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1.  Do Biophysical Properties of the Airway Smooth Muscle in Culture Predict Airway Hyperresponsiveness? 
Airway hyperresponsiveness is a cardinal feature of asthma but remains largely unexplained. In asthma, the key end-effector of acute airway narrowing is the airway smooth muscle (ASM) cell. Here we report novel biophysical properties of the ASM cell isolated from the relatively hyporesponsive Lewis rat versus the relatively hyperresponsive Fisher rat. We focused upon the ability of the cytoskeleton (CSK) of the ASM cell to stiffen, to generate contractile forces, and to remodel. We used optical magnetic twisting cytometry to measure cell stiffness and traction microscopy to measure contractile forces. To measure remodeling dynamics, we quantified spontaneous nanoscale motions of a microbead tightly anchored to the CSK. In response to a panel of contractile and relaxing agonists, Fisher ASM cells showed greater stiffening, bigger contractile forces, and faster CSK remodeling; they also exhibited higher effective temperature of the CSK matrix. These physical differences measured at the level of the single cell in vitro were consistent with strain-related differences in airway responsiveness in vivo. As such, comprehensive biophysical characterizations of CSK dynamics at the level of the cell in culture may provide novel perspectives on the ASM and its contributions to the excessive airway narrowing in asthma.
doi:10.1165/rcmb.2005-0453OC
PMCID: PMC2553364  PMID: 16484685
airway hyperresponsiveness; airway smooth muscle; cytoskeleton dynamics; inbred strains; remodeling
2.  Bioelectric Properties of Chloride Channels in Human, Pig, Ferret, and Mouse Airway Epithelia 
The development of effective therapies for cystic fibrosis (CF) requires animal models that can appropriately reproduce the human disease phenotype. CF mouse models have demonstrated cAMP-inducible, non–CF transmembrane conductance regulator (non-CFTR) chloride transport in conducting airway epithelia, and this property is thought to be responsible for the lack of a spontaneous CF-like phenotype in the lung. Thus, an understanding of species diversity in airway epithelial electrolyte transport and CFTR function is critical to developing better models for CF. Two species currently being used in attempts to develop better animal models of CF include the pig and ferret. In the study reported here, we sought to comparatively characterize the bioelectric properties of in vitro polarized airway epithelia—from human, mouse, pig and ferret—grown at the air–liquid interface (ALI). Bioelectric properties analyzed include amiloride-sensitive Na+ transport, 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid (DIDS)-sensitive Cl− transport, and cAMP-sensitive Cl− transport. In addition, as an index for CFTR functional conservation, we evaluated the ability of four CFTR inhibitors, including glibenclamide, 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid, CFTR inh-172, and CFTRinh-GlyH101, to block cAMP-mediated Cl− transport. Compared with human epithelia, pig epithelia demonstrated enhanced amiloride-sensitive Na+ transport. In contrast, ferret epithelia exhibited significantly reduced DIDS-sensitive Cl− transport. Interestingly, although the four CFTR inhibitors effectively blocked cAMP-mediated Cl− secretion in human airway epithelia, each species tested demonstrated unique differences in its responsiveness to these inhibitors. These findings suggest the existence of substantial species-specific differences at the level of the biology of airway epithelial electrolyte transport, and potentially also in terms of CFTR structure/function.
doi:10.1165/rcmb.2006-0286OC
PMCID: PMC1894945  PMID: 17008635
cystic fibrosis; airway biology; species; ion transport; airway epithelium
3.  Dissection of the hyper-adhesive phenotype of airway eosinophils in asthma 
Asthma is characterized by appearance of eosinophils in the airway. Eosinophils purified from the airway 48 hours after segmental antigen challenge are described as exhibiting greater adhesion to albumin-coated surfaces via an unidentified β2 integrin and increased expression of αMβ2 (CD11b/18) in comparison to purified blood eosinophils. We have investigated the determinants of this hyper-adhesive phenotype. Airway eosinophils exhibited increased reactivity with the CBRM1/5 anti-αM activation-sensitive antibody as well as enhanced adhesion to VCAM-1 (CD106) and diverse ligands, including albumin, ICAM-1 (CD54), fibrinogen, and vitronectin. Purified blood eosinophils did not adhere to the latter diverse ligands. Enhanced adhesion of airway eosinophils was blocked by anti-αMβ2. Podosomes, structures implicated in cell movement and proteolysis of matrix proteins, were larger and more common on airway eosinophils adherent to VCAM-1 when compared to blood eosinophils. Incubation of blood eosinophils with IL-5 replicated the phenotype of airway eosinophils. That is, IL-5 enhanced recognition of αM by CBRM1/5; stimulated αMβ2-mediated adhesion to VCAM-1, albumin, ICAM-1, fibrinogen, and vitronectin; and increased podosome formation on VCAM-1. Thus, the hyper-adhesion of airway eosinophils after antigen challenge is mediated by upregulated and activated αMβ2.
