bluuebunny/crossref_metadata_2025 huggingface dataset:
Maintenance of surfactant protein A and D secretion by rat alveolar type II cells in vitro
Authors: Mason Robert J. , Lewis Michele C. , Edeen Karen E. , McCormick-Shannon Kathleen , Nielsen Larry D. , Shannon John M.
Published: March 2015

Secretion of surfactant proteins A and D (SP-A and SP-D) has been difficult to study in vitro because a culture system for maintaining surfactant secretion has been difficult to establish. We evaluated several growth factors, corticosteroids, rat serum, and a fibroblast feeder layer for the ability to produce and maintain a polarized epithelium of type II cells that secretes SP-A and SP-D into the apical medium. Type II cells were plated on a filter insert coated with an extracellular matrix and were cultured at an air-liquid interface. Keratinocyte growth factor (KGF) stimulated type II cell proliferation and secretion of SP-A and SP-D more than fibroblast growth factor-10 (FGF-10), hepatocyte growth factor (HGF), or heparin-binding epidermal-like growth factor (HB-EGF). Cells cultured in the presence of KGF and rat serum with or without fibroblasts had high surfactant protein mRNA levels and exhibited a high level of SP-A and SP-D secretion. Dexamethasone inhibited type II cell proliferation but increased expression of SP-B. In the presence of KGF, rat serum, and dexamethasone, the mRNAs for the surfactant proteins were maintained at high levels. Secretion of SP-A and SP-D was found to be independent of phospholipid secretion.
Stretch-induced IL-8 depends on c-Jun NH2-terminal and nuclear factor-κB-inducing kinases
Authors: Li Li-Fu , Ouyang Bin , Choukroun Gabriel , Matyal Robina , Mascarenhas Marcella , Jafari Behrouz , Bonventre Joseph V. , Force Thomas , Quinn Deborah A.
Published: March 2015

Positive pressure ventilation with large tidal volumes has been shown to cause release of cytokines, including interleukin (IL)-8. The mechanisms regulating lung stretch-induced cytokine production are unclear. We hypothesized that stretch-induced IL-8 production is dependent on the activation of the mitogen-activated protein kinases, c-Jun NH2-terminal kinases (JNK), p38, and/or extracellular signal-regulated kinase (ERK) 1/2. We exposed A549 cells, a type II-like alveolar epithelial cell line, to cyclic stretch at 20 cycles/min for 5 min–2 h. Cyclic stretch induced IL-8 protein production, IL-8 mRNA expression, and JNK activation, but only transient activation of p38 and ERK1/2. Inhibition of stretch-induced JNK activation by adenovirus-mediated gene transfer of stress-activated protein kinase (SEK-1), a dominant-negative mutant of SEK-1, the immediate upstream activator of the JNKs, and pharmacological JNK inhibitor II SP-600125 blocked IL-8 mRNA expression and attenuated IL-8 production. Inhibition of p38 and ERK1/2 did not affect stretch-induced IL-8 production. Stretch-induced activation NF-κB and activator protein (AP)-1 was blocked by NF-κB inhibitor and JNK inhibitor, respectively. An NF-IL-6 site was not essential for cyclic stretch-induced IL-8 promoter activity. Stretch also induced NF-κB-inducing kinase (NIK) activation, and inhibition of NF-κB attenuated IL-8 mRNA expression and IL-8 production. We conclude that stretch-induced transcriptional regulation of IL-8 mRNA and IL-8 production was via activation of AP-1 and NF-κB and was dependent on JNK and NIK activation, respectively.
Variable ventilation induces endogenous surfactant release in normal guinea pigs
Authors: Arold Stephen P. , Suki Béla , Alencar Adriano M. , Lutchen Kenneth R. , Ingenito Edward P.
Published: March 2015

Variable or noisy ventilation, which includes random breath-to-breath variations in tidal volume (Vt) and frequency, has been shown to consistently improve blood oxygenation during mechanical ventilation in various models of acute lung injury. To further understand the effects of variable ventilation on lung physiology and biology, we mechanically ventilated 11 normal guinea pigs for 3 h using constant-Vt ventilation ( n = 6) or variable ventilation ( n = 5). After 3 h of ventilation, each animal underwent whole lung lavage for determination of alveolar surfactant content and composition, while protein content was assayed as a possible marker of injury. Another group of animals underwent whole lung lavage in the absence of mechanical ventilation to serve as an unventilated control group ( n = 5). Although lung mechanics did not vary significantly between groups, we found that variable ventilation improved oxygenation, increased surfactant levels nearly twofold, and attenuated alveolar protein content compared with animals ventilated with constant Vt. These data demonstrate that random variations in Vt promote endogenous release of biochemically intact surfactant, which improves alveolar stability, apparently reducing lung injury.
Cyclic mechanical stretch induces VEGF and FGF-2 expression in pulmonary vascular smooth muscle cells
Authors: Quinn Timothy P. , Schlueter Marielle , Soifer Scott J. , Gutierrez Jorge A.
Published: March 2015

Vascular endothelial growth factor (VEGF) and basic (b) fibroblast growth factor (FGF-2/bFGF) are involved in vascular development and angiogenesis. Pulmonary artery smooth muscle cells express VEGF and FGF-2 and are subjected to mechanical forces during pulsatile blood flow. The effect of stretch on growth factor expression in these cells is not well characterized. We investigated the effect of cyclic stretch on the expression of VEGF and FGF-2 in ovine pulmonary artery smooth muscle cells. Primary confluent cells from 6-wk-old lambs were cultured on flexible silicon membranes and subjected to cyclic biaxial stretch (1 Hz; 5–25% stretch; 4–48 h). Nonstretched cells served as controls. Expression of VEGF and FGF-2 was determined by Northern blot analysis. Cyclic stretch induced expression of both VEGF and FGF-2 mRNA in a time- and amplitude-dependent manner. Maximum expression was found at 24 h and 15% stretch (VEGF: 1.8-fold; FGF-2: 1.9-fold). These results demonstrate that mechanical stretch regulates VEGF and FGF-2 gene expression, which could play a role in pulmonary vascular development or in postnatal pulmonary artery function or disease.
Disrupted pulmonary vascular development and pulmonary hypertension in transgenic mice overexpressing transforming growth factor-α
Authors: Le Cras Timothy D. , Hardie William D. , Fagan Karen , Whitsett Jeffrey A. , Korfhagen Thomas R.
Published: March 2015

