bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanisms of anti-influenza activity of surfactant proteins A and D: comparison with serum collectins
Authors: Hartshorn Kevan L. , White Mitchell R. , Shepherd Virginia , Reid Ken , Jensenius Jens C. , Crouch E. C.
Published: December 2017

The present study provides the first direct comparison of anti-influenza A virus (IAV) activities of the collectins surfactant protein (SP) A and SP-D, mannose-binding lectin (MBL), and conglutinin. SP-D, MBL, and conglutinin inhibited IAV hemagglutination activity with a greater potency than and by a distinct mechanism from SP-A. Although isolated trimeric SP-D carbohydrate recognition domains inhibited hemagglutination activity, preparations of SP-D also containing the collagen domain and NH2terminus caused greater inhibition. In contrast to SP-A (or nonmultimerized SP-D), absence of the N-linked attachment did not effect interactions of multimerized SP-D with IAV. SP-D, SP-A, and conglutinin caused viral precipitation through formation of massive viral aggregates, whereas MBL formed aggregates of smaller size that did not precipitate. All of the collectins enhanced IAV binding to neutrophils; however, in the case of MBL, this effect was modest compared with the binding enhancement induced by SP-D or conglutinin. These studies clarify the structural requirements for viral inhibition by SP-D and reveal significant differences in the mechanisms of anti-IAV activity among the collectins.
Perinatal expression of IGFBPs in rat lung and its hormonal regulation in fetal lung explants
Authors: Van De Wetering J. Koenraad , Elfring Robert H. , Oosterlaken-Dijksterhuis Marja A. , Mol Jan A. , Haagsman Henk P. , Batenburg Joseph J.
Published: December 2017

To gain more insight into the regulation of the expression of insulin-like growth factor (IGF) binding proteins (IGFBPs) in the lung, the developmental patterns of the abundance of the mRNAs encoding IGFBPs were measured in the perinatal rat lung and in explant cultures of fetal rat lung. In hormone-free explant cultures, the levels of the mRNAs encoding IGFBP-2 through -5 changed with a pattern similar to that occurring in vivo (although in the case of IGFBP-3 to -5 at a faster rate), indicating that the developmental regulation of the expression of these IGFBPs in perinatal lung is mimicked in the explants. For the IGFBP-6 mRNA level, the pattern in vitro differed from that in vivo. In the explant cultures, dexamethasone decreased the production of IGFBP-3 and -4 and decreased the abundance of the mRNAs encoding IGFBP-2 to -5 but increased the abundance of IGFBP-6 mRNA. These observations indicate that glucocorticoids may be involved in the developmental regulation of the expression of these components of the IGF system and that the IGF system may be involved in the physiological effects of glucocorticoids on lung development. No appreciable effects of 3,3′,5-triiodothyronine on the expression of the IGFBPs were observed.
Glutamine synthetase gene expression in the lungs of endotoxin-treated and adrenalectomized rats
Authors: Lukaszewicz Gregory , Abcouwer Steve F. , Labow Brian I. , Souba Wiley W.
Published: December 2017

During sepsis, the lung responds by exporting increased amounts of the amino acid glutamine. This response is accompanied by increased enzymatic activity of glutamine synthetase (GS), which catalyzes the synthesis of glutamine from glutamate and ammonia. It is also known that GS expression in the rat lung can be induced by glucocorticoid hormones. To determine whether the septic response and the response to glucocorticoids are related, we have characterized the induction of GS expression during lipopolysaccharide (LPS)-induced endotoxemia in normal, neutropenic, and adrenalectomized rats. Normal rats exhibited a time- and dose-dependent induction of GS mRNA levels after a single intraperitoneal dose of LPS. Responses to LPS were maximal at doses of 0.1 mg/kg body wt and above. A single 10 mg/kg body wt dose of LPS led to a rapid, transient sevenfold increase in GS mRNA ( P ≤ 0.1) and a twofold increase in GS protein level 8 h postinjection. Induction of lung GS mRNA 4 h after LPS injection was approximately fivefold in neutropenic ( P ≤ 0.1) and fourfold in nonneutropenic control rats ( P ≤ 0.1), suggesting that infiltrating neutrophils or neutrophil-derived factors are not required for GS induction. In response to high-dose, short-term endotoxemia, adrenalectomized rat lung GS mRNA increased twofold ( P ≤ 0.02) compared with sixfold in sham-operated control rats ( P ≤ 0.02). However, in response to low-dose, long-term endotoxemia, adrenalectomized rat lung GS mRNA increased threefold ( P ≤ 0.02) compared with fourfold in sham-operated control rats ( P ≤ 0.02). Adrenalectomy did not affect the elevation of lung GS mRNA levels in response to dexamethasone. In addition, GS mRNA was induced four- and sixfold in rat microvascular pulmonary endothelial cells exposed to plasma from control and septic rats, respectively. The addition of a glucocorticoid antagonist, RU-38486, completely blocked GS gene induction in the presence of control plasma but only attenuated the response to plasma from septic animals by 30%. These results suggest that GS gene induction during sepsis is only partially mediated by adrenal-derived glucocorticoid hormones.
Role of oxidants in influenza virus-induced gene expression
Authors: Knobil Katharine , Choi Augustine M. K. , Weigand Gordon W. , Jacoby David B.
Published: December 2017

