bluuebunny/crossref_metadata_2025 huggingface dataset:
AP-1 and STAT mediate hyperoxia-induced gene transcription of heme oxygenase-1
Authors: Lee Patty J. , Camhi Sharon L. , Chin Beek Yoke , Alam Jawed , Choi Augustine M. K.
Published: December 2017

We have previously shown marked induction of the stress-inducible gene heme oxygenase-1 ( HO-1) in vivo and in vitro after hyperoxia. In RAW 264.7 cells, HO-1 induction is transcriptionally regulated and dependent on cooperation between the HO-1 gene promoter and the 5′ distal enhancer element SX2. In our present study, further deletional and mutational analyses demonstrate that signal transducer and activator of transcription (STAT) DNA binding sites located in the promoter of HO-1 and activator protein (AP)-1 DNA binding sites in the distal enhancer element SX2 are necessary for optimal HO-1 gene activation after hyperoxia. Interestingly, a second 5′ distal enhancer element, AB1, located 10 kb upstream from the HO-1 promoter, alone is activated after hyperoxia but cannot confer maximal hyperoxia-induced HO-1gene transcription. Mutational analysis of the AB1 enhancer shows that AP-1 is essential for AB1-mediated HO-1 gene transcription after hyperoxia. Electromobility shift assays show increased STAT1, STAT3, STAT5, and AP-1 DNA binding activity in RAW 264.7 cells after hyperoxia. Taken together, our data suggest that the 5′ distal enhancer elements of the HO-1 gene in concert with the promoter regulate HO-1 gene induction and highlight the complexity of HO-1 gene transcription in response to hyperoxia.
Retinoic acid stimulates immature lung fibroblast growth via a PDGF-mediated autocrine mechanism
Authors: Liebeskind Abraham , Srinivasan Suseela , Kaetzel David , Bruce Margaret
Published: December 2017

all trans-retinoic acid (RA) enhances alveolarization in neonates and reinitiates alveolarization in emphysematous adult rat lungs, suggesting that RA may stimulate cell proliferation by upregulating growth factor ligand and/or receptor expression either indirectly or directly by acting on RA-responsive genes encoding growth factors. We report that RA and 1,25-dihydroxyvitamin D3(Vit D), alone and in combination, significantly increase [3H]thymidine incorporation in cultured fetal and postnatal rat lung fibroblasts ( P < 0.05). The greatest increase (11-fold) was seen in 4-day cells treated with the two agents in combination ( P < 0.0001). [3H]thymidine incorporation was age dependent. The greatest response to RA occurred in 4-day fibroblasts ( P < 0.01), whereas the response to Vit D was greatest in embryonic day 20 fibroblasts ( P < 0.001). Neutralizing antibody to platelet-derived growth factor (PDGF)-AB decreased [3H]thymidine incorporation in response to RA alone or in combination with Vit D, indicating a role for PDGF. Expression of mRNAs for PDGF-A and PDGF receptor (PDGFR)-α and -β was upregulated at the transcriptional level in an age- and treatment-dependent manner. Thus exogenous RA may influence alveolarization by stimulating fibroblast proliferation through a PDGF-mediated autocrine mechanism, which is enhanced when RA and Vit D are administered in combination.
Effects of hypoxia in porcine pulmonary arterial myocytes: roles of KVchannel and endothelin-1
Authors: Sham James S. K. , Crenshaw Benjamin R. , Deng Li-Hua , Shimoda Larissa A. , Sylvester J. T.
Published: December 2017

Effects of acute hypoxia on intracellular Ca2+concentration ([Ca2+]i) and cell length were recorded simultaneously in proximal and distal pulmonary (PASMCs) and femoral (FASMCs) arterial smooth muscle cells. Reducing Po2from normoxia to severe hypoxia (Po2< 10 mmHg) caused small but significant decreases in length and a reversible increase in [Ca2+]iin distal PASMCs and a small decrease in length in proximal PASMCs but had no effect in FASMCs, even though all three cell types contracted significantly to vasoactive agonists. Inhibition of voltage-dependent K+(KV) channel with 4-aminopyridine produced a greater increase in [Ca2+]iin distal than in proximal PASMCs. In distal PASMCs, severe hypoxia caused a slight inhibition of KVcurrents; however, it elicited further contraction in the presence of 4-aminopyridine. Endothelin-1 (10−10M), which itself did not alter cell length or [Ca2+]i, significantly potentiated the hypoxic contraction. These results suggest that hypoxia only has small direct effects on porcine PASMCs. These effects cannot be fully explained by inhibition of KVchannels and were greatly enhanced via synergistic interactions with the endothelium-derived factor endothelin-1.
Dexamethasone enhances ras-recision gene expression in cultured murine fetal lungs: role in development
Authors: Chinoy Mala R. , Zgleszewski Steven E. , Cilley Robert E. , Krummel Thomas M.
Published: December 2017

We have shown that dexamethasone (Dex) accelerates maturation and differentiation of cultured fetal murine lungs (Cilley RE, Zgleszewski SE, Krummel TM, and Chinoy MR. Surg Forum 47: 692–695, 1996). We now demonstrate that although Dex inhibits thinning of acinar walls and secondary septa formation, it does, however, promote lung growth. CD-1 murine fetal lungs were cultured for 7 days in the presence and absence of 10 nM Dex. Dex-modulated genes were investigated and identified by differential display of mRNAs performed with specific anchor primer H-T11G and 24 arbitrary primers. Thirty-five differentially expressed cDNAs were isolated, subcloned, sequenced, and identified through BLAST searches. One of these cDNAs, termed Dex2, with enhanced expression in Dex-treated lungs, had 100% similarity with ras-recision gene ( rrg), also known as the lysyl oxidase ( LOX) gene that encodes lysyl oxidase. LOX gene is very highly conserved, with significant sequence similarity among mouse, rat, and human. Two other cDNAs, termed Dex1 and Dex4, were also identified as rrg, with 92 and 97% sequence similarity with the existing data bank sequence of rrg. LOX enzyme is known to downregulate p21rasprotein and play a central role in the maturation of collagen and elastin in the extracellular matrix as well as modulate the cytoskeletal elements. Thus LOX may be important in lung developmental processes involving epithelial-mesenchymal interactions.
CFTR modulates lung secretory cell proliferation and differentiation
Authors: Larson Janet E. , Delcarpio Joseph B. , Farberman Michelle M. , Morrow Susan L. , Cohen J. Craig
Published: December 2017

