bluuebunny/crossref_metadata_2025 huggingface dataset:
A urokinase receptor mRNA binding protein from rabbit lung fibroblasts and mesothelial cells
Authors: Shetty Sreerama , Idell Steven
Published: December 2017

The urokinase receptor (uPAR) influences several biological functions relevant to lung injury and repair, including proteolysis, cell migration, and adhesion. In malignant mesothelioma cells, we recently found that a posttranscriptional mechanism involving a cis- transinteraction between a uPAR mRNA sequence and a cytoplasmic uPAR mRNA binding protein (mRNABP) regulates uPAR gene expression (S. Shetty, A. Kumar, and S. Idell. Mol. Cell Biol. 17: 1075–1083, 1997). In this study, we sought to determine if uPAR expression in lung and pleural cells involves a similar posttranscriptional pathway. We first identified and characterized the uPAR mRNABP in rabbit tissues using gel mobility shift, ultraviolet (UV) cross-linking, and RNase protection assays and detected it in liver, heart, brain, spleen, colon, and lung. Phorbol 12-myristate 13-acetate, lipopolysaccharide, transforming growth factor-β, tumor necrosis factor-α, or cycloheximide induced uPAR and uPAR mRNA expression in cultured rabbit pleural mesothelial cells and lung fibroblasts and concurrently reduced the uPAR mRNA-uPAR mRNABP interaction. Using conventional and affinity chromatography, we purified a 50-kDa uPAR mRNABP that selectively binds to a 51-nucleotide fragment of the uPAR coding region. This protein migrates as a monomer when analyzed by SDS-PAGE and UV cross-linking and does not possess intrinsic RNase activity in vitro. A uPAR mRNABP physicochemically and functionally similar to that of human malignant mesothelioma is constitutively expressed in the rabbit lung and other nonneoplastic tissues. In rabbit lung fibroblasts and mesothelial cells, expression of uPAR involves posttranscriptional regulation whereby the uPAR mRNABP appears to interact with a specific coding region cis-element to decrease the stability of uPAR mRNA.
Endothelial dysfunction in pulmonary arteries of patients with mild COPD
Authors: Peinado Víctor I. , Barberà Joan A. , Ramírez Josep , Gómez Federico P. , Roca Josep , Jover Lluís , Gimferrer Josep M. , Rodriguez-Roisin Robert
Published: December 2017

To investigate whether endothelial dysfunction of pulmonary arteries (PA) is present in patients with mild chronic obstructive pulmonary disease (COPD) and to what extent it is related to the morphological abnormalities of PA, we studied 41 patients who underwent lung resection. Patients were divided into the following groups: nonsmokers ( n = 7), smokers with normal lung function ( n = 13), and COPD ( n = 21). Endothelium-dependent relaxation mediated by nitric oxide was evaluated in vitro in PA rings exposed to cumulative concentrations of acetylcholine (ACh) and ADP. Structural abnormalities of PA were assessed morphometrically. PA of COPD patients developed lower maximal relaxation in response to ADP than both nonsmokers and smokers ( P < 0.05 each) and a trend to reduced relaxation in response to ACh ( P = 0.08). Maximal relaxation to ADP correlated with the degree of airflow obstruction ( r = 0.48, P < 0.01). Morphometrical analysis of PA revealed thicker intimas, especially in small arteries, in both smokers and COPD compared with nonsmokers ( P < 0.05 each). We conclude that endothelial dysfunction of PA is already present in patients with mild COPD. In these patients, as well as in smokers with normal lung function, small arteries show thickened intimas, suggesting that tobacco consumption may play a critical role in the pathogenesis of pulmonary vascular abnormalities in COPD.
Effect of a novel covalent antithrombin-heparin complex on thrombin generation on fetal distal lung epithelium
Authors: Chan A. K. C. , Berry Leslie , Mitchell Lesley , Baranowski Bryan , O’Brodovich Hugh , Andrew Maureen
Published: December 2017

Respiratory distress syndrome is characterized by fibrin deposition in the lung. Fibrin adversely affects surfactant function and stimulates proliferation of fibroblasts. There is evidence that these properties may be important to the development of bronchopulmonary dysplasia. Despite successful initial treatment of neonatal respiratory distress syndrome with surfactant, the incidence of bronchopulmonary dysplasia has not decreased. In previous studies, it has been demonstrated that rat fetal distal lung epithelium (FDLE) possesses both procoagulant and anticoagulant properties. In this report, we have demonstrated (using factor VII-deficient plasma) that tissue factor is expressed on the FDLE surface and promotes thrombin generation. To regulate thrombin within this procoagulant environment, we have developed a novel anticoagulant, antithrombin-heparin covalent complex (ATH) that can be retained within the lung after intrapulmonary instillation. We have demonstrated that ATH was superior to antithrombin plus standard heparin in suppressing thrombin generation ( P < 0.001) and prothrombin consumption ( P < 0.01) in recalcified defibrinated plasma on the surface of FDLE. Further studies with ATH in vivo need to be performed.
Clearance of SP-C and recombinant SP-C in vivo and in vitro
Authors: Ikegami Machiko , Horowitz Ann D. , Whitsett Jeffrey A. , Jobe Alan H.
Published: December 2017

