TGF-β/BMP [TGF-beta-BMP] system in experimental and idiopathic pulmonary hypertension [Elektronische Ressource] / vorgelegt von Oana Veronica Amarie

Amarie, Oana Veronica

Sujets

• Gesundheit

Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2009
• Nombre de lectures: 51
• Poids de l'ouvrage: 5 Mo

Extrait

TGF-β/BMP system in experimental and
idiopathic pulmonary hypertension





Inaugural-Dissertation
zur Erlangung des Grades eines
Doktors der Humanbiologie
des Fachbereichs Medizin der


Justus-Liebig-Universität Gießen


vorgelegt von

Oana Veronica Amarie
aus Vaslui, Rumänien






Gießen, 2009
1 Aus dem Zentrum für Innere Medizin
Medizinische Klinik II und Poliklinik
Director: Prof. Dr. W. Seeger
Universitätsklinikum Gießen und Marburg GmbH, Standort Gießen













2 Gutachter: Prof. Dr. Ludger Fink
Gutachter: Prof. Dr. Irwin Reiss
Tag der Disputation: 10.12.2009
3 Table of contents
TABLE OF CONTENTS
TABLE OF CONTENTS.........................................................................................................I
LIST OF FIGURES .............................................................................................................. III
LIST OF TABLES ................................................................................................................. V
LIST OF ABREVIATIONS .................................................................................................VI
SUMMARY............................................................................................................................IX
ZUSAMMENFASSUNG ......................................................................................................XI

1 INTRODUCTION................................................................................................................1
1.1 GENERAL FEATURES OF THE PULMONARY CIRCULATION ............................1
1.2 PULMONARY HYPERTENSION.................................................................................3
1.2.1 Definition and Incidence..........................................................................................3
1.2.2 Classification ...........................................................................................................3
1.2.3 Pulmonary Arterial Hypertension (PAH) ................................................................6
1.2.4 Pathology of Pulmonary Hypertension....................................................................9
1.3 ANIMAL MODELS OF PULMONARY HYPERTENSION/VASCULAR
REMODELING...................................................................................................................11
1.3.1 Monocrotaline-induced pulmonary hypertension..................................................11
1.3.2 Hypoxia-induced pulmonary hypertension/vascular remodelling.........................12
1.3.3 Transgenic mice.....................................................................................................13
1.4 TRANSFORMING GROWTH FACTOR-BETA (TGF-β) SIGNALING ...................14
1.4.1 The TGF-β family..................................................................................................15
1.4.2 The BMP family ....................................................................................................17
1.5 PULMONARY VASCULAR REMODELING............................................................20
1.5.1 Chronic hypoxia and pulmonary vascular remodeling..........................................21
1.5.2 Transforming growth factor-beta family and pulmonary vascular remodeling.....23

2 AIM OF THE STUDY .......................................................................................................29

3 MATERIALS AND METHODS.......................................................................................30
3.1 MATERIALS ................................................................................................................30
3.1.1 Equipment..............................................................................................................30
3.1.2 Reagents.................................................................................................................31
3.2 METHODS....................................................................................................................33
3.2.1Cell culture..............................................................................................................33
3.2.1.1 Cell lines .......................................................................................................33
3.2.2 Tissue prelevation..................................................................................................33
3.2.2.1 Hypoxia-induced Pulmonary Hypertension Mouse Model ..........................33
3.2.2.2 Human tissue.................................................................................................34
3.2.3 RNA isolation, cDNA synthesis and PCR.............................................................34
3.2.3.1 RNA isolation from cells and tissue .............................................................34
3.2.3.2 RNA isolation from picked cells ..................................................................34
3.2.3.3 Reverse Transcription Reaction (RT-PCR) ..................................................35
3.2.3.4 The Polymerase Chain Reaction (PCR)........................................................36
3.2.3.4.1 Semi-quantitative PCR .....................................................................36
3.2.3.4.2 Real-time polymerase chain reaction................................................38
3.2.3.5 Agarose gel electrophoresis..........................................................................39
3.2.4 Protein isolation .....................................................................................................40
3.2.4.1 Protein isolation from tissue .........................................................................40
I Table of contents
3.2.4.2 Protein isolation from cells...........................................................................40
3.2.4.3 Protein quantification....................................................................................40
3.2.4.4 Separation of proteins by SDS......................................................................41
3.2.4.5 Western blotting............................................................................................42
3.2.4.6 Densitometry.................................................................................................42
3.2.5 Immunohistochemistry ..........................................................................................42
3.2.6 Immunocytochemistry ...........................................................................................43
3.2.7 Laser-microdissection............................................................................................44

4 RESULTS............................................................................................................................46
4.1 Chronic hypoxia-induced PH ........................................................................................46
4.2 Gene expression of TGF-β receptors and Smads during chronic hypoxia-induced PH 46
4.3 Protein expression of TGF-β receptors and Smads during chronic hypoxia-induced PH
.............................................................................................................................................48
4.4 Immunolocalization of TGF-β receptors and Smads in mouse lung tissue...................51
4.5 TGF-β receptors and Smads in lungs from iPAH patients compared to healthy donor
lungs.....................................................................................................................................52
4.6 ALK1 protein expression in iPAH versus donoir lung homogenates ...........................57
4.7 TGF-β receptor immunolocalization in iPAh patients versus donor lung homogenates58
4.8 ALK1 expression in different lung cell types................................................................61
4.9 ALK1 expression and activity in cultured human primary PASMC.............................62

5 DISCUSSION .....................................................................................................................65
5.1 The mouse model of hypoxia-induced PH ....................................................................66
5.2 TGFβ/BMP family components exhibit altered expression in hypoxia-induced PH ....67
5.3 TGFβ/BMP family component expression in iPAH patients ........................................68
5.4 ALK1 expression ..........................................................................................................70
5.4.1 ALK1 expression altered in hypoxia-induced PH and iPAH patients .................71
5.4.2 ALK1 expression and function on human primary smooth muscle cells ............72

6 REFERENCES ..................................................................................................................74

7 DECLARATION................................................................................................................90

8 CURRICULUM VITAE .................................