bluuebunny/crossref_metadata_2025 huggingface dataset:
Partial HIF-1α deficiency impairs pulmonary arterial myocyte electrophysiological responses to hypoxia
Authors: Shimoda Larissa A. , Manalo Dominador J. , Sham James S. K. , Semenza Gregg L. , Sylvester J. T.
Published: December 2017

Chronic hypoxia depolarizes and reduces K+ current in pulmonary arterial smooth muscle cells (PASMCs). Our laboratory previously demonstrated that hypoxia-inducible factor-1 (HIF-1) contributed to the development of hypoxic pulmonary hypertension. In this study, electrophysiological parameters were measured in PASMCs isolated from intrapulmonary arteries of mice with one null allele at the Hif1a locus encoding HIF-1α [ Hif1a(+/−)] and from their wild-type [ Hif1a(+/+)] littermates after 3 wk in air or 10% O2. Hematocrit and right ventricular wall and left ventricle plus septum weights were measured. Capacitance, K+ current, and membrane potential were measured with whole cell patch clamp. Similar to our laboratory's previous results, hypoxia-induced right ventricular hypertrophy and polycythemia were blunted in Hif1a(+/−) mice. Hypoxia increased PASMC capacitance in Hif1a(+/+) mice but not in Hif1a(+/−) mice. Chronic hypoxia depolarized and reduced K+ current density in PASMCs from Hif1a(+/+) mice. In PASMCs from hypoxic Hif1a(+/−) mice, no reduction in K+ current density was observed, and depolarization was significantly blunted. Thus partial deficiency of HIF-1α is sufficient to impair hypoxia-induced depolarization, reduction of K+ current density, and PASMC hypertrophy.
Dopamine regulates Na-K-ATPase in alveolar epithelial cells via MAPK-ERK-dependent mechanisms
Authors: Guerrero Carmen , Lecuona Emilia , Pesce Liuska , Ridge Karen M. , Sznajder Jacob I.
Published: December 2017

Dopamine (DA) increases lung edema clearance by regulating vectorial Na+transport and Na-K-ATPase in the pulmonary epithelium. We studied the role of the mitogen-activated protein kinase (MAPK)-extracellular signal-regulated kinase (ERK) pathway in the DA regulation of Na-K-ATPase in alveolar epithelial cells (AEC). Incubation of AEC with DA resulted in a rapid stimulation of ERK activity via dopaminergic type 2 receptors. Analysis of total RNA and protein showed a 1.5-fold increase in the Na-K-ATPase β1-subunit mRNA levels and up to a fivefold increase in β1-subunit protein abundance after DA stimulation, which was blocked by the MAPK kinase (MEK) inhibitors PD-98059 and U-0126. Also, the DA-ERK pathway stimulated the synthesis of a green fluorescent protein reporter gene driven by the β1-subunit promoter, which indicates that DA regulates the Na-K-ATPase β1-subunit at the transcriptional level. The DA-mediated increase in β1-subunit mRNA protein resulted in an increase in functional Na pumps in the basolateral membranes of alveolar type II cells. These results suggest that the MAPK-ERK pathway is an important mechanism in the regulation of Na-K-ATPase by DA in the alveolar epithelium.
Endocytosed SP-A and surfactant lipids are sorted to different organelles in rat type II pneumocytes
Authors: Wissel Heide , Lehfeldt Andrea , Klein Petra , Müller Torsten , Stevens Paul A.
Published: December 2017

Intracellular transport of endocytosed surfactant protein A (SP-A) and lipid was investigated in isolated rat type II cells. After internalization, SP-A and lipid are taken up via the coated-pit pathway and reside in a common compartment, positive for the early endosomal marker EEA1 but negative for the lamellar body marker 3C9. SP-A then recycles rapidly to the cell surface via Rab4-associated recycling vesicles. Internalized lipid is transported toward a Rab7-, CD63-, 3C9-positive compartment, i.e., lamellar bodies. Inhibition of calmodulin led to inhibition of uptake and transport out of the EEA1-positive endosome and thus of resecretion of both components. Inhibition of intravesicular acidification (bafilomycin A1) led to decreased uptake of both surfactant components. It inhibited transport out of early endosomes for lipid only, not for SP-A. We conclude that in type II cells, endocytosed SP-A and lipid are transported toward a common early endosomal compartment. Thereafter, both components dissociate. SP-A is rapidly recycled to the cell surface and does not enter classic lamellar bodies. Lipid is transported toward lamellar bodies.
C-type natriuretic peptide system in fetal ovine pulmonary vasculature
Authors: Lakshminrusimha Satyan , D'Angelis Christopher A. , Russell James A. , Nielsen Lori C. , Gugino Sylvia F. , Nickerson Peter A. , Steinhorn Robin H.
Published: December 2017

C-type natriuretic peptide (CNP) is a recently described endothelium-derived relaxing factor. CNP relaxes vascular smooth muscle and inhibits smooth muscle proliferation by binding to natriuretic peptide receptor (NPR) type B (NPR-B) and producing cGMP. Lung parenchyma and fifth-generation pulmonary arteries (PA) and veins (PV) were isolated from late-gestation fetal lambs. All three types of NPR mRNA were detected in PA and PV by RT-PCR. CNP and NPR-B immunostaining was positive in pulmonary vascular endothelium and medial smooth muscle. CNP concentration-response curves of PA and PV were compared with those of atrial natriuretic peptide (ANP) by use of standard tissue bath techniques. CNP relaxed PV significantly better than PA. ANP relaxed PA and PV equally, but ANP relaxed PA significantly better than CNP. Pretreating PA and PV with natriuretic peptide receptor blocker (HS-142-1) or cGMP-dependent protein kinase inhibitor Rp-β-phenyl-1- N2-etheno-8-bromoguanosine 3′,5′-cyclic monophosphorothionate significantly inhibited the CNP relaxation response, indicating that the response was mediated through the NPR-cGMP pathway. We conclude that CNP is important in mediating pulmonary venous tone in the fetus.
Upregulation of lung soluble guanylate cyclase during chronic hypoxia is prevented by deletion of eNOS
Authors: Li Dechun , Laubach Victor E. , Johns Roger A.
Published: December 2017

