Hyperoxia-induced Cell Research Articles

Role of Nox4 and Nox2 in Hyperoxia-Induced Reactive Oxygen Species Generation and Migration of Human Lung Endothelial Cells

In vascular endothelium, the major research focus has been on reactive oxygen species (ROS) derived from Nox2. The role of Nox4 in endothelial signal transduction, ROS production, and cytoskeletal reorganization is not well defined. In this study, we show that human pulmonary artery endothelial cells (HPAECs) and human lung microvascular endothelial cells (HLMVECs) express higher levels of Nox4 and p22(phox) compared to Nox1, Nox2, Nox3, or Nox5. Immunofluorescence microscopy and Western blot analysis revealed that Nox4 and p22(phox), but not Nox2 or p47(phox), are localized in nuclei of HPAECs. Further, knockdown of Nox4 with siRNA decreased Nox4 nuclear expression significantly. Exposure of HPAECs to hyperoxia (3-24 h) enhanced mRNA and protein expression of Nox4, and Nox4 siRNA decreased hyperoxia-induced ROS production. Interestingly, Nox4 or Nox2 knockdown with siRNA upregulated the mRNA and protein expression of the other, suggesting activation of compensatory mechanisms. A similar upregulation of Nox4 mRNA was observed in Nox2 2(-/-) ko mice. Downregulation of Nox4, or pretreatment with N-acetylcysteine, attenuated hyperoxia-induced cell migration and capillary tube formation, suggesting that ROS generated by Nox4 regulate endothelial cell motility. These results indicate that Nox4 and Nox2 play a physiological role in hyperoxia-induced ROS production and migration of ECs.

Heat Shock Protein 27 Protects Lung Epithelial Cells From Hyperoxia-Induced Apoptotic Cell Death

Oxygen toxicity or hyperoxia is one of the major contributing factors in the development of bronchopulmonary dysplasia. Heat shock protein 27 (Hsp27) is an important chaperone protein in the postnatal lung development. However, the role of Hsp27 in lung epithelial cells during hyperoxia is unclear. Our studies by cDNA array and immunohistochemistry revealed that hyperoxia decreased Hsp27 expression in newborn rat lungs. Western blot showed that hyperoxic treatment significantly decreased Hsp27 protein expression in cultured human lung epithelial cells (A549). The expression of Hsp27 was decreased approximately twofold after 24-h and threefold after 48- and 72-h hyperoxic exposure compared with that of the A549 cells exposed to normoxia (p < 0.05, n = 3). Knockdown of Hsp27 expression by siRNA resulted in more apoptotic cell death in A549 cells. Overexpression of Hsp27 reduced hyperoxia-induced apoptotic cell death to 9.2% in Hsp27 overexpressing A549 cells from 12.6% in control A549 cells after 72-h hyperoxic exposure (p < 0.01, n = 8-9). Overexpression of Hsp27 also diminished hyperoxia-induced caspase-9 activation in A549 cells. Our results demonstrated that hyperoxia decreased Hsp27 expression in newborn rat lung and cultured human lung epithelial cells. Overexpression of Hsp27 could reduce hyperoxia-induced apoptosis in cultured human lung epithelial cells.

IL-6 Protects against Hyperoxia-Induced Mitochondrial Damage via Bcl-2–Induced Bak Interactions with Mitofusions

