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In vitro examination of vitronectin, insulin-like growth factor, insulin-like growth
factor binding protein complexes as treatments to accelerate the healing of diabetic
ulcers.
By
Anthony Michael Noble
Bachelor of Applied Science (Hons.)
School of Life Sciences
Queensland University of Technology
Brisbane, Australia
A thesis submitted for the degree of Doctor of Philosophy of the Queensland
University of Technology
2007
STATEMENT OF ORIGINALITY
The work contained in this thesis has not been previously submitted for a degree or
diploma at any other higher education institution. This thesis does not contain any
material which has been previously published or written by another person except where
due references are made.
Signed:
Date:
Acknowledgements
I would firstly like to thank my family; Gary, Lorelle and Joshua, Alanna and Zak
for all their support over the long course of my studies.
I would importantly, like to acknowledge Drs Mark Ray, Tom Daley and Harry Gibbs of
the Vascular Surgery Unit of the Princess Alexandra Hospital, Brisbane, Australia and
their patients who generously donated their time and energy in obtaining our diabetic
patient-derived skin samples from amputated limbs. They obtained my tissue samples
immediately after performing major surgery and I whole-heartedly appreciate their efforts
which made this project possible. I would also like to thank Dr Tony Kane, Wickham
Terrace, Brisbane City and Dr Phil Richardson, Fortitude Valley, Brisbane and their
patients, who generously donated our non-diabetic skin samples.
My thanks also go to the Australian Red Cross Blood Service, Queen St, Brisbane, who
kindly irradiated my 3T3 fibroblast feeder cells. I would further like to acknowledge the
technical advice and guidance provided by Dr Damien Harkin, Dr Gary Shooter, Dr
Jenny Kricker, Dr Carolyn Hyde, Dr Jos Malda and in particular my associate supervisor
Dr David Leavesley, QUT.
I would also like to thank the Queensland University of Technology, The Diabetes
Australia Research Trust and Tissue Therapies Limited who funded aspects of my
project.
My special thanks must also go to Rebecca Dawson who facilitated the normal skin
samples used in this project and instructed me in the isolation and growth of primary skin
cells and in many cases provided me with the invaluable expertise that kept my cells
alive.
Lastly and most of all, I would like to thank my supervisor Prof. Zee Upton. I have been
working or studying in Zee’s laboratory for most of my adult life. Over this time as well
as fostering my curiosity and passion for knowledge, Zee has come to be more than just a
mentor and boss to me but a friend as well. Her enthusiasm, drive and courage are only
outweighed by her well-deserved success. Thanks for always believing in me. Abstract
It has previously been shown that VN can form complexes with IGF-II or IGF-I in
combination with its binding proteins IGFBP-3 or -5. This study aimed to determine the
efficacy of using these complexes as a treatment designed to accelerate wound healing,
particularly in diabetic ulcers. The primary functions of skin cells in wound healing are
attachment, proliferation and migration, thus these functions were assessed in response to
these complexes in skin cells derived from patients with diabetic ulcers and from non-
diabetic patients. These studies examined responses to the complexes in both skin
keratinocyte and fibroblast cells. Furthermore, in order to investigate the mechanisms that
underlie the responses observed, I also examined the ability of skin cells to retain these
functional responses when the complexes incorporated an IGF-I analogue that does not
activate the IGF receptor or when the cells had been pre-incubated with an anti-αv-
integrin function blocking antibody. In addition, the ability of the cells to survive and
grow when treated with the complexes under conditions mimicking the diabetic wound
was assessed using growth assays in which the media contained elevated concentrations
of glucose and calcium. I found that cells derived from skin from normal patients showed
enhanced proliferation in response to these complexes, whereas only the presence of IGF-
I and IGFBP seemed to be important in stimulating the proliferation of cells derived from
diabetic patients. I also found that enhanced migration was observed in fibroblasts from
diabetic ulcers in response to the complexes but these responses only required the
presence of VN in normal cells. Both normal and diabetic keratinocytes showed
enhanced migration in response to the complexes and the responses involved the
interaction of both IGF-I and VN with their respective cell surface receptors. However
the enhanced migration observed in diabetic ulcer derived keratinocytes was
approximately half the level seen in normal keratinocytes. Furthermore, I showed that
cells derived from skin from normal patients exhibited greater proliferation when treated
with complexes in the presence of high concentrations of glucose and calcium ion
compared to cells that were not treated with the complexes. Likewise, cells derived from
skin surrounding diabetic ulcers were able to grow in media containing high levels of
glucose and calcium when treated with VN:IGFBP:IGF-I complexes. In particular
diabetic skin derived fibroblasts grown in high calcium media demonstrated enhanced
proliferation when treated with the complexes, whereas diabetic keratinocyte cells
seemed less affected by these conditions than their normal counterparts were.
The findings in this thesis show that VN:IGFBP:IGF-I complexes can elicit enhanced
growth and migration in cells derived from skin from both normal and diabetic patients.
Further, these responses are maintained in conditions found in the diabetic wound
microenvironment, namely in the presence of high glucose and high calcium. Together
these findings demonstrate the potential of the VN:IGFBP:IGF complexes as wound
healing agents to treat wounds, especially diabetic ulcers. Such delayed healing wounds
represent a significant burden to health care systems and are one of the primary
conditions that leads to the amputation of limbs. Current treatments do not address the
co-ordination of ECM and growth factor action on cells that is here demonstrated to
stimulate multiple wound healing related functional effects in skin cells. The data
presented here represents important new information that may guide the design of new
integrated therapeutics that may enhance the healing of recalcitrant diabetic ulcers.
Table of Contents
Chapter 1 – Literature Review……………………………………………………………1
The Impact of Diabetes in Australia………………………………………….…….…….2
Delayed Healing Diabetic Ulcers……………………………………………….………...3
Existing Therapies ……………………………………………………………….……….6
An Inflammatory Wound Environment ……………………………..……..…….……….8
Insulin-like Growth Factors ………………………………………..………..….…..…...10
Growth Factor Dysfunction ………………………………………..………..….……….12
Vitronectin, IGF Complexes ………………………………………..………...…..……..14
Calcium and the Skin ……………………………………………..………………….....18
Calcium in Wound Healing ……………………………………………………....…….21
Calcium and Diabetes ………………………………………………..…………..……..22
Oxidation Stress ………………………………………………………………..…...…..24
Insulin: Glucose Metabolism in Normal and Diabetic Wounds …………………......…26
IGF-I and Insulin: Glucose Metabolism ………………………………..……….……...29
Excessive Glycosylation……………………………………………….……….……….31
Other Diabetic Pathologies …………………………………………………..……........33
Growth Factors and ECM as a Strategy to Enhance Wound Healing………..…..……..34
Aims and Hypothesis………………………………………………….……….………..37
Chapter 2 – Materials and Methods……………………………………………………39
2.1 Keratinocyte Isolation and Culture …………………………………………..……40
2.2 Fibroblast Isolation and Culture …………………………………………………...41
2.3 3T3 Cell Culture ………………………………………………………………..…42
2.4 Treatments Tested …………………………………………………………………42
2.5 Preparation of Wells Containing IGF-I, IGF-II, IGFBPs and VN…………………43
2.6 Anti-αv Integrin Antibody Treated Cells ………………………………………….44
2.7 Attachment Assays ………………………………………………………………...45
2.8 Protein Synthesis Assays ………………………………………………………….45
2.9 Trizolium Salt (MTT) Assay ……………………………………………………...46
2.10 Migration Assay ……………………………………………………………….....47
2.11 Proliferation of Skin Derived Cells in the Presence of Elevated
Concentrations of Calcium Ion or Glucose……………………………………….48
2.12 Statistical Analysis ……………………………………………………………….49
2.13 Rationale for Design of Experiments in Chapters 3 and 4 ……………………….50
2.14 Rationale for Selection of IGFBPs Incorporation into Treatments ……………...51
2.15 Rationale for Selection of Doses of Factors Examined ………………………….51
2.16 Rationale for Exclusion of EGF in Serum Free Media …………………………..52
2.17 Rationale for Using Insulin in Culture Media ……………………………………53
Chapter 3 – Functional Responses of Normal Keratinocyte and Fibroblast
Primary Cultured Cells To VN:IGFBP:IGF Complexes……………………...55
