bluuebunny/crossref_metadata_2025 huggingface dataset:
Maintenance of the mouse type II cell phenotype in vitro
Authors: Rice Ward R. , Conkright Juliana J. , Na Cheng-Lun , Ikegami Machiko , Shannon John M. , Weaver Timothy E.
Published: March 2015

The purpose of this study was to identify culture conditions for maintenance of isolated mouse type II cells with intact surfactant protein (SP) and phospholipid production. Type II cells were isolated from 6-wk-old mice and cultured on Matrigel matrix-rat tail collagen (70:30 vol/vol) in bronchial epithelial cell growth medium minus hydrocortisone plus 5% charcoal-stripped FBS and 10 ng/ml keratinocyte growth factor. Under these conditions, type II cells actively produced surfactant phospholipids and proteins for at least 7 days. Synthesis and secretion of surfactant phospholipids and SP-A, -B, -C, and -D declined on day 1 of culture but recovered by day 3, reaching levels comparable to or exceeding freshly isolated cells by day 5. Abundant lamellar bodies were readily apparent in cells examined on days 5 and 7, and a surfactant pellet was recovered by centrifugation of media harvested on each day of culture. Secretion of SP-B, SP-C, and phosphatidylcholine was stimulated by phorbol 12-myristate 13-acetate and was inhibited by compound 48/80. When tested with a bubble surfactometer, surfactant secreted by type II cells on day 5 of culture lowered surface tension to 5.2 ± 2.3 mN/m. This is the first description of the synthesis and secretion of a functional surfactant complex by mouse type II cells after 7 days in primary culture.
Classical isoforms of PKC as regulators of CAT-1 transporter activity in pulmonary artery endothelial cells
Authors: Krotova Karina Y. , Zharikov Sergey I. , Block Edward R.
Published: March 2015

We examined which isoforms of protein kinase C (PKC) may be involved in the regulation of cationic amino acid transporter-1 (CAT-1) transport activity in cultured pulmonary artery endothelial cells (PAEC). An activator of classical and novel isoforms of PKC, phorbol 12-myristate-13-acetate (PMA; 100 nM), inhibited CAT-1-mediatedl-arginine transport in PAEC after a 1-h treatment and activated l-arginine uptake after an 18-h treatment of cells. These changes in l-arginine transport were not related to the changes in the expression of the CAT-1 transporter. The inhibitory effect of PMA on l-arginine transport was accompanied by a translocation of PKCα (a classical PKC isoform) from the cytosol to the membrane fraction, whereas the activating effect of PMA on l-arginine transport was accompanied by full depletion of the expression of PKCα in PAEC. A selective activator of Ca2+-dependent classical isoforms of PKC, thymeleatoxin (Thy; 100 nM; 1-h and 18-h treatments), induced the same changes inl-arginine uptake and PKCα translocation and depletion as PMA. The effects of PMA and Thy on l-arginine transport in PAEC were attenuated by a selective inhibitor of classical PKC isoforms Go 6976 (1 μM). Phosphatidylinositol-3,4,5-triphosphate-dipalmitoyl (PIP; 5 μM), which activates novel PKC isoforms, did not affectl-arginine transport in PAEC after 1-h and 18-h treatment of cells. PIP (5 μM; 1 h) induced the translocation of PKCε (a novel PKC isoform) from the cytosolic to the particulate fraction and did not affect the translocation of PKCα. These results demonstrate that classical isoforms of PKC are involved in the regulation of CAT-1 transport activity in PAEC. We suggest that translocation of PKCα to the plasma membrane induces phosphorylation of the CAT-1 transporter, which leads to inhibition of its transport activity in PAEC. In contrast, depletion of PKCα after long-term treatment with PMA or Thy promotes dephosphorylation of the CAT-1 transporter and activation of its activity.
Interleukin-13 induces a hypersecretory ion transport phenotype in human bronchial epithelial cells
Authors: Danahay Henry , Atherton Hazel , Jones Gareth , Bridges Robert J. , Poll Christopher T.
Published: March 2015

Interleukin (IL)-13 has been associated with asthma, allergic rhinitis, and chronic sinusitis, all conditions where an imbalance in epithelial fluid secretion and absorption could impact upon the disease. We have investigated the effects of IL-13 on the ion transport characteristics of human bronchial epithelial cells cultured at an apical-air interface. Ussing chamber studies indicated that 48 h pretreatment with IL-13 or IL-4 significantly reduced the basal short-circuit current ( Isc) and inhibited the amiloride-sensitive current by >98%. Furthermore, the Iscresponses were increased by more than six- and twofold over control values when stimulated with UTP or forskolin, respectively, after cytokine treatment. The IL-13-enhanced response to UTP/ionomycin was sensitive to bumetanide and DIDS and was reduced in a low-chloride, bicarbonate-free solution. Membrane permeablization studies indicated that IL-13 induced the functional expression of an apical Ca2+-activated anion conductance and that changes in apical or basolateral K+conductances could not account for the increased Iscresponses to UTP or ionomycin. The results indicate that IL-13 converts the human bronchial epithelium from an absorptive to a secretory phenotype that is the result of loss of amiloride-sensitive current and an increase in a DIDS-sensitive apical anion conductance.
Role of the JAK-STAT pathway in PDGF-stimulated proliferation of human airway smooth muscle cells
Authors: Simon Amy R. , Takahashi Satoe , Severgnini Mariano , Fanburg Barry L. , Cochran Brent H.
Published: March 2015

