Role of dimethylarginine dimethylaminohydrolases (DDAH) in pulmonary arterial hypertension [Elektronische Ressource] / vorgelegt von Soni Pullamsetti

SONI PULLAMSETTI
ROLE OF DIMETHYLARGININE
DIMETHYLAMINOHYDROLASES
(DDAH) IN PULMONARY
ARTERIAL HYPERTENSION
INAUGURALDISSERTATION
zur Erlangung des Grades eines
édition scientifique
Doktors der HumanbiologieVVB LAUFERSWEILER VERLAG
des Fachbereichs Medizin der
VVB LAUFERSWEILER VERLAG ISBN 3-8359-5060-6 Justus-Liebig-Universität Gießen
STAUFENBERGRING 15
D-35396 GIESSEN
Tel: 0641-5599888 Fax: -5599890
redaktion@doktorverlag.de
www.doktorverlag.de 9 7 8 3 8 3 5 9 5 0 6 0 3
édition scientifique
VVB VVB LAUFERSWEILER VERLAG
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1. Auflage 2006
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édition scientifique
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Tel: 0641-5599888 Fax: 0641-5599890
email: redaktion@doktorverlag.de
www.doktorverlag.de

ROLE OF
DIMETHYLARGININE DIMETHYLAMINOHYDROLASES
(DDAH) IN PULMONARY ARTERIAL HYPERTENSION






INAUGURALDISSERTATION
zur Erlangung des Grades eines
Doktors der Humanbiologie
des Fachbereichs Medizin der
Justus-Liebig-Universität Giessen




vorgelegt von



SONI PULLAMSETTI
(M.Sc in Biotechnology)

aus Tenali, India





Giessen 2005



Aus der Medizinischen Klinik II
Direktor: Prof. Dr. med. Werner Seeger
des Universitätsklinikums Gießen und Marburg
Standort Gießen












Gutachter: PD Dr. rer nat Ralph Theo Schermuly
Gutachter: PD Dr. Jürgen Bauer
Tag der Disputation: 22.03.2006




My parents
Poorna, Subbarao
Brother Ramesh
Fiancé Raj

. i
Table of Contents
Chapters Page(s)

Table ofcontes i
List ofFigures v
List of Tables viii
Glossary of symbols ix
1. Introduction 1
1.1 Definition and classification of pulmonary arterial hypertension 1
1.2 Histopathology 4
1.3 Pathophysiology/Pathological mechanisms 6
1.3.1 Endothelial dysfunction/Vasoconstriction 6
1.3.2 Remodeling 9
1.3.3 In situ thrombosis 11
1.4 Diagnosis and evaluation 11
1.4.1 Clinical presentation
1.4.2 Diagnostic evaluation 12
1.5 Treatment 12
1.5.1 Prostacyclin analogues 13
1.5.2 Inhaled nitric oxide 14
1.5.3 Endothelin antagonists
1.5.4 Phosphodiesterase inhibitors 15
1.6 Nitric Oxide 15
1.6.1 Nitric oxide synthase 16 ii

Chapters Page(s)

1.6.2 Regulation of NO 19
1.6.3 NO and pulmonary hypertension 25
2. Aims of the study 27
3. Materials and Methods 29
3.1. Materials 29
3.2 Methods 33
3.2.1 Patient characteristics and measurements 33
3.2.2 Animal experiments 33
3.2.3 MCT treatment 34
3.2.4 Chronic dosing study
3.2.5 Surgical preparation and tissue preparation 34
3.2.6 RNA isolation 35
3.2.7 cDNA Synthesis 36
3.2.8 Polymerase chain reaction 37
3.2.9 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) 37
3.2.10 Agarose gel electrophoresis and PCR product purification 39
3.2.11 Western blotting 40
3.2.12 Dot Blotting 41
3.2.13 Immunohistochemical staining
3.2.14 Histological analysis 42
3.2.15 Measurement of ADMA and SDMA
3.2.16 DDAH activity assay 43 iii


Chapters Page(s)

3.2.17 NOx measurements 43
3.2.18 Culture of human HUVEC cells 44
3.2.19 Data analysis
4. Results 45
4.1 IPAH patients 45
4.1.2 IPAH patient characteristics 45
4.1.2 ADMA and SDMA elaboration in IPAH patients 45
4.1.3 Localization of dimethylarginines in lungs from patients with IPAH 46
4.1.4 Increased biosynthesis of dimethylarginines in lungs from patients with
IPAH 47
4.1.5 Localization of DDAH isoforms in lungs from patients with IPAH 48
4.1.6 Decreased metabolism of dimethylarginines in lungs from patients with
IPAH 50
4.1.7 Protein expression of DDAH isoforms 50
4.2 Monocrotaline (MCT) treated rats 52
4.2.1 Hemodynamics and right heart hypertrophy 52
4.2.3 Increased plasma levels of ADMA and SDMA in MCT-PAH rats 53
4.2.3 Localization of dimethylated arginine proteins in MCT-PAH rat lungs 54
4.2.4 Augmentation of dimethylated arginine proteins in MCT-PAH rat lungs 55
4.2.5 Localization of DDAH isoforms in MCT rat lungs 56
4.2.6 MCT inhibits expression of DDAH isoforms at mRNA level 57
4.2.7 MCT inhibits expr DDAH isoforms at protein and activity level 58
4.2.8 TNF- α and IFN-γ mediates DDAH dysregulation 60
4.3 Chronic effects of aerosolized tolafentrine in MCT treated rats 60 iv

Chapters Page(s)

4.3 1 Acute vasodilatory effects 60

4.3.2 Hemodynamics 61
4.3.3 Right ventricular hypertrophy 63

4.3.4 Histopathology 64
4.3.5 Effect on methylarginine production 65
4.3.6 Effect of tolafentrine on DDAH expression 66
4.3.7 Effect rine on DDAH Activity 68
4.3.8 Effect of tolafentrine on NO synthesis
5 Discussion 70
5.1 Increased levels and reduced catabolism of asymmetric and
symmetric dimethylarginines in pulmonary hypertension 70
5.2 Tolafentrine increases nitric oxide synthesis in MCT induced
pulmonary hypertension: a role for the induction of DDAH2 75
6. Summary 79
7. Zusammenfassung 81
8.References 83
9. Erklärung 103
10 Acknowledgments 104
11. Curriculum Vitae 106
12. Publications 109