bluuebunny/crossref_metadata_2025 huggingface dataset:
In vitro cellular effects of perfluorochemicals correlate with their lipid solubility
Authors: Obraztsov Viktor V. , Neslund Gerald G. , Kornbrust Elisabeth S. , Flaim Stephen F. , Woods Catherine M.
Published: December 2017

Preclinical studies comparing perflubron partial liquid ventilation with conventional mechanical ventilation have indicated that perflubron partial liquid ventilation may exert some anti-inflammatory effects. To assess whether these effects were related to the lipid solubility properties of perflubron rather than to nonspecific biophysical properties of the perfluorocarbon (PFC) liquid phase, we studied the effects of PFCs with varying lipid solubilities on the platelet aggregation response to various procoagulants and the erythrocyte hemolytic response to osmotic stress. In both cases, the degree of the response was directly related to the lipid solubility of the PFC. All the perflubron content of erythrocytes was found to be associated with the membrane compartment. The time to reach a maximum effect on hemolysis with perflubron was relatively slow (2–4 h), which paralleled the time for perflubron to accumulate in erythrocyte membranes. The rate and extent of perflubron partitioning into lecithin liposomes were similar to those of erythrocyte membranes, supporting the hypothesis that perflubron was partitioning into the lipid component of the membranes. Thus some of the potential modulatory effects of perflubron on excessive inflammatory responses that occur during acute lung injury and acute respiratory distress syndrome may be influenced in part by the extent of PFC partitioning into the lipid bilayers of cellular membranes.
Retinoic acid treatment partially rescues failed septation in rats and in mice
Authors: Massaro Gloria D. , Massaro Donald
Published: December 2017

Pulmonary alveoli are formed in part by subdivision (septation) of the gas-exchange saccules of the immature lung. Septation results in smaller, more numerous structures (alveoli) and is developmentally regulated in mammals including humans, rats, and mice; if it fails to occur at the appropriate time, there is no spontaneous post hoc septation nor has there been a means of inducing septation after it has failed to occur. We measured lung volume, the volume of individual alveoli, and alveolar surface area and calculated alveolar number in neonatal rats in which septation had been blocked by treatment with a glucocorticosteroid hormone and in adult tight-skin mice that have a genetic failure of septation. We tested the hypothesis that treatment with all- trans retinoic acid induces post hoc septation. In both models of failed septation, hence in two species, and in immature and adult animals, treatment with all- trans retinoic acid induced post hoc septation, offering the possibility of a similar effect in premature infants.
Characterization of mucins from cultured normal human tracheobronchial epithelial cells
Authors: Thornton David J. , Gray Thomas , Nettesheim Paul , Howard Marj , Koo Ja Seok , Sheehan John K.
Published: December 2017

Early-passage normal human tracheobronchial epithelial (NHTBE) cells grown in air-liquid interface cultures in medium containing retinoids differentiate into a mucociliary epithelium over a 2- to 3-wk period and express increasing mRNA levels of the airway mucin genes MUC5AC and MUC5Bas the cultures age; the levels of MUC2 mRNA were very low throughout the study. Using specific antibodies to MUC5AC and MUC5B mucins, we noted a gradual increase in these two mucins in the intracellular and apically secreted pools as a function of time. A low level of MUC2 mucin was detected, which did not change with time. The intracellular and apically secreted mucins isolated from day 14and day 21 cultures by density gradient centrifugation were similar in density to those previously isolated from human respiratory mucus secretions. The sedimentation rate of the apically secreted mucins indicated that they were highly oligomerized, polydisperse macromolecules similar to those previously documented from in vivo secretions. In contrast, the cell-associated mucins from the cultured NHTBE cells were much smaller, possibly only monomers and dimers. Anion-exchange chromatography detected no differences in charge density between the reduced and carboxymethylated cell-associated and secreted forms of the MUC5AC and MUC5B mucins. The MUC5AC mucin was of similar charge density to its in vivo counterpart; however, MUC5B was more homogeneous than that found in vivo. Finally, evidence is presented for an intracellular NH2-terminal cleavage of the MUC5B mucins. These studies indicate that the mucins produced by cultured NHTBE cells are similar to those found in human airways, suggesting that this cell culture model is suited for studies of respiratory mucin biosynthesis, processing, and assembly.
Airway surface liquid composition in mice
Authors: Cowley Elizabeth A. , Govindaraju Karuthapillai , Guilbault Claudine , Radzioch Danuta , Eidelman David H.
Published: December 2017

Airway surface liquid (ASL) lines the conducting airways of the respiratory tract. We collected small samples of this liquid from the lower tracheae of anesthetized C57BL/6 mice and determined its ionic composition (in mM: 87.2 Na+, 4.7 K+, and 57.0 Cl−). Intravenous methacholine produced significant increases in the concentrations of Na+, K+, and Cl− within ASL. A limited analysis of liquid from cystic fibrosis transmembrane conductance regulator (CFTR) knockout mice revealed no significant differences compared with littermate controls; however, Pseudomonas aeruginosa infection led to an increase in the salt concentration of ASL in cftr(+/+) mice. Morphometric measurements of tracheal submucosal gland volume revealed significant differences between inbred mouse strains, corresponding to ease of ASL collection. We conclude that although submucosal glands may be responsible for the production of some ASL, the ionic composition of this liquid is actively regulated by the underlying epithelial cells.
Ontogeny and localization of TGF-β type I receptor expression during lung development
Authors: Zhao Yun , Young Stephen L. , McIntosh J. Clarke , Steele Mark P. , Silbajoris Robert
Published: December 2017