Influenza virus-induced epithelial damage may be mediated, in part, by reactive oxygen intermediates (ROIs). In this study, we investigated the role of ROIs in the influenza virus-induced gene expression of antioxidant enzymes and in the activation of nuclear factor-κB (NF-κB), an oxidant-sensitive transcriptional factor. Influenza virus infection increased production of intracellular ROIs in A549 pulmonary epithelial cells. Induction of manganese superoxide dismutase (MnSOD) mRNA correlated with increased MnSOD protein and enzyme activity. Influenza virus infection also activated NF-κB binding as determined by an electrophoretic mobility shift assay. Pretreatment of A549 cells with N-acetyl-l-cysteine attenuated virus-induced NF-κB activation and interleukin (IL)-8 mRNA induction but did not block induction of MnSOD mRNA. In contrast, pyrrolidine dithiocarbamate blocked activation of NF-κB and induction of MnSOD and IL-8 mRNAs. Treatment with pyrrolidine dithiocarbamate also markedly decreased virus-induced cell death. Thus oxidants are involved in influenza virus-induced activation of NF-κB, in the expression of IL-8 and MnSOD, and in virus-induced cell death.
Na+-K+-ATPase expression in alveolar epithelial cells: upregulation of active ion transport by KGF
Authors: Borok Zea , Danto Spencer I. , Dimen Luis L. , Zhang Xiao-Ling , Lubman Richard L.
Published: December 2017

We evaluated the effects of keratinocyte growth factor (KGF) on alveolar epithelial cell (AEC) active ion transport and on rat epithelial Na channel (rENaC) subunit and Na+-K+-adenosinetriphosphatase (ATPase) subunit isoform expression using monolayers of AEC grown in primary culture. Rat alveolar type II cells were plated on polycarbonate filters in serum-free medium, and KGF (10 ng/ml) was added to confluent AEC monolayers on day 4 in culture. Exposure of AEC monolayers to KGF on day 4 resulted in dose-dependent increases in short-circuit current ( Isc) compared with controls by day 5, with further increases occurring through day 8. Relative Na+-K+-ATPase α1-subunit mRNA abundance was increased by 41% on days 6 and 8 after exposure to KGF, whereas α2-subunit mRNA remained only marginally detectable in both the absence and presence of KGF. Levels of mRNA for the β1-subunit of Na+-K+-ATPase did not increase, whereas cellular α1- and β1-subunit protein increased 70 and 31%, respectively, on day 6. mRNA for α-, β-, and γ-rENaC all decreased in abundance after treatment with KGF. These results indicate that KGF upregulates active ion transport across AEC monolayers via a KGF-induced increase in Na pumps, primarily due to increased Na+-K+-ATPase α1-subunit mRNA expression. We conclude that KGF may enhance alveolar fluid clearance after acute lung injury by upregulating Na pump expression and transepithelial Na transport across the alveolar epithelium.
Airway smooth muscle cell proliferation: characterization of subpopulations by sensitivity to heparin inhibition
Authors: Halayko Andrew J. , Rector Edward , Stephens Newman L.
Published: December 2017

Growth and maturation state of airway smooth muscle cells (SMCs) are determinants of asthma pathophysiology. Heparin reduces airway SMC proliferation and arterial SMC replication and phenotypic modulation. Distinct arterial SMC subtypes, differing in heparin sensitivity, have been characterized. We assessed the cellular mechanisms underlying the growth and phenotype of heparin-treated canine tracheal myocytes in primary culture. Heparin reduced replication by 40%. Immunoblot assay of myosin, actin, and myosin light chain kinase revealed heparin had no effect on rapid spontaneous phenotypic modulation after the cells were plated. Heparin increased cellular protein and vimentin contents in confluent cultures, suggesting that it may induce hypertrophic growth. Cell cycle analysis revealed that heparin decreased serum-stimulated replicating myocyte number by 40%. Also, G2-M transit was 20% slower for the set of SMCs that proceeded past G1in the presence of heparin. These data indicate that heparin does not inhibit airway SMC replication by blocking modulation from the contractile state. Moreover, airway smooth muscle is composed of distinct SMC populations differing in mitogen and antiproliferative mediator responsiveness. Identification of functionally divergent subgroups suggests that distinct sets of SMCs may contribute differentially to airway physiology and pathophysiology.
Chemokine regulation of ozone-induced neutrophil and monocyte inflammation
Authors: Zhao Qiyu , Simpson Leigh G. , Driscoll Kevin E. , Leikauf George D.
Published: December 2017

Pulmonary inflammation has been observed in humans and in many animal species after ozone exposure. Inflammatory cell accumulation involves local synthesis of chemokines, including neutrophil chemoattractants such as macrophage inflammatory protein-2 (MIP-2), and monocyte chemoattractants, such as monocyte chemoattractant protein-1 (MCP-1). To better understand the mechanism of ozone-induced inflammation, we exposed mice and rats to ozone for 3 h and measured MIP-2 and MCP-1 gene expression. In C57BL/6 mice, steady-state mRNA levels for MCP-1 in the lung increased at 0.6 parts/million (ppm) ozone and were maximal at 2.0 ppm ozone. After exposure to 2 ppm ozone, MIP-2 mRNA levels peaked at 4 h postexposure, whereas MCP-1 mRNA levels peaked at 24 h postexposure. Neutrophils and monocytes recovered in bronchoalveolar lavage fluid peaked at 24 and 72 h, respectively. The accumulation of monocytes was thus delayed relative to that of neutrophils, consistent with the sequential expression of the corresponding chemokines. The role of MCP-1 in monocyte accumulation was evaluated in greater detail in rats. Ozone caused an increase in monocyte chemotactic activity in bronchoalveolar fluid that was inhibited by an antibody directed against MCP-1. Ozone-induced MCP-1 mRNA levels were higher in lavage cells than in whole lung tissue, indicating that lavage cells are an important source of MCP-1. In these cells, nuclear factor-κB, a nuclear transcription factor implicated in MCP-1 gene regulation, was also activated 20–24 h after ozone exposure. These findings indicate that monocyte accumulation subsequent to acute lung injury can be mediated through MCP-1 and that nuclear factor-κB may play a role in ozone-induced MCP-1 gene expression.
Differences between rat alveolar and interstitial macrophages 5 wk after quartz exposure
Authors: Zetterberg Göran , Johansson Anne , Lundahl Joachim , Lundborg Margot , Sköld Carl Magnus , Tornling Göran , Camner Per , Eklund Anders
Published: December 2017

