bluuebunny/crossref_metadata_2025 huggingface dataset:
Pulmonary phosphatidic acid phosphatase and lipid phosphate phosphohydrolase
Authors: Nanjundan Meera , Possmayer Fred
Published: March 2015

The lung contains two distinct forms of phosphatidic acid phosphatase (PAP). PAP1 is a cytosolic enzyme that is activated through fatty acid-induced translocation to the endoplasmic reticulum, where it converts phosphatidic acid (PA) to diacylglycerol (DAG) for the biosynthesis of phospholipids and neutral lipids. PAP1 is Mg2+dependent and sulfhydryl reagent sensitive. PAP2 is a six-transmembrane-domain integral protein localized to the plasma membrane. Because PAP2 degrades sphingosine-1-phosphate (S1P) and ceramide-1-phosphate in addition to PA and lyso-PA, it has been renamed lipid phosphate phosphohydrolase (LPP). LPP is Mg2+independent and sulfhydryl reagent insensitive. This review describes LPP isoforms found in the lung and their location in signaling platforms (rafts/caveolae). Pulmonary LPPs likely function in the phospholipase D pathway, thereby controlling surfactant secretion. Through lowering the levels of lyso-PA and S1P, which serve as agonists for endothelial differentiation gene receptors, LPPs regulate cell division, differentiation, apoptosis, and mobility. LPP activity could also influence transdifferentiation of alveolar type II to type I cells. It is considered likely that these lipid phosphohydrolases have critical roles in lung morphogenesis and in acute lung injury and repair.
β-Adrenergic agonists inhibit corticosteroid-induced apoptosis of airway epithelial cells
Authors: Tse Roberta , Marroquin Bertha A. , Dorscheid Delbert R. , White Steven R.
Published: March 2015

Airway epithelial damage is a feature of persistent asthma. Treatment with inhaled and oral corticosteroids may suppress inflammation and gain clinical control despite continued epithelial damage. We have previously demonstrated that corticosteroids elicit apoptosis of airway epithelial cells in culture. β-Adrenergic receptor agonists are commonly used in asthma therapy and can inhibit corticosteroid-induced apoptosis of eosinophils. We tested the hypothesis that β-adrenergic agonists would inhibit corticosteroid-induced airway epithelial cell apoptosis in cultured primary airway epithelial cells and in the cell line 1HAEo-. Albuterol treatment inhibited dexamethasone-induced apoptosis completely but did not inhibit apoptosis induced by Fas receptor activation. The protective effect of albuterol was duplicated by two different analogs of protein kinase A. The protective effect was not associated with increased translocation of the glucocorticoid receptor to the nucleus nor with changes in glucocorticoid receptor-mediated transcriptional activation or repression. We demonstrate that β-adrenergic agonists can inhibit corticosteroid-induced apoptosis but not apoptosis induced by Fas activation. These data suggest that one potential deleterious effect of corticosteroid therapy in asthma can be prevented by concomitant β-adrenergic agonist treatment.
Distinct patterns of apoptosis in the lung during liquid ventilation compared with gas ventilation
Authors: Mantell Lin L. , Shaffer Thomas H. , Horowitz Stuart , Foust Ray , Wolfson Marla R. , Cox Cindy , Khullar Poonam , Zakeri Zahra , Lin Lin , Kazzaz Jeffrey A. , Palaia Tom , Scott William , Davis Jonathan M.
Published: March 2015

To determine whether liquid ventilation (LV) causes less cell injury and improves lung function compared with conventional gas ventilation (GV), we analyzed pulmonary physiological profiles, lung histology, and cell death in 110- and 120-day preterm lambs, which were randomized to receive either ventilation modality on Fi O2 = 1. LV lungs were well expanded with adequate pulmonary function, whereas GV animals exhibited marked atelectasis, poor pulmonary function, and increased mortality. Both ventilatory strategies induced marked lung cell apoptosis, but with distinct patterns of distribution. Although GV induced apoptosis of epithelium primarily in the lining and within the lumina of bronchioles, LV induced significant apoptosis much more homogeneously throughout lung parenchyma including alveoli and interstitial spaces. These studies suggest that although both forms of ventilation cause regional apoptosis, LV more effectively delivers oxygen and recruits the lung more homogeneously than GV.
Dexamethasone stimulates transcription of the Na+-K+-ATPase β1gene in adult rat lung epithelial cells
Authors: Hao Hong , Wendt Christine H. , Sandhu Gurpreet , Ingbar David H.
Published: March 2015

Na+-K+-ATPase plays an essential role in active alveolar epithelial fluid resorption. In fetal and adult alveolar epithelial cells, glucocorticoids (GC) increase Na+-K+-ATPase activity and mRNA levels. We sought to define the mechanism of Na+-K+-ATPase gene upregulation by GC. In a rat alveolar epithelial cell line (RLE), dexamethasone (Dex) increased β1-subunit Na+-K+-ATPase mRNA expression two- to threefold within 3 h after exposure to the GC. The increased gene expression was due to increased transcription as demonstrated by nuclear run-on assays, whereas mRNA stability remained unchanged. Transient transfection of 5′ deletion mutants of a β1promoter-reporter construct demonstrated a 1.5- to 2.2-fold increase in promoter activity by Dex. All of the 5′ deletion constructs contained partial or palindromic GC regulatory elements (GRE) and responded to GC. The increased expression of promoter reporter was inhibited by RU-486, a GC receptor (GR) antagonist, suggesting the involvement of GR. The palindromic GRE at -631 demonstrated Dex induction in a heterologous promoter construct. Gel mobility shift assays using RLE nuclear extracts demonstrated specific binding to this site and the presence of GR. We conclude that GC directly stimulate transcription of Na+-K+-ATPase β1gene expression in adult rat lung epithelial cells through a GR-dependent mechanism that can act at multiple sites.
Effect of cigarette smoke on fibroblast-mediated gel contraction is dependent on cell density
Authors: Wang Hangjun , Liu Xiangde , Umino Takeshi , Kohyama Tadashi , Zhu Yun Kui , Wen Fu-Qiang , Spurzem John R. , Romberger Debra J. , Kim Hui Jung , Rennard Stephen I.
Published: March 2015