3.1 Introduction………………………………………………………………………...56
3.2 Results……………………………………………………………………………...60
3.2.1 Attachment of Dermal Derived Fibroblasts ……………………………………..60
3.2.2 Attachment of Dermal Derived Keratinocytes ………………………………….62
3.2.3 Proliferation of Dermal Derived Fibroblasts ……………………………………64
3.2.4 Protein Synthesis of Dermal Derived Keratinocytes ……………………...…….67
3.2.5 Migration of Dermal Derived Fibroblasts ……………………………………….70
3.2.6 Migration of Dermal Derived Keratinocytes ……………………………………73
3.3 Discussion………………………………………………………………………….76
Chapter 4 – Functional Responses of Keratinocyte and Fibroblast Cells ……………..80
Derived from Diabetic Ulcers to VN:IGFBP:IGF Complexes………………...81
4.1 Introduction ………………………………………………………………………..82
4.2 Results ……………………………………………………………………………..85
4.2.1 Attachment of Dermal Fibroblasts Derived from Diabetic Skin ………………..85
4.2.2 Attachment of Dermal Keratinocytes Derived from Diabetic Skin ……………..85
4.2.3 Proliferation of Dermal Fibroblasts Derived from Diabetic Skin ……………….89
4.2.4 Proliferation of Dermal Keratinocytes Derived from Diabetic Skin ……………92
4.2.5 Migration of Dermal Fibroblasts Derived from Diabetic Skin ………………….95
4.2.6 Migration of Dermal Keratinocytes Derived from Diabetic Skin ………………98
4.3 Discussion………………………………………………………………………...101
Chapter 5 – Mechanisms Underlying the Functional Responses Observed
In Cultures of Skin Cells Treated with VN:IGFBP:IGF Complexes…………105
5.1 Introduction ………………………………………………………………………106
5.2 Results ……………………………………………………………………………109
5.2.1 Proliferation of Normal Keratinocytes …………………………………………109
5.2.2 Migration of Normal Keratinocytes ……………………………………………114
5.2.3 Proliferation of Diabetic Ulcer Derived Keratinocytes ………………………..119
5.2.4 Migration of Diabetic Ulcer Derived Keratinocytes …………………………...124
5.3 Discussion ………………………………………………………………………..130
Chapter 6 – Long Term Proliferation of Normal and Diabetic Skin-Derived
Keratinocytes and Fibroblasts Cultured in Hyperglycemic and
Differentiation Inducing Conditions………………………………………….133
6.1 Introduction ……………………………………………………………………....134
6.2 Results ……………………………………………………………………………137
6.2.1 Preliminary Assays …………………………………………………………….137
6.2.2 Proliferation of Fibroblasts Derived From Normal Skin
Cultured in Normal Media…………………………………………………....138
6.2.3 Proliferation of Fibroblasts Derived From Normal Skin
Cultured in Hyperglycemic Media……………………………………………139
6.2.4 Proliferation of Fibroblasts Derived From Normal Skin
Cultured in Differentiation Media……………………………………………142
6.2.5 Proliferation of Keratinocytes Derived From Normal Skin
Cultured in Normal Media…………………………………………………....144
6.2.6 Proliferation of Keratinocytes Derived From Normal Skin
Cultured in Hyperglycemic Media……………………………………………145
6.2.7 Proliferation of Keratinocytes Derived From Normal Skin
Cultured in Differentiation Media………………………………...………….146
6.2.8 Proliferation of Fibroblasts Derived From Diabetic Ulcer Skin
Cultured in Normal Media……………………………………………………150
6.2.9 Proliferation of Fibroblasts Derived From Diabetic Ulcer Skin
Cultured in Hyperglycemic Media……………………………………………151
6.2.10 Proliferation of Fibroblasts Derived From Diabetic Ulcer Skin
Cultured in Differentiation Media……………………………………………152
6.2.11 Proliferation of Keratinocytes Derived From Diabetic Ulcer Skin
Cultured in Normal Media……………………………………………………156
6.2.12 Proliferation of Keratinocytes Derived From Diabetic Ulcer Skin
Cultured in Hyperglycemic Media……………………………………………157
6.2.13 Proliferation of Keratinocytes Derived From Diabetic Ulcer Skin
Cultured in Differentiation Media……………………………………………158
6.3 Discussion………………………………………………………………………...162
Chapter 7 – General Discussion ……………………………………………………...168
Chapter 8 – References ………………………………………………………………181
Chapter 9 – Appendix ………………………………………………………………..212
Appendix I – Preliminary assays …………………………………………….213
Appendix II – Morphology Photographs…………………………………….220
List of Abbreviations
ABAM- Antibiotic Antimycotic
AGE - Advanced Glycation End Product
ALS - Acid Labile Subunit
bFGF - Basic Fibroblast Growth Factor
BSA - Bovine Serum Albumin
Ca - Calcium
cAMP - Cyclic Adenosine Mono Phosphate
CaR - Calcium Receptor
DAG - Di-acyl Glycerol
ddH20 - Double distilled water (sterile)
DM - Differentiation Media (1.5mM CaCl2)
DMEM - Dullbecco’s Modified Eagle Media
DMSO - Di-methyl Sulphoxide
DNA - De-oxy Ribonucleic Acid
ECM - Extracellular Matrix
EDTA - Ethylene-di-amine-tetra-acetic acid
EGF - Epidermal Growth Factor
eNOS - Endothelial Nitric Oxide Synthase
EPO - Erythropoietin
FBS - Foetal Bovine Serum
FGF - Fibroblast Growth Factor
FN - Fibronectin
GC - Glucocorticoid
GH - Growth Hormone
HBB - Hepes Binding Buffer
HBSS - Hanks Balanced Salt Solution
HIF - Hypoxia Inducible Factor
HM - Hyperglycemic Media (100 mM Glucose)
HSP - Heat Shock Protein
HSPG - Heparin Sulphate ProteoGlycans
IGF - Insulin-like Growth Factor
IGF1-R - The Type 1 Insulin-like Growth Factor Receptor
IGFBP - Insulin-like Growth Factor Binding Protein
IL - Interleukin
IR - Insulin Receptor
IRS - Insulin Receptor Substrate
kDa - Kilo Dalton
KGF - Keratinocyte Growth Factor
LN - Laminin
MAP-K - Mitogen activated protein kinase
MMP - Matrix Metalloproteinase
mRNA - Messenger Ribonucleic Acid
MTT - Trizolium Salt
Na - Sodium
NFκB - Nuclear Factor Kappa B
NY - New York
PBS - Phosphate Buffered Saline
PDGF - Platelet Derived Growth Factor
PI - Phosphatidyl Inositol
PK - Protein Kinase
PKC - Protein Kinase C
RAGE - Receptor for Advanced Glycation End Products
RIA - Radio Immuno-Assay
RT-PCR - Reverse Transcription Polymerase Chain Reaction
SFM - Serum Free Media
TGF - Transforming Growth Factor
TNF - Tumor Necrosis Factor
TIMP - Tissue Inhibitor of Matrix Metalloproteinases
TK - Tyrosine Kinase
TM - Trade Mark
US FDA - United States Food and Drug Administration
VEGF - Vascular Endothelial Growth Factor
VN - Vitronectin
CHAPTER 1
Literature Review: Vitronectin and Insulin-like Growth Factors and their Binding
Proteins in Skin Homeostasis and Delayed Healing Diabetic Ulcers.
2
1.1 The Impact of Diabetes Mellitus in Australia
Approximately 7% of the Australian population currently have diabetes in their
lifetime. Currently more than 940,000 (7.5%) Australians over the age of 25 years have
diabetes. Those most at risk are the elderly, obese individuals and those of Aboriginal,
Torres Straight Islander, Pacific Islander or Asian descent. It is estimated that by 2010,
1.3 million Australians will have diabetes mellitus, 85-95% of which will be type 2
(Figure 1.1). More worrying than the prevalence, however, is that it is estimated that for
every known case of diabetes, there is one undiagnosed case. In fact, almost one in
four Australians aged 25 years and over has diabetes or a pre-diabetic condition of
impaired glucose metabolism (glucose intolerance).
Figure 1.1 - reproduced from http://www.diabetes .com.au/diabesity.htm
Shows the estimated number of cases of diabetes mellitus in Australia in 1981, 1983, 1990 and 2000 and
estimated number of cases for 2010, in thousands of persons.
3
1.2 Delayed Healing Diabetic Ulcers
The statistics above underline the fact that delayed wound healing and the chronic
ulceration that it causes is a major problem for diabetic individuals. Many other
disorders of the skin are associated with diabetes and these include:
Diabetic dermopathy – or skin spots that occur on the shins, thighs, forearms and lateral
malleolus;
Necrobiosis Lipoidica Diabeticorum - typified by thickened blood vessel walls and the
presence of extracellular lipids in the skin;
Diabetic Bullae – swellings due the accumulation of fluid in the cuticle;
Increased skin Candida albicans infection;
Neuropathy;
Thickening of the skin on the hands and feet; and
Scleroderma diabeticorum – a hardening of the skin. (Feingold et al. 1987).
In fact, the vast majority of lower limb amputations worldwide are attributed to a non-
healing ulcer on the skin. The major reasons for the delayed healing were initially
thought to be hypoxia, infection, lack of moisture and nutritional deficit (MacFarlane
and Jeffcoate 1997). However, the persistence of the impaired healing seen, even in
diabetic patients who achieve good metabolic control, reveals that delayed healing may
also be due to a number of cellular dysfunctions in diabetic patients’ skin keratinocytes
and underlying fibroblasts. Fibroblasts in diabetic skin are less sensitive to growth
4
factor stimulation, that is, larger doses of growth factors are needed to elicit wound-
healing functional effects in these cells compared to those derived from non-diabetic
people (Loots et al. 2002). Diabetic fibroblasts have also been shown to exhibit
impaired migration and growth factor secretion (Lerman et al. 2002). Microvascular
complications in diabetic patients, that may be due to abnormal synthesis of the
basement membrane by diabetic fibroblasts, lead to impaired binding of endothelial
cells to integrins on these membranes (Werthiemer et al. 2001).
