The Role of Endoglin in Endothelial and Mesenchymal cells during Development, Maintenance and Repair of the Heart Esha Singh Thesis submitted for the degree of Doctor of Philosophy Institute of Genetic Medicine Faculty of Medical Sciences Newcastle University December 2019 Abstract Mutations in the endoglin gene lead to Haemorrhagic Telangiectasia Type 1 (HHT1), an inherited vascular disease, demonstrating the importance of endoglin in the vasculature. Haplo- deficiency of endoglin also leads to reduced angiogenesis in mice in several pathologies, indicating a pro-angiogenic role for endoglin. However, the precise role of endoglin in development and maintenance of the vasculature is still unclear. The aim of this project therefore was to investigate the role of endoglin during vascular development and maintenance and whether its pro-angiogenic properties can be used to enhance cardiac recovery following myocardial infarction (MI). Using immunofluorescence staining of mouse cardiac tissue, I showed high endoglin expression in coronary veins endothelial cells (ECs), capillaries & endocardium and weak expression in coronary arteries ECs, valve mesenchyme & in epicardially derived cells (EPDCs) during development. I hypothesised that during cardiac development endoglin is essential for EC proliferation and migration, and plays a key role during EPDC proliferation, migration and differentiation. Induced knockdown of endoglin in murine epicardial cells using Wt1-Cre led to a reduced number of EPDCs in E15.5 hearts but did not lead to any detectable vascular defects in E17.5 hearts. In contrast, EC-specific endoglin knockdown in the first week of postnatal life (which is known to lead to retinal AVMs) was associated with the development of eccentric cardiomyopathy and cardiomyocytes (CM) hypertrophy, but no obvious coronary vasculature defects. Deletion of endoglin in venous ECs and capillaries in first week of postnatal life also led to development of AVMs in P11 retinas coupled with the development of cardiac hypertrophy. A similar eccentric cardiomyopathy phenotype with CM hypertrophy was observed after endoglin deletion in adult ECs (Eng-iKOEC). Microbeads were used to show increased vascular shunting in the systemic but not the pulmonary vasculature of Eng-iKOEC mice. Ultimately Eng-iKOEC mice developed high output heart failure due to reduced vascular resistance cause by systemic AVMs. This data confirms that endoglin is vital for maintaining the adult vasculature. Finally, I analysed the role of endoglin in a heterogeneous population of cardiac derived stem cells known as cardiosphere derived cells (CDCs). CDCs express endoglin which I showed is vital for their pro-angiogenic paracrine effect when delivered to the infarcted heart. Furthermore, endoglin not only promotes paracrine mediated proliferation of ECs in the infarct zone but also helps in maturation of the neo-vasculature by recruitment of smooth muscle cells. In conclusion, the results from this thesis show the critical importance of endoglin in the development and maintenance of the vasculature. ii Declaration The work described in this thesis was undertaken in the laboratory of Prof. Helen M. Arthur at the Institute of Genetic Medicine, Newcastle University for the fulfilment of the requirements for the degree of Doctor of Philosophy. The results described in the thesis was my own work and I have correctly acknowledged the contributions of others where appropriate in the text. No material within this thesis has been submitted by me for a degree or diploma or other qualification in this or any other University. This thesis does not exceed the word limit prescribed by the Faculty of Medical Sciences. Esha Singh December 2019 iii Acknowledgement I would like to first thank my supervisor Prof. Helen Arthur for the opportunity to perform this research in in her laboratory. I would also like to thank Prof. Arthur for her continuous support, guidance and expertise which helped me to be a better scientist today. Next, I would like to thank my co-supervisor Dr. Helen Phillips for her expertise and support throughout my PhD. Next, I would like to thank Dr. Rachael Redgrave for her encouragement and support, for all the coffees and lunches. It meant a lot. I would also like to thank Rachael for all discussions and ideas which helped me understand my work better, especially for the contributions for the work during CDCs mediated heart repair. Also thank you so much for performing the MI surgeries and the intraocular injections. I would next like to thank Dr. Simon Tual-Chalot for collaborations and help especially during the endothelial endoglin study in adult and also for teaching me neonatal retina dissections. Next, I would like to thank Dr. Darroch Hall for preparing CDCs during MI surgeries and genotyping mice. I would next like to thank Dr. Kate Bailey for her help with Wt1-Cre optimisation and for her guidance during staining protocol optimisations. I would also like to thank Dr. Rebecca Dodds for her help with optimisation of Sm22a staining on wax sections. I would next like to thank Ms. Elizabeth Greally for her the useful tips on sectioning and Ms. Kathleen Alison for her help in lab. I would also like to thank Dr. Gavin Richardson for showing me how to use Stero SV 11 microscope. Next, I would like to pass my sincere thanks to all the staff in the mouse unit especially Mr. Steve Smith for the endless help and guidance with mice. I would also like to give a big thank you to Ms. Lisa Hodgson for answering my continuous questions on microscopy. I learnt a lot from her as well as from the Microscope team at Carl Zeiss. Finally, I would like to thank my family, my dad, my mum, my wife, brothers, aunts, cousins and all my dear friends (Monish, Avinash, Niki, Elena and Rachael) for giving me the strength and courage to be here today. I would especially like to thank two very important people in my life, my dad and my wife. Thank you, my sweet and wonderful father for being my inspiration, my rock and support throughout my life. And thank you to my beautiful wife, Jelena for being here for me, for listening to me endlessly, for holding me when I was sad, for making me laugh and for all love. This thesis is dedicated to you both because without you it would not be possible. I love you both so much!! iv Table of Contents Abstract ....................................................................................................................................... ii Declaration................................................................................................................................. iii Acknowledgement ..................................................................................................................... iv Table of Contents ....................................................................................................................... v List of Figures ............................................................................................................................. x List of Tables .......................................................................................................................... xvii List of Abbreviations ............................................................................................................... xix Chapter 1. Introduction ........................................................................................................ 23 1.1. Heart Development .................................................................................................... 24 1.2. Vasculogenesis and Angiogenesis during Development ........................................... 25 1.2.1. Anatomy of Mature Coronary Circulation ......................................................... 28 1.3. Cells in Coronary Vessels Development ................................................................... 31 1.3.1. Role of Epicardium in Coronary Vessel Development ...................................... 32 1.3.2. Origin of the Endothelial Component of Coronary Vessels ............................... 37 1.4. TGFβ and Endoglin in Coronary Vasculature Development ..................................... 40 1.4.1. TGFβ Ligands and its Receptors ........................................................................ 40 1.4.2. Endoglin: TGFβ type 3 co-receptor .................................................................... 42 1.5. TGFβ and Endoglin in Coronary Vessel Development ............................................. 45 1.5.1. TGFβ and Endoglin in Endothelial Cells ........................................................... 45 1.5.2. TGFβ Ligands and Receptors in Vasculature Development .............................. 48 1.5.3. TGFβ Signalling during EMT ............................................................................ 50 1.6. Hereditary Haemorrhagic Telangiectasia (HHT)....................................................... 51 Role of TGFβ Signalling in HHT ....................................................................... 53 Treatment for HHT ............................................................................................. 54 1.7. Myocardial Infarction ................................................................................................ 56 1.7.1. Importance of TGFβ during Heart Repair .......................................................... 57 1.7.2. Cell Mediated Therapy in Post-MI ..................................................................... 59 1.8. Aims