bluuebunny/crossref_metadata_2025 huggingface dataset:
NO2-induced generation of extracellular reactive oxygen is mediated by epithelial lining layer antioxidants
Authors: Velsor Leonard W. , Postlethwait Edward M.
Published: December 2017

Nitrogen dioxide (NO2) is an environmental oxidant that causes acute lung injury. Absorption of this aqueous insoluble gas into the epithelial lining fluid (ELF) that covers air space surfaces is, in part, governed by reactions with ELF constituents. Consequently, NO2absorption is coupled to its chemical elimination and the formation of ELF-derived products. To investigate mechanisms of acute epithelial injury, we developed a model encompassing the spatial arrangements of the lung surface wherein oxidation of cell membranes immobilized below a chemically defined aqueous compartment was assessed after NO2exposures. Because aqueous-phase unsaturated fatty acids displayed minimal NO2absorptive activity, these studies focused on glutathione (GSH) and ascorbic acid (AH2) as the primary NO2absorption substrates. Results demonstrated that membrane oxidation required both gas-phase NO2and aqueous-phase GSH and/or AH2. Membrane oxidation was antioxidant concentration and exposure duration dependent. Furthermore, studies indicated that GSH- and AH2-mediated NO2absorption lead to the production of the reactive oxygen species (ROS)[Formula: see text] and H2O2but not to ⋅ OH and that Fe-O2complexes likely served as the initiating oxidant. Similar results were also observed in combined systems (GSH + AH2) and in isolated rat ELF. These results suggest that the exposure-induced prooxidant activities of ELF antioxidants generate extracellular ROS that likely contribute to NO2-induced cellular injury.
Nitric oxide and peroxynitrite-mediated pulmonary cell death
Authors: Gow Andrew J. , Thom Stephen R. , Ischiropoulos Harry
Published: December 2017

Nitric oxide (⋅ NO) can be produced within the lung, and recently inhaled nitric oxide has been used as a therapeutic agent. Peroxynitrite1(ONOO−), the product of the nearly diffusion-limited reaction between ⋅ NO and superoxide, may represent the proximal reactive species mediating ⋅ NO injury to pulmonary cells. To investigate the physiological and pathological reactivities of ⋅ NO and ONOO−at the molecular and cellular levels, bovine pulmonary artery endothelial cells (BPAEC) and rat type II epithelial cells were exposed to ⋅ NO (0.01–2.5 μM/min for 2 h) generated by spermine-NONOate and papa-NONOate and to the same fluxes of ONOO−generated by 1,3-morpholinosydnonimine (SIN-1). Exposure to SIN-1 resulted in cellular injury and death in both cell types. Epithelial cells displayed a concentration-dependent loss of cellular viability within 8 h of exposure. In contrast, BPAEC loss of cellular viability was evident after 18 h postexposure. Events preceding cell death in BPAEC include depolarization of the mitochondrial membrane, evident as early as 6 h postexposure, loss of cellular redox activity at 16 h, and DNA fragmentation detected by in situ staining at 18 h after exposure. Exposure of BPAEC to ⋅ NO did not affect the cellular viability, but type II cells were injured in a manner similar to ONOO−exposure. ⋅ NO-mediated cellular injury within type II cells was reduced by preincubation with N-acetylcysteine. The data imply that the pathological and physiological effects of ⋅ NO may be regulated by its reactions with superoxide and reduced thiols.
Regulation of lung fibroblast tropoelastin expression by alveolar epithelial cells
Authors: Mariani Thomas J. , Dunsmore Sarah E. , Li Qinglang , Ye Xueming , Pierce Richard A.
Published: December 2017

Epithelial-mesenchymal interactions are of critical importance during tissue morphogenesis and repair. Although the cellular and molecular aspects of many of these interactions are beginning to be understood, the ability of epithelial cells to regulate fibroblast interstitial matrix production has not been extensively studied. We report here that cultured alveolar epithelial cells are capable of modulating the expression of tropoelastin, the soluble precursor of the interstitial lung matrix component elastin, by lung fibroblasts. Phorbol ester-stimulated alveolar epithelial cells secrete a soluble factor that causes a time- and dose-dependent repression of lung fibroblast tropoelastin mRNA expression. This alveolar epithelial cell-mediated repressive activity is specific for tropoelastin, is effective on lung fibroblasts from multiple stages of development, and acts at the level of transcription. Partial characterization of the repressive activity indicates it is an acid-stable, pepsin-labile protein. Gel fractionation of alveolar epithelial cell conditioned medium revealed two peaks of activity with relative molecular masses of ∼25 and 50 kDa. These data support a role for epithelial cells in the regulation of fibroblast interstitial matrix production.
Degradation of surfactant protein D by alveolar macrophages
Authors: Dong Qun , Wright Jo Rae
Published: December 2017

Surfactant protein (SP) D is a pulmonary surfactant-associated protein that may function in lung host defense. SP-D is produced by alveolar type II cells and nonciliated bronchiolar epithelial (Clara) cells of the airway and is secreted into the air space. Here we investigated whether alveolar macrophages degraded SP-D in vitro. We also examined the effects of SP-A and lipids on SP-D metabolism. The results showed that alveolar macrophages bound and degraded SP-D in a time- and temperature-dependent fashion. After 100 min of incubation, the formation of trichloroacetic acid-soluble degradation products increased 4-fold in the medium and 30-fold in the cells. The degradation of SP-D was via a cell-associated process because SP-D was not degraded when incubated in medium previously conditioned by alveolar macrophages. Gel autoradiography of cell lysate samples after incubation with125I-labeled SP-D demonstrated an increase in degradation products, further confirming the degradation of SP-D by alveolar macrophages. In addition, the degradation of SP-D was not affected by coincubation with SP-A or surfactant-like liposomes containing either phosphatidylglycerol or phosphatidylinositol. In conclusion, alveolar macrophages rapidly degrade SP-D and may play an important role in SP-D turnover and clearance.
Differential regulation of SP-A1 and SP-A2 genes by cAMP, glucocorticoids, and insulin
Authors: Kumar A. R. , Snyder J. M.
Published: December 2017

