Growth factor expression in normal and wounded skin: an ...

Growth factor expression in normal and wounded skin: an investigation of scar-free wound

healing in the leopard gecko (Eublepharis macularius)

by

Noeline Subramaniam

A Thesis

presented to

The University of Guelph

In partial fulfilment of the requirements

for the degree of

Master of Science

in

Biomedical Sciences

Guelph, Ontario, Canada

© Noeline Subramaniam, August, 2016

ii

Abstract

GROWTH FACTOR EXPRESSION IN NORMAL AND WOUNDED SKIN: AN

INVESTIGATION OF SCAR-FREE WOUND HEALING IN THE LEOPARD

GECKO (EUBLEPHARIS MACULARIUS)

Noeline Subramaniam Advisors:

University of Guelph, 2016 Dr. M.K. Vickaryous

Dr. J.J. Petrik

The skin is the primary interface between an organism and its environment.

Following injury, there are two possible outcomes: scar formation or scar-free wound

healing. Although the epidermis is understood to play a role during scar-free skin repair,

few details are currently available. Here I conducted a spatio-temporal characterization of

vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF-2), and

transforming growth factor β (TGFβ) in the epidermis of the leopard gecko (Eublepharis

macularius). I show that each VEGF, FGF-2 and TGFβ are widely expressed within the

normal and injured epidermis. My data indicate that these growth factors may play various

non-angiogenic roles, including keratinocyte proliferation, protect against ultraviolet

photodamage, and facilitate in photobiosynthesis of vitamin D3. Co-localization or

overlapping expression patterns with VEGF and FGF-2 receptors suggests that both

paracrine and autocrine signalling may participate in homeostatic and injury-mediated

functions of the epidermis.

iii

Acknowledgements

First and foremost, I would like to thank my supervisor, Dr. Matt Vickaryous. It pains me

to write kind things about you when I’ve spent the last two years trying to sass you, but if

there was any a time, it would be at the end of a chapter. You have been an amazing advisor

throughout this process and the scientist that I have become has largely been shaped by

you. You’ve created an environment in the lab (i.e. the Eublephosphere) that is conducive

to both developing critical and scientific skills while still having fun. I am grateful for your

mentorship, but more importantly your friendship.

I would also like to thank my co-advisor, Dr. Jim Petrik. Thank you for your

continued support and guidance throughout my program. You always had an open door

and helped me troubleshoot through many difficult times during my degree and I appreciate

your patience and willingness to help. I would like to thank the final member of my

advisory committee, Dr. Neil MacLusky. You have been such a kind and caring advisor

and mentor to me since my days as an undergrad. I’ve probably spent more hours in your

office contemplating my future than I can count and changed my mind a dozen times about

what I want to do with my life. Thanks for always lending an ear and making time for your

students. Additionally, I would like to thank the members of the exam committee including

Dr. Matt Vickaryous, Dr. Jim Petrik, Dr. Coral Murrant and my chair, Dr. Tami Martino.

Next, I would like to thank Jalene Lumb. Thank you for inviting us into your home

and letting us spend so much time with your kids! You have been incredibly fun to get to

know and you have also been very supportive throughout our degree. Thank you to Isla

and Piper - not only have you girls beautified our lab with your creative illustrations (I

never thought I’d look so good as a potato person and I don’t think you can ever have a

surplus of geckos, hearts or rainbows), you have often brightened our day when you have

dropped in unexpectedly to the lab, at lab socials and of course when we go to conferences!

Thanks for being our little buddies!

Thank you to Emily Gilbert aka Mama Bear. You were a great mentor and

confidante at the start of my Master’s program when I was still navigating through an

intensive course load. You trained us how to keep the lab running even in your absence,

iv

how to exude confidence during presentations and generally taught us to be conscientious

scientists. Besides the occasional trip to Aberfoyle, it has been incredibly fun to be one of

the cubs. #foreveracub, #cub33

I would next like to thank the Trifecta! First, Kathy Jacyniak: when I first met you,

I thought, ‘Wow, this girl’s got a lot to say.’ But by the time our first conversation ended,

I knew I wanted to be friends with you. We have really experienced the full breadth of grad

school together; from taking six graduate courses spanning topics from cell biology to

neuroscience to public health and policy, to sharing our insecurities about our (often)

failing projects and everything in between. It goes without saying, but I factually could not

have survived grad school without you. Second, Rebecca McDonald: when I first met you,

I thought, ‘Does this girl speak?’ Oh, how the times have changed. As my partner in crime,

there is no one else I’d like to mischievous with. From TA-ing anatomy, to playing

harmless pranks on our fellow lab members (actually mostly just Katrusia), I am really glad

that I got to know you. From our first lab Christmas party, to adventures in Boston, San

Diego and just day-to-day in the lab, we’ve spent probably hundreds of hours together.

We’ve talked about our projects ad nauseum, gone on shopping trips, gone on so many

impromptu adventures, right down to coordinating themed weeks in order to survive thesis

season. In between the science, I know that as the Trifecta, our dynamic is undeniable.

I would also like to thank the other members of the lab that I have had the pleasure

of working with, including Alaina McDonald, Sarah Donato and Rachel Tari. I would also

like to thank Kata Osz, and Helen Coates for their technical support. An extended thank

you also to Steph Delorme for her mentorship at the start of the program and for bestowing

her vet knowledge upon us when she dropped in on the lab. Moreover, I would like to thank

the bio-med department including staff, faculty and my fellow graduate students. Finally,

I would like to thank my friends and family for their continued support throughout this

process.

v

Table of Contents

ABSTRACT ...................................................................................................................... II

ACKNOWLEDGEMENTS ........................................................................................... III

TABLE OF CONTENTS ................................................................................................ V

LIST OF FIGURES ...................................................................................................... VII

LIST OF TABLES ....................................................................................................... VIII

LIST OF APPENDICES ................................................................................................ IX

LIST OF ABBREVIATIONS ......................................................................................... X

DECLARATION OF WORK PERFORMED ........................................................... XII

CHAPTER 1: LITERATURE REVIEW – WOUND HEALING ................................ 1

1.1 SKIN: STRUCTURE AND FUNCTION .............................................................................. 1

1.1.1 Two modes of acute cutaneous wound healing .................................................. 3

1.2 THE PHASES OF MAMMALIAN WOUND HEALING: SCAR FORMATION ............................ 4

1.2.1 Hemostasis and inflammation ............................................................................ 4

1.2.2 Proliferation ....................................................................................................... 5

1.2.3 Remodelling ........................................................................................................ 7

1.3 SCAR-FREE CUTANEOUS WOUND HEALING ................................................................. 8

1.4 ENDOGENOUS GROWTH FACTORS ............................................................................. 11

1.4.1 Vascular Endothelial Growth Factor (VEGF) ................................................. 11

1.4.2 Fibroblast Growth Factor (FGF) ..................................................................... 12

1.4.3 TGFβ Signalling Pathway ................................................................................ 13

1.5 THE LEOPARD GECKO, A MODEL OF SCAR-FREE CUTANEOUS WOUND HEALING ........ 15

RATIONALE .................................................................................................................. 17

CHAPTER 1 FIGURES ................................................................................................. 18

CHAPTER 2: GROWTH FACTOR EXPRESSION IN NORMAL AND

WOUNDED SKIN: AN INVESTIGATION OF SCAR-FREE WOUND HEALING

IN THE LEOPARD GECKO (EUBLEPHARIS MACULARIUS) ............................. 20

2.2 METHODS ................................................................................................................. 23

2.2.1 Animal Care ...................................................................................................... 23

2.2.2 Biopsies ............................................................................................................. 24

2.2.3 Tissue Collection .............................................................................................. 25

2.2.4 Haematoxylin and Eosin ................................................................................... 26

2.2.5 Modified Masson’s Trichrome .......................................................................... 26

2.2.6 Immunohistochemistry ...................................................................................... 27

2.2.7 Modified immunohistochemistry....................................................................... 28

2.2.8 Immunofluorescence ......................................................................................... 28

vi

2.3 RESULTS ................................................................................................................... 29

2.3.1 Original/uninjured skin .................................................................................... 29

2.3.2 Gross morphology ............................................................................................ 30

2.3.3 Keratinocytes of the Original Epidermis and Wound Epithelium Express VEGF

and FGF-2 ................................................................................................................. 31

2.3.4 VEGF Expression in the Dermis and Wound Bed ............................................ 34

2.3.5 Canonical TGFβ Signalling in the Wound Epithelium ..................................... 35

2.4 DISCUSSION .............................................................................................................. 36

2.4.1 Novel Expression of VEGF Receptors by Keratinocytes .................................. 37

2.4.2 FGF-2 and FGFR1 Expression by Keratinocytes ............................................ 40

2.4.3 Canonical TGFβ Signalling .............................................................................. 42

CHAPTER 2 TABLES ................................................................................................... 45

CHAPTER 2 FIGURES ................................................................................................. 48

CHAPTER 3: CONCLUDING STATEMENTS.......................................................... 61

3.1 SUMMARY ................................................................................................................ 61

3.2 GROWTH FACTOR EXPRESSION DURING RE-EPITHELIALIZATION ............................... 62

3.3 NOVEL FUNCTIONS FOR GROWTH FACTORS IN THE GECKO EPIDERMIS ...................... 63

3.4 CONCLUSIONS .......................................................................................................... 64

REFERENCES ................................................................................................................ 65

APPENDIX 1: SUPPLEMENTARY TABLE FOR CHAPTER 2 ............................. 90

APPENDIX 2: DETAILED HISTOCHEMICAL PROTOCOLS AND SOLUTIONS

........................................................................................................................................... 91

APPENDIX 3: ANTIBODY VALIDATION ................................................................ 97

vii

List of Figures

Chapter 1 Figures:

Figure 1: Schematic comparison of the epidermis in mammals and reptiles

Chapter 2 Figures:

Figure 1: Scar-free wound healing in the gecko.

Figure 2: Vascular endothelial growth factor A (VEGF) and VEGF receptor 2 (VEGFR2)

are expressed by keratinocytes of the gecko epidermis.

Figure 3: Vascular endothelial growth factor receptor 1 (VEGFR1) and VEGFR2 are

expressed by keratinocytes of the gecko epidermis.

Figure 4: Fibroblast growth factor 2 (FGF-2) and FGF receptor 1 (FGFR1) are expressed

by keratinocytes of the gecko epidermis.

Figure 5: Vascular endothelial growth factor A (VEGF) and VEGF receptor 2 (VEGFR2)

are co-localized within cells of the gecko dermis and wound bed.

Figure 6: Vascular endothelial growth factor receptor 1 (VEGFR1) and VEGFR2 are co-

localized within cells of the dermis and wound bed.

Figure 7: Transforming growth factor β (TGFβ) signalling in keratinocytes of the gecko

epidermis.

Figure 8: Summary schematic of growth factors expression in the normal and regenerating

epidermis.

Appendix Figures:

A3: Blocking peptide neutralizes vascular endothelial growth factor A (VEGF) expression.

viii

List of Tables

Chapter 2 Tables:

Table 1: A summary of all optimized immunohistochemistry and immunofluorescence

protocols for proteins of interest (vwf, α-sma, VEGF, VEGFR2 (flk-1), VEGFR1 (flt-1),

FGF-2, FGFR1, PCNA, pSMAD2, TGFβ1 and ActivinβA)

Appendix Tables:

Table A1: Weight at collection points in biopsy study

ix

List of Appendices

Appendix 1: Supplementary Table for Chapter 2

Appendix 2: Detailed Histochemical Protocols and Solutions

Appendix 3: Antibody Validation

x

List of Abbreviations

α-SMA alpha-smooth muscle actin

AUP Animal Usage Protocols

BMP Bone Morphogenetic Protein

BSA bovine serum albumin

DAB 3,3’-diaminobenzidine

DAPI 4’-6’-Diamidino-2-Phenylindole

DNA deoxyribonucleic acid

DPW days post-wounding

ECM extracellular matrix

EGF epidermal growth factor

EMT epithelial-to-mesenchymal transition

FGF fibroblast growth factor

FGFR fibroblast growth factor receptor

GDF growth differentiation factor

HRP horseradish peroxidase-conjugated streptavidin

H2O2 hydrogen peroxide

IF immunofluorescence

IHC immunohistochemistry

IL interleukin

MS22 ethyl 3-aminobenzoate methanesulfonic acid

NBF neutral buffered formalin

NGS normal goat serum

NRP-2 neuropilin-1

PBS phosphate buffered saline

PCNA proliferating cell nuclear antigen

PDGF platelet derived growth factor

pSMAD phosphorylated SMAD

RTK receptor tyrosine kinase

xi

SMAD Small Body and Mothers Against Decepentaplegic Homolog

TBST tris-buffered saline with Tween20®

TGF transforming growth factor

TNF tumour necrosis factor

UV ultraviolet

VEGF vascular endothelial growth factor

VEGFR vascular endothelial growth factor receptor

VEGFR1 flt-1

VEGFR2 flk-1

vwf von Willebrand factor

WE wound epithelium

xii

Declaration of Work Performed

I declare that I performed all the work in this thesis with the exception of the

following items: Hanna Peacock assisted with the collection of biopsy time points; Emily

Gilbert, Kathy Jacyniak and Rebecca McDonald assisted with perfusions of the animals;

and some immunostaining protocols were run by Rachel Tari.

1

Chapter 1: Literature Review – Wound Healing

1.1 Skin: structure and function

In addition to important roles in thermoregulation, somatosensation, and

maintaining homeostasis, the skin also functions to limit various forms of environmentally-

mediated damage including dehydration, solar radiation and chemical insults. As the

primary physical barrier surrounding the body and the most frequently injured organ, the

integument must also function to protect against microbial invasion, mechanical abrasion

and trauma (Pastar et al, 2014; Seifert & Maden, 2014). Despite these diverse functions,

the basic structure of the skin is comparatively simple: an outermost epidermis, primarily

composed of keratinocytes; and a deeper dermis, which is rich in connective tissues

(Martin, 1997).

The epidermis is a stratified squamous epithelium that is dominated by keratin-

producing keratinocytes. In mammals, as many as five major layers are recognized: the

deepest layer is the stratum basale, followed by the stratum spinosum, stratum granulosum,

stratum lucidum (present only in thick skin), and finally the most superficial layer, the

stratum corneum (Fig. 1A; Natajaran et al, 2014). The epidermis undergoes constant

physiological renewal, with new keratinocyte generation by cells of the stratum basale,

which is the source of the progenitor population in the epidermis (Pastar et al, 2014).

Specifically, the stratum basale is a highly proliferative, monolayer primarily composed of

cuboidal-shaped cells, which are bound to the basement membrane and act as the interface

between the epidermal and dermal compartments (Natarajan et al, 2014; Pastar et al, 2014).

Following proliferation, neo-keratinocytes begin to outwardly migrate (towards the stratum

corneum), differentiating along the way (Pastar et al, 2014). Importantly, proteins and

2

lipids need to be transported to the stratum corneum; they are first packaged into lamellar

bodies/granules in the stratum spinosum, and then they are further enriched by lipids in the

stratum granulosum (Natarajan et al, 2014; Pastar et al, 2014). Once these lamellar

bodies/granules release their contents, they create a lipid envelope that covers the cells

(corneocytes) of the stratum corneum (Hogan et al, 2012; Natarajan et al, 2014).

Keratinocytes also begin to dehydrate and flatten as they terminally differentiate (Eckert,

1989). Once keratinocytes have reached the stratum corneum, they are flattened and

physiologically dead cells known as corneocytes (Hogan et al, 2012). Ultimately, they are

desquamated and replaced by the next generation.

The dermis is comprised of two layers: the papillary layer (loose connective tissue);

and the reticular layer (dense connective tissue) (Freinkel & Woodley, 2001). The dermis

supports the overlying epidermis and is the site of many important tissue-level events

(Freinkel & Woodley, 2001). Approximately 70% of the dermis is composed of collagen,

which confers both elasticity and tensile strength to the skin (Sampson, 1983, Cooper et al,

1985). The main cell type within the dermal compartment are fibroblasts, which are formed

from mesenchymal-like cells (Freinkel & Woodley, 2001). Fibroblasts participate in the

synthesis of both fibrous and non-fibrous connective tissue and a number of growth factors

(Freinkel & Woodley, 2001). In addition, there are a number of important structures within

the dermal compartment including blood vessels, nerves, sebaceous glands, sweat glands,

melanocytes and isolated inflammatory cells (Freinkel & Woodley, 2001).

Although the composition of the skin is broadly conserved across vertebrates, there

are notable differences in the organization of the epidermis and epidermal-derived

appendages between major groups. For example, while mammals develop hair/hair

3

follicles and various types of glands (mammary, sebaceous, sweat), in reptiles the primary

epidermal appendage is the scale. Reptile epidermis is similarly made up of five main

layers: Oberhaütchen, β-keratin layer, α-keratin layer, intermediate zone and stratum

germinativum (Fig. 1B; Jensen-Jarolim, 2013; Allam et al, 2016). The Oberhaütchen, β-

keratin layer and α-keratin layer can collectively be referred to as the stratum corneum

(Jensen-Jarolim, 2013). The stratum germinativum, like the stratum basale is a single layer

of highly proliferative cuboidal cells, and it is likewise the source of keratinocyte

progenitors (Jensen-Jarolim, 2013). The keratinocytes differentiate as they move towards

the stratum corneum, and as such, the intermediate zone, (made up of two or more cell

layers) is made up of keratinocytes at different developmental stages (Jensen-Jarolim,

2013). The α-layer is a soft, elastic layer that has connections/projections to the scales

(Jensen-Jarolim, 2013). Superficial to this layer is the β-layer, which is a hard, but

simultaneously brittle layer and it forms the surface of the scales (Jensen-Jarolim, 2013).

When shedding or ecdysis occurs, the epidermis is duplicated, (via replication of the cells

of the intermediate zone) resulting in an inner and outer epidermal generation (Maderson,

1964, 1965; Jensen-Jarolim, 2013). As such, the outer epidemis and the Oberhaütchen are

sloughed off through this process (Stewart and Daniel, 1975). Deep to the epidermis is the

dermis, which is mainly composed of connective tissue with many important structures

including blood vessels, lymphatic vessels, nerves and chromatophores (Jensen-Jarolim,

2013).

1.1.1 Two modes of acute cutaneous wound healing

Corresponding with its numerous barrier and homeostatic functions, injuries to the

skin must be rapidly addressed (Martin, 1997). Acute wound healing involves one of two

4

modes of repair: either scar formation or scar-free wound healing. When healing fails, the

result is a chronic wound or a non-healing ulceration (Diegelmann & Evans, 2004).

Mammals typically heal via scar formation, a fibrotic process that does re-establish barrier

and homeostatic functions to the skin. However, scars are non-specific replacements that

are both structurally and functionally imperfect (Martin, 1997). Compared to the original

organ, scarred skin fails to reproduce epidermal appendages (such as hair follicles and

glands); it has a reduced tensile strength, diminished thermoregulatory abilities, and is less

elastic leading to a more limited range of motion (Seifert and Maden, 2014).

In contrast to scar formation, some vertebrates (including urodeles, amphibians and

some lizards) are able to heal cutaneous wounds without scarring. Significantly, this scar-

free mode of wound healing virtually replicates the original structure and function of

uninjured skin (Seifert and Maden, 2014). Unlike species that scar, those that heal scar-

free can regenerate epidermal appendages (e.g., glands and scales); regain pigmentation;

and restore a near perfect cytoarchitecture. Notwithstanding the obvious differences in

outcome between these two modes of healing, it is interesting to note that both scar

formation and scar-free wound healing are characterized by a highly similar sequence of

overlapping events (Seifert and Maden, 2014). Broadly stated, these include: 1) hemostasis

and inflammation; 2) re-epithelialization and tissue proliferation; and 3) tissue remodelling

(Singer and Clark, 1999; Diegelmann & Evans, 2004; Seifert and Maden, 2014).

1.2 The phases of mammalian wound healing: scar formation

1.2.1 Hemostasis and inflammation

Following injury to the skin, damage to blood vessels attracts platelets

(thrombocytes), which aggregate and adhere, thus initiating the formation of a temporary

5

platelet plug. Platelet degranulation promotes a vasoconstrictive response; minimize blood

flow to the lesion; and promotes secretion of cytokines (e.g., platelet-derived growth factor

(PDGF) and transforming growth factor β (TGFβ)) as well as the coagulation cascade.

