bluuebunny/crossref_metadata_2025 huggingface dataset:
Early high expression of IP-10 in F344 rats resistant to Sendai virus-induced airway injury
Authors: Cai Xuezhong , Castleman William L.
Published: March 2015

Weanling F344 and BN rats differ markedly in their susceptibility to Sendai virus-induced airway injury. Early gene expression that controls their differences in susceptibility remains poorly understood. In this study we combined suppressive subtractive hybridization and cDNA library array hybridization to identify genes differentially expressed in virus-susceptible BN and virus-resistant F344 rats during the first 3 days after inoculation. Differential expression of selected clones was further verified by quantitative RT-PCR. Seven virus-induced gene segments were identified. Of them, interferon-γ-inducible protein 10 (IP-10), Mx1, and guany-late-binding protein-2 mRNA abundance in infected F344 rats was 201.5, 188.2, and 281.7% higher, respectively, than that of infected BN rats at 2 days after inoculation. In situ hybridization indicated that virus-induced IP-10 was expressed mainly in airway epithelial cells of F344 rats. Sendai virus infection can directly induce IP-10 expression in rat tracheal epithelial cells in vitro. IP-10 early high expression might contribute to the resistance to virus-induced airway disease in F344 rats by promoting Th1 responses and increasing antiviral activity.
Pressure-induced endothelial Ca2+oscillations in lung capillaries
Authors: Kuebler Wolfgang M. , Ying Xiaoyou , Bhattacharya Jahar
Published: March 2015

Endothelial second messenger responses may contribute to the pathology of high vascular pressure but remain poorly understood because of the lack of direct in situ quantification. In lung venular capillaries, we determined endothelial cytosolic Ca2+concentration [Ca2+]iby the fura 2 ratioing method. Pressure elevation increased mean endothelial [Ca2+]iby Ca2+influx through gadolinium-inhibitable channels and amplified [Ca2+]ioscillations by Ca2+release from intracellular stores. Endothelial [Ca2+]itransients were induced by pressure elevations of as little as 5 cmH2O and increased linearly with higher pressures. Heptanol inhibition of [Ca2+]ioscillations in a subset of endothelial cells indicated that oscillations originated from pacemaker endothelial cells and were propagated to adjacent nonpacemaker cells by gap junctional communication. Our findings indicate the presence of a sensitive, active endothelial response to pressure challenge in lung venular capillaries that may be relevant in the pathogenesis of pressure-induced lung microvascular injury.
Increased fragility of pulmonary capillaries in newborn rabbit
Authors: Fu Zhenxing , Heldt Gregory P. , West John B.
Published: March 2015

The pulmonary capillaries of neonatal lungs are potentially vulnerable to stress failure because of the complex changes in the pulmonary circulation that occur at birth. We perfusion fixed the lungs from nine anesthetized newborn rabbits at capillary transmural pressures (Ptm) of 5 ± 5, 10 ± 5, and 15 ± 5 cmH2O. Normal microscopic appearances were seen at Ptm values of 5 ± 5 and 10 ± 5 cmH2O, but massive airway edema was observed in lungs perfused at a Ptm of 15 ± 5 cmH2O. Consistent with this, no disruptions of the alveolar epithelium were observed at Ptm values of 5 ± 5 cmH2O, but mean values of 0.11 and 1.22 breaks/mm epithelium were found at Ptm of 10 ± 5 and 15 ± 5 cmH2O, respectively ( P < 0.05 for 5 ± 5 vs. 15 ± 5 cmH2O). These pressures are in striking contrast to those in the adult rabbit in which, by a similar procedure, a Ptm of 52.5 cmH2O, is required before stress failure is consistently seen. We conclude that stress failure of pulmonary capillaries in newborn rabbit lungs can occur at Ptm values of less than one-third of those that are required in adult lungs.
Histone acetylation regulates epithelial IL-8 release mediated by oxidative stress from environmental particles
Authors: Gilmour Peter S. , Rahman Irfan , Donaldson Kenneth , MacNee William
Published: March 2015

Increases in the levels of environmental particulate matter with a diameter of <10 μm diameter (PM10) in the air are associated with a variety of adverse health effects, particularly chronic lung and cardiovascular diseases. The expression of many inflammatory genes involves the remodeling of the chromatin structure provided by histone proteins. Histone acetylation causes the unwinding of chromatin structure, therefore allowing transcription factor access to promoter sites. Acetylation is reversible and is regulated by histone acetyltransferases (HATs), which promote acetylation, and deacetylases, which promote deacetylation. PM10 and H2O2 increased IL-8 protein release from A549 cells after 24-h treatment, and this was enhanced by histone deacetylase inhibition by trichostatin A (cotreatment). PM10 and H2O2 treatment also increased HAT activity as well as the level of acetylated histone 4 (H4). PM10 enhanced H4 acetylation that was mediated by oxidative stress as shown by thiol antioxidant inhibition. Acetylation of H4 mediated by PM10 was associated with the promoter region of the IL-8 gene. These data suggest that remodeling of chromatin by histone acetylation plays a role in PM10-mediated responses in the lungs.
Eosinophil adhesion to cholinergic nerves via ICAM-1 and VCAM-1 and associated eosinophil degranulation
Authors: Sawatzky Deborah A. , Kingham Paul J. , Court Emma , Kumaravel Bharathy , Fryer Allison D. , Jacoby David B. , McLean W. Graham , Costello Richard W.
Published: March 2015

