A Role for the Receptor for Advanced Glycation End Products in Idiopathic Pulmonary Fibrosis
2008; Elsevier BV; Volume: 172; Issue: 3 Linguagem: Inglês
10.2353/ajpath.2008.070569
ISSN1525-2191
AutoresJudson M. Englert, Lana E. Hanford, Naftali Kaminski, Jacob M. Tobolewski, Roderick J. Tan, Cheryl L. Fattman, Lasse Ramsgaard, Thomas J. Richards, Inna Loutaev, Peter P. Nawroth, Michael Kasper, Angelika Bierhaus, Tim D. Oury,
Tópico(s)Chronic Obstructive Pulmonary Disease (COPD) Research
ResumoIdiopathic pulmonary fibrosis (IPF) is a severely debilitating disease associated with a dismal prognosis. There are currently no effective therapies for IPF, thus the identification of novel therapeutic targets is greatly needed. The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface receptors whose activation has been linked to various pathologies. In healthy adult animals, RAGE is expressed at the highest levels in the lung compared to other tissues. To investigate the hypothesis that RAGE is involved in IPF pathogenesis, we have examined its expression in two mouse models of pulmonary fibrosis and in human tissue from IPF patients. In each instance we observed a depletion of membrane RAGE and its soluble (decoy) isoform, sRAGE, in fibrotic lungs. In contrast to other diseases in which RAGE signaling promotes pathology, immunohistochemical and hydroxyproline quantification studies on aged RAGE-null mice indicate that these mice spontaneously develop pulmonary fibrosis-like alterations. Furthermore, when subjected to a model of pulmonary fibrosis, RAGE-null mice developed more severe fibrosis, as measured by hydroxyproline assay and histological scoring, than wild-type controls. Combined with data from other studies on mouse models of pulmonary fibrosis and human IPF tissues indicate that loss of RAGE contributes to IPF pathogenesis. Idiopathic pulmonary fibrosis (IPF) is a severely debilitating disease associated with a dismal prognosis. There are currently no effective therapies for IPF, thus the identification of novel therapeutic targets is greatly needed. The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily of cell surface receptors whose activation has been linked to various pathologies. In healthy adult animals, RAGE is expressed at the highest levels in the lung compared to other tissues. To investigate the hypothesis that RAGE is involved in IPF pathogenesis, we have examined its expression in two mouse models of pulmonary fibrosis and in human tissue from IPF patients. In each instance we observed a depletion of membrane RAGE and its soluble (decoy) isoform, sRAGE, in fibrotic lungs. In contrast to other diseases in which RAGE signaling promotes pathology, immunohistochemical and hydroxyproline quantification studies on aged RAGE-null mice indicate that these mice spontaneously develop pulmonary fibrosis-like alterations. Furthermore, when subjected to a model of pulmonary fibrosis, RAGE-null mice developed more severe fibrosis, as measured by hydroxyproline assay and histological scoring, than wild-type controls. Combined with data from other studies on mouse models of pulmonary fibrosis and human IPF tissues indicate that loss of RAGE contributes to IPF pathogenesis. Idiopathic pulmonary fibrosis (IPF) is a debilitating disease with a dismal prognosis. Mean survival time after biopsy-confirmed diagnosis is 3 to 5 years.1Crystal RG Bitterman PB Rennard SI Hance AJ Keogh BA Interstitial lung diseases of unknown cause. Disorders characterized by chronic inflammation of the lower respiratory tract.N Engl J Med. 1984; 310: 235-244Crossref PubMed Scopus (259) Google Scholar, 2Collard HR King Jr, TE Demystifying idiopathic interstitial pneumonia.Arch Intern Med. 2003; 163: 17-29Crossref PubMed Scopus (78) Google Scholar Traditional therapy involves the use of corticosteroids as nonspecific anti-inflammatory agents. This treatment produces an objective response in only 10 to 20% of patients and has a minimal effect on the fatal course of IPF.2Collard HR King Jr, TE Demystifying idiopathic interstitial pneumonia.Arch Intern Med. 2003; 163: 17-29Crossref PubMed Scopus (78) Google Scholar, 3Turner-Warwick M Burrows B Johnson A Cryptogenic fibrosing alveolitis: response to corticosteroid treatment and its effect on survival.Thorax. 