Physiological roles of the peptide
Prokineticin 1
Thesis for the degree of Doctor of Philosophy
Dr Ali Abbara MBBS BSc MRCP
2014
Department of Investigative Medicine Imperial College
Abstract
Prokineticin-1 (PK-1) and prokineticin-2 (PK-2) are two closely related cysteine-rich peptides which both bind to the prokineticin-1 and prokineticin-2 receptors (PKR-1 and
PKR-2). The prokineticins were originally named due to their ability to stimulate gastrointestinal motility. Since that time, these peptides have been found to play a role in other biological processes including reproduction and nociception. Prokineticin-2 is known to be expressed in regions of the central nervous system involved in food intake.
Consistent with this, work from our own department has shown that administration of prokineticin-2 to rodents reduces food intake both in lean and obese rodents.
Prokineticin-2 is predominantly expressed within the central nervous system, whereas prokineticin-1 is heavily expressed in the gut. My pilot data showed that prokineticin 1 reduces food intake in rodents. I therefore hypothesised that prokineticin 1 may be a novel gut hormone. In this thesis, I investigate the role of prokineticin-1 on appetite in rodents and also compare the effects of prokineticin-1 with prokineticin-2. I also measured the changes in plasma levels of prokineticin in humans during feeding to establish the potential physiological relevance of prokineticin-1 as an anorectic gut hormone.
Prokineticin-1 is also a potent angiogenic mitogen on endocrine vascular epithelium and hence it is also known as ‘Endocrine Gland-Vascular Endothelial Factor’. Placental angiogenesis plays an important role in placental function and risk of pregnancy complications such as miscarriage. I therefore hypothesised that prokineticin-1 is a biomarker of pregnancy complications in humans. In this thesis, I have measured circulating levels of prokineticin-1 found in pregnancy and correlated these with the occurrence of pregnancy complications. I have also compared the utility of prokineticin
1 with other potential novel markers of pregnancy complications. Together this body of work investigates the potential physiological roles of prokineticin-1.
2 Acknowledgements
I would like to gratefully acknowledge Professor Waljit Dhillo and Dr Channa Jayasena for supervising me and supporting me throughout my PhD. I would also like to thank all members of our group for all their support and assistance with this work. I am also grateful to the Wellcome Trust for funding my Clinical Training Fellowship.
3 Declaration of contributors
The majority of the work carried out in this thesis was performed by the author.
All collaborations and assistance are described below:
Chapter 2: Assistance with animal studies was provided by Dr Gardiner, Professor
Dhillo and members of the hypothalamic group.
Chapter 3: I would like to thank my colleagues Dr Izzi-Engbeaya, Ms Ganiyu-Dada for their help in collecting the pregnancy samples from the maternity unit. I would also like to thank my colleagues Dr Jayasena, Dr Comninos, Dr Sarang, Ms Malik, Dr
Narayanaswamy, Ms Ng, Dr Buckley, for their help in carrying out the measurement of
PK-1 and other pregnancy markers. In-house radioimmunoassay for the measurement of plasma kisspeptin used in this programme of research were established and maintained by Professor Mohammad Ghatei.
The copyright of this thesis rests with the author and is made available under a Creative
Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it.
For any reuse or redistribution, researchers must make clear to others the licence terms of this work.
Declaration signature Ali Abbara .
4 Table of Contents:
Abstract ...... 2
Acknowledgements ...... 3
Declaration of contributors ...... 4
Abbreviations ...... 14
Chapter 1 ...... 17
The obesity epidemic ...... 18
The Hypothalamus ...... 20
The Arcuate Nucleus (ARC) ...... 23
The Paraventricular nucleus (PVN) ...... 25
The Lateral Hypothalamus (LH) ...... 26
The Ventromedial Nucleus (VMN) ...... 27
The Dorsomedial Nucleus (DMN) ...... 29
Circulating Factors which act within the hypothalamus to regulate food intake and body
weight ...... 30
Prokineticins- a potential novel regulator of appetite ...... 33
Prokineticins (PKs): novel hypothalamic regulators of appetite and placental
angiogenesis ...... 41
Chapter 2 ...... 43
Introduction ...... 44
Hypothesis ...... 47
Aims ...... 47
Material and methods ...... 48
5 1. Effect of ICV administration of 15, 50, 150 pmol of PK-1 during Dark Phase in male
Wistar rats ...... 48
Materials ...... 48
Rodent models ...... 48
2. Effect of ICV administration of 15, 150 and 1500 pmol PK-1 ...... 50
3. Comparison of peripheral administration of IP PK-1 and PK-2 at doses of 20, 60 and
540 nmol/kg between 0-24hrs in male C57BL/6 mice...... 51
Materials ...... 51
4. Effect of IP PK-1 7 nmol/kg for up to 1 hour in mice (n=6 per group)...... 52
5. Determination of the effect of fasting on expression of PK-1 in the stomach of Fed and
Fasted mice ...... 53
Total RNA extraction ...... 54
Reverse Transcription (RT) ...... 56
6. Human plasma PK1 levels fed fasted ...... 58
Methods: Plasma PK-1 measurement by ELISA kit (ABNOVA) ...... 61
Reconstitution of the human PK-1 standard: ...... 61
Preparation of 0.01 M PBS wash buffer: ...... 62
1. Results: ...... 63
2. Results: ...... 65
3. Results: ...... 67
4. Results: ...... 71
5. Results: ...... 74
6. Results: ...... 76
6 Discussion: ...... 80
Chapter 3 ...... 83
Introduction: ...... 84
Recurrent Pregnancy Loss (RPL) ...... 86
The placenta ...... 88
The Placenta as an endocrine organ: ...... 89
Prokineticins during Pregnancy: ...... 91
Other novel markers of pregnancy complications: Kisspeptin ...... 97
Kisspeptin as an anti-metastatic marker ...... 100
Kisspeptin in the placenta ...... 101
Kisspeptin expression in Recurrent Pregnancy Loss ...... 102
Circulating levels of kisspeptin in pregnancy ...... 103
Circulating kisspeptin levels and Threatened Miscarriage ...... 105
Other angiogenic markers of pregnancy complications: ...... 106
Hypothesis ...... 108
Aims ...... 108
Material and methods ...... 109
Methods ...... 111
Dating of pregnancy ...... 112
Measurement of circulating markers of pregnancy complications levels ...... 113
Collection and processing of blood samples ...... 113
Measurement of PK-1 immuno-reactivity ...... 114
7 Measurement of total Kisspeptin immuno-reactivity ...... 114
Measurement of Total hCG immunoreactivity ...... 115
Measurment of PLGF by ELISA ...... 115
Measurment of Endoglin by ELISA ...... 116
Measurement of FLT-1 by ELISA ...... 117
Data Analysis and Statistics ...... 118
Patient details and outcomes: ...... 119
Baseline characteristics of pregnant subjects attending their antenatal booking visit . 120
Results: ...... 123
Levels of plasma Prokineticin-1 (PK-1) in non-pregnant women ...... 123
Correlation of Prokineticin-1 (PK-1) with serum BHCG levels ...... 125
Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG, Serum
Endoglin, Serum FLT-1 and Serum PLGF with gestation: ...... 127
Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG, Serum
Endoglin, Serum FLT-1 and Serum PLGF with multiple pregnancy and miscarriage: 131
Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG, Serum
Endoglin, Serum FLT-1 and Serum PLGF with singleton miscarriage: ...... 135
Relationship of plasma kisspeptin with time to diagnosis of miscarriage ...... 139
Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG, Serum
Endoglin, Serum FLT-1 and Serum PLGF with BMI: ...... 141
Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG, Serum
Endoglin, Serum FLT-1 and Serum PLGF with smoking: ...... 143
8 Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG, Serum
Endoglin, Serum FLT-1 and Serum PLGF with recurrent miscarriage in pregnant women
whose current singleton pregnancy did not result in miscarriage:...... 145
Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG, Serum
Endoglin, Serum FLT-1 and Serum PLGF with foetal sex: ...... 148
Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG, Serum
Endoglin, Serum FLT-1 and Serum PLGF with Birth weight: ...... 150
Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG, Serum
Endoglin, Serum FLT-1 and Serum PLGF with head circumference: ...... 153
Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG, Serum
Endoglin, Serum FLT-1 and Serum PLGF with prematurity: ...... 156
Discussion ...... 158
Chapter 4 ...... 160
General Discussion...... 160
Reference List ...... 166
Appendices ...... 202
Appendix 1: Solutions ...... 203
Appendix 2: Amino Acid Codes ...... 204
Appendix 3: List of Suppliers ...... 205
9 List of Figures
Figure 1: Diagrammatic representation of a sagittal section through a rodent brain showing the hypothalamic nuclei involved in the regulation of food intake...... 21
Figure 2: The amino acid sequence of Prokineticin-2 (PK-2)...... 35
Figure 3: Amino Acid Sequence of Prokineticin-1 and Prokineticin-2 ...... 36
Figure 4: Comparison of ICV PK-1 and PK-2 at doses of 15, 50, 150 pmol between 0-
24hrs during dark phase in adult male Wistar rats (n=6-8/group)...... 64
Figure 5: Comparison of ICV PK-1 and PK-2 at doses of 15, 150 and 1500 pmol between 0-24hrs during dark phase in adult male Wistar rats (n=7-10 per group)...... 66
Figure 6: Comparison of IP PK-1 and PK-2 at doses of 20-540 nmol/kg between 0-
24hrs in male C57BL/6 mice (n=6 per dosing group)...... 68
Figure 7: Comparison of IP PK-1 and PK-2 at doses of 20-540 nmol/kg between 0-
24hrs in male C57BL/6 mice (n=6 per dosing group)...... 69
Figure 8: Change in Body weight in grams over 24 hours after doses of 20, 60 and 540 nmol/kg of PK-1 or PK-2 in male C57BL/6 mice...... 70
Figure 9: Effect of IP PK1 7 nmol/kg for up to 1 hour in mice (n=6 per group) on food intake over the first hour in Male C57BL/6 mice...... 72
Figure 10: Serum PK-1 levels during the first hour following injection with 7 nmol/kg of PK-1 given IP to male C57BL/6 mice...... 73
Figure 11: Expression of PK-1 in the stomach of male C57BL/6 mice in fasting (n= 12) and in fed state (n=8)...... 75
Figure 12: Standard Curves used for the interpolation of plasma PK-1 levels...... 77
Figure 13: Circulating plasma PK1 levels in humans whilst fasted and following feeding...... 78
10 Figure 14: Change in circulating plasma PK-1 levels in humans whilst fasted and following feeding...... 79
Figure 15: Outcome of Conceptions ...... 85
Figure 16: A diagragm of chorionic villi showing localisation for PK-1 (EG-VEGF) and its receptors, PROKR1 and PROKR2...... 92
Figure 17: Kisspeptin isoforms ...... 99
Figure 18: Kisspeptin-54 levels are raised during normal human pregnancy ...... 104
Figure 19: Protocol for Study...... 110
Figure 20: Flowchart of pregnant women recruited to the study and main outcomes .. 121
Figure 21: Levels of plasma PK-1 in non-pregnant women...... 124
Figure 22: Correlation of Prokineticin-1 (PK-1) with serum BHCG levels ...... 126
Figure 23: Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG,
Serum Endoglin, Serum FLT-1 and Serum PLGF with gestation...... 128
Figure 24: Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG,
Serum Endoglin, Serum FLT-1 and Serum PLGF with multiple pregnancy and miscarriage...... 133
Figure 25: Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG,
Serum Endoglin, Serum FLT-1 and Serum PLGF with singleton miscarriage: ...... 137
Figure 26: ROC of plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG,
Serum Endoglin, Serum FLT-1 and Serum PLGF with singleton miscarriage: ...... 138
Figure 27: Changes in plasma levels of plasma Kisspeptin, Serum BHCG and Time to miscarriage: ...... 140
Figure 28: Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG,
Serum Endoglin, Serum FLT-1 and Serum PLGF with BMI: ...... 142
Figure 29: Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG,
Serum Endoglin, Serum FLT-1 and Serum PLGF with smoking: ...... 144
11 Figure 30: Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG,
Serum Endoglin, Serum FLT-1 and Serum PLGF with number of previous miscarriages in women whose current singleton pregnancy did not result in miscarriage: ...... 146
Figure 31: Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG,
Serum Endoglin, Serum FLT-1 and Serum PLGF with history of recurrent miscarriages in women whose current singleton pregnancy did not result in miscarriage: ...... 147
Figure 32: Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG,
Serum Endoglin, Serum FLT-1 and Serum PLGF with foetal sex: ...... 149
Figure 33: Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG,
Serum Endoglin, Serum FLT-1 and Serum PLGF with Birth weight in boys: ...... 151
Figure 34: Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG,
Serum Endoglin, Serum FLT-1 and Serum PLGF with Birth weight in girls...... 152
Figure 35: Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG,
Serum Endoglin, Serum FLT-1 and Serum PLGF with head circumference in boys... 154
Figure 36: Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG,
Serum Endoglin, Serum FLT-1 and Serum PLGF with head circumference in girls. .. 155
Figure 37: Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG,
Serum Endoglin, Serum FLT-1 and Serum PLGF with premature delivery...... 157
12 List of Tables
Table 1: Neuropeptides and hormones implicated in regulation of appetite...... 32
Table 2: Distribution of expression of prokineticins and their receptors ...... 38
Table 3: Study Protocol for measurement of plasma PK-1 levels in humans...... 59
Table 4: Details of contents of Macaroni Cheese packets used to induce satiety in humans ...... 60
Table 5: Cause of early Recurrent Pregnancy Loss and Risk factors for Miscarriage. .. 87
Table 6: Substances produced by the Placenta during Pregnancy...... 90
Table 7: Circulating PK-1 levels during pregnancy ...... 94
Table 8: Clinical characteristics of study participants ...... 122
Table 9: Correlation of levels of plasma Prokineticin-1, plasma Kisspeptin, Serum
BHCG, Serum Endoglin, Serum FLT-1 and Serum PLGF with gestation...... 129
Table 10: Gestation of pregnancy during which study participants with singleton pregnancy were recruited to the study...... 130
13 Abbreviations
AFP α-Feto Protein
α-MSH alpha Melanocyte Stimulating Hormone
AHA Anterior Hypothalamic Area
AMH Anti-Mullerian Hormone
AP Area Postrema
APC Activated Protein C
AVP Arginine VasoPressin
BMI Body Mass Index
BSA Bovine Serum Albumin
CART Cocaine and Amphetamine Regulated Transcript
CCK CholeCystoKinin
CNS Central Nervous System
CXCR4 Chemokine Receptor 4
DIO Diet Induced Obese mouse
DMN Dorsomedial Nucleus
DNA Deoxy-riboNucelic Acid
EG-VEGF Endocrine Gland derived Vascular Endothelial Growth Factor
GLP-1 Glucagon-Like Peptide 1
GnRH Gonadotrophin Releasing Hormone h or hr hour hCG human Chorionic Gonadotrophin hCG-H hyperglycosylated human Chorionic Gonadotrophin
HPL Human Placental Lactogen
ICV Intra-Cerebro-Ventricular
IP IntraPeritoneal
14 IUGR Intra-Uterine Growth Restriction
IV IntraVenous kDa Kilo Dalton
Kg Kilo-Gram kisspeptin-IR kisspeptin Immuno-Reactivity
LHA Lateral Hypothalamic Area
LV Lateral Ventricle
MCH Melanin Concentrating Hormone
MCIP1 Myocyte-enriched Calcineurin Interacting Protein 1
ME Median Eminence
µl Micro Litres mg Milligrams ml Millilitres
MMA Methylmalonic acid
MMP-9 Metalloproteinase-9
MOM Multiples of Median
MTHFR Methylenetetrahydrofolate reductase gene
Nmol Nano-Moles
NTS Nucleus Tractus Solitarus
NTS Nucleus Tractus Solitarus
PAPP-A Pregnancy Associated Plasma Protein A
PBS Phosphate Buffered Saline
PE Pre-eclampsia
PIH Pregnancy Induced Hypertension
PK-1 Prokineticin 1
PK-2 Prokineticin 2
15 PKR-1 Prokineticin Receptor 1
PKR-2 Prokineticin Receptor 2
PlGF Placental Growth Factor
Pmol Pico-Moles
POA Preoptic Area
POMC Pro-Opio-Melano-Cortin
PVN Para-Ventricular Nucleus
PVT Para-Ventricular Thalamic Nuclei
RIA Radio-Immuno Assay
RNA Ribo-Nucleic Acid
RPL Recurrent Pregnancy Loss
SC Subcutaneous
SCN SupraChiasmatic Nucleus
SEM Standard Error of Mean
Eng Solube Endoglin
FLT-1 Soluble FMS-like Tyrosine Kinase-1
SON Supraoptic Nucleus
TEG Thromboelastogram
3V Third Ventricle
VEGF Vascular Endothelial Growth Factor
VMH ventromedial hypothalamus
16
Chapter 1
Introduction
17 The obesity epidemic
According to the CDC (Centers for Disease Control and Prevention), 35.7% of adults in the USA are obese, as defined by a BMI (Body Mass Index) greater than 30 kg/m2
(Ogden et al., 2010). The WHO define a normal body weight as a BMI of 20-25 kg/m2,
25-30 kg/m2 as overweight, over 30 kg/m2 as ‘obese’, and a BMI of greater than 35 kg/m2 morbid obesity. A recent survey published in March 2014 by the Gallup-
Healthways Well-Being Index suggests that the prevalence of obesity in the USA is increasing year upon year. Importantly, the survey also reported that people living in the
10 states with the highest levels of obesity are more likely to carry a diagnosis of a chronic diseases such as high blood pressure, high cholesterol, depression, diabetes, cancer, and heart attacks, than are those living in the 10 states with the lowest obesity rates in data from 2009-2010.
In Britain the incidence and prevalence of obesity is rising rapidly; in 1980 eight percent of women and six percent of men were classified as obese, which rose to over half of women and two thirds of men being classified as overweight or obese in 2001.
Furthermore, the prevalence of childhood overweight and obesity in the U.K. has increased sharply over the last two decades affecting almost fourteen percent of boys and seventeen percent of girls aged two to fifteen years old. A prospective epidemiologic study of more than one million individuals has established that obesity is an independent risk factor for increased mortality and in addition, overweight and obesity cause significant morbidity including type II diabetes mellitus, certain cancers, coronary heart disease, hypertension, sleep apnoea, and psychological morbidity and societal stigmatisation (Calle et al. 1999; National audit office report; 2001). It has been estimated that the cost to the National Health Service (NHS) of treating obesity and its associated sequlae is 3.7 billion pounds per year which equates to just under three
18 percent of the total annual NHS budget. Obesity is a multifactorial condition in which genetic, environmental, psychological and socio-economic conditions influence food intake and body weight (Bray et al. 1992). However apetite regulation is the safest and likely to be the most effective therapeutic target for control of obesity, as therapies directed towards energy expenditure may carry a greater risk of adverse events. Apetite is tightly regulated by a complex interaction of neuronal and endocrine pathways many of which converge within the hypothalamus. The disruption of these hypothalamic pathways can result in changes in feeding behaviour and body weight and these findings have provided significant insights into the pathophysiology of obesity. Following the recent withdrawal of two anti-obesity drugs, there now exists only one agent (Orlistat) licensed for the treatment of obesity in the U.K. The patient drop out rate at one year for this sole remaining anti-obesity therapy is 70% which highlights the imperative to better understand appetite pathways in order to develop novel therapies for the treatment of obesity. The hypothalamic mechanisms controlling appetite are complex and plentiful; therefore a great deal of research is required in order to fully appreciate all of the different mechanisms and their interactions, such that new therapeutics for treating obesity can be developed.
19 The Hypothalamus
The hypothalamus is a midline structure situated at the base of the brain with symmetrical right and left halves traversing each lateral wall of the third ventricle. The hypothalamus contains populations of neurones organised into discrete nuclear aggregations with inter and intra-nuclear connections (see Figure 1).
20 Figure 1: Diagrammatic representation of a sagittal section through a rodent brain showing the hypothalamic nuclei involved in the regulation of food intake.
