bluuebunny/crossref_metadata_2025 huggingface dataset:
ERK activation and mitogenesis in human airway smooth muscle cells
Authors: Lee Jin-Hee , Johnson Peter R. A. , Roth Michael , Hunt Nicholas H. , Black Judith L.
Published: December 2017

Asthmatic airways are characterized by an increase in smooth muscle mass, due mainly to hyperplasia. Many studies suggest that extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2, respectively), one group of the mitogen-activated protein (MAP) kinase superfamily, play a key role in the signal transduction pathway leading to cell proliferation. PGE2and forskolin inhibited mitogen-induced ERK activation. Inhibition of MAP kinase kinases 1 and 2 (MEK1 and MEK2, respectively), which are upstream from ERK, with the specific MEK inhibitor U-0126 blocked both cell proliferation and ERK activation. In addition, U-0126 inhibited mitogen-induced activation of p90 ribosomal S6 kinase and expression of c-Fos and cyclin D1, all of which are downstream from ERK in the signaling cascade that leads to cell proliferation. Antisense oligodeoxynucleotides directed to ERK1 and -2 mRNAs reduced ERK protein and cell proliferation. These results indicate that ERK is required for human airway smooth muscle cell proliferation. Thus targeting the control of ERK activation may provide a new therapeutic approach for hyperplasia seen in asthma.
Dual signaling by the αvβ3-integrin activates cytosolic PLA2in bovine pulmonary artery endothelial cells
Authors: Bhattacharya Sunita , Patel Rashmi , Sen Namita , Quadri Sadiqa , Parthasarathi Kaushik , Bhattacharya Jahar
Published: December 2017

Vitronectin, which ligates the αvβ3-integrin, increases both lung capillary permeability and lung endothelial Ca2+. In stable monolayers of bovine pulmonary artery endothelial cells (BPAECs) viewed with confocal microscopy, multimeric vitronectin aggregated the apically located αvβ3-integrin. This caused arachidonate release that was inhibited by pretreating the monolayers with the anti-αvβ3monoclonal antibody (MAb) LM609. No inhibition occurred in the presence of the isotypic MAb PIF6, which recognizes the integrin αvβ5. Vitronectin also caused membrane translocation and phosphorylation of cytosolic phospholipase A2(cPLA2) as well as tyrosine phosphorylation of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK) 2. The cPLA2inhibitor arachidonyl trifluoromethylketone, the tyrosine kinase inhibitor genistein, and the MAPK kinase inhibitor PD-98059 all blocked the induced arachidonate release. PD-98059 did not inhibit the increase of cytosolic Ca2+or cPLA2translocation, although it blocked tyrosine phosphorylation of ERK2. Moreover, although the intracellular Ca2+chelator MAPTAM also inhibited arachidonate release, it did not inhibit tyrosine phosphorylation of ERK2. These findings indicate that ligation of apical αvβ3in BPAECs caused ERK2 activation and an increase of intracellular Ca2+, both conjointly required for cPLA2activation and arachidonate release. This is the first instance of a tyrosine phosphorylation-initiated “two-hit” signaling pathway that regulates an integrin-induced proinflammatory response.
Capacitative Ca2+entry in agonist-induced pulmonary vasoconstriction
Authors: McDaniel Sharon S. , Platoshyn Oleksandr , Wang Jian , Yu Ying , Sweeney Michele , Krick Stefanie , Rubin Lewis J. , Yuan Jason X.-J.
Published: December 2017

Agonist-induced increases in cytosolic Ca2+concentration ([Ca2+]cyt) in pulmonary artery (PA) smooth muscle cells (SMCs) consist of a transient Ca2+release from intracellular stores followed by a sustained Ca2+influx. Depletion of intracellular Ca2+stores triggers capacitative Ca2+entry (CCE), which contributes to the sustained increase in [Ca2+]cytand the refilling of Ca2+into the stores. In isolated PAs superfused with Ca2+-free solution, phenylephrine induced a transient contraction, apparently by a rise in [Ca2+]cytdue to Ca2+release from the intracellular stores. The transient contraction lasted for 3–4 min until the Ca2+store was depleted. Restoration of extracellular Ca2+in the presence of phentolamine produced a contraction potentially due to a rise in [Ca2+]cytvia CCE. The store-operated Ca2+channel blocker Ni2+reduced the store depletion-activated Ca2+currents, decreased CCE, and inhibited the CCE-mediated contraction. In single PASMCs, we identified, using RT-PCR, five transient receptor potential gene transcripts. These results suggest that CCE, potentially through transient receptor potential-encoded Ca2+channels, plays an important role in agonist-mediated PA contraction.
Effect of protein kinase C inhibition on hypoxic pulmonary vasoconstriction
Authors: Barman Scott A.
Published: December 2017

The current study was done to test the hypothesis that protein kinase C (PKC) inhibitors prevent the increase in pulmonary vascular resistance and compliance that occurs in isolated, blood-perfused dog lungs during hypoxia. Pulmonary vascular resistances and compliances were measured with vascular occlusion techniques. Hypoxia significantly increased pulmonary arterial resistance, pulmonary venous resistance, and pulmonary capillary pressure and decreased total vascular compliance by decreasing both microvascular and large-vessel compliances. The nonspecific PKC inhibitor staurosporine (10−7M), the specific PKC blocker calphostin C (10−7M), and the specific PKC isozyme blocker Gö-6976 (10−7M) inhibited the effect of hypoxia on pulmonary vascular resistance and compliance. In addition, the PKC activator thymeleatoxin (THX; 10−7M) increased pulmonary vascular resistance and compliance in a manner similar to that in hypoxia, and the L-type voltage-dependent Ca2+channel blocker nifedipine (10−6M) inhibited the response to both THX and hypoxia. These results suggest that PKC inhibition blocks the hypoxic pressor response and that the pharmacological activation of PKC by THX mimics the hypoxic pulmonary vasoconstrictor response. In addition, L-type voltage-dependent Ca2+channel blockade may prevent the onset of the hypoxia- and PKC-induced vasoconstrictor response in the canine pulmonary vasculature.
Alveolar epithelial barrier functions in ventilated perfused rabbit lungs
Authors: Ghofrani Hossein Ardeschir , Kohstall Markus Gerhard , Weissmann Norbert , Schmehl Thomas , Schermuly Ralph Theo , Seeger Werner , Grimminger Friedrich
Published: December 2017