doi:10.1165/rcmb.2006-0027OC
PMCID: PMC1550734  PMID: 16601240
human; eosinophils; adhesion molecules; cell trafficking
4.  Adenosine A1 receptors mediate mobilization of calcium in human bronchial smooth muscle cells 
Adenosine stimulates contraction of airway smooth muscle, but the mechanism is widely considered indirect, depending on release of contractile agonists from mast cells and nerves. The goal was to determine whether adenosine, by itself, directly regulates calcium signaling in human bronchial smooth muscle cells (HBSMC). Primary cultures of HBSMC from normal subjects were loaded with fura 2-AM and cytosolic calcium concentrations ([Ca2+]i) were determined ratiometrically by imaging single cells. The non-selective adenosine receptor agonist, 5'-N-ethylcarboxamidoadenosine (NECA), and the adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA), both stimulated rapid, transient increases in [Ca2+]i. In contrast, there were no calcium responses to 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido-adenosine (100 nM) or N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (100 nM), selective agonists at adenosine A2A receptors and adenosine A3 receptors, respectively. Calcium responses to NECA and CPA were inhibited by 8-cyclopentyl-1,3-dipropylxanthine, an adenosine A1 receptor antagonist, and by pertussis toxin (PTX). In other experiments, NECA stimulated calcium transients in the absence of extracellular calcium, but not when cells were preincubated in cyclopiazonic acid or thapsigargin to empty intracellular calcium stores. Calcium responses were attenuated by xestospongin C and 2-aminoethoxydiphenylborane, inhibitors of inositol trisphosphate (IP3) receptors, and by U73122, an inhibitor of phospholipase C. It was concluded that stimulation of adenosine A1 receptors on HBSMC rapidly mobilizes intracellular calcium stores by a mechanism dependent on PTX-sensitive G proteins, and IP3 signaling. These findings suggest that, in addition to its well-established indirect effects on HBSMC, adenosine also has direct effects on contractile signaling pathways.
doi:10.1165/rcmb.2005-0290OC
PMCID: PMC2065849  PMID: 16709961
Adenosine; calcium; human bronchial smooth muscle; adenosine A1 receptor; cAMP; insulin
5.  Adenosine A1 Receptors Mediate Mobilization of Calcium in Human Bronchial Smooth Muscle Cells 
Adenosine stimulates contraction of airway smooth muscle, but the mechanism is widely considered indirect, depending on release of contractile agonists from mast cells and nerves. The goal was to determine whether adenosine, by itself, directly regulates calcium signaling in human bronchial smooth muscle cells (HBSMC). Primary cultures of HBSMC from normal subjects were loaded with fura 2-AM, and cytosolic calcium concentrations ([Ca2+]i) were determined ratiometrically by imaging single cells. The nonselective adenosine receptor agonist, 5′-N-ethylcarboxamidoadenosine (NECA), and the adenosine A1 receptor agonist, N6-cyclopentyladenosine (CPA), both stimulated rapid, transient increases in [Ca2+]i. In contrast, there were no calcium responses to 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamido-adenosine (100 nM) or N6-(3-iodobenzyl)-adenosine-5′-N-methyluronamide (100 nM), selective agonists at adenosine A2A receptors and adenosine A3 receptors, respectively. Calcium responses to NECA and CPA were inhibited by 8-cyclopentyl-1,3-dipropylxanthine, an adenosine A1 receptor antagonist, and by pertussis toxin (PTX). In other experiments, NECA stimulated calcium transients in the absence of extracellular calcium, but not when cells were preincubated in cyclopiazonic acid or thapsigargin to empty intracellular calcium stores. Calcium responses were attenuated by xestospongin C and 2-aminoethoxydiphenylborane, inhibitors of inositol trisphosphate (IP3) receptors, and by U73122, an inhibitor of phospholipase C. It was concluded that stimulation of adenosine A1 receptors on HBSMC rapidly mobilizes intracellular calcium stores by a mechanism dependent on PTX-sensitive G proteins, and IP3 signaling. These findings suggest that, in addition to its well-established indirect effects on HBSMC, adenosine also has direct effects on contractile signaling pathways.
doi:10.1165/rcmb.2005-0290OC
PMCID: PMC2065849  PMID: 16709961
adenosine A1 receptor; calcium; cAMP; human bronchial smooth muscle; insulin
6.  Matrix Metalloproteinases Promote Inflammation and Fibrosis in Asbestos-Induced Lung Injury in Mice 
Inhalation of asbestos fibers causes pulmonary inflammation and eventual pulmonary fibrosis (asbestosis). Although the underlying molecular events are poorly understood, protease/antiprotease and oxidant/antioxidant imbalances are believed to contribute to the disease. Implicated in other forms of pulmonary fibrosis, the matrix metalloproteinases (MMPs) have not been examined in asbestosis. We therefore hypothesized that MMPs play a pathogenic role in asbestosis development. Wild-type C57BL/6 mice were intratracheally instilled with 0.1 mg crocidolite asbestos, causing an inflammatory response at 1 d and a developing fibrotic response at 7, 14, and 28 d. Gelatin zymography demonstrated an increase in MMP-9 (gelatinase B) during the inflammatory phase, while MMP-2 (gelatinase A) was profoundly increased in the fibrotic phase. Immunohistochemistry revealed MMP-9 in and around bronchiolar and airspace neutrophils that were often associated with visible asbestos fibers. MMP-2 was found in fibrotic regions at 7, 14, and 28 d. No increases in RNA levels of MMP-2, MMP-9, or MMP-8 were found, but levels of MMP-7, MMP-12, and MMP-13 RNA did increase at 14 d. The MMP inhibitors, TIMP-1 and TIMP-2, were also increased at 7–28 d after asbestos exposure. To confirm the importance of MMP activity in disease progression, mice exposed to asbestos were given daily injections of the MMP inhibitor, GM6001. MMP inhibition reduced inflammation and fibrosis in asbestos-treated mice. Collectively, these data suggest that MMPs contribute to the pathogenesis of asbestosis through effects on inflammation and fibrosis development.