Cigarette smoke exposure has been associated with a variety of diseases, including emphysema. The current study evaluated the interaction of cell density and cigarette smoke extract (CSE) on fibroblast contraction of collagen gels. Protein levels of transforming growth factor (TGF)-β1, fibronectin, PGE2, and TGF-β1 mRNA were quantified. Although both 5 and 10% CSE inhibited contraction by low-density fibroblasts (1 × 105cell/ml), only 5% CSE augmented contraction in higher-density cultures (3–5 × 105cells/ml). CSE also inhibited fibronectin and TGF-β1 production in low-density cultures but stimulated fibronectin production in high-density cultures. Active TGF-β1 was readily detectable only in higher-density cultures and was markedly augmented by 5% CSE. In contrast, although TGF-β1 mRNA expression was inhibited in high-density cultures by 10% CSE, expression was increased in the presence of 5% CSE. These results suggest that CSE-induced inhibition of low-density fibroblast contraction is due to inhibition of fibronectin production, whereas CSE's stimulatory effect on high-density cells is the result of increased release of TGF-β1. These effects may help explain the varied pathologies associated with exposure to cigarette smoke.
Gap junction-microtubule associations in rat alveolar epithelial cells
Authors: Guo Yihe , Martinez-Williams Cara , Rannels D. Eugene
Published: March 2015

Connexin 43 (Cx43) is a predominant gap junction (GJ) protein expressed by alveolar epithelial cells (AEC) in primary cell culture. Cx43 trafficking, assembly, and turnover are regulated by multiple mechanisms, including those mediated by integrins, by extracellular matrix, and by the cytoskeleton. Immunocytochemical double labeling demonstrates association of microtubules with internalization of Cx43-positive GJ plaques. Antibodies against the α5-integrin subunit block cell-matrix interactions without effect on tubulin expression, whereas inhibition of MAP kinase kinase by PD-98059 reduces tubulin expression, based on both Western blot and immunostaining. To examine direct association of microtubules (MT) with GJ plaques, we treated day 3 AEC for 0.5-24 h with colchicine, an inhibitor of tubulin polymerization. After 60 min, MTs were disassembled, whereas Western blot analysis showed no change in tubulin expression. In parallel, colchicine initiated redistribution of immunopositive Cx43 from the membrane to the cytosol. These observations support the premise that direct association of the cytoskeleton with gap junctions plays a significant role in regulation of Cx43 expression and distribution through integrin-mediated signal transduction pathways.
Total extracellular surfactant is increased but abnormal in a rat model of gram-negative bacterial pneumonia
Authors: Russo Thomas A. , Bartholomew Lori A. , Davidson Bruce A. , Helinski Jadwiga D. , Carlino Ulrike B. , Knight Paul R. , Beers Michael F. , Atochina Elena N. , Notter Robert H. , Holm Bruce A.
Published: March 2015

An in vivo rat model was used to evaluate the effects of Escherichia coli pneumonia on lung function and surfactant in bronchoalveolar lavage (BAL). Total extracellular surfactant was increased in infected rats compared with controls. BAL phospholipid content in infected rats correlated with the severity of alveolar-capillary leak as reflected in lavage protein levels ( R2= 0.908, P < 0.0001). Western blotting showed that levels of surfactant protein (SP)-A and SP-D in BAL were significantly increased in both large and small aggregate fractions at 2 and 6 h postinstillation of E. coli. SP-B was also increased at these times in the large aggregate fraction of BAL, whereas SP-C levels were increased at 2 h and decreased at 6 h relative to controls. The small-to-large (S/L) aggregate ratio (a marker inversely proportional to surfactant function) was increased in infected rats with >50 mg total BAL protein. There was a significant correlation ( R2= 0.885, P < 0.0001) between increasing S/L ratio in BAL and pulmonary damage assessed by total protein. Pulmonary volumes, compliance, and oxygen exchange were significantly decreased in infected rats with >50 mg of total BAL protein, consistent with surfactant dysfunction. In vitro surface cycling studies with calf lung surfactant extract suggested that bacterially derived factors may have contributed in part to the surfactant alterations seen in vivo.
Cigarette smoke exposure potentiates bleomycin-induced lung fibrosis in guinea pigs
Authors: Cisneros-Lira José , Gaxiola Miguel , Ramos Carlos , Selman Moisés , Pardo Annie
Published: March 2015

The role of tobacco smoking in the development and outcome of pulmonary fibrosis is uncertain. To approach the effects of cigarette smoke on bleomycin-induced lung fibrosis, we studied five groups of guinea pigs: 1) controls, 2) instilled with bleomycin (B), 3) exposed to tobacco smoke for 6 wk (TS), 4) bleomycin instillation plus tobacco smoke exposure for 6 wk (B+TS), and 5) tobacco smoke exposure for 6 wk and bleomycin after smoking (TS/B). Guinea pigs receiving bleomycin and tobacco smoke exposure exhibited higher fibrotic lesions including a significant increase in the number of positive α-smooth muscle actin cells compared with bleomycin alone (B+TS, 3.4 ± 1.2%; TS/B, 3.7 ± 1.5%; B, 2.3 ± 1.5%; P < 0.01). However, only the TS/B group reached a significant increase in lung collagen compared with the bleomycin group (TS/B, 3.5 ± 0.7; B ± TS, 2.9 ± 0.4; B, 2.4 ± 0.2 mg hydroxyproline/lung; P < 0.01). Bronchoalveolar lavage (BAL) from TS/B showed an increased number of eosinophils and higher levels of IL-4 and tissue inhibitor of metalloproteinase-2 ( P < 0.01 for all comparisons) and induced a significant increase in fibroblast proliferation ( P < 0.05). Importantly, smoke exposure alone induced an increase in BAL neutrophils, matrix metalloproteinase-9, and fibroblast proliferation compared with controls, suggesting that tobacco smoke creates a profibrotic milieu that may contribute to the increased bleomycin-induced fibrosis.
Pulmonary vasoconstriction by serotonin is inhibited byS-nitrosoglutathione
Authors: Nozik-Grayck Eva , McMahon Timothy J. , Huang Yuh-Chin T. , Dieterle Christine S. , Stamler Jonathan S. , Piantadosi Claude A.
Published: March 2015

