bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of mucin release from airway goblet cells by polycationic peptides
Authors: Ko Kwang Ho , Lee Choong Jae , Shin Chan Young , Jo Mijeong , Kim K. Chul
Published: December 2017

In the present study, we investigated whether polycationic peptides affect mucin release from cultured airway goblet cells. Confluent primary hamster tracheal surface epithelial cells were metabolically radiolabeled with [3H]glucosamine for 24 h and chased for 30 min in the presence of varying concentrations of either poly-l-arginine (PLA) or poly-l-lysine (PLL) to assess the effects on [3H]mucin release. Possible cytotoxicity by the polycations was assessed by measuring lactate dehydrogenase release,51Cr release, and cell exfoliation. The results were as follows: 1) both PLA and PLL inhibited mucin release in a dose-dependent fashion; 2) there was no significant difference in either lactate dehydrogenase release,51Cr release, or the number of floating cells between control and treatment groups; 3) the effects of both PLA and PLL on mucin release were completely blocked by neutralizing the positive charges either by pretreatment with heparin or by N-acetylation of the polycations; and 4) both PLA and PLL completely masked the stimulatory effect of ATP on mucin release. We conclude that these polycationic peptides can inhibit mucin release from airway goblet cells without any apparent cytotoxicity, and the inhibitory effect seems to be attributable to their positive charges. These are the first nonsteroidal agents, to the best of our knowledge, that have been shown to inhibit mucin release from airway goblet cells.
Differential NF-κB activation after intratracheal endotoxin
Authors: Blackwell Timothy S. , Lancaster Lisa H. , Blackwell Thomas R. , Venkatakrishnan Annapurna , Christman John W.
Published: December 2017

We examined the relationship between nuclear factor (NF)-κB DNA binding activity, cytokine gene expression, and neutrophilic alveolitis in rats after intratracheal (IT) instillation of endotoxin [lipopolysaccharide (LPS)]. NF-κB activation in lung tissue mirrored neutrophilic alveolitis after IT LPS instillation, with NF-κB activation and neutrophilic influx beginning 2 h after IT LPS doses of 0.01 mg/kg or greater. In lung lavage fluid cells, however, transient NF-κB activation was present in alveolar macrophages by 15 min after IT LPS instillation, followed by a second peak of NF-κB activation corresponding to the onset on neutrophilic alveolitis. For cytokines thought to be NF-κB dependent, two different patterns of mRNA expression were found. Interleukin (IL)-1α, IL-1β, and tumor necrosis factor-α showed increased mRNA by 30 min after IT LPS instillation, but IL-6- and cytokine-induced neutrophil chemoattractant mRNAs were not substantially increased until 2 h after IT LPS instillation. Therefore, IT LPS causes differential NF-κB activation in air space cells and lung tissue, which likely determines production of key cytokines and directs the evolution of neutrophilic alveolitis.
Induction of stress proteins in human endothelial cells by heavy metal ions and heat shock
Authors: Wagner M. , Hermanns I. , Bittinger F. , Kirkpatrick C. J.
Published: December 2017

In the present study, we compared the induction of heat shock proteins (HSPs) by heat and heavy metal ions in three different endothelial cell types, namely, human umbilical vein endothelial cells, human pulmonary microvascular endothelial cells, and the cell line EA.hy 926. Our results show that especially Zn2+and Cd2+are inducers of 70-kDa (HSP70), 60-kDa (HSP60), 32-kDa (HSP32), and 27-kDa (HSP27) HSPs. The strength of inducibility is specific for each HSP. Ni2+and Co2+only show an inducible effect at very high concentrations, that is, in the clearly cytotoxic range. Furthermore, we investigated the time course of HSP expression and the involvement of heat shock factor-1. Our study demonstrates that the three endothelial cell types that were under investigation show comparable stress protein expression when treated with heavy metal ions or heat shock. The expression of stress proteins may be used as an early marker for the toxic damage of cells. This damage can be an inducer of acute respiratory distress syndrome in which microvascular endothelial lesions occur early. Our study provides evidence that human umbilical vein endothelial cells or EA.hy 926 cells, which are much more easily isolated and/or cultivated than pulmonary microvascular endothelial cells, could be used as alternative cell culture systems for studies on cellular dysfunction in the lung caused by toxic substances, certainly with respect to the expression of HSPs.
Endothelial permeability and IL-6 production during hypoxia: role of ROS in signal transduction
Authors: Ali Mir H. , Schlidt Scott A. , Chandel Navdeep S. , Hynes Karen L. , Schumacker Paul T. , Gewertz Bruce L.
Published: December 2017

Prolonged hypoxia produces reversible changes in endothelial permeability, but the mechanisms involved are not fully known. Previous studies have implicated reactive oxygen species (ROS) and cytokines in the regulation of permeability. We tested whether prolonged hypoxia alters permeability to increasing ROS generation, which amplifies cytokine production. Human umbilical vein endothelial cell (HUVEC) monolayers were exposed to hypoxia while secretion of tumor necrosis factor-α (TNF-α), interleukin (IL)-1α, IL-6, and IL-8 was measured. IL-6 and IL-8 secretion increased fourfold over 24 h in a pattern corresponding to changes in HUVEC permeability measured by transendothelial electrical resistance (TEER). Addition of exogenous IL-6 to normoxic HUVEC monolayers caused time-dependent changes in TEER that mimicked the hypoxic response. An antibody to IL-6 significantly attenuated the hypoxia-induced changes in TEER (86 ± 4 vs. 63 ± 3% with hypoxia alone at 18 h), whereas treatment with anti-IL-8 had no effect. To determine the role of hypoxia-induced ROS on this response, HUVEC monolayers were incubated with the antioxidants ebselen (50 μM) and N-acetyl-l-cysteine (NAC, 1 mM) before hypoxia. Antioxidants attenuated hypoxia-induced IL-6 secretion (13 ± 2 pg/ml with ebselen and 19 ± 3 pg/ml with NAC vs. 140 ± 15 pg/ml with hypoxia). Ebselen and NAC prevented changes in TEER during hypoxia (94 ± 2% with ebselen and 90 ± 6% with NAC vs. 63 ± 3% with hypoxia at 18 h). N-nitro-l-arginine (500 μM) did not decrease hypoxia-induced changes in dichlorofluorescin fluorescence, IL-6 secretion, or TEER. Thus ROS generated during hypoxia act as signaling elements, regulating secretion of the proinflammatory cytokines that lead to alterations of endothelial permeability.
Expression of type II Na-Picotransporter in alveolar type II cells
Authors: Traebert Martin , Hattenhauer Olaf , Murer Heini , Kaissling Brigitte , Biber Jürg
Published: December 2017