1980; 35: 593-599Crossref PubMed Scopus (196) Google Scholar, 4Selman M King TE Pardo A Idiopathic pulmonary fibrosis: prevailing and evolving hypotheses about its pathogenesis and implications for therapy.Ann Intern Med. 2001; 134: 136-151Crossref PubMed Scopus (1546) Google Scholar Thus, the need for new therapeutic modalities is evident. The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin super family of cell surface receptors.5Schmidt AM Vianna M Gerlach M Brett J Ryan J Kao J Esposito C Hegarty H Hurley W Clauss M Wang F Pan Y-CE Tsang TC Stern D Isolation and characterization of two binding proteins for advanced glycosylation end products from bovine lung which are present on the endothelial cell surface.J Biol Chem. 1992; 267: 14987-14997Abstract Full Text PDF PubMed Google Scholar In most healthy adult animal tissues, RAGE is expressed at low to undetectable levels.6Brett J Schmidt AM Yan SD Zou YS Weidman E Pinsky D Nowygrod R Neeper M Przysiecki C Shaw A Migheli A Stern D Survey of the distribution of a newly characterized receptor for advanced glycation end products in tissues.Am J Pathol. 1993; 143: 1699-1712PubMed Google Scholar, 7Hanford LE Fattman CL Shaefer LM Enghild JJ Valnickova Z Oury TD Regulation of receptor for advanced glycation end products during bleomycin-induced lung injury.Am J Respir Cell Mol Biol. 2003; 29: S77-S81PubMed Google Scholar Activation of membrane-bound RAGE (mRAGE) by its ligands (including advanced glycation end products, HMGB1/amphoterin, S100/calgranulins, and amyloid-β peptide) often leads to proinflammatory signaling as well as up-regulation of RAGE itself.8Li J Schmidt AM Characterization and functional analysis of the promoter of RAGE, the receptor for advanced glycation end products.J Biol Chem. 1997; 272: 16498-16506Crossref PubMed Scopus (450) Google Scholar This signaling by mRAGE is believed to play an important role in disease progression for several nonpulmonary diseases, including various diabetic complications, chronic inflammation, and Alzheimer's disease, among others.9Schmidt AM Yan SD Yan SF Stern DM The multiligand receptor RAGE as a progression factor amplifying immune and inflammatory responses.J Clin Invest. 2001; 108: 949-955Crossref PubMed Scopus (1058) Google Scholar, 10Schmidt AM Yan SD Yan SF Stern DM The biology of the receptor for advanced glycation end products and its ligands.Biochim Biophys Acta. 2000; 1498: 99-111Crossref PubMed Scopus (607) Google Scholar In contrast to other healthy adult tissues, RAGE mRNA and sRAGE protein are highly expressed in normal adult lungs.6Brett J Schmidt AM Yan SD Zou YS Weidman E Pinsky D Nowygrod R Neeper M Przysiecki C Shaw A Migheli A Stern D Survey of the distribution of a newly characterized receptor for advanced glycation end products in tissues.Am J Pathol. 