bluuebunny/crossref_metadata_2025 huggingface dataset:
Depletion of pulmonary EC-SOD after exposure to hyperoxia
Authors: Oury Tim D. , Schaefer Lisa M. , Fattman Cheryl L. , Choi Augustine , Weck Karen E. , Watkins Simon C.
Published: March 2015

Extracellular superoxide dismutase (EC-SOD) is highly expressed in lung tissue. EC-SOD contains a heparin-binding domain that is sensitive to proteolysis. This heparin-binding domain is important in allowing EC-SOD to exist in relatively high concentrations in specific regions of the extracellular matrix and on cell surfaces. EC-SOD has been shown to protect the lung against hyperoxia in transgenic and knockout studies. This study tests the hypothesis that proteolytic clearance of EC-SOD from the lung during hyperoxia contributes to the oxidant-antioxidant imbalance that is associated with this injury. Exposure to 100% oxygen for 72 h resulted in a significant decrease in EC-SOD levels in the lungs and bronchoalveolar lavage fluid of mice. This correlated with a significant depletion of EC-SOD from the alveolar parenchyma as determined by immunofluorescence and immunohistochemistry. EC-SOD mRNA was unaffected by hyperoxia; however, there was an increase in the ratio of proteolyzed to uncut EC-SOD after hyperoxia, which suggests that hyperoxia depletes EC-SOD from the alveolar parenchyma by cutting the heparin-binding domain. This may enhance hyperoxic pulmonary injury by altering the oxidant-antioxidant balance in alveolar spaces.
Hypoxic but not anoxic stabilization of HIF-1α requires mitochondrial reactive oxygen species
Authors: Schroedl Clara , McClintock David S. , Budinger G. R. Scott , Chandel Navdeep S.
Published: March 2015

The molecular mechanisms by which cells detect hypoxia (1.5% O2), resulting in the stabilization of hypoxia-inducible factor 1α (HIF-1α) protein remain unclear. One model proposes that mitochondrial generation of reactive oxygen species is required to stabilize HIF-1α protein. Primary evidence for this model comes from the observation that cells treated with complex I inhibitors, such as rotenone, or cells that lack mitochondrial DNA (ρ0-cells) fail to generate reactive oxygen species or stabilize HIF-1α protein in response to hypoxia. In the present study, we investigated the role of mitochondria in regulating HIF-1α protein stabilization under anoxia (0% O2). Wild-type A549 and HT1080 cells stabilized HIF-1α protein in response to hypoxia and anoxia. The ρ0-A549 cells and ρ0-HT1080 cells failed to accumulate HIF-1α protein in response to hypoxia. However, both ρ0-A549 and ρ0-HT1080 were able to stabilize HIF-1α protein levels in response to anoxia. Rotenone inhibited hypoxic, but not anoxic, stabilization of HIF-1α protein. These results indicate that a functional electron transport chain is required for hypoxic but not anoxic stabilization of HIF-1α protein.
Alveolar macrophage-mediated elastolysis: roles of matrix metalloproteinases, cysteine, and serine proteases
Authors: Russell Richard E. K. , Thorley Andrew , Culpitt Sarah V. , Dodd Sara , Donnelly Louise E. , Demattos Carmen , Fitzgerald Mary , Barnes Peter J.
Published: March 2015

Chronic obstructive pulmonary disease (COPD) is a common lung disease with cigarette smoking as the major etiological factor, but only 15% of smokers develop COPD. Destruction of lung elastin observed in COPD is mediated by many enzymes, including cysteine, serine, and matrix metalloproteinases (MMP). The contribution of these enzymes to the lung elastolytic load, released from alveolar macrophages collected from nonsmokers, healthy smokers, and COPD patients, was examined by radiolabeled elastin as substrate in the presence of specific enzyme inhibitors. The activity of MMP was further examined by zymography and Western blotting. COPD macrophages degraded more elastin than either of the other groups. Elastolysis was greatest in the initial 24 h. Through the 72-h culture period, the contribution to elastolysis of serine elastases decreased, MMP increased, and cysteine elastases remained constant. The increased release of elastolytic enzymes in COPD subjects may explain why some smokers develop COPD. This difference may be due to unknown susceptibility factors. Serine proteases play a significant role; however, other enzymes, particularly the MMP, deserve further investigation.
PM10-exposed macrophages stimulate a proinflammatory response in lung epithelial cells via TNF-α
Authors: Jiménez L. A. , Drost E. M. , Gilmour P. S. , Rahman I. , Antonicelli F. , Ritchie H. , MacNee W. , Donaldson K.
Published: March 2015

There is now considerable evidence for an association between the levels of particulate air pollution [particulate matter <10 μm in aerodynamic diameter (PM10)] and various adverse health endpoints. The release of proinflammatory mediators from PM10-exposed macrophages may be important in stimulating cytokine release from lung epithelial cells, thus amplifying the inflammatory response. A549 cells were treated with conditioned media from monocyte-derived macrophages stimulated with PM10, titanium dioxide (TiO2), or ultrafine TiO2. We demonstrate that only conditioned media from PM10-stimulated macrophages significantly increased nuclear factor-κB and activator protein-1 DNA binding, enhanced interleukin-8 (IL-8) mRNA levels as assessed by RT-PCR, and augmented IL-8 protein levels, over untreated controls. Furthermore, PM10-conditioned media also caused transactivation of IL-8 as determined by an IL-8-chloramphenicol acetyl transferase reporter. Analysis of these conditioned media revealed marked increases in tumor necrosis factor-α (TNF-α) and protein levels and enhanced chemotactic activity for neutrophils. Preincubation of conditioned media with TNF-α-neutralizing antibodies significantly reduced IL-8 production. These data suggest that PM10-activated macrophages may amplify the inflammatory response by enhancing IL-8 release from lung epithelial cells, in part, via elaboration of TNF-α.
Transcriptional regulation of pulmonary elastin gene expression in elastase-induced injury
Authors: Rich Celeste B. , Carreras Isabel , Lucey Edgar C. , Jaworski Julie A. , Buczek-Thomas Jo Ann , Nugent Matthew A. , Stone Phillip , Foster Judith Ann
Published: March 2015