ARC, Arcuate nucleus; PVN, paraventricular nucleus; DMH, dorsomedial hypothalamus; VMH, ventromedial hypothalamus; LH, lateral hypothalamus.
21 The importance of the hypothalamus in body weight regulation came to the fore in the middle of the last century when it was reported that the lesioning of discrete hypothalamic nuclei could induce hyperphagia and obesity or aphagia and weight loss depending on the targeted nucleus (Hetherington et al. 1940). Since this time numerous studies have not only confirmed the importance of these hypothalamic nuclei in the regulation of body weight, but have also delineated many of the hypothalamic signals and pathways which control energy homeostasis (see Table 1). I shall briefly introduce the most prominent hypothalamic nuclei in turn and their role in appetite control, namely: i) The arcuate nucleus (ARC) ii) The paraventricular nucleus (PVN) iii) The lateral hypothalamus (LH) iv) The ventromedial nucleus (VMN) v) The dorsomedial nucleus
22 The Arcuate Nucleus (ARC)
The arcuate nucleus (ARC) is located near the median eminence, where the blood brain barrier is incomplete. This renders the ARC susceptible to the effects of circulating factors, which signal information about nutritional status and the metabolic milieu. One such factor is the anorectic peptide hormone leptin, which is synthesised and secreted by adipocytes signalling information about energy stores and nutritional status, circulating at levels proportional to fat mass (Zhang et al. 1994).
One population of ARC neurones co-expresses the orexigenic peptides neuropeptide Y
(NPY) and agouti-related protein (AgRP), whilst a second subpopulation expresses the anorectic peptides alpha-melanocyte-stimulating hormone (α-MSH) derived from pro- opiomelanocortin (POMC) and cocaine-and amphetamine-regulated transcript (CART).
Both NPY/AgRP and POMC/CART neurones express the leptin receptor and the synthesis and release of these peptides is regulated by the level of circulating leptin.
NPY is a potent orexigenic neuropeptide. The intracerebroventricular (ICV) injection of
NPY into the third ventricle of rats results in a marked increase in food intake and NPY administration over a sustained period leads to weight gain (Vettor et al. 1994). When serum leptin levels are high indicating adequate energy storage in adipose tissue the synthesis and release of NPY is inhibited. Conversely when circulating leptin levels fall,
NPY expression increases.
The NPY expressing neurones of the ARC also express the orexigenic peptide AgRP, which shows a similar expression pattern in response to the level of circulating leptin as that of NPY. The agouti viable yellow mouse (Avy) which over expresses agouti protein is hyperphagic and obese. Furthermore, ICV administration of AgRP to rats increases food intake (Klebig et al. 1995; Rossi et al. 1998). Both AgRP and the ARC anorectic peptide α-MSH act upon the melanocortin 4 receptor (MC4R).
23 Physiologically, α-MSH mediates a basal tonic inhibition of food intake by agonist action at MC4R, whilst AgRP acts as an antagonist at the MC4R and stimulates food intake (Ollmann et al. 1997).
The importance of the melanocortin system in the regulation of body weight in humans can be seen in the early onset obesity of children with mutations of MC4R, which may account for up to six percent of cases of morbid paediatric obesity (Yeo et al. 1998).
Furthermore individuals with homozygous mutations of the POMC gene are obese and heterozygotes also have an increased prevalence of overweight and obesity (Krude et al.
1998).
The POMC neurones of the ARC also express the neuropeptide cocaine- and amphetamine regulated transcript (CART). CART has been shown to have both an anorectic and orexigenic effect depending on the site of action. The expression of
CART mRNA in the ARC is reduced in food deprived animals, but can be increased by the administration of leptin. The ICV administration of CART reduces food intake which may be mediated in the hindbrain since plugging the aqueduct between the third and fourth ventricle prevents this anorectic effect (Kristensen et al. 1998). The orexigenic effect of CART may be mediated in the hypothalamus since the direct injection of CART into the ARC or the ventromedial nucleus stimulates feeding and the transgene mediated over expression of CART in the ARC leads to a significant increase in food intake (Kong et al. 2003).
24 The Paraventricular nucleus (PVN)
The paraventricular nucleus (PVN) is composed of a group of heterogeneous neurones situated on either side of the top of the third cerebral ventricle. Both NPY and melanocortin neurones of the ARC project to the PVN where MC4R and receptors for
NPY are expressed. The direct injection of NPY and AgRP in to the PVN induces hyperphagia whilst the intra-PVN administration of an MSH analogue inhibits feeding
(Kim et al. 2000). The leptin receptor is not highly expressed in the PVN and it is likely that the effect of leptin on PVN outflow is via projections from the leptin responsive neurones of other hypothalamic nuclei such as the ARC and the ventromedial nucleus.
Therefore, the PVN may act as a final common pathway of the neuronal, autonomic and endocrine response to circulating leptin with projections to numerous sites outside the hypothalamus including higher centres known to modulate motivational behaviours, the pituitary gland and the sympathetic nervous system.
25 The Lateral Hypothalamus (LH)
The lateral hypothalamus (LH) is a nucleus of the lateral zone of the hypothalamus. To date, two key groups of LH peptides, the orexins and melanin concentrating hormone
(MCH) have been identified as having a marked orexigenic effect. Levels of orexin pre- pro mRNA increase during fasting and the central administration of orexin stimulates food intake (Sakurai et al. 1998). Whilst the orexins were originally named for their stimulatory effect on food intake from the Greek ‘orexis’ meaning appetite, subsequent studies have demonstrated that the orexins play a significant role in behavioural arousal and in human narcolepsy, a condition of sudden irresistible day time somnolence has been shown to be associated with a deficiency of orexin or its receptor (Lin et al. 1999).
Like the orexins, MCH mRNA expression in the LH also increases during starvation and returns to baseline after re-feeding (Qu et al. 1996). Furthermore, the injection of
MCH into the LH results in an increase in food intake in rats whilst MCH knock out mice are hypophagic and have a lower body weight than wild type controls (Shimada et al. 1998). Within the hypothalamus the MCH receptor is expressed in the ARC and the ventromedial nucleus and following lesioning of the ventromedial nucleus in rats there is a marked increase in MCH expression in the LH, which may partly explain the hyperphagia of ventromedial nucleus lesioned animals.
26 The Ventromedial Nucleus (VMN)
The ventromedial nucleus (VMN) of the hypothalamus is a bilateral group of encapsulated neuronal cell bodies and their dendritic branches that sits adjacent to the third ventricle above the ARC and below the dorsomedial nucleus (DMN).
The importance of the VMN in the regulation of food intake was first demonstrated in the middle of the last century following experimental lesioning of the VMN in rats which resulted in hyperphagia and obesity (Hetherington et al. 1940). Soon after, it was reported that lesions of the LH resulted in aphagia and weight loss. The suggestion that the LH was a ‘feeding centre’ and the VMN a ‘satiety centre’ led to the ‘dual-centre’ hypothesis for motivated behaviour, which although now considered overly simplistic, nevertheless provided a framework for early studies investigating the hypothalamic regulation of food intake.
The transcription factor steroidogenic factor-1 (SF-1) is an orphan member of the nuclear hormone receptor family and is expressed by VMN neurones which also express the leptin receptor. SF-1 knockout mice develop late onset obesity predominantly due to a reduction in locomotor activity (Majdic et al. 2002).
Furthermore the targeted deletion of the leptin receptor from these neurones results in obesity which is not due to hyperphagia but attributable to reduced energy expenditure and impaired diet induced thermogenesis (Dhillon et al. 2006). The obesity of these mice with targeted VMN deletion of the leptin receptor is less marked compared to that of global receptor null mice but is augmented by the additional deletion of the leptin receptor on ARC POMC neurones (Dhillon et al. 2006).
Brain derived neurotrophic factor (BDNF) is a mediator of brain development and plasticity and also has a well defined role as an anorectic neuropeptide (Xu et al. 2003).
Infusion of BDNF into the lateral ventricle of rats results in a dose dependent reduction
27 in food intake and body weight. Within the hypothalamus, BDNF and its receptor are most abundantly expressed in the VMN where BDNF expression is co-localised with
SF-1 expressing neurones. BDNF expression in the VMN but not in other brain regions is regulated by nutritional status with food deprivation resulting in a 60% reduction in
BDNF mRNA in the VMN (Xu et al. 2003). When BDNF is selectively deleted in the
VMN and DMN the mutant animals are hyperphagic and obese with normal energy expenditure (Xu et al. 2003). The importance of BDNF signalling in the regulation of human body weight is suggested by the phenotype of severe hyperphagic obesity in subjects with mutations in the gene for BDNF or its receptor (Gray et al. 2006, Yeo et al. 2004).
28 The Dorsomedial Nucleus (DMN)
The dorsomedial nucleus (DMN) lies above the VMN in the mediobasal hypothalamus.
The DMN receives afferent input from the VMN and LH and efferent fibres project to the PVN. The leptin receptor is expressed by DMN neurones which highly express the orexigenic peptide NPY. The tissue specific over expression of NPY in the DMN of rats using adeno-associated virus (AAV) results in an increase in food intake and body weight whilst knockdown of NPY in the DMN normalises the food intake of Otsuka
Long-Evans Tokushima Fatty rats, a strain which is hyperphagic and obese due to the congenital absence of the receptor for the anorectic gut peptide cholecystokinin CCK
(Yang et al. 2009).
29 Circulating Factors which act within the hypothalamus to regulate food intake and body weight
In addition to leptin, several other circulating factors, produced in the periphery, act within the hypothalamus to regulate food intake and body weight. These signals include hormones produced by the gastro-intestinal tract and the thyroid hormone, tri- iodothyronine (T3).
The orexigenic peptide ghrelin is produced by the stomach and circulating levels rise during fasting and fall following food intake. The administration of ghrelin to rats results in the stimulation of feeding which is partly mediated by activation of
NPY/AgRP neurones of the ARC (Nakazato et al. 2001). Obese subjects have been found to have reduced ghrelin suppression after a meal compared with normal weight controls and ghrelin levels have been found to increase after diet-induced weight loss which could jeopardise the maintenance of weight reduction (Cummings et al. 2002).
Peptide YY (PYY) is a peptide hormone released from by the colon following food ingestion. It exerts an anorectic effect in the ARC through the inhibition of orexigenic
NPY neurones and disinhibition of anorectic POMC neurones (Batterham et al. 2002).
Obese humans have been found to have a reduced post-prandial rise in plasma PYY compared with lean controls and this is associated with an attenuated satiety response
(le Roux et al. 2006). Furthermore, circulating PYY levels rise following gastric bypass surgery which may partly explain the reduced appetite reported by some subjects following this procedure.
The anorectic gut peptide glucagon-like peptide-1 (GLP-1) is released following food intake and levels of expression fall with fasting. The direct injection of GLP-1 into the
PVN reduces food intake in rats and evidence for the physiological importance of GLP-
30 1 is suggested by the orexigenic effect of the central administration of the specific a
GLP-1 receptor antagonist to satiated rats which when given for ten days significantly increases body weight (Turton et al. 2006). Obese humans have been found to have an attenuated postprandial release of GLP-1 and reduced circulating levels of the hormone which increases with weight loss.
Oxyntomodulin (OXM) is co-secreted with GLP-1 and PYY following a meal. OXM promotes satiety in both rodents and humans and this effect may partly be through the augmentation of ARC α-MSH signalling (Dakin et al. 2006). In addition, part of OXM’s suppressive effect on food intake may be due to a reduction in plasma ghrelin.
Circulating OXM levels are raised after gastric bypass surgery which may contribute to the weight reducing effect of the procedure.
The importance of thyroid hormones in the regulation of energy homeostasis is attested by the hyperphagia and weight loss seen in patients with hyperthyroidism and the reduced metabolic rate and weight gain of hypothyroidism. During fasting T3 is produced within the hypothalamus from circulating thyroxine (T4) and in rodents the administration of T3 directly into the VMN stimulates food intake although the mechanism by which this occurs is not known (Kong et al. 2004; Diano et al. 1998).
Appetite regulation is thought to be coordinated at the ARC, whereby peripheral signals such as leptin act to modulate the release of orexigenic and anorectic neuropeptides via the MC4R and control appetite and thus body weight. This model allows for the communication of peripheral signals of energy stores to the hypothalamus to regulate energy stores.
31 Table 1: Neuropeptides and hormones implicated in regulation of appetite.
Orexigenic Peptides Anorectic Peptides
Neuropeptide Y (NPY) α-melanocyte stimulating hormone (α-MSH)
Agouti related peptide (AgRP) Corticotrophin releasing hormone (CRH)
Melanin concentrating hormone (MCH) Thyrotrophin releasing hormone (TRH)
Galanin Cocaine-and amphetamine-related transcript
Orexin-A and –B (CART)
-endorphin Glucagon-like peptide-1 (GLP-1)
Neuromedin U / S Oxyntomodulin (Oxm)
Bombesin
Circulating Factors: Somatostatin
Ghrelin Amylin
Circulating Factors:
Insulin
Leptin
Glucagon
Cholecystokinin (CCK)
Pancreatic polypeptide (PP)
Peptide YY (PYY)
32 Prokineticins- a potential novel regulator of appetite
A recently discovered family of peptides called the prokineticins may be involved in a variety of physiological functions including appetite regulation and reproduction.
Prokineticins
In 2000, Lie et al. isolated two human cDNAs and their encoded proteins, which were named ‘Prokineticins’ (PKs) due to their ability to stimulate gastrointestinal (GI) smooth muscle in guinea pig ileum (Li et al., 2001). The two forms of prokineticin
(PK), prokineticin-1 (PK-1) and prokineticin-2 (PK-2), share approximately 44% amino acid homology (Li et al. 2001; LeCouter et al. 2001). Prokineticin 1 (PK-1) and prokineticin 2 (PK-2) were first identified as the mammalian homologues of the amphibian peptide mamba intestinal toxin (MIT-1; a non-toxic constituent in the venom of the black mamba snake Dendroaspis polylepis) and Bv8 (Bombina variegate molecular mass-8 kDa: found in the skin secretion of the toad Bombina variegate) respectively. Prokineticin-1 (PK-1) is also known as endocrine gland vascular endothelial growth factor (EGVEGF) (Ngan et al. 2008).
More recently, molecular cloning analysis has identified a longer form peptide of PK-2,
PK-2L. PK-2L is an alternatively spliced product of the PK-2, which contains 21 additional amino acids. Proteolytic cleavage of PK-2L generates the smaller active form of PK-2 protein termed PK-2β (Chen et al., 2005).
The genes encoding PK-1 and PK-2 are located on human chromosome 1p21 and
3p21.1, respectively. The six amino acid sequence at the N-terminal domain of the PKs
(AVITGA) are highly conserved between mammalian and non-mammalian species.
The C-terminal domain contains 10 cysteine residues which form five pairs of
33 disulphide bonds (see Figure 2 and 3) and fold the molecule into a globular form (Li et al. 2001; Mollay et al. 1999; Boisouvier et al. 1998).
These prokineticins have been found to play a role in a number of biological processes including appetite and reproduction. Prokineticin 2 has been shown to be required for the formation of the olfactory bulb. Patients lacking prokineticin 2 are unable to secrete
GnRH with resultant hypogonadotrophic hypogonadism. Data from our own department has shown that administration of prokineticin 2 to rodents reduces food intake both in lean and obese rodents.
34 Figure 2: The amino acid sequence of Prokineticin-2 (PK-2).
Ten conserved cysteine residues are highlighted in colour and pairs of disulphide bonds are based on the sequence of mamba intestinal toxin 1 (MIT 1) (Wen et al., 2005). See
Appendix 2 for amino acid codes.
35 Figure 3: Amino Acid Sequence of Prokineticin-1 and Prokineticin-2
Amino acid sequences of prokineticin 1 (A), prokineticin 2 (B), Frog Bv8 (C), and partial sequence of MIT 1 (D). The ten conservative cysteine residues are marked (*).
Signal peptides are underlined (Li et al. 2001).
Human PK-1 sequence (86 αα) is :
(N-terminal) AVITGACERDVQCGAGTCCAISLWLRGLRMCTPLGREGEECHP
GSHKIPFFRKRKHHTCPCLPNLLCSRFPDGRYRCSMDLKNINF (C-terminal)
and Human PK-2 (81 αα) sequence is :
AVITGACDKDSQCGGGMCCAVSIWVKSIRICTPMGKLGDSCHPLTRKVPFFGRR
MHHTCPCLPGLACLRTSFNRFICLAQK
36 The PK’s are the ligands for two closely related cognate G-protein coupled receptors
(GPCRs), termed prokineticin receptor-1 (PKR-1) and prokineticin receptor-2 (PKR-2)
(Lin et al., 2002, Masuda et al., 2002). PKR-1 and PKR-2 are encoded by genes located on human chromosome region 2q14 and 20p13 respectively (Kaser et al., 2003). These receptors share 85% amino acid identity and differ mainly in their N-terminal sequences
(Kaser et al., 2003). Receptors couple to Gq, Gi and Gs to mediate the intracellular calcium mobilisation, phosphorylation of p44/p42 mitogen-activated protein kinase, serine/threonine kinase Akt and cAMP accumulation, respectively (Chen et al., 2005).
Receptor binding and functional assays have revealed that PK’s exhibit different affinities for the receptors and potencies in activating calcium mobilisation. PK-2 has greater affinity than PK1 (and PK2β) for both PK receptors 1 and 2 (Chen et al. 2005).
PK2β has greater affinity for PKR-1 rather than PKR-2 (Chen et al. 2005). The N- terminal amino acid sequence (AVITGA) is essential for activation of PKR’s, since invertebrate homologues of PK’s which lack AVITGA do not activate mammalian
PKR’s (Negri et al., 2007). Furthermore, PK’s with mutant N-terminal domains fail to activate the PKR’s (Bullock et al., 2004). The C-terminal cysteine residues are equally important, since mutant PK’s with substitutions of cysteine residues from the C- terminal also result in failure of activation of PKR’s (Bullock et al., 2004). PK-1 and
PKR-1 are expressed predominantly in peripheral tissues, whereas expression of PK-2 and PKR-2 is highest in the brain (Li et al., 2001, Masuda et al., 2002).
The wide expression of PK’s within the central nervous system (CNS) and the periphery suggests that they may play a role in several biological functions. The distribution and possible biological roles are summarised in Table 2.
37 Table 2: Distribution of expression of prokineticins and their receptors
Expression of prokineticins (PKs) and prokineticin receptors (PKRs) throughout the
body (adpated from Ngan et al. 2008).
Tissues PK-1 PK-2 PK-R1 PK-R2 Reference
Granulosa; Not detected Present Present LeCouter et al. (2001) Ovary corpus luteum Maldonado-Perez et al. (2007)
Endothelial cells Endothelial cells Leydig cells Spermatocytes Maldonado-Perez et al.(2007) Testis of testis of testis interstitium interstitium
Glomerulosa Glomerulosa Glomerulosa cell; Adrenal Glomerulosa cell; cell; cell; fasciculate cells; Keramidas et al. 2008) fasciculate cells gland fasciculate cells fasciculate cells; endothelial cells endothelial cells
Chen et al. (2005) Placenta Present Present Present Not detected Maldonado-Perez et al. (2007)
Glandular Glandular Glandular Glandular epithelium; epithelium; epithelium; epithelium; Maldonado-Perez et al. (2007) uterus endothelial; endothelial; endothelial; endothelial; Ngan et al. (2006) stromal cells; stromal cells; stromal cells; stromal cells; smooth muscle smooth muscle smooth muscle smooth muscle cells cells cells cells
Subventricular Subventricular Cerebral cortex; zone; zone; Cheng et al. (2002) Brain Low expression olfactory bulb; rostal migratory rostal migratory Ng et al. (2005) Purkinje cells stream; olfactory stream; olfactory bulb; bulb; hippocampus hippocampus
Enteric plexus; Hoogerwerf (2006), Intestinal Enteric plexus; Enteric plexus of Kaser et al. (2003), mucosa of Enteric plexus enteric neural ileocecum Ngan et al. (2007) tract embryonic gut crest cells Ngan et al. (2008)
Cardiovascular Cardiovascular Cardiovascular Heart No detected Urayama et al. (2007) tissue; cardiac tissue; tissue; cardiac
cell cardiac cell cell
Bone Hematopoietic Hematopoietic Hematopoietic stem cells; stem cells; stem cells; marrow; LeCouter et al. (2004) B and T cells monocytes; mature blood mature blood Lin et al. (2002) peripheral neutrophils and cells including cells including blood dendritic cells lymphocytes lymphocytes
38 In rodents, PK-1 expression has been identified in the gastrointestinal tract, ovaries, testes, adrenals, the placenta and the nucleus tractus solitarius (NTS) (Cheng et al.