We employed ultrasonic nebulization for homogeneous alveolar tracer deposition into ventilated perfused rabbit lungs.22Na and 125I-albumin transit kinetics were monitored on-line with gamma detectors placed around the lung and the perfusate reservoir. [3H]mannitol was measured by repetitive counting of perfusion fluid samples. Volume of the alveolar epithelial lining fluid was estimated with bronchoalveolar lavage with sodium-free isosmolar mannitol solutions. Sodium clearance rate was −2.2 ± 0.3%/min. This rate was significantly reduced by preadministration of ouabain/amiloride and enhanced by pretreatment with aerosolized terbutaline. The 125I-albumin clearance rate was −0.40 ± 0.05%/min. The appearance of [3H]mannitol in the perfusate was not influenced by ouabain/amiloride or terbutaline but was markedly enhanced by pretreatment with aerosolized protamine. An epithelial lining fluid volume of 1.22 ± 0.21 ml was calculated in control lungs. Fluid absorption rate was 1.23 μl · g lung weight−1 · min−1, which was blunted after pretreatment with ouabain/amiloride. We conclude that alveolar tracer loading by aerosolization is a feasible technique to assess alveolar epithelial barrier properties in aerated lungs. Data on active and passive sodium flux, paracellular solute transit, and net fluid absorption correspond well to those in previous studies in fluid-filled lungs; however, albumin clearance rates were markedly higher in the currently investigated aerated lungs.
Hyperoxia upregulates the NO pathway in alveolar macrophages in vitro: role of AP-1 and NF-κB
Authors: Pepperl Sonja , Dörger Martina , Ringel Florian , Kupatt Christian , Krombach Fritz
Published: December 2017

The inducible nitric oxide (NO) synthase gene in alveolar macrophages (AMs) is a stress response gene that may contribute to tissue injury in the lung after respiration with high O2concentrations through extensive production of NO. In this study, we investigated the influence of hyperoxia on the NO pathway in rat AMs in vitro, its regulation by the transcription factors nuclear factor (NF)-κB and activator protein (AP)-1, and the role of reactive oxygen species (ROS). AMs were treated with lipopolysaccharide (LPS) and/or interferon (IFN)-γ and incubated under 21 or 85% O2. Stimulation with LPS and IFN-γ led to induction of the NO pathway that was further upregulated by hyperoxia. The binding activity of NF-κB, in contrast to that of AP-1, was activated on stimulation with LPS and IFN-γ, and both were further increased under hyperoxia. The antioxidants pyrrolidine dithiocarbamate and N-acetyl-l-cysteine inhibited intracellular ROS production and the NO pathway under both normoxic and hyperoxic conditions but had diverse effects on the transcription factors. The results presented here indicate that hyperoxia can upregulate the NO pathway in stimulated AMs through increased production of intracellular ROS and activation of NF-κB and AP-1.
p38 MAP kinase negatively regulates cyclin D1 expression in airway smooth muscle cells
Authors: Page Kristen , Li Jing , Hershenson Marc B.
Published: December 2017

We have demonstrated that platelet-derived growth factor (PDGF) stimulates p38 mitogen-activated protein (MAP) kinase activation in bovine tracheal myocytes, suggesting that p38 is involved in growth regulation. We therefore examined whether p38 regulates expression of cyclin D1, a G1cyclin required for cell cycle traversal. The chemical p38 inhibitors SB-202190 and SB-203580 each increased basal and PDGF-induced cyclin D1 promoter activity and protein abundance. Overexpression of a dominant negative allele of MAP kinase kinase-3 (MKK3), an upstream activator of p38α, had similar effects. Conversely, active MKK3 and MKK6, both of which increase p38α activity, each decreased transcription from the cyclin D1 promoter. Together, these data demonstrate that p38 negatively regulates cyclin D1 expression. We tested whether p38 regulates cyclin D1 expression via inhibition of extracellular signal-regulated kinase (ERK) activation. Chemical inhibitors of p38 induced modest ERK phosphorylation and activation. However, dominant negative MKK3 was insufficient to activate ERK, and active MKK3 and MKK6 did not attenuate platelet-derived growth factor-mediated ERK activation. These data are consistent with the notion that p38α negatively regulates cyclin D1 expression via an ERK-independent pathway.
Release of biologically active TGF-β from airway smooth muscle cells induces autocrine synthesis of collagen
Authors: Coutts Amanda , Chen Gang , Stephens Newman , Hirst Stuart , Douglas Deborah , Eichholtz Thomas , Khalil Nasreen
Published: December 2017

In severe or chronic asthma, there is an increase in airway smooth muscle cell (ASMC) mass as well as an increase in connective tissue proteins in the smooth muscle layer of airways. Transforming growth factor-β (TGF-β) exists in three isoforms in mammals and is a potent regulator of connective tissue protein synthesis. Using immunohistochemistry, we had previously demonstrated that ASMCs contain large quantities of TGF-β1–3. In this study, we demonstrate that bovine ASMC-derived TGF-β associates with the TGF-β latency binding protein-1 (LTBP-1) expressed by the same cells. The TGF-β associated with LTBP-1 localizes TGF-β extracellularly. Furthermore, plasmin, a serine protease, regulates the secretion of a biologically active form of TGF-β by ASMCs as well as the release of extracellular TGF-β. The biologically active TGF-β released by plasmin induces ASMCs to synthesize collagen I in an autocrine manner. The autocrine induction of collagen expression by ASMCs may contribute to the irreversible fibrosis and remodeling seen in the airways of some asthmatics.
Characterization of mucins in human middle ear and Eustachian tube
Authors: Lin Jizhen , Tsuprun Vladimir , Kawano Hirokazu , Paparella Michael M. , Zhang Zhiqiang , Anway Ruth , Ho Samuel B.
Published: December 2017