Hypoxia upregulates endothelial (e) nitric oxide synthase (NOS), but how eNOS affects soluble guanylate cyclase (sGC) protein expression in hypoxia-induced pulmonary hypertension is unknown. Wild-type (WT), eNOS-deficient [eNOS(−/−)], and inducible NOS (iNOS)-deficient [iNOS(−/−)] mice were used to investigate the effects of lack of NO from different NOS isoforms on sGC activity and protein expression and its relationship to the muscularization of the pulmonary vasculature. After 6 days of hypoxic exposure (10% O2), the ratios of the right ventricle to left ventricle + septum weight (RV/LV+S) and right ventricle weight to body weight, the lung sGC activity, and vascular muscularization were determined, and protein analysis for eNOS, iNOS, and sGC was performed. Results demonstrated that there were significant increases of RV/LV+S in all animals treated with hypoxia. In hypoxic WT and iNOS(−/−) mice, eNOS and sGC α1- and β1-protein increased twofold; cGMP levels and the number of muscularized vessels also increased compared with hypoxic eNOS(−/−) mice. There was a twofold increase of iNOS protein in WT and eNOS(−/−) mice, and the basal iNOS protein concentration was higher in eNOS(−/−) mice than in WT mice. In contrast, the eNOS(−/−) mouse lung showed no eNOS protein expression, lower cGMP concentrations, and no change of sGC protein levels after hypoxic exposure compared with its normoxic controls ( P > 0.34). These results suggest that eNOS, but not iNOS, is a major regulator of sGC activity and protein expression in the pulmonary vasculature.
Chronic ethanol ingestion potentiates TNF-α-mediated oxidative stress and apoptosis in rat type II cells
Authors: Brown Lou Ann S. , Harris Frank L. , Guidot David M.
Published: December 2017

In septic patients, chronic alcohol abuse increases the incidence of the acute respiratory distress syndrome (ARDS). Because alveolar type II cell viability is critical for epithelial repair, our objective was to determine if chronic ethanol ingestion increased the sensitivity of type II cells to the inflammatory mediators upregulated during sepsis. In rats chronically fed ethanol, type II cell mitochondrial GSH was depleted, and tumor necrosis factor-α (TNF-α)-induced generation of mitochondrial reactive oxygen species (ROS) and apoptosis were potentiated. When added to the ethanol diet, the GSH precursor (−)-2-oxo-4-thiazolidinecarboxylic acid (Procysteine; Pro) but not N-acetylcysteine (NAC) normalized type II cell mitochondrial GSH. Likewise, Pro but not NAC normalized TNF-α-induced mitochondrial ROS and apoptosis. This suggested that chronic ethanol ingestion potentiated TNF-α-induced apoptosis in type II cells via mitochondrial GSH depletion. This may be particularly relevant in ARDS when type II cell viability is critical to repair of the damaged alveolar epithelium and may have important ramifications for the treatment of ARDS patients with a history of alcohol abuse.
Human SLPI inactivation after cigarette smoke exposure in a new in vivo model of pulmonary oxidative stress
Authors: Cavarra Eleonora , Lucattelli Monica , Gambelli Federica , Bartalesi Barbara , Fineschi Silvia , Szarka Andras , Giannerini Fabiola , Martorana Piero A. , Lungarella Giuseppe
Published: December 2017

The role of oxidative stress in inactivating antiproteases is the object of debate. To address this question, we developed an in vivo model of pulmonary oxidative stress induced by cigarette smoke (CS) in mice. The major mouse trypsin inhibitor contrapsin is not sensitive to oxidation, and the mouse secretory leukoprotease inhibitor (SLPI) does not inhibit trypsin. Instead, human recombinant (hr) SLPI inhibits trypsin and is sensitive to oxidation. Thus we determined the effect of CS in vivo on hrSLPI antiproteolytic function in the airways of mice. CS caused a significant decrease in total antioxidant capacity in bronchoalveolar lavage fluid (BALF) and significant changes in oxidized glutathione, ascorbic acid, protein thiols, and 8-epi-PGF2α. Intratracheal hrSLPI significantly increased BALF antitryptic activity. CS induced a 50% drop in the inhibitory activity of hrSLPI. Pretreatment with N-acetylcysteine prevented the CS-induced loss of hrSLPI activity, the decrease in antioxidant defenses, and the elevation of 8-epi-PGF-2α. Thus an inactivation of hrSLPI was demonstrated in this model. This is a novel model for studying in vivo the effects of CS oxidative stress on human protease inhibitors with antitrypsin activity.
Role of p38 MAP kinase in diperoxovanadate-induced phospholipase D activation in endothelial cells
Authors: Natarajan Viswanathan , Scribner William M. , Morris Andrew J. , Roy Shukla , Vepa Suryanarayana , Yang Jianbin , Wadgaonkar Raj , Reddy Sekhar P. M. , Garcia Joe G. N. , Parinandi Narasimham L.
Published: December 2017