Macrophages play an essential role in pulmonary host defense. We investigated differences between rat alveolar (AM) and interstitial (IM) macrophages after exposure in vivo to quartz, an inducer of intensive alveolitis. Rats were exposed to 0.5 ml of saline without ( n = 8) or with ( n = 8) 10 mg of quartz by intratracheal instillation. In a third group ( n = 8), 10 mg of surfactant were added to the quartz mixture. Five weeks later, AM were recovered by bronchoalveolar lavage and IM by mechanical fragmentation of the lung, followed by enzymatic treatment. Contamination of AM to the IM fraction was calculated to be 12–15%. After quartz exposure, the expression of major histocompatibility complex class Ia was increased in both AM and IM fractions. The receptor corresponding to human complement receptor 3 increased in AM after quartz exposure, and AM from quartz-exposed animals had a lower metabolic activation. Our findings indicate that IM are immunocompetent cells and that differences between AM and IM fractions occur upon quartz-induced inflammation. This response is not affected by addition of surfactant.
Metallothionein, glutathione, and cystine transport in pulmonary artery endothelial cells and NIH/3T3 cells
Authors: Susanto Irawan , Wright Shawn E. , Lawson Richard S. , Williams Charnae E. , Deneke Susan M.
Published: December 2017

Both glutathione (γ-glutamylcysteinylglycine; GSH) and the metalloprotein metallothionein (MT) are composed of approximately one-third cysteine. Both have antioxidant activity and are induced by oxidant stresses and heavy metals. Intracellular cysteine levels may depend on uptake and reduction of extracellular cystine. GSH synthesis can be limited by the activity of the [Formula: see text] cystine transport system, which is induced by oxidants and other stresses. MT is induced by treatments that also increase GSH levels and may compete with GSH for intracellular cysteine. We investigated the induction of MT and GSH and cystine transport in NIH/3T3 cells and bovine pulmonary artery endothelial cells exposed to cadmium (Cd) or arsenite. Cd and arsenite increased MT and GSH in both cells. Increases in MT and GSH were accompanied by increases in cystine uptake. Inhibition of cystine transport by glutamate decreased GSH levels and blocked Cd-induced GSH increases in both cell types. MT levels were not significantly affected, suggesting that MT synthesis is less sensitive to intracellular cysteine levels than GSH synthesis.
Fluoride stimulates cystic fibrosis transmembrane conductance regulator Cl−channel activity
Authors: Berger Herbert A. , Travis Sue M. , Welsh Michael J.
Published: December 2017

While studying the regulation of the cystic fibrosis transmembrane conductance regulator (CFTR), we found that addition of F−to the cytosolic surface of excised, inside-out membrane patches reversibly increased Cl−current in a dose-dependent manner. Stimulation required prior phosphorylation and the presence of ATP. F−increased current even in the presence of deferoxamine, which chelates Al3+, suggesting that stimulation was not due to A[Formula: see text]. F−also stimulated current in a CFTR variant that lacked a large part of the R domain, suggesting that the effect was not mediated via this domain. Studies of single channels showed that F−increased the open-state probability by slowing channel closure from bursts of activity; the mean closed time between bursts and single-channel conductance was not altered. These results suggested that F−influenced regulation by the cytosolic domains, most likely the nucleotide-binding domains (NBDs). Consistent with this, we found that mutation of a conserved Walker lysine in NBD2 changed the relative stimulatory effect of F−compared with wild-type CFTR, whereas mutation of the Walker lysine in NBD1 had no effect. Based on these and previous data, we speculate that F−interacts with CFTR, possibly via NBD2, and slows the rate of channel closure.
Identification of a putative surfactant convertase in rat lung as a secreted serine carboxylesterase
Authors: Barr Frederick , Clark Howard , Hawgood Samuel
Published: December 2017

In the alveolar lumen, pulmonary surfactant converts from the contents of secreted lamellar bodies to tubular myelin to apoprotein-depleted vesicles during respiration. Using an in vitro system, researchers have reported that the conversion of tubular myelin to vesicles is blocked by inhibitors of serine hydrolase activity and have tentatively ascribed “convertase” activity to a diisopropyl fluorophosphate (DFP)-binding protein in mouse bronchoalveolar lavage (BAL). We purified and sequenced the homologous enzyme from rat BAL fluid. Amino acid sequence from the amino terminus and an internal cyanogen bromide peptide of the purified rat DFP-binding protein perfectly match the sequence of the carboxylesterase ES-2. Although ES-2 was initially cloned from liver, we found a 1.8-kilobase mRNA for ES-2 in decreasing relative abundance in rat liver, kidney, and lung but not in heart or spleen. Although further studies are required to establish the identity between “convertase” and ES-2 or a homologous member of the carboxylesterase family, our results raise the possibility that a protein with esterase/lipase activity plays a role in extracellular surfactant metabolism.
Localization of clearance receptor in rat lung and trachea: association with chondrogenic differentiation
Authors: Fujishige Kotomi , Yanaka Noriyuki , Akatsuka Hiroyuki , Omori Kenji
Published: December 2017

