The role of the receptor for advanced glycation end products (RAGE) in idiopathic pulmonary fibrosis [Elektronische Ressource] / vorgelegt von Markus Alexander Queisser

Queisser, Jean-Paul Markus

Sujets

• Biologie

Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2009
• Nombre de lectures: 6
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

The Role of the Receptor for Advanced

Glycation End Products (RAGE) in

idiopathic Pulmonary Fibrosis

  Inaugural Dissertation Zur Erlangung des Doktorgrades der Naturwissenschaften  -Dr. rer. nat.- vorgelegt von   Markus Alexander Queisser aus Berlin, Deutschland     angefertigt am Institut für Biochemie Fachbereich Medizin und dem Fachbereich für Biologie und Chemie Justus-Liebig-Universität Giessen

  From the Department of Medicine Institute of Biochemistry Director: Prof. Dr. Klaus T. Preissner at the Justus Liebig University Giessen                                         First Supervisor and Committee Member Prof. Dr. A. Pingoud

Second Supervisor and Committee Member Prof. Dr. K.T. Preissner

Committee members: Prof. Dr. W. Clauss and Prof. Dr. R. Dammann

Date of Doctoral Defense ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙15.05.2009

“Forschung ist nie zu Ende, sie lebt von der Kritik und der intelligenten Skepsis, die ni cht die Arroganz des Ignoranten ist “ .

Prof. Dr. Lothar Jaenicke

Table of contents Table of contents ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ I List of figures ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ III List of abbreviations ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ IV Summary ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ VII Zusammenfassung ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ VIII 1. Introduction∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 1 1.2 Ligands of the recetor for advanced glycation end products ∙∙∙∙∙∙∙∙∙∙∙1 1.2.1 Advanced glycation end products (AGE) ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙2 1.2.2 Amyloid∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ides∙∙∙∙-tpep∙∙∙∙∙∙∙∙∙∙3∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 1.2.3 High mobility group box -protein B1 (HMGB1) ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙3 1.2.4 S100/Calgranulins ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙4 1.2.5 Mac-1 (CD11b/CD 18) ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙4 1.3 Physiological function of RAGE ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙5 1.4 RAGE expression and its involvement in pathogeneses∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙5 1.4.1 RAGE in vascular and renal complications of diabetes mellitus∙∙∙5 1.4.2 RAGE in tumor progr ession and metastasis ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙7 1.4.3 RAGE in innate and adapted immunity ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙8 1.5 Physiology and pathophysiology of the lung ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙9 1.5.1 Anatomy of the pu lmonary system ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙10 1.5.2 Interstitial lung diseases ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙12 1.5.3 Idiopathic pulmonary fibrosis∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙12 1.5.4 Pathogenesis of IPF∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙12 1.5.4.2 Chronic injury hypothesis ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙13 1.5.4.3 Sequential injury hypothesis ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙13 1.5.4.4 Circulating fibrocyte-hy pothesis ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙14 1.5.4.5 Epithelial-mesenchymal transition (EMT) hypothesis∙∙∙∙∙∙∙∙∙∙∙∙∙∙15 1.5.5 Genetic factors ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙16 1.6 Animal models of pulmonary fibrosis ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙17 1.6.1 Bleomycin model∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙18 1.6.2 Asbestos, silicia model ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙18 1.6.3 Fluorescein isothiocyanate -model∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙18 1.6.4 Irradiation model ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙19 1.6.5 Transgenic model∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙19 1.7 Hypothesis ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙20 1.8 Aims ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙20 2. Materials ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 21 2.1. Chemicals ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙21 2.1.2 Enzymes ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙23 2.1.3 Cytokines ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙23 2.1.4 Antibodies ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙23 2.1.5 DNA-Primers ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙24 2.1.6 Small interfering RNA (siRNA) ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙24 2.1.7 General consumabl e ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙24 2.1.8 Cell culture ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙25 2.1.9 Machines and systems∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙25 2.2 Patient Population ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙26 3 Methods∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 26 3.1 Animal Treatment∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙26 I