Transforming growth factor (TGF)-β is a family of multifunctional cytokines controlling cell growth, differentiation, and extracellular matrix deposition in the lung. The biological effects of TGF-β are mediated by type I (TβR-I) and II (TβR-II) receptors. Our previous studies show that the expression of TβR-II is highly regulated in a spatial and temporal fashion during lung development. In the present studies, we investigated the temporal-spatial pattern and cellular expression of TβR-I during lung development. The expression level of TβR-I mRNA in rat lung at different embryonic and postnatal stages was analyzed by Northern blotting. TβR-I mRNA was expressed in fetal rat lungs in early development and then decreased as development proceeded. The localization of TβR-I in fetal and postnatal rat lung tissues was investigated by using in situ hybridization performed with an antisense RNA probe. TβR-I mRNA was present in the mesenchyme and epithelium of gestational day 14 rat lungs. An intense TβR-I signal was observed in the epithelial lining of the developing bronchi. In gestational day 16 lungs, the expression of TβR-I mRNA was increased in the mesenchymal tissue. The epithelium in both the distal and proximal bronchioles showed a similar level of TβR-I expression. In postnatal lungs, TβR-I mRNA was detected in parenchymal tissues and blood vessels. We further studied the expression of TβR-I in cultured rat lung cells. TβR-I was expressed by cultured rat lung fibroblasts, microvascular endothelial cells, and alveolar epithelial cells. These studies demonstrate a differential regulation and localization of TβR-I that is different from that of TβR-II during lung development. TβR-I, TβR-II, and TGF-β isoforms exhibit distinct but overlapping patterns of expression during lung development. This implies a distinct role for TβR-I in mediating TGF-β signal transduction during lung development.
pH-regulated chloride secretion in fetal lung epithelia
Authors: Blaisdell Carol J. , Edmonds Rebecca D. , Wang Xi-Tao , Guggino Sandra , Zeitlin Pamela L.
Published: December 2017

The fetal lung actively transports chloride across the airway epithelium. ClC-2, a pH-activated chloride channel, is highly expressed in the fetal lung and is located on the apical surface of the developing respiratory epithelium. Our goal was to determine whether acidic pH could stimulate chloride secretion in fetal rat distal lung epithelial cells mounted in Ussing chambers. A series of acidic solutions stimulated equivalent short-circuit current ( I eq) from a baseline of 28 ± 4.8 (pH 7.4) to 70 ± 5 (pH 6.2), 114 ± 12.8 (pH 5.0), and 164 ± 19.2 (pH 3.8) μA/cm2. These changes in I eq were inhibited by 1 mM cadmium chloride and did not result in large changes in [3H]mannitol paracellular flux. Immunofluorescent detection by confocal microscopy revealed that ClC-2 is expressed along the luminal surface of polarized fetal distal lung epithelial cells. These data suggest that the acidic environment of the fetal lung fluid could activate chloride channels contributing to fetal lung fluid production and that the changes in I eqseen in these Ussing studies may be due to stimulation of ClC-2.
Conditional Clara cell ablation reveals a self-renewing progenitor function of pulmonary neuroendocrine cells
Authors: Reynolds Susan D. , Hong Kyung U. , Giangreco Adam , Mango Gregory W. , Guron Charanjeet , Morimoto Yasuo , Stripp Barry R.
Published: December 2017

The neuroepithelial body (NEB) is a highly dynamic structure that responds to chronic airway injury through hyperplasia of associated pulmonary neuroendocrine (PNE) cells. Although NEB dysplasia is correlated with preneoplastic conditions and PNE cells are thought to serve as a precursor for development of small cell lung carcinoma, mechanisms regulating expansion of the PNE cell population are not well understood. Based on studies performed in animal models, it has been suggested that NEB-associated progenitor cells that are phenotypically distinct from PNE cells contribute to PNE cell hyperplasia. We have previously used a Clara cell-specific toxicant, naphthalene, to induce airway injury in mice and have demonstrated that naphthalene-resistant Clara cells, characterized by their expression of Clara cell secretory protein (CCSP), and PNE cells contribute to airway repair and associated hyperplasia of NEBs. This study was conducted to define the contribution of NEB-associated CCSP-expressing progenitor cells to PNE cell hyperplasia after Clara cell ablation. Transgenic ( CCtk) mice were generated in which herpes simplex virus thymidine kinase was expressed within all CCSP-expressing cells of the conducting airway epithelium through the use of transcriptional regulatory elements from the mouse CCSP promoter. Chronic administration of ganciclovir (GCV) to CCtk transgenic mice resulted in selective ablation of CCSP-expressing cells within conducting airways. Proliferation and hyperplasia of PNE cells occurred in the absence of detectable proliferation among any other residual airway epithelial cell populations. These results demonstrate that PNE cells function as a self-renewing progenitor population and that NEB-associated Clara cells are not necessary for PNE cell hyperplasia.
E. colihemolysin-induced lipid mediator metabolism in alveolar macrophages: impact of eicosapentaenoic acid
Authors: Rose F. , Kiss L. , Grimminger F. , Mayer K. , Grandel U. , Seeger W. , Bieniek E. , Sibelius U.
Published: December 2017

