bluuebunny/crossref_metadata_2025 huggingface dataset:
Carbon monoxide promotes hypoxic pulmonary vascular remodeling
Authors: Carraway Martha Sue , Ghio Andrew J. , Suliman Hagir B. , Carter Jacqueline D. , Whorton A. Richard , Piantadosi Claude A.
Published: March 2015

CO is a biologically active gas that produces cellular effects by multiple mechanisms. Because cellular binding of CO by heme proteins is increased in hypoxia, we tested the hypothesis that CO interferes with hypoxic pulmonary vascular remodeling in vivo. Rats were exposed to inspired CO (50 parts/million) at sea level or 18,000 ft of altitude [hypobaric hypoxia (HH)], and changes in vessel morphometry and pulmonary pressure-flow relationships were compared with controls. Vascular cell single strand DNA (ssDNA) and proliferating cell nuclear antigen (PCNA) were assessed, and changes in gene and protein expression of smooth muscle α-actin (sm-α-actin), β-actin, and heme oxygenase-1 (HO-1) were evaluated by Western analysis, RT-PCR, and immunohistochemistry. After 21 days of HH, vascular pressure at constant flow and vessel wall thickness increased and lumen diameter of small arteries decreased significantly. The presence of CO, however, further increased both pulmonary vascular resistance (PVR) and the number of small muscular vessels compared with HH alone. CO + HH also increased vascular PCNA and nuclear ssDNA expression compared with hypoxia, suggesting accelerated cell turnover. CO in hypoxia downregulated sm-α-actin and strongly upregulated β-actin. CO also increased lung HO activity and HO-1 mRNA and protein expression in small pulmonary arteries during hypoxia. These data indicate an overall propensity of CO in HH to promote vascular remodeling and increase PVR in vivo.
Killing ofKlebsiella pneumoniaeby human alveolar macrophages
Authors: Hickman-Davis Judy M. , O'Reilly Philip , Davis Ian C. , Peti-Peterdi Janos , Davis Glenda , Young K. Randall , Devlin Robert B. , Matalon Sadis
Published: March 2015

We investigated putative mechanisms by which human surfactant protein A (SP-A) effects killing of Klebsiella pneumoniae by human alveolar macrophages (AMs) isolated from bronchoalveolar lavagates of patients with transplanted lungs. Coincubation of AMs with human SP-A (25 μg/ml) and Klebsiella resulted in a 68% decrease in total colony forming units by 120 min compared with AMs infected with Klebsiella in the absence of SP-A, and this SP-A-mediated effect was abolished by preincubation with NG-monomethyl-l-arginine. Incubation of transplant AMs with SP-A increased intracellular Ca2+concentration ([Ca2+]i) by 70% and nitrite and nitrate (NOx) production by 45% (from 0.24 ± 0.02 to 1.3 ± 0.21 nmol · 106AMs−1· h−1). Preincubation with 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid-acetoxymethyl ester inhibited the increase in [Ca2+]iand abrogated the SP-A-mediated Klebsiella phagocytosis and killing. In contrast, incubation of AMs from normal volunteers with SP-A decreased both [Ca2+]iand NOxproduction and did not result in killing of Klebsiella. Significant killing of Klebsiella was also seen in a cell-free system by sustained production of peroxynitrite (>1 μM/min) at pH 5 but not at pH 7.4. These findings indicate that SP-A mediates pathogen killing by AMs from transplant lungs by stimulating phagocytosis and production of reactive oxygen-nitrogen intermediates.
Clarithromycin inhibits overproduction of muc5ac core protein in murine model of diffuse panbronchiolitis
Authors: Kaneko Yukihiro , Yanagihara Katsunori , Seki Masafumi , Kuroki Misuzu , Miyazaki Yoshitsugu , Hirakata Yoichi , Mukae Hiroshi , Tomono Kazunori , Kadota Jun-ichi , Kohno Shigeru
Published: March 2015

Long-term treatment of macrolide antibiotics is considered an effective treatment for diffuse panbronchiolitis (DPB). Although hypersecretion is a common feature of this disease, and it is known that macrolides inhibit mucin production, the mechanism of the effect on mucin production is unclear. The aim of our study was to determine the production of muc5ac core protein, a major core protein of mucin in airway secretion, and the effect of clarithromycin treatment on such production in a mouse model mimicking DPB. Alcian blue-periodic acid-Schiff-positive cells were detected in the lungs of Pseudomonas aeruginosa-infected mice. Western blots of these mice showed muc5ac glycoprotein at day 1 and increased progressively from day 4 to day 14 after inoculation of bacteria. Clarithromycin (10 mg · kg-1· day-1for 7 days) significantly reduced the muc5ac expression at both the mRNA and protein levels. To investigate the role of molecules upstream in muc5ac regulation, we examined the role of mitogen-activated protein kinase. Extracellular signal-regulated kinase 1/2 phosphorylation increased in the infected lung and decreased after treatment. Our results suggest that overproduction of muc5ac plays an important role in the pathogenesis of DPB and that clinical improvement following macrolide therapy seems to involve, at least in part, its inhibition of mucin overproduction, through modulation of intracellular signal transduction.
DMT1 expression is increased in the lungs of hypotransferrinemic mice
Authors: Ghio Andrew J. , Wang Xinchao , Silbajoris Robert , Garrick Michael D. , Piantadosi Claude A. , Yang Funmei
Published: March 2015

