Adenosine Receptors and Endothelial Cell Mediated Wound Healing

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Adenosine Receptors And Endothelial Cell Mediated Wound Healing

Author Bonyanian, Zeinab

Published 2017

Thesis Type Thesis (PhD Doctorate)

School School of Medical Science

DOI https://doi.org/10.25904/1912/1773

Downloaded from http://hdl.handle.net/10072/367912

Griffith Research Online https://research-repository.griffith.edu.au

Adenosine Receptors And Endothelial Cell Mediated Wound Healing

Zeinab Bonyanian BSc, MSc

School of Medical Science Griffith Health Griffith University

Submitted in fulfilment of the requirements of the degree of Doctor of Philosophy

February 2017

STATEMENT OF ORIGINALITY

This work has not previously been submitted for a degree or diploma in any

university. To the best of my knowledge and belief, the thesis contains no

material previously published or written by another person except where due

reference is made in the thesis itself.

Zeinab Bonyanian (Zina)

ACKNOWLEDGEMENT

Firstly, I would like to express my sincere gratitude to my principal advisor,

Associate Professor Roselyn Rose’Meyer, for the continuous support of my

Ph.D study and related research, for her patience, motivation, and immense

knowledge. Her guidance helped me during both the research and writing of

this thesis. I could not imagine having a better supervisor and mentor for my

Ph.D study. I would also like to acknowledge Associate Professor Joss Du

Toit, my associate supervisor, and Professor David Shum for their support.

I am very thankful to Dr. Janet Hussein for providing me with professional

guidance and useful advice on my thesis writing. Many thanks to my friend

Ms. Lana Bivol for her guidance and support especially during my lab work.

Also, special thanks to Ms. Mina Kohandel Shirazi for her emotional support.

I am sincerely grateful to my parents, Akbar Bonyanian and Fereshteh

Heshmaty, and my little sister (Boshra), for their unconditional love,

motivation, encouragement and financial support. I am deeply indebted to

them for being beside me through stressful and difficult times, assisting and

encouraging me to persue my dreams.

My last but foremost appreciation is to my dearest husband, Mr. Morteza

Ziaeirad, who joined my life from the 3rd year of my PhD journey. He has

given me unconditional love, patience, encouragement, time and support

during this journey. I am truly thankful for having him in my life.

ABSTRACT

Impaired wound healing is a common and serious complication of diabetes

mellitus causing an increased risk of infection and tissue damage. Chronic

wounds associated with diabetes may be caused by peripheral neuropathy,

vascular complications and alterations in immune function and contributes to

non-traumatic amputations. Consequently, therapeutic management of wounds

in diabetes is centred on infection control and stimulating revascularization.

This research will focus on the latter and investigate the effects of high

glucose on endothelial function with a view of finding new strategies to

improve wound healing in diabetes. This project identified the adenosine

receptor subtypes in EA.hy926 endothelial cell lines that are involved in

stimulating wound healing and determined whether their roles are affected in

the hyperglycaemic environment associated with diabetes. As caffeine is a

non-selective antagonist of the adenosine receptors, this study also

investigated the effects of acute and 48 hour chronic caffeine treatments on

adenosine receptor populations and on wound closure and cell proliferation.

Caffeine has a crucial role in wound healing through various mechanisms such

as antioxidant activity or increasing the level of cyclic adenosine

monophosphate (cAMP). Therefore, it is hypothesised that a chronic caffeine

treatment could speed up wound healing in endothelial cells through up

regulation of adenosine receptor subtypes or increased levels of cAMP. Also,

4 it should be noted that any truly new tests done- showing your research is very innovation and has not been done before.

The aim of this research was to determine the effect of selective adenosine A1,

A2A and A2B receptor agonists on the rate of wound healing and cell proliferation in EA.hy926 endothelial cells in a high glucose condition (4500 mg/L or 25 mM) and a low glucose condition (1000 mg/L or 5.5 mM). This study also investigated the effects of acute caffeine, on the speed of wound closure, angiogenesis and proliferation of EA.hy926 endothelial cells, using the wound healing scratch assay, proliferation assay and tube formation assay.

Moreover, the effects of 48 hour caffeine treatment (chronic treatment) on the speed of wound healing and proliferation of EA.hy926 endothelial cells were presented using the wound healing scratch assay, proliferation assay expect tube formation assay. Note that the tube formation assay was not used for chronic caffeine treatment as the tube formation assay required 18 hours pre- incubation and the EA.hy926 endothelial cells did not survive a further 48 hour caffeine treatment. Finally this study compared the effect of a 48 hour caffeine treatment on adenosine receptor subtype protein expression in a high glucose condition, using the Western blot assay.

The results have demonstrated that under a high glucose condition, the selective adenosine A1, A2A and A2B receptors agonists, namely 30 nM CPA,

CGS21680 and NECA, significantly increased the rate of wound healing by

42.29%, 49% and 20.57%, respectively in EAhy926 endothelial cells compared to untreated cells. Also, the results showed that selective agonists of the adenosine A1 and A2B receptors have high efficacy in increasing the rate of wound closure under a high glucose condition. EAhy926 endothelial cell

5 proliferation was significantly stimulated by the selective adenosine A1 and

A2B receptor agonists, CPA and NECA. Furthermore, the selective adenosine

A1, A2A and A2B receptor antagonists, 10 nM DPCPX, ZM241385 and

MRS1754, reversed the effects of their agonists and decreased the rate of wound healing. The protein levels of the adenosine A2A and A1 receptors were highly expressed when compared to the adenosine A2B receptor in the

EAhy926 endothelial cell lines in a high glucose condition.

Acute caffeine did not affect the rate of wound healing or cell proliferation of

EA.hy926 endothelial cells. However, caffeine blocked the effects of selective adenosine A1, A2A and A2B receptor agonists and decreased the rate of wound healing in EA.hy926 endothelial cells. Nevertheless, caffeine treatment increased angiogenesis by stimulating tube formation in EA.hy926 endothelial cells. As this work has been completed on cells in a high glucose environment, acute caffeine may have a role in stimulating angiogenesis in diabetic patients.

Moreover, the 48 hour caffeine treatment significantly elevated the rate of wound healing in EA.hy926 endothelial cells when compared with untreated and acute caffeine treatment groups. The effect of chronic caffeine was attenuated by the adenylyl cyclase inhibitor MDL-12330A. Therefore, chronic caffeine treatment may alter cAMP signalling pathways in EA.hy926 endothelial cell lines. The protein expression of adenosine A1 and A2B receptors did not significantly change with chronic caffeine treatment in

EA.hy926 endothelial cell lines but the expression of adenosine A2A receptor in the chronic caffeine treatment was considerably lower than the untreated group.

The rate of wound healing in a low glucose condition was significantly accelerated with selective adenosine A1, A2A and A2B receptor agonists in

EA.hy926 endothelial cells when compared to a high glucose condition.

Furthermore, under a low glucose condition, the efficacy of selective adenosine A1, A2A and A2B receptor agonists and antagonists on EA.hy926 endothelial cell wound healing was greater than their effects under a high glucose condition. However, EA.hy926 endothelial cell proliferation was decreased in the low glucose condition. Moreover, these experiments show that EA.hy926 endothelial cells are not able to remain viable and functional in a low glucose condition after 48 hours. Cell morphology and confluency changes were observed after 48 hour cell growth in EA.hy926 endothelial cells under the low glucose conditions.

Consequently, this hypothesis has been approved that the rates of wound healing in EA.hy926 endothelial cells were increased in chronic caffeine treatment.

In conclusion, there may be a potential therapeutic role for synthetic analogues of adenosine to increase wound healing in the diabetic patient. Furthermore, chronic caffeine also elevated wound healing rates during hyperglycaemic conditions. An effect that appears to be a separate mechanism from its ability to antagonise adenosine receptors.

TABLE OF CONTENT

STATEMENT OF ORIGINALITY ...... 2

ACKNOWLEDGEMENT ...... 3

ABSTRACT ...... 4

TABLES ...... 14

FIGURES ...... 15

LIST OF ABBREVIATIONS ...... 20

LIST OF PUBLICATIONS AND CONFERENCES ...... 22

CHAPTER 1: LITERATURE REVIEW ...... 24

1.1. Significance ...... 24

1.2. Definition of Diabetes and classification ...... 28

1.3. Complications of diabetes ...... 30

1.3.1. Diabetic Atherosclerosis ...... 30

1.3.2. Diabetic Nephropathy ...... 30

1.3.3. Diabetic Retinopathy ...... 30

1.3.4. Diabetes-Impaired Wound healing ...... 31

1.4. Skin wound healing process ...... 32

1.4.1. Inflammatory Phase ...... 32

1.4.2. Proliferative Phase ...... 33

1.4.3. Maturation Phase ...... 34

1.5. Angiogenesis ...... 34

1.6. Adenosine ...... 35

1.6.1. Adenosine Metabolism ...... 37

1.6.2. Adenosine Receptors ...... 39

1.6.3. Signalling pathways of the adenosine receptors ...... 42

1.6.4. Association of adenosine receptors with wound healing ...... 44

1.7. Chemical Structure of Caffeine ...... 45

1.7.1. Caffeine Consumption ...... 47

1.7.2. Caffeine Absorption ...... 47

1.7.3. Caffeine Metabolism ...... 48

1.7.4. Caffeine and Ryanodine Receptors ...... 49

1.7.5. Various Effects of Caffeine ...... 49

1.7.6. Caffeine and Wound Healing ...... 51

1.8. Association of caffeine with diabetes and effect on Adenosine receptors ...... 52

1.9. Cytokines ...... 54

1.10. Conclusion ...... 59

1.11. Hypotheses AND AIMs ...... 59

CHAPTER 2: METHODOLOGY ...... 63

2.1. Human endothelial hybrid cell line (EA.hy926) ...... 63

2.1.1. Cell Culture Technique ...... 64

2.1.2. Cell Passaging ...... 65

2.1.3. Cryopreservation (Cell Freezing) ...... 66

2.1.4. Cell Counting ...... 67

2.2. The Scratch Wound Healing Assay ...... 69

2.2.1. Data Collection from the Wound Healing Scratch Assay ...... 70

2.3. Cell Proliferation Assays ...... 73

2.4. Western Blotting Technique ...... 79

2.4.1. Sample Preparation ...... 79

2.4.2. Cell Lysis ...... 79

2.4.3. BCA Assay ...... 81

2.4.4. Western Blot Assay ...... 83

2.4.5. Preparation of Gel Electrophoresis ...... 84

2.4.6. Electrotransfer Of Proteins Onto Membrane ...... 86

2.4.7. Membrane Blocking And Antibody Incubation ...... 87

2.4.8. Data Collection From Western Blot Assay ...... 88

2.5. Tube Formation Assay (Angiogenesis) ...... 91

2.5.1. Data Collection Of The Tube Formation Assay ...... 93

CHAPTER 3: ROLE OF ADENOSINE A1, A2A AND A2B RECEPTOR IN WOUND HEALING ... 96

3.1. Introduction ...... 96

3.1.1. Adenosine ...... 96

3.1.2. Signalling pathways of adenosine receptors in wound healing ...... 97

3.1.3. The Role of Adenosine A1 Receptor Agonists in Wound Healing ...... 97

3.1.4. The Role of Adenosine A2a and A2b Receptor Agonists in Wound Healing ...... 98

3.1.5. The Role of Adenosine A3 Receptor Agonists in Wound Healing ...... 99

3.2. Aims ...... 101

3.3. Methods and materials ...... 101

3.3.1. Preparation of Adenosine Receptor Agonists And Antagonists ...... 101

3.3.2. Wound Healing Scratch Assay ...... 103

3.3.3. Cell Proliferation Assay ...... 103

3.3.4. Western Blotting Technique ...... 106

3.4. Results ...... 107

3.4.1. The Effect Of Selective Adenosine Receptor Agonists On EA.hy926 Endothelial Cells Using

The Wound Healing Scratch Assay ...... 107

3.4.2. Wound Healing Scratch Assay Using The Selective Adenosine A1 Receptor Agonist ...... 109

3.4.3. Wound Healing Scratch Assay Using The Selective Adenosine A2A Receptor Agonist ...... 112

3.4.4. Wound Healing Scratch Assay Using The Selective Adenosine A2B Receptor Agonist ...... 115

3.4.5. The Effect Of Selective Adenosine Receptor Antagonists On Agonist Induced Increases In

Rates Of Wound Closure ...... 118

3.4.6. Effect Of Selective Adenosine Receptor Agonists And Antagonists On Cell Proliferation ...... 120

3.4.7. Protein Expression Of Adenosine Receptor Subtypes On EA.Hy926 Cells ...... 122

3.5. Discussion ...... 125

3.6. Conclusion ...... 130

CHAPTER 4: EFFECT OF ACUTE CAFFEINE ON ADENOSINE RECEPTOR MEDIATED

WOUND HEALING ...... 133

4.1. Introduction ...... 133

4.1.1. Acute caffeine ...... 133

4.2. Aims ...... 137

4.3. Methods and materials ...... 137

4.3.1. Treatment preparations (drugs) ...... 137

4.3.2. Wound healing scratch assay ...... 138

4.3.3. Cell proliferation assay ...... 140

4.3.4. Tube Formation Assay ...... 142

4.4. Results ...... 145

4.4.1. The Effect Of Caffeine Treatment On Adenosine Receptor Mediated Rates Of Wound Closure

In Ea.hy926 Endothelial Cells ...... 145

4.4.2. The Role Of Adenylyl Cyclase On Caffeine Treatment And Rates Of Wound Closure In

Ea.hy926 Endothelial Cells ...... 148

4.4.3. The Effect Of Caffeine On Adenosine A1, A2A And A2B Receptor Mediated Cell Proliferation 152

4.4.4. The Effect Of Acute Caffeine Treatment On Angiogenesis And Tube Formation In EA.hy926

Endothelial Cells ...... 154

4.5. Discussion ...... 157

4.6. Conclusion ...... 159

CHAPTER 5: EFFECT OF CHRONIC CAFFEINE ON ADENOSINE RECEPTOR MEDIATED

WOUND HEALING ...... 162

5.1. Introduction ...... 162

5.1.1. Chronic Caffeine ...... 162

5.2. Aims ...... 164

5.3. Methods and materials ...... 165

5.3.1. Treatment Preparations (Drugs) ...... 165

5.3.2. Wound Healing Scratch Assay ...... 167

5.3.3. Western Blotting Technique ...... 167

5.4. RESULTS ...... 168

5.4.1. The Effect Of Selective Adenosine Receptor Agonists On Rate Of Wound Closure In Ea.hy926

Endothelial Cells Following Chronic Caffeine Treatment ...... 168

5.4.2. Effect Of Chronic Caffeine Treatment On Rates Of Wound Closure In Ea.Hy926 Endothelial

Cells; Role Of cAMP ...... 172

5.4.3. Comparing The Effect Of Chronic And Acute Caffeine Treatment On The Rate Of Wound

Healing In EA.hy926 Endothelial Cells ...... 175

5.4.4. Expression Of The Adenosine Receptor Subtypes On EA.hy926 Endothelial Cells Following

Chronic Caffeine Treatment ...... 177

5.5. Discussion ...... 182

5.6. Conclusion ...... 185

CHAPTER 6: EFFECT OF LOW GLUCOSE CONCENTRATION ON ADENOSINE RECEPTOR

MEDIATED WOUND HEALING ...... 187

6.1. Introduction ...... 187

6.1.1. Diabetes Mellitus and Wound Healing ...... 187

6.1.2. Adenosine Receptors and Diabetes ...... 188

6.2. Aims ...... 189

6.3. Methods and materials ...... 190

6.3.1. Treatment preparations (drugs) ...... 190

6.3.2. Wound healing scratch assay ...... 190

6.3.3. Cell proliferation assay ...... 191

6.4. Results ...... 194

6.4.1. The effect of selective adenosine A1 receptor agonist and antagonist on rate of wound closure

in the low glucose condition ...... 194

6.4.2. The effect of selective adenosine A2A receptor agonist and antagonist on rate of wound

closure in the low glucose condition ...... 197

6.4.3. The effect of selective adenosine A2B receptor agonist and antagonist on rate of wound

closure in the low glucose condition ...... 200

6.4.4. Effect of adenosine agonists and anagonists on cell proliferation in the low glucose condition

...... 203

6.4.5. Comparing the effect of adenosine A1 receptor agonist and antagonist on the rate of wound

healing in low and high glucose conditions ...... 205

6.4.6. Comparing the effect of adenosine A2A receptor agonist and antagonist on the rate of wound

healing in low and high glucose conditions ...... 207

6.4.7. Comparing the effect of adenosine A2B receptor agonist and antagonist on the rate of wound

healing in low and high glucose conditions ...... 209

6.4.8. Comparing the effect of adenosine A1, A2A and A2B receptor agonists and antagonists on

EA.hy926 endothelial cell proliferation in low and high glucose conditions ...... 212

6.5. Discussion ...... 214

6.6. Conclusion ...... 216

CHAPTER 7: DISCUSSION ...... 218

7.1. General discussion ...... 218

7.2. Conclusion ...... 227

CHAPTER 8: REFERENCES ...... 229

APPENDIX 1 ...... 250

...... 251

TABLES

TABLE 1.6.1. ADENOSINE RECEPTORS CLASSIFICATION AND FUNCTIONS...... 41

TABLE 2.4.1.STANDARD SAMPLE PREPARATION FOR THE BCA STANDARD CURVE...... 82

TABLE 2.4.2.MATERIALS REQUIRED FOR THE SDS-PAGE ELECTROPHORESIS GELS ...... 85

TABLE 2.4.3. SHOWING EXAMPLE CALCULATIONS OF NORMALIZED DATA ...... 90

TABLE 3.3.1. TREATMENT CONDITIONS AND THE CONCENTRATIONS ...... 105

TABLE 4.3.1. PREPARATION OF THE ACUTE CAFFEINE TREATMENT ...... 139

TABLE 4.3.2. ACUTE CAFFEINE TREATMENT CONDITIONS AND CONCENTRATIONS ...... 141

TABLE 4.3.3. ACUTE CAFFEINE TREATMENT CONDITIONS AND CONCENTRATIONS ...... 144

TABLE 5.3.1. PREPARATION OF DRUG TREATMENTS FOR THE WOUND HEALING SCRATCH

ASSAY ...... 166

TABLE 6.3.1. TREATMENT CONDITIONS AND THEIR CONCENTRATIONS ...... 193

FIGURES

FIGURE 1.6.1.THE CHEMICAL STRUCTURE OF ADENOSINE ...... 36

FIGURE 1.6.2. ADENOSINE METABOLISM IN THE INTRACELLULAR AND EXTRACELLULAR ..... 38

FIGURE 1.7.1.CHEMICAL STRUCTURE OF CAFFEINE ...... 46

FIGURE 1.9.1. FIGURE SHOWING THE ROLE OF PRO-INFLAMMATORY CYTOKINE RESPONSES IN

199 WOUND HEALING . INTERLEUKIN-8 (CXCL8), INTERLEUKIN-1α (IL-1α),

INTERLEUKIN-1β (IL-1β), (C-C MOTIF) LIGAND 2 (CCL2), VASCULAR ENDOTHELIAL

GROWTH FACTOR (VEGF), TRANSFORMING GROWTH FACTOR-Β (TGF-β), TUMOUR

NECROSIS FACTOR (TNF) AND PLATELET-DERIVED GROWTH FACTOR (PDGF) ...... 58

FIGURE 2.1.1. THE FIGURE IS SHOWING GRID LINES OF THE HAEMOCYTOMETER CHAMBER. .. 68

FIGURE 2.2.1.GRAPH SHOWING THE EFFECT OF ADENOSINE RECEPTOR AGONIST (30NM) OR

ANTAGONIST (10NM) ON THE RATE OF WOUND CLOSURE (ΜM/HOUR) OVER TIME

(HOUR). THE CONTROL GROUP HAD NO DRUG TREATMENT...... 72

FIGURE 2.3.1.THE CHEMICAL STRUCTURE OF WST-8 IN CCK-8 AND FORMATION OF ITS

221 FORMAZAN DYE ...... 74

FIGURE 2.3.2. A STANDARD CURVE FOR THE CCK8 CELL PROLIFERATION ASSAY AT 450 NM

ABSORBANCE...... 78

FIGURE 2.4.1.IMAGE SHOWING AN EXAMPLE OF MEASURING DENSITY OF THE PROTEIN BAND

...... 89

FIGURE 2.5.1. IMAGE SHOWING AN EXAMPLE OF MONITORING THE TUBE FORMATION ...... 94

FIGURE 3.4.1.CONCENTRATION RESPONSE CURVES (CRC) SHOWING THE EFFECT OF

SELECTIVE ADENOSINE RECEPTOR AGONISTS ON EA.HY926 ENDOTHELIAL CELLS AND

THE RATE OF WOUND HEALING, N= 6 PER GROUP ...... 108

FIGURE 3.4.2.WOUND HEALING SCRATCH ASSAY USING DIFFERENT CONCENTRATIONS OF

CPA ...... 110

FIGURE 3.4.3. GRAPH SHOWING THE RATE OF THE WOUND HEALING WITH INCREASING CPA

...... 111

.FIGURE 3.4.4.WOUND HEALING SCRATCH ASSAY USING DIFFERENT CONCENTRATIONS OF

CGS21680 (CGS) ON EA.HY926 ENDOTHELIAL CELLS ...... 113

FIGURE 3.4.5.GRAPH ILLUSTRATING THE RATE OF THE WOUND HEALING WITH INCREASING

CGS21680 CONCENTRATIONS COMPARED TO THE CONTROL...... 114

FIGURE 3.4.6.WOUND HEALING SCRATCH ASSAY USING DIFFERENT CONCENTRATIONS OF

NECA ...... 116

FIGURE 3.4.7.GRAPH SHOWING THE RATE OF THE WOUND HEALING WITH INCREASING NECA

CONCENTRATIONS COMPARED TO THE CONTROL. DATA IS REPRESENTED AS MEAN ± SE,

N= 6, *P < 0.05, **P < 0.01, *** P < 0.001 AND **** P < 0.0001 VERSUS CONTROL (0)

...... 117

FIGURE 3.4.8. GRAPH SHOWING THE EFFECT OF ADENOSINE RECEPTOR AGONIST, ANTAGONIST

AND THEIR COMBINATION ON RATE OF WOUND HEALING ...... 119

FIGURE 3.4.9.GRAPH SHOWING THE EFFECT OF DIFFERENT TREATMENTS ON CELL

PROLIFERATION ...... 121

FIGURE 3.4.10. WESTERN BLOT IMAGES SHOWING PROTEIN BANDS ...... 123

FIGURE 3.4.11. GRAPH DEMONSTRATING THE RELATIVE EXPRESSION OF ADENOSINE A1, A2A

AND A2B RECEPTOR SUBTYPES ON E.AHY926 ENDOTHELIAL CELLS. THE DATA WAS

ANALYSED USING ONE WAY ANOVA. DATA IS EXPRESSED AS MEAN ± SEM (N=6), *P <

0.05, **P < 0.01 AND *** P < 0.001 ...... 124

FIGURE 4.4.1.IMAGES SHOWING THE WOUND HEALING SCRATCH ASSAY IN THE ABSENCE

(PANEL 1) AND PRESENCE OF ACUTE CAFFEINE (30 ΜM, PANEL 2) ...... 146

FIGURE 4.4.2. GRAPH COMPARING THE EFFECTS OF ADENOSINE RECEPTOR AGONISTS ON THE

RATE OF WOUND HEALING IN UNTREATED (DARK BLUE BARS; CONTROL, CPA,

CGS21680 AND NECA (30 NM)) AND CAFFEINE (30 µM) TREATED (LIGHT BLUE BARS;

CONTROL, CPA, CGS21680 AND NECA (30 NM)) EA.HY926ENDOTHELIAL CELLS.

TWO-WAY ANOVA WAS USED TO ANALYSE DATA. DATA ARE PRESENTED AS MEAN ±

SEM, N= 6. **** P < 0.0001 ...... 147

FIGURE 4.4.3. THE EFFECT OF VARIOUS TREATMENTS ON EA.HY926ENDOTHELIAL CELLS IN

THE WOUND HEALING SCRATCH ASSAY AT 0 AND 6 HR ...... 150

FIGURE 4.4.4. GRAPH SHOWING THE EFFECT OF ACUTE CAFFEINE, MDL-122330A AND

FORSKOLIN TREATMENTS ON THE RATES OF THE WOUND HEALING ...... 151

FIGURE 4.4.5. GRAPH COMPARING THE EFFECT OF ACUTE CAFFEINE ON ADENOSINE RECEPTOR

MEDIATED CELL PROLIFERATION AS INDICATED BY ABSORBANCE FOR: CONTROL GROUP

(NO DRUG) (CTRL); ADENOSINE A1 RECEPTOR AGONIST (CPA 30 NM); ADENOSINE A2A

RECEPTOR AGONIST (CG21680 30 NM); AND ADENOSINE A2B RECEPTOR AGONIST

(NECA 30 NM). THE DATA ARE PRESENTED AS MEAN ± SEM, N= 6 AND ANALYSED

USING TWO-WAY ANOVA, **** P < 0.0001 ...... 153

FIGURE 4.4.6. IMAGES SHOWING THE TUBE FORMATION ASSAY ...... 155

FIGURE 4.4.7. THE EFFECT OF DRUG TREATMENTS ON TUBE FORMATION IN EA.HY926

ENDOTHELIAL CELLS FOR: CONTROL (DMEM); ACUTE CAFFEINE (30 µM); POSITIVE

CONTROL (25% BFGF); AND NEGATIVE CONTROL 0.3% SURAMIN. DATA ARE

PRESENTED AS MEAN ± SEM, N= 12. DATA WERE ANALYSED USING TWO-WAY ANOVA

*P < 0.05, **P < 0.01, *** P < 0.001 AND **** P < 0.0001 VERSUS CONTROL (DMEM)

...... 156

FIGURE 5.4.1. IMAGES SHOWING THE EFFECTS OF SELECTIVE ADENOSINE RECEPTOR

AGONISTS ...... 170

FIGURE 5.4.2. GRAPH COMPARING THE EFFECTS OF ADENOSINE RECEPTOR AGONISTS ...... 171

FIGURE 5.4.3. IMAGE SHOWING EFFECT OF MDL-12330A (10 ΜM) AND FORSKOLIN (3 ΜM)

ON WOUND HEALING RATES FOLLOWING CHRONIC CAFFEINE TREATMENT IN

EA.HY926ENDOTHELIAL CELLS AT 0 AND 6 HOURS USING WOUND HEALING SCRATCH

ASSAY...... 173

FIGURE 5.4.4. GRAPH SHOWING THE EFFECT OF THE 48 HOURS CAFFEINE PRE-TREATMENT (P,

TOP PANEL) OR UNTREATED CELLS (U, LOWER PANEL) ON RATES OF WOUND HEALING

AND THE EFFECT OF MDL-12330A AND FORSKOLIN (VERSUS THEIR RESPECTIVE

CONTROL GROUP).DATA ARE PRESENTED AS MEAN ± SEM, N= 6, ***P < 0.001, ****P <

0.0001 VERSUS CONTROL ...... 174

FIGURE 5.4.5.GRAPH COMPARING THE EFFECT OF ACUTE CAFFEINE (30 µM) ON CHRONIC

CAFFEINE TREATED CELLS (48 HOURS) AND NO CAFFEINE PRE-TREATMENT ON RATE OF

THE WOUND HEALING...... 176

FIGURE 5.4.6. WESTERN BLOT IMAGES SHOWING PROTEIN BANDS OF ADENOSINE RECEPTORS

...... 179

FIGURE 5.4.7. HISTOGRAM SHOWING THE EXPRESSION OF ADENOSINE RECEPTOR SUBTYPES

...... 180

FIGURE 5.4.8. GRAPH SHOWING A COMPARISON OF THE PROTEIN EXPRESSION OF ADENOSINE

RECEPTOR SUBTYPES ...... 181

FIGURE 6.4.1. IMAGES SHOWING WOUND HEALING SCRATCH ASSAY IN LOW GLUCOSE

CONDITIONS ...... 195

FIGURE 6.4.2. GRAPH SHOWING THE EFFECT OF SELECTIVE ADENOSINE A1 RECEPTOR AGONIST

(CPA 30 NM), ANTAGONIST (DPCPX 10 NM) AND ITS COMBINATION (CPA+ DPCPX)