2002; LeCouter et al. 2003; Melchiorri et al. 2001; Wechselberger et al. 1999).
Interestingly, the expression of PK-1 is dynamic in the reproductive organs (ovary, uterus and placenta) and changes in response to changes in the hormonal milieux
(oestrogen, progesterone, human chorionic gonadotropin (hCG) and hypoxia-inducible factor (HIF-1α)) across the menstrual cycle and during pregnancy (Maldonado-Perez et al., 2007; Ngan et al., 2006).
Like PK-1, PK-2 mRNA has been reported in the small intestine and testes (Li et al.,
2002; Wechselberger et al., 1999), but PK-2 is mainly localised to the brain. PK-2 in the brain is present in the olfactory bulb, regions of the cerebral cortex, medial pre-optic area (MPOA) of the hypothalamus, the ARC, suprachiasmatic nucleus (SCN), some thalamic nuclei, Purkinje cells of the cerebellum, sensory and motor nuclei of the brain stem and spinal cord. PK-2 mRNA is rhythmically expressed in the SCN, according to the 24-h cycle of the circadian clock (Cheng et al., 2002).
Both PKR’s are present in the testis and several regions of the brain. In the mouse brain, PKR-2 mRNA is expressed in the lateral septal nucleus (LS), paraventricular, parataenial and paracentral thalamic nuclei (PVT / PT / PC), paraventricular nucleus
(PVN), dorso medial hypothalamus (DMH), arcuate (ARC), suprachiasmatic nucleus
(SCN), subfornical organ (SFO), lateral globus pallidus, amygdala, nucleus tractus solitarus (NTS) and area postrema (AP) (Cheng et al., 2002, Cheng et al., 2006). PKR-
1 mRNA has limited distribution within the CNS, with only moderate expression in the olfactory regions, dentate gyrus, zona incerta and dorsal motor vagal nucleus (Cheng et al. 2006).
PK-2 has been shown to be required for normal GnRH secretion and fertility. Mice lacking either PK-2 or the PKR-2 have a marked reduction in olfactory bulb size (Ng et
39 al. 2005; Matsumoto et al. 2006). Furthermore, the absence of either PK-2 or PKR-2 in mice or humans results in hypogonadotrophic hypogonadism, and a failure of gonadotrophin releasing hormone (GnRH) neuronal migration (Cole et al. 2008; Dode et al. 2006; Pitteloud et al. 2007; Matsumoto et al. 2006). PK-2 also plays a role in nociception, as absence of PK-2 in mice results in impaired perception of pain and administration of PK-2 to mice results in hyperalgesia (Hu et al., 2006).
PK-2 is expressed at high levels in the SCN of the hypothalamus and is therefore believed to play an important role in the regulation of the circadian cycle. Mice lacking
PK-2 or PKR-2 show disrupted sleep-wake cycles, locomotor activity and thermogenesis (Li et al. 2006, Prosser et al. 2007). The regulation of food intake is influenced by the circadian rhythms (Froy et al. 2007) and thus PK-2 signalling may influence food intake. Neurons from the SCN project to various hypothalamic regions important in the control of food intake that express PKR mRNA, including the ARC,
PVN and the DMH (Negri et al. 2004; Cheng et al. 2002). Consistent with this, PK-2 expression in the SCN is highest in the early light phase (when rats are satiated) and lowest at the beginning of the dark phase (natural feeding period) suggesting that PK-2 may act to physiologically inhibit food intake (Negri et al. 2004).
The frog homologue of the mammalian PK-2 (BV8) binds with high affinity to the
PKR-2 and inhibits food intake following intracerebroventricular (ICV) or direct ARC injection in food-deprived rats (Negri et al. 2004). The ARC receives neuronal inputs from the SCN, MPO and NTS, neurones all of which express PK-2 (Negri et al. 2004).
Interestingly, PK-2 has been shown to directly activate neurones in the PVN using whole-cell patch-clamp techniques (Yuill et al. 2007). PK-2 directly activated magnocellular and parvocellular neurones of the PVN and this activation was inhibited with a PK-2 inhibitor in rats (Yuill et al. 2007).
40 Prokineticins (PKs): novel hypothalamic regulators of appetite and placental angiogenesis
Prokineticin 2 is abundantly expressed in the suprachiasmatic nucleus of the hypothalamus. The expression of PK-2 mRNA falls markedly during fasting and the
ICV administration of PK-2 to rats potently reduces food intake (Gardiner et al. 2010).
Further evidence for the physiological role of PK-2 in the regulation of food intake comes from the increase in food intake which occurs following the ICV administration of an anti-PK-2 antibody to rats (Gardiner et al. 2010). The PK-2 receptor is highly expressed in the ARC and the direct injection of PK-2 into the ARC reduces food intake. Part of the anorectic effect of PK-2 is mediated through the melanocortin system. Ex vivo, PK-2 potentiates the release of α-MSH from hypothalamic explants.
Furthermore, in vivo, ICV administration of the MC4R antagonist, agouti, blocks the anorectic effect of PK-2. The peripheral administration of PK-2 to mice reduces food intake and furthermore, the chronic peripheral administration of PK-2 reduces body weight in both lean and obese mice. This demonstrates both that tachyphylaxis to the anorectic effect of PK-2 does not occur with repeated administration and that obese animals remain sensitive to the effects of PK-2. Both these findings identify the PK-2 signalling system as a potential novel therapeutic target for the treatment of obesity.
However PK-2 is predominantly expressed centrally within the CNS, where as PK-1 is expressed peripherally including within the gut. Both PK’s stimulate the PKRs, albeit
PK-2 with greater affinity during in vitro receptor binding studies. Therefore it is possible that PK-1 may represent a novel gut hormone, which interacts with PKR’s located centrally within the hypothalamus to mediate an anorectic effect on appetite. If this were to be the case then PK-1 may represent a more suitable target for therapeutic interventions, as these would need to be administered peripherally. In this thesis I have
41 investigated the role of prokineticin-1 on appetite in rodents and also compared the effects to prokineticin-2. I also determine the changes in plasma levels of prokineticin-1 in humans following satiation to establish the potential physiological relevance of prokineticin-1 as an anorectic gut hormone.
Prokineticin-1 is also a potent angiogenic mitogen on endocrine vascular epithelium and hence it is also known as ‘Endocrine Gland derived-Vascular Endothelial Factor’.
Placental angiogenesis plays an important role in placental function and risk of pregnancy complications such as miscarriage. I therefore hypothesised that prokineticin-
1 is a biomarker of pregnancy complications in humans. In this thesis, I have measured circulating levels of PK-1 found in pregnancy and correlated these with the occurrence of pregnancy complications. I have also compared the utility of PK-1 with other potential novel markers of pregnancy complications. Together this body of work investigates the potential physiological roles of PK-1.
42
Chapter 2
Role of PK-1 in
Appetite Regulation
43 Introduction
Both PK1 and PK2 stimulate the PKRs. Prokineticin-2 is predominantly expressed centrally within the CNS, whereas prokineticin-1 is expressed peripherally including within the gut. PK-2 administration has been shown to potently suppress food intake in rodent models as detailed below.
Hypothalamic PK-2 expression is significantly reduced by 45% in rats fasted for 12 or 24 h when compared with ad libitum fed rats (Gardiner et al. 2010). ICV PK-2 administration to ad libitum fed male rats significantly reduced food intake during the first hour following administration at doses greater than 0.05 nmol/rat at the beginning of the dark phase
(Gardiner et al. 2010). Indeed doses of 0.15-1.5 nmol/rat maximally reduced food intake by
85% when compared with vehicle treated rats (Gardiner et al. 2010). A reduction in food intake over the first hour was also seen in rats fasted for 24 h following injection with ICV with 0.15-4.5 nmol/rat of PK-2 in the early light phase (Gardiner et al. 2010). PK-2 reduced the number of feeding episodes and did not result in abnormal behavioural patterns suggesting a specific effect on appetite. Furthermore, there was no alteration in energy expenditure as assessed by O2 consumption and additionally pair-fed animals lost a similar amount of weight to PK-2 treated animals, suggesting that weight loss occurred through a direct effect on appetite (Gardiner et al. 2010).
Additionally, ICV administration of a PK-2 blocking antibody to satiated rats at the beginning of the light phase increased food intake (Gardiner et al. 2010). ICV administration of PK2 resulted in a significant increase in c-fos immuno-reactivity in the supraoptic nucleus (SON), arcuate (ARC), paraventricular nucleus (PVN), and anterior hypothalamic area (AHA), but not in the ventromedial hypothalamus (VMH), dorosmedial hypothalamus (DMN), suprachiasmatic nucleus (SCN), or lateral hypothalamic area (LHA)
44 (Gardiner et al. 2010). Furthermore co-localisation studies confirmed that ICV PK-2 activated arcuate (ARC) POMC neurons (Gardiner et al. 2010).
Intraperitoneal (IP) injection of 20 nmol/kg of PK-2 at the beginning of the dark phase significantly reduced food intake in rats (Gardiner et al. 2010). IP injections of 7, 20, 60,
180, or 540 nmol/kg dose-dependently reduced food intake in C57BL/6 mice (Gardiner et al. 2010). The highest dose of PK2 administered (540 nmol/kg) resulted in a 20% reduction in 24-h food intake (Gardiner et al. 2010). Twice daily IP injection of 180 nmol/kg of PK-2 for 5 days in lean mice significantly decreased cumulative food intake and body weight with no evidence of tachyphylaxis (Gardiner et al. 2010). Importantly, PK-2 was equally effective in DIO (diet induced obese) mice, which has important implications for therapeutic use (Gardiner et al. 2010).
IP administration of 540nmol/kg of PK-2 to C57Bl/6 mice significantly increased c-fos-like immuno-reactivity in the dorsal motor vagal nucleus (DMV) in the brainstem, but not in other areas of the brainstem or hypothalamus (Beale et al. 2013). Peripheral PK-2 administration retains its anorectic effects in PKR-2 knock-out mice, but not in PKR-1 knock out mice (Beale et al. 2013). This suggests that the anorectic effects of peripheral
PK-2 occur via PKR-1 in the brainstem (Beale et al. 2013). PK2 administered at 60 nmol/kg/h or 120 nmol/kg/h by continuous subcutaneous (SC) infusion to DIO mice significantly reduced body weight gain between days 1 and 13 post-surgical implantation of an osmotic minipump when compared with vehicle treated controls (Beale et al. 2013).
Mice pair-fed to each dose of PK2 lost a similar amount of weight to those administered
PK2, suggesting that these effects occurred via alterations in food intake alone (Beale et al.
2013).
45 Thus, PK2 has potent anorectic effects and both PK1 and PK2 stimulate the PKRs. Since
PK-1 is distributed peripherally (especially in the gut), one could hypothesise that PK-1 may be a physiological gut hormone which inhibits food intake. However no studies to date have determined the effects of PK-1 in food intake. In this chapter I will investigate the role of PK-1 on appetite regulation and compare this with PK-2.
46 Hypothesis and aims
Hypothesis
PK-1 is a gut hormone acting as a physiological inhibitor of food intake.
Aims
1. Comparison of ICV PK-1 and PK-2 at doses of 15-150 pmol between 0-24hrs
during dark phase in adult male Wistar rats
2. Comparison of ICV PK-1 and PK-2 at doses of 15-1500 pmol between 0-24hrs
during dark phase in adult male Wistar rats
3. Comparison of IP PK-1 and PK-2 at doses of 20-540 nmol/kg between 0-24hrs
4. Serum levels of PK-1 and food intake at 1 hour following administration of IP PK-1
7 nmol/kg on food intake in mice
5. Expression of PK-1 mRNA in the stomach of Fed and Fasted mice
6. Assessment of circulating levels of PK-1 in humans in fasted and fed states
47 Material and methods
1. Effect of ICV administration of 15, 50, 150 pmol of PK-1 during Dark Phase in male Wistar rats
Materials
PK-1 was purchased from PeproTech Ltd. (New Jersey, USA). Cannulation materials were purchased from Plastics One, Inc. (Roanoke, VA, USA). NDP-melanocyte stimulating hormone (NDP-MSH) was obtained from by Bachem (St Helen’s, UK).
Rodent models
Male Wistar rats
Male Wistar rats (specific pathogen free; Charles River, Margate, UK) weighing between
250-300g were maintained in individual cages under controlled temperature (21-23 °C), light (12:12 light-dark cycle on at 0700 h), with ad libitum access to food (RMI diet, SDS
UK Ltd) and water. All animal procedures were conducted under the British Home Office
Animals (Scientific Procedures) Act 1986 (Project Licence 70/5516).
48 Intracerebroventricular (ICV) cannulation and injections
Male Wistar rats were anaesthetised with an intraperitoneal (IP) injection of a mixture of xylazine (12mg/kg; Rompun, Bayer UK Ltd, Bury St Edmunds, UK) and ketamine
(60mg/kg; ketalar, Parke-Davis, Pontypool, UK). Prophylactic antibiotics, flucloxacillin
(37.5 mg/kg) and amoxicillin (37.5 mg/kg) were administered prior to surgery.
The rat was placed in a stereotaxic frame (David Kopf Instruments, supplied by Clark
Electromedical Instruments, Reading, UK), with the incisor bar positioned 3mm below the interaural line. The surgical area was cleaned with 10% (weight/volume) bovidine-iodine solution (Betadine, Seton Healthcare, Oldham, UK). A longitudinal incision of approximately 1.5 cm was made in the skin over the vertex of the skull and the periosteum removed from the underlying bone. A permanent 22-gauge stainless steel cannula (Plastics
One Inc., Roanoke, VA, USA.) was stereotactically placed 0.8mm posterior to bregma in the midline and a hole was made in the skull using an electric drill at this position to accommodate the cannula. The cannula was then stereotactically implanted 6.5 mm below the surface of the skull into the third cerebral ventricle. The cannula was secured and the wound sealed with dental cement (Associated Dental Products Ltd., Kemdent Works,
Swindon, U.K.) anchored to three stainless steel jewellers’ screws secured into the cranium.
The co-ordinates of the cannula position were calculated using the atlas of Paxinos and
Watson (Paxinos and Watson, 1998).
After surgery, a plastic topped wire stylet (Plastics One Inc.) was inserted into the cannula to prevent occlusion. The rats were given 0.9% sodium chloride IP (5ml/rat) for circulatory support and given subcutaneous (SC) buprenorphine 45 μg/kg (Schering-Plough Ltd.
Welwyn Garden City, U.K.) for postoperative analgesia. Following a 7 day recovery period the animals were handled and weighed on a daily basis. Only animals with correct
49 cannula placement, as confirmed by a sustained drinking response to ICV angiotensin II
(50ng /rat), were included in the studies. Animals were acclimatised to the injection process by a subsequent saline injection. Peptides were dissolved in 0.9% saline and administered in a 5 l volume via a stainless steel injector projecting 1 mm beyond the tip of the cannula.
The injector was connected by polyethylene tubing (inner diameter, 0.5 mm; outer diameter, 1mm) to a Hamilton syringe (Reno, NV, USA) in a syringe pump set to dispense
5l solution/min.
To determine whether PK-1 affects food intake during the natural rat feeding period of these nocturnal animals, ad-libitum fed rats (n=6-8 per group) were ICV injected with 0.9% saline or PK-1 or PK-2 at 15, 50 or 150 pmol immediately before the onset of dark phase
(19:00). NDP-MSH at 3nmols was injected as a positive control (n=3). Rats were returned to their home cages with a pre-weighed amount of chow and free access to water. The remaining food was reweighed at 1, 2, 4, 8 and 24 h post-injection.
2. Effect of ICV administration of 15, 150 and 1500 pmol PK-1
This study was conducted in the same manner as experiment 1, but the doses of PK-1 and
PK-2 injected were of a greater dosing range (15, 150 and 1500 pmol) in order to determine a more complete dose response of the effect of ICV administered PK-1 on nocturnal food intake in rats.
50 3. Comparison of peripheral administration of IP PK-1 and PK-2 at doses of 20, 60 and 540 nmol/kg between 0-24hrs in male C57BL/6 mice.
This experiment was performed in order to assess the effects of peripheral administration of
PK-1 on food intake in mice.
Materials
PK-1 was purchased from PeproTech, Inc. (New Jersey, USA), Oxyntomodulin was obtained from Peninsula Laboratories (RnDSystems, UK) and NDP-αMSH was purchased from Sigma-Aldrich (Dorset, UK).
C57BL/6 mice
Male C57BL/6 mice (specific pathogen free; Harlan, UK) weighing between 20-30g were maintained in individual cages under controlled temperature (21-23°C) and light (12:12 light-dark cycle on at 0700 h) with ad libitum access to food (RMI diet, SDS UK Ltd) and water.
All animal procedures were conducted under the British Home Office Animals (Scientific
Procedures) Act 1986 (Project Licence 70/5516 and 70/6402). All animals were randomised by weight in to treatment groups.
51 IP injections
Animals were handled daily and acclimatised to the injection procedure by two saline IP injections administered during the week before the study. On the day of the study, peptides were dissolved in 10% water and 90% saline (0.9%) immediately prior to injection. The injection volume was 0.1 ml per mouse.
I investigated the dosing range and time-frame of action on food intake in male mice at doses of 20, 60 and 540 nmol/kg over the first 24 hours following IP injection and compared this with equimolar doses of PK-2.
4. Effect of IP PK-1 7 nmol/kg for up to 1 hour in mice (n=6 per group).
As the lowest dose of PK1 tested of 20 nmol/kg reduced 1 hour food intake by approximately 50% during the previous experiment, I went on to determine if a smaller dose of PK-1 of 7 nmol/kg could still reduce food intake, but by a smaller amount which might be expected physiologically if PK-1 is an endogenous anorectic gut hormone. This would allow me to determine plasma PK-1 levels by ELISA following PK-1 administration to assess the circulating levels of PK-1 required for its anorectic effects.
To determine whether 7 nmol/kg of PK-1 effects food intake during the natural feeding period, ad-libitum fed mice (n = 6 per group) were injected IP immediately before the onset of the dark-phase (19:00) with either 0.9% saline or PK-1 at 7nmol/kg. Mice were returned to their home cages with a pre-weighed amount of chow and free access to water. The remaining food was reweighed at 1 hr post-injection as in experiment 3.
52 5. Determination of the effect of fasting on expression of PK-1 in the stomach of Fed and Fasted mice
Whilst PK-2 has been shown to be localised to the central nervous system (CNS), PK-1 is predominantly expressed peripherally including within the gut. It is therefore reasonable to hypothesise that PK-2 may influence appetite in response to circadian rhythms centrally, whilst PK-1 is a gut hormone regulating appetite in response to food intake.
If PK-1 was a physiological gut hormone regulating appetite in response to food intake, one might expect that expression of PK-1 in the stomach would be low during fasting, but would be increased in response to feeding. Therefore I investigated changes in PK-1 expression with feeding in male C57BL/6 mice.
Male C57BL/6 mice were either fed ad libitum (n=8) or fasted for 24 hours (n=12). Mice were killed by decapitation and the brains removed. The stomach was dissected out, snap frozen in liquid nitrogen and stored at -70°C. Total gut PK-1 mRNA expression determined by real-time quantitative PCR as described below.