Escherichia coli hemolysin (HlyA) is a prototype of a large family of pore-forming proteinaceous exotoxins that have been implicated in the pathogenetic sequelae of severe infection and sepsis, including development of acute lung injury. In the present study in rabbit alveolar macrophages (AMs), subcytolytic concentrations of purified HlyA evoked rapid synthesis of platelet-activating factor, with quantities approaching those in response to maximum calcium ionophore challenge. In parallel, large quantities of leukotriene (LT) B4and 5-, 8-, 9-, 12-, and 15-hydroxyeicosatetraenoic acid (HETE) were liberated from HlyA-exposed AMs depending on exogenous arachidonic acid (AA) supply. Coadministration of eicosapentaenoic acid (EPA) dose dependently suppressed generation of the proinflammatory lipoxygenase products LTB4and 5-, 8-, 9-, and 12-HETE in parallel with the appearance of the corresponding EPA-derived metabolites LTB5and 5-, 8-, 9-, and 12–hydroxyeicosapentaenoic acid (HEPE). At equimolar concentrations, EPA turned out to be the preferred substrate over AA for these AM lipoxygenase pathways, with the sum of LTB5and 5-, 8-, 9-, and 12-HEPE surpassing the sum of LTB4and 5-, 8-, 9-, and 12-HETE by >80-fold. In contrast, coadminstration of EPA did not significantly reduce HlyA-elicited generation of the anti-inflammatory AA lipoxygenase product 15-HETE. We conclude that AMs are sensitive target cells for HlyA attack, resulting in marked proinflammatory lipid mediator synthesis. In the presence of EPA, lipoxygenase product formation is shifted from a pro- to an anti-inflammatory profile.
Incorporation of biotinylated SP-A into rat lung surfactant layer, type II cells, and Clara cells
Authors: Savov Jordan , Wright J. R. , Young S. L.
Published: December 2017

The goal of this study was to compare the functions of Clara and type II cells during alveolar clearance and recycling of surfactant protein (SP) A, a secretory product of both cell types. We examined the incorporation of instilled biotinylated SP-A (bSP-A) into rat lung type II and Clara cells as a measure of clearance and recycling of the protein. Ultrastructural localization of bSP-A was accomplished by an electron-microscopic immunogold technique at 7, 30, and 120 min after intratracheal instillation. Localization of bSP-A was quantitatively evaluated within extracellular surfactant components (lipid-rich forms: myelin figures, vesicles, and tubular myelin; and lipid-poor hypophase) and in compartments of type II and Clara cells. bSP-A was incorporated into myelinic and vesicular forms of extracellular surfactant, but tubular myelin and hypophase had little bSP-A. Lamellar bodies of type II cells demonstrated a significant time-dependent increase in their incorporation of bSP-A. There was a concentration of bSP-A in the secretory granules and mitochondria of Clara cells, but no Clara cell compartment showed a pattern of time-dependent change in immunolabeling. Our immunolabeling data demonstrated a time-dependent movement of exogenous SP-A from extracellular components into type II cells and their secretory granules. Clara cells did not demonstrate a time-dependent incorporation of bSP-A into their secretory granules during the period of this study. If Clara cells recycle SP-A, they must reach a steady state very quickly or very slowly.
Abrogation of bleomycin-induced epithelial apoptosis and lung fibrosis by captopril or by a caspase inhibitor
Authors: Wang Rongqi , Ibarra-Sunga Olivia , Verlinski Luba , Pick Ruth , Uhal Bruce D.
Published: December 2017

Angiotensin-converting enzyme is involved in apoptosis of alveolar epithelial cells (Wang R, Zagariya A, Ang E, Ibarra-Sunga O, and Uhal BD. Am J Physiol Lung Cell Mol Physiol 277: L1245–L1250, 1999). This study tested the ability of the angiotensin-converting enzyme inhibitor captopril or the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone (ZVAD-fmk) to block alveolar epithelial cell apoptosis and lung fibrosis in vivo in response to bleomycin (Bleo). Male Wistar rats received 8 U/kg of Bleo (bleomycin sulfate) or vehicle intratracheally. Subgroups of Bleo-treated rats received captopril, ZVAD-fmk, or vehicle alone. Lung collagen was assessed by picrosirius red or hydroxyproline assay at 1, 7, and 14 days post-Bleo, and apoptosis was detected by in situ end labeling (ISEL). Bleo increased alveolar septal and peribronchial collagen by 100 and 133%, respectively (both P < 0.01), by day 14 but not earlier. In contrast, ISEL was increased in alveolar and airway cells at all time points. Captopril or ZVAD-fmk inhibited collagen accumulation by 91 and 85%, respectively ( P < 0.01). Both agents also inhibited ISEL in alveoli by 99 and 81% and in airways by 67 and 63%, respectively. These data suggest that the efficacy of captopril to inhibit experimental lung fibrogenesis is related to inhibition of apoptosis. They also demonstrate the antifibrotic potential of a caspase inhibitor.
Transglutaminase-mediated fibronectin multimerization in lung endothelial matrix in response to TNF-α
Authors: Chen Ruihua , Gao Baochong , Huang Cancan , Olsen Byron , Rotundo Robert F. , Blumenstock Frank , Saba Thomas M.
Published: December 2017

Exposure of lung endothelial monolayers to tumor necrosis factor (TNF)-α causes a rearrangement of the fibrillar fibronectin (FN) extracellular matrix and an increase in protein permeability. Using calf pulmonary artery endothelial cell layers, we determined whether these changes were mediated by FN multimerization due to enhanced transglutaminase activity after TNF-α (200 U/ml) for 18 h. Western blot analysis indicated that TNF-α decreased the amount of monomeric FN detected under reducing conditions. Analysis of125I-FN incorporation into the extracellular matrix confirmed a twofold increase in high molecular mass (HMW) FN multimers stable under reducing conditions ( P < 0.05). Enhanced formation of such HMW FN multimers was associated with increased cell surface transglutaminase activity ( P < 0.05). Calf pulmonary artery endothelial cells pretreated with TNF-α also formed nonreducible HMW multimers of FN when layered on surfaces precoated with FN. Inhibitors of transglutaminase blocked the TNF-α-induced formation of nonreducible HMW multimers of FN but did not prevent either disruption of the FN matrix or the increase in monolayer permeability. Thus increased cell surface transglutaminase after TNF-α exposure initiates the enhanced formation of nonreducible HMW FN multimers but did not cause either the disruption of the FN matrix or the increase in endothelial monolayer permeability.
Platelet-activating factor contributes to acute lung leak in rats given interleukin-1 intratracheally
Authors: Lee Young M. , Hybertson Brooks M. , Cho Hyun G. , Terada Lance S. , Cho Okyong , Repine Alexander J. , Repine John E.
Published: December 2017