Airway remodeling, as manifested by an increase in airway smooth muscle mass, mucous gland hyperplasia, and subepithelial fibrosis, contributes to the airway hyperresponsiveness and fixed obstruction seen in some asthmatic patients. Here we investigated whether the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway contributes to platelet-derived growth factor (PDGF)-stimulated mitogenesis of human airway smooth muscle cells (HASMC). PDGF treatment of quiescent HASMC resulted in the rapid tyrosine phosphorylation and DNA binding of STAT1 and STAT3. This phosphorylation was blocked by inhibition of Src and JAK2 kinases. In addition, STAT activation by PDGF was found to be redox dependent. Moreover, PDGF-induced thymidine uptake was completely blocked by pretreatment of HASMC with the STAT kinase inhibitors AG-490, SU-6656, and PP2. Interestingly, the JAK pathway was required for HASMC mitogenesis independently of mitogen-activated protein kinase activation. Inhibition of the Src and JAK kinases blocked PDGF-stimulated gene expression of the STAT target genes cyclin D1 and c- myc. These results indicate that the JAK-STAT pathway contributes to PDGF-induced mitogenesis, and thus this pathway may be important in the airway remodeling seen in some asthmatic patients.
Th2 cytokine regulation of type I collagen gel contraction mediated by human lung mesenchymal cells
Authors: Liu Xiangde , Kohyama Tadashi , Wang Hangjun , Zhu Yun Kui , Wen Fu-Qiang , Kim Hui Jung , Romberger Debra J. , Rennard Stephen I.
Published: March 2015

Asthma is characterized by chronic inflammation of the airway wall with the presence of activated T helper 2 (Th2) lymphocytes. The current study assessed the ability of Th2 cytokines to modulate fibroblast-mediated contraction of collagen gels to determine if Th2 cytokines could contribute to tissue remodeling by altering mesenchymal cell contraction. Human fetal lung fibroblasts, human adult bronchial fibroblasts and human airway smooth muscle cells were cast into native type I collagen gels and allowed to contract in the presence or absence of IL (interleukin)-4, IL-5, IL-10, or IL-13. IL-4 and IL-13 but not IL-5 and IL-10 augmented collagen gel contraction in a concentration-dependent manner. Neither IL-4 nor IL-13 altered fibroblast production of transforming growth factor-β or fibronectin. Both, however, decreased fibroblast prostaglandin (PG) E2 release. Decreased PGE2 release was associated with a decreased expression of cyclooxygenase 1 and 2 protein and mRNA. Indomethacin completely inhibited PGE2release and also augmented contraction. IL-4 and IL-13, however, added together with indomethacin further augmented contraction suggesting both a PGE-dependent and a PGE-independent effect. These findings suggest that IL-4 and IL-13 may modulate airway tissue remodeling and, therefore, could play a role in the altered airway connective tissue which characterizes asthma.
Mechanical strain increases cell stiffness through cytoskeletal filament reorganization
Authors: Smith Paul G. , Deng Linhong , Fredberg Jeffrey J. , Maksym Geoffrey N.
Published: March 2015

We tested the hypothesis that cytoskeletal reorganization induced by cyclic strain increases cytoskeletal stiffness (G′). G′ was measured by optical magnetic twisting cytometry in control cells and cells that had received mechanical strain for 10–12 days. G′ was measured before and after both contractile and relaxant agonists, and in the strained cells both parallel (Para) and perpendicular (Perp) to the aligned cytoskeleton. Before activation, G′ Para was 24 ± 5% (± SE) greater compared with Perp ( P < 0.05), and 35% ± 6 greater compared with control (Cont, P < 0.01). The difference between strained and control cells was enhanced by KCl, increasing G′ 171 ± 7% Para compared with 125 ± 6% Perp and 129 ± 8% Cont ( P < 10-5 both cases). The decrease in G′ from baseline due to relaxant agonists isoproterenol and dibutyryl cAMP was similar in all groups. Long-term oscillatory loading of airway smooth muscle (ASM) cells caused stiffness to increase and become anisotropic. These findings are consistent with the hypothesis that cytoskeletal reorganization can enhance ASM stiffness and contractility. They imply, furthermore, that oscillatory loading of ASM may contribute to airway narrowing and failure of airway dilation in asthma.
ET-1 receptor gene expression and distribution in L1 and L2 cells from hypertensive sheep pulmonary artery
Authors: Balyakina Elena V. , Chen Daohong , Lawrence Mayme L. , Manning Suzanne , Parker Richard E. , Shappell Scott B. , Meyrick Barbara
Published: March 2015

We examined gene and surface expression and activity of the endothelin (ET)-1 receptors (ETA and ETB) in subendothelial (L1) and inner medial (L2) cells from the main pulmonary artery of sheep with continuous air embolization (CAE)-induced chronic pulmonary hypertension (CPH). According to quantitative real-time RT-PCR, basal gene expression of both receptors was significantly higher in L2 than L1 cells, and hypertensive L2 cells showed significantly higher gene expression of ETB than controls. Expression of both genes in hypertensive L1 cells was similar to controls. Fluorescence-activated cell sorter analysis confirmed the increased distribution of ETB in hypertensive L2 cells. Although only the ETA receptors in control L2 cells showed significant binding of [125I]-labeled ET-1 at 1 h, both receptors bound ET-1 to hypertensive cells. Exposure to exogenous ET-1 for 18 h revealed that only the L2 cells internalized ET-1, and internalization by hypertensive L2 cells was significantly reduced when compared with controls. Treatment with ETA (BQ-610) and ETB (BQ-788) receptor antagonists demonstrated that both receptors contributed to internalization of ET-1 in control L2 cells, whereas in hypertensive cells only when both receptor antagonists were used in combination was significant suppression of ET-1 internalization found. We conclude that in sheep receiving CAE, alterations in ETB receptors in cells of the L2 layer may contribute to the maintenance of CPH via alterations in their expression, distribution, and activity.
Hypoxic constriction and reactive oxygen species in porcine distal pulmonary arteries
Authors: Liu J. Q. , Sham J. S. K. , Shimoda L. A. , Kuppusamy P. , Sylvester J. T.
Published: March 2015