53 Total RNA extraction
Materials
Lysis/Binding Solution
64% Ethanol
100% Ethanol
Wash Solution-1
Wash Solution-2/3
Elution Solution (0.1mM EDTA)
10x DNase-1 buffer
DNase-1 (RNase-free)
DNase Inactivation Reagent
5M Ammonium Acetate
Linear Acrylamide (5mg/ml)
Glass Distilled Water (GDW)
Method
Total RNA was extracted using an RNAqueous®-4PCR kit (Applied Biosystems,
California, USA) according to the manufacturer’s protocol. Stomachs were homogenised in lysis/binding solution (750µl of lysis/binding solution per stomach). An equal volume of
64% ethanol was added and gently mixed. The mixture was then placed in a filter cartridge and passed through the cartridge by centrifugation at 20,800 x g for one minute. The flow- through was disposed of and 700µl of wash solution-1 was then added to the filter
54 cartridge. The wash solution was passed through the filter cartridge as described previously and the flow-through again discarded. This step was repeated a further two times using wash solution-2/3 at a volume of 500µl each time. The filter was then placed in a fresh collection tube and 50µl of elution solution, preheated to 70-80ºC, was added to the filter cartridge. The solution was passed through the cartridge into the collection tube by centrifugation at 20,800 x g for 30 seconds. 5µl 10x DNase 1 buffer and 1.1µl DNase 1 was then added to the collected solution and the mixture incubated at 37ºC for 25 minutes.
Following this, 0.1 volumes of DNase inactivation agent was added, gently mixed and incubated for 1-2 minutes at room temperature. The mixture was then centrifuged for 1 minute at 20,800 x g and the supernatant collected. The RNA solution was concentrated by adding 0.1 volumes of 5M ammonium acetate, 0.01 volumes of linear acrylamide and 2.5 volumes 100% ethanol and incubating at -20ºC for at least one hour. The mixture was then centrifuged at 20,800 x g for 15 minutes and the pellet resuspended in 20µl GDW (glass distilled water). The RNA concentration was determined spectrophotometrically. The RNA was diluted 1:100 in GDW and placed into a quartz cuvette. The absorbance was measured at 280nm (UV-160 spectrophotometer, Shimadzu, Kyoto, Japan). The total amount of
RNA recovered was calculated using the following formula:
Concentration of RNA (µg/ml) = A260 x dilution factor x 40
The RNA was then diluted in GDW accordingly. RNA used for normalisation to 18S RNA was diluted to a concentration of 0.1µg/µl, whereas all other RNA samples were diluted to
0.05µg/µl.
55 Reverse Transcription (RT)
Reverse transcription of the extracted RNA was carried out in order to produce cDNA.
Materials
Mouse stomach RNA (at a concentration of either 0.1 or 0.5µg/ml)
2x RT Master Mix (per 20µl reaction):
2µl 10x RT Buffer
0.8µl 25x dNTP mix (100mM)
2µl 10x RT Random Primers
1µl MultiScribe™ Reverse Transcriptase
4.2µl GDW
GDW
Method
cDNA was synthesised from RNA using a High Capacity cDNA Reverse Transcription Kit
(Applied Biosystems, California, USA) according to the manufacturer’s protocol. 10µl of
2x RT master mix was pipetted to each well of a 96-well reaction well plate. 10µl of
0.05µg/µl RNA sample was added to each well in triplicate and pipetted up and down twice to ensure thorough mixing. RNA used for 18S RNA normalisation (at 0.1µg/µl) was first diluted 1:10 in GDW before adding to the reaction well plate. The 96-well plate was sealed with aluminium film and loaded into a thermal cycler under the following conditions: 1)
56 25ºC for 10 minutes, 2) 37ºC for 120 minutes, 3) 85ºC for 5 seconds, and 4) 4ºC until transferred to -20ºC for storage.
Real-time quantitative PCR
Materials
TaqMan Universal PCR Master Mix (Applied Biosystems)
20x TaqMan Gene Expression Assay (Applied Biosystems)
cDNA synthesised from mice stomach RNA
Method
10µl of TaqMan Universal PCR Master Mix, 1µl TaqMan Gene Expression Assay (18S
RNA primer assay ID: 4310893E-0608033, PK-1 primer assay ID-4351372) and 7µl GDW was added to each well in a 384-well reaction plate. 2µl of cDNA (approximately 50ng) was then added in triplicate to each well and pipetted up and down twice to ensure thorough mixing. The 384-well reaction well plate covered with optical adhesive film and centrifuged briefly to minimise air bubbles. The reaction well plate was then loaded into a quantitative PCR machine (7900HT quantitative PCR machine, Applied Biosystems,
California, USA) under the following conditions: 1) 50ºC for 2 minutes, 2) 95ºC for 10 minutes, 3) 95ºC for 15 seconds, 4) 60ºC for 1 minute (steps 3) and 4) were repeated for 40
-C cycles). Gene expression was quantified relative to 18S RNA and analysed using the 2 T method (Livak and Schmittgen, 2001).
57 6. Human plasma PK1 levels fed fasted
In order to investigate whether PK-1 is a physiological gut hormone in humans, I investigated whether plasma levels of PK-1 would change with feeding in humans.
If PK-1 was a physiological gut hormone then it would be expected that plasma PK-1 levels would be low during fasting and rise following food intake signalling satiety.
Therefore to investigate whether plasma levels of PK-1 are altered by feeding, 4 healthy men and 4 healthy women, aged 19-34 years old, were recruited and asked to fast from
10pm (only having sips of water thereafter). They attended the clinical research unit at
10am the following morning and were intravenously cannulated and fasting blood was sampled. At each time point 6mls of blood was taken into lithium heparin tubes each containing 200 µls of trasylol (see Table 3). The samples were immediately spun at 6,400 x g for 4 minutes. Plasma was separated into 3 eppendorfs and immediately frozen at -20○C until analysis. Two packets of macaroni cheese were prepared and participants were allowed to eat and drink until satiated from time 0 (see Table 4 for details of food used for ingestion). All participants ate at least one packet of macaroni cheese and some ate 2 packets. Blood was sampled at 15 minutely intervals until 2 hours following commencing eating at time 0 (see Table 3).
Plasma PK-1 was measured using a commercially available ELISA kit (ABNOVA) as described below.
58 Table 3: Study Protocol for measurement of plasma PK-1 levels in humans.
Participants were intravenously cannulated at Time -30. Participants were asked to start eating from Time 0. Plasma was measured every 15 minutes for 2 hours.
Blood Time point (min) Time (6ml) Well? Baseline Sampling -30 Start Eating Food 0 15 30 45 60 75 90 105 120
59 Table 4: Details of contents of Macaroni Cheese packets used to induce satiety in humans (Sainsbury’s Ltd, UK)
A mature Cheddar cheese sauce on a bed of macaroni pasta topped with grated mature
Cheddar cheese 400g pack.
Typical values per 100g per pack
Energy 201 kcal 780 kcal
Energy 844 kj 3266 kj
Protein 8.50 g 32.90 g
Carbohydrate 20.57 g 79.61 g
Sugars 1.13 g 4.37 g
Fat 9.13 g 35.33 g
Saturates 5.03 g 19.47 g
Fibre 1.50 g 5.81 g
Sodium 0.24 g 0.929 g
Ingredients:
Water, milk, mature Cheddar cheese (20%), durum wheat semolina, pasteurised free range egg, single cream, wheat flour, rapeseed oil, cornflour, mustard flour, salt, sugar, white pepper, acidity regulator citric acid, turmeric
60 Methods: Plasma PK-1 measurement by ELISA kit (ABNOVA)
Reconstitution of the human PK-1 standard:
One tube of 10,000 pg of standard was used to generate standards by dilution.
• 10,000 pg/ml of human PK-1 standard solution: 10,000 pg vial of PK-1 standard was reconstituted using 1 ml of sample diluent buffer. The standard was kept at room temperature for 10 minutes and thoroughly mixed.
• 1,000 pg/ml of human PK-1standard solution: 0.1 ml of the above 10,000 pg/ml PK-1 standard solution was added into 0.9 ml sample diluent buffer and thoroughly mixed.
• 500 pg/ml - 15.6 pg/ml of human PK-1 standard solutions: 6 Eppendorf tubes were labelled with 500 pg/ml, 250 pg/ml, 125 pg/ml, 62.5 pg/ml, 31.2 pg/ml, 15.6 pg/ml, respectively. 0.3 ml of the sample diluent buffer was aliquoted into each tube. 0.3 ml of the above 1000pg/ml PK-1 standard solution was added into 1st tube and thoroughly mixed.
0.3 ml from 1st tube was transferred to 2nd tube and thoroughly mixed. 0.3 ml from the
2nd tube was transferred to the 3rd tube and mixed, and so on.
0.1 ml per well of the 1000 pg/ml, 500 pg/ml, 250 pg/ml, 125 pg/ml, 62.5 pg/ml, 31.2 pg/ml, 15.6 pg/ml human PK-1 standard solutions were aliquoted into the pre-coated 96- well plate. 0.1ml of each sample of human plasma was added to each empty well.
The plate was sealed with a cover and incubated at 37°C for 90 minutes.
The cover was then removed, plate content discarded, and blotted onto paper towels. The wells were not allowed to completely dry at any time.
61 Then 0.1 ml of biotinylated anti-human PK-1 antibody working solution was added into each well using a multichannel pipette and the plate was incubated in the dark at 37°C for
60 minutes.
The plate was washed 3 times using an automated washer: The plate was blotted onto paper towels or other absorbent material.
Preparation of 0.01 M PBS wash buffer:
8.5 g sodium chloride, 1.4 g Na2HPO4 and 0.27 g NaH2PO4.2H20 were added to 1000 ml distilled water and pH adjusted to 7.2-7.6.
0.1 ml of prepared ABC working solution (secondary antibody) was added into each well and the plate incubated at 37°C for 30 minutes.
The plate was then washed 5 times with 0.01 M PBS, with a soak step of 90 seconds.
90 μl of prepared TMB colour developing agent was added into each well using a multichannel pipette and the plate incubated at 37°C in dark for 25 minutes.
Shades of blue can then be seen in the wells with the four most concentrated human PK-1 standard solutions; whereas the other wells show no obvious colour.
0.1 ml of prepared TMB stop solution was then added using multichannel pipette into each well. The colour then changes to yellow immediately.
O.D. absorbance was read at 450nm in a microplate reader within 30 minutes of adding the stop solution.
62 1. Results:
ICV administration of PK-1 administration significantly reduced food intake during the first 24 hrs when compared with saline administration (see Figure 4). Doses of 50 and 150 pmol of PK-1 similarly reduced food intake by 75% during the first hour following administration during the dark phase when compared with the saline group. This reduction in food intake was similar to that seen following PK-2 administration. Interestingly, PK-1 also significantly reduced food intake during the light phase during 8-24 hrs following ICV administration. ICV administration of PK-1 was at least as effective as equimolar doses of
PK-2 at reducing food intake. There was a tendency for the higher doses tested of 50 and
150 pmol of PK-1 and PK-2 to rapidly and immediately reduce food intake during the first hour following administration to a greater degree than the lower 15 pmol dose. The lower
15 pmol dose appears to have a slower onset of action and did not significantly reduce food intake until 2-8 hrs following administration than higher doses tested (see Figure 4).
The reduction in food intake during the first 24 hrs following ICV PK-1 at all three doses tested between 15 and 150 pmols was similar at approximately 60%, therefore I went on to investigate whether higher doses of PK-1 were able to reduce food intake to an even greater degree in experiment 2.
63 Figure 4: Comparison of ICV PK-1 and PK-2 at doses of 15, 50, 150 pmol between 0-
24hrs during dark phase in adult male Wistar rats (n=6-8/group).
NDP-MSH 3nmol was used as a positive control. Groups were compared by one way
ANOVA with post hoc Dunnet's test against Saline Group. *P<0.05,**P<0.01,***P<0.001.
4 3.5
3.0 3 2.5 ** ** ** ** 2.0 2 1.5
1.0 1 ***
0-1hr Food Intake (g) 1-2hr Food Intake (g) 0.5 ** 0 0.0
Saline Saline PK-1 15 PK-1 50 PK-2 15 PK-2 50 PK-1 15 PK-1 50 PK-2 15 PK-2 50 PK-1 150 PK-2 150 PK-1 150 PK-2 150
NDP-MSH 3nmol NDP-MSH 3nmol pmol pmol
8 10
8 6
6 4 4 * 2 2 2-4hr Food Intake (g) 4-8hr Food Intake (g) ** 0 0
Saline Saline PK-1 15 PK-1 50 PK-2 15 PK-2 50 PK-1 15 PK-1 50 PK-2 15 PK-2 50 PK-1 150 PK-2 150 PK-1 150 PK-2 150
NDP-MSH 3nmol NDP-MSH 3nmol pmol pmol
13 40 12 11 ** 10 * 30 * 9 * ** ** ** 8 ** ** 7 20 6 ** 5 4 3 10 2 8-24hr Food Intake (g) 1 0-24hr Food Intake (g) 0 0
Saline Saline PK-1 15 PK-1 50 PK-2 15 PK-2 50 PK-1 15 PK-1 50 PK-2 15 PK-2 50 PK-1 150 PK-2 150 PK-1 150 PK-2 150
NDP-MSH 3nmol NDP-MSH 3nmol pmol pmol
64 2. Results:
ICV PK-1 or PK-2 administration significantly reduced food intake during the first hour following administration down to 22% following 1500 pmol, which was greater than the reduction seen following 150 pmol (see Figure 5).
However, there were not significant reductions in food intake beyond this time frame.
This may partly have been due to the saline group not having eaten as much food as they would normally do (as in experiment 1), making some of the reductions less significant in comparison to saline.
Results from study 1 and 2 suggested that PK-1 appears to have similar efficacy to PK-2 when administered centrally. If PK-1 were to be administered as a therapeutic to target obesity, then it would be administered peripherally. Hence it was important to study the peripheral effects of PK-1 administration on food intake. As the cost of PK-1 is very high, and larger doses are required for peripheral administration, peripheral experiments were carried out in mice rather than rats.
65 Figure 5: Comparison of ICV PK-1 and PK-2 at doses of 15, 150 and 1500 pmol between 0-24hrs during dark phase in adult male Wistar rats (n=7-10 per group).
Oxyntomodulin (OXM) 3 nmol was used as a positive control (n=4). Groups were compared by one way ANOVA with post hoc Dunnet’s test with Saline Group.
* P<0.05, **P<0.01, ***P<0.001.
5.5 3.0 5.0 4.5 2.5 4.0 3.5 2.0 3.0 * 1.5 2.5 *** 2.0 1.0 1.5 *** *** 1.0
1-2hr Food Intake (g) 0.5 0-1hr Food Intake (g) 0.5 0.0 0.0
Saline Saline PK-1 15 PK-2 15 PK-1 15 PK-2 15 PK-1 150 PK-2 150 PK-1 150 PK-2 150 PK-1 1500 PK-2 1500 PK-1 1500 PK-2 1500 OXM 3nmol OXM 3nmol pmol pmol
6 6
4 4
2 2 2-4hr Food Intake (g) 4-8hr Food Intake (g)
0 0
Saline Saline PK-1 15 PK-2 15 PK-1 15 PK-2 15 PK-1 150 PK-2 150 PK-1 150 PK-2 150 PK-1 1500 PK-2 1500 PK-1 1500 PK-2 1500 OXM 3nmol OXM 3nmol pmol pmol
22 40 20 18 16 30 14 12 20 10 8 6 10 4 8-24hr Food Intake (g) 2 0-24hr Food Intake (g) 0 0
Saline Saline PK-1 15 PK-2 15 PK-1 15 PK-2 15 PK-1 150 PK-2 150 PK-1 150 PK-2 150 PK-1 1500 PK-2 1500 PK-1 1500 PK-2 1500 OXM 3nmol OXM 3nmol pmol pmol
66 3. Results:
Doses of 20 and 60 nmol/kg of both PK-1 and PK-2 similarly reduced food intake over the first hour by approximately half when compared with saline (see Figure 6). Impressively a dose of 540 nmol/kg of either PK-1 or PK-2 reduced food intake by over 90% during the first hour. However the effect on food intake had worn off by 4 hrs post administration, with no reduction in food intake observed between 4 and 24 hrs post administration. Hence only the highest dose of PK-1 resulted in a significant reduction over the first 24hrs following administration.
This was confirmed by observing the food intake over time, such that food intake was similar to saline after 8 hours following administration (see Figure 7). Only the highest dose of 540 nmol/kg of PK-1 or PK-2 resulted in a fall in body weight at 24 hrs (-0.66 g following the highest dose of PK-2 at 24 hours) (see Figure 8).
Importantly this demonstrates that both PK-1 and PK-2 are able to reduce food intake in mice following peripheral administration. Furthermore the highest doses of both peptides resulted in reduction in body weight at 24 hrs following administration. Given the short duration of action it would seem likely that more frequent administration may have more profound effects on food intake over a prolonged period.
67 Figure 6: Comparison of IP PK-1 and PK-2 at doses of 20-540 nmol/kg between 0-
24hrs in male C57BL/6 mice (n=6 per dosing group).
Mean ±SEM is shown for food intake and compared by one way ANOVA with post hoc
Dunnet’s test *P<0.05, **P<0.01, ***P<0.001. Separate graphs are shown for food intake between 0-1 hr, 1-2hr, 2-4hr, 4-8hr, 8-24hr and 0-24hr.
0.40
0.25 0.35
0.30 0.20 0.25 0.15 ** * ** 0.20
0.10 0.15 *** *** ** 0.10 0.05 1-2hr Food weight (g) 0-1 hr Food weight (g) *** 0.05 *** 0.00 0.00
Saline PK-1 20 PK-2 20 PK-1 60 PK-2 60 Saline PK-1 540 PK-2 540 PK-120 PK-220 PK-160 PK-260
PK-1540 PK-2540 nmol/kg nmol/kg
0.9 1.8 0.8 * 1.6 0.7 * ** 1.4 0.6 1.2 0.5 1.0 0.4 *** 0.8 0.3 0.6 0.2 2-4hr Food weight (g) 0.4 4-8hr Food weight (g) 0.1 0.2 0.0 0.0
Saline PK-1 20 PK-2 20 PK-1 60 PK-2 60 Saline PK-1 540 PK-2 540
PK-120 PK-220 PK-160 PK-260 PK-1540
nmol/kg 540 PK-2 nmol/kg 2.2 5 2.0 * *** 1.8 ** 4 1.6 1.4 * 3 1.2 1.0 0.8 2 0.6 8-24hr Food weight (g)
0.4 0-24hr Food weight (g) 1 0.2 0.0 0 68 Saline Saline PK-120 PK-220 PK-160 PK-260 PK-1 20 PK-2 20 PK-1 60 PK-2 60 PK-1540 PK-2540 PK-1 540 PK-2 540 nmol/kg nmol/kg Figure 7: Comparison of IP PK-1 and PK-2 at doses of 20-540 nmol/kg between 0-24hrs in male C57BL/6 mice (n=6 per dosing group).
Mean ±SEM of cumulative food intake expressed as a percentage of that seen following control saline administration is shown and compared by Two way ANOVA with post hoc bonferrroni correction. *P<0.05, **P<0.01, ***P<0.001.
100
90
80
70
60 *** 50
40 *** saline PK-1 540 30 PK-1 60 PK-1 20 20 PK-2 540 PK-2 60 10 *** PK-2 20 Cumulative food intake % saline Cumulativefood % intake 0 *** 0 *** 2 4 6 8 10 12 14 16 18 20 22 24
69 Figure 8: Change in Body weight in grams over 24 hours after doses of 20, 60 and
540 nmol/kg of PK-1 or PK-2 in male C57BL/6 mice.
Mean ±SEM of change in body weight from baseline until 24hrs following 20, 60 and 540 nmol/kg of PK-1 or PK-2 is shown.
0.50
0.35
0.20
0.05
24 -0.10 -0.25 Saline -0.40 PK-1 540nmol/kg PK-1 60 nmol/kg PK-1 20 nmol/kg -0.55 Time (hours) PK-2 540nmol/kg
Bodyweight Bodyweight change (g) PK-2 60nmol/kg -0.70 PK-2 20 nmol/kg -0.85
70 4. Results:
IP injection of PK-1 reduced food intake by just over 50% over the first hour following administration (see Figure 9). I measured the serum PK-1 levels by a commercial ELISA kit (Peprotech) during this experiment. Serum PK-1 levels were highest at 20 minutes following injection at 40 pg/ml, before falling to 10 pg/ml at 60 minutes following administration (see Figure 10).
