The role of microRNA 21 in the development of in-stent restenosis Crawford Alasdair Halliday

275 Pages·2016·23.05 MB·English

Halliday, Crawford Alasdair (2015) The role of microRNA 21 in the
development of in-stent restenosis. PhD thesis.

http://theses.gla.ac.uk/7062/

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The role of microRNA 21 in the development of
in-stent restenosis

Crawford Alasdair Halliday
BSc(Med Sci)(Hons), MB ChB(Hons), MRCP (UK)

Submitted in fulfilment of the requirements for the Degree
of Doctor of Philosophy

Institute of Cardiovascular and Medical Sciences

College of Medicine, Veterinary & Life Sciences

University of Glasgow

July 2015

© Crawford Alasdair Halliday 2015
2

Authors’s Declaration
I declare that this thesis has been written entirely by myself and is a record of
work performed by myself with the exception of in situ hybridisation presented
in Figure 4-5 which was performed by Dr Robert A McDonald. This thesis has not
been submitted previously for a higher degree. The research was conducted at
the Institute of Cardiovascular and Medical Sciences, University of Glasgow and
the Golden Jubilee National Hospital, Clydebank under the supervision of
Professor Andrew H Baker, Professor Keith G Oldroyd and Dr Simon Kennedy.
Crawford Halliday
July 2015
3

Acknowledgements
I would like to start by thanking my supervisors, Professor Andrew H Baker,
Professor Keith G Oldroyd and Dr Simon Kennedy for their guidance throughout
this project. I would also like to thank the British Heart Foundation for funding
this project.
Many thanks to all those at the University of Glasgow who have been extremely
helpful over the course of this project. In particular, the staff in the GCRC
should be given a special mention for their patience and support, especially
Robert, Nic, Gregor, Ashley and Iona who taught me many of the laboratory
techniques used within this thesis. Without the assistance of both Nosrat and
Alastair, from the bioelectronics unit, the building of the electrolysis equipment
would not have been possible. Thank you both very much for the random wire,
power packs and cables which you happily lent me.
Thank you to Hollie and Gillian who showed me the micro surgical procedure and
provided me with encouragement even when it seemed impossible to master. In
addition, I would like to thank the staff at the CRF who provided excellent
anaesthetic support and banter.
This project was driven not only by enthusiasm but also caffeine. Thank you to
all those in our office, with whom I shared in numerous cups of tea, coffee and
cake. In particular, thank you to Keith, Stacy, Ange, Margaret, Emily, Karine and
Mounia. Your encouragement and advice has been invaluable. Merci.
Throughout this PhD my family have been my greatest supporters. I would like to
thank my parents for their never ending support. However, I would like to say
my biggest thank you to my amazing wife Claire, whose relentless support and
encouragement has kept me going through some very challenging periods. Words
cannot express my gratitude. Your patience and love knows no bounds and I
could not have done this without you. The birth of our beautiful daughter
Isabella Iona has been the most wonderful experience to happen to us and I
would like to dedicate this thesis to you both.
Crawford
July 2015
4