Nitric oxide (NO) functions as an endothelium-derived relaxing factor by activating guanylate cyclase to increase cGMP levels. However, NO and related species may also regulate vascular tone by cGMP-independent mechanisms. We hypothesized that naturally occurring NO donors could decrease the pulmonary vascular response to serotonin (5-HT) in the intact lung through chemical interactions with 5-HT2receptors. In isolated rabbit lung preparations and isolated pulmonary artery (PA) rings, 50–250 μM S-nitrosoglutathione (GSNO) inhibited the response to 0.01–10 μM 5-HT. The vasoconstrictor response to 5-HT was mediated by 5-HT2receptors in the lung, since it could be blocked completely by the selective inhibitor ketanserin (10 μM). GSNO inhibited the response to 5-HT by 77% in intact lung and 82% in PA rings. In PA rings, inhibition by GSNO could be reversed by treatment with the thiol reductant dithiothreitol (10 mM). 3-Morpholinosydnonimine (100–500 μM), which releases NO and O[Formula: see text] simultaneously, also blocked the response to 5-HT. Its chemical effects, however, were distinct from those of GSNO, because 5-HT-mediated vasoconstriction was not restored in isolated rings by dithiothreitol. In the intact lung, neither NO donor altered the vascular response to endothelin, which activates the same second-messenger vasoconstrictor system as 5-HT. These findings, which did not depend on guanylate cyclase, are consistent with chemical modification by NO of the 5-HT2G protein-coupled receptor system to inhibit vasoconstriction, possibly by S-nitrosylation of the receptor or a related protein. This study demonstrates that GSNO can regulate vascular tone in the intact lung by a reversible mechanism involving inhibition of the response to 5-HT.
IL-13-induced changes in the goblet cell density of human bronchial epithelial cell cultures: MAP kinase and phosphatidylinositol 3-kinase regulation
Authors: Atherton Hazel C. , Jones Gareth , Danahay Henry
Published: March 2015

In addition to a direct proinflammatory role, IL-13 has been demonstrated to induce a goblet cell metaplastic phenotype in the airway epithelium in vivo. We have studied the direct effects of IL-13 (and IL-4) on well-differentiated, air-liquid interface cultures of human bronchial epithelial cells (HBEs) and provide a quantitative assessment of the development of a mucus hypersecretory phenotype induced by these cytokines. Using Alcian blue staining of goblet cells and immunohistochemical detection of MUC5AC, we found that IL-13 (and IL-4) induced increases in the goblet cell density (GCD) of the HBE cultures. The effects of these cytokines were critically dependent on concentration: 1 ng/ml routinely induced a 5- to 10-fold increase in GCD that was associated with a hypersecretory ion transport phenotype. Paradoxically, 10 ng/ml of either cytokine induced a profound reduction in GCD. Removal of EGF from the culture media or treatment of the cells with AG-1478 [a potent inhibitor of EGF receptor tyrosine kinase (EGFR-TK)] demonstrated that the EGFR-TK pathway was key to the regulation of the basal GCD but that it was not involved in the IL-13-driven increase. The IL-13-driven increase in GCD was, however, sensitive to inhibition of MEK (PD-98059, U-0126), p38 MAPK (SB-202190), and phosphatidylinositol (PtdIns) 3-kinase (LY-294002). These data support the concept that IL-13 is in part able to induce a mucus hypersecretory phenotype through a direct interaction with the airway epithelium and that MAP kinase and PtdIns 3-kinase signaling pathways are involved.
Sustained changes in lung expansion alter tropoelastin mRNA levels and elastin content in fetal sheep lungs
Authors: Joyce Belinda J. , Wallace Megan J. , Pierce Richard A. , Harding Richard , Hooper Stuart B.
Published: March 2015

Our objective was to determine the effects of sustained alterations in fetal lung expansion on pulmonary elastin synthesis. In fetal sheep, lung expansion was either decreased between 111 and 131 days' gestation (term ∼147 days) by tracheal drainage or increased for 2, 4, 7, or 10 days by tracheal obstruction, ending at 128 days' gestation. Lung tropoelastin mRNA levels were assessed by Northern blot analysis, total elastin content was measured biochemically, and staining of lung sections was used to assess the localization and form of elastic fibers. Tracheal obstruction significantly elevated pulmonary tropoelastin mRNA levels 2.5-fold at 2 days, but values were not different from controls at 4, 7, and 10 days; elastin content tended to be increased at all time points. A sustained decrease in lung expansion by tracheal drainage reduced pulmonary tropoelastin mRNA levels 2.5-fold; elastin content was also decreased compared with controls, and tissue localization was altered. Our results indicate that the degree of lung expansion in the fetus influences elastin synthesis, content, and tissue deposition.
Urokinase potentiates PDGF-induced chemotaxis of human airway smooth muscle cells
Authors: Carlin Stephen M. , Roth Michael , Black Judith L.
Published: March 2015