Type II Na-Picotransporters (type IIa and type IIb) represent apically located Na-Picotransporters in epithelia of proximal tubules (type IIa) and small intestine (type IIb). Here we provide evidence that the type IIb (but not the type IIa) Na-Picotransporter is also expressed in the lung. With the use of immunohistochemistry, location of the type IIb protein was found exclusively in the apical membrane of type II cells of the alveolar epithelium. Such a location of the type IIb cotransporter suggests an involvement in the reuptake of phosphate necessary for the synthesis of surfactant. A possible regulation of the abundance of the type IIb cotransporter in the lung was studied after adaptation of mice to a low-Pidiet. After a chronic adaptation to a low-Pidiet, no changes in the type IIb protein and the type IIb transcript were observed. These results exclude dietary intake of phosphate as a regulatory factor of the type IIb Na-Picotransporter in alveolar type II cells.
Newborn intrapulmonary veins are more reactive than arteries in normal and hypertensive piglets
Authors: Arrigoni F. I. , Hislop A. A. , Haworth S. G. , Mitchell J. A.
Published: December 2017

The reactivity of pulmonary veins during adaptation from pre- to postnatal life is not well characterized. With an in vitro organ bath technique, the responses to the contractile and relaxant agonists U-46619 (10−10 to 3 × 10−6 M) and acetylcholine (10−9 to 10−4 M) were compared in adjacent conduit pulmonary vein and artery rings from 66 piglets aged 1 wk preterm to 14 days of postnatal life and from adult tissue. Five additional piglets were made hypertensive by exposure to chronic hypoxia for 3 days after birth. Both arteries and veins showed smaller contractile and relaxant responses before birth than after. By 5 min after birth, the contraction by arteries and relaxation by veins had increased ( P < 0.05). By 3 days of age, arterial relaxation increased, but in all animals, venous relaxation exceeded that in arteries ( P < 0.05). Veins contracted more than arteries in animals aged 3–14 days. Neonatal hypoxia diminished the responses to both agonists in the veins ( P < 0.05), whereas the response in the arteries remained similar to that in the normal newborn. We speculate that veins may be more important in postnatal adaptation than previously suggested.
Role of ERK MAP kinases in responses of cultured human airway smooth muscle cells to IL-1β
Authors: Laporte Johanne D. , Moore Paul E. , Abraham Joseph H. , Maksym Geoffrey N. , Fabry Ben , Panettieri Reynold A. , Shore Stephanie A.
Published: December 2017

We have previously reported that interleukin (IL)-1β causes β-adrenergic hyporesponsiveness in cultured human airway smooth muscle cells by increasing cyclooxygenase-2 (COX-2) expression and prostanoid formation. The purpose of this study was to determine whether extracellular signal-regulated kinases (ERKs) are involved in these events. Levels of phosphorylated ERK (p42 and p44) increased 8.3- and 13-fold, respectively, 15 min after treatment with IL-1β (20 ng/ml) alone. Pretreating cells with the mitogen-activated protein kinase kinase inhibitor PD-98059 or U-126 (2 h before IL-1β treatment) decreased ERK phosphorylation. IL-1β (20 ng/ml for 22 h) alone caused a marked induction of COX-2 and increased basal PGE2release 28-fold ( P < 0.001). PD-98059 (100 μM) and U-126 (10 μM) each decreased COX-2 expression when administered before IL-1β treatment. In control cells, PD-98059 and U-126 had no effect on basal or arachidonic acid (AA; 10 μM)-stimulated PGE2release, but both inhibitors caused a significant decrease in bradykinin (BK; 1 μM)-stimulated PGE2release, consistent with a role for ERK in the activation of phospholipase A2by BK. In IL-1β-treated cells, prior administration of PD-98059 caused 81, 92 and 40% decreases in basal and BK- and AA-stimulated PGE2release, respectively ( P < 0.01), whereas administration of PD-98059 20 h after IL-1β resulted in only 38 and 43% decreases in basal and BK-stimulated PGE2release, respectively ( P < 0.02) and had no effect on AA-stimulated PGE2release. IL-1β attenuated isoproterenol-induced decreases in human airway smooth muscle stiffness as measured by magnetic twisting cytometry, and PD-98059 or U-126 abolished this effect in a concentration-dependent manner. These results are consistent with the hypothesis that ERKs are involved early in the signal transduction pathway through which IL-1β induces PGE2synthesis and β-adrenergic hyporesponsiveness and that ERKs act by inducing COX-2 and activating phospholipase A2.
Induction of fibrogenic mediators by fine and ultrafine titanium dioxide in rat tracheal explants
Authors: Churg A. , Gilks B. , Dai J.
Published: December 2017