We have permanently reversed the lethal phenotype in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-deficient (knockout) mouse after in utero gene therapy with an adenovirus containing the cftr gene. The gene transfer targeted somatic stem cells in the developing lung and intestine, and these epithelial surfaces demonstrated permanent developmental changes after treatment. The survival statistics from the progeny of heterozygote-heterozygote matings after in utero cftr gene treatment demonstrated an increased mortality in the homozygous normal pups, indicating that overexpression during development was detrimental. The lungs of these pups revealed accelerated secretory cell proliferation and differentiation. The extent of proliferation and differentiation in the secretory cells of the lung parenchyma after in utero transfer of the cftr gene was evaluated with morphometric and biochemical analyses. These studies provide further support of the regulatory role of the cftr gene in the development of the secretory epithelium.
Transgenic overexpression of β2-adrenergic receptors in airway epithelial cells decreases bronchoconstriction
Authors: McGraw Dennis W. , Forbes Susan L. , Mak Judith C. W. , Witte David P. , Carrigan Patricia E. , Leikauf George D. , Liggett Stephen B.
Published: December 2017

Airway epithelial cells express β2-adrenergic receptors (β2-ARs), but their role in regulating airway responsiveness is unclear. With the Clara cell secretory protein (CCSP) promoter, we targeted expression of β2-ARs to airway epithelium of transgenic (CCSP-β2-AR) mice, thereby mimicking agonist activation of receptors only in these cells. In situ hybridization confirmed that transgene expression was confined to airway epithelium, and autoradiography showed that β2-AR density in CCSP-β2-AR mice was approximately twofold that of nontransgenic (NTG) mice. Airway responsiveness measured by whole body plethysmography showed that the methacholine dose required to increase enhanced pause to 200% of baseline (ED200) was greater for CCSP-β2-AR than for NTG mice (345 ± 34 vs. 157 ± 14 mg/ml; P < 0.01). CCSP-β2-AR mice were also less responsive to ozone (0.75 ppm for 4 h) because enhanced pause in NTG mice acutely increased to 77% over baseline ( P < 0.05) but remained unchanged in the CCSP-β2-AR mice. Although both groups were hyperreactive to methacholine 6 h after ozone exposure, the ED200for ozone-exposed CCSP-β2-AR mice was equivalent to that for unexposed NTG mice. These findings show that epithelial cell β2-ARs regulate airway responsiveness in vivo and that the bronchodilating effect of β-agonists results from activation of receptors on both epithelial and smooth muscle cells.
Mechanisms regulating endothelial cell barrier function
Authors: Stevens Troy , Garcia Joe G. N. , Shasby D. Michael , Bhattacharya Jahar , Malik Asrar B.
Published: December 2017

Endothelium forms a physical barrier that separates blood from tissue. Communication between blood and tissue occurs through the delivery of molecules and circulating substances across the endothelial barrier by directed transport either through or between cells. Inflammation promotes macromolecular transport by decreasing cell-cell and cell-matrix adhesion and increasing centripetally directed tension, resulting in the formation of intercellular gaps. Inflammation may also increase the selected transport of macromolecules through cells. Significant progress has been made in understanding the molecular and cellular mechanisms that account for constitutive endothelial cell barrier function and also the mechanisms activated during inflammation that reduce barrier function. Current concepts of mechanisms regulating endothelial cell barrier function were presented in a symposium at the 2000 Experimental Biology Conference and are reviewed here.
Protein kinase C isozymes and the regulation of diverse cell responses
Authors: Dempsey Edward C. , Newton Alexandra C. , Mochly-Rosen Daria , Fields Alan P. , Reyland Mary E. , Insel Paul A. , Messing Robert O.
Published: December 2017

Individual protein kinase C (PKC) isozymes have been implicated in many cellular responses important in lung health and disease, including permeability, contraction, migration, hypertrophy, proliferation, apoptosis, and secretion. New ideas on mechanisms that regulate PKC activity, including the identification of a novel PKC kinase, 3-phosphoinositide-dependent kinase-1 (PDK-1), that regulates phosphorylation of PKC, have been advanced. The importance of targeted translocation of PKC and isozyme-specific binding proteins (like receptors for activated C-kinase and caveolins) is well established. Phosphorylation state and localization are now thought to be key determinants of isozyme activity and specificity. New concepts on the role of individual PKC isozymes in proliferation and apoptosis are emerging. Opposing roles for selected isozymes in the same cell system have been defined. Coupling to the Wnt signaling pathway has been described. Phenotypes for PKC knockout mice have recently been reported. More specific approaches for studying PKC isozymes and their role in cell responses have been developed. Strengths and weaknesses of different experimental strategies are reviewed. Future directions for investigation are identified.
VEGF, fetal liver kinase-1, and permeability increase during unilateral lung ischemia
Authors: Kazi Armina A. , Lee Won S. , Wagner Elizabeth , Becker Patrice M.
Published: December 2017

