bluuebunny/crossref_metadata_2025 huggingface dataset:
Hypoxia reversibly inhibits epithelial sodium transport but does not inhibit lung ENaC or Na-K-ATPase expression
Authors: Carpenter Todd C. , Schomberg Stacey , Nichols Christopher , Stenmark Kurt R. , Weil John V.
Published: March 2015

Hypoxia reduces alveolar liquid clearance and the nasal potential difference, a marker of airway epithelial sodium transport. The mechanisms underlying this impaired epithelial sodium transport in vivo remain uncertain. We hypothesized that epithelial sodium transport impaired by hypoxia would recover quickly with reoxygenation and that hypoxia decreases the expression of lung epithelial sodium channels and Na,K-ATPases. We studied adult rats exposed to normoxia, hypoxia (Fi O2 = 0.1) for 24 h, or hypoxia followed by recovery in normoxia. Nasal potential differences decreased by 40% with hypoxia ( P < 0.001), returning to baseline levels with reoxygenation. Lung Na,K-ATPase activity decreased by 40% with hypoxia ( P = 0.003), recovering to baseline levels with reoxygenation. Lung expression of mRNA encoding for epithelial sodium channel (ENaC)-α, -β, and -γ or for Na,K-ATPase-α1 did not change significantly with hypoxia or recovery nor did lung expression of ENaC-α, ENaC-β, Na,K-ATPase-α1, or Na,K-ATPase-β1 protein. We conclude that subacute exposure to moderate hypoxia reversibly impairs airway epithelial sodium transport and lung Na,K-ATPase activity but that those changes are not due to changes in the lung expression of sodium-transporting proteins.
Peroxisome proliferator ameliorates endothelial dysfunction in a murine model of hyperhomocysteinemia
Authors: Sood Harpreet S. , Hunt Matthew J. , Tyagi Suresh C.
Published: March 2015

To test the hypothesis that endothelial dysfunction in hyperhomocysteinemia was due to increased levels of nitrotyrosine and matrix metalloproteinase (MMP) activity in response to antagonism of peroxisome proliferator-activated receptor-α (PPAR-α), cystathionine β-synthase (CBS) −/+ mice were bred, tail tissue was analyzed for genotype by PCR, and tail vein blood was analyzed for homocysteine (Hcy) by spectrofluorometry. To induce PPAR-α, mice were administered 8 μg/ml of ciprofibrate (CF) and grouped: 1) wild type (WT), 2) WT + CF, 3) CBS, 4) CBS + CF ( n = 6 in each group). In these four groups of mice, plasma Hcy was 3.0 ± 0.2, 2.5 ± 1.2, 15.2 ± 2.6 ( P < 0.05 compared with WT), 11.0 ± 2.9 μmol/l. Mouse urinary protein was 110 ± 11, 86 ± 6, 179 ± 13, 127 ± 9 μg · day−1· kg−1by Bio-Rad dye binding assay. Aortic nitrotyrosine was 0.099 ± 0.012, 0.024 ± 0.004, 0.132 ± 0.024 ( P < 0.01 compared with WT), 0.05 ± 0.01 (scan unit) by Western analysis. MMP-2 activity was 0.053 ± 0.010, 0.024 ± 0.002, 0.039 ± 0.009, 0.017 ± 0.006 (scan unit) by zymography. MMP-9 was specifically induced in CBS −/+ mice and inhibited by CF treatment. Systolic blood pressure (SPB) was 90 ± 2, 88 ± 16, 104 ± 8 ( P < 0.05 compared with WT), 96 ± 3 mmHg. Aortic wall stress [(SPB · radius2/wall thickness)/2(radius + wall thickness)] was 10.2 ± 1.9, 9.7 ± 0.2, 16.6 ± 0.8 ( P < 0.05 compared with WT), 13.1 ± 2.1 dyn/cm2. The results suggest that Hcy increased aortic wall stress by increasing nitrotyrosine and MMP-9 activity.
Activation of type II alveolar epithelial cells during acute endotoxemia
Authors: Sunil Vasanthi R. , Connor Agnieszka J. , Guo Yan , Laskin Jeffrey D. , Laskin Debra L.
Published: March 2015

Lung injury induced by acute endotoxemia is associated with increased generation of inflammatory mediators such as nitric oxide and eicosanoids, which have been implicated in the pathophysiological process. Although production of these mediators by alveolar macrophages (AM) has been characterized, the response of type II cells is unknown and was assessed in the present studies. Acute endotoxemia caused a rapid (within 1 h) and prolonged (up to 48 h) induction of nitric oxide synthase-2 (NOS-2) in type II cells but a delayed response in AM (12–24 h). In both cell types, this was associated with increased nitric oxide production. Although type II cells, and to a lesser extent AM, constitutively expressed cyclooxygenase-2, acute endotoxemia did not alter this activity. Endotoxin administration had no effect on mitogen-activated protein kinase or protein kinase B-α (PKB-α) expression. However, increases in phosphoinositide 3-kinase and phospho-PKB-α were observed in type II cells. The finding that this was delayed for 12–24 h suggests that these proteins do not play a significant role in the regulation of NOS-2 in this model. After endotoxin administration to rats, a rapid (within 1–2 h) activation of nuclear factor-κB was observed. This response was transient in type II cells but was sustained in AM. Interferon regulatory factor-1 (IRF-1) was also activated rapidly in type II cells. In contrast, IRF-1 activation was delayed in AM. These data demonstrate that type II cells, like AM, are highly responsive during acute endotoxemia and may contribute to pulmonary inflammation.
Inhibition of the p38 MAP kinase pathway destabilizes smooth muscle length during physiological loading
Authors: Lakser Oren J. , Lindeman Robert P. , Fredberg Jeffrey J.
Published: March 2015