In vivo, eosinophils localize to airway cholinergic nerves in antigen-challenged animals, and inhibition of this localization prevents antigen-induced hyperreactivity. In this study, the mechanism of eosinophil localization to nerves was investigated by examining adhesion molecule expression by cholinergic nerves. Immunohistochemical and functional studies demonstrated that primary cultures of parasympathetic nerves express vascular cell adhesion molecule-1 (VCAM-1) and after cytokine pretreatment with tumor necrosis factor-α and interferon-γ intercellular adhesion molecule-1 (ICAM-1). Eosinophils adhere to these parasympathetic neurones after cytokine pretreatment via a CD11/18-dependent pathway. Immunohistochemistry and Western blotting showed that a human cholinergic nerve cell line (IMR-32) expressed VCAM-1 and ICAM-1. Inhibitory experiments using monoclonal blocking antibodies to ICAM-1, VCAM-1, or CD11/18 and with the very late antigen-4 peptide inhibitor ZD-7349 showed that eosinophils adhered to IMR-32 cells via these adhesion molecules. The protein kinase C signaling pathway is involved in this process as a specific inhibitor-attenuated adhesion. Eosinophil adhesion to IMR-32 cells was associated with the release of eosinophil peroxidase and leukotriene C4. Thus eosinophils adhere to cholinergic nerves via specific adhesion molecules, and this leads to eosinophil activation and degranulation; this may be part of the mechanism of eosinophil-induced vagal hyperreactivity.
Effects of hyperoxia on VEGF, its receptors, and HIF-2α in the newborn rat lung
Authors: Hosford Gayle E. , Olson David M.
Published: March 2015

Signaling through the hypoxia inducible factor (HIF)-VEGF-VEGF receptor system (VEGF signaling system) leads to angiogenesis and epithelial cell proliferation and is a key mechanism regulating alveolarization in lungs of newborn rats. Hyperoxia exposure (>95% O2days 4–14) arrests lung alveolarization and may do so through suppression of the VEGF signaling system. Lung tissue mRNA levels of HIF-2α and VEGF increased from days 4–14 in normoxic animals, but hyperoxia suppressed these increases. Levels of HIF-2α and VEGF mRNA were correlated in the air but not the O2-treated group, suggesting that the low levels of HIF-2α observed at high O2concentrations are not stimulating VEGF expression. VEGF164protein levels increased with developmental age, and with hyperoxia to day 9, but continuing hyperoxia decreased levels by day 12. VEGFR1 and VEGFR2 mRNA expression also increased in air-exposed animals, and these, too, were significantly decreased by hyperoxia by day 9 and day 12, respectively. Receptor protein levels did not increase with development; however, O2did decrease protein to less than air values. Hyperoxic suppression of VEGF signaling from days 9–14 may be one mechanism by which alveolarization is arrested.
cAMP activates BKCachannels in pulmonary arterial smooth muscle via cGMP-dependent protein kinase
Authors: Barman Scott A. , Zhu Shu , Han Guichan , White Richard E.
Published: March 2015

The signal transduction mechanisms defining the role of cyclic nucleotides in the regulation of pulmonary vascular tone is currently an area of great interest. Normally, signaling mechanisms that elevate cAMP and guanosine-3′,5′-cyclic monophosphate (cGMP) maintain the pulmonary vasculature in a relaxed state. Modulation of the large-conductance, calcium- and voltage-activated potassium (BKCa) channel is important in the regulation of pulmonary arterial pressure, and inhibition (closing) of the BKCachannel has been implicated in the development of pulmonary hypertension. Accordingly, studies were done to determine the effect of cAMP-elevating agents on BKCachannel activity using patch-clamp studies in pulmonary arterial smooth muscle cells (PASMC) of the fawn-hooded rat (FHR), a recognized animal model of pulmonary hypertension. Forskolin (10 μM), a stimulator of adenylate cyclase and an activator of cAMP-dependent protein kinase (PKA), and 8–4-chlorophenylthio (CPT)-cAMP (100 μM), a membrane-permeable derivative of cAMP, opened BKCachannels in single FHR PASMC. Treatment of FHR PASMC with 300 nM KT5823, a selective inhibitor of cGMP-dependent protein kinase (PKG) activity inhibited the effect of both forskolin and CPT-cAMP. In contrast, blocking PKA activation with 300 nM KT5720 had no effect on forskolin or CPT-cAMP-stimulated BKCachannel activity. These results indicate that cAMP-dependent vasodilators activate BKCachannels in PASMC of FHR via PKG-dependent and PKA-independent signaling pathways, which suggests cross-activation between cyclic nucleotide-dependent protein kinases in pulmonary arterial smooth muscle and therefore, a unique signaling pathway for cAMP-induced pulmonary vasodilation.
Elevated levels of plasminogen activator inhibitor-1 in pulmonary edema fluid are associated with mortality in acute lung injury
Authors: Prabhakaran Priya , Ware Lorraine B. , White Kimberly E. , Cross Michael T. , Matthay Michael A. , Olman Mitchell A.
Published: March 2015

The alveolar fibrinolytic system is altered in acute lung injury (ALI). Levels of the fibrinolytic protease inhibitor, plasminogen activator inhibitor-1 (PAI-1), are too low in bronchoalveolar lavage to address its prognostic significance. This study was performed to assess whether PAI-1 antigen in undiluted pulmonary edema fluid levels can identify patients with ALI and predict their outcome. PAI-1 antigen levels in both plasma and edema fluid were higher in ALI compared with hydrostatic edema, and edema fluid PAI-1 values identified those with ALI with high sensitivity and specificity. Both the high plasma and edema fluid PAI-1 antigen values were associated with a higher mortality rate and fewer days of unassisted ventilation in patients with ALI. Differences in PAI-1 activity were concordant with levels of PAI-1 antigen. Although the fibrin-derived alveolar D-dimer levels were strikingly similar in both groups, ALI patients had a higher relative proportion of D-monomer. In conclusion, PAI-1 levels in edema fluid and plasma identify those with ALI that have a poor prognosis. The data indicate that fibrin turnover in early ALI is a consequence of a rapid fibrinogen influx and fractional fibrinolytic inhibition.
Amino-terminal and midmolecule parathyroid hormone-related protein, phosphatidylcholine, and type II cell proliferation in silica-injured lung
Authors: Hastings Randolph H. , Quintana Rick A. , Sandoval Rebeca , Burton Douglas W. , Deftos Leonard J.
Published: March 2015