We investigated the chemotactic action of PDGF and urokinase on human airway smooth muscle (HASM) cells in culture. Cells were put in collagen-coated transwells with 8-μm perforations, incubated for 4 h with test compounds, then fixed, stained, and counted as migrated nuclei by microscopy. Cells from all culture conditions showed some basal migration (migration in the absence of stimuli during the assay), but cells preincubated for 24 h in 10% FBS or 20 ng/ml PDGF showed higher basal migration than cells quiesced in 1% FBS. PDGFBB, PDGFAA, and PDGFABwere all chemotactic when added during the assay. PDGF chemotaxis was blocked by the phosphatidyl 3′-kinase inhibitor LY-294002, the MEK inhibitor U-0126, PGE2, formoterol, pertussis toxin, and the Rho kinase inhibitor Y-27632. Urokinase alone had no stimulatory effect on migration of quiescent cells but caused a dose-dependent potentiation of chemotaxis toward PDGF. Urokinase also potentiated the elevated basal migration of cells pretreated in 10% FBS or PDGF. This potentiating effect of urokinase appears to be novel. We conclude that PDGF and similar cytokines may be important factors in airway remodeling by redistribution of smooth muscle cells during inflammation and that urokinase may be important in potentiating the response.
Effects of eosinophils on nerve cell morphology and development: the role of reactive oxygen species and p38 MAP kinase
Authors: Kingham Paul J. , McLean W. Graham , Walsh Marie-Therese , Fryer Allison D. , Gleich Gerald J. , Costello Richard W.
Published: March 2015

The adhesion of eosinophils to nerve cells and the subsequent release of eosinophil products may contribute to the pathogenesis of conditions such as asthma and inflammatory bowel disease. In this study we have separately examined the consequences of eosinophil adhesion and degranulation for nerve cell morphology and development. Eosinophils induced neurite retraction of cultured guinea pig parasympathetic nerves and differentiated IMR32 cholinergic neuroblastoma cells. Inhibition of eosinophil adhesion to IMR32 cells attenuated this retraction. Eosinophil adhesion to IMR32 cells led to tyrosine phosphorylation of a number of nerve cell proteins, activation of p38 MAP kinase, and generation of neuronal reactive oxygen species (ROS). Inhibition of tyrosine kinases with genistein prevented both the generation of ROS in the nerve cells and neurite retraction. The p38 MAP kinase inhibitor SB-239063 prevented neurite retraction but had no effect on the induction of ROS. Thus eosinophils induced neurite retraction via two distinct pathways: by generation of tyrosine kinase-dependent ROS and by p38 MAP kinase. Eosinophils also prevented neurite outgrowth during differentiation of IMR32 cells. In contrast to their effect on neurite retraction, this effect was mimicked by medium containing products released from eosinophils and by eosinophil major basic protein. These results indicate that eosinophils modify the morphology of nerve cells by distinct mechanisms that involve adhesion and released proteins.
Kv1.3 potassium channels in human alveolar macrophages
Authors: Mackenzie Amanda B. , Chirakkal Hari , North R. Alan
Published: March 2015

Human alveolar macrophages were obtained from macroscopically normal lung tissue obtained at surgical resections, isolated by adherence, and identified by morphology. Whole cell recordings were made from cells 1-3 h in culture, using electrodes containing potassium chloride. From a holding potential of -100 mV, depolarizing pulses to -40 mV or greater activated an outward current. Tail current reversals showed that this current was potassium selective. Margatoxin completely blocked the current; the concentration giving half-maximal block was 160 pM. In current clamp recordings, the resting membrane potential was -34 mV; margatoxin depolarized cells to close to 0 mV. A pure macrophage population was isolated by fluorescence-activated cell sorting, using the phagocytosis of BODIPY-labeled zymosan particles. Reverse transcription-polymerase chain reaction showed that, of 13 voltage-gated K+(Kv) potassium channels sought, only Kv1.3 mRNA was present. Margatoxin (1 nM) did not affect the percentage of cells showing phagocytosis sorted from the total population. Under these experimental conditions Kv1.3 sets the resting potential of the cells, but it is not required for Fc receptor-mediated phagocytosis.
Hypothermia attenuates iNOS, CAT-1, CAT-2, and nitric oxide expression in lungs of endotoxemic rats
Authors: Scumpia Philip O. , Sarcia Paul J. , DeMarco Vincent G. , Stevens Bruce R. , Skimming Jeffrey W.
Published: March 2015

Endotoxemia stimulates endogenous nitric oxide formation, induces transcription of arginine transporters, and causes lung injury. Hypothermia inhibits nitric oxide formation and is used as a means of organ preservation. We hypothesized that hypothermia inhibits endotoxin-induced intrapulmonary nitric oxide formation and that this inhibition is associated with attenuated transcription of enzymes that regulate nitric oxide formation, such as inducible nitric oxide synthase (iNOS) and the cationic amino acid transporters 1 (CAT-1) and 2 (CAT-2). Rats were anesthetized and randomized to treatment with hypothermia (18–24°C) or normothermia (36–38°C). Endotoxin was administered intravascularly. Concentrations of iNOS, CAT-1, CAT-2 mRNA, iNOS protein, and nitrosylated proteins were measured in lung tissue homogenates. We found that hypothermia abrogated the endotoxin-induced increase in exhaled nitric oxide and lung tissue nitrotyrosine concentrations. Western blot analyses revealed that hypothermia inhibited iNOS, but not endothelial nitric oxide synthase, protein expression in lung tissues. CAT-1, CAT-2, and iNOS mRNA concentrations were lower in the lungs of hypothermic animals. These findings suggest that hypothermia protects against intrapulmonary nitric oxide overproduction and nitric oxide-mediated lung injury by inhibiting transcription of iNOS, CAT-1, and CAT-2.
Bleomycin-induced lung fibrosis in IL-4-overexpressing and knockout mice
Authors: Izbicki Gabriel , Or Reuven , Christensen Thomas G. , Segel Michael J. , Fine Alan , Goldstein Ronald H. , Breuer Raphael
Published: March 2015

