AJP: Lung Cellular and Molecular Physiology recent issues

Role of lung iron in determining the bacterial and host struggle in cystic fibrosis

Reid, D. W., Anderson, G. J., Lamont, I. L. — Mon, 26 Oct 2009 14:24:03 PDT

Cystic fibrosis (CF) is the most common lethal genetic disorder in Caucasian populations. It is a multiorgan system disease that affects the lungs, gastrointestinal tract, liver, and pancreas. The majority of morbidity and mortality in CF relates to chronic airway infection with a variety of bacterial species, commencing in very early infancy, which results in lung destruction and ultimately organ failure (41, 43). This review focuses on iron homeostasis in the CF lung and its role in determining the success and chronicity of Pseudomonas aeruginosa infection. There have been previous excellent reviews regarding iron metabolism in the lower respiratory tract and mechanisms of P. aeruginosa iron acquisition, and we direct readers to these articles for further background reading (31, 53, 58, 77, 96). In this review, we have brought the "two sides of the coin" together to provide a holistic overview of the relationship between host and bacterial iron homeostasis and put this information into the context of current understanding on infection in the CF lung.

Could N-acetylcysteine slow progression of idiopathic pulmonary fibrosis by inhibiting EMT?

Wolters, P. J. — Mon, 26 Oct 2009 14:24:03 PDT

N-acetylcysteine inhibits alveolar epithelial-mesenchymal transition

Felton, V. M., Borok, Z., Willis, B. C. — Mon, 26 Oct 2009 14:24:03 PDT

The ability of transforming growth factor-β1 (TGF-β1) to induce epithelial-mesenchymal transition (EMT) in alveolar epithelial cells (AEC) in vitro and in vivo, together with the demonstration of EMT in biopsies of idiopathic pulmonary fibrosis (IPF) patients, suggests a role for TGF-β1-induced EMT in disease pathogenesis. We investigated the effects of N-acetylcysteine (NAC) on TGF-β1-induced EMT in a rat epithelial cell line (RLE-6TN) and in primary rat alveolar epithelial cells (AEC). RLE-6TN cells exposed to TGF-β1 for 5 days underwent EMT as evidenced by acquisition of a fibroblast-like morphology, downregulation of the epithelial-specific protein zonula occludens-1, and induction of the mesenchymal-specific proteins -smooth muscle actin (-SMA) and vimentin. These changes were inhibited by NAC, which also prevented Smad3 phosphorylation. Similarly, primary alveolar epithelial type II cells exposed to TGF-β1 also underwent EMT that was prevented by NAC. TGF-β1 decreased cellular GSH levels by 50–80%, whereas NAC restored them to ~150% of those found in TGF-β1-treated cells. Treatment with glutathione monoethyl ester similarly prevented an increase in mesenchymal marker expression. Consistent with its role as an antioxidant and cellular redox stabilizer, NAC dramatically reduced intracellular reactive oxygen species production in the presence of TGF-β1. Finally, inhibition of intracellular ROS generation during TGF-β1 treatment prevented alveolar EMT, but treatment with H₂O₂ alone did not induce EMT. We conclude that NAC prevents EMT in AEC in vitro, at least in part through replenishment of intracellular GSH stores and limitation of TGF-β1-induced intracellular ROS generation. We speculate that beneficial effects of NAC on pulmonary function in IPF may be mediated by inhibitory effects on alveolar EMT.

Mechanisms of alveolar epithelial chloride absorption

Ingbar, D. H., Bhargava, M., O'Grady, S. M. — Mon, 26 Oct 2009 14:24:03 PDT

Characteristics of Cl- uptake in rat alveolar type I cells

Johnson, M., Allen, L., Dobbs, L. — Mon, 26 Oct 2009 14:24:03 PDT

Although Cl^– transport in fetal lung is important for fluid secretion and normal lung development, the role of Cl^– transport in adult lung is not well understood. In physiological studies, the cystic fibrosis transmembrane regulator (CFTR) plays a role in fluid absorption in the distal air spaces of adult lung, and alveolar type II cells cultured for 5 days have the capacity to transport Cl^–. Although both alveolar type I and type II cells express CFTR, it has previously not been known whether type I cells transport Cl^–. We studied Cl^– uptake in isolated type I cells directly, using either radioisotopic tracers or halide-sensitive fluorescent indicators. By both methods, type I cells take up Cl^–. In the presence of β-adrenergic agonist stimulation, Cl^– uptake can be inhibited by CFTR antagonists. Type I cells express both the Cl^–/HCO₃^– anion exchanger AE2 and the voltage-gated Cl^– channels CLC5 and CLC2. Inhibitors of AE2 also block Cl^– uptake in type I cells. Together, these results demonstrate that type I cells are capable of Cl^– uptake and suggest that the effects seen in whole lung studies establishing the importance of Cl^– movement in alveolar fluid clearance may be, in part, the result of Cl^– transport across type I cells.

The triterpenoid CDDO limits inflammation in preclinical models of cystic fibrosis lung disease

Nichols, D. P., Ziady, A. G., Shank, S. L., Eastman, J. F., Davis, P. B. — Mon, 26 Oct 2009 14:24:03 PDT

Excessive inflammation in cystic fibrosis (CF) lung disease is a contributor to progressive pulmonary decline. Effective and well-tolerated anti-inflammatory therapy may preserve lung function, thereby improving quality and length of life. In this paper, we assess the anti-inflammatory effects of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid (CDDO) in preclinical models of CF airway inflammation. In our experiments, mice carrying the R117H Cftr mutation have significantly reduced airway inflammatory responses to both LPS and flagellin when treated with CDDO before inflammatory challenge. Anti-inflammatory effects observed include reduced airway neutrophilia, reduced concentrations of proinflammatory cytokines and chemokines, and reduced weight loss. Our findings with the synthetic triterpenoids in multiple cell culture models of CF human airway epithelia agree with effects previously described in other disease models (e.g., neoplastic cells). These include the ability to reduce NF-B activation while increasing nuclear factor erythroid-related factor 2 (Nrf2) activity. As these two signaling pathways appear to be pivotal in regulating the net inflammatory response in the CF airway, these compounds are a promising potential anti-inflammatory therapy for CF lung disease.

Intermedin/adrenomedullin-2 is a hypoxia-induced endothelial peptide that stabilizes pulmonary microvascular permeability

Mon, 26 Oct 2009 14:24:03 PDT

Accumulating evidence suggests a pivotal role of the calcitonin receptor-like receptor (CRLR) signaling pathway in preventing damage of the lung by stabilizing pulmonary barrier function. Intermedin (IMD), also termed adrenomedullin-2, is the most recently identified peptide targeting this receptor. Here we investigated the effect of hypoxia on the expression of IMD in the murine lung and cultured murine pulmonary microvascular endothelial cells (PMEC) as well as the role of IMD in regulating vascular permeability. Monoclonal IMD antibodies were generated, and transcript levels were assayed by quantitative RT-PCR. The promoter region of IMD gene was analyzed, and the effect of hypoxia-inducible factor (HIF)-1 on IMD expression was investigated in HEK293T cells. Isolated murine lungs and a human lung microvascular endothelial cell monolayer model were used to study the effect of IMD on vascular permeability. IMD was identified as a pulmonary endothelial peptide by immunohistochemistry and RT-PCR. Hypoxia caused an upregulation of IMD mRNA in the murine lung and PMEC. As shown by these results, HIF-1 enhances IMD promoter activity. Our functional studies showed that IMD abolished the increase in pressure-induced endothelial permeability. Moreover, IMD decreased basal and thrombin-induced hyperpermeability of an endothelial cell monolayer in a receptor-dependent manner and activated PKA in these cells. In conclusion, IMD is a novel hypoxia-induced gene and a potential interventional agent for the improvement of endothelial barrier function in systemic inflammatory responses and hypoxia-induced vascular leakage.

Modulation of reactive oxygen species by Rac1 or catalase prevents asbestos-induced pulmonary fibrosis

Mon, 26 Oct 2009 14:24:04 PDT

The release of reactive oxygen species (ROS) and cytokines by alveolar macrophages has been demonstrated in asbestos-induced pulmonary fibrosis, but the mechanism linking alveolar macrophages to the pathogenesis is not known. The GTPase Rac1 is a second messenger that plays an important role in host defense. In this study, we demonstrate that Rac1 null mice are protected from asbestos-induced pulmonary fibrosis, as determined by histological and biochemical analysis. We hypothesized that Rac1 induced pulmonary fibrosis via generation of ROS. Asbestos increased TNF- and ROS in a Rac1-dependent manner. TNF- was elevated only 1 day after exposure, whereas ROS generation progressively increased in bronchoalveolar lavage cells obtained from wild-type (WT) mice. To determine whether ROS generation contributed to pulmonary fibrosis, we overexpressed catalase in WT monocytes and observed a decrease in ROS generation in vitro. More importantly, administration of catalase to WT mice attenuated the development of fibrosis in vivo. For the first time, these results demonstrate that Rac1 plays a crucial role in asbestos-induced pulmonary fibrosis. Moreover, it suggests that a simple intervention may be useful to prevent progression of the disease.

EphA2 receptor mediates increased vascular permeability in lung injury due to viral infection and hypoxia

Cercone, M. A., Schroeder, W., Schomberg, S., Carpenter, T. C. — Mon, 26 Oct 2009 14:24:04 PDT

Ephrin family receptor tyrosine kinases are mediators of angiogenesis that may also regulate endothelial barrier function in the lung. Previous work has demonstrated that stimulation of EphA ephrin receptors causes increased vascular leak in the intact lung and increased permeability in cultured endothelial cells. Whether EphA receptors are involved in the permeability changes associated with lung injury is unknown. We studied this question in young rats exposed to viral respiratory infection combined with exposure to moderate hypoxia, a previously described lung injury model. We found that the EphA2 receptor is expressed in normal lung and that EphA2 expression is markedly upregulated in the lungs of hypoxic infected (HV) rats compared with normal control animals. Immunohistochemistry showed increased EphA2 expression principally in areas of edematous alveolar septae. In HV rats, EphA2 antagonism with either the soluble decoy receptor EphA2/Fc or with monoclonal anti-EphA2 antibody reduced albumin extravasation and histological evidence of edema formation (P < 0.01). Vascular leak in HV rats is mediated in large part by increased lung endothelin (ET) levels. In HV rats, ET receptor antagonism with bosentan resulted in reduced EphA2 mRNA and protein expression (P < 0.01). Experiments with cultured rat lung microvascular endothelial cells demonstrated that ET increases endothelial EphA2 expression. These results suggest that EphA2 expression is increased in lung injury, contributes to vascular leak in the injured lung, and is regulated in endothelial cells by ET. EphA2 may be a previously unrecognized contributor to the pathophysiology of lung injury.