Respirable ambient particles [particulate matter <10 μm (PM10)] are associated with both acute and chronic adverse health effects including chronic airflow obstruction. PM10 can induce expression of inflammatory and fibrogenic mediators, but there is controversy about the types and/or sizes of particles involved and, in particular, whether ultrafine particles are the major toxic agents. To examine whether particle size affects mediator generation, we exposed rat tracheal explants, an inflammatory cell-free model of the airway wall, to various concentrations up to 500 μg/cm2 of fine (0.12 μm) or ultrafine (0.021 μm) titanium dioxide (anatase), maintained the explants in an organ culture in air for 1–7 days, and used RT-PCR to examine the expression of fibrogenic mediators and procollagen. No increase in gene expression was seen at 1 or 3 days, but at 5 days, ultrafine dust induced a small increase in procollagen. At 7 days, fine titanium dioxide produced significantly greater increases for platelet-derived growth factor (PDGF)-B, transforming growth factor-α, and transforming growth factor-β compared with those by ultrafine dust; both dusts produced similar increases for PDGF-A; and ultrafine dust produced increases in procollagen expression, whereas fine dust had no effect. Expression levels were dose related. Both dusts produced a similar decrease in expression of PDGF receptor-α and a similar increase in PDGF receptor-β. These observations suggest that ultrafine particles are intrinsically able to induce procollagen expression even in the absence of inflammatory cells; that chronic exposure to PM10 may result in chronic airflow obstruction, in part because of ultrafine particle-mediated increases in airway wall fibrosis; and that chemically identical dusts of differing size can produce quite different patterns of gene expression in the airway wall. Differential upregulation of PDGF receptors does not appear to explain dust-induced fibrosis in this model.
Expression of C-type natriuretic peptide during development of rat lung
Authors: Nakanishi Kuniaki , Tajima Fumiko , Itoh Hiroshi , Nakata Yasuko , Hama Norio , Nakagawa Osamu , Nakao Kazuwa , Kawai Toshiaki , Torikata Chikao , Suga Tatsuko , Takishima Kunio , Aurues Takashi , Ikeda Tomosumi
Published: December 2017

C-type natriuretic peptide (CNP), recently found to be secreted from vascular endothelial cells, is now viewed as a novel endothelium-derived relaxing peptide. However, the distribution and expression of CNP during cardiopulmonary development is unclear. To follow changes in the expression of CNP during lung development, we examined rat embryos and neonates using Northern blot analysis and in situ hybridization for CNP mRNA and radioimmunoassay and immunohistochemistry for CNP protein. A substantial expression of CNP mRNA was first detected on postnatal day 2, and it thereafter remained fairly steady. The level of CNP protein also increased rapidly after postnatal day 1, reaching a settled level on postnatal day 4. CNP protein and mRNA were detected in the endothelium and smooth muscle cells of blood vessels and in bronchial airway and alveolar epithelia. Immunoreactivity for CNP protein in the endothelium of blood vessels increased to an intense level after the saccular stage. These results suggest that the changes in CNP levels may be related to the occurrence of pulmonary vasodilation after birth.
Autocrine production of matrix metalloproteinase-2 is required for human airway smooth muscle proliferation
Authors: Johnson Simon , Knox Alan
Published: December 2017

Airway smooth muscle proliferation is important in asthma and is dependent on pro- and antimitogenic factors and cell-matrix interactions. Here we show an antiproliferative effect of protease inhibitors on human airway smooth muscle due to inhibition of autocrine-derived matrix metalloproteinase (MMP)-2. Proliferation in response to fetal bovine serum, thrombin, and platelet-derived growth factor was inhibited by the broad-spectrum protease inhibitor Complete and the MMP inhibitors EDTA and Ro-31-9790 but not by cysteine or serine protease inhibitors. Conditioned medium from airway smooth muscle cells contained 72-kDa gelatinase that was secreted by growth-arrested cells and increased by fetal bovine serum but not by thrombin or platelet-derived growth factor. Immunostaining of cultured human airway smooth muscle cells and normal lung biopsies confirmed this gelatinase to be MMP-2. Our results suggest a novel role for MMP-2 as an important autocrine factor required for airway smooth muscle proliferation. Inhibition of MMPs could provide a target for the prevention of smooth muscle hyperplasia and airway remodeling in asthma.
Differential signaling pathways ofHO-1gene expression in pulmonary and systemic vascular cells
Authors: Hartsfield Cynthia L. , Alam Jawed , Choi Augustine M. K.
Published: December 2017

Heme oxygenase-1 (HO-1) is induced by oxidative stress and plays an important role in cellular protection against oxidant injury. Increasing evidence also suggests that HO-1 is markedly modulated by hypoxia in vitro and in vivo. Our group has previously demonstrated that the transcription factor hypoxia-inducible factor (HIF)-1 mediates hypoxia-induced HO-1 gene transcription and expression in systemic (aortic) vascular smooth muscle (AoVSM) cells (P. J. Lee, B.-H. Jiang, B. Y. Chin, N. V. Iyer, J. Alam, G. L. Semenza, and A. M. K. Choi. J. Biol. Chem. 272: 5375–5381, 1997). Because the pulmonary circulation is an important target of hypoxia, this study investigated whether HO-1gene expression in pulmonary arterial vascular smooth muscle was differentially regulated by hypoxia in comparison to AoVSM cells. Interestingly, hypoxia neither induced HO-1 gene expression nor increased HIF-1 DNA binding activity in pulmonary arterial vascular smooth muscle cells. Conversely, pulmonary arterial endothelial cells (PAECs) demonstrated a marked induction of HO-1 gene expression after hypoxia. Electrophoretic mobility shift assays detected an increase in activator protein-1 rather than in HIF-1 DNA binding activity in nuclear extracts of hypoxic PAECs. Analyses of the promoter and 5′-flanking regions of the HO-1 gene were performed by transiently transfecting PAECs with either the hypoxia response element (HIF-1 binding site) or the HO-1 gene distal enhancer element (AB1) linked to a chloramphenicol acetyltransferase reporter gene. Increased chloramphenicol acetyltransferase activity was observed only in transfectants containing the AB1 distal enhancer, and mutational analysis of this enhancer suggested that the activator protein-1 regulatory element was critical for hypoxia-induced HO-1 gene transcription. Collectively, our data demonstrate that the molecular regulation of HO-1 gene transcription during hypoxia differs between the systemic and pulmonary circulations and also provide evidence that hypoxia-induced HO-1 gene expression in PAECs and AoVSM cells is regulated through two discrete signaling pathways.
Fas-induced apoptosis of alveolar epithelial cells requires ANG II generation and receptor interaction
Authors: Wang Rongqi , Zagariya Alex , Ang Edmund , Ibarra-Sunga Olivia , Uhal Bruce D.
Published: December 2017

