Molecular mechanisms of nitrogen dioxide induced epithelial injury in the lung

The lung can be exposed to a variety of reactive nitrogen intermediates through the inhalation of environmental oxidants and those produced during inflammation. Reactive nitrogen species (RNS) include, nitrogen dioxide (·NO₂) and peroxynitrite (ONOO^–). Classically known as a major component of both indoor and outdoor air pollution, ·NO₂ is a toxic free radical gas. ·NO₂ can also be formed during inflammation by the decomposition of ONOO^– or through peroxidase-catalyzed reactions. Due to their reactive nature, RNS may play an important role in disease pathology. Depending on the dose and the duration of administration, ·NO₂ has been documented to cause pulmonary injury in both animal and human studies. Injury to the lung epithelial cells following exposure to ·NO₂ is characterized by airway denudation followed by compensatory proliferation. The persistent injury and repair process may contribute to airway remodeling, including the development of fibrosis. To better understand the signaling pathways involved in epithelial cell death by ·NO₂ or other RNS, we routinely expose cells in culture to continuous gas-phase ·NO₂. Studies using the ·NO₂ exposure system revealed that lung epithelial cell death occurs in a density dependent manner. In wound healing experiments, ·NO₂ induced cell death is limited to cells localized in the leading edge of the wound. Importantly, ·NO₂-induced death does not appear to be dependent on oxidative stress per se. Potential cell signaling mechanisms will be discussed, which include the mitogen activated protein kinase, c-Jun N-terminal Kinase and the Fas/Fas ligand pathways. During periods of epithelial loss and regeneration that occur in diseases such as asthma or during lung development, epithelial cells in the lung may be uniquely susceptible to death. Understanding the molecular mechanisms of epithelial cell death associated with the exposure to ·NO₂ will be important in designing therapeutics aimed at protecting the lung from persistent injury and repair.     

Nitric oxide-enhanced caspase-3 and acidic sphingomyelinase interaction: a novel mechanism by which airway epithelial cells escape ceramide-induced apoptosis

Reactive nitrogen species (RNS) are implicated in the pathophysiology of inflammatory lung diseases such as asthma and chronic obstructive pulmonary disease. The molecular mechanisms and signaling events involved in lung cell injury by RNS are still poorly understood. In the current study, we observe a novel anti-apoptotic response to nitric oxide (NO) exposure (via the NO donors 3-morpholine-syndnonimine (SIN1) or papa-NONOate) of human airway epithelial (HAE) cells. NO exposure via the NO donors increased cellular ceramide levels via ceramide synthase but did not trigger an apoptotic response. Rather, exposure to the NO donors promoted an increase in the protein-protein interaction between acidic sphingomyelinase (aSMase) and caspase-3, with aSMase sequestering caspase-3 and preventing its cleavage. In contrast, when aSMase was silenced in HAE cells or was knocked out in mice, an increase in cleaved caspase-3 was observed. This elevated caspase-3 cleavage was further augmented upon NO exposure (via SIN1 or papa-NONOate) of HAE cells and could be prevented by an inhibitor to ceramide synthase. These results demonstrate a novel mechanism of NO modulation of apoptosis, in which HAE cells exposed to NO via an NO donor induces ceramide generation via ceramide synthase. However, this ceramide induction does not lead to apoptosis unless aSMase is knocked down, allowing the release of caspase-3, its activation and execution of apoptosis.     

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.     

Integrin-β4 regulates the dynamic changes of phenotypic characteristics in association with epithelial-mesenchymal transition (EMT) and RhoA activity in airway epithelial cells during injury and repair

