Assessment of Novel Drugs for Treating Preterm Labour Using a Translational Model

Assessment of novel drugs for treating preterm labour using a translational model

A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health

2020

Ammar Ahmed Mohammed

School of Health Sciences

Division of Pharmacy and Optometry

Table of contents Table of contents ...... 2

List of Figures ...... 9

List of Tables ...... 20

List of abbreviations ...... 22

Publications ...... 27

Abstract ...... 28

Declaration ...... 29

Acknowledgements ...... 30

Dedication ...... 32

1 Chapter 1: ...... 33

Introduction ...... 33

1.1 The female reproductive system ...... 34

1.2 The human female reproductive system ...... 34

1.2.1 The human uterus ...... 34

1.3 The mouse female reproductive system ...... 36

1.3.1 The mouse uterus ...... 36

1.3.2 The mouse placenta ...... 38

1.4 The role of mouse uterus during oestrous cycle ...... 40

1.5 The role of the uterus during pregnancy ...... 41

1.6 Normal parturition: The common pathways of labour ...... 41

1.7 Preterm Labour (PTL) ...... 42

1.7.1 Risk factors ...... 44

1.7.2 Pathophysiology of PTL ...... 45

1.7.3 Role of steroid hormones in the induction of labour...... 45

1.7.4 Prostanoids ...... 50

1.8 The ROCK enzymes ...... 54

1.8.1 Role of ROCK pathway in uterine contraction ...... 55

1.9 Feasibility and translation of mouse model of PTL in humans ...... 56

1.10 Treatments for preterm labour ...... 59

1.10.1 Drugs used in the treatment of PTL ...... 60

1.10.2 Prophylactic therapy of premature rupture of the membranes in PTL ..... 71

1.11 Nitric Oxide (NO) and Nitric Oxide Synthase (NOS) isoforms ...... 71

1.11.1 The role of NO in the uterus ...... 72

1.11.2 The potential use of NO-donor compounds during pregnancy and labour

…………………………………………………………………………...74

1.11.3 The interaction between the NO and lipid mediators...... 75

1.11.4 SE175 (NO-donor), structure and pharmacology ...... 75

1.12 Drug targeting and liposomes ...... 76

1.12.1 Liposomes as a drug delivery system ...... 77

1.12.2 Liposomes composition ...... 77

1.13 Aims and objectives ...... 79

2 Chapter 2: ...... 80

Materials and Methods ...... 80

2.1 Source of the tissues ...... 81

2.1.1 Non-pregnant mice ...... 81

2.2 Methods for investigating myometrial contractility ...... 83

2.2.1 Preparation of isolated tissue for immersion ...... 83

2.2.2 Solutions and drug compounds ...... 85

2.2.3 Immersion ...... 87

2.2.4 Administration of drugs ...... 89

2.2.5 Preliminary experiments to determine dosing regimens ...... 91

2.2.6 Measuring contractile force relative to spontaneous activity ...... 94

2.3 Investigation of contractile proteins in mouse myometrium ...... 95

2.3.1 Western Blot Analysis...... 95

2.3.2 Isolation of protein ...... 97

2.3.3 Sample preparation for Western Blotting ...... 99

2.3.4 Immunoblotting to investigate protein expression ...... 103

2.4 Immunocytochemistry (ICC) ...... 106

2.4.1 Isolation of uterine myometrial cells for immunofluorescence studies .. 106

2.4.2 Cell plating and treatment ...... 109

2.4.3 Cell fixation ...... 109

2.4.4 Permeabilisation ...... 109

2.4.5 Blocking ...... 110

2.4.6 Immunofluorescence (IF) ...... 110

2.4.7 Cell immunostaining ...... 111

2.4.8 Imaging analysis...... 114

2.5 Enzyme-linked Immunosorbent Assay (ELISA) ...... 116

2.5.1 Overview of ELISA ...... 116

2.5.2 Collection of plasma samples...... 116

2.5.3 ELISA measurement of 17β-oestradiol ...... 117

2.6 Mass spectrometry analysis of total fatty acid composition ...... 120

2.6.1 Overview of mass spectrometry ...... 120

2.6.2 LC/ESI-MS/MS protocol ...... 121

2.6.3 Materials for LC/ESI-MS/MS ...... 123

2.6.4 Preparation of internal standards ...... 124

2.6.5 LC/ESI-MS/MS ...... 126

2.7 Statistical analysis ...... 137

3 Chapter 3: ...... 138

Effect of Ripasudil, a ROCK inhibitor on U46619-, PGF2α- and

5-HT-induced myometrial contraction in non-pregnant C57 WT

mice...... 138

3.1 Introduction ...... 139

3.2 Spontaneous activity during dioestrous stage ...... 140

3.3 Effect of PGF2α on spontaneous myometrial contraction in non-pregnant C57

WT mice ...... 142

3.4 Effect of 5-HT on spontaneous myometrial contraction in non-pregnant C57 WT

mice ...... 143

3.5 Effect of ripasudil on spontaneous myometrial contractions in non-pregnant C57

WT mice ...... 144

3.6 Effect of ripasudil on U46619-, PGF2α- and 5-HT-induced myometrial

contraction in non-pregnant C57 WT mice ...... 148

3.7 The effect of cumulative concentrations of ripasudil on spontaneous myometrial

contractility in non-pregnant C57 WT mice ...... 158

3.8 The effect of ripasudil on myometrial contractility induced by cumulative

concentrations of U46619, PGF2α or 5-HT ...... 162

3.9 Expression of contractile proteins in myometrium after treatment with U46619,

PGF2α, and 5-HT in the presence and absence of ripasudil ...... 174

3.10 Discussion ...... 191

4 Chapter 4: ...... 203

Effects of Ripasudil, a ROCK inhibitor on Oxytocin, U46619- and

5-HT-induced myometrial contractions in pregnant C57 wild type

mice at term ...... 203

4.1 Introduction ...... 204

4.2 Spontaneous activity in pregnant mouse uterus at term (E19) ...... 206

4.3 Plasma concentration of 17β-oestradiol ...... 208

4.4 Litter size and spontaneous activity ...... 209

4.5 The effect of cumulative doses of ripasudil on spontaneous myometrial

contractility in pregnant C57 mice at term (E19) ...... 210

4.6 The effect of ripasudil on the myometrial contractility induced by cumulative

concentrations of oxytocin, U46619 or 5-HT in pregnant C57 mice at term (E19)

……………………………………………………………………………….212

4.7 Expression of contractile proteins in myometrium after treatment with OXT,

U46619 and 5-HT in the presence and absence of ripasudil ...... 219

4.8 Discussion ...... 232

5 Chapter 5: ...... 249

Effect of NO-donor containing liposomes on the composition of

lipid mediators in the myometrium and placenta of pregnant C57

WT and eNOS knockout mice ...... 249

5.1 Introduction ...... 250

5.2 Myometrial contractility of the uterus of pregnant C57 WT and eNOS KO mice

at term ...... 253

5.3 The effect of SE175 on the concentration of lipid mediators in the myometrium

and placenta of pregnant C57 WT and eNOS KO mice at term ...... 254

5.3.1 Arachidonic Acid Mediators ...... 254

5.3.2 Linoleic Acid Mediators ...... 262

5.3.3 Dihomo-gamma-Linolenic Acid Mediators ...... 266

5.3.4 Alpha-Linolenic Acid Mediators ...... 270

5.3.5 Eicosapentaenoic Acid Mediators ...... 272

5.3.6 Docosahexaenoic Acid Mediators ...... 274

5.4 The effect of liposome and SE175 exposure on the lipid proportions in the

reproductive tissues according to the synthetic pathway ...... 278

5.4.1 Myometrium ...... 278

5.4.2 Placenta ...... 278

5.5 Discussion ...... 281

6 Chapter 6: ...... 293

Conclusion and Future work ...... 293

6.1 Conclusion ...... 294

6.2 Future work ...... 296

7 Chapter 7: ...... 298

References ...... 298

Appendices ...... 3399

Word count: 61,690

List of Figures

Figure 1.1. Human uterus, vagina, fallopian tubes, ovaries and uterine layers (Szmelskyj et al., 2015)………………………………………………………………...35

Figure 1.2. A dissected genital tract of the female mouse (Knoblaugh and Randolph- Habecker, 2018)……………………………………………………………37

Figure 1.3. A histological diagram of the mouse uterus (Bhartiya and James, 2017)….38

Figure 1.4. Overview of mature mouse placenta (Latos and Hemberger, 2016)………39

Figure 1.5. Diagram of arterial blood supply to the mouse placenta during gestation (Raz et al., 2012)…………………………………………………………...39

Figure 1.6. The role of myometrial ER and PR systems in modulating human labour (Mesiano et al., 2002)……………………………………………………...47

Figure 1.7. Interactions between oestrogens, progesterone and CAPs during human pregnancy and parturition………………………………………………….48

Figure 1.8. The possible pathway of progesterone withdrawal enhanced by dinoprostone and induction of labour…………………………………………………….49

Figure 1.9. The biosynthetic and metabolic processes of PGs (Fischer, 2010)………..51

Figure 1.10. The role of thromboxane in muscle contraction………………………….53

Figure 1.11. Structure and binding sites of ROCKI/ROCKII (Schmandke et al., 2007)…………………………………………………………………….54

Figure 1.12. The role of ROCK in the mono- and di-phosphorylation steps of MLC in uterine myocytes………………………………………………………...56

Figure 1.13. The chemical structure of ripasudil (Isobe et al., 2014)…………………..69

Figure 1.14. An overview of mechanism of smooth muscle contraction and relaxation and the role of NO………………………………………………………73

Figure 1.15. The chemical structure of SE175…………………………………………76

Figure 1.16. A diagram showing the composition of a liposome (Cureton, 2017)……78

Figure 2.1. Photomicrographs of vaginal smears from mice…………………………..82

Figure 2.2. Intact uterus isolated from pregnant and non-pregnant mouse in dioestrus..84

Figure 2.3. The immersion apparatus including the equipment and PowerLab used in the study……………………………………………………………………….88

Figure 2.4. The immersion equipment used to test functional receptors in the isolated mouse uterus (Fischer, 2010)………………………………………………88

Figure 2.5. Representative traces demonstrating the duration and variability in spontaneous activity of myometrial strips taken from (A) the upper, (B) the lower segments of uterine horn of a non-pregnant mouse in dioestrus and (C) the upper segments of a pregnant mouse (E19) when using the immersion technique…………………………………………………….90

Figure 2.6. Traces showing the effect of washout on the spontaneous activity of myometrial strips from (A) non-pregnant and (B) pregnant mice………91

Figure 2.7. Trace showing the restoration of myometrial spontaneous activity in pregnant mouse uterus after being inhibited by ripasudil 10-5M and then

washed with Krebs solution, which was aerated with 95% O2, 5% CO2 at 37ºC using immersion technique………………………………………..92

Figure 2.8. Trace showing typical responses to ripasudil (10-6M) and 5-HT (10-9M to 10- 5M) in immersed myometrial strips from non-pregnant mouse uterus taken in dioestrus and tested using immersion technique……………………….93

Figure 2.9. Traces showing the variability in spontaneous activity from immersed myometrial strips isolated from the same uterus of a non-pregnant mouse in dioestrus. Strips were taken from the upper segment (A and B) and lower segment (C and D) of the uterine horn of the same non-pregnant mouse…94

Figure 2.10. Example standard curve and equation of the line for protein content determination……………………………………………………………....99

Figure 2.11. Diagram of transfer cassette set-up……………………………………..102

Figure 2.12. Diagram showing the principles of protein detection by chemiluminescence...………………………………………………….105

Figure 2.13. Representative image of western blot bands using the ChemiDoc MP imaging system……..…………………………………………………...105

Figure 2.14. Cell counting using Neubauer haemocytometric slide………………….108

Figure 2.15. Indirect immunofluorescent staining…………………………………….111

Figure 2.16. An example of immunofluorescent image channels before and after the creation of a merged image…..…………………………………………115

Figure 2.17. The outline of the sandwich ELISA…………………………………….118

Figure 2.18. Exemplification of sandwich ELISA assay and binding sequence of antibodies and reagents from BioVision.………………………………119

Figure 2.19. BioVision ELISA assay for the standard curve of 17β-oestradiol……..119

Figure 2.20. Schematic overview of LC/ESI-MS/MS method……………………….121

Figure 2.21. A flow chart showing the main extraction step and lipidomics analysis of tissue specimens for eicosanoids and related hydroxy fatty acids in pregnant wild type and eNOS KONos3tm1Unc/J mice…...……………127

Figure 2.22. Representative chromatogram and standard curve for 11 HETE in TargetLynx……………………………………………………………...135

Figure 2.23. Standard curve and equation of the line for protein content determination……………………………………………………………136

Figure 3.1. Regional variation in spontaneous activity……………………………….140

Figure 3.2. Representative traces demonstrating the duration and variability in spontaneous activity of two myometrial strips taken from (A) the upper and (B) the lower segments of the uterine horn of a non-pregnant C57 WT mouse in dioestrus and mounted in organ baths using the immersion technique as explained in the method chapter (Section 2.2.4)………….141

-6 Figure 3.3. Representative traces showing the effect of PGF2α (10 M) on myometrial contractility of uterine tissue taken from (A) the upper and (B) the lower uterine horn of a non-pregnant C57 WT mouse in dioestrus……….…..142

-6 Figure 3.4. The response of different uterine regions to PGF2α (10 M)…………….142

Figure 3.5. Representative traces showing the effect of 5-HT (10-6M) on spontaneous myometrial contractility of immersed uterine tissue taken from (A) the upper and (B) the lower uterine horn of a non-pregnant mouse in dioestrus……………………………………………………………...….143

Figure 3.6. The response of different uterine regions to 5-HT (10-6M)………………143

Figure 3.7. Effect of ripasudil (10-9M - 10-5M) on spontaneous myometrial contractility of the upper segment of the uterine horn………………………………..145

Figure 3.8. Representative traces showing the inhibitory effect of different concentrations of ripasudil (10-9M - 10-5M) on spontaneous myometrial contractility of uterine tissue taken from the upper segment of the uterine horn of a non-pregnant C57 WT mouse in dioestrus…………………..145

Figure 3.9. Effect of ripasudil (10-9M - 10-5M) on spontaneous myometrial contractility of the lower segment of the uterine horn………………………………146

Figure 3.10. Representative traces showing the inhibitory effect of different concentrations of ripasudil (10-9M - 10-5M) on spontaneous myometrial contractility of uterine tissue taken from the upper segment of the uterine horn of a non-pregnant C57 WT mouse in dioestrus…………………...146

Figure 3.12. Effect of ripasudil (10-9M - 10-5M) on U46619-induced myometrial contractility in upper segment uterine horn……………………………148

Figure 3.13. Representative traces showing the effect of different concentrations of ripasudil (10-9M - 10-5M) on U46619 (10-6M)-induced contractions in upper segment uterine horn of non-pregnant C57 WT mice in dioestrus…………………………………………………………………149

Figure 3.14. Effect of ripasudil (10-9M - 10-5M) on U46619-induced myometrial contractility in lower segment uterine horn…………………………….150

Figure 3.15. Representative traces showing the effect of different concentrations of ripasudil (10-9M - 10-5M) on U46619 (10-6M)-induced contractions in lower segment uterine horn of non-pregnant C57 WT mice in dioestrus…………………………………………………………………151

Figure 3.16. Effect of ripasudil (10-8M - 10-5M) on 5-HT-induced myometrial contractility in upper segment uterine horn……………………………..152

Figure 3.17. Representative traces showing the effect of different concentrations of ripasudil (10-8M - 10-5M) on 5-HT (10-6M)-induced contractions in upper segment uterine horn of non-pregnant C57 WT mice in dioestrus……..153

Figure 3.18. Effect of ripasudil (10-8M - 10-5M) on 5-HT-induced myometrial contractility in lower segment uterine horn……………………………..154

Figure 3.19. Representative traces showing the effect of different concentrations of ripasudil (10-8M - 10-5M) on 5-HT (10-6M)-induced contractions in lower segment uterine horn of non-pregnant C57 WT mice in dioestrus…….154

Figure 3.20. Effect of ripasudil (10-8M - 10-5M) on PGF2α-induced myometrial contractility in upper segment uterine horn……………………………156

Figure 3.21. Representative traces showing the effect of different concentrations of -8 -5 -6 ripasudil (10 M - 10 M) on PGF2α (10 M)-induced contractions in upper segment uterine horn of non-pregnant C57 WT mice in dioestrus…… 156

-8 -5 Figure 3.22. Effect of ripasudil (10 M - 10 M) on PGF2α-induced myometrial contractility in lower segment uterine horn……………………………157

Figure 3.23. Representative traces showing the effect of different concentrations of -8 -5 -6 ripasudil (10 M - 10 M) on PGF2α (10 M)-induced contractions in lower segment uterine horn of non-pregnant C57 WT mice in dioestrus……157

Figure 3.24. Effect of cumulative concentrations of ripasudil on myometrial spontaneous activity in upper segment uterine horn……………………158

Figure 3.25. Representative traces showing concentration-effect curves for vehicle and ripasudil (10-9M - 10-5M) in isolated myometrial strips from the upper segment uterine horn of non-pregnant C57 WT mouse in dioestrus……159

Figure 3.26. Effect of cumulative concentrations of ripasudil on the myometrial spontaneous activity of lower segment uterine horn………………….. 160

Figure 3.27. Representative traces showing concentration-effect curves for vehicle and ripasudil (10-9M - 10-5M) in isolated myometrial strips from lower segment uterine horn of non-pregnant C57 WT mouse in dioestrus…………….161

Figure 3.28. Effect of cumulative concentrations of U46619 on the spontaneous activity of upper segment uterine horn in the presence and absence of ripasudil (10- 6M)………………………………………………………………………162

Figure 3.29. Representative traces showing concentration-effect curves for U46619 (10- 9M - 10-5M) in isolated myometrial strips from the upper segment uterine horn of non-pregnant C57 WT mouse in dioestrus in the presence and absence of ripasudil (10-6M).…………… ……………………………...163

Figure 3.31. Representative traces showing concentration-effect curves for U46619 (10- 9M - 10-5M) in immersed isolated myometrial strips from the lower segment uterine horn of a non-pregnant C57 WT mouse in dioestrus in the presence and absence of ripasudil………………….…………………..165

Figure 3.33. Representative traces showing concentration-effect curves for 5-HT (10-9M - 10-5M) in isolated myometrial strips from the upper segment uterine horn of a non-pregnant C57 WT mouse in dioestrus in the presence and absence of ripasudil…….………………………………………………………..167

Figure 3.35. Representative traces showing concentration-effect curves for 5-HT (10-9M - 10-5M) in isolated myometrial strips from the lower segment uterine horn of non-pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil (10-6M)……………………………………………………… 169

- Figure 3.37. Representative traces showing concentration-effect curves for PGF2α (10 9M - 10-5M) in isolated myometrial strips from the upper segment uterine horn in non-pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil……………………………………………………..171

- Figure 3.39. Representative traces showing concentration-effect curves for PGF2α (10 9M - 10-5M) in isolated myometrial strips from the lower segment uterine horn of non-pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil……………………………………………………..173

Figure 3.40. Effect of ripasudil on non-phosphoryated MLC expression in myometrial tissue from non-pregnant C57 WT mice in dioestrus. …………………175

Figure 3.41. Effect of ripasudil on non-phosphorylated MLC expression in myometrial cells from non-pregnant C57 WT mice in dioestrus……………………175

Figure 3.42. Effect of ripasudil on pMLC expression in myometrial tissue from non- pregnant C57 WT mice in dioestrus. ………………………………… 176

Figure 3.43. Effect of ripasudil on pMLC expression in myometrial cells from non- pregnant C57 WT mice in dioestrus………………………………….. 177

Figure 3.44. Effect of ripasudil on ppMLC expression in myometrial tissue from non- pregnant C57 WT mice in dioestrus. …………………………………..178

Figure 3.45. Effect of ripasudil on ppMLC expression in myometrial cells from non- pregnant C57 WT mice in dioestrus…………………………………… 178

Figure 3.46. Effect of U46619 on MLC expression in myometrial tissue from non- pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil………………………………………………………………. .179

Figure 3.47. Effect of U46619 on MLC expression in myometrial cells from non- pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil………………..………………………………………………..180

Figure 3.48. Effect of U46619 on pMLC expression in myometrial tissue from non- pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil. …………………………….…………………………………181

Figure 3.49. Effect of U46619 on pMLC expression in myometrial cells from non- pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil (10-6M).………………………………………..……………...181

Figure 3.50. Effect of U46619 on ppMLC expression in myometrial tissue from non- pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil…………………………………..……………………………..182

Figure 3.51. Effect of U46619 on ppMLC expression in myometrial cells from non- pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil…………………………………..……………………………..183

Figure 3.52. Effect of 5-HT on MLC expression in myometrial tissue from non- pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil……………………………………….………………………..184

Figure 3.53. Effect of 5-HT on pMLC expression in myometrial tissue from non- pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil……………………………………..…………………………..185

Figure 3.54. Effect of 5-HT on ppMLC expression in myometrial tissue from non- pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil…………………………………..……………………………..186

Figure 3.55. Effect of PGF2α on MLC expression in myometrial tissue from non- pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil…………………………………………………………………187

Figure 3.56. Effect of PGF2α on pMLC expression in myometrial tissue from non- pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil………………………..………………………………………..188

Figure 3.57. Effect of PGF2α on ppMLC expression in myometrial tissue from non- pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil………………………..………………………………………..189

Figure 4.1. Comparing spontaneous activity between pregnant and non-pregnant mouse uterine samples……………………………………...……….…………206

Figure 4.2. Representative traces demonstrating the duration and variability in spontaneous activity of myometrial strips taken from the upper segments of uterine horn of (A) non-pregnant mouse in dioestrus and (B) pregnant mouse (E19) when using the immersion technique……………………207

Figure 4.3. 17β-oestradiol concentrations (pg/ml) measured in the plasma of non- pregnant (in dioestrus) and pregnant (E19) C57 mice…………………208

Figure 4.4. Influence of litter size on uterine spontaneous activity in pregnant C57 mice at term (E19)……………………………………………………………209

Figure 4.5. Effect of cumulative doses of ripasudil on myometrial spontaneous activity in pregnant C57 mouse uterus at term (E19)……………………………210

Figure 4.6. Representative traces showing concentration-effect curves for ripasudil (10- 9M - 10-5M) and control in isolated myometrial strips from the upper segment of the uterine horn in pregnant C57 mouse at term (E19) when using the immersion technique……..………………………...... 211

Figure 4.7. Effect of cumulative concentrations of OXT on the myometrial spontaneous activity of the upper segment of uterine horn in pregnant C57 mice at term (E19) in the presence and absence of ripasudil………………………...213

Figure 4.8. Representative traces showing concentration-effect curves for OXT (10-12M - 10-6M) in the presence and absence of ripasudil (10-6M and 10-5M) in isolated myometrial strips from the upper segment of the uterine horn in pregnant C57 mouse at term (E19) when using the immersion technique...... 214

Figure 4.10. Representative traces showing concentration-effect curves for U46619 (10- 9M - 10-5M) in the presence and absence of ripasudil (10-6M and 10-5M) in isolated myometrial strips from the upper segment of the uterine horn in pregnant C57 mouse at term (E19) when using the immersion technique...... 216

Figure 4.12. Representative traces showing concentration-effect curves for 5-HT (10-9M - 10-5M) in the presence and absence of ripasudil (10-6M and 10-5M) in isolated myometrial strips from the upper segment of the uterine horn in pregnant C57 mouse at term (E19) when using the immersion technique...... 218

Figure 4.13. Effect of ripasudil on MLC expression in myometrial tissue from pregnant C57 mice at term (E19). ……………………………………………… 219

Figure 4.14. Effect of ripasudil on pMLC expression in myometrial tissue from pregnant C57 mice at term (E19)……………………….…………….. 220

Figure 4.15. Effect of ripasudil on ppMLC expression in myometrial tissue from pregnant C57 mice at term (E19)………………………………………221

Figure 4.16. Effect of OXT on MLC expression in myometrial tissue from pregnant C57 mice at term (E19) in the presence and absence of ripasudil………….222

Figure 4.17. Effect of OXT on pMLC expression in myometrial tissue from pregnant C57 mice at term (E19) in the presence and absence of ripasudil…….223

Figure 4.18. Effect of OXT on ppMLC expression in myometrial tissue from pregnant C57 mice at term (E19) in the presence and absence of ripasudil…..…224

Figure 4.19. Effect of U46619 on MLC expression in myometrial tissue from pregnant C57 mice at term (E19) in the presence and absence of ripasudil……..225

Figure 4.20. Effect of U46619 on pMLC expression in myometrial tissue from pregnant C57 mice at term (E19) in the presence and absence of ripasudil.……226

Figure 4.21. Effect of U46619 on ppMLC expression in myometrial tissue from pregnant C57 mice at term (E19) in the presence and absence of ripasudil…………………………………………………………………227

Figure 4.22. Effect of 5-HT on MLC expression in myometrial tissue from pregnant C57 mice at term (E19) in the presence and absence of ripasudil.……..228

Figure 4.23. Effect of 5-HT on pMLC expression in myometrial tissue from pregnant C57 mice at term (E19) in the presence and absence of ripasudil……..229

Figure 4.24. Effect of 5-HT on ppMLC expression in myometrial tissue from pregnant C57 mice at term (E19) in the presence and absence of ripasudil..……230

Figure 5.1. Comparison of spontaneous myometrial activities in myometrium from pregnant C57 WT and eNOS KO mice at term (E19)…………………..253

Figure 5.2. Arachidonic acid derived mediators quantified in myometrium from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI-MS/MS………………………………………258

Figure 5.3. Arachidonic acid derived mediators quantified in placentas from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI-MS/MS…………………..……………………………………..261

Figure 5.4. Linoleic acid derived mediators quantified in myometrium from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI-MS/MS…………………………..……………………………..263

Figure 5.5. Linoleic acid derived mediators quantified in placentas from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI-MS/MS…………………………………………………………265

Figure 5.6. Dihomo-gamma-Linolenic Acid mediators quantified in myometrium from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI-MS/MS………………………………………267

Figure 5.7. Dihomo-gamma-Linolenic Acid mediators quantified in placentas from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI-MS/MS……………...……………………….269

Figure 5.8. Alpha-Linolenic Acid mediators quantified in myometrium from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI-MS/MS…..……………………………………………………..270

Figure 5.9. Alpha-Linolenic Acid mediators quantified in placentas from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI-MS/MS…………..……………………………………………..271

Figure 5.10. Eicosapentaenoic Acid mediators quantified in myometrium from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI-MS/MS………………...……………………………………….272

Figure 5.11. Eicosapentaenoic Acid mediators quantified in placenta from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI-MS/MS………………..…………………………………….….273

Figure 5.12. Docosahexaenoic Acid mediators quantified in myometrium from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI-MS/MS…………………………………………………………276

Figure 5.13. Docosahexaenoic Acid mediators quantified in placentas from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI-MS/MS……………………..…………………………………..277

List of Tables

Table 1.1. Main reproductive physiological distinctions between human and mouse (Mitchell and Taggart, 2009)………………………………………………57

Table 1.2. The structural and physiological properties of ripasudil (Garnock-Jones, 2014)……………………………………………………………………….70

Table 2.1. Chemical composition of Krebs-Heinseleit solution……………………….85

Table 2.2. Stimulants and inhibitors used to investigate myometrial contractility in mice. The key indicates line colour on concentration-effect curves and Western Blotting graphs……………………...……………………………86

Table 2.3. Standard dilutions of BSA prepared from 1.5mg/ml stock in 1M NaOH to generate a standard curve for protein content analysis…………………….98

Table 2.4. Reagents required for preparing stacking gel…………………………….100

Table 2.5. Reagents required for preparing resolving gel…………………………….101

Table 2.6. Reagents required for preparing 10X running and Transfer buffers……..102

Table 2.7. Reagents required for preparing TBST buffer……………………………103

Table 2.8. List of primary antibodies used in Western Blotting……………………..104

Table 2.9. Composition of the permeabilising buffer in 100ml milli-Q water………110

Table 2.10. List of primary antibodies used in the immunofluorescence work………112

Table 2.11. List of secondary antibodies used in the immunofluorescence work…….112

Table 2.12. Illustration of the fixation, permeabilisation and immunostaining processes….…………………………………………………………...113

Table 2.13. List of prostanoids, leukotrienes and hydroxy fatty acids and their main polyunsaturated fatty acids (PUFA) precursors………………….……..122

Table 2.14. Solvent gradient for COX assay…………………………………………130

Table 2.15. Solvent gradient for LOX/CYP assay………………………………..….130

Table 2.16. Summary of individual MRM transition, cone voltage, collision energy and indicative retention times for COX assay……………………………….131

Table 2.17. Summary of individual MRM transition, cone voltage, collision energy and indicative retention times for the LOX/CYP assay…………………….132

Table 2.18. Standard dilutions of BSA prepared from 1.5mg/ml stock in 1M NaOH to generate standard curve for protein content analysis……………….….136

Table 3.1. Effect of U46619, PGF2α, 5-HT and ripasudil on MLC family expression in myometrial tissue from non-pregnant C57 WT mice in dioestrus……..190

Table 4.1. Effect of OXT, U46619 and 5-HT on MLC family expression in myometrial tissue from pregnant C57 mice at term (E19)…………………………….231

List of abbreviations

15-hydroxyprostaglandin dehydrogenase (15-HPGD) 17-α-hydroxypregesterone (17-P) 4′,6-Diamidino-2-phenylindole (DAPI) Adenosine triphosphate (ATP) Adenylate cyclase (AC) Aldo-keto-reductase (AKR) Amphotericin B (Amp B) Analysis of variance (ANOVA) Antibody (Ab) Arachidonic acid (AA) Area under the curve (AUC) Bovine serum albumen (BSA) Calcium (Ca2+) Calmodulin (Cal) Catalytic kinase domain (KD) Chemokine ligand (CCL) Chemokine receptor 7 (CCR7) Coiled-coil region (CCR) Connexion-43 (CX-43) Cyclic adenosine monophosphate (cAMP) Cyclooxygenase (COX) Cysteine-rich domain (CRD) Cytochromes P450 (CYP) Dehydroepiandrosterone (DHEA) Deoxyribonuclease I (DNase I) Deoxyribonucleotide triphosphates (dNTPs) Diacylglycerol (DAG) Dihomo γ linolenic acid (DGLA) Dihydroxydocosapentaenoic acid (DiHDPA) Dihydroxyeicosatetraenoic acid (DiHETE) Dihydroxyeicosatrienoic acid (DHET) Dihydroxyoctadecaenoic acid (DiHOME) Dimethyl sulphoxide (DMSO) Di-phosphorylated Myosin light chain (ppMLC)

Docosahexaenoic acid (DHA) Dulbecco’s modified eagles medium (DMEM) Dulbecco’s phosphate buffered saline (DPBS) Eicosapentaenoic acid (EPA) Electrospray ionisation liquid chromatography tandem mass spectrometry (LC/ESI-MS/MS) Endothelial nitric oxide synthase (eNOS) Enzyme-linked immunosorbent Assay (ELISA) Epoxydocosapentaenoic acid (EpDPE) Epoxyeicosatrienoic acid (EET) Epoxy-keto-octadecanoic acid (EKODE) Epoxyoctadecanoic acid (EpOME) Ethylendiamine tetra-acetic acid (EDTA) Extracellular signal-regulated kinase (ERK) Foetal bovine serum (FBS) Follicle stimulating hormone (FSH) Gram (g) Grams/second (g.s.) Growth factor receptor bound protein 2 (GRB2) Guanosine diphosphate (GDP) Guanosine triphosphate (GTP) Haematoxylin and eosin (H&E) Hank's balanced salt solution (HBSS) Heparan sulfate proteoglycan (HPSG) High performance liquid chromatography (HPLC) High-density lipoprotein (HDL) Horeseradish peroxidase (HRP) Hydrogen cloride (HCl) Hydrooctadecatrienoic acid (HOTrE) Hydroxydocosahexaenoic acid (HDHA) Hydroxyeicosapentaenoic acid (HEPE) Hydroxyeicosatetraenoic acids (HETE) Hydroxyeicosatrienoic acid (HETrE) Hydroxyoctadecadienoic acid (HODE) Hydroxysteroid dehydrogenase (HSD) Immunofluorescence (IF) Immunohistochemistry (IHC)

Inducible nitric oxide synthase (iNOS)

Inositol-1,4,5-trisphosphate (IP3). Intramuscularly (IM) Intrauterine systems (IUS) Krüppel-like factor 9 (KLF9) Leukotriene (LT) Limit of detection (LOD) Limit of quantification (LOQ) Linoleic acid (LA) Lipoic acid (ALA) Lipopolysaccharides (LPS) Lipoxygenase (LOX) Low-density lipoprotein cholesterol (LDL) Luteinisinghormone (LH) Magnesium (Mg2+) Maresin 1 (Mar1) Matrix metalloproteinases (MMPs) Maximum velocity (Vmax) Medroxyprogesterone acetate (MPA) Messenger ribonucleic acid (mRNA) Mitogen-activated protein kinases (MAPK) Molar (M) Mono-phosphorylated Myosin light chain (pMLC) Multiple reaction monitoring (MRM) Myosin light chain (MLC, MLC20) myosin light chain kinase (MLCK) myosin light chain phosphatase (MLCP), Natural killer (NK) Neuronal nitric oxide synthase (nNOS) Nicotinamide adenine dinucleotide phosphate (NADP) Nitric oxide (NO) Nitric oxide donor (NO-donor) Nitric oxide synthase (NOS) Non-obese diabetic (NOD) Nonsteroidal anti-inflammatory drugs (NSAIDs) Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) Number of contraction cycles (No)

Oestrogen (E2) Oestrogen receptors-α (ERα) Oestrogen receptors-β (ERβ) Optical densities (OD) Oxo octadecadienoic acid (OxoODE) Oxytocin (OXT) Oxytocin Receprots (OXR) Paraformaldehyde (PFA) Pleckstrin-homology domain (PHD) Penicillin-streptomycin solution (Pen-Strep) Peroxisome proliferator-activated receptor (PPAR) Phosphate buffered saline (PBS) Phospholipase (PL) Phospholipase C (PLCβ) Polyunsaturated fatty acid (PUFA) Potential of hydrogen (pH) Premature rupture of membranes (PROM)

Progesterone (P4) Progesterone receptor (PR) Progesterone receptor-A (PR-A) Progesterone receptor-B (PR-B) Prostacyclin (PGI) Prostacyclin synthase (PGIS) Prostaglandin (PGs) Prostaglandin D receptor (PD) Prostaglandin D synthase (PGDS) Prostaglandin E receptor (EP) Prostaglandin E synthase (PGES) Prostaglandin F receptor (FP) Prostaglandin F synthase (PGFS) Protein kinase C (PKC) Quadrupole (Q) Ras homolog family member A (RhoA) Resolvin (Rv) Retention times (RT) Rho Kinase (ROCK) Rho Kinase I (ROCKI)

Rho Kinase II (ROCKII) Rho binding domain (RBD) Ribonucleic acid (RNA) Schedule 1 (S1) Sodium hydroxide (NaOH) Soluble epoxyhydrolase (sHE) Standard error of the mean (SEM) Subcutaneously (SC) Thiazolyl blue tetrazolium bromide (MTT) Thromboxane A synthase (TXAS)

Thromboxane A2 (TXA2) Thromboxane receptors (TP) Thromboxane receptors α (TPα) Thromboxane receptors β (TPβ) Tumor necrosis factor α (TNF-α) United Kingdom (UK) United States of America (USA)

Publications

Poster presentation:

Preliminary Assessment of Potential Uterine Tocolytics in an in vivo Model. Manchester Pharmacy School Postgraduate Research Conference. 16–17 May / 2016. University of Manchester, Manchester, UK

Abstract

The uterus and placenta are essential organs playing key roles in reproductive processes. Labour is modulated by several factors such as sex hormones, nitric oxide, prostaglandins and the ROCK pathway, which stimulate the uterus via the phosphorylation of myosin-light chain (MLC). Abnormal function of these factors may lead to preterm labour (PTL) which still has no effective management strategies. The aims of this project were to: (i) examine the regulation of myometrial contractility in non-pregnant and pregnant mice, (ii) assess the ability of ripasudil (a ROCK inhibitor) to diminish uterine contractions, (iii) evaluate the effect of NO on myometrial contractility, and (iv) investigate the influence of administrating empty and NO-loaded liposomes on the level of lipid mediators in the uterus and placenta from C57 and eNOS KO pregnant mice at term. Results showed a significant higher contractility of the upper segment of uterus as compared to the lower segment (p<0.05). During pregnancy, stimulation of the uterus by oxytocin, U46619 and 5-HT upregulates the production of the di-phosphorylated MLC by 378.1%, 379.6% and 271%, respectively. Ripasudil was able to inhibit spontaneous and drug-induced myometrial contractility in non-pregnant and pregnant mice. Ripasudil inhibited the mono- and di-phosphorylation of MLC in the myometrium from both mice; it was more effective on ppMLC formation (88.61% and 86.76%). The lack of NO caused a significant increase in myometrial (by 132.5%) and amplitude of contractions (by 132.1%) in eNOS KOs. Vehicle- and SE175-Liposome treatments led to a significant reduction in the level of Cyclooxygenase (COX) and Lipoxygenase (LOX) derived lipid mediators in the myometrium and placentas from pregnant C57 WT and eNOS KO mice. Exposure to the SE175-Liposome significantly increased the level of myometrial DHETs (by 199.1%, 153% and 450.6%) and placental DiHDPA (by >4×108) compared with the Free SE175. SE175 significantly increased the concetration of the 15 HETrE. The majority of lipid mediators detected were the LOX- derived (myometrium) and COX-derived (placenta). In conclusion, the data support the higher myogenic activity of uterus in pregnant mice. It demonstrated for the first time that ppMLC is expressed in mouse myometrium and that ROCK inhibition is a promising tocolytic candidate for the treatment of PTL. Targeted liposomes were effective in modulating the level of certain lipid mediators. Administration of NO- donors at late pregnancy can regulate uterine activity and control placental blood flow.

Declaration

I declare that no portion of the work referred to in the thesis has been submitted in support of an application for another degree or qualification of this or any other university or other institutes of learning.

I. The author of this thesis (including any appendices and/or schedules to this

thesis) owns certain copyright or related rights in it (the “Copyright”) and s/he has given The University of Manchester certain rights to use such Copyright, including for administrative purposes. II. Copies of this thesis, either in full or in extracts and whether in hard or electronic copy, may be made only in accordance with the Copyright, Designs and Patents Act 1988 (as amended) and regulations issued under it or, where appropriate, in accordance with licensing agreements which the University has from time to time. This page must form part of any such copies made. III. The ownership of certain Copyright, patents, designs, trademarks and other intellectual property (the “Intellectual Property”) and any reproductions of copyright works in the thesis, for example graphs and tables (“Reproductions”), which may be described in this thesis, may not be owned by the author and may be owned by third parties. Such Intellectual Property and Reproductions cannot and must not be made available for use without the prior written permission of the owner(s) of the relevant Intellectual Property and/or Reproductions. IV. Further information on the conditions under which disclosure, publication and commercialisation of this thesis, the Copyright and any Intellectual Property and/or Reproductions described in it may take place is available in the University IP Policy (see http://documents.manchester.ac.uk/DocuInfo.aspx?DocID=24420), in any relevant Thesis restriction declarations deposited in the University Library, The University Library’s regulations (see http://www.manchester.ac.uk/library/aboutus/regulations) and in The University’s policy on Presentation of Theses.

Acknowledgements

I would like to thank several contributors to the work in this thesis. Firstly, I could not have hoped for a more approachable, enthusiastic, thoughtful and generous supervisor than Prof. Kay Marshall. She has been instrumental in developing my interest in reproductive pharmacology and research, which I hope to continue throughout my career. She has been an excellent leader, a valued colleague and friendly over this time. Honestly, without her help and support, this work couldn't have been done. I am also highly indebted to Dr.Debbie Fischer, my lovely second supervisor who has offered all the support and being very kind and cooperative throughout the whole study period. Really, I can’t forget her generosity and being always available to help. I am very grateful to Dr.Lynda Harris, my kind third supervisor for her valuable comments and being very supportive all the time. I would also thank her for providing mouse samples from her lab. I would also thank Prof. Anna Nicolauo for her scientific support and allowing us to join her group meetings and for permitting to use her lab facilities to conduct mass spectrometry. A special thank to Mrs. Sharon Farell for being very kind, highly cooperative and available whenever we need and always with a smiley face. Thanks as well to our lab colleagues Dr.osman Zarroug, Dr.Matthew Rosser and Dr.Orsi Kiss for being around and supportive. I am also very happy to have a nice friend who we started our studies together, Dr.Sara Santorelli. Thanks as well to Dr.Alexandra Kendall, Dr.Kathryn McGurk and Dr.Marta Koszyczarek for the support in the mass spectrometry work and data analysis. Thanks to Dr.Adel Alghaith, Yousef, Noora, Dr.Megan Uttley and Dr.Anggit Sunarwidhi for being kind and around during the study period. I would like to thank Dr.Jeff Penny and his group for their support and permission to use their lab facilities, especially Dr.Maryam Shubbar and Yu Siong for their time and help, thanks as well to Atheer Al-zurfi, and the kind Kuwaiti friend, Amna Alrubaee.

My thanks extend to Dr.Mike Harte and Dr.Ben Grayson for their help to order mice and using their lab machines. Thanks for the kind colleagues and friends for being around throughout these years Dr.Dina, Dr.Mai, Amira, Hend, Nuha, Abdul-Hamid, Dr.Osama Abu Sara, Dr.Sarmad, Dr.Hayat and Dr.Farah El mohtadi. Thanks as well to all Staff of Stopford reception; Ismaeel, Tom, Shirly and Carmel and all others who I can’t remember their names for being very kind with us all the time. I would like also to thank all my teachers through my whole life extending from primary school until today. A big thank to all professors and lecturers at Mosul College of Pharmacy, Duhok College of Medicine and my academic family at Duhok College of Pharmacy. I also greatly appreciate the kind support of Prof.Fouad Mohammad, Dr.Hishyar, Dr.Muyad Aghali, Dr.Ahmed M.Salih Bamerni, Prof.Kawa Dizaye and Dr.Ghazwan M.Raouf. To my lovely brother and life companion Dr.Husein M. Rashid, thanks for being around always. Thanks to my second family here in the UK kaka Ari and dear sister Juwan for being supportive while I am in Manchester. Thanks to my dear friends Dr.Muammar and Dr. Laith for the nice friendship in the UK. Huge thanks to: my inspiration in life, my Grandfather (Haj Mohammed Brifkani), your soul is still living with me; my lovely mum (The symbol of sacrifice and dedication); the respectful dad; my brothers and sisters, especially the faithful Ali; my uncle Haji Abdul-Rahman and my second mum, Hajia Layla for their love and support. Deep and many thanks to the centre of my life, my little family, the wonderful wife, Jinan, my smart son, Yasir, the sweaty star, Sima and the British-born baby, Adam. Thanks as well for everyone who was around and supportive to complete this work.

Dedication

I dedicate this thesis to my beloved wife and the wonderful

companion of my heart

♥ Jinan♥

For her unconditional support during the past 15 years,

especially during this study

Thank you my love… ♡♡♡

1 Chapter 1:

Introduction

1.1 The female reproductive system

The female reproductive system is specialised and adapted to accommodate the processes of oogenesis, the menstrual cycle and pregnancy. The uterus is a structural organ of the reproductive system that plays an essential role in the female genital tract and reproductive processes. Problems of the uterus can lead to various gynaecological disorders such as abnormal uterine bleeding and premature labour. Therefore, in order to discover and develop novel drugs for the treatment of female reproductive diseases, it is important to understand the normal physiology of the uterus.

1.2 The human female reproductive system

The human reproductive tract is made up of the ovaries, fallopian tubes, uterus and vagina which are located in the pelvis between the urinary bladder and the rectum (Marieb and Hoehn, 2016). The ovaries are located on either side of the uterus and are regarded as the major site of sex hormone production including oestrogen (E2) and progesterone (P4) (Narayanan and Cohen, 2019, Laven and Fauser, 2006). The fallopian tubes are crucial structures of the reproductive system, located on the sides of the uterus and they play an important role transporting the oocyte from the ovaries to the uterus (Briceag et al., 2015, Boehme and Donat, 1992). The vagina is a tube that connects the uterus to the outside of the body, which allows the flow of blood during the menstrual cycle and permits the passage of the foetus during labour (Anderson and Paterek, 2019, Stables and Rankin, 2010, Memon and Hand, 2013). Figure shows all components of the human uterus.

1.2.1 The human uterus

The human uterus is a pear-shaped structure and consists of three portions (Figure 1.1): the fundus, which the upper part of the uterus above fallopian tubes; the body or the corpus, which is the main and middle part of the uterus; The cervix, which is the lower and narrowest part connecting the uterus to the vagina (Waugh and Grant, 2018).

Figure 1.1. Human uterus, vagina, fallopian tubes, ovaries and uterine layers (Szmelskyj et al., 2015)

The uterine wall is composed of three layers (as seen in Figure 1.3): the perimetrium, myometrium, and the endometrium (Jividen et al., 2014). The endometrium represents the mucous membrane lining of the uterus and is composed of simple columnar epithelial cells and the lamina propria (Elchalal and Abramov, 1995). The myometrium is a muscular layer that makes up most of the uterine wall. It is composed of longitudinal and circular smooth muscle fibres. These fibres are arranged in flat or

35 cylindrical bundles and are separated by thin septa of connective tissue that is rich in blood vessels (Elchalal and Abramov, 1995, Lutton et al., 2018). As the myometrium reaches the cervix, its muscular content decreases and replaced by connective tissues, so the cervix becomes harder with a lower contractile ability than the other parts of the uterus (Vink and Mourad, 2017, Petersen et al., 1991). The outer serous layer of the uterus, the perimetrium (serona), covers the whole uterine surface except the cervical part. The uterine and ovarian arteries originate from the internal and iliac arteries and the aorta and supply the uterus with blood (Sabar, 2012).

1.3 The mouse female reproductive system

Similar to humans, the reproductive tract in the female mouse is composed of several anatomically and functionally specialised organs. These organs undergo frequent changes by the effect of female sex hormones leading to a periodic pattern of fertility (Bertolin and Murphy, 2014). The reproductive tract of the female mouse consists of paired ovaries and oviducts, a uterus and vagina (Figure 1.2). During pregnancy, the mouse undergoes hemochorial placentation, whereby maternal blood comes into direct contact with the foetal chorion (Hickman et al., 2016). The female mouse also possesses five pairs of mammary glands to produce milk (Chai and Dickie, 1966, Hammond et al., 1996).

1.3.1 The mouse uterus

The mouse uterus has a biocornuate shape and is composed of a pair of independent lateral horns. Each horn extends to connect a separate oviduct to the cervix and both horns are suspended from the dorsal body wall by specific ligaments (Griffiths, 2007, Boyd et al., 2018). Like other species, the wall of the mouse uterus consists of three layers: the perimetrium, myometrium, and endometrium (Bhartiya and James, 2017). Figure displays the three layers of the mouse uterus.

Figure 1.2. A dissected genital tract of the female mouse (Knoblaugh and Randolph- Habecker, 2018).

The endometrium is a specialised mucosal membrane which compromises the inner layer of the uterine wall. The endometrial glands, which are located inside the endometrial tissue, are directly connected to the myometrium and this connection is known as the uterine junctional zone (Fusi et al., 2006). The myometrium consists of smooth muscle cells and is regarded as the source of uterine force and contractile activity. The myometrium of the mouse uterus is composed of two layers of smooth muscle: the thick inner circular layer and the thinner outer longitudinal layer. There is a diffuse network of blood vessels between the two layers of the smooth muscle. The blood supply to the uterus is provided by the uterine and ovarian arteries, which anastomose extensively with each other (Griffiths, 2007). The endometrium is supplied with blood by the uterine artery, which branches into the arcuate arteries upon entering the myometrium. The uterus is also supplied by multiple veins and lymphatic vessels (Vom Saal and Dhar, 1992, Red-Horse, 2008) (Red-Horse, 2008).

Figure 1.3. A histological diagram of the mouse uterus (Bhartiya and James, 2017). The endometrium is surrounded by the myometrium and perimetrium.

1.3.2 The mouse placenta

In pregnancy, the placenta is the primary barrier between the embryo and the mother. It is considered as the main modulator regulating the nutrient and oxygen supply to the growing foetus (Zhang et al., 2015, Woods et al., 2018). Therefore, sufficient placental function is necessary for the development and growth of the foetus inside the uterus. foetal growth restriction is a prevalent pregnancy complication that is characterized by inadequate foetal growth and is often attributed to reduced placental development (Woods et al., 2018). The mature placenta of the mouse is composed of three layers; the maternal decidua, the middle junctional zone spongiotrophoblast layer, and the inner labyrinth layer (Zhu et al., 2017). The labyrinth layer is the biggest layer of the placenta and is the site of exchange of nutrients and gases between the maternal and the embryo blood circulations (Simmons et al., 2008). The mouse placenta is supplied with blood via two separate arteries, one which originates from the uterine artery and the other from the ovarian artery. Figures 1.4 and 1.5 show the structure and blood supply of the mature mouse placenta.

Figure 1.4. Overview of mature mouse placenta (Latos and Hemberger, 2016). The three major layers of the mature placenta: the labyrinth, the junctional zone consisting of spongiotrophoblast and glycogen cells and a layer of parietal trophoblast giant cells (TGCs), and the maternal decidua. The exchange interface in the labyrinth layer consists of three trophoblast cell types: a discontinuous layer of sinusoidal trophoblast giant cells, syncytiotrophoblast I (SynT-I), syncytiotrophoblast II (SynT-II) and an endothelial cell layer of the foetal capillary vessels.

Figure 1.5. Diagram of arterial blood supply to the mouse placenta during gestation (Raz et al., 2012). Both uterine and ovarian arteries supply blood to the mouse placenta. (Kidney (K); Ovary (Ov.); Foetus (F); Placenta (P); Uterine (Ut.).

Recently, the unique biological characteristics of the placenta have been exploited to identify placental homing peptides and create nanoparticles for targeted delivery of drugs to the placenta. This approach was developed to minimize drug exposure and adverse reactions in the developing foetus and decrease off-target effects in the maternal tissues (King et al., 2016). In addition, researchers have also developed nanoparticles for targeted delivery of drugs to the pregnant uterus to treat preterm labour (PTL) which displayed promising outcomes (Refuerzo et al., 2016).

1.4 The role of mouse uterus during oestrous cycle

Upon sexual maturity in the female mouse (after 6-8 weeks of birth), the uterus undergoes physiological alterations during the oestrous cycle. Unlike other species, the endometrium of mouse uterus is reabsorbed if no fertilization takes place. Mice are considered as polyoestrous mammals and the normal duration of their oestrous cycle is approximately 4-5 days. Interruption of the oestrous cycle is a condition characterized by quiescence and is termed as anoestrous phase, where there is an absence of ovarian activity (Griffiths, 2007). The mouse oestrous cycle is divided into four stages: pro- oestrus, oestrus, metoestrus and dioestrus and these stages are correlated with the variation in the concentration of sex hormones (Champlin et al., 1973, Caligioni, 2009).

Throughout the oestrous cycle, the level of oestrogen (E2) and progesterone (P4) are fluctuating according to the oestrous stage. In pro-oestrus, the concentration of E2 is gradually elevated, endometrial cells proliferate, leukocytes are depleted and uterine contractility is increased. During the oestrous stage, the increased level of E2 is maintained and the epithelial cells are predominantly cornified, while leukocytes remain at a minimal level. This is the only stage where the mouse is sexually receptive as there is hormonal-induced follicle maturation and ovulation. In metoestrus, the E2 level starts to decline with the formation of corpus luteum and a reduction in uterine size and vascular bed occurs. Metoestrus is characterized by a mixture of all cells types. Among the four stages of the oestrous cycle, dioestrus is regarded as the longest stage with a duration of more than 48 hours. Vaginal smears during dioestrus contain predominantly leukocytes, where P4 is high and E2 begins to increase. (Byers et al., 2012, Caligioni, 2009, Walmer et al., 1992, Tan et al., 2003, Griffiths, 2007).

1.5 The role of the uterus during pregnancy

The uterus performs multiple functions and undergoes several changes in shape and muscular strength throughout the gestational period and during labour. During pregnancy, and in order to tolerate the rapidly developing embryo, the uterus grows and the myometrium stretches and becomes thinner (Kim et al., 2018). In mice, the endometrial glands of the uterus play an important role in the process of decidualization by facilitating the implantation of blastocyst. This implantation occurs when the endometrium becomes receptive to blastocyst on day 4 of gestation (Spencer, 2014). The cervix keeps its structural integrity during pregnancy to retain the growing foetus inside the uterus and to protect it against microbial invasion through the vagina (Nott et al., 2016). During foetal maturation, the myometrium remains in a quiescent state until the time of labour where it produces powerful contractile forces to expel the foetus

(Zakar and Mesiano, 2011). P4 exhibits an essential role in maintaining uterine quiescence and suppressing myometrial contractility during gestation (Garfield et al., 1998, Zakar and Mesiano, 2011). Investigations have demonstrated the differential expression and functional characterization of distinct genes profile that associated with the regulation of uterine activity throughout the whole pregnancy and labour. Dysfunctional modulation of these gene products can lead to PTL or failure to begin or progress labour process (MacDougall et al., 2003).

1.6 Normal parturition: The common pathways of labour

Human labour can be defined as a multifactorial process that includes anatomical, biochemical, physiological, immunological, endocrinological and clinical events that occur in the mother and the foetus. The examination of uterine changes, such as myometrial contraction, cervical ripening and decidual induction can assist in clearly elucidating the common pathway of labour, which consists of different mechanisms that regulate the induction of parturition (Romero et al., 2006, Kota et al., 2013). Labour (or parturition) is also associated with important alterations in non-uterine or extrauterine components of the common pathway. Examples of extrauterine biomarkers of labour include alterations in the plasma concentrations of corticotropin-releasing factor, urocortin and cortisol (Koucky et al., 2009, Florio et al., 2002).

Correspondingly, adaptations of foetal physiology correlated to threatened spontaneous labour are likely to occur, including changes in foetal lung fluid distribution (Kalache et al., 2002). These alterations are mostly impossible to investigate in humans, thus the majority of studies are restricted to animal research (Romero et al., 2006). During labour, the relaxatory state of uterus is turned into to a stimulatory condition due to the forceful myogenic contractility (Griffiths, 2007). Labour involves a series of biological and physiological changes and further investigations are needed to explore the exact mechanism (Lopez Bernal, 2003). The main variation between human and mouse labour is progesterone withdrawal which is a major event for the initiation of labour in rodents (Sugimoto et al., 1997), while humans do not show significant changes in the serum concentrations of P4 on the progress of the first stage of labour (Astle et al., 2003).

1.7 Preterm Labour (PTL)

The normal duration of pregnancy in women ranges between 37 and 42 weeks, starting on the first day of the last ordinary menstrual cycle (Liao et al., 2005). Preterm labour (PTL) can be defined as childbirth before 37 weeks of gestation and it is either due to the spontaneous rupture of membranes or multiple contractions of the uterus causing dilatation and effacement of the cervix. PTL is regarded as a major cause of neonatal morbidity and mortality in the world (van Vliet et al., 2014). It contributes to 75% perinatal mortality (Goldenberg et al., 2008) and about 34.3% infant morbidity (Callaghan et al., 2006). Although many interventions have been undertaken to prevent and treat PTL, no declining incidence trends have been observed around the globe (Marinov et al., 2014), and moreover, an approximate 25% increase in PTL has been recorded in the United States since 1990 (Heron et al., 2010).

Most of the preterm births occur between 32-36 weeks of gestation (MacDorman et al., 2014). It has been shown that there is a sharp decrease in the prevalence of negative outcomes in the neonate, from 77% to less than 2%, when the gestational age is prolonged from 24-27 weeks to 34 weeks (Lim et al., 2007). In 2010, the rate of preterm birth between 24 and 36 weeks of gestation was 9.8% in the USA, 7-7.1% in the UK and around 5.6-8.9% in other European countries (MacDorman et al., 2014). PTL is associated with a high mortality rate, costly medical intervention, and a

42 requirement for particular educational programmes and special services for disabled children (Petrou et al., 2011). In 1997, it was concluded that the hospital cost for the initial hospitalization per each extremely preterm survivor infant is about £317,166 (Kilpatrick et al., 1997). PTL has been also regarded as a predisposing factor to a high incidence of chronic pathological complications, such as neuropsychiatric disorders, respiratory diseases, visual problems and hearing dysfunction in affected offspring (Murray and Lopez, 1997).

Birth requires a complex switch of the uterus from a quiescent state to a contractile state, sufficient for expulsion of the foetus (Ratajczak et al., 2010). Insufficient information is available to understand the factors and mechanisms that control this dynamic phase. About 50% of preterm births are idiopathic, with no distinguishing underlying factors (Muglia and Katz, 2010). More studies are required to investigate the aetiology of PTL in order to address this global health issue.

PTL can be categorized into three main groups; group 1, which represents nearly 33% of the cases, is due to the intrauterine infection. Recently, obstetricians have recommended the termination of these pregnancies as soon as possible, ignoring gestational age. This is because of the serious negative outcomes, such as maternal tachycardia or foetal death (Mitchell et al., 2013, van Kamp et al., 2005, Tita and Andrews, 2010). However, the role of antibiotics in decreasing the incidence of PTL is still controversial, as some studies have shown the effectiveness of antibiotics as a treatment (Lamont et al., 2003), while others have indicated that these drugs neither treat PTL nor decrease morbidity and mortality (van den Broek et al., 2009). The majority of the cases are within the second group; the iatrogenic group, where the health concerns of both, the mother and foetus indicate that positive foetal outcomes can be achieved with clinical intervention leading to immediate delivery, regardless of gestational age. Furthermore, the foetal outcome in this group is better than that of the first one. The third group is spontaneous PTL, which requires more investigations to identify any associated maternal or foetal factors that could prolong gestation. In these pregnancies, efforts to prevent PTL or arrest prematurely uterine contractions could hopefully reduce potential complications, extend time in utero and improve infant outcomes (Mitchell et al., 2013).

1.7.1 Risk factors

The precise components associated with elevated risk of PTL are mostly unknown (Di Renzo et al., 2011). However, previous research has attempted to identify risk factors correlated to PTL. PTL is a complicated multifactorial event which may be caused by a number of broad pathological and genetic mechanisms, including maternal stress, infection and/or alterations in obstetric practices, such as indicated induced-parturition, and assisted reproductive procedures, such as intrauterine insemination and in vitro fertilization (Ratajczak et al., 2010). In addition, psychological stress and social factors, such as physically demanding work can play a role in the development of PTL, as well as a contribution of geographic and demographic characteristics (Beck et al., 2010, Behrman and Butler, 2007). Moreover, ethnic origin is also associated with PTL particularly in the USA, where the rate of PTL is 9.7% among all singleton births. The percentage of PTL is 100% higher in people of Afro-Caribbean descent than in the white population and is 25% higher in Hispanics than in white women (Reagan and Salsberry, 2005). Recently, it has also been found that the rate of PTL is higher in US- born non-Hispanic Afro-Caribbeans than in non-Hispanic Afro-Caribbean immigrants (Mason et al., 2010).

Personal and family histories of PTL, maternal cigarette smoking and opiate use have been regarded as considerable risk factors for PTL. However, no significant association has been found between PTL and maternal job, gravidity, educational status, history of abortion and periodontal or urinary infection (Mirzaie and Mohammah-Alizadeh, 2007). According to Mahmoodi et al (2010), there was no significant relationship between PTL and mother’s age, education and pre-pregnancy body mass index. But their study has concluded that premature rupture of membranes (PROM) was a significant cause of PTL (Mahmoodi et al., 2010). The incidence of PTL has been suggested to be more than double in mothers with history of thyroid, cardiac or diabetic diseases compared to healthy women (Nabavizadeh et al., 2012). Nevertheless, multiple studies have been conducted worldwide, showing an association of PTL with various other risk factors, such as low socioeconomic status, employment, body mass index, smoking, anaemia, primiparity, congenital abnormalities, foetal growth retardation, spontaneous rupture of membranes, multiple pregnancies, cervical malfunction, antepartum haemorrhage, stress and malnutrition (Lo et al., 2007, Di Renzo et al., 2011, Raisanen et al., 2013).

Furthermore, hypertension during pregnancy represents a high risk for PTL (Weaver et al., 2015). This gestational elevation of blood pressure is hypothesised to be due to the increased concentrations of thromboxane A2 (TXA2), which is a potent vasoconstrictor and platelet aggregator (Van Assche et al., 1992). TXA2 induces uterine contraction through acting on thromboxane receptors (TP) (Fischer et al., 2008).

Measuring plasma P4 and corticotrophin releasing hormone (CRH) levels during the third trimester can be used as an indicative biochemical marker in women at high risk of

PTL. A 30% decrease in mean P4 level was observed at 28 to 34 gestational weeks in woman who delivered prematurely, compared to those delivered at term. At the same time, mean CRH level at 28 to 34 gestational weeks was six times higher in women who delivered prematurely than in those admitted to labour unit at term (Stamatelou et al., 2009). In addition, identifying and treating disorders among pregnant women particularly hypertension, oligohydramnios, preeclampsia, bleeding, hyperemesis gravidarum, urinary tract infection and low diastolic blood pressure, besides providing effective medical care for specific risks can reduce the delivery rate of preterm babies and any associated complications (Alijahan et al., 2014, Leonard et al., 2015).

1.7.2 Pathophysiology of PTL

The pathogenesis of PTL may involve one or more of the following mechanisms: infection, uteroplacental ischaemia, impaired maternal tolerance to the foetus, allergic reactions, increased uterine distention, cervical insufficiency (Koucky et al., 2009) and disorders of hormonal metabolism, such as premature P4 withdrawal (Holt et al., 2011). Inflammation is regarded as the primary pathogenic process that contributes to PTL, and different studies have been published, indicating a correlation of the above processes with the triggering of the inflammatory response that promotes PTL (Koucky et al., 2009).

1.7.3 Role of steroid hormones in the induction of labour

P4 plays a key role in the maintenance of uterine quiescence during pregnancy, and a reduction of P4 activity coincides with the initiation of labour in most mammalian species. This can be mediated by a decrease in blood concentration of P4, metabolic 45 local alterations that enhance P4 clearance, and/or changes in expression progesterone receptor (PR) isoforms: PR-A and PR-B (Condon et al., 2006). Both PR-A and PR-B are expressed in myometrial cells, and when the cells are PR-B dominant, PR-B mediates the anti-inflammatory effects of P4, thereby enhancing myometrial quiescence. During labour, there is an elevation of PR-A expression, and this induces parturition by blocking the anti-inflammatory effects of PR-B and increasing pro-inflammatory gene expression in response to P4 (Tan et al., 2012). Accordingly, cervical ripening, spontaneous abortion and parturition can be stimulated by taking mifepristone (RU486), a PR antagonist. This supports the concept that a reduction in P4 level or its activity may contribute to some cases of PTL (Holt et al., 2011, Gawron and Kiley, 2013).

Undoubtedly, every step in the normal process of labour is impacted by P4 in balance with E2. During cervical softening, which is the slow progressive stage of cervical remodeling starting early in gestation, there are elevated P4 concentrations and comparatively low E2 levels. Thereafter, this early phase interlinks with the accelerated remodeling phase that occurs toward the end of gestation, which is known as cervical ripening. Cervical ripening occurs when P4 bioactivity is reduced and E2 concentration is elevated (Holt et al., 2011). Despite the distinctions between species regarding the mechanisms responsible for reducing P4 to a level required for onset of labour, most of the subsequent events that contribute to the cervical ripening are common between human and animal models (Timmons et al., 2010). In human labour, P4 withdrawal and

E2 stimulation are not due to alterations in their hormonal concentrations. Instead, these outcomes could be attributed to the changes in the myometrial responsiveness to P4 and

E2, through their receptors; progesterone receptors (PR) and oestrogen receptors (ER).

Mesiano et al., hypothesized that the decline in P4 activity is caused by the elevated PR-

A isoform expression, which inhibits P4 responsiveness, and that the increase in the PR-

A/PR-B ratio brings about functional P4 withdrawal at term. They also showed that the functional effects of E2 are caused by the elevated myometrial expression of ERα, not

ERβ, and this was associated with increased PR-A/PR-B expression and functional P4 withdrawal as shown in Figure 1.6. Ultimately, P4 responsiveness is inhibited to a degree that leads to adequate ERα expression, to enhance the responsiveness of myometrium to systemic E2, which results in E2 activation and myometrial contraction (Mesiano et al., 2002). This supports the idea that human labour can be induced by using PR antagonists, such as RU486 to enhance artificial P4 withdrawal, but it is not

46 influenced by elevated E2 synthesis. Therefore, the interaction between PR and ER systems might be an essential pathway modulating myometrial function during gestation and delivery (Mesiano et al., 2002, Mesiano, 2004).

Figure 1.6. The role of myometrial ER and PR systems in modulating human labour (Mesiano et al., 2002).

E2 stimulates myometrial contraction through several mechanisms, such as increasing the concentration of circulating prostaglandin (PG) (Challis et al., 2002), enhancing myometrial expression of PG receptors (Challis, 2000), elevating expression of oxytocin receptors (OTR), enhancing connexin-43 (CX-43) and myometrium gap junction formation and by up-regulating myosin light chain kinase. Collectively, all these events induce consistent uterine contractions (Behrman and Butler, 2007). Figure

1.7 describes the interrelationship between E2, P4 and contraction-associated proteins (CAPs) with their actions on human parturition. CAPs include CX-43, OTR and PG receptors (Lye et al., 1998).

Figure 1.7. Interactions between oestrogens, progesterone and CAPs during human pregnancy and parturition. 11βHSDs = 11β hydroxysteroid dehydrogenases; CRH = corticotropin-releasing hormone; PR = progesterone receptors; ERα = oestrogen receptor α; CAPs = contraction-associated proteins; OTr = oxytocin receptors; CX43 = connexin43; COX-2 = cyclooxygenase-2 (Smith et al., 2002).

The myometrium exists in a relaxed state throughout the whole of pregnancy until term.

PG, such as PGE2 and PGF2α are known to regulate myometrial contractility and onset of parturition via specific receptors (EP1, EP3 and FP) (Matsumoto et al., 1997, Lebel et al., 2004). Additionally, PGs are thought to stimulate cervical ripening through increasing glycosaminoglycan levels. P4 was found to play a critical role in modulating

PG effects, as the stimulatory effect of PG (particularly PGE2) on glycosaminoglycan production by cervical cells could be inhibited by high levels of P4 (Carbonne et al., 2000). Accordingly, PGs are believed to induce delivery via different mechanisms, including the inhibition of uterine PR-A expression (in guinea pig), and this may lead to functional P4 withdrawal, enhanced E2 dominance and a further increase in myometrial contractions (Welsh et al., 2014). Konopka et al. hypothesized that in spite of the difficulty in demonstrating the causal correlation between P4 decline and a successful induction of delivery using dinoprostone (a synthetic PGE2), dinoprostone might be associated with functional P4 withdrawal and stimulation of labour (Figure 1.8) (Konopka et al., 2013).

Figure 1.8. The possible pathway of progesterone withdrawal enhanced by dinoprostone and induction of labour. An elevated PR-A/PR-B ratio inhibits progesterone (P4) activity by increasing NF-κB-mediated responses, including COX-2 stimulation and increased PGE2 production. PR= progesterone receptors; NF-kB = transcription nuclear factor-κB; COX-2 = cyclooxygenase-2 (Konopka et al., 2013).

During gestation, P4 enhances myometrial quiescence via decreasing the expression of inflammatory cytokines (e.g. IL-1, IL-8) (Tan et al., 2012) and CAPs (MacIntyre et al., 2012). Near term, elevated expression of microRNA family members (such as miR- 200) in the myometrium, which are involved in gene regulation, opposes most of progesterone’s effects, accelerating its metabolism and stimulating the expression of inflammatory mediators, such as cytokines, chemokines and COX-2 (Renthal et al., 2013).