The lung is rich in atrial natriuretic peptide binding sites, and the majority of them are considered to be the natriuretic peptide clearance receptor (NPR-C). In this study, localization of NPR-C in the rat lung and trachea was investigated by immunohistochemical analysis with the specific antibody. Positive staining was observed in the epithelial cell layers of the trachea and bronchiole and the myocardium surrounding the pulmonary vein. Moreover, expression of NPR-C was seen in mesenchymal cells; it was especially strong in cells in the perichondrium and decreased in chondrocytes in the cartilage. Because mesenchymal cells in the perichondrium differentiate to chondrocytes, NPR-C expression is suggested to be associated with chondrogenic differentiation. The chondrogenic cell line ATDC5 was used to study NPR-C expression during chondrogenic differentiation in vitro. The undifferentiated ATDC5 cells expressed NPR-C at a much higher level than the differentiated ATDC5 cells, in accordance with the observation of the immunohistochemical analysis in the cartilage. These findings suggest that NPR-C expression is differentially regulated in chondrocytes and that the natriuretic peptides may play a role in regulating chondrocyte development in the lung.
Lung matrix deposition of normal and alkylated plasma fibronectin: response to postsurgical sepsis
Authors: Brien Thomas P. , Reddy Pramod P. , Vincent Peter A. , Lewis Edward P. , Ross Jeffrey S. , Saba Thomas M.
Published: December 2017

Plasma fibronectin (Fn) can both enhance phagocytic clearance of microparticulate debris by macrophages as well as incorporate it into the lung extracellular matrix (ECM). The goal of this study was to document that N-ethylmaleimide (NEM)-treated human plasma Fn (HFn) would lose its ability to incorporate into the lung ECM in vivo even though it would retain its ability to stimulate test particle phagocytosis and bind to fibrin. Using dual-label immunofluorescence, we compared the lung deposition of purified normal HFn and NEM-alkylated HFn (NEM-HFn) after their intravenous injection into postoperative nonbacteremic and bacteremic sheep in relationship to the localization of endogenous sheep Fn. Two days after a sterile surgical thoracotomy, sheep were infused with either 5 × 108Pseudomonas aeruginosa (postsurgical bacteremic model) or the diluent (nonbacteremic model). They also received a bolus 100-mg injection (5 min) of either HFn or NEM-HFn. Analysis of serial lung biopsies harvested at 2-h intervals demonstrated little deposition of NEM-HFn compared with HFn in the lung interstitial matrix of postoperative nonbacteremic sheep. In contrast, enhanced deposition of both HFn and NEM-HFn was observed in the lungs of postoperative bacteremic sheep. However, in the lungs of bacteremic sheep, HFn displayed a diffuse fibrillar deposition pattern in the lung characteristic of ECM incorporation, whereas the enhanced NEM-HFn deposition, especially in the interstitial ECM region of the lung, was primarily focal and punctate, with very little fibrillar incorporation. Immunofluorescent analysis with antibodies specific to fibrinogen, Fn, and lung macrophage surface antigens coupled with immunoperoxidase staining for HFn antigen revealed that the punctate fluorescence pattern was due to both the binding of HFn to fibrin and its colocalization with inflammatory cells. Thus treatment of plasma Fn with low concentrations of NEM will limit its normal in vivo fibrillar incorporation into the interstitial ECM region of the lung.
Nitric oxide inhibits lung sodium transport through a cGMP-mediated inhibition of epithelial cation channels
Authors: Jain Lucky , Chen Xi-Juan , Brown Lou Ann , Eaton Douglas C.
Published: December 2017

We used the patch-clamp technique to study the effect of nitric oxide (NO) on a cation channel in rat type II pneumocytes [alveolar type II (AT II) cells]. Single-channel recordings from the apical surface of AT II cells in primary culture showed a predominant cation channel with a conductance of 20.6 ± 1.1 (SE) pS ( n = 9 cell-attached patches) and Na+-to-K+selectivity of 0.97 ± 0.07 ( n = 7 cell-attached patches). An NO donor, S-nitrosoglutathione (GSNO; 100 μM), inhibited the basal cation-channel activity by 43% [open probability ( Po), control 0.28 ± 0.05 vs. GSNO 0.16 ± 0.03; P < 0.001; n = 16 cell-attached patches], with no significant change in the conductance. GSNO reduced the Poby reducing channel mean open and increasing mean closed times. GSNO inhibition was reversed by washout. The inhibitory effect of NO was confirmed by using a second donor of NO, S-nitroso- N-acetylpenicillamine (100 μM; Po, control 0.53 ± 0.05 vs. S-nitroso- N-acetylpenicillamine 0.31 ± 0.04; −42%; P < 0.05; n = 5 cell-attached patches). The GSNO effect was blocked by methylene blue (a blocker of guanylyl cyclase; 100 μM), suggesting a role for cGMP. The permeable analog of cGMP, 8-bromo-cGMP (8-BrcGMP; 1 mM), inhibited the cation channel in a manner similar to GSNO ( Po, control 0.38 ± 0.06 vs. 8-BrcGMP 0.09 ± 0.02; P < 0.05; n = 7 cell-attached patches). Pretreatment of cells with 1 μM KT-5823 (a blocker of protein kinase G) abolished the inhibitory effect of GSNO. The NO inhibition of channels was not due to changes in cell viability. Intracellular cGMP was found to be elevated in AT II cells treated with NO (control 13.4 ± 3.6 vs. GSNO 25.4 ± 4.1 fmol/ml; P < 0.05; n = 6 cell-attached patches). We conclude that NO suppresses the activity of an Na+-permeant cation channel on the apical surface of AT II cells. This action appears to be mediated by a cGMP-dependent protein kinase.
Hypoxic exposure time dependently modulates endothelininduced contraction of pulmonary artery smooth muscle
Authors: Bialecki Russell A. , Fisher Carol S. , Murdoch Wallace W. , Barthlow Herbert G. , Stow Richard B. , Mallamaci Michael , Rumsey William
Published: December 2017

