validation and mechanism studies of novel therapeutic compounds modulating angiogenesis ·...
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VALIDATION AND MECHANISM STUDIES OF NOVEL THERAPEUTIC
COMPOUNDS MODULATING ANGIOGENESIS
By Jennifer Tat
A thesis submitted in conformity with the requirements for the degree of Master of Science
Department of Physiology
University of Toronto
Supervisor: Dr. Xiao-Yan Wen
Co-supervisor: Dr. Mingyao Liu
© Copyright by Jennifer Tat 2013
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ABSTRACT
Validation and mechanism studies of novel therapeutic compounds modulating angiogenesis
Jennifer Tat
Master of Science 2013 Department of Physiology
University of Toronto
Discovering novel compounds that stimulate or abrogate angiogenesis can lead to development of new therapeutic agents that may effectively treat diseases with pathological angiogenesis. The zebrafish model allows for a whole-organism approach to drug discovery. Advantages over other animal models include small embryo size, fecundity, rapid embryonic development, optical clarity and easy accessibility of the embryos. My goal is to validate the therapeutic efficacy and identify the molecular mechanisms of action of three compounds identified from our previous chemical genetic screens. Fenretinide promoted angiogenesis in zebrafish embryos but inhibited the angiogenesis-dependent process of fin regeneration. The pro-angiogenic effects of fenretinide appear secondary to the stimulation of somitogenesis. I3M potently inhibited angiogenesis and fin regeneration, and may act partially through the notch pathway. Lastly, I validated the anti-angiogenic effect of a novel compound DHM. Comprehensively, my studies support the utility of zebrafish as a versatile tool for anti-angiogenic drug discovery.
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ACKNOWLEDGEMENTS
I was forewarned about the isolative nature of research prior to my entry into graduate school. This is partly true, as I can account for dozens of hours of solitary data collection and analysis. However, I can also affirm that I have received a wealth of advice, support and sense of community in the course of this program, which has made the past two years fruitful as well as enjoyable.
I am grateful to my supervisor Dr. Xiao-Yan Wen for accepting me into his laboratory, for providing essential guidance during my training and for giving me opportunities to present my work and to act as a mentor to others. Additionally, I would like to express gratitude to my co-supervisor Dr. Mingyao Liu for his kind advice and critical feedback throughout my research training. I would also like to thank my committee members, Drs. Philip Marsden and Haibo Zhang, for their valued input, offered resources and words of encouragement.
I would like to recognize past and present Wen lab members - Wing, Dave, Suzan, Youdong, Dingyan, Rei, Antonio and Filip – and 5th floor LKS researchers for their technical assistance, advice and reagents. Thanks to Dave and Pamela for their critical reading of abstracts and manuscripts. Thanks to Wing, Pamela and Dingyan for their help with cell and molecular work. Thanks to Suzan for enduring RNA synthesis with me, and Youdong for his expertise and patience with the innumerable amount of microinjections we did together. There are a lot of beautiful zebrafish images throughout my thesis that I cannot entirely take credit for, as I received microscope training and general help from Youdong, Judy and Antonio. I would also like to express appreciation for the camaraderie, the laughs and the counsel of my fellow occupants in grad room 512 (A.K.A the fish bowl).
The external research community has been very open and supportive. Within the University of Toronto, I would like to thank Dr. Jason Fish for his vital tips on ISH and notch, Dr. Ian Scott for providing the Tg(flk1:EGFP) and Tg(GATA1:dsRED) strains, James (Medin lab) for transfecting our cancer cell lines, and Keisha (Gerlai lab) and Angela (Scott lab) for their tips on improving the survival of larval zebrafish. Additional thanks to Dr. Sarah Childs and Christian Lawrence (Zon lab) for providing the Tg(fli1:nEGFP)y7 and Tg(fli1:EGFP;casper) strains respectively, and Dr. Konstatin Stoletov for the MDA435 cell line and advice with xenograft fish models.
Lastly, I would like to thank my family and friends for putting up with me through my master’s degree and beyond. I am greatly indebted to their abundant support during the high and low points of the past two years.
I must also recognize the sacrifice of thousands of zebrafish for their essential contributions to my project and the field of biomedical research as a whole.
I am not certain what the future holds but aspire to continue contributing to research knowledge and its applications to improve outcomes in clinical practice.
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TABLE OF CONTENTS
ABSTRACT ................................................................................................................................... ii
ACKNOWLEDGEMENTS ........................................................................................................ iii
TABLE OF CONTENTS ............................................................................................................ iv
LIST OF ABBREVIATIONS .................................................................................................... vii
LIST OF FIGURES .................................................................................................................... xii
LIST OF APPENDICES ........................................................................................................... xiii
CHAPTER 1 – GENERAL INTRODUCTION ......................................................................... 1
CHAPTER 2 – RATIONALE and HYPOTHESES ................................................................ 33 2.1 Rationale .......................................................................................................................................... 33 2.2 Objective and General Hypothesis ................................................................................................ 38 2.3 Specific Hypotheses ......................................................................................................................... 39
CHAPTER 3: ROLES OF 4-HYDROXYPHENYL RETINAMIDE IN ANGIOGENESIS AND ITS POTENTIAL CELLULAR AND MOLECULAR MECHANISMS .................... 40
3.1 Introduction ..................................................................................................................................... 40 3.1.1 Retinoids: Physiology and pharmaceutical development ....................................................... 40 3.1.2 Fenretinide as an anti-cancer agent ......................................................................................... 42 3.1.3 Additional applications of fenretinide .................................................................................... 44 3.1.4 Rationale ................................................................................................................................. 45
3.2 Materials and Methods ................................................................................................................... 46 3.2.1 Preparation of drugs and reagents ........................................................................................... 46 3.2.2 Zebrafish strains and husbandry ............................................................................................. 46 3.2.3 Zebrafish developmental angiogenesis assay ......................................................................... 47 3.2.4 In vivo quantification of EC number ...................................................................................... 48 3.2.5 Fin regeneration assay ............................................................................................................. 49 3.2.6 Whole-mount immunohistochemistry (IHC) .......................................................................... 50 3.2.7 Whole-mount RNA in situ hybridization (ISH) ...................................................................... 51
3.3 Results .............................................................................................................................................. 56 3.3.2 Fenretinide alters the number of endothelial cells in blood vessels ....................................... 57 3.3.3 Fenretinide inhibits fin regeneration ...................................................................................... 58 3.3.5 Fenretinide increases RAR expression and may decrease FGFR expression ........................ 62
3.4 Discussion ......................................................................................................................................... 64 3.4.1 Fenretinide promotes developmental angiogenesis but inhibits EC migration ...................... 64 3.4.2 Fenretinide inhibits the process of fin regeneration ................................................................ 65 3.4.3 Fenretinide promotes angiogenesis through somitogenesis .................................................... 66 3.4.4 Fenretinide’s effect on somitogenesis ..................................................................................... 67 3.4.5 The teratology of retinoids and fenretinide ............................................................................. 69 3.4.6 Conclusions and future directions ........................................................................................... 71
3.5 Appendix .......................................................................................................................................... 73
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CHAPTER 4: ANTI-ANGIOGENIC ACTIVITY AND POTENTIAL MOLECULAR MECHANISMS OF INDIRUBIN-3-MONOXIME ................................................................. 75
4.1 Introduction ..................................................................................................................................... 75 4.1.1 Traditional Chinese medicine and the discovery of indirubin ................................................ 75 4.1.2 Applications and properties of indirubin ................................................................................ 76 4.1.3 Molecular mechanisms of indirubins ...................................................................................... 77 4.1.4 Indirubin-3-monoxime as an anti-cancer agent ...................................................................... 79 4.1.5 Additional applications of I3M ............................................................................................... 79 4.1.6 Rationale ................................................................................................................................. 80
4.2 Materials and Methods ................................................................................................................... 81 4.2.1 Preparation of drugs and reagents ........................................................................................... 81 4.2.2 Zebrafish strains and husbandry ............................................................................................. 81 4.2.3 Cell lines ................................................................................................................................. 81 4.2.4 Zebrafish developmental angiogenesis assay ......................................................................... 81 4.2.4 In vivo quantification of EC number ...................................................................................... 81 4.2.5 Fin regeneration assay ............................................................................................................. 83 4.2.6 Embryonic tumour xenograft model ....................................................................................... 83
4.3 Results .............................................................................................................................................. 86 4.3.1 I3M decreases ISV number in developing zebrafish embryos ............................................... 86 4.3.2 I3M inhibits endothelial cells migration in vivo ..................................................................... 87 4.3.3 I3M inhibits larval and adult fin regeneration ........................................................................ 88 4.3.4 Development of zebrafish tumour angiogenesis model and preliminary test of I3M ............. 89 4.3.5 DAPT partially rescues the I3M chemical genetic phenotype ................................................ 93
4.4 Discussion ......................................................................................................................................... 95 4.4.1 I3M demonstrates specific inhibition of developmental angiogenesis ................................... 95 4.4.2 I3M inhibits fin regeneration .................................................................................................. 96 4.4.3 I3M efficacy in tumour models ............................................................................................... 97 4.4.4 I3M inhibits zebrafish ISV development partially through the notch pathway ...................... 97 4.4.5 Conclusions and future directions ......................................................................................... 100
Appendix .............................................................................................................................................. 102
CHAPTER 5: THE ROLE OF DHM IN ANGIOGENESIS ................................................ 105 5.1 Introduction ................................................................................................................................... 105
5.1.1 Rotenoids .............................................................................................................................. 105 5.1.2 The anti-cancer activities of rotenoids .................................................................................. 106 5.1.3 Dihydromunduletone ............................................................................................................ 108
5.2 Materials and Methods ................................................................................................................. 109 5.2.1 Preparation of drugs and reagents ......................................................................................... 109 5.2.2 Zebrafish strains and husbandry ........................................................................................... 109 5.2.3 Zebrafish developmental angiogenesis assay ....................................................................... 109 5.2.4 In vivo quantification of EC number .................................................................................... 109 5.2.5 Fin regeneration assay ........................................................................................................... 109
5.3 Results ............................................................................................................................................ 110 5.3.1 DHM decreases ISV number in developing zebrafish embryos ........................................... 110
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5.3.2 DHM alters the number of endothelial cells in ISVs ............................................................ 111 5.3.3 DHM inhibits adult fin regeneration ..................................................................................... 112
5.4 Discussion ....................................................................................................................................... 114 5.4.1 DHM inhibits developmental angiogenesis .......................................................................... 114 5.4.2 DHM inhibits adult fin regeneration ..................................................................................... 114 5.4.3 DHM toxicity ........................................................................................................................ 114 5.4.4 DHM future studies ............................................................................................................... 115 5.4.5 Conclusions and future directions ......................................................................................... 116
5.5 Appendix ........................................................................................................................................ 117
CHAPTER 6 – GENERAL DISCUSSION AND FUTURE DIRECTIONS ....................... 118
CHAPTER 7 – GENERAL CONCLUSIONS ........................................................................ 122
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LIST OF ABBREVIATIONS
4HPR 4-hydroxyphenyl retinamide (or fenretinide)
ADME absorption, distribution, metabolism and excretion
ALS amyotrophic lateral sclerosis
AMD age-related macular degeneration
Ang-1 angiopoietin-2
Ang-2 angiopoietin-2
ANOVA analysis of variance
ATP adenosine triphosphate
bFGF basic fibroblast growth factor
BSA bovine serum albumin
CAD coronary artery disease
CAM chorioallantoic membrane
CDKs cyclin-dependent kinases
cDNA complementary DNA
CF cystic fibrosis
CLSM confocal laser scanning microscopy
CNV copy number variation
COPD chronic obstructive pulmonary diseases
CRABP cellular retinoic acid binding proteins
DA dorsal aorta
DAPT γ-secretase (notch) inhibitor
DHM dihydromunduletone
DLAV dorsal longitudinal anastomatic vessel
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Dll4 delta-like ligand 4 (notch ligand)
DMEM Dulbecco’s modified eagle medium
DMSO dimethyl sulfoxide
DNA deoxyribonucleic acid
Dpf days post fertilization
Dpa days post amputation
Dpi days post injection
DsRed red fluorescence protein
DU145 prostate cancer cell line
ECs endothelial cells
ECM extracellular matrix
EDTA ethylene diamine tetraacetic acid
EGF epidermal growth factor
eNOS endothelial nitric oxide synthase
FBS fetal bovine serum
FDA food and drug administration (American)
FGF fibroblast growth factor
FGF2 fibroblast growth factor 2
FGFR1a fibroblast growth factor receptor 1 alpha
G-CSF granulocyte colony-stimulating growth factor
GFP green fluorescence protein
GSK-3 glycogen synthase kinase-3
HIF-1 hypoxia inducible factor 1
HM hybridization mix
Hpf hours post fertilization (age of zebrafish)
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HSCs hematopoietic stem cells
Hsp90 heat shock protein 90
HTS high-throughput system
HUVECs human umbilical vein endothelial cells
I3M indirubin-3-monoxime
IC50 half maximal inhibitory concentration
IGF-1 insulin-like growth factor-1
IHC immunohistochemistry
IL-6 interleukin-6
IL-8 interleukin-8
ISH in situ hybridization
ISVs intersegmental vessels
iTRAQ isobaric tags for relative adn absolute quantitation
LB luria broth
LOPAC library of pharmacologically active compounds
mCherry red fluorescence protein
MDA435 breast cancer cell line
MMPs matrix metalloproteinases
MO morpholino
MRI magnetic resonance imaging
MTT methylthiazol tetrazolium
MyHC myosin heavy chain
NBT-BCIP nitro blue tetrazolium with 5-bromo-4-chloro-3-indoylphosphate
nEGFP nuclear enhanced green fluorescence protein
NF-H2O nuclease-free water
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NF-κB nuclear factor kappa B
NICD notch intracellular domain
NO nitric oxide
PAD peripheral artery disease
PBS phosphate buffered saline
PBT phosphate buffer saline with Triton-X
PC-3 prostate cancer cell line
PCR polymerase chain reaction
PCV posterior cardinal vein
PDGF platelet-derived growth factor
PFA paraformaldehyde
PI propidium iodide
PlGF placental growth factor
PPC-1 prostate cancer cell line
PTK787 vatalanib (VEGFR2 inhibitor)
PTU phenylthiourea
qRT-PCR real-time polymerase chain reaction
RA retinoic acid
RAR retinoic acid receptor
RALDH retinaldehyde dehydrogenases
RXR retinoic acid X receptors
RBP retinol binding protein
RNA ribonucleic acid
ROS reactive oxygen species
RPMI Roswell Park Memorial Institute (type of cell media)
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RTKIs receptor tyrosine kinase inhibitors
SCORE specimen in a corrected optical rotational enclosure (imaging)
SILAC stable isotope labeling by amino acids in cell culture
SIVs subintestinal vessels
SMCs smooth muscle cells
SNP single nucleotide polymorphism
SU5416 semaxanib (VEGR2 inhibitor)
SU5402 FGF inhibitor
TALEN transcription activator-like effector nuclease
TCM traditional Chinese medicine
TGFα transforming growth factor alpha
TGF-β transforming growth factor beta
TILLING targeting induced local lesions in genomes
TIMP-3 tissue inhibitor of metalloproteinase
TSP-1 thrombospondin-1
UTR untranslated region
VEGF vascular endothelial growth factor
VEGFR2 vascular endothelial growth factor receptor 2 (or Flk1)
ZFIN zebrafish model organism database
ZIRC zebrafish international resource center
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LIST OF FIGURES
Figure 1.1.1 Cells and factors in physiological angiogenesis..........................................4
Figure 1.1.3 The angiogenic balance in pathological angiogenesis.................................6
Figure 1.3.2 Fluorescent transgenic zebrafish lines and key vessels.............................18
Figure 3.2.3 Typical protocol for zebrafish developmental angiogenesis assay............47
Figure 3.2.5 The procedure for a fin regeneration assay................................................49
Figure 3.3.1 Effect of fenretinide on ISV development in developing zebrafish..........57
Figure 3.3.2 Average cell number in ISVs of fenretinide-treated zebrafish..................58
Figure 3.3.3.1 Effect of fenretinide on larval zebrafish fin regeneration..........................59
Figure 3.3.3.2 Effect of fenretinide on adult zebrafish fin regeneration...........................60
Figure 3.3.4 Labeling of somite and vessels in 4HPR and SU5416-treated embryos.. 62
Figure 3.3.5 Preliminary ISH of RARg labeling ...........................................................63
Figure 4.1.2 The structures of indirubin and its derivatives...........................................77
Figure 4.2.4 Images generated from MATLAB cell counting program........................83
Figure 4.2.6 Workflow of a zebrafish embryonic tumour xenograft assay....................85
Figure 4.3.1 Effect of I3M on ISV development in developing zebrafish.....................86
Figure 4.3.2 Average cell number in ISVs and DA of I3M-treated zebrafish...............87
Figure 4.3.3.1 Effect of I3M on larval zebrafish fin regeneration....................................88
Figure 4.3.3.2 Effect of I3M on adult zebrafish fin regeneration.....................................89
Figure 4.3.4 Zebrafish embryonic tumour xenograft model..........................................92
Figure 4.3.5 Effect of DAPT and I3M on cell numbers in DLAVs.............................. 94
Figure 5.1.3 Chemical structure of dihydromunduletone and related rotenoids..........105
Figure 5.3.1 Effect of DHM on ISV development in developing zebrafish................111
Figure 5.3.2 Average cell number in ISVs and DA of DHM-treated zebrafish...........112
Figure 5.3.3 Effect of DHM on adult zebrafish fin regeneration.................................113
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LIST OF APPENDICES
Appendix 3.4.2.1 MTT assay of HUVEC cells treated with fenretinide................................73
Appendix 3.4.2.2 Fenretinide decreases zebrafish fin regeneration in adults........................73
Appendix 3.4.3.1 Albino 2.5 dpf zebrafish treated with 4HPR and 0.5% DMSO.................74
Appendix 4.3.4.1 Xenograft of MDA435 breast cancer cells..............................................102
Appendix 4.3.4.2 Xenograft of PPC-2 prostate cancer cells................................................103
Appendix 4.3.4.3 Xenograft of DU145 prostate cancer cells...............................................104
Appendix 4.4.1 Bright field image of I3M-treated zebrafish embryos.............................104
Appendix 5.4.3 Bright field images of control and DHM-treated zebrafish embryos......117
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CHAPTER 1 – GENERAL INTRODUCTION
1.1 Angiogenesis: physiology and pathology
Angiogenesis, the formation of new blood vessels from existing ones, is a physiological
process that occurs during embryonic development. Soon after vasculogenesis establishes a
primary circulatory system formed from differentiating cells of mesodermal origin, angiogenesis
commences to support the metabolic demands of the rapidly growing embryo. Angiogenesis is
mostly quiescent throughout adulthood, though it plays a significant role in female reproduction
and tissue repair. Pathological angiogenesis underlies a variety of conditions, such as obesity,
asthma, diabetes, endometriosis and macular degeneration [1]. Angiogenesis is also a hallmark
of cancer and metastasis – tumour growth cannot be supported beyond a few millimetres if the
tumour cannot establish a link to a blood supply [2] . Considering that over 500 million people
could benefit from pro- and anti-angiogenic therapy [3], a great deal of research has been
undertaken in hopes of developing therapeutic strategies to target this process.
1.1.1 Mechanisms of angiogenesis
Studies of aberrant angiogenesis in these disorders have led to an understanding of its
mechanisms, which facilitate the development of disease treatment methods. Early during
embryonic development, the primary vascular plexus forms from the differentiation of precursor
angioblast cells in the process of vasculogenesis [4]. Secondary vessels arise from these
primitive vessels via angiogenesis; sprouting, branching and recombining in a variety of ways to
form an intricate vascular network. During development, angiogenesis is stimulated to meet the
increasing metabolic demands of growing tissues. Endothelial cells (ECs) from an existing vessel
will destabilize, proliferate and migrate to form a sprout. The cells in the stalk of the sprout
elongate and join lumens to form a tube while a basement membrane reforms. Smooth muscle
cells (SMCs) or pericytes are recruited to encircle nascent ECs. Genetic studies performed in
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vitro and in vivo in mice (Mus musculus) and zebrafish (Danio rerio) have elucidated the major
players involved in the sequence of events that occur during angiogenesis [5].
The process of angiogenesis is tightly regulated by a balance between stimulatory and
inhibitory factors in the cell environment (Fig.1.1.1). Angiogenesis is activated by a shift in the
concentration of growth factors that favour the activity of angiogenesis promoters. Vascular
endothelial growth factor (VEGF) is the predominant stimulator of angiogenesis and VEGF
signalling has been identified as a rate-limiting component of the angiogenic in states of health
and disease [6].
Angiogenesis commences with the destabilization of ECs and their subsequent invasion
into the stroma of surrounding tissues. Proteolytic enzymes plasmin and matrix
metalloproteinases (MMPs) are secreted to degrade the extracellular matrix (ECM) of ECs [7].
The vessel is further destabilized by the loosening of cell-cell contacts through VEGF-mediated
dissociation of VE-cadherin and angiopoietin-2 (Ang2)-mediated detachment of SMCs and
pericytes [7]. Vascular permeability is potentiated with VEGF activation of endothelial nitric
oxide synthase (eNOS) leading to vasodilation and EC migration [7]. Plasma proteins (such as
fibrin) extravasate to form a temporary matrix for migrating ECs [7]. Integrins found on the
provisional ECM scaffold mediate the adhesion of ECs to the ECM while EC proliferation is
stimulated by growth factors such as VEGF [8], fibroblast growth factor (FGF), platelet derived
growth factor (PDGF), transforming growth factor alpha (TGFα), epidermal growth factor (EGF)
and granulocyte colony-stimulating growth factor (G-CSF) [7].
Interactions between the VEGF and notch pathways are responsible for the coordination
of vessel sprouting which involves two types of endothelial cells; tip cells and stalk cells [9]. Tip
cells express the notch ligand Dll4 and VEGF receptor 2 (VEGFR2) while another notch ligand,
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Jagged1, is expressed in the proliferating stalk cells that form the body of the elongating sprout.
Tip cells guide the migration of the developing sprout along a gradient of soluble and matrix-
bound VEGF isoforms produced by stromal cells [10]. The reproducible vascular pattern
established during development is also driven by guidance signals that attract and repel
migrating ECs to their destination: UNC-5, plexin, slit, ephrin, neuropilin, netrin, and
semaphorin [11]. In neuronal development, Eph receptors inhibit axonal growth [12]. In vascular
development, repulsive ephrin signalling demarcates artery-vein boundaries and navigates tip
cells through somite boundaries in the trunk [11]. Neuronal and vascular cell migration share
many guidance signalling molecules, which is valid considering the similarities in the circuitry of
the two systems [11].
A lumen forms from the fusion of intracellular vacuoles and extracellular channels to
produce a patent vessel that permits blood flow [13]. Stabilization of the growing vessel occurs
through TGF-β and VE-cadherin activity, which inhibits EC proliferation [14]. TGF-β also
stimulates the ECM scaffold during tube formation and VE-cadherin strengthens the junctions
between cells to maintain a stable layer of ECs that make up the vessel wall [15]. Lastly, PDGF
and Ang-1 promote the recruitment and proliferation of pericytes and SMCs respectively, which
actively maintain the structure, survival and perfusion of the mature vessel [6].
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Figure.1.1.1 A) Cells and factors involved in the process of physiological angiogenesis [7]. B) Cells and factors involved in stabilization of the nascent vessel. Reprinted by permission from Macmillan Publishers Ltd: [Nature Medicine], [6], copyright (2003).