1993; 143: 1699-1712PubMed Google Scholar, 7Hanford LE Fattman CL Shaefer LM Enghild JJ Valnickova Z Oury TD Regulation of receptor for advanced glycation end products during bleomycin-induced lung injury.Am J Respir Cell Mol Biol. 2003; 29: S77-S81PubMed Google Scholar, 11Demling N Ehrhardt C Kasper M Laue M Knels L Rieber EP Promotion of cell adherence and spreading: a novel function of RAGE, the highly selective differentiation marker of human alveolar epithelial type I cells.Cell Tissue Res. 2006; 323: 475-488Crossref PubMed Scopus (77) Google Scholar Most recently it has been suggested that RAGE is a marker of type I alveolar epithelial cells12Fehrenbach H Kasper M Tschernig T Shearman MS Schuh D Muller M Receptor for advanced glycation endproducts (RAGE) exhibits highly differential cellular and subcellular localisation in rat and human lung.Cell Mol Biol. 1998; 44: 1147-1157PubMed Google Scholar and type II alveolar epithelial cell transdifferentiation, a component of pulmonary re-epithelialization and repair.13Shirasawa M Fujiwara N Hirabayashi S Ohno H Iida J Makita K Hata Y Receptor for advanced glycation end-products is a marker of type I lung alveolar cells.Genes Cells. 2004; 9: 165-174Crossref PubMed Scopus (158) Google Scholar, 14Dahlin K Mager EM Allen L Tigue Z Goodglick L Wadehra M Dobbs L Identification of genes differentially expressed in rat alveolar type I cells.Am J Respir Cell Mol Biol. 2004; 31: 309-316Crossref PubMed Scopus (101) Google Scholar Although RAGE expression is high in the lung compared to other tissues there have been few studies examining its role in pulmonary diseases such as IPF. Notably, there are several reasons to suggest that there may be an important role for RAGE in the pathogenesis of pulmonary fibrosis: 1) mRAGE ligation by AGEs in rat kidney epithelial cells results in myofibroblast metaplasia15Oldfield MD Bach LA Forbes JM Nikolic-Paterson D McRobert A Thallas V Atkins RC Osicka T Jerums G Cooper ME Advanced glycation end products cause epithelial-myofibroblast transdifferentiation via the receptor for advanced glycation end products (RAGE).J Clin Invest. 2001; 108: 1853-1863Crossref PubMed Scopus (394) Google Scholar; 2) AGE levels are increased in pulmonary fibrotic lungs16Matsuse T Ohga E Teramoto S Fukayama M Nagai R Horiuchi S Ouchi Y Immunohistochemical localisation of advanced glycation end products in pulmonary fibrosis.J Clin Pathol. 1998; 51: 515-519Crossref PubMed Scopus (54) Google Scholar; 3) mRAGE signaling results in transforming growth factor-β and collagen synthesis in renal cells17Li JH Wang W Huang XR Oldfield M Schmidt AM Cooper ME Lan HY Advanced glycation end products induce tubular epithelial-myofibroblast transition through the RAGE-ERK1/2 MAP kinase signaling pathway.Am J Pathol. 2004; 164: 1389-1397Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar; 4) mRAGE ligation leads to oxidant generation in endothelial cells18Wautier MP Chappey O Corda S Stern DM Schmidt AM Wautier JL Activation of NADPH oxidase by AGE links oxidant stress to altered gene expression via RAGE.Am J Physiol. 2001; 280: E685-E694PubMed Google Scholar; and 5) RAGE expression is essential for cellular adherence and spreading in type I alveolar epithelial cells.19Demling N Ehrhardt C Kasper M Laue M Knels L Rieber EP Promotion of cell adherence and spreading: a novel function of RAGE, the highly selective differentiation marker of human alveolar epithelial type I cells.Cell Tissue Res. 2006; 323: 475-488Crossref PubMed Scopus (187) Google Scholar Thus, RAGE signaling has the capacity to activate most of the components believed to play a role in the pathogenesis of pulmonary fibrosis, but whether or not RAGE has profibrotic responses in the lung has not been determined. Furthermore, RAGE expression is essential for type I alveolar epithelial cell morphology and transdifferentiation of type II cells, both of which are believed to play a role in the maintenance of normal lung physiology and repair.13Shirasawa M Fujiwara N Hirabayashi S Ohno H Iida J Makita K Hata Y Receptor for advanced glycation end-products is a marker of type I lung alveolar cells.Genes Cells. 