bluuebunny/crossref_metadata_2025 huggingface dataset:
In vitro sensitization of human bronchus by β2-adrenergic agonists
Authors: Faisy Christophe , Naline Emmanuel , Diehl Jean-Luc , Emonds-Alt Xavier , Chinet Thierry , Advenier Charles
Published: March 2015

Incubation of human distal bronchi from 48 patients for 15 h with 10−7 M fenoterol induced sensitization characterized by an increase in maximal contraction to endothelin-1 (ET-1) and acetylcholine (ACh). Incubation of human bronchi with 10−6, 3 × 10−6, and 10−5 M forskolin (an adenyl cyclase activator) reproduced sensitization to ET-1 and ACh. The sensitizing effect of fenoterol was inhibited by coincubation with gliotoxine (a nuclear factor-κB inhibitor), dexamethasone, indomethacin (a cyclooxygenase inhibitor), GR-32191 (a TP prostanoid receptor antagonist), MK-476 (a cysteinyl leukotriene type 1 receptor antagonist), SR-140333 + SR-48968 + SR-142801 (neurokinin types 1, 2, and 3 tachykinin receptor antagonists) with or without HOE-140 (a bradykinin B2receptor antagonist), SB-203580 (an inhibitor of the 38-kDa mitogen-activated protein kinase, p38MAPK), or calphostin C (a protein kinase C blocker). Our results suggest that chronic exposure to fenoterol induces proinflammatory effects mediated by nuclear factor-κB and pathways involving leukotrienes, prostanoids, bradykinin, tachykinins, protein kinase C, and p38MAPK, leading to the regulation of smooth muscle contraction to ET-1 and ACh.
Nitric oxide inhibits ADP-ribosyl cyclase through a cGMP-independent pathway in airway smooth muscle
Authors: White Thomas A. , Walseth Timothy F. , Kannan Mathur S.
Published: March 2015

There is evidence for a role of cyclic ADP-ribose (cADPR) in intracellular Ca2+regulation in smooth muscle. cADPR is synthesized and degraded by ADP-ribosyl cyclase and cADPR hydrolase, respectively, by a bifunctional protein, CD38. Nitric oxide (NO) inhibits intracellular Ca2+mobilization in airway smooth muscle. The present study was designed to determine whether this inhibition is due to regulation of ADP-ribosyl cyclase and/or cADPR hydrolase activity. Sodium nitroprusside (SNP) and S-nitroso- N-acetylpenicillamine, NO donors, produced a concentration-dependent decrease in ADP-ribosyl cyclase, but not cADPR hydrolase, activity. The NO scavenger carboxy-PTIO prevented and reversed, and reduced glutathione prevented, the inhibition of ADP-ribosyl cyclase by SNP, suggesting S-nitrosylation by NO as a mechanism. N-ethylmaleimide, which covalently modifies protein sulfhydryl groups, making them incapable of nitrosylation, produced a marked inhibition of ADP-ribosyl cyclase, but not cADPR hydrolase, activity. SNP and N-ethylmaleimide significantly inhibited the ADP-ribosyl cyclase activity in recombinant human CD38 without affecting the cADPR hydrolase activity. These results provide a novel mechanism for differential regulation of CD38 by NO through a cGMP-independent pathway involving S-nitrosylation of thiols.
Altered surfactant protein A gene expression and protein metabolism associated with repeat exposure to inhaled endotoxin
Authors: George Caroline L. S. , White Misty L. , O'Neill Marsha E. , Thorne Peter S. , Schwartz David A. , Snyder Jeanne M.
Published: March 2015

Chronically inhaled endotoxin, which is ubiquitous in many occupational and domestic environments, can adversely affect the respiratory system resulting in an inflammatory response and decreased lung function. Surfactant-associated protein A (SP-A) is part of the lung innate immune system and may attenuate the inflammatory response in various types of lung injury. Using a murine model to mimic occupational exposures to endotoxin, we hypothesized that SP-A gene expression and protein would be elevated in response to repeat exposure to inhaled grain dust and to purified lipopolysaccharide (LPS). Our results demonstrate that repeat exposure to inhaled endotoxin, either in the form of grain dust or purified LPS, results in increased whole lung SP-A gene expression and type II alveolar epithelial cell hyperplasia, whereas SP-A protein levels in lung lavage fluid are decreased. Furthermore, these alterations in SP-A gene activity and protein metabolism are dependent on an intact endotoxin signaling system.
Mucosal immunotherapy with CpG oligodeoxynucleotides reverses a murine model of chronic asthma induced by repeated antigen exposure
Authors: Jain Vipul V. , Businga Thomas R. , Kitagaki Kunihiko , George Caroline L. , O'Shaughnessy Patrick T. , Kline Joel N.
Published: March 2015

Murine models of acute atopic asthma may be inadequate to study the effects of recurrent exposure to inhaled allergens, such as the epithelial changes seen in asthmatic patients. We developed a murine model in which chronic airway inflammation is maintained by repeated allergen [ovalbumin (OVA)] inhalation; using this model, we examined the response to mucosal administration of CpG DNA (oligonucleotides) and specific antigen immunotherapy. Mice repeatedly exposed to OVA developed significantly greater airway hyperresponsiveness and goblet cell hyperplasia, but not airway eosinophilia, compared with those exposed only twice. CpG-based immunotherapy significantly reversed both acute and chronic markers of inflammation as well as airway hyperresponsiveness. We further examined the effect of mucosal immunotherapy on the response to a second, unrelated antigen. Mice sensitized to both OVA and schistosome eggs, challenged with inhaled OVA, and then treated with OVA-directed immunotherapy demonstrated significant reduction of airway hyperresponsiveness and a moderate reduction in eosinophilia, after inhalation challenge with schistosome egg antigens. In this model, immunotherapy treatment reduced bronchoalveolar lavage (BAL) levels of Th2 cytokines (IL-4, IL-5, IL-13, and IL-10) without changing BAL IFN-γ. Antigen recall responses of splenocytes from these mice demonstrated an antigen-specific (OVA) enhanced release of IL-10 from splenocytes of treated mice. These results suggest that CpG DNA may provide the basis for a novel form of immunotherapy of allergic asthma. Both antigen-specific and, to a lesser extent, antigen-nonspecific responses to mucosal administration of CpG DNA are seen.
In vivo tracheal occlusion in fetal mice induces rapid lung development without affecting surfactant protein C expression
Authors: Maltais France , Seaborn Tommy , Guay Stéphane , Piedboeuf Bruno
Published: March 2015

