bluuebunny/crossref_metadata_2025 huggingface dataset:
Endogenous pulmonary nitric oxide in the regulation of airway microvascular leak
Authors: Mehta Sanjay , Boudreau Jacques , Lilly Craig M. , Drazen Jeffrey M.
Published: December 2017

Endogenous nitric oxide (NO) is an important modulator of airway function, but its role in the regulation of airway microvascular leak (AMVL) remains unclear. Thus we assessed the effects of NO synthase (NOS) inhibition on expired NO (ENO) levels and on AMVL measured by the Evans blue dye technique in guinea pigs. In control unsensitized animals, systemic N G-nitro-l-arginine methyl ester (l-NAME) reduced ENO by 70 ± 8% ( P < 0.01) and reduced AMVL by 92 ± 1 and 44 ± 17% ( P < 0.05 for both) in the extrapulmonary and intrapulmonary airways, respectively. In animals sensitized and challenged with intratracheal antigen, markedly increased levels of AMVL and ENO were similarly attenuated byl-NAME. In contrast, aminoguanidine, a relatively selective type II NOS inhibitor, reduced ENO in both antigen-sensitized and control unsensitized animals by 39 ± 3% ( P < 0.01) but had no effect on AMVL. These data indicate that endogenous pulmonary NO contributes to both basal and antigen-stimulated levels of AMVL in guinea pigs and that this NO-dependent activity does not appear to be derived from type II NOS.
Activation of human macrophages by mechanical ventilation in vitro
Authors: Pugin Jérôme , Dunn Irène , Jolliet Philippe , Tassaux Didier , Magnenat Jean-Luc , Nicod Laurent P. , Chevrolet Jean-Claude
Published: December 2017

Positive-pressure mechanical ventilation supports gas exchange in patients with respiratory failure but is also responsible for significant lung injury. In this study, we have developed an in vitro model in which isolated lung cells can be submitted to a prolonged cyclic pressure-stretching strain resembling that of conventional mechanical ventilation. In this model, cells cultured on a Silastic membrane were elongated up to 7% of their initial diameter, corresponding to a 12% increase in cell surface. The lung macrophage was identified as the main cellular source for critical inflammatory mediators such as tumor necrosis factor-α, the chemokines interleukin (IL)-8 and -6, and matrix metalloproteinase-9 in this model system of mechanical ventilation. These mediators were measured in supernatants from ventilated alveolar macrophages, monocyte-derived macrophages, and promonocytic THP-1 cells. Nuclear factor-κB was found to be activated in ventilated macrophages. Synergistic proinflammatory effects of mechanical stress and molecules such as bacterial endotoxin were observed, suggesting that mechanical ventilation might be particularly deleterious in preinjured or infected lungs. Dexamethasone prevented IL-8 and tumor necrosis factor-α secretion in ventilated macrophages. Mechanical ventilation induced low levels of IL-8 secretion by alveolar type II-like cells. Other lung cell types such as endothelial cells, bronchial cells, and fibroblasts failed to produce IL-8 in response to a prolonged cyclic pressure-stretching load. This model is of particular value for exploring physical stress-induced signaling pathways, as well as for testing the effects of novel ventilatory strategies or adjunctive substances aimed at modulating cell activation induced by mechanical ventilation.
Angiotensin IV receptor-mediated activation of lung endothelial NOS is associated with vasorelaxation
Authors: Patel Jawaharlal M. , Martens Jeffrey R. , Li Yong D. , Gelband Craig H. , Raizada Mohan K. , Block Edward R.
Published: December 2017

The hexapeptide angiotensin (ANG) IV, a metabolic product of ANG II, has been reported to play a functional role in the regulation of blood flow in extrapulmonary tissues. Here, we demonstrate that ANG IV-specific (AT4) receptors are present in porcine pulmonary arterial endothelial cells (PAECs) and that the binding of ANG IV to AT4receptors can be blocked by its antagonist divalinal ANG IV but not by the ANG II-, AT1-, and AT2-receptor blockers [Sar1,Ile8]ANG II, losartan, and PD-123177, respectively. ANG IV significantly increased endothelial cell constitutive nitric oxide synthase (ecNOS) activity ( P < 0.05) as well as cellular cGMP content ( P < 0.001). Western blot analysis revealed that ecNOS protein expression was comparable in control and ANG IV-stimulated cells. Divalinal ANG IV but not [Sar1,Ile8]ANG II, losartan, or PD-123177 inhibited the ANG II- and ANG IV-stimulated increases in ecNOS activity and cGMP content in PAECs. Incubation in the presence of N-nitro-l-arginine methyl ester (l-NAME) or methylene blue but not of indomethacin significantly diminished ANG IV-stimulated as well as basal levels of cGMP ( P < 0.001). Similarly, in situ studies with precontracted porcine pulmonary arterial rings showed that ANG IV caused an endothelium-dependent relaxation that was blocked byl-NAME or methylene blue. Collectively, these results demonstrate that ANG IV binds to AT4receptors, activates ecNOS by posttranscriptional modulation, stimulates cGMP accumulation in PAECs, and causes pulmonary arterial vasodilation, suggesting that ANG IV plays a role in the regulation of blood flow in the pulmonary circulation.
Superoxide released from neutrophils causes a reduction in nitric oxide gas
Authors: Jones Kimberly L. , Bryan Ty W. , Jinkins Patricia A. , Simpson Keith L. , Grisham Matthew B. , Owens Michael W. , Milligan Shawn A. , Markewitz Boaz A. , Robbins Richard A.
Published: December 2017