Surfactant protein (SP) C metabolism was evaluated in vivo by measurements of the clearance of bovine native SP-C (nSP-C) and a recombinant SP-C (rSP-C) in rabbits and mice and in vitro by the uptake into MLE-12 cells. rSP-C is the 34-amino acid human sequence with phenylalanine instead of cysteine in positions 4 and 5 and isoleucine instead of methionine in position 32. Alveolar clearances of iodinated SP-C and rSP-C after tracheal instillation were similar and slower than those for dipalmitoyl phosphatidylcholine (DPC) in the rabbit. nSP-C and rSP-C were cleared from rabbit lungs similarly to DPC, each with a half-life ( t1/2) of ∼11 h. In mice, the clearance of rSP-C from the lungs was slower ( t1/228 h) than the clearance of DPC ( t1/212 h). Liposome-associated dinitrophenyl-labeled rSP-C was taken up by MLE-12 cells, and the uptake was inhibited by excess nSP-C. The pattern of inhibition of dinitrophenyl-rSP-C uptake by SP-B, but not by SP-A, was similar to that previously reported for nSP-C. Clearance kinetics of nSP-C were similar to previous measurements of pulmonary clearance of SP-B in rabbits and mice. rSP-C has clearance kinetics and uptake by cells similar to those of nSP-C.
Altered ETB- but not ETA-receptor density and function in sheep airway smooth muscle cells in culture
Authors: Maxwell Michael J. , Goldie Roy G. , Henry Peter J.
Published: December 2017

The possibility that significant changes in endothelin (ET)A- and ETB-receptor density and function occur in airway smooth muscle cells (ASMCs) during cell growth and extended cell culture was investigated in sheep tracheal ASMCs. As in intact tracheal smooth muscle tissue from this species, early-passage sheep ASMCs contained a homogeneous population of ETAreceptors. However, growth of ASMCs from seeding to postconfluence and repeated passage of ASMCs (6th to 14th passages) was associated with a substantial increase in ETB-receptor density, with no change in ETA-receptor density. ET-1-induced stimulation of ETBreceptors increased the intracellular Ca2+concentration in single ASMCs. Interestingly, a 2-day period of serum deprivation completely eliminated the increase in ETB-receptor density and the ETBreceptor-mediated change in intracellular Ca2+concentration. In summary, growth and repeated passage of sheep ASMCs were associated with a profound and selective increase in the density and function of the ETBreceptor, a receptor subtype not present in early-passage ASMCs and not detected in intact sheep tracheal airway smooth muscle.
Pulmonary apoptosis in aged and oxygen-tolerant rats exposed to hyperoxia
Authors: Otterbein Leo E. , Chin Beek Yoke , Mantell Lin L. , Stansberry Leah , Horowitz Stuart , Choi Augustine M. K.
Published: December 2017

Accumulating evidence demonstrates that genotoxic and oxidant stress can induce programmed cell death or apoptosis in cultured cells. However, little is known about whether oxidative stress resulting from the deleterious effects of hyperoxia can induce apoptosis in vivo and even less is known regarding the functional significance of apoptosis in vivo in response to hyperoxia. Using hyperoxia as a model of oxidant-induced lung injury in the rat, we show that hyperoxic stress results in marked apoptotic signals in the lung. Lung tissue sections obtained from rats exposed to hyperoxia exhibit increased apoptosis in a time-dependent manner by terminal transferase dUTP nick end labeling assays. To examine whether hyperoxia-induced apoptosis in the lung correlated with the extent of lung injury or tolerance (adaptation) to hyperoxia, we investigated the pattern of apoptosis with a rat model of age-dependent tolerance to hyperoxia. We show that apoptosis is associated with increased survival of aged rats to hyperoxia and with decreased levels of lung injury as measured by the volume of pleural effusion, wet-to-dry lung weight, and myeloperoxidase content in aged rats compared with young rats after hyperoxia. We also examined this relationship in an alternate model of tolerance to hyperoxia. Lipopolysaccharide (LPS)-treated young rats not only demonstrated tolerance to hyperoxia but also exhibited a significantly lower apoptotic index compared with saline-treated rats after hyperoxia. To further separate the effects of aging and tolerance, we show that aged rats pretreated with LPS did not exhibit a significant level of tolerance against hyperoxia. Furthermore, similar to the hyperoxia-tolerant LPS-pretreated young rats, the nontolerant LPS-pretreated aged rats also exhibited a significantly reduced apoptotic index compared with aged rats exposed to hyperoxia alone. Taken together, our data suggest that hyperoxia-induced apoptosis in vivo can be modulated by both aging and tolerance effects. We conclude that there is no overall relationship between apoptosis and tolerance.
Spontaneous transient outward currents and delayed rectifier K+ current: effects of hypoxia
Authors: Vandier C. , Delpech M. , Bonnet P.
Published: December 2017

Single smooth muscle cells of rabbit intrapulmonary artery were voltage clamped using the perforated-patch configuration of the patch-clamp technique. We observed spontaneous transient outward currents (STOCs) and a steady-state outward current. Because STOCs were tetraethylammonium sensitive and activated by Ca2+ influx, they were believed to represent activation of Ca2+-activated K+ channels. The steady-state outward current, which was sensitive to 4-aminopyridine, was the delayed rectifier K+ current. In cells voltage clamped at 0 mV, we found that STOCs were not randomly distributed in amplitude but were composed of multiples of 1.57 ± 0.56 pA/pF. The mean frequency of STOCs was 5.51 ± 3.49 Hz. Ryanodine (10 μM), caffeine (5 mM), thapsigargin (200 nM), and hypoxia [Formula: see text] = 10 mmHg) decreased STOCs. The effect of hypoxia on STOCs was partially reversible only if the experiment was conducted in the presence of thapsigargin. Hypoxia and thapsigargin decrease steady-state outward current. Thapsigargin and removal of external Ca2+abolished the effect of hypoxia, suggesting that hypoxia decreases steady-state outward current by a Ca2+-dependent mechanism.
Porcine origin of human sputum trypsin?
Authors: Caughey George H. , Raymond Wilfred W. , Fang Kenneth C.
Published: December 2017