1.1.2 Physiological angiogenesis
Blood vessel development is a major component of embryogenesis but also has critical
roles in wound healing and female reproduction.
Wound healing is dependent on neovascularisation, as vessels are required to sustain the
metabolic demands of regenerating tissue. Wound healing occurs in four stages: coagulation,
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inflammation, proliferation and remodeling [16]. Angiogenesis is initiated immediately
following tissue damage that is augmented by inflammation and suppressed once the nascent
vasculature is stabilized. Basic fibroblast growth factor (bFGF), an angiogenesis stimulator
normally sequestered inside a cell, spills out into the ECM when a cell has been damaged [17].
Platelets and thrombin, early responders to blood clotting, promote the release and expression of
angiogenic growth factors and their receptors: VEGF, PDGF, bFGF, TGF and Ang-1 [16].
Phagocytes arrive at the wound site as part of the inflammatory response, and release more
angiogenic growth factors and cytokines such as interleukin-8 (IL-8) and tumour necrosis factor
alpha (TNF-α) to activate angiogenesis [17]. Angiogenesis is prolonged by the hypoxic state of
injured tissue, leading to the production of VEGF and NO to induce EC proliferation and
enhance local blood flow respectively [18]. Once inflammation has been resolved and oxygen
levels are normal, the levels of pro-angiogenic factors decrease and angiogenesis inhibitors are
secreted [17].
Physiological angiogenesis routinely takes place in the endometrium of the uterus during
menstruation and pregnancy. The endometrium synthesizes and secretes VEGF in a spatially and
temporally regulated manner [19]. FGF, EGF, placental growth factor (PlGF) and angiopoietins
also contribute, though VEGF is considered to be the main mediator of endometrial angiogenesis
[20]. Angiogenesis is carefully modulated during the phases of endometrial growth and
regression through changes in the levels of growth factors (e.g VEGF, PlGF, etc) and hormones
(progesterone, estrogen and human chorionic gonadotropin) [19]. An imbalance of these
components can lead to abnormal bleeding, miscarriage, defective placentation and increased
risk of numerous pregnancy disorders [19, 21].
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The complexity of physiological angiogenesis is evident from the spatial and temporal
coordination of growth factors, proteins and cell types in embryogenesis, tissue repair and
reproduction. Normal angiogenesis is tightly regulated so that it only proceeds to the extent that a
tissue requires. In fact, endothelial cells are one of the longest-lived cells in the body and EC
turnover is normally on the scale of years [22]. Angiogenesis pathologies arise from the
deregulation of elements involved in the physiological angiogenesis.
1.1.3 Pathologies with insufficient angiogenesis
Abnormal angiogenesis is a signature feature in a plethora of health conditions and can be
categorized into two classes; diseases characterized by an insufficiency or excess of angiogenesis
(Fig.1.1.3).
Figure 1.1.3 The angiogenic balance in physiological angiogenesis. Pathologies characterized by excessive and insufficient angiogenesis correspond to a change in this relative amounts of angiogenic regulators [6, 23].
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Angiogenesis can be insufficient in two ways: not enough vessel growth or abnormal
vessel deterioration. Ischemic diseases such as stroke, peripheral artery and coronary artery
diseases (PAD, CAD) are characterized by the former. Ischemia occurs in tissues that receive
inadequate blood flow, most commonly by means of physical injury or a narrowing of blood
vessels due to atherosclerosis [24]. HIF-1 expression in hypoxic tissues activates VEGF
expression, initiating the angiogenic cascade. However, the HIF-VEGF response is reduced due
to a deficiency in VEGF expression and sensitivity that is likely attributed to age [25], genetics
[26] and co-morbidity with diabetes [27].
Similarly, chronic ulcers often develop from conditions such as diabetes, varicose veins,
atherosclerosis and pressure ulcers [24]. As a result, the pathophysiology of conditions with
vascular insufficiency appears to be multifactorial and complex. For instance, wound healing
angiogenesis is disrupted in foot ulcers because of a variety of physiological states associated
with diabetes: leukocyte abnormality, bacterial infection leading to a prolonged inflammatory
response, arterial insufficiency and microangiopathy caused by increased glucose uptake in ECs
[28].
The impairment of angiogenic processes contributes to the pathophysiology of a variety
of diseases. The recession of bone formation observed in osteoporosis may develop, in part, due
to an age-dependent decline in VEGF [29]. Glomerular damage in the kidney is associated with
decreased VEGF and increased expression of the angiogenic inhibitor, glycoprotein
thrombospondin-1 (TSP-1) [30]. Endothelial dysfunction in the kidney and placenta is thought to
mediate the pathophysiology of hypertension in pre-eclampsia [31]. EC apoptosis caused by
cytokines and oxidative stress contributes to the loss of alveolar cells in chronic obstructive
pulmonary diseases (COPD) like emphysema [32]. VEGF is significantly lower in patients with
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amyotrophic lateral sclerosis (ALS) [33]. Hypoxic neural tissue produces insufficient quantities
of VEGF, leading to neuron degradation [33]. Microvascular degeneration of ECs in the brain
also occurs in Alzheimer’s disease. Accumulations of amyloid-β diminishes nitric oxide levels
and EC toxicity develops into vascular abnormalities that cause neurodegeneration [34].
Therapeutic management of these disorders may be possible through the development of
interventions that induce angiogenesis, which are discussed in section 1.2. Although a lot of
progress has been made, there remains a need for the effective treatment of millions of people
affected with insufficient angiogenesis worldwide.
1.1.4 Pathologies with excessive angiogenesis
Pathological angiogenesis occurs with the loss of regulation in angiogenic homeostasis.
In disorders featuring excessive angiogenesis, the balance is shifted towards a surplus of
angiogenic activators accompanied sometimes by the suppression or reduction of angiogenic
inhibitors.
Angiogenesis is sometimes activated in tissues that do not require it. The growth of new
blood vessels in the eye occurs in a variety of ocular diseases that result in severe vision loss
[35]. Neovascular retinal vessels displace normal retinal tissue and cause leakages in the blood-
retinal barrier, which has analogous functions to the blood-brain-barrier [35]. The resultant
ischemia and inflammation leads to considerable tissue damage [35]. VEGF, PlGF, PDGF, Ang-
2, NO and IL-6 have all been implicated in the pathogenesis of neovascularisation in the retina
and choroid of the eye [36-38]. Ocular neovascularisation can also occur from the loss of
angiogenic inhibition. For instance, mutations in a tissue inhibitor of metalloproteinase (TIMP-3)
is reported in patients with choroidal neovascularisation [39].
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Other disorders in this category feature abnormalities in the progression of angiogenesis.
Teleangiectasia, a condition characterized by severe bleeding in multiple organs, is caused by
genetic mutations in receptors of TGF-β. Decreased TFG-β signalling allows for the continuous
activation of angiogenic factors that result in the development of fragile blood vessels [40].
Studies are also underway to explore the possibility of using anti-angiogenic therapy to impede
the progression of obesity and endometriosis [41, 42].
The most prominent disease in this category is cancer, one of the leading causes of death
worldwide. Consequently, tumour angiogenesis has been extensively studied to characterize its
mechanisms and to develop targeted and more effective methods of therapy. Although cancer
comes in many forms, angiogenesis is a process to which all cancer progression is dependent.
Two distinguishing features of cancer, uncontrollable cell growth and metastasis, cannot be
sustained in the absence of neovascularisation [2].
Tumour cells induce vessel growth in several ways. Oxygen and nutrient deprivation
result in the release of factors involved in angiogenic signalling; VEGF, PDGF, FGF, Ang, and
SD1α [43]. Oncogenes such as src [44] and Kras [45] upregulate growth factors like VEGF and
HIF-1. Oncogenes also enhance the production of cytokines and proteolytic enzymes [46]. Cell
irregularity and necrosis within a developing tumour is detected as a perceived wound, and an
inflammatory response is mounted. Immune cells are recruited to the site and secrete cytokines
that further amplify the angiogenic signal [43]. Endogenous inhibitors of angiogenesis, such as
TSP-1, are also suppressed in human cancers. In a mouse overexpression model, TSP-1
suppresses tumourigenesis [47] while a null model demonstrates increased vessel density and
tumour cell proliferation [48]. This correlates to the expression levels of TSP-1 in melanoma
and breast and lung carcinomas, which are inversely proportional to malignancy [49].
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The vessels derived from excessive, tumour-induced angiogenesis are immature and
poorly constructed. The characteristic morphology of tumour vasculature is leaky and
convoluted, producing inconsistent blood flow and oxygen delivery [50]. The irregularities in
perfusion produce pockets of hypoxic tissue, which results in more angiogenic signalling. The
constant presence of angiogenesis-stimulating factors in the tumour environment not only
enhances vessel growth but also prevents its stabilization. Persistently high levels of VEGF
disrupts the development of a stable vascular barrier by producing fenestrations in ECs,
loosening cell-cell adhesion, reducing pericyte coverage and activating proteases that degrade the
basement membrane [50].
1.2 Models of angiogenesis
The investigation of the physiological and pathological processes of angiogenesis and the
identification of its modulators could not have been accomplished without the development of in
vitro and in vivo bioassays.
1.2.1 In vitro assays of angiogenesis
In vitro studies are versatile and allow for the isolation and manipulation of specific
processes and molecular events. The actions of a newly identified growth factor or anti-
angiogenic agent is routinely tested in standard assays of proliferation, migration and tube
formation in cultures of human umbilical vein endothelial cells (HUVECs), though other cell
types (e.g. fibroblasts, smooth muscle cells) can also be used.
Colourimetric MTT assays are commonly employed to evaluate relative EC growth in
microwell culture plates. The tetrazolium compound MTT is reduced in metabolically active
cells to an insoluble purple dye that is measured with a spectrophotometer [51]. The readout of
11
an MTT assay allows you to quickly infer dose-dependent effects of an agent on EC viability.
Alternative assays such as Ki-67 antibody labelling, 3H-thymidine or 5-bromo-2’-deoxyuridine
(BrdU) incorporation provide more accurate measurements of cell proliferation by quantifying
DNA content in cells.
Cell migration is often studied in scratch assays and Boyden chambers. Scratch assays are
simple, inexpensive assays that evaluate the migratory response stimulated by denuding a region
on a monolayer of ECs. Migration is measured by monitoring the rate at which the region is
repopulated [52]. Comparatively, Boyden chamber assays require less time and allow
investigators to clearly distinguish between cell proliferation and migration [53]. ECs are loaded
in a chamber and migrate across a porous membrane towards chemoattractants in the adjacent
compartment.
Tube formation can be modelled in vitro by culturing ECs on 2 or 3-dimensional matrices
comprised of fibrin, collagen or Matrigel, a gelatinous protein mixture secreted from mouse
sarcoma cells. The presence of the matrix scaffold, growth factors, and (sometimes) stromal cells
stimulate ECs to attach, migrate and differentiate into a capillary-like network of tubules [54].
1.2.2 Ex vivo assays of angiogenesis
The aortic ring assay is an organ-culture system widely used in angiogenesis research. An
explant cross-section of the aorta is imbedded in an extracellular substrate containing fibrin or
Matrigel. Vessels proliferate from the ring as ECs and stromal cells in the aorta respond to
dissection-induced injury and factors administered in the culture [55].
1.2.3 In vivo assays of angiogenesis
In vivo assays are used to study angiogenesis in a manner that incorporates the complex
regulatory environment of an organism. In the corneal micropocket assay, a polymer pellet
12
containing the bioactive agent of interest is implanted into the cornea of rodents [56]. Corneas
are transparent and normally avascular. The development of new vessels can be easily observed
and attributed to the agent that diffuses from the pellet. The chick embryo chorioallantoic
membrane (CAM) assay works in a similar manner. Pro- or anti-angiogenic agents are applied
to the CAM and vascularisation is either stimulated or suppressed. Modifications of the CAM
assay have embedded the test agent into nylon mesh to differentiate between developmental and
agent-induced angiogenesis (which grow vertically through the mesh) [57].
Matrigel plug assays incorporate elements of micropocket and CAM assays. A
preparation of Matrigel containing the test agent is injected subcutaneously and solidifies into an
avascular plug. Matrigel itself induces vessel growth into the plug (as Matrigel contains some
growth factors), so the ability of the test agent to induce or inhibit angiogenesis must be
relatively assessed. A major drawback to this technique is caused by variation in the three-
dimensional structure of the Matrigel plug, though a Plexiglas-nylon chamber has been
developed to improve the reproducibility of the assay [58].
In vivo tumour angiogenesis is mainly studied in animal transplant models. Human or
murine tumour cell lines are grafted into immune-suppressed rodents, usually mice or rats
(Rattus norvegicus). The host animal’s entire angiogenic machinery (i.e. extracellular matrix,
growth factors, stromal cells, immune and inflammatory components) is available to interact
with the tumour. Parameters such as microvascular density and tumour size are used to measure
the effect of the test agent on tumour-induced angiogenesis. Xenograft models can incorporate
transgenic animals or cell lines to study specific processes. For example, luminescent OKD48
transgenic mice can be employed to assess the oxidative stress response in vivo [59]. Anti-
angiogenic drugs are routinely assessed preclinically in xenograft models. However, the efficacy
13
of xenograft models is challenged by “species differences and inherent circumstances” that are
responsible in part, for the difficulties in translating preclinical benefits to patients [55].
Organism-scale angiogenesis can also be studied in zebrafish embryos and tadpoles
(Xenopus laevis), though the former is predominantly used. Test agents can be added directly
into the water or injected into the yolk sac in the zebrafish developmental angiogenesis assay.
The optical transparency of the embryos facilitates visual analysis of the progression of
angiogenesis and potential toxicities. Transgenic zebrafish with fluorescently-labelled ECs are
used in high-throughput screening for angiogenic modulators [60]. Models of tumour and retinal
angiogenesis have also been developed in zebrafish (see section 1.3) [61, 62]. Additionally, a
variety of transgenic or gene knockout strains and technologies such as morpholino gene
knockdown are available for mechanism studies.
Mouse and other mammalian animal models are useful in validating compounds that
modulate angiogenesis. However, because of their size and relatively small number of offspring,
mammals are unfavourable to use in large-scale screens. Zebrafish has recently emerged as an
advanced in vivo model organism for high throughput screens (HTS) for angiogenesis drug
discovery.
1.3 Zebrafish angiogenesis and cancer models in drug discovery1
1.3.1 Zebrafish as an advanced model for chemical genetic screens
The conventional process of drug development is time-consuming and expensive. The
low success rate of traditional cell-based screens demonstrates a need for whole-organism
screening strategies. Zebrafish combine the biological complexity of a whole animal model with
1 Portions of this section have been published in a review that is currently in press. Tat, J., Liu, M., Wen, XY. Zebrafish cancer and metastasis models for in vivo drug discovery. Drug Discovery Today Technologies. 2012. Figures and excerpts reprinted with permission.
14
the versatility and throughput of in vitro models. Zebrafish demonstrate many conserved
vertebrate characteristics, sharing genes and physiology with mammals despite the 400 million
years that set us apart in evolution [63]. In fact, many genes and regulatory processes involved in
angiogenesis appear to be conserved in zebrafish and mammals [64]. Yet, zebrafish retain many
invertebrate features that facilitate experimental study: small size, fecundity (high number of
offspring), oviparity (eggs hatch outside of the mother), rapid development (major organs form
within three days of hatching) and relatively low maintenance costs. Hundreds of optically
transparent embryos can be generated per mating pair and development occurs externally so that
embryos can be easily (noninvasively) observed and manipulated (using techniques such as
microinjection, cell transplantation). Small molecules can be added directly into the fish water of
multiwell plates and absorbed via diffusion, while proteins can be injected directly into the yolk
sac. The quantity of the tested drug, and technical expertise required for its evaluation are much
less than that of an equivalent study performed in mice [65]. Chemical libraries can be applied to
a large number of embryos to systematically screen for a phenotype of interest. Employing a
phenotype-based approach permits the identification of new drug candidates for diseases in
which therapeutic targets are not known.
Zebrafish are highly accessible to genetic manipulation, and a collection of genetic tools
has been developed as a consequence. Transgenesis has been particularly useful in generating
reporter lines and disease models that are used in screening. Transgenic gene overexpression,
TALEN gene knockout and morpholino knockdown technologies permits in vivo evaluation of
gene functions in specific physiological processes, which are helpful in characterizing the
mechanisms of action and target of a compound [66].
15
The zebrafish genome has almost been completely sequenced by the Sanger Center and
has been extensively annotated by the Trans-National Institutes of Health Zebrafish Genome
Initiative. There is high genetic variability, in the form of copy number variants (CNVs) and
single nucleotide polymorphisms (SNPs) in zebrafish [67]. This may be advantageous for drug
screening, as genetic factors are known to contribute to phenotypic differences. In a behavioural
zebrafish screen, the basal locomotor activity of embryos and their locomotor response to alcohol
were significantly different depending on the genetic background (strain) of the fish [68].
Conventional preclinical animal models are often inbred and may not be able to predict drug
responses that occur in a more genetically heterogeneous human population [69]. Zebrafish can
be advantageously employed in screening, since they are observed to be four times more
genetically diverse than mice [67]. However, the genetic variation in zebrafish actually exceeds
the percentage observed in humans as well [67]. While genetic diversity may be desirable in
regards to screening, it has the potential to contribute to phenotypic differences in screens
employing disease models [67]. Fortunately, highly homogeneous zebrafish (such as the inbred
India strain) [70] and genetically uniform zebrafish lines [71] are available for these applications.
Thousands of mutant lines, generated through genome-wide mutagenesis such as
chemical, targeting induced local lesions in genomes (TILLING) [72], and insertional gene
trapping, have been catalogued and are readily accessible on online databases such as Sanger,
ZFIN, ZIRC and zfishbook.org. Zebrafish gene expression can also be analyzed using tools such
as real-time polymerase chain reaction (qRT-PCR) [73] and in situ hybridization [74]. RNA
extraction, followed by analysis with DNA microarray is available for gene expression profiling
[75].
16
Additionally, assessments of drug pharmacology can be determined at an earlier stage of
drug development. Within a whole-organism system, the effects of a drug and its metabolites can
be simultaneously evaluated. Assays of teratogenicity, cardiotoxicity and neuro-sensory organ
toxicity have been also developed in zebrafish and have demonstrated good predictive ability of
compound toxicology in humans [76]. While zebrafish pharmacokinetics for absorption,
distribution, metabolism and excretion (ADME) are not well characterized, efforts are currently
underway to assess the similarities and differences in drug metabolism and bioactivation between
animal models [77]. Preliminary findings have demonstrated shared gene expression and liver
enzyme activity between human and zebrafish in response to a panel of drug compounds,
suggesting conservation in drug pharmacodynamics [78, 79].
In addition to its utility in screening, zebrafish chemical genetics can also help analyze
the target and mechanism of action of a test compound through chemical rescue experiments,
knockdowns and phenotype comparisons.
Zebrafish are uniquely qualified for use in large-scale screening and the availability of
numerous genetic tools facilitates the detailed study of candidate drug effects in vivo, prior to
preclinical testing in mammalian models. The zebrafish chemical genetic screen is a time and
cost-effective method for direct in vivo drug discovery and serves as an advanced system in drug
development.
1.3.2 Drug Mechanisms and drug target deconvolution in zebrafish
Computational and experimental methods can be employed to identify the mechanisms of
action of a compound. The computational approach uses similarities between phenotypes or
chemical structures of compounds [80]. In a proof of concept study, Chan et al. studied the
signalling pathways disrupted by VEGFR2 inhibitor vatalanib (PTK787) by performing rescue
17
experiments with AKT/PKB and eNOS mRNA injections [81]. Experimental comparisons of the
phenotypes of an unknown drug with compounds of specific, known targets (like PTK787) allow
investigators to discern the implicated pathways of drug action. Subsequent rescue experiments
of specific pathway members, alongside in vitro assays, can be performed in zebrafish to identify
the effected pathways. Genistein, a phytoestrogen isolated from soybeans, potently inhibits
subintetinal vessel (SIV) formation in a manner that phenocopies VEGFR2 inhibitors PTK787
and semaxanib (SU5416) [82]. This led the authors to state that genistein was likely to be
exerting its inhibitory angiogenic effects through VEGF signalling [82].
Other target deconvolution techniques include affinity-based approaches, which use
mammalian or zebrafish protein lysates to evaluate potential binding partners of a small-
molecule [83]. PPA was identified as a stimulator of pigmentation from a zebrafish embryo
screen of a triazine small-molecule library [84]. Affinity chromatography was able to identify its
target as the mitochondrial F1F0-ATP synthase [84]. Proteomic technologies such as mass
spectrometric-based methods SILAC (stable isotope labelling) [85] and iTRAQ (tags of equal
mass) [86] are capable of simultaneously identifying all the binding partners of a compound,
though additional experimental verification of the candidate targets is required.
1.3.3 Zebrafish angiogenesis: process and models
Zebrafish angiogenesis occurs shortly after vasculogenesis, in a manner that is conserved
in most vertebrates. Intersegmental vessels (ISVs) sprout dorsally from the dorsal aorta (DA) at
the boundary of each somite and join to form the dorsal longitudinal anastomatic vessel (DLAV)
above the neural tube along the trunk of the embryo [64]. The temporal and spatial expression of
zebrafish homologs for essential molecules involved in the process of angiogenesis (VEGF,
angiopoietins, ephrins) is comparable to their mammalian counterparts [87-89].
18
Figure 1.3.2. Three fluorescent transgenic zebrafish lines with key vessels labelled. [A] A 2.5 dpf Tg(flk1:EGFP) embryo with green fluorescence expressed in the cytoplasm of ECs. [B] A 2.5 dpf Tg(fli1:nEGFP) embryo expressing green fluorescence in the nuclei of ECs. [C] A 6 dpf Tg(GATA1:dsRed) embryo with red fluorescence in erythrocytes.
The generation of transgenic zebrafish lines with fluorescent vasculature has enabled
real-time study of angiogenesis within an intact organism throughout its vascular development
(Fig. 1.3.2). Enhanced green fluorescent protein (EGFP) is expressed under the control of
promoters that are specifically expressed in ECs (e.g. Fli, Flk, mTie2) enabling visualization of
the entire developing vasculature under fluorescent microscopy [90-92]. The number of ECs in
specific vessels can be determined by employing the Tg(Fli:nEGFP)y7 line, which has nuclear-
specific expression of EGFP [93]. The quantification of EC number allows investigators to
resolve the specific process of angiogenesis being affected by a drug. The functionality of the
blood vessels can also be evaluated in real-time using a transgenic line with fluorescence
expressed under the GATA1 promoter, which is expressed in circulating erythrocytes.
Alternatively, blood flow can be assessed using microangiography, with the injection of
fluorescent labeling agents such as quantum dots [94]. The effects of a drug on vessel patterning
19
and blood flow can be tested in isolation, or together, in the double transgenic line
Tg(Fli:EGFP;GATA1-dsRED) [95]. The zebrafish developmental angiogenesis assay is simpler,
faster and easier to quantify than CAM, corneal micropocket or other in vivo angiogenesis
assays. Consequently, these embryos have been used in chemical screens for compounds
modulating angiogenesis [96].
In addition to optical transparency and accessibility, zebrafish have an advantage over
small rodent models, in that the heart rate of zebrafish embryos is closer to humans than the high
heart rate of mice [97]. Zebrafish embryos also have the remarkable capacity to survive for
several days in the absence of heart function or circulation [97]. These features have bolstered
the vigorous and detailed study of zebrafish cardiovascular development. For example, confocal
time-lapse microscopy of double transgenic zebrafish embryos permitted in vivo monitoring of
ECs during stages of angiogenesis in a study conducted by Jakobsson et al. The investigators
were able to observe ECs dynamically competing for the role of a tip cell during angiogenic
sprouting, in a process that is governed by VEGF-notch signalling [98]. Zebrafish research has
made substantial contributions to cellular, molecular and genetic studies of vascular development
in vertebrates, which has been reviewed elsewhere [97].