Exhaled nitric oxide (NO) is increased in some inflammatory airway disorders but not in others such as cystic fibrosis and acute respiratory distress syndrome. NO can combine with superoxide ([Formula: see text]) to form peroxynitrite, which can decompose into nitrate. Activated polymorphonuclear neutrophils (PMNs) releasing[Formula: see text] could account for a reduction in exhaled NO in disorders such as cystic fibrosis. To test this hypothesis in vitro, we stimulated confluent cultures of LA-4 cells, a murine lung epithelial cell line, to produce NO. Subsequently, human PMNs stimulated to produce [Formula: see text] were added to the LA-4 cells. A gradual increase in NO in the headspace above the cultures was observed and was markedly reduced by the addition of PMNs. An increase in nitrate in the culture supernatant fluids was measured, but no increase in nitrite was detected. Superoxide dismutase attenuated the PMN effect, and xanthine/xanthine oxidase reproduced the effect. No changes in epithelial cell inducible NO synthase protein or mRNA were observed. These data demonstrate that [Formula: see text]released from PMNs can decrease NO by conversion to nitrate and suggest a potential mechanism for modulation of NO levels in vivo.
CsA-sensitive purine-box transcriptional regulator in bronchial epithelial cells contains NF45, NF90, and Ku
Authors: Aoki Yosuke , Zhao Guohua , Qiu Daoming , Shi Lingfang , Kao Peter N.
Published: December 2017

Human bronchial epithelial (HBE) cells express interleukin (IL)-2 [Y. Aoki, D. Qiu, A. Uyei, and P. N. Kao. Am. J. Physiol. 272 ( Lung Cell. Mol. Physiol. 16): L276–L286, 1997]. 16HBE-transformed cells contain constitutive and inducible nuclear DNA-binding activity for the purine-box/nuclear factor (NF) of activated T cell (NFAT) target DNA sequence in the human IL-2 enhancer. Transcriptional activation through the purine-box DNA sequence requires stimulation with phorbol 12-myristate 13-acetate + ionomycin, and this activation is inhibited by cyclosporin A. Immunohistochemical staining of 16HBE cells demonstrates nuclear expression of the purine-box DNA-binding proteins NF45 and NF90 and no expression of NFATp or NFATc. NF90 and NF45 associate with the DNA-dependent protein kinase catalytic subunit and the DNA-targeting subunits Ku80 and Ku70 (N. S. Ting, P. N. Kao, D. W. Chan, L. G. Lintott, and S. P. Lees-Miller. J. Biol. Chem. 273: 2136–2145, 1998). Antibodies to Ku potently inhibit the purine-box DNA-binding complex. The purine-box transcriptional regulator in 16HBE cells likely comprises NF45, NF90, Ku80, Ku70, and the DNA-dependent protein kinase catalytic subunit.
Equibiaxial deformation-induced injury of alveolar epithelial cells in vitro
Authors: Tschumperlin Daniel J. , Margulies Susan S.
Published: December 2017

Deformation of the alveolar epithelial basement membrane with lung inflation has been implicated in blood-gas barrier breakdown during the development of ventilator-induced lung injury. To determine the vulnerability of alveolar epithelial cells to deformation-induced injury, we developed a cell-stretching device that subjects cells to cyclic, equibiaxial strains. Alveolar epithelial type II cells from primary culture were tested 1 and 5 days after seeding, during which time the cells underwent major morphological and phenotypic changes. Cells were subjected to changes in surface area of 12, 24, 37, and 50%, which corresponded to lung inflation of ∼60, 80, 100, and >100% of total lung capacity. Deformation-induced injury of alveolar epithelial cells, assessed with a fluorescent cell viability assay, increased with deformation magnitude and decreased with time elapsed after seeding. In cells stretched after 1 day in culture, the percentage of dead cells after a single deformation ranged from 0.5 to 72% over the range of deformations used. In cells stretched at 5 days, the percentage of dead cells ranged from 0 to 9% when exposed to identical deformation protocols. These results suggest that morphological and phenotypic changes with time in culture fundamentally change the vulnerability of alveolar epithelial cells to deformation.
Alveolar epithelial cell death adjacent to underlying myofibroblasts in advanced fibrotic human lung
Authors: Uhal Bruce D. , Joshi Iravati , Hughes W. Frank , Ramos Carlos , Pardo Annie , Selman Moises
Published: December 2017

Earlier work from this laboratory showed that abnormal fibroblast phenotypes isolated from fibrotic human lung produce factor(s) capable of inducing apoptosis and necrosis of alveolar epithelial cells in vitro [B. D. Uhal, I. Joshi, A. True, S. Mundle, A. Raza, A. Pardo, and M. Selman. Am. J. Physiol. 269 ( Lung Cell. Mol. Physiol. 13): L819–L828, 1995]. To determine whether epithelial cell death is associated with proximity to abnormal fibroblasts in vivo, the spatial distribution of epithelial cell loss, DNA fragmentation, and myofibroblasts was examined in the same tissue specimens used previously for fibroblast isolation. Paraffin sections of normal and fibrotic human lung were subjected to in situ end labeling (ISEL) of fragmented DNA and simultaneous immunolabeling of α-smooth muscle actin (α-SMA); replicate samples were subjected to electron microscopy and detection of collagens by the picrosirius red technique. Normal human lung exhibited very little labeling except for positive α-SMA immunoreactivity of smooth muscle surrounding bronchi and vessels. In contrast, fibrotic human lung exhibited moderate to heavy ISEL of interstitial, cuboidal epithelial, and free alveolar cells. ISEL of the alveolar epithelium was not distributed uniformly but was most intense immediately adjacent to underlying foci of α-SMA-positive fibroblast-like interstitial cells. Both electron microscopy and picrosirius red confirmed epithelial cell apoptosis, necrosis, and cell loss adjacent to foci of collagen accumulation surrounding fibroblast-like cells. These results demonstrate that the cuboidal epithelium of the fibrotic lung contains dying as well as proliferating cells and support the hypothesis that alveolar epithelial cell death is induced by abnormal lung fibroblasts in vivo as it is in vitro.
Effects of endogenous and exogenous catecholamines on LPS-induced neutrophil trafficking and activation
Authors: Abraham Edward , Kaneko Debra J. , Shenkar Robert
Published: December 2017