The role of IL-4 in the development of lung fibrosis is as yet unclear. Bleomycin (Bleo) or saline (Sal) was injected intratracheally into three groups of C57BL/6J mice: transgenic animals that overexpressed IL-4 (IL-4 TG, n = 14), mice with a targeted knockout mutation of the IL-4 gene (IL-4 KO, n = 11), and wild-type (WT, n = 13) mice. At 14 days, lung fibrosis was evaluated by hydroxyproline measurement and by quantitative image analysis of fibrosis fraction and alveolar wall area fraction. Bronchoalveolar lavage cell counts in all Bleo-treated groups demonstrated an increased percentage of lymphocytes with a corresponding decrease in the percentage of macrophages. Comparing Bleo- to Sal-treated controls within each group of mice showed increases in all lung fibrosis parameters in IL-4 KO and WT, but not in any of the parameters in IL-4 TG mice. The severity of Bleo-induced fibrotic response was decreased in overexpressed IL-4 TG compared with IL-4 KO mice. These data negate a critical profibrotic role for IL-4 in Bleo-induced lung fibrosis.
Thoughts on the pulmonary blood-gas barrier
Authors: West John B.
Published: March 2015

The pulmonary blood-gas barrier is an extraordinary structure because of its extreme thinness, immense strength, and enormous area. The essential components of the barrier were determined early in evolution and have been highly conserved. For example, the barriers of the African, Australian, and South American lungfish that date from as much as 400 million years ago have essentially the same structure as in the modern mammal or bird. In the evolution of vertebrates from bony fishes through amphibia, reptiles, and ultimately mammals and birds, changes in the pulmonary circulation occurred to limit the stresses in the blood-gas barrier. Only in mammals and birds is there a complete separation of the pulmonary and systemic circulations, which is essential to protect the extremely thin barrier from the necessary high-vascular pressures. To provide the blood-gas barrier with its required strength, evolution has exploited the high ultimate tensile strength of type IV collagen in basement membrane. Nevertheless, stress failure of the barrier occurs under physiological conditions in galloping Thoroughbred racehorses and also apparently in elite human athletes at maximal exercise. The human blood-gas barrier maintains its integrity during all but the most extreme physiological conditions. However, many pathological conditions cause stress failure. The structure of the blood-gas barrier is apparently continually regulated in response to wall stress, and this regulation is essential to maintain the extreme thinness but adequate strength. The mechanisms of this regulation remain to be elucidated and constitute one of the fundamental problems in lung biology.
Hepatocyte growth factor is produced by blood and alveolar neutrophils in acute respiratory failure
Authors: Jaffré Sandrine , Dehoux Monique , Paugam Catherine , Grenier Alain , Chollet-Martin Sylvie , Stern Jean-Baptiste , Mantz Jean , Aubier Michel , Crestani Bruno
Published: March 2015

We tested the novel hypothesis that neutrophils in the lung or the airspaces may produce hepatocyte growth factor (HGF) in ventilated patients with acute respiratory failure. Neutrophils were purified from blood and bronchoalveolar lavage (BAL) fluid samples from 16 mechanically ventilated patients who underwent BAL for a diagnostic workup of ventilator-acquired pneumonia. Most of the patients had pneumonia ( n = 11). Ten nonventilated patients served as controls. Both blood and BAL neutrophils released HGF in vitro. Basal HGF secretion by blood neutrophils from controls was 823 (666) pg · ml−1· 10−7neutrophils (median, 25th–75th percentile) and doubled to 1,730 (1,684–2,316) pg · ml−1· 10−7neutrophils ( P = 0.001) with lipopolysaccharide (LPS) stimulation. Basal HGF secretion by blood neutrophils from patients was similar [956 (655–2,140) pg · ml−1· 10−7neutrophils, P = 0.4] and doubled with LPS stimulation [2,767 (2,165–3,688) pg · ml−1· 10−7neutrophils, P < 0.0001 vs. controls]. Alveolar neutrophils released HGF in vitro [653 (397–1,209) pg · ml−1· 10−7neutrophils]. LPS stimulation did not significantly increase the HGF release from alveolar neutrophils [762 (434–1,305) pg · ml−1· 10−7neutrophils]. BAL HGF positively correlated with the BAL neutrophil count ( P = 0.01, R = 0.58). We conclude that blood and alveolar neutrophils from patients with acute respiratory failure can produce HGF, a mitogenic factor that may enhance the alveolar repair process.
Surfactant protein A differentially regulates peripheral and inflammatory neutrophil chemotaxis
Authors: Schagat Trista L. , Wofford Jessica A. , Greene Kelly E. , Wright Jo Rae
Published: March 2015

Surfactant protein A (SP-A), a pulmonary lectin, plays an important role in regulating innate immune cell function. Besides accelerating pathogen clearance by pulmonary phagocytes, SP-A also stimulates alveolar macrophage chemotaxis and directed actin polymerization. We hypothesized that SP-A would also stimulate neutrophil chemotaxis. With the use of a Boyden chamber assay, we found that SP-A (0.5–25 μg/ml) did not stimulate chemotaxis of rat peripheral neutrophils or inflammatory bronchoalveolar lavage (BAL) neutrophils isolated from LPS-treated lungs. However, SP-A affected neutrophil chemotaxis toward the bacterial peptide formyl-met-leu-phe (fMLP). Surprisingly, the effect was different for the two neutrophil populations: SP-A reduced peripheral neutrophil chemotaxis toward fMLP (49 ± 5% fMLP alone) and enhanced inflammatory BAL neutrophil chemotaxis (277 ± 48% fMLP alone). This differential effect was not seen for the homologous proteins mannose binding lectin and complement protein 1q but was recapitulated by type IV collagen. SP-A bound both neutrophil populations comparably and did not alter formyl peptide binding. These data support a role for SP-A in regulating neutrophil migration in pulmonary tissue.
Statin-mediated correction of STAT1 signaling and inducible nitric oxide synthase expression in cystic fibrosis epithelial cells
Authors: Kreiselmeier Norman E. , Kraynack Nathan C. , Corey Deborah A. , Kelley Thomas J.
Published: March 2015