In the human fetal lung, surfactant protein A (SP-A) is encoded by two highly similar genes, SP-A1 and SP-A2, which are developmentally and hormonally regulated. Using primer extension analysis, we evaluated the levels of SP-A1 and SP-A2 mRNA transcripts in human fetal lung explants and in a human adult lung adenocarcinoma cell line (H441 cells) cultured in the absence or presence of either dibutyryladenosine 3′,5′-cyclic monophosphate (DBcAMP, 1 mM), dexamethasone (10−7M), or insulin (2.5 μg/ml). In the human fetal lung explants, the content of SP-A1 mRNA was approximately four times that of SP-A2 mRNA. DBcAMP increased SP-A1 mRNA levels by 100% and SP-A2 mRNA levels by 500%, thus reducing the ratio of SP-A1 mRNA to SP-A2 mRNA to ∼1:1. Dexamethasone inhibited all of the SP-A1 and SP-A2 mRNA transcripts to the same extent, by ∼70%, whereas insulin inhibited all SP-A mRNA transcripts by ∼60%. The ratio of SP-A1 to SP-A2 mRNA in dexamethasone- or insulin-treated explants was the same as the ratio observed in controls. In the H441 cells, SP-A1 mRNA levels were ∼1.5 times that of SP-A2 mRNA levels. DBcAMP increased both SP-A1 and SP-A2 mRNA levels by 100%. Dexamethasone inhibited SP-A1 mRNA levels in the cell line by 60%, whereas SP-A2 mRNA levels were not significantly affected. Insulin inhibited SP-A1 mRNA levels in the cell line by 40% without affecting SP-A2 mRNA levels. These findings suggest that the two human SP-A genes are regulated differently in the two model systems.
Mechanical distension modulates pulmonary alveolar epithelial phenotypic expression in vitro
Authors: Gutierrez Jorge A. , Gonzalez Robert F. , Dobbs Leland G.
Published: December 2017

The pulmonary alveolar epithelium is composed of two distinct types of cells, type I and type II cells, both of which are critical for normal lung function. On the basis of experiments of both nature and in vivo studies, it has been hypothesized that expression of the type I or type II phenotype is influenced by mechanical factors. We have investigated the effects of mechanical distension on the expression of specific markers for the type I and type II cell phenotypes in cultured alveolar type II cells. Rat alveolar type II cells were tonically mechanically distended in culture. Cells were analyzed for a marker for the type I phenotype (rTI40, an integral membrane protein specific for type I cells) and for markers for the type II phenotype [surfactant protein (SP) A, SP-B, and SP-C] as well as for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Mechanical distension caused a 68 ± 25% ( n = 3) increase in mRNA content of rTI40relative to undistended controls. In contrast, mechanical distension resulted in a decrease in mRNA content of SP-B to 35 ± 19% ( n = 3) and of SP-C to 20 ± 6.7% ( n = 3) of undistended controls. There was no effect on mRNA content of SP-A or GAPDH. The differences in mRNA content of SP-B and SP-C were found to be primarily due to changes at the transcriptional level by nuclear run-on assays. The effects on rTI40appear to be due to posttranscriptional events. These data show that mechanical distension influences alveolar epithelial phenotypic expression in vitro, at least in part, at the transcriptional level.
Proteoglycan involvement during development of lesional pulmonary edema
Authors: Negrini Daniela , Passi Alberto , De Luca Giancarlo , Miserocchi Giuseppe
Published: December 2017

We evaluated the effect of pancreatic elastase (7 IU iv) on pulmonary interstitial pressure (Pip) in in situ rabbit lungs by a micropuncture technique through the intact parietal pleura. Pipwas −10.8 ± 2.2 (SD) cmH2O in the control condition, increased to +5.1 ± 1.7 cmH2O at ∼60 min [condition referred to as mild edema (ME)], and subsequently decreased to −0.15 ± 0.8 cmH2O, remaining steady from 80 up to 200 min with a marked increase in lung wet-to-dry weight ratio [condition referred to as severe edema (SE)], suggesting an increase in tissue compliance. We functionally correlated the measured Pipto structural modifications of proteoglycans, the major interfibrillar component of the extracellular matrix (ECM). The strength of the noncovalent bonds linking proteoglycans to other ECM components decreased with increasing severity of edema, as indicated by the increased extractability of proteoglycans with guanidine hydrochloride. Total proteoglycan recovery (expressed as μg hexuronate/g dry tissue) increased from 436.8 ± 14 in the control condition to 495.3 ± 23 and 547.0 ± 10 in ME and SE, respectively. Gel-filtration chromatography showed in ME a fragmentation of heparan sulfate proteoglycans, suggesting that elastase treatment first affected basement membrane integrity, whereas large chondroitin sulfate proteoglycans were degraded only in SE. Elastase caused a fragmentation only of the core protein of proteoglycans, the binding properties of which to collagens, fibronectin, and hyaluronic acid were markedly decreased, as indicated by a solid-phase binding assay. The sequential degradation of heparan sulfate and chondroitin sulfate proteoglycans may account for the initial increase in microvascular permeability, followed by a loss of the native architecture of the ECM, which may be responsible for the increase in tissue compliance.
Release of epithelium-derived PGE2from canine trachea after antigen inhalation
Authors: McGrogan I. , Janssen L. J. , Wattie J. , O’Byrne P. M. , Daniel E. E.
Published: December 2017