The delayed healing phenomenon is not limited to the skin, as indicated by Barr and
Joyce (1989) who studied the delayed healing mechanism in diabetic endothelium. By
showing that impaired healing also occurred in the repair of microvascular
anastamoses, they demonstrated that the delayed healing pathology persisted
throughout multiple tissue types and is not localized to the epithelium. Further, Tyndall
et al. (2003) showed that a range of growth factors, including insulin-like growth factor
(IGF)-I and -II, platelet derived growth factor (PDGF), transforming growth factor
(TGF) β and fibroblast growth factor (FGF), were down regulated (protein expression)
in the healing of diabetic fractures compared to non-diabetic patients. Taken together
with the extensively characterized delayed dermal wound healing observed, these
findings suggest a systemic healing failure in diabetic patients persisting beyond the
skin into bones and blood vessels, at least.
5
The major manifestation of the delayed healing in diabetic patients is as foot and leg
ulcers. The reasons given for the initial presentation of foot ulcers in diabetic patients
are poor footwear, accidents, podiatric illness and foot surgery (Macfarlane and
Jeffcoate 1997). There are also many biological factors that contribute to the
presentation of such diabetic ulcers. Primary among them is neuropathy and
subsequent loss of tone in the muscles of the foot. This leads to the development of a
classic “clawed foot” appearance, typified by a high arch and curved toes. (1)
Peripheral neuropathy is an ongoing loss of sensation in the foot. Neuropathy leads to
insensitivity to poorly fitting shoes or injury as well as the continuation of walking on
severe wounds (Boulton et al. 2000). Other contributing factors are peripheral micro-
vascular occlusion and other macrovascular causes, including arthrosclerosis. (2) Lack
of blood flow reduces the availability of elements of the blood such as growth factors
and immune cells (Keyser 1992). The lack of these factors also encourages infection,
another major factor influencing the slow healing of diabetic ulcers. (3) Infection can
be treated with antibiotics, however, most topically applied antibiotics can have further
adverse effects including dehydration of the skin and destruction of fibroblasts (Keyser
1992). A further factor promoting the formation of chronic ulcers in diabetic patients is
an underlying dysfunction in the cellular wound healing processes themselves. The
dysfunction manifests as a lack of cytokine factors and an increase in proteolytic
activity in the wound microenvironment (Keyser 1992). The focus of most current
treatment strategies for delayed healing ulcers have dealt with the first three factors, for
6
example, by reducing pressure to the wound by immobilization with negative pressure
bandages or by controlling infection or maintaining a moist environment. However,
these therapies do not address the underlying impaired cellular healing mechanisms and
the lack of pro-healing chemokines reaching cells either via impaired local production
by fibroblasts or transport in the circulation.
1.3 Existing Therapeutics
Therapies such as “Dermagraft” have sought to solve the problem of impaired cellular
healing by adding exogenous cell populations (neonatal fibroblasts) to the wound area
in the hope that these cells will reinstate the ECM and cytokine production of the
impaired native fibroblasts (Mansbridge et al. 1999). However, clinical trials of
products such as these, some using cadaveric skin or expanded exogenous cell
populations, have shown only limited promise. In addition, these products are also
associated with the risk of contamination through the use of allogeneic, and often
xenobiotic, material ( for example, ApligfraftTM, Sibbald et al. 1998).
Another group of therapies have focused on specific replacement of growth factors
whose production is inhibited in fibroblasts, or that if secreted do not retain activity
long enough to exert paracrine effects (due to proteolytic degradation), or have become
hyper-glycosylated and therefore non-functional. To date, the United States Food and
Drug Administration (US FDA) has only approved one “growth factor therapy” for
7
clinical use in the treatment of diabetic ulcers; recombinant PDGF therapy. Some
promising results in humans have been seen in response to this product, including faster
healing and the formation of more granular tissue and thicker scarring (Sibbald et al.
1998). However, all the trials have been conducted in parallel with other treatment
strategies (that is, debridement, bed rest, antibiotics and negative pressure bandages)
and as such do not accurately dissect the actual effect of the growth factor
supplementation (Robson et al. 1998). Even so, this treatment is successful and is in use
clinically. The treatment uses multiple growth factors in the latest iteration.
Several groups have attempted to re-instate growth factor activity by the transfection of
target tissues with vectors expressing growth factors. These include; Galeano et al.
(2003) who used an adenovirus vector to express vascular endothelial growth factor
(VEGF) in the wound epithelium; Chesnoy et al. (2003) who transfected wound
keratinocytes with a plasmid expressing TGFβ; and Byrnes et al. (2001) who
transfected keratinocyte growth factor (KGF) into wounds with a similar plasmid. All
of these transgenic approaches have had success in accelerating wound closure in vivo.
However, the last group identified significant detrimental effects on cell metabolism
associated with the DNA transfection process in control and KGF expressing plasmids,
indicating that while efficacious, these strategies are poorly understood and therefore
are far from yielding safe and effective therapies.
8
Many other non-growth factor based therapeutics have also been employed with some
success in rodent diabetic wound models. These include crude extracts from
Lithosperm roots (Fujita et al. 2003), diferuloyl-methane (from Curcuma longa root)
(Sidhu et al. 1999), adenosine (Montesinos et al. 2002), angiotensin analogues
(Rodgers et al. 2003), neuropeptide substance P (Gibran et al. 2002) and leptin (Frank
et al. 2000). All of these therapies have had some success in the murine models
through either metabolic or anti-inflammatory activity.
1.4 An Inflammatory Wound Environment
Normal wound healing occurs in three general stages; inflammation, proliferation and
maturation/angiogenesis. Diabetic ulcers tend to stay stalled in the inflammatory stage
and never progress to the cell proliferation phase. In the normal wound, a range of
cytokines (including PDGF, IGFs, TGF, Interleukin (IL)-6 and IL-8) signal cells to
proliferate and encourage a different subset of immune cells to migrate into the wound
than those that are present in the inflammation stage. In diabetes the excessive
glycosylation of proteins and the altered phenotype of the high glucose exposed
fibroblasts and keratinocytes (Spravchikov et al. 2001), as well as the increased activity
of extracellular proteases, leads to a failure in the induction of the proliferative phase.
An example of this was discovered by Nissen et al. (1999) who showed that the
glycosylation of growth factors, such as FGF, leads to a significant decrease in their
activity with respect to receptor stimulation and therefore mitogenic effects in diabetic
9
wounds. Another relevant study by Clarke et al. (2001) dissected the ability of IGF and
EGF to move throughout diabetic patients’ circulation. Intriguingly, these tests showed
that the transport of IGF and EGF molecules in diabetic people was equal or better than
that of normal subjects following both oral and topical administration. This finding
demonstrates that exogenous growth factors administered to diabetic people are taken
into the system and are transported through the circulation as they would be in normal
subjects. This then raises the question of why these effectively transported molecules
do not elicit a wound healing response once at the site of injury.
Increased glucocorticoid (GC) expression as a result of diabetes has been proposed as
another mechanism by which growth factor (IGF-I) action in diabetic ulcers is inhibited
(Bitar 2000). Earlier, Bitar (1998) linked stress-induced changes in GC expression
associated with diabetes to an increase in inflammatory response and reduced white
blood cell migration and angiogenic activity. The study is particularly interesting since
all of these characteristics are highly typical of the delayed healing wound. Rosger et
al. (1995) showed that increased endogenous corticosterone levels also had a key role
in the development of IGF-I resistance in diabetes. They found that increases in
corticosterone caused a reduction in the level of circulating IGFBP-3 and IGF-I and that
insulin deficiency exacerbated these effects.
Further contributing to this prolonged inflammatory environment is dysfunction in
10
cytokine production by immune cells. Down regulation of IL-6 and Tumor Necrosis
Factor Alpha (TNFα), and a lack of leukocyte activity have been correlated with
persistent inflammation in diabetic wounds (Fahey et al. 1991). Further immunological
investigations by Wetzler et al. (2000) showed a persistence of macrophages and
neutrophils in diabetic wounds beyond the normal inflammation phase. Moreover, the
removal of over-active neutrophils has been shown to reverse the delayed healing
phenotype in diabetic mice (Dovi et al. 2003). One reason proposed for the persistence
of these cells and factors, may be altered expression of heat shock proteins (HSPs) that
mediate the activities of ILs and TNFα. Furthermore, Heat Shock Proteins (HSPs) are
expressed extremely late in experimental models of diabetic wound healing (McMurtry
et al. 1999). A lack of normal apoptotic activity at the wound edge has also been
implicated in the persistence of inflammation and delayed healing. Interestingly, this
delay has been reversed by the administration of IGF-II and PDGF to wounds in
diabetic mice (Brown et al. 1997).
