bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein kinase C modulates pulmonary endothelial permeability: a paradigm for acute lung injury
Authors: Siflinger-Birnboim Alma , Johnson Arnold
Published: March 2015

The intracellular serine/threonine kinase protein kinase C (PKC) has an important role in the genesis of pulmonary edema. This review discusses the PKC-mediated mechanisms that participate in the pulmonary endothelial response to agents involved in lung injury characteristic of the respiratory distress syndrome. Thus the paradigms of PKC-induced lung injury are discussed within the context of pulmonary transvascular fluid exchange. We focus on the signal transduction pathways that are modulated by PKC and their effect on lung endothelial permeability. Specifically, α-thrombin, tumor necrosis factor (TNF)-α, and reactive oxygen species are discussed because of their well-established roles in both human and experimental lung injury. We conclude that PKC, most likely PKC-α, is a primary supporter for lung endothelial injury in response to α-thrombin, TNF-α, and reactive oxygen species.
Prior burn insult induces lethal acute lung injury in endotoxemic mice: effects of cytokine inhibition
Authors: Sasaki Junichi , Fujishima Seitaro , Iwamura Hiroyuki , Wakitani Korekiyo , Aiso Sadakazu , Aikawa Naoki
Published: March 2015

Many patients who experience surgical stress, including burn injury, become susceptible to severe sepsis and septic organ dysfunction, which remains the primary contributor to morbidity and mortality in burn patients. In the present study, we developed a murine model of burn-primed sublethal endotoxemia by producing a 15% BSA full-thickness burn on the dorsum of BALB/c mice under ether anesthesia and administering 10 mg/kg of LPS intravenously on day 11 to model endotoxemia. The prior burn injury in this model induced two-peaked production of cytokines, TNF-α, and macrophage inflammatory protein-2 at 2 and 12 h after LPS administration, and it was associated with increased mortality. We also assessed the effect of pharmacological modulation with cytokine synthesis inhibitors prednisolone and JTE-607 and found that pretreatment with them attenuated later cytokine production and decreased mortality after LPS administration. We speculate that the prior burn injury primed the mice for the secondary insult via cytokine production. These results also suggested that an anticytokine strategy might serve as a novel prophylactic therapy for septic organ dysfunction in burn-primed patients.
Pulmonary-specific expression of tumor necrosis factor-α alters surfactant lipid metabolism
Authors: Carroll James L. , McCoy Diann M. , McGowan Stephen E. , Salome Ronald G. , Ryan Alan J. , Mallampalli Rama K.
Published: March 2015

Tumor necrosis factor (TNF)-α is a major cytokine implicated in inducing acute and chronic lung injury, conditions associated with surfactant phosphatidylcholine (PtdCho) deficiency. Acutely, TNF-α decreases PtdCho synthesis but stimulates surfactant secretion. To investigate chronic effects of TNF-α, we investigated PtdCho metabolism in a murine transgenic model exhibiting lung-specific TNF-α overexpression. Compared with controls, TNF-α transgenic mice exhibited a discordant pattern of PtdCho metabolism, with a decrease in PtdCho and disaturated PtdCho (DSPtdCho) content in the lung, but increased levels in alveolar lavage. Transgenics had lower activities and increased immunoreactive levels of cytidylyltransferase (CCT), a key PtdCho biosynthetic enzyme. Ceramide, a CCT inhibitor, was elevated, and linoleic acid, a CCT activator, was decreased in transgenics. Radiolabeling studies revealed that alveolar reuptake of DSPtdCho was significantly decreased in transgenic mice. These observations suggest that chronic expression of TNF-α results in a complex pattern of PtdCho metabolism where elevated lavage PtdCho may originate from alveolar inflammatory cells, decreased surfactant reuptake, or altered surfactant secretion. Reduced parenchymal PtdCho synthesis appears to be attributed to CCT enzyme that is physiologically inactivated by ceramide or by diminished availability of activating lipids.
Hyperoxia-induced NAD(P)H oxidase activation and regulation by MAP kinases in human lung endothelial cells
Authors: Parinandi Narasimham L. , Kleinberg Michael A. , Usatyuk Peter V. , Cummings Rhett J. , Pennathur Arjun , Cardounel Arturo J. , Zweier Jay L. , Garcia Joe G. N. , Natarajan Viswanathan
Published: March 2015

Hyperoxia increases reactive oxygen species (ROS) production in vascular endothelium; however, the mechanisms involved in ROS generation are not well characterized. We determined the role and regulation of NAD(P)H oxidase in hyperoxia-induced ROS formation in human pulmonary artery endothelial cells (HPAECs). Exposure of HPAECs to hyperoxia for 1, 3, and 12 h increased the generation of superoxide anion, which was blocked by diphenyleneiodonium but not by rotenone or oxypurinol. Furthermore, hyperoxia enhanced NADPH- and NADH-dependent and superoxide dismutase- or diphenyleneiodonium-inhibitable ROS production in HPAECs. Immunohistocytochemistry and Western blotting revealed the presence of gp91, p67 phox, p22 phox, and p47 phox subcomponents of NADPH oxidase in HPAECs. Transfection of HPAECs with p22 phox antisense plasmid inhibited hyperoxia-induced ROS production. Exposure of HPAECs to hyperoxia activated p38 MAPK and ERK, and inhibition of p38 MAPK and MEK1/2 attenuated the hyperoxia-induced ROS generation. These results suggest a role for MAPK in regulating hyperoxia-induced NAD(P)H oxidase activation in HPAECs.
Negative impact of tissue inhibitor of metalloproteinase-3 null mutation on lung structure and function in response to sepsis
Authors: Martin Erica L. , Moyer Brent Z. , Pape M. Cynthia , Starcher Barry , Leco Kevin J. , Veldhuizen Ruud A. W.
Published: March 2015