Previously we have shown that treatment of confluent, pulmonary fibroblast cultures with elastase results in upregulation of elastin mRNA and protein levels. In the present study we focused on determining the level at which elastin expression is upregulated after elastase exposure. We examined as models for this investigation elastin gene expression in primary pulmonary fibroblast cells during the transition from subconfluent to confluent cultures and in confluent, matrix-laden cultures treated briefly with elastase. In addition, we extended our studies to mice that were given an intratracheal dose of elastase; the effects on lung elastin mRNA and elastin promoter activity levels were measured and compared with results from in vitro cell models. The results demonstrate that upregulation of elastin gene expression during the transition of subconfluent to confluent cultures and after elastase injury is associated with an increase in the level of transcription both in vitro and in vivo. Furthermore, intratracheal administration of elastase to transgenic mice illustrates that the increased levels of elastin mRNA are accompanied by increased activity of the elastin gene promoter in cells spatially positioned near major sites of tissue injury.
Na+-K+-ATPase activity in alveolar epithelial cells increases with cyclic stretch
Authors: Fisher Jacob L. , Margulies Susan S.
Published: March 2015

Na+-K+-ATPase pumps (Na+ pumps) in the alveolar epithelium create a transepithelial Na+ gradient crucial to keeping fluid from the pulmonary air space. We hypothesized that alveolar epithelial stretch stimulates Na+ pump trafficking to the basolateral membrane (BLM) and, thereby, increases overall Na+ pump activity. Alveolar type II cells were isolated from Sprague-Dawley rats and seeded onto elastic membranes coated with fibronectin or 5-day-conditioned extracellular matrix. After 2 days in culture, cells were uniformly stretched for 1 h in a custom-made device. Na+ pump activity was subsequently assessed by ouabain-inhibitable uptake of 86Rb+, a K+ tracer, and BLM Na+ pump abundance was measured. In support of our hypothesis, cells increased Na+pump activity in a “dose-dependent” manner when stretched to 12, 25, or 37% change in surface area (ΔSA), and cells stretched to 25% ΔSA more than doubled Na+ pump abundance in the BLM. Cells on 5-day matrix tolerated higher strain than cells on fibronectin before the onset of Na+ pump upregulation. Treatment with Gd3+, a stretch-activated channel blocker, amiloride, a Na+ channel blocker, or both reduced but did not abolish stretch-induced effects. Sustained tonic stretch, unlike cyclic stretch, elicited no significant Na+ pump response.
Mechanisms of bradykinin-mediated dilation in newborn piglet pulmonary conducting and resistance vessels
Authors: Aschner Judy L. , Smith Thuy K. , Kovacs Nora , Pinheiro Joaquim M. B. , Fuloria Mamta
Published: March 2015

Bradykinin (BK) is a potent dilator of the perinatal pulmonary circulation. We investigated segmental differences in BK-induced dilation in newborn pig large conducting pulmonary artery and vein rings and in pressurized pulmonary resistance arteries (PRA). In conducting pulmonary arteries and veins, BK-induced relaxation is abolished by endothelial disruption and by inhibition of nitric oxide (NO) synthase with nitro-l-arginine (l-NA). In PRA, two-thirds of the dilation response isl-NA insensitive. Charybdotoxin plus apamin and depolarization with KCl abolish the l-NA-insensitive dilations, findings that implicate the release of endothelium-derived hyperpolarizing factor (EDHF). However, endothelium-disrupted PRA retain the ability to dilate to BK but not to ACh or A-23187. In endothelium-disrupted PRA, dilation was inhibited by charybdotoxin. Thus in PRA, BK elicits dilation by multiple and duplicative signaling pathways. Release of NO and EDHF contributes to the response in endothelium-intact PRA; in endothelium-disrupted PRA, dilation occurs by direct activation of vascular smooth muscle calcium-dependent potassium channels. Redundant signaling pathways mediating pulmonary dilation to BK may be required to assure a smooth transition to extrauterine life.
Relationship between asynchronous Ca2+ waves and force development in intact smooth muscle bundles of the porcine trachea
Authors: Kuo Kuo-Hsing , Dai Jiazhen , Seow Chun Yong , Lee Cheng-Han , van Breemen Cornelis
Published: March 2015

Fluctuations in intracellular calcium concentration ([Ca2+]i) constitute the main link in excitation-contraction coupling (E-C coupling) in airway smooth muscle cells (ASMC). It has recently been reported that ACh induces asynchronous recurring Ca2+ waves in intact ASMC of murine bronchioles. With the use of a novel technique allowing us to simultaneously measure subcellular [Ca2+]i and force generation in ASMC located within an intact tracheal muscle bundle, we examined a similar pattern of Ca2+ signaling in the trachea. We found that application of ACh resulted in the generation of recurring intracellular Ca2+ waves progressing along the longitudinal axis of the ribbon-shaped intact ASMC. These Ca2+ waves were not synchronized between neighboring cells, and induction of wave-like [Ca2+]i oscillations was temporally associated with development of force by the tracheal muscle bundle. By comparing the concentration dependence of force generation and the parameters characterizing the [Ca2+]i oscillations, we found that the concentration-dependent increase in ACh-induced force development by the tracheal smooth muscle bundle is achieved by differential recruitment of intact ASMC to initiate Ca2+ waves and by enhancement in the frequency of [Ca2+]i oscillations and elevation of interspike [Ca2+]i once the cells are recruited. Our findings demonstrate that asynchronous recurring Ca2+ waves underlie E-C coupling in ACh-induced contraction of the intact tracheal smooth muscle bundle. Furthermore, in contrast to what was reported in enzymatically dissociated ASMC, Ca2+ influx through the L-type voltage-gated Ca2+ channel was not an obligatory requirement for the generation of [Ca2+]i oscillations and development of force in ACh-stimulated intact ASMC.
GATA-6 is required for maturation of the lung in late gestation
Authors: Liu Cong , Morrisey Edward E. , Whitsett Jeffrey A.
Published: March 2015