doi:10.1165/rcmb.2005-0471OC
PMCID: PMC1820635  PMID: 16574944
asbestos; extracellular superoxide dismutase; matrix metalloproteinase; pulmonary fibrosis
7.  Dissection of the Hyperadhesive Phenotype of Airway Eosinophils in Asthma 
Asthma is characterized by appearance of eosinophils in the airway. Eosinophils purified from the airway 48 h after segmental antigen challenge are described as exhibiting greater adhesion to albumin-coated surfaces via an unidentified β2 integrin and increased expression of αMβ2 (CD11b/18) compared with purified blood eosinophils. We have investigated the determinants of this hyperadhesive phenotype. Airway eosinophils exhibited increased reactivity with the CBRM1/5 anti-αM activation-sensitive antibody as well as enhanced adhesion to VCAM-1 (CD106) and diverse ligands, including albumin, ICAM-1 (CD54), fibrinogen, and vitronectin. Purified blood eosinophils did not adhere to the latter diverse ligands. Enhanced adhesion of airway eosinophils was blocked by anti-αMβ2. Podosomes, structures implicated in cell movement and proteolysis of matrix proteins, were larger and more common on airway eosinophils adherent to VCAM-1 when compared with blood eosinophils. Incubation of blood eosinophils with IL-5 replicated the phenotype of airway eosinophils. That is, IL-5 enhanced recognition of αM by CBRM1/5; stimulated αMβ2-mediated adhesion to VCAM-1, albumin, ICAM-1, fibrinogen, and vitronectin; and increased podosome formation on VCAM-1. Thus, the hyperadhesion of airway eosinophils after antigen challenge is mediated by upregulated and activated αMβ2.
doi:10.1165/rcmb.2006-0027OC
PMCID: PMC1550734  PMID: 16601240
adhesion molecules; cell trafficking; eosinophils; human
8.  Matrix Metalloproteinases Promote Inflammation and Fibrosis in Asbestos-Induced Lung Injury in Mice 
Inhalation of asbestos fibers causes pulmonary inflammation and eventual pulmonary fibrosis (asbestosis). Although the underlying molecular events are poorly understood, protease/antiprotease and oxidant/antioxidant imbalances are believed to contribute to the disease. Implicated in other forms of pulmonary fibrosis, the matrix metalloproteinases (MMPs) have not been examined in asbestosis. We therefore hypothesized that MMPs play a pathogenic role in asbestosis development. Wild-type C57BL/6 mice were intratracheally instilled with 0.1 mg crocidolite asbestos, causing an inflammatory response at 1 d and a developing fibrotic response at 7, 14, and 28 d. Gelatin zymography demonstrated an increase in MMP-9 (gelatinase B) during the inflammatory phase, while MMP-2 (gelatinase A) was profoundly increased in the fibrotic phase. Immunohistochemistry revealed MMP-9 in and around bronchiolar and airspace neutrophils that were often associated with visible asbestos fibers. MMP-2 was found in fibrotic regions at 7, 14, and 28 d. No increases in RNA levels of MMP-2, MMP-9, or MMP-8 were found, but levels of MMP-7, MMP-12, and MMP-13 RNA did increase at 14 d. The MMP inhibitors, TIMP-1 and TIMP-2, were also increased at 7–28 d after asbestos exposure. To confirm the importance of MMP activity in disease progression, mice exposed to asbestos were given daily injections of the MMP inhibitor, GM6001. MMP inhibition reduced inflammation and fibrosis in asbestos-treated mice. Collectively, these data suggest that MMPs contribute to the pathogenesis of asbestosis through effects on inflammation and fibrosis development.
doi:10.1165/rcmb.2005-0471OC
PMCID: PMC1820635  PMID: 16574944
asbestos; extracellular superoxide dismutase; matrix metalloproteinase; pulmonary fibrosis
9.  Do Biophysical Properties of the Airway Smooth Muscle in Culture Predict Airway Hyperresponsiveness? 
Airway hyperresponsiveness is a cardinal feature of asthma but remains largely unexplained. In asthma, the key end-effector of acute airway narrowing is the airway smooth muscle (ASM) cell. Here we report novel biophysical properties of the ASM cell isolated from the relatively hyporesponsive Lewis rat versus the relatively hyperresponsive Fisher rat. We focused upon the ability of the cytoskeleton (CSK) of the ASM cell to stiffen, to generate contractile forces, and to remodel. We used optical magnetic twisting cytometry to measure cell stiffness and traction microscopy to measure contractile forces. To measure remodeling dynamics, we quantified spontaneous nanoscale motions of a microbead tightly anchored to the CSK. In response to a panel of contractile and relaxing agonists, Fisher ASM cells showed greater stiffening, bigger contractile forces, and faster CSK remodeling; they also exhibited higher effective temperature of the CSK matrix. These physical differences measured at the level of the single cell in vitro were consistent with strain-related differences in airway responsiveness in vivo. As such, comprehensive biophysical characterizations of CSK dynamics at the level of the cell in culture may provide novel perspectives on the ASM and its contributions to the excessive airway narrowing in asthma.