Matrix metalloproteinases (MMPs) are degradative enzymes, which act to remodel tissue. Their activity is regulated by the tissue inhibitors of metalloproteinases (TIMPs). An imbalance in the degradation/inhibition activities has been associated with many diseases, including sepsis. We have previously shown that TIMP-3 knockout animals develop spontaneous, progressive air space enlargement. The objectives of this study were to determine the effects of a septic lung stress induced by cecal ligation and perforation (CLP) on lung function, structure, pulmonary surfactant, and inflammation in TIMP-3 null mice. Knockout and wild-type animals were randomized to either sham or CLP surgery, allowed to recover for 6 h, and then euthanized. TIMP-3 null animals exposed to sham surgery had a significant increase in lung compliance when compared with sham wild-type mice. Additionally, the TIMP-3 knockout mice showed a significant increase in compliance following CLP. Rapid compliance changes were accompanied by significantly decreased collagen and fibronectin levels and increased gelatinase (MMP-2 and -9) abundance and activation. Additionally, in situ zymography showed increased airway-associated gelatinase activity in the knockout animals enhanced following CLP. In conclusion, exposing TIMP-3 null animals to sepsis rapidly enhances the phenotypic abnormalities of these mice, due to increased MMP activity induced by CLP.
Modulation of Na-K-2Cl cotransport by intracellular Cl− and protein kinase C-δ in Calu-3 cells
Authors: Liedtke Carole M. , Papay Robert , Cole Thomas S.
Published: March 2015

In this study, we tested the hypothesis that intracellular Cl−(Cl[Formula: see text]) regulates the activity of protein kinase C (PKC)-δ and thus the activation of Na-K-Cl cotransport (NKCC1) in a Calu-3 cell line. The α1-adrenergic agonist methoxamine (MOX) and hypertonic sucrose increased Cl[Formula: see text] and increased or decreased intracellular volume, respectively, without changing Cl[Formula: see text] concentration ([Cl−]i). Titration of [Cl−]i from 20–140 mM in nystatin-permeabilized cell monolayers did not affect the baseline activity of PKC-δ, PKC-ζ, or rottlerin-sensitive NKCC1. At 200 mM Cl−, rottlerin-sensitive NKCC1 was activated, and PKC isotypes were localized predominantly to a particulate fraction. MOX induced a biphasic increase in NKCC1 activity and PKC-δ in activity and particulate localization of PKC-δ and -ζ. Activity of NKCC1 and PKC-δ decreased with increasing Cl[Formula: see text] from 20 to 80 mM Cl[Formula: see text] then increased at 140–200 mM Cl[Formula: see text] apparently as an additive effect to high [Cl−]i levels. Rottlerin inhibited the effects of MOX, which indicates that PKC-δ was required for activation of NKCC1. The results indicate that, in airway epithelial cells, a Cl[Formula: see text] electrochemical gradient alone is not sufficient to stimulate NKCC1 activity; rather, elevated activity of PKC-δ is necessary. Further, high Cl[Formula: see text] levels induce a subcellular redistribution of PKC-δ, which results in increased enzyme activity.
Mesenchymal-epithelial interactions in lung development and repair: are modeling and remodeling the same process?
Authors: Demayo F. , Minoo P. , Plopper C. G. , Schuger L. , Shannon J. , Torday J. S.
Published: March 2015

We propose that lung morphogenesis and repair are characterized by complex cell-cell interactions of endodermal and mesodermal origin, leading to (or returning back to) an alveolar structure that can effectively exchange gases between the circulation and the alveolar space. We provide the developmental basis for cell/molecular control of lung development and disease, what is known about growth and transcription factors in normal and abnormal lung development, and how endodermal and mesodermal cell origins interact during lung development and disease. The global mechanisms that mediate mesenchymal-epithelial interactions and the plasticity of mesenchymal cells in normal lung development and remodeling provide a functional genomic model that may bring these concepts closer together. We present a synopsis followed by a vertical integration of the developmental and injury/repair mechanisms.
Role of poly(ADP-ribose) synthetase in pulmonary leukocyte recruitment
Authors: Kiefmann Rainer , Heckel Kai , Dörger Martina , Schenkat Sonja , Stoeckelhuber Mechthild , Węsierska-Gądek Józefa , Goetz Alwin E.
Published: March 2015

During systemic inflammation, recruitment and activation of leukocytes in the pulmonary microcirculation may result in a potentially life-threatening acute lung injury. We elucidated the role of the poly(ADP-ribose) synthetase (PARS), a nucleotide-polymerizing enzyme, in the regulation of leukocyte recruitment within the lung with regard to the localization in the pulmonary microcirculation and in correlation to hemodynamics in the respective vascular segments and expression of intercellular adhesion molecule 1 during endotoxemia. Inhibition of PARS by 3-aminobenzamide reduced the endotoxin-induced leukocyte recruitment within pulmonary arterioles, capillaries, and venules in rabbits as quantified by in vivo fluorescence microscopy. Microhemodynamics and thus shear rates in all pulmonary microvascular segments remained constant. Simultaneously, inhibition of PARS with 3-aminobenzamide suppressed the endotoxin-induced adhesion molecules expression as demonstrated for intercellular adhesion molecule 1 by immunohistochemistry and Western blot analysis. We confirmed this result with the use of PARS knockout mice. The inhibitory effect of 3-aminobenzamide on leukocyte recruitment was associated with a reduction of pulmonary capillary leakage and edema formation. We first provide evidence that PARS activation mediates the leukocyte sequestration in pulmonary microvessels through upregulation of adhesion molecules. As reactive oxygen species released from leukocyte are supposed to cause an upregulation of adhesion molecules we conclude that PARS inhibition contributes to termination of this vicious cycle and inhibits the inflammatory process.
Chronic bronchial allergic inflammation increases alveolar liquid clearance by TNF-α-dependent mechanism
Authors: Tillie-Leblond Isabelle , Guery Benoit P. H. , Janin Anne , Leberre Rozenn , Just Nicolas , Pittet Jean-François , Tonnel André-Bernard , Gosset Philippe
Published: March 2015

