bluuebunny/crossref_metadata_2025 huggingface dataset:
Neutrophil elastase stimulates human airway epithelial cells to produce PGE2through activation of p44/42 MAPK and upregulation of cyclooxygenase-2
Authors: Perng Diahn-Warng , Wu Yu-Chung , Tsai Mei-Chuan , Lin Ching-Ping , Hsu Wen-Hu , Perng Reury-Perng , Lee Yu-Chin
Published: March 2015

The responses of airway epithelium following exposure to neutrophil elastase (NE) were investigated. Human bronchial epithelial cells were explanted on insert surfaces of a modified air-liquid interface culture system to which NE was added to stimulate epithelial cells. PGE2release significantly increased within 10 min of incubation with NE and peaked 3 h after NE (20 μg/ml) stimulation. This action required proteolytic activity as α1-antitrypsin blocked NE-induced PGE2release. The production of PGE2was also inhibited by indomethacin; a selective cyclooxygenase (COX)-2 inhibitor, celecoxib; and dexamethasone. Moreover, the mRNA expression for COX-2 relative to that for a housekeeping gene was approximately eightfold that of the unstimulated cells. Dexamethasone inhibited COX-2 gene transcription. We further observed that NE-induced PGE2release involved activation of p44/42, but not p38, MAP kinases. Such p44/42 MAP kinases were rapidly phosphorylated, with the concentration of phosphorylated p44/42 MAP kinases peaking at 10 min after stimulation and declining in culture at 90 min. The specific p44/42 MAP kinase inhibitor UO126 completely blocked p44/42 phosphorylation and, subsequently, PGE2production. The airway epithelium may play important bronchoprotective and immunomodulatory roles in chronic neutrophilic inflammation.
Role of claudin interactions in airway tight junctional permeability
Authors: Coyne Carolyn B. , Gambling Todd M. , Boucher Richard C. , Carson Johnny L. , Johnson Larry G.
Published: March 2015

Airway epithelial tight junctions (TJs) serve to separate the external and internal environments of the lung. However, the members of the claudin family that mediate this function have not been fully delineated. We characterized the claudin expression in normal airways removed from human donors during lung transplantation and determined the contribution of each claudin to airway barrier function. Stable cell lines in NIH/3T3 and human airway (IB3.1) cells were constructed expressing the claudin components found in the human airway, claudin-1, -3, or -5. The effects of claudin expression on transepithelial resistance, permeability coefficients, and claudin-claudin interactions were assessed. Claudin-1 and -3 decreased solute permeability, whereas claudin-5 increased permeability. We also detected oligomerization of claudin-5 in cell lines and in freshly excised human airways. Coimmunoprecipitation studies revealed heterophilic interactions between claudin species in both cell lines and human airway epithelium. These suggest that airway TJs are regulated by claudinclaudin interactions that confer the selectivity of the junction.
Vanadium-induced HB-EGF expression in human lung fibroblasts is oxidant dependent and requires MAP kinases
Authors: Ingram Jennifer L. , Rice Annette B. , Santos Janine , Van Houten Bennett , Bonner James C.
Published: March 2015

Vanadium pentoxide (V2O5) is a transition metal derived from the burning of petrochemicals that causes airway fibrosis and remodeling. Vanadium compounds activate many intracellular signaling pathways via the generation of hydrogen peroxide (H2O2) or other reactive oxygen species. In this study, we investigated the regulation of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in human lung fibroblasts after V2O5treatment. V2O5-induced HB-EGF mRNA expression was abolished by N-acetyl-l-cysteine, suggesting an oxidant-mediated effect. Exogenous H2O2(>10 μM) mimicked the effect of V2O5in upregulating HB-EGF expression. Fibroblasts spontaneously released low levels of H2O2(1–2 μM), and the addition of V2O5depleted the endogenous H2O2pool within minutes. V2O5caused a subsequent increase of H2O2into the culture medium at 12 h. However, the burst of V2O5-induced H2O2occurred after V2O5-induced HB-EGF mRNA expression at 3 h, indicating that the V2O5-stimulated H2O2burst did not mediate HB-EGF expression. Either V2O5or H2O2activated ERK-1/2 and p38 MAP kinase. Inhibitors of the ERK-1/2 pathway (PD-98059) or p38 MAP kinase (SB-203580) significantly reduced either V2O5- or H2O2-induced HB-EGF expression. These data indicate that vanadium upregulates HB-EGF via ERK and p38 MAP kinases. The induction of HB-EGF is not related to a burst of H2O2in V2O5treated cells, yet the action of V2O5in upregulating HB-EGF is oxidant dependent and could be due to the reaction of V2O5with endogenous H2O2.
Laser-assisted microdissection and real-time PCR detect anti-inflammatory effect of perfluorocarbon
Authors: von der Hardt Katharina , Kandler Michael Andreas , Fink Ludger , Schoof Ellen , Dötsch Jörg , Bohle Rainer Maria , Rascher Wolfgang
Published: March 2015

