an interesting role in human abdominal aortic aneurysm

258 Pages·2013·3.58 MB·English

by  Theophilus Ikenna Emeto

ResearchOnline@JCU

This file is part of the following reference:

Emeto, Theophilus Ikenna (2013) Urocortin 2: an
interesting role in human abdominal aortic aneurysm.
PhD thesis, James Cook University.

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http://eprints.jcu.edu.au/29838/

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UROCORTIN 2:
AN INTERESTING ROLE IN HUMAN
ABDOMINAL AORTIC ANEURYSM

Thesis submitted by
Theophilus Ikenna Emeto
MSc. (Distinction) Nottingham
in June 2013

for the degree of Doctor of Philosophy
in the School of Medicine and Dentistry
James Cook University, QLD
AUSTRALIA
PRINCIPAL SUPERVISOR
Professor Jonathan Golledge, BA, MB Bchir, M chir, FRCS, FRACS

CO-SUPERVISOR
Dr Catherine M Rush, BSc (Hons), PhD

ASSOCIATE SUPERVISORS
Dr Lynn Woodward, BSc, MSc, MA, PhD
Dr Joseph V Moxon, BSc, MSc, PhD

FINANCIAL SUPPORT
James Cook University Postgraduate Research Scholarship
School of Medicine and Dentistry Top-up Scholarship
CONTENTS
CONTENTS……… ............................................................................................................... i
DECLARATION .................................................................................................................. v
STATEMENT OF ACCESS TO THESIS ............................................................................ vi
ACKNOWLEDGEMENTS ................................................................................................ vii
COMMUNICATIONS, AWARDS AND GRANTS............................................................. ix
STATEMENT ON THE CONTRIBUTION OF OTHERS ................................................. xiv
LIST OF ABBREVIATIONS ........................................................................................... xvii
ABSTRACT…… ............................................................................................................. xxvi
CHAPTER 1 ........................................................................................................................ 1
INTRODUCTION AND LITERATURE REVIEW .......................................................... 1
1.1 General Introduction..................................................................................................... 2
1.2 Literature Review .......................................................................................................... 6
1.2.4.1 Chronic Inflammation and the immune response in AAA ........................................ 12
1.2.4.2 Proteolytic degradation of the aortic wall ................................................................. 13
1.2.4.3 Genetic predisposition to AAA and stresses within the aortic wall ........................... 16
1.2.7.1 Chemically induced AAA mouse models ................................................................. 19
1.2.7.2 Genetically modified AAA mouse models ............................................................... 19
1.2.7.3 Combined gene knockout and chemically induced AAA .......................................... 20
1.2.10.1 Synthesis and localisation of UCNs ....................................................................... 28
1.2.10.2 Urocortin receptors ................................................................................................ 29
1.2.10.3 Urocortin signalling ............................................................................................... 30
1.2.10.4 Urocortins: Protective or destructive force in the cardiovascular system? ............... 32
1.2.10.5 Studies in animal models ....................................................................................... 33
1.2.10.6 In vitro investigations of the UCNs ........................................................................ 35
1.2.10.7 Circulating UCNs concentration in CVD ............................................................... 37
1.2.10.8 The effect of UCNs administration in human subjects ............................................ 39
CHAPTER 2 ...................................................................................................................... 45
MATERIALS AND METHODS ...................................................................................... 45
2.1 Materials....................................................................................................................... 46
2.1.1 Mice……. .................................................................................................................. 46
2.1.2 Western blot and immunoprecipitation ....................................................................... 46
2.1.3 Real time PCR ............................................................................................................ 46
2.1.4 Histology and immunohistochemistry ......................................................................... 47
2.1.5 ELISA….. .................................................................................................................. 47
2.1.6 Tissue culture ............................................................................................................. 48
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2.1.7 Proliferation/viability, apoptosis and cell cycle assays ................................................ 48
2.2 Mouse Studies - The role of urocortin 3 (UCN3) in a mouse model of AAA ................ 48
2.3 Human studies .............................................................................................................. 49
2.3.1 Study Design .............................................................................................................. 49
2.3.2 Patients…. .................................................................................................................. 50
2.3.3 Plasma samples .......................................................................................................... 51
2.3.4 Aortic and atheroma samples ...................................................................................... 51
2.3.5 Gene expression studies .............................................................................................. 53
2.3.6 Histology. ................................................................................................................... 55
2.3.7 Immunohistochemistry ............................................................................................... 56
2.3.8 Assessment of UCN2 by ELISA ................................................................................. 57
2.4 In vitro investigations ................................................................................................... 58
2.4.1 Explant culture ........................................................................................................... 58
2.4.2 Cell culture ................................................................................................................. 58
2.4.3 Determination of human aortic smooth muscle cell counts .......................................... 59
2.4.4 Akt signalling assessment ........................................................................................... 60
2.4.5 Examination of cytokine secretion .............................................................................. 61
2.4.6 Determination of HASMC proliferation ...................................................................... 63
2.4.7 Apoptosis investigations ............................................................................................. 63
2.4.8 Cell cycle investigations using flow cytometry ........................................................... 65
2.5 Statistical analysis ........................................................................................................ 66
CHAPTER 3 ...................................................................................................................... 68
THE ASSOCIATION OF UROCORTIN 3 (UCN3) WITH ABDOMINAL AORTIC
ANEURYSM ..................................................................................................................... 68
3.1 Introduction ................................................................................................................. 69
3.2 Materials and methods ................................................................................................ 72
3.2.1 Effects of angiotensin II (AII) infusion on ApoE-/- mouse aortic diameter and blood
pressure……. ...................................................................................................................... 72
3.2.1.1 Mice….. .................................................................................................................. 72
3.2.1.2 Angiotensin II treatment to induce AAA .................................................................. 72