The expression of the inducible form of nitric oxide synthase (NOS2) is reduced in cystic fibrosis (CF) epithelium despite the presence of aggressive inflammation. A potential mechanism for reduced NOS2 expression in CF is diminished signal transducer and activator of transcription-1 (STAT1) activity, possibly due to an increase in expression of protein inhibitor of activated STAT1 (PIAS1). Previous evidence also suggests that NOS2 expression can be negatively regulated by increased activation of the GTPase RhoA, leading to the hypothesis that CF-related increases in PIAS1 expression and altered STAT1 signaling may be mediated by Rho GTPase function. Consistent with this hypothesis, data demonstrate increased expression of RhoA in two models of CF epithelium with a proportional increase in the active GTP-bound RhoA. Mouse embryonic fibroblasts null for p190B Rho GTPase-activating protein exhibit increased RhoA protein content and activation, similar to what is observed in CF models, and also exhibit CF-like alterations in STAT1 regulation, including decreased STAT1 activation, increased PIAS1 protein expression, and reduced NOS2 induction, implicating RhoA-mediated signaling in CF-related STAT1 alterations. Inhibition of the Rho GTPase pathway at the level of isoprenoid/cholesterol synthesis with mevastatin reduces PIAS1 expression, increases STAT1 activation, and restores NOS2 expression in models of CF epithelium, suggesting that pharmacological inhibition of the isoprenoid synthesis/Rho GTPase pathway may represent a potential avenue for therapeutic intervention for CF.
Parathyroid hormone-related protein response to hyperoxic lung injury
Authors: Hastings Randolph H. , Ryan Rita M. , D'Angio Carl T. , Holm Bruce A. , Patel Alka , Quintana Rick , Biederman Elana , Burton Douglas W. , Deftos Leonard J.
Published: March 2015

Parathyroid hormone-related protein (PTHrP) is a growth inhibitor for alveolar type II cells. Type II cell proliferation after lung injury from 85% oxygen is regulated, in part, by a fall in lung PTHrP. In this study, we investigated lung PTHrP after injury induced by >95% oxygen in rats and rabbits. In adult rats, lung PTHrP rose 10-fold over controls to 6,356 ± 710 pg/ml (mean ± SE) at 48 h of hyperoxia. Levels fell to 299 ± 78 pg/ml, and staining for PTHrP mRNA was greatly reduced at 60 h ( P < 0.05), the point of most severe injury and greatest pneumocyte proliferation. In adult rabbits, lung PTHrP peaked at 3,289 ± 230 pg/ml after 64 h of hyperoxia with 24 h of normoxic recovery and then dropped to 1,629 ± 153 pg/ml at 48 h of recovery ( P < 0.05). Type II cell proliferation peaked shortly after the fall in PTHrP. In newborn rabbits, lavage PTHrP increased by 50% during the first 8 days of hyperoxia, whereas type II cell growth decreased. PTHrP declined at the LD50, concurrent with increased type II cell division. In summary, lung PTHrP initially rises after injury with >95% hyperoxia and then falls near the peak of injury. Changes in PTHrP are temporally related to type II cell proliferation and may regulate repair of lung injury.
ATP stimulates Ca2+oscillations and contraction in airway smooth muscle cells of mouse lung slices
Authors: Bergner Albrecht , Sanderson Michael J.
Published: March 2015

In airway smooth muscle cells (SMCs) from mouse lung slices, ≥10 μM ATP induced Ca2+oscillations that were accompanied by airway contraction. After ∼1 min, the Ca2+oscillations subsided and the airway relaxed. By contrast, ≥0.5 μM adenosine 5′- O-(3-thiotriphosphate) (nonhydrolyzable) induced Ca2+oscillations in the SMCs and an associated airway contraction that persisted for >2 min. Adenosine 5′- O-(3-thiotriphosphate)-induced Ca2+oscillations occurred in the absence of external Ca2+but were abolished by the phospholipase C inhibitor U-73122 and the inositol 1,4,5-trisphosphate receptor inhibitor xestospongin. Adenosine, AMP, and α,β-methylene ATP had no effect on airway caliber, and the magnitude of the contractile response induced by a variety of nucleotides could be ranked in the following order: ATP = UTP > ADP. These results suggest that the SMC response to ATP is impaired by ATP hydrolysis and mediated via P2Y2or P2Y4receptors, activating phospholipase C to release Ca2+via the inositol 1,4,5-trisphosphate receptor. We conclude that ATP can serve as a spasmogen of airway SMCs and that Ca2+oscillations in SMCs are required to sustain airway contraction.
Role and regulation of activator protein-1 in toxicant-induced responses of the lung
Authors: Reddy Sekhar P. M. , Mossman Brooke T.
Published: March 2015