Endotoxemia produces elevations in catecholamine levels in the pulmonary and systemic circulation as well as rapid increases in neutrophil number and proinflammatory cytokine expression in the lungs. In the present experiments, we examined the effects of endogenous and exogenous adrenergic stimulation on endotoxin-induced lung neutrophil accumulation and activation. Levels of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and macrophage inflammatory protein (MIP)-2 mRNAs were increased in lung neutrophils from endotoxemic mice compared with those present in lung neutrophils from control mice or in peripheral blood neutrophils from endotoxemic or control mice. Treatment with the β-adrenergic antagonist propranolol before endotoxin administration did not affect trafficking of neutrophils to the lungs or the expression of IL-1β, TNF-α, or MIP-2 by lung neutrophils. Administration of the α-adrenergic antagonist phentolamine before endotoxemia did not alter lung neutrophil accumulation as measured by myeloperoxidase (MPO) levels but did result in significant increases in IL-1β, TNF-α, and MIP-2 mRNA expression by lung neutrophils compared with endotoxemia alone. Administration of the α1-adrenergic agonist phenylephrine before endotoxin did not affect trafficking of neutrophils to the lungs but was associated with significantly increased expression of TNF-α and MIP-2 mRNAs by lung neutrophils compared with that found after endotoxin alone. In contrast, treatment with the α2-adrenergic agonist UK-14304 prevented endotoxin-induced increases in lung MPO and lung neutrophil cytokine mRNA levels. The suppressive effects of UK-14304 on endotoxin-induced increases in lung MPO were not affected by administration of the nitric oxide synthase inhibitor N-nitro-l-arginine methyl ester. These data demonstrate that the initial accumulation and activation of neutrophils in the lungs after endotoxemia can be significantly diminished by α2-adrenergic stimulation. Therapy with α2-adrenergic agents may have a role in modulating inflammatory pulmonary processes associated with sepsis-induced acute lung injury.
Lung type II cell and macrophage annexin I release: differential effects of two glucocorticoids
Authors: Hall S. E. , Lim S. , Witherden I. R. , Tetley T. D. , Barnes P. J. , Kamal A. M. , Smith S. F.
Published: December 2017

Annexin I (lipocortin 1) is abundant in lung secretions. Concentrations rise after oral glucocorticoid, but the effect of inhaled budesonide on annexin I release is unknown. Extracellular annexin I in bronchoalveolar lavage fluid (BALF) from 11 asthmatic patients was unaffected by inhaled budesonide (800 μg twice daily for 4 wk; mean after budesonide, 110 ng/mg albumin; after placebo, 107 ng/mg albumin). Rat alveolar macrophages (AMs) and alveolar epithelial type II (ATII) cells were cultured alone and with budesonide or dexamethasone. Mean basal concentrations of cellular (3.5 ng/106 AMs; 4.4 ng/106 ATII cells) and secreted (1.4 ng/106 AMs; 1.8 ng/106 ATII cells) annexin I were similar in AMs and ATII cells. Although budesonide subdued annexin I secretion from both cell types, dexamethasone stimulated annexin I release. Annexin I release from ATII cells peaked at 10−7 M dexamethasone but at 10−3 M dexamethasone from AMs. Thus, at low concentrations of dexamethasone, ATII cells probably contribute more annexin I to respiratory tract secretions than AMs, although at high concentrations, both cells probably contribute. The study demonstrates previously undescribed differences between glucocorticoids and between AMs and ATII cells with respect to annexin I regulation.
Pulmonary intravascular macrophages: their contribution to the mononuclear phagocyte system in 13 species
Authors: Brain Joseph D. , Molina Ramon M. , DeCamp Malcolm M. , Warner Angeline E.
Published: December 2017

The organ uptake of intravenously injected particles was examined in 13 species. All animals were injected intravenously with198Au colloid and magnetic iron oxide particles. Vascular clearance kinetics of198Au colloid was similar in all species. Pulmonary uptake of198Au colloid ranged from 17 to 60% in sheep, calves, pigs, and cats but was <1.1% in monkeys, hyraxes, rabbits, guinea pigs, rats, mice, and chickens. For iron oxide particles, pulmonary uptake ranged from 80 to 99% in sheep, calves, pigs, goats, and cats and 15 to 18% in hamsters, hyraxes, and monkeys and was <10% in rabbits, chicken, mice, rats, and guinea pigs. In all species, the bulk of the remainder of particle uptake was in the liver. Pulmonary intravascular macrophages are the cellular site of lung uptake in calves, cats, pigs, goats, and sheep, whereas monocytes and neutrophils predominate in other species. Kupffer cells were the site of uptake in the liver. Our data show marked species differences in the fate of circulating particles; ruminants, pigs, and cats have extensive pulmonary localization due to phagocytosis by pulmonary intravascular macrophages.
NK1-receptor activation prevents hydrocarbon-induced lung injury in mice
Authors: Robledo Raymond F. , Witten Mark L.
Published: December 2017

Recent evidence suggests that neurokinin (NK)-receptor activation may have a protective role in maintaining lung integrity when challenged by airborne toxicants such as sulfur dioxide, ozone, acrolein, or hydrocarbons. To investigate the effect of NK1-receptor activation on hydrocarbon-induced lung injury, B6.A.D. ( Ahrd/ Nats) mice received subchronic exposures to JP-8 jet fuel (JP-8). Lung injury was assessed by the analysis of pulmonary physiology, bronchoalveolar lavage fluid, and morphology. Hydrocarbon exposure to target JP-8 concentrations of 50 mg/m3, with saline treatment, was characterized by enhanced respiratory permeability to99mTc-labeled diethylenetriaminepentaacetic acid, alveolar macrophage toxicity, and bronchiolar epithelial damage. Mice administered [Sar9,Met(O2)11]substance P, an NK1-receptor agonist, after each JP-8 exposure had the appearance of normal pulmonary values and tissue morphology. In contrast, endogenous NK1-receptor antagonism by CP-96345 administration exacerbated JP-8-enhanced permeability, alveolar macrophage toxicity, and bronchiolar epithelial injury. These data indicate that NK1-receptor activation may have a protective role in preventing the development of hydrocarbon-induced lung injury, possibly through the modulation of bronchiolar epithelial function.
Determination of low-molecular-mass antioxidant concentrations in human respiratory tract lining fluids
Authors: van der Vliet Albert , O’Neill Charles A. , Cross Carroll E. , Koostra John M. , Volz William G. , Halliwell Barry , Louie Samuel
Published: December 2017