Mucins are important glycoproteins in the mucociliary transport system of the middle ear and Eustachian tube. Little is known about mucin expression within this system under physiological and pathological conditions. This study demonstrated the expression of MUC5B, MUC5AC, MUC4, and MUC1 in the human Eustachian tube, whereas only MUC5B mucin expression was demonstrated in noninflamed middle ears. MUC5B and MUC4 mucin genes were upregulated 4.2- and 6-fold, respectively, in middle ears with chronic otitis media (COM) or mucoid otitis media (MOM). This upregulation of mucin genes was accompanied by an increase of MUC5B- and MUC4-producing cells in the middle ear mucosa. Electron microscopy of the secretions from COM and MOM showed the presence of chainlike polymeric mucin. These data indicate that the epithelium of the middle ear and Eustachian tube expresses distinct mucin profiles and that MUC5B and MUC4 mucins are highly produced and secreted in the diseased middle ear. These mucins may form thick mucous effusion in the middle ear cavity and compromise the function of the middle ear.
Differential effect of MLC kinase in TNF-α-induced endothelial cell apoptosis and barrier dysfunction
Authors: Petrache Irina , Verin Alexander D. , Crow Michael T. , Birukova Anna , Liu Feng , Garcia Joe G. N.
Published: December 2017

Tumor necrosis factor (TNF)-α is released in acute inflammatory lung syndromes linked to the extensive vascular dysfunction associated with increased permeability and endothelial cell apoptosis. TNF-α induced significant decreases in transcellular electrical resistance across pulmonary endothelial cell monolayers, reflecting vascular barrier dysfunction (beginning at 4 h and persisting for 48 h). TNF-α also triggered endothelial cell apoptosis beginning at 4 h, which was attenuated by the caspase inhibitor Z-Val-Ala-Asp-fluoromethylketone. Exploring the involvement of the actomyosin cytoskeleton in these important endothelial cell responses, we determined that TNF-α significantly increased myosin light chain (MLC) phosphorylation, with prominent stress fiber and paracellular gap formation, which paralleled the onset of decreases in transcellular electrical resistance and enhanced apoptosis. Reductions in MLC phosphorylation by the inhibition of either MLC kinase (ML-7, cholera toxin) or Rho kinase (Y-27632) dramatically attenuated TNF-α-induced stress fiber formation, indexes of apoptosis, and caspase-8 activity but not TNF-α-induced barrier dysfunction. These studies indicate a central role for the endothelial cell cytoskeleton in TNF-α-mediated apoptosis, whereas TNF-α-induced vascular permeability appears to evolve independently of contractile tension generation.
Tobacco smoke reduces viability in human lung fibroblasts: protective effect of glutathioneS-transferase P1
Authors: Ishii Takeo , Matsuse Takeshi , Igarashi Hiroko , Masuda Michiaki , Teramoto Shinji , Ouchi Yasuyoshi
Published: December 2017

Cigarette smoking is thought to be a major risk factor in various lung diseases including lung cancer and emphysema. However, the direct effect of cigarette smoke on the viability of lung-derived cells has not been fully elucidated. In this study, we investigated the viability of human lung fibroblast-derived (HFL1) cells to different concentrations of cigarette smoke extract (CSE). CSE induced apoptosis at lower concentrations (10–25%) and necrosis at higher concentrations (50–100%). We also examined the effects of glutathione S-transferase P1 (GSTP1), one of the xenobiotic metabolizing and antioxidant enzymes in the lung, against the cytotoxicity of CSE. Our results indicated that the level of HFL1 cell death was decreased by transfection with a GSTP1 expression vector and was increased by GSTP1 antisense vector transfection. Therefore, transient overexpression and underexpression of GSTP1 appeared to inhibit and enhance the cytotoxic effects of CSE on HFL1 cells, suggesting that GSTP1 may have protective effects against cigarette smoke in the airway cells.
Macrophage and type II cell catabolism of SP-A and saturated phosphatidylcholine in mouse lungs
Authors: Gurel Okyanus , Ikegami Machiko , Chroneos Zissis C. , Jobe Alan H.
Published: December 2017

Type II cells and macrophages are the major cells involved in the alveolar clearance and catabolism of surfactant. We measured type II cell and macrophage contributions to the catabolism of saturated phosphatidylcholine and surfactant protein A (SP-A) in mice. We used intratracheally administered SP-A labeled with residualizing125I-dilactitol-tyramine, radiolabeled dipalmitoylphosphatidylcholine ([3H]DPPC), and its degradation-resistant analog [14C]DPPC-ether. At 15 min and 7, 19, 29, and 48 h after intratracheal injection, the mice were killed; alveolar lavage was then performed to recover macrophages and surfactant. Type II cells and macrophages not recovered by the lavage were subsequently isolated by enzymatic digestion of the lung. Radioactivity was measured in total lung, lavage fluid macrophages, alveolar washes, type II cells, and lung digest macrophages. Approximately equal amounts of125I-dilactitol-tyramine-SP-A and [14C]DPPC-ether associated with the macrophages (lavage fluid plus lung digest) and type II cells when corrected for the efficiency of type II cell isolation. Eighty percent of the macrophage-associated radiolabel was recovered from lung digest macrophages. We conclude that macrophages and type II cells contribute equally to saturated phosphatidylcholine and SP-A catabolism in mice.
Role of cAMP-dependent protein kinase A activity in endothelial cell cytoskeleton rearrangement
Authors: Liu Feng , Verin Alexander D. , Borbiev Talai , Garcia Joe G. N.
Published: December 2017