States of pathological angiogenesis can also be studied in zebrafish. Zebrafish can be
raised in fish water that is perfused with nitrogen gas, creating systemic hypoxia in tissues [99].
This has led to the development of an embryonic metastasis model [100] and an adult
retinopathy model [62]. Myocardial infarction (MI) can also be studied in adult zebrafish. MI
can be induced by cryoinjury and subsequent cardiac regeneration can be analyzed with
histology, immunostaining and in situ hybridization (ISH) [101]
20
Angiogenesis can also be studied during zebrafish fin regeneration. Zebrafish have much
greater regenerative capacity than humans, but the underlying mechanisms (e.g.
functional role of Pu.1 gene) of tissue regeneration appear to be conserved [102]. In this
assay, half of the caudal fin is amputated and allowed to grow back. Test agents are
applied to the fish water during the allotted time for regeneration (3 days). The
percentage of fin regeneration in adults is an indirect measure of physiological
angiogenesis, as only 1 mm of tissue can be regenerated with the inhibition of
angiogenesis [103]. Chemical screening for fin regeneration can be performed in larval
[102] and adult zebrafish [104]. This assay and the developmental angiogenesis assay are
highly flexible, as there are a variety of options to select for the phenotypic readout.
Transgenic lines or immunostaining can be used to label specific cell types and
morpholinos or chemicals can be applied to block the function of specific genes and
pathways. The major disadvantages to fin regeneration models is that they are not very
amenable to large-scale screening and must be validated with additional studies, as
angiogenesis cannot be differentiated from inhibition of other parts of the regenerative
process.
1.3.4 Zebrafish cancer models
Zebrafish has also emerged as a promising experimental system for modelling human
cancers, through genetic manipulation or cell transplantation (refer to Liu and Leach 2011 for a
thorough review on zebrafish models of cancer [105]).
Transgenic and mutant zebrafish lines have been developed to mimic many key features
of tumourigenesis. A Gal4-UAS system produces HRAS oncogene overexpression in
melanocytes and faithfully reproduces phenotypes of human melanoma in zebrafish larvae [106].
21
Nguyen et al. have developed a system to induce and track the growth of fluorescent-labelled,
RAS-overexpressing tumours in the livers of larval zebrafish [107]. Mutant lines are also
available to model cancers with loss-of-function mutations. TP53, is a tumour suppressor gene
that is frequently mutated in the majority of human cancers [108]. A zebrafish line containing a
mutation in tp53 exhibits abnormal apoptosis and cell-cycle phenotypes and develops neural
sheath tumours at 8-9 months of age [109]. Conducting compound screens on mutant lines can
lead to the identification of therapeutic agents that can restore gene function and prevent
subsequent tumourigenesis [107].
Cell transplantation models have been used to study tumour angiogenesis and metastasis
in zebrafish. An embryonic xenograft model has been developed to investigate tumour
angiogenesis in vivo. The injection of cancer cells into the yolk sac of 2-day-old embryos
stimulates vessel growth from the subintestinal vessels (SIVs). This process is monitored by
fluorescence microscopy, since vessels and tumour cells are fluorescently labelled. Tumour
angiogenesis can be abrogated by the application of chemical inhibitors [61].
Injected cancer cells can be genetically modified to overexpress FGF2 or TGF-β to
stimulate angiogenic and metastatic behaviour [110, 111]. Metastasis and cell invasion can also
be studied by injecting cancer cells overexpressing the pro-metastatic gene twist directly into the
circulation of embryos [112]. The embryo strain and its environmental conditions can also be
altered. Vasculature-labelled transgenic embryos are commonly used in these studies, and they
can also be treated with morpholinos to inhibit specific pathways. Nicoli et al. treated transgenic
embryos with a morpholino targeting VE-cadherin and observed a decrease in tumour vascularity
[110]. Hypoxia-induced tumour activity can also be modelled by incubating xenografted
embryos in normoxic or hypoxic water. Under hypoxic conditions, there is increased
22
neovascularisation, and tumour cells disseminate and invade neighbouring tissues [113]. The
hypoxic system models the early stages of metastasis and it would be valuable to study and
develop therapies for this particular process.
The strength and weakness of embryo xenograft experiments is its duration of study,
which is limited to one week post fertilization. Embryos younger than a week old tolerate
xenograft transplants due to the immaturity of the immune system [114]. However, adult
zebrafish permit long-term evaluation of tumour development. Immunocompromised zebrafish
lines are not available at present, though immunosuppression is possible through sub-lethal doses
of radiation [115].
The prevalent use of zebrafish embryos in research faces some criticism. Embryo models
may depict processes that are specific to developmental biology, which may not adequately
translate to adult physiology. Juvenile and adult fish are not routinely used for practical reasons:
tissue opacity, longer experimental timeline, and the maturity of their nervous and immune
system hinder experimental study. A number of techniques can be employed to overcome these
issues, to improve the use of older zebrafish in cardiovascular research. Pigment formation can
be pharmacologically inhibited with propylthiouracil, though transparency can only be
maintained for several weeks [99]. Additionally, a doubly mutant zebrafish line (casper) has
been created to prolong tissue transparency into adulthood [116]. In zebrafish tumour xenograft
models, this has allowed for the in vivo assessment of tumour grafts for up to 5 weeks post-
transplant [116].
Zebrafish offer a diversity of options for modeling cancers. Different stages of cancer
progression can be recapitulated in transgenic and xenograft zebrafish. Genetically engineered
23
zebrafish also depict disease- and pathway-specific models of cancer. Zebrafish have great
potential in contributing to the advancement of cancer research and therapeutics.
1.3.5 Technological advances in zebrafish drug discovery
Zebrafish embryonic screens can greatly streamline the process of drug discovery by
automating the laborious phases of drug screening; sorting, sample processing, drug dosing,
image acquisition, analysis and interpretation. The COPAS XL (Union Biometrica) is a large
particle flow cytometer that is capable of sorting embryos and hatchlings based on optical light
and fluorescence (up to 3 fluorophores at once) and dispensing them into multiwell plates [117].
This device can significantly accelerate embryo selection and sorting for drug treatment by
identifying embryos positive for fluorescence. Automating slow and tedious tasks like drug
dosing and microinjection can further increase throughput. Liquid handling robots such as
Sciclone G3 (Caliper Life Sciences) can dispense reagents and drug compounds in series or in
parallel into multiwell plates [118]. With automated batch microinjection, up to 15 embryos can
be injected per minute with genetic material such as MO [119]. Although automated
microinjection is not substantially faster than manual microinjection, it reduces the variability
between injections and errors due to operator fatigue [119].
The transparency and small size of zebrafish embryos allows ease of light-based imaging.
Stereo- and confocal microscopies have been widely used in zebrafish studies. Confocal
microscopy is capable of high resolution cellular imaging with well-developed software analysis
tools, but its application is limited by tissue penetration and the transparency and size of the
zebrafish being analyzed [120]. Most zebrafish screens are performed in multiwell plates, and
phenotypic analysis within the plates is possible using Confocal Laser Scanning Microscopy
(CLSM), such as the ImageXpress Ultra (Molecular Devices), with point-by-point image
24
acquisition allowing for three-dimensional reconstructions. MicroMRI has been applied to detect
and characterize melanomas in adult zebrafish [121], and microscopic ultrasound has been
utilized to assess liver tumours in adult zebrafish in response to chemotherapeutic treatment
[122].
A number of methods have been devised to expedite image processing outside of
multiwell plates to overcome the problems caused by the large working distance and random
movement and orientation of zebrafish [117]. Capillaries [123], agarose-coated plates [124],
round-bottom plates (Corning COSTAR) and rectangular microplates with prisms (Physical
Sciences Inc) are several strategies that have been conceived to manage embryo orientation for
quick image capture. High-throughput histology is possible for larval and adult zebrafish using
several methods to accelerate sample handling: agarose arrays, automated tissue processors,
rotary microtomes and automated slide stainers [125]. An automated imaging system developed
by Gehrig et al. combines embryo recognition software with a high content microscope such that
fluorescent gene expression patterns in up to 2,000 embryos may be acquired within 4 hours
[124]. Analysis programs have been built to handle the quantity of data produced by rapid image
acquisition technologies while also reducing the burden of visual scoring and eliminating
observation bias [126]. Image analysis software such as Cognitive Network Technology
(Definiens) and MetaMorph software application modules (Molecular Devices) can be custom-
designed to detect and quantify specific structures such as intersegmental vessel number in
Tg(fli1:EGFP) zebrafish [126]. The development of these technologies has greatly increased the
efficiency of screening. However, in most zebrafish screenings reported thus far, automation is
not continuous and embryos must be manually manipulated at several steps (semi-automation).
Although zebrafish are an emerging model organism in biomedical research, it is a
25
powerful system for in vivo drug discovery that is being employed in many disciplines. The
increasing popularity of zebrafish has led to the development of numerous technologies to
improve handling, imaging and processing. Techniques for molecular, histological, behavioural
and genetic analyses are improving, and as a result the body of literature continues to grow. The
utility and versatility of zebrafish genetic tools and disease models will allow researchers to
continue to make waves in biomedical research and accelerate the process of drug development.
1.4 Angiogenesis-targeted therapies
1.4.1 Pro-angiogenic therapies
Traditionally, the clinical interventions for disorders with impaired circulation have
largely been surgical in nature. Clinical treatment of ischemic diseases involve lifestyle changes,
management of co-morbidities and macrovascular interventions such as surgical
revascularization, angioplasty and amputation [127]. Chronic wounds are managed similarly,
with the removal of necrotic tissue, surgical bypass, subcutaneous angioplasty and amputation
[16]. Microvascular therapies have been recently developed to directly promote angiogenesis in
these tissues.
Therapeutic angiogenesis induces the growth of blood vessels, to restore blood perfusion
and enhance tissue repair in disorders that feature a deficiency in angiogenesis [128].
Therapeutic induction of angiogenesis is achieved through the application of growth factors and
pharmacological agents. Cell-based therapies [129, 130], tissue engineered products [131] and
mechanochemical technologies (such as negative pressure [132], low-frequency ultrasound [133]
and hyperbaric oxygen systems [134]) are alternative strategies to stimulate angiogenesis.
However, I will focus on the development of bioactive agents.
26
Several of these therapies are based on the growth factors FGF, VEGF and PDGF, and
are administered locally as protein-laden formulations [135-137] or incorporated into vectors for
gene therapy delivery [138-140]. Recombinant PDGF (Becaplermin gel) is the only pro-
angiogenic agent approved for clinical use [136]. It has significantly improved the healing rates
of chronic diabetic ulcers [136] and has been successfully used off-label for great variety of
wounds [24]. Angiogenesis stimulation has also been achieved with VEGF and FGF gene
therapy in ischemic heart disease [141]. While gene therapy offers longer term treatment of
ischemia, it has not demonstrated the level of efficacy in clinical trials necessary for approved
usage [142].
Pharmacological agents in the form of peptides [143] and small molecules [144] are also
in development as stimulators of angiogenesis. These agents act as antagonists or agonists,
activating or inhibiting components of the angiogenic cascade to promote angiogenesis. One
method used by the peptide PR39 and synthetic compound TM6008 activates the HIF pathway
through inhibition of HIF degradation, to induce angiogenesis through hypoxic signalling [145].
Applications of therapeutic angiogenesis have mostly been studied in chronic wounds
and ischemic diseases in the heart and limbs. The efficacy of these agents is currently being
evaluated for relevance in other conditions with insufficient or faulty angiogenesis such as
infertility [19], neurodegeneration [146] and sepsis [147]. Substantial work has also been
conducted to stimulate neovascularisation in tissue engineering [148].
1.4.2 Anti-angiogenic therapy
Traditionally, general strategies have been employed to destroy defective tissues in
diseases with excessive angiogenesis. Routine cancer treatment involves chemotherapy, radiation
or surgical excision of a cancerous mass. Photodynamic therapy (PDT) uses a similar but more
27
refined approach to treat ocular tumours and neovascularisation. Photosensitized agents are
systemically injected and sequester in abnormal ocular vessels until a laser activates them,
resulting in local damage that seals off the vessel. [149]. The side effects and shortcomings of
these strategies have produced a great need for treatment modalities that are more specific, safe
and effective. In the past few decades, a multitude of agents have been developed to specifically
target the molecular mechanisms of the aberrant angiogenesis that characterizes these conditions
[150]. Anti-angiogenesis strategies are employed to inhibit or normalize abnormal angiogenesis.
Inhibitory agents are available in the following varieties: antibodies, growth factors, peptides and
small molecule compounds. The efficacy of these therapies has primarily been studied in ocular
and tumour angiogenesis.
VEGF has been well characterized as a critical component of the angiogenesis cascade in
normal and pathological tissues. Consequently, many anti-angiogenic strategies have been
developed to target VEGF signalling. The first anti-angiogenic agent to be approved for clinical
usage, bevacizumab, is a monoclonal antibody that targets and neutralizes VEGF protein.
Bevacizumab demonstrated improved patient survival in a number of cancers when used in
conjunction with chemotherapy [151]. A high affinity fusion protein for VEGF, aflibercept
(VEGF-Trap), is currently in Phase III clinical trials for colorectal cancer, retinal vein occlusions
and diabetic macular edema [152]. The aptamer (synthetic oligonucleotide), pegaptanib was the
first anti-VEGF agent to be approved for clinical use in age-related macular degeneration (AMD)
[153]. The long-term application of both protein and small-molecule VEGF-based therapies
revealed that positive responses were transient and tumours would develop resistance and
continue to grow vessels even with the loss of VEGF signalling [151].
28
While these agents target a single growth factor and its downstream effectors, second-
generation agents, are capable of targeting multiple angiogenic signalling pathways at once.
Some investigators have referred to this strategy as “magic shrapnel” in reference to the “magic
bullet” of single-target therapies [154]. RTKIs are a class of small molecule compounds that are
able to block signalling via receptor tyrosine kinases. RTKIs target receptors of VEGF, FGF,
PDGF and some oncogenes. Sunitinib is a RTKI that blocks VEGFR2, PDGFR-α and β, raf
kinase, FLT2 and c-Kit [155]. Another RTKI, sorafenib blocks VEGFR2, PDGF-β, FLT3 and c-
Kit [156]. Sunitinib and sorafenib have both been approved for clinical use for first-line
monotherapy of kidney and liver cancer [155-157]. However, their potency and systemic
administration (as oral agents) can cause serious toxicities related to the disruption vascular
homeostasis. A fraction of patients treated with bevacizumab, sunitinib or sorafenib have
reported higher incidences of bleeding, thrombotic events, hypertension, edema and delayed
post-operative wound healing [158].
Broad-spectrum angiogenesis agents, like angiostatin and endostatin, are endogenous
protein fragments produced by proteolytic processing [159]. Primary tumours are able to keep
distal metastases dormant by blocking angiogenesis and tumour growth through the action of
endogenous circulating inhibitors [160, 161]. Angiostatin and endostatin potently suppresses
angiogenesis and tumour growth in mice without detectable toxicity [162, 163]. Endostar,
recombinant human endostatin, is currently approved in China for the first-line treatment of lung,
gastric and colorectal cancer [164]. Although the mechanism of action is not known, endostatin
prevents ECs from responding to angiogenic signals by downregulating VEGF and FGF
signalling, EGFR, MMPs, c-myc, HIF-1α and simultaneously upregulating anti-angiogenic
factors such as TSP-1 and maspin [165, 166]. The range of this promising class of inhibitors has
29
allowed it to succeed where other anti-angiogenic therapies have failed. Animals treated with
endostatin do not appear to develop drug-induced resistance [167] and Endostar treatment
demonstrates the potential to reverse multi-drug resistance in a pilot study in patients with
colorectal and gastric cancer [164]. However, there are practical limitations to protein-based
therapy. Angiostatin and endostain are physically difficult to produce in active form, require
high-dose local administration and have short half-lives [168].
Anti-angiogenic agents have also been developed to block specific steps in the
angiogenesis process. Vitaxin is an antibody directed against αvβ3 integrin, which is involved in
cell adhesion and exclusively found in activated ECs [169]. Phase I clinical trials in patients with
advanced cancer determined that while Vitaxin was unable to cause tumours to recede, it did
delay tumour progression [170]. Broad and selective MMP inhibitors are also available to
impede proteolysis in the process of EC migration and tumour cell invasion and metastasis.
Clinical trials show increased survival in long-term use of marimastat [171], a broad spectrum
MMP inhibitor, though treatment benefits were offset by musculoskeletal toxicities [172].
1.4.3 Limitations and future directions in angiogenesis-targeting therapies
Presently, there are eight anti-angiogenic drugs approved for cancer therapy [150], three
approved for the treatment of AMD [173], and one pro-angiogenic drug approved for diabetic
ulcers [136]. Several dozen angiogenesis-targeting agents are making their way through the drug
development pipeline. Targeting angiogenesis is a rational approach to treating cancer, ocular
and ischemic diseases. However, a substantial body of clinical research have accumulated
evidence that the potent benefits observed in preclinical models do not translate to same level of
efficacy in the clinic. Not all cancer patients respond to anti-angiogenic therapy and resistance
quickly develops in those who do [174]. The effectiveness of bevacizumab has been modest; it
30
prolongs life on the order of months and that is only when it is used in combination with
chemotherapy [175]. Pro-angiogenic therapy has not fared any better, with no clinical benefit
observed in double-blind randomized placebo-controlled trials in ischemic cardiovascular
disease [176]. Assuming the validity of the premise, there are a number of issues that are
contributing to the overall lack of efficacy of these agents: associated side effects, drug
resistance, and the timing and specificity of the drug.
One of the major concerns that must be considered for the next generation of
angiogenesis-modulating therapies is drug resistance. So far, investigators have reasoned, “all
that needs to be done is initiate [or limit] the process of vascular growth and nature will take its
course. Thus far nature has perversely refused to cooperate [177].” It has become clear that a
systems biology approach must be considered. The short-lived efficacies of VEGF inhibitors
suggest that resistance stems from the ability of tumours to use alternative pathways of
angiogenesis such as the FGF, PDGF, PIGF, notch, TIE2/angiopoietin, and integrin pathways
[178, 179]. Breast cancer, and the majority of human cancers, has demonstrated the upregulation
of 5-6 proangiogenic proteins [180].
One method to overcome angiogenic redundancy is to employ broad-spectrum
angiogenesis inhibitors like angiostatin or endostatin. Another way to overpower built-in
compensatory mechanisms is to combine the use of multiple inhibitors to block many pathways
at once or in sequence [178]. Combinatorial strategies should be more effective in countering the
interacting effects of a numerous cell types and signalling pathways. Preclinical studies have
recently demonstrated that combination therapy may be more effective than monotherapy.
Combined delivery of VEGF and Ang-1 was able to increase the capillary density of a hindlimb
ischemia model greater than either agent alone [181]. Co-administration of systemic (angiostatin)
31
and local (bevacizumab) anti-angiogenic inhibitors has demonstrated additive effects in animal
models, strongly inhibiting angiogenesis while reducing bevacizumab-induced tumour invasion
[182]. Another potential benefit to employing combination therapy is the ability to reduce the
dosage of agents, which are known to produce side effects. Therefore, identification of novel
compounds targeting various pathways of angiogenesis is very important in both pro- and anti-
angiogenesis therapies.
Some pharmacologically active compounds could produce effects comparable to
combinatorial therapy. Established drugs such as celecoxib (Celebrex) [183] and rosiglitazone
[184] are able to upregulate endogenous anti-angiogenesis proteins (such as endostatin and TSP-
1 respectively) and are currently undergoing clinical trials for the treatment of cancer [185, 186].
Finding new functions for old drugs expedites the process of drug development since their
pharmacology and toxicology profiles are already known. The next generation of angiogenesis
inhibitors and stimulators could come from the identification of new small molecules that are
able to exert similar effects.
The efficacy of a drug may depend on the time at which it is administered. MMP and
VEGF inhibitors and endostatin are most successful at treating early-stage cancers due to their
roles in switching off tumour angiogenesis in the initial stages of tumourigenesis [22, 171, 187].
Comparably, an inhibitor of EC growth and SMC migration TNP470 is more suited for the
treatment of late-stage tumours [188]. Multi-target inhibitors are necessary to override the
numerous pathways activated by aggressive, end-stage tumours. Our knowledge of the
physiology of tumour development and angiogenesis should be considered when selecting and
applying appropriate anti-angiogenic agents for therapy.
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It is also important to conduct more detailed studies of pathological angiogenesis. The
identification of therapeutic targets will enable the development of angiogenesis therapies that
target specific components of the problematic physiology. The stage, treatment history and
genotype of a tumour may eschew molecular resistance to certain treatments [189]. Ideally, one
would employ an inhibitor specific to the angiogenic profile of a patient’s tumour for optimized,
personalized treatment, though the lack of biomarkers and patient screening limits this strategy at
present [179]. The discovery of novel angiogenesis modulators and thorough investigation of
their molecular mechanisms of action will greatly contribute to efforts in improving treatment
outcomes and future applications in general and personalized medicine.
As research in angiogenesis therapies is still in its infancy, it is imperative that we
continue to find ways to improve its study and clinical application. The zebrafish model system
presents an opportunity to enhance drug discovery in the field of angiogenesis. Judah Folkman,
the researcher credited as the father of angiogenesis therapy, once asserted that the outcomes of
angiogenesis therapy would one day reclassify cancer as a “chronic manageable disease” [190].
Therapeutic modulation of angiogenesis could lead to momentous changes in the management of
an expansive list of diseases that significantly affect human health worldwide.
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CHAPTER 2 – RATIONALE AND HYPOTHESES
2.1 Rationale
2.1.1 The need for new drugs targeting angiogenesis
Pathological angiogenesis underlies a variety of conditions, such as obesity, asthma,
diabetes, endometriosis, macular degeneration and cancer. The critical role of aberrant
angiogenesis in these disorders has led to a detailed investigation of its mechanisms to facilitate
the development of treatments that stimulate or abrogate it.
Angiogenesis is tightly regulated by a balance of pro- and anti-angiogenic factors in the
cell environment. Many therapeutic strategies have been devised to rectify the imbalance of
angiogenic factors present in disease conditions. VEGF is a well characterized growth factor
responsible for switching on the angiogenesis cascade and many angiogenesis-targeting therapies
have been developed to target VEGF signalling. However, the long-term application of both
protein and small-molecule based VEGF-targeting therapies has demonstrated short-lived
efficacies due to the development of drug resistance caused by multiple angiogenesis pathways.
Angiogenesis therapeutics must accommodate the physiology of angiogenic redundancy.
Stimulating or inhibiting multiple pathways, through the use of broad-spectrum agents or the
application of multiple single-target agents in combination, may overcome drug resistance.
Therefore, there is an urgent need for the identification of novel compounds targeting different
components and processes of angiogenesis.
The conventional process of drug development is time consuming and has a low rate of
success. In the initial phase of classic in vitro-based drug discovery, high-throughput screening
of compound libraries is performed in biochemical targets or cell-based assays in microtiter plate
formats that range from the standard 96- to 3456-well plates [191]. Several thousand of
34
compounds can be screened within a week at a typical pharmaceutical company such as Pfizer
[191]. Yet, many drug candidates do not enter clinical trials due to poor translation of in vitro
findings into animal models. To address this particular bottleneck, HTS methods were developed
to evaluate the effect of screened compounds on ADME targets such as microsomal P450
enzymes, protein binding, serum stability and cytotoxicity [191], to predict adverse drug
reactions. Despite the advances in drug profiling and the availability of sophisticated resources in
pharmaceutical technology, the number of FDA-approved drugs has declined in the past two
decades [192]. Scannell et al documented a reduction in the total number of new drugs approved
between 1996 and 2010 and diminishing cost return in respect to the billions of dollars expended
in research and development [192].
