Foxc1 regulates Pecam-1 expression in embryonic endothelial progenitor cells [Elektronische Ressource] / eingereicht von Mathias Lamparter

Mathias Lamparter

Description

Dissertation der Fakultät für Biologie der Ludwig-Maximilians-Universität München Foxc1 regulates Pecam-1 Expression in embryonic Endothelial Progenitor Cells Eingereicht von Mathias Lamparter München, 15. Januar 2008 Angefertigt am Institut für Klinische Molekularbiologie und Tumorgenetik, Helmholtz Zentrum München – Deutsches Forschungszentrum für Gesundheit und Umwelt, und Division of Cardiovascular Medicine, Vanderbilt University Medical Center, Nashville, TN, USA Erster Gutachter: Prof. Dr. Dirk Eick Zweiter Gutachter: Prof. Dr. Manfred Schliwa Tag der mündlichen Prüfung: 02. Juli 2008 aaaa SUMMARY ................................................................................................................. 1 1. INTRODUCTION.................................................................................................... 3 1.1 The Vascular System........................................................................................ 3 1.2 De novo formation of the vasculature – Vasculogenesis................................... 3 1.3 Expansion and maturation of the vasculature – Angiogenesis.......................... 4 1.4 The vasculature in disease states..................................................................... 5 1.5 Characteristics of adult EPCs ........................................................................... 5 1.

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2008
• Nombre de lectures: 38
• Langue: Deutsch
• Poids de l'ouvrage: 1 Mo

Extrait

Dissertation der Fakultät für Biologie der
Ludwig-Maximilians-Universität München








Foxc1 regulates Pecam-1 Expression in embryonic
Endothelial Progenitor Cells





Eingereicht von Mathias Lamparter
München, 15. Januar 2008






Angefertigt am Institut für Klinische Molekularbiologie und Tumorgenetik,
Helmholtz Zentrum München – Deutsches Forschungszentrum für Gesundheit
und Umwelt, und Division of Cardiovascular Medicine, Vanderbilt University
Medical Center, Nashville, TN, USA