We previously demonstrated that diperoxovanadate (DPV), a synthetic peroxovanadium compound and cell-permeable oxidant that acts as a protein tyrosine phosphatase inhibitor and insulinomimetic, increased phospholipase D (PLD) activation in endothelial cells (ECs). In this report, the regulation of DPV-induced PLD activation by mitogen-activated protein kinases (MAPKs) was investigated. DPV activated extracellular signal-regulated kinase, c-Jun NH2-terminal kinase (JNK), and p38 MAPK in a dose- and time-dependent fashion. Treatment of ECs with p38 MAPK inhibitors SB-203580 and SB-202190 or transient transfection with a p38 dominant negative mutant mitigated the PLD activation by DPV but not by phorbol ester. SB-202190 blocked DPV-mediated p38 MAPK activity as determined by activated transcription factor-2 phosphorylation. Immunoprecipitation of PLD from EC lysates with PLD1 and PLD2 antibodies revealed both PLD isoforms associated with p38 MAPK. Similarly, PLD1 and PLD2 were detected in p38 immunoprecipitates from control and DPV-challenged ECs. Binding assays demonstrated interaction of glutathione S-transferase-p38 fusion protein with PLD1 and PLD2. Both PLD1 and PLD2 were phosphorylated by p38 MAPK in vitro, and DPV increased phosphorylation of PLD1 and PLD2 in vivo. However, phosphorylation of PLD by p38 failed to affect PLD activity in vitro. These results provide evidence for p38 MAPK-mediated regulation of PLD in ECs.
Optical method for quantifying rates of mucus secretion from single submucosal glands
Authors: Joo Nam Soo , Wu Jin V. , Krouse Mauri E. , Saenz Yamil , Wine Jeffrey J.
Published: December 2017

We describe an optical method to quantify single- gland secretion. Isolated tracheal mucosa were mounted at the air-Krebs interface and coated with oil. Gland secretions formed spherical bubbles that were digitally imaged at intervals, allowing rates of secretion to be calculated. We monitored 340 glands in 54 experiments with 12 sheep. Glands secreted basally at low rates (0.57 ± 0.04 nl · min−1 · gland−1, 123 glands) in tissues up to 9 h postharvest and at lower rates for up to 3 days. Carbachol (10 μM) stimulated secretion with an early transient and a sustained or oscillating phase. Peak secretion was 15.7 ± 1.2 nl · min−1 · gland−1 (60 glands); sustained secretion was 4.5 ± 0.5 nl · min−1 · gland−1 (10 glands). Isoproterenol and phenylephrine (10 μM each) stimulated only small, transient responses. We confirmed that cats have a large secretory response to phenylephrine (11.6 ± 3.7 nl · min−1 · gland−1, 12 glands), but pigs, sheep, and humans all have small responses (<2 nl · min−1 · gland−1). Carbachol-stimulated peak secretion was inhibited 56% by bumetanide, 67% by HCO[Formula: see text] replacement with HEPES, and 92% by both. The distribution of secretion rates was nonnormal, suggesting the existence of subpopulations of glands.
EP2receptors mediate airway relaxation to substance P, ATP, and PGE2
Authors: Fortner Christopher N. , Breyer Richard M. , Paul Richard J.
Published: December 2017

Substance P (SP) and ATP evoke transient, epithelium-dependent relaxation of constricted mouse tracheal smooth muscle. Relaxation to either SP or ATP is blocked by indomethacin, but the specific eicosanoid(s) involved have not been definitively identified. SP and ATP are reported to release PGE2from airway epithelium in other species, suggesting PGE2as a likely mediator in epithelium-dependent airway relaxation. Using mice homozygous for a gene-targeted deletion of the EP2receptor [EP2(−/−)], one of the PGE2receptors, we tested the hypothesis that PGE2is the primary mediator of relaxation to SP or ATP. Relaxation in response to SP or ATP was significantly reduced in tracheas from EP2(−/−) mice. There were no differences between EP2(−/−) and wild-type tracheas in their physical dimensions, contraction to ACh, or relaxation to isoproterenol, thus ruling out any general alterations of smooth muscle function. There were also no differences between EP2(−/−) and wild-type tracheas in basal or stimulated PGE2production. Exogenous PGE2produced significantly less relaxation in EP2(−/−) tracheas compared with the wild type. Taken together, this experimental evidence supports the following two conclusions: EP2receptors are of primary importance in airway relaxation to PGE2and relaxation to SP or ATP is mediated through PGE2acting on EP2receptors.
eNOS and prostanoid enzymes in lungs of newborn piglets with chronic aortopulmonary shunts
Authors: Fike Candice D. , Kaplowitz Mark R. , Bousamra Michael
Published: December 2017

Our purpose was to determine if abundance of proteins underlying nitric oxide (NO) and prostanoid production is altered in lungs of piglets with aortopulmonary shunts. We also evaluated whether shunted piglets exhibit abnormal pulmonary vascular responses to ACh, an endothelium-dependent agent that mediates dilation in part by NO and prostanoid release. At age 4–5 days, piglets underwent either a sham operation or placement of an aortopulmonary shunt. At age 5–6 wk, pulmonary arterial pressure (Ppa) and cardiac output by the thermodilution technique were measured in anesthetized piglets. Ppa responses to the endothelium-dependent agent, ACh, and to a non-endothelium-dependent agent, papaverine, were measured in perfused lungs. An immunoblot technique was applied to homogenates of whole lung tissue and two size groups of pulmonary arteries. In shunted piglets, Ppa and cardiac output were elevated, and Ppa responses to papaverine were reduced. ACh responses were not decreased when expressed relative to Ppa dilation with papaverine. Endothelial nitric oxide synthase (eNOS), cyclooxygenase-1, cyclooxygenase-2, prostacyclin synthase, and thromboxane synthase amounts were unaltered in all lung tissue homogenates. Altered abundance of eNOS and/or prostanoid enzymes does not contribute to the blunted dilation and the elevation in Ppa associated with aortopulmonary shunts in newborn piglets.
Activation of the EGF receptor signaling pathway in airway epithelial cells exposed to Utah Valley PM
Authors: Wu Weidong , Samet James M. , Ghio Andrew J. , Devlin Robert B.
Published: December 2017

