Molekulare und funktionelle Charakterisierung von biglykan während pathologischem kardialen Remodelling [Elektronische Ressource] / Ariane Melchior-Becker. Gutachter: Verena Jendrossek ; Bodo Levkau. Betreuer: Jens W. Fischer

Ariane Melchior-Becker

Sujets

• Biologie

Informations

• Publié par: universitat_duisburg-essen
• Publié le: 01 janvier 2011
• Nombre de lectures: 30
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Molecular and functional characterization of biglycan
during pathologic cardiac remodelling


Inaugural-Dissertation
zur Erlangung des Doktorgrades
Dr. rer. nat.

der Fakultät für Biologie und Geografie
an der

Universität Duisburg-Essen

vorgelegt von

Ariane Melchior-Becker

aus Lörrach





































Supervisor: Prof. Dr. rer. nat. Jens W. Fischer
Review 1: Prof. Dr. rer. nat. Verena Jendrossek
Review 2: Prof. Dr. med. Bodo Levkau

Date of Disputation: 12.04.2011







Abbreviations 1

Abbreviations
Bgn biglycan
BMP bone morphogenic protein
BSA bovine serum albumine
CTGF connective tissue growth factor
Dcn decorin
ECM extracellular matrix
ERK extracellular signal regulated kinase
et al. et alii
FAK focal adhesion kinase
bFGF basal fibroblast growth factor
g gramm
GAG glycosaminoglycan
kDa kilodalton
kg kilogram
l litre
LDL Low density Lipoprotein
MAPK mitogen activated protein kinase
MI myocardial infarction
μl micro-litres
ml milli-litre
μM micro-molar
mM mili-molar
MMP matrix metalloproteinase
mRNA messenger RNA
NFκB nuclear factor kappa β
PARP poly [Adenosine-diphosphate]-polymerase
PBS phosphate buffered saline
PCR polymerase chain reaction
PDGF platelet derived growth factor
rpm rotations per minute
SMAD mothers against decapentaplegic homolog
SMURF E2 ubiquitin ligase SMURF2

Abbreviations 2

TAK1 transforming growth factor β associated kinase 1
TEMED N,N,N′,N′-Tetramethylethan-1,2-diamin
TGF-β transforming growth factor-β
TGF-β RI/II transforming growth factor-β receptor type I/II
TLR toll- like receptor
TBS tris buffered saline
WT wild-type

Directories 3

Directory
1 INTRODUCTION ................................................................................................. 9
1.1 Cardiac Remodelling ........................................................................................................... 9
1.1.1 Cardiac fibroblasts and their differentiation into myofibroblasts ............................... 9
1.2 Small leucine-rich repeat proteoglycans: biglycan ....................................................... 13
1.2.1 Biglycan interacts with growth factors, surface complexes, receptors and ECM
proteins ……………………………………………………………………………………………..14
1.2.2 TGF-β signalling and regulation ............................................................................. 15
1.3 Biglycan and its role in cardiac system .......................................................................... 18
1.4 Aim of Investigation .......................................................................................................... 20
2 MATERIAL AND METHODS .............................................................................22
2.1 Material and Substances................................................................................................... 22
2.1.1 Mice ........................................................................................................................ 22
2.1.2 Substances ............................................................................................................. 22
2.2 Methods .............................................................................................................................. 27
2.2.1 Mice ........................................................................................................................ 27
2.2.2 Isolation of primary murine cardiac fibroblasts ....................................................... 27
2.2.3 Cell growth and proliferation assays ...................................................................... 28
-/0
2.2.4 Proliferation on cell-derived ECM from WT- or bgn -fibroblasts ........................... 28
2.2.5 Immunocytochemistry............................................................................................. 29
2.2.6 Oil supported collagen Retraction Assay ............................................................... 29
2.2.7 Protein Analysis ...................................................................................................... 30
2.2.8 Protein concentration ............................................................................................. 30
2.2.9 Proteoglycan isolation from culture supernatants .................................................. 31
2.2.10 RNA-extraction and quantitative Real-Time RT-PCR ........................................ 31
2.2.11 Lentiviral overexpression of biglycan ................................................................. 32
2.2.12 Experimental myocardial infarction .................................................................... 32
2.2.13 Cryomicrotomy and immunohistochemistry ....................................................... 33
2.2.14 Statistical Analysis ............................................................................................. 34
3 RESULTS ...........................................................................................................35
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3.1 Morphology of bgn -fibroblasts revealed a distinct phenotype .................................. 35

Directories 4

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3.2 Bgn -fibroblasts showed a pro-proliferative phenotype .............................................. 37
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3.2.1 Proliferation on cell-derived ECM from either WT- or bgn -fibroblasts ................. 41
3.2.2 Overexpression of murine biglycan protein reverts pro-proliferative phenotype of
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bgn -fibroblasts .................................................................................................................. 43
3.3 Oil-supported collagen retraction (OSCR) assay revealed strong contractile
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phenotype of bgn -fibroblasts ..................................................................................................... 44
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3.4 Immunocytochemical analysis of bgn -fibroblasts phenotype ................................... 46
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3.5 Biochemical and molecular comparison of bgn -fibroblasts to WT-fibroblasts ....... 50
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3.5.1 ECM crosslinking and degrading enzymes are differentially regulated in bgn -
fibroblasts compared to WT-fibroblasts ............................................................................... 52
3.5.2 Induction of matrix receptors and cytoskeleton-membrane-anchoring complex in
-/0
bgn -fibroblasts .................................................................................................................. 53
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3.5.3 Characterization of TGF-β system in bgn -fibroblasts .......................................... 55
3.6 The neutralization of TGF-β rescues the pro-proliferative phenotype and
-/0
differentiation of bgn -fibroblasts ............................................................................................... 56
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3.7 TGF-β signalling in bgn -fibroblasts .............................................................................. 59
3.8 Translation of in vitro results to post infarct remodelling in vivo. ............................... 64
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3.8.1 Bgn -myofibroblasts fail to activate nNOS despite DAPC up-regulation in vivo
after experimental MI ........................................................................................................... 68
4 DISCUSSION .....................................................................................................73
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4.1 Morphological differences between WT and bgn -fibroblasts .................................... 74
4.2 Pro-proliferative phenotype of cardiac fibroblasts in the absence of biglycan .......... 74
4.3 Differentiation in the absence of biglycan ...................................................................... 75
4.4 Constitutive rate of apoptosis in the absence of biglycan is part of unleashed TGF-β
signalling ......................................................................................................................................... 83
4.5 Translation of bgn function in vitro into in vivo situation ............................................. 85
5 SUMMARY .........................................................................................................89
6 REFERENCES ...................................................................................................91

Directories 5

7 CIRICULUM VITAE ............................................................................................96
8 ORIGINAL PUBLICATIONS: .............................................................................98
9 ACKNOWLEDGEMENT ..................................................................................102
10 AFFIDAVIT ................