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Both high tidal volume mechanical ventilation (HV) and hyperoxia (HO) have been implicated in ventilator-induced lung injury. However, patients with acute lung injury are often exposed to HO before the application of mechanical ventilation. The potential priming of the lungs for subsequent injury by exposure to HO has not been extensively studied. We provide evidence that HO (90%) for 12 h followed by HV (25 [micro]l/g) combined with HO for 2 or 4 h (HO-12h+HVHO-2h or -4h) induced severe lung injury in mice. Analysis of lung homogenates showed that lung injury was associated with cleavage of executioner caspases, caspases-3 and -7, and their downstream substrate poly(ADP-ribose) polymerase-1 (PARP-1). No significant lung injury or caspase cleavage was seen with either HO for 16 h or HV for up to 4 h. Ventilation for 4 h with HO (HVHO) did not cause significant lung injury without preexposure to HO. Twelve-hour HO followed by lower tidal volume (6 [micro]l/g) mechanical ventilation failed to produce significant injury or caspase cleavage. We also evaluated the initiator caspases, caspases-8 and -9, to determine whether the death receptor or mitochondrial-mediated pathways were involved. Caspase-9 cleavage was observed in HO-12h+HVHO-2h and -4h as well as HO for 16 h. Caspase-8 activation was observed only in HO-12h+HVHO-4h, indicating the involvement of both pathways. Immunohistochemistry and in vitro stretch studies showed caspase cleavage in alveolar epithelial cells. In conclusion, preexposure to HO followed by HV produced severe lung injury associated with alveolar epithelial cell apoptosis. mechanical ventilation; mouse doi: 10.1152/ajplung.00072.2010.

Patients with acute respiratory distress syndrome undergoing mechanical ventilation may be exposed to both high levels of stretch and high levels of oxygen. We hypothesized that the combination of high stretch and hyperoxia promotes loss of epithelial adhesion and impairs epithelial repair mechanisms necessary for restoration of barrier function. We utilized a model of high tidal volume mechanical ventilation (25 ml/kg) with hyperoxia (50% 02) in rats to investigate alveolar type II (AT2) cell adhesion and focal adhesion signaling. AT2 cells isolated from rats exposed to hyperoxia and high tidal volume mechanical ventilation (MVHO) exhibited significantly decreased cell adhesion and reduction in phosphotyrosyl levels of local adhesion kinase (FAK) and paxillin compared with control rats, rats exposed to hyperoxia without ventilation (HO), or rats ventilated with normoxia (MV). MV alone increased phosphorylation of [p130.sup.Cas] RhoA activation was increased by MV, HO, and the combination of MV and HO. Treatment of MVHO cells with keratinocyte growth factor (KGF) for 1 h upon isolation reduced RhoA activity and restored attachment to control levels. Attachment and migration of control AT2 cells was significantly decreased by constitutively active RhoA or a kinase inactive form of FAK (FRNK), whereas expression of dominant negative RhoA in cells from MVHO-treated rats restored cell adhesion. Mechanical ventilation with hyperoxia promotes changes in focal adhesion proteins and RhoA in AT2 cells that may be deleterious for cell adhesion and migration. keratinocyte growth factor; RhoA; focal adhesion kinase

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 ([O.sub.2]) was measured by dihydroethidium fluorescence. CMS stimulated increased production of [O.sup.-.sub.2] 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 [O.sup.-.sub.2] production in 16HBE cells, whereas 15, 20, and 30% significantly increased generation of [O.sup.-.sub.2]. Rotenone, a mitochondrial complex I inhibitor, partially abrogated the stretch-induced generation of [O.sup.-.sub.2] after 2 h CMS in 16HBE cells. NADPH oxidase activity was increased after 2 h of CMS, contributing to the production of [O.sup.-.sub.2]. Increased ROS production in lung epithelial cells in response to elevated stretch may contribute to the onset of VILI. mechanotransduction; ventilator-induced lung injury

Alveolar type II (ATII) epithelial cells play a crucial role in the repair and remodeling of the lung following injury. ATII cells have the capability to proliferate and differentiate into alveolar type I (ATI) cells in vivo and into an ATI-like phenotype in vitro. While previous reports indicate that the differentiation of ATII cells into ATI cells is a complex biological process, the underlying mechanism responsible for differentiation is not fully understood. To investigate factors involved in this differentiation in culture, we used a PCR array and identified several genes that were either up- or downregulated in ATI-like cells (day 6 in culture) compared with day 2 ATII cells. Insulin-like growth factor-I (IGF-I) mRNA was increased nearly eightfold. We found that IGF-I was increased in the culture media of ATI-like cells and demonstrated a significant role in the differentiation process. Treatment of ATII cells with recombinant IGF-I accelerated the differentiation process, and this effect was abrogated by the IGF-I receptor blocker PQ401. We found that Wnt5a, a member of the Wnt-Frizzled pathway, was activated during IGF-I-mediated differentiation. Both protein kinase C and catenin were transiently activated during transdifferentiation. Knocking down Wnt5a using smallinterfering RNA abrogated the differentiation process as indicated by changes in the expression of an ATII cell marker (prosurfactant protein-C). Treatment of wounded cells with either IGF-I or Wnt5a stimulated wound closure. These results suggest that IGF-I promotes differentiation of ATII to ATI cells through the activation of a noncanonical Wnt pathway. [ABSTRACT FROM AUTHOR]

