http://ajplung.physiology.org AJP: Lung Cellular and Molecular Physiology RSS feed -- current issue 1522-1504 Jul 1 2009 12:00:00:000AM AJP: Lung Cellular and Molecular Physiology 1040-0605 http://ajplung.physiology.org/icons/banner/title.gif http://ajplung.physiology.org http://ajplung.physiology.org/cgi/content/abstract/297/1/L1?rss=1 Receptor for advanced glycation end-products (RAGE) is a marker of alveolar type I cells and is elevated in the pulmonary edema fluid of patients with acute lung injury (ALI). We tested the hypothesis that RAGE in the bronchoalveolar lavage (BAL) would be elevated in experimental models of direct ALI characterized by alveolar epithelial cell injury. We developed ELISA measurements for RAGE and studied ALI (direct and indirect) mouse models and collected BAL at specified endpoints to measure RAGE. We also tested whether levels of BAL RAGE correlated 1) with the severity of lung injury in acid and hyperoxia-induced ALI and 2) with the beneficial effect of a novel treatment, mesenchymal stem cells (MSC), in LPS-induced ALI. In ALI models of direct lung injury induced by intratracheal instillation of acid, LPS, or Escherichia coli, the BAL RAGE was 58-, 22-, and 13-fold elevated, respectively. In contrast, BAL RAGE was not detectable in indirect models of ALI induced by an intraperitoneal injection of thiourea or by an intravenous injection of MHC I monoclonal antibody that produces a mouse model of transfusion-related ALI. BAL RAGE did correlate with the severity of lung injury in acid and hyperoxia-induced ALI. In addition, with LPS-induced ALI, BAL RAGE was markedly reduced with MSC treatment. In summary, BAL RAGE is an indicator of ALI, and it may be useful in distinguishing direct from indirect models of ALI as well as assessing the response to specific therapies.

]]> 2009-06-26 info:doi/10.1152/ajplung.90546.2008 American Physiological Society 1 297 L5 2009-07-01 L1 TRANSLATIONAL PHYSIOLOGY http://ajplung.physiology.org/cgi/content/abstract/297/1/L6?rss=1 IL-6 overexpression protects mice from hyperoxic acute lung injury in vivo, and treatment with IL-6 protects cells from oxidant-mediated death in vitro. The mechanisms of protection, however, are not clear. We characterized the expression, localization, and regulation of Bax, a proapoptotic member of the Bcl-2 family, in wild-type (WT) and IL-6 lung-specific transgenic (Tg⁺) mice exposed to 100% O₂ and in human umbilical vein endothelial cells (HUVEC) treated with H₂O₂ and IL-6. In control HUVEC treated with H₂O₂ or in WT mice exposed to 100% O₂, a marked induction of Bax translocation and dimerization was associated with increased JNK and p38 kinase activity. In contrast, specific JNK or p38 kinase inhibitors or treatment with IL-6 inhibited Bax mitochondrial translocation and apoptosis of HUVEC. IL-6 Tg⁺ mice exposed to 100% O₂ exhibited enhanced phosphatidylinositol 3-kinase (PI3K)/Akt kinase and increased serine phosphorylation of Bax at Ser¹⁸⁴ compared with WT mice. The PI3K-specific inhibitor LY-2940002 blocked this IL-6-induced Bax phosphorylation and promoted cell death. Furthermore, IL-6 potently blocked hyperoxia- or oxidant-induced Bax insertion into mitochondrial membranes. Thus IL-6 functions in a cytoprotective manner, in part, by suppressing Bax translocation and dimerization through PI3K/Akt-mediated Bax phosphorylation.

]]> 2009-06-26 info:doi/10.1152/ajplung.90381.2008 American Physiological Society 1 297 L16 2009-07-01 L6 ARTICLES http://ajplung.physiology.org/cgi/content/abstract/297/1/L17?rss=1 Stromal interaction molecule 1 (STIM1) is a recently discovered membrane-spanning protein thought to sense lumenal Ca²⁺ in sarco(endo)plasmic reticulum (SR/ER) and transduce activation of Ca²⁺-permeable store-operated channels (SOC) in plasmalemma in response to SR/ER Ca²⁺ depletion. To evaluate the role of STIM1 and a closely related protein, STIM2, in Ca²⁺ signaling of rat distal pulmonary arterial smooth muscle cells (PASMC) during hypoxia, we used fluorescent microscopy and the Ca²⁺-sensitive dye, fura 2, to measure basal intracellular Ca²⁺ concentration ([Ca²⁺]_i), store-operated Ca²⁺ entry (SOCE), and increases in [Ca²⁺]_i caused by acute hypoxia (4% O₂) or depolarization (60 mmol/l KCl) in cells treated with small interfering RNA targeted to STIM1 (siSTIM1) or STIM2 (siSTIM2). As determined by real-time quantitative PCR analysis (qPCR), STIM1 mRNA was 200-fold more abundant than STIM2 in untreated control PASMC. siSTIM1 and siSTIM2 caused specific and significant knockdown of both mRNA measured by qPCR and protein measured by Western blotting. siSTIM1 markedly inhibited SOCE and abolished the sustained [Ca²⁺]_i response to hypoxia but did not alter the initial transient [Ca²⁺]_i response to hypoxia, the [Ca²⁺]_i response to depolarization, or basal [Ca²⁺]_i. The only effect of siSTIM2 was a smaller inhibition of SOCE. We conclude that STIM1 was quantitatively more important than STIM2 in activation of SOC in rat distal PASMC and that the increase in [Ca²⁺]_i induced by acute hypoxia in these cells required SR Ca²⁺ release and STIM1-dependent activation of SOC.

