bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of dexamethasone-mediated interleukin-8 gene suppression in cultured airway epithelial cells
Authors: Chang Mary Mann-Jong , Juarez Maya , Hyde Dallas M. , Wu Reen
Published: December 2017

The effects of dexamethasone, a glucocorticoid analog, on interleukin 8 (IL-8) gene expression were studied in cultures of primary human tracheobronchial epithelial cells and an immortalized human bronchial epithelial cell line, HBE1 cells. Dexamethasone inhibited IL-8 mRNA and protein expression in a concentration- and time-dependent manner. The inhibition did not occur at the transcriptional level since both nuclear run-on activity and IL-8 promoter-reporter gene expression assay revealed no significant effect. Instead, there was a change in IL-8 mRNA stability in dexamethasone-treated cultures. Under actinomycin D treatment, IL-8 mRNA was quite stable in dexamethasone-depleted cultures, while in dexamethasone-pretreated cultures, IL-8 message was rapidly degraded within the first hour, then leveled off. When dexamethasone and actinomycin D were added simultaneously to dexamethasone-depleted cultures, IL-8 mRNA remained rather stable. When cycloheximide was used to inhibit new protein synthesis, dexamethasone-dependent inhibition was not observed. These results suggest that a posttranscriptional mechanism, which requires dexamethasone-dependent new protein synthesis, is involved in the regulation of IL-8 mRNA by dexamethasone in airway epithelial cells.
Nitric oxide attenuates H2O2-induced endothelial barrier dysfunction: mechanisms of protection
Authors: Gupta Mahesh P. , Ober Michael D. , Patterson Carolyn , Al-Hassani Mohammed , Natarajan Viswanathan , Hart C. Michael
Published: December 2017

Nitric oxide (·NO) attenuates hydrogen peroxide (H2O2)-mediated injury in porcine pulmonary artery endothelial cells (PAECs) and modulates intracellular levels of cGMP and cAMP. We hypothesized that ·NO attenuates H2O2-induced PAEC monolayer barrier dysfunction through cyclic nucleotide-dependent signaling mechanisms. To examine this hypothesis, cultured PAEC monolayers were treated with H2O2, and barrier function was measured as transmonolayer albumin clearance. H2O2caused significant PAEC barrier dysfunction that was attenuated by intracellular as well as extracellular ·NO generation. ·NO increased PAEC cGMP and cAMP levels, but treatment with inhibitors of soluble guanylate cyclase or protein kinase G did not abrogate ·NO-mediated barrier protection. In contrast, H2O2decreased protein kinase A activity, and inhibiting protein kinase A abrogated the protective effect of ·NO. H2O2-induced barrier dysfunction was not associated with decreased levels of cGMP or cAMP. 3-Isobutyl-1-methylxanthine and the cGMP analog 8-bromo-cGMP had little effect on H2O2-mediated endothelial barrier dysfunction, whereas 8-bromo-cAMP plus 3-isobutyl-1-methylxanthine was protective. These results indicate that ·NO modulates vascular endothelial barrier function through cAMP-dependent signaling mechanisms.
Extracellular cyclic ADP-ribose potentiates ACh-induced contraction in bovine tracheal smooth muscle
Authors: Franco Luisa , Bruzzone Santina , Song Pinfang , Guida Lucrezia , Zocchi Elena , Walseth Timothy F. , Crimi Emanuele , Usai Cesare , De Flora Antonio , Brusasco Vito
Published: December 2017

Cyclic ADP-ribose (cADPR), a universal calcium releaser, is generated from NAD+by an ADP-ribosyl cyclase and is degraded to ADP-ribose by a cADPR hydrolase. In mammals, both activities are expressed as ectoenzymes by the transmembrane glycoprotein CD38. CD38 was identified in both epithelial cells and smooth myocytes isolated from bovine trachea. Intact tracheal smooth myocytes (TSMs) responded to extracellular cADPR (100 μM) with an increase in intracellular calcium concentration ([Ca2+]i) both at baseline and after acetylcholine (ACh) stimulation. The nonhydrolyzable analog 3-deaza-cADPR (10 nM) elicited the same effects as cADPR, whereas the cADPR antagonist 8-NH2-cADPR (10 μM) inhibited both basal and ACh-stimulated [Ca2+]ilevels. Extracellular cADPR or 3-deaza-cADPR caused a significant increase of ACh-induced contraction in tracheal smooth muscle strips, whereas 8-NH2-cADPR decreased it. Tracheal mucosa strips, by releasing NAD+, enhanced [Ca2+]iin isolated TSMs, and this increase was abrogated by either NAD+-ase or 8-NH2-cADPR. These data suggest the existence of a paracrine mechanism whereby mucosa-released extracellular NAD+plays a hormonelike function and cADPR behaves as second messenger regulating calcium-related contractility in TSMs.
Association between airway hyperreactivity and bronchial macrophage dysfunction in individuals with mild asthma
Authors: Alexis Neil E. , Soukup Joleen , Nierkens Stefan , Becker Susanne
Published: December 2017

Little is known about the functional capabilities of bronchial macrophages (BMs) and their relationship to airway disease such as asthma. We hypothesize that BMs from asthmatics may be modulated in their function compared with similar cells from healthy individuals. BMs obtained by induced sputum from mild asthmatics ( n = 20) and healthy individuals ( n = 20) were analyzed using flow cytometry for CD16, CD64, CD11b, CD14, and human leukocyte antigen-DR expression, phagocytosis of IgG opsonized yeast, and oxidant production. Asthma status was assessed by lung function [percent predicted forced vital capacity and forced expiratory volume in 1 s (FEV1)], percent sputum eosinophils, and nonspecific airway responsiveness [provocative concentration that produces a 20% fall in FEV1(PC20,FEV1)]. Asthmatics with >5% airway eosinophils (AEo+) had decreased BM CD64 expression and phagocytosis compared with asthmatics with <5% eosinophils (AEo−). Among asthmatics, a significant correlation was found between CD64 expression and BM phagocytosis ( R = 0.7, P < 0.009). Phagocytosis was also correlated with PC20,FEV1( R = 0.6, P < 0.007), lung function (%predicted FEV1, R= 0.7, P < 0.002) and percent eosinophils ( R= −0.6, P < 0.01). In conclusion, BM from asthmatics are functionally modulated, possibly by Th2 cytokines involved in asthma pathology.
Cytokine-induced changes in chromatin structure and in vivo footprints in the inducible NOS promoter
Authors: Mellott Jane K. , Nick Harry S. , Waters Michael F. , Billiar Timothy R. , Geller David A. , Chesrown Sarah E.
Published: December 2017