Exposure to ambient particulate matter (PM) in the Utah Valley has previously been associated with a variety of adverse health effects. To investigate intracellular signaling mechanisms for pulmonary responses to Utah Valley PM inhalation, human primary airway epithelial cells were exposed to aqueous extracts of PM collected from the year before (Y1), during (Y2), and after (Y3) the closure of a local steel mill located in the Utah Valley in this study. Transfection with kinase-deficient extracellular signal-regulated kinase (ERK) 1 constructs partially blocked Utah Valley PM-induced interleukin (IL)-8 promoter reporter activity. The mitogen-activated protein kinase/ERK kinase (MEK) activity inhibitor PD-98059 significantly abolished IL-8 released in response to Utah Valley PM, as did the epidermal growth factor (EGF) receptor kinase inhibitor AG-1478. Western blotting showed that Utah Valley PM induced phosphorylation of EGF receptor tyrosine, MEK1/2, and ERK1/2, which could be ablated with AG-1478 or PD-98059. For all findings, the potency of Utah Valley PM collected during Y2 was found to be lower relative to that of Y1 and Y3. These data demonstrate that Utah Valley PM can induce IL-8 expression partially through the activation of the EGF receptor signaling.
Microtubule disassembly increases endothelial cell barrier dysfunction: role of MLC phosphorylation
Authors: Verin Alexander D. , Birukova Anna , Wang Peiyi , Liu Feng , Becker Patrice , Birukov Konstantin , Garcia Joe G. N.
Published: December 2017

Endothelial cell (EC) barrier regulation is critically dependent on cytoskeletal components (microfilaments and microtubules). Because several edemagenic agents induce actomyosin-driven EC contraction tightly linked to myosin light chain (MLC) phosphorylation and microfilament reorganization, we examined the role of microtubule components in bovine EC barrier regulation. Nocodazole or vinblastine, inhibitors of microtubule polymerization, significantly decreased transendothelial electrical resistance in a dose-dependent manner, whereas pretreatment with the microtubule stabilizer paclitaxel significantly attenuated this effect. Decreases in transendothelial electrical resistance induced by microtubule disruption correlated with increases in lung permeability in isolated ferret lung preparations as well as with increases in EC stress fiber content and MLC phosphorylation. The increases in MLC phosphorylation were attributed to decreases in myosin-specific phosphatase activity without significant increases in MLC kinase activity and were attenuated by paclitaxel or by several strategies (C3 exotoxin, toxin B, Rho kinase inhibition) to inhibit Rho GTPase. Together, these results suggest that microtubule disruption initiates specific signaling pathways that cross talk with microfilament networks, resulting in Rho-mediated EC contractility and barrier dysfunction.
Adenoviral E1A primes alveolar epithelial cells to PM10-induced transcription of interleukin-8
Authors: Gilmour Peter S. , Rahman Irfan , Hayashi Shizu , Hogg James C. , Donaldson Kenneth , MacNee William
Published: December 2017

The presence of the adenoviral early region 1A (E1A) protein in human lungs has been associated with an increased risk of chronic obstructive pulmonary disease (COPD), possibly by a mechanism involving amplification of proinflammatory responses. We hypothesize that enhanced inflammation results from increased transcription factor activation in E1A-carrying cells, which may afford susceptibility to environmental particulate matter < 10 μm (PM10)-mediated oxidative stress. We measured interleukin (IL)-8 mRNA expression and protein release in human alveolar epithelial cells (A549) transfected with the E1A gene (E1A+ve). Both E1A+ve and −ve cells released IL-8 after incubation with TNF-α, but only E1A+ve cells were sensitive to LPS stimulation in IL-8 mRNA expression and protein release. E1A+ve cells showed an enhanced IL-8 mRNA and protein response after treatment with H2O2and PM10. E1A-enhanced induction of IL-8 was accompanied by increases in activator protein-1 and nuclear factor-κB nuclear binding in E1A+ve cells, which also showed higher basal nuclear binding of these transcription factors. These data suggest that the presence of E1A primes the cell transcriptional machinery for oxidative stress signaling and therefore facilitates amplification of proinflammatory responses. By this mechanism, susceptibility to exacerbation of COPD in response to particulate air pollution may occur in individuals harboring E1A.
Nickel requires hypoxia-inducible factor-1α, not redox signaling, to induce plasminogen activator inhibitor-1
Authors: Andrew Angeline S. , Klei Linda R. , Barchowsky Aaron
Published: December 2017

Human epidemiological and animal studies have associated inhalation of nickel dusts with an increased incidence of pulmonary fibrosis. At the cellular level, particulate nickel subsulfide inhibits fibrinolysis by transcriptionally inducing expression of plasminogen activator inhibitor (PAI)-1, an inhibitor of the urokinase-type plasminogen activator. Because nickel is known to mimic hypoxia, the present study examined whether nickel transcriptionally activates PAI-1 through the hypoxia-inducible factor (HIF)-1α signaling pathway. The involvement of the NADPH oxidase complex, reactive oxygen species, and kinases in mediating nickel-induced HIF-1α signaling was also investigated. Addition of nickel to BEAS-2B human airway epithelial cells increased HIF-1α protein levels and elevated PAI-1 mRNA levels. Pretreatment of cells with the extracellular signal-regulated kinase inhibitor U-0126 partially blocked HIF-1α protein and PAI-1 mRNA levels induced by nickel, whereas antioxidants and NADPH oxidase inhibitors had no effect. Pretreating cells with antisense, but not sense, oligonucleotides to HIF-1α mRNA abolished nickel-stimulated increases in PAI-1 mRNA. These data indicate that signaling through extracellular signal-regulated kinase and HIF-1α is required for nickel-induced transcriptional activation of PAI-1.
Mechanism of substance P-induced liquid secretion across bronchial epithelium
Authors: Trout Laura , Corboz Michel R. , Ballard Stephen T.
Published: December 2017