71 Figure 9: Effect of IP PK1 7 nmol/kg for up to 1 hour in mice (n=6 per group) on food intake over the first hour in Male C57BL/6 mice.
No significant difference was seen following comparison by unpaired Students’ t test although mean food intake was reduced by more than 50%.
0.24
0.20
0.16
0.12
0.08
0-1hr Food Intake (g) 0.04
0.00 Saline PK-1 7nmol/kg
72 Figure 10: Serum PK-1 levels during the first hour following injection with 7 nmol/kg of PK-1 given IP to male C57BL/6 mice.
PK-1 was measured by Peprotech ELISA kit in pg/ml (n=6 per group). Serum levels of PK-
1 peaks at 20 minutes following administration of exogenous PK-1 in rodents suggesting that PK-1 has a short half-life.
50
40
30
20
10
0 Serum PK-1 (pg/ml) Serum 0 20 40 60 Time (minutes)
73 5. Results:
Mean ±SEM of expression of PK-1 mRNA was higher in fasted mice (0.83 ±0.13) when compared with fed mice (0.58 ±0.07), although this was not statistically significant when compared by Mann Whitney test (P=0.15), this would be consistent with the pattern of expression which would be expected if PK-1 was a physiological gut hormone, being low in fasted mice and increasing with food intake to signal satiety to the appetite centres in the brain (see Figure 11).
It is possible that the results of this experiment would be significant should a larger sample size be employed.
74 Figure 11: Expression of PK-1 in the stomach of male C57BL/6 mice in fasting (n= 12) and in fed state (n=8).
Mean ±SEM of each group is shown. Groups were not significantly different when compared by Mann Whitney test (P=0.15).
1.0 P value 0.1535 RQ 0.8 (expression of 0.6 PK-1 mRNA in stomach) 0.4 0.2 0.0 Fed Fasted
75 6. Results:
Plasma PK-1 levels varied widely between different individuals. The standard curves for each plate during the assay were reasonably straight suggesting that poor assay technique is unlikely to observation (see Figure 12).
One female participant in the study had a plasma PK-1 level of approximately 400 pg/ml and a further male paritcipant had a PK-1 level between 100 and 200 pg/ml. However the majority of patients had levels below 100 pg/ml (see Figure 13).
There was no significant difference in plasma PK-1 levels following feeding and certainly no significant rise in PK-1 levels following feeding (see Figure 14).
Some participants had much higher baseline levels of PK-1 than other participants, suggesting the influence of other unidentified dominant factors beyond satiety on PK-1 levels. Another possibility for high levels of PK-1 seen in some individuals is the presence of other interfering substances interacting with the performance of the assay. Although the highest level was seen in a female there was no clear sex differential between participants.
76 Figure 12: Standard Curves used for the interpolation of plasma PK-1 levels.
Standard concentrations of 1000 pg/ml, 500 pg/ml, 250 pg/ml, 125 pg/ml, 62.5 pg/ml, 31.2 pg/ml, 15.6 pg/ml of human PK-1 standard solutions were aliquoted into the pre-coated 96- well plates and OD (optical density) Absorbance was read at 450 nm in a microplate reader.
3 plate 1 standard 3 plate 2 standard 3 plate 3 standard
2 2 2
1 1 1 Absorbance Absorbance Absorbance 0 0 0 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 standard conc (pg/ml) standard conc (pg/ml) standard conc (pg/ml)
3 plate 4 standard 3 plate 5 standard 3 plate 6 standard
2 2 2
1 1 1 Absorbance Absorbance Absorbance 0 0 0 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 standard conc (pg/ml) standard conc (pg/ml) standard conc (pg/ml)
plate 7 standard 3 plate 8 standard 3 plate 9 standard 3
2 2 2
1 1 1 Absorbance Absorbance Absorbance 0 0 0 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 standard conc (pg/ml) standard conc (pg/ml) standard conc (pg/ml)
3 plate 10 standard 3 plate 11 standard 3 plate 12 standard
2 2 2
1 1 1 Absorbance Absorbance Absorbance 0 0 0 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 standard conc (pg/ml) standard conc (pg/ml) standard conc (pg/ml)
77 Figure 13: Circulating plasma PK1 levels in humans whilst fasted and following feeding.
4 female and 4 male participants were fasted for 12 hours and then were asked to start eating at T=0. Plasma Pk-1 levels were then assessed for 120 minutes. Individual plasma prokineticin-1 (PK-1) levels as measured by ELISA (Abnova) for each participant is shown.
500 400 300 200 100 Plasma PK1 (pg/ml) PK1 Plasma
-30 0 30 60 90 120 Time (minutes) Start of Feeding
78 Figure 14: Change in circulating plasma PK-1 levels in humans whilst fasted and following feeding.
4 female and 4 male participants were fasted for 12 hours and then were asked to start eating at T=0. Plasma prokineticin-1 (PK-1) levels were then assessed for 120 minutes.
Mean ±SEM of change in plasma PK-1 levels from fasting levels as measured by ELISA
(Abnova) is shown. There was no significant difference in PK-1 levels as analysed by two way ANOVA.
Start of Feeding 40 20 0
PK1 -20 20 40 60 80 100 120 -40 Time (minutes)
Change in Plasma in Change -60 from baseline(pg/ml) from
79 Discussion:
In this chapter I have investigated the role of PK-1 as a potential novel gut hormone and I have compared its anorectic effects to those observed with PK-2.
Central administration of PK-1 administration significantly reduced food intake during the first 24 hrs when compared with saline administration. Doses of 50 and 150 pmol of PK-1 similarly reduced food intake by 75% during the first hour following administration during the dark phase when compared with the saline group. This reduction in food intake was similar to that seen following PK-2 administration.
Central administration of PK-1 was at least as effective as equimolar doses of PK-2 at reducing food intake. There was a tendency for the higher doses tested of 50 and 150 pmol of PK-1 and PK-2 to reduce food intake during the first hour following administration to a greater degree than the lower 15 pmol dose, whilst conversely, there was a trend for the lower 15 pmol dose to have a slower onset of action reducing food intake significantly between 2-8 hrs following administration.
The reduction in food intake during the first 24 hrs following central PK-1 at all three doses tested between 15 and 150 pmols was similar at approximately 60%, therefore I went on to investigate whether higher doses of PK-1 was able to reduce food intake to a greater degree.
Central PK-1 or PK-2 administration significantly reduced food intake during the first hour following administration down to 22% following 1500 pmol, which was greater than that seen following 150 pmol. However there were not significant reductions in food intake beyond this time frame suggesting a short duration of action.
80 PK-1 had a similar efficacy to PK-2 when administered centrally. However, if PK-1 were to be administered as a therapeutic to target obesity, then it would be important to study the peripheral effects of PK-1 administration. As the cost of PK-1 is very high, and larger doses are required for peripheral administration, peripheral experiments were carried out in mice.
Peripheral IP injection of PK-1 reduced food intake by just over half during the first hour following administration. I also measured the serum PK-1 levels by a commercial ELISA kit (Peprotech) in mice. Serum PK-1 levels were highest at 20 minutes following injection at 40 pg/ml, before falling to 10 pg/ml at 60 minutes following administration suggesting a short half life of approximately half an hour. I went on to investigate the dosing range and time frame of action on food intake in male mice at doses of 20, 60 and 540 nmol/kg over the first 24 hours following IP injection and compared this with equimolar doses of PK-2.
Doses of 20 and 60 nmol/kg of both PK-1 and PK-2 similarly reduced food intake over the first hour by approximately half when compared with saline. Impressively a dose of 540 nmol/kg of either PK-1 or PK-2 reduced food intake by over 90% during the first hour.
However the effect on food intake had worn off by 4 hrs post administration, with no reduction in food intake observed between 4 and 24 hrs post administration. Hence only the highest dose of PK-1 resulted in a significant reduction over the first 24hrs following administration.
Importantly this shows that both PK-1 and PK-2 are able to reduce food intake in mice following peripheral administration. Furthermore, the highest doses of these peptides resulted in a reduction in body weight at 24 hrs following administration highlighting its potential as a target for the development of anti-obesity therapeutics. Given the short duration of action it would seem reasonable that more frequent administration may have more profound effects on body weight over a longer period and similar experiments with
81 PK-2 have suggested that tachyphylaxis to the prokineticins with repeated administration does not occur.
PK-1 expression was also lower in the stomachs of fasted mice although this finding did not achieve statistical significance. This would be consistent with the pattern of expression which would be expected if PK-1 was a physiological gut hormone, being low in fasted mice and increasing with food intake to signal satiety to the appetite centres in the brain.
If PK-1 was a physiological gut hormone then it would be expected that plasma PK-1 levels would be low during fasting and rise following food intake signalling satiety. However I was not able to demonstrate a clear effect on circulating PK-1 levels in humans following feeding. Possible reasons for not seeing a rise in plasma PK-1 levels may be due to difficulties in measuring circulating PK-1 with current commercially available ELISA kits, as many participants had levels which were towards the lower end of the sensitivity of the assay. However even participants with higher endogenous levels did not show a clear effect on PK-1 following feeding (see Figure 13). The most likely possibility is that other unidentified factors may influence plasma PK-1 levels to a greater degree than those seen following feeding.
In summary, I have demonstrated that both central and peripheral administration of PK-1 potently reduces food intake and that expression of PK-1 levels in the stomachs of mice change in manner consistent with that of a physiological gut hormone. PK-1 therefore represents a novel target for the development of anti-obesity therapies.
82
Chapter 3
Prokineticin-1 and other
markers of complications of pregnancy
83 Introduction:
The commonest and perhaps the most distressing of pregnancy complications is miscarriage. Miscarriage occurs with a cumulative risk of miscarriage of 20% by 20 weeks of gestation (Ammon Avalos et al. 2012). Miscarriage occurs most commonly in early pregnancy with a weekly miscarriage rate of >20 miscarriages per thousand woman-weeks up to 12 weeks of gestation, falling sharply thereafter down to a weekly miscarriage rate of only 1 miscarriage per 1000 woman-weeks between 16-20 weeks (Ammon Avalos et al.
2012). Indeed most early miscarriages may not even be clinically apparent, with many pregnancies being lost within the first week after conception prior to implantation being established and many more during the following week prior to the start of the next menstrual cycle. Hence 6 in 7 miscarriages are clinically silent, occurring within the first 2 weeks of conception and may not result in delay of the next menstrual cycle (see Figure 15)
(Macklon et al. 2002). As patients are not scheduled for any routine antenatal appointments until their antenatal booking visit, which usually occurs after 8 weeks of pregnancy, it can be difficult to screen for miscarriage during the time when it is most prevalent (prior to 8 weeks of pregnancy). After 12 weeks of pregnancy, approximately 4% of pregnancies will still result in miscarriage (Ammon Avalos et al. 2012).
84 Figure 15: Outcome of Conceptions
Only 30% of conceptions result in live births and 60% of pregnancy loss is ‘subclinical’, occurring prior to the next period being delayed (within the first 2 weeks following conception). In fact only 1 in 7 miscarriages are clinically apparent occurring after 2 weeks post conception (adapted from Macklon et al. 2002).
85 Recurrent Pregnancy Loss (RPL)
Recurrent pregnancy loss (RPL), where miscarriage occurs in 3 or more consecutive pregnancies, occurs in ~1% of fertile couples trying to conceive and is termed ‘early’ if this occurs before 12 weeks of gestation. Chromosomal abnormality in the miscarried embryo is present in up to 70% of early pregnancy losses in the first trimester, but in only 20% when the pregnancy loss is between 13 and 20 weeks of gestation (Tang et al. 2010). Abnormal karyotype is an important positive prognostic indicator for women with recurrent pregnancy loss, with a more than 75% chance of a successful outcome with a subsequent pregnancy. Despite a wide range of investigations no cause is found in 50% of cases (see table 5). However the prognosis following RPL is good with over 75% subsequently achieving a live birth (Tang et al. 2010).
86 Table 5: Cause of early Recurrent Pregnancy Loss and Risk factors for
Miscarriage.
Causes adapted from (Tang et al. 2010) and risk factors for miscarriage adapted
from (Campbell et al. 2011).
Causes of early Recurrent Pregnancy Loss Risk Factors for Miscarriage
Parental and foetal Chromosomal abnormalities Increased Maternal Age
Structural / congenital Uterine abnormalities ‘Risky’ Alcohol use
Autoimmune disease E.g. anti-phospholipid syndrome Sexually transmitted infection
Increased Maternal Immunity
E.g. increased Natural Killer cells
Thrombophilia E.g. Factor V Leiden
Endocrinological disorders
E.g. Polycystic ovarian syndrome, untreated Diabetes mellitus
87 The placenta
Two thirds of miscarriages are attributed to defective placentation (Kaitu'u-Lino et al.
2012). The placenta is the organ which allows the developing foetus to safely interact with the maternal blood system for gaseous exchange, nutrient provision and waste disposal. The word ‘placenta’ originates from the Latin word for ‘cake’ which is derived from the greek for a flat round surface (plakóeis/plakoús – πλακόεις, πλακούς) (Lidell et al. 1940).
After fertilisation, the zygote (fertilised ovum) divides. Once it reaches 32 cells in size, it is termed a ‘morula’ as it resembles a mulberry. The morula consists of early foetal cells called blastomeres in a solid ball contained within the zona pellucida (outer glycoprotein membrane of an ovum). Within a few days of formation, cells on the outer aspect of the morula bind tightly together in a process known as ‘compaction’. These cells secrete a viscous liquid such that a central cavity (known as a ‘blastocoele’) is formed in a process known as ‘hatching’ and the entire structure is termed a ‘blastocyst’. A blastocyst comprises of 70-100 cells and the hatching process occurs at day 5 after fertilisation. The outer cell layer of the blastomere forms the trophoblasts which develops into the placenta and other extra-embryonic tissues, whereas cells from the inner part of the blastomere form the ‘inner cell mass’ or ‘embryoblast’ which form the ‘embryo proper’.
The placenta begins to form when the blastocyst implants in the maternal endometrium at day 7 post fertilisation. The outer part of the trophoblast layer forms the syncitiotrophoblasts (or syncytium), whilst the inner trophoblast layer becomes cytotrophoblasts. The syncytium forms a continuous layer covering the whole of the placenta and contributes to the barrier function between the foetus and mother. In preparation for implantation the maternal endometrium undergoes a process called
‘decidualisation’, during which maternal spiral arteries become less convoluted and
88 increase in diameter such that maternal blood flow to the placenta is maximised. Although the foetal chorion is bathed in maternal blood to allow gaseous and nutrient exchange to occur, no fluid is exchanged. Development of the placental blood flow is complete by 12-
13 weeks of gestation. The placenta continues to grow throughout pregnancy and typically weighs 400-1000 g in the third trimester (Pardi et al. 2002).
The Placenta as an endocrine organ:
As a pregnancy advances, the relative number of trophoblasts increases and the secretory function of the placenta begins to supersede that of foeto-maternal exchange. As gestation progresses towards term, the number of cytotrophoblasts is reduced and the syncitiotrophoblast layer at the foeto-maternal interface becomes very thin. During implantation the embryo actively secretes human chorionic gonadotrophin hCG (hCG), which is readily detectable in the maternal blood from 8 days after ovulation. hCG has an important role in maintaining secretion of progesterone from the corpus luteum, which in turn sustains a decidualised endometrium. The decidua is the endometrium of pregnancy and secretes a number of steroids and peptides including cortisol, prolactin and insulin-like growth factor binding protein-1 which help protect the foetus from the maternal immune system. The placenta is the main source of progesterone during pregnancy, which in addition to an important role in decidualisation of the placenta, also inhibits smooth muscle contractility, reduces prostaglandin production and contributes to the limitation of maternal immune responses (Feldt-Rasmussen et al. 2011) (see table 6).
89 Table 6: Substances produced by the Placenta during Pregnancy.
During pregnancy a number of substances and hormones are produced by the placenta and some of these are found at greatly increased circulating levels than those seen outside of pregnancy (adapted from Feldt-Rasmussen et al. 2011 and Kaitu'u-Lino et al. 2012).
Substances found at increased circulating levels in pregnancy
Human Chorionic Gonadotrophin (HCG)
Oestrogen
Progesterone
Sex Hormone Binding Globulin (SHBG)
Corticotrophic releasing hormone (CRH)
Adrenocorticotrophic hormone (ACTH)
Growth Hormone (GH)
Parathyroid Hormone related peptide (PTHrp)
Prolactin
Human Placental Lactogen (HPL)
Vascular Endothelial Growth Factor A (VEGF A)
Other Angiogenic Factors : Placental Growth Factor (PlGF) and Angiopoietins
90 Prokineticins during Pregnancy:
In 2001, Li et al. isolated and two peptides which potently stimulate rodent gastrointestinal tract smooth muscle and named them prokineticins (Li et al. 2001). In the same year,
LeCouter et al. identified a new peptide, highly expressed in human testis, adrenal gland, ovary, and placenta which appeared to be a specific growth factor for endocrine gland- derived endothelial cells (EC) (LeCouter et al. 2001). This protein was therefore termed endocrine gland derived vascular endothelial growth factor (EG-VEGF) to reflect its similarities to vascular endothelial growth factor (VEGF). However, EG-VEGF shared no sequence homology to VEGF and was instead found to be identical to PK-1 (LeCouter et al. 2001). For the remainder of this thesis I will use the term PK-1 (rather than EG-VEGF) for clarity.
In endothelial cells (ECs) isolated from steroidogenic tissues such as the placenta, PK-1 was shown to promote proliferation, survival, and chemotaxis. PK-1, but not Bv8/PK2, was found to be expressed in human placenta. PK-1 and its receptor PROKR1 are highly expressed in human placenta and are present in the syncytiotrophoblasts (ST), cytotrophoblasts (CT), fetal endothelium, and Hofbauer cells (Ho) (oval eosinophilic histiocytes found in the placenta) (see Figure 16) (Brouillet et al. 2012).
91 Figure 16: A diagragm of chorionic villi showing localisation for PK-1 (EG-VEGF) and its receptors, PROKR1 and PROKR2.
EG-VEGF (PK-1) is mainly expressed by the syncytiotrophoblast (ST). PROKR1 is expressed by the cytotrophoblast (CT) and PROKR2 by the syncytiotrophoblast (ST), extravillous trophoblast (EVT) and endothelial cells. Abbreviations: DC, decidual cells;
FBV, fetal blood vessel; Ho, Hofbauer cells; MBV, maternal blood vessel; Plug, aggregates of proliferative trophoblasts (Brouillet et al. 2012).
92 PK-1 is mainly localised to the syncytiotrophoblast layer of the placenta with the highest expression identified during 8-10th week of gestation (Hoffmann et al. 2006). Between 7–8 weeks of gestation PK-1 expression is restricted to the syncytiotrophoblast layer, the endocrine unit of the placental villi and the Hofbauer cells. However, at more advanced gestational stages (9-12th week of gestation), PK-1 expression is strongest in the syncytiotrophoblast and Hofbauer cells, with only mild expression in the cytotrophoblast layer (Hoffmann et al. 2006). Placental PK-1 expression peaks at 8–11th week of gestation, before gradually decreasing by the end of the first trimester (Hoffmann et al. 2006). This change in expression with gestation was also found to be reflected by serum levels of PK-1 during pregnancy (see Table 7). Serum PK-1 levels are elevated during pregnancy, and are highest during the first trimester which is thought to be the critical period during which placentogenesis is thought to be established (Hoffmann et al. 2009).
93 Table 7: Circulating PK-1 levels during pregnancy
Mean serum PK-1 levels (pg/ml) in non-pregnant and healthy pregnant women throughout gestation, and in pre-eclamptic women during the second and third trimester (n=9/group in non-pregnant, first trimester and second trimester; n=15-20 per group in third trimester pregnancy) (adapted from Brouillet et al. 2012 based on data from Hoffman et al. 2009).