From purulent cystic fibrosis (CF) sputum, previous investigators partially purified a trypsinlike protease. A similar purified enzyme is available commercially as “human sputum trypsin.” To explore the nature and origin of this preparation, we purified and NH2terminally sequenced its major protein component. The resulting sequence, Ile-Val-Gly-Gly-Tyr-Thr-(Cys)-Ala-Ala-Asn-Ser-Val/Ile-Pro-Tyr-Gln-Val-Ser-Leu-Asn-Ser, differs from known human proteins but is identical to porcine trypsin, including the Val/Ile polymorphism at residue 12. Specific activity and electrophoretic and inhibition profiles and immunoreactivity of sputum and porcine pancreatic trypsin are nearly identical. Because porcine trypsin is a major ingredient of digestive enzyme supplements taken by CF patients with pancreatic dysfunction, we propose that one or more lots of human sputum trypsin derive from enzyme supplements and are of porcine origin. The path by which trypsin ends up in sputum is unknown. Because sputum trypsin is active but susceptible to inactivation by plasma α1-proteinase inhibitor, it is unlikely to derive from trypsin absorbed into the bloodstream. However, it may originate from tracheally aspirated stomach contents or from digestive supplement-contaminated saliva mixed with expectorated sputum. The imbalance between proteases and antiproteases in CF bronchial secretions allows trypsin to remain active despite sensitivity to serpins and secretory leukocyte proteinase inhibitor. Furthermore, because sputum trypsin activates human progelatinase B, it may be responsible in part for the reported presence of activated matrix metalloproteinases in CF sputum.
Cellular mechanisms and role of endothelin-1-induced calcium oscillations in pulmonary arterial myocytes
Authors: Hyvelin Jean-Marc , Guibert Christelle , Marthan Roger , Savineau Jean-Pierre
Published: December 2017

The effect of endothelin (ET)-1 on both cytosolic Ca2+concentration ([Ca2+]i) and membrane current in freshly isolated myocytes, as well as on the contraction of arterial rings, was investigated in rat main pulmonary artery (RMPA) and intrapulmonary arteries (RIPA). ET-1 (5–100 nM, 30 s) induced a first [Ca2+]ipeak followed by 3–5 oscillations of decreasing amplitude. In RMPA, the ET-1-induced [Ca2+]iresponse was fully abolished by BQ-123 (0.1 μM). In RIPA, the response was inhibited by BQ-123 in only 21% of the cells, whereas it was abolished by BQ-788 (1 μM) in 70% of the cells. In both types of arteries, the response was not modified in the presence of 100 μM La3+or in the absence of external Ca2+but disappeared after pretreatment of the cells with thapsigargin (1 μM) or neomycin (0.1 μM). In RPMA myocytes clamped at −60 mV, ET-1 induced an oscillatory inward current, the reversal potential of which was close to the equilibrium potential for Cl−. This current was unaltered by the removal of external Ca2+but was abolished by niflumic acid (50 μM). In arterial rings, the ET-1 (100 nM)-induced contraction was decreased by 35% in the presence of either niflumic acid (50 μM) or nifedipine (1 μM). These results demonstrate that ET-1 via the ETAreceptor only in RMPA and both ETAand ETBreceptors in RIPA induce [Ca2+]ioscillations due to iterative Ca2+release from an inositol trisphosphate-sensitive Ca2+store. Ca2+release secondarily activates an oscillatory membrane Cl−current that can depolarize the cell membrane, leading to an influx of Ca2+, this latter contributing to the ET-1-induced vasoconstrictor effect.
Thioredoxin overexpression prevents NO-induced reduction of NO synthase activity in lung endothelial cells
Authors: Zhang Jianliang , Li Yong D. , Patel Jawaharlal M. , Block Edward R.
Published: December 2017

We recently reported that nitric oxide (NO) induces posttranscriptional modulation of lung endothelial cell NO synthase (ecNOS) that results in loss of activity. The loss of activity can be reversed by the redox regulatory proteins thioredoxin (Thx)/thioredoxin reductase (Thx-R). The present study was designed to examine whether diminished expression of endogenous Thx and Thx-R may account for regulation of ecNOS activity in NO-exposed cells and whether overexpression of Thx can prevent NO-induced reduction of ecNOS activity in cultured porcine pulmonary artery endothelial cells (PAEC). Exposure to 8.5 ppm NO gas for 24 h resulted in an 80% decrease of Thx and a 27% decrease of Thx-R mRNA expression. Similarly, NO exposure caused 30 and 50% reductions in Thx and Thx-R protein mass, respectively. This NO-induced decrease in the expression of Thx-R mRNA and protein was accompanied by a significant ( P < 0.05) decrease in the catalytic activity of Thx-R but not of glutaredoxin or the cellular levels of reduced glutathione and oxidized glutathione. Overexpression of Thx gene in PAEC was achieved by transient transfection of these cells with pcDNA 3.1 vector inserted in sense or antisense (native) orientation in a human Thx cDNA. Thx mRNA and protein contents in transfected cells were four- and threefold higher, respectively, than those in native PAEC. Exposure of native cells to 10 μM NO solution for 30 min resulted in a significant ( P < 0.01) loss of ecNOS activity, whereas ecNOS activity was comparable in Thx-overexpressed cells with or without NO exposure. These results demonstrate that NO exposure results in diminished expression of Thx and Thx-R in PAEC. Endogenous levels of Thx are critical to restoring the NO-induced loss of ecNOS activity because overexpression of Thx prevented the NO-induced loss of ecNOS catalytic activity. These results also demonstrate that NO modulation of ecNOS and Thx proteins is regulated by a physiologically relevant redox mechanism.
Altered expression and localization of 5-lipoxygenase accompany macrophage differentiation in the lung
Authors: Covin Randal B. , Brock Thomas G. , Bailie Marc B. , Peters-Golden Marc
Published: December 2017