Despite a lack of transferrin, hypotransferrinemic (Hp) mice demonstrate an accumulation of iron in peripheral organs including the lungs. One potential candidate for such transferrin-independent uptake of iron is divalent metal transporter-1 (DMT1), an established iron transporter. We tested the hypothesis that increased concentrations of iron in the lungs of Hp mice are associated with elevations in DMT1 expression. With the use of inductively coupled plasma emission spectroscopy, measurements of nonheme iron confirmed significantly elevated concentrations in the lung tissue of Hp mice relative to the wild-type mice. Western blot analyses for the expression of two isoforms of DMT1 in the Hp mice relative to the wild-type animals demonstrated an elevation for the isoform that lacks an iron-responsive element (IRE) with significant decrements in the expression of +IRE DMT1. With the use of immunohistochemistry, −IRE DMT1 was localized to both airway epithelial cells and alveolar macrophages in wild-type mice. Staining appeared increased in both types of cells in the Hp mice. Elevated concentrations of both tissue nonheme iron and expression of −IRE DMT1 in the Hp mice were associated with increased quantities of −IRE mRNA. There was no difference between wild-type and homozygotic Hp mice in the amount of mRNA for DMT1 +IRE. We conclude that differences between Hp and wild-type mice in nonheme iron concentrations were accompanied by increases in the expression of −IRE DMT1. Increased expression of −IRE DMT1 in the lungs of the Hp mice could be responsible for elevated concentrations of the metal in these tissues.
Recovery of rat type II cell surfactant components during primary cell culture
Authors: Bates Sandra R. , Gonzales Linda W. , Tao Jian-Qin , Rueckert Peter , Ballard Philip L. , Fisher Aron B.
Published: March 2015

A culture system designed to maintain the differentiated characteristics of rat type II cells based on protocols used for human fetal lung pneumocytes was investigated. Type II cells were isolated either from adult rats with elastase (adult type II cells) or from young rats (4–11 days postnatal) with collagenase and trypsin (young type II cells) and were incubated with dexamethasone (Dex, 10 nM) and cAMP (0.1 mM). By day 4 of culture with hormone treatment, the mRNA levels in adult type II cells were less than 3% of day 0 values, whereas surfactant protein (SP)-A protein content was 26%. However, young type II cells maintained lamellar bodies and microvilli and secreted phospholipid in response to ATP. SP-A, -B, and -C mRNA levels were elevated to 159, 350, and 39%, respectively, of day 0 values with a synergistic response to Dex and cAMP, whereas SP-A protein content rose to 119%. Surfactant mRNA and protein did not recover in cells cultured without hormones. This cell culture system restored surfactant components in rat type II cells.
Arachidonic acid metabolites and an early stage of pulmonary hypertension in chronically hypoxic newborn pigs
Authors: Fike Candice D. , Kaplowitz Mark R. , Pfister Sandra L.
Published: March 2015

Our purpose was to determine whether production of arachidonic acid metabolites, particularly cyclooxygenase (COX) metabolites, is altered in 100–400-μm-diameter pulmonary arteries of piglets at an early stage of pulmonary hypertension. Piglets were raised in either room air (control) or hypoxia for 3 days. A cannulated artery technique was used to measure responses of 100–400-μm-diameter pulmonary arteries to arachidonic acid, a prostacyclin analog, or the thromboxane mimetic U46619 . Radioimmunoassay was used to determine pulmonary artery production of thromboxane B2 (TxB2) and 6-keto-prostaglandin F1α (6-keto-PGF1α), the stable metabolites of thromboxane and prostacyclin, respectively. Assessment of abundances of COX pathway enzymes in pulmonary arteries was determined by immunoblot technique. Arachidonic acid induced less dilation in pulmonary arteries from hypoxic than in pulmonary arteries from control piglets. Pulmonary artery responses to prostacyclin and U46619 were similar for both groups. 6-Keto-PGF1αproduction was reduced, whereas TxB2 production was increased in pulmonary arteries from hypoxic piglets. Abundances of both COX-1 and prostacyclin synthase were reduced, whereas abundances of both COX-2 and thromboxane synthase were unaltered in pulmonary arteries from hypoxic piglets. At least partly due to altered abundances of COX pathway enzymes, a shift in production of arachidonic acid metabolites, away from dilators toward constrictors, may contribute to the early phase of chronic hypoxia-induced pulmonary hypertension in newborn piglets.
Putrescine transport in hypoxic rat main PASMCs is required for p38 MAP kinase activation
Authors: Ruchko Mykhaylo , Gillespie Mark N. , Weeks Reitha S. , Olson Jack W. , Babal Pavel
Published: March 2015

Hypoxic pulmonary vascular remodeling in rats is associated with increased polyamine transport in pulmonary artery smooth muscle cells (PASMCs). We therefore defined constitutive and hypoxia-induced polyamine transport properties of rat cultured PASMCs and determined the impact of polyamine transport blockade on hypoxia-induced accumulation of p38 MAP kinase. PASMCs exhibited polyamine transport pathways that were characterized by Michaelis-Menten kinetics. RNA synthesis inhibition attenuated while inhibition of protein synthesis increased polyamine uptake, thus suggesting regulation by ornithine decarboxylase-antizyme. The presence of two transporters with overlapping selectivities, one for putrescine and another for all three polyamines, was inferred by cross-competition studies and by findings that only putrescine uptake was sodium dependent and that hypoxia caused a selective, time-dependent induction of putrescine transport. The pathophysiological significance of augmented putrescine import was suggested by the observation that polyamine transport inhibition suppressed hypoxia-induced p38 MAP kinase phosphorylation. These results indicate that rat PASMCs express two polyamine transporters and that a specific increase in the putrescine uptake pathway is necessary for hypoxia-induced activation of p38 MAP kinase.
GM-CSF expression by human lung microvascular endothelial cells: in vitro and in vivo findings
Authors: Burg Jürgen , Krump-Konvalinkova Vera , Bittinger Fernando , Kirkpatrick Charles James
Published: March 2015