Background: In airway disease such as asthma a hyperactive cellular event of epithelial-mesenchymal transition (EMT) is considered as the mechanism of pathological airway tissue remodeling after injury to the airway epithelium. And the initiation of EMT in the airways depends on the epithelial disruption involving dissolution and/or destabilization of the adhesive structures between the cells and ECM. Previously, we have shown that integrin-β4, an epithelial adhesion molecule in bronchial epithelium is an important regulator of cell proliferation and wound repair in human airway epithelial cells. Therefore, in this study we aimed to investigate whether integrin-β4 also regulates EMT phenotypes during injury and repair in airway epithelial cells of both wild type/integrin-β4^-/- mice in vivo and cultured cells treated with integrin-β4/nonsense siRNA in vitro.Methods: We induced injury to the airway epithelial cells by either repeated exposure to ozone and mechanical scratch wound, and subsequently examined the EMT-related phenotypic features in the airway epithelial cells including biomarkers expression, adhesion and cytoskeleton reorganization and cell stiffness.Results: The results show that in response to injury (ozone exposure/scratch wound) and subsequent spontaneous repair (ozone withdrawal/wound healing) both in vivo and in vitro, the airway epithelial cells underwent dynamic changes in the epithelial and mesenchymal biomarkers expression, adhesion and cytoskeleton structures as well as cell stiffness, all together exhibiting enhanced EMT phenotypic features after injury and reversal of the injury-induced effects during repair. Importantly, these injury/repair-associated EMT phenotypic changes in airway epithelial cells appeared to be dependent on integrin-β4 expression. More specifically, when integrin-β4 was deficient in mice (integrin-β4^-/-) the repair of ozone-injured airway epithelium was impaired and the recovery of ozone-enhanced EMT biomarkers expression in the airway epithelium was delayed. Similarly, in the scratch wounded airway epithelial cells with integrin-β4 knockdown, the cells were impaired in all aspects related to EMT during wound and repair including cell proliferation, wound closure rate, adhesion and cytoskeleton protein expression (vinculin and vimentin), mesenchymal-like F-actin reorganization, cell stiffness and RhoA activation.Conclusion: Taken together, these results suggested that integrin-β4 may be essential in regulating the effects of injury and repair on EMT in airway epithelial cells via influencing both the cell adhesion to ECM and cells'' physical phenotypes through RhoA signaling pathway.     

Hog barn dust slows airway epithelial cell migration in vitro through a PKCalpha-dependent mechanism

Agricultural work and other occupational exposures are responsible for approximately 15% of chronic obstructive pulmonary disease (COPD). COPD involves airway remodeling in response to chronic lung inflammatory events and altered airway repair mechanisms. However, the effect of agricultural dust exposure on signaling pathways that regulate airway injury and repair has not been well characterized. A key step in this process is migration of airway cells to restore epithelial integrity. We have previously shown that agents that activate the critical regulatory enzyme protein kinase C (PKC) slow cell migration during wound repair. Based on this observation and direct kinase measurements that demonstrate that dust extract from hog confinement barns (HDE) specifically activates the PKC isoforms PKCalpha and PKCepsilon, we hypothesized that HDE would slow wound closure time in airway epithelial cells. We utilized the human bronchial epithelial cell line BEAS-2B and transfected BEAS-2B cell lines that express dominant negative (DN) forms of PKC isoforms to demonstrate that HDE slows wound closure in BEAS-2B and PKCepsilon DN cell lines. However, in PKCalpha DN cells, wound closure following HDE treatment is not significantly different than media-treated cells. These results suggest that the PKCalpha isoform is an important regulator of cell migration in response to agricultural dust exposure.     

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.     

Inflammatory levels of nitric oxide inhibit airway epithelial cell migration by inhibition of the kinase ERK1/2 and activation of hypoxia-inducible factor-1 alpha

Increased synthesis of NO during airway inflammation, caused by induction of nitric-oxide synthase 2 in several lung cell types, may contribute to epithelial injury and permeability. To investigate the consequence of elevated NO production on epithelial function, we exposed cultured monolayers of human bronchial epithelial cells to the NO donor diethylenetriaamine NONOate. At concentrations generating high nanomolar levels of NO, representative of inflammatory conditions, diethylenetriaamine NONOate markedly reduced wound closure in an in vitro scratch injury model, primarily by inhibiting epithelial cell migration. Analysis of signaling pathways and gene expression profiles indicated a rapid induction of the mitogen-activated protein kinase phosphatase (MPK)-1 and decrease in extracellular signal-regulated kinase (ERK)1/2 activation, as well as marked stabilization of hypoxia-inducible factor (HIF)-1alpha and activation of hypoxia-responsive genes, under these conditions. Inhibition of ERK1/2 signaling using U0126 enhanced HIF-1alpha stabilization, implicating ERK1/2 dephosphorylation as a contributing mechanism in NO-mediated HIF-1alpha activation. Activation of HIF-1alpha by the hypoxia mimic cobalt chloride, or cell transfection with a degradation-resistant HIF-1alpha mutant construct inhibited epithelial wound repair, implicating HIF-1alpha in NO-mediated inhibition of cell migration. Conversely, NO-mediated inhibition of epithelial wound closure was largely prevented after small interfering RNA suppression of HIF-1alpha. Finally, NO-mediated inhibition of cell migration was associated with HIF-1alpha-dependent induction of PAI-1 and activation of p53, both negative regulators of epithelial cell migration. Collectively, our results demonstrate that inflammatory levels of NO inhibit epithelial cell migration, because of suppression of ERK1/2 signaling, and activation of HIF-1alpha and p53, with potential consequences for epithelial repair and remodeling during airway inflammation.     