To determine whether reactive oxygen species (ROS) play an essential role in hypoxic pulmonary vasoconstriction (HPV) and the cellular locus of ROS production and action during HPV, we measured internal diameter (ID) at constant transmural pressure, lucigenin-derived chemiluminescence (LDCL), and electron paramagnetic resonance (EPR) spin adduct spectra in small distal porcine pulmonary arteries, and dichlorofluorescein (DCF) fluorescence in myocytes isolated from these arteries. Hypoxia (4% O2) decreased ID, increased DCF fluorescence, tended to increase LDCL, and in some preparations produced EPR spectra consistent with hydroxyl and alkyl radicals. Superoxide dismutase (SOD, 150 U/ml) or SOD + catalase (CAT, 200 U/ml) did not alter ID during normoxia but reduced or abolished the constriction induced by hypoxia. SOD also blocked HPV in endotheliumdenuded arteries after restoration of the response by exposure to 10-10M endothelin-1. Confocal fluorescence microscopy demonstrated that labeled SOD and CAT entered pulmonary arterial myocytes. SOD, SOD + CAT, and CAT blocked the increase in DCF fluorescence induced by hypoxia, but SOD + CAT and CAT also caused a stable increase in fluorescence during normoxia, suggesting that CAT diminished efflux of DCF from cells or oxidized the dye directly. We conclude that HPV required increased concentrations of ROS produced by and acting on pulmonary arterial smooth muscle rather than endothelium.
Mechanisms of bacterial lipopolysaccharide-induced endothelial apoptosis
Authors: Bannerman Douglas D. , Goldblum Simeon E.
Published: March 2015

Gram-negative bacterial sepsis remains a common, life-threatening event. The prognosis for patients who develop sepsis-related complications, including the development of acute respiratory distress syndrome (ARDS), remains poor. A common finding among patients and experimental animals with sepsis and ARDS is endothelial injury and/or dysfunction. A component of the outer membrane of gram-negative bacteria, lipopolysaccharide (LPS) or endotoxin, has been implicated in the pathogenesis of much of the endothelial cell injury and/or dysfunction associated with these disease states. LPS is a highly proinflammatory molecule that elicits a wide array of endothelial responses, including the upregulation of cytokines, adhesion molecules, and tissue factor. In addition to activation, LPS induces endothelial cell death that is apoptotic in nature. This review summarizes the evidence for LPS-induced vascular endothelial injury and examines the molecular signaling pathways that activate and inhibit LPS-induced endothelial apoptosis. Furthermore, the role of apoptotic signaling molecules in mediating LPS-induced activation of endothelial cells will be considered.
Postreceptor defects in alveolar epithelial β-adrenergic signaling after prolonged isoproterenol infusion
Authors: Morgan Eric E. , Stader Sonya M. , Hodnichak Cheryl M. , Mavrich Kate E. , Folkesson Hans G. , Maron Michael B.
Published: March 2015

We previously found that prolonged isoproterenol (Iso) infusion in rats impaired the ability of β-adrenoceptor (β-AR) agonists to increase alveolar liquid clearance (ALC). Here, we determined if postreceptor defects in β-AR signaling contribute to this impairment. Iso was infused using subcutaneous miniosmotic pumps (4, 40, or 400 μg · kg-1· h-1) in rats for 48 h. At this time, forskolin-stimulated ALC was measured by mass balance. Forskolin-stimulated ALC [33.4 ± 2.1%/h (mean ± SE) in vehicle-infused rats] was reduced by 25 and 38%, respectively, after the 40 and 400 μg · kg-1· h-1Iso infusions. The ability of forskolin to increase cAMP was reduced by 70% in alveolar type II (ATII) cells isolated from rats infused with 400 μg · kg-1· h-1Iso. Additionally, the ability of the stable cAMP analog 8-bromoadenosine-3′,5′-cyclic monophosphorothioate, Sp-isomer, to increase ALC (48.7 ± 3.0% in vehicle-infused rats) was reduced by 25 and 51%, respectively, after the 40 and 400 μg · kg-1· h-1infusions. Finally, the ability of cAMP to increase protein kinase A activity was eliminated in ATII cells isolated from rats infused with Iso at 400 μg · kg-1· h-1. These data demonstrate that prolonged β-AR agonist exposure can impair alveolar epithelial β-AR signaling downstream of the β-AR.
Secretion of acid and base equivalents by intact distal airways
Authors: Inglis S. K. , Wilson S. M. , Olver R. E.
Published: March 2015

Secretion of HCO[Formula: see text] by airway submucosal glands is essential for normal liquid and mucus secretion. Because the liquid bathing the airway surface (ASL) is acidic, it has been proposed that the surface epithelium may acidify HCO[Formula: see text]-rich glandular fluid. The aim of this study was to investigate the mechanisms by which intact distal bronchi, which contain both surface and glandular epithelium, modify pH of luminal fluid. Distal bronchi were isolated from pig lungs, cannulated in a bath containing HCO[Formula: see text]-buffered solution, and perfused continually with an aliquot of similar, lightly buffered solution (LBS) in which NaCl replaced NaHCO[Formula: see text] (pH 7 with NaOH). The pH of this circulating LBS initially acidified (by 0.053 ± 0.0053 pH units) and transepithelial potential difference (PD) depolarized. The magnitude of acidification was increased when pHLBS was higher. This acidification was unaffected by luminal dimethylamiloride (DMA, 100 μM) but was inhibited by 100 nM bafilomycin A1 (by 76 ± 13%), suggesting involvement of vacuolar-H+ ATPase. Addition of ACh (10 μM) evoked alkalinization of luminal LBS and hyperpolarization of transepithelial PD. The alkalinization was inhibited in HCO[Formula: see text]-free solutions containing acetazolamide (1 mM) and by DMA and was enhanced by bumetanide (100 μM), an inhibitor of Cl− secretion. The hyperpolarization was unaffected by these maneuvers. The anion channel blocker 5-nitro-2-(3-phenylpropylamino)benzoate (300 μM) and combined treatment with DMA and bumetanide blocked both the alkalinization and hyperpolarization responses to ACh. These results are consistent with earlier studies showing that ACh evokes glandular secretion of HCO[Formula: see text] and Cl−. Isolated distal airways thus secrete both acid and base equivalents.
Progressive adult-onset emphysema in transgenic mice expressing human MMP-1 in the lung
Authors: Foronjy Robert F. , Okada Yasunori , Cole Randolph , D'Armiento Jeanine
Published: March 2015

