bluuebunny/crossref_metadata_2025 huggingface dataset:
Acid aspiration increases sensitivity to increased ambient oxygen concentrations
Authors: Knight Paul R. , Kurek Carlos , Davidson Bruce A. , Nader Nader D. , Patel Alka , Sokolowski June , Notter R. H. , Holm Bruce A.
Published: December 2017

Previously we have demonstrated that prolonged exposure to 100% ambient oxygen leads to a marked loss in functional lung volume and lung compliance, hypoxemia, and surfactant system abnormalities similar to acute respiratory distress syndrome (ARDS). However, 50% oxygen administration is believed to be safe in most clinical settings. In the present study, we have evaluated the effects of a 24-h exposure to 50% oxygen in rabbits immediately following experimental gastric acid aspiration. Mild hypoxemia, but no changes in mortality, lung volume, lung compliance, surfactant metabolism, or edema formation occurred after 24 h of normoxia postacid aspiration. Conversely, a relatively short (24-h) exposure to 50% oxygen after acid aspiration results in increased pulmonary edema, physical signs of respiratory distress, and mortality, as well as decreased arterial oxygenation, lung volume, lung compliance, and type II alveolar cell surfactant synthesis. These results suggest that acid aspiration alters the “set point” for oxygen toxicity, possibly by “priming” cells through activation of inflammatory pathways. This pathogenic mechanism may contribute to the progression of aspiration pneumonia to ARDS.
Reduced lung endothelial angiotensin-converting enzyme activity in Watanabe hyperlipidemic rabbits in vivo
Authors: Orfanos Stylianos E. , Parkerson James B. , Chen Xilin , Fisher Eugene L. , Glynos Constantinos , Papapetropoulos Andreas , Gerrity Ross G. , Catravas John D.
Published: December 2017

We investigated pulmonary endothelial function in vivo in 12- to 18-mo-old male Watanabe heritable hyperlipidemic (WHHL; n = 7) and age- and sex-matched New Zealand White ( n = 8) rabbits. The animals were anesthetized and artificially ventilated, and the chest was opened and put in total heart bypass. The single-pass transpulmonary utilizations of the angiotensin-converting enzyme (ACE) substrate [3H]benzoyl-Phe-Ala-Pro (BPAP) and the 5′-nucleotidase (NCT) substrate [14C]AMP were estimated, and the first-order reaction parameter A max/ K m, where A max is the product of enzyme mass and the catalytic rate constant and K m is the Michaelis-Menten constant, was calculated. BPAP transpulmonary utilization and A max/ K m were reduced in WHHL (1.69 ± 0.16 vs. 2.9 ± 0.44 and 599 ± 69 vs. 987 ± 153 ml/min in WHHL and control rabbits, respectively; P < 0.05 for both). No differences were observed in the AMP parameters. BPAP K m and A max values were estimated separately under mixed-order reaction conditions. No differences in K m values were found (9.79 ± 1 vs. 9.9 ± 1.31 μM), whereas WHHL rabbit A max was significantly decreased (5.29 ± 0.88 vs. 7.93 ± 0.8 μmol/min in WHHL and control rabbits, respectively; P < 0.05). We conclude that the observed pulmonary endothelial ACE activity reduction in WHHL rabbits appears related to a decrease in enzyme mass rather than to alterations in enzyme affinity.
Comparison of SP-A and LPS effects on the THP-1 monocytic cell line
Authors: Song Mingchen , Phelps David S.
Published: December 2017

Surfactant protein A (SP-A) increases production of proinflammatory cytokines by monocytic cells, including THP-1 cells, as does lipopolysaccharide (LPS). Herein we report differences in responses to these agents. First, polymyxin B inhibits the LPS response but not the SP-A response. Second, SP-A-induced increases in tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-8 are reduced by >60% if SP-A is preincubated with Survanta (200 μg/ml) for 15 min before addition to THP-1 cells. However, the LPS effects on TNF-α and IL-8 are inhibited by <20% and the effect on IL-1β by <50%. Third, at Survanta levels of 1 mg/ml, SP-A-induced responses are reduced by >90%, and although the inhibitory effects on LPS action increase, they still do not reach those seen with SP-A. Finally, we tested whether SP-A could induce tolerance as LPS does. Pretreatment of THP-1 cells with LPS inhibits their response to subsequent LPS treatment 24 h later, including TNF-α, IL-1β, and IL-8. Similar treatment with SP-A reduces TNF-α, but IL-1β and IL-8 are further increased by the second treatment with SP-A rather than inhibited as with LPS. Thus, whereas both SP-A and LPS stimulate cytokine production, their mechanisms differ with respect to inhibition by surfactant lipids and in ability to induce tolerance.
Regenerative growth of respiratory bronchioles in dogs
Authors: Hsia C. C. W. , Zhou X. S. , Bellotto D. J. , Hagler H. K.
Published: December 2017

Loss of lung units due to pneumonectomy stimulates growth of the remaining lung. It is generally believed that regenerative lung growth involves only alveoli but not airways, a dissociated response termed “dysanaptic growth.” We examined the structural response of respiratory bronchioles in immature dogs raised to maturity after right pneumonectomy. In another group of adult dogs, we also examined the effect of preventing mediastinal shift after right pneumonectomy on the response of respiratory bronchioles. In immature dogs after pneumonectomy, the volume of the remaining lung increased twofold, with no change in volume density, numerical density, or mean diameter of respiratory bronchiole, compared with that in the control lung. The number of respiratory bronchiole segments and branch points increased proportionally with lung volume. In adult dogs after pneumonectomy, prevention of mediastinal shift reduced lung strain at a given airway pressure, but lung expansion and regenerative growth of respiratory bronchiole were not eliminated. We conclude that postpneumonectomy lung growth is associated with proliferation of intra-acinar airways. The proportional growth of acinar airways and alveoli should optimize gas exchange of the regenerated lung by enhancing gas conductance and mixing efficiency within the acinus.
Integrin α3-subunit expression modulates alveolar epithelial cell monolayer formation
Authors: Lubman Richard L. , Zhang Xiao-Ling , Zheng Jie , Ocampo Leah , Lopez Melissa Z. , Veeraraghavan Srihari , Zabski Stephanie M. , Danto Spencer I. , Borok Zea
Published: December 2017