Overexpression of IL-6 markedly diminishes hyperoxic lung injury, hyperoxia-induced cell death, and DNA fragmentation, and enhances Bcl-2 expression. We hypothesized that changes in the interactions between Bcl-2 family members play an important role in the IL-6-mediated protective response to oxidative stress. Consistent with this hypothesis, we found that IL-6 induced Bcl-2 expression, both in vivo and in vitro, disrupted interactions between proapoptotic and antiapoptotic factors, and suppressed H(2)O(2)-induced loss of mitochondrial membrane potential in vitro. In addition, IL-6 overexpression in mice protects against hyperoxia-induced lung mitochondrial damage. The overexpression of Bcl-2 in vivo prolonged the survival of mice exposed to hyperoxia and inhibited alveolar capillary protein leakage. In addition, apoptosis-associated DNA fragmentation was substantially reduced in these animals. This IL-6-mediated protection was lost when Bcl-2 was silenced, demonstrating that Bcl-2 is an essential mediator of IL-6 cytoprotection. Finally, Bcl-2 blocked the dissociation of Bak from mitofusin protein (Mfn) 2, and inhibited the interaction between Bak and Mfn1. Taken together, our results suggest that IL-6 induces Bcl-2 expression to perform cytoprotective functions in response to oxygen toxicity, and that this effect is mediated by alterations in the interactions between Bak and Mfns.

A critical role for Fas/CD‐95 dependent signaling pathways in the pathogenesis of hyperoxia‐induced brain injury

Prematurely born infants are at risk for development of neurocognitive impairment in later life. Oxygen treatment has been recently identified as a trigger of neuronal and oligodendrocyte apoptosis in the developing rodent brain. We investigated the role of the Fas death receptor pathway in oxygen-triggered developmental brain injury. Six-day-old Wistar rats were exposed to 80% oxygen for various periods (2, 6, 12, 24, 48, and 72 hours), and mice deficient in either Fas (B6.MRL-Tnfrsf6(lpr)) or Fas ligand (B6Smn.C3-Fasl(gld)) and control mice (C57BL/6J) were exposed to 80% oxygen for 24 hours. Polymerase chain reaction, Western blotting, and caspase activity assays of thalamus and cortex tissue were performed. Fas and Fas ligand messenger RNA and protein were upregulated. Furthermore, hyperoxia resulted in induction of downstream signaling events of Fas, such as Fas-associated death domain (FADD), the long and short form of FADD-like interleukin-1beta-converting enzyme (FLICE) inhibitory protein (FLIP-L, FLIP-S), and cleavage of caspase-8 and caspase-3. Injection of a selective caspase-8 inhibitor (TRP801, 1mg/kg) at the beginning of hyperoxia blocked subsequent caspase-3 cleavage in this model. B6.MRL-Tnfrsf6(lpr) mice were protected against oxygen-mediated injury, confirming Fas involvement in hyperoxia-induced cell death. Mice deficient in Fas ligand did not differ from control animals in the amount of cell death. We conclude that neonatal hyperoxia triggers Fas receptor and its downstream signaling events in a Fas ligand-independent fashion. Lack of functional Fas receptors and selective pharmacological inhibition of caspase-8 prevents activation of caspase-3 and provides significant neuroprotection.

Caveolin‐1 regulates the secretion and cytoprotection of Cyr61 in hyperoxic cell death

Cysteine-rich 61 (Cyr61) belongs to the CCN family and mediates cell proliferation, survival, and apoptosis. Our previous studies showed that Cyr61 protected against hyperoxia-induced lung cell death via Akt phosphorylation. Caveolin-1 (cav-1), a 22-kDa transmembrane scaffolding protein, is the principal structural component of caveolae. Emerging data show that cav-1 regulates signal transduction-associated proteins that reside in the caveolae. Numerous integrin-related pathways, including PI3K/Akt-induced cell survival are controlled by cav-1-mediated signaling. Our data showed that recombinant Cyr61 promoted cell proliferation and resistance to hyperoxia-induced cell death in vitro. Neutralizing antibodies reversed the above effects, indicating functional role of secreted Cyr61 in response to hyperoxic stress. While deletion of cav-1 protected cells from hyperoxia-induced cell death, Cyr61-neutralizing antibodies abolished this protective effect. Furthermore, Cyr61 and cav-1 colocalized and physically interacted via integrins in bronchial epithelial cells. Deletion of cav-1 increased extracellular and decreased cytosolic Cyr61, both in vitro and in vivo. Pretreatment with Brefeldin A increased intracellular Cyr61 in cav-1(-/-) cells, while decreasing extracellular Cyr61. Taken together, Cav-1/Cyr61 interaction via integrins represents a novel pathway of Cyr61 signaling involving cav-1-dependent processes, which play a critical role in regulating hyperoxia-induced cell death.