Endothelins (ETs) have been implicated in the pathogenesis of hypoxia-induced pulmonary hypertension. We determined whether hypoxic exposure of rats (10% O2-90% N2, 1 atm, 1–48 days) altered contraction to ET in isolated segments of endothelium-denuded extralobar branch pulmonary artery (PA) and aorta. Hypoxic exposure increased hematocrit, right ventricular hypertrophy, and ET-1 plasma concentration. Hypoxia also caused a sustained decrease in PA but not in aorta sensitivity to ET-1. In comparison, hypoxic exposure throughout 12 days decreased time dependently the maximum contraction of PA to ET-1, BaCl2, and KCl. The hypoxia-induced decrease in maximum contraction of PA to ET-1 returned toward normal levels by 21 days and approximated control levels by 48 days. After 14 days of hypoxia, right ventricular hypertrophy correlated with decreased sensitivity of PA to ET-1. After 21 days of hypoxia, PA sensitivity to ET-2 and ET-3 was decreased, and sarafotoxin S6c-induced contraction was abolished. In conclusion, hypoxic exposure time dependently modulates the responsiveness of PA smooth muscle to ETs, BaCl2, and KCl. The hypoxia-induced changes in tissue responsiveness to ET-1 may be associated with increased plasma concentrations of this peptide.
Cell-specific expression of fibronectin and EIIIA and EIIIB splice variants after oxygen injury
Authors: Maniscalco William M. , Watkins Richard H. , Chess Patricia R. , Sinkin Robert A. , Horowitz Stuart , Toia Liana
Published: December 2017

Cellular fibronectin (cFN) expression is characteristic of injured tissues. Unlike plasma FN, cFN mRNA often contains the EIIIA or EIIIB domains. We examined the lung cell-specific expression of total cFN mRNA and the EIIIA and EIIIB splice variants in rabbits after acute oxygen injury. By in situ hybridization, control lung had low cFN mRNA. After exposure to >95% oxygen, mRNAs for total cFN and EIIIA were noted primarily in alveolar macrophages and large-vessel endothelial cells. By 3–5 days recovery, cFN and EIIIA mRNA abundance was increased in alveolar septal cells (i.e., alveolar epithelial, interstitial, or endothelial cells) and in some large-vessel endothelial cells but was low in bronchial epithelial cells. During recovery, EIIIB mRNA was low in alveolar septal cells but was noted mainly in chondrocytes. Immunostaining for EIIIA increased during recovery, paralleling the in situ hybridizations. Because FN may modulate alveolar type II cell phenotype, we investigated type II cell cFN mRNA expression in vivo. During recovery, neither isolated type II cells nor cells with surfactant protein C mRNA in vivo contained FN mRNA. In summary, these data suggest that cFN with the EIIIA domain has a role in alveolar cell recovery from oxygen injury and that type II cells do not express cFN during recovery.
Insulin-like growth factor binding proteins in air- and 85% oxygen-exposed adult rat lung
Authors: Han Robin N. N. , Han Victor K. M. , Buch Shilpa , Freeman Bruce A. , Post Martin , Tanswell A. Keith
Published: December 2017

Expression of insulin-like growth factor (IGF) I and its type I receptor is increased in the adult rat lung exposed to 85% O2. We hypothesized that there would be a parallel up- and downregulation of growth-stimulating and growth-inhibiting IGF binding proteins (IGFBPs), respectively. The normal adult rat lung expresses mRNAs for IGFBP-2, -3, -4, -5, and -6 but not for IGFBP-1. O2exposure for 6 or 14 days reduced IGFBP-3 and -6 and increased IGFBP-4 mRNA abundance. IGFBP-5 mRNA was reduced at 6 days but increased at 14 days. IGFBP-4 mRNA was localized to perivascular and peribronchial interstitial cells and IGFBP-5 mRNA to airway and alveolar epithelial cells. IGFBP-2, -4, and -5 immunolocalized to airway epithelial cells in normal lung and to perivascular exudates after 6 days in 85% O2. IGFBP-2 was diffusely increased throughout the lung tissue only after a 6-day exposure. IGFBP-5 was reduced after a 6-day exposure but was increased and widely distributed after 14 days. IGFBP-4 increased over airway epithelium and subepithelial cells after 6 days and over perivascular interstitial cells after 14 days of 85% O2. These data are consistent with the predicted changes for IGFBPs on O2exposure except that the generally growth-inhibitory IGFBP-4 was increased at sites of active cell proliferation.
Ascorbate-mediated transplasma membrane electron transport in pulmonary arterial endothelial cells
Authors: Merker Marilyn P. , Olson Lars E. , Bongard Robert D. , Patel Meha K. , Linehan John H. , Dawson Christopher A.
Published: December 2017