GATA-6, a member of the GATA family of zinc finger proteins, is the only family member known to be expressed in the epithelial cells of the developing airway epithelium. To determine the role of GATA-6 in lung morphogenesis, a chimeric fusion protein containing GATA-6 and the strong transcriptional inhibitor, engrailed, was conditionally expressed in mice under control of a doxycycline-inducible transgene. Expression of GATA-6-engrailed was initiated at embryonic day ( E) 6–7 by treatment of the dam with doxycycline. Although branching morphogenesis of the proximal airways proceeded normally to E16.5, maturation of terminal airways and alveoli that normally occurs before birth was inhibited. At E17.5–18.5, aquaporin-5 mRNA and type I cell marker-α staining, both markers of type I cells, were decreased. Homogenous distribution of the thyroid transcription factor-1, decreased expression of surfactant proteins, delayed thinning of the walls of the peripheral airways, and lack of squamous differentiation of epithelial cells were observed in the lung periphery after expression of GATA-6-engrailed. Activity of GATA-6 is required for maturation of the gas exchange area before birth.
Insulin-like growth factor I receptor is downregulated after alveolarization in an apoptotic fibroblast subset
Authors: Srinivasan Suseela , Strange Jennifer , Awonusonu Feyisola , Bruce Margaret C.
Published: March 2015

After alveolar formation, >20% of interstitial lung fibroblasts undergo apoptosis, a process that is of critical importance for normal lung maturation. The immature lung contains two morphologically distinct fibroblast populations, lipid-filled interstitial fibroblasts (LIF) and non-LIF (NLIF), which differ with respect to contractile protein content, proliferative capacity, and expression of mRNAs for fibronectin and types I and III collagen, but not tropoelastin. After alveolarization, apoptosis occurs in only one fibroblast population, the LIF. Using flow cytometry to analyze fibroblasts stained with a lipophilic, fluorescent dye, we identified a subset, designated LIF(−), that contained fewer lipid droplets. Unlike LIF that retain lipid, LIF(+), the LIF(−) do not undergo apoptosis after alveolarization. In LIF(+), apoptosis was correlated with downregulation of insulin-like growth factor I receptor (IGF-IR) mRNA and cell surface protein expression. Treatment with anti-IGF-IR decreased total lung fibroblast survival ( P = 0.05) as did treatment with the phosphatidylinositol 3-kinase inhibitor LY-294002 and the ras-raf-mitogen-activated protein kinase inhibitor PD-98059 ( P < 0.002), which block IGF-I/insulin receptor survival pathways. These observations implicate downregulation of IGF-IR expression in fibroblast apoptosis after alveolar formation.
Increased epithelial cell proliferation in very premature baboons with chronic lung disease
Authors: Maniscalco William M. , Watkins Richard H. , O'Reilly Michael A. , Shea Colleen P.
Published: March 2015

Coordinated proliferation of lung cells is required for normal lung growth and differentiation. Chronic injury to developing lung may disrupt normal patterns of cell proliferation. To examine patterns of cell proliferation in injured developing lungs, we investigated premature baboons delivered at 125 days gestation (∼67% of term) and treated with oxygen and ventilation for 6, 14, or 21 days (PRN). Each PRN treatment group contained 3 or 4 animals. During normal in utero lung development, the proportion of proliferating lung cells declined as measured by the cell-cycle marker Ki67. In the PRN group, the proportion of proliferating lung cells was 2.5–8.5-fold greater than in corresponding gestational controls. By 14 days of treatment, the proportion of cells that expressed pro-surfactant protein B (proSP-B) was ∼2.5-fold greater than in gestational controls. In the PRN group, 41% of proliferating cells expressed proSP-B compared with 5.8% in the gestational controls. By 21 days of treatment, proliferation of proSP-B-expressing epithelial cells declined substantially, but the proportion of proliferating non-proSP-B-expressing cells increased approximately sevenfold. These data show that the development of chronic lung disease is associated with major alterations in normal patterns of lung-cell proliferation.
Lung and salivary scavenger receptor glycoprotein-340 contribute to the host defense against influenza A viruses
Authors: Hartshorn Kevan L. , White Mitchell R. , Mogues Tirsit , Ligtenberg Toon , Crouch Erika , Holmskov Uffe
Published: March 2015

The lung scavenger receptor-rich protein glycoprotein-340 (gp-340) is present in bronchoalveolar lavage (BAL) fluids and saliva and mediates specific adhesion to and aggregation of bacteria. It also binds to surfactant proteins A and D (SP-A and -D). Prior studies demonstrated that SP-A and SP-D contribute to innate defense against influenza A virus (IAV). We now show that lung and salivary gp-340 inhibit the hemagglutination activity and infectivity of IAV and agglutinate the virions through a mechanism distinct from that of SP-D. As in the case of SP-A, the antiviral effects of gp-340 are mediated by noncalcium-dependent interactions between the virus and sialic acid-bearing carbohydrates on gp-340. Gp-340 inhibits IAV strains that are resistant to SP-D. Concentrations of gp-340 present in saliva and BAL fluid of healthy donors are sufficient to bind to IAV and inhibit viral infectivity. On the basis of competition experiments using competing saccharide ligands, it appears that SP-D does not entirely mediate that anti-IAV activity of BAL fluid and contributes little to that of saliva. Furthermore, removal of gp-340 from BAL fluid and saliva significantly reduced anti-IAV activity. Hence, gp-340 contributes to defense against IAV and may be particularly relevant to defense against SP-D-resistant viral strains.
Role of extracellular superoxide dismutase in bleomycin-induced pulmonary fibrosis
Authors: Bowler Russell P. , Nicks Mike , Warnick Karrie , Crapo James D.
Published: March 2015