To examine signaling mechanisms relevant to cAMP/protein kinase A (PKA)-dependent endothelial cell barrier regulation, we investigated the impact of the cAMP/PKA inhibitors Rp diastereomer of adenosine 3′,5′-cyclic monophosphorothioate ( Rp-cAMPS) and PKA inhibitor (PKI) on bovine pulmonary artery and bovine lung microvascular endothelial cell cytoskeleton reorganization. Rp-cAMPS as well as PKI significantly increased the formation of actin stress fibers and intercellular gaps but did not alter myosin light chain (MLC) phosphorylation, suggesting that the Rp-cAMPS-induced contractile phenotype evolves in an MLC-independent fashion. We next examined the role of extracellular signal-regulated kinases (ERKs) in Rp-cAMPS- and PKI-induced actin rearrangement. The activities of both ERK1/2 and its upstream activator Raf-1 were transiently enhanced by Rp-cAMPS and linked to the phosphorylation of the well-known ERK cytoskeletal target caldesmon. Inhibition of the Raf-1 target ERK kinase (MEK) either attenuated or abolished Rp-cAMPS- and PKI-induced ERK activation, caldesmon phosphorylation, and stress fiber formation. In summary, our data elucidate the involvement of the p42/44 ERK pathway in cytoskeletal rearrangement evoked by reductions in PKA activity and suggest the involvement of significant cross talk between cAMP- and ERK-dependent signaling pathways in endothelial cell cytoskeletal organization and barrier regulation.
Identification of a novel antigen on the apical surface of rat alveolar epithelial type II and Clara cells
Authors: Boylan Gráinne M. , Pryde James G. , Dobbs Leland G. , McElroy Mary C.
Published: December 2017

Here we describe a monoclonal antibody (MMC4) that recognizes a novel antigen on the apical surface of rat alveolar epithelial type II and Clara cells in the lung, proximal tubule epithelial cells in the kidney, and villus epithelial cells in the small intestine. Biochemical analysis showed that the MMC4 antigen was sensitive to heating and proteinase K digestion and that it is distributed in the detergent-rich phase after Triton X-114 phase separation. These data suggest that the MMC4 antigen is an integral membrane protein. Glycerol gradient sedimentation identified two forms of the MMC4 antigen: one with a sedimentation coefficient of 10.1 and one with a sedimentation coefficient of 1.66, suggesting that the antigen may be part of a multiprotein complex. During rat development (fetal day 16 to adult), the MMC4 antigen increased 12-fold in the lung and 200-fold in the kidney. In the intestine, the MMC4 antigen increased 150-fold by neonatal day 1 and then decreased to adult values. Our data demonstrate that the MMC4 antigen is unlike known type II cell- and Clara cell-associated proteins. The MMC4 monoclonal antibody will be useful as a marker of epithelial cell phenotype in development and injury studies.
Developmental expression of neurokinin A and functional neurokinin-2 receptors in lung
Authors: Haley Kathleen J. , Sunday Mary E. , Osathanondh Rapin , Du Juan , Vathanaprida Charie , Karpitsky Vladimir V. , Krause James E. , Lilly Craig M.
Published: December 2017

Peribronchial smooth muscle constriction causes airway stretch, an important mechanical force in developing lung. Little is known about factors influencing these spontaneously active muscle elements. We measured contractile activity of neurokinin (NK) receptors on fetal intrapulmonary smooth muscle by tracheal perfusion assay ( n = 11). Injecting either capsaicin or the NK2receptor agonist [NLE10]NKA resulted in significant ( P < 0.05) bronchoconstriction. A specific NK2receptor antagonist inhibited constriction caused by endogenous tachykinins released by capsaicin. We then examined NK2receptor ( n = 44) and NKA ( n = 23) ontogeny in human lung. NKA immunostaining was identified in peribronchial nerves in samples with gestational age >12 wk. NK2receptor protein was identified in peribronchial and perivascular smooth muscle. These results indicate that endogenous tachykinins released by the developing lung act via NK2receptors to cause smooth muscle constriction. We speculate that tachykinins could modulate lung development.
Characterization of adenylyl cyclase isoforms in rat peripheral pulmonary arteries
Authors: Jourdan Karen B. , Mason Nicola A. , Long Lu , Philips Peter G. , Wilkins Martin R. , Morrell Nicholas W.
Published: December 2017

Activation of adenylyl cyclase (AC), of which there are 10 diversely regulated isoforms, is important in regulating pulmonary vascular tone and remodeling. Immunohistochemistry in rat lungs demonstrated that AC2, AC3, and AC5/6 predominated in vascular and bronchial smooth muscle. Isoforms 1, 4, 7, and 8 localized to the bronchial epithelium. Exposure of animals to hypoxia did not change the pattern of isoform expression. RT-PCR confirmed mRNA expression of AC2, AC3, AC5, and AC6 and demonstrated AC7 and AC8 transcripts in smooth muscle. Western blotting confirmed the presence of AC2, AC3, and AC5/6 proteins. Functional studies provided evidence of cAMP regulation by Ca2+and protein kinase C-activated but not Gi-inhibited pathways, supporting a role for AC2 and a Ca2+-stimulated isoform, AC8. However, NKH-477, an AC5-selective activator, was more potent than forskolin in elevating cAMP and inhibiting serum-stimulated [3H]thymidine incorporation, supporting the presence of AC5. These studies demonstrate differential expression of AC isoforms in rat lungs and provide evidence that AC2, AC5, and AC8 are functionally important in cAMP regulation and growth pathways in pulmonary artery myocytes.
CTP:phosphocholine cytidylyltransferase inhibition by ceramide via PKC-α, p38 MAPK, cPLA2, and 5-lipoxygenase
Authors: Awasthi Shanjana , Vivekananda Jeevalatha , Awasthi Vibhudutta , Smith Dolphin , King Richard J.
Published: December 2017