2004; 9: 165-174Crossref PubMed Scopus (158) Google Scholar, 14Dahlin K Mager EM Allen L Tigue Z Goodglick L Wadehra M Dobbs L Identification of genes differentially expressed in rat alveolar type I cells.Am J Respir Cell Mol Biol. 2004; 31: 309-316Crossref PubMed Scopus (101) Google Scholar, 19Demling N Ehrhardt C Kasper M Laue M Knels L Rieber EP Promotion of cell adherence and spreading: a novel function of RAGE, the highly selective differentiation marker of human alveolar epithelial type I cells.Cell Tissue Res. 2006; 323: 475-488Crossref PubMed Scopus (187) Google Scholar, 20Bierhaus A Humpert PM Morcos M Wendt T Chavakis T Arnold B Stern DM Nawroth PP Understanding RAGE, the receptor for advanced glycation end products.J Mol Med. 2005; 83: 876-886Crossref PubMed Scopus (1012) Google Scholar Therefore, we investigated the hypothesis that RAGE plays a central role in IPF pathogenesis by using animal models of pulmonary fibrosis, human IPF tissues, and RAGE-null mice. C57BL/6 and RAGE-null mice were treated with 0.1 mg of crocidolite asbestos or titanium dioxide (inert particulate control) intratracheally as previously described.21Tan RJ Fattman CL Watkins SC Oury TD Redistribution of pulmonary EC-SOD after exposure to asbestos.J Appl Physiol. 2004; 97: 2006-2013Crossref PubMed Scopus (68) Google Scholar A 24-gauge feeding needle was inserted endotracheally under anesthesia and a 70-μl suspension containing 1.43 mg/ml of either asbestos or titanium dioxide was then introduced. Mice were sacrificed at indicated time points. Genotyping of RAGE−/− mice was performed by Southern blot according to standard methods. In brief, DNA isolated from tail tip was digested with KpnI (Promega, Heidelberg, Germany) and blotted on nitrocellulose membranes (GE Health Care, München, Germany) before genotyping as described.22Constien R Forde A Liliensiek B Grone HJ Nawroth P Hammerling G Arnold B Characterization of a novel EGFP reporter mouse to monitor Cre recombination as demonstrated by a Tie2 Cre mouse line.Genesis. 2001; 30: 36-44Crossref PubMed Scopus (232) Google Scholar Soluble lung homogenates were prepared as described previously.7Hanford LE Fattman CL Shaefer LM Enghild JJ Valnickova Z Oury TD Regulation of receptor for advanced glycation end products during bleomycin-induced lung injury.Am J Respir Cell Mol Biol. 2003; 29: S77-S81PubMed Google Scholar Lungs were homogenized in an isotonic buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.4) to obtain all soluble proteins. The supernatant containing the soluble portion was removed and the insoluble portion was then pelleted by centrifugation and resuspended in a buffer containing CHAPS detergent (50 mmol/L Tris-HCl, pH 7.4, 150 mmol/L NaCl, 10 mmol/L CHAPS), rocked for 2 hours at 4°C, then centrifuged at 20,000 × g for 20 minutes at 4°C. The membrane fraction supernatant was removed and stored at −80°C until use. Fresh bovine lungs were obtained from McDonald's Meats (Girard, PA) and used as a starting material. One kg of lung tissue was homogenized in 2 L of homogenization buffer (50 mmol/L potassium phosphate, pH 7.4, 0.3 mol/L potassium bromide, 3 mmol/L diethylenetriamine-pentaacetic acid, 0.5 mmol/L phenylmethyl sulfonyl fluoride). Fast protein liquid chromatography was performed as previously described.23Hanford LE Enghild JJ Valnickova Z Petersen SV Schaefer LM Schaefer TM Reinhart TA Oury TD Purification and characterization of mouse soluble receptor for advanced glycation end products (sRAGE).J Biol Chem. 2004; 279: 50019-50024Crossref PubMed Scopus (187) Google Scholar Purified sRAGE was dialyzed into phosphate-buffered saline (PBS) and assayed for endotoxin using the Kinetic-QCL kinetic chromogenic assay (Lonza Group, Basel, Switzerland). sRAGE containing less than 0.05 ng/ml was stored at −20°C until use. RAGE knockout mice were treated daily by intraperitoneal injection with 50 μg of bovine sRAGE or bovine serum albumin control.24Wendt TM Tanji N Guo J Kislinger TR Qu W Lu Y Bucciarelli LG Rong LL Moser B Markowitz GS Stein G Bierhaus A Liliensiek B Arnold B Nawroth PP Stern DM D'Agati VD Schmidt AM RAGE drives the development of glomerulosclerosis and implicates podocyte activation in the pathogenesis of diabetic nephropathy.Am J Pathol. 