Both prolonged exposure to hyperoxia and large tidal volume mechanical ventilation can each independently cause lung injury. However, the combined impact of these insults is poorly understood. We recently reported that preexposure to hyperoxia for 12 h, followed by ventilation with large tidal volumes, induced significant lung injury and epithelial cell apoptosis compared with either stimulus alone (Makena et al. Am J Physiol Lung Cell Mol Physiol 299:L711-L719, 2010). The upstream mechanisms of this lung injury and apoptosis have not been clearly elucidated. We hypothesized that lung injury in this model was dependent on oxidative signaling via the c-Jun NH2-terminal kinases (JNK). We, therefore, evaluated lung injury and apoptosis in the presence of N-acetyl-cysteine (NAC) in both mouse and cell culture models, and we provide evidence that NAC significantly inhibited lung injury and apoptosis by reducing the production of ROS, activation of JNK, and apoptosis. To confirm JNK involvement in apoptosis, cells treated with a specific JNK inhibitor, SP600125, and subjected to preexposure to hyperoxia, followed by mechanical stretch, exhibited significantly reduced evidence of apoptosis. In conclusion, lung injury and apoptosis caused by preexposure to hyperoxia, followed by high tidal volume mechanical ventilation, induces ROS-mediated activation of JNK and mitochondrial-mediated apoptosis. NAC protects lung injury and apoptosis by inhibiting ROS-mediated activation of JNK and downstream proapoptotic signaling. [ABSTRACT FROM AUTHOR]

Airway narrowing due to hyperresponsiveness severely limits gas exchange in patients with asthma. Imaging studies in humans and animals have shown that bronchoconstriction causes patchy patterns of ventilation defects throughout the lungs, and several computational models have predicted that these regions are due to constriction of smaller airways. However, these imaging approaches are often limited in their ability to capture dynamic changes in small airways, and the patterns of constriction are heterogeneous. To directly investigate regional variations in airway narrowing and the response to deep inspirations (DIs), we utilized tantalum dust and microfocal X-ray imaging of rat lungs to obtain dynamic images of airways in an intact animal model. Airway resistance was simultaneously measured using the flexiVent system. Custom-developed software was used to track changes in airway diameters up to generation 19 (~0.3-3 mm). Changes in diameter during bronchoconstriction were then measured in response to methacholine (MCh) challenge. In contrast with the model predictions, we observed significantly greater percent constriction in larger airways in response to MCh challenge. Although there was a dose-dependent increase in total respiratory resistance with MCh, the percent change in airway diameters was similar for increasing doses. A single DI following MCh caused a significant reduction in resistance but did not cause a significant increase in airway diameters. Multiple DIs did, however, cause significant increases in airway diameters. These measurements allowed us to directly quantify dynamic changes in airways during bronchoconstriction and demonstrated greater constriction in larger airways.

Alveolar type II (ATII) epithelial cells play a crucial role in the repair and remodeling of the lung following injury. ATII cells have the capability to proliferate and differentiate into alveolar type I (ATI) cells in vivo and into an ATI-like phenotype in vitro. While previous reports indicate that the differentiation of ATII cells into ATI cells is a complex biological process, the underlying mechanism responsible for differentiation is not fully understood. To investigate factors involved in this differentiation in culture, we used a PCR array and identified several genes that were either up- or downregulated in ATI-like cells (day 6 in culture) compared with day 2 ATII cells. Insulin-like growth factor-I (IGF-I) mRNA was increased nearly eightfold. We found that IGF-I was increased in the culture media of ATI-like cells and demonstrated a significant role in the differentiation process. Treatment of ATII cells with recombinant IGF-I accelerated the differentiation process, and this effect was abrogated by the IGF-I receptor blocker PQ401. We found that Wnt5a, a member of the Wnt-Frizzled pathway, was activated during IGF-I-mediated differentiation. Both protein kinase C and β-catenin were transiently activated during transdifferentiation. Knocking down Wnt5a using small-interfering RNA abrogated the differentiation process as indicated by changes in the expression of an ATII cell marker (prosurfactant protein-C). Treatment of wounded cells with either IGF-I or Wnt5a stimulated wound closure. These results suggest that IGF-I promotes differentiation of ATII to ATI cells through the activation of a noncanonical Wnt pathway.