Recently, many findings indicate that granulocyte-macrophage colony-stimulating factor (GM-CSF) plays an important role in the pathogenesis of acute and chronic lung diseases. In the present paper, the production of this cytokine in human pulmonary microvascular endothelial cells (HPMEC) is investigated. In an in vitro study, quiescent HPMEC did not express GM-CSF, either at the transcriptional or at the protein level. After activation for 4 h with tumor necrosis factor (TNF)-α (30/300 U/ml), lipopolysaccharide (LPS; 0.1/1 μg/ml), or interleukin (IL)-1β (100 U/ml), a significant release of GM-CSF was measured by enzyme-linked immunosorbent assay, with a time-dependent increase over 72 h. IL-8 (4, 16, or 64 ng/ml) or IL-1β at a concentration of 10 U/ml did not induce the release of GM-CSF. Human umbilical vein endothelial cells (HUVEC) and the angiosarcoma cell line HAEND served as reference cell lines. GM-CSF release in HPMEC was significantly ( P < 0.025–0.05) less inducible by IL-1β than in HUVEC. A constitutive expression of GM-CSF by HAEND was observed. Additionally, GM-CSF expression in vivo by the lung microvasculature was confirmed by immunohistochemistry in lung tissue. To our knowledge, this is the first report of the ability of human pulmonary endothelial cells to synthesize and release GM-CSF. These results support the hypothesis that the lung microvasculature via the production of GM-CSF is a potential contributor to the cytokine network in lung diseases. This could be of particular importance in the pathogenesis of the acute respiratory distress syndrome in which endothelial dysfunction plays a central pathogenetic role.
Ablation of eosinophils leads to a reduction of allergen-induced pulmonary pathology
Authors: Justice J. Paul , Borchers Michael T. , Crosby Jeffrey R. , Hines Edith M. , Shen Huahao H. , Ochkur Sergei I. , McGarry Michael P. , Lee Nancy A. , Lee James J.
Published: March 2015

A strategy to deplete eosinophils from the lungs of ovalbumin (OVA)-sensitized/challenged mice was developed using antibody-mediated depletion. Concurrent administration [viz. the peritoneal cavity (systemic) and as an aerosol to the lung (local)] of a rat anti-mouse CCR3 monoclonal antibody resulted in the abolition of eosinophils from the lung such that the airway lumen was essentially devoid of eosinophils. Moreover, perivascular/peribronchial eosinophil numbers were reduced to levels indistinguishable from saline-challenged animals. This antibody-mediated depletion was not accompanied by effects on any other leukocyte population, including, but not limited to, T cells and mast cells/basophils. In addition, no effects were observed on other underlying allergic inflammatory responses in OVA-treated mice, including OVA-specific immunoglobulin production as well as T cell-dependent elaboration of Th2 cytokines. The ablation of virtually all pulmonary eosinophils in OVA-treated mice (i.e., without concurrent effects on T cell activities) resulted in a significant decrease in mucus accumulation and abolished allergen-induced airway hyperresponsiveness. These data demonstrate a direct causative relationship between allergen-mediated pulmonary pathologies and eosinophils.
Leukocyte recruitment in the airways: an intravital microscopic study of rat tracheal microcirculation
Authors: Lim Lina H. K. , Bochner Bruce S. , Wagner Elizabeth M.
Published: March 2015

Because of its relative inaccessibility, inflammatory cell extravasation within the airway circulation in vivo has been difficult to investigate in real time. A new method has been established using intravital microscopy in the anesthetized rat to visualize leukocytes in superficial postcapillary venules of the trachea. This technique has been validated using local superfusion of lipopolysaccharide (LPS) and N-formyl-methionyl-leucyl-phenylalanine (FMLP). Basal leukocyte rolling velocity (55.4 ± 9.3 μm/s) and adhesion (1.4 ± 0.3 cells/100 μm) were monitored in postcapillary venules (33.9 ± 1.3 μm diameter). At all time points up to 90 min, these parameters were unaltered in control rats ( n= 7). In contrast, vessels exposed to 1 μg/ml of LPS ( n = 6) exhibited a 57% reduction in leukocyte rolling velocity and an increase in the number of adherent cells (4.7 ± 1 cells/100 μm, P < 0.05). Superfusion with 0.1 μM of FMLP ( n = 6) also resulted in a 45% reduction in rolling velocity and an increase in adherent cells (4 ± 0.7 cells/100 μm, P < 0.05). Histological evaluation confirmed local stimulus-induced leukocyte extravasation. These results demonstrate leukocyte recruitment in the airway microvasculature and provide an important new method to study airway inflammation in real time.
Nitric oxide-induced changes in intracellular zinc homeostasis are mediated by metallothionein/thionein
Authors: St. Croix Claudette M. , Wasserloos K. J. , Dineley K. E. , Reynolds I. J. , Levitan E. S. , Pitt B. R.
Published: March 2015