Abrogation of TGF-{beta}1-induced fibroblast-myofibroblast differentiation by histone deacetylase inhibition

Guo, W., Shan, B., Klingsberg, R. C., Qin, X., Lasky, J. A. — Mon, 26 Oct 2009 14:24:04 PDT

Idiopathic pulmonary fibrosis (IPF) is a devastating disease with no known effective pharmacological therapy. The fibroblastic foci of IPF contain activated myofibroblasts that are the major synthesizers of type I collagen. Transforming growth factor (TGF)-β1 promotes differentiation of fibroblasts into myofibroblasts in vitro and in vivo. In the current study, we investigated the molecular link between TGF-β1-mediated myofibroblast differentiation and histone deacetylase (HDAC) activity. Treatment of normal human lung fibroblasts (NHLFs) with the pan-HDAC inhibitor trichostatin A (TSA) inhibited TGF-β1-mediated -smooth muscle actin (-SMA) and ₁ type I collagen mRNA induction. TSA also blocked the TGF-β1-driven contractile response in NHLFs. The inhibition of -SMA expression by TSA was associated with reduced phosphorylation of Akt, and a pharmacological inhibitor of Akt blocked TGF-β1-mediated -SMA induction in a dose-dependent manner. HDAC4 knockdown was effective in inhibiting TGF-β1-stimulated -SMA expression as well as the phosphorylation of Akt. Moreover, the inhibitors of protein phosphatase 2A and 1 (PP2A and PP1) rescued the TGF-β1-mediated -SMA induction from the inhibitory effect of TSA. Together, these data demonstrate that the differentiation of NHLFs to myofibroblasts is HDAC4 dependent and requires phosphorylation of Akt.

Identification of the amino acid sequence that targets peroxiredoxin 6 to lysosome-like structures of lung epithelial cells

Sorokina, E. M., Feinstein, S. I., Milovanova, T. N., Fisher, A. B. — Mon, 26 Oct 2009 14:24:04 PDT

Peroxiredoxin 6 (Prdx6), an enzyme with glutathione peroxidase and PLA₂ (aiPLA₂) activities, is highly expressed in respiratory epithelium, where it participates in phospholipid turnover and antioxidant defense. Prdx6 has been localized by immunocytochemistry and subcellular fractionation to acidic organelles (lung lamellar bodies and lysosomes) and cytosol. On the basis of their pH optima, we have postulated that protein subcellular localization determines the balance between the two activities of Prdx6. Using green fluorescent protein-labeled protein expression in alveolar epithelial cell lines, we showed Prdx6 localization to organellar structures resembling lamellar bodies in mouse lung epithelial (MLE-12) cells and lysosomes in A549 cells. Localization within lamellar bodies/lysosomes was in the luminal compartment. Targeting to lysosome-like organelles was abolished by the deletion of amino acids 31–40 from the Prdx6 NH₂-terminal region; deletion of the COOH-terminal region had no effect. A green fluorescent protein-labeled peptide containing only amino acids 31–40 showed lysosomal targeting that was abolished by mutation of S32 or G34 within the peptide. Studies with mutated protein indicated that lipid binding was not necessary for Prdx6 targeting. This peptide sequence has no homology to known organellar targeting motifs. These studies indicate that the localization of Prdx6 in acidic organelles and consequent PLA₂ activity depend on a novel 10-aa peptide located at positions 31–40 of the protein.

Influence of cytoskeletal structure and mechanics on epithelial cell injury during cyclic airway reopening

Mon, 26 Oct 2009 14:24:04 PDT

Although patients with acute respiratory distress syndrome require mechanical ventilation, these ventilators often exacerbate the existing lung injury. For example, the cyclic closure and reopening of fluid-filled airways during ventilation can cause epithelial cell (EpC) necrosis and barrier disruption. Although much work has focused on minimizing the injurious mechanical forces generated during ventilation, an alternative approach is to make the EpC less susceptible to injury by altering the cell's intrinsic biomechanical/biostructural properties. In this study, we hypothesized that alterations in cytoskeletal structure and mechanics can be used to reduce the cell's susceptibility to injury during airway reopening. EpC were treated with jasplakinolide to stabilize actin filaments or latrunculin A to depolymerize actin and then exposed to cyclic airway reopening conditions at room temperature using a previously developed in vitro cell culture model. Actin stabilization did not affect cell viability but significantly improved cell adhesion primarily due to the development of more numerous focal adhesions. Surprisingly, actin depolymerization significantly improved both cell viability and cell adhesion but weakened focal adhesions. Optical tweezer based measurements of the EpC's micromechanical properties indicate that although latrunculin-treated cells are softer, they also have increased viscous damping properties. To further investigate the effect of "fluidization" on cell injury, experiments were also conducted at 37°C. Although cells held at 37°C exhibited no changes in cytoskeletal structure, they did exhibit increased viscous damping properties and improved cell viability. We conclude that fluidization of the actin cytoskeleton makes the EpC less susceptible to the injurious mechanical forces generated during cyclic airway reopening.

Cigarette smoke extract induces COX-2 expression via a PKC{alpha}/c-Src/EGFR, PDGFR/PI3K/Akt/NF-{kappa}B pathway and p300 in tracheal smooth muscle cells

Mon, 26 Oct 2009 14:24:04 PDT

Exposure to cigarette smoke extract (CSE) leads to airway or lung inflammation, which may be mediated through cyclooxygenase-2 (COX-2) expression and its product prostaglandin E₂ (PGE₂) synthesis. The aim of this study was to investigate the molecular mechanisms underlying CSE-induced COX-2 expression in human tracheal smooth muscle cells (HTSMCs). Here, we describe that COX-2 induction is dependent on PKC/c-Src/EGFR, PDGFR/PI3K/Akt/NF-B signaling in HTSMCs. CSE stimulated the phosphorylation of c-Src, EGFR, PDGFR, and Akt, which were inhibited by pretreatment with the inhibitor of PKC (Gö6976 or Gö6983), c-Src (PP1), EGFR (AG1478), PDGFR (AG1296), or PI3K (LY294002). Moreover, CSE induced a significant increase in COX-2 expression, which was reduced by pretreatment with these inhibitors or transfection with siRNA of PKC, Src, or Akt. Furthermore, CSE-stimulated NF-B p65 phosphorylation and translocation were also attenuated by pretreatment with Gö6976, PP1, AG1478, AG1296, or LY294002. CSE-induced COX-2 expression was also mediated through the recruitment of p300 associated with NF-B in HTSMCs, revealed by coimmunoprecipitation and Western blot analysis. In addition, pretreatment with the inhibitors of NF-B (helenalin) and p300 (garcinol) or transfection with p65 siRNA and p300 siRNA markedly inhibited CSE-regulated COX-2 expression. However, CSE-induced PGE₂ generation was reduced by pretreatment with the inhibitor of COX-2 (NS-398). These results demonstrated that in HTSMCs, CSE-induced COX-2-dependent PGE₂ generation was mediated through PKC/c-Src/EGFR, PDGFR/PI3K/Akt leading to the recruitment of p300 with NF-B complex.

In vitro translocation of quantum dots and influence of oxidative stress

Geys, J., De Vos, R., Nemery, B., Hoet, P. H. M. — Mon, 26 Oct 2009 14:24:04 PDT

In vivo, translocation of inhaled nanoparticles to the circulation has been demonstrated. However, the interaction of nanoparticles with the lung epithelium is not understood. In this study, we investigated, in vitro, the translocation of nano-sized quantum dots (QDs; 25 pmol/ml) through a tight monolayer of primary isolated rat alveolar epithelial cells. The influence of surface charge on translocation was examined using nonfunctionalized QDs, amine-QDs, and carboxyl-QDs. The interaction between nanoparticles and the lung epithelium was monitored by repeatedly measuring the transepithelial electrical resistance (TEER) and by examining the cell layer with confocal microscopy. The effect of oxidative stress was tested by incubating the cells with tert-butyl hydroperoxide (t-BOOH; 75 µM or 1 or 10 mM); the antioxidant N-acetyl-l-cysteine was also used to assess the role of particle-mediated oxidative stress. No translocation through a tight monolayer of primary rat alveolar epithelial cells was observed for any of the different types of QDs. In general, an increase in TEER was found after incubation with QDs. A condition of low oxidative stress did not enhance translocation. In contrast, conditions of high stress (1 or 10 mM t-BOOH or due to QDs toxicity) with disruption of the cell layer, as shown in a decreased TEER, resulted in substantial translocation. In conclusion, no translocation of QDs was found through a tight monolayer of primary rat alveolar epithelial cells, regardless of the QDs surface charge. QDs did not impair the barrier function of the epithelial cells. In conditions with disruption of the cell-cell barrier, translocation was demonstrated.

