Mitogen-activated protein kinases modulate H(2)O(2)-induced apoptosis in primary rat alveolar epithelial cells

Increasing evidence suggests a role for apoptosis in the maintenance of the alveolar epithelium under normal and pathological conditions. However, the signaling pathways modulating alveolar type II (ATII) cell apoptosis remain poorly defined. Here we investigated the role of MAPKs as modulators of oxidant-mediated ATII cell apoptosis using in vitro models of H(2)O(2)-stress. H(2)O(2), delivered either as a bolus or as a flux, lead to time- and concentration-dependent increases in ATII cells apoptosis. Increased apoptosis in primary rat ATII cells was detected at H(2)O(2) concentrations and production rates in the physiological range (1 microM) and peaked at 100 microM H(2)O(2). Immortalized rat lung epithelial cells (RLE), in contrast, required millimolar concentration of H(2)O(2) for maximal responses. H(2)O(2)-induced apoptosis was preceded by rapid activation of all three classes of mitogen-activated protein kinases (MAPKs): ERK, JNK, and p38. Specific inhibition of JNK using antisense oligonucleotides and ERK and p38 using PD98059 or SB202190, respectively, indicated a pro-apoptotic role for JNK pathway and an anti-apoptotic role for ERK- and p38-initiated signaling events. Our data show that the balance between the activation of JNK, ERK, and p38 is a critical determinant of cell fate, suggesting that pharmacological interventions on the MAPK pathways may be useful in the treatment of oxidant-related lung injury.     

Green tea polyphenol blocks h(2)o(2)-induced interleukin-8 production from human alveolar epithelial cells

Reactive oxygen species (ROS) play crucial roles in ischemia-reperfusion (IR) injury of lung transplants. Reactive oxygen species may stimulate the production of neutrophil chemotactic factors such as interleukin-8 (IL-8), from alveolar epithelial cells, causing recruitment and activation of neutrophils in the reperfused tissue. Green tea polyphenol has potent anti-oxidative activities and anti-inflammatory effects by decreasing cytokine production. In the present study, we found that green tea polyphenol significantly inhibited IL-8 production induced by hydrogen peroxide (H(2)O(2)) in human lung alveolar epithelial cells (A549 line). It has been shown that mitogen activated protein kinases, such as Jun N-terminal kinase (JNK), p38 and p44/42, could mediate IL-8 production from a variety of cell types. We further investigated the effect of green tea polyphenol on these protein kinases, and demonstrated that H(2)O(2)-induced phosphorylation of JNK and p38 but not p44/42 was inhibited by green tea polyphenol in A549 cells. We speculate that green tea polyphenol may inhibit H(2)O(2)-induced IL-8 production from A549 cells through inactivation of JNK and p38.     

Gene transfer of hepatocyte growth factor by electroporation reduces bleomycin-induced lung fibrosis

Abnormal alveolar wound repair contributes to the development of pulmonary fibrosis after lung injury. Hepatocyte growth factor (HGF) is a potent mitogenic factor for alveolar epithelial cells and may therefore improve alveolar epithelial repair in vitro and in vivo. We hypothesized that HGF could increase alveolar epithelial repair in vitro and improve pulmonary fibrosis in vivo. Alveolar wound repair in vitro was determined using an epithelial wound repair model with HGF-transfected A549 alveolar epithelial cells. Electroporation-mediated, nonviral gene transfer of HGF in vivo was performed 7 days after bleomycin-induced lung injury in the rat. Alveolar epithelial repair in vitro was increased after transfection of wounded epithelial monolayers with a plasmid encoding human HGF, pCikhHGF [human HGF (hHGF) gene expressed from the cytomegalovirus (CMV) immediate-early promoter and enhancer] compared with medium control. Electroporation-mediated in vivo HGF gene transfer using pCikhHGF 7 days after intratracheal bleomycin reduced pulmonary fibrosis as assessed by histology and hydroxyproline determination 14 days after bleomycin compared with controls treated with the same vector not containing the HGF sequence (pCik). Lung epithelial cell proliferation was increased and apoptosis reduced in hHGF-treated lungs compared with controls, suggesting increased alveolar epithelial repair in vivo. In addition, profibrotic transforming growth factor-beta1 (TGF-beta1) was decreased in hHGF-treated lungs, indicating an involvement of TGF-beta1 in hHGF-induced reduction of lung fibrosis. In conclusion, electroporation-mediated gene transfer of hHGF decreases bleomycin-induced pulmonary fibrosis, possibly by increasing alveolar epithelial cell proliferation and reducing apoptosis, resulting in improved alveolar wound repair.     