We hypothesized that metallothionein (MT), a cysteine-rich protein with a strong affinity for Zn2+, plays a role in nitric oxide (NO) signaling events via sequestration or release of Zn2+by the unique thiolate clusters of the protein. Exposing mouse lung fibroblasts (MLF) to the NO donor S-nitrosocysteine resulted in 20–30% increases in fluorescence of the Zn2+-specific fluorophore Zinquin that were rapidly reversed by the Zn2+chelator N,N,N′, N′-tetrakis-(2-pyridylmethyl)ethylenediamine. The absence of a NO-mediated increase in labile Zn2+in MLF from MT knockouts and its restoration after MT complementation by adenoviral gene transfer inferred a critical role for MT in the regulation of Zn2+homeostasis by NO. Additional data obtained in sheep pulmonary artery endothelial cells suggested a role for the apo form of MT, thionein (T), as a Zn2+-binding protein in intact cells, as overexpression of MT caused inhibition of NO-induced changes in labile Zn2+that were reversed by Zn2+supplementation. Furthermore, fluorescence-resonance energy-transfer data showed that overexpression of green fluorescent protein-modified MT prevented NO-induced conformational changes, which are indicative of Zn2+release from thiolate clusters. This effect was restored by Zn2+supplementation. Collectively, these data show that MT mediates NO-induced changes in intracellular Zn2+and suggest that the ratio of MT to T can regulate Zn2+homeostasis in response to nitrosative stress.
Chronic hypoxia attenuates cGMP-dependent pulmonary vasodilation
Authors: Jernigan Nikki L. , Resta Thomas C.
Published: March 2015

Chronic hypoxia (CH) augments endothelium-derived nitric oxide (NO)-dependent pulmonary vasodilation; however, responses to exogenous NO are reduced following CH in female rats. We hypothesized that CH-induced attenuation of NO-dependent pulmonary vasodilation is mediated by downregulation of vascular smooth muscle (VSM) soluble guanylyl cyclase (sGC) expression and/or activity, increased cGMP degradation by phosphodiesterase type 5 (PDE5), or decreased VSM sensitivity to cGMP. Experiments demonstrated attenuated vasodilatory responsiveness to the NO donors S-nitroso- N-acetylpenicillamine and spermine NONOate and to arterial boluses of dissolved NO solutions in isolated, saline-perfused lungs from CH vs. normoxic female rats. In additional experiments, the sGC inhibitor, 1H-[1,2,4]oxadiazolo[4,3- a]quinoxalin-1-one, blocked vasodilation to NO donors in lungs from each group. However, CH was not associated with decreased pulmonary sGC expression or activity as assessed by Western blotting and cGMP radioimmunoassay, respectively. Consistent with our hypothesis, the selective PDE5 inhibitors dipyridamole and T-1032 augmented NO-dependent reactivity in lungs from CH rats, while having little effect in lungs from normoxic rats. However, the attenuated vasodilatory response to NO in CH lungs persisted after PDE5 inhibition. Furthermore, CH similarly inhibited vasodilatory responses to 8-bromoguanosine 3′5′-cyclic monophosphate. We conclude that attenuated NO-dependent pulmonary vasodilation after CH is not likely mediated by decreased sGC expression, but rather by increased cGMP degradation by PDE5 and decreased pulmonary VSM reactivity to cGMP.
Liquid secretion inhibitors reduce mucociliary transport in glandular airways
Authors: Ballard Stephen T. , Trout Laura , Mehta Anil , Inglis Sarah K.
Published: March 2015

Because of its possible importance in cystic fibrosis (CF) pulmonary pathogenesis, the effect of anion and liquid secretion inhibitors on airway mucociliary transport was examined. When excised porcine tracheas were treated with ACh to induce gland liquid secretion, the rate of mucociliary transport was increased nearly threefold from 2.5 ± 0.5 to 6.8 ± 0.8 mm/min. Pretreatment with both bumetanide and dimethylamiloride (DMA), to respectively inhibit Cl−and HCO[Formula: see text]secretion, significantly reduced mucociliary transport in the presence of ACh by 92%. Pretreatment with the anion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid similarly reduced mucociliary transport in ACh-treated airways by 97%. These agents did not, however, reduce ciliary beat frequency. Luminal application of benzamil to block liquid absorption significantly attenuated the inhibitory effects of bumetanide and DMA on mucociliary transport. We conclude that anion and liquid secretion is essential for normal mucociliary transport in glandular airways. Because the CF transmembrane conductance regulator protein likely mediates Cl−, HCO[Formula: see text], and liquid secretion in normal glands, we speculate that impairment of gland liquid secretion significantly contributes to defective mucociliary transport in CF.
Regulation of ion channel structure and function by reactive oxygen-nitrogen species
Authors: Matalon Sadis , Hardiman Karin M. , Jain Lucky , Eaton Douglas C. , Kotlikoff Michael , Eu Jerry P. , Sun Junhui , Meissner Gerhard , Stamler Jonathan S.
Published: March 2015

Ion channels subserve diverse cellular functions. Reactive oxygen and nitrogen species modulate ion channel function by a number of mechanisms including 1) transcriptional regulation of gene expression, 2) posttranslational modifications of channel proteins, i.e. nitrosylation, nitration, and oxidation of key amino acid residues, 3) by altering the gain in other signaling pathways that may in turn lead to changes in channel activity or channel gene expression, and 4) by modulating trafficking or turnover of channel proteins, as typified by oxygen radical activation of NF-kB, with subsequent changes in proteasomal degradation of channel degradation. Regardless of the mechanism, as was discussed in a symposium at the 2003 Experimental Biology Meeting in San Diego, CA, changes in the cellular level of reactive oxygen and nitrogen species can have profound effects on the activity of ion channels and cellular function.
Phenotypic comparison of allergic airway responses to house dust mite in three rat strains
Authors: Singh Pramila , Daniels Mary , Winsett Darrell W. , Richards Judy , Doerfler Donald , Hatch Gary , Adler Kenneth B. , Gilmour M. Ian
Published: March 2015