The alveolar macrophage (AM) exhibits a greater capacity to synthesize bioactive leukotrienes from arachidonic acid than does its circulating precursor the peripheral blood monocyte. Macrophage differentiation in the lung entails cellular residence within both the pulmonary interstitial and alveolar compartments. In the present study, we sought to determine 1) whether this enhanced metabolic activity was acquired during maturation within the alveolar space and 2) the underlying mechanisms responsible for this upregulation. Rat AMs were separated by Percoll gradient centrifugation into four density-defined subpopulations thought to reflect their degree of maturation. On stimulation with a calcium ionophore, synthesis of leukotriene B4increased with the degree of maturation, although it was diminished in the oldest subpopulation. This maturation-dependent upregulation was not explained by increases in arachidonic acid release but was associated with increased expression of 5-lipoxygenase (5-LO) protein as determined by immunoblot analysis. Whereas 5-LO is primarily cytosolic in monocytes, it is known to be primarily intranuclear in unfractionated AMs. Here, the localization of 5-LO was investigated by immunofluorescence microscopy and was found to be predominantly nuclear in all AM subpopulations; by contrast, the protein was cytosolic in interstitial macrophages isolated by mechanical and enzymatic lung digestion. These divergent localization patterns in AMs and interstitial macrophages were verified in situ by immunohistochemical staining of sections of normal rat lung. When unfractionated AMs were isolated and maintained in culture for 3 days, a shift in 5-LO distribution from nucleus to cytosol was observed. We conclude that 1) nuclear import of 5-LO occurs within the alveolar space and is reversible on removal from the alveolar milieu and 2) leukotriene synthetic capacity increases further during AM residence within the alveolar space as a result of a progressive increase in the amount of 5-LO protein.
Impaired cAMP production in human airway smooth muscle cells by bradykinin: role of cyclooxygenase products
Authors: Pang Linhua , Holland Elaine , Knox Alan J.
Published: December 2017

Interleukin (IL)-1β impairs human airway smooth muscle (ASM) cell cAMP responses to isoproterenol (Iso). We investigated if bradykinin (BK) could cause a similar effect and the role of cyclooxygenase (COX) products in this event, since we have recently reported that BK, like IL-1β, also causes COX-2 induction and prostanoid release in human ASM cells. BK pretreatment significantly attenuated Iso-induced cAMP generation in a time- and concentration-dependent manner. cAMP generation by prostaglandin (PG) E2but not by forskolin was also impaired. The COX inhibitor indomethacin completely prevented the impairment, whereas the selective COX-2 inhibitors NS-398 and nimesulide, protein synthesis inhibitors cycloheximide and actinomycin D, and steroid dexamethasone were all partially effective. The impairment was mimicked by the B2agonist [Tyr(Me)8]BK, the Ca2+ionophore A-23187, and PGE2and prevented by the B2antagonist HOE-140, but anti-IL-1β serum was ineffective. The results indicate that BK impairs human ASM cell responses to Iso, and the effect is largely mediated by B2receptor-related COX product release via both COX isoforms and is independent of IL-1β.
Connective tissue growth factor mRNA expression is upregulated in bleomycin-induced lung fibrosis
Authors: Lasky Joseph A. , Ortiz Luis A. , Tonthat Boihoang , Hoyle Gary W. , Corti Miriam , Athas Grace , Lungarella Giuseppe , Brody Arnold , Friedman Mitchell
Published: December 2017

Connective tissue growth factor (CTGF) is a newly described 38-kDa peptide mitogen for fibroblasts and a promoter of connective tissue deposition in the skin. The CTGF gene promotor contains a transforming growth factor-β1 (TGF-β1) response element. Because TGF-β1 expression is upregulated in several models of fibroproliferative lung disease, we asked whether CTGF is also upregulated in a murine lung fibrosis model and whether CTGF could mediate some of the fibrogenic effects associated with TGF-β1. A portion of the rat CTGF gene was cloned and used to show that primary isolates of both murine and human lung fibroblasts express CTGF mRNA in vitro. There was a greater than twofold increase in CTGF expression in both human and murine lung fibroblasts 2, 4, and 24 h after the addition of TGF-β1 in vitro. A bleomycin-sensitive mouse strain (C57BL/6) and a bleomycin-resistant mouse strain (BALB/c) were given bleomycin, a known lung fibrogenic agent. CTGF mRNA expression was upregulated in the sensitive, but not in the resistant, mouse strain after administration of bleomycin. In vivo differences in the CTGF expression between the two mouse strains were not due to an inherent inability of BALB/c lung fibroblasts to respond to TGF-β1 because fibroblasts from untreated BALB/c mouse lung upregulated their CTGF message when treated with TGF-β1 in vitro. These data demonstrate that CTGF is expressed in lung fibroblasts and may play a role in the pathogenesis of lung fibrosis.
Expression of matrix-degrading enzymes in pulmonary vascular remodeling in the rat
Authors: Thakker-Varia Smita , Tozzi Carol A. , Poiani George J. , Babiarz Joanne P. , Tatem Linda , Wilson Frank J. , Riley David J.
Published: December 2017

Exposure of rats to hypoxia causes pulmonary arterial remodeling, which is partly reversible after return to air. We hypothesized that degradation of excess collagen in remodeled pulmonary arteries in the posthypoxic period is mediated by endogenous matrix metalloproteinases (MMPs). Total proteolytic, collagenolytic, and gelatinolytic activities, levels of stromelysin-1 and tissue inhibitor of metalloprotease-1 (TIMP-1), and immunolocalization of stromelysin-1 in main pulmonary arteries were determined after exposure of rats to 10% O2for 10 days followed by normoxia. We observed transient increases in total proteolytic, collagenolytic, and gelatinolytic activities and expression of ∼72-, 68-, and 60-kDa gelatinases by zymography within 3 days of cessation of hypoxic exposure. The level of TIMP-1 increased as the stromelysin-1 level increased. Immunoreactive stromelysin-1 was localized predominantly in the luminal region of normal and hypertensive pulmonary arteries. These results are consistent with the notion that endogenous MMPs may mediate the breakdown of excess collagen in remodeled pulmonary arteries during the early posthypoxic period.
Impact of β-adrenergic agonist on Na+channel and Na+-K+-ATPase expression in alveolar type II cells
Authors: Minakata Yoshiaki , Suzuki Satoshi , Grygorczyk Czeslawa , Dagenais André , Berthiaume Yves
Published: December 2017