Bronchial inflammation in allergic asthma is associated with active exudation from the bronchial tree into the interstitial space of both mucosa and submucosa. The aim of this study was to evaluate epithelial and endothelial permeability as well as alveolar fluid movement in a model of chronic allergic inflammation in Brown-Norway rats sensitized and challenged with ovalbumin (OA). Control groups were challenged with saline solution (C), and rats were immunized by OA but not challenged (Se). Lung sections showed a marked inflammatory infiltrate associated with perivascular and peribronchiolar edema in OA. To measure alveolar liquid clearance, a 5% bovine albumin solution with 1 μCi of125I-labeled human albumin was instilled into the air spaces. Alveolar-capillary barrier permeability was evaluated by intravascular injection of 1 μCi of 131I-labeled albumin. Endothelial permeability was significantly increased in OA, from 0.08 ± 0.01 in the C group to 0.19 ± 0.03 in OA group ( P < 0.05). Final-to-initial protein ratio was also statistically higher in OA (1.6 ± 0.05) compared with C (1.38 ± 0.03, P = 0.01) and Se groups (1.42 ± 0.03, P = 0.04). Administration of anti-tumor necrosis factor-α antibodies within the instillate significantly decreased this ratio (1.32 ± 0.08, P = 0.003 vs. OA). To conclude, we demonstrated a tumor necrosis factor-α-dependent increase in alveolar fluid movement in a model of severe bronchial allergic inflammation associated with endothelial and epithelial leakage.
Ragweed-induced expression of GATA-3, IL-4, and IL-5 by eosinophils in the lungs of allergic C57BL/6J mice
Authors: Justice J. Paul , Borchers M. T. , Lee J. J. , Rowan W. H. , Shibata Y. , Van Scott M. R.
Published: March 2015

Allergen-induced recruitment of T lymphocytes and eosinophils to the airways is associated with increased expression of the transcription factor GATA-3. In this study, the relationship between airway inflammation and GATA-3 expression in the lungs was investigated using ragweed-sensitized C57BL/6J mice. Intratracheal ragweed challenge increased both the number of GATA-3-expressing cells in the perivascular and peribronchial regions and the amount of expression per cell. Interleukin (IL)-4 and IL-5 levels in bronchoalveolar lavage fluid were upregulated in parallel with GATA-3 expression. GATA-3 mRNA and protein colocalized to eosinophils. Eosinophils isolated from the lungs and stimulated with phorbol 12-myristate 13-acetate and/or A-23187 released IL-5. The release was inhibited by actinomycin D, which indicates that de novo synthesis of the cytokine was involved. Western blot analysis of proteins from isolated eosinophils demonstrated expression of the p50 subunit of nuclear factor-κB, a transcription factor that is implicated in control of GATA-3 expression. These data provide evidence that allergen challenge increases GATA-3 and proinflammatory cytokine expression by pulmonary eosinophils, which could provide positive feedback for the inflammatory response.
Regulation of RANTES promoter activation in alveolar epithelial cells after cytokine stimulation
Authors: Casola Antonella , Henderson Allyne , Liu Tianshuang , Garofalo Roberto P. , Brasier Allan R.
Published: March 2015

Regulated on activation, normal T cell expressed, and presumably secreted (RANTES) is a member of the CC chemokine family of proteins implicated in a variety of diseases characterized by lung eosinophilia and inflammation, strongly produced by stimulated airway epithelial cells. Because such cytokines as tumor necrosis factor (TNF)-α and interferon-γ (IFN-γ) have been shown to enhance RANTES induction in airway epithelial cells and RANTES gene expression appears to be differentially regulated depending on the cell type and the stimulus applied, in this study we have elucidated mechanisms that operate to control RANTES induction on exposure to TNF-α and/or IFN-γ. Our results indicate that TNF-α and IFN-γ synergistically induce RANTES protein secretion and mRNA expression. RANTES transcription is activated only after stimulation with TNF-α, but not IFN-γ, which affects RANTES mRNA stabilization. Promoter deletion and mutagenesis experiments indicate that the nuclear factor (NF)-κB site is the most important cis-regulatory element controlling TNF-induced RANTES transcription, although NF-interleukin-6 binding site, cAMP responsive element (CRE), and interferon-stimulated responsive element (ISRE) also play a significant role. TNF-α stimulation induces nuclear translocation of interferon regulatory factor (IRF)-3, which in viral infection binds the RANTES ISRE and is necessary for activation of RANTES transcription. However, TNF-induced IRF-3 translocation does not result in IRF-3 binding to the RANTES ISRE. Although viral infection can activate an ISRE-driven promoter, TNF cannot, indicating that RANTES gene enhancers are controlled in a stimulus-specific fashion. Identification of molecular mechanisms involved in RANTES gene expression is fundamental for developing strategies to modulate lung inflammatory responses.
Ubiquitous expression of phosphodiesterase 7A in human proinflammatory and immune cells
Authors: Smith Susan J. , Brookes-Fazakerley Steven , Donnelly Louise E. , Barnes Peter J. , Barnette Mary S. , Giembycz Mark A.
Published: March 2015