Recent works from this laboratory demonstrated potent inhibition of Fas-induced apoptosis in alveolar epithelial cells (AECs) by the angiotensin-converting enzyme (ACE) inhibitor captopril [B. D. Uhal, C. Gidea, R. Bargout, A. Bifero, O. Ibarra-Sunga, M. Papp, K. Flynn, and G. Filippatos. Am. J. Physiol. 275 ( Lung Cell. Mol. Physiol. 19): L1013–L1017, 1998] and induction of dose-dependent apoptosis in AECs by purified angiotensin (ANG) II [R. Wang, A. Zagariya, O. Ibarra-Sunga, C. Gidea, E. Ang, S. Deshmukh, G. Chaudhary, J. Baraboutis, G. Filippatos and B. D. Uhal. Am. J. Physiol. 276 ( Lung Cell. Mol. Physiol. 20): L885–L889, 1999]. These findings led us to hypothesize that the synthesis and binding of ANG II to its receptor might be involved in the induction of AEC apoptosis by Fas. Apoptosis was induced in the AEC-derived human lung carcinoma cell line A549 or in primary AECs isolated from adult rats with receptor-activating anti-Fas antibodies or purified recombinant Fas ligand, respectively. Apoptosis in response to either Fas activator was inhibited in a dose-dependent manner by the nonthiol ACE inhibitor lisinopril or the nonselective ANG II receptor antagonist saralasin, with maximal inhibitions of 82 and 93% at doses of 0.5 and 5 μg/ml, respectively. In both cell types, activation of Fas caused a significant increase in the abundance of mRNA for angiotensinogen (ANGEN) that was unaffected by saralasin. Transfection with antisense oligonucleotides against ANGEN mRNA inhibited the subsequent induction of Fas-stimulated apoptosis by 70% in A549 cells and 87% in primary AECs (both P < 0.01). Activation of Fas increased the concentration of ANG II in the serum-free extracellular medium 3-fold in primary AECs and 10-fold in A549 cells. Apoptosis in response to either Fas activator was completely abrogated by neutralizing antibodies specific for ANG II ( P < 0.01), but isotype-matched nonimmune immunoglobulins had no significant effect. These data indicate that the induction of AEC apoptosis by Fas requires a functional renin-angiotensin system in the target cell. They also suggest that therapeutic control of AEC apoptosis is feasible through pharmacological manipulation of the local renin-angiotensin system.
Decreased synthesis and vasodilation to nitric oxide in piglets with hypoxia-induced pulmonary hypertension
Authors: Berkenbosch John W. , Baribeau Johanne , Perreault Thérèse
Published: December 2017

Nitric oxide (NO) is thought to play an important role in the regulation of neonatal pulmonary vasculature. It has been suggested that neonates with pulmonary hypertension have a defective NO pathway. Therefore, we measured in 1-day-old piglets exposed to hypoxia (fraction of inspired O2 = 0.10) for 3 or 14 days to induce pulmonary hypertension 1) the activity of NO synthase (NOS) via conversion of l-arginine to l-citrulline and the concentration of the NO precursorl-arginine in isolated pulmonary vessels, 2) the vasodilator response to the NO donor 3-morpholinosydnonimine- N-ethylcarbamide (SIN-1) and the cGMP analog 8-bromo-cGMP in isolated perfused lungs, and 3) the production of cGMP in response to SIN-1 in isolated perfused lungs. After 3 days of exposure to hypoxia, endothelial NOS (eNOS) activity was unaffected, whereas, after 14 days of hypoxia, eNOS activity was decreased in the cytosolic fraction of pulmonary artery ( P < 0.05) but not of pulmonary vein homogenates. Inducible NOS activity was decreased in the cytosolic fraction of pulmonary artery homogenates after both 3 ( P < 0.05) and 14 ( P < 0.05) days of hypoxia but was unchanged in pulmonary veins. Pulmonary artery levels ofl-arginine were unaffected by hypoxic exposure. After 3 days of exposure to hypoxia, the reduction in the dilator response to SIN-1 ( P < 0.05) coincided with a decrease in cGMP production ( P < 0.005), suggesting that soluble guanylate cyclase activity may be altered. When the exposure was prolonged to 14 days, dilation to SIN-1 remained decreased ( P < 0.05) and, although cGMP production normalized, the dilator response to 8-bromo-cGMP decreased ( P < 0.05), suggesting that, after prolonged exposure to hypoxia, cGMP-dependent mechanisms may also be impaired. In conclusion, neonatal hypoxia-induced pulmonary hypertension is associated with multiple disruptions in the NO pathway.
Mechanism of hypoxic pulmonary vasoconstriction involves ETA receptor-mediated inhibition of KATPchannel
Authors: Sato Koichi , Morio Yoshiteru , Morris Kenneth G. , Rodman David M. , McMurtry Ivan F.
Published: December 2017

There is controversy on the role of endothelin (ET)-1 in the mechanism of hypoxic pulmonary vasoconstriction (HPV). Although HPV is inhibited by ET-1 subtype A (ETA)-receptor antagonists in animals, it has been reported that ETA-receptor blockade does not affect HPV in isolated lungs. Thus we reassessed the role of ET-1 in HPV in both rats and isolated blood- and physiological salt solution (PSS)-perfused rat lungs. In rats, the ETA-receptor antagonist BQ-123 and the nonselective ETA- and ETB-receptor antagonist PD-145065, but not the ETB-receptor antagonist BQ-788, inhibited HPV. Similarly, BQ-123, but not BQ-788, attenuated HPV in blood-perfused lungs. In PSS-perfused lungs, either BQ-123, BQ-788, or the combination of both attenuated HPV equally. Inhibition of HPV by combined BQ-123 and BQ-788 in PSS-perfused lungs was prevented by costimulation with angiotensin II. The ATP-sensitive K+(KATP)-channel blocker glibenclamide also prevented inhibition of HPV by BQ-123 in both lungs and rats. These results suggest that ET-1 contributes to HPV in both isolated lungs and intact animals through ETA receptor-mediated suppression of KATP-channel activity.
Adenoviral E3-14.7K protein in LPS-induced lung inflammation
Authors: Harrod Kevin S. , Mounday Amber D. , Whitsett Jeffrey A.
Published: December 2017