Bleomycin administration results in well-described intracellular oxidative stress that can lead to pulmonary fibrosis. The role of alveolar interstitial antioxidants in this model is unknown. Extracellular superoxide dismutase (EC-SOD) is the primary endogenous extracellular antioxidant enzyme and is abundant in the lung. We hypothesized that EC-SOD plays an important role in attenuating bleomycin-induced lung injury. Two weeks after intratracheal bleomycin administration, we found that wild-type mice induced a 106 ± 25% increase in lung EC-SOD. Immunohistochemical staining revealed that a large increase in EC-SOD occurred in injured lung. Using mice that overexpress EC-SOD specifically in the lung, we found a 53 ± 14% reduction in bleomycin-induced lung injury assessed histologically and a 17 ± 6% reduction in lung collagen content 2 wk after bleomycin administration. We conclude that EC-SOD plays an important role in reducing the magnitude of lung injury from extracellular free radicals after bleomycin administration.
Regulation of human airway epithelial cell IL-8 expression by MAP kinases
Authors: Li Jing , Kartha Sreedharan , Iasvovskaia Svetlana , Tan Alan , Bhat Rajesh K. , Manaligod Joel M. , Page Kristen , Brasier Allan R. , Hershenson Marc B.
Published: March 2015

Recent studies indicate that maximal IL-8 protein expression requires activation of NF-κB as well as activation of the MAP kinases ERK, JNK, and p38. However, the precise relationship between NF-κB transactivation and MAP kinase activation remains unclear. We examined the requirements of NF-κB, ERK, JNK, and p38 for TNF-α-induced transcription from the IL-8 promoter in a human bronchial epithelial cell line. Treatment with TNF-α induced activation of all three MAP kinases. Using a combination of chemical and dominant-negative inhibitors, we found that inhibition of NF-κB, ERK, and JNK, but not p38, each decreased TNF-α-induced transcription from the IL-8 promoter. Inhibition of JNK signaling also substantially reduced TNF-α-induced NF-κB transactivation, whereas inhibition of ERK and p38 had no effect. On the other hand, ERK was required and sufficient for TNF-α-induced activation of activator protein (AP)-1 promoter sequences, which together function as a basal level enhancer. JNK activation was also required for AP-1 transactivation. Finally, inhibition of p38 attenuated IL-8 protein abundance, suggesting that p38 regulates IL-8 expression in a posttranscriptional manner. We conclude that, in human airway epithelial cells, MAP kinases may regulate IL-8 promoter activity by NF-κB-dependent (in the case of JNK) and -independent (ERK) processes, as well as by posttranscriptional mechanisms (p38).
Proteoglycan expression in bleomycin lung fibroblasts: role of transforming growth factor-β1and interferon-γ
Authors: Venkatesan Narayanan , Roughley Peter J. , Ludwig Mara S.
Published: March 2015

Bleomycin (BM)-induced pulmonary fibrosis involves excess production of proteoglycans (PGs). Because transforming growth factor-β1(TGF-β1) promotes fibrosis, and interferon-γ (IFN-γ) inhibits it, we hypothesized that TGF-β1treatment would upregulate PG production in fibrotic lung fibroblasts, and IFN-γ would abrogate this effect. Primary lung fibroblast cultures were established from rats 14 days after intratracheal instillation of saline (control) or BM (1.5 units). PGs were extracted and subjected to Western blot analysis. Bleomycin-exposed lung fibroblasts (BLF) exhibited increased production of versican (VS), heparan sulfate proteoglycan (HSPG), and biglycan (BG) compared with normal lung fibroblasts (NLF). Compared with NLF, BLF released significantly increased amounts of TGF-β1. TGF-β1(5 ng/ml for 48 h) upregulated PG expression in both BLF and NLF. Incubation of BLF with anti-TGF-β antibody (1, 5, and 10 μg/ml) inhibited PG expression in a dose-dependent manner. Treatment of BLF with IFN-γ (500 U · ml−1× 48 h) reduced VS, HSPG, and BG expression. Furthermore, IFN-γ inhibited TGF-β1-induced increases in PG expression by these fibroblasts. Activation of fibroblasts by TGF-β1promotes abnormal deposition of PGs in fibrotic lungs; downregulation of TGF-β1by IFN-γ may have potential therapeutic benefits in this disease.
Bovine distal pulmonary arterial media is composed of a uniform population of well-differentiated smooth muscle cells with low proliferative capabilities
Authors: Stiebellehner Leopold , Frid Maria G. , Reeves John T. , Low Robert B. , Gnanasekharan Meena , Stenmark Kurt R.
Published: March 2015

The media of the normal bovine main pulmonary artery (MPA) is composed of phenotypically heterogeneous smooth muscle cells (SMC) with markedly different proliferative capabilities in response to serum, mitogens, and hypoxia. Little, however, is known of the SMC phenotype in distal pulmonary arteries (PA), particularly in arterioles, which regulate the pulmonary circulation. With a panel of muscle-specific antibodies against α-smooth muscle (SM)-actin, SM-myosin heavy chains (SM-MHC), SM-MHC-B isoform, desmin, and meta-vinculin, we demonstrate a progressive increase in phenotypic uniformity and level of differentiation of SMC along the proximal-to-distal axis of normal adult bovine pulmonary circulation so that the media of distal PA (1,500- to 100-μm diameter) is composed of a phenotypically uniform population of “well-differentiated” SMC. Similarly, when isolated and assessed in vitro, distal PA-SMC is composed of a single, uniform population of differentiated SMC that exhibited minimal growth responses to a variety of mitogens while their cell size increased substantially in response to serum. Their growth was inhibited by hypoxic exposure under all conditions tested. Distal PA-SMC also differed from MPA-SMC by exhibiting a distinct pattern of DNA synthesis in response to serum and mitogens. Thus, in contrast to the MPA, distal PA media is composed of an apparently uniform population of well-differentiated SMC that are proliferation resistant and have a substantial capacity to hypertrophy in response to growth-promoting stimuli. We thus speculate that distinct SMC phenotypes present in distal vs. proximal PA may confer different response mechanisms during remodeling in conditions such as hypertension.
Gelatinase activities in the airways of premature infants and development of bronchopulmonary dysplasia
Authors: Danan Claude , Jarreau Pierre-Henri , Franco Marie-Laure , Dassieu Gilles , Grillon Christophe , Alsamad Issam Abd , Lafuma Chantal , Harf Alain , Delacourt Christophe
Published: March 2015