We have determined the expression of phosphodiesterase (PDE) 7A1 and PDE7A2 in human cells that have been implicated in the pathogenesis of chronic obstructive pulmonary disease and asthma. Messenger RNA transcripts were detected by RT-PCR in T lymphocytes, monocytes, neutrophils, airway and vascular smooth muscle cells, lung fibroblasts, epithelial cells, and cardiac myocytes. Human epithelial, T cell, eosinophil, and lung fibroblast cell lines were also positive for PDE7A1 and PDE7A2 mRNA transcripts. By Western immunoblot analyses the amount of PDE7A1 was greatest in T cell lines, peripheral blood T lymphocytes, epithelial cell lines, airway and vascular smooth muscle cells, lung fibroblasts, and eosinophils but was not detected in neutrophils. In contrast, PDE7A2 protein, which was identified in human cardiac myocytes, was not found in any of the other cell types investigated. Immunoconfocal analyses showed that PDE7A was expressed in neutrophils and alveolar macrophages. As the expression of PDE7A mirrors the distribution of PDE4 we speculate that this enzyme could be a target for novel anti-inflammatory drugs.
Altered hemodynamics controls matrix metalloproteinase activity and tenascin-C expression in neonatal pig lung
Authors: Jones Peter Lloyd , Chapados Rene , Baldwin H. Scott , Raff Gary W. , Vitvitsky Eugene V. , Spray Thomas L. , Gaynor J. William
Published: March 2015

Tenascin-C (TN-C) expression and matrix metalloproteinase (MMP) activity are induced within remodeling pulmonary arteries (PAs), where they promote cell growth. Because pulmonary vascular disease in children with congenital heart defects is commonly associated with changes in pulmonary hemodynamics, we hypothesized that changes in pulmonary blood flow regulate TN-C and MMPs. To test this, we ligated the left PAs of neonatal pigs. After 12 wk, we evaluated the levels of TN-C and MMPs in control and ligated lung tissue. Modifying pulmonary hemodynamics increased TN-C mRNA and protein expression, MMP activity, and the DNA-binding activity of Egr-1, a transcription factor that has been shown to activate TN-C expression. To link MMP-mediated remodeling of the extracellular matrix to increased TN-C expression and Egr-1 activity, porcine PA smooth muscle cells were cultivated either on denatured type I collagen, which supported TN-C expression and Egr-1 activity, or on native collagen, which had the opposite effect. These data provide a framework for understanding how changes in pulmonary blood flow in the neonate modify the tissue microenvironment and cell behavior.
Oxygen-dependent signaling in pulmonary vascular smooth muscle
Authors: Raj Usha , Shimoda Larissa
Published: March 2015

The pulmonary circulation constricts in response to acute hypoxia, which is reversible on reexposure to oxygen. On exposure to chronic hypoxia, in addition to vasoconstriction, the pulmonary vasculature undergoes remodeling, resulting in a sustained increase in pulmonary vascular resistance that is not immediately reversible. Hypoxic pulmonary vasoconstriction is physiological in the fetus, and there are many mechanisms by which the pulmonary vasculature relaxes at birth, principal among which is the acute increase in oxygen. Oxygen-induced signaling mechanisms, which result in pulmonary vascular relaxation at birth, and the mechanisms by which chronic hypoxia results in pulmonary vascular remodeling in the fetus and adult, are being investigated. Here, the roles of cGMP-dependent protein kinase in oxygen-mediated signaling in fetal pulmonary vascular smooth muscle and the effects of chronic hypoxia on ion channel activity and smooth muscle function such as contraction, growth, and gene expression were discussed.
Variation of lung volume after fixation when measured by immersion or Cavalieri method
Authors: Yan Xiao , Polo Carbayo Juan Jose , Weibel Ewald R. , Hsia Connie C. W.
Published: March 2015

Organ volume is a critical parameter in morphometric analysis. The special problems of the lung as a nonsolid organ are overcome by tracheal instillation of fixatives at a constant airway pressure (Paw). Lung volume can change significantly after fixation as Paw change. To determine the variation of lung volume after fixation, we measured the volume of intact fixed lungs by serial immersion in saline (Vimm) at selected time points, compared with measurements obtained by point counting [Cavalieri Principle (Vcav)] after tissue sectioning to release Paw. Vimm was systematically higher than Vcav by 25% in dog lungs and 13% in guinea pig lungs ( P = 0.0003 between species). This size-dependent variability reflects residual elastic recoil, refolding and/or crumpling of alveolar septa after fixation. Vimm remained 14% higher than Vcav in dog lungs even after pressure release. Vcav/Vimmwas systematically lower in the upper than the lower strata of the same lung. We conclude that Vcav measured on lung slices after relaxation of Paw more precisely represents the state of the tissue to be used for subsequent morphometric analysis, particularly for large lungs.
Temporal/spatial expression of retinoid binding proteins and RAR isoforms in the postnatal lung
Authors: Hind Matthew , Corcoran Jonathan , Maden Malcolm
Published: March 2015

Endogenous retinoids have been implicated in alveologenesis in both the rat and the mouse, and exogenous retinoic acid (RA) can reverse or partially reverse experimental emphysema in adult rat and mouse models by an unknown mechanism. In this study, we examine the cellular and molecular biology of retinoid signaling during alveologenesis in the mouse. We describe the temporal and spatial expression of the retinoid binding proteins CRBP-I, CRBP-II, and CRABP-I using RT-PCR and immunohistochemistry. We identify the retinoic acid receptor isoforms RAR-α1, RAR-β2, RAR-β4, and RAR-γ2 and describe their temporal and spatial expression using RT-PCR and in situ hybridization. We demonstrate that both retinoid binding proteins and RAR isoforms are temporally regulated and found within the alveolar septal regions during alveologenesis. These data support a role of dynamic endogenous RA signaling during alveolar formation.
ThePseudomonassecretory product pyocyanin inhibits catalase activity in human lung epithelial cells
Authors: O'Malley Yunxia Q. , Reszka Krzysztof J. , Rasmussen George T. , Abdalla Maher Y. , Denning Gerene M. , Britigan Bradley E.
Published: March 2015