We tested the hypothesis that mechanical plasticity of airway smooth muscle may be mediated in part by the p38 mitogen-activated protein (MAP) kinase pathway. Bovine tracheal smooth muscle (TSM) strips were mounted in a muscle bath and set to their optimal length, where the active force was maximal (Fo). Each strip was then contracted isotonically (at 0.32 Fo) with ACh (maintained at 10−4 M) and allowed to shorten for 180 min, by which time shortening was completed and the static equilibrium length was established. To simulate the action of breathing, we then superimposed on this steady distending force a sinusoidal force fluctuation with zero mean, at a frequency of 0.2 Hz, and measured incremental changes in muscle length. We found that TSM strips incubated in 10 μM SB-203580-HCl, an inhibitor of the p38 MAP kinase pathway, demonstrated a greater degree of fluctuation-driven lengthening than did control strips, and upon removal of the force fluctuations they remained at a greater length. We also found that the force fluctuations themselves activated the p38 MAP kinase pathway. These findings are consistent with the hypothesis that inhibition of the p38 MAP kinase pathway destabilizes muscle length during physiological loading.
Cytokines and oxygen radicals after hyperoxia in preterm and term alveolar macrophages
Authors: Rozycki Henry J. , Comber Paul G. , Huff Thomas F.
Published: March 2015

To determine if the alveolar macrophage inflammatory cytokine response to oxygen differs in premature cells, macrophages were obtained from litters of premature (27 days) and term (31 days) rabbits. The majority of these cells were nonspecific esterase positive and actively phagocytosed latex particles. The cells that expressed cytokines also reacted with a monoclonal antibody against rabbit macrophages. After incubation overnight in 5 or 95% oxygen, the amount of interleukin (IL)-1β and IL-8 mRNA was assessed by RT-PCR and the amount of cytokine protein by quantitative immunofluorescence microscopy. The preterm macrophage showed a significant increase in cytokine mRNA and protein after overnight incubation in 95% oxygen. This response was not seen in the term cells. Only premature macrophages had a significant increase in intracellular oxygen radical content, measured by 2′,7′-dichlorofluorescin analysis, after incubation in 95% oxygen. This enhanced inflammatory cytokine response to oxygen may be one mechanism involved in the early development of chronic lung disease in premature infants.
Gene expression profiling in inflammatory airway disease associated with elevated adenosine
Authors: Banerjee Suman K. , Young Hays W. J. , Volmer Jonathan B. , Blackburn Michael R.
Published: March 2015

Adenosine has been implicated as a modulator of inflammatory processes central to asthma. However, the molecular mechanisms involved are poorly understood. We used Atlas mouse cDNA arrays to analyze differential gene expression in association with lung inflammation resulting from elevated adenosine in adenosine deaminase (ADA)-deficient mice. We report that of the 1,176 genes on the array, the expression patterns of 280 genes were consistently altered. Of these genes, the steady-state levels of 93 genes were upregulated and 29 were downregulated. We also show that lowering adenosine levels with ADA enzyme therapy has striking effects on gene expression that may be associated with resolution of pulmonary eosinophilia. In addition, we confirmed the nucleic acid and protein expression of vascular endothelial growth factor and monocyte chemoattractant protein-3, two candidate genes that may be regulated by adenosine. In conclusion, high-throughput profiling of gene expression by cDNA array hybridization has provided an overview of critical regulatory genes involved in airway inflammation in ADA-deficient mice. These mice will serve as a useful in vivo model for characterizing molecular mechanisms of adenosine-mediated lung damage.
IL-13 and IL-4 cause eotaxin release in human airway smooth muscle cells: a role for ERK
Authors: Moore Paul E. , Church Trudi L. , Chism David D. , Panettieri Reynold A. , Shore Stephanie A.
Published: March 2015

Human airway smooth muscle (HASM) cells express interleukin (IL)-13 and IL-4 receptors and respond to these cytokines with signal transducer and activator of transcription-6 and extracellular signal-regulated kinase (ERK) activation. The purpose of this study was to determine whether IL-13 and/or IL-4 influence eotaxin release in HASM cells and whether the ERK mitogen-activated protein (MAP) kinase pathway is involved in these events. Eotaxin release into HASM cell supernatants was assayed by ELISA, and eotaxin mRNA expression was determined by Northern blot analysis. Pretreatment with either IL-13 or IL-4 resulted in a concentration- and time-dependent release of eotaxin, although IL-4 was more effective. Eotaxin release was approximately twice baseline after treatment with 50 ng/ml IL-13 or IL-4 ( P < 0.001). IL-13 and IL-4 also acted synergistically with tumor necrosis factor (TNF)-α to induce eotaxin release: TNF-α alone (10 ng/ml for 24 h) resulted in an approximately fourfold increase in eotaxin release, whereas TNF-α in combination with IL-13 or IL-4 resulted in 10- or 20-fold increases ( P < 0.05). Similar results were obtained for eotaxin mRNA expression. Pretreatment with either U-0126 (10 μM) or PD-98059 (30 μM), both inhibitors of MAP/ERK kinase, the enzyme upstream of ERK, inhibited IL-13- or IL-4-induced eotaxin release ( P < 0.05). U-0126 also inhibited IL-13, and TNF-α induced mRNA expression. Our results indicate that IL-13 and IL-4 cause eotaxin release in HASM cells through a mechanism that, in part, involves ERK activation and suggest that the smooth muscle may be an important source of chemokines leading to eosinophil recruitment in asthma.
Exhaled NO is reduced at an early stage of hypoxia-induced pulmonary hypertension in newborn piglets
Authors: Turley Joyce E. , Nelin Leif D. , Kaplowitz Mark R. , Zhang Yongmei , Fike Candice D.
Published: March 2015