Normal women
Non-pregnant women 35 ± 7
First trimester of pregnancy 225 ± 39
Second trimester of pregnancy 73 ± 11
Third trimester of pregnancy 77 ± 5
Pre-eclamptic women
Second trimester of pregnancy 99 ± 10
Third trimester of pregnancy 120 ± 13
94 The placenta is one of the most densely vascularized organs in humans. During the first trimester, the placenta invades into the maternal decidua (endometrium of pregnancy) and this process involves vascular remodelling, which peaks by the end of the first trimester before declining rapidly thereafter (Aplin 1991). At 10–12 weeks of gestation, cytotrophoblasts in the anchoring villi generate multi-layered columns of extravillous trophoblasts which colonise the interstitium of the maternal decidua, the inner third of the myometrium and the uterine blood vessels (Aplin 1991). The depth of invasion by placental trophoblasts into the maternal decidua is finely controlled. A defect in this process is thought to result in disordered placentogenesis and an increase in the risk of pregnancy complications (Hoffman et al. 2009). Shallow invasion of the maternal decidua
(endometrium of pregnancy) by placental extravillous trophoblasts is thought to predispose to pre-eclampsia and intra-uterine growth restriction (IUGR); whereas excessive invasion to choriocarinoma, placenta accreta (abnormally deep attachment of the placenta to the myometrium) and invasive mole (Hoffman et al. 2009; Brouillet et al. 2012). PK-1 has an inhibitory effect on placental invasion as supported by a decrease in matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) production (Hoffman et al. 2009).
Furthermore, interference with PKR2 expression using specific siRNAs, reverses the PK-1- induced inhibition (Hoffman et al. 2009). The involvement of PK-1 in human placental angiogenesis has been demonstrated by data looking at the placental microvascular endothelial cells (HPEC) which cover foetal capillaries in the chorionic villi, ensure foeto- maternal exchanges and provide a framework for placental angiogenesis (Brouillet et al.
2012). These data demonstrate that PK-1 stimulates a number of angiogenic processes including endothelial proliferation, survival, migration, tube organization, sprouting, permeability, and paracellular transport (Brouillet et al. 2010). Furthermore, PK-1 has a
95 stronger effect on placental microvascular endothelial cell sprouting and permeability than the more established angiogenic factor VEGF (Brouillet et al. 2010).
The expression of PK-1 and its receptor have been shown to be up-regulated by hypoxia
(Hoffmann et al. 2006). Early embryonic development occurs under a hypoxic environment
(∼20 mm Hg or 3% O2), which protects developing organs from the harmful effects of radical oxygen species, as embryonic tissues express low levels of antioxidant enzymes during early pregnancy (Brouillet et al. 2012). The hypoxic environment in the decidua lasts throughout the first trimester. Thus, the peak in PK-1 and PKR-1 expression observed at 8–11 weeks of gestation cannot be explained solely by regulation by hypoxia. Other regulators of placental PK-1 expression may include hCG, which has similar temporal expression to PK-1 (peak hCG levels occur at 8-9 weeks of gestation). Exposure to hCG has been shown to induce a five-fold increased secretion of PK-1 from placentas collected at 6-8 weeks of gestation, but only 1.5 fold from placentas collected at 9-11 weeks
(Hoffmann et al. 2006).
Furthermore, PK-1 expression in the endometrium peaks during the mid-luteal window of embryonic implantation and PK-1 has been identified as a marker of human endometrial receptivity (Brouillet et al. 2012). PK-1 directly regulates implantation related genes and increases human trophoblast cell adhesion to extracellular matrix proteins via the induction of leukaemia inhibitory factor (LIF) (Evans et al. 2009).
To summarise PK-1 plays an important role in placental angiogenesis, especially during the first trimester. Lower levels of PK-1 may therefore be seen in disordered placental invasion, and may predispose to pregnancy complications such as miscarriage.
96 Other novel markers of pregnancy complications: Kisspeptin
Invasion of foetal trophoblasts into the maternal endometrium is a process, which can be regarded as analogous to the metastatic invasion of tumour cells into surrounding tissues.
Kisspeptin is a hormone, which was first discovered in 1996 as a peptide with anti- metastatic properties and was termed ‘metastin’. Kisspeptin is highly expressed in the placenta and circulating levels of kisspeptin are markedly elevated during pregnancy
(Horikoshi et al. 2004). Kisspeptin has been proposed to play an important role in the invasion of foetal trophoblasts into the maternal endometrium. Circulating plasma kisspeptin levels may provide a marker of placental function and are a strong candidate for investigation as a novel predictive marker of miscarriage in pregnancy.
The kisspeptins are a family of RF amide peptides with arginine–phenylalanine residues present at the amino terminal. The 145 amino acid gene product from the KiSS1 gene is cleaved into kisspeptin isoforms of different sizes (kisspeptin-54, kisspeptin-10, kisspeptin-
13, kisspeptin-14) with the suffix denoting the number of amino acids (see Figure 17). All kisspeptin isoforms have a C-terminal decapeptide moiety, which is critical for their biological action. Kisspeptin mRNA is peripherally expressed in the testis, ovary, pancreas and small intestine, however highest levels of kisspeptin expression are found in the placenta (Ohtaki et al. 2001). Exogenous administration of kisspeptin stimulates gonadotrophin releasing hormone (GnRH) release and subsequently gonadotrophin release.
Sustained exposure to continuous administration of kisspeptin results in desensitisation to its effects in some species, including humans. This is thought to be mediated at the level of the kisspeptin receptor as response to GnRH is maintained. Circulating kisspeptin levels are elevated 7,000-fold during pregnancy and it has been postulated that the hypothalamo-
97 pituitary axis may be desensitised to effects of kisspeptin as a result of this prolonged high level exposure. However exogenous administration of kisspeptin-10 to pregnant rodents is still able to stimulate the hypothalamo-pituitary axis (Hameed et al. 2010).
98 Figure 17: Kisspeptin isoforms
The 145 amino acid peptide product of the KiSS1 gene is cleaved into kisspeptin isoforms of length denoted by their suffix e.g. kisspeptin-54 (Kp-54), kisspeptin-10 (Kp-10),
Kisspeptin-14 (Kp14), and Kisspeptin-10 (Kp-10). The C-terminal decapeptide sequence is critical for the biological action of the kisspeptins. Kisspeptin-54 is the major circulating isoform during pregnancy (adapted from Bilban et al. 2004).
99 Kisspeptin as an anti-metastatic marker
Kisspeptin-54 has anti-metastatic properties in malignant melanoma cells and was hence initially termed ‘metastin’ (Lee et al 1996). Melanoma cells transfected with kisspeptin receptors have reduced growth and invasiveness in vitro (Ohtaki et al. 2001). Kisspeptin-10 and activation of the kisspeptin receptor have been shown to negatively regulate the chemotactic activity of another G-protein coupled receptor, the chemokine receptor 4
(CXCR4 receptor), which has a role in promoting metastasis. Kisspeptin is thought to play a role in a number of malignancies including breast, melanoma, gastric, hepatocellular, endometrial, and gestational trophoblastic disease (Makri et al. 2008). Kisspeptin negatively regulates matrix metalloproteinases (MMPs) in ovarian and hepatocellular carcinoma. These enzymes degrade the extracellular matrix and play an essential role in tumour cell invasion. Importantly MMPs also inactivate kisspeptins and the balance between kisspeptin and MMP activity may be an important factor in determining cancer cell invasion. Kisspeptin-54 treatment in thyroid cancer cells increases the calcineurin inhibitor Myocyte-enriched Calcineurin Interacting Protein 1 (MCIP1). MCIP1 inhibits
VEGF induced cell motility and angiogenesis in vascular endothelial cells (Makri et al.
2008).
100 Kisspeptin in the placenta
The invasion into and proliferation of trophoblasts in the maternal endometrium is often considered analogous to an invasive neoplastic process. However importantly and in contrast to a neoplastic process, the invasion of trophoblasts is highly regulated and kisspeptin has been proposed as a putative regulator of trophoblastic invasion.
Choriocarcinoma cells have reduced kisspeptin receptor mRNA expression in keeping with kisspeptin’s role as a metastasis suppressor gene (Janneau et al. 2002).
Bilban et al. 2004 localised kisspeptin mRNA expression and kisspeptin peptide to the syncitiotrophoblasts of anchoring and floating villi. Kisspeptin peptide distribution was undetectable in the maternal decidua and in villous/extra-villous cytotrophoblasts (Bilban et al. 2004). Hence kisspeptin expression appears to correlate with trophoblastic cells which have fused and are no longer invasive, whereas cytotrophoblasts only express the kisspeptin receptor. Therefore kisspeptin production by syncitiotrophoblasts may have an important paracrine role in regulating cytotrophoblastic invasiveness (Bilban et al. 2004).
Furthermore incubation with kisspeptin-10 (but not other isoforms of kisspeptin) lowers in vitro trophoblast migration by 70%, without affecting cytotrophoblast proliferation (Bilban et al. 2004).
Angiogenesis is a complex process involving multiple steps such as endothelial cell proliferation, migration, and tube formation. Kisspeptin has been shown to have vasoconstrictor properties in vitro. Both kisspeptin and its receptor are found in the endothelial and smooth muscle cells of human umbilical vein, placental blood vessels, aorta, and coronary arteries. Incubation of placental artery explants with kisspeptin-10 in vitro inhibited new vessel sprouting and tube-like structure formation by human umbilical
101 vein endothelial cells (HUVEC) in a concentration dependent manner. Although HUVEC cell viability was unaffected, cell proliferation and migration were reduced in a concentration dependent manner (Ramaesh et al. 2010).
Kisspeptin-10 inhibits in vitro cell migration of a human placental cell line derived from first trimester extra-villous trophoblasts (Roseweir et al. 2012). Kisspeptin also inhibits chemokine receptor 4 (CXCR4) and metalloproteinase-9 (MMP-9) in vitro (Yan et al.
2001). CXCR4 is believed to have an important role in the implantation of the blastocyst in the decidua and MMP 9 degrades the extracellular matrix making cell migration and invasion possible (Bilban et al. 2004).
Kisspeptin expression in Recurrent Pregnancy Loss
Park et al. 2012 collected trophoblastic and decidual tissues from women with at least 2 miscarriages of a genetically normal foetus and women who had had an elective termination (controls). In controls, kisspeptin protein was most abundantly found in syncytiotrophoblasts. Kisspeptin peptide distribution was reduced in the syncitiotrophoblasts of women with recurrent miscarriage (Park et al. 2012).
102 Circulating levels of kisspeptin in pregnancy
Horikoshi et al. 2004 were the first to assess circulating levels of kisspeptin in pregnancy.
They used a sensitive and specific two-site enzyme immunoassay to investigate kisspeptin-
54 levels in human plasma (Horikoshi et al. 2004). Mean plasma concentrations of kisspeptin-54 in non-pregnant females was 1.31 pmol/l (n = 10). The plasma kisspeptin-54 level was 1230 pmol/l (n = 11) in the 1st trimester, 4590 pmol/l (n = 16) in the 2nd trimester, and 9590 pmol/l (n = 12) in the 3rd trimester of pregnancy. Five days after delivery, kisspeptin-54 levels returned almost to the non-pregnant levels (7.63 pmol/l) suggesting that kisspeptin is predominantly derived from the placenta (Horikoshi et al.
2004) (see Figure 18). Kisspeptin is therefore secreted by the placenta into the circulation at high levels, making it an ideal candidate biomarker for detecting complications of pregnancy.
103 Figure 18: Kisspeptin-54 levels are raised during normal human pregnancy
Serum Kisspeptin-54 was measured in the first, second and third trimesters and was found to be raised several thousand fold those found in non-pregnant women (adapted from
Horikoshi et al. 2004).
104 Circulating kisspeptin levels and Threatened Miscarriage
Kavvasoglu et al. 2012 measured kisspeptin levels in 20 pregnant women with threatened miscarriage (PV bleeding) and 20 healthy controls matched for gestation. Kisspeptin levels were found to be significantly lower in the study group compared to the control group (391 vs. 5783 pg/mL; P <0.01). Birth weight was lower in the study group than that of the control group (2100g vs. 2860g, p = 0.06). A greater percentage of the study group required caesarean section or curettage (75%) as compared with the control group (45%, P=0.04)
(Kavvasoglu et al. 2012).
105 Other angiogenic markers of pregnancy complications:
Maternal blood flow to the placental inter-villous space becomes established towards the end of the first trimester. Between 11 and 12 weeks the trophoblast plugs dislodge and there is sudden perfusion of the placental inter-villous space. Prior to this, the development of the human embryo takes place in a relatively hypoxic environment. It has been proposed that hypoxia plays an important role in protecting dividing foetal and placental cells from exposure to free radicals during the critical early pregnancy growth period (Kaitu'u-Lino et al. 2012).
Soluble fms-like tyrosine kinase-1 (sFlt1) is an endogenous anti-angiogenic protein that is made by the placenta and acts to neutralize pro-angiogenic proteins including vascular endothelial growth factor (VEGF) and placental growth factor (PlGF). Free VEGF is undetectable in maternal serum, which may be due to it being bound by sFlt1. sFlt1 binds to PLGF and therefore high circulating sFlt1 has been associated with lower circulating
PLGF. sFlt1 and soluble endoglin (sEng) (a glycoprotein which is part of the TGF-beta1 receptor complex) are up-regulated by placental hypoxia. sFlt1 is up-regulated by cytotrophoblasts in hypoxia, whilst placental growth factor (PLGF) is the most abundantly regulated angiogenic factor in uncomplicated first trimester decidua. PLGF is predominantly expressed in the syncytiotrophoblast, and is secreted directly into the maternal circulation in a similar manner to kisspeptin. Circulating PLGF levels increase as placental mass increases (Kaitu'u-Lino et al. 2012).
Recently Kaitu'u-Lino et al. 2012 measured soluble FMS-like tyrosine kinase-1 (sFlt1), placental growth factor (PLGF), and soluble endoglin (sEng) in the serum of an asymptomatic cohort of pregnant women in the first trimester of pregnancy. Whilst serum levels of sFlt1 and PLGF rose with increasing gestation, serum endoglin levels remained 106 stable throughout the first trimester. Women who went on to miscarry had significantly reduced maternal sFlt-1 (decreased by 35%, P <0.05), but not PLGF or sEng when compared with women with healthy pregnancies. However due to over-lap in levels between women who miscarried and those who did not, serum Flt-1 cannot be used in isolation to predict miscarriage (Kaitu'u-Lino et al. 2012).
107 Hypothesis and aims
Hypothesis
PK-1 (also known as endocrine-gland vascular endothelial growth factor) is an angiogenic peptide, which plays an important role in the vascular invasion of the placenta into the maternal endometrium. Abnormal placentation may occur as a result of abnormal angiogenesis. Alterations in plasma PK-1 levels may thus correlate with the risk of pregnancy related complications such as miscarriage.
Aims
1. Characterise the levels of PK-1 in the plasma of pregnant women and correlate the
levels of PK-1 with the occurrence of pregnancy related complications.
2. Characterise the levels of other novel markers of pregnancy complications such as
kisspeptin (KP), soluble FMS-like tyrosine kinase-1 (FLT-1), placental growth
factor (PLGF), and soluble endoglin (ENG) in the same cohort.
108 Material and methods
Ethical approval was granted by the Hammersmith and Queen Charlotte’s and Chelsea
Hospitals Research Ethics Committee (registration number: Q0406/80). This study was performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants. In order to reduce selection bias, all women attending routine antenatal booking visit at this centre (between 1 January 2010 and 31 December
2012) were approached and invited to participate in the study. Women with renal failure were excluded due to possible interference with kisspeptin measurement. Women attending clinic as an emergency or with suspected miscarriage were excluded. Nine hundred and ninety-three pregnant women attending their routine antenatal booking clinic appointment at Queen Charlotte’s and Chelsea Hospital (a tertiary obstetric centre), London, UK, were recruited. Twelve women were lost to follow-up, and staff at the obstetric centre attempted to contact these women. The remaining 981 women were followed-up until the outcome of pregnancy was known (see Figure 19).
109 Figure 19: Protocol for Study.
Asymptomatic pregnant women were recruited in early pregnancy and blood was sampled for measurement of plasma prokineticin 1 (PK1), kisspeptin (KP), serum BHCG (BHCG), serum endoglin (ENG), serum FLT-1 (FLT-1) and placental growth factor (PLGF).
Women were then prospectively followed up for assessment for pregnancy outcomes.
110 Methods
I collected blood samples from pregnant women (n=981) attending Queen Charlotte and
Chelsea hospital outpatient antenatal clinic. As part of standard NHS care for pregnant women a routine booking visit is scheduled to occur between 8-10 weeks and involves an assessment of BMI, blood pressure and an evaluation by a midwife to identify women at risk of complications. At booking, a blood test is taken for blood group determination, previous infections with importance in pregnancy such as rubella and HIV, iron status, routine full blood count and renal and liver function measurement. Women return at ~12 weeks for an ultrasound scan which is used to date the pregnancy and assess the nuchal thickness (NT). The NT is combined with a blood test to identify women at higher risk of trisomy 21, who may then opt for a chorionic villus sampling (CVS) (or amniocentesis if later in the pregnancy) to confirm a genetic diagnosis if found to be at high risk. Women next visit at 20 weeks for an anomaly scan to assess for normal foetal development and the risk of a low lying placenta. Thereafter no routine scans are undertaken unless there are concerns regarding foetal growth, placental position or there is multiple pregnancy. One further routine blood test occurs at 28 weeks and women may have a further blood test prior to a caesarean section.
I also obtained blood samples from non-pregnant women (n=50) attending outpatient phlebotomy at Hammersmith Hospital. Written consent was obtained from all participants in the study. Participants were asked details such as name, date of birth, hospital number, gestation, parity, previous history of miscarriages and medical history.
111 Dating of pregnancy
Gestational age is historically derived from knowledge of last menstrual period (LMP), however in approximately 40% of cases, this is either not known or inaccurate (Verburg et al. 2008). Ultrasound dating of pregnancy is usually calculated based on crown–rump length (CRL) or biparietal diameter (BPD). Median gestational age can be calculated using
Crown–rump length = exp(1.4653 + 0.001737 × CRL + 0.2313 × log (CRL)). For example a CRL of 5mm corresponds to a gestational age of 6+2 and 40mm to 10+1 (Verburg et al.
2008). In the absence of serious pathology such as foetal chromosomal abnormality, or severe maternal malnutrition, foetal growth in early pregnancy is fairly uniform. Therefore ultrasound is more accurate than using LMP at deriving gestational age particularly in early pregnancy (<20 weeks gestation) (Verburg et al. 2008). Hence I have used gestational ages derived from ultrasounds carried out at 10-14 weeks where possible to calculate gestational age at time of sampling. Women were classified as first trimester from 0-14 weeks, second trimester from 14-28 weeks and third trimester if >28 weeks.
112 Measurement of circulating markers of pregnancy complications levels
Collection and processing of blood samples
All patients donated 5mls of blood in a serum clot activator tube and 5mls in a lithium heparin tube (Beckton Dickson, Franklin Lakes, NJ, USA) with 2000 units/tube containing
5,000 kallikrein units of aprotinin (0.2 ml; trasylol, Bayer, Newbury, UK). The lithium heparin tube with 2000 units/tube of trasylol was used for measurement of plasma levels
(kisspeptin and PK-1). Lithium heparin tube with 2000 units/tube of trasylol samples were immediately spun following collection at 6,400 x g for 4 minutes and plasma supernatant was aliquoted into eppendorf tubes. Plasma samples were then immediately placed in dry ice until transfer to a -20oC freezer. Serum samples collected in a serum clot activator tube were allowed to sit at room temperature until a clot had formed (at least 1 hour) and then spun at 4,800 x g for 10 minutes. Serum supernatant was separated and stored at -20oC for measurement of BHCG, PLGF, FLT-1 and ENG. All samples were analysed together in order to minimise assay variation.
113 Measurement of PK-1 immuno-reactivity
Plasma PK-1 was measured using a commercially available ELISA kit (ABNOVA) as described in Chapter 2.