3.2.1.3 Blood pressure and heart rate determination............................................................. 73
3.2.1.4 Ultrasound and plasma assessment of aneurysm progression ................................... 73
3.2.1.5 Aorta morphometry ................................................................................................. 74
3.2.2 Comparison of urocortin 3 (UCN3) levels in aneurysmal versus non-aneurysmal mice76
3.2.2.1 Extraction of aortic proteins ..................................................................................... 76
3.2.2.2 Mouse UCN3 ELISA ............................................................................................... 77
3.2.3 Expression of the UCN3 receptor CRFR2 expression in aortas from aneurysmal and
non-aneurysmal mice .......................................................................................................... 78
3.2.3.1 Extraction of aortic proteins ..................................................................................... 78
3.2.3.2 Immunoprecipitation ............................................................................................... 78
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3.2.3.3 Western blotting ...................................................................................................... 79
3.2.4 Statistics .................................................................................................................... 80
3.3 Results .......................................................................................................................... 82
3.3.1 Blood pressure is increased in ApoE-/- mice following AII infusion ............................ 82
3.3.2 Aneurysm progression in angiotensin II (AII) infused ApoE-/- mice ............................ 84
3.3.3 Aortic diameter morphometry in the aneurysmal and non-aneurysmal mice ................ 87
3.3.4 UCN3 protein is not upregulated in mice with AAA ................................................... 90
3.3.5 Expression of CRFR2 in mouse aorta ......................................................................... 92
3.4 Discussion .................................................................................................................... 97
CHAPTER 4 .................................................................................................................... 101
UROCORTIN 2 AND THE PRESENCE OF HUMAN ABDOMINAL AORTIC
ANEURYSM ................................................................................................................... 101
4.1 Introduction ............................................................................................................... 102
4.2 Materials and methods .............................................................................................. 105
4.2.1 Study design and patients ......................................................................................... 105
4.2.2 Aortic and atheroma samples: ................................................................................... 106
4.2.3 Expression of the UCNs and CRFR1/ 2 genes in human AAA biopsies .................... 106
4.2.4 Immunohistochemical staining for UCN2, lymphocytes and neutrophils .................. 106
4.2.5 UCN2 secretion from AAA biopsies ......................................................................... 107
4.2.6 Circulating concentration of UCN2 in AAA ............................................................. 110
4.2.7 Determination of plasma UCN2 by ELISA ............................................................... 110
4.2.7 Statistics ................................................................................................................... 111
4.3 Results ........................................................................................................................ 112
4.3.1 Primer design and real time PCR optimisation .......................................................... 112
4.3.2 UCN2 and its receptor, CRFR2 expression are upregulated in human AAA.............. 115
4.3.3 Assessment of UCN2 protein expression in AAA biopsies ....................................... 117
4.3.4 UCN2 is secreted by AAA explant biopsies .............................................................. 120
4.3.5 Circulating UCN2 is significantly elevated in AAA and correlates with aortic
diameter……. ................................................................................................................... 123
4.4 Discussion .................................................................................................................. 127
CHAPTER 5 .................................................................................................................... 132
UROCORTIN 2 (UCN2) FUNCTIONAL EFFECT ON SMOOTH MUSCLE CELLS
IN VITRO IN HUMAN ABDOMINAL AORTIC ANEURYSM .................................. 132
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5.1 Introduction ............................................................................................................... 133
5.2 Materials and methods .............................................................................................. 136
5.2.1 Determination of UCN2 secretion from AAA thrombus explant cultures and
concentration used in the in vitro investigations ................................................................ 136
5.2.2 Cell culture ............................................................................................................... 136
5.2.3 Determination of the efficacy of the AlamarBlue® cell proliferation and viability
assay……….. .................................................................................................................... 137
5.2.4 Determination of the optimal concentration range of UCN2 and Ast-2B for in vitro
studies……… ................................................................................................................... 137
5.2.5 HASMC proliferation assay ...................................................................................... 138
5.2.6 Akt signaling in HASMC ......................................................................................... 139
5.2.7 Cytokine assays ........................................................................................................ 140
5.2.8 Apoptosis investigations ........................................................................................... 140
5.2.9 Cell cycle progression analysis ................................................................................. 141
5.2.10 Statistics ................................................................................................................. 142
5.3 Results ........................................................................................................................ 143
5.3.1 Efficacy of the AlamarBlue® cell proliferation and viability assay ............................ 143
5.3.2 Optimal concentration range of UCN2 and Ast-2B for in vitro studies ...................... 144
5.3.3 UCN2 inhibited Akt phosphorylation in HASMC ..................................................... 145
5.3.4 Effect of UCN2 on HASMC cytokine secretion ........................................................ 148
5.3.5 UCN2 inhibited HASMC proliferation ..................................................................... 150
5.3.6 AAA thrombus conditioned media inhibits HASMC proliferation ............................ 152
5.3.8 UCN2 had no significant effect on HASMC apoptosis .............................................. 155
5.3.10 UCN2 induces a G1 arrest in HASMC .................................................................... 157
5.4 Discussion .................................................................................................................. 161
CHAPTER 6 .................................................................................................................... 169
GENERAL DISCUSSION AND FUTURE DIRECTIONS ........................................... 169
6.1 General Discussion .................................................................................................... 170
6.2 Limitations and future directions ............................................................................. 176
6.3 Summary ................................................................................................................... 178
APPENDIX 1- ETHICS APPROVAL ............................................................................... 181
APPENDIX 2- STANDARD CURVES ............................................................................ 189
APPENDIX 3- LIST OF SUPPLIERS .............................................................................. 193
APPENDIX 4- REAGENTS AND SOLUTIONS .............................................................. 196
REFERENCES ................................................................................................................. 200