Lung lavage fluid of patients with acute lung injury (ALI) has increased levels of interleukin-1 (IL-1) and neutrophils, but their relationship to the lung leak that characterizes these patients is unclear. To address this concern, we investigated the role of the neutrophil agonist platelet-activating factor [1- O-alkyl-2-acetyl- sn-glycero-3-phosphocholine (PAF)] in the development of the acute neutrophil-dependent lung leak that is induced by giving IL-1 intratracheally to rats. We found that PAF acetyltransferase and PAF activities increased in lungs of rats given IL-1 intratracheally compared with lungs of sham-treated rats given saline intratracheally. The participation of PAF in the development of lung leak and lung neutrophil accumulation after IL-1 administration was suggested when treatment with WEB-2086, a commonly used PAF-receptor antagonist, decreased lung leak, lung myeloperoxidase activity, and lung lavage fluid neutrophil increases in rats given IL-1 intratracheally. Additionally, neutrophils recovered from the lung lavage fluid of rats given IL-1 intratracheally reduced more nitro blue tetrazolium (NBT) in vitro than neutrophils recovered from control rats or rats that had been given WEB-2086 and then IL-1. Histological examination indicated that the endothelial cell-neutrophil interfaces of cerium chloride-stained lung sections of rats given IL-1 contained increased cerium perhydroxide (the reaction product of cerium chloride with hydrogen peroxide) compared with lungs of control rats or rats treated with WEB-2086 and then given IL-1 intratracheally. These in vivo findings were supported by parallel findings showing that WEB-2086 treatment decreased neutrophil adhesion to IL-1-treated cultured endothelial cells in vitro. We concluded that PAF contributes to neutrophil recruitment and neutrophil activation in lungs of rats given IL-1 intratracheally.
Gsprotein dysfunction in allergen-challenged human isolated passively sensitized bronchi
Authors: Song Pingfang , Milanese Manlio , Crimi Emanuele , Bruzzone Santina , Zocchi Elena , Rehder Kai , Brusasco Vito
Published: December 2017

We studied the intracellular mechanisms of allergen-induced β2-adrenoceptor dysfunction in human isolated passively sensitized bronchi. Sensitization was obtained by overnight incubation of bronchial rings with serum containing a high specific IgE level to Dermatophagoides but a low total IgE level. Allergen challenge was done by incubation with a Dermatophagoidesmix. The Gsprotein stimulant cholera toxin (2 μg/ml) displaced the carbachol (CCh) concentration-response curves of control and sensitized but not of challenged rings to the right. Cholera toxin (10 μg/ml) displaced the concentration-response curves to CCh of control, sensitized, and challenged rings to the right, but this effect was less in challenged rings. The effects of the Giprotein inhibitor pertussis toxin (250 ng/ml or 1 μg/ml) on salbutamol concentration-relaxation curves did not differ significantly between challenged and sensitized rings. The adenylyl cyclase activator forskolin and the Ca2+-activated K+-channel opener NS-1619 relaxed CCh-contracted bronchial rings without significant differences between control, sensitized, and challenged rings. Neither Ginor Gsα-subunit expression differed between control, sensitized, and challenged tissues. We conclude that Gsprotein dysfunction may be a mechanism of allergen-induced β2-adrenoceptor dysfunction in human isolated passively sensitized bronchi.
P2Y2receptor-stimulated phosphoinositide hydrolysis and Ca2+mobilization in tracheal epithelial cells
Authors: Yang Chuen-Mao , Wu Wen-Bin , Pan Shiow-Lin , Tsai Yih-Jeng , Chiu Chi-Tso , Wang Chuan-Chwan
Published: December 2017

Extracellular nucleotides have been implicated in the regulation of secretory function through the activation of P2 receptors in the epithelial tissues, including tracheal epithelial cells (TECs). In this study, experiments were conducted to characterize the P2 receptor subtype on canine TECs responsible for stimulating inositol phosphate (Ins Px) accumulation and Ca2+mobilization using a range of nucleotides. The nucleotides ATP and UTP caused a concentration-dependent increase in [3H]Ins Pxaccumulation and Ca2+mobilization with comparable kinetics and similar potency. The selective agonists for P1, P2X, and P2Y1receptors, N6-cyclopentyladenosine and AMP, α,β-methylene-ATP and β,γ-methylene-ATP, and 2-methylthio-ATP, respectively, had little effect on these responses. Stimulation of TECs with maximally effective concentrations of ATP and UTP showed no additive effect on [3H]Ins Pxaccumulation. The response of a maximally effective concentration of either ATP or UTP was additive to the response evoked by bradykinin. Furthermore, ATP and UTP induced a cross-desensitization in [3H]Ins Pxaccumulation and Ca2+mobilization. These results suggest that ATP and UTP directly stimulate phospholipase C-mediated [3H]Ins Pxaccumulation and Ca2+mobilization in canine TECs. P2Y2receptors may be predominantly mediating [3H]Ins Pxaccumulation, and, subsequently, inositol 1,4,5-trisphosphate-induced Ca2+mobilization may function as the transducing mechanism for ATP-modulated secretory function of tracheal epithelium.
Early abnormalities of pulmonary vascular development in the Fawn-Hooded rat raised at Denver's altitude
Authors: Le Cras Timothy D. , Kim Dug-Ha , Markham Neil E. , Abman And Steven H.
Published: December 2017