]]> 2009-06-26 info:doi/10.1152/ajplung.00063.2009 American Physiological Society 1 297 L25 2009-07-01 L17 ARTICLES http://ajplung.physiology.org/cgi/content/abstract/297/1/L26?rss=1 Airway inflammation leads to increased intracellular Ca²⁺ ([Ca²⁺]_i) levels in airway smooth muscle (ASM) cells. Sarcoplasmic reticulum Ca²⁺ release and reuptake are key components of ASM [Ca²⁺]_i regulation. Ca²⁺ reuptake occurs via sarcoendoplasmic reticulum Ca²⁺ ATPase (SERCA) and is regulated by the inhibitory protein phospholamban (PLB) in many cell types. In human ASM, we tested the hypothesis that inflammation increases PLB, thus inhibiting SERCA function, and leading to maintained [Ca²⁺]_i levels. Surprisingly, we found that human ASM does not express PLB protein (although mRNA is detectable). Overnight exposure to the proinflammatory cytokines TNF and IL-13 did not induce PLB expression, raising the issue of how SERCA is regulated. We then found that direct SERCA phosphorylation (via CaMKII) occurs in human ASM. In fura-2-loaded human ASM cells, we found that the CaMKII antagonist KN-93 significantly slowed the rate of fall of [Ca²⁺]_i transients induced by ACh or bradykinin (in zero extracellular Ca²⁺), suggesting a role for CaMKII-mediated SERCA regulation. SERCA expression was decreased by cytokine exposure, and the rate of fall of [Ca²⁺]_i transients was slowed in cells exposed to TNF and IL-13. Cytokine effects on Ca²⁺ reuptake were unaffected by additional exposure to KN-93. These data indicate that in human ASM, SERCA is regulated by mechanisms such as CaMKII and that airway inflammation maintains [Ca²⁺]_i levels by decreasing SERCA expression and slowing Ca²⁺ reuptake.

]]> 2009-06-26 info:doi/10.1152/ajplung.00026.2009 American Physiological Society 1 297 L34 2009-07-01 L26 ARTICLES http://ajplung.physiology.org/cgi/content/abstract/297/1/L35?rss=1 Inflammatory cytokines, particularly the neutrophil chemoattractant IL-8, are elevated in the cystic fibrosis (CF) airway, even in the absence of detectable infection. The transcriptional regulation of many inflammatory genes, including IL8 (CXCL8), involves chromatin remodeling through histone acetylation. NF-B is known to facilitate histone acetylation of IL8 and other proinflammatory gene promoters, but we find that increased NF-B activation cannot explain the elevated IL8 expression and promoter acetylation seen in CFTR-deficient cells. Recognized components of the NF-B-coactivator complex, acetyltransferase CBP, p300, and the histone deacetylase HDAC1, are unchanged by CFTR activity. However, we find that the histone acetyltransferase (HAT)/HDAC balance is sensitive to CFTR function, as cells with reduced or absent CFTR function have decreased HDAC2 protein, resulting in hyperacetylation of the IL8 promoter and increased IL8 transcription. Reduced HDAC2 and HDAC2 activity, but not HDAC2 mRNA, is observed in cells deficient in CFTR. Suppressing HDAC2 expression with HDAC2 short hairpin RNA (shRNA) results in increased IL8 expression and promoter acetylation comparable with CFTR-deficient cells. Treating CFTR-deficient cells with N-acetyl-cysteine (NAC) increases HDAC2 expression to near control levels. Our data suggest that there is an intrinsic alteration in the HAT/HDAC balance in cells lacking CFTR function in vitro and in native CF tissue and that oxidative stress is likely contributing to this alteration. This mechanism, found in other inflammatory airway diseases, provides an explanation for the apparent dysregulation of inflammatory mediators seen in the CF airway, as reduced histone deacetylation would potentially influence many genes.

