EFFECTS OF GLUCOSE ON ENDOTHELIAL FUNCTION IN …...1.1.11 Other specific types of diabetes 36 1.2 Animal models of diabetes 36 1.2.1 Animal models of type 1 diabetes 37 1.2.2 Animal

EFFECTS OF GLUCOSE ON ENDOTHELIAL FUNCTION IN

PREGNANCY AND THE INFLUENCE OF DIABETES

A thesis submitted to The University of Manchester for the degree of PhD in the

Faculty of Medical and Human Sciences

2006

Haiju Henry Chirayath

School of Medicine

2

LIST OF CONTENTS

ABSTRACT 17

CHAPTER 1:

INTRODUCTION 26

1.1 Diabetes Mellitus 27

1.1.1 Background 27

1.1.2 Rationale of study 28

1.1.3 Definition of diabetes 28

1.1.4 Classification of diabetes 29

1.1.5 Diagnosis of diabetes 29

1.1.6 Type 1 diabetes 30

1.1.7 Type 1 diabetes in pregnancy 31


1.1.9 Type 2 diabetes in pregnancy 32

1.1.10 Gestational diabetes 32

1.1.10.1 Definition and diagnosis 32

1.1.10.2 Features of gestational diabetes 35

1.1.11 Other specific types of diabetes 36

1.2 Animal models of diabetes 36

1.2.1 Animal models of type 1 diabetes 37

1.2.2 Animal models of type 2 diabetes 37

1.2.3 Animal models of diabetes in pregnancy 37

1.2.4 Advantages of animal models of diabetes 38

1.2.5 Limitations of animal models of diabetes 38

1.3 Pregnancy 39

1.3.1 Carbohydrate metabolism in pregnancy 39

1.3.2 Cardiovascular changes in pregnancy 40

1.4 Arteries 41

1.4.1 Blood flow to the fetus 41

1.4.2 Resistance arteries 41

3

1.5 The Endothelium 42

1.5.1 Endothelial function 42

1.5.2 Endothelium-dependent mediators of relaxation 43

1.5.2.1 Nitric Oxide 44

1.5.2.2 Prostacyclin 45

1.5.2.3 Endothelium Derived Hyperpolarizing Factor 46

1.5.3 Endothelium-derived vasoconstrictors 47

1.5.4 Other factors affecting endothelial function 48

1.5.4.1 Age 48

1.5.4.2 Smoking 48

1.5.4.3 Ethnicity 48

1.5.4.4 Cholesterol 48

1.5.5 Endothelial function in pregnancy 48

1.5.6 Methods of measuring endothelial function 49

1.5.6.1 Venous occlusion plethysmography 50

1.5.6.2 Brachial artery flow-mediated vasodilation 50

1.5.6.3 Limitations of brachial artery FMD 51

1.6 Endothelial dysfunction in diabetes 51

1.6.1 Animal studies of endothelial dysfunction in diabetes 52

1.6.2 Human studies of endothelial dysfunction in diabetes 52

1.6.3 Mechanisms of endothelial dysfunction in diabetes 53

1.6.4 Endothelial dysfunction and regional variations 55

1.6.5 Other factors influencing endothelial dysfunction in diabetes 56

1.6.5.1 Control of diabetes 56

1.6.5.2 Obesity 56

1.6.5.3 Lipids 57

1.6.5.4 Insulin resistance 58

1.6.5.5 Gender differences in diabetes 58

1.7 Endothelial dysfunction of diabetes in pregnancy 58


1.7.2 Gestational diabetes 59

1.7.3 Previous gestational diabetes 59

1.8 Glucose levels 61

1.8.1 HbA1C 62

4

1.9 Hyperglycaemia 62

1.9.1 Definition 62

1.9.2 Estimating hyperglycaemia 62

1.9.3 Degree of hyperglycaemia 63

1.9.4 Duration of hyperglycaemia 63

1.9.5 Pathophysiology of hyperglycaemia 64

1.9.5.1 Oxidative stress 64

1.9.5.2 Hyperglycaemia and endothelial mediators 66

1.9.6 Acute Hyperglycaemia 66

1.9.7 Hyperglycaemia and endothelium-dependent relaxation 67

1.10 Hypoglycaemia 69

1.10.1 Hypoglycaemia and pregnancy 69

1.10.2 Hypoglycaemia and endothelial function 70

1.11 Blood glucose in pregnancy 70

1.12 Summary of Introduction 72

1.13 Hypotheses 73

1.14 Aims 73

CHAPTER 2

MATERIALS AND METHODS 74

2.1 Ethics 75

2.2 Human subjects 75

2.2.1 Healthy women 75

2.2.1.1 Non-pregnant women 75

2.2.1.2 Pregnant women 75

2.2.2 Diabetic patients 75

2.2.2.1 Type 1 and type 2 diabetes in pregnancy 75

2.2.2.2 Gestational diabetes 76

2.3 Mice 76

2.4 Tissue Collection 76

2.4.1 Non-pregnant women 76

2.4.2 Pregnant women 77

2.4.3 Mice 77

5

2.5 Stereomicroscopic dissection 77

2.6 Resistance arteries 77

2.7 Choice of methodology 77

2.8 Wire myography 78

2.8.1 Multi Myograph System 610M 79

2.8.2 Advantages of wire myography 80

2.8.3 Limitations of wire myography 81

2.9 Normalisation of vessel lumen 81

2.10 Drugs 82

2.10.1 Potassium chloride 82

2.10.2 Bradykinin 82

2.10.3 Phenylephrine 83

2.10.4 Acetylcholine 83

2.10.5 U46619 83

2.10.6 L-NNA 83

2.10.7 Indomethacin 83

2.10.8 Streptozotocin 83

2.10.9 Choice of drugs 84

2.10.9.1 Vasoconstriction 84

2.10.9.2 Endothelium-dependent relaxation 84

2.11 Solutions 85

2.11.1 Glucose physiological saline solutions 85

2.11.2 60 mmol/L KPSS 85

2.11.3 Hyper-osmolar solution 86

2.11.4 Modified Krebs’ buffer 86

2.12 Experimental protocols 86

2.12.1 Protocol 1: The effect of glucose levels on maximum

constriction 86

2.12.2 Protocol 2: The effect of glucose levels on

endothelium-dependent relaxation 88

2.12.3 Protocol 3: The effect of hyper-osmolarity 89

2.12.4 Protocol 4: The effect of blockers to


6

2.13 Optimising experiments 92

2.13.1 General measures 92

2.13.2 Optimising mice experiments 92

2.14 Creating an animal model of diabetes 93

2.14.1 Restrictions in human research 93

2.14.2 Rationale for creating an animal model of diabetes 94

2.14.3 Obtaining a Home Office Licence 94

2.14.4 Species and strain 94

2.14.5 Duration of diabetes 95

2.14.6 Diabetogenic drug 95

2.14.7 Procedure 95

2.15.8 Impediments in creating an animal model of diabetes 96

2.15 Data analysis 97

2.15.1 Constriction 97

2.15.2 Endothelium-dependent relaxation 97

2.16 Statistical analysis 97

2.16.1 General Principles 97

2.16.2 Statistical analysis of data from patients 98

2.16.3 Statistical analysis of myography data 98

2.16.3.1 Constriction 98


CHAPTER 3

THE GLUCOSE LEVELS OF WOMEN WITH

DIABETES IN PREGNANCY 99

3.1 Introduction 100

3.2 Aims 101

3.3 Methodology 101

3.3.1 Inclusion criteria 102

3.3.2 Exclusion criteria 102

3.3.3 Data collection 103

3.3.4 Statistical analysis 103

7

3.4 Results 103

3.4.1 Clinical characteristics 103

3.4.2 Glucose levels 106

3.4.3 Glucose levels of type 1 diabetes in pregnancy 107

3.4.4 Glucose levels of type 2 diabetes in pregnancy 108

3.4.5 Glucose levels of gestational diabetes 108

3.4.6 HbA1C levels: comparison across types 112

3.4.7 HbA1C levels in each group 112

3.4.7.1 Type 1 diabetes 112

3.4.7.2 Type 2 diabetes 112

3.4.7.3 Gestational diabetes. 112

3.4.8 Glucose levels in GDM: effect of insulin 114

3.4.9 Glucose levels and outcomes in type 1 diabetes 116

3.4.10 HbA1C and outcomes in type 1 diabetes 116

3.5 Discussion 119

3.5.1 Glucose levels of women with diabetes in pregnancy 119

3.5.2 Glucose levels in type 1 diabetes 120

3.5.3 Glucose levels in type 2 diabetes 120

3.5.4 Glucose levels in gestational diabetes 120

3.5.5 Adverse outcomes 120

3.5.5.1 Type 1 and 2 diabetes 121

3.5.5.2 Gestational diabetes 122

3.5.6 HbA1C and glucose levels 122

3.5.7 Limitations of study 123

3.5.8 Summary 123

CHAPTER 4

THE EFFECT OF GLUCOSE ON ENDOTHELIAL FUNCTION

IN HEALTHY NON-PREGNANT AND PREGNANT WOMEN 125


4.2 Aims 127

4.3 Materials and Methods 127

4.4 Results 128

8

4.4.1 Clinical characteristics 128

4.4.2 Arterial diameters 129

4.4.3 Constriction: effect of glucose (non-pregnant group - KCl) 131

4.4.4 Endothelium-dependent relaxation: effect of glucose

(non-pregnant group – KCl) 131

4.4.5 Constriction: effect of glucose

(non-pregnant group - U46619) 131


(non-pregnant group – U46619) 131

4.4.7 Constriction: effect of glucose (pregnant group - KCl) 136


(pregnant group – KCl) 136

4.4.9 Constriction: effect of glucose (pregnant group–U46619) 139


(pregnant group – U46619) 139

4.4.11 Comparison of endothelium-dependent relaxation in

non-pregnant and pregnant groups 144

4.4.12 Effect of prolonged exposure (2 hours) of

2, 5, 8 and 12 mmol/L glucose 146

4.5 Discussion 148

4.5.1 Summary of results 148

4.5.2 Low glucose concentration and impaired relaxation 148

4.5.2.1 The effect of vasoconstrictors 149

4.5.2.2 The effect of pregnancy 150

4.5.3 Other findings 150


4.5.3.2 Effect of pregnancy on endothelium-dependent

relaxation 150

4.5.3.3 Effect of prolonged incubation 151

4.5.3.4 High glucose concentrations 152

4.5.4 Explanations for the lack of effect of high glucose levels 152

4.5.4.1 Inadequate duration of high glucose levels 152

4.5.4.2 Inadequate degree of high glucose levels 152

4.5.4.3 Systemic factors 153

9

4.5.4.4 Endothelial vasodilators 154

4.5.4.5 Bradykinin 154

4.5.4.6 Branch-order 155

4.5.4.7 Age 155

4.5.4.8 Nature of vascular bed 156

4.5.4.9 Gender 156

4.5.4 Summary 157

CHAPTER 5

THE EFFECT OF GLUCOSE ON ENDOTHELIAL FUNCTION IN

DIABETES COMPLICATING PREGNANCY 158


5.2 Aims 160

5.3 Materials and methods 161

5.4 Results 161

5.4.1 Clinical characteristics – gestational diabetes 161

5.4.2 Diameter of arteries – gestational diabetes 163

5.4.3 Constriction: effect of glucose (GDM-KCl) 163


(GDM-KCl) 166

5.4.5 Constriction: effect of glucose (GDM-U46619) 166


(GDM-U46619) 166

5.4.7 GDM: effect of vasoconstrictor on endothelium-dependent

relaxation 170

5.4.8 Constriction: effect of glucose (type 1 and 2 diabetes) 171


(type 1 and 2 diabetes) 171

5.4.10 Comparison between GDM and healthy pregnant women

constriction 175

5.4.10.1 Using KCl 175

5.4.10.2 Using U46619 175

10

5.4.11 Comparison between GDM and healthy pregnant women:


5.4.11.1 Using KCl 175

5.4.11.2 Using U46619 175

5.5 Discussion 179

5.5.1 Summary of results 179

5.5.2 Endothelial dysfunction in GDM 180

5.5.2.1 Explanations for impaired relaxation 180

5.5.2.2 Implications of findings 182

5.5.2.3 Limitations of study 183

5.5.3 Patient characteristics 183

5.5.4 The effect of glucose levels on vascular function 184



5.5.5 Type 1 and type 2 diabetes 185

5.5.6 Summary 186

CHAPTER 6

THE EFFECT OF GLUCOSE ON ENDOTHELIAL

FUNCTION IN NON-PREGNANT AND PREGNANT MICE 187


6.2 Aims 189

6.3 Materials and methods 189

6.4 Results 190

6.4.1 Arterial diameters 190

6.4.2 Constriction: normal non-pregnant mice 191

6.4.3 Endothelium-dependent relaxation: normal non-pregnant mice 191

6.4.4 Constriction: normal pregnant mice 195

6.4.5 Endothelium-dependent relaxation: normal pregnant mice 195

6.4.6 Effect of pregnancy on constriction and


6.4.7 Effect of hyper-osmolarity – pregnant mice 199

11

6.4.8 Normal pregnant mice: effect of endothelial blockers at

5 and 12 mmol/L glucose 202

6.4.9 Normal pregnant mice: effect of glucose levels on

endothelial blockers 202

6.4.10 Blood glucose in mice 206

6.4.11 Pregnant streptozotocin-control mice 207

6.4.11.1 Constriction: effect of glucose levels

(pregnant streptozotocin-controls) 207

6.4.11.2 Relaxation: effect of glucose levels


6.5 Discussion 212

6.5.1 Summary of findings 212

6.5.2 Enhanced vasodilation at 12 mmol/L glucose 212

6.5.3 Glucose requirements in different vascular beds 214

6.5.4 Hyperglycaemia and hyper-osmolarity 214

6.5.5 Hyperglycaemia and systemic factors 215

6.5.6 Effect of pregnancy 215

6.5.7 Effect of glucose on constriction 216

6.5.8 Endothelial mediators of vasodilation 216

6.5.9 Streptozotocin treated non-diabetic mice


6.5.10 Summary 219

CHAPTER 7

DISCUSSION 220

7.1 Overview of study 221

7.2 Key findings. 224

7.3 Hypothesis 1 224



7.6 Limitations of study 229

7.7 Future avenues of research 231

12

REFERENCES 232

APPENDIX A: Consent form 259

APPENDIX B: Patient Information sheet 260

APPENDIX C: Pro forma for study of diabetes in pregnancy 261

APPENDIX D: Review Article: Diabetes management in pregnancy 263

APPENDIX E: Published Paper 272

FINAL WORD COUNT = 43144

13

LIST OF FIGURES Figure 1.1 The Ebers Papyrus 27

Figure 1.2 Endothelium-dependent mediators of vasodilation 44

Figure 1.3 Brachial artery flow-mediated dilatation 50

Figure 1.4 Overview of glucose metabolism 60

Figure 1.5 Oxidative stress and endothelial dysfunction 65

Figure 2.1 Schematic representation of a blood vessel mounted on a

wire myograph 78

Figure 2.2 Myograph with data recordings on computer 79

Figure 2.3: Multi Myograph System 610M and an individual

myograph chamber 80

Figure 2.4: Schematic representation of concentration-response curve 87

Figure 2.5: Raw data trace 87

Figure 2.6: Experimental protocol 1 and 2 88

Figure 2.7: Protocol 3 89

Figure 2.8: Protocol 4: Raw data trace 90

Figure 2.9: Protocol 4 (5 mmol/L glucose) 91

Figure 2.10: Protocol 3 (12 mmol/L glucose) 91

Figure 3.1: Glucose levels: type 1 diabetes 109

Figure 3.2: Glucose levels: type 2 diabetes. 110

Figure 3.3: Glucose levels: gestational diabetes 111

Figure 3.4: HbA1C: type 1 diabetes 113

Figure 3.5: HbA1C: type 2 diabetes 113

Figure 3.6: HbA1C: gestational diabetes 114

Figure 3.7: Glucose levels in diet-controlled GDM 115

Figure 3.8: Glucose levels in insulin-controlled GDM 115

Figure 3.9: Glucose levels in type 1 diabetes – normal outcome 117

Figure 3.10: Glucose levels in type 1 diabetes – adverse outcome 117

Figure 3.11: HbA1C in type 1 diabetes – normal outcome 118

Figure 3.12: HbA1C in type 1 diabetes – adverse outcome 118

Figure 4.1: Arterial diameters (non-pregnant group) 130

Figure 4.2: Arterial diameters (healthy pregnant group) 130

14

Figure 4.3 (A-D): Constriction: effect of glucose

(non-pregnant group – KCl) 132

Figure 4.4 (A-D): Relaxation: effect of glucose

(non-pregnant group - KCl) 133



Figure 4.6 (A-D): Relaxation: effect of glucose on relaxation




Figure 4.8 (A-D): Relaxation: effect of glucose




Figure 4.10 (A-D): Relaxation: effect of glucose on relaxation


Figure 4.11: Relaxation at 5 mmol/L glucose


Figure 4.12: Relaxation at 2, 5 and 12 mmol/L glucose


Figure 4.13 (A-C): Relaxation: non-pregnant and pregnant groups 145

Figure 4.14: Two hour incubation: aberrant responses 147

Figure 5.1 (A & B): Arterial diameters – GDM 163

Figure 5.2 (A-D): Constriction: effect of glucose (GDM- KCl) 164

Figure 5.3 (A-D): Relaxation: effect of glucose levels (GDM- KCl) 166

Figure 5.4 (A-D): Constriction: effect of glucose levels

(GDM- U46619) 167

Figure 5.5 (A-D): Relaxation: effect of glucose levels

(GDM – U46619) 168

Figure 5.6: Relaxation: effect of vasoconstrictor 169

Figure 5.7: Constriction: effect of glucose levels, type 1 diabetes – KCl 171

Figure 5.8: Constriction: effect of glucose levels, type 2 diabetes – KCl 171

Figure 5.9 (A-D): Relaxation: effect of glucose (type 1 diabetes – KCl) 172

Figure 5.10 (A-D): Relaxation: effect of glucose (type 1 diabetes – KCl) 173

15

Figure 5.11: KCl Constriction: Normal vs GDM 175

Figure 5.12: U46619 Constriction: Normal vs GDM 175

Figure 5.13: Relaxation – Normal vs GDM: KCl 176

Figure 5.14: Relaxation – Normal vs GDM: U46619 177

Figure 6.1: Arterial diameters – non-pregnant and pregnant mice 189

Figure 6.2 (A-D): Constriction: effect of glucose (non-pregnant mice) 191

Figure 6.3 (A-D): Relaxation: effect of glucose (non-pregnant mice) 192

Figure 6.4: Relaxation: effect of glucose (non-pregnant mice) 193

Figure 6.5 (A-D): Constriction: effect of glucose (pregnant mice) 195

Figure 6.6 (A-D): Relaxation: effect of glucose (pregnant mice) 196

Figure 6.7: Relaxation: effect of glucose levels (pregnant group) 197

Figure 6.8: Constriction: non-pregnant vs pregnant mice 198

Figure 6.9: Relaxation: non-pregnant vs pregnant mice 199

Figure 6.10: The effect of hyper-osmolarity 200

Figure 6.11: The effect of blockers on relaxation – 5 mmol/L glucose 202

Figure 6.12: The effect of blockers on relaxation – 12 mmol/L glucose 203

Figure 6.13 (A-D): The effect of blockers at 5 and 12 mmol/L glucose 204

Figure 6.14: Blood glucose: mice 206

Fig. 6.15 (A-D): Constriction: effect of glucose levels

(pregnant STZ-controls) 208

Fig. 6.16 (A-D): Relaxation: the effect of glucose


Fig. 6.17: Relaxation: effect of glucose levels


Fig 7.1: Overview of study 222

16

LIST OF TABLES

Table 1.1: WHO classification of Diabetes Mellitus 29

Table 1.2: Vasoactive products synthesized by the endothelium 43

Table 1.3: Studies demonstrating impaired relaxation with hyperglycaemia 68

Table 1.4: Studies demonstrating vasodilation with hyperglycaemia 68

Table 1.5: Studies demonstrating no effect of hyperglycaemia

on vascular function 69

Table 2.1: Composition of solutions used (in mmol/L) 85

Table 3.1: Clinical characteristics 104

Table 3.2: Range of glucose levels seen in diabetes in pregnancy 107

Table 4.1: Clinical characteristics of healthy individuals 129

Table 4.2: Summary of results – Chapter 4 148

Table 5.1: Clinical characteristics – gestational diabetes 162

Table 5.2: Summary of results - effect of change in glucose levels-DM 178

Table 5.3: Summary of results – comparison between normal and GDM 178

17

THE UNIVERSITY OF MANCHESTER

ABSTRACT OF THESIS submitted by HAIJU HENRY CHIRAYATH

for the Degree of PhD and entitled “Effects of glucose on endothelial function

in pregnancy and the influence of diabetes”

September 2006

Diabetes in pregnancy is a potentially serious disease for both mother and fetus and

its prevalence is increasing globally. Poorly controlled diabetes, with recurrent

episodes of hypoglycaemia and hyperglycaemia, is associated with adverse

pregnancy outcomes. As diabetes is characterised by abnormalities in vascular

function, it is possible that these episodes of aberrant glucose levels may affect the

arteries regulating blood flow to the fetus, thereby playing a role in the

complications of this condition. The endothelium forms the innermost lining of

arteries and has been shown to exert important vasoregulatory effects which can

alter the haemodynamics of blood flow. Although endothelial dysfunction has been

demonstrated in various vascular beds in diabetes, it is uncertain whether the crucial

uterine circulation is similarly affected. The main aim of this study was to examine

whether aberrant glucose levels can affect endothelial function in these arteries.

Clinically relevant glucose levels were identified in this study to range from 2 to12

mmol/L by assessing the blood glucose levels in 111 patients with diabetes in

pregnancy. Patients with type 1 diabetes were noted to have wide fluctuations of

blood glucose as well as a high rate of pregnancy complications. Constriction and

endothelium-dependent relaxation were examined in myometrial arteries from three

groups of women: healthy non-pregnant, healthy pregnant and pregnant diabetic

women. The effect of changing glucose concentrations from 5 mmol/L to 2, 8 and

12 mmol/L glucose was studied using wire myography. An agonist-specific

impairment of endothelium-dependent relaxation was present when arteries from

healthy pregnant women were exposed to 2 mmol/L glucose for 30 minutes.

Arteries from healthy non-pregnant as well as pregnant diabetic women exhibited no

difference in constriction or endothelium-dependent relaxation. Myometrial arteries

from women with gestational diabetes had significantly impaired endothelium-

dependent relaxation compared to those from healthy pregnant women. When

similar studies were performed in the uterine artery of normal pregnant mice,

exposure to 12 mmol/L glucose for 30 minutes was associated with enhanced

endothelium-dependent relaxation. This effect was not present in uterine arteries of

non-pregnant mice, indicating a pregnancy-specific modulation of vascular function.

The contribution of the endothelial mediators of vasodilation was also assessed in

the uterine artery of pregnant mice. Prostacyclin did not contribute to endothelium-

dependent relaxation, which was mediated by nitric oxide and to a greater extent by

a non-NO/non-prostacyclin component. The contribution of these mediators was not

significantly affected by changes in glucose levels. Due to the limited availability of

tissue from diabetic patients, an attempt was made to create an animal model of

diabetes to enable further studies. After injection of streptozotocin, only a minority

of mice developed diabetes; as assessed by serial measurements of blood glucose.

Mice were divided into vehicle-controls, strepozotocin-controls and diabetic mice.

Uterine arteries from streptozotocin-controls did not demonstrate enhancement of

endothelium-dependent relaxation with 12 mmol/L glucose, as was seen in normal

mice. It is hoped that the findings of this study and the development of an animal

model of diabetes in pregnancy will help to increase our understanding of the

endothelial dysfunction seen in pregnant women with diabetes.

18

DECLARATION

No portion of the work referred to in this thesis has been submitted in support of an

application for another degree or qualification of this or any other university or other

institute of learning.

19

COPYRIGHT STATEMENT

(i) Copyright in text of this thesis rests with the author. Copies (by any process)

either in full, or of extracts, may be made only in accordance with instructions

given by the author and lodged in the John Rylands University Library of

Manchester. Details may be obtained from the Librarian. This page must form

part of any such copies made. Further copies (by any process) of copies made

in accordance with such instructions may not be made without the permission

(in writing) of the author.

(ii) The ownership of any intellectual property rights which may be described in

this thesis is vested in The University of Manchester, subject to any prior

agreement to the contrary, and may not be made available for use by third

parties without the written permission of the University, which will prescribe

the terms and conditions of any such agreement.

(iii) Further information on the conditions under which disclosures and

exploitation may take place is available from the Head of School of Medicine.

20

ACKNOWLEDGEMENTS

This endeavour is not a solo effort. It has been made possible by a number of

individuals and institutions to whom I owe a debt of gratitude.

I would like to express my sincere gratitude to Professor Philip Baker, Dr. Michael

Taggart and Dr. Mark Wareing for their support, supervision and advice throughout

my project. This study would not have been possible without them.

I am extremely grateful to Professor Graham Dunn (Head of Biomedical Statistics,

University of Manchester) and Professor Michael Campbell (Professor of Medical

Statistics, University of Sheffield) for their advice and help on the statistical analysis

of data presented in this thesis.

I would like to thank all my friends and colleagues at the Maternal and Fetal Health

Research Centre, the BSU and the University of Manchester for all their help over

the years. In particular, I am grateful to the Novo Nordisk Research Foundation, UK

for funding this project and supporting me through the course of this study.

I thank Dr. Nicholas Ashton, Professor David Tomlinson, Joanna Stanley and Mike

Jackson for helping me create an animal model of diabetes in pregnancy. I also

thank Professor Kennedy Cruickshank and Professor David Owens for their

valuable guidance and support.

I would also like to thank the staff of the diabetes in pregnancy clinic at St. Mary’s

Hospital, particularly Dr. Peter Selby and Dr. Michael Maresh, for opening my eyes

to diabetes in pregnancy and enabling me to learn more about this disorder.

Lastly, and perhaps most importantly, I would like to thank all the women who

agreed to take part in this study. Their selfless support of medical research has been

a constant source of inspiration for me.

21

PRESENTATIONS AND PUBLICATIONS

Papers

Chirayath H.H. (2006) Diabetes management in pregnancy, Reviews in

Gynaecological and Perinatal Practice. 6, pp.106-114 (Appendix D)

Chirayath H.H., Wareing M., Taggart M.J, Baker P.N. (2007) Acute

hyperglycemia in uterine arteries from pregnant, but not non-pregnant mice,

enhances endothelium-dependent relaxation, Vascular Pharmacology

46, 137-143 (Appendix E)

Chirayath H.H., Wareing M., Taggart M.J, Baker P.N. (2006) “ Impaired

endothelium-dependent relaxation in myometrial arteries of women with

gestational diabetes”

Submitted

Presentations

I. Annual Professional Conference (2005) - Diabetes UK

Poster Presentation

Acute hypoglycaemia impairs relaxation of uterine arteries in pregnant mice

II. 65th

Scientific Sessions (2005) –American Diabetes Association

Poster Presentation

Hypoglycaemia and hyperglycaemia impair myometrial artery relaxation in

pregnancies complicated by gestational diabetes

III. Annual Professional Conference (2006) - Diabetes UK

Poster Presentation

Hyperglycaemia enhances endothelium-dependent relaxation in uterine

arteries of pregnant mice

IV. 66th

Scientific Sessions (2006) –American Diabetes Association

Oral Presentation

Acute hyperglycemia in uterine arteries of pregnant, but not non-pregnant

mice, enhances endothelium-dependent relaxation

22

DEDICATION

This thesis is dedicated to my loving wife and family for their constant support and

also to all the patients and individuals who have helped me throughout this study.

23

LIST OF ABBREVIATIONS

AEP: Active Effective Pressure

ANOVA: Analysis of Variance

ATP: Adenosine Triphosphate

BH4: Tetrahydro biopterin

BMI: Body Mass Index

cAMP: Cyclic Adenosine Monophosphate

cGMP: Cyclic Guanosine Monophosphate

CEMACH: Confidential Enquiry into Maternal and Child Health

COX: Cyclo-oxygenase

DM: Diabetes Mellitus

EDCF: Endothelium-Derived Constricting Factor

EDHF: Endothelium-Derived Hyperpolarising Factor

EDRF: Endothelium-Derived Relaxing Factor

eNOS: Endothelial Nitric Oxide Synthase

ET-1: Endothelin - 1

FMD: Flow Mediated Dilatation

GDM: Gestational Diabetes

GTP: Guanosine Triphosphate

GTT: Glucose Tolerance Test

HAPO: Hyperglycaemia and Adverse Pregnancy Outcome study

HDL: High Density Lipoprotein

ICAM: Intercellular Adhesion Molecule

IFG: Impaired Fasting Glucose

IGT: Impaired Glucose Tolerance

Ind.: Indomethacin

KCl: Potassium Chloride

KPSS: Potassium Physiological Solution

LDL: Low Density Lipoprotein

L-NNA: Nω –Nitro-L-Arginine

NO: Nitric Oxide

OGTT: Oral Glucose Tolerance Test

24

PGH2: Prostaglandin H2

PGI2: Prostacyclin

PSS: Physiological Saline Solution

SD: Standard Deviation

SEM: Standard Error of Mean

sICAM-1: soluble Intercellular Adhesion Molecule-1

SMBG: Self-monitored blood glucose

STZ: Streptozotocin

TGF: Transforming Growth Factor

tPA: Tissue Plasminogen Activator

TXA2: Thromboxane A2

VEGF: Vascular Endothelial Growth Factor

vWF: von Willebrand Factor

25

“Diabetic women were discouraged from becoming pregnant.

They often died during the course of their pregnancy or their

babies often died before birth or as infants. Any successful

pregnancy was remarkable.”

Dr. Priscilla White

Pioneer in the treatment of diabetes in pregnancy;

quoted prior to the discovery of insulin

26

CHAPTER 1

INTRODUCTION

27

INTRODUCTION

1.1 Diabetes Mellitus

1.1.1 Background

Diabetes Mellitus is one of the oldest diseases known to mankind, the first record of

its symptoms having been documented on papyrus scrolls by ancient Egyptians

approximately 3500 years ago [King and Rubin, 2003]. The Ebers papyrus contains

a description of a disease characterised by excessive urination, which is believed to

be the very first description of diabetes (Fig. 1.1).

Fig 1.1: The Ebers Papyrus: the first record of diabetes, circa 1500 BC

Diabetes can be considered to be a state of reduced insulin action which occurs as a

result of decreased insulin availability or diminished insulin effectiveness [Bell and

Hockaday, 1996]. This leads to unduly high glucose levels and clinical symptoms

such as thirst and increased urine production, which occur secondarily to the

osmotic effect of raised glucose levels. The term diabetes originates from the Greek

“diabainein” meaning “to go through”; a reference to the excessive urine output. A

high blood glucose level is the hallmark of diabetes and plays a central role in the

diagnosis of this disease and the pathophysiology of its complications. However, the

deleterious effects of diabetes are a result of multiple factors such as alterations in

lipid metabolism, changes in cellular metabolism, and the production of metabolites

28

that are harmful to the vasculature. Diabetes Mellitus is not a single disease, but a

genetically heterogeneous group of disorders that share a common factor: glucose

intolerance. It is a true multi-system disorder which can affect almost all organ

systems of the body, causing a variety of debilitating and potentially fatal

complications. The multi-system nature of this disease, the lack of a cure, its

chronicity and rising prevalence combine to pose a serious threat to global health in

the 21st century.

1.1.2 Rationale of study

The total number of people with diabetes is projected to rise from 171 million in

2000 to 366 million in 2030 [Wild et al., 2004]. Diabetes in pregnancy

(incorporating type 1, type 2 and gestational diabetes) is a potentially serious

medical disorder in pregnancy, affecting approximately 1 in 250 pregnancies in UK

[CEMACH, 2005]. Patients with diabetes have a higher risk of cerebro-vascular

disease, ischaemic heart disease, retinopathy, amputations, and renal failure. These

myriad complications all share the common feature of abnormal vascular function,

an important cause of which is the effect of glucose on vasculature. Despite its

pivotal role in the pathophysiology of diabetes, the effect of aberrant glucose levels

on vessels is incompletely understood. Studies in this field have produced

conflicting results, which make the true effects of glucose difficult to discern.

Furthermore, it is unknown if the irregular glucose levels seen in pregnancies

complicated by diabetes are associated with vascular dysfunction in arteries

supplying blood to the fetus. The aim of this study therefore, was to directly

examine the effects of abnormal glucose levels on the function of arteries involved

in the blood flow to the fetus.

1.1.3 Definition of diabetes

The WHO has defined diabetes as a metabolic disorder of multiple aetiology,

characterized by chronic hyperglycaemia with disturbances of carbohydrate, fat and

protein metabolism, resulting from defects in insulin secretion, insulin action or both

[Alberti and Zimmet, 1999].

29

1.1.4 Classification of diabetes

The current WHO classification system is based on the aetiology of diabetes

mellitus [Alberti and Zimmet, 1999]. It divides diabetes into 4 main groups: type1,

type 2, gestational diabetes and other types (Table 1).

TYPE 1 DIABETES MELLITUS Auto-immune

Idiopathic

TYPE 2 DIABETES MELLITUS

GESTATIONAL DIABETES

OTHER SPECIFIC TYPES

Genetic Defects of -cell function

Genetic defects of insulin action

Diseases of the exocrine pancreas

Endocrinopathies

Drug- or Chemical-induced diabetes

Infections

Uncommon forms of immune-mediated diabetes

Other genetic syndromes

Table 1.1: WHO classification of Diabetes Mellitus

1.1.5 Diagnosis of diabetes

The diagnosis of diabetes is made with a fasting plasma glucose level ≥ 7.0 mmol/L

on two separate occasions or an oral glucose tolerance test (GTT). The GTT is more

sensitive for the diagnosis of diabetes than fasting plasma glucose [Reinauer et al.,

2002].

The GTT is used for diagnosis when blood glucose levels are equivocal, during

pregnancy or in epidemiological studies [Alberti and Zimmet, 1999]. The test is

preceded by fasting overnight until the commencement of the test, during which

only water may be drunk. After the collection of the fasting sample, the subject

30

drinks 75 g glucose dissolved in water. Blood samples are collected 2 hours after the

test load.

A diagnosis of diabetes mellitus is made if the fasting venous plasma glucose is ≥

7.0 mmol/L or the 2-hour post glucose load value is ≥ 11.1 mmol/L. The diagnosis

of gestational diabetes is made during pregnancy if a patient (with no previous

diagnosis of diabetes) fulfils this criteria, or the criteria for Impaired Glucose

Tolerance (IGT); which is fasting venous plasma glucose < 7.0 mmol/L and 2 hour

post glucose load ≥ 7.8 mmol/L.

1.1.6 Type 1 diabetes

Type 1 diabetes is characterised by cell-mediated auto-immune destruction of islet

-cells, which leads to a complete or nearly complete absence of endogenous insulin

secretion [Alberti and Zimmet, 1999]. Insulin is required for survival to prevent the

development of keto-acidosis, coma and death. Type 1 diabetes is predominantly a

disease of childhood and usually presents before the age of 35 [Reinauer et al.,

2002]. A large multi-centre study in Europe has shown that the incidence of this

disorder is much higher in the 0-14 age group than the 15-29 age group [Kyvik et

al., 2004]. The incidence of type 1 diabetes in Europe has been shown to be rising at

an average rate of 3.4%, with the largest rate of increase seen in children aged 0-4

years [EURODIAB Group, 2000]. There is a marked ethnic and geographic

variation of type 1 diabetes; its incidence being high in Finland, Sweden and

Norway and low in China and South America [Karvonen et al., 2000]. There is a

low genetic predisposition to develop this type of diabetes and antibodies to -cells

are often seen at the time of diagnosis [Alberti and Zimmet, 1999].

Laboratory findings include hyperglycaemia, ketonuria, low or undetectable serum

insulin and C-peptide levels, and auto-antibodies against components of the islet -

cells [Reinauer et al., 2002].

31

Type 1 diabetes has been subdivided into two sub-types: immune mediated and

idiopathic type 1 diabetes. The immune-mediated sub-type is distinguished by the

presence of auto-antibodies to glutamic acid decarboxylase, islet cell or insulin

antibodies. Idiopathic type 1 diabetes has a similar clinical presentation as immune–

mediated type 1 diabetes, although it is distinguished from it by a lack of

demonstrable antibodies to -cells. The idiopathic sub-type has a marked familial

pattern of inheritance and is more common in Asians and Africans than Caucasians.

1.1.7 Type 1 diabetes in pregnancy

A large population study in Denmark has demonstrated a higher rate of perinatal

mortality, pre-term delivery, stillbirths, congenital malformations and Caesarean

delivery in patients with type 1 diabetes [Jensen et al., 2004]. Another study in

Netherlands corroborated these findings, additionally reporting a higher maternal

mortality rate [Evers et al., 2004]. In both studies, better glycaemic control was

associated with an improved outcome, although the latter study reported

complications even on attaining near-optimal control of blood glucose levels. In

addition to the above complications, type 1 diabetes may also influence birth

weight. A study in Scotland has reported a higher mean birth weight in 216 babies

of women with type 1 diabetes [Penney et al., 2003].


Type 2 diabetes is due to insulin insensitivity combined with a failure of insulin

secretion to overcome this by hyper-secretion, resulting in relative insulin deficiency

[Reinauer et al., 2002]. This is the most common form of diabetes and there is a

strong genetic predisposition. The specific reasons for developing these

abnormalities remain unknown. Patients with this condition are usually obese and

tend to be older, although the disease can occur at any age and in lean individuals.

However, a recent trend for an earlier onset of type 2 diabetes has been noticed; as

obesity in children and adolescents becomes more prevalent [Rosenbloom et al.,

1999].