The Fawn-Hooded rat (FHR) is a genetic strain that has been extensively studied as a model of primary pulmonary hypertension in adult rats. Based on our recent observations that alveolar number and pulmonary arterial density are reduced in FHRs raised at Denver's altitude, we hypothesized that early abnormalities in pulmonary vascular development contribute to the progression of pulmonary hypertension in the FHR. We found that endothelial nitric oxide synthase (eNOS) protein content was lower in the lungs of fetal, 1- and 7-day-old, 3-week-old, and adult FHRs compared with that in the normal Sprague-Dawley (SDR) and Fischer rat strains, all raised at Denver's altitude. In contrast, lung expression of the endothelial proteins kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/Flk-1) and platelet endothelial cell adhesion molecule-1 (CD31) was not different between strains. Barium arteriograms showed that pulmonary arterial density was reduced in 3-week-old FHRs compared with SDRs. Perinatal treatment of FHRs with mild hyperbaria to simulate sea-level alveolar Po 2 improved lung eNOS content and pulmonary vascular growth and reduced right ventricular hypertrophy. We conclude that the development of pulmonary hypertension in Denver-raised FHRs is characterized by reductions in lung eNOS expression and abnormal pulmonary vascular growth during the fetal, neonatal, and postnatal periods.
Expression and regulation of vascular endothelial growth factor in human pulmonary epithelial cells
Authors: Boussat Sandrine , Eddahibi Saadia , Coste André , Fataccioli Virginie , Gouge Mallaury , Housset Bruno , Adnot Serge , Maitre Bernard
Published: December 2017

Vascular endothelial growth factor (VEGF) is a potent endothelial cell growth and permeability factor highly expressed in rodent alveolar epithelium after injury and repair. To investigate VEGF synthesis in human lung epithelial cells, we examined VEGF expression by cultured cells under basal conditions and after cytokine treatment or oxidative stress. Basal VEGF expression was detected in transformed human epithelial cell lines (A549 and 1HAEo−) and in primary human bronchial epithelial cells with RT-PCR, Western blot, and immunocytochemistry. Among the cytokines tested, only transforming growth factor-β1 increased the levels of excreted VEGF165as measured by ELISA. Under hypoxia (0% O2for 24 h), the VEGF165level increased fivefold, and this effect was O2concentration dependent. VEGF concentrations in the medium of all the cell types studied reached values similar to those found in bronchoalveolar lavage fluids from normal patients. Endothelial cells (human umbilical vein endothelial cells) exposed to conditioned medium from primary bronchial epithelial cell cultures showed an increased growth rate, which was inhibited in the presence of a specific neutralizing antibody to VEGF. These results suggest that lung epithelial cells participate in the endothelial repair and angiogenesis that follow lung injury through the synthesis of VEGF.
Dietary glycine blunts lung inflammatory cell influx following acute endotoxin
Authors: Wheeler Michael D. , Rose Michelle L. , Yamashima Shunhei , Enomoto Nobuyuki , Seabra Vitor , Madren Jonathan , Thurman Ronald G.
Published: December 2017

Mortality associated with endotoxin shock is likely mediated by Kupffer cells, alveolar macrophages, and circulating neutrophils. Acute dietary glycine prevents mortality and blunts increases in serum tumor necrosis factor-α (TNF-α) following endotoxin in rats. Furthermore, acute glycine blunts activation of Kupffer cells, alveolar macrophages, and neutrophils by activating a glycine-gated chloride channel. However, in neuronal tissue, glycine rapidly downregulates chloride channel function. Therefore, the long-term effects of a glycine-containing diet on survival following endotoxin shock were investigated. Dietary glycine for 4 wk improved survival after endotoxin but did not improve liver pathology, decrease serum alanine transaminase, or effect TNF-α levels compared with animals fed control diet. Interestingly, dietary glycine largely prevented inflammation and injury in the lung following endotoxin. Surprisingly, Kupffer cells from animals fed glycine for 4 wk were no longer inactivated by glycine in vitro; however, isolated alveolar macrophages and neutrophils from the same animals were sensitive to glycine. These data are consistent with the hypothesis that glycine downregulates chloride channels on Kupffer cells but not on alveolar macrophages or neutrophils. Importantly, glycine diet for 4 wk protected against lung inflammation due to endotoxin. Chronic glycine improves survival by unknown mechanisms, but reduction of lung inflammation is likely involved.
Metabolism of phosphatidylglycerol by alveolar macrophages in vitro
Authors: Quintero Omar A. , Wright Jo Rae
Published: December 2017

In whole animal studies, it has been shown that turnover of surfactant dipalmitoylphosphatidylglycerol (DPPG) is faster than that of dipalmitoylphosphatidylcholine (DPPC). The goal of this investigation was to characterize the metabolism of DPPG by alveolar macrophages and to determine whether they contribute to the faster alveolar clearance of DPPG. Isolated rat alveolar macrophages were incubated with liposomes colabeled with [3H]DPPG and [14C]DPPC. Macrophages internalized both lipids in a time- and temperature-dependent manner. The uptake of both lipids was increased by surfactant protein (SP) A and by adherence of the macrophages to plastic slides. The isotope ratio of DPPC to DPPG internalized by macrophages in suspension in the absence of SP-A was significantly lower than the isotope ratio in liposomes, suggesting that macrophages preferentially internalize DPPG when SP-A is absent. Phospholipase activity in macrophage homogenate was higher toward sn-2-labeled DPPG than toward sn-2-labeled DPPC. These studies show that alveolar macrophages play an important role in catabolizing surfactant lipids and may be partially responsible for the relatively faster clearance of DPPG from the lung.
Free radical production in hypoxic pulmonary artery smooth muscle cells
Authors: Killilea David W. , Hester Raymond , Balczon Ronald , Babal Pavel , Gillespie Mark N.
Published: December 2017