Transcription of the human inducible nitric oxide synthase ( iNOS) gene is regulated by inflammatory cytokines in a tissue-specific manner. To determine whether differences in cytokine-induced mRNA levels between pulmonary epithelial cells (A549) and hepatic biliary epithelial cells (AKN-1) result from different protein or DNA regulatory mechanisms, we identified cytokine-induced changes in DNase I-hypersensitive (HS) sites in 13 kb of the iNOS 5′-flanking region. Data showed both constitutive and inducible HS sites in an overlapping yet cell type-specific pattern. Using in vivo footprinting and ligation-mediated PCR to detect potential DNA or protein interactions, we examined one promoter region near −5 kb containing both constitutive and cytokine-induced HS sites. In both cell types, three in vivo footprints were present in both control and cytokine-treated cells, and each mapped within a constitutive HS site. The remaining footprint appeared only in response to cytokine treatment and mapped to an inducible HS site. These studies, performed on chromatin in situ, identify a portion of the molecular mechanisms regulating transcription of the human iNOS gene in both lung- and liver-derived epithelial cells.
Adenovirus infection increases iNOS and peroxynitrite production in the lung
Authors: Zsengellér Zsuzsanna K. , Ross Gary F. , Trapnell Bruce C. , Szabó Csaba , Whitsett Jeffrey A.
Published: December 2017

Host inflammatory and immune responses limit viral gene expression after administration of replication-deficient adenoviruses to the lung. The current study asks whether inducible nitric oxide synthase (iNOS) expression and peroxynitrite generation accompanied the inflammatory response following intratracheal administration of adenovirus. Pulmonary iNOS mRNA and protein were increased 2, 7, and 14 days following administration of 2 × 109plaque-forming units of recombinant adenovirus (Av1Luc1) to BALB/c mice. Adenovirus infection was associated with a marked increase in nitrotyrosine staining. Intense nitrotyrosine staining was observed in alveolar macrophages, respiratory epithelial cells, conducting airways, and alveolar spaces 2 days postinfection. Two weeks after exposure to adenovirus, nitrotyrosine staining was detected within alveolar macrophages, suggesting adenovirus enhanced the nitration of proteins that were subsequently taken up by alveolar macrophages. Western blot analysis using anti-nitrotyrosine antibody did not demonstrate accumulation of nitrated surfactant protein A (SP-A), although a small fraction of aggregated SP-A comigrated with a nitrotyrosine-positive protein. iNOS expression, peroxynitrite, and nitrotyrosine generation accompany and may contribute to inflammatory responses to adenovirus in the lung.
mRNA expression of novel CGRP1 receptors and their activity-modifying proteins in hypoxic rat lung
Authors: Qing Xin , Svaren John , Keith Ingegerd M.
Published: December 2017

Calcitonin gene-related peptide (CGRP) is a potent vasodilator. Our group has reported that exogenous CGRP may prevent or reverse hypoxic pulmonary hypertension in rats. The vasodilatory action of CGRP is mediated primarily by CGRP1 receptors. The calcitonin receptor-like receptor (CRLR) and the orphan receptor RDC-1 have been proposed as CGRP1 receptors, and recent evidence suggests that CRLR can function as either a CGRP1 receptor or an adrenomedullin (ADM) receptor. Receptor activity-modifying proteins (RAMPs) determine the ligand specificity of CRLR: coexpression of CRLR and RAMP1 results in a CGRP1 receptor, whereas coexpression of CRLR and RAMP2 or -3 results in an ADM receptor. We used qualitative, semiquantitative, and real-time quantitative RT-PCR to detect and quantitate the relative expression of these agents in the lungs of rats exposed to normoxia ( n = 3) and 1 and 2 wk of chronic hypobaric hypoxia (barometric pressure 380 mmHg, equivalent to an inspired O2 level of 10%; n = 3/time period). Our results show upregulation of RDC-1, RAMP1, and RAMP3 mRNAs in hypoxic rat lung and no change in CRLR and RAMP2 mRNAs. These findings support a functional role for CGRP and ADM receptors in regulating the adult pulmonary circulation.
17β-Estradiol increases nitric oxide-dependent dilation in rat pulmonary arteries and thoracic aorta
Authors: Gonzales Rayna J. , Walker Benjimen R. , Kanagy Nancy L.
Published: December 2017