2.1.2 Zebrafish as an advanced model system for drug discovery
Screening candidate compounds in a whole-organism allows for the evaluation of drug
specificity and toxicity at an earlier stage of drug development. Zebrafish have numerous
qualities that are well suited to drug screening: accessibility for manipulation and observation,
high fecundity, small size, rapid development, and a physiology and pharmacology that is
analogous to mammals. The optical transparency of zebrafish embryos, coupled with the
availability of transgenic zebrafish lines with fluorescent labelling of specific tissues or organs,
facilitates real-time visualization of drug-induced, cardiovascular phenotypes in a high-
throughput format.
Zebrafish screens are able to identify new hits from libraries of small compounds that
have already been heavily screened with mammalian cell-based assays. For example, Murphey et
al demonstrated the sensitivity of zebrafish screening by identifying 14 small molecule cell cycle
inhibitors not previously identified from in vitro mitotic screens of a 16320-compound library
35
[193]. Cell-based approaches are limited in their capacity to recapitulate the complexities of the
targeted physiological process and cannot model the metabolism of a drug in the way a whole-
organism model system does. Furthermore, drug metabolites may contribute to the observed
efficacy of a drug in vivo.
Zebrafish screening is able to quickly bring compounds into clinical trials. Zon and
colleagues identified prostaglandin signalling (PGE2) as a regulator of hematopoietic stem cell
(HSC) homeostasis through a small molecule zebrafish embryo screen [194]. Further analysis in
embryo knockdown and rescue experiments and subsequent studies in adult zebrafish and murine
in vitro and in vivo models supported PGE2 as an essential and druggable target for regulating
HSC number [194]. A bioactive PGE2 derivative has been developed and is currently being
evaluated as a therapeutic patients receiving cord blood transplants [194]. It only took two years
for the Zon lab to move the PGE2 compound to a (human) Phase I clinical trial since the first
article was published in 2007 [194].
The zebrafish model possesses specific advantages over existing cell-based and
mammalian models in respect to its use in vascular drug development. As a phenotype-based
screening model, it allows for the identification of compounds without prior knowledge of the
therapeutic target. Although VEGF has been implicated as a critical component of the angiogenic
cascade, it has become evident from existing VEGF-targeted therapies that it alone is not an ideal
therapeutic target. A phenotype-based approach is a practical method for discovering potent
modifiers of angiogenesis and may help identify unknown therapeutic targets for angiogenesis
pathologies.
The molecular and genetic processes of developmental angiogenesis are well understood
in zebrafish. Many of the mechanism studies routinely performed in vitro can be accomplished
36
within the context of a whole organism, though validation is often necessary in mammalian
models. The physiology of angiogenesis is conserved in vertebrates and a diverse array of
genetic tools is available for target validation and pharmacological studies within the zebrafish
model.
2.1.3 Our pilot zebrafish angiogenesis screens and my personal research interest
The advent of chemical genetic screens using zebrafish has enabled an efficient and
physiologically relevant method of identifying novel therapeutic compounds [195]. In addition,
new applications for established drugs may also be discovered using this screening platform. Our
lab has previously performed chemical genetic screens of two compound libraries using
endothelial-cell labelled Tg(Flk1:EGFP) zebrafish embryos.
The Spectrum Collection contains 2000 compounds, including existing drugs (60%),
compounds derived from natural plant products (25%) and other bioactive compounds (15%).
The screen of the Spectrum Collection produced seven hits that were classified into three groups:
rotenoids (isorotenone, dihydromunduletone; a class of natural pesticides), statins (simvastatin,
mevastatin, lovastatin, rosuvastatin; drugs used in the management of high cholesterol), and the
carcinogen aristolochic acid [96]. Rosuvastatin was selected for detailed cellular and animal
studies because of the potency of its effects in the zebrafish assay, its status as a clinically
established drug, and its controversial role in angiogenesis. My predecessor Chunyang Wang
evaluated the anti-angiogenic and anti-tumour effects of rosuvastatin and his findings were
published in European Urology in 2010 [96].
The LOPAC library contains 1280 bioactive compounds and our screen identified three
anti-angiogenic (SP600123, indirubin-3-monoxime, and mevastatin) and three pro-angiogenic
(retinoic acid, 4-hydroxyphenyl retinamide, and arotinoid acid) compounds (unpublished).
37
I have selected three compounds for validation and mechanism studies to evaluate their
potential as drugs for therapies targeting angiogenesis. The compound 4-hydroxyphenyl
retinamide (4HPR) was selected due to its incongruous ability to promote angiogenesis in
zebrafish when it is currently in clinical trials for use as an anti-cancer agent with reported anti-
angiogenic activities. Indirubin-3-monoxime (I3M) was chosen for study because it was the most
potent and well tolerated of the anti-angiogenic lead compounds. My last compound of study,
dihydromunduletone (DHM) was picked for its complete novelty as a therapeutic compound.
The unique qualities of the zebrafish model provide us with an advanced and cost-
effective method of screening and dissecting the molecular mechanisms of compounds identified
from high-throughput screening for therapeutic applications in angiogenesis pathologies.
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2.2 Objective and General Hypothesis
The zebrafish angiogenesis screen has the capacity to efficiently identify new compounds
that can modulate angiogenesis. My studies will validate the efficacy of three small molecule
compounds selected from our pilot zebrafish angiogenesis screens. I will also utilize the
versatility of the zebrafish model to investigate the molecular mechanisms of drug action of these
compounds in vivo.
I hypothesize that the whole-organism model system of my zebrafish studies provides
useful information for drug testing across different developmental stages. When used in
conjunction with transgenic techniques and other interventions, this system provides potential
mechanisms of the therapeutic functions as well as the side effects of potential drugs that may
affect angiogenesis process in vivo.
The overall objective of this study is to use zebrafish as an in vivo model to expedite the
development of therapeutic compounds that will effectively treat angiogenesis-dependent
pathologies.
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2.3 Specific Hypotheses
Since I chose to study three compounds and each of them may have different mechanisms
in modulating angiogenesis activities, I have formed individual hypotheses for each compound.
Hypothesis 1 – 4HPR
4-hydroxyphenyl retinamide (4HPR; fenretinide) was identified in our developmental
angiogenesis screen as a stimulator of angiogenesis. I hypothesize that 4HPR achieves this effect
by stimulating somitogenesis and that angiogenesis is secondary to somitogenesis.
Hypothesis 2 – I3M
Indirubin-3-monoxime (I3M) is a potent inhibitor of angiogenesis and has great potential
for development as an anti-cancer agent. I hypothesize that it affects the process of EC migration
in vivo by acting on the notch-VEGF pathway.
Hypothesis 3 - DHM
Dihydromunduletone (DHM) is a novel compound that is identified from our screen as an
inhibitor of angiogenesis. I hypothesize that it suppresses angiogenesis by modulating EC
function in vivo.
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CHAPTER 3: ROLES OF 4-HYDROXYPHENYL RETINAMIDE IN ANGIOGENESIS AND ITS POTENTIAL CELLULAR AND MOLECULAR
MECHANISMS
3.1 Introduction
3.1.1 Retinoids: Physiology and pharmaceutical development
Vitamin A (retinol) is an essential nutrient involved in physiological development and
homeostasis. The World Health Organization has documented weight loss, and eye and immune
disorders in impoverished countries as a consequence of insufficient Vitamin A intake [196].
Vitamin A is a small lipophilic molecule found naturally in foods such as carrots,
potatoes, poultry and fish. It is absorbed in the small intestine where it binds to retinol binding
protein (RBP) and enters the circulation. Retinol-RBP is transported into the liver for storage,
and delivered to tissues where it is metabolized into its functional forms: retinaldehyde and
retinoic acid (RA) [197]. Retinaldehyde is a chromatophore with critical roles in low-light and
colour vision and RA regulates a number of developmental and homeostatic processes [198].
Unlike most signalling molecules, RA is transported into the nucleus by cellular retinoic acid
binding proteins (CRABP) and interacts directly with nuclear retinoic acid receptors (RARs) and
retinoic acid X receptors (RXRs) to regulate gene transcription in various tissues [198].
Retinol is provided from the mother during mammalian development. It crosses the
placenta and is metabolized to retinaldehyde in the embryo [199]. In zebrafish, maternally
derived retinaldehyde is stored in the yolk [200], though both mammals and zebrafish rely on
retinaldehyde dehydrogenases (RALDH) to oxidize retinaldehyde into its active form. RA exerts
its actions in a paracrine manner during embryogenesis, repressing the action of growth factor
signalling pathways (such as FGF and Wnt) in a spatiotemporal manner [201]. RA signalling
41
also governs many developmental processes: neurogenesis, cardiogenesis, body axis extension,
developmental patterning and organogenesis [202].
RA signalling has important functions in maintaining normal function in organs such as
the heart, lungs and skin [203]. The role of RA in the differentiation and growth of epithelial
tissues was documented as early as 1925, when metaplasias were observed in animals deficient
in RA. These morphological changes, induced by RA-deficiency or chemical carcinogenesis,
could be offset with dietary RA treatment [204] [205]. The link between RA and cancer has
additionally been validated in an epidemiological study. Individuals reporting less Vitamin A
intake in their diet have a higher risk of developing cancer [206]. In the past few decades, RA
has been evaluated as a therapeutic agent for the treatment and prevention of cancer.
RA’s poor bioavailability and tendency to render chemotherapy-resistance to tumours has
limited its application as an anti-cancer agent [207]. RA is also associated with a number of
toxicities, which is to be expected when we consider the essential functions of RA in physiology
and development. These side effects include anemia, weight loss, birth defects and bone fractures
[208].
Nonetheless, a closely related derivative of RA is commonly used as a topical agent for
dermatologic disorders such as acne, under the generic name isotretinoin [209]. It is also
uniquely effective as a primary treatment for acute premyelocytic leukemia, a fatal blood
disorder caused by the inability of hematopoietic stem cells (HSCs) to mature. Oral
administration of isotretinoin is able to induce terminal differentiation of HSCs, resulting in 90-
95% complete remission rate [210]. This is a fantastic example demonstrating the potential of
molecular-targeted therapies to not only treat a disease, but also cure it.
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Retinoids are a group of natural and synthetic analogs of Vitamin A. Natural analogs of
Vitamin A, such as retinoic acid (RA) are produced from metabolic processes in the body.
Synthetic retinoids are created by modifying functional groups on RA. Over 10 000 retinoids
have been identified for a variety of biomedical applications [211]. Synthetic retinoids have
greater potency and more favourable pharmacokinetic and toxicity profiles than their parent
compounds [204]. My first compound of study, 4-hydroxyphenyl retinamide (4HPR or
fenretinide) is a derivative of RA that was synthesized in the late seventies by the pharmaceutical
company Johnson and Johnson [212]. It is currently being investigated for applications in the
management of cancer.
3.1.2 Fenretinide as an anti-cancer agent Fenretinide is a cytotoxic agent, arresting proliferation and inducing cell death in many
cancer cell lines: breast, ovarian, cervical, prostate, lung, renal, bladder, glioma, head and neck,
lymphoma, neuroblastoma and Ewing’s sarcoma [213].
The proposed mechanisms for fenretinide’s apoptotic effects can be divided into
receptor-mediated and receptor-independent mechanisms. RAR expression is increased in
fenretinide-induced cell differentiation and apoptosis in some cell types [214]. Curiously,
fenretinide has been shown to bind poorly to RARs [215]. It is possible that fenretinide is
increasing RA release from intracellular stores or is converted to a metabolite that may activate
RARs [216]. The apoptotic effect of 4HPR appears to be distinct from classical retinoids. It is
mainly driven by receptor-independent mechanisms, which involve the increase of signalling
molecules: ceramides, caspases, NO and reactive oxygen species (ROS) [217] [218].
Fenretinide-mediated release of ROS promotes apoptosis through the activation of JNK, p38,
ERK and PKC [219]. In addition, fenretinide acts in synergy with chemotherapeutic agents to
43
induce apoptosis in vitro [220]. Fenretinide shows the ability to complement the action of
chemotherapy agents by activating apoptosis independently of the p53 pathway, which is
commonly mutated in most cancers [221].
Another mechanism by which fenretinide is able to reduce tumour growth is through the
inhibition of angiogenesis. Studies have demonstrated that fenretinide is able to reduce in vivo
neovascularisation in CAM and Matrigel plug assays [222, 223]. Fenretinide disrupts the
function of ECs in a number of ways that affect the progression of angiogenesis: downregulation
of components of the VEGF and FGF pathways (proliferation), upregulation of TGF-β (to induce
apoptosis), decreased MMP activity (migration), and repression of differentiation (required in
tube formation) [223, 224].
The investigation of fenretinide’s mechanism to reduce tumour growth is an ongoing but
necessary process in determining the optimal applications of the drug.
The anti-proliferative and anti-angiogenic effects of fenretinide contributes to the
reduction of tumour growth observed in animal models of colon, prostate, bladder, pancreas,
liver and skin cancer [225]. However, the route of administration affects the absorption and
distribution of the drug, as mice with ovarian carcinomas survived much longer when fenretinide
was administered intraperitoneally rather than orally [226]. Additionally, not all cancers are
responsive to treatment. Fenretinide treatment did not inhibit total tumour formation in a
chemoprevention model of lung cancer [227]. Fenretinide’s potential to cause abnormal
embryonic development has been assessed in rats and rabbits (Oryctolagus cuniculus) during the
early stages of the drug’s development, where it was classified as a weak teratogen [228].
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The results of preclinical studies demonstrated that fenretinide was an effective candidate
for chemoprevention and was less toxic than alternative treatments. These factors encouraged the
evaluation of fenretinide in clinical trials. A 15-year clinical study has demonstrated the
sustained ability of fenretinide to significantly reduce the incidence of second breast cancer
[229]. The use of fenretnide for treatment and chemoprevention has also been tested in other
cancers; brain cancer [230], kidney cancer [231], prostate cancer [232] and bladder cancer [233].
While the drug is generally well tolerated, there does not appear to be any beneficial effect in the
chemoprevention of most cancers. The chemoprotective effect of fenretinide observed with
breast cancer is dependent on age and menopausal status, suggesting that fenretinide acts in
concert with hormones such as IGF-1 [234]. To make use of this window of opportunity,
fenretinide is presently undergoing clinical evaluation as prophylactic therapy for young women
with high-risk for breast cancer [235]. At present, there are over 25 clinical trials in progress to
evaluate the efficacy of fenretinide in chemoprevention and intervention of a variety of cancers
[224]. Long term evaluations of fenretinide in the clinic have established it to be relatively safe.
Eye conditions (diminished dark adaptation), dermatologic disorders, and gastrointestinal
symptoms have been reported as adverse events associated with 4HPR [236].
3.1.3 Additional applications of fenretinide The application of fenretinide is currently being explored in the treatment of a variety of
conditions. Fenretinide has recently been proposed for cystic fibrosis (CF) treatment. Fenretinide
administration was shown to normalize ceramide deficiencies that lead to lipid imbalance and
osteoporosis in a mouse model of CF [237]. Fenretinide is also being considered for the
treatment of diabetes and dyslipidemia. Intraperitoneal fenretinide administration reduced insulin
resistance and decreased plasmid lipid levels in genetically obese mice fed with a high-fat diet
45
[238]. The outcome of a recent Phase II clinical trial has established the potential use of
fenretinide in the treatment of late stage age-related macular degeneration. The accumulation of
retinol in the eye causes toxic effects that can lead to blindness. Fenretinide competes with
retinol to bind with RBP thereby reducing retinol’s mode of transport to the eye. Results of a
clinical trial demonstrated that oral fenretinide treatment reduced lesion growth and decreased
incidences of choroidal neovascularization in patients in two years [239].
3.1.4 Rationale New applications are constantly being explored to take advantage of the fact that
fenretinide has proven itself as a safe and effective therapeutic agent after decades of
pharmaceutical research. Fenretinide was identified in our pilot zebrafish screen as a compound
promoting angiogenesis, which contradicts published data supporting the anti-angiogenic
properties of fenretinide as an anti-cancer agent. The inhibitory effects of fenretinide on
angiogenesis have been studied in vitro and in vivo; in CAM and Matrigel plug assays. I intend
to examine the pro-angiogenic effects of fenretinide and explore its potential mechanism of
action within the zebrafish model system. I hope to either reconcile these contradictory findings
or provide evidence for a potentially novel application of fenretinide as a promoter of
angiogenesis.
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3.2 Materials and Methods
3.2.1 Preparation of drugs and reagents
Fenretinide (4HPR) and semaxanib (SU5416) were purchased from Tocris Bioscience
(Ellisville, MI, USA). Phenylthiourea (PTU) was purchased from Sigma-Aldrich (Oakville, ON,
Canada). Drugs were dissolved in 100% DMSO (Sigma-Aldrich, Oakville, Canada) and serially
diluted to a 10x stock solution stored at -20°C. Embryos and larvae were treated with 4HPR at
final concentrations of 1, 2, 4, 8, and 16 µM and adults were treated with 5 and 10 µM. Embryos
were not exposed to a concentration of DMSO that exceeded 1%. Embryo medium, containing
Millipore water and a mixture of salts, was prepared at half the strength of the protocol described
by Nusslein-Volhard and Dahm [240]. The anesthetic was prepared by mixing eugenol in
embryo water, containing 1% ethanol to a stock concentration of 10 parts per million (ppm).
2.5% methyl cellulose used in SCORE imaging was prepared by combining methyl cellulose and
clove oil in heated nanopure water, mixed until dissolution and left to stand in ice for several
hours to remove bubbles that formed.
3.2.2 Zebrafish strains and husbandry
The following strains were employed for experimental study: wild-type striped and
spotted adult zebrafish (mixed genetic background) and transgenic lines: Tg(flk1:EGFP),
Tg(GATA1:dsRED) both provided by Dr. Ian Scott (University of Toronto), Tg(fli1:nEGFP)y7
provided by Dr. Sarah Childs (University of Calgary), and Tg(fli1:EGFP;casper) provided by
Christian Lawrence (Dr. Leonard Zon, Harvard University) and a mutant albino line produced
through selective breeding in the Wen lab. Fish were maintained at 28°C in a circulating
aquaculture system and fed twice daily at our facilities in the Li Ka Shing Knowledge Institute at
47
St. Michael’s Hospital. Embryos were obtained using standard mating conditions and incubated
in embryo medium.
3.2.3 Zebrafish developmental angiogenesis assay
Figure 3.2.3 Typical protocol for a zebrafish developmental angiogenesis assay. Eggs produced by natural breeding were collected and dechorionated 10 hpf. The eggs were arrayed into 96-well plate and drugs were added directly into the fish water at 12 hpf. After 2 days of incubation, fish were imaged, and ISV number was used to evaluate the drug’s effect on angiogenesis.
The transgenic zebrafish lines Tg(flk1:EGFP) and Tg(fli1:EGFP;casper) were utilized in
this assay of developmental angiogenesis. Increasing concentrations of the selected compound
were used to evaluate dose-dependent effects on ISV development. Both lines express enhanced
green fluorescence protein (EGFP) in the cytoplasm of endothelial cells (ECs), allowing for
visualization of the entire vasculature under fluorescence microscopy. Following natural
breeding, the chorions of healthy embryos were manually removed (dechorionated) using
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Almedic #7 forceps at the bud stage (10 hpf) and 4-5 embryos were dispensed into a single well
of a 96-well plate, with each well containing 90 µL of 0.5x E2 embryo medium. 10 µL of a 10x
stock solution of fenretinide was added into embryo medium at the 6-somite stage (12 hpf), prior
to the commencement of angiogenesis. Control embryos were treated with 0.5% DMSO. After
48 hours of incubation at 28°C, the embryos were anesthetized with 100 ppm eugenol, mounted
on a 1% agarose-coated, 100 mm tissue culture plate and imaged with a Leica fluorescence
stereomicroscope at 32-40X magnification. Samples were analyzed with the following
specifications: only fully formed intersegmental vessels (ISVs) with attachment to the dorsal
longitudinal anastomotic vessel (DLAV) were counted and embryos without a heart beat were
excluded from analysis. Data was compiled in Microsoft excel and statistically analyzed using
ANOVA and a two-tailed student t-test. Error bars denote standard error (SE).
3.2.4 In vivo quantification of EC number Tg(fli1:nEGFP)y7
expresses EGFP in the nucleus of ECs, permitting the quantification of
EC number in specific vascular structures like the DA, DLAV, and ISV. Embryos were treated
as indicated in the angiogenesis screen. 0.22 mM of propylthiourea (PTU) was added into media
at 12 hpf to inhibit pigment formation to improve resolution for cell counting. Images of
embryos were acquired at a magnification of 40X after 2 days of incubation, using a Leica
fluorescent stereomicroscope and prepared using agarose or Specimen in a Corrected Optical
Rotational Enclosure (SCORE) mounting techniques. Embryos were immersed in a solution of
2.5% methylcellulose and 100 ppm eugenol, drawn into a capillary, and rotated in an optimal
orientation. For the manual counting of cell number in the ISV and DA, nuclei were counted and
averaged across 10 segments of each embryo, then expressed as an average of all samples of
each condition. Manual cell counting in the DLAV determined a total number of DLAV cells in
49
each fish, which was averaged for each condition. The data was compiled in Microsoft Excel and
statistically analyzed using ANOVA and a two-tailed student t-test. Error bars denote SE.
3.2.5 Fin regeneration assay
Figure 3.2.5. The procedure of a fin regeneration assay. Larval and young adult zebrafish were anesthetized and half of the caudal fin was transected with a blade. Fish were incubated in cups or wells with drugs dissolved in the fish water for 3 days. Images before and after amputation, and 3 days post amputation were compared to evaluate the amount of fin regenerated. The notochord is used as a reference point for larval fish. Newly regenerated tail fins are not pigmented in adult zebrafish. [241, 242].
Larval model: I collected the eggs produced by natural breeding of Tg(flk1:EGFP)
vascular-labelled zebrafish. I anaesthetized embryos aged 2 dpf with 100 ppm of eugenol and
transected the tail primordium, just posterior to the end of the notochord with a 26-gauge needle
50
tip. Each embryo was dispensed into a single well of a 96-well plate and a quantity of drug was
added into the well water. After 3 days of incubation, the larvae were imaged and the amount of
fin regenerated was measured using ImageJ (National Institutes of Health, Bethesda, Maryland)
with the notochord as a reference point. Data was compiled and statistically analyzed in
Microsoft Excel using ANOVA and a two-tailed student t-test. Error bars denote SE.
Adult model: Wild-type young adult zebrafish (3 months old) were anaesthetized with
10 ppm of eugenol and amputated midway through the caudal fin with a clean razor blade. Fish
were imaged at 12-15X magnification before and after amputation using a stereomicroscope.
After 15 minutes of recovery, the fish were distributed into containers each with 2-3 fish and 100
mL solution of water and drug/DMSO. The fish were incubated at 28°C, fed once a day, and
imaged three days post-amputation (3 dpa). Original, amputated, and regenerated fin lengths
were measured using ImageJ and analyzed using Microsoft Excel. The compiled and averaged
data were statistically analyzed using ANOVA and a two-tailed student t-test. Error bars denote
SE.