Mice with lung-specific expression of human matrix metalloproteinase-1 (MMP-1) develop emphysematous changes similar to those seen in smoking-induced emphysema in humans. Morphometric analyses of three transgenic lines [homozygous colony ( Col) 34, Col 50, and Col 64] with varying temporal expression of MMP-1 were undertaken to determine the validity of this animal as a model of adult-onset emphysema. Line 50 mice, which have early expression of MMP-1 (14 days postconception), exhibited morphometric changes by 5 days of age. In contrast, homozygous line 34 and 64 with delayed expression (birth and 2 wk of age) were normal up until 4 wk of age when progressive changes in their mean linear intercept were first noted. In contrast, heterozygous mice from line 34 with lower transgene expression did not develop emphysema until 1 yr of age. The changes in mean linear intercept coincided with an increase in lung compliance. Emphysema in these mice was associated with decreased immunostaining for type III collagen within the alveolar septa. This study provides evidence that MMP-1 induces progressive adult-onset emphysema by the selective degradation of type III collagen within the alveolar wall.
Allergic inflammation-induced neuropeptide production in rapidly adapting afferent nerves in guinea pig airways
Authors: Myers Allen C. , Kajekar Radhika , Undem Bradley J.
Published: March 2015

In the vagal-sensory system, neuropeptides such as substance P and calcitonin gene-related peptide (CGRP) are synthesized nearly exclusively in small-diameter nociceptive type C-fiber neurons. By definition, these neurons are designed to respond to noxious or tissue-damaging stimuli. A common feature of visceral inflammation is the elevation in production of sensory neuropeptides. Little is known, however, about the physiological characteristics of vagal sensory neurons induced by inflammation to produce substance P. In the present study, we show that allergic inflammation of guinea pig airways leads to the induction of substance P and CGRP production in large-diameter vagal sensory neurons. Electrophysiological and anatomical evidence reveals that the peripheral terminals of these neurons are low-threshold Aδ mechanosensors that are insensitive to nociceptive stimuli such as capsaicin and bradykinin. Thus inflammation causes a qualitative change in chemical coding of vagal primary afferent neurons. The results support the hypothesis that during an inflammatory reaction, sensory neuropeptide release from primary afferent nerve endings in the periphery and central nervous system does not require noxious or nociceptive stimuli but may also occur simply as a result of stimulation of low-threshold mechanosensors. This may contribute to the heightened reflex physiology and pain that often accompany inflammatory diseases.
Development of cystic fibrosis and noncystic fibrosis airway cell lines
Authors: Zabner Joseph , Karp Phil , Seiler Michael , Phillips Stacia L. , Mitchell Calista J. , Saavedra Mimi , Welsh Michael , Klingelhutz Aloysius J.
Published: March 2015

In this study, we utilized the reverse transcriptase component of telomerase, hTERT, and human papillomavirus type 16 (HPV-16) E6 and E7 genes to transform normal and cystic fibrosis (CF) human airway epithelial (HAE) cells. One cell line, designated NuLi-1 (normal lung, University of Iowa), was derived from HAE of normal genotype; three cell lines, designated CuFi (cystic fibrosis, University of Iowa)-1, CuFi-3, and CuFi-4, were derived from HAE of various CF genotypes. When grown at the air-liquid interface, the cell lines were capable of forming polarized differentiated epithelia that exhibited transepithelial resistance and maintained the ion channel physiology expected for the genotypes. The CF transmembrane conductance regulator defect in the CuFi cell lines could be corrected by infecting from the basolateral surface using adenoviral vectors. Using nuclear factor-κB promoter reporter constructs, we also demonstrated that the NuLi and CuFi cell lines retained nuclear factor-κB responses to lipopolysaccharide. These cell lines should therefore be useful as models for studying ion physiology, therapeutic intervention for CF, and innate immunity.
Liposomal NAD+prevents diminished O2consumption by immunostimulated Caco-2 cells
Authors: Khan Abrar U. , Delude Russell L. , Han Yong Y. , Sappington Penny L. , Han Xianonan , Carcillo Joseph A. , Fink Mitchell P.
Published: March 2015

Accumulating data support the view that sepsis is associated with an acquired intrinsic derangement in the ability of cells to consume O2, a phenomenon that has been termed “cytopathic hypoxia.” We sought to use an in vitro “reductionist” model system using cultured cells stimulated with proinflammatory cytokines to test the hypothesis that cytopathic hypoxia is mediated, at least in part, by depletion of intracellular levels of NAD+/NADH secondary to activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP). We measured O2consumption by Caco-2 enterocytes growing on microcarrier beads after cells were incubated for 24 h under control conditions or with cytomix, a mixture of tumor necrosis factor-α, interleukin-1β, and interferon-γ. Immunostimulated cells consumed O2at about one-half the rate of control cells, but this effect was largely prevented if any one of the following pharmacological agents was present during the period of incubation with cytomix: 4,5-dihydroxy-1,3-benzene disulfonic acid, a superoxide radical anion scavenger; 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a nitric oxide scavenger; 5,10,15,20- tetrakis-[4-sulfonatophenyl]-porphyrinato-iron[III], a peroxynitrite (ONOO−) decomposition catalyst; urate, an ONOO−scavenger; 3-aminobenzamide, a PARP inhibitor; or N-(6-oxo-5,6-dihydrophenanthridin-2-yl)- N,N-dimethylacetamide HCl, a chemically dissimilar and more potent PARP inhibitor. The decrease in O2uptake induced by cytomix was associated with decreased cellular levels of NAD+/NADH. The decrease in cellular NAD+/NADH content and the decrease in O2uptake induced by cytomix were completely abrogated if liposome-encapsulated NAD+was added to the cultures during immunostimulation. Empty liposomes also increased O2uptake by immunostimulated Caco-2 cells, but much less effectively than liposomes containing NAD+. These data are consistent with the view that enterocytes exposed to proinflammatory cytokines consume less O2due to NAD+/NADH depletion secondary to activation of PARP by ONOO−or other oxidants.
Interleukin-1β-induced airway hyperresponsiveness enhances substance P in intrinsic neurons of ferret airway
Authors: Wu Z.-X. , Satterfield B. E. , Fedan J. S. , Dey R. D.
Published: March 2015