We investigated expression of the α3-integrin subunit by rat alveolar epithelial cells (AECs) grown in primary culture as well as the effects of monoclonal antibodies with blocking activity against the α3-integrin subunit on AEC monolayer formation. α3-Integrin subunit mRNA and protein were detectable in AECs on day 1 and increased with time in culture. α3- and β1-integrin subunits coprecipitated in immunoprecipitation experiments with α3- and β1-subunit-specific antibodies, consistent with their association as the α3β1-integrin receptor at the cell membrane. Treatment with blocking anti-α3monoclonal antibody from day 0 delayed development of transepithelial resistance, reduced transepithelial resistance through day 5 compared with that in untreated AECs, and resulted in large subconfluent patches in monolayers viewed by scanning electron microscopy on day 3. These data indicate that α3- and β1-integrin subunits are expressed in AEC monolayers where they form the heterodimeric α3β1-integrin receptor at the cell membrane. Blockade of the α3-integrin subunit inhibits formation of confluent AEC monolayers. We conclude that the α3-integrin subunit modulates formation of AEC monolayers by virtue of the key role of the α3β1-integrin receptor in AEC adhesion.
TGF-β1 stimulates IL-8 release, COX-2 expression, and PGE2release in human airway smooth muscle cells
Authors: Fong Choong Yi , Pang Linhua , Holland Elaine , Knox Alan J.
Published: December 2017

We have recently shown that endogenous prostanoids are critical in bradykinin-stimulated interleukin (IL)-8 release from human airway smooth muscle (ASM) cells. In this study, we tested the ability of transforming growth factor (TGF)-β1 to stimulate IL-8 release, cyclooxygenase (COX)-2 expression and PGE2generation in cultured human ASM cells and explored the role of COX products and COX-2 induction on IL-8 release. TGF-β1 stimulated IL-8 release, COX-2 induction, and PGE2generation in a concentration- and time-dependent manner. Maximal IL-8 release was achieved with 10 ng/ml of TGF-β1 after 16 h of incubation, which was inhibited by the transcription inhibitor actinomycin D and the corticosteroid dexamethasone but was not affected by the nonselective COX inhibitor indomethacin and the selective COX-2 inhibitor NS-398 despite their inhibition on TGF-β1-induced PGE2release. These results show for the first time that TGF-β1 stimulates IL-8 release, COX-2 induction, and PGE2generation in human ASM cells and that PGE2generation is not critical for TGF-β1-induced IL-8 release. These findings suggest that TGF-β1 may play an important role in the pathophysiology of asthma.
Mechanical strain-induced proliferation and signaling in pulmonary epithelial H441 cells
Authors: Chess Patricia R. , Toia Liana , Finkelstein Jacob N.
Published: December 2017

Pulmonary epithelial cells are exposed to mechanical strain during physiological breathing and mechanical ventilation. Strain regulates pulmonary growth and development and is implicated in volutrauma-induced fibrosis. The mechanisms of strain-induced effects are not well understood. It was hypothesized that mechanical strain induces proliferation of pulmonary epithelial cells and that this is mediated by signals initiated within seconds of strain. To test this hypothesis, human pulmonary adenocarcinoma H441 cells were strained in vitro. Cyclic as well as tonic strain resulted in increased cellular proliferation. Western blot analysis of strained cells demonstrated three newly phosphorylated tyrosine residues within 30 s of strain. Phosphorylation of mitogen-activated protein kinases p42/44 increased, electrophoretic mobility shift assay demonstrated activation of transcription factor activating protein-1, and immunohistochemistry demonstrated increased phosphorylation of c- jun in response to strain. The tyrosine kinase inhibitor genistein blocked the strain-induced proliferation. We conclude that strain induces proliferation in pulmonary epithelial cells and that tyrosine kinase activity is necessary to signal the proliferative response to mechanical strain.
[Ca2+]i oscillations regulate type II cell exocytosis in the pulmonary alveolus
Authors: Ashino Yugo , Ying Xiaoyou , Dobbs Leland G. , Bhattacharya Jahar
Published: December 2017

Pulmonary surfactant, a critical determinant of alveolar stability, is secreted by alveolar type II cells by exocytosis of lamellar bodies (LBs). To determine exocytosis mechanisms in situ, we imaged single alveolar cells from the isolated blood-perfused rat lung. We quantified cytosolic Ca2+ concentration ([Ca2+]i) by the fura 2 method and LB exocytosis as the loss of cell fluorescence of LysoTracker Green. We identified alveolar cell type by immunofluorescence in situ. A 15-s lung expansion induced synchronous [Ca2+]i oscillations in all alveolar cells and LB exocytosis in type II cells. The exocytosis rate correlated with the frequency of [Ca2+]i oscillations. Fluorescence of the lipidophilic dye FM1-43 indicated multiple exocytosis sites per cell. Intracellular Ca2+ chelation and gap junctional inhibition each blocked [Ca2+]i oscillations and exocytosis in type II cells. We demonstrated the feasibility of real-time quantifications in alveolar cells in situ. We conclude that in lung expansion, type II cell exocytosis is modulated by the frequency of intercellularly communicated [Ca2+]i oscillations that are likely to be initiated in type I cells. Thus during lung inflation, type I cells may act as alveolar mechanotransducers that regulate type II cell secretion.
Relationship between force and regulatory myosin light chain phosphorylation in airway smooth muscle
Authors: Kai Tetsuya , Yoshimura Hayashi , Jones Keith A. , Warner David O.
Published: December 2017