It has been shown that short-term (hours) treatment with β-adrenergic agonists can stimulate lung liquid clearance via augmented Na+transport across alveolar epithelial cells. This increase in Na+transport with short-term β-agonist treatment has been explained by activation of the Na+channel or Na+-K+-ATPase by cAMP. However, because the effect of sustained stimulation (days) with β-adrenergic agonists on the Na+transport mechanism is unknown, we examined this question in cultured rat alveolar type II cells. Na+-K+-ATPase activity was increased in these cells by 10−4M terbutaline in an exposure time-dependent manner over 7 days in culture. This increased activity was also associated with an elevation in transepithelial current that was inhibited by amiloride. The enzyme’s activity was also augmented by continuous treatment with dibutyryl-cAMP (DBcAMP) for 5 days. This increase in Na+-K+-ATPase activity by 10−4M terbutaline was associated with an increased expression of α1-Na+-K+-ATPase mRNA and protein. β-Adrenergic agonist treatment also enhanced the expression of the α-subunit of the epithelial Na+channel (ENaC). These increases in gene expression were inhibited by propranolol. Amiloride also suppressed this long-term effect of terbutaline and DBcAMP on Na+-K+-ATPase activity. In conclusion, β-adrenergic agonists enhance the gene expression of Na+-K+-ATPase, which results in an increased quantity and activity of the enzyme. This heightened expression is also associated with augmented ENaC expression. Although the cAMP system is involved, the inhibition of enhanced enzyme activity with amiloride suggests that increased Na+entry at the apical surface plays a role in this process.
Epoxyeicosatrienoic acids relax airway smooth muscles and directly activate reconstituted KCachannels
Authors: Dumoulin Marc , Salvail Dany , Gaudreault Sophie B. , Cadieux Alain , Rousseau Eric
Published: December 2017

Epoxyeicosatrienoic acids (EETs) relax various smooth muscles by increasing outward K+movement, but the molecular mode of action of EET regioisomers remains to be clarified. The effects of EETs were investigated on bovine airway smooth muscle tone and on reconstituted Ca2+-activated K+(KCa) channels. 5,6-EET and 11,12-EET induced dose-dependent relaxations of precontracted bronchial spirals. These effects were partly abolished by 10 nM iberiotoxin. Bilayer experiments have shown that 0.1–10 μM 11,12-EET produced up to fourfold increases in the open probability of KCachannels from the cis (extracellular) side by enhancing the mean open time constant and reducing the long closed time constant, without affecting the unitary conductance. EET-induced activations were blocked by 10 nM iberiotoxin. Addition of vehicles or other lipids as well as of GTP and guanosine 5′- O-(3-thiotriphosphate) in the absence of EET had no effect on channel activity. Thus EETs directly activate KCachannels from airway smooth muscle through an interaction with the extracellular face of the channel. We propose that EETs could represent candidate molecules as epithelium-derived hyperpolarizing factors.
Liposome-mediated transfection of fetal lung epithelial cells: DNA degradation and enhanced superoxide toxicity
Authors: Tanswell A. Keith , Staub Olivier , Iles Richard , Belcastro Rosetta , Cabacungan Judy , Sedlackova Larisa , Steer Brent , Wen Yanxia , Hu Jim , O’Brodovich Hugh
Published: December 2017

Cationic liposomes, 1:1 (mol/mol) 1,2-dioleoyldimethylammonium chloride-1,2-dioleoyl- sn-glycero-3-phosphoethanolamine, were used to transfect primary cultures of distal rat fetal lung epithelial cells with pCMV4-based plasmids. A DNA-to-lipid ratio of 1:10 to 1:15 (wt/wt) optimized DNA uptake over a 24-h exposure. At a fixed DNA-to-lipid ratio of 1:15, chloramphenicol acetyltransferase (CAT) reporter gene expression declined at lipid concentrations > 2.5 nmol/cm2 cell surface area, whereas DNA uptake remained concentration dependent. CAT expression peaked 48 h after removal of the liposome-DNA complex, declining thereafter. Reporter gene expression was increased, and supercoiled cDNA degradation was reduced by the addition of 0.2 mM nicotinamide and 10 μM chloroquine. Rat fetal lung epithelial cells transfected with two different expression cassettes had an increased susceptibility to superoxide-mediated cytotoxicity. This could be attributed to a nonspecific delivery of exogenous DNA or some other copurified factor. The DNA-dependent increase in superoxide-mediated cytotoxicity, but not basal levels of cytotoxicity, was inhibited by the addition of 0.2 mM nicotinamide and 10 μM chloroquine.
Hypoxia-specific upregulation of calpain activity and gene expression in pulmonary artery endothelial cells
Authors: Zhang Jianliang , Patel Jawaharlal M. , Block Edward R.
Published: December 2017