Past studies have demonstrated that 17β-estradiol (E2β) increases endothelial nitric oxide (NO) synthase (eNOS) activity in uterine, heart, and skeletal muscle and in cultured human endothelial cells. However, little is known about E2β regulation of NO synthesis in the pulmonary vasculature. The present study evaluated E2β regulation of eNOS function in pulmonary arteries and thoracic aortas. We hypothesized that E2β upregulates vascular NO release by increasing eNOS expression. To test this, NO-dependent vasodilation was assessed in isolated perfused lungs and aortic rings from ovariectomized Sprague-Dawley rats treated for 1 wk with 20 μg/24 h of E2β or vehicle. Expression of eNOS was evaluated by Western blot and immunohistochemistry. Also, a RNase protection assay determined eNOS mRNA levels in lung and aortic homogenates from control and treated rats. Vasodilation to ionomycin in lungs from the E2β-treated group was enhanced compared with that in control animals. Endothelium-intact aortic rings from E2β-treated animals also demonstrated augmented endothelium-dependent dilation. Both responses were blocked with NOS inhibition. Immunostaining for eNOS was greater in pulmonary arteries and aortas from E2β-treated compared with control rats. However, mRNA levels did not differ between groups. Thus we conclude that in vivo E2β treatment augments endothelium-dependent dilation in aorta and lung, increasing expression of eNOS independently of sustained augmented gene transcription.
Tidal midexpiratory flow as a measure of airway hyperresponsiveness in allergic mice
Authors: Glaab Thomas , Daser Angelika , Braun Armin , Neuhaus-Steinmetz Ulrich , Fabel Helmut , Alarie Yves , Renz Harald
Published: December 2017

A method for the noninvasive measurement of airway responsiveness was validated in allergic BALB/c mice. With head-out body plethysmography and the decrease in tidal midexpiratory flow (EF50) as an indicator of airway obstruction, responses to inhaled methacholine (MCh) and the allergen ovalbumin were measured in conscious mice. Allergen-sensitized and -challenged mice developed airway hyperresponsiveness as measured by EF50to aerosolized MCh compared with that in control animals. This response was associated with increased allergen-specific IgE and IgG1 production, increased levels of interleukin-4 and interleukin-5 in bronchoalveolar lavage fluid and eosinophilic lung inflammation. Ovalbumin aerosol challenge elicited no acute bronchoconstriction but resulted in a significant decline in EF50baseline values 24 h after challenge in allergic mice. The decline in EF50to MCh challenge correlated closely with simultaneous decreases in pulmonary conductance and dynamic compliance. The decrease in EF50was partly inhibited by pretreatment with the inhaled β2-agonist salbutamol. We conclude that measurement of EF50to inhaled bronchoconstrictors by head-out body plethysmography is a valid measure of airway hyperresponsiveness in mice.
Changes in airway resistance by simultaneous exposure to TNF-α and IL-1β in perfused rat lungs
Authors: Martin Christian , Wohlsen Andrea , Uhlig Stefan
Published: December 2017

Tumor necrosis factor (TNF)-α and interleukin (IL)-1β are formed simultaneously under inflammatory conditions such as asthma and acute respiratory distress syndrome. Here we investigated the effects of TNF-α (10 ng/ml) and/or IL-1β (10 ng/ml) in isolated blood-free perfused rat lungs. In lungs precontracted with methacholine, IL-1β alone and IL-1β/TNF-α decreased airway resistance 10 min after administration, whereas TNF-α alone had no effect. In untreated lungs, airway resistance was unaltered by either cytokine alone but started to increase 40 min after treatment with both cytokines together, indicating bronchoconstriction. The bronchoconstriction was accompanied by a steroid-sensitive increase in cyclooxygenase (COX)-2 mRNA expression and thromboxane formation. The cytokine-induced bronchoconstriction was blocked by the thromboxane receptor antagonist SQ-29548, indomethacin, the selective COX-2 inhibitor NS-398, and the steroid dexamethasone. We conclude that IL-1β has an early bronchodilatory effect (after 10 min) that is unchanged by TNF-α. However, at later time points (after 40 min), IL-1β and TNF-α in concert cause a COX-2- and thromboxane-dependent bronchoconstriction. Our findings show that TNF-α and IL-1β exert complex and time-dependent effects on lung functions that cannot be predicted by studying each cytokine alone.
Alveolar fluid clearance in late-gestational guinea pigs after labor induction: mechanisms and regulation
Authors: Norlin Andreas , Folkesson Hans G.
Published: December 2017

We tested the hypothesis that labor-induced epinephrine release would stimulate alveolar fluid clearance in preterm fetuses. Preterm fetuses were obtained by cesarean section from timed-pregnant guinea pigs at 61–69 days postconception. Fetal guinea pigs were euthanized and placed on continuous positive airway pressure oxygenation, and an isosmolar 5% albumin solution was instilled. Alveolar fluid clearance was measured over 1 h. The fetal lung began to absorb fluid at 64–66 days postconception, and at birth, alveolar fluid clearance quadrupled. Baseline alveolar fluid clearance when present was sensitive to propranolol inhibition and depended on β-adrenergic stimulation. Measurements of plasma epinephrine in fetal animals confirmed high epinephrine levels in 66- to 69-day postconception fetuses. Prenatal alveolar fluid clearance when present was highly amiloride sensitive, suggesting that amiloride-sensitive Na+channels were critical. Oxytocin-induced labor initiated an amiloride- and propranolol-sensitive net alveolar fluid clearance in 61-day-gestation animals. Moreover, oxytocin induced significant epinephrine release in all fetuses. These results have clinical implications for infants delivered by cesarean section before the onset of labor. Use of pharmacological agents to induce labor may reduce the occurrence and severity of perinatal respiratory distress.
The role of p21CIP1/WAF1 in growth of epithelial cells exposed to hyperoxia
Authors: Rancourt Raymond C. , Keng Peter C. , Helt Christopher E. , O'Reilly Michael A.
Published: December 2017

