bluuebunny/crossref_metadata_2025 huggingface dataset:
Keratinocyte growth factor promotes alveolar epithelial cell DNA repair after H2O2exposure
Authors: Wu Kristina I. , Pollack Nani , Panos Ralph J. , Sporn Peter H. S. , Kamp David W.
Published: December 2017

Alveolar epithelial cell (AEC) injury and repair are important in the pathogenesis of oxidant-induced lung damage. Keratinocyte growth factor (KGF) prevents lung damage and mortality in animals exposed to various forms of oxidant stress, but the protective mechanisms are not yet established. Because DNA strand break (DNA-SB) formation is one of the earliest cellular changes that occurs after cells are exposed to an oxidant stress, we determined whether KGF reduces H2O2-induced pulmonary toxicity by attenuating AEC DNA damage. KGF (10–100 ng/ml) decreased H2O2(0.05–0.5 mM)-induced DNA-SB formation in cultured A549 and rat alveolar type II cells measured by an alkaline unwinding, ethidium bromide fluorometric technique. The protective effects of KGF were independent of alterations in catalase, glutathione (GSH), or the expression of bcl-2 and bax, two protooncogenes known to regulate oxidant-induced apoptosis. Actinomycin D and cycloheximide abrogated protective effects of KGF. Furthermore, protection by KGF was completely blocked by 1) genistein, a tyrosine kinase inhibitor; 2) staurosporine and calphostin C, protein kinase C (PKC) inhibitors; and 3) aphidicolin, butylphenyl dGTP, and 2′,3′-dideoxythymidine 5′-triphosphate, inhibitors of DNA polymerase. We conclude that KGF attenuates H2O2-induced DNA-SB formation in cultured AECs by mechanisms that involve tyrosine kinase, PKC, and DNA polymerases. These data suggest that the ability of KGF to protect against oxidant-induced lung injury is partly due to enhanced AEC DNA repair.
Captopril inhibits apoptosis in human lung epithelial cells: a potential antifibrotic mechanism
Authors: Uhal Bruce D. , Gidea Claudia , Bargout Raed , Bifero Antonio , Ibarra-Sunga Olivia , Papp Michael , Flynn Kevin , Filippatos Gerasimos
Published: December 2017

The angiotensin-converting enzyme inhibitor captopril has been shown to inhibit fibrogenesis in the lung, but the mechanisms underlying this action are unclear. Apoptosis of lung epithelial cells is believed to be involved in the pathogenesis of pulmonary fibrosis. For these reasons, we studied the effect of captopril on Fas-induced apoptosis in a human lung epithelial cell line. Monoclonal antibodies that activate the Fas receptor induced epithelial cell apoptosis as detected by chromatin condensation, nuclear fragmentation, DNA fragmentation, and increased activities of caspase-1 and -3. Apoptosis was not induced by isotype-matched nonimmune mouse immunoglobulins or nonactivating anti-Fas monoclonal antibodies. When applied simultaneously with anti-Fas antibodies, 50 ng/ml of captopril completely abrogated apoptotic indexes based on morphology, DNA fragmentation, and inducible caspase-1 activity and significantly decreased the inducible activity of caspase-3. Inhibition of apoptosis by captopril was concentration dependent, with an IC50 of 70 pg/ml. These data suggest that the inhibitory actions of captopril on pulmonary fibrosis may be related to prevention of lung epithelial cell apoptosis.
Maternal exposure to environmental tobacco smoke alters Clara cell secretory protein expression in fetal rat lung
Authors: Ji Chun-Mei , Royce Fred H. , Truong Uyen , Plopper Charles G. , Singh Gurmukh , Pinkerton Kent E.
Published: December 2017

We have previously demonstrated that aged and diluted sidestream cigarette smoke (ADSS) alters the development of bronchiolar epithelial cells in postnatal animals (C. M. Ji, C. G. Plopper, H. P. Witschi, and K. E. Pinkerton. Am. J. Respir. Cell Mol. Biol. 11: 312–320, 1994). This study was designed to examine the effects of maternal exposure to ADSS on the development of fetal Clara cells in rats with Clara cell 10-kDa protein (CC10; also designated Clara cell secretory protein) and CC10 mRNA as differentiation markers. Immunohistochemistry, Northern blots, and in situ hybridization were used to determine the abundance and distribution of CC10 at gestational days 14, 18, and 21. CC10 and CC10 mRNA were absent at gestational day 14 but were detectable at gestational day 18 and further increased by gestational day 21. Maternal exposure to ADSS was found to significantly increase fetal expression of CC10 and CC10 mRNA by gestational day 21 but not by gestational day 14 or 18. These findings demonstrate that in utero exposure to ADSS alters the normal developmental expression of CC10 in the fetal rat lung.
Glutamine synthetase expression in rat lung is regulated by protein stability
Authors: Labow Brian I. , Abcouwer Steve F. , Lin Cheng-Mao , Souba Wiley W.
Published: December 2017