Antioxidants present within lung epithelial lining fluids (ELFs) constitute an initial line of defense against inhaled environmental oxidants such as ozone, nitrogen oxides, and tobacco smoke, but the antioxidant composition of human ELFs is still incompletely characterized. We analyzed ELF concentrations of the low-molecular-mass antioxidants ascorbate, urate, glutathione (GSH), and α-tocopherol by obtaining bronchoalveolar lavage (BAL) and nasal lavage fluids from healthy nonsmoking volunteers and compared two different BAL procedures. ELF dilution by the lavage procedures was estimated by measurement of urea in recovered BAL fluids in comparison with those in blood plasma from the same subjects. The results indicated that a recently developed single-cycle BAL procedure minimizes influx of non-ELF urea into the instilled fluid and thus allows for a more accurate determination of ELF antioxidant concentrations. Using this procedure, we determined that bronchoalveolar ELF contains 40 ± 18 (SD) μM ascorbate, 207 ± 167 μM urate, 109 ± 64 μM GSH, and 0.7 ± 0.3 μM α-tocopherol ( n = 12 subjects). Similar analysis of nasal lavage fluid yielded nasal ELF levels of 28 ± 19 μM ascorbate and 225 ± 105 μM urate ( n = 12 subjects), whereas GSH was undetectable (<0.5 μM). Our results demonstrate that ascorbate and urate are major low-molecular-mass ELF antioxidants in both the upper and lower respiratory tract, whereas GSH is present at significant concentrations only in bronchoalveolar ELF.
Variable expression of endothelial NO synthase in three forms of rat pulmonary hypertension
Authors: Tyler Robert C. , Muramatsu Masashi , Abman Steven H. , Stelzner Thomas J. , Rodman David M. , Bloch Kenneth D. , McMurtry Ivan F.
Published: December 2017

Endothelial nitric oxide (NO) synthase (eNOS) mRNA and protein and NO production are increased in hypoxia-induced hypertensive rat lungs, but it is uncertain whether eNOS gene expression and activity are increased in other forms of rat pulmonary hypertension. To investigate these questions, we measured eNOS mRNA and protein, eNOS immunohistochemical localization, perfusate NO product levels, and NO-mediated suppression of resting vascular tone in chronically hypoxic (3–4 wk at barometric pressure of 410 mmHg), monocrotaline-treated (4 wk after 60 mg/kg), and fawn-hooded (6–9 mo old) rats. eNOS mRNA levels (Northern blot) were greater in hypoxic and monocrotaline-treated lungs (130 and 125% of control lungs, respectively; P < 0.05) but not in fawn-hooded lungs. Western blotting indicated that eNOS protein levels increased to 300 ± 46% of control levels in hypoxic lungs ( P < 0.05) but were decreased by 50 ± 5 and 60 ± 11%, respectively, in monocrotaline-treated and fawn-hooded lungs ( P < 0.05). Immunostaining showed prominent eNOS expression in small neomuscularized arterioles in all groups, whereas perfusate NO product levels increased in chronically hypoxic lungs (3.4 ± 1.4 μM; P < 0.05) but not in either monocrotaline-treated (0.7 ± 0.3 μM) or fawn-hooded (0.45 ± 0.1 μM) lungs vs. normotensive lungs (0.12 ± 0.07 μM). All hypertensive lungs had increased baseline perfusion pressure in response to nitro-l-arginine but not to the inducible NOS inhibitor aminoguanidine. These results indicate that even though NO activity suppresses resting vascular tone in pulmonary hypertension, there are differences among the groups regarding eNOS gene expression and NO production. A better understanding of eNOS gene expression and activity in these models may provide insights into the regulation of this vasodilator system in various forms of human pulmonary hypertension.
Pirfenidone inhibits PDGF isoforms in bleomycin hamster model of lung fibrosis at the translational level
Authors: Gurujeyalakshmi G. , Hollinger M. A. , Giri S. N.
Published: December 2017

Pirfenidone (PD) is known for its antifibrotic effects in the bleomycin (BL) hamster model of lung fibrosis. We evaluated whether pretreatment of hamsters with PD could influence the effects of BL-induced overexpression of platelet-derived growth factor (PDGF)-A and PDGF-B genes and proteins in the same model of lung fibrosis. We demonstrate elevated levels of PDGF-A and PDGF-B mRNAs in bronchoalveolar lavage (BAL) cells from lungs of BL-treated compared with saline control hamsters by RT-PCR analysis. However, these levels were not altered in BAL cells obtained from BL-treated hamsters on diets containing 0.5% PD. Western blot analysis of BAL fluid for PDGF isoforms demonstrated that PD treatment inhibited the synthesis of both PDGF-A and PDGF-B isoforms. PD treatment also decreased the mitogenic activity in the BAL fluid from BL-treated hamster lungs. Taken together, these data provide evidence that the protective effects of PD against BL-induced lung fibrosis may be mediated by a reduction in PDGF isoforms produced by lung macrophages.
Inhibition of hSP-B promoter in respiratory epithelial cells by a dominant negative retinoic acid receptor
Authors: Ghaffari Manely , Whitsett Jeffrey A. , Yan Cong
Published: December 2017

