bluuebunny/crossref_metadata_2025 huggingface dataset:
Surfactant protein A and surfactant protein D in health and disease
Authors: Mason Robert J. , Greene Kelly , Voelker Dennis R.
Published: December 2017

Surfactant protein (SP) A and SP-D are collagenous glycoproteins with multiple functions in the lung. Both of these proteins are calcium-dependent lectins and are structurally similar to mannose-binding protein and bovine conglutinin. Both form polyvalent multimeric structures for interactions with pathogens, cells, or other molecules. SP-A is an integral part of the surfactant system, binds phospholipids avidly, and is found in lamellar bodies and tubular myelin. Initially, most research interest focused on its role in surfactant homeostasis. Recently, more attention has been placed on the role of SP-A as a host defense molecule and its interactions with pathogens and phagocytic cells. SP-D is much less involved with the surfactant system. SP-D appears to be primarily a host defense molecule that binds surfactant phospholipids poorly and is not found in lamellar inclusion bodies or tubular myelin. Both SP-A and SP-D bind a wide spectrum of pathogens including viruses, bacteria, fungi, and pneumocystis. In addition, both molecules have been measured in the systemic circulation by immunologic methods and may be useful biomarkers of disease. The current challenges are characterization of the three-dimensional crystal structure of SP-A and SP-D, molecular cloning of their receptors, and determination of their precise physiological functions in vivo.
TNF-α-induced matrix Fn disruption and decreased endothelial integrity are independent of Fn proteolysis
Authors: Curtis Theresa M. , Rotundo Robert F. , Vincent Peter A. , McKeown-Longo Paula J. , Saba Thomas M.
Published: December 2017

Exposure of confluent pulmonary arterial endothelial monolayers to tumor necrosis factor (TNF)-α causes both a reorganization and/or disruption of fibronectin (Fn) in the extracellular matrix and an increase in transendothelial protein permeability. However, the factors initiating this response to TNF-α have not been defined. Because TNF-α can induce proteinase expression in endothelial cells, we determined whether proteinases cause both the alteration of the Fn matrix and the permeability increase as is often speculated. Incubation of calf pulmonary arterial endothelial monolayers with TNF-α (200 U/ml) for 18 h caused a disruption of the Fn matrix and an increase in transendothelial protein permeability. A reduced colocalization of cell-surface α5β1-Fn integrins with the Fn fibers in focal contacts was also observed. TNF-α treatment of endothelial monolayers with matrices prelabeled with125I-human Fn (hFn) did not cause the release of Fn fragments or alter the content of Fn antigen in the medium as analyzed by SDS-PAGE coupled with autoradiography. Both the content and fragmentation pattern of Fn within the cell layer and the insoluble Fn matrix also appeared unchanged after TNF-α exposure as confirmed by Western immunoblot. Fn-substrate zymography revealed that TNF-α increased the expression of two proteinases within the conditioned medium in which activity could be blocked by aprotinin but not by EDTA, 1,10-phenanthroline, leupeptin, or pepstatin. However, inhibition of the Fn proteolytic activity of these two serine proteinases did not prevent either the TNF-α-induced disruption of the Fn matrix or the increase in permeability. Thus the reorganization and/or disruption of the Fn matrix and the temporally associated increase in endothelial permeability caused by TNF-α appear not to be due to proteolytic degradation of Fn within the extracellular matrix. In contrast, decreased α5β1-Fn integrin interaction with Fn fibers in the matrix may be important in the response to TNF-α exposure.
Mitochondrial respiration after sepsis and prolonged hypoxia
Authors: Taylor David E. , Kantrow Stephen P. , Piantadosi Claude A.
Published: December 2017

Recently, marked oxygen dependence of respiration by isolated mitochondria after exposure to prolonged hypoxia has been described. Because mitochondrial oxygen-dependent respiration could significantly influence oxygen consumption during critical illness, we sought to confirm the oxygen-dependent behavior of mitochondria. We hypothesized that mitochondria isolated during sepsis would exhibit increased oxygen dependence. We isolated rat liver mitochondria 16 h after cecal ligation and puncture and found a 30–40% greater oxygen uptake compared with control rats under state 3 conditions. Mitochondria incubated in deoxygenated buffer were studied for oxygen dependence at 10-min intervals for 90 min. Mitochondrial respiration after reoxygenation was stable over a 60-min period of hypoxia for control rats and decreased slightly for septic rats (10–15%). State 3 respiration was 10% lower when mitochondria were reoxygenated at low (15–25 Torr) versus high (90–100 Torr) and low (10–15 Torr) versus intermediate (40–45 Torr) oxygen tension. Oxygen consumption with ascorbate+ N, N, N′, N′-tetramethyl- p-phenylenediamine was 20% lower at low versus high oxygen tension. No increase in oxygen dependence was observed during 1 h of hypoxic incubation. Our data indicate only a modest oxygen dependence of respiration between 10 and 100 Torr, which is similar for septic and control mitochondria. Additionally, oxygen dependence did not increase significantly during a 1-h hypoxic exposure for well-coupled mitochondrial preparations.
Modulation of T1α expression with alveolar epithelial cell phenotype in vitro
Authors: Borok Zea , Danto Spencer I. , Lubman Richard L. , Cao Yuxia , Williams Mary C. , Crandall Edward D.
Published: December 2017