Modulation of cell and DNA damage by poly(ADP)ribose polymerase in lung cells exposed to H(2)O(2) or asbestos fibres

Poly(ADP)ribose polymerase (PARP) may participate in cell survival, apoptosis and development of DNA damage. We investigated the role of PARP in transformed human pleural mesothelial (MeT-5A) and alveolar epithelial (A549) cells exposed from 0.05 to 5mM hydrogen peroxide (H(2)O(2)) or crocidolite asbestos fibres (1-10 microg/cm(2)) in the presence and absence of 3-aminobenzamide (ABA), a PARP inhibitor. The cells were investigated for the development of cell injury, DNA single strand breaks and depletion of the cellular high-energy nucleotides. Compared to H(2)O(2), fibres caused a minor decrease in cell viability and effect on the cellular high-energy nucleotide depletion, and a marginal effect on the development of DNA strand breaks when assessed by the single cell gel electrophoresis (the Comet assay). Inhibition of PARP transiently protected the cells against acute H(2)O(2) related irreversible cell injury when assessed by microculture tetrazolium dye (XTT) assay and potentiated oxidant related DNA damage when assessed by the Comet assay. However, PARP inhibition had no significant effect on fibre-induced cell or DNA toxicity with the exception of one fibre concentration (2 microg/cm(2)) in MeT-5A cells. Apoptosis is often associated with PARP cleavage and caspase activation. Fibres did not cause PARP cleavage or activation of caspase 3 further confirming previous results about relatively low apoptotic potential of asbestos fibres. In conclusion, maintenance of cellular high-energy nucleotide pool and high viability of asbestos exposed cells may contribute to the survival and malignant conversion of lung cells exposed to the fibres.     

Balance of life and death in alveolar epithelial type II cells: proliferation, apoptosis, and the effects of cyclic stretch on wound healing

After acute lung injury, repair of the alveolar epithelium occurs on a substrate undergoing cyclic mechanical deformation. While previous studies showed that mechanical stretch increased alveolar epithelial cell necrosis and apoptosis, the impact of cell death during repair was not determined. We examined epithelial repair during cyclic stretch (CS) in a scratch-wound model of primary rat alveolar type II (ATII) cells and found that CS altered the balance between proliferation and cell death. We measured cell migration, size, and density; intercellular gap formation; cell number, proliferation, and apoptosis; cytoskeletal organization; and focal adhesions in response to scratch wounding followed by CS for up to 24 h. Under static conditions, wounds were closed by 24 h, but repair was inhibited by CS. Wounding stimulated cell motility and proliferation, actin and vinculin redistribution, and focal adhesion formation at the wound edge, while CS impeded cell spreading, initiated apoptosis, stimulated cytoskeletal reorganization, and attenuated focal adhesion formation. CS also caused significant intercellular gap formation compared with static cells. Our results suggest that CS alters several mechanisms of epithelial repair and that an imbalance occurs between cell death and proliferation that must be overcome to restore the epithelial barrier.     