ON RATE OF WOUND HEALING IN EA.HY926 ENDOTHELIAL CELLS IN THE LOW GLUCOSE

CONDITION, ...... 196

FIGURE 6.4.3. IMAGE SHOWING THE WOUND HEALING SCRATCH ASSAY IN THE LOW GLUCOSE

...... 198

FIGURE 6.4.4. GRAPH SHOWING THE EFFECTS OF SELECTIVE ADENOSINE A2A RECEPTOR .... 199

FIGURE 6.4.5.IMAGE SHOWING THE WOUND HEALING SCRATCH ASSAY IN THE LOW GLUCOSE

...... 201

FIGURE 6.4.6.GRAPH SHOWING THE EFFECTS OF SELECTIVE ADENOSINE A2A RECEPTOR ..... 202

FIGURE 6.4.7. GRAPH SHOWING THE EFFECT OF DIFFERENT TREATMENTS ON CELL

PROLIFERATION IN THE LOW GLUCOSE CONDITIONS, ...... 204

FIGURE 6.4.8. GRAPH COMPARING THE EFFECT OF SELECTIVE ADENOSINE A1 RECEPTOR .... 206

FIGURE 6.4.9. GRAPH COMPARING THE EFFECT OF SELECTIVE ADENOSINE A2A RECEPTOR .. 208

FIGURE 6.4.10.GRAPH COMPARING THE EFFECTS OF SELECTIVE ADENOSINE A1 RECEPTOR . 211

FIGURE 6.4.11.GRAPHS COMPARING THE EFFECT OF SELECTIVE ADENOSINE A1, A2A AND A2B

RECEPTOR ...... 213

LIST OF ABBREVIATIONS

CPA N6 -cyclopentyladenosine CCPA 2-Chloro-N6-cyclopentyladenosine (S)-ENBA (2S)-N6-[2-endo-Norbornyl]adenosine R-PIA R-N6 -(phenylisopropyl)-adenosine CHA N6- cyclohexyladenosine CGS-21680 2-[p-(2-carbonyl-ethyl)-phenylethylamino]-5’-N- ethylcarboxamidoadenosine ATL-146e 4-{3-[6-amino-9-(5-ethylcarbamoyl-3,4-dihydroxy-tetrahydro-furan-2- yl)-9H-purin-2-yl]-prop-2-ynyl}-cyclohexanecarboxylic acid methyl ester PAPA-APEC 2-(4-[2-[(4-aminophenyl) methylcarbonyl]ethyl]phenyl)ethylamino-5’- N-ethylcarboxamidoadenosine NECA 5’-N-ethyl-carboxamidoadenosine BAY-60-6583 2-[[6-Amino-3,5-dicyano-4-[4-(cyclopropylmethoxy)phenyl]-2- pyridinyl]thio]-acetamide LUF5834 2-Amino-4-(4-hydroxyphenyl)-6-[(1H-imidazol-2-ylmethyl)thio]-3,5- pyridinecarbonitrile IB-MECA N6 -(3-iodobenzyl)adenosine-5’-N-methyluronamide C1-IB-MECA 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methyluronamide APNEA N6-2-(4-aminophenyl) ethyladenosine DPCPX 1,3-dipropyl-8-cyclopentylxanthine WRC-0571 8-(N-Methylisopropyl)amino-N-(5’-endohydroxy-endonorbornyl)-9- methyladenine N-0840 N6-Cyclopentyl-9-methyladenine ZM-241, 385 4-[2-(7-Amino-2-(2-furyl)[1,2,4-triazolo[2,3-a] [1,3,5]triazin-5-ylamino]ethyl phenol SCH58261 5-Amino-7-(β-phenylethyl)-2-(8-furyl)pyrazolo(4,3-e)-1,2,4- triazolo(1,5-c)pyrimidine CSC 8-(3-chlorostyryl)caffeine KW6002 (E)-1,3-Diethyl-8-(3,4-dimethoxyphenylethyl)-7-methyl-3,7-dihydro- 1H-purine-2,6-dione MRS1754 8-[4-[[(4-Cyano)phenylcarbamoylmethyl]oxy]phenyl]-1,3-di-(n- propyl)xanthine MRE2029-F20 N-Benzo[1,3]dioxol-5-yl-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7 -tetrahydro-1H- purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy]-acetamide CGS15943 9-Chloro-2-(2-furanyl)-[1,2,4]triazolo[1,5-c]quinazolin-5-amine BW A-1433 1,3-dipropyl-8-(4-acrylate)phenylxanthine MRS1191 3-Ethyl 5-benzyl 2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)- dihydropyridine-3,5-dicarboxylate MRS1220 9-chloro-2-(2-furanyl)-5-[(phenylacetyl)amino][1,2,4]-triazolo[1,5- c]quinazoline MRS1523 2,3-Diethyl-4,5-dipropyl-6-phenylpyridine-3-thiocarboxylate-5- carboxylate MRE3008-F20 5N-(4-methoxyphenylcarbamoyl)amino-8-propyl-2-(2-furyl) pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidine

IL-1 Interleukin-1

IL-6 Interleukin-6

CXCL8 Interleukin-8 IL-10 Interleukin-10 IL-12 Interleukin-12 TNF-α Tumour necrosis factor-α PDGF Platelet-derived growth factor EGF Epidermal growth factor TGF-β Transforming growth factor-β CCL2 (C-C motif) ligand 2 VEGF Vascular endothelial growth factor bFGF2 Basic fibroblast growth factor ECM Extracellular matrix components HbA1c Glycated haemoglobin A1c levels CAM Chick chorioallantoic membranes TSP-1 Thrombospondin-1 ATP Adenosine triphosphate, ADP Adenosine diphosphate cAMP Cyclic adenosine monophosphate SAH S-adenosylhomocysteine 5’-AMP 5’-adenosine monophosphate ENT1 Equilibrative nucleoside transporter 1 CD39 Nucleoside triphosphate phosphohydrolase CD73 Ecto-5’-nucleotidase DAG Diacylglycerol MAPK Mitogen-activated protein kinase PLC Phospholipase C CHO Chinese hamster ovary cells PKA Protein kinase A PLC Phospholipase C IP3 1,4,5-trisphosphate EA.hy926 Permanent human endothelial hybrid cell HUVEC Primary human umbilical vein endothelial cells DMEM Dulbecco’s Modified Eagle’s Medium FBS Foetal Bovine Serum DPBS Dulbecco’s Phosphate-Buffered Saline DMSO Dimethyl sulfoxide PMSF Phenylmethanesulfonyl fluoride NaPP Sodium Pyrophosphate BCA Bicinchoninic acid assay BSA Bovine serum albumin SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

LIST OF PUBLICATIONS AND CONFERENCES

Bonyanian, Z and Rose’Meyer, R. Caffeine and its potential role in

attenuating impaired wound healing in diabetes, November 2015, 5(4):

141-148. doi:10.1089/jcr.2015.0011, number JCR-2015-0011, Journal of

caffeine research

Bonyanian, Z., Slevin, M., Rose'Meyer, R and Ahmed, N. (2013) Glycation

of monomeric C-reactive protein is associated with impaired wound

healing in diabetes, The Gold Coast Health and Medical Research

Conference

Bonyanian, Z and Rose’Meyer, R. (2015) Effect of acute and chronic

caffeine on cell proliferation and wound healing assays, The Gold Coast

Health and Medical Research Conference, Chronic Diseases – Preventing and Improving Health Outcomes

CHAPTER 1

LITERATURE REVIEW

CHAPTER 1: LITERATURE REVIEW

1.1. SIGNIFICANCE

Many people experience some type of injury or wound during their life. In the

majority of cases, wounds heal in a simple and rapid process, though this can

leave noticeable scars on the skin. In some cases wound healing may be

delayed or impaired due to health factors, leading to complications that

adversely affect the patient’s quality of life. For instance, impaired wound

healing is a common and serious complication of diabetes mellitus. It causes

dysregulation of the process at multiple levels, increasing the risk of infection

and tissue damage 1. Chronic wounds associated with diabetes may be caused

by peripheral neuropathy, vascular complications and alterations in immune

function and increases the risk of non-traumatic amputations 1-3. Some stages

of the normal wound healing processes are disturbed in diabetes such as

changes in the bactericidal activity of granulocytes, the excessive

accumulation of extracellular matrix basement membrane and decreased tissue

perfusion 1, 2. Consequently, therapeutic management of wounds in diabetes is

essentially focused on infection control and stimulating revascularization 1, 2.

Despite this knowledge, amputation rates still remain unacceptably high in

diabetic patients with impaired wound healing. Hence, recent studies have

focussed on finding new strategies to improve wound healing in diabetes 1, 2.

Adenosine is a purine nucleoside that can bind four receptor subtypes; A1, A2A,

A2B and A3. Adenosine mediates most of its intracellular effects through the

regulation of the second messenger system adenylyl cyclase and cyclic

adenosine monophosphate (cAMP) production 4-11. Adenosine can induce

wound healing through increased angiogenesis and deposition of the collagen 24 together with stimulation of extracellular matrix production in the wound site

4-11. Several studies have reported that adenosine and adenosine receptor agonists can increase proliferation and cell migration, which promotes wound

3, 12, 13 healing . Animal model studies have shown that adenosine A2A receptor agonists increase dermal wound closure in healthy rats and in streptozotocin- induced diabetic rats with wound healing complications 14, 15. Therefore, the adenosine A2A receptor, by accelerating wound healing, could be important for diabetic patients with impaired wound healing and microvascular complications 16. While adenosine may appear to be beneficial with the process of wound healing, there is controversy regarding the protective role of adenosine in tissue repair. Alternatively, another study reported that the deposition of collagen as a main function of wound healing, may lead to pathological remodelling of organs such as the kidney and heart, which causes significant morbidity and mortality 17. This study found that adenosine signalling, mainly via adenosine A2A and A2B receptors, contributed to the pathogenesis of fibrosis 17. Although, adenosine receptors may decrease inflammation and accelerate wound healing, loss of these receptors can lead to disruption of the organ structure and the loss of its functional integrity in different type of fibrosing disorders 17. For instance, following hypoxia, stimulation of the adenosine A2B receptors causes the expression of renal endothelin-1 (ET-1), which increases the proliferation of mesangial cells and collagen production in the kidney 17-19. Furthermore, activation of adenosine

A2B receptors may modulate the release of ET-1 and Interleukin-6 (IL-6) in interstitial lung disease to cause deposition of collagen, vascular remodeling

25 and eventually pulmonary hypertension. Consequently, the adenosine A2B receptor is a common feature for inducing fibrosis of these two organs 17, 20.

Common sources of caffeine include tea, coffee, cocoa beans and cola nuts.

Caffeine is now also being added to beverages such as soft drinks and energy drinks, bottled water, chocolates, chewing gum and medications 21-25. Several studies have been reported that caffeine consumption has beneficial and protective effects on various diseases such as chronic liver disease, Type 2 diabetes and Parkinson’s disease 26-29. A study observed that the consumption of caffeinated beverages such as coffee and tea decreased the risk of type 2 diabetes in humans 30. Furthermore, another study showed that coffee intake of more than two cups daily was associated with lower alanine aminotransferase levels, which decreases the risk of chronic liver disease and reduce liver fibrosis 29, 31, 32. Topical coffee powder has been used as a wound covering in humans to treat acute and chronic wounds that occur in diabetes mellitus, sharp cuts and burns 33. As a result, this study determined that coffee improved the wound healing in the patients with all type of wounds. It had many advantages such as its bactericidal activity, antioxidant properties, deodorizing effects, allowed longer times to change the wound dressing, maintained a moistened wound, increased absorptive capacity, enhanced autolytic debridement, was cost effective and caused no adverse reactions 33.

Furthermore, caffeine consumption can decrease the risk of Type 2 diabetes 34,

35. Caffeine has been reported to have many different effects in cells that may account for its wound healing properties. Studies have confirmed that antioxidants may accelerate wound closure 36-38. Caffeine stimulates a variety of cellular and pharmacologic reactions in the central nervous system such as

26 stimulation of motor activity and antioxidant activity 36-38. According to reports, caffeine can enhance the release of Ca2+ from internal Ca2+ stores via activation of ryanodine receptor 39, 40.

Caffeine is a non-selective antagonist of adenosine receptors 6. Through blocking adenosine receptors, caffeine decreases fibrosis and collagen deposition in the lung and liver 41-44. Caffeine also has low affinity for the adenosine receptor subtypes being more effective in a high dose caffeine treatment (25 mg/kg per day) compared to a lower dose of caffeine (2.5 mg/kg per day) in the rat ventral prostate model 42. Moreover, an in vivo study using mice, showed that caffeine not only blocked the adenosine A2A receptor but also acted as a cAMP phosphodiesterase inhibitor 45. As a result, caffeine inhibits intracellular cAMP stimulation through antagonising adenosine A2A receptors, however, it increases immunosuppressive cAMP accumulation by preventing its degradation when it acts as a phosphodiesterase inhibitor to reduce inflammatory responses 45. Studies have reported that chronic caffeine treatment up-regulates adenosine A2A receptors to increase the anti- inflammatory property of adenosine and decrease the liver damage in mice 45-

47. Also, the brain damage associated with ischaemic episodes can be attenuated by chronic caffeine intake 45, 48-50. Marangos and colleagues (1984) reported that chronic caffeine treatment may have different effects on brain adenosine receptor subtypes according to the duration of caffeine treatment 51.

Their animal study, with 11, 16 and 23 days of chronic caffeine treatment, found that after 11 days of treatment, the number of adenosine receptors were significantly changed compared to the control conditions; at 16 days chronic caffeine treatment the number of adenosine receptors significantly increased

only in the cerebellum and brain stem regions 51. Subsequently, after 23 days

treatment, the numbers of adenosine receptors increased significantly in all

regions of the brain 51. Overall, this study found that the level of adenosine

receptor subtypes in adult animals following chronic caffeine treatment was

increased 20-30% in comparison with control models 51. Overall, the effects of

caffeine on wound healing could be altered if given acutely or chronically.

This thesis will consider the different effects of chronic and acute caffeine on

the wound healing and the potential role for this compound’s use in diabetes.

Please note that sections of this review have been published in “Caffeine and

its potential role in attenuating impaired wound healing in diabetes”

Bonyanian Zeinab and Rose'Meyer Roselyn B. Journal of Caffeine Research.

November 2015, 5(4): 141-148. doi:10.1089/jcr.2015.0011. (Appendix 1 for

paper)

1.2. DEFINITION OF DIABETES AND CLASSIFICATION

Diabetes mellitus is a chronic disease that is characterized by hyperglycaemia

and abnormalities in the metabolism of carbohydrates, proteins and lipids. It is

caused by a defect in insulin function or pancreatic secretions or both 3, 52.

According to the World Health Organisation (WHO), 346 million people

worldwide have diabetes and it estimates that the number of deaths from

diabetes will double between the years 2005 and 2030 53. Diabetes mellitus is

diagnosed using the classic symptoms of hyperglycemia (deficiency of the

insulin hormone that produced by the pancreas), glycosuria (secretion of

glucose into the urine) and polydipsia (excessive thirst or drinking excessive

amounts of liquids) 53. In addition, blood tests that are used to diagnose

diabetes mellitus include fasting plasma glucose levels >=7 mmol/L or

>=11.1mmol/L 2 hours after a 75g glucose drink. Glycated haemoglobin A1c levels (HbA1c), provides information regarding blood glucose levels over the previous 120 days with a normal measure of HbA1c being less than 6% 53.

Diabetes mellitus is classified into four different subtypes including Type 1,

Type 2, gestational diabetes and impaired glucose tolerance (pre-diabetes) 10.

Type 1 diabetes occurs as the result of the destruction of the pancreatic β-cells which prevents insulin secretion whereas Type 2 diabetes is a common form of diabetes mellitus that is the outcome of impaired insulin activity or secretion 10. Gestational diabetes is defined as glucose intolerance associated with pregnancy and is associated with alterations in reproductive and stress hormone levels that rise significantly during gestation and its symptoms are similar to Type 2 diabetes 15,36,37. Gestational diabetes is regularly diagnosed via prenatal screening of glucose tolerance rather than following the occurrence of its symptoms 16. Prolonged hyperglycaemia caused by diabetes mellitus may lead to several complications such as neuropathy, a high risk of foot ulceration and amputation, impaired wound healing, cataract development, retinopathy and associated blindness and nephropathy 3, 52. The development of complications of the vascular system occur in diabetic patients more often than in non-diabetic people and this is a main contributor to morbidity and mortality 12, 13. In addition, vascular complications may contribute to some diabetic pathogenesis such as retinopathy, nephropathy, neuropathy and impaired wound healing 13.

1.3. COMPLICATIONS OF DIABETES

1.3.1. Diabetic Atherosclerosis

Atherosclerosis is one of the more serious complications of vascular disease

that could occur through long-term diabetes mellitus 3, 54. The risk of vascular

disorders such as coronary, cerebrovascular and peripheral arterial disease

could be increased by diabetes mellitus; also, atherosclerosis is a main cause

of mortality or disability in the patient 3, 54. Atherosclerosis may occur via

deposition of lipid and cholesterol in the vascular walls leading to the

formation of stiff structures called plaques that block the blood flow 3.

1.3.2. Diabetic Nephropathy

Diabetic nephropathy is another common diabetes complication, which is

characterized by deposition of extracellular matrix protein in the mesangium,

thickness of the basement membrane, albuminuria, decrease of filtration and

renal failure 3, 14. The lack of balance between the synthesis and destruction of

extracellular matrix components causes the pathologic deposition of laminins

and collagen together with fibronectins 14.

1.3.3. Diabetic Retinopathy

Diabetic retinopathy is a common microvascular disorder that is a major cause

of blindness in the diabetic population of many developed countries 3, 15, 55.

Diabetes was originally considered a disease of the affluent and treatment

options for diabetics has allowed individuals to live long enough to develop

retinopathy. However, as the prevalence of this condition rises in both

developed and developing countries it would be expected that the occurrence

of retinopathy would increase across the world. Diabetic retinopathy could be

classified as non-proliferative or proliferative 56. Also, retinopathy is

characterized by vascular blocking, angiogenesis, developing of blood vessels

proliferation, microaneurysms and infarction influence on the retina of the eye

3. The pathological features of retinopathy are: thickening of the retinal

capillary basement membrane; loss of tight junctions in the retinal

endothelium; enhanced permeability of capillaries; and loss of pericytes so

that the capillaries can be coated by the contractile cells which will control the

vessels’ content and perfusion 3 .

1.3.4. Diabetes-Impaired Wound healing

Cardiovascular complications associated with diabetes occur due to

endothelial damage, which impairs angiogenesis and then delays wound

healing, contributing to atherosclerosis, thrombosis and hypertension 57.

Impaired wound healing is a serious diabetic complication that leads to

approximately 50% of non-traumatic amputations in diabetic patients 3, 37, 58-60.

Furthermore, a reduced capacity for angiogenesis, decreased

lymphangiogenesis and the destruction of endothelial cells increases the risk

of cardiovascular disease and impaired diabetic wound healing 57, 59. It is

noteworthy that both angiogenesis and lymphangiogenesis are essential factors

in the wound healing process 57, 59. Diabetics develop vascular complications

such as arteritis stiffness and poor blood circulation particularly to the lower

limbs that lead to hypoxia and reduced blood flow to areas of tissue damage,

which may impair wound healing 3, 38. Peripheral neuropathy is another

component associated with delayed wound healing through reduced sensorial

nerve function and nerve transmission. Lack of pain perception increases the

risk of wound development and delayed wound healing in diabetic patients as

they do not perceive the onset and development of ulcers or infections in their

extremities 34, 38. In diabetes mellitus, wound healing is also compromised due

to a delay of inflammatory cell influx into the damaged area and therefore the

inflammatory response 38. However, once the inflammatory cells penetrate the

wound they contribute to chronic inflammatory processes that inhibit the

extracellular matrix components (ECM) deposition, tissue remodelling and

wound healing 3, 34.

1.4. SKIN WOUND HEALING PROCESS

Wounds in the skin heal through three essential phases, namely the

inflammatory, proliferative and maturation stages 35, 39. In addition, growth

factors and cytokines together with chemokines have crucial roles in the

different phases of wound healing process 39, 61.

1.4.1. Inflammatory Phase

The destruction of blood vessels during tissue injury instigates platelet

accumulation and activation and the coagulation process that initiates the

inflammatory reaction 39. As the inflammatory process proceeds keratinocytes

release pre-stored interleukin-1 (IL-1) and tumour necrosis factor-α (TNF-α) 39.

In addition, other growth factors are secreted from the platelet α-granules.

These include platelet-derived growth factor (PDGF), epidermal growth factor

(EGF) and transforming growth factor-β (TGF-β) 39. Furthermore, TGF-β

stimulates the conversion of monocytes to macrophages to increase the

inflammatory reaction and tissue debridement 39.

1.4.2. Proliferative Phase

This phase includes epithelialisation, the formation of granulation tissues and

revascularization 39, 61. Migration of the epidermal cell has a key role in

reepithelialisation of wounds and these cells advance from the surrounding

wound margins towards the centre of the wound 62. Furthermore, epidermal

growth factor, TGF-α and keratinocyte growth factor have a major role in

stimulating the reepithelialisation process 63. Macrophages release different

types of pro-inflammatory cytokines such as IL-1 and IL-6 and growth factors,

which initiate the formation of granulation tissue 39. Granulation tissue is

created through the proliferation of fibroblast cells and vascular endothelial

cells, which initiate the repair process 35. In addition, macrophages release

TGF-β and PDGF to promote the proliferation of fibroblasts to generate new

ECM 63-65. Macrophages also release vascular endothelial growth factor

(VEGF) and basic fibroblast growth factor (bFGF2) to stimulate the formation

of new blood vessels through angiogenesis. This process depends on other

factors such as ECM formation and the proliferation and migration of

endothelial cells 63.

1.4.3. Maturation Phase

The maturation phase consists of wound contraction, the removal of excess

blood vessels via apoptosis and the conversion of collagen with its cross

linking and rearrangement along stress lines to strengthen the tissue 66.

Granulation tissues are removed during revascularization to generate a matrix

and collagen is replaced with elastin fibers 63. In tissue remodelling and wound

contraction, the TGF-β family which includes TGF-β1, TGF-β2, TGF-β3 and

TGF-βs, stimulates the synthesis of fresh collagen type III to replace old

collagen type I that is broken down by PDGF following up-regulation of

matrix metalloproteinases 63. Newly formed collagen then binds to fibroblasts

through integrin receptors to lead to scar tissue formation and healing of the

wound 63, 67.

1.5. ANGIOGENESIS

Angiogenesis is a process by which new capillary blood vessels are formed

from primary vessels 68, 69. This process is controlled through interactions

between growth factors that either induce or inhibit the growth of blood

vessels 68. Angiogenesis has an essential role in normal processes such as fetal

development, tissue repair, and the female reproductive cycle and in abnormal

processes including arthritis, stroke, ulcer development, tumour growth and

diabetic retinopathy 68, 70. Several studies have shown that adenosine and

adenosine receptor agonists up-regulate the expression of angiogenic factors

and anti-angiogenic factor thrombospondin-1 (TSP-1) to stimulate

angiogenesis 10, 68, 71-76. Furthermore, several in vitro and in vivo studies

reported that adenosine and its receptor agonists stimulate blood vessel growth

in pulmonary endothelial cells, the corneas of amphibian, mammalian

embryos, rat skeletal muscle and chick chorioallantoic membranes (CAM) 68,

73, 77-82. Studies have reported that adenosine plays an essential role in

angiogenesis and the wound healing process 41, 69, 83, 84 through elevating the

release of angiogenic factors 69, 85. Subsequently, a reported in vivo study

found that caffeine can induce defects in the angiogenesis process in zebrafish

embryos through blocking the adenosine receptors, an effect which was

reversed after stopping caffeine treatment 69.

1.6. ADENOSINE

Adenosine is a purine nucleoside which comprises an adenine molecule bound

to a ribose sugar and it has a short half-life of 0.6-10 seconds 73, 86, 87 (Figure

1.6.1). Adenosine is key local modulator of tissue function, particularly when

there is loss of oxygen delivery, leading to a severe reduction in cellular

energy production 88. Adenosine induces vasodilation, which increases

substrate and oxygen delivery to damaged tissue 89. Research has found that

adenosine generated during inflamed, ischaemic and hypoxic conditions

decreases damage to tissues and stimulates recovery through a number of

receptor-medicated processes 89. During low oxygen conditions, ATP is

degraded to adenosine 90. As intracellular levels of adenosine rise the

nucleotide then follows a concentration gradient out of the cell via carrier

mechanisms 90. Adenosine regulates organ and cellular activity through

binding of cell surface receptors, which are all members of the 7-

transmembrane spanning family, heterotrimeric G protein related receptors 9, 91.

The concentration of adenosine in the extracellular environment is usually

kept at minimal levels due to reuptake processes using transporters that are

selective for adenosine 9. Following its movement back to the cell, adenosine

is phosphorylated with adenosine kinase to generate AMP (adenosine

monophosphate) and ultimately ATP, or it may be broken down by the

enzyme adenosine deaminase to produce inosine 9.

Figure 1.6.1.The chemical structure of adenosine

1.6.1. Adenosine Metabolism

Adenosine is formed through the metabolism of the bioactive nucleotides

adenosine triphosphate (ATP), adenosine diphosphate (ADP) and cyclic

adenosine monophosphate (cAMP) together with S-adenosylhomocysteine

(SAH) 92. There are several metabolic pathways that utilize adenosine. This

includes the intracellular and extracellular phosphorylation of 5’-adenosine

monophosphate (5’-AMP) to 5’- nucleotides and the hydrolysis of S-

adenosylhomocysteine (SAH) to adenosine and homocysteine via S-

adenosylhomocysteine hydrolase in intracellular space 92. The extracellular

adenosine concentration is maintained through carrying adenosine from the

intracellular space to the extracellular space by the bi-directional equilibrative

nucleoside transporter 1 (ENT1). There are two enzymes located on the cell

surface, CD39 (nucleoside triphosphate phosphohydrolase) and CD73 (ecto-

5’-nucleotidase) that catalyze break down of ATP to form AMP, which is then

converted to adenosine in the extracellular space (Figure 1.6.2) 4, 93. Moreover,

adenosine can be further broken down by moving back into the cell to form

inosine through the enzyme adenosine deaminase, or adenosine may be

converted to 5’-AMP by adenosine kinase 89.

Figure 1.6.2. Adenosine metabolism in the intracellular and extracellular environment of the cell 4

1.6.2. Adenosine Receptors

Adenosine receptors consist of four subtypes A1, A2A, A2B and A3, which are

members of the G protein-coupled receptors and as such are recognised as

seven-transmembrane domain receptors 86, 88, 94-96. The adenosine receptors are

widely expressed on various cell types such as endothelial cells, fibroblasts,

neutrophils and macrophages. Therefore, adenosine as an effective

endogenous mediator, regulates physiological effects such as stimulation of

angiogenesis and wound healing through interaction its receptors 5, 73, 97, 98.

Originally the pharmacological properties of the adenosine receptors were

used to characterize and differentiate the four subtypes of receptor 88, 99. More

recently the genes that encode the adenosine receptor subtypes have been

identified and published, as well as their primary amino acid sequence 88, 99.

These four subtypes of adenosine receptors have different functions and

molecular weights 88, 99. The adenosine receptors are coupled to their particular

G proteins and are well recognized to inhibit and/or stimulate adenylyl cyclase

88, 91 . The adenosine A1 and A3 receptors comprise a molecular mass of

approximately 38 kDa and 52 kDa respectively and are coupled to Gi/o to

mediate adenylyl cyclase inhibition, attenuating the cyclic adenosine

monophosphate (cAMP) dependent pathway and decreasing cAMP

production. They also activate K+ channels, inhibit Ca2+ channels and mediate

catabolism of phospholipids (Table 1.6.1) 88, 91, 100-102.

A study investigating the avian heart has reported that activation of both

adenosine A1 receptors may stimulate an accumulation of diacylglycerol

103 (DAG) . Furthermore, this study showed that adenosine A1 receptors

stimulate phospholipase C (PLC) to cause a concomitant accumulation of

39 phosphoinositides 103. U-73122, a PLC inhibitor, reversed DAG production after the activation of adenosine A1 receptors and suppressed adenosine A1 receptor mediated cardioprotection 88, 103.

The adenosine A2A receptor subtype has a molecular mass of around 45 kDa and is coupled to the Gs protein. The adenosine A2B receptor has a molecular mass of around 53 kDa and can be coupled to both Gs and Gq proteins to stimulate adenylyl cyclase, increased stimulation of intracellular cAMP production, intracellular calcium mobilization, activation of mitogen-activated protein kinase (MAPK) and phospholipase C (PLC) (Table 1.6.1) 9, 88, 91, 102, 104,

105. There are several potent and selective agonists and antagonists for adenosine receptors that have a high affinity to its related subtype; some of these agonists and antagonist were utilised in this research study and are listed in Table 1.6.1.

Adenosine Number of G protein Mechanism Agonists Antagonist Receptor amino Coupling subtype acids A1 326 Gi/o AC inhibition, v Selective v Selective Decrease CPA DPCPX cAMP CCPA WRC-0571 productions, (S)-ENBA N-0840 Activation K+ R-PIA v Non- channels, CHA Selective inhibition of Caffeine Ca2+ channels A2A 410 Gs AC v Selective v Selective stimulation, CGS-21680 ZM-241, 385 Increase ATL-146e SCH58261 cAMP PAPA- APEC CSC productions KW6002 v Non- Selective Caffeine Theophylline

A2B 320 Gs & Gq AC stimulation v Selective v Non- Increase None due to Selective cAMP low agonist MRS1754 productions, affinity Caffeine Activation v Non- Theophylline MAPK and Selective Alloxazine PLC, Ca2+ NECA MRE2029-F20 mobilization BAY-60-6583 CGS15943 LUF5834 A3 ** 320 Gi/o AC inhibition, v Selective** v Selective** Decrease IB-MECA BWA-1433 cAMP C1-IB-MECA MRS1191 productions, APNEA MRS1220 Activation K+ v Non- MRS1523 channels, Selective MRE 3008-F20 Deactivation NECA v Non- Ca2+ channels Selective** Theophylline

Table 1.6.1. Adenosine receptors classification and functions. Adenylate cyclase (AC), mitogen-activated protein kinase (MAPK), phospholipase C (PLC), ** Adenosine A3 receptor agonist and antagonist were not utilised in this research study 7, 86, 88, 92.

1.6.3. Signalling pathways of the adenosine receptors

Activation of each adenosine receptor by its agonist causes different signalling

46, 106 events . Adenosine A1 and A3 receptors, through binding to Gi/o proteins,

inhibit the activity of adenylate cyclase thus decreasing the activation and

production of cyclic AMP (cAMP) 46. One study using Chinese hamster ovary

cells (CHO) demonstrated that inhibition of adenylyl cyclase through

functional binding of adenosine A3 receptors could be observed in CHO cells

107 transfected with the receptor cDNA . The adenosine A2A and A2B receptors

bind to Gs to stimulate adenylyl cyclase and increase cAMP accumulation to

activate protein kinase A (PKA) and eventually stimulate cell proliferation,

and is the primary signalling pathway associated with these receptors 46. Also,

activation of protein kinase C (PKC) and increasing intracellular calcium

levels occur following elevation of cAMP levels and activation of

46 phospholipase C (PLC) through the Gq protein . The Gq protein mediated

signalling is the second main signalling pathway of adenosine A2A and A2B

receptors 86, 108. A study has shown that adenosine at low concentrations can

stimulate the high affinity adenosine A1, A2A and A3 receptors whereas high

46 concentrations of adenosine activates the low affinity A2B receptors .

The adenosine A1 receptors can bind to the Gi/0 protein, which leads to

decreased intracellular cAMP levels 84, 109, 110. The interaction of adenosine

2+ with the adenosine A1 receptor can elevate intracellular Ca levels through

the stimulation of phospholipase C (PLC) 110. PLC breaks down

phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG) and

inositol 1,4,5-trisphosphate (IP3) 110. In addition, increased levels of

intracellular Ca+2 activate enzymes such as protein kinase C (PKC) and

84, 95 phospholipase A2 (PLA2) together with phospholipase D (PLD) . A study using selective adenosine receptor agonists and antagonists showed that adenosine A1 receptors can have pro-inflammatory effects in different tissues

110. For example, in a study on treatment of experimental lung injury in mice, it was demonstrated that DPCPX (1,3-dipropyl-8-cyclopentylxanthine), a selective adenosine A1 receptor antagonist, could prevent endothelial damage, migration of neutrophils and damage of alveolus in reperfusion in the lungs 111.