3.2.6 Whole-mount immunohistochemistry (IHC)
Tg(flk1:EGFP) embryos were treated with 4 µM 4HPR, 7.5 µM SU5416 (alone and
together), and 0.5% DMSO (vehicle) as indicated in the angiogenesis screen. 0.22 mM of PTU
was added to the well solution to inhibit pigmentation. 2.5 dpf embryos were fixed overnight in
4% paraformaldehyde (PFA) and processed using the IHC protocol provided by Dr. Jason Fish.
Embryos were washed 2-3 times (5 minutes each) with PBSTw (1x PBS containing 0.1%
Tween-20) to remove PFA. For permeabilization, the embryos were washed 3 times (10 minutes
each) in PBST solution, 1x PBS containing 1% Triton-X. An additional 20 minutes of washing
(10 minutes each) was performed with PBT solution, which contains 1xPBS, 1% Triton-X and
51
2% bovine serum albumin (BSA). The embryos were left at room temperature in blocking
solution (containing 1x PBS, 1% Triton-X, 2% BSA, 10% goat or sheep serum, and 1% DMSO)
for 3-4 hours.
The mouse F59 (Developmental Studies Hybridoma Bank, Iowa City, IO, USA) antibody
recognizes slow myosin heavy chain (MyHC) in somites and the rabbit α-GFP (Molecular
Probes, Invitrogen, Burlington, ON, Canada) antibody recognizes the GFP contained in ECs of
transgenic embryos. These primary antibodies were added (at 1:5 and 1:400 respectively) and
left overnight at 4°C. On the second day, the embryos were washed extensively with PBT for at
least 2 hours (10-15 minutes each wash). The secondary antibodies, anti-mouse Alexa-Fluor 647
and anti-rabbit Alexa-Fluor 488 (both raised in goat; Invitrogen, Burlington, Canada) were used
to label F59 in red and α-GFP in green. A 1:200 solution of secondary antibodies were added
into blocking solution and left to incubate overnight at 4°C.
On the third day, embryos were transferred to a new tube and washed extensively in PBT
at least 10 times, 10 minutes each wash. Embryos were cleared in a glycerol solution and
mounted on glass plates with a thin layer of 1% agarose to restrict motion during image capture.
At least 5 embryos were treated in each condition. Representative images of fully intact embryos
were acquired using a Leica confocal microscope and stacked to generate a 3-dimensional image
of the tail at 25X magnification.
3.2.7 Whole-mount RNA in situ hybridization (ISH) Primer preparation
RARg-a: A pCR4-TOPO plasmid containing RARg was provided by Ellie Melancon
(Postlethwait lab, University of Oregon). The plasmid contained a T3 RNA polymerase promoter
52
fused to a RARg gene fragment and was sequenced to confirm the identity of the RARg-a gene.
The plasmid was dissolved into nuclease-free water (NF-H2O) and OneShot TOP-10 competent
E-coli cells were transformed for the purification of more plasmid DNA. 5 µL of plasmid was
added to competent cells, gently mixed and incubated on ice for 30 minutes. The cells were heat-
shocked in a 42°C waterbath and placed in ice. 250 µL of pre-warmed LB medium was added
and incubated in for an hour at 225 revolutions per minute (rpm) in a 37°C rotary shaker-
incubator. 50 µL of the transformation mix was spread onto LB /ampicillin agar plates and
incubated overnight at 37°C. Three colonies were selected and cultured in LB/amp media
overnight in a 37°C 225 rpm rotary shaker-incubator. Plasmid DNA was isolated from
transformants using a minpreparation kit (Qiagen). 20 µL (6 µg) of purified DNA was digested
with 5 µL of the restriction enzyme NotI for 2 hours at 37°C. The digested product was purified
using a PCR purification kit (Qiagen). In vitro transcription using T3 polymerase and a DIG
RNA labelling kit (MEGAscript, Roche) was employed under the stringent RNAse-free
conditions of a Captair rolling fume hood. The labelling mixture combined 2 µL of each
nucleotide (ATP, GTP, CTP, DIG-UTP), 2 µL of 10x reaction buffer, 1 µL of SuperR (RNAse
inhibitor), 2 µL of T3 RNA polymerase and NF-H2O up to a total volume of 20 µL. The mixture
was incubated for 2 hours at 37°C. The RNA was precipitated with 30 µL of LiCl precipitation
solution and chilled for 45 minutes at -80°C. The sample was centrifuged for 15 minutes at 4°C
to pellet the RNA, washed with 70% ethanol and re-centrifuged. The pellet was then resuspended
in 30 µL of NF-H2O, aliquoted and stored at -20°C in either NF-H2O or hybridization mix until
usage. Nanodrop and 1% agarose gel electrophoresis of the product confirms RNA probes 500bp
in length and of good quality.
53
FGFR1a: PCR primers were designed using the electronic Primer3 program to flank the
5’ and 3’ untranslated regions (UTRs) of the FGFR1a and BLASTed for specificity. DNA
fragments were PCR amplified using Taq polymerase and a cDNA template generated from 7
day old embryo mRNA by reverse transcriptase (obtained from Dingyan Wang of the Gramolini
lab, University of Toronto). The solution was run in a thermocycler (Eppendorf Mastercycler
gradient) under the following conditions: 93°C for 3 minutes, *93°C for 30 seconds, 58°C for 45
seconds, 70°C for 1 minute,* with the asterisked settings repeating 40x, then 70°C for 10
minutes and 4°C for short term storage. Products were purified using a PCR purification kit
(Qiagen) and ligated into a TOPO 2.1 vector containing a T7 RNA polymerase promoter. 1 µL of
the PCR purified product was added to 1 µL of TOPO vector in a total volume of 7 µL NF-H2O
at room temperature for 15 minutes. Plasmids were cloned by transforming OneShot TOP-10
competent E-coli cells using a modified heat shock protocol. 1 µL of ligation mix was added to
thawed competent cells and incubated on ice for 15 minutes. The mixture was heat-shocked at
42°C for 45 seconds and iced for 2 minutes. 250 µL of pre-warmed LB media was added and
cells were incubated for 1 hour in a 37°C, 225 rpm rotary shaker-incubator. 150 µL of the
transformation mix was spread onto LB/ampicillin plates coated with 40 µL of 2% X-gal and
incubated overnight at 37°C. 3-4 isolated white colonies were selected to be cultured in
LB/ampicillin media overnight in a 37°C, 225 rpm rotary shaker-incubator. Vector DNA was
isolated from transformants using a minpreparation kit (Qiagen). A sample of the DNA was
digested with restriction enzyme EcoRI to confirm ligation of the insert by 1% agarose gel
electrophoresis. The remainder of the vector was linearized using restriction enzyme EcoRV.
RNA probes were generated by in vitro transcription using T7 polymerase and a DIG RNA
54
labelling kit (MEGAscript, Roche) under the stringent RNAse-free conditions of a Captair
rolling fume hood.
Embryo preparation
Embryos of the albino strain were treated with 4 µM 4HPR, 4 µM retinoic acid (RA) or
0.5% DMSO at 90% epiboly (9 hpf) and incubated overnight at 28°C. 24 hpf embryos were
fixed in 4% paraformaldehyde (PFA) overnight at 4°C, washed with PBS and dehydrated in
methanol for at least 24 hours at -20°C.
In situ hybridization
RNA in situ hybridization was performed as described by Thisse et al. (2010) with some
deviations. Dehydrated embryos stored at -20°C were rehydrated with successive dilutions of
methanol in 1x PBS, then washed 4 times, 5 minutes per wash, in PBT. Embryos were carefully
dechorionated using Almedic #7 forceps after rehydration to decrease tissue damage. Embryos
were permeabilized by digestion with 10 µg/mL Proteinase K for 10 minutes at room
temperature. This is followed with a 20 minute 4% PFA-PBS wash to stop the digestion. The
embryos are washed with PBT 4 times, 5 minutes per wash to remove PFA and transferred to
new 1.5 mL eppendorf tubes. Embryos are prehybridized in 300 µL of Hybridization Mix (HM)
containing tRNA and heparin for 3 hours in a 70°C hybridization chamber. HM is removed and
replaced with 200 µL of HM containing 1 ng of the DIG-labelled RNA probe, then left overnight
in the 70°C hybridization chamber. On day 2, HM (that does not contain tRNA and heparin) is
successively diluted to a solution of 2x SSC with a series of 10 minute washes at 70°C: 75%,
50%, 25% HM and 100% 2x SSC. Two 0.2x SSC washes, 30 minutes each, were performed at
70°C. 0.05x SSC was progressively replaced with PBT with a series of 10 minute washes at
55
room temperature: 75%, 50%, 25% of 0.05x SSC and 100% PBT. Embryos were incubated in
preincubation buffer (containing 2mg/mL BSA and 2% sheep serum) for 3 hours at room
temperature under agitation. The solution was replaced with 300 µL preincubation buffer
containing a 1:5000 dilution of anti-DIG antibody, and incubated overnight at 4°C under
agitation. On day 3, embryos were transferred to new 1.5 mL tubes and additional PBT washes
were performed at room temperature, 10-15 minutes each wash, at least 20 times. Embryos were
left in PBT to wash at 4°C overnight to decrease background staining caused by residual anti-
DIG antibodies. On day 4, the alkaline tris buffer and NBT-BCIP staining solutions were freshly
prepared without MgCl2 to prevent salt formation and the staining solution was kept in the dark.
Embryos were washed 3 times; 5 minutes each wash, in alkaline tris buffer at room temperature
with agitation. After adding staining solution, the embryos are periodically monitored, with
limited exposure to light, until the appropriate amount of staining is present. The embryos were
rinsed in stop solution (containing 5 mM EDTA) and cleared in glycerol for another 30 minutes.
A coverslip bridge platform was constructed to facilitate imaging by rolling the “sandwich-ed”
embryos into a planar orientation. Embryos were mounted and images were captured the same
day using z-stack compilation on a Leica stereomicroscope.
56
3.3 Results 3.3.1 Fenretinide increases ISV number in developing zebrafish embryos
In our pilot angiogenesis screen, fenretinide was identified to promote the development
of additional intersegmental vessels (ISVs) in developing zebrafish embryos. To validate this
result and establish a dose-response curve, I administered a range of 4HPR concentrations to
developing zebrafish embryos. Increasing concentrations of 4HPR in transgenic vasculature-
labelled embryos Tg(Flk1:EGFP) resulted in an overall increase in the number of ISVs of each
fish (Fig. 3.3.1). Control embryos treated with vehicle (0.5% DMSO) develop an average of 27.5
± 0.1 ISVs. Embryos treated with 1 µM 4HPR develop 30.2 ± 0.2 ISVs and 4 µM 4HPR develop
32.8 ± 0.3 ISVs (p<0.01). The additional ISVs arise by the formation of extra ISVs in the tail
region, an area absent of ISVs in control embryos.
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Figure 3.3.1 [A] Dose-dependent effect of 4HPR on intersegmental vessel (ISV) development in Tg(flk1:EGFP) zebrafish embryos (treated with PTU) after 48h of drug treatment. 4HPR increases the amount of ISV that develop in 2.5 dpf zebrafish embryos. Fluorescent images are shown of embryos treated with [B] a control of 0.5% DMSO (27.5 ± 0.1 ISVs), and [C] 4 µM 4HPR (32.6 ± 0.4 ISVs). 4HPR-treated embryos characteristically form ISVs that extend into the tail tip. *p < 0.05. ** p < 0.01. N=11-14. Error bars denote SE.
3.3.2 Fenretinide alters the number of endothelial cells in blood vessels
Tg(Fli1:nEGFP)y7 embryos were employed to observe changes in the number of
endothelial cells in specific vascular structures in response to 4HPR treatment (Fig.3.3.2).
Tg(Fli1:nEGFP)y7 is a transgenic zebrafish line with nuclear-localized GFP expression [93].
Embryos were treated with PTU to inhibit pigment formation to facilitate cell counting. The
average cell number decreases in each ISV from 7.5 ± 0.2 cells in control embryos to 3.8 ± 0.4
cells in embryos treated with 4 µM 4HPR. The average cell number in each segment of the DA
58
increases though it is difficult to quantify, as the increase in cell number causes cells to overlap
to the extent that manual and digital cell counting is not possible. There is also a diffusion of the
nuclear EGFP signal along the trunk of 4HPR-treated embryos, which may indicate cellular
apoptosis.
Figure 3.3.2. The average cell number in the [A] intersegmental vessel (ISV) in Tg(fli1:nEGFP) zebrafish embryos after 48h of IO treatment. Fluorescent images of zebrafish embryos at 2.5 dpf show embryos treated with [B] a control of 0.5% DMSO and [C] 4 µM 4HPR. Treatment with 4HPR decreases cell number in the ISV and increases cell number in the dorsal aorta. *p < 0.05. ** p < 0.01. N=6-14. Error bars denote SE.
3.3.3 Fenretinide inhibits fin regeneration Fin regeneration assays were performed in adult zebrafish to assess the effect of 4HPR on
adult angiogenesis. The process of adult tissue regeneration is dependent on angiogenesis and
cell proliferation. Larval fin regeneration is an avascular process, so the assay was performed in
larval and adult zebrafish to differentiate between drug effects on angiogenesis and cell
proliferation. 4HPR dose-dependently inhibits fin regeneration in larval (Fig. 3.3.3.1) and adult
59
zebrafish (Fig.3.3.3.2) as measured 3 days post amputation (dpa). Larval fish were treated with a
greater range of 4HPR as they can tolerate higher concentrations. At 16 µM, larval fish were able
to regenerate an average of 2 µm relative to the 86 µm regenerated by control-treated fish
(p<0.01). Adult fish treated with 0.5% DMSO regenerate 13.8 ± 0.6 % of their original fin length
3 days post amputation. 5 µM of 4HPR reduces the amount of fin regenerated to 9.8 ± 0.6 %
(p<0.01) and 10 µM decreases regeneration to 8.1 ± 0.7% of the original fin length (p<0.01).
Figure 3.3.3.1 [A] Effect of 4HPR on larval zebrafish fin regeneration. Fins were transected at the end of the notochord and fish were incubated in drug for 3 days. Bright field images of caudal fins 3 days post amputation reveal that fish treated with [B] 0.5% DMSO regenerate an average of 86 ± 6 µm of fin bud while fish treated with [C] 16 µM 4HPR regenerate 2 ± 2 µm of the original fin length. 4HPR dose-dependently inhibits fin regeneration. *p < 0.05. ** p < 0.01. N=6-9. Error bars denote SE.
60
Figure 3.3.3.2 [A] Effect of 4HPR on adult zebrafish fin regeneration. Fins are transected at the site of the red arrow and fish are incubated in drug for 3 days. Bright field images of caudal fins 3 days post amputation reveal that fish treated with [B] 0.5% DMSO regenerate an average of 13.8 ± 0.6% of the original fin length while fish treated with [C] 10 µM 4HPR regenerate 8.1 ± 0.7% of the original fin length. 4HPR dose-dependently inhibits fin regeneration. *p < 0.05. ** p < 0.01. N=9. Error bars denote SE.
61
3.3.4 Fenretinide affects somitogenesis during development
The presence of abnormal somites in the trunk and tail were observed in bright field
images of fenretinide-treated fish (appendix 3.4.3.1). Immunohistochemistry (IHC) labeling of
blood vessels (anti-GFP antibodies) and somites (F59, labeling myosin filaments) allows us to
clearly visualize the changes in these structures to distinguish the specificity of fenretinide’s
effects on somitogenesis or angiogenesis. Control embryos have a consistent phenotype:
chevron-shaped somite bundling and a tail tip region that contains a forked somite structure that
is absent of blood vessels (Fig. 3.3.4A). IHC confirmed the presence of somites and vessels in
the tail tip in embryos treated with 4HPR (Fig 3.3.4B). The drug phenotype characteristically
includes a tail curvature phenotype visible in bright field, with a distinct somite bundling pattern
to the tail tip observed with IHC labeling (Fig 3.3.4A and 3.3.4D). Embryos treated with 7.5 µM
of the VEGFR inhibitor, SU5416, develop fewer vessels but have a forked somite pattern in the
tail that is similar to control embryos (Fig. 3.3.4C). The combined treatment of 4HPR and
SU5416 is tested to evaluate the effects of 4HPR while angiogenesis is being suppressed with
inhibition of VEGF signaling. The presence of the somite pattern in the tail tip is apparent with
the combined dosing of these agents (Fig. 3.3.4D). The ability of 4HPR to extend the somite
boundaries into the tail tip persists in a reproducible manner (100% incidence observed).
62
Figure 3.3.4. Somite and vessel IHC labeling of 2.5 dpf Tg(flk1:EGFP) zebrafish embryos treated with [A] 0.5% DMSO, [B] 4 µM 4HPR, [C] 7.5 µM SU5416 and [D] co-treated with 10 µM SU5416 and 4 µM 4HPR. Images are confocal stacks that show fluorescent immunodetection of slow MyHC (F59, somite-specific marker) in red and GFP (endothelial cells) in green. 4HPR appears to affect somite patterning in the tail tip region independently of angiogenic vessel development. Representative images are shown. N=4-5.
3.3.5 Fenretinide increases RAR expression and may decrease FGFR expression
The curved tail phenotype of 4HPR is similar to that of its parent compound, RA, which
exerts its actions on somitogenesis through the RA receptor (RAR). I have performed a RNA in
situ hybridization experiment with a labeled RNA probe of RAR to examine the changes in RAR
expression in response to 4HPR treatment, compared to RA and vehicle treatments. My
preliminary experiment showed an increase in RAR in fenretinide- and RA-treated fish as
63
compared to the control (Fig.3.3.5). The intensity of RAR signal is greater in fenretinide-treated
fish than RA-treated fish, though the concentrations of 4HPR and RA are not equivalent in this
preliminary experiment.
Figure 3.3.5. Preliminary ISH of retinoid acid receptor G (RARg) performed in 24 hpf albino embryos. [A] RARg staining of 24 hpf embryo published in literature [243]. I performed an ISH for RARg in [B] embryos treated with 0.5% DMSO, [C] 4 µM 4HPR and [D] 0.5 µM RA.
64
3.4 Discussion
3.4.1 Fenretinide promotes developmental angiogenesis but inhibits EC migration
Fenretinide has been studied as an anti-cancer drug with documented anti-proliferative
and anti-angiogenic properties. Thus, it was surprising to discover in the initial screen of the
LOPAC compound library that it did not act as an angiogenic inhibitor but as a promoter. In this
study, we confirmed that fenretinide dose-dependently increased the number of ISVs that
developed in zebrafish embryos (Fig.3.3.1). More specifically, it stimulated the growth of ISVs
into the tail tip, an area that is normally absent of ISVs in control fish.
Nascent ISVs are generated from the proliferation and migration of ECs in the DA. To
evaluate the changes in the number of ECs in certain compartments, I performed an angiogenesis
assay with Tg(Fli1:nEGFP)y7 embryos, which have EGFP expression localized to the nucleus of
each EC. The quantification of cell number in the DA was difficult, as the high density of ECs
made it almost impossible to accurately count by manual or digital means. Nonetheless, the
relative increase in fluorescent signals in the DA and decrease in the ISVs (Fig 3.3.2) suggested
that fenretinide was inhibiting the process of cell migration in vivo. Qualitatively, I was also able
to observe EC toxicity in some embryos treated with 4 µM of 4HPR. The expression of EGFP in
the nucleus of 4HPR-treated embryos (Fig.3.3.2C) was much more diffuse than the distinct
nuclear-localized signal of controls (Fig.3.3.2B), suggesting 4HPR-induction of EC death. These
results correlate with in vitro experiments that demonstrate fenretinide’s ability to reduce cell
proliferation [244] and migration [223] in HUVECs.
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3.4.2 Fenretinide inhibits the process of fin regeneration
To observe the effects of fenretinide on tissue regeneration, I performed a fin
regeneration assay in larval and adult zebrafish. I noted a significant decrease in the amount of
fin regenerated in both assays (Figs.3.3.3.1 and 3.3.3.2). In the larval assay, I was able evaluate a
greater number of fish and wider range of concentrations. I also observed more variability, which
can be attributed to the high regenerative capacity of larval fish.
Although the process of wound healing depends on the initiation of physiological
angiogenesis, it also depends on the proliferation and differentiation of other regenerating tissues
(such as epithelial cells). Normally, it is difficult to distinguish on the basis of a tissue
regeneration assay if a drug is achieving this effect through the inhibition of angiogenesis, cell
proliferation, cell differentiation or all of the above. However, the larval assay specifically
evaluates avascular tissue regeneration, since regeneration of the larval fin bud does not involve
blood vessel growth [104]. Fenretinide is able to inhibit proliferative processes involved in
avascular larval fin regeneration and angiogenesis-dependent adult fin regeneration. The most
probable mechanism utilized by fenretinide is the inhibition of FGF signalling, which has been
documented in vitro [224]. The FGF signalling pathway induces proliferation of many cell types;
stimulating the proliferation of ECs as well as mesenchymal cells during wound healing [245].
A MTT assay was performed in HUVEC cells by my predecessor, who noted a biphasic
response in cell viability with increasing concentrations of fenretinide (appendix 3.4.2.1). I
expanded the range of my assay to determine if it was possible for fenretinide to promote fin
regeneration at low concentrations (as low as 0.1 µM; appendix 3.4.2.2). Beneath a concentration
of 2.5 µM, there was no distinguishable difference between fenretinide treatment and the control.
66
I repeated the fin regeneration experiment with transgenic vascular-labelled fish
Tg(Flk1:EGFP) to try to visualize the growth of new blood vessels. I was unable to observe any
fluorescence in the tail in either the control or the treated fish due to the limitations of the
microscope and the opacity of the tissue. Using transparent adult fish Tg(fli1:EGFP;casper) in
this assay may ameliorate these problems. Extending the endpoint to 7 dpa may also improve the
assay, since adult fish require 7 days to completely grow back their tail fins [104]. Nonetheless,
the fin regeneration and EC migration results are in line with literature reports of the anti-
angiogenic and anti-proliferative actions of fenretinide [223]. These findings dismiss the use of
fenretinide in stimulating tissue regeneration but demonstrate that fenretinide’s ability to
promote of angiogenesis is limited to a particular developmental window.
3.4.3 Fenretinide promotes angiogenesis through somitogenesis
The chevron-like arrangement of somites in the trunk of developing zebrafish is a distinct
and invariable pattern. Control fish can be easily distinguished from fenretinide-treated fish due
to the unusual curved-tail phenotype of the latter, even without the aid of a microscope (appendix
3.4.3.1). Under high magnification (bright field microscopy), the presence of small chevrons can
be seen in the tail bud of fenretinide-treated embryos (appendix 3.4.3.2). An
immunohistochemistry (IHC) stain was performed on embryos to compare the arrangements of
somites (Fig.3.3.4). The segmental bundling of fibers coincides with the presence of blood
vessels in control embryos (Fig.3.3.4A). The avascular region of the tail bud is normally
unsegmented and muscle fibers are split into two branches. Fenretinide-treated embryos have
somite segments and blood vessels extending to the tail tip (Fig.3.3.4B and D). Furthermore, this
aberrant pattern prevails even with the inhibition of blood vessel growth, via the application of
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VEGF inhibitor SU5416 (Fig.3.3C and D). These results suggest that fenretinide’s promotion of
angiogenesis in developing embryos is actually secondary to somitogenesis.
3.4.4 Fenretinide’s effect on somitogenesis
If fenretinide is able to stimulate somitogenesis in the tail bud, the most plausible
mechanism is through RA signaling, which coordinates embryo patterning during development.