Suramin induces and enhances apoptosis in a model of hyperoxia-induced oligodendrocyte injury

Recent evidence suggests oxygen as a powerful trigger for cell death in the immature white matter, leading to periventricular leukomalacia (PVL) as a cause of adverse neurological outcome in survivors of preterm birth. This oligodendrocyte (OL) death is associated with oxidative stress, upregulation of apoptotic signaling factors (i.e., Fas, caspase-3) and decreased amounts of neurotrophins. In search of neuroprotective strategies we investigated whether the polysulfonated urea derivative suramin, recently identified as a potent inhibitor of Fas signaling, affords neuroprotection in an in vitro model of hyperoxia-induced injury to immature oligodendrocytes. Immature OLs (OLN-93) were subjected to 80% hyperoxia (48 h) in the presence or absence of suramin (0, 30, 60, 120 microM). Cell death was assessed by flow cytometry (Annexin V, caspase-3 activity assay) and immunohistochemistry for activated caspase-3. Immunoblotting for the death receptor Fas, cleaved caspase-8 and the phosphorylated isoform of the serine-threonin kinase Akt (pAkt) was performed. Suramin lead to OL apoptosis and potentiated hyperoxia-induced injury in a dose-dependent manner. Immunoblotting revealed increased Fas and caspase-8 expression by suramin treatment. This effect was significantly enhanced when suramin was combined with hyperoxia. Furthermore, pAkt levels decreased following suramin exposure, indicating interference with neurotrophin-dependent growth factor signaling. These data indicate that suramin causes apoptotic cell death and aggravates hyperoxia-induced cell death in immature OLs. Its mechanism of action includes an increase of previously described hyperoxia-induced expression of pro-apoptotic factors and deprivation of growth factor dependent signaling components.

Deletion of Caveolin-1 Protects against Oxidative Lung Injury via Up-Regulation of Heme Oxygenase-1

Acute lung injury (ALI) is a major cause of morbidity and mortality in critically ill patients. Hyperoxia causes lung injury in animals and humans, and is an established model of ALI. Caveolin-1, a major constituent of caveolae, regulates numerous biological processes, including cell death and proliferation. Here we demonstrate that caveolin-1-null mice (cav-1(-/-)) were resistant to hyperoxia-induced death and lung injury. Cav-1(-/-) mice sustained reduced lung injury after hyperoxia as determined by protein levels in bronchoalveolar lavage fluid and histologic analysis. Furthermore, cav-1(-/-) fibroblasts and endothelial cells and cav-1 knockdown epithelial cells resisted hyperoxia-induced cell death in vitro. Basal and inducible expression of the stress protein heme oxygenase-1 (HO-1) were markedly elevated in lung tissue or fibroblasts from cav-1(-/-) mice. Hyperoxia induced the physical interaction between cav-1 and HO-1 in fibroblasts assessed by co-immunoprecipitation studies, which resulted in attenuation of HO activity. Inhibition of HO activity with tin protoporphyrin-IX abolished the survival benefits of cav-1(-/-) cells and cav-1(-/-) mice exposed to hyperoxia. The cav-1(-/-) mice displayed elevated phospho-p38 mitogen-activated protein kinase (MAPK) and p38beta expression in lung tissue/cells under basal conditions and during hyperoxia. Treatment with SB202190, an inhibitor of p38 MAPK, decreased hyperoxia-inducible HO-1 expression in wild-type and cav-1(-/-) fibroblasts. Taken together, our data demonstrated that cav-1 deletion protects against hyperoxia-induced lung injury, involving in part the modulation of the HO-1-cav-1 interaction, and the enhanced induction of HO-1 through a p38 MAPK-mediated pathway. These studies identify caveolin-1 as a novel component involved in hyperoxia-induced lung injury.