Fetal tracheal occlusion (TO) reverses lung hypoplasia by inducing rapid lung growth. Although increases in lung size accompanied by increased numbers of alveoli and capillaries have been reported, effects of TO on lung development have not been formally assessed. In the present study, the objective was to verify our prediction that the main effect of TO would be to accelerate fetal lung development. We have developed and characterized a new fetal mouse model of TO to best realize this goal. At embryonic day 16.5, pregnant CD1 mice were operated under general anesthesia. One fetus per dam was selected to undergo surgical TO with a surgical clip or a sham operation. The fetuses were delivered 24 or 36 h postsurgery. The maturation of lung parenchyma, evaluated by counting the generations of alveolar saccules from the terminal bronchiole to the pleura, was significantly accelerated in the TO group with a complexity of the gas exchange region comparable with postnatal days 1 and 3after 24 or 36 h of TO. Cellular proliferation and apoptosis peaks, assessed by immunohistochemistry directed against PCNA and the active form of caspase-3, were significantly increased 24 h after surgery in the TO group compared with the sham group. However, in situ hybridization showed no significant difference in the density of type II pneumocytes expressing surfactant protein C mRNA. Our results show that brief TO during late gestation in fetal mice induces accelerated lung development with minimal effects on surfactant protein C mRNA expression.
Nitric oxide inhibits surfactant protein B gene expression in lung epithelial cells
Authors: Salinas Darrell , Sparkman Loretta , Berhane Kiflu , Boggaram Vijayakumar
Published: March 2015

Surfactant protein B (SP-B) is an essential constituent of pulmonary surfactant. In a number of inflammatory diseases of the lung, elevated nitric oxide (NO) levels are associated with decreased SP-B levels, suggesting that reduced SP-B levels contribute to lung injury. In this study, we investigated the effects of NO on SP-B gene expression in H441 and MLE-12 cells, cell lines with characteristics of bronchiolar (Clara) and alveolar type II epithelial cells, respectively. Results show that NO donors decreased SP-B mRNA levels in a concentration- and time-dependent manner in H441 and MLE-12 cells. The NO donors also antagonized dexamethasone induction of SP-B mRNA in H441 cells. NO donor inhibition of SP-B mRNA was blocked by the transcriptional inhibitor 5,6-dichloro-1-β-d-ribofuranozyl-benzimidazole. NO donors decreased luciferase expression from a reporter plasmid containing -911/+41 bp of human SP-B 5′-flanking DNA in H441 and MLE-12 cells, indicating inhibitory effects on SP-B promoter activity. NO inhibition of SP-B mRNA levels was not blocked by LY-83583 and KT-5823, inhibitors of soluble guanylate cyclase and protein kinase G, respectively. Furthermore, cell-permeable cGMP analog 8-bromo-cGMP had no effect on SP-B mRNA levels. These data indicate that elevated NO levels negatively regulate SP-B gene expression by inhibiting gene transcription and that NO inhibits SP-B gene expression independently of cGMP levels. These data imply that reduced SP-B expression due to elevated NO levels can contribute to lung injury.
GM-CSF increases airway smooth muscle cell connective tissue expression by inducing TGF-β receptors
Authors: Chen Gang , Grotendorst Gary , Eichholtz Thomas , Khalil Nasreen
Published: March 2015

Fibrosis around the smooth muscle of asthmatic airway walls leads to irreversible airway obstruction. Bronchial epithelial cells release granulocyte/macrophage colony-stimulating factor (GM-CSF) in asthmatics and are in close proximity to airway smooth muscle cells (ASMC). The findings in this study demonstrate that GM-CSF induces confluent, prolonged, serum-deprived cultures of ASMC to increase expression of collagen I and fibronectin. GM-CSF also induced ASMC to increase the expression of transforming growth factor (TGF)-β receptors type I, II, and III (TβR-I, TβR-II, TβR-III), but had no detectable effect on the release of TGF-β1 by the same ASMC. The presence of GM-CSF also induced the association of TGF-β1 with TβR-III, which enhances binding of TGF-β1 to TβR-II. The induction of TβRs was parallel to the increased induction of phosphorylated Smad2 (pSmad2) and connective tissue growth factor (CTGF), indicative of TGF-β-mediated connective tissue synthesis. Dexamethasone decreased GM-CSF-induced TβR-I, TβR-II, TβR-III, pSmad2, CTGF, collagen I, and fibronectin. In conclusion, GM-CSF increases the responsiveness of ASMC to TGF-β1-mediated connective tissue expression by induction of TβRs, which is inhibited by corticosteroids.
Coordinate expression of fibulin-5/DANCE and elastin during lung injury repair
Authors: Kuang Ping-Ping , Goldstein Ronald H. , Liu Yue , Rishikof David C. , Jean Jyh-Chang , Joyce-Brady Martin
Published: March 2015