Arsenic upregulates MMP-9 and inhibits wound repair in human airway epithelial cells

As part of the innate immune defense, the polarized conducting lung epithelium acts as a barrier to keep particulates carried in respiration from underlying tissue. Arsenic is a metalloid toxicant that can affect the lung via inhalation or ingestion. We have recently shown that chronic exposure of mice or humans to arsenic (10-50 ppb) in drinking water alters bronchiolar lavage or sputum proteins consistent with reduced epithelial cell migration and wound repair in the airway. In this report, we used an in vitro model to examine effects of acute exposure of arsenic (15-290 ppb) on conducting airway lung epithelium. We found that arsenic at concentrations as low as 30 ppb inhibits reformation of the epithelial monolayer following scrape wounds of monolayer cultures. In an effort to understand functional contributions to epithelial wound repair altered by arsenic, we showed that acute arsenic exposure increases activity and expression of matrix metalloproteinase (MMP)-9, an important protease in lung function. Furthermore, inhibition of MMP-9 in arsenic-treated cells improved wound repair. We propose that arsenic in the airway can alter the airway epithelial barrier by restricting proper wound repair in part through the upregulation of MMP-9 by lung epithelial cells.     

Nitric oxide and reactive nitrogen species in airway epithelial signaling and inflammation

Nitric oxide (NO(.-)) is produced by many diverse cell types as a cellular or intracellular signaling molecule, by the activation of nitric oxide synthases (NOSs). All three known NOS isoforms are expressed within the respiratory tract and mediate various airway functional properties such as airway smooth muscle tone, ciliary function, epithelial electrolyte transport, and innate host defense. The respiratory epithelium is a major source of NO(.-), in which it regulates normal epithelial cell function and signaling as well as signaling pathways involved in airway inflammation. In addition to its normal physiological properties, increased airway NO(.-) production in inflammatory respiratory tract diseases such as asthma may activate additional signaling mechanisms to regulate inflammatory-immune pathways, and epithelial barrier (dys)function or repair. The biological actions of NO(.-) are controlled at various levels, including mechanisms that regulate NOS localization and activation, and variable oxidative metabolism of NO(.-), resulting in generation of bioactive reactive nitrogen species (RNS). Moreover, in addition to altered production of NO(.-) or RNS, the presence of various target enzymes and/or metabolic regulators of NO(.-)/RNS can be dramatically altered during airway inflammatory conditions, and contribute to alterations in NO(.-)-mediated signaling pathways in disease. This review summarizes current knowledge regarding NO(.-)-mediated epithelial signaling, as well as disease-related changes in airway NOS biology and target enzymes that affect NO(.-)/RNS signaling mechanisms. A detailed understanding of these various changes and their impact on NO(.-) signaling pathways are needed to fully appreciate the contributions of NO(.-)/RNS to airway inflammation and to develop suitable therapeutic approaches based on regulating NO(.-) function.     

Free radicals and the pancreatic acinar cells: role in physiology and pathology

Reactive oxygen and nitrogen species (ROS and RNS) play an important role in signal transduction and cell injury processes. Nitric oxide synthase (NOS)-the key enzyme producing nitric oxide (NO)-is found in neuronal structures, vascular endothelium and, possibly, in acinar and ductal epithelial cells in the pancreas. NO is known to regulate cell homeostasis, and its effects on the acinar cells are reviewed here. ROS are implicated in the early events within the acinar cells, leading to the development of acute pancreatitis. The available data on ROS/RNS involvement in the apoptotic and necrotic death of pancreatic acinar cells will be discussed.     