Peroxisome proliferator-activated receptor-{gamma} ligands induce heme oxygenase-1 in lung fibroblasts by a PPAR{gamma}-independent, glutathione-dependent mechanism

Mon, 26 Oct 2009 14:24:04 PDT

Oxidative stress plays an important role in the pathogenesis of pulmonary fibrosis. Heme oxygenase-1 (HO-1) is a key antioxidant enzyme, and overexpression of HO-1 significantly decreases lung inflammation and fibrosis in animal models. Peroxisome proliferator-activated receptor- (PPAR) is a transcription factor that regulates adipogenesis, insulin sensitization, and inflammation. We report here that the PPAR ligands 15d-PGJ₂ and 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), which have potent antifibrotic effects in vitro, also strongly induce HO-1 expression in primary human lung fibroblasts. Pharmacological and genetic approaches are used to demonstrate that induction of HO-1 is PPAR independent. Upregulation of HO-1 coincides with decreased intracellular glutathione (GSH) levels and can be inhibited by N-acetyl cysteine (NAC), a thiol antioxidant and GSH precursor. Upregulation of HO-1 is not inhibited by Trolox, a non-thiol antioxidant, and does not involve the transcription factors AP-1 or Nrf2. CDDO and 15d-PGJ₂ contain an /β unsaturated ketone that acts as an electrophilic center that can form covalent bonds with free reduced thiols. Rosiglitazone, a PPAR ligand that lacks an electrophilic center, does not induce HO-1. These data suggest that in human lung fibroblasts, 15d-PGJ₂ and CDDO induce HO-1 via a GSH-dependent mechanism involving the formation of covalent bonds between 15d-PGJ₂ or CDDO and GSH. Inhibiting HO-1 upregulation with NAC has only a small effect on the antifibrotic properties of 15d-PGJ₂ and CDDO in vitro. These results suggest that CDDO and similar electrophilic PPAR ligands may have great clinical potential as antifibrotic agents, not only through direct effects on fibroblast differentiation and function, but indirectly by bolstering antioxidant defenses.

Therapeutic effects of hypercapnia on chronic lung injury and vascular remodeling in neonatal rats

Mon, 26 Oct 2009 14:24:04 PDT

Permissive hypercapnia, achieved using low tidal volume ventilation, has been an effective protective strategy in patients with acute respiratory distress syndrome. To date, no such protective effect has been demonstrated for the chronic neonatal lung injury, bronchopulmonary dysplasia. The objective of our study was to determine whether evolving chronic neonatal lung injury, using a rat model, is resistant to the beneficial effects of hypercapnia or simply requires a less conservative approach to hypercapnia than that applied clinically to date. Neonatal rats inhaled air or 60% O₂ for 14 days with or without 5.5% CO₂. Lung parenchymal neutrophil and macrophage numbers were significantly increased by hyperoxia alone, which was associated with interstitial thickening and reduced secondary crest formation. The phagocyte influx, interstitial thickening, and impaired alveolar formation were significantly attenuated by concurrent hypercapnia. Hyperoxic pups that received 5.5% CO₂ had a significant increase in alveolar number relative to air-exposed pups. Increased tyrosine nitration, a footprint for peroxynitrite-mediated reactions, arteriolar medial wall thickening, and both reduced small peripheral pulmonary vessel number and VEGF and angiopoietin-1 (Ang-1) expression, which were observed with hyperoxia, was attenuated by concurrent hypercapnia. We conclude that evolving chronic neonatal lung injury in a rat model is responsive to the beneficial effects of hypercapnia. Inhaled 5.5% CO₂ provided a significant degree of protection against parenchymal and vascular injury in an animal model of chronic neonatal lung injury likely due, at least in part, to its inhibition of a phagocyte influx.

Elastase- and LPS-exposed mice display altered responses to rhinovirus infection

Mon, 26 Oct 2009 14:24:04 PDT

Viral infection is associated with approximately one-half of acute exacerbations of chronic obstructive pulmonary disease (COPD), which in turn, accelerate disease progression. In this study, we infected mice exposed to a combination of elastase and LPS, a constituent of cigarette smoke and a risk factor for development of COPD, with rhinovirus serotype 1B, and examined animals for viral persistence, airway resistance, lung volume, and cytokine responses. Mice exposed to elastase and LPS once a week for 4 wk showed features of COPD such as airway inflammation and obstruction, goblet cell metaplasia, reduced lung elastance, increased total lung volume, and increased alveolar chord length. In general, mice exposed to elastase or LPS alone showed intermediate effects. Compared with rhinovirus (RV)-infected PBS-exposed mice, RV-infected elastase/LPS-exposed mice showed persistence of viral RNA, airway hyperresponsiveness, increased lung volume, and sustained increases in expression of TNF, IL-5, IL-13, and muc5AC (up to 14 days postinfection). Furthermore, virus-induced IFNs, interferon response factor-7, and IL-10 were deficient in elastase/LPS-treated mice. Mice exposed to LPS or elastase alone cleared virus similar to PBS-treated control mice. We conclude that limited exposure of mice to elastase/LPS produces a COPD-like condition including increased persistence of RV, likely due to skewing of the immune response towards a Th2 phenotype. Similar mechanisms may be operative in COPD.

Deletion of caveolin-1 protects hyperoxia-induced apoptosis via survivin-mediated pathways

Zhang, M., Lin, L., Lee, S.-J., Mo, L., Cao, J., Ifedigbo, E., Jin, Y. — Mon, 26 Oct 2009 14:24:04 PDT

Hyperoxia-induced lung injury is an established model that mimics human acute respiratory distress syndrome. Cell death is a prominent feature in lungs following prolonged hyperoxia. Caveolae are omega-shaped invaginations of the plasma membrane. Caveolin-1 (cav-1), a 22-kDa transmembrane scaffolding protein, is the principal structural component of caveolae. We have recently shown that deletion of cav-1 (cav-1^–/–) protected against hyperoxia-induced cell death and lung injury both in vitro and in vivo; however, the mechanisms remain unclear. Survivin, a member of the inhibitor of apoptosis protein family, inhibits apoptosis in tumor cells. Although emerging evidence suggests that survivin is involved in wound healing, especially in vascular injuries, its role in hyperoxia-induced lung injury has not been investigated. Our current data demonstrated that hyperoxia induced apoptosis via suppressing survivin expression. Deletion of cav-1 abolished this suppression and subsequently protected against hyperoxia-induced apoptosis. Using "gain" and "loss" of function assays, we determined that survivin protected lung cells from hyperoxia-induced apoptosis via the inhibition of apoptosis executor caspase-3. Overexpression of survivin by deletion of cav-1 was regulated by Egr-1. Egr-1 functioned as a negative regulator of survivin expression. Deletion of cav-1 upregulated survivin via decreased Egr-1 binding of the survivin promoter region. Together, this study illustrates the effect of hyperoxia on survivin expression and the role of survivin in hyperoxia-induced apoptosis. We also demonstrate that deletion of cav-1 protects hyperoxia-induced apoptosis via modulation of survivin expression.

PI3K, Rho, and ROCK play a key role in hypoxia-induced ATP release and ATP-stimulated angiogenic responses in pulmonary artery vasa vasorum endothelial cells

Mon, 26 Oct 2009 14:24:04 PDT

We recently reported that vasa vasorum expansion occurs in the pulmonary artery (PA) adventitia of chronically hypoxic animals and that extracellular ATP is a pro-angiogenic factor for isolated vasa vasorum endothelial cells (VVEC). However, the sources of extracellular ATP in the PA vascular wall, as well as the molecular mechanisms underlying its release, remain elusive. Studies were undertaken to explore whether VVEC release ATP in response to hypoxia and to determine signaling pathways involved in this process. We found that hypoxia (1–3% O₂) resulted in time- and O₂-dependent ATP release from VVEC. Preincubation with the inhibitors of vesicular transport (monensin, brefeldin A, and N-ethylmaleimide) significantly decreased ATP accumulation in the VVEC conditioned media, suggesting that hypoxia-induced ATP release occurs through vesicular exocytosis. Additionally, both hypoxia and exogenously added ATP resulted in the activation of PI3K and accumulation of GTP-bound RhoA in a time-dependent manner. Pharmacological inhibition of PI3K and ROCK or knockout of RhoA by small interfering RNA significantly abolished hypoxia-induced ATP release from VVEC. Moreover, RhoA and ROCK play a critical role in ATP-induced increases in VVEC DNA synthesis, migration, and tube formation, indicating a functional contribution of PI3K, Rho, and ROCK to both the autocrine mechanism of ATP release and ATP-mediated angiogenic activation of VVEC. Taken together, our findings provide novel evidence for the signaling mechanisms that link hypoxia-induced increases in extracellular ATP and vasa vasorum expansion.

Pulmonary effects of keratinocyte growth factor in newborn rats exposed to hyperoxia

Mon, 26 Oct 2009 14:24:04 PDT

Acute lung injury and compromised alveolar development characterize bronchopulmonary dysplasia (BPD) of the premature neonate. High levels of keratinocyte growth factor (KGF), a cell-cell mediator with pleiotrophic lung effects, are associated with low BPD risk. KGF decreases mortality in hyperoxia-exposed newborn rodents, a classic model of injury-induced impaired alveolarization, although the pulmonary mechanisms of this protection are poorly defined. These were explored through in vitro and in vivo approaches in the rat. Hyperoxia decreased by 30% the rate of wound closure of a monolayer of fetal alveolar epithelial cells, due to cell death, which was overcome by recombinant human KGF (100 ng/ml). In rat pups exposed to >95% O₂ from birth, increased viability induced by intraperitoneal injection of KGF (2 µg/g body wt) every other day was associated with prevention of neutrophil influx in bronchoalveolar lavage (BAL), prevention of decreases in whole lung DNA content and cell proliferation rate, partial prevention of apoptosis increase, and a markedly increased proportion of surfactant protein B-immunoreactive cells in lung parenchyma. Increased lung antioxidant capacity is likely to be due in part to enhanced CAAT/enhancer binding protein expression. By contrast, KGF neither corrected changes induced by hyperoxia in parameters of lung morphometry that clearly indicated impaired alveolarization nor had any significant effect on tissue or BAL surfactant phospholipids. These findings evidence KGF alveolar epithelial cell protection, enhancing effects on alveolar repair capacity, and anti-inflammatory effects in the injured neonatal lung that may account, at least in part, for its ability to reduce mortality. They argue in favor of a therapeutic potential of KGF in the injured neonatal lung.