This study used an inexpensive and versatile environmental exposure system to test the hypothesis that hypoxia promoted free radical production in primary cultures of rat main pulmonary artery smooth muscle cells (PASMCs). Production of reactive species was detected by fluorescence microscopy with the probe 2′,7′-dichlorodihydrofluorescein, which is converted to the fluorescent dichlorofluorescein (DCF) in the presence of various oxidants. Flushing the airspace above the PASMC cultures with normoxic gas (20% O2, 75% N2, and 5% CO2) resulted in stable Po2values of ∼150 Torr, whereas perfusion of the airspace with hypoxic gas (0% O2, 95% N2, and 5% CO2) was associated with a reduction in Po2values to stable levels of ∼25 Torr. Hypoxic PASMCs became increasingly fluorescent at ∼500% above the normoxic baseline after 60 min. Hypoxia-induced DCF fluorescence was attenuated by the addition of the antioxidants dimethylthiourea and catalase. These findings show that PASMCs acutely exposed to hypoxia exhibit a marked increase in intracellular DCF fluorescence, suggestive of reactive oxygen or nitrogen species production.
Surfactant metabolism in SP-D gene-targeted mice
Authors: Ikegami Machiko , Whitsett Jeffrey A. , Jobe Alan , Ross Gary , Fisher James , Korfhagen Thomas
Published: December 2017

Mice with surfactant protein (SP)-D deficiency have three to four times more surfactant lipids in air spaces and lung tissue than control mice. We measured multiple aspects of surfactant metabolism and function to identify abnormalities resulting from SP-D deficiency. Relative to saturated phosphatidylcholine (Sat PC), SP-A and SP-C were decreased in the alveolar surfactant and the large-aggregate surfactant fraction. Although large-aggregate surfactant from SP-D gene-targeted [(−/−)] mice converted to small-aggregate surfactant more rapidly, surface tension values were comparable to values for surfactant from SP-D wild-type [(+/+)] mice.125I-SP-D was cleared with a half-life of 7 h from SP-D(−/−) mice vs. 13 h in SP-D(+/+) mice. Although initial incorporation and secretion rates for [3H]palmitic acid and [14C]choline into Sat PC were similar, the labeled Sat PC was lost from the lungs of SP-D(+/+) mice more rapidly than from SP-D(−/−) mice. Clearance rates of intratracheal [3H]dipalmitoylphosphatidylcholine were used to estimate net clearances of Sat PC, which were approximately threefold higher for alveolar and total lung Sat PC in SP-D(−/−) mice than in SP-D(+/+) mice. SP-D deficiency results in multiple abnormalities in surfactant forms and metabolism that cannot be attributed to a single mechanism.
Substance P-induced airway hyperreactivity is mediated by neuronal M2receptor dysfunction
Authors: Evans Christopher M. , Belmonte Kristen E. , Costello Richard W. , Jacoby David B. , Gleich Gerald J. , Fryer Allison D.
Published: December 2017

Neuronal muscarinic (M2) receptors inhibit release of acetylcholine from the vagus nerves. Hyperreactivity in antigen-challenged guinea pigs is due to blockade of these M2autoreceptors by eosinophil major basic protein (MBP) increasing the release of acetylcholine. In vivo, substance P-induced hyperactivity is vagally mediated. Because substance P induces eosinophil degranulation, we tested whether substance P-induced hyperreactivity is mediated by release of MBP and neuronal M2receptor dysfunction. Pathogen-free guinea pigs were anesthetized and ventilated. Thirty minutes after intravenous administration of [Sar9,Met(O2)11]- substance P, guinea pigs were hyperreactive to vagal stimulation and M2receptors were dysfunctional. The depletion of inflammatory cells with cyclophosphamide or the administration of an MBP antibody or a neurokinin-1 (NK1) receptor antagonist (SR-140333) all prevented substance P-induced M2dysfunction and hyperreactivity. Intravenous heparin acutely reversed M2receptor dysfunction and hyperreactivity. Thus substance P releases MBP from eosinophils resident in the lungs by stimulating NK1receptors. Substance P-induced hyperreactivity is mediated by blockade of inhibitory neuronal M2receptors by MBP, resulting in increased release of acetylcholine.
Role of diminished epithelial GM-CSF in the pathogenesis of bleomycin-induced pulmonary fibrosis
Authors: Christensen Paul J. , Bailie Marc B. , Goodman Richard E. , O'Brien Aidan D. , Toews Galen B. , Paine Robert
Published: December 2017

Evidence derived from human and animal studies strongly supports the notion that dysfunctional alveolar epithelial cells (AECs) play a central role in determining the progression of inflammatory injury to pulmonary fibrosis. We formed the hypothesis that impaired production of the regulatory cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) by injured AECs plays a role in the development of pulmonary fibrosis. To test this hypothesis, we used the well-characterized model of bleomycin-induced pulmonary fibrosis in rats. GM-CSF mRNA is expressed at a constant high level in the lungs of untreated or saline-challenged animals. In contrast, there is a consistent reduction in expression of GM-CSF mRNA in the lung during the first week after bleomycin injury. Bleomycin-treated rats given neutralizing rabbit anti-rat GM-CSF IgG develop increased fibrosis. Type II AECs isolated from rats after bleomycin injury demonstrate diminished expression of GM-CSF mRNA immediately after isolation and in response to stimulation in vitro with endotoxin compared with that in normal type II cells. These data demonstrate a defect in the ability of type II epithelial cells from bleomycin-treated rats to express GM-CSF mRNA and a protective role for GM-CSF in the pathogenesis of bleomycin-induced pulmonary fibrosis.
Attenuation of lung reperfusion injury after transplantation using an inhibitor of nuclear factor-κB
Authors: Ross Scott D. , Kron Irving L. , Gangemi James J. , Shockey Kimberly S. , Stoler Mark , Kern John A. , Tribble Curtis G. , Laubach Victor E.
Published: December 2017