1.5 Insulin-like Growth Factors IGF-I is the most abundant growth factor or cytokine found in acute wound fluid. It
occurs at approximately 20 - 40ng/mL; a level that is approximately half that found in
the circulation and IGFBP-3 is also present at approximately half its plasma
concentration (Vogt et al. 1998). Both IGF-I and -II are reduced in the diabetic wound
(Figure 1.2 from Brown et al. 1997) and the primary effect of this is thought to be
11
delayed migration of cells into the wound.
Figure 1.2 - IGF-I mRNA and protein expression after wounding in diabetic and
normal skin. From Brown et al. (1997)
Bitar (1996a) conducted the original study demonstrating that IGF-I supplementation
could reduce the impairment of healing seen in diabetic rat wounds. This researcher
found increases in collagen deposition, DNA and protein synthesis and the formation of
granulation tissue in response to topical administration of IGF-I to diabetic rat wounds.
These criteria were previously impaired in these wounds compared to non-diabetic rats.
Other studies have also shown that IGF-I in combination with IGFBPs (IGFBP-1 or -3)
can increase this enhanced healing effect (Muoller et al. 1991, Zhao et al. 1993). This
correlates with findings that IGFBPs are reduced in diabetic serum and wound fluid
(Wadlbilig et al. 1994) and is further supported by findings that systemic administration
IGF-I mRNA levels in diabetic and non-diabetic wounds over time as determined by RT-PCR. IGF-I mRNA was detected within days in normal mice whereas a significant delay was seen in the wounds of diabetic
IGF-I protein levels in diabetic and non-diabetic wounds over time as determined by RIA. The protein levels parallel findings for IGF-I mRNA expression.
12
of IGF-I and IGFBPs to diabetic rats experiencing delayed healing wounds can increase
the rate of healing (Bitar 2000). Indeed, an interesting study by Tsuboi et al. (1995)
showed that co-administration of IGFBP-1 and IGF-I had synergistic effects in
accelerating wound healing.
Bereket et al. (1996) showed that diabetic patients have reduced systemic expression of
the acid labile subunit (ALS) which facilitates the formation of a ternary complex
between IGF-I and certain IGFBPs. The study also showed an increase in insulin.
Although IGF-I would still be the rate-limiting factor in the formation of the ternary
complex at the levels detected in this study, these findings underlie the system-wide
changes among the IGF system elements that occur with diabetes. The differential
activity between normal and diabetic patient’s fibroblasts with respect to the IGF
system was further dissected by Giannini et al. (1994), who examined the expression of
IGFBPs in fibroblasts isolated from the skin of diabetic, obese and normal patients.
They found that IGFBP-2 and -3 were down regulated in both the diabetic and obese
patients fibroblasts compared to those from non-diabetic patients. Most interestingly,
they showed that IGFBP-5 expression was unchanged in obese patient-derived
fibroblasts, yet was down regulated in diabetic fibroblasts. This may point to IGFBP-5
having a key role in the terminal “dysregulation” of the healing processes in diabetic
patients.
13
1.6 Growth Factor Dysfunction
In normal healing, fibroblasts, macrophages and platelets associated with the wound
area secrete growth factors including IGF-I and IGFBPs into the wound environment.
Findings that diabetic patients have impaired IGF production from skin fibroblasts and
that inflammatory elements such as platelets and macrophages are impaired in their
action in diabetic wounds, highlights the importance of cytokines and growth factors in
the wound healing response (Stadelmann et al. 1998). Diabetic patients have
significantly reduced IGF-I, IGF-II and IGFBPs present in wound fluid (42%) and in
serum (48%). Furthermore, other growth factors such as Transforming Growth Factor
(TGF) β are also reduced by a similar amount (55%).
This reduction in growth factor expression is further exacerbated by an increase in
ECM protease activity (Bitar 1998). The gelatinases (MMP-2 and MMP-9) have a role
in normal healing, but are both over-expressed in the diabetic wound (Neeley et al.
2000). The research of Wall et al. (2002) further confirms that changes in matrix
metalloproteinases (MMPs) are strongly correlated with delayed healing. In addition
to these changes in proteases themselves, protease inhibitors, such as tissue inhibitors
of MMPs (TIMPs), have been shown to stimulate keratinocyte migration and stimulate
wound healing in the diabetic rat model (Terasaki et al. 2003), presumably by acting to
decrease the action of MMPs.
Many growth factors have been positively associated with improved healing, especially
PDGF, bFGF (Okumura et al. 1996), VEGF (Kirchner et al. 2003), TGFβ (Chesnoy et
14
al. 2003) and KGF. Werner et al. (1992) examined the role of KGF in wound healing
in rodents with experimentally induced diabetes. They showed a 150-fold increase in
KGF in the wound microenvironment 24 hours post wounding in normal mice. Diabetic
mice, however, showed a marked inhibition of this effect. FGF expression was also
studied and was found to occur earlier and for a shorter duration in diabetic wounds,
highlighting why improved healing are achieved by administration of this growth factor
(Bitar 2000). These findings indicate that the delayed and reduced expression of
growth factors, as previously established for IGFs (Brown et al. 1997), also occurs with
other growth factors involved in healing. However, many of the studies reporting that
growth factor treatments elicited increased epithelialisation and granulation tissue
formulation did not report improvements in the contractility of the healed skin.
Improvement in contractility is a feature of normal healing wounds (Greenhalgh et al.
1990, Albertson et al. 1993).
Combinations of growth factors have also been demonstrated to improve healing. Nath
and Gelati (1998) tested the ability of multiple growth factor combinations applied
sequentially and concurrently, for their ability to reverse delayed healing exhibited by
diabetic rats. They found administration of PDGF and IGF together resulted in an
improved rate of wound healing. They also tested the ability of other treatments in
combination with growth factor therapies and found that traditional debridement,
artificial skin bandages and electrical stimulation of the wound all contributed
positively in combination with the application of growth factors. These results suggest
that the efficacy of exogenous growth factors applied to delayed healing wounds is
15
highly dependent on the environment to which they are delivered and that multiple
endocrine and other factors are involved in the co-ordination of this process.
1.7 Vitronectin, IGF Complexes
The findings outlined above show that the regulation of IGF availability, such as via
binding proteins, is a key consideration in the use of IGFs to accelerate wound healing.
The identification of vitronectin (VN) as a novel IGF binding protein (Upton et al.
1999) and the further finding that IGFs and their traditional IGFBPs in complex with
VN can elicit functional effects on skin cells (Kricker et al. 2004, Hyde et al. 2005,
Hollier et al. 2005), taken together with the previously explained role of IGFs in wound
healing, led us to hypothesis that these complexes may have a role in remediating
delayed healing of diabetic wounds.
VN is a ubiquitous 75 kDa ECM protein found at high levels in the circulation and in
the peripheral ECM, including in the wound bed. Its primary role in normal physiology
is thought to be as an adhesive protein that binds to cells via surface integrins
containing the αv integrin subunit and thereby facilitates attachment, spreading and cell
migration, processes that are critical in wound healing (Schvartz et al. 1999). Jang et al.
(2000) examined the role of VN in wound healing in VN null mice. They found that
VN is important in two healing processes; the migration of cells into the wounded site
and in facilitating endothelial adhesion as part of the angiogenic response. These and
other findings highlighted in this chapter have led our research team to hypothesize that
in diabetes the increased glycosylation of proteins and protease activity inhibit the
16
ability of VN to mediate cell migration and attachment. Further evidence which
supports this hypothesis include the findings of Jang et al. (1999) who showed that the
αv integrins, vitronectin binding integrins, are crucial for the formation of new blood
vessels in wound healing.
ECM function is significantly impaired in the diabetic wound. Fibronectin (FN)
expression is down regulated in the diabetic wound (Fu et al. 2002). Furthermore, the
FN that is present persists longer (i.e. resists remodeling by proteases) (Loots et a.l
1998). A study undertaken by Grinnel et al. (1992) found that adhesive proteins such
as FN, and especially VN, are broken down in the diabetic wound fluid by proteolytic
activity. Furthermore, they found that the cell adhesive properties of such wound fluid
was greatly decreased compared to that of non-diabetic patients and that this was due
not only to destruction of adhesive proteins, but also, to expression of anti-adhesive
molecules. Moreover, Algenstaed et al. (2003) have shown that the impairment in the
growth of microvascular architecture in diabetic wounds is closely linked to the level of
hyperglycaemia in the wound environment. Further evidence in support of this
paradigm includes the work of Bobbink et al. (1997) who set out to determine why cell
adhesion and spreading were impaired in diabetic patients. They found that the primary
reason for these abnormalities was the glycosylation and subsequent loss of the
adhesive function of VN. These findings indicate that the extracellular environment is
highly altered in diabetic skin and that these alterations in ECM properties are
correlated with impairment in cell function, consistent with the observed properties of
delayed healing ulcers.