During the coagulation cascade, platelet alpha-granules release soluble fibrinogen (among

other factors), which is converted to insoluble fibrin. The fibrin clot serves as a provisional

extracellular matrix, permitting inflammatory cell infiltration and epithelial cell migration

(Epstein, 1999). Among the first inflammatory cells to infiltrate the wound site are

neutrophils, phagocytic cells that engulf any bacteria that may have entered the body during

the injury event, and monocytes (Seifert & Maden, 2014). Once the monocytes enter the

fibrin clot they differentiate into macrophages. Macrophages participate in pathogen and

neutrophil removal (Singer & Clark, 1999; Seifert & Maden, 2014), and secrete

collagenase and various proteinases, which degrade the provisional matrix to facilitate

macrophage motility (Riches, 1988). Macrophages are rich sources of numerous growth

factors, including PDGF, TGFβ1, fibroblast growth factors (FGFs), interleukin-1 (IL-1),

and tumour necrosis factor-α (TNFα) (Barrientos et al, 2008), which are required for the

proliferative phase of wound healing that subsequently follows (Clark, 1988). Related to

this, when macrophages are depleted, wound repair is adversely impacted (Singer & Clark,

1999; Godwin et al, 2013).

1.2.2 Proliferation

During the inflammatory phase, the next major event in wound healing begins: re-

epithelialization. In the uninjured integument, keratinocytes are bound to the basal lamina

via hemidesmosomes (Martin, 1997). During re-epithelialization, these attachments are

dissolved, and peripheral cytoplasmic actin filaments are formed, allowing for

6

keratinocytes to migrate across the wound (Gabbiani et al, 1978; Goliger & Paul, 1995;

Martin, 1997; Seifert & Maden, 2014). In mammals, keratinocyte migration begins

approximately 24 hours following injury (Woodley et al, 1993). In response to various

growth factors (including epidermal growth factor (EGF), TGF-α and FGFs) secreted by

macrophages, keratinocytes and fibroblasts, cells at the wound margin begin rapidly

proliferating (Woodley et al, 1993). Mammals also recruit keratinocytes for this neo-

epithelium from stem/progenitor populations in hair follicles (Martin, 1997; Ito et al, 2005).

Once re-epithelialization is complete, keratinocytes restore attachments to the newly

formed basement membrane via hemidesmosomes (Seifert & Maden, 2014).

One of the hallmark features of the scarring wound is a transient, vascularized

structure known as granulation tissue. Granulation tissue starts to form within the wound

bed ~4 days post-injury (Singer & Clark, 1999). The granular appearance of this tissue is

the result of a hypervascularized network of superficially looping capillaries (Martin,

1997). Granulation tissue is also rich in macrophages, which secrete growth factors to

promote angiogenesis, and fibroblasts, which begin synthesizing hyaluronic acid, fibrin,

fibronectin and type III collagen (Singer & Clark, 1999; Seifert & Maden, 2014). This new

ECM serves as a scaffold to facilitate continued cell migration, specifically endothelial

cells (contributing to angiogenesis) and fibroblasts (Eckes et al, 1988; Toole, 1991;

Greiling et al, 1997).

During granulation tissue formation, two of the most potent angiogenic factors are

FGF-2 and vascular endothelial growth factor (VEGF) (Martin, 1997). FGF-2 is secreted

by infiltrating macrophages and damaged endothelial cells, while VEGF is released by

macrophages and keratinocytes of the wound epithelium (Abraham & Klagsburn, 1988;

7

Broadley et al, 1989). Simultaneously, VEGF receptors are upregulated by endothelial cells

in the wound bed (Brown et al, 1992). Other factors contributing to injury-mediated

angiogenesis including TGFβ, angiogenin, angiotropin, and angiopoietin 1, which are all

reported to induce the release of VEGF and FGF-2 from macrophages and endothelial cells

(Guo et al, 1995; Paladini et al, 1996; Brock et al, 1996; Martin, 1997).

1.2.3 Remodelling

As wound healing continues, populations of recruited fibroblasts within the wound

bed acquire a contractile phenotype characterized by the expression of α-smooth muscle

actin (α-SMA). These newly formed myofibroblasts are involved in both wound

contraction and fibrogenesis (Desmoulière & Gabbiani, 1988; Welch et al, 1990).

Myofibroblast differentiation is mediated by a number of growth factors, including TGFβ1,

TGFβ2 and PDGF (Montesano & Orci, 1988; Clark et al, 1989), as well as

microenvironmental stimuli such as mechanical loading (Hinz, 2007). Simultaneously, the

once heavily vascularized and cell-rich granulation tissue is transformed into the fibrotic,

poorly vascularized and cell depleted scar tissue (Seifert & Maden, 2014). Remodelling of

granulation tissue involves a dysregulation of the normal balance between collagen

synthesis and catabolism, and the replacement of thin and loosely organized type III

collagen with thick, densely organized bundles of type I collagen (Singer & Clark, 1999;

Seifert & Maden, 2014). In stark contrast with the basket-weave architecture of original

dermis, scar tissue is characterized by type I collagen deposited in parallel (Yannas, 2001).

As a result of this distinct configuration, the tensile strength of scar tissue is only 70-80%

that of uninjured skin (Billingham & Russell, 1956). Furthermore, during scar formation

8

epidermally-derived organs, including hair follicles and glands, are not replaced (Seifert

and Maden, 2014).

1.3 Scar-free cutaneous wound healing

Although scar-formation is the most common response to injury in mammals, many

other vertebrates, as well as some mammals, are able to heal scar-free. In turn, scar-free

wound healing (or at least the absence of scar tissue) is associated with the ability to

specifically replace, or regenerate, lost or damaged tissues (Levesque et al., 2010). Various

explanations have been forwarded to account for the observed differences between scar-

forming and scarless wounds. One important difference is that regeneration-competent

species do not form hypervascular granulation tissue at the site of injury. Instead they

develop a blastema, a mass of proliferating mesenchymal-like cells that is only modestly

vascularized (Gilbert et al, 2015; Peacock et al., 2015). As such, it has been proposed that

exuberant vascularization promotes scarring and inhibits regeneration (e.g. Wilgus et al,

2008). Other key factors that likely contribute to scar formation include enhancing and

accelerating inflammation and ECM deposition (Desmouliere et al, 2005; Satish and

Kathju, 2010; Seifert et al., 2012)

Among the most commonly studied regeneration-competent species are zebrafish

and urodeles (newts and axolotls). It is widely understood that zebrafish (Danio rerio) can

spontaneously regenerate numerous tissues and organs, including spinal cord, heart, retina,

lesioned brain and amputated fins, amongst other tissue (Gemberling et al, 2013; Schmidt

et al, 2014; Rajaram et al, 2014). Recent work indicates that they are also able to heal

cutaneous wounds scar-free. Specifically, they can heal 2mm full-thickness wounds to the

skin, completely replacing the epidermis, dermis, scales and hypodermis within 28 days

9

(Richardson et al, 2013). Moreover, they can re-epithelialize the wound within 7 hours,

and restore the stratified architecture of the epidermis within 24 hours (Richardson et al,

2013, 2016). Interestingly, inhibiting blood clotting (through the use of Warfarin) or

inflammation (through the use of hydrocortisone) does not influence the rate of re-

epithelialization in zebrafish (Richardson et al, 2013). However, deeper wounds that

involve the underlying skeletal muscle take longer to resolve (Richardson et al., 2013).

Similar to zebrafish, urodeles demonstrate a robust capacity to regenerate a variety

of different organs and tissues including portions of the tail, limbs, spinal cord, jaw, heart,

and brain, as well as the lens and skin (Seifert et al, 2012a; Maden et al, 2013; McCusker

et al, 2015). As demonstrated by the administration of anti-proliferative agent bleomycin,

the skin of urodeles (specifically axololts, Ambystoma mexicanus) can scar, but normally

does not. Scar-free cutaneous wound healing in axolotls following a full-thickness

excisional biopsy wound is characterized by a relatively rapid re-epithelialization of the

lesion (8- 24 hours, depending on the diameter of the wound), with limited evidence of

either inflammation or fibrogenesis (Levesque et al., 2010; Seifert et al., 2012). Full

restoration of the skin, including glands and pigmentation takes 80-90 days (Levesque et

al., 2010; Seifert et al., 2012).

Although often overlooked, various examples of scar-free wound healing and

regeneration have been reported in mammals. For example, it has long been recognized

that through-and-through ear hole punches (1 cm2) in rabbits are not only closed, but fully

regenerate, complete with new epidermis, dermis, cartilage, hair follicles and associated

glands (Vorontsova and Liosner, 1960; Joseph and Dyson, 1966; Goss and Grimes, 1972;

Williams-Boyce and Daniel, 1980; Gawriluk et al., 2016). Re-epithelialization is complete

10

within 7 days, and full regeneration by 85 days (Gawriluk et al., 2016). Another example

of scar-free wound healing and regeneration in mammals are African spiny mice, Acomys

kempi, A. percivali and A. cahirinus (Seifert et al, 2012a; Gawriluk et al., 2016; Santos et

al., 2016). Not only can Acomys spp. regenerate large portions of skin (including hair

follicles), they can regenerate ear hole punches (Seifert et al, 2012a; Gawriluk et al., 2016;

Santos et al., 2016). Skin regeneration in these species appears to be related to an unusual

adaptation to avoid predation – skin autotomy. Similar to the tail of many lizards, the skin

of Acomys is able to self-detach when tension (such as that exerted by a grasping predator)

is applied (Seifert et al, 2012a). Within 30 days the skin is completely restored (Seifert et

al, 2012a).

Furthermore, mammals have the capacity to heal scar-free up until the beginning

or until the middle of the third trimester of gestation (Seifert et al, 2014). This includes

fetal mice, sheep, pigs, marsupials and rats (Lorenz and Adzick, 1993; Ferguson &

O’Kane, 2004; Larson et al, 2010; Satish and Kathju, 2010; Lo et al, 2012). The size of the

wound and the stage of gestation influences the success of wound healing in the embryo; a

smaller wound size and an early gestational stage correlates to reduced scarring (Cass et

al, 1997; Yang et al, 2003). An early idea regarding fetal wound healing was that the

aqueous environment in utero promoted a scar-free wound healing phenotype. However,

subsequent research examining marsupial embryos, who exit the uterus earlier during

gestation and continue to develop exposed to non-sterile conditions, still retained the

capacity to heal scar-free (Armstrong and Ferguson, 1995). Moreover, when adult skin is

grafted onto fetal skin in utero, it still scars, whereas the surrounding fetal skin can heal

11

scar-free (Longaker et al, 1994). Together this supports the notion that the ability to heal

scar-free is an intrinsic characteristic of the skin, independent of the environment.

Finally, many species of lizard can spontaneously drop their tails and regenerate a

replacement (Alibardi, 2009; McClean and Vickaryous, 2011; Delorme et al, 2012). To

date this includes, but is not limited to gekkotans (geckos), scincids (skinks), lacertids (wall

lizards) and anoles. Previously it was believed that the capacity to heal scar-free was

restricted to the tail (Barber, 1944). However it was recently found that the gecko

integument as a whole organ has an intrinsic capacity to heal scar-free (Peacock et al,

2015). In addition, there have also been some species of lizard that are able to regenerate

their scales following cutaneous injury, but details of this process and its contribution to

scar-free wound healing remain unclear (Wu et al, 2014).

1.4 Endogenous growth factors

1.4.1 Vascular Endothelial Growth Factor (VEGF)

It is well understood that various growth factors, including VEGF, FGF, and TGFβ,

play key roles in orchestrating wound healing and regeneration. VEGF is a heparin-binding

protein widely recognized as a potent pro-angiogenic, involved in endothelial cell

proliferation, migration and (ultimately) blood vessel formation, and hence is crucial for

restoring vasculature to regenerating tissues (Brown et al, 1992; Nissen et al, 1998; Gurtner

et al, 2008). VEGF also participates in increasing vascular permeability, recruiting

inflammatory cells, and recruiting bone marrow-derived hematopoetic progenitor cells

(Brown et al, 1992; Nissen et al, 1998; Galiano et al, 2004). Moreover, VEGF is a key pro-

survival factor for endothelial cells, especially of newly formed, immature blood vessels

(Benjamin et al, 1999; Yuan et al, 1996). The loss of VEGF dependence in established

12

vessels is thought to be associated with pericyte support that is associated with mature

blood vessels (Benjamin et al, 1999). It is also worth noting that VEGF’s mitogenic

properties extend to other cell types (Matsumoto & Claesson-Welsh, 2001), indicating

roles beyond its classical angiogenic functions.

During wound healing, the major sources of VEGF include keratinocytes, platelets

and macrophages (Brown et al, 1992). VEGF function is mediated through two main

receptor tyrosine kinases (RTK), both of which are primarily expressed by endothelial

cells: VEGFR2 (flk-1) and VEGFR1 (flt-1) (Brown et al, 1992; Ferrara, 2004). VEGF also

interacts with the non-RTK Neuropilin-1 (Nrp-1), likewise present on endothelial cells

(Soker et al, 1998; Ferrara, 2004). In response to injury, VEGFR1 and 2 are strongly

upregulated by endothelial cells, lining capillaries within the dermis, as well as infiltrating

macrophages (Lauer et al, 2000; Zhang et al, 2004). In vivo data indicates that VEGF

signals in a paracrine manner to induce neovascularization (Detmar et al, 1998), largely

through VEGFR2 (Banks et al, 1998).

1.4.2 Fibroblast Growth Factor (FGF)

FGF is a family of 22 polypeptides involved in various cell functions, including

differentiation, migration, proliferation and cytoprotective/cell survival roles following

stress (Abraham & Klagsburn, 1988; Basilico & Moscatelli, 1992; Werner, 1998; Ornitz

& Itoh, 2001; Werner & Grose, 2003; Yang et al., 2010). FGF signalling occurs through

four high affinity RTK, FGF receptors (FGFR) 1-4 (Ornitz & Itoh, 2001; Johnson &

Williams, 1992; Barrientos et al., 2008). Similar to VEGF, FGFs are also heparin-binding

proteins, with potent pro-angiogenic functions (Battegay, 1995; Tonnesen et al, 2000).

Although all FGF ligands and receptors have been detected in uninjured and regenerating

13

skin, FGF-2 (also known as basic FGF, bFGF) in particular appears to be important during

wound healing, inducing granulation tissue, re-epithelialization and tissue remodelling

(Werner & Grose, 2003; Barrientos et al., 2008). Specifically, members of the FGF family

have been reported to stimulate keratinocyte populations to both proliferate and migrate

during wound closure (Tsuboi et al, 1993). Related to this, FGF-2 null mice demonstrate a

significant delay in re-epithelialization, decreased collagen deposition and increased scab

thickness following injury (Ortega et al, 1998). Similarly, FGF-2 expression is reduced in

chronic wounds (Werner et al, 1994; Shukla et al, 1998; Swift et al, 1999). In addition to

this, FGF-2 has protective effects through which it promotes and regulates DNA repair

thereby maintaining the integrity of the genome (Harfouche et al, 2010).

1.4.3 TGFβ Signalling Pathway

The TGFβ superfamily includes the prototypic members, TGFβ1-3, along with the

activins, inhibins, bone morphogenic proteins (BMPs), growth differentiation factors

(GDFs), nodals and myostatin (Derynck and Miyazono, 2008). TGFβ/activin can signal

through the canonical pathway, specifically through serine/threonine kinase receptors

(Moustakas and Heldin, 2009; Wu and Hill, 2009; Ogunjimi et al, 2012). They then bind,

become activated and recruit and phosphorylate receptor regulated Small Body and

Mothers Against Decepentaplegic Homologs (R-SMAD) (Moustakas and Heldin, 2009;

Wu and Hill, 2009). There are multiple R-SMADs, but TGFβ and activin phosphorylate

SMAD2 and SMAD3 (Ross and Hill, 2008). Another factor, SMAD4 then creates a

complex with SMAD2/3 and are translated to the nucleus where they can elicit a variety of

effects by acting on and influencing gene expression (Ross and Hill, 2008). There is also a

14

non-canonical signalling pathway that can be directly or indirectly activated by SMAD or

non-SMAD-mediated signalling (She and Massague, 2003; Itoh et al, 2009).

TGFβ has pleiotropic effects across the body that influences homeostasis,

inflammation, wound healing, development and disease (Werner & Grose, 2003; Gilbert

et al., 2016). The known diversity of effects are context and cell-type dependent, making

precise characterizations of function problematic especially since TGFβ1, 2 and 3 have

specific, but also overlapping and even antagonistic functions (Roberts, 1998). For

example, while all three isoforms are known mitogens, they also suppress proliferation in

various cell types, including keratinocytes (Roberts, 1998). During wound healing, TGFβ1

and 2 are typically considered to be pro-fibrotic (and thus associated with scar formation),

whereas TGFβ3 has been shown to be anti-fibrotic/pro-regenerative (O’Kane & Ferguson,

1997; Jarvinen and Ruoslahti, 2010). For example, the exogenous application of TGFβ3

reduces scarring (Shah et al, 1995; Proetzel et al, 1995; Ferguson & O’Kane, 2004;

Occleston et al, 2008).

TGFβs can also induce re-epithelialization, by acting on the integrins to promote

keratinocyte migration (Coffey et al, 1988; Sellheyer et al, 1993; Gailit et al, 1994;

Zambruno et al, 1995); promote epithelial-to-mesenchymal transitions (EMT; Rasanen &

Vaheri, 2010; Weber et al, 2012; Lamouille et al, 2014); elicit granulation tissue formation

by stimulating neovascularization; and enhance the expression of ECM-associated genes

(Coffey et al, 1988; Sellheyer et al, 1993). TGFβ1 is abundantly released from platelets,

and thus participates in the initial recruitment of inflammatory cells (Pakyari et al, 2013;

Gilbert et al., 2016), as well as the hemostatic plug of mammals (Assoian et al, 1983). It

15

has been previously identified to be present in the epidermis of the regenerating lizard tail,

but absent in the wound epithelium during regenerative outgrowth (Gilbert et al, 2013).

Activins also signal through the canonical TGFβ pathway, and are dimer proteins

made up of the subunits activinβA and activinβB. In combination, these subunits create the

three activin ligands: activin A (βA, βA), activin B (βB, βB) and activin AB (βA, βB)

(Werner & Grose, 2003; Werner & Alzheimer, 2006). Although little is known about their

function during wound healing, activinβA is upregulated following injury in zebrafish

(Jazwinska et al, 2007) and geckos (Gilbert et al, 2013).

1.5 The leopard gecko, a model of scar-free cutaneous wound healing

Similar to many lizards, the leopard gecko (Eublepharis macularius; hereafter

‘gecko’) is able to voluntarily self-detach (or autotomize) its tail, to avoid predation, and

then regenerate a fully functional, multi-tissue replacement (McLean and Vickaryous,

2011). Previous work has demonstrated that this ability to regenerate is independent of

autotomy (Delorme et al, 2012), and that it extends beyond the tail (Peacock et al, 2015).

As has recently been shown, geckos can spontaneously and near perfectly heal full-

thickness (epidermis and dermis) excisional wounds to the skin of both the tail and body

(Peacock et al, 2015). Using a 3mm biopsy tool, full-thickness cutaneous wounds were

created on various locations across the dorsal body wall, the original (intact) tail, and the

fully regenerated tail. Regardless of location, all the wounds healed without scars within a

similar timeframe of 45 days (Peacock et al, 2015). Scar-free wound healing restored scales

and pigmentation, as well as the basket-weave architecture of the dermis (Peacock et al,

2015). Of note, the wound site failed to develop the hypervascularized appearance of

granulation tissue. Instead the wound bed (and neo-dermis) revealed only modest numbers

16

of structurally mature (mural cell-supported) blood vessels. Similar to most scar-free

wound healing species, re-epithelialization of the wound site in geckos is a relatively rapid

process, occurring within 5 days. Although available evidence indicates that re-

epithelialization is crucial for successful wound resolution, to date this process remains

poorly understood.

17

Rationale

To date, the majority of wound healing research has focused on characterizing the

tissue-level events of the dermis. For the epidermis, less is known. I sought to investigate

the spatio-temporal distribution of several key growth factors in the epidermis prior to and

following a full-thickness cutaneous injury in a species capable of spontaneous scar-free

wound healing, the leopard gecko (Eublepharis macularius). The goal of my research is to

characterize the pattern of growth factor expression in both the uninjured (homeostatic)

epidermis, and throughout the process of re-epithelialization and skin regeneration. These

findings will provide an important contribution to our understanding of the biology of scar-

free wound healing.

I hypothesize that there is dynamic expression of endogenous growth factors in the

newly formed (wound) epithelium during scar-free wound healing.

The objective of my study is:

1) To investigate the spatio-temporal distribution of the soluble growth factors VEGF

and FGF-2 in the epidermis prior to, during and following the completion of scar-

free wound healing.

2) To investigate the onset and pattern of distribution of TGFβ/Activin signalling

during scar-free wound healing.

18

Chapter 1 Figures

19

Figure 1: Schematic comparison of the epidermis in mammals and reptiles. (A) In

mammals five epidermal layers are commonly recognized (from deepest to superficial):

the stratum germinativum (= stratum basale), stratum spinosum, stratum granulosum,

stratum lucidum (thick skin only) and stratum corneum. (B) Reptilian epidermis is also

divided into five layers, although they are not necessarily homologous with those of

mammals. These layers include: the stratum germinativum (= stratum basale), intermediate

zone (= stratum spinosum), α-keratin layer, β-keratin layer and Oberhaütchen. The

combined α-keratin layer, β-keratin layer and Oberhaütchen are refered to as the stratum

corneum. Not to scale.