Effects of hypercapnia with and without acidosis on hypoxic pulmonary vasoconstriction

Mon, 26 Oct 2009 14:24:04 PDT

Acute respiratory disorders and permissive hypercapnic strategy may lead to alveolar hypoxia and hypercapnic acidosis. However, the effects of hypercapnia with or without acidosis on hypoxic pulmonary vasoconstriction (HPV) and oxygen diffusion capacity of the lung are controversial. We investigated the effects of hypercapnic acidosis and hypercapnia with normal pH (pH corrected with sodium bicarbonate) on HPV, capillary permeability, gas exchange, and ventilation-perfusion matching in the isolated ventilated-perfused rabbit lung. No alteration in vascular tone was noted during normoxic hypercapnia with or without acidosis compared with normoxic normocapnia. Hypercapnia with normal pH resulted in a transient increase in HPV during the course of consecutive ventilation maneuvers, whereas hypercapnic acidosis increased HPV over time. Hypercapnic acidosis decreased exhaled NO during hypoxia more than hypercapnia with normal pH and normocapnia, whereas intravascular NO release was unchanged. However, inhibition of NO synthesis by nitro-l-arginine (l-NNA) resulted in a loss of the increased HPV caused by hypercapnic acidosis but not that caused by hypercapnia with normal pH. Furthermore, capillary permeability increased during hypoxic hypercapnia with normal pH but not hypoxic hypercapnic acidosis. This effect was NO-dependent because it disappeared during l-NNA administration. Ventilation-perfusion matching and arterial Po₂ were improved according to the strength of HPV in hypercapnia compared with normocapnia during Tween nebulization-induced lung injury. In conclusion, the increased HPV during hypercapnic acidosis is beneficial to lung gas exchange by improving ventilation-perfusion matching and preserving the capillary barrier function. These effects seem to be linked to NO-mediated pathways.

Inhibition of geranylgeranyltransferase inhibits bronchial smooth muscle hyperresponsiveness in mice

Chiba, Y., Sato, S., Hanazaki, M., Sakai, H., Misawa, M. — Mon, 26 Oct 2009 14:24:04 PDT

Recent studies revealed an involvement of RhoA/Rho-kinase in the contraction of bronchial smooth muscle (BSM), and this pathway has now been proposed as a new target for asthma therapy. A posttranslational geranylgeranylation of RhoA is required for its activation. Thus selective inhibition of geranylgeranyltransferase may be a novel strategy for treatment of the BSM hyperresponsiveness in asthmatics. To test this hypothesis, we investigated the effect of a geranylgeranyltransferase inhibitor, GGTI-2133, on antigen-induced BSM hyperresponsiveness by using mice with experimental asthma. Mice were sensitized and repeatedly challenged with ovalbumin antigen. Animals also were treated with GGTI-2133 (5 mg/kg ip) once a day before and during the antigen inhalation period. Repeated antigen inhalation caused a BSM hyperresponsiveness to acetylcholine with the increased expressions of RhoA and the anti-farnesyl-positive 21-kDa proteins, probably geranylgeranylated RhoA. The in vivo GGTI-2133 treatments significantly inhibited BSM hyperresponsiveness induced by antigen exposure. In another series of experiments, BSM tissues isolated from the repeatedly antigen-challenged mice were cultured for 48 h in the absence or presence of GGTI-2133. Under these conditions, the putative geranylgeranylated RhoA was decreased in a GGTI-2133 concentration-dependent manner. The in vitro incubation with GGTI-2133 also inhibited BSM hyperresponsiveness induced by antigen exposure. These findings suggest that GGTI-2133 inhibits antigen-induced BSM hyperresponsiveness, probably by reducing downstream signal transduction of RhoA. Selective geranylgeranyltransferase inhibitors may be beneficial for the treatment of airway hyperresponsiveness, one of the characteristic features of allergic bronchial asthma.

Persistent bronchiolar remodeling following brief ventilation of the very immature ovine lung

Mon, 26 Oct 2009 14:24:04 PDT

Children and adults who were mechanically ventilated following preterm birth are at increased risk of reduced lung function, suggesting small airway dysfunction. We hypothesized that short periods of mechanical ventilation of very immature lungs can induce persistent bronchiolar remodeling that may adversely affect later lung function. Our objectives were to characterize the effects of brief, positive-pressure ventilation per se on the small airways in very immature, surfactant-deficient lungs and to determine whether the effects persist after the cessation of ventilation. Fetal sheep (0.75 of term) were mechanically ventilated in utero with room air (peak inspiratory pressure 40 cmH₂O, positive end-expiratory pressure 4 cmH₂O, 65 breaths/min) for 6 or 12 h, after which tissues were collected; another group was studied 7 days after 12-h ventilation. Age-matched unventilated fetuses were controls. The mean basement membrane perimeter of airways analyzed was 548.6 ± 8.5 µm and was not different between groups. Immediately after ventilation, 21% of airways had epithelial injury; in airways with intact epithelium, there was more airway smooth muscle (ASM) and less collagen, and the epithelium contained more mucin-containing and apoptotic cells and fewer proliferating cells. Seven days after ventilation, epithelial injury was absent but the epithelium was thicker, with greater cell turnover; there were increased amounts of bronchiolar collagen and ASM and fewer alveolar attachments. The increase in ASM was likely due to cellular hypertrophy rather than hyperplasia. We conclude that brief mechanical ventilation of the very immature lung induces remodeling of the bronchiolar epithelium and walls that lasts for at least 7 days; such changes could contribute to later airway dysfunction.

Mesenchymal stem cells produce Wnt isoforms and TGF-{beta}1 that mediate proliferation and procollagen expression by lung fibroblasts

Salazar, K. D., Lankford, S. M., Brody, A. R. — Mon, 26 Oct 2009 14:24:04 PDT

Studies have been carried out previously to determine whether mesenchymal stem cells (MSC) influence the progression of pulmonary fibrosis. Here, we asked whether MSC (derived from mouse bone marrow and human umbilical cord blood) produce factors that mediate lung fibroblast (LF) growth and matrix production. MSC-conditioned media (CM) were found by ELISA to contain significant amounts of PDGF-AA and transforming growth factor-β₁ (TGF-β₁). Proliferation was increased in a concentration-dependent manner in LF cell lines and primary cells cultured in MSC-CM, but neither anti-PDGF antibodies nor PDGF receptor-specific antibodies affected proliferation, nor did a number of other antibodies to well-known mitogenic factors. However, proliferation was significantly inhibited by the Wnt signaling antagonist, secreted frizzled related protein-1 (sFRP-1). In addition, anti-Wnt1 and anti-Wnt2 antibodies attenuated MSC-CM-induced proliferation, and increased expression of Wnt7b was identified. As would be expected in cells activated by Wnt, nuclear β-catenin was increased. The amount of TGF-β₁ in MSC-CM and its biological activity were revealed by activation at acidic pH. The stem cells synthesized and released TGF-β₁ that increased ₁-procollagen gene expression by LF target cells. Addition of anti-TGF-β to the MSC-CM blocked upregulation of collagen gene expression. These data demonstrate that MSC from mice and humans produce Wnt proteins and TGF-β₁ that respectively stimulate LF proliferation and matrix production, two hallmarks of fibroproliferative lung disease. It will be essential to determine whether these factors can play a role in attempts to use MSC for therapeutic approaches.

Measurement of extravascular lung water using the single indicator method in patients: research and potential clinical value

Brown, L. M., Liu, K. D., Matthay, M. A. — Fri, 25 Sep 2009 09:51:13 PDT

Extravascular lung water includes all of the fluid within the lung but outside of the vasculature. Lung water increases as a result of increased hydrostatic vascular pressure or from an increase in lung endothelial and epithelial permeability or both. Experimentally, extravascular lung water has been measured gravimetrically. Clinically, the chest radiograph is used to determine whether extravascular lung water is present but is an insensitive instrument for determining the quantity of lung water. Bedside measurement of extravascular lung water in patients is now possible using a single indicator thermodilution method. This review critically evaluates the experimental and clinical evidence supporting the potential value of measuring extravascular lung water in patients using the single indicator method.

Respiratory syncytial virus infection alters surfactant protein A expression in human pulmonary epithelial cells by reducing translation efficiency

Bruce, S. R., Atkins, C. L., Colasurdo, G. N., Alcorn, J. L. — Fri, 25 Sep 2009 09:51:13 PDT

Infection of neonatal lung by respiratory syncytial virus (RSV) is a common cause of respiratory dysfunction. Lung alveolar type II and bronchiolar epithelial (Clara) cells secrete surfactant protein A (SP-A), a collectin that is an important component of the pulmonary innate immune system. SP-A binds to the virus, targeting the infectious agent for clearance by host defense mechanisms. We have previously shown that while the steady-state level of SP-A mRNA increases approximately threefold after RSV infection, steady-state levels of cellular and secreted SP-A protein decrease 40–60% in human type II cells in primary culture, suggesting a mechanism where the virus alters components of the innate immune response in infected cells. In these studies, we find that changes in SP-A mRNA and protein levels in RSV-infected NCI-H441 cells (a bronchiolar epithelial cell line) recapitulate the results in SP-A expression observed in primary lung cells. While SP-A protein is normally ubiquitinated, there is no change in the level of SP-A protein ubiquitination or proteasome activity during RSV infection, suggesting that the reduced levels of SP-A protein are not due to degradation by activated proteasomes. SP-A mRNA is appropriately processed and exported from the nucleus to the cytoplasm during RSV infection. As evidenced by polysome analysis of SP-A mRNA and pulse-chase analysis of newly synthesized SP-A protein, we find a decrease in translational efficiency that is specific for SP-A mRNA. Therefore, the decrease in SP-A protein levels observed after RSV infection of pulmonary bronchiolar epithelial cells results from a mechanism that affects SP-A mRNA translation efficiency.

Regulation of intrapleural fibrinolysis by urokinase-{alpha}-macroglobulin complexes in tetracycline-induced pleural injury in rabbits

Komissarov, A. A., Mazar, A. P., Koenig, K., Kurdowska, A. K., Idell, S. — Fri, 25 Sep 2009 09:51:13 PDT

The proenzyme single-chain urokinase plasminogen activator (scuPA) more effectively resolved intrapleural loculations in rabbits with tetracycline (TCN)-induced loculation than a range of clinical doses of two-chain uPA (Abbokinase) and demonstrated a trend toward greater efficacy than single-chain tPA (Activase) (Idell S et al., Exp Lung Res 33: 419, 2007.). scuPA more slowly generates durable intrapleural fibrinolytic activity than Abbokinase or Activase, but the interactions of these agents with inhibitors in pleural fluids (PFs) have been poorly understood. PFs from rabbits with TCN-induced pleural injury treated with intrapleural scuPA, its inactive Ser195Ala mutant, Abbokinase, Activase, or vehicle, were analyzed to define the mechanism by which scuPA induces durable fibrinolysis. uPA activity was elevated in PFs of animals treated with scuPA, correlated with the ability to clear pleural loculations, and resisted (70–80%) inhibition by PAI-1. -macroglobulin (M) but not urokinase receptor complexes immunoprecipitated from PFs of scuPA-treated rabbits retained uPA activity that resists PAI-1 and activates plasminogen. Conversely, little plasminogen activating or enzymatic activity resistant to PAI-1 was detectable in PFs of rabbits treated with Abbokinase or Activase. Consistent with these findings, PAI-1 interacts with scuPA much slower than with Activase or Abbokinase in vitro. An equilibrium between active and inactive scuPA (k_on = 4.3 h^–1) limits the rate of its inactivation by PAI-1, favoring formation of complexes with M. These observations define a newly recognized mechanism that promotes durable intrapleural fibrinolysis via formation of M/uPA complexes. These complexes promote uPA-mediated plasminogen activation in scuPA-treated rabbits with TCN-induced pleural injury.

