Role of intrinsic coagulation pathway in the pathogenesis of idiopathic pulmonary fibrosis [Elektronische Ressource] / vorgelegt von Jabłońska, Ewa Danuta
109 Pages
English
Downloading requires you to have access to the YouScribe library
Learn all about the services we offer

Role of intrinsic coagulation pathway in the pathogenesis of idiopathic pulmonary fibrosis [Elektronische Ressource] / vorgelegt von Jabłońska, Ewa Danuta

-

Downloading requires you to have access to the YouScribe library
Learn all about the services we offer
109 Pages
English

Description

EWA JABLONSKARole of Intrinsic Coagulation Pathway in thePathogenesis of Idiopathic Pulmonary Fibrosis édition scientifiqueVVB LAUFERSWEILER VERLAG INAUGURALDISSERTATION zur Erlangung des Grades eines Doktors der Humanbiologie VVB LAUFERSWEILER VERLAG des Fachbereichs Medizin der Justus-Liebig-Universität Gießen STAUFENBERGRING 15 ISBN: 978-3-8359-5693-3D-35396 GIESSEN Tel: 0641-5599888 Fax: -5599890redaktion@doktorverlag.dewww.doktorverlag.de 9 7 8 3 8 3 5 9 5 6 9 3 3édition scientifiqueVVB LAUFERSWEILER VERLAGVVB© Sebastian Kaulitzki - Fotolia.comEWA JABLONSKA ROLE OF FXII IN THE PATHOGENESIS OF IPF . Das Werk ist in allen seinen Teilen urheberrechtlich geschützt. Jede Verwertung ist ohne schriftliche Zustimmung des Autors oder des Verlages unzulässig. Das gilt insbesondere für Vervielfältigungen, Übersetzungen, Mikroverfilmungen und die Einspeicherung in und Verarbeitung durch elektronische Systeme.1. Auflage 2010All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the Author or the Publishers.st1 Edition 2010© 2010 by VVB LAUFERSWEILER VERLAG, GiessenPrinted in Germany édition scientifiqueVVB LAUFERSWEILER VERLAGSTAUFENBERGRING 15, D-35396 GIESSENTel: 0641-5599888 Fax: 0641-5599890 email: redaktion@doktorverlag.dewww.doktorverlag.

Subjects

Informations

Published by
Published 01 January 2011
Reads 34
Language English
Document size 6 MB

Exrait

EWA JABLONSKA
Role of Intrinsic Coagulation Pathway in the
Pathogenesis of Idiopathic Pulmonary Fibrosis
édition scientifique
VVB LAUFERSWEILER VERLAG INAUGURALDISSERTATION zur Erlangung des Grades eines Doktors der Humanbiologie
VVB LAUFERSWEILER VERLAG des Fachbereichs Medizin der Justus-Liebig-Universität Gießen
STAUFENBERGRING 15 ISBN: 978-3-8359-5693-3
D-35396 GIESSEN
Tel: 0641-5599888 Fax: -5599890
redaktion@doktorverlag.de
www.doktorverlag.de 9 7 8 3 8 3 5 9 5 6 9 3 3
édition scientifique
VVB LAUFERSWEILER VERLAGVVB
© Sebastian Kaulitzki - Fotolia.com
EWA JABLONSKA ROLE OF FXII IN THE PATHOGENESIS OF IPF
. Das Werk ist in allen seinen Teilen urheberrechtlich geschützt.
Jede Verwertung ist ohne schriftliche Zustimmung des Autors
oder des Verlages unzulässig. Das gilt insbesondere für
Vervielfältigungen, Übersetzungen, Mikroverfilmungen
und die Einspeicherung in und Verarbeitung durch
elektronische Systeme.
1. Auflage 2010
All rights reserved. No part of this publication may be
reproduced, stored in a retrieval system, or transmitted,
in any form or by any means, electronic, mechanical,
photocopying, recording, or otherwise, without the prior
written permission of the Author or the Publishers.
st1 Edition 2010
© 2010 by VVB LAUFERSWEILER VERLAG, Giessen
Printed in Germany
édition scientifique
VVB LAUFERSWEILER VERLAG
STAUFENBERGRING 15, D-35396 GIESSEN
Tel: 0641-5599888 Fax: 0641-5599890
email: redaktion@doktorverlag.de
www.doktorverlag.de


Role of Intrinsic Coagulation Pathway in the Pathogenesis of Idiopathic
Pulmonary Fibrosis