Pulmonary vascular disease plays a major role in morbidity and mortality in infant and adult lung diseases in which increased levels of transforming growth factor (TGF)-α and its receptor EGFR have been associated. The aim of this study was to determine whether overexpression of TGF-α disrupts pulmonary vascular development and causes pulmonary hypertension. Lung-specific expression of TGF-α in transgenic mice was driven with the human surfactant protein (SP)-C promoter. Pulmonary arteriograms and arterial counts show that pulmonary vascular development was severely disrupted in TGF-α mice. TGF-α mice developed severe pulmonary hypertension and vascular remodeling characterized by abnormally extensive muscularization of small pulmonary arteries. Pulmonary vascular development was significantly improved and pulmonary hypertension and vascular remodeling were prevented in bitransgenic mice expressing both TGF-α and a dominant-negative mutant EGF receptor under the control of the SP-C promoter. Vascular endothelial growth factor (VEGF-A), an important angiogenic factor produced by the distal epithelium, was decreased in the lungs of TGF-α adults and in the lungs of infant TGF-α mice before detectable abnormalities in pulmonary vascular development. Hence, overexpression of TGF-α caused severe pulmonary vascular disease, which was mediated through EGFR signaling in distal epithelial cells. Reductions in VEGF may contribute to the pathogenesis of pulmonary vascular disease in TGF-α mice.
Dexamethasone prevents virus-induced hyperresponsiveness via multiple mechanisms
Authors: Moreno Liliana , Jacoby David B. , Fryer Allison D.
Published: March 2015

In the lungs, neuronal M2 muscarinic receptors inhibit acetylcholine release from the parasympathetic nerves. Parainfluenza virus infection causes loss of M2 receptor function, which increases acetylcholine release and vagally mediated bronchoconstriction. Because glucocorticoids are known to inhibit airway hyperresponsiveness, we tested whether dexamethasone (6.5 or 65 μg/kg ip) prevents virus-induced hyperresponsiveness and M2 receptor dysfunction in guinea pigs. In controls, pilocarpine, a muscarinic agonist, inhibited vagally induced bronchoconstriction, demonstrating functional M2 receptors. However, in virus-infected animals, pilocarpine failed to inhibit vagally induced bronchoconstriction, demonstrating M2 receptor dysfunction. Frequency-dependent bronchoconstriction was greater in virus-infected animals than in controls, indicating airway hyperresponsiveness. Low-dose dexamethasone (6.5 μg/kg ip) treatment prevented virus-induced airway hyperresponsiveness, ameliorated M2 receptor dysfunction, and decreased viral content in the lungs without inhibiting virus induced inflammation. High-dose dexamethasone (65 μg/kg ip) prevented virus-induced hyperresponsiveness, completely reversed M2 receptor dysfunction, decreased viral titers, and decreased virus-induced inflammation. This high-dose dexamethasone also increased M2 receptor function in uninfected animals. In conclusion, dexamethasone prevented virus-induced hyperresponsiveness and M2 receptor dysfunction via multiple mechanisms.
Angiogenic growth factors in the pathophysiology of a murine model of acute lung injury
Authors: Karmpaliotis Dimitrios , Kosmidou Ioanna , Ingenito Edward P. , Hong Kailin , Malhotra Atul , Sunday Mary E. , Haley Kathleen J.
Published: March 2015

Capillary leakage and alveolar edema are hallmarks of acute lung injury (ALI). Neutrophils and serum macromolecules enter alveoli, promoting inflammation. Vascular endothelial growth factor (VEGF) causes plasma leakage in extrapulmonary vessels. Angiopoietin (Ang)-1 and -4 stabilize vessels, attenuating capillary leakage. We hypothesized that VEGF and Ang-1 and -4 modulate vessel leakage in the lung, contributing to the pathogenesis of ALI. We examined a murine model of lipopolysaccharide (LPS)-induced ALI. C57BL/6 and 129/J mice were studied at baseline and 24, 48, and 96 h after single or multiple doses of aerosolized LPS. Both strains exhibited time- and dose-dependent increases in inflammation and a deterioration of lung mechanics. Bronchoalveolar lavage (BAL) protein levels increased significantly, suggesting capillary leakage. Increased BAL neutrophil and total protein content correlated with time-dependent increased tissue VEGF and decreased Ang-1 and -4 levels, with peak VEGF and minimum Ang-1 and -4 expression after 96 h of LPS challenge. These data suggest that changes in the balance between VEGF and Ang-1 and -4 after LPS exposure may modulate neutrophil influx, protein leakage, and alveolar flooding during early ALI.
Expression of epidermal growth factor and surfactant proteins during postnatal and compensatory lung growth
Authors: Foster David J. , Yan Xiao , Bellotto Dennis J. , Moe Orson W. , Hagler Herbert K. , Estrera Aaron S. , Hsia Connie C. W.
Published: March 2015