Measurement of total Kisspeptin immuno-reactivity
Plasma kisspeptin immuno-reactivity (kisspeptin IR) was measured using a manual radioimmunoassay. Antibody GQ2 was raised in sheep immunised with synthetic human kisspeptin-54 (Bachem UK Ltd.) conjugated to bovine serum albumin (BSA) by glutaraldehyde and used at a final dilution of 1:3,500,000. The antibody cross-reacted
100% with human kisspeptin-54, kisspeptin-14, and kisspeptin-10 and less than 0.01% with other related RF amide proteins, including prolactin-releasing peptide, RF amide-related peptide 1 (RFRP1, human and rat), RFRP2 (human), RFRP3 (human), QRFP43 (human), neuropeptide FF (human), and neuropeptide AF (human). The iodogen method was used to prepare the 125I-kisspeptin-54 label, which was subsequently purified by High
Performance Liquid Chromatography (Salacinski et al 1981). The assay was carried out in duplicate using dilutions of neat plasma in 0.7ml of 0.06M phosphate buffer (pH 7.2) containing 0.3% BSA. Incubation was for 3 days at 4oC. Subsequently free and antibody- bound label were separated by charcoal adsorption. The limit of detectability was 2 pmol/l of plasma kisspeptin with 95% confidence interval, and the intra- and inter-assay coefficients of variation were 8.3 and 10.2%, respectively (Ramachandaran et al. 2008,
Dhillo et al. 2005).
114 Measurement of Total hCG immunoreactivity
Measurement of plasma hCG was performed using the Siemens Immulite 2000 total immunometric hCG assay run on board the Siemens Immulite 2000 instrument (Siemens
Healthcare Diagnostics 1717 Deerfield Road, Deerfield, IL, USA). According to the manufacturer’s kit insert the analytical sensitivity of the assay is 0.4 IU/L. Performance is monitored daily and internal quality control samples ranging 5-250 IU/L yield intra- and interassay coefficients of variation (CV) of less than 10%. Studies using the latest WHO standard preparations for common hCG protein variants have demonstrated that the
Immulite 2000 assay detects all hCG isoforms, but is associated with relative slight overestimation of hCGβ and under-detection of the hCGβ core fragment isoforms
(Sturgeon et al. 2009; Harvey et al. 2010).
Measurment of PLGF by ELISA (RnDsystems, Minneapolis, USA)
The PLGF Standard was reconstitured with 1 mL of Calibrator Diluent. This reconstitution produces a stock solution of 1000 pg/mL. The standard is allowed to sit for a minimum of
15 minutes with gentle agitation prior to making dilutions. 500 μL of the Calibrator Diluent is then pipetted into each tube. The undiluted standard serves as the high standard (1000 pg/mL). The stock solution to produce a dilution series to generate standards of 500 pg/ml,
250 pg/ml, 125 pg/ml, 62.5 pg/ml, 31.2 pg/ml and 15.6 pg/ml. 100 μL of Assay Diluent
RD1-22 is added to each well. 100 μL of Standard, control, or sample is added to each well.
The plate is covered with an adhesive strip. The plate is incubated for 2 hours at room temperature. Each well is aspirated and washed with 400 µls wash buffer for 4 cycles. 200
115 μL of PLGF Conjugate is added to each well and incubated for 2 hours. Each well is again aspirated and washed with 400 µls wash buffer for 4 cycles. 200 μL of Substrate Solution is added to each well and incubated for 30 minutes at room temperature in the dark. 50 μL of
Stop Solution is added to each well. The optical density of each well is determined within
30 minutes, using a microplate reader set to 450 nm with wavelength correction set to 570 nm.
Measurment of Endoglin by ELISA (RnDsystems, Minneapolis, USA)
The Endoglin Standard was reconstitured with 1 mL of Calibrator Diluent. This reconstitution produces a stock solution of 100 ng/mL. The standard is allowed to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions. 100 μL of the standard solution is pipetted into 900 μL of Calibrator Diluent to generate a concentration of 10 ng/ml. Then 500 μL of the Calibrator Diluent is then pipetted into each tube. The stock solution to produce a dilution series to generate standards of 5 ng/ml, 2.5 ng/ml, 1.25 ng/ml, 0.625 ng/ml, 0.313 ng/ml and 0.156 ng/ml. 100 μL of Assay Diluent RD1S is added to each well. 100 μL of Standard, control, or sample is added to each well. The plate is covered with an adhesive strip. The plate is incubated for 2 hours at room temperature.
Each well is aspirated and washed with 400 µls wash buffer for 4 cycles. 200 μL of
Endoglin Conjugate is added to each well and incubated for 2 hours. Each well is again aspirated and washed with 400 µls wash buffer for 4 cycles. 200 μL of Substrate Solution is added to each well and incubated for 30 minutes at room temperature in the dark. 50 μL of
Stop Solution is added to each well. The optical density of each well is determined within
30 minutes, using a microplate reader set to 450 nm with wavelength correction set to 570 nm. 116 Measurement of FLT-1 by ELISA (RnDsystems, Minneapolis, USA)
High concentrations of FLT-1 is found in saliva and therefore a face mask and gloves to protect kit reagents from contamination was employed throughout. The FLT-1 Standard was reconstitured with 1 mL of deionised water to produces a stock solution of 20,000 pg/mL. The standard is allowed to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions. 100 μL of the standard solution is pipetted into 900 μL of
Calibrator Diluent to generate a concentration of 2000 pg/ml. Then 500 μL of the
Calibrator Diluent is then pipetted into each tube. The stock solution to produce a dilution series to generate standards of 1000 pg/ml, 500 pg/ml, 250 pg/ml, 125 pg/ml, 62.5 pg/ml and 31.2 pg/ml. 100 μL of Assay Diluent RD6-68 is added to each well. 100 μL of
Standard, control, or sample is added to each well. The plate is covered with an adhesive strip. The plate is incubated for 2 hours at room temperature. Each well is aspirated and washed with 400 µls wash buffer for 4 cycles. 200 μL of FLT-1 Conjugate is added to each well and incubated for 2 hours. Each well is again aspirated and washed with 400 µls wash buffer for 4 cycles. 200 μL of Substrate Solution is added to each well and incubated for 30 minutes at room temperature in the dark. 50 μL of Stop Solution is added to each well. The optical density of each well is determined within 30 minutes, using a microplate reader set to 450 nm with wavelength correction set to 570 nm.
117 Data Analysis and Statistics
Data was analysed using Prism version 5 (GraphPad, California). In order to account for variation in circulating blood markers with gestation, I calculated multiples of median
(MoM) levels of each hormone measurement correcting for the gestation at which the blood was collected. Pairs of means were analysed using the unpaired two-tailed t-test. Multiple means were compared using one-way ANOVA with post hoc bonferroni or Dunnet’s
Multiple Comparison Test. In all cases, P < 0.05 was considered statistically significant.
118 Patient details and outcomes:
All pregnancies were dated according to a dating ultrasound scan (typically at 8-14weeks gestation) routinely performed in the antenatal clinic. Ultrasound parameters from a 20- week fetal anomaly scan, patient-held clinical notes, and hospital electronic database records were collated for study participants to establish whether miscarriage or live birth had occurred. Miscarriage was either diagnosed using ultrasound scanning, or was self- reported by the patient. Patient clinical details are summarised in Table 8.
119 Baseline characteristics of pregnant subjects attending their antenatal booking visit
Nine hundred and ninety-three women attending antenatal clinic for a routine booking antenatal appointment were recruited to the study. Twelve women (1% of the sample) were lost to follow-up (therefore the outcome of their pregnancies could not be determined) and their data was excluded from all analyses (see Figure 20). The remaining 981 women had a mean gestation of 11.2 weeks (range 5.9-29.0), mean age 32.6 years old (see Table 8).
There was no missing data for each variable of interest for each of the 981 women included in the analyses. 50 of these women had singleton pregnancy which subsequently resulted in miscarriage. These parameters were similar between women with healthy pregnancies which did not result in miscarriage and women whose pregnancies subsequently resulted in miscarriage. Thirty-two of the pregnancies were multiple: 30 twin pregnancies (6 monochorionic and 24 dichorionic) and 2 triplet pregnancies. No participants attended clinic as an emergency, or presented with any symptoms of threatened miscarriage such as abdominal pain or vaginal bleeding at the time of recruitment.
Participants with singleton pregnancy were also classified by gestational week of pregnancy at time of recruitment. The majority of singleton pregnancies subsequently resulting in miscarriage (47/50) were recruited prior to the 12th gestational week of pregnancy. Most singleton pregnancies not resulting in miscarriage (554/899) were also recruited prior to the 12th gestational week of pregnancy. Thus this study provided a large pool of gestation-matched non-cases with which to compare levels of blood markers with miscarriage cases.
120 Figure 20: Flowchart of pregnant women recruited to the study and main outcomes
121 Table 8: Clinical characteristics of study participants
N number Mean Mean Mean Mean
Age BMI Parity Gestation
(years) (kg/m2) (weeks)
(±s.d.) (±s.d.) (±s.d.) (±s.d.)
Total number of women participating in 32.6 24.8 0.6 11.2 981 study (±2.0) (±5.5) (±0.8) (±2.0)
Number of women with 32.4 24.7 0.7 11.3 899 singleton pregnancy without miscarriage (±5.1) (±5.4) (±0.8) (±1.9)
Number of women 33.1 26.3 0.6 9.8 50 with singleton miscarriage (±4.8) (±7.4) (±0.8) (±3.1)
Number of women with multiple pregnancy 32 35.5 25.9 0.5 10.7
(±5.6) (±5.6) (±0.9) (±1.5)
Ethnicity (981) Caucasian 574 (58.5%)
South Asian 121 (12.3%)
Afrocaribbean 107 (10.9%)
Other 109 (11.1%)
Mixed 24 (2.4%)
Unknown 46 (4.7%)
122 Results:
Levels of plasma Prokineticin-1 (PK-1) in non-pregnant women
Plasma PK-1 levels in 50 non-pregnant women aged between 18 and 74 years old.
The majority of women had levels less than 200 pg/ml. The median plasma PK-1 level in non-pregnant women was 55 pg/ml with an interquartile range of 8.3 to 162 pg/ml. It is not clear why a small number of women had high detectable levels of PK-1 in the plasma
(some above the reference range of the assay) and there was no clear correlation with age
(r2 is only 0.05) (see Figure 21). It is possible that an as of yet unidentified factor results in raised plasma PK-1 in some individuals.
123 Figure 21: Levels of plasma PK-1 in non-pregnant women.
Plasma Prokineticin-1 (PK-1) levels in 50 non-pregnant women aged between 18 and 74 years old. There was no clear correlation with age (r2 only 0.05).
1000 800 Plasma PK1 600 (pg/ml) 400 200 0 20 40 60 80 Age (years)
124 Correlation of Prokineticin-1 (PK-1) with serum BHCG levels
It was previously suggested that as PK-1 is thought to show a similar temporal pattern of secretion to BHCG, that BHCG was a regulator of PK-1 secretion.
Therefore I analysed whether PK-1 levels correlated with serum BHCG levels.
However there was no significant correlation in this larger data set with r2 only 0.0034 and
P=0.084 (see Figure 22).
125 Figure 22: Correlation of Prokineticin-1 (PK-1) with serum BHCG levels
Linear correlation between plasma PK-1 levels and serum BHCG in women with singleton pregnancy uncomplicated by miscarriage. There is no significant correlation r2 is only
0.0034 and P=0.084.
1000 800 Plasma 600 PK1 (pg/ml) 400 200 0 0 100000 200000 300000 400000 serum HCG (iU/L)
126 Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG, Serum
Endoglin, Serum FLT-1 and Serum PLGF with gestation:
Levels of PK1 did not show a clear relationship with gestational week in singleton pregnancies uncomplicated by miscarriage (see Figure 23).
Levels of plasma kisspeptin in singleton pregnancies increased steadily during each week of gestation during the first trimester of pregnancy (Figure 23). Plasma kisspeptin levels were highly significantly and positively correlated with the gestational week of pregnancy
(r2=0.32; P<0.0001) (Table 9). By comparison, levels of serum hCG in single pregnancies were highest during the 8th week of gestation, then decreased steadily with increasing gestation (Figure 23). Thereafter serum hCG levels were significantly and negatively correlated with the gestational week of pregnancy (r2=-0.18; P<0.0001) (Figure 23). Serum endoglin levels increase until 10 weeks of gestation before remaining steady thereafter at higher gestations. Serum FLT-1 levels only changed marginally with gestation. Serum placental growth factor levels increased significantly with gestation (r2=0.51; P<0.0001) and was highly correlated.
In order to reduce the impact of gestation on further analyses, I calculated medians for each gestation and expressed blood markers as multiples of gestation specific median (MOM).
This is important as miscarriage often occurs at earlier gestation and if gestation were not corrected for, blood levels of markers associated with miscarriage may be be lower than control group pregnancies purely due to the effect of gestation (see Table 10).
127 Figure 23: Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum
BHCG, Serum Endoglin, Serum FLT-1 and Serum PLGF with gestation.
Mean and Standard error of mean of plasma PK1, plasma Kisspeptin, Serum BHCG, Serum
Endoglin, Serum FLT-1 and Serum PLGF is shown for each week of gestation in women with singleton pregnancy which did not end in miscarriage (n=899).
400 4000
300 3000 Plasma Plasma Pk1 200 kisspeptin 2000 (pg/ml) (pmol/L) 100 1000
0 0 <8.0 <8.0 >14.0 >14.0 8.0-8.99 9.0-9.99 8.0-8.99 9.0-9.99 10.0-10.99 11.0-11.99 12.0-12.99 13.0-13.99 10.0-10.99 11.0-11.99 12.0-12.99 13.0-13.99 Gestation (weeks) Gestation (weeks)
120000 8 100000 serum 6 serum 80000 endoglin HCG 60000 (ng/ml) 4 (iU/L) 40000 20000 2 0 0 <8.0 <8.0 >14.0 >14.0 8.0-8.99 9.0-9.99 8.0-8.99 9.0-9.99 10.0-10.99 11.0-11.99 12.0-12.99 13.0-13.99 10.0-10.99 11.0-11.99 12.0-12.99 13.0-13.99 Gestation (weeks) Gestation (weeks)
1200 150 1000 serum FLT-1 800 serum PLGF 100 (pg/ml) 600 (pg/ml) 400 50 200 0 0 <8.0 <8.0 >14.0 >14.0 8.0-8.99 9.0-9.99 8.0-8.99 9.0-9.99 10.0-10.99 11.0-11.99 12.0-12.99 13.0-13.99 10.0-10.99 11.0-11.99 12.0-12.99 13.0-13.99 Gestation (weeks) Gestation (weeks)
128 Table 9: Correlation of levels of plasma Prokineticin-1, plasma Kisspeptin, Serum
BHCG, Serum Endoglin, Serum FLT-1 and Serum PLGF with gestation.
Correlation of blood markers with gestation by linear regression in singleton pregnancy which did not result in miscarriage (n=899).
Plasma Plasma Serum Serum Serum Serum
Prokineticin-1 Kisspeptin BHCG Endoglin FLT-1 PLGF
level (pmol/l) (iU/L) (ng/ml) (pg/ml) (pg/ml)
(pg/ml)
R 1.82 305.4 -8618 0.073 -31.12 13.26 (slope of ± 5.66 ± 14.80 ± 620.6 ±0.029 ±6.441 ±0.4395 change with gestation)
R2 0.00011 0.32 0.18 0.0069 0.026 0.51
(goodness
of fit)
P Value 0.75 < 0.0001 < 0.0001 0.013 < 0.0001 < 0.0001
129 Table 10: Gestation of pregnancy during which study participants with singleton pregnancy were recruited to the study.
Plasma kisspeptin was measured on the day of recruitment, and pregnancies were classified by the subsequent diagnosis of miscarriage.
Gestation Number of participants with Number of participants with
(weeks) singleton pregnancy not resulting singleton pregnancy resulting in
in miscarriage (N=899) miscarriage (N=50)
<8 12 (1.3%) 2 (4.0%)
8-8.9 75 (8.3%) 15 (30.0%)
9-9.9 164 (18.2%) 17 (32.0%)
10-10.9 167 (18.6%) 7 (14.0%)
11-11.9 136 (15.1%) 6 (12.0%)
12-12.9 198 (22.0%) 3 (6.0%)
13-13.9 91 (10.1%) 0 (0%)
14-14.9 19 (2.1%) 0 (0%)
≥15 37 (4.1%) 0 (0%)
130 Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG, Serum
Endoglin, Serum FLT-1 and Serum PLGF with multiple pregnancy and miscarriage:
Thirty-two of the subjects participating in the study had multiple pregnancy (twins or triplets). I therefore investigated the observed relationship between blood markers and multiple pregnancy. Some of the participants with twin pregnancy (n=10) were also affected by foetal loss of one of the twins. I therefore investigated whether these participants had changes in their levels of blood markers in comparison to participants with multiple pregnancy unaffected by miscarriage.
Plasma levels of PK-1 did not show a clear relationship with multiple pregnancy, although appeared to be highest in twins affected by miscarriage (see Figure 24).
Plasma kisspeptin showed a more consistent and predictable change with multiple pregnancy and miscarriage. MoM of plasma kisspeptin was higher in twin and triplet pregnancies unaffected by miscarriage when compared with singleton pregnancies unaffected by miscarriage (median of MoM plasma kisspeptin: 1.0, singleton no miscarriage (n=899); 1.31, monochorionic twin no miscarriage (n=3); 1.7, dichorionic twin no miscarriage (n=17); 2.1, triplet no miscarriage (n=2)). However plasma kisspeptin levels were reduced in women with twin pregnancy affected by miscarriage (median of MoM plasma kisspeptin: 0.23, singleton with miscarriage (n=50); 0.82, monochorionic twin with miscarriage (n=3); 1.17, dichorionic twin with miscarriage (n=7)). Levels of kisspeptin in women with twin pregnancy affected by miscarriage were similar to those found in healthy singleton pregnancy.
MoM serum BHCG was higher in twin and triplet pregnancies unaffected by miscarriage when compared with singleton pregnancies unaffected by miscarriage (median of MoM
131 plasma kisspeptin: 0.99, singleton no miscarriage (n=899); 1.56, monochorionic twin no miscarriage (n=3); 1.52, dichorionic twin no miscarriage (n=17); 1.89, triplet no miscarriage (n=2)). However these were reduced in women with twin pregnancy affected by miscarriage (median of MoM plasma kisspeptin: 0.41, singleton with miscarriage
(n=50); 0.42, monochorionic twin with miscarriage (n=3); 1.36, dichorionic twin with miscarriage (n=7)).
MoM serum FLT-1 was higher in twin and triplet pregnancies unaffected by miscarriage when compared with singleton pregnancies unaffected by miscarriage (median of MoM plasma kisspeptin: 1.00, singleton no miscarriage (n=899); 1.35, monochorionic twin no miscarriage (n=3); 1.50, dichorionic twin no miscarriage (n=17); 1.87, triplet no miscarriage (n=2)). However these were reduced when women had a twin pregnancy affected by miscarriage (median of MoM plasma kisspeptin: 0.62, singleton with miscarriage (n=50); 0.77, monochorionic twin with miscarriage (n=3); 0.98, dichorionic twin with miscarriage (n=7)).
MoM serum PLGF was higher in dichorionic twin and triplet pregnancies unaffected by miscarriage when compared with singleton pregnancies unaffected by miscarriage (median of MoM plasma kisspeptin: 1.0, singleton no miscarriage (n=899); 1.00, monochorionic twin no miscarriage (n=3); 1.33, dichorionic twin no miscarriage (n=17); 2.01, triplet no miscarriage (n=2)). However these were reduced when women had a twin pregnancy affected by miscarriage (median of MoM plasma kisspeptin: 0.77, singleton with miscarriage (n=50); 0.77, monochorionic twin with miscarriage (n=3); 0.82, dichorionic twin with miscarriage (n=7)).
132 Figure 24: Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum
BHCG, Serum Endoglin, Serum FLT-1 and Serum PLGF with multiple pregnancy and miscarriage.