We tested the hypothesis that increases in force at a given cytosolic Ca2+concentration (i.e., Ca2+sensitization) produced by muscarinic stimulation of canine tracheal smooth muscle (CTSM) are produced in part by mechanisms independent of changes in regulatory myosin light chain (rMLC) phosphorylation. This was accomplished by comparing the relationship between rMLC phosphorylation and force in α-toxin-permeabilized CTSM in the absence and presence of acetylcholine (ACh). Forces were normalized to the contraction induced by 10 μM Ca2+in each strip, and rMLC phosphorylation is expressed as a percentage of total rMLC. ACh (100 μM) plus GTP (1 μM) significantly shifted the Ca2+-force relationship curve to the left (EC50: 0.39 ± 0.06 to 0.078 ± 0.006 μM Ca2+) and significantly increased the maximum force (104.4 ± 4.8 to 120.2 ± 2.8%; n = 6 observations). The Ca2+-rMLC phosphorylation relationship curve was also shifted to the left (EC50: 1.26 ± 0.57 to 0.13 ± 0.04 μM Ca2+) and upward (maximum rMLC phosphorylation: 70.9 ± 7.9 to 88.5 ± 5.1%; n = 6 observations). The relationships between rMLC phosphorylation and force constructed from mean values at corresponding Ca2+concentrations were not different in the presence and absence of ACh. We find no evidence that muscarinic stimulation increases Ca2+sensitivity in CTSM by mechanisms other than increases in rMLC phosphorylation.
Effects of neurokinin receptor antagonists in virus-infected airways
Authors: Jacoby David B. , Yost Bethany L. , Elwood Thomas , Fryer Allison D.
Published: December 2017

We investigated the effects of a neurokinin-1 (NK1) receptor antagonist (SR-140333) and a NK2receptor antagonist (SR-48968) on airway responsiveness and on the function of neuronal M2muscarinic receptors, which normally inhibit vagal acetylcholine release, in guinea pigs infected with parainfluenza virus. Antagonists were given 1 h before infection and daily thereafter. Four days later, bronchoconstriction induced by either intravenous histamine (which is partly vagally mediated) or electrical stimulation of the vagus nerves was increased by viral infection compared with control. In addition, the ability of the muscarinic agonist pilocarpine to inhibit vagally induced bronchoconstriction was lost in virus-infected animals, demonstrating loss of neuronal M2receptor function. Macrophage influx into the lungs was inhibited by pretreatment with both antagonists. However, only the NK1receptor antagonist prevented M2receptor dysfunction and inhibited hyperresponsiveness (measured as an increase in either vagally induced or histamine-induced bronchoconstriction). Thus virus-induced M2receptor dysfunction and hyperresponsiveness are prevented by a NK1receptor antagonist, but not by a NK2receptor antagonist, whereas both antagonists had similar anti-inflammatory effects.
Altered intracellular pH regulation in neutrophils from patients with cystic fibrosis
Authors: Coakley Raymond J. , Taggart Clifford , Canny Gerry , Greally Peter , O'Neill Shane J. , McElvaney Noel G.
Published: December 2017

Cystic fibrosis (CF) is a condition characterized by neutrophil-mediated lung damage and bacterial colonization. The physiological basis for reported functional alterations in CF neutrophils, including increased release of neutrophil elastase, myeloperoxidase, and oxidants, is unknown. These processes are, however, regulated by intracellular pH (pHi). We demonstrate here that pHiregulation is altered in neutrophils from CF patients. Although resting pHiis similar, pHiafter acid loading and activation ( N-formyl-methionyl-leucyl-phenylalanine and phorbol 12-myristate 13-acetate) is more acidic in CF cells than in normal cells. Furthermore, patients with non-CF-related bronchiectasis handle acid loading and activation in a fashion similar to subjects with normal neutrophils, suggesting that chronic pulmonary inflammation alone does not explain the difference in pHi. This is further supported by data showing that normal neutrophils exposed to the CF pulmonary milieu respond by increasing pHias opposed to decreasing pHias seen in activated CF neutrophils. These pHidifferences in activated or acid-loaded CF neutrophils are abrogated by ZnCl2but not by amiloride and bafilomycin A1, suggesting that passive proton conductance is abnormal in CF. In addition, DIDS, which inhibits HCO3−/Cl−exchange, causes alkalinization of control but not of CF neutrophils, suggesting that anion transport is also abnormal in CF neutrophils. In summary, we have shown that pHiregulation in CF neutrophils is intrinsically abnormal, potentially contributing to the pulmonary manifestations of the condition.
Different pathways of degradation of SP-A and saturated phosphatidylcholine by alveolar macrophages
Authors: Baritussio Aldo , Alberti Antonella , Armanini Decio , Meloni Federica , Bruttomesso Daniela
Published: December 2017

Alveolar macrophages degrade surfactant protein (SP) A and saturated phosphatidycholine [dipalmitoylphosphatidylcholine (DPPC)]. To clarify this process, using rabbit alveolar macrophages, we analyzed the effect of drugs known to affect phagocytosis, pinocytosis, clathrin-mediated uptake, caveolae, the cytoskeleton, lysosomal pH, protein kinase C, and phosphatidylinositol 3-kinase (PI3K) on the degradation of SP-A and DPPC. We found the following: 1) SP-A binds to the plasma membrane, is rapidly internalized, and then moves toward degradative compartments. Uptake could be clathrin mediated, whereas phagocytosis, pinocytosis, or the use of caveolae are less likely. An intact cytoskeleton and an acidic milieu are necessary for the degradation of SP-A. 2) Stimulation of protein kinase C increases the degradation of SP-A. 3) PI3K influences the degradation of SP-A by regulating both the speed of internalization and subsequent intracellular steps, but its inhibition does not prevent SP-A from reaching the lysosomal compartment. 4) The degradation of DPPC is unaffected by most of the treatments able to influence the degradation of SP-A. Thus it appears that DPPC is degraded by alveolar macrophages through mechanisms very different from those utilized for the degradation of SP-A.
Gene expression and function of adenosine A2A receptor in the rat carotid body
Authors: Kobayashi Shuichi , Conforti Laura , Millhorn David E.
Published: December 2017