T1α is a recently identified gene expressed in the adult rat lung by alveolar type I (AT1) epithelial cells but not by alveolar type II (AT2) epithelial cells. We evaluated the effects of modulating alveolar epithelial cell (AEC) phenotype in vitro on T1α expression using either soluble factors or changes in cell shape to influence phenotype. For studies on the effects of soluble factors on T1α expression, rat AT2 cells were grown on polycarbonate filters in serum-free medium (MDSF) or in MDSF supplemented with either bovine serum (BS, 10%), rat serum (RS, 5%), or keratinocyte growth factor (KGF, 10 ng/ml) from either day 0 or day 4 through day 8 in culture. For studies on the effects of cell shape on T1α expression, AT2 cells were plated on thick collagen gels in MDSF supplemented with BS. Gels were detached on either day 1(DG1) or day 4 (DG4) or were left attached until day 8. RNA and protein were harvested at intervals between days 1 and 8 in culture, and T1α expression was quantified by Northern and Western blotting, respectively. Expression of T1α progressively increases in AEC grown in MDSF ± BS between day 1 and day 8 in culture, consistent with transition toward an AT1 cell phenotype. Exposure to RS or KGF from day 0 prevents the increase in T1α expression on day 8, whereas addition of either factor from day 4 through day 8 reverses the increase. AEC cultured on attached gels express high levels of T1α on days 4 and 8. T1α expression is markedly inhibited in both DG1 and DG4 cultures, consistent with both inhibition and reversal of the transition toward the AT1 cell phenotype. These results demonstrate that both soluble factors and alterations in cell shape modulate T1α expression in parallel with AEC phenotype and provide further support for the concept that transdifferentiation between AT2 and AT1 cell phenotypes is at least partially reversible.
Resistance of pleural mesothelioma cell lines to apoptosis: relation to expression of Bcl-2 and Bax
Authors: Narasimhan Sudha Rani , Yang Lin , Gerwin Brenda I. , Broaddus V. Courtney
Published: December 2017

A failure of normal apoptosis, often due to mutant p53, may contribute to the formation of a cancer and to its resistance to therapy. Mesothelioma, an asbestos-induced tumor, is highly resistant to therapy but generally expresses wild-type p53. We asked whether mesothelioma was resistant to apoptosis and whether resistance was associated with altered expression of the antiapoptotic protein Bcl-2 or proapoptotic protein Bax. We found that three mesothelioma cell lines (1 with wild-type p53) were highly resistant to apoptosis induced by oxidant stimuli (asbestos, H2O2) or nonoxidant stimuli (calcium ionophore) compared with primary cultured mesothelial cells. By immunostaining, one of these three lines expressed Bcl-2 but only during mitosis. By immunoblotting, 3 of 14 additional mesothelioma lines (9 of 14 with wild type p53) expressed Bcl-2 but all 14 of 14 expressed the proapoptotic Bax, giving a low ratio of Bcl-2 to Bax. We conclude that mesothelioma cell lines are resistant to apoptosis and that the failure in apoptosis is not explained by Bcl-2 but by other mechanisms that counteract the proapoptotic effect of Bax.
Release of a leukocyte activation inhibitor by staurosporine-treated pulmonary artery endothelial cells
Authors: Chen Xilin , Catravas John D.
Published: December 2017

Bovine pulmonary arterial endothelial cells (BPAE) treated with the protein kinase C (PKC) inhibitor staurosporine inhibited[Formula: see text] generation by neutrophils exposed to phorbol myristate acetate (PMA) but did not affect[Formula: see text] generated enzymatically by xanthine/xanthine oxidase (X/XO). Similar results were obtained with conditioned medium from staurosporine-pretreated BPAE. The inhibitory effects of staurosporine-treated BPAE on[Formula: see text] generation were not altered by the superoxide dismutase inhibitor diethylcarbamazine. This BPAE-derived inhibitor was continuously released from staurosporine-pretreated BPAE for at least 5 h. The exact nature of the inhibitor remains unknown, but it appears to be a positively charged molecule with molecular weight <10,000. Treatment of either BPAE or neutrophils with staurosporine or conditioned medium from staurosporine-treated BPAE prevented the neutrophil-mediated decrease in endothelium-bound angiotensin-converting enzyme activity and cytotoxicity in BPAE. In contrast, staurosporine potentiated the H2O2- and X/XO-mediated endothelial cytotoxicity. These data suggest that staurosporine-treated endothelial cells release a soluble factor that inhibits neutrophil activation and protects endothelial cells from neutrophil-mediated injury.
Targeted identification of zinc finger genes expressed in rat lungs
Authors: Dovat Sinisa , Gilbert Kirk A. , Petrovic-Dovat Lidija , Rannels D. Eugene
Published: December 2017

Control of alveolar cell growth and differentiation after pneumonectomy likely involves changes in expression of regulatory genes, including those encoding zinc finger (ZF) proteins. To explore this premise, total RNA from the lungs of control and pneumonectomized rats was reverse transcribed; PCRs were performed with degenerate primers corresponding to amino acid sequences HTGEKP and CPECGK(N), which are evolutionarily conserved among ZF genes. Reaction products corresponding to three and four ZF units were isolated and cloned. Sixteen clones were sequenced and found to represent rat lung ZF genes: six clones were highly similar or identical to known ZF genes and ten clones showed lower homology to known ZF genes and thus appear to represent new members of the ZF family. Northern analysis demonstrated differential expression of some ZF genes after pneumonectomy. Thus a PCR-based strategy with primers derived from evolutionarily conserved ZF protein sequences efficiently identifies ZF genes expressed in lung, some of which may play a role in cellular growth and differentiation.
Increased secretion of urokinase-type plasminogen activator by human lung microvascular endothelial cells
Authors: Takahashi Kimiko , Uwabe Yasuhide , Sawasaki Yoshio , Kiguchi Toshio , Nakamura Hiroyuki , Kashiwabara Kosuke , Yagyu Hisanaga , Matsuoka Takeshi
Published: December 2017

