expression of distinct protein kinase C isoforms (alpha, eta, and zeta), and stabilized an interaction of PKC with Bax. In conclusion, FLIP exerted novel inhibitory effects on extrinsic and intrinsic apoptotic pathways, which significantly protected endothelial cells from the lethal effects of hyperoxia.     

Aerosolized human extracellular superoxide dismutase prevents hyperoxia-induced lung injury

An important issue in critical care medicine is the identification of ways to protect the lungs from oxygen toxicity and reduce systemic oxidative stress in conditions requiring mechanical ventilation and high levels of oxygen. One way to prevent oxygen toxicity is to augment antioxidant enzyme activity in the respiratory system. The current study investigated the ability of aerosolized extracellular superoxide dismutase (EC-SOD) to protect the lungs from hyperoxic injury. Recombinant human EC-SOD (rhEC-SOD) was produced from a synthetic cassette constructed in the methylotrophic yeast Pichia pastoris. Female CD-1 mice were exposed in hyperoxia (FiO2>95%) to induce lung injury. The therapeutic effects of EC-SOD and copper-zinc SOD (CuZn-SOD) via an aerosol delivery system for lung injury and systemic oxidative stress at 24, 48, 72 and 96 h of hyperoxia were measured by bronchoalveolar lavage, wet/dry ratio, lung histology, and 8-oxo-2'-deoxyguanosine (8-oxo-dG) in lung and liver tissues. After exposure to hyperoxia, the wet/dry weight ratio remained stable before day 2 but increased significantly after day 3. The levels of oxidative biomarker 8-oxo-dG in the lung and liver were significantly decreased on day 2 (P<0.01) but the marker in the liver increased abruptly after day 3 of hyperoxia when the mortality increased. Treatment with aerosolized rhEC-SOD increased the survival rate at day 3 under hyperoxia to 95.8%, which was significantly higher than that of the control group (57.1%), albumin treated group (33.3%), and CuZn-SOD treated group (75%). The protective effects of EC-SOD against hyperoxia were further confirmed by reduced lung edema and systemic oxidative stress. Aerosolized EC-SOD protected mice against oxygen toxicity and reduced mortality in a hyperoxic model. The results encourage the use of an aerosol therapy with EC-SOD in intensive care units to reduce oxidative injury in patients with severe hypoxemic respiratory failure, including acute respiratory distress syndrome (ARDS).     

Cytokines in tolerance to hyperoxia-induced injury in the developing and adult lung

Cytokines are peptides that are produced by virtually every nucleated cell type in the body, possess overlapping biological activities, exert different effects at different concentrations, can either synergize or antagonize the effects of other cytokines, are regulated in a complex manner, and function via cytokine cascades. Hyperoxia-induced acute lung injury (HALI) is characterized by an influx of inflammatory cells, increased pulmonary permeability, and endothelial and epithelial cell injury/death. Some of these effects are orchestrated by cytokines. There are significant differences in the response of the developing versus the adult lung to hyperoxia. We review here cytokines (and select growth factors) that are involved in tolerance toward HALI in animal models. Increased cytokine expression and release have a cascade effect in HALI. IL-1 precedes the increase in IL-6 and CINC-1/IL-8 and this seems to predate the influx of inflammatory cells. Inflammatory cells in the alveolar space amplify the lung damage. Other cytokines that are primarily involved in this inflammatory response include IFN-gamma, MCP-1, and MIP-2. Certain cytokines (and growth factors) seem to ameliorate HALI by affecting cell death pathways. These include GM-CSF, KGF, IL-11, IL-13, and VEGF. There are significant differences in the type and temporal sequence of cytokine expression and release in the adult and newborn lung in response to hyperoxia. The newborn lung is greatly resistant to hyperoxia compared to the adult. The delayed increase in lung IL-1 and IL-6 in the newborn could induce protective factors that would help in the resolution of hyperoxia-induced injury. Designing a therapeutic approach to counteract oxygen toxicity in the adult and immature lung first needs understanding of the unique responses in each scenario.     