Pyocyanin, produced by Pseudomonas aeruginosa, has many deleterious effects on human cells that relate to its ability to generate reactive oxygen species (ROS), such as superoxide and hydrogen peroxide. Human cells possess several mechanisms to protect themselves from ROS, including manganese superoxide dismutase (MnSOD), copper zinc superoxide dismutase (CuZnSOD), and catalase. Given the link between pyocyanin-mediated epithelial cell injury and oxidative stress, we assessed pyocyanin's effect on MnSOD, CuZnSOD, and catalase levels in the A549 human alveolar epithelial cell line and in normal human bronchial epithelial cells. In both cell types, CuZnSOD and MnSOD were unaltered, but over 24 h pyocyanin significantly decreased cellular catalase activity and protein content. Pyocyanin also decreased catalase mRNA. Overexpression of MnSOD in A549 cells prevented pyocyanin-mediated loss of catalase protein, but catalase activity still declined. Furthermore, pyocyanin decreased catalase activity, but not protein, in A549 cells overexpressing human catalase. These data suggest a direct effect of pyocyanin on catalase activity. Addition of pyocyanin to catalase in a cell-free system also decreased catalase activity. Mammalian catalase binds four NADPH molecules, helping maintain enzyme activity. Spin-trapping data suggest that pyocyanin directly oxidizes this NADPH, producing superoxide. We conclude that pyocyanin may decrease cellular catalase activity via both transcriptional regulation and direct inactivation of the enzyme. Decreased cellular catalase activity and failure to augment MnSOD could contribute to pyocyanin-dependent cytotoxicity.
Bulk shear viscosities of endogenous and exogenous lung surfactants
Authors: King David M. , Wang Zhengdong , Palmer Harvey J. , Holm Bruce A. , Notter Robert H.
Published: March 2015

Bulk shear viscosities were measured with a cone and plate microviscometer as a function of concentration, shear rate, and temperature for lavaged calf lung surfactant (LS), Exosurf, Infasurf, Survanta, and synthetic lipid mixtures dispersed in normal saline. Viscosity increased with phospholipid concentration for all surfactants, but its magnitude and shear dependence varied widely among the different preparations. Saline dispersions of Exosurf and synthetic phospholipids had low viscosities of only a few centipoise (cp) and exhibited minimal shear dependence. LS, Infasurf, Survanta, and lipid mixtures containing palmitic acid and tripalmitin had larger non-Newtonian viscosities that increased as shear rate decreased. At 35 mg of phospholipid/ml and 37°C, viscosity values were 52.3 cp (Survanta), 31.1 cp (LS), and 25 cp (Infasurf) at a shear rate of 77 s−1and 16.9 cp (Survanta), 10.1 cp (LS), and 6.6 cp (Infasurf) at 770 s−1. At 25 mg of phospholipid/ml and 37°C, viscosity values at 77 s−1were 28.8 cp (Survanta), 4.7 cp (LS), and 12.5 cp (Infasurf). At fixed shear rate, viscosity was substantially decreased at 23°C compared with 37°C for LS and Infasurf but was increased for Survanta. Calcium (5 mM) greatly reduced the viscosity of both Survanta and Infasurf at 37°C. Studies on synthetic mixtures indicated that phospholipid/apoprotein interactions were important in the rheology of lung-derived surfactants and that palmitic acid and tripalmitin contributed to the increased viscosity of Survanta. The viscous behavior of clinical exogenous surfactants potentially influences their delivery and distribution in lungs and varies significantly with composition, concentration, temperature, ionic environment, and physical formulation.
SP-A is necessary for increased clearance of alveolar DPPC with hyperventilation or secretagogues
Authors: Jain Deepika , Dodia Chandra , Bates Sandra R. , Hawgood Samuel , Poulain Francis R. , Fisher Aron B.
Published: March 2015

The role of surfactant protein-A (SP-A) in pulmonary uptake and metabolism of [3H]dipalmitoylphosphatidylcholine ([3H]DPPC) was studied in SP-A gene-targeted mice (SP-A −/−). Unilamellar liposomes were instilled into the trachea of anesthetized mice. Uptake was measured as dpm in lungs plus liver and kidney for in vivo experiments and in lungs and perfusate for isolated lung experiments. [3H]DPPC uptake increased with CO2-induced hyperventilation in wild-type mice (SP-A +/+) but was unchanged in SP-A −/−. Secretagogue treatment approximately doubled the uptake of [3H]DPPC in isolated lungs from SP-A +/+ but had no effect in SP-A −/−. Lungs degraded 23 ± 1.2% of internalized [3H]DPPC in SP-A +/+ and 36 ± 0.6% in SP-A −/−; degradation increased with 8-bromoadenosine 3′,5′-cyclic monophosphate in SP-A +/+ but was unchanged in SP-A −/−. Activity of lysosomal-type phospholipase A2(PLA2) was significantly greater in lungs from SP-A −/− compared with SP-A +/+. Thus SP-A is necessary for lungs to respond to hyperventilation or secretagogues with increased DPPC uptake and also modulates the PLA2-mediated degradation of internalized DPPC.
NADPH oxidase promotes NF-κB activation and proliferation in human airway smooth muscle
Authors: Brar Sukhdev S. , Kennedy Thomas P. , Sturrock Anne B. , Huecksteadt Thomas P. , Quinn Mark T. , Murphy Thomas M. , Chitano Pasquale , Hoidal John R.
Published: March 2015