Pulmonary endothelial cells are capable of reducing certain electron acceptors at the luminal plasma membrane surface. Motivation for studying this phenomenon comes in part from the expectation that it may be important both as an endothelial antioxidant defense mechanism and in redox cycling of toxic free radicals. Pulmonary arterial endothelial cells in culture reduce the oxidized forms of thiazine compounds that have been used as electron acceptor probes for studying the mechanisms of transplasma membrane electron transport. However, they reduce another commonly studied electron acceptor, ferricyanide, only very slowly by comparison. In the present study, we examined the influence of ascorbate [ascorbic acid (AA)] and dehydroascorbate [dehydroascorbic acid (DHAA)] on the ferricyanide and thiazine reductase activities of the bovine pulmonary arterial endothelial cell surface. The endothelial cells were grown on microcarrier beads so that the reduction of ferricyanide and methylene blue could be studied colorimetrically in spectrophotometer cuvettes and in flow-through cell columns. The ferricyanide reductase activity could be increased 80-fold by adding DHAA to the medium, with virtually no effect on methylene blue reduction. The DHAA effect persisted after the DHAA was removed from the medium. AA also stimulated the ferricyanide reductase activity but was less potent, and the relative potencies of AA and DHAA correlated with their relative rates of uptake by the cells. The results are consistent with the hypothesis that AA is an intracellular electron donor for an endothelial plasma membrane ferricyanide reductase and that the stimulatory effect of DHAA is the result of increasing intracellular AA. Adding sufficient DHAA to markedly increase extracellular ferricyanide reduction had little effect on the plasma membrane methylene blue reductase activity, suggesting that pulmonary arterial endothelial cells have at least two separate transplasma membrane electron transport systems.
PDGF-induced glycosaminoglycan synthesis is mediated via phosphatidylinositol 3-kinase
Authors: Liu Jason , Fitzli Dora , Liu Mingyao , Tseu Irene , Caniggia Isabella , Rotin Daniela , Post Martin
Published: December 2017

Platelet-derived growth factor (PDGF)-BB has been shown previously to increase glycosaminoglycan (GAG) synthesis but not DNA synthesis in freshly isolated fetal lung fibroblasts. In the present study, we found that PDGF-BB also enhanced35SO4incorporation into the small, soluble proteoglycan biglycan without affecting biglycan’s core protein mRNA expression, suggesting that PDGF-BB mainly affects GAG chain elongation and/or sulfation. PDGF-BB-stimulated GAG synthesis was abrogated by tyrphostin 9, a PDGF receptor-associated tyrosine kinase inhibitor, implying that the stimulatory effect is mediated via the PDGF β-receptor (PDGFR). The intracellular signal transduction pathways that mediate PDGF-BB-stimulated GAG synthesis in fetal lung fibroblasts were investigated. On ligand-induced tyrosine phosphorylation, PDGFR associated with phospholipase C (PLC)-γ1, Ras GTPase activating protein (RasGAP), and phosphatidylinositol 3-kinase (PI3K) but not with the Syp-growth factor receptor-bound protein 2-Son of Sevenless complex. Association of PDGFR with PLC-γ1 and RasGAP followed by their tyrosine phosphorylation failed, however, to activate PLC-γ1, protein kinase C (PKC), and Ras. Neither a PLC-γ inhibitor, U-73122; a PKC inhibitor, calphostin C; nor a mitogen-activated protein kinase kinase inhibitor, PD-98059, inhibited PDGF-BB-induced GAG synthesis. In contrast, PDGF-BB stimulation triggered PDGFR-associated PI3K activity. Both PDGF-BB-induced PI3K activation and GAG synthesis were abolished by the PI3K inhibitors wortmannin and LY-294002. The results suggest that PI3K is a downstream mediator of PDGF-BB-stimulated GAG synthesis in fetal rat lung fibroblasts.
Apoptosis and DNA damage in type 2 alveolar epithelial cells cultured from hyperoxic rats
Authors: Buckley Sue , Barsky Lora , Driscoll Barbara , Weinberg Kenneth , Anderson Kathryn D. , Warburton David
Published: December 2017

Apoptosis is a genetically controlled cellular response to developmental stimuli and environmental insult that culminates in cell death. Sublethal hyperoxic injury in rodents is characterized by a complex but reproducible pattern of lung injury and repair during which the alveolar surface is damaged, denuded, and finally repopulated by type 2 alveolar epithelial cells (AEC2). Postulating that apoptosis might occur in AEC2 after hyperoxic injury, we looked for the hallmarks of apoptosis in AEC2 from hyperoxic rats. A pattern of increased DNA end labeling, DNA laddering, and induction of p53, p21, and Bax proteins, strongly suggestive of apoptosis, was seen in AEC2 cultured from hyperoxic rats when compared with control AEC2. In contrast, significant apoptosis was not detected in freshly isolated AEC2 from oxygen-treated rats. Thus the basal culture conditions appeared to be insufficient to ensure the ex vivo survival of AEC2 damaged in vivo. The oxygen-induced DNA strand breaks were blocked by the addition of 20 ng/ml of keratinocyte growth factor (KGF) to the culture medium from the time of plating and were partly inhibited by Matrigel or a soluble extract of Matrigel. KGF treatment resulted in a partial reduction in the expression of the p21, p53, and Bax proteins but had no effect on DNA laddering. We conclude that sublethal doses of oxygen in vivo cause damage to AEC2, resulting in apoptosis in ex vivo culture, and that KGF can reduce the oxygen-induced DNA damage. We speculate that KGF plays a role as a survival factor in AEC2 by limiting apoptosis in the lung after acute hyperoxic injury.
Nitro blue tetrazolium inhibits but does not mimic hypoxic vasoconstriction in isolated rabbit lungs
Authors: Weissmann Norbert , Grimminger Friedrich , Voswinckel Robert , Conzen Jörg , Seeger Werner
Published: December 2017