Brown Norway (BN) rats develop a robust response to antigens in the lung, characterized by a large increase in allergen-specific immune function and pulmonary eosinophilia. The objective of this study was to investigate alternative models by determining whether other rat strains could be sensitized to house dust mite (HDM) antigen and whether the allergic disease process could be worsened with repeated allergen exposure. In general, BN rats sensitized by either subcutaneous or intratracheal routes exhibited increased pulmonary allergy compared with Sprague-Dawley (SD) and Lewis (L) rats. Multiple intratracheal allergen exposures incrementally increased HDM-specific immune function in BN rats but progressively decreased eosinophil recruitment and markers of lung injury. SD rats had more moderate responses, whereas L rats were relatively unresponsive. Because BN rats developed stronger clinical hallmarks of allergic asthma under various immunization regimes compared with SD and L rats, we conclude that the BN is the most appropriate strain for studying allergic asthma-like responses in rats. Phenotypic differences in response to HDM were associated with differences in the Th1/Th2 cytokine balance and antioxidant capacity.
Postnatal maturation in nitric oxide-induced pulmonary artery relaxation involving cyclooxygenase-1 activity
Authors: Pérez-Vizcaíno Francisco , López-López José G. , Santiago Rocío , Cogolludo Angel , Zaragozá-Arnáez Francisco , Moreno Laura , Alonso María J. , Salaices Mercedes , Tamargo Juan
Published: March 2015

The maturation in the vasodilator response to nitric oxide (NO) in isolated intrapulmonary arteries was analyzed in newborns and 15- to 20-day-old piglets. The vasodilator responses to NO gas but not to the NO donor sodium nitroprusside increased with age. The inhibitory effects of the superoxide dismutase inhibitor diethyldithiocarbamate and xanthine oxidase plus hypoxanthine and the potentiation induced by superoxide dismutase and MnCl2 of NO-induced vasodilatation were similar in the two age groups. Diphenyleneiodonium (NADPH oxidase inhibitor) potentiated the response to NO, and this effect was more pronounced in the older animals. The nonselective cyclooxygenase inhibitors indomethacin and meclofenamate and the preferential cyclooxygenase-1 inhibitor aspirin augmented NO-induced relaxation specifically in newborns, whereas the selective cycloxygenase-2 inhibitor NS-398 had no effect. The expressions of α-actin, cycloxygenase-1, and cycloxygenase-2 proteins were similar, whereas Cu,Zn-superoxide dismutase decreased with age. Therefore, the present data suggest that the maturational increase in the vasodilatation of NO in the pulmonary arteries during the first days of extrauterine life involves a cycloxygenase-dependent inhibition of neonatal NO activity.
Transcriptional regulation of the HO-1 gene in cultured macrophages exposed to model airborne particulate matter
Authors: Chin Beek Yoke , Trush Michael A. , Choi Augustine M. K. , Risby Terence H.
Published: March 2015

Respirable particulate matter generated during incomplete combustion of fossil fuels may principally target the cells found in the distal region of the lung. This study characterizes some of the effects that a model particulate matter has on the induction of heme oxygenase (HO)-1 in macrophages. HO-1 is a highly inducible stress response gene that has been demonstrated to modulate chemical, physical, and environmental stimuli. Cultured macrophages (RAW 264.7 cells) exposed continuously to a well-defined model of particulate matter (benzo[ a]pyrene adsorbed onto carbon black) induced HO-1 gene expression in a time-dependent manner. Likewise, the addition of benzo[ a]pyrene-1,6-quinone, a redox cycling metabolite of benzo[ a]pyrene, to RAW cells also induced HO-1. This particle-induced gene expression of HO-1 was found to correlate with a corresponding increase in protein levels. Gene regulation studies were performed to delineate the transcriptional regulation of HO-1 after exposure to model particulate matter. Deletional analysis of the HO-1 gene and mutational analysis of activator protein (AP)-1 regulatory element on both distal enhancers demonstrated the importance of this transcriptional factor in mediating HO-1 gene transcription in response to model particulate matter. These results were supported by gel shift analysis demonstrating increased AP-1 binding activity after exposure to particulate matter. In summary, this study demonstrates that model particulate matter enhanced the expression of HO-1. This inductive process may be mediated by AP-1 activation of the regulatory elements on both the 5′-distal enhancers.
Escherichia coliFPG and human OGG1 reduce DNA damage and cytotoxicity by BCNU in human lung cells
Authors: He Ying-Hui , Xu Yi , Kobune Masayoshi , Wu Min , Kelley Mark R. , Martin William J.
Published: March 2015

The pulmonary complications of 1,3- N, N′-bis(2-chloroethyl)- N-nitrosourea (BCNU) are among the most important dose-limiting factors of BCNU-containing cancer chemotherapeutic regimens. BCNU damages DNA of both cancer cells and normal cells. To increase the resistance of lung cells to BCNU, we employed gene transfer of Escherichia coliformamidopyrimidine-DNA glycosylase (FPG) and human 8-oxoguanine-DNA glycosylase (hOGG1) to A549 cells, a lung epithelial cell line, using a bicistronic retroviral vector, pSF91-RE, that encoded both FPG/hOGG1 and an enhanced green fluorescent protein. The transduced epithelial cells were sorted by flow cytometry, and expression of FPG/hOGG1 protein was determined by the level of FPG/hOGG1 RNA and enzyme activity. The single-cell gel electrophoresis (comet assay) measured DNA damage induced by BCNU. FPG/hOGG1-expressing A549 cells incubated with 40–500 μg/ml BCNU exhibited significantly less DNA damage than vector-transduced cells. In addition, FPG- and/or hOGG1-expressing cells incubated with 10–40 μg/ml BCNU showed at least a 25% increase in cell survival. Gene transfer of FPG/hOGG1 reduced BCNU-induced DNA damage and cytotoxicity of cultured lung cells and may suggest a new mechanism to reduce BCNU pulmonary toxicity.
Vitamin A deficiency promotes bronchial hyperreactivity in rats by altering muscarinic M2 receptor function
Authors: McGowan Stephen E. , Smith Jennifer , Holmes Amey Jo , Smith Lori A. , Businga Thomas R. , Madsen Mark T. , Kopp Ulla C. , Kline Joel N.
Published: March 2015