Acute silica lung injury is marked by alveolar phospholipidosis and type II cell proliferation. Parathyroid hormone-related protein (PTHrP) 1-34 could have a regulatory role in this process because it stimulates phosphatidylcholine secretion and inhibits type II cell growth. Other regions of the PTHrP molecule may have biological activity and can also exert pulmonary effects. This study examined the temporal pattern for expression of several regions of PTHrP after silica lung injury and evaluated the effects of changes in expression on cell proliferation and lung phospholipids. Expression of all PTHrP regions fell at 4 days after injury. Reversing the decline in PTHrP 1-34 or PTHrP 67-86 with one intratracheal dose and four daily subcutaneous doses of PTHrP 1-34 or PTHrP 67-86 stimulated bronchoalveolar lavage disaturated phosphatidylcholine (DSPC) levels. Cell culture studies indicate that the peptides exerted direct effects on DSPC secretion by type II cells. Neither peptide affected type II cell proliferation with this dosing regimen, but addition of an additional intratracheal dose resulted in significant inhibition of growth, consistent with previous effects of PTHrP 1-34 in hyperoxic lung injury. These studies establish a regulatory role for PTHrP 1-34 and PTHrP 67-86 in DSPC metabolism and type II cell proliferation in silica injury. Growth inhibitory effects of PTHrP could interact with phospholipid stimulation by affecting type II cell numbers. Further studies are needed to explore the complex interactions of PTHrP-derived peptides and the type II cell response at various stages of silica lung injury.
Subcellular localization ofPseudomonaspyocyanin cytotoxicity in human lung epithelial cells
Authors: O'Malley Yunxia Q. , Abdalla Maher Y. , McCormick Michael L. , Reszka Krzysztof J. , Denning Gerene M. , Britigan Bradley E.
Published: March 2015

The Pseudomonas aeruginosa secretory product pyocyanin damages lung epithelium, likely due to redox cycling of pyocyanin and resultant superoxide and H2O2generation. Subcellular site(s) of pyocyanin redox cycling and toxicity have not been well studied. Therefore, pyocyanin's effects on subcellular parameters in the A549 human type II alveolar epithelial cell line were examined. Confocal and electron microscopy studies suggested mitochondrial redox cycling of pyocyanin and extracellular H2O2release, respectively. Pyocyanin decreased mitochondrial and cytoplasmic aconitase activity, ATP levels, cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, and mitochondrial membrane potential. These effects were transient at low pyocyanin concentrations and were linked to apparent cell-mediated metabolism of pyocyanin. Overexpression of MnSOD, but not CuZnSOD or catalase, protected cellular aconitase, but not ATP, from pyocyanin-mediated depletion. This suggests that loss of aconitase activity is not responsible for ATP depletion. How pyocyanin leads to ATP depletion, the mechanism of cellular metabolism of pyocyanin, and the impact of mitochondrial pyocyanin redox cycling on other cellular events are important areas for future study.
Effect of poly(ADP ribose) synthetase inhibition on burn and smoke inhalation injury in sheep
Authors: Shimoda Katsumi , Murakami Kazunori , Enkhbaatar Perenlei , Traber Lillian D. , Cox Robert A. , Hawkins Hal K. , Schmalstieg Frank C. , Komjáti Katalin , Mabley Jon G. , Szabó Csaba , Salzman Andrew L. , Traber Daniel L.
Published: March 2015

We investigated the role of the nuclear enzyme poly (ADP ribose) synthetase (PARS) in the pathogenesis of combined burn and smoke inhalation (burn/smoke) injury in an ovine model. Eighteen sheep were operatively prepared for chronic study. PARS inhibition was achieved by treatment with a novel and selective PARS inhibitor INO-1001. The PARS inhibitor attenuated 1) lung edema formation, 2) deterioration of gas exchange, 3) changes in airway blood flow, 4) changes in airway pressure, 5) lung histological injury, and 6) systemic vascular leakage. Lipid oxidation and plasma nitrite/nitrate (stable breakdown products of nitric oxide) levels were suppressed with the use of INO-1001. We conclude that PARS inhibition attenuates various aspects of the pathophysiological response in a clinically relevant experimental model of burn/smoke inhalation injury.
Pattern of NOS2 and NOS3 mRNA expression in human A549 cells and primary cultured AEC II
Authors: Pechkovsky D. V. , Zissel G. , Goldmann T. , Einhaus M. , Taube C. , Magnussen H. , Schlaak M. , Müller-Quernheim J.
Published: March 2015

The human alveolar type II epithelium-like cell line A549 expresses nitric oxide synthase type 2 (NOS2), but not NOS3, and produces nitric oxide (NO) upon appropriate stimulation. However, relatively little is known regarding the NOS2 and NOS3 expression of type II human alveolar epithelial cells (AEC II) in primary culture. We detected NOS3 mRNA in freshly isolated AEC II and after 24 h of culture. NOS3 mRNA levels were much higher in AEC II cultured for 24 h with or without interferon-γ, interleukin-1β, and tumor necrosis factor-α, compared with freshly isolated cells. Cytokine stimulation did not change the NOS3 mRNA expression level in AEC II compared with unstimulated cells. NOS3 protein expression was verified by Western blot, and measuring nitrate/nitrite revealed that the protein is active. In contrast, neither NOS2 mRNA nor protein could be detected in freshly isolated, unstimulated or cytokine-stimulated human AEC II in 24- or 72-h primary cultures, whereas A549 cells expressed NOS2 message and protein upon stimulation with proinflammatory cytokines. In situ hybridization confirmed that AEC II express NOS3, but not NOS2 mRNA in vivo. These data demonstrate that there are significant differences between primary AEC II and A549 cells in NOS mRNA expression pattern.
Methacholine-induced airway hyperresponsiveness is dependent on Gαqsignaling
Authors: Borchers Michael T. , Biechele T. , Justice J. P. , Ansay T. , Cormier S. , Mancino V. , Wilkie T. M. , Simon M. I. , Lee N. A. , Lee J. J.
Published: March 2015