It has been suggested that hypoxic pulmonary vasoconstriction (HPV) may mainly proceed via loss of normoxic vasodilation, forwarded by tonic O2-dependent formation of nitric oxide and superoxide (23). Both agents may stimulate guanylate cyclase, the latter via conversion to hydrogen peroxide and formation of compound I with catalase. We probed this hypothesis in perfused rabbit lungs, employing the superoxide scavengers superoxide dismutase (SOD), 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), and nitro blue tetrazolium (NBT) and the catalase inhibitor aminotriazole (AT). NBT turned out to be a potent dose-dependent inhibitor of HPV in a concentration range of 200 nM to 1 μM, and superimposable dose-inhibition curves were obtained when lung synthesis of nitric oxide and vasodilatory prostanoids was preblocked by N G-monomethyl-l-arginine (l-NMMA) and acetylsalicylic acid (ASA). The NBT effect was specific because no inhibition in the vasoconstrictor responses to the stable thromboxane analog U-46619 and angiotensin II was observed. In contrast, SOD and Tiron were ineffective. AT exerted nonspecific inhibition of the hypoxia- and chemical vasoconstrictor-induced pressor responses. When applied under normoxic conditions, however, NBT alone or coapplied withl-NMMA or ASA, both for blockage of parallel vasodilatory pathways, did not mimic the hypoxia-induced vasoconstrictor response. In conclusion, the present study supports an important role for superoxide in the basic mechanism of HPV, but it questions the concept that loss of tonic vasorelaxation via this pathway is the underlying event in rabbit lungs. The mechanisms relating O2 tension-dependent superoxide and hydrogen peroxide generation to the vasoconstrictor event occurring in HPV remain to be further elucidated.
Metal-dependent expression of ferritin and lactoferrin by respiratory epithelial cells
Authors: Ghio Andrew J. , Carter Jacqueline D. , Samet James M. , Reed William , Quay Jacqueline , Dailey Lisa A. , Richards Judy H. , Devlin Robert B.
Published: December 2017

Increased availability of catalytically active metal has been associated with an oxidative injury. The sequestration of transition metals within intracellular ferritin confers an antioxidant function to this protein. Such storage by ferritin requires that the metal be transported across a cell membrane. We tested the hypothesis that, in response to in vitro exposures to catalytically active metal, respiratory epithelial cells increase the production of lactoferrin and ferritin to bind, transport, and store this metal with their coordination sites fully complexed. Residual oil fly ash is an emission source air pollution particle with biological effects that, both in vitro and in vivo, correspond with its metal content. Cell cultures were exposed to 0–200 μg/ml of oil fly ash for 2 and 24 h. Concentrations of ferritin and lactoferrin mRNA were estimated by reverse transcription-polymerase chain reaction, and concentrations of ferritin and lactoferrin proteins were measured in parallel. mRNA for ferritin did not change with exposure to oil fly ash. However, ferritin protein concentrations increased. Although mRNA for transferrin receptor decreased, mRNA for lactoferrin increased after incubation with the particle. Similar to changes in mRNA, transferrin concentration decreased, whereas that of lactoferrin increased. Deferoxamine, a metal chelator, inhibited these responses, and exposure of the cells to vanadium compounds alone reproduced elevations in lactoferrin mRNA. We conclude that increases in ferritin and lactoferrin expression can be metal dependent. This response can function to diminish the oxidative stress a metal chelate presents to a living system.
In chyloptysis, SP-A affects the clearance of serum lipoproteins entering the airways
Authors: Alberti Antonella , Ravenna Franco , Quaglino Daniela , Luisetti Maurizio , Muraca Maurizio , Previato Lorenzo , Enzi Goretta Baldo , Bruni Roberta , Baritussio Aldo
Published: December 2017

Serum lipoproteins may enter the airways and appear in sputum (chyloptysis) when the lymphatic circulation is impaired by inflammation, neoplasia, or an abnormal proliferation of smooth muscle cells. While analyzing the bronchoalveolar lavage fluid of a patient with chyloptysis, we noticed that surfactant could not be separated from contaminating serum lipoproteins and speculated that lipoproteins might interact with surfactant components. To clarify this point we immobilized surfactant protein (SP) A on microtiter wells and incubated it with125I-labeled very low density lipoproteins (VLDLs), low-density lipoproteins, and high-density lipoproteins. We found that SP-A binds lipoproteins. Studying in greater detail the interaction of SP-A with VLDLs, we found that the binding is time and concentration dependent; is inhibited by unlabeled lipoproteins, phospholipids, and antibodies to SP-A; is increased by Ca2+; and is unaffected by methyl α-d-mannopyranoside. Whole surfactant is a potent inhibitor of binding. Furthermore, we found that SP-A increases the degradation of VLDLs by alveolar macrophages and favors the association of VLDLs with alveolar surfactant. We conclude that SP-A influences the disposal of serum lipoproteins entering the airways and speculate that binding to alveolar surfactant might represent an important step in the interaction between exogenous substances and the lung.
Strain-associated differences in hypoxic chemosensitivity of the carotid body in rats
Authors: Weil J. V. , Stevens T. , Pickett C. K. , Tatsumi K. , Dickinson M. G. , Jacoby C. R. , Rodman D. M.
Published: December 2017

Studies in humans indicate genetic effects on the ventilatory response to hypoxia, but the site of these effects is unknown. The present study explores the question of whether there are genetically directed effects on the intrinsic hypoxic chemosensitivity of the carotid body. The approach was to study these responses in two inbred rat strains [spontaneously hypertensive rats (SHR) and Fischer 344 (F-344)] and to measure in vivo carotid chemosensitivity as the change in carotid sinus nerve (CSN) activity during progressive, isocapnic hypoxia and the isolated, in vitro responses of excised superfused carotid bodies, loaded with the fluorimetric indicator fura 2, measured as the cytosolic calcium response to moderate hypoxia ([Formula: see text] = 55 mmHg). CSN responses in F-344 rats ( n = 12) were uniformly low, with a shape parameter A of 13.8 ± 6.59 (SE), whereas responses in SHR ( n = 15) were sevenfold higher (108 ± 24.1; P < 0.002) and showed greater variation. In vitro, intracellular calcium responses of superfused carotid bodies estimated from the fluorimetric ratio (340/380 nm) showed a greater peak increase during hypoxia in carotid bodies from SHR (140 ± 4.7%) than from F-344 rats (114 6.0%; P < 0.01). Our results indicate strain-related differences in hypoxic chemosensitivity that are intrinsic to the carotid body and that could mediate genetic effects on ventilatory responsiveness to hypoxia.
Smooth muscle actin and myosin expression in cultured airway smooth muscle cells
Authors: Wong Jean Z. , Woodcock-Mitchell Janet , Mitchell John , Rippetoe Patricia , White Sheryl , Absher Marlene , Baldor Linda , Evans John , McHugh Kirk M. , Low Robert B.
Published: December 2017