Vitamin A deficiency (VAD) remains an important health problem among children in developing countries. Children living in these areas have a higher mortality from respiratory infections, which likely results in part from suboptimal nutrition, including VAD. Bronchial hyperreactivity can follow viral respiratory infections and may complicate the recovery. To investigate whether VAD promotes bronchial hyperreactivity, we have assessed methacholine-induced bronchoconstriction in VAD and vitamin A-sufficient rats. Bronchial constriction developed at lower concentrations of inhaled methacholine in VAD than in vitamin A-sufficient rats. This did not result from an increase in the bronchial wall thickness or the clearance of a small molecule (with a size similar to methacholine) from the air space. The function and abundance of the muscarinic M2 receptors in bronchial tissue were reduced in VAD rats, suggesting that this receptor may contribute to these animals' diminished ability to limit cholinergic-mediated bronchoconstriction. A similar reduction in muscarinic M2 receptor function has been observed in asthma. Vitamin A (retinol) and its congeners (retinoids) may be required to regulate bronchial responsiveness in addition to maintaining a normal bronchial epithelium.
Temporal correlation of measurements of airway hyperresponsiveness in ovalbumin-sensitized mice
Authors: Albertine Kurt H. , Wang Lu , Watanabe Suetaro , Marathe Gopal K. , Zimmerman Guy A. , McIntyre Thomas M.
Published: March 2015

Airway hyperresponsiveness, airway inflammation, and reversible airway obstruction are physiological hallmarks of asthma. These responses are increasingly being studied in murine models of antigen exposure and challenge, using whole body plethysmography to noninvasively assess airway hyperresponsiveness. This approach infrequently has been correlated with indexes of airway hyperresponsiveness measured by invasive means. Furthermore, correlation with quantitative histological data for tissue infiltration by inflammatory and immune cells, particularly in the wall of airways, during daily airway challenge is lacking. To address these uncertainties, we used C57BL/6 mice that were immunized with ovalbumin or vehicle (saline) and sensitized to aerosolized ovalbumin or vehicle 8 days later. The mice were subsequently exposed to aerosolized ovalbumin or vehicle, respectively, on days 14–22. We assessed airway hyperresponsiveness to methacholine noninvasively on days 14, 15, 18, or 22; we studied the same mice 24 h later while they were anesthetized for invasive analyses of airway hyperresponsiveness. Plasma total IgE concentration was significantly higher in the ovalbumin-treated mice compared with the vehicle-treated mice, but this did not correlate with eosinophil number. Peak airway hyperresponsiveness measured by either approach correlated early during daily antigen challenge ( days 14 and 15), but this correlation was lost later during subsequent daily antigen challenges ( days 18 and 22). On days 14 and 15, peak airway hyperresponsiveness correlated with transmigration of neutrophils and macrophages, but not lymphocytes, in the peribronchovascular connective tissue sheaths. This extravascular accumulation was found to be focal by three-dimensional microscopy. We conclude that, although ovalbumin treatment changed lung function in mice, correlation between noninvasive and invasive measures of peak airway hyperresponsiveness was inconsistent.
Maternal glucocorticoids increase endotoxin-induced lung inflammation in preterm lambs
Authors: Kallapur Suhas G. , Kramer Boris W. , Moss Timothy J. M. , Newnham John P. , Jobe Alan H. , Ikegami Machiko , Bachurski Cindy J.
Published: March 2015

Antenatal betamethasone (Beta) is widely used in women with asymptomatic chorioamnionitis at risk for preterm delivery, but its effects on fetal inflammation are unstudied. Groups of ewes at 109 ± 1 days of gestation received the following treatments: intra-amniotic (IA) saline (control), 0.5 mg/kg intramuscular Beta, 10 mg IA endotoxin (Endo), and Beta + 2 h later Endo (Beta + Endo). Beta suppressed Endo-induced lung inflammation at 1 day. However, compared with Endo 5 days after treatment, Beta + Endo lambs had increased alveolar neutrophils, proinflammatory cytokine mRNA expression, and serum amyloid A3 (SAA3) mRNA expression. IL-1β mRNA expression was localized to the inflammatory cells, whereas SAA3 mRNA expression was induced in the bronchial epithelium and the inflammatory cells. Compared with Endo, Beta + Endo lambs had increased lung inflammation but equivalent lung volumes 15 days after treatment. The late increase in inflammation in the Beta + Endo animals suggests that glucocorticoids impair the ability of the preterm lung to downregulate Endo-induced inflammation after fetal clearance of the glucocorticoids. These results have implications for lung inflammation and bronchopulmonary dysplasia in preterm infants exposed to chorioamnionitis and maternal glucocorticoids.
Regulation of ornithine decarboxylase and polyamine import by hypoxia in pulmonary artery endothelial cells
Authors: Babal Pavel , Ruchko Mykhaylo , Ault-Ziel Kathryn , Cronenberg Lauren , Olson Jack W. , Gillespie Mark N.
Published: March 2015