Fibulin-5, previously known as DANCE and EVEC, is a secreted extracellular matrix protein that functions as a scaffold for elastin fiber assembly and as a ligand for integrins αvβ3, αvβ5, and α9β1. Fibulin-5 is developmentally regulated in the lung, and lung air space enlargement develops in mice deficient in fibulin-5. Fibulin-5 is also induced in adult lung following lung injury by hyperoxia. To further examine the role of fibulin-5 during repair of lung injury, we assessed fibulin-5 expression during elastase-induced emphysema in C57/b mice. Mice were treated with either saline or elastase via the trachea, and the lung was examined 20 days after treatment. Fibulin-5 mRNA was induced almost fourfold, whereas elastin mRNA was minimally elevated. Immunohistochemistry studies showed that fibulin-5 was induced in cells within the alveolar wall following elastase treatment. Western analysis demonstrates that fibulin-5 was strongly expressed in isolated primary lung interstitial fibroblasts. Fibulin-5 protein was localized to the fibroblast cell layer in culture, and brief elastase treatment degraded the protein. Intact fibulin-5 did not accumulate in the culture media. Treatment of fibroblasts with the proinflammatory cytokine interleukin-1β abolished fibulin-5 mRNA expression. Our results indicate that fibulin-5 is coordinately expressed and regulated with elastin in lung fibroblasts and may serve a key role during lung injury and repair.
Repeated allergen inhalation induces phenotypic modulation of smooth muscle in bronchioles of sensitized rats
Authors: Moir Lyn M. , Leung Sum-Yee , Eynott Paul R. , McVicker Clare G. , Ward Jeremy P. T. , Chung K. Fan , Hirst Stuart J.
Published: March 2015

Repeated ovalbumin (OA) or saline exposure of sensitized Brown Norway rats was examined on agonist reactivity, airway smooth muscle (ASM) content, and contractile protein expression in small bronchioles at 24 h, 7 days, and 35 days after challenge. OA increased ASM content ( P < 0.05 vs. saline) at 24 h, which resolved by 7 days. Maximum developed tension (Tmax) to carbachol, KCl, and 4-β-phorbol 12,13-dibutyrate was increased ( P < 0.05) by OA in bronchioles at 24 h but was abrogated after correction for ASM. Differences in Tmaxwere not present at 7 days. In contrast, at 35 days, Tmaxwas increased ( P < 0.05) after correction for ASM. Smooth muscle (sm)-α-actin, sm-myosin heavy chain (MHC) isoform 1, calponin, smoothelin-A, and sm-myosin light chain kinase expression were reduced ( P < 0.05) by OA at 24 h in bronchioles but not in trachealis. Consistent with contraction findings, no difference in expression of these proteins was detected at 7 days. At 35 days, however, with the exception of sm-α-actin, their abundance was again reduced ( P < 0.05) by OA. Nonmuscle MHC and β-actin were unchanged throughout by OA. These findings indicate persistent changes in contractile protein content, consistent with ASM phenotypic modulation in vivo, which occur in response to repeated OA inhalation. Thus, OA exposure induces structural changes in bronchiole ASM content and in agonist responsiveness ex vivo that resemble remodeling in asthma.
Eosinophil-induced release of acetylcholine from differentiated cholinergic nerve cells
Authors: Sawatzky Deborah A. , Kingham Paul J. , Durcan Niamh , McLean W. Graham , Costello Richard W.
Published: March 2015

One immunological component of asthma is believed to be the interaction of eosinophils with parasympathetic cholinergic nerves and a consequent inhibition of acetylcholine muscarinic M2 receptor activity, leading to enhanced acetylcholine release and bronchoconstriction. Here we have used an in vitro model of cholinergic nerve function, the human IMR32 cell line, to study this interaction. IMR32 cells, differentiated in culture for 7 days, expressed M2 receptors. Cells were radiolabeled with [3H]choline and electrically stimulated. The stimulation-induced release of acetylcholine was prevented by the removal of Ca2+. The muscarinic M1/M2 receptor agonist arecaidine reduced the release of acetylcholine after stimulation (to 82 ± 2% of control at 10-7 M), and the M2 receptor antagonist AF-DX 116 increased it (to 175 ± 23% of control at 10-5 M), indicating the presence of a functional M2 receptor that modulated acetylcholine release. When human eosinophils were added to IMR32 cells, they enhanced acetylcholine release by 36 ± 10%. This effect was prevented by inhibitors of adhesion of the eosinophils to the IMR32 cells. Pretreatment of IMR32 cells with 10 mM carbachol, to desensitize acetylcholine receptors, prevented the potentiation of acetylcholine release by eosinophils or AF-DX 116. Acetylcholine release was similarly potentiated (by up to 45 ± 7%) by degranulation products from eosinophils that had been treated with N-formyl-methionyl-leucyl-phenylalanine or that had been in contact with IMR32 cells. Contact between eosinophils and IMR32 cells led to an initial increase in expression of M2 receptors, whereas prolonged exposure reduced M2 receptor expression.
Developmental changes in nitric oxide synthase isoform expression and nitric oxide production in fetal baboon lung
Authors: Shaul Philip W. , Afshar Sam , Gibson Linda L. , Sherman Todd S. , Kerecman Jay D. , Grubb Peter H. , Yoder Bradley A. , McCurnin Donald C.
Published: March 2015