Previous studies have shown that hyperoxia inhibits proliferation and increases the expression of the tumor suppressor p53 and its downstream target, the cyclin-dependent kinase inhibitor p21CIP1/WAF1, which inhibits proliferation in the G1 phase of the cell cycle. To determine whether growth arrest was mediated through activation of the p21-dependent G1 checkpoint, the kinetics of cell cycle movement during exposure to 95% O2 were assessed in the Mv1Lu and A549 pulmonary adenocarcinoma cell lines. Cell counts, 5-bromo-2′-deoxyuridine incorporation, and cell cycle analyses revealed that growth arrest of both cell lines occurred in S phase, with A549 cells also showing evidence of a G1 arrest. Hyperoxia increased p21 in A549 but not in Mv1Lu cells, consistent with the activation of the p21-dependent G1 checkpoint. The ability of p21 to exert the G1 arrest was confirmed by showing that hyperoxia inhibited proliferation of HCT 116 colon carcinoma cells predominantly in G1, whereas an isogenic line lacking p21 arrested in S phase. The cell cycle arrest in S phase appears to be a p21-independent process caused by a gradual reduction in the rate of DNA strand elongation. Our data reveal that hyperoxia inhibits proliferation in G1 and S phase and demonstrate that p53 and p21 retain their ability to affect G1 checkpoint control during exposure to elevated O2 levels.
NO and reactive oxygen species are involved in biphasic hypoxic vasoconstriction of isolated rabbit lungs
Authors: Weissmann Norbert , Winterhalder Stefan , Nollen Matthias , Voswinckel Robert , Quanz Karin , Ghofrani Hossein Ardeschir , Schermuly Ralph Theo , Seeger Werner , Grimminger Friedrich
Published: December 2017

Hypoxic pulmonary vasoconstriction (HPV) matches lung perfusion with ventilation but may also result in chronic pulmonary hypertension. It has not been clarified whether acute HPV and the response to prolonged alveolar hypoxia are triggered by identical mechanisms. We characterized the vascular response to sustained hypoxic ventilation (3% O2for 120–180 min) in isolated rabbit lungs. Hypoxia provoked a biphasic increase in pulmonary arterial pressure (PAP). Persistent PAP elevation was observed after termination of hypoxia. Total blockage of lung nitric oxide (NO) formation by N G-monomethyl-l-arginine caused a two- to threefold amplification of acute HPV, the sustained pressor response, and the loss of posthypoxic relaxation. This amplification was only moderate when NO formation was partially blocked by the inducible NO synthase inhibitor S-methylisothiourea. The superoxide scavenger nitro blue tetrazolium and the superoxide dismutase inhibitor triethylenetetramine reduced the initial vasoconstrictor response, the prolonged PAP increase, and the loss of posthypoxic vasorelaxation to a similar extent. The NAD(P)H oxidase inhibitor diphenyleneiodonium nearly fully blocked the late vascular responses to hypoxia in a dose that effected a decrease to half of the acute HPV. In conclusion, as similarly suggested for acute HPV, lung NO synthesis and the superoxide-hydrogen peroxide axis appear to be implicated in the prolonged pressor response and the posthypoxic loss of vasorelaxation in perfused rabbit lungs undergoing 2–3 h of hypoxic ventilation.
Expression of highly selective sodium channels in alveolar type II cells is determined by culture conditions
Authors: Jain Lucky , Chen Xi-Juan , Ramosevac Semra , Brown Lou Ann , Eaton Douglas C.
Published: December 2017

Alveolar fluid clearance in the developing and mature lungs is believed to be mediated by some form of epithelial Na channels (ENaC). However, single-channel studies using isolated alveolar type II (ATII) cells have failed to demonstrate consistently the presence of highly selective Na+channels that would be expected from ENaC expression. We postulated that in vitro culture conditions might be responsible for alterations in the biophysical properties of Na+conductances observed in cultured ATII cells. When ATII cells were grown on glass plates submerged in media that lacked steroids, the predominant channel was a 21-pS nonselective cation channel (NSC) with a Na+-to-K+selectivity of 1; however, when grown on permeable supports in the presence of steroids and air interface, the predominant channel was a low-conductance (6.6 ± 3.4 pS, n = 94), highly Na+-selective channel (HSC) with a PNa/ PK>80 that is inhibited by submicromolar concentrations of amiloride ( K0.5= 37 nM) and is similar in biophysical properties to ENaC channels described in other epithelia. To establish the relationship of this HSC channel to the cloned ENaC, we employed antisense oligonucleotide methods to inhibit the individual subunit proteins of ENaC (α, β, and γ) and used patch-clamp techniques to determine the density of this channel in apical membrane patches of ATII cells. Overnight treatment of cells with antisense oligonucleotides to any of the three subunits of ENaC resulted in a significant decrease in the density of HSC channels in the apical membrane cell-attached patches. Taken together, these results show that when grown on permeable supports in the presence of steroids and air interface, the predominant channels expressed in ATII cells have single-channel characteristics resembling channels that are associated with the coexpression of the three cloned ENaC subunits α-, β-, and γ-ENaC.
Excitation-contraction coupling in pulmonary vascular smooth muscle involves tyrosine kinase and Rho kinase
Authors: Janssen Luke J. , Lu-Chao Hwa , Netherton Stuart
Published: December 2017

We investigated the mechanisms that underlie the responses to norepinephrine (NE) and thromboxane (Tx) A2(TxA2) in the canine pulmonary vasculature with fura 2 fluorimetric, intracellular microelectrode, and force transduction techniques. KCl, caffeine, and cyclopiazonic acid elevated intracellular Ca2+concentration levels and tone, indicating that Ca2+mobilization is sufficient to produce contraction. However, contractions evoked by NE or the TxA2mimetic U-46619 were unaffected by nifedipine or by omitting external Ca2+and were reduced only partially by depleting the internal Ca2+store; furthermore, NE-evoked depolarization was subthreshold for voltage-dependent Ca2+currents. Agonist-evoked contractions were insensitive to inhibitors of protein kinase C (calphostin C and chelerythrine), mitogen-activated protein kinase kinase (PD-98059), and p38 kinase (SB-203580) but were abolished by the tyrosine kinase inhibitor genistein and the Rho kinase inhibitor Y-27632. We conclude that, although Ca2+influx and Ca2+release are sufficient for contraction, they are not necessary for adrenergic or TxA2contractions. Instead, excitation-contraction coupling involves the activation of tyrosine kinase and Rho kinase, leading to enhanced Ca2+sensitivity of the contractile apparatus.
Extracellular matrix fibronectin alters connexin43 expression by alveolar epithelial cells
Authors: Alford Andrea I. , Rannels D. Eugene
Published: December 2017