17
There are multiple findings that suggest ECM adhesive molecules, such as VN, have a
role in delayed healing in diabetic wounds via reduced angiogenic activity. As well as
alterations in the ECM, the reduced level of angiogenesis in delayed healing wounds
has also been attributed to a lack of growth factor expression, particularly VEGF and
FGF-2 (Colville-Nash et al. 1997). This was earlier shown by Cooper et al. (1994) who
demonstrated that reduced levels of many hormones, including PDGF, FGFs, EGF,
IGFs and TGF, in diabetic ulcer wound fluid also influenced angiogenesis. These
findings highlight the fact that there are multiple aberrations in cell and ECM function
that contribute to the delayed healing. The reduction in the expression of IGF-II found
by Cooper et al. (1994) is particularly relevant to this study. IGF-II, while being
expressed at a lower level than IGF-I protein in the adult human, seems to have a key
role in wound healing, where it is expressed at relatively high levels in the normal
dermis following injury (Brown et al. 1997). Interestingly IGF-II, unlike IGF-I, has
been reported to stimulate angiogenesis (Lee et al. 2000). These findings, coupled with
the known role of VN in angiogenesis suggested that the binding and thus, co-
ordination of these molecules may be of particular relevance in this process.
The differing properties of the normal wound environment compared to that
experienced by cells in delayed healing ulcers in diabetic patients has been a recurring
theme in studies that aimed to elucidate the underlying cause of non-healing lesions.
We have identified three key factors that impair wound healing that can be investigated
in combination with the effect of the novel IGF:IGFBP:VN complexes that we
18
hypothesize will be beneficial to wound healing. These are lack of oxygen due to
microvascular alterations, hyperglycemia and rapid differentiation as is controlled in
skin cells by calcium ion gradient.
1.8.1 Calcium and the Skin
The calcium ion and its binding proteins are intimately involved in the progression of
the cell cycle. Calcium ion concentration spikes, signaled from integrin-activated focal
adhesion complexes, as well as calcium receptors on the cell surface along with cAMP,
begin the cycle of cyclin dependent Protein Kinases (PKs) that in turn control
replication of DNA. Another calcium ion surge then triggers the mitotic prophase,
while then another surge at the end of metaphase triggers the destruction of prophase
PKs. Ca2+ also triggers cytoplasmic cell division. In the skin the role of Ca2+ is even
more crucial. Only a small amount of extracellular calcium is required to initiate DNA
replication in these cells and integrins and calcium receptors stimulate differentiation
and apoptosis at a set point somewhere above 1.0mM (Whitfield et al. 1995).
Calcium is a key mediator of skin metabolism and differentiation. In the skin there is a
gradient of calcium that exists from the deep basal cells where there is a low calcium
concentration to the upper cornified keratinocyte layers where calcium ion
concentration is relatively high. The exact concentrations of calcium in these layers is
not consistently reported, but is in the range 0.1 mM - 0.7 mM in the basal cell region
and 1.0 - 2.8 mM in the upper keratinocyte layer (Tsao et al. 1982, Sacks et al. 1985,
Al-Ani et al. 1988, Yuspa et al. 1988, Witfield et al. 1995, Landsdown 2002). Low
19
calcium concentration allows proliferation of the basal stem cell population and high
calcium induces differentiation in the upper keratinocyte layers. Interestingly, low
Ca2+ concentration also increases melanocyte growth in the basal region (Abdel-Naser
1999). This extracellular Ca2+ gradient translates to similar intracellular calcium ion
concentration changes as demonstrated by Tu et al. (2004), who showed that the
calcium ion concentration in the ECM mirrors the intracellular calcium concentration
and is a good predictor of cellular differentiation.
The localization of calcium ions in the cytoplasm and in the ECM is very tightly
regulated in space, frequency and amplitude (Missiaen et al. 2000). Calcium ions come
into the cell via various channels including the Na+ / Ca2+ exchanger and are extruded
from the cell by Ca2+ pumps, as well as by the exchanger. There are at least three other
transporters of calcium ions into the cell and they are calcium receptor (CaR),
Calmodulin and skin calmodulin related factor(s) (Hwang et al. 2005). Of particular
relevance to this study is CaR, a G-protein coupled receptor that is linked to
chemotaxis, proliferation and cell death (Riccardi et al. 1999). The importance of
calcium metabolism in the skin is demonstrated by the fact that mutations in Ca2+
pumps in epidermal keratinocytes and fibroblasts are the primary causes of major
genetic dermatological diseases such as Darrier and Hailey-Hailey diseases (Missiaen
et al. 2000). Skin calcium ion concentration is regulated by parathyroid hormone (via
CaR), calcitonin and vitamin D. There is a range of calcium binding proteins expressed
in the skin and these include S100, calmodulin, calbindin, cadherins and calpain
(Landsdown 2002).
20
Ca2+ mediated keratinocyte differentiation occurs via a pathway that includes effectors
such as tyrosine kinase and Protein Kinase C (PKC) (Denning et al. 2000). Another
mechanism reported to control skin cell differentiation via increased Ca2+ involves
Phosphatydl Inositol (PI) stimulating production of Di-acyl Glycerol (DAG) and
therefore increasing the activity of PKC leading to increased differentiation (Yuspa et
al. 1988). Ca2+ can also arrest cell growth via S100/A1 phosphorylation that results in
translocation to the nucleus and stimulates growth arresting transcription (Sakagucci et
al 2003). Interestingly calcium levels are low in the shed keratinocyte layer, indicating
the recycling of the cations in skin (Menon et al. 1985, 1994).
Unlike the well-defined effects of calcium on keratinocytes, differing reports on the
effect of Ca2+ concentration on dermal fibroblasts have been reported. For example,
the higher range of Ca2+ concentrations have been reported to stimulate MAP-kinase in
fibroblasts (via calmodulin and PKC) and increase fibroblast DNA synthesis (Hwang et
al. 2005) and pro-collagen expression (Huang et al. 1999). Varani (1998) showed that
fibroblasts require 1.0 mM or higher Ca2+ to grow in culture and this is consistent with
the physiological level in the skin. This contrasts the previously discussed findings that
the dermal Ca2+ concentration may be as low as 0.1mM in the peri-dermal region in
which fibroblast are found. Several other reports have confirmed that a relatively high
level of Ca2+ is required for the normal growth and maturation of fibroblasts (Kulesz-
Martin et al. 1984, Hovis et al. 1993, Weimann et al. 1999).
21
Higher levels of extracellular Ca2+ increase the proliferation of fibroblasts, but reduce
that of keratinocytes. No changes in migration are observed (Blair et al. 1988). It is
interesting to note that despite this high calcium in vitro paradigm, calcium antagonists
can improve the outcomes for several dermatologic diseases such as erythromelalgia,
idiopathic-related calcinosis cutis, primary and secondary Raynaud’s phenomenon,
chilblains, chronic anal fissures, keloids, and burn scars (Palaramis and Kyriakis 2005).
1.8.2 Calcium in Wound Healing
Unsurprisingly, calcium plays a role in many stages of wound healing. In the
inflammatory stage of healing, histamine is released and this leads to an increase in
available Ca2+ which activates phospholipases and protein kinases (Koizumi and
Ohkawara 1999). Cellular Ca2+ concentration spikes in the keratinocytes and then
undergoes a transient fall that may be associated with clotting, since calcium is factor
IV in the haemostatic cascade which follows injury to tissues and blood vessels. The
Ca2+ concentration then remains at a high level throughout the initial phases of wound
healing; that is, for up to 5 days (Koizumi and Ohkawara 1999).
Improved wound healing is observed when calcium is added to the wound via calcium
alginate dressings. However, the increased wound healing is functionally related to
increases in fibroblast metabolic activity (proliferation/protein synthesis), rather than
stimulation of keratinocytes (Doyle et al. 1996). This activity can be attributed to the
addition of exogenous calcium which can be absorbed pericutaneously in small
amounts in whole skin, but is absorbed more effectively in the wound since the skin
22
barrier function is removed (Landsdown 2002). Increases in migration of keratinocytes
into the wound are instead reported to be associated with reduced Ca2+ in the wound
environment, compared to the upper layers of unwounded skin (Grzesiak et al. 1995).
This indicates that in a wound bed, the increased Ca2+ concentration relative to the
basal layer stimulates increased protein synthesis and proliferation of the fibroblasts
from the dermis. Concomitantly, the reduction of Ca2+ concentration compared to the
upper layer increases the migration of keratinocytes, demonstrating the very fine
control of skin metabolism that changes in Ca2+ concentration can elicit.