Moreover, influences of P4 on the decidua and chorioamniotic membranes lead to the suppression of basal- and Tumor necrosis factor α (TNF-α)-induced apoptosis (Luo et al., 2010), which prevents calcium-mediated cell death (Murtha et al., 2007) and alleviates cytokine-induced matrix metalloproteinases expression and action (Keller et al., 2000), which would ultimately lead to membrane rupture. P4 also regulates cervical ripening through modulating the metabolism of extracellular matrix (Mahendroo, 2012).

Randomised clinical trials have concluded that use of P4 by pregnant women with a history of PTL lowers the rate of frequent preterm parturition and decreases the probability of multiple health consequences in their newborns (Meis et al., 2003). An

49 ongoing UK-based randomized trial called 'Does progesterone prophylaxis to prevent preterm labour improve outcome?' (OPPTIMUM) has been designed to examine and provide further evidence on the activity of daily prophylactic vaginal natural progesterone from 22-34 gestational weeks in prolonging gestation and improving maternal and neonatal health consequences (Norman et al., 2012). The trial started in

2008, but its results have not been published yet. The effectiveness of P4 to minimize preterm birth has been reported to be due to its pharmacological action rather than replacing deficient P4 (Romero et al., 2014).

1.7.4 Prostanoids

Prostanoids are classified as lipid mediators and consist of prostaglandins (PG) and thromboxane (TX). Prostanoids are liberated from cells to maintain various homeostatic processes in different tissues. PG are further subdivided into PGD, PGE, PGF and PGI, according to their chemical structure (Tsuboi et al., 2002). In addition to their functions in inflammation, ovulation and luteolysis, prostanoids have important roles in initiating and maintaining labour (Jabbour and Sales, 2004, Giannoulias et al., 2002). PG are synthesized and metabolized via a common pathway (Figure 1.9). PG can be classified into three series: 1, 2 or 3. The pro-inflammatory series 2 PG are derived from arachidonic acid (Murray et al., 2005 ) by cyclooxygenase (COX), which is present in three isoforms: COX-1, COX-2 and COX-3 (Chandrasekharan et al., 2002). Although, these three isoforms share certain structural and catalytic properties, most human cells constitutively express COX-1, whereas expression of COX-2 and COX-3 are induced by hormones, growth factors and cytokines (Morita, 2002, Fischer, 2010). The enzymes responsible for the synthesis of different PG and TX are named according to the prostanoid produced as synthases; they include PGDS for PGD2, PGES for PGE2, PGFS for PGF2α, PGIS for PGI2 and TXS for TXA2. Prostanoids exert their actions via binding to high affinity G protein-coupled receptors named as DP, EP, FP,

IP and TP receptors, which are responsible for the signalling transmission of PGD2,

PGE2, PGF2α, PGI2 and TXA2 respectively (Narumiya et al., 1999). EP are further subdivided into EP1, EP2, EP3 and EP4, which transmit different signalling pathways and exert different pharmacological effects (Fischer, 2010).

Figure 1.9. The biosynthetic and metabolic processes of PGs (Fischer, 2010).

1.7.4.1 Role of prostaglandins in labour

PG mediate uterine contractions via their actions on receptors expressed in the myometrium in both, the non-pregnant and pregnant states (Senior et al., 1993). The expression profile of prostanoid receptors depends on the type of tissue. For example, TP is largely expressed in platelets, DP in the ileum, lung, stomach, and uterus, FP in renal cells, EP1 in fibroblasts and EP2 and EP4 in smooth muscle cells (Bos et al., 2004).

The prostanoid receptors expressed in the myometrium of pregnant women are DP, EP2,

EP3, FP, IP and TP. Excitation is mediated via EP3, FP, TP receptors, while inhibition is

DP-, EP2- and IP-receptor-mediated (Senior et al., 1993).

The inhibitory effect exerted by EP2 is very important to maintain pregnancy and uterine quiescence via increasing cAMP formation (Brodt-Eppley and Myatt, 1999). At the initiation of parturition, the level of cAMP declines and the myometrium becomes more responsive to contractile stimulation (Bernal et al., 1995). Furthermore, high levels of uterine PG were observed during labour, and this elevation, especially in PGE2 and PGF2α synthesis by the foetal membranes and decidua is synchronized with increased expression of COX-2 (Erkinheimo et al., 2000). PGE2 exerts diverse effects on the uterus depending on the gestational period and receptors expression; its potency toward EP receptors occurs in the following order: EP3>EP4≫EP2>EP1 (Abramovitz et al., 2000). Moreover, PGE2 is essential for stimulating myometrial contraction and

PGE1 and PGE2 analogues are used to induce parturition and enhance cervical ripening (Di Lieto et al., 1991). Further investigations are required to examine the expression and role of different PG receptors in female genital tract, and their influences on the latency period of gestation and labour process.

1.7.4.2 Role of thromboxane in labour

In spite of the lack of knowledge regarding the association between thromboxane receptor (TP receptor) and the initiation of labour, researchers have demonstrated the expression of TP in human myometrium and placenta, and they proposed that TP could play a role in the modulation of myometrial contractility and labour onset (Swanson et al., 1992). TP receptors are present in two isoforms; TPα and TPβ, which vary in their carboxyl-terminal domains. The two isoforms produce opposite effects; TPα stimulates adenylyl cyclase and enhance cAMP release. Conversely, TPβ enhances contractility 52 via two cumulative effects, by inhibiting adenylyl cyclase, which reduces cAMP production, and stimulating intracellular Ca2+ release through the inositol triphosphate

(IP3) pathway (Hirata et al., 1996, Fischer, 2010). Both isoforms of TP stimulate a small GTPase, the Ras homolog family member A (RhoA), which in turn activates two proteins; the rho-associated protein kinase ROCKI and its isoform ROCKII. These two proteins indirectly prevent the dephospholylation of myosin light chain (MLC20), which potentiates uterine contractility (Fischer, 2010, Amano et al., 2000). Figure 1.10 describes the role of thromboxane in myometrial contraction.

Figure 1.10. The role of thromboxane in muscle contraction. Thromboxane activates both the phospholipase C (PLCβ)-mediated release of inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 increases the release of Ca2+ from the sarcoplasmic reticulum intracellular stores. After the binding of Ca2+ with calmodulin (Cal), the combination activates myosin light chain kinase (MLCK), which catalyses MLC20 phosphorylation and stimulate uterine contractility. Stimulation of the rho-associated coiled coil-forming protein kinase (ROCK) pathway inhibits MLC20 phosphatase (MLCP), denoted by X, blocking MLC20 dephosphorylation; this increases the sensitivity of the uterus to Ca2+ and induces contractility. (Fischer 2010).

1.8 The ROCK enzymes

Rho-associated protein kinases (ROCKs) are the major and extensively investigated downstream targets of RhoA (Leung et al., 1995, Matsui et al., 1996). Two genes express these enzymes which encode two protein isoforms: ROKα (also known as ROCKII or Rho kinases) and ROKβ (also termed as ROCKI or p16ROK) (Leung et al., 1996, Nakagawa et al., 1996, Fujisawa et al., 1996). Rho-associated protein kinases (ROCKs) exert an essential role in regulating the effect of the Rho family GTPases on the actin cytoskeleton after external stimulations. The ROCK enzymes composed of three chief domains: a RhoA binding domain (RBD) situated in a coiled-coil region (CCR), a kinase domain which possesses the catalytic activity and a cysteine-rich domain which is involved in the localization of protein (Riento et al., 2003). The two isoforms of ROCK can be stimulated by RhoA binding to the RBD or by intracellular second messengers such as sphingosylphosphorylcholine or arachidonic acid (Fu et al., 1998, Shirao et al., 2002). ROCKI can be inhibited by certain molecules which include the Gem which alters the substrate specificity through binding to CCR, and RhoE/Rnd3 which inhibit ROCKI by binding to the kinase domain. On the other hand, ROCKII is inhibited by the Rad through its binding to the CCR of the protein (Ward et al., 2002, Riento et al., 2003). Figure 1.11 shows structure and modulation of ROCKI and ROCKII.

Figure 1.11. Structure and binding sites of ROCKI/ROCKII (Schmandke et al., 2007). The upper and lower numbers represent the amino acids of ROCKI and ROCKII, respectively. The main domains are: the catalytic kinase domain (KD), the pleckstrin-homology domain (PHD) that encompasses a cysteine-rich domain (CRD), and the Rho binding domain (RBD) situated within the coiled-coil region. RhoA stimulates ROCKs by binding to the RBD, while Gem and RhoE/Rnd3 inhibit ROCKI by binding to the CCR and KD, respectively. Rad inhibits ROCKII by binding to CCR. N = N-terminal end; C = C-terminal end.

The ROCK signalling pathway is associated with multiple neuronal processes including cell migration, regeneration and survival. The Rho GTPases are widely investigated in scientific research for their contribution in the biological processes involved in the development of cancer as well as their role in cardiovascular physiology (Schmandke et al., 2007).

1.8.1 Role of ROCK pathway in uterine contraction

Recently, ROCKs were also examined for their role in mediating smooth muscle contraction and particularly uterine activity (Aguilar et al., 2012, Tahara et al., 2002). It has been demonstrated that activation of RhoA/ROCK pathway can lead to Ca2+ sensitization and more forceful myometrial contractility in the pregnant human (Moran et al., 2002). ROCKI and ROCKII inhibit the enzyme myosin light chain phosphatase (MLCP) and so inhibit the dephosphorylation of myosin light chain (MLC20), which leads to Ca2+ sensitization and excessive uterine contraction. The same mechanism was confirmed in the mouse, rat and rabbit uteri during gestation (Harrod et al., 2011, Cario- Toumaniantz et al., 2003, Riley et al., 2005, Tahara et al., 2002). In addition, studies have concluded that aberrant myometrial expression of ROCKI contributes to impaired uterine contraction, as observed in women with PTL and prolonged full term labour (Moore and Lopez Bernal, 2003).

Interestingly, recent studies have demonstrated the role of ROCK in the di- phosphorylation of MLC20, which causes further contraction of the smooth muscle cells. Drugs which inhibit ROCK have been exploited to target this di-phosphorylation step and prevent the second step of myometrial contraction, avoiding certain complications such as PTL with minimal effects on the vascular smooth muscle (Aguilar et al., 2012). Further investigations are required to define the precise role of ROCKs in the labour process and the therapeutic potential of RhoA inhibitors in PTL. Figure 1.12 displays the role of ROCK in the mono- and di-phosphorylation steps of MLC in the uterine smooth muscle.

Figure 1.12. The role of ROCK in the mono- and di-phosphorylation steps of MLC in uterine myocytes. After the production of mono-phosphorylated Myosin light chain (pMLC) by the enzyme MLCK, the ROCK activates and second step of MLC di-phosphorylation through inhibiting MLCP. Myosin light chain (MLC); Myosin light chain kinase (MLCK); Myosin light chain phosphatase (MLCP); rho-associated coiled coil-forming protein kinase (ROCK).

1.9 Feasibility and translation of mouse model of PTL in humans

Rodent models of parturition are the main resources for research of PTL; they suggest two major pathways that control the timing of parturition: the withdrawal of P4 and pro- inflammatory reactions by the immune system. Nonetheless, current data largely suggest that there are different essential regulators and mediators between humans and most animals, in terms of the labour process (Mitchell and Taggart, 2009).

There are considerable variations between the human and mouse reproductive systems and gestation processes, as shown in Table 1.1. The mouse is regarded as a fascinating mammalian model system for studying human parturition due to the ability to produce

56 knockout (KO) and transgenic mice for examining the function of certain genes in different pathways. Development of specific KO models makes it possible to investigate individual genes that may affect the labour cascade. An additional advantage that makes mice a good experimental model is its short gestational period (Ratajczak and Muglia, 2008).

At the initiation of normal murine labour, PGs (e.g. PGF2α) induce luteolysis, the structural and functional degradation of corpora lutea, and P4 withdrawal (Sugimoto et al., 1997). Humans do not show such serum P4 withdrawal in late pregnancy (Challis et al., 2000). However, there is a decline in PR transcriptional effectiveness regulating the functional P4 withdrawal and playing a role in human labour (Condon et al., 2003).

Moreover, it has been demonstrated that P4 withdrawal is not the fundamental cause of delivery in genetically modified mice (Hirsch and Muhle, 2002). This highlights the significance of other contributing processes in the labour pathway that exist in both humans and mice (Ratajczak and Muglia, 2008).

Table 1.1. Main reproductive physiological distinctions between human and mouse (Mitchell and Taggart, 2009).

Factors Mouse Human Gestational period 20±1 days 37±2 weeks

Usual litter size 10±5 1 (no.)

Placental Hemotrichorial, labyrinthine Hemotrichorial, villous, discoid morphology Uterine shape Bicornuate uterus Pyriforms uterus Number of embryos Multiple gestations (6-10) pups Single gestation (1 foetus) Source of Corpus luteum Corpus luteum then placenta progesterone Progesterone Yes No withdrawal Induction of preterm Antiprogestin, ovariectomy, LPS Cervical ripening (PGE2) or birth antiprogestin) + oxytocin Values: means ± SD, LPS: lipopolysaccharides

In certain instances, such as unfavourable cervix at term, oral administration of mifepristone, a PR antagonist, increases cervical ripening and induces parturition in term women (McGill and Shetty, 2007), suggesting the functional role of P4 withdrawal in human parturition (Ratajczak and Muglia, 2008). In 2008, an animal study using mice demonstrated the significance of PR at term. Mice deficient for Krüppel- like factor 9 (KLF9), a PR co-regulator, showed postponed delivery with corresponding aberrant PR isoform-A expression in the myometrium and insensitivity to PR antagonists at late pregnancy (Zeng et al., 2008). Other researchers have investigated myometrial expression ratio of various PR isoforms in humans at parturition (Condon et al., 2006), but according to the available evidence, there was no convincing proof for PR modulation as being a component of functional withdrawal (Ratajczak and Muglia, 2008). However, some human and mouse data indicates that a reduction in the expression of PR coactivator and histone acetylation within the uterus at late gestation might weaken the role of PR by inducing functional withdrawal of P4 (Condon et al., 2003). In early studies, Skarnes and Harper identified that PTL, which is induced in mice by ovariectomy, can be inhibited by the administration of P4 (Skarnes and Harper, 1972).

The myometrium is an essential organ in the common pathway of labour that has to be receptive at the initiation of the process (Ratajczak and Muglia, 2008). In both humans and mice, there is a uterine-specific elevation in expression of contractile-associated proteins (CAPs), such as OTR (Oxytocin receptors), CX-43 (Connexin-43) and PGF2α receptors (FP) at term parturition (Cook et al., 2000, Brodt-Eppley and Myatt, 1999). CAPs and PG play a fundamental role in converting the uterus from the quiescent state throughout most of pregnancy, to the active co-ordinately contracting state required to expel the foetus (Ratajczak and Muglia, 2008).

Oxytocin (OXT) has powerful uterotonic activity (Yang et al., 2014) and is used recurrently to enhance parturition (Stubbs, 2000). The myometrial expression of OTR is highly elevated at term (Helmer et al., 1998). In addition to the uterotonic effects of OXT, it has been shown to play a role in maintaining the corpus luteum. In mice, the luteotrophic activity induced by OXT in late pregnancy can be inhibited by the expression of COX-1 (Gross et al., 1998). Moreover, Imamura et al, have suggested that the downregulation of corpus luteal OTR and activation of myometrial OTR help to

58 switch over the main complement of OT effects throughout mouse gestation from delivery suppression to advancement (Imamura et al., 2000).

The main enzyme responsible for PGF2α degradation is known as 15- hydroxyprostaglandin dehydrogenase (15-HPGD) (Okita and Okita, 1996). In humans, there is a reduction in 15-HPGD mRNA level in chorion trophoblast cells in women at either term or PTL (Sangha et al., 1994). Similarly, this decrease has been observed in labouring mice (Wang and Hirsch, 2003).

Taking all the above considerations into account, it seems that mice are an appropriate model for studying human parturition and identifying the existence and function of different biomarkers and processes that play a role in PTL.

1.10 Treatments for preterm labour

The development of successful prophylactic treatments to prevent the occurrence of PTL still requires more research. This is because it is unclear whether spontaneous PTL is a result of premature stimulation of the common labour pathways or a result of a pathological process that is responsible for the propulsion of uterine quiescence toward parturition; certain evidence supports the latter process (de Laat et al., 2013). Recent trials in women at high risk of PTL have concentrated on preventing premature contractions at the last stages of the pathological sequence. Treating pregnant women with threatened PTL involves the administration of tocolytic agents for 48h prior to giving corticosteroids. This is to facilitate the maturation of foetal lungs and transportation of the mother to an infirmary with a neonatal intensive care unit if the gestation was less than 37 weeks (van Vliet et al., 2014).

Multiple kinds of tocolytic drugs are usually used to treat PTL. These drugs include β- adrenomimetics, calcium channel blockers, OTR antagonists, non-steroidal anti- inflammatory drugs (NSAIDs) and magnesium sulphate. The characteristics of an optimal tocolytic drug are that it is: effective in deferring PTL, appropriately safe in both mother and foetus and has a powerful capability in minimizing neonatal diseases and death rates. However, there is no unanimity on the kind of tocolytic medication that is used, and there are differences worldwide (van Vliet et al., 2014). Current UK

59 guidelines recommend a calcium channel blocker (nifedipine) and oxytocin (OT) antagonist (Worldwide Atosiban versus Beta-agonists Study) as the first line tocolytic therapy in the prevention and treatment of PTL (Brown and MacIntyre, 2014, Tara and Thornton, 2004).

1.10.1 Drugs used in the treatment of PTL

1.10.1.1 β-adrenoceptor agonists

Ritodrine, terbutaline and isoxsuprine are examples of β-adrenoceptor agonists (Feely et al., 1983). This group of drugs slow the myometrial contractions through activating adenyl cyclase to produce cyclic adenosine monophosphate (cAMP). This elevation in cellular concentration of cAMP reduces myosin light-chain kinase activity by two mechanisms: firstly, via phosphorylating myosin light-chain kinase itself, and secondly, via decreasing intracellular calcium by enhancing calcium uptake by sarcoplasmic reticulum (Bernal, 2003). β-adrenoceptor agonists have been used for a long time to prevent PTL (van Vliet et al., 2014). β-adrenoceptor agonists are able to delay labour for 48 h, and their use has been associated to an improved neonatal morbidity, but not mortality (Anotayanonth et al., 2004). No superiority has been found for β-mimetics over OTR antagonists in relation to tocolytic activity or infant outcomes (Flenady et al., 2014).

1.10.1.2 Calcium channel blockers

Nifedipine and nicardipine are calcium channel blockers and nonselective muscle relaxants. They are used largely in the therapeutic management of hypertension, especially in adults. They act as tocolytic agents through inhibiting the influx of calcium from extracellular spaces into the myometrial cells. Their absolute effectiveness is still ambiguous (van Vliet et al., 2014), but they exert a significant tocolytic activity through preventing preterm birth within a week of taking treatment and before 34 weeks of gestation. Their use also has shown to significantly reduce adverse neonatal consequences, such as Respiratory Distress Syndrome (RDS), necrotizing enterocolitis, intraventicular haemorrhage (IVH) and jaundice (King et al., 2003). Calcium channel

60 blockers together with PGs inhibitors (e.g. indomethacin and ketorolac) and oxytocin antagonists (e.g. atosiban), were considered the best and most highly ranked tocolytics for evoking a 48 h delay in labour, infant death rate and maternal adverse reactions (Haas et al., 2012, Tara and Thornton, 2004). Atosiban has been found better than nifedipine in relation to the rate of mothers who did not give birth and those who did not need an alternative tocolytic drug within 48 h from the initiation of treatment. Furthermore, atosiban was better tolerated by women compared to nifedipine (Salim et al., 2012). Nifedipine showed the same activity of the non-selective COX inhibitor indomethacin in delaying labour (Klauser et al., 2014).

1.10.1.3 Oxytocin receptor antagonists

This group of drugs blocks OTR in the myometrium, prohibiting the elevation in intracellular calcium and thus causing myometrial relaxation. Atosiban, which is a mixed oxytocin/vasopressin (V1a) receptor antagonist, was developed to prevent PTL (van Vliet et al., 2014). Atosiban is as effective a tocolytic as β-adrenoceptor agonists in relation to the number of women giving birth within 48 h and 7 days (Papatsonis et al., 2005). However, atosiban does not decrease the incidence of perinatal adverse events, such as RDS and admission to neonatal intensive care unit in comparison to β- mimetics (van Vliet et al., 2014). Although atosiban has shown an effectiveness in delaying delivery by 48 h (van Vliet et al., 2014), in an indirect study, it was inferior to nifedipine in decreasing the incidence of RDS (Coomarasamy et al., 2003) or perinatal death (van Vliet et al., 2014). Comparing the timing of atosiban administration, research has concluded that early treatment increases the ratio of undelivered women without the need for alternative tocolysis for 48 h (Husslein et al., 2006). However, OTR desensitization can play a beneficial role in delaying parturition (Phaneuf et al., 1998). The probability of considering atosiban as the most efficient tocolytic agent is very weak (Haas et al., 2012).

Atosiban has the lowest incidence of adverse reactions in both the mother and the foetus among all tocolytics (van Vliet et al., 2014). A prospective cohort study on adverse drug reactions to tocolytic therapy for preterm delivery demonstrated no serious adverse effects after treating 575 women with a single dose of atosiban. The study also indicated that the only tocolytic agents causing no serious adverse reactions were 61 indomethacin and atosiban (de Heus et al., 2009). In spite of its relative safety and lower maternal cardiovascular adverse reactions compared to β-mimetics (Worldwide Atosiban versus Beta-agonists Study, 2001), atosiban therapy might result in pulmonary oedema if is used in a multiple pregnancy (Seinen et al., 2013). Economically, the significance of the lack of adverse effects explains the cost-saving benefit of atosiban in comparison to β-adrenoceptor agonists in the prevention of PTL (Wex et al., 2011).

In addition to atosiban, other OTR antagonists have been studied recently, such as barusiban, which is a competitive OTR antagonist. Barusiban has a high affinity for the OTR and exerts an inhibitory effect on myometrial contractions of both term and preterm myometrium (Pierzynski et al., 2004). Clinical research has demonstrated that barusiban is well tolerated and safe for mother, foetus, neonate and infant. However, no significant differences have been found between barusiban and placebo in preventing preterm myometrial contractions (Thornton et al., 2009). It is unclear if the weak clinical activity of barusiban might be associated with its selectivity toward OTR. The inhibitory action of atosiban on vasopressin receptors might contribute to its tocolytic capability. In fact, the V1a receptor antagonist, relcovaptan, which is administered orally, has shown some tocolytic action (van Vliet et al., 2014). In a double-blind study involving 18 women with preterm uterine contractions between 32 and 36 weeks of gestation, relcovaptan showed a significant decrease in the frequency of contractions compared to placebo-treated women, and indicated its effectiveness in preventing PTL. Nevertheless, there could be an involvement of OTR, and the comparable significance of oxytocin (OT) and V1a receptors is yet to be elucidated (Steinwall et al., 2005).

Although many trials have been undertaken to develop efficient OTR inhibitors, and atosiban has shown a good safety and effectiveness in delaying PTL, there is still no available clinical data reporting the impact of these tocolytics on neonatal morbidity and mortality (van Vliet et al., 2014).

1.10.1.4 Cyclooxygenase inhibitors and prostaglandin receptor antagonists

PG play an important role in uterine contractions via inducing the formation of myometrial gap junctions and elevating the intracellular levels of calcium (van Vliet et al., 2014). Cyclooxygenase (COX), which is also known as PG endoperoxidase

62 synthase, is the enzyme responsible for PG production (Yazawa et al., 2013). There are two isoforms of COX: COX-1 and COX-2. COX-1 is a constitutively expressed isoform supporting different homeostatic processes, whereas COX-2 is a mostly an inducible isoform, participating in various inflammatory disorders (Khan and Fraser, 2012). Myometrial contraction is predominantly regulated by COX-2. COX inhibitors lower PG synthesis, and thus modulate the common pathway of parturition.

In clinical trials, COX inhibitors, such as indomethacin, sulindac, rofecoxib, nimesulide and ketorolac have contributed to increased birth weight and lower delivery rate before 37 gestational weeks, but they did not show any superiority over a placebo in terms of perinatal morbidity or death rate. However, these outcomes should be taken cautiously, as among 13 trials on PTL, only three studies compared PG synthase inhibitors with a placebo. When selective COX-2 inhibitors were compared to nonselective COX inhibitors, they did not show any differences in perinatal mortality, pulmonary hypertension of the neonate, premature closure of ductus arteriosus, neonatal renal failure, intraventricular haemorrhage (IVH) and maternal outcomes (van Vliet et al., 2014, Reinebrant et al., 2015). Therefore, further studies are needed to investigate the superiority of COX-2 inhibitors over nonselective COX inhibitors. A new network meta-analysis has reported that the anti-COX tocolytics are efficacious to delay labour by 48 h, but they have little effect in reducing RDS or neonatal death rates. The study also demonstrated that PG inhibitors and calcium channel blockers have the best probability of being the most effective treatment of PTL (Haas et al., 2012).

COX inhibitors, and indomethacin as an example, can easily cross the placenta (Moise et al., 1990), and thus interfere with foetal PG homeostasis. Different adverse reactions have been listed under the use of PG inhibitors, such as oligohydramnios, antenatal constriction of the ductus arteriosus and renal impairment. A Cochrane review involving 403 women taking COX inhibitors showed that only one incidence of antenatal closure of ductus arteriosus had occurred. The review also showed no elevation in the incidence of postnatal patent ductus arteriosus was reported in comparison to placebo. Accordingly, the role of COX inhibitors for women at risk of PTL requires more investigation (King et al., 2005).

PG receptor antagonists, such as THG113.31 have shown a significant activity in inhibiting PG-induced uterine contractility and preventing PTL in sheep (Hirst et al.,

2005). Lack or disruption of a PG receptor gene, such as that of PGF2α receptor (FP) can lead to delivery failure in mice (Sugimoto et al., 1997); also PG receptor antagonists, such as THG113.31, a selective FP antagonist have been indicated to potentially prolong gestation and prevent preterm labour in a mouse model of endotoxin-induced preterm labour (Peri et al., 2002). Moreover, selective prostaglandin

E2 receptor (EP2) antagonists have been recently reported to delay preterm cervical ripening in humans and may be beneficially added to the treatment regimen of PTL (Kishore et al., 2014). It has been concluded that PF-04418948 has a powerful and selective antagonistic effect at the EP2 receptor in different organs and species (Forselles et al., 2011). In addition, AS-604872 is a selective FP receptor antagonist. It also inhibits uterine contractility and increases the gestational period in rodents (Cirillo et al., 2007, Chollet et al., 2007). Furthermore, AS-604872 has exhibited some selectivity toward EP2 receptors as an antagonist (Jones et al., 2009). Collectively, this evidence provides a plan to further study the implications of PG receptor antagonists in tocolytic therapy.

1.10.1.5 Antimicrobials

Many studies have tested the effects of different antimicrobial both as empiric and targeted therapies to prevent or treat various infections associated with PTL. Antibiotics were used either as monotherapy, such as beta-lactams, clindamycin and macrolides, or as combination regimens (Subramaniam et al., 2012).

A double-blind trial by Newton et al, used a combination of ampicillin-sulbactam and indomethacin to improve the tocolytic effect of magnesium sulfate. The study failed to indicate any benefit of this treatment strategy in terms of gestational age or neonatal consequences (Newton et al., 1991). Similarly, other conducted randomized trials did not show any efficiency of ampicillin-sulbactam, amoxicillin-clavulanic acid (Cox et al., 1996) or ceftizoxime (Gordon et al., 1995) in preventing PTL.

Clindamycin was shown to decrease the incidence of preterm premature rupture of the membranes (PPROM). It also elevated the interval to labour and showed well-tolerated safety margins by women enrolled before 33 weeks gestational age (McGregor et al., 1991). Moreover, clindamycin vaginal cream could decrease the occurrence of PTL by

60% in women with unhealthy genital tract flora (Lamont et al., 2003). In contrast, some studies have concluded that vaginal clindamycin does not prevent PTL or minimize the risk of puerperal infectious events, such as caesarean section wound infection and endometritis (Vermeulen and Bruinse, 1999, Kurkinen-Raty et al., 2000). According to all these findings, the role of clindamycin in preventing PTL is still controversial. Macrolides, such as azithromycin have been investigated in only a few studies, but they did not exert any beneficial role in preventing PTL or reducing maternal morbidity or perinatal mortality (van den Broek et al., 2009).

In randomised controlled trials, antibiotic therapy correlated with low rates of chorioamnionitis, labour within 48 h and labour within 7 days of randomisation. It was also associated with an improvement of some adverse neonatal events (Kenyon et al., 2010). Similar usefulness was observed with the use of antibiotics for asymptomatic bacteriuria in a 14 study meta-analysis (Smaill and Vazquez, 2007), where antibiotic therapy showed a potential decrease in the risk of pyelonephritis and the incidence of low birthweight, but it did not show any prevention of PTL. Among all investigated antibiotics, co-amoxiclav contributed to high risk of neonatal necrotising enterocolitis, and therefore should be avoided in women at risk of PTL (Kenyon et al., 2010).

1.10.1.6 Magnesium Sulfate

The precise mode of tocolytic action of magnesium sulfate for both initial and maintenance treatment is not completely clear. In general, magnesium sulfate reduces recurrent smooth muscle depolarization through interfering with calcium uptake, binding and movement in smooth muscle cells. Collectively, this leads to uterine relaxation (van Vliet et al., 2014).

A Cochrane review determined that both small and large doses of magnesium sulfate as a tocolytic agent did not show any usefulness in preventing delivery by over 48 h from the beginning of therapy, and also neither in preterm (< 37 weeks) nor in very preterm (< 34 weeks) labours. Studies concluded high neonatal mortality rates with the use of magnesium sulfate, particularly when the maintenance dose of magnesium sulfate was high, but no clear reason has been suggested for this finding, which needs further investigations (Crowther et al., 2002).

1.10.1.7 Progestagens

Progestagens, such as progesterone have been shown to inhibit uterine smooth muscle contractions and reduce expression of myometrial gap-junction proteins (Ruddock et al., 2008, Hendrix et al., 1995). Some studies have been performed using 17-α- hydroxypregesterone (17-P) and vaginal progesterone, and their uses were assessed for tocolytic activity in treating PTL, prolonging the latency preceding parturition or as prophylactic therapy (van Vliet et al., 2014, Borna and Sahabi, 2008). Keirse found that using 17-P therapy in women at risk of preterm birth decreased the incidence of early delivery (Keirse, 1990).

Vaginal progesterone showed a powerful tocolytic action to delay PTL through restraining PGs cascades (Saleh Gargari et al., 2012). Vaginal progesterone also increased the gestational age at birth, time of delaying labour and birth weight, and it reduced admissions to the neonatal intensive care unit (Saleh Gargari et al., 2012). Moreover, vaginal progesterone provided additional usefulness as a maintenance therapy in PTL after effective parenteral magnesium sulfate tocolysis (Borna and Sahabi, 2008). However, these outcomes were not observed using intramuscular injections of 17-P. It has been proposed that progestagens might not inhibit PTL, but increase the sensitivity of uterus to tocolytic agents (Jayasooriya and Lamont, 2009). The administration of high-dosage progesterone with β-agonists produces a synergistic action and reduced the need for high doses of β-agonists, which exert harmful adverse reactions (Di Renzo et al., 2005). Other studies indicated a reduction in PTL when progesterone is used with other tocolytic agents, such as atosiban and ritodrine (Facchinetti et al., 2007, Noblot et al., 1991). An in vitro study on isolated myometrial strips taken from pregnant women at low risk showed that natural progesterone enhanced the relaxation produced by ritodrine by decreasing the maximal response, amplitude, and frequency of myometrial contraction to 50% (Chanrachakul et al., 2005). Collectively, further studies are essential to investigate the conceivable role of progesterone in sensitizing the uterus by other tocolytic agents (van Vliet et al., 2014).

1.10.1.8 Drugs interfering with 5-HT pathway

Serotonin or 5-hydroxytryptamine (5-HT) is a monoamine neurotransmitter that is associated with pathophysiological processes of multiple clinical conditions such as irritable bowel syndrome, carcinoid diarrhoea and chemotherapy-induced vomiting (De Ponti, 2004). 5-HT binds to and activates various 5-HT receptors which are expressed in different subtypes in the body organs. Stimulation of 5-HT receptors by agonists increases the hydrolysis of membrane phospholipids to produce the two second messengers, inositol-trisphosphate (IP3) and diacylglycerol (DAG). IP3 activates the Ca2+-release from the intracellular stores and subsequent smooth muscle contractions (Pauwels, 2000).

These 5-HT receptor subtypes include 5-HT1, 5-HT3, 5-HT4, and 5-HT7 (De Ponti and Tonini, 2001). The effect produced by 5-HT depends on the receptor subtype it binds to and the location of the receptor. For example, 5-HT1A is expressed in the central nervous system and its activation by 5-HT will regulate blood pressure; 5-HT2A is expressed in the platelets, and the smooth muscle cells of the blood vessel wall and heart; stimulation of the 5-HT2A receptor leads to 5-HT release, smooth muscle contraction and tachycardia (Watts et al., 2012). Furthermore, 5-HT4 receptors are localized in the gut and agonists to these receptors increase the gastric emptying rate (De Ponti, 2004).

Activation of 5-HT2A induces visual hallucinations (Zhang and Stackman Jr, 2015).

It has been demonstrated that 5-HT receptors are also expressed in the uterus of humans and mice (Osman and Ammar, 1975, Cordeaux et al., 2009), and stimulation of 5-HT2A receptors in the myometrium of pregnant women at term increased uterine contractions. Furthermore, the role of 5-HT receptors was studied in the mouse uterus and it was demonstrated that three receptor subtypes are expressed in the uterine tissue: 5-HT1D, 5-

HT2A and 5-HT2C receptors. Activation of these receptors resulted in increased myometrial contractility (Xiu-Kun et al., 2011). The downstream events of 5-HT receptor activation also involve the ROCK pathway and studies have concluded that stimulation of 5-HT1B receptors in the pulmonary fibroblast in mice can activate the RhoA/Rho-kinase system leading to stimulation of nuclear transcription factors (Mair et al., 2008).

1.10.1.9 ROCK inhibitors

Although many scientific studies have investigated ROCK activity and have shown its involvement in different physiological disorders, the molecular mechanism(s) that lead to the elevation of ROCK levels in these disorders is still under investigation. In addition, ROCK inhibitors are not selective for the vascular wall and other body tissues, or for the different ROCK isoforms. However, tissue-specific gene knockdown of specific ROCK isoforms may help us to understand the unique pathophysiological roles of individual ROCKs (Liao et al., 2007). Due to the essential functions of ROCK in the development of cardiovascular and central nervous systems, deficiencies in the two isoforms of ROCK were fatal in developing mouse foetuses (Thumkeo et al., 2003, Rikitake et al., 2005). However, researchers in the field of drug discovery have developed selective and non-selective ROCK inhibitors to advance understanding of the contribution of ROCK isoforms to various vascular and inflammatory processes. Recently, ROCK inhibitors such as Y-27632 and fasudil that target their ATP- dependent kinase domains have shown no selectivity toward ROCK1 and ROCK2. These inhibitors can also inhibit other protein kinases at higher concentrations (Rikitake et al., 2005). Non-selective ROCK inhibitors have been shown to prevent cerebral vasospasm after subarachnoid haemorrhage (Sato et al., 2000) and inhibit the development of atherosclerosis after vascular injury in animals (Mallat et al., 2003). Fasudil has exerted useful effects in several human pathological conditions such as hypertension, angina and chronic heart failure (Masumoto et al., 2001, Masumoto et al., 2002, Kishi et al., 2005). This evidence indicates the contribution of the ROCK pathway in the pathogenesis of cardiovascular diseases.

Furthermore, the effect of ROCK inhibitors on the uterine activity and myometrial contractility has also been examined. Inhibitors of the ROCK pathway such as g-H- 1152 and Y-27632 have shown significant efficiency in diminishing myometrial contraction in different animal species such as humans and rodents (Hudson et al., 2012, Woodcock et al., 2004, Harrod et al., 2011). Moreover, other ROCK inhibitors (fasudil hydrochloride and AS1892802) have also exhibited tocolytic properties in pregnant rats (Ergul et al., 2016). Nevertheless, the exact mechanism by which ROCK inhibitors reduce uterine contraction is not fully understood and requires further investigation (Sakamoto et al., 2003). Furthermore, inhibiting myometrial contractility through targeting different pathways may exacerbate existing cardiovascular problems (Aguilar

68 et al., 2012, Lopez Bernal, 2007, Norwitz and Robinson, 2001) and thus, research is ongoing to develop new therapies with maximal beneficial effects on the uterine activity and minimal complications on the peripheral resistance and other body organs.

1.10.1.10 Ripasudil: history, structure and pharmacology

Ripasudil hydrochloride hydrate (or K-115) or 4-fluoro-5-{[(2S)-2-methyl-1,4- diazepan-1-yl]sulfonyl}cisoquinolin (Figure 1.13) is a newly developed specific ROCK inhibitor. It was primarily approved in 2014 in Japan as an ophthalmic solution to treat glaucoma and ocular hypertension (Garnock-Jones, 2014). Ripasudil is a highly selective and effective ROCK inhibitor (Isobe et al., 2014) and has 50% inhibitory concentrations (IC50) of 0.051 and 0.019 μmol/l for ROCKI and ROCKII, respectively on trabecular meshwork cells in rabbits (Isobe et al., 2014). Table 1.2 displays the structural and physiological properties of ripasudil.

Figure 1.13. The chemical structure of ripasudil (Isobe et al., 2014). 4-fluoro-5-{[(2S)-2-methyl-1,4-diazepan-1-yl]sulfonyl}isoquinolin

All available studies on ripasudil have focussed on its effect on ocular blood flow and macular oedema in patients with diabetic retinopathy. In rabbits and monkeys, ripasudil has been shown to reduce intraocular pressure and enhance outflow facility (Kaneko et al., 2016). The effect of ripasudil on smooth muscle contraction in the myometrium has

69 not been investigated in pregnant nor non-pregnant animals. Therefore, we found it necessary to investigate the influence of this new ROCK inhibitor on the myometrial contractility during the oestrous cycle and last stages of pregnancy in mice.

Table 1.2. The structural and physiological properties of ripasudil (Garnock-Jones, 2014).

Alternative names Glanatec®; K 115; K-115; K115; ripasudil hydrochloride hydrate

Class Azepines, fluorine-compounds, isoquinolines, small- molecules, sulphonamides

Mechanism of action Rho-associated kinase inhibitor

Route of Administration Ophthalmic

Pharmacodynamics Highly selective and potent ROCK inhibitor; significantly reduces IOP in a dose-dependent manner in rabbits, monkeys and humans; maximum IOP reduction at 1–2 h; significantly greater reduction than with latanoprost in monkeys; has high intraocular permeability in rabbits

Pharmacokinetics

Maximum plasma 0.622 ng/mL concentration (Cmax)

Time to Cmax 0.083 h Area under plasma 0.231 ng·h/mL concentration-time curve

Plasma protein binding rate 55.4–59.8 %

Renal clearance 7.112 L/h

Elimination half-life 0.455 h

Most frequent adverse event Conjunctival hyperaemia

ATC codes

WHO ATC code S01E (antiglaucoma preparations and miotics), S01L-A (antineovascularisation agents)

EphMRA ATC code S1E (miotics and antiglaucoma preparations), S1P (ocular antineovascularisation products)

Chemical name 4-fluoro-5-{[(2S)-2-methyl-1,4-diazepan-1- yl]sulfonyl}isoquinoline

1.10.2 Prophylactic therapy of premature rupture of the membranes in PTL

Preterm premature rupture of the membranes (PPROM) is responsible for about 33% of PTL (Aagaard-Tillery et al., 2005). Prophylactic treatment, that is, tocolysis before the signalling of uterine contractions might prohibit PTL. Data on using prophylactic tocolysis in women with PPROM are still controversial (van Vliet et al., 2014). Some recent studies have indicated the usefulness of prophylactic therapy in prolonging the latency period (time from rupture of membranes to the onset of labour) (Mackeen et al., 2014), which disagrees with other previous studies (Decavalas et al., 1995). Researchers also did not demonstrate any improvement in maternal or neonatal outcomes (Mackeen et al., 2014). A new trial has investigated the role of indomethacin as a prophylactic therapy in PPROM and found that indomethacin has no benefit over placebo in terms of latency and neonatal outcomes (Ehsanipoor et al., 2011). A retrospective analysis of 99 cases of PPROM compared two separate treatment approaches: using tocolytic agents to prevent PTL until diagnosis of clinical chorioamnionitis or induction of labour or caesarean section, when either oligohydramnios was diagnosed or amnion elastase was increased (Shinjo et al., 2012). The analysis indicated a significant increase in pathological chorioamnionits and funisitis in the group treated with tocolytics. Recently, a large randomized trial is being performed to measure the activity of nifedipine versus placebo in women with PPROM and no contractions (van Vliet et al., 2014).

1.11 Nitric Oxide (NO) and Nitric Oxide Synthase (NOS) isoforms

Nitric oxide (NO) is a messenger molecule which is produced from L-arginine via the three specified nitric oxide synthase (NOS) isoforms: neuronal NOS (nNOS); endothelial NOS (eNOS) and inducible NOS (iNOS) (Moncada et al., 1991, Moodley, 2002, Sica et al., 2009). NO plays an important role in the modulation of multiple physiological processes in different body organs such as the vascular system, the nervous system, the endocrine system and the reproductive system (Craig et al., 2018, Moncada et al., 1991). All NOS isoforms bind to calmodulin and haem. nNOS is mainly expressed in the central and peripheral neurons and certain other cells. It is involved in synaptic plasticity, central modulation of blood pressure and relaxation of smooth muscle. Inducible NOS (iNOS) is released by various cell types after exposure to 71 cytokines, lipopolysaccharides or other molecules. iNOS generates high amounts of NO which exert cytostatic effects on parasitic target cells and this isoform of NOS is also associated with the pathophysiological processes involved in certain inflammatory disorders and septic shock. eNOS is largely expressed in endothelial cells, where it controls vascular dilatation and regulates blood pressure. In addition, eNOS-derived NO possesses other vasoprotective properties through minimizing atherosclerotic events (Förstermann and Sessa, 2012). The NO produced by NOS affects different target tissues and proteins in the body. The major essential signalling mechanism activated by NO is the induction of soluble guanylyl cyclase and the formation of cGMP (Rapoport et al., 1983, Forstermann et al., 1986). cGMP stimulates the myosin light chain phosphatase enzyme (MLCP), which in turn catalyzes the dephosphorylation of myosin light chain (MLC) and causes smooth muscle relaxation (Sauzeau et al., 2000). The vasodilation produced by certain NO-producing medications such as organic nitrates and sodium nitroprusside is believed to be due to the elevation of cGMP concentrations in the smooth muscle cells of the blood vessels (Forstermann et al., 1986). NO can also relax smooth muscle in different tissues such as trachea (Perez-Zoghbi et al., 2010) and uterus (Riemer et al., 1997). Figure 1.14 illustrates the mechanism of smooth muscle relaxation induced by NO.

1.11.1 The role of NO in the uterus

NO plays an important role during pregnancy through its vasodilatory effect in the maternal circulation, and also contributes to the regulation of blood supply to the uterine and foetoplacental tissues (Sladek et al., 1997). NO has shown to contribute to the maintenance of the rat uterine quiescence throughout gestation via the L-arginine- nitric oxide-cGMP system (Figure 1.14), but not during parturition (Yallampalli et al., 1994). During pregnancy, the activity of eNOS is elevated and cGMP release is increased in the uterine arteries (Sladek et al., 1997). Moreover, eNOS is also expressed in the human placental syncytiotrophoblast and in the foetoplacental and umbilical vascular endothelium (Ariel et al., 1998, Rossmanith et al., 1999). Some controversial data have demonstrated the placental expression of different NOS isoforms (eNOS and iNOS) in preeclampsia (Schiessl et al., 2005). However, this placental NOS may devoid of biological activity. Interestingly, iNOS has exerted its activity in the uterus

72 during pregnancy through controlling myometrial contraction as well as regulating blood flow, while its efficiency is reduced before the time of labour indicated by the reduction of its protein expression (Purcell et al., 1997), however, the exogenous NO inhibits myometrial contractility (Sladek et al., 1997). Until today, no evidence is available on the association between the endogenous NO and uterine quiescence during gestation. Studies have shown that during labour, endogenously released NO can exert a tocolytic action on the human myometrium (Buhimschi et al., 1995) and a deficiency in NO may lead to an induction of the labour process (Ledingham et al., 2000). In addition, NO-donor agents were able to diminish the rat myometrial contraction in vitro where this condition is desired as in preterm labour (Norman, 1996).

Figure 1.14. An overview of mechanism of smooth muscle contraction and relaxation and the role of NO. Myosin light chain kinase (MLCK); Myosin light chain kinase phosphatas (MLCP); cyclic adenosine monophosphate (cAMP); cyclic guanidine monophosphate (cGMP); nitric oxide (NO); adenosine triphosphate (ATP); calmodulin (CM); inositol triphosphate (IP3); calcium (Ca2+).

1.11.2 The potential use of NO-donor compounds during pregnancy and labour

Previous investigations have demonstrated that when the labour process is initiated, the myometrial sensitivity to the inhibitory effect of NO is decreased (Norman, 1996). Understanding the mechanism by which this decrease in sensitivity takes place is very important to illustrate the efficiency of NO-donors as potential treatments for premature contraction of the uterus in late pregnancy and in PTL. In vitro studies have shown that myometrial exposure to NO precursors in pregnant rats before the initiation of labour resulted in reduced spontaneous contraction when compared to the labouring animals (Yallampalli et al., 1993). In addition, L-arginine supplementation caused a total suppression of myometrial contractions induced by carbachol during mid-gestation, but did not show any effect on samples isolated from labouring rats (Izumi et al., 1993). The reduction in the sensitivity of the myometrium to the tocolytic properties of NO after the initiation of labour was also evident in mice (Buhimschi et al., 1995). The reason for this change in the myometrial sensitivity to NO between gestational stages is still unknown, and it is not obvious if this is due to NO itself or other factors. However, this may be attributed to the structural changes in the myometrium that affect the response of the tissue to the different agents with the initiation of labour. These changes include an increase in the gap junction formation and a reduction in the membrane potential (Garfield and Yallampalli, 1994). These findings suggest that compounds with the potential to influence PTL may be useful at stages before the onset of labour.

NO-donors can also decrease the amplitude and frequency of contractions and increase the interval between contractions in the human myometrium (Lee and Chang, 1995, Norman et al., 1997). In contrast, other studies observed no effect on myometrial contractility after treatment with NO inhibitors, suggesting there is no activity of endogenous NO in the myometrium in vitro (Buhimschi et al., 1995, Lee and Chang, 1995). It has also been demonstrated that to stop the process of labour, the concentrations of NO-donors required are higher than those needed to delay the onset of labour (Ledingham et al., 2000). Further research is needed to explore the exact role of NO during pregnancy and labour, and also to examine the effects of other NO-donors on myometrial function before and during parturition.

1.11.3 The interaction between the NO and lipid mediators

Studies have demonstrated the relationship between the NO release of and production of fatty acid-derived lipid mediators, as the high level of NO has resulted in COX activation and thus, has elevated the synthesis of PGs (Salvemini et al., 1993). No research is available on the direct connection between the NO and PGJ2, but it was demonstrated that the NO release is stimulated by 15-deoxy PGJ2, a PGJ2 metabolite in human aortic endothelial cells (Calnek et al., 2003). In addition, NO-donors such as sodium nitroprusside and L-arginine were able to elevate the synthesis of PGs in endothelial calls from the vein of human umbilical cord. Moreover, NG-monomethyl-L- arginine (an inhibitor of NOS) has been concluded to reduce the level of 6-keto-PGF1α but it did inhibit the release of PGE2. Researchers have also demonstrated that inhibition of COX with Indomethacin can increase the concentration of NO and eNOS in the same cells (Vassalle et al., 2003).

In murine, the administration of NO has increased the cardiac ischaemic injury through activating the soluble epoxyhydrolase (sEH) enzyme (Ding et al., 2017). Furthermore, decreased concentrations of DiHOME have been observed with the inhibition of NO in mouse brown adipose tissue (Park et al., 2018). It has been concluded that certain factors can play a role in the kind of NO influence on the pathway of lipid mediator production. These factors include the level of released NO, the type of organ in which the lipid mediator is produced and the degree of stimulation received by the affected organ (Mollace et al., 2005). No studies are available on the effect of NO on the production and activity of lipid mediators in the myometrial and placental tissues and thus, further examinations are needed to study their interaction in these organs.

1.11.4 SE175 (NO-donor), structure and pharmacology

SE175 or 2-[[4-[(nitrooxy)methyl]benzoyl]thio]-benzoic acid methyl ester, is a nitroxyacylated thiosalicylate with a (NO)-donating nitrate group attached to a NO- liberating thiosalicylate, so it is an organic nitrate similar to nitroglycerine (Endres et al., 1999). SE175 is a NO-donor which has been developed to release NO spontaneously when it comes in to contact with cell surface esterases, when the free thiol (SH) is able to liberate NO from nitrate groups (Lundberg et al., 2008). The compound is stable in buffer and saline solution. SE175 was able to activate endothelial 75 soluble guanylate cyclase and provoke aortic vasorelaxation with an EC50 of 20 μM, so it has an intermediate potency between that of isosorbide dinitrate and nitroglycerine. SE175 is a novel vasodilator that exerts a powerful effect to relax uterine arteries from pregnant mice and increases murine foetal growth when administered in the second half of pregnancy (Cureton et al., 2017). Figure 1.15 shows the chemical structure of SE175.

Figure 1.15. The chemical structure of SE175. 2-[[4-[(nitrooxy) methyl] benzoyl] thio]-benzoic acid methyl ester.

1.12 Drug targeting and liposomes

Tissue-specific drug targeting is a relatively new technique designed to deliver drugs to certain sites within the body in order to achieve an acceptable therapeutic concentration with minimal exposure of other tissues or organs. Targeted delivery systems possess two main advantages over the traditional system of drug administration: first, they substantially decrease the adverse effects of medications and secondly, lower doses of drugs can be administered (Tiwari et al., 2012). Tissue-specific receptors which are expressed on the cells or tissue of interest can be exploited to develop targeted drug delivery systems; peptides or antibodies which bind to these tissue-specific epitopes can be identified and used to decorate the surface of drug-loaded nanoparticles, such as liposomes (Rezler et al., 2007, Wang et al., 2015). The NO-donor, SE175 has been encapsulated in liposomes decorated with peptides that selectively bind to the placenta

76 and uterine vasculature of pregnant mice; intravenous administration of these targeted liposomes increased foetal weights, reduced placental oxidative stress and improved placental efficiency in eNOS knockout mice, a model of foetal growth restriction (Cureton et al., 2017). In this study we have examined the efficiency of SE175 as a free agent and as incorporated in liposomes in modulating the myometrial contractility. We have also examined the effect of free- and liposomally encapsulated SE175 on expression of lipid mediators in the myometrium and placental tissues from pregnant C57 wild type and eNOS knockout mice at term. This approach has been successfully applied to deliver chemotherapeutic agents to cancerous tissues in mice (Hatakeyama et al., 2007).

1.12.1 Liposomes as a drug delivery system

The placenta-binding homing peptides have been exploited to prepare a drug delivery system with targeting characteristics, as this method has sown its effectiveness in other pathological conditions such as cancer (Hatakeyama et al., 2007). Previous investigations have concluded that liposomes are the best carriers to be utilized with the use of targeting peptides as they are biocompatible and biodegradable and the lipid facilitates their conjugation with peptides (Rezler et al., 2007, Lee et al., 2007, Wang et al., 2015). Researchers have examined the distribution of nanoparticle formulation to the various body tissues and have demonstrated the accumulation of these preparations in placental trophoblasts as well as in foetal brain and liver after injection of silica nanoparticles. They also found that these nanoparticles caused foetal resorption and restricted foetal growth (Yamashita et al., 2011).

1.12.2 Liposomes composition

Liposomes are regarded as carriers which are made of a phospholipid bilayer which contains the hydrophilic tail regions. These regions interact and make the hydrophilic head groups facing the outside environment to form a conformation with the lowest possible entropy. The phospholipid bilayer can be altered for the purpose to make a spherical liposome in which the hydrophobic bilayer region and aqueous core are incorporated as shown in Figure 1.16 (Cureton, 2017).

Figure 1.16. A diagram showing the composition of a liposome (Cureton, 2017). The phospholipids interact to form the spherical bilayer with the hydrophobic bilayer region and aqeous core inside.

The surface of liposomes can be decorated with tissue-specific homing peptides via a suitable reactive group which should be added during the production of liposomes. This step can be accomplished by incorporating a PEG-maleimide into the lipid bilayer. The PEG-maleimide has an electro-rich double bond which facilitates electrophilic addition reactions, especially those with a thiol group (Nobs et al., 2004, Cureton, 2017). Liposomes can also be designed to liberate the incorporated medication at a certain pH range. The destabilisation can be facilitated by the addition of phosphatidylethanolamine into the structure of the liposome. Studies have demonstrated the benefit of this step to target and deliver drugs directly to the placenta (Simões et al., 2004, Kendall et al., 2011, Cureton, 2017).

1.13 Aims and objectives

The aims of this project were to:

(i) Investigate regulation of myometrial contractility in non-pregnant and pregnant mice;

Immersion was used to mimic in vivo uterine contractions according to anatomical location, the oestrous cycle and term pregnancy. This well characterised technique enabled selective pharmacological agents to be used to elucidate the role of PGs and oxytocin that are subject to hormonal and gestational-dependent regulation.

(ii) Assess the ability of the new ROCK inhibitor, ripasudil to inhibit murine uterine contractility in non-pregnant and pregnant mice;

Rho kinase inhibitors are shown to decrease cross bridge formation and myogenic contraction (Martinsen et al., 2012). This appears to be regulated at two sites of phosphorylation (Aguilar et al., 2011), which has yet to be examined in the mouse uterus. By targeting ROCK with ripasudil at dioestrus and term pregnancy, the functional expression of these proteins (non-, mono- and di-phosphorylated myosin light chain) and their relative effects could be explored. Greater understanding and control of Rho kinases could improve tocolytics for labour-associated disorders.

(iii) Assess the effect of NO on myometrial contraction forces in pregnant mice at term using eNOS Knockout and wild type mice.

To assess whether NO and/ or its method of delivery has a functional effect on the uterus.

(iv) Examine whether administration of empty or drug-loaded peptide-decorated liposomes alters the concentration of lipid mediators in the uterus or placenta of C57 wild type or eNOS KO pregnant mice

Overall effect of NO on lipidome, indicating mechanisms that play a role in pregnancy outcome. The ultimate goal was to determine whether these agents could be used to delay preterm uterine contraction and labour in humans.

2 Chapter 2:

Materials and Methods

2.1 Source of the tissues

The tissues used in this study were isolated uterus and placenta taken from non-pregnant (dioestrus) and pregnant mice (day 19 gestation). Female, mature C57BL/6J and eNOS knockout (KO; strain B6.129P2-Nos3tm1Unc/J) mice were used. Mice were obtained from Jackson Laboratories (UK). According to the information given by the providing company, the eNOS KO mice have the same genetic background of C57BL/6J, and the only difference is that the eNOS KO mice devoid of the endothelial nitric oxide synthase enzyme (eNOS). Experiments were performed in accordance with the UK Animals (Scientific Procedures) Act 1986. The mice were obtained from Envigo UK Limited. Animals were housed in cages containing up to 5 animals under a 12h light/dark cycle at 21-23°C with food (Beekay Mouse Diet, Bantin & Kingman, Hull, UK) and water ad libitum.

2.1.1 Non-pregnant mice

Non-pregnant sexually mature (8-12 weeks old) C57BL/6 mice (25-30g) were used. To determine the stage of the oestrous cycle, a vaginal smear was carried out by means of gently performing a vaginal lavage with 100µl PBS using a fine tip plastic pipette. Smears were then made on microscope slides and the stage of the cycle was determined by visual identification using a light microscope. As described by Caligioni (2009), the proestrous phase is characterised by the vaginal smear containing mostly nucleated epithelial cells. In smears obtained at oestrus, mainly cornified epithelial cells are observed. The vaginal smears taken at metoestrus display a mixture of cell types with a predominance of leukocytes, while dioestrus is distinctively characterised by leukocytes (Caligioni, 2009) (Figure 2.1). Vaginal smears were carried out at regular intervals before the mice were killed to confirm that the mice were sexually mature and cycling.

2.1.2 Pregnant mice

To obtain tissue samples from pregnant mice, C57BL/6 or eNOS KO mice (8-12 weeks old) were respectively mated and discovery of a copulation plug was designated as embryonic day 1 (E1) of pregnancy. The gestation period of the mouse is 19-20 days

81 with parturition usually occurring on day 20 (E20) (Peters et al., 2007). In this study, mice at term (E19 days), not in labour, were euthanized by cervical dislocation. They weighed within a range of 30-39g. Uteri were removed intact, the myometrium and placentas were collected, and foetuses were euthanized by decapitation with surgical scissors under Schedule 1, the Animal (Scientific Procedures) Act 1986.

Figure 2.1. Photomicrographs of vaginal smears from mice. Vaginal smears were taken at (A) proestrus: predominance of nucleated epithelial cells; (B) oestrus: anucleated cornified cells; (C) metoestrus: leukocytes, cornified, and nucleated epithelial cells; (D) dioestrus: predominance of leucocytes. [▲ N]: Nucleated epithelial cells, [⌂ L]: leucocytes, [∆ C]: cornified cells (Caligioni, 2009).

2.2 Methods for investigating myometrial contractility

It is difficult to study the direct effect of different substances on myometrial physiology in vivo. Therefore, in vitro techniques are applied (Verhoff, 1985). Both, immersion and superfusion methods can be conducted in vitro. The in vitro studies using isolated strips of myometrial tissues can provide significant knowledge of myometrial physiology. The disadvantage of these methods is that they only provide an assessment of the immediate state of the tissue but never the former or subsequent state of that tissue (Wagner, 1974); they also require removal of the uterus from its physiological environment and hormonal milieu.

2.2.1 Preparation of isolated tissue for immersion

Mouse uteri were obtained from pregnant or non-pregnant mice. Figure 2.2 shows the anatomical differences between the uterus from non-pregnant and pregnant mice.

The uteri were removed from the mouse immediately after the Schedule 1 procedure and placed in a dissecting tray containing Krebs-Heinseleit physiological salt (Krebs) solution (Table 2.1). After trimming off fatty tissues surrounding the uterus, strips of uterine smooth muscle with a length of 8-10mm and width of 2-3mm were dissected from each uterine horn. Samples taken from the uterus were cut parallel to the longitudinal fibres along the length of the uterus and taken from two anatomical regions of each horn and marked according to the region and the horn side. In experiments on non-pregnant mice, one sample was taken from the upper part of the horn close to the ovaries (U) and the other sample was taken from the lower part of the horn directly above the cervix (L) (Figure 2.2). Whereas for pregnant tissues, 8 samples were isolated from the upper segment of the uterine horns. The reason behind this kind of isolation is the big size of the myometrium taken from pregnant mice compared to the small size of the non-pregnant myometrial tissue. This allowed obtaining several strips from the upper segment of the pregnant uterus and then to compare the effect of various treatments on the same segment (upper) of the horn in each single immersion experiment. Due to the long duration of experiments on pregnant mouse tissues which lasted for several hours, it was impossible to use and investigate the lower segment of the uterus from pregnant mice where these strips started to lose their spontaneous

83 activity after an extended period of time. After isolation, the dissected strips of smooth muscle were immediately mounted in organ baths containing Krebs-Heinseleit solution at 37ºC and bubbled with 95% O2/CO2.

Figure 2.2. Intact uterus isolated from pregnant and non-pregnant mouse in dioestrus. The uterus from (A) a pregnant mouse at term (E19) and (C) the E19 isolated uterus compared to (B) a non-pregnant mouse in dioestrus and (D) its isolated uterus. The ovaries, cervix, mid- uterus (M) and fat were stored at -80°C. The upper (U) and lower (L) sections of each uterine horn were used for functional studies using immersion apparatus.

2.2.2 Solutions and drug compounds

Prior to immersion assay, Krebs-Heinseleit physiological salt (Krebs) solution (pH 7.4) was freshly prepared as shown in Table 2.1.

Table 2.1. Chemical composition of Krebs-Heinseleit solution. Chemical material Concentration (mM)

118.9 Sodium Chloride (NaCl) 4.7 Potassium Chloride (KCl) 1.2 Magnesium dihydrogen phosphate (KH2PO4) 1.2 Magnesium sulphate (MgSO4) 2.5 Calcium chloride (CaCl2) 25.0 Sodium hydrogen carbonate (NaHCO3) 10.0 D-glucose 1.0 L Distilled water

Stock solutions of different drug compounds were prepared according to the manufacturer's instructions (Table 2.2) and serial dilutions were made with 0.9% w/v normal saline and placed on ice throughout the experiment.

Table 2.2. Stimulants and inhibitors used to investigate myometrial contractility in mice. The key indicates line colour on concentration-effect curves and Western Blotting graphs.

Target Stock Compound Key Action Chemical name Source receptor Vehicle

Oxytocin OTR Agonist (2S)-1- dH2O Sigma- (Kubota et al., [(4R,7S,10S,13S,1 Aldrich 1996) 6S,19R)-19-amino- (Germany) 7-(2-amino-2- oxoethyl)-10-(3- amino-3- oxopropyl)-13- [(2S)-butan-2-yl]- 16-[(4- hydroxyphenyl)met hyl]-6,9,12,15,18- pentaoxo-1,2- dithia-5,8,11,14,17- pentazacycloicosan e-4-carbonyl]-N- [(2S)-1-[(2-amino- 2-oxoethyl)amino]- 4-methyl-1- oxopentan-2- yl]pyrrolidine-2- carboxamide Prostaglandin FP Agonist 9α,11α,15S- Ethanol Cayman F2α trihydroxy-prosta- Chemical. (Coleman et al., 5Z,13E-dien-1-oic (USA) 1994) acid

Ripasudil (K- ROCK Inhibitor 4-fluoro-5-[[(2S)- DMSO Selleck 115) 2-methyl-1,4- Chemicals (Isobe et al., diazepan-1- . (USA) 2014) yl]sulfonyl]isoquin oline Serotonin 5-HT Agonist 3-(2-aminoethyl)- Ethanol Sigma- hydrochloride receptors 1H-indol-5- Aldrich (5-HT) ol;hydrochloride (UK) (Kitazawa et al., 1998)

U46619 TP Agonist 9,11-Dideoxy- DMSO Tocris (Coleman et al., 9α,11α- (UK) 1994) methanoepoxy- prosta-5Z,13E- dien-1-oic acid

Distilled water (dH2O); PGF2α receptors (FP); Oxytocin receptors (OTR); Rho-associated protein kinase (ROCK); Thromboxane receptors (TP).

2.2.3 Immersion

Immersion is regarded as an important technique used to measure myometrial contractility in vitro. The technique involves the suspension of an isolated part of smooth muscle in a chamber filled with a nutrient fluid. It was first reported by Vilhelm Magus (1871-1929). This technique possesses some advantages such as its operating system makes the formation of the curve between the cumulative concentration effect and other parameters convenient for measurement and maintenance of tissue contraction. These parameters may include incubation times and dosing interval. A distinctive feature of the immersion method is its ability to preserve an equilibrium between the receptor and its ligand at the receptor-binding site (Hein et al., 2005). In this study, the immersion system was used, where the isolated muscle tissue was suspended in a chamber containing a nutrient fluid (Krebs solution).

After the uterine horn was split into strips, both ends of each dissected strip (8-10mm x 2mm) of the uterine horn were sutured using a cotton thread; the cervical end (lower) was secured to the metal tissue holder, mounted longitudinally in a separated 8ml water- jacketed tissue bath containing Krebs solution, which was aerated with 95% O2, 5%

CO2 at 37ºC. The ovarian end (upper) was connected to the isometric force transducer (AD Instruments, UK) which converts the contractile force (tension) of the uterine horn into electrical signals. The immersed tissues were maintained at a stable pH (7.4) and temperature (37ºC), and the volume of Krebs solution was adjusted by a tap and overflow system. Isometric force transducers were connected to a computer via bridge amplifiers. Variations in the tension were recorded digitally using a PowerLab data acquisition system (AD Instruments, UK) running Microsoft Chart v5.4 software. The components of the immersion equipment are described in Figures 2.3 and 2.4.

Figure 2.3. The immersion apparatus including the equipment and PowerLab used in the study.

Figure 2.4. The immersion equipment used to test functional receptors in the isolated mouse uterus (Fischer, 2010).

2.2.4 Administration of drugs

Tissue baths were refilled three times with Krebs solution during the first 15 min and an initial and optimal resting tension of 2g was applied to each strip (Slattery et al., 2001). The isolated tissue strips from non-pregnant and pregnant mice were randomly assigned treatments and equilibrated for 30 min or until regular contractions had started, and this spontaneous activity was recorded over a 30-min period before adding the drug to be investigated (Griffiths, 2007). The duration of equilibrium allowed the tissue to develop regular spontaneous contractions (myogenic activity). Cumulative concentration-effect curves were performed after the equilibrium period using log unit concentration increases. The myogenic activity remained stable for the duration of the experiment, which indicated the viability of the tested tissues inside the immersion baths.

In preliminary experiments, the duration of spontaneous activity of myometrial contractility was measured in the uterus of non-pregnant and pregnant mice. The myometrial strips taken from the upper and lower segments of the uterine horn of non- pregnant mouse and from the upper segment of pregnant mouse uterus maintained spontaneous contractile activity. This activity varied over time and lasted for about 5h in non-pregnant and around 48h in pregnant samples (Figure 2.5).

After tissue equilibration, drugs and vehicle were added directly to immersion baths every 12 min at a maximum volume of 8µl to avoid any pH or temperature fluctuation. When performing concentration-effect curves using myometrium from non-pregnant and pregnant mice, and in order to avoid any disruption in spontaneous activity pattern, drugs were added in a cumulative manner to negate the need for a washing step between increasing concentrations. Figure 2.6 shows how the washout of myometrial strips can decrease spontaneous activity for a short period of time. However, myometrial activity ultimately resumed even though in some samples it did not achieve the same levels observed before washing. These variations in the level of spontaneous activity may cause issues when measuring and explicating the mouse myometrial response to uterine stimulants and inhibitors compared to myogenic activity. Thus, it was decided to exclude the washing steps between the additions of different drug concentrations and administer drugs in a cumulative manner.

Figure 2.6. Traces showing the effect of washout on the spontaneous activity of myometrial strips from (A) non-pregnant and (B) pregnant mice.

2.2.5 Preliminary experiments to determine dosing regimens

Firstly, all concentrations stated in this section and all the following experiments are the final drug concentrations in the organ baths and not the stock concentrations. For example, when it is intended to expose a myometrial strip to a 10-9M of a drug as a final concentration in the organ bath, a 10-6M concentration of a drug is prepared as a stock solution, then 8µl is added to the 8ml of Krebs solution in the organ bath to produce a 10-9M concentration of the drug as a final concentration.

In order to determine the concentration of ripasudil to be used in the subsequent experiments to inhibit stimulant-induced uterine contraction, the effect of ripasudil 10- 9M - 10-5M, a ROCK inhibitor (Isobe et al., 2014), was assessed in preliminary experiments (Isobe et al., 2014) (Section 3.5). The concentrations of ripasudil that were able to significantly inhibit spontaneous activity were the 10-6M and 10-5M. Therefore, these two concentrations of ripasudil were used to inhibit the myometrial contraction in

91 the experiments of concentration-effect curves. The inhibitory effect of ripasudil on myometrial contractility of the uterus was reversible because the spontaneous activity can be restored after washing out ripasudil with Krebs solution as shown in Figure 2.7.

Figure 2.7. Trace showing the restoration of myometrial spontaneous activity in pregnant mouse uterus after being inhibited by ripasudil 10-5M and then washed with Krebs solution, which was aerated with 95% O2, 5% CO2 at 37ºC using immersion technique.