Retinoic acid (RA) receptors (RARs) belong to the nuclear hormone receptor superfamily and play important roles in lung differentiation, growth, and gene regulation. Surfactant protein (SP) B is a small hydrophobic protein synthesized and secreted by respiratory epithelial cells in the lung. Expression of the SP-Bgene is modulated at the transcriptional and posttranscriptional levels. In the present work, immunohistochemical staining revealed that RAR-α is present on day 14.5 of gestation in the fetal mouse lung. To assess whether RAR is required for SP-B gene transcription, a dominant negative mutant human (h) RAR-α403 was generated. The hRAR-α403 mutant was transcribed and translated into the truncated protein product by reticulocyte lysate in vitro. The mutant retained DNA binding activity in the presence of retinoid X receptor-γ to an RA response element in the hSP-B promoter. When transiently transfected into pulmonary adenocarcinoma epithelial cells (H441 cells), the mutant hRAR-α403 was readily detected in the cell nucleus. Cotransfection of the mutant hRAR-α403 repressed activity of the hSP-B promoter and inhibited RA-induced surfactant proprotein B production in H441 cells, supporting the concept that RAR is required for hSP-Bgene transcription in vitro.
Modulation of human airway smooth muscle proliferation by type 3 phosphodiesterase inhibition
Authors: Billington Charlotte K. , Joseph Sunil K. , Swan Caroline , Scott Mark G. H. , Jobson Timothy M. , Hall Ian P.
Published: December 2017

Elevation in cell cAMP content can inhibit mitogenic signaling in cultured human airway smooth muscle (HASM) cells. We studied the effects of the type 3-selective phosphodiesterase inhibitor siguazodan, the type 4-selective phosphodiesterase inhibitor rolipram, and the nonselective inhibitor 3-isobutyl-1-methylxanthine (IBMX) on proliferation of cultured HASM cells. At concentrations selective for the type 3 phosphodiesterase isoform, siguazodan inhibited both [3H]thymidine incorporation (IC502 μM) and the increase in cell number (10 μM; 64% reduction) induced by platelet-derived growth factor-BB (20 ng/ml). These effects were mimicked by IBMX. At concentrations selective for type 4 phosphodiesterase inhibition, rolipram was without effect. A 20-min exposure to siguazodan and rolipram did not increase whole cell cAMP levels. However, in HASM cells transfected with a cAMP-responsive luciferase reporter (p6CRE/Luc), increases in cAMP-driven luciferase expression were seen with siguazodan (3.9-fold) and IBMX (16.5-fold). These data suggest that inhibition of the type 3 phosphodiesterase isoform present in airway smooth muscle results in inhibition of mitogenic signaling, possibly through an increase in cAMP-driven gene expression.
Critical role of glass fiber length in TNF-α production and transcription factor activation in macrophages
Authors: Ye Jianping , Shi Xianglin , Jones William , Rojanasakul Yon , Cheng Ningli , Schwegler-Berry Diane , Baron Paul , Deye Gregory J. , Li Changhong , Castranova Vincent
Published: December 2017

Recent studies have demonstrated that dielectrophoresis is an efficient method for the separation of fibers according to fiber length. This method allows the investigation of fiber-cell interactions with fiber samples of the same composition but of different lengths. In the present study, we analyzed the effects of length on the interaction between glass fibers and macrophages by focusing on production of the inflammatory cytokine tumor necrosis factor (TNF)-α in a mouse macrophage cell line (RAW 264.7). The underlying molecular mechanisms controlling TNF-α production were investigated at the gene transcription level. The results show that glass fibers induced TNF-α production in macrophages and that this induction was associated with activation of the gene promoter. Activation of the transcription factor nuclear factor (NF)-κB was responsible for this induced promoter activity. The inhibition of both TNF-α production and NF-κB activation by N-acetyl-l-cysteine, an antioxidant, indicates that generation of oxidants may contribute to the induction of this cytokine and activation of this transcription factor by glass fibers. Long fibers (17 μm) were significantly more potent than short fibers (7 μm) in inducing NF-κB activation, the gene promoter activity, and the production of TNF-α. This fiber length-dependent difference in the stimulatory potency correlated with the fact that macrophages were able to completely engulf short glass fibers, whereas phagocytosis of long glass fibers was incomplete. These results suggest that fiber length plays a critical role in the potential pathogenicity of glass fibers.
Nitric oxide-mediated regulation of transepithelial sodium and chloride transport in murine nasal epithelium
Authors: Elmer Heather L. , Brady Kristine G. , Drumm Mitchell L. , Kelley Thomas J.
Published: December 2017

Transepithelial ion transport is regulated by a variety of cellular factors. In light of recent evidence that nitric oxide (NO) production is decreased in cystic fibrosis airways, we examined the role of NO in regulating sodium and chloride transport in murine nasal epithelium. Acute intervention with the inducible NO synthase (iNOS)-selective inhibitor S-methylisothiourea resulted in an increase of amiloride-sensitive sodium absorption observed as a hyperpolarization of nasal transepithelial potential difference. Inhibition of iNOS expression with dexamethasone also hyperpolarized transepithelial potential difference, but only a portion of this increase proved to be amiloride sensitive. Chloride secretion was significantly inhibited in C57BL/6J mice by the addition of both S-methylisothiourea and dexamethasone. Mice lacking iNOS expression [NOS2(−/−)] also had a decreased chloride-secretory response compared with control mice. These data suggest that constitutive NO production likely plays some role in the downregulation of sodium absorption and leads to an increase in transepithelial chloride secretion.
Expression of heat shock protein 72 by alveolar macrophages in hypersensitivity pneumonitis
Authors: Racine Claudia , Israël-Assayag Evelyne , Cormier Yvon
Published: December 2017