Airway function in health and disease as well as in response to bronchospastic stimuli (i.e., irritants, allergens, and inflammatory mediators) is controlled, in part, by cholinergic muscarinic receptor regulation of smooth muscle. In particular, the dependence of airway smooth muscle contraction/relaxation on heterotrimeric G protein-coupled receptor signaling suggests that these events underlie the responses regulating airway function. Gαq-containing G proteins are proposed to be a prominent signaling pathway, and the availability of knockout mice deficient of this subunit has allowed for an investigation of its potential role in airway function. Airway responses in Gαq-deficient mice (activities assessed by both tracheal tension and in vivo lung function measurements) were attenuated relative to wild-type controls. Moreover, ovalbumin sensitization/aerosol challenge of Gαq-deficient mice also failed to elicit an allergen-induced increase in airway reactivity to methacholine. These findings indicate that cholinergic receptor-mediated responses are dependent on Gαq-mediated signaling events and identify Gαqas a potential target of preventative/intervening therapies for lung dysfunction.
Leukotrienes mediate neurogenic inflammation in lungs of young rats infected with respiratory syncytial virus
Authors: Wedde-Beer Katrin , Hu Chengping , Rodriguez Maria M. , Piedimonte Giovanni
Published: March 2015

Respiratory syncytial virus (RSV) infection potentiates neurogenic inflammation in rat airways. Because some vascular effects of sensory nerves are mediated by cysteinyl leukotrienes (cysLTs), we studied whether the receptor antagonist montelukast inhibits neurogenic plasma extravasation in RSV-infected rats. Pathogen-free rats were inoculated at 2 wk (weanlings) or 12 wk (adults) of age with RSV or virus-free medium and treated with montelukast or its vehicle starting 1 day before inoculation. Five days postinoculation, we measured the extravasation of Evans blue-labeled albumin in the respiratory tract after stimulation of sensory nerves with capsaicin. Montelukast had no effect in the extrapulmonary airways but abolished albumin extravasation in the intrapulmonary airways of RSV-infected rats, with a larger effect in weanlings than in adults. Increased concentrations of 5-lipoxygenase-encoding mRNA and cysLTs, as well as numerous mast cells, were detected in the lung tissues of RSV-infected weanling rats. These observations suggest that the release of neuropeptides from capsaicin-sensitive sensory nerves and nonneuronal cells in the lungs of RSV-infected young rats increases vascular permeability by promoting the release of leukotrienes from mast cells.
Histamine stimulates phosphorylation of adherens junction proteins and alters their link to vimentin
Authors: Shasby D. Michael , Ries Dana R. , Shasby Sandra S. , Winter Michael C.
Published: March 2015

Histamine increases microvascular permeability by creating small transitory (100–400 nm) gaps between adjacent endothelial cells at sites of vascular endothelial (VE)-cadherin-based adhesion. We examined the effects of histamine on the proteins within the VE-cadherin-based adherens junction in primary human umbilical vein endothelial cells. VE-cadherin is linked not only by β- and α-catenin to cortical actin but also by γ-catenin to the intermediate filament vimentin. In mature human umbilical vein cultures, the VE-cadherin immunoprecipitate contained equivalent amounts of α- and β-catenin, 130% as much β- as γ-catenin, and 50% as much actin as vimentin. Within 60 s, histamine decreased the fraction of VE-cadherin in the insoluble portion of the cell lysate by 35 ± 1.5%. At the same time, histamine decreased the amount of vimentin that immunoprecipitated with VE-cadherin by 50 ± 6%. Histamine did not affect the amount of actin or the amount of α-, β-, or γ-catenin that immunoprecipitated with VE-cadherin. Within 60 s, histamine simulated a doubling in the phosphorylation of VE-cadherin and β- and γ-catenin. The VE-cadherin immunoprecipitate contained kinase activity that phosphorylated VE-cadherin and γ-catenin in vitro.
Role of cyclooxygenase activation and prostaglandins in antigen-induced excitability changes of bronchial parasympathetic ganglia neurons
Authors: Kajekar Radhika , Undem Bradley J. , Myers Allen C.
Published: March 2015

In vitro antigen challenge has multiple effects on the excitability of guinea pig bronchial parasympathetic ganglion neurons, including depolarization, causing phasic neurons to fire with a repetitive action potential pattern and potentiating synaptic transmission. In the present study, guinea pigs were passively sensitized to the antigen ovalbumin. After sensitization, the bronchi were prepared for in vitro electrophysiological intracellular recording of parasympathetic ganglia neurons to investigate the contribution of cyclooxygenase activation and prostanoids on parasympathetic nerve activity. Cyclooxygenase inhibition with either indomethacin or piroxicam before in vitro antigen challenge blocked the change in accommodation. These cyclooxygenase inhibitors also blocked the release of prostaglandin D2 (PGD2) from bronchial tissue during antigen challenge. We also determined that PGE2 and PGD2 decreased the duration of the action potential after hyperpolarization, whereas PGF2α potentiated synaptic transmission. Thus prostaglandins released during antigen challenge have multiple effects on the excitability of guinea pig bronchial parasympathetic ganglia neurons, which may consequently affect the output from these neurons and thereby alter parasympathetic tone in the lower airways.
Pulmonary NO synthase expression is attenuated in a fetal baboon model of chronic lung disease
Authors: Afshar Sam , Gibson Linda L. , Yuhanna Ivan S. , Sherman Todd S. , Kerecman Jay D. , Grubb Peter H. , Yoder Bradley A. , McCurnin Donald C. , Shaul Philip W.
Published: March 2015