Time-matched vehicle controls were used alongside 10-5M or 10-6M of ripasudil, in the pre-treatment period before addition of uterotonic agents. Ripasudil was administered 30 min before the beginning of any concentration-effect curves to allow time for equilibration as preliminary experiments demonstrated that this equilibration period was sufficient for the inhibitor to a reproducible effect. Cumulative concentration-effect curves reduced the required volume of inhibitor as replacement doses were not necessary after each wash step and myometrial strips exhibited stable contraction patterns. This is also useful as when novel compounds are used they are made and prepared in small amounts. In addition, it would affect the length of the experiment as after every washout the tissue would need to recover its own activity, which would require to be stabilised prior to the addition of any further concentration of a drug. Another characteristic of the cumulative dosing system is that, a precise measure of the inhibitor quantity in each organ bath is achieved. If a high lipophilic inhibitor was used

92 in a non-cumulative system, it is possible that not all the drug would be removed after each wash step. After the antagonist incubation period, a series of incremental concentrations of oxytocin (10-12M - 10-6M) and 10-9M - 10-5M of U46619, 5-HT or

PGF2α (Hutchinson, 2005, Griffiths, 2007, Fischer, 2010, Darios et al., 2012, Sabar, 2012) were added at 12-min intervals in a cumulative manner. Figure 2.8 shows a typical response of a myometrial tissue sample to the pre-treatment with ripasudil at 10- 6M and then increasing doses of a uterine stimulant (5-HT).

When testing the effect of U46619, PGF2α and 5-HT on spontaneous activity or on ripasudil-inhibited smooth muscle contraction in isolated myometrium from non- -6 pregnant mice, U46619, PGF2α or 5-HT at 10 M were added separately and directly to the tissue in the organ bath. This concentration was chosen as previous studies have demonstrated that this concentration can induce a submaximal excitatory response and also can maintain a consistent myometrial contraction for over 4h (Hutchinson, 2005, Griffiths, 2007).

The stock (distilled water, ethanol and dimethyl sulfoxide) and dilution vehicles (0.9% w/v saline) prepared for each drug used in the experiments (according to Table 2.2) did not have a significant effect on myometrial activity of non-pregnant and pregnant mouse uterus.

2.2.6 Measuring contractile force relative to spontaneous activity

The activity of myometrium was measured as the integrated area under the contraction curve, which represented the variation in the frequency (number of contraction cycles; No), period and amplitude (grams; g) of contraction. In order to set the lower base tension for calculating the area under the curve (AUC) (grams/second; g.s.), integral above mean settings were utilized using Chart software v5.4. A time period of 12 min was allowed between doses and data were calculated at 10-min intervals starting immediately after the addition of a drug as this time duration of activity characterized drug-induced effects from spontaneous contractions.

Every single myometrial strip showed a unique intrinsic activity and there was variation between different strips taken from the same mouse Figure 2.9.

Because of the large variations in myogenic activity between uterine tissues, it is preferable to normalise the AUC after each treatment against a reference contraction (Crankshaw, 2001). Previous studies in human and animal tissue samples have applied a hypotonic shock as a reference contraction where Krebs’ solution surrounding the muscle strips in the organ bath is replaced with distilled water at the end of experiment leading to a big contraction (Griffiths, 2007, Fischer, 2010, Sabar, 2012). Expressing concentration-response as a percentage of spontaneous activity was more appropriate for this study. Myometrial strips needed to be removed from the organ baths and immediately processed and frozen at -80ºC in order to be used later in a subsequent investigation of contractile protein expression in these treated tissues using the western blotting technique. The data were expressed as % spontaneous activity:

y AUC of the myometrial contraction at 10 M of y drug x100 AUC of the spontaneous activity

2.3 Investigation of contractile proteins in mouse myometrium

2.3.1 Western Blot Analysis

Western blotting is an important method used commonly in the field of molecular biology to investigate the expression of various proteins (Bolt and Mahoney, 1997). This technique is characterised by its ability to separate a mixture of several proteins on the basis of their molecular weights via gel electrophoresis. Thereafter, the separated proteins are transferred to a membrane which then undergoes several steps of treatment and incubation with different reagents and antibodies specific to the target proteins in order to produce bands for these proteins. The membrane is then washed out to remove any unbound antibody and leave the bound antibody to the target protein. The bound antibodies are then detected by treating the membrane with ECL reagents using a ChemDioc MP imaging system. Antibodies used will bind only to the target protein so just one band should be seen for each protein. Band thickness represents the quantity of the intended protein available in the membrane (Mahmood and Yang, 2012).

2.3.1.1 Phos-Tag-Based Analysis

Protein phosphorylation is an essential cellular regulatory mechanism which occurs in response to different stimuli (Hunter, 2000) and thus, multiple biochemical methods are developed and utilised to investigate this important pattern of cell signalling (Thingholm et al., 2009). Western blotting is one of the most commonly used methods in the determination and measurement of phosphorylated proteins in different laboratory cells and tissues using 1-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Recently, the strength and usage of this method have been augmented through developing a dinuclear metal complex ‘phosphate-binding tag’ (phos-tag) that can be mixed with the polyacrylamide gel matrix just before conducting SDS-PAGE (Kinoshita et al., 2006, Kinoshita et al., 2009). This alteration of the classical SDS-PAGE facilitates the separation of proteins according to their degree of phosphorylation. Therefore, one protein can separate into numerous bands and every band is relevant to a different phospho-level of the tested protein (Aguilar et al., 2011).

In the classical phospho-protein western blotting, the signal elicited by an antibody (Ab) toward the protein of interest is normalised to a reference protein or to the whole target protein utilizing an Ab that does not distinguish between protein isoforms whether they are phosphorylated or not. This kind of investigation is generally restricted to the determination of changes at a single step of phosphorylation per assay. Contrary to the above, Mn2+-phos-tag SDS-PAGE allows the impact of different treatments on the protein of interest to be examined over its multiple phospho-levels via probing a replica membrane with an anti-total-target protein Ab and permits testing of protein phosphorylation in the absence of Ab specific to the phospho-protein. When Abs for the phospho-protein-specific are available, the Mn2+-phos-tag SDS-PAGE method can provide data to identify specific phosphorylation sites on the target proteins (Aguilar et al., 2011). Researchers have demonstrated the efficiency of this method to estimate the phosphorylation of myosin regulatory light chain (MLC) in the renal arterioles of the rats (Takeya et al., 2008) and human uterine myocytes (Aguilar et al., 2012).

After completing each functional experiment, myometrial strips where immediately removed from the organ baths, separated from tissue holders and directly homogenised in 500µl of TRI reagent (as samples weighed  40mg) using a Polytron X-1020 (The Scientific Instrument Centre Germany) according to the TRI protocol (Sigma-Aldrich,

United Kingdom). After homogenization, strips were immediately frozen at -80ºC to be utilized later to isolate protein and to measure protein concentrations as well as to investigate MLC phosphorylation using Mn2+-phos-tag SDS-PAGE method of the quantitative western blotting technique.

2.3.2 Isolation of protein

The TRI protocol was used for protein isolation with some modifications. On the day of protein isolation and concentration measurement, myometrial tissues were taken from the -80ºC freezer, thawed and allowed to stand for 5 min at room temperature to ensure complete dissociation of nucleoprotein complexes. 200µl chloroform (Sigma, UK) was added per 1ml of TRI Reagent and the mixture was vigorously shaken for 15 sec, and allowed to stand for 10–15 min at room temperature. The suspension was then centrifuged at 12,000g for 15 min at 4°C. Centrifugation separated the suspension into 3 phases: a red organic phase (containing protein), an interphase (containing DNA), and a colourless upper aqueous phase (containing RNA). The upper aqueous phase was transferred to a fresh tube to be used for subsequent isolation of RNA (protocol is discussed in qRT-PCR method). The remaining organic phase and interphase layers were directly processed for protein isolation. Firstly, the interphase layer was carefully removed and discarded as this is a critical step to avoid protein contamination with DNA.

To the organic phase, 150µl of ethanol (Pure 200 prof, Honeywell, France) per 500µl of TRI Reagent was added and mixed by inversion for 15 sec and allowed to stand for 2-3 min at room temperature. The samples were centrifuged (Benchtop centrifuge, Heraeus Fresco 17, Thermo Fisher Scientific, UK) at 2,000g for 5 min at 4°C and then the supernatants were transferred to new tubes and the precipitates were discarded as they contain any remaining DNA. To the isolated supernatants, 750µl of isopropanol (molecular grade, Fisher Bioreagents, UK) per 500µl of TRI Reagent was added and mixed by inversion for 15 sec and incubated at room temperature for 10 min. Then, the mixtures were centrifuged at 12,000g for 10 min at 4°C to pellet the protein. The supernatants were discarded and the protein pellets were washed 3 times with 1ml of 0.3M guanidine-HCl (≥99%, Sigma-Aldrich, UK) in 95% ethanol (Pure 200 prof, Honeywell, France), mixed by inversion several times and incubated in the wash

97 solution at room temperature for 20 min. The solutions were then centrifuged at 7,500g for 5 min at 4°C and the supernatants were discarded after each wash. After the last wash, 2ml ethanol (Pure 200 prof, Honeywell, France) was added and the samples were vortexed (vortex mixer, Fisher Scientific, UK) and allowed to stand at room temperature for 20 min, then the samples were centrifuged at 7,500g for 5 min at 4°C and the supernatants were discarded. The tubes containing the pellets were left upside down and tilted against the rack for 5-10 min for airflow to dry the pellets, and then pellets were dissolved in 200µl of 1% SDS (≥99.0%, Sigma-Aldrich, UK) in UltraPure DEPC Treated Water (Invitrogen, UK) with continuous ultrasonication using an Ultrasonic Cleaner Bath USC300TH (VWR, Malaysia) at 50°C for 30 min. Any insoluble material was removed by centrifugation at 10,000g for 10 min at 4°C and the supernatant was transferred to a new tube for measurements of protein concentration using the Bio-Rad DC protein assay. The remaining protein was stored at -20°C until used for western blotting.

2.3.2.1 Measurement of protein concentration

To measure protein concentration, bovine serum albumin (BSA) (Sigma-Aldrich, UK) standards were prepared from 1.5mg/ml stock as described in Table 2.3.

Table 2.3. Standard dilutions of BSA prepared from 1.5mg/ml stock in 1M NaOH to generate a standard curve for protein content analysis.

In a 96 well plate, 5μl of each standard and 5μl of each sample in triplicate were added followed by the addition 25μl of a mixture of Protein Assay Reagents A and S (Bio- Rad, USA) at a ratio of 50:1 (A:S) and then 200μl of Protein Assay Reagent B (Bio- Rad, USA) was added. Plates were left in the dark at room temperature for 15 min and then read on a microplate reader (Multiskan™ FC, Thermo Scientific, Finland) at 650nm.

The standards were used to obtain a standard curve (Figure 2.10) and the protein concentration of the samples was estimated using the equation of the line of best fit. Data were normalised by dividing the lipid mediator concentration by the protein content of the samples.

Figure 2.10. Example standard curve and equation of the line for protein content determination. BSA concentrations were plotted against the absorbance value measured at 650nm.

2.3.3 Sample preparation for Western Blotting

Protein concentration was optimised (see Appendix 1), and then 25-50µg of protein sample was used for protein detection by western blotting. The concentration was selected depending on the protein of interest. The sample volume containing 25-50µg

99 protein was calculated by dividing 25-50 with the measured protein concentration and the result was divided by 5 to calculate the loading buffer volume, which was then added to the protein as 5X of the sample buffer was as used in order to give a final sample buffer concentration of 1X. The sample buffer was prepared by mixing Laemmli SDS sample buffer (Sigma-Aldrich, UK) with 2-mercaptoethanol (1:9) (Alfa Aesar, UK). Thereafter, samples containing sample buffer were mixed well, heated at 95°C for 5 min, centrifuged for 1 min and loaded into the stacking gel (Table 2.4) that sat on top of a 12% SDS-PAGE as a resolving gel which contained the phos-tag (Phos-tag™ Acrylamide AAL-107) (NRAD Institute Ltd, Japan) (Table 2.5). The resolving gel is regarded as the separating gel, which separates proteins out according to their molecular weight. 10µl of protein ladder (Bio-Rad Laboratories Ltd, UK) was loaded into the stacking gel in order to determine the protein of interest by molecular weight.

Gels were attached to electrodes and placed in a tank filled with 1X running buffer, which was prepared by adding 100ml of 10X running buffer (Table 2.6) to 900ml of dH2O. The electrophoresis machine was run at 25mA for about 2.5 h (until samples had run the full length of the gel).

Table 2.4. Reagents required for preparing stacking gel. Reagent Total 10ml of 4.5% Company (w/v) acrylamide 30% (w/v) Acrylamide solution 1.5ml Sigma-Aldrich, UK

0.5M Tris/HCl solution pH 6.8 2.5ml Fischer Scientific, UK

10% (w/v) SDS 0.1ml Sigma-Aldrich, UK

TEMED (Tetramethylethylenediamine) 10ml Sigma-Aldrich, UK

Distilled water 5.84ml Invitrogen, UK

10% (w/v) APS (Diammonium Sigma-Aldrich, UK 50µl Peroxydisulfate Solution (freshly prepared)

100

Table 2.5. Reagents required for preparing resolving gel. Reagent Total 10ml of 12% SDS- Company PAGE and 50µM Phos-tag 30% (w/v) Acrylamide solution 4ml Sigma-Aldrich, UK

1.5M Tris/HCl solution pH 8.8 2.5ml Fischer Scientific, UK

5mM Phos-tag solution 0.1ml NRAD Institute Ltd, Japan

10mM MnCl2 solution 0.1ml Sigma-Aldrich, UK

10% (w/v) SDS 0.1ml Sigma-Aldrich, UK

TEMED 10ml Sigma-Aldrich, UK (Tetramethylethylenediamine) Distilled water 3.15ml Invitrogen, UK

10% (w/v) APS (Diammonium Sigma-Aldrich, UK Peroxydisulfate Solution (freshly 50µl prepared)

After completion of the electrophoresis and before the transfer process, the gel was removed from the machine and incubated in transfer buffer which was supplemented with 2mM EDTA (Sigma-Aldrich, UK) at room temperature for 20 min with continuous shaking in order to chelate and remove Mn2+ ions. The gel was then re-incubated for another 20 min in transfer buffer without EDTA.

Transfer buffer (1X ice-cold) was prepared by the addition of 200ml methanol and

700ml dH2O to 100ml 10X transfer buffer (see Table 2.6). The transfer cassette was prepared by placing the cassette transparent side down first, then adding a sponge, filter paper, nitrocellulose membrane (Cell Signaling Technology, USA), the separating gel with samples after removing the stacking gel, filter paper and another sponge in ice-cold transfer buffer as shown in Figure 2.11. The cassette was then sat up by placing it inside the transfer cell with the transfer side of the cassette facing the red side of the transfer

101 cell. The transfer cell was put in the tank which was filled with 1X ice-cold transfer buffer and an ice pack. The transfer cell was then connected to electrodes and the tank was covered and left at 100V for 90 min.

Table 2.6. Reagents required for preparing 10X running and Transfer buffers. 10X Running buffer 10X Transfer buffer Company

30.3g Tris base (25mM) 30.3g Tris base (25mM) Fischer Scientific, UK

144g Glycine (200mM) 144g Glycine (200mM) Sigma-Aldrich, UK

10g SDS (0.1% w/v) 200ml Methanol (SDS) Sigma-Aldrich, UK (Methanol) Fischer Scientific, UK

Distilled water (upto 1L) Distilled water (upto 1L) Invitrogen, UK

Figure 2.11. Diagram of transfer cassette set-up

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2.3.4 Immunoblotting to investigate protein expression

After the transfer process, the nitrocellulose membrane was removed from the cassette and gel was checked for bands transfer. The membrane was washed in TBST buffer (Table 2.7), then non-specific binding sites were blocked by incubation with 5% (w/v) fat-free milk in TBST with continuous shaking at room temperature for 1 h. Membranes were then washed twice with TBST for 10 min and one time for 5 min.

Table 2.7. Reagents required for preparing TBST buffer. TBST (washing buffer) Company

1.21g Tris base Fischer Scientific, UK

8.78g NaCl Fischer Scientific, UK

0.5ml tween 20 Sigma-Aldrich, UK

Dissolve in distilled water to 1L Invitrogen, UK

Thereafter and depending on the target protein, the membrane was incubated with the primary antibody whilst shaking at room temperature and left in the cold room (4°C) overnight. Diluted concentrations of all primary antibodies were prepared using 2% fat- free milk in TBST as shown in Table 2.8, except the MLC antibody which was diluted in 5% fat-free milk in PBS buffer as recommended by the manufacturing company. All antibody concentrations have been optimised in preliminary experiments as shown in the Appendix 1.

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Table 2.8. List of primary antibodies used in Western Blotting. Protein Species Dilution Incubation Company period Mouse 1:5000 RT, 1 h Santa Cruz α-actin monoclonal Biotechnology, Inc., USA Rabbit 1:500 4°C, overnight LifeSpan Myosin Light Chain Antibody polyclonal BioSciences, (MLC) Inc., USA Mouse 1:500 4°C, overnight Cell Signaling Mono-phospho Myosin Light monoclonal Technology, Chain (Ser19) Antibody (pMLC) USA Di-phospho Myosin Light Chain Rabbit 1:100 4°C, overnight Cell Signaling (Thr18/Ser19) Antibody polyclonal Technology, (ppMLC) USA

Following incubation, the membrane was washed with TBST twice for 10 min and one time for 5 min. The secondary antibodies used in western blotting were conjugated to Horseradish Peroxidase (HRP) for protein detection by chemiluminescence. An anti- mouse IgG or anti-rabbit polyclonal secondary antibody (Cell Signaling Technology, USA) was diluted 1:5000 or 1:10000 in TBST. The membrane was incubated with the secondary antibody solution for 1 h at room temperature with continuous shaking and then washed with TBST as described above. Active chemiluminescence (ECL) substrate solution (Bio-Rad, UK) was prepared by mixing constituents A and B (1:1). These constituents contained H2O2 and the chemiluminescence substrate luminol along with phenols to enhance the luminescence signal. The H2O2 is utilized by HRP to stimulate luminol oxidation which leads to the luminescence and production of light as shown in Figure 2.12. Western blots were developed by using the ECL and the imaged were visualised by the ChemiDoc MP imaging system (Bio-Rad, UK). Figure 2.13 shows how protein bands are detected and visualised by the ChemiDoc MP imaging system.

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Figure 2.12. Diagram showing the principles of protein detection by chemiluminescence. The primary antibody binds to the target protein. The secondary antibody conjugated to HRP binds to the primary antibody. The ECL substrate stimulates the HRP to oxidize the luminol for the production of luminescence and light.

Figure 2.13. Representative image of western blot bands using the ChemiDoc MP imaging system.

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2.3.4.1 Measuring bands and normalization

In order to quantify protein expression, densitometry was conducted using ImageJ software (National Institutes of Health, USA). The free band selection tool was used to draw around the bands of the target protein and α-actin which was used as a control protein. The expression (density) of the target protein was measured and then normalised to the density of α-actin and presented as a percentage of untreated control samples.

2.4 Immunocytochemistry (ICC)

2.4.1 Isolation of uterine myometrial cells for immunofluorescence studies

Uterine tissue was obtained from non-pregnant C57 sexually mature female mice in different stages of the oestrous cycle. As the number of cells isolated from one mouse uterus was not enough to conduct the proposed work, cells were isolated from multiple uteri in a single preparation. As it was difficult to ensure that all of the mice were in the same stages of the oestrous cycle, uteri were collected from mice in different phases of the cycle and the isolated uterine cells were pooled together. Mice were euthanised by cervical dislocation and uteri were removed. Using forceps, the uterine horns were held at the cervix to easily allow a longitudinal incision to be made along the both horns. After making an incision, the uterine tissues were placed on a flat surface and spread open to expose the endometrial layer in each uterine horn. The uterine tissue was then transferred to a fresh petri dish and placed inverted open side up, then the decidua, fat and connective tissues were removed from the uterus and the myometrium was washed several times in sterile Hanks’ balanced salt solution (HBSS) (Sigma, UK) to remove any remaining residual mesentery and blood. The explant technique was used to isolate primary cells from the separated myometrium.

2.4.1.1 Myometrial explant and primary myometrial smooth muscle cell cultures

Under sterile conditions, the isolated myometrial tissue (as described above, section 2.4.1) was finely minced into pieces (<1mm3) using sterile surgical scissors and washed with HBSS. The homogenized tissue explants were placed in 1ml Dulbecco's

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Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F12 GlutaMAX-I) (Gibco, USA) containing 10% v/v foetal bovine serum (FBS) (Sigma, UK), 1% v/v L-glutamine (Sigma, UK) and 0.2% v/v amphotericin B (Amp B) (Sigma, UK). The explant suspensions were aliquoted into 75cm2 culture flasks and the flasks were inverted to improve explant adhesion; 14ml of DMEM/F12 GlutaMAX-I was added beneath to humidify the atmosphere and the flasks were incubated at 37°C (5% CO2) for 24 h. The next day, the flasks containing the cells were returned to the normal upright position and explants were submerged in fresh medium. The explants which were rich in myocytes but would also have had supporting stromal cell which grew out of the adherent explants were maintained under the same conditions until they reached sub-confluence and were monitored under the microscope routinely to ensure the quality of the culture. Culture media was changed at 48- 72 h intervals to provide fresh nutrients and remove non-adherent dead cells.

2.4.1.2 Cell passage

When the cells reached 80-90% confluence in the cell culture flask, they were dissociated from the surface by the process of trypsinisation. Firstly, the medium was removed and discarded and the cells were washed with HBSS 2-3 times. About 8ml of 0.05% w/v trypsin, 0.02% w/v ethylenediaminetetraacetic acid (EDTA) (Sigma, UK) were added to cover the bottom of the flask. The flask was then incubated for 2 min at 37°C. The cells were monitored regularly under the microscope to assess cell dissociation. After the cells had lifted off, the suspension was collected in a centrifuge tube and the flask was washed with 3-4ml of the culture medium, then this media was added to the tube and the tube was centrifuged at 1200rpm for 5 min at room temperature. The supernatant was discarded, by decanting initially and then by pipetting. The cell pellet was resuspended in 1ml of fresh media. A small volume (10μl) was taken from the suspension to quantify cell number, while the rest was divided between two 75cm2 plastic culture flasks or seeded onto 8-well glass chamber slides (Section 2.4.2).

2.4.1.3 Cell count and viability

Before plating, a trypan blue exclusion assay was performed to determine cell number and viability. The cell suspension was diluted 1:1 with DMEM/F12 GlutaMAX-I and 107

0.4% v/v trypan blue solution and 10µl was added to each chamber of a Neubauer haemocytometric slide with a coverslip in place. Using the 10x lens on the Olympus CK40 microscope (Olympus Co. LTD., Japan), live (unstained) and dead (trypan blue positive) cells were counted in five 1mm2 grids per chamber and an average was calculated per grid (Figure 2.14). Since the depth was 0.1 mm, each grid represented a total volume of 0.1 mm3 or 10-4 cm3.

The total number of cells and cell viability was therefore determined using the following calculations:

Cells/ ml = average cell count per grid x 104 x dilution factor

Total cells = Cells/ ml x volume of original cell suspension (ml)

% cell viability = total viable cells (unstained)/ total cells x 100

If cells were too concentrated to count, the cell suspension was diluted further and the dilution factor was adjusted accordingly.

Figure 2.14. Cell counting using Neubauer haemocytometric slide. Cells (live and dead) were counted within the squares indicated by the red circles, to estimate the total number of cells in the suspension. The area of each square is 1mm2 (The central area of the chamber is divided into 25 small squares with an area of 0.04 mm2 and each one is further divided into smaller squares 0.0025 mm2. The depth of the chamber is 0.1 mm).

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2.4.2 Cell plating and treatment

Uterine cells (2 x 104 cells/ well) diluted in culture medium were seeded into an 8-well glass chamber slide (Corning Incorporated Life Sciences, USA), and allowed to adhere and grow at 37°C. When cell confluency reached about 60-70%, the culture medium was aspirated and cells were washed with 500µl of culture medium (without phenol red). Throughout the protocol, all reagents were removed by aspiration flicking followed by careful blotting with Whatman filter paper (Sigma, UK).

In order to examine the effect of ripasudil on the expression of the target proteins (MLC, pMLC and ppMLC) in the presence and absence of U46619, smooth muscle cells were treated with DMSO (as vehicle control; concentration matched), U46619 (10- 6M), ripasudil (10-6M) or ripasudil (10-6M) + U46619 (10-6M) for 30 sec (after optimisation of the treatment period). Concentrations of U46619 and ripasudil (10-6M) were selected based on previous studies on pulmonary arterial myocytes in piglets and trabecular meshwork cells in monkeys (Fediuk et al., 2014, Kaneko et al., 2016).

2.4.3 Cell fixation

After treatment, vehicle and drugs were removed from the wells and cells were fixed with 250µl of 4% (w/v) paraformaldehyde (PFA; Sigma-Aldrich, UK) in PBS for 5 min at 37°C. The PFA was removed and cells were washed three times with 500µl PBS- Tween 20 (0.5% v/v) (Sigma-Aldrich, UK). Wells were covered with parafilm and the cells were stored at 4°C overnight.

2.4.4 Permeabilisation

Following fixation, the cells were taken out from the fridge and washed three times with 500µl of PBS-Tween 20 (0.5% v/v) for 5 min at 37°C. Cells were then permeabilised by incubation with 500µl of permeabilising buffer (Table 2.9) per well for 5 min at 4°C. The slide was then washed two times with PBS-Tween 20 (0.5% v/v) for 5 min at 37°C.

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Table 2.9. Composition of the permeabilising buffer in 100ml milli-Q water. Permeabilising buffer Volume Source Catalogue N°

Sucrose 10.3g Merck, Germany 107687

Sodium chloride (NaCl) 0.292g Fisher Scientific, UK S/1360/63

Magnesium chloride (MgCl2) 0.06g Sigma-Aldrich, UK M8266

HEPES 0.476g Sigma-Aldrich, UK H3784

Triton X-100 0.5ml Promega, UK H5142

2.4.5 Blocking

Non-specific binding sites were blocked by incubation with 3% (w/v) of blocking buffer for 1 h at room temperature. The blocking buffer was prepared as a 10% solution by adding 10g bovine serum albumen (BSA, Fraction V; Sigma-Aldrich, UK) to 100ml PBS-Tween 20 (0.05% v/v), then this mixture was inactivated by heating at 65C for 15 min and stored at 4°C. From this 10% solution, 3% and 1% blocking buffer were prepared by dilution in more PBS-Tween 20 (0.05% v/v).

2.4.6 Immunofluorescence (IF)

Immunofluorescence (IF) is a useful imaging technique that is commonly used to determine the expression of a specific target antigen in biological cells and tissues. This technique is based on immunological and biochemical reactions where the target antigen interacts with a specific antibody, and this interaction is amplified and visualised under a fluorescent microscope.

There are multiple methods of IF and the most common one is the indirect method which uses a primary unconjugated antibody, followed by a secondary antibody conjugated to a fluorophore, directed toward the primary antibody as illustrated in Figure 2.15.

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Figure 2.15. Indirect immunofluorescent staining. The unconjugated primary antibody binds to the antigen of interest and then the secondary antibody conjugated with a fluorophore is directed toward the primary antibody.

2.4.7 Cell immunostaining

Following blocking, cells were incubated with 100µl of primary antibody (concentrations are shown in Table 2.10) for 1 h at 37°C. Antibody concentrations were optimised in preliminary experiments. Thereafter, the primary antibody was removed and cells were gently washed three times with 500µl of PBS-Tween 20 (0.5% v/v) for 5 min at 37°C. The cells were then incubated with secondary antibody for 1 h in the dark at room temperature (Table 2.11). Following this, cells were rinsed three times with 500µl of PBS for 5 min at room temperature. Then, 150µl of phalloidin– tetramethylrhodamine B isothiocyanate (Phalloidin-TRITC) solution (Sigma-Aldrich, UK) was added per well and the cells were incubated for 1 h in the dark at room temperature. Phalloidin-TRITC solution is a toxin with an ability to bind to polymeric filamentous actin; it was diluted to 1µg/ml in PBS. Following this incubation, cells were washed three times with 500µl of PBS in the dark for 5 min at room temperature with gentle shaking. Afterwards, the silicone gasket adherent to the slide was removed by pulling with a tweezer from the edge, and the slide was rinsed with milli-Q purified water. Any remaining drops of water were removed by gently tapping the back of slide against the bench and wiping with filter paper; the slide was left for 5-10 min at room temperature to dry. A mounting medium that contained DAPI (4',6-diamidino-2- phenylindole, dihydrochloride) (Life Technologies, USA) was applied to the slide and a

111 coverslip was then mounted gently to avoid air bubbles entrapment. DAPI is a nuclear counterstain used in immunochemistry techniques. Subsequently, the edges of the slide were sealed by nail varnish and left to dry overnight in the dark, and then left at room temperature until microscopic examination. Table 2.12 illustrates the steps of fixation, permeabilisation and immunostaining

Table 2.10. List of primary antibodies used in the immunofluorescence work. Protein Species Dilution Company Myosin Light Chain Antibody Rabbit 1:200 LifeSpan BioSciences, (MLC) polyclonal Inc., USA

Mono-phospho Myosin Light Mouse 1:200 Cell Signaling Chain (Ser19) Antibody (pMLC) monoclonal Technology, USA

Di-phospho Myosin Light Chain Rabbit 1:200 Cell Signaling (Thr18/Ser19) Antibody (ppMLC) polyclonal Technology, USA

Table 2.11. List of secondary antibodies used in the immunofluorescence work. Antibody Dilution Company

Goat anti-Rabbit IgG H&L (Alexa Fluor® 488) 1:200 Life Technologies, USA

Rabbit anti-Mouse IgG (Alexa Fluor® 488) 1:500 Thermo Scientific, USA

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Table 2.12. Illustration of the fixation, permeabilisation and immunostaining processes. Step Procedure Temperature Time

1 Fixation with 4% paraformaldehyde solution 37°C 5 min

2 Washing with PBS-Tween (0.5%) (3 times) 37°C 5 min x 3

3 Incubation with permeabilising solution 4°C 5 min

4 Washing with PBS-Tween (0.5%) (2 times) 37°C 5 min x 2

5 Incubation with 3% blocking solution 37°C 1 h

6 Incubation with primary antibody solution 37°C 1 h

7 Washing with PBS-Tween (0.5%) (3 times) 37°C 5 min x 3

8 Incubation with secondary antibody in dark 37°C 1 h

Room 9 Washing with PBS on shaker in dark (3 times) 5 min x 3 temperature

Washed with milli-Q purified water. DAPI was used to mount the coverslips on to slides. After Room 10 drying, slides were left in dark at room temperature temperature until microscopic examination.

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2.4.8 Imaging analysis

Following immunostaining, the cells were examined using a ZEISS Axio Imager 2 Research Upright Microscope (Carl Zeiss Microscopy, Germany) coupled with a CCD camera (CoolSNAP HQ2, Photometrics, USA). The Micro-Manager software (v.1.46, Vale Lab, USA) was used to adjust the microscope and the camera, as well as to capture images using a 20x objective lens. The exposure time for DAPI, FITC (fluorescein isothiocyanate) and TRITC were adjusted individually to avoid any signal value above 12,000 (according to the machine protocol). Images were then processed using ImageJ software (v.1.48, National Institutes of Health, USA) by splitting image channels, adjusting thresholds and re-merging channels to create an integrated image. Figure 2.16 shows how individual images of cell components were taken and then merged using Micro-Manager and ImageJ software.

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Figure 2.16. An example of immunofluorescent image channels before and after the creation of a merged image. The diagram displays the triple staining at different cell components (red TRITC actin filaments, blue DAPI nuclear staining and green FITC for ppMLC proteins) in uterine myometrial cells isolated from non-pregnant mice. Magnification x20.

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2.5 Enzyme-linked Immunosorbent Assay (ELISA)

2.5.1 Overview of ELISA

Enzyme-linked Immunosorbent Assay (ELISA) is a commonly used biochemical technique which is designed to detect and measure different biomarkers such as antigens, hormones and proteins in biological liquids. It is characterised by being simple, versatile and rapid, and multiple specimens can be analysed in each individual experiment. Like other immunological assays, ELISA is based on the interaction between a specific antibody and its target antigen. The advantages of ELISA encouraged the development of various methods of the technique. Among these, the sandwich ELISA is one of the broadly used methods. It is most convenient for detecting cytokines, hormones and growth factors through using a target capture and detection antibody pair. The basic concept behind the “sandwich” is that the location of the analyte is between the antibody that coats the well and the antibody used for detection. After the addition of an HRP (Horseradish Peroxidase) substrate, such as 3,3',5,5'- tetramethylbenzidine reagent (TMB), the oxidation of the substrate is stimulated by HRP and this can be visualised by the formation of a blue chromagen that turn a yellow colour by adding a strongly acidic stop solution that inactivates HRP. In a sandwich ELISA, increased concentrations of the analyte in the sample directly increase the intensity of the colour produced.

2.5.2 Collection of plasma samples

For plasma collection, the method described by Roszkowski was used (Roszkowski, 2014). The plasma was collected from the heart following the euthanasia of mice and prior to the removal of the uteri and placentas. The mice were placed on their back and the chest was opened using surgical scissors. Then, the heart was opened with different surgical scissors and approximately 300µl of blood was collected from the ventricle using a fine tip plastic pipette and the blood was added into Microvette CB300 LH Lithium-Heparin collection tubes (Sarstedt, Germany) for plasma extraction. Blood was spun for 5 minutes at 2,000g to obtain plasma at 20°C. Plasma was immediately frozen at -80ºC until analysed for measurement of 17β-oestradiol concentration.

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2.5.3 ELISA measurement of 17β-oestradiol

The concentration of 17β-oestradiol (17β-E2) was measured in the plasma of pregnant and non-pregnant (dioestrus) C57 mice using a sandwich oestradiol mouse ELISA kit (BioVision, USA). The kit was provided with a 96 well plate coated with anti-mouse oestradiol antibody, mouse oestradiol standard (72ng/L), HRP conjugate reagent, standard diluent, sample diluent, chromogen solution A, chromogen solution B, stop solution and wash solution (30x stock).

At the beginning, plasma samples (see section 2.5.2) were taken from the freezer, left to thaw on ice and vortexed, then were centrifuged at 3000rpm for 20 min at 20°C to remove particulates. In the meantime, all reagents were left to reach room temperature. To create the standard curve, serial dilutions of standards were prepared at concentrations of 72, 48, 32, 16, 8, 4 and 0 ng/L. For the preparation of 1x working wash solution, the wash solution (30x) (20ml) was diluted 1:30 with 600 ml distilled water.

As outlined in Figure 2.17, 50μl of standard or sample was added in duplicate to individual wells of the 96-well plate, and the plate was then sealed with an adhesive cover and incubated at 37°C for 30 min. Thereafter, the content of each well was aspirated from the plate, which was washed 5 times with 350μl of working wash solution and incubated at room temperature (2 min each time). When removing the wash solution, it was essential to avoid drying of the plate as this would inactivate the enzyme reactions. In the dark, 50μl of HRP-conjugate antibody reagent was added to each well and the plate was sealed, incubated at 37°C for 30 min and kept away from light. The HRP-conjugate was then discarded and wells were washed 5 times with the working wash solution and incubated at room temperature (2 min each time). Then, 50μl of chromogen solution A and B were added to each well and the plate was incubated again in the dark at 37°C for 15 min. To each well, 50μl of stop solution was added and this step changed the colour of the wells from blue to yellow. Figure 2.18 illustrates the binding sequence of antibodies and the target biomarker (17β-E2) as well as the colour change in sandwich ELISA from BioVision.

The absorbance at 450nm was then read in a microplate reader (Multiskan™ FC, Thermo Scientific, Finland) within 15 min after addition of the stop solution. The mean absorbance value (A450) was used to create the standard curve (as shown in Figure 2.19)

117 by plotting the mean absorbance of each standard against its concentration. Samples were quantified by interpolating from the standard curve, which was expressed in ng/L using GraphPad Prism 7.03 software.

Figure 2.17. The outline of the sandwich ELISA.

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Figure 2.18. Exemplification of sandwich ELISA assay and binding sequence of antibodies and reagents from BioVision.

1 7  - E 2 (n g /L ) S ta n d a r d 2 .5

2 .0 )

m 1 .5

2 n

( r = 0 .9 9 8 6

D 1 .0 O

0 .5

0 .0 1 1 0 1 0 0 1 0 0 0

1 7  - E 2 (n g /L )

Figure 2.19. BioVision ELISA assay for the standard curve of 17β-oestradiol.

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2.6 Mass spectrometry analysis of total fatty acid composition

2.6.1 Overview of mass spectrometry

Electrospray ionisation liquid chromatography tandem mass spectrometry (LC/ESI- MS/MS) is regularly used in lipidomics studies as it is characterized by its accuracy and high sensitivity. The precision of this analysis requires combined use of different methods. Mass spectrometry (MS) allows the identification of analytes such as proteins and lipids in various biological materials by measuring their mass-to-charge ratio, so it provides information about their structures and molecular weights. However, its accuracy is impaired with low concentrations of analytes, especially when ions overlap in the mass spectrum, from the different compounds present in the sample. Therefore, MS is usually combined with High Performance Liquid Chromatography (HPLC), a technique which splits the components of a mixture according to their retention times along a mobile and stationary phase. If this technique is used separately, it will be difficult to provide an accurate identification of analytes, as various compounds may have a similar retention time. Combining of HPLC and MS techniques ensures the separation of compounds within the mixture prior to entry into the mass spectrometer. Electrospray ionisation (ESI) is used to link HPLC to MS through ionising molecules and evaporating the solvent. ESI is a mild ionization technique and is applied for the ionization of large biological molecules such as lipids and lipid mediators without inducing extensive fragmentation prior to MS analysis (Ardrey, 2003).

In order to increase the sensitivity and specificity of this method, tandem mass spectrometry (MS/MS) is employed. Tandem MS uses either a “Selected Decomposition Monitoring” (SDM) or “Selected Reaction Monitoring” (SRM) mode to analyse the fragmentation of a precursor ion of interest into product ions. The “Multiple Reaction Monitoring” (MRM) mode helps in analysing the fragmentation of multiple precursor ions. Therefore, the analytes are exposed to an ionisation process which is regulated by the ionisation source and after that the analytes can enter into the triple quadrupole system of the MS-MS device: initially, the ion of interest is selected according to its m/z ratio by the first quadrupole (Q1); then, it is exposed to a fragmentation mechanism by the second quadrupole (Q2), which acts as a collision cell device rather than a mass separation device, and thereafter, the third quadrupole (Q3)

120 will identify the products of the pre-selected ion according to their m/z ratio and this can be detected and visualised using as a mass spectrometry software (as seen in Figure 2.20).

Figure 2.20. Schematic overview of LC/ESI-MS/MS method. HPLC separates the components of the lipid extract according to their retention times. ESI induces the ionisation of the analytes before entering the MS triple quadruple analyser. The first quadruple (Q1) selects the precursor ion (ion of interest) according to its m/z ratio. The second quadruple (Q2) facilitates the process of collision induced dissociation (CID) which leads to its fragmentation. The third quadruple (Q3) selects the product ion fragments produced by the CID process according to the m/z ratio. The product ion hits the detector which converts its signal to a mass spectrum.

2.6.2 LC/ESI-MS/MS protocol

In this study, the method developed by Masoodi and Nicolaou to measure eicosanoids and hydroxy fatty acids was used (Masoodi and Nicolaou, 2006, Masoodi et al., 2008). 79 lipid species were quantified in biological tissues. Table 2.13 shows a list of the lipid mediators investigated, classified by their precursor fatty acids.

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Table 2.13. List of prostanoids, leukotrienes and hydroxy fatty acids and their main polyunsaturated fatty acids (PUFA) precursors.

DGLA/LA/ALA-derived AA-derived EPA-derived DHA-derived

PGD1 PGD2 ± 5-HEPE RvD1

PGE1 PGE2 ± 8-HEPE RvD2

PGF1α 15-keto PGE2 ± 9-HEPE MaR1

13,14-dihydro-15-keto PGE1 13,14-dihydro-15-keto PGE2 ± 11-HEPE PDX

13,14-dihydro-15-keto PGF1α PGI2 (as 6-keto PGF1α) ± 12-HEPE ± 4-HDHA

15-HETrE PGF2α ± 15-HEPE ± 7-HDHA

13,14-dihydro PGE1 15 keto-PGF2α ± 18-HEPE ± 10-HDHA

13,14-dihydro PGF2α TXB3 ± 11-HDHA

13,14-dihydro-15-keto PGF2α PGD3 ± 13-HDHA

± 5-HETE RvE1 ± 14-HDHA

LA-derived ± 8-HETE PGE3 ± 17-HDHA

9-HODE ± 9-HETE ± 20-HDHA 13-HODE ± 11-HETE 19,20 DiHDPA 9,10 DiHOME ± 12-HETE 16(17) EpDPE 12,13 DiHOME ± 15-HETE 19(20) EpDPE

9 OxoODE ± 20-HETE

13 OxoODE LTB4

9(10) EpOME PGJ2 12 12(13) EpOME Δ -PGJ2 12 Trans EKODE 15-deoxy-Δ ,14-PGJ2 ± 14,15-DHET ± 11,12-DHET ± 8,9-DHET ± 5,6-DHET

ALA-derived ± 5(6)-EET

9 HOTrE ± 11(12)-EET

13 HOTrE ± 14(15)-EET ± 8(9)-EET 5-oxo-ETE

TXB2 HXA3 5,15 DiHETE 8,15 DiHETE

Dihomo-gamma-linolenic acid (DGLA); linoleic acid (LA); Alpha-linolenic acid (ALA); Arachidonic acid (AA); Eicosapentaenoic acid (EPA); Docosahexaenoic acid (DHA); Prostaglandin (PG); hydroxyeicosatrienoic acid (HETrE); hydroxyoctadecadienoic acid (HODE); dihydroxyoctadecenoic acid (diHOME); oxooctadecadieonic acid (oxoODE); epoxyoctadecenoic acid (EpOME); transepoxyketooctadecenoic acid (Trans EKODE); hydroxyoctadecatrienoic acid (HOTrE); hydroxyeicosatetraenoic acid (HETE); leukotriene (LT); dihydroxyeicosatrienoic acid (DHET); epoxyeicosatrienoic acid (EET); thromboxane (TX); hepoxilin (HX); dihydroxyeicosatetraenoic acid (DiHETE); hydroxyeicosapentaenoic acid; resolvins (RV); maresin (MaR); protectin (PDX); hydroxydocosahexaenoic acid (HDHA); dihydroxydocosapentaenoic acid (DiHDPA); epoxydocosapentaenoic acid (EpDPE).

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2.6.3 Materials for LC/ESI-MS/MS

In this work, we assessed the effect of targeted delivery of the nitric oxide donor (NO- donor), SE175 on the tissue concentration (myometrial vasculature and placenta) of lipid mediators in both pregnant C57BL/6J wild-type (C57 WT) mice and endothelial nitric oxide synthase knockout (eNOS KO) Nos3tm1Unc/J mice at term, E19).

2.6.3.1 Preparation of liposomes

Liposomes were prepared in the lab of Dr. Lynda Harris and her group at the University of Manchester / UK (Cureton, 2017). The method used to prepare liposomes was the lipid rehydration method (Nallamothu et al., 2006). Several materials were weighed and dissolved in methanol:chloroform at a ratio of 9:1 ratio. These materials included 1,2 distearoyl-sn-glycero-3-phosphocholine at 32.5 µmol, cholesterol at 15 µmol and 1,2 diaster diastearoyl-sn-glycero-3-phosphoethanolamine-N-[PEG] at 2.5 µmol. In order to produce a lipid film, the solvent was removed via rotatory evaporation (40°C, 270 mbar), thereafter, the lipid film was incubated in a vacuum drier at 250°C and 270 mbar overnight. To make large multilameller vesicles, the film was rehydrated in PBS (1 ml) and the solution was incubated for 3 h in an oven (62°C). Then, the solution was extruded 11 times through a thermobarrel Mini-Extruder of 1 ml with the use of a polycarbonate membrane (0.1 µm, 19 mm) which was surrounded by a filter support of 10 mm to produce monodisperse unilamellar liposomes. The resultant liposomes were kept at 4°C until usage.

To prepare PBS-encapsulated liposomes and enhance peptide conjugation, 10% of the 1,2 distearoyl-sn-glycero-3-phosphocholine substituted with 1,2 diaster diastearoyl-sn- glycero-3-phosphoethanolamine-N-[PEG]. Then, the lipid film was synthesized and rehydrated with PBS and then extruded as explained above and stored at 4°C. Whereas to prepare SE175-encapsulated liposomes, 1 ml of 320 µM SE175 was added after the preparation and rehydration of the lipid film. Other steps were the same as above. The produced liposomes were then stored at 4°C.

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2.6.3.2 Treatment of animals

Treatment of mice was conducted by Natalie Cureton, a PhD student within Dr. Lynda Harris’s group. After the incorporation of PBS or SE175 into the targeted liposomes, mice were intravenously injected with 100 µl four times during the second and third trimester of pregnancy. Doses of either PBS as a control (as labelled as PBS control), PBS-encapsulated liposomes (PBS liposomes), SE175-encapsulated liposomes (SE175 liposomes) or free SE175 were given. Treatments were administered on E12, E14, E16 and E18; mice were sacrificed at E19 of gestational age and myometrial tissues and placentas were collected and mass spectrophotometric analysis was performed using two separate LC/ESI-MS/MS protocols. The COX assay consisted of 24 prostanoids and related lipid mediators, while the LOX/CYP assay detected 55 metabolites of lipoxygenase and cytochrome P450 derived hydroxy fatty acids and related compounds.

2.6.4 Preparation of internal standards

All standards were prepared by Dr. Alexandra Kendall, a Research Associate within Prof. Anna Nicolaou’s group at the University of Manchester / UK according to the method developed and modified by Masoodi and Nicolaou to quantify eicosanoids and hydroxy fatty acids (Masoodi and Nicolaou, 2006, Masoodi et al., 2008).

Internal calibration standard

1ng/μl of internal standards (PGB2-d4, 12-HETE-d8, 8(9) EET-d11 and 8,9 DHET- d11) were purchased from Cayman Chemical (USA) and prepared as the following:

Using a 100μl Hamilton syringe (SGE, Australia), 100μl of 10ng/μl stock of each of the above standards was added to an amber vial (Phenomenex, Macclesfield, United Kingdom). During preparation, the Hamilton syringe was rinsed 6 times with HPLC grade ethanol (Thermo Fischer, UK) to avoid cross-contamination. 600μl of HPLC- grade ethanol was added before standards were sealed with Parafilm and stored at -20°C for up to three months.

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Prostanoid calibration standard

For the COX calibration line, 100pg/μl of prostanoid cocktail was prepared as follows:

10μl of the 10ng/μl PGE1; PGD1; 13,14-dihydro-15-keto PGE1; 6-keto-PGF1α; TXB2;

13,14-dihydro PGF1α; PGF1α; 13,14-dihydro PGE1; 13,14-dihydro PGF1α; PGE2; PGD2; 12,14 12 15-keto-PGE2; 15-deoxy-Δ -PGJ2; PGJ2; Δ -PGJ2; 13,14-dihydro-15-keto PGE2;

13,14-dihydro-15-keto PGF2α; PGF2α; 8-iso PGF2α; 13,14-dihydro PGF2α; PGE3; PGD3;

PGF3α; TXB3 (Cayman Chemical, USA) stocks were added to an amber vial using a 20μl Hamilton syringe, rinsing between standards with HPLC grade ethanol. 760μl of ethanol was added to obtain the final volume of 1ml of 100pg/μl COX standard- cocktail. Standards were mixed, sealed with Parafilm and stored at -80°C for up to three months.

Hydroxyl fatty acid calibration standard

For the LOX/CYP calibration line, 100pg/μl hydroxy fatty acid cocktail was prepared as follows:

10μl of the 10ng/μL 9 HODE; 13 HODE; RvE1; RvD1; RvD2; MaR1; PDX; ± 11 HDHA; ± 4 HDHA; ± 7 HDHA; ± 10 HDHA; ± 13 HDHA; ± 14 HDHA; ± 17 HDHA; ± 20 HDHA; LTB4; ± 14,15 DHET; ± 11,12 DHET; ± 8,9 DHET; ± 5,6 DHET; ± 5(6) EET; ± 11(12) EET; ± 14(15) EET; ± 8(9) EET; 5oxoETE; 5 HETE; 8 HETE; ± 9HETE; 11 HETE; 12HETE; 15- HETE; 20 HETE; 5 HEPE; ± 8 HEPE; ± 9 HEPE; ± 11 HEPE; ± 15 HEPE; ± 18 HEPE; 12 HEPE; 9 HOTrE; 13 HOTrE; 15 HETrE; HXA3; 19(20) DiHDPA; 9(10) EpOME; 12(13) EpOME; 9oxoODE; 13-oxoODE; 5(15) DiHETE; 8(15) DiHETE; 19(20) EpDPE; 16(17) EpDPE; trans-EKODE; 9,10- DiHOME (Cayman Chemical, USA) stocks were added to an amber vial, using a 20μl Hamilton syringe, rinsing between standards with HPLC grade ethanol. 460μl of ethanol was added to the 100pg/μl LOX/CYP standard cocktail to obtain the final volume of 1ml. Standards were mixed, sealed with Parafilm and stored at -80°C for up to three months.

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2.6.5 LC/ESI-MS/MS

2.6.5.1 Lipid extraction

Mass spectrometry to detect eicosanoids and hydroxy fatty acids was performed on whole tissues (mouse uteri and placentas). Figure 2.21 shows the main steps of lipid extraction from whole tissues.

Uterine and placental tissue samples were weighed and placed into a dounce tissue grinder (Wheaton, USA) for homogenisation. 500μl ice cold absolute methanol (LC-MS grade Sigma-Aldrich, UK) was added to each sample and a pestle was used to break up the tissue until fully homogenised. The homogenised samples were transferred into flat- bottomed tubes (Fisher Scientific, Loughborough, UK) using a glass Pasteur pipette (Fisher Scientific, Loughborough, UK). Ice cold methanol (200μl) was used to rinse the tissue grinder walls and the pestle to fully collect the remaining tissue, which was then added to the flat-bottomed tube using a glass Pasteur pipette. In order to avoid cross contamination, the tissue grinder and pestle were washed with analytical grade water (purified using PURELAB Flex, ElgaLabWater, High Wycombe, UK) and ethanol (HPLC grade, ≥99.8% v/v, Fisher Scientific, Loughborough, UK) between each sample. Then, 4ml ice cold water was added to the tubes to bring the concentration to 15% methanol in water (v/v). Using a 50μl Hamilton syringe, 20μl of the 1ng/μl internal standard cocktail was added to the solution. Thereafter, the tubes were gently shaken and then incubated on ice in the dark for 30 minutes and centrifuged (refrigerated centrifuge (Sorvall; RT6000B), 3000 rpm, 10 min, 4ºC).

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2.6.5.2 Solid phase extraction

During centrifugation, SPE cartridges (C18-E 500mg, 6ml, Phenomenex, UK) were preconditioned with 6ml 100% methanol (to facilitate the penetration of the highly aqeuos solvents through the hyrophobic surface and wet the sorbent), and 6ml deionised water (this process should be completed no more than 10 minutes before adding the samples to the cartridges). After centrifugation, the lipid supernatants were transferred to new extraction (flat-bottomed) tubes using a glass Pasteur pipette.

The pellet was saved and stored at -20°C to measure protein content ( 2.6.5.8). The pH of the lipid supernatant was adjusted to pH3 by adding 3-4 drops of 0.1M hydrochloric acid (ACS grade, Sigma-Aldrich, UK). To measure the degree of acidity, pH indicator strips (Merck, UK) were used. After reaching pH3, the samples were passed through the SPE cartridges. Once the samples had completely run through the columns, the cartridges were washed with 6ml 15% (v/v) methanol in water under a low vacuum (vacuum pump: 1c Vacuumbrand, Germany), with 6ml deionised water (using low vacuum) and with 6ml hexane (HPLC grade, Fisher Scientific, UK), using higher vacuum. Any remaining bubbles or water drops on the surface of the cartridges were removed with a Pasteur pipette. Round-bottomed tubes (Fisher Scientific, UK) were then placed underneath the cartridges and 6ml methyl formate (HPLC grade, Sigma- Aldrich, UK) was then added to the cartridges using a moderate vacuum (starting with a very high vacuum to initiate the elution of methyl formate through the cartridges and later finishing with a very high vacuum again to completely drain the cartridges).

2.6.5.3 Drying and reconstituting samples

After extraction, tubes were transferred to the drying cabinet and exposed to a nitrogen stream whose intensity was increased until the drying step was complete. When the solvent was completely evaporated, the pellet was resuspended in 100μl ethanol (HPLC grade) using a 100μl Hamilton syringe, which had been rinsed with HPLC grade ethanol.

Tubes were then vortexed (vortex mixer, Fisher Scientific, UK) and centrifuged at 2,000g at 4°C for 1 minute. Samples were transferred to amber sample vials, containing a 100μl insert (Phenomenex, UK) using a 100μl Hamilton syringe and stored at -20°C

128 until LC/ESI-MS/MS was run, which was conducted using a Xevo TQ-S electrospray ionisation triple quadrupole mass spectrometer (Waters, UK) associated with a Waters Alliance 2695 ultra-high-performance liquid chromatography (UPLC) pump (Acquity, Waters, UK).

2.6.5.4 Preparation of standards for quantification

To precisely determine the concentration of each compound in the samples, calibration lines for all the compounds of interest were generated. A 100pg/μl of either an internal prostanoid standard for the COX calibration line or a hydroxy fatty acid standard for the LOX/CYP calibration line was made up from 10ng/μl of each of the compound of interest (in a 1:100 dilution of each stock). COX and LOX/CYP standards were subjected to a two-fold serial dilution to generate five-point calibration lines. The standard points were 0.625pg/μl, 1.25pg/μl, 2.5pg/μl, 5pg/μl and 10pg/μl.

Internal standards were added to each standard point and were exposed to the drying process as with the samples. The dried standards were reconstituted in 100μl ethanol and stored at -20ºC for up to a week before LC/ESI-MS/MS analysis was performed.

2.6.5.5 Liquid chromatography

Chromatographic analysis was carried out (by Prof. Anna Nicolaou’s group at the University of Manchester / UK) on a C18 column (Acuity UPLC BEH, 1.7μm, 2.1x50mm; Waters, UK). The temperature of the autosampler chamber (Waters, UK) was set at 8°C to keep the analytes cold during the assay, while for the column, it was set at 25°C. COX and LOX/CYP assays were performed using two separated chromatographic runs; 5.8 minutes assay for COX and 5 minutes assay for LOX/CYP. Standards and analytes were injected in duplicate with the injection volume at 3µl, while ethanol blanks were run in between each sample to avoid sample contamination. The water-acetonitrile gradient was used at a flow rate of 0.6ml/min to separate the analytes. Mobile phase A was ultrapure water:acetic acid (HPLC grade, Sigma-Aldrich, UK) (100:0.02), mobile phase B was acetonitrile (LC-MS grade, Sigma-Aldrich, UK):

129 glacial acetic acid (HPLC grade) (100:0.02). The proportions of gradients are shown in Tables 2.14 and 2.15.

Table 2.14. Solvent gradient for COX assay.

The table shows the concentration ratios of mobile phases A and B used for the COX assay. Mobile phases A is HPLC grade ultrapure water:acetic acid (100:0.02), mobile phase B is LC- MS grade acetonitrile:HPLC grade glacial acetic acid (100:0.02).

Table 2.15. Solvent gradient for LOX/CYP assay.

The table shows the concentration ratios of mobile phases A and B used for the LOX/CYP assay. Mobile phases A is HPLC grade ultrapure water:acetic acid (100:0.02), mobile phase B is LC-MS grade acetonitrile:HPLC grade glacial acetic acid (100:0.02).

2.6.5.6 ESI-MS/MS

The instrument was operated in a negative ionisation mode and different MS/MS settings were used for COX and LOX/CYP assays. For the COX assay, the capillary voltage was 3100V, the source temperature was 150°C, the desolvation temperature was

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500°C, the cone voltage was 35V, and the dwell time was 0.005s. For the LOX/CYP assay, the capillary voltage was 1500V, the source temperature was 150°C, the desolvation temperature was 500°C, the cone voltage was 35V, and the dwell time was 0.003s. In order to determine the multiple reaction monitoring (MRM) transitions, the IntelliStart protocol was used. Tables 2.16 and 2.17 show the MRM, cone voltage and collision energy for each lipid, whereas Appendix 2 and Appendix 3 show representative chromatograms of LC-ESI MS/MS for COX and LOX/CYP respectively.

Table 2.16. Summary of individual MRM transition, cone voltage, collision energy and indicative retention times for COX assay.

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Table 2.17. Summary of individual MRM transition, cone voltage, collision energy and indicative retention times for the LOX/CYP assay.

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PDX and MaR1 share the same MRM transition so they cannot be separated by retention time. This indicates that a peak appearing in either transition could be either compound or a combination of both.

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2.6.5.7 Quantification of compounds using TargetLynx

TargetLynx extension (Waters, UK) running under MassLynx 4.0 software was used to process data and quantify the concentration of the analytes of interest in biological specimens. The use of deuterated internal standards which were added to samples and standards allowed the normalisation of peak integrals against the prepared internal standards. In every case, the most related internal standard was used for the normalization, e.g. 12-HETE-d8 was used for the quantification of hydroxy fatty acid metabolites. A calibration line was generated by plotting the normalised peak area versus concentration. The calibration lines were used to accurately measure the concentration of the compounds of interest in our samples. The calculated concentrations were expressed as pg/μl. In order to calculate the concentration in each extract, the mean of the two duplicate injections was calculated, this was multiplied by 100 (dilution factor of reconstitution after nitrogen drying) to give the total concentration of each compound in the extract. This total concentration (in pg) was normalised against the protein content. The limit of detection (LOD) was set to a signal to noise ratio of 3, whist the limit of quantification (LOQ) was set to a signal to noise ratio of ˃5. Peak area under the samples below 150pg/μl were excluded and considered to be noise. Figure 2.22 illustrates the standard curve and a representative chromatogram of an analyte of interest in TargetLynx.

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Figure 2.22. Representative chromatogram and standard curve for 11 HETE in TargetLynx. Figures (A) and (B) show retention times (RT), area underneath the peak, internal standard area (IS area), response factor (the ratio between the peak area and the concentration), S/N ratio. Figure (C) shows the calibration line from the standards and R2 value of the curve.

2.6.5.8 Protein content

The protein pellet was collected after centrifuging the sample homogenate and stored at -20°C until analysis. 1ml of 1M NaOH (Sigma-Aldrich, UK) was added to each sample tube, which was then heated in a water bath at 60°C for 90 minutes. Bovine serum albumin (BSA, Sigma-Aldrich, UK) standards were prepared from 1.5mg/ml stock as described in Table 2.18.

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Table 2.18. Standard dilutions of BSA prepared from 1.5mg/ml stock in 1M NaOH to generate standard curve for protein content analysis.

In a 96 well plate, 5μl of each standard and 5μl of each sample in triplicate were added followed by the addition of 25μl Protein Assay Reagent A (Bio-Rad, USA) and 200μl Protein Assay Reagent B (Bio-Rad, USA). Plates were left the dark for 15 minutes and then read on a plate reader at 650nm.

The standards were plotted to obtain a standard curve as shown in Figure 2.23 and the protein content of the samples was estimated using the equation of the line of the best fit. Data were normalised by dividing the lipid mediator concentration by the protein content of the samples.

Figure 2.23. Standard curve and equation of the line for protein content determination. BSA concentrations were blotted against the absorbance value measured at 650nm. 136

2.7 Statistical analysis

In functional studies work, data analysis and comparison between different spontaneous activities and dose-response curves were performed using unpaired student t-test, one- way ANOVA or two-way ANOVA and a Bonferroni post-hoc test depending on the number of groups being compared.

In western blotting, each experiment was repeated for three times and data were expressed as means ± SEM and were analysed using unpaired student t-test or one-way ANOVA and a Bonferroni post-hoc test depending on the number of groups being compared.

For ELISA derived data relating to plasma 17β-oestradiol levels from different groups these were expressed as mean ± SEM and statistics were performed using unpaired Student t-test.

In mass spectrometry, data analysis and comparison between different groups were performed using two-way ANOVA and a Bonferroni post-hoc test.

GraphPad Prism version 7.03 for Windows (GraphPad Software, La Jolla California, USA) was used to conduct the statistical analysis. All values were presented as means ± S.E.M. and differences were considered significant when p < 0.05.

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3 Chapter 3:

Effect of Ripasudil, a ROCK inhibitor on U46619-,

PGF2α- and 5-HT-induced myometrial contraction in non-pregnant C57 WT mice

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3.1 Introduction

The mouse uterus is a muscular and contractile tissue and is extensively rich in uterine smooth muscle cells (myocytes) that support stromal and vascular tissue. These cells have the ability to contract in the absence of any hormonal or nervous stimuli. Isolated uterine pieces from both, pregnant and non-pregnant animals exhibit spontaneous contraction over a period of time (Wray, 1993). This type of contraction and in the absence of external inducers is called smooth muscle tone (Griffiths, 2007). The contractility of smooth muscle cells is mainly regulated by the elevation of intracellular Ca2+ and it takes place via the interaction of myosin and actin filaments. A complex is formed by binding of Ca2+ and calmodulin. This complex then stimulates an enzyme known as myosin light chain kinase (MLCK), which in turn phosphorylates myosin regulatory light chain (MLC20). As a consequence of this stimulation and phosphorylation, the formation of actin-myosin cross-bridges occur which is combined with ATP hydrolysis (Word, 1995). RhoA, a small GTPase, is also involved in the process of smooth muscle contraction through activating two proteins, the rho- associated protein kinase (ROCKl) and its isoform ROCKII. These two proteins indirectly prevent the dephosphorylation of the phosphorylated MLC20, which potentiates uterine contractility (Amano et al., 2000) (see Figure ).

It has been demonstrated that FP and TP receptors are expressed and show physiological effects in the mouse uterus and ovary (Namba et al., 1992, Sugimoto et al., 1994). 5-HT receptors are expressed in the myometrium of non-pregnant mice and involved in the process of uterine contraction (Xiu-Kun et al., 2011).

The human uterus shows different myogenicity during the menstrual cycle, pregnancy and labour (Hutchinson, 2005). In order to examine whether the mouse is an applicable model for human uterine activity, spontaneous myometrial contractility of mouse uterus taken during dioestrus was measured and compared, as this is the most representative phase of the oestrous cycle, being the longest stage which lasts for more than two days (Byers et al., 2012, Faqi, 2012). The dioestrous stage in human is equivalent to late luteal phase in human, which is charactewrized by high levels of P4 (Walmer et al., 1992, Mihm et al., 2011).

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In addition, to investigate the effect of ripasudil, a ROCK inhibitor (Garnock-Jones, 2014) on spontaneous uterine activity and also on the contraction induced by U46619,

5-HT, and PGF2α, myometrial contractility of uterine tissue taken from non-pregnant mice was recorded and compared.

3.2 Spontaneous activity during dioestrous stage

Mouse uterine tissue samples showed spontaneous contractility. Both upper and lower segments of uterine horn taken from non-pregnant C57 WT mice displayed variable activities that lasted for about 5 h as shown in Figure 3.2. These traces demonstrate the duration of and variability in spontaneous activity of two myometrial strips taken from (A) the upper and (B) the lower segments of the uterine horn of a non-pregnant C57 WT mouse in dioestrus. In both segments, the myometrium was highly active during the first hour and then started to decline gradually until most of its activity was lost after the fourth hour.

Regional variation in uterine contraction was obvious as seen in Figure 3.1, where spontaneous activity was significantly higher in the upper segment of the uterine horn than the lower segment during the dioestrous stage (p0.05) due to greater frequency of contraction as shown in Figure 3.2.

Figure 3.1. Regional variation in spontaneous activity. Comparison between spontaneous activity measured as area under the curve (AUC) (g.s.), in isolated upper (n=33) and lower (n=33) uterine segments from non-pregnant C57 WT mice in dioestrus. Data are not normalised and are measured as AUC for a 10 minute period after equilibrium. Data are expressed as mean ± SEM, and were analysed using unpaired Student t- test, (*p0.05). 140

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3.3 Effect of PGF2α on spontaneous myometrial contraction in non- pregnant C57 WT mice

-6 Addition of PGF2α at 10 M to the bath increased the myometrial contractility of both upper and lower segments of the uterine horn as shown in Figure 3.3. PGF2α induced a higher mean level of activity in the lower segment than the upper segment; however, this increase in activity was not statistically significant (Figure 3.4).

-6 Figure 3.4. The response of different uterine regions to PGF2α (10 M). -6 Comparing the effect of PGF2α (10 M) on myometrial contractility between the upper (n=23) and lower (n=23) segments of uterine horn in non-pregnant C57 WT mice in dioestrus. Responses were measured over a 10 minute period (AUC) and expressed as a percentage of 10 minutes (AUC) initial spontaneous activity. Data are expressed as mean ± SEM, and were analysed using unpaired Student t-test, (ns) not significant.

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3.4 Effect of 5-HT on spontaneous myometrial contraction in non- pregnant C57 WT mice

Uterine tissue samples isolated from the upper or lower horn displayed an increased contractile response with the addition of 5-HT (10-6M), as the frequency of contraction was highly increased in the lower segment compared with the upper segment as shown in Figure 3.5. This difference in tissue response to 5-HT was statistically significant (p0.05). Figure 3.6 shows the effect of 5-HT on spontaneous activity of myometrium from both segments.

Figure 3.6. The response of different uterine regions to 5-HT (10-6M). Comparing the effect of 5-HT (10-6M) on myometrial contractility between the upper (n=8) and lower (n=8) segments of uterine horn in non-pregnant C57 WT mice in dioestrus. Responses were measured over a 10 minute period (AUC) and expressed as a percentage of (AUC) initial spontaneous activity. Data are expressed as mean ± SEM, and were analysed using unpaired Student t-test, *p<0.05.

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3.5 Effect of ripasudil on spontaneous myometrial contractions in non-pregnant C57 WT mice

The effect of ripasudil concentrations (10-9M to 10-5M) on myometrial contractility was measured in the upper and lower segments of the uterine horn. In both segments, ripasudil at a concentration of 10-9M and 10-8M did not change spontaneous myometrial contraction when compared to time-matched vehicle controls. At higher concentrations (10-6M and 10-5M), it significantly inhibited spontaneous activity (p0.01 and p0.001, respectively for the upper segment and p0.05 and p0.01, respectively for the lower segment) as shown in Figures 3.7 and 3.9. These inhibitory effects were concentration related. Representative traces of responses of upper and lower segments of uterine tissue to ripasudil at different concentrations are shown in Figures 3.8 and 3.10. Significant variations were found among samples exposed to different concentrations of ripasudil in both, the upper and lower segments of the uterine horn where spontaneous contractility decreased with increasing ripasudil concentration as seen in Figure 3.11.

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Figure 3.7. Effect of ripasudil (10-9M - 10-5M) on spontaneous myometrial contractility of the upper segment of the uterine horn. Effect of ripasudil (10-9M - 10-5M) on spontaneous myometrial contractility of the upper segment of uterine horn in non-pregnant C57 WT mice in dioestrus. Ripasudil or vehicle was added after 30 min of equilibrium in organ baths. Responses were measured over a 10 minute period (AUC) and expressed as a percentage of initial spontaneous activity. Data are expressed as mean ± SEM, n=4, and were analysed using one-way ANOVA with Bonferroni’s adjustment; **p<0.01; ***p<0.001, significantly different from the control.