Developmental patterning is conserved in vertebrates and has been studied in detail. In
zebrafish, somites form in pairs from 10-24 hpf, with about 30 somite pairs forming in total
[246]. RA signaling stimulates differentiation and FGF signaling promotes the proliferation of
stem cells in the anterior and posterior regions of the zebrafish trunk respectively [247]. There is
mutual, lateral inhibition between these two pathways. FGF8 represses RA synthesis by
downregulating the expression of the raldh2 enzyme and upregulating cyp26-mediated RA
degradation [248]. Likewise, RA has been shown to attenuate the expression levels of FGF8
[249]. The sequential formation of somite pairs is coordinated by timed fluctuations in the
gradients of RA and FGF.
Disturbing the dimensions of RA and FGF signaling can easily alter the segmentation
program. Increasing FGF or decreasing RA leads to the “caudalization” of the embro.
Implantation of FGF-soaked beads [250] and RALDH2 knockdown [251] leads to the production
of smaller somites and the enlargement of the undifferentiated presomitic mesoderm. In
comparison, decreasing FGF and increasing RA lead to “anteriorization” of the embryo. The
inhibition of FGF signaling shifts the boundary of anterior signaling lower, resulting in the
development of large somites [252]. Embryos treated with exogenous RA during somitogenesis
68
end up with severely truncated bodies, with the complete loss of the posterior features beyond
the yolk sac [253].
The formation of extra somites I observed in fenretinide-treated embryos is compatible
with the “anteriorizing” effect of excess RA. It is likely that fenrentinide is acting as an
exogenous source of RA to promote somitogenesis in the tail. By extending somite development
into the tail tip, fenretinide is also causing vessels to develop along the extended somites
boundaries. If fenretinide is assuming the role of RA, I should be able to detect the changes in
the expression levels of receptors, RA-metabolizing enzymes and target genes.
I intended to assess the localization and intensity of RAR and FGFR expression in
fenretinide-treated embryos with RNA in situ hybridization (ISH). Normally, FGF signaling in
the posterior trunk excludes RA activity [248]. ISH labeling of FGFR should depict a decrease in
the tail in response to fenretinide treatment [254]. The corresponding RAR expression, which is
absent in control embryos [243], should increase in the tail of treated embryos. In my
preliminary ISH for RAR, I observed an increase in RAR in fenretinide- and RA-treated fish
relative to the control (Fig.3.3.5). The RAR expression is most intense in fenretinide-treated fish,
which may be attributed to the higher concentration, or the potency of the drug. This experiment
is to be repeated with equal concentrations of RA and fenretinide. Additionally, a glycerol-based
mounting method will be employed to improve the resolution and transparency of the ISH.
Fenretinide has reportedly hydrolyzed to RA in vivo [255]. If fenretinide (an amide) is
hydrolyzed to RA then we should be able observe an increase in RALDH expression in treated
embryos. The change in RALDH expression can be compared in response to systemic drug
treatment or localized implants of fenretinide- and RA-soaked beads. If RALDH expression does
69
not change, then it is possible that fenretinide is exerting its effects through other metabolites
such as N-(4-Methoxyphenyl)retinamide (4MPR) or 4-oxo-4HPR and their associated enzymes
[256]. Alternatively, I could also investigate the RA-degrading enzyme cyp26, which is one of
the gene targets of RAR activation [198]. If fenretinide is able to bind to RAR, a relative increase
in cyp26 should be observed in treated embryos.
The expression levels of cyclic genes and FGF target genes after fenretinide treatment can
also be examined to see whether they will be changed in a manner comparable to exogenous RA
treatment. Excess RA in the tail (knockdown cyp26a or exogenous application of RA)
contributes to asymmetric expression of all cyclic genes such as her1 and her7 [257, 258].
Her13.2 has been shown to be regulated by FGF signalling [258]. Using ISH we expect to see
reduced or absent expression of her13.2 in the tailbud of fish treated with 4HPR and RA
compared to vehicle treatment. Likewise, we expect to see a reduction in the expression of FGF
target genes (e.g. Hox genes c6-10) that are involved in caudalization signalling of body
extension during embryogenesis [247].
3.4.5 The teratology of retinoids and fenretinide
Levels of RA are maintained within a specific range during normal embryogenesis and
the wrong concentration at the wrong period in development can produce congenital
malformations in vertebrates [259]. Rats, whose mothers are Vitamin A-deficient, are born with
malformations in the eye, urogential tract, diaphragm, heart and lung [260]. Excess vitamin A
during gestation can cause resorption of the embryo, and craniofacial and ocular defects [261,
262]. These effects are likely mediated by RAR activation, since RAR was found to be induced
following RA treatment in a mouse developmental model [262]. There is a 40% risk for
spontaneous abortion and 26% risk for major malformations (of craniofacial, cardiac, thymic and
70
neural structures) reported in cases of embryonic exposure to exogenous RA during early
pregnancy in humans [263, 264].
During the preclinical phase of its development, the teratogenicity of fenretinide was
tested in rats and rabbits. Fenretinide was characterized as a weak teratogen, with low incidences
of craniofacial malformations such as hydrocephalus, micropthalmia and misshapen skull bones
[228]. However, pharmacokinetic studies reveal that fenretinide did not reach significant levels
in fetuses until 10 days of dosing, on gestational day 15 [228]. Considering that somite formation
and many other RA-dependent processes commence at gestational day 9-10 in rats, different
findings may be observed if the dosing regimen commenced at an earlier stage of development
[265]. In a clinical setting, 200 mg of 4HPR is administered orally per day [234]. A series of
maternal oral intake dosages of fenretinide should be studied to determine if RA-like teratogenic
effects would develop in humans.
A zebrafish teratogenicity assay has recently been developed by a group of investigators
affiliated with the pharmaceutical company Bristol-Myers Squibb. Guidelines for numerical
morphological scoring have been established, enabling the quantification of the teratogenicity of
a compound [266]. The authors of the study have tested a number of chemicals in this assay and
were able to identify 87% of the teratogens correctly [267]. RA was one of the compounds to be
scored and identified as a teratogen. The protocol for embryo treatment is virtually the same as
the developmental angiogenesis assay, although wild-type embryos are employed and embryos
are assessed later in development, at 5 dpf. This assay is a simple and convenient method of
assessing the teratogenicity of fenretinide. Using the bright-field images I have obtained from the
angiogenesis assays, I can already identify abnormalities in a number of the structures and organ
systems (e.g. eyes, skull, notochord, somites, and tail) in 2.5 dpf fenretinide-treated embryos
71
(appendix 3.4.3.2). It would be interesting to investigate the teratogen score of fenretinide
relative to RA and other retinoids.
Isotretinoin (a close relative of RA) is regarded as the biggest teratogenic threat since
thalidomide [268]. When you are dealing with teratogenicity, it is not the same as other
potentially adverse side effects. “There is no safe minimal dose” and health practitioners exercise
caution in their use of retinoids as a result [208]. By extension, all systemically administered
retinoids are considered to be teratogenic, especially if exposure occurs during the first trimester
[208]. Retinoid treatment presents a high risk for spontaneous abortion and the development of
retinoid embryopathy, a term used to describe the collection of craniofacial, cardiovascular,
thymic, nervous and limb anomalies that develop in children exposed to retinoids in utero [269].
Consequently, the clinical use of oral retinoids has been restricted against women with positive
pregnancy tests. In clinical trials for fenretinide, pregnancy is listed among the exclusion criteria
[231]. In addition, female patients of child-bearing age are advised to use contraceptives during
the course of RA treatment and programs are being implemented to raise awareness and increase
compliance [270]. In America, a FDA-mandated program (iPledge) has been employed to
monitor and prevent pregnancy in patients being treated with isotretinoin [270].
3.4.6 Conclusions and future directions
In a zebrafish angiogenesis chemical genetic screen, we identified fenretinide as a pro-
angiogenic agent. Through compound validation and drug mechanism studies, we characterized
that the “pro-angiogenic effect” of fenretinide is secondary to its effect in promoting
somitogenesis, and fenretinide is a potential teratogen
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So far, fenretinide’s clinical application has involved a patient population where
pregnancy is not a common occurrence (i.e. cancer patients). As its applications extend to
chemoprevention and the treatment of other diseases (e.g. diabetes, cystic fibrosis), the detailed
characterization of fenretinide’s teratogenic potential becomes a priority. If fenretinide
demonstrates the capacity to act as an exogenous source of RA during critical periods of
development and shows teratogenicity in an animal model of development, then it should be
regarded as a dangerous teratogen as RA. My studies in zebrafish models strongly support that
fenretinide is a teratogen and detailed studies in other mammalian model systems are needed to
validate this finding.
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3.5 Appendix
Appendix 3.4.2.1. MTT assay performed on HUVEC cells treated with fenretinide (Hit #4). HUVEC cells were seeded in 96-well plates and viable cells were measured in triplicate. A biphasic growth response was observed, with increased cell viability at low concentration and decreased viability at high concentration of fenretinide. These assays were performed by Chunyang Wang (unpublished).
Appendix 3.4.2.2. Fenretinide decreases adult zebrafish fin regeneration. At lower concentrations of fenretinide, the amount of fin regeneration is similar to the control. Contrary to the MTT results, a low concentration of fenretinide is unable to improve cell survival in vivo. N=2-6
**
**
0
5
10
15
20
0 0.1 0.25 0.5 1 2.5 5 10
% Fin Regen
erated
4HPR Concentra9on (μM)
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Appendix 3.4.3.1. [A] Bright field image of a 2.5 dpf albino zebrafish embryo treated with 0.5% DMSO, under high magnification (40x). [B] Bright field image of 2.5 dpf albino zebrafish embryo treated with 4 µM 4HPR under high magnification (40x). Note the curvature of the tail, smaller eyes and skull, and abnormalities in the somite and notochord of the tail.
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CHAPTER 4: ANTI-ANGIOGENIC ACTIVITY AND POTENTIAL MOLECULAR MECHANISMS OF INDIRUBIN-3-MONOXIME
4.1 Introduction Historically, natural products have been a rich source of therapeutic compounds [271]:
73% of cancer therapeutics approved to date are derived from natural sources [272]. In the
1970’s and 1980’s this trend surged as pharmaceutical companies modified many naturally-
derived compounds for a variety of therapeutic applications [273]. Two of the compounds I am
studying can trace their origin to this era.
4.1.1 Traditional Chinese medicine and the discovery of indirubin
Traditional Chinese medicine (TCM) has been practiced for millennia and is still used
today. Many of the herbal remedies used in TCM have been investigated within the paradigm of
Western medical research. The active components of medicinal plants have proven to be an
excellent resource for the development of new drugs.
Dang Gui Long Hui Wan is a traditional Chinese medical preparation employed in the
treatment of myelocytic leukemia. The powder derived from indigo naturalis was identified as
the active constituent out of the eleven herbal ingredients contained within this prescription,
[274]. I. naturalis has hemostatic, anti-pyretic, anti-inflammatory, sedative, anti-bacterial and
anti-viral properties [275]. The anti-leukemic property of the powder was attributed to indirubin;
a red indole (aromatic heterocyclic compound) found in minute quantities (0.11%) in the leaves
of indigo-producing plants such as indigofera tinctoria [276].
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4.1.2 Applications and properties of indirubin Indirubin was able to reduce the tumour burden of mice xenografted with Lewis lung
carcinoma and Walker carcinoma with minor toxicity [277]. Another preclinical study in dogs
assessed the relative safety of indirubin using 25 times the therapeutic dosage for half a year.
Although diarrhea and liver damage were noted side effects, there was no apparent
cardiovascular or renal toxicity [278]. In clinical trials, indirubin was able to produce beneficial
effects in patients with chronic myelocytic leukemia. Complete remission was observed in 26%
of patients and partial remission in 33% [279]. Indirubin was well tolerated with minor
gastrointestinal effects such as diarrhea, nausea, and vomiting [279].
Derivatives of indirubin have been synthesized to retain its useful properties while
improving its limitations (i.e. poor solubility and absorption and mild gastrointestinal toxicities
[280]. Among these analogues is my second compound of study, indirubin-3-monoxime (I3M;
Fig. 4.1.2).
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Figure 4.1.2 The discovery of indirubin and chemical structures indirubin and its derivative, I3M. Indirubin was isolated from Qing Dai, a medicinal powder contained within Dang Gui Long Hui Wan, a Traditional Chinese Medicinal preparation used in the treatment of myelocytic leukemia. Indirubin undergoes imine formation to produce I3M.
4.1.3 Molecular mechanisms of indirubins The anti-leukemic properties of indirubin have largely been attributed to two mechanisms
of action: cell cycle disruption and induction of cell death.
Indirubin demonstrated early in its development, the ability to block cell proliferation by
inhibiting DNA synthesis of leukemia cells in vitro [281] and in vivo [282]. Indirubin and I3M
selectively inhibit the phosphorylation of cyclin-dependent kinases (CDKs) [276]. The activity of
CDKs is essential to the regulation of the cell cycle; progression through all the phases of the cell
cycle is coordinated through the activation and inhibition of CDKs [283]. The advancement of a
normal cell cycle is regulated at three checkpoints to evaluate the integrity of DNA and the
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preparedness of the cell for imminent cell division [275]. Many cells that lack control
mechanisms for these checkpoints end up with deregulated expression of CDKs, deviant cell
cycle progression and uncontrollable cell proliferation that is characteristic of cancer [275]. The
ability of indirubins to inhibit CDKs allows them to target a specific feature that enables cancer
growth. Although the mechanisms are not well understood, indirubin and its derivatives have
demonstrated the ability to induce cell cycle arrest in a variety of cancer cell lines [284, 285].
Several studies have also demonstrated I3M’s ability to induce apoptosis. Tumour cells
treated with I3M experienced cell death, evident from the change in cell morphology (membrane
blebbing, chromatin condensation and DNA fragmentation), caspase activation, PARP cleavage,
and cytochrome c release [286]. I3M inhibition of the NF-κB pathway appears to sensitize cells
to apoptosis in response to TNF-activation or the application of chemotherapeutic agents such as
taxol [287].
I3M exerts effects on several other processes that are involved in tumourigenesis. It
inhibits c-Src kinase, which is involved in the STAT signalling that mediates cell proliferation,
tumourigenesis and metastasis [288]. Another intracellular target of I3M is the tyrosine kinase,
FGFR1. I3M-induced inhibition of FGF signalling reduces cell proliferation of a leukemia cell
line just as potently as FGFR1 inhibitor SU5402 [289]. Both indirubin and I3M have
demonstrated the ability to inhibit angiogenesis in vitro and in vivo [290, 291]. I3M also inhibits
glycogen synthase kinase-3 (GSK-3), a kinase involved in the regulation of apoptosis, cell
growth, differentiation, and inflammation [292, 293]. I3M’s negative regulation of cell
proliferation, inflammation and angiogenesis translates to its efficacy in vivo, as all three
processes are overactive during tumourigenesis.
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4.1.4 Indirubin-3-monoxime as an anti-cancer agent The 3’-monoxime modification of indirubin enhances its solubility, kinase inhibition and
cell permeability [276, 294]. Consequently, I3M is more potent and more soluble than indirubin
[284]. I3M inhibits the proliferation of many types of cancer cells with minimal toxicity to
normal cells [295]. I3M is a strong and selective inhibitor of CDKs and GSK-3, in addition to
many other intracellular kinases [296]. It has the ability to inhibit neovascularisation in the aortic
ring and Matrigel plug assay through the inhibition of VEGFR activity, though the mechanism is
currently unknown [291]. Furthermore, oral I3M treatment has demonstrated the capacity to
reduce tumour growth in a mouse model of lung cancer [297]. Short-term animal studies of I3M
have not observed any toxic side effects [298]. Collectively, literature regarding I3M indicates
that it is able to selectively target multiple pathological processes in cancer and is relatively safe
to use. Thus, it is a promising anti-cancer agent.
4.1.5 Additional applications of I3M Although I have indicated that I3M promotes the induction of apoptosis, it is also able to
prevent apoptosis through the inhibition of cellular kinases. CDK activation is implicated in the
induction of neuron death in Alzheimer’s disease. I3M, a selective CDK inhibitor, demonstrates
neuroprotective effects by preventing tau phosphorylation in vitro, through inhibition of
amyloid-β induced apoptosis of neuroblastoma cells [299]. These effects translate in vivo, as
I3M treatment was shown to reduce memory impairment and neuropathology in a transgenic
mouse model of Alzheimer’s disease [300]. I3M can also downregulate apoptosis through the
inhibition of glycogen synthase kinase-3 (GSK-3), to exert protective effects in renal and hepatic
injury [301]. The inhibition of c-Jun NH2-terminal protein kinase (JNK) is another means of
preventing apoptosis, as demonstrated in cerebellar granule neurons [302].
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Indirubin and I3M have also demonstrated immunomodulatory activities. Indirubins
decrease production of the chemokine RANTES, implicated in inflammatory cell recruitment
during the pathogenesis of influenza [303]. Indirubin treatment suppresses the production of a
number of factors involved in the inflammatory response such as interferon γ, IL-6, and NF-κB
[287, 304, 305]. Similarly, I3M acts on NFκB and JNK to lower levels of NO, prostaglandin E2,
IL-1β and IL-6 in cells with lipopolysaccharide-induced inflammation [306]. These findings
support the potential application of I3M as a therapeutic agent for various inflammatory diseases.
Many of I3M’s kinase targets are implicated in the pathogenesis of a diversity of
diseases. Among the most recently proposed applications of I3M are its uses in treating type 2
diabetes and mood disorders [292]. Additionally, a brominated derivative of I3M has shown the
capacity to maintain pluripotency in embryonic stem cells through Wnt activation [307].
Indirubins can be used to prolong self-renewal in stem cell cultures, thereby overcoming a major
limitation in stem cell-related biomedical research and regenerative medicine [308].
4.1.6 Rationale I3M was identified in our initial zebrafish screen as a negative modulator of
angiogenesis. A literature review of indirubin activity has indicated that I3M has demonstrated
anti-angiogenic properties in HUVEC and Matrigel plug assays. However, the mechanism by
which I3M inhibits angiogenesis has not yet been identified. In my studies, I will evaluate the
anti-angiogenic properties and investigate the mechanisms of its actions in zebrafish models of
angiogenesis. These studies will help demonstrate I3M’s value as an anti-angiogenic agent in
vivo for future applications in the treatment of diseases characterized by excessive angiogenesis.
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4.2 Materials and Methods
4.2.1 Preparation of drugs and reagents
Indirubin-3-monoxime (I3M) and γ-secretase inhibitor DAPT were purchased from
Tocris Bioscience (Ellisville, MI, USA). Refer to text and section 3.2.1 for source and
preparation of other drugs and reagents respectively. Embryos and larvae were treated with 2, 4,
8, and 16 µM of I3M and/or 100 µM of DAPT and adults were treated with 5 and 10 µM of I3M.
4.2.2 Zebrafish strains and husbandry Refer to 3.2.1
4.2.3 Cell lines
Several human cancer cell lines were used in the zebrafish xenograft assay. Breast cancer
MDA435 DsRed-expressing cells that express normal and excess amounts of the gene RhoC
were provided by Dr. Konstantin Stoletov (University of San Diego). Several prostate cancer cell
lines (PPC-1, PC3 and DU145) were modified to express mCherry by James Wang (Dr. Jeff
Medin lab, University of Toronto). These cells were cultured in DMEM or RPMI media
containing 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Multicell, Wisent Inc)
and passaged biweekly.
4.2.4 Zebrafish developmental angiogenesis assay Refer to 3.2.3
4.2.4 In vivo quantification of EC number The transgenic zebrafish line, Tg(fli1:nEGFP)y7
expresses EGFP in the nucleus of ECs,
permitting the quantification of EC number in specific vascular structures like the DA, DLAV,
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and ISV. Embryos were treated with I3M and DAPT (alone and in combination) as described in
section 3.2.4 with the following modifications; cell counting of “good” fluorescent images (non-
blurry, with low background fluorescence) were performed with digital assistance and lower
quality images were analyzed by manual counting.
Cell number in the ISV and DA were manually counted and averaged across 10 segments
of each embryo, and then expressed as an average of all samples treated with each drug
concentration. Manual cell counting in the DLAV determined a total number of DLAV cells in
each fish, which was averaged for each condition.
A custom algorithm was developed to analyze and batch process the number of nuclei in
each vessel type within the Matlab software (MatLab V.7.6, The MathWorks, Natick, MA,
USA). The Matlab program analyzed each image under two specifications recognizing high or
low thresholds for fluorescent intensity. The average of the conservative and non-conservative
outputs produced cell counts that were within 10 nuclei of the manual count. The final cell
numbers were manually corrected to include missed nuclei (e.g. overlapping nuclei) by visually
assessing each Matlab output. The data was compiled in Microsoft Excel and statistically
analyzed using ANOVA and a two-tailed student t-test. Error bars denote SE.
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Figure 4.2.4. Sample images generated from MATLAB’s semi-automated cell counting algorithm, which was able to quantify the number of cells (overlay in blue) in the ISV, DA and DLAV.
4.2.5 Fin regeneration assay Refer to 3.2.5
4.2.6 Embryonic tumour xenograft model
Newly fertilized eggs were collected following natural breeding of Tg(Flk1:EGFP) or
Tg(Fli1:nEGFP)y7 fish. At 12 hpf, propylthiouracil (PTU) was added to inhibit the development
of embryonic pigmentation. Tg(fli1:EGFP;casper) transparent embryos were also used. At 2-3
dpf, embryos were immobilized in embryo medium containing 100ppm eugenol. In preparation
for microinjection, human cancer cells were washed with D-PBS and treated with non-enzymatic
cell-dissociation solution (Sigma-Aldrich, Oakville, ON, Canada) to remove adherent cells from
the surface of the tissue culture flask. Cell concentration was calculated by staining cells with
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0.4% trypan blue (Medstore, Toronto, Canada) and counted with the aid of a hematocytometer
(VWR Scientific, 15170-208) and a hand-held tally counter. The cell suspension was iced or
maintained at room temperature until microinjection.
Embryos were microinjected in the perivitelline space (yolk sac) using a Femtojet
microinjector (Eppendorf) and permitted to recover in embryo medium for 15-30 minutes.
Control embryos were injected with vehicle, containing cell dissociation solution. Healthy
embryos were injected with a moderate amount of cancer cells (between 10-100 cells) were
selected, anesthetized, mounted onto a layer of agarose, imaged and dispensed into 96-well
plates. Each well housed one embryo to prevent contamination in the event of embryo mortality.
I3M and DMSO were added into the embryo water and incubated at 28 or 32°C. Two days post-
injection, the embryos were imaged with a fluorescence stereomicroscope using the SCORE
mounting method. SIVs, endothelial and cancer cell numbers were compared to assess tumour
angiogenesis and metastasis.
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Figure 4.2.6 Workflow of a zebrafish embryonic tumour xenograft assay. Newly fertilized eggs are collected and dosed with PTU 12 hpf to block pigment formation. At 2-3 dpf, embryos are microinjected with cancer cells and dispensed into wells of a 96-well plate. Drugs are added directly into the fish water. The embryos are imaged with fluorescence 2 days post injection (dpi). SIV morphology was analyzed to evaluate the drug effect on tumour angiogenesis. Figure reprinted with permission from Elsevier Limited (review paper in press).
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4.3 Results
4.3.1 I3M decreases ISV number in developing zebrafish embryos
I3M was the most potent inhibitory compound identified in the pilot screen of the
LOPAC library. I administered increasing concentrations of I3M in the zebrafish angiogenesis
assay. I3M dose-dependently inhibits vessel development in Tg(Flk1:EGFP) zebrafish embryos
resulting in an decrease in the number of ISVs (Fig. 4.3.1). Control embryos (0.5% DMSO)
develop an average of 27.5 ± 0.1 ISVs. Embryos treated with 4 µM I3M develop 16.3 ± 1.2
ISVs, treatment with 8 µM results in 3.3 ± 1.1 ISVs (p<0.01). With 16 µM I3M treatment, vessel
development is almost completely inhibited, with 0.7 ± 0.4 ISVs developing (p<0.01).