In a companion paper (Vivekananda J, Smith D, and King RJ. Am J Physiol Lung Cell Mol Physiol 281: L98–L107, 2001), we demonstrated that tumor necrosis factor (TNF)-α inhibited the activity of CTP:phosphocholine cytidylyltransferase (CT), the rate-limiting enzyme in the de novo synthesis of phosphatidylcholine (PC), and that its actions were likely exerted through a metabolite of sphingomyelin. In this paper, we explore the signaling pathway employed by TNF-α using C2ceramide as a cell-penetrating sphingolipid representative of the metabolites induced by TNF-α. We found that in H441 cells, as reported in other cell types, cytosolic phospholipase A2(cPLA2) is activated by TNF-α. We also observed that the inhibiting action of C2ceramide on CT requires protein kinase C-α, p38 mitogen-activated protein kinase, and cPLA2. The actions of C2ceramide on CT activity can be duplicated by adding 2 μM lysoPC to these cells. Furthermore, we found that the effects of C2ceramide are dependent on 5-lipoxygenase but that cyclooxygenase II is unimportant. We hypothesize that CT activity is inhibited by the lysoPC generated as a consequence of the activation of cPLA2by protein kinase C-α and p38 mitogen-activated protein kinase. The other product of the activation of cPLA2, arachidonic acid, is a substrate for the synthesis of leukotrienes, which raise intracellular Ca2+levels and complete the activation of cPLA2.
Bacterial induction of pleural mesothelial monolayer barrier dysfunction
Authors: Mohammed Kamal A. , Nasreen Najmunnisa , Hardwick Joyce , Logie Carolyn S. , Patterson Carolyn E. , Antony Veena B.
Published: December 2017

Pneumonia remains one of the most common infectious causes of mortality. Patients with pneumonia develop parapneumonic effusions with a high neutrophil count as well as high protein concentrations. We hypothesized that pulmonary parenchymal bacterial infection causes a permeability change in the pleural mesothelium by inducing the production of vascular endothelial growth factor (VEGF). Complicated parapneumonic pleural effusions (empyema) have a 19-fold higher VEGF level than pleural fluids secondary to congestive heart failure and a 4-fold higher level than pleural fluids secondary to uncomplicated parapneumonic effusions. We also analyzed the influence of live Staphylococcus aureus on mesothelial barrier function using a model of confluent mesothelial monolayers. There was a significant drop in electrical resistance across S. aureus-infected pleural mesothelial cell (PMC) monolayers. Recombinant VEGF also decreases PMC electrical resistance. Neutralizing antibodies to VEGF significantly inhibited the drop in PMC electrical resistance caused by S. aureus. S. aureusinfection also caused a significant increase in protein leak across confluent mesothelial monolayers. Our results suggest that bacterial pathogens induce VEGF release in mesothelial cells and alter mesothelial permeability, leading to protein exudation in empyema.
Evaluation of basement membrane degradation during TNF-α-induced increase in epithelial permeability
Authors: Lacherade Jean-Claude , Van De Louw Andry , Planus Emmanuelle , Escudier Estelle , D'Ortho Marie-Pia , Lafuma Chantal , Harf Alain , Delclaux Christophe
Published: December 2017

We evaluated whether tumor necrosis factor (TNF)-α induces an increase in permeability of an alveolar epithelial monolayer via gelatinase secretion and basement membrane degradation. Gelatinase secretion and epithelial permeability to radiolabeled albumin under unstimulated and TNF-α-stimulated conditions of an A549 human epithelial cell line were evaluated in vitro. TNF-α induced both upregulation of a 92-kDa gelatinolytic activity (pro form in cell supernatant and activated form in extracellular matrix) and an increase in the epithelial permeability coefficient compared with the unstimulated condition (control: 1.34 ± 0.04 × 10−6cm/s; 1 μg/ml TNF-α: 1.47 ± 0.05 × 10−6cm/s, P < 0.05). The permeability increase in the TNF-α-stimulated condition involved both paracellular permeability, with gap formation visualized by actin cytoskeleton staining, and basement membrane permeability, with an increase in the basement membrane permeability coefficient (determined after cell removal; control: 2.58 ± 0.07 × 10−6cm/s; 1 μg/ml TNF-α: 2.82 ± 0.02.10−6× cm/s, P < 0.05). Because addition of gelatinase inhibitors [tissue inhibitor of metalloproteinase (TIMP)-1 or BB-3103] to cell supernatants failed to inhibit the permeability increase, the gelatinase-inhibitor balance in the cellular microenvironment was further evaluated by cell culture on a radiolabeled collagen matrix. In the unstimulated condition, spontaneous collagenolytic activity inhibited by addition to the matrix of 1 μg/ml TIMP-1 or 10−6M BB-3103 was found. TNF-α failed to increase this collagenolytic activity because it was associated with dose-dependent upregulation of TIMP-1 secretion by alveolar epithelial cells. In conclusion, induction by TNF-α of upregulation of both the 92-kDa gelatinase and its inhibitor TIMP-1 results in maintenance of the gelatinase-inhibitor balance, indicating that basement membrane degradation does not mediate the TNF-α-induced increase in alveolar epithelial monolayer permeability.
Modulation of α-ENaC and α1-Na+-K+-ATPase by cAMP and dexamethasone in alveolar epithelial cells
Authors: Dagenais André , Denis Christine , Vives Marie-France , Girouard Sonia , Massé Chantal , Nguyen Thao , Yamagata Toshiyuki , Grygorczyk Czeslawa , Kothary Rashmi , Berthiaume Yves
Published: December 2017