In rat lung and cultured lung vascular cells, hypoxia decreases ornithine decarboxylase (ODC) activity and increases polyamine import. In this study, we used rat cultured pulmonary artery endothelial cells to explore the mechanism of hypoxia-induced reduction in ODC activity and determined whether this event was functionally related to the increase in polyamine import. Two strategies known to suppress proteasome-mediated ODC degradation, lactacystin treatment and use of cells expressing a truncated ODC incapable of interacting with the proteasome, prevented the hypoxia-induced decrease in ODC activity. Interestingly, though, cellular abundance of the 24-kDa antizyme, a known physiological accelerator of ODC degradation, was not increased by hypoxia. These observations suggest that an antizyme-independent ODC degradation pathway contributes to hypoxia-induced reductions of ODC activity. When reductions in ODC activity in hypoxia were prevented by the proteasome inhibitor strategies, hypoxia failed to increase polyamine transport. The induction of polyamine transport in hypoxic pulmonary artery endothelial cells thus seems to require decreased ODC activity as an initiating event.
Characterization of adenosine receptor(s) involved in adenosine-induced bronchoconstriction in an allergic mouse model
Authors: Fan Ming , Qin Weixi , Mustafa S. Jamal
Published: March 2015

We recently reported that adenosine caused bronchoconstriction and enhanced airway inflammation in an allergic mouse model. In this study, we further report the characterization of the subtype of adenosine receptor(s) involved in bronchoconstriction. 5′-( N-ethylcarboxamido)adenosine (NECA), a nonselective adenosine agonist, elicited bronchoconstriction in a dose-dependent manner. Little effects of N6-cyclopentyladenosine (A1-selective agonist) and 2- p-(2-carboxyethyl)phenethylamino-5′- N-ethylcarboxamidoadenosine (A2A-selective agonist) compared with NECA were observed in this model. 2-Chloro- N6-(3-iodobenzyl)-9-[5-(methylcarbamoyl)-β-d-ribofuranosyl]adenosine, an A3-selective receptor agonist, produced a dose-dependent bronchoconstrictor response, which was blocked by selective A3antagonist 2,3-diethyl-4,5-dipropyl-6-phenylpyridine-3-thiocarboxylate-5-carboxylate (MRS1523). However, MRS1523 only partially inhibited NECA-induced bronchoconstriction. Neither selective A1nor A2Aantagonists affected NECA-induced bronchoconstriction. Enprofylline, a relatively selective A2Breceptor antagonist, blocked partly NECA-induced bronchoconstriction. Furthermore, a combination of enprofylline and MRS1523 completely abolished NECA-induced bronchoconstrictor response. Using RT-PCR, we found that all four adenosine receptor subtypes are expressed in control lungs. Allergen sensitization and challenge significantly increased transcript levels of the A2Band A3receptors, whereas the A1receptor message decreased. No change in transcript levels of A2Areceptors was observed after allergen sensitization and challenge. These findings suggest that A2Band A3adenosine receptors play an important role in adenosine-induced bronchoconstriction in our allergic mouse model. Finally, whether the airway effects of the receptor agonists/antagonists are direct or indirect needs further investigations.
Hypoxia decreases active Na transport across primary rat alveolar epithelial cell monolayers
Authors: Mairbäurl Heimo , Mayer Katrin , Kim Kwang-Jin , Borok Zea , Bärtsch Peter , Crandall Edward D.
Published: March 2015

L665, 2002. First published December 14, 2001; 10.1152/ajplung.00355.2001.—Hypoxia has been reported to inhibit activity and expression of ion transporters of alveolar epithelial cells. This study extended those observations by investigating the mechanisms underlying inhibition of active Na transport across primary cultured adult rat alveolar epithelial cell monolayers grown on polycarbonate filters. Cell monolayers were exposed to normoxia and hypoxia (1.5% and 5% O2, 5% CO2), and resultant changes in bioelectric properties [i.e., short-circuit current ( I sc) and transepithelial resistance ( R t)] were measured in Ussing chambers. Results showed that I sc decreased with duration of exposure to hypoxia, while relatively little change was observed for R t. In normoxia, amiloride inhibited ∼70% of I sc. The amiloride-sensitive portion of I sc decreased over time of exposure to hypoxia, whereas the magnitude of the amiloride-insensitive portion of I sc was not affected. Na pump capacity measured after permeabilization of the apical plasma membrane with amphotericin B decreased in monolayers exposed to 1.5% O2 for 24 h, as did the capacity of amiloride-sensitive Na uptake measured after imposing an apical to basolateral Na gradient and permeabilization of the basolateral membrane. These results demonstrate that exposure to hypoxia inhibits alveolar epithelial Na reabsorption by reducing the rates of both apical amiloride-sensitive Na entry and basolateral Na extrusion.
Fusion of lung lobes and vessels in mouse embryos heterozygous for theforkhead box f1targeted allele
Authors: Lim Lorena , Kalinichenko Vladimir V. , Whitsett Jeffrey A. , Costa Robert H.
Published: March 2015

Previously, we showed that newborn forkhead box ( Fox)f1(+/−) mice with diminished pulmonary FoxF1 levels died of severe lung hemorrhage and exhibited abnormal formation of alveolar sacs and capillaries. Another group recently reported that Foxf1(+/−) mouse embryos displayed a number of organ and skeletal defects including fusion of lung lobes. However, identification of pulmonary genes whose altered expression was associated with the lobular fusion defect in Foxf1(+/−) lungs remains uncharacterized. The present study was conducted to determine the nature of the malformations leading to lung fusions in the FoxF1 embryos and to identify potential signaling pathways influenced by FoxF1 haploinsufficiency. We show that Foxf1(+/−) embryos exhibit defects in formation and branching of primary lung buds, which causes fusion of the right lung lobes and vessels. The severity of the Foxf1(+/−) lung fusions was correlated with decreased levels of FoxF1 mRNA. In situ hybridization studies demonstrated that the defective primary lung-bud development in early Foxf1(+/−) embryos was associated with fewer pulmonary mesenchymal-epithelial interfaces. Defects in branching morphogenesis in the Foxf1(+/−) embryos were associated with altered expression of the fibroblast growth factor-10, bone morphogenetic protein-4, and the Gli3 transcription factor, which are known to influence primary lung-bud development.
Human SP-A 3′-UTR variants mediate differential gene expression in basal levels and in response to dexamethasone
Authors: Wang Guirong , Guo Xiaoxuan , Floros Joanna
Published: March 2015