During physiological stress, the lung increases production of the amino acid glutamine (Gln) using the enzyme Gln synthetase (GS) to maintain Gln homeostasis. Glucocorticoid hormones are considered the principal mediators of GS expression during stress. However, whereas animal studies have shown that glucocorticoids increase lung GS mRNA levels 500–700%, GS activity levels rise only 20–45%. This discrepancy suggests that a posttranscriptional control mechanism(s) ultimately determines GS expression. We hypothesized that the level of GS protein in the lung is governed by the intracellular Gln concentration through a mechanism of protein destabilization, a feedback regulatory mechanism that has been observed in vitro. To test this hypothesis, Sprague-Dawley rats were treated with a Gln-free diet and the GS inhibitor methionine sulfoximine (MSO) to deplete tissue Gln levels and prevent this feedback regulation. Exposure to Gln-free chow and MSO (100 mg/kg body wt) for 6 days decreased plasma Gln levels 50% ( P < 0.01) and decreased lung tissue Gln levels by 70% ( P < 0.01). Although lung GS mRNA levels were not influenced by Gln depletion, there was a sevenfold ( P < 0.01) increase in GS protein. A parenteral Gln infusion (200 mM, 1.5 ml/h) for the last 2 days of MSO treatment replenished lung Gln levels to 65% of control level and blunted the increase in GS protein levels by 33% ( P < 0.05) compared with rats receiving an isomolar glycine solution. The acute effects of glucocorticoid and MSO administration on lung GS expression were also measured. Whereas dexamethasone (0.5 mg/kg) and MSO injections individually augmented lung GS protein levels twofold and fourfold ( P < 0.05), respectively, the combination of dexamethasone and MSO produced a synergistic, 12-fold induction ( P < 0.01) in lung GS protein over 8 h. The data suggest that, whereas glucocorticoids are potent mediators of GS transcriptional activity, protein stability greatly influences the ultimate expression of GS in the lung.
Molecular cloning, characterization, and differential expression pattern of mouse lung surfactant convertase
Authors: Krishnasamy S. , Teng A. L. , Dhand R. , Schultz R. M. , Gross N. J.
Published: December 2017

We recently reported the purification and partial amino acid sequence of “surfactant convertase,” a 72-kDa glycoprotein involved in the extracellular metabolism of lung surfactant (S. Krishnasamy, N. J. Gross, A. L. Teng, R. M. Schultz, and R. Dhand. Biochem. Biophys. Res. Commun. 235: 180–184, 1997). We report here the isolation of a cDNA clone encoding putative convertase from a mouse lung cDNA library. The cDNA spans a 1,836-bp sequence, with an open reading frame encoding 536 amino acid residues in the mature protein and an 18-amino acid signal peptide at the NH2terminus. The deduced amino acid sequence matches the four partial amino acid sequences (68 residues) that were previously obtained from the purified protein. The deduced amino acid sequence contains an 18-amino acid residue signal peptide, a serine active site consensus sequence, a histidine consensus sequence, five potential N-linked glycosylation sites, and a COOH-terminal secretory-type sequence His-Thr-Glu-His-Lys. Primer-extension analysis revealed that transcription starts 29 nucleotides upstream from the start codon. Northern blot analysis of RNA isolated from various mouse organs showed that convertase is expressed in lung, kidney, and liver as a 1,800-nucleotide-long transcript. The nucleotide and amino acid sequences of putative convertase are 98% homologous with mouse liver carboxylesterase. It thus may be the first member of the carboxylesterase family (EC 3.1.1.1 ) to be expressed in lung parenchyma and the first with a known physiological function.
Sodium hydrosulfite contractions of smooth muscle are calcium and myosin phosphorylation independent
Authors: Yu Ming-Fu , Gorenne Isabelle , Su Xiaoling , Moreland Robert S. , Kotlikoff Michael I.
Published: December 2017

In an effort to further understand the processes underlying hypoxic pulmonary vasoconstriction, we examined the mechanism by which sodium hydrosulfite (Na2S2O4), a potent reducing agent and oxygen scavenger, induces smooth muscle contraction. In rat pulmonary arterial strips, sodium hydrosulfite (10 mM) induced contractions that were 65.9 ± 12.8% of the response to 60 mM KCl ( n = 9 segments). Contractions were not inhibited by nisoldipine (5 μM) or by repeated stimulation with caffeine (10 mM), carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (10 μM), or cyclopiazonic acid (10 μM), all of which eliminated responses to contractile agonists. Maximum force generation after exposure to sodium hydrosulfite was 0.123 ± 0.013 mN in the presence of 1.8 mM calcium and 0.127 ± 0.015 mN in the absence of calcium. Sodium hydrosulfite contractions in pulmonary arterial segments were not due to the generation of H2O2and occurred in the presence of chelerythrine (10 μM), which blocked phorbol ester contractions, and solution hyperoxygenation. Similar contractile responses were obtained in rat aortic and tracheal smooth muscles. Finally, contractions occurred in the complete absence of an increase in myosin light chain phosphorylation. Therefore sodium hydrosulfite-induced smooth muscle contraction is not specific to pulmonary arterial smooth muscle, is independent of calcium and myosin light chain phosphorylation, and is not mediated by either hypoxia or protein kinase C.
Cell size, cell cycle, and α-smooth muscle actin expression by primary human lung fibroblasts
Authors: Uhal Bruce D. , Ramos Carlos , Joshi Iravati , Bifero Antonio , Pardo Annie , Selman Moises
Published: December 2017

Primary human lung fibroblasts were separated into small ( group I), intermediate ( group II), and large ( group III) subpopulations by unit gravity sedimentation (1 G). The three subsets retained differences in cell size for up to 15 days of primary culture. Flow cytometric (fluorescence-activated cell sorter) measurements of forward-angle light scatter agreed well with fibroblast volume measured by image analysis and confirmed the utility of forward-angle light scatter for discriminating size subpopulations. Group II fibroblasts accumulated most rapidly by 8 days of culture and also contained the greatest proportion of S and G2/M phase cells as determined by fluorescence-activated cell sorter. Fibroblasts that were immunoreactive with antibodies to α-smooth muscle actin (α-SMA) were found only in group III. In situ end labeling of fragmented DNA detected apoptotic cells in both groups II and III, but double labeling for in situ end labeling and α-SMA revealed apoptotic cells in both the α-SMA-positive and -negative populations. These results demonstrate that primary human lung fibroblasts behave as predicted by classic models of cell cycle progression and differentiation. However, they do not support the hypothesis that the expression of α-actin is related to apoptosis. We also describe a simple and reproducible method for the high-yield isolation of human lung fibroblast subsets of differing proliferative potential and phenotype.
A maturational shift in pulmonary K+ channels, from Ca2+ sensitive to voltage dependent
Authors: Reeve Helen L. , Weir E. Kenneth , Archer Stephen L. , Cornfield David N.
Published: December 2017