Enhanced resistance to oxidative lung injury by an Nrf2‐ARE inducer in mice

Nrf2 is essential in airway protection against oxidative insults via antioxidant response element (ARE)‐mediated induction of antioxidants. To test the hypothesis that enhanced Nrf2‐ARE responsiveness protects lungs from subsequent hyperoxia (&gt;95% O2) exposure, Nrf2+/+ and Nrf2−/− mice were fed with diet containing standardized broccoli sprout extract (SBE) or regular diet for 14 days before air or hyperoxia exposure. In a separate study, the mice were orally treated with sulforaphane (days ‐5, ‐3, ‐1) before hyperoxia exposure. In Nrf2+/+ mice, hyperoxia‐induced lung neutrophilic inflammation, protein edema, and cell necrosis were significantly decreased by SBE treatment. Hyperoxia‐induced body weight loss was significantly attenuated by SBE. Sulforaphane also significantly decreased lung inflammation caused by hyperoxia in these mice. Expression of lung Nrf2 and antioxidant mRNAs was highly elevated in Nrf2+/+ mice by sulforaphane after hyperoxia. SBE enhanced antioxidant expressions in air‐exposed mice but there was no significant effect of SBE after hyperoxia. Lung injury phenotypes of Nrf2−/− mice with suppressed antioxidant expressions were not significantly changed by SBE or sulforaphane treatment after hyperoxia. Results support a preventive role for fortified Nrf2‐ARE pathway in pulmonary oxygen toxicity. Supported by the Intramural Program of NIEHS

Heat Shock Protein 27 (Hsp27) Protects Lung Epithelial Cells from Hyperoxia‐Induced Cell Death

Supplemental oxygen therapy (hyperoxia) is a common life‐saving practice for preterm babies with respiratory distress. Prolonged hyperoxia is one of the major contributing factors in cell death and the development of bronchopulmonary dysplasia (BPD), a chronic lung disease, in preterm babies. Hsp27 is the smallest inducible heat shock chaperone protein that modulates the ability of cells to respond to oxidative stress. Our DNA Superarray analysis revealed that hyperoxia significantly decreased Hsp27 gene expression in newborn rat lungs. Immunohistochemistry of Hsp27 confirmed that Hsp27 protein in epithelial cells of neonatal rat lungs was markedly decreased after hyperoxic exposure. In cultured human lung epithelial cells (A549), prolonged hyperoxia significantly reduced Hsp27 expression and caused cell death. Overexpression of hHsp27 could prevent hyperoxia‐induced cell death in cultured human lung epithelial cells. Our studies suggest that hyperoxia reduces Hsp27 expression in lung epithelial cells and overexpression of hHsp27 confers cytoprotection against hyperoxia‐induced cell death in lung epithelial cells.

TGF-β signaling promotes survival and repair in rat alveolar epithelial type 2 cells during recovery after hyperoxic injury