Altered nitric oxide (NO) production could contribute to the pathogenesis of hypoxia-induced pulmonary hypertension. To determine whether parameters of lung NO are altered at an early stage of hypoxia-induced pulmonary hypertension, newborn piglets were exposed to room air (control, n = 21) or 10% O2 (hypoxia, n = 19) for 3–4 days. Some lungs were isolated and perfused for measurement of exhaled NO output and the perfusate accumulation of nitrite and nitrate (NOx−), the stable metabolites of NO. Pulmonary arteries (20–600-μm diameter) and their accompanying airways were dissected from other lungs and incubated for NOx− determination. Abundances of the nitric oxide synthase (NOS) isoforms endothelial NOS and neural NOS were assessed in homogenates of PAs and airways. The perfusate NOx− accumulation was similar, whereas exhaled NO output was lower for isolated lungs of hypoxic, compared with control, piglets. The incubation solution NOx− did not differ between pulmonary arteries (PAs) of the two groups but was lower for airways of hypoxic, compared with control, piglets. Abundances of both eNOS and nNOS proteins were similar for PA homogenates from the two groups of piglets but were increased in airway homogenates of hypoxic compared with controls. The NO pathway is altered in airways, but not in PAs, at an early stage of hypoxia-induced pulmonary hypertension in newborn piglets.
γ-Glutamyl transferase deficiency results in lung oxidant stress in normoxia
Authors: Jean Jyh Chang , Liu Yue , Brown Lou Ann , Marc Robert E. , Klings Elizabeth , Joyce-Brady Martin
Published: March 2015

γ-Glutamyl transferase (GGT) is critical to glutathione homeostasis by providing substrates for glutathione synthesis. We hypothesized that loss of GGT would cause oxidant stress in the lung. We compared the lungs of GGTenu1mice, a genetic model of GGT deficiency, with normal mice in normoxia to study this hypothesis. We found GGT promoter 3 (P3) alone expressed in normal lung but GGT P3 plus P1, an oxidant-inducible GGT promoter, in GGTenu1lung. Glutathione content was barely decreased in GGTenu1lung homogenate and elevated nearly twofold in epithelial lining fluid, but the fraction of oxidized glutathione was increased three- and fourfold, respectively. Glutathione content in GGTenu1alveolar macrophages was decreased nearly sixfold, and the oxidized glutathione fraction was increased sevenfold. Immunohistochemical studies showed glutathione deficiency together with an intense signal for 3-nitrotyrosine in nonciliated bronchiolar epithelial (Clara) cells and expression of heme oxygenase-1 in the vasculature only in GGTenu1lung. When GGTenu1mice were exposed to hyperoxia, survival was decreased by 25% from control because of accelerated formation of vascular pulmonary edema, widespread oxidant stress in the epithelium, diffuse depletion of glutathione, and severe bronchiolar cellular injury. These data indicate a critical role for GGT in lung glutathione homeostasis and antioxidant defense in normoxia and hyperoxia.
Developmental differences in the shear stress-induced expression of endothelial NO synthase: changing role of AP-1
Authors: Wedgwood Stephen , Mitchell Calista J. , Fineman Jeffrey R. , Black Stephen M.
Published: March 2015

Endothelial nitric oxide synthase (eNOS) mRNA and protein levels increase during late gestation and then decrease postnatally in sheep lung parenchyma. The increase in fluid shear stress at birth, resulting from increased pulmonary blood flow, is an important mediator of postnatal eNOS gene expression. Our objective was to identify factors stimulating eNOS expression in pulmonary arterial endothelial cells (PAEC) in response to shear stress and to determine if these factors are developmentally regulated. PAEC were isolated from fetal lambs and adult sheep. Transcriptional activity from a 1,600-bp eNOS promoter fragment increased in both fetal and adult PAEC exposed to 8 h of shear stress. Conversely, activity driven from an 840-bp promoter fragment containing a putative activator protein (AP)-1 binding site was increased only in fetal PAEC. This increase was completely abolished in an identical construct containing a mutant AP-1 sequence. The AP-1 protein c-Jun was localized to the cytosol in static adult PAEC and to the nucleus in static fetal PAEC. After shear, c-Jun was nuclear localized in both cell types. However, transcriptionally active phosphorylated c-Jun was elevated only in the nuclei of sheared fetal PAEC. Resting levels of eNOS and NO were 2- and 20-fold higher, respectively, in fetal cells. Shear increased eNOS and NO in both cell types: levels were ∼2.5-fold higher in fetal PAEC. Phosphorylation of Akt and eNOS was evident in sheared fetal but not adult PAEC. We have therefore identified mechanisms of eNOS regulation at the transcriptional level and to be enzyme activation specific to the fetal pulmonary arterial circulation.
Inflammatory cytokines can enhance CD44-mediated airway epithelial cell adhesion independently of CD44 expression
Authors: Leir Shih-Hsing , Holgate Stephen T. , Lackie Peter M.
Published: March 2015

In airways, the cell surface molecule CD44 is upregulated on bronchial epithelial cells in areas of damage. We have shown that a blocking standard CD44 (CD44s) antibody caused a 77% (± 19%) inhibition of cell migration at 3 h after mechanical damage and decreased epithelial cell repair of cells grown on cell culture filter inserts. With the use of primary human bronchial epithelial cells and the bronchial epithelial cell line 16HBE 14o-, a CD44s antibody inhibited >95% ( P < 0.01) of cell binding to hyaluronic acid (HA). The cytokines TNF-α, IFN-γ, IL-1β, and IL-4 stimulated a 2- to 3.5-fold increase in CD44-dependent cell binding to HA. IFN-γ treatment did not increase CD44 expression as assessed by flow cytometry, although phorbol myristate acetate treatment did. This indicates that IFN-γ-induced cell binding to HA did not require increased CD44 expression. These data indicate that CD44 is important for bronchial epithelial cell binding to HA and that cytokines known to be expressed in inflammation can increase HA binding independently of the level of CD44 expression.
Chloride and potassium channel function in alveolar epithelial cells
Authors: O'Grady Scott M. , Lee So Yeong
Published: March 2015