The aim of this study was to identify cell types involved in the anti-inflammatory effect of ventilation with perfluorocarbon in vivo. Fifteen anesthetized, surfactant-depleted piglets received either aerosolized perfluorocarbon (Aerosol-PFC), partial liquid ventilation (rLV) at functional residual capacity (FRC) volume (FRC-PLV), or intermittent mandatory ventilation (control). After laser-assisted microdissection of different lung cell types, mRNA expression of IL-8 and ICAM-1 was determined using TaqMan real-time PCR normalized to hypoxanthine phosphoribosyltransferase (HPRT). IL-8 mRNA expression (means ± SE; control vs. Aerosol-PFC) was 356 ± 142 copies IL-8 mRNA/copy HPRT mRNA vs. 3.5 ± 1.8 in alveolar macrophages ( P <0.01); 208 ± 108 vs. 2.7 ± 0.8 in bronchiolar epithelial cells ( P <0.05); 26 ± 11 vs. 0.7 ± 0.2 in alveolar septum cells ( P <0.01); 2.8 ± 1.0 vs. 0.8 ± 0.4 in bronchiolar smooth muscle cells ( P <0.05); and 1.1 ± 0.4 vs. 0.2 ± 0.05 in vascular smooth muscle cells ( P <0.05). With FRC-PLV, IL-8/HPRT mRNA expression was significantly lower in macrophages, bronchiolar epithelial, and vascular smooth muscle cells. ICAM-1 mRNA expression in vascular endothelial cells remained unchanged. Predominantly, alveolar macrophages and bronchiolar epithelial cells were involved in the inflammatory pulmonary process. The anti-inflammatory effect of Aerosol-PFC was most pronounced.
Cyclic stretch activates ERK1/2 via G proteins and EGFR in alveolar epithelial cells
Authors: Correa-Meyer Eduardo , Pesce Liuska , Guerrero Carmen , Sznajder Jacob I.
Published: March 2015

Mechanical stimuli are transduced into intracellular signals in lung alveolar epithelial cells (AEC). We studied whether mitogen-activated protein kinase (MAPK) pathways are activated during cyclic stretch of AEC. Cyclic stretch induced a rapid (within 5 min) increase in extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation in AEC. The inhibition of Na+, L-type Ca2+and stretch-activated ion channels with amiloride, nifedipine, and gadolinium did not prevent the stretch-induced ERK1/2 activation. The inhibition of Grb2-SOS interaction with an SH3 binding sequence peptide, Ras with a farnesyl transferase inhibitor, and Raf-1 with forskolin did not affect the stretch-induced ERK1/2 phosphorylation. Moreover, cyclic stretch did not increase Ras activity, suggesting that stretch-induced ERK1/2 activation is independent of the classical receptor tyrosine kinase-MAPK pathway. Pertussis toxin and two specific epidermal growth factor receptor (EGFR) inhibitors (AG-1478 and PD-153035) prevented the stretch-induced ERK1/2 activation. Accordingly, in primary AEC, cyclic stretch activates ERK1/2 via G proteins and EGFR, in Na+and Ca2+influxes and Grb2-SOS-, Ras-, and Raf-1-independent pathways.
ClC-5: ontogeny of an alternative chloride channel in respiratory epithelia
Authors: Edmonds Rebecca D. , Silva Ian V. , Guggino William B. , Butler Robert B. , Zeitlin Pamela L. , Blaisdell Carol J.
Published: March 2015

Chloride transport is critical to many functions of the lung. Molecular defects in the best-known chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR), lead to impaired function of airway defensins, hydration of airway surface fluid, and mucociliary clearance leading to chronic lung disease, and premature death, but do not cause defects in lung development. We examined the expression of one member of the ClC family of volume- and voltage-regulated channels using the ribonuclease protection assay and Western blot analysis in rats. ClC-5 mRNA and protein are most strongly expressed in the fetal lung, and expression is maintained although downregulated postnatally. In addition, using immunocytochemistry, we find that ClC-5 is predominantly expressed along the luminal surface of the airway epithelium, suggesting that ClC-5 may participate in lung chloride secretion. Identifying candidate genes for critical ion transport functions is essential for understanding normal lung morphogenesis and the pathophysiology of several lung diseases. In addition, the manipulation of non-CFTR chloride channels may provide a viable approach for treating cystic fibrosis lung disease.
Core 2 oligosaccharides mediate eosinophil and neutrophil peritoneal but not lung recruitment
Authors: Broide David H. , Miller Marina , Castaneda Diego , Nayar Jyothi , Cho Jae Youn , Roman Mark , Ellies Lesley G. , Sriramarao P.
Published: March 2015

We have investigated the importance of cell-surface serine- and/or threonine-linked oligosaccharide adhesion molecules synthesized by the Golgi enzyme core 2 β-1,6- N-acetylglucosaminyltransferase (C2GlcNAcT) in mediating eosinophil trafficking to the lung in studies utilizing C2GlcNAcT-I-deficient mice. The number of bronchoalveolar eosinophils, the number of lung eosinophils, and airway responsiveness to methacholine were not significantly different in C2GlcNAcT-I-deficient compared with wild-type mice sensitized and challenged by inhalation with ovalbumin. C2GlcNAcT-I-deficient mice do not demonstrate defects in neutrophil trafficking to the lung in response to lipopolysaccharide (LPS). In contrast, ragweed-sensitized C2GlcNAcT-I-deficient mice exhibit significantly reduced eosinophil trafficking to the peritoneal cavity in response to ragweed peritoneal challenge. C2GlcNAcT-I-deficient mice also have significantly reduced neutrophil trafficking to the peritoneal cavity in response to LPS challenge. Overall, these studies demonstrate an important role for serine/threonine-linked oligosaccharides synthesized by the Golgi enzyme C2GlcNAcT-I in eosinophil and neutrophil trafficking to the peritoneum but not for eosinophil or neutrophil trafficking to the lung.
Recombinant human SP-A1 and SP-A2 proteins have different carbohydrate-binding characteristics
Authors: Oberley Rebecca E. , Snyder Jeanne M.
Published: March 2015