The current study was done to look at a possible role of heat shock proteins (HSPs) in hypersensitivity pneumonitis (HP). The specific aims were to determine whether there was a difference in the expression of HSP72 in alveolar macrophages (AMs) between mice challenged with HP antigen and saline-treated control mice and between AMs obtained by bronchoalveolar lavage from 18 patients with HP and 11 normal subjects. The expression of HSP72 was studied under basal conditions and under a mild heat shock. HSP72 expression by AMs in response to in vitro stimulation with Saccharopolyspora rectivirgula was lower in AMs of control mice than in those of HP animals. HSP72 was constitutively expressed in AMs of both normal and HP subjects. Densitometric ratios showed that AMs from normal subjects responded to heat shock with a 39°C-to-37°C ratio of 1.72 ± 0.18 (mean ± SE), and AMs from HP patients responded with a ratio of 1.16 ± 0.16 ( P = 0.0377). This decreased induction by additional stress of AMs could lead to an altered immunoregulatory activity and account for the inflammation seen in HP.
Protein kinase C isoforms in human airway smooth muscle cells: activation of PKC-ζ during proliferation
Authors: Carlin Steve , Yang Kenny X. F. , Donnelly Richard , Black Judith L.
Published: December 2017

Protein kinase C (PKC) is implicated in the regulation of smooth muscle contractility and growth. We have previously described the pattern of isoform expression of PKC in canine airway smooth muscle. This study identified the isoforms present in human cultured airway smooth muscle cells and also addressed the question of whether mitogenesis in these cells is associated with changes in a specific isoform, PKC-ζ. Western blot analysis revealed the presence of PKC-α, -βI, and -βII of the conventional group; PKC-δ, -θ, -ε, and -η of the novel group; and PKC-ζ, -μ, and -ι of the atypical group. There was a significant increase in density of the Western blot for PKC-ζ in cells proliferating in response to 10% fetal bovine serum (FBS) to 372 ± 115% of control values ( P < 0.05; n = 3 patients) in the cytosolic fraction. Platelet-derived growth factor (PDGF) produced increases in PKC-ζ in both the cytosolic and membrane fractions to 210 ± 49 and 443 ± 227%, respectively, of control values ( P < 0.05; n = 4 patients). There was no change in expression of PKC-α, -βI, -βII, -θ, -ε, -η, -δ, or -ι in response to the same stimuli. PGE2(1 μM) added to the cells 30 min before PDGF reduced incorporation of [3H]thymidine from 5,580 ± 633 (SE) to 3,980 ± 126 dpm ( P < 0.05; n = 3 patients) and, in addition, reduced expression of PKC-ζ in the membrane fraction as determined by Western blotting from 266 ± 66 to 110 ± 4% of control values ( P < 0.05; n = 3 patients). PKC-ζ activity in stimulated cells (10% FBS), as assessed by immunoprecipitation and phosphorylation of glycogen synthase peptide, was ∼3-fold greater than that in unstimulated cells, and the amount of PKC-ζ protein correlated with isoenzyme activity ( r2= 0.91; P < 0.02; n = 4 patients). In conclusion, this study 1) provides the first description of which isoforms of PKC are present in human cultured airway smooth muscle cells and 2) shows that proliferation of these cells is associated with upregulation of PKC-ζ. Whether activation of PKC-ζ is a primary or secondary event in airway smooth muscle cell proliferation remains to be determined.
Induction of thioredoxin and thioredoxin reductase gene expression in lungs of newborn primates by oxygen
Authors: Das Kumuda C. , Guo Xiao-Ling , White Carl W.
Published: December 2017

Thioredoxin (TRX) is a potent protein disulfide oxidoreductase important in antioxidant defense and regulation of cell growth and signal transduction processes, among them the production of nitric oxide. We report that lung TRX and its reductase, TR, are specifically upregulated at birth by O2. Throughout the third trimester, mRNAs for TRX and TR were expressed constitutively at low levels in fetal baboon lungs. However, after premature birth (125 or 140 of 185 days gestation), lung TRX and TR mRNAs increased rapidly with the onset of O2or air breathing. Lung TRX mRNA also increased in lungs of term newborns with air breathing. Premature animals (140 days) breathing 100% O2develop chronic lung disease within 7–14 days. These animals had greater TRX and TR mRNAs after 1, 6, or 10 days of life than fetal control animals. In 140-day animals given lesser O2concentrations (as needed) who do not develop chronic lung disease, lung TRX and TR mRNAs were also increased on days 1 and 6 but not significantly on day 10. In fetal distal lung explant culture, mRNAs for TRX and TR were elevated within 4 h in 95% O2relative to 1% O2, and the response was similar at various gestations. In contrast, TRX protein did not increase in lung explants from premature animals (125 or 140 days) but did in those from near-term (175-day) fetal baboons after exposure to hyperoxia. However, lung TRX protein and activity, as well as TR activity, eventually did increase in vivo in response to hyperoxia (6 days). Increases in TRX and TR mRNAs in response to 95% O2also were observed in adult baboon lung explants. When TRX redox status was determined, increased O2tension shifted TRX to its oxidized form. Treatment of lung explants with actinomycin D inhibited TRX and TR mRNA increases in 95% O2, indicating transcriptional regulation by O2. The acute increase in gene expression for both TRX and TR in response to O2suggests an important role for these proteins during the transition from relatively anaerobic fetal life to O2breathing at birth.
Regulation of human airway mucins by acrolein and inflammatory mediators
Authors: Borchers Michael T. , Carty Michael P. , Leikauf George D.
Published: December 2017