Table of contents 3.2 Isolation and Culture of Human Alveolar Epithelial Cells type II∙∙∙∙26 3.3 Isolation and Culture of Human Pulmonary Fibroblasts ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙27 3.4 Cytokine Stimulation ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙27 3.5 Immunohistochemistry ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙28 3.6 Immunofluorescence∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙28 3.7 siRNA knock down ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙28 3.8 Reverse Transcriptase (RT) -PCR ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙29 3.9 Real-time PCR ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙29 3.10 Western Blot ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙30 3.11 Extracellular Matrix Preparation ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙31 3.12 Adhesion Assay ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙31 3.13 Proliferation Assa y ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙31 3.14 Migration (chemotaxis) Assay ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙31 3.15 Wound Healing Assay∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙32 3.16 Basolateral membrane isolation ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙32 4. Statistics∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙33 5. Results∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 34 5.1 Differential expression of RAGE in mouse tissue∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙34 5.2 Distribution of RAGE in donor and IPF lung tissue ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙34 5.3 RAGE expression in donor, IPF lungs, alveolar type II cells and fibroblasts ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙35 5.4 RAGE Expression in the bleomycin mouse model of lung fibrosis 38 5.5 Influence of Cytokines on RAGE Expression ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙38 5.6 Relation between RAGE and Cell Adhesion, Migration and Prolifer ation ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙39 6. Discussion ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 45 6.1 The role of RAGE in pulmonary fibrosis ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙45 6.2 RAGE as a biomarker for lung injury∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙47 6.3 RAGE-ligand signaling in the lung∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙48 6.4 potential mechanism of RAGE downregulation ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙49 6.4.1 RAGE downregulation by micro-RNA ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙50 6.4.2 RAGE downregulation by proteases ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙50 6.4.3 Downregulation of RAGE in relation to caveolae ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙51 6.4 Involvement of RAGE in epithelial-mesenchymal transition ∙∙∙∙∙∙∙∙∙∙51 6.5 RAGE as an adhesion molecule ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙52 7. Declaration ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 53 8. Curriculum vitae ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 54 9. Acknowledgements ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 57 10. References ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 59  

II

List of figures

List of figures

Figures Introduction

Figure 1: RAGE isoforms and signaling cascade ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 2 Figure 2: Endothelial dysfunction by AGE-RAGE interaction ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 6 Figure 3: RAGE dependent regulation of cellular invasion.∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 8 Figure 4: Schematic diagram of lung anatomy ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 10 Figure 5: Air-blood barrier ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 11 Figure 6 Hypothetical scheme of the abnormal wound healing model for idiopathic pulmonary fibros is.∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 14 Figure 7: Alveolar epithelial tr ansdifferentiation pathways. ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 16

Figures Results

Figure 8: Abundant RAGE Expression in the Lung ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 34 . Figure 9: RAGE distribution in IPF and Donor lungs. ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 35 Figure 10: RAGE Downregulation in IPF lung homogenate. ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 36 Figure 11: RAGE Downregulation in alveolar epithelial cells type II from IPF patients.∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 37   Figure 12: Cytokine-Dependent RAGE Downregulation in A549 Cells. ∙∙ 39 Figure 13:Cytokine-Dependent RAGE Downregulation in Pulmonary Fibroblasts∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 40 Figure 14: Impairement of Cell Adhesion on Collagen and Extracellular Matrix by RAGE Blocking. ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 41 Figure 15 Increased Cell Proliferation and Migration due to siRNAmediated RAGE knockdown ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 42 Figure 16: Increased Cell Migration in Wound Scratch Assay ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 43 . Figure 17: RAGE is associated with the Cytoskeleton ∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙∙ 44

III

List of abbreviations

List of abbreviations

A   ADAM   AEC AGE ALI ARDS AT ATP BAL BCA Bcl-2 CD CML CMPC   Col CT CXCR   DMEM DNA dNTP DTT EC ECL ECM EDTA Egr-1 EF EMT EN-RAGE 

= Amyloid-beta = A Disintegrin And Metalloproteinase protein = alveolar epithelial cells = Advanced Glycation End Products = Acute lung injury = Acute respiratory distress syndrome = Alveolar type = Adenosin triphosphate = Bronchoalveolar lavage fluid = Bicinchoninic acid = B-cell lymphoma 2 = Cluster of differentiation Carboxymethyl lysine = = Circulating mesenchymal progenitor cells = Collagen = Cycle of threshold = Chemokine-CXC-motif Receptor = Dulbecco’s modified Eagle medium = Deoxyribonucleic acid = Desoxy nucleotide triphosphate = DL-Dithiothreitol = Endothelial Cell = Enhanced Chemiluminescence = Extracellular matrix   = Eythelene diamino tetra acetic acid = Early growth factor-1   = Elongation factor = Epithelial-mesenchymal transition = Extracellular newly identified RAGE binding protein (S100A12) 

IV

List of Aberrations ERK = Extracellular signal-regulated kinase esRAGE = Endogenous soluble RAGE FBS = Fetal bovine serum FGF-2   = Fibroblast growth factor 2 FITC = Fluorescein isothiocyanate FSP-1   = Fibroblast specific protein GTPase = GTP hydrolase GTP = Guanosine triphosphate HBSS = Hank's Buffered Salt Solution HEPES = 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HGF = Hepatocyte growth factor Hmbs = Hydroxymethylbilane synthase HMGB1 = High mobility group binding-protein B1 HRP = Horse-radish peroxidase ICAM-1 = Inter-Cellular Adhesion Molecule 1 IgG = Immune globuline IL-1 Interleukin-1 =   IL-6 = Interleukin-6 ILD = Interstitial lung diseases IFN- = Interferon-   IPF = Idiopathic Pulmonary Fibrosis JNK = C-Jun-N-terminal kinase KGF = Keratinocyte growth factor MAPK   = Mitogen-activated protein kinase MCP-1 = Monocyte chemotactic protein-1 MMP = Matrix Metalloprotease    mRNA   = messenger RNA miRNA = micro RNA NADPH = Nicotinamide adenine dinucleotide phosphate NF-B   = Nuclear factor -B pDC = Plasmacytoid dendritic cells PAI-1 = Plasminogen-activator inhibitor 1 PCR = Polymerase chain reaction V