Endothelial cells have a large capacity to store and release adenosine 108. In addition, many of inflammatory processes of endothelial cells can be modulated by adenosine A2B receptors, which are expressed on endothelial

71, 108 cell surfaces . Interactions of adenosine with adenosine A2A and A2B receptors leads to increased cell proliferation, chemotaxis and capillary tube formation, therefore confirming a role for adenosine A2A and A2B receptors in

108, 112, 113 stimulating angiogenesis . Furthermore, activated adenosine A2B receptors in human microvascular endothelial cells stimulate the secretion of

VEGF and the insulin-like growth factor-I together with the basic fibroblast growth factor (bFGF) 71.

The binding of adenosine to the adenosine A3 receptors can initiate the classical second messenger pathway through adenylyl cyclase inhibition,

2+ 84, 114-116 increasing Ca levels and the stimulation of PLC . Adenosine A3 receptors are coupled to mitogen-activated protein kinase (MAPK) to stimulate extracellular signal-regulated kinases (ERK1/2). Hence adenosine

A3 receptors have a role in cell proliferation, differentiation, survival and death 84, 104. A study in melanoma cells demonstrated that activation of adenosine A3 receptors stimulates PI3K-dependent phosphorylation of protein

kinase B (PKB/Akt) to decrease levels of ERK1/2 phosphorylation and inhibit

cell proliferation 117. Angiogenesis and cell preservation can also be mediated

through adenosine A3 receptors, which enhance VEGF and HIF-1 α

production 118. Furthermore, through activation of Protein kinase C (PKC) or

phosphoinositide 3-kinase (PI3K), the adenosine A3 receptor can activate

degranulation and cell migration of rodent mast cells via increased

intracellular Ca2+ levels 84, 114-116.

Studies have noted that in comparison to other adenosine receptor subtypes,

the adenosine A3 receptor has the lowest peptide homology between species

110, 119.

1.6.4. Association of adenosine receptors with wound healing

Stimulation of the adenosine A2A and A2B receptors enhances migration of

fibroblasts and endothelial cells through cAMP- and PKA mediated signalling

pathways 5. Prior studies have also illustrated that adenosine and its analogues,

through binding adenosine A2A receptors, can enhance proliferation and

migration of endothelial cells as well as the release of vascular endothelial

growth factors (VEGF) 5, 120. This demonstrates that adenosine receptors

enhance wound healing through increased growth factor secretion into the

target area 5. On the other hand, occupation of adenosine receptors can impair

wound healing through inhibition of inflammatory cytokines secretion such as

TNF-α, IL-6, IL-8, IL-12 and vascular endothelial growth factor (VEGF) 121-

123 . Adenosine A1, A2A and A3 receptors can downregulate TNF-α and IL-12

production in monocytes and macrophages 46, 124. Also, the stimulation of

adenosine receptors, especially the adenosine A2 receptor in U-937 human

macrophages and human umbilical vein endothelial cells, inhibits the secretion

of vascular endothelial growth factor (VEGF) and IL-6, IL-8 respectively 46.

VEGF is a main factor that stimulates angiogenesis and vascular permeability.

Therefore adenosine may accelerate angiogenesis by increasing VEGF

46, 112, 125 production . According to Hashimoto et al., 1994, adenosine A2

receptor stimulation in U-937 human macrophages, leads to the accumulation

of VEGF mRNA 46, 72. Other studies have demonstrated that adenosine can

stimulate monocytes and macrophages to increase IL-10 production 46, 126-129.

As adenosine receptor agonists have been demonstrated to enhance wound

repair in diabetic animals, it has been proposed as a new innovation for the

advancement of agents that enhance wound healing in healthy people as well

as those with impaired wound healing 5.

1.7. CHEMICAL STRUCTURE OF CAFFEINE

Caffeine is a 1,3,7-trimethylxanthine and is synthesised from the purine

nucleotide xanthosine (Figure 1.7.1) 22, 130, 131.

Figure 1.7.1.Chemical structure of caffeine

1.7.1. Caffeine Consumption

Caffeine is present in several of dietary sources such as food and beverages,

mostly coffee and tea, that are widely consumed around the world 40. The

caffeine content in some popular dietary sources includes: coffee 40 to 180

mg/150 ml; tea 24 to 50 mg/150 ml; cola 15 to 29 mg/180 ml; cocoa 2 to 7

mg/150 ml; and 1 to 36 mg/28 g for chocolate 40, 132.

1.7.2. Caffeine Absorption

Caffeine is rapidly absorbed through the gastrointestinal tract in humans and

absorption reaches 99% within 45 minutes after ingestion. Caffeine is

metabolized in the liver by the microsomal enzyme CYP1A2 22, 40, 133. One

study observed that the absorption of caffeine from coffee is faster than from

cold drinks and suggested several reasons for this 22. The low temperature of

the cold beverage may reduce the blood flow rates within the intestine and the

rate of gastric emptying may be decreased by phosphoric acid and sugar in the

cold beverage 22, 134. The hydrophobic properties of caffeine allow it to diffuse

through the body and penetrate all cell membranes, the blood-brain barrier and

the placenta, however, caffeine does not accumulate in the tissues or organs 22,

24, 133. The peak plasma concentration of caffeine is reached between 15 and 20

minutes after oral ingestion in humans and this is the equivalent to 8 to 10

mg/l for 5 to 8 mg/kg doses 22, 40, 135, 136. Furthermore, smoking accelerates the

metabolism of caffeine thus the half-life of caffeine in a smoker adult male

may decrease by 30% to 50% 133. The half-life of caffeine doubles in women

who are taking oral contraceptives (OCPs) and can be increased in the last

three months of pregnancy. Also caffeine levels will be elevated in the patients

with chronic liver disorder 22, 133. Moreover, one study has shown that

ingestion of high doses of caffeine (doses more than 300 mg) can induce

anxiety, panic attacks and hallucinations in human 22. Acute caffeine

poisoning in humans occurs following ingestion of caffeine at the levels of a

few hundred micromoles per litre or 40–400 mg/L in plasma 40.

1.7.3. Caffeine Metabolism

Caffeine is metabolized via the liver to produce dimethylxanthines and

monomethylxanthines, dimethyl uric acids and monomethyl uric acids,

trimethylallantoin and dimethylallantoin, and derivatives of uracil 40, 137. Also,

several substances are formed by caffeine metabolism such as 1,3-

dimethylxanthine (theophylline) and 1,7-dimethylxanthine (paraxanthine)

which also have marked pharmacological activity 40. Millimolar

concentrations of caffeine cause the release of intracellular calcium through

binding to ryanodine receptors on the sarcoplasmic reticulum 40, 138. Moreover,

the inhibition of phosphodiesterases may occur at higher concentrations of

caffeine in humans than those usually reached during the consumption of

beverages 40, 139, 140. According to Fredholm et al., 1999, caffeine is able to

block adenosine A2A and A1 receptors at low concentrations, which can be

reached after a single cup of coffee. However, higher concentrations of 20

times this are required to inhibit the cyclic nucleotide breakdown by

phosphodiesterases; 40 times higher concentrations will block GABAA

receptors and 100 times higher concentrations will mobilize Ca2+ from

intracellular calcium depots 40. The highest two levels of caffeine referred to

above are improbable in humans through any form of normal usage of

caffeinated beverages 140. Furthermore, an in vivo study using rats

demonstrated that the effects of low doses of caffeine were similar to the

selective adenosine A2A receptor antagonist SCH 58261 but not the selective

141 adenosine A1 receptor antagonist DPCPX .

1.7.4. Caffeine and Ryanodine Receptors

Ryanodine receptors mediate rapid calcium (Ca2+) release from the

sarco/endoplasmic reticulum (SR/ER) and are responsible for activating

several Ca2+ -activated physiological processes 142. Caffeine is used commonly

as a ryanodine receptor agonist and stimulates Ca2+ release from intracellular

Ca2+ stores 143-148. Caffeine stimulates Ca2+ release via sensitizing the channel

to cytosolic Ca2+ activation 144, 149, 150. The ryanodine receptors channels can

be activated by the resting cytosolic Ca2+ upon the addition of caffeine 144.

Caffeine at submaximal concentrations has the ability to stimulate a partial

and temporary Ca2+ release from intracellular Ca2+ stores in cells expressing

ryanodine receptors or from sarcoplasmic reticulum membrane vesicles in a

phenomena recognized as “quantal” Ca2+ release 143-148.

1.7.5. Various Effects of Caffeine

Caffeine exerts “positive” subjective effects on mood in humans at low doses

of caffeine (20–200 mg) 40. Caffeine is however well recognized as increasing

anxious behaviour in human and animal models and can increase risk of panic

attacks 151, 152. It has been reported that patients who suffer from panic disorder,

a serious form of anxiety disorder, were particularly sensitive to low doses of

152, 153 caffeine (i.e. instant coffee at 65 mg caffeine per cup . Adenosine A1 receptors regulate the release of various neurotransmitters such as glutamate.

Therefore, excitatory transmission is increased when the adenosine A1 receptors are blocked by caffeine directly or indirectly when caffeine affects

GABAergic transmission 40.

Many studies using rats have shown that caffeine stimulates electrical activity in the cortex 154, 155. Also, cats treated with caffeine exhibited activation of the cortical electroencephalography (EEG) similar to activity recorded at physiological awakening times and comparable to the EEGs that are generated via direct stimulation of the reticular formation, a part of the brain that plays an essential role in vigilance and awakening 40. Methylxanthines increase the excitability of rat hippocampal sections through antagonizing the actions of adenosine and also activate the EEG theta rhythm in the hippocampus of rabbit 156-158. Adenosine decreases the development of long-term potentiation while xanthines have the opposite effect through blocking adenosine receptors in rat hippocampal slices 40, 159, 160.

Caffeine can promote different acute cardiovascular effects including the up- regulation of circulating catecholamines in the cardiovascular system 22, 161.

Arterial stiffness and endothelium-dependent vasodilation also occur, leading to elevated systolic and diastolic blood pressure 22, 161. In addition, an increase in the respiration rate is an effect that is dependent on the plasma caffeine levels 22, 133

Adenosine prevents catecholamine and renin release, and also inhibits lipolysis 133. Furthermore, caffeine as a nonselective adenosine receptor subtype antagonist, blocks the actions of adenosine 133. Consequently, caffeine

increases circulating catecholamine levels and via this mechanism elevates the

basal metabolic rate. Hence, lipolysis occurs, causing the release of free fatty

acids. This increases the levels of serum free fatty acids to 50% -100% above

normal (the reference range is 0.00–0.72 mmol/L) 133. Caffeine stimulates the

small intestine, causing secretion of water and sodium 22, 133. According to a

medical report, caffeine may stimulate apoptosis in UVB-damaged cells as

well as block adenosine receptors to regulate blood vessels contraction 22, 162.

Furthermore, caffeine may be utilized as a psychoactive drug to treat the

patients with Parkinson’s disease 22, 162.

1.7.6. Caffeine and Wound Healing

Caffeine is a methylxanthine and a member of an alkaloid group that is a main

component in coffee and other beverages such as cocoa and tea 6, 42, 163, 164.

Caffeine has the potential to alter wound healing through a variety of

mechanisms. It has been shown that caffeine can act as antioxidant in the body

and components including xanthine and theobromine may have antioxidant

roles 6, 163, 164. Following tissue injury, damaged cells in the wound generate

numerous amounts of reactive oxygen species (ROS), which are generated in

the early stage of wound healing, and antioxidants are needed to scavenge free

radical species and accelerate wound closure 33, 165.

In addition, some studies have demonstrated that antioxidant intake can

accelerate wound healing 6, 165, 166. For instance, acceleration of wound healing

has been demonstrated using different components containing antioxidant

compounds such as the methanolic and ethanolic extract of Boesenbergia

rotunda, green tea extract and coffee powder 33, 167, 168. Sol Gel has been

developed as an application method to apply antioxidants including a mixture

of vitamin C in Pluronic F127 165. The application of sol-gel on skin wounds

has been shown to promote wound closure and enhanced cell proliferation and

collagen synthesis as well as reducing apoptosis in streptozotocin induced

diabetic rats 165. Coffee powder has likewise been shown to improve and

support wound healing through its antibacterial and antioxidant properties in

human and animal models 33. Therefore, coffee powder is utilised directly, to

cover different types of wounds associated with diabetes mellitus, sharp cuts

and burns 169. On the other hand, an in vitro study in human keratinocytes and

epidermal cells has shown that caffeine limited cell proliferation and cell

migration in a dose- and time-dependent manner, thereby attenuating two

main functions for wound healing 6.

1.8. ASSOCIATION OF CAFFEINE WITH DIABETES AND EFFECT ON

ADENOSINE RECEPTORS

Caffeine can alter the activity of adenosine and its effects either through

modifying glucose levels, which are a fundamental cause of impaired wound

healing, or through acute inhibition of adenosine receptors or chronic

regulation of the adenosine receptor subtypes. The non-selective adenosine

A2B receptor agonist NECA has been shown to increase glucose production in

hepatocytes 170. Similarly, the application of NECA through oral gavage

caused an increase in fasting glucose levels in mice 171. The implementation of

adenosine A2B receptor knockout in mice and blockade of the adenosine A2B

receptors with the selective antagonist ATL-801 in this study has indicated

171 that the adenosine A2B receptor may act as an initial mediator of this effect .

Caffeine can act as a non-selective blocker of the adenosine A1, A2A and A2B adenosine receptors 172. However, the ATL-801 is around 5000 times more potent than caffeine 122. A study of diabetic KKAY mice model has shown that the intake of large amount of coffee decreased hyperglycaemia, fat mass, and the expression of TNF-α and IL-6 in white adipose tissue. It also reduced the synthesis of fatty acids through decreased hepatic expression of the SREBP-1

173 and FAS genes . By blocking the adenosine A2B receptor in diabetic mice, caffeine increased the sensitivity to insulin and the metabolism of glucose in the liver and skeletal muscle and brown adipose tissue 171.

Chronic caffeine treatment has the capacity to increase adenosine receptor populations. A study investigating chronic caffeine treatment over one week reported significant increases in the number of adenosine receptors in rat cerebral cortical membranes 174. A mouse model of acute lung injury demonstrated that an acute low dose caffeine treatment (5 or 15 mg/kg) increased lung injury by acting as an adenosine A2A receptor antagonist to inhibit the anti-inflammatory effect of adenosine A2A receptors, whereas an acute high dose of caffeine (50 mg/ kg) and chronic caffeine treatment decreased the lung damage and inhibited inflammation in an adenosine A2A receptor-independent manner 175. In addition, this study showed that acute low dose caffeine decreased the level cAMP in wild type (WT) mice but it did not have any effect on cAMP levels in adenosine A2A receptor knockout (KO) mice 175. Another in vivo study has shown that caffeine inhibits cAMP phosphodiesterase and can suppress immune activity by reducing cAMP levels in immune cells to decrease tissue injury during acute inflammation 45, 176, 177.

These results demonstrated that neutrophil infiltration and expression of

inflammatory cytokines were inhibited through chronic caffeine and acute

high dose caffeine (50 mg/ kg) treatments in both WT and KO mice.

Therefore the reduction in lung damage was not related to presence of

175 adenosine A2A receptors . Furthermore, other studies have shown that

chronic caffeine decreases tissue injury in the kidney and liver 175, 178, 179.

Additionally, chronic caffeine–induced up-regulation of adenosine A2A

receptors may increase the anti-inflammatory role of adenosine to decrease

180, 181 tissue injury. . Similarly up-regulation of adenosine A2B receptors with

chronic caffeine was shown to be protective against acute lung injury in mice.

However, there was no significant change in the expression of adenosine A2A,

A1, and A3 receptors in pre- and post-acute lung injury groups with chronic

and acute caffeine treatment in comparison with a control group. Activation of

the adenosine A2B receptor, which leads to increased cAMP levels as

previously described, can decrease acute inflammation, which protects tissues

from further injury or mitigates tissue damage 182-185. Overall, chronic caffeine

treatment upregulates adenosine A2B receptor numbers, which may enhance

this receptor’s anti-inflammatory role when activated via extracellular

adenosine 175.

1.9. CYTOKINES

As discussed previously, depending on the cell type inflammatory, endothelial,

or structural adenosine and caffeine can have different effects on the cytokines

released during the stages of wound healing (Figure 1.9.1). Pro-inflammatory

cytokines consist of IL-1, IL-6 and TNF-α which are the main elements

involved in the inflammatory phase of wound healing (Figure 1.9.1) 63. IL-1 is

54 generated immediately following tissue injury from macrophages, neutrophils, keratinocytes and monocytes 63. IL-1 and TNF-α stimulate fibroblast

39, 186 production and the release of FGF7 . The release of IL-6 and TNF-α from activated neutrophils and macrophages that infiltrate the site of damage are essential in initiating the wound healing process and improving reepithelialisation 39. IL-6 persists through the inflammatory process and it stimulates mitogenic and proliferative activity in keratinocytes and attracts neutrophils to the wound 39, 187. TNF-α indirectly improves reepithelialisation through increased stimulation of FGF7 production, however it has been shown that TNF-α itself can reduce re-epithelialization of wound 39. In addition,

TNF-α attenuates the synthesis of ECM proteins and tissue inhibitor of metalloproteinase (TIMP-1) and stimulates the synthesis of metalloproteinases

(MMP-1, MMP-2, MMP-3, MMP-9, MMP-13, and MT1-MMP) 39, 188-190.

A study using the mouse macrophage cell line RAW 264.7, reported that adenosine A1, A2 and A3 receptors have an essential role in the regulation of cytokines such as IL-10, TNF-α 128. Furthermore, this study showed that drugs such as methotrexate, sulfasalazine and GP-1-515, can elevate extracellular adenosine concentrations and are potent anti-inflammatory agents 128.

Moreover, there are several mechanisms that might contribute to the anti- inflammatory properties of these drugs including: stimulation of adenosine A2 and A3 receptors to increase the production of IL-10; and stimulation of

128 adenosine A1, A2 and A3 receptors to reduce the release of TNF-α . Another study demonstrated that adenosine can potently inhibit the expression of pro- inflammatory cytokines such as TNF-α from mouse peritoneal macrophages in response to Toll-like receptors (TLR) activation 191, 192. An effect mediated

55 through the adenosine A2A receptor acting by different mechanisms included

192-195 inhibition of nuclear factor-κB (NF- κB) . Adenosine A3 receptors are also able to suppress pro-inflammatory cytokine expression 126, 191, 192. The adenosine A2A receptor is more effective at inhibiting TNF-α production in

192 murine peritoneal macrophages than the adenosine A2B receptor . Moreover, the adenosine A2B receptor has been commonly considered to be a pro-

192 inflammatory receptor . It is reported that activation of the adenosine A2B receptors can stimulate the production of IL-8 in a human mast cell line HMC-

1 and also stimulate production of IL-6 and monocyte chemotactic protein-1 from human primary bronchial smooth muscle cells 192, 196, 197.

With respect to the effects of caffeine on cytokine release an in vivo study reported that the effect of caffeine on inflammatory responses is dose dependent in mice 45. For example, caffeine at low doses (10 and 20 mg/kg) increased pro-inflammatory cytokine release including TNF-α, IL-4 and IL-12 to enhance tissue damage, while caffeine at higher doses (100 mg/kg) reduced tissue destruction via the inhibition of pro-inflammatory cytokine responses and the release of an anti-inflammatory cytokine IL-10 45. Also, this study showed that lower doses of caffeine intensely exacerbated acute liver damage and elevated pro-inflammatory cytokines levels in mice 45. In contrast, caffeine at higher doses entirely inhibited liver damage and pro-inflammatory

45 cytokines responses via the adenosine A2A receptor-independent mechanism .

Another study reported that plasma caffeine levels were strongly associated with reduced inflammatory cytokine levels of Aβ 1-40, IL-12 (p70), IFN-γ and

TFN-α in the hippocampus 198. Also, a significant reduction in hippocampal

56 inflammatory cytokines was observed in caffeine treated mice with higher plasma caffeine levels in comparison with lower caffeine levels 198

Figure 1.9.1. Figure showing the role of pro-inflammatory cytokine responses in wound healing 199. Interleukin-8 (CXCL8), interleukin-1α (IL-1α), interleukin-1β (IL-1β), (C-C motif) ligand 2 (CCL2), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), tumour necrosis factor (TNF) and platelet- derived growth factor (PDGF)

1.10. CONCLUSION

Impaired wound healing and cardiovascular complications are significant

disorders in diabetes mellitus that could be caused by attenuated angiogenesis

together with endothelial cell dysfunction. Adenosine is a purine nucleoside

that could activate cell proliferation through the interaction with adenosine

receptor subtypes (A1, A2A, A2B, and A3). Adenosine A1 and A2 receptor

agonists improve wound healing, and adenosine can stimulate angiogenesis as

well. In addition, occupation of the adenosine receptor could reduce the

elements that impair wound healing through inhibition of inflammatory

cytokine secretion such as TNF-a, IL-6, and IL-8. While caffeine is a

nonselective antagonist of the adenosine receptor subtypes, it has also been

found to increase cell proliferation and wound healing. Caffeine may inhibit

the role of adenosine in the development of glucose intolerance and insulin

resistance, and alter the ability of adenosine to moderate the inflammatory

processes. In addition, caffeine acts as an antioxidant and phosphodiesterase

inhibitor, and may improve wound healing through these mechanisms.

1.11. HYPOTHESES AND AIMS

This project will identify the adenosine receptor subtypes involved in

stimulating wound healing and determine whether their roles are affected in

the hyperglycaemic environment associated with diabetes. The project will

also investigate the effects of acute and chronic caffeine treatments on

adenosine receptor populations and whether they subsequently alter the wound

closure and adenosine functions within the cellular and vascular systems.

Caffeine may have a crucial role in wound healing through various pathways such as: antioxidant pathways and increasing the level of cyclic adenosine monophosphate (cAMP). Therefore, it is hypothesised that a chronic caffeine treatment could speed up wound healing in endothelial cells through up- regulation of adenosine receptor subtypes and increased levels of cAMP.

This study will investigate several hypotheses as follows;

HA; Cell proliferation and wound healing in EAhy926 endothelial cells will be stimulated by selective adenosine receptor agonists

H0; Cell proliferation and wound healing in EAhy926 endothelial cells will not be stimulated by selective adenosine receptor agonists

HA; Acute caffeine has an inhibitory effect on the wound healing rate and cell proliferation in EAhy926 endothelial cells

H0; Acute caffeine does not have an inhibitory effect on the wound healing rate and cell proliferation in EAhy926 endothelial cells

HA; Chronic caffeine has a stimulatory effect on the wound healing rate and cell proliferation in EAhy926 endothelial cells

H0; Chronic caffeine will not have stimulatory effects on the wound healing rate and cell proliferation in EAhy926 endothelial cells

HA; There will be differences in rates of wound healing and cell proliferation in EAhy926 endothelial cells during high and low glucose conditions.

H0; There will be no differences in rates of wound healing and cell proliferation in EAhy926 endothelial cells during high and low glucose conditions.

The aims of this research project are to:

I. Identify the effect of adenosine receptor subtypes on signalling

pathways associated with EAhy926 endothelial cell proliferation and

wound healing process.

II. Determine the effect of chronic and acute caffeine treatment on the

speed of wound closure and angiogenesis in EAhy926 endothelial cells.

III. Compare the effect of chronic caffeine treatments on adenosine

receptor subtype population protein expression.

IV. To determine the effect of high and low glucose levels on EAhy926

endothelial cell proliferation and wound healing process.

CHAPTER 2

METHODOLOGY

CHAPTER 2: METHODOLOGY

This chapter describes the various methods and materials used in this study, ranging from cell culture techniques to tube formation assays and Western blotting techniques.

These methods have been implemented to determine the presence of adenosine A1,

A2A and A2B receptors in the endothelial cell population used for these studies and to investigate the effect of acute and chronic caffeine treatment on endothelial cell function such as wound healing, angiogenesis and calcium signalling. The results of these studies are presented in chapters 3, 4 and 5.

2.1. HUMAN ENDOTHELIAL HYBRID CELL LINE (EA.HY926)

The permanent human endothelial hybrid cell line (EA.hy926) was used for

the studies presented in this thesis. This cell line was created through the

hybridisation of primary human umbilical vein endothelial cells (HUVEC) and

a human lung carcinoma cell line (A549/8) in 1983 200, 201. Following the

establishment of these cells they have been used for a number of studies

related to vascular function including the biochemical pathways of coagulation

and fibrinolysis 202. Also, these endothelial cell have been reported to contain

tissue-type plasminogen activator (t-PA), which when released into the

bloodstream is the main enzyme that facilitates the process of fibrinolysis and

thrombolysis 202-208. In addition, t-PA and a plasminogen activator inhibitor

(PAI) were synthesised and secreted by human endothelial cells 202, 209-212.

Many studies using the EA.hy926 cell line have reported that these cells

maintain the expression of cell surface thrombomodulin proteins, the synthesis

of von Willebrand factor and prostacyclins and the release of platelet

activation factor 201, 202, 213, 214. Moreover, another study demonstrated the

fibrinolytic characteristics of the EAhy926 cell line which secretes both tissue

plasminogen activator (t-PA) and plasminogen activator inhibitor type 1 (PAI-

1) 202. Several studies have investigated the properties of EA.hy926

endothelial cells with respect to angiogenesis, cell proliferation, cell migration

and apoptosis. A study reported that down-regulation of

dihydrofolatereductase prevents the stimulation of G1 phase thus inhibiting

cell proliferation 215. Another study observed that cordycepin (3'-

deoxyadenosine)a derivative of adenosine, stimulated apoptosis and inhibited

EA.hy926 cell migration in a dose- and time -dependent manner 216.

Furthermore, homocysteine and adenosine impaired angiogenesis in EA.hy926

endothelial cells through activation of the MEK/ERK signalling pathway 217.

This study also reported that dipyridamole, a nucleoside transport inhibitor

prevented impaired angiogenesis in the EA.hy926 cell line through the

suppression of intracellular accumulation of S-adenosylhomocysteine 217.

2.1.1. Cell Culture Technique

The EA.hy926 endothelial cell line used for these studies was generously

donated to our research group by the Apoptosis Research Group, Griffith

University, Gold Coast campus. The EA.hy926 endothelial cells were seeded

into a 75 cm2 cell culture flask containing 13 µL of complete media. Complete

media comprised high glucose (4500 mg/L, 25 mM) Dulbecco’s Modified

Eagle’s Medium (DMEM) (ThermoFisher Scientific, VIC, Australia)

supplemented with a mixture of Hypoxanthine-Aminopterin-Thymidine

(HAT)(Sigma Aldrich, NSW, Australia), 10% Foetal Bovine Serum (FBS),

and 1% Antibiotic-Antimycotic (penicillin/streptomycin/amphotericin B)

(Invitrogen, VIC, Australia). Please note that the high glucose media was used

to grow the EA.hy926 endothelial cell line in culture for Chapters 3 to 5 to

mimic a diabetic condition. The low glucose media (5.5mM) was used for

experiments in Chapter 6 to create a normal low glucose condition (non

diabetes). The high glucose media is the recommended media for this cell line

as the cells do not grow well at the lower glucose concentration. Cells were

incubated in a humidified incubator at 37 ºC and 5 % carbon dioxide (CO2).

The cells were maintained under these conditions until they adhered to the

surface of the flask. They were grown for 4-5 days until they reached the

optimal confluency to commence experiments.

2.1.2. Cell Passaging

The cells were passaged after 2-3 days, once they had reached 90% confluence.

The remaining media was discarded from the 75 cm2cell culture flask and

replaced with 8-10mL pre-warmed Dulbecco’s Phosphate-Buffered Saline

(DPBS) (ThermoFisher Scientific, VIC, Australia) to wash the cells, then the

DPBS was discarded from the culture flask. Afterwards, the cells were

incubated with 2 mL pre-warmed trypsin (ThermoFisher Scientific, VIC,

Australia) to detach the cells from the surface of the culture flask and placed

for 5 minutes in a humidified incubator at 37 ºC, 5% CO2. Concurrently, a

culture flask containing 10mLof complete media was placed into the incubator

for 15 minutes to allow the solution to reach a temperature of 37 ºC and pH of

7 to 7.6. Then 4 mL of complete media was added to the detached cells,

denature the trypsin and the solution aspirated a few times to prevent

clumping of cells. The total 6 mL cell solution was then equally divided into 3

pre-incubated75 cm2 culture flasks containing 10 mL high glucose media,

giving 12 mL total solution in each flask. The cells and high glucose media

were gently mixed with a 10 mL pipette to obtain a uniform solution. The cells

were then incubated at 37 ºC, 5% CO2, for 2 days to reach 90% confluence.

Cells were tested for mycoplasma infections using the MycoAlert®Myco

Plasma Detection kit (Lonza, Basel, Switzerland). Mycoplasma-positive cell

lines underwent antimycoplasma treatment with antibiotics including

plasmocin, BM-Cyclin and baytril for periods of 7–21 days.

For the duration of these experiments, the cells would have undergone 20

passages for the wound healing experiments and another 20 passages for the

cell proliferation assays.

2.1.3. Cryopreservation (Cell Freezing)

When the cells reached 80% confluence, the existing media was discarded

from the 75cm2 cell culture flask and the cells washed with pre-warmed DPBS.

Then 2 mL pre-warmed trypsin was added to the 75 cm2flask and the flask

incubated for 5 minutes at 37 ºC, 5% CO2. After the incubation period, 8 mL

of high glucose media was added to the cells and mixed, then the 10 mL

suspension was transferred to a 15mLfalcon tube, and centrifuged for 5

minutes at 1200 RPM and 4 ºC. In the meantime, the media used to freeze the

cells was prepared by adding 50% filtered FBS to 40% high glucose media

and 10% dimethyl sulfoxide (DMSO). After 5 minutes of centrifuging, the

supernatant was discarded and the pellet or cell residue was retained. Then 4.5

mL of freshly prepared media was gradually added to the cells and mixed.

Subsequently, 1.5 mL of the cell solution was added to each of the three 2 mL

cryotubes. The cryotubes were stored in a -20 ºC freezer for 1 hour then

placed into a -80ºC freezer.