Alveolar type II epithelial cells undergo phenotypic changes and establish gap junction intercellular communication as they reach confluence in primary culture. The pattern of gap junction protein (connexin) expression changes in parallel. Although connexin (Cx)43 mRNA and protein increase significantly by culture day 2, Cx26 and Cx32 expression decline. Along with increasing Cx43 expression, the cells assemble fibronectin derived both from serum in the culture medium and from de novo synthesis into the extracellular matrix (ECM). The present studies indicate that this ECM regulates Cx43 expression. Culture of type II cells in DMEM containing 8–10% fetal bovine serum (FBS) promotes assembly of a fibronectin-rich ECM that stimulates expression of both Cx43 mRNA and protein. Although Cx43 protein expression increased in response to FBS in a dose-dependent manner, fibronectin also elevated Cx43 protein in the absence of FBS. Anti-fibronectin antibody significantly reduced the serum-dependent increase in Cx43 expression. These results support the premise that fibronectin in the ECM contributes to the regulation of Cx43 expression by alveolar epithelial cells in primary culture.
Cell-specific and developmental regulation of rabbit surfactant protein B promoter in transgenic mice
Authors: Adams Constantin C. , Alam M. Nurul , Starcher Barry C. , Boggaram Vijayakumar
Published: December 2017

Surfactant protein B (SP-B) is expressed tissue specifically in the lung and is developmentally regulated. To identify genomic regions that control SP-B expression, we analyzed SP-B promoter activity in transgenic mice containing rabbit SP-B 5′-flanking DNA fragments linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Results showed that whereas the −2,176/+39-bp fragment failed to express CAT, shorter fragments of −730/+39 and −236/+39 bp expressed CAT tissue specifically in the lung. Further deletion of 5′-flanking DNA to −136 bp resulted in no expression of CAT. Immunostaining demonstrated that both −730/+39- and −236/+39-bp regions expressed CAT specifically in alveolar type II and Clara cells. The −236/+39-bp region expressed CAT at a significantly lower level than the −730/+39-bp region. CAT expression in mice containing the −730/+39-bp region was detected in embryonic day 14 lung and attained maximum levels in day 18 lung, indicating that the developmental expression of CAT was similar to that of SP-B. These data show that the DNA elements necessary for cell type-specific expression are located within −236/+39 bp of the SP-B gene. Additionally, these data suggest that the −2,176/−730- and −730/−236-bp regions contain the DNA elements that repress and enhance SP-B gene transcription, respectively.
Inhibition of LPS-induced airway hyperresponsiveness and airway inflammation by LPS antagonists
Authors: Schwartz David A. , Christ William J. , Kleeberger Steven R. , Wohlford-Lenane Christine L.
Published: December 2017

To determine whether the inflammatory effects of inhaled endotoxin could be prevented, we pretreated mice with synthetic competitive antagonists (975, 1044, and 1287) for lipopolysaccharide (LPS) before a LPS inhalation challenge. In preliminary studies, we found that these LPS antagonists did not act as agonists in vitro (THP-1 cells) or in vivo (after intratracheal instillation of 10 μg) and that these compounds (at least 1 μg/ml) effectively antagonized the release of tumor necrosis factor-α by LPS-stimulated THP-1 cells. Pretreatment of mice with 10 μg of either 1044 or 1287 resulted in a decrease in the LPS-induced airway hyperreactivity. Moreover, pretreatment of mice with 10 μg of 975, 1044, or 1287 resulted in significant reductions in LPS-induced lung lavage fluid concentrations of total cells, neutrophils, and specific proinflammatory cytokines compared with mice pretreated with sterile saline. Using residual oil fly ash to induce airway inflammation, we found that the action of the LPS antagonists was specific to LPS-induced airway disease. These results suggest that LPS antagonists may be an effective and potentially safe treatment for endotoxin-induced airway disease.
Transcription factors in mouse lung development and function
Authors: Costa Robert H. , Kalinichenko Vladimir V. , Lim Lorena
Published: December 2017

Development of the mouse lung initiates on day 9.5postcoitum from the laryngotracheal groove and involves mesenchymal-epithelial interactions, in particular, those between the splanchnic mesoderm and epithelial cells (derived from foregut endoderm) that induce cellular proliferation, migration, and differentiation, resulting in branching morphogenesis. This developmental process mediates formation of the pulmonary bronchiole tree and integrates a terminal alveolar region with an extensive endothelial capillary bed, which facilitates efficient gas exchange with the circulatory system. The major function of the mesenchymal-epithelial signaling is to potentiate the activity or expression of cell type-specific transcription factors in the developing lung, which, in turn, cooperatively bind to distinct promoter regions and activate target gene expression. In this review, we focus on the role of transcription factors in lung morphogenesis and the maintenance of differentiated gene expression. These lung transcription factors include forkhead box A2 [also known as hepatocyte nuclear factor (HNF)-3β], HNF-3/forkhead homolog (HFH)-8 [also known as FoxF1 or forkhead-related activator-1], HNF-3/forkhead homolog-4 (also known as FoxJ1), thyroid transcription factor-1 (Nkx2.1), and homeodomain box A5 transcription factors, the zinc finger Gli (mouse homologs of the Drosophila cubitus interruptus) and GATA transcription factors, and the basic helix-loop-helix Pod1 transcription factor. We summarize the phenotypes of transgenic and knockout mouse models, which define important functions of these transcription factors in cellular differentiation and lung branching morphogenesis.
Identification of genes induced by inflammatory cytokines in airway epithelium
Authors: Cooper Paul , Potter Suzanne , Mueck Beatrice , Yousefi Shida , Jarai Gabor
Published: December 2017