The mechanism responsible for the abrupt decrease in resistance of the pulmonary circulation at birth may include changes in the activity of O2-sensitive K+ channels. We characterized the electrophysiological properties of fetal and adult ovine pulmonary arterial (PA) smooth muscle cells (SMCs) using conventional and amphotericin B-perforated patch-clamp techniques. Whole cell K+ currents of fetal PASMCs in hypoxia were small and characteristic of spontaneously transient outward currents. The average resting membrane potential (RMP) was −36 ± 3 mV and could be depolarized by charybdotoxin (100 nM) or tetraethylammonium chloride (5 mM; both blockers of Ca2+-dependent K+ channels) but not by 4-aminopyridine (4-AP; 1 mM; blocker of voltage-gated K+ channels) or glibenclamide (10 μM; blocker of ATP-dependent K+channels). In hypoxia, chelation of intracellular Ca2+ by 5 mM 1,2-bis(2-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid further reduced the amplitude of the whole cell K+ current and prevented spontaneously transient outward current activity. Under these conditions, the remaining current was partially inhibited by 1 mM 4-AP. K+ currents of fetal PASMCs maintained in normoxia were not significantly reduced by acute hypoxia. In normoxic adult PASMCs, whole cell K+ currents were large and RMP was −49 ± 3 mV. These 4-AP-sensitive K+ currents were partially inhibited by exposure to acute hypoxia. We conclude that the K+ channel regulating RMP in the ovine pulmonary circulation changes after birth from a Ca2+-dependent K+ channel to a voltage-dependent K+ channel. The maturational-dependent differences in the mechanism of the response to acute hypoxia may be due to this difference in K+ channels.
Nitrated SP-A does not enhance adherence ofPneumocystis cariniito alveolar macrophages
Authors: Zhu Sha , Kachel Diane L. , Martin William J. , Matalon Sadis
Published: December 2017

We investigated whether nitration of surfactant apoprotein (SP) A alters its ability to bind to mannose-containing saccharides on Pneumocystis carinii and its potential role in the mediation of P. carinii adherence to alveolar macrophages. Human SP-A was nitrated by incubation with tetranitromethane at pH 8.0 or synthetic peroxynitrite (ONOO−) at pH 7.4, which resulted in significant nitration of tyrosines in its carbohydrate recognition domain [0.63 ± 0.001 (SE) and 1.25 ± 0.02 mol nitrotyrosine/mol monomeric SP-A, respectively; n = 3 samples]. Binding of SP-A to P. carinii was calcium dependent and competitively inhibited by α-methyl-d-mannopyranoside. Nitration of SP-A by ONOO−or tetranitromethane decreases its binding to P. carinii by increasing its dissociation constant from 7.8 × 10−9to 1.6 × 10−8or 2.4 × 10−8M, respectively, without significantly affecting the number of binding sites (7.1 × 106/ P. carinii organisms, assuming that the native molecular mass of oligomeric SP-A is 650 kDa). Furthermore, ONOO−-nitrated SP-A failed to mediate the adherence and phagocytosis of P. carinii to rat alveolar macrophages as observed with normal SP-A. Binding of SP-A to rat alveolar macrophages was not altered by nitration. These results indicate that nitration of SP-A interferes with its ability to serve as a ligand for P. carinii adherence to alveolar macrophages at the site of the SP-A moleculeP. carinii interaction.
Differential regulation of baboonSP-A1andSP-A2genes: structural and functional analysis of 5′-flanking DNA
Authors: Li Jinxing , Gao Erwei , Seidner Steven R. , Mendelson Carole R.
Published: December 2017

Surfactant protein (SP) A gene transcription is developmentally regulated and stimulated by hormones and factors that increase intracellular cAMP. The baboon (b) genome contains two highly similar SP-A genes, bSP-A1 and bSP-A2. With the use of a ribonuclease protection assay with gene-specific probes, the two bSP-Agenes were found to be differentially regulated during baboon fetal lung development in that expression of the bSP-A2 gene appeared to be induced to a high level at a later time in gestation than that of the bSP-A1 gene. Both the bSP-A1 and bSP-A2 genes were found to be highly responsive to the inductive effects of cAMP in baboon fetal lung explants in culture. By DNase I footprinting and electrophoretic mobility shift assays with bacterially expressed thyroid transcription factor-1 (TTF-1) and type II cell nuclear extracts, three TTF-1 binding elements were identified within the 255-bp region flanking the 5′-end of each bSP-A gene; however, these differed in position and spacing for the two bSP-A genes. To functionally define the genomic regions that are required for cAMP regulation of bSP-A gene expression in type II cells, fusion genes composed of various amounts of 5′-flanking DNA from the bSP-A1 and bSP-A2 genes linked to the human growth hormone structural gene as a reporter were transfected into type II cells in primary culture. We found that 255 bp of 5′-flanking DNA, which contain three TTF-1 binding elements, from bSP-A1 and bSP-A2 genes were sufficient to mediate high basal and cAMP-inducible expression in type II cells. We also observed that there were no obvious differences in the magnitude of the responses of these fusion genes to cAMP treatment.
Depletion of nitric oxide causes cell cycle alterations, apoptosis, and oxidative stress in pulmonary cells
Authors: Janssen Yvonne M. W. , Soultanakis Rebecca , Steece Katharine , Heerdt Ellen , Singh Ravinder J. , Joseph Joy , Kalyanaraman Balaraman
Published: December 2017