The adenoviral E3-14.7K protein is a cytoplasmic protein synthesized after adenoviral infection. To assess the contribution of E3-14.7K-sensitive pathways in the modulation of inflammation by the respiratory epithelium, inflammatory responses to intratracheal lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-α were assessed in transgenic mice bearing the adenoviral E3-14.7K gene under the direction of the surfactant protein (SP) C promoter. When E3-14.7K transgenic mice were administered LPS intratracheally, lung inflammation as indicated by macrophage and neutrophil accumulation in bronchoalveolar lavage fluid was decreased compared with wild-type control mice. Lung inflammation and epithelial cell injury were decreased in E3-14.7K mice 24 and 48 h after LPS administration. Intracellular staining for surfactant proprotein (proSP) B, proSP-C, and SP-B was decreased and extracellular staining was markedly increased in wild-type mice after LPS administration, consistent with LPS-induced lung injury. In contrast, intense intracellular staining of proSP-B, proSP-C, and SP-B persisted in type II cells of E3-14.7K mice, whereas extracellular staining of proSP-B and proSP-C was absent. Inhibitory effects of intratracheal LPS on SP-C mRNA were ameliorated by expression of the E3-14.7Kgene. Similar to the response to LPS, lung inflammation after intratracheal administration of TNF-α was decreased in E3-14.7K transgenic mice. Levels of TNF-α after LPS administration were similar in wild-type and E3-14.7K-bearing mice. Cell-selective expression of E3-14.7K in the respiratory epithelium inhibited LPS- and TNF-α-mediated lung inflammation, demonstrating the critical role of respiratory epithelial cells in LPS- and TNF-α-induced lung inflammation.
IL-10 reduces Th2 cytokine production and eosinophilia but augments airway reactivity in allergic mice
Authors: van Scott Michael R. , Justice J. Paul , Bradfield John F. , Enright Edward , Sigounas Anastasia , Sur Sanjiv
Published: December 2017

We investigated the effects of interleukin (IL)-10 administration on allergen-induced Th2 cytokine production, eosinophilic inflammation, and airway reactivity. Mice were sensitized by intraperitoneal injection of ragweed (RW) adsorbed to Alum and challenged by intratracheal instillation of the allergen. Sensitization and challenge with RW increased concentrations of IL-10 in bronchoalveolar lavage (BAL) fluid from undetectable levels to 60 pg/ml over 72 h. Intratracheal instillation of 25 ng of recombinant murine IL-10 at the time of RW challenge further elevated BAL fluid IL-10 concentration to 440 pg/ml but decreased BAL fluid IL-4, IL-5, and interferon-γ levels by 40–85% and eosinophil numbers by 70% ( P < 0.0001). Unexpectedly, the same IL-10 treatment increased airway reactivity to methacholine in spontaneously breathing mice that had been sensitized and challenged with RW ( P < 0.001). IL-10 treatment in naive animals or RW-sensitized mice challenged with PBS failed to increase airway reactivity, demonstrating that IL-10 induces an increase in airway reactivity only when it is administered in conjunction with allergic sensitization and challenge. The results demonstrate that IL-10 reduces Th2 cytokine levels and eosinophilic inflammation but augments airway hyperreactivity. Thus, despite its potent anti-inflammatory activity, IL-10 could contribute to the decline in pulmonary function observed in asthma.
Residual oil fly ash and charged polymers activate epithelial cells and nociceptive sensory neurons
Authors: Oortgiesen Marga , Veronesi Bellina , Eichenbaum Gary , Kiser Patrick F. , Simon Sidney A.
Published: December 2017

Residual oil fly ash (ROFA) is an industrial pollutant that contains metals, acids, and unknown materials complexed to a particulate core. The heterogeneous composition of ROFA hampers finding the mechanism(s) by which it and other particulate pollutants cause airway toxicity. To distinguish culpable factors contributing to the effects of ROFA, synthetic polymer microsphere (SPM) analogs were synthesized that resembled ROFA in particle size (2 and 6 μm in diameter) and zeta potential (−29 mV). BEAS-2B human bronchial epithelial cells and dorsal root ganglion neurons responded to both ROFA and charged SPMs with an increase in intracellular Ca2+concentration ([Ca2+]i) and the release of the proinflammatory cytokine interleukin-6, whereas neutral SPMs bound with polyethylene glycol (0-mV zeta potential) were relatively ineffective. In dorsal root ganglion neurons, the SPM-induced increases in [Ca2+]iwere correlated with the presence of acid- and/or capsaicin-sensitive pathways. We hypothesized that the acidic microenvironment associated with negatively charged colloids like ROFA and SPMs activate irritant receptors in airway target cells. This causes subsequent cytokine release, which mediates the pathophysiology of neurogenic airway inflammation.
Increased production of nitrotyrosine in lung tissue of rats with radiation-induced acute lung injury
Authors: Tsuji Chizuko , Shioya Sumie , Hirota Yuki , Fukuyama Naoto , Kurita Daisaku , Tanigaki Toshimori , Ohta Yasuyo , Nakazawa Hiroe
Published: December 2017