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Figure 3.9. Effect of ripasudil (10-9M - 10-5M) on spontaneous myometrial contractility of the lower segment of the uterine horn. Effect of ripasudil (10-9M - 10-5M) on spontaneous myometrial contractility of the lower segment of uterine horn in non-pregnant C57 WT mice in dioestrus. Ripasudil or vehicle was added after 30 min of equilibrium in organ baths. Responses were measured over a 10 minute period (AUC) and expressed as a percentage of initial spontaneous activity. Data are expressed as mean ± SEM, n=4, and were analysed using one-way ANOVA with Bonferroni’s adjustment; *p<0.05; **p<0.01, significantly different from the control.

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Figure 3.11. A summative diagram comparing the effect of ripasudil (10-9M - 10-5M) on spontaneous myometrial contractions of the upper (n=4) and lower (n=4) segments of the uterine horn. Comparing the effect of ripasudil (10-9M - 10-5M) on spontaneous myometrial contractility of the upper and lower segments of uterine horn in non-pregnant C57 WT mice in dioestrus. Ripasudil or vehicle was added after 30 min equilibrium in organ baths. Responses were measured over a 10 minute period (AUC) and expressed as a percentage of initial spontaneous activity. Data are expressed as mean ± SEM, and were analysed using two-way ANOVA with Bonferroni’s adjustment; *p<0.05, **p<0.01; ***p<0.001, significantly different from the corresponding segmemt of a different ripasudil concentration.

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3.6 Effect of ripasudil on U46619-, PGF2α- and 5-HT-induced myometrial contraction in non-pregnant C57 WT mice

The effect of ripasudil (10-9M to 10-5M) on myometrial activity induced by different -6 uterine stimulants (U46619, PGF2α, and 5-HT), all used at a concentration of 10 M was also investigated in the upper and lower segments of the uterine horn.

Ripasudil at 10-9M and 10-8M was unable to inhibit U46619-induced myometrial contractions when compared to U46619 alone. At 10-6M and 10-5M, ripasudil significantly inhibited contraction induced by U46619 in the upper (p0.05 and p0.001 respectively as shown in Figure 3.12) and lower (p0.05 and p0.01 respectively as shown in Figure 3.14) segments of uterine horn. Figure 3.13 and 3.15 show traces of upper and lower segment uterine tissue responding to ripasudil at different concentrations before stimulating with U46619.

Figure 3.12. Effect of ripasudil (10-9M - 10-5M) on U46619-induced myometrial contractility in upper segment uterine horn. Effect of ripasudil (10-9M - 10-5M) on U46619 (10-6M)-induced contractions in upper segment uterine horn of non-pregnant C57 WT mice in dioestrus. Tissue was pre-incubated for 12 min with vehicle or ripasudil before addition of U46619. Responses were measured over a 10 minute period (area under the curve) and expressed as a percentage of initial spontaneous activity. Data are expressed as mean ± SEM, n=4, and were analysed using one-way ANOVA with Bonferroni’s adjustment; *p<0.05; ***p<0.001, significantly different from U46619 alone.

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Figure 3.14. Effect of ripasudil (10-9M - 10-5M) on U46619-induced myometrial contractility in lower segment uterine horn.

Effect of ripasudil (10-9M - 10-5M) on U46619 (10-6M)-induced contractions in lower segment of uterine horn of non-pregnant C57 WT mice in dioestrus. Tissue was pre-incubated for 12 min with vehicle or ripasudil before addition of U46619. Responses were measured over a 10 minute period (AUC) and expressed as a percentage of initial spontaneous activity. Data are expressed as mean ± SEM, n=4, and were analysed using one-way ANOVA with Bonferroni’s adjustment; *p<0.05; **p<0.01, significantly different from U46619 alone.

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The lower concentration of ripasudil (10-8M) did not inhibit myometrial contractions induced by 5-HT at 10-6M in both, the upper and lower uterine segments compared to 5- HT alone. Similar to the U46619 experiment, ripasudil at 10-6M and 10-5M had a significant inhibitory effect on the myometrial contractions induced by 5-HT in the upper segment (p0.05) as the reduction of myometrial contractility was by 32.2% and 35.3% by ripasudil at 10-6M and 10-5M, respectively as shown in Figure 3.16. Traces of this ripasudil effect on 5-HT-induced contraction can be observed in Figure 3.17.

Although ripasudil at a concentration range of 10-8M - 10-5M caused some inhibition in 5-HT-induced myometrial contractility in the lower segment (Figure 3.18), this effect did not reach significance. Figure 3.19 shows the representative traces of contraction of the lower uterine segment, which is inhibited by ripasudil at different concentrations and induced by 5-HT at 10-6M.

Figure 3.16. Effect of ripasudil (10-8M - 10-5M) on 5-HT-induced myometrial contractility in upper segment uterine horn. Effect of ripasudil (10-8M - 10-5M) on 5-HT (10-6M)-induced contractions in upper segment uterine horn of non-pregnant C57 WT mice in dioestrus. 5-HT was added after 12 min pre- incubation with vehicle or ripasudil. Responses were measured over a 10 minute period (AUC) and expressed as a percentage of 10 minute initial spontaneous activity. Data are expressed as mean ± SEM, n=4, and were analysed using one-way ANOVA with Bonferroni’s adjustment; *p<0.05, significantly different from 5-HT alone.

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Figure 3.18. Effect of ripasudil (10-8M - 10-5M) on 5-HT-induced myometrial contractility in lower segment uterine horn. Effect of ripasudil (10-8M - 10-5M) on 5-HT (10-6M)-induced contractions in lower segment uterine horn of non-pregnant C57 WT mice in dioestrus. 5-HT was added after 12 min pre- incubation with vehicle or ripasudil. Responses were measured over a 10 minute period (AUC) and expressed as a percentage of 10 minutes initial spontaneous activity. Data are expressed as mean ± SEM, n=4, and were analysed using one-way ANOVA with Bonferroni’s adjustment; (ns) not significant.

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The effect of ripasudil on myometrial contraction stimulated by PGF2α shows that ripasudil displayed similar patterns to those seen with U46619 and 5-HT. Despite their -6 capability to reduce the uterine activity induced by PGF2α at 10 M, concentrations of 10-8M and 10-6M of ripasudil did not significantly reduce uterine activity in either segment of the uterine horn. However, the only concentration of ripasudil that -5 significantly inhibited PGF2α-induced myometrial contraction was the 10 M in the upper and lower segments of the uterus (p0.01) and this can be seen in Figures 3.20 and 3.22. Representative traces of contraction are shown in Figure 3.21 (upper segment) and Figure 3.23 (lower segment).

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-8 -5 Figure 3.20. Effect of ripasudil (10 M - 10 M) on PGF2α-induced myometrial contractility in upper segment uterine horn. -8 -5 -6 Effect of ripasudil (10 M - 10 M) on PGF2α (10 M)-induced contractions in upper segment uterine horn of non-pregnant C57 WT mice in dioestrus. PGF2α was added after 12 min pre- incubation with vehicle or ripasudil. Responses were measured over a 10 minute period (AUC) and expressed as a percentage of 10 minutes initial spontaneous activity. Data are expressed as mean ± SEM, n=5, and were analysed using one-way ANOVA with Bonferroni’s adjustment; **p<0.01, significantly different from PGF2α alone.

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-8 -5 Figure 3.22. Effect of ripasudil (10 M - 10 M) on PGF2α-induced myometrial contractility in lower segment uterine horn. -8 -5 -6 Effect of ripasudil (10 M - 10 M) on PGF2α (10 M)-induced contractions in lower segment uterine horn of non-pregnant C57 WT mice in dioestrus. PGF2α was added after 12 min of adding ripasudil or vehicle. Responses were measured over a 10 minute period (AUC) and expressed as a percentage of 10 minutes initial spontaneous activity. Data are expressed as mean ± SEM, n=5, and were analysed using one-way ANOVA with Bonferroni’s adjustment; **p<0.01, significantly different from PGF2α alone.

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3.7 The effect of cumulative concentrations of ripasudil on spontaneous myometrial contractility in non-pregnant C57 WT mice

In upper uterine segment samples taken from non-pregnant C57 WT mouse uterus in dioestrus, ripasudil (10-9M - 10-5M) evoked an inhibitory effect on myogenicity via reducing the myometrial contraction (as AUC relative to initial spontaneous activity) compared to the control (vehicle). This inhibition was significant at higher ripasudil concentrations (10-6M and 10-5M) as shown in Figure 3.24. The effect of cumulative concentrations of ripasudil and control traces can be seen in Figure 3.25.

Figure 3.24. Effect of cumulative concentrations of ripasudil on myometrial spontaneous activity in upper segment uterine horn. Concentration-effect curves for ripasudil (10-9M - 10-5M) compared with the control (vehicle) in isolated myometrial strips from the upper segment of the uterine horn in non-pregnant C57 WT mouse in dioestrus. Responses were measured over a 10 minute period (AUC) and expressed as a percentage of 10 minutes initial spontaneous activity. Data are expressed as mean ± SEM, n=6, and were analysed using two-way ANOVA with Bonferroni’s adjustment; ***p<0.001, ****p<0.0001, significantly different from the control.

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Similarly, the effect of ripasudil (10-9M - 10-5M) on the lower segment uterus followed the same pattern as the upper segment and the concentrations that evoked a significant inhibition were 10-6M and 10-5M compared to the control (vehicle; p<0.0001) as shown in Figure 3.26, where the frequency of contraction became slower. Ripasudil and vehicle effects on contraction can be observed in Figure 3.27.

Figure 3.26. Effect of cumulative concentrations of ripasudil on the myometrial spontaneous activity of lower segment uterine horn. Concentration-effect curves for ripasudil (10-9M - 10-5M) compared with the control (vehicle) in isolated myometrial strips from the lower segment uterine horn of non-pregnant C57 WT mouse in dioestrus. Responses were measured over a 10 minute period (AUC) and expressed as a percentage of 10 minutes initial spontaneous activity. Data are expressed as mean ± SEM, n=6, and were analysed using two-way ANOVA with Bonferroni's adjustment; ****p<0.0001, significantly different from time-matched vehicle control.

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3.8 The effect of ripasudil on myometrial contractility induced by

cumulative concentrations of U46619, PGF2α or 5-HT

Figure 3.28 shows concentration-effect curves for U46619 in the presence and absence of the ROCK inhibitor, ripasudil at 10-6M (as explained in the methods chapter) on the upper segment uterine horn of non-pregnant mice. U46619 elicited a concentration- dependent excitatory response over the concentration range of 10-9M - 10-5M. Ripasudil attenuated this response at 10-6M and 10-5M. Figure 3.29 shows representative traces of U46619-induced contraction in the presence and absence of ripasudil.

Figure 3.28. Effect of cumulative concentrations of U46619 on the spontaneous activity of upper segment uterine horn in the presence and absence of ripasudil (10-6M). Concentration-effect curves for U46619 (10-9M - 10-5M) in the presence and absence of ripasudil (10-6M) in isolated myometrial strips from upper segment uterine horn of non-pregnant C57 WT mouse in dioestrus. Tissues were set up for immersion. Responses were measured over a 10 minute period (AUC) and expressed as a percentage of 10 minutes initial spontaneous activity. Data are expressed as mean ± SEM, n=5-6, and were analysed using two-way ANOVA with Bonferroni's adjustment; *p<0.05; **p<0.01, for U46619 compared to U46619 incubated with ripasudil.

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Figure 3.29. Representative traces showing concentration-effect curves for U46619 (10-9M - 10-5M) in isolated myometrial strips from the upper segment uterine horn of non-pregnant C57 WT mouse in dioestrus in the presence and absence of ripasudil (10-6M).

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Figure 3.30 shows concentration-effect curves for U46619 in the presence and absence of the ROCK inhibitor, ripasudil at 10-6M on lower segment uterine horn of non- pregnant mice. U46619 also evoked a concentration-dependent excitatory effect (10-9M - 10-5M). Similar to the upper segment, ripasudil significantly inhibited this response at U46619 concentrations of 10-6M and 10-5M (p<0.05). Representative traces of contractions in both cases are shown in Figure 3.31.

Figure 3.30. Effect of cumulative concentrations of U46619 on the myometrial spontaneous activity of lower segment uterine horn in the presence and absence of ripasudil (10-6M). Concentration-effect curves for U46619 (10-9M - 10-5M) in the presence and absence of ripasudil (10-6M) in isolated myometrial strips from the lower segment uterine horn of non- pregnant C57 WT mice in dioestrus. Tissues were set up for immersion. Responses were measured over a 10 minute period (AUC) and expressed as a percentage of 10 minutes initial spontaneous activity. Data are expressed as mean ± SEM, n=6, and were analysed using two- way ANOVA with Bonferroni's adjustment; *p<0.05, for U46619 compared to U46619 incubated with ripasudil.

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Figure 3.31. Representative traces showing concentration-effect curves for U46619 (10-9M - 10-5M) in immersed isolated myometrial strips from the lower segment uterine horn of a non-pregnant C57 WT mouse in dioestrus in the presence and absence of ripasudil.

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To investigate the effect of ripasudil on 5-HT-induced myometrial contractility in non- pregnant mice, strips of myometrium from the upper uterine segment were incubated with 5-HT in the presence and absence of the ROCK inhibitor ripasudil. 5-HT elicited a concentration-dependent excitatory response over a concentration range of 10-9M - 10- 5M with a reduction in myogenic activity at 10-5M compared to 10-6M but this reduction was not statistically significant. Interestingly, ripasudil at 10-6M significantly inhibited 5-HT-induced myometrial contractions at three agonist concentrations, 10-7M, 10-6M and 10-5M.

Figure 3.32 shows concentration-effect curves for 5-HT in the presence and absence of ripasudil on the upper segment uterine horn in non-pregnant mice. Representative traces of 5-HT-induced contraction in the presence and absence of ripasudil are shown in Figure 3.33.

Figure 3.32 Effect of cumulative concentrations of 5-HT on the myometrial spontaneous activity of the upper segment uterine horn in the presence and absence of ripasudil (10-6M). Concentration-effect curves for 5-HT (10-9M - 10-5M) in the presence and absence of ripasudil (10-6M) in isolated myometrial strips from the upper segment uterine horn of non-pregnant C57 WT mouse in dioestrus. Tissues were set up for immersion. Responses were measured over a 10 minute period (AUC) and expressed as a percentage of 10 minutes initial spontaneous activity. Data are expressed as mean ± SEM, n=5, and were analysed using two-way ANOVA with Bonferroni’s adjustment; *p<0.05; **p<0.01, for 5-HT compared to 5-HT incubated with ripasudil.

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Correspondingly, the effect of ripasudil on myometrial contraction elicited by 5-HT was tested on the lower uterine segment.

5-HT stimulated myometrial activity over a concentration range of 10-9M - 10-5M with the same reduction in myometrial activity at 10-5M which was observed with the upper uterine segment. Ripasudil at 10-6M significantly inhibited 5-HT-induced myometrial contractions at 10-7M and 10-6M and its inhibitory effect almost approached significance at 10-5M (p=0.66) when compared to the 5-HT alone group as shown in Figure 3.34.

Figure 3.35 shows a concentration-effect in representative traces for 5-HT in the presence and absence of ripasudil on the lower segment uterine horn of non-pregnant mice.

Figure 3.34. Effect of cumulative concentrations of 5-HT on the myometrial spontaneous activity of the lower segment uterine horn in the presence and absence of ripasudil (10-6M). Concentration-effect curves for 5-HT (10-9M - 10-5M) in the presence and absence of ripasudil (10-6M) in isolated myometrial strips from the lower segment uterine horn of non-pregnant C57 WT mouse in dioestrus. Tissues were set up for immersion. Responses were measured over a 10 minute period (AUC) and expressed as a percentage of 10 minutes initial spontaneous activity. Data are expressed as mean ± SEM, n=5, and were analysed using two-way ANOVA with Bonferroni's adjustment; (ns) not significant); *p<0.05, for 5-HT compared to 5-HT incubated with ripasudil.

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Similar to U46619 and 5-HT, PGF2α elicited myometrial excitation over a concentration range of 10-9M - 10-5M in upper segment uterine horn. Ripasudil at 10-6M significantly -7 -6 -5 inhibited PGF2α-induced myometrial contraction at 10 M, 10 M, and 10 M of PGF2α concentrations, as shown in Figure 3.36.

Representative traces for PGF2α-induced contractile response in the presence and absence of ripasudil on the upper segment uterine horn in non-pregnant mice are shown in Figure 3.37.

Figure 3.36. Effect of cumulative concentrations of PGF2α on myometrial spontaneous activity of the upper segment uterine horn in the presence and absence of ripasudil (10-6M). -9 -5 Concentration-effect curves for PGF2α (10 M - 10 M) in the presence and absence of ripasudil (10-6M) in isolated myometrial strips from the upper segment of the uterine horn in non- pregnant C57 WT mouse in dioestrus. Tissues were set up for immersion. Responses were measured over a 10 minute period (AUC) and expressed as a percentage of 10 minutes initial spontaneous activity. Data are expressed as mean ± SEM, n=6, and were analysed using two- way ANOVA with Bonferroni's adjustment; *p<0.05, for PGF2α, compared to PGF2α incubated with ripasudil.

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-9 -5 Figure 3.37. Representative traces showing concentration-effect curves for PGF2α (10 M - 10 M) in isolated myometrial strips from the upper segment uterine horn in non-pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil.

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In the same way, the effect of ripasudil on myometrial contraction induced by PGF2α was investigated on the lower segment uterine horn.

-9 -5 Figure 3.38 shows that PGF2α over a concentration range of 10 M - 10 M stimulated -6 myometrial contractions. The PGF2α response was significantly inhibited by the 10 M -6 -5 concentration of ripasudil at two PGF2α concentrations, 10 M, and 10 M.

Figure 3.39 shows concentration-effect representative traces for PGF2α in the presence and absence of ripasudil on the lower segment uterine horn in non-pregnant mice.

Figure 3.38. Effect of cumulative concentrations of PGF2α on spontaneous myometrial activity of the lower segment uterine horn in the presence and absence of ripasudil (10-6M). -9 -5 Concentration-effect curves for PGF2α (10 M - 10 M) in the presence and absence of ripasudil (10-6M) in isolated myometrial strips from the lower segment uterine horn of non-pregnant C57 WT mice in dioestrus. Tissues were set up for immersion. Responses were measured over a 10 minute period (AUC) and expressed as a percentage of 10 minutes initial spontaneous activity. Data are expressed as mean ± SEM, n=5, and were analysed using two-way ANOVA with Bonferroni's adjustment; *p<0.05; **p<0.01, for PGF2α, compared to PGF2α incubated with ripasudil.

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-9 -5 Figure 3.39. Representative traces showing concentration-effect curves for PGF2α (10 M - 10 M) in isolated myometrial strips from the lower segment uterine horn of non-pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil.

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3.9 Expression of contractile proteins in myometrium after treatment

with U46619, PGF2α, and 5-HT in the presence and absence of ripasudil

The expression level and activation status of contractile proteins in uterine tissue and cells which were isolated from non-pregnant C57 WT mice during dioestrus were examined after treatment of the uterine tissue and cells with different uterine stimulants and a ROCK inhibitor. The stimulants included U46619, PGF2α and 5-HT, while ripasudil was used as a ROCK pathway inhibitor. The proteins investigated were non- phosphorylated MLC (MLC), mono-phosphorylated MLC (pMLC) and the di- phosphorylated MLC (ppMLC); these were assessed using western blotting and immunocytochemistry (ICC) techniques.

In the tissues taken at the end of cumulative treatment using immersion techniques, ripasudil at 10-9M – 10-5M caused a significant elevation in the expression of non- phosphorylated MLC protein when compared to the control (p<0.05) (Figure 3.40). The figure also demonstrates that there is a slight conversion of MLC to the mono- phosphorylated form of MLC (pMLC) in the control group which was less pronounced in the ripasudil-treated tissue.

In addition to the data from western blotting, results from ICC indicate that, although MLC was expressed in both treated myometrial cells, its expression seemed to be higher in ripasudil-treated cells than in the cells treated with the vehicle (control) (Figure 3.41).

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Figure 3.40. Effect of ripasudil on non-phosphoryated MLC expression in myometrial tissue from non-pregnant C57 WT mice in dioestrus. Effect of ripasudil on the expression of MLC in non-pregnant C57 WT mouse myometrium in dioestrus. Tissues were treated with cumulative concentrations of ripasudil (10-9M - 10-5M) or vehicle (control) using immersion technique. Panel (A) is a representative western blot image and panel (B) is a densitometric analysis. Data are normalised to α-actin and are presented as a percentage of the control. Data were analysed using unpaired Student’s t-test and are presented as the mean ± SEM of 3 independent experiments, *p< 0.05.

Figure 3.41. Effect of ripasudil on non-phosphorylated MLC expression in myometrial cells from non-pregnant C57 WT mice in dioestrus. Immunofluorescent analysis of MLC in ripasudil (at 10-6M) and vehicle-treated myometrial cells (30 sec) using an MLC2 antibody (green). Actin filaments are labelled with TRITC (red). DNA is labelled using a DAPI (blue). Magnification x20.

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In contrast to the effect of ripasudil (10-9M – 10-5M) on MLC expression in myometrial tissue and cells, the expression level of the contractile protein, pMLC observed in uterine tissue after treatment with ripasudil was significantly lower compared with the control group (Figure 3.42; p<0.001).

Similar to the tissue data, myometrial cells taken from the uterus of non-pregnant mouse and treated with ripasudil at 10-6M showed lower expression of pMLC compared to the control as shown in Figure 3.43.

Figure 3.42. Effect of ripasudil on pMLC expression in myometrial tissue from non- pregnant C57 WT mice in dioestrus. Effect of ripasudil on the expression of pMLC in non-pregnant C57 WT mouse myometrium in dioestrus. Tissues were treated with cumulative concentrations of ripasudil (10-9M - 10-5M) or vehicle (control) using immersion technique. Panel (A) is a representative western blot image and panel (B) is a densitometric analysis. Data are normalised to α-actin and are presented as a percentage of the control. Data were analysed using unpaired Student’s t-test and are presented as the mean ± SEM of 3 independent experiments, ***p< 0.001.

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Figure 3.43. Effect of ripasudil on pMLC expression in myometrial cells from non- pregnant C57 WT mice in dioestrus. Immunofluorescent analysis of pMLC in ripasudil (at 10-6M) and vehicle-treated myometrial cells (30 sec) using a pMLC antibody (green). Actin filaments are labelled with TRITC (red). DNA is labelled using a DAPI (blue). Magnification x20.

The most significant effect of ripasudil was observed on ppMLC at concentrations of at 10-9M – 10-5M, where protein expression was significantly inhibited with the addition of cumulative concentrations of ripasudil when compared with the tissue treated with the vehicle. Figure 3.44 shows the level of ppMLC inhibition by ripasudil and also the representative protein bands.

On the cellular level, although the inhibition of ppMLC by ripasudil at 10-6M was not very clear compared to that observed with pMLC, a slight reduction in ppMLC expression can be seen (Figure 3.45).

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Figure 3.44. Effect of ripasudil on ppMLC expression in myometrial tissue from non- pregnant C57 WT mice in dioestrus. Effect of ripasudil on the expression of ppMLC in non-pregnant C57 WT mouse myometrium in dioestrus. Tissues were treated with cumulative concentrations of ripasudil (10-9M - 10-5M) or vehicle (control) using immersion technique. Panel (A) is a representative western blot image and panel (B) is a densitometric analysis. Data are normalised to α-actin and are presented as a percentage of the control. Data were analysed using unpaired Student’s t-test and are presented as the mean ± SEM of 3 independent experiments, ****p< 0.0001.

Figure 3.45. Effect of ripasudil on ppMLC expression in myometrial cells from non- pregnant C57 WT mice in dioestrus. Immunofluorescent analysis of ppMLC in ripasudil (at 10-6M) and vehicle-treated myometrial cells (30 sec) using a ppMLC antibody (green). Actin filaments are labelled with TRITC (red). DNA is labelled using a DAPI (blue). Magnification x20.

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Incubation of cumulative concentrations of U46619 (10-9M – 10-5M) to isolated myometrial tissues using immersion technique evoked a slight increase in the mean expression level of MLC when compared to the control; this elevation was not statistically significant. Ripasudil at 10-6M was able to increase MLC expression in U46619-treated tissue even though this increase was not statistically significant. Although the mean level of MLC expression in the ripasudil+U46619 group was higher than that of the control group, this elevation was non-significant (Figure 3.46). The bands from the blot also showed that in both control and U46619 groups, there was some expression of pMLC, which was not observed in the ripasudil+U46619 group.

In myometrial cells taken from uterus of non-pregnant mice and treated with U46619 at 10-6M, ripasudil (10-6M) was able to increase the expression of the non-contractile protein, MLC as shown in Figure 3.47.

Figure 3.46. Effect of U46619 on MLC expression in myometrial tissue from non-pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil. Effect of U46619 on the expression of MLC in non-pregnant C57 WT mouse myometrium in dioestrus in the presence and absence of ripasudil 10-6M. Tissues were either pre-treated with ripasudil 10-6M and then with cumulative concentrations of U46619 (10-9M - 10-5M), U46619 alone (10-9M - 10-5M), or with the vehicle (control) using immersion technique. Panel (A) is a representative western blot image and panel (B) is a densitometric analysis. Data are normalised to α-actin and are presented as a percentage of the control. Data were analysed using one-way ANOVA with Bonferroni’s adjustment and are presented as the mean ± SEM of 3 independent experiments.

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Figure 3.47. Effect of U46619 on MLC expression in myometrial cells from non-pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil. Immunofluorescent analysis of MLC in myometrial cells treated with U46619 at 10-6M (30 sec) in the presence and absence of ripasudil 10-6M using a MLC antibody (green). Actin filaments are labelled with TRITC (red). DNA is labelled using a DAPI (blue). Magnification x20.

The effect of ripasudil on U46619-stimulated pMLC expression was also studied. U46619 at 10-9M – 10-5M caused some reduction in the mean expression level of pMLC when compared with the control but this inhibition was not significant. Although ripasudil at 10-6M caused an obvious inhibition (47.8%) in the expression of the contractile protein pMLC in myometrial tissue treated with U46619 this inhibition was below the significant level (p=0.09). pMLC expression in the ripasudil+U46619 group was significantly lower than that in the control group as seen in Figure 3.48.

The ICC data demonstrates that ripasudil 10-6M decreased the expression of U46619- induced pMLC expression in myometrial cells isolated from non-pregnant mice in dioestrus (Figure 3.49).

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Figure 3.48. Effect of U46619 on pMLC expression in myometrial tissue from non- pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil. Effect of U46619 on the expression of pMLC in non-pregnant C57 WT mouse myometrium in dioestrus in the presence and absence of ripasudil 10-6M. Tissues were either pre-treated with ripasudil 10-6M and then with cumulative concentrations of U46619 (10-9M - 10-5M), U46619 alone (10-9M - 10-5M), or with the vehicle (control) using immersion technique. Panel (A) is a representative western blot image and panel (B) is a densitometric analysis. Data are normalised to α-actin and are presented as a percentage of the control. Data were analysed using one-way ANOVA with Bonferroni’s adjustment and are presented as the mean ± SEM of 3 independent experiments; (ns) not significant.

Figure 3.49. Effect of U46619 on pMLC expression in myometrial cells from non-pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil (10-6M). Immunofluorescent analysis of MLC in myometrial cells treated with U46619 at 10-6M (30 sec) in the presence and absence of ripasudil 10-6M using a pMLC antibody (green). Actin filaments are labelled with TRITC (red). DNA is labelled using a DAPI (blue). Magnification x20. 181

Furthermore, the expression of the contractile protein, ppMLC was also examined in myometrial tissues and cells after treatment with cumulative concentrations (10-9M – 10-5M) or single concentration (10-6M) of U46619 in the presence and absence of ripasudil at 10-6M.

U46619 alone did not change ppMLC expression in myometrial cells. Whereas, tissues treated with U46619 in the presence of ripasudil showed a significant reduction in ppMLC expression when compared to the U46619-alone group or control. Figure 3.50 shows the degree of inhibition in ppMLC expression as well as the representative bands.

In addition, ripasudil at 10-6M decreased ppMLC expression in U46619 treated myometrial cells taken from uterus of non-pregnant mouse compared to U46619 alone (Figure 3.51).

Figure 3.50. Effect of U46619 on ppMLC expression in myometrial tissue from non- pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil. Effect of U46619 on the expression of ppMLC in non-pregnant C57 WT mouse myometrium in dioestrus in the presence and absence of ripasudil 10-6M. Tissues were either pre-treated with ripasudil 10-6M and then with cumulative concentrations of U46619 (10-9M - 10-5M), U46619 alone (10-9M - 10-5M), or with the vehicle (control) using immersion technique. Panel (A) is a representative western blot image and panel (B) is a densitometric analysis. Data are normalised to α-actin and are presented as a percentage of the control. Data were analysed using one-way ANOVA with Bonferroni’s adjustment and are presented as the mean ± SEM of 3 independent experiments; *p<0.05.

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Figure 3.51. Effect of U46619 on ppMLC expression in myometrial cells from non- pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil. Immunofluorescent analysis of ppMLC in myometrial cells treated with U46619 at 10-6M (30 sec) in the presence and absence of ripasudil 10-6M using a ppMLC antibody (green). Actin filaments are labelled with TRITC (red). DNA is labelled using a DAPI (blue). Magnification x20.

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Data from the western blotting experiments showed that 5-HT followed the same pattern as U46619 regarding its activity on the non-contractile MLC protein expression in the myometrium. Tissue exposed to 10-9M – 10-5M concentrations of 5-HT in a cumulative manner exhibited a higher mean expression level of MLC in comparison to the control. However, this increase in protein expression was not statistically significant. Ripasudil at a concentration of 10-6M significantly elevated MLC expression in myometrial tissues treated with U46619 when compared to the control. Although this protein elevation with the addition of ripasudil was not significant compared to the U46619 group, it suggests that ripasudil caused a difference in protein expression (Figure 3.52).

Figure 3.52. Effect of 5-HT on MLC expression in myometrial tissue from non-pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil. Effect of 5-HT on the expression of MLC in non-pregnant C57 WT mouse myometrium in dioestrus in the presence and absence of ripasudil 10-6M. Tissues were taken at the end of immersion work and were either pre-treated with ripasudil 10-6M and then with cumulative concentrations of 5-HT (10-9M - 10-5M), 5-HT alone (10-9M - 10-5M), or with the vehicle (control) using immersion technique. Panel (A) is a representative western blot image and panel (B) is a densitometric analysis. Data are normalised to α-actin and are presented as a percentage of the control. Data were analysed using one-way ANOVA with Bonferroni’s adjustment and are presented as the mean ± SEM of 3 independent experiments; *p0.05.

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Further analyses have been conducted to test the effect of ripasudil on the expression of the pMLC protein in 5-HT-treated myometrial tissues taken from non-pregnant mice in dioestrus.

Figure 3.53 demonstrates that 5-HT at 10-9M – 10-5M decreased the mean level of pMLC expression in the above samples when compared with the control, but this reduction was not statistically significant. Ripasudil at 10-6M induced significant reduction in the pMLC level in 5-HT-treated myometrial tissues when compared to both the 5-HT-alone (p<0.01) and control groups (p<0.001).

Figure 3.53. Effect of 5-HT on pMLC expression in myometrial tissue from non-pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil. Effect of 5-HT on the expression of pMLC in non-pregnant C57 WT mouse myometrium in dioestrus in the presence and absence of ripasudil 10-6M. Tissues were either pre-treated with ripasudil 10-6M and then with cumulative concentrations of 5-HT (10-9M - 10-5M), 5-HT alone (10-9M - 10-5M), or with the vehicle (control) using immersion technique. Panel (A) is a representative western blot image and panel (B) is a densitometric analysis. Data are normalised to α-actin and are presented as a percentage of the control. Data were analysed using one-way ANOVA with Bonferroni’s adjustment and are presented as the mean ± SEM of 3 independent experiments; **p<0.01; ***p<0.001.

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In contrast to U46619, treatment of myometrial tissues isolated from non-pregnant mouse uterus with cumulative concentrations of 5-HT (10-9M – 10-5M) inhibited the di- phosphorylation of MLC, as observed through decreasing ppMLC expression in the treated samples, compared to the control; this inhibition was statistically significant. Furthermore, ripasudil at 10-6M produced a noticeable effect on ppMLC expression in tissue treated with 5-HT. Ripasudil significantly reduced the level of ppMLC expression when compared with 5-HT-alone and control groups as shown in Figure 3.54.

Figure 3.54. Effect of 5-HT on ppMLC expression in myometrial tissue from non-pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil. Effect of 5-HT on the expression of ppMLC in non-pregnant C57 WT mouse myometrium in dioestrus in the presence and absence of ripasudil 10-6M. Tissues were either pre-treated with ripasudil 10-6M and then with cumulative concentrations of 5-HT (10-9M - 10-5M), 5-HT alone (10-9M - 10-5M), or with the vehicle (control) using immersion technique. Panel (A) is a representative western blot image and panel (B) is a densitometric analysis. Data are normalised to α-actin and are presented as a percentage of the control. Data were analysed using one-way ANOVA with Bonferroni’s adjustment and are presented as the mean ± SEM of 3 independent experiments; *p<0.05, **p<0.01.

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-9 -5 PGF2α like U46619 and 5-HT at cumulative concentrations of 10 M - 10 M appeared to increase the level of MLC in the samples compared to the control but this elevation was not statistically significant. Western blotting bands also show that there was a reasonable expression of the contractile mono-phosphorylated MLC form in both the -6 control and PGF2α-alone groups. Ripasudil at a concentration of 10 M was able to increase MLC expression in samples treated with PGF2α and this elevation in protein level was significantly different from the control group (p0.05) and was trending toward significance when compared with the PGF2α-alone group (p0.152). No conversion of MLC to pMLC was observed in the ripasudil-treated samples as seen in Figure 3.55.

Figure 3.55. Effect of PGF2α on MLC expression in myometrial tissue from non-pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil.

Effect of PGF2α on the expression of MLC in non-pregnant C57 WT mouse myometrium in dioestrus in the presence and absence of ripasudil 10-6M. Tissues were either pre-treated with -6 -9 -5 ripasudil 10 M and then with cumulative concentrations of PGF2α (10 M - 10 M), PGF2α alone (10-9M - 10-5M), or with the vehicle (control) using immersion technique. Panel (A) is a representative western blot image and panel (B) is a densitometric analysis. Data are normalised to α-actin and are presented as a percentage of the control. Data were analysed using one-way ANOVA with Bonferroni’s adjustment and are presented as the mean ± SEM of 3 independent experiments; *p0.05.

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To compare the effect of ripasudil on PGF2α-regulated pMLC formation in uterine tissues from non-pregnant mice, samples were treated with cumulative concentrations of -9 -5 -6 PGF2α (10 M – 10 M) in the presence and absence of ripasudil at 10 M and protein expression was examined via western blotting.

PGF2α caused a significant reduction in expression of pMLC when compared to the control. Ripasudil at 10-6M produced an effect comparable to the previous experiments with U46619 and 5-HT as it inhibited pMLC expression in the tissue treated with PGF2α when compared to PGF2α-alone group, as well as the control. This inhibition was significantly different from the control group and also it was very close to the significant level when compared to the PGF2α-alone group (p=0.053) as shown in Figure 3.56.

Figure 3.56. Effect of PGF2α on pMLC expression in myometrial tissue from non-pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil.

Effect of PGF2α on the expression of pMLC in non-pregnant C57 WT mouse myometrium in dioestrus in the presence and absence of ripasudil 10-6M. Tissues were either pre-treated with -6 -9 -5 ripasudil 10 M and then with cumulative concentrations of PGF2α (10 M - 10 M), 5-HT alone (10-9M - 10-5M), or with the vehicle (control) using immersion technique. Panel (A) is a representative western blot image and panel (B) is a densitometric analysis. Data are normalised to α-actin and are presented as a percentage of the control. Data were analysed using one-way ANOVA with Bonferroni’s adjustment and are presented as the mean ± SEM of 3 independent experiments; (ns) non-significant, **p<0.01, ***p<0.001.

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As the final part of investigating the effect of ripasudil and uterine stimulants on contractile protein expression in myometrial strips isolated from non-pregnant mice in -6 dioestrus, the effect of ripasudil (10 M) on the expression of ppMLC in PGF2α-treated -9 -5 samples at PGF2α concentrations of 10 M – 10 M was examined.

As with the other investigated uterine stimulant 5-HT, PGF2α reduced the mean level of ppMLC expression in the treated samples compared to the control, but this reduction was not significant. In addition, ripasudil at 10-6M significantly altered ppMLC expression in tissues treated with PGF2α. Ripasudil significantly decreased the expression of ppMLC when compared with both, PGF2α-alone and control groups as seen in Figure 3.57.

Figure 3.57. Effect of PGF2α on ppMLC expression in myometrial tissue from non- pregnant C57 WT mice in dioestrus in the presence and absence of ripasudil.

Effect of PGF2α on the expression of ppMLC in non-pregnant C57 WT mouse myometrium in dioestrus in the presence and absence of ripasudil 10-6M. Tissues were either pre-treated with -6 -9 -5 ripasudil 10 M and then with cumulative concentrations of PGF2α (10 M - 10 M), PGF2α alone (10-9M - 10-5M), or with the vehicle (control) using immersion technique. Panel (A) is a representative western blot image and panel (B) is a densitometric analysis. Data are normalised to α-actin and are presented as a percentage of the control. Data were analysed using one-way ANOVA with Bonferroni’s adjustment and are presented as the mean ± SEM of 3 independent experiments; *p<0.05, **p<0.01.

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Comparing the effect of cumulative concentrations of U46619, PGF2α, 5-HT, which are used to stimulate the myometrium taken from non-pregnant C57 WT mouse uteri in dioestrus on the expression of MLC, pMLC and ppMLC showed that the expression of MLC was higher in the group treated with 5-HT followed by the samples exposed to

PGF2α and U46619, respectively. The lowest expression of pMLC was in the PGF2α- treated animals, whereas the higher expression of this protein was in the samples exposed to U46619. The most powerful effect on the expression of ppMLC was in the myometrial strips treated with U46619 followed by the tissues treated with PGF2α and 5-HT, respectively as seen in Table 3.1.

Table 3.1. Effect of U46619, PGF2α, 5-HT and ripasudil on MLC family expression in myometrial tissue from non-pregnant C57 WT mice in dioestrus.

Effect of cumulative concentration of U46619, PGF2α, 5-HT and ripasudil on the protein expression of MLC, pMLC and ppMLC in non-pregnant C57 WT mouse myometrium in dioestus. Tissues were treated either with cumulative concentrations of U46619 (10-9M - 10-5M), -9 -5 -9 -5 PGF2α (10 M - 10 M) or 5-HT (10 M - 10 M) using immersion technique and protein expression was investigated by western blotting. Band densities were measured and then normalised to α-actin and are presented as a percentage of the control (vehicle, 0.9% w/v normal saline). Data are presented as the mean ± SEM of 3 independent experiments.

U46619 PGF2α 5-HT Ripasudil

MLC 123.5 ± 30.3 127.1 ± 30.4 135.4 ± 23.2 151.7 ± 17.7

pMLC 80.3 ± 13.9 51.1 ± 10.2 69.5 ± 11.0 38.9 ± 5.9

ppMLC 96.3 ± 21.1 58.6 ± 14.5 52.8 ± 14.1 11.4 ± 5.4

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3.10 Discussion

Data from this study indicate that the myometrium isolated from non-pregnant mouse uterus during the dioestrous stage has the capability to contact spontaneously in vitro. These spontaneous contractions arise from the repeated and increasing depolarisations of resting membrane potential which occur as a result of changes in ion distribution across the membrane (Parkington and Coleman, 1990). Tissue strips were taken from the uterus during dioestrus as this stage is the most representative stage of the oestrous cycle as it is the longest stage which lasts for more than 2 days (Byers et al., 2012, Faqi, 2012). Tissues taken from the uterine horn were separated into two segments: upper (fundus) and lower (cervical end). In both segments, uterine strips exhibited spontaneous activity during the first hour of being set-up in the organ baths. This spontaneous activity started to decline gradually over the next hours until it lost most of its activity by the fifth hour of incubation in organ baths. The results show that variations in myogenicity are correlated with the site of excision.

It has been recognised that Ca2+ movement is involved with each spontaneous contraction in myometrial tissues. The myometrial spontaneous activity is dependent on external Ca2+ sources and Ca2+ enters the cell through voltage-gated L-type Ca2+ channels. This activity is not significantly associated with Ca2+ release from sarcoplasmic reticulum, while agonist-stimulated activity is highly dependent on intracellular stores of calcium. Ca2+ removal from the physiological Krebs’ solution which is in contact with the myometrial strips causes inhibition of the spontaneous activity, which indicates the dependence of spontaneous contraction on the Ca2+ entry from external sources (Matthew et al., 2004). Elevation in Ca2+ levels provokes stimulation of MLCK by Ca2+-calmodulin complex which leads to MLC20 phosphorylation and an increase in actin-activated myosin ATPase activity producing active contraction (Word, 1995). ROCKI and ROCKII, the two isoforms of rho- associated protein kinase, can further increase the contraction force through diminishing pMLC dephosphorylation (Amano et al., 2000). Furthermore, MLC20 can be phosphorylated at two sites, threonine 18 and serine 19 to form the di-phosphorylated myosin light chain (ppMLC; Figure 1.12). This ppMLC causes more potent activation of myosin ATPase (Ikebe et al., 1985, Ikebe et al., 1988). The process of ppMLC

191 formation is regulated by the ROCK-mediated phosphorylation of MLCK-derived pMLC (Aguilar et al., 2012). A reduction in Ca2+ and the dephosphorylation of MLC will lead to smooth muscle relaxation (Word, 1995, Wray, 1993).

In this study, the long duration of myometrial spontaneous activity in the organ bath was inconsistent with previous studies, where myometrial strips isolated from non- pregnant mouse uterus lost most of their spontaneous activity within two hours of being set-up (Hutchinson, 2005). This may be due to the tissue sampling as longitudinal strips were used in this study whilst others cut the tissue transversely. This also indicates that longitudinal and circular myometrial muscles can exhibit different contractile patterns. In addition, some species have shown region-related differences in prostaglandin receptor populations as observed with the TP receptor population in longitudinal muscle with isolated porcine uterine tissue (Cao et al., 2004).

The frequency of spontaneous contractions was greator in the upper segment uterine horn compared to the lower segment as shown in Figure 3.2. This regional variation in myogenicity, which is seen in the uterine horn from non-pregnant mice may be attribuatable to the release of ovarian steroids (Griffiths, 2007). The AUC during spontaneous contraction was also greater in the upper segment of the uterine horn when compared to the lower segment. The upper segment is closer to the ovaries, the site of hormonal production and the tissue sample was taken at the end of dioestrus where oestrogen levels start to increase (Walmer et al., 1992, Levine, 2015) and activate uterine ERα receptors in the upper segment of the uterine horn. However, these findings are inconsistent with previous researches on the same species (Griffiths, 2007). These contradictory results may be due to the time of sample collection during the oestrous stage and/or they have used different mouse strains.

The hormonal variations during the oestrous cycle in the non-pregnant state can modulate uterine function. Stimulation of both the upper and lower segments of uterine horn observed by the addition of PGF2α demonstrates the expression of functional FP receptors in non-pregnant mouse uterus (Hutchinson et al., 2003). In addition to stimulating FP receptors, PGF2α has the ability to activate other receptors such as the TP receptors and this may further increase the force of uterine contraction (Griffiths, 2007).

Studies have summarized the greatest affinity of PGF2α toward FP receptors, with rank order of affinity being FP>EP1=EP3=TP (Okada et al., 2000). Myometrial tissues were

192 responsive to PGF2α in dioestrus and along the length of the uterine horn. The trend towards regional variation in response to PGF2α suggests a role for the hormonal milieu in regulating and localizing the FP receptor population. Other studies have demonstrated a greater response to PGF2α in the upper segment than the lower segment of the uterine horn isolated from mice (Griffiths, 2007) and rats (Oropeza et al., 2002). Our samples might be taken during early dioestrous where progesterone concentrations are high so activation of the highly expressed progesterone (PR)-B receptors in the upper segment would lead to a reduction in contractile tone.

The myometrium from non-pregnant mice was also responsive to 5-HT, which indicates the presence of functional 5-HT receptors in these species in the non-pregnant state (Xiu-Kun et al., 2011). The subtypes and distribution of 5-HT receptors are discussed in details in the introduction chapter. Regional variation in response to uterine stimulants was also observed with 5-HT where the lower uterine segment was more responsive to 5-HT than the upper segment. Multiple factors may play a role in this difference between the two uterine segments, these include number of receptors, different G- protein subunits expressed in the tissue or the variance in coupling mechanisms for individual 5-HT receptor subtypes (Raymond et al., 2001). This topographical distribution of 5-HT receptors in the non-pregnant uterus has also been suggested in the rat uterus with 5-HT binding decreasing from the ovarian region to the cervical region which indicates that in the mouse uterus the population of 5-HT receptors might be higher in the lower segment compared to the upper segment of the uterine horn. These results are consistent with previous studies in rat uterus and greater contractile force activated by 5-HT in the lower segment suggest that 5-HT might be involved in the regulation of movement of spermatozoa from the cervical region toward the oviduct (Oropeza et al., 2002).

Ripasudil, the Rho-associated protein kinase (ROCK) inhibitor was firstly developed as an ophthalmic solution for the treatment of glaucoma and ocular hypertension in Japan (Garnock-Jones, 2014). It has exhibited an important role in the regulation of smooth muscle contraction through diminishing ROCK activity and potentiating contractile force (Isobe et al., 2014). Inhibition of the ROCK enzyme has two correlated physiological actions in the modulation of smooth muscle contraction. Firstly, it inhibits phosphorylation of the myosin light chain phosphatase (MLCP) enzyme and so potentiates transformation of pMLC to the non-contractile MLC form.

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Secondly, the inactivation of ROCK will abolish the conversion of pMLC to the di- phosphorylated form ppMLC. Altogether, this will transform the contractile state of the muscle to relaxation (Aguilar et al., 2012).

In this study, the concentration range of ripasudil was chosen according to previous studies in human, where its IC50 values were 51 and 19 nM for ROCKI and ROCKII respectively (Isobe et al., 2014). Ripasudil added incrementally was able to inhibit myometrial spontaneous activity in non-pregnant mice in the upper and lower segments of the uterine horn. It also decreased the amplitude of contraction in both segments at high concentrations. Furthermore, cumulative concentrations of ripasudil were able to inhibit myometrial contractions in the upper and lower segments and this inhibition was statistically significant at high ripasudil concentrations. Ripasudil was able to inhibit the frequency and amplitude of myometrial contractility in both uterine segments. This demonstrates that the ROCK pathway has an essential role in modulating myometrial contraction in the non-pregnant state (Arthur et al., 2007, Chen et al., 2019). This was the first study to investigate the effect of ripasudil on myometrial spontaneous activity and agonist-induced uterine contraction in non- pregnant mice.

Thromboxane A2 (TXA2) is the natural prostanoid acting on TP receptor (Coleman et al., 1994). TXA2 has a very short half-life of 30 sec (Verstraete, 1983) and is an unstable compound which is rapidly hydrolysed to the more stable and inactive metabolite, Thromboxane B2 (TXB2) (Wild, 2005). Due to the difficulty of using this short half-life prostanoid, the more stable analogue of thromboxane U46619 was used in this study in order to examine the functional TP distribution in the uterus of non- pregnant mice. U46619 is highly associated with ROCK pathway activation through stimulating TP receptors in the myometrium of non-pregnant human and mouse uteri (Kennedy et al., 1994, Senchyna and Crankshaw, 1999). U46619 stimulated myometrial contraction in non-pregnant mouse uterus and this confirms the presence of functional TP receptors.

One of the aims of this work was to examine the effect of multiple concentrations of ripasudil on myometrial contraction induced by different uterine stimulants. The data presented have demonstrated the ability of ripasudil to diminish U46619-induced myometrial contractions in non-pregnant mice with increasing concentrations of

194 ripasudil. The activity of ripasudil against U46619-induced contraction has followed the same pattern in both the upper and lower segments of the uterine horn. In addition to that and in both segments, uterotonic activity was stimulated by cumulative concentrations of U46619. These findings are consistent with previous research in the non-pregnant mouse uterus (Griffiths, 2007, Hutchinson, 2005). Ripasudil attenuated U46619-induced contractions as well as significantly decreasing the maximum myometrial response achieved by U46619. This indicates that U46619 excitation may involve the ROCK pathway. Previous studies have demonstrated that chronic stimulation of human myometrial cells with this thromboxane mimetic can lead to an elevation in ROCK concentrations (Moore and Lopez Bernal, 2003). We can conclude from this that ripasudil has the ability to inhibit myometrial contractility in the non-pregnant mouse uterus during both spontaneous activity and agonist-induced muscular stimulation.

Increasing concentrations of ripasudil were also capable of reducing 5-HT-induced uterine contractions in both segments of the non-pregnant mouse uterus. The effect of ripasudil was statistically significant in the upper segment, while it was non- significant in the lower segment even though the graph showed a certain degree of inhibition. Cumulative concentrations of 5-HT caused an increased stimulation of myometrial activity in both uterine segments. These results are consistent with other research in non-pregnant mouse uterus (Xiu-Kun et al., 2011). Ripasudil demonstrated its ability to inhibit contractions induced by 5-HT especially at high 5- HT concentrations. Surprisingly, the response to 5-HT above 10-6M showed some attenuation in both segments of the uterine horn and it happened in the presence and absence of ripasudil. This might be due to receptor desensitisation with the addition of repeated concentrations of 5-HT. Similar findings have been reported in previous work in the brain (Hanley and Hensler, 2002, Maroteaux et al., 2016) and the gut (Coates et al., 2004, Gershon, 2004, Mawe et al., 2006). It has also been reported that stimulation of 5-HT receptors by an agonist will stimulate the ROCK pathway during pulmonary hypertension in mice (Mair et al., 2008). Although the association between 5-HT and ROCK pathway in non-pregnant mouse uterus is still unclear and needs more investigation, according to these data, activation of 5-HT receptors may stimulate the ROCK mechanism and potentiate myometrial contractions.

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In order to further explore the action of ripasudil, myometrial strips from non-pregnant mice were stimulated by addition of PGF2α in the presence and absence of ripasudil.

PGF2α stimulates FP receptors and can also evoke the thromboxane A2 receptors (TP) as demonstrated by previous studies (Wong et al., 2009). Activation of TP receptors will stimulate the ROCK pathway and its downstream signals. PGF2α notably increased uterine contraction in both uterine segments and this indicates the distribution of FP receptors throughout the horn as discussed previously. Interestingly, increasing concentrations of ripasudil inhibited PGF2α-induced myometrial contractility in the non- pregnant mouse uterus and this inhibition reached a significant level at a 10-5M ripasudil -5 in both segments. The high concentration of PGF2α (10 M) may have led to some off- target activities of PGF2α, such as stimulation of other prostanoid receptors (EP1, EP3 and TP) (Breyer et al., 2001, Griffiths, 2007). Thus, the effect produced by ripasudil against PGF2α-stimulated samples may involve some of these off-target actions of

PGF2α. The addition of cumulative concentrations of PGF2α to the organ bath increased myometrial contractility with increasing concentrations in upper and lower segments of the uterine horn. These data are consistent with previous studies in the non-pregnant mouse uterus (Griffiths, 2007, Hutchinson, 2005).

As in previous experiments with U46619 and 5-HT, ripasudil inhibited PGF2α-induced myometrial contraction and this inhibition has achieved a significant level at three -5 PGF2α concentrations. Similar to 5-HT, the myometrial response to PGF2α at 10 M showed some attenuation, particularly in the lower uterine segment. This might be due to PGF2α inducing FP receptor desensitisation at the highest PGF2α concentrations, a phenomenon observed also previously in the human ciliary muscle (Kunapuli et al.,

1997). No previous studies have reported a correlation between of PGF2α and the ROCK signalling cascade in non-pregnant mouse uterus. However, some researchers have demonstrated an association between this prostaglandin and ROCK but in a pregnant state where the Rho/ROCK signalling pathway was mediated by PGF2α through activation of FP receptors in mouse uterus (Goupil et al., 2010). Further studies are required to confirm this mechanism in pregnant and non-pregnant samples.

A number of recent studies have investigated the effect of some ROCK inhibitors on the expression of contractile proteins in myometrial tissues after treatments with the uterine stimulant, oxytocin. ROCK inhibitors were able to inhibit MLC phosphorylation in the rat uterus (Tahara et al., 2002) and human uterine smooth muscle cells (Aguilar et al.,

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2012). This is the first study to examine the role of ripasudil as a ROCK inhibitor in the modulation of MLC-family expression in myometrium from a non-pregnant mouse uterus. This work found that ripasudil has the ability to regulate the three MLC-related proteins, the non-phosphorylated MLC, pMLC, and ppMLC. In myometrial tissues treated with cumulative concentrations of ripasudil using the immersion technique, ripasudil caused significant elevation in MLC expression when compared to the control. This interesting result indicates that ROCK inhibitors can prevent the phosphorylation of MLC into pMLC even in unstimulated tissues. Accumulation of MLC will lead to muscular relaxation and a decrease in muscular tension force (Somlyo and Somlyo, 2003). This also confirms the role of ROCK pathway in modulating the spontaneous activity of the uterus as ROCK inhibitors were able to reduce the amplitude of spontaneous contractions in human myometrium (Kupittayanant et al., 2001). Our findings are in agreement with other studies that used different ROCK inhibitors (glycyl-H-1152) with myometrial stimulants in human uterine myocytes (Aguilar et al., 2011). Looking at the representative western blot image (Figure 3.40) there is a partial conversion of MLC into pMLC in the control group which was much lower in ripasudil- treated samples. This further demonstrates the higher stimulatory state of myometrium in the control group over the ripasudil group. Immunocytochemistry demonstrated a similar change in MLC expression after treating myometrial cells with ripasudil: the expression of MLC was slightly higher in the ripasudil group, even though this observation was not very clear compared to what was seen in the western blot bands. This may be due to the single concentration of ripasudil used in immunofluorescence to treat myometrial cells, whereas myometrial strips treated with repeated concentrations of ripasudil were used for the western blot. It also might be due to the myometrial tissue retaining the drug for a longer period after repeated exposure, so its effect may have been prolonged and easier to detect than on a cellular level. Also the tissue would need more time to metabolise the infused drug, especially because lipophilic drugs may bind to tissue constituents, such as proteins and lipids (Di and Kerns, 2016)

On the other hand, ripasudil reduced the level of mono-phosphorylated MLC (pMLC) in myometrial tissue taken from the non-pregnant mouse uterus after administration of cumulative concentrations of ripasudil compared to the control. Both western blots and ICC confirmed this drop in pMLC expression, which was also previously shown using myometrial cells from pregnant women (Aguilar et al., 2011). All results together are

197 consistent with our findings from functional experiments to test the impact of ripasudil on spontaneous myometrial activity.

The most interesting result was the effect of ripasudil on the di-phosphorylation of MLC in myometrial cells. Western blotting showed that ripasudil caused a highly significant reduction in the expression of the di-phosphorylated form of MLC, ppMLC compared to the control. This was the first study to test this effect of ripasudil in mouse uterus. Previous studies have demonstrated the effect of other ROCK inhibitors on human uterine cells and rat aortic smooth muscles (Aguilar et al., 2011, Hsu et al., 2019). Although, the data from the ICC work did not obviously show such action of ripasudil, western blotting results were very clear and further confirmed the hypothesis of ROCK involvement in the transformation of pMLC into ppMLC, which has been demonstrated as an essential pathway in smooth muscle contraction in human uterine but not vascular tissue (Aguilar et al., 2012). Further investigations are needed to confirm this mechanism on a cellular level and also to check if ripasudil can distinguish between the two isoforms, ROCKI, and ROCKII. All the above work was performed using non- pregnant mouse in dioestus and so, further investigations at different stages of the oestrous cycle are needed to examine impact of hormonal milieu and sex steroids on the ROCK-modulated myometrial contractility.

As described above, U46619 activates the ROCK pathway, which in turn induces the phosphorylation of MLC and so the formation of pMLC (Amano et al., 2000). Surprisingly, our results show a non-significant elevation of MLC by U46619 when compared with the control. In addition to that, western blotting work shows that there is a slight reduction in pMLC occurrence when myometrial tissue is treated with U46619 compared to the control, although in functional studies, the myometrial contraction induced by U46619 was much higher than that of the control. This highlights the possibility of protein phosphorylation recycling mechanism; this phenomenon could be due to the partial dephosphorylation of pMLC to its precursor MLC. It was also hypothesised that this recycling might be due to several factors. Firstly, the phasic nature of uterine contraction might affect the rapid protein phosphorylation processes as observed previously in studies on human myometrium (Paul et al., 2011). The second factor is that protein phosphorylation recycling might be implicated by the removal of myometrial strips from the organ bath containing the uterine stimulant until tissue was homogenised and frozen, and thirdly, it might be a protective mechanism by the

198 myometrium against further tissue stimulation. Western blot bands in Figure 3.46 and Figure 3.40 support this hypothesis as smaller pMLC bands were observed with U46619 treatment compared to its sizes in the control group. However, no significant difference in ppMLC expression was observed between U46619 and control groups and this might be due to the same hypothesis above.

On the other hand, and although ripasudil at 10-6M caused a slight increase in MLC expression in the U46619-treated samples as seen in Figure 3.46, this increase was not remarkably signiﬁcant as the p value did not approach the borderline of significance (p=0.2). We found similar results on a cellular level where uterine myocytes showed a higher expression of MLC after the U46619 group was pre-treated with ripasudil at 10- 6M. Moreover, ripasudil at 10-6M did not significantly inhibit phosphorylation of MLC into pMLC on both tissues and cells but the inhibition was experimentally meaningful (p=0.09). However, the effect of ripasudil on inhibition of MLC was more obvious when compared to the control group. This demonstrates the effectiveness of ripasudil as a ROCK inhibitor to abolish the U46619 downstream action in stimulating the phosphorylation of MLC by MLCK through activating TP receptors and the Rho/ROCK signalling cascade in the myometrium of non-pregnant mouse uterus. Similar findings were observed with other ROCK inhibitors and TP receptor agonists in rat tail arteries (Tsai and Jiang, 2006). The most interesting finding was the high efficacy of ripasudil at 10-6M to diminish the di-phosphorylation of MLC into ppMLC in myometrial strips treated with U46619, which was significant when compared with U46619 (p=0.035) or control (p=0.029) group. These effects were also found in uterine myometrial cells treated U46619 in the presence and absence of ripasudil at 10-6M. This was the first study to demonstrate the effect of ROCK inhibitors on the TP receptor agonists- stimulated MLC di-phosphorylation. Comparing the degree of inhibition in MLC mono and di-phosphorylation by ripasudil in U46619-treated myometrial strips of non- pregnant mice, it is evident that the di-phosphorylation process is highly dependent on ROCK pathway as inhibition of this pathway by ripasudil as a ROCK inhibitor had much more effect than on the mono-phosphorylation process. This can be clearly seen in graph columns as well as western blot image bands (Figures 3.48 and 3.50). ROCK pathway involvement in MLC di-phosphorylation has recently been demonstrated in few studies in human cultured uterine cells (Aguilar et al., 2012) and myometrium (Hudson et al., 2012) as well as rat hippocampal neurons (Newell-Litwa et al., 2015).

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MLC phosphorylation in myometrial tissue from non-pregnant mouse uterus at both, single and double MLC subunits after treatment with 5-HT in the presence and absence of ripasudil at 10-6M followed the same pattern as U46619 experiments. The relative high MLC and low pMLC and ppMLC expressions in 5-HT-treated samples compared to the control, can be attributed the same protein phosphorylation recycling events that were hypothesized with U46619. In addition, it might be also due to the receptor desensitisation that was observed at high 5-HT concentrations (10-5M) in functional studies experiments in both upper and lower segments of the uterine horn. Nevertheless, ripasudil (10-6M) significantly inhibited the expression of both phosphorylated forms of MLC in myometrial strips treated with 5-HT which brings to light the contribution of 5- HT in stimulating ROCK pathway in non-pregnant uterine tissues which is inhibited here by ripasudil. Other studies have demonstrated that 5-HT receptors are expressed in uterine smooth muscles of human (Kelly et al., 2006), mice (Xiu-Kun et al., 2011), guinea-pigs (Masson et al., 2012) and rabbits (Lychkova et al., 2014) and the stimulation of these receptors can activate Rho/ROCK cascade in mouse pulmonary and porcine coronary arteries, an effect which can be suppressed by ROCK inhibitors (Mair et al., 2008, Kandabashi et al., 2000). Interestingly, our findings are in agreement with other researchers who have previously demonstrated the effectiveness of ROCK inhibitors in abolishing 5-HT-induced MLC mono and di-phosphorylation in porcine coronary arteries (Shimokawa et al., 1999). Further investigations are needed to examine the role of ROCK inhibitors on 5-HT-stimulated myometrial contraction in the human uterus.

To determine the contribution of FP receptors and its natural ligand, PGF2α on MLC phosphorylation events in non-pregnant mouse uterus, isolated myometrial strips were -9 -5 stimulated with cumulative concentrations of PGF2α (10 M – 10 M) after pre-treatment -6 with ripasudil at 10 M using functional studies. Mounted strips were removed after all treatments and examined later for the occurrence of the three forms of MLC protein, MLC, pMLC and ppMLC. Similar to the above two uterine stimulants, U46619 and 5-

HT, PGF2α showed a relative elevation in MLC, a significant reduction in pMLC as well as a slight decline in ppMLC expression compared to the control. These effects are encouraging and follow the same hypotheses for 5-HT including protein phosphorylation recycling processes and also receptor desensitization, as observed from the functional studies experiments that there is attenuation in myometrial activity at

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-5 high concentrations of PGF2α (10 M) in the upper and lower segments of the uterine horn. However, PGF2α effects on MLC phosphorylation events were different in the presence of ripasudil at 10-6M, as ripasudil caused a relative but not significant increase in MLC in samples treated with PGF2α when compared to the PGF2α-alone group (p=0.15). On the other hand, an approximately significant reduction in pMLC (p=0.0536) and a highly significant inhibition in ppMLC expression (p=0.0006) were -6 found in myometrial strips treated with PGF2α in the presence of ripasudil at 10 M. Functional FP receptors were observed in the uterus of non-pregnant mice and their activities have been tested by the ability of PGF2α to stimulate these myometrial tissues (Hutchinson et al., 2003). As described previously, there is no evidence indicating the contribution of PGF2α or its FP receptors in Rho/ROCK signalling in the myometrium of the non-pregnant mouse. Nevertheless, this correlation has been demonstrated in pregnant human and mouse uterine tissues (Goupil et al., 2010, Tahara et al., 2005, Woodcock et al., 2006). To the best of our knowledge, this is the first work to examine the impact of PGF2α on MLC phosphorylation levels in the uterine tissues.

Comparing the concentration-effect of U46619, PGF2α, 5-HT and ripasudil treatments on the phosphorylation level of MLC protein in the myometrial strips isolated from non- pregnant C57 mice uterus in dioestrus as observed in Table 3.1 shows the uterine stimulant which has the most powerful effect in keeping the MLC in its non- phosphorylated form is the 5-HT followed by U46619 and then PGF2α. It can be concluded from this that either there are a high population of 5-HT receptors in the myometrium in dioestrous stage or these receptors more sensitive to stimulation than TP and FP receptors. Ripasudil has elevated the expression of MLC when compared to the above three uterine stimulants as well as to the control-treated samples. This indicates the effectiveness of ripasudil in preventing attenuating uterine contractility via abolishing the MLC phosphorylation. On the other hand, U46619 has caused the most considerable increase in the expression of the mono- and di-phosphorylated forms of

MLC (pMLC and ppMLC) when compared with 5-HT and PGF2α. It can be hypothesized from this that both levels of MLC phosphorylation in the non-pregnant mouse uterus during dioestrus are more and highly responsive to TP receptor activation than to FP and 5-HT receptors stimulation. This activation of TP receptors increases the ROCK signalling pathway and thus, enhances the level of MLC phosphorylation. No previous research is available on the population and responsiveness of these receptors in

201 the myometrial stips of non-pregnant mouse uterus. However, some studies have examined and demonstrated the functional activity of these receptors in the uterus of non-pregnant mice (Hutchinson, 2005, Griffiths, 2007, Xiu-Kun et al., 2011). Further examinations are required to investigate the number of these receptors in the non- pregnant mouse uterus at different oestrous stages.

In addition, the effect of cumulative concentrations of ripasudil on the level of MLC phosphorylation was also studied in this work. Ripasudil was able to prevent the phosphorylation of MLC and keep the protein at its high non-phosphorylated level (151%) when compared to the control. Furthermore, the inhibition of MLC di- phosphorylation step by ripasudil (11.4%) was much higher than that on the mono- phosphorylation level (38.9%) of the protein as shown in Table 3.1. This demonstrates that the transformation of pMLC to ppMLC is predominantly induced by the ROCK signalling pathway, and this di-phosphorylation process is extensively expressed in the myometrium of non-pregnant mouse uterus. To the best of our knowledge, this is the first study to investigate the expression of such kind of phosphorylation in the mouse uterus. However, researchers have recently confirmed the expression of this di- phosphorylation step in the myometrial strips taken from the pregnant human uterus at term. They also have demonstrated that this di-phosphorylation is essentially dependent on the ROCK pathway and that ROCK inhibitors can block the conversion of pMLC to ppMLC (Aguilar et al., 2011, Aguilar et al., 2012).

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4 Chapter 4:

Effects of Ripasudil, a ROCK inhibitor on Oxytocin, U46619- and 5-HT-induced myometrial contractions in pregnant C57 wild type mice at term

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4.1 Introduction

During pregnancy, the uterus is more quiescent / or has a decreased myogenic tone and is stretched to accommodate the foetus and placenta, while the cervix is firm and stiff enough to maintain pregnancy. At term, combined biochemical, hormonal and physical changes occur to enhance the transformation of the uterus from a quiescent state into a contractile one. This conversion is facilitated by the increased expression of the genes for contractile-associated proteins (CAPs). These CAPs include connexin-43, ion channel proteins and receptors activated by uterine stimulants (Challis et al., 2000). Consequently, the uterus is being prepared for labour.

Oxytocin (OXT) plays an important role in timing the onset of labour in mice. It activates oxytocin receptors (OXR) in the uterus, which are highly expressed at the late stages of pregnancy, and this activation also leads to an increase in the production of prostanoids in the uterine tissues (Blanks and Thornton, 2003). In addition, prostanoid receptors such as the FP receptors are highly expressed in mouse uterus at term and their activation by PGF2α induces uterine contractility (Cook et al., 2003). Furthermore, the uterine thromboxane receptors (TP) are highly reactive to the thromboxane-synthetic analogue U46619 during pregnancy in mice (Griffiths et al., 2006). 5- hydroxytryptamine (5-HT) receptor subtypes have been reported to be upregulated in the myometrium of human and rat uteri at late gestation, leading to 5-HT-stimulated uterine contraction in these species (Cruz et al., 1989, Minosyan et al., 2007). Furthermore, it has been demonstrated that 5-HT receptors are involved in parturition via activating myometrial contractility and that antagonists to these receptors possess tocolytic properties that are useful to treat PTL (Cordeaux et al., 2009). Additionally, the RhoA/ROCK pathway is activated in the late stages of pregnancy in the myometrium of human, rats and mice, which results in RhoA-activated calcium sensitization and so enhanced uterine contractility (Niiro et al., 1997, Moore et al., 2000, Riley et al., 2005).

In this chapter, myometrial tissues were isolated from C57 WT mice at term (E19) and were exposed to different treatments to examine the effect of the selective ROCK inhibitor ripasudil (Garnock-Jones, 2014) on uterine contractility, which is induced by

204 various stimulants. In this chapter OXT was used instead of to stimulate the myometrial contractility to as the population of OXR is very high in the uterine tissues at the late stages of pregnancy (50-100 times) as compared to the non-pregnant uterus and so, the responsiveness of myometrium is much higher in pregnant samples at term (Fuchs et al, 1985). E19 was considered as the term stage of gestation; the first day of gestation (E1) was recorded upon the detection of a vaginal plug. The normal gestation period of the mouse is 19-20 days, and labour normally takes place on day 20 (E20) (Peters et al., 2007). In the mouse gestation day 20 (E20) is the equivalent of 38 weeks of gestation or term in the human, based on the last menstrual period, thus, E19 of mouse gestation will correspond to 36.1 weeks of gestation in a human pregnancy and any labour occurring before 37 completed weeks in human pregnancy is considered as preterm labour (Quinn et al., 2016, Griffiths, 2007).