32

Important associations of type 2 diabetes [Reinauer et al., 2002] include:

Family history of diabetes (in particular parents or siblings with diabetes)

Obesity (≥ 20% over ideal body weight or BMI ≥ 25 kg/m²)

Ethnicity (Africans, Asians, Hispanic and Native American)

Age ≥ 45 years

Previously identified impaired fasting glucose (IFG) or IGT

Hypertension (≥ 140/90 mmHg in adults)

HDL cholesterol level <1.0 mmol/L (< 0.38 g/L)

and/or a triglyceride level ≥ 2.3 mmol/L (≥ 2.0 g/L)

Reduced physical activity

Past history of gestational diabetes or delivery of babies > 4.5 kg

1.1.9 Type 2 diabetes in pregnancy

A UK study demonstrated that infants of women with type 2 diabetes in pregnancy

have a 2-fold greater risk of stillbirth, a 2.5-fold greater risk of perinatal mortality

and an 11-fold greater risk of congenital malformations [Dunne et al., 2003].

Another study demonstrated that the percentage of pregnancies having a serious

adverse outcome was higher in type 2 patients than type 1 (16.4 vs. 6.4%), with

more congenital abnormalities in the type 2 group [Roland et al., 2005]. One

possible explanation cited for this was that these women had less pre-conception

care. In UK, it has been estimated that only 37% of women with type 2 diabetes in

pregnancy have had a measurement of glycaemic control before pregnancy

[CEMACH, 2005]. As noted above, a past history of gestational diabetes is a risk

factor for type 2 diabetes, emphasising the association between these two

conditions.

1.1.10 Gestational diabetes

1.1.10.1 Definition and diagnosis

Compared to type 1 and 2 diabetes, the recognition of the disorder termed

gestational diabetes has been relatively recent. In the first half of the 20th

century, it

was perceived that women who developed diabetes years after pregnancy had

33

suffered from abnormally high fetal and neonatal mortality [Miller, 1946]. By the

1950s the term "gestational diabetes" was applied to what was thought to be a

transient condition that affected fetal outcomes adversely, then abated after delivery

[Carrington et al., 1957]. In 1964, O’Sullivan found that the degree of glucose

intolerance during pregnancy was related to the risk of developing diabetes after

pregnancy. He proposed statistical criteria for the interpretation of oral glucose

tolerance tests during pregnancy, establishing cut-off values (approximately

2

standard deviations) for diagnosing glucose intolerance

during pregnancy

[O'Sullivan and Mahan, 1964]. These cut-off points were adapted to modern

methods for measuring glucose and applied to the WHO definition of gestational

diabetes: carbohydrate intolerance resulting in hyperglycaemia of varying severity

with onset or first recognition during pregnancy [Alberti and Zimmet, 1999]. This

definition does not exclude the possibility that glucose intolerance was present, but

unrecognized, before pregnancy. The definition is independent of the treatment

modality used during pregnancy.

The WHO definition of gestational diabetes remains controversial. The earlier

definition by O’Sullivan was “a transient abnormality of glucose tolerance during

pregnancy.” This definition implies that the glucose intolerance reverts back to

normal after pregnancy. The objection to the WHO definition is that patients with

pre-existing type 1 or type 2 diabetes may be wrongly classified as having

gestational diabetes [Omori and Jovanovic, 2005]. In patients with pre-existing

diabetes, abnormal carbohydrate metabolism is present for the entire duration of

pregnancy, whereas it is seen mainly in the second half of pregnancy in gestational

diabetes. The prevalence of maternal retinopathy and fetal congenital defects are

also higher in women with pre-existing diabetes. However, in practice, it is difficult

to clinically distinguish the three types of diabetes in pregnant women as there are

no diagnostic markers for type 2 diabetes and the antibody tests to detect type 1

diabetes are not routinely carried out. Furthermore, post-natal GTT results are not

performed in all women, which precludes the classification of their diabetes

[Beischer et al., 1997].

Treatment of women with gestational diabetes (by dietary advice, glucose

monitoring and insulin as needed) has been reported to improve their pregnancy

34

outcome [Crowther et al., 2005]. Therefore, screening of such women using a

glucose tolerance test is useful in identifying those who would benefit from

treatment during pregnancy. However, available data do not identify the threshold of

maternal glycaemia at which the risk begins or increases during pregnancy. To

resolve this issue, a large, international trial in a multi-ethnic population (the

Hyperglycaemia and Adverse Pregnancy Outcome study - HAPO) is currently

underway [HAPO Group, 2002]. As blood glucose levels represent a continuous

variable, it is possible that a continuum of increased risk to the baby is seen rather

than clear-cut distinctions between normal and abnormal groups [Scott et al., 2002].

The current lack of an evidence-based threshold for defining the glucose levels at

which increased risk occurs has resulted in different guidelines worldwide for the

diagnosis of gestational diabetes. Although glucose tolerance tests are used for

diagnosis, the point of time in pregnancy at which it is performed, the amount of

glucose taken (50, 75 and 100 grams), the duration of the test (1, 2 and 3 hours) and

the cut-off values for diagnosis vary from region to region. For example, studies in

Spain [Chico et al., 2005], China [Yang et al., 2005] and Mexico [Ramirez-Torres

et al., 2003] have used different glucose tolerance tests to diagnose gestational

diabetes. The WHO currently uses the term gestational diabetes to encompass both

gestational impaired glucose tolerance (GIGT) and gestational diabetes mellitus

(GDM), which were previously regarded as separate entities. Furthermore, the cut-

off values for these conditions are the same as in the non-pregnant state. The normal

physiological changes in pregnancy include increasing insulin resistance, resulting

in raised post-prandial blood glucose levels. Therefore, the milder degrees of GIGT

diagnosed later in pregnancy may actually represent normal values [Maresh, 2005].

Because of this, the European Association for the Study of Diabetes recommended

in 1989 that the lower cut-off for the 2-hour value defining GIGT be raised from 7.8

to 9.0 mmol/L in the third trimester on the basis of an epidemiological study [Lind,

1989]. However, this is at variance with the current WHO guidelines.

From the above discussion, it is clear that there is no consensus of expert opinion

regarding either the definition or the diagnostic criteria for gestational diabetes,

although this condition has been recognised for more than 50 years. It is expected

35

that the results of the HAPO trial will provide the required evidence-based data from

different populations around the world to clarify these issues.

1.1.10.2 Features of gestational diabetes

Risk factors for gestational diabetes [Alberti and Zimmet, 1999] include:

Age

Previous history of glucose intolerance

History of large for gestational age babies

Ethnicity (such as Asian)

Raised fasting or random blood glucose levels

A population-based study in Sweden demonstrated that raised BMI and advanced

maternal age were risk factors for impaired glucose tolerance (IGT) in pregnancy

[Aberg et al., 2001]. Untreated IGT was associated with higher rates of Caesarean

sections and large for gestational age infants [Ostlund et al., 2003]. It has been

demonstrated that Asian women were more likely to develop gestational diabetes

than Caucasian women, with GDM occurring at a lower BMI in this group [Gunton

et al., 2001]. For a given population and ethnicity, the risk of diabetes in pregnancy

has been demonstrated to reflect the underlying frequency of type 2 diabetes, with

insulin resistance being a possible link [Ben-Haroush et al., 2004]. Post-partum

studies of women with gestational diabetes have demonstrated defects in insulin

secretory response and decreased insulin sensitivity, indicating typical type 2

abnormalities in glucose metabolism [Catalano et al., 1986]. Furthermore, a study

reported that only 1–2% of women with previous gestational diabetes had specific

islet cell antibodies, indicating a low risk of type 1 diabetes [Catalano et al., 1990].

These findings suggest a strong link between gestational diabetes and type 2

diabetes. It remains unclear, however, if these ethnic and immunological findings

accurately reflect GDM, as studies have differed in methods of screening, oral and

intravenous glucose loads, and diagnostic criteria.

Women with gestational diabetes have been reported to be older at the age of

delivery than women without diabetes and more likely to have a Caesarean section

[Saydah et al., 2005]. Similar findings were obtained in another study which, in

36

addition, found a higher rate of large for gestational age babies in GDM [Johns et

al., 2006]. Thus, although the prevalence of complications in gestational diabetes is

lower than that of type 1 and 2 diabetes in pregnancy, it is still higher than that seen

in the normal population.

1.1.11 Other specific types of diabetes

Other types of diabetes are extremely rare in pregnancy. They include genetic

defects and syndromes, drug- or chemical-induced diabetes, endocrinopathies,

infections and diseases of the exocrine pancreas.

1.2 Animal models of diabetes

Animals have been used to study diabetes since the discovery in the 1880s of von

Mering and Minkowski that the removal of the pancreas in dogs causes diabetes

[Rees and Alcolado, 2005]. Research in dogs also led to the pioneering discovery of

insulin by Banting and Best [Banting et al., 1922]. In the United Kingdom, animal

research is currently controlled by the Animals (Scientific Procedures) Act 1986

which stipulates that research should involve animals with the lowest degree of

neuro-physiological sensitivity. This has resulted in the vast majority of animal

research in diabetes being restricted to rats and mice. Diabetes can be induced by

both surgical and non-surgical methods. Surgical methods involve partial or total

removal of the pancreas, whereas non-surgical methods include the injection of islet

cell toxins such as streptozotocin and alloxan. Murine models of diabetes have

enabled the study of hyperglycaemia, hyperlipidaemia, gestational diabetes, islet

cell transplantation and the assessment of new drugs [Rees and Alcolado, 2005].

Animal models of diabetes have also been invaluable in the study of vascular

function in diabetes. Streptozotocin-induced diabetes in rats has been associated

with impaired endothelium-dependent relaxation of the aorta [Cameron and Cotter,

1992].

37

1.2.1 Animal models of type 1 diabetes

As described previously, type 1 diabetes in humans is characterised by immune-

mediated destruction of the pancreatic cells. In animals, this destruction can be

induced by the injection of toxins, of which streptozotocin is the most widely used.

Streptozotocin is an N-nitroso-containing compound that acts as a nitric oxide donor

in pancreatic islet cells, thereby inducing death of insulin-secreting cells and

producing an animal model of diabetes [McEvoy et al., 1984]. A single large dose

of streptozotocin or multiple small doses (e.g. 40 mg/kg on five consecutive days)

can be used in rodents. In susceptible animals this induces an insulin-deficient

diabetes in which immune destruction plays a role, as in human type 1 diabetes

[Rees and Alcolado, 2005].

Spontaneously hyperglycaemic animal models of type 1 diabetes also exist, where

animals have been in-bred for many generations. A drawback to this model is that

various genes and phenotypes have been altered, which may not be relevant for

either mice or humans.

1.2.2 Animal models of type 2 diabetes

Unlike type 1 diabetes, the pathogenesis of type 2 diabetes is relatively obscure and

a multitude of causal factors are involved such as insulin resistance, obesity and islet

cell failure. This is reflected in animal models of this disease: insulin resistance

predominates in some and islet cell failure in others. Animal models of type 2

diabetes include Zucker fatty rats [Tokuyama et al., 1995], Goto-Kakizaki rats

[Goto et al., 1988], Psammomys obesus sand rats [Kalderon et al., 1986] and

various transgenic mice models with reduced -cell mass [Masiello, 2006].

1.2.3 Animal models of diabetes in pregnancy

Animal models of diabetes in pregnancy have been used to investigate the fetal

origins of adult disease. Using these models, it has been shown that diabetes in

pregnancy predisposes to the later development of diabetes amongst offspring [Van

Assche et al., 1991]. Intra-uterine malnutrition has also been shown to be associated

38

with a higher risk of diabetes in later life [Boloker et al., 2002]. However, studies

examining vascular function in pregnant animals with diabetes are extremely rare. It

is therefore unknown if aberrant vascular function is present in the uterine vascular

bed of pregnant rats with diabetes.

1.2.4 Advantages of animal models of diabetes

Animal models have been responsible for many breakthroughs in the field of

diabetes research, from the discovery of insulin itself to new drugs such as PPAR-

agonists [Finegood et al., 2001]. In the development of new drugs, both drug

efficacy and drug toxicity can be studied in the same model [Boelsterli, 2003].

Animal models provide unique opportunities for studying disorders such as fetal

origins of disease due to their short gestation and rapid turnover. The confounding

effects of concurrent diseases such as hypertension and hyperlipidaemia, which are

often present in human studies of diabetes, can be reduced by using animal models.

Knockout mice have provided valuable insights into the pathophysiology of diabetes

[LeRoith and Gavrilova, 2006]. Mouse and human genomes bear striking

similarities to each other. Approximately 99% of mouse genes have a homologue in

the human genome, making it a leading mammalian system for studying human

physiology and disease [Chinwalla et al., 2002]. When studying pregnancy, the

easier access to tissue from animals compared to humans enables a broader range of

experiments; an advantage which has been availed of in the present study.

1.2.5 Limitations of animal models of diabetes

The genetic homogeneity of inbred animals and the lack of environmental triggers

in a pathogen-free setting have raised concerns about the validity of animal models.

Some of the commonly used animal models have specific genetic defects that are

not seen in humans [Lohmann, 1998]. A lack of reproducible animal models of

certain human diabetic complications and the disappointing results of studies aimed

at preventing type 1 diabetes, based on strategies that were successful in rodents,

have also been noted [Rees and Alcolado, 2005].

39

1.3 Pregnancy

1.3.1 Carbohydrate metabolism in pregnancy

During pregnancy, a progressive rise in insulin resistance occurs that begins near

mid-pregnancy and progresses through to the third trimester [Buchanan and Xiang,

2005]. The insulin resistance appears to result from a combination of increased

maternal adiposity and the rise in hormones secreted by the growing fetoplacental

unit, which are antagonistic to the actions of insulin. These include human chorionic

somatomammotropin, progesterone, cortisol, and prolactin [Butte, 2000]. The fact

that placental hormones are responsible for insulin resistance is also suggested by

the observation that following delivery, this resistance is diminished [Sivan et al.,

1997].

Pancreatic cells normally increase their insulin secretion to compensate for the

insulin resistance of pregnancy. A progressive increase in basal and postprandial

insulin concentrations is seen with advancing pregnancy. By the third

trimester,

basal and 24-hour mean insulin concentrations may double [Lesser and Carpenter,

1994]. This ensures that maternal glucose levels do not increase to a large degree

compared with the large changes in insulin sensitivity. Thus glucose regulation

during pregnancy requires adequate cell function to overcome progressive insulin

resistance.

By the third trimester, postprandial glucose concentrations are significantly elevated

and the glucose peak is prolonged [Cousins et al., 1980]. This may be due to

impaired insulin-mediated glucose utilization, suppression of endogenous glucose

production, and inadequate increase in first-phase insulin secretion [Di Cianni et al.,

2003]. Women with gestational diabetes have decreased insulin sensitivity in

comparison with weight-matched control groups [Ryan et al., 1985; Xiang et al.,

1999]. Furthermore, decreased insulin secretion rates have also been noted in

women with GDM, making them unable to compensate for the increased insulin

resistance [Homko et al., 2001]. The ultimate effect of these numerous metabolic

changes in pregnancy is a pre-disposition for glucose intolerance; which in

individuals with risk factors for diabetes, can lead to GDM.

40

1.3.2 Cardiovascular changes in pregnancy

Pregnancy is associated with numerous cardio-vascular adaptations, which promote

healthy fetal development without compromising maternal circulation in other organ

systems. This includes a rise in blood volume; which reaches a plateau at

approximately 34 weeks and can exceed the non-pregnant value by up to 45-55% at

term [Rovinsky and Jaffin, 1965]. This is associated with an increase in the cardiac

volume by about 40%, mainly due to an increase in the stroke volume, but also due

to an increase in heart rate [Walters et al., 1966].

Despite this marked increase in blood volume, the blood pressure in normal

pregnancies does not rise correspondingly, due to a reduction in the peripheral

arterial resistance. Although studies have investigated whether this reduced

resistance is a result of vasorelaxants such as prostacyclin or nitric oxide (NO), its

precise cause remains unknown. Prostacyclin has been associated with attenuation

in angiotensin 2-induced uterine vasoconstriction in sheep [Magness et al., 1992].

Increased generation of NO has been demonstrated in hand blood flow of pregnant

women [Williams et al., 1997]. Increased urinary excretion of nitrate, which was

inhibited by chronic infusion of the nitric oxide synthase inhibitor L-N arginine

methyl ester (L-NAME), has been demonstrated during pregnancy in rats [Conrad et

al., 1993]. However, other studies in rats have suggested that increased NO

production is not responsible for this decrease in vascular resistance [Ahokas et al.,

1991; Umans et al., 1990].

It has been hypothesized that the elevated levels of urinary nitrate excretion may

reflect local increases in NO production in specific vascular beds [Poston et al.,

1995]. For example; in the sheep uterine circulation, oestrogen-induced vasodilation

has been demonstrated to be mediated by NO [Rosenfeld et al., 1996]. The above

studies indicate that the state of pregnancy is associated with important alterations to

vascular function in the uterine circulation. Although teleologically these changes

are aimed at improving blood flow to the fetus, they may also lead to altered effects

of glucose on vascular function. This possibility has been investigated further in

this study.

41

1.4 Arteries

1.4.1 Blood flow to the fetus

A substantial portion of cardiac output (approximately 10% at term) is directed to

the uterine circulation to meet the needs of the growing fetus [Assali et al., 1978].

To accommodate this increase in uterine blood flow, the uterine vasculature

undergoes marked morphologic changes: the diameters of the blood vessels (both

veins and arteries) increase resulting in a fall in resistance of the smaller vessels

[Moll, 2003]. Aberrant vascular function in the uterine arteries has been

demonstrated to have deleterious effects on the fetus. Reduction in uterine blood

flow was associated with fetal hypoxemia, acidemia and changes in fetal

cardiovascular function [Stevens and Lumbers, 1990]. An association between

increased uterine artery resistance and recurrent pregnancy loss has also been

demonstrated [Habara et al., 2002]. In pregnant streptozotocin-induced diabetic rats,

decreased basal prostacyclin production with reduced myometrial blood flow has

been reported [Takeda and Kitagawa, 1992]. However, little is known about

whether the vascular changes associated with diabetes in pregnant women affect

blood flow to the fetus.

1.4.2 Resistance arteries

Poiseuille’s equation states that in tubes of uniform diameter carrying a

homogeneous liquid (such as water) the flow is proportional to the fourth power of

radius [Badeer, 2001]. Although this equation cannot be directly applied to

biological systems (as arteries are not rigid tubes and blood is heterogeneous) it still

highlights the fact that even small changes in vessel diameter will result in

disproportionately large changes to blood flow. This concept forms the basis of

resistance arteries, which have been defined as pre-capillary vessels that contribute

both passively to the resting resistance and actively to the blood flow control during

altered demands [Duling, 1991]. Although most small pre-capillary arteries satisfy

the first part of this definition by contributing to resistance, it is less certain which

among these can control blood flow during altered demands. The limitations of the

42

various methodologies used to study vessels have resulted in heterogeneous data and

uncertainty regarding the location of true resistance arteries.

In one of the earliest studies, Sugiura et al. showed that in the dog mesentery the

average fall in blood pressure was 7.5 mm Hg between the aorta to 1mm-diameter

arteries, and 17 mm Hg between aorta to 200 µm-diameter arteries [Sugiura and

Freis, 1962]. This study concluded that in dogs, arterioles less than 200 µm in

diameter fulfilled the criteria for resistance vessels. Most of the available evidence

originates from measurements conducted in small animals during anaesthesia, which

in itself can alter the vascular resistance [DeLano et al., 1991]. It has been reported

that 100- to 300-µm mesenteric arteries from conscious rats also comply with the

criteria for resistance arteries [Christensen and Mulvany, 1993]. In the human

uterine circulation, myometrial arteries are likely to represent the prime site of

resistance because the pregnancy-associated increase in diameter and loss of

contractile function in the more distal spiral arteries eliminate their functional role in

vascular control [Kublickiene et al., 1997]. These myometrial arteries are important

in regulating uterine blood flow, as they are densely innervated and exhibit a more

pronounced effect to vasoconstrictors than the main branches of the uterine artery

[Akerlund, 1994]. Due to these reasons, the effects of glucose on myometrial

arteries have been examined in this study.

1.5 The Endothelium

1.5.1 Endothelial function

The endothelium is a single layer of cells which constitutes the inner–most surface

of the vessel wall. It forms the interface between blood flow and the blood vessel

wall. Previously thought to be an inert layer of cells, it is now known to regulate

vasomotor tone, by secreting various vasoactive factors which modulate both

relaxation and constriction (Table 1.2). Our understanding of its function has

improved dramatically over the past decades since Furchgott’s pioneering discovery

of the vasoregulatory role of the endothelium [Furchgott and Zawadzki, 1980]. The

interest in endothelial function is based on its pivotal role in various diseases such as

diabetes, coronary artery disease, hypertension and pre-eclampsia. Ischaemic heart

disease, cerebro-vascular disease and renal failure are leading causes of death in the

43

developed world. These disorders are often a result of atherosclerosis, hypertension

and diabetes. Research into endothelial function may pave the way for novel

therapies aimed at alleviating the morbidity and mortality from these conditions.

Furthermore, as endothelial dysfunction is a common feature of these inter-related

disorders, knowledge gained by studying one disease may be applicable to another.

Vasoregulation

Relaxation

Nitric Oxide

Prostacyclin

EDHF

Contraction

Endothelin - 1

PGH2

Thromboxane A2

Permeability VCAM-1, ICAM-1, PCAM-1, P and E selectin

Vasculogenesis VEGF, TGF

Haemostasis

Coagulation

Prostacyclin

Thromboxane A2

vWF

Fibrinolysis tPA

PAI-1

Table 1.2: Vasoactive products synthesized by the endothelium

Abbreviations listed on page 23.

1.5.2 Endothelium-dependent mediators of relaxation

The three mediators responsible for endothelium-dependent relaxation are nitric

oxide (NO), prostacyclin (PGI2) and the endothelium derived hyperpolarizing factor

(EDHF). However, not all endothelial cells release all of the mediators. Variation

with species is seen: endothelium-dependent relaxation in fish is mediated mainly

by prostanoids, whereas that in frogs by NO [Vanhoutte and Scott-Burden, 1994]. In

mammalian species, all three mediators are involved in endothelium-dependent

relaxation, although there are variations between vascular beds.

44

1.5.2.1 Nitric Oxide

Nitric oxide (NO) is an important endothelium derived relaxing agent that diffuses

easily across cell membranes, as it is lipophilic and has a small molecular weight.

NO is synthesized from L-arginine by the enzyme endothelial Nitric Oxide Synthase

(eNOS), which is stimulated by shear stress [Ayajiki et al., 1996]. The NO crosses

the endothelium and reaches the smooth muscular tissue of the arterial wall,

releasing cyclic Guanosine Monophosphate (cGMP) from GTP [Rapoport and

Murad, 1983]. This in turn regulates the cytosolic Ca2+

and causes smooth muscle

relaxation leading to vasodilation (Figure 1.2).

Figure 1.2: Endothelium-dependent mediators of vasodilation


In vessels stripped of endothelium, the response to stimulation by NO releasers such

as acetylcholine or shear stress is lost, despite the persistent response to exogenous

nitrates and sodium nitroprusside [Furchgott and Zawadzki, 1980]. Endothelium-

dependent vasodilation has become synonymous with intact or normal endothelial

function and preserved NO bioavailability.

NO

GTP

cGMP

RELAXATION

Endothelial Cell

Smooth Muscle Cell

Ca2+

Prostacyclin

ATP

cAMP

EDHF

K+

Hyperpolarization

45

NO production has been demonstrated to vary depending on gender. Hayashi et al.

demonstrated that basal NO release from endothelium-intact aortic rings in rabbits

depends on circulating estradiol concentration, as this basal release was greater in

females than males, but returned to the level of males after oophorectomy [Hayashi

et al., 1992]. In addition to the effects of oestrogen, another explanation for the

gender differences is that the main endothelial mediator of vasodilation may vary in

males and females. EDHF has been demonstrated to be the predominant

endothelium-derived relaxing factor in female mice, whereas NO and PGI2 were

more important in male mice [Scotland et al., 2005].

Despite reduced bioavailability of NO, vascular disease is not necessarily associated

with a complete loss of either flow-mediated or agonist-induced vasodilation,

because mediators which are thought to play a minor role in the regulation of tone in

healthy vessels may compensate (at least partially) for the lack of NO. For example,

depending on the vascular bed, either PGI2 [Sun et al., 1999] or EDHF [Huang et

al., 2001] can mediate flow-induced dilation in eNOS knockout mice. Exogenously

applied NO decreases EDHF-mediated responses in isolated arteries [Bauersachs et

al., 1996; Nishikawa et al., 2000]. After angioplasty, the regenerated endothelium in

the porcine coronary artery generates less NO; but relaxation to bradykinin is

maintained via an EDHF-dependent mechanism [Thollon et al., 2002]. These

studies reveal a more complicated inter-relationship between the endothelial

mediators than previously thought. They also suggest that the blocking of one

mediator, rather than isolating the remaining mediators, may actually increase their

role in inducing endothelium-dependent vasodilation.

1.5.2.2 Prostacyclin

Prostacyclin is a prostaglandin derivative produced by vascular endothelial cells

from arachidonic acid by the enzymes cyclo-oxygenase (COX) and prostacyclin

synthase [Parkington et al., 2004]. Its release is stimulated by bradykinin and

adenine nucleotides [Guerci et al., 2001]. Prostacyclin acts by stimulating adenylate

cyclase and by increasing intracellular cyclic adenosine monophosphate (cAMP) in

vascular smooth muscle [Kukovetz et al., 1979]. This in turn stimulates ATP

46

sensitive K+ channels to cause hyperpolarization of the cell membrane and inhibit

the development of contraction [Parkington et al., 2004]. cAMP also increases the

extrusion of Ca2+

from the cytosol in vascular smooth muscle and inhibits the

contractile machinery [Abe and Karaki, 1992; Bukoski et al., 1989]. Unlike NO, the

vasodilatory effect of prostacyclin depends on the expression of certain receptors in

vascular smooth muscle [Halushka et al., 1989]. Hence in arterial beds that do not

express such receptors, prostacyclin does not contribute to endothelium-dependent

relaxation. Prostacyclin facilitates the release of NO by endothelial cells

[Shimokawa et al., 1988]. Furthermore, the action of prostacyclin in vascular

smooth muscle is potentiated by NO, as the increase in cGMP in target cells inhibits

a phosphodiesterase that breaks down cAMP [Delpy et al., 1996]. Therefore, NO

indirectly prolongs the half-life of the second messenger of prostacyclin.

1.5.2.3 Endothelium Derived Hyperpolarising Factor (EDHF)

EDHF is defined as a mediator of vascular relaxation via a non-NO, non-prostanoid

mechanism [McGuire et al., 2001]. This mediator was so-named as these cyclo-

oxygenase and nitric oxide inhibitor-independent dilator responses are associated

with vascular smooth muscle hyperpolarisation. EDHF-mediated responses involve

an increase in the intracellular calcium concentration, the opening of calcium-

activated potassium channels of small and intermediate conductance and the

hyperpolarization of the endothelial cells [Feletou and Vanhoutte, 2006]. An

increase in potassium ion efflux from a cell results in a more negative resting

membrane potential, leading to hyperpolarisation. The amplitude of the

hyperpolarisation is inversely proportional to the extracellular concentration of K+

ions and the hyperpolarisation disappears totally in concentrations higher than

25 mmol/L [Chen and Suzuki, 1989]. The identity of this so-called EDHF is

controversial, probably because more than one type of EDHF exists with substantial

species and regional heterogeneity.

EDHF appears to play a more important role in mediating relaxation in smaller

arteries than larger arteries. A study in rats has demonstrated that the contribution of

NO was most prominent in the aorta, whereas that of EDHF was most prominent in

the distal mesenteric arteries [Shimokawa et al., 1996]. One explanation for this

may be the association between EDHF and gap junctions. Electrical conductance

47

can occur directly from endothelium to smooth muscle via gap junctions, formed

between the cells by connexin proteins. Gap junctions are conspicuously present in

small vessels, where the apposition of 2 cells is the closest. This may explain the

greater role for EDHF in small arteries [Pepper et al., 1992]. In murine resistance

vessels the predominant agonist-induced endothelium-dependent vasodilation in

vivo and in vitro is mediated by an EDHF-like principle that requires functional gap

junctions [Brandes et al., 2000]. This has been corroborated by Luksha et al. who

demonstrated that gap junctions are involved in the EDHF-mediated responses to

bradykinin in small subcutaneous arteries in normal pregnancy [Luksha et al.,

2004]. Non-NO/non-prostanoid endothelium dependent hyperpolarization has been

postulated to be the pre-dominant mechanism for endothelium-dependent relaxation

in the human sub-cutaneous vascular bed [Buus et al., 2000].

There also appears to be a gender variation in the effect of EDHF, as male mice

exhibit markedly reduced EDHF activity compared to females [Scotland et al.,

2005]. This effect may be mediated by the female sex hormone oestrogen. There is

evidence that oestrogen deficiency in ovariectomized rats specifically impairs

EDHF; possibly by reduced expression of connexin-43, a component of gap

junctions in resistance arteries [Liu et al., 2002]. Further evidence for a link between

oestrogen and EDHF comes from a study demonstrating variations in the

contribution of EDHF to relaxation depending on the day of the oestrus cycle in

perfused rat uterine vasculature [Lucca et al., 2000].

1.5.3 Endothelium-derived vasoconstrictors

The endothelium also secretes vasoconstrictors, which play a role in regulating

vascular tone (Table 1.2). Endothelin is present in healthy individuals in low

concentrations and is involved in vascular counter-regulation for preserving

peripheral resistance [Guerci et al., 2001]. Under certain conditions, the

endothelium also produces endothelium derived contracting factors such as

prostaglandin H2 and thromboxane A2, which activate specific receptors on the

vascular smooth muscle.

48

1.5.4 Other factors affecting endothelial function

1.5.4.1 Age

Endothelial function can deteriorate with age [Gerhard et al., 1996; Lyons et al.,

1997]. eNOS expression has been demonstrated to be up-regulated with age

[Briones et al., 2005]. However, the production of reactive oxygen species from

NADPH oxidase also increases with age, which may counter-act increased NO

production [Briones et al., 2005; van der Loo et al., 2004]. Endothelium-dependent

relaxation to acetylcholine has been demonstrated to be reduced with advancing age

in humans [Taddei et al., 1995].

1.5.4.2 Smoking

Both short-term smoking [Lekakis et al., 1997] and passive smoking [Celermajer et

al., 1996] have been reported to impair endothelium-dependent dilatation. The latter

study in healthy young adults demonstrated that the impairment was related to the

degree of exposure.

1.5.4.3 Ethnicity

Compared to Caucasians, endothelial cells from Afro-Caribbeans have been

described to have reduced release of bioactive NO, with an accompanying increase

in the release of superoxide anions [Kalinowski et al., 2004].

1.5.4.4 Cholesterol

Hypercholesterolaemia has been associated with impaired endothelium-dependent

relaxation in human resistance vessels [Shiode et al., 1996]. This impairment has

been demonstrated to be reversed by reducing serum cholesterol [Leung et al.,

1993].

1.5.5 Endothelial function in pregnancy

Enhanced sensitivity to acetylcholine-mediated relaxation in pregnancy, compared

to the non-pregnant state has been demonstrated in uterine arteries [Lucca et al.,

2000] and mesenteric arteries [Gerber et al., 1998] of rats. Another study has

reported enhanced NO-mediated relaxation in the uterine artery of pregnant rats [Ni

49

et al., 1997]. In healthy pregnant women, endothelium-dependent relaxation

(measured by flow mediated dilatation) has been found to be enhanced in the third

trimester of pregnancy, compared to the first and second trimester [Faber-Swensson

et al., 2004]. In contrast to these findings, McCarthy et al. demonstrated similar

endothelium-dependent relaxation in small subcutaneous arteries of both pregnant

and non-pregnant women [McCarthy et al., 1994]. This suggests that endothelial

function may vary in different vascular beds in pregnancy, emphasising the

importance of studying the relevant vascular bed directly.

The differences in endothelial function between vascular beds in pregnancy are

further illustrated by human studies examining subcutaneous and myometrial

arteries. Luksha et al. found that EDHF and NO contributed equally to endothelium-

dependent relaxation in subcutaneous arteries from healthy pregnant women

[Luksha et al., 2004]. However, in myometrial arteries from healthy pregnant

women, neither NO inhibition alone nor EDHF inhibition alone affected

endothelium-dependent relaxation [Kenny et al., 2002]. When both NO and EDHF

inhibition were combined, a significant impairment of relaxation was noted. This

implies a compensatory mechanism among the endothelial mediators which can

rectify the absence of one particular mediator in the myometrial vascular bed. This

feature was reported by Kenny et al. to be absent in arteries isolated from healthy

non-pregnant women. These studies indicate that the role of NO and EDHF varies in

subcutaneous and myometrial vascular beds during normal pregnancy. The inter-

relationship between these mediators may also be altered in pregnancy, with

changes in the amount of relaxation contributed by each mediator.

1.5.6 Methods for measuring endothelial function

The evaluation of endothelial function can theoretically be made by evaluating any

of its specific functions such as vasoregulation, haemostasis, vasculogenesis or

permeability. Of these, the most important is the vasoregulatory role of

endothelium, which has resulted in the measurement of endothelium-dependent

vasodilation becoming the main parameter in both basic science and clinical

research for assessing endothelial function. Early studies examined endothelial

50

function by catheterising coronary arteries [Ludmer et al., 1986]. More recent

studies have utilised forearm plethysmography and ultrasonography.

1.5.6.1 Venous occlusion plethysmography

The underlying principle of venous occlusion plethysmography involves the arrest

of venous outflow from the forearm such that it begins to swell. The rate and degree

of swelling reflects forearm vascular resistance, which is a function of normal

vascular endothelial function [Alam et al., 2004]. The increase in forearm volume is

taken to represent blood flow in the resistance vessels of the forearm (muscle, soft

tissues and skin). Venous occlusion plethysmography has been used to investigate

endothelial dysfunction in diabetes using intra-arterial infusions of drugs such as

acetylcholine and glyceryl trinitrate [McVeigh et al., 1992].

1.5.6.2 Brachial artery flow-mediated vasodilation

Most clinical studies have utilised this technique, making it the most common

method for assessing endothelial function. Ischaemia is induced by the inflation of

an arterial occlusion cuff, positioned on the proximal or mid-forearm. After cuff

deflation, brachial artery flow increases because of downstream vessel dilation, and

this augmented flow increases brachial artery shear stress, resulting in vasodilation

[Anderson and Mark, 1989]. The precise mechanism of flow-mediated vasodilation

is unknown, but the release of NO through shear stress-mediated stimulation of the

endothelium is thought to be involved [Faulx et al., 2003]. In patients with impaired

NO bioavailability (and therefore impaired endothelial function), the dilator

response is diminished, and in some patients a constrictor response is seen.

Although the use of this technique is widespread, it is subject to important

limitations, which throw into doubt the validity of some of the previous conclusions

reached about endothelial function. As these limitations are important in critically

evaluating such studies, they have been examined in detail.

51

Figure 1.3: Brachial artery flow-mediated dilatation

1.5.6.3 Limitations of brachial artery FMD

Although most reported inter-observer variability rates are low (< 5%), they are

usually expressed as a percentage of the baseline diameter. However, when

expressed as the percent increase above baseline diameter, variability is more

marked, with coefficients of variation up to 33% [Woodman et al., 2001]. Another

potential problem relates to image timing. Because of the wide baseline variability

in the normal response to increased flow, present techniques may fail to detect the

true peak of FMD [Bressler et al., 2000]. The location of the occlusion cuff may

also affect measurements, as FMD was more pronounced when the occlusion cuff

was placed on the proximal upper arm, as opposed to the forearm [Berry et al.,

2000].

Visualization of the vascular intima is often difficult in patients who are obese,

because the distance between the ultrasound probe and the vessel increases and this

attenuates the ultrasound signal. This is of particular relevance to diabetes, as a large

number of patients are either overweight or obese. Raised BMI has been associated

with diminished flow-mediated dilation [Olson et al., 2006].

Age, sex and ethnicity can also influence results. Although the compliance of elastic

arteries generally decreases with increasing age, brachial artery compliance in

women may actually increase [van der Heijden-Spek et al., 2000]. Young, healthy

52

African-American men and women have significantly reduced post-ischemic

brachial artery dilation compared with similar Caucasian subjects [Perregaux et al.,

2000].

Measurements of endothelium-dependent relaxation are based on shear stress in

large conduit arteries, which triggers the release of NO. It is uncertain how this

stimulus affects the other mediators of endothelium-dependent relaxation. The

degree to which sheer stress influences endothelium-dependent relaxation in

resistance arteries is also unknown.

From the above discussion, it can be inferred that the results obtained from FMD

studies often cannot be extrapolated to other systemic vessels or resistance arteries.

It is therefore necessary to examine the relevant vascular bed directly in order to

achieve a proper understanding of its endothelial function.

1.6 Endothelial dysfunction in diabetes

Impaired endothelium-dependent vasodilation has been demonstrated in various

vascular beds such as forearm, cerebral, coronary and subcutaneous arteries in

patients with diabetes as well as various animal models of this disease. However,

few studies have examined the uterine circulation in pregnancies complicated by

diabetes, either in animals or humans. It is therefore uncertain if abnormal

endothelial function in pregnancies complicated by diabetes is responsible for the

higher rate of complications seen in this condition.

1.6.1 Animal studies of endothelial function in diabetes

Studies investigating endothelium-dependent vasodilation in animal models of type

1 diabetes have yielded conflicting results. Early studies showed normal [Head et

al., 1987; Mulhern and Docherty, 1989] and even enhanced [Bhardwaj and Moore,

1988] endothelium-dependent relaxation in rat models of diabetes. However, more

recent studies have demonstrated impaired endothelium-dependent relaxation in

streptozotocin-induced diabetic rats [Otter and Chess-Williams, 1994; Pieper and

Peltier, 1995]. Endothelial dysfunction has been reported in large conduit arteries

53

such as the aorta in diabetic rabbits [Tesfamariam et al., 1989] as well as in smaller

vessels such as mesenteric arteries in diabetic rats [Palmer et al., 1998]. Some of the

discrepancies noted may be due to differences in the vascular bed, animal model and

agonists used. Damage to the endothelium during vessel preparation can also make

interpretation of results difficult. Nevertheless, the majority of studies have

demonstrated impaired endothelium-dependent relaxation with diabetes.

