Regulation and pathomechanistic role of matrix metalloproteinases in idiopathic pulmonary fibrosis [Elektronische Ressource] / by Eusebius Henry Nkyimbeng Takwi

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Informations

• Publié par: justus-liebig-universitat_giessen
• Publié le: 01 janvier 2009
• Nombre de lectures: 21
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

Regulation and pathomechanistic role of matrix metalloproteinases in
Idiopathic Pulmonary Fibrosis.




Dissertation submitted in partial fulfilment of the degree of Doctor of Philosophy of
Science in the Faculties of Veterinary Medicine and Medicine of the
Justus-Liebig University Giessen.



by

Eusebius Henry Nkyimbeng Takwi,
from Mankon, Cameroon.

Department of Internal Medicine II
Universitätklinikum Giessen und Marburg GmbH

Giessen, 2008 Aus dem Medizinischen Zentrum für Innere Medizin
Medizinische Klinik und Poliklinik II
Direktor: Prof. Dr. Med. Werner Seeger
des Fachbereichs Medizin der Justus-Liebig-Universität Giessen






Dekan: Prof. Dr. Dr. Georg Baljer (Faculty of Veterinary Medicine)
Dekan: Prof. Dr. Wolfgang Weidner (Faculty of Medicine)

1. Gutachter: Prof. Dr. Andreas Günther
2. Gutachter: Prof. Dr. Jeanine D’Armiento

Prüfungsvorsitzender: Prof. Dr. Martin Diener
Beisitzer: Priv.-Doz. Dr. Konstantin Mayer

Tag der Disputation: 28th August, 2008
Regulation and pathomechanistic role of matrix metalloproteinases in idiopathic pulmonary fibrosis 1 Index of contents

Index of contents............................................................................................................…......2

Index of figures..............................….......5
Index of tables…………………………………………………………………………………..........6

1 INTRODUCTION...................................................................................................….......7

1.1 Physiology of the lung………………………………………………………………..........7
1.1.1 Functional anatomy of the lung ……………………………………….….............7

1.2 Interstitial lung disease……………………………………………………………...........9
1.2.1 Classification of interstitiall lung disease (ILD)……………..............................11
1.2.2 Prevalence of interstitial lung diseases.......................................13
1.3 Idiopathic pulmonary fibrosis (IPF)……………………………………………….........13
1.3.1 Clinical and histological features of IPF…………………………………...........14
1.3.2 Diagnosis of IPF.............................................................................................15
1.3.3 The initial trigger of IPF................................….......16
1.3.4 Mediators of distorted epithelial-mesencymal interactions in
lung fibrosis…………………………………………………………………..........18
1.3.5 Origin of the activated (myo)-fibroblast in IPF................................................19
1.3.6 Major signaling pathways underlying matrix remodeling in the lung…...........20
1.3.7 Influence of genetic background in IPF pathogenesis..........................…......23
1.3.7.1 Surfactant protein-C (SP-C) mutations………....................................23
1.3.7.2 Telomerase mutation……………………………………........................24
1.3.8 Treatment of lung fibrois-translational approaches……………………….....25

1.4 Animal models of pulmonary fibrosis……………………………………………......…26

1.5 Matrix metalloproteinases (MMPs)………………………………………....................28
1.5.1 Structure and function of matrix metalloproteinases…………………...........32
1.5.1.1 Propeptide domain……………………………………………………......34
1.5.1.2 Catalytic domain………………………………………………………......35
1.5.1.3 Hinge region, hemopexin domain and other domains……………......36
.
1.6 MMP gene arrangement..........................................................................................36

1.7 Activation of matrix metalloproteinases...........................37
1.7.1 Regulation of MMP activity ………………………………………………........38

1.8 Genetic knockout of matrix metalloproteinases…………………………………........39

1.9 Tissue inhibitors of metalloproteinases (TIMPs.......................................................40
1.9.1 Structure and biological functions of TIMPs………………………….............41