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4.2 Spontaneous activity in pregnant mouse uterus at term (E19)

Pregnant mouse uterine tissue samples exhibited spontaneous contractility. Strips taken from the upper horn of the uterine segment of pregnant C57 WT mice at term (E19) displayed higher activity when compared to the equivalent samples from non-pregnant mice in dioestrus, in terms of amplitude and frequency as shown in Figure 4.1. The tissues from pregnant mice also exhibited a longer duration of spontaneous contractile activity (48h) than the strips from non-pregnant mice (5h). Traces of contraction demonstrate that both tissues were viable and maintained their activity during the whole period of the immersion experiment, which lasted for about 2.5h despite the gradual decline in activity after the first hour in the non-pregnant uterus (Figure 4.2).

Figure 4.1. Comparing spontaneous activity between pregnant and non-pregnant mouse uterine samples. Comparison between spontaneous activity measured as AUC in isolated upper uterine segments from non-pregnant (in dioestrus; n=13) and pregnant (at term, E19; n=13) C57 mice. Myometrial strips were incubated in an organ bath containing Krebs’ solution at 37ºC and bubbled with 95% O2/ 5% CO2. Data are not normalised and are measured as AUC for a 10 min period. Data are expressed as mean ± SEM, and were analysed using an unpaired Student t-test, (***p0.001).

206

Myometrial strips were incubated in an organ bath containing Krebs’ solution at 37ºC and bubbled with 95% O2/CO2 as described in methods. The interruption in recording traces seen between the 4th and 12th hours of contraction in pregnant sample (B) was due to the failure of connection between the immersion machine and the computer overnight while the tissue was still contracting and active.

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4.3 Plasma concentration of 17β-oestradiol

In order to examine the influence of the hormonal milieu on uterine tissue activity in both pregnant and non-pregnant states, the concentrations of 17β-oestradiol were measured in the plasma of non-pregnant (in dioestrus) and pregnant (at term, E19) C57 wild-type (WT) mice using a mouse ELISA assay. Figure 4.3 shows the level of 17β- oestradiol detected in the plasma of pregnant and non-pregnant mice and demonstrates that there was a slight elevation in the mean 17β-oestradiol concentration in the pregnant samples although this difference was not statistically significant (p=0.166).

Figure 4.3. 17β-oestradiol concentrations (pg/ml) measured in the plasma of non-pregnant (in dioestrus) and pregnant (E19) C57 mice. Comparison between plasma 17β-oestradiol levels (pg/ml) in non-pregnant (dioestrus; n=6) and pregnant (E19; n=6) C57 mice. Data are expressed as mean ± SEM and were analysed using unpaired Student t-test, (p=0.166), (ns) not significant.

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4.4 Litter size and spontaneous activity

The correlation between uterine spontaneous activity and litter size (number of pups) was examined in pregnant C57 mice at term (E19). Data show variable measurements and no correlation was found between uterine contractile activity and the litter size in C57 mice at term as seen in Figure 4.4. Sample size was low (n=1-2) within each group, so data could not be statistically analysed.

Figure 4.4. Influence of litter size on uterine spontaneous activity in pregnant C57 mice at term (E19). The relationship between litter size and spontaneous activity measured as AUC in the uterus of pregnant C57 mice at term (E19). There was no correlation between litter size and uterine spontaneous activity in these animals. Data are expressed as mean ± SEM, n=1-2/ group.

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4.5 The effect of cumulative doses of ripasudil on spontaneous myometrial contractility in pregnant C57 mice at term (E19)

In samples taken from the upper segment of the uterine horn of pregnant C57 mice at term (E19), ripasudil at high concentrations (10-6M and 10-5M) evoked a predominantly inhibitory effect on myogenicity via reducing the uterine contractile activity of myometrial strips compared to the control (vehicle, 0.9% w/v normal saline), even though there was some increase in the frequency of contraction a short time before the addition of ripasudil at 10-7M, which continued after adding this concentration. The frequency and amplitude of contraction decreased when the tissue was treated with higher ripasudil concentrations (10-6M and 10-5M) and this inhibition of contraction was significant at these two concentrations (see Figure 4.5). Traces of the effect of cumulative concentrations of ripasudil and control (vehicle) are shown in (Figure 4.6).

Figure 4.5. Effect of cumulative doses of ripasudil on myometrial spontaneous activity in pregnant C57 mouse uterus at term (E19). Concentration-effect curves for ripasudil (10-9M - 10-5M) compared with control (vehicle, 0.9% w/v normal saline) in isolated myometrial strips from the upper segment of the uterine horn in pregnant C57 mouse at term (E19). Myometrial strips were incubated in an organ bath containing Krebs solution at 37ºC and bubbled with 95% O2/5% CO2. Responses were measured over a 10 min period (area under the curve) and expressed as a percentage of initial spontaneous activity. Data are expressed as mean ± SEM, n=6, and were analysed using two-way ANOVA with Bonferroni’s adjustment; *p<0.05, ****p<0.0001, significantly different from the control (vehicle).

210

Figure 4.6. Representative traces showing concentration-effect curves for ripasudil (10-9M - 10-5M) and control in isolated myometrial strips from the upper segment of the uterine horn in pregnant C57 mouse at term (E19) when using the immersion technique.

Myometrial strips were incubated in an organ bath containing Krebs solution at 37ºC and bubbled with 95% O2/5% CO2 as described in methods.

211

4.6 The effect of ripasudil on the myometrial contractility induced by cumulative concentrations of oxytocin, U46619 or 5-HT in pregnant C57 mice at term (E19)

Figure 4.7 shows concentration-effect curves for OXT in the presence and absence of the ROCK inhibitor, ripasudil at two concentrations, 10-6M and 10-5M on the upper segment of the uterine horn in pregnant C57 mice at term (E19). OXT elicited a concentration-dependent excitatory response over the concentration range of 10-12M - 10-6M. Ripasudil at both concentrations, 10-6M and 10-5M attenuated this response to OXT and this was statistically significant at agonist concentrations of 10-8M, 10-7M and 10-6M (p-values are shown in the Figure legend). There was a slight decline in the uterine contractile activity at the maximum OXT concentration used (10-6M) in the absence and presence of ripasudil at both ripasudil concentrations. These declines were 13.8%, 17.5% and 22.9% in the OXT-alone, Ripasudil 10-6M+ OXT and Ripasudil 10- 5M+ OXT groups respectively. OXT concentrations were chosen according to previous studies in the mouse uterus (Patel et al., 2017), while ripasudil concentrations were selected based on the results of our preliminary experiments in the non-pregnant mouse uterus. Figure 4.8 shows representative traces of OXT-induced contraction in the presence and absence of ripasudil.

212

Effect of ripasu dil on oxytocin-induced uterine contractility in pregnant mice

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213

Myometrial strips were incubated in organ bath containing Krebs solution at 37ºC and bubbled with 95% O2/5% CO2 as described in methods. 214

The impact of ripasudil at 10-6M and 10-5M on the myometrial contractility which is stimulated by cumulative additions of concentrations of U46619 was also investigated in upper segment uterine tissues isolated from pregnant C57 mice at term (E19). U46619 induced a concentration-dependent excitatory response over the concentration range of 10-9M - 10-5M. Similar to the previous experiment with OXT, ripasudil at both concentrations, 10-6M and 10-5M attenuated the U46619-induced myometrial contractions and this attenuation was statistically significant at a U46619 concentration of 10-5M. Ripasudil at 10-5M was also able to significantly reduce the uterine contractile activity stimulated by 10-6M U46619 (p<0.05) as shown in Figure 4.9. U46619 concentrations were chosen according to previous studies in the mouse uterus (Griffiths, 2007). Representative traces of contraction show that U46619 at high concentration (10- 6M) increased the frequency of contraction in the U46619-alone and ripasudil 10-6M+ U46619 groups. U46619-induced myometrial contraction in the presence and absence of ripasudil at both concentrations are shown in Figure 4.10.

Figure 4.9. Effect of cumulative concentrations of U46619 on the myometrial spontaneous activity of the upper segment of uterine horn in pregnant C57 mice at term (E19) in the presence and absence of ripasudil. Concentration-effect curves for U46619 (10-9M - 10-5M) in the presence and absence of ripasudil (10-6M and 10-5M) in isolated myometrial strips from the upper segment of the uterine horn in pregnant C57 mouse at term (E19). Myometrial strips were incubated in an organ bath containing Krebs’ solution at 37ºC and bubbled with 95% O2/5% CO2. Responses were measured over a 10 min period (AUC) and expressed as a percentage of initial spontaneous activity. Data are expressed as mean ± SEM, n=6, and were analysed using two- way ANOVA with Bonferroni's adjustment; *p<0.05; **p<0.01, ***p<0.001; ****p<0.0001 for U46619 alone compared to tissues immersed with a ripasudil at 10-6M and b ripasudil at 10-5M.

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Figure 4.10. Representative traces showing concentration-effect curves for U46619 (10-9M - 10-5M) in the presence and absence of ripasudil (10-6M and 10-5M) in isolated myometrial strips from the upper segment of the uterine horn in pregnant C57 mouse at term (E19) when using the immersion technique.

Myometrial strips were incubated in an organ bath containing Krebs’ solution at 37ºC and bubbled with 95% O2/ 5% CO2 as described in methods.

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In order to examine the influence of ripasudil on 5-HT-induced myometrial contractility in pregnant C57 mice at term (E19), strips of myometrium from the upper uterine segment were incubated with 5-HT in the presence and absence of the ROCK inhibitor ripasudil. 5-HT elicited a concentration-dependent excitatory response over a concentration range of 10-9M - 10-5M. A reduction in the mean myogenic activity was observed with the addition of a 10-5M concentration of 5-HT when compared with 10- 6M. This decline in uterine activity at the maximum 5-HT concentration was obvious in the 5-HT alone-treated group and was less pronounced in the samples pre-treated with ripasudil at 10-6M. Interestingly, ripasudil at 10-6M and 10-5M significantly inhibited 5- HT-induced myometrial contraction in these pregnant samples at three agonist concentrations, 10-7M, 10-6M, and 10-5M as shown in Figure 4.11. The selection of 5- HT concentrations was according to other research in mice (Poyton et al., 2015). Representative traces of 5-HT-induced contraction in the presence and absence of ripasudil in pregnant myometrial tissue are shown in Figure 4.12.

Figure 4.11. Effect of cumulative concentrations of 5-HT on the myometrial spontaneous activity of the upper segment of uterine horn in pregnant C57 mice at term (E19) in the presence and absence of ripasudil. Concentration-effect curves for 5-HT (10-9M - 10-5M) in the presence and absence of ripasudil (10-6M and 10-5M) in isolated myometrial strips from the upper segment of the uterine horn in pregnant C57 mouse at term (E19). Myometrial strips were incubated in an organ bath containing Krebs’ solution at 37ºC and bubbled with 95% O2/5% CO2. Responses were measured over a 10 min period (AUC) and expressed as a percentage of initial spontaneous activity. Data are expressed as mean ± SEM, n=4-5/ group, and were analysed using two-way ANOVA with Bonferroni's adjustment; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001 for 5- HT alone compared to tissues immersed with a ripasudil at 10-6M and b ripasudil at 10-5M. 217

Myometrial strips were incubated in an organ bath containing Krebs solution at 37ºC and bubbled with 95% O2/5% CO2 as described in the methods. 218

4.7 Expression of contractile proteins in myometrium after treatment with OXT, U46619 and 5-HT in the presence and absence of ripasudil

Western blotting was used in order to investigate the effect of ripasudil, OXT, U46619 and 5-HT on expression of the following proteins, which are involved in the contraction process in the uterus, MLC, pMLC and ppMLC. The measurements were made using the tissues collected after completion of the immersion experiments.

Figure 4.13 demonstrates that treatment of myometrial strips with cumulative concentrations of ripasudil at 10-9M - 10-5M caused a significant increase in protein expression of non-phosphorylated MLC compared with the control (p<0.05). The representative western blotting image (panel A of Figure 4.13) shows the variation in band densities between the control and ripasudil-treated groups. Densitometry was used to quantify the changes in protein expression observed (panel B of Figure 4.13).

Figure 4.13. Effect of ripasudil on MLC expression in myometrial tissue from pregnant C57 mice at term (E19). Effect of ripasudil on protein expression of MLC in pregnant C57 mouse myometrium at term (E19). Tissues were treated with cumulative concentrations of ripasudil (10-9M - 10-5M) or control (vehicle, 0.9% w/v normal saline) and protein expression was investigated by western blotting. Panel (A) is a representative western blot image and panel (B) is a densitometric analysis. Data are normalised to α-actin and are presented as a percentage of the control. Data were analysed using unpaired Student’s t-test and are presented as the mean ± SEM of 3 independent experiments, *p< 0.05.

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Figure 4.14 demonstrates the effect of cumulative concentrations of ripasudil on pMLC expression in the above tissues and the representative image (panel A) shows the difference in the density of bands between the two treated groups. A reduction in the expression of the mono-phosphorylated MLC (pMLC) in the strips treated with ripasudil compared with the control (vehicle, 0.9% w/v normal saline) group was observed. This reduction in protein expression was statistically significant (p< 0.05).

Figure 4.14. Effect of ripasudil on pMLC expression in myometrial tissue from pregnant C57 mice at term (E19). Effect of ripasudil on the protein expression of pMLC in pregnant C57 mouse myometrium at term (E19). Tissues were treated with cumulative concentrations of ripasudil (10-9M - 10-5M) or control (vehicle, 0.9% w/v normal saline) using immersion technique and protein expression was investigated by western blotting. Panel (A) is a representative western blot image and panel (B) is a densitometric analysis. Data are normalised to α-actin and are presented as a percentage of the control. Data were analysed using unpaired Student’s t-test and are presented as the mean ± SEM of 3 independent experiments, *p< 0.05.

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Similar to the previous work on non-pregnant mouse uterine tissues (Figure 3.44), the most significant effect of ripasudil was seen on the di-phosphorylated form of MLC (ppMLC) when the myometrial strips were treated with cumulative concentrations of ripasudil (10-9M - 10-5M). Ripasudil induced a profound inhibition of ppMLC expression in the treated tissue samples (86.76%) and this inhibition was significant when compared to the control (vehicle, 0.9% w/v normal saline) group (p< 0.0001).

These effects of ripasudil at (10-9M - 10-5M) can be clearly seen in Figure 4.15 and the representative western blotting image shows the variation in the band densities between ripasudil and control groups.

Figure 4.15. Effect of ripasudil on ppMLC expression in myometrial tissue from pregnant C57 mice at term (E19). Effect of ripasudil on the protein expression of ppMLC in pregnant C57 mouse myometrium at term (E19). Tissues were treated with cumulative concentrations of ripasudil (10-9M - 10-5M) or control (vehicle, 0.9% w/v normal saline) using immersion technique and protein expression was investigated by western blotting. Panel (A) is a representative western blot image and panel (B) is a densitometric analysis. Data are normalised to α-actin and are presented as a percentage of the control. Data were analysed using unpaired Student’s t-test and are presented as the mean ± SEM of 3 independent experiments, ****p< 0.0001.

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The impact of the interaction between oxytocin and ripasudil on the expression of proteins involved in myometrial contraction in the pregnant C57 mouse uterus at term (E19) was also tested. Cumulative concentrations of OXT (10-12M – 10-6M) applied to isolated myometrial tissues immersed in organ baths did not evoke a remarkable change in the expression of the non-phosphorylated MLC when compared to the control. Pre- treatment with ripasudil at 10-6M was shown to cause a slight increase in the mean level of MLC expression in OXT-treated myometrial strips; however, this elevation was not statistically significant. Furthermore, although MLC expression in the ripasudil+OXT group seemed to be higher than that of the control group, this elevation was non- significant (Figure 4.16). The bands from the representative blots also showed that in all groups there is low expression of the mono-phosphorylated form of MLC (pMLC) with little variations among treatment groups.

Figure 4.16. Effect of OXT on MLC expression in myometrial tissue from pregnant C57 mice at term (E19) in the presence and absence of ripasudil. Effect of OXT on the protein expression of MLC in pregnant C57 mouse myometrium at term (E19) in the presence and absence of ripasudil 10-6M. Tissues were treated either with the control (vehicle, 0.9% w/v normal saline), cumulative concentrations of OXT (10-12M - 10-6M) or pre-treated with ripasudil 10-6M and then with cumulative concentrations of OXT (10-12M - 10-6M) using immersion technique and protein expression was investigated by western blotting. Panel (A) is a representative western blot image and panel (B) is a densitometric analysis. Data are normalised to α-actin and are presented as a percentage of the control. Data were analysed using one-way ANOVA with Bonferroni’s adjustment and are presented as the mean ± SEM of 3 independent experiments, (ns) not significant.

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Treatment of myometrial strips isolated from pregnant C57 mouse uterus at term (E19) with cumulative concentrations (10-12M – 10-6M) of OXT evoked a statistically significant elevation in the expression of pMLC compared with the control (vehicle, 0.9% w/v normal saline) (p< 0.01). When the tissues were pre-treated with ripasudil at 10-6M before adding OXT, the expression of pMLC was inhibited by ripasudil when compared to the OXT alone-treated samples. This inhibition of pMLC by ripasudil was at a statistically significant level (p< 0.05). Figure 4.17 shows the variation in pMLC expression among all groups.

Figure 4.17. Effect of OXT on pMLC expression in myometrial tissue from pregnant C57 mice at term (E19) in the presence and absence of ripasudil. Effect of OXT on the protein expression of pMLC in pregnant C57 mouse myometrium at term (E19) in the presence and absence of ripasudil 10-6M. Tissues were treated either with the control (vehicle, 0.9% w/v normal saline), cumulative concentrations of OXT (10-12M - 10-6M) or pre-treated with ripasudil 10-6M and then with cumulative concentrations of OXT (10-12M - 10-6M) using immersion technique and protein expression was investigated by western blotting. Panel (A) is a representative western blot image and panel (B) is a densitometric analysis. Data are normalised to α-actin and are presented as a percentage of the control. Data were analysed using one-way ANOVA with Bonferroni’s adjustment and are presented as the mean ± SEM of 3 independent experiments, *p< 0.05; **p< 0.01.

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Moreover, the expression of the contractile di-phosphorylated MLC protein (ppMLC) was also examined in myometrial strips isolated from pregnant C57 mouse uterus at term (E19) after treatment with OXT at cumulative concentrations (10-12M – 10-6M). The tissues were also pre-treated with ripasudil at 10-6M before stimulation with OXT.

In these tissues, OXT caused a three-fold elevation in ppMLC expression which was statistically significant (p=0.013) compared to the control group. Whereas, myometrial strips pre-treated with ripasudil at 10-6M, followed by the addition of OXT (10-12M – 10-6M) showed a decrease in the expression of ppMLC when compared to OXT alone- treated samples. This decrease in protein expression was at a three-fold level and was also significant (p=0.0159). No difference in ppMLC expression was found between the control and ripasudil+OXT treated groups (see Figure 4.18).

Figure 4.18. Effect of OXT on ppMLC expression in myometrial tissue from pregnant C57 mice at term (E19) in the presence and absence of ripasudil. Effect of OXT on the protein expression of ppMLC in pregnant C57 mouse myometrium at term (E19) in the presence and absence of ripasudil 10-6M. Tissues were treated either with the control (vehicle, 0.9% w/v normal saline), cumulative concentrations of OXT (10-12M - 10-6M) or pre-treated with ripasudil 10-6M and then with cumulative concentrations of OXT (10-12M - 10-6M) using immersion technique and protein expression was investigated by western blotting. Panel (A) is a representative western blot image and panel (B) is a densitometric analysis. Data are normalised to α-actin and are presented as a percentage of the control. Data were analysed using one-way ANOVA with Bonferroni’s adjustment and are presented as the mean ± SEM of 3 independent experiments, *p< 0.05; **p< 0.01.

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The effect of pre-treatment of ripasudil on the phosphorylation level of MLC protein in myometrial tissues treated with U46619 was also examined in pregnant mice. Exposure of myometrial strips isolated from the uterus of pregnant C57 mouse at term (E19) to cumulative concentrations of U46619 (10-9M – 10-5M) in the organ baths caused a slight but not significant reduction (p=0.11) in the mean expression level of MLC compared with the control (vehicle, 0.9% w/v normal saline). When the same tissues were pre- treated with ripasudil at 10-6M before adding the U46619 (10-9M – 10-5M), there was a small elevation in the mean expression level of the non-phosphorylated form of MLC when compared to the samples treated with the U46619 alone, but this increase was below the level of significance. Furthermore, no difference in MLC expression was observed between the control and ripasudil+U46619 groups as shown in Figure 4.19. The blot image from the Figure shows the variation in band densities among U46619 alone, ripasudil+U46619 and control groups, taking into account that band densities are normalised to α-actin.

Figure 4.19. Effect of U46619 on MLC expression in myometrial tissue from pregnant C57 mice at term (E19) in the presence and absence of ripasudil. Effect of U46619 on the protein expression of MLC in pregnant C57 mouse myometrium at term (E19) in the presence and absence of ripasudil 10-6M. Tissues were treated either with the control (vehicle, 0.9% w/v normal saline), cumulative concentrations of U46619 (10-9M - 10-5M) or pre-treated with ripasudil 10-6M and then with cumulative concentrations of U46619 (10-9M - 10-5M) using immersion technique and protein expression was investigated by western blotting. Panel (A) is a representative western blot image and panel (B) is a densitometric analysis. Data are normalised to α-actin and are presented as a percentage of the control. Data were analysed using one-way ANOVA with Bonferroni’s adjustment and are presented as the mean ± SEM of 3 independent experiments, (ns) not significant.

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Treatment of myometrial tissues taken from pregnant C57 mouse uteri at term (E19) with cumulative concentrations of U46619 (10-9M – 10-5M) elicited a high elevation in pMLC expression when compared to the control (vehicle, 0.9% w/v normal saline or DMSO at ripasudil 10-5M). The elevation was extremely significant with a p-value of less than 0.0001. Pre-treatment of myometrial strips with ripasudil at 10-6M prior to the addition of the cumulative concentrations of U46619 (10-9M – 10-5M) caused a statistically significant decrease in the expression of pMLC compared with the sample treated with the U46619 alone (p<0.01). However, no significant variation of expression was found in pMLC between the ripasudil+U46619 and control groups. Figure 4.20 displays the level of variation in pMLC expression and band densities among all groups.

Figure 4.20. Effect of U46619 on pMLC expression in myometrial tissue from pregnant C57 mice at term (E19) in the presence and absence of ripasudil. Effect of U46619 on the protein expression of pMLC in pregnant C57 mouse myometrium at term (E19) in the presence and absence of ripasudil 10-6M. Tissues were treated either with the control (vehicle, 0.9% w/v normal saline), cumulative concentrations of U46619 (10-9M - 10- 5M) or pre-treated with ripasudil 10-6M and then with cumulative concentrations of U46619 (10- 9M - 10-5M) using immersion technique and protein expression was investigated by western blotting. Panel (A) is a representative western blot image and panel (B) is a densitometric analysis. Data are normalised to α-actin and are presented as a percentage of the control. Data were analysed using one-way ANOVA with Bonferroni’s adjustment and are presented as the mean ± SEM of 3 independent experiments, **p< 0.01; ****p< 0.0001.

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The most significant difference in protein expression in U46619 experiments in pregnant mice was observed with the di-phosphorylated MLC (ppMLC), where treating myometrial strips isolated from pregnant C57 mouse uterus at term (E19) with the U46619 at cumulative concentrations of 10-9M – 10-5M induced an extremely significant increase in ppMLC expression compared to the control (vehicle, 0.9% w/v normal saline) group (p<0.0001). The level of ppMLC was about three-fold higher in the U46619 group than in the controls. Similar differences in the degree of significance and protein levels were seen between the U46619 alone-group and the group pre-treated with ripasudil at 10-6M and then with the same cumulative concentrations of U46619, where the U46619 alone-group showed higher expression of the protein (p<0.0001). The level of expression of ppMLC in the ripasudil+U46619 group was approximately comparable to that of the control group as shown in Figure 4.21.

Figure 4.21. Effect of U46619 on ppMLC expression in myometrial tissue from pregnant C57 mice at term (E19) in the presence and absence of ripasudil. Effect of U46619 on the protein expression of ppMLC in pregnant C57 mouse myometrium at term (E19) in the presence and absence of ripasudil 10-6M. Tissues were treated either with the control (vehicle, 0.9% w/v normal saline), cumulative concentrations of U46619 (10-9M - 10- 5M) or pre-treated with ripasudil 10-6M and then with cumulative concentrations of U46619 (10- 9M - 10-5M) using immersion technique and protein expression was investigated by western blotting. Panel (A) is a representative western blot image and panel (B) is a densitometric analysis. Data are normalised to α-actin and are presented as a percentage of the control. Data were analysed using one-way ANOVA with Bonferroni’s adjustment and are presented as the mean ± SEM of 3 independent experiments, ****p<0.0001.

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The expression of the MLC protein family in the myometrium of pregnant mouse uterus has also been investigated following exposure to the serotonin receptor agonist, 5-HT in the presence and absence of ripasudil.

Treatment of myometrial strips taken from pregnant C57 mouse uteri at term (E19) with cumulative concentrations of 5-HT (10-9M – 10-5M) using the immersion technique did not cause a significant change in the expression of MLC when compared to the control (vehicle, 0.9% w/v normal saline) strips. At the same time, pre-exposing the pregnant mouse myometrial strips to 10-6M of ripasudil before treatment with 5-HT at cumulative concentrations of 10-9M – 10-5M did not yield to any significant variation in MLC expression compared with the 5-HT treatment in the absence of ripasudil (Figure 4.23). Similarly, the control and ripasudil+5-HT groups exhibited comparable expression of MLC in these tissues as seen in the graph and the bands of the western blotting image of Figure 4.22.

Figure 4.22. Effect of 5-HT on MLC expression in myometrial tissue from pregnant C57 mice at term (E19) in the presence and absence of ripasudil. Effect of 5-HT on the protein expression of MLC in pregnant C57 mouse myometrium at term (E19) in the presence and absence of ripasudil 10-6M. Tissues were treated either with the control (vehicle, 0.9% w/v normal saline), cumulative concentrations of 5-HT (10-9M - 10-5M) or pre-treated with ripasudil 10-6M and then with cumulative concentrations of 5-HT (10-9M - 10-5M) using immersion technique and protein expression was investigated by western blotting. Panel (A) is a representative western blot image and panel (B) is a densitometric analysis. Data are normalised to α-actin and are presented as a percentage of the control. Data were analysed using one-way ANOVA with Bonferroni’s adjustment and are presented as the mean ± SEM of 3 independent experiments, (ns) not significant.

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The expression of pMLC protein, after treating the tissue with increasing concentrations of 5-HT (10-9M – 10-5M), was significantly increased (p<0.05) as seen in Figure 4.24. The presence of ripasudil at 10-6M led to a reduction in the expression of pMLC when compared to the samples exposed to 5-HT alone (p<0.05). Similar to U46619 experiments, no significant alterations in pMLC protein density was found in the control and ripasudil+5-HT-treated samples. Figure 4.23 displays the difference in band densities and protein levels of pMLC among all groups.

Figure 4.23. Effect of 5-HT on pMLC expression in myometrial tissue from pregnant C57 mice at term (E19) in the presence and absence of ripasudil. Effect of 5-HT on the expression of pMLC in pregnant C57 mouse myometrium at term (E19) in the presence and absence of ripasudil 10-6M. Tissues were treated either with the control (vehicle, 0.9% w/v normal saline), cumulative concentrations of 5-HT (10-9M - 10-5M) or pre- treated with ripasudil 10-6M and then with cumulative concentrations of 5-HT (10-9M - 10-5M) using immersion technique and protein expression was investigated by western blotting. Panel (A) is a representative western blot image and panel (B) is a densitometric analysis. Data are normalised to α-actin and are presented as a percentage of the control. Data were analysed using one-way ANOVA with Bonferroni’s adjustment and are presented as the mean ± SEM of 3 independent experiments, *p< 0.05.

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The myometrium of pregnant C57 mouse uteri at term (E19) was also investigated for level of di-phosphorylation of MLC following incubation with 5-HT in challenge with ripasudil. The results demonstrated that the increasing concentrations of 5-HT (10-9M – 10-5M) evoked a significant elevation in ppMLC expression when compared to the control (vehicle, 0.9% w/v normal saline) (p<0.05). Pre-treating these tissues with ripasudil at 10-6M prior to the exposure to cumulative concentrations of 5-HT (10-9M – 10-5M) decreased the expression of ppMLC compared to the 5-HT alone group. Although the decrease did not achieve statistical significance (p=0.0539), the graph and blot image of Figure 4.25 show that the ripasudil has caused some inhibition in the mean level of protein expression. No significant difference in the expression of ppMLC was detected between the control and ripasudil+5-HT-treated groups (Figure 4.24).

Figure 4.24. Effect of 5-HT on ppMLC expression in myometrial tissue from pregnant C57 mice at term (E19) in the presence and absence of ripasudil. Effect of 5-HT on the protein expression of ppMLC in pregnant C57 mouse myometrium at term (E19) in the presence and absence of ripasudil 10-6M. Tissues were treated either with the control (vehicle, 0.9% w/v normal saline), cumulative concentrations of 5-HT (10-9M - 10-5M) or pre-treated with ripasudil 10-6M and then with cumulative concentrations of 5-HT (10-9M - 10-5M) using immersion technique and protein expression was investigated by western blotting. Panel (A) is a representative western blot image and panel (B) is a densitometric analysis. Data are normalised to α-actin and are presented as a percentage of the control. Data were analysed using one-way ANOVA with Bonferroni’s adjustment and are presented as the mean ± SEM of 3 independent experiments, *p<0.05; (ns) not significant.

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Table 4.1 displays the comparison of the effect of the three uterine stimulants used, OXT, U46619 and 5-HT in cumulative concentrations on the expression of the MLC family in the myometrium obtained from pregnant C57 mouse uteri at term (E19). The protein expression of MLC was higher in the OXT-treated samples followed by those exposed to 5-HT and U46619, respectively. The level of pMLC expression was comparable in uterine tissues treated with OXT and U46619, but was lower than that of 5-HT-exposed strips. The most marked effect of the three drugs was on the expression of ppMLC, where both OXT and U46619 caused a similar and very high expression of the protein when compared to the 5-HT group.

Table 4.1. Effect of OXT, U46619, 5-HT and ripasudil on MLC family expression in myometrial tissue from pregnant C57 mice at term (E19). Effect of cumulative concentration of OXT, U46619, 5-HT and ripasudil on the protein expression of MLC, pMLC and ppMLC in pregnant C57 mouse myometrium at term (E19). Tissues were treated either with cumulative concentrations of OXT (10-12M - 10-6M), U46619 (10-9M - 10-5M) or 5-HT (10-9M - 10-5M) using immersion technique and protein expression was investigated by western blotting. Band densities were measured and then normalised to α-actin and are presented as a percentage of the control (vehicle, 0.9% w/v normal saline). Data are presented as the mean ± SEM of 3 independent experiments.

OXT U46619 5-HT Ripasudil

MLC 91.6 ± 11.4 77.7 ± 10.9 85 ± 15.75 124.6 ± 5.7

pMLC 137.8 ± 3.5 132.7 ± 1.0 177.6 ± 27.7 82.4 ± 4.2

ppMLC 378.1 ± 69.3 379.6 ± 8.2 271.7 ± 39.4 13.2 ± 5.9

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4.8 Discussion

In this part of the study, uterine tissue was taken from pregnant mice at term (E19; 19th day of embryonic age or gestation), which represents week 36.1 in human pregnancy. During pregnancy, the uterus undergoes several physiological and anatomical changes in order to nurture and accommodate the developing foetus. This study demonstrates that the myometrial tissues taken from these pregnant mice were able to contract spontaneously in vitro. The reason behind this spontaneous activity, which is seen in uterine myocytes, is the development of and frequent depolarisations of resting membrane potential which happen as a consequence of the unequal distribution of ions across the both sides of the membrane (Parkington and Coleman, 1990).

The size of the myometrium of pregnant mice was much larger than that of non- pregnant mice and thus, it was possible to isolate several strips from the upper segment of the uterine horn and the number of these isolated strips was enough to occupy all the organ baths available in the immersion apparatus for each experiment. This was also helpful to compare the effect of different treatments on the same part (upper segment) of the horn in one immersion experiment. Lower segments of uterine horn of pregnant mice were not examined in this study due to the above reason and also due to the duration of immersion method, which lasted for several hours when samples from pregnant mice were used and strips from the lower segment were found to lose their spontaneous activity when left in aerated physiological solution for an extended period.

Myometrial strips taken from pregnant mice at term (E19) showed higher levels of spontaneous activity when compared with the non-pregnant mouse uterus in dioestrus. These results are consistent with previous studies on mice where spontaneous myometrial activity was greater in pregnant mice at late pregnancy than that in non- pregnant mice (Patel et al., 2017, Gravina et al., 2014). In addition, the duration of spontaneous contraction in pregnant mouse uterus at term was longer than that of the non-pregnant mouse as shown in Figure 4.2 where the duration was 9 times longer in the pregnant samples. As discussed in Chapter 3, the movement of external Ca2+ through the voltage-gated L-type Ca2+ channels plays an essential role in mediating the spontaneous contractile activity of the myometrium (Matthew et al., 2004). Increased intracellular Ca2+ concentration stimulates the formation the Ca2+-calmodulin complex

232 and MLCK which in turn will induce the phosphorylation of MLC20 and stimulate actin-activated myosin ATPase activity leading to spontaneous contractile activity (Word, 1995). The high activity of myometrial tissue seen in the pregnant uterus compared to the non-pregnant uterus might be attributed to the elevated levels of oestrogen which increase during late pregnancy and term, while the progesterone levels tend to decrease toward term. The concentrations of 17β-oestradiol start to increase on day 16 of pregnancy and go on until term, whereas progesterone levels starts to decrease on day 17 of gestation and reaches the lowest concentration at term (McCormack and Greenwald, 1974). Oestrogen has the ability to regulate multiple processes which lead to uterine stimulation at term, these include release of oxytocin, expression of oxytocin receptors (Fang et al., 1996), occurrence of PG receptors (Blesson et al., 2012) and gap junction proteins (Petrocelli and Lye, 1993). On the other hand, dioestrus is characterized by high progesterone to oestrogen ratio (Walmer et al., 1992), and this high ratio in the non-pregnant samples of this study could be regarded as another factor that contributes to the lower spontaneous myometrial activity observed in non-pregnant samples, compared to the pregnant uterine tissues.

In order to assess the effect of the hormonal milieu on the activity of uterine tissue in pregnant and non-pregnant mice, plasma 17β-oestradiol concentrations were measured and compared. Although the result in this study showed no significant variation (p=0.166) in 17β-oestradiol concentrations between the plasma of pregnant and non- pregnant mice, data showed some elevation in the mean level of this hormone in the pregnant mice at term when compared to the non-pregnant mice in dioestrus as observed in Figure 4.3. Individual data indicates that there are some variations among non-pregnant samples which may interpret the reason behind the non-significant statistical difference between the two groups. The high plasma levels of oestradiol observed during late pregnancy and at term has been also reported in previous studies in human and mouse (McCormack and Greenwald, 1974, Kaludjerovic et al., 2012). Furthermore, some plasma samples from non-pregnant mice may have been collected at the late dioestrous stage where the progesterone concentration is decreased, and the ovaries begin to release oestrogen which then reaches the blood circulation and elevates plasma oestrogen levels (Walmer et al., 1992). As discussed above, the high concentration of oestrogen can stimulate certain receptors and potentiate contractile proteins which result in increased myometrial activity in pregnant mouse uterus at term.

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The effect of litter size or number of pups on spontaneous myometrial activity in pregnant C57 mice at term was also examined in this study. Variable measurements of spontaneous contraction were recorded and data showed no correlation between litter size and uterine contraction. Due to the small number of samples in each group (n=1-2), it was difficult to conduct statistical analysis to conclude any relation between the litter size and spontaneous activity. However, high litter size can increase the stretch of uterine tissue (Patel et al., 2017) which in turn will stimulate a transient contractile activity due to the induction of Ca2+ entry into the cells. This process is myogenic and relies on the speed and direction of the stretch caused. In addition to that, oxytocin- stimulated rhythmic contractions can be regulated by stretch (Kasai et al., 1995).

Furthermore, mechanical stretch increases uterine contractile activity as seen in the traces of initial contraction of myometrial strips in both pregnant and non-pregnant mouse uteri after the tissues were held in organ baths and washed with Krebs’ solution (Figure 4.2). Then, the level of contraction decreased gradually until it reached an approximately constant frequency. This is in agreement with previous studies in animals which demonstrated that stretch is a main modulator of smooth muscle contraction and can potentiate uterine contractile activity, and stretch is also correlated with the elevation in the release and expression of certain contraction-associated proteins such as COX-2, OTR and connexion-43 (Ou et al., 1997, Ou et al., 1998, Sun et al., 2018). Similar results have been found in human studies where stretch was able to regulate PG release and induce uterine contractile activity (Sooranna et al., 2004, Manbe et al., 1982).

As described in the introduction and discussed in the previous Chapter, the ROCK inhibitor, ripasudil was discovered and used in the management of glaucoma (Garnock- Jones, 2014) and it has been demonstrated that it can modulate the contraction of smooth muscle via inhibiting the activity of ROCK pathway (Isobe et al., 2014). Blocking the ROCK pathway enhances the transformation of pMLC to MLC and also it prevents the di-phosphorylation of pMLC to ppMLC (Aguilar et al., 2012). Both actions lead to the conversion of muscle state from contraction into relaxation (see Figure 1.12).

To examine the effect of cumulative concentrations of ripasudil on the spontaneous activity of myometrium isolated from pregnant mouse at term, a series of ripasudil concentrations were used. The concentration range of ripasudil used in the treatment of

234 pregnant myometrial strips was similar to the range used with non-pregnant mouse uterine samples. These concentrations were selected according to previous investigations in human tissues (Isobe et al., 2014). Incremental concentrations of ripasudil added into the organ baths were able to diminish the myometrial spontaneous activity of pregnant mouse uterus at term. The amplitude of muscular contraction was also reduced at high ripasudil concentrations. This action of ripasudil demonstrates the involvement of Rho/ROCK signalling pathway in the regulation of myogenic activity of the mouse uterus in late gestation and also suggests the ability of ripasudil to block this pathway and control myometrial contraction at term. Previous research has reported the occurrence and activity of the ROCK pathway in myometrial contraction at term in humans (Friel et al., 2005), mice (Riley et al., 2005) and rats (Taggart et al., 2012). Contradictory findings concerning the up-regulation of the ROCK pathway in late pregnancy have been reported in WT mice (Harrod et al., 2011). This was the first study to examine the influence of ripasudil on the spontaneous activity of myometrium of pregnant mouse uterus at term. The results of this work are in agreement with previous studies in humans, where other ROCK inhibitors have exerted their ability to inhibit human spontaneous myometrial contraction at term (Kupittayanant et al., 2001, Hudson et al., 2011). To the best of our knowledge, there have been no studies showing the effect of ROCK inhibitors on spontaneous uterine contractility in pregnant mice.

Oxytocin (OXT) exerts central and peripheral effects on body organs; it has several functions in multiple physiological and pathophysiological mechanisms, such as labour, lactation, erectile dysfunction and other processes which are associated with social attitude (Bethlehem et al., 2013, Arrowsmith and Wray, 2014). One of the main roles of OXT during labour is the induction of uterine contraction; therefore, its effects have been widely investigated in pregnant human and animal samples. OXT acts through binding and activating OXT receptors (OTR) which are classified as typical class I G protein-coupled receptors and their high-affinity situation is mainly regulated by Mg2+ and cholesterol. The principle physiological functions of OXT are induction of smooth muscle contractility of the uterus during parturition and the release of milk during breastfeeding (Gimpl and Fahrenholz, 2001). OXT induces myometrial contractions through several mechanisms which include the release of Ca2+ from intracellular stores and increased tissue responsiveness to the released Ca2+ which is known as Ca2+-

235 sensitization, a mechanism leading to a higher contraction (Somlyo and Somlyo, 1998, Shmygol et al., 2006).

To determine the role of functional myometrial OXR receptor populations, the effect of ripasudil on OXT-induced uterine contraction, in vitro examinations were conducted using strips isolated from pregnant mice at term (E19). OXT gradually increased uterine contractility in the upper uterine segments reaching a peak effect at 10-7M OXT. However, a reduction in the response to OXT was observed in all treatment groups at the maximum OXT concentration used (10-6M). This pattern of OXT action was similar in the presence and absence of ripasudil. The effect of OXT on these isolated myometrial strips indicates the expression and activity of OXR in the mouse uterus at late pregnancy. Similar findings were observed in with previous studies in mice (Padol et al., 2017), rats (Edwards et al., 1986) and human (Yin et al., 2018).

Interestingly, ripasudil and at its two concentrations (10-5M and 10-6M) demonstrated its ability to inhibit OXT-induced myometrial contractility in the pregnant mouse uterus at term especially at high OXT concentrations. These data demonstrate involvement of the ROCK pathway in OXT-induced myometrial contractility at late pregnancy. The traces show a decrease in the contraction force in the samples pre-treated with ripasudil 10-6M and 10-5M (in particular 10-5M) and OXT compared to the OXT-alone treated group. These results are consistent with previous research on rat uterus at late pregnancy where other ROCK inhibitors have inhibited OXT-stimulated contraction (Tahara et al., 2002). Recently, this correlation of oxytocin and ROCK pathways has also been demonstrated in the vascular smooth muscle of the Wistar rats (Soti, 2019).

Surprisingly, the response to OXT above 10-7M showed some attenuation in the examined uterine segments and this reduction in contraction has followed the same pattern in the presence and absence of ripasudil. This reduction in the myometrial activity at high OXT concentration is likely to be due to receptor desensitisation. Similar findings were reported in previous studies in human myometrial cells where long exposure to OXT has caused a reduction in OXT binding to cell membrane (Phaneuf et al., 1997, Robinson et al., 2003). Receptor desensitization comprises a series of mechanisms which have been recognized as uncoupling of G-protein from receptors, receptor phosphorylation, receptor sequestration and its down-regulation (Kelly et al., 2008, Ferguson, 2001, Hanania and Cazzol, 2008). The OXR

236 desensitisation which is evoked by OXT involves a reduction in receptor population in the human myometrial cells and this receptor desensitisation can be almost entirely overcome by the addition of the synthetic OXR antagonist, atosiban (Phaneuf et al., 1994).

The expression and role of TP receptors in modulating myometrial contractility during pregnancy and labour have been studied previously and the activity of TXA2 analogues on these receptors and on the uterine contraction was also examined in various species (Fischer et al., 2008, Griffiths et al., 2006, Griffiths, 2007, Smith et al., 2001). In order to test the function of TP receptors in myometrial strips isolated from pregnant mouse uterus at term and also to examine the activity of ROCK inhibitors against TP agonists, the stable analogue of TXA2, U46619 was challenged with ripasudil. Concentrations of U46619 were chosen depending on previous investigations in mouse uterus (Griffiths, 2007), whereas ripasudil concentrations were selected depending on preliminary experiments in this work. Previous research demonstrated the ability of U46619 to stimulate the ROCK pathway via activating TP receptors in uterus of pregnant human (Senior et al., 1993, Duckworth et al., 2002, Fischer, 2010) and mouse (Griffiths et al., 2006) samples at late gestation and at term. It was also demonstrated that responses to TP receptor activation may include the Ca2+ signalling pathway through hydrolysing phosphatase C-catalysed phosphoinositol, which leads to the mobilization of intracellular Ca2+ (Nakahata, 2008, Nakahata et al., 1989). Data also showed that U46619 at high concentration (10-6M) increased the frequency of contraction as observed in the representative traces (Figure 4.11). We speculate that this may be due to the effect of this TXA2 agonist on targets other than the TP receptors. Studies have demonstrated that FP receptor antagonists were able to inhibit the stimulation induced by the U46619, which indicates the off-target effects of this drug (Vysniauskiene et al., 2006). The results of this study showed that U46619 with increasing concentration can induce myometrial contractility in pregnant mouse uterus at term, which demonstrates the expression of TP receptors in the myometrium during this stage of gestation. Our findings are in agreement with other studies on human and murine species (Fischer, 2010, Griffiths, 2007).

Uterine tissue pre-incubated with vehicle or ripasudil at either concentration (10-6M and 10-5M) and then challenged with U46619 showed that ripasudil was able to inhibit the U46619-stimulated myometrial contraction of pregnant mouse uterus. This action of

237 ripasudil further indicates that U46619 stimulation may involve the ROCK pathway and that ROCK inhibitors can be exploited to diminish this effect of TP receptor agonists. To date, no published research has examined the effect of ROCK inhibitors on the myometrial contraction which is induced by TP receptor agonists. However, some similar findings were reported in human studies on myometrial and placental arteries isolated from pregnant women at term where ROCK inhibitors abolished U46619- induced contraction of these tissues (Wareing et al., 2005).

To investigate the expression and function of 5-HT receptors in myometrial strips isolated from the pregnant mouse at term and also to examine the relationship between these receptors and the ROCK pathway, uterine tissues were treated with 5-HT in the presence and absence of ripasudil. 5-HT concentrations were selected according to previous studies in human, mice and rats (Griffiths, 2007, Cordeaux et al., 2009, Osman and Ammar, 1975), whereas those of ripasudil were determined after several preliminary experiments in this study (Figures 3.7 and 3.9). Cumulative concentrations of 5-HT resulted in increased stimulation of spontaneous myometrial activity in isolated upper uterine segments. These data are in agreement with other studies on non-pregnant mice uterus (Xiu-Kun et al., 2011) but at the same time the data are contrary to previous work on pregnant human myometrium at term that have suggested that 5-HT has no effect on spontaneous uterine contractions under normal physiological conditions and that the high responsiveness to 5-HT is due to the aberrant 5-HT signalling, which may also participate in the development of preterm delivery (Cordeaux et al., 2009). Possibly this inconsistency between their work and our study might be due to species variation. As with non-pregnant uterine samples, the response of myometrial strips to 5-HT at high concentrations (10-6M) showed attenuation in tissue activity. This might be due to receptor desensitization after the long exposure of the tissue to 5-HT. Desensitisation of 5-HT receptors have been also reported in previous studies in the brain and the gut (Hanley and Hensler, 2002, Maroteaux et al., 2016).

Interestingly, ripasudil at both concentrations, 10-6M and 10-5M, was able to inhibit 5- HT-induced myometrial contraction as well as significantly reduce the maximum uterine stimulation produced by 5-HT; this can also be seen in the traces from all treatment groups. This effect of ripasudil demonstrates the involvement of 5-HT on the relationship between 5-HT receptor stimulation and ROCK pathways in pregnant uterine tissue. Activation of 5-HT receptors can induce Rho/Rho kinase and

238 subsequently stimulates nuclear transcription factors in pulmonary fibroblasts in the mice (Mair et al., 2008). More investigations are needed to explore the role of 5-HT receptors and transporters in the uterine tissues during gestation and parturition and the interaction of this system with the ROCK pathway.

Recently, research has focused on examining the expression and activation of Rho/Rho kinase system in pregnant and non-pregnant human and animal species. The research has also investigated the influence of certain inhibitors of the ROCK pathway on the expression and activity of contractile proteins in uterine tissues during spontaneous myogenic activity and after treating the myometrium with certain uterine stimulants (Tahara et al., 2002, Wray, 1993, Aguilar and Mitchell, 2010, Ergul et al., 2016). To the best of our knowledge, this is the first study to investigate the role of the ROCK pathway inhibitor, ripasudil in modulating the expression and transformation of the contractile proteins associated with Rho/Rho kinase system in uterine tissues isolated from pregnant mouse at term after stimulation of the tissue with various agents. The ROCK pathway is known to be activated during the last stages of gestation which then helps to facilitate the process of delivery and termination of pregnancy at term. Stimulation of ROCK will increase the transformation of the non-contractile MLC protein into the phosphorylated and contractile forms of MLC (Tahara et al., 2002). Upon the activation of Rho kinase, MLC can be phosphorylated into two MLC isoforms, pMLC and ppMLC. Both, pMLC and ppMLC enhance uterine contraction in isolated myometrium at term (Aguilar et al., 2012). In this work, ripasudil was able to regulate the protein expression of the three MLC form, MLC, pMLC and ppMLC. Myometrial strips isolated from pregnant mice at term and treated with cumulative concentrations of ripasudil after immersion experiments showed that ripasudil had the ability to cause a significant elevation in the expression of the non-phosphorylated form of MLC when compared with the control group. This interesting result demonstrates that inhibition of ROCK pathway can abolish MLC phosphorylation even in tissues not treated with uterine stimulants. Accumulation of MLC decreases the relative levels of pMLC and ppMLC, and subsequently will facilitate relaxation of smooth muscle and reduce muscular contractile force (Word et al., 1994). The effect which was exerted by ripasudil further confirms the role of the ROCK pathway in the regulation of uterine spontaneous contractility, as ROCK inhibitors have shown their ability to reduce the amplitude of myogenic contraction in the myometrium isolated from pregnant women

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(Hudson et al., 2012) and rabbits (Cario-Toumaniantz et al., 2003) at term. These data are consistent with previous studies when ROCK inhibitors were able to inhibit the conversion of MLC to its phosphorylated forms in myometrial cells isolated from pregnant human at term (Aguilar et al., 2011). A representative western blot image (Figure 4.14) shows that there was a slight increase in the level of pMLC in the control group when compared with the ripasudil treated samples and this further suggests the higher activated state of the myometrium which contracting spontaneously over the one treated with ripasudil.

Moreover, treatment of myometrial strips isolated from pregnant mouse uterus at term with cumulative concentrations of ripasudil decreased the level of mono-phosphorylated MLC (pMLC) expression compared to the control. This significant outcome can further demonstrate the reason behind the reduction in spontaneous activity of the myometrium in non-pregnant mouse uterus and the decrease in the amplitude and frequency of contraction observed in immersion after treating these strips with ripasudil. This also substantiates ripasudil as a ROCK inhibitor and that the phosphorylation of MLC is an essential step in stimulating uterine contraction. Similar findings were seen in previous studies on the myometrium of human and rats at late pregnancy and term (Aguilar et al., 2011, Oh et al., 2003).

The most interesting and significant finding from this work was the influence of ripasudil on the di-phosphorylation step of MLC. The data from western blotting experiments showed that ripasudil led to a significant reduction in the expression of the di-phosphorylated form of MLC, ppMLC in the myometrial strips of pregnant uterus when compared to the control group. The action of ripasudil on MLC phosphorylation more profoundly affected MLC di-phosphorylation than MLC mono-phosphorylation. This suggests that the di-phosphorylation of MLC is a very sensitive and critical step in pregnant uterine tissue at term and thus, blocking this mechanism may be crutial to modulate uterine contractions at late stages of pregnancy and to prevent preterm labour. This is the first work to investigate the role of ripasudil on the expression of MLC protein family in the mouse uterus. There have been some similar studies on the effect of other ROCK inhibitors on pregnant human myometrial cells at term and on rat aortic smooth muscle cells (Aguilar et al., 2011, Hsu et al., 2019). The di-phosphorylation of MLC is only a recent discovery and very few studies document this phosphorylation event. There is still no evidence on the expression and activity of the di-phosphorylated

240 form of MLC in the mouse uterus and more investigations are required to explore this mechanism and its function during gestation and labour.

The oxytocin signalling pathway involves activation of the RhoA/Rho kinase system and phosphorylation of MLC during pregnancy and at term, which in turn induces myometrial contraction (Tahara et al., 2002, Arthur et al., 2007). Our data showed that treating myometrial strips isolated from term pregnant mouse uterus with cumulative concentrations of OXT using immersion technique did not change the level of MLC expression when compared to the control group. However, when these OXT-treated tissues were exposed to ripasudil at 10-6M the level of protein expression appeared to be elevated even though this increase was below the level of significance. Similarities in MLC expression between the OXT-treated and control groups might be because OXT predominantly exerts its regulatory effect on the phosphorylation process, rather than on the accumulation of the non-contractile form of MLC. In addition to that, the phasic type of contraction in the uterine tissue might influence the rapid protein phosphorylation and dephosphorylation processes described in other studies on human myometrial samples (Paul et al., 2011). Furthermore, the slight elevation in MLC expression in the OXT-treated samples which were pre-treated with ripasudil supports the possibility of blocking MLC phosphorylation by the ROCK inhibitor, ripasudil. This idea can be further confirmed when observing the western blotting data assessing the expression of the mono-phosphorylated form of MLC, pMLC where cumulative concentrations of OXT caused a statistically significant increase in pMLC expression compared with the control group. This effect supports the idea of OXT activating the ROCK cascade and transformation of MLC protein to its contractile form. Similar findings were reported in other research in pregnant human and rat myometrium at term (Shojo and Kaneko, 2001, Tahara et al., 2002, Aguilar et al., 2011).

Moreover, investigating the effect of cumulative concentrations of oxytocin using immersion on the expression of pMLC in the presence and absence of ripasudil showed that OXT was able to significantly increase the expression of this protein in the treated myometrial strips when compared to the control. Conversion of MLC to pMLC is associated with enhanced uterine muscular contractility and this can further confirm the involvement of OXT through activating OXR in the stimulation of RhoA/Rho kinase- mediated Ca2+ sensitization and downstream effects (Kupittayanant et al., 2001). The data are consistent with previous research on the mono-phosphorylation of MLC in

241 pregnant human and rat myometrium (Aguilar et al., 2011, Shojo and Kaneko, 2001). On the other hand, pre-treatment of the above OXT-treated myometrial strips with ripasudil resulted in a significant inhibition of pMLC expression compared to the OXT- treated alone samples. Ripasudil has shown its ability to block the OXT-activated ROCK pathway through inhibiting the downstream actions associated with this pathway which is represented by the transformation of MLC into pMLC. This demonstrates the activity of ripasudil to regulate myometrial contractility in tissues stimulated with certain uterine stimulants. Similar results were found with other ROCK inhibitors after stimulating pregnant human and rat myometrial strips with OXT at term (Tahara et al., 2002, Hudson et al., 2012).

Di-phosphorylation of MLC has been investigated recently in different human tissues and it has been demonstrated that di-phosphorylation occurs at Ser19 and Thr18 sites on MLC. This process was confirmed in human vascular and uterine smooth muscles (Aguilar et al., 2012) as well as in the caudal and thoracic arterial smooth muscle of rats (Takeya et al., 2014, Matsui and Deguchi, 2019). Until recently, no evidence has been reported on the expression of this di-phosphorylation event in the uterus of pregnant or non-pregnant mice; we found it necessary to examine the occurrence of this mechanism in these mouse tissues and also to test the influence of different uterine stimulants on the process and the role of ROCK inhibitor in its regulation. In the myometrial strips isolated from pregnant mouse uterus at term and treated with cumulative concentrations of OXT in the organ baths, the level of ppMLC expression was significantly higher than that of the control group. This demonstrates that activation of the ROCK signalling pathway by OXT is involved in the di-phosphorylation of MLC and the production of ppMLC will lead to a greater stimulation of uterine contraction. These data are in agreement with previous work on human uterine smooth muscle (Aguilar et al., 2012, Aguilar et al., 2011). Interestingly, pre-treatment of the myometrial strips mounted in immersion with ripasudil before administration of OXT resulted in a significant reduction in ppMLC expression compared to the samples treated with OXT in the absence of ripasudil. This is a very important result as it demonstrates that the ROCK cascade system plays an essential role in the di-phosphorylation on MLC in uterine tissue and in the modulation of myometrial contractility at the late stages of gestation. Therefore, blocking the ROCK pathway with inhibitors such as ripasudil could be used to control uterine activity during pregnancy and labour. These data are consistent with

242 the work of other researchers who used other ROCK inhibitors (selective ROCKI and ROCKII inhibitors) to inhibit OXT-induced MLC di-phosphorylation and myometrial contractility in human uterus at term (Aguilar et al., 2012). They have also concluded that blocking of ppMLC expression in human myometrial cells is 100 times more responsive to the action of ROCK inhibitors than the effect of these agents on pMLC expression in the human vascular myocytes. Therefore low doses of ROCK inhibitors might be required to attenuate myometrial contractions without evoking profound adverse reactions on vascular smooth muscle contractile force.

U46619, the synthetic analogue of TXA2 is known to act through TP receptors, which in turn leads to stimulation of RhoA/Rho kinase signalling pathway. This mechanism has been demonstrated in human and rats (Wareing et al., 2005, Grann et al., 2016). Activation of the ROCK pathway induces the phosphorylation of MLC and enhances uterine contractility (Lartey and Bernal, 2009). To further explore the role of ripasudil as a ROCK inhibitor in the phosphorylation of MLC in the pregnant mouse uterus at term after exposure to TP receptor agonists, myometrial strips were treated with cumulative concentrations of U46619 in the presence and absence of ripasudil. Data showed that even though no significant difference was seen in the expression of MLC between the U46619 and control group, the expression of the protein seems to be slightly lower in the U46619-treated samples. It is generally expected that TP agonists reduce the expression of the non-phosphorylated MLC and increase its transformation into the phosphorylated forms which enhance smooth muscle contractility (Jiang et al., 1994). In addition, ripasudil at 10-6M evoked elevation in MLC expression in the U46619-treated strips compared to the group treated with U46619 alone, although this elevation was below a significant level. This further demonstrates the hypothesis that stimulation of myometrial contraction which is induced by MLC phosphorylation through activating TP receptors can be inhibited by the ROCK inhibitors and thus, ROCK inhibitors can modulate the phosphorylation of contractile proteins and regulate uterine contractility during gestation and at term. This interaction between TP agonists and ROCK inhibitors has been illustrated in human myometrial and placental arteries at term pregnancy (Wareing et al., 2005).

The effect of U46619 was more evident for mono-phosphorylation of MLC after myometrial strips were treated with cumulative concentrations of U46619 in the presence and absence of ripasudil. U46619 caused a significant increase in the

243 expression of pMLC compared to the control group (p0.0001). This demonstrates the effectiveness of TP agonists in stimulating uterine contractility at the late stages of pregnancy (Griffiths et al., 2006) through phosphorylation of the MLC (Dorn and Becker, 1993). Interestingly, pre-incubation of myometrial strips from pregnant mice with ripasudil prior to administration of cumulative concentration of U46619 in the organ baths resulted in a significant inhibition of pMLC expression when compared to the U46619-treated alone group. This can be regarded as a promising finding and it indicates the activity of ripasudil as a ROCK inhibitor to prevent the downstream consequences of TP activation at late pregnancy and term gestation through blocking the undesired uterine contractions. This is the first study to examine the effect of ROCK inhibitors on TP-induced MLC phosphorylation in pregnant mouse uterus. However, these data are in agreement with previous studies on rat arteries where ROCK inhibitors were able to attenuate the U46619-stimulated smooth muscle contractions and MLC phosphorylation (Tsai and Jiang, 2006). Further investigations are needed to explore the thromboxane-ROCK-MLC interaction in other species using various ROCK inhibitors.

The more interesting and promising finding was the effectiveness of ripasudil at 10-6M to abolish the di-phosphorylation process of MLC in the myometrial strips isolated from pregnant mouse uterus and treated with cumulative concentrations of U46619. U46619 alone caused a significant increase in the expression of ppMLC when compared to the control group. This is the first time that pregnant mouse uteri have been used to examine MLC di-phosphorylation at late stages of gestation. Furthermore, this finding demonstrates the contribution of the ROCK pathway in phosphorylating two subunits on the MLC in pregnant mouse uterus. A similar mechanism was confirmed in human myometrial cells at term after inducing the ROCK pathway by OXT and calpeptin (Aguilar et al., 2011, Aguilar et al., 2012). The doubly phosphorylated MLC was also expressed in the smooth muscle of the basilar artery of guinea pigs (Bao et al., 2002). The first report describing MLC di-phosphorylation in uterine tissues was conducted in rats in 1986 (Dokhac et al., 1986). Ripasudil has shown astonishing results through its efficacy to diminish the expression of ppMLC protein in the myometrium exposed to cumulative concentrations of compared to the U46619-treated alone samples. The effect of ripasudil was highly significant and it was comparable to that of the control group. This has promising outcomes as it was confirmed previously that the myometrial tissues are remarkably sensitive to ROCK inhibitors and thus, low doses of these agents can

244 inhibit uterine contractions without evoking recognised adverse reactions on the vascular bed (Aguilar et al., 2012). Moreover, ROCK inhibitors were able to abolish MLC di-phosphorylation in the coronary arteries of pigs which demonstrates the activity of ROCK inhibitors in preventing di-phosphorylation in different organ tissues (Shimokawa et al., 1999). As mentioned above, no previous evidence is available on the expression of the di-phosphorylation process in the pregnant mouse uterus. Therefore, interactions between the ROCK pathways and the degree of MLC phosphorylation as well as the role of different uterine stimulants in various species during the middle and late stages of pregnancy warrant further investigation. Future studies can help in identifying new therapies for the prevention and management of preterm labour.

Analysing the phosphorylation of MLC in myometrial strips isolated from term pregnant mouse uterus at the single and double subunits of MLC after pre-incubation with and without ripasudil and then repeated treatment with 5-HT has shown the same pattern of U46619-exposed samples. 5-HT-treated strips did cause a significant variation in MLC expression when compared to the control. It is hypothesised that either a little or no change in the expression/ activity of myometrial MLC protein can happen after activation with 5-HT receptor stimulants, or it can be attributed to the protein phosphorylation recycling events that was discussed in the previous chapter. The results here are comparable to those observed in the U46619 experiments on pregnant uterus. Furthermore, ripasudil caused a very slight and non-significant elevation in MLC expression in the 5-HT-treated tissues compared to those treated with 5-HT alone. This suggests that the ROCK inhibitors can still keep MLC in its non- phosphorylated form even though its phosphorylation is not highly stimulated by ROCK pathway inducers. The remarkable effects of 5-HT and its challenge with ripasudil were shown after examining the expression of pMLC in the above samples. Myometrial tissues treated with cumulative concentrations of 5-HT using the immersion technique have shown a significant elevation in pMLC expression compared with the control group. These results are consistent with those from the functional studies experiments where 5-HT caused strong stimulation of myometrial contractile force in comparison to the strips treated with the vehicle (vehicle, 0.9% w/v normal saline). Ripasudil at 10-6M was able to significantly reduce the expression of pMLC in the tissues treated with 5- HT when compared to the samples exposed to 5-HT alone. This indicates the contribution of 5-HT receptors in activating the ROCK pathway in uterine tissues from

245 pregnant mice and thus, ROCK inhibitors can attenuate myometrial contractions through diminishing MLC phosphorylation in these tissues. Expression of 5-HT2A receptors has been proven in myometrium from pregnant women and their potential activity in stimulating the uterine contractions was identified previously (Cordeaux et al., 2009). Other 5-HT receptor subtypes such as 5-HT1D, 5-HT2A and 5-HT2C were identified in the mouse uterine tissues and their activities to induce myometrial contractility have been also demonstrated (Xiu-Kun et al., 2011).

Furthermore, investigating the contribution of 5-HT receptor agonists and the degree of MLC di-phosphorylation showed that 5-HT caused a significant increase in the expression of ppMLC in myometrial strips of pregnant uterus after exposure to cumulative concentrations of the agonist compared to the control samples. This further demonstrates the involvement of 5-HT receptors in the stimulation of MLC phosphorylation which results in enhanced uterine contractility. Ripasudil was effective as seen with U46619 in abolishing the ppMLC expression in the 5-HT-treated uterine tissues when compared with samples exposed to 5-HT alone. Even so, this inhibition in protein expression was at a borderline level of statistical significance (p=0.0539). This is a very interesting effect of ripasudil and the results are promising as it confirms the effectiveness of ROCK inhibitors in diminishing the di-phosphorylation of MLC in pregnant uterine tissues and myometrial contractile force after its stimulation by 5-HT receptor signalling through ROCK cascade pathway. Similar findings of interaction between 5-HT agonism and the ROCK pathway were observed in previous studies on mouse pulmonary and porcine coronary arteries (Kandabashi et al., 2000, Mair et al., 2008). In addition, our data are in agreement with researchers who have examined the ability of ROCK inhibitors to reduce mono- and di-phosphorylation of MLC in 5-HT- exposed porcine coronary arteries (Shimokawa et al., 1999).

Comparing the concentration-effect of OXT, U46619, 5-HT and ripasudil treatments on the degree of MLC phosphorylation in the myometrium of pregnant C57 mice uterus as shown in Table 4.1 indicates that among the uterine stimulants, OXT has the strongest activity in keeping the MLC in its non-phosphorylated form among all tested agents followed by 5-HT and then U46619. This suggests that either OXR are more numerous in the myometrial tissues at term or these receptors are more responsive to stimuli than TP and 5-HT receptors. Whereas, ripasudil has increased the level of MLC compared with the stimulants or the control, and this indicates the ability of ripasudil to maintain

246 the quiescent state of the uterus through inhibiting the phosphorylation of MLC. The mono-phosphorylation level of MLC was considerably higher following 5-HT exposure, compared to OXT and U46619, which were comparable to each other. We can hypothesize from this that stimulating murine uterine 5-HT receptors is more specifically connected with a single subunit phosphorylation of MLC than the signalling pathway of the two other agents. This hypothesis can be further demonstrated by examining double MLC phosphorylation. Treatment with OXT and U46619 have resulted in comparable outcomes which were extensively higher than that of 5-HT and therefore, it can be assumed that stimulation of ROCK pathway in the term pregnant uterine tissues through OXR and TP receptors can lead to remarkable degree of MLC di-phosphorylation. No studies are available to compare the number and sensitivity of these receptors in the uterine tissues of pregnant animals, however, OXR, TP and 5-HT receptors were demonstrated to be expressed extensively in the uterine tissues of pregnant human and rodents at term (Cruz et al., 1989, Senior et al., 1993, Kimura et al., 1996, Kubota et al., 1996, Garfield et al., 2000, Minosyan et al., 2007). Further investigations are needed to test the interaction of the above receptors with the uterine contraction mechanisms during gestation and parturition. Nonetheless, the inhibitory effect of ripasudil on di-phosphorylation was much higher than that on the mono- phosphorylation level of MLC as indicated by the percentage of both, pMLC and ppMLC expressions (13.24% for ppMLC versus 82.42% for pMLC) as seen in Table 4.1. Accordingly, it can be concluded that the conversion of pMLC to ppMLC is predominately (if not exclusively) dependent on the ROCK activity and this transformation of pMLC is highly expressed in the uterine tissues. Interestingly, recent studies have demonstrated that such a di-phosphorylation process is strongly expressed in the myometrium of term pregnant human and that is it substantially controlled by the ROCK pathway, as the ROCK inhibitors were able to diminish this process (Aguilar et al., 2012, Aguilar et al., 2011).

In conclusion, mouse myometrium obtained from pregnant uterus at term is highly active and it is responsive to different stimuli which can induce various signalling pathways through activating several receptor types such as OXR, TP and 5-HT receptors. Induction of these receptors results in the stimulation of labour and passage of the foetus. The ROCK pathway plays an essential role in the stimulation of uterine contractility and parturition through sensitizing the myometrial tissues to the released

247

Ca2+, heightening contractions. ROCK inhibitors can function as effective controllers of these contractions and as discussed above they can be used in low concentrations to regulate the contraction of the uterus with minimal consequences on the vascular smooth muscle. Further examinations are required to test the efficacy and safety of ripasudil and other ROCK inhibitors in the reproductive and cardiovascular systems during and after labour.

248

5 Chapter 5:

Effect of NO-donor containing liposomes on the composition of lipid mediators in the myometrium and placenta of pregnant C57 WT and eNOS knockout mice

249

5.1 Introduction

The myometrium and placenta play important functions in the growth and maintenance of a healthy foetus. The myometrium has a structural role in the uterus but also produces the contractions required for labour via its smooth muscle cells (Wu and DeMayo, 2017). The placenta acts as a mediator to facilitate the exchange of nutrients, gases and waste products between maternal and foetal circulations (Avagliano et al., 2012). In addition, it also acts as a defensive layer to protect the growing foetus from the adverse effects of various conditions and stimuli, such as the invasion by microorganisms (Robbins and Bakardjiev, 2012).