Nitric oxide (NO), produced by NO synthase (NOS), plays a critical role in multiple processes in the lung during the perinatal period. To better understand the regulation of pulmonary NO production in the developing primate, we determined the cell specificity and developmental changes in NOS isoform expression and action in the lungs of third-trimester fetal baboons. Immunohistochemistry in lungs obtained at 175 days (d) of gestation (term = 185 d) revealed that all three NOS isoforms, neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), are primarily expressed in proximal airway epithelium. In proximal lung, there was a marked increase in total NOS enzymatic activity from 125 to 140 d gestation due to elevations in nNOS and eNOS, whereas iNOS expression and activity were minimal. Total NOS activity was constant from 140 to 175 d gestation, and during the latter stage (160–175 d gestation), a dramatic fall in nNOS and eNOS was replaced by a rise in iNOS. Studies done within 1 h of delivery at 125 or 140 d gestation revealed that the principal increase in NOS during the third trimester is associated with an elevation in exhaled NO levels, a decline in expiratory resistance, and greater pulmonary compliance. Thus, there are developmental increases in pulmonary NOS expression and NO production during the early third trimester in the primate that may enhance airway and parenchymal function in the immediate postnatal period.
Cell-specific and developmental expression of phospholipase C-γ and diacylglycerol in fetal lung
Authors: Ramadurai Sujatha M. , Chen Wu-Yuan , Yerozolimsky George B. , Zagami Michelle , Dammann Christiane E. L. , Nielsen Heber C.
Published: March 2015

Epidermal growth factor (EGF) receptor (EGFR) regulates development of cell-cell communication in fetal lung, but the signal transduction mechanisms involved are unknown. We hypothesized that, in late-gestation fetal rat lung, phospholipase C-γ (PLC-γ) expression and activation by EGF is cell specific and developmentally regulated. PLC-γ immunolocalized to cuboidal epithelium and mesenchymal clusters underlying developing saccules. PLC-γ protein increased from day 17 to day 19 and then decreased. In cultured fetal lung fibroblasts, EGF stimulated PLC-γ phosphorylation 2.6-fold ( day 17), 10.8-fold ( day 19), and 4.2-fold ( day 21). EGF stimulated3H-labeled diacylglycerol production in fibroblasts (beginning on day 18 in female and on day 19 in male rats), but not in type II cells at any time during gestation. EGFR blockade abrogated the observed stimulation of PLC-γ phosphorylation by EGF. In conclusion, PLC-γ expression and activation by EGF in fetal lung are cell specific, corresponding to the development of EGFR expression. EGF induces diacylglycerol production in a cell- and gestation-specific manner. PLC-γ activation by EGFR in fetal lung fibroblasts may be involved in EGF control of lung development.
Sequential targeted deficiency of SP-A and -D leads to progressive alveolar lipoproteinosis and emphysema
Authors: Hawgood Samuel , Ochs Matthias , Jung Anja , Akiyama Jennifer , Allen Lennell , Brown Cindy , Edmondson Jess , Levitt Stacey , Carlson Elaine , Gillespie Anne Marie , Villar Angela , Epstein Charles J. , Poulain Francis R.
Published: March 2015

Surfactant proteins-A and -D (SP-A and SP-D) are members of the collectin protein family. Mice singly deficient in SP-A and SP-D have distinct phenotypes. Both have altered inflammatory responses to microbial challenges. To further investigate the functions of SP-A and SP-D in vivo, we developed mice deficient in both proteins by sequentially targeting the closely linked genes in embryonic stem cells using graded resistance to G-418. There is a progressive increase in bronchoalveolar lavage phospholipid, protein, and macrophage content through 24 wk of age. The macrophages from doubly deficient mice express high levels of the matrix metalloproteinase MMP-12 and develop intense but patchy lung inflammation. Stereological analysis demonstrates significant air space enlargement and reduction in alveolar septal tissue per unit volume, consistent with emphysema. These changes qualitatively resemble the lung pathology seen in SP-D-deficient mice. These doubly deficient mice will be useful in dissecting the potential overlap in function between SP-A and SP-D in host defense.
JNK1 and AP-1 regulate PMA-inducible squamous differentiation marker expression in Clara-like H441 cells
Authors: Vuong Hue , Patterson Tricia , Adiseshaiah Pavan , Shapiro Paul , Kalvakolanu Dhananjaya V. , Reddy Sekhar P. M.
Published: March 2015

Exposure of distal bronchiolar region to various toxicants and pollutants suppresses Clara cell differentiation marker expression and greatly enhances the induction of squamous cell differentiation (SCD). Here, we demonstrate for the first time phorbol 13-myristate 12-acetate (PMA)-inducible expression of SCD markers, SPRRs, in Clara-like H441 cells. The transcriptional stimulation of human SPRR1B expression is mainly mediated by a −150- to −84-bp region that harbors two critical activator protein (AP)-1 sites. In unstimulated cells, the −150- to −84-bp region is weakly bound by AP-1 proteins, mainly JunD and Fra1. However, PMA prominently induced the binding of JunB and Fra1. Consistent with this, overexpression of wild-type Jun proteins upregulated the SPRR1B promoter activity. Conversely, a c- jun mutant suppressed both basal and PMA-inducible reporter gene expression. Intriguingly, overexpression of fra2 suppressed PMA-inducible reporter activity, whereas fra1 significantly enhanced basal level activity, indicating an opposing role for these proteins in SPRR1B expression in a manner similar to that observed in proximal tracheobronchial epithelial cells (BEAS-2B clone S6). Interestingly, unlike in S6 cells, a catalytically inactive c-Jun NH2-terminal kinase (JNK) 1 mutant significantly reduced the PMA-inducible SPRR1B promoter activity in H441 cells. Thus either temporal expression and/or spatial activation of AP-1 proteins by JNK1 might contribute to the induction of SCD in Clara cells.
Surface-lining layer of airways in cystic fibrosis mice
Authors: Geiser Marianne , Bastian Sara
Published: March 2015