The present study was undertaken to identify and determine the mechanism of noncholinergic pathways for the induction of liquid secretion across airway epithelium. Excised porcine bronchi secreted substantial and significant quantities of liquid when exposed to acetylcholine, substance P, or forskolin but not to isoproterenol, norepinephrine, or phenylephrine. Bumetanide, an inhibitor of Na+-K+-2Cl−cotransport, reduced the liquid secretion response to substance P by 69%. Approximately two-thirds of bumetanide-insensitive liquid secretion was blocked by dimethylamiloride (DMA), a Na+/H+exchange inhibitor. Substance P responses were preserved in airways after surface epithelium removal, suggesting that secreted liquid originated from submucosal glands. The anion channel blockers diphenylamine-2-carboxylate (DPC) and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) inhibited >90% of substance P-induced liquid secretion, whereas DIDS had no effect. DMA, DPC, and NPPB had greater inhibitory effects on net HCO[Formula: see text] secretion than on liquid secretion. Although preserved relative to liquid secretion, net HCO[Formula: see text]secretion was reduced by 39% in the presence of bumetanide. We conclude that substance P induces liquid secretion from bronchial submucosal glands of pigs through active transport of Cl−and HCO[Formula: see text]. The pattern of responses to secretion agonists and antagonists suggests that the cystic fibrosis transmembrane conductance regulator mediates this process.
Intrinsic AHR in IL-5 transgenic mice is dependent on CD4+cells and CD49d-mediated signaling
Authors: Borchers Michael T. , Crosby J. , Justice P. , Farmer S. , Hines E. , Lee J. J. , Lee N. A.
Published: December 2017

Overexpression of interleukin (IL)-5 by the airway epithelium in mice using the rat CC10 promoter (NJ.1726 line) leads to several histopathologies characteristic of human asthma, including airway hyperreactivity (AHR). We investigated the contribution of B and T cells, as well as CD4 expression, to the development of AHR in IL-5 transgenic mice. NJ.1726 mice on a T cell or CD4 knockout background, but not on a B cell knockout background, lost intrinsic AHR. These effects occurred without decreases in IL-5 or eosinophils. We further investigated the contribution of α4-integrin signaling to the development of AHR in IL-5 transgenic mice through the administration of anti-CD49d (α4-integrin) antibody (PS/2). Administration of PS/2 resulted in immediate (16-h) inhibition of AHR. The inhibition of AHR was not associated with a decrease in airway eosinophils. These studies demonstrate that, despite the presence of increased levels of IL-5 and eosinophils in the lungs of NJ.1726 mice, CD4+cells and α4-integrin signaling are necessary for the intrinsic AHR that develops in IL-5 transgenic mice.
Inhibition of geranylgeranylation blocks agonist-induced actin reorganization in human airway smooth muscle cells
Authors: Lesh Ryan E. , Emala Charles W. , Lee H. Thomas , Zhu Defen , Panettieri Reynold A. , Hirshman Carol A.
Published: December 2017

To determine whether RhoA isoprenylation (geranylgeranylation) is required for agonist-induced actin cytoskeleton reorganization (measured by an increase in the filamentous F- to monomeric G-actin ratio), human airway smooth muscle cells were treated for 72 h with inhibitors of geranylgeranyltransferase I. Geranylgeranyltransferase inhibitor (GGTI)-2147 or -286 pretreatment completely blocked the increase in the F- to G-actin fluorescence ratio when cells were stimulated with lysophosphatidic acid (LPA), endothelin, or carbachol. In contrast, LPA or endothelin induced actin cytoskeletal reorganization in cells treated with farnesyltransferase inhibitor (FTI)-277 to inactivate Ras. Forskolin-induced adenylyl cyclase activity was inhibited by carbachol in GGTI-2147-pretreated cells, demonstrating that the effect of geranylgeranyltransferase I inhibition on stress fiber formation was not due to uncoupling of signaling between the heterotrimeric Giprotein (the Gγ subunit is isoprenylated) and distal effectors. These results demonstrate that selective GGTIs can inhibit agonist-induced actin reorganization.
Changes in proteoglycans and lung tissue mechanics during excessive mechanical ventilation in rats
Authors: Al-Jamal Rehab , Ludwig Mara S.
Published: December 2017

Excessive mechanical ventilation results in changes in lung tissue mechanics. We hypothesized that changes in tissue properties might be related to changes in the extracellular matrix component proteoglycans (PGs). The effect of different ventilation regimens on lung tissue mechanics and PGs was examined in an in vivo rat model. Animals were anesthetized, tracheostomized, and ventilated at a tidal volume of 8 (Vt8), 20, or 30 (Vt30) ml/kg, positive end-expiratory pressure of 0 (PEEP0) or 1.5 (PEEP1.5) cmH2O, and frequency of 1.5 Hz for 2 h. The constant-phase model was used to derive airway resistance, tissue elastance, and tissue damping. After physiological measurements, one lung was frozen for immunohistochemistry and the other was reserved for PG extraction and Western blotting. After 2 h of mechanical ventilation, tissue elastance and damping were significantly increased in rats ventilated at Vt30PEEP0compared with control rats (ventilated at Vt8PEEP1.5). Versican, basement membrane heparan sulfate PG, and biglycan were all increased in rat lungs ventilated at Vt30PEEP0compared with control rats. At Vt30PEEP0, heparan sulfate PG and versican staining became prominent in the alveolar wall and airspace; biglycan was mostly localized in the airway wall. These data demonstrate that alterations in lung tissue mechanics with excessive mechanical ventilation are accompanied by changes in all classes of extracellular matrix PG.
Dose-dependent lung remodeling in transgenic mice expressing transforming growth factor-α
Authors: Hardie William D. , Piljan-Gentle Alyssa , Dunlavy Michelle R. , Ikegami Machiko , Korfhagen Thomas R.
Published: December 2017