1.8.3 Calcium and Diabetes
Calcium metabolism is systemically impaired in diabetic patients and is strongly
implicated in renal diabetic complications (Ling et al. 1995). Similarly, calcium
homeostasis disorders in the diabetic heart are a major cause of the cardiovascular
complications associated with diabetes (Solini et al. 2000). Tight regulation of Ca2+
concentration is important, as highlighted by the finding that deficiencies or imbalances
in calcium ion regulation are associated with non-diabetic skin disorders (Moynahan
1974, Landsdown et al. 1997, 2001). Similarly, calcium regulation associated with
wound healing is altered in the diabetic patient and may be associated with the delayed
healing of diabetic ulcers. For example, Somogyi et al. (2001) found that multiple
Ca2+transport systems are impaired in diabetic patients. Levy (1999) confirms that there
are defects in calcium metabolism in diabetic skin cells in particular and that these
defects result in increased intracellular calcium. Excesses in Ca2+ are confirmed as a
feature of delayed healing by Blair et al. (1988) who showed that while Ca2+ is reduced
23
in the proliferative phase of healing in normal wounds, Ca2+ persists at relatively high
concentration in this phase in chronic wounds. Counter-intuitively, wound healing in
diabetic patients is enhanced with the use of calcium alginate dressings that supplement
calcium in the wound (Lalau et al. 2002). These findings are reconciled when
considered in the context of the different effects of calcium reported between skin
fibroblasts and keratinocytes discussed earlier in this section.
Molecular evidence for changes in calcium metabolism in diabetic patients includes
findings that insulin reduces the Ca2+ influx into skin cells from the ECM and that
insulin resistance and diabetes retard this process. Conversely, a high level of calcium
can interfere with insulin signaling (Zemel et al. 1995). This is confirmed by the
findings of Trevisan et al. (1996) who showed that insulin treatment cannot increase the
intracellular routing of calcium from the extracellular environment. Further,
hyperglycemia causes increased cytosolic calcium concentration by inducing influx of
the ion and the mobilization of intracellular stores to the cytoplasm, as well as
decreased calcium exit from cells (Massry and Smogorsewski 1997) .
Calcium has also been shown to be a modulator of growth factor activity in the skin.
KGF and EGF stimulation of skin keratinocytes is abrogated at 1.0 mM Ca2+ (Marchese
et al. 1990). Increased Ca2+ concentration (above 1.5mM) in combination with EGF
administration to keratinocytes leads to phosphorylation of integrins and reduced
proliferation (Carey et al. 1992). Importantly for my studies, however, IGF-I and the
type one IGF receptor (IGF-1R) are detected at functional levels in skin keratinocytes at
24
both high and low concentrations of Ca2+ (Tavakkol et al. 1999). Interestingly, calcium
supplements are administered with Regranex (PDGF) treatment of diabetic lesions and
presumably improve the efficacy of this growth factor treatment (Tarronni et al. 2002),
although controlled data is not presented by the manufacturers to confirm this.
Of further relevance, Ca2+ concentration is also intimately linked to interactions
between cells and the extracellular matrix. Integrin adhesion motifs, such as RGDS
from vitronectin and DGEA from collagen, stimulate Ca2+ influx into the cell when
they associate with their integrin receptors (Mineur et al. 2005). This is unsurprising
given that calcium modulates integrin adhesion to vitronectin (Kirschofer et al. 1991)
and is involved in the Calcineurin (phosphatase 2B)-mediated ‘caterpillar-like’ traction
of cells migrating via integrin-VN dislocation and re-attachment at the leading edge of
migrating cells (Lawson and Maxfield 1995).
It is clear from these findings that Ca2+ plays an important role in the growth and repair
of skin and that abnormalities in the responses to, and metabolism of, calcium are
numerous within diabetic skin pathophysiology. Furthermore, the constituent proteins
of the IGF:IGFBP:VN complexes examined in this thesis are also dependent on, and
linked to, Ca2+ metabolism. Changes in Ca2+concentrations that are seen in the diabetic
wound could be reasonably expected to interact with any therapeutic, such as the
complexes tested in these studies, when administered to a wound.
25
1.9 Oxidative Stress
Oxidative stress is another suggested underlying mechanism of delayed healing via
altered redox enzyme metabolism. Glutathione, a key redox regulator, is down
regulated in delayed healing wounds in diabetic patients and the elderly (Rasik and
Shukla 2000), and administration of glutathione and other anti-oxidants has been
demonstrated to enhance the healing of these wounds (Galeano et al. 2000, Rasik and
Shukla 2000). Further studies by Mudge et al. (2002) showed that delayed healing due
to redox imbalance was caused by disruption of growth factor activity in this
environment. Similarly, Hehenburger et al. (1997) explored strategies to address the
so-called “high glucose growth factor resistance” in diabetic patient skin-derived
fibroblasts. They found that administration of anti-oxidants or protein kinase inhibitors
could reverse growth factor ‘resistance’ and restore fibroblast function. Rather than the
general environmental and metabolic roles of oxygen that are disrupted in wounding
per se these findings illustrate the role played by chemical and specific degradation of
protein factors in the extracellular milieu of the diabetic wound and role of oxidation in
these processes.
Of particular relevance to the diabetic wound, hypoxia reduces glycogen levels in skin
cells via a calcium ion mediated process and furthermore, administration of a calcium
chelating complex will reverse these changes in cells (Escourbet et al. 1986). Also
relevant to this study is the finding that hypoxia in the diabetic foot causes impaired
leukocyte bacteria killing and decreases the amount of underlying collagen that
interacts with endothelial cells when initiating angiogenesis and this is due to impaired
26
synthesis of collagen by fibroblasts (Davis 1987). Another interesting interrelationship
between calcium and hypoxia in wound healing is the finding that hypoxia induced
VEGF expression, which occurs via Hypoxia Inducible Factor – I (HIF-I), requires
Ca2+ as a cofactor (Salnikow et al. 2002). Similarly the amount of HIF-1 is reduced
associated with hyperglycemia in the diabetic wound compared to chronic ulcers in
non-diabetic patients (Catrina et al. 2004). These findings demonstrate that the response
of diabetic skin to wounding-induced hypoxia is not the same as that in normal cells.
Microvascular dysfunction in diabetic cells also complicates this since diabetic skin
cells may be permanently exposed to oxygen levels that normal cells would only
experience in wounding. Hence, the destruction of vasculature in the diabetic patient
via wounding may not present a sufficient environmental change for skin cells to
stimulate metabolic changes important for healing.
1.10 Insulin: Glucose Metabolism in Normal and Diabetic Wounds
Insulin also plays a role in the healing of wounds in normal skin by stimulating
fibroblast proliferation and collagen synthesis. Yet, even type 1 diabetic patients who
achieve strict control of insulin, by regulating diet and insulin injections, show impaired
wound healing. Contrary to this, however, Eshragi et al. (1995) found that insulin has
no effect on the repair of aortic endothelial wounds. Given that the ability of IGF to
exert its effect on keratinocytes is dependent on their differentiation state, Wertheimer
et al. (2001) contend that the lack of IGF activity to mediate wound closure seen in
diabetic wounds may be due to abnormal insulin receptor (IR) activity influencing the
differentiation of keratinocytes in diabetic patients. A lack of IR stimulation leads to
27
cells becoming less responsive to IGF stimulation due to a lack of type-1 IGF receptor
(IGF-1R) expression or due to insulin signaling through IGF-1R in order to compensate
for lack of signaling through IR. These findings are further supported by Spravchikov
et al. (2001) who showed that the high glucose environment as found in diabetic
tissues, leads to a Ca2+-mediated rapid differentiation of keratinocytes, and that in their
more differentiated form, these cells showed lower IGF-1R auto-phosphorylation.
Abnormal glucose metabolism is the defining feature of diabetes. The result of this is
systemic extracellular hyperglycaemia. Studies which examined glucose metabolism of
diabetic animal models have shown that glucose metabolism is altered in the skin of
diabetic rats through alterations in crucial metabolic intermediaries such as hexokinase,
lactose dehydrogenase, citrate synthase and glucose–6-P dehydrogenase (Gupta et al.
2005). Hyperglycaemia induces non-reversible changes in functional cellular
responses; for example, exposure to high levels of glucose has been shown to reduce
the contractile response of fibroblasts in vitro (Howard et al. 1996, Deveci et al. 2005).
Furthermore, restoration of normoglycaemic conditions fails to restore these cells to
normal function (Blazer et al. 2002). This suggests that while temporal control of
glucose metabolism can be maintained with insulin therapy or diet the effects of
extended periods of hyperglycemia on cells and proteins may well be irreversible.
Further evidence of defects in epidermal cell metabolism induced by hyperglycemia
include the finding that corneal epithelium adhesion and proliferation is significantly
reduced in high glucose (Mc Dermott et al. 1998) and the previously discussed findings
of Spravchikov et al. (2001) who showed changes in skin cell function were induced by
28
growth in high glucose culture conditions.
Several deleterious effects are correlated with hyperglycemia; that is, the severity of the
complications are proportional to the level of systemic glucose. Some examples of this
include the well-studied microvascular complications that are modeled in the diabetic
mouse. These microvascular abnormalities are exacerbated by hyperglycemia and the
severity of ischemia is correlated with blood glucose level (Anglstead et al. 2003). A
molecular basis for this dysfunction may be that the glycation of FGF leads to a
significantly reduced cellular action on diabetic endothelial cells and that this is linked
to decreased angiogenesis in the diabetic wound (Duraisamy et al. 2001). Furthermore,
Nathan et al. (2005) demonstrated that improved glucose control reduced the risk of
both micro- and macrovascular disease. Similarly, Home et al. (2005) showed that a
doubling of HbA1c ( a measure of glucose clearance) was correlated with a ten-fold
increase in microvascular disease.