20

Chapter 2: Growth factor expression in normal and wounded skin: an investigation

of scar-free wound healing in the leopard gecko (Eublepharis macularius)

2.1 Introduction

The skin is the primary interface between an organism and its environment. In

addition to participating in the maintenance of homeostasis (e.g., Slominski et al, 1993),

skin serves a variety of barrier functions related to protection against mechanical abrasion,

ultraviolet radiation (UV), and microorganisms (Seifert and Maden, 2014). It is also

capable of structural self-repair following injury, undergoing one of two alternative modes

of wound healing: scar formation or scar-free wound healing. With few exceptions, most

mammals resolve injuries to the skin with the formation of a scar (Hardy, 1989). Although

scars restore homeostasis and prevent pathogen entry, they are non-specific, fibrous

replacements of the original organ. As a result of the altered cytoarchitecture and structural

composition, scar tissue is functionally imperfect with decreased tensile strength,

diminished range of motion (particularly across joints), and reduced thermoregulatory

abilities (owing to the failed restoration of glands and hair follicles) (Yannas, 2001). In

stark contrast to the fibrotic replacement resulting from scar formation, many vertebrates

are able to accurately heal lost or damaged tissues without scarring. This so-called scar-

free mode of wound healing is best known for urodeles (Kragl et al, 2009; Lévesque et al.,

2010; Seifert et al., 2012a; Godwin & Rosenthal, 2014), zebrafish (e.g., Azevedo et al,

2011; Stewart & Stankunas, 2012; Richardson et al., 2013), and fetal mammals (up until

mid-gestation; Lorenz & Adzick, 1993; Ferguson & O’Kane, 2004; Larson et al, 2010;

Satish and Kathju, 2010). Of recent, a number of other examples have been identified

including African spiny mice (Acomys spp.; Seifert et al., 2012b) and various species of

21

lizard (Wu et al., 2014; Peacock et al., 2015). In each species, cutaneous wounds are healed

with a near-perfect restoration of tissue architecture, pigmentation, and the re-

establishment of integumentary organs (i.e., glands, hair, scales).

Regardless of outcome, wound healing is a highly conserved process that involves

an overlapping cascade of events. These events are generally summarized as: (i) hemostasis

and inflammation; (ii) re-epithelialization and tissue proliferation; and (iii) tissue

remodelling (Martin, 1997; Gurtner et al, 2008). The magnitude and duration of these

events appears to determine whether a wound will exhibit a scar-forming or scar-free

phenotype. For example, compared to scar formation, scarless wound healing involves a

decrease in the magnitude of inflammation (as measured by neutrophil abundance), a delay

and overall decrease in fibrogenesis, and modulation of the angiogenic response (Seifert et

al., 2012; Peacock et al., 2015). Moreover, scarring wounds are characterized by the

transient development of a heavily vascularized and cell-rich organ known as granulation

tissue (Gurtner et al, 2008). During the remodelling phase of wound healing, this dense

assembly of cells and blood vessels are replaced with acellular and avascular scar tissue

(Mera, 1997; Xu & Clark, 2000). Similar to scar formation, a transient, cell-rich organ is

also created during scar-free wound healing. However unlike granulation tissue, this organ

– called a blastema – never becomes hypervascularized. Instead, the number of blood

vessels present appears to match the vascular density of the surrounding uninjured skin

(Peacock et al., 2015).

In addition to the reparative events involving the wound bed and surrounding

dermis, successful wound healing also requires the re-establishment of the overlying

epidermis (Pastar et al, 2014). Re-epithelialization is a structural repair that involves the

22

proliferation and migration of keratinocytes across the site of injury. In the absence of re-

epithelialization, wound resolution fails, leading to chronic non-healing lesions and/or

ulcers (Seifert and Maden, 2014). Among regeneration-competent species, rapid re-

epithelialization is frequently correlated with scar-free wound healing (Seifert et al, 2012a;

Richardson, 2013). Interestingly, emerging evidence indicates that the rate of repair is not

necessarily an influential factor (Seifert et al., 2014). For example, axolotls are an aquatic

species of salamander that, as adults, resemble the immature (larval) form of other

salamander species (i.e., they are paedomorphic; retain juvenile characteristics as sexually

mature adults). Axolotls treated with the hormone thyroxine undergo a metamorphic

transformation into a distinct terrestrial morph (Page & Voss, 2009; also Monaghan et al.,

2014). Both untreated and metamorphosed axolotls undergo scar-free wound healing

following excisional injuries to the skin. However, whereas untreated axolotls can re-

epithelialize a 4mm diameter cutaneous wound in ~18 hours, metamorphosed axolotls take

~3 days, comparable to the timeframe for wound closure in scarring mice (3-7 days)

(Seifert et al., 2012).

In addition to obvious barrier functions, the epidermis may also serve as an

important source of growth factors and cytokines. Throughout wound healing, pleiotropic

roles have been recognized for members of several different growth factor families

including vascular endothelial growth factors (VEGF), fibroblast growth factors (FGF),

and transforming growth factor β (TGFβ). Although VEGF and FGF are best known as

potent pro-angiogenic factors in the dermal compartment (Clark, 1988; Risau, 1990)

emerging evidence indicates that they may also play a role in re-epithelialization. A study

conducted by Wilgus et al (2005), found that VEGF and VEGFR1 were present in mouse

23

epidermis prior to and during wound closure. When VEGFR1 was neutralized (using an

antibody) the authors observed a pronounced delayed re-epithelialization and a notable

reduction in keratinocyte proliferation (Wilgus et al, 2005). Among lizards, there is also

evidence of FGF-2 (also known as basic FGF, bFGF) expression in the newly formed

(wound) epithelium capping the regenerating tail (Alibardi & Lovicu, 2010; Alibardi,

2012). Together, these data support a role for these growth factors beyond angiogenesis,

particularly during re-epithelialization.

Here I performed a detailed investigation of growth factor expression in the

epidermis prior to and during scar-free cutaneous wound healing. The model for my

investigations is the leopard gecko (Eublepharis macularius), a lab-amenable lizard

capable of spontaneous scar-free wound healing (Delorme et al., 2012; Peacock et al,

2015). I found that keratinocytes of the epidermis expressed an unexpected diversity of

growth factors during wound healing as well as under homeostatic conditions. Moreover,

this work suggests that there may be novel autocrine roles for keratinocyte-mediated

growth factor expression during re-epithelialization.

2.2 Methods

2.2.1 Animal Care

Captive bred Eublepharis macularius (leopard geckos; hereafter ‘geckos’) were

acquired from a commercial supplier (Global Exotic Pets, Kitchener, Ontario, Canada). At

the beginning of the experiment, all animals were sexually immature and less than one year

old, with an body mass range of 8.3g - 29.7g. Animal Usage Protocols (AUPs) were

approved by the University of Guelph Animal Care Committee (Protocol Number 2493)

and followed the procedures of the Canadian Council on Animal Care. Geckos were housed

24

and maintained following the work of Vickaryous and McLean (2011). Briefly, the gecko

colony was kept in an isolated, temperature-controlled environmental chamber with a

12:12 photoperiod and an average room temperature of 27.5˚C. Individual geckos were

housed in 5 gallon polycarbonate containers, with a subsurface heating cable (Hagen Inc.,

Baie d’Urfe, Quebec, Canada) set to 32˚C placed under one end to create a temperature

gradient. Geckos were fed 3-5 larval Tenebrio spp. (mealworm) dusted with powdered

calcium and vitamin D3 (cholecalciferol) (Zoo Med Laboratories Inc., San Luis Obispo,

California, USA) daily and had free access to clean drinking water. A total of 20 geckos

(n=4 for each of 4 time points, plus 4 sentinel or environmental controls) were used to

characterize the spatio-temporal expression of endogenous growth factors throughout

wound healing and regeneration. Geckos were randomly assigned into one of five groups:

sentinel; original uninjured tissue; biopsy and tissue collection at 2 days, 8 days and 45

days following injury.

2.2.2 Biopsies

In order to create the biopsies, the geckos were first anesthetized using a 30mg/kg

intramuscular injection of Alfaxan (diluted to 2mg/mL in sterile injectable 0.9% sodium

chloride, using a 0.5cc insulin syringe; Abbott Laboratories, Saint-Laurent, Quebec,

Canada). The injections were done bilaterally into the cervical epaxial musculature. Geckos

were considered to have reached the surgical plane of anesthesia once the righting reflex

was lost (Schumacher and Yelen, 2006). A biopsy punch tool (Integra Miltex, Burlington,

Ontario, Canada) was then used to create a 3mm full-thickness (epidermis and dermis)

wound into the dorsal skin of the tail. Each gecko received two parasagittal biopsy wounds:

one proximal (along the proximal third of the tail); one distal (along the middle third of the

25

tail). Proximal and distal wounds were on alternate sides of the midline. Each biopsy

wound was separated by 5cm. Excised tissue was removed with the use of both a #11

scalpel blade and forceps. Both biopsies on each tail healed in a similar manner and as such

they were both treated the same and not as independent groups for the purposes of this

study.

2.2.3 Tissue Collection

Experimental gecko tissues were collected at 4 time points either prior to (original

tissue/biopsy controls) or following biopsy (2 days, 8 days and 45 days); tissues were not

collected from sentinel geckos. Geckos were euthanized with an intra-abdominal injection

of 250-500 mg ethyl 3-aminobenzoate methane sulfonic acid (tricaine methansulfonate,

MS222), then in 10% NBF (neutral buffered formalin; Fisher Scientific, Waltham,

Massachusetts, USA) for approximately 24 hours (using either transcardial perfusion

followed by immersion, or the tail tissues were dissected into regions of interest and these

were directly immersed). Following fixation, the tissue was then rinsed with distilled water

and transferred to 70% isopropanol. Regions of interest were dissected/trimmed as

necessary, before being de-calcified with Cal-Ex® (Fisher Scientific, Waltham,

Massachusetts, USA) for 30 minutes. Tissues were then put in 100% isopropanol, cleared

in xylene and infiltrated with paraffin wax using an automated processor (Shandon

Excelsior ES Tissue Processor, Thermo Fisher Scientific). Tissue samples were then

embedded in paraffin blocks and sectioned at 5μm using a rotary microtome (Shandon

Finesse ME+,Thermo Fisher Scientific), before being mounted on charged slides

(Surgipath® X-tra®, Leica Microsystems, Concord, Ontario, Canada), and baked at 60˚C

overnight.

26

2.2.4 Haematoxylin and Eosin

Representative sections from each tissue sample were stained with haematoxylin

and eosin in order to examine tissue structure. Briefly, slide-mounted tissue sections were

first rehydrated to water: three rinses of xylene (2 minutes each); three rinses of 100%

isopropanol (2 minutes each); one rinse in 70% isopropanol (2 minutes); and one rinse in

deionized water (2 minutes). Once rehydrated, sections were stained with modified Harris

hematoxylin (Fisher Scientific, Waltham, Massachusetts, USA) for 10 minutes then dipped

6-10 times in a 1% hydrochloric acid in 70% isopropanol solution, before being rinsed in

deionized water. Next slides were blued in ammonia water for about 15 seconds rinsed in

deionized water, and then dipped in 70% isopropanol 6 times, followed by staining with

eosin (1 minute). Stained sections were dehydrated with four rinses of absolute isopropanol

(2 minutes each), and then cleared with three rinses of xylene (2 minutes each). Finally,

slides were cover slipped using Cytoseal (Fisher Scientific, Waltham, Massachusetts,

USA).

2.2.5 Modified Masson’s Trichrome

To differentiate fibrous connective tissue, representative sections were stained with

a modified Masson’s trichrome (McLean and Vickaryous, 2011). After rehydration (see

above), slide-mounted sections were stained with Mayer’s hematoxylin (10 minutes), blued

in ammonia water for approximately 15 seconds and rinsed with deionized water. Sections

were stained in 0.5% ponceau xylidine/0.5% acid fuschin in 1% acetic acid solution (2

minutes); rinsed in deionized water; stained in 1% phosphomolybdic acid (10 minutes);

rinsed in deionized water; stained in 2% light green (90 seconds); and rinsed in deionized

water. Slides were then dehydrated (one rinse in 95% isopropanol (2 minutes); three rinses

27

in absolute isopropanol (2 minutes)) and cleared with three rinses in xylene (2 minutes

each). Finally, slides were cover slipped using Cytoseal (Fisher Scientific, Waltham,

Massachusetts, USA).

2.2.6 Immunohistochemistry

Immunohistochemistry was performed to identify localized protein expression of

VEGF, VEGFR1, VEGFR2, FGF-2, FGFR1, pSMAD2, TGFβ1 and ActivinβA. Once

rehydrated, slide-mounted sections were quenched in 3% hydrogen peroxide (20 minutes),

then rinsed three times in phosphate buffered saline (PBS) (2 minutes each). For five

proteins (VEGF, VEGFR2, VEGFR1, FGF-2 and ActivinβA), heat-induced antigen

retrieval was employed to unmask the epitope of interest (citrate buffer at 90˚C for 12

minutes, after which the buffer was allowed to cool for 20 minutes). Sections were then

rinsed three times in PBS (2 minutes each), and blocked using 3% normal goat serum

(Vector Laboratories, Burlingame, California, USA) diluted in sterile PBS for one hour at

room temperature. Sections were then incubated with the primary antibody diluted in sterile

PBS overnight at 4˚C; omission (negative) controls were incubated without the primary

antibody. The next day sections were rinsed three times in PBS (2 minutes each), then

incubated with the secondary antibody diluted in sterile PBS for one hour at room

temperature. Sections were then rinsed three times in PBS (2 minutes each), before being

incubated with horseradish peroxidase conjugated streptavidin (Jackson ImmunoResearch

Laboratories, Inc. West Grove, Pennsylvania, USA, code: 016-030-084) diluted in sterile

PBS for one hour at room temperature. Sections were then rinsed three times in PBS (2

minutes each) and then 3,3’-diaminobenzidine peroxidase substrate (DAB; Vector

Laboratories, Burlingame, California, USA) was applied for a time optimized to each

28

antibody (Table 2.1). The chromogenic reaction was stopped by immersing the sections in

deionized water, after which the section were counterstained with Mayer’s hematoxylin (1

minute), rinsed in deionized water, blued in ammonia water, and rinsed again in deionized

water. Slides are then dehydrated (see above) and cover slipped using Cytoseal (Fisher

Scientific, Waltham, Massachusetts, USA).

2.2.7 Modified immunohistochemistry

For ActivinβA, a modified immunohistochemistry protocol was utilized to detect

the epitope. There are three primary differences between the above-mentioned

immunohistochemistry protocol and the modified protocol: 1) tris-buffered saline with

Tween20® (TBST; Sigma-Aldrich, Oakville, ON) rinses are used in replacement of PBS

rinses; 2) a blocking buffer comprised of 3% bovine serum albumin (BSA; Santa Cruz

BioTechnology, Santa Cruz, California, USA), 10% normal goat serum in TBST is used

instead of the 3% NGS block; 3) the secondary antibody is only incubated for 30 minutes

at room temperature as opposed to one hour.

2.2.8 Immunofluorescence

Immunofluorescence was performed in order to co-localize four pairs of proteins:

vWF and α-SMA; VEGF and VEGFR2; VEGFR1 and VEGFR2; and VEGFR2 and PCNA.

Sections were then rinsed three times in PBS (2 minutes each), and blocked using 3%

normal goat serum (Vector Laboratories, Burlingame, California, USA) diluted in sterile

PBS for one hour at room temperature. Sections were then incubated with the primary

antibodies diluted in sterile PBS overnight at 4˚C; omission (negative) controls were

incubated without either primary antibody. The next day sections were rinsed three times

in PBS (2 minutes each), then incubated with the secondary antibodies diluted in sterile

29

PBS for one hour at room temperature. Sections were then rinsed three times in PBS (2

minutes each) before being incubated with 4’-6’-Diamidino-2-Phenylindole (DAPI; Life

Technologies, ThermoFisher Scientific, D1306 Waltham, MA, USA) diluted 1:10000 in

PBS (2 minutes). Sections were then rinsed three times in PBS (2 minutes each) and

coverslipped with Dako Fluorescent Mounting Medium (Dako Canada S3022, Burlington,

ON, Canada).

2.3 Results

2.3.1 Original/uninjured skin

Prior to biopsy, the scalation pattern of the skin is organized into a pavement of

small scales (~2mm in diameter) interrupted at regular intervals by larger, conical-shaped

tubercles (each approximately 1mm in diameter and ~5 times larger than the pavement

scales). The arrangement of pavement scales surrounding each tubercle forms a rosette.

Although the pigmentation pattern of geckos can be highly variable, it typically involves

countershading with a dorsal pattern of brown-black spots on a background of white,

orange and yellow, and a near solid white ventral surface.

As for other vertebrates, the integument is composed of a superficial epidermis

overlying a deeper dermis. The epidermis is a stratified, squamous, keratinized epithelium

that, in lizards and snakes, is commonly described as having five main layers: a superficial

Oberhaütchen, β-keratin layer, α-keratin layer, intermediate zone and a deeply nested

stratum germinativum (Jensen-Jarolim, 2013; Allam et al, 2016). Combined, the

Oberhaütchen, β-keratin layer and α-keratin layer are referred to as the stratum corneum

(Jensen-Jarolim, 2013). Portions of the stratum corneum are sometimes lost during

histological preparation (particularly, the Oberhaütchen and β-keratin layers; Lang, 1989).

30

The layers of the stratum corneum are composed of flattened, anucleated (and thus

terminally differentiated) keratinocytes. Deep to the stratum corneum is the intermediate

zone. The intermediate zone includes two or more layers of differentiating keratinocytes

with variable morphologies, and represents the transition between the mitotically active

cuboidal or columnar cells of the stratum germinativum, and the anucleated and squamous

cells of the stratum corneum. The stratum germinativum (= stratum basale) is the source of

the progenitor population and participates in continuous renewal of the epidermis.

Deep to the epidermis is the dermis, a dense connective tissue network that includes

adipocytes, blood vessels, nerves, lymphatics, resident inflammatory cells and two types

of pigment cells (chromatophores): xanthophores (yellow pigments) and melanophores

(black pigements) (Szydłowski et al., 2015). The dermis is organized into two

compartments, the superficial dermis, dominated by loose connective tissue, and the more

compact and densely arranged deep dermis. In general, the deep dermis is thicker and less

cellular than the superficial dermis.

2.3.2 Gross morphology

In order to characterize the epidermis/wound epithelium before, during and after

re-epithelialization in scar-free wound healing, I created 3mm full-thickness (epidermis

and dermis) biopsy wounds to the dorsal surface of the original tail (Fig. 1A-B). Tail tissues

were then collected at four time points post-biopsy (days 2, 8 and 45), and compared with

control (uninjured) tissues. In general, the pattern of wound healing and regeneration

observed matched previous reports (Peacock et al., 2015). In this study, I chose to focus on

three time points post-biopsy: day 2 (incomplete re-epithelialization; Fig. 1C), day 8

(complete re-epithelialization; Fig. 1D) and day 45 (complete wound healing; Fig. 1E).

31

Blood loss following excision was minimal, and all wounds healed through

secondary intention (similar to humans but unlike mice) (Galiano et al, 2004; Christenson

et al, 2005; Dunn et al, 2013). Overall, there were no observed changes in gecko behaviour

or growth, nor were there any signs of infection or inflammation at the site of biopsy. The

pattern and mode of wound healing was identical across biopsy wounds created on

proximal and distal locations along the tail, and therefore these data are presented together.

At the level of gross morphology, an exudate clot was present within 12 hours post-

wounding (Fig. 1C). By 8 days post-wounding (DPW), the clot had fallen off, revealing a

shiny, smooth wound epithelium (WE; Fig. 1D). The WE remains unpigmented until ~14

DPW (Peacock et al, 2015), but by 45 DPW the pattern of scalation and pigmentation are

fully restored (Fig. 1E). Newly formed/regenerate scales are virtually identical in

morphology to those of the uninjured skin, even though their organization does differ

(specifically, the tubercles are not regenerated).

2.3.3 Keratinocytes of the Original Epidermis and Wound Epithelium Express VEGF and

FGF-2

Although VEGF is best known as a potent pro-angiogenic factor and mitogen of

endothelial cells, it is also expressed by keratinocytes during wound healing in mammals

(Brown et al., 1992; see also Frank et al., 1995). To spatio-temporally characterize VEGF

expression in keratinocytes of the gecko, I performed immunostaining at four key time

points: prior to injury (original tissue); at the start of re-epithelialization (2 DPW); at the

completion of re-epithelialization (8 DPW); and at the completion of regeneration (45

DPW). Original (uninjured) epidermis ranges in thickness from 3-4 cell layers, and

virtually all keratinocytes demonstrate robust co-localization of VEGF and VEGFR2 (Fig.