Interleukin-1beta augments in vitro alveolar epithelial repair

Biologically active interleukin (IL)-1beta is present in the pulmonary edema fluid obtained from patients with acute lung injury and has been implicated as an important early mediator of nonpulmonary epithelial wound repair. Therefore, we tested the hypothesis that IL-1beta would enhance wound repair in cultured monolayers from rat alveolar epithelial type II cells. IL-1beta (20 ng/ml) increased the rate of in vitro alveolar epithelial repair by 118 +/- 11% compared with that in serum-free medium control cells (P < 0.01). IL-1beta induced cell spreading and migration at the edge of the wound but not proliferation. Neutralizing antibodies to epidermal growth factor (EGF) and transforming growth factor-alpha or inhibition of the EGF receptor by tyrphostin AG-1478 or genistein inhibited IL-1beta-induced alveolar epithelial repair, indicating that IL-1beta enhances in vitro alveolar epithelial repair by an EGF- or transforming growth factor-alpha-dependent mechanism. Moreover, the mitogen-activated protein kinase pathway is involved in IL-1beta-induced alveolar epithelial repair because inhibition of extracellular signal-regulated kinase activation by PD-98059 inhibited IL-1beta-induced alveolar epithelial repair. In conclusion, IL-1beta augments in vitro alveolar epithelial repair, indicating a possible novel role for IL-1beta in the early repair process of the alveolar epithelium in acute lung injury.     

Protectin DX promotes epithelial injury repair and inhibits fibroproliferation partly via ALX/PI3K signalling pathway

Acute respiratory distress syndrome/acute lung injury (ARDS/ALI) is histologically characterized by extensive alveolar barrier disruption and excessive fibroproliferation responses. Protectin DX (PDX) displays anti‐inflammatory and potent inflammation pro‐resolving actions. We sought to investigate whether PDX attenuates LPS (lipopolysaccharide)‐induced lung injury via modulating epithelial cell injury repair, apoptosis and fibroblasts activation. In vivo, PDX was administered intraperitoneally (IP) with 200 ng/per mouse after intratracheal injection of LPS, which remarkedly stimulated proliferation of type II alveolar epithelial cells (AT II cells), reduced the apoptosis of AT II cells, which attenuated lung injury induced by LPS. Moreover, primary type II alveolar cells were isolated and cultured to assess the effects of PDX on wound repair, apoptosis, proliferation and transdifferentiation in vitro. We also investigated the effects of PDX on primary rat lung fibroblast proliferation and myofibroblast differentiation. Our result suggests PDX promotes primary AT II cells wound closure by inducing the proliferation of AT II cells and reducing the apoptosis of AT II cells induced by LPS, and promotes AT II cells transdifferentiation. Furthermore, PDX inhibits transforming growth factor‐β₁ (TGF‐β₁) induced fibroproliferation, fibroblast collagen production and myofibroblast transformation. Furthermore, the effects of PDX on epithelial wound healing and proliferation, fibroblast proliferation and activation partly via the ALX/ PI3K signalling pathway. These data present identify a new mechanism of PDX which targets the airway epithelial cell and fibroproliferation are potential for treatment of ARDS/ALI.     

Poly(ADP-ribose) polymerase inhibition prevents both apoptotic-like delayed neuronal death and necrosis after H(2)O(2) injury

Toxic reactive oxygen species (ROS) such as hydrogen peroxide, nitric oxide, superoxide, and the hydroxyl radical are generated in a variety of neuropathological conditions and cause significant DNA damage. We determined the effects of 3-aminobenzamide (AB), an inhibitor of the DNA repair enzyme poly(ADP-ribose) polymerase (PARP), on cell death in differentiated PC12 cells, a model of sympathetic neurons, after H(2) O(2) injury. Exposure to 0.5 mm H(2) O(2) resulted in a significant decrease in intracellular NAD(H), NADP(H), and ATP levels. This injury resulted in the death of 90% of the cells with significant necrosis early (2 h) after injury and increased apoptosis (12-24 h after injury), as measured by PS exposure and the presence of cytoplasmic oligonucleosomal fragments. Treatment with 2.5 mm AB restored pyridine nucleotide and ATP levels and ameliorated cell death (65% versus 90%) by decreasing the extent of both necrosis and apoptosis. Interestingly, we observed that H(2) O(2) -induced injury caused a delayed cell death exhibiting features of apoptosis but in which caspase-3 like activity was absent. Moreover, pretreatment with AB restored caspase-3-like activity. Our results suggest that apoptosis and necrosis are both triggered by PARP overactivation, and that maintenance of cellular energy levels after injury by inhibiting PARP shifts cell death from necrosis to apoptosis.     