]]> 2009-06-26 info:doi/10.1152/ajplung.90399.2008 American Physiological Society 1 297 L43 2009-07-01 L35 ARTICLES http://ajplung.physiology.org/cgi/content/abstract/297/1/L44?rss=1 Supplemental oxygen therapy (hyperoxia) in preterm babies with respiratory stress is associated with lung injury and the development of bronchopulmonary dysplasia. Endoplasmic reticulum (ER) homeostasis plays critical roles in maintaining cellular functions such as protein synthesis, folding, and secretion. Interruption of ER homeostasis causes ER stress and triggers the unfolded protein response, which can lead to apoptosis in persistently stressed cells. ERp57 is an ER protein and is associated with calreticulin and calnexin in protein glycosylation. In this study, we found hyperoxia downregulated ERp57 in neonatal rat lungs and cultured human endothelial cells. Transient transfection of ERp57 small interfering RNA significantly knocked down ERp57 expression and reduced hyperoxia- or tunicamycin-induced apoptosis in human endothelial cells. Apoptosis was decreased from 26.8 to 9.9% in hyperoxia-exposed cells and from 37.8 to 5.0% in tunicamycin-treated cells. The activation of caspase-3 induced by hyperoxia or tunicamycin was diminished and immunoglobulin heavy chain-binding protein/glucose-regulated protein 78-kDa (BiP/GRP78) induction was increased in ERp57 knockdown cells. Overexpression of ERp57 exacerbated hyperoxia- or tunicamycin-induced apoptosis in human endothelial cells. Apoptosis was increased from 10.1 to 14.3% in hyperoxia-exposed cells and from 14.0 to 21.2% in tunicamycin-treated cells. Overexpression of ERp57 also augmented tunicamycin-induced caspase-3 activation and reduced BiP/GRP78 induction. Our results demonstrate that ERp57 can regulate apoptosis in human endothelial cells. It appears that knockdown of ERp57 confers cellular protection against hyperoxia- or tunicamycin-induced apoptosis by inhibition of caspase-3 activation and stimulation of BiP/GRP78 induction.

]]> 2009-06-26 info:doi/10.1152/ajplung.90626.2008 American Physiological Society 1 297 L51 2009-07-01 L44 ARTICLES http://ajplung.physiology.org/cgi/content/abstract/297/1/L52?rss=1 Toll-like receptors (TLRs) of the innate immune system contribute to noninfectious inflammatory processes. We employed a murine model of hilar clamping (1 h) with reperfusion times between 15 min and 3 h in TLR4-sufficient (C3H/OuJ) and TLR4-deficient (C3H/HeJ) anesthetized mice with additional studies in chimeric and myeloid differentiation factor 88 (MyD88)- and TLR4-deficient mice to determine the role of TLR4 in lung ischemia-reperfusion injury. Human pulmonary microvascular endothelial monolayers were subjected to simulated warm ischemia and reperfusion with and without CRX-526, a competitive TLR4 inhibitor. Functional TLR4 solely on pulmonary parenchymal cells, not bone marrow-derived cells, mediates early lung edema following ischemia-reperfusion independent of MyD88. Activation of MAPKs and NF-B was significantly blunted and/or delayed in lungs of TLR4-deficient mice as a consequence of ischemia-reperfusion injury, but edema development appeared to be independent of activation of these signaling pathways. Pretreatment with a competitive TLR4 inhibitor prevented edema in vivo and reduced actin cytoskeletal rearrangement and gap formation in pulmonary microvascular endothelial monolayers subjected to simulated warm ischemia and reperfusion. In addition to its well-accepted role to alter gene transcription, functioning TLR4 on pulmonary parenchymal cells plays a key role in very early and profound pulmonary edema in murine lung ischemia-reperfusion injury. This may be due to a novel mechanism: regulation of endothelial cell cytoskeleton affecting microvascular endothelial cell permeability.