To investigate the role of prostaglandin (PG) E2in allergen-induced hyperresponsiveness, dogs inhaled either the allergen Ascaris suum or vehicle (Sham). Twenty-four hours after inhalation, some animals exposed to allergen demonstrated an increased responsiveness to acetylcholine challenge in vivo (Hyp-Resp), whereas others did not (Non-Resp). Strips of tracheal smooth muscle, either epithelium intact or epithelium denuded, were suspended on stimulating electrodes, and a concentration-response curve to carbachol (10−9to 10−5M) was generated. Tissues received electrical field stimulation, and organ bath fluid was collected to determine PGE2content. With the epithelium present, all three groups contracted similarly to 10−5M carbachol, whereas epithelium-denuded tissues from animals that inhaled allergen contracted more than tissues from Sham dogs. In response to electrical field stimulation, Hyp-Resp tissues contracted less than Sham tissues in the presence of epithelium and more than Sham tissues in the absence of epithelium. PGE2release in the muscle bath was greater in Non-Resp tissues than in Sham or Hyp-Resp tissues when the epithelium was present. Removal of the epithelium greatly inhibited PGE2release. We conclude that tracheal smooth muscle is hyperresponsive in vitro after in vivo allergen exposure only when the modulatory effect of the epithelium, largely through PGE2release, is removed.
O2regulates surfactant protein A mRNA transcription and stability in human fetal lung in vitro
Authors: Acarregui Michael J. , Kumar Ashish R. , Penisten Scott T. , Snyder Jeanne M.
Published: December 2017

The effect of O2on surfactant protein (SP) A mRNA transcription and half-life was determined in midtrimester human fetal lung tissue cultured in either 20 (control) or 70% O2. Incubation of tissues in 70% O2resulted in a 133% increase in SP-A mRNA transcription rate compared with control tissues. The SP-A mRNA half-life was increased by 54% in lung tissues cultured in 70% O2vs. control tissues. Western blot analysis indicated a threefold increase in SP-A in the 70% O2condition, demonstrating that O2regulation of SP-A mRNA levels results in corresponding changes in SP-A levels. Primer extension assays were performed to determine whether the observed increase in SP-A mRNA levels is secondary to the preferential expression of one of the human SP-A genes, SP-A1 or SP-A2. Transcripts of both the SP-A1 and SP-A2 genes were increased ∼100% in tissues maintained in 70% O2compared with control tissues. These data demonstrate that O2regulates human SP-A mRNA levels by both transcriptional and posttranscriptional mechanisms. Furthermore, because there is no differential effect of O2on the expression of SP-A1 vs. SP-A2 mRNA, the properties of these genes that mediate regulation by O2must be conserved between the two genes.
Nitric oxide inhibits Na+absorption across cultured alveolar type II monolayers
Authors: Guo Yi , Duvall Michael D. , Crow John P. , Matalon Sadis
Published: December 2017

We examined the mechanisms by which nitric oxide (⋅ NO) decreased vectorial Na+transport across confluent monolayers of rat alveolar type II (ATII) cells grown on permeable supports. Amiloride (10 μM) applied to the apical side of monolayers inhibited ∼90% of the equivalent ( Ieq) and the short-circuit ( Isc) current, with an half-maximal inhibitory concentration (IC50) of 0.85 μM, indicating that Na+entry into ATII cells occurred through amiloride-sensitive Na+channels. ⋅ NO generated by spermine NONOate and papa NONOate added to both sides of the monolayers decreased Ieqand increased transepithelial resistance in a concentration-dependent fashion (IC50= 0.4 μM ⋅ NO). These changes were prevented or reversed by addition of oxyhemoglobin (50 μM). Incubation of ATII monolayers with 8-bromoguanosine 3′,5′-cyclic monophosphate (400 μM) had no effect on transepithelial Na+transport. When the basolateral membranes of ATII cells were permeabilized with amphotericin B (10 μM) in the presence of a mucosal-to-serosal Na+gradient (145:25 mM), ⋅ NO (generated by 100 μM papa NONOate) inhibited ∼60% of the amiloride-sensitive Isc. In addition, after permeabilization of the apical membranes, ⋅ NO inhibited the Isc[a measure of Na+-K+-adenosinetriphosphatase (ATPase) activity] by ∼60%. We concluded that ⋅ NO at noncytotoxic concentrations decreased Na+absorption across cultured ATII monolayers by inhibiting both the amiloride-sensitive Na+channels and Na+-K+-ATPase through guanosine 3′,5′-cyclic monophosphate-independent mechanisms.
Regulation of the depth of surface liquid in bovine trachea
Authors: Wu D. X.-Y. , Lee C. Y. C. , Uyekubo S. N. , Choi H. K. , Bastacky S. J. , Widdicombe J. H.
Published: December 2017

The luminal surface of airways is lined by a thin film of airway surface liquid (ASL). Physiological regulation of the depth of ASL has not been reported previously. In this paper, we have used low-temperature scanning electron microscopy of rapidly frozen specimens of bovine tracheal epithelium to demonstrate alterations in the depth of ASL in response to the cholinergic agonist methacholine. We first established that methacholine selectively stimulated airway glands, with maximal secretion at ∼2 min and a return to baseline within ∼5 min. A 2-min exposure to methacholine increased the depth of ASL from 23 to 78 μm. Thereafter, depth decreased linearly with time, reaching 32 μm at 30 min. The initial increase in depth was blocked by bumetanide, an inhibitor of active chloride secretion, whereas the slow decline back to baseline was inhibited by amiloride, a blocker of active sodium absorption. We conclude that the methacholine-induced changes in ASL depth reflect transient gland secretion followed by liquid absorption across the surface epithelium.
Synergistic cytotoxicity from nitric oxide and hyperoxia in cultured lung cells
Authors: Narula Pramod , Xu Jing , Kazzaz Jeffrey A. , Robbins Carolyn G. , Davis Jonathan M. , Horowitz. Stuart
Published: December 2017