Human surfactant protein A (SP-A) is encoded by two genes ( SP-A1, SP-A2), and each is identified with several alleles. SP-A is involved in normal lung function, innate immunity, inflammatory processes, and is regulated by glucocorticoids. We investigated the role of 3′-untranslated region (UTR) of 10 SP-A variants on gene expression using transient transfection of 3′-UTR constructs in the human lung adenocarcinoma cell line NCI-H441. We found: 1) both basal mRNA and protein levels of the reporter gene of SP-A 3′-UTR constructs are significantly ( P < 0.01) reduced compared with controls (vector pGL3 and surfactant protein B pGL3) and that differences exist among alleles; and 2) after dexamethasone (Dex) treatment (100 nM for 16 h), mRNA was reduced (31–51%). Seven alleles showed a significant decrease ( P < 0.05) in mRNA, and three did not. Reporter activity was also decreased, from 17% (1A1) to 38% (1A), with six alleles showing a significant decrease. The data indicate that the 3′-UTR of SP-As play a differential role in SP-A basal expression and in response to Dex. Therefore, a careful consideration of individual use of steroid treatment may be considered.
Modulation of ion conductance and active transport by TGF-β1 in alveolar epithelial cell monolayers
Authors: Willis Brigham C. , Kim Kwang-Jin , Li Xian , Liebler Janice , Crandall Edward D. , Borok Zea
Published: March 2015

Transforming growth factor-β1 (TGF-β1) may be a critical mediator of lung injury and subsequent remodeling during recovery. We evaluated the effects of TGF-β1 on the permeability and active ion transport properties of alveolar epithelial cell monolayers. Rat alveolar type II cells plated on polycarbonate filters in defined serum-free medium form confluent monolayers and acquire the phenotypic characteristics of alveolar type I cells. Exposure to TGF-β1 (0.1-100 pM) from day 0 resulted in a concentration- and time-dependent decrease in transepithelial resistance ( Rt) and increase in short-circuit current ( Isc). Apical amiloride or basolateral ouabain on day 6 inhibited Iscby 80 and 100%, respectively. Concurrent increases in expression of Na+-K+-ATPase α1- and β1-subunits were observed in TGF-β1-treated monolayers. No change in the α-subunit of the rat epithelial sodium channel (α-rENaC) was seen. Exposure of confluent monolayers to TGF-β1 from day 4 resulted in an initial decrease in Rtwithin 6 h, followed by an increase in Iscover 72-96 h. These results demonstrate that TGF-β1 modulates ion conductance and active transport characteristics of the alveolar epithelium, associated with increased Na+-K+-ATPase, but without a change in α-rENaC.
Combined effect of chronic hypoxia and in vitro exposure to gas pollutants on airway reactivity
Authors: Roux Etienne , Duvert Michel , Marthan Roger
Published: March 2015

This study investigated the interaction between exposure to air pollutants and chronic hypoxia (CH). We used a hypobaric chamber (14 days at barometric pressure 380 mmHg) to produce CH in rats. Exposure to various doses of acrolein or ozone did not modify the mechanical response to cholinergic agonists. Exposure to 3 μM/min acrolein did not alter epithelium-free trachea responsiveness. In contrast, direct exposure of freshly isolated myocytes to 2 and 3 μM/min acrolein enhanced the amplitude of the first intracellular [Ca2+] rise in response to 0.1 μM ACh and the calcium oscillation frequency in response to 10 μM ACh. CH alone did not alter smooth muscle cross-sectional area (SMA) or epithelium-plus-submucosa thickness. CH decreased maximal contractile response (maximal force normalized to SMA) but increased sensitivity (pEC50) to cholinergic agonists. We conclude that unlike in normoxic rats, exposure to air pollutants does not induce airway hyperresponsiveness in CH rats, although it increased calcium signaling. These results cannot be explained by change in smooth muscle accessibility, but may be linked to the effect of CH on calcium-contraction coupling.
Deficiency in the c-Jun NH2-terminal kinase signaling pathway confers susceptibility to hyperoxic lung injury in mice
Authors: Morse Danielle , Otterbein Leo E. , Watkins Simon , Alber Sean , Zhou Zhihong , Flavell Richard A. , Davis Roger J. , Choi Augustine M. K.
Published: March 2015

Hyperoxia generates an oxidative stress in the mouse lung, which activates the major stress-inducible kinase pathways, including c-Jun NH2-terminal kinase (JNK). We examined the effect of Jnk1 gene deletion on in vivo responses to hyperoxia in mice. The survival of Jnk1-/-mice was reduced relative to wild-type mice after exposure to continuous hyperoxia. Jnk1- /-mice displayed higher protein concentration in bronchoalveolar lavage (BAL) fluid and increased expression of heme oxygenase-1, a stress-inducible gene, after 65 h of hyperoxia. Contrary to other markers of injury, the leukocyte count in BAL fluid of Jnk1- /-mice was markedly diminished relative to that of wild-type mice. The decrease in BAL leukocyte count was not associated with any decrease in lung myeloperoxidase activity at baseline or after hyperoxia treatment. Pretreatment with inhaled lipopolysaccharide increased BAL neutrophil content and extended hyperoxia survival time to a similar extent in Jnk1- /-and wild-type mice. Associated with increased mortality, Jnk1- /-mice had increased pulmonary epithelial cell apoptosis after exposure to hyperoxia compared with wild-type mice. These results indicate that JNK pathways participate in adaptive responses to hyperoxia in mice.