Distinct patterns of insulin-like growth factor binding protein (IGFBP)-2 and IGFBP-3 expression in oxidant exposed lung epithelial cells

Oxygen (O(2)) species are involved in a large variety of pulmonary diseases. Among the various cell types that compose the lung, the epithelial cells of the alveolar structure appear to be a major target for oxidant injury. Despite their importance in the repair processes, the mechanisms which regulate the replication of the stem cells of the alveolar epithelium, the type 2 cells, remain poorly understood. Based on the results of several studies which have documented the involvement of the insulin-like growth factor (IGF) system in lung epithelial cell replication, and which have also suggested a role for IGF binding proteins (IGFBPs) in the control of cell proliferation, the aim of the present work was to determine whether IGFBPs could be involved in the modulation of growth of human lung epithelial cells exposed to oxidants. Experiments were performed using a human lung adenocarcinoma cell line (A549) which was exposed for various durations to hyperoxia (95% O(2)). We observed a rapid and reversible growth arrest of the cells after only 24 h of O(2) exposure. When oxidant injury was prolonged, growth arrest was followed by induction of apoptosis with activation of the Fas pathway. These effects were associated with an increased expression of IGFBP-2 and IGFBP-3. In addition, study of localization of these proteins revealed distinct patterns of distribution. IGFBP-3 was mainly present in the extracellular compartment. In comparison, the fraction of IGFBP-2 secreted was less abundant whereas the IGFBP-2 fraction in the intracellular compartment appeared stronger. In addition, analysis of the subcellular localization provided data indicating the presence of IGFBP-2 in the nucleus. Taken together these data support a role for IGFBP-2 and IGFBP-3 in the processes of growth arrest and apoptosis in lung epithelial cells upon oxidant exposure. They also suggest that distinct mechanisms may link IGFBP-2 and IGFBP-3 to the key regulators of the cell cycle.     

Keratinocyte growth factor can enhance alveolar epithelial repair by nonmitogenic mechanisms

Pretreatment with keratinocyte growth factor (KGF) ameliorates experimentally induced acute lung injury in rats. Although alveolar epithelial type II cell hyperplasia probably contributes, the mechanisms underlying KGF's protective effect remain incompletely described. Therefore, we tested the hypothesis that KGF given to rats in vivo would enhance alveolar epithelial repair in vitro by nonproliferative mechanisms. After intratracheal instillation (48 h) of KGF (5 mg/kg), alveolar epithelial type II cells were isolated for in vitro alveolar epithelial repair studies. KGF-treated cells had markedly increased epithelial repair (96 +/- 22%) compared with control cells (P < 0.001). KGF-treated cells had increased cell spreading and migration at the wound edge but no increase in in vitro proliferation compared with control cells. KGF-treated cells were more adherent to extracellular matrix proteins and polystyrene. Inhibition of the epidermal growth factor (EGF) receptor with tyrosine kinase inhibitors abolished the KGF effect on epithelial repair. In conclusion, in vivo administration of KGF augments the epithelial repair rate of alveolar epithelial cells by altering cell adherence, spreading, and migration and through stimulation of the EGF receptor.     

