The interleukin 13 receptor system [Elektronische Ressource] : a novel pathomechanism involved in pulmonary arterial hypertension / by Hecker, Matthias
98 Pages
English
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The interleukin 13 receptor system [Elektronische Ressource] : a novel pathomechanism involved in pulmonary arterial hypertension / by Hecker, Matthias

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98 Pages
English

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The Interleukin 13 Receptor System: A Novel Pathomechanism Involved in 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 Hecker, Matthias of Kreuztal Germany Giessen 2008 From the Department of Medicine Director: Prof. Dr. Werner Seeger of the Faculty of Medicine of the Justus Liebig University Giessen First Supervisor and Committee Member: Prof. Dr. Oliver Eickelberg Second Supervisor and Committee Member: Prof. Dr. Susetta Finotto Committee Members: Prof. Dr. Heinz-Jürgen Thiel Priv.-Doz. Dr. Sandip Kanse Date of Doctoral Defence: 11.05.2009 I declare that I have completed this dissertation single-handedly without the unauthorized help of a second party and only with the assistance acknowledged therein. I have appropriately acknowledged and referenced all text passages that are derived literally from or are based on the content of published of unpublished work of others, and all information that related to verbal communications.

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Published 01 January 2008
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The Interleukin 13 Receptor System:
A Novel Pathomechanism Involved in 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
Hecker, Matthias

of
Kreuztal
Germany


Giessen 2008




From the Department of Medicine
Director: Prof. Dr. Werner Seeger
of the Faculty of Medicine of the Justus Liebig University Giessen





















First Supervisor and Committee Member: Prof. Dr. Oliver Eickelberg
Second Supervisor and Committee Member: Prof. Dr. Susetta Finotto
Committee Members: Prof. Dr. Heinz-Jürgen Thiel
Priv.-Doz. Dr. Sandip Kanse

Date of Doctoral Defence: 11.05.2009




I declare that I have completed this dissertation single-handedly without the
unauthorized help of a second party and only with the assistance
acknowledged therein. I have appropriately acknowledged and referenced all
text passages that are derived literally from or are based on the content of
published of unpublished work of others, and all information that related to
verbal communications. I have abided by the principles of good scientific
conduct laid down in the charter of the Justus Liebig University of Giessen
in carrying of the investigations described in the dissertation.


























I. Table of contents

I. Table of contents 4
II. List of figures and tables 8
III. List of abbreviations 10

1 Introduction
1.1 Pulmonary arterial hypertension 12
1.1.1 Characteristics of pulmonary arterial hypertension 12
1.1.2 Histopathological changes 12
1.1.3 Pathogenesis of pulmonary hypertension and therapy 14
1.1.3.1 Prostacyclin/prostaglandin I2 15
1.1.3.2 Endothelins 16
1.1.3.3 Nitric oxide 17
+1.1.3.4 K channels 18
1.1.3.5 Serotonin (5-Hydroxytryptamine) 19
1.1.3.6 Natriuretic peptides 19
1.1.3.7 BMPR2 and Alk/endoglin mutations 19
1.2 Interleukin 13 and its receptors 21
1.2.1 T helper cell type 1 and 2 immune response 21
1.2.2 Interleukin-13 21
1.2.2.1 Biological activities of IL-13 22
1.2.3 IL-13 Receptor complexes 24
1.2.4 Pathobiological relevance of IL-13 and its receptors 26
1.2.4.1 Resistance to gastrointestinal nematodes 26
1.2.4.2 Allergic asthma and airway hyperresponsiveness 27
1.2.4.3 Tissue remodeling and fibrosis 28
1.3 Aims of the study 30