2003; 162: 1123-1137Abstract Full Text Full Text PDF PubMed Scopus (513) Google Scholar, 25Park L Raman KG Lee KJ Lu Y Ferran Jr, LJ Chow WS Stern D Schmidt AM Suppression of accelerated diabetic atherosclerosis by the soluble receptor for advanced glycation endproducts.Nat Med. 1998; 4: 1025-1031Crossref PubMed Scopus (1023) Google Scholar Treatment with sRAGE was initiated 1 day before intratracheal injection of asbestos. Twenty μg of each protein sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted as described previously.7Hanford LE Fattman CL Shaefer LM Enghild JJ Valnickova Z Oury TD Regulation of receptor for advanced glycation end products during bleomycin-induced lung injury.Am J Respir Cell Mol Biol. 2003; 29: S77-S81PubMed Google Scholar Mouse mRAGE/sRAGE was detected using an anti-RAGE antibody previously described7Hanford LE Fattman CL Shaefer LM Enghild JJ Valnickova Z Oury TD Regulation of receptor for advanced glycation end products during bleomycin-induced lung injury.Am J Respir Cell Mol Biol. 2003; 29: S77-S81PubMed Google Scholar; for human samples, anti-RAGE H-300 (Santa Cruz Biotechnology, Santa Cruz, CA) was used. Mouse aquaporin-5 was used as a marker of type I alveolar epithelial cells and was detected using anti-AQP5 antibody (Sigma, St. Louis, MO). Mouse anti-β-actin (Sigma) and mouse anti-β-tubulin (Sigma) were used for loading controls. To visualize antibody binding, enhanced chemiluminescence was used (ECL, Amersham Biosciences, Buckinghamshire, UK; and ECL Plus, GE Health Care). Image capture and densitometric analysis were performed on a Kodak Gel Logic 2200 imaging system and Kodak molecular imaging software, respectively (Eastman Kodak, Rochester, NY). Samples were obtained from the tissue bank of the Department of Pathology at the University of Pittsburgh. The use of archived tissue has been approved by the institutional review board. Diagnosis of IPF was supported by history, physical examination, pulmonary function studies, chest high-resolution computed tomography, and corroborated by open lung biopsy. The morphological diagnosis of IPF was based on typical microscopic findings consistent with this disease.26Katzenstein AL Myers JL Idiopathic pulmonary fibrosis: clinical relevance of pathologic classification.Am J Respir Crit Care Med. 1998; 157: 1301-1315Crossref PubMed Scopus (1178) Google Scholar The patients fulfilled the criteria of the American Thoracic Society and European Respiratory Society.27American Thoracic Society Idiopathic pulmonary fibrosis: diagnosis and treatment. International consensus statement. American Thoracic Society (ATS), and the European Respiratory Society (ERS).Am J Respir Crit Care Med. 2000; 161: 646-664Crossref PubMed Scopus (993) Google Scholar Control samples included histologically normal lung samples resected from patients with lung cancer obtained from the University of Pittsburgh Department of Pathology Tissue Bank. Total RNA was extracted from 13 IPF samples (surgical remnants of biopsies or lungs explanted from patients with IPF that underwent pulmonary transplant) and 11 control samples (normal histology lung samples resected from patients with lung cancer) and was used for microarray analysis according to manufacturer's recommendations (CodeLink UniSet Human I Bioarray, Amersham