Human lung microvascular endothelial cells (HLMECs) secreted 1.5–15 times more urokinase-type plasminogen activator (uPA) antigen than human hepatic microvascular endothelial cells, human umbilical vein endothelial cells (HUVECs), angioma endothelial cells, and lung fibroblasts. All of these cells also secreted a 100-fold greater amount of plasminogen activator inhibitor-1 than of uPA antigen, and uPA activities were not detected in the culture medium. The expression of uPA mRNA in HLMECs was higher (100-fold) compared with HUVECs, angioma endothelial cells, and lung fibroblasts. HLMECs secreted uPA antigen on both the luminal and basal sides of the cells. On the other hand, HLMECs secreted a 10- to 15-fold lower amount of tissue-type plasminogen activator than HUVECs, mostly on the luminal side. After stimulation with interleukin (IL)-1β, HLMECs secreted a six- to ninefold amount of uPA antigen. In contrast, no stimulatory effect was observed in HUVECs even under high IL-1β concentrations. The secretion of uPA and plasminogen activator inhibitor-1 from HLMECs was also enhanced by tumor necrosis factor-α and IL-2. These results suggest that HLMECs may contribute not only to the patency of lung vessels but also to the maintenance of alveolar functions through the production and secretion of uPA, especially in the presence of inflammatory cytokines.
Retinoic acid-induced proliferation of lung alveolar epithelial cells: relation with the IGF system
Authors: Nabeyrat Elodie , Besnard Valérie , Corroyer Sophie , Cazals Véronique , Clement Annick
Published: December 2017

Retinoids, including retinol and retinoic acid (RA) derivatives, are important molecules for lung growth and homeostasis. The presence of RA receptors and of RA-binding proteins in the alveolar epithelium led to suggest a role for RA on alveolar epithelial cell replication. In the present study, we examined the effects of RA on proliferation of the stem cells of the alveolar epithelium, the type 2 cells. We showed that treatment of serum-deprived type 2 cells with RA led to a stimulation of cell proliferation, with an increase in cell number in a dose-dependent manner. To gain some insights into the mechanisms involved, we studied the effects of RA on the expression of several components of the insulin-like growth factor (IGF) system that have been shown to be associated with the growth arrest of type 2 cells, mainly the IGF-binding protein-2 (IGFBP-2), IGF-II, and the type 2 IGF receptor. We documented a marked decrease in the expression of these components upon RA treatment. Using conditioned media from RA-treated cells, we provided evidence that the proliferative response of type 2 cells to RA was mediated through production of growth factor(s) distinct from IGF-I. We also showed that RA was able to reduce the decrease in cell number observed when type 2 cells were treated with transforming growth factor (TGF)-β1. These results together with the known stimulatory effect of TGF-β1 on IGFBP-2 expression led to suggest that RA may be associated with type 2 cell proliferation through mechanisms interfering with the TGF-β1 pathway.
Signal transduction and regulation of lung endothelial cell permeability. Interaction between calcium and cAMP
Authors: Moore Timothy M. , Chetham Paul M. , Kelly John J. , Stevens Troy
Published: December 2017

Pulmonary endothelium forms a semiselective barrier that regulates fluid balance and leukocyte trafficking. During the course of lung inflammation, neurohumoral mediators and oxidants act on endothelial cells to induce intercellular gaps permissive for transudation of proteinaceous fluid from blood into the interstitium. Intracellular signals activated by neurohumoral mediators and oxidants that evoke intercellular gap formation are incompletely understood. Cytosolic Ca2+concentration ([Ca2+]i) and cAMP are two signals that importantly dictate cell-cell apposition. Although increased [Ca2+]ipromotes disruption of the macrovascular endothelial cell barrier, increased cAMP enhances endothelial barrier function. Furthermore, during the course of inflammation, elevated endothelial cell [Ca2+]idecreases cAMP to facilitate intercellular gap formation. Given the significance of both [Ca2+]iand cAMP in mediating cell-cell apposition, this review addresses potential sites of cross talk between these two intracellular signaling pathways. Emerging data also indicate that endothelial cells derived from different vascular sites within the pulmonary circulation exhibit distinct sensitivities to permeability-inducing stimuli; that is, elevated [Ca2+]ipromotes macrovascular but not microvascular barrier disruption. Thus this review also considers the roles of [Ca2+]iand cAMP in mediating site-specific alterations in endothelial permeability.
Asbestos-induced lung epithelial permeability: potential role of nonoxidant pathways
Authors: Peterson Michael W. , Kirschbaum Jennifer
Published: December 2017

Asbestos fibers are an important cause of lung fibrosis; however, the biological mechanisms are incompletely understood. The lung epithelium serves an important barrier function in the lung, and disrupting the epithelial barrier can contribute to lung fibrosis. Lung epithelial permeability is increased in patients with asbestosis, and asbestos fibers increase permeability across cultured human lung epithelium. However, the mechanism of this increased permeability is not known. Many of the biological effects of asbestos are postulated to be due to its ability to generate oxidants, and oxidants are known to increase epithelial permeability. However, we previously reported that altering the iron content of asbestos (important in oxidant generation) had no effect on its ability to increase permeability. For that reason, we undertook these studies to determine whether asbestos increases epithelial permeability through nonoxidant pathways. Both extracellular (H2O2) and intracellular (menadione) oxidants increase paracellular permeability across human lung epithelial monolayers. Extracellular catalase but not superoxide dismutase prevented increased permeability after both oxidant exposures. However, catalase offered no protection from asbestos-induced permeability. We next depleted the cells of glutathione or catalase to determine whether depleting normal cellular antioxidants would increase the sensitivity to asbestos. Permeability was the same in control cells and in cells depleted of these antioxidants. In addition to generating oxidants, asbestos also activates signal transduction pathways. Blocking protein kinase C activation did not prevent asbestos-induced permeability; however, blocking tyrosine kinase with tyrophostin A25 did prevent asbestos-induced permeability, and blocking tyrosine phosphatase with sodium vanadate enhanced the effect of asbestos. These data demonstrate that asbestos may increase epithelial permeability through nonoxidant pathways that involve tyrosine kinase activation. This model offers an important system for studying pathways involved in regulating lung epithelial permeability.
Glucocorticoid regulation of GM-CSF: evidence for transcriptional mechanisms in airway epithelial cells
Authors: Adkins Karissa K. , Levan Tricia D. , Miesfeld Roger L. , Bloom John W.
Published: December 2017