2 Materials and Methods
2.1 Materials 31
2.1.1 Equipment 31
2.1.2 Chemicals and reagents 31
2.1.3 Antibodies 33 2.2 Methods 34
2.2.1 Polymerase chain reaction 34
2.2.1.1 Quantitative RT-PCR 34
2.2.1.2 Reverse-transcription PCR 35
2.2.2 RNA isolation 35
2.2.3 Cloning of PCR products 36
2.2.3.1 PCR product purification 36
2.2.3.2 Ligation of PCR products into pGEM-T Easy 36
2.2.3.3 Transformation and propagation of plasmids 36
2.2.3.4 Subcloning in expression vectors 37
2.2.4 Western blot 37
2.2.4.1 Cell lysis and protein extraction 37
2.2.4.2 SDS-polyacrylamid gel electrophoresis 38
2.2.4.3 Protein blotting and detection 38
2.2.5 Proliferations assay 39
2.2.6 Apoptosis assay 40
2.2.7 Flow cytometric cell cycle analysis 40
2.2.8 Flow cytometry 40
2.2.9 Immunofluorescence 40
2.2.10 Immunohistochemistry 41
2.2.11 Laser-captured microdissection 41
2.2.12 Agarose gel electrophoresis 42
2.2.13 Cell culture of pulmonary artery smooth muscle cells 42
2.2.13.1 Isolation of pulmonary artery smooth muscle cells 42
2.2.13.2 Culture of pulmonary artery smooth muscle cells 42
2.2.13.3 Cell culture under hypoxic conditions 43
2.2.14 Enzyme-linked immunosorbant assay 43
2.2.15 Transfection of paSMC 44
2.2.16 Microarray experiments 44
2.2.17 Animal models of PAH 45
2.2.17.1 The monocrotaline rat model of PH 45
2.2.17.2 The model of hypoxia-induced PH 45

3 Results
3.1 IL-13 receptor gene expression 47
3.2 IL-13 receptor gene expression in IPAH 48
3.3 IL-13 receptor localization in IPAH patients 50
3.4 IL-13 receptor expression in experimental PH 52
3.5 Effect of IL-13 on paSMC growth and apoptosis 54
3.6 IL-13 serum levels in IPAH 58
3.7 IL-13-induced signaling in paSMC 58
3.8 Effect of IL-13Rα2 overexpression on paSMC 59
3.9 Analysis of IL-13-induced genes by DNA microarray 61
3.9.1 IL-13-regulated genes after 2 h of stimulation 63
3.9.2 IL-13-regulated genes after 6 h of stimulation 63
3.9.3 Classification of genes according to biological processes 64
3.10 IL-13 induces down-regulation of endothelin-1 65

4 Discussion
4.1 IL-13Rα2 68
4.2 IL-13Rα2: Decoy or signaling receptor? 69
4.3 Role of IL-13Rα2 in fibrotic disease 70
4.3.1 Pulmonary granuloma formation 70
4.3.2 Liver fibrosis in schistosomiasis 71
4.3.3 Current model of involvement of Th1/2 response and
IL-13Rα2 in tissue remodeling 73
4.4 Role of IL-13 and IL-13Rα2 in IPAH 75
4.5 Outlook and future directions 76

5 Summary 78

6 Zusammenfassung 79

7 References 80

8 Appendix 88
9 Curriculum vitae 97

10 Acknowledgements 100

II. List of figures and tables

Figure 1.1 Histopathological changes in PAH
Figure 1.2 Histopathological changes in PAH II
Figure 1.3 Regulation of pulmonary vascular tone and structure by cAMP
Figure 1.4 Schematic overview of the different endothelins, endothelin
receptors and their respective biological effects
Figure 1.5 Role of Kv channels in the regulation of pulmonary vascular tone
Figure 1.6 The polarization of Th0 cells into Th1 and Th2 response
Figure 1.7 Schematic representation of some major activities of IL-13 on
allergic and inflammatory processes
Figure 1.8 Schematic overview of IL-4 and IL-13 receptor complexes
Figure 1.9 Proposed helminth model
Figure 1.10 Proposed asthma model
Figure 1.11 Opposing roles for Th1 andTh2 cytokines in fibrosis
Figure 1.12 IL-13 promotes collagen production by three mechanisms