Lung disease is the major cause of death in individuals suffering from cystic fibrosis (CF), with abnormal lung-lining fluids occurring as early as early infancy. However, the precise etiology of CF lung disease is still poorly understood. We investigated the structural components of the airway surface-lining layer in targeted Cftrtm1HGU/ Cftrtm1HGU mutant mice and non-CF controls. Five lungs per animal group were fixed by intravascular triple perfusion. The ultrastructure of the surface-lining layer of large and small intrapulmonary conducting airways was systematically investigated according to a standard protocol in transmission and scanning electron micrographs. In both animal groups, the surface-lining layer consisted of an aqueous phase and an osmiophilic film of variable thickness at the air-fluid interface. The aqueous phase usually did extend <1 μm beyond the uppermost tips of the epithelial cells in both animal groups. The aqueous phase of the small airways was slightly more electron dense in Cftrtm1HGU/ Cftrtm1HGU than in non-CF mice. Neither the ultrastructure of the surfactant film at the air-fluid interface nor the forms assumed by the osmiophilic structures associated with surfactant turnover in the aqueous layer differed significantly in Cftrtm1HGU/ Cftrtm1HGU and non-CF mice. Hence, there were no signs of any ultrastructural abnormalities in the surface-lining layer of young adult Cftrtm1HGU/ Cftrtm1HGU mice before infection with CF-related pathogens.
Attenuation of antigen-induced airway hyperresponsiveness in CGRP-deficient mice
Authors: Aoki-Nagase Tomoko , Nagase Takahide , Oh-hashi Yoshio , Shindo Takayuki , Kurihara Yukiko , Yamaguchi Yasuhiro , Yamamoto Hiroshi , Tomita Tetsuji , Ohga Eijiro , Nagai Ryozo , Kurihara Hiroki , Ouchi Yasuyoshi
Published: March 2015

Bronchial hyperresponsiveness and eosinophilia are major characteristics of asthma. Calcitonin gene-related peptide (CGRP) is a neuropeptide that has various biological actions. In the present study, we questioned whether CGRP might have pathophysiological roles in airway hyperresponsiveness and eosinophilia in asthma. To determine the exact roles of endogenous CGRP in vivo, we chose to study antigen-induced airway responses using CGRP gene-disrupted mice. After ovalbumin sensitization and antigen challenge, we assessed airway responsiveness and measured proinflammatory mediators. In the sensitized CGRP gene-disrupted mice, antigen-induced bronchial hyperresponsiveness was significantly attenuated compared with the sensitized wild-type mice. Antigen challenge induced eosinophil infiltration in bronchoalveolar lavage fluid, whereas no differences were observed between the wild-type and CGRP-mutant mice. Antigen-induced increases in cysteinyl leukotriene production in the lung were significantly reduced in the CGRP-disrupted mice. These findings suggest that CGRP could be involved in the antigen-induced airway hyperresponsiveness, but not eosinophil infiltration, in mice. The CGRP-mutant mice may provide appropriate models to study molecular mechanisms underlying CGRP-related diseases.
Intrauterine hypertension decreases lung VEGF expression and VEGF inhibition causes pulmonary hypertension in the ovine fetus
Authors: Grover Theresa R. , Parker Thomas A. , Zenge Jeanne P. , Markham Neil E. , Kinsella John P. , Abman Steven H.
Published: March 2015

Although vascular endothelial growth factor (VEGF) plays a vital role in lung vascular growth in the embryo, its role in maintaining endothelial function and modulating vascular structure during late fetal life has not been studied. We hypothesized that impaired lung VEGF signaling causes pulmonary hypertension, endothelial dysfunction, and structural remodeling before birth. To determine whether lung VEGF expression is decreased in an experimental model of persistent pulmonary hypertension of the newborn (PPHN), we measured lung VEGF and VEGF receptor protein content from fetal lambs 7–10 days after ductus arteriosus ligation (132–140 days gestation; term = 147 days). In contrast with the surge in lung VEGF expression during late gestation in controls, chronic intrauterine pulmonary hypertension reduced lung VEGF expression by 78%. To determine whether VEGF inhibition during late gestation causes pulmonary hypertension, we treated fetal lambs with EYE001, an aptamer that specifically inhibits VEGF165. Compared with vehicle controls, EYE001 treatment elevated pulmonary artery pressure and pulmonary vascular resistance by 22 and 50%, respectively, caused right ventricular hypertrophy, and increased wall thickness of small pulmonary arteries. EYE001 treatment reduced lung endothelial nitric oxide synthase protein content by 50% and preferentially impaired the pulmonary vasodilator response to ACh, an endothelium-dependent agent. We conclude that chronic intrauterine pulmonary hypertension markedly decreases lung VEGF expression and that selective inhibition of VEGF165 mimics the structural and physiological changes of experimental PPHN. We speculate that hypertension downregulates VEGF expression in the developing lung and that impaired VEGF signaling may contribute to the pathogenesis of PPHN.
Enalapril protects mice from pulmonary hypertension by inhibiting TNF-mediated activation of NF-κB and AP-1
Authors: Ortiz Luis A. , Champion Hunter C. , Lasky Joseph A. , Gambelli Federica , Gozal Evelyn , Hoyle Gary W. , Beasley Mary B. , Hyman Albert L. , Friedman Mitchell , Kadowitz Philip J.
Published: March 2015

The present study was undertaken to investigate the effects of treatment with the angiotensin-converting enzyme (ACE) inhibitor enalapril in a mouse model of pulmonary hypertension induced by bleomycin. Bleomycin-induced lung injury in mice is mediated by enhanced tumor necrosis factor-α (TNF) expression in the lung, which determines the murine strain sensitivity to bleomycin, and murine strains are sensitive (C57BL/6) or resistant (BALB/c). Bleomycin induced significant pulmonary hypertension in C57BL/6, but not in BALB/c, mice; average pulmonary arterial pressure (PAP) was 26.4 ± 2.5 mmHg ( P < 0.05) vs. 15.2 ± 3 mmHg, respectively. Bleomycin treatment induced activation of nuclear factor (NF)-κB and activator protein (AP)-1 and enhanced collagen and TNF mRNA expression in the lung of C57BL/6 but not in BALB/c mice. Double TNF receptor-deficient mice (in a C57BL/6 background) that do not activate NF-κB or AP-1 in response to bleomycin did not develop bleomycin-induced pulmonary hypertension (PAP 14 ± 3 mmHg). Treatment of C57BL/6 mice with enalapril significantly ( P < 0.05) inhibited the development of pulmonary hypertension after bleomycin exposure. Enalapril treatment inhibited NF-κB and AP-1 activation, the enhanced TNF and collagen mRNA expression, and the deposition of collagen in bleomycin-exposed C57BL/6 mice. These results suggest that ACE inhibitor treatment decreases lung injury and the development of pulmonary hypertension in bleomycin-treated mice.
Low-dose carbon monoxide reduces airway hyperresponsiveness in mice
Authors: Ameredes Bill T. , Otterbein Leo E. , Kohut Lauryn K. , Gligonic Amber L. , Calhoun William J. , Choi Augustine M. K.
Published: March 2015