cAMP and dexamethasone are known to modulate Na+transport in epithelial cells. We investigated whether dibutyryl cAMP (DBcAMP) and dexamethasone modulate the mRNA expression of two key elements of the Na+transport system in isolated rat alveolar epithelial cells: α-, β-, and γ-subunits of the epithelial Na+channel (ENaC) and the α1- and β1-subunits of Na+-K+-ATPase. The cells were treated for up to 48 h with DBcAMP or dexamethasone to assess their long-term impact on the steady-state level of ENaC and Na+-K+-ATPase mRNA. DBcAMP induced a twofold transient increase of α-ENaC and α1-Na+-K+-ATPase mRNA that peaked after 8 h of treatment. It also upregulated β- and γ-ENaC mRNA but not β1-Na+-K+-ATPase mRNA. Dexamethasone augmented α-ENaC mRNA expression 4.4-fold in cells treated for 24 h and also upregulated β- and γ-ENaC mRNA. There was a 1.6-fold increase at 8 h of β1-Na+-K+-ATPase mRNA but no significant modulation of α1-Na+-K+-ATPase mRNA expression. Because DBcAMP and dexamethasone did not increase the stability of α-ENaC mRNA, we cloned 3.2 kb of the 5′ sequences flanking the mouse α-ENaC gene to study the impact of DBcAMP and dexamethasone on α-ENaC promoter activity. The promoter was able to drive basal expression of the chloramphenicol acetyltransferase (CAT) reporter gene in A549 cells. Dexamethasone increased the activity of the promoter by a factor of 5.9. To complete the study, the physiological effects of DBcAMP and dexamethasone were investigated by measuring transepithelial current in treated and control cells. DBcAMP and dexamethasone modulated transepithelial current with a time course reminiscent of the profile observed for α-ENaC mRNA expression. DBcAMP had a greater impact on transepithelial current (2.5-fold increase at 8 h) than dexamethasone (1.8-fold increase at 24 h). These results suggest that modulation of α-ENaC and Na+-K+-ATPase gene expression is one of the mechanisms that regulates Na+transport in alveolar epithelial cells.
Role of zinc in pulmonary endothelial cell response to oxidative stress
Authors: Tang Zi-Lue , Wasserloos Karla , St. Croix Claudette M. , Pitt Bruce R.
Published: December 2017

Although zinc is a well-known inhibitor of apoptosis, it may contribute to oxidative stress-induced necrosis. We noted that N, N, N′, N′- tetrakis(2-pyridylmethyl)ethylenediamine (TPEN; >10 μM), a zinc chelator, quenched fluorescence of the zinc-specific fluorophore Zinquin and resulted in an increase in spontaneous apoptosis in cultured sheep pulmonary artery endothelial cells (SPAECs). Addition of exogenous zinc (in the presence of pyrithione, a zinc ionophore) to the medium of SPAECs caused an increase in Zinquin fluorescence and was associated with a concentration-dependent increase in necrotic cell death. Exposure of SPAECs to TPEN (10 μM) resulted in enhanced apoptosis after lipopolysaccharide or complete inhibition of t-butyl hydroperoxide (tBH)-induced necrosis. We further investigated the role of two zinc-dependent enzymes, poly(ADP-ribose) polymerase (PARP) and protein kinase (PK) C, in tBH toxicity. tBH toxicity was only affected by the PARP inhibitors 4-amino-1,8-naphthalimide or 3-aminobenzamide over a narrow range, whereas the PKC inhibitors bisindolylmaleimide and staurosporine significantly reduced tBH toxicity. tBH caused translocation of PKC to the plasma membrane of SPAECs that was partially inhibited by TPEN. Thus pulmonary endothelial cell zinc inhibits spontaneous and lipopolysaccharide-dependent apoptosis but contributes to tBH-induced necrosis, in part, via a PKC-dependent pathway.
Novel mechanisms in murine nitrofen-induced pulmonary hypoplasia: FGF-10 rescue in culture
Authors: Acosta Juan M. , Thébaud Bernard , Castillo Carmenza , Mailleux Arnaud , Tefft Denise , Wuenschell Carol , Anderson Kathryn D. , Bourbon Jacques , Thiery Jean-Paul , Bellusci Savério , Warburton David
Published: December 2017

We evaluated the role of the key pulmonary morphogenetic gene fibroblast growth factor-10 ( Fgf10) in murine nitrofen-induced primary lung hypoplasia, which is evident before the time of diaphragm closure. In situ hybridization and competitive RT-PCR revealed a profound disturbance in the temporospatial pattern as well as a 10-fold decrease in mRNA expression level of Fgf10 but not of the inducible inhibitor murine Sprouty2 (mSpry2) after nitrofen treatment. Exogenous FGF-10 increased branching not only of control lungs [13% (right) and 27% (left); P < 0.01] but also of nitrofen-exposed lungs [23% (right) and 77% (left); P < 0.01]. Expression of mSpry2increased 10-fold with FGF-10 in both nitrofen-treated and control lungs, indicating intact downstream FGF signaling mechanisms after nitrofen treatment. We conclude that nitrofen inhibits Fgf10 expression, which is essential for lung growth and branching. Exogenous FGF-10 not only stimulates FGF signaling, marked by increased mSpry2 expression, in both nitrofen-treated and control lungs but also substantially rescues nitrofen-induced lung hypoplasia in culture.
Regulation of murine airway responsiveness by endothelial nitric oxide synthase
Authors: Feletou Michel , Lonchampt Michel , Coge Francis , Galizzi Jean-Pierre , Bassoullet Claire , Merial Christelle , Robineau Pascale , Boutin Jean A. , Huang Paul L. , Vanhoutte Paul M. , Canet Emmanuel
Published: December 2017