The purposes of this study were 1) to identify the nitric oxide (NO) synthase (NOS) isoform responsible for NO-mediated radiation-induced lung injury, 2) to examine the formation of nitrotyrosine, and 3) to see whether nitrotyrosine formation and lung injury are reduced by an inducible NOS (iNOS) inhibitor, aminoguanidine. The left hemithorax of rats was irradiated (20 Gy), and the degree of lung injury, the expression of NOS isoforms, and the formation of nitrotyrosine and superoxide were examined after 2 wk. iNOS mRNA was induced, and endothelial NOS mRNA was markedly increased in the irradiated lung. Nitrotyrosine was detected biochemically and immunohistochemically. Aminoguanidine prevented acute lung injury as indicated by decreased protein concentration and lactate dehydrogenase activity in bronchoalveolar lavage fluid and improved NMR parameters and histology. Furthermore, the formation of nitrotyrosine was significantly reduced in the aminoguanidine group. We conclude that iNOS induction is a major factor in radiation-induced lung injury and that nitrotyrosine formation may participate in the NO-induced pathogenesis.
Immunotargeting of glucose oxidase to endothelium in vivo causes oxidative vascular injury in the lungs
Authors: Christofidou-Solomidou Melpo , Pietra Giuseppe G. , Solomides Charalambos C. , Arguiris Evgenia , Harshaw David , Fitzgerald Garret A. , Albelda Steven M. , Muzykantov Vladimir R.
Published: December 2017

Vascular immunotargeting is a novel approach for site-selective drug delivery to endothelium. To validate the strategy, we conjugated glucose oxidase (GOX) via streptavidin with antibodies to the endothelial cell surface antigen platelet endothelial cell adhesion molecule (PECAM). Previous work documented that 1) anti-PECAM-streptavidin carrier accumulates in the lungs after intravenous injection in animals and 2) anti-PECAM-GOX binds to, enters, and kills endothelium via intracellular H2O2generation in cell culture. In the present work, we studied the targeting and effect of anti-PECAM-GOX in animals. Anti-PECAM-GOX, but not IgG-GOX, accumulated in the isolated rat lungs, produced H2O2,and caused endothelial injury manifested by a fourfold elevation of angiotensin-converting enzyme activity in the perfusate. In intact mice, anti-PECAM-GOX accumulated in the lungs (27 ± 9 vs. 2.4 ± 0.3% injected dose/g for IgG-GOX) and caused severe lung injury and 95% lethality within hours after intravenous injection. Endothelial disruption and blebbing, elevated lung wet-to-dry ratio, and interstitial and alveolar edema indicated that anti-PECAM-GOX damaged pulmonary endothelium. The vascular injury in the lungs was associated with positive immunostaining for iPF2α-III isoprostane, a marker for oxidative stress. In contrast, IgG-GOX caused a minor lung injury and little (5%) lethality. Anti-PECAM conjugated with inert proteins induced no death or lung injury. None of the conjugates caused major injury to other internal organs. These results indicate that an immunotargeting strategy can deliver an active enzyme to selected target cells in intact animals. Anti-PECAM-GOX provides a novel model of oxidative injury to the pulmonary endothelium in vivo.
Expression of heme oxygenase-1 in the lung in chronic hypoxia
Authors: Carraway Martha Sue , Ghio Andrew J. , Carter Jacqueline D. , Piantadosi Claude A.
Published: December 2017

Heme oxygenase (HO)-1 is an oxygen-dependent enzyme that may regulate vascular tone and cell proliferation through the production of carbon monoxide (CO). We tested the hypothesis that HO-1 is upregulated in the lung in chronic hypoxia by exposing male Sprague-Dawley rats to 17,000 feet (395 Torr) for 0, 1, 3, 7, 14, or 21 days. After exposure, blood gases, carboxyhemoglobin (COHb) levels, and hematocrit were measured, and the lungs were either inflation fixed for immunohistochemistry or frozen for later measurement of HO enzyme activity, Western blot for HO-1 protein, and RT-PCR for HO-1 mRNA. The heart was excised and weighed, and the right-to-left heart weight ratio was determined. During hypoxia, the hematocrit increased progressively, reaching significantly higher values than the control value after 3 days. COHb levels increased above the control value after 1 day of hypoxia and increased progressively between 14 and 21 days, whereas arterial[Formula: see text] and arterial[Formula: see text] did not vary significantly. HO-1 protein determined by Western blot increased for the first 7 days and declined thereafter; however, enzyme activity was elevated only after 1 day. Changes in HO-1 during hypoxia were localized by immunohistochemistry to inflammatory cells (early) and newly muscularized arterioles (later). Lung HO-1 mRNA normalized to glyceraldehyde-3-phosphate dehydrogenase was increased after 1 and 21 days. The data indicate that lung HO-1 protein and activity are upregulated only during early chronic hypoxia, whereas persistent COHb elevations indicate high endogenous CO production rates at nonpulmonary sites. If CO has antiproliferative properties, the lack of HO enzyme activity in the lung may be permissive for pulmonary vascular proliferation in hypoxia.
Biophysical properties and molecular characterization of amiloride-sensitive sodium channels in A549 cells
Authors: Lazrak A. , Samanta A. , Matalon S.
Published: December 2017

Amiloride-sensitive Na+channels, present in fetal and adult alveolar epithelial type II (ATII) cells, play a critical role in the reabsorption of fetal fluid shortly after birth and in limiting the extent of alveolar edema across the adult lung. Because of the difficulty in isolating and culturing ATII cells, there is considerable interest in characterizing the properties of ion channels and their response to injury of ATII cell-like cell lines such as A549 that derive from a human alveolar cell carcinoma. A549 cells were shown to contain α-, β-, and γ-epithelial Na+channel mRNAs. In the whole cell mode of the patch-clamp technique (bath, 145 mM Na+; pipette, 145 mM K+), A549 cells exhibited inward Na+currents reversibly inhibited by amiloride, with an inhibition constant of 0.83 μM. Ion substitution studies showed that these channels were moderately selective for Na+(Na+-to-K+permeability ratio = 6:1). Inward Na+currents were activated by forskolin (10 μM) and inhibited by nitric oxide (300 nM) and cGMP. Recordings in cell-attached mode revealed the presence of an amiloride-sensitive Na+channel with a unitary conductance of 8.6 ± 0.04 (SE) pS. Channel activity was increased by forskolin and decreased by nitric oxide and the cGMP analog 8-bromo-cGMP. These data demonstrate that A549 cells contain amiloride-sensitive Na+channels with biophysical properties similar to those of ATII cells.
Distinct changes in pulmonary surfactant homeostasis in common β-chain- and GM-CSF-deficient mice
Authors: Reed Jacquelyn A. , Ikegami Machiko , Robb Lorraine , Begley C. Glenn , Ross Gary , Whitsett Jeffrey A.
Published: December 2017