Exogenous nitric oxide (NO) is being tested clinically for the treatment of pulmonary hypertension in infants and children. In most cases, these patients receive simultaneous oxygen (O2) therapy. However, little is known about the combined toxicity of NO+hyperoxia. To test this potential toxicity, human alveolar epithelial cells (A549 cells) and human lung microvascular endothelial lung cells were cultured in room air (control), hyperoxia (95% O2), NO (derived from chemical donors), or combined hyperoxia+ NO. Control cells grew normally over a 6-day study period. In contrast, cell death from hyperoxia was evident after 4–5 days, whereas cells neither died nor divided in NO alone. However, cells exposed to both NO and hyperoxia began to die on day 2 and died rapidly thereafter. This cytotoxic effect was clearly synergistic, and cell death did not occur via apoptosis. As an indicator of peroxynitrite formation, nitrotyrosine-containing proteins were assayed using anti-nitrotyrosine antibodies. Two protein bands, at molecular masses of 25 and 35 kDa, were found to be increased in A549 cells exposed to NO or NO+hyperoxia. These results indicate that combined NO+hyperoxia has a synergistic cytotoxic effect on alveolar epithelial and lung vascular endothelial cells in culture.
Airway epithelial Fas ligand expression: potential role in modulating bronchial inflammation
Authors: Gochuico Bernadette R. , Miranda Kathleen M. , Hessel Edith M. , De Bie Joris J. , Van Oosterhout Antoon J. M. , Cruikshank William W. , Fine Alan
Published: December 2017

Epithelium-derived Fas ligand is believed to modulate inflammation within various tissues. In this paper, we report findings that suggest a similar immunoregulatory role for Fas ligand in the lung. First, Fas ligand was localized to nonciliated, cuboidal airway epithelial cells (Clara cells) throughout the airways in the normal murine lung by employing nonisotopic in situ hybridization and immunohistochemistry. Second, gldmutant mice, which express a dysfunctional Fas ligand protein, were noted to develop prominent infiltration of inflammatory cells in submucosal and peribronchial regions of the upper and lower airways. Third, during allergic airway inflammation induced by ovalbumin in mice, cell-associated staining for Fas ligand mRNA and protein was markedly reduced in the airway epithelium. These data suggest that Clara cell-derived Fas ligand may control immune activity in the airway; thus alterations in this protective mechanism may be involved in the pathogenesis of certain inflammatory conditions of the airway, such as asthma.
Chronic hypoxia decreases nitric oxide production and endothelial nitric oxide synthase in newborn pig lungs
Authors: Fike Candice D. , Kaplowitz Mark R. , Thomas Carol J. , Nelin Leif D.
Published: December 2017

To examine the effect of chronic hypoxia on nitric oxide (NO) production and the amount of the endothelial isoform of nitric oxide synthase (eNOS) in lungs of newborn piglets, studies were performed using 1- to 3-day-old piglets raised in room air (control) or 10% O2 (chronic hypoxia) for 10–12 days. Exhaled NO output and plasma nitrites and nitrates (collectively termed[Formula: see text]) were measured in anesthetized animals. [Formula: see text]concentrations were measured in the perfusate of isolated lungs. eNOS amounts were assessed in whole lung homogenates. In the intact piglets, exhaled NO outputs and plasma [Formula: see text]were lower in the chronically hypoxic (exhaled NO output = 0.2 ± 0.1 nmol/min; plasma [Formula: see text] = 10.3 ± 3.7 nmol/ml) than in control animals (exhaled NO output = 0.8 ± 0.2 nmol/min; plasma [Formula: see text] = 22.3 ± 4.3 nmol/ml). In perfused lungs, the perfusate accumulation of [Formula: see text] was lower in chronic hypoxia (1.0 ± 0.3 nmol/min) than in control (2.6 ± 0.6 nmol/min) piglets. The amount of whole lung homogenate eNOS from the chronic hypoxia piglets was 40 ± 8% less than that from the control piglets. The reduced NO production observed in anesthetized animals or perfused lungs of chronically hypoxic newborn piglets is consistent with the finding of reduced lung eNOS protein amounts. Decreased NO production might contribute to the development of chronic hypoxia-induced pulmonary hypertension in newborns.
Lung-specific induction of heme oxygenase-1 and hyperoxic lung injury
Authors: Taylor Jennifer L. , Carraway Martha Sue , Piantadosi Claude A.
Published: December 2017

Heme oxygenase (HO)-1, which catalyzes heme breakdown, is induced by oxidative stress and may protect against oxidative injury. We hypothesized that induction of HO-1 by hemoglobin (Hb) in the lung would protect the rat from pulmonary O2toxicity. Rats given intratracheal (IT) Hb showed lung-specific induction of HO-1 by 8 h by Western analysis. Rats were then pretreated for 8 h before 60 h of exposure to 100% O2with either IT normal saline, Hb, or Hb plus the HO-1 inhibitor tin-protoporphyrin (SnPP). Both the Hb+O2and Hb+O2+ SnPP animals had less lung injury than normal saline controls as indicated by lower pleural fluid volumes and wet-to-dry weight ratios ( P < 0.01). The improvement in injury in the two Hb-treated groups was the same despite a 61% decrease in HO enzyme activity in the Hb+SnPP group after 60 h of O2. In addition, inhibition of HO activity with SnPP alone before O2exposure did not augment the extent of hyperoxic lung injury. These results demonstrate that IT Hb induces lung HO-1 in the rat and protects against hyperoxia; however, the protection is not mediated by increased HO enzyme activity.
Cigarette smoke extract inhibits fibroblast-mediated collagen gel contraction
Authors: Carnevali S. , Nakamura Y. , Mio T. , Liu X. , Takigawa K. , Romberger D. J. , Spurzem J. R. , Rennard S. I.
Published: December 2017