Effects of hypoxia and hypercapnia on surfactant protein expression proliferation and apoptosis in A549 alveolar epithelial cells

During lung injury alveolar epithelial cells are directly exposed to changes in PO(2) and PCO(2). Integrity of alveolar epithelial type II cells (AECII) is critical in lung injury but the effect of hypoxia and hypercapnia on AECII function, viability and proliferation has not been clearly investigated. Aim of the present work was to determine the direct effect of hypoxia and hypercapnia on surfactant protein expression, proliferation and apoptosis of lung epithelial cells in vitro. A549 alveolar epithelia cells were subjected to hypoxia (1%O(2)-5% CO(2)) or hypercapnia (21% O(2-) 15% CO(2)) and expression of surfactant protein C was measured and compared to normal conditions (21% O(2)- 5% CO(2)). Cell cycle progression and apoptosis were measured by flow cytometric analysis. RESULTS: A549 alveolar epithelial cells produce surfactant proteins, including surfactant protein C, when cultured under normal conditions, which is reduced under hypoxic conditions. Specifically, pro-SpC expression is moderately decreased after 8 h of culture in hypoxia, and is completely attenuated after 48 h. Hypercapnia decreases pro-SpC expression only after 48 h of exposure. Stimulation with TNF-alpha partly reverses pSPC decrease observed under hypoxic and hypercapnic conditions. Hypoxic culture of A549 cells results in progressive arrest of cells in the G1 phase of the cell cycle and increased apoptosis first observed 4 h following exposure and peaking at 24 h. In contrast hypercapnia has no significant effect on alveolar epithelial cell proliferation or apoptosis. CONCLUSIONS: Taken together we can conclude that hypoxia rapidly and severely affects AECII function and viability while hypercapnia has an inhibitory effect on pro-SpC production only after prolonged exposure.     

The Axonal Guidance Cue Semaphorin 3C Contributes to Alveolar Growth and Repair

Lung diseases characterized by alveolar damage such as bronchopulmonary dysplasia (BPD) in premature infants and emphysema lack efficient treatments. Understanding the mechanisms contributing to normal and impaired alveolar growth and repair may identify new therapeutic targets for these lung diseases. Axonal guidance cues are molecules that guide the outgrowth of axons. Amongst these axonal guidance cues, members of the Semaphorin family, in particular Semaphorin 3C (Sema3C), contribute to early lung branching morphogenesis. The role of Sema3C during alveolar growth and repair is unknown. We hypothesized that Sema3C promotes alveolar development and repair. In vivo Sema3C knock down using intranasal siRNA during the postnatal stage of alveolar development in rats caused significant air space enlargement reminiscent of BPD. Sema3C knock down was associated with increased TLR3 expression and lung inflammatory cells influx. In a model of O₂-induced arrested alveolar growth in newborn rats mimicking BPD, air space enlargement was associated with decreased lung Sema3C mRNA expression. In vitro, Sema3C treatment preserved alveolar epithelial cell viability in hyperoxia and accelerated alveolar epithelial cell wound healing. Sema3C preserved lung microvascular endothelial cell vascular network formation in vitro under hyperoxic conditions. In vivo, Sema3C treatment of hyperoxic rats decreased lung neutrophil influx and preserved alveolar and lung vascular growth. Sema3C also preserved lung plexinA2 and Sema3C expression, alveolar epithelial cell proliferation and decreased lung apoptosis. In conclusion, the axonal guidance cue Sema3C promotes normal alveolar growth and may be worthwhile further investigating as a potential therapeutic target for lung repair.     