There are very few therapeutic agents for treating preterm labour; the available ones are not very effective and may cause adverse reactions in delivering mothers and their newborns (Miyazaki et al., 2016). To improve the range of clinical treatments available, one approach is to develop targeted nanomedicines to improve drug delivery, safety and efficacy, and reduce side effects. In addition, it is essential to gather more details on the general effects of targeted liposomes (irrespective of their contents) on normal uterine biology and activity. Therefore, a set of experiments were designed and performed to explore the role and activity of new compounds as tocolytics either as a free agent or incorporated into targeted liposomes. The expression of tissue-specific markers has been recently exploited to facilitate drug targeting to individual tissues and these markers can be regarded as tissue-specific receptors. In one approach, antibody fragments to a cell surface matrix metalloproteinase were used to produce doxorubicin- containing immunoliposomes which selectively delivered chemotherapeutic agents to tumours in mice (Hatakeyama et al., 2007). Taking a slightly different approach, our group has utilised peptide-decorated liposomes to selectively deliver payloads to the placentas and uterine vasculature of pregnant mice, as a means to create new therapies to treat pre-eclampsia and foetal growth restriction (King et al., 2016, Cureton, 2017). In the study by Cureton et al. (Cureton et al., 2017), the nitric oxide donor SE175 was encapsulated in targeted liposomes and administered intravenously to pregnant mice during the second half of pregnancy. A subset of myometrial and placental tissues from these animals were collected at term (E19), and the effects of the liposomal carrier and

250 the SE175 payload on uterine contractility and the local concentrations of lipid mediators were investigated.

Studies have demonstrated the role of NO in modulating smooth muscle contractility and neurotransmission in various body tissues (Moncada, 1991, Förstermann and Kleinert, 1995). However, no investigations are available on the effect of NO on myometrial activity in pregnant samples. It has also been concluded that the NO play a role in the production of different lipid mediators via regulating the activity of the enzymes involved in the synthesis of these mediators (Salvemini et al., 1993, Ding et al., 2017). Thesefore, we found that it is essential to move from the direct stimulation and inhibition of the myometrial contractility using uterine stimulants and ROCK inhibitors to an indirect approach through investigating and further exploring the influence of NO on uterine activity at the late stage of pregnancy and also to test interaction between the NO and lipid mediators in the placenta and uterus in pregnancy at term.

The interaction between the NO and release of lipid mediators has been studied and it was indicated that the PG pathway is stimulated via inducing the COX by increased NO concentrations (Salvemini et al., 1993). It was also found that NOS inhibitors can decrease the release of certain PGs without changing the level of other lipid mediators (Vassalle et al., 2003). In addition, the production of sEH-catalysed lipid mediators is stimulated by the administration of NO in murine cardiac cells. (Ding et al., 2017). The concentration of NO, the tissue type and the degree of tissue stimulation can determine the type of NO impact on the synthesis of lipid mediators (Mollace et al., 2005). Investigations are required to examine the effect of NO on the release and effectiveness of lipid mediators in gestational tissues during pregnancy and labour.

SE175 is an organic nitrate (nitroxyacylated thiosalicylate) which has a nitric oxide (NO)-releasing nitrate group attached to a NO-liberating thiosalicylate (Endres et al., 1999). This class of compound was synthesised to create a molecule which spontaneously released NO from nitrate groups in vivo, based on the hypothesis that the free thiol has the ability to release NO from nitrate groups, thus SE175 acts as an effective vasodilator (Lundberg et al., 2008). As part of the pathophysiology of preeclampsia and foetal growth restriction includes impaired uteroplacental blood flow and placental oxidative stress, and SE175 induced vasodilation of mouse uterine arteries in

251 vitro, it was hypothesised that SE175 may represent a candidate therapeutic for the treatment of these conditions (King et al., 2016).

Lipid mediators display multiple biological functions in different physiological and pathological conditions. Despite the wide variety of oxygenated lipid mediators, only a few sub-groups have been investigated in scientific studies. These include the prostaglandins and leukotrienes. Furthermore, the composition of COX- and LOX- derived lipid mediators in the myometrium and placenta of the pregnant uterus, and how it alters following therapeutic intervention, remains mostly unknown.

Two different mouse models were used in this study: C57BL/6J wild-type (C57 WT) mice and endothelial nitric oxide synthase knockout (eNOS KO) mice. The C57 WT was used as a control and to represent normal pregnancy, whereas the eNOS KO represents a model of foetal growth restriction and pre-eclampsia (Li et al., 2012).

In this study, we assessed the effect of targeted delivery of SE175 to the myometrial vasculature and placentas of pregnant mice (at term, E19) on the tissue concentration of lipid mediators in both C57 WT mice and eNOS KO mice. The aims in this chapter are threefold: firstly, to evaluate the effect of eNOS on the spontaneous uterine contractility in pregnant mice at term, by using mice deficient in this enzyme and comparing outcomes with wild type mice. Secondly, to investigate the effect of liposomal delivery on the local concentration of lipid mediators in the myometrium and placenta from pregnant C57 WT and eNOS KO mouse strains, and finally to examine the influence of SE175 on the concentration of lipid mediators in these tissues.

252

5.2 Myometrial contractility of the uterus of pregnant C57 WT and eNOS KO mice at term

Mouse uterine tissue samples isolated from the control group (PBS control) of pregnant C57 WT and eNOS KO mice at term (E19) showed spontaneous contractility when mounted in organ baths using the immersion technique. Strain variation in uterine contractions was clear, as displayed in Figure 5.1, where spontaneous activity and amplitude of contraction were significantly higher in the eNOS KO mice than the C57 WT samples (p0.05). However, the frequency of contraction between both species was not significantly different (p=0.0683).

Figure 5.1. Comparison of spontaneous myometrial activities in myometrium from pregnant C57 WT and eNOS KO mice at term (E19). Comparing the (A) spontaneous myometrial contractility expressed as AUC, (B) Amplitude of contraction and (C) Frequency of contraction between the PBS control groups of pregnant C57 WT (n=4) and eNOS KO (n=4) mice at term (E19) . Responses were measured over a 10 minute period. Data are expressed as mean ± SEM, and statistics were performed using unpaired Student t-test, ns=not significant, *=p<0.05.

253

5.3 The effect of SE175 on the concentration of lipid mediators in the myometrium and placenta of pregnant C57 WT and eNOS KO mice at term

5.3.1 Arachidonic Acid Mediators

5.3.1.1 Arachidonic acid mediators in the myometrium

Comparing the concentration of arachidonic acid (AA) mediators in myometrial samples from pregnant C57 WT and eNOS KO mice at term (E19) subjected to different treatments highlighted the following differences: the concentration of 13,14 dihydro 15-keto PGE2 was significantly higher in the myometrium from the PBS control group of eNOS KO mice when compared to the PBS liposomes-treated group of the same strain. The concentration of 6-keto PGF1α showed several significant variations in the myometrium among mouse strains and treatment groups, as its level was significantly elevated in the PBS control groups of both C57 WT and eNOS KO mice, when compared to all other treatment groups of both mouse strains. It was also higher in the eNOS KO than the C57 WT mice within the PBS control group. The levels of 5,6 DHET, 11,12 DHET and 14,15 DHET displayed significant elevation in the SE175 liposome group of the C57 WT mice when compared to the PBS control group of the same strain. In addition, the 14,15 DHET concentration in the SE175 liposome group in the WT mice also showed a significant increase, when compared to the free SE175 and PBS liposome groups of the same strain, and also when compared to the same treatment group in the eNOS KO mice. No significant differences were seen in the myometrial level of other AA derived mediators among treatment groups or between the pregnant C57 WT and eNOS KO mice as observed in Figure 5.2.

The concentrations of other AA mediators in the isolated myometrial tissues were below the lower limit of detection, these include 13,14 dihydro 15-keto PGF2α, PGE3 and PGF3α, whereas, 15-keto PGE2 and D12 PGJ2 were under the detection level in majority of samples.

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4000 g 20000

g p p 2000 10000

0 0 l 5 l o 7 s s o 75 s tr 1 e e tr 1 e es n E m m n E m o S o o o S o m c s s c s so S ee o o S ee o o r ip p B r ip p B F l li P F l li P S 5 S 5 B 17 B 17 P E P E S S Treatment Treatment

Figure 5.2. Arachidonic acid derived mediators quantified in myometrium from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using

LC/ESI-MS/MS.

Mice treated with either PBS as a control (PBS control) (100 μl), PBS liposomes (100 μl), SE175 liposomes (100 µl, approximately 0.44 mg/kg) or free SE175 (320 µM, approximately 0.44 mg/kg). Data are expressed as mean ± SEM and were analysed using two-way ANOVA with Bonferroni’s adjustment; *significant compared to C57 WT mice, #significant compared to eNOS KO mice. *,#=p<0.05, **,##=p<0.01, ***,###=p<0.001, ****,####=p<0.0001, significantly different according to the compared groups. Undetected values were not plotted on the graphs. N=(3-4).

258

5.3.1.2 Arachidonic acid mediators in the placenta

Comparing the concentration of AA mediators in placental tissues isolated from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI-MS/MS revealed no significant differences in mean concentration across most treatment groups or mouse strains. 11(12) EET showed a significant increase in the PBS control of the C57 WT mice when compared to all other treatment groups or its corresponding treated eNOS KO mice. Besides, 14,15 DHET was significantly elevated in the PBS control group of the C57 WT mice as compared to the same strain in the free

SE175 treatment group. However, 15-keto PGE2 showed an overall significant variation between the C57 WT and eNOS KO mice, but it did not exhibit any significant differences among treatment groups (see Figure 5.3). The placental tissues did not show any detection of other AA mediators such as PGD2, 15-deoxy D12,14 PGJ2, D12 PGJ2,

PGJ2, PGD3, PGF3α and 13,14 dihydro 15-keto PGF1α. 13,14 dihydro PGF1α was detected in the majority of samples but was under the limit of quantification.

PGE2 15-keto PGE2 250000 6000 C57 WT C57 WT

200000 eNOS KO eNOS KO

e t

t 4000 o

150000 o

100000

g g

m 2000

g p 50000 p

0 0

l l 5 o 5 s s o 7 s s tr 17 e e tr 1 e e n E m n E m m o o m o o o c S s so c S s s e o o e o o S re ip p S re ip p B F l li B F l li P P S 5 S 5 7 B 17 B 1 P E P E S S Treatment Treatment

13,14 dihydro 15-keto PGE2 PGF2 25000 8000 C57 WT C57 WT 20000 eNOS KO eNOS KO

n 6000

o o

15000 r

f 4000

10000 g

/ g

g 2000 p p 5000 0 0

l l 5 o 5 s s o 7 s s tr 17 e e tr 1 e e n m n E m m o E o m o S o o c S s so c s s e o o e o o S re p p S re ip p B F li li B F l li P P S 5 S 5 7 B 17 B 1 P E P E S S Treatment Treatment 259

6-keto PGF1 13,14 dihydro 15-keto PGF2 80000 800 C57 WT C57 WT eNOS KO eNOS KO

n 60000 n i

i 600

f 40000

f 400

/ /

g 20000

g 200

p p

0 0

l 5 l 5 o 7 s s o 7 s s tr 1 e e tr 1 e e n E m m n E m m o S o o o S o o c s s c s s e o o S ee o o S re ip ip r ip ip B F l l B F l l P 5 P S 5 S 7 7 B 1 B 1 P E P E S S Treatment Treatment

TXB2 12 HETE

6000 60000 C57 WT C57 WT

eNOS KO eNOS KO

i i e 4000

e 40000

2000 g 20000

p p

0 0

l l o 5 s s o 75 s s tr 17 e e tr 1 e e n E m m n E m m o S o o o S o o c s s c e s s S e o o S e o o re ip p r ip p B F l li B F l li P S P S 5 75 B 7 B 1 P 1 P E E S S Treatment Treatment

8-iso PGF 2 15 HETE

600 8000 C57 WT C57 WT eNOS KO eNOS KO

n 6000

n i

e 400

p f

4000

200 g

/ g

g 2000

p p

0 0

l 5 l ro 7 s s o 5 s s t 1 e e tr 17 e e n E m m n m o S o o o E m c s s c S so o e o o e o s S re ip p S re p o B F l li B F li lip P S P 75 S 5 B 1 B 17 P E P E S S Treatment Treatment

260

11(12) EET 11,12 DHET

2000 400 C57 WT C57 WT

n i

n eNOS KO eNOS KO i

1500 e 300

f 1000 200

/ g

g 500 100

p p

0 0 l l 5 s s o 5 s s o 7 e e r 7 e e tr 1 t 1 m m n m m n E o o o E o o o S s s c S s s c e o o e o o S e ip ip S e ip ip B r l l B r l l P F 5 P F S 5 S 7 7 B 1 B 1 P E P E S S Treatment Treatment

14,15 DHET 5 HETE 600 2500 * C57 WT C57 WT 2000 eNOS KO

n eNOS KO

t e

400 t o

r 1500

1000

g m

m 200

/ g

g p

p 500

0 0 l 5 s s l 5 o 7 e o 7 s s tr 1 e tr 1 e e n E m m n E m m o o o o S o o c S s s c s s e o o S ee o o S e ip ip r ip ip B r l l B F l l P F 5 P S 5 S 7 B 7 B 1 P 1 P E E S S Treatment Treatment

LTB4 11 HETE

800 8000 C57 WT C57 WT eNOS KO eNOS KO

600 6000

i e

4000

f 400

/ g

g 200 2000

p p

0 0

l 5 l o 7 s s o 75 s tr 1 e e tr 1 e es n E m m n E m o S o o o S o m c s s c s so S ee o o S ee o o r ip p r p p B F l li B F li li P S P 75 S 5 B 1 B 17 P E P E S S Treatment Treatment

Figure 5.3. Arachidonic acid derived mediators quantified in placentas from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI-MS/MS. Mice treated with either PBS as a control (PBS control) (100 μl), PBS liposomes (100 μl), SE175 liposomes (100 µl, approximately 0.44 mg/kg) or free SE175 (320 µM, approximately 0.44 mg/kg). Data are expressed as mean ± SEM and were analysed using two-way ANOVA with Bonferroni’s adjustment; *significant compared to C57 WT mice. *=p<0.05, significantly different according to the compared groups Undetected values were not plotted on the graphs. N=(3-4).

261

5.3.2 Linoleic Acid Mediators

5.3.2.1 Linoleic acid mediators in the myometrium

Although the mean concentration of the linoleic acid (LA) derived mediators, 9 OxoODE, 13 OxoODE, 9(10) EpOME, 12(13) EpOME and 12(13) DiHOME showed an overall elevation in the C57 WT over the eNOS KO mice, no significant variations in the level of these lipids or other LA derived lipids were seen among individual treatment groups or between mouse strains as displayed in Figure 5.4.

9 HODE 9 OxoODE

50000 2000 C57 WT C57 WT 40000 eNOS KO eNOS KO

1500

e t

30000 e

r r

1000

20000 g

g p 10000 p 500

0 0 l o 75 s s l tr 1 e e o 75 s s n E m m tr 1 e e o S o o n E m m c e s s o S o o S e o o c e s s B r lip ip S e o o P F l B r ip ip S 5 P F l l B 17 S 5 P E B 17 S P E S Treatment Treatment

13 HODE 13 OxoODE 60000 4000 C57 WT C57 WT eNOS KO eNOS KO

3000

n i

40000 i

o r r

p 2000

m /

20000 /

g p p 1000

0 0 l o 75 s s l 5 tr 1 e e ro 7 es s n E m m t 1 e o S o n E m m c s so o S o o e o o c e s s S re ip p S e o o B F l li B r lip ip P S 5 P F l B 7 S 5 P 1 B 17 E P E S S Treatment Treatment

262

9(10) EpOME 9,10 DiHOME 3000 4000 C57 WT C57 WT eNOS KO eNOS KO 3000

n 2000

o r

r 2000

g m

m /

1000 /

g p p 1000

0 0

l o 5 s l 5 tr 17 e es ro 7 s s n E m t 1 e e o S o m on E m m c s so c S o o S ee o o e os s B r ip p S re p o F l li B F li lip P S 5 P B 7 S 5 P 1 B 17 E P E S S Treatment Treatment

12(13) EpOME 12,13 DiHOME 2500 4000 C57 WT C57 WT 2000 eNOS KO eNOS KO

3000

e t

t 1500

o r

r 2000

g 1000

g p p 1000 500

0 0

l l 5 o 5 s o 7 s s tr 17 e es tr 1 e e n E n E m m o m m o S o o c S so o c s s e o s S ee o o S re p o r ip p B F li lip B F l li P P S S 5 75 B 17 B 1 P E P E S S Treatment Treatment

Trans EKODE 1500 C57 WT

eNOS KO n

i 1000

g m

/ 500

g p

l o 5 s s tr 17 e e n E m o S o m c s so e o o S re p p B F li li P S 5 B 17 P E S Treatment

Figure 5.4. Linoleic acid derived mediators quantified in myometrium from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI-MS/MS. Mice treated with either PBS as a control (PBS control) (100 μl), PBS liposomes (100 μl), SE175 liposomes (100 µl, approximately 0.44 mg/kg) or free SE175 (320 µM, approximately 0.44 mg/kg). Data are expressed as mean ± SEM and were analysed using two-way ANOVA with Bonferroni’s adjustment. Undetected values were not plotted on the graphs. N=(3).

263

5.3.2.2 Linoleic acid mediators in the placenta

The concentration of LA mediators was also measured and compared in placental samples collected from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments. Data indicated that the C57 WT mice treated with SE175 liposomes showed a significant increase (p=0.0326) in 9(10) DiHOME concentration when compared to the PBS control group. Similar findings were observed with 12,13 DiHOME (p=0.0094). No significant differences were observed in the mean placental concentration of other LA mediators, across treatment groups or mouse strains. Figure 5.5 shows the differences in mean levels of LA lipid mediators in the investigated placental tissues.

9 HODE 9 OxoODE

8000 3000 C57 WT C57 WT eNOS KO

6000 eNOS KO

i i

e 2000

4000

g 1000

/ g

2000 g

p p

0 0

l l o 5 s o 75 s s tr 17 e es tr 1 e e n E m n E m m o S o m o S o o c s so c s s ee o o S ee o o S r p p r ip p B F li li B F l li P P S S 5 75 B 17 B 1 P E P E S S Treatment Treatment

13 HODE 13 OxoODE 10000 2000 C57 WT C57 WT 8000 eNOS KO eNOS KO

1500

e t

6000 o

1000

4000

/ g

g 500 p 2000 p

0 0

l l 5 o 5 s o 7 s s tr 17 e s tr 1 e e n E e n E m m o S m m o S o o c so o c s s e o s S ee o o S re p o r ip p B F li lip B F l li P P S S 5 75 B 17 B 1 P E P E S S Treatment Treatment

264

9(10) EpOME 9,10 DiHOME

1500 * C57 WT 600 C57 WT

eNOS KO eNOS KO

e e 1000 t

t 400

g 500 m

m 200

p p

0 0

l 5 l 5 o 7 s s o 7 s s tr 1 e e tr 1 e e n E m n m o S o m o E o m c s so c S s o e o o e o s S re p p S e p o B F li li B r li lip P S 5 P F B 7 S 5 P 1 B 17 E P E S S Treatment Treatment

12,13 DiHOME Trans EKODE 800 2000 ** C57 WT C57 WT

eNOS KO eNOS KO

n i

n 600 1500

f 400 1000

/ g

g 200 500

p p

0 0 l l 5 o 5 s s o 7 s s r 7 e e tr 1 e e t 1 m m n E m m n E o o o S o o o S s s c s s c e o o e o o S e p p S re ip p B r li li B F l li P F P S 5 S 5 7 B 7 B 1 P 1 P E E S S Treatment Treatment

Figure 5.5. Linoleic acid derived mediators quantified in placentas from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI-MS/MS. Mice treated with either PBS as a control (PBS control) (100 μl), PBS liposomes (100 μl), SE175 liposomes (100 µl, approximately 0.44 mg/kg) or free SE175 (320 µM, approximately 0.44 mg/kg). Data are expressed as mean ± SEM and were analysed using two-way ANOVA with Bonferroni’s adjustment; *significant compared to C57 WT mice. *=p<0.05, significantly different according to the compared groups Undetected values were not plotted on the graphs. N=(3).

265

5.3.3 Dihomo-gamma-Linolenic Acid Mediators

5.3.3.1 Dihomo-gamma-Linolenic acid mediators in the myometrium

Data from LC/ESI-MS/MS work showed that Dihomo-gamma-Linolenic (DGLA) Acid mediators were not detected in the myometrium of most treatment groups of pregnant C57 WT and eNOS KO mice. However, results also showed that significant variations were only found in the concentrations of PE1 and PD1, where the concentration of PE1 was significantly increased in the PBS control groups of both strains when compared to other groups of their corresponding treatment regimens. In addition, PE1 was also significantly higher in the eNOS KO as compared to the C57 WT mice. Results from

PGE1 reflected in its metabolites 13,14 dihydro 15-keto PGE1 and 13,14 dihydro PGE1

(see Appendix 4). PD1 showed multiple variations among groups as its concentration showed a higher elevation in the PBS control group of either mouse type when compared to its same strain in all other treatment groups. The level of PD1 was also significantly higher in the free SE175-treated sample of both C57 WT and eNOS KO mice when compared to the PBS- and SE175-liposomes groups. Moreover, in the PBS control group, eNOS KO mice displayed a significantly increased PGD1 concentration as compared with wild type mice. No significant differences were seen in 15 HETrE concentrations in the myometrium from any treatment group in either pregnant mouse strain at term (E19), as observed in Figure 5.6.

266

PGE1 PGD1 , #### , ### ** 400 * , #### C57 WT 8000 ** **, ### C57 WT *** eNOS KO *** , #### 300 **** eNOS KO

n 6000 i

n ****, ###

o r

p 200

p 4000

g g p 100 p 2000

0 0 l o 5 s s l 5 tr 17 e e o 7 s s n m tr 1 e e o E o m n E m m c S s so o S o o e o o c e s s S re ip p S e o o B F l li B r lip ip P S 5 P F l B 7 S 5 P 1 B 17 E P E S S Treatment Treatment

15 HETrE 20000 C57 WT eNOS KO

15000

o r

p 10000

/ g

p 5000

l 5 o 7 s s tr 1 e e n E m m o S o o c s s S e o o re ip ip B F l l P 5 S 7 B 1 P E S Treatment

Figure 5.6. Dihomo-gamma-Linolenic Acid mediators quantified in myometrium from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI-MS/MS. Mice treated with either PBS as a control (PBS control) (100 μl), PBS liposomes (100 μl), SE175 liposomes (100 µl, approximately 0.44 mg/kg) or free SE175 (320 µM, approximately 0.44 mg/kg). Data are expressed as mean ± SEM and were analysed using two-way ANOVA with Bonferroni’s adjustment; *significant compared to C57 WT mice, #significant compared to eNOS KO mice. *,#=p<0.05, **,##=p<0.01, ***,###=p<0.001, ****,####=p<0.0001, significantly different according to the compared groups. Undetected values were not plotted on the graphs. N=(3-4).

267

5.3.3.2 Dihomo-gamma-Linolenic acid mediators in the placenta

LC/ESI-MS/MS analysis indicated that DGLA mediators were not detected in the placentas from some treatment groups of either strain. Data also showed that the concentration of PGD1 in the PBS control group of the eNOS KO mice was significantly increased when compared to the same strain in all other treatment groups. Whereas, 15 HETrE showed several variations among treatment groups; its concentration in the SE175 liposome group of the eNOS KO mice was significantly higher than the PBS control and free SE175 treatment regimens of the same strain, and was also higher than that of the same treatment groups in C57 wild type mice. Furthermore, eNOS KO mice exposed to free SE175 exhibited significantly elevated levels of 15 HETrE when compared to the PBS control and PBS liposome groups, and also when compared to the same treatment groups in C57 WT mice, as seen in Figure 5.7. PGE1, PGF1α and 6-keto

PGF1α did not show any significant change in concentration across treatment groups or mouse strain.

268

PGE1 PGD1

20000 800 ### C57 WT C57 WT eNOS KO

15000 eNOS KO n

i 600

10000 o

400

f o

/ g

5000 g

m /

g 200 0 p l o 5 s s tr 17 e e 0 n E m o S o m c s so e o o S re p p l B F li li o 75 s P S 5 tr 1 e es B 7 n E m P 1 o S o m E c s so S ee o o S r p p B F li li Treatment P S 5 B 17 P E S Treatment

15 HETrE PGF1 #### #### 400 1000 C57 WT **** C57 WT #### #### eNOS KO

800 **** eNOS KO 300

e o

t 600

o p

r 200

400 g

m /

g 100

g p

p 200

0 0

l l o 75 s o 75 s tr 1 e es tr 1 e es on E m m n E m m c S o o co S o o e os s e s s S re p o S re o o B F li lip B F lip ip P S P l B 75 S 5 P 1 B 17 E P E S S Treatment Treatment

Figure 5.7. Dihomo-gamma-Linolenic Acid mediators quantified in placentas from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI-MS/MS. Mice treated with either PBS as a control (PBS control) (100 μl), PBS liposomes (100 μl), SE175 liposomes (100 µl, approximately 0.44 mg/kg) or free SE175 (320 µM, approximately 0.44 mg/kg). Data are expressed as mean ± SEM and were analysed using two-way ANOVA with Bonferroni’s adjustment; *significant compared to C57 WT mice, #significant compared to eNOS KO mice. ***,###=p<0.001, ****,####=p<0.0001, significantly different according to the compared groups. Undetected values were not plotted on the graphs. N=(3-4).

269

5.3.4 Alpha-Linolenic Acid Mediators

5.3.4.1 Alpha-Linolenic Acid mediators in the myometrium

Figure 5.8 shows that no significant differences in the concentration of Alpha-Linolenic Acid (ALA) mediators, 9 HOTrE and 13 HOTrE were found in the myometrium from any treatment group in pregnant C57 WT and eNOS KO mice at term (E19).

9 HOTrE 13 HOTrE 500 2500 C57 WT C57 WT

400 eNOS KO 2000 eNOS KO

n i

300 i 1500

200 g 1000

m /

g p p 100 500

0 0

l 5 l 5 ro 7 s s ro 7 s nt E1 e e t 1 e es o S m m on SE m c o o c o m S ee os s S ee s so B r p o B r po o P F li ip P F li ip S l S l B 75 B 75 P E1 P 1 S SE Treatment Treatment

Figure 5.8. Alpha-Linolenic Acid mediators quantified in myometrium from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI-MS/MS. Mice treated with either PBS as a control (PBS control) (100 μl), PBS liposomes (100 μl), SE175 liposomes (100 µl, approximately 0.44 mg/kg) or free SE175 (320 µM, approximately 0.44 mg/kg). Data are expressed as mean ± SEM and were analysed using two-way ANOVA with Bonferroni’s adjustment. Undetected values were not plotted on the graphs. N=(3).

270

5.3.4.2 Alpha-Linolenic Acid mediators in the placenta

Among all ALA mediators, only 9 HOTrE was detected in the placental tissues from pregnant C57 WT and eNOS KO mice at term (E19). LC/ESI-MS/MS data showed that despite the overall variation in the mean placental concentration of 9 HOTrE between the two mouse strains, no specific differences were identified between treatment groups of both C57 WT and eNOS KO mice. Differences can be seen in Figure 5.9.

9 HOTrE 1000 C57 WT

800 eNOS KO

t o

r 600

g 400

/ g

p 200

l 5 o 7 s s tr 1 e e n E m m o S o o c s s S e o o re ip ip B F l l P 5 S 7 B 1 P E S Treatment

Figure 5.9. Alpha-Linolenic Acid mediators quantified in placentas from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI-MS/MS. Mice treated with either PBS as a control (PBS control) (100 μl), PBS liposomes (100 μl), SE175 liposomes (100 µl, approximately 0.44 mg/kg) or free SE175 (320 µM, approximately 0.44 mg/kg). Data are expressed as mean ± SEM and were analysed using two-way ANOVA with Bonferroni’s adjustment. Undetected values were not plotted on the graphs. N=(3).

271

5.3.5 Eicosapentaenoic Acid Mediators

5.3.5.1 Eicosapentaenoic Acid mediators in the myometrium

Quantification of Eicosapentaenoic Acid (EPA) mediators showed no significant variations in the level of EPA lipid mediators in the myometrium of either pregnant C57 WT or eNOS KO mice within all treatment groups. However, the mean concentration of 11 HEPE exhibited an overall increase in the eNOS KO mice as compared to the C57 WT mice. Figure 5.10 shows the variation in the EPA mediator concentrations in all treated groups.

PGD3 11 HEPE

1500 1500 C57 WT C57 WT

eNOS KO eNOS KO

n i

i 1000 e

e 1000

m /

/ 500 500

p p

0 0 l 5 l o 7 s s o 5 s s tr 1 e e r 7 e e n m m t 1 o E o o n E m m c S s s o S o o e o o c s s S e p p S e o o B r li li re ip ip F B F l l P S 5 P 5 B 7 S 7 P 1 B 1 E P E S S Treatment Treatment

12 HEPE 15 HEPE 6000 1500 C57 WT C57 WT

eNOS KO eNOS KO

i i

e 4000 1000

m /

2000 / 500

p p

0 0

l 5 l 5 o 7 s s o 7 s s tr 1 e e tr 1 e e n E m m n E m m o S o o o S o o c s s c s s S e o o S e o o re ip ip re ip ip B F l l B F l l P 5 P 5 S 7 S 7 B 1 B 1 P E P E S S Treatment Treatment

Figure 5.10. Eicosapentaenoic Acid mediators quantified in myometrium from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI- MS/MS. Mice treated with either PBS as a control (PBS control) (100 μl), PBS liposomes (100 μl), SE175 liposomes (100 µl, approximately 0.44 mg/kg) or free SE175 (320 µM, approximately 0.44 mg/kg). Data are expressed as mean ± SEM and were analysed using two-way ANOVA with Bonferroni’s adjustment. Undetected values were not plotted on the graphs. N=(3). 272

5.3.5.2 Eicosapentaenoic Acid mediators in the placenta

Data from LC/ESI-MS/MS showed that the only EPA derived lipids detected in the placental samples from pregnant C57 WT and eNOS KO mice were PGE3 and 12 HEPE. Nevertheless, these two lipids did not show any significant variations in their mean concentrations across the treatment groups or mouse strains. In addition, comparing the concentration of PGE3 in the PBS control group between the C57 WT and eNOS KO mice showed a slight elevation in the WT samples, however, this was not statistically significant (p=0.092) as observed in Figure 5.11.

PGE3 12 HEPE

3000 6000 C57 WT C57 WT

eNOS KO eNOS KO

i i

2000 e 4000

g 1000 2000

p p

0 0

l l o 75 s s o 5 s s tr 1 e e tr 17 e e n E m n E m o S o m o o m c s so c S s o S ee o o e o s r ip p S re p o B F l li B F li lip P S 5 P B 7 S 5 P 1 B 17 E P E S S Treatment Treatment

Figure 5.11. Eicosapentaenoic Acid mediators quantified in placenta from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI-MS/MS. Mice treated with either PBS as a control (PBS control) (100 μl), PBS liposomes (100 μl), SE175 liposomes (100 µl, approximately 0.44 mg/kg) or free SE175 (320 µM, approximately 0.44 mg/kg). Data are expressed as mean ± SEM and were analysed using two-way ANOVA with Bonferroni’s adjustment. Undetected values were not plotted on the graphs. N=(3).

273

5.3.6 Docosahexaenoic Acid Mediators

5.3.6.1 Docosahexaenoic Acid mediators in the myometrium

Figure 5.12 shows that although the myometrium from pregnant C57 WT mice at term (E19) exerted an overall increased mean concentration of 16(17) EpDPE and 4 HDHA when compared to the same gestational age eNOS KO mice, this increase was below significance. In addition, the mean concentration of 11 HDHA also showed an overall difference among treatment groups with no specific significant variation between any two groups. Other Docosahexaenoic Acid (DHA) mediators did not show any difference among all treatment groups or mouse strains as displayed in Figure 5.12.

4 HDHA 7 HDHA

4000 600 C57 WT C57 WT eNOS KO eNOS KO

3000

n i

i 400

o r

r 2000

m /

/ 200

g p 1000 p

0 0

l l o 5 s s o 5 s s tr 17 e e tr 17 e e n E m n E m o S o m o S o m c s so c s so S ee o o e o o r ip p S re p p B F l li B F li li P S 5 P S B 7 75 P 1 B 1 E P E S S Treatment Treatment

274

10 HDHA 11 HDHA

2000 800 C57 WT C57 WT eNOS KO eNOS KO

1500 600

r r

1000 p 400

g p p 500 200

0 0

l l 5 o 5 s s ro 7 es s tr 17 e e t 1 e n E m m n E m m o S o o o S o o c s s c e s s S ee o o S e o o r ip p B r ip ip B F l li P F l l P S S 5 75 B 7 B 1 P 1 P E E S S Treatment Treatment

13 HDHA 14 HDHA

10000 40000 C57 WT C57 WT 8000 eNOS KO eNOS KO

30000

i e

6000 e

o r

r 20000

4000 g

g p p 10000 2000

0 0

l 5 l o 7 s o 75 s tr 1 e es tr 1 e es on E m m n E m m c S o o co S o o e os s e s s S re p o S re po o B F li lip B F li ip P S P l B 75 S 5 P 1 B 17 E P E S S Treatment Treatment

17 HDHA 20 HDHA

15000 4000 C57 WT C57 WT eNOS KO

eNOS KO 3000

n i

n 10000

o r

r 2000

m /

/ 5000

g p

p 1000

0 0

l l 5 o 5 s o 7 s s tr 17 e es tr 1 e e n E m n E m m o S o m o S o o c s so c e s s S ee o o S e o o B r ip p B Fr lip ip F l li P l P S 5 S 5 B 7 B 17 P 1 P E E S S Treatment Treatment

275

16(17) EpDPE

4000 C57 WT eNOS KO

3000

r p

2000

/ g

p 1000

l 5 s s ro 7 e e t 1 m m n E o o o S s s c e o o S re ip ip B l l P F 5 S 7 B 1 P E S Treatment

19,20 DiHDPA 19(20) EpDPE

1500 4000 C57 WT C57 WT eNOS KO eNOS KO

3000

n i

i 1000

o r

r 2000

m /

/ 500

g p p 1000

0 0

l l 5 o 5 s s o 7 s s tr 17 e e tr 1 e e n n E m m o E m m o S o o c S so o c s s e o s S ee o o S re p o r ip p B F li lip B F l li P P S 5 S 5 B 7 B 17 P 1 P E E S S Treatment Treatment

Figure 5.12. Docosahexaenoic Acid mediators quantified in myometrium from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI-MS/MS. Mice treated with either PBS as a control (PBS control) (100 μl), PBS liposomes (100 μl), SE175 liposomes (100 µl, approximately 0.44 mg/kg) or free SE175 (320 µM, approximately 0.44 mg/kg). Data are expressed as mean ± SEM and were analysed using two-way ANOVA with Bonferroni’s adjustment. Undetected values were not plotted on the graphs. N=(3).

276

5.3.6.2 Docosahexaenoic Acid mediators in the placenta

Among all investigated DHA mediators, only three were detected in the placental tissues isolated from pregnant C57 WT and eNOS KO mice at term (E19). These mediators are: 13 HDHA, 14 HDHA and 19,20 DiHDPA. No significant variations were found in 13 HDHA or 14 HDHA among any of the treatment groups or mouse strains. Whereas the placentas from C57 WT mice exhibited a significant elevation in the concentration of 19,20 DiHDPA in the SE175 liposome-treated samples when compared to the same strain in all other treatment regimens, and also when compared to the eNOS KO mice subjected to the same treatment protocol. Figure 5.13 shows the differences among all groups and mouse types. The concentrations of other lipid mediators in the placental tissues can be seen in Appendix 5.

13 HDHA 14 HDHA 800 4000

C57 WT C57 WT

i i

eNOS KO e

e eNOS KO t

t 600 3000

f o

o 400 2000

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0 0 l l s s 5 s s 5 e ro 7 e e ro 7 e t 1 m m t 1 m m n E o n E o o o o o s c S s s c S s e o o e o o S e ip ip S e ip ip B r l l B r l l F 5 F 5 P S 7 P S 7 B 1 B 1 P E P E S S Treatment Treatment 19,20 DiHDPA

800 C57 WT

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t 600

o 400

m /

g 200 p

0 l 5 s s ro 7 e e t 1 m n E m o o o c S s s e o o S e ip ip B r l l P F 5 S 7 B 1 P E S Treatment

Figure 5.13. Docosahexaenoic Acid mediators quantified in placentas from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI-MS/MS. Mice treated with either PBS as a control (PBS control) (100 μl), PBS liposomes (100 μl), SE175 liposomes (100 µl, approximately 0.44 mg/kg) or free SE175 (320 µM, approximately 0.44 mg/kg). Data are expressed as mean ± SEM and were analysed using two-way ANOVA with Bonferroni’s adjustment; *significant compared to C57 WT mice ****=p<0.0001, significantly different according to the compared groups. Undetected values were not plotted on the graphs. N=(3). 277

5.4 The effect of liposome and SE175 exposure on the lipid proportions in the reproductive tissues according to the synthetic pathway

5.4.1 Myometrium

The level of different lipid mediators in the myometrial tissues isolated from the uterus of pregnant C57 WT and eNOS KO mice at term (E19) were grouped according to the synthetic pathway that produces each lipid, and these data were expressed as a percentage of total lipid content to produce a clear illustration of the contribution of each synthetic pathway to total lipid mediator production. Figure 5.14 displays the proportions of each lipid family in the myometrium of both C57 WT and eNOS KO mice. LOX-derived lipid mediators constituted most of the total lipids in all treatment groups and in both mouse strains (41.08% - 62.65%), followed by COX-derived lipids (19.96% - 43.28%), non-enzymatically-derived lipids (12.41% - 18.50%), cytochrome (CYP)-derived lipids (1.90% - 3.46%), soluble epoxyhydrolase (sEH)-derived lipids (0.37% - 1.11%) and peroxidase-derived lipids (0.05% - 0.16%), respectively.

5.4.2 Placenta

Similar to the myometrium, lipid mediator levels in placentas were also grouped according to the synthetic pathway that yields each lipid, and values were expressed as a percentage of the total lipids, to examine the contribution of each synthetic pathway to total lipid production. Figure 5.15 shows the relative lipid percentages in the placentas of both mouse strains. Unlike the myometrium, the majority of lipids produced across all treatment groups in both mouse strains were the COX-derived lipid mediators (74.43% - 83.97%) followed by LOX-derived lipids (10.21% - 19.01), non-enzymatic- derived lipids (3.25% - 6.29%), cytochrome (CYP)-derived lipids (0.34% - 0.84%), soluble epoxyhydrolase (sEH)-derived lipids (0.19% - 0.88%) and peroxidase-derived lipids (0.15% - 0.41%), respectively.

278

Figure 5.14. Proportions of lipids derived from cyclooxygenase (COX), lipoxygenase (LOX), cytochrome (CYP), peroxidase and non-enzymatic synthetic pathways in myometrium from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI-MS/MS. Mice treated with either PBS as a control (PBS control) (100 μl), PBS liposomes (100 μl), SE175 liposomes (100 µl, approximately 0.44 mg/kg) or free SE175 (320 µM, approximately 0.44 mg/kg). Data are collected from the investigated myometrial tissues of (A) C57 WT and (B) eNOS KO mice. Data represent average values and are expressed as a percentage of total lipids in each treatment group and are presented as mean % of the total. N=(3-4).

279

Figure 5.15. Proportions of lipids derived from cyclooxygenase (COX), lipoxygenase (LOX), cytochrome (CYP), peroxidase and non-enzymatic synthetic pathways in placentas from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI-MS/MS. Mice treated with either PBS as a control (PBS control) (100 μl), PBS liposomes (100 μl), SE175 liposomes (100 µl, approximately 0.44 mg/kg) or free SE175 (320 µM, approximately 0.44 mg/kg). Data are collected from the investigated myometrial tissues of (A) C57 WT and (B) eNOS KO mice. Data represent average values and are expressed as a percentage of total lipids in each treatment group and are presented as mean % of the total. N=(3-4).

280

5.5 Discussion

The first aim in this chapter was to assess the influence of eNOS deficiency on the spontaneous myometrial contractility in pregnant mice at term (E19). Mice lacking the eNOS enzyme are a valuable model for studying labour, as previous studies have reported preterm labour in mice after injection of NOS inhibitors (Tiboni and Giampietro, 2000). Both mouse strains (C57 WT and eNOS mice) were obtained from the same source (Jackson Laboratories, UK) and they have the same genetic background. The eNOS KO mice differ in lacking the eNOS enzyme. In this study, mice deficient in eNOS exhibited higher contraction forces when compared to the WT strain in terms of AUC and the amplitude of contraction. However, the contraction frequencies were non-significantly (p=0.0683) higher in the eNOS mice than the C57 WT mice, as observed in Figure 5.1. These findings are in agreement with previous studies on the uterine arteries in eNOS KO mice, where those animals showed a significant decrease in the endothelium-dependent arterial vasodilation in the uterus when compared to WT mice (Kusinski et al., 2012). NO is a short-lived free radical involved in the modulation of vasoactivity, contraction of smooth muscle and neurotransmission in many cell types (Moncada, 1991, Förstermann and Kleinert, 1995), with a half-life of approximately 2 minutes (Liu et al., 1998). This molecule can be produced in the myometrium of humans (Telfer et al., 1995) and mice (Naghashpour and Dahl, 2000). The production of NO from L-arginine is catalysed by NOS (Palmer et al., 1988). The NO released from vascular endothelial cells diffuses into the underlying smooth muscle cells and induces guanylate cyclase, which causes an elevation in cyclic guanosine monophosphate (cGMP), and this, in turn, stimulates protein kinase G leading to relaxation of the smooth muscle (Lincoln, 1989, Schmidt et al., 1993, Lincoln et al., 2001).

There are three main isoforms of NOS which have been identified in multiple tissues including the myometrium (Forstermann et al., 1994, Bartlett et al., 1999). These isoforms include neuronal NOS (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS). Studies on the expression and localization of NOS isoforms are conflicting as findings depend on the quality of laboratory methods utilized, experimental procedures

281 or the animal strains used in the research. However, during pregnancy, the only isoform expressed in mouse myometrial tissues is eNOS (Naghashpour and Dahl, 2000).

The myometrial strips from eNOS KO mice also showed a significantly higher amplitude of contraction and a non-significant (p=0.0683) increase in the frequency of contraction when compared to the WT control mice. We speculate that this effect may be due to the Ca2+ oscillations induced by the reduction in NO production in the myometrial smooth muscle from the pregnant eNOS KO mice samples. Studies have demonstrated that eNOS plays an essential role in Ca2+ release in smooth muscles cells and thus, lack of this enzyme results in an augmentation of the amplitude and frequency of contraction. These findings are in agreement with previous research on eNOS KO mouse urinary bladder (Zheng et al., 2016).

As explained in the introduction, the myometrium and placenta have vital roles in the development of the foetus and during pregnancy by providing structural support and protection to the uterus and foetus, respectively (Avagliano et al., 2012, Cureton, 2017, Wu and DeMayo, 2017). Pregnancy is associated with the production and storage of different lipid-derived mediators in the uterus and placenta of humans and mice, and these mediators can modulate the myometrial activity and placental function (Pearson et al., 2009, Durn et al., 2010, Brown et al., 2016).

The second and third aims in this chapter were to identify the influences of targeted liposomes, whether as drug-incorporated or as empty liposomes, on the biological processes and physiological functions of the uterus. For this purpose, different treatment delivery systems were used: PBS control, PBS-loaded liposomes, SE175-loaded liposomes and systemically administered SE175. LC/ESI-MS/MS analysis showed that myometrial and placental strips taken from pregnant C57 WT and eNOS KO mice at term (E19) exhibited different levels of lipid mediators after exposure to the above treatments. Mass spectrometry conducted on myometrium and placenta revealed no significant variations in the level of most lipid mediators among treatment groups and mouse strains, however, some mediators exhibited significant differences in their mean concentrations between C57 WT and eNOS KO mice and/or after subjecting the animals to different treatments. Nevertheless, the mean concentration of several lipid mediators in certain groups was increased or decreased when compared other corresponding groups but the change was below the significance threshold.

282

The data from isolated myometrial and placental tissues showed that no significant variations were found in the level of the following AA mediators: PGE2, PGF2α, PGD2,

15-deoxy D12,14 PGJ2, 13,14 dihydro 15-keto PGE2, 13,14 dihydro 15-keto PGF2α, 8- iso PGF2α, TXB2, TBX3, PGD3, LTB4, 8,9 DHET, 5,6 DHET, 11,12 DHET, 12 HETE, 8 HETE, 9HETE, 11 HETE, 12 HETE, 15 HETE, 8(9) EET, 11(12) EET, 14(15) EET and 5,15 DiHETE. However, these mediators were not detected in some samples and therefore, no graphs are shown for these groups. Among all AA derived mediators,

13,14 dihydro 15-keto PGE2 showed an increased mean concentration in the myometrium from eNOS KO mice compared with the C57 WT mice. In addition, the mean level of this lipid was significantly elevated in the PBS control group of eNOS KO mice when compared to the same mouse strain in the PBS liposomes group.

However this metabolite of PGE2 was not detected in the placental tissues from both mouse strains. 13,14 dihydro 15-keto PGE2 is a metabolite of PGE2 and has shown both pro- and anti-inflammatory properties in different body tissues (Lee et al., 2019, Kaushal et al., 2014). Our results demonstrate that loss of the eNOS enzyme is associated with the elevation of this lipid mediator, and it also demonstrates that treatment with PBS liposomes, SE175 liposomes or free SE175 can reduce 13,14 dihydro 15-keto PGE2 levels in the eNOS KO mice, which is potentially indicative of a lower inflammatory response in these treatment groups, compared with the control group. We assume that 13,14 dihydro 15-keto PGE2 has an inflammatory action in our samples and the increased concentration of 13,14 dihydro 15-keto PGE2 in the PBS control group of eNOS KO mice further support the results of our functional studies where the C57 WT mice showed lower myometrial contractions when compared to the eNOS KO mice (see Figure 5.1). 13,14 dihydro 15-keto PGE2 is a metabolite of PGE2, which is known to induce uterine contractility in pregnant human at term (Arulkumaran et al., 2012). In addition, previous investigations have demonstrated elevated plasma concentrations of 13,14 dihydro 15-keto PGE2 after the onset of uterine contraction in pregnant women at term (Lackritz et al., 1978). However, some studies have observed no effect of 13,14 dihydro 15-keto PGE2 on myometrial contractility in pregnant women

(Gordon-Wright and Elder, 1980). In the placenta, the other metabolite of PGE2, 15- keto PGE2 showed an increase in its mean concentration in C57 WT mice, particularly those treated with SE175 liposomes or free SE175, when compared to the same treatment groups in the eNOS KO mice. Nevertheless, no significant variations were observed in 15-keto PGE2 levels across treatment groups. 15-keto PGE2 shares the same 283 pro- and anti-inflammatory properties of its precursor, PGE2 (Chen et al., 2018, Chou et al., 2007). This metabolite has been shown to exert a relaxatory effect on tracheal tissues from guinea-pigs whilst not affecting the uterine activity in the same animals

(Crutchley and Piper, 1975). It has also been demonstrated that 15-keto PGE2 has a high affinity to EP4 receptors (Nishigaki et al., 1996) and that those receptors can activate cAMP through inducing adenylate cyclase and thereby initiating a relaxatory effect on the uterus during pregnancy (Arrowsmith et al., 2010). These findings further support our results from the functional studies on the C57 WT and eNOS KO mice. Interestingly, previous studies have demonstrated an increased synthesis of PGs with other NO-donors (sodium nitroprusside and L-arginine) (Vassalle et al., 2003).

However, the same studies did not find any effect of PGE2 on NO levels.

Conversely, no significant differences were found in the concentration of PGJ2 between the myometrial tissues isolated from the C57 WT and eNOS KO mice or among treatment groups. Moreover, this lipid was not detected in the placental tissues of either mouse strain. Nevertheless, PGJ2 and its metabolites are prostanoids with anti- inflammatory properties, which act through inhibiting the NF-kB pathway via stimulation of PPARγ (Straus et al., 2000). Research indicated that there is a direct association between NO synthesis and PG production and that an increase in NO is associated with the stimulation of COX, which increases the release of PG (Salvemini et al., 1993). Although there is no available evidence on the direct effect of eNOS and NO on PGJ2, studies have demonstrated that the PGJ2 metabolite, 15-deoxy PGJ2 induces NO production from human aortic endothelial cells (Calnek et al., 2003). Furthermore,

15-deoxy PGJ2 has proven anti-inflammatory actions and has been shown to inhibit lipopolysaccharide-induced PTL in mice by attenuating various pathways such as NF-

κB, cytosolic phospholipase A2 and inflammatory protein synthesis. In our study, 15- deoxy PGJ2 was detected in the myometrial tissues but did not show any variation among all groups (see Appendix 4).

Interestingly, the most significant variations seen within the AA derived mediators was with 6-keto PGF1α, where it displayed multiple differences between both C57 WT and eNOS KO mice, and among treatments groups. Data show that injection of liposomes resulted in a significant reduction in the concentration of 6-keto PGF1α (irrespective of content), as PBS control and free SE175 treatment groups of both mouse strains exhibited significant elevation in 6-keto PGF1α concentration when compared to their 284 corresponding liposome treatments. In addition, PBS control samples from eNOS KO mice showed a significant increase in the myometrial level of 6-keto PGF1α as compared with the free SE175-treated eNOS KO mice or with the C57 WT mice of PBS control. These results indicate that liposome composition could have a suppressant effect on the production and/or release of 6-keto PGF1α and this means that liposomes which are used as vehicles to deliver targeted drugs can interfere with certain biological and physiological processes in body organs. Further investigations are required to examine the influence of liposomes themselves on the targeted tissues. The decrease in 6-keto

PGF1α concentration observed in the NO-donor, free SE175 group of the eNOS KO mice compared with the control samples of the same strain is inconsistent with previous studies on rat cardiac endothelial cells, where NO-donors have augmented the synthesis of prostacyclin (PGI2), which is the precursor of 6-keto-PGF1 (Sievi et al., 1997). We suppose these contradictory outcomes could be due to the species, time points and tissue variations between the studies. Another possibility is that the conversion of PGI2 to 6- keto-PGF1 might be attenuated in the samples used in the free SE175 group. Therefore, further investigations are required to measure PGI2 concentrations after exposure to free SE175.

Furthermore, another AA derived lipid mediator exhibited different myometrial mean concentrations among treatment groups and mouse strains. This lipid is 14,15 DHET, which showed elevations in its mean levels in the pregnant C57 WT mice over the pregnant eNOS KO mice. It was also found in significantly higher concentrations in the myometrium of C57 WT mice treated with SE175 liposomes, when compared to eNOS KO mice receiving the same treatment, or C57 WT mice treated with free SE175. Similar findings with 11,12 DHET were observed between the myometrial tissues from pregnant C57 WT mice treated with SE175 liposomes and PBS control groups. In addition, placental tissues showed the same variation in 14,15 DHET concentration within the C57 WT mice but the difference was between the free SE175 and PBS control treatment groups. In general, DHETs are products of the Cytochrome 450 (CYP) epoxygenases pathway which are expressed in the endothelium of the vascular tissues and their role is comprehensively examined in circulatory disorders (Fleming, 2011). These results suggest that when the NO-donor SE175 is encapsulated in liposomes it has the ability to induce upregulation of the CYP pathway in the myometrium and so increase the level of the downstream products (EETs) in the treated

285 samples. EETs are then converted to DHETs by the soluble epoxide hydrolase (sEH). However, the effect produced by SE175 liposomes in the C57 WT mice was not observed in the eNOS KO mice. DHETs are involved in the regulation of blood pressure and their expression is associated with increased blood pressure, therefore, sEH inhibitors exhibit vasodilatory properties by preventing the conversion of the vasodilators, EETs into DHETs (Imig et al., 2002). Our findings are consistent with previous work on rodents, where the administration of NO has stimulated the sEH and increased the cardiac ischaemic injury (Ding et al., 2017). However, other studies on murine pulmonary arteries have concluded that DHETs are not associated with smooth muscle contraction (Keseru et al., 2008). Concentrations of EETs did not vary between myometrial tissues from the C57 WT mice and eNOS KO mice or among treatment groups. Whereas, placenta exerted a significant elevation of 12(12) EET in the PBS control when compared to all other treatment groups and eNOS KO PBS control. It has been previously reported that 11(12) EET can augment the isometric tension in isolated rat uterus (Gonzalez et al., 1997), while other EETs such as 8,9-EET and 5,6-EET have exerted a uterine relaxatory effect in isolated myometrium from pregnant women at term (Pearson et al., 2009). These results are in agreement with our findings in the LC/ESI-MS/MS and also in the functional studies experiments where C57 WT mice exhibited lower uterine contractility and amplitude of contraction. The roles of sEH pathway and DHETs in the modulation of smooth muscle contraction of the uterus are not well known and so, further investigations are required to explore the exact function of this pathway and the impact of its downstream metabolites on gestational tissues.

However, both the myometrial and placental tissues did not exhibit any significant differences in the level of other AA derived lipid mediators between C57 WT and eNOS KO mice. In addition, liposome and SE175 treatments had no influence on the concentrations of these lipids including the HETEs, although several members of these hydroxyeicosanoids were examined in this study. Nevertheless, research on uterine tissues from pregnant women showed that HETEs possess a myometrial stimulant effect which was identical to that of oxytocin, as well as a vasoconstrictor effect on myometrial arteries (Pearson et al., 2009). The researchers have speculated that the 20- HETE may function to maintain the depolarization of smooth muscle that is already in a contractile state, such as the labouring one.

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The concentrations of LA derived lipid mediators in the myometrium from pregnant C57 WT and eNOS KO mice taken at term showed no significant differences between treatments groups or between mouse strains. However, some significant variations were observed in the level of LA derived mediators in the isolated placental tissues. Despite the overall difference in the mean concentration of 9 OxoODE and 13 OxoODE between the myometrium from C57 WT and eNOS KO mice, no significant variations in their levels and in their precursors’ concentrations (9 HODE and 13 HODE, respectively) were observed in any treatment groups of myometrial or placental samples. However, C57 WT mice exhibited a slight increase in the mean concentration of 9 OxoODE when compared to the eNOS KO mice, particularly in the samples treated with PBS liposomes where the p value was close the threshold of significance (p=0.077). OxoODEs are metabolic products of HODEs and they are known for their anti-inflammatory properties and this has been demonstrated through the ability of 13 OxoODE to bind to peroxisome proliferator-activated receptor γ (PPARγ) and exert its inhibitory action against inflammation in colonic epithelial cells (Altmann et al., 2007). Cytokines and pro-inflammatory proteins are involved in PTL and stimulation of uterine contraction (Goldenberg et al., 2000) and PPARγ can have a beneficial role through suppressing the inflammatory responses evoked by these factors (Wang et al., 2017). As the uterine contraction can be induced by inflammation (Konishi et al., 2019), we assume that the increased mean concentration of 9 OxoODE observed in the C57 WT mice can be considered as another factor behind the lower myometrial contractility seen in these strains. Furthermore, the lack of NO in the eNOS KO mice may further enhance the inflammatory process as NO plays a key role as an anti-inflammatory agent, however, several studies have confirmed its activity in inducing inflammation particularity in abnormal physiological situations (Sharma et al., 2007). Our results failed to demonstrate any activity of SE175 or liposomes to modulate OxoODEs or HODEs expression.

The LA metabolic products of CYP-, sEH- and peroxidase-catalysed pathways did not show any significant difference in their concentration in the myometrium from pregnant C57 WT and eNOS KO mice in any treatment groups. However, the mean concentration of 9(10) EpOME, 12(13) EpOME and 12,13 DiHOME was increased in the myometrium from C57 WT mice when compared to the eNOS KO mice, whereas in placenta, significant variations were found with 9(10) DiHOME and12,13 DiHOME, as

287 their levels were significantly higher in the SE175 liposome group as compared with the PBS control. We can conclude from this that first, there is a slight reduction in the concentration of these mediators in the mice lacking the eNOS enzyme, and secondly, SE175 and/or the liposomes may have a positive effect on targeting the placenta as the SE175 liposome treatment caused an increased accumulation of 9(10) DiHOME and 12,13 DiHOME in these tissues when compared to other treatment protocols. In relation to the correlation between the DiHOMEs and NO, these outcomes are consistent with previous studies in mouse brown adipose tissue, where NO inhibitors have reduced the level of 12,13 DiHOME (Park et al., 2018). 12,13 DiHOME has also showed a role in the inflammation and it has been involved in the in the mediation of thermal hyperalgesia during pain and the inflammatory response and through inducing Ca2+ oscillation in sensory neurons. Therefore, sEH inhibitors have exerted potent antihyperalgesic properties (Zimmer et al., 2018). No evidence is available regarding the role of CYP-, sEH products derived from LA on the uterine or placenta functions and so, more investigations are required to explore their effects on the gestational tissues.

Interestingly, the DGLA lipids have exhibited several statistically significant variations between C57 WT and eNOS KO mice and among treatments groups. PGD1 data demonstrated that treatment of the myometrium with liposomes induced a significant reduction the lipid concentration in both mouse strains whether the liposomes contained PBS or SE175. It can be concluded from this that the composition of the liposome itself may interfere with the PG synthesis or other pathways that are connected to this biological process. In addition, within the PBS control group, the PGD1 level was significantly elevated in the eNOS KO mice when compared to the WT samples. The same effect of liposomes was observed in the placental tissues of both strains. However, free SE175 has a significant attenuating effect on the level of PGD1 in the myometrial samples when compared with the PBS control group. Similarly, the liposome and free

SE175 treatments significantly reduced the concentration of PGE1 in the treated myometrial, but not placental, tissues from both C57 WT and eNOS KO mice. It has been shown that NO regulates PG production, and the type of NO effect on the pathway depends on the concentration of NO produced, the type of the tissue in which PG is synthesized and the intensity of the stimulus received by the tissue (Mollace et al.,

2005). Researchers have reported the stimulant effect of PGE1 on the uterine contraction

288 in pregnant women at term, and uterine rupture is highly correlated to the administration of PGE1 (Chiossi et al., 2012). These reports can provide a better explanation for the high myometrial levels of this lipid in the PBS control group of the eNOS KO mice as compared with the C57 WT sample, and it further supports the functional studies in our samples where eNOS KO mice showed higher contractile forces. Nevertheless, this finding was not observed in the placental tissues. PGD1 and PGE1 are metabolites of the same precursor, PGH1 (Rouzer and Marnett, 2003) and they exhibit pro-inflammatory properties (Bharat et al., 2008, Schröder et al., 2012). In addition, these two PG were found to be produced at low to moderate concentrations in the myometrium from labouring and non-labouring women at term (Durn et al., 2010). PGD1 can stimulate PPARγ receptors (Cizkova et al., 2012), which are expressed in the pregnant human gestational organs (Wieser et al., 2008) and are associated with the physiological changes in the uterine tissue before the initiation of labour, proposing the role of these prostanoids in the process. Although the concentration of DGLA derived lipid, 15 HETrE did not show any significant variation between the myometrial tissues from the C57 WT and eNOS KO mice or across treatment groups, its placental level exhibited a significant increase in the eNOS KO mice treated with the free- or liposome incorporated-SE175 when compared to all other treatment groups and C57 WT mice. 15 HETrE is known to have an indirect anti-inflammatory effect in human neutrophils via inhibiting the 15-LOX, the enzyme catalysing the synthesis of LB4, which is a powerful inflammatory lipid, from AA (Iversen et al., 1991, Vasudevan et al., 2013) (Vasudevan et al., 2013). Our findings of the placental tissues show that the NO-donor, SE175 was able to elevate the level of 15 HETrE, regardless of the type of delivery system used. As discussed previously, the NO has double actions in inflammatory processes, either acting as pro-inflammatory or anti-inflammatory agent (Tallet et al., 2002, Sharma et al., 2007). We speculate that one of the characteristics that makes the NO to act as an anti-inflammatory compound is the enhancement of 15 HETrE release, thus, more studies are required to investigate the role of NO-donors in suppressing the inflammatory response in the late pregnancy and before the initiation of labour through potentiating the production of DGLA derived lipids mediators with anti-inflammatory properties, and how these mediators can modulate uterine activity at term.

Furthermore, measurement of the concentration of the ALA derived lipid mediators exhibited that, despite the increased mean level of 9 HOTrE in the myometrium treated

289 with non-liposome therapies in both mouse strains, when compared to the PBS- and SE175-liposome treatments and also despite the increase of its mean concentration in the C57 WT mice as compared with the eNOS KO mice in the placenta, no significant variations were observed in the level of the ALA derived mediators across treatment groups or between the pregnant C57 WT and eNOS KO mice at term (E19). It has been demonstrated that HOTrEs are released in the mouse uterus (Santorelli, 2018) and these lipids have shown their anti-inflammatory efficiencies in the macrophages isolated from the mouse peritoneum through deactivating NLRP3 inflammasome complex via stimulating the PPAR-γ pathway (Kumar et al., 2016). No evidence is available regarding the role of HOTrEs during pregnancy and labour and so more investigations are required to better understand their contribution in modulating the function of gestational tissues in these conditions.

Similar to ALA, the lipid mediators derived from EPA showed no significant differences in their mean concentrations across treatment groups or mouse strains in both the myometrium and placenta. However, there was an overall non-significant increase in the mean concentration of 11 HEPE in the myometrium from the eNOS KO mice when compared to the C57 WT strain. In addition, treating the pregnant C57 WT mice with the free SE175 caused a non-significant elevation in the concentration of

PGE3 when compared to the eNOS KO mice, nevertheless, the p value approached the level of signiﬁcance (p=0.092). In general, previous studies have shown that EPA derived lipids are produced in the myometrium of pregnant mice and these lipids have exhibited their anti-inflammatory and protective properties in delaying LPS-induced PTL (Yamashita et al., 2013). The results of these studies also demonstrate the contribution of inflammation to the development of PTL. Interestingly, researchers have demonstrated the anti-inflammatory effect of PGE3 (Oyelowo, 2007) and their promising usefulness in the treatment endometriosis (Netsu et al., 2008). Furthermore, NO can be produced by the endothelial cells during normal physiological situations to maintain resting vasodilator tone. Inflammation promotes increased production of NO by the endothelial tissue and this further elevates the level of NO synthesis (Rote and Huether, 2018). These results further support our findings of the functional studies and LC/ESI-MS/MS.

Lipidomic analysis of the DHA derived mediators in the myometrial isolated from the pregnant C57 WT and eNOS KO mice showed the following measurements: an overall

290 increase in the myometrial concentration of 4 HDHA, 16(17) EpDPE and 19(20) EpDPE in the C57 WT mice when compared to the eNOS KO samples, with an overall difference in the mean level of 4 HDHA and 11 HDHA among treatment groups; however, there was no significant variation was observed between any individual groups in regard to these two lipids. The placental tissues exhibited the presence of just three DHA derived mediators and among those showed differences in their concentrations were the 13 HDHA which showed an overall elevation in its mean concertation in the C57 WT when compared to the eNOS KO mice, and the 19,20 DiHDPA which exhibited a significant increase in its level in the free SE175-treated C57 WT mice when compared to the eNOS KO mice or any other treatment group. DHA have been characterized by their local anti-inflammatory effects (Mozaffarian and Wu, 2011) and their metabolites have exerted useful cardiovascular impacts (Arnold et al., 2010). Studies have demonstrated the ability of DHA to attenuate the production of

PGE2 and PGF2α in the placental and uterine tissues from pregnant women (Kim et al., 2012), and as these two PG are increased prior to the initiation of labour and induce cervical ripening and myometrial contractility in at term and PTL (O'Brien et al., 1986, Karim et al., 1971), therefore, DHA can exert a beneficial role in tocolysis through this pathway. These studies are in agreement with our findings of DHA derived lipids as certain lipids have shown increased concentration in the C57 WT mice as compared with the eNOS KO samples, and the lower uterine contraction forces seen in the functional studies. However, DHA has shown its ability to increase the release of NO through iNOS, not eNOS. Although most of the differences seen with the DHA derived lipids were between mouse strains and liposomes and/or SE175 therapies did not show any effect on the levels of most lipids, surprisingly, SE175-liposome treatment in the C57 WT mice has shown an interesting outcome of the drug targeting delivery in the gestational organs.

Collective grouping of all investigated lipid mediators (Figures 5.14 and 5.15) in gestational tissues isolated from the uterus of pregnant C57 WT and eNOS KO mice at term (E19) as per the synthetic pathway that yields each mediator (see Appendix 6), and after data are expressed as a percentage of the total lipid composition showed the participation of each pathway to the total synthesis of these mediators. Results exhibited different contribution ratio of pathways between the myometrial and placental tissues in both the C57 WT and eNOS KO mice, where the LOX pathway comprised the highest

291 percentage in the myometrium and second ratio in the placenta. Conversely, the COX lipid mediators encompassed the highest percentage in the placenta samples and the second-highest ratio in the myometrium. In both tissues, the non-enzymatic pathway was the third percentage followed by sEH and peroxidase, respectfully. These data indicate that the lipids produced by the COX and LOX pathways represent the majority of mediators in the gestational tissue in the pregnant mouse at term. It also indicates that within each tissue type, no obvious differences were found in the percentages of these pathways among different treatment groups, except the ratios of COX and LOX in the PBS control group of the eNOS KO mice, where the COX percentage was increased and LOX ration was decreased as compared to all other treatment protocols. Neither liposomes nor SE175 has exhibited any effect of these ratios in both mouse strains. To the best of our knowledge, no previous studies have been conducted to investigate the percentage of the above pathways in the tissues involved in mouse pregnancy or labour. However, studies have concluded that developing new therapies with COX/LOX dual inhibitory properties and targeting enzyme downstream of these two pathways will provide an alternative pharmacotherapeutic strategy to treat inflammation and its associated pathophysiological complications and symptoms (Hwang et al., 2013). We propose that developing these strategies will also provide better understanding of the pathophysiology of PTL and the role of NO and NOS in modulating the uterine activity and placental function during gestation and labour.

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6 Chapter 6:

Conclusion and Future work

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6.1 Conclusion

There were four aims of this study: Firstly to investigate and compare the modulations of uterine contraction in non-pregnant (in dioestrus) and pregnant (at term, E19) C57 WT mice using functional studies experiments. The second aim was to evaluate the activity of the recently developed ROCK pathway inhibitor, ripasudil in abolishing the myometrial contractility in the uterus isolated from both, non-pregnant and pregnant C57 WT mice through interfering with the mono- and di-phosphorylation processes of MLC. Thirdly, to assess the influence of presence and absence NO on myometrial contractility in the uterus of pregnant mice at term using two different types of mouse strains, C57 WT and eNOS KO mice. The final aim was to examine the effect of liposome administration (whether empty or NO-loaded) on the levels of various lipid mediators in the uterine and placental tissues taken from pregnant C57 WT and eNOS KO mice at term (E19) and to compare the percentage of each group of lipid mediators between the myometrium and placenta as well as between both mouse strains.

Data displayed that the myometrium isolated from the uterus of both, non-pregnant and pregnant C57 WT mice have the ability to spontaneously contract for several hours in vitro, which indicates the continuous depolarisation of resting membrane potential due to the differences in ion movement across the cell membrane. This also demonstrates the viability of these isolated tissues to conduct functional studies experiments. However, the myometrial strips from pregnant mice at term maintained their contractile activity for approximately 24h compared to the 5h for samples from non-pregnant mice. In addition, the upper segment of the uterine horn from non-pregnant mice (in dioestrus) has a significantly elevated myometrial contractility when compared with the lower segment of the same tissue. This further demonstrates the release of E2 from ovaries which are close to the upper segment of the uterus and this sex hormone increases in the expression of other CAPs, such as CX-43, OTR and PG receptors at the late stages of gestation.