Mitigation of chlorine gas lung injury in rats by postexposure administration of sodium nitrite

Nitrite (NO(2)(-)) has been shown to limit injury to the heart, liver, and kidneys in various models of ischemia-reperfusion injury. Potential protective effects of systemic NO(2)(-) in limiting lung injury or enhancing repair have not been documented. We assessed the efficacy and mechanisms by which postexposure intraperitoneal injections of NO(2)(-) mitigate chlorine (Cl(2))-induced lung injury in rats. Rats were exposed to Cl(2) (400 ppm) for 30 min and returned to room air. NO(2)(-) (1 mg/kg) or saline was administered intraperitoneally at 10 min and 2, 4, and 6 h after exposure. Rats were killed at 6 or 24 h. Injury to airway and alveolar epithelia was assessed by quantitative morphology, protein concentrations, number of cells in bronchoalveolar lavage (BAL), and wet-to-dry lung weight ratio. Lipid peroxidation was assessed by measurement of lung F(2)-isoprostanes. Rats developed severe, but transient, hypoxemia. A significant increase of protein concentration, neutrophil numbers, airway epithelia in the BAL, and lung wet-to-dry weight ratio was evident at 6 h after Cl(2) exposure. Quantitative morphology revealed extensive lung injury in the upper airways. Airway epithelial cells stained positive for terminal deoxynucleotidyl-mediated dUTP nick end labeling (TUNEL), but not caspase-3. Administration of NO(2)(-) resulted in lower BAL protein levels, significant reduction in the intensity of the TUNEL-positive cells, and normal lung wet-to-dry weight ratios. F(2)-isoprostane levels increased at 6 and 24 h after Cl(2) exposure in NO(2)(-)- and saline-injected rats. This is the first demonstration that systemic NO(2)(-) administration mitigates airway and epithelial injury.     

Cigarette Smoke Modulates Repair and Innate Immunity following Injury to Airway Epithelial Cells

Cigarette smoking is the main risk factor associated with chronic obstructive pulmonary disease (COPD), and contributes to COPD development and progression by causing epithelial injury and inflammation. Whereas it is known that cigarette smoke (CS) may affect the innate immune function of airway epithelial cells and epithelial repair, this has so far not been explored in an integrated design using mucociliary differentiated airway epithelial cells. In this study, we examined the effect of whole CS exposure on wound repair and the innate immune activity of mucociliary differentiated primary bronchial epithelial cells, upon injury induced by disruption of epithelial barrier integrity or by mechanical wounding. Upon mechanical injury CS caused a delayed recovery in the epithelial barrier integrity and wound closure. Furthermore CS enhanced innate immune responses, as demonstrated by increased expression of the antimicrobial protein RNase 7. These differential effects on epithelial repair and innate immunity were both mediated by CS-induced oxidative stress. Overall, our findings demonstrate modulation of wound repair and innate immune responses of injured airway epithelial cells that may contribute to COPD development and progression.     

Epithelial cell senescence impairs repair process and exacerbates inflammation after airway injury

Background

Genotoxic stress, such as by exposure to bromodeoxyuridine (BrdU) and cigarette smoke, induces premature cell senescence. Recent evidence indicates that cellular senescence of various types of cells is accelerated in COPD patients. However, whether the senescence of airway epithelial cells contributes to the development of airway diseases is unknown. The present study was designed to test the hypothesis that premature senescence of airway epithelial cells (Clara cells) impairs repair processes and exacerbates inflammation after airway injury.

Methods

C57/BL6J mice were injected with the Clara-cell-specific toxicant naphthalene (NA) on days 0, 7, and 14, and each NA injection was followed by a daily dose of BrdU on each of the following 3 days, during which regenerating cells were allowed to incorporate BrdU into their DNA and to senesce. The p38 MAPK inhibitor SB202190 was injected 30 minutes before each BrdU dose. Mice were sacrificed at different times until day 28 and lungs of mice were obtained to investigate whether Clara cell senescence impairs airway epithelial regeneration and exacerbates airway inflammation. NCI-H441 cells were induced to senesce by exposure to BrdU or the telomerase inhibitor MST-312. Human lung tissue samples were obtained from COPD patients, asymptomatic smokers, and nonsmokers to investigate whether Clara cell senescence is accelerated in the airways of COPD patients, and if so, whether it is accompanied by p38 MAPK activation.

Results

BrdU did not alter the intensity of the airway epithelial injury or inflammation after a single NA exposure. However, after repeated NA exposure, BrdU induced epithelial cell (Clara cell) senescence, as demonstrated by a DNA damage response, p21 overexpression, increased senescence-associated β-galactosidase activity, and growth arrest, which resulted in impaired epithelial regeneration. The epithelial senescence was accompanied by p38 MAPK-dependent airway inflammation. Senescent NCI-H441 cells impaired epithelial wound repair and secreted increased amounts of pro-inflammatory cytokines in a p38 MAPK-dependent manner. Clara cell senescence in COPD patients was accelerated and accompanied by p38 MAPK activation.