]]> 2009-06-26 info:doi/10.1152/ajplung.90406.2008 American Physiological Society 1 297 L63 2009-07-01 L52 ARTICLES http://ajplung.physiology.org/cgi/content/abstract/297/1/L64?rss=1 Patients with mutations in the pulmonary surfactant protein C (SP-C) gene develop interstitial lung disease and pulmonary exacerbations associated with viral infections including respiratory syncytial virus (RSV). Pulmonary infection with RSV caused more severe interstitial thickening, air space consolidation, and goblet cell hyperplasia in SP-C-deficient (Sftpc^–/–) mice compared with SP-C replete mice. The RSV-induced pathology resolved more slowly in Sftpc^–/– mice with lung inflammation persistent up to 30 days postinfection. Polymorphonuclear leukocyte and macrophage counts were increased in the bronchoalveolar lavage (BAL) fluid of Sftpc^–/– mice. Viral titers and viral F and G protein mRNA were significantly increased in both Sftpc^–/– and heterozygous Sftpc^+/– mice compared with controls. Expression of Toll-like receptor 3 (TLR3) mRNA was increased in the lungs of Sftpc^–/– mice relative to Sftpc^+/+ mice before and after RSV infection. Consistent with the increased TLR3 expression, BAL inflammatory cells were increased in the Sftpc^–/– mice after exposure to a TLR3-specific ligand, poly(I:C). Preparations of purified SP-C and synthetic phospholipids blocked poly(I:C)-induced TLR3 signaling in vitro. SP-C deficiency increases the severity of RSV-induced pulmonary inflammation through regulation of TLR3 signaling.

]]> 2009-06-26 info:doi/10.1152/ajplung.90640.2008 American Physiological Society 1 297 L72 2009-07-01 L64 ARTICLES http://ajplung.physiology.org/cgi/content/abstract/297/1/L73?rss=1 Soluble adenylyl cyclase toxins, such as Pseudomonas aeruginosa exoY, generate a cAMP pool that retracts cell borders. However, the cytoskeletal basis by which this cAMP signal retracts cell borders is not known. We sought to determine whether activation of chimeric, soluble adenylyl cyclase I/II (sACI/II) reorganizes either microtubules or peripheral actin. Endothelial cells were stably transfected with either green fluorescent protein-labeled -tubulin or β-actin, and then infected with adenovirus to express sACI/II. Forskolin, which stimulates both the endogenously expressed transmembrane adenylyl cyclases and sACI/II, induced cell retraction accompanied by the reorganization of peripheral microtubules. However, cortical filamentous-actin (f-actin) did not reorganize into stress fibers, and myosin light-chain-20 phosphorylation was decreased. Isoproterenol, which activates endogenous adenylyl cyclases but does not activate sACI/II, did not induce endothelial cell gaps and did not influence microtubule or f-actin architecture. Thus, sACI/II generates a cAMP signal that reorganizes microtubules and induces cell retraction, without inducing f-actin stress fibers. These findings illustrate that endothelial cell gap formation can proceed without f-actin stress fiber formation, and provide mechanistic insight how bacterial adenylyl cyclase toxins reorganize the cytoskeleton to induce cell rounding.

]]> 2009-06-26 info:doi/10.1152/ajplung.90577.2008 American Physiological Society 1 297 L83 2009-07-01 L73 ARTICLES http://ajplung.physiology.org/cgi/content/abstract/297/1/L84?rss=1 Angiotensin-converting enzyme 2 (ACE2) is a terminal carboxypeptidase and the receptor for the SARS and NL63 coronaviruses (CoV). Loss of ACE2 function is implicated in severe acute respiratory syndrome (SARS) pathogenesis, but little is known about ACE2 biogenesis and activity in the airways. We report that ACE2 is shed from human airway epithelia, a site of SARS-CoV infection. The regulation of ACE2 release was investigated in polarized human airway epithelia. Constitutive generation of soluble ACE2 was inhibited by DPC 333, implicating a disintegrin and metalloprotease 17 (ADAM17). Phorbol ester, ionomycin, endotoxin, and IL-1β and TNF acutely induced ACE2 release, further supporting that ADAM17 and ADAM10 regulate ACE2 cleavage. Soluble ACE2 was enzymatically active and partially inhibited virus entry into target cells. We determined that the ACE2 cleavage site resides between amino acid 716 and the putative transmembrane domain starting at amino acid 741. To reveal structural determinants underlying ACE2 release, several mutant and chimeric ACE2 proteins were engineered. Neither the juxtamembrane stalk region, transmembrane domain, nor the cytosolic domain was needed for constitutive ACE2 release. Interestingly, a point mutation in the ACE2 ectodomain, L584A, markedly attenuated shedding. The resultant ACE2-L584A mutant trafficked to the cell membrane and facilitated SARS-CoV entry into target cells, suggesting that the ACE2 ectodomain regulates its release and that residue L584 might be part of a putative sheddase "recognition motif." Thus ACE2 must be cell associated to serve as a CoV receptor and soluble ACE2 might play a role in modifying inflammatory processes at the airway mucosal surface.