Vascular endothelial growth factor (VEGF) is a potent mediator of increased vascular permeability and an endothelial cell mitogen. Because VEGF is upregulated during ventilated ischemia of isolated lungs and may lead to both increased vascular permeability and neovascularization, we hypothesized that VEGF and kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/flk-1) expression would increase acutely after unilateral pulmonary arterial (PA) ischemia in vivo in association with evidence of endothelial cell barrier dysfunction. To test this hypothesis, VEGF and KDR/flk-1 mRNA and protein expression were measured after 4, 8, and 24 h of left PA ligation in mice. Permeability was assessed at the same time points by measurement of bronchoalveolar lavage protein concentration and lung wet-to-dry weight ratios. Results were compared with those from uninstrumented and sham-operated mice. VEGF and KDR/flk-1 protein in the left lung both increased by 4 h and then returned to baseline, whereas increased VEGF and KDR/flk-1 mRNA expression was sustained throughout 24 h of unilateral ischemia. Bronchoalveolar lavage protein concentration increased transiently during ischemia, whereas wet-to-dry weight ratio of the left lung increased more slowly and remained elevated after 24 h of left PA ligation. These results suggest that increased expression of VEGF and KDR/flk-1 during unilateral PA occlusion in mice may contribute to the development of acute lung injury in this model.
The PDE inhibitor zaprinast enhances NO-mediated protection against vascular leakage in reperfused lungs
Authors: Schütte Hartwig , Witzenrath Martin , Mayer Konstantin , Weissmann Norbert , Schell Alexander , Rosseau Simone , Seeger Werner , Grimminger Friedrich
Published: December 2017

Disruption of endothelial barrier properties with development of noncardiogenic pulmonary edema is a major threat in lung ischemia-reperfusion (I/R) injury that occurs under conditions of lung transplantation. Inhaled nitric oxide (NO) reduced vascular leakage in lung I/R models, but the efficacy of this agent may be limited. We coadministered NO and zaprinast, a cGMP-specific phosphodiesterase inhibitor, to further augment the NO-cGMP axis. Isolated, buffer-perfused rabbit lungs were exposed to 4.5 h of warm ischemia. Reperfusion provoked a transient elevation in pulmonary arterial pressure and a negligible rise in microvascular pressure followed by a massive increase in the capillary filtration coefficient and severe lung edema formation. Inhalation of 10 parts/million of NO or intravascular application of 100 μM zaprinast on reperfusion both reduced pressor response and moderately attenuated vascular leakage. Combined administration of both agents induced no additional vasodilation at constant microvascular pressures, but additively protected against capillary leakage paralleled by a severalfold increase in perfusate cGMP levels. In conclusion, combining low-dose NO inhalation and phosphodiesterase inhibition may be suitable for the maintenance of graft function in lung transplantation by amplifying the beneficial effect of the NO-cGMP axis and avoiding toxic effects of high NO doses.
Matrix metalloproteinases and tissue inhibitor of metalloproteinase-2 in fetal rabbit lung
Authors: Fukuda Yuh , Ishizaki Masamichi , Okada Yasunori , Seiki Motoharu , Yamanaka Nobuaki
Published: December 2017

Cell-extracellular matrix interaction and extracellular matrix remodeling are known to be important in fetal lung development. We investigated the localization of matrix metalloproteinases (MMPs) in fetal rabbit lungs. Immunohistochemistry for type IV collagen, MMP-1, MMP-2, MMP-9, membrane type (MT) 1 MMP, and tissue inhibitor of metalloproteinase (TIMP)-2 and in situ hybridization for MMP-9 mRNA were performed. Gelatin zymography and Western blotting for MT1-MMP in lung tissue homogenates were also studied. MMP-1 and MT1-MMP were detected in epithelial cells, and MMP-2 and TIMP-2 were detected in epithelial cells and some mesenchymal cells in each stage. MMP-9 was found in epithelial cells mainly in the late stage. Gelatin zymography revealed that the ratio of active MMP-2 to latent MMP-2 increased dramatically during the course of development. MT1-MMP was detected in tissue homogenates, especially predominant in the late stage. These findings suggest that MMPs and their inhibitors may contribute to the formation of airways and alveoli in fetal lung development and that activated MMP-2 of alveolar epithelial cells may function to provide an extremely wide alveolar surface.
β-Adrenergic agonists exert their “anti-inflammatory” effects in monocytic cells through the IκB/NF-κB pathway
Authors: Farmer Pierre , Pugin Jérôme
Published: December 2017

In addition to their well-studied bronchodilatory and cardiotonic effects, β-adrenergic agonists carry anti-inflammatory properties by inhibiting cytokine production by human mononuclear cells. In a model of human promonocytic THP-1 cells stimulated with lipopolysaccharide (LPS), we showed that β-agonists inhibited tumor necrosis factor-α and interleukin-8 production predominantly via the β2-adrenergic receptor through the generation of cAMP and activation of protein kinase A. This effect was reproduced by other cAMP-elevating agents such as prostaglandins and cAMP analogs. Activation and nuclear translocation of the transcription factor nuclear factor-κB induced by LPS were inhibited with treatment with β-agonists, an effect that was prominent at late time points (>1 h). Although the initial IκB-α degradation induced by LPS was minimally affected by β-agonists, the latter induced a marked rebound of the cytosolic IκB-α levels at later time points (>1 h), accompanied by an increased IκB-α cytoplasmic half-life. This potentially accounts for the observed nuclear factor-κB sequestration in the cytoplasmic compartment. We postulate that the anti-inflammatory effects of β-agonists reside in their capacity to increase cytoplasmic concentrations of IκB-α, possibly by decreasing its degradation.
Receptor-dependent activation of store-operated calcium entry increases endothelial cell permeability
Authors: Moore Timothy M. , Norwood Natalie R. , Creighton Judy R. , Babal Pavál , Brough George H. , Shasby D. Michael , Stevens Troy
Published: December 2017