Nitric oxide (NO), produced by NO synthase (NOS), serves multiple functions in the perinatal lung. In fetal baboons, neuronal (nNOS), endothelial (eNOS), and inducible NOS (iNOS) are all primarily expressed in proximal respiratory epithelium. In the present study, NOS expression and activity in proximal lung and minute ventilation of NO standard temperature and pressure (V˙eNOSTP) were evaluated in a model of chronic lung disease (CLD) in baboons delivered at 125 days (d) of gestation (term = 185 d) and ventilated for 14 d, obtaining control lung samples from fetuses at 125 or 140 d of gestation. In contrast to the normal 73% increase in total NOS activity from 125 to 140 d of gestation, there was an 83% decline with CLD. This was related to marked diminutions in both nNOS and eNOS expression and enzymatic activity. nNOS accounted for the vast majority of enzymatic activity in all groups. The normal 3.3-fold maturational rise in iNOS protein expression was blunted in CLD, yet iNOS activity was elevated in CLD compared with at birth. The contribution of iNOS to total NOS activity was minimal in all groups.V˙eNOSTPremained stable in the range of 0.5–1.0 nl · kg−1· min−1from birth to day 7 of life, and it then rose by 2.5-fold. Thus the baboon model of CLD is characterized by deficiency of the principal pulmonary isoforms, nNOS and eNOS, and enhanced iNOS activity over the first 2 wk of postnatal life. It is postulated that these alterations in NOS expression and activity may contribute to the pathogenesis of CLD.
Meconium aspiration produces airway hyperresponsiveness and eosinophilic inflammation in a murine model
Authors: Khan Amir M. , Elidemir Okan , Epstein Cynthia E. , Lally Kevin P. , Xue Hasen , Blackburn Michael , Larsen Gary L. , Colasurdo Giuseppe N.
Published: March 2015

Meconium aspiration syndrome is a cause of significant morbidity and mortality in the perinatal period and has been implicated in the pathogenesis of airway dysfunction. In this study, we developed a murine model to evaluate the effects of meconium aspiration on airway physiology and lung cellular responses. Under light anesthesia, BALB/c mice received a single intratracheal instillation of meconium or physiological saline. Respiratory mechanics were measured in unrestrained animals and expressed as percent increase in enhanced pause to increasing concentrations of methacholine (MCh). Furthermore, we assessed the changes in cells and cytokines into the bronchoalveolar lavage fluid (BALF). We found meconium aspiration produced increased airway responsiveness to MCh at 7 days. These functional changes were associated with lymphocytic/eosinophilic inflammation, goblet cell metaplasia, and increased concentrations of IL-5 and IL-13 in the BALF. Our findings suggest meconium aspiration leads to alterations of airway function, lung eosinophilia, goblet cell metaplasia, and cytokine imbalance, thus providing the first evidence of meconium-induced airway dysfunction in a mouse model.
Magnitude-dependent regulation of pulmonary endothelial cell barrier function by cyclic stretch
Authors: Birukov Konstantin G. , Jacobson Jeffrey R. , Flores Alejandro A. , Ye Shui Q. , Birukova Anna A. , Verin Alexander D. , Garcia Joe G. N.
Published: March 2015

Ventilator-induced lung injury syndromes are characterized by profound increases in vascular leakiness and activation of inflammatory processes. To explore whether excessive cyclic stretch (CS) directly causes vascular barrier disruption or enhances endothelial cell sensitivity to edemagenic agents, human pulmonary artery endothelial cells (HPAEC) were exposed to physiologically (5% elongation) or pathologically (18% elongation) relevant levels of strain. CS produced rapid (10 min) increases in myosin light chain (MLC) phosphorylation, activation of p38 and extracellular signal-related kinase 1/2 MAP kinases, and actomyosin remodeling. Acute (15 min) and chronic (48 h) CS markedly enhanced thrombin-induced MLC phosphorylation (2.1-fold and 3.2-fold for 15-min CS at 5 and 18% elongation and 2.1-fold and 3.1-fold for 48-h CS at 5 and 18% elongation, respectively). HPAEC preconditioned at 18% CS, but not at 5% CS, exhibited significantly enhanced thrombin-induced reduction in transendothelial electrical resistance but did not affect barrier protective effect of sphingosine-1-phosphate (0.5 μM). Finally, expression profiling analysis revealed a number of genes, including small GTPase rho, apoptosis mediator ZIP kinase, and proteinase activated receptor-2, to be regulated by CS in an amplitude-dependent manner. Thus our study demonstrates a critical role for the magnitude of CS in regulation of agonist-mediated pulmonary endothelial cell permeability and strongly suggests phenotypic regulation of HPAEC barrier properties by CS.
Tumor necrosis factor-α activation by adenovirus E1A 13S CR3 occurs in a cell-dependent and cell-independent manner
Authors: Sanchez Traci A. , Booth J. Leland , Metcalf Jordan P.
Published: March 2015

The adenovirus (Ad) early gene product 13S transactivates the tumor necrosis factor (TNF)-α promoter in inflammatory cells. We examined both the subdomains of E1A and the upstream TNF promoter elements involved. In both Jurkat and U-937 cells, zinc finger or carboxyl region mutation of Ad E1A 13S conserved region 3 resulted in a significant loss of transactivation of the TNF promoter (≥69%). For both cell types there was a TNF-negative regulatory element in the −242 to −199 region and a positive regulatory element between −199 and −118. In contrast, an upstream positive regulatory element was detected in different regions in both cell types. In U-937 cells the positive regulatory unit was between −600 and −576, whereas in Jurkat cells it was between −576 and −242. The U-937 upstream element was dependent on a site previously designated epsilon in cooperation with an adjacent nuclear factor-κB-2a site. Therefore, transactivation of the TNF promoter by Ad 13S in lymphocyte and monocyte cell types involves similar subdomains of the E1A protein, but cell-specific TNF promoter elements.
Impaired synthesis of prostaglandin E2by lung fibroblasts and alveolar epithelial cells from GM-CSF−/−mice: implications for fibroproliferation
Authors: Charbeneau Ryan P. , Christensen Paul J. , Chrisman Cara J. , Paine Robert , Toews Galen B. , Peters-Golden Marc , Moore Bethany B.
Published: March 2015