doi:10.1165/rcmb.2005-0453OC
PMCID: PMC2553364  PMID: 16484685
airway hyperresponsiveness; airway smooth muscle; cytoskeleton dynamics; inbred strains; remodeling
10.  Bioelectric Properties of Chloride Channels in Human, Pig, Ferret, and Mouse Airway Epithelia 
The development of effective therapies for cystic fibrosis (CF) requires animal models that can appropriately reproduce the human disease phenotype. CF mouse models have demonstrated cAMP-inducible, non–CF transmembrane conductance regulator (non-CFTR) chloride transport in conducting airway epithelia, and this property is thought to be responsible for the lack of a spontaneous CF-like phenotype in the lung. Thus, an understanding of species diversity in airway epithelial electrolyte transport and CFTR function is critical to developing better models for CF. Two species currently being used in attempts to develop better animal models of CF include the pig and ferret. In the study reported here, we sought to comparatively characterize the bioelectric properties of in vitro polarized airway epithelia—from human, mouse, pig and ferret—grown at the air–liquid interface (ALI). Bioelectric properties analyzed include amiloride-sensitive Na+ transport, 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid (DIDS)-sensitive Cl− transport, and cAMP-sensitive Cl− transport. In addition, as an index for CFTR functional conservation, we evaluated the ability of four CFTR inhibitors, including glibenclamide, 5-nitro-2-(3-phenylpropyl-amino)-benzoic acid, CFTR inh-172, and CFTRinh-GlyH101, to block cAMP-mediated Cl− transport. Compared with human epithelia, pig epithelia demonstrated enhanced amiloride-sensitive Na+ transport. In contrast, ferret epithelia exhibited significantly reduced DIDS-sensitive Cl− transport. Interestingly, although the four CFTR inhibitors effectively blocked cAMP-mediated Cl− secretion in human airway epithelia, each species tested demonstrated unique differences in its responsiveness to these inhibitors. These findings suggest the existence of substantial species-specific differences at the level of the biology of airway epithelial electrolyte transport, and potentially also in terms of CFTR structure/function.
doi:10.1165/rcmb.2006-0286OC
PMCID: PMC1894945  PMID: 17008635
cystic fibrosis; airway biology; species; ion transport; airway epithelium
11.  Retinoic Acid Inhibits Airway Smooth Muscle Cell Migration 
Airway remodeling in chronic asthma is characterized by increased smooth muscle mass that is associated with the reduction of the bronchial lumen as well as airway hyperresponsiveness. The development of agents that inhibit smooth muscle growth is therefore of interest for therapy to prevent asthma-associated airway remodeling. All-trans retinoic acid (ATRA) suppresses growth of vascular smooth muscle cells (SMCs) from the systemic and pulmonary circulation. The present study investigated the effects of ATRA on human bronchial (airway) SMCs. Human bronchial SMCs were found to express mRNAs for retinoic acid receptor (RAR)-α, -β, -γ, and retinoid X receptor (RXR)-α, -β, but not RXR-γ. Although ATRA was not effective in inhibiting proliferation or in inducing apoptosis in airway SMCs, we found that ATRA (0.2–2 μM) inhibited the SMC migration in response to platelet-derived growth factor (PDGF), as determined in a modified Boyden chamber assay. Both RAR and RXR agonists also blocked PDGF-induced airway SMC migration. ATRA also inhibited PDGF-induced actin reorganization associated with migration. PDGF-induced actin reorganization and migration were blocked by inhibitors of phosphatidylinositol 3 kinase (PI3K) and Akt. However, migration was blocked by inhibitors of the MEK/ERK pathway, with no effect on cytoskeletal reorganization. ATRA suppressed PDGF-induced Akt activation without influencing ERK activation. RAR was found to form protein–protein interactions with the p85 PI3K subunit. These results suggest that retinoic acid inhibits airway SMC migration through the modulation of the PI3K/Akt pathway.
doi:10.1165/rcmb.2005-0306OC
PMCID: PMC1831536  PMID: 16456186
airway; migration; retinoic acid; signal transduction; smooth muscle
12.  Retinoic Acid Inhibits Airway Smooth Muscle Cell Migration 
Airway remodeling in chronic asthma is characterized by increased smooth muscle mass that is associated with the reduction of the bronchial lumen as well as airway hyperresponsiveness. The development of agents that inhibit smooth muscle growth is therefore of interest for therapy to prevent asthma-associated airway remodeling. All-trans retinoic acid (ATRA) suppresses growth of vascular smooth muscle cells (SMCs) from the systemic and pulmonary circulation. The present study investigated the effects of ATRA on human bronchial (airway) SMCs. Human bronchial SMCs were found to express mRNAs for retinoic acid receptor (RAR)-α, -β, -γ, and retinoid X receptor (RXR)-α, -β, but not RXR-γ. Although ATRA was not effective in inhibiting proliferation or in inducing apoptosis in airway SMCs, we found that ATRA (0.2–2 μM) inhibited the SMC migration in response to platelet-derived growth factor (PDGF), as determined in a modified Boyden chamber assay. Both RAR and RXR agonists also blocked PDGF-induced airway SMC migration. ATRA also inhibited PDGF-induced actin reorganization associated with migration. PDGF-induced actin reorganization and migration were blocked by inhibitors of phosphatidylinositol 3 kinase (PI3K) and Akt. However, migration was blocked by inhibitors of the MEK/ ERK pathway, with no effect on cytoskeletal reorganization. ATRA suppressed PDGF-induced Akt activation without influencing ERK activation. RAR was found to form protein–protein interactions with the p85 PI3K subunit. These results suggest that retinoic acid inhibits airway SMC migration through the modulation of the PI3K/Akt pathway.