Epithelial cells lining the airways are thought to play a prominent role in respiratory diseases. We utilized cDNA representational difference analysis to identify the genes in which expression is induced by the proinflammatory cytokines tumor necrosis factor-α and interleukin-1β in primary human bronchial epithelial cells and hence are relevant to airway inflammation. Hybridization of the subtraction product to arrayed cDNAs indicated that known tumor necrosis factor-α- and interleukin-1β-inducible genes such as B94, Zfp36, and regulated on activation normal T cell expressed and secreted were represented, confirming the success of the subtraction experiment. A 1,152-clone library potentially representing genes with higher transcript levels in cytokine-treated human bronchial epithelial cells was generated and sequenced. Sequence similarity searches indicated that these clones represented 57 genes of known function, 1 gene of unknown function, 6 expressed sequence tags, and 2 novel sequences. The expression of 19 of these clones was studied by a combination of Northern blotting and RT-PCR analyses and confirmation of differential expression for 10 known genes, 2 expressed sequence tags, and a novel sequence not represented in any of the public databases was obtained. Thus cDNA representational difference analysis was utilized to isolate known and novel differentially expressed genes, which putatively play a role in airway inflammation.
Pulmonary interstitial pressure and tissue matrix structure in acute hypoxia
Authors: Miserocchi Giuseppe , Passi Alberto , Negrini Daniela , Del Fabbro Massimo , De Luca Giancarlo
Published: December 2017

Pulmonary interstitial pressure was measured via micropuncture in anesthetized rabbits in normoxia and after breathing 12% O2. In normoxia [arterial Po 2 = 88 ± 2 (SD) mmHg], pulmonary arterial pressure and pulmonary interstitial pressure were 16 ± 8 and −9.6 ± 2 cmH2O, respectively. After 6 h of hypoxia (arterial Po 2 = 39 ± 16 mmHg), the corresponding values were 30 ± 8 and 3.5 ± 2.5 cmH2O ( P < 0.05). Pulmonary interstitial proteoglycan extractability, evaluated by hexuronate assay after 0.4 M guanidinium hydrochloride extraction, was 12.3, 32.4, and 60.6 μg/g wet tissue in normoxia and after 3 and 6 h of hypoxia, respectively, indicating a weakening of the noncovalent bonds linking proteoglycans to other extracellular matrix components. Gel filtration chromatography showed an increased fragmentation of chondroitin sulfate- and heparan sulfate-proteoglycans during hypoxic exposure, accounting for a loss of extracellular matrix native architecture and basement membrane structure. Gelatin zymography demonstrated increased amounts of the proteolytically activated form of gelatinase B (matrix metalloproteinase-9) after hypoxic exposure, providing evidence that the activation of proteinases may play a role in hypoxia-induced lung injury.
Role of ecNOS-derived NO in mediating TNF-induced endothelial barrier dysfunction
Authors: Bove Kathleen , Neumann Paul , Gertzberg Nancy , Johnson Arnold
Published: December 2017

We tested the hypothesis that endothelial cell nitric oxide synthase (ecNOS) mediates the tumor necrosis factor (TNF)-α-induced increase in nitric oxide (NO) and albumin permeability in pulmonary microvessel endothelial monolayers (PEM). PEM lysates were analyzed for ecNOS mRNA (RT-PCR), ecNOS protein (Western immunoblot), NO levels (NO[Formula: see text], the oxidation product of NO), and barrier function (albumin clearance rate). PEM were incubated with TNF (50 ng/ml) for 0.5, 2, 4, and 24 h. TNF induced a decrease in ecNOS mRNA at 2, 4, and 24 h. TNF induced an acute (0.5 h) increase followed by a protracted decrease (4–24 h) in ecNOS protein levels. The other NOS isotypes, inducible and brain NOS, could not be detected in the PEM using RT-PCR and Western blot assay. ecNOS antisense oligonucleotide decreased ecNOS protein, which prevented the increase in NO and albumin permeability at TNF-4 h. Spermine-NONOATE, the NO agonist, ablated the protective effect of ecNOS antisense oligonucleotide on albumin permeability in response to TNF-4 h. However, ecNOS antisense oligonucleotide had no effect on the TNF-induced increase in albumin permeability at 24 h despite prevention of the increase in NO. The data indicate that the isotype ecNOS mediates generation of NO and the acute (i.e., 4 h) barrier dysfunction; however, the prolonged (i.e., 24 h) increase in the TNF-induced increase in endothelial permeability is independent of NO.
Deformation-induced lipid trafficking in alveolar epithelial cells
Authors: Vlahakis Nicholas E. , Schroeder Mark A. , Pagano Richard E. , Hubmayr Rolf D.
Published: December 2017

Mechanical ventilation with a high tidal volume results in lung injury that is characterized by blebbing and breaks both between and through alveolar epithelial cells. We developed an in vitro model to simulate ventilator-induced deformation of the alveolar basement membrane and to investigate, in a direct manner, epithelial cell responses to deforming forces. Taking advantage of the novel fluorescent properties of BODIPY lipids and the fluorescent dye FM1-43, we have shown that mechanical deformation of alveolar epithelial cells results in lipid transport to the plasma membrane. Deformation-induced lipid trafficking (DILT) was a vesicular process, rapid in onset, and was associated with a large increase in cell surface area. DILT could be demonstrated in all cells; however, only a small percentage of cells developed plasma membrane breaks that were reversible and nonlethal. Therefore, DILT was not only involved in site-directed wound repair but might also have served as a cytoprotective mechanism against plasma membrane stress failure. This study suggests that DILT is a regulatory mechanism for membrane trafficking in alveolar epithelia and provides a novel biological framework within which to consider alveolar deformation injury and repair.
Cdc42, but not RhoA, regulates cyclin D1 expression in bovine tracheal myocytes
Authors: Bauerfeld Christian P. , Hershenson Marc B. , Page Kristen
Published: December 2017