Interleukin (IL)-1β causes airway inflammation, enhances airway smooth muscle responsiveness, and alters neurotransmitter expression in sensory, sympathetic, and myenteric neurons. This study examines the role of intrinsic airway neurons in airway hyperresponsiveness (AHR) induced by IL-1β. Ferrets were instilled intratracheally with IL-1β (0.3 μg/0.3 ml) or saline (0.3 ml) once daily for 5 days. Tracheal smooth muscle contractility in vitro and substance P (SP) expression in tracheal neurons were assessed. Tracheal smooth muscle reactivity to acetylcholine (ACh) and methacholine (MCh) and smooth muscle contractions to electric field stimulation (EFS) both increased after IL-1β. The IL-1β-induced AHR was maintained in tracheal segments cultured for 24 h, a procedure that depletes SP from sensory nerves while maintaining viability of intrinsic airway neurons. Pretreatment with CP-99994, an antagonist of neurokinin 1 receptor, attenuated the IL-1β-induced hyperreactivity to ACh and MCh and to EFS in cultured tracheal segments. SP-containing neurons in longitudinal trunk, SP innervation of superficial muscular plexus neurons, and SP nerve fiber density in tracheal smooth muscle all increased after treatment with IL-1β. These results show that IL-1β-enhanced cholinergic airway smooth muscle contractile responses are mediated by the actions of SP released from intrinsic airway neurons.
Appetizing rancidity of apoptotic cells for macrophages: oxidation, externalization, and recognition of phosphatidylserine
Authors: Kagan V. E. , Borisenko G. G. , Serinkan B. F. , Tyurina Y. Y. , Tyurin V. A. , Jiang J. , Liu S. X. , Shvedova A. A. , Fabisiak J. P. , Uthaisang W. , Fadeel B.
Published: March 2015

Programmed cell death (apoptosis) functions as a mechanism to eliminate unwanted or irreparably damaged cells ultimately leading to their orderly phagocytosis in the absence of calamitous inflammatory responses. Recent studies have demonstrated that the generation of free radical intermediates and subsequent oxidative stress are implicated as part of the apoptotic execution process. Oxidative stress may simply be an unavoidable yet trivial byproduct of the apoptotic machinery; alternatively, intermediates or products of oxidative stress may act as essential signals for the execution of the apoptotic program. This review is focused on the specific role of oxidative stress in apoptotic signaling, which is realized via phosphatidylserine-dependent pathways leading to recognition of apoptotic cells and their effective clearance. In particular, the mechanisms involved in selective phosphatidylserine oxidation in the plasma membrane during apoptosis and its association with disturbances of phospholipid asymmetry leading to phosphatidylserine externalization and recognition by macrophage receptors are at the center of our discussion. The putative importance of this oxidative phosphatidylserine signaling in lung physiology and disease are also discussed.
Systemic arteriovenous fistula leads to pulmonary artery remodeling and abnormal vasoreactivity in the fetal lamb
Authors: Jouannic Jean-Marie , Roussin Régine , Hislop Alison A. , Lanone Sophie , Martinovic Jelena , Boczkowski Jorge , Dumez Yves , Dinh-Xuan A. Tuan
Published: March 2015

Several cases of systemic arteriovenous fistula diagnosed in the human fetus have been associated with the postnatal development of persistent pulmonary hypertension. The aim of this study was to determine the effects of a prenatally created systemic arteriovenous fistula on the structure and reactivity of the pulmonary circulation in the fetal lamb. A fistula between the jugular vein and carotid artery was created in fetal lambs at 119-124 days of gestation. At delivery (134-139 days), left pulmonary artery (LPA) pressure was increased in the fistula group ( n = 12) compared with controls ( n = 11, P < 0.01). The pulmonary vascular resistance was significantly higher in the fistula group ( P < 0.05), whereas mean LPA blood flow was not statistically different between the two groups. Morphometric analysis of the pulmonary vascular bed revealed an increase in the number of peripheral muscular arteries, together with an increase in pulmonary arterial medial thickness in the fistula group. There was no difference in the relative number or size of intraacinar arteries. In vitro organ bath studies on pulmonary arterial rings showed impaired endothelium-dependent relaxation in the fistula group compared with controls. However, endothelial nitric oxide synthase protein expression was similar in both groups, whereas endothelium-independent relaxation to sodium nitroprusside was greater in the fistula group compared with controls. A systemic arteriovenous fistula leads to both structural and functional alteration of the pulmonary vasculature, which might lead to the development of persistent pulmonary hypertension after birth.
cAMP regulation of Cl−and HCO 3 − secretion across rat fetal distal lung epithelial cells
Authors: Lazrak Ahmed , Thome Ulrich , Myles Carpantanto , Ware Janice , Chen Lan , Venglarik Charles J. , Matalon Sadis
Published: March 2015