The present study evaluated the necessity of store-operated Ca2+entry in mediating thrombin-induced 20-kDa myosin light chain (MLC20) phosphorylation and increased permeability in bovine pulmonary artery endothelial cells (BPAECs). Thrombin (7 U/ml) and thapsigargin (1 μM) activated Ca2+entry through a common pathway in confluent BPAECs. Similar increases in MLC20phosphorylation were observed 5 min after thrombin and thapsigargin challenge, although thrombin produced a sustained increase in MLC20phosphorylation that was not observed in response to thapsigargin. Neither agonist increased MLC20phosphorylation when Ca2+influx was inhibited. Thrombin and thapsigargin induced inter-endothelial cell gap formation and increased FITC-dextran (molecular radii 23 Å) transfer across confluent BPAEC monolayers. Activation of store-operated Ca2+entry was required for thapsigargin and thrombin receptor-activating peptide to increase permeability, demonstrating that activation of store-operated Ca2+entry is coupled with MLC20phosphorylation and is associated with intercellular gap formation and increased barrier transport of macromolecules. Unlike thrombin receptor-activating peptide, thrombin increased permeability without activation of store-operated Ca2+entry, suggesting that it partly disrupts the endothelial barrier through a proteolytic mechanism independent of Ca2+signaling.
Control of the mode of excitation-contraction coupling by Ca2+ stores in bovine trachealis muscle
Authors: Tao Liang , Huang Yu , Bourreau Jean-Pierre
Published: December 2017

Full muscarinic stimulation in bovine tracheal smooth muscle caused a sustained contraction and increase in intracellular Ca2+concentration ([Ca2+]i) that was largely resistant to inhibition by nifedipine. Depletion of internal Ca2+ stores with cyclopiazonic acid resulted in an increased efficacy of nifedipine to inhibit this contraction and the associated increase in [Ca2+]i. Thus internal Ca2+ store depletion promoted electromechanical coupling between full muscarinic stimulation and muscle contraction to the detriment of pharmacomechanical coupling. A similar change in coupling mode was induced by ryanodine even when it did not significantly modify the initial transient increase in [Ca2+]iinduced by this stimulation, indicating that depletion of internal stores was not necessary to induce the change in excitation-contraction coupling mode. Blockade of the Ca2+-activated K+ channel by tetraethylammonium, charybdotoxin, and iberiotoxin all induced the change in excitation-contraction coupling mode. These results suggest that in this preparation, Ca2+released from the ryanodine-sensitive Ca2+ store, by activating Ca2+-activated K+ channels, plays a central role in determining the expression of the pharmacomechanical coupling mode between muscarinic excitation and the Ca2+influx necessary for the maintenance of tone.
A primary culture model of differentiated murine tracheal epithelium
Authors: Davidson Donald J. , Kilanowski Fiona M. , Randell Scott H. , Sheppard David N. , Dorin Julia R.
Published: December 2017

The goal of this study was to develop a primary culture model of differentiated murine tracheal epithelium. When grown on semipermeable membranes at an air interface, dissociated murine tracheal epithelial cells formed confluent polarized epithelia with high transepithelial resistances (∼12 kΩ · cm2) that remained viable for up to 80 days. Immunohistochemistry and light and electron microscopy demonstrated that the cells were epithelial in nature (cytokeratin positive, vimentin and α-smooth muscle actin negative) and differentiated to form ciliated and secretory cells from day 8 after seeding onward. With RT-PCR, expression of the cystic fibrosis transmembrane conductance regulator ( Cftr) and murine β-defensin ( Defb) genes was detected ( Defb-1 was constitutively expressed, whereas Defb-2 expression was induced by exposure to lipopolysaccharide). Finally, Ussing chamber experiments demonstrated an electrophysiological profile compatible with functional amiloride-sensitive sodium channels and cAMP-stimulated CFTR chloride channels. These data indicate that primary cultures of murine tracheal epithelium have many characteristics similar to those of murine tracheal epithelium in vivo. This method will facilitate the establishment of primary cultures of airway epithelium from transgenic mouse models of human diseases.
Complement-mediated host defense in the lung
Authors: Watford Wendy T. , Ghio Andrew J. , Wright Jo Rae
Published: December 2017

Complement is a system of plasma proteins that aids in the elimination of pathogens from the body. We hypothesized that there is a functional complement system present in the lung that aids in the removal of pathogens. Western blot analysis revealed complement proteins of the alternative and classical pathways of complement in bronchoalveolar lavage fluids (BALF) from healthy volunteers. Functional classical pathway activity was detected in human BALF, but there was no significant alternative pathway activity in lavage fluid, a finding that correlates with the low level of the alternative pathway protein, factor B, in these samples. Although the classical pathway of complement was functional in lavage fluid, the level of the classical pathway activator C1q was very low. We tested the ability of the lung- specific surfactant proteins, surfactant protein A (SP-A) and surfactant protein D (SP-D), to substitute for C1q in classical pathway activation, since they have structural homology to C1q. However, neither SP-A nor SP-D restored classical pathway activity to C1q-depleted serum. These data suggest that the classical pathway of complement is functionally active in the lung where it may play a role in the recognition and clearance of bacteria.
Synergistic and additive killing by antimicrobial factors found in human airway surface liquid
Authors: Singh Pradeep K. , Tack Brian F. , McCray Paul B. , Welsh Michael J.
Published: December 2017