Matrix-degrading metalloproteinases may play a role in the pathophysiology of bronchopulmonary dysplasia (BDP). We, therefore, evaluated correlations between gelatinase activities [metalloproteinase (MMP)-2 and MMP-9] or tissue inhibitor of metalloproteinase (TIMP)-1 levels present in the airways during the initial phase of hyaline membrane disease and the onset of BPD. Tracheal aspirates were obtained within 6 h of birth ( day 0) from 64 intubated neonates with a gestational age ≤30 wk. Forty-five neonates were resampled on day 3 or 5. Total MMP-2 level measured by zymography fell with time, whereas total MMP-9 level and TIMP-1 levels, assayed by ELISA, increased; the MMP-9 increase correlated with the increase in airway inflammatory cell numbers. Among the parameters measured on day 0, 3, or 5, lower total MMP-2 level, lower birth weight, and higher fraction of inspired oxygen on day 0 were significantly and independently associated with the development of BPD. In conclusion, MMP-9 level and TIMP-1 levels increased after birth but are not linked to BPD outcome. In contrast, low MMP-2 level at birth is strongly associated with the development of BPD.
Calcium-independent contraction and sensitization of airway smooth muscle by p21-activated protein kinase
Authors: McFawn P. K. , Shen L. , Vincent S. G. , Mak A. , Van Eyk J. E. , Fisher J. T.
Published: March 2015

In Triton-skinned phasic ileal smooth muscle, constitutively active recombinant p21-activated kinase (PAK3) has been shown to induce Ca2+-independent contraction, which is accompanied by phosphorylation of caldesmon and desmin (Van Eyk JE, Arrell DK, Foster DB, Strauss JD, Heinonen TY, Furmaniak-Kazmierczak E, Cote GP, and Mak AS. J Biol Chem 273: 23433–23439, 1998). In the present study, we investigated whether PAK has a broad impact on smooth muscle in general by testing the hypothesis that PAK induces Ca2+-independent contractions and/or Ca2+sensitization in tonic airway smooth muscle and that the process is mediated via phosphorylation of caldesmon. In the absence of Ca2+(pCa > 9), constitutively active glutathione- S-transferase-murine PAK3 (GST-mPAK3) caused force generation of Triton-skinned canine tracheal smooth muscle (TSM) fibers to ∼40% of the maximal force generated by Ca2+at pCa 4.4. In addition, GST-mPAK3 enhanced Ca2+sensitivity of contraction by increasing force generation by 80% at intermediate Ca2+concentrations (pCa 6.2), whereas it had no effect at pCa 4.4. Catalytically inactive GST-mPAK3K297Rhad no effect on force production. Using antibody against one of the PAK-phosphorylated sites (Ser657) on caldesmon, we showed that a basal level of phosphorylation of caldesmon occurs at this site in skinned TSM and that PAK-induced contraction was accompanied by a significant increase in the level of phosphorylation. Western blot analyses show that PAK1 is the predominant PAK isoform expressed in murine, rat, canine, and porcine TSM. We conclude that PAK causes Ca2+-independent contractions and produces Ca2+sensitization of skinned phasic and tonic smooth muscle, which involves an incremental increase in caldesmon phosphorylation.
Protein kinase C modifications of VE-cadherin, p120, and β-catenin contribute to endothelial barrier dysregulation induced by thrombin
Authors: Konstantoulaki Maria , Kouklis Panos , Malik Asrar B.
Published: March 2015

The adherens junction is a multiprotein complex consisting of the transmembrane vascular endothelial cadherin (VEC) and cytoplasmic catenins (p120, β-catenin, plakoglobin, α-catenin) responsible for the maintenance of endothelial barrier function. Junctional disassembly and modifications in cadherin/catenin complex lead to increased paracellular permeability of the endothelial barrier. However, the mechanisms of junctional disassembly remain unclear. In this study, we used the proinflammatory mediator thrombin to compromise the barrier function and test the hypothesis that phosphorylation-induced alterations of VEC, β-catenin, and p120 regulate junction disassembly and mediate the increased endothelial permeability response. The study showed that thrombin induced dephosphorylation of VEC, which is coupled to disassembly of cell-cell contacts, but VEC remained in aggregates at the plasma membrane. The cytoplasmic catenins dissociated from the VEC cytoplasmic domain in thin membrane projections formed in interendothelial gaps. We also showed that thrombin induced dephosphorylation of β-catenin and phosphorylation of p120. Thrombin-induced interendothelial gap formation and increased endothelial permeability were blocked by protein kinase C inhibition using chelerythrine and Gö-6976 but not by LY-379196. Chelerythrine also prevented thrombin-induced phosphorylation changes of the cadherin/catenin complex. Thus the present study links posttranslational modifications of VEC, β-catenin, and p120 to the mechanism of thrombin-induced increase in endothelial permeability.
NF-κB inhibition by ω-3 fatty acids modulates LPS-stimulated macrophage TNF-α transcription
Authors: Novak Todd E. , Babcock Tricia A. , Jho David H. , Helton W. Scott , Espat N. Joseph
Published: March 2015