We isolated and cultured fetal distal lung epithelial (FDLE) cells from 17- to 19-day rat fetuses and assayed for anion secretion in Ussing chambers. With symmetrical Ringer solutions, basal short-circuit currents ( Isc) and transepithelial resistances were 7.9 ± 0.5 μA/cm2and 1,018 ± 73 Ω · cm2, respectively (means ± SE; n = 12). Apical amiloride (10 μM) inhibited basal Iscby ∼50%. Subsequent addition of forskolin (10 μM) increased Iscfrom 3.9 ± 0.63 μA/cm2to 7.51 ± 0.2 μA/cm2( n = 12). Basolateral bumetanide (100 μM) decreased forskolin-stimulated Iscfrom 7.51 ± 0.2 μA/cm2to 5.62 ± 0.53, whereas basolateral 4,4′-dinitrostilbene-2,2′-disulfonate (5 mM), an inhibitor of HCO[Formula: see text] secretion, blocked the remaining Isc. Forskolin addition evoked currents of similar fractional magnitudes in symmetrical Cl−- or HCO[Formula: see text]-free solutions; however, no response was seen using HCO[Formula: see text]- and Cl−-free solutions. The forskolin-stimulated Iscwas inhibited by glibenclamide but not apical DIDS. Glibenclamide also blocked forskolin-induced Iscacross monolayers having nystatin-permeablized basolateral membranes. Immunolocalization studies were consistent with the expression of cystic fibrosis transmembrane conductance regulator (CFTR) protein in FDLE cells. In aggregate, these findings indicate the presence of cAMP-activated Cl−and HCO[Formula: see text] secretion across rat FDLE cells mediated via CFTR.
mSprouty2 inhibits FGF10-activated MAP kinase by differentially binding to upstream target proteins
Authors: Tefft D. , Lee M. , Smith S. , Crowe D. L. , Bellusci S. , Warburton D.
Published: March 2015

Murine Sprouty2 ( mSpry2) is a conserved ortholog of DrosophilaSprouty, a gene that inhibits several tyrosine kinase receptor pathways, resulting in net reduction of mitogen-activated protein (MAP) kinase activation. However, the precise mechanism mediating mSpry2 function as a negative regulator in tyrosine kinase growth factor pathways that regulate diverse biological functions remains incompletely characterized. Fibroblast growth factor 10 (FGF10) is a key positive regulator of lung branching morphogenesis and induces epithelial expression of mSpry2 adjacent to mesenchymal sites of FGF10. Herein, we demonstrate that FGF10 stimulation of mouse lung epithelial cells (MLE15) overexpressing mSpry2 results in both mSpry2 tyrosine phosphorylation and differential binding of mSpry2 to several key upstream target proteins in the MAP kinase-activating pathway. Thus FGF receptor (FGFR) activation results in increased association of mSpry2 with growth factor receptor-binding protein 2, suc-1-associated nuerotrophic factor target 2, and Raf but decreased binding to protein tyrosine phosphatase 2 and GTPase-activating protein 1, resulting in a net reduction of MAP kinase activation. mSpry2 also spatially translocates to the plasma membrane and intracellular membrane structures in response to FGF10 stimulation. Our data demonstrate novel intracellular mechanisms mediating mSpry2 function as a negative regulator of uncontrolled FGF-induced MAP kinase signaling.
Synthesis and anti-inflammatory activity of a chimeric recombinant superoxide dismutase: SOD2/3
Authors: Gao Bifeng , Flores Sonia C. , Leff Jonathan A. , Bose Swapan K. , McCord Joe M.
Published: March 2015

External surfaces of cells are normally protected by extracellular superoxide dismutase, SOD3, which binds to polyanions such as heparan sulfate. We constructed a fusion gene encoding a chimeric SOD consisting of the mature human mitochondrial SOD2 plus the COOH-terminal 26-amino acid heparin-binding “tail” from SOD3. This tail is responsible for the enzyme's affinity for endothelial surfaces. The fusion gene was expressed in Escherichia coli, and the fully active enzyme SOD2/3 was purified. Although native SOD2 has no affinity for heparin, SOD2/3 binds to a heparin-agarose column. In a rat model of acute lung injury induced by intratracheal instillation of IL-1, SOD2/3, SOD2, and denatured SOD2/3 showed 92%, 13.8%, and 0% reduction of lung leak, respectively. Only SOD2/3 prevented neutrophil accumulation. In the carrageenan-induced foot edema model in the rat, SOD2/3 reduced edema by 62% ( P < 0.003) at a dose in which native SOD2 produced no significant effect. Thus SOD2/3 appears to have properties as a therapeutic anti-inflammatory agent that are greatly superior to other available forms of the enzyme.
Thyroid hormone stimulates Na-K-ATPase activity and its plasma membrane insertion in rat alveolar epithelial cells
Authors: Lei Jianxun , Nowbar Sogol , Mariash Cary N. , Ingbar David H.
Published: March 2015

Na-K-ATPase protein is critical for maintaining cellular ion gradients and volume and for transepithelial ion transport in kidney and lung. Thyroid hormone, 3,3′,5-triiodo-l-thyronine (T3), given for 2 days to adult rats, increases alveolar fluid resorption by 65%, but the mechanism is undefined. We tested the hypothesis that T3stimulates Na-K-ATPase in adult rat alveolar epithelial cells (AEC), including primary rat alveolar type II (ATII) cells, and determined mechanisms of the T3effect on the Na-KATPase enzyme using two adult rat AEC cell lines (MP48 and RLE-6TN). T3at 10-8and 10-5M increased significantly hydrolytic activity of Na-K-ATPase in primary ATII cells and both AEC cell lines. The increased activity was dose dependent in the cell lines (10-9-10-4M) and was detected within 30 min and peaked at 6 h. Maximal increases in Na-K-ATPase activity were twofold in MP48 and RLE-6TN cells at pharmacological T3of 10-5and 10-4M, respectively, but increases were statistically significant at physiological T3as low as 10-9M. This effect was T3specific, because reverse T3(3,3′,5′-triiodo-l-thyronine) at 10-9-10-4M had no effect. The T3-induced increase in Na-K-ATPase hydrolytic activity was not blocked by actinomycin D. No significant change in mRNA and total cell protein levels of Na-K-ATPase were detected with 10-9-10-5M T3at 6 h. However, T3increased cell surface expression of Na-K-ATPase α1- or β1-subunit proteins by 1.7- and 2-fold, respectively, and increases in Na-K-ATPase activity and cell surface expression were abolished by brefeldin A. These data indicate that T3specifically stimulates Na-K-ATPase activity in adult rat AEC. The upregulation involves translocation of Na-K-ATPase to plasma membrane, not increased gene transcription. These results suggest a novel nontranscriptional mechanism for regulation of Na-K-ATPase by thyroid hormone.
Leukotrienes mediate part of Ova-induced lung effects in mice via EGFR
Authors: Vargaftig B. Boris , Singer Monique
Published: March 2015