Airway surface liquid contains multiple factors thought to provide a first line of defense against bacteria deposited in the airways. Although the antimicrobial action of individual factors has been studied, less is known about how they work in combination. We examined the combined action of six antimicrobial peptides found in airway surface liquid. The paired combinations of lysozyme-lactoferrin, lysozyme-secretory leukocyte protease inhibitor (SLPI), and lactoferrin-SLPI were synergistic. The triple combination of lysozyme, lactoferrin, and SLPI showed even greater synergy. Other combinations involving the human β-defensins, LL-37, and tobramycin (often administered to cystic fibrosis patients by inhalation) were additive. Because the airway surface liquid salt concentration may be elevated in cystic fibrosis patients, we examined the effect of salt on the synergistic combinations. As the ionic strength increased, synergistic interactions were lost. Our data suggest that the antibacterial potency of airway surface liquid may be significantly increased by synergistic and additive interactions between antimicrobial factors. These results also suggest that increased salt concentrations that may exist in cystic fibrosis could inhibit airway defenses by diminishing these synergistic interactions.
Surfactant phospholipid catabolic rate is pool size dependent in mice
Authors: Kramer Boris W. , Ikegami Machiko , Jobe Alan H.
Published: December 2017

We increased surfactant pool size by surfactant treatment in mice to test if the catabolism of the major component of surfactant, saturated phosphatidylcholine (Sat PC), was rate limited. By intratracheal instillation, we gave mice trace doses, doses of 45 or 110 μmol/kg, or three doses of 110 μmol/kg of Sat PC in surfactant that contained radiolabeled dipalmitoylphosphatidylcholine (DPPC) and a radiolabeled phospholipase A-resistant ether analog of DPPC. Two strains of mice with 2-fold differences in alveolar and total Sat PC pool sizes were used; the mice with the higher pool sizes had a 2.3-fold higher steady-state catabolic rate. Acute increases in alveolar surfactant given by intratracheal instillation increased catabolic rates ∼2-fold over the steady-state rates in both strains. There was minimal loss of the ether analog of DPPC from the lungs, and the alveolar macrophages did not accumulate more than 10% of the ether analog. In these two strains of mice, the catabolism of Sat PC was not rate limited because catabolic rate increased when alveolar pool sizes were increased.
Angiotensin IV-mediated pulmonary artery vasorelaxation is due to endothelial intracellular calcium release
Authors: Chen Sifeng , Patel Jawaharlal M. , Block Edward R.
Published: December 2017

Angiotensin (ANG) IV stimulation of pulmonary artery (PA) endothelial cells (PAECs) but not of PA smooth muscle cells (PASMCs) resulted in significant increased production of cGMP in PASMCs. ANG IV receptors are not present in PASMCs, and PASMC nitric oxide synthase activity was not altered by ANG IV. ANG IV caused a dose-dependent vasodilation of U-46619-precontracted endothelium-intact but not endothelium-denuded PAs, and this response was blocked by the ANG IV receptor antagonist divalinal ANG IV but not by ANG II type 1 and 2 receptor blockers. ANG IV receptor-mediated increased intracellular Ca2+concentration ([Ca2+]i) release from intracellular stores in PAECs was blocked by divalinal ANG IV as well as by the G protein, phospholipase C, and phosphoinositide (PI) 3-kinase inhibitors guanosine 5′- O-(2-thiodiphosphate), U-73122, and LY-294002, respectively, and was regulated by both PI 3-kinase- and ryanodine-sensitive Ca2+stores. Basal and ANG IV-mediated vasorelaxation of endothelium-denuded PAs was restored by exogenous PAECs but not by exogenous PAECs pretreated with the intracellular Ca2+chelator 1,2-bis( o-aminophenoxy)ethane- N, N, N′, N′-tetraacetic acid-AM. These results demonstrate that ANG IV-mediated vasodilation of PAs is endothelium dependent and regulated by [Ca2+]irelease through receptor-coupled G protein-phospholipase C-PI 3-kinase signaling mechanisms.
Free radical-mediated transgene inactivation of macrophages by endotoxin
Authors: Dokka Sujatha , Toledo David , Wang Liying , Shi Xianglin , Huang Chuanshu , Leonard Stephen , Rojanasakul Yon
Published: December 2017

Endotoxin, the lipopolysaccharide component of gram-negative bacteria, is a common contaminant of plasmid DNA preparations. The present study investigated the effect of endotoxin on gene transfection efficiency and the role of reactive oxygen species (ROS) in this process. Gene transfection studies were performed in various cell types with cytomegalovirus-luciferase as a reporter plasmid and cationic liposome as a transfecting agent. The presence of endotoxin in plasmid DNA preparations severely limited transgene expression in macrophages but had little or no effect in other cell types tested. This decreased transfection was dependent on ROS-mediated cellular toxicity induced by endotoxin. Neutralizing the endotoxin by the addition of polymyxin B effectively increased transfection efficiency and reduced toxicity. Electron spin resonance studies confirmed the formation of ROS in endotoxin-treated cells and their inhibition by free radical scavengers. The ROS scavenger N- t-butyl-α-phenylnitrone, the H2O2scavenger catalase, and the ·OH scavenger sodium formate effectively inhibited endotoxin-induced effects, whereas the O2−scavenger superoxide dismutase had lesser effects. These results indicate that multiple oxidative species are involved in the transfection inactivation process and that ·OH formed by H2O2-dependent, metal-catalyzed Fenton reaction play a major role in this process.
SB 239063, a p38 MAPK inhibitor, reduces neutrophilia, inflammatory cytokines, MMP-9, and fibrosis in lung
Authors: Underwood David C. , Osborn Ruth R. , Bochnowicz Steven , Webb Edward F. , Rieman David J. , Lee John C. , Romanic Anne M. , Adams Jerry L. , Hay Douglas W. P. , Griswold Don E.
Published: December 2017