Hepatocyte growth factor improves viability after H2O2-induced toxicity in bile duct epithelial cells

Intracellular defence mechanisms against oxidative stress may play an important role in the progression of liver diseases, including cholangiopathies. The multifunctional anti-apoptotic hepatocyte growth factor (HGF) has been suggested to have antioxidant functions. The effect of HGF upon cell viability, the generation of ROS, the expression of genes that play a role in ROS defence, and the activation of caspase-3 were measured in bile duct epithelial (BDE) cells in the presence or absence of H(2)O(2). HGF reduced H(2)O(2)-induced loss of viability, diminished H(2)O(2)-mediated ROS generation and abrogated H(2)O(2)-triggered changes in GSH/GSSG ratio. Furthermore, HGF increased the gene-expression of gamma-glutamylcysteine synthetase (GCLC) and glutathione reductase (GSR), while no effect was seen upon the gene-expression of superoxide dismutase 1 (SOD1), catalase (CAT), glutathione peroxidase (GPX1), and glutathione synthetase (GSR). Finally, HGF diminished the proteolytical activation of the key mediator of apoptosis (caspase-3) after H(2)O(2) exposure. Together, HGF may improve viability in bile duct epithelia cells after H(2)O(2) induced toxicity by proliferation, strengthening the intrinsic antioxidant defences, and/or by an attenuation of apoptosis. These in vitro results support the evaluation of HGF as antioxidative factor in hepatobiliary pathologies.     

Alveolar Type II Epithelial Cell Dysfunction in Rat Experimental Hepatopulmonary Syndrome (HPS)

The hepatopulmonary syndrome (HPS) develops when pulmonary vasodilatation leads to abnormal gas exchange. However, in human HPS, restrictive ventilatory defects are also observed supporting that the alveolar epithelial compartment may also be affected. Alveolar type II epithelial cells (AT2) play a critical role in maintaining the alveolar compartment by producing four surfactant proteins (SPs, SP-A, SP-B, SP-C and SP-D) which also facilitate alveolar repair following injury. However, no studies have evaluated the alveolar epithelial compartment in experimental HPS. In this study, we evaluated the alveolar epithelial compartment and particularly AT2 cells in experimental HPS induced by common bile duct ligation (CBDL). We found a significant reduction in pulmonary SP production associated with increased apoptosis in AT2 cells after CBDL relative to controls. Lung morphology showed decreased mean alveolar chord length and lung volumes in CBDL animals that were not seen in control models supporting a selective reduction of alveolar airspace. Furthermore, we found that administration of TNF-α, the bile acid, chenodeoxycholic acid, and FXR nuclear receptor activation (GW4064) induced apoptosis and impaired SP-B and SP-C production in alveolar epithelial cells in vitro. These results imply that AT2 cell dysfunction occurs in experimental HPS and is associated with alterations in the alveolar epithelial compartment. Our findings support a novel contributing mechanism in experimental HPS that may be relevant to humans and a potential therapeutic target.     

Acid contact in the rodent pulmonary alveolus causes proinflammatory signaling by membrane pore formation

Although gastric acid aspiration causes rapid lung inflammation and acute lung injury, the initiating mechanisms are not known. To determine alveolar epithelial responses to acid, we viewed live alveoli of the isolated lung by fluorescence microscopy, then we microinjected the alveoli with HCl at pH of 1.5. The microinjection caused an immediate but transient formation of molecule-scale pores in the apical alveolar membrane, resulting in loss of cytosolic dye. However, the membrane rapidly resealed. There was no cell damage and no further dye loss despite continuous HCl injection. Concomitantly, reactive oxygen species (ROS) increased in the adjacent perialveolar microvascular endothelium in a Ca(2+)-dependent manner. By contrast, ROS did not increase in wild-type mice in which we gave intra-alveolar injections of polyethylene glycol (PEG)-catalase, in mice overexpressing alveolar catalase, or in mice lacking functional NADPH oxidase (Nox2). Together, our findings indicate the presence of an unusual proinflammatory mechanism in which alveolar contact with acid caused membrane pore formation. The effect, although transient, was nevertheless sufficient to induce Ca(2+) entry and Nox2-dependent H(2)O(2) release from the alveolar epithelium. These responses identify alveolar H(2)O(2) release as the signaling mechanism responsible for lung inflammation induced by acid and suggest that intra-alveolar PEG-catalase might be therapeutic in acid-induced lung injury.     