Nitric oxide (NO) is a potent vasodilator, but it can also modulate contractile responses of the airway smooth muscle. Whether or not endothelial (e) NO synthase (NOS) contributes to the regulation of bronchial tone is unknown at present. Experiments were designed to investigate the isoforms of NOS that are expressed in murine airways and to determine whether or not the endogenous release of NO modulates bronchial tone in wild-type mice and in mice with targeted deletion of eNOS [eNOS(−/−)]. The presence of neuronal NOS (nNOS), inducible NOS (iNOS), and eNOS in murine trachea and lung parenchyma was assessed by RT-PCR, immunoblotting, and immunohistochemistry. Airway resistance was measured in conscious unrestrained mice by means of a whole body plethysmography chamber. The three isoforms of NOS were constitutively present in lungs of wild-type mice, whereas only iNOS and nNOS were present in eNOS(−/−) mice. Labeling of nNOS was localized in submucosal airway nerves but was not consistently detected, and iNOS immunoreactivity was observed in tracheal and bronchiolar epithelial cells, whereas eNOS was expressed in endothelial cells. In wild-type mice, treatment with N-nitro-l-arginine methyl ester, but not with aminoguanidine, potentiated the increase in airway resistance produced by inhalation of methacholine. eNOS(−/−) mice were hyperresponsive to inhaled methacholine and markedly less sensitive to N-nitro-l-arginine methyl ester. These results demonstrate that the three NOS isoforms are expressed constitutively in murine lung and that NO derived from eNOS plays a physiological role in controlling bronchial airway reactivity.
Enhanced clearance of surfactant protein D during LPS-induced acute inflammation in rat lung
Authors: Herbein Joel F. , Wright Jo Rae
Published: December 2017

Pulmonary surfactant participates in the regulation of alveolar compliance and lung host defense. Surfactant homeostasis is regulated through a combination of synthesis, secretion, clearance, recycling, and degradation of surfactant components. The extracellular pool size of surfactant protein (SP) D fluctuates significantly during acute inflammation. We hypothesized that changes in SP-D levels are due, in part, to altered clearance of SP-D. Clearance pathways in rats were assessed with fluorescently labeled SP-D that was instilled into control lungs or lungs that had been treated with lipopolysaccharide (LPS) 16 h earlier. SP-D clearance from lavage into lung tissue was time dependent from 5 min to 1 h and 1.7-fold greater in LPS-treated lungs than in control lungs. Analysis of cells isolated by enzymatic digestion of lung tissue revealed differences in the SP-D-positive cell population between groups. LPS-treated lungs had 28.1-fold more SP-D-positive tissue-associated neutrophils and 193.6-fold greater SP-D association with those neutrophils compared with control lungs. These data suggest that clearance of SP-D into lung tissue is increased during inflammation and that tissue-associated neutrophils significantly contribute to this process.
Involvement of the MAP kinase ERK2 in MUC1 mucin signaling
Authors: Meerzaman Daoud , Shapiro Paul S. , Kim K. Chul
Published: December 2017

MUC1 mucin is a receptor-like glycoprotein expressed abundantly in various cancer cell lines as well as in glandular secretory epithelial cells, including airway surface epithelial cells. The role of this cell surface mucin in the airway is not known. In an attempt to understand the signaling mechanism of MUC1 mucin, we established a stable cell line from COS-7 cells expressing a chimeric receptor consisting of the extracellular and transmembrane domains of CD8 and the cytoplasmic (CT) domain of MUC1 mucin (CD8/MUC1 cells). We previously observed that treatment of these cells with anti-CD8 antibody resulted in tyrosine phosphorylation of the CT domain of the chimera. Here we report that treatment of CD8/MUC1 cells with anti-CD8 resulted in activation of extracellular signal-regulated kinase (ERK) 2 as assessed by immunoblotting, kinase assay, and immunocytochemistry. The activation of ERK2 was completely blocked either by a dominant negative Ras mutant or in the presence of a mitogen-activated protein kinase kinase (MEK) inhibitor. We conclude that tyrosine phosphorylation of the CT domain of MUC1 mucin leads to activation of a mitogen-activated protein kinase pathway through the Ras-MEK-ERK2 pathway. Combined with the existing data by others, it is suggested that one of the roles of MUC1 mucin may be regulation of cell growth and differentiation via a common signaling pathway, namely the Grb2-Sos-Ras-MEK-ERK2 pathway.
DNA damage and cell cycle checkpoints in hyperoxic lung injury: braking to facilitate repair
Authors: O'Reilly Michael A.
Published: December 2017

The beneficial use of supplemental oxygen therapies to increase arterial blood oxygen levels and reduce tissue hypoxia is offset by the knowledge that it injures and kills cells, resulting in increased morbidity and mortality. Although many studies have focused on understanding how hyperoxia kills cells, recent findings reveal that it also inhibits proliferation through activation of cell cycle checkpoints rather than through overt cytotoxicity. Cell cycle checkpoints are thought to be protective because they allow additional time for injured cells to repair damaged DNA and other essential molecules. During recovery in room air, the lung undergoes a burst of proliferation to replace injured and dead cells. Failure to terminate this proliferation has been associated with fibrosis. These observations suggest that growth-suppressive signals, which inhibit proliferation of injured cells and terminate proliferation when tissue repair has been completed, may play an important role in the pulmonary response to hyperoxia. Because DNA replication is coupled with DNA repair, activation of cell cycle checkpoints during hyperoxia may be a mechanism by which cells protect themselves from oxidant genotoxic stress. This review examines the effect of hyperoxia on DNA integrity, pulmonary cell proliferation, and cell cycle checkpoints activated by DNA damage.
Hypoxic release of calcium from the sarcoplasmic reticulum of pulmonary artery smooth muscle
Authors: Dipp Michelle , Nye Piers C. G. , Evans A. Mark
Published: December 2017