ω-3 Fatty acid (FA) emulsions reduce LPS-stimulated murine macrophage TNF-α production, but the exact mechanism has yet to be defined. The purpose of this study was to determine the mechanism for ω-3 FA inhibition of macrophage TNF-α production following LPS stimulation. RAW 264.7 cells were pretreated with isocaloric emulsions of ω-3 FA (Omegaven), ω-6 FA (Lipovenos), or DMEM and subsequently exposed to LPS. IκB-α and phospho-IκB-α were determined by Western blotting. NF-κB binding was assessed using the electromobility shift assay, and activity was measured using a luciferase reporter vector. RT-PCR and ELISA quantified TNF-α mRNA and protein levels, respectively. Pretreatment with ω-3 FA inhibited IκB phosphorylation and significantly decreased NF-κB activity. Moreover, ω-3-treated cells demonstrated significant decreases in both TNF-α mRNA and protein expression by 47 and 46%, respectively. These experiments demonstrate that a mechanism for proinflammatory cytokine inhibition in murine macrophages by ω-3 FA is mediated, in part, through inactivation of the NF-κB signal transduction pathway secondary to inhibition of IκB phosphorylation.
Heparan sulfates expressed in the distal lung are required for Fgf10 binding to the epithelium and for airway branching
Authors: Izvolsky Konstantin I. , Zhong Lin , Wei Lin , Yu Qiang , Nugent Matthew A. , Cardoso Wellington V.
Published: March 2015

Fibroblast growth factor (Fgf) 10 is a critical regulator of bud formation during lung morphogenesis. fgf10 is expressed in distal lung mesenchyme at sites of prospective budding from the earliest developmental stages and signals through its epithelial receptor Fgfr2b. Experiments in intact lung organ cultures demonstrate that Fgf10 is a chemotactic factor for distal, but not for proximal, epithelium. This differential response suggests the involvement of an additional mechanism regulating Fgf10-Fgfr2b interactions, because Fgfr2b is uniformly expressed throughout the respiratory tract. Here we use an immunohistochemistry-based binding assay to show that O-sulfated heparan sulfates (HS) are critical for Fgf10 binding to the distal epithelium. We show that altering endogenous gradients of HS sulfation with sodium chlorate or over- O-sulfated synthetic heparin in lung organ cultures dramatically decreases Fgf10 binding. Moreover, we show that under these conditions epithelial binding is not improved by providing exogenous FGF10. Our data suggest that, not only ligand availability, but also the presence of specific patterns of HS modification in the distal lung epithelium are critical determinants of Fgf10 binding to the epithelium and signaling.
Surfactant protein A increases matrix metalloproteinase-9 production by THP-1 cells
Authors: Vazquez de Lara Luis G. , Umstead Todd M. , Davis Sara E. , Phelps David S.
Published: March 2015

Matrix metalloproteinase (MMP)-9 from alveolar macrophages is a major source of elastolytic activity in the lung. It is increased in the bronchoalveolar lavage fluid of patients with emphysema. Although the importance of macrophage-derived elastolytic activity in the pathogenesis of emphysema is well established, questions remain about MMP-9 regulation and activity. Because surfactant protein A (SP-A) is capable of modulating other functions of human monocytic cells, we hypothesized that SP-A may regulate MMP-9 expression. Vitamin D3-differentiated THP-1 cells and peripheral blood mononuclear cells were stimulated in vitro with several concentrations of SP-A for different incubation times. MMP-9 mRNA expression was measured by dot-blot analysis, gelatinolytic activity in the medium was determined by gel zymography, protein expression was determined by ELISA, and a specific MMP-9 activity assay was used to measure the state of activation of this enzyme in the cell supernatants. SP-A induced the expression of MMP-9 in both cell types, the effect was time and dose dependent, and MMP-9 was released in its zymogen form. On the basis of results of neutralizing antibody studies, we believe that SP-A action is mediated through Toll-like receptor-2. Even though the biological meaning of these findings remains to be elucidated, these observations suggest the presence of a novel, locally controlled mechanism by which MMP-9 levels may be regulated in alveolar macrophages. We speculate that SP-A may influence the protease/antiprotease balance in the lungs of patients with quantitative and/or qualitative changes in surfactant constituents favoring an abnormal breakdown of extracellular matrix components.
Coordinate regulation of membrane cAMP by Ca2+-inhibited adenylyl cyclase and phosphodiesterase activities
Authors: Creighton Judy R. , Masada Nanako , Cooper Dermot M. F. , Stevens Troy
Published: March 2015

Activation of store-operated Ca2+entry inhibits type 6 adenylyl cyclase (EC 4.6.1.1 ; AC6; Yoshimura M and Cooper DM. Proc Natl Acad Sci USA 89: 6712–6720, 1992) activity in pulmonary artery endothelial cells. However, in lung microvascular endothelial cells (PMVEC), which express AC6and turn over cAMP at a rapid rate, inhibition of global (whole cell) cAMP is not resolved after direct activation of store-operated Ca2+entry using thapsigargin. Present studies sought to determine whether the high constitutive phosphodiesterase activity in PMVECs rapidly hydrolyzes cAMP so that Ca2+inhibition of AC6is difficult to resolve. Direct stimulation of adenylyl cyclase using forskolin and inhibition of type 4 phosphodiesterases using rolipram increased cAMP and revealed Ca2+inhibition of AC6. Enzyme activity was assessed using PMVEC membranes, where Ca2+and cAMP concentrations were independently controlled. Endogenous AC6activity exhibited high- and low-affinity Ca2+inhibition, similar to that observed in C6-2B cells, which predominantly express AC6. Ca2+inhibition of AC6in PMVEC membranes was observed after enzyme activation and inhibition of phosphodiesterase activity and was independent of the free cAMP concentration. Thus, under basal conditions, the constitutive type 4 phosphodiesterase activity rapidly hydrolyzes cAMP so that Ca2+inhibition of AC6is difficult to resolve, indicating that high phosphodiesterase activity works coordinately with AC6to regulate membrane-delimited cAMP concentrations, which is important for control of cell-cell apposition.
Muscarinic M2 receptors in acetylcholine-isoproterenol functional antagonism in human isolated bronchus
Authors: Sarria Benjamin , Naline Emmanuel , Zhang Yong , Cortijo Julio , Molimard Mathieu , Moreau Joelle , Therond Patrice , Advenier Charles , Morcillo Esteban J.
Published: March 2015