Evidence is rapidly accumulating that low-activity-reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases homologous to that in phagocytic cells generate reactive oxygen species as signaling intermediates in both endothelium and vascular smooth muscle. We therefore explored the possibility of such an oxidase regulating growth of airway smooth muscle (AWSM). Proliferation of human AWSM cells in culture was inhibited by the antioxidants catalase and N-acetylcysteine, and by the flavoprotein inhibitor diphenylene iodonium (DPI). Membranes prepared from human AWSM cells generated superoxide anion (O[Formula: see text]) measured by superoxide dismutase-inhibitable lucigenin chemiluminescence, with a distinct preference for NADPH instead of reduced nicotinamide adenine dinucleotide as substrate. Chemiluminescence was also inhibited by DPI, suggesting the presence of a flavoprotein containing oxidase generating O[Formula: see text] as a signaling molecule for cell growth. Examination of human AWSM cells by reverse transcriptase-polymerase chain reaction consistently demonstrated transcripts with sequences identical to those reported for p22phox. Transfection with p22phoxantisense oligonucleotides reduced human AWSM proliferation. Inhibition of NADPH oxidase activity with DPI prevented serum-induced activation of nuclear factor-κB (NF-κB), and overexpression of a superrepressor form of the NF-κB inhibitor IκBα significantly reduced human AWSM growth. These findings suggest that an NADPH oxidase containing p22phoxregulates growth-factor responsive human AWSM proliferation, and that the oxidase signals in part through activation of the prototypical redox-regulated transcription factor NF-κB.
Diesel particles increase phosphatidylcholine release through a NO pathway in alveolar type II cells
Authors: Juvin Philippe , Fournier Thierry , Grandsaigne Martine , Desmonts Jean-Marie , Aubier Michel
Published: March 2015

Diesel exhaust particles (DEPs) have been shown in vivo as well as in vitro to affect the respiratory function and in particular the immune response to infection and allergens. In the current study, we investigated the effect of DEPs on the production of phosphatidylcholine (PC), a major constituent of surfactant, by rat alveolar type II (ATII) primary cells in vitro. Our results demonstrate that incubation of ATII cells with DEPs lead to a time- and dose-dependent increase in labeled PC release. This effect was mimicked by nitric oxide (NO) donors and cGMP and was abolished by inhibitors of NO synthase (NOS). In addition, a NOS inhibitor inhibits by itself the basal secretion of PC. We next examined the effects of DEPs on NOS gene expression and showed that DEPs increase NO production and upregulate both protein content and mRNA levels of the inducible NOS (NOS II). Together our data demonstrate that DEPs alter the production of surfactant by ATII cells through a NO-dependent signaling pathway.
Beclomethasone rapidly ablates allergen-induced β2- adrenoceptor pathway dysfunction in human isolated bronchi
Authors: Brichetto Lorenzo , Milanese Manlio , Song Pingfang , Patrone Mauro , Crimi Emanuele , Rehder Kai , Brusasco Vito
Published: March 2015

Bronchial rings from nonatopic humans were passively sensitized with serum from allergic subjects. Allergen challenge significantly reduced the relaxant effect of salbutamol on carbachol-induced contractions, suggesting β2-adrenoceptor (β2-AR) pathway dysfunction. Incubation of challenged rings for 3 h with 3 × 10−6 M beclomethasone dipropionate (BDP) restored the relaxant effect, suggesting reversal of β2-AR pathway dysfunction. Incubation with the Gsα protein-stimulating cholera toxin attenuated contractile responses to carbachol significantly less in challenged than in unchallenged rings. Treatment of challenged rings with BDP resulted in an inhibitory effect of cholera toxin that was similar to the effect in unchallenged rings. Gsα protein expression was not significantly altered by BDP, suggesting that the activity of Gsα protein was increased. Relaxation of challenged rings by forskolin was not significantly affected by BDP, suggesting that β2-AR pathway dysfunction was proximal to the adenylyl cyclase. In conclusion, short-term (3-h) treatment with BDP after allergen challenge ablated β2-AR pathway dysfunction by increasing the activity of the Gsα protein in human isolated bronchi.
Leukotrienes, IL-13, and chemokines cooperate to induce BHR and mucus in allergic mouse lungs
Authors: Vargaftig B. Boris , Singer Monique
Published: March 2015

In mice, intratracheal challenges with antigen (ovalbumin) or recombinant murine interleukin-13 (IL-13) induce lung inflammation, bronchial hyperreactivity (BHR), and mucus accumulation as independent events (Singer M, Lefort J, and Vargaftig BB. Am J Respir Cell Mol Biol 26: 74–84, 2002), largely mediated by leukotrienes (LT). We previously showed that LTC4 was released 15 min after ovalbumin, and we show that it induces the expression of monocyte chemoattractant proteins 1 and 5 and KC in the lungs, as well as IL-13 mRNA. Instilled intratracheally, these chemokines induced BHR and mucus accumulation, which were inhibited by the 5-lipoxygenase inhibitor zileuton and by the cysteinyl-LT receptor antagonist MK-571, suggesting mediation by cysteinyl-LT. Because these chemokines also induced release of LT into the bronchoalveolar lavage fluid and IL-13 into the lungs, we hypothesize that LT- and chemokine-based loops for positive-feedback regulations cooperate to maintain and amplify BHR and lung mucus accumulation after allergic challenge and in situations where IL-13, LT, or chemokines are generated.
Chondroitin sulfate proteoglycans are required for lung growth and morphogenesis in vitro
Authors: Shannon John M. , McCormick-Shannon Kathleen , Burhans Michael S. , Shangguan Xiaofei , Srivastava Kalpana , Hyatt Brian A.
Published: March 2015