The hypoxic constriction of isolated pulmonary vessels is composed of an initial transient phase ( phase 1) followed by a slowly developing increase in tone ( phase 2). We investigated the roles of the endothelium and of intracellular Ca2+ stores in both preconstricted and unpreconstricted intrapulmonary rabbit arteries when challenged with hypoxia (Po 2 16–21 Torr). Removing the endothelium did not affect phase 1, but phase 2 appeared as a steady plateau. Removing extracellular Ca2+ had essentially the same effect as removing the endothelium. Depletion of sarcoplasmic reticulum Ca2+stores with caffeine and ryanodine abolished the hypoxic response. Omitting preconstriction reduced the amplitude of the hypoxic response but did not qualitatively affect any of the above responses. We conclude that hypoxia releases intracellular Ca2+ from ryanodine-sensitive stores by a mechanism intrinsic to pulmonary vascular smooth muscle without the need for Ca2+ influx across the plasmalemma or an endothelial factor. Our results also suggest that extracellular Ca2+ is required for the release of an endothelium-derived vasoconstrictor.
Cigarette smoke extract induces endothelin-1 via protein kinase C in pulmonary artery endothelial cells
Authors: Lee Sang-Do , Lee Dong-Soon , Chun Yong-Gam , Shim Tae-Sun , Lim Chae-Man , Koh Younsuck , Kim Woo-Sung , Kim Dong-Soon , Kim Won-Dong
Published: December 2017

We examined the mechanism of endothelin (ET)-1 regulation by cigarette smoke extract (CSE) and the effect of platelets on CSE-induced stimulation of ET-1 gene expression in human and bovine pulmonary artery endothelial cells (PAECs). Our data show that CSE (1%) induces ET-1 gene expression (after 1 h) and ET-1 peptide synthesis (after 4 h) in bovine PAECs. The induction of preproET-1 mRNA level was due to de novo transcription, and new protein synthesis was not required for this induction. The protein kinase C inhibitors staurosporine (10−8mol/l) and calphostin C (10−7mol/l) abolished the induction of ET-1 gene expression by CSE in bovine and human PAECs. Although a lower concentration of platelets (106cells/ml in bovine PAECs; 107cells/ml in human PAECs) did not significantly alter ET-1 gene expression in PAECs, incubation of platelets with CSE (1%) and PAECs produced a significant increase in preproET-1 mRNA and ET-1 peptide compared with the values in the presence of CSE (1%) alone. CSE (1%) induced platelet aggregation and increased the expression of platelet membrane glycoproteins ex vivo. Thus our data suggest that CSE stimulates ET-1 gene expression via PKC in PAECs. CSE and platelets showed a synergistic effect on ET-1 gene expression, possibly through the activation of platelets by CSE.
Intermittent hypoxia: cell to system
Authors: Prabhakar Nanduri R. , Fields R. Douglas , Baker Tracy , Fletcher Eugene C.
Published: December 2017

This symposium was organized to present research dealing with the effects of intermittent hypoxia on cardiorespiratory systems and cellular mechanisms. The pattern of neural impulse activity has been shown to be critical in the induction of genes in neuronal cells and involves distinct signaling pathways. Mechanisms associated with different patterns of intermittent hypoxia might share similar mechanisms. Chronic intermittent hypoxia selectively augments carotid body sensitivity to hypoxia and causes long-lasting activation of sensory discharge. Intermittent hypoxia also activates hypoxia-inducible factor-1. Reactive oxygen species are critical in altering carotid body function and hypoxia-inducible factor-1 activation caused by intermittent hypoxia. Blockade of serotonin function in the spinal cord prevents long-term facilitation in respiratory motor output elicited by episodic hypoxia and requires de novo protein synthesis. Chronic intermittent hypoxia leads to sustained elevation in arterial blood pressure and is associated with upregulation of catecholaminergic and renin-angiotensin systems and downregulation of nitric oxide synthases.
Endothelial cellular response to altered shear stress
Authors: Fisher Aron B. , Chien Shu , Barakat Abdul I. , Nerem Robert M.
Published: December 2017

Endothelial cells are normally exposed constantly to mechanical forces that significantly influence their phenotype. This symposium presented recent information concerning endothelial cell responses to shear stress associated with blood flow. Endothelial cell shear stress mechanosensors that have been proposed include membrane receptor kinases, integrins, G proteins, ion channels, intercellular junction proteins, membrane lipids (e.g., those associated with caveolae), and the cytoskeleton. These sensors are linked to signaling cascades that interact with or result in generation of reactive oxygen species, nitric oxide, and various transcription factors among other responses. Endothelial cells adapt to sustained shear stress, and either an increase or decrease from normal shear leads to signaling events. In vitro models for the study of endothelial cell responses must consider the pattern of shear stress (e.g., steady vs. oscillatory flow), the scaffold for cell growth (e.g., basement membrane or other cell types such as smooth muscle cells), and the extent of flow adaptation. These cellular responses have major relevance for understanding the pathophysiological effects of increased shear stress associated with hypertension or decreased shear stress associated with thrombotic occlusion.
Chronic ethanol downregulates PKA activation and ciliary beating in bovine bronchial epithelial cells
Authors: Wyatt T. A. , Sisson J. H.
Published: December 2017

Previously, we reported that ethanol (EtOH) stimulates a rapid increase in ciliary beat frequency (CBF) of bovine bronchial epithelial cells (BBEC). Agents activating cAMP-dependent protein kinase (PKA) also stimulate CBF. EtOH stimulates BBEC CBF through cyclic nucleotide kinase activation. However, EtOH-stimulated CBF is maximal by 1 h and subsides by 6 h, returning to baseline by 24 h. We hypothesized that the loss of EtOH-stimulated CBF was a result of downregulation of PKA activity. To determine the PKA activation state in response to EtOH, ciliated BBEC were stimulated for 0–72 h with various concentrations of EtOH and assayed for PKA. EtOH (100 mM) treatment of the cells for 1 h increased PKA activity threefold over unstimulated controls. PKA activity decreased with increasing time from 6 to 24 h. When BBEC were preincubated with 100 mM EtOH for 24 h, the stimulation of PKA by isoproterenol or 8-bromo-cAMP was abrogated. EtOH desensitizes BBEC to PKA-activating agents, suggesting that EtOH rapidly stimulates, whereas long-term EtOH downregulates, CBF via PKA in BBEC.