Electrolyte transport across the adult alveolar epithelium plays an important role in maintaining a thin fluid layer along the apical surface of the alveolus that facilitates gas exchange across the epithelium. Most of the work published on the transport properties of alveolar epithelial cells has focused on the mechanisms and regulation of Na+transport and, in particular, the role of amiloride-sensitive Na+channels in the apical membrane and the Na+-K+-ATPase located in the basolateral membrane. Less is known about the identity and role of Cl−and K+channels in alveolar epithelial cells, but studies are revealing important functions for these channels in regulation of alveolar fluid volume and ionic composition. The purpose of this review is to examine previous work published on Cl−and K+channels in alveolar epithelial cells and to discuss the conclusions and speculations regarding their role in alveolar cell transport function.
Regulation of nitric oxide production in limb and ventilatory muscles during chronic exercise training
Authors: Vassilakopoulos T. , Deckman G. , Kebbewar M. , Rallis G. , Harfouche R. , Hussain S. N. A.
Published: March 2015

In this study, we evaluated the differential influence of chronic treadmill training (30 m/min, 15% incline, 1 h/day, 5 days/wk) on nitric oxide (NO) production and NO synthase (NOS) isoform expression as well as 3-nitrotyrosine formation (footprint of peroxynitrite) both in limb (gastrocnemius) and ventilatory (diaphragm) muscles. A group of exercise-trained rats and a control group (no training) were examined after a 4-wk experimental period. Exercise training elicited an approximate fourfold rise in gastrocnemius NOS activity and augmented protein expression of the endothelial (eNOS) and neuronal (nNOS) isoforms of NOS to ∼480% and 240%, respectively. Qualitatively similar but quantitatively smaller elevations in NOS activity and eNOS and nNOS expression were observed in the diaphragm. No detectable inducible NOS (iNOS) protein expression was found in any of the muscle samples. Training increased the intensity of 3-nitrotyrosine only in the gastrocnemius muscle. We conclude that whole body exercise training enhances both limb and ventilatory muscle NO production and that constitutive and not iNOS isoforms are responsible for increased protein tyrosine nitration in trained limb muscles.
Absence of SP-A modulates innate and adaptive defense responses to pulmonary influenza infection
Authors: LeVine Ann Marie , Hartshorn Kevan , Elliott James , Whitsett Jeffrey , Korfhagen Thomas
Published: March 2015

Mice lacking surfactant protein SP-A [SP-A(−/−)] and wild type SP-A(+/+) mice were infected with influenza A virus (IAV) by intranasal instillation. Decreased clearance of IAV was observed in SP-A(−/−) mice and was associated with increased pulmonary inflammation. Treatment of SP-A(−/−) mice with exogenous SP-A enhanced viral clearance and decreased lung inflammation. Uptake of IAV by alveolar macrophages was similar in SP-A(−/−) and SP-A(+/+) mice. Myeloperoxidase activity was reduced in isolated bronchoalveolar lavage neutrophils from SP-A(−/−) mice. B lymphocytes and activated T lymphocytes were increased in the lung and spleen, whereas T helper (Th) 1 responses were increased [interferon-γ, interleukin (IL)-2, and IgG2a] and Th2 responses were decreased (IL-4, and IL-10, and IgG1) in the lungs of SP-A(−/−) mice 7 days after IAV infection. In the absence of SP-A, impaired viral clearance was associated with increased lung inflammation, decreased neutrophil myeloperoxidase activity, and increased Th1 responses. Because the airway is the usual portal of entry for IAV and other respiratory pathogens, SP-A is likely to play a role in innate defense and adaptive immune responses to IAV.
Inhibition of prostaglandin synthesis during polystyrene microsphere-induced pulmonary embolism in the rat
Authors: Jones Alan E. , Watts John A. , Debelak Jacob P. , Thornton Lisa R. , Younger John G. , Kline Jeffrey A.
Published: March 2015

Our objective was to test the effect of inhibition of thromboxane synthase versus inhibition of cyclooxygenase (COX)-1/2 on pulmonary gas exchange and heart function during simulated pulmonary embolism (PE) in the rat. PE was induced in rats via intrajugular injection of polystyrene microspheres (25 μm). Rats were randomized to one of three posttreatments: 1) placebo (saline), 2) thromboxane synthase inhibition (furegrelate sodium), or 3) COX-1/2 inhibition (ketorolac tromethamine). Control rats received no PE. Compared with controls, placebo rats had increased thromboxane B2(TxB2) in bronchoalveolar lavage fluid and increased urinary dinor TxB2. Furegrelate and ketorolac treatments reduced TxB2and dinor TxB2to control levels or lower. Both treatments significantly decreased the alveolar dead space fraction, but neither treatment altered arterial oxygenation compared with placebo. Ketorolac increased in vivo mean arterial pressure and ex vivo left ventricular pressure (LVP) and right ventricular pressure (RVP). Furegrelate improved RVP but not LVP. Experimental PE increased lung and systemic production of TxB2. Inhibition at the COX-1/2 enzyme was equally as effective as inhibition of thromboxane synthase at reducing alveolar dead space and improving heart function after PE.
Effects of IL-13 on airway responses in the guinea pig
Authors: Morse Brian , Sypek Joseph P. , Donaldson Debra D. , Haley Kathleen J. , Lilly Craig M.
Published: March 2015