Biosciences) and as previously described.28Kaminski N Friedman N Practical approaches to analyzing results of microarray experiments.Am J Respir Cell Mol Biol. 2002; 27: 125-132Crossref PubMed Scopus (83) Google Scholar, 29Pardo A Gibson K Cisneros J Richards TJ Yang Y Becerril C Yousem S Herrera I Ruiz V Selman M Kaminski N Up-regulation and profibrotic role of osteopontin in human idiopathic pulmonary fibrosis.PLoS Med. 2005; 2: e251Crossref PubMed Scopus (362) Google Scholar Fragmented cRNA was hybridized to CodeLink UniSet Human I Bioarray (Amersham Biosciences) slides. After hybridization, arrays were washed and stained with streptavidin-Alexa Fluor 647. The arrays were scanned using a Genepix 4000B microarray scanner. Images were analyzed using CodeLink Expression II Analysis Suite (Amersham Biosciences). They were visually inspected for visual defects and QC parameters as recommended by the manufacturer. Data files were imported into a microarray database and linked with updated gene annotations using SOURCE (http://genome-www5.stanford.edu/cgi-bin/SMD/source/sourceSearch) and then median scaled. Statistical analysis was performed using Scoregene gene expression package (http://www.cs.huji.ac.il/labs/compbio/scoregenes), and data visualization was performed using Genexpress (http://genexpress.stanford.edu) and Spotfire Decision Suite 8.0 (Spotfire Inc., Göteborg, Sweden). The complete set of gene array data has been deposited in the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo) with GEO serial accession number GSE2052. The general approach to analysis has been previously described.28Kaminski N Friedman N Practical approaches to analyzing results of microarray experiments.Am J Respir Cell Mol Biol. 2002; 27: 125-132Crossref PubMed Scopus (83) Google Scholar The oligo probe designed to detect human RAGE expression recognizes both mRAGE and sRAGE transcripts. Sirius Red was used to stain collagen and reticulin fibers as previously described.30Kasper M Seidel D Knels L Morishima N Neisser A Bramke S Koslowski R Early signs of lung fibrosis after in vitro treatment of rat lung slices with CdCl2 and TGF-beta1.Histochem Cell Biol. 2004; 121: 131-140Crossref PubMed Scopus (56) Google Scholar Sections of lung were deparaffinized with xylene and hydrated with an ethanol series, washed in tap water, and stained with a Sirius Red/fast green solution for 30 minutes at room temperature. They were washed again and dehydrated in an ethanol series and xylene. Paraffin sections (4 μm thick) layered on silane-coated slides were used for immunohistochemical staining with primary antibodies against RAGE (Merck and Dhome, Cramlington, UK) (1:4000) and collagen type I (Quartett, Germany) (1:10). Before immunostaining using the Vectastain Elite Kit (Vector/Alexis, Grunberg, Germany), the sections were treated with 10 mmol/L sodium citrate buffer (pH 6.0) in a microwave (750 W, 2 × 5 minutes). Endogenous peroxidase in the tissue was blocked by incubation using 1% H2O2 in PBS, pH 7.4. Nonspecific sites were blocked with 10% goat serum in PBS (15 minutes at room temperature). After the incubation with primary antibody (1 hour, 37°C), the sections were washed with PBS, then incubated with the corresponding biotinylated link antibody (30 minutes, 37°C) and with the ABC complex (30 minutes, 37°C). After further washes with PBS, bound ABC complexes were detected with 0.06% diaminobenzidine tetrahydrochloride in PBS. The sections were counterstained with hematoxylin. Controls for immunospecificity were included in all experiments by omission of the primary antibody and its replacement by phosphate-buffered saline and matching concentrations of normal mouse, rabbit, or goat serum (data not illustrated). Lungs were dried at 110°C for at least 24 hours, then acid hydrolyzed using 6 N hydrochloric acid in the absence of oxygen. Vials were then vacuum-sealed and incubated at 110°C for 24 hours. Hydroxyproline content was quantified as described previously.21Tan RJ Fattman CL Watkins SC Oury TD Redistribution of pulmonary EC-SOD after exposure to asbestos.J Appl Physiol. 