On the genetics of intracranial aneurysm and on growth - Helda

152 Pages·2012·3.42 MB·English

by  Emilia Gaal

On the Genetics of Intracranial
Aneurysms and on
Growth Factor Induced Angiogenesis
in the Murine Brain

Emília Ilona Gaál

ACADEMIC DISSERTATION

To be publicly discussed, with the permission of the Faculty of Medicine
of the University of Helsinki, in the Lecture Hall 1 of Töölö Hospital
on the 30th of November, 2012 at 12 o´clock noon.

The Neurosurgery Research Group
National Institute for Health and Welfare
Institute for Molecular Medicine Finland
Molecular/Cancer Biology Laboratory
Haartman Institute and Biomedicum Helsinki,
Faculty of Medicine, University of Helsinki, Finland
and
The Yale Neurogenetics Program
Yale School of Medicine, New Haven, CT, USA

Helsinki 2012
Supervisors
Mika Niemelä, M.D., Ph.D.
Associate Professor of Neurosurgery
Department of Neurosurgery
Helsinki University Central Hospital
Helsinki, Finland

Aarno Palotie, M.D., Ph.D.
Professor
Wellcome Trust Sanger Institute
Cambridge, United Kingdom
Institute for Molecular Medicine Finland (FIMM) and
University of Helsinki
Helsinki, Finland

Kari Alitalo, M.D., Ph.D.
Research Professor of the Finnish Academy of Sciences
Molecular/Cancer Biology Laboratory
Haartman Institute and Biomedicum Helsinki
University of Helsinki
Helsinki, Finland

Reviewers
Maris Laan, Ph.D.
Professor at the Institute of Molecular and Cell Biology
Faculty of Biology and Geography
University of Tartu
Tartu, Estonia

Ville Leinonen, M.D., Ph.D.
Associate Professor of Neurosurgery
Department of Neurosurgery
University of Eastern Finland
Kuopio, Finland

Discussed with
Iver A. Langmoen M.D., Ph.D.
Professor of Neurosurgery
Vilhelm Magnus Laboratory for Neurosurgical Research
Institute for Surgical Research
Department of Neurosurgery
Oslo University Hospital and University of Oslo
Oslo, Norway

Cover photos: Jere Paavola & Emília Ilona Gaál

ISBN 978-952-10-8368-6 (paperback)
ISBN 978-952-10-8369-3 (pdf)

Helsinki University Print
Helsinki, Finland 2012
“I think that only daring speculation can lead
us further and not accumulation of facts.”

Albert Einstein

“Judah Folkman (who was both a surgeon and
a basic scientist) once said that the difference
between surgeons and basic scientists is that
when someone can’t reproduce the results of a
basic scientist, the scientist becomes alarmed.
When someone can’t reproduce the results of a
surgeon, the surgeon takes it as a compliment to
their superior skills.”

Brent R. Stockwell: The Quest for the Cure
Author’s contact information:

Emília Ilona Gaál
Department of Neurosurgery
Helsinki University Central Hospital
Topeliuksenkatu 5
00260 Helsinki
Finland
mobile: [phone]
fax: [phone]
e-mail: [email protected]
Table of Contents
Table of Contents ........................................................................................... 6
List of Original Publications .......................................................................... 11
Abstract........................................................................................................ 13
Abbreviations ............................................................................................... 15
Introduction ................................................................................................. 19
Review of Literature ...................................................................................... 21
1 Special Features of Cerebral Circulation ............................................................. 21
1.1 General Features ......................................................................................... 21
1.2 Cerebral Blood Supply ................................................................................ 22
1.3 The Blood-Brain Barrier ............................................................................. 23
1.4 The Cerebral Artery Wall ........................................................................... 24
2 Common Pathologies of the Cerebral Vasculature ................................................. 25
2.1 General Classification of Cerebrovascular Disorders .................................. 25
2.2 Intracranial Aneurysms ............................................................................... 25
2.2.1 Epidemiology of IA ............................................................................ 26
2.2.2 Rupture of IA causes SAH.................................................................. 26
2.2.3 Epidemiology of SAH ......................................................................... 26
2.2.4 Risk Factors of IA and SAH ............................................................... 27
2.2.5 Histopathology of IA .......................................................................... 27
2.2.6 Diagnosis and Treatment of IA and SAH ........................................... 28
2.2.7 Prevention of IA and SAH ................................................................. 29
2.3 Cerebral Hypoperfusion ............................................................................. 30
2.3.1 Acute Cerebral Hypoperfusion ........................................................... 30
2.3.2 Chronic Cerebral Hypoperfusion ....................................................... 31
2.3.3 Risk Factors of Cerebral Hypoperfusion ............................................. 33
3 Genetics of Intracranial Aneurysm .................................................................... 34
3.1 The Human Genome ................................................................................. 34
3.1.1 The Structure of the Human Genome ............................................... 34
3.1.2 Common and Rare Variations in the Genome .................................. 35
3.1.3 Heritability ......................................................................................... 37
3.2 Mendelian Genetics .................................................................................... 39
3.2.1 Mendelian Diseases ............................................................................ 39
3.2.2 Linkage and Linkage Disequilibrium .................................................. 40
3.3 Genetics of Complex Diseases .................................................................... 44
3.3.1 Complex Diseases ............................................................................... 44
3.3.2 Susceptibility ...................................................................................... 45
3.3.3 Association and GWAS...................................................................... 45
3.4 Studies on the Genetics of IA Before the “GWAS Era” .............................. 49
3.4.1 Linkage Studies .................................................................................. 50
3.4.2 Candidate Gene Studies ..................................................................... 52
3.4.3 Associations with Other Disorders ..................................................... 54
3.5 9p21, the First Common Susceptibility Locus for IA .................................. 54
3.6 Genetics of SAH ......................................................................................... 55
4 Aiming to Tame Angiogenesis for Therapy ........................................................... 56
4.1 Functions of the Cardiovascular System ..................................................... 56
4.2 Vasculogenesis and Angiogenesis ................................................................ 57
4.3 Biology of Angiogenesis .............................................................................. 58
4.3.1 VEGFs and Their Receptors ............................................................... 58
4.3.2 The Angiopoietins and their Tie Receptors ........................................ 61
4.4 Distinctive Angiogenic Features of the Central Nervous System................. 62
4.4.1 Brain Angiogenesis During Development .......................................... 62
4.4.2 Neuronal Effects of Angiogenic Factors.............................................. 63
4.5 Anti-Angiogenic Therapy ............................................................................ 64
4.5.1 In Cancer ........................................................................................... 64
4.5.2 In Neovascular Age-Related Macular Degeneration ............................ 65
4.6 Towards Pro-Angiogenic Therapy ............................................................... 66
Aims of the Study ......................................................................................... 67
Materials and Methods .................................................................................. 69
1 Association Studies (I-III) ........................................................................................ 69
1.1 Genome-wide Association Studies (I,II) ...................................................... 69
1.1.1 Population samples in the GWASs .................................................... 69
1.1.2 Methods in the GWASs ..................................................................... 70
1.1.3 Copy Number Variation Analysis in the GWAS cohorts ................... 72
1.2 Association Analysis of IA Risk Loci with Blood Pressure (III) ................... 74
1.2.1 Population Samples ............................................................................ 74
1.2.2 Methods of IA Risk Loci Association Analysis with BP ...................... 74