doi:10.1165/rcmb.2005-0306OC
PMCID: PMC1831536  PMID: 16456186
airway; migration; retinoic acid; signal transduction; smooth muscle
13.  ALK-5 Mediates Endogenous and TGF-β1–Induced Expression of Connective Tissue Growth Factor in Embryonic Lung 
Transforming growth factor-β1 (TGF-β1) has been implicated as a major negative regulator of lung branching morphogenesis. Since connective tissue growth factor (CTGF) is a downstream mediator of TGF-β1 effects on mesenchymal cells, we hypothesized that TGF-β1 induces CTGF expression in mouse embryonic lung explants and that CTGF mediates TGF-β1 inhibition of branching morphogenesis. We show that addition of TGF-β1 to the serum-free medium of embryonic day (E)12.5 lung explant cultures inhibited branching morphogenesis and induced CTGF mRNA expression in time- and dose-dependent manners. In contrast to basal endogenous CTGF protein, which was exclusively localized in the distal airway epithelium, TGF-β1–induced CTGF protein was localized in both the epithelium and the mesenchyme. Addition of exogenous CTGF to culture medium directly inhibited branching morphogenesis. To identify the signal transduction pathway through which TGF-β1 induces CTGF, we used SB431542, a specific inhibitor for TGF-β type I receptor (TβRI)/ALK-5 to block TGF-β1–induced Smad2/3 phosphorylation. Consequently, SB431542 stimulated normal branching morphogenesis and blocked TGF-β1 inhibition of branching. Furthermore, SB-431542 blocked both endogenous and TGF-β1–induced expression of CTGF mRNA and protein. These results demonstrate for the first time that TGF-β1 induces CTGF expression in mouse embryonic lung explants, that CTGF inhibits branching morphogenesis, and that both endogenous and TGF-β1–induced CTGF expression are mediated by the TβRI/ALK-5–dependent Smad2 signaling pathway.
doi:10.1165/rcmb.2006-0320OC
PMCID: PMC1899336  PMID: 17197570
connective tissue growth factor; TGF-β; 1; ALK-5; Smad2; lung
14.  Asymmetric Dimethylarginine Induces Oxidative and Nitrosative Stress in Murine Lung Epithelial Cells 
Reactive oxygen species (ROS) and reactive nitrogen species (RNS) produced by epithelial and inflammatory cells are key mediators of the chronic airway inflammation of asthma. Low L-arginine levels can result in the uncoupling of nitric oxide synthase (NOS) leading to production of both ROS and RNS. Asymmetric dimethylarginine (ADMA) is a competitive endogenous inhibitor of all NOS isoforms and has been demonstrated to inhibit NO formation and increase oxidative stress in vascular endothelial and smooth muscle cells. The effect of ADMA on inducible NOS (iNOS) activity in epithelial cells has not been explored. In this study, we investigated whether addition of exogenous ADMA alters the generation of NO and superoxide anion (\documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{-}\end{equation*}\end{document}), leading to peroxynitrite (ONOO−) formation in a mouse epithelial cell line. In stimulated LA-4 cells, ADMA dose-dependently inhibited nitrite accumulation after 24 h of treatment. In addition, ADMA concentrations as low as 10 μM induced rapid increases in \documentclass[10pt]{article} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{pmc} \pagestyle{empty} \oddsidemargin -1.0in \begin{document} \begin{equation*}{\mathrm{O}}_{2}^{-}\end{equation*}\end{document} production as measured by dihydroethidium oxidation. Furthermore, using dihydrorhodamine to monitor ONOO− formation, ADMA caused a dose-dependent increase in ONOO− after treatment for 24 h. Similar effects of ADMA were seen using purified iNOS protein in a cell-free system. Together, these data indicate that elevated ADMA may contribute to the production of ROS and RNS in airway inflammation.
doi:10.1165/rcmb.2006-0302SM
PMCID: PMC1899333  PMID: 17158357
ADMA; epithelial cells; LA-4 cells; NOS; oxidative stress
15.  Silica Induces Macrophage Cytokines through Phosphatidylcholine-Specific Phospholipase C with Hydrogen Peroxide 
Silica particle–associated inflammation is implicated in the genesis of several pulmonary diseases, including silicosis and lung cancer. In this study we investigated the role of phosphatidylcholine-specific phospholipase C (PC-PLC) in silica-stimulated induction of TNF-α and IL-1β and how PC-PLC activity is regulated by silica in a rat alveolar macrophage model. We demonstrated that inhibition of PC-PLC, which was achieved with tricychodecan-9-yl-xanthate (D609), blocked the silica-stimulated induction of TNF-α and IL-1β in alveolar macrophage, suggesting that PC-PLC is involved in the silica-associated inflammatory response. PC-PLC activity was increased significantly by silica exposure, and this could be inhibited by MnTBAP, which catalyzes both the dismutation of O2.- to O2 and H2O2 and the dismutation of H2O2 to O2 and H2O, revealing that PC-PLC activity is regulated in a redox-dependent manner. This is further confirmed by the finding that PC-PLC activity was increased by exogenous H2O2. The intracellular calcium chelator BAPTA blocked the H2O2-increased PC-PLC activity, while the calcium ionophore, A23187, enhanced PC-PLC activity. The data indicate that PC-PLC plays critical roles in the silica-associated inflammatory response and that PC-PLC is regulated through redox- and calcium-dependent manners in alveolar macrophages.