Antigen induces murine bronchial hyperreactivity (BHR), inflammation, mucus accumulation, and airway remodeling. To investigate whether leukotrienes (LT) mediate the effects of antigen [ovalbumin (Ova)], we studied 5-lipoxygenase (5-LO) expression in immunized BP2 mice and blocked LT synthesis with the 5-LO inhibitor zileuton or antagonized their effects with receptor antagonists [cysteinyl leukotriene (Cys-LT)-ra MK-571, LY-171883; LTB4-ra PH-163]. Cys-LT content increased in the bronchoalveolar lavage fluid (BALF) as early as 15 min after the intratracheal instillation of Ova. Zileuton inhibited LT release in the BALF and eosinophil recruitment in the lungs, and dose dependently reduced BHR, mucus accumulation, and remodeling, as did the LT-ra. Thus LT, released just after antigen challenge, might constitute the first step in accounting for the effects of Ova. Because mucus accumulation is regulated via the EGF receptor (EGFR), which is also implicated in the effects of LT, we studied this pathway with AG-1478, an EGFR tyrosine kinase inhibitor given at 0.5, 4, and 20 mg/kg. AG-1478 inhibited BHR, inflammation, and lung remodeling induced by Ova or by molecules themselves generated by Ova, such as LT, IL-13, and monocyte chemoattractant protein-1, which promote identical effects, suggesting the involvement of the EGFR pathway in the asthma-like syndrome observed.
Laminin α-chain expression and basement membrane formation by MLE-15 respiratory epithelial cells
Authors: Nguyen Nguyet M. , Bai Yushi , Mochitate Katsumi , Senior Robert M.
Published: March 2015

Basement membranes have a critical role in alveolar structure and function. Alveolar type II cells make basement membrane constituents, including laminin, but relatively little is known about the production of basement membrane proteins by murine alveolar type II cells and a convenient system is not available to study basement membrane production by murine alveolar type II cells. To facilitate study of basement membrane production, with particular focus on laminin chains, we examined transformed murine distal respiratory epithelial cells (MLE-15), which have many structural and biochemical features of alveolar type II cells. We found that MLE-15 cells produce laminin-α5, a trace amount of laminin-α3, laminins-β1 and -γ1, type IV collagen, and perlecan. Transforming growth factor-β1 significantly induces expression of laminin-α1. When grown on a fibroblast-embedded collagen gel, MLE-15 cells assemble a basement membrane-like layer containing laminin-α5. These findings indicate that MLE-15 cells will be useful in modeling basement membrane production and assembly by alveolar type II cells.
20-HETE inotropic effects involve the activation of a nonselective cationic current in airway smooth muscle
Authors: Cloutier Martin , Campbell Shirley , Basora Nuria , Proteau Sonia , Payet Marcel D. , Rousseau Eric
Published: March 2015

20-Hydroxyeicosatetraenoic acid (20-HETE) controls several mechanisms such as vasoactivity, mitogenicity, and ion transport in various tissues. Our goal was to quantify the effects of 20-HETE on the electrophysiological properties of airway smooth muscle (ASM). Isometric tension measurements, performed on guinea pig ASM, showed that 20-HETE induced a dose-dependent inotropic effect with an EC50 value of 1.5 μM. This inotropic response was insensitive to GF-109203X, a PKC inhibitor. The sustained contraction, requiring Ca2+ entry, was partially blocked by either 100 μM Gd3+ or 1 μM nifedipine, revealing the involvement of noncapacitative Ca2+ entry and L-type Ca2+ channels, respectively. Microelectrode measurements showed that 3 μM 20-HETE depolarized the membrane potential in guinea pig ASM by 13 ± 2mV( n = 7), as did 30 μM 1-oleoyl-2-acetyl- sn-glycerol. Depolarizing effects were also observed in the absence of epithelium. Patch-clamp recordings demonstrated that 1 μM 20-HETE activated a nonselective cationic inward current that may be supported by the activation of transient receptor potential channels. The presence of canonical transient receptor potential mRNA was confirmed by RT-PCR in guinea pig ASM cells.
Regulation of heme oxygenase-1 by nitric oxide during hepatopulmonary syndrome
Authors: Carter Ethan P. , Hartsfield Cynthia L. , Miyazono Motoaki , Jakkula Malathi , Morris Kenneth G. , McMurtry Ivan F.
Published: March 2015