Bronchitis, asthma, and cystic fibrosis, marked by inflammation and mucus hypersecretion, can be caused or exacerbated by airway pathogens or irritants including acrolein, an aldehyde present in tobacco smoke. To determine whether acrolein and inflammatory mediators alter mucin gene expression, steady-state mRNA levels of two airway mucins, MUC5AC and MUC5B, were measured (by RT-PCR) in human lung carcinoma cells (NCI-H292). MUC5AC mRNA levels increased after ≥0.01 nM acrolein, 10 μM prostaglandin E2or 15-hydroxyeicosatetraenoic acid, 1.0 nM tumor necrosis factor-α (TNF-α), or 10 nM phorbol 12-myristate 13-acetate (a protein kinase C activator). In contrast, MUC5B mRNA levels, although easily detected, were unaffected by these agonists, suggesting that irritants and associated inflammatory mediators increase mucin biosynthesis by inducing MUC5ACmessage levels, whereas MUC5B is constitutively expressed. When transcription was inhibited, TNF-α exposure increased MUC5AC message half-life compared with control level, suggesting that transcript stabilization is a major mechanism controlling increased MUC5AC message levels. Together, these findings imply that irritants like acrolein can directly and indirectly (via inflammatory mediators) increase airway mucin transcripts in epithelial cells.
Rearrangement of adherens junctions by transforming growth factor-β1: role of contraction
Authors: Hurst Victor , Goldberg Peter L. , Minnear Fred L. , Heimark Ronald L. , Vincent Peter A.
Published: December 2017

The signal transduction pathways that lead to disruption of pulmonary endothelial monolayer integrity by transforming growth factor-β1 (TGF-β1) have not been elucidated. The purpose of this investigation was to determine whether disassembly of the adherens junction is temporally associated with the TGF-β1-induced decrease in pulmonary endothelial monolayer integrity. Measurement of albumin clearance and electrical resistance showed that monolayer integrity started to decrease between 1 and 2 h post-TGF-β1 treatment and continued to slowly decrease over the next 6 h. Immunofluorescence microscopy of monolayers between 2 and 3 h post-TGF-β1 showed that β-catenin, plakoglobin, α-catenin, and cadherin-5 were colocalized both at the cell periphery and in newly formed bands that are perpendicular to the cell-cell border. At 4 h post-TGF-β1, cells began separating; however, β- and α-catenin, plakoglobin, and cadherin-5 could still be found at the cell periphery at areas of cell separation and in strands between separated cells. By 8 h, these junctional proteins were no longer present at the cell periphery at areas of cell separation. The myosin light chain kinase inhibitor KT-5926 prevented the TGF-β1-induced change in integrity but did not inhibit the formation of actin stress fibers or the formation of bands containing adherens junction proteins that were perpendicular to the cell-cell junction. Overall, these results suggest that adherens junction disassembly occurs after cell separation during TGF-β1-induced decreases in pulmonary endothelial monolayer integrity and that the loss of integrity may be due to the activation of a myosin light chain kinase-dependent signaling cascade.
Regulation of 15-lipoxygenase expression and mucus secretion by IL-4 in human bronchial epithelial cells
Authors: Jayawickreme S. P. , Gray T. , Nettesheim P. , Eling T.
Published: December 2017

Our laboratory has recently shown that mucus differentiation of cultured normal human tracheobronchial epithelial (NHTBE) cells is accompanied by the increased expression of 15-lipoxygenase (15-LO). We used differentiated NHTBE cells to investigate the regulation of 15-LO expression and mucus secretion by inflammatory cytokines. Interleukin (IL)-4 and IL-13 dramatically enhanced the expression of 15-LO, whereas tumor necrosis factor-α, IL-1β, and interferon (IFN)-γ had no effect. These cytokines did not increase the expression of cyclooxygenase-2, with the exception of a modest induction by IL-1β. The IL-4-induced 15-LO expression was concentration dependent, and mRNA and protein expression increased within 3 and 6 h, respectively, after IL-4 treatment. In metabolism studies with intact cells, 15-hydroxyeicosatetraenoic acid (15-HETE) and 13-hydroxyoctadecadienoic acid (13-HODE) were the major metabolites formed from exogenous arachidonic acid and linoleic acid. No prostaglandins were detected. IL-4 treatment dramatically increased the formation of 13-HODE and 15-HETE compared with that in untreated NHTBE cells, and several additional 15-LO metabolites were observed. Pretreatment of NHTBE cells with IFN-γ or dexamethasone did not inhibit the IL-4-induced expression of 15-LO except at high concentrations (100 ng/ml of IFN-γ and 10 μM dexamethasone). IL-4 treatment inhibited mucus secretion and attenuated the expression of the mucin genes MUC5AC and MUC5B at 12–24 h after treatment. Addition of 15-HETE precursor and 13-HODE precursor to the cultures did not alter mucin secretion or mucin gene expression. On the basis of the data presented, we conclude that the increase in 15-LO expression by IL-4 and attenuation of mucus secretion may be independent biological events.
Inhibition of factor XIIIa-mediated incorporation of fibronectin into fibrin by pulmonary surfactant
Authors: Elssner Andreas , Mazur Gertraud , Vogelmeier Claus
Published: December 2017

Intra-alveolar deposition of exudated plasma proteins is a hallmark of acute and chronic inflammatory lung diseases. In particular, fibrin and fibronectin may provide a primary matrix for fibrotic lung remodeling in the alveolar compartment. The present study was undertaken to explore the effect of two surfactant preparations on the incorporation of fibronectin into fibrin. We observed that surfactant phospholipids are associated with insoluble fibrin, factor XIIIa-cross-linked fibrin, and cross-linked fibrin with incorporated fibronectin. Factor XIIIa-mediated binding of fibronectin to fibrin was noticeably altered in the presence of surfactant. Coincubation with two different commercially available surfactants but not with dipalmitoylphosphatidylcholine alone resulted in a reduction of fibronectin incorporation into fibrin clots by approximately one-third. This effect was not dependent on the calcium concentration. We conclude that 1) factor XIIIa-cross-linked fibrin-fibronectin is able to incorporate surfactant phospholipids in amounts comparable to fibrin clots without fibronectin and 2) the binding of fibronectin to fibrin is partially inhibited in the presence of pulmonary surfactant.
Effects of lisofylline on hyperoxia-induced lung injury
Authors: George Caroline L. S. , Fantuzzi Giamila , Bursten Stuart , Leer Laura , Abraham Edward
Published: December 2017