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DECLARATION
I, the undersigned declare that this research investigation is carried out on my own and it has
not been previously submitted anywhere for another degree or diploma at any university or
institution of tertiary education in or out of Australia. Information derived from the published
or unpublished works of others has been acknowledged in the text and a list of references is
given.
Theophilus I Emeto June 2013

v
STATEMENT OF ACCESS TO THESIS
I, the undersigned, author of this thesis, understand that James Cook University will make
this work available for use within the university library and, by microfilm or other
photographic means, allow access to users in other approved libraries. All users consulting
this thesis will have to sign the following statement.

‘In consulting this thesis, I agree not to copy or closely paraphrase it in whole or in part
without written consent of the author, and to make proper written acknowledgement for any
assistance which I have obtained from it.’

Theophilus I Emeto June 2013

vi
ACKNOWLEDGEMENTS
First, all glory to God for mercies and grace and for teaching me to appreciate the important
things in life. Most PhD candidature involves a convoluted dance balancing between rewards
and pitfalls, whilst hoping that one has more gains than frustration. Unlike most PhD
candidature, mine came with extra obstacles which I call ‘stepping stones’ towards greatness.
The last two years of my candidature was undertaken whilst being the sole carer for my
darling wife who had been diagnosed with Philadelphia positive B-cell acute lymphoblastic
leukaemia just before our first wedding anniversary. She went on to have an allogeneic stem
cell transplant from a generous and kind hearted German girl. Emma is more than one year
cancer free today due to this kind girl, the amazing doctors and to the glory of God. I could
not have completed this PhD without the amazing support, love and prayers from everyone I
know.

My sincere thanks to my supervisors: Prof. Jonathan Golledge, Drs Catherine Rush, Lynn
Woodward and Joseph Moxon for their constant encouragement, guidance and the confidence
they have in me to complete this work. Thanks to the School of Medicine and Dentistry,
James Cook University, for providing a friendly, stimulating and enjoyable environment that
allowed me undertake a worthwhile research project within the doctoral program and for
financial and administrative support. I also thank all the members of the Vascular Biology
Unit both past and present for help and support within the confines of my PhD candidature
and beyond. I can’t fail to express my profound gratitude to all the friends who made
Townsville home.

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