In this study, the expression of smooth muscle actin and myosin was examined in cultures of rat tracheal smooth muscle cells. Protein and mRNA analyses demonstrated that these cells express α- and γ-smooth muscle actin and smooth muscle myosin and nonmuscle myosin-B heavy chains. The expression of the smooth muscle specific actin and myosin isoforms was regulated in the same direction when growth conditions were changed. Thus, at confluency in 1 or 10% serum-containing medium as well as for low-density cells (50–60% confluent) deprived of serum, the expression of the smooth muscle forms of actin and myosin was relatively high. Conversely, in rapidly proliferating cultures at low density in 10% serum, smooth muscle contractile protein expression was low. The expression of nonmuscle myosin-B mRNA and protein was more stable and was upregulated only to a small degree in growing cells. Our results provide new insight into the molecular basis of differentiation and contractile function in airway smooth muscle cells.
Thrombin regulation of endothelin-1 gene in isolated human pulmonary endothelial cells
Authors: Golden Catherine L. , Nick Harry S. , Visner Gary A.
Published: December 2017

Endothelin (ET)-1 is a potent vasoconstrictor elicited from endothelial cells in response to a variety of stimuli and an important mediator for a variety of vascular diseases including pulmonary hypertension. In this paper, we describe the molecular regulation of the ET-1 gene in response to a vasoactive mediator, thrombin, in human pulmonary endothelial cells. Thrombin induces preproET-1 mRNA through a transcriptionally dependent mechanism, with a peak induction after 1 h of exposure. Analysis of chromatin structure identified several DNase I-hypersensitive regions under both basal and thrombin-stimulated conditions that reside in the 5′-promoter region, indicating that the ET-1 promoter is a constitutive promoter. Deletion analysis was employed as a functional assay to identify regions of the ET-1 promoter that are important in transcriptional regulation. We found that sites between −141 and −378 bp are essential for basal activity and that those between −378 and −484 bp are essential for thrombin-stimulated activity. However, full expression under both conditions required an element(s) within −952 bp.
Interferon-γ stimulates human Clara cell secretory protein production by human airway epithelial cells
Authors: Yao X. L. , Ikezono T. , Cowan M. , Logun C. , Angus C. W. , Shelhamer J. H.
Published: December 2017

Clara cell secretory protein (CCSP) is an inhibitor of secretory phospholipase A2. It is produced by airway epithelial cells and is present in airway secretions. Because interferon (IFN)-γ can induce gene expression in airway epithelial cells and may modulate the inflammatory response in the airway, it was of interest to study the effect of this cytokine on epithelial cell CCSP mRNA expression and CCSP protein synthesis. A human bronchial epithelial cell line (BEAS-2B) was used for this study. CCSP mRNA was detected by ribonuclease protection assay. IFN-γ was found to increase CCSP mRNA expression in a time- and dose-dependent manner. The CCSP mRNA level increased after IFN-γ (300 U/ml) treatment for 8–36 h, with the peak increase at 18 h. Immunobloting of CCSP protein also demonstrated that IFN-γ induced the synthesis and secretion of CCSP protein in a time-dependent manner. Nuclear run-on, CCSP reporter gene activity assay, and CCSP mRNA half-life assay demonstrated that IFN-γ-induced increases in CCSP gene expression were mediated, at least in part, at the posttranscriptional level. The present study demonstrates that IFN-γ can induce increases in steady-state mRNA levels and protein synthesis of human CCSP protein in airway epithelial cells and may modulate airway inflammatory responses in this manner.
cAMP protects endothelial barrier function independent of inhibiting MLC20-dependent tension development
Authors: Moy Alan B. , Bodmer James E. , Blackwell Ken , Shasby Sandy , Shasby D. Michael
Published: December 2017

Exposure of cultured human umbilical vein endothelial cells to the cAMP agonists theophylline and forskolin decreased constitutive isometric tension of a confluent monolayer inoculated on a collagen membrane, but it did not prevent increased tension in cells exposed to thrombin. The inability of cAMP agonists to prevent tension development correlated with an inability of cAMP stimulation to prevent increased 20-kDa myosin light chain (MLC20) phosphorylation in response to thrombin. Although cAMP did not prevent tension development or increased MLC20phosphorylation, cAMP attenuated the effect of thrombin on transendothelial electrical resistance across a confluent monolayer inoculated on a gold microelectrode. Activation of cAMP-dependent signal transduction did not prevent a decline in resistance in thrombin-treated cells, but it more promptly restored transendothelial resistance to initial basal levels (10 min) compared with thrombin only (60 min). ML-7, an MLC kinase antagonist, at doses that attenuate increased MLC20phosphorylation and tension development, did not prevent a decline in resistance in thrombin-treated cells. Yet, ML-7 also restored transendothelial resistance more rapidly than thrombin alone (20 min) but at a slower rate than cAMP. These data demonstrate that activation of cAMP-dependent signal transduction protects barrier function independent of inhibition of MLC20-dependent tension development.