We examined whether lung growth after pneumonectomy (PNX) invokes normal signaling pathways of postnatal development. We qualitatively and quantitatively assessed the immunoexpression of epidermal growth factor (EGF), its receptor (EGFR), surfactant proteins (SP) [SP-A and -D and surfactant proproteins (proSP)-B and -C] and proliferating cell nuclear antigen (PCNA) in immature and mature dog lung. We also assayed these proteins in lungs of immature dogs 3 wk or 10 mo after they underwent right PNX compared with simultaneous matched sham controls. During maturation, alveolar cell proliferation is regionally regulated in parallel with EGF and EGFR levels and inversely correlated with SP-A and proSP-C levels. In contrast, post-PNX lung growth is not associated with EGF or EGFR upregulation but with markedly increased SP-A level and moderately increased SP-D level; proSP-B and proSP-C levels did not change. We conclude that 1) signaling of EGF axis and differential regulation of SPs persist during postnatal lung development, 2) post-PNX lung growth is not a simple recapitulation of maturational responses, and 3) SP-A and SP-D may modulate post-PNX lung growth.
Norepinephrine transport by the extraneuronal monoamine transporter in human bronchial arterial smooth muscle cells
Authors: Horvath Gabor , Sutto Zoltan , Torbati Aliza , Conner Gregory E. , Salathe Matthias , Wanner Adam
Published: March 2015

Inhaled glucocorticosteroids (GSs) cause acute, α1-adrenoreceptor (AR)-mediated bronchial vasoconstriction. After release from sympathetic nerves, norepinephrine (NE) must be taken up into cells for deactivation by intracellular enzymes. Because postsynaptic cellular NE uptake is steroid sensitive, GSs could increase NE concentrations at α1-AR, causing vasoconstriction. We therefore evaluated mRNA expression of different NE transporters in human bronchial arterial smooth muscle and pharmacologically characterized NE uptake into these cells. RT-PCR demonstrated mRNA expression of the extraneuronal monoamine transporter (EMT) and organic cation transporter 1 (OCT-1). Fluorometric uptake assay showed time (within minutes)- and concentration-dependent NE uptake by freshly isolated bronchial arterial smooth muscle cells (SMC) with an estimated Km of 240 μM. Corticosterone and O-methylisoprenaline (1 μM each), but not desipramine, inhibited NE uptake, a profile indicative of NE uptake by EMT, but not OCT-1. Budesonide and methylprednisolone inhibited uptake with IC50 values of 0.9 and 5.6 μM, respectively. Corticosterone's action was reversible and not sensitive to RU-486 (GS receptor antagonist), actinomycin D (transcription inhibitor), or cycloheximide (protein synthesis inhibitor). Corticosterone made membrane impermeant by coupling to BSA also blocked NE uptake. Immunocytochemistry indicated a specific membrane binding site for corticosterone on bronchial arterial SMC. These data demonstrate that although human bronchial arterial SMC express OCT-1 and EMT, EMT is the predominant plasma membrane transporter for NE uptake. This process can be inhibited by GSs, likely via a specific membrane binding site. This nongenomic GS action (increasing NE concentrations at α1-AR) could explain acute bronchial vasoconstriction caused by inhaled GSs.
ET-1 activates Ca2+ sparks in PASMC: local Ca2+ signaling between inositol trisphosphate and ryanodine receptors
Authors: Zhang Wei-Min , Yip Kay-Pong , Lin Mo-Jun , Shimoda Larissa A. , Li Wen-Hong , Sham James S. K.
Published: March 2015

Ca+ sparks originating from ryanodine receptors (RyRs) are known to cause membrane hyperpolarization and vasorelaxation in systemic arterial myocytes. By contrast, we have found that Ca2+ sparks of pulmonary arterial smooth muscle cells (PASMCs) are associated with membrane depolarization and activated by endothelin-1 (ET-1), a potent vasoconstrictor that mediates/modulates acute and chronic hypoxic pulmonary vasoconstriction. In this study, we characterized the effects of ET-1 on the physical properties of Ca2+ sparks and probed the signal transduction mechanism for spark activation in rat intralobar PASMCs. Application of ET-1 at 0.1-10 nM caused concentration-dependent increases in frequency, duration, and amplitude of Ca2+ sparks. The ET-1-induced increase in spark frequency was inhibited by BQ-123, an ETA-receptor antagonist; by U-73122, a PLC inhibitor; and by xestospongin C and 2-aminoethyl diphenylborate, antagonists of inositol trisphosphate (IP3) receptors (IP3Rs). However, it was unrelated to sarcoplasmic reticulum Ca2+ content, activation of L-type Ca2+ channels, PKC, or cADP ribose. Photorelease of caged-IP3 indicated that Ca2+ release from IP3R could cross-activate RyRs to generate Ca2+ sparks. Immunocytochemistry showed that the distributions of IP3Rs and RyRs were similar in PASMCs. Moreover, inhibition of Ca2+ sparks with ryanodine caused a significant rightward shift in the ET-1 concentration-tension relationship in pulmonary arteries. These results suggest that ET-1 activation of Ca2+ sparks is mediated via the ETA receptor-PLC-IP3 pathway and local Ca2+ cross-signaling between IP3Rs and RyRs; in addition, this novel signaling mechanism contributes significantly to the ET-1-induced vasoconstriction in pulmonary arteries.
Apparent normal lung architecture in protein tyrosine phosphatase-ς-deficient mice
Authors: Batt Jane , Cutz Ernest , Fladd Chris , Rotin Daniela
Published: March 2015

Protein tyrosine phosphatase-ς (PTP-ς) is a member of the mammalian LAR family of phosphatases, which is characterized by a cell adhesion-like ectodomain, a single transmembrane segment, and two tandemly repeated intracellular catalytic domains. The expression of PTP-ς is developmentally regulated in epithelial, neuronal, and neuroendocrine tissues. We previously showed that PTP-ς is strongly expressed within the fetal, but not adult, rat lung and is localized to the Clara cells and type II pneumocytes. In view of the developmentally regulated pulmonary expression of PTP-ς, we performed a detailed histological and ultrastructural study of the lungs of PTP-ς knockout mice we have generated. Our findings indicate no apparent structural abnormalities in the lungs of PTP-ς−/−mice, including airway and alveolar epithelium. In addition, pulmonary neuroendocrine cells also appear normal, in contrast to pituitary, pancreatic, and gastrointestinal endocrine cells, in the knockout mice, suggesting different developmental regulation of these neuroendocrine cells. These observations suggest compensation for the absence of PTP-ς during development by related family member phosphatases, such as LAR.
Effect of bradykinin, TGF-β1, IL-1β, and hypoxia on COX-2 expression in pulmonary artery smooth muscle cells
Authors: Bradbury D. A. , Newton R. , Zhu Y.-M. , Stocks J. , Corbett L. , Holland E. D. , Pang L. H. , Knox A. J.
Published: March 2015

