The role of Src-homology 2 domain containing tyrosine phosphatase 2 in growth factor dependent endothelial signalling and angiogenesis [Elektronische Ressource] / Hanna Karin Mannell

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Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2007
• Nombre de lectures: 16
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
Der Fakultät für Chemie und Pharmazie
Der Ludwig-Maximilians-Universität München



The role of Src-homology 2 domain containing
tyrosine phosphatase 2 in growth factor
dependent endothelial signalling and
angiogenesis





Hanna Karin Mannell
aus
Vallentuna, Schweden




2007 Erklärung

Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung
vom 29. Januar 1998 von Herrn Prof. Ulrich Pohl betreut und von Herrn Prof. Ernst
Wagner vor der Fakultät für Chemie und Pharmazie vertreten.



Ehrenwörtliche Versicherung

Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.


München, am …………………………




…………………………………………….
Unterschrift des Autors






Dissertation eingereicht am …09.03.2007………...
1. Gutachter …Prof. Ernst Wagner..
2. Gutachter …Prof. Ulrich Pohl……
Mündliche Prüfung am …24.05.2007………….
1


CONTENTS
2 1. INTRODUCTION 6
1.1. THE PROCESS OF ANGIOGENESIS .............................................................. 7
1.1.1. VEGF and angiogenesis ..................................................................................................... 11
1.1.2. FGF and angiogenesis........................................................................................................ 13
1.1.3. PDGF and angiogenesis 13
1.2. ANGIOGENIC PATHWAYS ............................................................................ 14
1.2.1. The PI3-Kinase pathway 14
1.2.2. The Raf-MEK-ERK Pathway............................................................................................... 19
1.3. THE SRC-HOMOLOGY 2 DOMAIN CONTAINING TYROSINE
PHOSPHATASE 2........................................................................................... 23
1.3.1. The biochemistry of SHP-2 ................................................................................................. 24
1.3.2. SHP-2 and disease ............................................................................................................. 26
1.3.3. SHP-2 and growth factor signalling..................................................................................... 28
1.3.4. SHP-2 and endothelial physiology ...................................................................................... 31
1.4. A NOVEL STRATEGY OF INHIBITING SHP-2 ............................................... 32
1.4.1. Posttranscriptional gene silencing....................................................................................... 32
1.4.2. Magnetofection- a highly efficient tool for transfection of endothelial cells......................... 35
1.5. OBJECTIVES OF THIS STUDY...................................................................... 37
1.5.1. SHP-2 and angiogenesis initiation 37
1.5.2. SHP-2 and vessel formation ............................................................................................... 38
1.5.3. SHP-2 and endothelial signalling ........................................................................................ 38

2. MATERIALS AND METHODS 39
2.1. CHEMICALS ................................................................................................... 40
2.2. CELL CULTURE AND CELL LINES................................................................ 41
2.2.1. Isolation and cultivation of human umbilical vein endothelial cells ..................................... 42
2.2.2. Isolation and cultivation of porcine arterial endothelial cells ............................................... 43
2.2.3. Cultivation of human microvascular endothelial cells ......................................................... 43
2.2.4. Passaging of cells ............................................................................................................... 44
2.2.5. Freezing of cells .................................................................................................................. 44
2.3. MOLECULAR BIOLOGY................................................................................. 45
2.3.1. AS-oligodesoxynucleotide transfer...................................................................................... 45
2.3.2. Assessment of transfer efficiency ....................................................................................... 47
2.3.3. Cell extracts for immunoprecipitations, western blot analysis and SHP-2 activity
measurement ...................................................................................................................... 49
2.3.4. Assessment of protein concentration.................................................................................. 51
2.4. PROTEIN CHEMISTRY 52
2.4.1. Western Blot analysis.......................................................................................................... 52
2.4.2. Immunoprecipitation of p-85 and SHP-2............................................................................. 56
3 2.5. FUNCTIONAL ASSAYS .................................................................................. 57
2.5.1. Treatment with growth factors and pharmacological inhibitors........................................... 57
2.5.2. Protein activity assays.........................................................................................................58
2.5.3. In vitro wound healing assay............................................................................................... 60
2.5.4. Cell proliferation assay........................................................................................................61
2.5.5. Fluorescent-activating cell scanning analyses.................................................................... 63
2.5.6. Cell cycle measurement...................................................................................................... 64
2.5.7. Apoptosis measurements.................................................................................................... 65
2.5.8. Capillary like structure assay .............................................................................................. 66
2.5.9. Aortic ring sprouting ............................................................................................................ 67
2.6. STATISTICAL ANALYSIS ............................................................................... 68

3. RESULTS 69
3.1. SHP-2 PROTEIN KNOCK-DOWN................................................................... 70
3.1.1. Efficiency of AS-ODN transfer............................................................................................. 70
3.1.2. Kinetics of SHP-2 knock-down............................................................................................ 72
3.2. SHP-2 AFFECTS IMPORTANT STEPS OF ANGIOGENESIS INITIATION.... 74
3.2.1. SHP-2 inhibition suppresses basal wound healing in vitro ................................................. 74
3.2.2. SHP-2 protein knock-down inhibits endothelial basal proliferation..................................... 75
3.2.3. SHP-2 protein knock-down impairs endothelial cell viability and induces apoptosis.......... 78
3.2.4. Growth factors enhance SHP-2 protein activity and induce phosphorylation of SHP-2 ..... 80
3.2.5. Inhibition of SHP-2 impairs growth factor enhanced endothelial wound healing in vitro .... 82
3.2.6. Loss of SHP-2 prevents bFGF and PDGF induced endothelial cell proliferation ............... 85
3.3. SHP-2 IS NECESSARY FOR COMPLETE VESSEL FORMATION................ 87
3.3.1. Loss of SHP-2 impairs the ability of endothelial cells to form capillary like structures ....... 87
3.3.2. Inhibition of SHP-2 negatively affects vessel sprouting ex vivo.......................................... 90
3.4. SHP-2 AND ENDOTHELIAL SIGNALLING ..................................................... 92
3.4.1. SHP-2 inhibition abrogates bFGF, PDGF-BB and VEGF-A induced activation of the PI3-
Kinase in endothelial cells................................................................................................... 92
3.4.2. SHP-2 interacts with the p85 subunit of PI3-Kinase and Gab-1 upon growth factor
stimulation ........................................................................................................................... 94
3.4.3. SHP-2 activates Akt upon bFGF and PDGF-BB but not VEGF-A stimulation.................... 96
3.4.4. Raf inhibition impairs endothelial cell proliferation and migration....................................... 98
3.4.5. Growth factor dependent PI3-Kinase regulation of Raf and ERK activation .................... 102
3.4.6. SHP-2 knock-down reduces growth factor induced ERK activation ................................. 104
3.4.7. The effects of SHP-2 knock-down are specific ................................................................. 106



4 4. DISCUSION 107
4.1. SILENCING OF SHP-2 PROTEIN................................................................. 108
4.1.1. Why AS-ODN? ...............................................................................................