Neuroprotective Effect of Fucoidan on H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Apoptosis in PC12 Cells Via Activation of PI3K/Akt Pathway

One of the plausible ways to prevent the reactive oxygen species (ROS)-mediated cellular injury is dietary or pharmaceutical augmentation of endogenous antioxidant defense capacity. In this study, we investigated the neuroprotective effect of fucoidan on H(2)O(2)-induced apoptosis in PC12 cells and the possible signaling pathways involved. The results showed that fucoidan inhibited the decrease of cell viability, scavenged ROS formation and reduced lactate dehydrogenase release in H(2)O(2)-induced PC12 cells. These changes were associated with an increase in superoxide dismutase and glutathione peroxidase activity, and reduction in malondialdehyde. In addition, fucoidan treatment inhibited apoptosis in H(2)O(2)-induced PC12 cells by increasing the Bcl-2/Bax ratio and decreasing active caspase-3 expression, as well as enhancing Akt phosphorylation (p-Akt). However, the protection of fucoidan on cell survival, p-Akt, the Bcl-2/Bax ratio and caspase-3 activity were abolished by pretreating with phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002. In consequence, fucoidan might protect the neurocytes against H(2)O(2)-induced apoptosis via reducing ROS levels and activating PI3K/Akt signaling pathway.     

Cyclic mechanical strain increases reactive oxygen species production in pulmonary epithelial cells

Overdistention of lung tissue during mechanical ventilation may be one of the factors that initiates ventilator-induced lung injury (VILI). We hypothesized that cyclic mechanical stretch (CMS) of the lung epithelium is involved in the early events of VILI through the production of reactive oxygen species (ROS). Cultures of an immortalized human airway epithelial cell line (16HBE), a human alveolar type II cell line (A549), and primary cultures of rat alveolar type II cells were cyclically stretched, and the production of superoxide (O2-) was measured by dihydroethidium fluorescence. CMS stimulated increased production of O2- after 2 h in each type of cell. 16HBE cells exhibited no significant stimulation of ROS before 2 h of CMS (20% strain, 30 cycles/min), and ROS production returned to control levels after 24 h. Oxidation of glutathione (GSH), a cellular antioxidant, increased with CMS as measured by a decrease in the ratio of the reduced GSH level to the oxidized GSH level. Strain levels of 10% did not increase O2- production in 16HBE cells, whereas 15, 20, and 30% significantly increased generation of O2-. Rotenone, a mitochondrial complex I inhibitor, partially abrogated the stretch-induced generation of O2- after 2 h CMS in 16HBE cells. NADPH oxidase activity was increased after 2 h of CMS, contributing to the production of O2-. Increased ROS production in lung epithelial cells in response to elevated stretch may contribute to the onset of VILI.     

Mitochondrial localization of catalase provides optimal protection from H2O2-induced cell death in lung epithelial cells

Reactive oxygen species (ROS) can cause cell injury and death via mitochondrial-dependent pathways, and supplementation with antioxidants has been shown to ameliorate these processes. The c-Jun NH(2)-terminal kinase (JNK) pathway has been shown to play a critical role in ROS-induced cell death. To determine if targeting catalase (CAT) to the mitochondria provides better protection than cytosolic expression against H(2)O(2)-induced injury, the following two approaches were taken: 1) adenoviral-mediated transduction was performed using cytosolic (CCAT) or mitochondrial (MCAT) CAT cDNAs and 2) stable cell lines were generated overexpressing CAT in mitochondria (n = 3). Cells were exposed to 250 microM H(2)O(2), and cell survival, mitochondrial function, cytochrome c release, and JNK activity were analyzed. Although all viral transduced cells had a transient twofold increase in CAT activity, MCAT cells had significantly higher survival rates, the best mitochondrial function, and lowest JNK activity compared with CCAT and LacZ controls. The improved protection with MCAT was observed in primary type II lung epithelial cells and in transformed lung epithelial cells. In the three stable cell lines, cell survival directly correlated with extent of mitochondrial localization (r = 0.60572, P < 0.05) and not overall CAT activity (r = -0.45501, P < 0.05). Data indicate that targeting of antioxidants directly to the mitochondria is more effective in protecting lung epithelial cells against ROS-induced injury. This has important implications in antioxidant supplementation trials to prevent ROS-induced lung injury in critically ill patients.     