2.1.4. Cell Counting

The harvested cells were centrifuged for 5 minutes at 1200 RPM and 4 ºC then

the pellet was collected and resuspended in 5-10 mL pre-warmed high glucose

media. An aliquot of 10 µL cell suspension was mixed with 10 µL of trypan

blue (0.4%)(Sigma Aldrich, NSW, Australia).Then using a haemocytometer

chamber in a Nikon ECLIPSE TS100 light microscope (Nikon, USA)under 40

x magnification, cells were counted in the grids. Then after calculations, the

required number of cells were seeded into the designated plates. To calculate

the viable number of the cells per mL, the cells were counted from each set of

16 corner squares and divided by 4 as there are 4 sets of 16 corner squares

(Figure 2.1.1). The result was multiplied by 104 (10,000), and by dilution

factor of 2 as the cell suspension was diluted 1: 1 with trypan blue.

Total cells counted × Dilution factor ×104 = Total number of the viable cells /mL Number of squares

i.e. 125×2 × 104 = 625,000 cells /mL 4

Figure 2.1.1. The figure is showing grid lines of the haemocytometer chamber. Each red square indicates a corner square (a set of 16 squares)

2.2. THE SCRATCH WOUND HEALING ASSAY

The wound-healing assay used throughout this thesis is a straightforward and

low-cost method and is one of the advanced techniques used to examine cell

migration in vitro 218, 219. This simple method imitates the migration of cells in

the wound healing process observed during in vivo conditions 218, 219. The

primary stages of an in vitro wound healing assay includes creating a wound

or scratch on a confluent cell monolayer, collecting the images at the starting

point and at certain time intervals of cell migration until wound closure occurs.

The images are then viewed to measure and determine the rate of cell

migration 218, 219. Furthermore, this method may determine the efficacy of

different type of treatments or drugs on the rates of cell migration and wound

healing.

The EA.hy926 endothelial cells (≈2x105) were seeded in 12-well plates, in

high glucose media, and once they had reached 80-85% confluence, the

scratch wound healing assay was performed. The EA.hy926 cell monolayer

was then scratched with a 200 µL pipette tip (yellow tip) using a ruler as a

guide, to create a straight line through the centre of the well. Then the media

was removed and the cells rinsed once gently with pre-warmed DPBS, to

remove any cellular debris or remaining media 6. The cells were subsequently

treated with fresh media combined with drugs in different concentrations and

timeframes in accordance with the designed experiments. The preparation of

the drugs used for these experiments is described in Chapter 3.

Cultured cells reaching Incubate for 3 days 80-85% confluence at 37 oC

Cell monolayer Capture an Image scratched with 200 µL at 0,2,4 and 6 hrs pipette tip Incubation

Experimental timeline

2.2.1. Data Collection from the Wound Healing Scratch Assay

Cell migration was monitored using the Nikon ECLIPSE TS100 light

microscope (Nikon, USA) with attached camera. The wells were

photographed at the starting point of the experiment (0 hours) then at 2, 4 and

6 hours. Rates of cell migration were then evaluated by measuring the width

of the wound at each 2 hour time period until the end of the experiment, using

Image J software 6. Data values (µm/hour) were collected for each sample at 0,

2, 4 and 6 hours then the data was transferred to Microsoft Excel to determine

the slope of the line, for example see figure 2.2.1. The slope of the line has

been used to measure the speed of wound healing (µm/hour). The data from

single doses of agonists or antagonists was analysed to calculate the mean and

SEM which was then represented in histograms.

Statistical analysis was completed using GraphPad Prism software (version

6.0) (CA, USA to examine the rates of wound healing in a dose and time

dependent manner. The significance of differences between groups was

evaluated using one-way analysis of variance (ANOVA). Values are presented

(Chapter 3) as mean± SEM and differences were considered statistically significant at P < 0.05.

Figure 2.2.1.Graph showing the effect of adenosine receptor agonist (30nM) or antagonist (10nM) on the rate of wound closure (µM/hour) over time (hour). The control group had no drug treatment.

2.3. CELL PROLIFERATION ASSAYS

Cells are basic structural and functional parts of an organism 220.Cell

proliferation and differentiation plays an essential role in the development and

maintenance of organisms from embryogenesis to mature tissue homoeostasis

by replacing the old, dead or damaged cells with new ones 220. Cell

proliferation assays may include metabolic activity analysis, determination of

DNA replication rates or the detection of antigens on cell surfaces 220.The Cell

Counting Kit-8 (CCK-8) from Sigma Aldrich (NSW, Australia) was utilized to

measure the cell viability and proliferation in this study. CCK-8 contains a

highly water-soluble tetrazolium salt, WST-8 [2-(2-methoxy-4-nitrophenyl)-3-

(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt]

which generates a yellow colour due to the formation of water-soluble

formazan upon cellular reduction (Figure 2.3.1) 221, 222. In live cells, a

dehydrogenase enzyme in the cytosol of cells reduces WST-8 to produce a

formazan dye that is soluble in the tissue culture media and is directly related

to the number of viable cells 221.

Figure 2.3.1.The chemical structure of WST-8 in CCK-8 and formation of its formazan dye 221

The CCK-8 assay has several advantages when compared to other colorimetric assays such as the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-

Diphenyltetrazolium Bromide), XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-

Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) and WST-1 (2-(4-

Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) methods. CCK-8 is a convenient and simple assay and does not require any further steps regarding premixing. The water-soluble formazan dyes are non-radioactive and also have no toxic effects on cells. Detection of

CCK-8 formazn dye from absorbance measurements is also more sensitive than for other conventional tetrazolium salts 221, 222. Unlike CCK-8 or WST-8,

MTT produces a formazan dye, which is extremely insoluble in water. Its residue damages the cells and an accurate absorbance measurement cannot be obtained 222. Therefore, to prevent this problem the MTT assay requires an additional step to dissolve the formazan 222.

The ATP detection assay is a reliable and sensitive luminescence method that can be utilized as an alternative to the tetrazolium reduction or colorimetric assays such as MTT, MTS, XTT, WST-1, and WST-8 223, 224. The ATP assay detects viable cells by measuring ATP as a marker of live cells. Performing the luminescence assay has advantages in that the protocol is simple, highly sensitive and fast 223, 224.

For most of the experiments, the EA.hy926 endothelial cells were seeded in triplicate at 5000 cells/ well in 96 well plates. For the purpose of creating a calibration curve other wells were prepared with duplicate known numbers of viable cells (including4000, 5000, 6000,7000,8000 and 9000 cells per well).

Hence, cell numbers were compared to the average absorbance of each well to

75 create a standard curve (to see the calibration curve refer to Chapter 3). Once cells were seeded, the plate was incubated for 24 hours at 37 ºC, 5%

CO2.CCK-8 was either thawed at room temperature for 30 mins, or defrosted in the water bath at 37 ºC for 5 mins. After 24 hours incubation, the high glucose complete media was discarded from each well and subsequently replaced with freshly prepared high glucose complete media (200 µL) containing10 µL of various drugs. Then 10 µL of the CCK-8 solution was carefully loaded into each well of the plate to avoid producing any air bubbles

(air bubbles in the wells could interfere with the optical density O.D. reading).

The 96 well culture plate was then incubated at 37 ºC, 5% CO2, for 4 hours.

Then the plate was placed into the Infinite M200 PRO- TECAN microplate reader (Tecan group, Switzerland) to measure absorbance at 450 nm. The standard curve can be seen in Figure 2.3.2. The standard curve was used to determine cell numbers, to allow an accurate quantification of cell proliferation.

Statistical analysis for the cell proliferation was performed using a one-way analysis of variance (ANOVA) with version 6.0 of GraphPad Prism software.

Statistical differences in mean values of cell proliferation with different drug treatments and also compare means with the control group. A P-value of <

0.05 considered statistically significant.

Timeline for experimental protocol

Cultured Incubate cells in 96 for 24hrs well plate at 37 oC

Add 10 µL Incubate CCK-8 for 4hrs at solution 37 oC

Then measure absorbance at 450 nm

Calibration Curve 3

2.5

1.5

Absorbance 1

0.5

0 4000 5000 6000 7000 8000 9000 Cell Number / Well

Figure 2.3.2. A standard curve for the CCK8 cell proliferation assay at 450 nm absorbance.

2.4. WESTERN BLOTTING TECHNIQUE

2.4.1. Sample Preparation

The EA.hy926 cells were seeded in a75 cm2 cell culture flask. Once the cells

reached 80% to 85% confluency, the cells were detached from the surface of

the flask using a rotatable Techno Plastic Products (TTP) cell scraper

(Zellkultur Lab, Switzerland). It should be noted that the cells were not

trypsinized to separate them from the surface of the flask, as trypsin breaks

down the proteins and cell membrane and the whole cell (with its membrane)

was required for this experiment. The harvested cells were transferred to a 15

mL falcon tube, then counted as previously described and prepared for cell

lysis.

2.4.2. Cell Lysis

The cells were lysed using lysis buffer (1mM Phenylmethanesulfonyl fluoride

(PMSF), 10µM Leupeptin, 3mM Benzamidine, 5µM Pepstatin A, 1mM

Sodium orthovanadate, 0.1% Triton-X and Kinexus Buffer*), which was

freshly prepared using the recommended formula from the Heart Foundation

Research centre at Griffith University. Kinexus Buffer was prepared from

mixture of Mops Buffer, 2mM EGTA, 5mM EDTA, 30mM NaF, 40mM B-

Glycerophosphate and 20mM Sodium Pyrophosphate (NaPP). The cell

suspension was centrifuged at 1200 RPM, for 5 minutes at 4 oC and then the

supernatant was removed, then the cells were washed with ice-cold DPBS.

The cells were then re-centrifuged at 1200 RPM, for 5 minutes at 4 oC. The

supernatant was subsequently removed, leaving only the cell pellet. The cells

79 were agitated with the lysis buffer using the pipette tip, then transferred into an eppendorf tube and maintained on ice, with constant agitation for 30 minutes. Afterwards, the cell and lysis buffer mixture was centrifuged at

13200 RPM, for 10 minutes at 4 oC. The supernatant was then aspirated and preserved in a fresh eppendorf tube and kept on ice. To determine the protein concentration, the bicinchoninic acid assay (BCA) (Pierce BCA protein assay kit, ThermoFisher Scientific, VIC, Australia) was applied.

Experimental timeline

Cultured cells reaching 80-85% Incubate for 3 days at 37 oC confluence

Cells centrifuged at 1200 RPM 5 minutes

Mixture of cells and lysis buffer were agitated 30 minutes and kept in ice

Mixture centrifuged at 10 minutes 13200 RPM

Protein sample mixed with BCA Incubate for 30 minutes at 37 oC reagent

Then measure the absorbance at 540 nm

2.4.3. BCA Assay

The BCA protocol was used to measure protein concentrations of cell lysates

and compare these to known standard sample concentrations 225. Protein

samples and protein standards (Table 2.4.1) were combined with the BCA

reagents using the published company protocol for this kit and the final

product absorbance was measured using a spectrophotometer 225.

A clear 96 well plate (Sarstedt, Germany) was used to load the samples. The

BCA standard samples were prepared using bovine serum albumin (BSA) in

Kinexus buffer, standard samples (10 µL) were loaded in duplicate into each

allocated well. The protein samples were prepared in 1:5 dilution (5 µL

samples in 20 µL Kinexus buffer) then 10 µL of the protein samples loaded

into the empty wells. Afterward, 200 µL of BCA mixture of reagent part A

(BCA) and reagent part B (cupric sulphate) were added to standard and

protein samples. The 96 well plate was covered with aluminium foil to avoid

light exposure and incubated for 30 minutes at 37 oC. After the incubation

period, the plate was placed into the Infinite M200 PRO- TECAN microplate

reader (Tecan group, Switzerland) to measure the absorbance at 540 nm. Once

the sample protein concentration was determined using the standard curve, the

samples were diluted and aliquoted. The aliquots were frozen at -80 oC to

avoid protein degradation.

PCR BSA (µg/µL) Diluent Volume BSA From Vial tubes (µL) Volume Kinexus buffer (µL) A 2.5 0 21 2.0 Stock

B 1.5 13 39 2.0 Stock

C 1.0 14 28.0 From

sample

D 0.5 19.5 19.5 From

sample

E 0.25 16.5 16.5 From

sample

F 0.125 11 11.0 From

sample

G Blank 21 0.0 ---

Table 2.4.1.Standard sample preparation for the BCA standard curve.

2.4.4. Western Blot Assay

The Western blot assay is a common technique used to separate and detect

specific proteins from a complex mixture. This technique separates proteins by

molecular weight and type using gel electrophoresis. Proteins are then

transferred or blotted onto a membrane and the target protein detected using

specific antibodies 226-228. Western blot techniques allow the semi-

quantification of proteins 228.

A sample of the total protein (50 µg) was mixed in 6:1 loading buffer (a

mixture of β-mercaptoethanol and 2x trypan blue loading dye) and the samples

placed in a dry block heater (Ratek Instruments, Boronia, Australia) and

denatured at 97 oC for 5 minutes. Samples were then immediately transferred

onto ice in a cooler box (esky) and maintained on ice for another 5 minutes.

In addition, a loading control in the Western blot assay is essential to

normalize protein expression levels across all wells 229. In selecting a proper

loading control for each experiment, there are a few factors that need to be

considered including the protein molecular weight and sample type (tissue) 229,

230. For instance, a loading control should have a different molecular weight to

the target protein either small or large enough to be detected from the target

protein. Additionally, a selected loading control should be highly expressed in

the sample 229, 230. Beta (β)-Actin (42 kDa) and GAPDH (36 kDa) are the most

common loading controls due to their stable expression across all eukaryotic

cell types 229. Moreover, GAPDH is essential for glycolysis and has numerous

roles in nuclear function including apoptosis and regulation of transcription 229.

2.4.5. Preparation of Gel Electrophoresis

Proteins were separated according their molecular weight using 10% sodium

dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which is

suitable for medium molecular weight proteins up to 100 kDa. The SDS-

PAGE gel consists of two parts: resolving gel and stacking gel. The resolving

gel was prepared (Table 2.4.2) and loaded into 2/3 of the gel cassette (BioRad,

NSW, Australia) and allowed to solidify for 45 minutes. Meanwhile, the

stacking gel was prepared (Table 2.4.2) and added on top of the resolving gel,

then the 15 well combs were inserted into the gels and left to solidify for

another 35 minutes. The gels were then placed into the electrophoresis tank,

which had been filled with 1Χ running buffer (3g/L Trizma Base, 14.4g/L

Glysine, 1g/L SDS). The combs were subsequently removed and pre-stained

protein ladder or molecular weight marker (ThermoFisher Scientific, VIC,

Australia) was loaded in the first well and 28 µL of samples into the remaining

wells. The gel electrophoresis was run at 150 volts for approximately 50

minutes until the samples reached the bottom of the gel.

Reagents Resolving Gel Stacking Gel

Volume (µL) Volume (µL)

40% Acrylamide 10000 1250

0.5M Tris-HCl pH 6.8 ------3150

1.5M Tris-HCl pH 8.8 10000 ------

10% SDS 400 125

N,N,N′,N′Tetramethylethylenediamine 20 12.5

(TEMED)

10% Ammonium Persulfate (APS) 200 62.5

Distilled deionised water 19400 7.950

Table 2.4.2.Materials required for the SDS-PAGE electrophoresis gels used in the Western blot studies

2.4.6. Electrotransfer Of Proteins Onto Membrane

Blotting paper and sponge pads were immersed in the transfer buffer (Trizma

Base 3g/L, Glysine 20g/L, Methanol 25%) for 10 minutes. The SDS-PAGE

gels were cut from the top and bottom where there were no protein bands. In

the meantime, the Immobilon-FL polyvinylidinedifluoride (PVDF) transfer

membranes (Merck Millipore, VIC, Australia) were activated by soaking in

methanol for 1 minute then rinsing in milli-Q water for a further 1 minute.

Afterward, the cassettes were placed into the Western blot tray filled with

transfer buffer and the cassette was prepared including a sponge pad, blotting

paper, SDS-PAGE gels, PVDF transfer membranes, blotting paper and sponge

pad. Once the components were assembled, the cassette was firmly closed.

The tank was filled with transfer buffer and a block of ice was placed into the

tank to keep it cool. The cassette was placed into the tank and the transfer gel

was run at 75 volts for 1 hour and 30 minutes.

2.4.7. Membrane Blocking And Antibody Incubation

Following protein transfer, the PVDF transfer membranes were soaked in1Χ

TBST (Trizma Base 2.43g/L, NaCl 9g/L, HCl (for adjusting 7.6 pH) and 0.1%

Tween 20) for 5 minutes, and then membranes were blocked with Odyssey

Blocking Buffer (ThermoFisher Scientific, VIC, Australia) for one hour on a

rocking platform at room temperature. The PVDF transfer membranes were

washed with 1X TBS (Trizma Base 2.43g/L, NaCl 9g/L and HCl (for

adjusting 7.6 pH)) for 5 minutes to remove the residue of the blocking buffer

and then the PVDF transfer membranes incubated with the primary antibody

(5 mL), on a rocking platform at 4 oC overnight. Tween 20 (0.1-0.2%) was

added to the Odyssey blocking buffer, which was used to dilute antibodies.

The same protocol was applied in preparing the secondary antibody, however,

0.02% SDS was also added to the mixture. More information regarding

primary and secondary antibody suppliers and concentrations is presented in

Chapter 3.

To remove the residual antibody, the PVDF transfer membranes were washed

3 times with 1X TBST for 5 minutes using a rocking platform at room

temperature. The PVDF transfer membranes were incubated in 5 mL of

secondary antibody for 1 hour on a rocking platform away from light sources

at room temperature. After the incubation period, the PVDF transfer

membrane was washed 3 times with 1X TBST for 5 minutes on a rocking

platform at room temperature in order to remove the remainder of the

secondary antibody. The rinsed membranes were then washed with 1X TBS

for 2 minutes, and the membranes were subsequently ready to scan on the Li-

Cor Odyssey Western blotting system (LI-COR Biotechnology, USA).

Timeline for experimental protocol

Electrophoresis 1hr and 20 process minutes

Electrotransfer of proteins onto 1hr and 40 minutes membrane process

Membrane 1hr incubation at TBS washed 5 blocking room tempreture minutes

Membrane Incubate overnight in antibody primary antibody at TBST washed 15 minutes incubation 4oC

Membrane Incubate 1hr in secondary antibody antibody at room TBST washed 15 minutes incubation tempreture

2.4.8. Data Collection From Western Blot Assay

Images of the membranes were analysed using Image Studio application

(Version 5.2.5 by Li-Cor Odyssey, USA). Data was collected by measuring

the density and size of each protein band (Figure 2.4.1). The loading control β-

Actin was used to normalize all the data. As a result, the signals from each

band of the protein samples were divided by normalization factor of the same

band in the normalization channel (Table 2.4.3).

Statistical analyses were performed for Western blot assay using an unpaired,

two-tailed Student’s t-test for a single comparison and a one way-ANOVA for

more than two or multiple comparisons. Data analysis was performed using

version 6.0 of GraphPad Prism software. Values are presented as mean± SEM.

A P-value of < 0.05 was considered to be statistically significant.

Figure 2.4.1.Image showing an example of measuring density of the protein band with the yellow numbers in lanes 1 and 2 indicating the signal strength of each band.

Signal Normalization Target Signal Normalized Normalization Channel Factor Protein Signals (β-Actin)

Control 1 17100 17100÷26400* Control 1 3170 31700 ÷ 0.741 =0.741

Control 2 19600 19600÷26400* Control 2 2740 2740 ÷ 0.841 =0.841

Control 3 26400* 26400*÷26400* Control 3 2730 2730 ÷ 1 =1

Table 2.4.3. Showing example calculations of normalized data using the normalization channel (which is β-Actin). The red font (*) indicates the highest signal value and normalization factor values are indicated in blue font.

2.5. TUBE FORMATION ASSAY (ANGIOGENESIS)

Angiogenesis is an essential process required to develop new blood vessels

from the existing vasculature to support wound healing, tissue and embryonic

development. However, angiogenesis may play a pathological role in

conditions such as metastasis and tumour growth and impaired wound healing

as observed in diabetics 231, 232. The tube formation assay is a quick,

quantitative and highly reliable method to demonstrate in vitro angiogenic

activity of endothelial cells 231, 233. Also, the tube formation technique can

examine the effect of various compounds on angiogenic and anti-angiogenic

activity 234.

For more than 20 years, studies have demonstrated that the implementation of

a basement membrane extracellular matrix is an appropriate environment to

stimulate angiogenesis in endothelial cells 235-237. Endothelial cells rapidly

attach to the reconstructed basement membrane extracellular matrix after

seeding and form capillary-like structures in vitro 235, 237.

Basement membrane extracellular matrices have been prepared from extracts

of the engelbreth-holm-swarm tumours (EHS) 238, three-dimensional fibrin gel

matrices 239, collagen gels from the collagen of rat tail tendons 58 or

alternatively are available from laboratory suppliers such as BD Biosciences

(USA), Invitrogen (Australia) and others commercial sources 235, 237, 238.

Commercially produced Matrigel is the most commonly reported basement

membrane extracellular matrix over past 15 years. It is made from an

extracellular gelatine form mixture and basement membrane proteins isolated

from the murine Engelbreth Holm Swarm (EHS) tumour 237. Furthermore,

growth factor reduced Matrigel (GFRM) is another form of the Matrigel

91 matrix, in which the level of stimulatory cytokines and growth factors have been significantly reduced to prevent overstimulation of endothelial cells that may occur when using standard Matrigel 237, 240.

It is important to note that the formation of capillary-like structures are specific for the various types of endothelial cells that are derived from different tissues. Other cell types can form different structures 235, 237, 241. For example, metastatic tumour cells will migrate and invade the basement membrane matrix while salivary gland cells and mammary epithelial cells form spheres with a central lumen on the matrix 235, 237. Furthermore, tube formation assays using different types of endothelial cells such as human umbilical vein endothelial cells (HUVEC), EA hy926 cells, calf pulmonary artery endothelial cells (CPAECs) and bovine aortic endothelial cells (BAEC) have been reported 58, 216, 239, 242.

The tube formation protocol was performed to examine the angiogenic activity of the EA.hy926 cell line. The cell line was grown in a 75 cm2 tissue culture flask until it reached 80% confluence. Geltrex (Reduced Growth Factor

Basement Membrane Matrix) (Invitrogen, VIC, Australia) was utilized to coat a 96 well, flat clear bottom plate. The pipette tips and the 96 well plate were maintained in the -20oC freezer for a day prior to the assay, to prevent any solidification of the Geltrex during the experiment and when loading into the wells 216.

Geltrex (50 µL) was loaded into the each well of the plate and incubated at

37 ºC, 5% CO2, for 30-40 minutes to allow the gel to solidify. Afterwards, the cells were harvested using 2 mL trypsin incubated in the flask for 5 minutes at

37 ºC, 5% CO2. After the incubation period, 8 mL high glucose complete

media were added to the cells and mixed, then the cell suspension centrifuged

for 5 minutes at 300 RCF at 4 ºC. The supernatant was discarded and a

sufficient amount of DMEM added to the cells to resuspend the cells. Cells

(50,000) were loaded gently into the each well of a 96 well plate and the high

glucose compete media together with the drug treatment were then added to

give a final volume of 100 µL (more information about the treatment and its

concentration in presented in subsequent chapters). The plate was incubated

for a maximum of 18 hours at 37 ºC, 5% CO2.

2.5.1. Data Collection Of The Tube Formation Assay

The angiogenesis process was monitored through the Nikon ECLIPSE TS100

light microscope (Nikon, USA) with attached camera. The tube networks were

photographed in six random selected microscopic fields after 4 hours

incubation at the starting point of the tube formation, and after 18 hours at the

end of incubation. The quantity of total number of junctions, nodes and

branches in each individual image was determined using Angiogenesis

Analyzer of Image J software (Figure 2.5.1) 216.

Statistical analysis was completed using one-way ANOVA to determine

significance of differences between groups and means of each group were

compared to the control group. Values are presented as mean± SEM.

Differences were considered statistically significant at P < 0.05.

Figure 2.5.1. Image showing an example of monitoring the tube formation after 18 hours and analysing the quantity of total number of junctions, nodes and branches

CHAPTER 3

ROLE OF ADENOSINE A1, A2A AND A2B

RECEPTOR IN WOUND HEALING

CHAPTER 3: ROLE OF ADENOSINE A1, A2A AND A2B

RECEPTOR IN WOUND HEALING

3.1. INTRODUCTION

3.1.1. Adenosine

Adenosine is a ubiquitous purine nucleoside that is produced following the

dephosphorylation of ATP and adenine nucleotides and it protects the tissues

and the cells during various conditions such as tissue damage or hypoxic-

ischaemic situations 4, 88. Its role becomes important during times of metabolic

stress and cellular energy imbalance caused by the difference in the amount of

energy required and energy supplied to cells 4, 88. Research has shown that the

generation of adenosine can induce wound healing and angiogenesis in

endothelial cells. Adenosine can also contribute to the development of fibrosis

including pulmonary fibrosis, hepatic fibrosis, peritoneal fibrosis and cardiac

fibrosis 4. Adenosine levels vary between 20-300 nM under physiological

conditions and these levels may rise during conditions including intense

exercise or in a low oxygen environment such as being at high altitude 243.

Under severe pathological conditions such as ischaemia, the concentration of

adenosine can rise to 30 µM 243, 244.

3.1.2. Signalling pathways of adenosine receptors in wound healing

Repair of damaged tissues is a vital homeostatic mechanism. It comprises

several phases including inflammation, neovascularization, tissue regeneration,

and tissue reorganization 41 (Chapter 1).

Adenosine and its receptors have essential roles in the production of matrix

and stimulation of neovascularization, which both crucial process for tissue

repair and wound healing 4, 41. Conversely, adenosine by binding to its

receptors, promotes the development of fibrosis in the skin, lungs and liver 41.

3.1.3. The Role of Adenosine A1 Receptor Agonists in Wound Healing

Cyclopentyladenosine (CPA) has a similar structure to adenosine and is

classified as a highly selective agonist for the adenosine A1 receptor as noted

245 in Table 1.6.1 . CPA, through binding to the adenosine A1 receptor located

on many cell types, can reduce damage associated with ischaemia, attenuate

calcium levels in the cytosol or reduce coronary vasodilation 245-250. In

addition, CPA through the activation of adenosine A1 receptors in macrophage

cell lines mediates nitric oxide (NO) release as well as the production of

inflammatory cytokines including IL-10 and TNF-α, which are essential for

tissue repair 128, 189, 245, 251, 252. In vivo studies using mice have shown that CPA

stimulates the proliferation of endothelial cells, keratinocytes and fibroblasts

245, 250. Also CPA acts as a mitogen and has strong vasodilator properties,

which support cell proliferation and wound healing 245, 250. Also, a dose–

response relationship for the efficacy of CPA on human dermal microvascular

endothelial cell proliferation has been reported 245.

3.1.4. The Role of Adenosine A2a and A2b Receptor Agonists in Wound

Healing

Adenosine A2A and A2B receptor agonists both cooperate to promote

angiogenesis and migration of endothelial cells and fibroblasts through cAMP-

and PKA (protein kinase A) - dependent pathways 5, 97. A study using human

coronary endothelial cells reported the presence of the adenosine A2A

89 receptors on the cell surface that stimulates an increase in cAMP production .

Also, another study using human aortic endothelial cells in culture, observed

253, 254 messenger RNA encoding adenosine A2A and A2B receptors . Activation

of the adenosine A2B and A3 receptors in human mast cells stimulates

angiogenesis through the release of vascular endothelial growth factor (VEGF)

and interleukin-8 (IL-8) while in microvascular endothelial cells they elevate

255 the expression of angiopoietin 2 . Adenosine A2B receptors through Gq

protein signaling activate the expression of angiogenic factors IL-8, VEGF

and basic fibroblast growth (bFGF) in the human microvascular endothelial

cell lines (HMEC-1) 71.

Moreover, the rate of cell proliferation has been reported to be enhanced by

different concentrations of adenosine when applied to human umbilical vein

endothelial cell lines (HUVEC) through the stimulation of VEGF production

256. Endothelial cells in the vasculature generate adenosine from adenine

nucleotides and respond to adenosine through increased proliferation and

migration 4, 120, 257-260. Adenosine also causes vascular leakage through

coronary vasodilation 4, 120, 257-260. In addition to the promotion of wound

healing following activation of adenosine receptors, stimulation of the

adenosine A2A receptors may also enhance extracellular matrix production in

healing wounds5. Human foreskin dermal fibroblasts were recognized to

express adenosine A1 and A2 receptors that produce matrix through regulating

4, 261 intracellular levels of cAMP . Also, the stimulation of adenosine A2A

receptors in these cells directly increases collagen production 4, 262. A study

using a bleomycin-induced dermal fibrosis model reported that stimulation of

adenosine A2A receptors is responsible for increased collagen production in the

skin. Adenosine A2A receptor knockout mice or mice treated with the

adenosine A2A receptor antagonist, such as ZM241385, were protected from

increased diffuse dermal fibrosis 4. While the receptor populations on the

endothelial cells have been identified there is limited published research

investigating selective adenosine A2A and A2B receptor agonists or antagonists.

3.1.5. The Role of Adenosine A3 Receptor Agonists in Wound Healing

The adenosine A3 receptor couples to several G proteins such as Gi α2, Gi α3

263-265 and Gq α . The adenosine A3 receptor has been reported to induce

selective RhoA-dependent activation of phospholipase D (PLD). Also

adenosine A3 receptor activation modulates mitogen-activated protein kinase

(MAPK) signalling and stimulates protective extracellular signal-regulated

kinases (ERK1/2) in cardiomyocytes 103, 104, 265-267 and contributes to a

265 protective function in cardiovascular diseases . The adenosine A3 receptor

signalling plays a principally anti-inflammatory role in bleomycin-treated

mice, however it also acts to elevate the levels of matrix metallopeptidase-9

(MMP-9) that can stimulate the activation of transforming growth factor

(TGF)-β1 and downstream fibrotic pathways 268. Moreover, this study

99 reported that stimulation of the adenosine A3 receptors can contribute to the intensification of pulmonary fibrosis through regulation of MMP-9, and/or

268 mediating eosinophil activation . Therefore, the adenosine A3 receptors can contribute to fibrotic and inflammatory processes and can adjust the balance between normal wound closure and the development of tissue fibrosis 268.