The effects of a second generation p38 mitogen-activated protein kinase (MAPK) inhibitor, SB 239063 [ trans-1-(4-hydroxycyclohexyl)-4-(4-fluorophenyl)-5-(2-methoxypyridimidin-4-yl)imidazole; IC50= 44 nM vs. p38α], were assessed in models that represent different pathological aspects of chronic obstructive pulmonary disease (COPD) [airway neutrophilia, enhanced cytokine formation and increased matrix metalloproteinase (MMP)-9 activity] and in a model of lung fibrosis. Airway neutrophil infiltration and interleukin (IL)-6 levels, assessed by bronchoalveolar lavage 48 h after lipopolysaccharide (LPS) inhalation, were inhibited dose dependently by 3–30 mg/kg of SB 239063 given orally twice a day. In addition, SB 239063 (30 mg/kg orally) attenuated IL-6 bronchoalveolar lavage fluid concentrations (>90% inhibition) and MMP-9 activity (64% inhibition) assessed 6 h after LPS exposure. In guinea pig cultured alveolar macrophages, SB 239063 inhibited LPS-induced IL-6 production (IC50of 362 nM). In a bleomycin-induced pulmonary fibrosis model in rats, treatment with SB 239063 (2.4 or 4.8 mg/day via osmotic pump) significantly inhibited bleomycin-induced right ventricular hypertrophy (indicative of secondary pulmonary hypertension) and increases in lung hydroxyproline synthesis (indicative of collagen synthesis and fibrosis). Therefore, SB 239063 demonstrates activity against a range of sequelae commonly associated with COPD and fibrosis, supporting the therapeutic potential of p38 MAPK inhibitors such as SB 239063 in chronic airway disease.
Anti-inflammatory effects of triptolide in human bronchial epithelial cells
Authors: Zhao Guohua , Vaszar Laszlo T. , Qiu Daoming , Shi Lingfang , Kao Peter N.
Published: December 2017

Triptolide (PG490, 97% pure) is a diterpenoid triepoxide with potent anti-inflammatory and immunosuppressive effects in transformed human bronchial epithelial cells and T cells (Qiu D, Zhao G, Aoki Y, Shi L, Uyei A, Nazarian S, Ng JC-H, and Kao PN. J Biol Chem 274: 13443–13450, 1999). Triptolide, with an IC50of ∼20–50 ng/ml, inhibits normal and transformed human bronchial epithelial cell expression of interleukin (IL)-6 and IL-8 stimulated by phorbol 12-myristate 13-acetate (PMA), tumor necrosis factor-α, or IL-1β. Nuclear runoff and luciferase reporter gene assays demonstrate that triptolide inhibits IL-8 transcription. Triptolide also inhibits the transcriptional activation, but not the DNA binding, of nuclear factor-κB. A cDNA array and clustering algorithm analysis reveals that triptolide inhibits expression of the PMA-induced genes tumor necrosis factor-α, IL-8, macrophage inflammatory protein-2α, intercellular adhesion molecule-1, integrin β6, vascular endothelial growth factor, granulocyte-macrophage colony-stimulating factor, GATA-3, fra-1, and NF45. Triptolide also inhibits constitutively expressed cell cycle regulators and survival genes cyclins D1, B1, and A1, cdc-25, bcl-x, and c-jun. Thus anti-inflammatory, antiproliferative, and proapoptotic properties of triptolide are associated with inhibition of nuclear factor-κB signaling and inhibition of genes known to regulate cell cycle progression and survival.
Adoptive transfer of acute lung injury
Authors: Moxley Michael A. , Baird Tracey L. , Corbett John A.
Published: December 2017

In this study, we describe a novel adoptive transfer protocol to study acute lung injury in the rat. We show that bronchoalveolar lavage (BAL) cells isolated from rats 5 h after intratracheal administration of lipopolysaccharide (LPS) induce a lung injury when transferred to normal control recipient rats. This lung injury is characterized by increased alveolar-arterial oxygen difference and extravasation of Evans blue dye (EBD) into lungs of recipient rats. Recipient rats receiving similar numbers of donor cells isolated from healthy rats do not show adverse changes in the alveolar-arterial oxygen difference or in extravasation of EBD. The adoptive transfer-induced lung injury is associated with increased numbers of neutrophils in the BAL, the levels of which are similar to the numbers observed in BAL cells isolated from rats treated for 5 h with LPS. As an indicator of BAL cell activation, donor BAL cell inducible nitric oxide synthase (iNOS) expression was compared with BAL cell iNOS expression 48 h after adoptive transfer. BAL cells isolated 5 h after LPS administration expressed iNOS immediately after isolation. In contrast, BAL cells isolated 48 h after adoptive transfer did not express iNOS immediately after isolation but expressed iNOS following a 24-h ex vivo culture. These findings indicate that the activation state of donor BAL cells differs from BAL cells isolated 48 h after adoptive transfer, suggesting that donor BAL cells may stimulate migration of new inflammatory cells into the recipient rats lungs.
Hypercapnia induces injury to alveolar epithelial cells via a nitric oxide-dependent pathway
Authors: Lang John D. , Chumley Phillip , Eiserich Jason P. , Estevez Alvaro , Bamberg Thad , Adhami Ahmad , Crow John , Freeman Bruce A.
Published: December 2017