Differential Effects of Hypoxic and Hyperoxic Stress-Induced Hypertrophy in Cultured Chick Fetal Cardiac Myocytes

The adult heart responds to contraction demands by hypertrophy, or enlargement, of cardiac myocytes. Adaptive hypertrophy can occur in response to hyperoxic conditions such as exercise, while pathological factors that result in hypoxia ultimately result in heart failure. The difference in the outcomes produced by pathologically versus physiologically induced hypertrophy suggests that the cellular signaling pathways or conditions of myocytes may be different at the cellular level. The structural and functional changes in myocytes resulting from hyperoxia (simulated using hydrogen peroxide) and hypoxia (using oxygen deprivation) were tested on fetal chick cardiac myocytes grown in vitro. Structural changes were measured using immunostaining for α-sarcomeric actin or MyoD, while functional changes were assessed using immunostaining for calcium/calmodulin-dependent kinase (CaMKII) and by measuring intracellular calcium fluxes using live cell fluorescence imaging. Both hypoxic and hyperoxic stress resulted in an upregulation of actin and MyoD expression. Similarly, voltage-gated channels governing myocyte depolarization and the regulation of CaMK were unchanged by hyperoxic or hypoxic conditions. However, the dynamic features of calcium fluxes elicited by caffeine or epinephrine were different in cells subjected to hypoxia versus hyperoxia, suggesting that these different conditions differentially affect components of ligand-activated signaling pathways that regulate calcium. Our results suggest that changes in signaling pathways, rather than structural organization, may mediate the different outcomes associated with hyperoxia-induced versus hypoxia-induced hypertrophy, and these changes are likely initiated at the cellular level.     

Superoxide dismutase moderates basal and induced bacterial adherence and interleukin-8 expression in airway epithelial cells

Bacterial infection of the tracheobronchial tree is a frequent, serious complication in patients receiving treatment with oxygen and mechanical ventilation, resulting in increased morbidity and mortality. Using human airway epithelial cell culture models, we examined the effect of hyperoxia on bacterial adherence and the expression of interleukin-8 (IL-8), an important mediator involved in the inflammatory process. A 24-h exposure to 95% O(2) increased Pseudomonas aeruginosa (PA) adherence 57% in A549 cells (P < 0.01) and 115% in 16HBE cells (P < 0.01) but had little effect on Staphylococcus aureus (SA) adherence. Exposure to hyperoxia, followed by a 1-h incubation with SA, further enhanced PA adherence (P < 0.01), suggesting that hyperoxia and SA colonization may enhance the susceptibility of lung epithelial cells to gram-negative infections. IL-8 expression was also increased in cells exposed to both hyperoxia and PA. Stable or transient overexpression of manganese superoxide dismutase reduced both basal and stimulated levels of PA adherence and IL-8 levels in response to exposure to either hyperoxia or PA. These data indicate that hyperoxia increases susceptibility to infection and that the pathways are mediated by reactive oxygen species. Therapeutic intervention strategies designed to prevent accumulation of intracellular reactive oxygen species may reduce opportunistic pulmonary infections.     

Effect of high oxygen on placental function in short-term explant cultures

Ex situ culture of human gestational tissues has been routinely used as a model to investigate tissue function. The objective of this study was to determine the effect of varying oxygen concentrations on human term placental explants over a 24-h time period. Specifically, the effect of incubating placental explants in oxygen concentrations of 8%, 21% or 95% on tissue viability, metabolism and cell death was measured by assessing glucose consumption, lactate production, release of lactate dehydrogenase, parathyroid hormone-related protein (PTHrP), tumour necrosis factor-alpha (TNF-α) and 8-isoprostane, immunoreactivity for cleaved-caspase-9 and immunohistochemistry for the caspase-3-cleaved cytokeratin-18 neoepitope, M30. Exposure to higher oxygen concentrations significantly increased the rates of glucose consumption and lactate production. Apoptosis was significantly increased under conditions of higher oxygen as evidenced by increased M30 in placental explant sections. Similarly, hyperoxia significantly increased the releases of PTHrP, TNF-α and 8-isoprostane. Thus, incubation of placental explants with oxygen concentrations of 95% and, to a lesser extent, 21% oxygen was associated with the modulation of multiple cellular response pathways including those associated with tissue viability and cell death. These data are consistent with the hypothesis that hyperoxia activates pathways and mechanisms involved in cellular metabolism, necrosis and apoptosis, thereby shifting the balance from a steady state towards cell death.     