1.6.2 Human studies of endothelial function in diabetes

Human research on endothelial function in diabetes has similarly led to divergent

results. Patients with type 1 diabetes and normoalbuminuria have been demonstrated

to have both impaired [Lekakis et al., 1997] and normal [Lambert et al., 1996; Smits

et al., 1993] endothelium-dependent relaxation. Type 1 patients with a long duration

of disease, but good glycaemic control were reported to have normal endothelial

function [Enderle et al., 1998], whereas patients with uncomplicated type 2 diabetes

demonstrated endothelial dysfunction [Gazis et al., 1999].

The reasons for the above discrepancies may involve differences in the type of

diabetes, duration and glycaemic control; as well as the presence of clinical

characteristics affecting endothelial function such as hypertension, hyperlipidaemia

and obesity. These factors are of particular relevance in type 2 diabetes and may

confound results. Most of these studies were performed using brachial artery flow-

mediated dilation, the limitations of which have been previously discussed. Another

reason for the discordant results may be small study sizes. For example, the findings

of Smits et al. were based on 11 diabetic patients in the study group.

1.6.3 Mechanisms of endothelial dysfunction in diabetes

A) Decreased production of vasorelaxants

Elevated glucose levels have been demonstrated to inhibit insulin-stimulated NO

release in human endothelial cells [Salt et al., 2003]. Hyperglycaemia has been

associated with depletion of L-arginine, a precursor of NO, in mesangial cells of

rats [Trachtman et al., 1997]. Reduced serum arginine levels have also been

54

observed in diabetic rats [Pieper and Peltier, 1995]. However, another study

demonstrated that although serum arginine was diminished in diabetic rats, NO

synthase was increased [Rosen et al., 1996]. This increased NO generation may

reflect a compensatory mechanism to balance the enhanced inactivation of NO

[Angulo et al., 1998]. Exogenous L-arginine improved the endothelium-dependent

vasodilation in diabetes in some studies [Matsunaga et al., 1996; Pieper and Peltier,

1995], whereas this effect was not seen in others [Mayhan et al., 1997; Zsofia Koltai

et al., 1997]. The different results may be due to certain systemic effects of arginine

that have been noticed in patients with diabetes: high doses of arginine were

associated with increased plasma insulin [MacAllister et al., 1995]. Insulin itself is

associated with vasodilation that is mediated by NO [Steinberg et al., 1994].

B) Enhanced inactivation of vasorelaxants

Endothelial dysfunction has been prevented by anti-oxidants, suggesting that

impaired vasodilation may be due to an accelerated inactivation of NO by

superoxide anions [Rosen et al., 1996]. Reduced NO availability

through the

interaction between NO and superoxide anion has been demonstrated to be a causal

factor for the impaired vasodilation seen in a rat model of type 2 diabetes [Kim et

al., 2002].

C) Decreased responsiveness of smooth muscle to vasorelaxants

An impaired response to nitrovasodilators has been noted in patients with diabetes,

suggesting a generalized reduction in the sensitivity of the smooth muscle to NO

[Calver et al., 1992; McVeigh et al., 1992].

D) Enhanced generation of vasoconstrictors

Endothelium-derived constricting factors (Table 1.2) may be released along with the

mediators of relaxation, opposing their effect on smooth muscle cells. The impaired

relaxation with acetylcholine in diabetic rats has been associated with stimulation of

the thromboxane A2 - prostaglandin H2 receptor [Mayhan et al., 1991]. In the aorta

of diabetic animals, the impaired relaxations have been demonstrated to be restored

by non-specific cyclo-oxygenase blockade, but not by thromboxane A2 synthase

blockers; suggesting that the culprit is a prostaglandin endoperoxide [Shimizu et al.,

1993; Tesfamariam et al., 1989]. In contrast to these findings, cyclo-oxygenase

55

inhibition did not restore impaired endothelium-dependent relaxations in mesenteric

arteries of diabetic rats [Taylor et al., 1992]. These studies suggest that the role of

constrictor prostanoids in diabetes varies depending on the vascular bed. The

relative potency of the vasoconstrictor effect may also differ at individual sites.

1.6.4 Endothelial dysfunction and regional variations

Regional variations in endothelial dysfunction exist, which may be an important

reason for the conflicting reports in diabetes vascular research. A study in diabetic

rats has demonstrated impaired endothelium-dependent relaxation in the mesenteric

circulation which was absent in the aorta of the same animals [Taylor et al., 1994].

Similarly, loss of bradykinin-mediated vasodilation in the hindquarters vascular bed

of diabetic rats has been reported with normal renal and mesenteric vasodilatations

[Kiff et al., 1991]. A possible explanantion for these findings could be that

endothelial cells from individual vascular beds exhibit metabolic differences, and

may be affected differentially by hyperglycaemia [Sobrevia and Mann, 1997]. A

recent study looking at the femoral, mesenteric and carotid vascular beds in diabetic

rats has demonstrated heterogeneity in endothelial responses in these arteries [Shi et

al., 2006]. This study also illustrated that under physiological conditions, the

production of EDHF can be curtailed by the production of NO. As NO synthesis is

impaired in diabetes, this inhibition is alleviated; resulting in improved EDHF-

mediated vasodilation.

The above studies indicate that endothelium-dependent relaxation varies in different

vascular beds in diabetes. As mentioned previously, studies have demonstrated a

gradient in the release of endothelial mediators, with a progressively increasing

contribution of EDHF in the more distal vessels (Section 1.5.2.3). The overall

picture is further complicated by the fact that in diabetes, reduced bioavailability of

NO can affect other mediators such as EDHF. Most studies have been performed in

animals, which restricts conclusions from being drawn regarding these changes in

humans. This emphasizes the importance of studying relevant vascular beds directly

to assess the effect of diabetes in humans.

56

1.6.5 Other factors influencing endothelial dysfunction in

diabetes

1.6.5.1 Control of diabetes

Endothelial dysfunction associated with diabetes is closely related to metabolic

control of the disease; endothelial function improving with better control. This has

been demonstrated in both animal and human studies of diabetes. Streptozotocin-

induced diabetic rats, with good metabolic control (HbA1C 5.5 – 7.4%) had similar

endothelium-dependent relaxation as non-diabetic rats, whereas rats with HbA1C

levels greater than 7.5% showed a significantly impaired response [Angulo et al.,

1998]. In contrast to type 2 diabetic patients with cardiovascular complications, type

1 patients with a long duration of diabetes and good long-term metabolic control

have been demonstrated to have similar endothelial function as control subjects

[Enderle et al., 1998]. In a study of ten patients with type 1 diabetes, poorly

controlled diabetes over 48 hours resulted in reduced flow-mediated dilation

[Sorensen et al., 2005]. However, a concurrent impairment of endothelium-

independent vasodilation with nitroglycerin was also seen with hyperglycaemia.

This restricts the assessment of endothelial function in this study and raises the

possibility of aberrant smooth muscle function. Von Willebrand factor, a marker of

endothelial function, was found to be elevated during poor glycaemic control in this

study; although other markers such as soluble intercellular adhesion molecule

(sICAM) were found to be normal. In contrast, both of these endothelial markers

were found to be unaffected by improved glycaemic control in patients with type 2

diabetes [Yudkin et al., 2000]. Another study in patients with type 2 diabetes

demonstrated improved flow-mediated dilation with improved glycaemic control

[Gaenzer et al., 2002]. These studies indicate that glucose levels can influence

endothelial function, although the precise association between the two remains

poorly characterised.

1.6.5.2 Obesity

Obesity is commonly seen in patients with type 2 and gestational diabetes.

However, conflicting reports exist regarding the relationship between

overweight/obesity and endothelial dysfunction. Obesity has been independently

associated with endothelial dysfunction which was related to insulin resistance

57

[Steinberg et al., 1996]. Various studies have reported that weight reduction in

obese individuals improved endothelium-dependent vasodilation [Sasaki et al.,

2002; Sciacqua et al., 2003]. This improvement has also been linked to a reduction

in plasma glucose levels [Raitakari et al., 2004]. However, it has also been

demonstrated that basal release of endothelial nitric oxide is maintained in

overweight and obese adults [DeSouza et al., 2005]. Furthermore, a study

examining monozygotic twins reported that endothelial dysfunction was not

correlated with obesity, but with the accompanying metabolic abnormality of

adiponectin deficiency [Pietilainen et al., 2006]. From the above, it is evident that

the link between obesity and endothelial dysfunction is complex; with involvement

of factors such as insulin resistance, hyperglycaemia and hypoadiponectinemia.

1.6.5.3 Lipids

Diabetes is associated with alterations in lipid profile, which may have an effect on

endothelial function. In type 1 diabetes, the cholesterol lowering agent pravastatin

has been demonstrated to improve endothelial function [Joyce et al., 2004].

Lowering LDL cholesterol has been associated with improvement in endothelial

function in obese women with previous gestational diabetes [Bergholm et al., 2003].

Fatty acids have been demonstrated to impair endothelium-dependent relaxation in

animal conduit arteries [Edirisinghe et al., 2006] and resistance arteries [Sainsbury

et al., 2004].

Another pathway for endothelial dysfunction in diabetes involves LDL oxidation. In

a study where patients with type 2 diabetes were given two different meals,

(resulting in two different levels of post-prandial hyperglycaemia), LDL was more

susceptible to oxidation after the meal that produced greater hyperglycaemia

[Ceriello et al., 1999]. Oxidized LDL is degraded through lectin-like oxidized LDL

receptor-1 (LOX-1), which is expressed in vascular endothelium [Sawamura et al.,

1997]. Oxidized LDL uptake by LOX-1 reduces eNOS expression [Mehta et al.,

2001], leading to diminished NO bioavailability. This mechanism may represent a

link between hyperglycaemia and endothelial dysfunction.

58

1.6.5.4 Insulin resistance

Endothelial dysfunction has been associated with insulin resistance [Kim et al.,

2006]. An enhancement in endothelial function was noticed when insulin resistance

was reduced with rosiglitazone [Pistrosch et al., 2004] Responses of

angiographically normal coronary arteries to acetylcholine were impaired in subjects

with insulin resistance, which was linked to oxidative stress [Shinozaki et al., 2001].

These studies indicate the multi-factorial aetiology of endothelial dysfunction in

diabetes. Obesity and insulin resistance are closely associated with type 2 diabetes

but not with type 1 diabetes, suggesting a distinct difference in the pathogenesis of

endothelial dysfunction in these two disorders.

1.6.5.5 Gender differences in diabetes

There is evidence to suggest that gender differences exist in the effects of diabetes

on vascular function. An early study demonstrated that diabetic women had a higher

relative mortality from coronary heart disease than men [Garcia et al., 1974].

Relaxation to acetylcholine in the aorta of diabetic rats was described to be impaired

in females, but not in males [Pinna et al., 2001]. In this study the vascular tissue

from female rats was more susceptible to oxidative damage, but also more

responsive to antioxidant treatment. Gender differences in vascular function,

although subtle, may be of importance in the pathophysiology of diabetes in

pregnancy.

1.7 Endothelial dysfunction of diabetes in pregnancy


In pregnant rats with streptozotocin-induced diabetes, enhanced endothelium-

dependent relaxation to acetylcholine has been reported [Omer et al., 1999]. In this

study, the activity of NOS was raised in renal, cardiac, aortic and uterine vascular

beds, but not in the placenta. Using a microvascular skin perfusion technique,

Ramsay et al. demonstrated improved endothelial function during pregnancy

compared to the post-natal period in both healthy women and women with type 1

diabetes [Ramsay et al., 2003]. However, women with diabetes had impaired

59

responses compared to healthy women, both during pregnancy and in the postnatal

period. Another study in pregnant women with type 1 diabetes using flow-mediated

dilation of the brachial artery described impaired vasodilation, which correlated with

the duration of diabetes [Savvidou et al., 2002]. In contrast, a small study

investigating subcutaneous arteries from 9 pregnant women with type 1 diabetes

demonstrated normal endothelial function in this vascular bed [Ang et al., 2002].

Apart from this study, type 1 diabetes in pregnancy has generally been associated

with endothelial dysfunction.

1.7.2 Gestational diabetes

Subcutaneous arteries from women with gestational diabetes displayed impaired

relaxation to acetylcholine compared to healthy pregnant women [Knock et al.,

1997]. This study also suggested that in this vascular bed, prostacyclin may be of

more importance to relaxation than NO. Using the novel technique of transcranial

Doppler during visual stimulation, altered endothelial function has also been

demonstrated in the cerebral circulation of women with GDM [Rosengarten et al.,

2004]. Gestational diabetes has been associated with pre-eclampsia [Bryson et al.,

2003]: both disorders share endothelial dysfunction as a common factor [Savvidou

et al., 2003]. Furthermore, as in diabetes, the endothelial dysfunction of pre-

eclampsia has been reported to be reversed by anti-oxidants [Chambers et al., 2001].

1.7.3 Previous gestational diabetes

Endothelial dysfunction has been demonstrated in patients with impaired glucose

tolerance in pregnancy [Paradisi et al., 2002] and in women with previous

gestational diabetes [Anastasiou et al., 1998]. Hannemann et al. used both laser

Doppler fluximetry of maximum skin microvascular hyperaemia and brachial artery

flow-mediated dilatation to assess endothelial function in seventeen patients with

previous gestational diabetes [Hannemann et al., 2002]. Although microvascular

hyperaemia was impaired compared to healthy controls, flow-mediated dilation was

similar in both groups. This suggests aberrant function in the microvascular

circulation that was not evident in the larger arteries. A similar study using the laser

Doppler technique demonstrated that acetylcholine induced vasodilation was

reduced in seventeen women with previous gestational diabetes compared with

controls [Hu et al., 1998]. In contrast to the findings of Hannemann et al., both

60

Aberrant Homeostasis

Digestion

Glycogenolysis

Gluconeogenesis

macro- and microvascular function were found to be abnormal in this study. Oral

vitamin C has been demonstrated to improve endothelial function in women with

previous GDM [Lekakis et al., 2000]. The small number of studies makes

interpretation of vascular function in women with previous gestational diabetes

challenging. Studies investigating GDM are also often constrained by relatively low

number of study participants. Nevertheless, a general trend of impaired

endothelium-dependent relaxation in these women has been demonstrated.

Figure 1.4: Overview of glucose metabolism

CO2 H2O

PHOTOSYNTHESIS

PLANT STARCH

ANIMALS

DIABETES

HYPERGLYCAEMIA

CITRIC ACID CYCLE GLYCOLYSIS

ENERGY

GLUCOSE

GLYCOGEN HUMANS

61

1.8 Glucose levels

Glucose is the key molecule for energy production in the biological world. The

oxidation of glucose represents the culmination of a series of steps by which energy

from the sun is directly or indirectly harnessed by organisms as diverse as anaerobic

bacteria, plants and human beings (Fig. 1.4). The levels of this molecule are

therefore closely regulated with varying degrees of complexity in all life forms. The

breakdown of the physiological mechanisms controlling the homeostasis of glucose

results in diabetes.

Considerable controversy exists over the precise levels of glucose which represent

normoglycaemia, hypoglycaemia and hyperglycaemia. Data for these levels are

based on population studies and the risk for developing complications of diabetes at

each glycaemic level. Furthermore, numerous methods are currently in use for

measuring glucose levels. The term “blood glucose” can refer to capillary blood,

arterial blood, venous whole blood or plasma, all of which can vary in their values

from the same individual, albeit to slight degrees. The overall uncertainty of glucose

measurement has been reported to be about 5% during serial measurement

[Reinauer et al., 2002].

The WHO has defined a fasting venous plasma glucose concentration less than 6.1

mmol/L as normal [Alberti and Zimmet, 1999]. Although this choice is arbitrary,

such levels have been observed in people with normal glucose tolerance. Values

above this are associated with a progressively greater risk of developing micro- and

macro-vascular complications [Alberti, 1996; Engelgau et al., 1997]. In healthy

pregnant women, random blood glucose levels at various stages in gestation have

been reported to vary from 4.3 (median) to 7.3 (95th

percentile) mmol/L [Ostlund

and Hanson, 2004]. Another study of healthy pregnant women demonstrated a range

of blood glucose (mean ± SD) from 3.0 ± 0.3 to 5.8 ± 0.3 mmol/L during pregnancy

[Parretti et al., 2001]. Furthermore, daily mean glucose values increased slightly

between 28 (4 ± 0.3 mmol/L) and 38 (4.3 ± 0.3 mmol/L) weeks of pregnancy, with

the mean post-prandial glucose levels reaching a maximum of 5.8 mmol/L. The

small rise in blood glucose values normally seen in the third trimester has been

attributed to increasing insulin resistance, as discussed previously. A third study of

healthy non-obese pregnant women reported a blood glucose level (mean ± SD) of

62

4.6 ± 1.0 mmol/L with a peak post-prandial level of 5.9 ± 0.9 mmol/L; this peak

being reached in 71.4 ± 30 minutes [Yogev et al., 2004]. These three studies provide

evidence that in healthy pregnant women, the blood glucose level varies

approximately between 3 to 6.8 mmol/L.

1.8.1 HbA1C

HbA1C (also referred to as glycated or glycosylated haemoglobin) is the sub-fraction

of haemoglobin formed as a result of irreversible, non-enzymatic glycation of the

haemoglobin -chain [Schnedl et al., 2001]. The measurement of HbA1C is

expressed as a percentage of total haemoglobin. The formation of glycosylated

haemoglobin occurs over the average life span of erythrocytes (approximately 120

days) [Bunn et al., 1976]. Therefore, HbA1C can serve as a retrospective indicator of

the average glucose concentration over the previous 8 to 10 weeks [Reinauer et al.,

2002]. HbA1C concentration has been demonstrated to fall when diabetic control is

improved by treatment [Koenig et al., 1976]. Although HbA1C levels have been

extensively used in epidemiological studies looking at the chronic effects of

diabetes, its value in pregnancy is less certain. This is because adverse outcomes

have been demonstrated in pregnancy despite achieving a near optimal HbA1C

concentration [Evers et al., 2004].

1.9 Hyperglycaemia

1.9.1 Definition

For the purpose of diagnosing diabetes, a fasting venous plasma glucose of ≥ 7.0

mmol/L has been considered to be hyperglycaemic and in the diabetic range [Alberti

and Zimmet, 1999].

1.9.2 Estimating hyperglycaemia

Currently, self-monitored blood glucose (SMBG) is the most prevalent method for

measuring blood glucose levels during pregnancy, where a drop of capillary blood

from the fingertip is analysed using a glucometer. However, the timing of this test

varies; with both pre-and postprandial values being measured. In the UK, pre-

prandial levels taken three times a day and an additional fourth reading before bed

63

are usually advised for pregnant women with diabetes who are on insulin.

Limitations of this method include poor patient compliance and errors due to

improper techniques. Levels are normally measured before meals; however these

values may also be affected by snacks between meals, leading to errors in

interpretation. SMBG may not detect diurnal fluctuations of glucose levels, as a

study has estimated that in pregnant women with poorly controlled type 1 diabetes,

ten daily SMBG determinations may be required to detect these fluctuations

[Kerssen et al., 2006]. Concern has also been raised about the reliability of self-

generated data, as falsification has been reported [Kendrick et al., 2005].

The recent advent of a new technique, called the continuous glucose monitoring

system (CGMS) has simplified the estimation of glucose levels. The blood glucose

is monitored continuously by a subcutaneous implant, allowing precise estimation of

glucose fluctuations. This new method has revealed that standard measures for

glycaemic control may markedly underestimate the frequency of both

hyperglycaemia [Praet et al., 2006] and hypoglycaemia [Cheyne and Kerr, 2002] in

patients with diabetes. CGMS is of particular value in estimating the duration of

each episode of hyperglycaemia and hypoglycaemia; a measurement which is

difficult to make using SMBG. However, CGMS is primarily used as a research tool

at present due to its cost and limited availability.

1.9.3 Degree of hyperglycaemia

A study using CGMS in women with diabetes in pregnancy demonstrated a mean

peak glucose value (± SD) of 10.1 ± 3.2 mmol/L in type 1 diabetes patients, 7.2 ±

1.6 mmol/L in patients with diet-controlled GDM and 8.2 ± 2.5 mmol/L in patients

with insulin-treated GDM. [Ben-Haroush et al., 2004].

1.9.4 Duration of hyperglycaemia

Buhling et al. examined the duration of hyperglycaemia present in women with

diabetes in pregnancy using CGMS. The mean duration (± SD) of hyperglycaemia

above 8.9 mmol/L varied from 7.5 ± 14 minutes in healthy pregnant women to 14 ±

21 minutes in women with gestational diabetes [Buhling et al., 2004]. The peak of

postprandial hyperglycaemia in women with GDM and type 1 diabetes has been

estimated to occur at approximately 90 minutes [Ben-Haroush et al., 2004].

64

1.9.5 Pathophysiology of hyperglycaemia

1.9.5.1 Oxidative stress

Reactive oxygen species are atoms or molecules capable of independent existence

that contain one or more unpaired electrons [Mehta et al., 2006]. These include

various derivatives of oxygen and nitrogen such as superoxide (O2−), hydroxyl

(OH−), peroxide (H2O2) and peroxynitrite (ONOO

−). These molecules can damage

protein, RNA and DNA structure, but are normally neutralized by innate antioxidant

defence mechanisms, thus maintaining a stable intracellular environment. Oxidative

stress implies a disturbance of this delicate balance, resulting in an excess of free

radicals and its sequelae.

Hyperglycaemia exerts part of its deleterious effects by promoting oxidative stress

[Monnier et al., 2006]. Hyperglycaemia has been reported to cause an

overproduction

of superoxide by the mitochondrial electron-transport chain

[Nishikawa et al., 2000]. In normal pregnant women, it has been demonstrated that

hyperglycaemia is associated with increased production of reactive oxygen species

in neutrophils [Petty et al., 2005].

The link between reactive oxygen species and endothelial dysfunction involves, in

part, NO metabolism (Fig. 1.5). The enzyme endothelial nitric oxide synthase

(eNOS) regulates vascular tone through production of NO. Upon electron transfer

from various sources, eNOS oxidises L-arginine to L-citrulline with the

simultaneous release of a single molecule of NO. (6R)-5,6,7,8-tetrahydrobiopterin

(BH4) is required as a co-factor for these electron transfer reactions [Vasquez-Vivar

et al., 1998]. When L-arginine and BH4 are available in adequate quantities, these

reactions generate NO. However, if either or both are deficient, eNOS switches from

a coupled state (generating NO) to an uncoupled state, producing superoxide (O2−)

[Alp and Channon, 2004]. The resultant O2− can react further with existing NO and

generate peroxynitrite (ONOO−). Diabetes is linked to this process by causing

depletion of L-arginine and BH4 [Cai et al., 2005].

65

Figure 1.5: Oxidative stress and endothelial dysfunction


Anti-oxidants have been shown to attenuate some of the deleterious effects of

hyperglycaemia. In intestinal arterioles of normal rats, hyperglycaemia of 16.6

mmol/L (200 mg/dl) suppressed acetylcholine mediated vasodilation; this was

prevented by superoxide dismutase [Bohlen and Lash, 1993]. Treatment with anti-

oxidants has been demonstrated to protect against impaired endothelium-dependent

relaxation caused by elevated glucose in the aorta of diabetic rabbits [Tesfamariam

and Cohen, 1992]. Vitamin C (which is an anti-oxidant) has been reported to reverse

diabetes-induced impairment in endothelium-dependent relaxation in mesenteric

arteries of diabetic rats [Sridulyakul et al., 2006].

eNOS

NO

SUPEROXIDE

BH4 BH4

PEROXYNITRITE

ENDOTHELIAL

DYSFUNCTION

L-ARGININE

DIABETES

↓ BIO-AVAILABILITY

66

1.9.5.2 Hyperglycaemia and endothelial mediators

Hyperglycaemia has been associated with the generation of vasoconstrictor

prostanoids in normal rabbit aorta [Tesfamariam et al., 1990]. Ozkan et al.

demonstrated that during hyperglycaemia, reactive oxygen species such as

superoxide play a role in the impairment of EDHF-mediated relaxations [Ozkan and

Uma, 2005]. NO availability has been reported to be reduced when the blood

glucose levels were elevated above normal levels in diabetic women [Konukoglu et

al., 2003]. However, the plasma nitrite and nitrate measured in this study are not

accurate markers of vascular NO. In the rat spinotrapezius muscle,

acute

hyperglycaemia has been shown to suppress arteriolar NO production [Lash et al.,

1999]. The association between hyperglycaemia and the endothelial mediators of

vasodilation is unresolved, as it is unknown which of the three endothelial mediators

is affected the most by alterations in glucose concentration.

1.9.6 Acute hyperglycaemia

Short periods of hyperglycaemia, referred to as “glucose spikes”, usually seen after

a meal, are becoming increasingly recognised as an important factor in the

pathogenesis of complications in diabetes. Flow-mediated vasodilation in the post-

prandial state was found to be diminished in patients with type 2 diabetes; the

reduction being inversely correlated to the magnitude of post-prandial

hyperglycaemia [Shige et al., 1999].

Indirect evidence for the deleterious effects of glucose spikes also comes from

studies which have demonstrated improvement in endothelial function when such

spikes were suppressed. Acarbose, an -glucosidase inhibitor that limits glucose

spikes, improves endothelial dysfunction in rats exhibiting repetitive blood glucose

fluctuation [Azuma et al., 2006]. As acarbose also improves the lipid profile

[Ogawa et al., 2004], some of the benefits may be due to indirect effects on the

endothelium. Another study has demonstrated that the avoidance of spikes in type 2

diabetes patients by intensive hospital treatment of hyperglycaemia improved

endothelial function [Yasuda et al., 2006].

67

The mechanism of how glucose spikes mediate endothelial dysfunction is believed

to be related to free radical production. Glucose fluctuations during post-prandial

periods have been demonstrated to be a more effective trigger for oxidative stress

than chronic sustained hyperglycaemia [Monnier et al., 2006]. Constant as well as

intermittent high glucose levels have also been shown to enhance endothelial cell

apoptosis through mitochondrial superoxide overproduction [Piconi et al., 2006].

1.9.7 Hyperglycaemia and endothelium-dependent relaxation

Studies examining the association between hyperglycaemia and endothelium-

dependent relaxation have produced contradictory results. Impaired, unaltered and

enhanced endothelium-dependent relaxation has been demonstrated in both animal

and human vascular beds. These studies have been summarised in Table 1.3

(impaired), Table 1.4 (enhanced) and Table 1.5 (unaltered). These conflicting results

may be attributable to differences in the species, vascular bed, methodology and

both the degree and duration of hyperglycaemia. However, different results were

obtained even in the same vascular bed (upper limb circulation) using similar

methods. The conflicting results of these studies suggest that the actions of glucose

on endothelial function are complex. They also indicate that the glucose

concentration, its duration of exposure, the vascular bed studied and the

methodology used are four distinct variables that require standardisation in order to

understand the intricate relationship between glucose and endothelial function.

68

Author Vascular

Bed Method Duration

Level of Hyperglycaemia

Result

Bohlen and Lash,

1993

Intestinal arterioles of normal

rats

Direct microscopic visualization

1 hour 16.6 mmol/L Suppression

of vasodilation

Williams et al., 1998

Brachial artery of healthy humans

Plethysmo-graphy

6 hours 16.6 mmol/L

Impaired endothelium-dependent relaxation

Renaudin et al., 1998

Trapezius muscle of

normal rats

Intravital microscopy

45 min 16.9 ± 0.5

mmol/L Vaso-

constriction

Kawano et al., 1999

Brachial artery in normal, IGT and

type 2 DM patients

FMD 1-2 hours

OGTT 75 g Norm 8.5 mmol/L IGT 12.2 mmol/L DM 13.3 mmol/L

Impaired dilatation at 1

hour in all groups

Title et al., 2000


FMD Up to 4 hours

75 g OGTT

Reduced FMD which

was restored with Vitamin

C and E

Beckman et al., 2001


Plethysmo-graphy

6 hours 16.6 mmol/L

Reduced endothelium-dependent

vasodilation, which was restored by Vitamin C

Affonso Fde et al.,

2003

Renal artery of normal rabbits

Tissue perfusion

3 hours 15 mmol/L

Impaired endothelium-dependent relaxation

Table 1.3: Studies demonstrating impaired relaxation with hyperglycaemia

Author Vascular

Bed Method Duration


Result

Cipolla et al., 1997

Posterior cerebral artery of

rats

Pressure Myography

1-2 hours 44 mmol/L Endothelium-

dependent vasodilation

van Veen et al., 1999

Forearm blood flow of healthy humans

Forearm blood Flow

5 minutes

20% glucose infusion

Vasodilation not modified by

hyperinsulinemia

Hoffman et al., 1999

Forearm blood flow of healthy humans

Plethysmo-graphy

1 hour 20% glucose

infusion

Sympatho-excitation and

peripheral vasodilation

Oomen et al., 2002

Skin of type 1 DM patients

Skin micro-vascular

flow 1 hour 12 mmol/L

Increased blood flow

Table 1.4: Studies demonstrating vasodilation with hyperglycaemia

69

Author Vascular

Bed Method Duration


Result

Houben et al., 1993

Brachial artery

Laser Doppler

flow-metry

24 hours 15 mmol/L

No effect on basal blood flow or endothelium-dependent or independent

relaxation

Brands and

Fitzgerald, 1998

Hind limb blood flow

of streptozo-

tocin treated diabetic

rats

Arterial catheter

6 days 22 mmol/L

No effect on endothelium-dependent relaxation

Charkoudian et al.,

2002

Leg skin blood flow in healthy individuals

Laser Doppler Flow-metry

6 hours 11.1 mmol/L No effect on vasodilation

Reed et al., 2004

Forearm of healthy

individuals

Forearm blood flow

6 hours 11.1 mmol/L No effect on endothelial

function

Oomen et al., 2004

Skin of type 2 DM patients

Laser Doppler

flow-metry

3.5 hours 12 mmol/L No change in

micro-circulation

Table 1.5: Studies demonstrating no effect of hyperglycaemia on

vascular function

1.10 Hypoglycaemia

1.10.1 Hypoglycaemia and pregnancy

Hypoglycaemia has been defined as a clinical and biological syndrome, caused by

an abnormal decrease in plasma glucose levels to below 3 mmol/L [Virally and

Guillausseau, 1999]. However, the definition of hypoglycaemia is controversial, as

it has both a quantitative as well as a clinical aspect. In contrast to hyperglycaemia,

hypoglycaemia can be associated with more pronounced symptoms such as tremors,

headaches and behavioural changes. The threshold for developing these features can

vary from patient to patient. As a result, clinically relevant hypoglycaemia may not

correlate with its quantitative measurement and vice versa. A study in ten pregnant

women with type 1 diabetes demonstrated an association between hypoglycaemia

and an increase in frequency and amplitude of fetal heart rate accelerations, as well

as a rise in maternal catecholamine levels [Bjorklund et al., 1996]. This is in

contrast to a similarly sized study (also in type 1 diabetic patients) which reported

70

that hypoglycaemia had no significant effect on fetal movements, heart rate or

breathing [Reece et al., 1995]. Safety and ethical concerns limit the study of

hypoglycaemia in women during pregnancy, making its precise effects uncertain.

Nevertheless, hypoglycaemia is a common occurrence in poorly controlled diabetes,

its true frequency now becoming evident in pregnancy with new techniques such as

continuous glucose monitoring (CGMS) [Yogev et al., 2003]. Asymptomatic

hypoglycaemic events in women with gestational diabetes have been reported to be

particularly common overnight in insulin-treated women [Yogev et al., 2004].

Hypoglycaemia approximately 160 minutes after a meal has also been noted in

women with diabetes in pregnancy [Ben-Haroush et al., 2004].

1.10.2 Hypoglycaemia and endothelial function

A study investigating the effect of hypoglycaemia on rat middle cerebral artery

demonstrated that reducing the glucose concentration from 5.5 mmol/L to 1.0 or 0.5

mmol/L for 1.5 hours each had no significant effect on arterial diameter [Swafford

et al., 1998]. The production of prostacyclin has been found to be increased in

cultured human endothelial cells exposed to a low concentration of glucose

[Watanabe and Jaffe, 1995]. Little is known about the effect of hypoglycaemia on

endothelial function, as research in diabetes has concentrated on the vascular effects

of hyperglycaemia. The suprising paucity of published studies examining

endothelial function during hypoglycaemia prevents any meaningful analysis of its

effects.

1.11 Blood glucose in pregnancy

Karlsson and Kjellmer were among the first to demonstrate a relationship between

fetal outcome and glycaemic control; reporting a linear correlation between the third

trimester mean maternal blood glucose and the perinatal mortality rate [Karlsson

and Kjellmer, 1972]. Since then, various studies have shown the beneficial aspects

of glucose control on pregnancy outcome in diabetes [Crowther et al., 2005; Drexel

et al., 1988]. However¸ the criteria for satisfactory metabolic control vary widely,

and studies showing a low perinatal morbidity and mortality differ in their details.

For example, in the study by Crowther et al., although outcome improved with

treatment in GDM patients, the actual glucose levels these women attained were not

assessed. Karlsson and Kjellmer reported a low perinatal morbidity at a mean third

71

trimester blood glucose value of less than 5.5 mmol/l. These mean values were

obtained by averaging daily measurements that were taken at three unspecified

times during the day, without considering the effect of meals on these levels. When

estimating blood glucose levels, the lack of taking into account the time since the

last meal is a recurring problem in many studies. This makes interpreting the true

degree of glycaemic control difficult.

Most studies currently use HbA1C as a marker for glycaemic control. HbA1C

measures long-term glycaemic control, reflecting a time-weighted mean over the

previous 3 to 4 months. Improvement of HbA1C is associated with a reduction of

complications associated with type 2 diabetes [Stratton et al., 2000]. However, there

is uncertainty regarding the precise correlation between HbA1C and the various

phases of glycaemia such as basal, post-prandial and fasting. Other limitations of

HbA1C include differences in predictive value between type 1 and type 2 diabetes, a

considerable degree of biological variability between subjects and analytical

variability [Jeffcoate, 2004].

Pregnancy complications have been demonstrated to be reduced in diabetic women

with good glycaemic control, as evaluated using HbA1C [Boulot et al., 2003].

However, a large study in Netherlands surveying women with type 1 diabetes in

pregnancy reported increased maternal and perinatal complications, despite

achieving satisfactory HbA1C levels of less than 7.0% [Evers et al., 2004]. Rebound

hyperglycaemia elicited by hypoglycaemia has been put forward as a possible

explanation for this outcome, as hypoglycaemia is more common in well controlled

type 1 patients [ter Braak et al., 2002]. In women with type 1 diabetes, HbA1C

levels during pregnancy were found to have no correlation with neonatal

hypoglycaemia or macrosomia [Taylor et al., 2002]. These studies signify that using

HbA1C to assess glycaemic control may not be useful in predicting the

complications of diabetes in pregnancy.

Numerous studies have examined Doppler flow parameters during hyperglycaemia.

In healthy pregnant women, acute hyperglycaemia did not affect Doppler flow in the

umbilical or uterine arteries at any stage of oral glucose tolerance testing [Yogev et

al., 2003]. Three studies have demonstrated no correlation between uterine and

72

umbilical artery Doppler waveforms and the degree of glycaemic control [Dicker et

al., 1990; Ishimatsu et al., 1991; Kofinas et al., 1991]. Although these studies

indicate that Doppler flow is unaffected by maternal blood glucose levels, it is

uncertain whether blood flow in smaller resistance arteries are affected by glucose

levels during pregnancy.

The above discussion outlines the limitations of using HbA1C and absolute glucose

levels in assessing control during diabetes in pregnancy. It can be surmised that

other parameters, such as fluctuation in the levels of glucose, may be of importance

in diabetes complicating pregnancy. As previously described, glucose fluctuations

during post-prandial periods may be a more effective trigger for oxidative stress

than chronic sustained hyperglycaemia. However, the effects of acute

hypoglycaemia and hyperglycaemia on resistance artery function in pregnancy are

unknown.

1.12 Summary of Introduction

Poorly controlled diabetes in pregancy, with recurrent hypo- and hyperglycaemia, is

associated with an increased incidence of complications. Hyperglycaemia has been

linked to aberrant endothelial function, which may have an important role in the

pathophysiology of these complications, as controlling hyperglycaemia improves

pregnancy outcome. However, studies investigating the effects of glucose on

endothelial function have produced conflicting results, raising the possibility of

heterogeneity of these effects depending on the vascular site and degree of

hyperglycaemia. To discern the true effects of glucose concentrations on endothelial

function, it is therefore necessary to examine the relevant vascular bed directly. In

diabetes complicating pregnancy, the myometrial arterial bed is important in

determining blood supply to the fetus. However, the effects of hypo- and

hyperglycaemia in this vascular bed are not known, either in healthy individuals or

diabetic patients. It has been demonstrated that fluctuations in blood glucose may be

more important than absolute values in the pathogenesis of aberrant vascular

function. However, the value of certain studies has been limited by the high glucose

levels examined, which are not seen clinically. Defining clinically relevant glucose

concentrations and characterising their effect on endothelial function in myometrial

73

arteries of healthy women and pregnant women with diabetes is therefore important

in advancing our limited knowledge of this disorder.

1.13 Hypotheses

I. The effects of glucose levels on endothelial function vary in different

vascular beds

II. Acute change in glucose level from the normal range is associated with

alterations in endothelial function

III. Diabetes in pregnancy is associated with endothelial dysfunction in

myometrial arteries

1.14 Aims

1. Identify clinically relevant glucose levels in diabetes complicating pregnancy

2. Characterise the effect of glucose levels in myometrial arteries of healthy

pregnant and non-pregnant individuals

3. Examine the effect of acute changes in glucose levels in myometrial arteries in

pregnancies complicated by diabetes

4. Examine the effect of acute changes in glucose levels in the uterine artery of

pregnant and non-pregnant mice

5. Discern the contributions of the endothelial mediators of vasodilation in the

uterine artery of pregnant mice

6. Enable further characterisation of the vascular effects of diabetes in pregnancy

by creating an animal model of diabetes

74

CHAPTER 2

MATERIALS AND METHODS

75

MATERIALS AND METHODS

2.1 Ethics

The study conformed to the Declaration of Helsinki (2000) and was approved by the

Central Manchester Local Research Ethics Committee under the title “Endothelial

vascular behaviour in pregnancies complicated by diabetes”. Informed written

consent was obtained from all participating patients (sample information sheet and

consent form is given in the Appendix). Diabetic patients were recruited from the

three hospitals in Greater Manchester having the largest number of patients with

diabetes in pregnancy (St. Mary’s Hospital, Hope Hospital and North Manchester

Hospital). All animal studies were carried out in accordance with the UK Animals

(Scientific Procedures) Act 1986.