Cell cycle actions of parathyroid hormone-related protein in non-small cell lung carcinoma

Fri, 25 Sep 2009 09:51:13 PDT

Parathyroid hormone-related protein (PTHrP), a paraneoplastic protein expressed by two-thirds of human non-small cell lung cancers, has been reported to slow progression of lung carcinomas in mouse models and to lengthen survival of patients with lung cancer. This study investigated the effects of ectopic expression of PTHrP on proliferation and cell cycle progression of two human lung adenocarcinoma cell lines that are normally PTHrP negative. Stable transfection with PTHrP decreased H1944 cell DNA synthesis, measured by thymidine incorporation, bromodeoxyuridine uptake, and MTT proliferation assay. A substantial fraction of PTHrP-positive cells was arrested in or slowly progressing through G1. Cyclin D2 and cyclin A2 protein levels were 60–70% lower in PTHrP-expressing cells compared with control cells (P < 0.05, N = 3 independent clones per group), while expression of p27^Kip1, a cyclin-dependent kinase inhibitor, was increased by 35 ± 9% (mean ± SE, P < 0.05) in the presence of PTHrP. Expression of other cyclins, including cyclins D1 and D3, and cyclin-dependent kinases was unaffected by PTHrP. PTHrP did not alter the phosphorylation state of Rb, but decreased cyclin-dependent kinase (CDK) 2-cyclin A2 complex formation. Ectopic expression of PTHrP stimulated ERK phosphorylation. In MV522 cells, PTHrP had similar effects on DNA synthesis, cyclin A2 expression, pRb levels, CDK2-cyclin A2 association, and ERK activation. In summary, PTHrP appears to slow progression of lung cancer cells into S phase, possibly by decreasing activation of CDK2. Slower cancer cell proliferation could contribute to slower tumor progression and increased survival of patients with PTHrP-positive lung cancer.

TNF{alpha} inhibits apoptotic cell clearance in the lung, exacerbating acute inflammation

Fri, 25 Sep 2009 09:51:13 PDT

Efficient removal of apoptotic cells is essential for resolution of inflammation. Failure to clear dying cells can exacerbate lung injury and lead to persistent inflammation and autoimmunity. Here we show that TNF blocks apoptotic cell clearance by alveolar macrophages and leads to proinflammatory responses in the lung. Compared with mice treated with intratracheal TNF or exogenous apoptotic cells, mice treated with the combination of TNF plus apoptotic cells demonstrated reduced apoptotic cell clearance from the lungs and increased recruitment of inflammatory leukocytes to the air spaces. Treatment with intratracheal TNF had no effect on the removal of exogenous apoptotic cells from the lungs of TNF receptor-1 (p55) and -2 (p75) double mutant mice and no effect on leukocyte recruitment. Bronchoalveolar lavage from mice treated with TNF plus apoptotic cells contained increased levels of proinflammatory cytokines IL-6, KC, and MCP-1, but exhibited no change in levels of anti-inflammatory cytokines IL-10 and TGF-β. Administration of TNF plus apoptotic cells during LPS-induced lung injury augmented neutrophil accumulation and proinflammatory cytokine production. These findings suggest that the presence of TNF in the lung can alter the response of phagocytes to apoptotic cells leading to inflammatory cell recruitment and proinflammatory mediator production.

NADPH oxidases and reactive oxygen species at different stages of chronic hypoxia-induced pulmonary hypertension in newborn piglets

Fri, 25 Sep 2009 09:51:13 PDT

Recently, we reported that reactive oxygen species (ROS) generated by NADPH oxidase (NOX) contribute to aberrant responses in pulmonary resistance arteries (PRAs) of piglets exposed to 3 days of hypoxia (Am J Physiol Lung Cell Mol Physiol 295: L881–L888, 2008). An objective of the present study was to determine whether NOX-derived ROS also contribute to altered PRA responses at a more advanced stage of pulmonary hypertension, after 10 days of hypoxia. We further wished to advance knowledge about the specific NOX and antioxidant enzymes that are altered at early and later stages of pulmonary hypertension. Piglets were raised in room air (control) or hypoxia for 3 or 10 days. Using a cannulated artery technique, we found that treatments with agents that inhibit NOX (apocynin) or remove ROS [an SOD mimetic (M40403) + polyethylene glycol-catalase] diminished responses to ACh in PRAs from piglets exposed to 10 days of hypoxia. Western blot analysis showed an increase in expression of NOX1 and the membrane fraction of p67phox. Expression of NOX4, SOD2, and catalase were unchanged, whereas expression of SOD1 was reduced, in arteries from piglets raised in hypoxia for 3 or 10 days. Markers of oxidant stress, F₂-isoprostanes, measured by gas chromatography-mass spectrometry, were increased in PRAs from piglets raised in hypoxia for 3 days, but not 10 days. We conclude that ROS derived from some, but not all, NOX family members, as well as alterations in the antioxidant enzyme SOD1, contribute to aberrant PRA responses at an early and a more progressive stage of chronic hypoxia-induced pulmonary hypertension in newborn piglets.

Genome-wide transcriptional profiling of mononuclear phagocytes recruited to mouse lungs in response to alveolar challenge with the TLR2 agonist Pam3CSK4

Fri, 25 Sep 2009 09:51:13 PDT

Compared with the Toll-like receptor 4 (TLR4) ligand LPS restricted to Gram-negative bacteria, few studies have addressed induction of lung inflammation and concomitant leukocyte recruitment in response to TLR2 ligands. This study is the first report showing that selective TLR2 stimulation by its ligand Pam₃-Cys-Ser-Lys-Lys-Lys-Lys-OH (Pam₃CSK₄) within the alveolar compartment promoted lung inflammation in mice and induced the migration of circulatory immune cells including mononuclear phagocytes into the inflamed alveolar space. By using the transgenic CX₃CR1^+/GFP mouse strain for high-purity sorting of circulating and alveolar recruited mononuclear phagocytes together with SMART preamplification and whole genome oligonucleotide microarray techniques, we found that alveolar trafficking of mononuclear phagocytes was associated with profound changes of their gene expression profiles (~900 differentially regulated genes postrecruitment). In particular, alveolar recruited mononuclear phagocytes showed upregulated transcripts of genes encoding cytokines/chemokines and pattern recognition receptor (PRR)-associated molecules. Notably, we observed a dynamic change of the genetic program of recruited mononuclear phagocytes obtained from bronchoalveolar lavage fluid at different time points (24 vs. 48 h) post-Pam₃CSK₄ challenge. In early alveolar recruited mononuclear phagocytes, mRNA levels of both proinflammatory (e.g., TNF-, CCL2, and IL-6) and central anti-inflammatory/ proresolution [e.g., IL-1-receptor antagonist (IL-1RN), CD200 receptor (CD200R), IL-1 receptor-associated kinase (IRAK-M), IL-10, and Bcl-2-associated X protein (Bax)] mediators were found to be highly upregulated simultaneously. In corresponding cells recruited until later time points, transcript levels of anti-inflammatory/proresolution molecules persisted at the same level, whereas mRNA levels of proinflammatory mediators were found to decline. Collectively, our in vivo study identifies genetic programs by which alveolar recruited mononuclear phagocytes may contribute to the development and termination of pneumonia caused by Gram-positive bacteria.

Maturation of O2 sensing and signaling in the chicken ductus arteriosus

Fri, 25 Sep 2009 09:51:13 PDT

The increase in O₂ tension after birth is a major factor stimulating ductus arteriosus (DA) constriction and closure. Here we studied the role of the mitochondrial electron transport chain (ETC) as sensor, H₂O₂ as mediator, and voltage-gated potassium (K_V) channels and Rho kinase as effectors of O₂-induced contraction in the chicken DA during fetal development. Switching from 0% to 21% O₂ contracted the pulmonary side of the mature DA (mature pDA) but had no effect in immature pDA and relaxed the aortic side of the mature DA (mature aDA). This contraction of the pDA was attenuated by inhibitors of the mitochondrial ETC and by the H₂O₂ scavenger polyethylene glycol (PEG)-catalase. Moreover, O₂ increased reactive oxygen species (ROS) production, measured with the fluorescent probes dihydroethidium and 2',7'-dichlorofluorescein, only in mature pDA. The H₂O₂ analog t-butyl-hydroperoxide mimicked the responses to O₂ in the three vessels. In contrast to immature pDA cells, mature pDA cells exhibited high-amplitude O₂-sensitive potassium currents. The K_V channel blocker 4-aminopyridine prevented the current inhibition elicited by O₂. The L-type Ca²⁺ (Ca_L) channel blocker nifedipine and the Rho kinase inhibitors Y-27632 and hydroxyfasudil induced a similar relaxation when mature pDA were stimulated with O₂ or H₂O₂. Moreover, the sensitivity to these drugs increased with maturation. Our results indicate the presence of a common mechanism for O₂ sensing/signaling in mammalian and nonmammalian DA and favor the idea that, rather than a single mechanism, a parallel maturation of the sensor and effectors is critical for O₂ sensitivity appearance during development.