Surfactant protein (SP)-A is a member of the collectin family of proteins and plays a role in innate host defense of the lung. SP-A binds to the carbohydrates of lung pathogens via its calcium-dependant carbohydrate-binding domain. Native human alveolar SP-A consists of two distinct gene products: SP-A1 and SP-A2; however, only SP-A2 is expressed in the submucosal glands of the conducting airways. The function of the isolated SP-A2 protein is unknown. We hypothesized that SP-A1 and SP-A2 might have different carbohydrate-binding properties. In this study, we characterized the carbohydrate-binding specificities of native human alveolar SP-A and recombinant human SP-A1 and SP-A2 in the presence of either 1 or 5 mM Ca2+. We found that all of the SP-A proteins bind carbohydrates but with different affinities. All of the SP-A proteins bind to fucose with the greatest affinity. SP-A2 binds with a higher affinity to a wider variety of sugars than SP-A1 at either 1 or 5 mM Ca2+. These findings are suggestive that SP-A2 may interact with a greater variety of pathogens than native SP-A.
Impairment of macrophage survival by NaCl: implications for early pulmonary inflammation in cystic fibrosis
Authors: Kerby Gwendolyn S. , Cottin Vincent , Accurso Frank J. , Hoffmann Fukun , Chan Edward D. , Fadok Valerie A. , Riches David W. H.
Published: March 2015

Inflammation, characterized by the presence of proinflammatory chemokines and neutrophils, is a hallmark of early airway disease in infants with cystic fibrosis (CF), although the underlying mechanisms remain poorly defined. In this study, we evaluated the role of NaCl and the ensuing hyperosmolar effect on tumor necrosis factor (TNF)-α signaling and apoptosis in macrophages. Incubation of mouse macrophages with NaCl activated p38mapkand the p46jnkand p54jnkc- jun NH2-terminal kinase isoforms, but not p42mapk/erk2or Akt. Similar results were obtained with sorbitol, suggesting a general response to hyperosmolarity. Strikingly, the activation of p42mapk/erk2and Akt by TNF-α was also inhibited in the presence of NaCl. Because the activation of p42mapk/erk2and Akt has been associated with survival responses, we investigated the effect of NaCl on macrophage apoptosis. The results indicated a synergistic increase in apoptosis when macrophages were exposed to TNF-α in the presence of NaCl compared with stimulation with TNF-α alone or NaCl alone. Furthermore, pharmacological inhibition of p42mapk/erk2and Akt mimicked the effect of NaCl. Collectively, these findings indicate that modest elevations in NaCl differentially regulate the activation of mitogen-activated protein kinases and Akt and potentiate macrophage apoptosis. We speculate that augmentation of macrophage apoptosis in CF airways may result in decreased clearance of neutrophils and in deficiencies in the elimination of common CF pathogens.
Bronchial epithelial compression regulates MAP kinase signaling and HB-EGF-like growth factor expression
Authors: Tschumperlin Daniel J. , Shively Jonathan D. , Swartz Melody A. , Silverman Eric S. , Haley Kathleen J. , Raab Gerhard , Drazen Jeffrey M.
Published: March 2015

Airway smooth muscle constriction leads to the development of compressive stress on bronchial epithelial cells. Normal human bronchial epithelial cells exposed to an apical-to-basal transcellular pressure difference equivalent to the computed stress in the airway during bronchoconstriction demonstrate enhanced phosphorylation of extracellular signal-regulated kinase (ERK). The response is pressure dependent and rapid, with phosphorylation increasing 14-fold in 30 min, and selective, since p38 and c-Jun NH2-terminal kinase phosphorylation remains unchanged after pressure application. Transcellular pressure also elicits a ninefold increase in expression of mRNA encoding heparin-binding epidermal growth factor-like growth factor (HB-EGF) after 1 h, followed by prominent immunostaining for pro-HB-EGF after 6 h. Inhibition of the ERK pathway with PD-98059 results in a dose-dependent reduction in pressure-induced HB-EGF gene expression. The magnitude of the HB-EGF response to transcellular pressure and tumor necrosis factor (TNF)-α (1 ng/ml) is similar, and the combined mechanical and inflammatory stimulus is more effective than either stimulus alone. These results demonstrate that compressive stress is a selective and potent activator of signal transduction and gene expression in bronchial epithelial cells.
Regulation of surfactant proteins by LPS and proinflammatory cytokines in fetal and newborn lung
Authors: Väyrynen Outi , Glumoff Virpi , Hallman Mikko
Published: March 2015

Intra-amniotic lipopolysaccharide (LPS) and cytokines may decrease respiratory distress syndrome (RDS) and increase chronic lung disease in the newborn. The aim was to identify the primary inflammatory mediators regulating the expression of surfactant proteins (SP) in explants from immature (22-day-old fetus) and mature (30-day term fetus and 2-day-old newborn) rabbits. In immature lung, interleukin (IL)-1α and IL-1β upregulated the expression of SP-A and SP-B. These effects of IL-1 were diminished, and SP-C mRNA was suppressed additively in the presence of tumor necrosis factor (TNF)-α and either LPS or interferon (IFN)-γ. LPS, TNF-α, or IFN-γ had no effect alone. In explants from the term fetus and the newborn, LPS, IL-1α, and TNF-α additively suppressed the SPs. LPS acutely induced IL-1α in alveolar macrophages in mature lung but not in the immature lung. IFN-γ that generally has low expression in intrauterine infection decreased the age dependence of the other agonists' effects on SPs. The present study serves to explain the variation of the pulmonary outcome after an inflammatory insult. We propose that IL-1 from extrapulmonary sources induces the SPs in premature lung and is responsible for the decreased risk of RDS in intra-amniotic infection.
Phosphatidylinositol 3-kinase but not tuberin is required for PDGF-induced cell migration
Authors: Irani Carla , Goncharova Elena A. , Hunter Deborah S. , Walker Cheryl L. , Panettieri Reynold A. , Krymskaya Vera P.
Published: March 2015

