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Asymmetric Dimethylarginine Metabolism and Its Involvement in the Pathogenesis of Pulmonary Arterial Hypertension

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Asymmetric Dimethylarginine Metabolism and Its Involvement in the Pathogenesis of Pulmonary Arterial Hypertension Asymmetric dimethylarginine metabolism and its involvement in the pathogenesis of pulmonary arterial hypertension Inaugural Dissertation submitted to the Faculty of Medicine in partial fulfillment of the requirements for the PhD-Degree of the Faculties of Veterinary Medicine and Medicine of the Justus Liebig University Giessen by Zakrzewicz, Dariusz of Poznan, Poland Giessen 2008 From the Department of Medicine Director / Chairman: Prof. Dr. Werner Seeger of Medicine of the Justus Liebig University Giessen First Supervisor and Committee Member: Dr. Oliver Eickelberg Second Supervisor and Committee Member: Prof. Dr. James Leiper Committee Members: Prof. Dr. Martin Diener Privatdozent Dr. Konstantin Mayer Date of Doctoral Defense: 17.09.2008 Tables of contents I I Tables of contents I TABLES OF CONTENTS ........................................................................................... I II LIST OF FIGURES .................................................................................................. VII III LIST OF TABLES .................................................................................................... IX IV LIST OF ABBREVIATIONS .................................................................................... X V SUMMARY ........................................................................................................... XIV VI ZUSAMMENFASSUNG ....................................................................................... XVI 1 INTRODUCTION ....................................................................................................... 1 1.1 ADMA metabolism ................................................................................................. 1 1.1.1 ADMA synthesis ................................................................................................. 1 1.1.1.1 The PRMT family of enzymes: biological function ................................... 2 1.1.2 Role of cellular ADMA ....................................................................................... 7 1.1.3 Elimination of ADMA by DDAH ....................................................................... 8 1.2 Alterations to methylarginine metabolism in human .............................................. 9 1.2.1 Human cancer ...................................................................................................... 9 1.2.2 Viral pathogenesis ............................................................................................... 9 1.2.3 Multiple sclerosis ................................................................................................ 9 1.2.4 Abnormal ADMA levels in human disorders ................................................... 10 1.3 Pulmonary arterial hypertension ........................................................................... 11 1.3.1 Characteristics of pulmonary arterial hypertension .......................................... 11 1.3.2 Histopathological abnormalities ........................................................................ 11 Tables of contents II 1.3.3 Genetic determinants of pulmonary arterial hypertension ................................ 13 1.3.3.1 The bone morphogenetic protein pathway ................................................ 13 1.3.3.2 Activin receptor-like kinase 1 ................................................................... 14 1.3.3.3 Serotonin ................................................................................................... 14 1.3.4 Humoral regulators in pulmonary arterial hypertension ................................... 14 1.3.4.1 Prostacyclin ............................................................................................... 14 1.3.4.2 Endothelins ................................................................................................ 16 1.3.5 Nitric oxide generation in PAH ......................................................................... 16 1.3.5.1 Nitric oxide production ............................................................................. 16 1.3.5.2 L-arginine content in patients with PAH .................................................. 17 1.3.6 ADMA metabolism in PAH .............................................................................. 17 2 AIM OF THE STUDY .............................................................................................. 19 3 MATERIALS AND METHODS .............................................................................. 20 3.1 Materials ................................................................................................................ 20 3.1.1 Equipment ......................................................................................................... 20 3.1.2 Reagents ............................................................................................................ 22 3.2 Cells ....................................................................................................................... 24 3.3 Animals ................................................................................................................. 24 3.4 Samples from mice ................................................................................................ 25 3.4.1 Broncho-alveolar lavage fluid, serum and tissues ............................................. 25 3.5 Samples from human origin .................................................................................. 25 3.5.1 Lung tissue ........................................................................................................ 25 3.5.2 Serum and broncho-alveolar lavage fluid ......................................................... 25 3.6 Methods ................................................................................................................. 26 3.6.1 RNA isolation .................................................................................................... 26 Tables of contents III 3.6.2 RNA and DNA determination ........................................................................... 26 3.6.3 Reverse transcription reaction ........................................................................... 26 3.6.4 Polymerase chain reaction ................................................................................. 27 3.6.4.1 Semi-quantitative PCR .............................................................................. 27 3.6.4.2 Real-time PCR .......................................................................................... 28 3.6.5 Gel electrophoresis ............................................................................................ 29 3.6.5.1 DNA gel electrophoresis ........................................................................... 29 3.6.5.2 Protein gel electrophoresis ........................................................................ 30 3.6.6 Protein isolation ................................................................................................ 31 3.6.6.1 Protein isolation from cell culture ............................................................. 31 3.6.6.2 Protein isolation from tissue ...................................................................... 31 3.6.6.3 TCA precipitation ...................................................................................... 32 3.6.6.4 Hydrolysis ................................................................................................. 32 3.6.6.5 Protein determination ................................................................................ 33 3.6.7 Subcellular fractionation ................................................................................... 33 3.6.8 Analysis of basic amino acids ........................................................................... 34 3.6.8.1 Isolation of basic amino acids ................................................................... 34 3.6.8.2 Derivatization with OPA reagent .............................................................. 34 3.6.8.3 Chromatographic separation by HPLC ..................................................... 35 3.6.8.4 DDAH activity assay ................................................................................. 35 3.6.9 Western blot analysis ........................................................................................ 36 3.6.9.1 Immunoblotting ......................................................................................... 36 3.6.9.2 Protein visualization .................................................................................. 36 3.6.9.3 Densitometric analysis .............................................................................. 37 3.6.10 Immunohistochemistry .................................................................................. 37 3.6.11 Cell culture .................................................................................................... 38 3.6.11.1 Culture of mammalian cells ...................................................................... 38 3.6.11.1.1 A549 cells ............................................................................................ 38 3.6.11.1.2 Pulmonary artery smooth muscle cells ................................................ 38 3.6.11.2 Transient transfection ................................................................................ 39 3.6.11.2.1 siRNA technique .................................................................................. 39 Tables of contents IV 3.6.11.3 Proliferation
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