Role of HIF-1{alpha} in the regulation ACE and ACE2 expression in hypoxic human pulmonary artery smooth muscle cells

Fri, 25 Sep 2009 09:51:14 PDT

Angiotensin-converting enzyme (ACE) enhances the proliferation and migration of pulmonary artery smooth muscle cells (PASMCs), which contribute to the pathogenesis of hypoxic pulmonary hypertension (HPH). Previous reports have demonstrated that hypoxia upregulates ACE expression, but the underlying mechanism is unknown. Here, we found that ACE is persistently upregulated in PASMCs on the transcriptional level during hypoxia. Hypoxia-inducible factor 1 (HIF-1), a key transcription factor activated during hypoxia, was able to upregulate ACE protein expression under normoxia, whereas knockdown of HIF-1 expression in PASMCs inhibited hypoxia-induced ACE upregulation. Furthermore, HIF-1 can bind and transactivate the ACE promoter directly. Therefore, we report that ACE is a novel target of HIF-1. Recently, a homolog of ACE, ACE2, was reported to counterbalance the function of ACE. In contrast to ACE, we found that ACE2 mRNA and protein levels increased during the early stages of hypoxia and decreased to near-baseline levels at the later stages after HIF-1 accumulation. Thus HIF-1 inhibited ACE2 expression, and the accumulated ANG II catalyzed by ACE is a key mediator in the downregulation of ACE2 by HIF-1. Moreover, a reduction of ACE2 expression in PASMCs by RNA interference was accompanied by significantly enhanced proliferation and migration during hypoxia. We conclude that ACE is directly regulated by HIF-1, whereas ACE2 is regulated in a bidirectional way during hypoxia and may play a protective role during the development of HPH. In sum, these findings contribute to the understanding of the pathogenesis of HPH.

Neonatal oxygen adversely affects lung function in adult mice without altering surfactant composition or activity

Fri, 25 Sep 2009 09:51:14 PDT

Despite its potentially adverse effects on lung development and function, supplemental oxygen is often used to treat premature infants in respiratory distress. To understand how neonatal hyperoxia can permanently disrupt lung development, we previously reported increased lung compliance, greater alveolar simplification, and disrupted epithelial development in adult mice exposed to 100% inspired oxygen fraction between postnatal days 1 and 4. Here, we investigate whether oxygen-induced changes in lung function are attributable to defects in surfactant composition and activity, structural changes in alveolar development, or both. Newborn mice were exposed to room air or 40%, 60%, 80%, or 100% oxygen between postnatal days 1 and 4 and allowed to recover in room air until 8 wk of age. Lung compliance and alveolar size increased, and airway resistance, airway elastance, tissue elastance, and tissue damping decreased, in mice exposed to 60–80% oxygen; changes were even greater in mice exposed to 100% oxygen. These alterations in lung function were not associated with changes in total protein content or surfactant phospholipid composition in bronchoalveolar lavage. Moreover, surface activity and total and hydrophobic protein content were unchanged in large surfactant aggregates centrifuged from bronchoalveolar lavage compared with control. Instead, the number of type II cells progressively declined in 60–100% oxygen, whereas levels of T1, a protein expressed by type I cells, were comparably increased in mice exposed to 40–100% oxygen. Thickened bundles of elastin fibers were also detected in alveolar walls of mice exposed to ≥60% oxygen. These findings support the hypothesis that changes in lung development, rather than surfactant activity, are the primary causes of oxygen-altered lung function in children who were exposed to oxygen as neonates. Furthermore, the disruptive effects of oxygen on epithelial development and lung mechanics are not equivalently dose dependent.

Ca2+ entry via {alpha}1G and TRPV4 channels differentially regulates surface expression of P-selectin and barrier integrity in pulmonary capillary endothelium

Fri, 25 Sep 2009 09:51:14 PDT

Pulmonary vascular endothelial cells express a variety of ion channels that mediate Ca²⁺ influx in response to diverse environmental stimuli. However, it is not clear whether Ca²⁺ influx from discrete ion channels is functionally coupled to specific outcomes. Thus we conducted a systematic study in mouse lung to address whether the _1G T-type Ca²⁺ channel and the transient receptor potential channel TRPV4 have discrete functional roles in pulmonary capillary endothelium. We used real-time fluorescence imaging for endothelial cytosolic Ca²⁺, immunohistochemistry to probe for surface expression of P-selectin, and the filtration coefficient to specifically measure lung endothelial permeability. We demonstrate that membrane depolarization via exposure of pulmonary vascular endothelium to a high-K⁺ perfusate induces Ca²⁺ entry into alveolar septal endothelial cells and exclusively leads to the surface expression of P-selectin. In contrast, Ca²⁺ entry in septal endothelium evoked by the selective TRPV4 activator 4-phorbol-12,13-didecanoate (4-PDD) specifically increases lung endothelial permeability without effect on P-selectin expression. Pharmacological blockade or knockout of _1G abolishes depolarization-induced Ca²⁺ entry and surface expression of P-selectin but does not prevent 4-PDD-activated Ca²⁺ entry and the resultant increase in permeability. Conversely, blockade or knockout of TRPV4 specifically abolishes 4-PDD-activated Ca²⁺ entry and the increase in permeability, while not impacting depolarization-induced Ca²⁺ entry and surface expression of P-selectin. We conclude that in alveolar septal capillaries Ca²⁺ entry through _1G and TRPV4 channels differentially and specifically regulates the transition of endothelial procoagulant phenotype and barrier integrity, respectively.

The soluble guanylate cyclase activator HMR1766 reverses hypoxia-induced experimental pulmonary hypertension in mice

Fri, 25 Sep 2009 09:51:14 PDT

Severe pulmonary hypertension (PH) is a disabling disease with high mortality, characterized by pulmonary vascular remodeling and right heart hypertrophy. In mice with PH induced by chronic hypoxia, we examined the acute and chronic effects of the soluble guanylate cyclase (sGC) activator HMR1766 on hemodynamics and pulmonary vascular remodeling. In isolated perfused mouse lungs from control animals, HMR1766 dose-dependently inhibited the pressor response of acute hypoxia. This dose-response curve was shifted leftward when the effects of HMR1766 were investigated in isolated lungs from chronic hypoxic animals for 21 days at 10% oxygen. Mice exposed for 21 or 35 days to chronic hypoxia developed PH, right heart hypertrophy, and pulmonary vascular remodeling. Treatment with HMR1766 (10 mg·kg^–1·day^–1), after full establishment of PH from day 21 to day 35, significantly reduced PH, as measured continuously by telemetry. In addition, right ventricular (RV) hypertrophy and structural remodeling of the lung vasculature were reduced. Pharmacological activation of oxidized sGC partially reverses hemodynamic and structural changes in chronic hypoxia-induced experimental PH.

Inhibition of mTOR attenuates store-operated Ca2+ entry in cells from endarterectomized tissues of patients with chronic thromboembolic pulmonary hypertension

Fri, 25 Sep 2009 09:51:14 PDT

Pulmonary vascular remodeling occurs in patients with chronic thromboembolic pulmonary hypertension (CTEPH). One factor contributing to this vascular wall thickening is the proliferation of pulmonary artery smooth muscle cells (PASMC). Store-operated Ca²⁺ entry (SOCE) and cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) in PASMC are known to be important in cell proliferation and vascular remodeling in pulmonary hypertension. Rapamycin is widely known for its antiproliferative effects in injured coronary arteries. Although several reports have suggested favorable effects of rapamycin in animal models of pulmonary hypertension, no reports have been published to date in human tissues. Here we report that rapamycin has an inhibitory effect on SOCE and an antiproliferative effect on PASMC derived from endarterectomized tissues of CTEPH patients. Cells were isolated from endarterectomized tissues obtained from patients undergoing pulmonary thromboendarterectomy (PTE). Immunohistochemical analysis indicated high deposition of platelet-derived growth factor (PDGF) in tissue sections from PTE tissues and increased PDGF receptor expression. PDGF transiently phosphorylated Akt, mammalian target of rapamycin (mTOR), and p70S6 kinase in CTEPH cells from CTEPH patients. Acute treatment (30 min) with rapamycin (10 nM) slightly increased cyclopiazonic acid (10 µM)-induced Ca²⁺ mobilization and significantly reduced SOCE. Chronic treatment (24 h) with rapamycin reduced Ca²⁺ mobilization and markedly inhibited SOCE. The inhibitory effects of rapamycin on SOCE were less prominent in control cells. Rapamycin also significantly reduced PDGF-stimulated cell proliferation. In conclusion, the data from this study indicate the importance of the mTOR pathway in the development of pulmonary vascular remodeling in CTEPH and suggest a potential therapeutic benefit of rapamycin (or inhibition of mTOR) in these patients.

Dysfunctional cystic fibrosis transmembrane conductance regulator inhibits phagocytosis of apoptotic cells with proinflammatory consequences

Fri, 25 Sep 2009 09:51:14 PDT

Cystic fibrosis (CF) is caused by mutated CF transmembrane conductance regulator (CFTR) and is characterized by robust airway inflammation and accumulation of apoptotic cells. Phagocytosis of apoptotic cells (efferocytosis) is a pivotal regulator of inflammation, because it prevents postapoptotic necrosis and actively suppresses release of a variety of proinflammatory mediators, including IL-8. Because CF is associated with accumulation of apoptotic cells, inappropriate levels of IL-8, and robust inflammation, we sought to determine whether CFTR deficiency specifically impairs efferocytosis and its regulation of inflammatory mediator release. Here we show that CFTR deficiency directly interferes with efferocytosis by airway epithelium, an effect that is not due to altered binding of apoptotic cells to epithelial cells or altered expression of efferocytosis receptors. In contrast, expression of RhoA, a known negative regulator of efferocytosis, is substantially increased in CFTR-deficient cells, and inhibitors of RhoA or its downstream effector Rho kinase normalize efferocytosis in these cells. Impaired efferocytosis appears to be mediated through an amiloride-sensitive ion channel, because amiloride restores phagocytic competency in CFTR-deficient cells. Finally, ineffective efferocytosis in CFTR-deficient cells appears to have proinflammatory consequences, because apoptotic cells enhance IL-8 release by these cells, but not by wild-type controls. Therefore, in CF, dysregulated efferocytosis may lead to accumulation of apoptotic cells and impaired regulation of the inflammatory response and, ultimately, may suggest a new therapeutic target.