Inflammation plays a central role in the pathogenesis of asthma. Glucocorticoids are first-line anti-inflammatory therapy in the treatment of asthma and are effective inhibitors of inflammatory cytokines. Clinical data demonstrate that granulocyte-macrophage colony-stimulating factor (GM-CSF) production by airway epithelial cells may be an important target of inhaled glucocorticoid therapy. We examined the regulatory mechanisms of GM-CSF expression by interleukin-1β (IL-1β) and the synthetic glucocorticoid dexamethasone in the BEAS-2B human bronchial epithelial cell line. IL-1β stimulation resulted in a 15-fold induction of GM-CSF protein, which was associated with a corresponding 47-fold maximal induction of GM-CSF mRNA levels. Treatment with the transcriptional inhibitor actinomycin D before IL-1β stimulation completely abolished induction of GM-CSF mRNA, whereas incubation with cycloheximide had no effect. Taken together, these data demonstrate that IL-1β induction of GM-CSF is mediated through transcriptional mechanisms. Dexamethasone treatment of BEAS-2B cells produced an 80% inhibition of IL-1β-induced GM-CSF protein and a 51% inhibition of GM-CSF mRNA. GM-CSF mRNA was rapidly degraded in these cells, and dexamethasone treatment did not significantly affect this decay rate. We conclude that, in the BEAS-2B bronchial epithelial cell line, IL-1β induction and dexamethasone repression of GM-CSF expression are mediated predominantly through transcriptional mechanisms.
Mechanism of extracellular ATP- and adenosine-induced apoptosis of cultured pulmonary artery endothelial cells
Authors: Rounds Sharon , Yee Winnie Lin , Dawicki Doloretta D. , Harrington Elizabeth , Parks Nancy , Cutaia Michael V.
Published: December 2017

Apoptosis may be important in the exacerbation of endothelial cell injury or limitation of endothelial cell proliferation. We have found that extracellular ATP (exATP) and adenosine cause endothelial apoptosis and that the development of apoptosis is linked to intracellular metabolism of adenosine [Dawicki, D. D., D. Chatterjee, J. Wyche, and S. Rounds. Am. J. Physiol. 273 ( Lung Cell Mol. Physiol. 17): L485–L494, 1997]. In the present study, we investigated the mechanism of this effect. We found that exATP, adenosine, and the S-adenosyl-l-homocysteine (SAH) hydrolase inhibitor MDL-28842 caused apoptosis and decreased the ratio of S-adenosyl-l-methionine to SAH compared with untreated control cells. Using release of soluble [3H]thymidine as a measure of DNA fragmentation, we found that the effect of adenosine on soluble DNA release was potentiated by coincubation with homocysteine. These results suggest that the mechanism of exATP- and adenosine-induced endothelial cell apoptosis involves inhibition of SAH hydrolase. exATP-induced apoptosis was enhanced by an inhibitor of adenosine deaminase, whereas exogenous adenosine-induced apoptosis was partially inhibited by an adenosine deaminase inhibitor. These results suggest that adenosine deaminase may also be involved in the mechanism of adenosine-induced endothelial cell apoptosis. Adenosine and MDL-28842 caused intracellular acidosis as assessed with the fluorescent probe 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. The cell-permeant base chloroquine prevented adenosine-induced acidosis but not apoptosis. Thus, although intracellular acidosis is associated with adenosine-induced apoptosis, it is not necessary for this effect. We speculate that exATP- and adenosine-induced endothelial cell apoptosis may be due to an inhibition of methyltransferase(s) activity. Purine-induced endothelial cell apoptosis may be important in limiting endothelial cell proliferation after vascular injury.
IL-8 is one of the major chemokines produced by monkey airway epithelium after ozone-induced injury
Authors: Chang Mary Mann-Jong , Wu Reen , Plopper Charles G. , Hyde Dallas M.
Published: December 2017

A rhesus monkey interleukin (IL)-8 cDNA clone with >94% homology to the human IL-8 gene was isolated by differential hybridization from a cDNA library of distal airways after ozone inhalation. In situ hybridization and immunohistochemistry showed increased IL-8 mRNA and protein levels in epithelial cells at 1 h but not at 24 h after inhalation of ozone. The appearance of IL-8 in airway epithelial cells correlated well with neutrophil influx into airway epithelia and lumens. Air-liquid interface cultures of tracheobronchial epithelial cells were exposed to ozone in vitro. We observed a transient increase in IL-8 secretion in culture medium immediately after ozone exposure and a dose-dependent increase in IL-8 secretion and mRNA production. In vitro neutrophil chemotaxis showed a parallel dose and time profile to epithelial cell secretion of IL-8. Treatment with anti-IL-8 neutralizing antibody blocked >80% of the neutrophil chemotaxis in vitro. These results suggest that IL-8 is a key chemokine in acute ozone-induced airway inflammation in primates.
Activation of MAPKs in human bronchial epithelial cells exposed to metals
Authors: Samet James M. , Graves Lee M. , Quay Jacqueline , Dailey Lisa A. , Devlin Robert B. , Ghio Andrew J. , Wu Weidong , Bromberg Philip A. , Reed William
Published: December 2017