Cigarette smoking, the major cause of pulmonary emphysema, is characterized by destruction of alveolar walls. Because tissue destruction represents a balance between injury and repair, we hypothesized that cigarette smoke exposure may contribute to the development of emphysema through the inhibition of tissue contraction during the repair process. To partially evaluate this hypothesis, we investigated the effects of cigarette smoke extract (CSE) on the ability of cultured fibroblasts to mediate collagen gel contraction in vitro: CSE inhibited fibroblast-mediated gel contraction in a concentration-dependent manner ( P < 0.01). Production of prostaglandin E2, a known inhibitor of fibroblast contraction, was unchanged by CSE as was cell surface integrin expression. In contrast, fibronectin production by fibroblasts was inhibited ( P < 0.01), and addition of exogenous fibronectin partially restored the contractile activity, thus suggesting at least one mechanism to explain inhibition of gel contraction by CSE. When CSE was treated to remove volatile components, it showed less inhibitory activity on fibroblast-mediated gel contraction. Therefore, we also examined the effects of acrolein and acetaldehyde, two volatile components of cigarette smoke. Inhibition of contraction was observed at 5 μM acrolein and at 0.5 mM acetaldehyde. In conclusion, cigarette smoke inhibited fibroblast-mediated gel contraction, and this inhibition was due, at least in part, to the volatile components of cigarette smoke and may be mediated, at least in part, by a decrease in fibroblast fibronectin production. By inhibition of repair, these smoke components may contribute to the development of pulmonary emphysema.
Growth factors and dexamethasone regulate Hoxb5 protein in cultured murine fetal lungs
Authors: Chinoy Mala R. , Volpe Maryann V. , Cilley Robert E. , Zgleszewski Steven E. , Vosatka Robert J. , Martin Ana , Nielsen Heber C. , Krummel Thomas M.
Published: December 2017

Studies on lung morphogenesis have indicated a role of homeobox( Hox) genes in the regulation of lung development. In the present study, we attempted to modulate the synthesis of Hoxb5 protein in cultured murine fetal lungs after mechanical or chemical stimuli. Murine fetuses at gestational day 14 (GD14) were removed from pregnant CD-1 mice, and lungs were excised and cultured for 7 days in BGJb media. The experimental groups were 1) untreated, unligated; 2) tracheal ligation; 3) supplemented media with either epidermal growth factor (EGF; 10 ng/ml), transforming growth factor (TGF)-β1 (2 ng/ml), dexamethasone (10 nM), EGF+TGF-β1, or EGF+TGF-β1+dexamethasone. After 3 or 7 days, the cultured lungs were compared with in vivo lungs. Immunoblotting signals at 3 days in culture were stronger than those at 7 days. Western blot analyses showed that ligation, EGF, TGF-β1, and EGF+TGF-β1 downregulated Hoxb5 protein to ∼20–70% of Hoxb5 protein levels in unligated, untreated cultured lungs. Furthermore, dexamethasone alone or in combination with EGF and TGF-β1 downregulated Hoxb5 protein by >90% ( P < 0.05) signal strength, similar to that seen in GD19 or in neonatal lungs. Immunostaining showed that Hoxb5 protein was expressed strongly in the lung mesenchyme at early stages in gestation. However, by GD19 and in neonates, it was present only in specific epithelial cells. A persistent level of Hoxb5 protein in the mesenchyme after EGF or TGF-β1 treatments or tracheal ligation was noted. Hoxb5 protein was significantly downregulated by EGF+TGF-β1, and it was least in lungs after dexamethasone or EGF+TGF-β1+dexamethasone treatment. The decrease in Hoxb5 protein was significant only in the groups with dexamethasone added to the media. Thus immunostaining results parallel those of immunoblotting. The degree of Hoxb5 downregulation by dexamethasone or EGF+TGF-β1+dexamethasone was similar to that seen in vivo in very late gestation, which correlated to the advancing structural development of the lung.
VEGF induces hyperpermeability by a direct action on endothelial cells
Authors: Hippenstiel S. , Krüll M. , Ikemann A. , Risau W. , Clauss M. , Suttorp Norbert
Published: December 2017

Vascular endothelial growth factor (VEGF) is a key regulator of vasculo- and angiogenesis. Earlier studies demonstrated a permeability-increasing effect of VEGF in skin tests, leading to its other name, vascular permeability factor. We wondered whether VEGF-induced hyperpermeability was a direct effect of VEGF on endothelial cells and studied the permeability of human and porcine endothelial cell monolayers in a well-characterized in vitro system. VEGF increased the hydraulic conductivity up to 20-fold and simultaneously decreased the albumin reflection coefficient. This effect occurred after a delay of 150 min, although VEGF-induced early endothelial cell activation was verified by enhanced inositol phosphate accumulation within 5 min and increased P-selectin expression within 15 min. Platelet-derived growth factor and granulocyte-macrophage colony-stimulating factor, two endothelial cell nonspecific mitogens, also stimulated phosphatidylinositol metabolism and P-selectin expression; however, they had no effect on endothelial permeability. The increase in intracellular cyclic nucleotide levels of human endothelial monolayers abolished VEGF-induced endothelial hyperpermeability. In summary, VEGF increased endothelial permeability by a direct action on endothelial cells. Based on the pattern of endothelial cell activation by growth factors, VEGF appears to be a unique stimulus.
Modulation of lung liquid clearance by isoproterenol in rat lungs
Authors: Saldías F. , Lecuona E. , Friedman E. , Barnard M. L. , Ridge K. M. , Sznajder J. I.
Published: December 2017