Furthermore, adenosine A3 receptor agonists have the capacity to provide protective responses as well as adverse inflammatory responses in the heart and vasculature 265. Several studies using in vivo mice models or in vitro

(neuronal cells, macrophages and bone marrow cells) protocols have demonstrated that the exogenous activation of adenosine A3 receptors leads to considerable cardioprotection and tissue injury prevention during and

265, 269-272 following ischaemia reperfusion . Adenosine A3 receptors can induce a vasoprotective response and have a significant role in vascular injury and modifying the post-ischaemic myocardial phenotype 265, 273. During tissue injury, tumor growth, ischaemia and inflammation, adenosine is released into the extracellular space and stimulates the secretion of vascular endothelial growth factor (VEGF) and interleukin 8 (IL8) through adenosine A2B receptors and it also stimulates angiopoietin-2 secretion through adenosine A3

255 receptors . As a result, the adenosine A3 receptor on mast cells and human umbilical vein endothelial cells (HUVEC) acts as a promoter of angiogenic signals 255.

While all the adenosine receptor subtypes have been implicated in cell proliferation and angiogenesis, this study focuses on the adenosine A1, A2A and A2B receptors as mRNA of the adenosine A3 receptor has not been found in previous studies using the EA.hy926 endothelial cells.

100

3.2. AIMS

The aims of this research are as follows:

1. To measure the effect of selective adenosine A1, A2A and A2B receptor agonists

and antagonists on the rate of wound closure in EA.hy926 endothelial cells,

using the wound healing scratch assay in vitro.

2. To determine the effect of selective adenosine receptor agonists and

antagonists in stimulating EA.hy926 endothelial cell proliferation.

3. To establish the protein expression level of adenosine A1, A2A and A2B

receptor subtypes in EA.hy926 endothelial cells, using Western-blotting

assays.

3.3. METHODS AND MATERIALS

3.3.1. Preparation of Adenosine Receptor Agonists And Antagonists

The selective adenosine receptor agonists and antagonists used in this study

included:

• N6-cyclopentyladenosine (CPA, agonist) and 8-cyclopentyl-1,3-

dipropylxanthine (DPCPX, antagonist) for the adenosine A1 receptor;

• 2-p-(2-caroboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine

(CGS21680, agonist) and 4-(-2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-

a][1,3,5]triazin-5-ylamino]ethyl)phenol (ZM 241385, antagonist) for the

adenosine A2A receptor: and

• 5’-N-ethylcarboxamidoadenosine (NECA, agonist) and N-(4-cyanophenyl)-2-

[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)phenoxy]-

acetamide (MRS1754, antagonist) for the adenosine A2B receptor.

101

The adenosine A3 receptor agonist and antagonist were not utilised for these studies as previous work in our laboratory (unpublished data), have shown that there is no mRNA expression for the adenosine A3 receptors in the EA.hy926 endothelial cell line 274. Furthermore, this work showed that the addition of the non-selective adenosine receptor antagonist 8-sulphylphenyltheophylline did not affect the rate of wound closure in the scratch assay, indicating that residual adenosine (if it is released) in cell culture conditions does not affect cell function in this assay.

It should also be noted that NECA is a non-selective agonist for the adenosine

A2 receptors, hence ZM241385 (10 nM) was added to experiments exploring adenosine A2B receptor function to exclude the adenosine A2A receptor.

Different concentrations of agonists were utilised (1-300 nM) in the initial scratch assay experiments to obtain concentration response curves, determine the EC50 value and establish an appropriate concentration of agonist required for rest of the experiments. The Ki values for adenosine receptor antagonists are as follows, DPCPX (adenosine A1 receptor, 3.8 nM), ZM241385

(adenosine A2A receptor, 1.4 nM) and MRS1754 (adenosine A2B receptor, 1.97 nM). A final concentration of 10 nM was used for all the adenosine receptor antagonists in this study.

Stock solutions of the adenosine agonists and antagonists (1 mM) were dissolved in DMSO and stored as aliquots in the -20 oC freezer until further use. All drugs were freshly prepared on the day of the experiments from thawed stock solution by diluting with high glucose complete media to reach the concentration required for experiments. The reagents used in this experiment are DMSO, CPA, DPCPX, NECA, MRS1754 and CGS21680

102

from Sigma Aldrich, NSW, Australia and ZM241385 from Tocris Bioscience,

Bristol, UK.

3.3.2. Wound Healing Scratch Assay

EA.hy926 endothelial cells (2x105 cells) in high glucose complete media were

seeded into the 12-well plates in triplicate, then incubated until the cells

reached 80-85% confluence. The cell layer was then scratched with a 200 µL

pipette tip and prepared agonists and antagonists were loaded into the wells.

The wounds were photographed at the starting point of the treatment (0 hours)

and at 2, 4 and 6 hours (end of the treatment). For more detail on this assay

refer to Chapter 2. For the vehicle control, diluted DMSO was added to the

scratch assay. In these experiments DMSO (0.03%) did reduce wound closure

rates when compared to control conditions. Analysis of the concentration

response curves to the selective adenosine receptors agonists were performed

using GraphPad Prism software (CA, USA) (version 6.0).

3.3.3. Cell Proliferation Assay

The EA.hy926 endothelial cells were seeded in triplicate at 5000 cells/ well in

96 well plates and also, 4000, 5000, 6000, 7000, 8000 and 9000 cells were

seeded in duplicate per well, respectively, to form the standard curve. They

were all incubated for 24 h at 37 ºC, 5% CO2 (Chapter 2). The adenosine

receptor agonists and antagonists were prepared using high glucose complete

media at 30 nM and 10 nM concentrations respectively. In addition, for

control experiments cells were treated with normal high glucose complete

103 media and vehicle controls using DMSO at two different dilutions of 0.004% and 0.03% v/v (Table 3.3.1). The plate was placed into the micro-plate reader and the absorbance measured at 450 nm (Chapter 2).

104

Conditions Concentration

CPA CPA 30 nM + high glucose Complete Media

DPCPX DPCPX 10 nM + high glucose Complete Media

CPA+ DPCPX CPA 30 nM + DPCPX 10 nM + high glucose

Complete Media

CGS21680 CGS21680 30 nM + high glucose Complete Media

ZM 241385 ZM241385 10 nM + high glucose Complete Media

CGS21680 + ZM CGS21680 30 nM + ZM241385 10 nM + high glucose Complete Media 241385

NECA NECA 30 nM + ZM241385 10 nM + high glucose

Complete Media

MRS1754 MRS1754 10 nM + high glucose Complete Media

NECA+ MRS1754 NECA 30 nM + ZM241385 10 nM + MRS1754 10 nM

+ high glucose Complete Media

Control Cells + high glucose Complete Media

Vehicle Control 1 0.004% DMSO + high glucose Complete Media

Vehicle Control 2 0.03% DMSO + high glucose Complete Media

Negative Control No cells –only high glucose Complete Media

Table 3.3.1. Treatment conditions and the concentrations

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3.3.4. Western Blotting Technique

The EA.hy926 endothelial cells were harvested, lysed and prepared for the

Western blotting technique as explained in Chapter 2. Protein samples (50 µg)

were loaded into the gels and run at 150 volts for approximately 50 minutes

until the samples reached the bottom of the gel. The protein samples were then

transferred to PVDF membranes using 75 volts for 1 hour and 30 minutes. The

membranes were treated with blocking buffer for 1 hour then they were

incubated overnight at 4°C with rabbit polyclonal primary antibody for

adenosine A1 receptor (1:500) (Abcam, Cambridge, UK), adenosine A2A

receptor (1:500) (Santa Cruz Biotechnology, CA, USA) or adenosine A2B

receptor (1:200) (Merck Millipore, VIC, Australia). Afterward, the

membranes were incubated in horseradish peroxidase-conjugated goat anti-

rabbit secondary antibody (1:10000) for adenosine A1 and A2A receptor and (1:

500) for adenosine A2B receptor (Santa Cruz Biotechnology, CA, USA) for 1

hour at room temperature. Furthermore, beta (β)-Actin antibody was using as a

loading control (1:1000) (Thermo Fisher, VIC, Australia) for all three

adenosine receptor subtypes and a horseradish peroxidase-conjugated goat

anti-rabbit antibody was used as the secondary antibody (1:3000).

The expression of the proteins was examined by comparing their size with

protein ladder or molecular weight markers.

106

3.4. RESULTS

3.4.1. The Effect Of Selective Adenosine Receptor Agonists On

EA.hy926 Endothelial Cells Using The Wound Healing Scratch Assay

The data for the wound healing scratch assay was obtained from measuring

the width of the scratch at 2 hourly intervals, using Image J software. The

average speed of wound healing was examined by comparing the effect of

different concentrations of adenosine receptor agonists. The results were

analysed using a one way ANOVA. The effective concentration producing

50% maximal effective concentration response (EC50) to agonist (log

concentration) was calculated based on curve fitting using GraphPad Prism

(Figure 3.4.1).

107

40 M/hrs)

µ 30

CGS21680 20 CPA

NECA 10

Rate of wound closure ( 0 -10 -9 -8 -7 -6 Log Agonist [M]

Figure 3.4.1.Concentration response curves (CRC) showing the effect of selective adenosine receptor agonists on EA.hy926 endothelial cells and the rate of wound healing, n= 6 per group

108

3.4.2. Wound Healing Scratch Assay Using The Selective Adenosine

A1 Receptor Agonist

The adenosine A1 receptor agonist CPA caused a significant concentration

dependant increase in the rate of wound healing at all concentrations of CPA

when compared to the control group (P< 0.0001) (Figures 3.4.2 and 3.4.3).

The EC50 value for CPA in the wound healing scratch assay of EA.hy926

endothelial cell was 7.16 x 10-9 M (confidence intervals 4.75 x 10-9 – 1.08 x

10-8 M). The data also shows that the efficacy of CPA and its ability to

stimulate wound healing was greater than the two other selective adenosine

receptor agonists, CGS21680 and NECA (adenosine A2A receptor and A2B

receptor agonists respectively) (Figure 3.4.1). However, CPA was not the

most potent agonist in stimulating the wound healing scratch assay with the

order of potency being CGS21680 < CPA < NECA (Figure 3.4.1).

109

Figure 3.4.2.Wound healing scratch assay using different concentrations of CPA on EA.hy926 endothelial cells. CPA concentrations from left to right are control (0 nM), 1 nM, 3 nM, 10 nM, 30 nM, 100 nM and 300 nM CPA. Magnification x40

110

60 **** **** **** m/hrs) µ **** 40 **** ****

20 Rate of wound healing ( healing wound of Rate

0 0 1 3 10 30 100 300 CPA concentrations (nM)

Figure 3.4.3. Graph showing the rate of the wound healing with increasing CPA concentrations compared to the control Data is represented as mean ± SE, n= 6, **P < 0.01, ***P < 0.001 and **** P < 0.0001 versus control (0)

111

3.4.3. Wound Healing Scratch Assay Using The Selective Adenosine

A2A Receptor Agonist

CGS21680 was used as a selective agonist for the adenosine A2A receptors.

The rates of wound healing at all concentrations of CGS21680 were

significantly increased when compared to the control group (P< 0.0001)

(Figures 3.4.4 and 3.4.5). The EC50 value for CGS21680 in the wound healing

scratch assay of EA.hy926 endothelial cells was 3.25 x 10-9 M (confidence

intervals 2.83 x 10-10 – 3.74 x 10-8 M). The CGS21680 was more potent than

the other two adenosine A1 and A2B receptor agonists (CPA and NECA) used

in this assay (Figure 3.4.1). While CGS21680 was the most potent of the

selective agonists used in this assay, its efficacy was low when compared to

the other two agonists with respect to rates of wound healing.

112

.Figure 3.4.4.Wound healing scratch assay using different concentrations of CGS21680 (CGS) on EA.hy926 endothelial cells CGS concentrations from left to right are: control group (0 nM), 1 nM, 3 nM, 10 nM, 30 nM, 100 nM, 300 nM. Magnification x40

113

80 **** **** **** **** **** ****

m/hrs) 60 µ

20 Rate of wound healing ( healing wound of Rate

0 0 1 3 10 30 100 300 CGS 21680 concentrations (nM)

Figure 3.4.5.Graph illustrating the rate of the wound healing with increasing CGS21680 concentrations compared to the control. Data is represented as mean ± SE, n= 6, *P < 0.05, **P < 0.01 and **** P < 0.0001 versus control (0)

114

3.4.4. Wound Healing Scratch Assay Using The Selective Adenosine

A2B Receptor Agonist

NECA was used as an agonist for adenosine A2B receptor in this study, but as

previously explained, NECA is a non-selective agonist for the adenosine A2

receptor. Therefore, NECA was combined with the adenosine A2A receptor

antagonist ZM241385 (10 nM) to block the adenosine A2A subtype. The rates

of wound healing at all concentrations of NECA were significantly increased

when compared to the control group (P < 0.05) to (P< 0.0001). (Figures 3.4.6

and 3.4.7). The EC50 value for NECA in the wound healing scratch assay of

EA.hy926 endothelial cells was 1.48 x 10-8 M (confidence intervals 2.83 x 10-

10 – 3.74 x 10-8 M). Of the three agonists tested, NECA was the least potent on

EA.hy926 endothelial cells in stimulating wound healing in the scratch assay

(Figure 3.4.1).

115

Figure 3.4.6.Wound healing scratch assay using different concentrations of NECA combined with ZM241385 (10 nM) on EA.hy926 endothelial cells. NECA concentrations from left to right are: control group (0 nM), 1 nM, 3 nM, 10 nM, 30 nM, 100 nM and 300 nM. Magnification x40

116

60 **** **** **** * ** m/hrs) µ 40

20 Rate of wound healing ( healing wound of Rate

0 0 1 3 10 30 100 300 NECA Concentrations (nM)

Figure 3.4.7.Graph showing the rate of the wound healing with increasing NECA concentrations compared to the control. Data is represented as mean ± SE, n= 6, *P < 0.05, **P < 0.01, *** P < 0.001 and **** P < 0.0001 versus control (0)

117

3.4.5. The Effect Of Selective Adenosine Receptor Antagonists On

Agonist Induced Increases In Rates Of Wound Closure

Selective adenosine receptor antagonists were used in the EA.hy926

endothelial cell wound healing scratch assay. Single concentrations of

selective adenosine receptor agonists (30 nM) were used in the wound healing

scratch assay and tested against selective adenosine receptor antagonists at 10

nM. The rates of wound healing with all the adenosine receptor agonists

increased as follows; adenosine A1 receptor agonist (CPA 30 nM) increased

by 42.29%, adenosine A2A receptor agonist (CGS21680 30 nM) rose by 49%

and adenosine A2B receptor agonist (NECA 30 nM) was elevated by 20.57%

when compared with their respective control group (P < 0.0001) (Figure 3.4.8).

The selective adenosine A1 and A2B receptor antagonists (DPCPX 10 nM) and

(MRS1754 10 nM) considerably decreased the rate of wound healing in

comparison with the control group respectively (P < 0.0001) (Figure 3.4.8).

Furthermore, the wound closure rate was significantly reduced by the selective

adenosine A2A receptor antagonist (ZM241385 10 nM) when compared to its

control group (P < 0.05) (Figure 3.4.8). The combined group included a

mixture of the selective adenosine receptor agonist (30 nM) with its relevant

selective adenosine receptor antagonist (10 nM) such as (CPA+DPCPX),

(CGS21680+ ZM241385) and (NECA+MRS1754). These results show the

rate of wound healing has been significantly decreased with the combined

group in comparison with its relevant selective adenosine agonist alone as

CPA, CGS21680 and NECA, respectively (P<0.0001) (Figure 3.4.8).

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Adenosine A1 Receptor Agonist & Antagonist 80 **** **** 60

** 40 **** **** 20

Rate of wound healing (µm/hrs) healing wound of Rate 0

Control CPA 30nM DMSO 0.03% DPCPX 10nM

CPA 30nM + DPCPX 10nM Adenosine A2A Receptor Agonist & Antagonist 80 **** 60 ****

40 *** 20

Rate of wound healing (µm/hrs) healing wound of Rate 0

Control ZM 10nM CGS 30nM DMSO 0.03%

CGS 30nM +ZM 10nM

Adenosine A2B Receptor Agonist & Antagonist 80 **** 60 ***

40 **** **** 20

Rate of wound healing (µm/hrs) healing wound of Rate 0

Control MRS 10nM NECA 30nM DMSO 0.03%

NECA 30nM + MRS 10nM

Figure 3.4.8. Graph showing the effect of adenosine receptor agonist, antagonist and their combination on rate of wound healing One-Way ANOVA was applied to analyse data. Data is expressed as mean ± SEM, n= 6, **P < 0.01, *** P < 0.001 and **** P < 0.0001 versus the control. CGS21680 (CGS), ZM241385 (ZM) and MRS1754 (MRS)

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3.4.6. Effect Of Selective Adenosine Receptor Agonists And

Antagonists On Cell Proliferation

A standard curve was generated to convert sample absorbance values into cell

numbers using 4000, 5000, 6000, 7000, 8000 and 9000 cells per well.

Analysis of the standard curve showed that absorbance values were

proportional to cell numbers, as indicated by figure 2.3.2 in Chapter 2.

The results show that cell proliferation was significantly decreased by the

vehicle control treatment (0.004% and 0.03% DMSO) when compared with

the control group (P < 0.0001) (Figure 3.4.9). Also, increased cell proliferation

was observed in cells treated with the adenosine A1 and A2B receptor agonists

(CPA 30 nM and NECA 30 nM) when compared with the control groups (P <

0.01) and (P < 0.05) respectively (Figure 3.4.9). However, in comparison with

control groups, the other treatment groups did not show any changes (P >

0.05). Figure 3.4.9 shows that all the adenosine receptor subtype agonists

(CPA, CGS21680 and NECA 30 nM) increased cell proliferation but they did

not have any significant differences when compared with each other (P > 0.05).

Moreover, the results showed that agonist-induced cell proliferation was

reversed by antagonist treatment; (CPA+DPCPX), (CGS21680+ ZM241385)

and (NECA+MRS1754). As a result, the effects of the adenosine receptor

agonists were blocked by their selective adenosine receptor antagonists.

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3 ** *

Absorbance 1 *** ****

Ctrl

ZM 10 nMCGS + ZM CPA 30 nM CGS 30nM MRS 10 nM NECA 30nM NECA + MRS 0.03% DMSO DPCPXCPA 10 nM + DPCPX Blank (no cells) 0.004% DMSO Treatment

Figure 3.4.9.Graph showing the effect of different treatments on cell proliferation Control group or untreated (Ctrl), vehicle control group (0.004% and 0.03% DMSO), Adenosine receptor agonists 30 nM (CPA, CGS21680 and NECA), Adenosine receptor antagonists 10nM (DPCPX, ZM241385 and MRS1754) and combination group (mixture of adenosine receptor agonist 30 nM with antagonist 10 nM). The data was obtained through one way ANOVA. Data is expressed as mean ± SEM n= 6, *P < 0.05, **P < 0.01, *** P < 0.001 and **** P < 0.0001 versus control

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3.4.7. Protein Expression Of Adenosine Receptor Subtypes On

EA.Hy926 Cells

The results of Western blotting techniques confirmed the presence of

adenosine A1, A2A and A2B receptors of expected molecular size A1 (≈ 37 kDa),

A2A (≈ 45 kDa) and A2B (≈ 50-52 kDa) in EA.hy926 endothelial cells.

Furthermore, β-actin with a molecular size of (≈ 42 kDa) was used as a

loading control (Figure 3.4.10). As figure 3.4.10 shows, three different blots

were utilized for each adenosine A1, A2A and A2B receptor antibody and they

were individually analysed against internal controls.

Data analysis shows that the protein expression of adenosine A2A receptor was

considerably higher in EA.hy926 endothelial cells when compared with

adenosine A1 and A2B receptors (P < 0.05) and (P < 0.001) respectively

(Figure 3.4.11). Moreover, the adenosine A1 receptor had higher expression

when compared with adenosine A2B receptors (P < 0.01) (Figure 3.4.11).

122

Figure 3.4.10. Western blot images showing protein bands of (A) A1 ≈ 37 kDa, (B) A2A ≈ 45 kDa, (C) A2B ≈ 50-52 kDa and (D) β-actin ≈ 42 kDa. In all images the first lane contains the molecular weight marker. Each protein sample is loaded in the subsequent wells in triplicate

123

** 10000 * *** 8000

6000

4000

Relative Density Density Relative 2000

1 A 2A 2B A A Adenosine Receptor Subtypes

Figure 3.4.11. Graph demonstrating the relative expression of adenosine A1, A2A and A2B receptor subtypes on E.Ahy926 endothelial cells. The data was analysed using one way ANOVA. Data is expressed as mean ± SEM (n=6), *P < 0.05, **P < 0.01 and *** P < 0.001

124

3.5. DISCUSSION

This study measured the effect of selective adenosine A1, A2A and A2B receptor

agonists and antagonists on the rate of wound closure in EA.hy926 endothelial

cells. This project also determined the effect of selective adenosine receptor

agonists on cell proliferation and tube formation and determined the protein

expression level of the adenosine A1, A2A and A2B receptor subtypes in

EA.hy926 endothelial cells. Many studies have used cultured endothelial cells

to model different physiological and pathological conditions, particularly in

the study of angiogenesis 275. Primary endothelial cells have a restricted

lifespan and they can demonstrate characteristics that vary due to their multi-

donor origin 275. Immortalized cell lines have been reported to be generally

better characterized and more constant in their endothelial properties than

primary endothelial cells that were given an extended life span 275. This study

reported that the best characterized human macro vascular endothelial cell

lines and human micro vascular endothelial cell lines are EA.hy926 and

HMEC-1, respectively 275. The permanent human endothelial hybrid cell line

(EA.hy926) was generated by hybridisation of primary human umbilical vein

endothelial cells (HUVEC) and the human lung carcinoma cell line (A549/8)

200, 201. Many studies have used the EA.hy926 endothelial cell line. For

example, one study using these cells reported that the compound cordycepin

(3'-deoxyadenosine) significantly decreased cell proliferation and

angiogenesis and inhibited cell migration 216.

The results of this study illustrate that the rates of wound healing in EAhy926

endothelial cells were significantly increased by the selective adenosine A1,

A2A and A2B receptor agonists (CPA, CGS21680 and NECA respectively)

125 when compared with their control group. Also, the concentration response curves to the selective adenosine A1, A2A and A2B receptor agonists show that the rate of wound healing was concentration dependent. Adenosine exerts its effects via G-protein coupled cell surface receptors that can be divided into four subtypes: adenosine A1 and A3 receptors that inhibit adenylyl cyclase and

245 adenosine A2A and A2B receptors that stimulate adenylyl cyclase . Other research has determined that stimulation of adenosine receptors increased endothelial cell migration via cAMP- and PKA-dependent pathways 5.

Previous studies investigating cells in the wound area observed that the cells were distributed into two cell cycle phases; the log phase (G1) and the quiescent phase (G0) 245. A study using human fibroblasts and endothelial cells reported that both cell types in G1 and G0 phases responded in a dose– dependent manner to the adenosine A1 receptor agonist (CPA). Also, studies using human endothelial cells confirmed that adenosine is a mitogen for endothelial cells 245, 256, 276. Another study observed that the topical application of the selective adenosine A2A receptor agonist (CGS21680) in mice, significantly increased the wound closure rates when compared with mice treated with vehicle 1.5% methylcellulose (control group) 5.

The efficacy and potency of the three selective adenosine receptor agonists

(CPA, CGS21680 and NECA) on the rate of wound healing varies in the

EAhy926 endothelial cell line. For instance, the adenosine A2A receptor agonist (CGS21680) was more potent than CPA and NECA (adenosine A1 and

A2B receptors agonist respectively) on wound closure. On the other hand, the efficacy of CPA in rate of wound healing in EAhy926 endothelial cells was greater than CGS21680 and NECA. Similar to previous literature, the

126 adenosine A2B receptors agonist (NECA) were the least potent and had reduced efficacy in stimulating wound healing when compared to the other

two adenosine receptor agonists (CPA and CGS21680). The adenosine A2B receptor has the lowest affinity for selective agonists, that is, the agonists for the other receptor subtypes have potencies in the low nanomolar range while

the most potent of adenosine A2B receptor agonists have affinities around the 1

μM range 91. Similarly, a study using human umbilical vein endothelial cells

reported that the selective adenosine A2A receptor agonist CGS 21680 was more potent in activating adenylyl cyclase production than the non-selective

adenosine A2B receptor agonist (NECA). Although NECA was less potent than

CGS21680, it exhibited higher efficacy in the activation of adenylyl cyclase 71.

The rates of wound healing in EAhy926 endothelia cells were significantly increased by all three adenosine A1, A2A and A2B receptor agonists (CPA,

CGS21680 and NECA) when compared to their control groups and relevant selective adenosine receptor antagonists (DPCPX, ZM241385 and MRS1754 respectively). Also, when the selective agonists and antagonists were used together, the antagonists decreased wound closure rates indicating that all the adenosine receptor antagonists were able to reverse the effect of adenosine receptor agonists on the rate of wound healing in EAhy926 endothelial cells.

This indicates that all three adenosine receptor subtypes studied in these cells induce wound healing. Moreover, an in vivo study using mice showed that the topical application of the selective adenosine A2 receptor antagonist DMPX

(3,7-dimethyl-1- propargyl xanthine) did not affect the rate of wound healing itself, however it reversed the effect of the selective adenosine A2 receptor agonist (CGS21680) on the rate of wound closure 5. It should be noted that

127

DMPX and ZM 241385 are highly selective adenosine A2A receptor antagonists. Thus both of them are able to reverse the effect of the selective

277 adenosine A2A receptor agonist (CGS-21680) .

A decrease in rates of cell proliferation and wound healing was also caused by the addition of the vehicle control DMSO, which was used to make the stock solutions of the adenosine receptor agonists and antagonists. As observed in the proliferation assay, DMSO (vehicle control) significantly decreased cell proliferation in EA.hy926. This study also showed that the adenosine A1, A2A and A2B receptor agonists stimulated the proliferation of EAhy926 endothelial cells, an effect inhibited by their respective adenosine receptor antagonists.

The adenosine receptor subtypes have been reported to stimulate cell proliferation and wound healing in other in vitro studies, as well as in vivo studies. The adenosine A1 and A2B receptors have been reported to regulate proliferation and cell differentiation in vascular smooth muscle cells and to promote cell proliferation and migration of endothelial cells in wound healing

17, 91, 278 studies using mice . Moreover, the selective adenosine A1 receptor agonist (CPA) has been shown to significantly increase the proliferation of keratinocytes, fibroblasts and endothelial cells using bromodeoxyuridine labelled cells in an in vivo wound healing study in mice 245. The non-selective adenosine A2B receptor agonist (NECA) stimulates retinal angiogenesis, cell proliferation and cell migration as well as stimulating cAMP response element-binding protein (CREB) and extracellular signal-regulated kinase

(ERK) signalling pathways associated with cell survival and proliferation 113.

This same study also reported that selective adenosine A2B receptor antagonists blocked the effect of NECA and attenuated the proliferation of

128 human retinal endothelial cells, leading to abnormal angiogenesis as observed in diabetic retinopathy 113.

Western blotting results of this study determined that the protein expression of adenosine A1, A2A and A2B receptor subtypes varies in the EAhy926 endothelial cells. This study showed that the adenosine A2A receptors are predominantly expressed in EAhy926 endothelial cells when compared to the adenosine A1 and A2B receptors. The expression of adenosine receptor subtypes have been reported to vary in different cell lines such as isolated cerebral arterial muscle cells (CAMCs), dermal fibroblast and human umbilical vein endothelial cells (HUVECs), human microvascular endothelial cells-1 (HMEC-1) and human and porcine coronary artery endothelial cells 5.

The presence of the A2A, A2B, and A3 receptors subtypes were observed in cultured dermal fibroblasts and human umbilical vein endothelial cells using the Western blotting technique and reverse transcriptase–PCR were expressed

5 in both cell lines . In contrast, the adenosine A1 receptor was only expressed in human umbilical vein endothelial cells (HUVECs) but not in dermal

5 fibroblasts . Previous studies investigating the expression of adenosine A2A and A2B receptors reported that the adenosine A2A receptors were the predominant subtype and highly expressed in human umbilical vein endothelial cells (HUVECs), human and porcine coronary artery endothelial

71, 279 cells . In contrast, the expression of adenosine A2B receptors was higher in human retinal microvascular endothelial cells and human microvascular endothelial cells-1 (HMEC-1) 71, 253, 280. Feoktistov et al., 2002 suggested that the adenosine receptor populations might depend on location in the vasculature, with the adenosine A2A receptor subtypes predominantly

129

expressed in endothelial cells lining large conduit vessels while the adenosine

A2B receptor subtypes were mainly expressed in endothelial cells lining

capillaries 71.

These experiments were completed in high glucose cell culture media and the

results observed in this study may be relevant to the high glucose environment

observed in humans with Diabetes Mellitus. A comparison of the effects of

high and low glucose concentrations in the media on EAhy926 endothelial cell

function in the wound healing assay can be seen in Chapter 6.

3.6. CONCLUSION

In summary, the selective adenosine A1, A2A and A2B receptors agonists (CPA,

CGS21680 and NECA) significantly increased the rate of the wound healing

and cell proliferation in EAhy926 endothelial cells. In contrast, the selective

adenosine receptors antagonists DPCPX (A1), ZM241385 (A2A) and

MRS1754 (A2B) considerably reduced wound healing rates and proliferation of

EAhy926 endothelial cells and blocked the effect of their relevant adenosine

receptor agonists. Furthermore, the selective adenosine A2A receptor agonist

(CGS21680) was more potent than the other adenosine receptor agonists but

less efficacious than selective adenosine A1 receptor agonists (CPA). This

study reported the protein levels of the adenosine A2A and A1 receptors are

highly expressed in EAhy926 endothelial cell lines. However, the adenosine

A2B receptor protein is also expressed in EAhy926 endothelial cells but in

reduced levels compared to the other two subtypes. This work suggests that all

three adenosine receptors can individually stimulate cell proliferation and

130 wound healing in the EA.hy926 endothelial cells and probably work together when the non-selective physiological agonist adenosine is released.