]]> 2009-06-26 info:doi/10.1152/ajplung.00071.2009 American Physiological Society 1 297 L96 2009-07-01 L84 ARTICLES http://ajplung.physiology.org/cgi/content/abstract/297/1/L97?rss=1 Fibroblasts from patients with pulmonary fibrosis express higher levels of the receptor for urokinase, and the extent of fibrosis in some animal models exhibits a dependence on the urokinase receptor. Recent observations have identified the urokinase receptor as a trans-interacting receptor with consequences on signaling and cell responses that vary depending on its interacting partner, the relative levels of expression, and the state of cellular transformation. We undertook this study to define the urokinase-type plasminogen activator cellular receptor (u-PAR)-integrin interactions and to determine the functional consequences of such interactions on normal human lung fibroblast attachment and migration. u-PAR colocalizes in lammelipodia/filopodia with relevant integrins that mediate fibroblast attachment and spreading on the provisional matrix proteins vitronectin, fibronectin, and collagens. Inhibitory antibody studies have revealed that human lung fibroblasts utilize _vβ₅ to attach to vitronectin, predominantly ₅β₁ (and _vβ₃) to attach to fibronectin, and ₁β₁, ₂β₁, and ₃β₁ to attach to collagen. Blocking studies with -integrin subunit decoy peptides and u-PAR neutralizing antibodies indicate that u-PAR modulates the integrin-mediated attachment to purified provisional matrix proteins, to anti-integrin antibodies, or to fibroproliferative lesions from fibrotic lungs. Furthermore, these decoy peptides blunt fibroblast spreading and migration. We show that u-PAR can interact with multiple -integrins but with a preference for ₃. Taken together, these data demonstrate that u-PAR may interact with multiple integrins in normal human lung fibroblasts thereby promoting attachment, spreading, and migration. Modulation of fibroblast invasion would be expected to lead to amelioration of fibroproliferative diseases of the lung.

]]> 2009-06-26 info:doi/10.1152/ajplung.90283.2008 American Physiological Society 1 297 L108 2009-07-01 L97 ARTICLES http://ajplung.physiology.org/cgi/content/abstract/297/1/L109?rss=1 Reactive oxygen species (ROS) present in cigarette smoke (CS) are thought to contribute to the development of COPD. Although CS-ROS can hardly enter airway epithelial cells, and certainly not the circulation, systemic levels of ROS have been found to be elevated in COPD patients. We hypothesize that lipophilic components present in CS can enter airway epithelial cells and increase intracellular ROS production by disturbing mitochondrial function. Different airway epithelial cells were exposed to CS extract (CSE), hexane-treated CSE (CSE without lipophilic components), gaseous-phase CS, and water-filtered CS (gaseous-phase CS without ROS). Mitochondrial membrane potential (_m) and ATP levels were assessed using the bronchial epithelial cell line Beas-2b. ROS generation measured directly by DCF fluorescence and indirectly by measuring free thiol groups (-SH) upon exposure to CS was assessed using lung alveolar epithelial cells devoid of functional mitochondria (A549-0), with normal A549 cells serving as controls. In Beas-2b cells, CSE (4 h) caused a dose-dependent decrease in _m and ATP levels, whereas hexane-treated CSE did not. DCF fluorescence in A549 cells increased in response to CSE, whereas this was not the case in A549-0 cells. Exposure of A549 cells to CS resulted in a rapid decrease in free -SH, whereas exposure to ROS-depleted CS only resulted in a delayed decrease. This delayed decrease was less pronounced in A549-0 cells. Lipophilic components in CS disturb mitochondrial function, which contributes to increased intracellular generation of ROS. Our results are of importance in understanding the systemic effects of smoking observed in patients with COPD.

]]> 2009-06-26 info:doi/10.1152/ajplung.90461.2008 American Physiological Society 1 297 L114 2009-07-01 L109 ARTICLES http://ajplung.physiology.org/cgi/content/abstract/297/1/L115?rss=1 Idiopathic pulmonary fibrosis (IPF) is a disease of unknown etiology characterized by the development of subpleural foci of myofibroblasts that contribute to the exuberant fibrosis noted in the pulmonary parenchyma. Pleural mesothelial cells (PMC) are metabolically dynamic cells that cover the lung and chest wall as a monolayer and are in intimate proximity to the underlying lung parenchyma. The precise role of PMC in the pathogenesis of pulmonary parenchymal fibrosis remains to be identified. Transforming growth factor (TGF)-β1, a cytokine known for its capacity to induce proliferative and transformative changes in lung cells, is found in significantly higher quantities in the lungs of patients with IPF. High levels of TGF-β1 in the subpleural milieu may play a key role in the transition of normal PMC to myofibroblasts. Here we demonstrate that PMC activated by TGF-β1 undergo epithelial-mesenchymal transition (EMT) and respond with haptotactic migration to a gradient of TGF-β1 and that the transition of PMC to myofibroblasts is dependent on smad-2 signaling. The EMT of PMC was marked by upregulation of -smooth muscle actin (-SMA), fibroblast specific protein-1 (FSP-1), and collagen type I expression. Cytokeratin-8 and E-cadherin expression decreased whereas vimentin remained unchanged over time in transforming PMC. Knockdown of smad-2 gene by silencing small interfering RNA significantly suppressed the transition of PMC to myofibroblasts and significantly inhibited the PMC haptotaxis. We conclude that PMC undergo EMT when exposed to TGF-β1, involving smad-2 signaling, and PMC may be a possible source of myofibroblasts in IPF.