Levels of interleukin (IL)-13 are increased in asthmatic airways. IL-13 has been shown to be necessary and sufficient for allergen-induced airway hyperresponsiveness and increased inflammatory cell counts in bronchoalveolar lavage (BAL) fluid in a murine model of asthma but is thought to protect against airway inflammation when low doses are provided to the guinea pig lung. To determine the role of IL-13 in the guinea pig, we studied the effects of a 360-μg/kg dose of nebulized IL-13 in naive animals and of IL-13 abrogation after airway challenge of sensitized animals. Nebulized IL-13 significantly decreased the dose of histamine required to double baseline respiratory system resistance (ED100, 22 ± 3 vs. 13 ± 2 nmol/kg; P < 0.05) and was associated with recovery of significantly greater numbers of macrophages, lymphocytes, eosinophils, and neutrophils in BAL fluid. Guinea pigs pretreated with a fusion protein that binds IL-13 [soluble IL-13 receptor α2 (sIL-13Rα2)] were protected from developing antigen-induced airway hyperresponsiveness (ED100, 210 ± 50 vs. 20 ± 10 nmol/kg; P <0.01). sIL-13Rα2 (2 doses of 20 mg/kg) significantly reduced the histological grade of allergen-induced lung eosinophil accumulation, whereas the effects of two doses of 10 mg/kg were not significant. These findings demonstrate that the tissue levels of IL-13 induced by allergen challenge of sensitized animals induce airway hyperresponsiveness and inflammation and that IL-13 is required for the expression of allergen-induced airway hyperresponsiveness in the guinea pig ovalbumin model.
Susceptibility to ozone-induced acute lung injury in iNOS-deficient mice
Authors: Kenyon Nicholas J. , van der Vliet Albert , Schock Bettina C. , Okamoto Tatsuya , McGrew Gabrielle M. , Last Jerold A.
Published: March 2015

Mice deficient in inducible nitric oxide synthase (iNOS; C57Bl/6Ai-[KO] NOS2 N5) or wild-type C57Bl/6 mice were exposed to 1 part/million of ozone 8 h/night or to filtered air for three consecutive nights. Endpoints measured included lavagable total protein, macrophage inflammatory protein (MIP)-2, matrix metalloproteinase (MMP)-9, cell content, and tyrosine nitration of whole lung proteins. Ozone exposure caused acute edema and an inflammatory response in the lungs of wild-type mice, as indicated by significant increases in lavage protein content, MIP-2 and MMP-9 content, and polymorphonuclear leukocytes. The iNOS knockout mice showed significantly greater levels of lung injury by all of these criteria than did the wild-type mice. We conclude that iNOS knockout mice are more susceptible to acute lung damage induced by exposure to ozone than are wild-type C57Bl/6 mice and that protein nitration is associated with the degree of inflammation and not dependent on iNOS-derived nitric oxide.
Activation of the G2 cell cycle checkpoint enhances survival of epithelial cells exposed to hyperoxia
Authors: O'Reilly Michael A. , Staversky Rhonda J. , Finkelstein Jacob N. , Keng Peter C.
Published: March 2015

Reactive oxygen species produced during hyperoxia damage DNA, inhibit proliferation in G1- through p53-dependent activation of p21Cip1/WAF1/Sdi1, and kill cells. Because checkpoint activation protects cells from genotoxic stress, we investigated cell proliferation and survival of the murine type II epithelial cell line MLE15 during hyperoxia. These cells were chosen for study because they express Simian large and small-T antigens, which transform cells in part by disrupting the p53-dependent G1 checkpoint. Cell counts, 5-bromo-2′-deoxyuridine labeling, and flow cytometry revealed that hyperoxia slowed cell cycle progression after one replication, resulting in a pronounced G2 arrest by 72 h. Addition of caffeine, which inactivates the G2 checkpoint, diminished the percentage of hyperoxic cells in G2 and increased the percentage in sub-G1 and G1. Abrogation of the G2 checkpoint was associated with enhanced oxygen-induced DNA strand breaks and cell death. Caffeine did not affect DNA integrity or viability of cells exposed to room air. Similarly, caffeine abrogated the G2 checkpoint in hyperoxic A549 epithelial cells and enhanced oxygen-induced toxicity. These data indicate that hyperoxia rapidly inhibits proliferation after one cell cycle and that the G2 checkpoint is critical for limiting DNA damage and cell death.
Interleukin-9 influences chemokine release in airway smooth muscle: role of ERK
Authors: Baraldo Simonetta , Faffe Deborah S. , Moore Paul E. , Whitehead Timothy , McKenna Matthew , Silverman Eric S. , Panettieri Reynold A. , Shore Stephanie A.
Published: March 2015

Interleukin (IL)-9 is a pleiotropic cytokine that has been proposed as a candidate gene for asthma. As IL-9 expression is correlated with airway hyperresponsiveness in animals, we examined the effects of IL-9 on cultured human airway smooth muscle (HASM) cells. IL-9 alone had no effect on IL-8 release, but at concentrations of ≥30 ng/ml, IL-9 significantly increased IL-8 release induced by TNF-α. IL-9 increased phosphorylation of extracellular signal-regulated protein kinase (ERK, p42 and p44) in a concentration- and time-dependent fashion, and U-0126 (10 μM), which inhibits ERK phosphorylation, abolished the synergism between TNF-α and IL-9 on IL-8 release. IL-9 alone had no effect on eotaxin release into HASM cell supernatants but at concentrations of ≥10 ng/ml caused an ∼50% increase in release of eotaxin evoked by IL-13 (10 ng/ml). U-0126 blocked the synergism between IL-9 and IL-13 on eotaxin release. IL-9 had no effect on cyclooxygenase-2 (COX-2) expression or PGE2release and did not augment the COX-2 expression that was induced by IL-1β. Our results indicate that airway smooth muscle is a target for IL-9 and that IL-9 amplifies the potential for these cells to recruit eosinophils and neutrophils into the airways by a mechanism involving ERK.
Changes in alveolar epithelial cell proportions during fetal and postnatal development in sheep
Authors: Flecknoe S. J. , Wallace M. J. , Cock M. L. , Harding R. , Hooper S. B.
Published: March 2015