2.2 Human Subjects

2.2.1 Healthy women

2.2.1.1 Non-pregnant women

Healthy women under the age of 60 with no co-existing disease and not on

medication were selected. All patients had elective hysterectomies, the most

common indication being menorrhagia.

2.2.1.2 Pregnant women

Healthy women with no documented disease and not on medication, who had

uncomplicated singleton pregnancies were selected. All patients had elective

Caesarean section deliveries at term, the most common indications being previous

Caesarean section or breech presentation.

2.2.2 Diabetic Patients

2.2.2.1 Type 1 and type 2 diabetes in pregnancy

Patients with type 1 or type 2 diabetes diagnosed before pregnancy and not on

medication during pregnancy (apart from insulin) were selected. Type 2 diabetes

76

patients with co-morbidities such as hypertension were excluded. Type 1 patients in

the ongoing Diabetes And Pre-eclampsia Intervention Trial (DAPIT) were also

excluded as they were taking vitamins A and E or placebo.

2.2.2.2 Gestational diabetes

All the patients with GDM fulfilled the WHO criteria for having gestational diabetes

i.e.; the glucose intolerance was first recognized during pregnancy with a fasting

blood glucose ≥ 7.0 mmol/L and/or 2 hour ≥ 7.8 mmol/L. Patients with GDM who

had no other co-existing disease and were not on medication during pregnancy

(except insulin) were selected. GDM was diagnosed either in the second or third

trimester of pregnancy, with a glucose tolerance test (GTT) usually being performed

between 24 to 28 weeks. Each hospital had different policies regarding post-natal

glucose tolerance tests. Of the 16 patients, 8 had a normal post-natal GTT and 8

patients had normal post-natal blood glucose readings, but no formal GTT.

2.3 Mice

Female C57BL/6JOla mice (Harlan, UK) were used as they have been reported to

have similar blood glucose levels as healthy humans. Fasting glucose levels of 4.4 ±

0.2 mmol/L [Schreyer et al., 1998] and mean levels of 5.3 ± 1.3 mmol/L [Keren et

al., 2000] have been reported in these mice. Furthermore, this strain has been

extensively used over the years in studies dealing with vascular research examining

normal mice [Laufs et al., 2005], knockout mice [Waldron et al., 1999] and

steptozocin-induced animal models of diabetes [Cai et al., 2005]. Non-pregnant

mice, pregnant mice at term (day 19) and their offspring were humanely killed by

cervical dislocation, as per the Animals (Scientific Procedures) Act 1986.

2.4 Tissue collection

All tissue was collected in cold Modified Krebs’ buffer (Table 2.1) and immersed in

this solution until dissection of the arteries was completed.

2.4.1 Non-pregnant women

Tissue samples were taken from the lower segment of the hysterectomy specimens,

in order to correspond to the anatomically equivalent site of Caesarean section

biopsies.

77

2.4.2 Pregnant women

After the fetus was delivered by Caesarean section, myometrial biopsies were

obtained from the upper lip of the transverse lower segment incision of the uterus.

2.4.3 Mice

After the mice had been humanely killed, the main uterine arteries were dissected

bilaterally.

2.5 Stereomicroscopic dissection

Samples obtained were examined under a light microscope and small arteries were

dissected out using fine forceps and small dissecting scissors, taking care not to

damage the vessel wall. The length of arteries was measured using a calibrated

microscope eyepiece. Vessels were cut into lengths of about 2 mm and mounted in a

wire myograph chamber, where their diameter was assessed.

2.6 Resistance arteries

As discussed in Section 1.4.2, mesenteric arteries in rats with a diameter of 100 to

300 µm have been demonstrated to be resistance arteries [Christensen and Mulvany,

1993]. The diameter of the uterine arteries of both pregnant and non-pregnant mice

studied fell in this range. The diameter of resistance arteries in humans is uncertain;

however because of the reasons discussed in Section 1.4.2, myometrial arteries with

diameters in the approximate range of 200 to 500 µm were used in this study.

2.7 Choice of methodology

The decision regarding which technique to use for studying vascular function is of

considerable importance. This decision should be made by weighing the relative

benefits and drawbacks of each methodology in the context of the particular

experiments envisaged. In this study, vessel function at four different glucose levels

was examined in a vascular region inaccessible by techniques such as

plethysmography and flow-mediated dilation. As four glucose levels were

investigated, single-vessel techniques such as pressure myography were unsuitable.

78

It was therefore decided that investigating vascular function using wire myography

would be the most appropriate way to achieve the aims of the study.

2.8 Wire myography

Wire myography is an in vitro technique for studying the mechanical,

morphological and pharmacological properties of small vessels. It was first

proposed by Bevan et al. [Bevan and Osher, 1972] and subsequently developed by

Halpern and Mulvany [Halpern et al., 1978]. In this technique, segments of vessels

are mounted as ring preparations, with two fine wires through their lumen which are

then secured at both ends. During the experiment, the circumference of the vessel is

kept constant; therefore the vessels are examined under isometric conditions (Fig.

2.1).

Fig. 2.1: Schematic representation of a blood vessel mounted on a wire myograph

This is necessary as the force production is dependent on the extent of stretch,

according to the active tension-length relation of all muscles. Also, the sensitivity of

vessels to different agonists is dependent on stretch [Aalkjaer and Mulvany, 1983].

The force exerted on the wire during constriction and relaxation is measured by a

sensitive strain gauge, transmitted to a computer and graphically represented as a

curve (Fig. 2.2). Mounted vessels have been demonstrated to maintain their

functional characteristics ex-vivo [Mulvany and Aalkjaer, 1990]. Refinement of the

79

mechanistic aspects of this technique has occurred over the decades and a

considerable body of work has developed around this methodology. It has proven to

be a robust and reproducible method in the study of vascular function.

2.8.1 Multi Myograph System 610M

All experiments described were performed on a four-chamber Multi Myograph

System 610M (Danish Myo Technology A/S, Aarhus, Denmark: Fig. 2.3). The

artery preparations in all four chambers were kept under physiological conditions at

37° C, and aerated with a gas mixture containing air / 5% CO2 (British Oxygen

Company, Surrey, UK).

Fig. 2.2: Myograph with data recordings on computer

80

2.8.2 Advantages of wire myography

1. Four arteries can be studied in parallel, enabling a more comprehensive

study of vessel function from a single subject. This is of particular relevance

to this study as it enables the study of 4 different glucose levels.

2. Vessels are studied ex-vivo, maintaining their functional integrity

3. By studying isolated vessels, greater control of experimental conditions

(such as glucose levels, solutions and oxygenation) is possible than studying

perfusion of a tissue region in vivo.

4. It allows resistance arteries from any vascular bed to be studied, irrespective

of whether they were obtained from humans or animals.

Fig 2.3: Multi Myograph System 610M (above) and an

individual myograph chamber (left)

81

2.8.3 Limitations of wire myography

Mounted vessels in wire myography have a flattened, rather than the customary

cylindrical shape. Other techniques such as plethysmography or pressure

myography are therefore considered to approximate physiological function more

closely than wire myography, as vessels maintain their normal shape. The delicate

endothelial cell layer is prone to damage if the two wires are not carefully inserted.

The wires exert pressure on the ends of the vessel wall, rather than distributing it

evenly along the circumference of the artery. Furthermore, vessels tend to retract as

they are stretched, due to elasticity. By studying isolated arteries, tissue-specific

systemic factors (such as neuronal and hormonal input) which may influence vessel

function are abolished. Additionally, vessels are exposed to solutions bereft of blood

cells and plasma. Shear stress, an important stimulus for NO release in vivo

[Kublickiene et al., 1997], cannot be reproduced using wire myography. However,

these limitations are outweighed by the benefit wire myography confers in enabling

four arteries to be studied concurrently in the present study.

2.9 Normalisation of vessel lumen

Normalisation is a process whereby the vessel circumference is calculated and set to

a standardized level, enabling comparison of responses across different vessels. The

rationale of this procedure is three-fold. The behaviour of elastic structures like

vessels can only be compared if the conditions are standardised and uniform.

Furthermore, the sensitivity of preparations to agonists is dependent of the degree of

stretch, so it is important that these conditions be clearly defined. Finally, because

the active response of a vessel is dependent on the degree of stretch, it is useful to

set vessels to an internal circumference which gives the maximum response.

Normalisation was performed using the Myodaq 2.01 Multi + software program

(Myonic Software, National Instruments Corporation, USA).

In practice, the vessel is stretched in a series of small steps at two minute intervals,

and the resting tension determined for each stretch. The wall tension is the measured

force divided by the wall length and is expressed in mN/mm (milli-Newtons per

82

millimetre). From the wall tension, the Laplace relation can be used to determine the

Active Effective Pressure (Pi):

Active Effective Pressure (Pi) = Wall Tension / 2 π

Internal Circumference

Active effective pressure is thus an estimate of the pressure which would be

necessary to extend the vessel to the measured internal circumference.

The active effective pressure is calculated at each step until a pressure of 13.3 kPa

(100 mm Hg) is reached. An exponential curve is then fitted to the internal

circumference pressure data and the point on the curve corresponding to 100 mm Hg

is determined and denoted IC100. Having found this, the internal circumference is set

to 90% of this, as previous studies have shown that a maximal force is generated at

this setting [Mulvany and Aalkjaer, 1990].

2.10 Drugs

All drugs and solutes were obtained from Sigma-Aldrich (UK). All drugs were

newly re-constituted from powder form on each day of experimentation apart from

bradykinin and U46619 which were made from the frozen stock solution.

2.10.1 Potassium chloride

At concentrations of 60 mmol/L, potassium chloride constricts vessels by

depolarization of smooth muscle cells. This property has been utilised in previous

studies to induce arterial constriction [Lagaud et al., 2002; Vial and Evans, 2005].

An advantage of using potassium to constrict vessels is that it produces a regular

and sustained constriction, enabling greater reproducibility when analysing

constriction and relaxation.

2.10.2 Bradykinin

Bradykinin has been demonstrated to cause relaxation of blood vessels that is

endothelium-dependent, as denudation of the endothelium abolished this response

[Cherry et al., 1982; Whalley et al., 1987]. Bradykinin is a stimulator of eNOS and

NO production through Ca

2+-mediated mechanisms in endothelial cells via the B2

receptor [Leeb-Lundberg et al., 2005]. It has also been demonstrated to be

83

associated with prostacyclin release [Lamontagne et al., 1992] as well as relaxation

mediated by EDHF [Ohlmann et al., 1997].

2.10.3 Phenylephrine

Phenylephrine is structurally related to epinephrine and induces constriction of

vessels by its action as an agonist [Sofowora et al., 2004].

2.10.4 Acetylcholine

Acetylcholine mediates endothelium-dependent relaxation by the activation of

muscarinic receptors located on endothelial cells [Walch et al., 2001].

2.10.5 U46619

U46619 (9, 11-dideoxy-11α, 9α-epoxymethanoprostaglandin F2α) is a prostanoid

which acts as a specific thromboxane receptor agonist. This biologically relevant

vasoconstrictor has previously been demonstrated to provide a sustained and

reproducible constriction in myometrial arteries [Ong et al., 2003].

2.10.6 L-NNA

L-NNA (Nω –Nitro-L-arginine) is an inhibitor of the enzyme Nitric Oxide Synthase

(NOS) [Lamontagne et al., 1991; Ding and Triggle, 2000]. Exposure to a

concentration of 100 µM L-NNA has been previously used in the aorta of C57BL/6

mice to block the action of NO [Rabelo et al., 2003].

2.10.7 Indomethacin

Indomethacin is a cyclo-oxygenase inhibitor that inhibits both COX-1 and COX-2

isoforms [Mitchell and Warner, 2006]. In the carotid artery of C57BL/6 mice,

concentrations of 10 µM indomethacin have been used to block cyclo-oxygenase

activity [Quan et al., 2006].

2.10.8 Streptozotocin

Streptozotocin is a nitrosurea derivative isolated from Streptomyces achromogenes

with broad-spectrum antibiotic and anti-neoplastic activity [Bono, 1976]. It is a

powerful alkylating agent that has been shown to interfere with glucose transport

[Wang and Gleichmann, 1998], glucokinase function [Zahner and Malaisse, 1990]

84

and induce multiple DNA strand breaks [Bolzan and Bianchi, 2002]. A single large

dose of streptozotocin or multiple small doses can induce an insulinopaenic diabetes

due to its toxic effects on pancreatic cells [Rees and Alcolado, 2005].

2.10.9 Choice of drugs

2.10.9.1 Vasoconstriction

60 mmol/L potassium was used to constrict myometrial arteries as it produced a

sustained and reproducible constriction in these vessels. However, at this

concentration, the effect of EDHF on subsequent endothelium-dependent relaxation

was blocked, as discussed in Section 1.5.2.3. Therefore, experiments were also

performed using U46619 as a vasoconstrictor in myometrial arteries. Initial

experiments in mice uterine arteries demonstrated diminished constriction in this

vascular bed with 60 mmol/L potassium, but greater constriction with

phenylephrine. Similar enhanced constriction with phenylephrine compared with

potassium has been previously noted in mouse arteries [Nobe et al., 2006].

Preliminary experiments using U46619 in these arteries resulted in less reproducible

constriction compared with phenylephrine. It was therefore decided to use

phenylephrine as the vasoconstrictor in experiments investigating mice uterine

arteries.

2.10.9.2 Endothelium-dependent relaxation

Bradykinin was used in myometrial arteries, as it has been noted to produce a more

reproducible relaxation in these vessels than acetylcholine [Kenny et al., 2002].

Preliminary experiments with bradykinin in mice uterine arteries demonstrated

minimal relaxation with bradykinin. A possible explanation for this result is that

there are diminished amounts of endothelial cells responsive to bradykinin in this

vascular bed. Using confocal microscopy, it has been demonstrated that individual

endothelial cells in the aorta of mice respond in different ways to endothelium-

dependent vasodilators such as acetylcholine and bradykinin [Marie and Beny,

2002]. Endothelial cells which responded to acetylcholine were found to be different

from those activated by bradykinin. Furthermore, these endothelial cells were not

homogeneously distributed, with a significantly higher percentage of cells

85

responding to acetylcholine compared with bradykinin. This may explain why

acetylcholine produced more reproducible relaxations than bradykinin in mice

uterine arteries. Subsequent mice experiments were therefore performed using

acetylcholine.

2.11 Solutions

All solutions were freshly made up each day, titrated to a pH of 7.4 and stored in a

fridge. The composition of the various solutions in mmol/L is given in Table 2.1.

Solute Glucose

Solutions KPSS

Hyper-

osmolar

Solution

Modified

Krebs’

Buffer

NaCl 128 72.45 128 136.9

NaHCO3 25 25 25 11.9

KCl 4.7 60 4.7 2.7

MgSO4 2.4 2.4 2.4 0.6

CaCl2 1.6 1.6 1.6 1.8

KH2PO4 1.18 1.18 1.18 0.5

EDTA 0.034 0.034 0.034 -

Glucose 2, 5, 8 or 12 2, 5, 8 or 12 5 11.5

Mannitol - - 7 -

Table 2.1: Composition of solutions used (in mmol/L)

2.11.1 Glucose physiological saline solutions

The glucose solutions used consisted of Physiological Saline Solution (PSS) to

which 2, 5, 8 or 12 mmol/L of glucose was added.

2.11.2 60 mmol/L KPSS

Potassium Physiological Saline Solution (KPSS) consisted of the previously

described PSS with an equi-osmolar substitution of NaCl with KCl. 2, 5, 8 or 12

mmol/L of glucose was added to the solution as before.

86

2.11.3 Hyper-osmolar solution

This solution consisted of PSS to which 5 mmol/L glucose and 7 mmol/L mannitol

were added. This resulted in a solution equi-osmolar to PSS containing 12 mmol/L

glucose (equi-osmolar substitution of glucose with mannitol).

2.11.4 Modified Krebs’ buffer

This solution was used to temporarily preserve tissue while arteries were dissected

out, because of its ability to maintain its pH at room temperature without having to

be aerated. Although this solution contained a relatively high concentration of

glucose (11.5 mmol/L), the vessels were only exposed to this for less than 30

minutes.

2.12 Experimental protocols

2.12.1 Protocol 1: The effect of glucose levels on maximum

constriction

For each experiment, arteries were all initially placed inside individual chambers in

PSS with a glucose concentration of 5 mmol/L (representing normoglycaemia).

Arteries were constricted with a vasoconstrictor (KCl, U46619 or phenylephrine)

and allowed to plateau. The maximum active effective pressure in the first two

curves at 5 mmol/L glucose for each artery was taken and the data averaged. After

relaxation (described in Protocol 2), vessels were subsequently exposed to PSS

containing 2, 8 or 12 mmol/L glucose for 30 minutes, leaving a control vessel at 5

mmol/L glucose. To avoid methodological error, vessels were randomly allocated to

a particular glucose level. Arteries were washed with fresh glucose solutions three

times in these 30 minutes. The maximum active effective pressure after change in

glucose concentration in two subsequent curves was similarly taken and the data

averaged (Fig. 2.4 and 2.5).

87

Fig. 2.4: Schematic representation of concentration-response curve Fixed dose of vasoconstrictor added (green arrow). Endothelium-dependent vasodilator

added at maximum constriction of artery in incremental concentrations (red arrows) at two

minute intervals. After five doses of vasodilator, drugs were washed off (blue arrow).

Protocol 1, 2 and 3

0

1

2

3

4

5

30 min

Ten

sio

n (

mN

/mm

)

Fig. 2.5: Raw data trace Actual raw data trace of an experiment, illustrating the scheme of concentration-response

curves in Protocol 1, 2 and 3. The red arrow represents the change of solution to one of

different glucose concentration or osmolarity. Similar traces were obtained simultaneously

from four myograph chambers for each experiment.

Vasodilator

Wash

Vasoconstrictor

2 Minute Intervals

Maximum

Constriction

88

2

5

8

12

Fig. 2.6: Experimental protocol 1 and 2

A schematic representation of experimental protocol 1 and 2. Glucose

concentrations represented as: 2 = 2 mmol/l (blue), 5 = 5 mmol/L (red), 8 = mmol/L

(green) and 12 = 12 mmol/L (orange).

2.12.2 Protocol 2: The effect of glucose levels on endothelium-

dependent relaxation

As described in Protocol 1, arteries were all initially placed inside individual

chambers in PSS with a glucose concentration of 5 mmol/L. Vessels were

constricted and subsequently an endothelium-dependent vasodilator (bradykinin or

acetylcholine) was added in incremental concentrations from 10-10

to 10-6

Mol/L at

two-minute intervals. The drugs were then washed away with fresh PSS. Two such

constriction-relaxation curves were performed at 5 mmol/L glucose and the data was

averaged. Vessels were subsequently exposed to PSS containing 2, 5, 8 or 12

mmol/L glucose for 30 minutes. Vessels were washed with fresh PSS glucose

solutions three times in these 30 minutes. Two further constriction-relaxation curves

were performed as before and the data averaged. This has been schematically

represented in Fig. 2.6. A paired comparison was made of the endothelium-

dependent relaxation before and after the change in glucose levels.

89

2.12.3 Protocol 3: The effect of hyper-osmolarity

This was similar to Protocol 2, except an artery kept was kept as control at 5

mmol/L glucose and another artery incubated in a hyper-osmolar solution with

mannitol (Fig 2.8). The hyper-osmolar solution contained 5 mmol/L glucose to

which 7 mmol/L mannitol had been added to make it equi-osmolar to a 12 mmol/L

glucose solution. Endothelium-dependent relaxation was assessed in the same

manner as in Protocol 2.

Hyper-osmolar

Solution

Normal Solution

Fig. 2.7: Protocol 3

Normal Solution (red): glucose solution (with 5 mmol/L glucose)

Hyper-osmolar solution (light blue): glucose solution (with 5 mmol/L glucose) +

7 mmol/L mannitol

90

2.12.4 Protocol 4: The effect of blockers to endothelium-


Arteries were initially placed in a glucose solution containing PSS with 5 mmol/L

glucose and a constriction-relaxation curve performed as normal. Vessels were then

incubated in either a 5 (Fig. 2.6) or 12 (Fig. 2.7) mmol/L glucose solution for 30

minutes and a second curve performed. Subsequently, the four arteries were treated

as follows:

1) Control

2) 10-4

M L-NNA added

3) 10-5

M Indomethacin added

4) Both 10-4

M L-NNA and 10-5

M Indomethacin added

After a 20 minute exposure to these drugs, a final concentration-response curve was

performed in the previous manner.

Protocol 4

0

1

2

3

30 min 20 min

Ten

sio

n (

mN

/mm

)

Fig. 2.8: Protocol 4: Actual raw data trace illustrating Protocol 4. Red arrow

represents change of solution to 12 mmol/L glucose (or 5 mmol/L in control). Green

arrow represents addition of one of the following in each vessel (no drug in control):

L-NNA (10-4

M), indomethacin (10-5

M), L-NNA + indomethacin.

91

Control

+ L-NNA

+ Ind

+ L-NNA

+ Ind

Fig. 2.9: Protocol 4 (5 mmol/L glucose)

A schematic representation of Protocol 3 at 5 mmol/L glucose.

Control= no drugs added, L-NNA=10-4

M L-NNA added,

Ind=10-5

M Indomethacin added added for 20 minutes

Control

+ L-NNA

+ Ind

+ L-NNA

+ Ind

Fig. 2.10: Protocol 4 (12 mmol/L glucose)

A schematic representation of Protocol 3 at 5 (red) and 12 mmol/L glucose (orange)

Control= no drugs added, L-NNA=10-4

M L-NNA added,

Ind=10-5

M Indomethacin added added for 20 minutes

92

2.13 Optimising experiments

2.13.1 General measures

As the main aim was to investigate the effect of acute changes in glucose levels, all

experiments were immediately performed as soon as tissue was obtained. This

helped to avoid storage for prolonged periods at a fixed glucose level: overnight

storage of tissue for up to 19 hours has been reported [Ang et al., 2002]. All

solutions were freshly made on each day of experimentation to prevent deterioration

in glucose levels of solutions from storage over time. Reduction in the potency of

drugs over time was minimised by freshly re-constituting drugs immediately prior to

the experiments.

When dissecting the arteries, care was taken not to damage the vessel wall by undue

stretching, with handling restricted to the ends of the artery. When inserting wires

inside the vessel, particular care was taken not to damage the endothelial layer:

vessels where such damage occurred were discarded. Damage to vessels was also

offset by ensuring wires had blunt edges, which prevented endothelial damage due

to scraping of the inner wall. The Multi Myograph Systems were calibrated at

regular intervals to ensure accurate responses. The average of two concentration-

response curves was taken to avoid the effect of any drug sensitization.

2.13.2 Optimising mice experiments

Uterine arteries from mice were found to have a limited ability to maintain

constriction, with occasional spontaneous relaxation to the baseline. This was

prevented by the following measures:

1. Smaller incremental stretches when normalising

As mouse uterine arteries were more delicate than human myometrial

arteries, performing smaller increments while stretching the vessel during

normalisation was found to be beneficial. This was found to prevent damage

to the vessel wall from excessive tension.

93

2. Extra washing of vessels after each constriction/relaxation curve

It was surmised that spontaneous relaxation could be due to acetylcholine

remaining in the vessel or in the vessel chamber despite a single wash; as

such relaxation was not present during the first curve. By washing the vessel

three times after each run, this problem was alleviated.

3. Longer interval between curves

It was observed that constriction was not maintained if the artery was

subjected to multiple constriction and relaxation curves without an adequate

time interval in between. Spontaneous relaxation to baseline was minimised

by allowing at least 10 minutes between each curve.

Vessels that continued to relax spontaneously to the baseline despite the above

measures were excluded from analysis.

2.14 Creating an animal model of diabetes

2.14.1 Restrictions in human research

The availability of tissue samples from both healthy individuals and diabetic

patients was found to be limited. Only a minority of patients underwent Caesarean

section, restricting myometrial biopsies from both healthy and diabetic pregnant

women. A large number of patients had co-existing disease or were on regular

medication and hence could not be included in the study. This was particularly true

of type 2 diabetes patients who often had co-existing hypertension. Most type 1

diabetes patients were recruited to the Diabetes And Pre-eclampsia Intervention

Trial (DAPIT) and were taking vitamins A and E or placebo, excluding them from

the present study. Of the women who fulfilled the inclusion criteria, consent was

often declined as the biopsies were perceived by the patients to be obtained by an

invasive procedure. As a result, only a small group of type 1 and 2 patients could be

enrolled in the study, despite recruiting from the hospital with the highest number of

pregnant diabetic patients in the Greater Manchester region (St. Mary’s Hospital).

To increase the number of patients in the study, approval was obtained to recruit

diabetic patients from two further hospitals (Hope Hospital, Salford and North

Manchester Hospital, Crumpsall) which had the second and third highest number of

94

patients with diabetes complicating pregnancy in the region. Despite this, the overall

number of patients recruited was still less than anticipated. The difficulties in

attempting to study diabetes in pregnancy using clinical research protocols have

been acknowledged in the past [Farrell et al., 1982]. It was therefore decided to

create an animal model of diabetes to enable progress of the study.

2.14.2 Rationale for creating an animal model of diabetes

The advantages of an animal model of diabetes have been previously discussed

(Section 1.2.4). Research in the field of pregnant diabetic animals has focused on

fetal defects and the fetal origins of adult disease. Little is known about endothelial

function in uterine arteries of diabetic rodents. Characterisation of uterine artery

endothelial function in diabetic rodents would therefore be valuable in discerning

the role of vascular responses in the pathogenesis of these conditions. If endothelial

function in diabetic animals was comparable to that seen in human pregnancies

complicated by diabetes, it would provide a basis for future work in this field. It

would also enable a broader range of studies to be undertaken, which would not

have been possible if research had been confined to human subjects.

2.14.3 Obtaining a Home Office Licence

In collaboration with Dr. Nicholas Ashton, a Home Office Project Licence was

obtained to create an animal model of diabetes (Project licence no: PPL 40/2916).

The proposal for a project licence was written by the author, with assistance from

Dr.Ashton. The abstract describing the purpose of the study has been posted by the

Home Office on the Internet at:

http://scienceandresearch.homeoffice.gov.uk/animal-research/publications/001-

abstracts/march-2006/097?view=Html

2.14.4 Species and strain

Both rat and mouse models of diabetes can be produced by injection of

streptozotocin (see Section 1.2). C57BL/6JOla mice (Harlan, UK) were chosen

mainly because this was the same strain used in earlier work, thus enabling

comparison with previous results. Other advantages of this strain have been

discussed in Section 2.3. Furthermore, the use of mice to establish baseline

95

characteristics of endothelial function enables the future utilization of genetic

manipulation technologies, such as knock-out models.

2.14.5 Duration of diabetes

Endothelium-dependent relaxation in the aorta of rats injected with streptozotocin is

affected by the duration of diabetes [Pieper, 1999]. Relaxation to acetylcholine was

increased at 24 hours following injection with streptozotocin compared with

controls, normal after 1 and 2 weeks of disease and impaired at 8 weeks of disease.

Therefore all experiments in the present study were performed 8 weeks after

injection, with regular testing of blood glucose in the intervening period.

2.14.6 Diabetogenic drug

Streptozotocin was used to induce diabetes as it is the most commonly used drug for

diabetogenesis. A single intra-peritoneal injection of streptozotocin was used as

described in other studies [Alp et al., 2003; Soriano et al., 2001]. A dose of 200

mg/kg body weight has been demonstrated to induce diabetes [Sango et al., 2002].

Other research groups in Manchester have successfully utilised murine models of

diabetes, which has facilitated local experience with this technique [Burnand et al.,

2004].

2.14.7 Procedure

Normal female C57 BL/6JOla mice of approximately 8 weeks of age were given a

single intra-peritoneal injection of 200mg/kg body weight of streptozotocin. Blood

glucose was measured from a superficial vein by the tail-prick method, using an

Accu-chek advantage glucose meter (Roche Diagnostics, UK).

All mice were provided with food and water ad libitum and their general condition

monitored on a daily basis over 8 weeks. Body weight and blood glucose were

measured periodically. The initial batch of 18 mice did not develop diabetes, as

random blood glucose levels measured over 8 weeks were persistently lower than

the accepted threshold for diabetes of 11.1 mmol/L (200mg/dl) [Soriano et al.,

2001]. Only 5 out of 10 mice developed diabetes in the second batch, of which one

subsequently died.

96

Mice were divided into 3 categories:

Category 1: vehicle controls: injected with vehicle alone

Category 2: streptozotocin-controls: injected with streptozotocin, but blood

glucose levels remained in the normal range

Category 3: diabetic mice: injected with streptozotocin, with glucose levels

in the diabetic range.

After an interval of 8 weeks from the date of injection, mice were allowed to

become pregnant. At term (day 19) mice were humanely killed and the main uterine

artery carefully dissected out. Subsequent steps were similar to that described

previously for normal pregnant mice.

2.14.8 Impediments in creating an animal model of diabetes

The poor success rate for inducing diabetes in mice, the stipulation of waiting 8

weeks prior to experimentation and the lack of any diabetic mice in the initial batch

limited the range of studies possible during the allotted time. Furthermore, diabetic

mice were noted to have an impaired ability to conceive. This effect has been

previously noted in mice with uncontrolled streptozotocin-induced diabetes

[Diamond et al., 1989]. Deterioration in the health of diabetic mice during

pregnancy was also noticed. Therefore, only results from Category 2 mice

(streptozotocin injected, but not diabetic) have been presented here. However, these

results can be used in future comparisons of endothelial function in the three

categories of mice, as this work is being continued by the post-graduate student

Joanna Stanley. Despite these impediments, the initiation of the above steps in

creating an animal model of diabetes will enable future investigations into vascular

function in diabetes complicating pregnancy.

97

2.15 Data analysis

Data obtained as above was analysed using Myodata 2.02 software (Myonic

Software, National Instruments Corporation, USA).

2.15.1 Constriction

The maximum active effective pressures obtained in the two constriction curves at 5

mmol/L glucose were averaged. Maximum active effective pressures in the two

curves after change of glucose concentration were similarly averaged.

2.15.2 Endothelium-dependent relaxation

The baseline for calculating the amount of relaxation was taken as the resting level

before constriction. The average force per unit length between markers of drug

addition on the concentration response curve was taken. This ensured

reproducibility of results and enabled comparison between experiments. The active

tension produced by the vessels was calculated in mN/mm to eliminate variations

arising due to different vessel lengths. The maximum active tension obtained when

the vessels constricted and reached a plateau was taken as 100%, and the reduction

in tension on adding vasodilators was calculated as a percentage of this. This

eliminated variation in the numerical value of tension developed in different vessels,

thereby allowing comparison between arteries.

2.16 Statistical analysis

2.16.1 General Principles

All results were analysed using GraphPad Prism version 4.00 for Windows,

(GraphPad Software, San Diego USA) and plotted graphically using this software.

Data was tested for normality using the Kolmogorov-Smirnov Test. Normally

distributed data were analysed using the appropriate parametric test. At 95%

confidence intervals, a p value of less than 0.05 was taken to be statistically

significant in all analyses. Bonferroni post-hoc testing was performed on all

statistically significant results. 95% confidence intervals have been stated for major

comparisons. All data were expressed as mean ± standard error (SEM = standard

deviation / √n ) except where stated.

98

2.16.2 Statistical analysis of data from patients

To compare normally distributed data between groups of women, results were

analysed using a one-way ANOVA. Bonferroni post tests were used to discern

differences between groups.

Serial measurements of blood glucose and HbA1C in women were also compared

during pregnancy. For each woman, blood glucose and HbA1C levels at a fixed point

in pregnancy were matched with their corresponding levels at a subsequent time

point. Missing values were excluded and paired t-tests were performed on normally

distributed data.

2.16.3 Statistical analysis of myography data

In the myography studies, “N” refers to the number of subjects or animals studied

and “n” indicates the number of arteries analysed.

2.16.3.1 Constriction

Paired t-tests (two-tailed) were used to assess constriction before and after changes

in glucose concentration.


Repeated measures ANOVA was used to compare relaxation in the same artery after

changing the glucose concentration. Ordinary two-way ANOVA was used to

calculate differences in endothelium-dependent relaxation when comparing

relaxation between groups.

99

CHAPTER 3

The glucose levels of women with

diabetes in pregnancy

100

THE GLUCOSE LEVELS OF WOMEN WITH

DIABETES IN PREGNANCY

3.1 Introduction

The glucose levels of women with diabetes in pregnancy play an important role in

determining the outcome of pregnancy. Karlsson and Kjellmer were among the first

to demonstrate the relationship between fetal outcome and glucose control, showing

that the third-trimester mean maternal blood glucose was linearly correlated with the

perinatal mortality rate [Karlsson and Kjellmer, 1972]. Improved control of diabetes

has been associated with a reduction in the perinatal complication rate [DCCT

Group, 1996; Drexel et al., 1988].

Studies examining the control of diabetes in pregnancy are often subject to

important limitations. Instead of examining glucose levels, most of these studies

have taken HbA1C as the marker for glycaemic control [DCCT Group, 1996], the

limitations of which have been discussed in Section 1.11. Furthermore, HbA1C has

been demonstrated to be a poor predictor of adverse outcome in pregnant women

with diabetes [Brustman et al., 1987; Evers et al., 2004]. Studies on diabetes in

pregnancy have often focused on a particular class: type 1, type 2 or gestational

diabetes. As a result, variations in glucose levels between the three types of diabetes

have not been adequately characterised. The effect of hypoglycaemia during

diabetes in pregnancy is not as well defined as that of hyperglycaemia, as research

in this field has concentrated on high levels of glucose.

As discussed in Section 1.9.7, conflicting results have been obtained in studies

examining the effect of hyperglycaemia on endothelial function. Some of these

studies were based on theoretical rather than empirical values of hyperglycaemia.

This has led to investigations at levels as high as 44 mmol/L glucose [Cipolla et al.,

1997], which is not seen clinically. Furthermore, although hypoglycaemia is

frequently seen in patients on insulin during pregnancy (see Section 1.10.1), its

frequency has not been adequately characterised. It is therefore important to define

clinically relevant glucose levels in pregnant women with diabetes before examining

its effects on endothelial function.

101

3.2 Aims

The main aim of this section was to define a clinically relevant range for

blood glucose levels in women undergoing treatment at a tertiary centre for

diabetes in pregnancy.

Secondary aims include:

a) Retrospectively examine glucose levels and HbA1C values at fixed points

through pregnancy in women with type 1, type 2 and gestational diabetes.

b) Examine diabetes control in women with normal and adverse outcome in

pregnancy.

3.3 Methodology

Ethical approval was obtained from the audit department of the Central Manchester

NHS Trust. The study was instigated in discussion with Dr. Moulinath Banerjee

(Department of Medicine, Manchester Royal Infirmary). Pregnancy records of

patients with type 1, type 2 and gestational diabetes who had undergone treatment at

the diabetes ante-natal clinic of St.Mary’s Hospital, Manchester during the period

2001-2006 were retrospectively analysed. It was envisaged to include a time-

matched group of healthy pregnant women for comparison. However, glucose levels

in healthy pregnant women were found to be measured infrequently, which

precluded further study of this group. All case note evaluation and data collection

were performed by the author. The results were collated by the audit department of

the Central Manchester NHS Trust. Statistical advice was obtained from Professor

Graham Dunn (Professor of Biomedical Statistics, University of Manchester) on

analysis of the data, which was performed by the author.

102

3.3.1 Inclusion criteria:

1) Patients with a proven diagnosis of diabetes

a) Documented glucose intolerance:

Positive GTT or

Fasting blood glucose ≥ 7.0 mmol/L on at least two separate

occasions or

Random blood glucose readings ≥ 11.1 mmol/L on at least two

separate occasions.

b) Patients with type 1 or 2 diabetes diagnosed before pregnancy.

c) Patients with no previous diabetes but found to have gestational diabetes with

documented glucose intolerance (as above) during pregnancy.

2) Patients with a clearly classified type of diabetes

a) Patients classified as either type 1 or type 2 before pregnancy

b) Patients with no diabetes prior to pregnancy, but classified as having gestational

diabetes during pregnancy with documented glucose intolerance.

3.3.2 Exclusion criteria:

a) Patients being monitored for marginally elevated glucose levels but not having

diabetes.

b) Patients with risk factors for diabetes (such as previous large for gestational age

baby) but not having blood glucose levels in the diabetic range.

c) Patients considered to have gestational diabetes, but without documented

glucose intolerance.

d) Patients who were diagnosed to have gestational diabetes in a previous

pregnancy, but without documentation of normal glucose tolerance test in the

subsequent post-natal period.

e) Patients where there was uncertainty regarding the type of diabetes.

f) Patients on medication during pregnancy, such as heparin. Unfractioned heparin

has been demonstrated to have vasoactive properties such as causing

endothelium-dependent vasodilation [Tasatargil et al., 2005].

103

Patients diagnosed to have gestational diabetes, but subsequently found to have

a positive glucose tolerance test in the post-natal period were re-classified as

having type 2 diabetes.

3.3.3 Data collection

Pregnant women whose expected date of delivery fell between January 2001 and

March 2006 were included in the study. 131 case notes of patients were randomly

selected, of which 111 fulfilled the inclusion criteria. Blood glucose readings

recorded during each clinic appointment were analysed. Readings obtained at the

following time points were recorded: pre-conception, first documented value at the

start of pregnancy, 10 ± 1 week, 20 ± 1 week, 34 ± 1 week and the last recorded

reading during pregnancy. The blood glucose readings represented the maximum

and minimum values obtained by the patient (from self-monitoring of blood

glucose) in the week preceding the clinic appointment. In patients with gestational

diabetes, the first blood glucose reading represented the initial value after the

diagnosis of GDM had been confirmed. The diagnosis of GDM was made between

22 and 32 weeks of gestation in the patients studied.

3.3.4 Statistical analysis

Data was analysed as discussed previously in Section 2.16.1 and 2.16.2.