]]> 2009-06-26 info:doi/10.1152/ajplung.90587.2008 American Physiological Society 1 297 L124 2009-07-01 L115 ARTICLES http://ajplung.physiology.org/cgi/content/abstract/297/1/L125?rss=1 Cigarette smoke (CS) induces a rapid, sustained upregulation of ceramide production in human bronchial epithelial cells, leading to increased apoptosis. Using loss-of-function and overexpression analyses, we show that neutral sphingomyelinase 2 (nSMase2) is required for CS-mediated ceramide generation and apoptosis. Glutathione (GSH), a crucial antioxidant in lung defense, blocks nSMase2 activity and thus inhibits apoptosis triggered by CS. We found that the exposure to CS, as with exposure to H₂O₂, results in increased nSMase2 activation leading to ceramide generation and therefore increased apoptosis. Interestingly, exposure of cells to GSH abolishes nSMase2 activation caused by CS and leads to a decrease in CS-induced apoptosis. This suggests that the effects of CS oxidants on nSMase2 are counteracted by GSH. Our data support a model where CS induces nSMase2 activation thereby increasing membrane-sphingomyelin hydrolysis to ceramide. In turn, elevated ceramide enhances airway epithelial cell death, which causes bronchial and alveolar destruction and lung injury in pulmonary diseases.

]]> 2009-06-26 info:doi/10.1152/ajplung.00031.2009 American Physiological Society 1 297 L133 2009-07-01 L125 ARTICLES http://ajplung.physiology.org/cgi/content/abstract/297/1/L134?rss=1 Hyperoxia disrupts postnatal lung development in part through inducing inflammation. To determine the contribution of leukocyte-derived reactive oxygen species, we exposed newborn wild-type and NADPH oxidase p47^phox subunit null (p47^phox–/–) mice to air or acute hyperoxia (95% O₂) for up to 11 days. Hyperoxia-induced pulmonary neutrophil influx was similar in wild-type and p47^–/– mice at postnatal days (P) 7 and 11. Macrophages were decreased in wild-type hyperoxia-exposed mice compared with p47^phox–/– mice at P11. Hyperoxia impaired type II alveolar epithelial cell and bronchiolar epithelial cell proliferation, but depression of type II cell proliferation was significantly less in p47^–/– mice at P3 and P7, when inflammation was minimal. We found reciprocal results for the expression of the cell cycle inhibitor p21^cip/waf in type II cells, which was induced in 95% O₂-exposed wild-type mice, but significantly less in p47^phox–/– littermates at P7. Despite partial preservation of type II cell proliferation, deletion of p47^phox did not prevent the major adverse effects of hyperoxia on alveolar development estimated by morphometry at P11, but hyperoxia impairment of elastin deposition at alveolar septal crests was significantly worse in wild-type vs. p47^phox–/– mice at P11. Since we found that p47^phox is expressed in a subset of alveolar epithelial cells, its deletion may protect postnatal type II alveolar epithelial proliferation from hyperoxia through effects on epithelial as well as phagocyte-generated superoxide.

]]> 2009-06-26 info:doi/10.1152/ajplung.00112.2009 American Physiological Society 1 297 L142 2009-07-01 L134 ARTICLES http://ajplung.physiology.org/cgi/content/abstract/297/1/L143?rss=1 In many organs, integrins and cadherins are partly regulated by Hox genes, but their interactions in airway morphogenesis and congenital lung diseases are unknown. We previously showed that the Hox protein HoxB5 is abnormally increased in bronchopulmonary sequestration (BPS) and congenital cystic adenomatoid malformation (CCAM), congenital lung lesions with abnormal airway branching. We now report on ₂-, ₃-, and β₁-integrin and E-cadherin expression in normal human lung and in BPS and CCAM tissue previously shown to have abnormal HoxB5 expression and on the relationship of cell adhesion molecule expression to Hoxb5 regulation. ₂-, ₃-, and β₁-integrins and E-cadherin expression in normal human lung and BPS and CCAM were evaluated using Western blot and immunohistochemistry. Fetal mouse lung fibroblasts with Hoxb5-specific siRNA downregulation were evaluated for ₂-integrin protein levels by Western blot. Compared with normal human lung, a previously undetected ₂-integrin isoform potentially lacking essential cytoplasmic sequences was significantly increased in BPS and CCAM, and ₂-integrin spatial and cellular expression was more intense. E-cadherin protein levels were also significantly increased, whereas ₃ increased in CCAM compared with canalicular, but not with alveolar, stage lung. β₁-integrin levels were unchanged. We conclude that in BPS and CCAM, altered ₂-integrin cytoplasmic signaling contributes to abnormal cellular behavior in these lung lesions. Aberrant cell adhesion molecule and Hox protein regulation are likely part of the mechanism involved in the development of BPS and CCAM.