doi:10.1165/rcmb.2006-0297OC
PMCID: PMC1899332  PMID: 17158358
phosphatidylcholine-specific phospholipase C; redox signaling; calcium; cytokine; hydrogen peroxide
16.  Molecular Pathogenesis of Lymphangioleiomyomatosis 
Lymphangioleiomyomatosis (LAM) is a rare progressive cystic lung disease affecting young women. The pivotal observation that LAM occurs both spontaneously and as part of the tuberous sclerosis complex (TSC) led to the hypothesis that these disorders share common genetic and pathogenetic mechanisms. In this review we describe the evolution of our understanding of the molecular and cellular basis of LAM and TSC, beginning with the discovery of the TSC1 and TSC2 genes and the demonstration of their involvement in sporadic (non-TSC) LAM. This was followed by rapid delineation of the signaling pathways in Drosophila melanogaster with confirmation in mice and humans. This knowledge served as the foundation for novel therapeutic approaches that are currently being used in human clinical trials.
doi:10.1165/rcmb.2006-0372TR
PMCID: PMC2176113  PMID: 17099139
tuberous sclerosis; TSC1; TSC2; mTOR; signal transduction; estrogen
17.  NPAS1 Regulates Branching Morphogenesis in Embryonic Lung 
Drosophila trachealess (Trl), master regulator of tracheogenesis, has no known functional mammalian homolog. We hypothesized that genes similar to trachealess regulate lung development. Quantitative (Q)RT-PCR and immunostaining were used to determine spatial and temporal patterns of npas1 gene expression in developing murine lung. Immunostaining for α-smooth muscle actin demonstrated myofibroblasts, and protein gene product (PGP)9.5 identified neuroendocrine cells. Branching morphogenesis of embryonic lung buds was analyzed in the presence of antisense or sense oligodeoxynucleotides (ODN). Microarray analyses were performed to screen for changes in gene expression in antisense-treated lungs. QRT-PCR was used to validate the altered expression of key genes identified on the microarrays. We demonstrate that npas1 is expressed in murine embryonic lung. npas1 mRNA peaks early at Embryonic Day (E)10.5–E11.5, then drops to low levels. Sequencing verifies the identity of npas1 transcripts in embryonic lung. NPAS1 immunostaining occurs in nuclei of parabronchial mesenchymal cells, especially at the tracheal bifurcation. Arnt, the murine homolog of Tango (the heterodimerization partner for Trl) is also expressed in developing lung but at constant levels. npas1- or arnt-antisense ODN inhibit lung branching morphogenesis, with altered myofibroblast development and increased pulmonary neuroendocrine cells. On microarrays, we identify > 50 known genes down-regulated by npas1-antisense, including multiple genes regulating cell migration and cell differentiation. QRT-PCR confirms significantly decreased expression of the neurogenic genes RBP-Jk and Tle, and three genes involved in muscle development: β−ig-h3, claudin-11, and myocardin. Npas1 can regulate myofibroblast distribution, branching morphogenesis, and neuroendocrine cell differentiation in murine embryonic lung.
doi:10.1165/rcmb.2006-0314OC
PMCID: PMC1899329  PMID: 17110583
branching morphogenesis; myofibroblasts; smooth muscle actin; cell migration; neuroendocrine cells
18.  Mitochondrial Localization and Function of Heme Oxygenase-1 in Cigarette Smoke–Induced Cell Death 
Cigarette smoke–induced apoptosis and necrosis contribute to the pathogenesis of chronic obstructive pulmonary disease. The induction of heme oxygenase-1 provides cytoprotection against oxidative stress, and may protect in smoking-related disease. Since mitochondria regulate cellular death, we examined the functional expression and mitochondrial localization of heme oxygenase-1 in pulmonary epithelial cells exposed to cigarette smoke extract (CSE), and its role in modulating cell death. Heme oxygenase-1 expression increased dramatically in cytosolic and mitochondrial fractions of human alveolar (A549), or bronchial epithelial cells (Beas-2b) exposed to either hemin, lipopolysaccharide, or CSE. Mitochondrial localization of heme oxygenase-1 was also observed in a primary culture of human small airway epithelial cells. Furthermore, heme oxygenase activity increased dramatically in mitochondrial fractions, and in whole cell extracts of Beas-2b after exposure to hemin and CSE. The mitochondrial localization of heme oxygenase-1 in Beas-2b was confirmed using immunogold-electron microscopy and immunofluorescence labeling on confocal laser microscopy. CSE caused loss of cellular ATP and rapid depolarization of mitochondrial membrane potential. Apoptosis occurred in Beas-2b at low concentrations of cigarette smoke extract, whereas necrosis occurred at high concentrations. Overexpression of heme oxygenase-1 inhibited CSE-induced Beas-2b cell death and preserved cellular ATP levels. Finally, heme oxygenase-1 mRNA expression was elevated in the lungs of mice chronically exposed to cigarette smoke. We demonstrate the functional compartmentalization of heme oxygenase-1 in the mitochondria of lung epithelial cells, and its potential role in defense against mitochondria-mediated cell death during CSE exposure.
doi:10.1165/rcmb.2006-0214OC
PMCID: PMC1899328  PMID: 17079780
cigarette smoke; COPD; heme oxygenase-1; mitochondria
19.  Gata4 Is Necessary for Normal Pulmonary Lobar Development 
Mutations of Fog2 in mice result in a phenotype that includes pulmonary lobar defects. To determine whether formation of the accessory lobe bronchus is mediated by a Gata family cofactor, we evaluated embryonic lungs from mice carrying missense mutations that cause loss of FOG–GATA protein interaction. Lungs from embryos carrying a missense mutation in Gata6 were structurally normal, while lungs from embryos carrying mutations of either Gata4 or of both Gata4 and Gata6 had a structural phenotype that matched the Fog2 mutant phenotype. Expression analysis showed that Gata4 and Fog2 are expressed in the ventral and medial pulmonary mesenchyme during secondary budding. Although Gata4 has not previously been suspected as playing a role in lung development, we have found that a Fog2–Gata4 interaction is critical for the development of normal pulmonary lobar structure, and this phenotype is not influenced by the additional loss of Gata6 interaction. Fog2 and Gata4 in the early pulmonary mesenchyme participate in patterning the secondary bronchus of the accessory lobe.