We previously demonstrated that Rac1 increased cyclin D1 promoter activity in an extracellular signal-regulated kinase (ERK)-independent, antioxidant-sensitive manner. Here, we examined the regulation of cyclin D1 expression by Cdc42 and RhoA. Overexpression of active Cdc42, but not of RhoA, induced transcription from the cyclin D1 promoter. Furthermore, dominant negative Cdc42, but not RhoA, attenuated platelet-derived growth factor-mediated activation of the cyclin D1 promoter. Overexpression of active Cdc42 increased cyclin D1 protein abundance in COS cells. Cdc42-induced cyclin D1 promoter activation was independent of ERK as evidenced by insensitivity to PD-98059, an inhibitor of mitogen-activated protein kinase/ERK kinase (MEK). Furthermore, Cdc42 was neither sufficient nor required for activation of ERK. Similar to Rac1-induced cyclin D1 expression, pretreatment with the antioxidants catalase and ebselen inhibited Cdc42-mediated transcription from the cyclin D1 promoter. Finally, like Rac1, active Cdc42 induced transactivation of the cyclin D1 promoter cAMP response element binding protein/activating transcription factor-2 binding site. Together, these data suggest that in airway smooth muscle cells, Cdc42 and Rac1 share a common signaling pathway to cyclin D1 promoter activation.
Interleukin-10-mediated inhibition of free radical generation in macrophages
Authors: Dokka Sujatha , Shi Xianglin , Leonard Stephen , Wang Liying , Castranova Vincent , Rojanasakul Yon
Published: December 2017

Interleukin-10 (IL-10) is a pleiotropic cytokine that controls inflammatory processes by suppressing the production of proinflammatory cytokines that are known to be transcriptionally regulated by nuclear factor-κB (NF-κB). Although still controversial, IL-10 has been shown to inhibit NF-κB activation through a process that involves proteolytic degradation of inhibitory subunit IκB-α. What is not known, however, is the mechanism by which IL-10 exerts its effect on IκB-α degradation. The present study investigates the possible role of reactive oxygen species (ROS) and their inhibition by IL-10 in NF-κB activation and IκB-α degradation in macrophages. Treatment of the cells with lipopolysaccharide (LPS) caused activation of NF-κB and rapid proteolysis of IκB-α as determined by the electrophoretic mobility shift assay, gene transfection, and Western blot. IL-10 pretreatment inhibited both NF-κB activation and IκB-α degradation. Both of these processes were also inhibited by ROS scavengers, catalase (H2O2scavenger), and sodium formate (·OH scavenger) but were minimally affected by superoxide dismutase (O[Formula: see text] scavenger). These results suggests that ·OH radicals, formed by an H2O2-dependent, metal-catalyzed Fenton reaction, play a major role in this process. Electron spin resonance studies confirmed the formation of ·OH radicals in LPS-treated cells. Addition of IL-10 inhibited both IκB-α degradation and generation of ·OH radicals in response to LPS stimulation. These results demonstrate, for the first time, direct evidence for the role of IL-10 in ROS-dependent NF-κB activation.
Regulation of lung epithelial cell morphology by cAMP-dependent protein kinase type I isozyme
Authors: Porter Stephanie E. , Dwyer-Nield Lori D. , Malkinson Alvin M.
Published: December 2017

Cell shape is mediated in part by the actin cytoskeleton and the actin-binding protein vinculin. These proteins in turn are regulated by protein phosphorylation. We assessed the contribution of cAMP-dependent protein kinase A isozyme I (PKA I) to lung epithelial morphology using the E10/E9 sibling cell lines. PKA I concentration is high in flattened, nontumorigenic E10 cells but low in their round E9 transformants. PKA I activity was lowered in E10 cells by stable transfection with a dominant negative RIα mutant of the PKA I regulatory subunit and was raised in E9 cells by stable transfection with a wild-type Cα catalytic subunit construct. Reciprocal changes in morphology ensued. E10 cells became rounder and grew in colonies, their actin microfilaments were disrupted, and vinculin localization at cell-cell junctions was diminished. The converse occurred in E9 cells on elevating their PKA I content. Demonstration that PKA I is responsible for the dichotomy in these cellular behaviors suggests that manipulating PKA I concentrations in lung cancer would provide useful adjuvant therapy.
Oxygen radical-induced mitochondrial DNA damage and repair in pulmonary vascular endothelial cell phenotypes
Authors: Grishko Valentina , Solomon Marie , Wilson Glenn L. , LeDoux Susan P. , Gillespie Mark N.
Published: December 2017

Mitochondrial (mt) DNA is damaged by free radicals. Recent data also show that there are cell type-dependent differences in mtDNA repair capacity. In this study, we explored the effects of xanthine oxidase (XO), which generates superoxide anion directly, and menadione, which enhances superoxide production within mitochondria, on mtDNA in pulmonary arterial (PA), microvascular (MV), and pulmonary venous (PV) endothelial cells (ECs). Both XO and menadione damaged mtDNA in the EC phenotypes, with a rank order of sensitivity of (from most to least) PV > PA > MV for XO and MV = PV > PA for menadione. Dimethylthiourea and deferoxamine blunted menadione- and XO-induced mtDNA damage, thus supporting a role for the iron-catalyzed formation of hydroxyl radical. Damage to the nuclear vascular endothelial growth factor gene was not detected with either XO or menadione. PAECs and MVECs, but not PVECs, repaired XO-induced mtDNA damage quickly. Menadione-induced mtDNA damage was avidly repaired in MVECs and PVECs, whereas repair in PAECs was slower. Analysis of mtDNA lesions at nucleotide resolution showed that damage patterns were similar between EC phenotypes, but there were disparities between XO and menadione in terms of the specific nucleotides damaged. These findings indicate that mtDNA in lung vascular ECs is damaged by XO- and menadione-derived free radicals and suggest that mtDNA damage and repair capacities differ between EC phenotypes.
Proteoglycans decorin and biglycan differentially modulate TGF-β-mediated fibrotic responses in the lung
Authors: Kolb Martin , Margetts Peter J. , Sime Patricia J. , Gauldie Jack
Published: December 2017