]]> 2009-06-26 info:doi/10.1152/ajplung.90618.2008 American Physiological Society 1 297 L152 2009-07-01 L143 ARTICLES http://ajplung.physiology.org/cgi/content/abstract/297/1/L153?rss=1 A family of gas-transporting proteins, the Mep/Amt/Rh glycoprotein family, has been identified recently. These are integral membrane proteins, are widely expressed in sites of gas transport, and are known to transport the gaseous molecule, NH₃, and recent evidence indicates they can transport CO₂. Because the mammalian lung is a critical site for gas transport, the current studies examine the expression of the nonerythroid members of this extended family, Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg), in the normal mouse lung. Real-time RT-PCR and immunoblot analysis demonstrated both Rhbg and Rhcg mRNA and protein expression, respectively. Immunohistochemistry demonstrated both Rhbg and Rhcg were expressed in bronchial and bronchiolar epithelial cells. Rhbg was expressed by Clara cells, specifically, whereas all bronchial/bronchiolar epithelial cells, with the exception of goblet cells, expressed Rhcg. Rhbg expression was basolateral, whereas Rhcg exhibited apical and intracellular immunolabel, polarized expression similar to that observed in Rhbg- and Rhcg-expressing epithelial cells in other organs. There was no detectable expression of either Rhbg or Rhcg in alveolar endothelial or epithelial cells, in pneumocytes or in vascular tissue. In vitro studies using cultured bronchial epithelial cells confirm Rhbg and Rhcg expression, demonstrate that saturable, not diffusive, transport is the primary mechanism of ammonia/methylammonia transport, and show that the saturable transport mechanism has kinetics similar to those demonstrated previously for Rhbg and Rhcg. These findings suggest Rhbg and Rhcg may contribute to bronchial epithelial cell ammonia metabolism and suggest that they do not contribute to pulmonary CO₂ transport.

]]> 2009-06-26 info:doi/10.1152/ajplung.90524.2008 American Physiological Society 1 297 L163 2009-07-01 L153 ARTICLES http://ajplung.physiology.org/cgi/content/abstract/297/1/L164?rss=1 Migration of airway epithelial cells (AEC) is an integral component of airway mucosal repair after injury. The inflammatory cytokine IL-4, abundant in chronic inflammatory airways diseases such as asthma, stimulates overproduction of mucins and secretion of chemokines from AEC; these actions enhance persistent airway inflammation. The effect of IL-4 on AEC migration and repair after injury, however, is not known. We examined migration in primary human AEC differentiated in air-liquid interface culture for 3 wk. Wounds were created by mechanical abrasion and followed to closure using digital microscopy. Concurrent treatment with IL-4 up to 10 ng/ml accelerated migration significantly in fully differentiated AEC. As expected, IL-4 treatment induced phosphorylation of the IL-4 receptor-associated protein STAT (signal transducer and activator of transcription)6, a transcription factor known to mediate several IL-4-induced AEC responses. Expressing a dominant negative STAT6 cDNA delivered by lentivirus infection, however, failed to block IL-4-stimulated migration. In contrast, decreasing expression of either insulin receptor substrate (IRS)-1 or IRS-2 using a silencing hairpin RNA blocked IL-4-stimulated AEC migration completely. These data demonstrate that IL-4 can accelerate migration of differentiated AEC after injury. This reparative response does not require STAT6 activation, but rather requires IRS-1 and/or IRS-2.