3.4 Results

3.4.1 Clinical characteristics

A summary of clinical characteristics is given in Table 3.1. Parameters where a

significant difference was noted between groups have been described below. Data

has been expressed as mean ± SEM.

104

Parameters Type 1

diabetes

Type 2

diabetes

Gestational

diabetes

Number of Patients 44 16 51

Age at delivery (years) 30.5 ± 0.8 34.5 ± 1.1 33.6 ± 0.7

Ethnicity Caucasian

Asian Afro-Caribbean

Unspecified

37 3 1 3

4 8 4 0

9 31 9 2

BMI (kg/m2) 26.6 ± 0.8 30.9 ± 1.9 30.3 ± 0.9

Treatment Insulin

Diet Alone

44 0

16 0

30 21

Vascular Complications

Retinopathy Nephropathy Neuropathy

Ischaemic Heart Disease

5 3 0 1

0 0 0 0

0 0 0 0

Mean Gestation (weeks)

37.4 ± 0.2 38.3 ± 0.3 38.7 ± 0.2

Mode of Delivery Normal Vaginal Assisted Vaginal

Elective Caesarean Emergency Caesarean

Unknown

16 (38%) 6 (14.2 %) 7 (16.6 %)

11 (26.1 %) 2 (4.7 %)

4 (33.3%) 1 (8.3%) 3 (25%)

4 (33.3%) 0

29 (56.8%) 7 (13.7%) 6 (11.7%) 8 (15.6%) 1 (1.9%)

Termination of Pregnancy

2 1 0

Mean birth weight (g) 3511 ± 94 3499 ± 250 3575 ± 86

Birth weight > 4000 g 9 (20%) 3 (19%) 14 (27%)

Adverse Outcome Stillbirth

SCBU admission > 24 hrs

Early Pregnancy Loss

1

11 0

0

1 4

0

4 0

Congenital Malformations

4 1 1

Table 3.1: Clinical characteristics

Data represented as mean ± SEM. BMI was based on weight at initial ante-natal

clinic booking.

105

Patient Numbers:

The numbers of patients studied in the type 1, type 2 and gestational diabetes group

were 44, 16 and 51 respectively.

Age:

The age at delivery of patients with type 1 diabetes was significantly lower (30.5 ±

0.8 years) compared to both type 2 and gestational diabetes (one-way ANOVA,

Bonferroni post test: p < 0.05).

Ethnicity:

Most type 1 patients were Caucasian, whereas most patients with gestational

diabetes were Asian. Ethnicity was unspecified in five patients.

Body Mass Index (BMI):

The BMI was based on the weight at the initial ante-natal clinic booking. BMI was

significantly lower in type 1 diabetes (26.6 ± 0.8) compared to gestational diabetes

(30.3 ± 0.9) (one-way ANOVA, Bonferroni post test: p < 0.05).

Treatment:

Most patients with gestational diabetes (30 out of 51) and all women with type 1 and

2 diabetes were treated with insulin during pregnancy.

Vascular complications:

Vascular complications were noticed only in patients with type 1 diabetes. Three

patients had pre-existing retinopathy, of which one worsened during pregnancy.

Two had new onset retinopathy diagnosed during pregnancy. Proteinuria was

present in one patient with type 1 diabetes and microalbuminuria in a further two;

all of which had preceded pregnancy and did not worsen subsequently. One patient

with type 1 diabetes had pre-existing ischaemic heart disease.

Gestational Age:

The mean gestation period was significantly lower in women with type 1 diabetes

(37.4 ± 0.2 weeks) compared to gestational diabetes (38.7 ± 0.2 weeks) (one-way

ANOVA, Bonferroni post test: p < 0.001).

Mode of delivery:

The pre-dominant mode of delivery was normal vaginal in type 1 (38%) and

gestational diabetes (56.8%). In type 2 diabetes, emergency Caesarean section was

as common as normal vaginal delivery (33.3% each). In all groups, emergency

Caesarean deliveries were higher than elective Caesarean deliveries. The mode of

106

delivery was unknown in 3 pregnancies due to a change in residence or transfer of

care with loss of follow-up.

Termination of pregnancy:

Termination of pregnancy was performed in one woman with type 2 diabetes due to

severe congenital defects (cerebral ventricle defects). The reason for termination

was not specified in two patients with type 1 diabetes.

Birthweight:

No significant difference was noticed in birth weight between patients with type 1

(3511 ± 94 g), type 2 (3499 ± 250 g) or gestational diabetes (3575 ± 86 g) (one-way

ANOVA, Bonferroni post test: p > 0.05). However, women with GDM were noted

to have the highest rate of babies born with a birth weight greater than 4000 g.

Congenital Malformations:

These included two cases of ventricular septal defect, one case of ventricular septal

hypertrophy and one case of Down’s syndrome in patients with type 1 diabetes. A

case of cerebral ventricle defect in type 2 diabetes and a case of ventricular

hypertrophy was noted in gestational diabetes.

3.4.2 Glucose levels

The analysis of 792 glucose readings recorded from women with type 1, type 2 and

gestational diabetes is summarised in Table 2. As expected, more variability was

seen among maximum values than minimum values. The overall range of values

was 1 to 28 mmol/L glucose. The lower quartile for minimum glucose levels was 3

mmol/L glucose whereas the upper quartile for maximum glucose levels was 11

mmol/L glucose. Mean ± SD values for minimum glucose levels were 4.0 ± 1.5 and

for maximum glucose levels 9.7 ± 3.4 mmol/L. These results indicate that most

glucose levels were in the range of 2 to 12 mmol/L glucose.

107

Parameters Minimum Glucose

(mmol/L)

Maximum Glucose

(mmol/L)

Number of values 396 396

Minimum 1 5

Lower Quartile < 3 < 7.2

Median 4 9

Upper Quartile > 4.7 > 11

Maximum 12 28

Mean ± SD 4.0 ± 1.5 9.7 ± 3.4

Table 3.2: Range of glucose levels seen in diabetes in pregnancy

Collective glucose levels of women with type 1, type 2 and gestational diabetes.

3.4.3 Glucose levels of type 1 diabetes in pregnancy

Glucose readings were plotted from the pre-conception period to the end of

pregnancy in 44 women with type 1 diabetes (Fig. 3.1). Narrower ranges of

minimum and maximum glucose levels with less hyperglycaemia were seen as

pregnancy progressed; indicating improved glycaemic control. The mean minimum

and maximum pre-conception values were 4.3 and 13.1 mmol/L respectively. These

values improved to 3.1 and 8.8 mmol/L respectively for the last recorded value in

pregnancy. The very high levels of hyperglycaemia seen at the beginning of

pregnancy (23, 26 and 28 mmol/L glucose) were absent in the last recorded

readings, where the highest reading was 14 mmol/L. Paired maximum glucose

levels from women at week 10 were significantly higher than corresponding values

at week 34 (paired t-test, mean difference = 2.88, 95% confidence interval = 1.32 to

4.44, p < 0.001).

108

3.4.4 Glucose levels of type 2 diabetes in pregnancy

In 16 women with type 2 diabetes, glucose readings were plotted from the pre-

conception period to the end of pregnancy (Fig. 3.2). The mean minimum and

maximum pre-conception values were 5.1 and 11.3 mmol/L, although these readings

could be obtained from only 3 patients in the group. The mean first recorded lowest

and highest readings in pregnancy were 6.0 and 9.6 mmol/L whereas the

cooresponding values at the end of pregnancy were 4.5 and 9.0 respectively. The

very high glucose readings noticed in type 1 patients were absent in type 2 patients

during pregnancy. In contrast to type 1 diabetes, no significant difference was seen

between paired glucose values in women at week 10 and week 34, (paired t-test, p >

0.05).

3.4.5 Glucose levels of gestational diabetes

Glucose readings were examined from the time of diagnosis to the end of pregnancy

in 51 women with gestational diabetes (Fig. 3.3). The diagnosis of gestational

diabetes was made at a mean ± SEM of 29.4 ± 1.4 weeks with an interquartile range

of 28 to 30.5 weeks. Therefore, the first recorded readings in this group were taken

later in pregnancy compared to the first trimester readings in type 1 and 2 diabetes.

The mean first-recorded minimum and maximum blood glucose values were 4.6 and

8.9 mmol/L, measured at a mean of 29 ± 1 weeks. Mean minimum and maximum

glucose levels at the end of pregnancy were 3.8 and 8.2 mmol/L. Paired maximum

glucose levels at the time of diagnosis were not significantly different from those

recorded at the end of pregnancy (paired t-test, p > 0.05).

109

Fig 3.1 Glucose Levels: Type 1 Diabetes

Pre

-Con M

in

Pre

-Con M

ax

First M

in

First M

ax

Wee

k 10

Min

Wee

k 10

Max

Wee

k 20

Min

Wee

k 20

Max

Wee

k 34

Min

Wee

k 34

Max

Last M

in

Last M

ax

0

5

10

15

20

25

30

* p < 0.01G

luco

se m

mo

l/L

Fig 3.1: Glucose levels: type 1 diabetes

Maximum and minimum glucose levels in patients with type 1 diabetes (N = 44) at

different time points of pregnancy. Maximum values indicated by green dots,

minimum values by blue dots and mean values by horizontal red bars.

Pre-Con: Pre-conception values

First: First recorded values in pregnancy

Last: Last recorded values before delivery

110

Fig 3.2 Glucose Levels: Type 2 Diabetes

Pre

-Con M

in

Pre

-Con M

ax

First M

in

First M

ax

Wee

k 10

Min

Wee

k 10

Max

Wee

k 20

Min

Wee

k 20

Max

Wee

k 34

Min

Wee

k 34

Max

Last M

in

Last M

ax

0

5

10

15

20

25

30

Glu

co

se m

mo

l/L

Fig 3.2: Glucose levels: type 2 diabetes

Maximum and minimum glucose levels in patients with type 2 diabetes (N = 16) at

different time points of pregnancy. Maximum values indicated by green dots,





111

Fig 3.3 Glucose Levels: Gestational Diabetes

First M

in

First M

ax

Wee

k 34

Min

Wee

k 34

Max

Last M

in

Last M

ax

0

5

10

15

20

25

30

Glu

co

se m

mo

l/L

Fig 3.3: Glucose levels: gestational diabetes

Maximum and minimum glucose levels in patients with gestational diabetes (N =

51) at different time points in pregnancy. Maximum values indicated by green dots,


First: First recorded values in pregnancy (28 to 30.5 weeks interquartile range)


112

3.4.6 HbA1C levels: comparison across types

The first recorded HbA1C levels represent first trimester levels in type 1 and 2

diabetes, but second or third trimester levels in gestational diabetes, depending on

the time of diagnosis. Therefore only the 34th week values can be considered to be

time-matched across all three groups. There was no significant difference in HbA1C

values between the three groups at week 34 (one-way ANOVA, Bonferroni post

test: p > 0.05).

3.4.7 HbA1C levels in each group

3.4.7.1 Type 1 diabetes

Paired data from women at week 10 and week 34 were compared. HbA1C levels

were significantly higher at week 10 compared to week 34 (Fig. 3.4, paired t-test,

mean difference = 1.56, 95% confidence interval = 0.79 to 2.34, p < 0.001).

3.4.7.2 Type 2 diabetes

Paired results from women at week 10 and week 34 showed no significant

improvement in HbA1C levels (Fig. 3.5, paired t-test, p > 0.05).


In women with gestational diabetes, no significant difference was noted on

comparing their first recorded HbA1C with the corresponding final values (Fig. 3.6,

paired t-test, p > 0.05).

113

Fig 3.4 HbA1c: Type 1 Diabetes

Pre-Con First Week 10 Week 20 Week 34 Last4

5

6

7

8

9

10

11

12

* p < 0.001

Time of Estimation

Hb

A1C

%

Fig 3.4: HbA1C: type 1 diabetes

HbA1C levels in type 1 diabetes at different time points, from pre-conception to end

of pregnancy. Horizontal lines represent median values.




Fig 3.5 HbA1c: Type 2 Diabetes

First Week 10 Week 20 Week 34 Last4

5

6

7

8

9

10

11

12

Time of Estimation

Hb

A1C %

Fig 3.5: HbA1C: type 2 diabetes

HbA1C levels at different time points during pregnancy in type 2 diabetes.

Horizontal lines represent median values.



114

Fig 3.6 HbA1c: Gestational Diabetes

First Week 34 Last4

5

6

7

8

9

10

11

12

Time of Estimation

Hb

A1C

%

Fig 3.6: HbA1C: gestational diabetes

HbA1C levels at different time points during pregnancy in gestational diabetes.

Horizontal lines represent median values.



3.4.8 Glucose levels in GDM: effect of insulin

Of a total of 51 patients, 21 were diet-controlled and 30 were treated with insulin.

Diet-controlled: Paired maximum blood glucose levels measured at the time of

diagnosis of GDM (first maximum) were compared with that at the end of

pregnancy (last maximum). No significant difference was seen between the two

glucose levels. (Fig. 3.7, paired t-test, p > 0.05).

Insulin-controlled: In this group, the first glucose levels measured at the time of

diagnosis of GDM represented values before the start of insulin. As expected, this

first set of maximum glucose values was significantly higher than the last set, where

patients were on insulin (paired t-test, mean difference = 1.45, 95% confidence

interval = 0.32 to 2.59, p < 0.05). Mean ± SD of first and last maximum glucose

levels were 10.0 ± 2.1 and 8.5 ± 1.9 mmol/L.

115

Fig 3.7 GDM: Diet-controlled

First

Min

.

First

Max

.

34 W

eek

Min

.

34 W

eek

Max

.

Last M

in.

Last M

ax.

0

5

10

15

20

Time of Estimation

Glu

co

se

mm

ol/L

Fig 3.8 GDM: Insulin-controlled

First

Min

.

First

Max

.

34 W

eek

Min

.

34 W

eek

Max

.

Last M

in.

Last M

ax.

0

5

10

15

20 * p < 0.05

Time of Estimation

Glu

co

se

mm

ol/L

Fig 3.7 & 3.8: Treatment and glucose levels in GDM

Glucose levels in patients with diet-controlled (N=21, fig 3.7) and insulin-controlled

(N=30, fig 3.8) gestational diabetes at different time points in pregnancy. Red

horizontal bars represent mean values.



116

3.4.9 Glucose levels and outcomes in type 1 diabetes:

Adverse outcome was defined as stillbirth or special care baby unit (SCBU)

admission for greater than 24 hours. In both normal outcome (N=32) and adverse

outcome (N=12) groups, paired maximum glucose values were significantly higher

at week 10 compared to week 34 (paired t-test, p < 0.05).

3.4.10 HbA1C and outcomes in type 1 diabetes:

In patients with a normal outcome, as well as those with an adverse outcome, a

significant reduction in mean HbA1C levels was seen between 10 and 34 weeks

(paired t-test, p < 0.05).

117

Fig 3.9 Type 1 Diabetes: Normal Outcome

First M

in

First M

ax

Wee

k 10

Min

Wee

k 10

Max

Wee

k 20

Min

Wee

k 20

Max

Wee

k 34

Min

Wee

k 34

Max

Last M

in

Last M

ax

0

5

10

15

20

25

30 * p < 0.05

Glu

co

se m

mo

l/L

Fig 3.10 Type 1 Diabetes: Adverse Outcome

First M

in

First M

ax

Wee

k 10

Min

Wee

k 10

Max

Wee

k 20

Min

Wee

k 20

Max

Wee

k 34

Min

Wee

k 34

Max

Last M

in

Last M

ax

0

5

10

15

20

25

30

* p < 0.05

Glu

co

se m

mo

l/L

Fig 3.9 & 3.10: Adverse outcome and glucose levels in type 1 diabetes

Blood glucose levels in patients with type 1 diabetes who had a normal (N=32, fig

3.9) and an adverse (N=12, Fig 3.10) pregnancy outcome. Maximum values

indicated by green dots, minimum values by blue dots and mean values by

horizontal red bars.



118

Fig 3.11 Type 1 Diabetes: Normal Outcome

First Week 10 Week 20 Week 344

5

6

7

8

9

10

11

12 * p < 0.01

Time of Estimation

Hb

A1C %

Fig 3.12 Type 1 Diabetes: Adverse Outcome

First Week 10 Week 20 Week 344

5

6

7

8

9

10

11

12

* p < 0.01

Time of Estimation

Hb

A1C %

Fig 3.11 & 3.12: Outcomes and HbA1C in type 1 diabetes

HbA1C levels in patients with type 1 diabetes who had a normal (N=32, Fig 3.11)

and an adverse (N=12, Fig 3.12) pregnancy outcome.


119

3.5 Discussion

3.5.1 Glucose levels of women with diabetes in pregnancy

The main aim of this section was to identify a clinically relevant range for glucose

values in women with diabetes complicating pregnancy. These results indicate that

such a range would be between 2 and 12 mmol/L glucose. This range is markedly

lower than the glucose levels that have been used to study vascular function in the

past.

This section has demonstrated that women with type 1 diabetes in pregnancy have

wide fluctuations in their blood glucose level (between 1 and 28 mmol/L) even

when treated at a tertiary care centre. In comparison, a narrower range of glucose

levels was seen in women with type 2 diabetes (2 to18 mmol/L) and gestational

diabetes (2 to17 mmol/L). The lack of ante-natal blood glucose measurements in

healthy pregnant women prevented comparison with a non-diabetic group.

However, as described in Section 1.8, previous studies have reported a range of 3 to

6.8 mmol/L glucose in healthy pregnant women. A small rise in mean blood glucose

values in the third trimester due to increasing insulin resistance has been observed in

healthy pregnant women [Parretti et al., 2001]. However, in the present study, a

similar rise in glucose levels towards the end of pregnancy was not seen in the

diabetic patients. This was probably due to insulin treatment and also because only

the range of glucose levels was recorded.

The results of this section demonstrate heterogeneity in glucose levels between the

three types of diabetes in pregnancy. In the group studied, type 1 diabetes patients

had a glycaemic profile distinct from both type 2 and gestational diabetes. Type 1

diabetes was characterised by extremes of hyperglycaemia and hypoglycaemia,

which were absent in type 2 and gestational diabetes. This can be attributed to the

relative lack of endogenous insulin production in type 1 diabetes and its

inappropriate replacement with insulin therapy; although other mechanisms such as

a deficient glucagon and epinephrine response may also play a role [Cryer, 1991].

120

3.5.2 Glucose levels in type 1 diabetes complicating pregnancy

Patients with type 1 diabetes had the poorest glycaemic control, with recurrent

episodes of hypo- and hyperglycaemia. The hypoglycaemic episodes frequently

reached the level of 2 mmol/L, although the lowest value obtained was 1.1 mmol/L.

Hyperglycaemia was more frequent in patients with type 1 diabetes, although the

values improved as pregnancy progressed. This resulted in a narrower mean range

of glucose levels towards the end of pregnancy compared to the beginning. Using

the continuous glucose monitoring system (CGMS), high variability in glucose

levels have been previously demonstrated in pregnant women with type 1 diabetes

[Kerssen et al., 2004].

3.5.3 Glucose levels in type 2 diabetes complicating pregnancy

Patients with type 2 diabetes had lower levels of hyperglycaemia compared to type 1

diabetes, resulting in a narrower range of glucose levels throughout pregnancy. In

contrast to type 1 patients, no significant improvement in hyperglycaemia occurred

as pregnancy progressed. Hypoglycaemic levels of 2 mmol/L were also seen in

patients with type 2 diabetes.

3.5.4 Glucose levels in gestational diabetes

Of the 51 patients with gestational diabetes, 30 patients were treated with insulin

and 21 patients by dietary measures alone. All newly diagnosed GDM patients were

on diet control initially and started on insulin if subsequent blood glucose levels

remained high. This explains the small but significant lowering of hyperglycaemia

in the last readings compared to the first in women treated with insulin. Insulin

requirement has been associated with the time of diagnosis of GDM; women

diagnosed earlier being more likely to require insulin than those diagnosed later on

in their pregnancy [Svare et al., 2001].

3.5.5 Adverse outcomes

Adverse outcome was defined as stillbirth or admission in the special care baby unit

(SCBU) for greater than 24 hours. Reasons for admission included neonatal

hypoglycaemia, jaundice, surfactant-deficient lung disease, low Apgar score and

congenital defects.

121

3.5.5.1 Type 1 and 2 diabetes

27% of patients with type 1 diabetes had an adverse outcome compared to 31% of

patients with type 2 diabetes. Although glucose levels improved throughout

pregnancy in women with type 1 diabetes, adverse pregnancy outcomes were still

high in this group. In women with type 1 diabetes, high levels of HbA1C early in

pregnancy have been associated with a raised fetal malformation rate in numerous

studies [Hanson et al., 1990; Nielsen et al., 1997; Temple et al., 2002; Ylinen et al.,

1984].

In addition to elevated glucose levels in women with type 1 diabetes, this group also

had the greatest degree of fluctuations between high and low blood glucose levels.

However, it is unknown if these fluctuations contribute to the complications of

diabetes in pregnancy. When women with normal and adverse outcome were

compared, the glucose and HbA1C levels demonstrated a significant drop from week

10 to week 34 in both groups. However, the relatively small number of patients

studied prevented statistical or epidemiological conclusions from being reached.

Adverse outcomes in pregnancy have been previously compared between type 1 and

2 diabetes. A large study examining 389 type 1 and 146 type 2 diabetes patients

demonstrated a higher rate of serious adverse pregnancy outcome in type 2

compared to type 1 diabetes [Roland et al., 2005]. However, a larger study with

2359 patients has recently reported similar prevalence of congenital anomalies and

perinatal mortality in patients with type 1 and 2 diabetes [Macintosh et al., 2006].

Comparing outcome measures between studies is often challenging due to

differences in the level of care (both pre-conception and ante-natal), ethnicity and

the socio-economic background of patients. The Diabetes Control and

Complications Trial has reported that in pregnant women with type 1 diabetes,

intensive control of glucose levels was associated with lowering of the congenital

malformation rate to normal levels [DCCT Group, 1996]. As a corollary to this,

daily self-monitoring of glucose levels in patients with type 1 diabetes has been

associated with a reduction in adverse outcomes [Jensen et al., 2004].

122


In this study, patients with GDM had the lowest rate of adverse pregnancy outcome

(7.8%). Additionally, these women had more stable glucose levels than women with

type 1 diabetes. Unlike type 1 and 2 diabetes, considerable debate exists regarding

whether better control of blood glucose improves pregnancy outcome in gestational

diabetes and Impaired Glucose Tolerance (IGT). It has been demonstrated that

dietary advice, blood glucose monitoring, and insulin therapy (as required) in

women with gestational diabetes reduced the rate of serious perinatal morbidity, but

increased the rate of admission to the special care baby unit [Crowther et al., 2005].

An important limitation of this study by Crowther et al. was that no assessment was

made of the actual glucose levels attained by GDM patients in the treated group. In

contrast to type 1 diabetes [Jensen et al., 2004], self-monitoring of blood glucose

has not been associated with improved outcome in gestational diabetes [Homko et

al., 2002]. A study has shown no adverse outcome for impaired glucose tolerance

(defined as a fasting plasma glucose between 6.1 and 7 mmol/L), but increased

macrosomia in GDM [Nordin et al., 2006]. However, a larger study in China has

found an association between IGT and high fetal birth weight [Yang et al., 2002]. It

is hoped that the ongoing HAPO study will clarify the association between

hyperglycaemia in GDM and adverse pregnancy outcome [HAPO Group, 2002].

3.5.6 HbA1C and glucose levels

In type 1 diabetes, HbA1C levels improved as pregnancy progressed; reflecting a

similar trend in blood glucose levels. As described previously, raised HbA1C values

in early pregnancy may play an important role in the pathogenesis of complications

in pregnancy. HbA1C has been used as the surrogate marker for assessing blood

glucose levels in the vast majority of epidemiological studies in diabetes. An

important reason for its predominance in the literature is its reproducibility and ease

of analysis compared to collating individual blood glucose levels. This also enables

cross- comparisons between studies. However, its importance in the field of diabetes

in pregnancy is less certain. In the current study, a significant drop was noticed

between 10 and 34 weeks in type 1 patients with both a normal and adverse outcome

in pregnancy.

123

In contrast to type 1 diabetes, no improvement in HbA1C levels was noted in women

with type 2 and gestational diabetes on performing matched comparisons during

pregnancy. A lack of improvement was also seen in the maximum blood glucose

levels of these two groups.

3.5.7 Limitations of study

The relatively small number of patients examined (compared with population

studies) prevents associations from being drawn between the parameters measured

and pregnancy outcome. Furthermore, maximum and minimum glucose levels were

assessed, which do not represent typical blood glucose levels in these patients.

However, the main aim of this study was to identify a clinically relevant range of

glucose levels to guide subsequent analysis of vascular function in pregnancy. The

majority of studies have used HbA1C as a surrogate marker for actual blood glucose

levels, necessitating the present investigation of glucose levels in a representative

study population. Larger epidemiological studies measuring actual glucose levels

(as opposed to HbA1C) are required to examine a possible link between diurnal

variations in blood glucose levels and pregnancy outcome.

Although this study has identified a clinically relevant range of glucose levels in

women with diabetes in pregnancy, the duration of these aberrant glucose levels

could not be assessed due to limitations of the SMBG method. The recently

developed continuous glucose monitoring system (CGMS) has distinct advantages

over SMBG in measuring the time span of these abnormal glucose levels. Using

CGMS, Buhling et al. demonstrated the mean duration of hyperglycaemia (±

standard deviation) above 8.9 mmol/L/24 h to be 9.3 ± 25 minutes in healthy non-

pregnant women, 7.5 ± 14 minutes in healthy pregnant women and 14 ± 21 minutes

in GDM [Buhling et al., 2004].

3.5.8 SUMMARY

Diabetes in pregnancy, despite being treated at a tertiary centre, was associated with

aberrant blood glucose levels, which were more marked in type 1 diabetes. A high

rate of adverse pregnancy outcome was noticed in patients with type 1 diabetes, who

124

also had the greatest fluctuation in glucose levels. The lower quartile of minimum

glucose levels was 3 mmol/L, whereas the upper quartile of the maximum glucose

levels was 11 mmol/L. These findings suggest that clinically-relevant glucose

concentrations range from 2 to 12 mmol/L glucose in diabetes complicating

pregnancy. Identifying this range provided a basis for all subsequent experiments

investigating the effect of glucose levels on endothelial function in pregnancy.

125

CHAPTER 4

The effect of glucose on endothelial

function in healthy non-pregnant and

pregnant women

126

THE EFFECT OF GLUCOSE ON ENDOTHELIAL FUNCTION IN HEALTHY NON-PREGNANT AND PREGNANT WOMEN

4.1 Introduction

The previous chapter demonstrated fluctuating levels of both hypoglycaemia and

hyperglycaemia in women with diabetes in pregnancy. As discussed in Section

1.9.7, studies investigating hyperglycaemia have yielded conflicting results, which

may be attributable to differences in the degree and duration of hyperglycaemia,

agonists used, techniques employed and vascular beds studied. Therefore, the ideal

method to investigate the effects of glucose on endothelial function would be to

study the effect of a fixed number of glucose levels using a single technique on a

variety of vascular beds. This would eliminate some ambiguity in this area of

research by allowing comparison of effects between vascular beds at particular

levels of glucose.

Clinically relevant glucose levels in women with diabetes in pregnancy have been

identified in the preceding section. In healthy pregnant women, levels have been

demonstrated to vary approximately between 3 to 6.8 mmol/L glucose (see Section

1.8). Using the new technique of continuous glucose monitoring system (CGMS),

Buhling et al. recently illustrated that the prevalence of hyperglycaemia in healthy

pregnant women was higher than previously reported, with levels greater than 8.9

mmol/L seen for short periods of 7.5 ± 14 minutes (mean ± standard deviation, over

24 hours) [Buhling et al., 2004]. In non-pregnant healthy individuals, similar levels

of hyperglycaemia lasted for 9.3 ± 25 minutes. These novel findings signify that

marked fluctuations in blood glucose level are seen even in normal pregnancy.

Myometrial arteries of the uterus play a vital role in facilitating blood flow to the

growing fetus. However, the effects of the aforementioned glucose levels on these

vessels are unknown in both normal and diabetic women. Furthermore, it is unclear

whether the state of pregnancy or the agonist used for vasoconstriction influences

the response to altered glucose levels. It is therefore necessary to characterise

127

vascular responses in myometrial arteries from both healthy pregnant and non-

pregnant women prior to examining these effects in women with diabetes in

pregnancy.

In this study, the effects of four clinically relevant glucose concentrations (2, 5, 8

and 12 mmol/L) were studied for a single duration of time (30 minutes), using wire

myography with two vasoconstrictors (60 mmol/L potassium and U46619).

Constriction and endothelium-dependent relaxation were evaluated in myometrial

arteries isolated from both healthy pregnant and non-pregnant women.

4.2 Aims

1. To examine the effect of a 30 minute exposure to 2, 5, 8 and 12 mmol/L

glucose on maximal constriction in myometrial arteries.

2. To examine the effect of a 30 minute exposure to 2, 5, 8 and 12 mmol/L

glucose on endothelium-dependent relaxation in myometrial arteries.

3. To study whether any effect is agonist-dependent by using two different

vasoconstrictors:

A) 60 mmol/L KCl

B) U46619

4. To study whether any effect is pregnancy-related by performing these

studies in both non-pregnant and pregnant women.

5. To investigate the effects of a prolonged exposure of 2, 5, 8 and 12 mmol/L

glucose lasting 2 hours on constriction and relaxation.

4.3 Materials and methods

These have been discussed in Chapter 2. The experimental protocols used were

Protocol 1 and 2 (see Section 2.12.1 and 2.12.2). Arteries with maximum

128

constriction less than 1 mN/mm were excluded from analysis due to poor

constriction.

4.4 Results

4.4.1 Clinical Characteristics

A summary of the clinical characteristics of healthy pregnant and non-pregnant

women are given in Table 4.1. Subjects were divided into normal non-pregnant

(N=15) and normal pregnant (N=30) groups. The normal pregnant group was further

subdivided into the KCl group (N=16) and the U46619 group (N=14) depending on

the vasoconstrictor used during experimentation. Patients were allocated these

groups randomly. The BMI was based on the weight at the initial ante-natal clinic

booking. Women in the non-pregnant group were significantly older and had a

higher BMI than those in both pregnant KCl and U46619 groups (one-way

ANOVA: Bonferroni post test p < 0.001). However, pregnant women in the KCl

and U46619 groups had similar ages and BMI (one-way ANOVA: Bonferroni post

test p > 0.05). The BMI of the non-pregnant women was in the obese range (> 30

kg/m2) and significantly higher than that of the pregnant women (one-way ANOVA:

Bonferroni post test p < 0.05). The BMI of the healthy pregnant women was in the

overweight range (25-30 kg/m2). Both gestational age at delivery and birthweight of

the offspring were similar in the two groups of pregnant women (unpaired t-test, p >

0.05). In all groups, the blood pressure was in the normal range.

129

PARAMETERS

NORMAL NON-

PREGNANT WOMEN (N = 15)

NORMAL PREGNANT

WOMEN: KCL GROUP

(N = 16)

NORMAL PREGNANT

WOMEN: U46619 GROUP (N = 14)

Age (years) 43.0 ± 2.4 32.7 ± 1.6 30.6 ± 1.1

BMI (kg/m2) 34.4 ± 3.4 26.6 ± 1.0 26.6 ± 1.3

Max. Systolic BP (mm Hg)

120 ± 9 122 ± 2 120 ± 2

Max. Diastolic BP (mm Hg)

77 ± 5 73 ± 2 70 ± 2

Gravidity 2 ± 0.4 2.3 ± 0.3 2.2 ± 0.2

Parity 1.7 ± 0.4 0.9 ± 0.2 0.9 ± 0.2

Gestational Age at Delivery (weeks)

- 38.8 ± 0.2 39.0 ± 0.3

Birth Weight (g) - 3663 ± 130 3546 ± 144

Individualised Birth Ratio (centile)

- 66 ± 9 52 ± 8

Table 4.1: Clinical characteristics of healthy individuals

All data expressed as mean ± SEM. The individual birthweight ratio is a centile

which is dependent on maternal ethnicity, height, weight, parity and gestation at

delivery, in addition to fetal birthweight and sex [Sanderson et al., 1994].

4.4.2 Arterial Diameters

The mean diameter of arteries from non-pregnant women had a diameter (mean ±

SEM) of 426 ± 23m in the KCl group (N=9, 32 arteries) and 392 ± 22m in the

U46619 group (N=6, 22 arteries). No significant difference was noted in the mean

diameters of these two groups (Fig. 4.1; unpaired t-test, mean difference = 34.09 ±

34.01, 95% confidence interval = -34.22 to 102.4, p > 0.05). Arteries from healthy

pregnant women in the KCl group (N= 16, 50 arteries) had a significantly larger

diameter than that in the U46619 group (N=14, 50 arteries) (377 ± 15 m vs. 335 ±

13 m) [Fig. 4.2; unpaired t-test, mean difference = 42.72 ± 20.06, 95% confidence

interval = 2.854 to 82.59, p < 0.05].

130

4.1 Arterial Diameter: Non-pregnant group

KCl U 466190

100

200

300

400

500

600

700

800

900

Vasoconstrictor

Art

eri

al D

iam

ete

r

m

Fig. 4.1: Arterial diameters (non-pregnant group)

Myometrial arteries isolated from healthy non-pregnant women in two

vasoconstrictor groups: 60 mmol/L KCl (circles; N=9, artery segments=32) and

U46619 (triangles; N=6, artery segments =22). Horizontal bars represent mean

values.Unpaired t-test: mean difference = 34.09 ± 34.01, 95% confidence interval =

-34.22 to 102.4

4.2 Arterial Diameter: Pregnant group

KCl U 466190

100

200

300

400

500

600

700

800

900

p < 0.05*

Vasoconstrictor

Art

eri

alD

iam

ete

r

m

Fig. 4.2: Arterial diameters (normal pregnant group)

Myometrial arteries isolated from pregnant women in two vasoconstrictor groups:

60 mmol/L KCl (N=16, artery segments = 50) and U46619 (N=14, artery segments

= 50). Horizontal bars represent mean values. Unpaired t-test: p < 0.05

131


(non-pregnant group - KCl)

Myometrial arteries from healthy non-pregnant women were constricted with 60

mmol/L potassium. No significant difference in active effective pressure was seen at

2, 5, 8 or 12 mmol/L glucose (Fig. 4.3, paired t-test: p > 0.05).


(non-pregnant group - KCl)

No significant difference was noted in endothelium dependent relaxation of

myometrial arteries of non-pregnant women after incubation at 2, 5, 8 and 12

mmol/L glucose for 30 minutes (Fig. 4.4, repeated measures ANOVA: p > 0.05)


(non-pregnant group - U46619)

Myometrial arteries from healthy non-pregnant women were constricted with 10-6

M

U46619. No significant difference in active effective pressure was seen at 2, 5, 8 or

12 mmol/L glucose (Fig. 4.5, paired t-test: p > 0.05).


(non-pregnant group – U46619)

Exposure to glucose levels of 2, 8 and 12 mmol/L for 30 minutes had no effect on

endothelium-dependent relaxation when U46619 was used to constrict the arteries

(Fig. 4.6, repeated measures ANOVA: p > 0.05).

132

4.3A Constriction: KCl5 vs 2 mmol/L Glucose

5 mmol/L 2 mmol/L0

10

20

30

40

Glucose Concentration

Acti

ve E

ffecti

ve P

ressu

re

kP

a

4.3B Constriction: KClControl: 5 mmol/L Glucose

5 mmol/L: Part 1 5 mmol/L: Part 20

10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a

4.3C Constriction: KCl5 vs 8 mmol/L Glucose

5 mmol/L 8 mmol/L 0

10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a

4.3D Constriction: KCl5 vs 12 mmol/L Glucose

5 mmol/L 12 mmol/L0

10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a

Fig. 4.3A-D: Constriction: effect of glucose (non-pregnant group – KCl)

The effect of changes in glucose concentration from 5 mmol/L glucose on active

effective pressure in myometrial arteries from healthy non-pregnant women (N=9).

Vessels constricted with 60 mmol/L KCl. Active effective pressure assessed at:

Fig. 4.3A: 5 and 2 mmol/L glucose (n=7 arteries)

Fig. 4.3B: 5 mmol/L glucose (n=9 arteries)

Fig. 4.3C: 5 and 8 mmol/L glucose (n=8 arteries)

Fig. 4.3D: 5 and 12 mmol/L glucose (n=8 arteries)

Paired t-test: p > 0.05

133

4.4A 5 vs 2 mmol/L Glucose

-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

2 mmol/L Glucose

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on

4.4B Control: 5 mmol/L Glucose

-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose: Part 1


Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on

4.4C 5 vs 8 mmol/L Glucose

-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

8 mmol/L Glucose

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on

4.4D 5 vs 12 mmol/L Glucose

-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

12 mmol/L Glucose

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on

Fig. 4.4A-D: Relaxation: effect of glucose (non-pregnant group – KCl)

Endothelium-dependent relaxation at 5 mmol/L glucose and after 30 minute

incubation at 2, 5, 8, and 12 mmol/L glucose in myometrial arteries from healthy

non-pregnant women (N=9). Vessels constricted with 60 mmol/L potassium and

relaxed with 10-10

to 10-6

M bradykinin. Relaxation assessed at:

Fig. 4.4A: 5 (circles) and 2 (triangles) mmol/L glucose (n=7 arteries)

Fig. 4.4B: 5 mmol/L glucose (circles) (n=9 arteries)

Fig. 4.4C: 5 (circles) and 8 (inverted triangles) mmol/L glucose (n=8 arteries)

Fig. 4.4D: 5 (circles) and 12 (squares) mmol/L glucose (n=8 arteries)

Repeated measures ANOVA: p > 0.05

134

4.5A Constriction: U466195 vs 2 mmol/L Glucose

5 mmol/L 2 mmol/L0

10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a

4.5B Constriction: U46619Control: 5 mmol/L Glucose


10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a

4.5C Constriction: U446195 vs 8 mmol/L Glucose

5 mmol/L 8 mmol/L0

10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a

4.5D Constriction: U466195 vs 12 mmol/L Glucose

5 mmol/L 12 mmol/L0

10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a

Fig. 4.5A-D: Constriction: effect of glucose (non-pregnant group – U46619)


effective pressure in myometrial arteries from healthy non-pregnant women (N=6).