1.10 Hypothesis……………………………………………………………………………......44
1.10.1 Aims of the study …………………………………………………………….....44



Regulation and pathomechanistic role of matrix metalloproteinases in idiopathic pulmonary fibrosis 2 Index of contents
2 MATERIALS …………………………………………………………………………….........45

2.1 Chemicals......................................................................................................…......45

2.2 Injecting solutions and substances...........................…...47

2.3 Consumables……………………………………………………………………….....…48

2.4 Histology...........................................................................................................…...50

2.5 Antibodies…………………………………………………………………………….......52

2.6 Machines, systems and software………………………………………………….......54

3 METHODS……………………………………………………………………………….…...56

3.1 Human lung…………………………………………………………………………….....56
3.1.1 Study population and specimen collection……………………………….......56

3.2 Animals……………………………………………………………………………….......58

3.3 Induction of pulmonary fibrosis in mice by bleomycin treatment……………….......59

3.4 Quasi-static lung compliance…………………………………………………….....….60

3.5 Bronchoalveolar lavage....................................................................................…...61

3.6 Processing of human and murine lungs…………………………………………........61

3.7 Gelatin zymography in murine BALF samples…………………………………….....62

3.8 Macrophage chemotaxis of BALF samples............................................................63

3.9 Analysis of collagenase activity in human lung homogenates…………………......63

3.10 Hydroxyproline determination in human lungs…………………………………....….64

3.11 ination in murine lungs………………………………….…....66

3.12 Quantitative real- time reverse transcription polymerase chain reaction
(RT-PCR) analysis………………………………………………………………….......67

3.13 Western blot analysis of MMPs and TIMPs in human lung tissue……………....…70

3.14 Histopathology and immunohistochemistry………………………………………......72

3.15 Immunohistochemistry combined with in situ zymography………………………....73

3.16 Data analysis…………………………………………………………………………......75


Regulation and pathomechanistic role of matrix metalloproteinases in idiopathic pulmonary fibrosis 3 Index of contents


4 RESULTS…..................................................................................................................76

4.1 IPF patient lungs manifest the typical UIP histological pattern
with marked collagen deposition in the matrix……………………………………......76
4.1.1 Histological phenotype of patient lungs…………………………………........76
4.1.2 Hydroxyproline levels are increased in IPF lungs……………………….......78

4.2 Expression of MMPs and TIMPs in IPF and control lungs……………………….....79
4.2.1 mRNA expression of collagenases and matrilysin are upregulated in
IPF lungs compared to control lungs………………………………................79
4.2.2 Increased protein content of MMP-1,-2,-7,-9,-13 in IPF lungs
compared to controls……………………………………………………….......81
4.2.3 Increased collagenase and gelatinase activities in IPF versus
control lungs…………………………………………………………………......83
4.2.4 Spatial distribution of MMP antigen and collagenolytic
and gelatinolytic activity in IPF and controls……………………………........85

4.3 Role of MMP-13 in the pathogenesis of fibrotic lung disease……………………....87
-/- 4.3.1 MMP-13 mice develop exaggerated inflammation following
bleomycin challenge……………………………………………………............88
-/- 4.3.2 Regulation of chemotactic activity in macrophages from MMP-13 mice...90
-/- 4.3.3 MMP-13 mice show more extensive lung fibrosis in response
to bleomycin administration…………………………………………………....92
-/- 4.3.4 Expression of MMPs and TIMPs in MMP-13 and WT mice……………....95


5 DISCUSSION………………………………………………………………………………...99
5.1 Role of MMPs and TIMPs in pulmonary fibrosis…………………………….............99

5.2 Regulation of MMP and TIMP expression and activity in human lungs……….....102

5.3 Upregulation of MMP-13 protein in human IPF lungs………………………….......103

5.4 Role of MMP-13 in pulmonary fibrosis………………………………………............104


6 SUMMARY………………………………………………………………………………......108

7 ZUSAMMENFASSUNG………………………………………………………………........111

8 ABBREVIATION………………………………………………………………………….....115

9 REFERENCE LIST………………………………………………………………………....119

10 ERKLÄRUNG…………………………………………………………………………….....146

11