Nitric oxide (NO ⋅) is important in the regulation of mitochondrial function, cell signaling, and gene expression. To elucidate how endogenous NO ⋅ regulates the function of airway epithelial cells, we used carboxy-PTIO, a hydrophilic, negatively charged NO ⋅ trap, to scavenge NO ⋅ from rat lung epithelial (RLE) and rat pleural mesothelial (RPM) cells and to determine the elicitation of cell cycle alterations, apoptosis, and oxidative stress. The reaction of NO ⋅ with PTIO causes the formation of PTI, which is measured by electron spin resonance (ESR) and is a quantitative measure of NO ⋅ formation. ESR spectroscopy revealed the production of NO ⋅ in RLE or RPM cells over a period from 1 to 24 h of exposure, indicating scavenging of NO ⋅ by PTIO. Cycle analyses in confluent RLE or RPM cells revealed two- to threefold increases in S and G2/M phases after exposure to 100–200 μM PTIO as well as increases in the fraction of cells undergoing apoptosis. Direct addition of PTI to cells failed to elicit cell cycle perturbations or apoptosis. The guanylyl cyclase inhibitor ODQ mimicked the effects of PTIO. 8-Bromo-cGMP but not 8-bromo-cAMP ameliorated the PTIO- or ODQ-mediated cell cycle perturbations and apoptosis, suggesting that cGMP-dependent pathways are involved in these cell cycle perturbations. Treatment of log-phase cells with PTIO resulted in more dramatic cell cycle perturbations compared with cells treated at confluence. Assessment of 5-bromo-2′-deoxyuridine incorporation to measure DNA synthesis demonstrated decreases in PTIO-treated compared with sham cells in addition to a cell cycle arrest in late S or G2/M phase. Last, incubation with dichlorofluorescin diacetate revealed oxidative stress in PTIO- but not in PTI-exposed RLE or RPM cells. We conclude that the depletion of endogenous NO ⋅ induces oxidative stress, cell cycle perturbations, and apoptosis. Our findings illustrate the importance of endogenous NO ⋅ in the control of cell cycle progression and survival of pulmonary and pleural cells and that a critical balance between NO ⋅ and superoxide may be necessary for these physiological events.
Silica-induced chemokine expression in alveolar type II cells is mediated by TNF-α
Authors: Barrett Edward G. , Johnston Carl , Oberdörster Günter , Finkelstein Jacob N.
Published: December 2017

Recent evidence has suggested that epithelial cells may contribute to the inflammatory response in the lung after exposure to crystalline silica through the production of and response to specific growth factors, chemokines, and cytokines. However, the exact cellular and molecular responses of epithelial cells to silica exposure remains unclear. Using a murine alveolar type II cell line [murine lung epithelial (MLE)-15 cell line], we measured the early changes in various cytokine and chemokine mRNA species after exposure of the cells to 4–35 μg/cm2of silica (cristobalite), interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and lipopolysaccharide (LPS) alone or in combination. Total mRNA was isolated and assayed with an RNase protection assay after 6 and 24 h of exposure. Cristobalite exposure alone led to an increase in monocyte chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-2, and regulated on activation normal T cells expressed and secreted (RANTES) mRNAs. Treatment with IFN-γ alone increased MCP-1 mRNA levels. Treatment with TNF-α or LPS alone led to an increase in MCP-1 and MIP-2 mRNA. The combination of cristobalite plus TNF-α led to an additive increase in MCP-1 and MIP-2, whereas cristobalite plus IFN-γ or LPS had a synergistic effect. We also found with a TNF-α-neutralizing antibody that TNF-α plays a major role in mediating the type II cell chemokine response to cristobalite exposure. The results indicate that the cristobalite-induced chemokine response in the lung epithelium is mediated in part by TNF-α and can be enhanced by macrophage- and lymphocyte-derived inflammatory mediators in an additive and synergistic fashion.
Changes in alveolar septal border lengths with postnatal lung growth
Authors: Wood Jonathan P. , Kolassa John E. , McBride John T.
Published: December 2017

Evaluation of alveolar development beyond the postnatal period of rapid septation has generally involved alveolar counting. We used an alternate approach to assess postseptation parenchymal development: measurement of the lengths of various types of alveolar septal borders. This technique directly addresses changes in the elastin fiber network that determines parenchymal complexity. Lungs from weanling and adult ferrets, inflated to 15 cmH2O, were perfusion fixed and dehydrated, and 2-μm sections were stained with Miller’s elastin stain for light microscopy. We used standard morphometric methods to measure the lengths of the various types of alveolar septal borders. Three types comprised >90% of all septal borders: 1) free septal ends (“ends”) containing an elastin cable; 2) angled meetings of two alveolar septa (“bends”), also with a cable; and 3) the near-symmetrical intersections of three septa (“junctions”) devoid of elastin. When scaled for lung volume, ends and bends were 23 and 37% greater in adults ( P < 0.001), reflecting the increase in parenchymal complexity with growth. The 17% difference in scaled junction lengths was not significant ( P = 0.10). Bends increased out of proportion to the increase in ends, and both bends and ends increased to a greater degree than any possible increase in junctions ( P < 0.001 for all comparisons). Although the interpretation of changes in the distribution of alveolar border lengths is not straightforward, an increase in bends resulting in an increase in the complexity of individual alveoli may contribute to the increase in alveolar gas-exchanging surface area with growth. Septation, the process responsible for the rapid early postnatal increase in parenchymal complexity in many species, should tend to increase the lengths of ends and junctions and decrease the lengths of bends. Therefore, these data suggest that septation is not the predominant mechanism of later postnatal parenchymal development in the ferret.
Regulation of alveolar macrophage and type II cell DNA synthesis: effects of ozone inhalation
Authors: Prokhorova Svetlana , Patel Naimish , Laskin Debra L.
Published: December 2017