Prostaglandin E2(PGE2) is a potent suppressor of fibroblast activity. We previously reported that bleomycin-induced pulmonary fibrosis was exaggerated in granulocyte-macrophage colony-stimulating factor knockout (GM-CSF−/−) mice compared with wild-type (GM-CSF+/+) mice and that increased fibrosis was associated with decreased PGE2levels in lung homogenates and alveolar macrophage cultures. Pulmonary fibroblasts and alveolar epithelial cells (AECs) represent additional cellular sources of PGE2within the lung. Therefore, we examined fibroblasts and AECs from GM-CSF−/−mice, and we found that they elaborated significantly less PGE2than did cells from GM-CSF+/+mice. This defect was associated with reduced expression of cyclooxygenase-1 and -2 (COX-1 and COX-2), key enzymes in the biosynthesis of PGE2. Additionally, proliferation of GM-CSF−/−fibroblasts was greater than that of GM-CSF+/+fibroblasts, and GM-CSF−/−AECs were impaired in their ability to inhibit fibroblast proliferation in coculture. The addition of GM-CSF to fibroblasts from GM-CSF−/−mice increased PGE2production and decreased proliferation. Similarly, AECs isolated from GM-CSF−/−mice with transgenic expression of GM-CSF under the surfactant protein C promoter (SpC-GM mice) produced more PGE2than did AEC from control mice. Finally, SpC-GM mice were protected from fluorescein isothiocyanate-induced pulmonary fibrosis. In conclusion, these data demonstrate that GM-CSF regulates PGE2production in pulmonary fibroblasts and AECs and thus plays an important role in limiting fibroproliferation.
β-Adrenergic regulation of amiloride-sensitive lung sodium channels
Authors: Chen Xi-Juan , Eaton Douglas C. , Jain Lucky
Published: March 2015

We investigated the mechanism by which cAMP increases sodium transport in lung epithelial cells. Alveolar type II (ATII) cells have two types of amiloride-sensitive, cation channels: a nonselective cation channel (NSC) and a highly selective channel (HSC). Exposure of ATII cells to cAMP, β-adrenergic agonists, or other agents that increase adenylyl cyclase activity increased activity of both channel types, albeit by different mechanisms. NSC open probability ( P o) increased severalfold when exposed to terbutaline, isoproterenol, forskolin, or cAMP analogs without any change in NSC number. In contrast, terbutaline increased HSC number with no significant change in HSC P o. For both channels, the effect of terbutaline was blocked by propranolol and H-89, suggesting a protein kinase A (PKA) requirement for β-adrenergic-induced changes in channel activity. Terbutaline increased cAMP levels in ATII cells, but intracellular calcium also increased. Calcium sequestration with BAPTA blocked β-adrenergic-induced increases in NSC P o but did not alter HSC activity. These observations suggest that β-adrenergic stimulation increases intracellular cAMP and activates PKA. PKA increases HSC number and increases intracellular calcium. The increase in calcium increases NSC P o. Thus increased cAMP levels are likely to increase lung sodium transport regardless of which channel type is present.
Targeted blocking of gene expression for CGRP receptors elevates pulmonary artery pressure in hypoxic rats
Authors: Qing Xin , Keith Ingegerd M.
Published: March 2015

We previously described the protection by calcitonin gene-related peptide (CGRP) against hypoxic pulmonary hypertension. Here, we examine the roles of its putative receptor RDC-1 and receptor activity-modifying protein (RAMP) 1 in mediating this protection by selectively inhibiting their synthesis. RAMP1 is an accessory protein for another putative CGRP receptor, calcitonin receptor-like receptor. Antisense oligodeoxyribonucleotides (ASODNs, 5 mg·kg-1·day-1 or 5 and 10 mg·kg-1·day-1 for RDC-1) targeting RAMP1 and RDC-1 mRNAs were chronically infused to the pulmonary circulation of male Sprague-Dawley rats during 7 days of normoxia or hypobaric hypoxia (380 mmHg), and α-CGRP ASODN was used as a technical control. CGRP, RAMP1, and RDC-1 ASODNs significantly elevated pulmonary artery pressure (PPA) in chronic hypoxic rats compared with hypoxic mismatched ASODN (MMODN) and saline vehicle controls. CGRP and RAMP1 ASODNs raised PPA in normoxic rats briefly exposed to 10% O2 above MMODN and saline controls. Moreover, normoxic rats treated with CGRP ASODN had higher basal pulmonary vascular tone compared with controls. These data confirm the protective role of CGRP in the pulmonary circulation and suggest that endogenous RAMP1 and RDC-1 are essential in regulation of PPA in hypoxia. This is the first in vivo evidence supporting RDC-1 and RAMP1 as functional CGRP receptor and receptor component.
Surfactant protein A enhances the phagocytosis of C1q-coated particles by alveolar macrophages
Authors: Watford Wendy T. , Smithers Molly B. , Frank Michael M. , Wright Jo Rae
Published: March 2015

