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The Long Noncoding RNA H19 Controls Endothelial Cell Functions by STAT3 Repression

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The Long Noncoding RNA H19 Controls Endothelial Cell Functions by STAT3 Repression The long noncoding RNA H19 controls endothelial cell functions by STAT3 repression Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften Vorgelegt beim Fachbereich Biowissenschaften (FB15) der Johann Wolfgang Goethe-Universität in Frankfurt am Main von Patrick Hofmann aus Weinheim (Bergstraße) Frankfurt 2017 (D 30) Vom Fachbereich Biowissenschaften (FB 15) der Johann Wolfgang Goethe-Universität als Dissertation angenommen Dekan: Prof. Dr. Meike Piepenbring Gutachter: Prof. Dr. Stefanie Dimmeler Prof. Dr. Amparo Acker-Palmer Datum der Disputation: Table of contents 1. Introduction ..................................................................................................................................... 1 1.1 The mammalian cardiovascular system .................................................................................. 1 1.1.1 The circulatory system of mammals.................................................................................... 1 1.1.2 Angiogenesis ........................................................................................................................ 3 1.1.3 Shear Stress ......................................................................................................................... 5 1.1.4 Atherosclerosis .................................................................................................................... 6 1.2 Aging ........................................................................................................................................ 9 1.2.1 Systemic Aging ..................................................................................................................... 9 1.2.2 Senescence ........................................................................................................................ 10 1.2.3 Aging of the Cardiovascular System .................................................................................. 12 1.2.4 IL-6/JAK2/STAT3 Signaling ................................................................................................. 14 1.3 Noncoding RNAs .................................................................................................................... 17 1.3.1 Noncoding RNAs ................................................................................................................ 17 1.3.2 MicroRNAs ......................................................................................................................... 17 1.3.3 Long Noncoding RNAs ....................................................................................................... 20 1.3.4 The Long Noncoding RNA H19 .......................................................................................... 24 2. Objective........................................................................................................................................ 31 3. Materials and Methods ................................................................................................................. 32 3.1 Materials ................................................................................................................................ 32 3.2 Methods ................................................................................................................................ 40 3.2.1 Cell culture ......................................................................................................................... 40 3.2.2 In vitro assays .................................................................................................................... 42 3.2.3 Molecular Biology .............................................................................................................. 44 3.2.4 Animal Experiments........................................................................................................... 48 3.2.5 Statistics ............................................................................................................................. 49 4. Results ........................................................................................................................................... 50 4.1 H19 is repressed by aging and induced by KLF2 .................................................................... 50 4.2 H19 can be pharmacologically inhibited with siRNAs and LNA GapmeRs and overexpressed with lentivirus .................................................................................................................................... 52 4.3 H19 does not function via known mechanisms in endothelial cells ..................................... 53 4.4 Depletion of H19 delays proliferation and promotes senescence in endothelial cells ......... 57 4.5 H19 does not influence migration in vitro ............................................................................. 64 4.6 H19 depletion impairs endothelial cell function ex vivo and in vivo ..................................... 65 4.7 H19 induces inflammatory signaling in vitro ......................................................................... 71 4.8 H19 exerts its function mainly through inhibition of STAT3 activation ................................ 73 5. Discussion ...................................................................................................................................... 77 5.1 Expression of H19 is tightly regulated ................................................................................... 77 5.2 Loss of H19 promotes senescence and counteracts proliferation ........................................ 77 5.3 H19 is required for proper endothelial cell function ex vivo and in vivo .............................. 81 5.4 Loss of H19 promotes inflammatory activation of endothelial cells ..................................... 84 5.5 H19 does not regulate endothelial cell function via previously described mechanisms ...... 84 5.6 H19 mainly exerts its functions via regulation of STAT3 activation ...................................... 86 5.7 H19 overexpression partially confirms loss-of-function studies ........................................... 89 5.8 Therapeutic perspectives of H19 ........................................................................................... 90 5.9 Conclusion ............................................................................................................................. 90 6. Summary........................................................................................................................................ 92 7. Zusammenfassung ......................................................................................................................... 95 8. References ................................................................................................................................... 101 Abbreviations ...................................................................................................................................... 125 Eidesstattliche Erklärung ..................................................................................................................... 128 Acknowledgements ............................................................................................................................. 129 Curriculum Vitae .................................................................................................................................. 130 1. Introduction 1.1 The mammalian cardiovascular system 1.1.1 The circulatory system of mammals In the mammalian organism, two vascular systems provide oxygen, liquids, and nutrients to all of its cells. The cardiovascular system forms a closed network, while the lymphatic vasculature is a blind end network of vessels. The lymphatic system maintains fluid homeostasis and transports immune cells 1,2. The cardiovascular system comprises the heart and all blood vessels. Muscular contraction of the left heart ventricle pumps oxygenized blood into the aorta and through branching arteries into capillaries, in which oxygen (O2), carbon dioxide (CO2), and nutrient exchange with the surrounding tissue takes place along the gradient 3. Blood with low oxygen content is then transported back to the heart through veins and pumped by the right ventricle into the pulmonary circulation, where CO2 diffuses from the blood into the lung alveoli and O2 diffuses into the blood. The oxygenized blood then enters the body circulation through the left ventricle again (Fig. 1). 1 Figure 1: The human circulatory system. Blood is oxygenized in the capillary bed of the lungs, enters the heart through the left atrium and is pumped into the aorta and all body tissues by the left ventricle. Oxygen and nutrient exchange happens in the capillary bed of all body tissues and CO2-rich blood flows back to the heart through the systemic veins and the finally the vena cava into the right atrium. The right ventricle pumps the blood into the pulmonary circuit, where it is oxygenized again. Modified from Guyton and Hall 2016, Textbook of Medical Physiology, Philadelphia, USA, Elsevier. Arteries are the most complex blood vessels in the human body; they consist of the tunica intima as the most inner and thinnest layer, which comprises a single layer of endothelial cells surrounded by subendothelial connective tissue interlaced with the internal elastic membrane and the basement membrane. The tunica media mainly consists of smooth muscle cells and elastic tissue and is the thickest
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