Conclusions

Senescence of airway epithelial cells impairs repair processes and exacerbates p38 MAPK-dependent inflammation after airway injury, and it may contribute to the pathogenesis of COPD.     

Reactive nitrogen species-induced cell death requires Fas-dependent activation of c-Jun N-terminal kinase

Nitrogen dioxide is a highly toxic reactive nitrogen species (RNS) recently discovered as an inflammatory oxidant with great potential to damage tissues. We demonstrate here that cell death by RNS was caused by c-Jun N-terminal kinase (JNK). Activation of JNK by RNS was density dependent and caused mitochondrial depolarization and nuclear condensation. JNK activation by RNS was abolished in cells lacking functional Fas or following expression of a truncated version of Fas lacking the intracellular death domain. In contrast, RNS induced JNK potently in cells expressing a truncated version of tumor necrosis factor receptor 1 or cells lacking tumor necrosis factor receptor 1 (TNF-R1), illustrating a dependence of Fas but not TNF-R1 in RNS-induced signaling to JNK. Furthermore, Fas was oxidized, redistributed, and colocalized with Fas-associated death domain (FADD) in RNS-exposed cells, illustrating that RNS directly targeted Fas. JNK activation and cell death by RNS occurred in a Fas ligand- and caspase-independent manner. While the activation of JNK by RNS or FasL required FADD, the cysteine-rich domain 1 containing preligand assembly domain required for FasL signaling was not involved in JNK activation by RNS. These findings illustrate that RNS cause cell death in a Fas- and JNK-dependent manner and that this occurs through a pathway distinct from FasL. Thus, avenues aimed at preventing the interaction of RNS with Fas may attenuate tissue damage characteristic of chronic inflammatory diseases that are accompanied by high levels of RNS.     

Effects of corticosteroid-induced apoptosis on airway epithelial wound closure in vitro

Damage to the airway epithelium is common in asthma. Corticosteroids induce apoptosis in and suppress proliferation of airway epithelial cells in culture. Whether apoptosis contributes to impaired epithelial cell repair after injury is not known. We examined whether corticosteroids would impair epithelial cell migration in an in vitro model of wound closure. Wounds (approximately 0.5-1.3 mm2) were created in cultured 1HAEo- human airway epithelial cell monolayers, after which cells were treated with up to 10 microM dexamethasone or budesonide for 24 h. Cultured cells were pretreated for 24 or 48 h with dexamethasone to observe the effect of long-term exposure on wound closure. After 12 h, the remaining wound area in monolayers pretreated for 48 h with 10 microM dexamethasone was 43+/-18% vs. 10+/-8% for untreated control monolayers. The addition of either corticosteroid immediately after injury did not slow closure significantly. After 12 h the remaining wound area in monolayers treated with 10 microM budesonide was 39+/-4% vs. 43+/-3% for untreated control monolayers. The proportion of apoptotic epithelial cells as measured by terminal deoxynucleotidyltransferase-mediated dUTP biotin nick end labeling both at and away from the wound edge was higher in monolayers treated with budesonide compared with controls. However, wound closure in the apoptosis-resistant 1HAEo-.Bcl-2+ cell line was not different after dexamethasone treatment. We demonstrate that corticosteroid treatment before mechanical wounding impairs airway epithelial cell migration. The addition of corticosteroids after injury does not slow migration, despite their ability to induce apoptosis in these cells.     

H(2)O(2) inhibits alveolar epithelial wound repair in vitro by induction of apoptosis

Reactive oxygen species (ROS) are released into the alveolar space and contribute to alveolar epithelial damage in patients with acute lung injury. However, the role of ROS in alveolar repair is not known. We studied the effect of ROS in our in vitro wound healing model using either human A549 alveolar epithelial cells or primary distal lung epithelial cells. We found that H(2)O(2) inhibited alveolar epithelial repair in a concentration-dependent manner. At similar concentrations, H(2)O(2) also induced apoptosis, an effect seen particularly at the edge of the wound, leading us to hypothesize that apoptosis contributes to H(2)O(2)-induced inhibition of wound repair. To learn the role of apoptosis, we blocked caspases with the pan-caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp (zVAD). In the presence of H(2)O(2), zVAD inhibited apoptosis, particularly at the wound edge and, most importantly, maintained alveolar epithelial wound repair. In H(2)O(2)-exposed cells, zVAD also maintained cell viability as judged by improved cell spreading and/or migration at the wound edge and by a more normal mitochondrial potential difference compared with cells not treated with zVAD. In conclusion, H(2)O(2) inhibits alveolar epithelial wound repair in large part by induction of apoptosis. Inhibition of apoptosis can maintain wound repair and cell viability in the face of ROS. Inhibiting apoptosis may be a promising new approach to improve repair of the alveolar epithelium in patients with acute lung injury.     