β-Adrenergic agonists have been reported to increase lung liquid clearance by stimulating active Na+transport across the alveolar epithelium. We studied mechanisms by which β-adrenergic isoproterenol (Iso) increases lung liquid clearance in isolated perfused fluid-filled rat lungs. Iso perfused through the pulmonary circulation at concentrations of 10−4to 10−8M increased lung liquid clearance compared with that of control lungs ( P < 0.01). The increase in lung liquid clearance was inhibited by the β-antagonist propranolol (10−5M), the Na+-channel blocker amiloride (10−4M), and the antagonist of Na-K-ATPase, ouabain (5 × 10−4M). Colchicine, which inhibits cell microtubular transport of ion-transporting proteins to the plasma membrane, blocked the stimulatory effects of Iso on active Na+transport, whereas the isomer lumicolchicine, which does not affect cell microtubular transport, did not inhibit Na+transport. In parallel with these changes, the Na-K-ATPase α1-subunit protein abundance and activity increased in alveolar type II cells stimulated by 10−6M Iso. Colchicine blocked the stimulatory effect of Iso and the recruitment of Na-K-ATPase α1-protein to the basolateral membrane of alveolar type II cells. Accordingly, Iso increased active Na+transport and lung liquid clearance by stimulation of β-adrenergic receptors and probably by upregulation of apical Na+channels and basolateral Na-K-ATPase mechanisms. Recruitment from intracellular pools and microtubular transport of Na+pumps to the plasma membrane participate in β-adrenergic stimulation of lung liquid clearance in rat lungs.
Effect of anion secretion inhibitors on mucin content of airway submucosal gland ducts
Authors: Inglis Sarah K. , Corboz Michel R. , Ballard Stephen T.
Published: December 2017

In porcine bronchi, inhibition of both Cl− and[Formula: see text] transport is required to block the anion secretion response to ACh and to cause mucus accumulation within ACh-treated submucosal gland ducts [S. K. Inglis, M. R. Corboz, A. E. Taylor, and S. T. Ballard. Am. J. Physiol. 272 ( Lung Cell. Mol. Physiol. 16): L372–L377, 1997]. In this previous study, a combination of three potential [Formula: see text] transport inhibitors [1 mM acetazolamide, 1 mM DIDS, and 0.1 mM dimethylamiloride (DMA)] was used to block carbonic anhydrase, Cl−/[Formula: see text]exchange, and Na+/H+exchange, respectively. The aim of the present study was to obtain a better understanding of the mechanism of ACh-induced[Formula: see text] secretion in airway glands by determining which of the three inhibitors, in combination with bumetanide, is required to block anion secretion and so cause ductal mucin accumulation. Gland duct mucin content was measured in distal bronchi isolated from domestic pigs. Addition of either bumetanide alone, bumetanide plus acetazolamide, or bumetanide plus DIDS had no significant effect on ACh-induced mean gland duct mucin content. In contrast, glands treated with bumetanide plus DMA as well as glands treated with all four anion transport blockers were almost completely occluded with mucin after the addition of ACh. These data suggest that mucin is cleared from the ducts of bronchial submucosal glands by liquid generated from Cl−- and DMA-sensitive [Formula: see text] transport.
Myosin isoform shifts and decreased reactivity in hypoxia-induced hypertensive pulmonary arterial muscle
Authors: Packer C. S. , Roepke J. E. , Oberlies N. H. , Rhoades R. A.
Published: December 2017

The principal stimulus that evokes pulmonary hypertension is chronic alveolar hypoxia. Pulmonary hypertension is associated with remodeling of the vessel walls, involving hypertrophy and hyperplasia of pulmonary arterial smooth muscle (PASM) and a concomitant increase in the deposition of connective tissue, resulting in increased wall thickness. The purpose of the present study was to determine the effect of hypoxia-induced hypertension on the structure and function of PASM. Experiments were designed to determine whether hypoxia-induced pulmonary hypertension is associated with alterations in PASM: 1) reactivity to a variety of agonists, 2) contractile protein proportions and isoforms, and 3) structural properties. Young adult male rats were made hypoxic by lowering the fraction of inspired O2(10%) for 14 days. Pulmonary arterial segments were isolated and dose-response curves to various agonists (high K+, norepinephrine, serotonin, angiotensin II, and adenosine) were generated. Gel electrophoresis was used to measure changes in the relative amounts of actin or myosin and of myosin heavy chain (MHC) isoforms. Structural changes were correlated with the pharmacological and biochemical data. Hypoxia-induced pulmonary hypertension caused a general decreased reactivity, an increase in the proportion of nonmuscle to muscle MHC isoforms in PASM, and an increase in arterial wall thickness with PASM hypertrophy or hyperplasia.
Pulmonary microvascular and macrovascular endothelial cells: differential regulation of Ca2+and permeability
Authors: Kelly J. J. , Moore T. M. , Babal P. , Diwan A. H. , Stevens T. , Thompson W. J.
Published: December 2017