Transforming growth factor (TGF)-β is a key cytokine in the pathogenesis of pulmonary fibrosis, and pharmacological interference with TGF-β can ameliorate the fibrotic tissue response. The small proteoglycans decorin and biglycan are able to bind and inhibit TGF-β activity in vitro. Although decorin has anti-TGF-β properties in vivo, little is known about the physiological role of biglycan in vivo. Adenoviral gene transfer was used to overexpress active TGF-β, decorin, and biglycan in cell culture and in murine lungs. Both proteoglycans were able to interfere with TGF-β bioactivity in vitro in a dose-dependant manner. In vivo, overexpression of TGF-β resulted in marked lung fibrosis, which was significantly reduced by concomitant overexpression of decorin. Biglycan, however, had no significant effect on lung fibrosis induced by TGF-β. The data suggest that differences in tissue distribution are responsible for the different effects on TGF-β bioactivity in vivo, indicating that decorin, but not biglycan, has potential therapeutic value in fibrotic disorders of the lung.
Uptake of a natural surfactant and increased delivery of small organic anions into type II pneumocytes
Authors: Griese Matthias , Baatz Astrid , Beck Julia , Deubzer Barbara
Published: December 2017

The uptake of natural lung surfactant into differentiated type II cells may be used for the targeted delivery of other molecules. The fluorescent anion pyranine [hydroxypyren-1,3,6-trisulfonic acid, sodium salt (HPTS)] was incorporated into a bovine surfactant labeled with [3H]dipalmitoylphosphatidylcholine ([3H]DPPC). The uptake of [3H]DPPC and of HPTS increased with time of incubation and concentration, decreased with the size of the vesicles used, and was stimulated by 8-bromo-cAMP and partially inhibited by hypertonic sucrose. However, the amount of HPTS uptake was ∼100 times smaller than that of [3H]DPPC. This large difference was due to a more rapid regurgitation of some of the HPTS from the cells but not to leakage from the surfactant before uptake. The acidification of the internalized surfactant increased linearly over 90 min to 7.13, and after 24 h, a pH of 6.83 was measured. In conclusion, after internalization of a double-labeled natural surfactant, the lipid moieties were accumulated in relation to the anions, which were targeted to a compartment not very acidic and in part rapidly expelled from the cells.
Bradykinin augments fibroblast-mediated contraction of released collagen gels
Authors: Mio Tadashi , Liu Xiangde , Toews Myron L. , Adachi Yuichi , Romberger Debra J. , Spurzem John R. , Rennard Stephen I.
Published: December 2017

Bradykinin is a multifunctional mediator of inflammation believed to have a role in asthma, a disorder associated with remodeling of extracellular connective tissue. Using contraction of collagen gels as an in vitro model of wound contraction, we assessed the effects of bradykinin tissue on remodeling. Human fetal lung fibroblasts were embedded in type I collagen gels and cultured for 5 days. After release, the floating gels were cultured in the presence of bradykinin. Bradykinin significantly stimulated contraction in a concentration- and time-dependent manner. Coincubation with phosphoramidon augmented the effect of 10−9and 10−8M bradykinin. A B2receptor antagonist attenuated the effect of bradykinin, whereas a B1receptor antagonist had no effect, suggesting that the effect is mediated by the B2receptor. An inhibitor of intracellular Ca2+mobilization abolished the response; addition of EGTA to the culture medium attenuated the contraction of control gels but did not modulate the response to bradykinin. In contrast, the phospholipase C inhibitor U-73122 and the protein kinase C inhibitors staurosporine and GF-109203X attenuated the responses. These data suggest that by augmenting the contractility of fibroblasts, bradykinin may have an important role in remodeling of extracellular matrix that may result in tissue dysfunction in chronic inflammatory diseases, such as asthma.
Chronic hypoxia-induced spontaneous and rhythmic contractions in the rat main pulmonary artery
Authors: Bonnet Sébastien , Hyvelin Jean-Marc , Bonnet Pierre , Marthan Roger , Savineau Jean-Pierre
Published: December 2017

The effect of chronic hypoxia (CH; 1–4 wk) on the electromechanical properties of the rat main pulmonary artery (MPA) was investigated. MPA rings obtained from rats exposed for 14 days to hypobaric (50.5 kPa) CH exhibited spontaneous and rhythmic contractions (SRCs) that were never observed in control (normoxic) rats. SRCs were unaffected by tetrodotoxin, phentolamine, BQ-123 and BQ-788, N-nitro-l-arginine methyl ester, or endothelium removal. CH depolarized smooth muscle cells from −58.8 ± 9 to −38.6 ± 5.4 mV and increased the resting cytosolic Ca2+concentration from 67.3 ± 11.9 to 112.5 ± 16.4 nM. CH also induced spontaneous spikelike depolarizations. All of these effects were inhibited by external Ca2+removal or nifedipine (1 μM). Moreover, depletion of intracellular Ca2+stores with ryanodine (1–5 μM) or cyclopiazonic acid (3 μM) progressively attenuated SRCs. This study demonstrates that CH switches the MPA from a quiescent to a spontaneously active mechanical state. Finally, the fact that SRCs precede the development of right ventricle hypertrophy and disappear when this hypertrophy reaches a maximal value (after 3–4 wk of CH) suggests that SRCs may play a role in the adaptive process of the pulmonary circulation to CH.