Systemic hyperglycemia is a response to wounding in the normal patient and is also
observed in patients with moderate and severe burns immediately following wounding
(in the blood and wound exudates). However, the level of hyperglycemia observed in
these patients is positively correlated with increased healing time and increased
mortality (Holm et al. 2004). Circulatory hyperglycemia, as is observed in the diabetic
patient, is preserved as hyperglycemia in these patients’ wound fluid. Furthermore,
levels of blood glucose are proportional to the healing time of wounds and to the level
of hyperglycemia in the wound fluid (Lu et al. 2005). The reverse also seems to be true
29
,as demonstrated by Furnary et al. (2004), who showed that surgical wounds have been
shown to heal faster in patients who had their blood glucose reduced prior to wounding.
As well as having elevated glucose in the blood and interstitium, which may impede the
action of factors within the wound fluid, wound fluid from diabetic patients has been
shown to have specific proteolytic activities. Among the most relevant of these is the
finding that diabetic wound fluid degrades insulin (Duckworth et al. 2004). These
changes in proteolytic action of wound fluid are also linked to glucose level in some
cases. For example, elevated glucose has indirect influence on the healing of wounds
via increasing MMP-9 in diabetic epithelium (i.e. MMP-9 level correlates with
hyperglycemia). In terms of vascularisation of the healing skin, it has been shown that
high glucose can inhibit the migration of endothelial cells in the skin capillary. This
phenomenon is linked to glucose affecting NFκB and thus eNOS and nitric oxide, both
of which are key mediators of angiogenesis (Hamuro et al. 2002).
1.11 IGF-I and Insulin-Glucose Metabolism
As well as having homology with proinsulin, IGF-I is interlinked with insulin and
glucose metabolism. Insulin stimulates IGF-I secretion from the liver and regulates
IGFBP-3 production (Baxter 1990). Similarly as a primary effector of the Growth
Hormone (GH) mediated growth axis, IGF-I stimulates mitogenesis in a range of
tissues. Indeed, GH reduces insulin sensitivity (Zierler and Rabinomitz 1963) in the
diabetic patient. Systemic administration of IGF-I stimulates hypoglycemia (Zenobi et
al. 1992) and this may occur by down regulating GH. This reduction in sensitivity may
also may be due to direct action of IGF-I on the insulin receptor or hybrid insulin IGF-I
30
receptors, a mechanism that is very poorly understood. However, these studies also
found that in the non-IGF-I-treated cases, IGF-I is reduced in diabetic patients and this
leads to increased GH due to the lack of positive feedback via IGF-I and further
exacerbates insulin insensitivity (Maes et al. 1986). In view of this, IGF-I has been a
candidate treatment for diabetes in the past. Clinical trials, however, revealed that a
range of deleterious side effects manifest in patients treated with systemic IGF-I and
hence IGF-I is not a viable insulin replacement candidate. Interestingly, a study has
shown that these side effects are significantly ameliorated by the co-administration of
IGF-I with IGFBP-3 (Clemmons et al. 2000, 2001).
Aside from interactions with the liver / GH axis in stimulating insulin production or
increasing insulin sensitivity, IGF-I clearly has independent effects on glucose
metabolism (Simpson et al. 2001). This is confirmed by the fact that IGF-1R
phosphorylation stimulates the IRS-1 as well as tyrosine kinase (TK), and is further
demonstrated by the ability of IGF-I to stimulate glucose uptake by a Phosphoinositide-
3 Kinase (P-I-3K) dependent mechanisms. Ranke et al. (2005) have shown that the
mechanism by which glucose metabolism is stimulated by IGF-I may differ from those
detailed for metabolism stimulated by insulin; specifically that insulin induces the
glucose transporters GLUT-1 and -5, whereas IGF-I induces GLUT-2 and -3 in the
skin. In fact, IGF-I increases glucose metabolism and decreases liver glucose
production, as well as increasing protein metabolism and the lypolysis of non-esterified
fatty acids via the IR in adipocytes (Bolinder et al 1987). Furthermore, IGF-I causes a
reduction of tri-acyl-glycerol, lipoprotein and cholesterol in adipocytes (Oscarsson et
31
al. 1995).
Primary fibroblasts derived from diabetic ulcers have been reported to have decreased
detectable IR and IGF1-R (Chisalita and Arnqvist 2004) while diabetic derived
endothelial cells have reduced IR but increased IGF1-R. This has been confirmed
morphologically, at least for fibroblasts, by Solini et al. (2000) who showed that skin
fibroblasts grown in high glucose conditions undergo ATP mediated phenotypic
changes and an increased rate of apoptosis. Similarly Rai et al. (2005) have observed
that hyperglycemia increases apoptosis in skin cells from diabetic ulcers.
Some functional changes in diabetic skin, though, seem to be independent of glucose
level. For example, the finding that collagen deposition in the skin is impaired in type 1
and type 2 diabetes, however, glycemic control and blood glucose appears to be
irrelevant in the aetiology of this reduced collagen deposition (Black et al. 2003). This
data should be considered in the context of the previously discussed findings that
periodic hyperglycemia causes irreversible changes in diabetic skin cells.
1.12 Excessive Glycosylation
Excessive glycosylation affects proteins in the ECM that are exposed to elevated
glucose in their microenvironment. Interestingly, one of the most relevent ECM
proteins to this study that undergoes this modification is VN. Hammes et al. (1996)
showed that the pathology of diabetic retinopathy is linked to the modification of VN
by glycosylation. The process of modification creates advanced glycation end products
32
(AGEs). The formation of AGEs involves extensive glycosylation of amino acids
followed by further reactions that produce irreversible ketone derivatives from the
glycosylated proteins (Figure 1.3). Conversion of proteins into glycation end products
greatly reduces the biological activity of the converted proteins. In the case of VN, this
process retards the ability of cells to attach to, and thus migrate on AGE-VN substrates,
compared to native VN.
Figure1.3 A.G.E. FORMATION PATHWAY (from Brownlee et al. 1992)
Brownlee et al. (1992) showed that the AGE-modified ECM proteins (including VN) in
the basement membrane interact differently with cells. The administration of inhibitors
of AGE formation, such as amino guanidine, inhibit the development of diabetic
pathologies such as delayed healing, retinopathy and microvascular disease in vivo. A
specific VN based alteration in association with the basement membrane was also
Formation of Advanced Glycation End-products (AGEs) from glucose. Reversible early products can give rise to irreversible advanced products through generation of highly reactive carbonyl compounds such as 3-deoxy-0-glucosone. Reductase enzymes may retard A.G.E. formation in vivo.
33
elucidated in this study. Furthermore, the ability of AGE-VN to bind heparin sulphate
proteoglycans (HSPGs) is greatly reduced. HSPGs play an important role in the
regulation of growth factor: ECM interactions, especially with respect to the
extracellular presentation of growth factors and particularly bFGF. Another interesting
finding in this study was that macrophages, an immune cell type associated with the
persistence of the inflammatory response, have a receptor specific for AGE modified
proteins and that ligand binding to this receptor stimulates altered IL-1, TNFα and IGF-
I expression. This finding fits well with the general axiom that failure of the healing
response corresponds to a persistent inflammatory response that impairs the re-
epithelialisation process and with the finding that hyperglycemia correlates with the
severity of healing impairment. The specific receptor for AGEs (RAGE) has been
identified. In fact there are multiple AGE adducts and multiple RAGE receptors that
have ligands other than AGE adducts only. Interestingly, this is the first receptor that
differentially recognizes the glycosylation states of its protein ligand. Of particular
interest, RAGE is up regulated in the wound healing process of diabetic bone injury
(Santana 2003), which, as discussed earlier, is also impaired. Furthermore, Goova et al.
(2001) demonstated the important role that the AGE-RAGE interactions can play in
delayed healing. They showed that blockade of RAGE reversed delayed healing in rats
with experimental diabetes, back to a healing time similar to that found with normal
control rodents.
34
1.13 Other Diabetic Pathologies
Both the IGF system, and VN have been identified as contributing to other diabetic
pathologies. For example, Price et al. (1997) conducted studies on elements of the IGF
system (i.e. IGFs and IGFBPs) in diabetic rat nephropathy. They found expression of
IGF-I and IGFBPs was increased in diabetic rats compared to normal, further
demonstrating that up regulation of multiple IGF system components is associated with
this pathology. Reduced VN protein expression in the glomerulus has also been
associated with increased nephropathy (Yoon et al. 2001). Furthermore, Feldman et al.
(2000) showed that although IGF and IGFBPs are still expressed in the diabetic retina,
the level of bio-available IGF in the eye is much lower than in the non-disease state, as
evidenced by lack of activity in the tissues. Marano et al. (1995) studied the relative
expression of VN and other ECM adhesion proteins in the diabetic retina. Their study
also examined the expression of the cellular receptors for these proteins, integrins.