The present study was undertaken to determine whether rat carotid bodies express adenosine (Ado) A2A receptors and whether this receptor is involved in the cellular response to hypoxia. Our results demonstrate that rat carotid bodies express the A2A and A2B Ado receptor mRNAs but not the A1 or A3 receptor mRNAs as determined by reverse transcriptase-polymerase chain reaction. In situ hybridization confirmed the expression of the A2A receptor mRNA. Immunohistochemical studies further showed that the A2A receptor is expressed in the carotid body and that it is colocalized with tyrosine hydroxylase in type I cells. Whole cell voltage-clamp studies using isolated type I cells showed that Ado inhibited the voltage-dependent Ca2+ currents and that this inhibition was abolished by the selective A2A receptor antagonist ZM-241385. Ca2+ imaging studies using fura 2 revealed that exposure to severe hypoxia induced elevation of intracellular Ca2+ concentration ([Ca2+]i) in type I cells and that extracellularly applied Ado significantly attenuated the hypoxia-induced elevation of [Ca2+]i. Taken together, our findings indicate that A2A receptors are present in type I cells and that activation of A2Areceptors modulates Ca2+ accumulation during hypoxia. This mechanism may play a role in regulating intracellular Ca2+homeostasis and cellular excitability during hypoxia.
Activation of NF-κB induced by H2O2and TNF-α and its effects on ICAM-1 expression in endothelial cells
Authors: True Andrea L. , Rahman Arshad , Malik Asrar B.
Published: December 2017

Reactive oxygen species have been proposed to signal the activation of the transcription factor nuclear factor (NF)-κB in response to tumor necrosis factor (TNF)-α challenge. In the present study, we investigated the effects of H2O2and TNF-α in mediating activation of NF-κB and transcription of the intercellular adhesion molecule (ICAM)-1 gene. Northern blot analysis showed that TNF-α exposure of human dermal microvascular endothelial cells (HMEC-1) induced marked increases in ICAM-1 mRNA and cell surface protein expression. In contrast, H2O2added at subcytolytic concentrations failed to activate ICAM-1 expression. Challenge with H2O2also failed to induce NF-κB-driven reporter gene expression in the transduced HMEC-1 cells, whereas TNF-α increased the NF-κB-driven gene expression ∼10-fold. Gel supershift assay revealed the presence of p65 (Rel A), p50, and c-Rel in both H2O2- and TNF-α-induced NF-κB complexes bound to the ICAM-1 promoter, with the binding of the p65 subunit being the most prominent. In vivo phosphorylation studies, however, showed that TNF-α exposure induced marked phosphorylation of NF-κB p65 in HMEC-1 cells, whereas H2O2had no effect. These results suggest that reactive oxygen species generation in endothelial cells mediates the binding of NF-κB to nuclear DNA, whereas TNF-α generates additional signals that induce phosphorylation of the bound NF-κB p65 and confer transcriptional competency to NF-κB.
Mice that overexpress Cu/Zn superoxide dismutase are resistant to allergen-induced changes in airway control
Authors: Larsen Gary L. , White Carl W. , Takeda Katsuyuki , Loader Joan E. , Nguyen Dee Dee H. , Joetham Anthony , Groner Yoram , Gelfand Erwin W.
Published: December 2017

Within the respiratory epithelium of asthmatic patients, copper/zinc-containing superoxide dismutase (Cu/Zn SOD) is decreased. To address the hypothesis that lung Cu/Zn SOD protects against allergen-induced injury, wild-type and transgenic mice that overexpress human Cu/Zn SOD were either passively sensitized to ovalbumin (OVA) or actively sensitized by repeated airway exposure to OVA. Controls included nonsensitized wild-type and transgenic mice given intravenous saline or airway exposure to saline. After aerosol challenge to saline or OVA, segments of tracheal smooth muscle were obtained for in vitro analysis of neural control. In response to electrical field stimulation, wild-type sensitized mice challenged with OVA had significant increases in cholinergic reactivity. Conversely, sensitized transgenic mice challenged with OVA were resistant to changes in neural control. Stimulation of tracheal smooth muscle to elicit acetylcholine release showed that passively sensitized wild-type but not transgenic mice released more acetylcholine after OVA challenge. Function of the M2muscarinic autoreceptor was preserved in transgenic mice. These results demonstrate that murine airways with elevated Cu/Zn SOD were resistant to allergen-induced changes in neural control.
Involvement of c-Src in diperoxovanadate-induced endothelial cell barrier dysfunction
Authors: Shi Shu , Garcia Joe G. N. , Roy Shukla , Parinandi Narasimham L. , Natarajan Viswanathan
Published: December 2017

Reactive oxygen species (ROS) generated by activated leukocytes play an important role in the disruption of endothelial cell (EC) integrity, leading to barrier dysfunction and pulmonary edema. Although ROS modulate cell signaling, information remains limited regarding the mechanism(s) of ROS-induced EC barrier dysfunction. We utilized diperoxovanadate (DPV) as a model agent to explore the role of tyrosine phosphorylation in the regulation of EC barrier function. DPV disrupted EC barrier function in a dose-dependent manner. Tyrosine kinase inhibitors, genistein, and PP-2, a specific inhibitor of Src, reduced the DPV-mediated barrier dysfunction. Consistent with these results, DPV-induced Src activation was attenuated by PP-2. Furthermore, DPV increased the association of Src with cortactin and myosin light chain kinase, indicating their potential role as cytoskeletal targets for Src. Transient overexpression of either wild-type Src or a constitutively active Src mutant potentiated the DPV-mediated decline in barrier dysfunction, whereas a dominant negative Src mutant attenuated the response. These studies provide the first direct evidence for Src involvement in DPV-induced EC barrier dysfunction.
Regulation and function of CCSP during pulmonaryPseudomonas aeruginosainfection in vivo
Authors: Hayashida Shinya , Harrod Kevin S. , Whitsett Jeffrey A.
Published: December 2017