Figure 3.1 Gene expression of IL-13R isotypes in multiple tissues
Figure 3.2 Relative expression patterns of IL-13R isotypes in the lung
Figure 3.3 Localization of IL-13Rα2 in the lung
Figure 3.4 Analysis of IL-13 receptor isotype expression in PAH
Figure 3.5 Quantitative analysis of IL-13R expression in IPAH
Figure 3.6 In vivo expression of IL-13Rα2 analyzed by LCM
Figure 3.7 Quantitative analysis of IL-13Rα2 in microdissected arteries
Figure 3.8 Immunohistochemical localization of IL-13 receptors
Figure 3.9 IL-13Rα2 and IL-13 expression in IPAH lesions
Figure 3.10 IL-13R expresion in hypoxia-induced pulmonary hypertension
Figure 3.11 Quantitative analysis of IL-13R expression in hypoxia-induced
pulmonary hypertension
Figure 3.12 IL-13R expression in monocrotaline-induced pulmonary
hypertension
Figure 3.13 IL-13Rα2 expression in paSMC exposed to hypoxia
Figure 3.14 Effect of IL-13 on paSMC proliferation I
Figure 3.15 Effect of IL-13 on paSMC proliferation II
Figure 3.16 Effect of IL-4 on paSMC proliferation
Figure 3.17 Effect of IL-13 on apoptosis
Figure 3.18 Effect of IL-13 on paSMC cell-cycle progression Figure 3.19 Effect of IL-13 on STAT phosphorylation in paSMC
Figure 3.20 Effect of IL-13 on STAT phosphorylation and translocation in
paSMC
Figure 3.21 Analysis of transfection efficiency on GFP-transfected paSMC
Figure 3.22 Effect of IL-13Rα2 overexpression on paSMC proliferation
Figure 3.23 Effect of IL-13Rα2 overexpression on paSMC signaling
Figure 3.24 Overview of IL-13 regulated genes
Figure 3.25 Heat map analysis of IL-13 regulated genes
Figure 3.26 Cluster analysis of IL-13 regulated biological processes
Figure 3.27 Cluster analysis of IL-13 regulated signaling pathways
Figure 3.28 IL-13 induced downregulation of endothelin-1 mRNA expression
Figure 3.29 IL-13 induced downregulation of endothelin-1 protein levels

Figure 4.1 Involvement of Th1/Th2 responses and IL-13α2 in tissue fibrosis
Figure 4.2 Proposed model of IL-13 involvement in the pathogenesis of PAH



Table 3.1 Hypoxic parameters from mice subjected to chronic hypoxia
Table 3.2 Most regulated genes 2 h after IL-13 stimulation
Table 3.3 Most regulated genes 6 h after IL-13 stimulation















III. List of abbreviations

5-HT 5-Hydroxytryptamine
ANP Atrial natriuretic peptide
AP-1 Activator protein 1
APS Ammonium persulfate
BMPR Bone morphogenic protein receptor
BNP Brain natriuretic peptide
BSA Bovine serum albumin
cAMP Cyclic andenosine monophosphate
cDNA Complementary deoxyribonucleic acid
CD Cluster of differentiation
DAB Diaminobenzidine
DAPI 4,6-diamidino-2-phenylindole
DNA Deoxyribonucleic acid
dpm disintegrations per minute
ECE Endothelin-converting enzyme
ECM Extracellular matrix
EDTA Ethylenedinitrilo-N,N,N´,N´,-tetra acetate
ELISA Enzyme linked immunosorbent assay
EMSA Electrophoretic mobility shift assay
ET Endothelin
FCS Fetal calf serum
FITC Fluorescein isothiocyanate
GFP Green fluorescent protein
HRP Horseradish peroxidase
IFN Interferon
Ig Immunoglobulin
IL Interleukin
IPF Idiopathic pulmonary fibrosis
IPAH Idiopathic pulmonary arterial hypertension
LB Luria Bertani
LCM Laser-captured microdissection
LPS Lipopolysaccharide
MAP Mitogen-activated protein
MCT Monocrotaline
MCTP Monocrotaline pyrole