Prostanoids are major regulators of smooth muscle function that are generated by cyclooxygenase (COX). Here we hypothesized that cytokines and mediators that regulate the pulmonary circulation would alter COX expression and prostanoid generation in pulmonary artery smooth muscle cells. Bradykinin, transforming growth factor-β1 (TGF-β1), and interleukin-1β (IL-1β) increased inducible COX-2 expression and prostaglandin E2 (PGE2) release. Transfection studies using a COX-2 promoter construct demonstrated that all three agents acted transcriptionally. Constitutive COX-1 protein expression was unchanged. The COX inhibitor indomethacin, the COX-2 inhibitor NS-398, the protein synthesis inhibitor cycloheximide, and the glucocorticoid dexamethasone abrogated the increased PGE2levels. Dexamethasone and cycloheximide prevented COX-2 induction. Hypoxia (3% O2-5% CO2-92% N2) for 24 h selectively augmented TGF-β1-stimulated PGE2 production and COX-2 induction but had no effect alone. Prolonged hypoxic culture alone for 48 and 72 h enhanced COX-2 induction and increased PGE2. These studies show that a number of stimuli are capable of inducing COX-2 in pulmonary artery smooth muscle cells. The interaction between hypoxia and TGF-β1 may be particularly relevant to pulmonary hypertension.
Identification and characterization of rodent ABCA1 in isolated type II pneumocytes
Authors: Bortnick Anna E. , Favari Elda , Tao Jian-Qin , Francone Omar L. , Reilly Muredach , Zhang Yuzhen , Rothblat George H. , Bates Sandra R.
Published: March 2015

ATP-binding cassette transporter A1 (ABCA1) promotes transfer of cholesterol and phospholipid from cells to lipid-free serum apolipoproteins. ABCA1 mRNA and protein expression in primary cultures of rodent type II cells was sensitive to upregulation with 5 μM9- cis-retinoic acid ( 9cRA ) and 6.2 μM 22-hydroxycholesterol (22-OH). The increase in ABCA1 protein levels was time dependent and was maximal after 16 h of exposure to 9cRA + 22-OH. Inducible ABCA1 was also found in transformed cell lines of lung origin: WI38/VA13, A549, and NIH-H441 cells. Stimulation of ABCA1 in rat type II cells by 9cRA + 22-OH resulted in a four- or fivefold enhancement of efflux of radioactive phospholipid or cholesterol, respectively, from the pneumocytes to apolipoprotein AI (apo AI), whereas cAMP (0.3 mM) had no effect. ABCA1-mediated lipid efflux to apo AI was independent of the surfactant secretion pathway, inasmuch as upregulation of ABCA1 resulted in a reduction of secretagogue-stimulated surfactant phospholipid release. These studies demonstrate the presence of functional ABCA1 in type II cells from the lung.
Induction of 1-cys peroxiredoxin expression by oxidative stress in lung epithelial cells
Authors: Kim Han-Suk , Manevich Yefim , Feinstein Sheldon I. , Pak Jhang Ho , Ho Ye Shih , Fisher Aron B.
Published: March 2015

1-Cys peroxiredoxin (1-cysPrx), a member of the peroxiredoxin family that contains a single conserved cysteine residue, reduces a broad spectrum of hydroperoxides. We studied changes in 1-cysPrx expression in rat lungs and lung cell lines in response to oxidative stress due to hyperoxia, H2O2, or paraquat. After 60 h of hyperoxia (>95% O2), mRNA and protein levels of 1-cysPrx and peroxidase activity were significantly elevated in rat lungs by ∼1.5- to 2-fold compared with the control ( P < 0.05). A similar induction of 1-cysPrx was observed in mouse lungs following exposure to O2 for 63 or 72 h; enzyme induction in mouse lungs was similar for wild-type and glutathione peroxidase 1 gene-targeted mice. H2O2 and paraquat treatment induced 1-cysPrx gene expression in L2 cells. Enzyme induction was attenuated by pretreatment with Trolox or N-acetylcysteine. Actinomycin D treatment showed that stability of 1-cysPrx mRNA was not altered in the presence of H2O2 or paraquat, indicating that increased expression with oxidative stress is regulated at the transcriptional level. These data indicate that the antioxidant enzyme 1-cysPrx is induced in lung cells by oxidative stress.
Signaling intermediates required for NF-κB activation and IL-8 expression in CF bronchial epithelial cells
Authors: Li Jing , Johnson Xa Dwight , Iazvovskaia Svetlana , Tan Alan , Lin Anning , Hershenson Marc B.
Published: March 2015

Ligation of the asialoGM1 Pseudomonas aeruginosa pilin receptor has been demonstrated to induce IL-8 expression in airway epithelial cells via an NF-κB-dependent pathway. We examined the signaling pathways required for asialoGM1-mediated NF-κB activation in IB3 cells, a human bronchial epithelial cell line derived from a cystic fibrosis (CF) patient, and C-38 cells, the rescued cell line that expresses a functional CF transmembrane regulator. Ligation of the asialoGM1 receptor with specific antibody induced greater IL-8 expression in IB3 cells than C-38 cells, consistent with the greater density of asialoGM1 receptors in CF phenotype cells. AsialoGM1-mediated activation of NF-κB, IκB kinase (IKK), and ERK was also greater in IB3 cells. With the use of genetic inhibitors, we found that IKK-β and NF-κB-inducing kinase are required for maximal NF-κB transactivation and transcription from the IL-8 promoter. Finally, although ERK activation was required for maximal asialoGM1-mediated IL-8 expression, inhibition of ERK signaling had no effect on IKK or NF-κB activation, suggesting that ERK regulates IL-8 expression in an NF-κB-independent manner.
Chronic intrauterine pulmonary hypertension compromises fetal pulmonary artery smooth muscle cell O2sensing
Authors: Linden Bradley C. , Resnik Ernesto R. , Hendrickson Kristine J. , Herron Jean M. , O'Connor Timothy J. , Cornfield David N.
Published: March 2015