Transgenic mice overexpressing human transforming growth factor-α (TGF-α) develop emphysema and fibrosis during postnatal alveologenesis. To assess dose-related pulmonary alterations, four distinct transgenic lines expressing different amounts of TGF-α in the distal lung under control of the surfactant protein C (SP-C) promoter were characterized. Mean lung homogenate TGF-α levels ranged from 388 ± 40 pg/ml in the lowest expressing line to 1,247 ± 33 pg/ml in the highest expressing line. Histological assessment demonstrated progressive alveolar airspace size changes that were more severe in the higher expressing TGF-α lines. Pleural and parenchymal fibrosis were only detected in the highest expressing line ( line 28), and increasing terminal airspace area was associated with increasing TGF-α expression. Hysteresis on pressure-volume curves was significantly reduced in line 28 mice compared with other lines of mice. There were no differences in bronchoalveolar lavage fluid cell count or differential that would indicate any evidence of lung inflammation among all transgenic lines. Proliferating cells were increased in line 28 without alterations of numbers of type II cells. We conclude that TGF-α lung remodeling in transgenic mice is dose dependent and is independent of pulmonary inflammation.
Apoptosis by pan-caspase inhibitors in lipopolysaccharide-activated macrophages
Authors: Kim Sung Ouk , Ono Koh , Han Jiahuai
Published: December 2017

Although apoptosis has been observed in macrophages during the course of infections, the mechanism of apoptosis in activated macrophages is not fully understood. This study shows that pan-caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (ZVAD) or t-butyloxycarbonyl-Asp-fluoromethylketone (Boc-D) caused the death of lipopolysaccharide (LPS)-activated macrophages and RAW 264.7 cells with apoptotic features. The apoptosis was also observed in lipoprotein-treated bacteria but not in CpG oligonucleotide- or flagellin-treated macrophages, indicating a difference of cellular responses downstream of different Toll-like receptors. Consistent with the induction of cell death by pan-caspase inhibitors, no activation of known caspases was detected in LPS-ZVAD-treated cells, suggesting an involvement of unknown proapoptotic caspases in the cell death. ZVAD inhibited the activation of extracellular signal-regulated kinase (ERK) and p38 but not of nuclear factor (NF)-κB induced by LPS, suggesting that the ZVAD-sensitive molecule lies upstream of the ERK and p38 pathways but downstream of the divergent site of NF-κB and mitogen-activated protein kinases. Our results demonstrate that apoptosis of macrophages induced by LPS+ZVAD is independent from the known proapoptotic caspases and suggest that activity of an unidentified ZVAD-sensitive molecule(s) is involved in the survival of LPS-activated macrophages.
Hyperoxia increases leptin production: a mechanism mediated through endogenous elevation of corticosterone
Authors: Barazzone-Argiroffo Constance , Muzzin Patrick , Donati Yves R. , Kan Chen-Da , Aubert Michel L. , Piguet Pierre-François
Published: December 2017

Leptin, a cytokine involved in the regulation of food intake, has been reported to be decreased in lung diseases such as chronic obstructive pulmonary disease and cystic fibrosis and increased in critically ill patients with sepsis. We investigated the role of leptin during hyperoxia in mice, which results in alveolar edema, severe weight loss, and death within 3–4 days. In oxygen-breathing mice, serum leptin was increased six- to sevenfold and its mRNA was upregulated in white adipose tissue. Leptin elevation could not be attributed to changes in circulating tumor necrosis factor-α but was completely dependent on endogenous corticosterone elevation because adrenalectomized mice did not exhibit any increase in leptin levels. Using leptin-deficient mice and wild-type mice treated with anti-leptin antibody, we demonstrate that weight loss was leptin independent. Lung damage was moderately attenuated in leptin-deficient mice but was not modified by anti-leptin antibody or leptin administration, suggesting that leptin does not play an essential role in the direct and short-term effects of oxygen-induced injury.
NO and prostaglandin interactions during hemodynamic stress in the fetal ovine pulmonary circulation
Authors: Zenge Jeanne P. , Rairigh Robyn L. , Grover Theresa R. , Storme Laurent , Parker Thomas A. , Kinsella John P. , Abman Steven H.
Published: December 2017

Nitric oxide (NO) and prostacyclin (PGI2) are potent fetal pulmonary vasodilators, but their relative roles and interactions in the regulation of the perinatal pulmonary circulation are poorly understood. We compared the separate and combined effects of nitric oxide synthase (NOS) and cyclooxygenase (COX) inhibition during acute hemodynamic stress caused by brief mechanical compression of the ductus arteriosus (DA) in chronically prepared fetal lambs. Nitro-l-arginine (l-NNA; NOS antagonist), meclofenamate (Mec; COX inhibitor), combined drugs (l-NNA-Mec), or saline (control) was infused into the left pulmonary artery (LPA) before DA compression. In controls, DA compression decreased pulmonary vascular resistance (PVR) by 43% ( P < 0.01). l-NNA, but not Mec, treatment completely blocked vasodilation and caused a paradoxical increase in PVR (+31%; P < 0.05). The effects ofl-NNA-Mec and l-NNA on PVR were similar. To determine if the vasodilator effect of PGI2 is partly mediated by NO release, we studied PGI2-induced vasodilation before and after NOS inhibition. l-NNA treatment blocked the PGI2-induced rise in LPA blood flow by 73% ( P < 0.001). We conclude that NO has a greater role than PGs in fetal pulmonary vasoregulation during acute hemodynamic stress and that PGI2-induced pulmonary vasodilation is largely mediated by NO release in the fetal lung.
Partial correction of defective Cl−secretion in cystic fibrosis epithelial cells by an analog of squalamine
Authors: Jiang Canwen , Lee Edward R. , Lane Mathieu B. , Xiao Yong-Fu , Harris David J. , Cheng Seng H.
Published: December 2017