]]> 2009-06-26 info:doi/10.1152/ajplung.90453.2008 American Physiological Society 1 297 L173 2009-07-01 L164 ARTICLES http://ajplung.physiology.org/cgi/content/abstract/297/1/L174?rss=1 Mucous hypersecretion is a serious feature of chronic airway diseases such as asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. Although mucins are produced via activation of an EGF receptor (EGFR) signaling cascade, the mechanisms leading to exaggerated mucin production in mucous hypersecretory diseases are unknown. Because expression of ICAM-1 and of the ICAM-1 ligand fibrinogen is increased in the airways of subjects with mucous hypersecretory diseases, we hypothesized that fibrinogen binding to ICAM-1 could increase EGFR-dependent mucin production in human airway (NCI-H292) epithelial cells. Consistent with this hypothesis, we found that an ICAM-1 neutralizing antibody and an ICAM-1(8–22) peptide that binds fibrinogen decreased mucin production induced by the EGFR ligand transforming growth factor (TGF)- dose-dependently. Exogenous fibrinogen and a fibrinogen(117–133) peptide that binds ICAM-1 rescued mucin production in cells treated with the ICAM-1(8–22) peptide. Surprisingly, the ICAM-1(8–22) peptide increased EGFR phosphotyrosine and phospho-ERK1/2 in cells treated with TGF-. The ICAM-1(8–22) peptide-induced increases in EGFR phosphotyrosine and phospho-ERK1/2 were prevented by exogenous fibrinogen, by the fibrinogen(117–133) peptide, and by selective inhibitors of phospholipase C (PLC), protein kinase C (PKC)-/β, and metalloproteases. These results suggest that fibrinogen binding to ICAM-1 promotes mucin production by decreasing TGF--induced EGFR and ERK1/2 activation and that the fibrinogen-ICAM-1-dependent decrease in EGFR and ERK1/2 activation occurs via inhibition of an early positive feedback pathway involving PLC- and PKC-/β-dependent metalloprotease activation and subsequent metalloprotease-dependent EGFR reactivation.

]]> 2009-06-26 info:doi/10.1152/ajplung.00032.2009 American Physiological Society 1 297 L183 2009-07-01 L174 ARTICLES http://ajplung.physiology.org/cgi/content/abstract/297/1/L184?rss=1 Persistent pulmonary hypertension of newborn (PPHN) is associated with impaired pulmonary vasodilation at birth. Previous studies demonstrated that a decrease in angiogenesis contributes to this failure of postnatal adaptation. We investigated the hypothesis that oxidative stress from NADPH oxidase (Nox) contributes to impaired angiogenesis in PPHN. PPHN was induced in fetal lambs by ductus arteriosus ligation at 85% of term gestation. Pulmonary artery endothelial cells (PAEC) from fetal lambs with PPHN (HTFL-PAEC) or control lambs (NFL-PAEC) were compared for their angiogenic activities and superoxide production. HTFL-PAEC had decreased tube formation, cell proliferation, scratch recovery, and cell invasion and increased cell apoptosis. Superoxide (O₂^–) production, measured by dihydroethidium epifluorescence and HPLC, were increased in HTFL-PAEC compared with NFL-PAEC. The mRNA levels for Nox2, Rac1, p47^phox, and Nox4, protein levels of p67^phox and Rac1, and NADPH oxidase activity were increased in HTFL-PAEC. NADPH oxidase inhibitor, apocynin (Apo), and antioxidant, N-acetyl-cysteine (NAC), improved angiogenic measures in HTFL-PAEC. Apo and NAC also reduced apoptosis in HTFL-PAEC. Our data suggest that PPHN is associated with increased O₂^– production from NADPH oxidase in PAEC. Increased oxidative stress from NADPH oxidase contributes to the impaired angiogenesis of PAEC in PPHN.

]]> 2009-06-26 info:doi/10.1152/ajplung.90455.2008 American Physiological Society 1 297 L195 2009-07-01 L184 ARTICLES http://ajplung.physiology.org/cgi/content/abstract/297/1/L196?rss=1 15-Lipoxygenase-1 (15-LOX-1) has been proposed to be involved in various physiological and pathophysiological activities such as inflammation, atherosclerosis, cell maturation, and tumorigenesis. Asthma and chronic obstructive pulmonary disease are associated with increased expression of 15-LOX-1 in bronchial epithelial cells, but the potential functions of 15-LOX-1 in airway epithelial cells have not been well clarified. To study the function of 15-LOX-1 in bronchial epithelial cells, we ectopically expressed 15-LOX-1 in the human lung epithelial cell line A549. We found that overexpression of 15-LOX-1 in A549 cells leads to increased release of the chemokines MIP-1, RANTES, and IP-10, and thereby to increased recruitment of immature dendritic cells, mast cells, and activated T cells. These results suggest that an increased expression and activity of 15-LOX-1 in lung epithelial cells is a proinflammatory event in the pathogenesis of asthma and other inflammatory lung disorders.

]]> 2009-06-26 info:doi/10.1152/ajplung.00036.2008 American Physiological Society 1 297 L203 2009-07-01 L196 ARTICLES http://ajplung.physiology.org/cgi/content/full/297/1/L204?rss=1 2009-06-26 info:doi/10.1152/ajplung.zh5-5479-corr.2009 American Physiological Society 1 297 L204 2009-07-01 L204 CORRIGENDA