To test the hypothesis that chronic intrauterine pulmonary hypertension (PHTN) compromises pulmonary artery (PA) smooth muscle cell (SMC) O2sensing, fluorescence microscopy was used to study the effect of an acute increase in Po2on the cytosolic Ca2+concentration ([Ca2+]i) of chronically hypoxic subconfluent monolayers of PA SMC in primary culture. PA SMCs were derived from fetal lambs with PHTN due to intrauterine ligation of the ductus arteriosus. Acute normoxia decreased [Ca2+]iin control but not PHTN PA SMC. In control PA SMC, [Ca2+]iincreased after Ca2+-sensitive (KCa) and voltage-sensitive (Kv) K+channel blockade and decreased after diltiazem treatment. In PHTN PA SMC, KCablockade had no effect, whereas Kvblockade and diltiazem increased [Ca2+]i. Inhibition of sarcoplasmic reticulum Ca2+ATPase activity caused a greater increase in [Ca2+]iin controls compared with PHTN PA SMC. Conversely, ryanodine caused a greater increase of [Ca2+]iin PHTN compared with control PA SMC. KCachannel mRNA is decreased and Kvchannel mRNA is unchanged in PHTN PA SMC compared with controls. We conclude that PHTN compromises PA SMC O2sensing, alters intracellular Ca2+homeostasis, and changes the predominant ion channel that determines basal [Ca2+]ifrom KCato Kv.
Fibrinolysis in LPS-induced chronic airway disease
Authors: Savov Jordan D. , Brass David M. , Berman Katherine G. , McElvania Erin , Schwartz David A.
Published: March 2015

To examine the role of the fibrinolytic system in LPS-induced airway disease, we compared the effect of a chronic LPS challenge in plasminogen activator inhibitor-deficient (C57BL/6JPAI-1-/-) mice and wild-type (WT) C57BL/6J mice. Physiological and biological assessments were performed, immediately after, and 4 wk after an 8-wk exposure to LPS or saline. Immediately after the LPS exposure, WT mice had increased estimates of airway reactivity to methacholine compared with C57BL/6JPAI-1-/-mice; however, airway inflammation was similar in both LPS-exposed groups. Significant increases in both active transforming growth factor (TGF)-β1 and active matrix metalloproteinase (MMP)-9 was detected after LPS exposure in WT but not C57BL/6JPAI-1-/-mice. C57BL/6JPAI-1-/-mice showed significantly less TGF-β1 in the lavage and higher MMP-9 in the lung tissue than WT mice at the end of exposure and 4 wk later. After LPS exposure, both WT and C57BL/6JPAI-1-/-mice had substantial expansion of the subepithelial area of the medium [diameter (d) = 90-129 μm]- and large (d > 129 μm)-size airways when compared with saline-exposed mice. Subepithelial fibrin deposition was prevalent in WT mice but diminished in C57BL/6JPAI-1-/-. PAI-1 expression by nonciliated bronchial epithelial cells was enhanced in LPS-exposed WT mice compared with the saline-exposed group. Four weeks after LPS inhalation, airway hyperreactivity and the expansion of the subepithelial area in the medium and large airways persisted in WT but not C57BL/6JPAI-1-/-mice. We conclude that an active fibrinolytic system can substantially alter the development and resolution of the postinflammatory airway remodeling observed after chronic LPS inhalation.
Platelets inhibit the lysis of pulmonary microemboli
Authors: Murciano Juan-Carlos , Harshaw David , Neschis David G. , Koniaris Lauren , Bdeir Khalil , Medinilla Sandra , Fisher Aron B. , Golden Michael A. , Cines Douglas B. , Nakada Marian T. , Muzykantov Vladimir R.
Published: March 2015

Using tracings of125I-labeled fibrin(ogen) in rodents, we examined the hypothesis that platelets impede the lysis of pulmonary emboli.125I-Microemboli (ME, 3–10 micron diameter) lodged homogeneously throughout the lungs after intravenous injection in both rats and mice (60% of injected dose), caused no lethality, and underwent spontaneous dissolution (50 and 100% within 1 and 5 h, respectively). Although lung homogenates displayed the most intense fibrinolytic activity of all the major organs, dissolution of ME was much slower in isolated perfused lungs (IPL) than was observed in vivo. Addition of rat plasma to the perfusate facilitated ME dissolution in IPL to a greater extent than did addition of tissue-type plasminogen activator alone, suggesting that permeation of the clot by plasminogen is the rate-limited step in lysis. Platelet-containing ME injected in rats lysed much more slowly than did ME formed from fibrin alone.125I-Thrombi, formed in the pulmonary vasculature of mice in response to intravascular activation of platelets by injection of collagen and epinephrine, were essentially resistant to spontaneous dissolution. Moreover, injection of the antiplatelet glycoprotein IIb/IIIa antibody 7E3 F(ab′)2facilitated spontaneous dissolution of pulmonary ME and augmented fibrinolysis by a marginally effective dose of Retavase (10 μg/kg) in rats. These studies show that platelets suppress pulmonary fibrinolysis. The mechanism(s) by which platelets stabilize ME and utility of platelet inhibitors to facilitate their dissolution deserves further study.
Alterations in lung collectins in an adaptive allergic immune response
Authors: Haley Kathleen J. , Ciota Alex , Contreras Johanna P. , Boothby Mark R. , Perkins David L. , Finn Patricia W.
Published: March 2015