Defective cystic fibrosis (CF) transmembrane conductance regulator (CFTR)-mediated Cl−transport across the apical membrane of airway epithelial cells is implicated in the pathophysiology of CF lungs. A strategy to compensate for this loss is to augment Cl−transport through alternative pathways. We report here that partial correction of this defect could be attained through the incorporation of artificial anion channels into the CF cells. Introduction of GL-172, a synthetic analog of squalamine, into CFT1 cells increased cell membrane halide permeability. Furthermore, when a Cl−gradient was generated across polarized monolayers of primary human airway or Fischer rat thyroid cells in an Ussing chamber, addition of GL-172 caused an increase in the equivalent short-circuit current. The magnitude of this change in short-circuit current was ∼30% of that attained when CFTR was maximally stimulated with cAMP agonists. Patch-clamp studies showed that addition of GL-172 to CFT1 cells also increased whole cell Cl−currents. These currents displayed a linear current-voltage relationship and no time dependence. Additionally, administration of GL-172 to the nasal epithelium of transgenic CF mice induced a hyperpolarization response to perfusion with a low-Cl−solution, indicating restoration of Cl−secretion. Together, these results demonstrate that in CF airway epithelial cells, administration of GL-172 is capable of partially correcting the defective Cl−secretion.
Examining basal chloride transport using the nasal potential difference response in a murine model
Authors: Brady Kristine G. , Kelley Thomas J. , Drumm Mitchell L.
Published: December 2017

Epithelia of humans and mice with cystic fibrosis are unable to secrete chloride in response to a chloride gradient or to cAMP-elevating agents. Bioelectrical properties measured using the nasal transepithelial potential difference (TEPD) assay are believed to reflect these cystic fibrosis transmembrane conductance regulator (CFTR)-dependent chloride transport defects. Although the response to forskolin is CFTR mediated, the mechanisms responsible for the response to a chloride gradient are unknown. TEPD measurements performed on inbred mice were used to compare the responses to low chloride and forskolin in vivo. Both responses show little correlation between or within inbred strains of mice, suggesting they are mediated through partially distinct mechanisms. In addition, these responses were assayed in the presence of several chloride channel inhibitors, including DIDS, diphenylamine-2-carboxylate, glibenclamide, and 5-nitro-2-(3-phenylpropylamino)-benzoic acid, and a protein kinase A inhibitor, the Rp diastereomer of adenosine 3′,5′-cyclic monophosphothioate ( Rp-cAMPS). The responses to low chloride and forskolin demonstrate significantly different pharmacological profiles to both DIDS and Rp-cAMPS, indicating that channels in addition to CFTR contribute to the low chloride response.
Impaired functional activity of alveolar macrophages from GM-CSF-deficient mice
Authors: Paine Robert , Morris Susan B. , Jin Hong , Wilcoxen Steven E. , Phare Susan M. , Moore Bethany B. , Coffey Michael J. , Toews Galen B.
Published: December 2017

We hypothesized that pulmonary granulocyte-macrophage colony-stimulating factor (GM-CSF) is critically involved in determining the functional capabilities of alveolar macrophages (AM) for host defense. To test this hypothesis, cells were collected by lung lavage from GM-CSF mutant mice [GM(−/−)] and C57BL/6 wild-type mice. GM(−/−) mice yielded almost 4-fold more AM than wild-type mice. The percentage of cells positive for the β2-integrins CD11a and CD11c was reduced significantly in GM(−/−) AM compared with wild-type cells, whereas expression of CD11b was similar in the two groups. The phagocytic activity of GM(−/−) AM for FITC-labeled microspheres was impaired significantly compared with that of wild-type AM both in vitro and in vivo (after intratracheal inoculation with FITC-labeled beads). Stimulated secretion of tumor necrosis factor-α (TNF-α) and leukotrienes by AM from the GM(−/−) mice was greatly reduced compared with wild-type AM, whereas secretion of monocyte chemoattractant protein-1 was increased. Transgenic expression of GM-CSF exclusively in the lungs of GM(−/−) mice resulted in AM with normal or supranormal expression of CD11a and CD11c, phagocytic activity, and TNF-α secretion. Thus, in the absence of GM-CSF, AM functional capabilities for host defense were significantly impaired but were restored by lung-specific expression of GM-CSF.
Prostaglandin E2inhibits fibroblast chemotaxis
Authors: Kohyama Tadashi , Ertl Ronald F. , Valenti Vincenzo , Spurzem John , Kawamoto Masashi , Nakamura Yoichi , Veys Tom , Allegra Luigi , Romberger Debra , Rennard Stephen I.
Published: December 2017

Fibroblasts are the major source of extracellular connective tissue matrix, and the recruitment, accumulation, and stimulation of these cells are thought to play important roles in both normal healing and the development of fibrosis. Prostaglandin E2(PGE2) can inhibit this process by blocking fibroblast proliferation and collagen production. The aim of this study was to investigate the inhibitory effect of PGE2on human plasma fibronectin (hFN)- and bovine bronchial epithelial cell-conditioned medium (BBEC-CM)-induced chemotaxis of human fetal lung fibroblasts (HFL1). Using the Boyden blind well chamber technique, PGE2(10−7M) inhibited chemotaxis to hFN 40.8 ± 5.3% ( P < 0.05) and to BBEC-CM 49.7 ± 11.7% ( P < 0.05). Checkerboard analysis demonstrated inhibition of both chemotaxis and chemokinesis. The effect of PGE2was concentration dependent, and the inhibitory effect diminished with time. Other agents that increased fibroblast cAMP levels, including isoproterenol (10−5M), dibutyryl cAMP (10−5M), and forskolin (3 × 10−5M) had similar effects and inhibited chemotaxis 54.1, 95.3, and 87.0%, respectively. The inhibitory effect of PGE2on HFL1 cell chemotaxis was inhibited by the cAMP-dependent protein kinase (PKA) inhibitor KT-5720, which suggests a cAMP-dependent effect mediated by PKA. In summary, PGE2appears to inhibit fibroblast chemotaxis, perhaps by modulating the rate of fibroblast migration. Such an effect may contribute to regulation of the wound healing response after injury.
Decreased surfactant proteins in lambs with pulmonary hypertension secondary to increased blood flow
Authors: Gutierrez Jorge A. , Parry Andrew J. , McMullan D. Michael , Chapin Cheryl J. , Fineman Jeffrey R.
Published: December 2017