131

CHAPTER 4

EFFECT OF ACUTE CAFFEINE ON

ADENOSINE RECEPTOR MEDIATED

WOUND HEALING

132

CHAPTER 4: EFFECT OF ACUTE CAFFEINE ON

ADENOSINE RECEPTOR MEDIATED WOUND HEALING

4.1. INTRODUCTION

4.1.1. Acute caffeine

Caffeine (1,3,7-trimethylxanthine) is one of the most commonly used

psychostimulants that is consumed in the form of beverages and food by almost

70% of world’s population with an average consumption of 200-250 mg per day

per person 45, 175. Following ingestion of a single cup of coffee (which contains 80

mg of caffeine) by a 70 kg adult, the peak plasma caffeine concentration will

occur between 15 and 120 minutes after oral ingestion and equates to a plasma

concentration 8 to 10 mg/L 40, 135, 136. Caffeine is mostly present in coffee, which

includes trace amount of theophylline 281. Studies have reported that caffeine acts

as a non-selective antagonist of all the adenosine receptor subtypes 40, 281.

Caffeine blocks the actions of endogenously released adenosine which can

tonically activates its receptors 40. Therefore, caffeine potentially affects all areas

of the brain where adenosine receptors are expressed and endogenous adenosine

40 can exert its effects . Caffeine can also reduce adenosine A2A receptor mediated

activity during various pathological conditions and has been reported to attenuate

brain damage in Parkinson's disease, ischaemic brain injury associated

excitotoxicity and experimental allergic encephalitis 6, 45, 91, 282-285. Conversely,

caffeine can exacerbate acute liver damage and elevate the level of pro-

inflammatory cytokines such as IFN-ϒ, TFN-α and IL-4 6, 45, 91, 282-285.

133

Previous studies have demonstrated that caffeine has many actions other than

40, 45, 175, 176 blocking the adenosine receptors (especially adenosine A2A receptor) .

Caffeine can act on many other targets such as cAMP phosphodiesterase and phosphatidylinositol 3-kinase (PI3K) 40, 45, 175, 176. Caffeine, at higher concentrations, is a phosphodiesterase inhibitor and through increasing cAMP content in immune cells can exert immunosuppressive effects to attenuate tissue damage during an acute inflammatory injury 45, 175-177. Studies have reported that cAMP is a key cellular regulator that mediates various effects including cell adhesion, cytoskeletal dynamics and cell migration and it is a unique biofactor associated with tissue repair and regeneration 286-289. Caffeine can increase cytosolic Ca2+ and cAMP via ryanodine receptor activation and phosphodiesterase (PDE) inhibition, respectively 286-289. Moreover, studies have demonstrated that intracellular cAMP levels can be increased by forskolin which associates with adenylyl cyclase while, cis-N-(2-phenylcyclopentyl)- azacyclotridec-1-en-2-amine hydrochloride (MDL-12330A) is an adenylyl cyclase inhibitor that can decrease the levels of cAMP 290-294.

It has been reported that the protective effects of caffeine on tissues occurs via

45 inhibition of the adenosine A2A receptors . The therapeutic or detrimental effects of caffeine are significantly different, depending on whether it is consumed chronically or acutely 152. For instance, high doses of acute caffeine suppresses liver damage via inhibition of cAMP-phosphodiesterase 45. Also, chronic caffeine consumption can be neuroprotective by elevating plasma adenosine levels and up-regulating the adenosine A1 receptors. This is in direct contrast to the effects

295, 296 of acutely antagonising the adenosine A1 receptors .

134

Several researches have demonstrated that caffeine can have an effect on cutaneous wound healing procedures, particularly wound epithelialisation and closure, through its antioxidant properties and antagonism of the adenosine receptors 6. Human skin fibroblasts pre-treated with caffeine at low doses (0.01 mM) and intermediate doses (0.1 mM) were protected from apoptosis and necrosis induced by hydrogen peroxide 297. Caffeine also stimulated an increase in cell number and altered cell morphology, an effect which was mediated by a mechanism other than its antioxidant property 297. Furthermore, an in vivo study reported that caffeine increased cell proliferation in the ventral prostatic lobe in

5-week-old male Wistar rats 42.

The varied results observed following caffeine treatment may be caused by differences in tissue types, use of in vivo or in vitro models, differences in caffeine concentrations or duration of treatment 6.

Several in vivo and in vitro studies have reported that caffeine can attenuate angiogenesis in human umbilical vein endothelial cells (HUVEC), the chorioallantoic membranes (CAM) of chick embryos and zebrafish embryos 68, 69,

298. These studies demonstrated that caffeine inhibited in vitro proliferation of

HUVECs in a time and dose dependent manner through the stimulation of apoptosis. This suggested that the inhibitory effect of caffeine on angiogenesis could be related to its ability to induce apoptosis in these cells 68, 299, 300.

Angiogenesis is regulated via a balance between pro-angiogenic and anti- angiogenic factors. Another study reported that adenosine and adenosine receptor agonists can stimulate angiogenesis through down regulating the production of the anti-angiogenic matrix protein thrombospondin-1 (TSP-1) 68, 73. Caffeine, through blocking adenosine receptors, increased the expression of TSP-1 in

135

HUVECs 10, 68, 71, 73. Consequently, this study reported that caffeine mediated inhibition of angiogenesis was associated with cell apoptosis related to the elevated expression of TSP-1, an effect mediated by the adenosine A2B receptor subtype 68. High doses of caffeine (4,200–5,800 mg/kg) were able to inhibit the angiogenesis in zebrafish embryos at a dose which is much higher than standard daily consumption of caffeine in the adult human (150–300 mg per day) 69. This study also indicated that daily caffeine consumption would not cause defects in angiogenesis 69. Another study reported that exposure to caffeine (5–15 µmol per egg) reduced angiogenesis in chick embryo chorioallantoic membranes and chick yolk sacs by altering the expression of angiogenesis-associated genes, some of which are involved in the regulation of excess ROS production 298. For example, caffeine treatment was found to down-regulate the expression of vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor-2

(VEGFR-2), placental growth factor (PlGF), insulin-like growth factor-2 (IGF-2) and neuropilin-1 (NRP1) in chick embryo chorioallantoic membranes and chick yolk sacs, causing a decrease in angiogenesis with an associated negative impact on fetal development 298.

The purpose of this work was to investigate the effects of caffeine (applied acutely) on the EA.h926 endothelial cells in wound healing, cell proliferation and tube formation (angiogenesis) assays. Furthermore, this study investigated whether acute caffeine modified the cell function assays through affecting adenosine receptor function or cAMP levels.

136

4.2. AIMS

The aims of this study were as follows;

1. To determine the effect of acute caffeine treatment alone and on adenosine

A1, A2A and A2B receptor mediated stimulation of wound closure using the

EA.h926 endothelial cells in the wound healing scratch assay.

2. To study the role of adenylyl cyclase in the wound healing scratch assay.

3. To investigate the effects of acute caffeine treatment on EA.hy926

endothelial cell proliferation and angiogenesis in vitro using the cell

proliferation assay and tube formation assay.

4.3. METHODS AND MATERIALS

4.3.1. Treatment preparations (drugs)

All drugs were freshly prepared from stock solution (1 mM) as described in

Chapter 3. Caffeine solutions were prepared from stock solution by serial

dilutions with high glucose complete media to reach a final concentration of

30 µM. The selective adenosine A1, A2A and A2B receptor agonists (CPA,

CGS21680 and NECA) were prepared from stock solution by diluting with

caffeine mixture to reach a final concentration of 30 nM and 10 nM,

respectively (Table 4.3.1). The 1 mM stock solutions of MDL-12330A and

forskolin (Sigma Aldrich, NSW, Australia) were dissolved in DMSO and

stored as aliquots in the -20 oC freezer until further use. Then, MDL-12330A

and forskolin were prepared from stock solution by diluting with high glucose

complete media to reach a final concentration of 10 µM (Table 4.3.1). Also, a

137

mixture of MDL-12330A and caffeine was prepared through dilution of MDL-

12330A from stock solution into caffeine 30 µM treated high glucose

complete media to reach a final concentration of 10 µM (Table 4.3.1).

4.3.2. Wound healing scratch assay

EA.hy926 endothelial cells (2x105 cells) were seeded into the 12-well plates,

in triplicate in high glucose complete media and incubated until the cells

reached 80-85% confluence. The cell layer was then scratched with a 200 µL

pipette tip and high glucose complete media with caffeine (30 µM) were

loaded into the wells (Table 4.3.1). The wells were photographed at the

starting point of the treatment (0 hours) and at 2, 4 and 6 hours (end of the

treatment). For more detail, refer to Chapter 2. The data from the wound

healing scratch assay was obtained by measuring the width of the scratch at 2

hours intervals, using Image J software. The average speed of wound healing

was examined by comparing the effect of acute caffeine treatment on

adenosine receptor agonists. The results were analysed using One Way

ANOVA via GraphPad Prism software (CA, USA -version 6.0).

138

Conditions Concentrations

Control Cells + high glucose Complete Media

Acute Caffeine Caffeine 30 µM + high glucose Complete Media

CPA CPA 30 nM + Caffeine Mixture (30 µM)

CGS21680 CGS21680 30 nM + Caffeine Mixture (30 µM)

NECA NECA 30 nM + ZM241385 10 nM + Caffeine Mixture (30 µM)

MDL-12330A &Caffeine MDL-12330A 10 µM + Caffeine Mixture (30 µM)

MDL-12330A MDL-12330A 10 µM + high glucose Complete

Media

Forskolin Forskolin 10 µM + high glucose Complete Media

MDL-12330A & Forskolin MDL-12330A 10 µM + Forskolin 10 µM

Table 4.3.1. Preparation of the acute caffeine treatment and test drugs for the wound healing scratch assay

139

4.3.3. Cell proliferation assay

The EA.hy926 endothelial cells were seeded in triplicate at 5000 cells/ well in

96 well plates. Additionally, 4000, 5000, 6000, 7000, 8000 and 9000 cells per

well, were seeded in duplicate respectively, to form a standard curve. The

plates were then incubated for 24 h at 37 ºC, 5% CO2 as described in Chapters

2 and 3. The adenosine receptor agonists were prepared with the caffeine

mixture 30 µM (caffeine + high glucose complete media) at final

concentrations of 30 nM and incubated for 4 hours. In addition, this

experiment included the control conditions, where cells were treated with

normal high glucose complete media and vehicle controls conditions using

DMSO at two different dilutions of 0.004% and 0.03% (Table 4.3.2). The

plate was then placed into the micro-plate reader and the absorbance at 450

nm measured. For more detail refer to Chapter 2.

140

Conditions Concentrations

Acute Caffeine treatment Caffeine 30 µM + high glucose Complete Media

CPA CPA 30 nM + Caffeine Mixture (30 µM)

CGS21680 CGS21680 30 nM + Caffeine Mixture (30 µM)

NECA NECA 30 nM + Caffeine Mixture (30 µM)

Control Cells + high glucose Complete Media

Vehicle Control 1 0.004% DMSO + high glucose Complete Media

Vehicle Control 2 0.03% DMSO + high glucose Complete Media

Negative Control No cells + high glucose Just Complete Media

Table 4.3.2. Acute caffeine treatment conditions and concentrations

141

4.3.4. Tube Formation Assay

Geltrex (50 µL) was loaded into each well of the plate and incubated at 37 ºC,

5% CO2, for 30-40 minutes to allow the gel to solidify. Afterwards, 50,000

cells were loaded gently into each well of a 96 well plate and the drug

treatment added to give a final volume of 100 µL. Suramin (0.3%) inhibits

angiogenesis in the chick chorioallantoic membrane assay and was used as a

negative control in this study 301-303. The positive control for this work was the

basic fibroblast growth factor (bFGF, 25%), which is reported to stimulate

angiogenesis in isolated cells 301-303. Therefore, this experiment comprised the

following groups: control group containing cells treated with DMEM without

FBS; positive control group prepared from 25% basic fibroblast growth

factors (bFGFs) diluted in high glucose complete media with 10% FBS; a

negative control group prepared from 0.3% Suramin sodium salt, diluted in

high glucose complete media with 2% FBS; and acute caffeine treatment (30

µM) diluted in high glucose complete media with 2% FBS (Table 4.3.3). FBS

(2%) was used instead of 10% FBS to prepare acute caffeine conditions as

FBS can stimulate tube formation. Therefore, the FBS levels were reduced to

prevent interference of effect of FBS on acute caffeine treatment.

The plate was incubated for a maximum of 18 hours at 37 ºC, 5% CO2. At the

end of the incubation period tube formation was analysed using the Nikon

ECLIPSE TS100 light microscope (Nikon, USA) with attached camera. The

tube networks were photographed in twelve randomly selected microscopic

fields. The total number of junctions, nodes and branches in each individual

image was detected using the Angiogenesis Analyzer Image J software.

Statistical analysis was conducted using a two-way ANOVA to determine

142 significance of differences between groups and each mean of each group was compared to the control group. Values are presented as mean± SEM.

Differences were considered statistically significant at P < 0.05.

143

Conditions Concentrations

Control Cells + DMEM (No FBS)

Positive Control 25% bFGF + high glucose Complete Media (10% FBS)

Negative Control 0.3% Suramin Sodium Salt + high glucose Complete Media

(2% FBS)

Acute Caffeine treatment Caffeine 30 µM + high glucose Complete Media (2% FBS)

Table 4.3.3. Acute caffeine treatment conditions and concentrations

144

4.4. RESULTS

4.4.1. The Effect Of Caffeine Treatment On Adenosine Receptor

Mediated Rates Of Wound Closure In Ea.hy926 Endothelial Cells

The results of the wound healing scratching assay show that acute caffeine

treatment did not alter the rate of wound closure in EA.hy926 endothelial cells

in comparison with the control group (Figures 4.4.1 and 4.4.2).

The effect of caffeine (30 µM) was determined on the three selective

adenosine receptor agonists. The results showed that caffeine significantly

decreased the effect of the selective adenosine A1, A2A and A2B receptor

agonists (CPA, CGS21680 and NECA 30 nM) on wound healing rates by 28%

16% and 12%, respectively when compared with the untreated conditions (P <

0.0001) (Figure 4.4.2). Therefore, acute caffeine treatment can block the effect

of selective adenosine receptor agonists (CPA, CGS21680 and NECA) to

significantly reduce the rate of wound healing (Figure 4.4.2).

145

Figure 4.4.1.Images showing the wound healing scratch assay in the absence (panel 1) and presence of acute caffeine (30 μM, panel 2) The effect of caffeine (30 µM) on selective adenosine receptor agonists (CPA, CGS21680 and NECA) (30 nM) on the rate of wound healing in EA.hy926endothelial cells at 0 and 6 hrs are also presented (panels 3 to 5 respectively). Magnification x 40

146

80 ****

**** **** M/hour)

µ 60

Untreated 20 Acute Caffeine Treatment Rate of wound healing ( 0

Control CPA 30nM CGS 30nM NECA 30nM Treatments

Figure 4.4.2. Graph comparing the effects of adenosine receptor agonists on the rate of wound healing in untreated (dark blue bars; control, CPA, CGS21680 and NECA (30 nM)) and caffeine (30 µM) treated (light blue bars; control, CPA, CGS21680 and NECA (30 nM)) EA.hy926endothelial cells. Two-Way ANOVA was used to analyse data. Data are presented as mean ± SEM, n= 6. **** P < 0.0001

147

4.4.2. The Role Of Adenylyl Cyclase On Caffeine Treatment And Rates

Of Wound Closure In Ea.hy926 Endothelial Cells

The effects of forskolin and MDL-12330A were studied on the rate of wound

healing. The purpose of this work was to determine the potential role of cAMP

in the cellular effects of acute caffeine in wound closure. In this experiment

the EA.hy926 endothelial cells were treated with different drug treatments as

follows: acute caffeine treatment (30 µM); acute caffeine (30 µM)+ MDL-

12330A (10 µM); MDL-12330A group (10 µM); forskolin group (3 µM); and

forskolin (3 µM) and MDL-12330A group (10 µM).

The results demonstrated that the rate of wound healing in EA.hy926

endothelial cells increased by 32% with the caffeine treated control group

(T/control) when compared to the untreated control group (U/control),

however it was not significant (P > 0.05) (Figures 4.4.3 and 4.4.4). Also, the

wound-healing rate in EA.hy926 endothelial cells significantly increased by

85±4 µM/hour with the forskolin treatment in comparison with U/control

group (40±3 µM/hour) and T/control group (54±5 µM/hour) (P < 0.0001)

(Figure 4.4.4). In comparison with U/control and T/control group, the rates of

wound healing did not have any significant changes with the other treatment

group (P> 0.05) (Figure 4.4.4).

Consequently, while these results showed a non-significant increase in wound

healing rate with acute caffeine treatment, they also demonstrated a decrease

in caffeine stimulated rate of wound closure in the presence of the adenylyl

148 cyclase inhibitor MDL-12330A (which alone did not effect wound closure rates. Additionally, MDL-12330A significantly decreased the effect of forskolin on wound closure rate by 40% in EA.hy926 endothelial cells (P <

0.0001) (Figures 4.4.3 and 4.4.4). Therefore, the results with forskolin show that stimulation of adenylyl cyclase increases rates of wound closure in

EA.hy926 endothelial cells.

149

Figure 4.4.3. The effect of various treatments on EA.hy926endothelial cells in the wound healing scratch assay at 0 and 6 hr Panel 1 illustrates control conditions. Panel 2 shows the effect of acute caffeine treatment (30 μM) while Panel 3 demonstrates the effect of adenylyl cyclase inhibitor MDL-12330A (10 μM) alone and with acute caffeine (Panel 4). Panel 5 demonstrates the effect of adenylyl cyclase activator forskolin (3 μM) alone and in the presence of MDL-12330A (Panel 6). Magnification x 40

150

**** 100 ****

80 M/hour) µ

20 Rate of wound healing ( 0

U/ControlT/Control Forskolin MDL-12330A

MDL-12330A &Caffeine MDL-12330A & Forskolin Treatments

Figure 4.4.4. Graph showing the effect of acute caffeine, MDL-122330A and forskolin treatments on the rates of the wound healing Data are presented as mean ± SE, n= 6, **** P < 0.0001 versus Untreated control (U/Control) and Caffeine Treated control (T/Control) as indicated

151

4.4.3. The Effect Of Caffeine On Adenosine A1, A2A And A2B Receptor

Mediated Cell Proliferation

A standard curve was generated to convert sample absorbance values into cell

numbers (4000, 5000, 6000, 7000, 8000 and 9000 cells) as described in

Chapters 2 and 3. The calibration curve shows that absorbance values rose

with increasing cell numbers. Data for the proliferation assay were analysed

using one-way ANOVA and multiple comparisons.

EA.hy926 endothelial cell proliferation was considerebly decreased by vehicle

control treatments (0.004% and 0.03% DMSO) when compared with the

control group (P < 0.0001) (Chapter 3, Figure 3.4.9). Also acute caffeine

treatment did not alter the effects of DMSO in this assay (data not shown).

The effect of acute caffeine treatment (30 µM) on selective adenosine receptor

agonists CPA, CGS21680 and NECA (30 nM) mediated proliferation of

EA.hy926 endothelial cells was studied. The results show that acute caffeine

significantly decreased the cell proliferation stimulated by all the selective

adenosine A1, A2A and A2B receptor agonists (CPA, CGS21680 and NECA

30nM) when compared to the control (untreated conditions), (P < 0.0001)

(Figure 4.4.5). Consequently, these results show that the adenosine receptor

agonist mediated proliferation of EA.hy926 endothelial cell was non-

selectively blocked by acute caffeine .

152

**** **** ****

Absorbance 1 Untreated Acute Caffeine Treatment 0

Ctrl

CPA 30 nM CGS 30nM NECA 30nM Blank (no cells) Treatments (Adenosine Receptor Agonists)

Figure 4.4.5. Graph comparing the effect of acute caffeine on adenosine receptor mediated cell proliferation as indicated by absorbance for: control group (no drug) (Ctrl); Adenosine A1 receptor agonist (CPA 30 nM); adenosine A2A receptor agonist (CG21680 30 nM); and adenosine A2B receptor agonist (NECA 30 nM). The data are presented as mean ± SEM, n= 6 and analysed using two-Way ANOVA, **** P < 0.0001

153

4.4.4. The Effect Of Acute Caffeine Treatment On Angiogenesis And

Tube Formation In EA.hy926 Endothelial Cells

Tube formation was monitored using the Nikon ECLIPSE TS100 light

microscope (Nikon, USA) with attached camera as described in Chapter 2

(Figure 4.4.6). The total number of junctions, nodes and branches in each

individual image was determined using Angiogenesis Analyzer of Image J

software 216.

The addition of the positive control, 25% bFGF to EA.hy926 endothelial cells

considerably increased the formation of tubes when compared to the control

group (P < 0.0001) (Figure 4.4.7). Similarly, the addition of the negative

control suramin (0.3%) significantly decreased tube formation when compared

with the control group (P < 0.001). In these experiments, however, suramin

induced morphological changes in the EA.hy926 endothelial cells and the cells

did not survive in the presence of suramin after 18 hours incubation.

The results of the tube formation assay with acute caffeine treatment

demonstrated that caffeine significantly increased the number of nodes,

junction and branches when compared to control conditions, indicating that

acute caffeine induces angiogenesis and tube formation in EA.hy926

endothelial cells (Figure 4.4.7).

154

Figure 4.4.6. Images showing the tube formation assay with control (DMEM, Panel 1), acute caffeine treatment (30 μM, Panel 2), positive control (25% bFGF, Panel 3) and negative control (0.3% Suramin, Panel 4). Magnification x 40

155

600 **** **** Nb nodes

400 Nb Junctions

Nb Branches 200 ** ***

Calculated Quantity *

DMEM

Caffeine 30µM

25% bFGF (Possitive CTRL) 0.3% Suramin (Negative CTRL) Treatments

Figure 4.4.7. The effect of drug treatments on tube formation in EA.hy926 endothelial cells for: Control (DMEM); acute caffeine (30 µM); positive control (25% bFGF); and negative control 0.3% Suramin. Data are presented as mean ± SEM, n= 12. Data were analysed using two-way ANOVA *P < 0.05, **P < 0.01, *** P < 0.001 and **** P < 0.0001 versus control (DMEM)

156

4.5. DISCUSSION

This study determined the effects of acute caffeine treatment (30 µM) on

wound healing, cell proliferation and tube formation using EA.hy926

endothelial cells.

In this study using the immortalised EA.hy926 endothelial cell line, it was

found that acute caffeine (30 µM) did not significantly alter wound healing

rates or cell proliferation. Acute caffeine however did inhibit the stimulatory

effects of all three adenosine receptor agonists on the wound healing rate and

cell proliferation assays used in this study. Previous studies have reported that

adenosine is able to up-regulate the expression of vascular endothelial growth/

245, 304 vascular permeability factor . Selective adenosine A1, A2A and A2B

receptor agonists have been shown to stimulate the proliferation of various cell

types in vivo mice models and in vitro studies investigating keratinocytes,

fibroblasts, and endothelial cells as well as in normal and genetically modified

healing-impaired mice 5, 71, 113, 245.

This study demonstrated that the effects of all the selective adenosine receptor

agonists (CPA, CGS21680 and NECA, 30 nM) on rates of wound healing and

cell proliferation were reversed following caffeine treatment. Consequently,

these results support previous literature indicating the adenosine-antagonist

activity of caffeine. Caffeine has been reported to competitively inhibit

binding to adenosine receptors in brain membranes 51, 305. Another study

showed that caffeine was associated with reduced collagen deposition and

decreased fibrosis in the liver and lung, an effect due to the antagonist actions

of caffeine on adenosine receptors 41, 42, 262. Sarobo et al., 2012 reported that

inhibition of adenosine activity occurs with high doses of caffeine (25 mg/kg

157 per day) and that no changes were observed on adenosine activity at lower doses (2.5 mg/kg per day) 42.

In this study, the role of adenylyl cyclase in the wound healing assay and its effects on acute caffeine treatment were investigated. Forskolin was implemented as a positive control in these experiments. Forskolin directly stimulates the catalytic subunit of adenylyl cyclase to stimulate cAMP formation, which can affect cellular growth and morphology 306, 307. Also, forskolin is well recognised as a potent tool to study this enzyme and the role of cAMP in physiological and pathological processes 306, 307. A significant increase in the rate of wound closure was observed with the addition of forskolin to the assay. The effect of forskolin was reversed by the compound

MDL12330A, indicating that increased rates of wound healing were linked to stimulation of adenylyl cyclase and increases in intracellular cAMP levels.

Other studies have shown that exposure to forskolin considerably increased intracellular cAMP concentrations in different cell types such as endothelial cells, smooth muscle cells and glial cells 306. MDL-12330A, as an adenylyl cyclase inhibitor, significantly decreased the wound healing rate with acute caffeine treatment. Thus, the non-significant rise in rate of wound closure may be mediated in some part by cAMP in EA.hy926 endothelial cells. Another study has reported that caffeine inhibits cell proliferation in endothelial cells in both a time and dose dependent manner by inducing apoptosis 68. Moreover, a different study showed caffeine, in a dose-and time dependent manner, suppresses cell proliferation and impedes migration in a human keratinocyte

HaCaT cell line 6. Interestingly, other in vivo and in vitro studies have reported that caffeine can increase cell proliferation in human skin fibroblast, epithelial

158

cells and cell proliferation in the prostate gland of rats 6, 42, 297. The results of

tube formation assay demonstrated that acute caffeine treatment (30 µM)

significantly increased tube formation and angiogenesis in EA.hy926

endothelial cells and the changes observed were similar to the effect of 25%

bFGF on EA.hy926 endothelial cells. These results are contrary to several

other studies, which reported that caffeine treatment can inhibit angiogenesis

in different cell types. For example, adenosine receptor activation stimulates

the release of angiogenic factors to promote angiogenesis in zebrafish

embryos, an effect that was inhibited by caffeine 41, 69, 84, 85. Moreover, a study

using cultured human umbilical vein endothelial cells (HUVECs) reported that

caffeine at concentrations of 250 µM and 500 µM, inhibited HUVEC

proliferation and angiogenesis but when caffeine was applied at concentrations

lower than 100 µM, it had no effect on cell proliferation 68. Similarly, Li et al.,

2013 observed that caffeine had no effect on HUVEC induced tube formation

on Matrigel or cell migration in vitro 68.

4.6. CONCLUSION

In conclusion, caffeine did not affect the rate of wound healing or cell

proliferation of EA.hy926 endothelial cells. However, caffeine by its

adenosine receptor antagonist role blocked the effect of selective adenosine A1,

A2A and A2B receptor agonists and decreased the rate of wound healing in

EA.hy926 endothelial cells. The second messenger molecule cAMP does have

a role in increased wound healing rates, however caffeine at the concentration

tested in our assay did not increase wound healing rates. Nevertheless,

caffeine treatment did increase angiogenesis by stimulation of tube formation

159 in EA.hy926 endothelial cells. As this work has been completed on cells in a high glucose environment, acute caffeine may have a role in stimulating angiogenesis in diabetic patients.

160

CHAPTER 5

EFFECT OF CHRONIC CAFFEINE ON

ADENOSINE RECEPTOR MEDIATED

WOUND HEALING

161

CHAPTER 5: EFFECT OF CHRONIC CAFFEINE ON

ADENOSINE RECEPTOR MEDIATED WOUND HEALING

5.1. INTRODUCTION

5.1.1. Chronic Caffeine

A chronic caffeine treatment is defined as a continuous treatment of animal

models and /or cell lines with caffeine, lasting a few days up to a few weeks

using in vivo and in vitro conditions 6, 308-310. For example, an in vitro cell

proliferation study used primary human keratinocytes that were cultured and

treated with caffeine chronically for 3 and 5 days 6. Also, several in vivo

studies have investigated chronic caffeine treatments, administered to different

animals including rats and mice, with water containing caffeine for various

timeframes including 3 days, 14 days, 23 days and 6 or 12 weeks 308-310.

As described in Chapter 4, caffeine is a non-selective antagonist for all

subtypes of the adenosine receptor 152. This Chapter will investigate the role of

adenosine A1, A2A and A2B receptors following chronic caffeine treatment of

EA.hy926 endothelial cell on cell proliferation, migration and protein

expression.

Caffeine is ingested in beverages consumed daily and is taken chronically in

many humans 45. A single cup of coffee contains around 100 mg of caffeine 40,

175. Studies have examined the effect of chronic caffeine treatment on organ

and tissue function. An in vivo study demonstrated that chronic caffeine

inhibited the development of hepatic fibrosis in two mouse models of hepatic

injury and fibrosis 311. Another study using mice, reported that chronic caffeine

treatment reduced lung injury and inhibited inflammation in an adenosine A2A

162 receptor independent manner 175. This study also showed that the adenosine

A2A receptors were significantly up-regulated following chronic caffeine treatment when compared with the other adenosine receptor subtypes 175.

Moreover, another study showed that chronic caffeine consumption decreased liver tissue damage in mice, due to up-regulation of the adenosine A2A receptor and increasing adenosine mediated anti-inflammatory actions 45, 48.

It has been shown that the chronic consumption of caffeine can protect the brain from ishaemic damage as well 45, 49, 50. Chronic caffeine consumption can be neuroprotective by decreasing neuronal damage in the rat hippocampus in a model of global forebrain ischaemia 152, 295. Chronic adenosine receptor

antagonism with xanthines led to upregulation of the adenosine A1 receptor in

the rat brain, with no changes in the level of adenosine A2A receptor

152, 312 expression , leading to a shift in the balance of adenosine A1 and A2A receptor populations with prolonged caffeine consumption 152, 313. Furthermore, following chronic caffeine treatment in rats, a modified function of the

adenosine A1 and A2A receptor heteromer was reported. The authors suggested this altered function to be the cause of the potent tolerance to the psychomotor effects observed following the chronic consumption of caffeine 152, 308. As a result, upregulation of adenosine receptors by chronic caffeine treatment can cause increased levels of adenosine and adenosine mediated actions in the rat brain 152.

163

5.2. AIMS

Chronic caffeine is well documented to affect adenosine receptor subtype

populations in a range of cells and tissues. The purpose of this study was to

determine whether chronic caffeine affects endothelial cell function with

respect to wound healing and cell proliferation. This study will investigate the

effect of chronic caffeine treatment on adenosine A1, A2A and A2B receptor

populations in EA.h926 endothelial cell lines. As described previously, the

adenosine A3 receptor is not expressed in the EA.hy926 endothelial cells and

was not therefore studied in this Chapter. This study used a high glucose cell

culture solution, as previously described. While studies have reported the

effect of chronic caffeine treatment on adenosine receptors and their properties

in different types of endothelial cell lines, the EA.hy926 endothelial cells have

yet to be investigated. As a result, the aims of this study are to:

1. Determine the effect of chronic caffeine treatment on selective adenosine

A1, A2A and A2B receptor agonist stimulation of wound healing in

EA.hy926 endothelial cells.