Surfactant protein-A (SP-A) plays multiple roles in pulmonary host defense, including stimulating bacterial phagocytosis by innate immune cells. Previously, SP-A was shown to interact with complement protein C1q. Our goal was to further characterize this interaction and elucidate its functional consequences. Radiolabeled SP-A bound solid-phase C1q but not other complement proteins tested. The lectin activity of SP-A was not required for binding to C1q. Because C1q is involved in bacterial clearance but alone does not efficiently enhance the phagocytosis of most bacteria, we hypothesize that SP-A enhances phagocytosis of C1q-coated antigens. SP-A enhanced by sixfold the percentage of rat alveolar macrophages in suspension that phagocytosed C1q-coated fluorescent beads. Furthermore, uptake of C1q-coated beads was enhanced when either beads or alveolar macrophages were preincubated with SP-A. In contrast, SP-A had no significant effect on the uptake of C1q-coated beads by alveolar macrophages adhered to plastic slides. We conclude that SP-A may serve a protective role in the lung by interacting with C1q to enhance the clearance of foreign particles.
Fibronectin matrix polymerization regulates small airway epithelial cell migration
Authors: Hocking Denise C. , Chang Cecilia H.
Published: March 2015

The continuous conversion of soluble fibronectin into extracellular matrix fibrils occurs through a dynamic, cell-dependent process. As the extracellular matrix is assembled, changes in the conformation of matrix proteins may expose biologically active, matricryptic sites that alter cell behavior. In this study, an in vitro model of wound healing was used to determine the role of matrix fibronectin in airway epithelial cell motility. Our findings indicate that, under basal conditions, small airway epithelial cell (SAEC) migration requires active fibronectin matrix polymerization. Furthermore, SAEC migration is increased significantly by the interaction of cells with a recombinant construct containing fibronectin's matricryptic III-1 site. In contrast, addition of increasing amounts of fibronectin to SAECs significantly decreased the rate of cell migration. This fibronectin-induced inhibition of cell migration was overcome by blocking excess fibronectin matrix deposition. These data indicate that SAEC migration is regulated in a biphasic manner by the polymerization of fibronectin in the extracellular matrix and suggest a stimulatory role for fibronectin's matricryptic III-1 site in cell motility.
Invited Review: Pulmonary alveoli: formation, the “call for oxygen,” and other regulators
Authors: Massaro Donald , Massaro Gloria D.
Published: March 2015

The lung's only known essential function is to provide sufficient alveolar surface to meet the organism's need for oxygen and elimination of CO2. The importance of the magnitude of alveolar surface area (Sa) to O2uptake (V˙o2) is supported by the presence among mammals of a direct linear relationship between Sa and V˙o2. This match has been achieved, despite the higher body mass-specific V˙o2of small organisms compared with large, by a greater subdivision of alveolar surface, not by a larger relative lung volume in small organisms. This highly conserved relationship between alveolar architecture and V˙o2suggests the presence of similarly conserved mechanisms that control the onset, rate, and cessation of alveolus formation and alveolar size, which are also influenced by retinoids and thyroid and corticosteroid hormones. Furthermore, the “call for oxygen” is met at a breathing rate and tidal volume at which the work of breathing is lowest. Thus there is a complex, fascinating, but poorly understood, signaling relationship among V˙o2, the neural regulation of breathing, and lung architecture, composition, and mechanics.
Glutathione and other low-molecular-weight thiols relax guinea pig trachea ex vivo: interactions with nitric oxide?
Authors: Kloek Joris , van Ark Ingrid , Bloksma Nanne , De Clerck Fred , Nijkamp Frans P. , Folkerts Gert
Published: March 2015

The aim of this study was to determine the effects of glutathione (GSH) on trachea smooth muscle tension in view of previously reported interactions between GSH and nitric oxide (NO) (Gaston B. Biochim Biophys Acta 1411: 323–333, 1999; Kelm M. Biochim Biophys Acta 1411: 273–289, 1999; and Kharitonov VG, Sundquist AR, and Sharma VS. J Biol Chem 270: 28158–28164, 1995) and the high (millimolar) concentrations of GSH in trachea epithelium (Rahman I, Li XY, Donaldson K, Harrison DJ, and MacNee W. Am J Physiol Lung Cell Mol Physiol 269: L285–L292, 1995). GSH and other thiols (1.0–10 mM) dose dependently decreased the tension in isolated guinea pig tracheas. Relaxations by GSH were paralleled with sevenfold increased nitrite levels ( P < 0.05) in the tracheal effluent, suggesting an interaction between GSH and NO. However, preincubation with a NO scavenger did not reduce the relaxations by GSH or its NO adduct, S-nitrosoglutathione (GSNO). Inhibition of guanylyl cyclase inhibited the relaxations induced by GSNO, but not by GSH. Blocking potassium channels, however, completely abolished the relaxing effects of GSH ( P < 0.05). Preincubation of tracheas with GSH significantly ( P< 0.05) suppressed hyperreactivity to histamine as caused by removal of tracheal epithelium. These data indicate that GSH plays a role in maintaining tracheal tone. The mechanism is probably an antioxidative action of GSH itself rather than an action of NO or GSNO.
Activity of pulmonary edema fluid interleukin-8 bound to α2-macroglobulin in patients with acute lung injury
Authors: Kurdowska Anna K. , Geiser Thomas K. , Alden Susanne M. , Dziadek Bozena R. , Noble James M. , Nuckton Thomas J. , Matthay Michael A.
Published: March 2015

The formation of α2-macroglobulin (α2-M)/interleukin-8 (IL-8) complexes may influence the biological activity of IL-8 and the quantitative assessment of IL-8 activity. Therefore, in this study, concentrations of free IL-8 and IL-8 complexes with α2-M were measured in pulmonary edema fluid samples from patients with acute lung injury/acute respiratory distress syndrome (ALI/ARDS) and compared with control patients with hydrostatic pulmonary edema. Patients with ALI/ARDS had significantly higher concentrations of α2-M ( P < 0.01) as well as α2-M/IL-8 complexes ( P < 0.05). Because a substantial amount of IL-8 is complexed to α2-M, standard assays of free IL-8 may significantly underestimate the concentration of biologically active IL-8 in the distal air spaces of patients with ALI/ARDS. Furthermore, IL-8 bound to α2-M retained its biological activity, and this fraction of IL-8 was protected from proteolytic degradation. Thus complex formation may modulate the acute inflammatory process in the lung.
Stretch-stimulated surfactant synthesis is coordinated by the paracrine actions of PTHrP and leptin
Authors: Torday J. S. , Rehan V. K.
Published: March 2015