Hyperoxic rats treated with inosine during oxygen exposure have increased levels of active transforming growth factor (TGF)-beta in the bronchoalveolar lavage (BAL), yet alveolar epithelial type 2 cells (AEC2) isolated from these animals demonstrate less hyperoxia-induced DNA damage and increased expression of active Smad2. To determine whether TGF-beta1 signaling per se protected AEC2 against hyperoxic damage, freshly isolated AEC2 from hyperoxic rats were incubated with TGF-beta1 for 24 h and assayed for DNA damage by fluorescein-activated cell sorter analysis of TdT-mediated dUTP nick end labeling. TGF-beta1 was protective over a concentration range similar to that in BAL of inosine-treated hyperoxic animals (50-5,000 pg/ml). TGF-beta1 also augmented hyperoxia-induced DNA repair activity and cell migration, stimulated autocrine secretion of fibronectin, accelerated closure of a monolayer scratch wound, and restored hyperoxia-depleted VEGF secretion by AEC2 to normoxic levels. The TGF-beta receptor type I activin-like kinase-4, -5, and -7 inhibitor peptide SB-505124 abolished the protective effect of TGF-beta on hyperoxic DNA damage and increased TdT-mediated dUTP nick end labeling in normoxic cells. These data suggest that endogenous TGF-beta-mediated Smad signaling is required for AEC2 homeostasis in vitro, while exogenous TGF-beta1 treatment of hyperoxia-damaged AEC2 results in a cell that is equipped to survive, repair, migrate, secrete matrix, and induce new blood vessel formation more efficiently than AEC2 primed by hyperoxia alone.

Epidermal growth factor-like domain 7 protects endothelial cells from hyperoxia-induced cell death

Hyperoxia is one of the major contributors to the development of bronchopulmonary dysplasia (BPD), a chronic lung disease in premature infants. Emerging evidence suggests that the arrested lung development of BPD is associated with pulmonary endothelial cell death and vascular dysfunction resulting from hyperoxia-induced lung injury. A better understanding of the mechanism of hyperoxia-induced endothelial cell death will provide critical information for the pathogenesis and therapeutic development of BPD. Epidermal growth factor-like domain 7 (EGFL7) is a protein secreted from endothelial cells. It plays an important role in vascular tubulogenesis. In the present study, we found that Egfl7 gene expression was significantly decreased in the neonatal rat lungs after hyperoxic exposure. The Egfl7 expression was returned to near normal level 2 wk after discounting oxygen exposure during recovery period. In cultured human endothelial cells, hyperoxia also significantly reduced Egfl7 expression. These observations suggest that diminished levels of Egfl7 expression might be associated with hyperoxia-induced endothelial cell death and lung injury. When we overexpressed human Egfl7 (hEgfl7) in EA.hy926 human endothelial cell line, we found that hEgfl7 overexpression could partially block cytochrome c release from mitochondria and decrease caspase-3 activation. Further Western blotting analyses showed that hEgfl7 overexpression could reduce expression of a proapoptotic protein, Bax, and increase expression of an antiapoptotic protein, Bcl-xL. Theses findings indicate that hEGFL7 may protect endothelial cell from hyperoxia-induced apoptosis by inhibition of mitochondria-dependent apoptosis pathway.

Estradiol attenuates hyperoxia‐induced cell death in the developing white matter

Periventricular leukomalacia is the predominant type of brain injury in preterm infants underlying the development of cerebral palsy. Periventricular leukomalacia has its peak incidence at 23 to 32 weeks postconceptional age characterized by extensive oligodendrocyte migration and maturation. Oxygen toxicity has been identified as a possible contributing factor to the pathogenesis of cerebral palsy in survivors of preterm birth. 17beta-estradiol (E2) is important for the development and function of the central nervous system. Furthermore, neuroprotective properties have been attributed to estrogens. We examined the effect of E2 on hyperoxia-induced cell death in the developing white matter in the rat brain. Six-day-old (P6) rat pups, the immature oligodendroglial cell line (OLN-93), and primary oligodendrocyte cultures were subjected to 80% O(2) in the presence or absence of E2 (600 microg/kg intraperitoneally in vivo, 10(-6)-10(-10)M in vitro). Cell counts and lactate dehydrogenase assay were used to assess cell survival. Immunoblot analysis was used for detection of estrogen receptor expression and investigation of apoptotic signaling pathways. White matter injury was assessed by myelin basic protein immunocytochemistry at P11. E2 produced significant dose-dependent protection against oxygen-induced apoptotic cell death in primary oligodendrocytes. Treatment with E2 prevented hyperoxia-induced proapoptotic Fas-upregulation and caspase-3 activation. Finally, E2 antagonized hyperoxia-induced inactivation of extracellular signal-regulated kinase 1 and 2 and Akt, key kinases of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase cell survival promoting pathways, respectively. Loss of myelin basic protein labeling was seen in P11 pups after oxygen exposure, and E2 attenuated this injury. These results suggest a possible role for estrogens in the prevention of neonatal oxygen-induced white matter injury.