Basal lung expansion is an important determinant of alveolar epithelial cell (AEC) phenotype in the fetus. Because basal lung expansion increases toward term and is reduced after birth, we hypothesized that these changes would be associated with altered proportions of AECs. AEC proportions were calculated with electron microscopy in fetal and postnatal sheep. Type I AECs increased from 4.8 ± 1.3% at 91 days to 63.0 ± 3.6% at 111 days of gestation, remained at this level until term, and decreased to 44.8 ± 1.8% after birth. Type II AECs increased from 4.3 ± 1.5% at 111 days to 29.6 ± 4.1% at 128 days of gestation, remained at this level until term, and then increased to 52.9 ± 1.5% after birth. Surfactant protein (SP)-A, -B and -C mRNA levels increased with increasing gestational age before birth, but the changes in SP expression after birth were inconsistent. Thus before birth type I AECs predominate, whereas after birth type II AECs predominate, possibly due to the reduction in basal lung expansion associated with the entry of air into the lungs.
Parathyroid hormone-related protein-(38–64) regulates lung cell proliferation after silica injury
Authors: Hastings Randolph H. , Asirvatham Angela , Quintana Rick , Sandoval Rebeca , Dutta Ruchika , Burton Douglas W. , Deftos Leonard J.
Published: March 2015

Inhalation of silica leads to acute lung injury and alveolar type II cell proliferation. Type II cell proliferation after hyperoxic lung injury is regulated, in part, by parathyroid hormone-related protein (PTHrP). In this study, we investigated lung PTHrP and its effects on epithelial proliferation after injury induced by silica. Lung PTHrP decreased modestly 4 days after we instilled 10 mg of silica into rat lungs and then recovered from 4 to 28 days. The number of proliferating cell nuclear antigen (PCNA)-positive type II cells was increased threefold in silica-injured lungs compared with controls. Subsequently, rats were treated with four exogenous PTHrP peptides in the silica instillate, which were administered subcutaneously daily. One peptide, PTHrP-(38–64), had consistent and significant effects on cell proliferation. PTHrP-(38–64) increased the median number of PCNA-positive cells/field nearly fourfold above controls, 380 vs. 109 ( P < 0.05). Thymidine incorporation was 2.5 times higher in type II cells isolated from rats treated with PTHrP-(38–64) compared with PBS. PTHrP-(38–64) significantly increased the number of cells expressing alkaline phosphatase, a type II cell marker. This study indicates that PTHrP-(38–64) stimulates type II cell growth and may have a role in lung repair in silica-injured rats.
Morpho-functional analysis of lung tissue in mild interstitial edema
Authors: Conforti Elena , Fenoglio Carla , Bernocchi Graziella , Bruschi Ombretta , Miserocchi Giuseppe A.
Published: March 2015

Mild pulmonary interstitial edema was shown to cause fragmentation of interstitial matrix proteoglycans. We therefore studied compartmental fluid accumulation by light and electron microscopy on lungs of anesthetized rabbits fixed in situ by vascular perfusion after 0.5 ml · kg−1 · min−1 iv saline infusion for 180 min causing ∼6% increase in lung weight. Morphometry showed that a relevant portion (44%) of extravascular fluid is detected early in the alveolar septa, 85% of this fluid accumulating in the thick portion of the air-blood barrier. The arithmetic mean thickness of the barrier increased in interstitial edema from 1.06 ± 0.05 (SE) to 1.33 ± 0.06 μm. The harmonic mean thickness increased from 0.6 ± 0.03 to 0.86 ± 0.07 μm, mostly due to thickening of the thin portion causing an increase in gas diffusion resistance. Despite some structural damage, the air-blood barrier displays a relatively high structural resistance providing a safety factor against the development of severe edema. It is suggested that the increase in extra-alveolar perivascular space occurs as a consequence of fluid accumulation in the air-blood barrier.
Angiopoietin-1 and VEGF in vascular development and angiogenesis in hypoplastic lungs
Authors: Chinoy Mala R. , Graybill Megan M. , Miller Shane A. , Lang C. Max , Kauffman Gordon L.
Published: March 2015

We hypothesized that exposure of murine fetuses to environmental toxins, such as nitrofen, during early embryogenesis alters vasculogenesis. To address our hypothesis, we assessed protein levels of endothelial cell-selective angiogenic factors: angiopoietin (ANG)-1, vascular endothelial growth factor (VEGF), and mediator of VEGF signaling, VEGF receptor-2 [fetal liver kinase (Flk)-1], a transmembrane receptor tyrosine kinase. VEGF and Flk-1 proteins were lower in hypoplastic lungs from pseudoglandular to alveolar stages than in normal lungs at equivalent developmental time points significant for induction of pulmonary vasculogenesis and angiogenesis. ANG-1 protein was higher in hypoplastic lungs than in normal lungs at all the developmental stages considered in this study, i.e., pseudoglandular, canalicular, saccular, and alveolar stages. We assessed exogenous VEGF-mediated endothelial cell response on extracellular signal-regulated kinase (ERK) 1/2, also referred to as p44/42 mitogen-activated protein kinase. Hypoplastic lungs had more elevated ERK 1/2 protein than normal developing lungs. Exposure to exogenous VEGF activated ERK 1/2 in normal developing lungs but not in hypoplastic lungs. Our results suggest that in hypoplastic lungs: 1) low VEGF signifies negative effects on vasculogenesis/angiogenesis and indicates altered endothelial-mesenchymal interactions; 2) increased ANG-1 protein may be required to maintain vessel integrity and quiescence; and 3) regulation of ERK 1/2 protein is affected in hypoplastic lungs. We speculate that extensive remodeling of blood vessels in hypoplastic lungs may occur to compensate for structurally and functionally defective vasculature.
Receptors and pathways mediating the effects of prostaglandin E2 on airway tone
Authors: Tilley Stephen L. , Hartney John M. , Erikson Christopher J. , Jania Corey , Nguyen Mytrang , Stock Jeffrey , McNeisch John , Valancius Cathy , Panettieri Reynold A. , Penn Raymond B. , Koller Beverly H.
Published: March 2015