Cytosolic Ca2+concentration ([Ca2+]i) plays an important role in control of pulmonary vascular endothelial cell (ECs) barrier function. In this study, we investigated whether thapsigargin- and ionomycin-induced changes in cytosolic Ca2+induce permeability in rat pulmonary microvascular (RPMV) versus macrovascular (RPA) ECs. In Transwell cultures, RPMVECs formed a tighter, more restrictive barrier than RPAECs to 12,000-, 72,000-, and 150,000-molecular-weight FITC-labeled dextrans. Thapsigargin (1 μM) produced higher [Ca2+]ilevels in RPAECs than in RPMVECs and increased permeability in RPAEC but not in RPMVEC monolayers. Due to the attenuated [Ca2+]iresponse in RPMVECs, we investigated whether reduced activation of store-operated Ca2+entry was responsible for the insensitivity to thapsigargin. Addition of the drug in media containing 100 nM extracellular Ca2+followed by readdition media with 2 mM extracellular Ca2+increased RPMVEC [Ca2+]ito a level higher than that in RPAECs. Under these conditions, RPMVEC permeability was not increased, suggesting that [Ca2+]iin RPMVECs does not initiate barrier disruption. Also, ionomycin (1.4 μM) did not alter RPMVEC permeability, but the protein phosphatase inhibitor calyculin A (100 nM) induced permeability in RPMVECs. These data indicate that, whereas increased [Ca2+]ipromotes permeability in RPAECs, it is not sufficient in RPMVECs, which show an apparent uncoupling of [Ca2+]isignaling pathways or dominant Ca2+-independent mechanisms from controlling cellular gap formation and permeability.
O3-induced formation of bioactive lipids: estimated surface concentrations and lining layer effects
Authors: Postlethwait Edward M. , Cueto Rafael , Velsor Leonard W. , Pryor William A.
Published: December 2017

Recent evidence suggests that inhaled ozone (O3) does not induce toxicity via direct epithelial interactions. Reactions with epithelial lining fluid (ELF) constituents limit cellular contact and generate products, including lipid ozonation products, postulated to initiate pathophysiological cascades. To delineate specific aspects of lipid ozonation product formation and to estimate in situ surface concentrations, we studied the O3absorption characteristics of ELF constituent mixtures and measured hexanal, heptanal, and nonanal yields as a function of ascorbic acid (AH2) concentration. Exposures of isolated rat lungs, bronchoalveolar lavage fluid (BALF) and egg phosphatidylcholine (PC) liposomes were conducted. 1) O3absorption by AH2, uric acid, and albumin exceeded that by egg PC and glutathione. O3reaction with egg PC occurred when AH2concentrations were reduced. 2) Aldehydes were produced in low yield during lung and BALF exposures in a time- and O3concentration-dependent manner. 3) Diminishing BALF AH2content lowered O3uptake but increased aldehyde yields. Conversely, AH2addition to egg PC increased O3uptake but reduced aldehyde yields. Estimations of bioactive ozonation and autoxidation product accumulation within the ELF suggested possible nanomolar to low micromolar concentrations. The use of reaction products as metrics of O3exposure may have intrinsic sensitivity and specificity limitations. Moreover, due to the heterogenous nature of O3reactions within the ELF, dose-response relationships may not be linear with respect to O3absorption.
Rhinovirus infection induces mucus hypersecretion
Authors: Yuta Atsushi , Doyle William J. , Gaumond Ethan , Ali Mushtaq , Tamarkin Lawrence , Baraniuk James N. , Van Deusen Matthew , Cohen Sheldon , Skoner David P.
Published: December 2017

Rhinorrhea is a prominent symptom of the common cold. Although increases in vascular permeability and serous cell secretion have been demonstrated in human nasal mucus during active rhinovirus infections, changes in mucin constituents have not been quantified. Nonallergic ( n= 48) and asymptomatic allergic rhinitis ( n = 32) subjects were inoculated with rhinovirus type hanks before the spring allergy season. Nasal lavages were performed before inoculation ( day 0), then daily for 5 days afterward. The subjects were divided into infected and noninfected groups on the basis of evidence of successful rhinovirus infection (nasal shedding of virus or fourfold increases in specific serum antibodies). Concentrations of interleukin (IL)-8, markers of vascular leak (IgG), seromucous cells (lysozyme), and mucoglycoprotein exocytosis [7F10-immunoreactive mucin (7F10-irm) and Alcian blue staining of acidic mucoglycoproteins] were measured in lavage fluids. The infected subgroup had maximal increases in nasal lavage fluid concentrations of IL-8 (sevenfold), IgG (fourfold), total protein (twofold), and gel-phase 7F10-irm (twofold) on day 3. There were no differences between infected allergic and nonallergic subjects. IL-8 and gel-phase 7F10-irm were significantly higher in infected than in noninfected subjects. In addition to promoting plasma exudation, rhinovirus hanks infection increases IL-8 and gel-phase mucin secretion. These processes may contribute to a progression from watery rhinorrhea to mucoid discharge, with mild neutrophilic infiltration during the common cold.
Upregulation of urokinase in alveolar macrophages and lung tissue in response to silica particles
Authors: Lardot Cécile , Delos Monique , Lison Dominique
Published: December 2017

Impaired fibrinolytic activity and persistent fibrin deposits in lung tissue have been associated with lung fibrotic disorders. The present study examined the sources of plaminogen activator (PA) changes induced by a single intratracheal administration of silica particles (5 mg) in the mouse lung. We found in both control and silica-treated animals that amiloride almost totally abolished PA activity in bronchoalveolar lavage (BAL) fluid (BALF), indicating that initial upregulation (from day 1) as well as sustained PA activity (up to day 30) observed in response to silica is related to changes in urokinase-type PA (uPA). The upregulation of BALF uPA activity was associated with a marked and persistent increase in uPA mRNA levels in lung tissue. Changes in uPA expression were also reflected in the BAL cell fraction. A maximal and constant increase in cell uPA activity was associated with the early response to silica, whereas significant but lower upregulation was still noted at the fibrotic stage. From days 3 to 30, a progressive increase in uPA mRNA levels was noted in BAL inflammatory cells elicited by silica. Because the number of BAL neutrophils was strongly correlated with BALF and BAL cell-associated uPA activity, their involvement in uPA upregulation was addressed by inducing neutropenia with cyclophosphamide (200 mg/kg ip) before administration of the silica. Neutrophilic depletion did not, however, reduce, and even increased, the BAL cell-associated uPA activity. At the BALF level, neutropenia did not change PA activity in silica-treated mice, pointing to alveolar macrophages as the principal source of uPA in response to silica. Immunohistochemical stainings identified alveolar macrophages and pneumocytes as uPA-expressing cells in silica-treated animals ( day 30). Intense and heterogenous staining was observed in silicotic nodules. These findings indicate that urokinase produced by alveolar macrophages is operative not only at the alveolitis stage but also later in the fibrotic process, produced by silica particles, supporting the role of uPA in fibrogenesis.
eNOS expression is not altered in pulmonary vascular remodeling due to increased pulmonary blood flow
Authors: Everett Allen D. , Le Cras Timothy D. , Xue Chun , Johns Roger A.
Published: December 2017