Inauguraldissertation
zur Erlangung des Grades eines Doktors der Humanbiologie
des Fachbereichs Medizin
der Justus-Liebig-Universität Gießen




vorgelegt von
Jab łońska, Ewa Danuta
aus
Toru ń, Polen


Giessen 2010 Aus dem Institut für Biochemie
des Fachbereichs Medizin der Justus-Liebig-Universität Giessen
Leiter/Direktor: Prof. Dr. Klaus T. Preissner























Gutachter: Prof. Dr. K. T. Preissner rof. Dr. W. Kummer

Tag der Disputation: 29.11.10.Table of contents
________________________________________________________________________
I. Table of contents
I. Table of contents...............................................................................................................I
II. List of figures ............................................................................................................... IV
III. List of tables.... VI
IV. List of abbreviations ..................................................................................................VII
V. Summary ....................................................................................................................... X
VI. Zusammenfassung......................................................................................................XII
1. Introduction..................................................................................................................... 1
1.1. Blood Coagulation Pathways ................................................................................... 1
1.1.1. Structural and functional characteristics of the intrinsic coagulation pathway
factors.......................................................................................................................... 1
1.1.1.1. Factor XII ................................................................................................... 1
1.1.1.2. Factor XI .................................................................................................... 3
1.1.1.3. High molecular weight kininogen.............................................................. 4
1.1.1.4. Kallikrein 5
1.1.2. Activation of FXII............................................................................................. 6
1.1.2.1. Contact activation of FXII ......................................................................... 6
1.1.2.2. Activation of the contact system on the endothelial cell surface ............... 7
1.1.2.3. Inhibition of FXII activity.......................................................................... 7
1.1.2.3.1. Inhibition of FXII activity in vitro ...................................................... 7
1.1.2.3.2. Inhibition of FXII activity in vivo ....................................................... 8
1.1.3. Physiologic activities of FXII ........................................................................... 9
1.1.3.1. FXII and inflammatory reactions ............................................................... 9
1.1.3.1.1. FXII in hereditary angioedema ........................................................... 9
1.1.3.1.2. FXII in sepsis ...................................................................................... 9
1.1.3.2. Factor XII in thrombosis .......................................................................... 10
1.1.3.3. Factor XII in fibrinolysis 11
1.1.3.4. Mitogenic activities of FXII..................................................................... 11
1.1.4. Characterization of FXII promoter.................................................................. 12
1.2. Idiopathic pulmonary fibrosis ................................................................................ 12
1.2.1. Role of Transforming Growth Factor-  in the pathogenesis of lung fibrosis..13
ITable of contents
________________________________________________________________________
1.2.2. Bleomycin model of lung fibrosis................................................................... 14
1.2.3. Role of coagulation in the pathogenesis of idiopathic pulmonary fibrosis..... 15
2. Aim of the study............................................................................................................ 17
3. Materials and methods .................................................................................................. 18
3.1. Materials................................................................................................................. 18
3.1.1. Equipment ....................................................................................................... 18
3.1.2. Reagents .......................................................................................................... 19
3.2. Methods.................................................................................................................. 21
3.2.1. Intratracheal bleomycin administration........................................................... 21
3.2.2. Pulmonary compliance measurements............................................................ 21
3.2.3. Lung preparation ............................................................................................. 22
3.2.4. Isolation of murine and human lung fibroblasts and cell culture.................... 22
3.2.5. Microdissection of lung tissue and alveolar epithelial type II cells ................ 23
3.2.6. RNA isolation and reverse transcriptase reaction ........................................... 23
3.2.7. Real Time PCR ............................................................................................... 24
3.2.8. Protein isolation and quantification ................................................................ 25
3.2.9. SDS polyacrylamide gel electrophoresis ........................................................ 25
3.2.10. Immunoblotting............................................................................................. 25
3.2.11. Immunocytochemistry................................................................................... 26
3.2.12. Immunohistochemistry.................................................................................. 27
3.2.13. Proliferation assay......................................................................................... 27
3.2.14. Immunoprecipitation..................................................................................... 28
3.2.15. Generation of FXII promoter constructs and site-directed mutagenesis....... 28
3.2.16. Transient transfection and luciferase assay................................................... 29
3.2.17. Antisense Oligonucleotides........................................................................... 29
3.2.18. Chromatin immunoprecipitation ................................................................... 29
3.2.19. Streptavidin pull-down assay ........................................................................ 30
3.2.20. Statistics ........................................................................................................ 31
4. Results........................................................................................................................... 32
4.1. Expression of FXII, FXI and HMWK is altered in idiopathic pulmonary fibrosis
lungs .............................................................................................................................. 32
4.2. Expression of FXII, FXI and HMWK is elevated in bleomycin lungs.................. 34
IITable of contents
________________________________________________________________________
4.3. FXII knockout or FXIIa inhibition protects against bleomycin-induced lung
fibrosis........................................................................................................................... 37
4.4. Bradykinin receptor 1/2 knockout mice are not protected against bleomycin-
induced lung fibrosis..................................................................................................... 42
4.5. FXII stimulates proliferation of lung fibroblasts ................................................... 43
4.6. TGF- 1 regulates FXII expression in human lung fibroblasts .............................. 48
4.6.1. TGF- 1 upregulates FXII mRNA and protein levels in HLF......................... 48
4.6.2. TGF- 1 induces phosphorylation of MAPK, Akt and Smad3........................ 49
4.6.3. Smad 3 and JNK kinase regulate TGF- 1-induced FXII expression in HLF. 51
4.6.4. JNK kinase does not regulate Smad3 phosphorylation and translocation to the
nucleus....................................................................................................................... 51
4.6.5. TGF- β1 induces FXII promoter activity via SBE located at position – 272... 54
4.6.6. Smad 3 interacts with SBE within the FXII promoter ............................... 55 -272
4.6.7. JNK kinase affects binding of Smad 3 to SBE ........................................... 56 -272
5. Discussion ..................................................................................................................... 58
5.1. Expression of FXII, FXI and HMWK is elevated in lung fibrosis ........................ 58
5.2. Inhibition of FXIIa or knockout of FXII protects against lung fibrosis................. 60
5.3. FXII-induced proliferation of murine lung fibroblasts may contribute to lung
fibrosis development ..................................................................................................... 62
5.4. Regulation of FXII expression in human lung fibroblasts ..................................... 65
6. Conclusions ................................................................................................................... 70
7. References ..................................................................................................................... 72
8. Curriculum vitae............................................................................................................ 87
9. Declaration .................................................................................................................... 89
10. Acknowledgements ..................................................................................................... 90
IIIList of figures
________________________________________________________________________
II. List of figures