The loss of function of the tumor suppressor gene TSC2 and its protein product tuberin promotes the development of benign lesions by stimulating cell growth, although the role of tuberin in regulating cell migration and metastasis has not been characterized. In addition, the role of phosphatidylinositol 3-kinase (PI 3-kinase), an important signaling event regulating cell migration, in modulating tuberin-deficient cell motility remains unknown. Using a tuberin-deficient rat smooth muscle cell line, ELT3, we demonstrate that platelet-derived growth factor (PDGF) stimulates cell migration by 3.2-fold, whereas vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-α, and basic fibroblast growth factor (bFGF) increase migration by 2.1-, 2.1-, and 2.6-fold, respectively. Basal and PDGF-induced migration in tuberin-deficient ELT3, ELT4, and ERC15 cells was not significantly different from that of tuberin-positive transformed rat kidney epithelial 2, airway smooth muscle, and pulmonary arterial vascular smooth muscle cells. Expression of tuberin in tuberin-deficient ELT3 cells also had little effect on cell migration. In parallel experiments, the role of PI 3-kinase activation in ELT3 cell migration was investigated. LY-294002, a PI 3-kinase inhibitor, decreased PDGF-induced migration in a concentration-dependent manner with an IC50of ∼5 μM. LY-294002 also abrogated ELT3 cell migration stimulated by bFGF and TGF-α but not by VEGF and phorbol 12-myristate 13-acetate. Furthermore, transient expression of constitutively active PI 3-kinase (p110*) was sufficient to induce ELT3 cell migration. However, the migration induced by p110* was less than that induced by growth factors, suggesting other signaling pathways are also critically important in modulating growth factor-induced cell migration. These data suggest that PI 3-kinase is required for growth factor-induced cell migration and loss of tuberin appears to have little effect on cell migration.
Vascular regulation of type II cell exocytosis
Authors: Wang P. M. , Fujita E. , Bhattacharya J.
Published: March 2015

To determine whether lung capillary pressure regulates surfactant secretion, we viewed alveoli of the constantly inflated, isolated blood-perfused rat lung by fluorescence microscopy. By alveolar micropuncture we infused fura 2 and lamellar body (LB)-localizing dyes for fluorescence detection of, respectively, the alveolar cytosolic Ca2+concentration ([Ca2+]i) and type II cell exocytosis. Increasing left atrial pressure (Pla) from 5 to 10 cmH2O increased septal capillary diameter by 26% and induced marked alveolar [Ca2+]ioscillations that abated on relief of pressure elevation. The rate of loss of LB fluorescence that reflects the LB exocytosis rate increased fourfold after the pressure elevation and continued at the same rate even after pressure and [Ca2+]ioscillations had returned to baseline. In alveoli pretreated with either 1,2-bis(2-aminophenoxy)ethane- N,N,N′,N′-tetraacetic acid-AM, the intracellular Ca2+chelator, or heptanol, the gap junctional blocker, the pressure-induced exocytosis was completely inhibited. We conclude that capillary pressure and surfactant secretion are mechanically coupled. The secretion initiates in a Ca2+-dependent manner but is sustained by Ca2+-independent mechanisms.
Role of interferon-γ in the evolution of murine bleomycin lung fibrosis
Authors: Segel Michael J. , Izbicki Gabriel , Cohen Pazit Y. , Or Reuven , Christensen Thomas G. , Wallach-Dayan Shulamit B. , Breuer Raphael
Published: March 2015

IFN-γ production is upregulated in lung cells (LC) of bleomycin-treated C57BL/6 mice. The present study characterizes the time course, cellular source, and regulation of IFN-γ expression in bleomycin-induced lung injury. IFN-γ mRNA in LC from bleomycin-treated mice peaked 3 days after intratracheal instillation. IFN-γ protein levels were increased at 6 days, as was the percentage of LC expressing IFN-γ. CD4+, CD8+, and natural killer cells each contributed significantly to IFN-γ production. IL-12 mRNA levels were increased at 1 day in LC of bleomycin-treated mice. Anti-IL-12 and anti-IL-18 antibodies decreased IFN-γ production by these cells. To define the role of endogenous IFN-γ in the evolution of bleomycin lung injury, we compared the effect of bleomycin in mice with a targeted knockout mutation of the IFN-γ gene (IFN-γ knockout) and wild-type mice. At 14 days after intratracheal bleomycin, total bronchoalveolar lavage cell counts and lung hydroxyproline were decreased in IFN-γ knockouts compared with wild-type animals. There was no difference in morphometric parameters of fibrosis. Our data show that enhanced IFN-γ production in the lungs of bleomycin-treated mice is at least partly IL-12 and IL-18 dependent. Absence of IFN-γ in IFN-γ knockout mice does not increase pulmonary fibrosis. Endogenous IFN-γ may play a proinflammatory or profibrotic role in bleomycin-induced lung fibrosis.
Src modulates serotonin-induced calcium signaling by regulating phosphatidylinositol 4,5-bisphosphate
Authors: Tolloczko Barbara , Turkewitsch Petra , Choudry Sofia , Bisotto Sandra , Fixman Elizabeth D. , Martin James G.
Published: March 2015

We tested the hypothesis that, in airway smooth muscle cells, stimulation of G-protein-coupled receptors by contractile agonists activates Src kinase and that this kinase modulates cell contractility and Ca2+signaling by affecting the levels of the phospholipase C substrate phosphatidylinositol 4,5-bisphosphate (PIP2). Stimulation of cultured rat tracheal smooth muscle cells with serotonin (5-HT) induced an increase in Src activity, Ca2+mobilization, and contraction (decrease in cell area). 5-HT-evoked cell contraction was reduced by a specific inhibitor of Src family kinases, 4-amino-5(4-methylphenyl)-7-( t-butyl)pyrazolo[3,4 -d]pyrimidine (PP1). Peak Ca2+responses to 5-HT were attenuated by PP1 and an anti-Src-blocking antibody and augmented by expression of constitutively activated Y529F Src. Sustained phases of Ca2+responses to 5-HT and Ca2+influx resulting from emptying of Ca2+stores in the endoplasmic reticulum by thapsigargin were also decreased after PP1 treatment. PP1 significantly reduced the turnover of inositol phosphates produced on 5-HT stimulation and the amount of PIP2in the Triton X-100-insoluble lipid fraction. Overall, these data demonstrate that, in rat tracheal smooth muscle cells, Src kinase modulates 5-HT-evoked cell contractility and Ca2+signaling by regulating PIP2levels and Ca2+influx.
Increased expression of collagen-binding heat shock protein 47 in murine bleomycin-induced pneumopathy
Authors: Ishii Hiroshi , Mukae Hiroshi , Kakugawa Tomoyuki , Iwashita Tetsuji , Kaida Hideyuki , Fujii Takeshi , Hayashi Tomayoshi , Kadota Jun-ichi , Kohno Shigeru
Published: March 2015