2004; 97: 2006-2013Crossref PubMed Scopus (68) Google Scholar, 31Woessner JF The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid.Arch Biochem Biophys. 1961; 93: 440-447Crossref PubMed Scopus (3375) Google Scholar, 32Fattman CL Chu CT Kulich SM Enghild JJ Oury TD Altered expression of extracellular superoxide dismutase in mouse lung after bleomycin treatment.Free Radic Biol Med. 2001; 31: 1198-1207Crossref PubMed Scopus (62) Google Scholar Standard hematoxylin and eosin (H&E) staining was performed on 5-μm-thick lung sections as described previously.32Fattman CL Chu CT Kulich SM Enghild JJ Oury TD Altered expression of extracellular superoxide dismutase in mouse lung after bleomycin treatment.Free Radic Biol Med. 2001; 31: 1198-1207Crossref PubMed Scopus (62) Google Scholar The sections were scored by a pathologist (T.D.O.) who was blinded to both treatment and strain as previously described.21Tan RJ Fattman CL Watkins SC Oury TD Redistribution of pulmonary EC-SOD after exposure to asbestos.J Appl Physiol. 2004; 97: 2006-2013Crossref PubMed Scopus (68) Google Scholar Individual fields were examined with a light microscope at ×200 magnification. Every field in the entire lung was scored, starting peripherally. Each field had to contain >50% alveolar tissue/terminal bronchioles to be counted. Scoring was based on the extent of interstitial fibrosis according to the following scale: 0 = no fibrosis, 1 = 0 to 25%, 2 = 25 to 50%, 3 = 50 to 75%, and 4 = 75 to 100%. The pathological index score was then reported as ratio of the sum of all of the scores divided by the total number of fields counted for each sample. Group scores were averaged for statistical analyses. RT reactions were performed on 1 μg of RNA using 5 mmol/L MgCl2, PCR buffer II, 1 mmol/L nucleotide mix (Promega, Madison, WI), 1.0 U RNAsin, 2.5 U MuLV reverse transcriptase enzyme, and 3 μg of random primers. RT reactions were performed in a thermocycler programmed for 42°C for 40 minutes, 99°C for 5 minutes, and 5°C for 5 minutes. Quantitative PCR was performed by adding Universal PCR buffer and TaqMan primer/probe assay reagent for RAGE according to the manufacturer's protocol. Primers/probe for GAPDH were used as a loading control for normalization. The default program was performed on an ABI Prism 7300 (Applied Biosystems, Foster City, CA) and consisted of 50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles of 95°C for 15 seconds, and 60°C for 1 minute. Sequence Detection Software Version 1.4 (Applied Biosystems) was used to analyze the data and obtain relative quantities of mRNA expression for each sample based on the crossing threshold. The relative expressions for each treatment group were then averaged and the expression of the TiO2 treated controls was set to 100% for relative expression comparison. Paired samples were analyzed using Student's t-test. Variances of paired samples were analyzed by an F-test. When variances were determined to be significantly different, analysis was performed by t-test with Welch's correction. Analysis of variance followed by a Tukey test were used for multiple comparisons. Values are reported ±SEM, and P values <0.05 were considered significant. Although it has been shown that RAGE mRNA and sRAGE protein expression are highest in the lung among normal adult tissues,6Brett J Schmidt AM Yan SD Zou YS Weidman E Pinsky D Nowygrod R Neeper M Przysiecki C Shaw A Migheli A Stern D Survey of the distribution of a newly characterized receptor for advanced glycation end products in tissues.Am J Pathol. 