Vessels constricted with 10-6

M U46619. Active effective pressure assessed at:






135


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

2 mmol/L Glucose

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on


-10 -9 -8 -7 -60

25

50

75

100



Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

8 mmol/L Glucose

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

12 mmol/L Glucose

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on

Fig. 4.6A-D: Relaxation: effect of glucose (non-pregnant group – U46619)



non-pregnant women (N=6). Vessels constricted with 10-6

M U46619 and relaxed

with 10-10

to 10-6







136

4.4.7 Constriction: effect of glucose (pregnant group - KCl)

Myometrial arteries from healthy pregnant women were constricted using 60

mmol/L KCl. The active effective pressure was calculated at the peak of

constriction. No significant difference in active effective pressure was seen at 2, 5, 8

or 12 mmol/L glucose (Fig. 4.7, paired t-test: p > 0.05).

4.4.8 KCl - Endothelium-dependent relaxation: effect of glucose

(pregnant group - KCl)

Exposure to glucose levels of 2, 8 and 12 mmol/L for 30 minutes had no effect on

endothelium-dependent relaxation when 60 mmol/L KCl was used to constrict the

arteries (Fig. 4.8, repeated measures ANOVA: p > 0.05).

137


5 mmol/L 2 mmol/L0

10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a

4.7B Constriction: KClControl: 5 mmol/L Glucose


10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a


5 mmol/L 8 mmol/L0

10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a


5 mmol/L 12 mmol/L0

10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a

Fig. 4.7A-D: Constriction: effect of glucose (pregnant group - KCl)


effective pressure in myometrial arteries from healthy pregnant women (N=16).

Vessels constricted with 60 mmol/L KCl. Active effective pressure assessed at:

Fig. 4.7A: 5 and 2 mmol/L glucose (n=13 artery segments)

Fig. 4.7B: 5 mmol/L glucose (n=14 artery segments)

Fig. 4.7C: 5 and 8 mmol/L glucose (n=12 artery segments)

Fig. 4.7D: 5 and 12 mmol/L glucose (n=10 artery segments)


138


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

2 mmol/L Glucose

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on


-10 -9 -8 -7 -60

25

50

75

100



Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

8 mmol/L Glucose

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

12 mmol/L Glucose

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on

Fig. 4.8A-D: Relaxation: effect of glucose (pregnant group - KCl)



pregnant women (N=16). Vessels constricted with 60 mmol/L potassium and

relaxed with 10-10

to 10-6



Fig. 4.8B: 5 mmol/L glucose (circles)(n=15 arteries)




139

4.4.9 Constriction: effect of glucose (pregnant group – U46619)

Myometrial arteries from healthy pregnant women were constricted with 10-6

M

U46619. Similar to the results obtained using 60 mmol/L potassium, no significant

difference in active effective pressure was seen at 2, 5, 8 or 12 mmol/L glucose (Fig.

4.9, paired t-test: p > 0.05).


(pregnant group –U46619)

In the control arteries at 5 mmol/L glucose, an enhancement of endothelium-

dependent relaxation was observed with time (Fig. 4.10B, repeated measures

ANOVA: p < 0.05). Bonferroni post-hoc testing revealed significant differences in

relaxation at 10-8

M (p < 0.01) and 10-7

M (p < 0.05) bradykinin. Similarly,

significant enhancement of relaxation was also present after incubation at 8 and 12

mmol/L glucose (Fig. 4.10C and 4.10D, repeated measures ANOVA: p < 0.05).

Bonferroni post-hoc testing did not reveal a significant difference at any specific

concentration of bradykinin (p > 0.05). However after exposure to 2 mmol/L

glucose for 30 minutes, this enhancement was absent, with no difference in

relaxation (Fig 4.10A, repeated measures ANOVA: p > 0.05). This implies an

impairment of endothelium-dependent relaxation at 2 mmol/L. This was more

clearly illustrated on unpaired comparisons at different glucose levels. In Fig. 4.11,

similar endothelium-dependent relaxation was present in arteries incubated at 5

mmol/L glucose (two-way ANOVA, p > 0.05). However, when these arteries were

incubated at 2, 5, 8 and 12 mmol/L glucose for 30 minutes, impaired relaxation was

noted at 2 mmol/L glucose compared with control at 5 mmol/L glucose (Fig. 4.12;

two-way ANOVA, p < 0.05). Bonferroni post-hoc testing did not reveal a significant

difference at any specific concentration of bradykinin (at 10-6

M bradykinin, mean

difference = 1.28, 95% confidence interval = -41.98 to 16.72).

140

4.9AConstriction:U46619

5 vs 2 mmol/L

5 mmol/L 2 mmol/L0

10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a

4.9BConstriction:U46619Control at 5 mmol/L


10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a

4.9CConstriction:U46619

5 vs 8 mmol/L

5 mmol/L 8 mmol/L0

10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a

4.9DConstriction:U46619

5 vs 12 mmol/L

5 mmol/L 12 mmol/L0

10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a

Fig. 4.9A-D: Constriction: effect of glucose (pregnant group – U46619)


effective pressure in myometrial arteries from healthy pregnant women (N=14).








141


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

2 mmol/L Glucose

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on


-10 -9 -8 -7 -60

25

50

75

100



Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

8 mmol/L Glucose

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

12 mmol/L Glucose

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on

Fig.4.10A-D: Relaxation: effect of glucose (pregnant group – U46619)



pregnant women (N=14). Vessels constricted with 10-6

M U46619 and relaxed with

10-10

to 10-6






Repeated measures ANOVA: p < 0.05 in 5 (control), 8 and 12 mmol/L glucose

(time effect)

Repeated measures ANOVA: p > 0.05 at 2 mmol/L glucose

142

4.11 Relaxation at 5 mmol/L glucose

-10 -9 -8 -7 -60

25

50

75

100

Prior to 2 mmol/L Glucose



Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on

Fig. 4.11: Relaxation at 5 mmol/L glucose (pregnant group – U46619)

Endothelium-dependent relaxation in arteries all initially at 5 mmol/L glucose prior

to changing glucose levels

Two-way ANOVA: p > 0.05

143

4.12 Relaxation at 2, 5 and 12 mmol/L Glucose

-10 -9 -8 -7 -60

25

50

75

100

2 mmol/L Glucose

5 mmol/L Glucose

12 mmol/L Glucose

*

p < 0.05

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on

Fig. 4.12 Relaxation at 2, 5 and 12 mmol/L glucose (pregnant group – U46619)

Endothelium-dependent relaxation after change of glucose level to 2, 5 and 12

mmol/L glucose. Impaired relaxation at 2 mmol/L compared to control at 5 mmol/L

glucose. Relaxation at 8 mmol/L glucose overlapped that at 5 and 12 mmol glucose

and was omitted for clarity.

* Two-way ANOVA: p < 0.05

144

4.4.11: Comparison of endothelium-dependent relaxation in non-

pregnant and pregnant groups

The effect of pregnancy on myometrial artery function was assessed by comparing

endothelium-dependent relaxation in non-pregnant and pregnant groups at 5 mmol/L

glucose (Fig. 4.13 A, B, C). No significant difference was noted in the endothelium-

dependent relaxation between the two groups, irrespective of whether 60 mmol/L

KCl (Fig. 4.13A) or U46619 (Fig. 4.13B) was used as a vasoconstrictor (two-way

ANOVA: p > 0.05). Furthermore, analysis of maximum relaxation at the highest

concentration of bradykinin (10-6

M, Fig. 4.13C) revealed no significant difference

in relaxation between any of the groups (one-way ANOVA: p > 0.05).

145

4.13A KCl: Non-Pregnant vs Pregnant

-10 -9 -8 -7 -60

25

50

75

100

Non- Pregnant: KCl

Pregnant: KCl

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on

4.13B U46619: Non-Pregnant vs Pregnant

-10 -9 -8 -7 -60

25

50

75

100

Non-Pregnant: U46619

Pregnant: U46619

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on

4.13C Relaxation at 10-6 M Bradykinin

Non-Pregnant: KCl

Pregnant: KCl

Non-Pregnant: U46619

Pregnant: U46619

0

10

20

30

40

50

60

70

80

90

100

% M

axim

um

Co

nstr

icti

on

Fig. 4.13A-C: Relaxation: non-pregnant and pregnant groups

Comparison of endothelium-dependent relaxation in myometrial arteries from

healthy non-pregnant and pregnant women at 5 mmol.L glucose

Fig. 4.13A after constriction with KCl

Fig. 4.13B after constriction with U46619

Two-way ANOVA, p > 0.05

Fig. 4.13C Maximum relaxation at highest concentration of bradykinin (10-6

M)

One-way ANOVA: p > 0.05

146

4.4.12 Effect of prolonged exposure (2 hours) of 2, 5, 8 and 12

mmol/L glucose

To examine whether more prolonged exposures to altered glucose levels affected

vascular function, Protocols 1 and 2 were used (see Section 2.12) with the only

difference being that the vessels were exposed to 2, 5, 8 and 12 mmol/L glucose for

2 hours instead of 30 minutes, with periodic washes as before. The majority of

myometrial arteries from both healthy pregnant and non-pregnant women

demonstrated the following aberrant vascular responses after 2 hours:

a) marked reduction in maximum constriction (Fig. 4.13)

b) transient constriction with spontaneous relaxation

Atypical responses were obtained in both control arteries as well as arteries

incubated at different glucose levels. Spontaneous relaxation without addition of a

vasodilator prevented the assessment of endothelium-dependent relaxation.

However, arteries with responses similar to that seen after 30 minutes incubation

were also seen, as well as arteries with varying degrees of the above anomalous

responses.

147

0

1

2

3

4

5

6

2 Hours

A B

C D

Ten

sio

n (

mN

/mm

)

Fig. 4.14: Two hour incubation: aberrant responses

Actual raw data trace illustrating the effect of two hour incubation on vascular

function. The four concentration-response curves are marked by arrows (A-D).

After two hour incubation at 8 mmol/L glucose, arteries demonstrated markedly

diminished constriction and endothelium-dependent relaxation. This was illustrated

by the small amplitude of curves C and D compared to the initial two curves A and

B. Similar results were obtained in the control vessel.

148

4.5 Discussion

4.5.1 Summary of results

The results are summarised in Table 4.2

Group Parameter Effect of glucose

Healthy

non-pregnant

(N=15 women,

n=54 arteries)

KCl - Constriction No effect

KCl - Relaxation No effect

U46619 -

Constriction No effect

U46619 - Relaxation No effect

Healthy pregnant

(N=30 women,

n=100 arteries)

KCl - Constriction No effect

KCl - Relaxation No effect

U46619 -


U46619 - Relaxation Impaired relaxation

at 2 mmol/L glucose

Table 4.2: Summary of results

4.5.2 Low glucose concentration and impaired relaxation

Exposure to 2 mmol/L glucose for 30 minutes resulted in impaired endothelium-

dependent relaxation in myometrial arteries from healthy pregnant women when

constricted with U46619 (Fig. 4.12), an effect not present when the vessels were

constricted with 60 mmol/L KCl (Fig. 4.8). In contrast, constriction and relaxation

in myometrial arteries from healthy non-pregnant women were unaltered at 2, 8 or

12 mmol/L of glucose, irrespective of the vasoconstrictor used.

149

This represents the first demonstration that hypoglycaemia inhibits endothelium-

dependent relaxation in any vascular bed. Studies examining the effect of

hypoglycaemia on vascular function are extremely limited. Swafford et al. studied

rat middle cerebral artery and found that reducing the glucose concentration from

5.5 mmol/L to 1.0 or 0.5 mmol/L for 1.5 hours each had no significant effect on the

diameter of the arteries [Swafford et al., 1998]. A study by Watanabe and

colleagues postulated that hypoglycaemia stimulated prostacyclin production in

human umbilical vein endothelial cells [Watanabe and Jaffe, 1995]. However, the

incubation period of 24 hours and the cell culture model may not accurately reflect

the in vivo setting.

The mechanisms responsible for impairment of relaxation with hypoglycaemia were

not investigated in the present study. The diameter of arteries constricted with

U46619 was marginally lower than those constricted with KCl (Fig. 4.2), although it

is uncertain if this influenced the results.

4.5.2.1 The effect of vasoconstrictors

Impairment of endothelium-dependent relaxation was noted (at 2 mmol/L glucose)

when the arteries were constricted with U46619, but not with potassium, implying

that this effect may be agonist-specific. As discussed in Section 1.5.5, a

compensatory mechanism of the endothelial vasodilators has been demonstrated to

exist which can rectify the absence of one particular mediator. Endothelium-

dependent relaxation in myometrial arteries of healthy pregnant women has been

previously demonstrated to be preserved in the presence of 25 mmol/L potassium,

an inhibitor of EDHF [Kenny et al., 2002]. The explanation given for this result was

that NO-mediated relaxation was enhanced in such conditions, compensating for the

absence of EDHF-mediated relaxation. Furthermore, concurrent blockade of NO

and EDHF resulted in significantly impaired relaxation. These findings of Kenny et

al. in the myometrial vascular bed may also explain the results of the present study.

The preservation of endothelium-dependent relaxation noted here (when arteries

were constricted with 60 mmol/L potassium) may be due to increased NO-mediated

relaxation, which counterbalanced the lack of EDHF-mediated relaxation. This

enhancement of NO was present only when EDHF was blocked; on using U46619,

150

the lack of EDHF blockade may have prevented an increase in NO-mediated

relaxation. This may also explain why hypoglycaemia was not associated with

impaired relaxation in arteries from non-pregnant women as the aforementioned

compensatory mechanism has noted to be absent in this group [Kenny et al., 2002].

4.5.2.2 The effect of pregnancy

The impaired relaxation with hypoglycaemia was specific to pregnancy, as reducing

glucose concentrations had no effect on arteries from non-pregnant women

irrespective of the vasoconstrictor used (Fig. 4.10 and 4.12). This may represent a

physiological adaptation in pregnancy to counter-act the deleterious effects of

reduced blood glucose levels. During hypoglycaemia, shunting of blood into the

splanchnic circulation to maintain blood flow to vital organs such as the liver has

been previously reported [Braatvedt et al., 1991; Parker et al., 1999]. However, it is

uncertain if the impaired endothelium-dependent relaxation noted when myometrial

arteries were exposed to hypoglycaemia is an adaptive response to maintain visceral

circulation.

4.5.3 Other findings


Constriction was observed to be unaltered irrespective of the glucose level, the

vasoconstrictor used or whether vessels were obtained from non-pregnant (Fig. 4.3

and 4.5) or pregnant women (Fig. 4.7 and 4.9). This signifies that constriction, an

endothelium-independent vascular function, is not affected by acute hypo- and

hyperglycaemia.

4.5.3.2 Effect of pregnancy on endothelium-dependent relaxation

Irrespective of whether 60 mmol/L KCl or U46619 was used as the vasoconstrictor,

endothelium-dependent relaxation was similar in both pregnant and non-pregnant

groups. Although relaxation was greater with U46619 at 10-8

and 10-7

M bradykinin,

the maximum relaxation at 10-6

M was similar in all groups. These findings

corroborate those by Kenny et al., who found similar relaxation in myometrial

arteries of pregnant and non-pregnant women [Kenny et al., 2002]. The

151

physiological changes in vessel function during pregnancy have been discussed in

Section 1.3.2. It has been previously demonstrated that endothelium-dependent

relaxation is enhanced in normal pregnancies [Faber-Swensson et al., 2004;

Saarelainen et al., 2006]. However, these studies were performed using brachial

artery flow-mediated dilation: a limitation of this technique is that it assesses

function in larger conduit arteries (Section 1.5.6.2). Therefore, these results may not

be applicable to the resistance arteries in the uterine vascular bed. The present study

demonstrated no difference in maximal endothelium-dependent relaxation of

myometrial arteries from non-pregnant and pregnant women. This finding

emphasizes the importance of studying relevant vascular beds directly, as vascular

function can vary in different tissue regions.

4.5.3.3 Effect of prolonged incubation

Incubation of arteries for two hours resulted in aberrant vascular responses which

were not reproducible (Fig. 4.13). The defects in constriction and relaxation

signified a loss of viability of these arteries when mounted for a prolonged time in a

wire myograph. Dissected arteries stored at 4° C prior to mounting on a wire

myograph have been demonstrated to be viable [Ang et al., 2002]. However,

viability appears to be impaired after wire insertion and hours of experimentation at

37° C. The prolonged stretching of the vessel by the wires that occurs even in the

resting state during experimentation may have resulted in damage to the arterial

wall, leading to the abnormal responses. Viability after 2-hour incubation was

considerably less with mice uterine arteries, probably because these vessels had a

much thinner wall compared to human myometrial arteries. Although function was

preserved in approximately 50% of the myometrial arteries examined, the high

failure rate prevented four different glucose levels from being assessed, thereby

limiting the value of the study. Therefore the incubation period in subsequent

experiments was limited to 30 minutes, as no loss of viability was observed for this

duration of time.

152

4.5.3.4 High glucose concentrations

This study has also demonstrated that exposure to physiologically relevant high

glucose concentrations for a short period of time had no effect on endothelium-

dependent relaxation in healthy individuals (Fig. 4.4, 4.6, 4.8 and 4.10). This was

seen irrespective of the vasoconstrictor or whether arteries were from pregnant or

non-pregnant women.

4.5.4 Explanations for the lack of effect of high glucose levels

4.5.4.1 Inadequate duration of high glucose levels

Most studies have studied the effect of glucose levels for longer durations lasting up

to 24 hours [Houben et al., 1996]. However, as discussed in Section 1.9.4, such a

long duration of hyperglycaemia is not usually seen clinically, limiting the

physiological relevance of such studies. In healthy pregnant and non-pregnant

women, the mean duration of hyperglycaemia (± standard deviation) greater than

8.9 mmol/l over 24 hours has been reported to be 7.5 ± 14 minutes and 9.3 ± 25

minutes respectively [Buhling et al., 2004]. Due to this observation and also due to

methodological considerations, the time duration chosen for this study has been

limited to 30 minutes. However, this may have been an insufficient time for the

deleterious effects of a high glucose concentration to be manifested, as studies

demonstrating impaired relaxation with hyperglycaemia have used longer durations

of up to 6 hours (see Table 1.3).

4.5.4.2 Inadequate degree of high glucose levels

As discussed previously, most studies investigating the vascular effects of

hyperglycaemia have examined high levels such as 44 mmol/L [Cipolla et al.,

1997]. However, such high glycaemic levels are rarely seen clinically. In healthy

women, the blood glucose levels have been reported to range between 3 to 6.8

mmol/L (see Section 1.8.). Even in diabetic women, the mean level of

hyperglycaemia does not usually reach levels as high as 44 mmol/L; in the previous

chapter the highest reading was 28 mmol/L glucose. In another study, the mean

peak glucose level (± standard deviation) in women with type 1 diabetes in

153

pregnancy was reported to be 10.1 ± 3.2 mmol/L [Ben-Haroush et al., 2004]. The

aim of this study was to examine the effects of glucose at physiologically relevant

levels and extremely high concentrations of glucose were therefore not investigated.

However, it is possible that higher levels of hyperglycaemia may result in altered

endothelial function.

4.5.4.3 Systemic Factors

It is not yet clear if hyperglycaemia per se causes vascular dysfunction or whether it

causes dysfunction by triggering associated systemic factors. These factors could

include: neural mechanisms, hyperinsulinemia and lipid alterations, as discussed in

Chapter 1. In this study the in vitro method of wire myography was used, thereby

eliminating the effect of such systemic factors on vascular function. Systemic neuro-

humoral factors such as circulating hormones and vascular innervation may be

important in mediating vascular responses in the myometrial vascular bed. In

humans, the smaller branches of uterine arteries have been observed to be densely

innervated, emphasizing the importance of neural regulation in uterine blood flow

[Akerlund, 1994]. Shear stress from blood flow, an important trigger for NO-

mediated relaxation in vivo, is not reproduced in wire myography. Arteries may

exhibit different characteristics in vivo from those demonstrated here, due to the

various biological interactions influencing vascular function. An advantage of using

in vivo methods such as flow-mediated dilation is that such interactions are taken

into account when assessing vessel responses. However, a limitation of such studies

is that a precise level of hyperglycaemia is often difficult to maintain and

characterise. For example, studies by Kawano et al. and Title et al. used glucose

tolerance tests to induce hyperglycaemia [Kawano et al., 1999; Title et al., 2000].

However, this technique is associated with variable levels of hyperglycaemia over

different periods of time. An important advantage of wire myography is that it

allows four precise levels of glucose to be studied simultaneously for a specific time

period in the same patient.

154

4.5.4.4 Endothelial mediators of vasodilation

Previous studies have used 25 mmol/L potassium to block EDHF-mediated

relaxation [Kenny et al., 2002]. Although the rationale for using 60 mmol/L

potassium in the present study was to constrict arteries by depolarization, a corollary

effect of employing this agonist was the inhibition of EDHF-mediated relaxation. In

such arteries, the endothelium-dependent relaxation observed is therefore restricted

to the actions of NO and prostacyclin. Nevertheless, maximal relaxation in the

absence of EDHF was similar to that in the presence of EDHF, when U46619 was

used for constriction (Fig. 4.13C). Hyperglycaemia has been associated with

reduced bioavailability of NO and impaired endothelium-dependent relaxation

(Section 1.6.3). Therefore, vascular beds where NO contributes significantly to

relaxation may have a greater degree of impairment. The effect of hyperglycaemia

on prostacyclin and EDHF-mediated relaxation is yet to be clearly outlined.

Although some of the earlier studies attributed the impaired endothelium-dependent

relaxation with hyperglycaemia to reduced NO, the EDHF component was often not

adequately blocked [Williams et al., 1996]. There may also be a compensatory

mechanism occurring between the various pathways, as discussed previously,

making assessment of individual mediators of relaxation difficult. This mechanism

may circumvent the effect of hyperglycaemia on one particular pathway. The

proportion of relaxation due to NO, EDHF and prostacyclin has been demonstrated

to vary in different vascular beds (Section 1.6.4). In the myometrial vascular bed,

endothelium-dependent relaxation has been mainly attributed to NO and EDHF

[Kenny et al., 2002]. The present study has demonstrated that a high glucose

concentration was not associated with alterations in maximum relaxation, either in

the presence or absence of EDHF. However, it is possible that a compensatory

enhancement of NO-mediated relaxation may occur on blocking EDHF.

4.5.4.5 Bradykinin

Although bradykinin is an effective endothelium-dependent vasodilator in vitro its

physiological role in vivo is less well-characterised. In the physiological setting,

bradykinin is inactivated by a host of peptidases, of which angiotensinogen

converting enzyme is particularly important. Bradykinin has been demonstrated to

have little effect on the pulmonary vascular bed as a result of this [Bonner et al.,

155

1990]. Inactivation of bradykinin also occurs within the arterial wall [Skidgel,

1992], which may limit its effectiveness in certain vascular beds. In the myometrial

arterial bed, however, various studies have demonstrated reproducible efficacy in

using bradykinin as an endothelium-dependent relaxing agent [Kenny et al., 2002;

Myers et al., 2006]. A study in diabetic rats has reported that bradykinin can reduce

oxidative stress in hyperglycaemic conditions [Mikrut et al., 2001]. Therefore,

although bradykinin produces reproducible relaxation in myometrial arteries, it is

uncertain if aberrant glucose levels can affect its potency to mediate endothelium-

dependent relaxation.

4.5.4.6 Branch-order

Vascular function, whether constriction or relaxation, is largely determined by the

calibre of the artery. As previously discussed (Section 1.5.2.3), the relative

contribution to relaxation from NO, EDHF or prostacyclin varies depending on

vessel diameter. Shimokawa et al. has demonstrated in the rat that expression of

eNOS was the highest in the aorta and the lowest in the distal mesenteric arteries

[Shimokawa et al., 1996]. Similarly, in human arteries the contribution of EDHF

has been shown to increase as the vessel diameter decreased [Urakami-Harasawa et

al., 1997]. Therefore, vascular function of any artery needs to be considered in the

context of its location in the vascular tree. Most of the studies demonstrating

impaired endothelium-dependent relaxation with hyperglycaemia have been

performed on large vessels such as brachial arteries (Table 1.3). Studies

investigating hyperglycaemia in the smaller resistance arteries are rare. As the role

of the endothelium-dependent mediators NO and EDHF have been demonstrated to

vary depending on vessel size, it may not be possible to generalise the results

obtained in large-calibre arteries to resistance vessels. Therefore the effect of

hyperglycaemia at this branch-order of the arterial tree is uncertain.

4.5.4.7 Age

In Section 1.5.4.1, evidence for the deterioration of endothelial function with age

has been discussed. One limitation of this study is that the subjects in the pregnant

group and in the non-pregnant group were not age matched, as hysterectomies were

156

normally performed only in women who had completed their families. The mean (±

SEM) age of women in the pregnant and non-pregnant group was 31.5 (± 0.9) years

and 43 (± 2.4) years respectively. The age of individuals in studies that have

demonstrated impaired relaxation with hyperglycaemia vary from 25 years [Title et

al., 2000] to 62 years [Kawano et al., 1999]. The effect of hypo- and

hyperglycaemia on endothelial function at different age groups is yet to be

elucidated, as no known study has investigated this clinically important subject.

Endothelial function has been reported to deteriorate with age [Gerhard et al., 1996].

It is therefore possible that the effects of hyperglycaemia on endothelial function

become more pronounced with age. The lack of effect of high glucose

concentrations on endothelial function may be due to the relatively young age of the

patients in the present study.

4.5.4.8 Nature of vascular bed

As described in Section 1.6.4, aberrant glucose levels do not have the same effect in

all vascular beds. It is also uncertain whether the onset of endothelial dysfunction

occurs concurrently in all vascular systems of the body. It is possible to speculate

from the conflicting results of studies investigating hyperglycaemia (Section 1.9.7)

that the effect of hyperglycaemia may vary depending on the nature of the vascular

bed and its metabolic requirements. The majority of studies have examined upper-

limb circulation, an arterial bed with vastly different vessel characteristics and

metabolic demands than the uterine circulation in pregnancy. This highlights the

importance of studying hyperglycaemia in the myometrial arteries directly, instead

of extrapolating findings obtained in other vascular beds in pregnancy.

4.5.4.9 Gender

As noted in Section 1.5.2.1, gender plays an important role in NO-mediated

relaxation, which has been related to circulating estradiol [Hayashi et al., 1992]. In

female mice, EDHF was demonstrated to be more important than NO and PGI2 in

mediating relaxation, whereas the converse was true in males [Scotland et al.,

2005]. Studies investigating hyperglycaemia and vascular function have had a

mixed study population, which may obscure gender-specific differences in

157

endothelial function. As the current study is limited to females, it restricts

comparisons between studies. Hyperglycaemia may be affected by the type of

endothelial mediator pre-dominant in the gender examined.

4.5.5 SUMMARY

Incubation of myometrial arteries from healthy pregnant women at 2 mmol/L

glucose for 30 minutes was associated with impaired endothelium-dependent

relaxation when constricted with U46619. However, this effect was absent on

constricting arteries using 60 mmol/L potassium, signifying an agonist-specific

effect. Constriction of myometrial arteries was not affected by changes in glucose

level in either the pregnant or non-pregnant group. Furthermore, exposure to 2, 5, 8

and 12 mmol/L for 30 minutes had no effect on endothelium-dependent relaxation

in arteries from healthy non-pregnant women. These results suggest that acute

exposure to low glucose levels can impair endothelial function in healthy pregnant

women, an effect which is specific to both the agonist used and the state of

pregnancy. Characterisation of the effects of glucose levels on endothelial function

in normal pregnancy provides a basis for examining these effects in diabetes

complicating pregnancy, which is dealt with in the next chapter.

158

CHAPTER 5

The effect of glucose on endothelial

function in diabetes complicating

pregnancy

159

THE EFFECT OF GLUCOSE ON ENDOTHELIAL FUNCTION IN

DIABETES COMPLICATING PREGNANCY

5.1 Introduction:

The previous chapter demonstrated that reduced glucose levels can cause

endothelial dysfunction in myometrial arteries of healthy pregnant women. In

diabetes, endothelial dysfunction has been demonstrated in various vascular beds

such as the coronary [Nitenberg et al., 1993] and renal circulation [Shestakova et

al., 2005]. However, no known study has examined the myometrial arterial bed in

pregnancies complicated by diabetes. Studies in diabetes have often used the upper-

limb circulation as a marker for endothelial dysfunction. However, as discussed in

Section 1.6.4, the nature of the vascular bed plays a pivotal role in determining

endothelial responses. The preceding chapter demonstrated unique endothelial

responses in myometrial arteries distinct from those seen in other vascular beds. For

example, maximal vasodilation in these arteries was not found to be enhanced in

pregnancy, in contrast with reports in brachial arteries [Faber-Swensson et al.,

2004]. Furthermore, as the time of onset of endothelial dysfunction in diabetes and

its rate of progression in various vascular beds are unknown, it is possible that

normal endothelial function at one site may not be representative of vascular

responses at other sites. For example, the endothelial responses in mesenteric and

femoral arteries of diabetic rats were markedly different from that seen in the carotid

artery of the same animals [Shi et al., 2006]. Because of the heterogeneity of

responses, studying the relevant vascular bed directly is crucial in assessing vascular

function in diabetes.

The complications of diabetes in pregnancy are multi-factorial in nature. However,

as diabetes is a vascular disease, endothelial dysfunction in the arteries that regulate

blood flow to the growing fetus may be an aetiological factor in these

complications. Myometrial resistance arteries are pivotal in this regard as they

determine blood flow to the placenta, and unlike placental arteries have been

affected by maternal diabetes and aberrant glucose levels for the entire duration of

the disease.

160

For this study, patients with type 1, type 2 and gestational diabetes were recruited

using the criteria discussed in Section 2.2.2. Similar experimental protocols were

followed as in the previous chapter, to facilitate comparison of vascular function

between healthy and diabetic pregnant women.

The findings in Chapter 3 suggest that glucose levels between 2 to 12 mmol/L

glucose represent clinically relevant values for women undergoing treatment for

diabetes in pregnancy, although poorly controlled diabetes is associated with much

higher levels of hyperglycaemia. The duration of different glucose levels has been

limited to 30 minutes to assess whether acute changes in glucose concentration

influence vessel function. This time interval has been demonstrated to reflect a

clinically relevant duration of hyperglycaemia in pregnancies complicated by

diabetes [Buhling et al., 2004].

5.2 Aims

1. To examine the effect of exposure to 30 minutes of 2, 5, 8 and 12 mmol/L

glucose on maximal constriction (as measured by active effective pressure)

in myometrial arteries from women with diabetes complicating pregnancy.

2. To examine the effect of exposure to 30 minutes of 2, 5, 8 and 12 mmol/L

glucose on endothelium-dependent relaxation in these myometrial arteries.

6. To study whether effects are agonist-dependent by using two different

vasoconstrictors:

A) 60 mmol/L KCl

B) U46619

3. To compare constriction and endothelium dependent relaxation of

myometrial arteries between healthy pregnant women and women with

gestational diabetes

161


These have been discussed in Chapter 2. The experimental protocols used were

Protocol 1 and 2 (see Section 2.12.1 and 2.12.2). Data obtained from healthy

pregnant women described in the preceding chapter were used for comparison with

diabetic patients.

5.4 Results

5.4.1 Clinical characteristics - gestational diabetes

Details of patients with gestational diabetes recruited in this study are given in Table

5.1. To enable correlation of clinical characteristics with vascular function, GDM

patients have been sub-divided into KCl and U46619 groups; depending on the

vasoconstrictor used. Overlap of these groups was present, as arteries from the same

patient were used in both groups in five cases. Patients in both groups were obese

and also had high birth weights. These patients had near-normal HbA1C levels

(normal reference range < 6.5 %). Patients in the two groups had no significant

difference in their age, BMI, birth weight of babies or HbA1C (unpaired t-test, p >

0.05).

162

PARAMETERS GDM

KCl GROUP (N = 14)

GDM U46619 GROUP

(N = 8)

Age (years) 33.6 ± 1.2 34.1 ± 1.7

BMI (kg/m2) 35.0 ± 2.3 34.5 ± 2.5

Max. Systolic BP (mm Hg) 134 ± 5 133 ± 6

Max. Diastolic BP (mm Hg) 85 ± 4 83 ± 4

Gravidity 3 ± 0.3 3.4 ± 0.3

Parity 1.9 ± 0.3 2.2 ± 0.3

Antenatal Blood Glucose (low reading, mmol/L)

4.1 ± 0.2 4.5 ± 0.3

Antenatal Blood Glucose (high reading, mmol/L)

9.7 ± 0.7 11.4 ± 1.2

Mean Antenatal HbA1C (%) 6.4 ± 0.2 6.9 ± 0.3

Mother’s blood glucose at delivery (mmol/L)

4.9 ± 0.4 4.7 ± 0.5

Baby’s blood glucose at delivery (mmol/L)

2.2 ± 0.2 2.0 ± 0.05

Gestational Age at Delivery (weeks)

38.5 ± 0.2 38.8 ± 0.2

Birth Weight (gm) 4010 ± 143 4171 ± 257

Individualised Birth Ratio (centile)

81 ± 7 79 ± 10

Table 5.1: Clinical characteristics – gestational diabetes

Data shown as mean ± SEM. Normal reference range for HbA1C = < 6.5% .The

individual birthweight ratio is a centile which is dependent on maternal ethnicity,

height, weight, parity and gestation at delivery, in addition to fetal birthweight and

sex.

163

5.4.2 Diameter of arteries- gestational diabetes

The mean diameter (± SEM) of arteries from women with gestational diabetes was

398 (± 18) µm in the KCl group and 347 (± 21) µm in the U46619 group (Fig.

5.1A). There was no significant difference in diameter between the two groups

(unpaired t-test: p > 0.05). To assess whether this result was due to multiple

observations in arteries obtained from the same patient, the mean diameter of all

arteries obtained from a single patient was also plotted and compared (Fig. 5.1B).

Here also, no significant difference was seen between the KCl (N=14) and U46619

(N=8) groups (unpaired t-test: p > 0.05).

5.4.3 Constriction: effect of glucose (GDM - KCl)

Myometrial arteries from women with gestational diabetes were constricted using

60 mmol/L KCl, initially at 5 mmol/L glucose. The active effective pressure was

calculated at the peak of constriction. After 30 minutes incubation in 2, 5, 8 or 12

mmol/L glucose, constriction was calculated again (Fig. 5.2). No significant

difference in active effective pressure was present at any of the glucose levels

examined (paired t-test: p > 0.05 in all comparisons; at 12 mmol/L glucose: mean

difference = 0.65, 95% confidence interval = -0.42 to 1.72).

164

Fig 5.1A Arterial Diameter

KCl U466190

100

200

300

400

500

600

700

800

Agonist

Art

eri

al

Dia

mete

r

mic

rom

etr

es

Fig 5.1B Mean diameter of arteries from individual patients

KCl U466190

100

200

300

400

500

600

700

800

Agonist

Art

eri

al

Dia

mete

r

mic

rom

etr

es

Fig. 5.1A & B: Arterial diameters - GDM

Fig 5.1A: Myometrial arteries from women with gestational diabetes divided into

two groups based on the vasoconstrictor used: KCl (N=14, 51 arteries)

and U46619 (N=8, 24 arteries). Horizontal bars represent mean values.

Fig 5.1B: Mean of the diameter of all arteries obtained from individual patients

Unpaired t-test: p > 0.05

165


5 mmol/L 2 mmol/L0

10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a

5.2B Constriction: KClControl: 5 mmol Glucose


10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a


5 mmol/L 8 mmol/L0

10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a


5 mmol/L 12 mmol/L0

10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a

Fig. 5.2A-D: Constriction: effect of glucose (GDM- KCl)

The effect of changes in glucose concentration on active effective pressure in

myometrial arteries from women with Gestational Diabetes (N=14). Vessels were

constricted with 60 mmol/L KCl. Active effective pressure assessed at:


Fig. 5.2B: 5 (circles) mmol/L glucose (n=13 arteries)




166


(GDM - KCl)

Myometrial arteries were constricted using 60 mmol/L KCl and endothelium-

dependent relaxation of arteries at 5 mmol/L was compared to that after 30 minutes

of incubation at 2, 5, 8 and 12 mmol/L glucose (Fig. 5.3). Acute changes in glucose

concentration had no significant effect on endothelium-dependent relaxation in

arteries from patients with GDM at any glucose level studied (repeated measures

ANOVA: p > 0.05).

5.4.5 Constriction: effect of glucose (GDM - U46619)

When myometrial arteries from women with gestational diabetes were constricted

with U46619, no significant difference in active effective pressure was present at

any of the glucose levels examined (Fig. 5. 4, paired t-test: p > 0.05).


(GDM - U46619)

Acute changes in glucose concentration had no significant effect on endothelium-

dependent relaxation when myometrial arteries from patients with GDM were

constricted with U46619 (Fig. 5.5, repeated measures ANOVA: p > 0.05).

167


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

2 mmol/L Glucose

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on


-10 -9 -8 -7 -60

25

50

75

100



Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

8 mmol/L Glucose

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

12 mmol/L Glucose

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on

Fig. 5.3 A-D: Relaxation: effect of glucose (GDM-KCl)

Endothelium-dependent relaxation in myometrial arteries from women with

gestational diabetes (N=14). Arteries were constricted with 60 mmol/L KCl.

Relaxation assessed at:






168

5.4A Constriction: U466195 vs 2 mmol/L Glucose

5 mmol/L 2 mmol/L0

10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a

5.4B Constriction: U46619Control: 5 mmol/L Glucose


10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a

5.4C Constriction: U466195 vs 8 mmol/L Glucose

5 mmol/L 8 mmol/L0

10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a

5.4D Constriction: U466195 vs 12 mmol/L Glucose

5 mmol/L 12 mmol/L0

10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a

Fig. 5.4A-D: Constriction: effect of glucose (GDM- U46619)

The effect of changes in glucose concentration on active effective pressure in

myometrial arteries from women with gestational diabetes (N=8). Vessels were

constricted with 10-6







169


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

2 mmol/L Glucose

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose: Part 15 mmol/L Glucose: Part 2

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

8 mmol/L Glucose

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

12 mmol/L Glucose

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on

Fig. 5.5 (A-D): Relaxation: effect of glucose (GDM – U46619)


gestational diabetes (N=8). Arteries were constricted with 10-6

M U46619.