A characteristic reaction of the lung to inhaled ozone is an increase in the number of type II epithelial cells and alveolar macrophages (AMs). In the present study, we analyzed mechanisms regulating this response. Acute exposure of rats to ozone (2 parts/million, 3 h) induced expression of proliferating cell nuclear antigen, a marker of cellular proliferation, in both type II cells and AMs. This was maximum 48 h after ozone inhalation. Type II cells and AMs isolated from treated rats at this time also incorporated significantly more [3H]thymidine ([3H]TdR) than cells from control animals. When type II cells and AMs were cocultured, a synergistic increase in [3H]TdR uptake was observed. This appeared to be due to increased DNA synthesis by both cell types. Thus [3H]TdR incorporation by type II cells and AMs cocultured with mitomycin C-treated AMs and type II cells, respectively, was elevated compared with cells cultured alone. Type II cells and AMs plated onto tissue culture inserts, as well as culture supernatants from these cells, were found to stimulate DNA synthesis in AMs and type II cells, respectively. In addition, crude membrane preparations from these cells exhibited growth-promoting activity. Thus the mitogenic effects of both cell types appeared to be mediated by soluble factors and membrane-associated molecules. Ozone inhalation resulted in an increase in the mitogenic activity of AMs treated with mitomycin C and plated on tissue culture inserts toward type II cells and of type II cell culture supernatants toward AMs. These data suggest that type II cell and AM proliferation contributes to the regulation of the number of cells in the lung under normal homeostatic conditions and after ozone-induced injury. Moreover, type II cells and AMs produce paracrine mediators that contribute to cellular proliferative responses.
Alveolar macrophage apoptosis and TNF-α, but not p53, expression correlate with murine response to bleomycin
Authors: Ortiz Luis A. , Moroz Kryztof , Liu Jing-Yao , Hoyle Gary W. , Hammond Timothy , Hamilton Raymond F. , Holian Andrij , Banks William , Brody Arnold R. , Friedman Mitchell
Published: December 2017

Apoptosis is considered to be a protective mechanism that limits lung injury. However, apoptosis might contribute to the inflammatory burden present in the injured lung. The exposure of mice to bleomycin (BLM) is a well-established model for the study of lung injury. BLM exposure induces DNA damage and enhances tumor necrosis factor (TNF)-α expression in the lung. To evaluate the importance of alveolar macrophage (AM) apoptosis in the pathogenesis of lung injury, we exposed BLM-sensitive (C57BL/6) and BLM-resistant (BALB/c) mice to BLM (120 mg/kg) and studied the induction of apoptosis [by light-microscopy changes (2, 8, 12, 24, 48, and 72 h) and annexin V uptake by flow cytometry (24 h)], the secretion of TNF-α (measured by ELISA), and the expression of p53 (by immunoblotting) in AM retrieved from these mice. BLM, but not vehicle, induced apoptosis in AM from both murine strains. The numbers of apoptotic AM were significantly greater ( P < 0.001) in C57BL/6 mice (52.9%) compared with BALB/c mice (40.8%) as demonstrated by annexin V uptake. BLM induction of apoptosis in AM was preceded by an increased secretion of TNF-α in C57BL/6 but not in BALB/c mice. Furthermore, double TNF-α receptor-deficient mice, developed on a C57BL/6 background, demonstrated significantly ( P < 0.001) lower numbers of apoptotic AM compared with C57BL/6 and BALB/c mice. BLM also enhanced p53 expression in AM from both murine strains. However, p53-deficient mice developed BLM-induced lung injury, exhibited similar lung cell proliferation (measured as proliferating cell nuclear antigen immunostaining), and accumulated similar amounts of lung hydroxyproline (65 ± 6.9 μg/lung) as did C57BL/6 (62 ± 6.5 μg/lung) mice. Therefore, AM apoptosis is occurring during BLM-induced lung injury in a manner that correlates with murine strain sensitivity to BLM. Furthermore, TNF-α secretion rather than p53 expression contributes to the difference in murine strain response to BLM. tumor necrosis factor; strain susceptibility
Hyperoxia increases keratinocyte growth factor mRNA expression in neonatal rabbit lung
Authors: Charafeddine Lama , D’Angio Carl T. , Richards Julie L. , Stripp Barry R. , Finkelstein Jacob N. , Orlowski Craig C. , LoMonaco Michael B. , Paxhia Anna , Ryan Rita M.
Published: December 2017

Acute hyperoxic lung injury remains a major factor in the development of chronic lung disease in neonates. A critical step in the repair of acute lung injury is the proliferation of type II alveolar epithelial cells. Type II cell proliferation is stimulated by keratinocyte growth factor (KGF), an epithelial cell-specific mitogen. We sought to investigate KGF mRNA expression in relation to type II cell proliferation during hyperoxic lung injury. We studied a previously described newborn (NB) rabbit model of acute and chronic hyperoxic injury [C. T. D’Angio, J. N. Finkelstein, M. B. LoMonaco, A. Paxhia, S. A. Wright, R. B. Baggs, R. H. Notter, and R. M. Ryan. Am. J. Physiol. 272 ( Lung Cell. Mol. Physiol. 16): L720–L730, 1997]. NB rabbits were placed in 100% O2for 9 days and then recovered in 60% O2. RT-PCR was used to synthesize and amplify a 267-bp fragment of rabbit KGF cDNA from whole lung RNA. KGF mRNA expression was analyzed by ribonuclease protection assay, and mRNA abundance was quantified by phosphorimaging. Proliferating cell nuclear antigen immunohistochemistry was used on lung sections to identify proliferating cells. The rabbit partial cDNA sequenced was >95% homologous to human cDNA, and all amino acids were conserved. Whole lung KGF mRNA expression was increased 12-fold after 6 days of hyperoxia compared with control lungs, and remained increased throughout the 100% O2exposure period. Proliferating cell nuclear antigen immunohistochemistry showed an increase in type II cell proliferation after 8–12 days of hyperoxia. NB rabbits exposed to hyperoxic injury exhibit increased whole lung KGF mRNA expression preceding type II cell proliferation. KGF may be an important mitogen in the regulation of alveolar epithelial repair after hyperoxic lung injury.
Unaltered vasoconstrictor responsiveness after iNOS inhibition in lungs from chronically hypoxic rats
Authors: Resta Thomas C. , O’Donaughy Theresa L. , Earley Scott , Chicoine Louis G. , Walker Benjimen R.
Published: December 2017