Prostaglandin E2(PGE2) has complex effects on airway tone, and the existence of four PGE2 [E-prostanoid (EP)] receptors, each with distinct signaling characteristics, has provided a possible explanation for the seemingly contradictory actions of this lipid mediator. To identify the receptors mediating the actions of PGE2 on bronchomotor tone, we examined its effects on the airways of wild-type and EP receptor-deficient mice. In conscious mice the administration of PGE2 increased airway responsiveness primarily through the EP1 receptor, although on certain genetic backgrounds a contribution of the EP3 receptor was detected. These effects of PGE2 were eliminated by pretreatment with either atropine or bupivacaine and were undetectable in anesthetized mice or in denervated tracheal rings, where only EP2-mediated relaxation of airway smooth muscle was observed. Together, our findings are consistent with a model in which PGE2 modulates airway tone by activating multiple receptors expressed on various cell populations and in which the relative contribution of these receptors might depend on the expression of modifier alleles. PGE2/EP1/EP3-induced airway constriction occurs indirectly through activation of neural pathways, whereas PGE2-induced bronchodilation results from direct activation of EP2 receptors on airway smooth muscle. This segregation of EP receptor function within the airway suggests that PGE2 analogs that selectively activate the EP2 receptor without activating the EP1/EP3 receptors might prove useful in the treatment of asthma.
Activation of NF-κB-dependent gene expression by silica in lungs of luciferase reporter mice
Authors: Hubbard Andrea K. , Timblin Cynthia R. , Shukla Arti , Rincón Mercedes , Mossman Brooke T.
Published: March 2015

Occupational exposure to crystalline silica is associated with the development of pulmonary inflammation and silicosis, yet how silica initiates pulmonary fibrosis and which cell types are involved are unclear. In studies here, we hypothesized that silica particles interact initially with pulmonary epithelial cells and alveolar macrophages (AMs) to cause transcriptional activation of nuclear factor (NF)-κB-regulated genes encoding inflammatory cytokines. Exposure of NF-κB luciferase reporter mice intratracheally to silica or lipopolysaccharide (LPS), but not the nonfibrogenic particle titanium dioxide (TiO2), increased immunoreactivity of luciferase protein in bronchiolar epithelial cells and AMs. Ribonuclease protection assays revealed significant ( P ≤ 0.05) increases in mRNA levels of inducible nitric oxide synthase, tumor necrosis factor-α, macrophage inflammatory protein-2, macrophage chemotactic protein-1 (MCP-1), interferon-γ, interleukin (IL)-6, and IL-12 in lung homogenates of reporter mice after exposures to silica or LPS. Immunoreactivity of MCP-1 in these animals was localized to AMs and epithelial cells. These data are the first to show activation of NF-κB in situ by fibrogenic particles in pulmonary epithelial cells and AMs. Increased expression of NF-κB-related inflammatory cytokines by these cell types, which first encounter silica after inhalation, may be critical to the initiation of silica-associated lung diseases, thus providing a rationale for focusing on NF-κB in preventive and therapeutic strategies.
High tidal volume ventilation induces NOS2 and impairs cAMP- dependent air space fluid clearance
Authors: Frank James A. , Pittet Jean-François , Lee Hyon , Godzich Micaela , Matthay Michael A.
Published: March 2015

Tidal volume reduction during mechanical ventilation reduces mortality in patients with acute lung injury and the acute respiratory distress syndrome. To determine the mechanisms underlying the protective effect of low tidal volume ventilation, we studied the time course and reversibility of ventilator-induced changes in permeability and distal air space edema fluid clearance in a rat model of ventilator-induced lung injury. Anesthetized rats were ventilated with a high tidal volume (30 ml/kg) or with a high tidal volume followed by ventilation with a low tidal volume of 6 ml/kg. Endothelial and epithelial protein permeability were significantly increased after high tidal volume ventilation but returned to baseline levels when tidal volume was reduced. The basal distal air space fluid clearance (AFC) rate decreased by 43% ( P < 0.05) after 1 h of high tidal volume but returned to the preventilation rate 2 h after tidal volume was reduced. Not all of the effects of high tidal volume ventilation were reversible. The cAMP-dependent AFC rate after 1 h of 30 ml/kg ventilation was significantly reduced and was not restored when tidal volume was reduced. High tidal volume ventilation also increased lung inducible nitric oxide synthase (NOS2) expression and air space total nitrite at 3 h. Inhibition of NOS2 activity preserved cAMP-dependent AFC. Because air space edema fluid inactivates surfactant and reduces ventilated lung volume, the reduction of cAMP-dependent AFC by reactive nitrogen species may be an important mechanism of clinical ventilator-associated lung injury.
IL-13-dependent autocrine signaling mediates altered responsiveness of IgE-sensitized airway smooth muscle
Authors: Grunstein M. M. , Hakonarson H. , Leiter J. , Chen M. , Whelan R. , Grunstein J. S. , Chuang S.
Published: March 2015

In testing the hypothesis that interleukin-4 receptor α-subunit (IL-4Rα)-coupled signaling mediates altered airway smooth muscle (ASM) responsiveness in the atopic sensitized state, isolated rabbit tracheal ASM segments were passively sensitized with immunoglobulin E (IgE) immune complexes, both in the absence and presence of an IL-4Rα blocking antibody (anti-IL-4RαAb). Relative to control ASM, IgE-sensitized tissues exhibited enhanced isometric constrictor responses to administered ACh and attenuated relaxation responses to isoproterenol. These proasthmatic-like effects were prevented in IgE-sensitized ASM that were pretreated with anti-IL-4RαAb. In complementary experiments, IgE-sensitized cultured human ASM cells exhibited upregulated expression of IL-13 mRNA and protein, whereas IL-4 expression was undetected. Moreover, extended studies demonstrated that 1) exogenous IL-13 administration to naı̈ve ASM elicited augmented contractility to ACh and impaired relaxation to isoproterenol, 2) these effects of IL-13 were prevented by pretreating the tissues with an IL-5 receptor blocking antibody, and 3) IL-13 administration induced upregulated mRNA expression and release of IL-5 protein from cultured ASM cells. Collectively, these findings provide new evidence demonstrating that the altered responsiveness of IgE-sensitized ASM is largely attributed to activation of an intrinsic Th2-type autocrine mechanism involving IL-13/IL-4Rα-coupled release and action of IL-5 in the sensitized ASM itself.
Role of PKC, tyrosine kinases, and Rho kinase in α-adrenoreceptor-mediated PASM contraction
Authors: Damron Derek S. , Kanaya Noriaki , Homma Yasuyuki , Kim Si-Oh , Murray Paul A.
Published: March 2015