Aberrant cell proliferation and differentiation after toxic injury to airway epithelium can lead to the development of various lung diseases including cancer. The activator protein-1 (AP-1) transcription factor, composed of mainly Jun-Jun and Jun-Fos protein dimers, acts as an environmental biosensor to various external toxic stimuli and regulates gene expression involved in various biological processes. Gene disruption studies indicate that the AP-1 family members c- jun, junB , and fra1 are essential for embryonic development, whereas junD, c- fos, and fosB are required for normal postnatal growth. However, broad or target-specific transgenic overexpression of the some of these proteins gives very distinct phenotype(s), including tumor formation. This implies that, although they are required for normal cellular processes, their abnormal activation after toxic injury can lead to the pathogenesis of the lung disease. Consistent with this view, various environmental toxicants and carcinogens differentially regulate Jun and Fos expression in cells of the lung both in vivo and in vitro. Moreover, Jun and Fos proteins distinctly bind to the promoter regions of a wide variety of genes to differentially regulate their expression in epithelial injury, repair, and differentiation. Importantly, lung tumors induced by various carcinogens display a sustained expression of certain AP-1 family members. Therefore a better understanding of the mechanisms of regulation and functional role(s), as well as identification of target genes of members of the AP-1 family in airway epithelial cells, will provide additional insight into toxicant-induced lung diseases. These studies might offer a unique opportunity to use AP-1 family members and transactivation as potential diagnostic markers or drug targets for early detection and/or prevention of various lung diseases.
Role of gap junctions in CO2 chemoreception and respiratory control
Authors: Dean Jay B. , Ballantyne David , Cardone Daniel L. , Erlichman Joseph S. , Solomon Irene C.
Published: March 2015

Gap junctions are composed of connexins, which are organized into intercellular channels that form transmembrane pathways between neurons (cell-cell coupling), and in some cases, neurons and glia, for exchange of ions and small molecules (metabolic coupling) and ionic current (electrical coupling). Cell-cell coupling via gap junctions has been identified in brain stem neurons that function in CO2/H+ chemoreception and respiratory rhythmogenesis; however, the exact roles of gap junctions in respiratory control are undetermined. Here we review the methods commonly used to study gap junctions in the mammalian brain stem under in vitro and in vivo conditions and briefly summarize the anatomical, pharmacological, and electrophysiological evidence to date supporting roles for cell-cell coupling in respiratory rhythmogenesis and central chemoreception. Specific research questions related to the role of gap junctions in respiratory control are suggested for future research.
Activation of protein kinase A accelerates bovine bronchial epithelial cell migration
Authors: Spurzem John R. , Gupta Jitendrakumar , Veys Thomas , Kneifl Kristen R. , Rennard Stephen I. , Wyatt Todd A.
Published: March 2015

Bronchial epithelial cell migration is required for the repair of damaged airway epithelium. We hypothesized that bronchial epithelial cell migration during wound repair is influenced by cAMP and the activity of its cyclic nucleotide-dependent protein kinase, protein kinase A (PKA). We found that, when confluent monolayers of bronchial epithelial cells are wounded, an increase in PKA activity occurs. Augmentation of PKA activity with a cell-permeable analog of cAMP, dibutyryl adenosine 3′,5′-cyclic monophosphate, isoproterenol, or a phosphodiesterase inhibitor accelerated migration of normal bronchial epithelial cells in in vitro wound closure assays and Boyden chamber migration assays. A role for PKA activity was also confirmed with a PKA inhibitor, KT-5720, which reduced stimulated migration. Augmentation of PKA activity reduced the levels of active Rho and the formation of focal adhesions. These studies suggest that PKA activation modulates Rho activity, migration mechanisms, and thus bronchial epithelial repair mechanisms.
Ventilator-induced lung injury: in vivo and in vitro mechanisms
Authors: Matthay Michael A. , Bhattacharya Sunita , Gaver Donald , Ware Lorraine B. , Lim Lina H. K. , Syrkina Olga , Eyal Fabien , Hubmayr Rolf
Published: March 2015

A lung-protective ventilator strategy significantly reduces mortality in patients with acute lung injury. Substantial progress has been made in understanding how mechanical stress can injure the lung, both in terms of alterations in barrier properties of the pulmonary endothelium and epithelium as well as in stimulating proinflammatory responses of macrophages and neutrophils.
Mediator generation and signaling events in alveolar epithelial cells attacked byS. aureusα-toxin
Authors: Rose Frank , Dahlem Gabriele , Guthmann Bernd , Grimminger Friedrich , Maus Ulrich , Hänze Jörg , Duemmer Nils , Grandel Ulrich , Seeger Werner , Ghofrani Hossein Ardeschir
Published: March 2015

Staphylococcus aureus α-toxin is a pore-forming bacterial exotoxin that has been implicated as a significant virulence factor in human staphylococcal diseases. In primary cultures of rat pneumocyte type II cells and the human A549 alveolar epithelial cell line, purified α-toxin provoked rapid-onset phosphatidylinositol (PtdIns) hydrolysis as well as liberation of nitric oxide and the prostanoids PGE2, PGI2, and thromboxane A2. In addition, sustained upregulation of proinflammatory interleukin (IL)-8 mRNA expression and protein secretion occurred. “Priming” with low-dose IL-1β markedly enhanced the IL-8 response to α-toxin, which was then accompanied by IL-6 appearance. The cytokine response was blocked by the intracellular Ca2+-chelating reagent 1,2-bis(2-aminophenoxy)-ethane- N,N,N′ ,N′-tetraacetic acid, the protein kinase C inhibitor bis-indolyl maleimide I, as well as two independent inhibitors of nuclear factor-κB activation, pyrrolidine dithiocarbamate and caffeic acid phenethyl ester. We conclude that alveolar epithelial cells are highly reactive target cells of staphylococcal α-toxin. α-Toxin pore-associated transmembrane Ca2+flux and PtdIns hydrolysis-related signaling with downstream activation of protein kinase C and nuclear translocation of nuclear factor-κB are suggested to represent important underlying mechanisms. Such reactivity of the alveolar epithelial cells may be relevant for pathogenic sequelae in staphylococcal lung disease.
Decreased alveolar oxygen induces lung inflammation
Authors: Madjdpour C. , Jewell U. R. , Kneller S. , Ziegler U. , Schwendener R. , Booy C. , Kläusli L. , Pasch T. , Schimmer R. C. , Beck-Schimmer B.
Published: March 2015