Ventilator strategies allowing for increases in carbon dioxide (CO2) tensions (hypercapnia) are being emphasized to ameliorate the consequences of inflammatory-mediated lung injury. Inflammatory responses lead to the generation of reactive species including superoxide (O2−), nitric oxide (·NO), and their product peroxynitrite (ONOO−). The reaction of CO2and ONOO−can yield the nitrosoperoxocarbonate adduct ONOOCO2−, a more potent nitrating species than ONOO−. Based on these premises, monolayers of fetal rat alveolar epithelial cells were utilized to investigate whether hypercapnia would modify pathways of ·NO production and reactivity that impact pulmonary metabolism and function. Stimulated cells exposed to 15% CO2(hypercapnia) revealed a significant increase in ·NO production and nitric oxide synthase (NOS) activity. Cell 3-nitrotyrosine content as measured by both HPLC and immunofluorescence staining also increased when exposed to these same conditions. Hypercapnia significantly enhanced cell injury as evidenced by impairment of monolayer barrier function and increased induction of apoptosis. These results were attenuated by the NOS inhibitor N-monomethyl-l-arginine. Our studies reveal that hypercapnia modifies ·NO-dependent pathways to amplify cell injury. These results affirm the underlying role of ·NO in tissue inflammatory reactions and reveal the impact of hypercapnia on inflammatory reactions and its potential detrimental influences.
Neutrophils as early immunologic effectors in hemorrhage- or endotoxemia-induced acute lung injury
Authors: Abraham Edward , Carmody Aaron , Shenkar Robert , Arcaroli John
Published: December 2017

Acute lung injury is characterized by accumulation of neutrophils in the lungs, accompanied by the development of interstitial edema and an intense inflammatory response. To assess the role of neutrophils as early immune effectors in hemorrhage- or endotoxemia-induced lung injury, mice were made neutropenic with cyclophosphamide or anti-neutrophil antibodies. Endotoxemia- or hemorrhage-induced lung edema was significantly reduced in neutropenic animals. Activation of the transcriptional regulatory factor nuclear factor-κB after hemorrhage or endotoxemia was diminished in the lungs of neutropenic mice compared with nonneutropenic controls. Hemorrhage or endotoxemia was followed by increases in pulmonary mRNA and protein levels for interleukin-1β (IL-1β), macrophage inflammatory protein-2 (MIP-2), and tumor necrosis factor-α (TNF-α). Endotoxin-induced increases in proinflammatory cytokine expression were greater than those found after hemorrhage. The amounts of mRNA or protein for IL-1β, MIP-2, and TNF-α were significantly lower after hemorrhage in the lungs of neutropenic versus nonneutropenic mice. Neutropenia was associated with significant reductions in IL-1β and MIP-2 but not in TNF-α expression in the lungs after endotoxemia. These experiments show that neutrophils play a centrol role in initiating acute inflammatory responses and causing injury in the lungs after hemorrhage or endotoxemia.
Keratinocyte growth factor protects againstPseudomonas aeruginosa-induced lung injury
Authors: Viget Nathalie B. , Guery Benoit P. H. , Ader Florence , Nevière Remi , Alfandari Serge , Creuzy Colette , Roussel-Delvallez Micheline , Foucher Claude , Mason Carol M. , Beaucaire Gilles , Pittet Jean-François
Published: December 2017

We have previously reported that keratinocyte growth factor (KGF) attenuates α-naphthylthiourea-induced lung injury by upregulating alveolar fluid transport. The objective of this study was to determine the effect of KGF pretreatment in Pseudomonas aeruginosa pneumonia. A 5% bovine albumin solution with 1 μCi of125I-labeled human albumin was instilled into the air spaces 4 or 24 h after intratracheal instillation of P. aeruginosa, and the concentration of unlabeled and labeled proteins in the distal air spaces over 1 h was used as an index of net alveolar fluid clearance. Alveolocapillary barrier permeability was evaluated with an intravascular injection of 1 μCi of131I-albumin. In early pneumonia, KGF increased lung liquid clearance (LLC) compared with that in nonpretreated animals. In late pneumonia, LLC was significantly reduced in the absence of KGF but increased above the control value with KGF. KGF pretreatment increased the number of polymorphonuclear cells recovered in the bronchoalveolar lavage fluid and decreased bacterial pulmonary translocation. In conclusion, KGF restores normal alveolar epithelial fluid transport during the acute phase of P. aeruginosa pneumonia and LLC in early and late pneumonia. Host response is also improved as shown by the increase in the alveolar cellular response and the decrease in pulmonary translocation of bacteria.
NO protects alveolar type II cells from stretch-induced apoptosis. A novel role for macrophages in the lung
Authors: Edwards Yasmin S. , Sutherland Leanne M. , Murray Andrew W.
Published: December 2017

We have previously shown that mechanical distortion or stretch of alveolar type II (ATII) cells induces both surfactant release and the induction of apoptosis. We hypothesize that nitric oxide (NO) secreted from alveolar macrophages (AMs) prevents cyclic stretch-induced apoptosis. We show that S-nitroso- N-acetyl-d,l-penicillamine (SNAP), a chemical donor of NO, protects cells against nuclear condensation and DNA fragmentation induced by stretch (30% at 60 cycles/min) as well as by sorbitol. SNAP depleted of NO had no protective effect, and the NO scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide blocked the antiapoptotic effect of SNAP. We also show that AMs isolated from rat lung lavage fluid actively synthesize and secrete NO. Using a novel technique in which AMs were cocultured with ATII cells while adhered to floating membrane rafts, we found that NO released from AMs was effective in protecting ATII cells from undergoing apoptosis. We therefore propose that NO secreted by AMs may function as part of a physiological antiapoptotic mechanism that prevents ATII cells from undergoing stretch-induced cell death in the lung.
TNF-α and IL-1β are not essential to the inflammatory response in LPS-induced airway disease
Authors: Moreland Jessica G. , Fuhrman Robert M. , Wohlford-Lenane Christine L. , Quinn Timothy J. , Benda Erin , Pruessner Jonathan A. , Schwartz David A.
Published: December 2017