The 47-kDa heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that has been shown to play a major role during the processing and/or secretion of procollagen. Expression of HSP47 has been reported to increase in parallel with expression of collagens during the progression of various fibrosis models. The aim of the present study was to investigate the association between HSP47 expression and collagen accumulation in bleomycin (BLM)-induced murine fibrosis. We investigated the expression of HSP47 protein and mRNA using immunohistochemical analysis and semi-quantitative RT-PCR in murine BLM-induced pulmonary fibrosis. Immunohistochemical analysis showed that higher expression of HSP47 protein was present in BLM-induced pulmonary fibrosis compared with controls. HSP47 was localized predominantly in α-smooth muscle actin-positive myofibroblasts, F4/80 negative, surfactant protein-A-positive type II pneumocytes, and F4/80-positive macrophages. RT-PCR also demonstrated an increase of HSP47 mRNA expression in BLM-treated lungs. Moreover, the relative amounts of HSP47 mRNA correlated significantly with the lung hydroxyproline content as an indicator of pulmonary fibrosis in BLM-treated lungs ( r = 0.406, P <0.05). Our results suggest that these cells may play a role in the fibrotic process of BLM-treated lungs through upregulation of HSP47.
Role of sphingosine-1 phosphate in the enhancement of endothelial barrier integrity by platelet-released products
Authors: Schaphorst Kane L. , Chiang Eddie , Jacobs Keri N. , Zaiman Ari , Natarajan Viswanathan , Wigley Frederick , Garcia Joe G. N.
Published: March 2015

In vitro and in vivo evidence indicates that circulating platelets affect both vascular integrity and hemostasis. How platelets enhance the permeability barrier of the vascular endothelium is not well understood. We measured the effect of isolated human platelets on human pulmonary artery endothelial cell (EC) barrier integrity by monitoring transmonolayer electrical resistance. EC barrier function was significantly increased by the addition of platelets (∼40% maximum, 2.5 × 106platelets/ml). Platelet supernatants, derived from 2.5 × 106platelets/ml, reproduced the barrier enhancement and reversed the barrier dysfunction produced by the edemagenic agonist thrombin, which implicates a soluble barrier-promoting factor. The barrier-enhancing effect of platelet supernatants was heat stable but was attenuated by either charcoal delipidation (suggesting a vasoactive lipid mediator) or pertussis toxin, implying involvement of a Giα-coupled receptor signal transduction pathway. Sphingosine-1-phosphate (S1P), a sphingolipid that is released from activated platelets, is known to ligate G protein-coupled EC differentiation gene (EDG) receptors, increase EC electrical resistance, and reorganize the actin cytoskeleton (Garcia JG, Liu F, Verin AD, Birukova A, Dechert MA, Gerthoffer WT, Bamberg JR, and English D. J Clin Invest 108: 689–701, 2001). Infection of EC with an adenoviral vector expressing an antisense oligonucleotide directed against EDG-1 but not infection with control vector attenuated the barrier-enhancing effect of both platelet supernatants and S1P. These results indicate that a major physiologically relevant vascular barrier-protective mediator produced by human platelets is S1P.
Angiogenic factors and alveolar vasculature: development and alterations by injury in very premature baboons
Authors: Maniscalco William M. , Watkins Richard H. , Pryhuber Gloria S. , Bhatt Abhay , Shea Colleen , Huyck Heidie
Published: March 2015

Proper formation of the pulmonary microvasculature is essential for normal lung development and gas exchange. Lung microvascular development may be disrupted by chronic injury of developing lungs in clinical diseases such as bronchopulmonary dysplasia. We examined microvascular development, angiogenic growth factors, and endothelial cell receptors in a fetal baboon model of chronic lung disease (CLD). In the last third of gestation, the endothelial cell marker platelet endothelial cell adhesion molecule (PECAM)-1 increased 7.5-fold, and capillaries immunostained for PECAM-1 changed from a central location in airspace septa to a subepithelial location. In premature animals delivered at 67% of term and supported with oxygen and ventilation for 14 days, PECAM-1 protein and capillary density did not increase, suggesting failure to expand the capillary network. The capillaries of the CLD animals were dysmorphic and not subepithelial. The angiogenic growth factor vascular endothelial growth factor (VEGF) and its receptor fms-like tyrosine kinase receptor (Flt-1) were significantly decreased in CLD. Angiopoietin-1, another angiogenic growth factor, and its receptor tyrosine kinase with immunoglobulin and epidermal growth factor homology domains were not significantly changed. These data suggest that CLD impairs lung microvascular development and that a possible mechanism is disruption of VEGF and Flt-1 expression.
Pulmonary PKG-1 is upregulated following chronic hypoxia
Authors: Jernigan Nikki L. , Walker Benjimen R. , Resta Thomas C.
Published: March 2015