Repeated measures ANOVA, p > 0.05

170

5.4.7 GDM: Effect of vasoconstrictor on endothelium-dependent

relaxation

Endothelium-dependent relaxation was compared in myometrial arteries from

women with GDM constricted with KCl and U46619 at 5 mmol/L glucose (Fig.

5.6). Relaxation with KCl was significantly impaired compared to that with U46619

(two-way ANOVA: p < 0.001). Bonferroni post-hoc test revealed significant

differences at 10-8

, 10-7

and 10-6

M bradykinin (p < 0.001).

5.6 GDM: Effect of vasoconstrictor

-10 -9 -8 -7 -60

20

40

60

80

100

KCl

U46619

*

*

*

p < 0.001

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on

Fig. 5.6: Endothelium-dependent relaxation in GDM: effect of vasoconstrictor


gestational diabetes after constriction with 60 mmol/L potassium and 10-6

M

U46619. Significantly impaired relaxation when KCl used

Two-way ANOVA, p < 0.001

* Bonferroni post-hoc test: p < 0.001 at 10-8

, 10-7

and 10-6

M bradykinin

171


(Type 1 and 2 DM: KCl)

As suitable numbers of pregnant women with type 1 and 2 diabetes (N= 4 and 3

respectively) could not be obtained, only a limited study of vascular function was

possible in these two groups. Changes in glucose levels had no significant effect on

maximal constriction (paired t-test: p > 0.05), when myometrial arteries from

women with type 1 and 2 diabetes were constricted with 60 mmol/L KCl (Fig. 5.7

and 5.8).


(Type 1 and 2 DM: KCl)

No significant difference in endothelium-dependent relaxation was noted in either

type 1 or 2 diabetes groups on altering glucose levels to 2, 5, 8 or 12 mmol/L (Fig.

5.9; repeated measures ANOVA: p > 0.05).

172

5.7 Type 1 diabetes - Constriction: KCl

5 2 5 5 5 8 5 120

10

20

30

40

Glucose Concentration (mmol/L)

Acti

ve E

ffecti

ve P

ressu

re

kP

a

5.8 Type 2 diabetes - Constriction: KCl

5 2 5 5 5 8 5 120

10

20

30

40

Glucose Concentration (mmol/L)

Acti

ve E

ffecti

ve P

ressu

re

kP

a

Fig. 5.7 & 5.8: Constriction: effect of glucose (type 1 and 2 diabetes – KCl)


effective pressure in myometrial arteries from pregnant women with type 1 diabetes

(N=4, Fig 5.6) and type 2 diabetes (N=3, Fig 5.7). Vessels constricted with 60

mmol/L KCl, and those achieving constriction < 1 mN/mm excluded

Fig. 5.6: Type 1 diabetes Active Effective Pressure at 2, 5, 8 and 12 mmol/L glucose

Fig. 5.7: Type 2 diabetes Active Effective Pressure at 2, 5, 8 and 12 mmol/L glucose


173


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

2 mmol/L Glucose

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on


-10 -9 -8 -7 -60

25

50

75

100



Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on


-11 -10 -9 -8 -7 -6 -50

25

50

75

100

5 mmol/L Glucose

8 mmol/L Glucose

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

12 mmol/L Glucose

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on

Fig. 5.9 (A-D): Relaxation: effect of glucose (Type 1 diabetes – KCl)

Endothelium-dependent relaxation in myometrial arteries from women with type 1

diabetes (N=4). Arteries were constricted with 60 mmol/L KCl.







174


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

2 mmol/L Glucose

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on


-10 -9 -8 -7 -60

25

50

75

100



Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

8 mmol/L Glucose

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

12 mmol/L Glucose

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on

Fig. 5.10(A-D): Relaxation: effect of glucose levels (Type 2 diabetes – KCl)

Endothelium-dependent relaxation in myometrial arteries from women with type 2

diabetes. Arteries were constricted with 60 mmol/L KCl. Relaxation assessed at:






175


Constriction

The maximum constriction of myometrial arteries, expressed as active effective

pressure, was compared between healthy pregnant women and patients with GDM

at 5 mmol/L glucose (Fig. 5.11 and 5.12).

5.4.10.1 Using KCl

No significant difference was noted in the active effective pressure between the two

groups (Fig. 5.11; unpaired t-test: p > 0.05).

5.4.10.2 Using U46619

The active effective pressure was significantly higher in the GDM group compared

to healthy pregnant women (Fig. 5.12; unpaired t-test: p < 0.05).


Endothelium-dependent relaxation

5.4.11.1 Using KCl

Myometrial arteries from healthy pregnant individuals and women with GDM were

constricted with 60 mmol/L KCl and endothelium-dependent relaxation compared at

5 mmol/L glucose. Relaxation in arteries from women with GDM were significantly

impaired compared to normal (Fig. 5.13, two-way ANOVA: p < 0.001). Bonferroni

post-hoc tests showed significantly reduced relaxation (p < 0.001, mean difference =

11.74, 95% confidence intervals = 5.86 to 17.61) for the last concentration of

bradykinin (10-6

M). There was no significant difference in bradykinin sensitivity

between the two groups as EC50 values were similar (normal = 1.18 x 10-7

M and

GDM = 1.45 x 10-7

M).

5.4.11.2 Using U46619

Myometrial arteries from healthy pregnant women and women with GDM were


M U46619 and endothelium-dependent relaxation was

compared (Fig. 5.14). No significant difference was seen between the two groups

(Two-way ANOVA, p > 0.05)

176

5.11 KCl Constriction: Normal vs GDM

Normal GDM0

10

20

30

40

Acti

ve E

ffecti

ve P

ressu

re

kP

a

Fig 5.11: KCl Constriction: Normal vs GDM

Arterial constriction using KCl in normal pregnant (N=16, n=49 arteries) and GDM

(N=14, n=51 arteries) groups. Horizontal red bars represent mean values

Unpaired t-test, p > 0.05

5.12 U46619 Constriction: Normal vs GDM

Normal GDM0

10

20

30

40p < 0.05*

Acti

ve E

ffecti

ve P

ressu

re

kP

a

Fig 5.12: U46619 Constriction: Normal vs GDM

Arterial constriction using 10-6

M U46619 in normal pregnant (N=14, n=46 arteries)

and GDM (N=8, n=24 arteries) groups. Horizontal red bars represent mean values.

Unpaired t-test, p < 0.05

177

5.13 Normal vs GDM: KCl

-10 -9 -8 -7 -60

40

Normal

GDM

50

60

70

80

90

100

*

p < 0.001

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on

Fig. 5.13: Endothelium-dependent relaxation – Normal vs GDM: KCl


normal pregnant women (N=16) and women with gestational diabetes (N=14).

Arteries constricted with 60 mmol/L KCl at 5 mmol/L glucose. Relaxation

significantly impaired in GDM.

Two-way ANOVA, p < 0.001

Bonferroni post-hoc test: 10-6

M bradykinin * p < 0.001

178

5.14 Normal vs GDM: U46619

-10 -9 -8 -7 -60

25

50

75

100

Normal

GDM

p > 0.05

Bradykinin M [log]

% M

axim

um

Co

nstr

icti

on

Fig 5.14: Endothelium-dependent relaxation - GDM and normal: U46619


normal pregnant women and women with gestational diabetes. Arteries constricted

with 10-6

M U46619 at 5 mmol/L glucose. No significant difference was seen in

relaxation.

Two-way ANOVA, p > 0.05

179

5.5 Discussion

5.5.1 Summary of results

The findings of this section have been summarised in Table 5.2 (GDM, type 1 and 2

diabetes) and Table 5.3 (comparison between normal and GDM).

Table 5.2: Summary of results - effect of change in glucose levels

Parameter Vasoconstrictor Result

Constriction

KCl No difference

U46619 Enhanced constriction

GDM

Endothelium-


KCl Impaired relaxation

GDM

U46619 No difference

Table 5.3: Summary of results – comparison between normal and GDM

Diabetes Group Effect of change in glucose

Gestational Diabetes

(N=17 women, n=75 arteries)

Constriction: KCl & U46619


No effect

Type 1 and 2 Diabetes

(N=7 women, n=21 arteries)

Constriction: KCl


No effect

180

5.5.2 Endothelial dysfunction in GDM

The main finding of this section was that endothelium-dependent relaxation was

significantly impaired in myometrial arteries of women with gestational diabetes

compared to normal (Fig. 5.12). This aberrant function was present despite these

patients having a near-normal HbA1C, a marker for diabetes control. Endothelial

dysfunction has been previously demonstrated in the subcutaneous arteries of

women with GDM [Knock et al., 1997]. However the present study is the first

demonstration of abnormal vascular function in the myometrial vascular bed, which

is more relevant to foetal outcome.

The impairment in relaxation was evident only when the arteries were constricted

with 60 mmol/L KCl, and was absent when U46619 was used. This signifies that

these effects are specific to the agonist used. As demonstrated in the previous

chapter, myometrial arteries of healthy pregnant women also demonstrated agonist-

specific effects (see Section 4.4.10).

Hypoglycaemia was not associated with impaired endothelium-dependent relaxation

in GDM, a result at variance with that demonstrated in healthy pregnant women.

However, similar results were obtained in myometrial arteries from non-pregnant

women. This suggests that the pregnancy-associated alterations to the effects of

glucose in myometrial arteries may be inhibited in gestational diabetes.

5.5.2.1 Explanations for impaired relaxation

A) EDHF

An absence of EDHF-mediated relaxation may be responsible for the impaired

vasodilation with 60 mmol/L KCl, as it has been demonstrated that high

concentrations of potassium inhibit the EDHF response [Chen and Suzuki, 1989].

When vessels were constricted with U46619 the presence of EDHF-mediated

relaxation may have normalised endothelium-dependent relaxation. Diabetes has

been associated with impairment in both NO [Bolego et al., 2006] and EDHF

mediated relaxation [Matsumoto et al., 2006]. However, as the diameter of arteries

becomes smaller, EDHF plays a more important role in mediating endothelium-

dependent relaxation than NO [Shimokawa et al., 1996]. Thus, at the resistance

181

artery level, EDHF may play a greater role in endothelium-dependent relaxation

than nitric oxide. Furthermore, in the rat model of type 1 diabetes, EDHF has been

shown to compensate for reduced bio-availability of NO [Shi et al., 2006]. Kenny et

al. have demonstrated the importance of both NO and EDHF in the myometrial

arterial bed in healthy pregnant women [Kenny et al., 2002], although their relative

contribution in diabetes is uncertain. The present study suggests that EDHF may

play an important role in endothelium-dependent relaxation in the myometrial

vascular bed in women with gestational diabetes.

B) Obesity

Patients in the gestational diabetes group were obese, with a higher BMI than the

normal pregnant group. The impaired endothelium-dependent relaxation noted may

be due to the association between obesity and endothelial dysfunction (see Section

1.6.5.2). However, reports conflict on this association and it is not clear whether

obesity in GDM independently affects endothelial function. A reduction of visceral

fat in non-diabetic obese women has been associated with an improvement in

endothelial function [Park and Shim, 2005]. In contrast to this finding, two studies

examining endothelial function in obese women with previous GDM have not found

obesity to be associated with altered endothelial function. In the first study, equal

impairment in brachial artery flow-mediated dilation was observed in obese and

non-obese women with previous GDM [Anastasiou et al., 1998]. Another study

demonstrated that moderate weight loss and therapeutic lowering of LDL

cholesterol was associated with improvement of endothelial function in obese

women with previous GDM; whereas a similar improvement was not seen with

weight loss alone [Bergholm et al., 2003]. This suggests that LDL levels, rather than

obesity per se may have a stronger influence on endothelial function. It is therefore

uncertain whether the maternal obesity noted here is independently associated with

the endothelial dysfunction demonstrated in GDM patients. However, as obesity is

frequently associated with gestational diabetes, this distinction may not be clinically

relevant.

182

5.5.2.2 Implications of findings

Endothelial dysfunction is associated with functional impairment of target organs in

diabetes such as the heart [Esper et al., 2006] and kidney [Shestakova et al., 2005].

In humans, endothelial dysfunction in type 2 diabetes has been related to altered

hemodynamics in the renal circulation, with aberrant capillary blood flow [Futrakul

et al., 2006]. However, it is unknown whether the endothelial dysfunction

demonstrated in the present study is associated with aberrant uterine blood supply

during pregnancy in GDM. As discussed in Section 1.4.2, even minimal impairment

of arterial diameter can be associated with a marked reduction of blood flow, as

flow is proportional to the fourth power of radius. Reduced blood flow may also be

a factor in the pathogenesis of other complications of diabetes in pregnancy such as

infertility. A study has demonstrated that an absence of sub-endometrial blood flow

results in a poor success rate during in-vitro fertilization [Zaidi et al., 1995].

Endothelial dysfunction in diabetic rats has been positively correlated with the

duration of diabetes [Pieper, 1999]. However, a study has demonstrated that the

degree of diabetes control may be a more important factor influencing endothelial

dysfunction (as estimated by the concentration of asymmetric dimethylarginine)

than the duration of diabetes [Xiong et al., 2005]. Gestational diabetes has a

relatively shorter duration than type 1 diabetes, and in the patients studied the mean

glycated haemoglobin was close to normal levels. Nevertheless, the presence of

endothelial dysfunction in this group indicates that factors other than diabetes

duration and glycaemic control are important. One such factor is insulin resistance,

which has been noted even in lean women with gestational diabetes [Kautzky-Willer

et al., 1997]. The link between insulin resistance and endothelial dysfunction may

involve NO, as endothelial nitric oxide synthesis has been positively correlated with

insulin sensitivity [Petrie et al., 1996]. Insulin resistance has also been demonstrated

to be an important factor in the endothelial dysfunction of type 2 diabetes [Pistrosch

et al., 2004].

GDM is associated with a higher rate of pre-eclampsia [Nordin et al., 2006]. The

aberrant endothelial function reported in pre-eclampsia [Savvidou et al., 2003] may

form a link between these two conditions. Increased insulin resistance has also been

183

noted in pre-eclampsia [Berkowitz, 1998], further strengthening the association with

gestational diabetes.

5.5.2.3 Limitations of study

Compared to studies utilising techniques such as plethysmography, this study was

limited by a relatively small number of patients, despite recruitment from three large

hospitals (see Section 2.14.1). However, the number of patients studied exceeds that

of other reports investigating vascular function in diabetic patients [Ang et al., 2002;

Smits et al., 1993]. As noted in Chapter 3, only a small proportion (11.7 % in the

cohort studied) of women with gestational diabetes underwent elective Caesarean

section, which restricted tissue availability. Patients with potential confounders such

as hypertension and medication, pre-term deliveries and emergency Caesarean

sections were excluded, further reducing patient numbers. This resulted in an

inability to group these patients based on factors such as ethnicity, BMI and mode of

treatment (insulin or diet control). Endothelium-independent relaxation was not

systematically investigated in this study. However, previous studies have

demonstrated no association between hyperglycaemia and endothelium-independent

relaxation [Reed et al., 2004; Williams et al., 1998]. As cholesterol levels were not

routinely checked in all patients, it was uncertain if hypercholesterolaemia was

present, which may affect endothelial function (see Section 1.5.4.4). However,

hypercholesterolaemia was absent in patients whose cholesterol level had been

measured.

5.5.3 Patient characteristics

In this study, despite demonstrating impaired endothelial function in myometrial

arteries, the mean birth weight of babies in the GDM group was higher than normal.

One of the earliest explanations of macrosomia in diabetes is the Pedersen

hypothesis, which states that maternal hyperglycaemia stimulates the fetal pancreas

to secrete excess insulin, resulting in increased adiposity and macrosomia [Pedersen,

1954]. However, in the subsequent decades since this was first postulated, a more

complex picture for the aetiology of large babies in gestational diabetes has

emerged. In both diabetic and non-diabetic pregnant women, maternal obesity is

associated with fetal size [Ehrenberg et al., 2004; Mathew et al., 2005; Yogev et al.,

2005]. A study has demonstrated that macrosomia was not related to gestational age,

184

ethnic group or insulin treatment, but was associated with high fasting plasma

glucose levels, non-pregnant weight and weight gain during pregnancy [Hardy,

1999]. These findings have been corroborated by other studies [Bo et al., 2003;

Yang et al., 2004]. Factors such as maternal weight may therefore be of more

importance in the pathogenesis of macrosomia than uterine blood flow.

Furthermore, it has been shown that decreased uterine blood flow in the diabetic

pregnant rat does not modify the augmented glucose transfer to the fetus [Palacin et

al., 1985]. It is therefore possible that impaired endothelial function in myometrial

arteries does not influence the aetiology of macrosomia, which is multi-factorial and

may be attributable to the high BMI of the patients with GDM in this study.

5.5.4 The effect of glucose levels on vascular function


Changing glucose levels from 5 mmol/L to 2, 5, 8 and 12 mmol/L had no significant

effect on maximum constriction (Fig. 5.2 A-D). Identical results were obtained in

both healthy non-pregnant and pregnant women, signifying that myometrial artery

constriction (an endothelium-independent function) was not influenced by the

glucose levels studied.

No significant difference was noticed in maximum arterial constriction between

arteries from healthy individuals or those with GDM when constricted with KCl

(Fig. 5.10). This implies that myometrial arteries from this group of diabetic patients

had similar smooth muscle function as those from healthy pregnant women. This is

an important observation, as impairment in endothelium-dependent relaxation can

theoretically be attributed to aberrant smooth muscle function. Constriction was

significantly enhanced in the GDM group compared to normal when U46619 was

used (Fig. 5.11), although a smaller number of GDM patients was studied (N=8).


Irrespective of the vasoconstrictor used, exposure to glucose levels of 2, 5, 8 or 12

mmol/L had no effect on endothelium-dependent relaxation in myometrial arteries

185

from women with gestational diabetes (Fig. 5.3 and 5.5). This result is at variance

from that seen in healthy pregnant women, where levels of 2 mmol/L glucose were

associated with impaired endothelium-dependent relaxation when U46619 was used

(Fig. 4.8). It was postulated that blocking EDHF may have enhanced NO-mediated

relaxation in healthy pregnant women. However, this mechanism may be absent in

women with gestational diabetes. This suggests that arteries from women with

GDM respond differently to stimuli such as hypoglycaemia. However, the relatively

small number of patients in the GDM-U46619 group prevents firm conclusions from

being drawn.

Levels of 12 mmol/L glucose had no effect on endothelium-dependent relaxation in

both the normal pregnant group and the GDM group. In the preceding chapter,

possible explanations for the lack of effect of high glucose concentrations in the

normal pregnant group had been discussed (Section 4.5.4). Most of these arguments

may also be relevant in explaining the lack of effect of high glucose concentrations

in the GDM group. Vascular NO has been demonstrated to be reduced after

exposure to 16.6 mmol/L glucose for 1 hour in rat vessels [Lash et al., 1999]. The

level and duration of high glucose concentrations studied may be insufficient to

affect NO bio-availability. Alternatively, it is possible that impairment in NO may

be compensated for by other mediators such as EDHF. Such a compensatory

mechanism has been demonstrated to exist in myometrial arteries from normal

women [Kenny et al., 2002]. Little is known about the association between

hyperglycaemia and mediators such as EDHF and PGI2, as existing research has

mainly focused on NO. Increased production of PGI2 has been reported in the early

stages of diabetes in mice injected with streptozotocin [Shen et al., 2003]. The

association between aberrant glucose levels and the endothelial mediators has been

explored in the following chapter.

5.5.5 Type 1 diabetes and type 2 diabetes

The insufficient numbers of type 1 and 2 diabetes patients recruited (reasons for

which have been described in Section 2.14.1) prevent conclusions from being made

regarding the effect of glucose on endothelial function in these two types of

diabetes. However, data were consistent with a lack of effect of glucose levels on

186

either constriction or endothelium-dependent relaxation was seen, similar to the

results obtained in the GDM group.

5.5.6 SUMMARY

When constricted with 60 mmol/L potassium, myometrial arteries from women with

gestational diabetes demonstrated impaired endothelium-dependent relaxation

compared to those obtained from normal women. This effect was absent when

U46619 was used as an agonist. Exposure to 2, 5, 8 and 12 mmol/L glucose for 30

minutes had no effect on either arterial constriction or endothelium-dependent

relaxation in GDM irrespective of the vasoconstrictor used. Limited availability of

tissue from healthy women and patients with diabetes prevented investigation of the

mechanisms of endothelial function. This limitation was overcome and the

mechanisms examined by utilising an animal model of normal pregnancy and

diabetes in pregnancy, which is discussed in the next chapter.

187

CHAPTER 6

The effect of glucose on endothelial function in non-pregnant

and pregnant mice

188

THE EFFECT OF GLUCOSE ON ENDOTHELIAL FUNCTION IN NON-PREGNANT

AND PREGNANT MICE

6.1 Introduction

The preceding two chapters have illustrated that human research is often limited by

tissue availability, a restriction which can curtail the scope of a study. Researchers

have overcome this obstacle by utilising animal models to understand both

physiological and pathological processes. As discussed in Chapter 1, investigations

in animal models of normal pregnancy have resulted in a greater understanding of

the normal physiological changes occurring in pregnancy. Furthermore, animal

models of diabetes have facilitated investigations of the aetiology of diabetes and its

complications, including those occurring in pregnancy.

Animal studies have usually been conducted independent of human studies: this

often makes extrapolation of findings to humans difficult. Few studies have

concurrently examined the effects of an identical stimulus in both human and animal

physiology. This limits cross-comparison between species, hindering attempts at

understanding physiological differences which may be of importance in animal

models.

For the above reasons, a study of the effect of glucose levels on endothelial function

in animal models would provide valuable insight into whether inter-species

variations of glucose effects exist; as well as allowing investigations which could

not be performed in humans due to limited tissue availability. Little is known about

uterine vascular function in pregnant diabetic mice, as studies have concentrated on

other vascular beds such as mesenteric, carotid and the aorta. Constriction and

relaxation in the main uterine artery of normal pregnant and non-pregnant mice

were evaluated using similar methods as described in Chapter 2. This helped to

minimise methodological differences between human and animal studies.

Due to the limited number of diabetic patients in this study, the creation of an

animal model of diabetes was undertaken, as described in Section 2.14. However,

189

diabetes could be induced only in a minority of mice, whereas most were found to

be resistant to the diabetogenic actions of streptozotocin. Baseline experiments

using previously described protocols were carried out in the pregnant mice not

responding to streptozotocin (streptozotocin-controls), to act as a control group for

future studies in diabetic pregnant mice.

6.2 Aims

1. Examine the effect of 2, 5, 8 and 12 mmol/L glucose for 30 minutes

duration, on constriction and endothelium-dependent relaxation in uterine

arteries from non-pregnant, pregnant and streptozotocin-control mice

2. Examine the effect of pregnancy on constriction and relaxation by

comparing results from non-pregnant and pregnant mice

3. Examine the effect of hyper-osmolarity on endothelium-dependent

relaxation

4. Examine the role of the mediators of endothelium-dependent relaxation in

the uterine artery of normal pregnant mice

5. Examine whether aberrant glucose concentrations altered the contribution of

these mediators.


These were previously described in Chapter 2. The experimental protocols used

were Protocol 1, 2, 3 and 4 (see Section 2.12). Uterine arteries were constricted with

phenylephrine and relaxed with acetylcholine. Arteries with maximum constriction

less than 0.2 mN/mm were excluded from analysis.

190

6.4 Results

6.4.1 Arterial diameters

The mean diameter of uterine arteries from pregnant mice (250 ± 4 µm; N=18, 62

arteries) was significantly greater than that from non-pregnant mice (184 ± 3 µm;

N=15, 44 arteries) [Fig. 6.1, unpaired t-test: p < 0.0001].

6.1 Arterial Diameter: Mice

Non-Pregnant Pregnant100

150

200

250

300

350

p < 0.0001

Art

eri

al

Dia

mete

r

m

Fig. 6.1: Arterial diameters – normal non-pregnant and pregnant mice

Diameter of uterine arteries from normal non-pregnant and pregnant mice.

Horizontal red bars represent mean values. Mean diameter significantly greater in

pregnant mice.

Unpaired t-test: p < 0.0001

191

6.4.2 Constriction: normal non-pregnant mice

Exposure of uterine arteries from normal non-pregnant mice to 2, 5, 8 and 12

mmol/L glucose for 30 minutes had no effect on arterial constriction (Fig. 6.2(A-D);

paired t-test: p > 0.05 in all comparisons; at 12 mmol/L glucose: mean difference =

-0.95, 95% confidence intervals = -1.97 to 0.07).

6.4.3 Endothelium-dependent relaxation: normal non-pregnant

mice

Endothelium-dependent relaxation in uterine arteries of normal non-pregnant mice

was not altered by exposure to 2, 5, 8 or 12 mmol/L glucose for 30 minutes (Fig.

6.3(A-D); repeated measures ANOVA: p > 0.05). The similarity in relaxation was

also illustrated in Fig. 6.4 where the relaxation at all four glucose levels are shown.

192


5 mmol/L 2 mmol/L 0

10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

(kP

a)



10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

(kP

a)


5 mmol/L 8 mmol/L0

10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

(kP

a)


5 mmol/L 12 mmol/L0

10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

(kP

a)

Fig. 6.2A-D: Constriction: effect of glucose (normal non-pregnant mice)

Constriction at 5 mmol/L glucose and after 30 minute incubation at 2, 5, 8, and 12

mmol/L glucose in uterine arteries from normal non-pregnant mice (N = 15).


M phenylephrine. Constriction assessed at:





No significant difference in constriction noted.


193


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

2 mmol/L Glucose

Acetylcholine M [log]

% M

axim

um

Co

nstr

icti

on


-10 -9 -8 -7 -60

25

50

75

100



Acetylcholine [log]

% M

axim

um

Co

nstr

icti

on


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

8 mmol/L Glucose

Acetylcholine [log]

% M

axim

um

Co

nstr

icti

on


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

12 mmol/L Glucose

Acetylcholine [log]

% M

axim

um

Co

nstr

icti

on

Fig. 6.3A-D: Relaxation: effect of glucose (normal non-pregnant mice)


incubation at 2, 5, 8, and 12 mmol/L glucose in uterine arteries from normal non-

pregnant mice (N = 15). Vessels constricted with 10-5

M phenylephrine. Relaxation

assessed at:





No significant difference in relaxation noted.

Repeated measures ANOVA, p > 0.05

194

6.4 Relaxation: Non-Pregnant Mice

-10 -9 -8 -7 -60

25

50

75

100

2 mmol/L Glucose

5 mmol/L Glucose

8 mmol/L Glucose

12 mmol/L Glucose


% M

axim

um

Co

nstr

icti

on

Fig. 6.4: Relaxation: effect of glucose (normal non-pregnant mice)

Unpaired analysis of data in Fig 6.3. Endothelium-dependent relaxation at 2 (blue),

5 (red), 8 (green), and 12 (orange) mmol/L glucose in uterine arteries from normal

non-pregnant mice (N = 15). Vessels constricted with 10-5

M phenylephrine.

No difference in relaxation noted.


195

6.4.4 Constriction: normal pregnant mice

In contrast to findings in non-pregnant mice, a significant increase in constriction

was seen in arteries from normal pregnant mice when exposed for 30 minutes to 2, 8

or 12 mmol/L glucose for 30 minutes (Fig. 6.5(A-D); paired t-test: p < 0.05). This

effect was not seen in the control vessel at 5 mmol/L glucose (Fig. 6.5B; paired t-

test: p > 0.05). Mean values (± SEM) of active effective pressure were:

5 and 2 mmol/L glucose = 16.3 (± 1.2) and 17.3 (± 1.3) kPa




6.4.5 Endothelium-dependent relaxation: normal pregnant mice

Endothelium-dependent relaxation demonstrated impairment with time at 5 mmol/L

glucose (control vessel), 2 and 8 mmol/L glucose (Fig. 6.6(A-C); repeated measures

ANOVA: p < 0.05). However, at 12 mmol/L glucose, no significant impairment

with time was seen (Fig. 6.6D; repeated measures ANOVA: p > 0.05), signifying an

enhancement of endothelium-dependent relaxation at this glucose level. This effect

was more clearly demonstrated by plotting endothelium-dependent relaxation at the

four different glucose concentrations together (Fig. 6.7). In contrast to findings in

non-pregnant mice, relaxation was significantly enhanced at 12 mmol/L glucose

compared to 5 mmol/L (two-way ANOVA, p < 0.001). Bonferroni post-hoc testing

revealed a significant difference at 10-7

M acetylcholine (p < 0.01, mean difference

= -18.22, 95% confidence intervals = -36.71 to 0.27).

196


5 mmol/L 2 mmol/L 0

10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a



10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a


5 mmol/L 8 mmol/L0

10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a


5 mmol/L 12 mmol/L0

10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a

Fig. 6.5A-D: Constriction: effect of glucose levels (normal pregnant mice)


mmol/L glucose in uterine arteries from normal pregnant mice (N = 18). Vessels


M phenylephrine. Constriction assessed at:


p < 0.05, paired t-test

Fig. 6.5B: 5 mmol/L glucose (circles) (n=17 arteries) p > 0.05, paired t-test





197


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

2 mmol/L Glucose


% M

axim

um

Co

nstr

icti

on


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L: Part 1

5 mmol/L: Part 2


% M

axim

um

Co

nstr

icti

on


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

8 mmol/L Glucose


% M

axim

um

Co

nstr

icti

on


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

12 mmol/L Glucose


% M

axim

um

Co

nstr

icti

on

Fig. 6.6A-D: Relaxation: effect of glucose levels (normal pregnant mice)


incubation at 2, 5, 8, and 12 mmol/L glucose in uterine arteries from normal

pregnant mice (N = 18). Vessels constricted with 10-5

M phenylephrine.






198

6.7 Relaxation: Pregnant Mice

-10 -9 -8 -7 -60

20

40

60

80

100

2 mmol/L Glucose

5 mmol/L Glucose

8 mmol/L Glucose

12 mmol/L Glucose

*

p < 0.01


% M

axim

um

Co

nstr

icti

on

Fig. 6.7: Relaxation: effect of glucose (normal pregnant mice)

Unpaired representation of data from Fig. 6.6. Endothelium-dependent relaxation at

2 (blue), 5 (red), 8 (green), and 12 (orange) mmol/L glucose in uterine arteries from

normal pregnant mice (N = 18). Vessels constricted with 10-5

M phenylephrine. No

significant difference was seen at 2 or 8 mmol/L glucose compared to control (two-

way ANOVA: p > 0.05). Relaxation was significantly enhanced at 12 mmol/L

glucose compared to control at 5 mmol/L glucose (two-way ANOVA: p < 0.01).

Bonferroni post-hoc testing revealed significant difference at 10-7

M acetylcholine

(* p < 0.01)

199

6.4.6 Effect of pregnancy on constriction and endothelium-


Uterine arteries from pregnant mice demonstrated enhanced constriction (Fig. 6.8;

unpaired t-test: p < 0.05) as well as enhanced endothelium-dependent relaxation

(Fig. 6.9; two-way ANOVA: p < 0.05) compared to arteries from non-pregnant

mice.

6.4.7 Effect of hyper-osmolarity - pregnant mice

No difference in endothelium-dependent relaxation was seen in a normal osmolar

medium (at 5 mmol/L glucose) compared to a hyper-osmolar medium (with an

additional 7 mmol/L mannitol) (Fig. 6.10; two-way ANOVA: p > 0.05,)

6.8 Constriction: Non-pregnant vs pregnant mice

Non-Pregnant Mice Pregnant Mice0

10

20

30

40

p < 0.001*

Acti

ve E

ffecti

ve P

ressu

re

(kP

a)

Fig. 6.8: Constriction: normal non-pregnant vs pregnant mice

Comparison of constriction in uterine arteries at 5 mmol/L glucose in normal non-

pregnant (triangles; N=15, n=41arteries) and pregnant mice (inverted triangles;

N=18, n=62 arteries) and Vessels were constricted with 10-5

M phenylephrine.

Horizontal bars represent mean values. Constriction was significantly enhanced in

arteries from pregnant mice

* Unpaired t-test: p < 0.001

200

6.9 Relaxation: Non-pregnant vs Pregnant mice

-10 -9 -8 -7 -60

25

50

75

100

Pregnant Mice

Non-Pregnant Mice

*

p < 0.001


% M

axim

um

Co

nstr

icti

on

Fig. 6.9: Relaxation: non-pregnant vs pregnant mice

Comparison of endothelium-dependent relaxation in uterine arteries at 5 mmol/L

glucose in normal non-pregnant (triangles; N=15, n=41 arteries) and pregnant mice

(circles; N=18, n=62 arteries) and. Vessels were constricted with 10-5

M

phenylephrine. Endothelium-dependent relaxation was significantly enhanced in

arteries from pregnant mice

Two-way ANOVA: p < 0.001

Bonferroni post-hoc test: at 10-6

M acetylcholine * p <0.001

201

6.10 Effect of Hyper-osmolarity

-10 -9 -8 -7 -60

25

50

75

100

Hyper-osmolar

solution

Normal osmolar

solution


% M

axim

um

Co

nstr

icti

on

Fig. 6.10: The effect of hyper-osmolarity:

Endothelium-dependent relaxation in uterine arteries from pregnant mice (N=3)

compared in two different solutions

Normal osmolar solution (= 5 mmol/L glucose PSS; solid circle)

Hyper-osmolar mannitol solution (= 5 mmol/L glucose PSS + 7 mmol/L of

Mannitol; open square).

No significant difference was noted in endothelium-dependent relaxation

Two-way ANOVA: p < 0.05).

202

6.4.8 Normal pregnant mice: effect of endothelial blockers at 5

and 12 mmol/L glucose

At both 5 mmol/L (Fig. 6.11) and 12 (Fig. 6.12) mmol/L glucose, indomethacin had

no effect on endothelium-dependent relaxation compared to control (two-way

ANOVA: p > 0.05). L-NNA was associated with a significant impairment of

relaxation at both 5 and 12 mmol/L glucose (two-way ANOVA: p < 0.05). Impaired

relaxation compared with control was also present when L-NNA and indomethacin

were used in combination at 12 mmol/L glucose (two-way ANOVA: p < 0.05).

Endothelium-dependent relaxation was predominantly due to a non-NO/non-

prostanoid mechanism, most likely EDHF, although this was not specifically tested.

6.4.9 Normal pregnant mice: effect of glucose levels on

endothelial blockers

Endothelium-dependent relaxation mediated by NO (as blocked by L-NNA, Fig.

6.13B) and prostanoids (as blocked by indomethacin, Fig. 6.13C) was similar at

both 5 mmol/L and 12 mmol/L glucose (two-way ANOVA, p > 0.05). The non-

NO/non-prostacyclin component of relaxation was also similar at both levels (Fig.

6.13D, two-way ANOVA: p > 0.05).

203

6.11 Endothelial Blockers at 5 mmol/L Glucose

-10 -9 -8 -7 -60

20

40

60

80

100

Control

+ L-NNA

+ IND

+ L-NNA + IND

p < 0.05

*


% M

axim

um

Co

nstr

icti

on

Fig. 6.11: The effect of blockers on relaxation – 5 mmol/L glucose

The effects of specific blockers to endothelium-dependent relaxation in uterine

arteries of pregnant mice (N=13) were evaluated at 5 mmol/L glucose. Vessels


M phenylephrine.

Control (circles, n=8 arteries) L-NNA (inverted triangles, n=13 arteries)

indomethacin (squares, n=9 arteries) L-NNA & indomethacin (triangles, n=12

arteries)

Control vs indomethacin: two-way ANOVA, p > 0.05

Control vs L-NNA + indomethacin two-way ANOVA, p > 0.05

Control vs L-NNA two-way ANOVA, p < 0.05

204

6.12 Endothelial Blockers at 12 mmol/L Glucose

-10 -9 -8 -7 -60

20

40

60

80

100

Control

+ L-NNA

+ IND

+ L-NNA + IND

*

p < 0.05


% M

axim

um

Co

nstr

icti

on

Fig. 6.12: The effect of blockers on relaxation – 12 mmol/L glucose

The effects of specific blockers to endothelium-dependent relaxation in uterine

arteries of pregnant mice (N=15) were evaluated at 12 mmol/L glucose. Vessels


M phenylephrine.

Control (circles, n=11 arteries) L-NNA (inverted triangles, n=14 arteries)

indomethacin (squares, 13 arteries) L-NNA & indomethacin (triangles, n=15

arteries)

Control and indomethacin: two-way ANOVA, p > 0.05

Control and L-NNA: two-way ANOVA, p < 0.05

Control and L-NNA + indomethacin: two-way ANOVA, p < 0.05

(Bonferroni post-hoc test= 10-6

M ACh, p < 0.05)

205

6.13A Control

-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

12 mmol/L Glucose


% M

axim

um

Co

nstr

icti

on

6.13B L-NNA

-10 -9 -8 -7 -60

25

50

75

100

12 mmol/L Glucose

5 mmol/L Glucose


% M

axim

um

Co

nstr

icti

on

6.13C Indomethacin

-10 -9 -8 -7 -60

25

50

75

100

12 mmol/L Glucose

5 mmol/L Glucose


% M

axim

um

Co

nstr

icti

on

6.13D L-NNA + Indomethcin

-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

12 mmol/L Glucose


% M

axim

um

Co

nstr

icti

on

Fig. 6.13A-D: The effect of blockers at 5 and 12 mmol/L glucose

Comparison of endothelium-dependent relaxation at 5 and 12 mmol/L glucose after

addition of blockers

Fig. 6.13A: Control at 5 (solid circles) and 12 (open circles) mmol/L glucose

Fig. 6.13B: L-NNA at 5 (solid inverted triangles) and 12 (open inverted triangles)

mmol/L glucose

Fig. 6.13C: Indomethacin at 5 (solid squares) and 12 (open squares) mmol/L

glucose

Fig. 6.13D: L-NNA + Indomethacin at 5 (solid triangles) and 12 (open triangles)

mmol/L glucose

No significant difference in endothelium-dependent relaxation was seen at any

glucose level (two-way ANOVA, p > 0.05)

206

6.4.10 Blood glucose in mice

Mean (± SD) blood glucose of mice before streptozotocin injection was 6.8 (± 0.5)

mmol/L. Values obtained after injection of either streptozotocin or vehicle alone are

illustrated in Fig. 6.14. Very few mice (approximately 17%) developed diabetes

(two examples shown: D1-2), with mean (± SD) blood glucose levels of 16.2 (± 2.2)

mmol/L glucose. Mice not responding to streptozotocin (STZ-control, S1-S8) had

blood glucose levels of 7.4 (± 1.2) mmol/L, whereas mice injected with vehicle

alone (vehicle-control, two examples shown: V1-2) had levels of 6.7 (± 0.9) mmol/L

glucose. There was no significant difference in glucose levels between STZ-control

and vehicle-control mice (one-way ANOVA, Bonferroni post test: p > 0.05).