For Claire and Isabella,
5

Table of Contents

!
Authors’s Declaration ............................................................................................... 2!
Acknowledgements .................................................................................................. 3!
List of Publications ................................................................................................... 7!
*Joint first authors ................................................................................................... 7!
Appendix .................................................................................................................. 8!
Abbreviations ........................................................................................................... 9!
Abstract .................................................................................................................. 19!
1! Introduction. ..................................................................................................... 21!
1.1! Global burden of Cardiovascular Disease ................................................. 22!
1.2! Risk Factors for Coronary Disease ............................................................ 23!
1.3! Arterial wall structure ................................................................................. 24!
1.4! Atherosclerosis .......................................................................................... 24!
1.5! Clinical Manifestations of Coronary Heart Disease ................................... 29!
1.6! Management of Coronary Heart Disease .................................................. 29!
1.7! Coronary Angiography and Intervention .................................................... 32!
1.8! In-stent Restenosis .................................................................................... 33!
1.9! Animal Models of In-stent Restenosis ....................................................... 38!
1.10! Vascular Smooth Muscle Phenotype ....................................................... 40!
1.11! MicroRNAs .............................................................................................. 42!
1.12! Expression of microRNAs are essential for development ....................... 48!
1.13! MicroRNA expression and cardiovascular development ......................... 49!
1.14! Role of MicroRNAs following Vascular Injury .......................................... 51!
1.15! The role of microRNAs in macrophage function following vascular injury
66!
1.16! Aims ......................................................................................................... 72!
1.17! Hypotheses .............................................................................................. 73!
2! Methods ........................................................................................................... 74!
2.1! Animals ...................................................................................................... 75!
2.2! Genotyping ................................................................................................ 75!
2.3! RNA and miRNA Extraction from Cells and Tissues ................................. 80!
2.4! Quantification of RNA ................................................................................ 81!
2.5! Complementary Deoxyribonucleic Acid (cDNA) Synthesis ....................... 82!
2.6! TaqMan® quantitative real-time polymerase chain reaction (qRT-PCR) ... 83!
2.7! Cell Culture ................................................................................................ 86!
2.8! Murine Monocyte and Macrophages ......................................................... 92!
2.9! Luminex Assay .......................................................................................... 95!
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2.10! Conditioned Media Experiments .............................................................. 97!
2.11! Arterial Stenting and Grafting Operation ................................................. 98!
2.12! Tissue Processing ................................................................................. 101!
2.13! Histochemical Staining .......................................................................... 105!
2.14! Immunohistochemistry ........................................................................... 107!
2.15! In situ Hybridisation ............................................................................... 110!
2.16! Image Analysis ...................................................................................... 112!
2.17! Statistical analysis ................................................................................. 115!
3! Refinement of the Mouse Model of ISR and Stent Electrolysis ...................... 116!
3.1! Introduction .............................................................................................. 117!
3.2! Aims ......................................................................................................... 120!
3.3! Results ..................................................................................................... 121!
3.4! Discussion ............................................................................................... 141!
4! Investigation of Multiple Strategies to Manipulate MicroRNA-21 Expression and
the Impact on the Development of In-stent Restenosis. ...................................... 146!
4.1! Introduction .............................................................................................. 147!
4.2! Aims ......................................................................................................... 149!
4.3! Results ..................................................................................................... 150!
4.4! Discussion ............................................................................................... 185!
5! Effects of microRNA-21 on macrophage polarisation and function in the
development of in-stent restenosis. ..................................................................... 195!
5.1! Introduction .............................................................................................. 196!
5.2! Aims ......................................................................................................... 198!
5.3! Methods ................................................................................................... 199!
5.4! Results ..................................................................................................... 201!
5.5! Discussion ............................................................................................... 223!
6! General Discussion. ....................................................................................... 228!
References .......................................................................................................... 236!
Appendix .............................................................................................................. 259!
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List of Publications
Papers
Halliday CA*, McDonald RA*, Miller AM, Diver LA, Dakin RS, Montgomery J,
McBride MW, Kennedy S, McClure JD, Robertson KE, Douglas G, Channon KM,
Oldroyd KG, Baker AH (2015). Reducing In-Stent Restenosis: Therapeutic
Manipulation of miRNA in Vascular Remodeling and Inflammation. J Am Coll
Cardiol; 65(21): 2314-2327.
McDonald RA, White KM, Wu J, Cooley BC, Robertson KE, Halliday CA, McClure
JD, Francis S, Lu R, Kennedy S, George SJ, Wan S, van Rooij E, Baker AH (2013).
miRNA-21 is dysregulated in response to vein grafting in multiple models and
genetic ablation in mice attenuates neointima formation. Eur Heart J;
34(22):1636-43.

Abstracts
Halliday CA, Robertson KE, McDonald RA, Kennedy S, Oldroyd KG, Douglas
G,Channon KM, Baker AH (2013). MicroRNA 21 is Upregulated Following Coronary
Artery Stenting and Genetic Deletion Limits Neointima Formation. Eur Heart J 34
(suppl1):806. Oral presentation at European Society of Cardiology Congress 2013
Amsterdam.
Halliday CA, Robertson KE, McDonald RA, Kennedy S, Oldroyd KG, Douglas G,
Channon KM, Baker AH (2013). The Role of MicroRNA 21 in Porcine and Murine
Models of In-Stent Restenosis. Oral presentation at Scottish Cardiovascular
Forum 2013

*Joint first authors
8

Appendix
Halliday CA, et al. (2015). Reducing In-Stent Restenosis: Therapeutic
Manipulation of miRNA in Vascular Remodeling and Inflammation. J Am Coll
Cardiol; 65(21): 2314-2327.
9

Abbreviations
AAA Abdominal Aortic Aneurysm
ABCA1 ATP Binding Cassette A1
ACh Acetylcholine
AHA American Heart Association
AngII Angiotensin II
ANOVA Analysis of Variance
AP-1 Activator Protein - 1
ApoE Apolipoprotein E
Arg1 Arginase 1
Bcl-2 B-cell lymphoma 2
Bcl-6 B-cell lymphoma 6
BH 5,6,7,8-tetrahydrobiopterin
4
BMDM Bone Marrow Derived Macrophages
BMP Bone Morphogenetic Protein
BMPR Bone Morphogenetic Protein Receptor
BMS Bare Metal Stent
BrdU 5-Bromo-2’-deoxyuridine
BSA Bovine Serum Albumin