The Neurosurgery Research Group Intracranial aneurysm genetics and
7 brain angiogenesis
Emília Ilona Gaál/2012 growth factor induced CNS angiogenesis
2 Growth Factor Induced Angiogenesis in the Murine CNS (IV) ................................. 77
2.1 Methods Used in Model Organism ............................................................ 77
2.1.1 Generation of Adeno-Associated Virus Vectors and Intracranial
Injections .................................................................................................... 77
2.1.2 Immunohistochemistry ...................................................................... 77
2.1.3 Imaging Modalities and Statistical Analysis ........................................ 78
Results and Discussion .................................................................................. 81
1 Common Genetic Susceptibility to IA (I,II) ............................................................... 81
1.1 The First GWAS Identified Three IA Risk Loci at 2q33.1, 8q11.23-q12.1
and 9p21.3 ....................................................................................................... 81
1.1.1 The Genes at and Nearby 2q33.1, 8q11.23–q12.1 and 9p21.3 .......... 83
1.2 The Second GWAS Confirmed 8q12.1 and 9p21.3 and Identified Three
New Loci .......................................................................................................... 84
1.2.1 The Genes at and Nearby 10q24.32, 13q13.1 and 18q11.2 ............... 86
1.3 The Five Loci Together Explain Only Little of the Familial Risk ................ 86
1.4 No Top Loci Associated with Gender, Family History or Age .................... 86
1.5 No Loci Associated with SAH .................................................................... 87
1.6 No Association within Previously Identified Linkage Intervals ................... 87
1.7 CNVs in IA ................................................................................................ 87
1.7.1 Rare and Common Variants Affect the Same Pathways ..................... 88
1.8 Possible Mechanism of IA Formation Based on GWAS Results ................. 88
2 Suggestive IA Risk Locus is Associated with High Blood Pressure (III) ........................ 89
2.1 IA Risk Loci Association Analysis with BP ................................................. 89
2.2 Fine-Mapping the 5q23.2 Region ............................................................... 90
2.3 Possible Mechanism of IA and High SBP Risk ........................................... 91
2.4 Causality or Pleiotropy? .............................................................................. 91
3 CNS Angiogenesis in a Model Organism (IV) ...................................................... 93
3.1 Therapeutic Brain Revascularisation........................................................... 93
3.2 PlGF Promotes the Formation of Arterialised Microvessels ........................ 93
3.2.1 Significant Increase of Microvessel Density ........................................ 93
3.2.2 PlGF Induced Microvessels Arterialise ............................................... 94
3.3 PlGF Induced Angiogenesis Does Not Incite Significant Side Effects ......... 95
3.3.1 No Strong Inflammation .................................................................... 95
3.3.2 No Marked Gliosis or Neuronal Rearrangement ................................ 95
3.3.3 BBB is Intact and Functional ............................................................. 96
3.3.4 Intact In Vivo Anatomy ...................................................................... 96
3.4 PlGF Is the Prime Candidate Vascular Growth Factor for CNS
Revascularisation .............................................................................................. 97
Present State and Future Perspective.............................................................. 99
1 Overhauling the Status Quo of IA ........................................................................... 99
1.1 Is IA a Disease of its Own? Is IA Genetic?................................................... 99
2 Quo Vadis IA Research? ......................................................................................... 99
2.1 Search for Finnish Specific IA Loci........................................................... 100
2.2 Mendelian Boom No 2 and Beyond ......................................................... 100
2.3 Genetic Diagnostics in Sporadic IA is Unlikely ........................................ 101
2.4 Shaping Future Therapies ......................................................................... 101
2.5 Further Epidemiological Studies are Needed ............................................ 101
2.6 The Haves and the Have-Nots................................................................... 102
3 The $1000 Genome ............................................................................................. 103
3.1 Recreational Genetics ............................................................................... 103
4 Preventive Indirect Bypass Surgery in Local Anaesthesia: Go-go Bypass ....................... 104
4.1 Challenges of Current Cerebral Revascularisation Procedures ................. 104
4.2 PlGF Enhanced Multiple Bur Hole EC-IC Bypass .................................... 104
Conclusions ................................................................................................ 107
Acknowledgements ..................................................................................... 109
References .................................................................................................. 111

The Neurosurgery Research Group Intracranial aneurysm genetics and
9 brain angiogenesis
Emília Ilona Gaál/2012 growth factor induced CNS angiogenesis