Asymmetric dimethylarginine metabolism and its involvement in the pathogenesis of pulmonary arterial hypertension [Elektronische Ressource] / by Zakrzewicz, Dariusz

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Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2008
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

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 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 con