Carbon monoxide (CO) in expired gas has been shown to be elevated with asthma; however, its function is not known, and there is some potential that it may serve a bronchoprotective role to decrease airway hyperresponsiveness (AHR). Thus the ability of CO to reverse methacholine (MCh)-induced bronchoconstriction was evaluated in C57BL/6 (C57) and A/J mice with and without airway inflammation produced by ovalbumin (OVA). Acutely administered CO (1% in air, 10 min) reduced MCh-driven increases in lung resistance in OVA-challenged C57 mice by an average of 50% (from 14.5 to 7.1 cmH2O·ml-1·s-1), whereas no effect was observed in naïve C57 mice or OVA-challenged C57 mice inhaling air alone. Acutely inhaled CO (500 ppm = 0.05%, for 10 min) reduced MCh-induced airway reactivity (AR) by 20-60% in airway hyperresponsive naïve A/J mice, whereas repeated 10-min administrations of 500 ppm CO over a 5-day period decreased AR by 50%. Repeated administration of low-dose CO [250 (0.025%) and (0.05%) 500 ppm, 1 h/day, 5 days] to A/J mice with airway inflammation likewise resulted in a drop of AR by 50%, compared with those not receiving CO. Inhibition of guanylyl cyclase/guanosine 3′,5′-cyclic monophosphothioate (cGMP) using 1H-[1,2,4] oxydiazolo[4,3-a]quinoxalin-1-one or a competitive inhibitor, Rp diastereomers of 8-bromo-cGMP, resulted in inhibition of the effect of CO on AHR, suggesting that the effects of CO were mediated through this mechanism. These results indicate that low-dose CO can effectively reverse AHR in the presence and absence of airway inflammation in mice and suggest a potential role for CO in the modulation of AHR.
Quantitative analysis of albumin uptake and transport in the rat microvessel endothelial monolayer
Authors: John Theresa A. , Vogel Stephen M. , Tiruppathi Chinnaswamy , Malik Asrar B. , Minshall Richard D.
Published: March 2015

We determined the concentration dependence of albumin binding, uptake, and transport in confluent monolayers of cultured rat lung microvascular endothelial cells (RLMVEC). Transport of125I-albumin in RLMVEC monolayers occurred at a rate of 7.2 fmol · min−1· 106cells−1. Albumin transport was inhibited by cell surface depletion of the 60-kDa albumin-binding glycoprotein gp60 and by disruption of caveolae using methyl-β-cyclodextrin. By contrast, gp60 activation (by means of gp60 cross-linking using primary and secondary antibodies) increased125I-albumin uptake 2.3-fold. At 37°C,125I-albumin uptake had a half time of 10 min and was competitively inhibited by unlabeled albumin (IC50= 1 μM). Using a two-site model, we estimated by Scatchard analysis the affinity ( KD) and maximal capacity (Bmax) of albumin uptake to be 0.87 μM ( KD1) and 0.47 pmol/106cells (Bmax1) and 93.3 μM ( KD2) and 20.2 pmol/106cells (Bmax2). At 4°C, we also observed two populations of specific binding sites, with high ( KD1= 13.5 nM, 1% of the total) and low ( KD2= 1.6 μM) affinity. On the basis of these data, we propose a model in which the two binding affinities represent the clustered and unclustered gp60 forms. The model predicts that fluid phase albumin in caveolae accounts for the bulk of albumin internalized and transported in the endothelial monolayer.
Distinct cytokine production by lung and blood neutrophils from children with cystic fibrosis
Authors: Corvol Harriet , Fitting Catherine , Chadelat Katarina , Jacquot Jacky , Tabary Olivier , Boule Michele , Cavaillon Jean-Marc , Clement Annick
Published: March 2015

Inflammation plays a critical role in lung disease progression in cystic fibrosis (CF). This inflammatory process is dominated by a neutrophil influx in the airways. To determine whether the accumulation of neutrophils in the airways of CF patients is associated with an altered function, we analyzed the capacity of neutrophils isolated from the lung compartment and the blood to release the major neutrophil pro- and anti-inflammatory cytokines IL-8 and IL-1-receptor antagonist (ra) spontaneously and in the presence of LPS. Comparison of cytokine production by blood neutrophils from CF patients and from control subjects showed significantly increased IL-8 and decreased IL-1ra release by CF neutrophils. Comparison of cytokine production by airway and blood neutrophils from CF patients also documented distinct profiles: the spontaneous release of IL-8 and IL-1ra by airway neutrophils was significantly higher than that from blood neutrophils. Culture in the presence of LPS failed to further enhance cytokine production. Analysis of the effect of dexamethasone confirmed the difference in the responsiveness of lung and blood neutrophils in CF. Used at a concentration effective in reducing IL-8 production by blood neutrophils, dexamethasone (10−6M) was unable to repress secretion of IL-8 by airway neutrophils. In addition, comparison of cytokine production by airway neutrophils from children with CF and children with dyskinetic cilia syndrome also documented distinct profiles of secretion. These results are consistent with a dysregulated cytokine production by lung and blood neutrophils in CF. They provide support to the hypothesis that not only the CF genotype but also the local environment may modify the functional properties of the neutrophils.
A novel mechanism by which hydrogen peroxide decreases calcium sensitivity in airway smooth muscle
Authors: Perkins William J. , Lorenz Robert R. , Bogoger Michelle , Warner David O. , Cremo Christine R. , Jones Keith A.
Published: March 2015