Recent studies from our laboratory indicate that pulmonary vasodilatory responses to exogenous nitric oxide (NO) are attenuated following chronic hypoxia (CH) and that this NO-dependent vasodilation is mediated by cGMP. Similarly, we have demonstrated that CH attenuates vasodilatory responses to the cGMP analog 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP). We hypothesized that attenuated pulmonary vasodilation to 8-BrcGMP following CH is mediated by decreased protein kinase G-1 (PKG-1) expression/activity. Therefore, we examined vasodilatory responses to 8-BrcGMP (1 μM) in isolated, saline-perfused lungs from control and CH (4 wk at barometric pressure of 380 mmHg) rats in the presence of the competitive PKG inhibitor Rp-β-phenyl-1, N2-etheno-8-bromoguanosine 3',5'-cyclic monophosphorothionate (30 μM) or the highly specific PKG inhibitor KT-5823 (10 μM). PKG-1 expression and activity were determined in whole lung homogenates from each group, and vascular PKG-1 levels were assessed by quantitative immunohistochemistry. PKG inhibition with either Rp-8-Br-PET-cGMPS or KT-5823 diminished vasodilatory responses to 8-BrcGMP in lungs from both control and CH rats, thus indicating a role for PKG in mediating reactivity to 8-BrcGMP in each group. However, in contrast to our hypothesis, PKG-1 levels were approximately twofold greater in lungs from CH rats vs. controls, and furthermore, this upregulation was localized to the vasculature. This correlates with an increase in PKG activity following CH. We conclude that PKG-1 is involved in 8-BrcGMP-mediated vasodilation; however, attenuated pulmonary vasodilation following CH is not associated with decreased expression/activity of PKG-1.
Polarity of alveolar epithelial cell acid-base permeability
Authors: Joseph Dilip , Tirmizi Omar , Zhang Xiao-Ling , Crandall Edward D. , Lubman Richard L.
Published: March 2015

We investigated acid-base permeability properties of electrically resistive monolayers of alveolar epithelial cells (AEC) grown in primary culture. AEC monolayers were grown on tissue culture-treated polycarbonate filters. Filters were mounted in a partitioned cuvette containing two fluid compartments (apical and basolateral) separated by the adherent monolayer, cells were loaded with the pH-sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, and intracellular pH was determined. Monolayers in HCO[Formula: see text]-free Na+buffer (140 mM Na+, 6 mM HEPES, pH 7.4) maintained a transepithelial pH gradient between the two fluid compartments over 30 min. Replacement of apical fluid by acidic (6.4) or basic (8.0) buffer resulted in minimal changes in intracellular pH. Replacement of basolateral fluid by acidic or basic buffer resulted in transmembrane proton fluxes and intracellular acidification or alkalinization. Intracellular alkalinization was blocked ≥80% by 100 μM dimethylamiloride, an inhibitor of Na+/H+exchange, whereas acidification was not affected by a series of acid/base transport inhibitors. Additional experiments in which AEC monolayers were grown in the presence of acidic (6.4) or basic (8.0) medium revealed differential effects on bioelectric properties depending on whether extracellular pH was altered in apical or basolateral fluid compartments bathing the cells. Acid exposure reduced (and base exposure increased) short-circuit current from the basolateral side; apical exposure did not affect short-circuit current in either case. We conclude that AEC monolayers are relatively impermeable to transepithelial acid/base fluxes, primarily because of impermeability of intercellular junctions and of the apical, rather than basolateral, cell membrane. The principal basolateral acid exit pathway observed under these experimental conditions is Na+/H+exchange, whereas proton uptake into cells occurs across the basolateral cell membrane by a different, undetermined mechanism. These results are consistent with the ability of the alveolar epithelium to maintain an apical-to-basolateral (air space-to-blood) pH gradient in situ.
MAPK activation is involved in posttranscriptional regulation of RSV-induced RANTES gene expression
Authors: Pazdrak Konrad , Olszewska-Pazdrak Barbara , Liu Tianshuang , Takizawa Ryuta , Brasier Allan R. , Garofalo Roberto P. , Casola Antonella
Published: March 2015

Airway epithelial cells represent the primary cell target of respiratory syncytial virus (RSV) infection. They actively participate in the lung immune/inflammatory response that follows RSV infection by expressing chemokines, small chemotactic cytokines that recruit and activate leukocytes. Regulated on activation, normal T cell expressed, and presumably secreted (RANTES) is a member of the CC chemokine subfamily and is strongly chemotactic for T lymphocytes, monocytes, basophils, and eosinophils, cell types that are present or activated in the inflammatory infiltrate that follows RSV infection of the lung. RSV infection of airway epithelial cells induces RANTES expression by increasing gene transcription and stabilizing RNA transcripts. The signaling pathway regulating RANTES gene expression after RSV infection has not been determined. In this study, we examined the role of extracellular signal-regulated kinase (ERK) and p38, members of the mitogen-activated protein (MAP) kinase (MAPK) family, in RSV-induced RANTES production. RSV infection of alveolar epithelial cells induced increased phosphorylation and catalytic activity of ERK and the upstream kinases Raf-1 and MAP ERK kinase. Induction of the MAP signaling cascade required a replication-competent virus. RSV infection of alveolar epithelial cells also induced activation of p38 MAPK. Inhibition of ERK and p38 activation significantly reduced RSV-induced RANTES mRNA and protein secretion without affecting RANTES gene transcription or transcription factor activation. These results indicate that the MAPK signaling cascade regulates RANTES production in alveolar epithelial cells through a posttranscriptional mechanism.
Pulmonary lymphatics and edema accumulation after brief lung injury
Authors: Schraufnagel Dean E. , Agaram Narasimhan P. , Faruqui Aamir , Jain Sajal , Jain Leena , Ridge Karen M. , Sznajder Jacob Iasha
Published: March 2015