Parathyroid hormone-related protein response to hyperoxic lung injury

Parathyroid hormone-related protein (PTHrP) is a growth inhibitor for alveolar type II cells. Type II cell proliferation after lung injury from 85% oxygen is regulated, in part, by a fall in lung PTHrP. In this study, we investigated lung PTHrP after injury induced by >95% oxygen in rats and rabbits. In adult rats, lung PTHrP rose 10-fold over controls to 6,356 +/- 710 pg/ml (mean +/- SE) at 48 h of hyperoxia. Levels fell to 299 +/- 78 pg/ml, and staining for PTHrP mRNA was greatly reduced at 60 h (P < 0.05), the point of most severe injury and greatest pneumocyte proliferation. In adult rabbits, lung PTHrP peaked at 3,289 +/- 230 pg/ml after 64 h of hyperoxia with 24 h of normoxic recovery and then dropped to 1,629 +/- 153 pg/ml at 48 h of recovery (P < 0.05). Type II cell proliferation peaked shortly after the fall in PTHrP. In newborn rabbits, lavage PTHrP increased by 50% during the first 8 days of hyperoxia, whereas type II cell growth decreased. PTHrP declined at the LD(50), concurrent with increased type II cell division. In summary, lung PTHrP initially rises after injury with >95% hyperoxia and then falls near the peak of injury. Changes in PTHrP are temporally related to type II cell proliferation and may regulate repair of lung injury.     

Vitamin C prevents hyperoxia-mediated vasoconstriction and impairment of endothelium-dependent vasodilation

High arterial blood oxygen tension increases vascular resistance, possibly related to an interaction between reactive oxygen species and endothelium-derived vasoactive factors. Vitamin C is a potent antioxidant capable of reversing endothelial dysfunction due to increased oxidant stress. We tested the hypotheses that hyperoxic vasoconstriction would be prevented by vitamin C, and that acetylcholine-mediated vasodilation would be blunted by hyperoxia and restored by vitamin C. Venous occlusion strain gauge plethysmography was used to measure forearm blood flow (FBF) in 11 healthy subjects and 15 congestive heart failure (CHF) patients, a population characterized by endothelial dysfunction and oxidative stress. The effect of hyperoxia on FBF and derived forearm vascular resistance (FVR) at rest and in response to intra-arterial acetylcholine was recorded. In both healthy subjects and CHF patients, hyperoxia-mediated increases in basal FVR were prevented by the coinfusion of vitamin C. In healthy subjects, hyperoxia impaired the acetylcholine-mediated increase in FBF, an effect also prevented by vitamin C. In contrast, hyperoxia had no effect on verapamil-mediated increases in FBF. In CHF patients, hyperoxia did not affect FBF responses to acetylcholine or verapamil. The addition of vitamin C during hyperoxia augmented FBF responses to acetylcholine. These results suggest that hyperoxic vasoconstriction is mediated by oxidative stress. Moreover, hyperoxia impairs acetylcholine-mediated vasodilation in the setting of intact endothelial function. These effects of hyperoxia are prevented by vitamin C, providing evidence that hyperoxia-derived free radicals impair the activity of endothelium-derived vasoactive factors.     