Although surfactant apoproteins are known to be mediators of innate responses, their relationship to adaptive responses has not been examined extensively. We investigated possible links between surfactant apoproteins and responses to allergens by studying alterations in surfactant apoproteins A, B, and D in a murine model of allergic pulmonary inflammation. Three murine strains (BALB/c, C57BL/6, and 129J) demonstrated increased immunostaining of surfactant apoproteins A and D in nonciliated epithelial cells of noncartilaginous airways after aerosolized challenge. In contrast, surfactant apoprotein B immunostaining was unchanged. Immunoblotting demonstrated increased surfactant A in bronchoalveolar lavage fluid after allergen sensitization and challenge. Surfactant apoprotein A and D induction required T and/or B lymphocyte responses to allergen, since the induction was absent in recombinase-activating gene-deficient mice, which lack functional lymphocytes. We conclude that increased immunoreactivity of two collectins, surfactant apoproteins A and D, occurs within the response to allergen. Our findings support a model in which surfactant apoproteins A and D are important to both innate immunity and adaptive immune responses to allergens.
Inhibition of TNF-α gene expression and bioactivity by site-specific transcription factor-binding oligonucleotides
Authors: Ye Jianping , Wang Liying , Zhang Xiaoying , Tantishaiyakul Vimon , Rojanasakul Yon
Published: March 2015

The present study investigated transcriptional inactivation of TNF-α gene by nuclear factor-binding oligonucleotides (ON) and their effects on pulmonary inflammatory responses in mice. PCR-based gene mutation and gel shift assays were used to identify specific cis-acting elements necessary for nuclear factor binding and transactivation of TNF-α gene by lipopolysaccharide (LPS). LPS inducibility of TNF-α was shown to require transcriptional activation by NF-κB at multiple binding sites, including the −850 (κ1), −655 (κ2), and −510 (κ3) sites, whereas the −210 (κ4) site had no effect. Maximum inducibility was associated with the activation of κ3 site. The sequence-specific, double-stranded ON targeting this site was most effective in inhibiting TNF-α activity induced by LPS. The inhibitory effect of ON on TNF-α bioactivity was also investigated using a murine lung inflammation model. Pretreatment of mice with ON, but not its mutated sequence, inhibited LPS-induced inflammatory neutrophil influx and TNF-α production by lung cells. Effective inhibition by ON in this model was shown to require a liposomal agent for efficient cellular delivery of the ON. Together, our results indicate that transcriptional inactivation of TNF-α gene can be achieved by using ON that compete for nuclear factor binding to TNF-α gene promoter. This gene inhibition approach may be used as a research tool or as potential therapeutic modality for diseases with etiology dependent on aberrant gene expression.
E. colipneumonia induces CD18-independent airway neutrophil migration in the absence of increased lung vascular permeability
Authors: Ong Evan S. , Gao Xiao-Pei , Xu Ning , Predescu Dan , Rahman Arshad , Broman Michael T. , Jho David H. , Malik Asrar B.
Published: March 2015

We examined the relationship between neutrophil [polymorphonuclear leukocyte (PMN)] influx and lung vascular injury in response to Escherichia coli pneumonia. We assessed lung tissue PMN uptake by measuring myeloperoxidase and transvascular PMN migration by determining PMN counts in lung interstitium and bronchoalveolar lavage fluid (BALF) in mice challenged intratracheally with E. coli. Lung vascular injury was quantified by determining microvessel filtration coefficient ( Kf,c), a measure of vascular permeability. We addressed the role of CD18 integrin in the mechanism of PMN migration and lung vascular injury by inducing the expression of neutrophil inhibitory factor, a CD11/CD18 antagonist. In control animals, we observed a time-dependent sixfold increase in PMN uptake, a fivefold increase in airway PMN migration, and a 20-fold increase in interstitial PMN uptake at 6 h after challenge. Interestingly, Kf,cincreased minimally during this period of PMN extravasation. CD11/CD18 blockade reduced lung tissue PMN uptake consistent with the role of CD18 in mediating PMN adhesion to the endothelium but failed to alter PMN migration in the tissue. Moreover, CD11/CD18 blockade did not affect Kf,c. Analysis of BALF leukocytes demonstrated diminished oxidative burst compared with leukocytes from bacteremic mice, suggesting a basis for lack of vascular injury. The massive CD11/CD18-independent airway PMN influx occurring in the absence of lung vascular injury is indicative of an efficient host-defense response elicited by E. coli pneumonia.
Pro-EMAP II is not primarily cleaved by caspase-3 and -7
Authors: Zhang F. R. , Schwarz M. A.
Published: March 2015

Endothelial monocyte-activating polypeptide (EMAP) II is a unique cytokine, also known as p43, the active mature form of which exhibits antiangiogenic properties in vivo and in vitro. The proteolytic enzymes associated with the cleavage and release of the active mature form, however, remain unclear. Here we show that, in contrast to prior observations, purified pro-EMAP II is not cleaved by either caspase-3 or -7 in vivo or in vitro. Thus other proteolytic processes, which allow it to induce apoptosis via caspase-3 activation in migrating and dividing endothelium, may be involved in the release of the active mature EMAP II.
Cellular biomechanics in the lung
Authors: Waters Christopher M. , Sporn Peter H. S. , Liu Mingyao , Fredberg Jeffrey J.
Published: March 2015