Figure 4.3.1. [A] Dose-dependent effect of I3M on ISV development in Tg(flk1:EGFP) zebrafish embryos after 48h of treatment. I3M decreases ISV development in 2.5 dpf zebrafish embryos. Fluorescent images are shown of embryos treated with [B] a control of 0.5% DMSO (27.5 ± 0.1 ISVs), and [C] 4 µM I3M (16 ± 1.2 ISVs).*p < 0.05. ** p < 0.01. N=9-13. Error bars denote SE.
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4.3.2 I3M inhibits endothelial cells migration in vivo
Tg(Fli1:nEGFP)y7 embryos (nuclear-localized GFP expression in ECs) were treated with
I3M to evaluate changes in the number of endothelial cells in each vessel (Fig. 4.3.2). The
average cell number dose-dependently decreases in the ISVs from 7.4 ± 0.2 in controls to 3.6 ±
0.5 in 8 µM and 1.8 ± 0.3 in 16 µM of IO (p<0.01). Average cell number increases in the
segments of DA from 8.2 ± 0.2 in controls to 10.6 ± 0.4 in 8 µM and 12.8 ±0.6 in 16 µM of I3M
(p<0.01). It is interesting to note that the sum of DA and ISV ECs are similar between control
and various concentrations of I3M-treated embryos (Fig. 4.3.2 A&B), suggesting that I3M
affects endothelial cell migration from DA to ISV.
Figure 4.3.2. The average cell number in the [A] intersegmental vessel (ISV) and [B] dorsal aorta (DA) in Tg(fli1:nEGFP) zebrafish embryos after 48h of I3M treatment. Fluorescent images of zebrafish embryos at 2.5 dpf show embryos treated with [C] 0.5% DMSO (control) and [D] 16 µM I3M. Treatment with I3M decreases ISV cell number and increases DA cell number. *p < 0.05 **p< 0.01. N=9-14. Error bars denote SE.
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4.3.3 I3M inhibits larval and adult fin regeneration
A fin regeneration assay was performed in adult zebrafish to examine the anti-angiogenic
effect of I3M in an adult organism. Because tissue generation is dependent on other processes
(proliferation, migration, differentiation) as well as angiogenesis, a larval (avascular) model was
also employed. I3M dose-dependently inhibits fin regeneration in larvae (Fig. 4.3.3.1) and in
adults (Fig.4.3.3.2). 16 µM of I3M reduced the length of fin regenerated in the control, by more
than half (50 and 86 µm respectively; p<0.01). In the adult model, 5 µM reduces fin regeneration
to 7.5 ± 0.6% of the original fin length, and 10 µM decreases regeneration to 3.1± 0.4 %
compared to a control regeneration of 13.8 ± 0.6% (p<0.01).
Figure 4.3.3.1 [A] Effect of I3M on larval zebrafish fin regeneration. Fins are transected at the end of the notochord and fish are incubated in drug for 3 days. Bright field images of caudal fins 3 days post amputation reveal that fish treated with [B] 0.5% DMSO regenerate an average of 86 ± 6 µm of fin bud while fish treated with [C] 16 µM I3M regenerate 50 ± 10 µm of the original fin length. *p < 0.05. ** p < 0.01. N=9-12. Error bars denote SE.
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Figure 4.3.3.2 [A] Effect of I3M on adult zebrafish fin regeneration. Fins are transected at the site of the red arrow and fish are incubated in drug for 3 days. Bright field images of caudal fins 3 days post amputation reveal that fish treated with [B] a control of 0.5% DMSO regenerate an average of 13.8 ± 0.5% of their original fin length and fish treated with [C] 10 µM I3M regenerate an average of 3.1± 0.4%. IO dose-dependently inhibits fin regeneration. *p < 0.05. ** p < 0.01. N=9-10. Error bars denote SE.
4.3.4 Development of zebrafish tumour angiogenesis model and preliminary test of I3M
Tumour development is dependent on angiogenesis and consequently, many cancer cells
have the property to induce angiogenesis. Previously our lab used the mouse xenograft cancer
model to study cancer angiogenesis but this assay is time-consuming (2-3 months for each
experiment) and expensive (as it requires large quantities of chemical compounds). Therefore, I
intended to establish a zebrafish xenograft cancer angiogenesis model and test the drug efficacy
against cancer angiogenesis in 96-well plate format. This will facilitate the compound validation
process of drug development in our lab.
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I conducted transplantation experiments, grafting fluorescent cancer cells into embryo
recipients possessing fluorescent vasculature to observe the effect of a drug on cancer cell-
induced angiogenesis. The assay was time-consuming and technically demanding. Only 10-40
embryos could be injected and imaged per experiment. Embryo mortality was extremely high as
there were numerous technical challenges and variables to manipulate in this model.
Injected cells had the tendency to aggregate in the injection needle. I was able to solve
this problem by substituting a PBS injection solution with a non-enzymatic cell-dissociation
solution. The strain and age of zebrafish receiving the graft was another variable to consider. Of
the three strains used, Tg(fli1:EGFP;casper) was most convenient as PTU did not need to be
administered to inhibit pigment formation. However, Tg(Fli1:nEGFP)y7 was most effective as
nuclear fluorescent signals produced distinct visualization of the SIVs (which were intensely
fluorescent and more difficult to visualize in casper and flk embryos). The ideal age for injection
was 3 dpf, when SIVs have already formed and neovascular SIV growth is reported to occur
directionally towards the injection site. This was not observed experimentally. Instead, embryos
were injected at 2 dpf amidst SIV development. Embryos were observed 2-3 days post injection;
though at least half the injected embryos died 24 hours after injection. Comparatively, only about
a quarter of embryos injected with vehicle would die. Embryo mortality could be attributed to
three causes; injury during injection, infection of the injection site and inability to accommodate
the cancer cells injected. Embryos can reportedly tolerate injections of up to 200 cells for 3 days
of observation. The optimal cell number was 50-100 cells, such that there was an adequate
number to induce angiogenesis but not so much as to cause embryo mortality. The temperature
of incubation was also a variable factor; zebrafish are normally maintained at 28°C and cells at
36°C. I compromised between the two and incubated injected embryos at 33°C. Usually injected
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cells remain at the site of injection, though approximately 1% of embryos would show cancer
cells disseminated throughout the circulatory system.
Several cancer cell lines were evaluated to find a suitable cell type for xenografting.
MDA435 expressed red fluorescence poorly, was not particularly angiogenic and caused
significant embryo mortality (very few untreated embryos survived with MDA435; appendix
4.3.4.1). PPC-1 cells proved to be too malignant as well. Even though very small quantities of
PPC-1 were injected, the majority of embryos did not survive past 1.5 dpi (appendix 4.3.4.2).
PC3 cells were cultured but not used since cell growth was slow and media was contaminated
with particulate matter secreted by the cells. DU145 cells demonstrated the best qualities of the
cell lines used: strong mCherry expression, moderate proliferation and lower embryo mortality
(appendix 4.3.4.3).
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Figure 4.3.4.Zebrafish embryonic tumour xenograft model. [A] SCORE fluorescence imaging of Flk1:EGFP 5 day old zebrafish (vessels in green), 2 days after injection with mcherry labelled human prostate cancer cells (DU145; red). [B] Control injection with vehicle (cell dissociation solution) and treated with 0.5% DMSO. [C] Xenograft embryo treated with 0.5% DMSO. [D] Xenograft embryo treated with 8 µM IO. Note the inhibition of vessel growth from the subintestinal vessels (arrow). N=1 due to high embryo mortality.
I was able to observe an inhibitory effect of I3M treatment in a number of injected fish
(Fig. 4.3.4). However, statistical analysis was not an option considering the extremely small
sample size. After months of experimentation and over a thousand embryo injections, I stopped
trying to develop this assay. There were too many challenges to the xenograft; problems in
maintaining consistency in the injection, large variability in angiogenic response (which was
confounded by infection-induced angiogenesis) and high mortality of embryos (ranging from 50-
95%).
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4.3.5 DAPT partially rescues the I3M chemical genetic phenotype
Since I3M was shown to affect EC migration in our zebrafish cell counting assay, I
investigated a candidate pathway involved in angiogenic sprouting to elucidate I3M’s
mechanism of action. The interaction between the notch and VEGF pathways coordinates the
process of vessel sprouting during angiogenesis [98]. Notch activation has been shown to block
sprouting of ISVs in zebrafish [309] to produce a similar phenotype to a VEGF morphant [310].
The notch-activated embryos are also characterized by immature cytoplasmic extensions along
the dorsal aorta, which suggests that some ECs are able to sprout from the DA but not complete
their migration. This is reminiscent of I3M’s chemical genetic phenotype. The notch inhibitor
DAPT was co-administered with I3M in zebrafish embryos to determine if I3M is exerting its
effects through the activation of the notch pathway.
Nuclear fluorescent Tg(fli1:nEGFP)y7 zebrafish were used to quantify total endothelial
cell number in of 2.5dpf embryos after drug treatment (Fig. 4.3.5). Control embryos treated with
0.5% DMSO have an average total of 105 ± 2.7 cells in the DLAV, while 100 µM DAPT
treatment produces an average total of 100.4 ± 7.8 (p>0.05). Embryos treated with 4 µM and 8
µM I3M have an average total of 30.5 ± 4.4 and 22.2 ± 4.1 cells in the DLAV. Combined dosing
of I3M with 100 µM of the notch inhibitor DAPT significantly increased the average total
DLAV cell number to 44.3 ± 3.2 and 33.5 ± 3.4 at 4 µM and 8 µM of I3M respectively (p<0.05).
Embryos treated with I3M alone, DAPT alone and co-treated with I3M and DAPT did not have
statistically different cell numbers in the DA and ISV from those of the controls (p>0.05).
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Figure 4.3.5 Effect of notch inhibitor DAPT on cell numbers in the ISV and [A] DLAV in I3M-treated Tg(fli1:nEGFP) y7 zebrafish embryos after 48h of treatment. Fluorescent images of zebrafish embryos are shown at 2.5 dpf treated with [B] 0.5% DMSO, [C] 100 µM DAPT, [D] 4 µM I3M and [E] 4 µM I3M +100 µM DAPT. The treatment of DAPT increases the average cell number in the DLAV of I3M-treated embryos. *p < 0.05. ** p< 0.01. N=10-25. Error bars denote SE.
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4.4 Discussion
4.4.1 I3M demonstrates specific inhibition of developmental angiogenesis I3M was identified from our initial screen of the LOPAC library as an angiogenesis
inhibitor. I observed a potent, dose-dependent inhibition of angiogenesis in my assay of zebrafish
developmental angiogenesis (Fig.4.3.1). The blood vessels that make up the primary circulatory
loop, such as the DA and PCV, remained intact and functional with I3M treatment. This was
apparent even at the highest dose of I3M (16 µM), when very few angiogenesis-derived vessels
were observed. Thus, I3M may selectively affect the process of angiogenesis rather than
vasculogenesis, and the viability of ECs does not appear to be affected.
This assay was repeated using Tg(Fli1:nEGFP)y7 embryos to discern changes in EC
number within specific vessels. I observed a dose-dependent decrease in the number of ECs in
the ISV and a corresponding increase in DA cells in response to I3M treatment (Fig.3.3.2). The
increase in DA cells indicates that ECs proliferate but do not migrate from their original location.
I3M may be inhibiting developmental angiogenesis in zebrafish by acting on mechanisms that
prevent cell migration. I3M’s ability to inhibit this particular stage of angiogenesis has also been
observed in vitro. In an endothelial cell scratch assay, I3M treatment dose-dependently hindered
the migration of HUVECs [291].
During the course of these studies, I was also able to coarsely evaluate the safety of I3M.
I did not observe the diffusion of nuclear-GFP expression in Tg(Fli1:nEGFP)y7 embryos
(Fig.4.3.2D). Thus, I3M may not be inhibiting angiogenesis through generalized induction of cell
death. I also did not observe any obvious morphological malformations relative to control
embryos during the first 3 days of embryonic development. There was no curved body shape,
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enlarged yolk sac, no changes in skull shape or delayed pigmentation in I3M-treated embryos at
2.5 dpf (appendix 4.4.1). The teratogenicity of I3M can be more accurately evaluated with a
morphological scoring assay [266]. The embryo size decreases at high concentrations of I3M,
possibly due to a delay in development (toxicity). However, at its effective range of dosages, the
development of I3M-treated embryos is relatively normal, its effects on angiogenesis
notwithstanding. Additional assays for organ-specific toxicology can also be used to assess I3M
effects the function of the heart, liver, kidney, central nervous system, muscles and
gastrointestinal tract [76].
4.4.2 I3M inhibits fin regeneration Reportedly, adult zebrafish can only regenerate up to 1 mm of fin tissue when
angiogenesis is inhibited [104]. The results of my adult fin regeneration assay are indicative of
I3M’s ability to halt regenerative angiogenesis (Fig.4.3.3.2). However, as I noted in section
3.4.2, adult fin regeneration involves several other processes. I3M’s inhibition of fin regeneration
can be attributed to more than just the inhibition of physiological angiogenesis. The results of the
larval assay demonstrate that I3M is also able to affect avascular processes of regeneration, such
as cell proliferation, differentiation and migration (Fig. 4.3.3.1). It has been reported in literature
that I3M is able to directly target FGFR tyrosine kinase to inhibit cell proliferation as potently as
the FGFR1 inhibitor SU5402 [289]. The blockade of FGF, a prominent signalling pathway
involved in the proliferation of many tissue types, is the most likely mechanism of action for
I3M’s suppression of tissue regeneration. FGF is also a potent angiogenic factor that stimulates
angiogenesis.
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4.4.3 I3M efficacy in tumour models Despite the problems I encountered in setting up my xenograft assay, I was able to
observe that I3M treatment blocked SIV growths in embryos injected with DU145 (Fig. 4.3.3).
However, these findings must be interpreted with caution considering the numerous limitations
of my assay. The sample size was very small, due to substantial mortality of injected fish,
variability in the site of injection and presence of infection (which would stimulate angiogenesis
even in fish injected with injection solution). In addition, the timing of the assay (injection at 2
dpf) made it difficult to differentiate tumour-induced angiogenesis from developmental
angiogenesis. While it would be ideal to inject cells after SIV formation is complete (at 3dpf), I
was unable to observe tumour-induced angiogenesis from mature SIVs. Perhaps this would be
possible if I employed a cancer cell line that has been genetically modified to secrete
angiogenesis factors such as FGF [110].
I3M has demonstrated the ability to selectively inhibit the growth of cancer cells in vitro,
in a variety of cell lines [295]. I3M’s in vivo efficacy has also been evaluated in one study of a
[B(α)P]-induced (tobacco carcinogen) lung cancer model in mice. Oral I3M treatment dose-
dependently reduced the number of pulmonary tumours and alveolar damage by inducing
apoptosis in cancer cells [297]. While these results are encouraging, I3M needs to be tested in
more animal models of cancer to verify if I3M’s wide spread in vitro efficacy translates to anti-
tumour effects in vivo.
4.4.4 I3M inhibits zebrafish ISV development partially through the notch pathway The combined administration of I3M with the notch inhibitor DAPT increases the
number of ECs in the DLAV, partially restoring the vascular network of developing zebrafish
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embryos (Fig. 4.3.5). When notch ligands (e.g. Dll4, Jagged1) bind to a transmembrane notch
receptor, it undergoes a conformational change from a series of enzymatic cleavages. The
enzyme γ-secretase releases the intracellular domain of the notch receptor (NICD), which
translocates to the nucleus to activate the transcription of target genes. DAPT inhibits the notch
pathway in an early part of the signalling cascade by inhibiting γ-secretase activity. DAPT
partially rescues the I3M phenotype, which suggests that I3M may be achieving its effects
through the activation of the notch pathway. This can be verified by investigating relative
changes in the expression levels of notch ligands, receptors or target genes.
However, it is also possible that notch activation is indirectly stimulated by I3M. Notch
plays such an important role in the developmental process, as indicated by the role of notch
genes in somitogenesis, that a stimulator of notch would be expected to produce more deleterious
effects in embryos. This was observed in a transgenic zebrafish line that utilized a heat-shock
triggered Gal4-UAS system to overactivate the notch pathway. Heat-shock was applied to
stimulate the overexpression of NICD. Although vessel sprouting was inhibited, it was
accompanied by numerous morphological defects [309, 311]. In my angiogenesis assay, I did not
observe any obvious defects in I3M treated embryos (appendix 4.4.1). Curiously, other
investigators have reported inhibitory effects of I3M on notch signalling. I3M has been shown to
suppress the activation of the notch1 receptor, through GSK-3β [312]. This is not supported by
my in vivo results as the vascular phenotype of mutant zebrafish with deficient notch signalling
has increased vessel sprouting (with multiple filopodia resembling tree branches) [313].
I3M has been well characterized as a kinase inhibitor and there are numerous kinases
involved in angiogenic cascade. It would be most reasonable to speculate that I3M’s anti-
angiogenic effects occur through kinase inhibition. I3M acts on a variety of kinases because it is
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able to interact with the ATP-binding site of a kinase. Co-crystallization experiments have
revealed that I3M forms 3 hydrogen bonds with glutamate and leucine residues of CDK2’s ATP-
site [276]. The complementary shape and H-bonding allows I3M to fit into the ATP-binding
pocket more snugly than indirubin or ATP [276]. Similarly, co-crystal structures of I3M and
GSK-3β indicate that I3M also tightly fits into the ATP-site [314]. I3M forms 4 H-bonds with
aspartate, valine and glutamine residues and orients its apolar ring into the apolar residues found
within the hinge region of GSK-3’s binding pocket [314].
It is reasonable to speculate that I3M may also competitively inhibit ATP binding at the
catalytic sites of other kinases. I3M is able to inhibit the phosphorylation of FGFR1 in fibroblast
cells under FGF-1 stimulation, which subsequently blocked the phosphorylation of several
downstream effectors (e.g. ERK1, Src, STAT3) [289].
A recently published paper identified another tyrosine kinase receptor involved in
angiogenic signalling as a target of I3M. The phosphorylation of VEGFR2 was reported to be
dose-dependently reduced by I3M treatment in a kinase assay performed in HUVEC cells [291].
Furthermore, Kim et al (2011) were able to show that I3M inhibition of angiogenesis requires
VEGFR2-mediated signalling. I3M was unable to inhibit EC migration and tube formation in
HUVECs treated with VEGFR2-targeted siRNA [291]. The requirement of VEGFR2 in this
study strongly supports that I3M’s anti-angiogenic effects are attributed to the direct inhibition of
VEGFR2 activity.
A kinase assay can be performed with varying concentrations of I3M and ATP to verify
the kinetic mechanism of competitive inhibition. In addition, the rescue strategy Chan et al used
to study the VEGF inhibitor PTK787 can also be effectively employed by performing AKT/PKB
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or eNOS mRNA injections in I3M-treated embryos [81]. If the anti-angiogenic effects of I3M
diminish with the restoration of VEGFR2 downstream signaling in zebrafish, we can further
validate its role as a VEGFR2 inhibitor in vivo. The co-crystallization of I3M with VEGFR2 is
needed to ascertain if the structural-activity relationship of I3M’s inhibition occurs through the
ATP-site, as it is with CDK2 and GSK-3β. It is probable that the potency of I3M’s anti-
angiogenic effects is due to its ability to inhibit multiple kinase signalling pathways at once.
I believe that the findings of the DAPT rescue experiment can be re-evaluated in
consideration of I3M’s function as a competitive inhibitor of VEGFR2. There is considerable
crosstalk between the VEGF and notch pathways in the process of vessel sprouting during
angiogenesis. Notch activation has been shown to limit vessel branching through negative
regulation of VEGF [315]. This is achieved through a downstream target of notch signalling,
transcription factor HESR-1, which represses the transcription of VEGFR2 [316]. DAPT
treatment increases the expression of VEGFR2 by inhibiting notch signalling [98].
Consequently, in my experiment, DAPT may be able to overcome I3M’s competitive inhibition
and restore VEGF-mediated angiogenesis by increasing the amount of substrate (VEGFR2)
available. In essence, it is a systems biology illustration of enzyme-inhibitor kinetics. Within this
proposed framework, it would be plausible to observe full restoration of angiogenesis by
adjusting the relative quantities of DAPT and I3M (increasing and decreasing respectively).
4.4.5 Conclusions and future directions
At present, I3M is at an early point in drug development, with the majority of it studies
conducted in vitro. The next step is to test its efficacy in vivo, in animal models. Although rodent
studies remain the gold standard for preclinical testing, zebrafish are uniquely capable of
expediting drug development by permitting in vivo mechanism studies.
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The results of my studies in zebrafish have supported the efficacy of I3M as a therapeutic
agent for the treatment of cancer and angiogenesis disorders. I3M inhibition of cell proliferation
and angiogenesis allows it to target numerous aspects of tumour pathophysiology, rendering it
more resistant to drug resistance. I3M’s ability to inhibit angiogenesis occurs through
competitive inhibition of the kinase VEGFR2. Co-crystallization experiments will be able to
confirm the precise interaction of I3M with VEGFR2.
The abundance of VEGF-based angiogenesis agents and their lack of clinical efficacy
limit the utility of I3M in angiogenic therapy. However, I3M’s capacity to inhibit several
aberrant cell processes as a broad kinase inhibitor can be powerfully applied in anti-cancer
therapy. Thus far, I3M has demonstrated a protective effect in the progression of lung cancer in a
mouse model. Its utility in other cancers will need to be further characterized. The safety status
of I3M in mammalian models will also need to be confirmed in the future, though I have
observed that I3M is well tolerated in adult and larval zebrafish.
The culmination of I3M’s studies thus far reveals that it shows great potential as
therapeutic agent for applications in the treatment of cancer, angiogenesis disorders, and
neurodegenerative and immune diseases. The development of I3M and its potential successes are
additionally significant because it reflects positively on the combined efforts of traditional
medicine and modern biomedical science.
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Appendix
Appendix 4.3.4.1. [A] Bright field image and [B] fluorescent image of human breast cancer cells MDA435, expressing DsRed and the metastatic gene RhoC. Note the levels of DsRed expression. [C] Fluorescent image of 5 dpf Tg(fli1:nEGFP) y7 embryos. [D] Fluorescent image of 5 dpf Tg(fli1:nEGFP) y7 embryo treated with 16 µM of I3M. Most of the fish injected with MDA435 did not survive. MDA435 cells would proliferate/migrate, bursting out of the yolk sac and causing embryo death.
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Appendix 4.3.4.2. [A] Bright field image and [B] fluorescent image of human prostate cancer cells PPC-1, expressing mcherry. [C] Fluorescent image of 5 dpf Tg(fli1:nEGFP) y7 embryos mounted in a SCORE imaging apparatus to allow rotation of the embryo [D] Fluorescent image of 5 dpf Tg(fli1:nEGFP) y7 embryo injected with PBS. [E] Fluorescent image of 5 dpf Tg(fli1:nEGFP) y7 embryo injected with PPC-1. Most of the fish injected with PPC-1 did not survive.
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Appendix 4.3.4.3. [A] Bright field image and [B] fluorescent image of human prostate cancer cells DU145, strongly expressing mcherry. Fluorescent image of 5 dpf Tg(fli1:nEGFP) y7 embryo injected with DU145, treated with [C] 0.5% DMSO and [D] 8 µM of I3M
Appendix 4.4.1. Bright field image of 2.5 dpf Tg(fli1:nEGFP)y7 embryo treated with 16 µM of I3M, at 40x magnification.