Lisofylline [1-(5 R-hydroxyhexyl)-3,7-dimethylxanthine] decreases lipid peroxidation in vitro and in vivo suppresses proinflammatory cytokine expression in models of lung injury due to sepsis, blood loss, and oxidative damage. In the present experiments, we used a murine hyperoxia model to examine the effects of lisofylline on the activation of nuclear transcriptional regulatory factors [nuclear factor-κB and cAMP response element binding protein (CREB)], the expression of proinflammatory cytokines in the lungs, and the circulating levels of oxidized free fatty acids as well as on hyperoxia-induced lung injury and mortality. Treatment with lisofylline inhibited hyperoxia-associated increases in tumor necrosis factor-α, interleukin-1β, and interleukin-6 in the lungs as well as decreased the levels of hyperoxia-induced serum-oxidized free fatty acids. Although hyperoxic exposure produced activation of both nuclear factor-κB and CREB in lung cell populations, only CREB activation was reduced in the mice treated with lisofylline. Lisofylline diminished hyperoxia-associated increases in lung wet-to-dry weight ratios and improved survival in animals exposed to hyperoxia. These results suggest that lisofylline ameliorates hyperoxia-induced lung injury and mortality through inhibiting CREB activation, membrane oxidation, and proinflammatory cytokine expression in the lungs.
Effect of immune response on gene transfer to the lung via systemic administration of cationic lipidic vectors
Authors: Li Song , Wu Su-Ping , Whitmore Mark , Loeffert Eric J. , Wang Lin , Watkins Simon C. , Pitt Bruce R. , Huang Leaf
Published: December 2017

Cationic lipid-mediated intravenous gene delivery shows promise in treating pulmonary diseases including lung tumor metastases, pulmonary hypertension, and acute respiratory distress syndrome. Nevertheless, clinical applications of cationic lipidic vectors via intravenous administration are limited by their transient gene expression. In addition, repeated dosing is not effective at frequent intervals. In an effort to elucidate the mechanism of gene inactivation, we report in this study that cationic lipid-protamine-DNA (LPD) complexes, but not each component alone, can induce a high level of cytokine production, including interferon-γ and tumor necrosis factor-α. Furthermore, we demonstrate that LPD administration triggers apoptosis in the lung, a phenomenon that may be mediated in part by the two cytokines. Treatment of mice with antibodies against the two cytokines prolongs the duration of gene expression and also improves lung transfection on a second administration of LPD. Although the mechanism underlying LPD-induced cytokine production is unclear, methylation of the DNA significantly decreased the level of both interferon-γ and tumor necrosis factor-α, suggesting that unmethylated CpG sequences in plasmid DNA play an important role. These data suggest that decreasing the CpG-mediated immune response while not affecting gene expression may be a useful therapeutic strategy to improve cationic lipid-mediated intravenous gene delivery to the lung.
Phenotypic control of gap junctional communication by cultured alveolar epithelial cells
Authors: Abraham Valsamma , Chou Michael L. , DeBolt Kristine M. , Koval Michael
Published: December 2017

We examined phenotype-specific changes in gap junction protein [connexin (Cx)] expression and function by cultured rat alveolar type II cells. Type II cells cultured on extracellular matrix in medium containing keratinocyte growth factor (KGF) and 2% fetal bovine serum (FBS; KGF/2) retained expression of surfactant protein C and the 180-kDa lamellar body membrane protein (lbm180). These markers were lost when cells were cultured in medium containing 10% FBS (MEM/10). With RT-PCR, cells cultured in MEM/10 showed transient increases in Cx43 and Cx46 mRNA expression, whereas Cx32 and Cx26 decreased and Cx30.3 and Cx37 were unchanged. Transient changes in Cx32, Cx43, and Cx46 protein expression were confirmed by immunoblot. In contrast, cells cultured in KGF/2 retained expression of Cx32 and showed increased expression of Cx30.3 and Cx46 mRNAs, compared with that in day 0 cells. With immunofluorescence microscopy, Cx32 and Cx43 were at the plasma membrane of cells grown in KGF/2, whereas Cx46 was exclusively intracellular. Type II cells cultured in MEM/10 showed ∼3- to 4-fold more intercellular transfer of microinjected lucifer yellow through gap junctions than cells grown in 2% FBS. Thus type II cells dynamically alter gap junctional communication, and distinct alveolar epithelial cell phenotypes express different connexins.
Neutrophil elastase increasesMUC5ACmRNA and protein expression in respiratory epithelial cells
Authors: Voynow Judith A. , Young Lisa Rosenthal , Wang Yiqiong , Horger Teresa , Rose Mary C. , Fischer Bernard M.
Published: December 2017

Chronic neutrophil-predominant inflammation and hypersecretion of mucus are common pathophysiological features of cystic fibrosis, chronic bronchitis, and viral- or pollution-triggered asthma. Neutrophils release elastase, a serine protease, that causes increased mucin production and secretion. The molecular mechanisms of elastase-induced mucin production are unknown. We hypothesized that as part of this mechanism, elastase upregulates expression of a major respiratory mucin gene, MUC5AC. A549, a human lung carcinoma cell line that expresses MUC5AC mRNA and protein, and normal human bronchial epithelial cells in an air-liquid interface culture were stimulated with neutrophil elastase. Neutrophil elastase increased MUC5AC mRNA levels in a time-dependent manner in both cell culture systems. Neutrophil elastase treatment also increased MUC5AC protein levels in A549 cells. The mechanism of MUC5AC gene regulation by elastase was determined in A549 cells. The induction of MUC5AC gene expression required serine protease activity; other classes of proteases had no effect on MUC5AC gene expression. Neutrophil elastase increased MUC5AC mRNA levels by enhancing mRNA stability. This is the first report of mucin gene regulation by this mechanism.