Previous studies suggest that inducible (i) nitric oxide synthase (NOS) expression within the pulmonary vasculature is increased in rats with chronic hypoxia (CH)-induced pulmonary hypertension. We therefore hypothesized that enhanced iNOS expression associated with CH causes attenuated pulmonary vasoconstrictor responsiveness. To test this hypothesis, we examined the effect of selective iNOS blockade withl- N 6-(1-iminoethyl)lysine dihydrochloride (l-NIL) and nonselective NOS inhibition with N ω-nitro-l-arginine (l-NNA) on vasoconstrictor responses to U-46619 in isolated saline-perfused lungs from both control and CH (4 wk at 380 mmHg) rats. We additionally measured pulmonary hemodynamic responses tol-NIL in conscious CH rats (fraction of inspired O2 = 0.12). Finally, iNOS mRNA levels were assessed in lungs from each group of rats using ribonuclease protection assays. Despite a significant increase in iNOS mRNA expression after exposure to CH, responses to U-46619 were unaltered by l-NIL but augmented by l-NNA in lungs from both control and CH rats. Pulmonary hemodynamics were similarly unaltered by l-NIL in conscious CH rats. We conclude that iNOS does not modulate pulmonary vasoconstrictor responsiveness after long-term hypoxic exposure.
Dexamethasone regulation of lung epithelial cell and fibroblast interleukin-11 production
Authors: Wang Jingming , Zhu Zhou , Nolfo Robert , Elias Jack A.
Published: December 2017

Studies were undertaken to define the effects of corticosteroids on stromal cell interleukin (IL)-11 production. Unstimulated A549 epithelial-like cells produced modest amounts of IL-11, and transforming growth factor (TGF)-β1 was a potent, dose-dependent stimulator of A549 cell IL-11 elaboration. Dexamethasone inhibited the levels of basal and TGF-β1-stimulated IL-11 elaboration in a dose-dependent fashion. In the setting of TGF-β1 stimulation, dexamethasone caused a >90% decrease in IL-11 production at 10−6M, a 50% decrease in IL-11 production at ∼1 × 10−9M, and significant inhibition at 10−10M. This dexamethasone-induced inhibition was reversed by the glucocorticoid-receptor antagonist RU-486. Dexamethasone also inhibited respiratory syncytial virus, rhinovirus, and TGF-β1-stimulated IL-11 production by MRC-5 lung fibroblasts. In all cases, dexamethasone caused comparable changes in IL-11 mRNA accumulation. Nuclear run-on studies demonstrated that dexamethasone caused a modest (≤40%) decrease in TGF-β1-stimulated IL-11 gene transcription. Actinomycin D pulse-chase experiments demonstrated that dexamethasone simultaneously destabilized IL-11 mRNA. Dexamethasone also inhibited TGF-β1-stimulated IL-11 promoter-driven luciferase activity but did not diminish activator protein-1 binding to IL-11 promoter sequences. Glucocorticoids inhibit lung cell IL-11 production via a complex mechanism that involves the inhibition of IL-11 gene transcription and the destabilization of IL-11 mRNA.
Pulmonary surfactant inhibits LPS-induced nitric oxide production by alveolar macrophages
Authors: Miles P. R. , Bowman L. , Rao K. M. K. , Baatz J. E. , Huffman L.
Published: December 2017

The objectives of this investigation were 1) to report that pulmonary surfactant inhibits lipopolysaccharide (LPS)-induced nitric oxide (⋅ NO) production by rat alveolar macrophages, 2) to study possible mechanisms for this effect, and 3) to determine which surfactant component(s) is responsible. ⋅ NO produced by the cells in response to LPS is due to an inducible ⋅ NO synthase (iNOS). Surfactant inhibits LPS-induced ⋅ NO formation in a concentration-dependent manner; ⋅ NO production is inhibited by ∼50 and ∼75% at surfactant levels of 100 and 200 μg phospholipid/ml, respectively. The inhibition is not due to surfactant interference with the interaction of LPS with the cells or to disruption of the formation of iNOS mRNA. Also, surfactant does not seem to reduce ⋅ NO formation by directly affecting iNOS activity or by acting as an antioxidant or radical scavenger. However, in the presence of surfactant, there is an ∼80% reduction in the amount of LPS-induced iNOS protein in the cells. LPS-induced ⋅ NO production is inhibited by Survanta, a surfactant preparation used in replacement therapy, as well as by natural surfactant. ⋅ NO formation is not affected by the major lipid components of surfactant or by two surfactant-associated proteins, surfactant protein (SP) A or SP-C. However, the hydrophobic SP-B inhibits ⋅ NO formation in a concentration-dependent manner; ⋅ NO production is inhibited by ∼50 and ∼90% at SP-B levels of 1–2 and 10 μg/ml, respectively. These results show that lung surfactant inhibits LPS-induced ⋅ NO production by alveolar macrophages, that the effect is due to a reduction in iNOS protein levels, and that the surfactant component responsible for the reduction is SP-B.
Isoproterenol increases Na+-K+-ATPase activity by membrane insertion of α-subunits in lung alveolar cells
Authors: Bertorello Alejandro M. , Ridge Karen M. , Chibalin Alexander V. , Katz Adrian I. , Sznajder Jacob I.
Published: December 2017