A central role for nuclear factor-κB (NF-κB) in the induction of lung inflammatory injury is emerging. We hypothesized that NF-κB is a critical early regulator of the inflammatory response in lung ischemia-reperfusion injury, and inhibition of NF-κB activation reduces this injury and improves pulmonary graft function. With use of a porcine transplantation model, left lungs were harvested and stored in cold Euro-Collins preservation solution for 6 h before transplantation. Activation of NF-κB occurred 30 min and 1 h after transplant and declined to near baseline levels after 4 h. Pyrrolidine dithiocarbamate (PDTC), a potent inhibitor of NF-κB, given to the lung graft during organ preservation (40 mmol/l) effectively inhibited NF-κB activation and significantly improved lung function. Compared with control lungs 4 h after transplant, PDTC-treated lungs displayed significantly higher oxygenation, lower Pco2, reduced mean pulmonary arterial pressure, and reduced edema and cellular infiltration. These results demonstrate that NF-κB is rapidly activated and is associated with poor pulmonary graft function in transplant reperfusion injury, and targeting of NF-κB may be a promising therapy to reduce this injury and improve lung function.
Myeloperoxidase and protein oxidation in cystic fibrosis
Authors: Van der Vliet Albert , Nguyen Mai N. , Shigenaga Mark K. , Eiserich Jason P. , Marelich Gregory P. , Cross Carroll E.
Published: December 2017

Cystic fibrosis (CF) is associated with chronic pulmonary inflammation and progressive lung dysfunction, possibly associated with the formation of neutrophil myeloperoxidase (MPO)-derived oxidants. Expectorated sputum specimens from adult CF patients were analyzed for MPO characteristic protein modifications and found to contain large amounts of active MPO as well as high levels of protein-associated 3-chlorotyrosine and 3,3′-dityrosine, products that result from MPO activity, compared with expectorated sputum from non-CF subjects. Sputum levels of nitrite (NO2−) and nitrate (NO3−), indicating local production of nitric oxide (NO·), were not elevated but in fact were slightly reduced in CF. However, there was a slight increase in protein-associated 3-nitrotyrosine in CF sputum compared with controls, reflecting the formation of reactive nitrogen intermediates, possibly through MPO-catalyzed oxidation of NO2−. CF sputum MPO was found to contribute to oxidant-mediated cytotoxicity toward cultured tracheobronchial epithelial cells; however, peroxidase-dependent protein oxidation occurred primarily within sputum proteins, suggesting scavenging of MPO-derived oxidants by CF mucus and perhaps formation of secondary cytotoxic products within CF sputum. Our findings demonstrate the formation of MPO-derived oxidizing and possibly nitrating species within the respiratory tract of subjects with CF, which collectively may contribute to bronchial injury and respiratory failure in CF.
TNF-α increases tracheal epithelial asbestos and fiberglass binding via a NF-κB-dependent mechanism
Authors: Xie C. , Reusse A. , Dai J. , Zay K. , Harnett J. , Churg A.
Published: December 2017

Tumor necrosis factor (TNF)-α is released from alveolar macrophages after phagocytosis of mineral fibers. To determine whether TNF-α affects the binding of fibers to epithelial cells, we exposed rat tracheal explants to TNF-α or to culture medium alone, followed by a suspension of amosite asbestos or fiberglass (MMVF10). Loosely adherent fibers were removed from the surface with a standardized washing technique, and the number of bound fibers was determined by scanning electron microscopy. Increasing doses of TNF-α produced increases in fiber binding. This effect was abolished by an anti-TNF-α antibody, the proteasome inhibitor MG-132, and the nuclear factor (NF)-κB inhibitor pyrrolidine dithiocarbamate. Gel shift and Western blot analyses confirmed that TNF-α activated NF-κB and depleted IκB in this system and that these effects were prevented by MG-132 and pyrrolidine dithiocarbamate. These observations indicate that TNF-α increases epithelial fiber binding by a NF-κB-dependent mechanism. They also suggest that mineral particles may cause pathological lesions via an autocrine-like process in which the response evoked by particles, for example, macrophage TNF-α production, acts to enhance subsequent interactions of particles with tissue.
Connexin mimetic peptides reversibly inhibit Ca2+signaling through gap junctions in airway cells
Authors: Boitano Scott , Evans W. Howard
Published: December 2017

The effect of peptides with sequences derived from connexins, the constituent proteins of gap junctions, on mechanically stimulated intercellular Ca2+signaling in tracheal airway epithelial cells was studied. Three peptides with sequences corresponding to connexin extracellular loop regions reversibly restricted propagation of Ca2+waves to neighboring cells. Recovery of communication began within 10 min of removal of the peptides, with inhibition totally reversed by 20–40 min. The peptides were shown to be more effective in inhibiting Ca2+waves than glycyrrhetinic acid or oleamide. Inhibition of intercellular Ca2+waves by connexin mimetic peptides did not affect the Ca2+response to extracellular ATP. Although the intracellular Ca2+response of tracheal epithelial cells to ATP was greatly reduced by either pretreatment with high doses of ATP or application of apyrase, mechanically stimulated intercellular Ca2+signaling was not affected by these agents. We conclude that connexin mimetic peptides are effective and reversible inhibitors of gap junctional communication of physiologically significant molecules that underlie Ca2+wave propagation in tracheal epithelial cells and propose a potential mechanism for the mode of action of mimetic peptides.
Surface-expressed lamellar body membrane is recycled to lamellar bodies
Authors: Schaller-Bals S. , Bates S. R. , Notarfrancesco K. , Tao J. Q. , Fisher A. B. , Shuman H.
Published: December 2017