The effects of exposure to hypoxia on the catalytic activity and mRNA expression of calpain, a calcium-regulated neutral cysteine protease, were examined in porcine pulmonary artery endothelial cells (PAECs). Specificity of the response to hypoxia was determined by comparing the effects of hypoxic exposure with exposure to oxidants such as nitrogen dioxide (NO2) and nitric oxide (NO), as well as to the sulfhydryl reactive chemical acrolein. Exposure of cells to hypoxia (0% O2) for 1 and 12 h significantly increased catalytic activity ( P < 0.01 for both 1 and 12 h vs. control cells), as well as mRNA expression ( P < 0.01 for 1 h and P < 0.05 for 12 h vs. control cells) of calpain. With more prolonged exposure to 24 h of hypoxia, calpain activity remained significantly elevated, whereas calpain mRNA expression returned to the control level. Calpain activities in cells exposed to NO2[5 parts/million (ppm)] or NO (7.5 ppm) for 1 h or to acrolein (5 μM) for 1 and 24 h were unchanged. However, calpain activities in cells exposed to NO2or NO for 24 h were significantly ( P < 0.05) reduced compared with control cells. The hypoxia-induced increases in calpain mRNA content were prevented by the transcriptional inhibitor actinomycin D and by calpain inhibitor I. In addition, hypoxia increased the degradation of nuclear factor-κB (NF-κB) inhibitor IκB and enhanced the translocation of the p50 subunit of NF-κB to the nuclear membrane. Pretreatment with the calpain-specific inhibitor E-64d prevented hypoxia-induced mRNA expression and degradation of IκBα, as well as translocation of p50 subunit to the nuclear membrane. These results demonstrate for the first time that hypoxia upregulates calpain activity and mRNA expression in PAECs and that the upregulation is specific to hypoxia. Upregulation appears to involve activation of the transcription factor NF-κB.
Cigarette smoke increases amosite asbestos fiber binding to the surface of tracheal epithelial cells
Authors: Churg A. , Sun J.-P. , Zay K.
Published: December 2017

Binding of asbestos fibers to the cell surface appears to be important in the initiation of intracellular signaling events as well as in initiation of particle uptake by the cell. We have previously shown that cigarette smoke increases the uptake of asbestos fibers by tracheal epithelial cells in explant culture. Whether smoke acts by increasing surface binding of fibers is not known. In this study, we exposed rat tracheal explants to air or cigarette smoke and then to a suspension of amosite asbestos. Explants were harvested after 1 or 24 h of dust exposure and washed by repeated dips in culture medium to remove loosely bound fibers, and the number of fibers adhering to the apical cell surfaces was determined by scanning electron microscopy. Smoke-exposed explants retained significantly more surface fibers than air-exposed explants. After four washes, binding levels were similar at 1 and 24 h. The smoke effect was still present when incubations were carried out at 4°C, but binding was decreased ∼25%. Preincubation of the asbestos fibers with iron chloride to increase surface iron increased fiber binding in both air- and smoke-exposed explants, whereas preincubation of the fibers with the iron chelator deferoxamine decreased binding after air exposure and completely eliminated the smoke effect. Inclusion of mannitol or catalase in the medium or preincubation of the explants with GSH produced decreases in binding of 10–25% in air-exposed explants and generally greater decreases in smoke-exposed explants. We conclude that 1) amosite binding is a very rapid process that does not require active cellular metabolism, 2) cigarette smoke increases adhesion of fibers to the epithelial surfaces, and 3) iron on the asbestos fiber appears to play an important role in binding, probably through an active oxygen species-mediated process.
E-selectin expression in human endothelial cells by TNF-α-induced oxidant generation and NF-κB activation
Authors: Rahman Arshad , Kefer John , Bando Masashi , Niles Walter D. , Malik Asrar B.
Published: December 2017

Because reactive oxygen species (ROS) can function as second messengers and regulate the activation of the transcription factor nuclear factor (NF)-κB, we investigated the possible role of tumor necrosis factor-α (TNF-α)-induced ROS generation in endothelial cells in signaling E-selectin gene transcription. We demonstrated that stimulation of human pulmonary artery endothelial cells with TNF-α (100 U/ml) resulted in ROS production using the oxidant-sensitive dye 5 (and 6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate bis(acetoxymethyl)ester. Pretreatment with N-acetyl-l-cysteine (NAC) or pyrrolidine dithiocarbamate (PDTC) for 0.5 h inhibited TNF-α-induced generation of ROS as well as activation of NF-κB and E-selectin mRNA and the cell surface protein expression. These findings indicate that TNF-α induces NF-κB activation and the resultant E-selectin gene expression by a pathway that involves formation of ROS and that E-selectin expression can be inhibited by the antioxidant action of NAC or PDTC. The results support the hypothesis that generation of ROS in endothelial cells induced by proinflammatory cytokines such as TNF-α is a critical signal mediating E-selectin expression.
Induction of ferritin and heme oxygenase-1 by endotoxin in the lung
Authors: Carraway M. S. , Ghio A. J. , Taylor J. L. , Piantadosi C. A.
Published: December 2017

