Endothelial microRNA-24 contributes to capillary density in the infarcted heart [Elektronische Ressource] / Jan Fiedler. Betreuer: Johann Bauersachs

Jan Fiedler

Sujets

• Gesundheit

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2011
• Nombre de lectures: 49
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Endothelial microRNA-24 contributes to capillary
density in the infarcted heart



Dissertation zur Erlangung des
naturwissenschaftlichen Doktorgrades
der Julius-Maximilians-Universität Würzburg








Vorgelegt von

Jan Fiedler
aus Husum





Würzburg, 2010


Eingereicht am:



Mitglieder der Promotionskomission:


Vorsitzender: Prof. Dr. Thomas Dandekar

Gutachter: Prof. Dr. Johann Bauersachs

Gutachter: Prof. Dr. Thomas Dandekar

Betreuer: Prof. Dr. Dr. Thomas Thum











Tag des Promotionskolloquiums:


Doktorurkunde ausgehändigt am:



Dedicated to my family
Contents

Contents


Summary 1

Zusammenfassung 2


1. Introduction

1.1 Cardiac remodelling and heart failure 3

1.2 Angiogenesis and apoptotic signalling 7
1.2.1 General mechanisms of angiogenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.2.2 Apoptosis signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.2.3 Endothelial cell apoptosis in the cardiovascular system . . . . . . . . . . . . . . . . . . . 10

1.3 MicroRNAs (miRNAs): regulatory RNAs 12
1.3.1 MiRNAs in the cardiovascular system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
1.3.1.1 Cardiomyocyte miRNAs
1.3.1.2 Fibroblast miRNAs
1.3.1.3 Endothelial miRNAs (angiomiRs)
1.3.2 MiRNA antagonists (“antagomirs“) for in vivo application . . . . . . . . . . . . . . . . 23

1.4 MiR-24 genelocus and miR-24 function 25

1.5 Scope and aim of this study 28


2. Material and Methods

2.1 Material 30
2.1.1 Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.1.2 Consumable material and chemicals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.1.3 Provided kit systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.1.4 Solutions and buffers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.1.4.1 Cell culture media, components and used cells
2.1.4.2 Cardiomyocyte preparation and fractionation of cardiac cells
2.1.4.3 DNA electrophoresis
2.1.4.4 Chromatinimmunoprecipitation (ChIP)
2.1.4.5 SDS-PAGE and Western Blot
2.1.5 Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
2.1.6 Oligonucleotides and probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Contents

2.1.7 Antibodies, enzymes and standard markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
2.1.8 FACS solution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
2.1.9 Components for bacterial culture and bacteria strains . . . . . . . . . . . . . . . . . . . . 49
2.1.10 Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

2.2 Methods 51
2.2.1 Cell culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
2.2.1.1 Cultivation of endothelial cells (ECs)
2.2.1.2 Isolation and cultivation of neonatal rat cardiomyocytes
and cardiacfibroblasts
2.2.1.3 Cultivation of other applied cell types
2.2.1.4 Transfection assays
2.2.1.5 Luciferase reporter assay
2.2.1.6 In vitro tube formation assay for ECs
2.2.1.7 Viral transduction
2.2.2 Molecular biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
2.2.2.1 RNA isolation
2.2.2.2 DNA isolation
2.2.2.3 Determination of RNA and DNA concentration
2.2.2.4 Agarose gel electrophoresis
2.2.2.5 Luciferase pMIR-Report cloning
2.2.2.6 Real-time PCR analysis
2.2.2.7 ChIP
2.2.2.8 Affymetrix gene chips
2.2.3 Protein biochemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
2.2.3.1 Protein isolation
2.2.3.2 Determination of protein concentration
2.2.3.3 SDS-PAGE
2.2.3.4 Western Blot
2.2.3.5 Apoptosis array
2.2.3.6 phospho-Bad ELISA
2.2.4 Fluorescence-activated cell sorting (FACS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
2.2.4.1 Apoptosis staining
2.2.4.2 Cell cycle propidiume iodide (PI) stain
2.2.4.3 Reactive oxygen species (ROS) detection
2.2.5 Immunocytochemistry and immunohistochemistry . . . . . . . . . . . . . . . . . . . . . . 63
2.2.5.1 Immunocytochemistry
2.2.5.2 Immunohistochemistry
2.2.6 In vivo methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 64
2.2.6.1 Fractionation of cardiac cells from heart tissue
2.2.6.2 Antagomir injection
2.2.6.3 Myocardial infarction
2.2.6.4 Echocardiography
2.2.6.5 Matrigel implantation and determination of vasuclarization
2.2.7 MicroRNA target prediction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
2.2.8 Statistical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66