In non-pregnant samples, U46619, 5-HT and PGF2α in cumulative concentration were able to stimulate uterine activity and elevate the force of contraction through activating their receptors (TP, 5-HT receptors and FP, respectively). The activity of these uterine stimulants also involved the ROCK pathway via upregulating the synthesis of the di- phosphorylated form of MLC. The pregnant tissues have followed the same pattern of

294 myometrial contractility and MLC phosphorylation when the uterus was stimulated with oxytocin, U46619 and 5-HT through inducing their specific receptors.

The newly developed ROCK pathway inhibitor, ripasudil as a bolus dose and in cumulative concentrations produced a powerful activity to diminish myometrial spontaneous and drug- induced contractility in the uterus of both, non-pregnant and pregnant mice. Ripasudil was also able to inhibit the phosphorylation of MLC at two levels, single and double phosphorylations in the myometrium of both, non-pregnant and pregnant mouse uterus. Nevertheless, the inhibitory effect drug was more obvious on the di-phosphorylation step.

Results showed that the NO is involved in the uterine activity in pregnant mice at term where mice deficient in NO displayed a significant elevation in myometrial contractility and amplitude of contraction with a non-significant increase in the frequency of contraction compared to the WT mice. Furthermore, liposome treatment exerted their effects on the production of certain lipid mediators in the uterus and placenta of pregnant C57 WT and eNOS KO mice and both PBS- and the NO-donor SE175-liposome produced a significant decrease in the level 6-keto PGF1α), 11(12) EET, (PGD1 and PGE1 in the myometrium and/or placentas from both mouse strains. Moreover, liposomes incorporated with SE175 caused a significant elevation in the myometrial concentration of DHETs and placental level of DiHDPA in the C57 WT mice as compared to the animals treated with Free SE175. In the samples isolated from eNOS KO mice, the levels of 15 HETrE were significantly elevated with the exposure to the Free- and liposomes incorporated-SE175. The lipid mediators synthesized by the LOX pathway represented the higher percentage OF myometrial lipids in the C57 WT and eNOS KO mice, while the majority of lipid mediators in the placental tissues were those derived from the COX pathway in both, WT and KO mouse strains.

In conclusion, the results of this study further support the hypothesis of increased myometrial spontaneous activity in the uterus of pregnant mice when compared with the non-pregnant samples. The upper segment of the uterus is more active than the lower part of the uterus in non-pregnant mice. To the best of our knowledge, this is the first to demonstrate the expression of the double phosphorylation level of MLC in the uterus of non-pregnant and pregnant mice and that the ROCK pathway plays a key role in this di- phosphorylation. The data suggest that ROCK inhibitors are promising tocolytics for the management of PTL. However, more investigations are needed to further explore the function of the ROCK pathway in different gestational tissues during pregnancy and

295 labour. The study also showned that utilizing liposomes as a targeted drug delivery system to administer medications is effective in regulating the release of various lipid mediators. NO-donor can play an important role in modulating uterine activity and attenuating gestational diseases when used at late pregnancy through controlling the contractions of myometrium and placental circulation.

6.2 Future work

The findings presented in this study have raised several questions and created new ideas that require further research.

The non-prenant mice used in this study were in dioestus and thus, further Examinations are required to compare the uterine spontaneous activity in non-pregnant mice at different oestrous stages (pro-oestrus, oestrus, metoestrus and dioestrus) and investigate the expression of different receptors and proteins of contraction in each stage. In addition, more investigations are needed to elucidate the variations in the hormonal milieu during the whole oestrous cycle.

In this study, only the upper segment of the uterine horn of pregnant mice at term was used for investigations and thus, more studies to be performed to test the spontaneous activity and drug influence on the lower segment of the uterus and also to compare the role of CAPs on both segments.

Ripasudil was the only ROCK inhibitor used in this work to clarify the role of this pathway during dioestrus and at late in pregnancy. Research is needed to use other newly developed ROCK inhibitors to deeply explore the role of this signalling pathway in the uterus, placenta and other gestational tissues and its effect on MLC phosphorylation and expression of receptors and proteins involved in the regulations of the oestrous cycle, pregnancy and labour. In addition, ripasudil was investigated using mouse uterine tissue and thus, more studies are required to test the effect of this ROCK inhibitor on isolated human myometrium during pregnancy and labour. Furthermore, studies are required to examine the effect of ripasudil on MLC phosphorylation on a cellular level and also to test if ripasudil can distinguish between the two isoforms, ROCKI, and ROCKII.

296

The study did not investigate the complications of ripasudil on other body organs. Research is required to examine the efficacy and safety of ripasudil and other ROCK inhibitors (in the presence and absence of uterine stimulants) in reproductive and non- gestational tissues such as blood vessels, urinary organs and respiratory tissues.

The thesis has also raised the need for studying the effect of FP, TP, 5-HT and OXR receptor antagonists on the latency of gestational period and labour process and the role of these antagonists in the nodulation of ROCK pathway and MLC phosphorylation. Furthermore, the interaction between the sex hormones and the ROCK pathway and their impacts on pregnancy and labour would provide more explanation on the precise mechanism of gestation and the process of labour.

Data showed that the NO plays a role in uterine activity in pregnant mice at term through inhibiting myometrial contractility and amplitude of contraction. Thus, further research is required to define the exact role of NO during the whole pregnancy and labour, and also to investigate the influences of other NO-donors on uterine function before and during parturition.

The results displayed that liposomes composition can regulate certain biological and physiological processes in the body such as the production of PGs even though they are used just as carries to deliver targeted medications. This leads to the requirement for further examination to investigate the effect of liposomes themselves on the targeted tissues and if they interfere with organ functions.

The NO-donor, SE175 also showed some impacts on the concentration of certain lipid mediators in the uterine and placental tissues in pregnant mice at term and thus, more studies should be conducted to check the influence of SE175 and other NO donors on lipid and other biological mediators in the gestational tissues in human and various animal strains during pregnancy and labour and also to test whether these agents have any complications on foetal or maternal organs.

In addition, very little knowledge is available about the role of sEH pathway of lipid mediator production and DHETs in regulating the contraction of uterine smooth muscle and thus, more studies are needed to identify the precise function of this pathway and the effects of its derived lipid metabolites on different tissues involved in the labour process.

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7 Chapter 7:

References

AAGAARD-TILLERY, K. M., NUTHALAPATY, F. S., RAMSEY, P. S. & RAMIN, K. D. 2005. Preterm premature rupture of membranes: perspectives surrounding controversies in management. Am J Perinatol, 22, 287-97.

ABRAMOVITZ, M., ADAM, M., BOIE, Y., CARRIERE, M. C., DENIS, D., GODBOUT, C., LAMONTAGNE, S., ROCHETTE, C., SAWYER, N., TREMBLAY, N. M., BELLEY, M., GALLANT, M., DUFRESNE, C., GAREAU, Y., RUEL, R., JUTEAU, H., LABELLE, M., OUIMET, N. & METTERS, K. M. 2000. The utilization of recombinant prostanoid receptors to determine the affinities and selectivities of prostaglandins and related analogs. Biochim Biophys Acta, 1483, 285-293.

AGUILAR, H. N. & MITCHELL, B. F. 2010. Physiological pathways and molecular mechanisms regulating uterine contractility. Hum Reprod Update, 16, 725-44.

AGUILAR, H. N., TRACEY, C. N., TSANG, S. C., MCGINNIS, J. M. & MITCHELL, B. F. 2011. Phos-tag-based analysis of myosin regulatory light chain phosphorylation in human uterine myocytes. PLoS One, 6, e20903.

AGUILAR, H. N., TRACEY, C. N., ZIELNIK, B. & MITCHELL, B. F. 2012. Rho- kinase mediates diphosphorylation of myosin regulatory light chain in cultured uterine, but not vascular smooth muscle cells. J Cell Mol Med, 16, 2978-89.

ALIJAHAN, R., HAZRATI, S., MIRZARAHIMI, M., POURFARZI, F. & AHMADI HADI, P. 2014. Prevalence and risk factors associated with preterm birth in Ardabil, Iran. Iran J Reprod Med, 12, 47-56.

ALTMANN, R., HAUSMANN, M., SPOTTL, T., GRUBER, M., BULL, A. W., MENZEL, K., VOGL, D., HERFARTH, H., SCHOLMERICH, J., FALK, W. & ROGLER, G. 2007. 13-Oxo-ODE is an endogenous ligand for PPARgamma in human colonic epithelial cells. Biochem Pharmacol, 74, 612-22.

AMANO, M., FUKATA, Y. & KAIBUCHI, K. 2000. Regulation and functions of Rho- associated kinase. Exp Cell Res, 261, 44-51.

ANDERSON, J. & PATEREK, E. 2019. Vaginal Foreign Body Evaluation and Treatment. StatPearls [Internet]. StatPearls Publishing.

ANOTAYANONTH, S., SUBHEDAR, N. V., GARNER, P., NEILSON, J. P. & HARIGOPAL, S. 2004. Betamimetics for inhibiting preterm labour. Cochrane Database Syst Rev, CD004352.

298

ARDREY, R. E. 2003. Liquid chromatography-mass spectrometry: an introduction, John Wiley & Sons.

ARIEL, I., HOCHBERG, A. & SHOCHINA, M. 1998. Endothelial nitric oxide synthase immunoreactivity in early gestation and in trophoblastic disease. Journal of clinical pathology, 51, 427-431.

ARNOLD, C., MARKOVIC, M., BLOSSEY, K., WALLUKAT, G., FISCHER, R., DECHEND, R., KONKEL, A., VON SCHACKY, C., LUFT, F. C., MULLER, D. N., ROTHE, M. & SCHUNCK, W. H. 2010. Arachidonic acid-metabolizing cytochrome P450 enzymes are targets of {omega}-3 fatty acids. J Biol Chem, 285, 32720-33.

ARROWSMITH, S., KENDRICK, A. & WRAY, S. 2010. Drugs acting on the pregnant uterus. Obstet Gynaecol Reprod Med, 20, 241-247.

ARROWSMITH, S. & WRAY, S. 2014. Oxytocin: its mechanism of action and receptor signalling in the myometrium. J Neuroendocrinol, 26, 356-369.

ARTHUR, P., TAGGART, M. J. & MITCHELL, B. F. 2007. Oxytocin and parturition: a role for increased myometrial calcium and calcium sensitization? Front Biosci, 12, 619-33.

ARULKUMARAN, S., KANDOLA, M. K., HOFFMAN, B., HANYALOGLU, A. C., JOHNSON, M. R. & BENNETT, P. R. 2012. The roles of prostaglandin EP 1 and 3 receptors in the control of human myometrial contractility. The Journal of Clinical Endocrinology & Metabolism, 97, 489-498.

ASTLE, S., SLATER, D. M. & THORNTON, S. 2003. The involvement of progesterone in the onset of human labour. Eur J Obstet Gynecol Reprod Biol, 108, 177-81.

AVAGLIANO, L., GARO, C. & MARCONI, A. M. 2012. Placental amino acids transport in intrauterine growth restriction. Journal of pregnancy, 2012.

BAO, J., OISHI, K., YAMADA, T., LIU, L., NAKAMURA, A., UCHIDA, M. K. & KOHAMA, K. 2002. Role of the short isoform of myosin light chain kinase in the contraction of cultured smooth muscle cells as examined by its down- regulation. Proc Natl Acad Sci U S A, 99, 9556-9561.

BARTLETT, S. R., BENNETT, P. R., CAMPA, J. S., DENNES, W. J., SLATER, D. M., MANN, G. E., POSTON, L. & POSTON, R. 1999. Expression of nitric oxide synthase isoforms in pregnant human myometrium. J Physiol, 521 Pt 3, 705-16.

BECK, S., WOJDYLA, D., SAY, L., BETRAN, A. P., MERIALDI, M., REQUEJO, J. H., RUBENS, C., MENON, R. & VAN LOOK, P. F. 2010. The worldwide incidence of preterm birth: a systematic review of maternal mortality and morbidity. Bull World Health Organ, 88, 31-8.

299

BEHRMAN, R. E. & BUTLER, A. S. 2007. Behavioral and Psychosocial Contributors to Preterm Birth. Preterm Birth: Causes, Consequences, and Prevention. Washington, D.C., U.S.A.: The National Academies Press.

BERNAL, A. L. 2003. Mechanisms of labour - biochemical aspects. Bjog-an International Journal of Obstetrics and Gynaecology, 110, 39-45.

BERNAL, A. L., EUROPEFINNER, G. N., PHANEUF, S. & WATSON, S. P. 1995. Preterm Labor: a Pharmacological Challenge. Trends Pharmacol Sci, 16, 129- 133.

BERTOLIN, K. & MURPHY, B. D. 2014. Reproductive tract changes during the mouse estrous cycle. The Guide to Investigation of Mouse Pregnancy. Elsevier.

BETHLEHEM, R. A., VAN HONK, J., AUYEUNG, B. & BARON-COHEN, S. 2013. Oxytocin, brain physiology, and functional connectivity: a review of intranasal oxytocin fMRI studies. Psychoneuroendocrinology, 38, 962-974.

BHARAT, A., KUO, E., STEWARD, N., ALOUSH, A., HACHEM, R., TRULOCK, E. P., PATTERSON, G. A., MEYERS, B. F. & MOHANAKUMAR, T. 2008. Immunological link between primary graft dysfunction and chronic lung allograft rejection. Ann Thorac Surg, 86, 189-95; discussion 196-7.

BHARTIYA, D. & JAMES, K. 2017. Very small embryonic-like stem cells (VSELs) in adult mouse uterine perimetrium and myometrium. J Ovarian Res, 10, 29.

BLANKS, A. M. & THORNTON, S. 2003. The role of oxytocin in parturition. Bjog, 110 Suppl 20, 46-51.

BLESSON, C. S., BÜTTNER, E., MASIRONI, B., SAHLIN, L. 2012. Prostaglandin receptors EP and FP are regulated by estradiol and progesterone in the uterus of ovariectomized rats. Reprod Biol Endocrinol 10, 3.

BOEHME, M. & DONAT, H. 1992. Identification of lymphocyte subsets in the human fallopian tube. Am J Reprod Immunol, 28, 81-4.

BOLT, M. W. & MAHONEY, P. A. 1997. High-efficiency blotting of proteins of diverse sizes following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Anal Biochem, 247, 185-92.

BORNA, S. & SAHABI, N. 2008. Progesterone for maintenance tocolytic therapy after threatened preterm labour: a randomised controlled trial. Aust N Z J Obstet Gynaecol, 48, 58-63.

BOS, C. L., RICHEL, D. J., RITSEMA, T., PEPPELENBOSCH, M. P. & VERSTEEG, H. H. 2004. Prostanoids and prostanoid receptors in signal transduction. Int J Biochem Cell Biol, 36, 1187-1205.

BOYD, K. L., MUEHLENBACHS, A., RENDI, M. H., GARCIA, R. L. & GIBSON- CORLEY, K. N. 2018. Female reproductive system. Comparative Anatomy and Histology. 2nd edition ed. Chennai: Elsevier.

300

BREYER, R. M., BAGDASSARIAN, C. K., MYERS, S. A., BREYER, M. D. 2001. Prostanoid receptors: subtypes and signaling. Annu Rev Pharmacol Toxicol. 41, 661-690.

BRICEAG, I., COSTACHE, A., PURCAREA, V. L., CERGAN, R., DUMITRU, M., BRICEAG, I., SAJIN, M. & ISPAS, A. T. 2015. Fallopian tubes--literature review of anatomy and etiology in female infertility. J Med Life, 8, 129-31.

BRODT-EPPLEY, J. & MYATT, L. 1999. Prostaglandin receptors in lower segment myometrium during gestation and labor. Obstet Gynecol, 93, 89-93.

BROWN, R. G. & MACINTYRE, D. A. 2014. Calcium channel blockers are effective as first line for tocolysis in the management of preterm labour. Evid Based Med, 19, 214.

BROWN, S. H., EATHER, S. R., FREEMAN, D. J., MEYER, B. J. & MITCHELL, T. W. 2016. A lipidomic analysis of placenta in preeclampsia: evidence for lipid storage. PLoS One, 11.

BUHIMSCHI, I., YALLAMPALLI, C., DONG, Y. L. & GARFIELD, R. E. 1995. Involvement of a nitric oxide-cyclic guanosine monophosphate pathway in control of human uterine contractility during pregnancy. Am J Obstet Gynecol, 172, 1577-84.

BYERS, S. L., WILES, M. V., DUNN, S. L. & TAFT, R. A. 2012. Mouse estrous cycle identification tool and images. PLoS One, 7, e35538.

CALIGIONI, C. S. 2009. Assessing reproductive status/stages in mice. Curr Protoc Neurosci, Appendix 4, Appendix 4I.

CALLAGHAN, W. M., MACDORMAN, M. F., RASMUSSEN, S. A., QIN, C. & LACKRITZ, E. M. 2006. The contribution of preterm birth to infant mortality rates in the United States. Pediatrics, 118, 1566-73.

CALNEK, D. S., MAZZELLA, L., ROSER, S., ROMAN, J. & HART, C. M. 2003. Peroxisome proliferator-activated receptor gamma ligands increase release of nitric oxide from endothelial cells. Arterioscler Thromb Vasc Biol, 23, 52-7.

CAO, J., WAKATSUKI, A., YOSHIDA, M., KITAZAWA, T. & TANEIKE, T. 2004. Thromboxane A2 (TP) receptor in the non-pregnant porcine myometrium and its role in regulation of spontaneous contractile activity. Eur J Pharmacol, 485, 317-27.

CARBONNE, B., DALLOT, E., HADDAD, B., FERRE, F. & CABROL, D. 2000. Effects of progesterone on prostaglandin E-2-induced changes in glycosaminoglycan synthesis by human cervical fibroblasts in culture. Mol Hum Reprod, 6, 661-664.

CARIO-TOUMANIANTZ, C., REILLAUDOUX, G., SAUZEAU, V., HEUTTE, F., VAILLANT, N., FINET, M., CHARDIN, P., LOIRAND, G. & PACAUD, P. 2003. Modulation of RhoA-Rho kinase-mediated Ca2+ sensitization of rabbit myometrium during pregnancy - role of Rnd3. J Physiol, 552, 403-13.

301

CHAI, C. K. & DICKIE, M. M. 1966. Endocrine variations. Biology of the Laboratory Mouse. 2nd edition ed.: DOVER PUBLICATIONS, INC., NEW YORK.

CHALLIS, J. R. G. 2000. Mechanism of parturition and preterm tabor. Obstet Gynecol Surv, 55, 650-660.

CHALLIS, J. R. G., MATTHEWS, S. G., GIBB, W. & LYE, S. J. 2000. Endocrine and paracrine regulation of birth at term and preterm. Endocr Rev, 21, 514-50.

CHALLIS, J. R. G., SLOBODA, D. M., ALFAIDY, N., LYE, S. J., GIBB, W., PATEL, F. A., WHITTLE, W. L. & NEWNHAM, J. P. 2002. Prostaglandins and mechanisms of preterm birth. Reproduction, 124, 1-17.

CHAMPLIN, A. K., DORR, D. L. & GATES, A. H. 1973. Determining the stage of the estrous cycle in the mouse by the appearance of the vagina. Biol Reprod, 8, 491- 4.

CHANDRASEKHARAN, N. V., DAI, H., ROOS, K. L., EVANSON, N. K., TOMSIK, J., ELTON, T. S. & SIMMONS, D. L. 2002. COX-3, a cyclooxygenase-1 variant inhibited by acetaminophen and other analgesic/antipyretic drugs: cloning, structure, and expression. Proc Natl Acad Sci U S A, 99, 13926-31.

CHANRACHAKUL, B., BROUGHTON PIPKIN, F., WARREN, A. Y., ARULKUMARAN, S. & KHAN, R. N. 2005. Progesterone enhances the tocolytic effect of ritodrine in isolated pregnant human myometrium. Am J Obstet Gynecol, 192, 458-63.

CHEN, C.-J., XIAO, P., CHEN, Y. & FANG, R. 2019. Selenium Deficiency Affects Uterine Smooth Muscle Contraction Through Regulation of the RhoA/ROCK Signalling Pathway in Mice. Biological trace element research, 1-10.

CHEN, I. J., HEE, S. W., LIAO, C. H., LIN, S. Y., SU, L., SHUN, C. T. & CHUANG, L. M. 2018. Targeting the 15-keto-PGE2-PTGR2 axis modulates systemic inflammation and survival in experimental sepsis. Free Radic Biol Med, 115, 113-126.

CHIOSSI, G., COSTANTINE, M. M., BYTAUTIENE, E., KECHICHIAN, T., HANKINS, G. D., SBRANA, E., SAADE, G. R. & LONGO, M. 2012. The effects of prostaglandin E1 and prostaglandin E2 on in vitro myometrial contractility and uterine structure. Am J Perinatol, 29, 615-22.

CHOLLET, A., TOS, E. G. & CIRILLO, R. 2007. Tocolytic effect of a selective FP receptor antagonist in rodent models reveals an innovative approach to the treatment of preterm labor. BMC Pregnancy Childbirth, 7 Suppl 1, S16.

CHOU, W. L., CHUANG, L. M., CHOU, C. C., WANG, A. H., LAWSON, J. A., FITZGERALD, G. A. & CHANG, Z. F. 2007. Identification of a novel prostaglandin reductase reveals the involvement of prostaglandin E2 catabolism in regulation of peroxisome proliferator-activated receptor gamma activation. J Biol Chem, 282, 18162-72.

302

CIRILLO, R., TOS, E. G., PAGE, P., MISSOTTEN, M., QUATTROPANI, A., SCHEER, A., SCHWARZ, M. K. & CHOLLET, A. 2007. Arrest of preterm labor in rat and mouse by an oral and selective nonprostanoid antagonist of the prostaglandin F2alpha receptor (FP). Am J Obstet Gynecol, 197, 54 e1-9.

CIZKOVA, K., KONIECZNA, A., ERDOSOVA, B., LICHNOVSKA, R. & EHRMANN, J. 2012. Peroxisome proliferator-activated receptors in regulation of cytochromes P450: new way to overcome multidrug resistance? J Biomed Biotechnol, 2012:656428.

COATES, M. D., MAHONEY, C. R., LINDEN, D. R., SAMPSON, J. E., CHEN, J., BLASZYK, H., CROWELL, M. D., SHARKEY, K. A., GERSHON, M. D. & MAWE, G. M. 2004. Molecular defects in mucosal serotonin content and decreased serotonin reuptake transporter in ulcerative colitis and irritable bowel syndrome. Gastroenterology, 126, 1657-1664.

COLEMAN, R. A., SMITH, W. L. & NARUMIYA, S. 1994. International Union of Pharmacology classification of prostanoid receptors: properties, distribution, and structure of the receptors and their subtypes. Pharmacol Rev, 46, 205-29.

CONDON, J. C., HARDY, D. B., KOVARIC, K. & MENDELSON, C. R. 2006. Up- regulation of the progesterone receptor (PR)-C isoform in laboring myometrium by activation of nuclear Factor-kappa B may contribute to the onset of labor through inhibition of PR function. Molecular Endocrinology, 20, 764-775.

CONDON, J. C., JEYASURIA, P., FAUST, J. M., WILSON, J. W. & MENDELSON, C. R. 2003. A decline in the levels of progesterone receptor coactivators in the pregnant uterus at term may antagonize progesterone receptor function and contribute to the initiation of parturition. Proc Natl Acad Sci U S A, 100, 9518- 9523.

COOK, J. L., SHALLOW, M. C., ZARAGOZA, D. B., ANDERSON, K. I. & OLSON, D. M. 2003. Mouse placental prostaglandins are associated with uterine activation and the timing of birth. Biol Reprod, 68, 579-87.

COOK, J. L., ZARAGOZA, D. B., SUNG, D. H. & OLSON, D. M. 2000. Expression of myometrial activation and stimulation genes in a mouse model of preterm labor: myometrial activation, stimulation, and preterm labor. Endocrinology, 141, 1718-28.

COOMARASAMY, A., KNOX, E. M., GEE, H., SONG, F. & KHAN, K. S. 2003. Effectiveness of nifedipine versus atosiban for tocolysis in preterm labour: a meta-analysis with an indirect comparison of randomised trials. BJOG, 110, 1045-9.

CORDEAUX, Y., PASUPATHY, D., BACON, J., CHARNOCK-JONES, D. S. & SMITH, G. C. 2009. Characterization of serotonin receptors in pregnant human myometrium. J Pharmacol Exp Ther, 328, 682-91.

303

COX, S. M., BOHMAN, V. R., SHERMAN, M. L. & LEVENO, K. J. 1996. Randomized investigation of antimicrobials for the prevention of preterm birth. Am J Obstet Gynecol, 174, 206-10.

CRAIG, J. C., COLBURN, T. D., HIRAI, D. M., SCHETTLER, M. J., MUSCH, T. I. & POOLE, D. C. 2018. Sex and nitric oxide bioavailability interact to modulate interstitial Po2 in healthy rat skeletal muscle. J Appl Physiol (1985), 124, 1558- 1566.

CRANKSHAW, D. J. 2001. Pharmacological techniques for the in vitro study of the uterus. J Pharmacol Toxicol Methods, 45, 123-40.

CROWTHER, C. A., HILLER, J. E. & DOYLE, L. W. 2002. Magnesium sulphate for preventing preterm birth in threatened preterm labour. Cochrane Database Syst Rev, CD001060.

CRUTCHLEY, D. J. & PIPER, P. J. 1975. Comparative bioassay of prostaglandin E2 and its three pulmonary metabolites. Br J Pharmacol, 54, 397-9.

CRUZ, M. A., GONZALEZ, C., ACEVEDO, C. G., SEPULVEDA, W. H. & RUDOLPH, M. I. 1989. Effects of histamine and serotonin on the contractility of isolated pregnant and nonpregnant human myometrium. Gynecol Obstet Invest, 28, 1-4.

CURETON, N. 2017. Development of nanocarriers for targeted drug delivery to the placenta. PhD Thesis, The University of Manchester, UK.

CURETON, N., KOROTKOVA, I., BAKER, B., GREENWOOD, S., WAREING, M., KOTAMRAJU, V. R., TEESALU, T., CELLESI, F., TIRELLI, N., RUOSLAHTI, E., APLIN, J. D. & HARRIS, L. K. 2017. Selective Targeting of a Novel Vasodilator to the Uterine Vasculature to Treat Impaired Uteroplacental Perfusion in Pregnancy. Theranostics, 7, 3715-3731.

DARIOS, E. S., SEITZ, B. & WATTS, S. W. 2012. Smooth muscle pharmacology in the isolated virgin and pregnant rat uterus and cervix. J Pharmacol Exp Ther, 341, 587-96.

DE HEUS, R., MOL, B. W., ERWICH, J. J., VAN GEIJN, H. P., GYSELAERS, W. J., HANSSENS, M., HARMARK, L., VAN HOLSBEKE, C. D., DUVEKOT, J. J., SCHOBBEN, F. F., WOLF, H. & VISSER, G. H. 2009. Adverse drug reactions to tocolytic treatment for preterm labour: prospective cohort study. BMJ, 338, b744.

DE LAAT, M. W. M., PIEPER, P. G., OUDIJK, M. A., MULDER, B. J. M., CHRISTOFFELS, V. M., AFINK, G. B., POSTMA, A. V. & RIS-STALPERS, C. 2013. The Clinical and Molecular Relations Between Idiopathic Preterm Labor and Maternal Congenital Heart Defects. Reprod Sci, 20, 190-201.

DE PONTI, F. 2004. Pharmacology of serotonin: what a clinician should know. Gut, 53, 1520-35.

304

DE PONTI, F. & TONINI, M. 2001. Irritable bowel syndrome: new agents targeting serotonin receptor subtypes. Drugs, 61, 317-32.

DECAVALAS, G., MASTROGIANNIS, D., PAPADOPOULOS, V. & TZINGOUNIS, V. 1995. Short-term verus long-term prophylactic tocolysis in patients with preterm premature rupture of membranes. Eur J Obstet Gynecol Reprod Biol, 59, 143-7.

DI, L. & KERNS, E. 2016. Drug-Like Properties: Concepts, Structure Design and Methods from ADME to Toxicity Optimization. 2016, Londod, UK, Elsevier Science.

DI LIETO, A., CATALANO, D., ALBANO, G., UCCELLO, N., CIVETTA, A., FERRARA, R., FIMIANI, R., PONTILLO, M. & PALADINI, A. 1991. Uterine motility and cervical ripening in second trimester elective abortion by two different PGE analogues. Clin Exp Obstet Gynecol, 18, 251-9.

DI RENZO, G. C., GIARDINA, I., ROSATI, A., CLERICI, G., TORRICELLI, M., PETRAGLIA, F. & ITALIAN PRETERM NETWORK STUDY, G. 2011. Maternal risk factors for preterm birth: a country-based population analysis. Eur J Obstet Gynecol Reprod Biol, 159, 342-6.

DI RENZO, G. C., ROSATI, A., MATTEI, A., GOJNIC, M. & GERLI, S. 2005. The changing role of progesterone in preterm labour. BJOG, 112 Suppl 1, 57-60.

DING, Y., LI, Y., ZHANG, X., HE, J., LU, D., FANG, X., WANG, Y., WANG, J., ZHANG, Y., QIAO, X., GAN, L. M., CHEN, C. & ZHU, Y. 2017. Soluble epoxide hydrolase activation by S-nitrosation contributes to cardiac ischemia- reperfusion injury. J Mol Cell Cardiol, 110, 70-79.

DOKHAC, L., D'ALBIS, A., JANMOT, C. & HARBON, S. 1986. Myosin light chain phosphorylation in intact rat uterine smooth muscle. Role of calcium and cyclic AMP. J Muscle Res Cell Motil, 7, 259-68.

DORN, G. W., 2ND & BECKER, M. W. 1993. Thromboxane A2 stimulated signal transduction in vascular smooth muscle. J Pharmacol Exp Ther, 265, 447-56.

DUCKWORTH, N., MARSHALL, K. & CLAYTON, J. K. 2002. An investigation of the effect of the prostaglandin EP2 receptor agonist, butaprost, on the human isolated myometrium from pregnant and non-pregnant women. J Endocrinol, 172, 263-9.

DURN, J. H., MARSHALL, K. M., FARRAR, D., O'DONOVAN, P., SCALLY, A. J., WOODWARD, D. F. & NICOLAOU, A. 2010. Lipidomic analysis reveals prostanoid profiles in human term pregnant myometrium. Prostaglandins Leukot Essent Fatty Acids, 82, 21-6.

EDWARDS, D., GOOD, D. M., GRANGER, S. E., HOLLINGSWORTH, M., ROBSON, A., SMALL, R. C. & WESTON, A. H. 1986. The spasmogenic action of oxytocin in the rat uterus--comparison with other agonists. Br J Pharmacol, 88, 899-908.

305

EHSANIPOOR, R. M., SHRIVASTAVA, V. K., LEE, R. M., CHAN, K., GALYEAN, A. M., GARITE, T. J., RUMNEY, P. J. & WING, D. A. 2011. A randomized, double-masked trial of prophylactic indomethacin tocolysis versus placebo in women with premature rupture of membranes. Am J Perinatol, 28, 473-8.

ELCHALAL, U. & ABRAMOV, Y. 1995. Uterine biology and the intrauterine device. Advanced drug delivery reviews, 17, 151-164.

ENDRES, S., HACKER, A., NOACK, E., KOJDA, G. & LEHMANN, J. 1999. NO- Donors, part 3: nitrooxyacylated thiosalicylates and salicylates–synthesis and biological activities. European journal of medicinal chemistry, 34, 895-901.

ERGUL, M., TURGUT, N. H., SARAC, B., ALTUN, A., YILDIRIM, S. & BAGCIVAN, I. 2016. Investigating the effects of the Rho-kinase enzyme inhibitors AS1892802 and fasudil hydrochloride on the contractions of isolated pregnant rat myometrium. Eur J Obstet Gynecol Reprod Biol, 202, 45-50.

ERKINHEIMO, T. L., SAUKKONEN, K., NARKO, K., JALKANEN, J., YLIKORKALA, O. & RISTIMAKI, A. 2000. Expression of cyclooxygenase-2 and prostanoid receptors by human myometrium. J Clin Endocrinol Metab, 85, 3468-3475.

FACCHINETTI, F., PAGANELLI, S., COMITINI, G., DANTE, G. & VOLPE, A. 2007. Cervical length changes during preterm cervical ripening: effects of 17- alpha-hydroxyprogesterone caproate. Am J Obstet Gynecol, 196, 453 e1-4; discussion 421.

FANG, X., WONG, S. & MITCHELL, B. 1996. Relationships among sex steroids, oxytocin, and their receptors in the rat uterus during late gestation and at parturition. Endocrinology, 137, 3213-3219.

FAQI, A. S. 2012. A comprehensive guide to toxicology in preclinical drug development, Academic Press.

FEDIUK, J., SIKARWAR, A. S., NOLETTE, N. & DAKSHINAMURTI, S. 2014. Thromboxane-induced actin polymerization in hypoxic neonatal pulmonary arterial myocytes involves Cdc42 signaling. Am J Physiol Lung Cell Mol Physiol, 307, L877-87.

FEELY, J., DE VANE, P. J. & MACLEAN, D. 1983. New Drugs. Beta-blockers and sympathomimetics. Br Med J (Clin Res Ed), 286, 1043-7.

FERGUSON, S. S. 2001. Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol Rev, 53, 1-24.

FISCHER, D. P. 2010. The influence of the hormonal milieu on functional prostaglandin and oxytocin receptors and their downstream signal pathways in isolated human myometrium. PhD Thesis, University of Bradford, UK.

306

FISCHER, D. P., HUTCHINSON, J. A., FARRAR, D., O'DONOVAN, P. J., WOODWARD, D. F. & MARSHALL, K. M. 2008. Loss of prostaglandin F2α, but not thromboxane, responsiveness in pregnant human myometrium during labour. J Endocrinol, 197, 171-179.

FLEMING, I. 2011. Cytochrome P450-dependent eicosanoid production and crosstalk. Curr Opin Lipidol, 22, 403-9.

FLENADY, V., REINEBRANT, H. E., LILEY, H. G., TAMBIMUTTU, E. G. & PAPATSONIS, D. N. 2014. Oxytocin receptor antagonists for inhibiting preterm labour. Cochrane Database Syst Rev, 6, CD004452.

FLORIO, P., COBELLIS, L., WOODMAN, J., SEVERI, F. M., LINTON, E. A. & PETRAGLIA, F. 2002. Levels of maternal plasma corticotropin-releasing factor and urocortin during labor. J Soc Gynecol Investig, 9, 233-7.

FORSELLES, K. J., ROOT, J., CLARKE, T., DAVEY, D., AUGHTON, K., DACK, K. & PULLEN, N. 2011. In vitro and in vivo characterization of PF-04418948, a novel, potent and selective prostaglandin EP2 receptor antagonist. Br J Pharmacol, 164, 1847-1856.

FORSTERMANN, U., CLOSS, E. I., POLLOCK, J. S., NAKANE, M., SCHWARZ, P., GATH, I. & KLEINERT, H. 1994. Nitric oxide synthase isozymes. Characterization, purification, molecular cloning, and functions. Hypertension, 23, 1121-31.

FÖRSTERMANN, U. & KLEINERT, H. 1995. Nitric oxide synthase: expression and expressional control of the three isoforms. Naunyn-Schmiedeberg's archives of pharmacology, 352, 351-364.

FORSTERMANN, U., MULSCH, A., BOHME, E. & BUSSE, R. 1986. Stimulation of soluble guanylate cyclase by an acetylcholine-induced endothelium-derived factor from rabbit and canine arteries. Circ Res, 58, 531-8.

FÖRSTERMANN, U. & SESSA, W. C. 2012. Nitric oxide synthases: regulation and function. Eur Heart J, 33, 829-37, 837a-837d.

FRIEL, A. M., CURLEY, M., RAVIKUMAR, N., SMITH, T. J. & MORRISON, J. J. 2005. Rho A/Rho kinase mRNA and protein levels in human myometrium during pregnancy and labor. J Soc Gynecol Investig.12, 20-27.

FU, X., GONG, M. C., JIA, T., SOMLYO, A. V. & SOMLYO, A. P. 1998. The effects of the Rho-kinase inhibitor Y-27632 on arachidonic acid-, GTPgammaS-, and phorbol ester-induced Ca2+-sensitization of smooth muscle. FEBS Lett, 440, 183-7.

FUCHS, A.R., FUCHS, F., SOLOFF, M.S. 1985. Oxytocin receptors in nonpregnant human uterus. J Clin Endocrinol Metab.;60(1):37‐ 41. doi:10.1210/jcem-60-1- 37.

307

FUJISAWA, K., FUJITA, A., ISHIZAKI, T., SAITO, Y. & NARUMIYA, S. 1996. Identification of the Rho-binding domain of p160ROCK, a Rho-associated coiled-coil containing protein kinase. J Biol Chem, 271, 23022-8.

FUSI, L., CLOKE, B. & BROSENS, J. J. 2006. The uterine junctional zone. Best Pract Res Clin Obstet Gynaecol, 20, 479-91.

GARFIELD, R. & YALLAMPALLI, C. 1994. Structure and function of uterine muscle. The uterus, 54-93.

GARFIELD, R. E., BYTAUTIENE, E., VEDERNIKOV, Y. P., MARSHALL, J. S. & ROMERO, R. 2000. Modulation of rat uterine contractility by mast cells and their mediators. Am J Obstet Gynecol, 183, 118-25.

GARFIELD, R. E., SAADE, G., BUHIMSCHI, C., BUHIMSCHI, I., SHI, L., SHI, S. Q. & CHWALISZ, K. 1998. Control and assessment of the uterus and cervix during pregnancy and labour. Hum Reprod Update, 4, 673-95.

GARNOCK-JONES, K. P. 2014. Ripasudil: first global approval. Drugs, 74, 2211-5.

GAWRON, L. M. & KILEY, J. W. 2013. Labor induction outcomes in third-trimester stillbirths. Int J Gynaecol Obstet, 123, 203-6.

GERSHON, M. 2004. serotonin receptors and transporters—roles in normal and abnormal gastrointestinal motility. Aliment pharmacol Ther, 20, 3-14.

GIANNOULIAS, D., ALFAIDY, N., HOLLOWAY, A. C., GIBB, W., SUN, M., LYE, S. J. & CHALLIS, J. R. 2002. Expression of prostaglandin I(2) synthase, but not prostaglandin E synthase, changes in myometrium of women at term pregnancy. J Clin Endocrinol Metab, 87, 5274-82.

GIMPL, G. & FAHRENHOLZ, F. 2001. The oxytocin receptor system: structure, function, and regulation. Physiol Rev, 81, 629-83.

GOLDENBERG, R. L., CULHANE, J. F., IAMS, J. D. & ROMERO, R. 2008. Epidemiology and causes of preterm birth. Lancet, 371, 75-84.

GOLDENBERG, R. L., HAUTH, J. C. & ANDREWS, W. W. 2000. Intrauterine infection and preterm delivery. N Engl J Med, 342, 1500-7.

GONZALEZ, E., JAWERBAUM, A., NOVARO, V. & GIMENO, M. A. 1997. Influence of epoxyeicosatrienoic acids on uterine function. Prostaglandins Leukot Essent Fatty Acids, 56, 57-61.

GORDON-WRIGHT, A. P. & ELDER, M. G. 1980. Effect of prostaglandin E2 and its metabolites on lower segment myometrium in vitro. Eur J Obstet Gynecol Reprod Biol, 10, 297-302.

GORDON, M., SAMUELS, P., SHUBERT, P., JOHNSON, F., GEBAUER, C. & IAMS, J. 1995. A randomized, prospective study of adjunctive ceftizoxime in preterm labor. Am J Obstet Gynecol, 172, 1546-52.

308

GOUPIL, E., TASSY, D., BOURGUET, C., QUINIOU, C., WISEHART, V., PÉTRIN, D., LE GOUILL, C., DEVOST, D., ZINGG, H. H. & BOUVIER, M. 2010. A novel biased allosteric compound inhibitor of parturition selectively impedes the prostaglandin F2α-mediated Rho/ROCK signaling pathway. Journal of Biological Chemistry, 285, 25624-25636.

GRANN, M., COMERMA-STEFFENSEN, S., ARCANJO, D. D. & SIMONSEN, U. 2016. Mechanisms Involved in Thromboxane A2 -induced Vasoconstriction of Rat Intracavernous Small Penile Arteries. Basic Clin Pharmacol Toxicol, 119 Suppl 3, 86-95.

GRAVINA, F. S., VAN DDHELDEN, D. F., KERR, K. P., DE OLIVEIRA, R. B. & JOBLING, P. 2014. Phasic contractions of the mouse vagina and cervix at different phases of the estrus cycle and during late pregnancy. PLoS One. 9, e111307.

GRIFFITHS, A. L. 2007. The influence of the hormonal milieu on uterine prostaglandin EP, FP and TP-Receptor populations. PhD Thesis, University of Bradford, UK.

GRIFFITHS, A. L., MARSHALL, K. M., SENIOR, J., FLEMING, C. & WOODWARD, D. F. 2006. Effect of the oestrous cycle, pregnancy and uterine region on the responsiveness of the isolated mouse uterus to prostaglandin F-2 alpha and the thromboxane mimetic U46619. J Endocrinol, 188, 569-577.

GROSS, G. A., IMAMURA, T., LUEDKE, C., VOGT, S. K., OLSON, L. M., NELSON, D. M., SADOVSKY, Y. & MUGLIA, L. J. 1998. Opposing actions of prostaglandins and oxytocin determine the onset of murine labor. Proc Natl Acad Sci U S A, 95, 11875-9.

HAAS, D. M., CALDWELL, D. M., KIRKPATRICK, P., MCINTOSH, J. J. & WELTON, N. J. 2012. Tocolytic therapy for preterm delivery: systematic review and network meta-analysis. BMJ, 345, e6226.

HAMMOND, K. A., LLOYD, K. & DIAMOND, J. 1996. Is mammary output capacity limiting to lactational performance in mice? Journal of Experimental Biology, 199, 337-349.

HANANIA, N. A. & CAZZOLA, M. J. 2008. Bronchodilators: Beta 2-Agonists and CHAPTER. Clinical Asthma E-Book, p.241.

HANLEY, N. R. & HENSLER, J. G. 2002. Mechanisms of ligand-induced desensitization of the 5-hydroxytryptamine(2A) receptor. J Pharmacol Exp Ther, 300, 468-77.

HARROD, J. S., RADA, C. C., PIERCE, S. L., ENGLAND, S. K. & LAMPING, K. G. 2011. Altered contribution of RhoA/Rho kinase signaling in contractile activity of myometrium in leptin receptor-deficient mice. Am J Physiol Endocrinol Metab, 301, E362-9.

309

HATAKEYAMA, H., AKITA, H., ISHIDA, E., HASHIMOTO, K., KOBAYASHI, H., AOKI, T., YASUDA, J., OBATA, K., KIKUCHI, H., ISHIDA, T., KIWADA, H. & HARASHIMA, H. 2007. Tumor targeting of doxorubicin by anti-MT1- MMP antibody-modified PEG liposomes. Int J Pharm, 342, 194-200.

HEIN, P., MICHEL, M. C., LEINEWEBER, K., WIELAND, T., WETTSCHURECK, N. & OFFERMANNS, S. 2005. Receptor and binding studies. Practical Methods in Cardiovascular Research. Springer.

HELMER, H., HACKL, T., SCHNEEBERGER, C., KNOFLER, M., BEHRENS, O., KAIDER, A. & HUSSLEIN, P. 1998. Oxytocin and vasopressin 1a receptor gene expression in the cycling or pregnant human uterus. Am J Obstet Gynecol, 179, 1572-8.

HENDRIX, E. M., MYATT, L., SELLERS, S., RUSSELL, P. T. & LARSEN, W. J. 1995. Steroid hormone regulation of rat myometrial gap junction formation: effects on cx43 levels and trafficking. Biol Reprod, 52, 547-60.

HERON, M., SUTTON, P. D., XU, J., VENTURA, S. J., STROBINO, D. M. & GUYER, B. 2010. Annual summary of vital statistics: 2007. Pediatrics, 125, 4- 15.

HICKMAN, D., JOHNSON, J., VEMULAPALLI, T., CRISLER, J. & SHEPHERD, R. 2016. Commonly Used Animal Models. Principles of Animal Research for Graduate and Undergraduate Students. Amesterdam: Elsevier Inc.

HIRATA, T., USHIKUBI, F., KAKIZUKA, A., OKUMA, M. & NARUMIYA, S. 1996. Two thromboxane A2 receptor isoforms in human platelets. Opposite coupling to adenylyl cyclase with different sensitivity to Arg60 to Leu mutation. J Clin Invest, 97, 949-56.

HIRSCH, E. & MUHLE, R. 2002. Intrauterine bacterial inoculation induces labor in the mouse by mechanisms other than progesterone withdrawal. Biol Reprod, 67, 1337-1341.

HIRST, J. J., PARKINGTON, H. C., YOUNG, I. R., PALLISER, H. K., PERI, K. G. & OLSON, D. M. 2005. Delay of preterm birth in sheep by THG113.31, a prostaglandin F-2 alpha receptor antagonist. Am J Obstet Gynecol, 193, 256- 266.

HOLT, R., TIMMONS, B. C., AKGUL, Y., AKINS, M. L. & MAHENDROO, M. 2011. The Molecular Mechanisms of Cervical Ripening Differ between Term and Preterm Birth. Endocrinology, 152, 1036-1046.

HSU, C. R., CHEN, Y. H., LIU, C. P., CHEN, C. H., HUANG, K. K., HUANG, J. W., LIN, M. N., LIN, C. L., CHEN, W. R., HSU, Y. L., LEE, T. C., CHOU, S. H., TU, C. M., HWANG, C. S., HUANG, Y. C. & LU, D. W. 2019. A Highly Selective Rho-Kinase Inhibitor (ITRI-E-212) Potentially Treats Glaucoma Upon Topical Administration With Low Incidence of Ocular Hyperemia. Invest Ophthalmol Vis Sci, 60, 624-633.

310

HUDSON, C. A., HEESOM, K. J. & LOPEZ BERNAL, A. 2012. Phasic contractions of isolated human myometrium are associated with Rho-kinase (ROCK)- dependent phosphorylation of myosin phosphatase-targeting subunit (MYPT1). Mol Hum Reprod, 18, 265-79.

HUNTER, T. 2000. Signaling--2000 and beyond. Cell, 100, 113-27.

HUSSLEIN, P., ROURA, L. C., DUDENHAUSEN, J., HELMER, H., FRYDMAN, R., RIZZO, N., SCHNEIDER, D. & GROUP, T. S. 2006. Clinical practice evaluation of atosiban in preterm labour management in six European countries. BJOG, 113 Suppl 3, 105-10.

HUTCHINSON, J., MARSHALL, K. & SENIOR, J. 2003. Preliminary studies using a putative FP-receptor antagonist, AL-8810, on isolated mouse uterus. British Journal of Pharmacology, 1.

HUTCHINSON, J. A. 2005. A study into the roles of prostanoid receptors in the uterus. PhD Thesis, University of Bradford, UK

HWANG, S. H., WECKSLER, A. T., WAGNER, K. & HAMMOCK, B. D. 2013. Rationally designed multitarget agents against inflammation and pain. Curr Med Chem, 20, 1783-99.

IKEBE, M., INAGAKI, M., KANAMARU, K. & HIDAKA, H. 1985. Phosphorylation of smooth muscle myosin light chain kinase by Ca2+-activated, phospholipid- dependent protein kinase. J Biol Chem, 260, 4547-50.

IKEBE, M., KORETZ, J. & HARTSHORNE, D. J. 1988. Effects of phosphorylation of light chain residues threonine 18 and serine 19 on the properties and conformation of smooth muscle myosin. J Biol Chem, 263, 6432-7.

IMAMURA, T., LUEDKE, C. E., VOGT, S. K. & MUGLIA, L. J. 2000. Oxytocin modulates the onset of murine parturition by competing ovarian and uterine effects. Am J Physiol Regul Integr Comp Physiol, 279, R1061-7.

IMIG, J. D., ZHAO, X., CAPDEVILA, J. H., MORISSEAU, C. & HAMMOCK, B. D. 2002. Soluble epoxide hydrolase inhibition lowers arterial blood pressure in angiotensin II hypertension. Hypertension, 39, 690-4.

ISOBE, T., MIZUNO, K., KANEKO, Y., OHTA, M., KOIDE, T. & TANABE, S. 2014. Effects of K-115, a rho-kinase inhibitor, on aqueous humor dynamics in rabbits. Curr Eye Res, 39, 813-22.

IVERSEN, L., FOGH, K., BOJESEN, G. & KRAGBALLE, K. 1991. Linoleic acid and dihomogammalinolenic acid inhibit leukotriene B4 formation and stimulate the formation of their 15-lipoxygenase products by human neutrophils in vitro. Evidence of formation of antiinflammatory compounds. Agents Actions, 33, 286-91.

IZUMI, H., YALLAMPALLI, C. & GARFIELD, R. E. 1993. Gestational changes in L- arginine-induced relaxation of pregnant rat and human myometrial smooth muscle. Am J Obstet Gynecol, 169, 1327-37.

311

JABBOUR, H. N. & SALES, K. J. 2004. Prostaglandin receptor signalling and function in human endometrial pathology. Trends Endocrinol Metab, 15, 398-404.

JAYASOORIYA, G. S. & LAMONT, R. F. 2009. The use of progesterone and other progestational agents to prevent spontaneous preterm labour and preterm birth. Expert Opin Pharmacother, 10, 1007-1016.

JIANG, M. J., CHAN, C. F. & CHANG, Y. L. 1994. Intracellular calcium and myosin light chain phosphorylation during U46619-activated vascular contraction. Life Sci, 54, 2005-13.

JIVIDEN, K., MOVASSAGH, M. J., JAZAERI, A. & LI, H. 2014. Two methods for establishing primary human endometrial stromal cells from hysterectomy specimens. J Vis Exp.

JONES, R. L., GIEMBYCZ, M. A. & WOODWARD, D. F. 2009. Prostanoid receptor antagonists: development strategies and therapeutic applications. Br J Pharmacol, 158, 104-145.

KALACHE, K. D., CHAOUI, R., MARKS, B., WAUER, R. & BOLLMANN, R. 2002. Does fetal tracheal fluid flow during fetal breathing movements change before the onset of labour? Bjog-an International Journal of Obstetrics and Gynaecology, 109, 514-519.

KALUDJEROVIC, J. and WARD, W. 2012. The interplay between estrogen and fetal adrenal cortex. J Nutr Metab., 2012:837901.

KANDABASHI, T., SHIMOKAWA, H., MIYATA, K., KUNIHIRO, I., KAWANO, Y., FUKATA, Y., HIGO, T., EGASHIRA, K., TAKAHASHI, S. & KAIBUCHI, K. 2000. Inhibition of myosin phosphatase by upregulated Rho- kinase plays a key role for coronary artery spasm in a porcine model with interleukin-1β. Circulation, 101, 1319-1323.

KANEKO, Y., OHTA, M., INOUE, T., MIZUNO, K., ISOBE, T., TANABE, S. & TANIHARA, H. 2016. Effects of K-115 (Ripasudil), a novel ROCK inhibitor, on trabecular meshwork and Schlemm's canal endothelial cells. Sci Rep, 6, 19640.

KARIM, S., HILLIER, K. & TRUSSELL, R. 1971. The effects of prostaglandins E2 and F2α administered by different routes on uterine activity and the cardiovascular system in pregnant and non‐ pregnant women. BJOG: An International Journal of Obstetrics & Gynaecology, 78, 172-179.

KASAI, Y., TSUTSUMI, O., TAKETANI, Y., ENDO, M. & IINO, M. 1995. Stretch‐ induced enhancement of contractions in uterine smooth muscle of rats. J Physiol., 486, 373-384.

KAUSHAL, N., KUDVA, A. K., PATTERSON, A. D., CHIARO, C., KENNETT, M. J., DESAI, D., AMIN, S., CARLSON, B. A., CANTORNA, M. T. & PRABHU, K. S. 2014. Crucial role of macrophage selenoproteins in experimental colitis. J Immunol, 193, 3683-92.

312

KEIRSE, M. J. 1990. Progestogen Administration in Pregnancy May Prevent Preterm Delivery. Br J Obstet Gynaecol, 97, 149-154.

KELLER, N. R., SIERRA-RIVERA, E., EISENBERG, E. & OSTEEN, K. G. 2000. Progesterone exposure prevents matrix metalloproteinase-3 (MMP-3) stimulation by interleukin-1alpha in human endometrial stromal cells. J Clin Endocrinol Metab, 85, 1611-9.

KELLY, C. R., SHARIF, N. A. 2006. Pharmacological evidence for a functional serotonin-2B receptor in a human uterine smooth muscle cell line. J Pharmacol Exp Ther., 317, 1254-1261.

KELLY, E., BAILEY, C. P. & HENDERSON, G. 2008. Agonist‐ selective mechanisms of GPCR desensitization. Br J Pharmacol., 153, S379-S388.

KENDALL, G. S., HRISTOVA, M., ZBARSKY, V., CLEMENTS, A., PEEBLES, D. M., ROBERTSON, N. J. & RAIVICH, G. 2011. Distribution of pH changes in mouse neonatal hypoxic-ischaemic insult. Dev Neurosci, 33, 505-18.

KENNEDY, F., MARSHALL, K. & SENIOR, J. 1994. Characterization Of The Thromboxane (Tp) Receptor Population In Isolated Mouse Uterus. Br J Pharmacol, 112, pp. U218-U218.

KENYON, S., BOULVAIN, M. & NEILSON, J. P. 2010. Antibiotics for preterm rupture of membranes. Cochrane Database Syst Rev, CD001058.

KESERU, B., BARBOSA-SICARD, E., POPP, R., FISSLTHALER, B., DIETRICH, A., GUDERMANN, T., HAMMOCK, B. D., FALCK, J. R., WEISSMANN, N., BUSSE, R. & FLEMING, I. 2008. Epoxyeicosatrienoic acids and the soluble epoxide hydrolase are determinants of pulmonary artery pressure and the acute hypoxic pulmonary vasoconstrictor response. Faseb j, 22, 4306-15.

KHAN, M. & FRASER, A. 2012. Cox-2 inhibitors and the risk of cardiovascular thrombotic events. Ir Med J, 105, 119-21.

KILPATRICK, S. J., SCHLUETER, M. A., PIECUCH, R., LEONARD, C. H., ROGIDO, M. & SOLA, A. 1997. Outcome of infants born at 24-26 weeks' gestation .1. Survival and cost. Obstet Gynecol, 90, 803-808.

KIM, H. S., PARK, J. E., KIM, S. Y., KIM, J. E., CHAE, S. H., SOHN, I. S., HWANG, H. S. & KWON, H. S. 2018. Incarceration of early gravid uterus with adenomyosis and myoma: report of two patients managed with uterine reduction. Obstet Gynecol Sci, 61, 621-625.

KIM, P. Y., ZHONG, M., KIM, Y.-S., SANBORN, B. M. & ALLEN, K. G. 2012. Long chain polyunsaturated fatty acids alter oxytocin signaling and receptor density in cultured pregnant human myometrial smooth muscle cells. PLoS One, 7.

313

KIMURA, T., TAKEMURA, M., NOMURA, S., NOBUNAGA, T., KUBOTA, Y., INOUE, T., HASHIMOTO, K., KUMAZAWA, I., ITO, Y., OHASHI, K., KOYAMA, M., AZUMA, C., KITAMURA, Y. & SAJI, F. 1996. Expression of oxytocin receptor in human pregnant myometrium. Endocrinology, 137, 780-5.

KING, A., NDIFON, C., LUI, S., WIDDOWS, K., KOTAMRAJU, V. R., AGEMY, L., TEESALU, T., GLAZIER, J. D., CELLESI, F., TIRELLI, N., APLIN, J. D., RUOSLAHTI, E. & HARRIS, L. K. 2016. Tumor-homing peptides as tools for targeted delivery of payloads to the placenta. Sci Adv, 2, e1600349.

KING, J., FLENADY, V., COLE, S. & THORNTON, S. 2005. Cyclo-oxygenase (COX) inhibitors for treating preterm labour. Cochrane Database Syst Rev, CD001992.

KING, J. F., FLENADY, V. J., PAPATSONIS, D. N., DEKKER, G. A. & CARBONNE, B. 2003. Calcium channel blockers for inhibiting preterm labour. Cochrane Database Syst Rev, CD002255.

KINOSHITA, E., KINOSHITA-KIKUTA, E. & KOIKE, T. 2009. Separation and detection of large phosphoproteins using Phos-tag SDS-PAGE. Nat Protoc, 4, 1513-21.

KINOSHITA, E., KINOSHITA-KIKUTA, E., TAKIYAMA, K. & KOIKE, T. 2006. Phosphate-binding tag, a new tool to visualize phosphorylated proteins. Mol Cell Proteomics, 5, 749-57.

KISHI, T., HIROOKA, Y., MASUMOTO, A., ITO, K., KIMURA, Y., INOKUCHI, K., TAGAWA, T., SHIMOKAWA, H., TAKESHITA, A. & SUNAGAWA, K. 2005. Rho-kinase inhibitor improves increased vascular resistance and impaired vasodilation of the forearm in patients with heart failure. Circulation, 111, 2741- 7.

KISHORE, A. H., OWENS, D. & WORD, R. A. 2014. Prostaglandin E2 regulates its own inactivating enzyme, 15-PGDH, by EP2 receptor-mediated cervical cell- specific mechanisms. J Clin Endocrinol Metab, 99, 1006-18.

KITAZAWA, T., KUBO, O., SATOH, M. & TANEIKE, T. 1998. Involvement of 5- hydroxytryptamine7 receptors in inhibition of porcine myometrial contractility by 5-hydroxytryptamine. Br J Pharmacol, 123, 173-82.

KLAUSER, C. K., BRIERY, C. M., MARTIN, R. W., LANGSTON, L., MAGANN, E. F. & MORRISON, J. C. 2014. A comparison of three tocolytics for preterm labor: a randomized clinical trial. J Matern Fetal Neonatal Med, 27, 801-6.

KNOBLAUGH, S. E. & RANDOLPH-HABECKER, J. 2018. 3 - Necropsy and Histology. In: TREUTING, P. M., DINTZIS, S. M. & MONTINE, K. S. (eds.) Comparative Anatomy and Histology (Second Edition). San Diego: Academic Press.

314

KONISHI, H., URABE, S., MIYOSHI, H., TERAOKA, Y., MAKI, T., FURUSHO, H., MIYAUCHI, M., TAKATA, T., KUDO, Y. & KAJIOKA, S. 2019. Fetal Membrane Inflammation Induces Preterm Birth Via Toll-Like Receptor 2 in Mice With Chronic Gingivitis. Reprod Sci, 26, 869-878.

KONOPKA, C. K., MORAIS, E. N., NAIDON, D., PEREIRA, A. M., RUBIN, M. A., OLIVEIRA, J. F. & MELLO, C. F. 2013. Maternal serum progesterone, estradiol and estriol levels in successful dinoprostone-induced labor. Braz J Med Biol Res, 46, 91-7.

KOTA, S. K., GAYATRI, K., JAMMULA, S., KOTA, S. K., KRISHNA, S. V., MEHER, L. K. & MODI, K. D. 2013. Endocrinology of parturition. Indian J Endocrinol Metab, 17, 50-9.

KOUCKY, M., GERMANOVA, A., HAJEK, Z., PARIZEK, A., KALOUSOVA, M. & KOPECKY, P. 2009. Pathophysiology of preterm labour. Prague Med Rep, 110, 13-24.

KUBOTA, Y., KIMURA, T., HASHIMOTO, K., TOKUGAWA, Y., NOBUNAGA, K., AZUMA, C., SAJI, F. & MURATA, Y. 1996. Structure and expression of the mouse oxytocin receptor gene. Mol Cell Endocrinol, 124, 25-32.

KUMAR, N., GUPTA, G., ANILKUMAR, K., FATIMA, N., KARNATI, R., REDDY, G. V., GIRI, P. V. & REDDANNA, P. 2016. 15-Lipoxygenase metabolites of α- linolenic acid,[13-(S)-HPOTrE and 13-(S)-HOTrE], mediate anti-inflammatory effects by inactivating NLRP3 inflammasome. Scientific reports, 6, 31649.

KUNAPULI, P., LAWSON, J. A., ROKACH, J. & FITZGERALD, G. A. 1997. Functional Characterization of the Ocular Prostaglandin F2α (PGF2α) Receptor ACTIVATION BY THE ISOPROSTANE, 12-iso-PGF2α. Journal of Biological Chemistry, 272, 27147-27154.

KUPITTAYANANT, S., BURDYGA, T. & WRAY, S. 2001. The effects of inhibiting Rho-associated kinase with Y-27632 on force and intracellular calcium in human myometrium. Pflugers Arch, 443, 112-114.

KURKINEN-RATY, M., VUOPALA, S., KOSKELA, M., KEKKI, M., KURKI, T., PAAVONEN, J. & JOUPPILA, P. 2000. A randomised controlled trial of vaginal clindamycin for early pregnancy bacterial vaginosis. BJOG, 107, 1427- 32.

KUSINSKI, L. C., STANLEY, J. L., DILWORTH, M. R., HIRT, C. J., ANDERSSON, I. J., RENSHALL, L. J., BAKER, B. C., BAKER, P. N., SIBLEY, C. P., WAREING, M. & GLAZIER, J. D. 2012. eNOS knockout mouse as a model of fetal growth restriction with an impaired uterine artery function and placental transport phenotype. Am J Physiol Regul Integr Comp Physiol, 303, R86-93.

LACKRITZ, R., TULCHINSKY, D., RYAN, K. J. & LEVINE, L. 1978. Plasma prostaglandin metabolites in human labor. Am J Obstet Gynecol, 131, 484-9.

315

LAMONT, R. F., DUNCAN, S. L., MANDAL, D. & BASSETT, P. 2003. Intravaginal clindamycin to reduce preterm birth in women with abnormal genital tract flora. Obstet Gynecol, 101, 516-22.

LARTEY, J. & BERNAL, A. L. 2009. RHO protein regulation of contraction in the human uterus. Reproduction, 138, 407-424.

LATOS, P. A. & HEMBERGER, M. 2016. From the stem of the placental tree: trophoblast stem cells and their progeny. Development, 143, 3650-3660.

LAVEN, J. S. & FAUSER, B. C. 2006. What role of estrogens in ovarian stimulation. Maturitas, 54, 356-62.

LEBEL, W., RICCARDI, K., GRASSER, W. A., TERRY, K., THOMPSON, D. & PARALKAR, V. M. 2004. Prostaglandin E2 receptor subtype EP-2 is not involved in the induction of non-pregnant guinea pig uterine contractions associated with terminal pregnancy. Prostaglandins Leukot Essent Fatty Acids, 71, 399-404.

LEDINGHAM, M. A., THOMSON, A. J., GREER, I. A. & NORMAN, J. E. 2000. Nitric oxide in parturition. BJOG: An International Journal of Obstetrics & Gynaecology, 107, 581-593.

LEE, E. J., KIM, S.-J., HAHN, Y.-I., YOON, H.-J., HAN, B., KIM, K., LEE, S., KIM, K. P., SUH, Y. G. & NA, H.-K. 2019. 15-Keto prostaglandin E2 suppresses STAT3 signaling and inhibits breast cancer cell growth and progression. Redox biology, 23, 101175.

LEE, J. H. & CHANG, K. C. 1995. Different sensitivity to nitric oxide of human pregnant and non-pregnant myometrial contractility. Pharmacol Commun, 5, 147-154.

LEE, T. Y., LIN, C. T., KUO, S. Y., CHANG, D. K. & WU, H. C. 2007. Peptide- mediated targeting to tumor blood vessels of lung cancer for drug delivery. Cancer Res, 67, 10958-65.

LEONARD, S. A., CRESPI, C. M., GEE, D. C., ZHU, Y. & WHALEY, S. E. 2015. Prepregnancy Risk Factors for Preterm Birth and the Role of Maternal Nativity in a Low-Income, Hispanic Population. Matern Child Health J., 19(10):2295‐ 2302.

LEUNG, T., CHEN, X. Q., MANSER, E. & LIM, L. 1996. The p160 RhoA-binding kinase ROK alpha is a member of a kinase family and is involved in the reorganization of the cytoskeleton. Mol Cell Biol, 16, 5313-27.

LEUNG, T., MANSER, E., TAN, L. & LIM, L. 1995. A novel serine/threonine kinase binding the Ras-related RhoA GTPase which translocates the kinase to peripheral membranes. J Biol Chem, 270, 29051-4.

LEVINE, J. 2015. Neuroendocrine control of the ovarian cycle of the rat. Knobil and Neill’s Physiology of Reproduction. Elsevier Inc., 2, pp.1199-1257.

316

LI, F., HAGAMAN, J. R., KIM, H.-S., MAEDA, N., JENNETTE, J. C., FABER, J. E., KARUMANCHI, S. A., SMITHIES, O. & TAKAHASHI, N. 2012. eNOS deficiency acts through endothelin to aggravate sFlt-1–induced pre-eclampsia– like phenotype. Journal of the American Society of Nephrology, 23, 652-660.

LIAO, J. B., BUHIMSCHI, C. S. & NORWITZ, E. R. 2005. Normal labor: mechanism and duration. Obstet Gynecol Clin North Am, 32, 145-64, vii.

LIAO, J. K., SETO, M. & NOMA, K. 2007. Rho kinase (ROCK) inhibitors. J Cardiovasc Pharmacol, 50, 17-24.

LIM, A. C., BLOEMENKAMP, K. W., BOER, K., DUVEKOT, J. J., ERWICH, J. J., HASAART, T. H., HUMMEL, P., MOL, B. W., OFFERMANS, J. P., VAN OIRSCHOT, C. M., SANTEMA, J. G., SCHEEPERS, H. C., SCHOLS, W. A., VANDENBUSSCHE, F. P., WOUTERS, M. G., BRUINSE, H. W. & GROUP, A. S. 2007. Progesterone for the prevention of preterm birth in women with multiple pregnancies: the AMPHIA trial. BMC Pregnancy Childbirth, 7, 7.

LINCOLN, T. M. 1989. Cyclic GMP and mechanisms of vasodilation. Pharmacol Ther, 41, 479-502.

LINCOLN, T. M., DEY, N. & SELLAK, H. 2001. Invited review: cGMP-dependent protein kinase signaling mechanisms in smooth muscle: from the regulation of tone to gene expression. Journal of applied physiology, 91, 1421-1430.

LIU, X., MILLER, M. J., JOSHI, M. S., SADOWSKA-KROWICKA, H., CLARK, D. A. & LANCASTER, J. R., JR. 1998. Diffusion-limited reaction of free nitric oxide with erythrocytes. J Biol Chem, 273, 18709-13.

LO, C.-C., HSU, J.-J., HSIEH, C.-C. & HUNG, T.-H. 2007. Risk factors for spontaneous preterm delivery before 34 weeks of gestation among Taiwanese women. Taiwanese Journal of Obstetrics and Gynecology, 46, 389-394.

LOPEZ BERNAL, A. 2003. Mechanisms of labour--biochemical aspects. Bjog, 110 Suppl 20, 39-45.

LOPEZ BERNAL, A. 2007. Overview. Preterm labour: mechanisms and management. BMC Pregnancy Childbirth, 7 Suppl 1, S2.

LUNDBERG, J. O., WEITZBERG, E. & GLADWIN, M. T. 2008. The nitrate-nitrite- nitric oxide pathway in physiology and therapeutics. Nat Rev Drug Discov, 7, 156-67.

LUO, G., ABRAHAMS, V. M., TADESSE, S., FUNAI, E. F., HODGSON, E. J., GAO, J. & NORWITZ, E. R. 2010. Progesterone inhibits basal and TNF-alpha-induced apoptosis in fetal membranes: a novel mechanism to explain progesterone- mediated prevention of preterm birth. Reprod Sci, 17, 532-9.

LUTTON, E. J., LAMMERS, W., JAMES, S., VAN DEN BERG, H. A. & BLANKS, A. M. 2018. Identification of uterine pacemaker regions at the myometrial- placental interface in the rat. J Physiol, 596, 2841-2852.