Erster Gutachter: Prof. Dr. Dirk Eick

Zweiter Gutachter: Prof. Dr. Manfred Schliwa

Tag der mündlichen Prüfung: 02. Juli 2008


SUMMARY ................................................................................................................. 1
1. INTRODUCTION.................................................................................................... 3
1.1 The Vascular System........................................................................................ 3
1.2 De novo formation of the vasculature – Vasculogenesis................................... 3
1.3 Expansion and maturation of the vasculature – Angiogenesis.......................... 4
1.4 The vasculature in disease states..................................................................... 5
1.5 Characteristics of adult EPCs ........................................................................... 5
1.6 EPC promote neovascularization...................................................................... 6
1.7 Molecular control of blood vessel formation...................................................... 6
1.7.1 VEGF and VEGF-Receptors ...................................................................... 7
1.7.2 Angiopoietins and Tie-Receptors................................................................ 8
1.7.3 Ephrins and Eph Receptors........................................................................ 8
1.7.4 Extracellular Matrix and Cell Adhesion Molecules...................................... 8
1.7.5 Other signaling pathways involved in vascular development...................... 9
1.7.6 Cytokines.................................................................................................. 10
1.7.7 Angiogenic inhibitors ................................................................................ 10
1.7.8 Haemodynamic forces.............................................................................. 10
1.8 Transcriptional control of vascular formation................................................... 10
1.8.1 Ets transcription factors................................................................................ 11
1.8.2 Basic Helix-Loop-Helix Transcription Factors........................................... 11
1.8.3 Homeobox Transcription Factors.............................................................. 12
1.8.4 Further Transcription Factors ................................................................... 12
1.8.5 Hypoxia and HIF .................................................................................... 12
1.9 Embryonic endothelial progenitor cells (eEPCs) as a model system .............. 13
1.10 Foxc1 and Foxc2 are induced during eEPC in vitro differentiation................ 14
1.11 Forkhead (Fox) Transcription Factors........................................................... 14
1.11.1 General characteristics of Fox Genes .................................................... 14
1.11.2 Nomenclature of Fox Genes................................................................... 15
1.11.3 Chromosomal organization of Fox genes ............................................... 15
1.11.4 Fox Genes in Development .................................................................... 16
1.11.5 Fox Genes in Signaling Pathways.......................................................... 17
1.11.6 Fox Genes in Human Diseases.............................................................. 18
1.11.7 Fox Genes in the Adult Organism........................................................... 19
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1.12 The FoxC subfamily - Foxc1 and Foxc2........................................................ 19
1.12.1 Expression Patterns ............................................................................... 19
1.12.2 Abnormalities in FoxC mutant embryos.................................................. 20
1.12.3 Foxc1 and Foxc2 - signaling pathways and target gene activation......... 21
1.13 Aims of the Ph.D. Project.............................................................................. 22
2. MATERIAL and METHODS.................................................................................. 23
2.1 Tissue Culture................................................................................................. 23
2.1.1 Cell lines................................................................................................... 23
2.1.2 Tissue culture media ................................................................................ 24
2.1.3 Passage of cell cultures............................................................................ 24
2.1.4 Freezing and thawing of cell lines............................................................. 25
2.1.5 Transfection of cell lines ........................................................................... 25
2.2 Molecular biology techniques.......................................................................... 26
2.2.1 Total RNA isolation................................................................................... 26
2.2.2 Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) ............... 27
2.2.3 Polymerase Chain Reaction (PCR) .......................................................... 28
2.2.4 PCR Primer Design .................................................................................. 29
2.2.5 DNA Sequencing...................................................................................... 29
2.2.6 Plasmid DNA preparation ......................................................................... 31
2.2.7 Transformation of CaCl -competent DH5 E.coli ..................................... 32 2
2.2.8 DNA restriction digests ............................................................................. 33
2.2.9 DNA ligation ............................................................................................. 34
2.2.10 Plasmid Constructs................................................................................. 34
2.2.11 Agarose gel electrophoresis ................................................................... 37
2.2.12 Ethanol Precipitation of DNA .................................................................. 38
2.3 Quantitative Real-Time Amplification (qPCR) ................................................. 38
2.3.1 LightCycler Real-Time PCR System......................................................... 38
2.3.2 iQ5 Real-Time PCR System..................................................................... 40
2.4 Fluorescence Activated Cell Sorting (FACS) Analysis .................................... 42
2.5 Microscopy and Fluorescence Microscopy ..................................................... 43
2.6 Immunofluorescence....................................................................................... 43
2.7 Promoter-Luciferase Assays ........................................................................... 44
2.8 Western Blot ................................................................................................... 46
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2.9 Chromatin Immunoprecipiation ....................................................................... 48
2.10 Online Databases and Bioinformatics Programs........................................... 51
3. RESULTS............................................................................................................. 53
3.1 Expression of Fox genes in eEPCs................................................................. 53
3.2 Foxc1 and Foxc2 regulate expression of Pecam-1 in eEPCs ......................... 56
3.3 Pecam-1 as an endothelial target gene of Foxc1............................................ 60
3.4 Cis-regulatory areas of the Pecam-1 promoter ............................................... 61
3.5 Pecam-1 promoter and enhancer analysis in different cell lines ..................... 62
3.6 Foxc1 activates transcription through the 5’-flanking 3.5kb-fragment ............. 65
3.7 The distal 5’-flanking 3.5kb-fragment responds specifically to Foxc1 ............. 66
3.8 Foxc1 activates transcription specifically in eEPCs ........................................ 68
3.9 Endogenous Pecam-1 RNA analysis matches promoter activation studies.... 70
3.10 Dose-dependent activation of the Pecam-1 promoter by Foxc1 ................... 71
3.11 Activation of the Pecam-1 promoter takes place in multiple, independently
isolated eEPC clones............................................................................................ 72
3.12 Pecam-1 promoter activity – Summary .........