Clara cell secretory protein (CCSP) is a 16-kDa homodimeric polypeptide secreted by respiratory epithelial cells in the conducting airways of the lung. To assess the role of CCSP in bacterial inflammation and to discern whether CCSP expression is influenced by bacterial infection, CCSP-deficient [(−/−)] gene-targeted mice and wild-type mice were given Pseudomonas aeruginosa intratracheally. Infiltration by polymorphonuclear cells was significantly increased in the lungs of CCSP(−/−) mice 6 and 24 h after the administration of the bacteria. The number of viable bacteria isolated from the lungs in CCSP(−/−) mice was decreased compared with that in wild-type mice. Concentrations of the proinflammatory cytokines interleukin-1β and tumor necrosis factor-α were modestly increased after 6 and 24 h, respectively, in CCSP(−/−) mice. The concentration of CCSP protein in lung homogenates decreased for 1–5 days after infection and recovered by 14 days after infection. Likewise, CCSP mRNA and immunostaining for CCSP markedly decreased in respiratory epithelial cells after infection. CCSP deficiency was associated with enhanced pulmonary inflammation and improved killing of bacteria after acute pulmonary infection with P. aeruginosa. The finding that Pseudomonas infection inhibited CCSP expression provides further support for the concept that CCSP plays a role in the modulation of pulmonary inflammation during infection and recovery.
Endothelin receptor blockade decreases lung water in young rats exposed to viral infection and hypoxia
Authors: Carpenter Todd C. , Stenmark Kurt R.
Published: December 2017

Viral respiratory infections may increase the susceptibility of young animals to hypoxia-induced pulmonary edema. Because hypoxia stimulates endothelin production, we hypothesized that an increase in lung endothelin contributes to these alterations in lung water. Weanling rats were infected with Sendai virus, causing a mild respiratory infection. At day 7 after infection, animals were exposed to hypoxia (inspired O2fraction = 0.1) for 24 h. Exposure to virus plus hypoxia led to increases in lung water compared with control groups ( P< 0.001). Lung endothelin levels were significantly higher in the virus plus hypoxia group than in control groups ( P < 0.001). A second group of infected animals received bosentan, a nonselective endothelin receptor antagonist, during exposure to hypoxia. Bosentan-treated animals showed less lung water accumulation, less lung lavage fluid protein, and less perivascular fluid cuffing than untreated animals ( P < 0.01). We conclude that the combination of a recent viral respiratory infection and exposure to moderate hypoxia led to increases in endothelin in the lungs of young rats and that endothelin receptor blockade ameliorates the hypoxia-induced increases in lung water found in these animals.
Chemokine expression in Th1 cell-induced lung injury: prominence of IFN-γ-inducible chemokines
Authors: Dixon Anne E. , Mandac Janis B. , Madtes David K. , Martin Paul J. , Clark Joan G.
Published: December 2017

Proinflammatory responses generated by T helper type 1 (Th1) cells may contribute significantly to immune-mediated lung injury. We describe a murine model of Th1 cell-induced lung injury in which adoptive transfer of alloreactive Th1 cells produces pulmonary inflammation characterized by mononuclear cell vasculitis, alveolitis, and interstitial pneumonitis. To investigate the link between activation of Th1 cells in the lung and inflammatory cell recruitment, we characterized cytokine and chemokine mRNA expression in Th1 cells activated in vitro and in lung tissue after adoptive transfer of Th1 cells. Activated Th1 cells per se express mRNA for interferon (IFN)-γ and several members of the tumor necrosis factor family as well as the C-C chemokine receptor-5 ligands regulated on activation normal T cells expressed and secreted and macrophage inflammatory protein-1α and -1β. Additional chemokine genes were induced in the lung after Th1 cell administration, most notably IFN-γ-inducible protein (IP-10) and monokine induced by IFN-γ (MIG). Remarkable increases in IP-10- and MIG-immunoreactive proteins were present in inflammatory foci lung and identified in macrophages, endothelium, bronchial epithelium, and alveolar structures. The findings suggest that IFN-γ-inducible chemokines are an important mechanism for amplifying inflammation initiated by Th1 cells in the lung.
Inducible lung-specific expression of RANTES: preferential recruitment of neutrophils
Authors: Pan Zhong-Zong , Parkyn Lisa , Ray Anuradha , Ray Prabir
Published: December 2017

The chemokine regulated on activation normal T cells expressed and secreted (RANTES) has been implicated in eosinophil chemotaxis in asthma and allergic diseases, which are thought to be T helper (Th) type 2-dominated diseases. However, adoptive transfer of Th1 cells in mice upregulates RANTES gene expression in the lung, and increased RANTES expression has been documented in several Th1 cell-dominated conditions that are associated with neutrophilia. The in vivo role of RANTES in the pathogenesis of disease processes is not well understood. To determine the effect of RANTES expression alone in vivo, we generated transgenic mice that overexpress RANTES specifically in the lung in an inducible fashion. The airways of the transgenic mice overexpressing RANTES displayed a significant increase in neutrophil infiltration compared with that in control mice. The increased airway neutrophilia was also evident when the transgenic mice were tested in a murine model of allergic airway inflammation. RANTES expression also induced expression of the chemokine genes macrophage inflammatory protein-2, 10-kDa interferon-γ-inducible protein, and monocyte chemoattractant protein-1 in the lungs of the transgenic mice. Our studies highlight a hitherto unappreciated role for RANTES in neutrophil trafficking during inflammation. Thus increased RANTES expression, as observed during respiratory viral infections, may play an important role in the associated neutrophilia and exacerbations of asthma.
Hypoxic vasoconstriction in intact lungs: a role for NADPH oxidase-derived H2O2?
Authors: Weissmann Norbert , Tadić André , Hänze Jörg , Rose Frank , Winterhalder Stefan , Nollen Matthias , Schermuly Ralph Theo , Ghofrani Hossein Ardeschir , Seeger Werner , Grimminger Friedrich
Published: December 2017

Hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion with ventilation. Controversy exists whether decreased or increased reactive oxygen species may elicit HPV and from which source such oxygen metabolites are derived. In rabbit lungs, we detected transcripts of a nonphagocytic NADPH oxidase subunit homologous to mitogenic oxidase-1 (Mox1) or NADPH oxidase homolog 1 (NOH-1L). In perfused rabbit lungs, we employed 1) a new NADPH oxidase inhibitor [4-(2-aminoethyl)benzenesulfonyl fluoride (AEBSF; 100–600 μM)] and 2) the superoxide dismutase (SOD) inhibitors diethyldithiocarbamic acid (DETC; 100 μM to 10 mM) and triethylenetetramine (TETA; 1–25 mM). Specificity of these agents for HPV was investigated by comparison with U-46619-induced vasoconstrictions. AEBSF induced a transient increase in pulmonary arterial pressure with increased strength of HPV. Subsequent to this initial response, normoxic pulmonary arterial pressure was not affected and HPV was specifically suppressed. Whereas DETC turned out to act in a nonspecific fashion, TETA suppressed HPV specifically. These findings provide evidence of a role for a nonphagocytic NAD(P)H oxidase with superoxide and SOD-related hydrogen peroxide formation in HPV. Because HPV was inhibited but not mimicked by the inhibitors, increased rather than decreased superoxide and/or hydrogen peroxide formation is suggested as the hypoxia-provoked signaling event.
Regulation of plasminogen activator inhibitor-1 and urokinase by hyaluronan fragments in mouse macrophages
Authors: Horton Maureen R. , Olman Mitchell A. , Bao Clare , White Kimberly E. , Choi Augustine M. K. , Chin Beek-Yok , Noble Paul W. , Lowenstein Charles J.
Published: December 2017

Pulmonary inflammation and fibrosis are characterized by increased turnover and production of the extracellular matrix as well as an impairment of lung fibrinolytic activity. Although fragments of the extracellular matrix component hyaluronan induce macrophage production of inflammatory mediators, the effect of hyaluronan on the fibrinolytic mediators plasminogen activator inhibitor (PAI)-1 and urokinase-type plasminogen activator (uPA) is unknown. This study demonstrates that hyaluronan fragments augment steady-state mRNA, protein, and inhibitory activity of PAI-1 as well as diminish the baseline levels of uPA mRNA and inhibit uPA activity in an alveolar macrophage cell line. Hyaluronan fragments alter macrophage expression of PAI-1 and uPA at the level of gene transcription. Similarly, hyaluronan fragments augment PAI-1 and diminish uPA mRNA levels in freshly isolated inflammatory alveolar macrophages from bleomycin-treated rats. These data suggest that hyaluronan fragments influence alveolar macrophage expression of PAI-1 and uPA and may be a mechanism for regulating fibrinolytic activity during lung inflammation.
S-nitrosoglutathione breakdown prevents airway smooth muscle relaxation in the guinea pig
Authors: Fang Kezhong , Johns Roger , Macdonald Timothy , Kinter Michael , Gaston Benjamin
Published: December 2017

Airway levels of the endogenous bronchodilator S-nitrosoglutathione (GSNO) are low in children with near-fatal asthma. We hypothesized that GSNO could be broken down in the lung and that this catabolism could inhibit airway smooth muscle relaxation. In our experiments, GSNO was broken down by guinea pig lung homogenates, particularly after ovalbumin sensitization (OS). Two lung protein fractions had catabolic activity. One was NADPH dependent and was more active after OS. The other was NADPH independent and was partially inhibited by aurothioglucose. Guinea pig lung tissue protein fractions with GSNO catabolic activity inhibited GSNO-mediated guinea pig tracheal ring relaxation. The relaxant effect of GSNO was partially restored by aurothioglucose. These observations suggest that catabolism of GSNO in the guinea pig 1) is mediated by lung proteins, 2) is partially upregulated after OS, and 3) may contribute to increased airway smooth muscle tone. We speculate that enzymatic breakdown of GSNO in the lung could contribute to asthma pathophysiology by inhibiting the beneficial effects of GSNO, including its effect on airway smooth muscle tone.
Prolonged endothelin B receptor blockade causes pulmonary hypertension in the ovine fetus
Authors: Dunbar Ivy D. , Parker Thomas A. , Abman Steven H.
Published: December 2017

Endothelin (ET)-1 contributes to regulation of pulmonary vascular tone and structure in the normal ovine fetus and in models of perinatal pulmonary hypertension. The hemodynamic effects of ET-1 are due to activation of its receptors. The ETAreceptor mediates vasoconstriction and smooth muscle cell proliferation, whereas the ETBreceptor mediates vasodilation. In a lamb model of chronic intrauterine pulmonary hypertension, ETBreceptor activity and gene expression are decreased. To determine whether prolonged ETBreceptor blockade causes pulmonary hypertension, we studied the hemodynamic effects of selective ETBreceptor blockade with BQ-788. Animals were treated with an infusion of either BQ-788 or vehicle for 7 days. Prolonged BQ-788 treatment increased pulmonary arterial pressure and pulmonary vascular resistance ( P < 0.05). The pulmonary vasodilator response to sarafotoxin 6c, a selective ETBreceptor agonist, was attenuated after 7 days of BQ-788 treatment, demonstrating pharmacological blockade of the ETBreceptor. Animals treated with BQ-788 had greater right ventricular hypertrophy and muscularization of small pulmonary arteries ( P < 0.05). Lung ET-1 levels were threefold higher in the animals treated with BQ-788 ( P < 0.05). We conclude that prolonged selective ETBreceptor blockade causes severe pulmonary hypertension and pulmonary vascular remodeling in the late-gestation ovine fetus.
Contribution of R domain phosphoserines to the function of CFTR studied in Fischer rat thyroid epithelia
Authors: Baldursson Olafur , Berger Herbert A. , Welsh Michael J.
Published: December 2017