Involvement of the MAPK and PI3K pathways in chitinase 3-like 1-regulated hyperoxia-induced airway epithelial cell death

BackgroundExposure to 100% oxygen causes hyperoxic acute lung injury characterized by cell death and injury of alveolar epithelial cells. Recently, the role of chitinase 3-like 1 (CHI3L1), a member of the glycosyl hydrolase 18 family that lacks chitinase activity, in oxidative stress was demonstrated in murine models. High levels of serum CHI3L1 have been associated with various diseases of the lung, such as asthma, chronic obstructive pulmonary disease, and cancer. However, the role of CHI3L1 in human airway epithelial cells undergoing oxidative stress remains unknown. In addition, the signaling pathways associated with CHI3L1 in this process are poorly understood.PurposeIn this study, we demonstrate the role of CHI3L1, along with the MAPK and PI3K signaling pathways, in hyperoxia-exposed airway epithelial cells.MethodThe human airway epithelial cell line, BEAS-2B, was exposed to >95% oxygen (hyperoxia) for up to 72 h. Hyperoxia-induced cell death was determined by assessing cell viability, Annexin-V FITC staining, caspase-3 and -7 expression, and electron microscopy. CHI3L1 knockdown and overexpression studies were conducted in BEAS-2B cells to examine the role of CHI3L1 in hyperoxia-induced apoptosis. Activation of the MAPK and PI3K pathways was also investigated to determine the role of these signaling cascades in this process.ResultsHyperoxia exposure increased CHI3L1 expression and apoptosis in a time-dependent manner. CHI3L1 knockdown protected cells from hyperoxia-induced apoptosis. In contrast, CHI3L1 overexpression promoted cell death after hyperoxia exposure. Finally, phosphorylation of ERK1/2, p38, and Akt were affected by CHI3L1 knockdown.ConclusionThis study indicates that CHI3L1 is involved in hyperoxia-induced cell death, suggesting that CHI3L1 may be one of several cell death regulators influencing the MAPK and PI3K pathways during oxidative stress in human airway epithelial cells.     

Reactive oxygen species are required for hyperoxia-induced Bax activation and cell death in alveolar epithelial cells

Exposure of animals to hyperoxia results in respiratory failure and death within 72 h. Histologic evaluation of the lungs of these animals demonstrates epithelial apoptosis and necrosis. Although the generation of reactive oxygen species (ROS) is widely thought to be responsible for the cell death observed following exposure to hyperoxia, it is not clear whether they act upstream of activation of the cell death pathway or whether they are generated as a result of mitochondrial membrane permeabilization and caspase activation. We hypothesized that the generation of ROS was required for hyperoxia-induced cell death upstream of Bax activation. In primary rat alveolar epithelial cells, we found that exposure to hyperoxia resulted in the generation of ROS that was completely prevented by the administration of the combined superoxide dismutase/catalase mimetic EUK-134 (Eukarion, Inc., Bedford, MA). Exposure to hyperoxia resulted in the activation of Bax at the mitochondrial membrane, cytochrome c release, and cell death. The administration of EUK-134 prevented Bax activation, cytochrome c release, and cell death. In a mouse lung epithelial cell line (MLE-12), the overexpression of Bcl-XL protected cells against hyperoxia by preventing the activation of Bax at the mitochondrial membrane. We conclude that exposure to hyperoxia results in Bax activation at the mitochondrial membrane and subsequent cytochrome c release. Bax activation at the mitochondrial membrane requires the generation of ROS and can be prevented by the overexpression of Bcl-XL.     

Loss of Gadd45a does not modify the pulmonary response to oxidative stress

It is well established that exposure to high levels of oxygen (hyperoxia) injures and kills microvascular endothelial and alveolar type I epithelial cells. In contrast, significant death of airway and type II epithelial cells is not observed at mortality, suggesting that these cell types may express genes that protect against oxidative stress and damage. During a search for genes induced by hyperoxia, we previously reported that airway and alveolar type II epithelial cells uniquely express the growth arrest and DNA damage (Gadd)45a gene. Because Gadd45a has been implicated in protection against genotoxic stress, adult Gadd45a (+/+) and Gadd45a (-/-) mice were exposed to hyperoxia to investigate whether it protected epithelial cells against oxidative stress. During hyperoxia, Gadd45a deficiency did not affect loss of airway epithelial expression of Clara cell secretory protein or type II epithelial cell expression of pro-surfactant protein C. Likewise, Gadd45a deficiency did not alter recruitment of inflammatory cells, edema, or overall mortality. Consistent with Gadd45a not affecting the oxidative stress response, p21(Cip1/WAF1) and heme oxygenase-1 were comparably induced in Gadd45a (+/+) and Gadd45a (-/-) mice. Additionally, Gadd45a deficiency did not affect oxidative DNA damage or apoptosis as assessed by oxidized guanine and terminal deoxyneucleotidyl transferase-mediated dUTP nick-end labeling staining. Overexpression of Gadd45a in human lung adenocarcinoma cells did not affect viability or survival during exposure, whereas it was protective against UV-radiation. We conclude that increased tolerance of airway and type II epithelial cells to hyperoxia is not attributed solely to expression of Gadd45a.     