Molecular mechanisms of the inflammatory reaction in hypoxia-induced lung injury are not well defined. Therefore, effects of alveolar hypoxia were studied in rat lungs, exposing rats to 10% oxygen over periods of 1, 2, 4, 6, and 8 h. An increase in the number of macrophages in bronchoalveolar lavage fluid of hypoxic animals was shown between 1 and 8 h. Extravasation of albumin was enhanced after 1 h and remained increased throughout the study period. NF-κB-binding activity as well as mRNA for TNF-α, macrophage inflammatory protein (MIP)-1β, and monocyte chemoattractant protein (MCP)-1 were increased within the first 2 h of exposure to hypoxia. Hypoxia-inducible factor (HIF)-1α and intercellular adhesion molecule (ICAM)-1 mRNA were upregulated between 1 and 6 h. Elimination of alveolar macrophages by intratracheal application of liposome-encapsulated clodronate led to a decreased expression of NF-κB binding activity, HIF-1α, TNF-α, ICAM-1, and MIP-1β. In summary, alveolar hypoxia induced macrophage recruitment, an increase in albumin leakage, and enhanced expression of inflammatory mediators, which were mainly macrophage dependent. Alveolar macrophages appear to have a prominent role in the inflammatory response in hypoxia-induced lung injury and the related upregulation of inflammatory mediators.
Growth and differentiation of mouse tracheal epithelial cells: selection of a proliferative population
Authors: You Yingjian , Richer Edward J. , Huang Tao , Brody Steven L.
Published: March 2015

Highly regulated programs for airway epithelial cell proliferation and differentiation during development and repair are often disrupted in disease. These processes have been studied in mouse models; however, it is difficult to isolate and identify epithelial cell-specific responses in vivo. To investigate these processes in vitro, we characterized a model for primary culture of mouse tracheal epithelial cells. Small numbers of cells seeded at low density (7.5 × 104 cells/cm2) rapidly proliferated and became polarized. Subsequently, supplemented media and air-liquid interface conditions resulted in development of highly differentiated epithelia composed of ciliated and nonciliated cells with gene expression characteristic of native airways. Genetically altered or injured mouse tracheal epithelial cells also reflected in vivo patterns of airway epithelial cell gene expression. Passage of cells resulted in continued proliferation but limited differentiation after the first passage, suggesting that transit-amplifying cell populations were present but with independent programs for proliferation and differentiation. This approach provides a high-fidelity in vitro model for evaluation of gene regulation and expression in mouse airway epithelial cells.
Dopamine activates ERKs in alveolar epithelial cells via Ras-PKC-dependent and Grb2/Sos-independent mechanisms
Authors: Guerrero Carmen , Pesce Liuska , Lecuona Emilia , Ridge Karen M. , Sznajder Jacob I.
Published: March 2015

Recently it has been described that dopamine (DA), via dopaminergic type 2 receptors (D2R), activates the mitogen-activated protein kinase extracellular signal-regulated kinase (MAPK/ERK) proteins in alveolar epithelial cells (AEC), which results in the upregulation of Na+-K+-ATPase. In the present report, we used AEC to investigate the signaling pathway that links DA with ERK activation. Incubation of AEC with DA resulted in rapid and transient stimulation of ERK activity, which was mediated by Ras proteins and the serine/threonine kinase Raf-1. Pretreatment of AEC with Src homology 3 binding peptide, which blocks the interaction between Grb2 and Sos, did not prevent DA activation of ERK. Diacylglycerol (DAG)-dependent protein kinase C (PKC) isoenzymes, involved in the DA-mediated activation of ERK proteins as pretreatment with either bisindolylmaleimide or Ro-31-8220, prevented the phosphorylation of Elk-1, and quinpirole, a D2R activator, stimulates the translocation of PKCε. Together, the data suggest that DA activated MAPK/ERK via Ras, Raf-1 kinase, and DAG-dependent PKC isoenzymes, but, importantly and contrary to the classical model, this pathway did not involve the Grb2-Sos complex formation.
Effect of surfactant on pulmonary expression of type IIA PLA2 in an animal model of acute lung injury
Authors: Wu Yongzheng , Singer Monique , Thouron Françoise , Alaoui-El-Azher Mounia , Touqui Lhousseine
Published: March 2015

We previously showed that the seminatural surfactant Curosurf inhibits the in vitro synthesis of secretory type IIA phospholipase A2 (sPLA2-IIA) in alveolar macrophages (AM). These cells are the main source of sPLA2-IIA in a guinea pig model of lipopolysaccharide (LPS)-induced acute lung injury (ALI). Here, we investigate the effect of Curosurf on the pulmonary synthesis of sPLA2-IIA in this ALI model. Our results showed that intratracheal administration of LPS (330 μg/kg) induced an increase in pulmonary expression of sPLA2-IIA, which was inhibited when animals received Curosurf (16 mg/guinea pig) 30 min or 8 h after LPS instillation. When AM were isolated from LPS-treated animals and cultured in conditioned medium, they expressed higher levels of sPLA2-IIA than AM from saline-treated animals. This ex vivo sPLA2-IIA expression was significantly reduced when guinea pigs received Curosurf 30 min after LPS instillation. Finally, we examined the effect of Curosurf on pulmonary inflammation measured 8 or 24 h after LPS administration. Curosurf instillation 30 min or 8 h after LPS reversed the increase in tumor necrosis factor-α expression, polymorphonuclear cell extravasation, and protein concentration in bronchoalveolar lavage fluids. Curosurf also decreased the bronchial reactivity induced by LPS. We conclude that Curosurf inhibits the pulmonary expression of sPLA2-IIA and exhibits palliative anti-inflammatory effects in an animal model of ALI.