2. Examine the effect of chronic caffeine treatment on cAMP levels and the

rate of wound closure in EA.hy926 endothelial cells, using the wound

healing scratch assay and selective adenylyl cyclase compounds.

3. Compare the effect of chronic and acute caffeine treatment on the wound

healing rate in EA.hy926 endothelial cells using the wound healing scratch

assay.

4. Establish the protein expression level of adenosine A1, A2A and A2B

receptor subtypes in EA.hy926 endothelial cells under chronic caffeine

treatment, using Western-blotting assays.

164

5.3. METHODS AND MATERIALS

5.3.1. Treatment Preparations (Drugs)

All drugs were freshly prepared from stock solution (1mM) as described in

Chapter 3. Caffeine was prepared from stock solution by several dilutions with

high glucose complete media to reach a final concentration of 30 µM.

Selective adenosine A1, A2A and A2B receptor agonists (CPA, CGS21680 and

NECA) were prepared from stock solution by diluting with high glucose

complete media to reach a final concentration of 30 nM, as described in

Chapter 3 and presented in the following Table 5.3.1. MDL-12330A and

forskolin were prepared from stock solution by diluting with high glucose

complete media to reach a final concentration of 10 µM and 3 µM respectively

as explained in Chapter 4 (Table 5.3.1).

165

Conditions Concentration

Control Cells + high glucose Complete Media

Chronic Caffeine Caffeine 30 µM + high glucose Complete Media + 48 hours incubation (Caffeine mixture)

CPA CPA 30 nM + high glucose Complete media

CGS21680 CGS21680 30 nM + high glucose Complete media

NECA NECA 30 nM + ZM241385 10 nM + high glucose Complete media

MDL-12330A MDL-12330A 10 µM + high glucose Complete Media

Forskolin Forskolin 3 µM + high glucose Complete Media

MDL-12330A & Forskolin MDL-12330A 10 µM + Forskolin 3 µM

Table 5.3.1. Preparation of drug treatments for the wound healing scratch assay

166

5.3.2. Wound Healing Scratch Assay

EA.hy926 endothelial cells (2x105 cells) were seeded into12-well plates, in

triplicate in high glucose complete media, to reach 60-65% confluence. High

glucose complete media was then replaced with high glucose complete media

and caffeine (30 µM) and the cells underwent chronic caffeine treatment for

48 hours. At the end of the incubation period, the high glucose complete

media containing caffeine (30 µM) was removed from the wells. The cell layer

was then scratched with a 200 µL pipette tip, and subsequently the various

drug treatments were loaded into the wells (Table 5.3.1). The wounds were

photographed at the starting point of the treatment (0 hours) and at 2, 4 and 6

hours (end of the treatment). For more detail, refer to Chapter 2. The data for

the wound healing scratch assay was obtained by measuring the width of the

scratch at 2 hour intervals, using Image J software. The average speed of

wound healing was examined by comparing the effect of chronic caffeine

treatment on adenosine receptor agonists. The results were analysed using one

way ANOVA via GraphPad Prism software (CA, USA) (version 6.0).

5.3.3. Western Blotting Technique

Two groups of EA.hy926 endothelial cells (the control group and chronic

caffeine treated group) were harvested, then lysed and prepared for the

Western blotting technique as explained in Chapters 2 and 3. Protein samples

(50 µg) were loaded into the gels and run at 150 volts for approximately 50

minutes until the samples reached the bottom of the gel. The protein samples

167

were then transferred to PVDF membranes using 75 volts for 1 hour and 30

minutes. The membranes were treated with a blocking buffer for 1 hour.

Subsequently, they were incubated overnight at 4 °C with rabbit polyclonal

primary antibody for adenosine A1 receptor (1:500), adenosine A2A receptor

(1:500) and adenosine A2B receptor (1:200); details are in Chapter 3.

Afterward, the membranes were incubated in horseradish peroxidase-

conjugated goat anti-rabbit secondary antibody (1:10000) for adenosine A1

and A2A receptors and (1: 500) for adenosine A2B receptor for 1 hour at room

temperature. Beta (β)-Actin antibody was used as a loading control (1:1000)

for all three adenosine receptor subtypes and horseradish peroxidase-

conjugated goat anti-rabbit antibody was chosen for the secondary antibody

(1:3000) as described in Chapter 3.

The protein size was determined through comparison to the protein ladder or

molecular weight marker. The data were analysed using the unpaired student’s

t-test and two-way ANOVA via GraphPad Prism software (version 6.0).

5.4. RESULTS

5.4.1. The Effect Of Selective Adenosine Receptor Agonists On Rate

Of Wound Closure In Ea.hy926 Endothelial Cells Following Chronic

Caffeine Treatment

The results from the wound healing scratch assay showed that the rate of

wound closure in EA.HY926 endothelial cells after the 48 hours caffeine

treatment increased significantly to 74±3.0 µM/hour when compared with the

168 control group (no caffeine treatment, 48±0.5 µM/hour, P < 0.0001) (Figures

5.4.1 and 5.4.2).

A comparison of wound healing rates between the chronic caffeine treatment and untreated EA.hy926 endothelial cells, showed that the rates of wound closure with all the selective adenosine A1, A2A and A2B receptor agonists

(CPA, CGS21680 and NECA 30nM) increased significantly by 11%, 14% and

25% (P < 0.0001), respectively (Figure 5.4.2). However, compared to the control caffeine treated cells, the rate of wound closure did not change with the agonists CPA and NECA and was in fact reduced in the CGS21680 treated cells (P<0.05). Consequently, chronic caffeine treatment alone enhanced wound healing in the high glucose condition but had no effect on adenosine A1 or A2B receptor mediated increases in wound healing rates in EA.hy926 endothelial cells, and it attenuated the ability of adenosine A2A receptors to mediate wound closure.

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Figure 5.4.1. Images showing the effects of selective adenosine receptor agonists (CPA, CGS21680 and NECA) (30nM) on the rate of wound healing following chronic caffeine treatment (30 µM) in EA.hy926endothelial cells at 0 and 6 hours using a wound healing scratch assay. Panel 1 control untreated cells; panels 2-5 have been treated with caffeine (30 µM for 48 hours) prior to the experiments. Panel 3 shows CPA (30 nM), panel 4 CGS21680 (30 nM) and panel 5 NECA (30 nM +ZM241385 10 nM). Magnification x 40.

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100 *

**** M/hour) 80 **** **** µ **** 60 Untreated

48hrs Caffeine 40 Pre-treatment

Rate of wound healing ( 0

Control CPA 30nM CGS 30nM NECA 30nM Treatments

Figure 5.4.2. Graph comparing the effects of adenosine receptor agonists (CPA, CGS21680 and NECA) (30 nM) on the rate of wound healing with 48 hours caffeine pre-treatment and compared to untreated conditions,Two-way ANOVA was applied to analyse data. The data are presented as mean ± SEM, n= 6. ****P < 0.0001, *P < 0.05 versus 48 hours caffeine pre-treated control

171

5.4.2. Effect Of Chronic Caffeine Treatment On Rates Of Wound

Closure In Ea.Hy926 Endothelial Cells; Role Of cAMP

Forskolin and MDL-12330A were used to determine the effect of chronic

caffeine treatment on adenylyl cyclase activity in EA.hy926 endothelial cells

undergoing the wound healing assay. The wound healing rates in EA.hy926

endothelial cells in the wound healing assay following caffeine treatment (48

hours) were 74±3.0 µM/hour (control) compared to forskolin treatment (3 µM)

67±3.0 µM/hour showing that forskolin treatment had no effect in chronic

caffeine treated cells in this assay (Figures 5.4.3 and 5.4.4). Also, MDL-

12330A significantly attenuated the effect of chronic caffeine and reversed the

rate of wound healing by 47% (P < 0.0001) (Figures 5.4.3 and 5.4.4).

Additionally, MDL-12330A decreased the effect of forskolin on wound

closure rate by 32% in EA.hy926 endothelial cells (P < 0.001) (Figures 5.4.3

and 5.4.4). These results indicate that chronic caffeine treatment increases

wound healing rates through increased cAMP levels.

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Figure 5.4.3. Image showing effect of MDL-12330A (10 µM) and forskolin (3 µM) on wound healing rates following chronic caffeine treatment in EA.hy926endothelial cells at 0 and 6 hours using wound healing scratch assay. Conditions from left to right: Control, chronic Caffeine (30 µM), MDL-12330A (10 µM), chronic caffeine MDL-12330A (10µM), forskolin (3 µM), Mixture of forskolin (3 µM) and MDL-12330A (10 µM). Magnification x40

173

100 **** **** 80

20 Rate of wound healing (µm/hrs) 0

P/ Control P/ Forskolin P/ MDL-12330A

P/ MDL-12330A & Forskolin 100 ****

m/hrs) 80 µ *** 60

20 Rate of wound healing ( 0

U/ Control U/ Forskolin U/ MDL-12330A

U/ MDL-12330A & Forskolin

Figure 5.4.4. Graph showing the effect of the 48 hours caffeine Pre-treatment (P, top panel) or Untreated cells (U, lower panel) on rates of wound healing and the effect of MDL-12330A and Forskolin (versus their respective control group).Data are presented as mean ± SEM, n= 6, ***P < 0.001, ****P < 0.0001 versus control

174

5.4.3. Comparing The Effect Of Chronic And Acute Caffeine

Treatment On The Rate Of Wound Healing In EA.hy926 Endothelial

Cells

Figure 5.4.5 shows that the wound healing rate in the control group with 48

hours caffeine pre-treatment significantly increased compared to the control

group with no caffeine pre-treatment in EA.hy926 endothelial cells (P <

0.0001). Also, the addition of acute caffeine on EA.hy926 endothelial cells

with the 48 hours caffeine pre-treatment (30 µM) did not significantly alter the

rate of wound healing in chronic caffeine treated or no caffeine treated cells

(P> 0.05) (Figure 5.4.5).

175

100 **** ** 80 M/hour) µ

48hrs Caffeine 40 Pre-treatment No Caffeine Pre-treatment 20 Rate of wound healing ( 0

Control Caffeine

Treatments

Figure 5.4.5.Graph comparing the effect of acute caffeine (30 µM) on chronic caffeine treated cells (48 hours) and no caffeine pre-treatment on rate of the wound healing. Two-way ANOVA was applied to analyse data. Data are presented as mean ± SEM, n= 6, **P < 0.01, ****P < 0.0001

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5.4.4. Expression Of The Adenosine Receptor Subtypes On EA.hy926

Endothelial Cells Following Chronic Caffeine Treatment

The Western blotting technique showed the expression of the adenosine A1,

A2A and A2B receptors following the chronic caffeine treatment (48 hours) and

in untreated conditions (Figure 5.4.6). The expected molecular size of

adenosine receptor subtypes is A1 (≈ 37 kDa), A2A (≈ 45 kDa) and A2B (≈ 50-

52 kDa) in EA.hy926 endothelial cells as described in Chapter 3. Moreover, β-

actin (molecular size ≈ 42 kDa) was used as a loading control and used to

normalize the data for protein expression of receptors across the blots (Figure

5.4.6).

The expression of adenosine A1 and A2B receptors on EA.hy926 endothelial

cells following chronic caffeine treatment did not show any significant

changes compared to the untreated or control group (P > 0.05) (Figure 5.4.7).

However, the chronic caffeine treatment significantly decreased the expression

of the adenosine A2A receptor on EA.hy926 endothelial cells when compared

with the control group (P < 0.01) (Figure 5.4.7).

Furthermore, the expressions of the adenosine receptor subtypes (A1, A2A and

A2B) were compared between the chronic caffeine treatment and control

groups (Figure 5.4.8). This data showed that the expression of adenosine A2A

receptors was significantly higher than the other adenosine receptor subtypes

(A1 and A2B) in the control group (P < 0.05) and (P < 0.0001), respectively

(Figure 5.4.8). However, the expression of the adenosine A2A receptors

decreased with chronic caffeine treatment and was similar in expression levels

to the adenosine A1 receptor (Figure 5.4.8). Moreover, the adenosine A1 and

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A2B receptor expression did not change with chronic caffeine treatment (P >

0.05) (Figure 5.4.8).

178

Figure 5.4.6. Western blot images showing protein bands of adenosine receptors (A) A1 ≈ 37 kDa, (B) A2A ≈ 45 kDa, (C) A2B ≈ 50-52 kDa and (D) β-actin ≈ 42 kDa in the control / untreated condition (Ctrl) and (48 hours) chronic caffeine treatment (Caffeine). Also in all images, a lane contains the molecular weight marker

179

Protein Expresion of Adenosine A1 Receptors 10000

8000

6000

4000 Relative Density

2000

Control

ChronicTreatment Caffeine

Protein Expresion of Adenosine A2A Receptors 10000

8000

6000 **

4000 Relative Density 2000

Control

ChronicTreatment Caffeine

Protein Expresion of Adenosine A2B Receptors 10000

8000

6000

4000 Relative Density

2000

Control

ChronicTreatment Caffeine

Figure 5.4.7. Histogram showing the expression of adenosine receptor subtypes A1, A2A and A2B on EA.hy926 endothelial cells in untreated conditions (control) and (48 hours) chronic caffeine treatment. The data was analysed using unpaired student’s t-test, **(P < 0.01), caffeine treatment versus control. Data are presented as mean ± SEM (n=6)

180

10000

8000

Adenosine A 6000 1 Receptor

4000 Adenosine A2A Receptor Relative Density Adenosine A2B 2000 Receptor

Control

Chronic Caffeine Treatment

Figure 5.4.8. Graph showing a comparison of the protein expression of adenosine receptor subtypes A1, A2A and A2B on EA.hy926 endothelial cells in untreated condition (control) and (48 hours) chronic caffeine treatment.Two-way ANOVA was applied to analyse data. Data are presented as mean ± SE, n= 6

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5.5. DISCUSSION

This study determined the effect of chronic caffeine treatment on selective

adenosine A1, A2A and A2B receptor agonist stimulation of wound healing rates

in EA.hy926 endothelial cells. It also investigated the effect of acute caffeine

treatment on cells. Furthermore, the protein expression of adenosine A1, A2A

and A2B receptors in EA.hy926 endothelial cells was examined following the

chronic caffeine treatment.

The results of this study showed that chronic caffeine treatment significantly

increased the rate of wound healing in EA.hy926 endothelial cells when

compared with the untreated group. An in vivo study using humans and

animals (rats) reported that topical coffee powder treatment for 4 weeks and 8

weeks respectively increased wound tissue healing without causing adverse

effects 33. Studies have reported that coffee has numerous polyphenols that act

as antioxidants 33. High levels of radical oxygen species are produced in the

wound area during the early stages of the wound healing process. Thus

antioxidants are required to scavenge free radicals to increase the rate of

wound healing 33, 314. In addition, another study has shown that long-term low

dose caffeine consumption can stimulate modifications in the morphology and

homoeostasis of the rat ventral prostate 42. Also, chronic caffeine consumption

from puberty to adulthood elevated androgenic signalling and cell

proliferation in the prostate gland and could be related to the development of

benign prostatic hyperplasia (BPH) 42.

The results of this PhD study show that chronic caffeine treatment did not

significantly accelerate the rate of wound healing by the selective adenosine

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A1 and A2B receptor agonists (CPA and NECA) and reduced the effect of the adenosine A2A receptor agonist (CGS21680) in the EA.hy926 endothelial cells.

Chronic caffeine treatment of nigrostriatal neurons causes up-regulation of adenosine A1 receptors and reduces the toxic effects of psychostimulant drugs such as methamphetamine in the mouse 296. However, the same study also reported that methamphetamine-induced toxicity was exacerbated in the mouse with acute caffeine treatment 296, 315. Another in vivo study observed that chronic caffeine consumption up-regulated adenosine receptors and increased the levels of adenosine and adenosine actions in the rat brain 152.

Other studies have reported that caffeine decreases collagen deposition and reduced fibrosis in the liver and lung, an effect attributed to the antagonistic effects of caffeine on adenosine receptors 41, 42, 44, 311.

This recent study showed that chronic caffeine treatment significantly increased the rates of wound healing in the EA.hy926 endothelial cells, however the effect of selective adenosine receptor agonists was not enhanced and was depressed with the adenosine A2A receptor agonist CGS21680.

Protein levels of the adenosine receptors showed a reduction of the adenosine

A2A receptor expression with chronic caffeine treatment which may explain why there was a reduced rate of wound closure with EA.hy926 endothelial cells. The protein expression of the adenosine A1 and A2B receptors did not change with chronic caffeine treatment, however the selective agonist induced stimulation of wound closure rates did not increase further when compared to the chronic caffeine untreated cells. It is feasible that there is a maximum rate of wound closure (around 80 µM/hour) and further increases may not be possible.

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The present study showed that acute caffeine had no effect on caffeine pretreated cells, which may be an indication of tolerance occurring with the chronic caffeine treatment.

In the current study, MDL12330A, an inhibitor of adenylyl cyclase, significantly reduced the rates of wound healing in caffeine pretreated cells.

Forskolin, on the other hand, caused no increase in wound healing rates in caffeine treated EA.hy926 endothelial cells. Caffeine is a nonselective competitive inhibitor of the phosphodiesterase enzyme. This enzyme has the ability to degrade the phosphodiesterase bond in cAMP and cyclic guanosine monophosphate (cGMP) 316, 317. Therefore, through inhibition of cyclic nucleotide phosphodiesterase activity, caffeine causes an accumulation of cAMP 316, 318, 319. Also, it has been reported that inhibition of phosphodiesterase activity occurs at extremely high concentrations of caffeine such as plasma concentration 0.1–1 mM or caffeine doses 1520–15,200 mg 319.

A study investigating chronic caffeine and its effects in the mouse hippocampus reported that the Gi and Gs proteins were down-regulated in the hippocampus and suggested that chronic caffeine treatment could induce adaptive increases in G-protein stimulated adenylyl cyclase 320. While these effects occur in brain tissue, a similar effect may be occurring in the endothelial cells.

Western blotting results of the present study demonstrated that the protein expression of adenosine A1 and A2B receptors did not significantly change with chronic caffeine treatment in EA.hy926 endothelial cells. However, the expression of the adenosine A2A receptor subtype in EAhy926 endothelial cells was significantly decreased with chronic caffeine treatment when compared to

184

the control group. On the other hand, an in vivo study reported that adenosine

receptors significantly increased following chronic caffeine in the mice brain

stem 51. Also, their results suggested that the adenosine receptors required

longer exposure to caffeine to significantly change the expression of these

receptors in the cerebral cortex and the thalamus 51. For instance, in both these

areas, the expression of adenosine receptors was enhanced at 16 days caffeine

treatment but just reached statistical significance at 23 days 51. The results

from this study suggest that chronic caffeine does not increase adenosine

receptor subtype population in the endothelial cells. This may be due to

differences in cell type and changes in adenosine receptor population that can

be seen only in neuronal cells or alternatively, the duration of caffeine

treatment was not long enough to cause a change in adenosine receptors.

5.6. CONCLUSION

In summary, the 48 hours caffeine treatment significantly elevated the rate of

wound healing in EA.hy926 endothelial cells compared with untreated group

and acute caffeine treatment. Therefore, this shows that chronic caffeine

treatment may alter cAMP signalling pathways in EA.hy926 endothelial cell

lines. The protein expression of adenosine A1 and A2B receptors did not

significantly change with chronic caffeine treatment in EA.hy926 endothelial

cell lines but the expression of the adenosine A2A receptor chronic caffeine

treatment was considerably lower than untreated group.

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CHAPTER 6

EFFECT OF LOW GLUCOSE

CONCENTRATION ON ADENOSINE

RECEPTOR MEDIATED WOUND

HEALING

186

CHAPTER 6: EFFECT OF LOW GLUCOSE

CONCENTRATION ON ADENOSINE RECEPTOR

MEDIATED WOUND HEALING

6.1. INTRODUCTION

6.1.1. Diabetes Mellitus and Wound Healing

Diabetes mellitus is a chronic metabolic disorder that is characterized by

hyperglycaemia and caused by insulin deficiency and/or insulin resistance 3, 321.

Diabetes is now present in all countries and its prevalence continues to grow

in the world’s population 3, 321, 322. According to the World Health

Organization, the number of people with diabetes has increased from 108

million in 1980 to 422 million in 2014 53. Also, the USA National Diabetes

Statistics report has predicted that diabetes mellitus will affect approximately

600 million of the population worldwide by the year 2035 322, 323. Diabetic

patients are prone to prolonged multifaceted complications such as

cardiovascular diseases, retinopathy, neuropathy, nephropathy and impaired

wound healing 3, 322.

Normal wound healing in non-diabetic conditions is a complex coordinated

sequence of processes including cell migration into the wound area,

inflammation, cell proliferation, angiogenesis, formation of extracellular

matric components, remodeling and finally wound healing 3. However, the

wound healing sequelae in diabetes is dysregulated at several levels causing,

increased inflammation, impaired tissue repair and eventually loss of function

187

1. Furthermore, it has been confirmed that several process in normal wound

healing are disrupted by diabetes, such as changed bactericidal actions of

granulocytes and thickening of the extracellular matrix basement membrane

together with poor tissue perfusion 1, 2. Therefore, most diabetic patients suffer

from chronic ulceration due to vascular disease, peripheral neuropathy and

immune dysfunction 1-3, 37. Diabetes is reported to be the number one cause of

non-traumatic amputation 1-3, 37.

6.1.2. Adenosine Receptors and Diabetes

Adenosine has a critical role in the regulation of glucose homeostasis and the

pathophysiology of diabetes 322, 324-328. The concentration of adenosine

increases during energy starvation or environmental stress conditions to

regulate cellular energy metabolism and prevent cellular damage 321.

Furthermore, adenosine via activation of adenosine A2A and A3 receptors

affects insulin secretion and also regulates β-cell homeostasis via controlling

cell proliferation and β-cell regeneration 322, 329, 330. In addition, adenosine is a

regulator of cellular responses to insulin as it controls insulin signaling in

adipose tissue, muscles and liver 171, 322, 327, 331. Adenosine can indirectly

mediate the actions of inflammatory cells and/ or immune cells in the above

tissues 171, 322, 327, 331. These studies also show that diabetes is associated with

altered expression of adenosine receptors and their function 322. For example,

the levels of adenosine A1 and A3 receptors were elevated in isolated cardiac

myocytes of streptozotocin-treated diabetic rats and the levels of adenosine

A2A receptors increased in diabetic rat whole hearts but decreased in isolated

188

cardiac myocytes 322, 332. Several studies have reported that adenosine can

protect cells from damage and also adenosine receptors effectively improve

diabetic ischaemic wound repair 9, 97, 106, 321.

All experiments in the previous chapters were performed in diabetic

conditions, as the EA.hy926 endothelial cells were seeded in high glucose cell

culture solution (4500 mg/L glucose) as recommended in EA.hy926 (ATCC®

CRL-2922TM) product sheet information (ATCC, VA. USA). However, this

Chapter will examine the effect of low glucose or normal conditions (1000

mg/L) on EA.hy926 endothelial cell proliferation and rate of wound healing

and compare the results with EA.hy926 endothelial cell function in high

glucose conditions, as reported in Chapters 3 to 5.

6.2. AIMS

The purpose of this study is to determine the rate of wound healing and

proliferation of EA.h926 endothelial cells in low glucose cell culture solution

(1000 mg/L or 5.5 mM) and high glucose (4500 mg/L or 25 mM) conditions.

The aims of these experiments were to:

1. Determine the effect of selective adenosine A1, A2A and A2B receptor

agonists and antagonists on the rate of the wound closure in EA.h926

endothelial cells using wound healing scratch assay in vitro.

2. Measure the actions of selective adenosine A1, A2A and A2B receptor

agonists and antagonists on the proliferation of EA.h926 endothelial cells.

3. Investigate the effects of high and normal glucose levels on wound healing

rates and cell proliferation.

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6.3. METHODS AND MATERIALS

6.3.1. Treatment preparations (drugs)

The low glucose complete media were supplemented with the same mixture as

high glucose complete media in Chapter 2 however high glucose DMEM was

replaced with low glucose DMEM (1000 mg/L or 5.5 mM) (Dulbecco’s

Modified Eagle’s Medium) (ThermoFisher Scientific, VIC, Australia) for

more detail refer to Chapter 2.

All drugs were freshly prepared from stock solution (1 mM) by diluting with

low glucose complete media (1000 mg/L) to reach a final concentration of 30

µM for caffeine, 30 nM and 10 nM for selective adenosine A1, A2A and A2B

receptor agonists (CPA, CGS21680 and NECA) and antagonists (DPCPX,

ZM241385 and MRS1754), respectively (Table 6.3.1). Also, it is noteworthy

that due to NECA being non-selective for the adenosine A2B receptor agonist,

10 nM ZM241385 was added to exclude the adenosine A2A receptor as

described in Chapter 3.

6.3.2. Wound healing scratch assay

EA.hy926 endothelial cells (2x105 cells) were seeded into the 12-well plates,

in triplicate in low glucose complete media, and incubated until the cells had

reached 80-85% confluence.

The EA.hy926 endothelial cells are not able to remain viable and functional

under low glucose conditions after 48 hours. Cell morphology and confluency

190

changes were observed after 48 hours cell growth in EA.hy926 endothelial

cells under the low glucose condition.

The cell layer was then scratched with a 200 µL pipette tip and prepared

caffeine (30 µM), agonists (30 nM) and antagonists (10 nM) were loaded into

the wells. The EA.hy926 endothelial cells exhibited less adhesion to the plate

in low glucose condition and the scratch process caused a wider wound

scratch.

The wells were photographed at the starting point of the treatment (0 hours)

and at 2, 4 and 6 hours (end of the treatment). For more detail refer to Chapter

2. The data for the wound healing scratch assay was obtained by measuring

the width of the scratch at 2 hour intervals, using Image J software. The

average speed of wound healing was examined by comparing the effect of

caffeine and adenosine receptor agonists on EA.hy926 endothelial cells in the

low glucose condition with the diabetes condition (high glucose condition).

The results were analysed using unpaired student’s t-test, One Way ANOVA

and Two Way ANOVA via GraphPad Prism software (CA, USA) (version

6.0).

6.3.3. Cell proliferation assay

The EA.hy926 endothelial cells were seeded in triplicate at 5000 cells/ well in

96 well plates. Also, 4000, 5000, 6000, 7000, 8000 and 9000 cells were

seeded in duplicate per well respectively to form a calibration curve and all

incubated for 24 h at 37 ºC, 5% CO2 as described in Chapters 2 and 3.

Selective adenosine receptor agonists (30 nM) and antagonists (10 nM) were

191 prepared with low glucose complete media. In addition, this experiment comprised the control conditions where cells were treated with normal complete media and vehicle controls conditions using DMSO at two different dilutions of 0.004% and 0.03% (Table 6.3.1). The plate was placed into the micro-plate reader and the absorbance at 450 nm measured. For more detail refer to Chapter 2.

192

Treatments Concentrations

CPA CPA 30 nM + Low Glucose Complete Media

DPCPX DPCPX 10 nM + Low Glucose Complete Media

CPA+ DPCPX CPA 30 nM + DPCPX 10 nM + Low Glucose Complete Media

CGS21680 CGS21680 30 nM + Low Glucose Complete Media

ZM 241385 ZM241385 10n M + Low Glucose Complete Media

CGS21680 + ZM 241385 CGS21680 30 nM + ZM241385 10 nM + Low Glucose Complete Media

NECA NECA 30 nM+ZM241385 10 nM +Low Glucose Complete Media

MRS1754 MRS1754 10 nM + Low Glucose Complete Media

NECA+ MRS1754 NECA 30 nM +ZM241385 10 nM +MRS1754 10 nM +

Low Glucose Complete Media

Control Cells + Low Glucose Complete Media

Vehicle Control 1 0.004% DMSO + Low Glucose Complete Media

Vehicle Control 2 0.03% DMSO + Low Glucose Complete Media

Negative Control No cells + Just Low Glucose Complete Media

Table 6.3.1. Treatment conditions and their concentrations

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6.4. RESULTS

6.4.1. The effect of selective adenosine A1 receptor agonist and

antagonist on rate of wound closure in the low glucose condition

The results of the wound healing scratch assay showed that the selective

adenosine A1 receptor agonist (CPA 30 nM) significantly increased the rate of

wound healing by 51% in EA.hy926 endothelial cells when compared to the

control group in the low glucose condition (P < 0.0001) (Figures 6.4.1 and

6.4.2). However, there were no significant changes in wound healing rate

with selective adenosine A1 receptor antagonist (DPCPX 10nM) and combined

group (CPA+DPCPX) in comparison with the control group (P > 0.05)

(Figures 6.4.1 and 6.4.2).

Furthermore, the results showed that the effect of the selective adenosine A1

receptor agonist (CPA 30 nM) on wound healing rate was considerably

decreased by the selective adenosine A1 receptor antagonist (DPCPX 10 nM)

by 32% in the combined group (CPA+ DPCPX) (P < 0.0001) (Figures 6.4.1

and 6.4.2).

194

Figure 6.4.1. Images showing wound healing scratch assay in low glucose conditions Effect of selective adenosine A1 receptor agonist (CPA 30 nM), antagonist (DPCPX 10nM) and its combination (CPA+ DPCPX) on the rate of wound healing in EA.hy926 endothelial cells at 0 and 6 hrs. Magnification x 40

195

**** 150 **** M/hour) µ 100

50 Rate of wound healing ( 0

Control CPA 30nM DPCPX 10nM CPA+DPCPX

Figure 6.4.2. Graph showing the effect of selective adenosine A1 receptor agonist (CPA 30 nM), antagonist (DPCPX 10 nM) and its combination (CPA+ DPCPX) on rate of wound healing in EA.hy926 endothelial cells in the low glucose condition, One-Way ANOVA applied to analyse data. Data are presented as mean ± SEM, n= 6, **** P < 0.0001 versus control and versus CPA treated cells as indicated

196

6.4.2. The effect of selective adenosine A2A receptor agonist and antagonist on rate of wound closure in the low glucose condition

The results show that the selective adenosine A2A receptor agonist (CGS21680

30 nM) significantly enhanced the rate of wound healing in EA.hy926

endothelial cells in the low glucose condition by 16% in comparison with

control group (P < 0.01) (Figures 6.4.3 and 6.4.4). However, the selective

adenosine A2A receptor antagonist (ZM241385 10 nM) and combined group

(CGS21680+ZM241385) considerably reduced the wound healing rate in

EA.hy926 endothelial cells compared with control group (P < 0.001) and (P <

0.05) (Figures 6.4.3 and 6.4.4). Moreover, the data shows that the selective

adenosine A2A receptor antagonist (ZM241385 10nM) significantly decreased

the effect of CGS21680 (30 nM) on the rate of wound closure by 25% in the

low glucose condition (P < 0.0001) (Figures 6.4.3 and 6.4.4).