Monoclonal antibody (MAb) 3C9, an antibody generated to the lamellar body of rat lung type II pneumocytes, specifically labels the luminal face of the lamellar body membrane. To follow the retrieval of lamellar body membrane from the cell surface in these cells, MAb 3C9 was instilled into rat lungs. In vivo, it was endocytosed by type II cells but not by other lung cells. In type II cells that were isolated from rat lungs by elastase digestion and cultured on plastic for 24 h, MAb 3C9 first bound to the cell surface, then was found in endosomes, vesicular structures, and multivesicular bodies and, finally, clustered on the luminal face of lamellar body membranes. The amount internalized reached a plateau after 1.5 h of incubation and was stimulated with the secretagogue ATP. In double-labeling experiments, internalized MAb 3C9 did not completely colocalize with NBD-PC liposomes or the nonspecific endocytic marker TMA-DPH, suggesting that lamellar body membrane is retrieved back to existing lamellar bodies by a pathway different from that of bulk membrane and may be one pathway for surfactant endocytosis. The lamellar body membrane components are retrieved as subunits that are redistributed among the preexisting lamellar bodies in the cell.
Mechanical stretch stimulates macrophage inflammatory protein-2 secretion from fetal rat lung cells
Authors: Mourgeon Eric , Isowa Noritaka , Keshavjee Shaf , Zhang Xiaoming , Slutsky Arthur S. , Liu Mingyao
Published: December 2017

Ventilation-induced lung injury has been related to cytokine production. Immaturity and barotrauma are important contributors to the development of bronchopulmonary dysplasia in infants. In the present study, stretch of organotypic cultured fetal rat lung cells was used to simulate ventilation of preterm newborns. Cells were stimulated with lipopolysaccharide (LPS; 100 ng/ml) and/or mechanical stretch. After 4 h, stretch enhanced LPS-induced macrophage inflammatory protein (MIP)-2 production in a force- and frequency-dependent manner. The maximal effect of stretch was seen with 5% elongation at 40 cycles/min. In contrast, after 1 h of stimulation, stretch alone significantly increased MIP-2 production, which was not blocked by cycloheximide, an inhibitor of protein synthesis. At both the 1- and 4-h time points, only LPS increased MIP-2 mRNA levels. Stretch-induced MIP-2 release was associated with cell injury as measured by lactate dehydrogenase release and was not inhibited by gadolinium, a stretch-activated ion channel blocker. Taken together, these results suggest that the major effect of stretch on MIP-2 production from fetal rat lung cells is to stimulate its secretion.
Bleomycin-induced pulmonary injury in mice deficient in SPARC
Authors: Savani Rashmin C. , Zhou Zhao , Arguiri Evguenia , Wang Sunny , Vu Dinh , Howe Chin C. , DeLisser Horace M.
Published: December 2017

SPARC (secreted protein, acidic and rich in cysteine) is a component of the matrix that appears to regulate tissue remodeling. There is evidence that it accumulates in the lung in the setting of pulmonary injury and fibrosis, but direct evidence of its involvement is only now emerging. We therefore investigated the development of pulmonary fibrosis induced by bleomycin administered either intratracheally or intraperitoneally in mice deficient in SPARC. Bleomycin (0.15 U/mouse) given intratracheally induced significantly more pulmonary fibrosis in mice deficient in SPARC compared with that in wild-type control mice, with the mutant mice demonstrating greater neutrophil accumulation in the lung. However, in wild-type and SPARC-deficient mice given intraperitoneal bleomycin (0.8 U/injection × 5 injections over 14 days), the pattern and severity of pulmonary fibrosis, as well as the levels of leukocyte recruitment, were similar in both strains of mice. These findings suggest that the involvement of SPARC in pulmonary injury is likely to be complex, dependent on several factors including the type, duration, and intensity of the insult. Furthermore, increased neutrophil accumulation in the peritoneal cavity was also observed in SPARC-null mice after acute chemical peritonitis. Together, these data suggest a possible role for SPARC in the recruitment of neutrophils to sites of acute inflammation.
Fetal rabbit pulmonary artery smooth muscle cell response to ryanodine is developmentally regulated
Authors: Porter Valerie A. , Reeve Helen L. , Cornfield David N.
Published: December 2017

To study developmental changes in intracellular calcium handling in pulmonary artery smooth muscle cells (PASMCs), cells were isolated from distal and proximal pulmonary arteries from rabbits at different developmental stages: juvenile (4–6 wk old), newborn (<48 h), and full-term fetal. Isolated PASMCs were studied using the calcium-sensitive dye fura 2. Cells from each age group responded to caffeine with an increase in calcium; however, ryanodine (50 μM) only increased calcium in fetal distal PASMCs. The ryanodine-induced increase was due to influx of extracellular calcium because it was blocked by removal of extracellular calcium or by diltiazem. The calcium-sensitive potassium (KCa) channel blocker iberiotoxin produced a transient increase in calcium in the fetal distal PASMCs, which could be inhibited by prior application of ryanodine. Conversely, the ryanodine response was inhibited if iberiotoxin was given first. With the use of electrophysiology and confocal microscopy, fetal PASMCs were shown to exhibit spontaneous transient outward currents and calcium sparks, respectively. These observations suggest that ryanodine-sensitive release of calcium from the sarcoplasmic reticulum and KCa channels act together to control intracellular calcium only in fetal distal PASMCs.