Intrauterine lung development, culminating in physiological pulmonary surfactant production by epithelial type II (TII) cells, is driven by fluid distension through unknown mechanisms. Differentiation of alveolar epithelial and mesenchymal cells is mediated by soluble factors like parathyroid hormone-related protein (PTHrP), a stretch-sensitive TII cell product. PTHrP stimulates pulmonary surfactant production by a paracrine feedback loop mediated by leptin, a soluble product of the mature lipofibroblast (LF). When LFs and TIIs are stretched in coculture, there is a fivefold increase in surfactant phospholipid synthesis that can be “neutralized” by inhibitors of PTHrP or leptin, implicating a paracrine feedback loop in this mechanism. Stretching LFs stimulates PTHrP binding (2.5-fold) and downstream stimulation of triglyceride uptake quantitatively (15–25%) due to upregulation of adipose differentiation-related protein expression. Stretching TII cells increases leptin stimulation of their surfactant phospholipid synthesis threefold, suggesting that retrograde signaling by leptin to TII cells is also stretch sensitive. We conclude that the effect of stretch on alveolar LF and TII differentiation is coordinated by PTHrP, leptin, and their receptors.
Identification ofPseudomonas aeruginosaflagellin as an adhesin for Muc1 mucin
Authors: Lillehoj Erik P. , Kim Beom T. , Kim K. Chul
Published: March 2015

We reported previously that Muc1 mucin on the epithelial cell surface is an adhesion site for Pseudomonas aeruginosa (Lillehoj EP, Hyun SW, Kim BT, Zhang XG, Lee DI, Rowland S, and Kim KC. Am J Physiol Lung Cell Mol Physiol 280: L181–L187, 2001). The present study was designed to identify the adhesin(s) responsible for bacterial binding to Muc1 mucin using genetic and biochemical approaches. Chinese hamster ovary (CHO) cells stably transfected with a Muc1 cDNA (CHO-Muc1) or empty plasmid (CHO-X) were compared for adhesion of P. aeruginosa strain PAK. Our results showed that 1) wild-type PAK and isogenic mutant strains lacking pili (PAK/NP) or flagella cap protein (PAK/ fliD) demonstrated significantly increased binding to CHO-Muc1 cells, whereas flagellin-deficient (PAK/ fliC) bacteria were no more adherent to CHO-Muc1 than CHO-X cells, and 2) P. aeruginosa adhesion was blocked by pretreatment of bacteria with antibody to flagellin or pretreatment of CHO-Muc1 cells with purified flagellin. We conclude that flagellin is an adhesin of P. aeruginosa responsible for its binding to Muc1 mucin on the epithelial cell surface.
Keratinocyte growth factor can enhance alveolar epithelial repair by nonmitogenic mechanisms
Authors: Atabai Kamran , Ishigaki Masanobu , Geiser Thomas , Ueki Iris , Matthay Michael A. , Ware Lorraine B.
Published: March 2015

Pretreatment with keratinocyte growth factor (KGF) ameliorates experimentally induced acute lung injury in rats. Although alveolar epithelial type II cell hyperplasia probably contributes, the mechanisms underlying KGF's protective effect remain incompletely described. Therefore, we tested the hypothesis that KGF given to rats in vivo would enhance alveolar epithelial repair in vitro by nonproliferative mechanisms. After intratracheal instillation (48 h) of KGF (5 mg/kg), alveolar epithelial type II cells were isolated for in vitro alveolar epithelial repair studies. KGF-treated cells had markedly increased epithelial repair (96 ± 22%) compared with control cells ( P < 0.001). KGF-treated cells had increased cell spreading and migration at the wound edge but no increase in in vitro proliferation compared with control cells. KGF-treated cells were more adherent to extracellular matrix proteins and polystyrene. Inhibition of the epidermal growth factor (EGF) receptor with tyrosine kinase inhibitors abolished the KGF effect on epithelial repair. In conclusion, in vivo administration of KGF augments the epithelial repair rate of alveolar epithelial cells by altering cell adherence, spreading, and migration and through stimulation of the EGF receptor.
A potent inhibitor of cytosolic phospholipase A2, arachidonyl trifluoromethyl ketone, attenuates LPS-induced lung injury in mice
Authors: Nagase Takahide , Uozumi Naonori , Aoki-Nagase Tomoko , Terawaki Kan , Ishii Satoshi , Tomita Tetsuji , Yamamoto Hiroshi , Hashizume Kohei , Ouchi Yasuyoshi , Shimizu Takao
Published: March 2015

Acute respiratory distress syndrome (ARDS) is an acute lung injury of high mortality rate, and sepsis syndrome is one of the most frequent causes of ARDS. Metabolites of arachidonic acid, including thromboxanes and leukotrienes, are proinflammatory mediators and potentially involved in the development of ARDS. A key enzyme for the production of these inflammatory mediators is cytosolic phospholipase A2(cPLA2). Recently, it has been reported that arachidonyl trifluoromethyl ketone (ATK) is a potent inhibitor of cPLA2. In the present study, we hypothesized that pharmacological intervention of cPLA2could affect acute lung injury. To test this hypothesis, we examined the effects of ATK in a murine model of acute lung injury induced by septic syndrome. The treatment with ATK significantly attenuated lung injury, polymorphonuclear neutrophil sequestration, and deterioration of gas exchange caused by lipopolysaccharide and zymosan administration. The current observations suggest that pharmacological intervention of cPLA2could be a novel therapeutic approach to acute lung injury caused by sepsis syndrome.