doi:10.1165/rcmb.2006-0211RC
PMCID: PMC1899327  PMID: 17142311
lung; accessory lobe; Gata; Fog; branching
20.  Effect of Concentrated Ambient Particles on Macrophage Phagocytosis and Killing of Streptococcus pneumoniae 
Particulate air pollution is linked to increased pneumonia epidemiologically and diminished lung bacterial clearance experimentally. We investigated the effect of concentrated ambient particles (CAPs, ⩽ PM2.5) on the interaction of murine primary alveolar macrophages (AMs) and the murine macrophage cell line, J774 A.1, with Streptococcus pneumoniae. We found that CAPs increased binding of bacteria by both primary AMs and J774 cells (66.7 ± 10.6% and 58.9 ± 4.0%, respectively, n = 4). In contrast to bacterial binding, CAPs decreased internalization in both AMs and J774 (55.4 ± 8.5% and 54.7 ± 5.1%, respectively, n = 4). The rate of killing of internalized bacteria was similar, but CAPs caused a decrease in the absolute number of bacteria killed by macrophages, mainly due to decreased internalization. Additional analyses showed that soluble components of CAPs mediated the enhanced binding and decreased internalization of S. pneumoniae. Chelation of iron in soluble CAPs substantially reversed, while addition of iron as ferric ammonium citrate restored inhibition of phagocytosis of S. pneumoniae in vitro. The results identify phagocytic internalization as a specific target for toxic effects of air pollution particles on AMs.
doi:10.1165/rcmb.2006-0293OC
PMCID: PMC1899326  PMID: 17079778
concentrated ambient particles; macrophages; Streptococcus pneumoniae; phagocytosis; killing
21.  Nonphagocytic Oxidase 1 Causes Death in Lung Epithelial Cells via a TNF-RI–JNK Signaling Axis 
Airway epithelial cells are simultaneously exposed to and produce cytokines and reactive oxygen species (ROS) in inflammatory settings. The signaling events and the physiologic outcomes of exposure to these inflammatory mediators remain to be elucidated. Previously we demonstrated that in cultured mouse lung epithelial cells exposed to bolus administration of H2O2, TNF-α–induced NF-κB activity was inhibited, whereas c-Jun-N-terminal kinase (JNK) activation was enhanced via a mechanism involving TNF receptor-1 (TNF-RI). In this study we used the nonphagocytic NADPH oxidase (Nox1) to study the effects of endogenously produced ROS on a line of mouse alveolar type II epithelial cells. Nox1 expression and activation inhibited TNF-α–induced inhibitor of κB kinase (IKK), and NF-κB while promoting JNK activation and cell death. Nox1-induced JNK activation and cell death were attenuated through expression of a dominant-negative TNF-RI construct, implicating a role for TNF-RI in Nox1 signaling. Furthermore, Nox1 used the TNF-RI adaptor protein TNF-receptor–associated factor-2 (TRAF2), and the redox-regulated JNK MAP3K, apoptosis signal kinase-1 (ASK1), to activate JNK. In addition, ASK1 siRNA attenuated both Nox1-induced JNK activity and cell death. Collectively, these studies suggest a mechanism by which ROS produced in lung epithelial cells activate JNK and cause cell death using TNF-RI and the TRAF2–ASK1 signaling axis.
doi:10.1165/rcmb.2006-0109OC
PMCID: PMC1899325  PMID: 17079781
Nox1; c-Jun-N-terminal kinase; hydrogen peroxide; cell death; TNF-RI
22.  Novel Role of the Human Alveolar Epithelium in Regulating Intra-Alveolar Coagulation 
Intra-alveolar fibrin deposition is a common response to localized and diffuse lung infection and acute lung injury (ALI). We hypothesized that the alveolar epithelium modulates intra-alveolar fibrin deposition through activation of protein C. Our obejctives were to determine whether components of the protein C activation pathway are present in the alveolar compartment in ALI and whether alveolar epithelium is a potential source. In patients with ALI, pulmonary edema fluid levels of endothelial protein C receptor (EPCR) were higher than plasma, suggesting a source in the lung. To determine whether alveolar epithelial cells are a potential source, protein C activation by A549, small airway epithelial, and primary human alveolar epithelial type II cells was measured. All three cell types express thrombomodulin (TM) and EPCR, and activate protein C on the cell surface. Activation of protein C was inhibited by cytomix (TNF-α, IL-1β, and IFN-γ). Release of EPCR and TM into the conditioned medium was inhibited by the metalloproteinase inhibitors tumor necrosis factor protease inhibitor (TAPI) and GM6001, indicating that the shedding of EPCR and TM from the alveolar epithelium is mediated by a metalloproteinase. These findings provide new evidence that the alveolar epithelium can modulate the protein C pathway and thus could be an important determinant of alveolar fibrin deposition. Local fibrin deposition may be a fundamental mechanism for the lung to localize and confine injury, thus limiting the risk of dissemination of injury or infection to the systemic circulation.