Heme oxygenase (HO)-1 expression is increased by forms of oxidative stress that also induce ferritin. Even though this could result from release of iron by heme degradation, we hypothesized that ferritin expression in the lung after endotoxin [lipopolysaccharide (LPS)] would occur independently of HO-1 because iron sequestration is an important response to infection. We tested this hypothesis by instilling saline or LPS (1 mg) into lungs of rats and measuring ferritin expression, HO-1 expression and activity, and HO-1 and ferritin mRNAs at different times. Lungs were also inflation fixed for immunohistochemistry for HO-1 and ferritin. Studies were performed with and without the HO inhibitor tin protoporphyrin. Ferritin and HO-1 labeling were minimal (macrophages only) in control lungs. By 4 h after LPS instillation, ferritin staining was present in bronchial epithelium and macrophages, became diffuse at 16 h, and was nearly gone by 48–72 h. HO-1 was detectable in macrophages 4 and 16 h after LPS instillation, increased in macrophages and bronchial epithelium at 24 h, and diffusely increased in bronchial epithelium and the alveolar region at 48–72 h. Lung ferritin content increased significantly by 4 h and peaked at 16 h before declining. HO-1 protein was present by Western blot in control lung, stable at 4 h, and increased by 24 h after LPS instillation, whereas HO enzyme activity had increased by 4 h after LPS instillation. After complete inhibition of HO enzyme activity with tin protoporphyrin, ferritin increased threefold at 4 h and sixfold at 24 h after LPS instillation. HO-1 mRNA increased by 4 h and was sustained at 24 h, whereas ferritin mRNA did not change after LPS instillation. These results indicate that intratracheal LPS rapidly induces ferritin protein in the lung independently of its mRNA synthesis or HO enzyme activity. LPS induces HO-1 mRNA, which is followed by increased expression of protein.
Interleukin-6 production in hemorrhagic shock is accompanied by neutrophil recruitment and lung injury
Authors: Hierholzer Christian , Kalff Jörg C. , Omert Laurel , Tsukada Katsuhiko , Loeffert J. Eric , Watkins Simon C. , Billiar Timothy R. , Tweardy David J.
Published: December 2017

Hemorrhagic shock (HS) initiates an inflammatory cascade that includes the production of cytokines and recruitment of neutrophils (PMN) and may progress to organ failure, inducing acute respiratory distress syndrome (ARDS). To examine the hypothesis that interleukin-6 (IL-6) contributes to PMN infiltration and lung damage in HS, we examined the lungs of rats subjected to unresuscitated and resuscitated HS for the production of IL-6 and activation of Stat3. Using semiquantitative RT-PCR, we found a striking increase in IL-6 mRNA levels only in resuscitated HS, with peak levels observed 1 h after initiation of resuscitation. Increased IL-6 protein expression was localized to bronchial and alveolar cells. Electrophoretic mobility shift assay of protein extracts from shock lungs exhibited an increase in Stat3 activation with kinetics similar to IL-6 mRNA. In situ DNA binding assay determined Stat3 activation predominantly within alveoli. Intratracheal instillation of IL-6 alone into normal rats resulted in PMN infiltration into lung interstitium and alveoli, marked elevation of bronchoalveolar lavage cellularity, and increased wet-to-dry ratio. These findings indicate that IL-6 production and Stat3 activation occur early in HS and may contribute to PMN-mediated lung injury, including ARDS after HS.
Laminin 2 attachment selects myofibroblasts from fetal mouse lung
Authors: Flores-Delgado Guillermo , Bringas Pablo , Warburton David
Published: December 2017

Laminins (LNs) are extracellular matrix glycoproteins that are involved in cell adhesion, proliferation, and differentiation. So far, 11 LN variants (LN1 to LN11) have been described. In the lung, at least six LN variants have been identified. However, only the role of LN1 has been characterized to any extent. In this study, we hypothesized that the LN2 variant may play a role during lung development. We identified, by RT-PCR analysis, that the α2-chain mRNA of LN2 is expressed during mouse lung development. LN2 adhesion assays were then performed with cells from fetal mouse lung primary cultures. Our results showed that a specific subpopulation of fetal lung cells that expressed vimentin, α-smooth muscle actin, and desmin attached onto LN2, whereas the cells that did not adhere to LN2 as well as the total cell population were able to adhere readily on fibronectin. Cell attachment onto LN2 was inhibited by EDTA. In addition, we demonstrated, by RT-PCR and Western analysis, that the LN2-adherent cells autoexpressed the α2-chain of LN2. In the late pseudoglandular period, LN2 was localized by immunohistochemistry in the basement membrane of airways and blood vessels and around mesenchymal cells. We conclude that LN2 is expressed during lung development and that a specific subpopulation of fetal lung mesenchymal cells expressing a myofibroblastic phenotype can be selected by attachment to LN2 in primary culture. These findings lead us to speculate that LN2 may play a key role in the cell biology of myofibroblasts during lung development.
Involvement of lung interstitial proteoglycans in development of hydraulic- and elastase-induced edema
Authors: Passi Alberto , Negrini Daniela , Albertini Riccardo , De Luca Giancarlo , Miserocchi Giuseppe
Published: December 2017

We extracted and isolated proteoglycans from lung tissue samples obtained from three groups of anesthetized rabbits: 1) control animals (C; n= 8) killed by overdose after 180 min; 2) animals receiving an intravenous saline infusion (S; n = 4, 1.5 ml ⋅ kg-1⋅ min-1) for 180 min; 3) animals receiving an intravenous bolus of 200 μg of pancreatic elastase (E; n = 4), killed after 200 min. The lung dry weight-to-wet weight ratio in the three groups was 5.2 ± 0.2, 6.0 ± 0.4, and 5.6 ± 0.5, respectively. Gel-filtration analysis showed a massive fragmentation of the versican family of the extracellular matrix (ECM) in the S groups and a marked degradation of heparan sulfate-containing proteoglycans, including perlecan of the basement membrane, in the E group. The binding properties of total proteoglycans to other ECM components were lowered in both groups relative to control. The decrease in proteoglycan binding was more pronounced for collagen type IV in the E group relative to C (−93.5%, P < 0.05) and for hyaluronic acid in the S groups (−85.8%, P < 0.05). These findings suggest that elastase treatment produces a major degree of damage to the organization of basement membrane, whereas saline loading affects more markedly the architecture of interstitial ECM. Qualitative zymography performed on lung extracts showed increased gelatinase activities in both S and E groups, providing direct evidence that the activation of tissue proteinases may play a role in acute lung injury.
Surfactant protein A inhibits T cell proliferation via its collagen-like tail and a 210-kDa receptor
Authors: Borron Paul , McCormack Francis X. , Elhalwagi Baher M. , Chroneos Zissis C. , Lewis James F. , Zhu Sha , Wright Jo Rae , Shepherd Virginia L. , Possmayer Fred , Inchley Kevin , Fraher Laurence J.
Published: December 2017