Infants with increased pulmonary blood flow secondary to congenital heart disease suffer from tachypnea, dyspnea, and recurrent pulmonary infections. We have recently established a model of pulmonary hypertension secondary to increased pulmonary blood flow in lambs after in utero placement of an aortopulmonary vascular graft. The purpose of the present study was to utilize our animal model to determine the effects on the expression of surfactant proteins A (SP-A), B (SP-B), and C (SP-C). At age 4 wk, SP-A mRNA content in lambs decreased to 61.4 ± 8% of age-matched control value ( n = 5; P < 0.05). In addition, SP-A protein content was decreased to 50 ± 12% of control value ( n = 6; P < 0.0001). Although we did not observe statistically significant changes in SP-B mRNA content, SP-B protein was decreased to 74 ± 25% of control value ( n = 4; P < 0.02). There was no difference in SP-C mRNA. These data show that in a model of congenital heart disease with pulmonary hypertension secondary to increased pulmonary blood flow, there is a decrease in SP-A gene expression as well as a decrease in SP-A and SP-B protein contents.
IL-4 differentially regulates eotaxin and MCP-4 in lung epithelium and circulating mononuclear cells
Authors: Nakamura Hidetoshi , Luster Andrew D. , Tateno Hiroki , Jedrzkiewicz Sean , Tamura Gen , Haley Kathleen J. , Garcia-Zepeda Eduardo A. , Yamaguchi Kazuhiro , Lilly Craig M.
Published: December 2017

To investigate the mechanisms of eosinophil recruitment in allergic airway inflammation, we examined the effects of interleukin (IL)-4, a Th2-type cytokine, on eotaxin and monocyte chemoattractant protein-4 (MCP-4) expression in human peripheral blood mononuclear cells (PBMCs; n = 10), in human lower airway mononuclear cells ( n = 5), in the human lung epithelial cell lines A549 and BEAS-2B, and in human cultured airway epithelial cells. IL-4 inhibited eotaxin and MCP-4 mRNA expression induced by IL-1β and tumor necrosis factor-α in PBMCs but did not significantly inhibit expression in epithelial cells. Eotaxin and MCP-4 mRNA expression was not significantly induced by proinflammatory cytokines in lower airway mononuclear cells. IL-1β-induced eotaxin and MCP-4 protein production was also inhibited by IL-4 in PBMCs, whereas IL-4 enhanced eotaxin protein production in A549 cells. In contrast, dexamethasone inhibited eotaxin and MCP-4 expression in both PBMCs and epithelial cells. The divergent effects of IL-4 on eotaxin and MCP-4 expression between PBMCs and epithelial cells may create chemokine concentration gradients between the subepithelial layer and the capillary spaces that may promote the recruitment of eosinophils to the airway in Th2-type responses.
CCSP modulates airway dysfunction and host responses in an Ova-challenged mouse model
Authors: Wang Shan-Ze , Rosenberger Cynthia L. , Espindola Teresa M. , Barrett Edward G. , Tesfaigzi Yohannes , Bice David E. , Harrod Kevin S.
Published: December 2017

Clara cell secretory protein (CCSP) is synthesized by nonciliated bronchiolar cells in the lung and modulates lung inflammation to infection. To determine the role of CCSP in the host response to allergic airway disease, CCSP-deficient [(−/−)] mice were immunized twice with ovalbumin (Ova) and challenged by Ova (2 or 5 mg/m3) aerosol. After 2, 3, and 5 days of Ova aerosol challenge (6 h/day), airway reactivity was increased in CCSP(−/−) mice compared with wild-type [CCSP(+/+)] mice. Neutrophils were markedly increased in the bronchoalveolar lavage fluid of CCSP(−/−) Ova mice, coinciding with increased myeloperoxidase activity and macrophage inflammatory protein-2 levels. Lung histopathology and inflammation were increased in CCSP(−/−) compared with wild-type mice after Ova challenge. Mucus production, as assessed by histological staining, was increased in the airway epithelium of CCSP(−/−) Ova mice compared with that in CCSP(+/+) Ova mice. These data suggest a role for CCSP in airway reactivity and the host response to allergic airway inflammation and provide further evidence for the role of the airway epithelium in regulating airway responses in allergic disease.
Pulmonary microvascular and perivascular interstitial geometry during development of mild hydraulic edema
Authors: Negrini Daniela , Candiani Anna , Boschetti Federica , Crisafulli Beatrice , Del Fabbro Massimo , Bettinelli Dario , Miserocchi Giuseppe
Published: December 2017

To study pulmonary arteriolar vasomotion in control conditions and in the transition to hydraulic edema, changes in subpleural pulmonary arteriolar diameter and perivascular interstitial volume were evaluated in anesthetized spontaneously breathing rabbits. Images of subpleural pulmonary microvessels were recorded in control conditions and for up to 180 min during a 0.5 ml · kg−1 · min−1 intravenous saline infusion through an intact parietal pleural window. Images were digitized and analyzed with a semiautomatic procedure to determine vessel diameter and perivascular interstitial thickness from which interstitial fluid volume was derived. In control vessels, the diameter of ∼30-, ∼50-, and ∼80-μm arterioles and the perivascular interstitial thickness were fairly stable. During infusion, the diameter increased maximally by 20% in ∼30-μm vessels, was unchanged in ∼50-μm vessels, and decreased by 25% in ∼80-μm arterioles; the perivascular interstitial volume increased by 54% only around 30-μm microvessels. In papaverine-treated rabbits, all arterioles dilated and a larger increase in perivascular interstitial thickness was observed. The data suggest that the opposite vasomotor behavior of 30- and 80-μm arterioles during development of mild edema may represent a local specific response of the pulmonary microcirculation to reduce capillary pressure in the face of an increased transendothelial fluid filtration, thus counteracting progression toward severe edema.