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

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

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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 ...........................................................................................................

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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 contents IV
3.6.11.3 Proliferation assay ..................................................................................... 39
4 RESULTS ................................................................................................................. 40
4.1 Development of a method for quantification of methylarginine content in
biological samples ................................................................................................. 40
4.1.1 Standard curves and calibration ........................................................................ 41
4.1.2 Recovery ........................................................................................................... 43
4.1.3 Dynamic metabolism of L-Arg, ADMA and SDMA under proteosome
inhibition ........................................................................................................... 44
4.2 Analysis of ADMA metabolism ............................................................................ 46
4.2.1 Methylarginine content in mouse organs of the cardiovascular system ........... 46
4.2.1.1 Protein-incorporated L-arginine, ADMA and SDMA .............................. 46
4.2.1.2 Free-cellular methylarginines .................................................................... 47
4.2.2 L-Arg, ADMA and SDMA in serum and BALF .............................................. 48
4.2.2.1 Concentration of L-arginine derivatives in mouse and human serum ...... 48
4.2.2.2 Methylargine content in human and mouse BALF ................................... 50
4.2.3 Expression of PRMTs in mouse lung, heart, liver and kidney .......................... 51
4.2.4 Analysis of ADMA degradation ....................................................................... 53
4.2.4.1 Expression of mouse DDAH isoforms ...................................................... 53
4.2.4.2 Tissue-specific activity of DDAH ............................................................. 54
4.3 Protein arginine methylation in pulmonary arterial hypertension ......................... 55
4.3.1 Methylarginine content in tissue lung homogenates from PAH patients .......... 55
4.3.1.1 Protein-incorporated L-Arg, ADMA and SDMA ..................................... 55
4.3.1.2 Free-cellular methylarginines .................................................................... 56
4.3.2 Expression of PRMTs in the lungs from PAH patients and healthy donors ..... 57
4.3.3 Western blot analysis of asymmetric dimethylated proteins in lung
homogenates ...................................................................................................... 59
4.3.4 Immunohistochemical analysis of lung sections ............................................... 60
4.3.5 Subcellular fractionation of proteins from human PASMC .............................. 61 Tables of contents V
4.3.6 PRMT1 silencing in PASMC ............................................................................ 62
4.3.6.1 Optimalization of siRNA transfection ...................................................... 62
4.3.6.2 Specificity of PRMT1 knockdown in PASMC ......................................... 64
4.3.6.3 Effect of reduced PRMT1 expression on cell proliferation ...................... 65
5 DISCUSSION ........................................................................................................... 66
5.1 Protein arginine methylation in the cardiovascular system ................................... 66
5.1.1 Quantitative assessment of free versus protein-incorporated methylarginine in
vitro and in vivo ................................................................................................ 66
5.1.2 Analysis of methylarginine metabolism in the cardiovascular system ............. 68
5.2 Analysis of protein arginine methylation in pulmonary arterial hypertension ..... 71
5.2.1 Lung methylarginine content of patients with pulmonary arterial
hypertension ...................................................................................................... 71
5.2.2 Analysis of asymmetric dimethylated proteins in PAH compared to healthy
lungs .................................................................................................................. 72
5.2.3 Analysis of PRMT1 localization in the human lung ......................................... 73
5.2.3.1 Immunohistochemical analysis of PRMT1 tissue localization ................. 73
5.2.3.2 Cellular compartmentalization of PRMT1 in PASMC ............................. 73
5.2.4 Functional effects of transient PRMT1 knockout on smooth muscle cell
proliferation ....................................................................................................... 74
5.3 Conclusions and further perspectives .................................................................... 75
6 APPENDIX ............................................................................................................... 77
7 REFERENCES .......................................................................................................... 79
8 DECLARATION ...................................................................................................... 89
9 CURRICULUM VITAE ........................................................................................... 90 Tables of contents VI
10 ACKNOWLEDGEMENTS .................................................................................. 94

List of figures VII
II List of figures

Figure 1.1 Methylarginines are generated during methylation of protein L-arginine
residues
Figure 1.2 The protein arginine methyltransferase family
Figure 1.3 ADMA inhibits NOS activity and is metabolized by DDAH
Figure 1.4 Histopathological changes observed in PAH
Figure 1.5 Consequences of pulmonary artery endothelial cell dysfunction on
pulmonary artery smooth muscle cell tone and proliferation
Figure 1.6 Possible roles played by enzymes involved in ADMA metabolism in PAH
Figure 4.1 Strategy for the quantification of protein-incorporated and free-cellular
L-Arg, ADMA and SDMA in human and mouse tissue homogenates
Figure 4.2 Linear regression of standard curves
Figure 4.3 Protein precipitation does not alter protein methylation
Figure 4.4 Degree of protein methylation in protein hydrolysates (A) and crude
extracts (B) from A549 cells after treatment with proteosome inhibitor
Figure 4.5 Concentration of protein-incorporated L-Arg, ADMA and SDMA in
mouse lung, heart, kidney and liver
Figure 4.6 Concentration of free-cellular L-Arg, ADMA and SDMA in mouse lung,
heart, kidney and liver
Figure 4.7 Concentration (A) and ratios (B) of methylarginines in mouse and human
serum
Figure 4.8 ) of meouse and human
BAL fluid
Figure 4.9 Expression pattern of PRMT enzymes in mouse tissues
Figure 4.10 Expression of DDAH isoforms in mouse tissues
Figure 4.11 DDAH activity in mouse tissues
Figure 4.12 Box plots of protein-incorporated L-Arg, ADMA, and SDMA levels in the
lung tissue homogenates from PAH patients and healthy donors List of figures VIII
Figure 4.13 Box plots of free-cellular L-Arg, ADMA, and SDMA levels in the lung
tissue homogenates from PAH patients and healthy donors
Figure 4.14 Expression of PRMTs in the lung homogenates derived from PAH patients
and healthy donors
Figure 4.15 Analysis of asymmetric dimethylated proteins in PAH and donor lung
homogenates
Figure 4.16 Localization of PRMT1 in the human lung sections
Figure 4.17 Subcellular fractionation of human PASMC
Figure 4.18 Optimization of PRMT1 knockdown in PASMC
Figure 4.19 PRMT1 siRNA did not affect mRNA and protein level of PRMT4
Figure 4.20 Increased PASMC proliferation using siRNA directed against PRMT1