The regulatory domain of cystic fibrosis transmembrane conductance regulator (CFTR) regulates channel activity when several serines are phosphorylated by cAMP-dependent protein kinase. To further define the functional role of individual phosphoserines, we studied CFTR containing previously studied and new serine to alanine mutations. We expressed these constructs in Fischer rat thyroid epithelia and measured transepithelial Cl−current. Mutation of four in vivo phosphorylation sites, Ser660, Ser737, Ser795, and Ser813(S-Quad-A), substantially decreased cAMP-stimulated current, suggesting that these four sites account for most of the phosphorylation-dependent response. Mutation of either Ser660or Ser813alone significantly decreased current, indicating that these residues play a key role in phosphorylation-dependent stimulation. However, neither Ser660nor Ser813alone increased current to wild-type levels; both residues were required. Changing Ser737to alanine increased current above wild-type levels, suggesting that phosphorylation of Ser737may inhibit current in wild-type CFTR. These data help define the functional role of regulatory domain phosphoserines and suggest interactions between individual phosphoserines.
Secretion of extracellular superoxide dismutase in neonatal lungs
Authors: Nozik-Grayck Eva , Dieterle Christine S. , Piantadosi Claude A. , Enghild Jan J. , Oury Tim D.
Published: December 2017

Extracellular superoxide dismutase (EC-SOD), the only known enzymatic scavenger of extracellular superoxide, may modulate reactions of nitric oxide (NO) in the lungs by preventing reactions between superoxide and NO. The regulation of EC-SOD has not been examined in developing lungs. We hypothesize that EC-SOD plays a pivotal role in the response to increased oxygen tension and NO in the neonatal lung. This study characterizes rabbit EC-SOD and investigates the developmental regulation of EC-SOD activity, protein expression, and localization. Purified rabbit EC-SOD was found to have several unique biochemical attributes distinct from EC-SOD in other species. Rabbit lung EC-SOD contains predominantly uncleaved subunits that do not form disulfide-linked dimers. The lack of intersubunit disulfide bonds may contribute to the decreased heparin affinity and lower EC-SOD content in rabbit lung. EC-SOD activity in rabbit lungs is low before birth and increases soon after gestation. In addition, the enzyme is localized intracellularly in preterm and term rabbit lungs. Secretion of active EC-SOD into the extracellular compartment increases with age. The changes in EC-SOD localization and activity have implications for the neonatal pulmonary response to oxidative stress and the biological activity of NO at birth.
IL-1β induces eotaxin gene transcription in A549 airway epithelial cells through NF-κB
Authors: Jedrzkiewicz Sean , Nakamura Hidetoshi , Silverman Eric S. , Luster Andrew D. , Mansharamani Naresh , In Kwang Ho , Tamura Gen , Lilly Craig M.
Published: December 2017

Eotaxin is an asthma-related C-C chemokine that is produced in response to interleukin-1β (IL-1β). We detected an increase in newly transcribed eotaxin mRNA in IL-1β-stimulated airway epithelial cells. Transient transfection assays using promoter-reporter constructs identified a region as essential for IL-1β-induced increases in eotaxin transcription. Using site-directed mutagenesis, we found that a nuclear factor-κB (NF-κB) site located 46 bp upstream from the transcriptional start site was both necessary and sufficient for IL-1β induction of reporter construct activity. Electrophoretic mobility shift assay demonstrated that IL-1β-stimulated airway epithelial cells produced p50 and p65 protein that bound this site in a sequence-specific manner. The functional importance of the NF-κB site was demonstrated by coexpression experiments in which increasing doses of p65 expression vector were directly associated with reporter activity exclusively in constructs with an intact NF-κB site ( r2= 0.97, P = 0.002). Moreover, IL-1β-induced increases in eotaxin mRNA expression are inhibited by inhibitors of NF-κB. Our findings implicate NF-κB and its binding sequence in IL-1β-induced transcriptional activation of the eotaxin gene.
Lung function, permeability, and surfactant composition in oleic acid-induced acute lung injury in rats
Authors: Davidson Kate G. , Bersten Andrew D. , Barr Heather A. , Dowling Kay D. , Nicholas Terence E. , Doyle Ian R.
Published: December 2017

Although acute lung injury (ALI) is associated with inflammation and surfactant dysfunction, the precise sequence of these changes remains poorly described. We used oleic acid to study the pathogenesis of ALI in spontaneously breathing anesthetized rats. We found that lung pathology can occur far more rapidly than previously appreciated. Lung neutrophils were increased approximately threefold within 5 min, and surfactant composition was dramatically altered within 15 min. Alveolar cholesterol increased by ∼200%, and even though disaturated phospholipids increased by ∼30% over 4 h, the disaturated phospholipid-to-total phospholipid ratio fell. Although the alveolocapillary barrier was profoundly disrupted after just 15 min, with marked elevations in lung fluid (99mTc-labeled diethylenetriamine pentaacetic acid) and125I-labeled albumin flux, the lung rapidly began to regain its sieving properties. Despite the restoration in lung permeability, the animals remained hypoxic even though minute ventilation was increased approximately twofold and static compliance progressively deteriorated. This study highlights that ALI can set in motion a sequence of events continuing the respiratory failure irrespective of the alveolar surfactant pool size and the status of the alveolocapillary barrier.
Retinoic acid reduces p11 protein levels in bronchial epithelial cells by a posttranslational mechanism
Authors: Gladwin M. T. , Yao X. L. , Cowan M. , Huang X. L. , Schneider R. , Grant L. R. , Logun C. , Shelhamer J. H.
Published: December 2017