Figure 1.1. Blood coagulation pathways.
Figure 1.2. Structure of intrinsic coagulation pathway protease zymogens.
Figure 1.3. The KLK/kinin system.
Figure 4.1. mRNA level of FXII, FXI and HMWK is elevated in the lungs of IPF
patients.
Figure 4.2. Protein level of FXII, FXI and HMWK and FXII activity are elevated in
the lungs of IPF patients.
Figure 4.3. Expression and localization of FXII, FXI and HMWK in lung tissue of
donor and IPF patients.
Figure 4.4. mRNA level of FXII, FXI and HMWK is elevated in the lungs of
bleomycin-treated mice.
Figure 4.5. Increased protein level of FXII, FXI and HMWK in lung homogenates of
control and bleomycin challenged mice.
Figure 4.6. Expression and localization of FXII, FXI and HMWK in the lungs of
control and bleomycin-treated mice.
-/-Figure 4.7. FXII mice are protected against bleomycin-induced lung fibrosis.
-/-Figure 4.8. Fibrin deposition in the lungs of FXII mice is not impaired after
bleomycin application.
Figure 4.9. FXIIa inhibitor (CTI) attenuates bleomycin-induced lung fibrosis.
Figure 4.10. PCK administration does not improve bleomycin-induced lung fibrosis.
-/- Figure 4.11. B1B2 mice are not protected against bleomycin-induced lung fibrosis.
Figure 4.12. FXIIa stimulates proliferation of murine lung fibroblasts.
Figure 4.13. p44/42 kinase regulates FXII-induced proliferation of murine lung
fibroblasts.
Figure 4.14. uPAR mediates FXIIa-induced murine lung fibroblast proliferation.
Figure 4.15. uPAR is required for FXIIa mitogenic activities.
Figure 4.16. α5β1-integrin regulates FXIIa mediated murine lung fibroblast
proliferation.
Figure 4.17. TGF- 1 upregulates FXII expression in HLF.
Figure 4.18. TGF- 1 induces phosphorylation of MAPK, Akt and Smad3.
IVList of figures
________________________________________________________________________
Figure 4.19. Smad 3 and JNK kinase regulate TGF- 1-induced FXII expression in
HLF.
Figure 4.20. JNK1 kinase does not regulate Smad3 phosphorylation and translocation
to the nucleus.
Figure 4.21. TGF- β1 induces FXII promoter activity via SBE located at position
– 272.
Figure 4.22. TGF- βoter activity
– 272.
Figure 4.23. Smad 3 - SBE interaction is suppressed in the presence of JNK -272
inhibitor.
Figure 5.1. Factor XIIa may contribute to increased proliferation of fibroblasts in
lung fibrosis.
V