The purpose of this study was to test the hypothesis that H2O2decreases the amount of force produced by a given intracellular Ca2+concentration (i.e., the Ca2+sensitivity) in airway smooth muscle (ASM) in part by mechanisms independent of changes in regulatory myosin light chain (rMLC) phosphorylation. A new preparation was developed and validated in which canine ASM strips were first exposed to H2O2and then permeabilized with 10% Triton X-100 to assess the persistent effects of H2O2on Ca2+sensitivity. Experiments in which H2O2was administered before permeabilization revealed a novel mechanism that contributed to reduced Ca2+sensitivity independently of changes in rMLC phosphorylation, in addition to an rMLC phosphorylation-dependent mechanism. The mechanism depended on factors not available in the permeabilized ASM strip or in the buffer to which the strip was exposed, since there was no effect when H2O2was added to permeabilized strips. H2O2treatment of a maximally thiophosphorylated purified myosin subfragment (heavy meromyosin) significantly reduced actomyosin ATPase activity, suggesting one mechanism by which the phosphorylation-independent reduction in Ca2+sensitivity may occur.
Effects of oligohydramnios on lung growth and maturation in the fetal rat
Authors: Kitterman Joseph A. , Chapin Cheryl J. , Vanderbilt Jeff N. , Porta Nicolas F. M. , Scavo Louis M. , Dobbs Leland G. , Ertsey Robert , Goerke Jon
Published: March 2015

Oligohydramnios (OH) retards fetal lung growth by producing less lung distension than normal. To examine effects of decreased distension on fetal lung development, we produced OH in rats by puncture of uterus and fetal membranes at 16 days of gestation; fetuses were delivered at 21 or 22 days of gestation. Controls were position-matched littermates in the opposite uterine horn. OH lungs had lower weights and less DNA, protein, and water, but no differences in saturated phosphatidylcholine, surfactant proteins (SP)-A and -B, and mRNA for SP-A, -B, -C, and -D. To evaluate effects on epithelial differentiation, we used RTI40and RTII70, proteins specific in lung to luminal surfaces of alveolar type I and II cells, respectively. At 22 days of gestation, OH lungs had less RTI40mRNA ( P < 0.05) and protein ( P < 0.001), but RTII70did not differ from controls. With OH, type I cells (in proportion to type II cells) covered less distal air space perimeter ( P < 0.01). We conclude that OH, which retards lung growth, has little effect on surfactant and impedes formation of type I cells relative to type II cells.
Metalloproteinase inhibition by Batimastat attenuates pulmonary hypertension in chronically hypoxic rats
Authors: Herget Jan , Novotná Jana , Bíbová Jana , Povýšilová Viera , Vaňková Marie , Hampl Václav
Published: March 2015

Chronic hypoxia induces lung vascular remodeling, which results in pulmonary hypertension. We hypothesized that a previously found increase in collagenolytic activity of matrix metalloproteinases during hypoxia promotes pulmonary vascular remodeling and hypertension. To test this hypothesis, we exposed rats to hypoxia (fraction of inspired oxygen = 0.1, 3 wk) and treated them with a metalloproteinase inhibitor, Batimastat (30 mg/kg body wt, daily ip injection). Hypoxia-induced increases in concentration of collagen breakdown products and in collagenolytic activity in pulmonary vessels were inhibited by Batimastat, attesting to the effectiveness of Batimastat administration. Batimastat markedly reduced hypoxic pulmonary hypertension: pulmonary arterial blood pressure was 32 ± 3 mmHg in hypoxic controls, 24 ± 1 mmHg in Batimastat-treated hypoxic rats, and 16 ± 1 mmHg in normoxic controls. Right ventricular hypertrophy and muscularization of peripheral lung vessels were also diminished. Batimastat had no influence on systemic arterial pressure or cardiac output and was without any effect in rats kept in normoxia. We conclude that stimulation of collagenolytic activity in chronic hypoxia is a substantial causative factor in the pathogenesis of pulmonary vascular remodeling and hypertension.
Induced p21Cip1in premature baboons with CLD: implications for alveolar hypoplasia
Authors: O'Reilly Michael A. , Watkins Richard H. , Staversky Rhonda J. , Maniscalco William M.
Published: March 2015

Aberrant pulmonary epithelial and mesenchymal cell proliferation occurs when newborns are treated with oxygen and ventilation to mitigate chronic lung disease. Because the cyclin-dependent kinase inhibitor p21 inhibits proliferation of oxygen-exposed cells, its expression was investigated in premature baboons delivered at 125 days (67% of term) and treated with oxygen and ventilation pro re nata (PRN) for 2, 6, 14, and 21 days. Approximately 5% of all cells expressed p21 during normal lung development of which <1% of these cells were pro-surfactant protein (SP)-B-positive epithelial cells. The percentage of cells expressing p21 increased threefold in all PRN-treated animals, but different cell populations expressed it during disease progression. Between 2 and 6 days of treatment, p21 was detected in 30-40% of pro-SP-B cells. In contrast, only 12% of pro-SP-B cells expressed p21 by 14 and 21 days of treatment, by which time p21 was also detected in mesenchymal cells. Even though increased epithelial and mesenchymal cell proliferation occurs during disease progression, those cells expressing p21 did not also express the proliferative marker Ki67. Thus two populations of epithelial and mesenchymal cells can be identified that are either expressing Ki67 and proliferating or expressing p21 and not proliferating. These data suggest that p21 may play a role in disorganized proliferation and alveolar hypoplasia seen in newborn chronic lung disease.
Fibroblast growth factor-2 during postnatal development of the tracheal basement membrane zone
Authors: Evans Michael J. , Fanucchi Michelle V. , Van Winkle Laura S. , Baker Gregory L. , Murphy April E. , Nishio Susan J. , Sannes Philip L. , Plopper Charles G.
Published: March 2015