p11 is a member of the S100 family of proteins, is the cellular ligand of annexin II, and interacts with the carboxyl region of 85-kDa cytosolic phospholipase A2 (cPLA2), inhibiting cPLA2activity and arachidonic acid (AA) release. We studied the effect of retinoic acid (RA) on PLA2 activity in human bronchial epithelial cells and whether p11 contributes to these effects. The addition of 10−6 M RA resulted in reduced p11 protein levels at 4 days, with the greatest effect observed on days 6 and 7. This effect was dose related (10−6 to 10−9 M). RA treatment (10−6 M) had no effect on cPLA2 protein levels. p11 mRNA levels were unchanged at 6 and 8 days of treatment (correlating with maximum p11 protein reduction). Treatment with RA reduced p11 levels in control cells and in cells transfected with a p11 expression vector, suggesting a posttranslational mechanism. Lactacystin (10−6 M), an inhibitor of the human 26S proteasome, blocked the decrease in p11 observed with RA treatment. Compared with control cells ( n = 3), RA-treated cells ( n = 3) had significantly increased AA release after treatment with the calcium ionophore A-23187 ( P = 0.006). Therefore, RA reduces p11 protein expression and increases PLA2 activity and AA release.
Hypoxia decreases proteins involved in epithelial electrolyte transport in A549 cells and rat lung
Authors: Wodopia Ralf , Ko Hyun Soo , Billian Javiera , Wiesner Rudolf , Bärtsch Peter , Mairbäurl Heimo
Published: December 2017

Fluid reabsorption from alveolar space is driven by active Na reabsorption via epithelial Na channels (ENaCs) and Na-K-ATPase. Both are inhibited by hypoxia. Here we tested whether hypoxia decreases Na transport by decreasing the number of copies of transporters in alveolar epithelial cells and in lungs of hypoxic rats. Membrane fractions were prepared from A549 cells exposed to hypoxia (3% O2) as well as from whole lung tissue and alveolar type II cells from rats exposed to hypoxia. Transport proteins were measured by Western blot analysis. In A549 cells, α1- and β1-Na-K-ATPase, Na/K/2Cl cotransport, and ENaC proteins decreased during hypoxia. In whole lung tissue, α1-Na-K-ATPase and Na/K/2Cl cotransport decreased. α- and β-ENaC mRNAs also decreased in hypoxic lungs. Similar results were seen in alveolar type II cells from hypoxic rats. These results indicate a slow decrease in the amount of Na-transporting proteins in alveolar epithelial cells during exposure to hypoxia that also occurs in vivo in lungs from hypoxic animals. The reduced number of transporters might account for the decreased transport activity and impaired edema clearance in hypoxic lungs.
Time course of inflammatory and remodeling events in a murine model of asthma: effect of steroid treatment
Authors: Trifilieff Alexandre , El-Hashim Ahmed , Bertrand Claude
Published: December 2017

The kinetics of airway inflammation and remodeling processes following ovalbumin aerosol challenge in sensitized BALB/c mice was studied. Mice were exposed to either single or five ovalbumin challenges over 5 days. In both protocols, time-dependent increases in bronchoalveolar lavage (BAL) cellular fibronectin, neutrophils and eosinophils were observed. The kinetics of these events were similar in both protocols; however, the magnitude of the response was much greater following repeated challenges. BAL protein levels and lymphocyte numbers were increased only following repeated challenges, whereas interleukin (IL)-5 and IL-4 were increased in both protocols. Histological analysis revealed a time-dependent increase in epithelial cell proliferation and in mucus-producing epithelial cells. Proliferation of alveolar cells was observed only following repeated challenges. Airway hyperreactivity was observed in both protocols but was much greater following repeated challenges. Pretreatment with dexamethasone fully inhibited the inflammatory response and airway hyperreactivity but only partially inhibited the remodeling process. These data suggest that glucocorticoids, although potent anti-inflammatory agents, may not be potent in reducing the lung remodeling process associated with asthma.
Lung hypoplasia in the nitrofen model of congenital diaphragmatic hernia occurs early in development
Authors: Guilbert Theresa W. , Gebb Sarah A. , Shannon John M.
Published: December 2017

The teratogen nitrofen produces a congenital diaphragmatic hernia (CDH) and pulmonary hypoplasia in rodent fetuses that closely parallel observations made in humans. We hypothesized that these changes may be due to primary pulmonary hypoplasia and not herniation of the abdominal contents. Timed-pregnant rats were given nitrofen on day 9, and fetuses were harvested on days 13 through 21. Initial evagination of lung buds on gestational day 11 was not delayed in nitrofen-treated fetuses. On gestational day 13, however, there was a significant decrease in the number of terminal end buds in the lungs of nitrofen-exposed fetuses vs. controls. Thymidine-labeled lung epithelial and mesenchymal cells were significantly decreased in nitrofen-treated lungs. Lungs from nitrofen-treated fetuses exhibited wide septae with disorganized, compacted tissue, particularly around the air spaces. Expression of surfactant protein B and C mRNAs was significantly decreased in the nitrofen litters. In situ hybridization of fetal lung tissue at all gestational ages showed no difference in the expression of vascular endothelial growth factor, Flk-1, or Flt-1 mRNAs. Because closure of the diaphragm is completed on gestational day 16 in the rat, our results suggest that lung hypoplasia in this model of CDH is due at least in part to a primary effect of nitrofen on the developing lung.