32

2A-D). As expected, cell proliferation (demonstrated by immunoreactivity with PCNA) is

restricted to cells of the stratum germinativum (Fig. 2E); PCNA+ cells also co-localize

with VEGFR2.

Use of the biopsy tool creates a circular wound that removes a portion of the

epidermis and dermis. Restoration of the wound begins with the formation of an exudate

clot, followed by re-epithelization. By 2 DPW the WE has begun to form, starting at the

wound margins and progressing centripetally. In section the WE appears as a 1-4 cell layer

thick tongue-like outgrowth that gradually undercuts the overlying clot (Fig. 2F). Matching

the tapering thickness of the WE, neo-keratinocytes reveal a dynamic pattern of

VEGF/VEGFR2 expression. Regions of the WE that are stratified/multiple cell layers thick

(e.g., covering the wound margin) strongly co-localize VEGF and VEGFR2 (Fig. 2G-I)

whereas isolated cells spanning more medial positions are immunonegative (data not

shown). Isolated PCNA+ cells are present throughout the developing WE (Fig. 2J). By 8

DPW, re-epithelialization of the WE is complete, and the WE has achieved its maximal

thickness: 6-10 cell layers. Although not uniformly distributed, VEGF with VEGFR2 co-

localization is detected in the majority of WE keratinocytes (Fig. 2K-N), while PCNA

expression is primarily observed among cells of the intermediate zone and the stratum

germinativum (Fig. 2O). Epidermal regeneration is complete at 45 DPW, including the re-

establishment of both pigmentation and scalation. The WE has returned to its original

thickness (3-4 cell layers thick) and is essentially indistinguishable from the surrounding

uninjured tissue. This includes a near homogenous expression of VEGF/VEGFR2 by

keratinocytes (Fig. 2P-S), with evidence of proliferating cells limited to the stratum

germinativum (Fig. 2T).

33

Next I used immunofluorescence to investigate the expression pattern of VEGFR1.

In the uninjured integument, VEGFR1 is widespread among keratinocytes and strongly co-

localized with VEGFR2 (Fig. 3A-C). Although VEGFR2 is expressed by newly formed

keratinocytes by 2DPW (corresponding to the early initiation of the WE), VEGFR1 was

conspicuously absent (Fig. 3D-F). However by 8DPW (once re-epithelialization of the

wound is complete), VEGFR1 is again strongly co-localized with VEGFR2 and detected

amongst the majority of keratinocytes (Fig. 3G-I). By the time the wound has completely

healed (45DPW), the expression pattern of both receptors has returned to that observed

prior to injury, with VEGFR1+/VEGFR2+ keratinocytes being labeled throughout the (de

novo) epidermis (Fig. 3J-L).

To further investigate the expression pattern of growth factors by keratinocytes, I

then examined FGF-2. Similar to VEGF, FGF-2 is involved in regulating angiogenesis and

cell proliferation. In mammals, FGF-2 is also reported to play roles in self-renewal of

stem/progenitor populations and DNA repair of keratinocytes following radiation-induced

damage (Harfouche et al., 2010). Furthermore, exogenous treatment with FGF-2 stimulates

keratinocyte migration in vitro (Sogabe et al., 2006). Prior to injury, keratinocytes of the

gecko epidermis demonstrate strong immunoreactivity for both FGF-2 (Fig. 4A) and its

receptor FGFR1 (Fig. 4B). Prior to wound closure at 2DPW, FGF-2 expression is

discontinuous and restricted to the isolated neo-keratinocytes, primarily of the outermost

layers of the WE (Fig 4C), whereas FGFR1 was notably absent (Fig. 4D). Once wound

closure is complete (8DPW), FGF-2 expression was widespread throughout all cell layers

of the WE (Fig. 4E). Although FGFR1 was detected, immunopositive cells were primarily

restricted to the stratum corneum, and uppermost layer of the intermediate zone (Fig. 4F).

34

At the cessation of wound healing (45DPW), the expression patterns of both FGF-2 (Fig.

4G) and FGFR1 (Fig. 4H) have returned to near homogenous expression within the WE.

2.3.4 VEGF Expression in the Dermis and Wound Bed

To characterize VEGF expression within the dermal compartment, I performed

double immunofluorescence with VEGFR2. Prior to injury, the intact dermis is a fabric of

connective tissue populated by VEGF+/VEGFR2+ fibroblasts (Fig 5A-D). As evidenced

by the expression of the endothelial marker vWF and the mural cell marker αSMA, intrinsic

blood vessels are structurally mature (Fig 5E). At 2 DPW there is no evidence of tissue

restoration or new blood vessels within the wound bed (data not shown). By 8 DPW, the

wound bed has become infilled with a cell-rich aggregate, the blastema. Blastema cells are

mesenchymal-like in appearance and express VEGF, often co-localized with VEGFR2 (Fig

5F-I). Despite the abundance of VEGF/VEGFR2, the blastema only demonstrates modest

numbers of blood vessels (Fig. 5J), the majority of which are structurally mature. By 45

DPW, the structure of the dermis has returned to its pre-injury appearance and is essentially

indistinguishable from the surrounding original tissue, complete with VEFGF+/VEGFR2+

fibroblasts and vWF+/α-SMA+ blood vessels (Fig. 5K-O).

To further characterize the role of VEGF, I then used double immunofluorescence

for the receptors VEGFR1 and VEGFR2. In the uninjured dermis, 8DPW and 45DPW,

there are VEGFR1+ and VEGR2+ cells throughout, and a proportion of these cells also co-

localize (Figure 6A-I). VEGF and VEGFR1 could not be co-localized, but (based on

adjacent slides) it is almost certain that the expression patterns overlap.

35

2.3.5 Canonical TGFβ Signalling in the Wound Epithelium

Next, I sought to investigate the involvement of canonical (SMAD-mediated)

TGFβ signalling during the formation of the WE. I focused on the intracellular mediator

phosphorylated SMAD2 (pSMAD2), and the ligands TGβ1 and activinβA. SMAD2 is one

of two receptor-mediated SMADs (the other being SMAD3) and, once phosphorylated, it

translocates to the nucleus and participates in modulating gene expression. Hence,

pSMAD2 represents a readout of canonical TGFβ activation (Penn et al, 2012). In the

uninjured skin, pSMAD2 expression was detected throughout the stratum germinativum

and intermediate layers of the WE (Fig. 7A). Matching this pattern of distribution, I

observed near ubiquitous expression of TGFβ1 (Fig. 7B) and activinβA (Fig. 7C) within

the epidermis. At 2DPW, pSMAD2 expression within the WE was limited to isolated cells

(Fig. 7D). Similarly, TGFβ1 (Fig. 7E) and activinβA (Fig. 7F) expression was also reduced

and no longer widespread among keratinocytes. More specifically, expression was weakest

or even absent among cells in the center of the wound, and strongest amongst the stratified

populations adjacent to the original epidermis at the wound margins.

At 8DPW, pSMAD2 was observed among keratinocytes throughout the

intermediate layer and the stratum germinativum (Fig. 7G), corresponding with widespread

expression of TGFβ1 (Fig. 7H). In contrast, activinβA expression was not uniform. Instead,

this protein demonstrates an interrupted distribution, with some regions of the WE

demonstrating a near ubiquitous pattern of expression, while adjacent regions are

essentially devoid of immunoreactivity (Fig. 7I). However, by 45 DPW, the original

(uninjured) pattern of protein expression is restored to widespread expression of pSMAD2

36

(Fig. 7J), TGFβ1 (Fig. 7K), and activinβA (Fig. 7L), which are all essentially ubiquitous

in their expression throughout the newly formed epidermis.

2.4 Discussion

Scar-free wound healing represents the idealized outcome for tissue repair.

Although uncommon among mammals, the ability to near-perfectly restore tissue

architecture and function is widespread among teleost fish (Azevedo et al, 2011; Stewart

& Stankunas, 2012; Richardson et al., 2013), urodeles (Kragl et al, 2009; Seifert et al,

2012a; Godwin & Rosenthal, 2014) and some lizards (Delorme et al., 2012; Wu et al.,

2014; Peacock et al., 2015). As evidenced by comparative studies of scar-free wound

healing, tissue regeneration and the absence of scar tissue is associated with decreased or

suppression of inflammation, neovascularization and fibrogenesis (Seifert et al., 2012a;

Seifert and Maden, 2014). Emerging evidence also suggests an important role for

keratinocytes and the formation of the WE during cutaneous repair (e.g., Wilgus et al,

2005). This study reveals, for the first time, an unexpected diversity of growth factors,

including VEGF, FGF-2, TGFβ1 and activinβA, which are expressed by gecko

keratinocytes before, during and after scar-free wound healing. Functionally, VEGF and

FGF-2 are most commonly associated with neovascularization whereas TGFβ1 and

activinβA are members of the multifaceted SMAD-mediated TGFβ signalling pathway.

Combined, these data point towards keratinocyte-derived growth factors as participating in

both paracrine and autocrine signalling to elicit effects beyond their angiogenic capacities.

Simultaneously, it provides a novel avenue for further investigation into the differences

between scar formation and scar-free wound healing.

37

2.4.1 Novel Expression of VEGF Receptors by Keratinocytes

VEGF, VEGFR2 and VEGFR1 are all expressed by keratinocytes of the gecko

integument before, during and after wound repair. VEGF has been traditionally interpreted

to be a potent pro-angiogenic factor and endothelial cell mitogen in the dermis (Ferrara,

2004). The co-localization of VEGF and its receptors in cells of the WE point towards a

novel role for VEGF signalling during wound healing. Moreover, constitutive expression

of VEGF and its receptors in the uninjured epidermis points towards a largely unexplored

homeostatic function. Among mammals VEGF expression is normally minimal prior to

injury, and then strongly upregulated during skin repair (Eming & Krieg, 2006). VEGF is

expressed by macrophages, fibroblasts and keratinocytes (Barrientos et al, 2008) and then

signals through its receptors on endothelial cells in a paracrine manner (Maharaj &

D’Amore, 2007). This results in an increase in neo-vascularization (Brown et al, 1992;

Zhang et al, 2004; Lauer et al, 2000). Our data indicates that both autocrine and paracrine

VEGF signalling may be occurring in the gecko integument, and points towards a role

beyond blood vessel formation. Related to this, VEGFR expression by keratinocytes has

previously been reported for mice (VEGFR1 but not VEGFR2; Wilgus et al., 2005) and

humans (VEGFR1 and VEGFR2; Man et al., 2006) prior to wounding. When cutaneous

wounds in mice are treated with neutralizing antibodies against VEGFR1 there is a

significant delay in re-epithelialization and a decrease in proliferation of keratinocytes

(Wilgus et al., 2005). Moreover, among mutant mice whose keratinocytes no longer

express VEGF, the overall vascular density of the wound bed remains the same, but there

was an obvious delayed eschar (scab) shedding and wound closure (Rossiter et al, 2004).

38

Indirectly, various other lines of evidence also lend support to the prediction of a

non-angiogenic role for VEGF. While we observed robust evidence for widespread VEGF

signalling by virtually every keratinocyte of the WE and the majority of blastema cells, the

exuberant vascular phenotype characteristic of granulation tissue is never formed (Peacock

et al, 2015). Indeed, the vast majority of newly formed blood vessels are structurally mature

(i.e., mural cell supported). Related to this, it has been reported that mature blood vessels

are no longer VEGF-dependent (Alon et al, 1995; Banks et al, 1998). Furthermore, I

detected strong VEGF/VEGFR2 expression by keratinocytes even prior to the appearance

of any blood vessels (2 DPW). Other evidence comes from the study of murine models.

Following a full-thickness (epidermis and dermis) excisional injury in mice, VEGF

expression peaks with the onset of re-epithelialization (2-5DPW) but not with the onset of

angiogenesis (which begins once re-epithelialization is complete, 10-14DPW; Swift et al,

1999; Szpaderska et al, 2003; Egozi et al, 2003).

Constitutive VEGF signalling by epidermal keratinocytes may also play a role in

limiting the harmful effects of exposure to ultraviolet (UV) radiation. UV irradiation is a

well-known mutagen and damaging stressor, and can lead to erythema, inflammation and

carcinogenesis in the skin (Brauchle et al., 1996; Johnson and Wilgus, 2012). Various in

vivo and in vitro investigations have demonstrated that VEGF and VEGFRs are widely

expressed by keratinocytes in mammals (Brauchle et al., 1996; Wilgus et al., 2005; Man et

al., 2006; Zhu et al., 2012), and that exposure to UV radiation activates VEGFR-mediated

pro-survival (anti-apoptotic) mechanisms in these cells (Zhu et al., 2013).

Paradoxically, UV irradiation (at low doses) of the epidermis is beneficial,

promoting vitamin D3 (cholecalciferol) photobiosynthesis (Brauchle et al., 1996). Indeed,

39

for many reptiles cutaneous vitamin D3 synthesis and metabolism is an essential

physiological process (Holick et al., 1995; Acierno et al., 2008). In the absence of UV

(especially UVB; spectrum range 290-315 nm) exposure or dietary supplementation,

reptiles may develop metabolic bone disease, a complex spectrum of disorders that includes

rickets, lethargy and anorexia (Klaphake, 2010). Furthermore, as ectotherms, many

reptilian species behaviourally thermoregulate by either seeking shade (when too warm) or

basking (when too cold). Although details of the relationship between UVB irradiation and

vitamin D3 production remain to be elucidated, my data indicate that VEGFR expression

may participate in attenuating photodamage while faciliating photobiosynthesis.

VEGFR1 expression is dynamic during re-epithelialization. Whereas VEGF and

VEGFR2 are robustly expressed at all time points by keratinocytes, VEGFR1 is notably

absent at 2 DPW. At this time, the failure of keratinocytes to express VEGFR1 prior to

closure of the WE cannot be explained. In particular, it stands in stark contrast with

observations made on wound healing in mice. In these experiments, it was determined that

VEGFR1 expression by keratinocytes is necessary for epidermal cell proliferation and

rapid wound closure (Wilgus et al., 2005). However, one explanation could be that in part

VEGFR1 may be acting as a decoy receptor and is sequestering a proportion of VEGF-A

(Gerber et al, 1999; Rahimi et al, 2000; Zeng et al, 2001a; Zeng et al, 2001b; Roberts et al,

2004). VEGFR1 has been reported to have a ten-fold greater affinity to VEGF-A than

VEGFR2 (Takahashi & Shibuya, 2005; Shibuya, 2011). Another reason could be that anti-

angiogenic factors are playing a regulatory role. To support this, it has been previously

reported by Peacock et al (2015) that there is ubiquitous expression of TSP-1 in the dermis

prior to and during wound healing.

40

Taken together, my data underscores one or more non-angiogenic functions of

VEGF signalling by gecko keratinocytes during both the normal physiological

maintenance of the epidermis, as well as during injury-mediated re-epithelialization. My

study is the first to establish the presence and co-localization of VEGF, VEGFR2, and

VEGFR1 in the reptilian epidermis, and to provide evidence of an autocrine signalling loop

by keratinocytes in a non-mammalian model. By way of explanation, I suggest that

VEGF/VEGFR expression may be involved in promoting keratinocyte survival during

behaviours that involve UVB irradiation, including thermoregulation and vitamin D3

synthesis. VEGF signalling is also involved in keratinocyte proliferation and re-

epithelialization, with a preferential role for VEGFR2 prior to wound closure.

2.4.2 FGF-2 and FGFR1 Expression by Keratinocytes

Similar to VEGF, FGF-2 is a potent pro-angiogenic factor and endothelial cell

mitogen (Krufka et al, 1996; Takahashi & Shibuya, 2005) that is robustly expressed by

gecko keratinocytes prior to injury and throughout the process of wound healing. Although

I was unable to co-localize FGF-2 with its receptor FGFR1, data from adjacent sections

clearly demonstrates that both proteins are detected in the majority of keratinocytes (except

prior to re-epithelialization; see below). Hence, FGF-2/FGFR1 are almost certainly co-

expressed thus suggesting a possible autocrine signalling loop.

FGF-2 has a well-documented role in stimulating keratinocyte proliferation

(Vrabec et al, 1994; Werner & Steiling, 2003), and has previously been reported in the

epidermis of two distantly related species of tail-regenerating lizard (Lamprophilis

guichenoti and Podarcis sicula; Alibardi & Lovicu, 2010; Alibardi, 2012). Among

mammals, exogenous administration of FGF-2 not only promotes re-epithelialization in

41

vivo (Oda et al, 2004), but can even reverse the impaired mode of wound healing otherwise

characteristic of diabetic mice (Broadley et al, 1987; Broadley et al, 1988). FGF-2 may

also play a protective role by regulating DNA repair and maintaining genomic integrity in

keratinocyte progenitor populations (Harfouche et al., 2010). Treatment of keratinocyte

progenitor cells with exogenous FGF-2 prior to irradiation not only increased the rate of

double-strand break repair, but it also demonstrated a pro-survival effect (Harfouche et al.,

2010). Authors of this study postulated that the effects were mediated through both

autocrine and paracrine signalling loops (Harfouche et al, 2010).

In support of a role for FGF-2 expression by keratinocytes, a previous study found

that injecting FGF-2 following injury in rat palatal mucosa accelerated wound healing via

a more rapid re-epithelialization (Oda et al., 2004). Using immunohistochemistry, the

authors saw an increase in FGFR1 correlated to the increase in FGF-2 following injection

(Oda et al, 2004). Similar to my own findings, Oda et al (2004) reported a lack of FGFR1

expression in the stratum germinativum; at 8DPW, FGFR1 is also conspicuously absent

from the gecko stratum germinativum and lowermost layers of the intermediate zone.

Conspicuously, these FGFR1- layers demonstrate the highest levels of proliferation (as

evidenced by PCNA+ cells). Although not tested here, one explanation is that another FGF

receptor is being activated in these basal layers, or that other member(s) of the FGF family

are being expressed, or both. In addition to FGF-2, FGF-7 (also known as keratinocyte

growth factor) is known to stimulate keratinocyte proliferation and migration (Tsuboi et al,

1993). By 45 DPW, FGF-2 and FGFR1 are ubiquitously expressed within the neoepidermis

and as such, it is virtually indistinguishable from that of the uninjured epidermis. I propose

that this pattern of FGF-2 and FGFR1 expression before, during and after wound healing,

42

similar to that of VEGF and its receptors, is indicative of one or more homeostatic

functions.

Although I documented robust FGF-2/FGFR1 expression by keratinocytes of the

intact epidermis, detection of the proteins is more varied during wound healing. In

particular, at 2 DPW FGF-2 expression by neo-keratinocytes is restricted to isolated cells

nearest the wound margin, while FGFR1 expression is notably absent from the WE.

Although presently untested, it seems likely that FGF-2 could be signalling through another

one of its receptors, such as FGFR2 (which, after FGFR1, has the highest binding affinity

to FGF-2; Mansukhani et al, 1992; Ornitz et al, 1996).

2.4.3 Canonical TGFβ Signalling

TGFβ is a multi-functional cytokine superfamily with numerous roles related to

tissue homeostasis and wound healing (Penn et al, 2012). I determined that the canonical

TGFβ/Activin signalling pathway is constitutively activated in the gecko epidermis, based

on the near ubiquitous expression of pSMAD2, TGFβ1 and activinβA in both the uninjured

and wounded skin. Prior to injury, TGFβ1 is known to limit keratinocyte proliferation

(Ramirez et al., 2014). In TGFβ1 null mice, keratinocytes undergo hyperproliferation,

leading to an increased risk in skin carcinogenesis (Glick et al, 1993). Curiously, while

TGFβ1 is localized to the uppermost layers of keratinocytes in human epidermis (Gold et

al, 2000), I found it expressed throughout all epidermal strata in the original and fully

regenerated epidermis. One possible explanation is that geckos, as squamate reptiles,

undergo coordinated body wide skin-shedding or ecdysis events (Maderson, 1964, 1965).

This periodic process requires the synchronization of stratum germinativum proliferation

and keratinocyte differentiation to duplicate the epidermis, resulting in an inner and outer

43

epidermal generation (Maderson, 1964, 1965). During ecdysis, the outer generation is

sloughed off while the inner generation is retained. I propose that constitutive expression

of TGFβ1 functions in coordinating ecdysis by restricting keratinocyte proliferation.

TGFβ1 also participates in wound closure by promoting keratinocyte migration. More

specifically, TGFβ1 drives epithelial-to-mesenchymal transitions (Rasanen & Vaheri,

2010; Weber et al, 2012; Lamouille et al, 2014), and increases the expression of, while

acting on, the integrins (Gailit et al, 1994; Li et al, 2006; Margadant & Sonnenberg, 2010).