Hepatocyte growth factor and other fibroblast secretions modulate the phenotype of human bronchial epithelial cells

The luminal airway surface is lined with epithelial cells that provide a protective barrier from the external environment and clear inhaled pathogens from the lung. To accomplish this important function, human bronchial epithelial (HBE) cells must be able to rapidly regenerate a mucociliary layer of cells following epithelial injury. Whereas epithelial-fibroblast interactions are known to modulate the airway architecture during lung development and repair, little is known about how these two cells interact. Using a primary HBE and lung fibroblast coculture system, we demonstrate that 1) subepithelial fibroblasts provide a suitable environment for differentiation of HBE cells into a polarized ciliated phenotype despite being cultured in media that induces terminal squamous differentiation and growth arrest in the absence of fibroblasts, 2) HBE cells cocultured with subepithelial fibroblasts exhibit augmented ciliogenesis, accelerated wound repair, and diminished polarized ion transport compared with cells grown in control conditions, and 3) hepatocyte growth factor (HGF) is important for subepithelial fibroblast modulation of HBE cell differentiation. These results provide a model to study fibroblast modulation of epithelial phenotype and indicate that HGF secreted by subepithelial fibroblasts contributes to HBE cell differentiation.     

Time course of airway remodelling after an acute chlorine gas exposure in mice

Accidental chlorine (Cl₂) gas inhalation is a common cause of acute airway injury. However, little is known about the kinetics of airway injury and repair after Cl₂ exposure. We investigated the time course of airway epithelial damage and repair in mice after a single exposure to a high concentration of Cl₂ gas. Mice were exposed to 800 ppm Cl₂ gas for 5 minutes and studied from 12 hrs to 10 days post-exposure. The acute injury phase after Cl₂ exposure (≤ 24 hrs post-exposure) was characterized by airway epithelial cell apoptosis (increased TUNEL staining) and sloughing, elevated protein in bronchoalveolar lavage fluid, and a modest increase in airway responses to methacholine. The repair phase after Cl₂ exposure was characterized by increased airway epithelial cell proliferation, measured by immunoreactive proliferating cell nuclear antigen (PCNA), with maximal proliferation occurring 5 days after Cl₂ exposure. At 10 days after Cl₂ exposure the airway smooth muscle mass was increased relative to controls, suggestive of airway smooth muscle hyperplasia and there was evidence of airway fibrosis. No increase in goblet cells occurred at any time point. We conclude that a single exposure of mice to Cl₂ gas causes acute changes in lung function, including pulmonary responsiveness to methacholine challenge, associated with airway damage, followed by subsequent repair and airway remodelling.     

Dose dependent effects of reactive oxygen and nitrogen species on the function of neuronal nitric oxide synthase

Reactive nitrogen species (RNS) and oxygen species (ROS) have been reported to modulate the function of nitric oxide synthase (NOS); however, the precise dose-dependent effects of specific RNS and ROS on NOS function are unknown. Questions remain unanswered regarding whether pathophysiological levels of RNS and ROS alter NOS function, and if this alteration is reversible. We measured the effects of peroxynitrite (ONOO-), superoxide (O2.-), hydroxyl radical (.OH), and H2O2 on nNOS activity. The results showed that NO production was inhibited in a dose-dependent manner by all four oxidants, but only O2.- and ONOO- were inhibitory at pathophysiological concentrations (50muM). Subsequent addition of tetrahydrobiopterin (BH4) fully restored activity after O2.- exposure, while BH4 partially rescued the activity decrease induced by the other three oxidants. Furthermore, treatment with either ONOO- or O2.- stimulated nNOS uncoupling with decreased NO and enhanced O2.- generation. Thus, nNOS is reversibly uncoupled by O2.- (50muM), but irreversibly uncoupled and inactivated by ONOO-. Additionally, we observed that the mechanism by which oxidative stress alters nNOS activity involves not only BH4 oxidation, but also nNOS monomerization as well as possible degradation of the heme.