317

LYCHKOVA, A. E., DE PASQUALE, V., AVALLONE, L., PUZIKOV, A. M., PAVONE, L. M. 2014. Serotonin regulates contractile activity of the uterus in non-pregnant rabbits. Toxicology & Pharmacology, 165, 53-59.

LYE, S. J., OU, C.-W., TEOH, T.-G., ERB, G., STEVENS, Y., CASPER, R., PATEL, F. A. & CHALLIS, J. R. 1998. The molecular basis of labour and tocolysis. Fetal and Maternal Medicine Review, 10, 121-136.

MACDORMAN, M. F., MATTHEWS, T. J., MOHANGOO, A. D. & ZEITLIN, J. 2014. International comparisons of infant mortality and related factors: United States and Europe, 2010. Natl Vital Stat Rep, 63, 1-6.

MACDOUGALL, M. W., EUROPE-FINNER, G. N. & ROBSON, S. C. 2003. Human myometrial quiescence and activation during gestation and parturition involve dramatic changes in expression and activity of particulate type II (RII alpha) protein kinase A holoenzyme. J Clin Endocrinol Metab, 88, 2194-205.

MACINTYRE, D. A., SYKES, L., TEOH, T. G. & BENNETT, P. R. 2012. Prevention of preterm labour via the modulation of inflammatory pathways. J Matern Fetal Neonatal Med, 25 Suppl 1, 17-20.

MACKEEN, A. D., SEIBEL-SEAMON, J., MUHAMMAD, J., BAXTER, J. K. & BERGHELLA, V. 2014. Tocolytics for preterm premature rupture of membranes. Cochrane Database Syst Rev, 2, CD007062.

MAHENDROO, M. 2012. Cervical remodeling in term and preterm birth: insights from an animal model. Reproduction, 143, 429-38.

MAHMOOD, T. & YANG, P. C. 2012. Western blot: technique, theory, and trouble shooting. N Am J Med Sci, 4, 429-34.

MAHMOODI, Z., HOSEINI, F. H. F. H. F., GHODSI, Z., AMINI, L. & AMINI, M. S. L. 2010. The Association The Association between Maternal Factors een Maternal Factors een Maternal Factors and Preterm Birth Preterm Birth and Premature and Premature Rapture of Membranes Rapture of Membranes Rapture of Membranes. Journal of Family and Reproductive Health, 4, 135.

MAIR, K., MACLEAN, M., MORECROFT, I., DEMPSIE, Y. & PALMER, T. 2008. Novel interactions between the 5‐ HT transporter, 5‐ HT1B receptors and Rho kinase in vivo and in pulmonary fibroblasts. British Journal of Pharmacology, 155, 606-616.

MALLAT, Z., GOJOVA, A., SAUZEAU, V., BRUN, V., SILVESTRE, J. S., ESPOSITO, B., MERVAL, R., GROUX, H., LOIRAND, G. & TEDGUI, A. 2003. Rho-associated protein kinase contributes to early atherosclerotic lesion formation in mice. Circ Res, 93, 884-8.

MANBE, Y., MANABE, A. & TAKAHASHI, A. 1982. F prostaglandin levels in amniotic fluid during balloon-induced cervical softening and labor at term. Prostaglandins, 23, 247-256.

318

MARIEB, E. N. & HOEHN, K. 2016. Human anatomy & physiology, Essex, England, Pearson Education Limited.

MARINOV, B., ANDREEVA, A., KARAMISHEVA, V. & MUSEVA, A. 2014. [Preterm birth in Bulgaria--changes in aetiology and incidence in the last decade]. Akush Ginekol (Sofiia), 53 Suppl 1, 33-9.

MAROTEAUX, L., AYME-DIETRICH, E., AUBERTIN-KIRCH, G., BANAS, S., QUENTIN, E., LAWSON, R. & MONASSIER, L. 2016. New therapeutic opportunities for 5-HT2 receptor ligands. Pharmacol Ther, 170, 14-36.

MARTINSEN, A., YERNA, X., RATH, G., GOMEZ, E. L., DESSY, C. & MOREL, N. 2012. Different effect of Rho kinase inhibition on calcium signaling in rat isolated large and small arteries. Journal of vascular research, 49, 522-533.

MASON, S. M., KAUFMAN, J. S., EMCH, M. E., HOGAN, V. K. & SAVITZ, D. A. 2010. Ethnic density and preterm birth in African-, Caribbean-, and US-born non-Hispanic black populations in New York City. Am J Epidemiology, 172, 800-8.

MASOODI, M., MIR, A. A., PETASIS, N. A., SERHAN, C. N. & NICOLAOU, A. 2008. Simultaneous lipidomic analysis of three families of bioactive lipid mediators leukotrienes, resolvins, protectins and related hydroxy-fatty acids by liquid chromatography/electrospray ionisation tandem mass spectrometry. Rapid Commun Mass Spectrom, 22, 75-83.

MASOODI, M. & NICOLAOU, A. 2006. Lipidomic analysis of twenty-seven prostanoids and isoprostanes by liquid chromatography/electrospray tandem mass spectrometry. Rapid Commun Mass Spectrom, 20, 3023-9.

MASSON, J., EMERIT, M. B., HAMON, M., DARMON, M. 2012. Serotonergic signaling: multiple effectors and pleiotropic effects. Wiley Interdisciplinary Reviews: Membrane Transport and Signaling, 1, 685-713.

MASUMOTO, A., HIROOKA, Y., SHIMOKAWA, H., HIRONAGA, K., SETOGUCHI, S. & TAKESHITA, A. 2001. Possible involvement of Rho- kinase in the pathogenesis of hypertension in humans. Hypertension, 38, 1307- 10.

MASUMOTO, A., MOHRI, M., SHIMOKAWA, H., URAKAMI, L., USUI, M. & TAKESHITA, A. 2002. Suppression of coronary artery spasm by the Rho- kinase inhibitor fasudil in patients with vasospastic angina. Circulation, 105, 1545-7.

MATSUI, T., AMANO, M., YAMAMOTO, T., CHIHARA, K., NAKAFUKU, M., ITO, M., NAKANO, T., OKAWA, K., IWAMATSU, A. & KAIBUCHI, K. 1996. Rho-associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho. Embo j, 15, 2208-16.

319

MATSUI, T. S. & DEGUCHI, S. 2019. Spatially selective myosin regulatory light chain regulation is absent in dedifferentiated vascular smooth muscle cells but is partially induced by fibronectin and Klf4. Am J Physiol Cell Physiol, 316, C509- c521.

MATSUMOTO, T., SAGAWA, N., YOSHIDA, M., MORI, T., TANAKA, I., MUKOYAMA, M., KOTANI, M. & NAKAO, K. 1997. The prostaglandin E 2 and F 2α receptor genes are expressed in human myometrium and are down- regulated during pregnancy. Biochem Biophys Res Commun, 238, 838-841.

MATTHEW, A., KUPITTAYANANT, S., BURDYGA, T. & WRAY, S. 2004. Characterization of contractile activity and intracellular Ca2+ signalling in mouse myometrium. J Soc Gynecol Investig, 11, 207-12.

MAWE, G., COATES, M., MOSES, P. L. 2006. Intestinal serotonin signalling in irritable bowel syndrome. Alimentary pharmacology & therapeutics, 23, 1067- 1076.

MCCORMACK, J. & GREENWALD, G. 1974. Progesterone and oestradiol-17β concentrations in the peripheral plasma during pregnancy in the mouse. J Endocrinol., 62, 101-107.

MCGILL, J. & SHETTY, A. 2007. Mifepristone and misoprostol in the induction of labor at term. International Journal of Gynecology & Obstetrics, 96, 80-84.

MCGREGOR, J. A., FRENCH, J. I. & SEO, K. 1991. Adjunctive clindamycin therapy for preterm labor: results of a double-blind, placebo-controlled trial. Am J Obstet Gynecol, 165, 867-75.

MEIS, P. J., KLEBANOFF, M., THOM, E., DOMBROWSKI, M. P., SIBAI, B., MOAWAD, A. H., SPONG, C. Y., HAUTH, J. C., MIODOVNIK, M., VARNER, M. W., LEVENO, K. J., CARITIS, S. N., IAMS, J. D., WAPNER, R. J., CONWAY, D., O'SULLIVAN, M. J., CARPENTER, M., MERCER, B., RAMIN, S. M., THORP, J. M., PEACEMAN, A. M., GABBE, S., NATIONAL INSTITUTE OF CHILD, H. & HUMAN DEVELOPMENT MATERNAL- FETAL MEDICINE UNITS, N. 2003. Prevention of recurrent preterm delivery by 17 alpha-hydroxyprogesterone caproate. N Engl J Med, 348, 2379-85.

MEMON, H. U. & HANDA, V. L. 2013. Vaginal childbirth and pelvic floor disorders. Women’s health, 9, 265-277.

MESIANO, S. 2004. Myometrial progesterone responsiveness and the control of human parturition. J Soc Gynecol Investig, 11, 193-202.

MESIANO, S., CHAN, E. C., FITTER, J. T., KWEK, K., YEO, G. & SMITH, R. 2002. Progesterone withdrawal and estrogen activation in human parturition are coordinated by progesterone receptor A expression in the myometrium. J Clin Endocrinol Metab, 87, 2924-2930.

MIHM, M., GANGOOLY, S. & MUTTUKRISHNA, S. 2011. The normal menstrual cycle in women. Animal reproduction science, 124, 229-236.

320

MINOSYAN, T. Y., LU, R., EGHBALI, M., TORO, L. & STEFANI, E. 2007. Increased 5-HT contractile response in late pregnant rat myometrium is associated with a higher density of 5-HT2A receptors. J Physiol, 581, 91-7.

MIRZAIE, F. & MOHAMMAH-ALIZADEH, S. 2007. Contributing factors of preterm delivery in parturient in a University Hospital in Iran. Saudi Med J, 28, 400-4.

MITCHELL, B. F., AGUILAR, H. N., MOSHER, A., WOOD, S. & SLATER, D. M. 2013. The uterine myocyte as a target for prevention of preterm birth. Facts Views Vis Obgyn, 5, 72-81.

MITCHELL, B. F. & TAGGART, M. J. 2009. Are animal models relevant to key aspects of human parturition? Am J Physiol Regul Integr Comp Physiol, 297, R525-45.

MIYAZAKI, C., GARCIA, R. M., OTA, E., SWA, T., OLADAPO, O. T. & MORI, R. 2016. Tocolysis for inhibiting preterm birth in extremely preterm birth, multiple gestations and in growth-restricted fetuses: a systematic review and meta- analysis. Reproductive health, 13, 4.

MOISE, K. J., JR., OU, C. N., KIRSHON, B., CANO, L. E., ROGNERUD, C. & CARPENTER, R. J., JR. 1990. Placental transfer of indomethacin in the human pregnancy. Am J Obstet Gynecol, 162, 549-54.

MOLLACE, V., MUSCOLI, C., MASINI, E., CUZZOCREA, S. & SALVEMINI, D. 2005. Modulation of prostaglandin biosynthesis by nitric oxide and nitric oxide donors. Pharmacological reviews, 57, 217-252.

MONCADA, S. 1991. Nitric oxide: physiology, pathophysiology and pharmacology. Pharmacol rev, 43, 109-142.

MONCADA, S., PALMER, R. M. & HIGGS, E. A. 1991. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev, 43, 109-42.

MOODLEY, Y. 2002. The role of inducible nitric oxide in health and disease. Current Diagnostic Pathology, 8, 297-304.

MOORE, F., DA SILVA, C., WILDE, J. I., SMARASON, A., WATSON, S. P. & LOPEZ BERNAL, A. 2000. Up-regulation of p21- and RhoA-activated protein kinases in human pregnant myometrium. Biochem Biophys Res Commun, 269, 322-6.

MOORE, F. & LOPEZ BERNAL, A. 2003. Chronic exposure to TXA2 increases expression of ROCKI in human myometrial cells. Prostaglandins Other Lipid Mediat, 71, 23-32.

MORAN, C., FRIEL, A., SMITH, T., CAIRNS, M. & MORRISON, J. 2002. Expression and modulation of Rho kinase in human pregnant myometrium. Molecular human reproduction, 8, 196-200.

MORITA, I. 2002. Distinct functions of COX-1 and COX-2. Prostaglandins Other Lipid Mediat, 68-69, 165-75.

321

MOZAFFARIAN, D. & WU, J. H. 2011. Omega-3 fatty acids and cardiovascular disease: effects on risk factors, molecular pathways, and clinical events. Journal of the American College of Cardiology, 58, 2047-2067.

MUGLIA, L. J. & KATZ, M. 2010. The enigma of spontaneous preterm birth. N Engl J Med, 362, 529-35.

MURRAY, C. J. & LOPEZ, A. D. 1997. Global mortality, disability, and the contribution of risk factors: Global Burden of Disease Study. Lancet, 349, 1436- 42.

MURRAY, M., PIZZORNA, J. & PIZZORNA, L. 2005 the encyclopedia of healing food ATRIABOOKS, New York, USA, 86.

MURTHA, A. P., FENG, L., YONISH, B., LEPPERT, P. C. & SCHOMBERG, D. W. 2007. Progesterone protects fetal chorion and maternal decidua cells from calcium-induced death. Am J Obstet Gynecol, 196, 257 e1-5.

NABAVIZADEH, S. H., MALEKZADEH, M., MOUSAVIZADEH, A., SHIRAZI, H. R., GHAFFARI, P., KARSHENAS, N., MALEKZADEH, T. & ZOLADL, M. 2012. Retrospective study of factors related to preterm labor in Yasuj, Iran. Int J Gen Med, 5, 1013-7.

NAGHASHPOUR, M. & DAHL, G. 2000. Relaxation of myometrium by calcitonin gene-related peptide is independent of nitric oxide synthase activity in mouse uterus. Biol Reprod, 63, 1421-7.

NAKAGAWA, O., FUJISAWA, K., ISHIZAKI, T., SAITO, Y., NAKAO, K. & NARUMIYA, S. 1996. ROCK-I and ROCK-II, two isoforms of Rho-associated coiled-coil forming protein serine/threonine kinase in mice. FEBS Lett, 392, 189-93.

NAKAHATA, N. 2008. Thromboxane A2: physiology/pathophysiology, cellular signal transduction and pharmacology. J Pharmacol Ther., 118, 18-35.

NAKAHATA, N., MATSUOKA, I., ONO, T. & NAKANISHI, H. 1989. Thromboxane A2 activates phospholipase C in astrocytoma cells via pertussis toxin-insensitive G-protein. Eur J Pharmacol., 162, 407-417.

NALLAMOTHU, R., WOOD, G. C., PATTILLO, C. B., SCOTT, R. C., KIANI, M. F., MOORE, B. M. & THOMA, L. A. 2006. A tumor vasculature targeted liposome delivery system for combretastatin A4: design, characterization, and in vitro evaluation. AAPS PharmSciTech, 7, E32.

NAMBA, T., SUGIMOTO, Y., HIRATA, M., HAYASHI, Y., HONDA, A., WATABE, A., NEGISHI, M., ICHIKAWA, A. & NARUMIYA, S. 1992. Mouse thromboxane A2 receptor: cDNA cloning, expression and northern blot analysis. Biochem Biophys Res Commun, 184, 1197-203.

NARAYANAN, M. & COHEN, H. L. 2019. Pelvic Ultrasound. In StatPearls [Internet]. StatPearls Publishing. Treasure Island (FL): StatPearls Publishing. Available from: https://www.ncbi.nlm.nih.gov/books/NBK470360/

322

NARUMIYA, S., SUGIMOTO, Y. & USHIKUBI, F. 1999. Prostanoid receptors: structures, properties, and functions. Physiol Rev, 79, 1193-226.

NETSU, S., KONNO, R., ODAGIRI, K., SOMA, M., FUJIWARA, H. & SUZUKI, M. 2008. Oral eicosapentaenoic acid supplementation as possible therapy for endometriosis. Fertil Steril, 90, 1496-502.

NEWELL-LITWA, K. A., BADOUAL, M., ASMUSSEN, H., PATEL, H., WHITMORE, L. & HORWITZ, A. R. 2015. ROCK1 and 2 differentially regulate actomyosin organization to drive cell and synaptic polarity. J Cell Biol., 210, 225-242.

NEWTON, E. R., SHIELDS, L., RIDGWAY, L. E., 3RD, BERKUS, M. D. & ELLIOTT, B. D. 1991. Combination antibiotics and indomethacin in idiopathic preterm labor: a randomized double-blind clinical trial. Am J Obstet Gynecol, 165, 1753-9.

NIIRO, N., NISHIMURA, J., SAKIHARA, C., NAKANO, H. & KANAIDE, H. 1997. Up-regulation of rho A and rho-kinase mRNAs in the rat myometrium during pregnancy. Biochem Biophys Res Commun, 230, 356-9.

NISHIGAKI, N., NEGISHI, M. & ICHIKAWA, A. 1996. Two Gs-coupled prostaglandin E receptor subtypes, EP2 and EP4, differ in desensitization and sensitivity to the metabolic inactivation of the agonist. Mol Pharmacol, 50, 1031-7.

NOBLOT, G., AUDRA, P., DARGENT, D., FAGUER, B. & MELLIER, G. 1991. The Use of Micronized Progesterone in the Treatment of Menace of Preterm Delivery. European Journal of Obstetrics Gynecology and Reproductive Biology, 40, 203-209.

NOBS, L., BUCHEGGER, F., GURNY, R. & ALLÉMANN, E. 2004. Current methods for attaching targeting ligands to liposomes and nanoparticles. J Pharm Sci, 93, 1980-92.

NORMAN, J. 1996. Nitric oxide and the myometrium. Pharmacol Ther, 70, 91-100.

NORMAN, J., WARD, L., MARTIN, W., CAMERON, A., MCGRATH, J., GREER, I. & CAMERON, I. 1997. Effects of cGMP and the nitric oxide donors glyceryl trinitrate and sodium nitroprusside on contractions in vitro of isolated myometrial tissue from pregnant women. Reproduction, 110, 249-254.

NORMAN, J. E., SHENNAN, A., BENNETT, P., THORNTON, S., ROBSON, S., MARLOW, N., NORRIE, J., PETROU, S., SEBIRE, N., LAVENDER, T. & WHYTE, S. 2012. Trial protocol OPPTIMUM-Does progesterone prophylaxis for the prevention of preterm labour improve outcome? BMC Pregnancy Childbirth, 12.

NORWITZ, E. R. & ROBINSON, J. N. 2001. A systematic approach to the management of preterm labor. Semin Perinatol, 25, 223-35.

323

NOTT, J. P., BONNEY, E. A., PICKERING, J. D. & SIMPSON, N. A. 2016. The structure and function of the cervix during pregnancy. Translational Research in Anatomy, 2, 1-7.

O'BRIEN, W. F., KNUPPEL, R. A. & COHEN, G. R. 1986. Plasma prostaglandin metabolite levels after use of prostaglandin E2 gel for cervical ripening. American journal of obstetrics and gynecology, 155, 1037-1040.

OH, J. H., YOU, S. K., HWANG, M. K., AHN, D. S., KWON, S. C., TAGGART, M. J. & LEE, Y. H. 2003. Inhibition of rho-associated kinase reduces MLC20 phosphorylation and contractility of intact myometrium and attenuates agonist- induced Ca2+ sensitization of force of permeabilized rat myometrium. J Vet Med Sci, 65, 43-50.

OKADA, Y., HARA, A., MA, H., XIAO, C. Y., TAKAHATA, O., KOHGO, Y., NARUMIYA, S. & USHIKUBI, F. 2000. Characterization of prostanoid receptors mediating contraction of the gastric fundus and ileum: studies using mice deficient in prostanoid receptors. Br J Pharmacol, 131, 745-55.

OKITA, R. T. & OKITA, J. R. 1996. Prostaglandin-metabolizing enzymes during pregnancy: characterization of NAD(+)-dependent prostaglandin dehydrogenase, carbonyl reductase, and cytochrome P450-dependent prostaglandin omega-hydroxylase. Crit Rev Biochem Mol Biol, 31, 101-26.

OROPEZA, M. V., PONCE-MONTER, H., VILLANUEVA-TELLO, T., PALMA- AGUIRRE, J. A. & CAMPOS, M. G. 2002. Anatomical differences in uterine sensitivity to prostaglandin F(2alpha) and serotonin in non-pregnant rats. Eur J Pharmacol, 446, 161-6.

OSMAN, F. H. & AMMAR, E. M. 1975. 5-hydroxytryptamine receptors in uterine smooth muscle. Jpn J Pharmacol, 25, 631-7.

OU, C.-W., CHEN, Z.-Q., QI, S. & LYE, S. J. 1998. Increased expression of the rat myometrial oxytocin receptor messenger ribonucleic acid during labor requires both mechanical and hormonal signals. Biol Reprod., 59, 1055-1061.

OU, C.-W., ORSINO, A. & LYE, S. J. 1997. Expression of connexin-43 and connexin- 26 in the rat myometrium during pregnancy and labor is differentially regulated by mechanical and hormonal signals. Endocrinology, 138, 5398-5407.

OYELOWO, T. 2007. Mosby's Guide to Women's Health: A Handbook for Health Professionals, Elsevier Health Sciences, Missouri

PADOL, A. R., SUKUMARAN, S. V., SADAM, A., KESAVAN, M., ARUNVIKRAM, K., VERMA, A. D., SRIVASTAVA, V., PANIGRAHI, M., SINGH, T. U., TELANG, A. G., MISHRA, S. K. & PARIDA, S. 2017. Hypercholesterolemia impairs oxytocin-induced uterine contractility in late pregnant mouse. Reproduction, 153, 565-576.

PALMER, R. M., ASHTON, D. S. & MONCADA, S. 1988. Vascular endothelial cells synthesize nitric oxide from L-arginine. Nature, 333, 664-6.

324

PAPATSONIS, D., FLENADY, V., COLE, S. & LILEY, H. 2005. Oxytocin receptor antagonists for inhibiting preterm labour. Cochrane Database Syst Rev, CD004452.

PARK, K., LI, Q., LYNES, M., YOKOMIZO, H., SHINJO, T., ST-LOUIS, R., FU, J., RASK-MADSEN, C., TSENG, Y.-H. & KING, G. L. 2018. Endothelial/Nitric Oxide Regulation of Brown Adipose Tissue Activating Lipokine, 12, 13- diHOME and Its Antiatherogenic Actions. Am Diabetes Assoc.

PARKINGTON, H. C. & COLEMAN, H. A. 1990. The role of membrane potential in the control of uterine motility. Uterine Function. (pp. 195-248). Springer, Boston, MA.

PATEL, R., MOFFATT, J. D., MOURMOURA, E., DEMAISON, L., SEED, P. T., POSTON, L. & TRIBE, R. M. 2017. Effect of reproductive ageing on pregnant mouse uterus and cervix. The Journal of physiology, 595, 2065-2084.

PAUL, J., MAITI, K., READ, M., HURE, A., SMITH, J., CHAN, E. C. & SMITH, R. 2011. Phasic phosphorylation of caldesmon and ERK 1/2 during contractions in human myometrium. PLoS One, 6, e21542.

PAUWELS, P. J. 2000. Diverse signalling by 5-hydroxytryptamine (5-HT) receptors. Biochem Pharmacol, 60, 1743-50.

PEARSON, T., WARREN, A. Y., BARRETT, D. A. & KHAN, R. N. 2009. Detection of EETs and HETE-generating cytochrome P-450 enzymes and the effects of their metabolites on myometrial and vascular function. Am J Physiol Endocrinol Metab, 297, E647-56.

PEREZ-ZOGHBI, J. F., BAI, Y. & SANDERSON, M. J. 2010. Nitric oxide induces airway smooth muscle cell relaxation by decreasing the frequency of agonist- induced Ca2+ oscillations. J Gen Physiol, 135, 247-59.

PERI, K. G., QUINIOU, C., HOU, X., ABRAN, D., VARMA, D. R., LUBELL, W. D. & CHEMTOB, S. 2002. THG113: A novel selective FP antagonist that delays preterm labor. Semin Perinatol, 26, 389-397.

PETERS, L. L., ROBLEDO, R. F., BULT, C. J., CHURCHILL, G. A., PAIGEN, B. J. & SVENSON, K. L. 2007. The mouse as a model for human biology: a resource guide for complex trait analysis. Nat Rev Genet, 8, 58-69.

PETERSEN, L. K., OXLUND, H., ULDBJERG, N. & FORMAN, A. 1991. In vitro analysis of muscular contractile ability and passive biomechanical properties of uterine cervical samples from nonpregnant women. Obstet Gynecol, 77, 772-6.

PETROCELLI, T. & LYE, S. J. 1993. Regulation of transcripts encoding the myometrial gap junction protein, connexin-43, by estrogen and progesterone. Endocrinology, 133, 284-290.

325

PETROU, S., EDDAMA, O. & MANGHAM, L. 2011. A structured review of the recent literature on the economic consequences of preterm birth. Archives of Disease in Childhood-Fetal and Neonatal Edition, 96, F225-F232.

PHANEUF, S., ASBOTH, G., CARRASCO, M. P., EUROPE-FINNER, G. N., SAJI, F., KIMURA, T., HARRIS, A. & LOPEZ BERNAL, A. 1997. The desensitization of oxytocin receptors in human myometrial cells is accompanied by down-regulation of oxytocin receptor messenger RNA. J Endocrinol, 154, 7- 18.

PHANEUF, S., ASBOTH, G., CARRASCO, M. P., LINARES, B. R., KIMURA, T., HARRIS, A. & BERNAL, A. L. 1998. Desensitization of oxytocin receptors in human myometrium. Hum Reprod Update, 4, 625-33.

PHANEUF, S., ASBOTH, G., MACKENZIE, I. Z., MELIN, P. & LOPEZ BERNAL, A. 1994. Effect of oxytocin antagonists on the activation of human myometrium in vitro: atosiban prevents oxytocin-induced desensitization. Am J Obstet Gynecol, 171, 1627-34.

PIERZYNSKI, P., LEMANCEWICZ, A., REINHEIMER, T., AKERLUND, M. & LAUDANSKI, T. 2004. Inhibitory effect of barusiban and atosiban on oxytocin- induced contractions of myometrium from preterm and term pregnant women. J Soc Gynecol Investig, 11, 384-7.

POYTON, C., MANCHADI, M.-L., CHEESMAN, M. & LAVIDIS, N. 2015. Effects of Lavender and Linalool on Neurotransmission and Contraction of Smooth Muscle. Pharmacognosy Communications, 5(3).

PURCELL, T. L., BUHIMSCHI, I. A., GIVEN, R., CHWALISZ, K. & GARFIELD, R. E. 1997. Inducible nitric oxide synthase is present in the rat placenta at the fetal- maternal interface and decreases prior to labour. Mol Hum Reprod, 3, 485-91.

QUINN, J.-A., MUNOZ, F. M., GONIK, B., FRAU, L., CUTLAND, C., MALLETT- MOORE, T., KISSOU, A., WITTKE, F., DAS, M., NUNES, T., PYE, S., WATSON, W., RAMOS, A.-M. A., CORDERO, J. F., HUANG, W.-T., KOCHHAR, S., BUTTERY, J. & BRIGHTON COLLABORATION PRETERM BIRTH WORKING GROUP. 2016. Preterm birth: Case definition & guidelines for data collection, analysis, and presentation of immunisation safety data. Vaccine, 34, 6047-6056.

RAISANEN, S., GISSLER, M., SAARI, J., KRAMER, M. & HEINONEN, S. 2013. Contribution of Risk Factors to Extremely, Very and Moderately Preterm Births - Register-Based Analysis of 1,390,742 Singleton Births. Plos One, 8.

RAPOPORT, R. M., DRAZNIN, M. B. & MURAD, F. 1983. Endothelium-dependent relaxation in rat aorta may be mediated through cyclic GMP-dependent protein phosphorylation. Nature, 306, 174-6.

RATAJCZAK, C. K., FAY, J. C. & MUGLIA, L. J. 2010. Preventing preterm birth: the past limitations and new potential of animal models. Dis Model Mech, 3, 407- 14.

326

RATAJCZAK, C. K. & MUGLIA, L. J. 2008. Insights Into Parturition Biology From Genetically Altered Mice. Pediatr Res, 64, 581-589.

RAYMOND, J. R., MUKHIN, Y. V., GELASCO, A., TURNER, J., COLLINSWORTH, G., GETTYS, T. W., GREWAL, J. S., GARNOVSKAYA, M. N. 2001. Multiplicity of mechanisms of serotonin receptor signal transduction. Pharmacol Ther., 92, 179-212.

RAZ, T., AVNI, R., ADDADI, Y., COHEN, Y., JAFFA, A. J., HEMMINGS, B., GARBOW, J. R. & NEEMAN, M. 2012. The hemodynamic basis for positional- and inter-fetal dependent effects in dual arterial supply of mouse pregnancies. PLoS One, 7, e52273.

REAGAN, P. B. & SALSBERRY, P. J. 2005. Race and ethnic differences in determinants of preterm birth in the USA: broadening the social context. Soc Sci Med, 60, 2217-28.

RED-HORSE, K. 2008. Lymphatic vessel dynamics in the uterine wall. Placenta, 29 Suppl A, S55-9.

REFUERZO, J. S., LEONARD, F., BULAYEVA, N., GORENSTEIN, D., CHIOSSI, G., ONTIVEROS, A., LONGO, M. & GODIN, B. 2016. Uterus-targeted liposomes for preterm labor management: studies in pregnant mice. Sci Rep, 6, 34710.

REINEBRANT, H. E., PILEGGI-CASTRO, C., ROMERO, C. L., DOS SANTOS, R. A., KUMAR, S., SOUZA, J. P. & FLENADY, V. 2015. Cyclo-oxygenase (COX) inhibitors for treating preterm labour. Cochrane Database Syst Rev, 6, CD001992.

RENTHAL, N. E., WILLIAMS, K. C. & MENDELSON, C. R. 2013. MicroRNAs- mediators of myometrial contractility during pregnancy and labour. Nat Rev Endocrinol, 9, 391-401.

REZLER, E. M., KHAN, D. R., TU, R., TIRRELL, M. & FIELDS, G. B. 2007. Peptide-mediated targeting of liposomes to tumor cells. Methods Mol Biol, 386, 269-98.

RIEMER, R. K., BUSCHER, C., BANSAL, R. K., BLACK, S. M., HE, Y. & NATUZZI, E. S. 1997. Increased expression of nitric oxide synthase in the myometrium of the pregnant rat uterus. Am J Physiol, 272, E1008-15.

RIENTO, K., GUASCH, R. M., GARG, R., JIN, B. & RIDLEY, A. J. 2003. RhoE binds to ROCK I and inhibits downstream signaling. Mol Cell Biol, 23, 4219-29.

RIKITAKE, Y., KIM, H. H., HUANG, Z., SETO, M., YANO, K., ASANO, T., MOSKOWITZ, M. A. & LIAO, J. K. 2005. Inhibition of Rho kinase (ROCK) leads to increased cerebral blood flow and stroke protection. Stroke, 36, 2251-7.

327

RILEY, M., WU, X., BAKER, P. N. & TAGGART, M. J. 2005. Gestational-dependent changes in the expression of signal transduction and contractile filament- associated proteins in mouse myometrium. J Soc Gynecol Investig, 12, e33-43.

ROBBINS, J. R. & BAKARDJIEV, A. I. 2012. Pathogens and the placental fortress. Curr Opin Microbiol, 15, 36-43.

ROBINSON, C., SCHUMANN, R., ZHANG, P. & YOUNG, R. C. 2003. Oxytocin- induced desensitization of the oxytocin receptor. Am J Obstet Gynecol, 188, 497-502.

ROMERO, R., DEY, S. K. & FISHER, S. J. 2014. Preterm labor: one syndrome, many causes. Science, 345, 760-5.

ROMERO, R., ESPINOZA, J., KUSANOVIC, J. P., GOTSCH, F., HASSAN, S., EREZ, O., CHAIWORAPONGSA, T. & MAZOR, M. 2006. The preterm parturition syndrome. BJOG, 113 Suppl 3, 17-42.

ROSSMANITH, W. G., HOFFMEISTER, U., WOLFAHRT, S., KLEINE, B., MCLEAN, M., JACOBS, R. A. & GROSSMAN, A. B. 1999. Expression and functional analysis of endothelial nitric oxide synthase (eNOS) in human placenta. Mol Hum Reprod, 5, 487-94.

ROSZKOWSKI, M. 2014. The effects of acute stress on Apold1 gene expression and blood-brain barrier permeability. MSc Thesis, University of Zurich, Switzerland.

ROTE, N. S. & HUETHER, S. E. 2018. Pathophysiology-E-book: the biologic basis for disease in adults and children. In: MCCANCE, K. L. & HUETHER, S. E. (eds.) Pathophysiology-E-book: the biologic basis for disease in adults and children. Elsevier Health Sciences.

ROUZER, C. A. & MARNETT, L. J. 2003. Mechanism of free radical oxygenation of polyunsaturated fatty acids by cyclooxygenases. Chemical reviews, 103, 2239- 2304.

RUDDOCK, N. K., SHI, S.-Q., JAIN, S., MOORE, G., HANKINS, G. D., ROMERO, R. & GARFIELD, R. E. 2008. Progesterone, but not 17-alpha- hydroxyprogesterone caproate, inhibits human myometrial contractions. Am J Obstet Gynecol, 199, 391. e1-391. e7.

SABAR, U. J. 2012. The effect of prostaglandins in myometrial tissue; a functional and lipidomic study. PhD Thesis, University of Bradford, UK.

SAKAMOTO, K., HORI, M., IZUMI, M., OKA, T., KOHAMA, K., OZAKI, H. & KARAKI, H. 2003. Inhibition of high K+-induced contraction by the ROCKs inhibitor Y-27632 in vascular smooth muscle: possible involvement of ROCKs in a signal transduction pathway. Journal of pharmacological sciences, 92, 56- 69.

328

SALEH GARGARI, S., HABIBOLAHI, M., ZONOBI, Z., KHANI, Z., SARFJOO, F. S., KAZEMI ROBATI, A., ETEMAD, R. & KARIMI, Z. 2012. Outcome of vaginal progesterone as a tocolytic agent: randomized clinical trial. ISRN obstetrics and gynecology, 2012: 607906.

SALIM, R., GARMI, G., NACHUM, Z., ZAFRAN, N., BARAM, S. & SHALEV, E. 2012. Nifedipine compared with atosiban for treating preterm labor: a randomized controlled trial. Obstet Gynecol, 120, 1323-31.

SALVEMINI, D., MISKO, T. P., MASFERRER, J. L., SEIBERT, K., CURRIE, M. G. & NEEDLEMAN, P. 1993. Nitric oxide activates cyclooxygenase enzymes. Proc Natl Acad Sci U S A, 90, 7240-4.

SANGHA, R. K., WALTON, J. C., ENSOR, C. M., TAI, H. H. & CHALLIS, J. R. G. 1994. Immunohistochemical Localization, Messenger-Ribonucleic-Acid Abundance, and Activity of 15-Hydroxyprostaglandin Dehydrogenase in Placenta and Fetal Membranes during Term and Preterm Labor. Journal of Clinical Endocrinology & Metabolism, 78, 982-989.

SANTORELLI, S. 2018. Development of a novel controlled release pelvic drug delivery system to manage endometriosis. PhD Thesis, University of Manchester, UK.

SATO, M., TANI, E., FUJIKAWA, H. & KAIBUCHI, K. 2000. Involvement of Rho- kinase-mediated phosphorylation of myosin light chain in enhancement of cerebral vasospasm. Circ Res, 87, 195-200.

SAUZEAU, V., LE JEUNE, H., CARIO-TOUMANIANTZ, C., SMOLENSKI, A., LOHMANN, S. M., BERTOGLIO, J., CHARDIN, P., PACAUD, P. & LOIRAND, G. 2000. Cyclic GMP-dependent protein kinase signaling pathway inhibits RhoA-induced Ca2+ sensitization of contraction in vascular smooth muscle. Journal of Biological Chemistry, 275, 21722-21729.

SCHIESSL, B., MYLONAS, I., HANTSCHMANN, P., KUHN, C., SCHULZE, S., KUNZE, S., FRIESE, K. & JESCHKE, U. 2005. Expression of endothelial NO synthase, inducible NO synthase, and estrogen receptors alpha and beta in placental tissue of normal, preeclamptic, and intrauterine growth-restricted pregnancies. J Histochem Cytochem, 53, 1441-9.

SCHMANDKE, A., SCHMANDKE, A. & STRITTMATTER, S. M. 2007. ROCK and Rho: biochemistry and neuronal functions of Rho-associated protein kinases. Neuroscientist, 13, 454-69.

SCHMIDT, H. H., LOHMANN, S. M. & WALTER, U. 1993. The nitric oxide and cGMP signal transduction system: regulation and mechanism of action. Biochim Biophys Acta, 1178, 153-75.

329

SCHRÖDER, R., XUE, L., KONYA, V., MARTINI, L., KAMPITSCH, N., WHISTLER, J. L., ULVEN, T., HEINEMANN, A., PETTIPHER, R. & KOSTENIS, E. 2012. PGH1, the precursor for the anti-inflammatory prostaglandins of the 1-series, is a potent activator of the pro-inflammatory receptor CRTH2/DP2. PLoS One, 7, e33329.

SEINEN, L. H., SIMONS, S. O., VAN DER DRIFT, M. A., VAN DILLEN, J., VANDENBUSSCHE, F. P. & LOTGERING, F. K. 2013. [Maternal pulmonary oedema due to the use of atosiban in cases of multiple gestation]. Ned Tijdschr Geneeskd, 157, A5316.

SENCHYNA, M. & CRANKSHAW, D. J. 1999. Operational correlates of prostanoid TP receptor expression in human non‐ pregnant myometrium are unaffected by excision site or menstrual cycle status of the donor. Br J Pharmacol., 128, 1524- 1528.

SENIOR, J., MARSHALL, K., SANGHA, R. & CLAYTON, J. K. 1993. In vitro characterization of prostanoid receptors on human myometrium at term pregnancy. Br J Pharmacol, 108, 501-6.

SHARMA, J. N., AL-OMRAN, A. & PARVATHY, S. S. 2007. Role of nitric oxide in inflammatory diseases. Inflammopharmacology, 15, 252-9.

SHIMOKAWA, H., SETO, M., KATSUMATA, N., AMANO, M., KOZAI, T., YAMAWAKI, T., KUWATA, K., KANDABASHI, T., EGASHIRA, K., IKEGAKI, I., ASANO, T., KAIBUCHI, K. & TAKESHITA, A. 1999. Rho- kinase-mediated pathway induces enhanced myosin light chain phosphorylations in a swine model of coronary artery spasm. Cardiovasc Res, 43, 1029-39.

SHINJO, A., OTSUKI, K., SAWADA, M., OTA, H., TOKUNAKA, M., OBA, T., MATSUOKA, R. & OKAI, T. 2012. Retrospective cohort study: a comparison of two different management strategies in patients with preterm premature rupture of membranes. Arch Gynecol Obstet, 286, 337-45.

SHIRAO, S., KASHIWAGI, S., SATO, M., MIWA, S., NAKAO, F., KUROKAWA, T., TODOROKI-IKEDA, N., MOGAMI, K., MIZUKAMI, Y., KURIYAMA, S., HAZE, K., SUZUKI, M. & KOBAYASHI, S. 2002. Sphingosylphosphorylcholine is a novel messenger for Rho-kinase-mediated Ca2+ sensitization in the bovine cerebral artery: unimportant role for protein kinase C. Circ Res, 91, 112-9.

SHMYGOL, A., GULLAM, J., BLANKS, A. & THORNTON, S. 2006. Multiple mechanisms involved in oxytocin-induced modulation of myometrial contractility. Acta Pharmacol Sin, 27, 827-32.

SHOJO, H. & KANEKO, Y. 2001. Oxytocin-induced phosphorylation of myosin light chain is mediated by extracellular calcium influx in pregnant rat myometrium. J Mol Recognit, 14, 401-5.

330

SICA, M., MARTINI, M., VIGLIETTI-PANZICA, C. & PANZICA, G. 2009. Estrous cycle influences the expression of neuronal nitric oxide synthase in the hypothalamus and limbic system of female mice. BMC Neurosci, 10, 78.

SIEVI, E., LAHTEENMAKI, T. A., ALANKO, J., VUORINEN, P. & VAPAATALO, H. 1997. Nitric oxide as a regulator of prostacyclin synthesis in cultured rat heart endothelial cells. Arzneimittelforschung, 47, 1093-8.

SIMMONS, D. G., NATALE, D. R., BEGAY, V., HUGHES, M., LEUTZ, A. & CROSS, J. C. 2008. Early patterning of the chorion leads to the trilaminar trophoblast cell structure in the placental labyrinth. Development, 135, 2083-91.

SIMÕES, S., MOREIRA, J. N., FONSECA, C., DÜZGÜNEŞ, N. & DE LIMA, M. C. 2004. On the formulation of pH-sensitive liposomes with long circulation times. Adv Drug Deliv Rev, 56, 947-65.

SKARNES, R. C. & HARPER, M. J. 1972. Relationship between Endotoxin-Induced Abortion and Synthesis of Prostaglandin F. Prostaglandins, 1, 191-203.

SLADEK, S. M., MAGNESS, R. R. & CONRAD, K. P. 1997. Nitric oxide and pregnancy. Am J Physiol, 272, R441-63.

SLATTERY, M. M., FRIEL, A. M., HEALY, D. G. & MORRISON, J. J. 2001. Uterine relaxant effects of cyclooxygenase-2 inhibitors in vitro. Obstet Gynecol, 98, 563-9.

SMAILL, F. & VAZQUEZ, J. C. 2007. Antibiotics for asymptomatic bacteriuria in pregnancy. Cochrane Database Syst Rev, CD000490.

SMITH, G. C., WU, W. X. & NATHANIELSZ, P. W. J. B. O. R. 2001. Effects of gestational age and labor on expression of prostanoid receptor genes in baboon uterus. 64, 1131-1137.

SMITH, R., MESIANO, S. & MCGRATH, S. 2002. Hormone trajectories leading to human birth. Regul Pept, 108, 159-164.

SOMLYO, A. P. & SOMLYO, A. V. 1998. From pharmacomechanical coupling to G- proteins and myosin phosphatase. Acta Physiol Scand, 164, 437-48.

SOMLYO, A. P. & SOMLYO, A. V. 2003. Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiol Rev, 83, 1325-58.

SOORANNA, S., LEE, Y., KIM, L., MOHAN, A., BENNETT, P. & JOHNSON, M. 2004. Mechanical stretch activates type 2 cyclooxygenase via activator protein‐ 1 transcription factor in human myometrial cells. Mol Hum Reprod., 10, 109- 113.

SOTI, V. K. 2019. Protective Actions of Oxytocin on Aortic Wistar and SHR VSMCs. PhD Thesis, Nova Southeastern University, USA.

331

SPENCER, T. E. 2014. Biological roles of uterine glands in pregnancy. Semin Reprod Med, 32, 346-57.

STABLES, D. & RANKIN, J. 2010. Physiology in childbearing: With anatomy and related biosciences, Edinburg, Elsevier Health Sciences.

STAMATELOU, F., DELIGEOROGLOU, E., FARMAKIDES, G. & CREATSAS, G. 2009. Abnormal progesterone and corticotropin releasing hormone levels are associated with preterm labour. Ann Acad Med Singapore, 38, 1011-6.

STEINWALL, M., BOSSMAR, T., BROUARD, R., LAUDANSKI, T., OLOFSSON, P., URBAN, R., WOLFF, K., LE-FUR, G. & AKERLUND, M. 2005. The effect of relcovaptan (SR 49059), an orally active vasopressin V1a receptor antagonist, on uterine contractions in preterm labor. Gynecol Endocrinol, 20, 104-9.

STRAUS, D. S., PASCUAL, G., LI, M., WELCH, J. S., RICOTE, M., HSIANG, C.-H., SENGCHANTHALANGSY, L. L., GHOSH, G. & GLASS, C. K. 2000. 15- Deoxy-Δ12, 14-prostaglandin J2 inhibits multiple steps in the NF-κB signaling pathway. Proceedings of the National Academy of Sciences, 97, 4844-4849.

STUBBS, T. M. 2000. Oxytocin for labor induction. Clin Obstet Gynecol, 43, 489-94.

SUBRAMANIAM, A., ABRAMOVICI, A., ANDREWS, W. W. & TITA, A. T. 2012. Antimicrobials for preterm birth prevention: an overview. Infect Dis Obstet Gynecol, 2012, 157159.

SUGIMOTO, Y., HASUMOTO, K., NAMBA, T., IRIE, A., KATSUYAMA, M., NEGISHI, M., KAKIZUKA, A., NARUMIYA, S. & ICHIKAWA, A. 1994. Cloning and expression of a cDNA for mouse prostaglandin F receptor. J Biol Chem, 269, 1356-60.

SUGIMOTO, Y., YAMASAKI, A., SEGI, E., TSUBOI, K., AZE, Y., NISHIMURA, T., OIDA, H., YOSHIDA, N., TANAKA, T., KATSUYAMA, M., HASUMOTO, K., MURATA, T., HIRATA, M., USHIKUBI, F., NEGISHI, M., ICHIKAWA, A. & NARUMIYA, S. 1997. Failure of parturition in mice lacking the prostaglandin F receptor. Science, 277, 681-3.

SUN, X., GUO, J. H., ZHANG, D., CHEN, J. J., LIN, W. Y., HUANG, Y., CHEN, H., HUANG, W. Q., LIU, Y. & TSANG, L. L. 2018. Activation of the epithelial sodium channel (ENaC) leads to cytokine profile shift to pro‐ inflammatory in labor. EMBO Mol Med., 10(10):e8868..

SWANSON, M. L., LEI, Z. M., SWANSON, P. H., RAO, C. V., NARUMIYA, S. & HIRATA, M. 1992. The expression of thromboxane A2 synthase and thromboxane A2 receptor gene in human uterus. Biol Reprod, 47, 105-17.

SZMELSKYJ, I., AQUILINA, L. & SZMELSKYJ, A. O. 2015. Acupuncture for IVF and Assisted Reproduction: An Integrated Approach to Treatment and Management, Edinburg, UK, Elsevier Ltd.

332

TAGGART, M. J., ARTHUR, P., ZIELNIK, B., MITCHELL, B. F. 2012. Molecular pathways regulating contractility in rat uterus through late gestation and parturition. Am J Obstet Gynecol., 207, 76. e15-76. e24.

TAHARA, M., KAWAGISHI, R., SAWADA, K., MORISHIGE, K., SAKATA, M., TASAKA, K., MURATA, Y. 2005. Tocolytic effect of a Rho-kinase inhibitor in a mouse model of lipopolysaccharide-induced preterm delivery. American journal of obstetrics and gynecology, 192, 903-908.

TAHARA, M., MORISHIGE, K.-I., SAWADA, K., IKEBUCHI, Y., KAWAGISHI, R., TASAKA, K. & MURATA, Y. 2002. RhoA/Rho-kinase cascade is involved in oxytocin-induced rat uterine contraction. J Endocrinology, 143, 920-929.

TAKEYA, K., LOUTZENHISER, K., SHIRAISHI, M., LOUTZENHISER, R. & WALSH, M. P. 2008. A highly sensitive technique to measure myosin regulatory light chain phosphorylation: the first quantification in renal arterioles. Am J Physiol Renal Physiol, 294, F1487-92.

TAKEYA, K., WANG, X., SUTHERLAND, C., KATHOL, I., LOUTZENHISER, K., LOUTZENHISER, R. D. & WALSH, M. P. 2014. Involvement of myosin regulatory light chain diphosphorylation in sustained vasoconstriction under pathophysiological conditions. J Smooth Muscle Res, 50, 18-28.

TALLET, D., DEL SOLDATO, P., OUDART, N. & BURGAUD, J. L. 2002. NO- steroids: potent anti-inflammatory drugs with bronchodilating activity in vitro. Biochem Biophys Res Commun, 290, 125-30.

TAN, H. Q., YI, L. J., ROTE, N. S., HURD, W. W. & MESIANO, S. 2012. Progesterone Receptor-A and -B Have Opposite Effects on Proinflammatory Gene Expression in Human Myometrial Cells: Implications for Progesterone Actions in Human Pregnancy and Parturition. Journal of Clinical Endocrinology & Metabolism, 97, E719-E730.

TAN, Y., LI, F., PIAO, Y., SUN, X. & WANG, Y. 2003. Global gene profiling analysis of mouse uterus during the oestrous cycle. REPRODUCTION-CAMBRIDGE-, 126, 171-182.

TARA, P. N. & THORNTON, S. 2004. Current medical therapy in the prevention and treatment of preterm labour. Semin Fetal Neonatal Med, 9, 481-9.

TELFER, J., LYALL, F., NORMAN, J. & CAMERON, I. 1995. Identification of nitric oxide synthase in human uterus. Human Reproduction, 10, 19-23.

THINGHOLM, T. E., JENSEN, O. N. & LARSEN, M. R. 2009. Analytical strategies for phosphoproteomics. Proteomics, 9, 1451-68.

THORNTON, S., GOODWIN, T. M., GREISEN, G., HEDEGAARD, M. & ARCE, J. C. 2009. The effect of barusiban, a selective oxytocin antagonist, in threatened preterm labor at late gestational age: a randomized, double-blind, placebo- controlled trial. Am J Obstet Gynecol, 200, 627 e1-10.

333

THUMKEO, D., KEEL, J., ISHIZAKI, T., HIROSE, M., NONOMURA, K., OSHIMA, H., OSHIMA, M., TAKETO, M. M. & NARUMIYA, S. 2003. Targeted disruption of the mouse rho-associated kinase 2 gene results in intrauterine growth retardation and fetal death. Mol Cell Biol, 23, 5043-55.

TIBONI, G. M. & GIAMPIETRO, F. 2000. Inhibition of nitric oxide synthesis causes preterm delivery in the mouse. Hum Reprod, 15, 1838-42.

TIMMONS, B., AKINS, M. & MAHENDROO, M. 2010. Cervical remodeling during pregnancy and parturition. Trends Endocrinol Metab, 21, 353-361.

TITA, A. T. N. & ANDREWS, W. W. 2010. Diagnosis and Management of Clinical Chorioamnionitis. Clin Perinatol, 37, 339-354.

TIWARI, G., TIWARI, R., SRIWASTAWA, B., BHATI, L., PANDEY, S., PANDEY, P. & BANNERJEE, S. K. 2012. Drug delivery systems: An updated review. Int J Pharm Investig, 2, 2-11.

TSAI, M. H. & JIANG, M. J. 2006. Rho-kinase-mediated regulation of receptor- agonist-stimulated smooth muscle contraction. Pflugers Arch, 453, 223-32.

TSUBOI, K., SUGIMOTO, Y. & ICHIKAWA, A. 2002. Prostanoid receptor subtypes. Prostaglandins Other Lipid Mediat, 68-69, 535-56.

VAN ASSCHE, F. A., SPITZ, B., HANSSENS, M., R., P. & BOSTEELS, J. 1992. Prostacyclin and thromboxane in pregnancy Current Obstetrics and gynaecology, 2, 27-30

VAN DEN BROEK, N. R., WHITE, S. A., GOODALL, M., NTONYA, C., KAYIRA, E., KAFULAFULA, G. & NEILSON, J. P. 2009. The APPLe study: a randomized, community-based, placebo-controlled trial of azithromycin for the prevention of preterm birth, with meta-analysis. PLoS Med, 6, e1000191.

VAN KAMP, I. L., KLUMPER, F. J. C. M., OEPKES, D., MEERMAN, R. H., SCHERJON, S. A., VANDENBUSSCHE, F. P. H. A. & KANHAI, H. H. H. 2005. Complications of intrauterine intravascular transfusion for fetal anemia due to maternal red-cell alloimmunization. Am J Obstet Gynecol, 192, 171-177.

VAN VLIET, E. O., BOORMANS, E. M., DE LANGE, T. S., MOL, B. W. & OUDIJK, M. A. 2014. Preterm labor: current pharmacotherapy options for tocolysis. Expert Opin Pharmacother, 15, 787-97.

VASSALLE, C., DOMENICI, C., LUBRANO, V. & L'ABBATE, A. 2003. Interaction between nitric oxide and cyclooxygenase pathways in endothelial cells. J Vasc Res, 40, 491-9.

VASUDEVAN, D. M., SREEKUMARI, S. & VAIDYANATHAN, K. 2013. Textbook of biochemistry for medical students, JP Medical Ltd, New Delhi.

334

VERHOFF, A. 1985. MYOMETRIAL CONTRACTILITY AND GAP JUNCTIONS AN EXPERIMENTAL STUDY IN CHRONICALLY INSTRUMENTED EWES Netherlands Organization for the Advancement of Pure Research (ZWO), the Medical Research Council of Canada, and Sarva-Syntex Nederland.

VERMEULEN, G. M. & BRUINSE, H. W. 1999. Prophylactic administration of clindamycin 2% vaginal cream to reduce the incidence of spontaneous preterm birth in women with an increased recurrence risk: a randomised placebo- controlled double-blind trial. Br J Obstet Gynaecol, 106, 652-7.

VERSTRAETE, M. 1983. Introduction: thromboxane in biological systems and the possible impact of its inhibition. Br J Clin Pharmacol, 15 Suppl 1, 7s-11s.

VINK, J. & MOURAD, M. 2017. The pathophysiology of human premature cervical remodeling resulting in spontaneous preterm birth: Where are we now? Semin Perinatol, 41, 427-437.

VOM SAAL, F. S. & DHAR, M. G. 1992. Blood flow in the uterine loop artery and loop vein is bidirectional in the mouse: implications for transport of steroids between fetuses. Physiol Behav, 52, 163-71.

VYSNIAUSKIENE, I., ALLEMANN, R., FLAMMER, J. & HAEFLIGER, I. O. 2006. Vasoactive responses of U46619, PGF2alpha, latanoprost, and travoprost in isolated porcine ciliary arteries. Invest Ophthalmol Vis Sci, 47, 295-8.

WAGNER, G. 1974. Contractility of myometrial transplants. Basic Life Sci, 4, 203-18.

WALMER, D. K., WRONA, M. A., HUGHES, C. L. & NELSON, K. G. 1992. Lactoferrin expression in the mouse reproductive tract during the natural estrous cycle: correlation with circulating estradiol and progesterone. Endocrinology, 131, 1458-1466.

WANG, C., WANG, X., ZHONG, T., ZHAO, Y., ZHANG, W. Q., REN, W., HUANG, D., ZHANG, S., GUO, Y., YAO, X., TANG, Y. Q., ZHANG, X. & ZHANG, Q. 2015. The antitumor activity of tumor-homing peptide-modified thermosensitive liposomes containing doxorubicin on MCF-7/ADR: in vitro and in vivo. Int J Nanomedicine, 10, 2229-48.

WANG, D., SHI, L., XIN, W., XU, J., XU, J., LI, Q., XU, Z., WANG, J., WANG, G., YAO, W., HE, B., YANG, Y. & HU, M. 2017. Activation of PPARgamma inhibits pro-inflammatory cytokines production by upregulation of miR-124 in vitro and in vivo. Biochem Biophys Res Commun, 486, 726-731.

WANG, H. & HIRSCH, E. 2003. Bacterially-induced preterm labor and regulation of prostaglandin-metabolizing enzyme expression in mice: The role of toll-like receptor 4. Biol Reprod, 69, 1957-1963.

WARD, Y., YAP, S. F., RAVICHANDRAN, V., MATSUMURA, F., ITO, M., SPINELLI, B. & KELLY, K. 2002. The GTP binding proteins Gem and Rad are negative regulators of the Rho-Rho kinase pathway. J Cell Biol, 157, 291-302.

335

WAREING, M., O'HARA, M., SEGHIER, F., BAKER, P. N. & TAGGART, M. J. 2005. The involvement of Rho-associated kinases in agonist-dependent contractions of human maternal and placental arteries at term gestation. Am J Obstet Gynecol, 193, 815-24.

WATTS, S. W., MORRISON, S. F., DAVIS, R. P. & BARMAN, S. M. 2012. Serotonin and blood pressure regulation. Pharmacol Rev, 64, 359-88.

WAUGH, A. & GRANT, A. 2018. Ross and Wilson anatomy and physiology in health and illness, Edinburg, UK, Elisevier.

WEAVER, E. H., GIBBONS, L., BELIZAN, J. M. & ALTHABE, F. 2015. The increasing trend in preterm birth in public hospitals in northern Argentina. Int J Gynaecol Obstet., 130(2), 137-141.

WELSH, T. N., HIRST, J. J., PALLISER, H. & ZAKAR, T. 2014. Progesterone receptor expression declines in the guinea pig uterus during functional progesterone withdrawal and in response to prostaglandins. PLoS One, 9, e105253.

WEX, J., ABOU-SETTA, A. M., CLERICI, G. & DI RENZO, G. C. 2011. Atosiban versus betamimetics in the treatment of preterm labour in Italy: clinical and economic importance of side-effects. Eur J Obstet Gynecol Reprod Biol, 157, 128-35.

WIESER, F., WAITE, L., DEPOIX, C. & TAYLOR, R. N. 2008. PPAR Action in Human Placental Development and Pregnancy and Its Complications. PPAR Res, 2008, 527048.

WILD, D. 2005. The Immunoassay Handbook, Third Edition, Amesterdam, , Elsevier Ltd.

WONG, S. L., LEUNG, F. P., LAU, C. W., AU, C. L., YUNG, L. M., YAO, X., CHEN, Z.-Y., VANHOUTTE, P. M., GOLLASCH, M. & HUANG, Y. 2009. Cyclooxygenase-2–derived prostaglandin F2α mediates endothelium-dependent contractions in the aortae of hamsters with increased impact during aging. Circ Res., 104, 228-235.

WOODCOCK, N. A., TAYLOR, C. W. & THORNTON, S. 2004. Effect of an oxytocin receptor antagonist and rho kinase inhibitor on the [Ca++]i sensitivity of human myometrium. Am J Obstet Gynecol, 190, 222-8.

WOODCOCK, N. A., TAYLOR, C. W., THORNTON, S. 2006. Prostaglandin F2α increases the sensitivity of the contractile proteins to Ca2+ in human myometrium. American journal of obstetrics and gynecology, 195, 1404-1406.

WOODS, L., PEREZ-GARCIA, V. & HEMBERGER, M. 2018. Regulation of Placental Development and Its Impact on Fetal Growth-New Insights From Mouse Models. Front Endocrinol (Lausanne), 9, 570.

WORD, R. A. 1995. Myosin phosphorylation and the control of myometrial contraction/relaxation. Semin Perinatol, 19, 3-14.

336

WORD, R. A., TANG, D. C. & KAMM, K. E. 1994. Activation properties of myosin light chain kinase during contraction/relaxation cycles of tonic and phasic smooth muscles. J Biol Chem, 269, 21596-602.

WORLDWIDE ATOSIBAN VERSUS BETA-AGONISTS STUDY, G. 2001. Effectiveness and safety of the oxytocin antagonist atosiban versus beta- adrenergic agonists in the treatment of preterm labour. The Worldwide Atosiban versus Beta-agonists Study Group. BJOG, 108, 133-42.

WRAY, S. 1993. Uterine contraction and physiological mechanisms of modulation. Am J Physiol, 264, C1-18.

WU, S.-P. & DEMAYO, F. J. 2017. Progesterone receptor signaling in uterine myometrial physiology and preterm birth. Current topics in developmental biology. Elsevier.

XIU-KUN, W., FAN, L., YU-SHUANG, C., HONG-LEI, Z., DONG-MING, X., YU- GANG, W., XUE-FU, Y., HUI-YING, L. & LI-JUN, D. 2011. Brazilein, a selective 5-HT receptors antagonist in mouse uterus. Chinese journal of natural medicines, 9, 338-344.

YALLAMPALLI, C., GARFIELD, R. E. & BYAM-SMITH, M. 1993. Nitric oxide inhibits uterine contractility during pregnancy but not during delivery. Endocrinology, 133, 1899-902.

YALLAMPALLI, C., IZUMI, H., BYAM-SMITH, M. & GARFIELD, R. E. 1994. An L-arginine-nitric oxide-cyclic guanosine monophosphate system exists in the uterus and inhibits contractility during pregnancy. American journal of obstetrics and gynecology, 170, 175-185.

YAMASHITA, A., KAWANA, K., TOMIO, K., TAGUCHI, A., ISOBE, Y., IWAMOTO, R., MASUDA, K., FURUYA, H., NAGAMATSU, T., NAGASAKA, K., ARIMOTO, T., ODA, K., WADA-HIRAIKE, O., YAMASHITA, T., TAKETANI, Y., KANG, J. X., KOZUMA, S., ARAI, H., ARITA, M., OSUGA, Y. & FUJII, T. 2013. Increased tissue levels of omega-3 polyunsaturated fatty acids prevents pathological preterm birth. Sci Rep, 3, 3113.

YAMASHITA, K., YOSHIOKA, Y., HIGASHISAKA, K., MIMURA, K., MORISHITA, Y., NOZAKI, M., YOSHIDA, T., OGURA, T., NABESHI, H., NAGANO, K., ABE, Y., KAMADA, H., MONOBE, Y., IMAZAWA, T., AOSHIMA, H., SHISHIDO, K., KAWAI, Y., MAYUMI, T., TSUNODA, S., ITOH, N., YOSHIKAWA, T., YANAGIHARA, I., SAITO, S. & TSUTSUMI, Y. 2011. Silica and titanium dioxide nanoparticles cause pregnancy complications in mice. Nat Nanotechnol, 6, 321-8.

YANG, Y., LI, H., WARD, R., GAO, L., WEI, J. F. & XU, T. R. 2014. Novel oxytocin receptor agonists and antagonists: a patent review (2002 - 2013). Expert Opin Ther Pat, 24, 29-46.

337

YAZAWA, T., KAWABE, S., KANNO, M., MIZUTANI, T., IMAMICHI, Y., JU, Y. F., MATSUMURA, T., YAMAZAKI, Y., USAMI, Y., KURIBAYASHI, M., SHIMADA, M., KITANO, T., UMEZAWA, A. & MIYAMOTO, K. 2013. Androgen/androgen receptor pathway regulates expression of the genes for cyclooxygenase-2 and amphiregulin in periovulatory granulosa cells. Mol Cellular Endocrinol, 369, 42-51.

YIN, Z., HE, W., LI, Y., LI, D., LI, H., YANG, Y., WEI, Z., SHEN, B., WANG, X., CAO, Y. & KHALIL, R. A. 2018. Adaptive reduction of human myometrium contractile activity in response to prolonged uterine stretch during term and twin pregnancy. Role of TREK-1 channel. Biochem Pharmacol, 152, 252-263.

ZAKAR, T. & MESIANO, S. 2011. How does progesterone relax the uterus in pregnancy. N Engl J Med, 364, 972-973.

ZENG, Z., VELARDE, M. C., SIMMEN, F. A. & SIMMEN, R. C. 2008. Delayed parturition and altered myometrial progesterone receptor isoform A expression in mice null for Kruppel-like factor 9. Biol Reprod, 78, 1029-37.

ZHANG, G. & STACKMAN JR, R. 2015. The role of serotonin 5-HT2A receptors in memory and cognition. Front Pharmacol 6: 225.

ZHANG, S., REGNAULT, T. R., BARKER, P. L., BOTTING, K. J., MCMILLEN, I. C., MCMILLAN, C. M., ROBERTS, C. T. & MORRISON, J. L. 2015. Placental adaptations in growth restriction. Nutrients, 7, 360-89.

ZHENG, J., ZHAI, K., CHEN, Y., ZHANG, X., MIAO, L., WEI, B. & JI, G. 2016. Nitric oxide mediates stretch-induced Ca2+ oscillation in smooth muscle. J Cell Sci, 129, 2430-7.

ZHU, D., GONG, X., MIAO, L., FANG, J. & ZHANG, J. 2017. Efficient Induction of Syncytiotrophoblast Layer II Cells from Trophoblast Stem Cells by Canonical Wnt Signaling Activation. Stem Cell Reports, 9, 2034-2049.

ZIMMER, B., ANGIONI, C., OSTHUES, T., TOEWE, A., THOMAS, D., PIERRE, S. C., GEISSLINGER, G., SCHOLICH, K. & SISIGNANO, M. 2018. The oxidized linoleic acid metabolite 12, 13-DiHOME mediates thermal hyperalgesia during inflammatory pain. Biochimica et Biophysica Acta (BBA)- Molecular and Cell Biology of Lipids, 1863, 669-678.

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Appendices

Appendix 1. Optomization of protein and abtibody concentrations for western blotting

339

Appendix 2. Chromatograms illustrating the retention time and transitions of prostanoids detected in this analysis for the standards.

340

341

342

343

Peaks highlighted in blue represent the response given for each compound at a defined retention time and transition specified to each lipid. Other peaks represent co-eluting compounds from the UPLC that appear within the same transition of the MS/MS, these are considered ‘noise’ within each UPLC run.

344

Appendix 3. Chromatograms illustrating the retention time and transitions of hydroxy fatty acids detected in this analysis for the standards.

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346

347

348

349

350

Peaks highlighted in green represent the response given for each compound at a defined retention time and transition specified to each lipid. Other peaks represent co-eluting compounds from the UPLC that appear within the same transition of the MS/MS, these are considered ‘noise’ within each UPLC run.

351

Appendix 4. Fatty acid derived lipid mediators quantified in myometrium from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI-MS/MS.

13,14 dihydro 15-keto PGE1 13,14 dihydro PGE1 ***, ### 25 300 ** ## C57 WT C57 WT 20 eNOS KO eNOS KO

n 200

e e 15 t

g 10

m /

/ 100

g g

p p 5

0 0 l 5 l 5 o 7 s s ro 7 s s tr 1 e e t 1 e e n E m n E m m o S o m o S o o c s so c e s s S ee o o S re o o B r ip p B F lip ip P F l li P l S 5 S 5 B 7 B 17 P 1 P E E S S Treatment Treatment

15-deoxy D12,14 PGJ2 5,15 DiHETE 50 500 C57 WT C57 WT

40 eNOS KO 400 eNOS KO

n i

i e e 300

30 t

g 20 200

m /

g p p 10 100

0 0

l l o 75 s s o 5 s tr 1 e e tr 17 e s n E m n E e o S o m o S om m c s so c s o ee o o e o s S r p p S re p o B F li li B F li lip P S P 75 S 5 B 1 B 17 P E P E S S Treatment Treatment

PDX TXB3 1000 200 C57 WT C57 WT 800 eNOS KO eNOS KO

150

e t

600 t

o r

r p

p 100

400 g

g p p 50 200

0 0 l o 75 s s l 5 tr 1 e e ro 7 es s n E m m t 1 e o o o n E m m c S s s o S o o e o o c e s s S re p p S e o o B F li li B r lip ip P S 5 P F l B 7 S 5 P 1 B 17 E P E S S Treatment Treatment

352

Appendix 5. Fatty acid derived lipid mediators quantified in plcenta from pregnant C57 WT and eNOS KO mice at term (E19) after different treatments using LC/ESI- MS/MS.

9 HETE

1000 #

n 800

i C57 WT

e t

o eNOS KO

r 600

g 400

/ g

p 200

0 l 5 s s ro 7 e e t 1 m m n E o o o S s s c e o o S e ip ip B r l l P F 5 S 7 B 1 P E S Treatment

13,14 dihydro 15-keto PGE1 13,14 dihydro PGE1

800 300

C57 WT n

i eNOS KO

e n

i 600 C57 WT

e o

t 200

p r

eNOS KO

o f

400

g g

m 100

/ g

g 200

p p

0 0 l 5 s s ro 7 e e l 5 s s t 1 ro 7 e e n m m t 1 o E o o n E m m c S s s o S o o e o o c s s S e p ip e o o B r li l S re ip ip F B F l l P S 5 P 5 B 7 S 7 1 B 1 P E P E S S Treatment Treatment

TXB3 200 C57 WT

n eNOS KO

i 150

f 100

m /

g 50 p

0 l 5 s s ro 7 e e t 1 m m n E o o o S s s c e o o S e ip ip B r l l P F 5 S 7 B 1 P E S Treatment

353

Appendix 6. Oxygenated lipid mediator pathways (a & b).

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