Compared to TGFβ1, less is known about the function(s) of activin in the epidermis

although a role in resolving the epidermal defect is indicated (Wankell et al, 2001). Activin,

and the closely related inhibin, are dimer proteins best known for their roles in reproductive

organs. ActivinβA contributes to two activin homodimers, activin A (βA, βA) and activin

AB (βA, βB), and one heterodimer, activin AB (βA, βB) (Werner & Alzheimer, 2006);

therefore one or both could be expressed in geckos. Human (unpublished, but cited in

Werner & Alzheimer, 2006) and gecko (Gilbert et al., 2013) keratinocytes upregulate

activinβA in response to injury, while mouse keratinocytes upregulate activinβA and

activinβB and express activin A (Werner & Alzheimer, 2006). In mammals, inhibiting

activin (using a transgenic mouse line that overexpresses the antagonist follistatin) delays

wound healing, but also reduces the amount of granulation tissue formed resulting in a

smaller scar (Wankell et al., 2001).

Overall, my data reveals a close correspondence between pSMAD2, TGFβ1 and

activinβA expression: all are generally widespread throughout the original epidermis and

fully re-epithelialized WE (8DPW and onwards), but reveal limited expression prior to re-

epithelialization (2DPW). It is possible that during early wound healing, the non-canonical

44

signalling pathway is more strongly upregulated, or that other ligands of the canonical

TGFβ signalling pathway (e.g., TGFβ2) are expressed. Interestingly, and for reasons that

remain unclear, segments of the fully regenerated WE fail to express activinβA. Combined,

my findings indicate homeostatic and injury-mediated roles for TGFβ1 and activinβA,

likely related to the suppression of keratinocyte proliferation and facilitating cell migration

associated with re-epithelialization (Martin, 1997; Zhang et al, 2005).

Taken together, my data point towards both overlapping (functionally redundant

and/or synergistic) and unique roles for each of VEGF, FGF-2 and TGFβ1. For example,

both VEGF and FGF-2 have protective roles in response to UV irradiation (Oda et al, 2004;

Man et al, 2006; Harfouche et al, 2010; Zhu et al, 2013), while all three growth factors

induce angiogenesis (Seifert et al, 2014). A similar example of functional redundancy is

observed in tumours resistant to selective anti-angiogenic agents (Casanovas et al, 2005;

Batchelor et al, 2007; Ellis & Hicklin, 2008), leading to the emergence of drugs that target

multiple factors and combination therapies (Ribatti, 2011). This may explain why deleting

VEGF from keratinocytes in mice has no effect on the vascular density of the wound, but

results in a delayed eschar shedding and wound closure (Rossiter et al, 2004). Moreover,

although VEGF and FGF-2 stimulate keratinocyte proliferation, TGFβ1 has the opposite

effect. Combined, these effects suggest roles in fine-tuning and possibly coordinating the

generation of keratinocytes.

45

Chapter 2 Tables

Table 1: A summary of all optimized immunohistochemistry and immunofluorescence protocols for proteins of interest (vwf,

α-sma, VEGF, VEGFR2 (flk-1), VEGFR1 (flt-1), FGF-2, FGFR1, PCNA, pSMAD2, TGFβ1 and ActivinβA)

Antigen Type Retrieval Block Primary Secondary HRP DAB

vwf IF None 3% NGS 1 hr

RT

Rabbit anti-von

Willebrand

Factor 1:500

(Dako Canada,

A0082)

Cy3 Goat anti-

Rabbit

1:400(Jackson

ImmunoResearch

Laboratories,

Inc. 111-165-

144)

α-sma IF None 3% NGS 1 hr

RT

Mouse anti-α-

actin 1:400

(Santa Cruz

BioTechnology,

Inc sc-32252)

Goat anti-Mouse

AlexaFluor-488

1:400 (Life

Technologies,

A-11001)

VEGF IHC 12 min citrate

buffer

3% NGS 1 hr

RT

Rabbit anti-

VEGF 1:100

(Santa Cruz

BioTechnology,

Inc sc-152)

Biotinylated

Goat anti-Rabbit

1:500(Jackson

ImmunoResearch

Laboratories,

Inc. 111-066-

003)

1:200 25 sec

IF 12 min citrate

buffer

3% NGS 1 hr

RT

Rabbit anti-

VEGF 1:50

(Santa Cruz

BioTechnology,

Inc sc-152)

Cy3 Goat anti-

Rabbit 1:50)

(Jackson

ImmunoResearch

Laboratories,

Inc. 111-154-

144)

VEGFR2

(flk-1)

IHC 12 min citrate

buffer

3% NGS 1 hr

RT

Mouse anti-Flk-1

1:600 (Santa

Cruz

Biotinylated

Goat anti-Mouse

1:200 (Vector

1:200 40 sec

46

BioTechnology,

Inc sc-6251)

Laboratories BA-

9200)

IF 12 min citrate

buffer

3% NGS 1 hr

RT

Mouse anti-Flk-1

1:100 (Santa

Cruz

BioTechnology,

Inc sc-6251)

Goat anti-Mouse

AlexaFluor-488

1:100 (Life

Technologies,

A-11001)

VEGFR1

(flt-1)

IHC 12 min citrate

buffer

3% NGS 1 hr

RT

Rabbit anti-Flt-1

1:200 (Santa

Cruz

BioTechnology,

Inc sc-316)

Biotinylated

Goat anti-Rabbit

1:200 (Jackson

ImmunoResearch

Laboratories,

Inc. 111-165-

144)

1:200 50sec

IF 12 min citrate

buffer

3% NGS 1 hr

RT

Rabbit anti-Flt-1

1:50 (Santa Cruz

BioTechnology,

Inc sc-316)

Cy3 Goat anti-

Rabbit 1:200)

(Jackson

ImmunoResearch

Laboratories,

Inc. 111-154-

144)

FGF-2 IHC 12 min citrate

buffer

5% NGS 1 hr

RT

Rabbit anti-FGF-

2 1:100 (Santa

Cruz

BioTechnology,

Inc. sc-79)

Biotinylated

Goat anti-Rabbit

1:200 (Jackson

ImmunoResearch

Laboratories,

Inc. 111-165-

144)

1:200 40sec

FGFR1 IHC None 3% NGS 1 hr

RT

Rabbit anti-

FGFR1 1:50

(Cell Signaling

Technology,

#3472)

Biotinylated

Goat anti-Rabbit

1:200(Jackson

ImmunoResearch

Laboratories,

1:200 70sec

47

Inc. 111-066-

003)

PCNA IF None 3% NGS 1 hr

RT

Rabbit anti-

PCNA 1:100

(Santa Cruz

BioTechnology,

Inc. sc-7907)

Cy3 Goat anti-

Rabbit 1:200)

(Jackson

ImmunoResearch

Laboratories,

Inc. 111-154-

144)

pSMAD2 IHC None 3% NGS 1 hr

RT

Rabbit anti-

pSMAD2 1:800

(Cell

Signaling

Technology,

ser465/467)

Biotinylated

Goat anti-Rabbit

1:200(Jackson

ImmunoResearch

Laboratories,

Inc.111-066-003)

1:200 40sec

TGFβ1 IHC None 3% NGS 1 hr

RT

Rabbit anti-

TGFβ1 1:500

(Santa

Cruz

BioTechnology

Inc., sc-146)

Biotinylated

Goat anti-Rabbit

1:500(Jackson

ImmunoResearch

Laboratories,

Inc.111-066-003)

1:200 30sec

ActivinβA IHC 12 min citrate

buffer

Blocking buffer

1hr RT

Kind donation

from Dr. Paul

Sawchenko;1:500

Salk Institute for

Biological

Studies, La Jolla,

CA PBL #207-

234

Biotinylated

Goat anti-Rabbit

1:200(Jackson

ImmunoResearch

Laboratories,

Inc.111-066-003)

1:50 45sec

48

Chapter 2 Figures

Figure 1: Scar-free wound healing in the gecko. Macroscopic images taken: prior to biopsy (A), immediately following

biopsy (B), 2 days post-wounding (2DPW) (C), 8 DPW (D), and 45 DPW (E). Note the restoration of pigmentation and

scalation at 45 DPW. Hatched yellow lines represent the diameter of a 3mm biopsy punch.

49

50

Figure 2: Vascular endothelial growth factor A (VEGF) and VEGF receptor 2 (VEGFR2) are expressed by

keratinocytes of the gecko epidermis. Prior to injury, the epidermis is 3-4 nucleated cell layers thick (A) and

demonstrates widespread expression of VEGF (B) and VEGFR2 (C). Merge image reveals co-localization of the ligand

and receptor (D). Within the epidermis, proliferating cell nuclear antigen (PCNA) immunopositive cells (white

arrowheads) are restricted to the stratum germinativum (E) Note that PCNA+ cells co-express VEGFR2. At 2DPW, the

wound epithelium (WE) is 1-4 nucleated cell layers thick (F). Neo-keratinocytes of the WE continue to express VEGF

(G) and VEGFR2 (H), and these proteins co-localize (I). PCNA+ cells are relatively uncommon (J). At 8DPW, the WE

is 6-10 nucleated cell layers thick (K) and demonstrate discontinuous expression of VEGF (L) and VEGFR2 (M). The

overlay image (N) reveals most (but not all) keratinocytes co-localize both proteins. PCNA+ cells are primarily present

in the deepest layers of the epidermis (O), corresponding to the intermediate zone and the stratum germinativum. By 45

DPW, the neo-epidermis has returned to its pre-injury thickness (1-4 nucleated cell layers) (P), and demonstrates a pattern

of expression (Q-T) that closely resembles that of the original epidermis. Scale bar = 10μm.

51

52

Figure 3: Vascular endothelial growth factor receptor (VEGFR) 1 and

VEGFR2 are expressed by keratinocytes of the gecko epidermis. Prior to injury,

keratinocytes demonstrate widespread expression of both VEGFR1 (A) and

VEGFR2 (B) (merge: (C)). At 2DPW, VEGFR1 (D) expression is limited, whereas

VEGFR2 (E) is widespread (merge: (F)). At 8DPW VEGFR1 (G), and VEGFR2

(H) expression is discontinuous (merge: (I)). By 45DPW, VEGFR1 and 2

expression has returned to its original (pre-injury) appearance (J-L). Scale bar =

10μm.

53

54

Figure 4: Fibroblast growth factor 2 (FGF-2) and FGF receptor 1 (FGFR1)

are expressed by keratinocytes of the gecko epidermis. Prior to injury,

keratinocytes demonstrate widespread expression of both FGF-2 (A) and FGFR1

(B). At 2DPW, FGF-2 (C) is expressed by neo-keratinocytes near the wound

margins, but is absent from cells spanning the centre of the wound (to the left of

the image). FGFR1 (D) is not detected at this timepoint. At 8DPW, there is

widespread expression of FGF-2 (E) throughout the wound epithelium, whereas

FGFR1 (F) most strongly expressed within the newly formed stratum corneum and

the uppermost layers of the intermediate zone. At 45DPW, FGF-2 and FGFR1

expression has returned to its original (pre-injury) appearance (G-H). Scale bar =

10μm

55

56

Figure 5: Vascular endothelial growth factor A (VEGF) and VEGF receptor 2

(VEGFR2) are co-localized within cells of the gecko dermis and wound bed. Prior to

injury, the dermis is a fabric of connective tissue (A) with cells immunopositive for both

VEGF (B), and VEGFR2 (C) (merge: (D), whitearrow heads). Structurally mature blood

vessels, immunopositive for vonWillebrand (vWF) and α-smooth muscle actin (αSMA) are

also present. At 8DPW, the blastema (cell-rich aggregate) has formed (F). Blastema cells

also express VEGF (G) and VEGFR2 (H) (merge: (I)). Importantly, structurally mature

blood vessels are already present (J). By 45DPW, the newly formed dermis is virtually

indistinguishable from the surrounding uninjured dermis (K-O). Scale bar = 10μm

57

Figure 6: Vascular endothelial growth factor receptor (VEGFR) 1 and 2 are co-

localized within cells of the dermis and wound bed. Prior to injury, cells of the dermis

are immunopositive for both VEGFR1 (A) and VEGFR2 (B), and these proteins co-localize

(C) (white arrowheads). Similar patterns of VEGFR1 and VEGFR2 protein expression

were observed at 8DPW (D-F) and 45DPW (G-I).

58

59

Figure 7: Transforming growth factor β (TGFβ) signalling in keratinocytes of the

gecko epidermis. Prior to injury, keratinocytes demonstrate widespread expression of

phosphorylated SMAD2 (pSMAD2) (A), TGFβ1(B) and activinβA (C). At 2DPW,

pSMAD2 immunopositive cells are rare (D). At 2DPW, TGFβ1 (C) and activinβA (D) are

expressed by neo-keratinocytes near the wound margins, but are absent from cells spanning

the centre of the wound (to the left of the images). At 8DPW, pSMAD2+ cells are

distributed throughout the wound epithelium (G). Similarly, TGFβ1 (H) and activinβA (I)

are widely expressed by keratinocytes, although conspicuous activinβA- segments of the

wound epithelium are present. By 45 DPW, the expression pattern of pSMAD2 (J),

TGFβ1(K) and activinβA (L) closely matches that of the original epidermis. The scale bar

= 10μm.

60

Figure 8: Summary schematic of growth factors expression in the normal and regenerating

epidermis. Note: FGFR1 and VEGFR1 are notably absent early during re-epithelialization of

scar-free wound healing.

61

Chapter 3: Concluding Statements

3.1 Summary

This study sought to investigate growth factor expression in normal (pre-injury)

and regenerating skin. Previous work on wound healing has largely focused on the tissue-

level events of the dermis, including inflammation, angiogenesis, and fibrogenesis.

Although less is known about the epidermis, emerging evidence points towards important

roles for growth factors beyond strictly angiogenesis (Wilgus et al., 2005). Previous work

has demonstrated that leopard gecko (Eublepharis macularius) integument has an intrinsic

capacity to heal excisional wounds scar-free (Peacock et al, 2015). I took advantage of this

naturally evolved ability to test my hypothesis that endogenous growth factors are

dynamically expressed in the wound epithelium during scar-free wound healing.

Unexpectedly, I found that multiple growth factors were constitutively expressed by the

keratinocytes before, during and after wound healing. More specifically, I discovered that

VEGF, VEGFR1, VEGFR2, FGF-2, FGFR1, pSMAD2 TGFβ1 and activinβA were present

in both the normal (uninjured) epidermis and the neo-epidermis, indicating roles in

homeostasis and injury-mediated re-epithelialization. Only the receptor tyrosine kinases

VEGFR1 and FGFR1 were dynamically expressed, and only during the earliest

(incomplete) phase of wound closure. Taken together, my data shows that keratinocyte-

derived expression of growth factors likely signals through both autocrine and paracrine

actions to mediate not only angiogenic effects, but also re-epithelialization and possibly

photo-protective functions.

62

3.2 Growth factor expression during re-epithelialization

VEGF, VEGFR2 and FGF-2 are widely expressed by keratinocytes of the wound

epithelium (WE) during re-epithelialization. Although VEGFR1 and FGFR1 are notably

absent prior to re-epithelialization (day 2), their pattern of expression is restored once re-

epithelialization is complete (day 8). This suggests that, at least initially, VEGF is primarily

signalling through VEGFR2. Later during wound closure it may signal through either

VEGFR1 or 2. One possible explanation is that many stimulatory effects of VEGF-A are

mediated through VEGFR2, whereas VEGFR1 is hypothesized to participate in a more

regulatory capacity (Gerber et al, 1999; Rahimi et al, 2000; Zeng et al, 2001a; Zeng et al,

2001b; Roberts et al, 2004). Curiously, while FGF-2 is detected at day 2, FGFR1 is not.

Although currently untested, FGF-2 may be signalling through FGFR2, for which it also

has shares a high binding affinity. In the future it would be interesting to know if other

members of the FGF family, such as FGF-7 (also known as keratinocyte growth factor) are

also upregulated.

As evidenced by pSMAD2 expression, the canonical TGFβ pathway is also active

in the normal epidermis and during re-epithelialization. Since this pathway elicits a

multitude of pleiotropic effects, it is difficult to conclude, based on the data gathered, the

specific effects of this activation. However, it is likely that, as for other species, TGFβ1 is

playing an inhibitory role to prevent hyperproliferation of keratinocytes in geckos both

before and during re-epithelialization (Glick et al, 1993; Santoro & Gaudino, 2005).

Moreover, TGFβ1 may also participate in stimulating keratinocyte migration (Gailit et al,

1994; Li et al, 2006; Margadant & Sonnenberg, 2010; Richardson et al, 2016; Fong et al,

2010). Similar to TGFβ1, activin (whether activin A or activin AB, or both) functions are

63

also mediated through SMAD2 (Werner & Alzheimer, 2006). Available evidence suggests

that activin likely has a stimulatory effect on keratinocytes during wound closure (Werner

& Alzheimer, 2006). While activin is expressed at low levels in uninjured skin, it is

upregulated during wound healing (Werner & Alzheimer, 2006). Building on my findings,

it would also be worth investigating the expression of activin βB subunit in order to clarify

the specific activin dimer(s) participating in these actions.

3.3 Novel functions for growth factors in the gecko epidermis

Despite the robust expression of multiple potent pro-angiogenic factors (especially

VEGF and FGF-2) by cells of the epidermis and the dermis prior to and throughout wound

healing, we did not find evidence of hypervascularity or granulation tissue. Instead, the

newly formed blastema is only modestly vascularized. One explanation is that VEGFR1

may function as a decoy receptor sequestering VEGF, thus preventing it from binding to

VEGFR2 (Gerber et al, 1999; Rahimi et al, 2000; Zeng et al, 2001a; Zeng et al, 2001b;

Roberts et al, 2004). Another possibility is that VEGF and FGF-2 promote cell survival in

response to ultraviolet (UV) radiation exposure. As ectotherms, reptiles engage in

behavioural thermoregulation and often seek sunlight as a heat source. In addition, solar

radiation in the UVB spectrum (range 290-315 nm) is necessary for vitamin D3

(cholecalciferol) photobiosynthesis (Brauchle et al., 1996). However, UV radiation is also

a powerful DNA mutagen. Based on these data, I propose a novel hypothesize: that VEGF

and FGF-2 expression by gecko keratinocytes plays a photo-protective role associated with

the normal physiological functions of the skin in regulating body temperature and

synthesizing vitamin D3. More specifically, these growth factors promote cell survival by

regulating DNA repair in stem and progenitor populations (Harfouche et al., 2010).

64

3.4 Conclusions

Overall, my findings provide compelling evidence that keratinocyte-mediated

growth factors participate in roles beyond angiogenesis, before and after injury. In addition,

they support previous work indicating that both VEGF and FGF-2 function, (at least in

part) in an autocrine manner. Future work is needed to further characterize other

endogenous growth factors such as FGF-7 and other TGFβ/activin ligands and their

receptors in order to gain a clearer understanding of the spatio-temporal environment

before, during and after wound healing. Moreover, experimental manipulations can be

employed to further elucidate the effects of specific growth factors and/or their receptors.

65

References

Abraham, J. A., & Klagsbrun, M. (1988). Modulation of wound repair by members of the

fibroblast growth factor family. In The Molecular and Cellular Biology of Wound

Repair (pp. 195-248). Springer US.

Acierno, M. J., Mitchell, M. A., Zachariah, T. T., Roundtree, M. K., Kirchgessner, M. S.,

& Guzman, D. S. M. (2008). Effects of ultraviolet radiation on plasma 25-

hydroxyvitamin D3 concentrations in corn snakes (Elaphe guttata). American

Journal of Veterinary Research, 69(2), 294-297.

Alibardi, L. (2009). Morphological and Cellular Aspects of Tail and Limb Regeneration in

Lizards: a Model System with Implications for Tissue Regeneration in

Mammals (Vol. 207). Springer Science & Business Media.

Alibardi, L., & Lovicu, F. J. (2010). Immunolocalization of FGF1 and FGF2 in the

regenerating tail of the lizard Lampropholis guichenoti: implications for FGFs as

trophic factors in lizard tail regeneration. Acta Histochemica, 112(5), 459-473.

Alibardi, L. (2012). Observations on FGF immunoreactivity in the regenerating tail

blastema, and in the limb and tail scars of lizard suggest that FGFs are required for

regeneration. Belgian Journal of Zoology, 142(1), 23-38.

Allam, A. A., Daza, J. D., & Abo‐Eleneen, R. E. (2016). Histology of the skin of three

limbless squamates dwelling in mesic and arid environments. The Anatomical

Record.

Alon, T., Hemo, I., Itin, A., Pe'er, J., Stone, J., & Keshet, E. (1995). Vascular endothelial

growth factor acts as a survival factor for newly formed retinal vessels and has

implications for retinopathy of prematurity. Nature Medicine, 1(10), 1024-1028.

Armstrong, J. R., & Ferguson, M. W. J. (1995). Ontogeny of the skin and the transition

from scar-free to scarring phenotype during wound healing in the pouch young of

a marsupial, Monodelphis domestica. Developmental Biology, 169(1), 242-260.

