In vitro examination of vitronectin, insulin-like growth factor ......In vitro examination of...

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


Cultured in Hyperglycemic Media……………………………………………139


Cultured in Differentiation Media……………………………………………142

6.2.5 Proliferation of Keratinocytes Derived From Normal Skin

Cultured in Normal Media…………………………………………………....144




Cultured in Differentiation Media………………………………...………….146

6.2.8 Proliferation of Fibroblasts Derived From Diabetic Ulcer Skin

Cultured in Normal Media……………………………………………………150





6.2.11 Proliferation of Keratinocytes Derived From Diabetic Ulcer Skin

Cultured in Normal Media……………………………………………………156





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

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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

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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

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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.

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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.

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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

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