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CHAPTER 5: THE ROLE OF DHM IN ANGIOGENESIS
5.1 Introduction
5.1.1 Rotenoids Rotenoids are a group of natural and synthetic heterocyclic compounds with insecticide
properties. The core structure of a rotenoid is comprised of a linked ring system
(tetrahydrochromeno[3,4b] chromene rings) [317] (Fig. 5.1.3). Natural rotenoids have been
isolated from the seeds, fruits, leaves and stems of leguminous plants native to the southern
regions of Asia, America and Africa [318]. These plants have been historically utilized by
indigenous people for applications in traditional medicine and as pesticides [319].
Figure 5.1.3. Chemical structure of dihydromunduletone and related rotenoids.
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Rotenoids have been employed as household and industrial organic pesticides. Reports of
its bioactivity in mammals have stimulated the scientific community to investigate the potential
environmental dangers posed by rotenoids, as well as its potential use in drug discovery. The
World Health Organization has classified a common rotenoid, rotenone, as a moderately
hazardous chemical and many countries have placed restrictions on its use to curtail the
possibility of environmental damage [318]. Yet, rotenoids present a great opportunity for drug
discovery. Many extracted and modified rotenoids have been evaluated for applications in anti-
bacterial, anti-malarial, anti-fungal and anti-cancer therapies [317].
5.1.2 The anti-cancer activities of rotenoids Mundulone is a compound extracted from the plant Mundulea chaperlieri that is native to
Madagascar [320]. Mundulone was able to exert moderate cytotoxic effects (IC50 = 13 µg/ml) in
an ovarian cancer cell line [320], though further characterization of its anti-cancer activities have
not been evaluated.
Rotenone has been applied traditionally, environmentally and industrially as a natural
piscicide, a fish-killing substance. It is a lipophilic compound that is easily absorbed into the gills
of fish and enters the bloodstream to quickly produce toxic effects. Rotenone toxicity is
attributed to its ability to interfere with the transfer of electrons in the mitochondrial membrane
during cellular respiration [321]. Although its use is prohibited in several countries, its
environmental hazards are minor because it is light-sensitive and the mode of chemical exposure
(skin, gastrointestinal tract) in mammals curbs its toxicity [318]. Interestingly, rotenone has been
successfully applied in biomedical research to model Parkinson’s disease (PD). Rotenone is
injected into rats, crosses the blood-brain-barrier and damages dopaminergic neurons in a
manner that reproduces the symptoms and pathogenesis of PD [322]. While rotenone does not
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show carcinogenic or seriously toxic effects in mammals, it still poses a health risk. A recent
epidemiological study has demonstrated an association between rotenone use and the incidence
of Parkinson’s disease [323].
Deguelin is another naturally occurring rotenoid that is commercially used as an
insecticide. Actually, deguelin and rotenone can both be found in Indigofera tinctoria, which if
you recall, also contains small quantities of indirubin [324]. This plant appears to be a bountiful
source of anti-cancer chemicals since deguelin, like indirubin, exhibits anti-cancer properties.
Deguelin is able to limit the growth of breast, skin and colon cancer cells through apoptotic and
anti-proliferative effects [325, 326].
Deguelin was found to induce apoptosis in colon and gastric cancer cells by inhibiting
AKT and NF-κB signalling [327]. Deguelin’s reported inhibition of angiogenesis has also been
tested in vitro and in vivo. Deguelin is able to reduce angiogenesis in a scratch assay, tube
formation assay, a CAM assay [328]. Its anti-angiogenic effects may be attributed to the
inhibition of AKT and downregulation of VEGF and HIF-1α [329]. Deguelin also inhibits heat-
shock 90 (Hsp90), by directly binding to its ATP-site [330]. Hsp90 is overexpressed in many
cancer cells, so by targeting it, deguelin is able to downregulate Hsp-driven tumour progression,
angiogenesis and drug-induced resistance [331].
A reduction in tumour progression has also been observed in rodent models of cancer
(xenograft and carcinogen treatment) after the short term application of deguelin [330].
Deguelin’s activities appear to be selective, as pre-malignant and malignant cells respond more
sensitively than normal cells to its effects [329]. Additionally, no toxic effects have been
observed at therapeutic doses in animal models [330]. Unfortunately, long term or high doses of
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deguelin have produced neurotoxic effects resembling the PD-like symptoms produced by
rotenone [332]. Biomedical research continues to develop new rotenoid derivatives to search for
the balance between the efficacy and potential toxicity of deguelin and rotenone [333].
5.1.3 Dihydromunduletone My last compound of study, dihydromunduletone, is a rotenoid derivative identified from
the Spectrum Collection library as an inhibitor of angiogenesis in zebrafish [96].
DHM is a novel, semi-synthetic compound that has not been previously studied as an
anti-angiogenic or anti-cancer agent. In fact, a literature search on DHM only produces two hits.
Wetli et al identified DHM as a weak inhibitor of iron uptake in a cell-based screen and did not
characterize it any further [334]. The second paper, published from our lab, identified DHM as
an inhibitor of angiogenesis [96].
Although next to nothing is known about DHM, the documented activities of the
aforementioned, structurally related rotenoids could provide insights into DHM’s potential
biological activities (Fig 5.1.3).
I would like to evaluate DHM’s anti-angiogenic properties in order to assess its potential
uses in the treatment of cancer and other diseases characterized by excessive angiogenesis.
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5.2 Materials and Methods
5.2.1 Preparation of drugs and reagents DHM was purchased from MicroSource Discovery Systems Incorporated (Gaylordsville, CT,
USA). Embryos and larvae were treated with DHM at the final concentrations of 2, 4 and 6 µM,
and adults were treated with 0.5, 0.75 and 1 µM.
5.2.2 Zebrafish strains and husbandry Refer to 3.2.1
5.2.3 Zebrafish developmental angiogenesis assay Refer to 3.2.3
5.2.4 In vivo quantification of EC number Refer to 3.2.4
5.2.5 Fin regeneration assay Refer to 3.2.5
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5.3 Results
5.3.1 DHM decreases ISV number in developing zebrafish embryos
DHM reduced the number of ISVs that developed in zebrafish embryos in the pilot
screen so I administered a range of concentrations to study the dose responsiveness of the drug.
DHM dose-dependently inhibits ISV development in transgenic Tg(Flk1:EGFP) zebrafish
embryos resulting in an decrease in the number of mature ISVs (Fig. 5.3.1). Control embryos
treated with vehicle (0.5% DMSO) develop an average of 27.5 ± 0.1 ISVs. Embryos treated with
2 µM DHM develop 25.7.1 ± 0.7 ISVs (p<0.05), 4 µM develop 18.1 ± 2.1 ISVs (p<0.05), and
treatment with 6 µM results in 13.6 ± 2.6 ISVs (p<0.01). Survival of embryos treated with 2 µM
DHM was similar to controls, whereas treatment with 4 µM and 6 µM DHM resulted in the
death of 33% and 67% of treated embryos respectively.
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Figure 5.3.1. [A] Dose-dependent effect of DHM on ISV development in Tg(flk1:EGFP) zebrafish embryos after 48h of treatment. Fluorescent images are shown of 2.5 dpf zebrafish embryos treated with [B] a control of 0.5% DMSO (27.5 ± 0.1 ISVs), [C] 4 µM DHM (18.1 ± 2.1 ISVs). *p < 0.05. ** p< 0.01. N=6-19. Error bars denote SE.
5.3.2 DHM alters the number of endothelial cells in ISVs
Tg(Fli1:nEGFP) zebrafish embryos were treated with DHM and manually counted to
detect any changes in the number of endothelial cells in each vessel. The average cell number
dose-dependently decreases in the ISVs (Fig.5.3.2). Embryos treated with 2 µM of DHM have an
average of 5.4 ± 0.4 cells per ISV and 4 µM have 4.0 ± 0.4 compared to the control average of
7.5 ± 0.2 cells per ISV (p<0.01). Average cell number increases in the caudal segments of DA.
Cells were digitally counted using Matlab software with manual corrections. Embryos treated
with 4 µM of DHM have an average of 11.8 ± 0.3 compared to the control average of 8.2 ± 0.2
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cells per DA (p<0.05).
Figure 5.3.2. The average cell number in the [A] intersegmental vessel (ISV) and [B] dorsal aorta (DA) in Tg(fli1:nEGFP) zebrafish embryos after 48h of DHM treatment. Average cell number decreases in the ISV and increases in DA segments. Control embryos treated with 0.5% DMSO develop an average of 7.5 ± 0.2 cells per ISV and of 8.2 ± 0.2 cells per DA. Embryos treated with 4 µM DHM have an average of 4.0 ± 0.4 cells per ISV and 11.8 ± 0.3 cells per DA. *p < 0.05. ** p < 0.01. N=7-14. Error bars denote SE.
5.3.3 DHM inhibits adult fin regeneration A fin regeneration assay was performed in adult zebrafish to examine the anti-angiogenic
effect of DHM in an adult organism. For my other two compounds, I also assessed larval fin
regeneration to distinguish between angiogenesis and other tissue regenerative processes (such as
proliferation). I was not able to complete larval fin regeneration experiments for DHM due to
high embryo mortality. DHM dose-dependently inhibited adult fin regeneration (Fig.5.3.3). After
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3 days of drug treatment, 0.5 µM reduces fin regeneration to 9.8 ± 0.6% of the original fin
length, 0.75 µM decreases regeneration to 8.6 ± 0.7 % and 1 µM decreases regeneration to 5.9 ±
0.8 % compared to a control regeneration of 13.8 ± 0.5% (p<0.01). Survival of fish treated with
less than 1 µM DHM was similar to controls, whereas treatment with 1 µM DHM resulted in the
death of 5 fish.
Figure 5.3.3. [A] Effect of DHM on adult zebrafish fin regeneration. Fins are transected at the site of the red arrow and fish are incubated in drug for 3 days. Bright field images of caudal fins 3 days post amputation reveal that fish treated with [B] a control of 0.5% DMSO regenerate an average of 13.8 ± 0.5% of their original fin length and fish treated with [C] 10 µM 4HPR regenerate 8.1 ±0.7%. DHM dose-dependently inhibits fin regeneration. *p < 0.05 ** p < 0.01. N=9-10. Error bars denote SE.
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5.4 Discussion
5.4.1 DHM inhibits developmental angiogenesis DHM moderately inhibits angiogenesis, reducing the number of ISVs that develop in
zebrafish embryos (Fig 5.3.1). Vessels derived through vasculogenesis are intact, so DHM’s
action may be specific to the process of angiogenesis.
DHM treatment in Tg(Fli1:nEGFP)y7embryos demonstrated a decrease in the number of
ECs in the ISV and increase in the DA (Fig. 5.3.2). DHM may be inhibiting the process of EC
migration during developmental angiogenesis. The EGFP expression of control embryos is
localized to the nucleus, producing a distinct fluorescent signal (Fig.5.3.2c). I observed a diffuse
fluorescent signal in a proportion of embryos treated with 4 µM of DHM (Fig.5.3.2d), indicating
that DHM treatment has the potential to produce EC apoptosis at a higher concentration.
5.4.2 DHM inhibits adult fin regeneration DHM is able to inhibit fin regeneration in adult zebrafish as measured at 3 dpa
(Fig.3.4.2). This may be achieved through the inhibition of regenerative angiogenesis, or other
processes involved in fin regeneration, such as mesenchymal cell proliferation. On the basis of
these findings alone, it is difficult to delineate if a particular process or cell type is being affected
by DHM.
5.4.3 DHM toxicity
I observed a substantial amount of drug toxicity during the course of my experiments
with DHM in zebrafish. Although embryos can be treated with 4-6 µM of DHM, adults appear to
be more sensitive to the toxic effects of DHM. Very low concentrations of DHM were tolerated
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in adult zebrafish, and half of the fish would perish before the end of the assay. About half of
DHM-treated embryos exhibited features of toxicity and delayed development; less
pigmentation, change in body shape and enlarged yolk sacs (appendix 5.4.3). The therapeutic
window of this drug is very small in both embryos and adults, and it is accompanied by toxic and
possibly teratogenic effects.
The observed fatalities in DHM-treated fish could be associated with rotenoid fish-
specific toxicity. Compounds that are structurally related to DHM (rotenone, deguelin) have been
applied as piscicides due to the ease of chemical absorption - fish uptake rotenoids dissolved in
water more readily than mammals which absorb rotenoids through the skin and gastrointestinal
tract [318]. Rotenone inhibition of mitochondrial complex I reduces ATP and enhances ROS
production in fibrosarcoma cells [335][335]. If DHM is acting through the same mechanism, it
should produce similar changes in ATP and ROS levels in vitro.
5.4.4 DHM future studies
To assess its suitability for therapeutic use, I would first determine the specificity of
DHM’s toxicity, pertaining to its specificity of action in fish and particular cell types.
Mundulone, DHM’s most closely related compound, has demonstrated cytotoxic effects in
cancer cells, although its limited study does not distinguish between general and selective
toxicity [320]. To evaluate DHM’s anti-cancer, anti-angiogenic and cytotoxic properties, I would
propose a comparative cell survivability assay (e.g. MTT) in ECs, cancer cells and non-ECs (e.g.
epithelial cells or fibroblasts). If the in vitro studies determine DHM’s activity to be specific to
angiogenesis or cancer cells, it is necessary to validate these findings in a whole-organism
model. I would suggest a mammalian model, as previous studies performed with rotenone have
reported minimal toxicity in non-fish models, due to absorption and rapid metabolism [336].
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Several assays can be performed to validate the effect of DHM on angiogenesis. An in
vitro characterization of DHM using MTT, Ki-67 labelling or thymidine incorporation assays
can test EC viability and proliferation. PI staining and flow cytometry can analyze cell cycle
progression, and TUNEL staining can evaluate apoptosis in ECs. Cell viability or proliferation
can also be assessed in vivo in zebrafish using methods such as acridine orange staining TUNEL,
Ki-67 labeling, or a transgenic reporter line for apoptosis [337]. Additionally, a scratch assay and
tube formation assay can be performed in HUVECs to evaluate DHM effects on in vitro
angiogenesis. The next step involves the in vivo characterization of DHM’s anti-angiogenic
capacity, in a rodent Matrigel plug assay or corneal micropocket assay.
A commercially available PCR microarray specific to angiogenesis (containing 113
angiogenesis related genes) could help identify particular pathways affected by DHM (SA
Biosciences). I propose to treat HUVECs with DHM and vehicle and analyze the differential
gene expression to identify candidate pathways using whole genome microarray. Proteins in
affected pathways can then be examined as potential drug targets using target deconvolution
strategies such as affinity binding or mass spectrometry-based proteomic techniques.
Foreknowledge of DHM’s related compound, deguelin, can lead to more focused experiments
such as western blot analyses of the phosphorylation states of AKT, MAPK, NF-κB, VEGF and
Hsp90 [327, 329, 330].
5.4.5 Conclusions and future directions
DHM has demonstrated potential as an inhibitor of angiogenesis in my experiments with
zebrafish. A proper evaluation of its toxicity and specificity of action is warranted before more
detailed mechanism studies are conducted. To be considered as a potential therapeutic agent,
DHM must be well tolerated and selective in its effects. Chemical modification of the compound
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may be necessary to increase DHM’s anti-angiogenic efficacy while decreasing its general
toxicity.
5.5 Appendix
Appendix 5.4.3. Bright field images of 2.5 dpf Tg(Fli1:nEGFP)y7 treated with [A] 0.5% DMSO and [B] 4 µM DHM with 25x magnification. Note the morphological differences in the pigmentation, yolk sac size and body shape.
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CHAPTER 6 – GENERAL DISCUSSION AND FUTURE DIRECTIONS
Numerous drugs in use today were discovered through happenstance. Serendipity is well
documented in the history of medicine [338], though happy accidents are not a reliable means of
drug discovery. As the scientific community improved their understanding of the mechanisms of
disease, target-based drug discovery became the prevailing albeit ineffective strategy for the past
few decades [339]. This has been evident in the field of angiogenesis as VEGF monotherapies
(strategies directed only at VEGF signalling) have produced disappointing outcomes in the clinic
[340]. Phenotype-based approaches are able to identify new chemical candidates without prior
knowledge of a known target. Traditional cell-based phenotype assays can efficiently screen
large numbers of chemicals. However, a very limited number of drugs can translate their efficacy
from in vitro to in vivo systems. Performing screens in animal models allow for a more
comprehensive assessment of drug efficacy on the systems biology of a phenotype or disease.
The success rates of in vivo screens are higher than cell-based screens, which are unable to
simulate the complex physiology of a disease [341].
The advantage to employing zebrafish rather than mice in whole-organism chemical
screening lies in its amenability for high-throughput analysis. A large number of samples can be
evaluated at once and the phenotypic readouts are more simple and accessible. Compare the
angiogenesis assays of mice and zebrafish; Matrigel plugs and corneal micropocket assays
compared to the developmental angiogenesis assay. The former assays have more extensive
sample preparations (as they are invasive techniques), and require larger quantities of drug and
maintenance costs. ISV development in zebrafish is easy to quantify and visualize, with the
availability of transgenic and transparent lines, and hundreds of embryos can be handled together
in one assay.
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I encountered a few limitations to zebrafish screening in the course of my studies.
Incorporating a whole-organism into a chemical screen allowed for the combined assessment of
drug efficacy and toxicity. However, sometimes these phenotypes can overlap, making it
difficult to distinguish between good and bad drug candidates. Furthermore, the developmental
stage of the zebrafish may also contribute to the discovery of undesirable hits. Compounds like
fenretinide can modulate angiogenesis through the disruption of developmental processes. Other
compounds may impede angiogenesis by inducing nonspecific toxicity. DHM may be achieving
its effects this way or through species-specific rotenoid toxicity. Neither of these effects are
desired qualities of therapeutic compounds.
A thorough characterization of embryo phenotypes can help improve the identification of
hits that are poor candidates for angiogenesis therapy. Bristol-Myers Squibb has developed a
zebrafish teratogenicity assay that uses morphological scoring to quantify the teratogenic
potential of a compound. These studies have revealed a good correlation between zebrafish and
human teratogenicity [267]. A post-hoc evaluation of fenretinide-treated embryos reveals several
morphological features indicative of teratogenicity (eyes, skull, notochord, somites, and tail).
Zebrafish have also demonstrated the capacity to predict organ-specific toxicities of
environmental and pharmacological agents [76]. Large-scale genetic screens have employed a
phenoclustering strategy to computationally search for patterns between phenotypes of known
and unknown chemicals in invertebrates such as C. elegans [342]. Methods of automating
phenotype recognition have been developed to assess zebrafish angiogenesis [126] and toxicity
[343] more efficiently. Phenotype annotation will aid the characterization of drug activities and
help predict toxicities as well as mechanisms of action.
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My studies have demonstrated that zebrafish phenotypic screens can accelerate the
process of drug discovery by identifying in vivo efficacy and toxicity earlier in a drug’s
developmental timeline. The appropriate identification of toxic and teratogenic phenotypes will
be essential to distinguish the promising hits (such as I3M) from the less desirable ones (4HPR
and DHM). Although additional validation is required in cell-based assays and mouse models,
my studies support the relevance of the model in the development of human angiogenesis
therapeutics.
Chemical and genetic screens performed in zebrafish have demonstrated strong
correlations to the physiology observed in mice and men [76]. Zebrafish absorption, distribution,
metabolism and excretion (ADME) have been representative of higher vertebrates but systemic
characterization of ADME is necessary to improve the comparative analysis of drug
pharmacology. New technologies are continually being developed to improve the speed and
precision of zebrafish handling, imaging and processing.
Fully automated HTS platforms represent the pinnacle of efficiency for drug screening.
Our lab at St. Michael’s Hospital has recently acquired a fully automated zebrafish screening
facility that is capable of performing high-throughput screens for angiogenesis modulating drugs.
The system is able to automate embryo sorting and dispensing, drug dosing, imaging and image
analysis. Based on the estimated hit rate of the pilot screens, we hope to identify over 100
angiogenesis-modulating compounds in the next few years.
The strategies I have employed in my investigation of 4HPR, I3M and DHM will
hopefully serve as a guide for more detailed characterizations of new compounds identified in
future angiogenesis screens. The characteristics of a drug’s activity on a particular physiological
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process (such as wound healing or somitogenesis) or molecular pathway can also be probed in
zebrafish assays and methods of phenotype analysis (phenoclustering or morphological scoring).
The zebrafish model is a robust tool for drug discovery and its utility is continually being
improved and validated against other animal models. Zebrafish are expected to contribute
extensively to preclinical drug development, complementing data found in other model systems.
The power of the zebrafish model will allow biomedical science to optimize and “systematize the
serendipity of drug discovery” [344].
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CHAPTER 7 – GENERAL CONCLUSIONS
The traditional process of drug discovery has a very low success rate. In oncology, only
5% of preclinical anti-cancer agents are approved for clinical usage [345]. Drug attrition is
mainly caused by the lack of efficacy and safety of drug candidates [346]. Studying the effects of
a compound within the physiological context of a whole-organism allows for the characterization
of a drug’s efficacy and toxicity earlier in the drug development pipeline [347]. Small molecules
isolated from natural sources, and their derivatives, are a promising class of compounds for drug
discovery. These compounds are more likely to possess bioactivities that can be exploited for
therapeutic applications [273]. I have used zebrafish to characterize three angiogenesis-
modulating compounds identified through a small molecule screen of two compound libraries.
Although fenretinide was identified in our screen as a promoter of angiogenesis, I was
able to determine that it achieved this effect by interfering with somitogenesis in zebrafish.
Fenretinide is currently being evaluated in clinical trials for prophylactic and therapeutic
applications in a variety of health conditions. If these findings translate to mammalian models,
then patients undergoing fenretinide treatments should be strongly advised about the risk of
developmental defects in fetuses.
I3M was identified in our screen as a potent inhibitor of angiogenesis. My studies in
zebrafish angiogenesis and fin regeneration have characterized it as an agent that produces anti-
angiogenic effects. I have identified notch as a potential pathway involved in I3M’s anti-
angiogenic chemical genetic phenotype. Based on I3M’s kinase activity in the literature, it is also
likely that the anti-angiogenic activity and DAPT rescue I observed were produced through
modulation of the VEGF pathway, as the literature has documented cross-talk between these two
123
pathways. Although additional studies are required to validate this theory, my studies in
zebrafish have significantly contributed to the small body of research of I3M’s in vivo effects.
My last compound of study, DHM, was found to inhibit developmental angiogenesis and
fin regeneration in zebrafish. Due to time constraints, I did not have the opportunity to explore its
mechanisms or specificity of its actions. However, by studying DHM’s effects in a whole
organism, I have been able to identify anti-angiogenic and anti-proliferative activity as well as
indications of cellular, tissue and general toxicities. If DHM is confirmed as a generic cytotoxic
agent, then my zebrafish studies have assisted and streamlined the identification of an
incompetent drug early in the drug development pipeline.
Comprehensively, my studies have demonstrated the utility of zebrafish as a convenient
and versatile tool for advanced drug discovery. With 4HPR, I was able to learn new qualities
about a drug in clinical development. I was also able to contribute to the validation of a proposed
mechanism of action for the promising anti-cancer agent, I3M. Lastly, with DHM, I was able to
discover a novel use for a novel compound while simultaneously identifying its potential
toxicity. I hope that I have demonstrated in the course of my studies, that zebrafish are uniquely
suited to provide an efficient and effective systems-biology approach to improving the outcomes
of angiogenesis drug discovery.
124
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