Proteoglycans (PGs) have been shown to play a key role in the development of many tissues. We have investigated the role of sulfated PGs in early rat lung development by treating cultured tissues with 30 mM sodium chlorate, a global inhibitor of PG sulfation. Chlorate treatment disrupted growth and branching of embryonic day 13 lung explants. Isolated lung epithelium (LgE) migrated toward and invaded lung mesenchyme (LgM), and chlorate irreversibly suppressed this response. Chlorate also inhibited migration of LgE toward beads soaked in FGF10. Chlorate severely decreased branching morphogenesis in tissue recombinants consisting of LgM plus either LgE or tracheal epithelium (TrE) and decreased expression of surfactant protein C gene ( SP-C). Chlorate also reduced bone morphogenetic protein-4 expression in cultured tips and recombinants but had no effect on the expression of clara cell 10-kDa protein ( CC10), sonic hedgehog ( Shh), FGF10, and FGF receptor 2IIIb. Chlorate reduced the growth of LgE in mesenchyme-free culture but did not affect SP-C expression. In contrast, chlorate inhibited both rudiment growth and the induction of SP-C in mesenchyme-free cultured TrE. Treatment of lung tips and tissue recombinants with chondroitinase ABC abolished branching morphogenesis. Chondroitinase also suppressed growth of TrE in mesenchyme-free culture. Chondroitinase treatment, however, had no effect on the induction of SP-C expression in any of these cultures. These results demonstrate the overall importance of sulfated PGs to normal lung development and demonstrate a dynamic role for chondroitin sulfate PGs in embryonic lung growth and morphogenesis.
Caveolin regulation of endothelial function
Authors: Minshall Richard D. , Sessa William C. , Stan Radu V. , Anderson Richard G. W. , Malik Asrar B.
Published: March 2015

Caveolae are the sites in the cell membrane responsible for concentrating an array of signaling molecules critical for cell function. Recent studies have begun to identify the functions of caveolin-1, the 22-kDa caveolar protein that oligomerizes and inserts into the cytoplasmic face of the plasma membrane. Caveolin-1 appears to regulate caveolar internalization by stabilizing caveolae at the plasma membrane rather than controlling the shape of the membrane invagination. Because caveolin-1 is a scaffolding protein, it has also been hypothesized to function as a “master regulator” of signaling molecules in caveolae. Deletion of the caveolin-1 gene in mice resulted in cardiac hypertrophy and lung fibrosis, indicating its importance in cardiac and lung development. In the endothelium, caveolin-1 regulates nitric oxide signaling by binding to and inhibiting endothelial nitric oxide synthase (eNOS). Increased cytosolic Ca2+or activation of the kinase Akt leads to eNOS activation and its dissociation from caveolin-1. Caveolae have also been proposed as the vesicle carriers responsible for transcellular transport (transcytosis) in endothelial cells. Transcytosis, the primary means of albumin transport across continuous endothelia, occurs by fission of caveolae from the membrane. This event is regulated by tyrosine phosphorylation of caveolin-1 and dynamin. As Ca2+influx channels and pumps are localized in caveolae, caveolin-1 is also an important determinant of Ca2+signaling in endothelial cells. Many of these findings were presented in San Diego, CA, at the 2003 Experimental Biology symposium “Caveolin Regulation of Endothelial Function” and are reviewed in this summary.
Iron uptake and Nramp2/DMT1/DCT1 in human bronchial epithelial cells
Authors: Wang Xinchao , Ghio Andrew J. , Yang Funmei , Dolan Kevin G. , Garrick Michael D. , Piantadosi Claude A.
Published: March 2015

The capacity of natural resistance-associated macrophage protein-2 [Nramp2; also called divalent metal transporter-1 (DMT1) and divalent cation transporter-1 (DCT1)] to transport iron and its ubiquitous expression make it a likely candidate for transferrin-independent uptake of iron in peripheral tissues. We tested the hypothesis that non-transferrin-bound iron uptake by airway epithelial cells is associated with Nramp2/DMT1/DCT1 and that exposure to iron can increase Nramp2/DMT1/DCT1 mRNA and protein expression and transport of this metal. Exposure of BEAS-2B cells to ferric ammonium citrate (FAC) resulted in a decrease in Fe3+concentration in the supernatant that was dependent on time and initial iron concentration. In the presence of internalized calcein, FAC quenched the fluorescent signal, indicating intracellular transport of the metal. The Nramp2/DMT1/DCT1 mRNA isoform without an iron-response element (IRE) increased with exposure of BEAS-2B cells to FAC. RT-PCR demonstrated no change in the mRNA for the isoform with an IRE. Similarly, Western blot analysis for the isoform without an IRE confirmed an increased expression of this protein after FAC exposure, whereas the isoform with an IRE exhibited no change. Finally, immunohistochemistry revealed an increase in the isoform without an IRE in the rat lung epithelium after instillation of FAC. Comparable to mRNA and protein increases, iron transport was elevated after pretreatment of BEAS-2B cells with iron-containing compounds. We conclude that airway epithelial cells increase mRNA and expression of the Nramp2/DMT1/DCT1 without an IRE after exposure to iron. The increase results in an elevated transport of iron and its probable detoxification by these cells.
Mitochondrial complex I, aconitase, and succinate dehydrogenase during hypoxia-reoxygenation: modulation of enzyme activities by MnSOD
Authors: Powell Charles S. , Jackson Robert M.
Published: March 2015

Both NADH dehydrogenase (complex I) and aconitase are inactivated partially in vitro by superoxide ([Formula: see text]) and other oxidants that cause loss of iron from enzyme cubane (4Fe-4S) centers. We tested whether hypoxia-reoxygenation (H-R) by itself would decrease lung epithelial cell NADH dehydrogenase, aconitase, and succinate dehydrogenase (SDH) activities and whether transfection with adenoviral vectors expressing MnSOD (Ad.MnSOD) would inhibit oxidative enzyme inactivation and thus confirm a mechanism involving [Formula: see text]. Human lung carcinoma cells with alveolar epithelial cell characteristics (A549 cells) were exposed to <1% O2-5% CO2(hypoxia) for 24 h followed by air-5% CO2for 24 h (reoxygenation). NADH dehydrogenase activity was assayed in submitochondrial particles; aconitase and SDH activities were measured in cell lysates. H-R significantly decreased NADH dehydrogenase, aconitase, and SDH activities. Ad.MnSOD increased mitochondrial MnSOD substantially and prevented the inhibitory effects of H-R on enzyme activities. Addition of α-ketoglutarate plus aspartate, but not succinate, to medium prevented cytotoxicity due to 2,3-dimethoxy-1,4-naphthoquinone. After hypoxia, cells displayed significantly increased dihydrorhodamine fluorescence, indicating increased mitochondrial oxidant production. Inhibition of NADH dehydrogenase, aconitase, and SDH activities during reoxygenation are due to excess [Formula: see text] produced in mitochondria, because enzyme inactivation can be prevented by overexpression of MnSOD.
Transgenic expression of protein phosphatase 2A regulatory subunit B56γ disrupts distal lung differentiation
Authors: Everett Allen D. , Kamibayashi Craig , Brautigan David L.
Published: March 2015