Thickening of the basement membrane zone (BMZ) is a characteristic of several airway diseases; however, very little is known about how this process occurs. The purpose of this study was to define development of the BMZ in the trachea of growing rhesus monkeys at 1, 2, 3, and 6 mo of age. We measured immunoreactivity of collagen types I, III, and V to detect structural changes in the developing BMZ. To detect more dynamic, functional components of the epithelial-mesenchymal trophic unit, we evaluated the distribution of perlecan, fibroblast growth factor-2 (FGF-2), and fibroblast growth factor receptor-1 (FGFR-1). One-month-old monkeys had a mean collagen BMZ width of 1.5 ± 0.7 μm that increased to 4.4 ± 0.4 μm in 6-mo-old monkeys. Perlecan was localized in the BMZ of the epithelium at all ages. FGF-2 was strongly expressed in basal cells at 1–3 mo. At 6 mo, FGF-2 was expressed throughout the BMZ and weakly in basal cells. FGFR-1 immunoreactivity was expressed by basal cells and cilia and weakly in the nuclei of columnar cells at all time points. These data indicate that development of the BMZ is a postnatal event in the rhesus monkey that involves FGF-2.
Surfactant protein A modulates the differentiation of murine bone marrow-derived dendritic cells
Authors: Brinker Karen G. , Garner Hollie , Wright Jo Rae
Published: March 2015

Surfactant protein A (SP-A) is an innate immune molecule that regulates pathogen clearance and lung inflammation. SP-A modulates innate immune functions such as phagocytosis, cytokine production, and chemotaxis; however, little is known about regulation of adaptive immunity by SP-A. Dendritic cells (DCs) are the most potent antigen-presenting cell with the unique capacity to activate naı̈ve T cells and initiate adaptive immunity. The goal of this study was to test the hypothesis that SP-A regulates the differentiation of immature DCs into potent T cell stimulators. The data show that incubation of immature DCs for 24 h with SP-A inhibits basal- and LPS-mediated expression of major histocompatibility complex class II and CD86. Stimulation of immature DCs by SP-A also inhibits the allostimulation of T cells, enhances dextran endocytosis, and alters DC chemotaxis toward RANTES and secondary lymphoid tissue chemokine. The effects on DC phenotype and function are similar for the structurally homologous C1q, but not for SP-D. These studies demonstrate that SP-A participates in the adaptive immune response by modulating important immune functions of DCs.
Characterization of CD34+cells isolated from human fetal lung
Authors: Acarregui Michael J. , England Katherine M. , Richman Joshua T. , Littig Jennifer L.
Published: March 2015

The large capillary mass of the newborn lung demands the presence of endothelial cell precursors in lung tissue before development of the pulmonary capillary bed. The objective of this investigation was to isolate and characterize putative endothelial cell precursors from developing human lung. CD34, a cell surface marker for hematopoietic progenitor cells, endothelial precursor cells, and small vessel endothelial cells, was employed as an immunological “handle” for the selection of the desired cells. When CD34+cells were isolated from midtrimester human fetal lung tissue, then maintained in culture, the isolated cells expressed immunoreactivity for the endothelial cell marker von Willebrand factor and the vascular endothelial growth factor receptors KDR and Flt-1. However, only 5% or fewer of the cells expressed PECAM, an important factor in cell-cell interactions and a marker for endothelial cells associated with vessels. The CD34+cells endocytosed acetylated low-density lipoprotein and formed capillary-like structures when incubated in a cushion of Matrigel. RT-PCR analysis of mRNA for endothelial cell-related proteins Flt-1, Tie-2, and endothelial nitric oxide synthase demonstrated expression of these mRNAs by the isolated cells for at least 16 cell passages. These observations demonstrate that capillary endothelial cell precursors can be isolated from developing human lung and maintained in cell culture. These cells represent a potentially important tool for investigating the regulation of mechanisms governing development of the air-blood barrier in the human lung.
Role of adipocyte differentiation-related protein in surfactant phospholipid synthesis by type II cells
Authors: Schultz C. J. , Torres E. , Londos C. , Torday J. S.
Published: March 2015

Adipocyte differentiation-related protein (ADrP) is an intrinsic lipid storage droplet protein that is highly expressed in lung. ADrP localizes to lipid storage droplets within lipofibroblasts, pulmonary cells characterized by high triacylglycerol, which is a precursor for surfactant phospholipid synthesis by alveolar type II epithelial (EPII) cells. The developmental pattern of ADrP mRNA and protein expression in lung tissue parallels triacylglycerol accumulation in rat lung. ADrP mRNA levels are relatively high in isolated lipofibroblasts, accounting for the high ADrP expression in lung. Isolated EPII cells, which do not store neutral lipids but derive them from lipofibroblasts, have low levels of ADrP mRNA expression. ADrP is found around lipid droplets in cultured lipofibroblasts, but not in EPII cells isolated from developing rat lung. After coculture with lipofibroblasts, EPII cells acquired ADrP, which associates with lipid droplets. Furthermore,3H-labeled triolein in isolated ADrP-coated lipid droplets is a tenfold better substrate for surfactant phospholipid synthesis by cultured EPII cells than3H-labeled synthetic triolein alone. Antibodies to ADrP block transfer of neutral lipid. These data suggest a role for ADrP in this novel mechanism for the transfer of lipid between lipofibroblasts and EPII cells.