Ashcroft, G. S., Yang, X., Glick, A. B., Weinstein, M., Letterio, J. J., Mizel, D. E., Anzano,

M., Greenwell-Wild, T., Wahl, S.M., & Roberts, A. B. (1999). Mice lacking Smad3

66

show accelerated wound healing and an impaired local inflammatory

response. Nature Cell Biology, 1(5), 260-266.

Assoian, R. K., Komoriya, A., Meyers, C. A., Miller, D. M., & Sporn, M. B. (1983).

Transforming growth factor-beta in human platelets. Identification of a major

storage site, purification, and characterization. Journal of Biological

Chemistry, 258(11), 7155-7160.

Azevedo, A. S., Grotek, B., Jacinto, A., & Weidinger, G. (2011). The regenerative capacity

of the zebrafish caudal fin is not affected by repeated amputations. PLoS One, 6(7),

e22820.

Banks, R. E., Forbes, M. A., Kinsey, S. E., Stanley, A., Ingham, E., Walters, C., & Selby,

P. J. (1998). Release of the angiogenic cytokine vascular endothelial growth factor

(VEGF) from platelets: significance for VEGF measurements and cancer

biology. British Journal of Cancer,77(6), 956.

Barber, L. W. (1944). Correlations between wound healing and regeneration in fore‐limbs

and tails of lizards. The Anatomical Record, 89(4), 441-453.

Barrientos, S., Stojadinovic, O., Golinko, M. S., Brem, H., & Tomic‐Canic, M. (2008).

Growth factors and cytokines in wound healing. Wound Repair and

Regeneration, 16(5), 585-601.

Basilico, C., & Moscatelli, D. (1992). The FGF family of growth factors and

oncogenes. Advances in Cancer Research, 59, 115-165.

Batchelor, T. T., Sorensen, A. G., di Tomaso, E., Zhang, W. T., Duda, D. G., Cohen, K. S.,

Kozak, K.R., Cahill, D. P., Chen, P.J., Zhu, M., Ancukiewicz, M, Mrugala, M. M.,

Plotkin, S., Drappatz, J., Louis, D. N., Ivy, P., Scadden, D. T., Benner, T., Loeffler,

J.S., Wen, P.Y., and Jain, R. K. (2007). AZD2171, a pan-VEGF receptor tyrosine

kinase inhibitor, normalizes tumor vasculature and alleviates edema in glioblastoma

patients. Cancer Cell, 11(1), 83-95.

Battegay, E. J. (1995). Angiogenesis: mechanistic insights, neovascular diseases, and

therapeutic prospects. Journal of Molecular Medicine, 73(7), 333-346.

67

Benjamin, L. E., Golijanin, D., Itin, A., Pode, D., & Keshet, E. (1999). Selective ablation

of immature blood vessels in established human tumors follows vascular

endothelial growth factor withdrawal. The Journal of Clinical Investigation,

103(2), 159-165.

Billingham, R. E., & Russell, P. S. (1956). Incomplete wound contracture and the

phenomenon of hair neogenesis in rabbits' skin.

Boocock, C. A., Charnock-Jones, D. S., Sharkey, A. M., McLaren, J., Barker, P. J., Wright,

K. A., Twentyman, P.R., & Smith, S. K. (1995). Expression of Vascular Endothelial

Growth Factor and Its Receptors fit and KDR in Ovarian Carcinoma. Journal of the

National Cancer Institute, 87(7), 506-516.

Brauchle, M., Funk, J. O., Kind, P., & Werner, S. (1996). Ultraviolet B and H2O2 are

potent inducers of vascular endothelial growth factor expression in cultured

keratinocytes. Journal of Biological Chemistry, 271(36), 21793-21797.

Brem, H., Kodra, A., Golinko, M. S., Entero, H., Stojadinovic, O., Wang, V. M., Sheahan,

C.M., Weinberg, A.D., Woo, S.L. Ehrlich, H.P., & Tomic-Canic, M. (2009).

Mechanism of sustained release of vascular endothelial growth factor in

accelerating experimental diabetic healing. Journal of Investigative

Dermatology, 129(9), 2275-2287.

Broadley, K. N., Aquino, A. M., Hicks, B., Ditesheim, J. A., McGee, G. S., Demetriou, A.

A., Woodward, S.C. & Davidson, J. M. (1987). Growth factors bFGF and TGB beta

accelerate the rate of wound repair in normal and in diabetic rats. International

Journal of Tissue Reactions, 10(6), 345-353.

Broadley, K. N., Aquino, A. M., Hicks, B., Ditesheim, J. A., McGee, G. S., Demetriou, A.

A., Woodward, S.C., & Davidson, J. M. (1988). The diabetic rat as an impaired

wound healing model: stimulatory effects of transforming growth factor-beta and

basic fibroblast growth factor. Biotechnology Therapeutics, 1(1), 55-68.

Broadley, K. N., Aquino, A. M., Woodward, S. C., Buckley-Sturrock, A., Sato, Y., Rifkin,

D. B., & Davidson, J. M. (1989). Monospecific antibodies implicate basic fibroblast

68

growth factor in normal wound repair. Laboratory Investigation; a Journal of

Technical Methods and Pathology, 61(5), 571-575.

Brock, J., McCluskey, J., Baribault, H., & Martin, P. (1996). Perfect wound healing in the

keratin 8 deficient mouse embryo. Cell Motility and the Cytoskeleton, 35(4), 358-

366.

Brown, L. F., Yeo, K., Berse, B., Yeo, T. K., Senger, D. R., Dvorak, H. F., & Van De

Water, L. (1992). Expression of vascular permeability factor (vascular endothelial

growth factor) by epidermal keratinocytes during wound healing. The Journal of

Experimental Medicine, 176(5), 1375-1379.

Casanovas, O., Hicklin, D. J., Bergers, G., & Hanahan, D. (2005). Drug resistance by

evasion of antiangiogenic targeting of VEGF signaling in late-stage pancreatic islet

tumors. Cancer Cell, 8(4), 299-309.

Cass, D. L., Bullard, K. M., Sylvester, K. G., Yang, E. Y., Longaker, M. T., & Adzick, N.

S. (1997). Wound size and gestational age modulate scar formation in fetal wound

repair. Journal of Pediatric Surgery, 32(3), 411-415.

Chen, T. T., Luque, A., Lee, S., Anderson, S. M., Segura, T., & Iruela-Arispe, M. L. (2010).

Anchorage of VEGF to the extracellular matrix conveys differential signaling

responses to endothelial cells. The Journal of Cell Biology,188(4), 595-609.

Christenson, L. J., Phillips, P. K., Weaver, A. L., & Otley, C. C. (2005). Primary closure

vs second-intention treatment of skin punch biopsy sites: a randomized

trial. Archives of Dermatology, 141(9), 1093-1099.

Clark, R. A. (1988). Wound repair. In The Molecular and Cellular Biology of Wound

Repair (pp. 3-50). Springer US.

Clark, R. A., Folkvord, J. M., Hart, C. E., Murray, M. J., & McPherson, J. M. (1989).

Platelet isoforms of platelet-derived growth factor stimulate fibroblasts to contract

collagen matrices. Journal of Clinical Investigation, 84(3), 1036.

Coffey, R. J., Bascom, C. C., Sipes, N. J., Graves-Deal, R., Weissman, B. E., & Moses, H.

L. (1988). Selective inhibition of growth-related gene expression in murine

69

keratinocytes by transforming growth factor beta. Molecular and Cellular

Biology, 8(8), 3088-3093.

Cooper, K. D., Kang, K., Chan, S. C., & Hanifin, J. M. (1985). Phosphodiesterase

inhibition by Ro 20-1724 reduces hyper-IgE synthesis by atopic dermatitis cells in

vitro. Journal of Investigative Dermatology, 84(6), 477-482.

Coulombe, P. A. (1997). Towards a molecular definition of keratinocyte activation after

acute injury to stratified epithelia. Biochemical and Biophysical Research

Communications, 236(2), 231-238.

Delorme, S. L., Lungu, I. M., & Vickaryous, M. K. (2012). Scar‐Free Wound Healing and

Regeneration Following Tail Loss in the Leopard Gecko, Eublepharis

macularius. The Anatomical Record, 295(10), 1575-1595.

Derynck, R., & Miyazono, K. (Eds.). (2008). The TGF-[beta] Family (Vol. 50). CSHL

Press.

Desmoulière, A., & Gabbiani, G. (1988). The Role of the Myofibroblast in Wound Healing

and Fibrocontractive Diseases (pp. 391-423). Springer US.

Desmoulière, A., Chaponnier, C., & Gabbiani, G. (2005). Tissue repair, contraction, and

the myofibroblast. Wound Repair and Regeneration,13(1), 7-12.

Detmar, M., Yeo, K. T., Nagy, J. A., Van De Water, L., Brown, L. F., Berse, B., Elicker,

B.M., Ledbetter, S., & Dvorak, H. F. (1995). Keratinocyte-derived vascular

permeability factor (vascular endothelial growth factor) is a potent mitogen for

dermal microvascular endothelial cells. Journal of Investigative

Dermatology, 105(1), 44-50.

Detmar, M., Brown, L. F., Schön, M. P., Elicker, B. M., Velasco, P., Richard, L.,

Fukumara, D., Monsky, W., Claffey, K.P., & Jain, R. K. (1998). Increased

microvascular density and enhanced leukocyte rolling and adhesion in the skin of

VEGF transgenic mice. Journal of Investigative Dermatology, 111(1), 1-6.

Diegelmann, R. F., & Evans, M. C. (2004). Wound healing: an overview of acute, fibrotic

and delayed healing. Frontiers in Bioscience, 9(1), 283-289.

70

Dunk, C., & Ahmed, A. (2001). Vascular endothelial growth factor receptor-2-mediated

mitogenesis is negatively regulated by vascular endothelial growth factor receptor-

1 in tumor epithelial cells. The American Journal of Pathology,158(1), 265-273.

Dunn, L., Prosser, H. C., Tan, J. T., Vanags, L. Z., Ng, M. K., & Bursill, C. A. (2013).

Murine model of wound healing. JoVE (Journal of Visualized Experiments), (75),

e50265-e50265.

Eckert, R. L. (1989). Structure, function, and differentiation of the

keratinocyte. Physiological Reviews, 69(4), 1316-1346.

Eckes, B., Aumailley, M., & Krieg, T. (1988). Collagens and the reestablishment of dermal

integrity. In The molecular and Cellular Biology of Wound Repair (pp. 493-512).

Springer US.

Egozi, E. I., Ferreira, A. M., Burns, A. L., Gamelli, R. L., & Dipietro, L. A. (2003). Mast

cells modulate the inflammatory but not the proliferative response in healing

wounds. Wound Repair and Regeneration, 11(1), 46-54.

Ellis, L. M., & Hicklin, D. J. (2008). Pathways mediating resistance to vascular endothelial

growth factor–targeted therapy. Clinical Cancer Research, 14(20), 6371-6375.

Eming, S. A., & Krieg, T. (2006, September). Molecular mechanisms of VEGF-A action

during tissue repair. In Journal of Investigative Dermatology Symposium

Proceedings (Vol. 11, No. 1, pp. 79-86). Elsevier.

Eming, S. A., & Hubbell, J. A. (2011). Extracellular matrix in angiogenesis: dynamic

structures with translational potential. Experimental dermatology, 20(7), 605-613.

Fan, X., Krieg, S., Kuo, C. J., Wiegand, S. J., Rabinovitch, M., Druzin, M. L., Brenner,

R.M., Giudice, L.C., & Nayak, N. R. (2008). VEGF blockade inhibits angiogenesis

and reepithelialization of endometrium. Federation of American Societies for

Experimental Biology, 22(10), 3571-3580.

Ferguson, M. W., & O'Kane, S. (2004). Scar–free healing: from embryonic mechanisms to

adult therapeutic intervention. Philosophical Transactions of the Royal Society of

London B: Biological Sciences, 359(1445), 839-850.

71

Ferrara, N., Gerber, H. P., & LeCouter, J. (2003). The biology of VEGF and its

receptors. Nature Medicine, 9(6), 669-676.

Ferrara, N. (2004). Vascular endothelial growth factor: basic science and clinical progress.

Endocrine Reviews, 25(4), 581-611.

Fong, E., Tzlil, S., & Tirrell, D. A. (2010). Boundary crossing in epithelial wound healing.

Proceedings of the National Academy of Sciences, 107(45), 19302-19307.

Frank, S., Hübner, G., Breier, G., Longaker, M. T., Greenhalgh, D. G., & Werner, S.

(1995). Regulation of vascular endothelial growth factor expression in cultured

keratinocytes. Implications for normal and impaired wound healing. Journal of

Biological Chemistry, 270(21), 12607-12613.

Freinkel, R. K., & Woodley, D. T. (Eds.). (2001). The Biology of the Skin. CRC Press.

Fujio, Y., & Walsh, K. (1999). Akt mediates cytoprotection of endothelial cells by vascular

endothelial growth factor in an anchorage-dependent manner. Journal of Biological

Chemistry, 274(23), 16349-16354.

Gabbiani, G., Chaponnier, C., & Hüttner, I. (1978). Cytoplasmic filaments and gap

junctions in epithelial cells and myofibroblasts during wound healing. The Journal

of Cell Biology, 76(3), 561-568.

Gailit, J., Clark, R. A., & Welch, M. P. (1994). TGF-β1 stimulates expression of

keratinocyte integrins during re-epithelialization of cutaneous wounds. Journal of

Investigative Dermatology, 103(2), 221-227.

Galiano, R. D., Michaels, V., Dobryansky, M., Levine, J. P., & Gurtner, G. C. (2004).

Quantitative and reproducible murine model of excisional wound healing. Wound

Repair and Regeneration, 12(4), 485-492.

Gawriluk, T. R., Simkin, J., Thompson, K. L., Biswas, S. K., Clare-Salzler, Z., Kimani, J.

M., Kiama, S. G., Smith, J. J., Ezenwa, V. O., & Seifert, A. W. (2016). Comparative

analysis of ear-hole closure identifies epimorphic regeneration as a discrete trait in

mammals. Nature Communications, 7.

72

Gemberling, M., Bailey, T. J., Hyde, D. R., & Poss, K. D. (2013). The zebrafish as a model

for complex tissue regeneration. Trends in Genetics, 29(11), 611-620.

Gerber, H. P., Hillan, K. J., Ryan, A. M., Kowalski, J., Keller, G. A., Rangell, L., Wright,

B.D., Radtke, F., Aquet, M. & Ferrara, N. (1999). VEGF is required for growth and

survival in neonatal mice. Development, 126(6), 1149-1159.

Gilbert, R. W., Vickaryous, M. K., & Viloria‐Petit, A. M. (2013). Characterization of TGFβ

signaling during tail regeneration in the leopard gecko (Eublepharis

macularius). Developmental Dynamics, 242(7), 886-896.

Gilbert, R. W., Vickaryous, M. K., & Viloria‐Petit, A. M. (2016). Signalling by

transforming growth factor β isoforms in wound healing and tissue

regeneration. Journal of Developmental Biology, 242(7), 886-896.

Glick, A. B., Kulkarni, A. B., Tennenbaum, T., Hennings, H., Flanders, K. C., O'Reilly,

M., Sporn, M.B., Karlsson, S., & Yuspa, S. H. (1993). Loss of expression of

transforming growth factor beta in skin and skin tumors is associated with

hyperproliferation and a high risk for malignant conversion. Proceedings of the

National Academy of Sciences, 90(13), 6076-6080.

Godwin, J. W., Pinto, A. R., & Rosenthal, N. A. (2013). Macrophages are required for

adult salamander limb regeneration. Proceedings of the National Academy of

Sciences, 110(23), 9415-9420.

Godwin, J. W., & Rosenthal, N. (2014). Scar-free wound healing and regeneration in

amphibians: immunological influences on regenerative

success. Differentiation, 87(1), 66-75.

Gold, L. I., Jussila, T., Fusenig, N. E., & Stenbäck, F. (2000). TGF‐β isoforms are

differentially expressed in increasing malignant grades of HaCaT keratinocytes,

suggesting separate roles in skin carcinogenesis. The Journal of Pathology, 190(5),

579-588.

73

Goliger, J. A., & Paul, D. L. (1995). Wounding alters epidermal connexin expression and

gap junction-mediated intercellular communication. Molecular Biology of the

Cell, 6(11), 1491-1501.

Goss, R. J., & Grimes, L. N. (1972). Tissue interactions in the regeneration of rabbit ear

holes. American Zoologist, 12(1), 151-157.

Greiling, D., & Clark, R. A. (1997). Fibronectin provides a conduit for fibroblast

transmigration from collagenous stroma into fibrin clot provisional matrix. Journal

of Cell Science, 110(7), 861-870.

Guo, L., Degenstein, L., Dowling, J., Yu, Q. C., Wollmann, R., Perman, B., & Fuchs, E.

(1995). Gene targeting of BPAG1: abnormalities in mechanical strength and cell

migration in stratified epithelia and neurologic degeneration. Cell, 81(2), 233-243.

Gurtner, G. C., Werner, S., Barrandon, Y., & Longaker, M. T. (2008). Wound repair and

regeneration. Nature, 453(7193), 314-321.

Hardy, M. A. (1989). The biology of scar formation. Physical Therapy, 69(12), 1014-1024.

Harfouche, G., Vaigot, P., Rachidi, W., Rigaud, O., Moratille, S., Marie, M., Lemaitre, G.,

Fortunel, N.O., & Martin, M. T. (2010). Fibroblast growth factor type 2 signaling

is critical for DNA repair in human keratinocyte stem cells. Stem Cells, 28(9), 1639-

1648.

Hinz, B. (2007). Formation and function of the myofibroblast during tissue repair. Journal

of Investigative Dermatology, 127(3), 526-537.

Hiratsuka, S., Minowa, O., Kuno, J., Noda, T., & Shibuya, M. (1998). Flt-1 lacking the

tyrosine kinase domain is sufficient for normal development and angiogenesis in

mice. Proceedings of the National Academy of Sciences, 95(16), 9349-9354.

Hiratsuka, S., Maru, Y., Okada, A., Seiki, M., Noda, T., & Shibuya, M. (2001).

Involvement of Flt-1 tyrosine kinase (vascular endothelial growth factor receptor-

1) in pathological angiogenesis. Cancer Research, 61(3), 1207-1213.

74

Hogan, M. B., Peele, K., & Wilson, N. W. (2012). Skin barrier function and its importance

at the start of the atopic march. Journal of Allergy, 2012.

Holick, M. F. (1995). Environmental factors that influence the cutaneous production of

vitamin D.The American Journal of Clinical Nutrition, 61(3), 638S-645S.

Houck, K. A., Leung, D. W., Rowland, A. M., Winer, J., & Ferrara, N. (1992). Dual

regulation of vascular endothelial growth factor bioavailability by genetic and

proteolytic mechanisms. Journal of Biological Chemistry, 267(36), 26031-26037.

Hübner, G., Hu, Q., Smola, H., & Werner, S. (1996). Strong induction of activin expression

after injury suggests an important role of activin in wound repair. Developmental

Biology, 173(2), 490-498.

Ito, M., Liu, Y., Yang, Z., Nguyen, J., Liang, F., Morris, R. J., & Cotsarelis, G. (2005).

Stem cells in the hair follicle bulge contribute to wound repair but not to

homeostasis of the epidermis. Nature Medicine, 11(12), 1351-1354.

Itoh, S., Itoh, F., Goumans, M. J., & ten Dijke, P. (2000). Signaling of transforming growth

factor‐β family members through Smad proteins. European Journal of

Biochemistry, 267(24), 6954-6967.

Jacobi, J., Tam, B. Y., Sundram, U., von Degenfeld, G., Blau, H. M., Kuo, C. J., & Cooke,

J. P. (2004). Discordant effects of a soluble VEGF receptor on wound healing and

angiogenesis. Gene Therapy, 11(3), 302-309.

Järvinen, T. A., & Ruoslahti, E. (2010). Target-seeking antifibrotic compound enhances

wound healing and suppresses scar formation in mice. Proceedings of the National

Academy of Sciences, 107(50), 21671-21676.

Jaźwińska, A., Badakov, R., & Keating, M. T. (2007). Activin-βA signaling is required for

zebrafish fin regeneration. Current Biology, 17(16), 1390-1395.

Jensen-Jarolim, E. (Ed.). (2013). Comparative Medicine: Anatomy and Physiology.

Springer Science & Business Media.

75

Jiang, C. K., Tomic-Canic, M., Lucas, D. J., Simon, M., & Blumenberg, M. (1995). TGFβ

promotes the basal phenotype of epidermal keratinocytes: transcriptional induction

of K# 5 and K# 14 keratin genes. Growth Factors, 12(2), 87-97.

Johnson, K. E., & Wilgus, T. A. (2012). Multiple roles for VEGF in non-melanoma skin

cancer: angiogenesis and beyond. Journal of Skin Cancer,2012.

Johnson, D. E., & Williams, L. T. (1992). Structural and functional diversity in the FGF

receptor multigene family. Advances in Cancer Research, 60, 1-41.