Pulmonary alveolar proteinosis (PAP) is caused by inactivation of either granulocyte-macrophage colony-stimulating factor (GM-CSF) or GM receptor common β-chain (βc) genes in mice [GM(−/−), βc(−/−)], demonstrating a critical role of GM-CSF signaling in surfactant homeostasis. To distinguish possible phenotypic differences in GM(−/−) and βc(−/−) mice, surfactant metabolism was compared in βc(−/−), GM(−/−), and wild-type mice. Although lung histology in βc(−/−) and GM(−/−) mice was indistinguishable, distinct differences were observed in surfactant phospholipid and surfactant protein concentrations and clearance from lungs of βc(−/−) and GM(−/−) mice. At 1–2 days of age, lung saturated phosphatidylcholine (Sat PC) pool sizes were higher in wild-type, βc(−/−), and GM(−/−) mice compared with wild-type adult mice. In wild-type mice, Sat PC pool sizes decreased to adult levels by 7 days of age; however, Sat PC increased with advancing age in βc(−/−) and GM(−/−) mice. Postnatal changes in Sat PC pool sizes were different in GM(−/−) compared with βc(−/−) mice. After 7 days of age, the increased lung Sat PC pool sizes remained constant in βc(−/−) mice but continued to increase in GM(−/−) mice, so that by 56 days of age, lung Sat PC pools were increased three- and sixfold, respectively, compared with wild-type controls. After intratracheal injection, the percent recovery of [3H]dipalmitoylphosphatidylcholine and125I-recombinant surfactant protein (SP) C was higher in βc(−/−) compared with wild-type mice, reflecting decreased clearance in the receptor-deficient mice. The defect in clearance was significantly more severe in GM(−/−) than in βc(−/−) mice. The ratio of SP Sat PC to SP-A, -B, and -C was similar in bronchoalveolar lavage fluid (BALF) from adult mice of all genotypes, but the ratio of SP-D to Sat PC was markedly increased in βc(−/−) and GM(−/−) mice (10- and 5-fold, respectively) compared with wild-type mice. GM-CSF concentrations were increased in BALF but not in serum of βc(−/−) mice, consistent with a pulmonary response to the lack of GM-CSF signaling. The observed differences in surfactant metabolism suggest the presence of alternative clearance mechanisms regulating surfactant homeostasis in βc(−/−) and GM(−/−) mice and may provide a molecular basis for the range in severity of PAP symptoms. surfactant metabolism; alveolar macrophage; granulocyte-macrophage colony-stimulating factor
Augmentation of LTC4synthesis in human eosinophils caused by CD3-stimulated Th2-like cells in vitro
Authors: Muñoz Nilda M. , van Seventer Gijs A. , Semnani Roshanak T. , Leff Alan R.
Published: December 2017

We assessed the effect of anti-CD3-stimulated secretion of cultured human Th1- and Th2-like cells on leukotriene C4(LTC4) secretion in isolated human eosinophils. T helper (Th) cell subsets were generated from human naive CD4+T cells cocultured with irradiated human transformed B cells and either recombinant human interleukin (rhIL)-1β plus rhIL-6 plus rhIL-12 for Th1-like cells or rhIL-1β plus rhIL-6 plus rhIL-4 for Th2-like cells. Coincubation of eosinophils with 1:5 dilution of Th2-supernatant (Sup) caused an increase in LTC4secretion caused by 0.1 μM formyl-Met-Leu-Phe and 5 μg/ml cytochalasin B from 921 ± 238 to 3,067 ± 1,462 pg/106eosinophils ( P < 0.01). Th1-Sup at the same dilution had no augmenting effect on stimulated secretion of LTC4in eosinophils despite substantial concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the supernatant. Dilution of Th1-Sup caused increased LTC4that returned to baseline after immunoabsorption of GM-CSF, suggesting the presence of a possible inhibitory factor. We demonstrate that pretreatment of eosinophils with 1:5 dilution of Th2-Sup but not of Th1-Sup causes substantial augmentation of LTC4secretion in vitro and establishes that human Th2 cells cause direct augmentation of LTC4secretion within 15–30 min of exposure.
Modification of transepithelial ion transport in human cultured bronchial epithelial cells by interferon-γ
Authors: Galietta Luis J. V. , Folli Chiara , Marchetti Carla , Romano Luca , Carpani Daniela , Conese Massimo , Zegarra-Moran Olga
Published: December 2017

Human bronchial epithelial cells were treated in vitro with interferon-γ or tumor necrosis factor-α to assess their effect on transepithelial ion transport. Short-circuit current measurements revealed that Na+absorption was markedly inhibited by interferon-γ (10–1,000 U/ml). The cystic fibrosis transmembrane conductance regulator was also downregulated by interferon-γ as evident at the protein level and by the decrease in the cAMP-dependent current. On the other hand, interferon-γ caused an increase of the current elicited by apical UTP application, which is due to the activity of Ca2+-dependent Cl−channels. Tumor necrosis factor-α caused few changes in ion transport. Transepithelial fluid transport was measured in normal and cystic fibrosis cells. At rest, both types of cells showed an amiloride-sensitive fluid absorption that was inhibited by interferon-γ but not by tumor necrosis factor-α. Our results show that interferon-γ alters the transepithelial ion transport of cultured bronchial cells. This effect may change the ion composition and/or volume of periciliary fluid.
Molecular responses of rat tracheal epithelial cells to transmembrane pressure
Authors: Ressler Barbara , Lee Richard T. , Randell Scott H. , Drazen Jeffrey M. , Kamm Roger D.
Published: December 2017