To determine the role of tumor necrosis factor (TNF)-α and interleukin (IL)-1β in the lower respiratory tract inflammatory response after inhalation of lipopolysaccharide (LPS), we conducted inhalation exposure studies in mice lacking expression of TNF-α and/or IL-1 receptor type 1 and in mice with functional blockade of these cytokines using adenoviral vector delivery of soluble receptors to one or both cytokines. Alterations in airway physiology were assessed by pulmonary function testing before and immediately after 4 h of LPS exposure, and the cellular inflammatory response was measured by whole lung lavage and assessment of inflammatory cytokine protein and mRNA expression. Airway resistance after LPS exposure was similarly increased in all groups of mice without evidence that blockade of either or both cytokines was protective from this response. Additionally, all groups of mice demonstrated significant increases in lung lavage fluid cellularity with a complete shift in the population of cells to a predominantly neutrophilic infiltrate as well as elevation in inflammatory cytokine protein and mRNA levels. There were no significant differences between the groups in measures of lung inflammation. These results indicate that TNF-α and IL-1β do not appear to have an essential role in mediating the physiological or inflammatory response to inhaled LPS.
Reovirus triggers cell type-specific proinflammatory responses dependent on the autocrine action of IFN-β
Authors: Hamamdzic Damir , Phillips-Dorsett Taetia , Altman-Hamamdzic Sanja , London Steven D. , London Lucille
Published: December 2017

Resident cells of the respiratory and gastrointestinal tracts, including epithelial and fibroblast cells, are the initial sites of entry for many viral pathogens. We investigated the role that these cells play in the inflammatory process in response to infection with reovirus 1/L. In A549 human bronchial or HT-29 human colonic epithelial cells, interferon (IFN)-β, regulated on activation T cell expressed and secreted (RANTES), IFN-γ-inducible protein (IP)-10, and interleukin-8 were upregulated regardless of whether cells were infected with replication-competent or replication-deficient reovirus 1/L. However, in CCD-34Lu human lung fibroblast cells, IFN-β, IP-10, and RANTES were expressed only after infection with replication-competent reovirus 1/L. Expression of interleukin-8 in CCD-34Lu fibroblast cells was viral replication independent. This differential expression of IFN-β, RANTES, and IP-10 was shown to be due to the lack of induction of IFN regulatory factor-1 and -2 in CCD-34Lu fibroblast cells treated with replication-deficient reovirus 1/L. We have shown that cytokine and/or chemokine expression may not be dependent on viral replication. Therefore, treatment of viral infections with inhibitors of replication may not effectively alleviate inflammatory mediators because most viral infections result in the generation of replication-competent and replication-deficient virions in vivo.
Effect of acetylsalicylic acid on endogenous IκB kinase activity in lung epithelial cells
Authors: Yoo Chul-Gyu , Lee Seunghee , Lee Choon-Taek , Kim Young Whan , Han Sung Koo , Shim Young-Soo
Published: December 2017

The anti-inflammatory effect of acetylsalicylic acid (ASA) has been thought to be secondary to the inhibition of prostaglandin synthesis. Because doses of ASA necessary to treat chronic inflammatory diseases are much higher than those needed to inhibit prostaglandin synthesis, a prostaglandin-independent pathway has been emerging as the new anti-inflammatory mechanism of ASA. Here, we examined the effect of ASA on the interleukin (IL)-1β- and tumor necrosis factor (TNF)-α-induced proinflammatory cytokine expression and evaluated whether this effect is closely linked to the nuclear factor (NF)-κB/IκB-α pathway. A high dose of ASA blocked IL-1β- and TNF-α-induced TNF-α and IL-8 expression, respectively. ASA inhibited TNF-α-induced activation of NF-κB by preventing phosphorylation and subsequent degradation of IκB-α in a prostanoid-independent manner. TNF-α-induced activation of IκB kinase was also suppressed by ASA pretreatment. These observations suggest that the anti-inflammatory effect of ASA in lung epithelial cells may be due to suppression of IκB kinase activity, which thereby inhibits subsequent phosphorylation and degradation of IκB-α, activation of NF-κB, and proinflammatory cytokine expression in lung epithelial cells.
Secretion and gene expression of secretory leukocyte protease inhibitor by human airway submucosal glands
Authors: Saitoh Hiroki , Masuda Tohru , Shimura Sanae , Fushimi Toshiaki , Shirato Kunio
Published: December 2017

Submucosal glands were isolated within 4 h of death from tracheae and bronchi obtained from autopsied lungs, and the secretory response of secretory leukocyte protease inhibitor (SLPI) was examined with ELISA and a secretory index. Although human neutrophil elastase (HNE) at low concentrations increased SLPI secretion above the control level (i.e., 149% of control level at 10−11M), HNE at high concentrations significantly decreased it below the control level (i.e., 16% of control level at 10−7M). The decrease in SLPI concentration was shown to result from the degradation of SLPI by excessive HNE. Methacholine induced significant secretion (i.e., 363% of control level at 10−5M) that was abolished by both M1and M3receptor antagonists. A semiquantitative analysis of SLPI mRNA by RT-PCR and Southern blot showed that compared with the superficial epithelium, submucosal glands had a 30-fold or higher level of SLPI mRNA. Both HNE and methacholine significantly increased the level of SLPI mRNA in submucosal glands in a dose-dependent manner (i.e., 357% of control level at 10−7M and 175% of control level at 10−5M, respectively). These findings indicate that human airway submucosal glands can transcribe 30-fold or more SLPI mRNA than the superficial epithelium and that SLPI mRNA transcription and secretion are regulated by both HNE and muscarinic receptors.