Developmental differences in the responses of IL-6 and IL-13 transgenic mice exposed to hyperoxia

Our previous work has shown that adult mice with overexpression of IL-6 and IL-13 in the lung have enhanced survival in hyperoxia associated with reduced hyperoxia-induced lung injury and cell death. We hypothesized that there are developmental differences in these responses in the adult vs. the newborn (NB) animal, and these responses have clinical relevance in the human NB. We compared the responses to 100% O(2) of NB IL-6 and IL-13 transgenic mice with wild-type littermate controls by evaluating mortality, lung tissue TUNEL staining, and mRNA expression using RT-PCR. We used ELISA to measure IL-6 levels in tracheal aspirates from human neonates. Our results show that, in contrast to the cytoprotective effects in mature mice, IL-6 caused significantly increased mortality, DNA injury, caspases, cell death regulator and angiogenic factor expression in hyperoxia in the NB. Furthermore, tracheal aspirate levels of IL-6 were significantly increased in premature neonates with respiratory distress syndrome who had an adverse outcome (bronchopulmonary dysplasia/death). In contrast to the protective effects in adults, there was no survival advantage to the NB IL-13 mice in hyperoxia. These findings imply that caution should be exercised in extrapolating results from the adult to the NB.

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 p47phox 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 (α, η, and ζ), 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.

Bcl-2 protects against hyperoxia-induced apoptosis through inhibition of the mitochondria-dependent pathway

Bcl-2 is an antiapoptotic molecule that prevents oxidative stress damage and cell death. We investigated the possible protective mechanisms mediated by Bcl-2 during hyperoxia-induced cell death in L929 cells. In these cells, hyperoxia promoted apoptosis without DNA fragmentation. Overexpression of Bcl-2 significantly protected cells from oxygen-induced apoptosis, as shown by measurement of lactate dehydrogenase release, quantification of apoptotic nuclei, and detection of Annexin-V-positive cells. Bcl-2 partially prevented mitochondrial damage and interfered with the mitochondrial proapoptotic signaling pathway: it reduced Bax translocation to mitochondria, decreased the release of cytochrome c, and inhibited caspase 3 activation. However, treatment with the caspase inhibitor Z-VAD.fmk failed to rescue the cells from death, indicating that protection provided by Bcl-2 was due not only to caspase inhibition. Bcl-2 also prevented the release of mitochondrial apoptotic inducing factor, a mediator of caspase-independent apoptosis, correlating with the absence of oligonucleosomal DNA fragmentation. In addition, Bcl-2-overexpressing cells showed significantly higher intracellular amounts of glutathione after 72 h of oxygen exposure. In conclusion, our results demonstrate that the overexpression of Bcl-2 is able to prevent hyperoxia-induced cell death, by affecting mitochondria-dependent apoptotic pathways and increasing intracellular antioxidant compounds.

Carbon Monoxide Protects against Hyperoxia-induced Endothelial Cell Apoptosis by Inhibiting Reactive Oxygen Species Formation