List of Aberrations

PBS PDGF PI3K PMVEC PRP PDVF RAGE   RBC RNA RNP SDS siRNA   SMA SP-C sRAGE ROS RT TBS TBST TEMED TERT TF TIMP TGF-   TLR TNF-   TR TRIS UIP UTR VCAM-1 ZO-1

= Phosphate buffered saline Platelet derived growth factor = = Phosphoinositide kinase 3 = Pulmonary microvascular endothelial cells Pattern recognition receptor = = Polyvinylidene difluoride = Receptor for Advanced Glycation End Products = Red blood cells = Ribonucleic acid = Ribonucleoprotein = Dodecyl sodium salt = Small interfering RNA = Smooth muscle actin = Surfactant protein C = Soluble RAGE = Reactive oxygen species = Reverse Transcriptase = Tris buffered saline = Tris buffered saline tween = N,N,N',N'-Tetramethylethylenediamine = Telomerase reverse transcriptase = Tissue factor = Tissue inhibitors of MMP = Tumor growth factor-   = Toll-like receptor    = Tumor-necrosis factor-   = Telomerase RNA = Tris(hydroxymethyl)aminomethane = Usual interstitial pneumonia = Untranslated region = Vascular cellular adhesion molecule 1 = Zonula occludens-1

VI

Summary

Summary

The Receptor for Advanced Glycation End Products (RAGE) is a transmembrane receptor of the immunoglobulin superfamily. While vascular RAGE expression is associated with kidney and liver fibrosis, under physiological conditions high expression level of RAGE is found in the lung. In this work, RAGE expression in idiopathic pulmonary fibrosis (IPF) was assessed, and the relation of the receptor to functional changes of epithelial cells and pulmonary fibroblasts in the pathogenesis of the disease was investigated. Significant downregulation of RAGE was observed in lung homogenate and alveolar epithelial cells (AEC) type II from IPF patients as well as in bleomycin-treated mice, demonstrated by RT-PCR, western blotting and immunohistochemistry. RAGE downregulation was provoked by stimulation of primary human lung fibroblasts and A549 epithelial cells with the pro-inflammatory cytokines, transforming growth factor-1 or tumor necrosis factor-   in vitroof RAGE resulted in impaired cell. Blockade adhesion, and siRNA induced knock down of RAGE increased cell proliferation and migration of A549 cells and human primary fibroblastsin vitro. These results indicate that RAGE serves a protective role in the lung and that loss of the receptor is related with functional changes of pulmonary cell types with the consequences of fibrotic disease. The study provides evidence that the expression and regulation of RAGE in the pulmonary system differs from that in the vascular system. Here, a possible functional mechanism of RAGE in pulmonary fibrosis is described for the first time.

VII

Zusammenfassung

Zusammenfassung

Der Rezeptor für “Advanced glycation end products” (RAGE) ist ein Transmembranrezeptor aus der Superfamilie der Immunglobuline. Die vaskuläre RAGE Expression ist mit Nieren- und Leberfibrose assoziert, während eine hohe Expression von RAGE in der Lunge unter normalen physiologischen Bedingungen gefunden wurde. In dieser Studie wurde die Expression von RAGE in Patienten der idiopathischen Lungenfibrose (IPF) gemessen, und die Beziehung zwischen RAGE und die funktionellen Änderungen von Epithelzellen und pulmonalen Fibroblasten wurde untersucht. Signifikante Absenkung der Expression von RAGE wurde in Lungenhomogenaten und isolierten alveolaren Epithelzellen type II von IPF Patienten sowie auch in Bleomycin-behandelten Mäusen, nachgewiesen mittels RT-PCR, Western-blot und Immohistochemie.In vitro die wurde Repression von RAGE durch die pro-inflammatorischen Zytokine TGF-und TNF-in primären Fibroblasten und A549 Epithelzellen erreicht. Desweiteren führte die Blockade von RAGE mittels anti-RAGE Antikörpern zu reduzierter Zelladhäsion. siRNA-induzierte Inhibierung der Expression von RAGE in A549 und Fibroblasten führte zur vermehrten Zellproliferation und -Migration in vitro. Diese Ergebnisse deuten auf eine Schutzfunktion der RAGE Expression in der Lunge hin, hingegen trägt der Verlust an RAGE zu zellulären Änderungen und fibrotischen Erkrankungen bei. Diese Studie deckt molekulare Zusammenhänge auf, die zur Erklärung der Unterschiede in der Expression und Regulation von RAGE zwischen dem pulmonalen und vaskulären System führen können. Ein möglicher, funktioneller Mechanismus von RAGE in der pulmonalen Fibrose wurde hier zum ersten Mal beschrieben.

VIII