Patients with severe acute lung injury are frequently administered high concentrations of oxygen (>50%) during mechanical ventilation. Long-term exposure to high levels of oxygen can cause lung injury in the absence of mechanical ventilation, but the combination of the two accelerates and increases injury. Hyperoxia causes injury to cells through the generation of excessive reactive oxygen species. However, the precise mechanisms that lead to epithelial injury and the reasons for increased injury caused by mechanical ventilation are not well understood. We hypothesized that alveolar epithelial cells (AECs) may be more susceptible to injury caused by mechanical ventilation if hyperoxia alters the mechanical properties of the cells causing them to resist deformation. To test this hypothesis, we used atomic force microscopy in the indentation mode to measure the mechanical properties of cultured AECs. Exposure of AECs to hyperoxia for 24 to 48 h caused a significant increase in the elastic modulus (a measure of resistance to deformation) of both primary rat type II AECs and a cell line of mouse AECs (MLE-12). Hyperoxia also caused remodeling of both actin and microtubules. The increase in elastic modulus was blocked by treatment with cytochalasin D. Using finite element analysis, we showed that the increase in elastic modulus can lead to increased stress near the cell perimeter in the presence of stretch. We then demonstrated that cyclic stretch of hyperoxia-treated cells caused significant cell detachment. Our results suggest that exposure to hyperoxia causes structural remodeling of AECs that leads to decreased cell deformability.

Restoration of the epithelial barrier following acute lung injury is critical for recovery of lung homeostasis. After injury, alveolar type II epithelial (ATII) cells spread and migrate to cover the denuded surface and, eventually, proliferate and differentiate into type I cells. The chemokine CXCL12, also known as stromal cell-derived factor 1α, has well-recognized roles in organogenesis, hematopoiesis, and immune responses through its binding to the chemokine receptor CXCR4. While CXCL12/CXCR4 signaling is known to be important in immune cell migration, the role of this chemokine-receptor interaction has not been studied in alveolar epithelial repair mechanisms. In this study, we demonstrated that secretion of CXCL12 was increased in the bronchoalveolar lavage of rats ventilated with an injurious tidal volume (25 ml/kg). We also found that CXCL12 secretion was increased by primary rat ATII cells and a mouse alveolar epithelial (MLE12) cell line following scratch wounding and that both types of cells express CXCR4. CXCL12 significantly increased ATII cell migration in a scratch-wound assay. When we treated cells with a specific antagonist for CXCR4, AMD-3100, cell migration was significantly inhibited. Knockdown of CXCR4 by short hairpin RNA (shRNA) caused decreased cell migration compared with cells expressing a nonspecific shRNA. Treatment with AMD-3100 decreased matrix metalloproteinase-14 expression, increased tissue inhibitor of metalloproteinase-3 expression, decreased matrix metalloproteinase-2 activity, and prevented CXCL12-induced Rac1 activation. Similar results were obtained with shRNA knockdown of CXCR4. These findings may help identify a therapeutic target for augmenting epithelial repair following acute lung injury.

Hyperoxia can lead to a myriad of deleterious effects in the lung including epithelial damage and diffuse inflammation. The specific mechanisms by which hyperoxia promotes these pathological changes are not completely understood. Activation of ion channels has been proposed as one of the mechanisms required for cell activation and mediator secretion. The two-pore-domain K(+) channel (K2P) Trek-1 has recently been described in lung epithelial cells, but its function remains elusive. In this study we hypothesized that hyperoxia affects expression of Trek-1 in alveolar epithelial cells and that Trek-1 is involved in regulation of cell proliferation and cytokine secretion. We found gene expression of several K2P channels in mouse alveolar epithelial cells (MLE-12), and expression of Trek-1 was significantly downregulated in cultured cells and lungs of mice exposed to hyperoxia. Similarly, proliferation cell nuclear antigen (PCNA) and Cyclin D1 expression were downregulated by exposure to hyperoxia. We developed an MLE-12 cell line deficient in Trek-1 expression using shRNA and found that Trek-1 deficiency resulted in increased cell proliferation and upregulation of PCNA but not Cyclin D1. Furthermore, IL-6 and regulated on activation normal T-expressed and presumably secreted (RANTES) secretion was decreased in Trek-1-deficient cells, whereas release of monocyte chemoattractant protein-1 was increased. Release of KC/IL-8 was not affected by Trek-1 deficiency. Overall, deficiency of Trek-1 had a more pronounced effect on mediator secretion than exposure to hyperoxia. This is the first report suggesting that the K(+) channel Trek-1 could be involved in regulation of alveolar epithelial cell proliferation and cytokine secretion, but a direct association with hyperoxia-induced changes in Trek-1 levels remains elusive.

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.