Congenital heart lesions resulting in increased pulmonary blood flow are common and if unrepaired often lead to pulmonary hypertension and heart failure. Therefore, we hypothesized that increased pulmonary blood flow without changes in pressure would result in remodeling of the pulmonary arterial wall. Furthermore, because the vasodilator nitric oxide is produced by the lung, is regulated by flow in the systemic circulation, and has been associated with the regulation of smooth muscle cell proliferation, we hypothesized that increased pulmonary blood flow would result in altered expression of endothelial nitric oxide synthase (eNOS). To study this hypothesis, 42-day-old Sprague-Dawley rats had creation of an aortocaval shunt to increase pulmonary blood flow for 6 wk. The shunt resulted in a significant increase in the heart- and lung-to-body weight ratios (>2-fold; P < 0.05) without significant alteration of pulmonary or systemic blood pressures. Significant thickening of the pulmonary arterial medial wall developed, with increased muscularization of small (50–100 μm)- and medium (101–200 μm)-sized arteries as evidenced by α-actin smooth muscle staining. Proliferating cell nuclear antigen staining and bromodeoxyuridine labeling did not detect proliferating smooth muscle cells in the vascular wall. eNOS Western and Northern blot analyses and immunohistochemical staining demonstrated that eNOS protein and mRNA levels were not altered in the shunt lungs compared with sham controls. Therefore, increased pulmonary flow without increased pressure resulted in pulmonary artery medial thickening, without ongoing proliferation. Unlike chronic hypoxia-induced vascular remodeling, the pulmonary vascular remodeling resulting from increased pulmonary blood flow is not associated with changes in eNOS.
Mechanical strain inhibits repair of airway epithelium in vitro
Authors: Savla Ushma , Waters Christopher M.
Published: December 2017

The repair of airway epithelium after injury is crucial in restoring epithelial barrier integrity. Although the airway epithelium is stretched and compressed due to changes in both circumferential and longitudinal dimensions during respiration and may be overdistended during mechanical ventilation, the effect of cyclic strain on the repair of epithelial wounds is unknown. Human and cat airway epithelial cells were cultured on flexible membranes, wounded by scraping with a metal spatula, and subjected to cyclic strain using the Flexercell Strain Unit. Because the radial strain profile in the wells was nonuniform, we compared closure in regions of elongation and compression within the same well. Both cyclic elongation and cyclic compression significantly slowed repair, with compression having the greatest effect. This attenuation was dependent upon the time of relaxation (TR) during the cycle. When wells were stretched at 10 cycles/min (6 s/cycle) with TR = 5 s, wounds closed similarly to wounds in static wells, whereas in wells with TR = 1 s, significant inhibition was observed. As the TR during cycles increased (higher TR), wounds closed faster. We measured the effect of strain at various TRs on cell area and centroid-centroid distance (CD) as a measure of spreading and migration. While cell area and CD in static wells significantly increased over time, the area and CD of cells in the elongated regions did not change. Cells in compressed regions were significantly smaller, with significantly lower CD. Cell area and CD became progressively larger with increasing TR. These results suggest that mechanical strain inhibits epithelial repair.
Eosinophils, major basic protein, and polycationic peptides augment bovine airway myocyte Ca2+mobilization
Authors: Wylam Mark E. , Gungor Nesli , Mitchell Richard W. , Umans Jason G.
Published: December 2017

Previous studies in vivo or in isolated airway preparations have suggested that eosinophil-derived polycationic proteins enhance airway smooth muscle tone in an epithelium-dependent manner. We assessed the direct effects of activated human eosinophil supernatant, major basic protein (MBP), and polycationic polypeptides on basal and agonist-stimulated intracellular Ca2+concentrations ([Ca2+]i) in cultured bovine tracheal smooth muscle (TSM) cells. A 1-h incubation of myocytes with activated eosinophil buffer resulted in a doubling of basal [Ca2+]iand increased responsivity to histamine compared with myocytes that were exposed to sham-activated eosinophil buffer. In addition, concentration-dependent acute transient increases and subsequent 1-h sustained elevations of basal [Ca2+]iwere observed immediately after addition of MBP and model polycationic proteins. Finally, both peak and plateau [Ca2+]iresponses to bradykinin addition were augmented significantly in cultured myocytes that had been exposed to low concentrations of MBP or model polycationic proteins but were inhibited at greater concentrations. This elevated [Ca2+]ito polycationic proteins was manifest in epithelium-denuded bovine TSM strips as concentration-dependent increased basal tone. We conclude that activated eosinophil supernatant, MBP, and other polycationic proteins have a direct effect on both basal and subsequent agonist-elicited Ca2+mobilization in cultured TSM cells; TSM strips in vitro demonstrated, respectively, augmented and diminished responses to the contractile agonist acetylcholine. It is possible that alteration in myocyte Ca2+mobilization induced by these substances may influence clinical states of altered airway tone, such as asthma.