The muscarinic functional antagonism of isoproterenol relaxation and the contribution of muscarinic M2 receptors were examined in human isolated bronchus. In intact tissues, acetylcholine (ACh) precontraction decreased isoproterenol potency and maximal relaxation (−log EC50 shift = −1.49 ± 0.16 and Emax inhibition for 100 μM ACh = 30%) more than the same levels of histamine contraction. The M2receptor-selective antagonist methoctramine (1 μM) reduced this antagonism in ACh- but not histamine-contracted tissues. Similar results were obtained for forskolin-induced relaxation. After selective inactivation of M3 receptors with 4-diphenylacetoxy- N-(2-chloroethyl)piperadine hydrochloric acid (30 nM), demonstrated by abolition of contractile and inositol phosphate responses to ACh, muscarinic recontractile responses were obtained in U-46619-precontracted tissues fully relaxed with isoproterenol. Methoctramine antagonized recontraction, with pKB (6.9) higher than in intact tissues (5.4), suggesting participation of M2 receptors. In M3-inactivated tissues, methoctramine augmented the isoproterenol relaxant potency in U-46619-contracted bronchus and reversed the ACh-induced inhibition of isoproterenol cAMP accumulation. These results indicate that M2 receptors cause indirect contraction of human bronchus by reversing sympathetically mediated relaxation and contribute to cholinergic functional antagonism.
Glucocorticoid-stimulated lung epithelial Na+transport is associated with regulated ENaC andsgk1expression
Authors: Itani Omar A. , Auerbach Scott D. , Husted Russell F. , Volk Kenneth A. , Ageloff Shana , Knepper Mark A. , Stokes John B. , Thomas Christie P.
Published: March 2015

H441 cells, a bronchiolar epithelial cell line, develop a glucocorticoid-regulated amiloride-sensitive Na+transport pathway on permeable supports (R. Sayegh, S. D. Auerbach, X. Li, R. Loftus, R. Husted, J. B. Stokes, and C. P. Thomas. J Biol Chem 274: 12431–12437, 1999). To understand its molecular basis, we examined the effect of glucocorticoids (GC) on epithelial Na+channel (ENaC)-α, -β, and -γ and sgk1 expression and determined the biophysical properties of Na+channels in these cells. GC stimulated the expression of ENac-α, -β, and -γ and sgk1 mRNA, with the first effect seen by 1 h. These effects were abolished by actinomycin D, but not by cycloheximide, indicating a direct stimulatory effect on ENaC and sgk1 mRNA synthesis. The GC effect on transcription of ENaC-α mRNA was accompanied by a significant increase in ENaC-α protein levels. GC also stimulated ENaC-α, -β, and -γ and sgk1 mRNA expression in A549 cells, an alveolar type II cell line. To determine the biophysical properties of the Na+channel, single-channel currents were recorded from cell-attached H441 membranes. An Na+-selective channel with slow kinetics and a slope conductance of 10.8 pS was noted, properties similar to ENaC-α, -β, and -γ expressed in Xenopus laevis oocytes. These experiments indicate that amiloride-sensitive Na+transport is mediated through classic ENaC channels in human lung epithelia and that GC-regulated Na+transport is accompanied by increased transcription of each of the component subunits and sgk1.
Phenazine-1-carboxylic acid, a secondary metabolite ofPseudomonas aeruginosa, alters expression of immunomodulatory proteins by human airway epithelial cells
Authors: Denning Gerene M. , Iyer Shankar S. , Reszka Krzysztof J. , O'Malley Yunxia , Rasmussen George T. , Britigan Bradley E.
Published: March 2015

Pseudomonas aeruginosa is a gram-negative bacterium that causes both acute and chronic lung disease in susceptible patient populations. P. aeruginosa secretes numerous proteins and secondary metabolites, many of which have biological effects that likely contribute to disease pathogenesis. An unidentified small-molecular-weight factor was previously reported to increase IL-8 release both in vitro and in vivo. To identify this factor, we subjected the <3-kDa fraction from P. aeruginosa-conditioned medium to HPLC analysis. A peak fraction that stimulated IL-8 release was found by mass spectrometry to have a molecular mass (MM) of 224 Da. On the basis of this MM and other biochemical properties, we hypothesized that the factor was phenazine-1-carboxylic acid (PCA). Subsequent studies and comparison with purified PCA confirmed this hypothesis. Purified PCA exhibited a number of biological effects in human airway epithelial cells, including increasing IL-8 release and ICAM-1 expression, as well as decreasing RANTES and monocyte chemoattractant protein-1 (MCP-1) release. PCA also increased intracellular oxidant formation as measured by electron paramagnetic resonance and by an intracellular oxidant-sensitive probe. Antioxidants inhibited PCA-dependent increases in IL-8 and ICAM-1, suggesting that oxidants contributed to these effects. However, in contrast to the related phenazine compound pyocyanin, PCA did not oxidize NAD(P)H at physiologically relevant pH, providing preliminary evidence that PCA and pyocyanin may have distinct redox chemistries within the cell. Thus PCA is a biologically active factor secreted by P. aeruginosa that has several activities that could alter the host immune and inflammatory response and thereby contribute to bacterial disease pathogenesis.
Microarray analysis of lipopolysaccharide-treated human neutrophils
Authors: Malcolm Kenneth C. , Arndt Patrick G. , Manos Elizabeth J. , Jones David A. , Worthen G. Scott
Published: March 2015