A paradoxical protective role for the proinflammatory peptide substance P receptor (NK1R) in acute hyperoxic lung injury

Fri, 25 Sep 2009 09:51:14 PDT

The neuropeptide substance P manifests its biological functions through ligation of a G protein-coupled receptor, the NK1R. Mice with targeted deletion of this receptor reveal a preponderance of proinflammatory properties resulting from ligand activation, demonstrating a neurogenic component to multiple forms of inflammation and injury. We hypothesized that NK1R deficiency would afford a similar protection from inflammation associated with hyperoxia. Counter to our expectations, however, NK1R–/– animals suffered significantly worse lung injury compared with wild-type mice following exposure to 90% oxygen. Median survival was shortened to 84 h for NK1R–/– mice from 120 h for wild-type animals. Infiltration of inflammatory cells into the lungs was significantly increased; NK1R–/– animals also exhibited increased pulmonary edema, hemorrhage, and bronchoalveolar lavage fluid protein levels. TdT-mediated dUTP nick end labeling (TUNEL) staining was significantly elevated in NK1R–/– animals following hyperoxia. Furthermore, induction of metallothionein and Na⁺-K⁺-ATPase was accelerated in NK1R–/– compared with wild-type mice, consistent with increased oxidative injury and edema. In cultured mouse lung epithelial cells in 95% O₂, however, addition of substance P promoted cell death, suggesting the neurogenic component of hyperoxic lung injury is mediated by additional mechanisms in vivo. Release of bioactive constituents including substance P from sensory neurons results from activation of the vanilloid receptor, TRPV1. In mice with targeted deletion of the TRPV1 gene, acute hyperoxic injury is attenuated relative to NK1R–/– animals. Our findings thus reveal a major neurogenic mechanism in acute hyperoxic lung injury and demonstrate concerted actions of sensory neurotransmitters revealing significant protection for NK1R-mediated functions.

The effects of repeated allergen challenge on airway smooth muscle structural and molecular remodeling in a rat model of allergic asthma

Fri, 25 Sep 2009 09:51:14 PDT

The effects of remodeling of airway smooth muscle (SM) by hyperplasia on airway SM contractility in vivo are poorly explored. The aim of this study was to investigate the relationship between allergen-induced airway SM hyperplasia and its contractile phenotype. Brown Norway rats were sensitized with ovalbumin (OVA) or saline on day 0 and then either OVA-challenged once on day 14 and killed 24 h later or OVA-challenged 3 times (on days 14, 19, and 24) and killed 2 or 7 days later. Changes in SM mass, expression of total myosin, SM myosin heavy chain fast isoform (SM-B) and myosin light chain kinase (MLCK), tracheal contractions ex vivo, and airway responsiveness to methacholine (MCh) in vivo were assessed. One day after a single OVA challenge, the number of SM cells positive for PCNA was greater than for control animals, whereas the SM mass, contractile phenotype, and tracheal contractility were unchanged. Two days after three challenges, SM mass and PCNA immunoreactive cells were increased (3- and 10-fold, respectively; P < 0.05), but airway responsiveness to MCh was unaffected. Lower expression in total myosin, SM-B, and MLCK was observed at the mRNA level (P < 0.05), and total myosin and MLCK expression were lower at the protein level (P < 0.05) after normalization for SM mass. Normalized tracheal SM force generation was also significantly lower 2 days after repeated challenges (P < 0.05). Seven days after repeated challenges, features of remodeling were restored toward control levels. Allergen-induced hyperplasia of SM cells was associated with a loss of contractile phenotype, which was offset by the increase in mass.

Thrombin induces fibronectin-specific migration of pulmonary microvascular endothelial cells: requirement of calcium/calmodulin-dependent protein kinase II

Meoli, D. F., White, R. J. — Fri, 25 Sep 2009 09:51:14 PDT

Pulmonary arterial hypertension (PAH) is a progressive disease of excess vasoconstriction and vascular cell proliferation that results in increased pulmonary vascular resistance and right heart failure. We have previously shown (66) that tissue factor expression is increased in the abnormal vessels of patients and rats with PAH. We hypothesized that tissue factor and its downstream mediator, thrombin, would promote migration of endothelial cells (EC) and the vascular pathology of PAH. Immunostaining revealed EC and a fibronectin-enriched matrix within the "plexiform-like" lesions in a rat model of severe PAH. In a modified Boyden assay, protease-activated receptor 1 (PAR1; thrombin receptor) stimulation by agonist peptide or thrombin induced pulmonary microvascular EC (PMVEC) migration when the cells were interacting with fibronectin, but not with other extracellular matrix proteins. Thrombin/fibronectin-induced migration was confirmed in wound healing and angiogenesis assays and was abrogated by the PAR1 antagonist SCH79797 and soluble RGD peptide. This fibronectin dependence was unique to PAR1 activation; other EC agonists evaluated did not induce migration on any matrix, and 10% FBS stimulated similar levels of migration on all matrix proteins tested. Thrombin/fibronectin stimulated autophosphorylation of calcium/calmodulin dependent protein kinase II (CaMKII) in PMVEC, and inhibitors of CaMKII blocked thrombin-induced migration on fibronectin, but had no effect on migration induced by 10% FBS. In contrast, EC isolated from the proximal pulmonary artery migrated in response to most agonists independent of the matrix substrate. Our findings illustrate EC heterogeneity in a single tissue and indicate a novel role for CaMKII in mediating EC migration. Because PMVEC have been shown to have impressive proliferative potential, thrombin/fibronectin-stimulated migration of these cells to a site of injured endothelium is a potential mechanism by which thrombin contributes to the development of vascular lesions in PAH.

Golgi, trafficking, and mitosis dysfunctions in pulmonary arterial endothelial cells exposed to monocrotaline pyrrole and NO scavenging

Lee, J., Reich, R., Xu, F., Sehgal, P. B. — Fri, 25 Sep 2009 09:51:14 PDT

Although the administration of monocrotaline (MCT) into experimental animals is in widespread use today in investigations of pulmonary arterial hypertension (PAH), the underlying cellular and subcellular mechanisms that culminate in vascular remodeling are incompletely understood. Bovine pulmonary arterial endothelial cells (PAECs) in culture exposed to monocrotaline pyrrole (MCTP) develop "megalocytosis" 18–24 h later characterized by enlarged hyperploid cells with enlarged Golgi, mislocalization of endothelial nitric oxide synthase away from the plasma membrane, decreased cell-surface/caveolar nitric oxide (NO), and hypo-S-nitrosylation of caveolin-1, clathrin heavy chain, and N-ethylmaleimide-sensitive factor. We investigated whether MCTP did in fact affect functional intracellular trafficking. The NO scavenger (4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) and the NO donor diethylamine NONOate were used for comparison. Both MCTP and c-PTIO produced distinctive four- to fivefold enlarged PAECs within 24–48 h with markedly enlarged/dispersed Golgi, as visualized by immunostaining for the Golgi tethers/matrix proteins giantin, GM130, and p115. Live-cell uptake of the Golgi marker C₅ ceramide revealed a compact juxtanuclear Golgi in untreated PAECs, brightly labeled enlarged circumnuclear Golgi after MCTP, but minimally labeled Golgi elements after c-PTIO. These Golgi changes were reduced by NONOate. After an initial inhibition during the first day, both MCTP and c-PTIO markedly enhanced anterograde secretion of soluble cargo (exogenous vector-expressed recombinant horseradish peroxidase) over the next 4 days. Live-cell internalization assays using fluorescently tagged ligands showed that both MCTP and c-PTIO inhibited the retrograde uptake of acetylated low-density lipoprotein, transferrin, and cholera toxin B. Moreover, MCTP, and to a variable extent c-PTIO, reduced the cell-surface density of all receptors assayed (LDLR, TfnR, BMPR, Tie-2, and PECAM-1/CD31). In an important distinction, c-PTIO enhanced mitosis in PAECs but MCTP inhibited mitosis, even that due to c-PTIO, despite markedly exaggerated Golgi dispersal. Taken together, these data define a broad-spectrum Golgi and subcellular trafficking dysfunction syndrome in endothelial cells exposed to MCTP or NO scavenging.

Golgi dysfunction is a common feature in idiopathic human pulmonary hypertension and vascular lesions in SHIV-nef-infected macaques

Fri, 25 Sep 2009 09:51:14 PDT

Golgi dysfunction has been previously investigated as a mechanism involved in monocrotaline-induced pulmonary hypertension (PAH). In the present study, we addressed whether Golgi dysfunction might occur in pulmonary vascular cells in idiopathic PAH (IPAH) and whether there might be a causal relationship between trafficking dysfunction and vasculopathies of PAH. Quantitative immunostaining for the Golgi tethers giantin and p115 on human lung tissue from patients with IPAH (n = 6) compared with controls demonstrated a marked cytoplasmic dispersal of giantin- and p115-bearing vesicular elements in vascular cells in the proliferative, obliterative, and plexiform lesions in IPAH and an increase in the amounts of these Golgi tethers/matrix proteins per cell. The causality question was approached by genetic means using human immunodeficiency virus (HIV)-Nef, a protein that disrupts endocytic and trans-Golgi trafficking. Macaques infected with a chimeric simian immunodeficiency virus (SIV) containing the HIV-nef gene (SHIV-nef), but not the nonchimeric SIV virus containing the endogenous SIV-nef gene, displayed pulmonary arterial vasculopathies similar to those in human IPAH. Giantin and p115 levels and their subcellular distribution in pulmonary vascular cells in lungs of SHIV-nef infected macaques (n = 4) were compared with SIV-infected (n = 3) and an uninfected macaque control. Only macaques infected with chimeric SHIV-nef showed pulmonary vascular lesions containing cells with dramatic cytoplasmic dispersal and an increase in giantin and p115. Specifically, the HIV-Nef-positive cells showed increased giantin, p115, and the activated transcription factor PY-STAT3. These data represent the first test of the Golgi dysfunction hypothesis in IPAH and place trafficking and Golgi disruption in the chain of causality of pulmonary vasculopathies in the macaque model.