We have previously shown that in vitro exposure to metallic compounds enhances expression of interleukin (IL)-6, IL-8, and tumor necrosis factor-α in human bronchial epithelial cells. To characterize signaling pathways involved in metal-induced expression of inflammatory mediators and to identify metals that activate them, we studied the effects of As, Cr, Cu, Fe, Ni, V, and Zn on the mitogen-activated protein kinases (MAPK) extracellular receptor kinase (ERK), c-Jun NH2-terminal kinase (JNK), and P38 in BEAS cells. Noncytotoxic concentrations of As, V, and Zn induced a rapid phosphorylation of MAPK in BEAS cells. Activity assays confirmed marked activation of ERK, JNK, and P38 in BEAS cells exposed to As, V, and Zn. Cr and Cu exposure resulted in a relatively small activation of MAPK, whereas Fe and Ni did not activate MAPK under these conditions. Similarly, the transcription factors c-Jun and ATF-2, substrates of JNK and P38, respectively, were markedly phosphorylated in BEAS cells treated with As, Cr, Cu, V, and Zn. The same acute exposure to As, V, or Zn that activated MAPK was sufficient to induce a subsequent increase in IL-8 protein expression in BEAS cells. These data suggest that MAPK may mediate metal-induced expression of inflammatory proteins in human bronchial epithelial cells.
Differential regulation of eotaxin expression by TNF-α and PMA in human monocytic U-937 cells
Authors: Nakamura Hidetoshi , Haley Kathleen J. , Nakamura Toshiko , Luster Andrew D. , Lilly Craig M.
Published: December 2017

Regulation of eotaxin expression was investigated in U-937 cells, a human monocyte-like cell line. Eotaxin mRNA was induced by tumor necrosis factor-α (TNF-α; 0.1–100 ng/ml) and phorbol 12-myristate 13-acetate (PMA; 0.01–1 μM). PMA-induced eotaxin mRNA expression was of greater magnitude and was maximal at a later time point than TNF-α-induced expression (16 h vs. 2 h after stimulation), which was consistent with eotaxin protein expression detected by immunocytochemistry. Dexamethasone (0.01–10 μM) decreased eotaxin mRNA expression in both TNF-α- and PMA-stimulated U-937 cells. PMA-induced eotaxin mRNA expression was inhibited by cycloheximide (10 μg/ml), whereas TNF-α-induced expression was not. The protein kinase C (PKC) inhibitor staurosporine (10–50 nM) inhibited PMA-induced eotaxin mRNA expression, whereas TNF-α-induced expression was enhanced by this reagent. These results suggest that eotaxin expression can be induced by more than one mechanism: the PMA-triggered pathway is mediated by PKC activation and requires new protein synthesis, whereas the TNF-α-triggered pathway is independent of PKC and protein synthesis. TNF-α- and PMA-induced pathways are both associated with nuclear factor-κB, because its binding activity was enhanced in the presence of these stimuli, and both pathways were limited by its inhibitor, diethyldithiocarbamate.
C-type natriuretic peptide expression and pulmonary vasodilation in hypoxia-adapted rats
Authors: Klinger James R. , Siddiq Farjaad M. , Swift Richard A. , Jackson Cynthia , Pietras Linda , Warburton Rod R. , Alia Christopher , Hill Nicholas S.
Published: December 2017

Atrial and brain natriuretic peptides (ANP and BNP, respectively) are potent pulmonary vasodilators that are upregulated in hypoxia-adapted rats and may protect against hypoxic pulmonary hypertension. To test the hypothesis that C-type natriuretic peptide (CNP) also modulates pulmonary vascular responses to hypoxia, we compared the vasodilator effect of CNP with that of ANP on pulmonary arterial rings, thoracic aortic rings, and isolated perfused lungs obtained from normoxic and hypoxia-adapted rats. We also measured CNP and ANP levels in heart, lung, brain, and plasma in normoxic and hypoxia-adapted rats. Steady-state CNP mRNA levels were quantified in the same organs by relative RT-PCR. CNP was a less potent vasodilator than ANP in preconstricted thoracic aortic and pulmonary arterial rings and in isolated lungs from normoxic and hypoxia-adapted rats. Chronic hypoxia increased plasma CNP (15 ± 2 vs. 6 ± 1 pg/ml; P < 0.05) and decreased CNP in the right atrium (35 ± 14 vs. 65 ± 17 pg/mg protein; P < 0.05) and in the lung (3 ± 1 vs. 14 ± 3 pg/mg protein; P < 0.05) but had no effect on CNP in brain or right ventricle. Chronic hypoxia increased ANP levels fivefold in the right ventricle (49 ± 5 vs. 11 ± 2 pg/mg protein; P < 0.05) but had no effect on ANP in lung or brain. There was a trend toward decreased ANP levels in the right atrium (2,009 ± 323 vs. 2,934 ± 397 pg/mg protein; P = not significant). No differences in CNP transcript levels were observed between the two groups of rats except that the right atrial CNP mRNA levels were lower in hypoxia-adapted rats. We conclude that CNP is a less potent pulmonary vasodilator than ANP in normoxic and hypoxia-adapted rats and that hypoxia raises circulating CNP levels without increasing cardiopulmonary CNP expression. These findings suggest that CNP may be less important than ANP or BNP in protecting against hypoxic pulmonary hypertension in rats.
Human surfactant proteins A1 and A2 are differentially regulated during development and by soluble factors
Authors: Scavo Louis M. , Ertsey Robert , Gao Bi Qi
Published: December 2017

An RT-PCR method for the relative quantitation of the mRNAs for human surfactant protein (SP) A1 and SP-A2 was developed, verified, and then utilized to determine the relative levels of these mRNAs in fetal and adult lung samples in vivo, as well as in cultured human fetal lung explants and H441 cells. For the cultured tissue and cells, we assessed the effects of a variety of soluble factors known to modulate total SP-A. Comprehensive analysis revealed many significant findings, including the following: both mRNAs were expressed as early as 15 wk of gestation; throughout midgestation, SP-A1 was present at higher levels than SP-A2, with an average ratio of 30:1. In the adult lung, SP-A1 mRNA was present at lower levels than SP-A2, with a ratio of 0.4:1, whereas in H441 cells, the ratio was 0.85:1. In fetal lung cultured for 4 days, both mRNAs increased, with a greater increase in SP-A2 (97-fold) than in SP-A1 (15-fold), resulting in a final ratio of 4:1. Differential regulation was demonstrated for 8-(4-chlorophenylthio)-cAMP, interferon (IFN)-γ, tumor necrosis factor-α, and transforming growth factor (TGF)-β in the human fetal lung explant system, with SP-A2 being more affected, and for IFN-γ and TGF-β in the H441 cells, where SP-A1 showed greater regulation. Of the soluble factors tested, IFN-γ and TGF-β had the most potent and consistent effects in both systems.
O2-sensitive K+channels in neuroepithelial body-derived small cell carcinoma cells of the human lung
Authors: O’Kelly I. , Peers C. , Kemp P. J.
Published: December 2017