1993; 143: 1699-1712PubMed Google Scholar, 7Hanford LE Fattman CL Shaefer LM Enghild JJ Valnickova Z Oury TD Regulation of receptor for advanced glycation end products during bleomycin-induced lung injury.Am J Respir Cell Mol Biol. 2003; 29: S77-S81PubMed Google Scholar it is important to determine whether mRAGE is also expressed at high levels in the lung because this is the isoform of RAGE that mediates signaling. mRAGE expression was examined in membrane preparations from several untreated mouse tissues by Western blot analysis (Figure 1). Similar to RAGE mRNA and sRAGE protein, mRAGE protein is found at the highest levels in the lung. The relatively abundant expression of pulmonary mRAGE and sRAGE in the normal lung observed by us as well as others6Brett J Schmidt AM Yan SD Zou YS Weidman E Pinsky D Nowygrod R Neeper M Przysiecki C Shaw A Migheli A Stern D Survey of the distribution of a newly characterized receptor for advanced glycation end products in tissues.Am J Pathol. 1993; 143: 1699-1712PubMed Google Scholar, 7Hanford LE Fattman CL Shaefer LM Enghild JJ Valnickova Z Oury TD Regulation of receptor for advanced glycation end products during bleomycin-induced lung injury.Am J Respir Cell Mol Biol. 2003; 29: S77-S81PubMed Google Scholar, 33Katsuoka F Kawakami Y Arai T Imuta H Fujiwara M Kanma H Yamashita K Type II alveolar epithelial cells in lung express receptor for advanced glycation end products (RAGE) gene.Biochem Biophys Res Commun. 1997; 238: 512-516Crossref PubMed Scopus (81) Google Scholar suggests that it plays a homeostatic role unique to the lung. Thus, alterations in mRAGE and sRAGE expression may be a factor in pulmonary diseases such as pulmonary fibrosis. As indicated above, RAGE contributes to the pathogenesis of many nonpulmonary diseases. In such diseases, mRAGE expression has been found to be up-regulated. To investigate the regulation of mRAGE and sRAGE expression in pulmonary fibrosis, we used both the bleomycin and the asbestos mouse models of pulmonary fibrosis.21Tan RJ Fattman CL Watkins SC Oury TD Redistribution of pulmonary EC-SOD after exposure to asbestos.J Appl Physiol. 2004; 97: 2006-2013Crossref PubMed Scopus (68) Google Scholar, 32Fattman CL Chu CT Kulich SM Enghild JJ Oury TD Altered expression of extracellular superoxide dismutase in mouse lung after bleomycin treatment.Free Radic Biol Med. 2001; 31: 1198-1207Crossref PubMed Scopus (62) Google Scholar Because mouse sRAGE is not a product of alternative splicing,23Hanford LE Enghild JJ Valnickova Z Petersen SV Schaefer LM Schaefer TM Reinhart TA Oury TD Purification and characterization of mouse soluble receptor for advanced glycation end products (sRAGE).J Biol Chem. 2004; 279: 50019-50024Crossref PubMed Scopus (187) Google Scholar it is necessary to look at the protein level to examine differential expression of these two isoforms. The asbestos model recapitulates many of the salient features of IPF. In this model, we found that pulmonary mRAGE and sRAGE levels decrease significantly by 24 hours after asbestos injury (not illustrated) and remain depressed through 14 days (Figure 2, a and b). This loss corresponds with a 1.5-fold decrease in mRNA expression of the RAGE gene after 14 days of asbestos treatment as compared to a 24-hour titanium dioxide treatment (Figure 2c). Previous experiments have shown that titanium dioxide has no effect on the lungs at any time point.21Tan RJ Fattman CL Watkins SC Oury TD Redistribution of pulmonary EC-SOD after exposure to asbestos.J Appl Physiol. 2004; 97: 2006-2013Crossref PubMed Scopus (68) Google Scholar Bo
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