Romo1 expression contributes to oxidative stress-induced death of lung epithelial cells

Oxidant-mediated death of lung epithelial cells due to cigarette smoking plays an important role in pathogenesis in lung diseases such as idiopathic pulmonary fibrosis (IPF). However, the exact mechanism by which oxidants induce epithelial cell death is not fully understood. Reactive oxygen species (ROS) modulator 1 (Romo1) is localized in the mitochondria and mediates mitochondrial ROS production through complex III of the mitochondrial electron transport chain. Here, we show that Romo1 mediates mitochondrial ROS production and apoptosis induced by oxidative stress in lung epithelial cells. Hydrogen peroxide (H₂O₂) treatment increased Romo1 expression, and Romo1 knockdown suppressed the cellular ROS levels and cell death triggered by H₂O₂ treatment. In immunohistochemical staining of lung tissues from patients with IPF, Romo1 was mainly localized in hyperplastic alveolar and bronchial epithelial cells. Romo1 overexpression was detected in 14 of 18 patients with IPF. TUNEL-positive alveolar epithelial cells were also detected in most patients with IPF but not in normal controls. These findings suggest that Romo1 mediates apoptosis induced by oxidative stress in lung epithelial cells.     

Reactive nitrogen and oxygen species activate different sphingomyelinases to induce apoptosis in airway epithelial cells

Airway epithelial cells are constantly exposed to environmental insults such as air pollution or tobacco smoke that may contain high levels of reactive nitrogen and reactive oxygen species. Previous work from our laboratory demonstrated that the reactive oxygen species (ROS), hydrogen peroxide (H(2)O(2)), specifically activates neutral sphingomyelinase 2 (nSMase2) to generate ceramide and induce apoptosis in airway epithelial cells. In the current study we examine the biological consequence of exposure of human airway epithelial (HAE) cells to reactive nitrogen species (RNS). Similar to ROS, we hypothesized that RNS may modulate ceramide levels in HAE cells and induce apoptosis. We found that nitric oxide (NO) exposure via the NO donor papa-NONOate, failed to induce apoptosis in HAE cells. However, when papa-NONOate was combined with a superoxide anion donor (DMNQ) to generate peroxynitrite (ONOO(-)), apoptosis was observed. Similarly pure ONOO(-)-induced apoptosis, and ONOO(-)-induced apoptosis was associated with an increase in cellular ceramide levels. Pretreatment with the antioxidant glutathione did not prevent ONOO(-)-induced apoptosis, but did prevent H(2)O(2)-induced apoptosis. Analysis of the ceramide generating enzymes revealed a differential response by the oxidants. We confirmed our findings that H(2)O(2) specifically activated a neutral sphingomyelinase (nSMase2). However, ONOO(-) exposure did not affect neutral sphingomyelinase activity; rather, ONOO(-) specifically activated an acidic sphingomyelinase (aSMase). The specificity of each enzyme was confirmed using siRNA to knockdown both nSMase2 and aSMase. Silencing nSMase2 prevented H(2)O(2)-induced apoptosis, but had no effect on ONOO(-)-induced apoptosis. On the other hand, silencing of aSMase markedly impaired ONOO(-)-induced apoptosis, but did not affect H(2)O(2)-induced apoptosis. These findings support our hypothesis that ROS and RNS modulate ceramide levels to induce apoptosis in HAE cells. However, we found that different oxidants modulate different enzymes of the ceramide generating machinery to induce apoptosis in airway epithelial cells. These findings add to the complexity of how oxidative stress promotes lung cell injury.