Neutrophils respond to infection by degranulation, release of reactive oxygen intermediates, and secretion of chemokines and cytokines; however, activation of neutrophil transcriptional machinery has been little appreciated. Recent findings suggest that gene expression may represent an additional neutrophil function after exposure to lipopolysaccharide (LPS). We performed microarray gene expression analysis of 4,608 mostly nonredundant genes on LPS-stimulated human neutrophils. Analysis of three donors indicated some variability but also a high degree of reproducibility in gene expression. Twenty-eight verifiable, distinct genes were induced by 4 h of LPS treatment, and 13 genes were repressed. Genes other than cytokines and chemokines are regulated; interestingly, genes involved in cell growth regulation and survival, transcriptional regulation, and interferon response are among those induced, whereas genes involved in cytoskeletal regulation are predominantly repressed. In addition, we identified monocyte chemoattractant protein-1 as a novel LPS-regulated chemokine in neutrophils. Included in these lists are five clones with no defined function. These data suggest molecular mechanisms by which neutrophils respond to infection and indicate that the transcriptional potential of neutrophils is greater than previously thought.
Angiotensin receptor subtype AT1 mediates alveolar epithelial cell apoptosis in response to ANG II
Authors: Papp Michael , Li Xiaopeng , Zhuang Jiaju , Wang Rongqi , Uhal Bruce D.
Published: March 2015

Previous work from this laboratory demonstrated induction of apoptosis in lung alveolar epithelial cells (AEC) by purified angiotensin II (ANG II) and expression of mRNAs for both ANG II receptor subtypes AT1 and AT2(Wang R, Zagariya A, Ibarra-Sunga O, Gidea C, Ang E, Deshmukh S, Chaudhary G, Baraboutis J, Filippatos G, and Uhal BD. Am J Physiol Lung Cell Mol Physiol 276: L885–L889, 1999.). The present study was designed to determine the ANG II receptor subtype mediating AEC apoptosis in response to ANG II. Apoptosis was induced with purified ANG II applied to the human lung AEC-derived carcinoma cell line A549 or to primary AEC isolated from Wistar rats. In both cell types, the AT1-selective receptor antagonists L-158809 or losartan inhibited ANG II-induced apoptosis by 90% at concentrations of 10−8 M and 10−7 M, respectively. The inhibition was concentration dependent with IC50 of 10−12 M and 10−11 M on the primary rat AEC. In contrast, the AT2-selective antagonists PD-123319 or PD-126055 could not block ANG II-induced apoptosis in either cell type. In primary rat AEC, apoptosis in response to ANG II was blunted in a dose-dependent manner by the protein kinase C inhibitor chelerythrine but not by the tyrosine phosphatase inhibitor sodium orthovanadate. Together, these data indicate that AEC apoptosis in response to ANG II is mediated by receptor subtype AT1, despite the expression of mRNAs for both AT1 and AT2.
Graded response of K+current, membrane potential, and [Ca2+]ito hypoxia in pulmonary arterial smooth muscle
Authors: Olschewski Andrea , Hong Zhigang , Nelson Daniel P. , Weir E. Kenneth
Published: March 2015

Many studies indicate that hypoxic inhibition of some K+channels in the membrane of the pulmonary arterial smooth muscle cells (PASMCs) plays a part in initiating hypoxic pulmonary vasoconstriction. The sensitivity of the K+current ( Ik), resting membrane potential ( Em), and intracellular Ca2+concentration ([Ca2+]i) of PASMCs to different levels of hypoxia in these cells has not been explored fully. Reducing Po2levels gradually inhibited steady-state Ikof rat resistance PASMCs and depolarized the cell membrane. The block of Ikby hypoxia was voltage dependent in that low O2tensions (3 and 0% O2) inhibited Ikmore at 0 and −20 mV than at 50 mV. As expected, the hypoxia-sensitive Ikwas also 4-aminopyridine sensitive. Fura 2-loaded PASMCs showed a graded increase in [Ca2+]ias Po2levels declined. This increase was reduced markedly by nifedipine and removal of extracellular Ca2+. We conclude that, as in the carotid body type I cells, PC-12 pheochromocytoma cells, and cortical neurons, increasing severity of hypoxia causes a proportional decrease in Ikand Emand an increase of [Ca2+]i.
Expression of functional nicotinic acetylcholine receptors in neuroepithelial bodies of neonatal hamster lung
Authors: Fu Xiao Wen , Nurse Colin A. , Farragher Suzanne M. , Cutz Ernest
Published: March 2015

Pulmonary neuroepithelial bodies (NEB) are presumed airway chemoreceptors involved in respiratory control, especially in the neonate. Nicotine is known to affect both lung development and control of breathing. We report expression of functional nicotinic acetylcholine receptors (nAChR) in NEB cells of neonatal hamster lung using a combination of morphological and electrophysiological techniques. Nonisotopic in situ hybridization method was used to localize mRNA for the β2-subunit of nAChR in NEB cells. Double-label immunofluorescence confirmed expression of α4-, α7-, and β2-subunits of nAChR in NEB cells. The electrophysiological characteristics of nAChR in NEB cells were studied using the whole cell patch-clamp technique on fresh lung slices. Application of nicotine (∼0.1-100 μM) evoked inward currents that were concentration dependent (EC50= 3.8 μM; Hill coefficient = 1.1). ACh (100 μM) and nicotine (50 μM) produced two types of currents. In most NEB cells, nicotine-induced currents had a single desensitizing component that was blocked by mecamylamine (50 μM) and dihydro-β-erythroidine (50 μM). In some NEB cells, nicotine-induced current had two components, with fast- and slow-desensitizing kinetics. The fast component was selectively blocked by methyllcaconitine (MLA, 10 nM), whereas both components were inhibited by mecamylamine. Choline (0.5 mM) also induced an inward current that was abolished by 10 nM MLA. These studies suggest that NEB cells in neonatal hamster lung express functional heteromeric α3β2, α4β2, and α7nAChR and that cholinergic mechanisms could modulate NEB chemoreceptor function under normal and pathological conditions.