Neutrophil-mediated lung permeability and host defense proteins

Kantrow, S. P., Shen, Z., Jagneaux, T., Zhang, P., Nelson, S. — Fri, 25 Sep 2009 09:51:14 PDT

Neutrophil recruitment to the alveolar space is associated with increased epithelial permeability. The present study investigated in mice whether neutrophil recruitment to the lung leads to accumulation of plasma-derived host defense proteins in the alveolar space and whether respiratory burst contributes to this increase in permeability. Albumin, complement C1q, and IgM were increased in bronchoalveolar lavage (BAL) fluid 6 h after intratracheal LPS challenge. Neutrophil depletion before LPS treatment completely prevented this increase in BAL fluid protein concentration. Respiratory burst was not detected in neutrophils isolated from BAL fluid, and BAL proteins were increased in mice deficient in a key subunit of the respiratory burst apparatus, gp91^phox, similar to wild-type mice. Neutrophil recruitment elicited by intratracheal instillation of the chemokines macrophage inflammatory protein-2 and keratinocyte-derived chemokine was also accompanied by accumulation of albumin, C1q, and IgM. During neutrophil recruitment to the alveolar space, epithelial permeability facilitates delivery of host defense proteins. The observed increase in epithelial permeability requires recruitment of neutrophils, but not activation of the respiratory burst, and occurs with chemokine-induced neutrophil migration independent of LPS exposure.

Mechanism regulating proasthmatic effects of prolonged homologous {beta}2-adrenergic receptor desensitization in airway smooth muscle

Nino, G., Hu, A., Grunstein, J. S., Grunstein, M. M. — Fri, 25 Sep 2009 09:51:14 PDT

Use of long-acting β₂-adrenergic receptor (β2AR) agonists to treat asthma incurs an increased risk of asthma morbidity with impaired bronchodilation and heightened bronchoconstriction, reflecting the adverse effects of prolonged homologous β2AR desensitization on airway smooth muscle (ASM) function. Since phosphodiesterase 4 (PDE4) regulates ASM relaxation and contractility, we examined whether the changes in ASM function induced by prolonged homologous β2AR desensitization are attributed to altered expression and action of PDE4. Cultured human ASM cells and isolated rabbit ASM tissues exposed for 24 h to the long-acting β2AR agonist salmeterol exhibited impaired acute β2AR-mediated cAMP accumulation and relaxation, respectively, together with ASM constrictor hyperresponsiveness. These proasthmatic-like changes in ASM function were associated with upregulated PDE4 activity due to enhanced expression of the PDE4D5 isoform and were prevented by pretreating the ASM preparations with the PDE4 inhibitor rolipram or with inhibitors of either PKA or ERK1/2 signaling. Extended studies using gene silencing and pharmacological approaches demonstrated that: 1) the mechanism underlying upregulated PDE4D5 expression following prolonged β2AR agonist exposure involves PKA-dependent activation of G_i protein signaling via its β-subunits, which elicits downstream activation of ERK1/2 and its induction of PDE4D5 transcription; and 2) the induction of PDE4 activity and consequent changes in ASM responsiveness are prevented by pretreating the β2AR agonist-exposed ASM preparations with inhibitors of G_i-β signaling. Collectively, these findings identify that the proasthmatic changes in ASM function resulting from prolonged homologous β2AR desensitization are attributed to upregulated PDE4 expression induced by G_i-β-mediated cross-talk between the PKA and ERK1/2 signaling pathways.

Mutation of protein kinase C phosphorylation site S1076 on {alpha}-subunits affects BKCa channel activity in HEK-293 cells

Zhu, S., Browning, D. D., White, R. E., Fulton, D., Barman, S. A. — Fri, 25 Sep 2009 09:51:14 PDT

Large conductance, calcium- and voltage-activated potassium (BK_Ca) channels are important modulators of pulmonary vascular smooth muscle membrane potential, and phosphorylation of BK_Ca channels by protein kinases regulates pulmonary arterial smooth muscle function. However, little is known about the effect of phosphorylating specific channel subunits on BK_Ca channel activity. The present study was done to determine the effect of mutating protein kinase C (PKC) phosphorylation site serine 1076 (S1076) on transfected human BK_Ca channel -subunits in human embryonic kidney (HEK-293) cells, a heterologous expression system devoid of endogenous BK_Ca channels. Results showed that mutating S1076 altered the effect of PKC activation on BK_Ca channels in HEK-293 cells. Specifically, the phospho-deficient mutation BK_Ca-(S1076A)/β₁ attenuated the excitatory effect of the PKC activator phorbol myristate acetate (PMA) on BK_Ca channels, whereas the phospho-mimetic mutation BK_Ca-(S1076E)/β₁ increased the excitatory effect of PMA on BK_Ca channels. In addition, the phospho-null mutation S1076A blocked the activating effect of cGMP-dependent protein kinase G (PKG) on BK_Ca channels. Collectively, these results suggest that specific putative PKC phosphorylation site(s) on human BK_Ca channel -subunits influences BK_Ca channel activity, which may subsequently alter pulmonary vascular smooth muscle function and tone.

Organic dust exposure alters monocyte-derived dendritic cell differentiation and maturation

Fri, 25 Sep 2009 09:51:14 PDT

Organic dust exposure in agricultural animal environments results in airway diseases. Dendritic cells (DCs) orchestrate inflammatory immune response in the airways, but little is known about how organic dust affects differentiation and maturation of monocyte-derived immature and mature DCs (iDCs, mDCs). Peripheral blood monocytes were differentiated in vitro into iDCs with granulocyte-macrophage colony stimulating factor + IL-4 (6 days) with and without swine facility organic dust extract (ODE, 0.1%). Unlike control iDCs, ODE-conditioned iDCs maintained key monocyte properties (increased mCD14, increased phagocytic ability) while expressing DC features [increased mCD83, HLA-DR, CD80, CD86, diminished cytokine (TNF-, IL-6) responsiveness]. At day 6, iDCs were cultured for an additional 48 h (days 7 and 8) with lipopolysaccharide (LPS) to induce mDCs. ODE-conditioned mDCs maintained high expression of mCD14⁺ and elevated phagocytosis while their DC features weakened as evidenced by decreased CD11c, CD83, HLA-DR, CD86, and CCR7 expression and reduced lymphocyte-stimulating capacity. Similar results were observed when monocytes were exposed to ODE for only the first 48 h and with ODE depleted of endotoxin. Control iDCs exposed to ODE during the final 2 days of iDC maturation (days 7 and 8) did not differ from control (no ODE) iDCs in surface marker expression and phagocytic ability, but exhibited enhanced lymphocyte-stimulating capacity. Dust exposure alters monocyte differentiation to iDCs and prevents maturation of iDC to mDCs. The first 48 h of monocyte differentiation appears to be the susceptible period to exposure. Environmental exposures present during early monocyte differentiation may impact the critical balance of DCs in the lung.

Chronic hypercapnia downregulates arginase expression and activity and increases pulmonary arterial smooth muscle relaxation in the newborn rat

Fri, 25 Sep 2009 09:51:14 PDT

In rats, chronic hypercapnia has been reported to ameliorate hypoxia-induced pulmonary hypertension in newborn and adult and to enhance endothelium-dependent vasorelaxation in adult pulmonary arteries. The underlying mechanisms accounting for chronic hypercapnia-induced improvements in pulmonary vascular function are not understood. Hypothesizing that downregulation of arginase activity may be contributory, we examined relaxation responses and arginase activity and expression in pulmonary arteries from newborn rats that were exposed (from birth to 14 days) to either mild-to-moderate (5.5% inhaled CO₂) or severe (10% CO₂) hypercapnia with either normoxia or hypoxia (13% O₂). Pulmonary arteries from pups exposed to normoxia and chronic hypercapnia (5.5 or 10% CO₂) contracted less in response to a thromboxane A₂ analog, U-46619, and showed enhanced endothelium-dependent (but not independent) relaxation compared with arteries from normocapnic pups (P < 0.01). Parallel with these changes, arginase activity and arginase I (but not II) expression in lung and pulmonary arterial tissue were significantly decreased (P < 0.05). Exposure to 10% CO₂ significantly increased (P < 0.01) pulmonary arterial tissue nitric oxide (nitrite) generation. In pups chronically exposed to hypoxia (13% O₂), severe hypercapnia (10% CO₂) significantly (P < 0.05) enhanced endothelium-dependent relaxation, increased nitric oxide generation, and decreased arginase activity but not expression. We conclude that chronic hypercapnia-induced downregulation of lung arginase expression and/or activity may reduce pulmonary vascular resistance by enhancing nitric oxide generation and thus endothelium-dependent relaxation. This mechanism may explain some of the beneficial effects of chronic hypercapnia on experimental pulmonary hypertension.

Effect of ventilation pressure on alveolar fluid clearance and {beta}-agonist responses in mice

Yu, E. N. Z., Traylor, Z. P., Davis, I. C. — Fri, 25 Sep 2009 09:51:14 PDT

High tidal volume ventilation is detrimental to alveolar fluid clearance (AFC), but effects of ventilation pressure (P) on AFC are unknown. In anesthetized BALB/c mice ventilated at constant tidal volume (8 ml/kg), mean AFC rate was 12.8% at 6 cmH₂O P, but increased to 37.3% at 18 cmH₂O P. AFC rate declined at 22 cmH₂O P, which also induced lung damage. Increased AFC at 18 cmH₂O P did not result from elevated plasma catecholamines, hypercapnia, or hypocapnia, but was due to augmented Na⁺ and Cl^– absorption. PKA agonists and β-agonists stimulated AFC at 10 cmH₂O P by upregulating amiloride-sensitive Na⁺ transport. However, at 18 cmH₂O P, PKA agonists and β-agonists reduced AFC. At 15 cmH₂O P, the AFC rate was intermediate (mean 26.6%), and forskolin and β-agonists had no effect. Comparable P dependency of AFC and β-agonist responsiveness was found in C57BL/6 mice. The effect on AFC of increasing P to 18 cmH₂O was blocked by adenosine deaminase or an A_2b-adenosine receptor antagonist, and could be mimicked by adenosine in mice ventilated at 10 cmH₂O P. Modulation of adenosine signaling also resulted in altered responsiveness to β-agonists. These findings indicate that, in the normal mouse lung, basal AFC rates and responses to β-agonists are impacted by ventilation pressure in an adenosine-dependent manner.

Asthma and sarcoplasmic reticulum Ca2+ reuptake in airway smooth muscle

Prakash, Y. S., Sathish, V., Thompson, M. A., Pabelick, C. M., Sieck, G. C. — Fri, 25 Sep 2009 09:51:14 PDT