In a past study of hyperoxia-induced lung injury, the extensive lymphatic filling could have resulted from lymphatic proliferation or simple lymphatic recruitment. This study sought to determine whether brief lung injury could produce similar changes, to show which lymphatic compartments fill with edema, and to compare their three-dimensional structure. Tracheostomized rats were ventilated at high tidal volume (12–16 ml) or low tidal volume (3–5 ml) or allowed to breathe spontaneously for 25 min. Light microscopy showed more perivascular, interlobular septal, and alveolar edema in the animals ventilated at high tidal volume ( P< 0.0001). Scanning electron microscopy of lymphatic casts showed extensive filling of the perivascular lymphatics in the group ventilated at high tidal volume ( P < 0.01), but lymphatic filling was greater in the nonventilated group than in the group that was ventilated at low tidal volume ( P < 0.01). The three-dimensional structures of the cast interlobular and perivascular lymphatics were similar. There was little filling and no difference in pleural lymphatic casts among the three groups. More edema accumulated in the surrounding lymphatics of larger blood vessels than smaller blood vessels. Brief high-tidal-volume lung injury caused pulmonary edema similar to that caused by chronic hyperoxic lung injury, except it was largely restricted to perivascular and septal lymphatics and prelymphatic spaces.
Bleomycin upregulates expression of γ-glutamylcysteine synthetase in pulmonary artery endothelial cells
Authors: Day Regina M. , Suzuki Yuichiro j. , Lum Julie M. , White Alexander C. , Fanburg Barry L.
Published: March 2015

The chemotherapeutic agent bleomycin induces pulmonary fibrosis through the generation of reactive oxygen species (ROS), which are thought to contribute to cellular damage and pulmonary injury. We hypothesized that bleomycin activates oxidative stress response pathways and regulates cellular glutathione (GSH). Bovine pulmonary artery endothelial cells exposed to bleomycin exhibit growth arrest and increased cellular GSH content. γ-Glutamylcysteine synthetase (γ-GCS) controls the key regulatory step in GSH synthesis, and Northern blots indicate that the γ-GCS catalytic subunit [γ-GCS heavy chain (γ-GCSh)] is upregulated by bleomycin within 3 h. The promoter for human γ-GCShcontains consensus sites for nuclear factor-κB (NF-κB) and the antioxidant response element (ARE), both of which are activated in response to oxidative stress. Electrophoretic mobility shift assays show that bleomycin activates the transcription factor NF-κB as well as the ARE-binding factors Nrf-1 and -2. Nrf-1 and -2 activation by bleomycin is inhibited by the ROS quenching agent N-acetylcysteine (NAC), but not by U-0126, a MEK1/2 inhibitor that blocks bleomycin-induced MAPK activation. In contrast, NF-κB activation by bleomycin is inhibited by U-0126, but not by NAC. NAC and U-0126 both inhibit bleomycin-induced upregulation of γ-GCS expression. These data suggest that bleomycin can activate oxidative stress response pathways and upregulate cellular GSH.
Transcriptional regulation of the hamsterMuc1gene: identification of a putative negative regulatory element
Authors: Lee Insong James , Hyun Sang Won , Nandi Asit , Kim K. Chul
Published: March 2015

The mucin gene Muc1 is expressed in glandular epithelial cells and is involved in lubricative and protective functions. It is also overexpressed in many carcinomas including breast and lung cancer cells. To study the transcriptional regulation of Muc1, we cloned a 2.4-kb fragment containing the promoter region of the hamster Muc1 gene and analyzed it for its ability to mediate transcription. Transcriptional initiation was localized to 22 base pairs downstream of the TATA box. We performed functional analysis of the Muc1 promoter in hamster (HP-1 and Chinese hamster ovary) and human cells (MCF-7, A549, and BEAS-2B) using deletion/reporter constructs. A positive regulatory region between bases −555 and −252 and a putative negative regulatory element (P-NRE) between nucleotides −1,652 and −1,614 were found to be active in transfected cells. The P-NRE contains a yin yang 1 (YY1) transcription factor binding site, and electrophoretic mobility shift assays with HP-1 cell nuclear extract revealed the binding of YY1 to this site. Our data suggest that YY1 may play an inhibitory role in the transcription of the Muc1 gene.
CFTR activation in human bronchial epithelial cells by novel benzoflavone and benzimidazolone compounds
Authors: Caci Emanuela , Folli Chiara , Zegarra-Moran Olga , Ma Tonghui , Springsteel Mark F. , Sammelson Robert E. , Nantz Michael H. , Kurth Mark J. , Verkman A. S. , Galietta Luis J. V.
Published: March 2015

Activators of the CFTR Cl-channel may be useful for therapy of cystic fibrosis. Short-circuit current ( Isc) measurements were done on human bronchial epithelial cells to characterize the best flavone and benzimidazolone CFTR activators identified by lead-based combinatorial synthesis and high-throughput screening. The 7,8-benzoflavone UCcf-029 was a potent activator of Cl-transport, with activating potency (<1 μM) being much better than other flavones, such as apigenin. The benzimidazolone UCcf-853 gave similar Iscbut with lower potency (5–20 μM). In combination, the effect induced by maximal UCcf-029 and UCcf-029, UCcf-853, and apigenin increased strongly with increasing basal CFTR activity: for example, Kdfor activation by UCcf-029 decreased from >5 to <0.4 μM with increasing basal Iscfrom ∼4 μA/cm2to ∼12 μA/cm2. This dependence was confirmed in permeabilized Fischer rat thyroid cells stably expressing CFTR. Our results demonstrate efficacy of novel CFTR activators in bronchial epithelia and provide evidence that activating potency depends on basal CFTR activity.
Diesel exhaust particles upregulate eotaxin gene expression in human bronchial epithelial cells via nuclear factor-κB-dependent pathway
Authors: Takizawa Hajime , Abe Shinji , Okazaki Hitoshi , Kohyama Tadashi , Sugawara Isamu , Saito Yoshinobu , Ohtoshi Takayuki , Kawasaki Shin , Desaki Masashi , Nakahara Kazuhiko , Yamamoto Kazuhiko , Matsushima Kouji , Tanaka Mitsuru , Sagai Masaru , Kudoh Shoji
Published: March 2015