doi:10.1165/rcmb.2005-0425OC
PMCID: PMC1899324  PMID: 17099142
alveolar epithelium; endothelium; protein C; coagulation; acute lung injury
23.  Cigarette Smoke Stimulates Matrix Metalloproteinase-2 Activity via EGR-1 in Human Lung Fibroblasts 
Cigarette smoking is a major risk factor for chronic obstructive pulmonary disease (COPD). Recent reports of increased matrix metalloproteinase-2 (MMP-2) in lungs of patients with emphysema support the paradigm of proteinase/antiproteinase imbalance in the pathogenesis of COPD. We sought to define the signaling pathways activated by smoke and to identify molecules responsible for emphysema-associated MMP-2 expression. In this study, we show that cigarette smoke extract (CSE) induced MMP-2 protein expression and increased MMP-2 gelatinase activity of normal lung fibroblasts. We previously identified a transcription factor, early growth response 1 (EGR-1), with robust expression in the lung tissues of patients with COPD compared with control smokers. Here, the treatment of fibroblasts with CSE resulted in marked induction of EGR-1 mRNA and protein in a dose- and time-dependent manner, accompanied by increased EGR-1 binding activity. CSE-induced MMP-2 mRNA and protein expression and activity were significantly inhibited using EGR-1 small interfering RNA (siRNA) or in Egr-1–null−/− mouse fibroblasts. Furthermore, we observed induction of membrane type 1 matrix metalloproteinase (MT1-MMP), which has an EGR-1–binding site on its promoter, in CSE-treated primary normal lung fibroblasts. The concomitant MT1-MMP expression and MMP-2 activation by CSE are inhibited by EGR-1 siRNA. Rapid activation of mitogen-activated protein kinases was observed in CSE-treated fibroblasts. Chemical inhibitors of ERK1/2 MAPK, but not of p38 and JNK, decreased CSE-induced EGR-1 protein expression and MMP-2 activity of fibroblasts. The identification that induction of MMP-2 and MT1-MMP by CSE from lung fibroblasts is EGR-1–dependent reveals a molecular mechanism for matrix remodeling in cigarette smoke–related emphysema.
doi:10.1165/rcmb.2006-0106OC
PMCID: PMC1899323  PMID: 17099140
chronic obstructive pulmonary disease; EGR-1; cigarette smoke extract; MMP-2; MT1-MMP
24.  Integrin αvβ5 Regulates Lung Vascular Permeability and Pulmonary Endothelial Barrier Function 
Increased lung vascular permeability is an important contributor to respiratory failure in acute lung injury (ALI). We found that a function-blocking antibody against the integrin αvβ5 prevented development of lung vascular permeability in two different models of ALI: ischemia-reperfusion in rats (mediated by vascular endothelial growth factor [VEGF]) and ventilation-induced lung injury (VILI) in mice (mediated, at least in part, by transforming growth factor-β [TGF-β]). Knockout mice homozygous for a null mutation of the integrin β5 subunit were also protected from lung vascular permeability in VILI. In pulmonary endothelial cells, both the genetic absence and blocking of αvβ5 prevented increases in monolayer permeability induced by VEGF, TGF-β, and thrombin. Furthermore, actin stress fiber formation induced by each of these agonists was attenuated by blocking αvβ5, suggesting that αvβ5 regulates induced pulmonary endothelial permeability by facilitating interactions with the actin cytoskeleton. These results identify integrin αvβ5 as a central regulator of increased pulmonary vascular permeability and a potentially attractive therapeutic target in ALI.
doi:10.1165/rcmb.2006-0238OC
PMCID: PMC1899321  PMID: 17079779
integrin αvβ5; lung vascular permeability; pulmonary endothelial barrier function
25.  EGFR-Activated Signaling and Actin Remodeling Regulate Cyclic Stretch–Induced NRF2-ARE Activation 
Cyclic stretch (CS) associated with mechanical ventilation (MV) can cause excessive alveolar and endothelial distention, resulting in lung injury and inflammation. Antioxidant enzymes (AOEs) play a major role in suppressing these effects. The transcription factor Nrf2, via the antioxidant response element (ARE), alleviates pulmonary toxicant- and oxidant-induced oxidative stress by up-regulating the expression of several AOEs. Although gene expression profiling has revealed the induction of AOEs in the lungs of rodents exposed to MV, the mechanisms by which mechanical forces, such as CS, regulate the activation of Nrf2-dependent ARE-transcriptional responses are poorly understood. To mimic mechanical stress associated with MV, we have cultured pulmonary alveolar epithelial and endothelial cells on collagen I–coated BioFlex plates and subjected them to CS. CS exposure stimulated ARE-driven transcriptional responses and subsequent AOE expression. Ectopic expression of a dominant-negative Nrf2 suppressed the CS-stimulated ARE-driven responses. Our findings suggest that actin remodeling is necessary but not sufficient for high-level CS-induced ARE activation in both epithelial and endothelial cells. We also found that inhibition of EGFR activity by a pharmacologic agent ablated the CS-induced ARE transcriptional response in both cell types. Additional studies revealed that amphiregulin, an EGFR ligand, regulates this process. We further demonstrated that the PI3K-Akt pathway acts as the downstream effector of EGFR and regulates CS-induced ARE-activation in an oxidative stress–dependent manner. Collectively, these novel findings suggest that EGFR-activated signaling and actin remodeling act in concert to regulate the CS-induced Nrf2-ARE transcriptional response and subsequent AOE expression.
doi:10.1165/rcmb.2006-0131OC
PMCID: PMC1899320  PMID: 17008637
oxidative stress; MAP kinases; mechanical stress; antioxidant response element; lung

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