The distal epithelium of the developing lung exhibits high-level expression of protein phosphatase 2A (PP2A), a vital signaling enzyme. Here we report the discovery that in the lung, the PP2A regulatory subunit B56γ is expressed in a discrete developmental period, with the highest protein levels at embryonic day (e) 17, but no detectable protein in the newborn or adult. By in situ hybridization, B56γ was highly expressed in the distal epithelium of newly forming airways and in mesenchymal cells. In contrast, expression of B56γ was quite low in the bronchial epithelium and vascular smooth muscle. Transgenic expression of B56γ using the lung-specific promoter for surfactant protein C (SP-C) resulted in neonatal death. Examination of lungs from SP-C-B56γ transgenic e18 fetuses revealed proximal airways and normal blood vessels, but the tissue was densely populated with epithelial-type cells and was devoid of normal peripheral lung structure. A component of the Wnt signaling pathway, β-catenin, was developmentally regulated in the normal lung and was absent in lung tissue from B-56γ transgenic fetuses. We propose that B56γ is expressed at a particular stage of lung development to modulate PP2A action on the Wnt/β-catenin signaling pathway during lung airway morphogenesis.
β-Adrenergic agonists regulate Na-K-ATPase via p70S6k
Authors: Pesce Liuska , Comellas Alejandro , Sznajder Jacob I.
Published: March 2015

We have recently reported that the β-adrenergic agonist isoproterenol regulates the alveolar epithelial cell Na-K-ATPase via MAPK/extracellular signal-regulated kinase and rapamycin-sensitive pathways. Here we report that isoproterenol phosphorylated the protein S6 kinase (p70S6k) in alveolar epithelial cells, which was inhibited by both rapamycin and the MEK1/2 inhibitor U-0126. In alveolar epithelial cells transfected with a p70S6kdominant negative construct, isoproterenol did not increase Na-K-ATPase total protein expression, whereas in cells transfected with a rapamycin-resistant mutant, the isoproterenol-mediated increase in Na-K-ATPase was not prevented by rapamycin. Accordingly, we provide here first evidence that isoproterenol regulates Na-K-ATPase via p70S6kin alveolar epithelial cells.
Effect of surfactant protein A on granular pneumocyte surfactant secretion in vitro
Authors: Bates Sandra R. , Tao Jian-Qin , Notarfrancesco Kathleen , DeBolt Kristine , Shuman Henry , Fisher Aron B.
Published: March 2015

Surfactant secretion by lung type II cells occurs when lamellar bodies (LBs) fuse with the plasma membrane and surfactant is released into the alveolar lumen. Surfactant protein A (SP-A) blocks secretagogue-stimulated phospholipid (PL) release, even in the presence of surfactant-like lipid. The mechanism of action is not clear. We have shown previously that an antibody to LB membranes (MAb 3C9) can be used to measure LB membrane trafficking. Although the ATP-stimulated secretion of PL was blocked by SP-A, the cell association of iodinated MAb 3C9 was not altered, indicating no effect on LB movement. FM1-43 is a hydrophobic dye used to monitor the formation of fusion pores. After secretagogue exposure, the threefold enhancement of the number of FM1-43 fluorescent LBs (per 100 cells) was not altered by the presence of SP-A. Finally, there was no evidence of a large PL pool retained on the cell surface through interaction with SP-A. Thus SP-A exposure does not affect these stages in the surfactant secretory pathway of type II cells.
Exaggerated hypoxic pulmonary hypertension in endothelin B receptor-deficient rats
Authors: Ivy D. Dunbar , Yanagisawa Masashi , Gariepy Cheryl E. , Gebb Sarah A. , Colvin Kelley L. , McMurtry Ivan F.
Published: March 2015

Mechanisms by which endothelin (ET)-1 mediates chronic pulmonary hypertension remain incompletely understood. Although activation of the ET type A (ETA) receptor causes vasoconstriction, stimulation of ET type B (ETB) receptors can elicit vasodilation or vasoconstriction. We hypothesized that the ETB receptor attenuates the development of hypoxic pulmonary hypertension and studied a genetic rat model of ETB receptor deficiency (transgenic sl/sl). After 3 wk of severe hypoxia, the transgenic sl/sl pulmonary vasculature lacked expression of mRNA for the ETB receptor and developed exaggerated pulmonary hypertension that was characterized by elevated pulmonary arterial pressure, diminished cardiac output, and increased total pulmonary resistance. Plasma ET-1 was fivefold higher in transgenic sl/sl rats than in transgenic controls. Although mRNA for prepro-ET-1 was not different, mRNA for ET-converting enzyme-1 was higher in transgenic sl/sl than in transgenic control lungs. Hypertensive lungs of sl/sl rats also produced less nitric oxide metabolites and 6-ketoprostaglandin F1α, a metabolite of prostacyclin, than transgenic controls. These findings suggest that the ETB receptor plays a protective role in the pulmonary hypertensive response to chronic hypoxia.