Hyperoxia causes cell injury and death associated with reactive oxygen species formation and inflammatory responses. Recent studies show that hyperoxia-induced cell death involves apoptosis, necrosis, or mixed phenotypes depending on cell type, although the underlying mechanisms remain unclear. Using murine lung endothelial cells, we found that hyperoxia caused cell death by apoptosis involving both extrinsic (Fas-dependent) and intrinsic (mitochondria-dependent) pathways. Hyperoxia-dependent activation of the extrinsic apoptosis pathway and formation of the death-inducing signaling complex required NADPH oxidase-dependent reactive oxygen species production, because this process was attenuated by chemical inhibition, as well as by genetic deletion of the p47(phox) subunit, of the oxidase. Overexpression of heme oxygenase-1 prevented hyperoxia-induced cell death and cytochrome c release. Likewise, carbon monoxide, at low concentrations, markedly inhibited hyperoxia-induced endothelial cell death by inhibiting cytochrome c release and caspase-9/3 activation. Carbon monoxide, by attenuating hyperoxia-induced reactive oxygen species production, inhibited extrinsic apoptosis signaling initiated by death-inducing signal complex trafficking from the Golgi apparatus to the plasma membrane and downstream activation of caspase-8. We also found that carbon monoxide inhibited the hyperoxia-induced activation of Bcl-2-related proteins involved in both intrinsic and extrinsic apoptotic signaling. Carbon monoxide inhibited the activation of Bid and the expression and mitochondrial translocation of Bax, whereas promoted Bcl-X(L)/Bax interaction and increased Bad phosphorylation. We also show that carbon monoxide promoted an interaction of heme oxygenase-1 with Bax. These results define novel mechanisms underlying the antiapoptotic effects of carbon monoxide during hyperoxic stress.

Hyperoxia causes angiopoietin 2–mediated acute lung injury and necrotic cell death

The angiogenic growth factor angiopoietin 2 (Ang2) destabilizes blood vessels, enhances vascular leak and induces vascular regression and endothelial cell apoptosis. We considered that Ang2 might be important in hyperoxic acute lung injury (ALI). Here we have characterized the responses in lungs induced by hyperoxia in wild-type and Ang2-/- mice or those given either recombinant Ang2 or short interfering RNA (siRNA) targeted to Ang2. During hyperoxia Ang2 expression is induced in lung epithelial cells, while hyperoxia-induced oxidant injury, cell death, inflammation, permeability alterations and mortality are ameliorated in Ang2-/- and siRNA-treated mice. Hyperoxia induces and activates the extrinsic and mitochondrial cell death pathways and activates initiator and effector caspases through Ang2-dependent pathways in vivo. Ang2 increases inflammation and cell death during hyperoxia in vivo and stimulates epithelial necrosis in hyperoxia in vitro. Ang2 in plasma and alveolar edema fluid is increased in adults with ALI and pulmonary edema. Tracheal Ang2 is also increased in neonates that develop bronchopulmonary dysplasia. Ang2 is thus a mediator of epithelial necrosis with an important role in hyperoxic ALI and pulmonary edema.

Endothelial STAT3 is essential for the protective effects of HO‐1 in oxidant‐induced lung injury

Administering high levels of inspired oxygen, or hyperoxia, is commonly used as a life-sustaining measure in critically ill patients. Unfortunately, the oxidant stress generated by prolonged hyperoxia can lead to respiratory failure, multiorgan failure, and death. Although the endothelial cell is known to be a target for hyperoxia-induced injury, its precise role is unclear. Heme oxygenase-1 (HO-1) and "signal transducer and activator of transcription 3" (STAT3) have been found to confer protection against endothelial cell injury. We sought to elucidate the specific roles of HO-1 and STAT3 in hyperoxic lung and endothelial cell injury. Mice or murine lung endothelial cells (MLEC) administered HO-1 siRNA exhibited marked injury and death compared with nonspecific siRNA. Overexpression of either HO-1 or STAT3 confers protection. However, HO-1 and its reaction product carbon monoxide (CO) lose their protective effects in the presence of STAT3 siRNA in MLEC or in endothelial-specific, STAT3-deficient mice. STAT3 overexpression is able to partially rescue HO-1-deficient MLEC from hyperoxia-induced cell death. Our results demonstrate 1) the importance of the endothelium in lethal hyperoxic injury, 2) HO-1 and CO require endothelial STAT3 for their protective effects, and 3) STAT3 confers endothelial cell protection via both HO-1-dependent and independent mechanisms.