Diabetic mice had significantly higher blood glucose levels than both

streptozotocin-controls and vehicle controls (one-way ANOVA, Bonferroni post

test: p < 0.001). Due to the extremely poor success rate for diabetes induction and

the resultant time constraints, only results from streptozotocin-controls (S1-8) have

been demonstrated in subsequent experiments.

207

6.14 Blood Glucose: Mice

S1 S2 S3 S4 S5 S6 S7 S8 D1 D2 V1 V20

2

4

6

8

10

12

14

16

18

20

Mice

Blo

od

Glu

co

se:

mm

ol/

L

Fig. 6.14: Blood glucose: mice

Glucose levels measured in mice after injection of streptozotocin or vehicle:

S1 - 8 = Streptozotocin –controls: mice injected with streptozotocin, non-diabetic

D1-2 = Diabetic mice: mice injected with streptozotocin, diabetic

V1-2 = Vehicle-controls: mice injected with vehicle only

6.4.11 Pregnant streptozotocin-control mice

6.4.11.1 Constriction: effect of glucose levels (pregnant streptozotocin-

control mice)

Maximum active effective pressure was compared before and after acute changes in

glucose concentration in uterine arteries of pregnant mice (N=8) that had been

injected with streptozotocin, but did not develop diabetes (Fig. 6.15). A significant

increase of active effective pressure was noted in the control arteries (5 mmol/L

glucose) as well as after incubation at 8 and 12 mmol/L glucose (paired t-test: p <

0.05). However, no significant difference was noted after incubation at 2 mmol/L

208

glucose (paired t-test: p > 0.05). Mean values (± SEM) of active effective pressure

were:

5 and 2 mmol/L glucose = 14.6 (± 0.8) and 15.9 (± 0.6 kPa)

5 and 5 mmol/L glucose (control) = 18.2 (± 0.8) and 20.0 (± 0.6) kPa



6.4.11.2 Relaxation: effect of glucose levels (pregnant streptozotocin-

control mice)

Endothelium-dependent relaxation in uterine arteries of pregnant non-diabetic mice

treated with streptozotocin was not altered by exposure to 2, 5, 8 or 12 mmol/L

glucose for 30 minutes (Fig. 6.16, repeated measures ANOVA, p > 0.05). The

similarity in relaxation has also been demonstrated in Fig. 6.17 where relaxation at

all four glucose levels have been shown.

209


5 mmol/L 2 mmol/L0

10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a



10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a


5 mmol/L 8 mmol/L0

10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a


5 mmol/L 12 mmol/L0

10

20

30

40


Acti

ve E

ffecti

ve P

ressu

re

kP

a

Fig. 6.15A-D: Constriction: effect of glucose levels (pregnant STZ-controls)


mmol/L glucose in uterine arteries from pregnant mice not responsive to

streptozotocin (N = 8). Vessels constricted with 10-5

M phenylephrine.

Constriction assessed at:


p > 0.05, paired t-test

Fig. 6.15B: 5 mmol/L glucose (circles) (n=7 arteries) p < 0.05, paired t-test





210


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

2 mmol/L Glucose


% M

ax

imu

m C

on

str

icti

on


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/LGlucose: Part 1

5 mmol/LGlucose: Part 2


% M

ax

imu

m C

on

str

icti

on


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

8 mmol/L Glucose


% M

ax

imu

m C

on

str

icti

on


-10 -9 -8 -7 -60

25

50

75

100

5 mmol/L Glucose

12 mmol/L Glucose


% M

ax

imu

m C

on

str

icti

on

Fig. 6.16A-D: Relaxation: the effect of glucose (pregnant STZ-controls)


incubation at 2, 5, 8, and 12 mmol/L glucose in uterine arteries from pregnant mice

not responsive streptozotocin (N = 8). Vessels constricted with 10-5

M

phenylephrine. Relaxation assessed at:






211

6.17 Relaxation: Pregnant STZ-Controls

-10 -9 -8 -7 -60

25

50

75

1002 mmol/L Glucose

8 mmol/L Glucose

12 mmol/L Glucose

5 mmol/L Glucose


% M

axim

um

Co

nstr

icti

on

Fig. 6.17: Relaxation: effect of glucose levels (pregnant STZ-controls)

Unpaired representation of data from Fig. 6.14. Endothelium-dependent relaxation

at 2 (blue), 5 (red), 8 (green), and 12 (orange) mmol/L glucose in uterine arteries

from pregnant STZ non-responding mice (N=8). Vessels constricted with 10-5

M

phenylephrine.

Two-way ANOVA: p > 0.05

212

6.5 Discussion

6.5.1 Summary of findings

The findings in this section have been summarised in Table 6.1 below.

Group Parameter Effect of glucose levels

Normal non-pregnant

mice (N=15 mice, n=44

arteries)


Endothelium-dependent

relaxation No effect

Normal pregnant mice

(N=18 mice, n=62

arteries)

Constriction Enhanced at 2, 8 and 12

mmol/L glucose


relaxation

Enhanced at 12 mmol/L

glucose

STZ- control pregnant

mice (N=8 mice, n=31

arteries)

Constriction Attenuated at 2 mmol/L

glucose


relaxation No effect

Table 6.1: Summary of findings

6.5.2 Enhanced vasodilation at 12 mmol/L glucose

The main finding of this study was that 12 mmol/L glucose lasting 30 minutes was

associated with enhanced endothelium-dependent relaxation in the uterine artery of

normal pregnant mice. This effect was specific to pregnancy, as no such change was

noticed in arteries from non-pregnant mice. It was also independent of hyper-

osmolarity, as a hyper-osmolar mannitol solution had no effect on endothelium-

dependent relaxation in pregnant mice.

213

Studies reporting endothelium-dependent vasodilation with hyperglycaemia have

been previously discussed (Table 1.4). These studies varied in the precise glucose

levels examined; therefore the threshold above which raised glucose concentrations

start affecting vessel function is unknown. In the uterine arteries of pregnant mice,

vasodilation was noted at 12 mmol/L glucose but not at 8 mmol/L. This narrows the

range at which the vasoactive effects of glucose become evident in this vascular

bed. Exposure to 12 mmol/L glucose for 30 minutes represents one of the lowest

and shortest degrees of hyperglyacemia for which vascular effects have been

demonstrated. Interestingly, studies demonstrating vasodilation with

hyperglycaemia (Table 1.4) generally had a duration of hyperglycaemia of an hour

or less [Hoffman et al., 1999; van Veen et al., 1999], whereas those demonstrating

impaired relaxation (Table 1.3) had a duration of 1-6 hours [Affonso Fde et al.,

2003; Kawano et al., 1999; Williams et al., 1998]. Therefore, it may be possible that

hyperglycaemia has a biphasic effect on vascular function: enhancing vasodilation

during short-term exposures, but causing impairment during longer exposures. The

vasodilation observed in the uterine artery at high glucose concentrations may

enhance blood flow to the offspring in the post-prandial period. The longer

exposures to hyperglycaemia may uncouple the endothelial Nitric Oxide Synthase

(eNOS) enzyme; resulting in the generation of free radicals and diminished NO

availability [Santilli et al., 2004]. NO has been demonstrated to be reduced after

exposure to 16.6 mmol/L glucose for 1 hour in rat spinotrapezius arterioles [Lash et

al., 1999].

A limitation of the present study is that the effect of a more prolonged exposure to

high glucose concentrations was not examined. This was due to the methodological

limitations of using wire myography: after a 2-hour exposure to high glucose

concentrations, ex-vivo uterine arteries were found to have diminished viability with

erratic responses. Similar aberrant responses were seen in human myometrial

arteries (see Section 4.4.12). Therefore it is uncertain whether the enhanced

endothelium-dependent vasodilation continues to be present after a longer exposure

to high glucose concentrations.

214

6.5.3 Glucose requirements in different vascular beds

In normal individuals, hyperglycaemia has been associated with both vasodilation

[Hoffman et al., 1999; van Veen et al., 1999] and conversely, the impairment of

endothelium-dependent vasodilation [Title et al., 2000; Williams et al., 1998]. In

animals, Cipolla et al. has shown that hyperglycaemia (44 mmol/L for 1-2 hours)

induces endothelium-dependent vasodilation in cerebral arteries of rats [Cipolla et

al., 1997]. As glucose is a crucial substrate for many cellular processes,

hyperglycaemia may have different effects at different vascular beds depending on

the metabolic requirements of that tissue region. Therefore, studies in

physiologically relevant vascular beds may be of more value in understanding the

pathophysiology of hyperglycaemia. As the glucose requirements are vastly

different in the cerebral or uterine circulation compared to forearm musculature or

skin, these different effects may be an adaptive response to limited substrate

availability. The vasodilation observed in the uterine artery at high glucose

concentrations may therefore represent a vascular adaptation to promote blood flow

and the delivery of nutrients to the offspring in the post-prandial period.

6.5.4 Hyperglycaemia and hyper-osmolarity

It has been previously reported that hyper-osmolarity is associated with

vasodilation, mediated via the endothelium [Massett et al., 2000; Sasaki et al.,

1986]. Vasodilation seen in this study is an effect specific for high glucose

concentrations, as incubation in an equi-osmolar mannitol solution did not alter

endothelium-dependent relaxation. Furthermore, at 12 mmol/L glucose, vasodilation

was seen only in arteries from pregnant mice and not in those from non-pregnant

mice. The change in osmolarity induced by increasing glucose levels from 5 to 12

mmol/L is minimal: studies have shown the effect of hyper-osmolarity to become

evident only at much higher levels, such as 36 mmol/L glucose [Wang et al., 1998].

Other studies have also demonstrated that alterations in vascular function with

hyperglycaemia are not due to hyper-osmolarity [Affonso Fde et al., 2003; Gomes

et al., 2004; Sercombe et al., 2004].

215

6.5.5 Hyperglycaemia and systemic factors

Systemic factors have been implicated in the modulation of vascular function by

hyperglycaemia. These include alterations in neural activity and increased insulin

levels in the blood. Acute hyperglycaemia in normal individuals has been associated

with increased sympathetic neural activation and vasodilation [Hoffman et al.,

1999]. Hyperosmolarity may have been responsible for this vasodilation, as

mannitol was found to produce effects similar to glucose. This is in contrast to the

results demonstrated in this section, where mannitol had no vasoactive effect.

Hyperinsulinemia has been associated with vasodilation involving NO release

[Scherrer et al., 1994]. Renaudin et al. reported arteriolar vasodilation in a rat

trapezius muscle preparation with hyperinsulinaemia alone, but vasoconstriction

when hyperglycaemia and hyperinsulinaemia were present [Renaudin et al., 1998].

Systemic hyperinsulinaemia has been demonstrated to be more important than local

hyperinsulinaemia in mediating vasodilation [Cardillo et al., 1998]. These studies

suggest that the concurrent increase in insulin level with hyperglycaemia may play

an important role in mediating vasodilation in vivo. However, the present study has

demonstrated that high glucose concentrations can enhance vasodilation by a local

action independent of systemic factors such as hyperosmolarity, hyperinsulinaemia

and neural input.

6.5.6 Effect of pregnancy

Vasoconstriction, as measured by active effective pressure, was significantly higher

in uterine arteries from pregnant mice compared to non-pregnant mice, signifying

that the state of pregnancy is associated with an enhanced constrictor response to

phenylephrine. Enhanced endothelium-dependent relaxation was also seen in uterine

arteries from pregnant mice compared to those obtained from non-pregnant mice.

Similar augmentation of vasodilation in pregnancy has been previously reported in

human uterine arteries [Nelson et al., 1998] and the rat aorta [Honda et al., 1996].

As demonstrated in Chapter 4, no such enhanced vasodilation during pregnancy was

present in human myometrial arteries, highlighting the differences in artery function

at disparate vascular beds in pregnancy. These findings also raise the possibility of

inter-species variation in the effect of preganancy on vasculature.

216

6.5.7 Effect of glucose on constriction

Exposure to 30 minutes of 2, 8 and 12 mmol/L glucose was associated with an

increase in constriction in uterine arteries of normal pregnant mice; an effect not

present either in the control arteries at 5 mmol/L glucose or at any glucose level

examined in normal non-pregnant mice. Although this enhanced constriction was

statistically significant, the actual change in active effective pressure was small. The

physiological relevance of this finding is therefore uncertain.

The above findings signify that unique alterations occur in mouse uterine arteries

during pregnancy. Morphological changes include increased diameter whereas

functional changes comprise of alterations in the physiological effects of glucose.

Preganancy was also associated with an increase in maximal constriction and

endothelium-dependent relaxation of uterine arteries.

6.5.8 Endothelial mediators of vasodilation

This study demonstrated that in small-calibre arteries, endothelium-dependent

relaxation is mainly mediated by a non-NO/non-prostacyclin mediator; most likely

EDHF, although this was not specifically blocked. NO played a less important role

in this vascular bed, whereas prostacyclin did not mediate endothelium-dependent

relaxation in the uterine artery of pregnant mice. These findings corroborate other

studies that have documented the dominance of EDHF-mediated relaxation in small

arteries [Shimokawa et al., 1996; Urakami-Harasawa et al., 1997]. In contrast, NO

has been associated with relaxation in larger arteries such as the aorta in rats [Vizioli

et al., 2005]. Current research in the field of endothelium-dependent relaxation in

hyperglycaemia is predominantly directed at the contribution of NO, with relatively

few studies examining the role of EDHF. This is because hyperglycaemia is

associated with increased free radical formation and diminished bio-availability of

NO, which contributes to endothelial dysfunction in diabetes [Marfella et al., 2000;

Monnier et al., 2006]. The present study has demonstrated no alteration in NO-

mediated relaxation after exposure to mildly raised glucose concentrations for 30

minutes. As EDHF was not specifically blocked, the effects of glucose on this

mediator are uncertain. Resistance arteries play an important role in modulating

blood flow to target organs and EDHF has been demonstrated to be the predominant

217

mediator of endothelium-dependent relaxation in these vessels [Shimokawa et al.,

1996]. Reduced EDHF has been recently associated with endothelial dysfunction in

streptozotocin-induced diabetic mice [Matsumoto et al., 2006]. Hence

characterization of EDHF by future studies may help to discern its contribution to

endothelial function during hyperglycaemia.

As described in Section 1.6.4, there is considerable variation in the proportion of

endothelium-dependent relaxation mediated by the three endothelial mediators

depending on the vascular bed. When Crauwels et al. examined these mediators in

C57BL/6 mice (also used in the present study), endothelium-dependent relaxation

was demonstrated to be mediated almost exclusively by nitric oxide in the carotid

artery [Crauwels et al., 2000]. In the femoral artery of these mice, relaxation was

pre-dominantly mediated by NO with a smaller contribution from a non-prostanoid/

non-NO factor. No prostanoid-mediated relaxation was seen in the carotid or

femoral arteries. These results are at variance to those demonstrated in the present

section, where NO had a relatively small role in mediating relaxation in the uterine

arteries. This emphasizes the variation in activity of the endothelial mediators at

different vascular beds.

A number of studies have unravelled a more complex inter-relationship between the

endothelial vasorelaxants than originally envisaged. Nitric oxide and cyclo-

oxygenase derivatives can substitute for each other in producing relaxation in

human small omental arteries [Ohlmann et al., 1997]. Therefore, blocking one

pathway may enhance the vasodilation mediated by another pathway. As discussed

in Chapter 3, a similar mechanism has been observed in myometrial arteries of

normal pregnant women [Kenny et al., 2002]. In mesenteric arteries of eNOS-

knockout mice eNOS (-/-), endothelium-dependent relaxation was mediated by

upregulation of EDHF in the absence of NO [Waldron et al., 1999]. Similar results

have been reported in muscle arterioles of female eNOS-knockout mice [Huang et

al., 2001]. In addition, the proportion of relaxation mediated by prostacyclin, EDHF

and NO has been demonstrated to vary depending on gender; as discussed in Section

1.5.2.1. [Scotland et al., 2005]. Estimating the true role of a specific pathway in

relaxation can therefore be challenging.

218

The enhanced vasodilation demonstrated in the present study could not be attributed

to enhancement of a particular endothelium-dependent mediator. This may be

because EDHF was not specifically blocked. One of the reasons for this was that

previous studies had demonstrated an important role for NO in mediating aberrant

responses to hyperglycaemia [Giugliano et al., 1997; Trachtman et al., 1997],

making it the main area of interest in this study. Furthermore, the relative

importance of NO, PGI2 and EDHF in this vascular bed was unknown, as this has

not been previously reported in the literature. There were also technical limitations

of the myograph apparatus: the addition of 25 mmol/L potassium to block EDHF

would envisage a fifth artery studied in parallel, whereas a maximum of 4 arteries

can be studied in the myograph used. This preliminary characterisation of vascular

function in this vascular bed, has however pointed to the importance of examining

EDHF in future studies. This can be done by utilising specific blockers such as a

combination of apamin and charybdotoxin, which abolishes EDHF-mediated

relaxation [Coleman et al., 2004].

6.5.9 Streptozotocin treated non-diabetic mice (STZ-controls)

As discussed in Section 2.14.7, the majority of mice injected with streptozotocin

failed to develop diabetes. Failure to conceive was noted in diabetic mice, in

addition to deterioration in health during pregnancy. Due to these factors, only data

from vascular studies on streptozotocin treated non-diabetic mice has been

presented.

Possible reasons for failure to induce diabetes include differences in the cell

responsiveness to streptozotocin, as well as the age of mice. Streptozotocin has been

demonstrated to induce more beta-cell destruction in young animals than in older

mice [Riley et al., 1981]. The mice used in the present study were 8 weeks old at the

time of injection of streptozotocin. In C57BL/6 mice, diabetes has been induced by

streptozotocin from the age of 4 weeks [Shen et al., 2003] to 5 months [Fahim et al.,

1999]. It is therefore uncertain how important age is in determining resistance to the

diabetogenic actions of streptozotocin in C57BL/6 mice. Differences in cell

responsiveness to the action of streptozotocin has also been demonstrated between

different strains of mice [Abramovici and Agarwal, 1985; Hayashi et al., 2006].

219

At the control level of glucose (5 mmol/L), uterine arteries of STZ-control mice

demonstrated a small, but significant increase in constriction during the second part

of the experiment, implying a time effect. This was also seen at 8 and 12 mmol/L

glucose, although the increase was not significant at 2 mmol/L glucose. However,

these changes were minimal, and as noted in normal pregnant mice, may not be

biologically significant.

In these mice, endothelium-dependent relaxation was not affected by changes in

glucose levels, in contrast to the vasodilation noted in normal pregnant mice at 12

mmol/L glucose. The relatively small number of streptozotocin-control mice studied

(N=8) compared to the normal pregnant mice (N=18) prevents a valid interpretation

of these results. Furthermore, it is not known if streptozotocin alters vascular

function independent of its diabetogenic properties, due to the marked lack of

reports on vessel function in non-diabetic streptozotocin-control mice. These results

may therefore be important in examining whether streptozotocin can influence

vascular function independent of diabetes.

Postscript: Work on the mice model of diabetes in pregnancy has been

subsequently carried forward by the postgraduate student Joanna Stanley, with

experiments being performed in vehicle-control, streptozotocin-control and diabetic

mice.

6.5.10 SUMMARY

Pregnancy in normal mice is associated with changes in uterine vascular reactivity

to glucose. Exposure to 12 mmol/L glucose for 30 minutes enhanced endothelium-

dependent vasodilation. This effect was not seen in uterine arteries of non-pregnant

mice. The endothelium-dependent relaxation was mainly due to a non-NO/non-

prostanoid mediator and to a lesser degree NO, with no contribution from

prostacyclin in this vascular bed. This indicates that short exposures to elevated

glucose concentrations can influence vascular function of the uterine arteries during

pregnancy. This effect is mediated locally and is independent of osmotic, hormonal

or neural mechanisms. The development of the animal model of diabetes in

pregnancy will facilitate future investigations into the effects of glucose on vascular

function.

220

CHAPTER 7

DISCUSSION

221

DISCUSSION

7.1 Overview of study

The main purpose of this study was to examine if acute changes in glucose

concentration affected endothelial function in normal pregnancies and pregnancies

complicated by diabetes. A limitation of earlier studies was that theoretical rather

than empirical concentrations had been used to examine the effects of

hyperglycaemia. Furthermore, extremely few studies have examined the effect of

hypoglycaemia on endothelial function. The first part of this study was to therefore

identify clinically-relevant glucose concentrations during diabetes in pregnancy.

Although the duration of these aberrant glucose concentrations was not examined,

results from earlier studies of diabetes in pregnancy were used to select a clinically-

relevant duration of exposure. Arteries of the uterine vascular bed were directly

studied at these glucose levels, thereby overcoming limitations of previous studies

which examined vascular function in large conduit arteries at extremely high

glucose levels. By studying ex-vivo arteries using wire myography, vessel function

could be characterised in both human and animal vascular beds over a range of

glucose concentrations. This enabled the comparison of effects between species at

standardised experimental conditions.

This work represents the first study of the effects of glucose concentration on

endothelial function in human myometrial arteries and mice uterine arteries. Altered

vascular function was observed at both high and low glucose concentrations. At 2

mmol/L glucose, myometrial arteries from normal pregnant women exhibited

impaired endothelium-dependent relaxation when constricted with U46619; an

effect not seen in arteries from non-pregnant women. This represents the first report

of a possible link between hypoglycaemia and impaired endothelium-dependent

relaxation. At 12 mmol/L glucose, enhanced endothelium-dependent relaxation was

noted in uterine arteries of pregnant, but not non-pregnant mice. These findings

have demonstrated for the first time that short exposures of clinically-relevant

glucose levels in pregnant humans and mice can influence endothelial function, an

effect not evident in the non-pregnant state. The novel results obtained suggest that

222

the state of pregnancy is associated with a unique modulation of endothelial

responses to glucose in both animals and humans.

This study represents the first report on the role of NO, prostacyclin and EDHF in

the uterine artery of pregnant mice. Endothelium-dependent relaxation in the uterine

artery of pregnant mice was predominantly mediated by a non-nitric oxide/non-

prostanoid mechanism, with a smaller contribution from nitric oxide, and no

prostanoid-mediated relaxation. Another novel finding was that NO-mediated

endothelium-dependent relaxation in mice uterine arteries was similar at 5 and 12

mmol/L glucose.

Myometrial arteries from women with diabetes in pregnancy have been examined

for the first time in this study. Compared to healthy pregnant women, impaired

endothelium-dependent relaxation was observed in these arteries from women with

GDM. This effect was present when the arteries were constricted with 60 mmol/L

potassium, but absent with U46619; indicating the important role of EDHF in

normalising endothelial function in this vascular bed.

A unique feature of this study was that the glucose levels studied and their duration

were chosen based on results of women with diabetes in pregnancy. Previous studies

have examined the effects of hyperglycaemia on vascular function after prolonged

and markedly high glucose concentrations. Although changes in function have been

demonstrated by these studies, such conditions are rarely seen clinically. The aim of

the present study was to therefore examine if more subtle changes in glucose

concentrations altered endothelial function.

The findings of this study may be of clinical significance in the setting of diabetes in

pregnancy. The endothelial dysfunction of myometrial arteries in GDM may

influence blood supply to the fetus. Fluctuations in glucose level did not affect

endothelial function in women with GDM, although the effect of these fluctuations

in women with type 1 and 2 diabetes could not be examined fully due to a lack of

patients. This study has also demonstrated that fluctuations in blood glucose levels

as well as adverse outcomes were high in women with type 1 diabetes. These swings

in glucose level have been demonstrated to exert a more specific triggering effect on

223

oxidative stress than chronic sustained hyperglycaemia [Monnier et al., 2006].

Further studies are required to investigate whether fluctuations between high and

low glucose levels play an additional role in mediating the complications of diabetes

in pregnancy.

Figure 7.1: Overview of study

Previously established associations

Associations characterised further by this study

New associations demonstrated by this study

ENDOTHELIAL

FUNCTION

Diabetes

Pregnancy

Hypoglycaemia Hyperglycaemia

Vascular

Bed

Endothelial

Mediator

224

7.2 Key Findings

A summary of the key findings obtained from this project has been depicted in

Figure 7.1. Novel associations demonstrated for the first time in this study include

impaired endothelium-dependent relaxation at 2 mmol/L glucose and impaired

endothelial function in myometrial arteries of women with GDM. Another new

association was the enhancement of endothelium-dependent relaxation at 12

mmol/L glucose in uterine arteries of pregnant mice. In addition to these original

findings, a number of existing associations have been characterised further by this

study. These include the variation in endothelial function with the type of vascular

bed, the role of prostacyclin, NO and EDHF in small arteries as well as the effect of

pregnancy on endothelial function.

7.3 Hypothesis 1:

The effects of glucose concentrations on endothelial function

vary in different vascular beds.

Previous studies examining the effects of glucose have differed in the methodology,

species, concentration of glucose, duration of exposure and the vascular bed

investigated. The large number of variables inherent in these studies has predictably

led to heterogeneous results. This study has attempted to overcome some of these

intrinsic ambiguities by utilising a single methodology, and by keeping the duration

and concentration of glucose constant. By doing so, it has been demonstrated that

exposure to the same glucose concentration can modulate endothelial function in

disparate ways, depending on the vascular bed. The state of pregnancy and the

species examined were found to be important in determining the effect of glucose on

vascular function. This work therefore reveals a more complex relationship between

glucose concentrations and endothelial function than previously envisaged. It also

suggests that the effects of glucose should be considered in the context of the

vascular bed studied, helping to explain some of the inconsistencies of past studies.

225

Regional variations in vascular responses to various stimuli have been reported in a

number of studies. For example, both regional and species heterogeneity have been

demonstrated in the vascular responses to the action of muscarinic agonists in the rat

and frog [Leont'eva and Krivchenko, 2001]. The present study has demonstrated

inter-species variation in the effects of 2 and 12 mmol/L glucose on endothelial

function. These different responses may be due to the variations between species in

counter-acting the effects of oxidative stress. Studies in C57BL/6 mice have

suggested that there are differences in the response to oxidative stress, even between

strains of mice [Agardh et al., 2000]. Differences in the balance between NO and

superoxide generation have been demonstrated to be an important factor in the

variation in vascular responses between C57BL/6 mice and other strains [Bendall et

al., 2002]. Hyperglycaemia-related oxidative stress may induce endothelial

dysfunction due to variations in anti-oxidant capabilities. Inter-species differences in

endothelial mediators also exist, further influencing the interaction between glucose

concentrations, oxidant stress and endothelial dysfunction. Additionally, it has been

noted in humans that genetic variations between individuals can play a role in

determining vascular reactivity [Henrion et al., 2002].

The importance of glucose in metabolism is underscored by the fact that multiple

homeostatic mechanisms are in place to ensure an adequate supply of glucose to

different tissue regions depending on their metabolic activity. Glucose transport and

its concentrations are closely regulated with varying degrees of complexity in all

biological systems. This study has demonstrated differences in endothelial function

to glucose in the pregnant and non-pregnant state within the same vascular bed in

animals (uterine arteries) as well as humans (myometrial arteries). The

heterogeneity in these responses may be related to the altered glucose demands and

the vascular permutations occurring in pregnancy, which have been discussed in

Section 1.3.2. Differences in vascular function in various vascular beds of the same

animal have also been reported during pregnancy. The vascular sensitivity to

U46619 was attenuated in small myometrial arteries of pregnant sheep compared

with omental arteries, which may help preserve uterine blood flow [Anwar et al.,

1999].

226

Arterial diameter may play an important role in determining regional responses to

the effect of glucose concentrations. Endothelial mediators vary with the calibre of

arteries; however, the effects of glucose on the action of these mediators

(particularly EDHF and prostacyclin) are yet to be fully characterised. This study

has highlighted the importance of EDHF at the resistance artery level, corroborating

previous work [Shimokawa et al., 1996]. Furthermore, EDHF has been

demonstrated to be the predominant endothelium-derived mediator in female mice,

whereas NO and PGI2 were more important in male mice [Scotland et al., 2005].

Variations in the potency of endothelial mediators at various vascular beds as well

as differences in the response of these mediators to hypoglycaemia and

hyperglycaemia may account for the results obtained.

7.4 Hypothesis 2:

Acute changes in glucose concentration from normal are

associated with altered endothelial function

This study has demonstrated that short exposures to both high and low glucose

levels can affect endothelial function in pregnancy. The changes in constriction and

endothelium-dependent relaxation indicate that even subtle changes in glucose

concentration may influence vascular function. An implication of this finding is that

episodic rises in glucose levels (such as in the post-prandial state) may be associated

with aberrant vascular function at the resistance artery level. Most of the earlier

studies on the effects of glucose examined large conduit arteries, which differ from

smaller arteries in the relative contribution of the individual endothelial mediators of

relaxation. Results obtained in these studies cannot therefore be extrapolated to

smaller arteries, where EDHF has a more prominent role than NO.

The mechanisms responsible for the altered endothelial function with glucose

concentrations have been investigated in this study. No enhancement of endothelial

mediators was found at 12 mmol/L glucose in mice uterine arteries, most likely due

to a lack of specific EDHF blockade. Nevertheless, the findings provide a basis for

227

future studies investigating the mechanisms responsible for altered endothelial

responses.

In contrast to previous studies, a high glucose concentration was not associated with

impaired endothelium-dependent relaxation in any of the arterial beds studied. This

suggests that longer exposures or higher glucose concentrations may be required for

endothelial function to be disrupted by the deleterious effects of elevated glucose

levels. This finding may be of consequence in determining the time course of

hyperglycaemia-related changes to vascular function. Prolonged exposure to

hyperglycaemia (lasting 1- 6 hours) has been associated with impaired endothelium-

dependent relaxation in previous studies (Table 1.3). This study therefore suggests

that the effects of glucose should be considered in the context of the duration of

exposure.

7.5 Hypothesis 3:

Diabetes in pregnancy is associated with endothelial dysfunction

in myometrial arteries

When constricted with KCl, endothelium-dependent relaxation was impaired in

myometrial arteries from women with GDM compared with normal. However,

when U46619 was used as an agonist, endothelial function was preserved, indicating

that EDHF (which was blocked with KCl) may play an important role in

normalising endothelial function in diabetes. This study therefore brings in to focus

the need for specifically analysing the role of EDHF and its relationship with

changes in glucose concentrations.

Knock et al. demonstrated impaired relaxation to acetylcholine in subcutaneous

arteries from women with gestational diabetes compared with normal pregnant

women [Knock et al., 1997]. This study suggested that prostacyclin may be of more

importance to relaxation than NO in subcutaneous arteries. However, prostanoids

have been demonstrated to have little or no role in myometrial arteries of pregnant

women, a vascular bed where endothelium-dependent relaxation is mediated by NO

228

and EDHF [Ashworth et al., 1999; Kenny et al., 2002]. It is uncertain how diabetes

in pregnancy influences the role of these mediators in myometrial arteries. This

aspect could not be investigated further in the present study due to the limited

availability of suitable patients.

In streptozotocin-induced diabetic C57BL/6 mice, relaxation mediated by EDHF

has been reported to be impaired [Morikawa et al., 2005]. In rat mesenteric arteries,

the impaired endothelium-dependent relaxation after exposure to 22.2 mmol/L

glucose has been demonstrated to be due to impaired EDHF-mediated relaxation

[Ozkan and Uma, 2005]. The importance of EDHF is also underscored by the

demonstration that EDHF can substitute for NO in mediating relaxation in arteries

of female eNOS-knockout mice [Huang et al., 2001]. Future research directed at the

association between hyperglycaemia and EDHF may reveal important links between

it and the pathophysiology of endothelial dysfunction in diabetes.

Women in the GDM group studied had relatively well-controlled diabetes, with

near-normal mean HbA1C values. This may have influenced endothelial responses,

as well-controlled diabetes has been associated with normal endothelial function in

sub-cutaneous arteries of pregnant women with type 1 diabetes [Ang et al., 2002].

However, the fundamental differences between GDM and type 1 diabetes, as well as

the difference in the vascular beds examined limit comparisons of endothelial

function between these studies.

Obesity is associated with impaired endothelial function [Myers et al., 2006], and

the women with GDM had a higher BMI than those in the normal group. However,

this is unlikely to be the cause of impaired relaxation in myometrial arteries of

women with GDM, as impairment was not present when U46619 was used as an

agonist. Nevertheless, the elevated BMI of the GDM patients may explain the

increased birth weight of babies in this group, as an association between the two has

been previously demonstrated [Bo et al., 2003; Hardy, 1999].

Impaired endothelium-dependent relaxation has also been reported in the

myometrial vascular bed in pre-eclampsia [Ashworth et al., 1997], a condition with

a raised prevalence in gestational diabetes [Bryson et al., 2003]. This highlights the

229

importance of future investigations into endothelial dysfunction in GDM, which

may lead to valuable insights into the pathophysiology of complications seen in this

condition.

7.6 Limitations of study

This study was subject to certain limitations which need to be considered when

interpreting the results. The methodological limitations of wire myography have

been previously alluded to in Section 2.8.3. Studies performed in humans and mice

differed in the vasoconstrictor and vasodilator used; the reasons for this having been

discussed in Section 2.10.9. This may limit comparisons between species, although

the protocols were identical in other respects. A time-related impairment of

endothelium-dependent relaxation was observed in arteries from mice, but not from

humans. However, the inclusion of a time control at 5 mmol/L glucose enabled

accurate interpretation of the data despite this effect. Endothelium-independent

relaxation was not systematically investigated in this study, thereby restricting the

assessment of smooth muscle function. This was because the vast majority of

previous reports have found no association between hyperglycaemia and

endothelium-independent relaxation [Beckman et al., 2001; Reed et al., 2004;

Williams et al., 1998]. The preservation of endothelium-independent relaxation has

been demonstrated in disease states such as diabetes [Fukao et al., 1997; McNally et

al., 1994]. An estimation of the integrity of smooth muscle function can be made

from maximal constriction; which was found to be similar in myometrial arteries

from both normal pregnant women and women with gestational diabetes.

In Chapter 1, the influence of factors such as age, BMI and ethnicity on endothelial

function have been examined. The human subjects included in this study were not

matched for these variables. This was because the evidence for an association

between these factors and endothelial function is not conclusive. Acquiring

myometrial arteries entails an invasive biopsy, which severely restricts the number

of patients, even when recruiting simultaneously from three large hospitals.

Furthermore, only a minority of pregnant women deliver by Caesarean section.

Matching obese and non-obese patients with GDM is of questionable value, as

230

obesity and the subsequent insulin resistance are often necessary pre-requisites for

the development of GDM. The limited availability of human biopsies was the main

reason for attempting to create an animal model of diabetes.

When the endothelial mediators in uterine arteries of pregnant mice were examined,

a specific blocker was not used for EDHF. Previous studies in this strain of mice

had identified NO to be the most important mediator of relaxation in carotid and

femoral arteries [Crauwels et al., 2000]. Furthermore, hyperglycaemia has been

associated with reduced NO bioavailability [Giugliano et al., 1997; Trachtman et

al., 1997], making this mediator the focus of interest in the present study. Although

the results suggested that NO was unaffected by exposure to 12 mmol/L glucose,

they also revealed the importance of EDHF in this vascular bed. This preliminary

characterisation of the endothelial mediators in the uterine artery of mice may

facilitate future vascular studies.

The effects of a more prolonged exposure to altered glucose concentrations (such as

two hours) could not be fully investigated because of the loss of viability of arteries

as described in Section 4.4.12. Techniques such as pressure myography may offer

improved viability for prolonged durations of time, as vessels are not stretched as in

wire myography. However in pressure myography, only a single vessel can be

examined at a time; this limits its value in a study of this nature, where multiple

glucose concentrations need to be investigated concurrently.

A successful model of diabetes in pregnancy was created by the author with the

assistance of the University of Manchester staff. Although the initial batch of mice

failed to develop diabetes, results from this group have been presented as

streptozotocin-controls, to form a basis for further work. Diabetes was successfully

induced in the second batch of mice, despite a high failure rate. Although results

from diabetic mice could not be presented due to time constraints, it is hoped that

the ongoing work on this model of diabetes in pregnancy will lead to further insights

into this disorder.

231

7.7 Future avenues of research

The results obtained from this study have brought to light specific directions which

future research may be able to take in ascertaining the effects of glucose on

endothelial function in pregnancy and the influence of diabetes.

I. Determine the mechanisms responsible for impaired endothelium-dependent

relaxation in gestational diabetes, and examine whether such changes occur in

type 1 and type 2 diabetes in pregnancy.

Understanding the mechanisms responsible for endothelial

dysfunction may help in developing novel therapies aimed at

normalising vascular function in diabetes.

II. Investigate further the association between hypoglycaemia and endothelial

dysfunction.

This may help determine whether hypoglycaemia plays a role

in mediating vascular complications in diabetes

III. Use the animal model of diabetes in pregnancy developed in this study to:

examine the association of EDHF with hyperglycaemia and

hypoglycaemia

The response of EDHF to these stimuli may be more

important than NO in mediating endothelial dysfunction in

diabetes at the resistance artery level

ascertain the link between endothelial dysfunction and fetal

development.

Animal models would enable characterisation of how the

endothelial dysfunction demonstrated in diabetes can

influence the fetus.

232

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259

APPENDIX A: CONSENT FORM

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APPENDIX B: PATIENT INFORMATION SHEET

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APPENDIX C: PRO FORMA FOR STUDY OF DIABETES IN PREGNANCY

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APPENDIX C: PRO FORMA FOR STUDY OF DIABETES IN PREGNANCY

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