Our objectives were to identify the relative contributions of intracellular free Ca2+concentration ([Ca2+]i) and myofilament Ca2+sensitivity in the pulmonary artery smooth muscle (PASM) contractile response to the α-adrenoreceptor agonist phenylephrine (PE) and to assess the role of PKC, tyrosine kinases (TK), and Rho kinase (ROK) in that response. Our hypothesis was that multiple signaling pathways are involved in the regulation of [Ca2+]i, myofilament Ca2+sensitization, and vasomotor tone in response to α-adrenoreceptor stimulation of PASM. Simultaneous measurement of [Ca2+]iand isometric tension was performed in isolated canine pulmonary arterial strips loaded with fura 2-AM. PE-induced tension development was due to sarcolemmal Ca2+influx, Ca2+release from inositol 1,4,5-trisphosphate-dependent sarcoplasmic reticulum Ca2+stores, and myofilament Ca2+sensitization. Inhibition of either PKC or TK partially attenuated the sarcolemmal Ca2+influx component and the myofilament Ca2+sensitizing effect of PE. Combined inhibition of PKC and TK did not have an additive attenuating effect on PE-induced Ca2+sensitization. ROK inhibition slightly decreased [Ca2+]ibut completely inhibited myofilament Ca2+sensitization. These results indicate that PKC and TK activation positively regulate sarcolemmal Ca2+influx in response to α-adrenoreceptor stimulation in PASM but have relatively minor effects on myofilament Ca2+sensitivity. ROK is the predominant pathway mediating PE-induced myofilament Ca2+sensitization.
Surface activity of a synthetic lung surfactant containing a phospholipase-resistant phosphonolipid analog of dipalmitoyl phosphatidylcholine
Authors: Wang Z. , Schwan A. L. , Lairson L. L. , O'Donnell J. S. , Byrne G. F. , Foye A. , Holm B. A. , Notter R. H.
Published: March 2015

Surface activity and sensitivity to inhibition from phospholipase A2(PLA2), lysophosphatidylcholine (LPC), and serum albumin were studied for a synthetic C16:0 diether phosphonolipid (DEPN-8) combined with 1.5% by weight of mixed hydrophobic surfactant proteins (SP)-B/C purified from calf lung surfactant extract (CLSE). Pure DEPN-8 had better adsorption and film respreading than the major lung surfactant phospholipid dipalmitoyl phosphatidylcholine and reached minimum surface tensions <1 mN/m under dynamic compression on the Wilhelmy balance and on a pulsating bubble surfactometer (37°C, 20 cycles/min, 50% area compression). DEPN-8 + 1.5% SP-B/C exhibited even greater adsorption and had overall dynamic surface tension lowering equal to CLSE on the bubble. In addition, films of DEPN-8 + 1.5% SP-B/C on the Wilhelmy balance had better respreading than CLSE after seven (but not two) cycles of compression-expansion at 23°C. DEPN-8 is structurally resistant to degradation by PLA2, and DEPN-8 + 1.5% SP-B/C maintained high adsorption and dynamic surface activity in the presence of this enzyme. Incubation of CLSE with PLA2led to chemical degradation, generation of LPC, and reduced surface activity. DEPN-8 + 1.5% SP-B/C was also more resistant than CLSE to direct biophysical inhibition by LPC, and the two were similar in their sensitivity to biophysical inhibition by serum albumin. These findings indicate that synthetic surfactants containing DEPN-8 combined with surfactant proteins or related synthetic peptides have potential utility for treating surfactant dysfunction in inflammatory lung injury.
Adenosine A3receptor-mediated potentiation of mucociliary transport and epithelial ciliary motility
Authors: Taira Manako , Tamaoki Jun , Nishimura Kazuyuki , Nakata Junko , Kondo Mitsuko , Takemura Hisashi , Nagai Atsushi
Published: March 2015

To examine the effect of adenosine A3receptor stimulation on airway mucociliary clearance, we measured transport of Evans blue dye in rabbit trachea in vivo and ciliary motility of epithelium by the photoelectric method in vitro. Mucociliary transport was enhanced dose dependently by the selective A3agonist N6-(3-iodobenzyl)-5′- N-methylcarbamoyladenosine (IB-MECA) and to a lesser extent by the less-selective N6-2-(4-amino-3-iodophenyl)ethyladenosine, whereas the A1agonist N-cyclopentyladenosine (CPA) and the A2agonist CGS-21680 had no effect. The effect of IB-MECA was abolished by pretreatment with the selective A3antagonist MRS-1220 but not by the A1antagonist 1,3-dipropyly-8-cyclopentylxanthine or the A2antagonist 3,7-dimethyl-l-propargylxanthine. Epithelial ciliary beat frequency was increased by IB-MECA in a concentration-dependent manner, the maximal increase being 33%, and this effect was inhibited by MRS-1220. The IB-MECA-induced ciliary stimulation was not altered by the Rp diastereomer of cAMP but was greatly inhibited by Ca2+-free medium containing BAPTA-AM. Incubation with IB-MECA increased intracellular Ca2+contents. Therefore, A3agonist enhances airway mucociliary clearance probably through Ca2+-mediated stimulation of ciliary motility of airway epithelium.