197

Figure 6.4.3. Image showing the wound healing scratch assay in the low glucose condition Effect of selective adenosine A2A receptor agonist (CGS21680 (CGS) 30 nM), antagonist (ZM241385 (ZM) 10 nM) and its combination (CGS21680+ZM241385) on the rate of wound healing in EA.hy926 endothelial cells at 0 and 6 hrs. Magnification x40

198

150 **** M/hour) µ ** 100 * ***

Rate of wound healing ( 0

Control ZM 10nM CGS+ZM CGS 30nM Figure 6.4.4. Graph showing the effects of selective adenosine A2A receptor agonist (CGS21680 (CGS) 30 nM), antagonist (ZM241385 (ZM) 10 nM) and its combination (CGS21680+ZM241385) on rate of wound healing in EA.hy926 endothelial cells in the low glucose condition. One-Way ANOVA was applied to analyse data. Data are presented as mean ± SEM, n= 6, *P < 0.05, **P < 0.01, *** P < 0.001 and **** P < 0.0001 versus control and versus CGS21680 treated cells as indicated

199

6.4.3. The effect of selective adenosine A2B receptor agonist and antagonist on rate of wound closure in the low glucose condition

The data shows that the rate of wound healing in the low glucose condition

significantly increased by 30% with the selective adenosine A2B receptor

agonist (NECA 30 nM) when compared with control group (P < 0.0001)

(Figures 6.4.5 and 6.4.6). In comparison with the control group, the selective

adenosine A2B receptor antagonist (MRS1754) considerably decreased the

wound healing rate in EA.hy926 endothelial cells in the low glucose condition

(P < 0.0001) (Figures 6.4.5 and 6.4.6). Furthermore, the results demonstrated

that MRS1754 significantly reversed the effect of NECA and decreased the

rate of wound healing by 30% in EA.hy926 endothelial cells (P < 0.0001)

(Figures 6.4.5 and 6.4.6).

200

Figure 6.4.5.Image showing the wound healing scratch assay in the low glucose condition Effect of selective adenosine A2B receptor agonist (NECA 30 nM + ZM241385 10 nM), antagonist (MRS1754 (MRS) 10 nM) and its combination (NECA+ ZM + MRS) on the rate of wound healing in EA.hy926 endothelial cells at 0 and 6 hrs. Magnification x40

201

150 ****

M/hour) **** µ 100

**** 50

Rate of wound healing ( 0

Control MRS 10nM NECA 30nM NECA+MRS

Figure 6.4.6.Graph showing the effects of selective adenosine A2A receptor agonist (NECA 30 nM), antagonist (MRS1754 (MRS) 10 nM) and its combination (NECA+ MRS) on the rate of wound healing in EA.hy926 endothelial cells in the low glucose condition, One-Way ANOVA applied to analyse data. Data are presented as mean ± SEM, n= 6, **** P < 0.0001 versus control and NECA treated cells as indicated

202

6.4.4. Effect of adenosine agonists and anagonists on cell proliferation in the low glucose condition

The calibration curve was created for converting sample absorbance values

into cell numbers such as 4000, 5000, 6000, 7000, 8000 and 9000 cells per

well. The calibration curve demonstrated that absorbance values were

proportional to cell numbers see Figure 2.3.2 in Chapter 2.

The data shows that cell proliferation was significantly decreased by the

vehicle control (0.004% DMSO) and the selective adenosine A2A receptor

antagonist (ZM241385 10 nM) when compared with the control group (P <

0.05) and (P < 0.01), respectively (Figure 6.4.7). However, in comparison with

control group, the other treatment groups did not demonstrate any significant

changes (P > 0.05). During treatment, the other selective adenosine agonists

and antagonists did not affect cell proliferation in the low glucose condition (P

> 0.05).

203

1.5

1.0

* ** Absorbance 0.5

0.0

Ctrl

CPA 30 nM CGS 30nMZM 10CGS nM + ZM NECAMRS 30nM 10 nM Blank(no cells)0.03% DMSO DPCPXCPA 10 + nM DPCPX NECA + MRS 0.004% DMSO Treatments

Figure 6.4.7. Graph showing the effect of different treatments on cell proliferation in the low glucose conditions, Control group or untreated (Ctrl), vehicle control group (0.004% and 0.03% DMSO), adenosine receptor agonists 30 nM (CPA, CGS21680 and NECA), Adenosine receptor antagonists 10 nM (DPCPX, ZM241385 and MRS1754) and combined group (mixture of adenosine receptor agonist 30 nM with antagonist 10 nM). One-Way ANOVA applied to analyse data. Data are presented as mean ± SEM n= 6, *P < 0.05, **P < 0.01 versus control

204

6.4.5. Comparing the effect of adenosine A1 receptor agonist and antagonist on the rate of wound healing in low and high glucose conditions

The results show that the selective adenosine A1 receptor agonist and

antagonist in the low glucose condition are more effective than in the high

glucose condition in the wound healing process. Also, comparison of the two

control groups showed that the rate of wound healing significantly increased

in the low glucose condition in contrast to high glucose conditions by 78% (P

< 0.0001) (Figure 6.4.8). Furthermore, the selective adenosine A1 receptor

agonist (CPA 30 nM) significantly enhanced the wound healing rates by 51%

in the low glucose condition when compared with its control group in a same

condition (P < 0.0001). Also, the CPA treatment significantly increased

wound healing rates by 88% in the low glucose condition in comparison with

wound healing rates in high glucose (P < 0.0001) (Figure 6.4.8). Moreover,

the selective adenosine A1 receptor antagonist (DPCPX 10 nM) considerably

reduced the effect of the selective adenosine receptor agonist and decreased

the rate of wound closure in both conditions (low glucose and high glucose)

(Figure 6.4.8). However, the data showed that the rate of wound healing with

the selective adenosine A1 receptor antagonist (DPCPX 10 nM) in the high

glucose condition decrease significantly by 60% when compared to rates in

the low glucose condition (P < 0.0001) (Figure 6.4.8).

205

**** 150 **** M/hour) µ 100 **** **** ****

Low Glucose High Glucose

Rate of wound healing( 0

Control CPA 30nM DPCPX 10nM CPA+DPCPX

Treatments

Figure 6.4.8. Graph comparing the effect of selective adenosine A1 receptor agonist and antagonist (CPA 30 nM and DPCPX 10 nM), respectively on the rate of wound healing in EA.hy926 endothelial cells in low glucose and high glucose conditions, Two-Way ANOVA applied to analyse data. Data are presented as mean ± SEM, n= 6, **** P < 0.0001. CPA versus Control in the low glucose condition as indicated

206

6.4.6. Comparing the effect of adenosine A2A receptor agonist and antagonist on the rate of wound healing in low and high glucose conditions

The results show that the selective adenosine A2A receptor agonist and

antagonist (CGS21680 and ZM241385, respectively) in the low glucose

condition are more effective than in the high glucose condition in the wound

healing process. A comparison of the two control groups demonstrated that

the rate of wound healing significantly decreased by 57% in the high glucose

condition in contrast to the low glucose condition (P < 0.0001) (Figure 6.4.9).

Furthermore, the selective adenosine A2A receptor agonist (CGS21680 30 nM)

considerably increased the wound healing rates by 18% in the low glucose

condition when compared with its control group of cells under the same

conditions (P < 0.01). Also, CGS21680 significantly increased the wound

healing rate by 78% in the low glucose condition in comparison with high

glucose’s wound healing rate (P < 0.0001) (Figure 6.4.9). Moreover, the

selective adenosine A2A receptor antagonist (ZM241385 10 nM) significantly

reduced the effect of CGS21680 and decreased the rate of wound closure in

both conditions (low and high glucose) (Figure 6.4.9). However, the results

showed that the rate of wound healing with the selective adenosine A2A

receptor antagonist (ZM241385 10 nM) in the high glucose condition

significantly decreased by 45% when compared to its rate in the low glucose

condition (P < 0.0001) (Figure 6.4.9).

207

150

** M/hour)

µ **** 100 **** **** ****

Low Glucose

Rate of wound healing( 0 High Glucose

Control CGS+ZM CGS 30nM ZM 10nM Treatments

Figure 6.4.9. Graph comparing the effect of selective adenosine A2A receptor agonist and antagonist (CGS21680 (CGS) 30 nM and ZM241385 (ZM) 10 nM), respectively on the rate of wound healing in EA.hy926 endothelial cells in low and high glucose conditions,Two-Way ANOVA applied to analyse data. Data are presented as mean ± SEM, n= 6, ** P < 0.01, **** P < 0.0001. CGS21680 versus Control in the low glucose condition as indicated

208

6.4.7. Comparing the effect of adenosine A2B receptor agonist and antagonist on the rate of wound healing in low and high glucose conditions

Similar to the other two adenosine receptors, these results show that the rate of

wound healing with the selective adenosine A2B receptor agonist and

antagonist (NECA and MRS1754, respectively) was higher in the low glucose

condition than in the high glucose condition. For the control groups, the

wound healing rate in the low glucose condition was significantly increased by

75% compared to the wound healing rate in the high glucose condition (P <

0.0001) (Figure 6.4.10). In addition, the selective adenosine A2B receptor

agonist (NECA 30 nM) significantly increased the wound healing rate by 31%

in the low glucose condition when compared with its control group in the

same conditions (P < 0.0001). Also, NECA significantly increased the wound

healing rate by 89% in the low glucose condition compared to the high

glucose condition (P < 0.0001) (Figure 6.4.10). The selective adenosine A2B

receptor antagonist (MRS1754 10 nM) significantly reduced the effect of

NECA and decreased the rate of wound closure in both conditions (low and

high glucose) (Figure 6.4.10). However, the results showed that the rate of

wound healing with the selective adenosine A2B receptor antagonist

significantly reduced in high glucose condition by 44% when compared to the

wound healing rate in the low glucose condition (P < 0.0001) (Figure 6.4.10).

Overall, the results show that the effects of the selective adenosine A1 receptor

agonist (CPA) and its ability to improve wound healing in the low glucose

condition was greater than the selective adenosine A2A and A2B receptor

agonists (CGS21680 and NECA, respectively). The order of efficacy in the

209 low glucose condition was CPA< NECA< CGS21680 with no differences in the order of efficacy in the high glucose condition.

210

150 **** **** M/hour) µ 100 **** ****

**** 50

Low Glucose Rate of wound healing( 0 High Glucose

Control MRS 10nM NECA 30nM NECA+MRS Treatments

Figure 6.4.10.Graph comparing the effects of selective adenosine A1 receptor agonist and antagonist (NECA 30 nM and MRS1754 (MRS) 10 nM), respectively on the rate of wound healing in EA.hy926 endothelial cells in low and high glucose conditions, Two-Way ANOVA applied to analyse data. Data are presented as mean ± SEM, n= 6, **** P < 0.0001. NECA versus Control in the low glucose condition as indicated

211

6.4.8. Comparing the effect of adenosine A1, A2A and A2B receptor agonists and antagonists on EA.hy926 endothelial cell proliferation in low and high glucose conditions

The cell proliferation rates not have any significant differences in the low

glucose condition treatments (P > 0.05) (Figure 6.4.11). However, the

selective adenosine A1, A2A and A2B receptor agonists (CPA, CGS21680 and

NECA) significantly increased cell proliferation in the high glucose compared

with the low glucose condition (P < 0.0001) (Figure 6.4.11). Moreover, the

selective adenosine A1, A2A and A2B receptor antagonists (DPCPX, ZM241385

and MRS1754, respectively) reduced the effect of the selective adenosine

receptor agonists and decreased proliferation of EA.hy926 endothelial cells in

both low and high glucose conditions, however the data were not significantly

different (P > 0.05) (Figure 6.4.11).

212

** *

Absorbance 1

Low Glucose

0 High Glucose

Ctrl

CPA 30 nM DPCPX 10 nM CPA + DPCPX Treatments 3

**** 2 *** **

Absorbance 1

Low Glucose High Glucose 0

Ctrl

CGS 30nM ZM 10 nM CGS + ZM Treatments

3 ****

2 ** *

1 Absorbance Low Glucose

High Glucose 0

Ctrl

NECA 30nM MRS 10 nM NECA + MRS Treatments

Figure 6.4.11.Graphs comparing the effect of selective adenosine A1, A2A and A2B receptor agonists 30 nM (CPA, CGS21680 and NECA), antagonists 10 nM (DPCPX, ZM241385 and MRS1754), respectively and combined group (mixture of adenosine receptor agonists 30 nM with antagonists 10 nM) on EA.hy926 endothelial cell proliferation in low and high glucose conditions.Two-Way ANOVA applied to analyse data. Data are presented as mean ± SEM, n= 6, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 as indicated

213

6.5. DISCUSSION

This study determined the effect of selective adenosine A1, A2A and A2B

receptor agonists and antagonists on the rate of wound closure and

proliferation of EA.hy926 endothelial cells in a low glucose condition (1000

mg/L or 5.5 mM glucose). The results showed that the rates of wound healing

in the EA.hy926 endothelial cells were significantly lower in the high glucose

condition compared to the low glucose environment. However, the

proliferation rates of the EA.hy926 endothelial cells were slightly higher (not

significant) in cells under the high glucose condition.

Endothelial cell proliferation and migration are essential steps in angiogenesis

and wound healing processes 333. This study also reported that endothelial

cells were sensitive to hyperglycemia due to their poor ability to modulate

intracellular glucose levels 333. Another study observed that high glucose

concentrations (15-30 mmol/L) significantly inhibited proliferation and

migration of human umbilical vein endothelial cells (HUVECs) and that

angiogenesis and wound healing rates were reduced in high glucose conditions

333, 334. In diabetes mellitus, increased plasma glucose causes endothelial

dysfunction, which is characterized by the destruction of vasoactive factors

including nitric oxide (NO)-dependent vasorelaxation and this leads to

comorbidities such as retinopathy, peripheral neuropathy, renal failure,

atherosclerosis and heart disease 335.

The results of the current study showed that the wound healing rates of

EAhy926 endothelial cells in the low glucose condition were increased

significantly by the selective adenosine A1, A2A and A2B receptor agonists

214

(CPA, CGS21680 and NECA respectively) when compared with their control group and selective adenosine receptor agonists in the high glucose condition

(4500 mg/L or 25 mM glucose). In addition, these results demonstrated that the selective adenosine A1, A2A and A2B antagonists (DPCPX, ZM241385 and

MRS1754, respectively) in the low glucose condition significantly reduced the effect of selective adenosine receptors agonists on the rate of wound healing.

Similar outcomes were observed in the high glucose condition. It should be noted that there are no other studies on the effect of the adenosine receptors on wound healing in the EA.hy926 endothelial cell lines to compare with the current study results.

The results of the current study show that EA.hy926 endothelial cell proliferation did not show any significant changes under the low glucose compared to the high glucose condition, although generally the proliferation was lower in the low glucose condition. Therefore, due to this issue, the

EA.hy926 endothelial cell lines were grown in the high glucose condition as recommended, to improve the cell proliferation in studies using these cells.

Another study demonstrated that under prolonged (48 hour) high glucose (25 mM) conditions, modification of EA.hy926 endothelial cell morphology is accompanied by initiation of apoptosis 336. Moreover, previous studies showed that increased cell death, rather than reduced cell proliferation, is one of the main consequences associated with high glucose conditions 336-338.

215

6.6. CONCLUSION

In conclusion, the low glucose condition can accelerate the wound healing in

EA.hy926 endothelial cells when compared to the high glucose condition.

Under the low glucose condition, the selective adenosine A1, A2A and A2B

receptor agonists significantly increased the rate of wound healing in

EA.hy926 endothelial cells. Also, similar to high glucose conditions, the

selective adenosine A1, A2A and A2B receptor antagonists reversed the effect of

their agonist and decreased the wound healing rates in low glucose conditions

indicating that all three receptors can be involved in stimulating wound

healing. Furthermore, under the low glucose condition, the efficacy of

selective adenosine A1, A2A and A2B receptor agonists and antagonists on

EA.hy926 endothelial cell wound healing were greater than their effects under

the high glucose condition.

EA.hy926 endothelial cell proliferation was decreased in the low glucose

condition. Cell morphology and confluency changes in EA.hy926 endothelial

cells were observed after 48 hours of cell growth in the low glucose condition.

These experiments showed that EA.hy926 endothelial cells do not remain

viable and functional in the low glucose condition after 48 hours.

216

CHAPTER 7

DISCUSSION

217

CHAPTER 7: DISCUSSION

7.1. GENERAL DISCUSSION

Diabetes mellitus is a chronic metabolic disorder that is characterized by

hyperglycaemia and caused by insulin deficiency and or insulin resistance 3, 321.

Diabetic patients develop complications with this condition, which include

cardiovascular disease, retinopathy, neuropathy, nephropathy and impaired

wound healing 3, 322. Normal wound healing in non-diabetic conditions is a

complex coordinated sequence of processes including cell migration into the

wound area, inflammation, cell proliferation, angiogenesis, formation of

extracellular matric components, remodeling and finally wound healing 3.

Consequently, many diabetic patients suffer from chronic ulceration due to

vascular disease, peripheral neuropathy and immune dysfunction and diabetes

is known to be a main cause of non-traumatic amputation 1-3, 37. The

permanent human endothelial hybrid cell line (EA.hy926), which was used

throughout this study, was created through the hybridisation of primary human

umbilical vein endothelial cells (HUVEC) and a human lung carcinoma cell

line (A549/8) in 1983 200, 201. After the establishment of the EA.hy926

endothelial cell line, several studies have utilised these cell lines for research

related to vascular function. For instance, it was reported that vascular

endothelial growth factor (VEGF) stimulated angiogenesis by elevating

EA.hy926 endothelial cell proliferation, migration and micro-vascular

hyperpermeability 339. Adenosine is a ubiquitous purine nucleoside, which is

generated through the dephosphorylation of ATP and adenine nucleotides 4, 88.

Adenosine and adenosine receptors have essential roles in the processes of

218 matrix production and neovascularization or angiogenesis that are important for wound healing and tissue repair 41. This study showed that the selective adenosine A1, A2A and A2B receptor agonists, CPA, CGS21680 and NECA, significantly increased the rate of wound healing in EA.hy926 endothelial cells in both a high (4500 mg/L or 25 mM) and a low (1000 mg/L or 5.5 mM) glucose condition. Several in vivo studies have demonstrated that the selective adenosine A1 receptor agonist CPA reduced tissue damage associated with ischaemia and stimulated the proliferation of endothelial cells, keratinocytes and fibroblasts in mouse models 245-250. Also, CPA has a potent vasodilator property in conjunction with cell proliferation and wound healing activities 245,

250. Another in vivo study reported that the topical application of the selective adenosine A2A receptor agonist, CGS21680, significantly accelerated wound healing rates in mice 5. Moreover, in vitro studies show that selective adenosine receptor agonists of the adenosine A2A and A2B receptors enhanced migration of both human dermal fibroblasts and human umbilical vein endothelial cells (HUVEC) in an in vitro artificial wound via cAMP and PKA- dependent pathways 5. However, data from the wound healing scratch assay demonstrated that the selective adenosine A1, A2A and A2B receptor antagonists (DPCPX, ZM 241385 and MRS1754) reversed the effects of the selective adenosine receptor agonists and considerably decreased the rate of the wound healing in EA.hy926 endothelial cells. Furthermore, an in vivo study showed that the topical application of the selective adenosine A2 receptor antagonist DMPX (3,7-dimethyl-1- propargyl xanthine) blocked the effect of CGS21680 on the rate of wound closure in mice 5. Moreover,

Western blot data from this study showed the expression of adenosine A1, A2A

219 and A2B receptors in the EA.hy926 endothelial cell line. The adenosine receptors in order of expression in EA.hy926 endothelial cells were A2A > A1

> A2B from highest to lowest.

It has been well established that each adenosine receptor subtype has different effects on stimulation or inhibition of adenylyl cyclase. For instance, the adenosine A1 and A3 receptors inhibit adenylyl cyclase but adenosine A2A and

245 A2B receptors stimulate adenylyl cyclase . Moreover, several studies reported that intracellular cAMP levels can be elevated by forskolin that associates with adenylyl cyclase, while MDL-12330A is an adenylyl cyclase inhibitor that can decrease the levels of cAMP 290-294. This study observed that forskolin significantly increased the rate of wound healing in EA.hy926 endothelial cells. However, the effect of forskolin was reversed by the compound MDL12330A, demonstrating the increased rate of wound healing was linked to stimulation of adenylyl cyclase and increases in intracellular cAMP levels.

Caffeine (1,3,7-trimethylxanthine) is synthesised from the purine nucleotide xanthosine and is one of the most commonly used psychostimulants, usually consumed in the form of beverages and food 22, 45, 130, 131, 175. Several studies have demonstrated that caffeine is a non-selective antagonist against all of the adenosine receptor subtypes 152. Also, caffeine affects the cutaneous wound healing process, especially wound epithelialisation and closure, through its antioxidant properties and antagonism of the adenosine receptors6. Caffeine can have different effects if it is taken as an acute dose or chronically. This study found that acute caffeine significantly decreased the effect of the selective adenosine A1, A2A and A2B receptor agonists (CPA, CGS21680 and

220

NECA 30 nM) on wound healing rates by 28% 16% and 12%, respectively in

EA.hy926 endothelial cells. A study using mouse brain slices demonstrated that caffeine blocks the actions of endogenously released adenosine which can tonically activate its receptors 152. Furthermore, these studies demonstrated that the actions of caffeine are not only mediated through antagonism of adenosine receptors but also through increasing the second messenger cAMP levels by inhibition of phosphodiesterase activity and elevated cytosolic Ca2+ via activation of ryanodine receptors 340-343. Another study showed that high doses of acute caffeine suppressed liver damage by inhibition of cAMP- phosphodiesterase 45. These results demonstrated that acute caffeine treatment increased the rate of wound closure, but the value was not significant. Also, the effect of caffeine decreased the rate of the wound healing in the presence of the adenylyl cyclase inhibitor MDL-12330A in EA.hy926 endothelial cells.

In this study, proliferation rates decreased in the acute caffeine treated group and also caffeine significantly reduced the effect of selective adenosine A1,

A2A and A2B receptor agonists (CPA, CGS21680 and NECA) on EA.hy926 endothelial cell proliferation. Another in vivo study demonstrated that caffeine enhanced cell proliferation in the ventral prostatic lobe in Wistar rats 42.

The tube formation assay in this study showed that caffeine significantly increased tube formation, which promoted angiogenesis in the EA.hy926 endothelial cells. However, other studies have reported that caffeine attenuated angiogenesis in HUVEC, the chorioallantoic membranes (CAM) of chick embryos and in zebrafish embryos 68, 69, 298. These other studies demonstrated that caffeine inhibited the in vitro proliferation of HUVECs, in a time and dose dependent manner, through the stimulation of apoptosis, suggesting that

221 the inhibitory effect of caffeine on angiogenesis could be related to its ability to induce apoptosis in these cells 68, 299, 300.

In this study, the rate of wound healing after the 48 hour caffeine treatment was significantly increased in the EA.hy926 endothelial cell line. Also, the 48 hour caffeine pre-treatment considerably elevated the effects of adenosine A1,

A2A and A2B receptor agonists (CPA, CGS21680 and NECA) when compared with untreated and acute caffeine treated groups. Furthermore, other studies have reported that chronic caffeine treatment is associated with up-regulation of adenosine receptors in mouse brain tissue 291, 320. However, chronic consumption of caffeine was shown to decrease lung injury and inhibit

175 inflammation in an adenosine A2A receptor independent manner in mice . In this same study, chronic caffeine treatment significantly up-regulated the

175 adenosine A2A receptors . Also, other researchers have reported that up- regulation of the adenosine A2A receptor and increased adenosine levels mediated the anti-inflammatory actions through chronic consumption of caffeine to decrease liver tissue damage in mice 45, 48. Chronic caffeine consumption can protect the brain from ishaemic damage 45, 49, 50. The studies have demonstrated that caffeine treatment, due to its chronic blocking of the adenosine A1 receptors, lead to down-regulation of adenylyl cyclase

40, 344 expression . It has been reported that Gi and Gs proteins were down- regulated in the mouse hippocampus following chronic caffeine treatment which suggests that chronic caffeine treatment induces adaptive increases in

G-protein stimulated adenylyl cyclase 320. Furthermore, our Western blot study showed that following chronic caffeine treatment, the expression of adenosine

A1, A2B receptor subtypes did not change, but a significant decrease was

222 observed in the expression of the adenosine A2A receptor in EA.hy926 endothelial cells. Also, data from the wound healing scratch assay showed that the effect of the selective adenosine A2A receptor agonist, CGS21680, on wound healing rate was less than the effect of the other two selective adenosine A1 and A2B receptor agonists, CPA and NECA, on the rate of

EA.hy926 endothelial cell wound healing after chronic caffeine treatment.

This may be due to the low protein expression of the adenosine A2A receptor in

EA.hy926 endothelial cells. Another in vivo study has reported that adenosine receptors significantly increased expression in the mouse brain stem with chronic caffeine treatment 51. In addition, their data suggested that the adenosine receptors required longer exposure to caffeine to significantly change the expression of these receptors in the cerebral cortex and the thalamus, with 16-23 days of treatment examined 51. Further research could investigate the effect of chronic caffeine on G protein, adenylyl cyclase or phosphodiesterase expression in EA.hy926 endothelial cells. In Chapters 3 to

5 of this PhD study, EAhy926 endothelial cells were seeded in a high glucose cell culture solution (4500 mg/L glucose) to mimic diabetic conditions. In contrast, as presented in Chapter 6, the EAhy926 endothelial cell lines were seeded in a low glucose cell culture solution (1000 mg/L) to mimic the

‘normal’ condition (non-diabetes) and its effect on EAhy926 endothelial cell proliferation and rate of wound healing. However, the EA.hy926 endothelial cells did not remain viable and functional under the low glucose condition after 48 hours. Also, some changes in the cell morphology and confluency were observed in the EA.hy926 endothelial cells under the low glucose condition. These results support the recommendation in the product

223 information sheet for EAhy926 endothelial cells, which advises that a high glucose cell culture solution should be used for seeding and growing this type of cell line.

Several studies have demonstrated that cell proliferation and migration are crucial stages in endothelial cell angiogenesis and wound healing processes 333.

Also, they have reported that endothelial cells are very sensitive to hyperglycemia due to their poor ability to regulate intracellular glucose levels

333. This current study demonstrated that a low glucose condition significantly accelerated the rate of wound healing in the EAhy926 endothelial cell line.

Furthermore, stimulation of adenosine A1, A2A and A2B receptor subtypes with selective agonists increased wound healing rates, similar to that in the high glucose condition. Moreover, the order of efficacy of selective adenosine A1,

A2A and A2B receptor agonists in stimulating wound healing under both low and high glucose conditions was CPA< NECA< CGS21680 (Table 7.1.1).

224

Selective Low Glucose Condition High Glucose Condition Adenosine (1000 mg/L (4500 mg/L Receptor = 5.5 mM glucose) = 25 mM glucose) Agonists (30 nM)

CPA 130±2 µM/hour 70± 0.5 µM/hour 51% 46%

CGS21680 100±3 µM/hour 56± 0.4 µM/hour 18% 50%

NECA 111± 2 µM/hour 60± 0.03 µM/hour 31% 23%

Table 7.1.1. The rate of wound healing (µM/hour) as affected by selective adenosine A1, A2A and A2B receptor agonists in low and high glucose conditions. Data is presented as mean ± SEM, n= 6. Also shown are the percentage increases achieved with the selective adenosine receptor agonists versus their control

225

The cell proliferation assays in this study have shown that in the high glucose condition, increased cell proliferation occurred with the selective adenosine A1,

A2A and A2B receptor agonists in EA.hy926 endothelial cells. However, no changes were observed in cell proliferation rates under the low glucose condition. Several studies have reported that adenosine has a critical role in the regulation of glucose homeostasis and the pathophysiology of diabetes 322,

324-328. Furthermore, the studies have shown adenosine protects cells from any damage, and additionally, activation of adenosine receptors can efficiently accelerate diabetic wound healing 9, 97, 106, 345. Chen et al. 2002 showed that adenosine can increase diabetic ischaemic wound healing by elevating autophagy in endothelial progenitor cells (EPCs) grown on biomaterials 345.

Also, they produced a degradable biomaterial with high biocompatibility. This material was seeded with adenosine-stimulated EPCs and transplanted onto the surfaces of diabetic ischaemic wounds to accelerate healing of the wounds

345. Limitations of this study include the use of an immortalised endothelial cell line which may not represent natural endothelial cells which would have a limited lifespan. These cells would also not represent the various types of endothelial cells that come from different parts of body and so the results of these studies can not be generalised. This work has also been in an in vitro environment away from the complexity of tissues and organ systems. The variable results of manual method of wound healing scratching assay caused issues and inconsistent outcomes as did the high sensitivity of EA.hy926 endothelial cells to low glucose conditions.

226

7.2. CONCLUSION

In conclusion, diabetes mellitus is a chronic disease that is characterized by

hyperglycaemia and abnormalities in the metabolism of carbohydrates,

proteins and lipids 3, 52. Impaired wound healing is a common and serious

complication of diabetes mellitus. It causes dysregulation of the process at

multiple levels, increasing the risk of infection and tissue damage 1. The

adenosine A1, A2A and A2B receptors increase wound healing rates in high and

low glucose conditions. Also, the effect of adenosine receptor on wound

healing is antagonised by acute caffeine treatment and selective adenosine

receptor antagonists. In diabetes, activation of adenosine receptors may

enhance wound healing rates similar to the effect of chronic caffeine treatment

on wound healing in EA.hy926 endothelial cells.

227

CHAPTER 8

REFERENCES

228

CHAPTER 8: REFERENCES

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In order to comply with copyright the article has been removed. Caffeine and its Potential Role in Attenuating Impaired Wound Healing in Diabetes. By Zeinab Bonyanian and Roselyn B. Rose'Meyer.

Journal of Caffeine Research, Vol. 5, No. 4, 2015.

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