Smooth muscle constriction in asthma causes the airway to buckle into a rosette pattern, folding the epithelium into deep crevasses. The epithelial cells in these folds are pushed up against each other and thereby experience compressive stresses. To study the epithelial cell response to compressive stress, we subjected primary cultures of rat tracheal epithelial cells to constant elevated pressures on their apical surface (i.e., a transmembrane pressure) and examined changes in the expression of genes that are important for extracellular matrix production and maintenance of smooth muscle activation. Northern blot analysis of RNA extracted from cells subjected to transmembrane pressure showed induction of early growth response-1 (Egr-1), endothelin-1, and transforming growth factor-β1 in a pressure-dependent and time-dependent manner. Increases in Egr-1 protein were detected by immunohistochemistry. Our results demonstrate that airway epithelial cells respond rapidly to compressive stresses. Potential transduction mechanisms of transmembrane pressure were also investigated.
Voltage-gated K+-channel activity in ovine pulmonary vasculature is developmentally regulated
Authors: Cornfield David N. , Saqueton Connie B. , Porter Valerie A. , Herron Jean , Resnik Ernesto , Haddad Imad Y. , Reeve Helen L.
Published: December 2017

To examine mechanisms underlying developmental changes in pulmonary vascular tone, we tested the hypotheses that 1) maturation-related changes in the ability of the pulmonary vasculature to respond to hypoxia are intrinsic to the pulmonary artery (PA) smooth muscle cells (SMCs); 2) voltage-gated K+(Kv)-channel activity increases with maturation; and 3) O2-sensitive Kv2.1 channel expression and message increase with maturation. To confirm that maturational differences are intrinsic to PASMCs, we used fluorescence microscopy to study the effect of acute hypoxia on cytosolic Ca2+concentration ([Ca2+]i) in SMCs isolated from adult and fetal PAs. Although PASMCs from both fetal and adult circulations were able to sense an acute decrease in O2tension, acute hypoxia induced a more rapid and greater change in [Ca2+]iin magnitude in PASMCs from adult compared with fetal PAs. To determine developmental changes in Kv-channel activity, the effects of the K+-channel antagonist 4-aminopyridine (4-AP) were studied on fetal and adult PASMC [Ca2+]i. 4-AP (1 mM) caused PASMC [Ca2+]ito increase by 94 ± 22% in the fetus and 303 ± 46% in the adult. Kv-channel expression and mRNA levels in distal pulmonary arteries from fetal, neonatal, and adult sheep were determined through the use of immunoblotting and semiquantitative RT-PCR. Both Kv2.1-channel protein and mRNA expression in distal pulmonary vasculature increased with maturation. We conclude that there are maturation-dependent changes in PASMC O2sensing that may render the adult PASMCs more responsive to acute hypoxia.
Ethanol ingestion via glutathione depletion impairs alveolar epithelial barrier function in rats
Authors: Guidot David M. , Modelska Katharina , Lois Manuel , Jain Lucky , Moss I. Marc , Pittet Jean-François , Brown Lou Ann S.
Published: December 2017

We determined that rats fed a liquid diet containing ethanol (36% of calories) for 6 wk had decreased ( P < 0.05) net vectorial fluid transport and increased ( P < 0.05) bidirectional protein permeability across the alveolar epithelium in vivo compared with rats fed a control diet. However, both groups increased ( P < 0.05) fluid transport in response to epinephrine (10−5M) stimulation, indicating that transcellular sodium transport was intact. In parallel, type II cells isolated from ethanol-fed rats and cultured for 8 days formed a more permeable monolayer as reflected by increased ( P < 0.05) leak of [14C]inulin. However, type II cells from ethanol-fed rats had more sodium-permeant channels in their apical membranes than type II cells isolated from control-fed rats, consistent with the preserved response to epinephrine in vivo. Finally, the alveolar epithelium of ethanol-fed rats supplemented withl-2-oxothiaxolidine-4-carboxylate (Procysteine), a glutathione precursor, had the same ( P < 0.05) net vectorial fluid transport and bidirectional protein permeability in vivo and permeability to [14C]inulin in vitro as control-fed rats. We conclude that chronic ethanol ingestion via glutathione deficiency increases alveolar epithelial intercellular permeability and, despite preserved or even enhanced transcellular sodium transport, renders the alveolar epithelium susceptible to acute edematous injury.
Distribution of ion transport mRNAs throughout murine nose and lung
Authors: Rochelle Lori G. , Li Dong Chen , Ye Helen , Lee Eddie , Talbot Colleen R. , Boucher Richard C.
Published: December 2017

Evidence of absorptive or secretory ion transport in different respiratory regions of the mouse was sought by assessing the regional distribution of α-, β-, and γ-epithelial sodium channel (ENaC; Na+absorptive), cystic fibrosis transmembrane conductor regulator (CFTR), and Na+-K+-2Cl−cotransporter mRNAs. High levels of ENaC subunit expression were found in nasal surface epithelium and gland ducts. CFTR was expressed in both superficial nasal respiratory epithelium and glands. These results are consistent with basal amiloride-sensitive Na+absorption and cAMP-dependent Cl−secretion in murine nasal epithelia. Expression of all three ENaC subunits increased progressively from trachea to terminal bronchioles. Intermediate levels of CFTR and cotransporter expression in bronchial epithelium diminished in bronchioles. The low abundance of CFTR mRNA throughout murine pulmonary epithelium is consistent with functional data that attributes Cl−secretion predominantly to an alternative Cl−channel. α-ENaC as the only mRNA found in all regions of airway epithelia is consistent with the α-subunit as requisite for Na+absorption, and the increased expression of α-, β-, and γ-ENaC in distal airways suggests a greater absorptive capability in this region.