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