Mitochondrial aldehyde dehydrogenase attenuates hyperoxia-induced cell death through activation of ERK/MAPK and PI3K-Akt pathways in lung epithelial cells

Oxygen toxicity is one of the major risk factors in the development of the chronic lung disease or bronchopulmonary dysplasia in premature infants. Using proteomic analysis, we discovered that mitochondrial aldehyde dehydrogenase (mtALDH or ALDH2) was downregulated in neonatal rat lung after hyperoxic exposure. To study the role of mtALDH in hyperoxic lung injury, we overexpressed mtALDH in human lung epithelial cells (A549) and found that mtALDH significantly reduced hyperoxia-induced cell death. Compared with control cells (Neo-A549), the necrotic cell death in mtALDH-overexpressing cells (mtALDH-A549) decreased from 25.3 to 6.5%, 50.5 to 9.1%, and 52.4 to 15.1% after 24-, 48-, and 72-h hyperoxic exposure, respectively. The levels of intracellular and mitochondria-derived reactive oxygen species (ROS) in mtALDH-A549 cells after hyperoxic exposure were significantly lowered compared with Neo-A549 cells. mtALDH overexpression significantly stimulated extracellular signal-regulated kinase (ERK) phosphorylation under normoxic and hyperoxic conditions. Inhibition of ERK phosphorylation partially eliminated the protective effect of mtALDH in hyperoxia-induced cell death, suggesting ERK activation by mtALDH conferred cellular resistance to hyperoxia. mtALDH overexpression augmented Akt phosphorylation and maintained the total Akt level in mtALDH-A549 cells under normoxic and hyperoxic conditions. Inhibition of phosphatidylinositol 3-kinase (PI3K) activation by LY294002 in mtALDH-A549 cells significantly increased necrotic cell death after hyperoxic exposure, indicating that PI3K-Akt activation by mtALDH played an important role in cell survival after hyperoxia. Taken together, these data demonstrate that mtALDH overexpression attenuates hyperoxia-induced cell death in lung epithelial cells through reduction of ROS, activation of ERK/MAPK, and PI3K-Akt cell survival signaling pathways.

P21 Cip1 Protection against Hyperoxia Requires Bcl-X L and Is Uncoupled from Its Ability to Suppress Growth

The cyclin-dependent kinase inhibitor p21Cip1/Waf1/Sdi1 protects the lung against hyperoxia, but the mechanism of protection remains unclear because loss of p21 does not lead to aberrant cell proliferation. Because some members of the Bcl-2 gene family have been implicated in hyperoxia-induced cell death, the current study investigated their expression as well as p21-dependent growth suppression and cytoprotection. Conditional overexpression of full-length p21, its amino-terminal cyclin-binding (p211-82NLS) domain or its carboxy-terminal PCNA-binding (p2176-164) domain inhibited growth of human lung adenocarcinoma H1299 cells, but only the full-length protein was cytoprotective. Low levels of p21 inhibited cell proliferation, whereas higher levels were required for protection. Expression of the anti-apoptotic protein Bcl-XL declined during hyperoxia but was maintained in cells expressing p21. RNA interference (RNAi) knockdown of Bcl-XL enhanced hyperoxic death of cells expressing p21, whereas overexpression of Bcl-XL increased cell survival. Consistent with growth suppression and cytoprotection requiring different levels of p21, hyperoxia inhibited PCNA expression in p21+/+ and p21+/- mice but not in p21-/- mice. In contrast, p21 was haplo-insufficient for maintaining expression of Bcl-XL and protection against hyperoxia. Taken together, these data show that p21-mediated cytoprotection against hyperoxia involves regulation of Bcl-XL and is uncoupled from its ability to inhibit proliferation.