Neuroepithelial bodies act as airway O2sensors, but studies of their activity at the cellular level have been severely limited because they are present at such a low density in lung tissue. Small cell lung carcinoma (SCLC) cells are believed to be derived from neuroepithelial body cells and may represent a model system for investigating the mechanisms of airway chemoreception. Here we have used the whole cell patch-clamp technique to investigate the effects of acute hypoxia on voltage-gated ionic currents and membrane potential in H-146 SCLC cells. Step depolarizations evoked transient inward currents due to activation of Na+and Ca2+channels, followed by outward K+currents. K+currents were partially inhibited by 200 μM Cd2+(indicative of the presence of a Ca2+-dependent component of the K+current) and were inhibited by tetraethylammonium (TEA) in a concentration-dependent manner, although even at 100 mM TEA, a residual K+current could be detected. Hypoxia ([Formula: see text] 15–20 mmHg) caused a reversible inhibition of outward K+currents without affecting inward currents. Inhibition by hypoxia was also observed in the presence of Cd2+. Hypoxia and TEA caused membrane depolarization in H-146 cells, and their effects appeared additive. These findings indicate that H-146 cells possess O2-sensitive, Ca2+-independent K+channels that can influence cell membrane potential. SCLC cells may, therefore, represent a good model for investigating the mechanisms underlying O2sensing by airway chemoreceptor cells.
Hydrolysis of surfactant-associated phosphatidylcholine by mammalian secretory phospholipases A2
Authors: Hite R. Duncan , Seeds Michael C. , Jacinto Randy B. , Balasubramanian R. , Waite Moseley , Bass David
Published: December 2017

Hydrolysis of surfactant-associated phospholipids by secretory phospholipases A2is an important potential mechanism for surfactant dysfunction in inflammatory lung diseases. In these conditions, airway secretory phospholipase A2(sPLA2) activity is increased, but the type of sPLA2and its impact on surfactant function are not well understood. We examined in vitro the effect of multiple secretory phospholipases A2on surfactant, including their ability to 1) release free fatty acids, 2) release lysophospholipids, and 3) increase the minimum surface tension (γmin) on a pulsating bubble surfactometer. Natural porcine surfactant and Survanta were exposed to mammalian group I (recombinant porcine pancreatic) and group II (recombinant human) secretory phospholipases A2. Our results demonstrate that mammalian group I sPLA2hydrolyzes phosphatidylcholine (PC), producing free fatty acids and lysophosphatidylcholine, and increases γmin. In contrast, mammalian group II sPLA2demonstrates limited hydrolysis of PC and does not increase γmin. Group I and group II secretory phospholipases A2from snake venom hydrolyze PC and inhibit surfactant function. In summary, mammalian secretory phospholipases A2from groups I and II differ significantly from each other and from snake venom in their ability to hydrolyze surfactant-associated PC.
Tobacco smoke induces both apoptosis and necrosis in mammalian cells: differential effects of HSP70
Authors: Vayssier Muriel , Banzet Nathalie , François Dominique , Bellmann Kerstin , Polla Barbara S.
Published: December 2017

Tobacco smoke (TS) has been implicated as a major risk factor in human pulmonary diseases including cancer. In this study, we used TS as a model of oxidative stress. TS-mediated oxidative stress has been shown to induce protein oxidation, DNA damage, and cell death. Here we investigated, in human and rodent cell lines, whether TS induces cell death by apoptosis or by necrosis. As described for classic oxidants, TS induced apoptosis at low concentrations and necrosis at higher concentrations. We have previously described the induction of heat shock (HS) protein (HSP) (in particular, HSP70) in human monocytes exposed to TS. HSP70 is implicated in the regulation of cell injury and cell death and, in particular, modulates apoptosis, as does the antiapoptotic oncoprotein Bcl-2. At both apoptotic and necrotic concentrations, TS induced a dose-dependent HSP70 expression, whereas Bcl-2 was induced only at necrotic concentrations. TS- or HS-induced HSP had no protective effects either on apoptosis or on necrosis, but HSP70 overexpression prevented TS-induced necrosis and consequently led to increased apoptosis. These results might reconcile the apparently contradictory data previously reported on the effects of HSP on apoptosis.
Immunotargeting of catalase to ACE or ICAM-1 protects perfused rat lungs against oxidative stress
Authors: Atochina Elena N. , Balyasnikova Irina V. , Danilov Sergei M. , Granger D. Neil , Fisher Aron B. , Muzykantov Vladimir R.
Published: December 2017