They found that increased, and locally altered, expression of VN, fibronectin and
laminin and their integrin receptors all contributed to the aberrant growth of retinal
capillaries that typifies diabetic retinopathy. Conversely, another study, published in a
more obscure journal, by Esser et al. (1994) suggests that diabetic retinopathy may be
linked to a reduction in vitronectin expression.
1.14 Growth Factors and ECM as a Strategy to Enhance Wound Healing
As can be seen from the wide scope of research examining epithelial repair, the
mechanisms behind delayed healing are complex and interrelated. However, a
commonality seems to emerge in all the research and that is impairment of growth
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factor co-ordination. Whether this is due to failure to respond to hypoxia, altered
glucocorticoid dynamics, oxidation stress, excess extracellular glucose, systemic
hyperglycaemia, rapid differentiation of cells, insulin imbalance, uncontrolled glycation
or proteolysis of proteins, the delayed healing pathology is consistently linked back to a
failure of growth factors which in turn affects cells in the wound.
Growth factors act to enhance wound healing in a number of ways. Primarily they
promote cells to migrate into the wound, promote cellular proliferation and
angiogenesis, encourage further immune cells into the wound site and promote the
synthesis of new ECM. Many, if not all, healing processes are directly or indirectly
linked to ECM-mediated activities. Diabetic skin, however, appears to be impaired in
terms of its ECM interactions, possibly due to excess protease expression (Harding et
al. 2002).
As introduced earlier, VN:IGFBP:IGF complexes consist of growth factors bound to
VN. As such VN:IGFBP:IGF complexes have adhesive ECM substrate molecules, VN,
incorporated. It could be hypothesized that this facilitates immediate chemotaxis in
response to growth factors that chemokines alone cannot induce (Millington et al.
2000). The importance of ECM factors is highlighted by Livant et al. (2000) who
administered a peptide cell binding sequence from FN (PHSRN) to delayed healing
wounds. They showed that this fragment alone could significantly increase cell
migration into the wound and the rate of wound contraction in diabetic mice.
Previously, Lee et al. (1998) showed that IGF and IGFBP co-administration was also
36
able to increase contraction in a cardiac fibroblast-embedded collagen gel model.
Contraction is an important parameter in healed wound quality and has been obtained
using treatments that contain molecules with adhesive protein sequences such as
PHSRN (Livant et al. 2000) or the RGD sequence in IGFBP-1 which is also found in
VN. Importantly these studies suggest that administering cell binding ECM molecules
restores wound contraction, the property conspicuously absent from growth factor-
induced healing acceleration detailed in multiple studies described above.
Given that VN also acts to present IGFs to cells, the increased glycosylation of VN in
wounds may also represent a mechanism by which IGF function is impaired in healing.
VN:IGFBP:IGF complexes could represent a mechanism to address this impairment by
providing an exogenous source of normally glycosylated VN. Furthermore, research
conducted by Galiano et al. (1996) showed important links between the IGF system and
cell adhesion via integrins. They found that IGF-I binding to IGFBP-1, and IGFBP-1
in turn binding to the cell surface integrin αvβ5 (a VN receptor), were both required to
regulate wound healing in vivo. This demonstrates an explicit link between growth
factors and cell adhesion systems. Our laboratory’s finding that VN, the ligand for αv
integrins, also binds directly to IGF-II and indirectly to IGF-I via IGFBPs (International
Patent Application WO 02/242219 A1) further highlights the link between these
systems. Thus, VN:IGFBP:IGF complexes enable growth factors to be presented to
cells in a manner that may be more biologically relevant. At the same time the
complexes supplement adhesive proteins that have been degraded or rendered useless
due to environmental changes in the diabetic wound. As such, VN:IGFBP:IGF
37
complexes may prove to be particularly appropriate for wound healing, and especially
useful for the delayed healing state in diabetic patients. It is important though to
appreciate that skin expresses a wide range of growth factors and integrin adhesion
elements (Cole et al. 2001). Thus the ability to reinstate the delicate balance of growth
factors and ECM elements is likely to be beyond the reach of the current research
proposed here. However, a therapeutic designed in recognition of the importance of
growth factor and ECM signal co-ordination, such as VN:IGFBP:IGF complexes, is
likely to have greater potential than other therapeutics that are not yet addressing this
critical interplay of biological factors. In addition recent research within our team has
demonstrated that complexes can be formed which incorporate several growth factors at
the same time. (Hollier et al. 2005) This again, highlights that it may be possible to
develop a growth factor-ECM therapeutic that restores function to many of the
elements that have been altered in the diabetic wound environment and lead to a better
understanding of IGF biology if diabetic wound healing.
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1.15 Aims and Hypotheses
The aim of this project is to examine the potential of VN:IGFBP:IGF complexes to
stimulate parameters relevant to wound healing, in vitro, in cells derived from normal
patients and from diabetic ulcers.
In general, we hypothesize that the given the roles of VN and IGFs in wound healing,
that VN:IGFBP:IGF complexes stimulate the proliferation, migration and growth in
challenged conditions of skin cells derived from diabetic ulcers via mechanisms that
involve binding of IGF to IGF-1R and VN to αv-integrins and, further, that these
responses are retained in the presence of high concentrations of glucose and calcium.
Specifically we hypothesise:
- That the attachment, protein synthesis and migration responses to VN:IGFBP:IGF
complexes reported in the HaCAT keratinocyte cell line will be observed in
keratinocytes cultured from non-diabetic patient skin.
- That similar attachment, metabolic activity and migration responses to
VN:IGFBP:IGF complexes will be observed in fibroblasts cultured from non-diabetic
patient skin.
- That similar attachment, metabolic activity and migration responses to
VN:IGFBP:IGF complexes will be observed in fibroblasts cultured from diabetic
patient skin.
- That similar attachment, protein synthesis and migration responses to
39
VN:IGFBP:IGF complexes will be observed in keratinocytes cultured from diabetic
patient skin.
- That the mechanisms underlying the functional responses predicted in hypotheses 1-4
are facilitated by VN binding to αv- integrins and IGF-I binding to IGF-1R.
- That the responses detailed in hypotheses 1-4 are retained when differentiation of
skin cells is induced by culture in high concentrations of calcium.
- That the responses detailed in hypotheses 1-4 are retained when cells are cultured in
high concentrations of glucose, modelling systemic hyperglycaemia in the diabetic
patient.
CHAPTER 2
Materials and Methods.
41
2.1 Keratinocyte/Fibroblast isolation and culture
Ethics approval was obtained, after significant delays, for the provision of skin samples
obtained from type-2 diabetic patients from the Princess Alexandra Hospital Ethics
Committee as well as from QUT University Human Ethic Committee. Consent was
gained from patients by nursing staff and surgeons prior to amputation surgery or
cosmetic surgery. Skin samples from normal (non-diabetic) patients were derived from
excess skin resulting from cosmetic surgery. It is important to note that all primary
cultured skin cells were from adult donors, not foetal or neonatal derived skin. This is
in contrast to many studies in skin which are performed using these non-adult sources
for cells. Split thickness skin grafts were transported from surgery in Serum Free DME
Media (SFM) (Invitrogen, Mt Waverly, Victoria, Australia) containing 1% Antibiotic
Antimycotic (ABAM) Solution (Invitrogen) and 3 µg/mL Gentamicin (Invitrogen).
Skin samples from diabetic ulcers were obtained by removal of the nearest non-necrotic
tissue adjacent to the ulcer following amputation of a limb that was due to the non-
healing ulcer. Full thickness skin and subcutaneous tissue was transported from
surgery in the SFM with ABAM and Gentamicin solution. Upon arrival at the
laboratory the diabetic samples were dissected until they were of the same thickness as
a split thickness skin graft. That is, any attached adipose or muscle tissue was removed
and the dermis was then shaved with a scalpel until the darker coloration of the
epidermis was visible but not exposed. These ‘pared down’ samples were then treated
identically to the normal skin samples.
42
Both types of skin sample were then washed in SFM with 1% ABAM and 1 µg/mL
gentamycin. Samples were then washed twice for 10 min in SFM with 1% ABAM
Solution. Split thickness sections of skin were then digested overnight in
trypsin/EDTA (0.25%) (Invitrogen) to separate the epidermal and dermal layers. The
opposing faces of the separated skin layers were then scraped and the harvested cells
were washed in culture medium supplemented with 10% foetal bovine serum (FBS)
(Thermo Trace, Melbourne, Victoria, Australia). Cultures were then established and
propagated in the presence of gamma irradiated mouse 3T3 cells (i3T3; ~7 x 104/cm2)
(American Type Culture Collection #CCL-92). Greens’ culture medium (Reinwald and
Green. 1975) was used to culture these cells and consisted of DMEM/F12 medium
(Invitrogen) supplemented with 10% FBS (Thermo Trace), insulin (1 μg/ml) (Sigma
Aldrich), epidermal growth factor (10 ng/ml)(EGF) (Sigma Aldrich, Australia), adenine
(180 μM) (Sigma Aldri