Contents

3. Results

3.1 MiR-24 is induced post myocardial infarction (MI) 67
3.1.1 MiR-24 profiling reveals an ubiquitous expression panel . . . . . . . . . . . . . . . . . . 68
3.1.2 MiR-24 is induced in ECs post hypoxia and in cardiac ECs post MI . . . . . . . . . 69

3.2 MiR-24 modulation in different cell types 72
3.2.1 MiR-24 overexpression induces apoptosis specifically in ECs . . . . . . . . . . . . . 74
3.2.2 Apoptosis array post miR-24 modulation reveals dysregulation
of anti-apoptotic HMOX1 and Bad protein . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
3.2.3 Elevated reactive oxygen species (ROS) upon miR-24
overexpression in ECs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
3.2.4 Capillary tube formation is impaired in miR-24 overexpressing ECs . . . . . . . . 81

3.3 MiR-24 regulates endothelial GATA2, H2A.X,
PAK4 and RASA1 81
3.3.1 Luciferase reporter gene assays confirm miR-24 targets GATA2,
H2A.X, PAK4 and RASA1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
3.3.2 Transient knockdown of miR-24 targets GATA2, H2A.X, PAK4
and RASA1 induces apoptosis in ECs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
3.3.3 GATA2 is a key player for cell cycle progression in ECs . . . . . . . . . . . . . . . . . 88
3.3.4 Impairment in tube formation ability when silencing GATA2 or PAK . . . . . . . 89
3.3.5 PAK4 and RASA1 are downregulated under hypoxic conditions in ECs . . . . . 90

3.4 Affymetrix and ChIP data indicate GATA2-regulated
genes related to angiogenic processes 91
3.4.1 Overexpression of murine Gata2 in ECs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
3.4.2 Transcriptome analysis upon GATA2 modulation by Affymetrix gene chip . . 93
3.4.3 ChIP analysis for GATA2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
3.4.4 Pro-angiogenic HMOX1 and SIRT1 are regulated on protein level
by the miR-24 target GATA2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

3.5 Bad phosphorylation status is regulated
by the miR-24 target PAK4 99

3.6 In vivo treatment of myocardial infarction by a specific
miR-24 antagonist (antagomir) – antagomir-24 study 101
3.6.1 Antagomir-24 efficiently lowers cardiac miR-24 expression . . . . . . . . . . . . . . . 103
3.6.2 Heart function tests after MI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
3.6.3 Cardiac angiogenesis is improved upon antagomir-24 treatment . . . . . . . . . . . . 105
3.6.4 Implantation of matrigel plugs to characterize global
neovascularization in vitro. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107


Contents

4. Discussion 109

4.1 MiR-24 deregulation in cardiac disease 110

4.2 Induction of endothelial cell apoptosis by miR-24 112

4.3 Target-specific regulation for miR-24 in ECs 115

4.4 Antagonizing miR-24 as a therapeutic option in
treatment of cardiovascular disease 119

4.5 Concluding remarks 122



References I

Abbreviations II

Publications III

Curriculum Vitae IV

Acknowledgement V

Affidavit VI

Summary
Summary


Cardiovascular disease is the most common mortality risk in the industrialized world.
Myocardial infarction (MI) results in the irreversible loss of cardiac muscle, triggering