Mechanical forces affect both the function and phenotype of cells in the lung. In this symposium, recent studies were presented that examined several aspects of biomechanics in lung cells and their relationship to disease. Wound healing and recovery from injury in the airways involve epithelial cell spreading and migration on a substrate that undergoes cyclic mechanical deformation; enhanced green fluorescent protein-actin was used in a stable cell line to examine cytoskeletal changes in airway epithelial cells during wound healing. Eosinophils migrate into the airways during asthmatic attacks and can also be exposed to cyclic mechanical deformation; cyclic mechanical stretch caused a decrease in leukotriene C4synthesis that may be dependent on mechanotransduction mechanisms involving the production of reactive oxygen species. Recent studies have suggested that proinflammatory cytokines are increased in ventilator-induced lung injury and may be elevated by overdistention of the lung tissue; microarray analysis of human lung epithelial cells demonstrated that cyclic mechanical stretch alone profoundly affects gene expression. Finally, airway hyperresponsiveness is a basic feature of asthma, but the relationship between airway hyperresponsiveness and changes in airway smooth muscle (ASM) function remain unclear. New analysis of the behavior of the ASM cytoskeleton (CSK) suggests, however, that the CSK may behave as a glassy material and that glassy behavior may account for the extensive ASM plasticity and remodeling that contribute to airway hyperresponsiveness. Together, the presentations at this symposium demonstrated the remarkable and varied roles that mechanical forces may play in both normal lung physiology as well as pathophysiology.
Axonemal dynein expression in human fetal tracheal epithelium
Authors: Carson Johnny L. , Reed William , Lucier Thomas , Brighton Luisa , Gambling Todd M. , Huang Chien-Hui , Collier Albert M.
Published: March 2015

Ciliogenesis in human fetal airway epithelium occurs from 11 to 24 gestational weeks. Using genetic and antigenic markers specific for human axonemal dynein heavy chain 9, we characterized temporal aspects of axonemal dynein expression associated with large airway epithelial ciliogenesis during human fetal development. Late in the first trimester, an undifferentiated columnar epithelium is characteristic of the large airways, and immunocytochemical studies exhibited focal localization of axonemal dynein antigen on luminal epithelial cell borders at sites consistent with emergent ciliary beds. From 12 to 22 wk, immunocytochemical labeling of new ciliary beds was prominent, and localization within the cytoplasm of epithelial cells suggested avid synthesis of axonemal dynein in advance of ciliogenic events. Quantitative RT-PCR of tracheal RNA and in situ hybridization studies compared favorably with immunocytochemical findings with the earliest expression of axonemal dynein at 9–10 wk gestation. These studies have documented that axonemal dynein is expressed early in human fetal life during airway epithelial maturation and well before histological or ultrastructural evidence of ciliogenesis is apparent.
Essential role of complex II of the respiratory chain in hypoxia-induced ROS generation in the pulmonary vasculature
Authors: Paddenberg Renate , Ishaq Barat , Goldenberg Anna , Faulhammer Petra , Rose Frank , Weissmann Norbert , Braun-Dullaeus Ruediger C. , Kummer Wolfgang
Published: March 2015

In the pulmonary vasculature, the mechanisms responsible for oxygen sensing and the initiation of hypoxia-induced vasoconstriction and vascular remodeling are still unclear. Nitric oxide (NO) and reactive oxygen species (ROS) are discussed as early mediators of the hypoxic response. Here, we describe a quantitative analysis of NO- and ROS-producing cells within the vascular walls of murine lung sections cultured at normoxia or hypoxia. Whereas the number of NO-producing cells was not changed by hypoxia, the number of ROS-generating cells was significantly increased. Addition of specific inhibitors revealed that mitochondria were the source of ROS. The participation of the individual mitochondrial complexes differed in normoxic and hypoxic ROS generation. Whereas normoxic ROS production required complexes I and III, hypoxic ROS generation additionally demanded complex II. Histochemically demonstrable succinate dehydrogenase activity of complex II in the arterial wall decreased during hypoxia. Inhibition of the reversed enzymatic reaction, i.e., fumarate reductase, by application of succinate, specifically abolished hypoxic, but not normoxic, ROS generation. Thus complex II plays an essential role in hypoxic ROS production. Presumably, its catalytic activity switches from succinate dehydrogenase to fumarate reductase at reduced oxygen tension, thereby modulating the directionality of the electron flow.
TGF-β1stimulates HO-1 via the p38 mitogen-activated protein kinase in A549 pulmonary epithelial cells
Authors: Ning Wen , Song Ruiping , Li Chaojun , Park Edward , Mohsenin Amir , Choi Augustine M. K. , Choi Mary E.
Published: March 2015

In lung injury and progressive lung diseases, the multifunctional cytokine transforming growth factor-β1(TGF-β1) modulates inflammatory responses and wound repair. Heme oxygenase-1 (HO-1) is a stress-inducible protein that has been demonstrated to confer cytoprotection against oxidative injury and provide a vital function in maintaining tissue homeostasis. Here we report that TGF-β1is a potent inducer of HO-1 and examined the signaling pathway by which TGF-β1regulates HO-1 expression in human lung epithelial cells (A549). TGF-β1(1–5 ng/ml) treatment resulted in a marked time-dependent induction of HO-1 mRNA in A549 cells, followed by corresponding increases in HO-1 protein and HO enzymatic activity. Actinomycin D and cycloheximide inhibited TGF-β1-responsive HO-1 mRNA expression, indicating a requirement for transcription and de novo protein synthesis. Furthermore, TGF-β1rapidly activated the p38 mitogen-activated protein kinase (p38 MAPK) pathway in A549 cells. A chemical inhibitor of p38 MAPK (SB-203580) abolished TGF-β1-inducible HO-1 mRNA expression. Both SB-203580 and expression of a dominant-negative mutant of p38 MAPK inhibited TGF-β1-induced ho-1 gene activation, as assayed by luciferase activity of an ho-1enhancer/luciferase fusion construct (pMHO1luc-33+SX2). These studies demonstrate the critical intermediacy of the p38 MAPK pathway in the regulation of HO-1 expression by TGF-β1.
Role of macrophage migration inhibitory factor in bleomycin-induced lung injury and fibrosis in mice
Authors: Tanino Yoshinori , Makita Hironi , Miyamoto Kenji , Betsuyaku Tomoko , Ohtsuka Yoshinori , Nishihira Jun , Nishimura Masaharu
Published: March 2015