Joseph, J., & Dyson, M. (1966). Effect of anabolic androgens on tissue replacement in the

ear of the rabbit.

Kato, H., Yoshikawa, M., Miyazaki, T., Nakajima, M., Fukai, Y., Masuda, N., Fukuchi,

M., Manda, R., Tsukada, K., & Kuwano, H. (2001). Expression of vascular

endothelial growth factor (VEGF) and its receptors (Flt-1 and Flk-1) in esophageal

squamous cell carcinoma. Anticancer Research, 22(6C), 3977-3984.

Kieran, I., Knock, A., Bush, J., So, K., Metcalfe, A., Hobson, R., Mason, T., O’Kane, S.,

& Ferguson, M. (2013). Interleukin‐10 reduces scar formation in both animal and

human cutaneous wounds: Results of two preclinical and phase II randomized

control studies. Wound Repair and Regeneration, 21(3), 428-436.

Klaphake, E. (2010). A fresh look at metabolic bone diseases in reptiles and amphibians.

Veterinary Clinics of North America: Exotic Animal Practice, 13(3), 375-392.

Komine, M., Rao, L. S., Kaneko, T., Tomic-Canic, M., Tamaki, K., Freedberg, I. M., &

Blumenberg, M. (2000). Inflammatory Versus Proliferative Processes in Epidermis

Tumor Necrosis Factor α induces K6b keratin synthesis through a transcriptional

complex containing NFκB and C/EBPβ. Journal of Biological Chemistry, 275(41),

32077-32088.

Kragl, M., Knapp, D., Nacu, E., Khattak, S., Maden, M., Epperlein, H. H., & Tanaka, E.

M. (2009). Cells keep a memory of their tissue origin during axolotl limb

regeneration. Nature, 460(7251), 60-65.

76

Krufka, A., Guimond, S., & Rapraeger, A. C. (1996). Two hierarchies of FGF-2 signaling

in heparin: mitogenic stimulation and high-affinity binding/receptor

transphosphorylation. Biochemistry, 35(34), 11131-11141.

Kurschat, P., Bielenberg, D., Rossignol-Tallandier, M., Stahl, A., & Klagsbrun, M. (2006).

Neuron restrictive silencer factor NRSF/REST is a transcriptional repressor of

neuropilin-1 and diminishes the ability of semaphorin 3A to inhibit keratinocyte

migration. Journal of Biological Chemistry, 281(5), 2721-2729.

Larson, B. J., Longaker, M. T., & Lorenz, H. P. (2010). Scarless fetal wound healing: a

basic science review. Plastic and Reconstructive Surgery, 126(4), 1172.

Lamalice, L., Houle, F., & Huot, J. (2006). Phosphorylation of Tyr1214 within VEGFR-2

triggers the recruitment of Nck and activation of Fyn leading to SAPK2/p38

activation and endothelial cell migration in response to VEGF. Journal of

Biological Chemistry, 281(45), 34009-34020.

Lamouille, S., Xu, J., & Derynck, R. (2014). Molecular mechanisms of epithelial–

mesenchymal transition. Nature reviews. Molecular Cell Biology,15(3), 178.

Lang, M. (1989). The morphology of the Oberhäutchen with the description and

distribution of scale organs in basiliscine iguanians. Amphibia-Reptilia, 10(4), 423-

434.

Lauer, G., Sollberg, S., Cole, M., Flamme, I., Stürzebecher, J., Mann, K., Krieg,T., &

Eming, S. A. (2000). Expression and proteolysis of vascular endothelial growth

factor is increased in chronic wounds. Journal of Investigative Dermatology,

115(1), 12-18.

Lévesque, M., Villiard, É., & Roy, S. (2010). Skin wound healing in axolotls: a scarless

process.Journal of Experimental Zoology Part B: Molecular and Developmental

Evolution, 314(8), 684-697.

Li, Y., Fan, J., Chen, M., Li, W., & Woodley, D. T. (2006). Transforming growth factor-

alpha: a major human serum factor that promotes human keratinocyte

migration. Journal of Investigative Dermatology, 126(9), 2096-2105.

77

Li, C., Man, X., Li, W., Zhou, J., Chen, J., Cai, S., & Zheng, M. (2011). Regulation of

adhesion by vascular endothelial growth factor in HaCaT cells. Molecular and

Cellular Biochemistry, 346(1-2), 173-178.

Lorenz, H. P., & Adzick, N. S. (1993). Scarless skin wound repair in the fetus. Western

Journal of Medicine, 159(3), 350.

Lo, D. D., Zimmermann, A. S., Nauta, A., Longaker, M. T., & Lorenz, H. P. (2012).

Scarless fetal skin wound healing update. Birth Defects Research Part C: Embryo

Today: Reviews, 96(3), 237-247.

Longaker, M. T., Whitby, D. J., Ferguson, M. W., Lorenz, H. P., Harrison, M. R., &

Adzick, N. S. (1994). Adult skin wounds in the fetal environment heal with scar

formation. Annals of Surgery, 219(1), 65.

Maden, M., Manwell, L. A., & Ormerod, B. K. (2013). Proliferation zones in the axolotl

brain and regeneration of the telencephalon. Neural Development, 8(1), 1.

Maderson, P. F. A. (1964). The skin of lizards and snakes. J. Herpet, 3, 151-154.

Maderson, P. F. A. (1965, September). Histological changes in the epidermis of snakes

during the sloughing cycle. In Proceedings of the Zoological Society of

London (Vol. 146, No. 1, pp. 98-113). Blackwell Publishing Ltd.

Maharaj, A. S., & D'Amore, P. A. (2007). Roles for VEGF in the adult. Microvascular

Research, 74(2), 100-113.

Man, X. Y., Yang, X. H., Cai, S. Q., Yao, Y. G., & Zheng, M. (2006). Immunolocalization

and expression of vascular endothelial growth factor receptors (VEGFRs) and

neuropilins (NRPs) on keratinocytes in human epidermis. Molecular

Medicine, 12(7-8), 127.

Man, X. Y., Yang, X. H., Cai, S. Q., Bu, Z. Y., & Zheng, M. (2008). Overexpression of

vascular endothelial growth factor (VEGF) receptors on keratinocytes in psoriasis:

regulated by calcium independent of VEGF. Journal of Cellular and Molecular

Medicine, 12(2), 649-660.

78

Mansukhani, A., Dell'Era, P., Moscatelli, D., Kornbluth, S., Hanafusa, H., & Basilico, C.

(1992). Characterization of the murine BEK fibroblast growth factor (FGF)

receptor: activation by three members of the FGF family and requirement for

heparin. Proceedings of the National Academy of Sciences, 89(8), 3305-3309.

Margadant, C., & Sonnenberg, A. (2010). Integrin–TGF‐β crosstalk in fibrosis, cancer and

wound healing. EMBO reports, 11(2), 97-105.

Martin, P. (1997). Wound healing--aiming for perfect skin

regeneration. Science, 276(5309), 75-81.

Matsumoto, T., & Claesson-Welsh, L. (2001). VEGF receptor signal transduction. Science

Signaling, 2001(112), re21-re21.

McCusker, C., Bryant, S. V., & Gardiner, D. M. (2015). The axolotl limb blastema: cellular

and molecular mechanisms driving blastema formation and limb regeneration in

tetrapods. Regeneration, 2(2), 54-71.

McLean, K. E., & Vickaryous, M. K. (2011). A novel amniote model of epimorphic

regeneration: the leopard gecko, Eublepharis macularius. BMC Developmental

Biology, 11(1), 1.

Mera, S. L. (1997). Understanding Disease: Pathology and Prevention. Nelson Thornes.

Monaghan, J. R., Stier, A. C., Michonneau, F., Smith, M. D., Pasch, B., Maden, M., &

Seifert, A. W. (2014). Experimentally induced metamorphosis in axolotls reduces

regenerative rate and fidelity. Regeneration, 1(1), 2-14.

Montesano, R., & Orci, L. (1988). Transforming growth factor beta stimulates collagen-

matrix contraction by fibroblasts: implications for wound healing. Proceedings of

the National Academy of Sciences, 85(13), 4894-4897.

Moustakas, A., & Heldin, C. H. (2005). Non-Smad TGF-β signals. Journal of Cell

Science, 118(16), 3573-3584.

79

Natarajan, V. T., Ganju, P., Ramkumar, A., Grover, R., & Gokhale, R. S. (2014).

Multifaceted pathways protect human skin from UV radiation. Nature Chemical

Biology, 10(7).

Nayak, N. R., & Brenner, R. M. (2002). Vascular proliferation and vascular endothelial

growth factor expression in the rhesus macaque endometrium. The Journal of

Clinical Endocrinology & Metabolism, 87(4), 1845-1855.

Nissen, N. N., Polverini, P., Koch, A. E., Volin, M. V., Gamelli, R. L., & DiPietro, L. A.

(1998). Vascular endothelial growth factor mediates angiogenic activity during the

proliferative phase of wound healing. The American Journal of Pathology,152(6),

1445.

Occleston, N. L., Metcalfe, A. D., Boanas, A., Burgoyne, N. J., Nield, K., O'Kane, S., &

Ferguson, M. W. (2010). Therapeutic improvement of scarring: mechanisms of

scarless and scar-forming healing and approaches to the discovery of new

treatments. Dermatology Research and Practice, 2010.

Oda, Y., Kagami, H., & Ueda, M. (2004). Accelerating effects of basic fibroblast growth

factor on wound healing of rat palatal mucosa. Journal of Oral and Maxillofacial

Surgery, 62(1), 73-80.

Ogunjimi, A. A., Zeqiraj, E., Ceccarelli, D. F., Sicheri, F., Wrana, J. L., & David, L. (2012).

Structural basis for specificity of TGFβ family receptor small molecule

inhibitors. Cellular Signalling, 24(2), 476-483.

O'Kane, S., & Ferguson, M. W. (1997). Transforming growth factor βs and wound healing.

The International Journal of Biochemistry & Cell biology, 29(1), 63-78.

Olofsson, B., Korpelainen, E., Pepper, M. S., Mandriota, S. J., Aase, K., Kumar, V., Gunji,

Y., Jeltsch, M.M., Shibuya, M., Alitalo, K., & Eriksson, U. (1998). Vascular

endothelial growth factor B (VEGF-B) binds to VEGF receptor-1 and regulates

plasminogen activator activity in endothelial cells. Proceedings of the National

Academy of Sciences, 95(20), 11709-11714.

80

Ornitz, D. M., Xu, J., Colvin, J. S., McEwen, D. G., MacArthur, C. A., Coulier, F., Gao,

G., & Goldfarb, M. (1996). Receptor specificity of the fibroblast growth factor

family. Journal of Biological Chemistry, 271(25), 15292-15297.

Ornitz, D. M., & Itoh, N. (2001). Fibroblast growth factors. Genome biology, 2(3), 1.

Ortega, S., Ittmann, M., Tsang, S. H., Ehrlich, M., & Basilico, C. (1998). Neuronal defects

and delayed wound healing in mice lacking fibroblast growth factor 2. Proceedings

of the National Academy of Sciences, 95(10), 5672-5677.

Page, R. B., & Voss, S. R. (2009). Induction of metamorphosis in axolotls (Ambystoma

mexicanum). Cold Spring Harbor Protocols, 2009(8), pdb-prot5268.

Pakyari, M., Farrokhi, A., Maharlooei, M. K., & Ghahary, A. (2013). Critical role of

transforming growth factor beta in different phases of wound healing. Advances in

Wound Care, 2(5), 215-224.

Paladini, R. D., Takahashi, K., Bravo, N. S., & Coulombe, P. A. (1996). Onset of re-

epithelialization after skin injury correlates with a reorganization of keratin

filaments in wound edge keratinocytes: defining a potential role for keratin 16. The

Journal of Cell Biology, 132(3), 381-397.

Park, J. E., Chen, H. H., Winer, J., Houck, K. A., & Ferrara, N. (1994). Placenta growth

factor. Potentiation of vascular endothelial growth factor bioactivity, in vitro and in

vivo, and high affinity binding to Flt-1 but not to Flk-1/KDR. Journal of Biological

Chemistry, 269(41), 25646-25654.

Pastar, I., Stojadinovic, O., Yin, N. C., Ramirez, H., Nusbaum, A. G., Sawaya, A., Patel,

S.B., Khalid, L., Isseroff, R. R., & Tomic-Canic, M. (2014). Epithelialization in

wound healing: a comprehensive review. Advances in Wound Care, 3(7), 445-464.

Peacock, H. M., Gilbert, E. A., & Vickaryous, M. K. (2015). Scar‐free cutaneous wound

healing in the leopard gecko, Eublepharis macularius. Journal of Anatomy, 227(5),

596-610.

81

Penn, J. W., Grobbelaar, A. O., & Rolfe, K. J. (2012). The role of the TGF-β family in

wound healing, burns and scarring: a review. International Journal of Burns and

Trauma, 2(1), 18-28.

Proetzel, G., Pawlowski, S. A., Wiles, M. V., Yin, M., Boivin, G. P., Howles, P. N., Ding,

J., Ferguson, M.W., & Doetschman, T. (1995). Transforming growth factor-β3 is

required for secondary palate fusion. Nature Genetics, 11(4).

Rahimi, N., Dayanir, V., & Lashkari, K. (2000). Receptor chimeras indicate that the

vascular endothelial growth factor receptor-1 (VEGFR-1) modulates mitogenic

activity of VEGFR-2 in endothelial cells. Journal of Biological Chemistry, 275(22),

16986-16992.

Rajaram, K., Harding, R. L., Bailey, T., Patton, J. G., & Hyde, D. R. (2014). Dynamic

miRNA expression patterns during retinal regeneration in zebrafish: Reduced dicer

or miRNA expression suppresses proliferation of Müller Glia‐derived neuronal

progenitor cells. Developmental Dynamics, 243(12), 1591-1605.

Ramirez, H., Patel, S. B., & Pastar, I. (2014). The role of TGFβ signaling in wound

epithelialization. Advances in Wound Care, 3(7), 482-491.

Räsänen, K., & Vaheri, A. (2010). TGF-beta1 causes epithelial-mesenchymal transition in

HaCaT derivatives, but induces expression of COX-2 and migration only in benign,

not in malignant keratinocytes. Journal of Dermatological Science, 58(2), 97-104.

Relf, M., LeJeune, S., Scott, P. A., Fox, S., Smith, K., Leek, R., Moghaddam, A.,

Whitehouse, R., Bicknell, R., & Harris, A. L. (1997). Expression of the angiogenic

factors vascular endothelial cell growth factor, acidic and basic fibroblast growth

factor, tumor growth factor β-1, platelet-derived endothelial cell growth factor,

placenta growth factor, and pleiotrophin in human primary breast cancer and its

relation to angiogenesis. Cancer Research, 57(5), 963-969.

Ribatti, D. (2011). Novel angiogenesis inhibitors: addressing the issue of redundancy in

the angiogenic signaling pathway. Cancer Treatment Reviews, 37(5), 344-352.

82

Riches, D. W. (1988). Macrophage involvement in wound repair, remodeling, and fibrosis.

In The Molecular and Cellular Biology of Wound Repair (pp. 95-141). Springer

US.

Richardson, R., Slanchev, K., Kraus, C., Knyphausen, P., Eming, S., & Hammerschmidt,

M. (2013). Adult zebrafish as a model system for cutaneous wound-healing

research. Journal of Investigative Dermatology, 133(6), 1655-1665.

Richardson, R., Metzger, M., Knyphausen, P., Ramezani, T., Slanchev, K., Kraus, C.,

Schmelzer, Elmon, & Hammerschmidt, M. (2016). Re-epithelialization of

cutaneous wounds in adult zebrafish combines mechanisms of wound closure in

embryonic and adult mammals. Development, 143(12), 2077-2088.

Risau, W. (1990). Angiogenic growth factors. Progress in Growth Factor Research, 2(1),

71-79.

Roberts, A. B. (1998). Molecular and cell biology of TGF-β. Mineral and Electrolyte

Metabolism, 24(2-3), 111-119.

Roberts, D. M., Kearney, J. B., Johnson, J. H., Rosenberg, M. P., Kumar, R., & Bautch, V.

L. (2004). The vascular endothelial growth factor (VEGF) receptor Flt-1 (VEGFR-

1) modulates Flk-1 (VEGFR-2) signaling during blood vessel formation. The

American Journal of Pathology, 164(5), 1531-1535.

Roman, C. D., Choy, H., Nanney, L., Riordan, C., Parman, K., Johnson, D., & Beauchamp,

R. D. (2002). Vascular endothelial growth factor-mediated angiogenesis inhibition

and postoperative wound healing in rats. Journal of Surgical Research, 105(1), 43-

47.

Ross, S., & Hill, C. S. (2008). How the Smads regulate transcription. The International

Journal of Biochemistry & Cell Biology, 40(3), 383-408.

Rossiter, H., Barresi, C., Pammer, J., Rendl, M., Haigh, J., Wagner, E. F., & Tschachler,

E. (2004). Loss of vascular endothelial growth factor a activity in murine epidermal

keratinocytes delays wound healing and inhibits tumor formation. Cancer

Research, 64(10), 3508-3516.

83

Sampson, H. A. (1983). Role of immediate food hypersensitivity in the pathogenesis of

atopic dermatitis. Journal of Allergy and Clinical Immunology, 71(5), 473-480.

Santoro, M. M., & Gaudino, G. (2005). Cellular and molecular facets of keratinocyte

reepithelization during wound healing. Experimental Cell Research, 304(1), 274-

286.

Santos, D., Rita, A. M., Casanellas, I., Brito Ova, A., Araújo, I. M., Power, D., & Tiscornia,

G. (2016). Ear wound regeneration in the African spiny mouse Acomys cahirinus.

Regeneration, 3(1), 52-61.

Sase, H., Watabe, T., Kawasaki, K., Miyazono, K., & Miyazawa, K. (2009). VEGFR2-

PLCγ1 axis is essential for endothelial specification of VEGFR2+ vascular

progenitor cells. Journal of Cell Science, 122(18), 3303-3311.

Satish, L., & Kathju, S. (2010). Cellular and molecular characteristics of scarless versus

fibrotic wound healing. Dermatology Research and Practice, 2010.

Schmidt, R., Beil, T., Strähle, U., & Rastegar, S. (2014). Stab wound injury of the zebrafish

adult telencephalon: a method to investigate vertebrate brain neurogenesis and

regeneration. Journal of Visualized Experiments: JoVE, (90).

Schumacher, J., & Yelen, T. (2006). Anesthesia and analgesia. Reptile Medicine and

Surgery, 2, 442-452.

Seifert, A. W., Monaghan, J. R., Voss, S. R., & Maden, M. (2012a). Skin regeneration in

adult axolotls: a blueprint for scar-free healing in vertebrates. PloS One, 7(4),

e32875.

Seifert, A. W., Kiama, S. G., Seifert, M. G., Goheen, J. R., Palmer, T. M., & Maden, M.

(2012b). Skin shedding and tissue regeneration in African spiny mice

(Acomys). Nature, 489(7417), 561-565.

Seifert, A. W., & Maden, M. (2014). New insights into vertebrate skin

regeneration. International Review of Cell and Molecular Biology, 310, 129-169.

84

Sellheyer, K., Bickenbach, J. R., Rothnagel, J. A., Bundman, D., Longley, M. A., Krieg,

T., Roche, N.S., Roberts, A.B., & Roop, D. R. (1993). Inhibition of skin

development by overexpression of transforming growth factor beta 1 in the

epidermis of transgenic mice. Proceedings of the National Academy of

Sciences, 90(11), 5237-5241.

Shah, M., Foreman, D. M., & Ferguson, M. W. (1995). Neutralisation of TGF-beta 1 and

TGF-beta 2 or exogenous addition of TGF-beta 3 to cutaneous rat wounds reduces

scarring. Journal of Cell Science, 108(3), 985-1002.

Shi, Y., & Massagué, J. (2003). Mechanisms of TGF-β signaling from cell membrane to

the nucleus. Cell, 113(6), 685-700.

Shibuya, M. (2011). Vascular endothelial growth factor (VEGF) and its receptor (VEGFR)

signaling in angiogenesis a crucial target for anti-and pro-angiogenic

therapies. Genes & Cancer, 2(12), 1097-1105.

Shukla, A., Dubey, M. P., Srivastava, R., & Srivastava, B. S. (1998). Differential

expression of proteins during healing of cutaneous wounds in experimental normal

and chronic models. Biochemical and Biophysical Research

Communications, 244(2), 434-439.

Singer, A. J., & Clark, R. A. (1999). Cutaneous wound healing. New England Journal of

Medicine,341(10), 738-746.

Slominski, A., Paus, R., & Wortsman, J. (1993). On the potential role of

proopiomelanocortin in skin physiology and pathology. Molecular and Cellular

Endocrinology, 93(1), C1-C6.