Fine particles derived from diesel engines, diesel exhaust particles (DEP), have been shown to augment gene expression of several inflammatory cytokines in human airway epithelial cells in vitro. However, it remains unclear whether or not DEP have any effect on the expression and production of eotaxin, an important chemokine involved in eosinophil recruitment into the airways. We studied the effects of DEP by using a conventional suspended DEP and by a recently established in vitro cell exposure system to diesel exhaust (Abe S, Takizawa H, Sugawara I, and Kudoh S, Am J Respir Cell Mol Biol 22: 296–303, 2000). DEP showed a dose-dependent stimulatory effect on eotaxin production by normal human peripheral airway epithelial cells as well as by bronchial epithelial cell line BET-1A as assessed by specific ELISA. mRNA levels increased by DEP were shown by RT-PCR. DEP showed an additive effect on IL-13-stimulated eotaxin expression. DEP induced NF-κB activation by EMSA as previously reported but did not induce signal transducer and activator of transcription (STAT) 6 activation according to Western blot analysis. Finally, antioxidant agents ( N-acetyl cysteine and pyrrolidine dithiocarbamate), which inhibited NF-κB activation but failed to affect STAT6 activation, almost completely attenuated DEP-induced eotaxin production, whereas these agents failed to attenuate IL-13-induced eotaxin production. These findings suggested that DEP stimulated eotaxin gene expression via NF-κB-dependent, but STAT6-independent, pathways.
Mucin secretion and PKC isoforms in SPOC1 goblet cells: differential activation by purinergic agonist and PMA
Authors: Abdullah Lubna H. , Bundy Jason T. , Ehre Camille , Davis C. William
Published: March 2015

SPOC1 cells, which are a mucin-secreting model of rat airway goblet cells, possess a luminal P2Y2purinoceptor through which UTP, ATP, and ATPγS stimulate secretion with EC50values of ∼3 μM. PMA elicits mucin secretion with high EC50(75 nM) and saturation (300 nM) values. For the first time in airway mucin-secreting cells, the PKC isoforms expressed and activated by a secretagogue were determined using RT-PCR/restriction-enzyme mapping and Western blotting. Five isoforms were expressed: cPKCα, nPKCδ and -η, and aPKCζ and -ι/λ. PMA caused cPKCα and nPKCδ to translocate to the membrane fraction of SPOC1 cells; only nPKCδ so responded to ATPγS. Membrane-associated nPKCδ and mucin secretion increased in parallel with ATPγS concentration and yielded EC50values of 2–3 μM and maximum values of 100 μM. Effects of PMA to increase membrane-associated cPKCα and nPKCδ saturated at 30 nM, whereas mucin secretion saturated at 300 nM, which suggests a significant PKC-independent effect of PMA on mucin secretion. A prime alternate phorbol ester-receptor candidate is the C1-domain protein MUNC13. RT-PCR revealed the expression of ubiquitous (ub)MUNC13-2 and its binding partner, DOC2-γ. Hence, P2Y2agonists activate nPKCδ in SPOC1 cells. PMA activates cPKCα and nPKCδ at high affinity and stimulates a lower affinity PKC-independent pathway that leads to mucin secretion.
Thickness of the blood-gas barrier in premature and 1-day-old newborn rabbit lungs
Authors: Fu Zhenxing , Heldt Gregory P. , West John B.
Published: March 2015

The pulmonary capillaries of neonatal lungs are potentially vulnerable to stress failure because of the complex changes in the pulmonary circulation that occur at birth. We studied the ultrastructure of the blood-gas barrier (BGB) in premature and 1-day-old rabbit lungs and compared it with the ultrastructure of adult lungs. Normal gestation of rabbits is 30 days. After extensive pilot measurements, three premature (27 days gestation) and three newborn (1 day old) rabbit lungs were perfusion-fixed at arterial, venous, and airway pressures of 25, 0, and 10 cmH2O, respectively, and the measurements were compared with those of three adult lungs. The thickness of the capillary endothelium, alveolar epithelium, and interstitium of the BGB was measured at right angles to the barrier at random points. A striking finding was the large number of measurements of the interstitial thickness in 1-day-old lungs that were very thin (0–0.1 μm). The percentages of occurrence of very thin interstitium in premature, 1-day-old, and adult lungs were 35.3 ± 9.4, 71.7 ± 5.2, and 43.0 ± 2.6, respectively ( P < 0.02 for 1 day old vs. premature and adult). Given the previously found relationship between stress failure and interstitial thickness, this large proportion of very thin interstitial layers in the capillaries of 1-day-old lungs is a reasonable explanation for their previously demonstrated vulnerability to stress failure.
Protective role of retinoic acid from antiproliferative action of TNF-α on lung epithelial cells
Authors: Besnard Valérie , Nabeyrat Elodie , Henrion-Caude Alexandra , Chadelat Katarina , Perin Laurence , Le Bouc Yves , Clement Annick
Published: March 2015

Tumor necrosis factor (TNF)-α is a key molecule in lung inflammation. We have established the insulin-like growth factor binding protein 2 (IGFBP-2) as a marker associated with the growth arrest of lung alveolar epithelial cells (AEC). Here, we studied the effects of TNF-α on AEC proliferation and the putative protective role of retinoic acid (RA). We documented an antiproliferative action of TNF-α that was reversible only at 24 h and then became irreversible with induction of apoptosis. TNF-α treatment was associated with a dramatic induction of IGFBP-2. To discover the mechanism of action of IGFBP-2, we further tested the mitogenic potential of IGF-I to counteract TNF-α inhibition. Addition of IGF-I to the TNF-α containing medium did not stimulate proliferation, whereas des(1–3)IGF-I, an analog of IGF-I that bears low affinity for IGFBPs, was able to restore cell growth. Interestingly, we observed that RA abrogated TNF-α-induced growth arrest and that this effect was associated with a dramatic decrease in IGFBP-2 expression. These results suggest a protective role of RA from TNF-α antiproliferative action, through mechanisms involving modulation of IGFBP-2 production.