Macrophage migration inhibitory factor (MIF) is a unique cytokine that reportedly overrides the anti-inflammatory effect of endogenous glucocorticoids. MIF has been demonstrated to be involved in a variety of inflammatory diseases. In this study, we examined the role of MIF in bleomycin (BLM)-induced lung injury and fibrosis. The levels of MIF in lung tissues and bronchoalveolar lavage fluids were significantly increased in the period 5–10 days after intratracheal administration of BLM. Treatment with the anti-MIF antibody significantly reduced the mortality at 14 days and the histopathological lung injury score at 10 days. These effects were accompanied with significant suppression of the accumulation of inflammatory cells in the alveolar space and tumor necrosis factor-α in the lungs at 7 days. However, the anti-MIF antibody did not affect either the content of lung hydroxyproline or the histopathological lung fibrosis score at 21 days after BLM. These data provide further evidence for the crucial role of MIF in acute lung inflammation but do not support the involvement of MIF in lung fibrosis induced by BLM in mice.
Alterations in TGF-β1 expression in lambs with increased pulmonary blood flow and pulmonary hypertension
Authors: Mata-Greenwood Eugenia , Meyrick Barbara , Steinhorn Robin H. , Fineman Jeffrey R. , Black Stephen M.
Published: March 2015

The mechanisms responsible for pulmonary vascular remodeling in congenital heart disease with increased pulmonary blood flow remain unclear. We developed a lamb model of congenital heart disease and increased pulmonary blood flow utilizing an in utero placed aortopulmonary vascular graft (shunted lambs). Morphometric analysis of barium-injected pulmonary arteries indicated that by 4 wk of age, shunts had twice the pulmonary arterial density of controls ( P < 0.05), and their pulmonary vessels showed increased muscularization and medial thickness at both 4 and 8 wk of age ( P < 0.05). To determine the potential role of TGF-β1 in this vascular remodeling, we investigated vascular changes in expression and localization of TGF-β1 and its receptors TβRI, ALK-1, and TβRII in lungs of shunted and control lambs at 1 day and 1, 4, and 8 wk of life. Western blots demonstrated that TGF-β1 and ALK-1 expression was elevated in shunts compared with control at 1 and 4 wk of age ( P < 0.05). In contrast, the antiangiogenic signaling receptor TβRI was decreased at 4 wk of age ( P < 0.05). Immunohistochemistry demonstrated shunts had increased TGF-β1 and TβRI expression in smooth muscle layer and increased TGF-β1 and ALK-1 in endothelium of small pulmonary arteries at 1 and 4 wk of age. Moreover, TβRI expression was significantly reduced in endothelium of pulmonary arteries in the shunt at 1 and 4 wk. Our data suggest that increased pulmonary blood flow dysregulates TGF-β1 signaling, producing imbalance between pro- and antiangiogenic signaling that may be important in vascular remodeling in shunted lambs.
FAK blunts adenosine-homocysteine-induced endothelial cell apoptosis: requirement for PI 3-kinase
Authors: Bellas Robert E. , Harrington Elizabeth O. , Sheahan Kerri Lynn , Newton Julie , Marcus Caroline , Rounds Sharon
Published: March 2015

Treatment of cultured bovine pulmonary endothelial cells (BPAEC) with adenosine (Ado) alone or in combination with homocysteine (Hc) leads to disruption of focal adhesion complexes, caspase-dependent degradation of components of focal adhesion complexes, and subsequent apoptosis. Endothelial cells transiently overexpressing paxillin or p130CascDNAs underwent Ado-Hc-induced apoptosis to an extent similar to that of cells transfected with vector alone. However, overexpression of focal adhesion kinase (FAK) cDNA blunted Ado-Hc-induced apoptosis. FAK constructs lacking the central catalytic domain or containing a point mutation, rendering the catalytic domain enzymatically inactive, did not provide protection from apoptosis. Constructs containing a mutation in the major autophosphorylation site (tyrosine-397) similarly did not prevent cell death. A FAK mutant in amino acid 395, deficient in phosphatidylinositol 3-kinase (PI 3-kinase) binding, was not able to blunt apoptosis. Finally, overexpression of FAK did not provide protection from apoptosis in the presence of LY-294002, a PI 3-kinase inhibitor. Taken together, these data suggest that the survival signals mediated by overexpression of FAK in response to Ado-Hc-induced apoptosis require a PI 3-kinase-dependent pathway.
Mitogen-activated protein kinases mediate peroxynitrite-induced cell death in human bronchial epithelial cells
Authors: Nabeyrat Elodie , Jones Gina E. , Fenwick Peter S. , Barnes Peter J. , Donnelly Louise E.
Published: March 2015

Peroxynitrite, formed by the reaction of nitric oxide (NO · ) with superoxide anions (O[Formula: see text]·), may play a role in the pathophysiology of inflammation. The effects of 3-morpholinosydnonimine (SIN-1), a peroxynitrite generator, on the human bronchial epithelial cell line BEAS-2B, were examined. SIN-1 exposure resulted in cell death in a time- and dose-dependent manner. Depletion of intracellular glutathione increased the vulnerability of the cells. Pretreatment with Mn(III)tetrakis( N-methyl-4′-pyridyl)porphyrin (MnTMPyP) or hydroxocobalamin (HC), O[Formula: see text]· and NO · scavengers, respectively, reduced significantly SIN-1-induced cell death (18.66 ± 3.57 vs. 77.01 ± 14.07 or 82.20 ± 9.64, % cell viability SIN-1 vs. MnTMPyP or HC). Moreover, the mitogen-activated protein kinases (MAPK) p44/42 (ERK), p38, and p54/46 (JNK) were also activated in a time- and concentration-dependent manner. PD-98059 and SB-239063, specific inhibitors of ERK and p38 MAPK pathways, failed to protect cells against 1 mM SIN-1. However, PD-98059 partially inhibited (60% cell survival) SIN-1 effects at ≤0.25 mM, and this was increased with the inclusion of SB-239063. Therefore, MAPKs may mediate signal transduction pathways induced by peroxynitrite in lung epithelial cells leading to cell death.