Soker, S., Takashima, S., Miao, H. Q., Neufeld, G., & Klagsbrun, M. (1998). Neuropilin-

1 is expressed by endothelial and tumor cells as an isoform-specific receptor for

vascular endothelial growth factor. Cell, 92(6), 735-745.

Speirs, V., & Atkin, S. L. (1999). Production of VEGF and expression of the VEGF

receptors Flt-1 and KDR in primary cultures of epithelial and stromal cells derived

from breast tumours. British Journal of Cancer, 80(5-6), 898.

85

Steiling, H., & Werner, S. (2003). Fibroblast growth factors: key players in epithelial

morphogenesis, repair and cytoprotection. Current Opinion in

Biotechnology, 14(5), 533-537.

Stewart, G. R., & Daniel, R. S. (1972). Scales of the lizard Gekko gecko: surface structure

examined with the scanning electron microscope. Copeia, 252-257.

Stewart, S., & Stankunas, K. (2012). Limited dedifferentiation provides replacement tissue

during zebrafish fin regeneration. Developmental Biology, 365(2), 339-349.

Swift, M. E., Kleinman, H. K., & DiPietro, L. A. (1999). Impaired wound repair and

delayed angiogenesis in aged mice. Laboratory Investigation; a Journal of

Technical Methods and Pathology, 79(12), 1479-1487.

Szpaderska, A. M., Egozi, E. I., Gamelli, R. L., & DiPietro, L. A. (2003). The effect of

thrombocytopenia on dermal wound healing. Journal of Investigative

Dermatology, 120(6).

Szydłowski, P., Madej, J. P., & Mazurkiewicz‐Kania, M. (2015). Ultrastructure and

distribution of chromatophores in the skin of the leopard gecko (Eublepharis

macularius). Acta Zoologica.

Takahashi, T., Yamaguchi, S., Chida, K., & Shibuya, M. (2001). A single

autophosphorylation site on KDR/Flk‐1 is essential for VEGF‐A‐dependent

activation of PLC‐γ and DNA synthesis in vascular endothelial cells. The EMBO

Journal, 20(11), 2768-2778.

Takahashi, H., & Shibuya, M. (2005). The vascular endothelial growth factor

(VEGF)/VEGF receptor system and its role under physiological and pathological

conditions. Clinical Science, 109(3), 227-241.

Tan, W. H., Popel, A. S., & Mac Gabhann, F. (2013a). Computational model of VEGFR2

pathway to ERK activation and modulation through receptor trafficking. Cellular

Signalling, 25(12), 2496-2510.

Tan, W. H., Popel, A. S., & Mac Gabhann, F. (2013b). Computational model of Gab1/2-

dependent VEGFR2 pathway to Akt activation. PloS One, 8(6), e67438.

86

Tonnesen, M. G., Feng, X., & Clark, R. A. (2000, December). Angiogenesis in wound

healing. In Journal of Investigative Dermatology Symposium Proceedings (Vol. 5,

No. 1, pp. 40-46). Nature Publishing Group.

Toole, B. P. (1991). Proteoglycans and hyaluronan in morphogenesis and differentiation.

In Cell Biology of Extracellular Matrix (pp. 305-341). Springer US.

Tsou, R., Fathke, C., Wilson, L., Wallace, K., Gibran, N., & Isik, F. (2002). Retroviral

delivery of dominant‐negative vascular endothelial growth factor receptor type 2 to

murine wounds inhibits wound angiogenesis. Wound Repair and

Regeneration, 10(4), 222-229.

Tsuboi, R., Sato, C., Kurita, Y., Ron, D., Rubin, J. S., & Ogawa, H. (1993). Keratinocyte

growth factor (FGF-7) stimulates migration and plasminogen activator activity of

normal human keratinocytes. Journal of Investigative Dermatology, 101(1), 49-53.

Vorontsova, M. A., & Liosner, L. D. (1960). Asexual Propagation and Regeneration (No.

574.16 V67).

Vrabec, J. T., Schwaber, M. K., Davidson, J. M., & Clymer, M. A. (1994). Evaluation of

basic fibroblast growth factor in tympanic membrane repair. The

Laryngoscope, 104(9), 1059-1064.

Wankell, M., Munz, B., Hübner, G., Hans, W., Wolf, E., Goppelt, A., & Werner, S. (2001).

Impaired wound healing in transgenic mice overexpressing the activin antagonist

follistatin in the epidermis. The EMBO Journal, 20(19), 5361-5372.

Weber, C. E., Li, N. Y., Wai, P. Y., & Kuo, P. C. (2012). Epithelial-mesenchymal

transition, TGF-β, and osteopontin in wound healing and tissue remodeling after

injury. Journal of Burn Care & Research, 33(3), 311-318.

Welch, M. P., Odland, G. F., & Clark, R. A. (1990). Temporal relationships of F-actin

bundle formation, collagen and fibronectin matrix assembly, and fibronectin

receptor expression to wound contraction. The Journal of Cell Biology, 110(1),

133-145.

87

Werner, S., Breeden, M., Hübner, G., Greenhalgh, D. G., & Longaker, M. T. (1994a).

Induction of keratinocyte growth factor expression is reduced and delayed during

wound healing in the genetically diabetic mouse. Journal of Investigative

Dermatology, 103(4), 469-473.

Werner, S. (1998). Keratinocyte growth factor: a unique player in epithelial repair

processes. Cytokine & Growth Factor Reviews, 9(2), 153-165.

Werner, S., Smola, H., Liao, X., Longaker, M. T., Krieg, T., Hofschneider, P. H., &

Williams, L. T. (1994b). The function of KGF in morphogenesis of epithelium and

reepithelialization of wounds. Science, 266(5186), 819-822.

Werner, S., & Alzheimer, C. (2006). Roles of activin in tissue repair, fibrosis, and

inflammatory disease. Cytokine & Growth Factor Reviews, 17(3), 157-171.

Werner, S., & Grose, R. (2003). Regulation of wound healing by growth factors and

cytokines. Physiological Reviews, 83(3), 835-870.

Whitby, D. J., Longaker, M. T., Harrison, M. R., Adzick, N. S., & Ferguson, M. W. (1991).

Rapid epithelialisation of fetal wounds is associated with the early deposition of

tenascin. Journal of Cell Science, 99(3), 583-586.

Wilgus, T. A., Matthies, A. M., Radek, K. A., Dovi, J. V., Burns, A. L., Shankar, R., &

DiPietro, L. A. (2005). Novel function for vascular endothelial growth factor

receptor-1 on epidermal keratinocytes. The American Journal of Pathology,167(5),

1257-1266.

Wilgus, T. A., Ferreira, A. M., Oberyszyn, T. M., Bergdall, V. K., & DiPietro, L. A. (2008).

Regulation of scar formation by vascular endothelial growth factor. Laboratory

Investigation, 88(6), 579-590.

Williams‐Boyce, P. K., & Daniel, J. C. (1980). Regeneration of rabbit ear tissue. Journal

of Experimental Zoology, 212(2), 243-253.

Woodley, D. T., Chen, J. D., Kim, J. P., Sarret, Y., Iwasaki, T., Kim, Y. H., & O'Keefe, E.

J. (1993). Re-epithelialization. Human keratinocyte locomotion. Dermatologic

Clinics, 11(4), 641-646.

88

Wu, M. Y., & Hill, C. S. (2009). TGF-β superfamily signaling in embryonic development

and homeostasis. Developmental Cell, 16(3), 329-343.

Wu, P., Alibardi, L., & Chuong, C. M. (2014). Regeneration of reptilian scales after

wounding: neogenesis, regional difference, and molecular

modules. Regeneration, 1(1), 15-26.

Xu, J., & Clark, R. A. (2000). Integrin regulation in wound repair. Scarless Wound

Healing, 63.

Yannas, I. V. (2001). Tissue and Organ Regeneration in Adults. Springer.

Yang, G. P., Lim, I. J., Phan, T. T., Lorenz, H. P., & Longaker, M. T. (2003). From scarless

fetal wounds to keloids: molecular studies in wound healing. Wound Repair and

Regeneration, 11(6), 411-418.

Yang, X. H., Man, X. Y., Cai, S. Q., Yao, Y. G., Bu, Z. Y., & Zheng, M. (2006). Expression

of VEGFR-2 on HaCaT cells is regulated by VEGF and plays an active role in

mediating VEGF induced effects. Biochemical and Biophysical Research

Communications, 349(1), 31-38.

Yang, J., Meyer, M., Müller, A. K., Böhm, F., Grose, R., Dauwalder, T., Verrey, F., Kopf,

M., Partanen, J., Bloch, W., Ornitz, D. M., & Werner, S. (2010). Fibroblast growth

factor receptors 1 and 2 in keratinocytes control the epidermal barrier and cutaneous

homeostasis. The Journal of Cell Biology, 188(6), 935-952.

Yuan, F., Chen, Y., Dellian, M., Safabakhsh, N., Ferrara, N., & Jain, R. K. (1996). Time-

dependent vascular regression and permeability changes in established human

tumor xenografts induced by an anti-vascular endothelial growth factor/vascular

permeability factor antibody. Proceedings of the National Academy of

Sciences, 93(25), 14765-14770.

Zambruno, G., Marchisio, P. C., Marconi, A., Vaschieri, C., Melchiori, A., Giannetti, A.,

& De Luca, M. (1995). Transforming growth factor-beta 1 modulates beta 1 and

beta 5 integrin receptors and induces the de novo expression of the alpha v beta 6

89

heterodimer in normal human keratinocytes: implications for wound healing. The

Journal of Cell Biology, 129(3), 853-865.

Zeng, H., Dvorak, H. F., & Mukhopadhyay, D. (2001a). Vascular permeability factor

(VPF)/vascular endothelial growth factor (VEGF) receptor-1 down-modulates

VPF/VEGF receptor-2-mediated endothelial cell proliferation, but not migration,

through phosphatidylinositol 3-kinase-dependent pathways. Journal of Biological

Chemistry, 276(29), 26969-26979.

Zeng, H., Sanyal, S., & Mukhopadhyay, D. (2001b). Tyrosine residues 951 and 1059 of

vascular endothelial growth factor receptor-2 (KDR) are essential for vascular

permeability factor/vascular endothelial growth factor-induced endothelium

migration and proliferation, respectively. Journal of Biological

Chemistry, 276(35), 32714-32719.

Zhang, N., Fang, Z., Contag, P. R., Purchio, A. F., & West, D. B. (2004). Tracking

angiogenesis induced by skin wounding and contact hypersensitivity using a

Vegfr2-luciferase transgenic mouse. Blood, 103(2), 617-626.

Zhang, L., Deng, M., Parthasarathy, R., Wang, L., Mongan, M., Molkentin, J. D., Zheng,

Y., & Xia, Y. (2005). MEKK1 transduces activin signals in keratinocytes to induce

actin stress fiber formation and migration. Molecular and Cellular Biology, 25(1),

60-65.

Zhu, J. W., Wu, X. J., Luo, D., Lu, Z. F., Cai, S. Q., & Zheng, M. (2012). Activation of

VEGFR-2 signaling in response to moderate dose of ultraviolet B promotes survival

of normal human keratinocytes. The International Journal of Biochemistry & Cell

Biology, 44(1), 246-256.

Zhu, J. W., Wu, X. J., Lu, Z. F., Luo, D., Cai, S. Q., & Zheng, M. (2013). Role of VEGF

receptors in normal and psoriatic human keratinocytes: evidence from irradiation

with different UV sources. PloS One, 8(1), e55463.

90

Appendix 1: Supplementary Table for Chapter 2

Table A1: Weight at collection points in biopsy study.

Group Day Animal Weight at

collection

Controls Original Control-1 29.7

Original Control-2 24.6

Original 14-JA-08 10.5

Original 14-JA-17 12.4

Biopsy 2 14JA-22 10.1

2 14JA-12 10.1

2 14JA-21 9.1

2 14JA-20 8.3

8 8-9 19.6

8 8-13 18.5

8 April 21-15 12.5

8 April 21-17 10.3

45 OT-45-15 14.3

45 45-8 15.9

45 45-3 17.4

45 45-4 18.3

*Sentinels not collected

91

Appendix 2: Detailed Histochemical Protocols and Solutions

Anaesthesia

Dosage:

30 mg/kg body weight alfaxan diluted to 2 mg/mL in sterile injectable saline (0.9% NaCl)

Administration:

1. Restrain gecko

2. Inject appropriate dosage of diluted alfaxan as a divided dose into epaxial

musculature near forelimbs

3. Inject cranial, parallel to the spinal cord, not perpendicular to the skin

4. Place gecko in small aquarium with underlying heating cable set to 32˚C

5. Monitor for loss of reflexes

6. Righting reflex: the gecko should not be able to right itself when placed on its back

7. Pain reflex: the gecko should not respond when a toe is firmly pinched or when the

rip of the tail is pinched

8. Perform experimental procedure while gecko is under anaesthesia

9. Place gecko back into small aquarium and monitor for return of righting reflex

10. Place gecko in the heated side of a clean enclosure and cover with a hut

11. Monitor gecko until it resumes normal behaviour

Biopsy

Materials:

3 mm disposable biopsy punch

Number 11 scalpel blade

Number 3 scalpel handle

Small rounded tweezers

Small scissors

Procedure:

1. Anesthetize gecko

92

2. Mark the biopsy site with a non-toxic permanent marker

3. Using thumb and index finger on non-dominant hand, stretch the skin of the biopsy

site until taut

4. Centre the biopsy punch over the mark, holding punch between thumb, index finger

and middle finger of dominant hand; use ring finer of dominant hand to apply

tension to the skin

5. Firmly press the biopsy punch perpendicular to the skin and rotate between thumb

and forefinger until the epidermis is punctured all the way around the wound site

6. Create a wound to the depth that disrupts the dermis but does not puncture muscle

fascia

7. Use tweezers to remove the plug of tissue from the wound site

8. If necessary, use a scalpel or scissors to free any adherent dermis

9. DO NOT swab or dress the wound

Haematoxylin and Eosin

Protocol:

1. Absolute xylene (3 x 2 minutes)

2. Absolute isopropanol (3 x 2 minutes)

3. 70% isopropanol (2 minutes)

4. Deionized water (2 minutes)

5. Modified Harris Haematoxylin (10 minutes)

6. Rinse excess dye in running deionized water

7. Acid alcohol solution (6-10 dips)

8. Rinse in running deionized water

9. Blue in ammonia water (6 dips)

10. Rinse in running deionized water

11. 70% isopropanol (6 dips)

12. Eosin (1 minute)

13. Absolute isopropanol (3 x 2 minutes)

14. Absolute xylene (3 x 2 minutes)

15. Coverslip

93

Solutions:

Acid alcohol:

1% HCl in 70% isopropanol

Eosin Stock:

10 g Eosin Y

1 g Phloxine B

Dissolve in 1000 ml 80% ethanol (add dyes slowly dissolving)

Eosin Working:

200 mL stock Eosin

200 mL deionized water

600 mL absolute ethanol

5 mL glacial acetic acid

Masson’s Trichrome (modified)

Protocol:

1. Absolute xylene (3 x 2 minutes)

2. Absolute isopropanol (3 x 2 minutes)

3. 70% isopropanol (2 minutes)

4. Deionized water (2 minutes)

5. Mayer’s Haematoxylin (10 minutes)

6. Rinse off excess dye in running deionized water

7. Blue in ammonia water (6 dips)

8. Rinse off ammonia water in running deionized water

9. Ponceau Xylidine/Acid Fuchsin (2 minutes)

10. Rinse off excess dye in running deionized water

11. 1% phophomolybdic acid (10 minutes)

12. Rinse off excess phosphomolybdic acid in running deionized water

13. 2% Light Green (90 seconds)

94

14. Rinse in running deionized water

15. 95% isopropanol (2 minutes)

16. 100% isopropanol (3 x 2 minutes)

17. Absolute xylene (3 x 2 minutes)

18. Coverslip

Solutions:

0.5% Ponceau Xylidine/0.5% Acid Fuchsin in 1% acetic acid:

1.25 g Ponceau Xylidine 2R in 250mL 1% acetic acid solution

1.25 g Acid Fuchsin in 250mL 1% acetic acid solution

Mix together. Store at room temperature

1% Phosphomolybdic Acid

10 g phosphomolybdic acid in 1L deionized water

2% Light Green

2 g Light Green Yellowish SF in 100mL 2% citric acid solution

Mix 1:10 with deionized water

Ammonia water

5 drops ammonium hydroxide in 250mL deionized water

Immunohistochemical Protocol

Protocol:

1. Absolute xylene (3 x 2 minutes)

2. Absolute isopropanol (3 x 2 minutes)

3. 70% isopropanol (2 minutes)

4. Deionized water (2 minutes)

5. 3% hydrogen peroxide (20 minutes)

6. Phosphate buffered saline (3 x 2 minutes)

7. Citrate Buffer Retrieval (if required)

95

a. Citrate buffer 90˚C water bath (12 minutes)

b. Remove from water bath and let cool (20 minutes)

c. Phosphate buffered saline (3 x 2 minutes)

8. Block 3% normal goat serum for 1 hour at room temperature in a humidity chamber

9. Primary antibody overnight at 4˚C

10. Phosphate buffered saline (3 x 2 minutes)

11. Secondary antibody for 1 hour at room temperature

12. Phosphate buffered saline (3 x 2 minutes)

13. HRP-conjugate streptavidin for 1 hour at room temperature

14. Phosphate buffered saline (3 x 2 minutes)

15. DAB

16. Meyer’s haematoxylin (1 minute)

17. Rinse in running deionized water

18. Blue in ammonia water (6 dips)

19. Rinse in running deionized water

20. Absolute isopropanol (3 x 2 minutes)

21. Absolute xylene (3 x 2 minutes)

22. Coverslip

Citrate Buffer Retrieval:

Solution A (0.1 M citric acid)

1.92 g citric acid powder

100 mL deionized water

SolutionB (0.1 M sodium citrate dehydrate)

14.7 g sodium citrate dehydrate

500 mL deionized water

Citrate Buffer

450 mL deionized water

9 mL solution A

96

41 mL solution B

pH to 6.00

DAB:

Vector Laboratories DAB Peroxidase (HRP) Substrate Kit

5 mL deionized water

1 drop hydrogen peroxide stock

1 drop buffer stock

2 drops DAB stock

Immunofluorescence:

1. Absolute xylene (3 x 2 minutes)

2. Absolute isopropanol (3 x 2 minutes)

3. 70% isopropanol (2 minutes)

4. Deionized water (2 minutes)

5. Block 3% normal goat serum

6. Primary antibody overnight at 4˚C in humidity chamber

7. Phosphate buffered saline (3 x 2 minutes)

8. Secondary antibody 1 hour at room temperature

9. Phosphate buffered saline (3 x 2 minutes)

10. DAPI 1:10 000 (2 minutes)

11. Phosphate buffered saline (3 x 2 minutes)

12. Coverslip

97

Appendix 3: Antibody Validation

Introduction

In order to validate the specificity of our VEGF-A antibody (VEGF; A-20, Santa

Cruz, BioTechnology, Santa Cruz, California), we used a blocking peptide (sc-152 P, Santa

Cruz, BioTechnology, Santa Cruz, California). The antibody A-20 maps to the N-terminus

of human VEGF, and identifies three human splice variants: VEGF165, VEGF189 and

VEGF121. When sections are pre-treated with the blocking peptide the target epitope is

quenched, thus preventing immunoreactivity with the primary antibody. The result is the

absence of immunostaining.

Methods

Using previously collected original (uninjured) skin from the dorsal surface of the

gecko tail, I performed serial histology and immunostaining (both immunohistochemistry

and immunofluorescence as outlined in Chapter 2). I divided my samples into three

application groups, otherwise the protocols were unchanged: (1) primary antibody only;

(2) pre-treatment with blocking peptide followed by application of the primary antibody;

and (3) omission control (no pre-treatment, no primary antibody). For application group

(2), a 5 fold excess of blocking peptide (to antibody mass) was used, and then incubated

overnight prior to the application of the primary antibody.

Results

Application of VEGF alone (application group (1)) reveals in cytoplasmic

immunostaining of nearly all the keratinocytes of the epidermis, as well as many fibroblasts

of the dermis (Fig. 1A). Pre-treatment with the blocking peptide (followed by VEGF;

98

application group (2)) abolishes VEGF expression in all cells (Fig. 1B). Similarly, no

expression was observed in the omission control slides (Fig. 1C).

Discussion

Extinction of VEGF expression with pre-treatment using the blocking peptide

indicates that sc-152 P successfully competes with the antibody A-20. However, a

limitation of using this primary antibody is that VEGF-A is not specific to individual

isoforms.

Appendix 3 Figures:

A3: Blocking peptide neutralizes vascular endothelial growth factor A (VEGF)

expression. Trial comparing the pattern of VEGF expression by keratinocytes of the

epidermis and fibroblasts of the dermis without (A) and with (B) the blocking peptide. Note

the complete extinction of immunoreactivity with the application of the blocking peptide.

Omission (no primary antibody) control (C).