Catecholamines promote lung edema clearance via β-adrenergic-mediated stimulation of active Na+transport across the alveolar epithelium. Because alveolar epithelial type II cell Na+-K+-ATPase contributes to vectorial Na+flux, the present study was designed to investigate whether Na+-K+-ATPase undergoes acute changes in its catalytic activity in response to β-adrenergic-receptor stimulation. Na+-K+-ATPase activity increased threefold in cells incubated with 1 μM isoproterenol for 15 min, which also resulted in a fourfold increase in the cellular levels of cAMP. Forskolin (10 μM) also stimulated Na+-K+-ATPase activity as well as ouabain binding. The increase in Na+-K+-ATPase activity was abolished when cells were coincubated with a cAMP-dependent protein kinase inhibitor. This stimulation, however, was not due to protein kinase-dependent phosphorylation of the Na+-K+-ATPase α-subunit; rather, it was the result of an increased number of α-subunits recruited from the late endosomes into the plasma membrane. The recruitment of α-subunits to the plasma membrane was prevented by stabilizing the cortical actin cytoskeleton with phallacidin or by blocking anterograde transport with brefeldin A but was unaffected by coincubation with amiloride. In conclusion, isoproterenol increases Na+-K+-ATPase activity in alveolar type II epithelial cells by recruiting α-subunits into the plasma membrane from an intracellular compartment in an Na+-independent manner.
Time domain reflectometry: measurement of free water in normal lung and pulmonary edema
Authors: Miura Nobuhiro , Shioya Sumie , Kurita Daisaku , Shigematsu Teruyoshi , Mashimo Satoru
Published: December 2017

The free water content of lung tissue was investigated by dielectric spectroscopy in normal lungs and in pulmonary edema induced by oleic acid in rats. The dielectric relaxation in a frequency range of 107 to 1010 Hz was measured with the time domain reflectometry method at 25°C. Three dielectric relaxation processes were analyzed for the lung tissue. A high-frequency process around 10 GHz was attributed to the orientation of free water molecules based on the relaxation time [log τh (in s) = −11.03]. The dielectric strength (Δε) of this high-frequency peak (Δεh) should reflect the amount of free water in the tissue. Because the measured Δεh depended on the air content of the lung samples, the value of Δεh was corrected for the air content of each sample as determined by the point-counting method in the area where the time domain reflectometry data were obtained. The lungs of rats that received an injection of oleic acid had a significantly increased free water content [(Δεh of lung/Δε of pure water) × density of pure water] compared with that in the normal lung (0.76 vs. 0.59 g/cm3). These results indicate that free water occupies ∼60% of the total volume of normal lung tissue and that there is an increase in free water in pulmonary edema.
Effect of oxygen on lung superoxide dismutase activities in premature baboons with bronchopulmonary dysplasia
Authors: Morton Ronald L. , Das Kumuda C. , Guo Xiao-Ling , Iklé David N. , White Carl W.
Published: December 2017

We investigated the effects of gestational age and oxygen exposure on superoxide dismutase (SOD) activities in distal fetal lung tissue in primate models of bronchopulmonary dysplasia. During the final third of fetal life, lung coppper-zinc SOD (Cu,ZnSOD) specific activity decreased, whereas lung manganese SOD (MnSOD) specific activity tended to increase. In the premature newborn (140 days, 78% of term gestation), lung total SOD and Cu,ZnSOD specific activities decreased after 6–10 days of ventilation with as needed [pro re nada (PRN)] or 100% oxygen compared with fetal control animals. Neither Cu,ZnSOD mRNA nor protein expression changed after either oxygen exposure at this gestation (140 days) relative to fetal control animals. At this age (6–10 days), lung MnSOD specific activity did not change in oxygen-exposed relative to fetal control animals, even though lung expression of MnSOD mRNA and protein increased after PRN or 100% oxygen exposure. In the very premature 125-day newborn (69% of term), lung Cu,ZnSOD specific activity and protein decreased, whereas Cu,ZnSOD mRNA increased, after 6–10 days of ventilation with PRN oxygen compared with fetal control animals. In fetal lung explants, hyperoxia also decreased expression of SOD activity acutely (16-h exposure, 125 and 140 days gestation). To conclude, expression of SOD activity in the premature primate lung did not increase in response to elevated oxygen tension, apparently due to effects occurring subsequent to the expression of these mRNAs.
Effect of halothane on intracellular calcium oscillations in porcine tracheal smooth muscle cells
Authors: Pabelick Christina M. , Prakash Y. S. , Kannan Mathur S. , Jones Keith A. , Warner David O. , Sieck Gary C.
Published: December 2017

The effect of halothane on intracellular Ca2+ concentration ([Ca2+]i) regulation in porcine tracheal smooth muscle cells was examined with real-time confocal microscopy. Both 1 and 2 minimum alveolar concentration (MAC) halothane increased basal [Ca2+]iwhen Ca2+ influx and efflux were blocked, suggesting increased sarcoplasmic reticulum (SR) Ca2+ leak and/or decreased reuptake. In β-escin-permeabilized cells, heparin inhibition of inositol 1,4,5-trisphosphate-receptor channels blunted the halothane-induced increase in [Ca2+]i. Both 1 and 2 MAC halothane decreased the frequency and amplitude of ACh-induced [Ca2+]ioscillations (which represent SR Ca2+ release through ryanodine-receptor channels), abolishing oscillations in ∼20% of tracheal smooth muscle cells at 2 MAC. When Ca2+ influx and efflux were blocked, halothane increased the baseline and decreased the frequency and amplitude of [Ca2+]ioscillations, inhibiting oscillations in ∼70% of cells at 2 MAC. The fall time of [Ca2+]ioscillations and the rate of fall of the [Ca2+]iresponse to caffeine were both increased by halothane. These results suggest that halothane abolishes agonist-induced [Ca2+]ioscillations by 1) depleting SR Ca2+ via increased Ca2+ leak through inositol 1,4,5-trisphosphate-receptor channels, 2) decreasing Ca2+ release through ryanodine-receptor channels, and 3) inhibiting reuptake.
Differential monocyte adhesion and adhesion molecule expression in venous and arterial endothelial cells
Authors: Kalogeris Theodore J. , Kevil Christopher G. , Laroux F. Stephen , Coe Laura L. , Phifer Travis J. , Alexander J. Steven
Published: December 2017