Investigation of possible mechanisms to describe the hyporesponsiveness of pulmonary leukocytes has led to the study of pulmonary surfactant and its constituents as immune suppressive agents. Pulmonary surfactant is a phospholipid-protein mixture that reduces surface tension in the lung and prevents collapse of the alveoli. The most abundant protein in this mixture is a hydrophilic molecule termed surfactant-associated protein A (SP-A). Previously, we showed that bovine (b) SP-A can inhibit human T lymphocyte proliferation and interleukin-2 production in vitro. Results presented in this investigation showed that different sources of human SP-A and bSP-A as well as recombinant rat SP-A inhibited human T lymphocyte proliferation in a dose-dependent manner. A structurally similar collagenous protein, C1q, did not block the in vitro inhibitory action of SP-A. The addition of large concentrations of mannan to SP-A-treated cultures also did not disrupt inhibition, suggesting that the effect is not mediated by the carbohydrate recognition domain of SP-A. Use of recombinant mutant SP-As revealed that a 36-amino acid Arg-Gly-Asp (RGD) motif-containing span of the collagen-like domain was responsible for the inhibition of T cell proliferation. A polyclonal antiserum directed against an SP-A receptor (SP-R210) completely blocked the inhibition of T cell proliferation by SP-A. These results emphasize a potential role for SP-A in dampening lymphocyte responses to exogenous stimuli. The data also provide further support for the concept that SP-A maintains a balance between the clearance of inhaled pathogens and protection against collateral immune-mediated damage.
Nitric oxide inhibits lipopolysaccharide-induced apoptosis in pulmonary artery endothelial cells
Authors: Ceneviva Gary D. , Tzeng Edith , Hoyt Dale G. , Yee Emily , Gallagher Alicia , Engelhardt John F. , Kim Young-Myeong , Billiar Timothy R. , Watkins Simon A. , Pitt Bruce R.
Published: December 2017

Our group recently reported that cultured sheep pulmonary artery endothelial cells (SPAECs) became resistant to lipopolysaccharide (LPS)-induced apoptosis several days after constitutive synthesis of nitric oxide (NO) after adenoviral (Ad) transfer of inducible NO synthase (iNOS) or exposure to the NO donor S-nitroso- N-acetylpenicillamine (SNAP) (E. Tzeng, Y.-M. Kim, B. R. Pitt, A. Lizonova, I. Kovesdi, and T. R. Billiar. Surgery 122: 255–263, 1997). In the present study, we confirmed this observation by establishing stable transfectants after retroviral gene transfer [replication-deficient retrovirus (DFG)] of human iNOS (DFG-iNOS) SPAECs and then used all three approaches (Ad, DFG, and SNAP) to determine underlying mechanisms of this phenomenon. Continuous endogenous production of NO in itself did not cause apoptosis as assessed by phase-contrast microscopy, nuclear morphology, and internucleosomal DNA fragmentation. Prolonged (72–96 h) synthesis of NO, however, after DFG- or replication-deficient adenovirus (Ad.CMV)-iNOS or SNAP (100 μM, 96 h) inhibited LPS-induced apoptosis. The kinetics of such protection suggested that NO may be inducing other gene products. Ad-mediated transfer of manganese superoxide dismutase (MnSOD) decreased the sensitivity of wild-type SPAECs to LPS-induced apoptosis. MnSOD, however, was not induced in an NG-monomethyl-l-arginine (l-NMMA)-sensitive time-dependent fashion after Ad.CMV-iNOS. Other inducible genes that may be affected by NO and that may protect against potential oxidant-mediated LPS-induced apoptosis including 70-kDa heat shock protein, heme oxygenase-1, metallothionein, and Bcl-2 also were not elevated in an l-NMMA-sensitive, time-dependent fashion. Although the candidate gene product underlying NO-induced protection remains unclear, we did note that prolonged synthesis of NO inhibited LPS-induced activation of an interleukin-1β-converting enzyme-like cysteine protease (cysteine protease protein-32-like) in a dithiothreitol-sensitive fashion, suggesting that S-nitrosylation of an important downstream target of convergence of apoptotic signals may contribute to the sensitivity of SPAECs to LPS.
Involvement of cytochromeP-450 enzyme activity in the control of microvascular permeability in canine lung
Authors: Ivey Claire L. , Stephenson Alan H. , Townsley Mary I.
Published: December 2017

Products of cytochrome P-450 enzymes may play a role in capacitative Ca2+entry in endothelial cells, which can promote a rise in vascular permeability. Thapsigargin (150 nM) stimulated capacitative Ca2+entry and increased the capillary filtration coefficient ( Kf,c) in isolated normal canine lung lobes. Pretreatment of the lobes with cytochrome P-450 inhibitors clotrimazole (10 μM) or 17-octadecynoic acid (5 μM) abolished the thapsigargin-induced increases in Kf,c. Because clotrimazole also blocks Ca2+-activated K+channels, the K+-channel blocker tetraethylammonium (10 mM) was used to ensure that permeability was not influenced by this mechanism. Tetraethylammonium did not affect thapsigargin-induced permeability. The effects of the cytochrome P-450 arachidonic acid metabolite 5,6-epoxyeicosatrienoic acid (EET) were also investigated in lobes taken from control dogs and dogs with pacing-induced heart failure (paced at 245 beats/min for 4 wk). 5,6-EET (10 μM) significantly increased Kf,cin lobes from the control but not from the paced animals. We conclude that cytochrome P-450 metabolites are involved in mediating microvascular permeability in normal canine lungs, but an absence of 5,6-EET after heart failure does not explain the resistance of lungs from these animals to permeability changes.