The pulmonary endothelium is susceptible to oxidative insults. Catalase conjugated with monoclonal antibodies (MAbs) against endothelial surface antigens, angiotensin-converting enzyme (MAb 9B9) or intercellular adhesion molecule-1 (MAb 1A29), accumulates in the lungs after systemic injection in rats (V. Muzykantov, E. Atochina, H. Ischiropoulos, S. Danilov, and A. Fisher. Proc. Natl. Acad. Sci. USA 93: 5213–5218, 1996). The present study characterizes the augmentation of antioxidant defense by these antibody-catalase conjugates in isolated rat lungs perfused for 1 h with catalase conjugated with either MAb 9B9, MAb 1A29, or control mouse IgG. Approximately 20% of the injected dose of Ab-125I-catalase accumulated in the perfused rat lungs (vs. <5% for IgG-125I-catalase). After elimination of nonbound material, the lungs were perfused further for 1 h with 5 mM hydrogen peroxide (H2O2). H2O2induced an elevation in tracheal and pulmonary arterial pressures (126 ± 7 and 132 ± 5%, respectively, of the control level), lung wet-to-dry weight ratio (7.1 ± 0.4 vs. 6.0 ± 0.01 in the control lungs), and ACE release into the perfusate (436 ± 20 vs. 75 ± 7 mU in the control perfusates). Both MAb 9B9-catalase and MAb 1A29-catalase significantly attenuated the H2O2-induced elevation in 1) angiotensin-converting enzyme release to the perfusate (215 ± 14 and 217 ± 38 mU, respectively), 2) lung wet-to-dry ratio (6.25 ± 0.1 and 6.3 ± 0.3, respectively), 3) tracheal pressure (94 ± 4 and 101 ± 4%, respectively, of the control level), and 4) pulmonary arterial pressure (103 ± 3 and 104 ± 7%, respectively, of the control level). Nonconjugated catalase, nonconjugated antibodies, nonspecific IgG, and IgG-catalase conjugate had no protective effect, thus confirming the specificity of the effect of MAb-catalase. These results support a strategy of catalase immunotargeting for protection against pulmonary oxidative injury.
Increased expression of heat shock protein-70 protects A549 cells against hyperoxia
Authors: Wong Hector R. , Menendez Ingrid Y. , Ryan Marnie A. , Denenberg Alvin G. , Wispé Jonathan R.
Published: December 2017

Acute and chronic lung injury secondary to hyperoxia remains an important complication in critically ill patients, and, consequently, there is interest in developing strategies to protect the lung against hyperoxia. Heat shock proteins (HSPs) confer protection against a broad array of cytotoxic agents. In this study, we tested the hypothesis that increased expression of the 70-kDa HSP (HSP70) would protect cultured human respiratory epithelium against hyperoxia. Recombinant A549 cells were generated in which human HSP70 was increased by stable transfection with a plasmid containing human HSP70 cDNA under control of the cytomegalovirus promoter (A549-HSP70 cells). A549-HSP70 cells exposed to hyperoxia had greater acute survival rates and clonogenic capacity compared with wild-type A549 cells and with control cells stably transfected with the empty expression plasmid. Hyperoxia-mediated lipid peroxidation and ATP depletion were also attenuated in A549-HSP70 cells exposed to hyperoxia. Increased expression of HSP70 did not detectably alter mRNA levels of the intracellular antioxidants manganese superoxide dismutase, catalase, and glutathione peroxidase. Collectively, these data demonstrate a specific in vitro protective role for HSP70 against hyperoxia and suggest that potential mechanisms of protection involve attenuation of hyperoxia-mediated lipid peroxidation and ATP depletion.
Refilling of caffeine-sensitive intracellular calcium stores in bovine airway smooth muscle cells
Authors: Madison J. Mark , Ethier Michael F. , Yamaguchi Hiroshi
Published: December 2017

The goal of this study was to assess the mechanisms by which the caffeine-sensitive calcium stores of airway smooth muscle cells are refilled. Bovine trachealis cells were loaded with fura 2-AM (0.5 μM) for imaging of cytosolic calcium concentrations ([Ca2+]i) in the inner cytosol. After a first stimulation (S1) with caffeine, the response to a second stimulation (S2) depended on the presence of extracellular calcium during an intervening 80-s-long refilling phase. The S2-to-S1 ratio (S2/S1) was 0.11 ± 0.05 ( n = 13 cells) during calcium-free refilling but 0.72 ± 0.04 ( n = 36 cells) within 80 s of exposure to extracellular calcium. Maximum mean [Ca2+]iduring the 80 s of refilling was not different for calcium-free (116 ± 19 nM; n = 13 cells) versus extracellular calcium plus nickel (2 mM) (121 ± 12 nM; n = 21 cells); despite this, significantly greater refilling (S2/S1 0.58 ± 0.06; n = 24 cells) occurred in the presence of extracellular calcium plus nickel. The protein tyrosine kinase inhibitors genistein (100 μM) and ST-638 (50 μM) significantly decreased refilling over 80 s (S2/S1 0.35 ± 0.06, n = 14 cells and 0.51 ± 0.07, n = 14 cells, respectively). Daidzein (100 μM) had no effect on S2/S1. We concluded that [Ca2+]iof the inner cytosol during refilling correlated poorly with S2/S1 values and that, therefore, additional compartments not well detected by fura 2 contribute to refilling. The findings suggest that calcium influx for refilling is segregated from the inner cytosol of the cell, relatively insensitive to nickel, and regulated or modulated by protein tyrosine kinase activity.
γ-Glutamylcysteine synthetase: mRNA stabilization and independent subunit transcription by 4-hydroxy-2-nonenal
Authors: Liu Rui-Ming , Gao Lin , Choi Jinah , Forman Henry Jay
Published: December 2017

γ-Glutamylcysteine synthetase (GCS), the rate-limiting enzyme in de novo glutathione (GSH) synthesis, is composed of one catalytic (heavy) and one regulatory (light) subunit. Although both subunits are increased at the mRNA level by oxidants, it is not clear whether they are regulated through the same mechanism. 4-Hydroxy-2-nonenal (4HNE), a lipid peroxidation product, may act as a mediator for the induction of gene expression by oxidants. In the present study, 4HNE was used to study the mechanism of induction of the two GCS subunits in rat lung epithelial L2 cells. 4HNE increased both the transcription rates and the stability of mRNA for both GCS subunits, resulting in an increased mRNA content for both subunits. Both GCS subunit proteins and enzymatic activities also increased. Emetine, a protein synthesis inhibitor, blocked the increase in GCS light subunit mRNA but not the increase in GCS heavy subunit mRNA. This suggested that although 4HNE increased transcription and stabilization of both GCS subunit mRNAs, the signaling pathways involved in the induction of the two GCS subunits differed.
Flavonoids stimulate Cl conductance of human airway epithelium in vitro and in vivo
Authors: Illek Beate , Fischer Horst
Published: December 2017