We compared U-937 cell adhesion and adhesion molecule expression in human umbilical venous (HUVECs) and arterial (HUAECs) endothelial cells exposed to tumor necrosis factor (TNF), interleukin-1, and lipopolysaccharide (LPS). TNF and LPS stimulated vascular cell adhesion molecule (VCAM)-1 surface expression and adhesion of U-937 monocyte-like cells to HUVECs but not to HUAECs. Antibody studies demonstrated that in HUVECs at least 75% of the adhesion response is VCAM-1 mediated. Interleukin-1 stimulated U-937 cell adhesion to and VCAM-1 surface expression in both HUVECs and HUAECs. Pyrrolidinedithiocarbamate and the proteasome inhibitor MG-132 blocked TNF- and LPS-stimulated U-937 cell adhesion to HUVECs. These agents also significantly decreased TNF- and LPS-stimulated increases in HUVEC surface VCAM-1. TNF increased VCAM-1 protein and mRNA in HUVECs that was blocked by pyrrolidinedithiocarbamate. However, neither TNF or LPS stimulated VCAM-1 expression in HUAECs. TNF stimulated expression of both intercellular adhesion molecule-1 and E-selectin in HUVECs, but in HUAECs, only intercellular adhesion molecule-1 was increased. Electrophoretic mobility shift assays demonstrated no difference in the pattern of TNF-stimulated nuclear factor-κB activation between HUVECs and HUAECs. These studies demonstrate a novel and striking insensitivity of arterial endothelium to the effects of TNF and LPS and indicate a dissociation between the ability of HUAECs to upregulate nuclear factor-κB and VCAM-1.
Effects of fluoxetine, phentermine, and venlafaxine on pulmonary arterial pressure and electrophysiology
Authors: Reeve Helen L. , Nelson Daniel P. , Archer Stephen L. , Weir E. Kenneth
Published: December 2017

The anorexic agents dexfenfluramine and fenfluramine plus phentermine have been associated with outbreaks of pulmonary hypertension. The fenfluramines release serotonin and reduce serotonin reuptake in neurons. They also inhibit potassium current ( I K), causing membrane potential depolarization in pulmonary arterial smooth muscle cells. The recent withdrawal of the fenfluramines has led to the use of fluoxetine and phentermine as an alternative anorexic combination. Because fluoxetine and venlafaxine reduce serotonin reuptake, we compared the effects of these agents with those of phentermine and dexfenfluramine on pulmonary arterial pressure, I K, and membrane potential. Fluoxetine, venlafaxine, and phentermine caused minimal increases in pulmonary arterial pressure at concentrations < 100 μM but did cause a dose-dependent inhibition of I K. The order of potency for inhibition of I K at +50 mV was fluoxetine > dexfenfluramine = venlafaxine > phentermine. Despite the inhibitory effect on I K at more positive membrane potentials, fluoxetine, venlafaxine, and phentermine, in contrast to dexfenfluramine, had minimal effects on the cell resting membrane potential (all at a concentration of 100 μM). However, application of 100 μM fluoxetine to cells that had been depolarized to −30 mV by current injection elicited a further depolarization of >18 mV. These results suggest that fluoxetine, venlafaxine, and phentermine do not inhibit I K at the resting membrane potential. Consequently, they may present less risk of inducing pulmonary hypertension than the fenfluramines, at least by mechanisms involving membrane depolarization.
EGF activates ErbB-2 and stimulates phosphatidylinositol 3-kinase in human airway smooth muscle cells
Authors: Krymskaya Vera P. , Hoffman Rebecca , Eszterhas Andrew , Kane Sibyl , Ciocca Vincenzo , Panettieri Reynold A.
Published: December 2017

The epidermal growth factor (EGF)-receptor (EGFR) family includes four homologous transmembrane receptor protein tyrosine kinases, EGFR, ErbB-2, ErbB-3, and ErbB-4. This receptor family plays a pivotal role in regulating cell proliferation, differentiation, and transformation. Ligand-induced activation of these receptors results in formation of homo- and heterodimers, which undergo transphosphorylation and transactivation. The aim of this study was to characterize the role of EGFR family members in signaling EGF-induced human airway smooth muscle (HASM) cell proliferation. Here, we show that EGF stimulates activation of EGFR and transactivation of ErbB-2 in quiescent HASM cells. Phosphatidylinositol (PI) 3-kinase, a critical signaling enzyme that modulates HASM cell growth, is preferentially associated with ErbB-2, and EGF-stimulated transactivation of ErbB-2 induces PI 3-kinase activation. ErbB-3 and ErbB-4 are present in HASM cells; however, EGF has no effect on their activation. Betacellulin, a ligand for EGFR, ErbB-3, and ErbB-4, induced DNA synthesis of HASM cells and stimulated signaling through the activation of EGFR and ErbB-2 but not of ErbB-3 and ErbB-4. Heregulin, a specific ligand for ErbB-3 and ErbB-4, did not effect DNA synthesis and did not activate its specific receptors. These data suggest that EGF imparts signals that involve activation of ErbB-2 and are associated with ErbB-2 PI 3-kinase activation. Despite the mRNA and protein expression of all members of the EGFR family, ligand-stimulated signaling induced activation of EGFR and ErbB-2 but not of ErbB-3 and ErbB-4.