Cyclic stretch attenuates effects of hyperoxia on cell proliferation and viability in human alveolar epithelial cells

The treatment of severe lung disease often requires the use of high concentrations of oxygen coupled with the need for assisted ventilation, potentially exposing the pulmonary epithelium to both reactive oxygen species and nonphysiological cyclic stretch. Whereas prolonged hyperoxia is known to cause increased cell injury, cyclic stretch may result in either cell proliferation or injury depending on the pattern and degree of exposure to mechanical deformation. How hyperoxia and cyclic stretch interact to affect the pulmonary epithelium in vitro has not been previously investigated. This study was performed using human alveolar epithelial A549 cells to explore the combined effects of cyclic stretch and hyperoxia on cell proliferation and viability. Under room air conditions, cyclic stretch did not alter cell viability at any time point and increased cell number after 48 h compared with unstretched controls. After exposure to prolonged hyperoxia, cell number and [(3)H]thymidine incorporation markedly decreased, whereas evidence of oxidative stress and nonapoptotic cell death increased. The combination of cyclic stretch with hyperoxia significantly mitigated the negative effects of prolonged hyperoxia alone on measures of cell proliferation and viability. In addition, cyclic stretch resulted in decreased levels of oxidative stress over time in hyperoxia-exposed cells. Our results suggest that cyclic stretch, as applied in this study, can minimize the detrimental effects of hyperoxia on alveolar epithelial A549 cells.     

Reactive oxygen and nitrogen species induce cell apoptosis via a mitochondria-dependent pathway in hyperoxia lung injury

Hyperoxia-induced lung injury limits the application of mechanical ventilation on rescuing the lives of premature infants and seriously ill and respiratory failure patients, and its mechanisms are not completely understood. In this article, we focused on the relationship between hyperoxia-induced lung injury and reactive oxygen species (ROS), reactive nitrogen species (RNS), mitochondria damage, as well as apoptosis in the pulmonary epithelial II cell line RLE-6TN. After exposure to hyperoxia, the cell viability was significantly decreased, accompanied by the increase in ROS, nitric oxide (NO), inflammatory cytokines, and cell death. Furthermore, hyperoxia triggered the loss of mitochondrial membrane potential (▵Ψm), thereby promoting cytochrome c to release from mitochondria to cytoplasm. Further studies conclusively showed that the Bax/Bcl-2 ratio was enlarged to activate the mitochondria-dependent apoptotic pathway after hyperoxia treatment. Intriguingly, the effects of hyperoxia on the level of ROS, NO and inflammation, mitochondrial damage, as well as cell death were reversed by free radical scavengers N-acetylcysteine and hemoglobin. In addition, a hyperoxia model of neonatal Sprague-Dawley (SD) rats presented the obvious characteristics of lung injury, such as a decrease in alveolar numbers, alveolar mass edema, and disorganized pulmonary structure. The effects of hyperoxia on ROS, RNS, inflammatory cytokines, and apoptosis-related proteins in lung injury tissues of neonatal SD rats were similar to that in RLE-6TN cells. In conclusion, mitochondria are a primary target of hyperoxia-induced free radical, whereas ROS and RNS are the key mediators of hyperoxia-induced cell apoptosis via the mitochondria-dependent pathway in RLE-6TN cells.