The ability of the flavonoids genistein, apigenin, kaempferol, and quercetin to activate cystic fibrosis transmembrane conductance regulator-mediated Cl currents in human airway epithelium was investigated. We used the patch-clamp technique on single Calu-3 cells, transepithelial measurements in Calu-3 monolayers, and in vivo measurements of nasal potential difference. All flavonoids stimulated Cl currents in transepithelial experiments dose dependently. Half-maximal stimulatory concentrations were kaempferol (5.5 ± 1.7 μM) ≤ apigenin (11.2 ± 2.1 μM) ≤ genistein (13.6 ± 3.5 μM) ≤ quercetin (22.1 ± 4.5 μM). Stimulation of monolayers with forskolin significantly increased their sensitivity to flavonoids: kaempferol (2.5 ± 0.7 μM) ≤ apigenin (3.4 ± 0.9 μM) ≤ quercetin (4.1 ± 0.7 μM) ≤ genistein (6.9 ± 2.2 μM). Forskolin pretreatment significantly reduced the Hill coefficient ( nH) for all flavonoids. Control monolayers showed nH= 2.00 ± 0.21 (all flavonoids combined), and forskolin-stimulated monolayers showed nH= 1.07 ± 0.07, which was not different among the flavonoids. These data imply that the activation kinetics and the binding site(s) for flavonoids were significantly altered by forskolin stimulation. In whole cell patch-clamp experiments, maximal flavonoid-stimulated currents (percentage of forskolin-stimulated currents) were apigenin (429 ± 86%) ≥ kaempferol (318 ± 45%) ≥ genistein (258 ± 20%) = quercetin (256 ± 26%). Stimulation of the currents was caused by an increase in channel open probability. No other Cl conductances contributed significantly to the flavonoid-activated Cl currents in Calu-3 cells. In vivo, flavonoids significantly stimulated nasal potential difference by, on average, 27.8% of isoproterenol responses.
Chronic pulmonary hypertension increases fetal lung cGMP phosphodiesterase activity
Authors: Hanson Kimberly A. , Ziegler James W. , Rybalkin Sergei D. , Miller Jim W. , Abman Steven H. , Clarke William R.
Published: December 2017

An experimental ovine fetal model for perinatal pulmonary hypertension of the neonate (PPHN) was characterized by altered pulmonary vasoreactivity and structure. Because past studies had suggested impaired nitric oxide-cGMP cascade in this experimental model, we hypothesized that elevated phosphodiesterase (PDE) activity may contribute to altered vascular reactivity and structure in experimental PPHN. Therefore, we studied the effects of the PDE inhibitors zaprinast and dipyridamole on fetal pulmonary vascular resistance and PDE5 activity, protein, mRNA, and localization in normal and pulmonary hypertensive fetal lambs. Infusion of dipyridamole and zaprinast lowered pulmonary vascular resistance by 55 and 35%, respectively, in hypertensive animals. In comparison with control animals, lung cGMP PDE activity was elevated in hypertensive fetal lambs (150%). Increased PDE5 activity was not associated with either an increased PDE5 protein or mRNA level. Immunocytochemistry demonstrated that PDE5 was localized to vascular smooth muscle. We concluded that PDE5 activity was increased in experimental PPHN, possibly by posttranslational phosphorylation. We speculated that these increases in cGMP PDE activity contributed to altered pulmonary vasoreactivity in experimental perinatal pulmonary hypertension.
TGF-β1 inhibits surfactant component expression and epithelial cell maturation in cultured human fetal lung
Authors: Beers Michael F. , Solarin Kola O. , Guttentag Susan H. , Rosenbloom Joel , Kormilli Annapurna , Gonzales Linda W. , Ballard Philip L.
Published: December 2017

Transforming growth factor-β1 (TGF-β1) is a multifunctional cytokine shown to play a critical role in organ morphogenesis, development, growth regulation, cellular differentiation, gene expression, and tissue remodeling after injury. We examined the effect of exogenously administered TGF-β1 on the expression of surfactant proteins (SPs) and lipids, fatty acid synthetase, and ultrastructural morphology in human fetal lung cultured for 5 days with and without dexamethasone (10 nM). Expression of the type II cell-specific marker surfactant proprotein C (proSP-C), studied by [35S]Met incorporation and immunoprecipitation, increased sevenfold with dexamethasone treatment. TGF-β1 (0.1–100 ng/ml) in the presence of dexamethasone inhibited 21-kDa proSP-C expression in a dose-dependent manner (maximal inhibition 31% of control level at 100 ng/ml). There was no change in [35S]Met incorporation into total protein in any of the treatment groups vs. the control group. In immunoblotting experiments, TGF-β1 blocked culture-induced accumulation of SP-A and SP-B. Under the same conditions, TGF-β1 reduced mRNA content for SP-A, SP-B, and SP-C to 20, 38, and 41%, respectively, of matched control groups but did not affect levels of β-actin mRNA. SP transcription rates after 24 h of exposure to TGF-β1 were reduced to a similar extent (20–50% of control level). In both control and dexamethasone-treated explants, TGF-β1 (10 ng/ml) also decreased fatty acid synthetase mRNA, protein, and enzyme activity and the rate of [3H]choline incorporation into phosphatidylcholine. By electron microscopy, well-differentiated type II cells lining potential air spaces were present in explants cultured with dexamethasone, whereas exposure to TGF-β1 with or without dexamethasone resulted in epithelial cells lacking lamellar bodies. We conclude that exogenous TGF-β1 disrupts culture-induced maturation of fetal lung epithelial cells and inhibits expression of surfactant components through effects on gene transcription.