During hepatopulmonary syndrome caused by liver cirrhosis, pulmonary endothelial nitric oxide (NO) synthase (NOS) expression and NO production are increased. Increased NO contributes to the blunted hypoxic pressor response (HPR) during cirrhosis and may induce heme oxygenase-1 (HO-1) expression and carbon monoxide (CO) production, exacerbating the blunted HPR. We hypothesized that NO regulates the expression of HO-1 during cirrhosis, contributing to hepatopulmonary syndrome. Cirrhosis was induced in rats by common bile duct ligation (CBDL). Rats were studied 2 and 5 wk after CBDL or sham surgery. Lung HO-1 expression was elevated 5 wk after CBDL. Liver HO-1 was increased at 2 wk and remained elevated at 5 wk. In catheterized rats, the blunted HPR was partially restored by HO inhibition. Rats treated with the NOS inhibitor N G-nitro-l-arginine methyl ester for the entire 2- or 5-wk duration had normalized HO-1 expression and HPR. These data provide in vivo evidence for the NO-mediated upregulation of HO-1 expression and support the concept that hepatopulmonary syndrome is multifactorial, involving not only NO, but also HO-1 and CO.
Early surfactant administration protects against lung dysfunction in a mouse model of ARDS
Authors: Rasaiah Vijay P. A. , Malloy Jaret L. , Lewis James F. , Veldhuizen Ruud A. W.
Published: March 2015

Sepsis can predispose the lung to insults such as mechanical ventilation (MV). It was hypothesized that treating the lung with exogenous surfactant early in the development of sepsis will reduce the lung dysfunction associated with MV 18 h later. Mice underwent sham or cecal ligation and perforation (CLP) surgery. Immediately after surgery, mice were either untreated or given 100 mg/kg of bovine lipid extract surfactant intratracheally. Eighteen hours later, the lungs were removed and analyzed either immediately or following ventilation ex vivo for 2 h by an “injurious” mode of ventilation (20 ml/kg, 0 cm positive end-expiratory pressure). In nonventilated lungs, exogenous surfactant had no impact on compliance or IL-6 concentrations in the lungs. In the ventilated groups, the administered surfactant had a significant protective effect on the lung dysfunction induced by MV, but only in the CLP lungs. We conclude that administration of exogenous surfactant at the time of a systemic insult can protect the lung from the damaging effects of MV 18 h later.
Minimal lung and systemic responses to TNF-α in preterm sheep
Authors: Ikegami Machiko , Moss Timothy J. M. , Kallapur Suhas G. , Mulrooney Neil , Kramer Boris W. , Nitsos Ilias , Bachurski Cindy J. , Newnham John P. , Jobe Alan H.
Published: March 2015

TNF-α has been associated with chorioamnionitis and the subsequent development of bronchopulmonary dysplasia in preterm infants. We asked whether bioactive recombinant ovine TNF-α could induce chorioamnionitis, lung inflammation, lung maturation, and systemic effects in fetal sheep. We compared the responses to IL-1α, a cytokine known to induce these responses in preterm sheep. Intra-amniotic TNF-α caused no chorioamnionitis, no lung maturation, and a very small increase in inflammatory cells in the fetal lung after 5 h, 2 days (d), and 7 d. In contrast, IL-1α induced inflammation and lung maturation. TNF-α given into the airways at birth increased granulocytes in the bronchoalveolar lavage fluid of ventilated preterm lungs and decreased the mRNA for surfactant protein C but did not adversely effect postnatal lung function. An intravascular injection of IL-1α caused a systemic inflammatory response in fetal sheep, whereas there was no fetal response to intravascular TNF-α. Fetal and newborn preterm sheep are minimally responsive to TNF-α. Therefore, the presence of a mediator such as TNF-α in a developing animal does not necessarily mean that it is causing the responses anticipated from previous results in adult animals.
Oxidative stress activates anion exchange protein 2 and AP-1 in airway epithelial cells
Authors: Turi Jennifer L. , Jaspers Ilona , Dailey Lisa A. , Madden Michael C. , Brighton Luisa E. , Carter Jacqueline D. , Nozik-Grayck Eva , Piantadosi Claude A. , Ghio Andrew J.
Published: March 2015

Anion exchange protein 2 (AE2) is a membrane-bound protein that mediates chloride-bicarbonate exchange. In addition to regulating intracellular pH and cell volume, AE2 exports superoxide (O[Formula: see text]·) to the extracellular matrix in an HCO[Formula: see text]-dependent process. Given this ability to export O[Formula: see text]·, we hypothesized that expression of AE2 in the lung is regulated by oxidative stress. AE2 mRNA and protein expression was measured by RT-PCR and Western blot analysis, respectively, in differentiated human bronchial epithelial cells exposed to H2O2(100 μM). Alterations in in vivo AE2 protein expression were evaluated in lung tissue of rats exposed to 70% O2. The role of transcription factor activator protein (AP)-1 in oxidant regulation of AE2 was evaluated by EMSA and by immunoblotting of nuclear phospho-c- jun. Results show increased AE2 mRNA and protein expression after oxidant exposure. This was preceded by transient increases in DNA binding of AE2-specific AP-1 and phosphorylation of c- jun. This study demonstrates that AE2 expression is regulated by oxidative stress in airway epithelial cells and that this regulation correlates with activation of AP-1.
Treatment of newborn rats with a VEGF receptor inhibitor causes pulmonary hypertension and abnormal lung structure
Authors: Le Cras Timothy D. , Markham Neil E. , Tuder Rubin M. , Voelkel Norbert F. , Abman Steven H.
Published: March 2015

To determine whether disruption of vascular endothelial growth factor (VEGF)-VEGF receptor (VEGFR) signaling in the newborn has long-term effects on lung structure and function, we injected 1-day-old newborn rat pups with a single dose of Su-5416, a VEGFR inhibitor, or vehicle (controls). Lungs from infant (3-wk-old) and adult (3- to 4-mo-old) rats treated with Su-5416 as newborns showed reductions in arterial density (82 and 31%, respectively) and alveolar counts (45 and 29%) compared with controls. Neonatal treatment with Su-5416 increased right ventricle weight to body wt ratios (4.2-fold and 2.0-fold) and pulmonary arterial wall thickness measurements (2.7-fold and 1.6-fold) in infant and adult rats, respectively, indicating marked pulmonary hypertension. We conclude that treatment of newborn rats with the VEGFR inhibitor Su-5416 impaired pulmonary vascular growth and postnatal alveolarization and caused pulmonary hypertension and that these effects were long term, persisting well into adulthood.