Mean ±SEM is shown for S= singleton pregnancy (n=899 no miscarriage and 50 miscarriage), MC= monochorionic twin pregnancy (n=3 no miscarriage and 3 miscarriage), DC= dichroionic twin pregnancy (n=17 no miscarriage and 7 miscarriage), T=triplet pregnancy (n=2 no miscarriage) which did not (no miscarriage) and which did (miscarriage) result in foetal death. No significant difference was found when compared by one way ANOVA.
6 2.5 5 MOM of 2.0 4 MOM of plasma 1.5 3 plasma PK1 Kisspeptin 2 1.0 1 0.5
0 0.0 S MC DC T S MC DC S MC DC T S MC DC No Miscarriage Miscarriage No Miscarriage Miscarriage
2.5 1.5 2.0 MOM of MOM of 1.0 1.5 serum serum BHCG 1.0 Endoglin 0.5 0.5
0.0 0.0 S MC DC T S MC DC S MC DC T S MC DC No Miscarriage Miscarriage No Miscarriage Miscarriage
2.0 2.5
2.0 1.5 MOM of MOM of 1.5 serum 1.0 serum FLT-1 PLGF 1.0 0.5 0.5
0.0 0.0 S MC DC T S MC DC S MC DC T S MC DC No Miscarriage Miscarriage No Miscarriage Miscarriage
133 Serum Endoglin was only marginally higher in multiple pregnancy (1.1-1.2 in twins or triplets unaffected by miscarriage) and only marginally reduced by miscarriage (median
MOM in singleton miscarriage 0.9) suggesting that serum endoglin is not a good marker for identifying women with multiple pregnancy at increased risk of miscarriage.
This data helps to show that the levels of kisspeptin, BHCG, FLT-1 and PLGF were increased with the number of placentae and were reduced when women with twin pregnancy were affected by miscarriage of one twin. MOM of plasma kisspeptin appeared to be the most useful of the markers evaluated, as it increased from healthy singleton to healthy monochorionic to healthy dichorionic to healthy triplet pregnancy and reduced by the greatest degree by miscarriage within all of these settings. MOM of plasma kisspeptin in twin pregnancies affected by loss of one twin were similar to those seen in healthy singleton pregnancy unaffected by miscarriage (median of MoM plasma kisspeptin: 0.23, singleton with miscarriage (n=50); 0.82, monochorionic twin with miscarriage (n=3); 1.17, dichorionic twin with miscarriage (n=7)).
However, whilst suggestive, the number of patients with multiple pregnancy was small
(n=32) making it difficult to draw firm conclusions regarding the utility of these markers for the diagnosis of pregnancy complicated by miscarriage. That said, the data set contained a larger number of singleton pregnancies, some of whom resulted in miscarriage (n=50) whilst the majority resulted in live birth (n=899).
Therefore I went on to investigate which of the markers performed best in the identifying women at increased risk of subsequent miscarriage in asymptomatic singleton pregnancy.
134 Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG, Serum
Endoglin, Serum FLT-1 and Serum PLGF with singleton miscarriage:
Fifty of the 949 (5.2%) participants with singleton pregnancy were diagnosed with miscarriage after recruitment to the study. The 12th gestational week was the most common time for miscarriage to be diagnosed amongst the participants of this study.
Mean ±SD MOM of plasma PK-1 was 2.1 in women who did not miscarry and 1.83 ±2.0 in women who did go on to miscarry suggesting that it was not a good marker for identifying women at increased risk of miscarriage.
Kisspeptin fell the by the greatest proportion in asymptomatic pregnant women who later experienced miscarriage when compared with those who went on to have live births.
Multiples of Median (MoM; gestation-corrected) levels of plasma kisspeptin were 60% lower in women who later experienced miscarriage when compared with unaffected pregnancies (mean ±SD MoM plasma kisspeptin: 1.04±0.41, singleton no miscarriage,
0.41±0.41, singleton with miscarriage, P<0.001 vs. singleton no miscarriage) (Figure 25).
By comparison, MoM levels of serum BhCG were only 36% lower in women who later miscarried when compared with pregnancies which did not result in miscarriage (mean
±SD of MoM plasma kisspeptin: 1.06±0.47, singleton no miscarriage, 0.64±1.1, singleton with miscarriage, P<0.001 vs. singleton no miscarriage) (Figure 25).
Receiver operator characteristic (ROC) curve analysis was performed to determine the diagnostic performance of blood markers with respect to miscarriage (Figure 26). Plasma kisspeptin had the greatest diagnostic performance with respect to identifying asymptomatic women at increased risk of miscarriage (ROC area under curve: 0.86, plasma kisspeptin). Furthermore, kisspeptin levels increased to a significantly lesser extent with
135 gestational age, in the cohort of women who experienced miscarriage when compared with the cohort of women who had an uncomplicated pregnancy (slope coefficient for increase in plasma kisspeptin in pmol/L/week: 305±15, singleton without miscarriage; 94±30, singleton with miscarriage; P<0.0001). BHCG was the next best performing marker with an area under the ROC curve of 0.83, followed by FLT-1 and PLGF (AUC of ROC curve of
0.73).
PK-1 was the worst performing marker evaluaeted for indentifying asymptomatic pregnant women at increased risk of miscarriage with an area under the ROC curve of only 0.51 (see
Figure 26).
136 Figure 25: Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum
BHCG, Serum Endoglin, Serum FLT-1 and Serum PLGF with singleton miscarriage:
Mean ±SEM of Multiples of Median of plasma prokineticin-1 (PK-1), plasma kisspeptin
(KP), serum BHCG (HCG), serum endoglin (ENG), serum FLT-1 (FLT-1) and placental growth factor (PLGF) in healthy singleton pregnancy which did not end in miscarriage (H)
(n=899) and in women whose pregnancy resulted in miscarriage (M) (n=50).
Each pair of multiple of medians was compared by unpaired t test.
** P<0.01, *** P<0.001.
2.5 2.0 Multiples 1.5 *** *** ** of *** *** Median 1.0 0.5 0.0 H M . H M . H M . H M . H M . H M PK1 KP HCG ENG FLT-1 PLGF
137 Figure 26: ROC of plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG,
Serum Endoglin, Serum FLT-1 and Serum PLGF with singleton miscarriage:
Receiver Operator Characteristic curves of sensitivity vs 100%-specificity for plasma PK1, plasma Kisspeptin, Serum BHCG, Serum Endoglin, Serum FLT-1 and Serum PLGF. AUC
(area under curve) is indicated at the top of each graph.
ROC MoM PK1 AUC 0.51 100 ROC MoM Kisspeptin AUC 0.86 100 80 80 60 60 40 40
20 Sensitivity 20 Sensitivity (%) 0 0 0 20 40 60 80 100 0 20 40 60 80 100 100% - Specificity% 100% - Specificity%
ROC MOM BHCG AUC 0.83 ROC MOM Endoglin AUC 0.65 100 100 80 80 60 60 40 40 Sensitivity Sensitivity 20 20 0 0 0 20 40 60 80 100 0 20 40 60 80 100 100% - Specificity% 100% - Specificity%
ROC MOM FLT-1 AUC 0.73 ROC MOM PLGF AUC 0.73 100 100 80 80 60 60 40 40 Sensitivity Sensitivity 20 20 0 0 0 20 40 60 80 100 0 20 40 60 80 100 100% - Specificity% 100% - Specificity%
138 Relationship of plasma kisspeptin with time to diagnosis of miscarriage
Ultrasonography confirmed fetal viability in 13 out of 50 pregnancies following participation in the study in patients who subsequently miscarried. Miscarriage occurred a mean ±SD of 34.8 ±6.8 days (range 2 - 85) following participation in the study. I therefore investigated in more detail the length of time following participation in the study, when patients were diagnosed with miscarriage (Figure 27).
Plasma levels of kisspeptin were significantly lower in patients who miscarried within 8 days of participation in the study when compared with patients who miscarried further in the future. This suggests that plasma kisspeptin levels are even lower in women with imminent miscarriage than women who will miscarry further in the future.
139 Figure 27: Changes in plasma levels of plasma Kisspeptin, Serum BHCG and Time to miscarriage:
Mean ±SEM of Multiples of Median of plasma prokineticin 1 (PK1), kisspeptin (KP), serum BHCG (HCG), serum endoglin (ENG), serum FLT-1 (FLT-1) and placental growth factor (PLGF) in singleton pregnancy resulted in miscarriage within 8 days (<8) (n=16) and those resulting in miscarriage after 9 days following recruitment into the study (>9) (n=34).
Each pair of multiple of median was compared by unpaired t test. * P<0.05.
2.5 2.0 Multiples 1.5 of * Median 1.0 * 0.5 0.0 <8 >9 . <8 >9 . <8 >9 . <8 >9 . <8 >9 . <8 >9 PK1 KP HCG ENG FLT-1 PLGF Days to miscarriage diagnosis
140 Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG, Serum
Endoglin, Serum FLT-1 and Serum PLGF with BMI:
Obesity is a risk factor for the development of pre-eclampsia, a complication of pregnancy occurring during the third trimester which is thought to occur due to abnormal placentation.
Previous reports have suggested that obese women (BMI >40 kg/m2) had lower levels of circulating kisspeptin at 16 weeks of pregnancy than lean women ((BMI <25 kg/m2) and that obese women with pre-eclampsia had lower levels still of kisspeptin than obese women
(Logie et al. 2012). Therefore I investigated whether the levels of blood markers of pregnancy evaluated in this study showed a relationship with BMI (see Figure 28).
Plasma kisspeptin, serum BHCG, and serum endoglin showed significant falls in levels with increasing BMI. Mean MOM of plasma kisspeptin was 1.26 in pregnant women with a
BMI <20 kg/m2 and 0.80 in women with a BMI >35 kg/m2. Mean MOM of serum BHCG was 1.29 in pregnant women with a BMI <20 kg/m2 and 0.85 in women with a BMI >35 kg/m2. Mean MOM of serum endoglin was 1.10 in pregnant women with a BMI <20 kg/m2 and 0.86 in women with a BMI >35 kg/m2.
One might expect that if these plasma markers were merely reflecting placental size that women with higher BMI’s would have higher levels than in women with lower BMI’s.
Therefore it seems feasible that these markers are indeed reflective of placental function which is known to be disordered with increased BMI.
141 Figure 28: Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum
BHCG, Serum Endoglin, Serum FLT-1 and Serum PLGF with BMI:
Mean ±SEM of Multiples of Median (MOM) of plasma plasma PK1, plasma Kisspeptin,
Serum BHCG, Serum Endoglin, Serum FLT-1 and Serum PLGF were compared for different BMI categories by One way ANOVA with post hoc Dunnet’s multiple comparison test versus BMI 15-19.9. * P<0.05 , ** P<0.01 , *** P<0.001 .
3.0 1.4 2.5 1.2 MOM of MOM of ** 2.0 1.0 *** plasma plasma *** *** 1.5 0.8 PK-1 Kisspeptin 0.6 1.0 0.4 0.5 0.2 0.0 0.0 15-19.9 20-24.9 25-29.9 30-34.9 >35 15-19.9 20-24.9 25-29.9 30-34.9 >35 2 BMI (kg/m ) BMI (kg/m2)
1.4 1.2 1.2 MOM of ** 1.0 *** 1.0 *** MOM of *** *** *** *** 0.8 serum 0.8 serum BHCG 0.6 Endoglin 0.6 0.4 0.4 0.2 0.2 0.0 15-19.9 20-24.9 25-29.9 30-34.9 >35 0.0 15-19.9 20-24.9 25-29.9 30-34.9 >35 BMI (kg/m2) BMI (kg/m2)
1.2 1.4 * 1.2 MOM of 1.0 * MOM of 1.0 serum 0.8 serum 0.8 FLT-1 0.6 PLGF 0.6 0.4 0.4 0.2 0.2 0.0 0.0 15-19.9 20-24.9 25-29.9 30-34.9 >35 15-19.9 20-24.9 25-29.9 30-34.9 >35 2 BMI (kg/m ) BMI (kg/m2)
142 Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG, Serum
Endoglin, Serum FLT-1 and Serum PLGF with smoking:
Smoking is known to increase the risk of a number of pregnancy complications, so therefore I assessed whether women who were smoking at the time of the antenatal booking visit during the first trimester had altered levels of blood markers evaluated when compared with non-smoking women in women with singleton pregnancy not resulting in miscarriage.
Multiple of gestation specific medians of plasma kisspeptin, serum BHCG, and serum endoglin were all significantly reduced in pregnant women who smoked when compared with women who did not smoke. Placental growth factor was significantly increased in non-pregnant women who were smoking at booking when compared with non-smoking pregnant women (see Figure 29).
It is therefore reasonable to speculate that women with increased BMI and women who smoked during early pregnancy had lower levels of these blood markers reflecting disordered placentation.
143 Figure 29: Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum
BHCG, Serum Endoglin, Serum FLT-1 and Serum PLGF with smoking:
Mean ±SEM of multiples of median of plasma prokineticin 1 (PK1), kisspeptin (KP), serum BHCG (HCG), serum endoglin (ENG), serum FLT-1 (FLT-1) and placental growth factor (PLGF) in mothers with singleton pregnancy who were smoking at the time of their booking antenatal clinic visit (S) (n=34) and in mothers who did not smoke (N) (n=829).
Each pair of multiple of median was compared by unpaired t test. * P<0.05 , ** P<0.01 ,
*** P<0.001 .
2.4 2.0 ** ** * *** Multiples 1.6 of 1.2 Median 0.8 0.4 0.0 N S . N S . N S . N S . N S . N S PK-1 KP HCG ENG FLT-1 PLGF
144 Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG, Serum
Endoglin, Serum FLT-1 and Serum PLGF with recurrent miscarriage in pregnant women whose current singleton pregnancy did not result in miscarriage:
Women with a history of 3 or more consecutive miscarriage are termed as having recurrent miscarriage. Recurrent miscarriage occurs in ~1% of fertile couples trying to conceive.
Chromosomal abnormality is the commonest cause of miscarriage and hence most miscarriage is not due to maternal factors. However in recurrent miscarriage, it becomes fruitful to screen women for rarer causes of miscarriage including autoimmune or thrombotic causes which can then be identified in 50% of women (see table 5 for full list of seconday causes of miscarriage).
Kisspeptin peptide distribution previously found to be reduced in the syncitiotrophoblasts of women with recurrent miscarriage (Park et al. 2012). So therefore I investigated whether blood levels of pregnancy markers were reduced in women with at least 3 or more miscarriages when compared with women a fewer number of previous miscarriage.
Levels of pregnancy markers were similar in women between 0-2 previous miscarriages
(see Figure 30). However levels of plasma kisspeptin were significantly reduced in women with recurrent miscarriage (at least 3 previous miscarriages) when compared with women with a fewer number of miscarriages (mean ±SD MOM of plasma kisspeptin in women with <2 previous miscarriages is 1.046 ±0.4067 and in women with 3 or more miscarriages is 0.8886 ±0.3822) (see Figure 30). This is consistent with previous data suggesting a reduced kisspeptin peptide distribution in syncitiotrophoblasts of women with recurrent miscarriage (Park et al. 2012).
145 Figure 30: Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum
BHCG, Serum Endoglin, Serum FLT-1 and Serum PLGF with number of previous miscarriages in women whose current singleton pregnancy did not result in miscarriage:
Mean ±SEM of Multiples of Median (MOM) of plasma plasma PK1, plasma Kisspeptin, Serum
BHCG, Serum Endoglin, Serum FLT-1 and Serum PLGF were compared by categories of previous miscarriage by One way ANOVA with post hoc Dunnet’s multiple comparison test versus no previous miscarriage group. N= 608 for no previous miscarriage, 170 for 1 previous miscarriage, 55 for 2 previous miscarriage and 31 for at least 3 previous miscarriages.
2.5 1.2 2.0 1.0 MOM of MOM of 0.8 1.5 plasma plasma 0.6 1.0 PK-1 Kisspeptin 0.4 0.5 0.2 0.0 0.0 0 1 2 >3 0 1 2 >3 Number of previous miscarriage Number of previous miscarriage
1.2 1.2 1.0 1.0 MOM of 0.8 MOM of 0.8 plasma 0.6 plasma 0.6 BHCG 0.4 ENG 0.4 0.2 0.2 0.0 0.0 0 1 2 >3 0 1 2 >3 Number of previous miscarriage Number of previous miscarriage
1.2 1.4 1.0 1.2 MOM of 0.8 MOM of 1.0 0.8 plasma 0.6 plasma FLT-1 PLGF 0.6 0.4 0.4 0.2 0.2 0.0 0.0 0 1 2 >3 0 1 2 >3 Number of previous miscarriage Number of previous miscarriage
146 Figure 31: Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum
BHCG, Serum Endoglin, Serum FLT-1 and Serum PLGF with history of recurrent miscarriages in women whose current singleton pregnancy did not result in miscarriage:
Mean ±SEM of Multiples of Median (MOM) of plasma plasma PK1, plasma Kisspeptin,
Serum BHCG, Serum Endoglin, Serum FLT-1 and Serum PLGF were compared by history of recurrent miscarriage (3 or more previous miscarriage) (n=31) versus women with up to
2 previous miscarriages (n=833) by unpaired t test. * P<0.05
2.5 2.0 Multiples 1.5 of * Median 1.0 0.5 0.0 0-2 >3 . 0-2 >3 . 0-2 >3 . 0-2 >3 . 0-2 >3 . 0-2 >3 PK1 KP HCG ENG FLT-1 PLGF Recurrent miscarriage
147 Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG, Serum
Endoglin, Serum FLT-1 and Serum PLGF with foetal sex:
Kisspeptin is often regarded as a gate-keeper of puberty and the influence of androgens in the early neonatal period is thought to be important in influencing with sexual dimorphism found in hypothalamic kisspeptin expression (Tena-Sempere 2010).
It was therefore interesting to see whether maternal kisspeptin levels during pregnancy were altered whilst carrying a foetus of either sex. It was interesting to note that both kisspeptin and BHCG were significantly higher in women with a female foetus when compared with those bearing a male foetus (see Figure 32). However the difference was small (mean ±SD MOM of plasma kisspeptin in women carrying a male foetus is 0.99
±0.39 and in women carrying a female foetus is 1.08 ±0.41). PK-1 was also higher in women bearing a female foetus when compared with women bearing a male foetus, although this was not statistically significant. None of the remaining markers were significantly different in women depending on the sex of the foetus they were carrying (see
Figure 32).
148 Figure 32: Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum
BHCG, Serum Endoglin, Serum FLT-1 and Serum PLGF with foetal sex:
Multiples of median (MOM) of plasma prokineticin 1 (PK1), kisspeptin (KP), serum
BHCG (HCG), serum endoglin (ENG), serum FLT-1 (FLT-1) and placental growth factor
(PLGF) in mothers with singleton pregnancy who gave birth to male babies (M) and in mothers who gave birth to female babies (F). Each pair of multiple of medians was compared by unpaired t test. * P<0.05, ** P<0.01.
2.5 2.0 Multiples 1.5 ** * of Median 1.0 0.5 0.0 M F . M F . M F . M F . M F . M F PK-1 KP HCG ENG FLT-1 PLGF
149 Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG, Serum
Endoglin, Serum FLT-1 and Serum PLGF with Birth weight:
Previous reports have suggested that maternal kisspeptin levels were lower in women with intra-uterine growth restriction (IUGR) (Armstrong et al. 2009).
Therefore it was interesting to see whether mothers who gave birth to neonates with low body weight had altered levels of pregnancy markers when compared with women who gave birth to neonates with normal body weight.
Low birth weight is often defined as a birth weight less than 2500g (Mcallister et al. 2014).
As foetal sex independently had an effect on kisspeptin and BHCG levels and foetal sex may have an impact on neonatal weight, I analysed the association of these markers with birthweight in each foetal sex separately. Plasma kisspeptin was the best performing of the pregnancy markers in identifying women who will have babies with low body weight.
Kisspeptin was significantly lower in women with boys born weighing less than 2500g, than in women with boys born weighing 2500-4000g. Furthermore plasma kisspeptin was significantly higher still in women with baby boys born weighing more than 4000g. Plasma kisspeptin was also lower in women with female neonates weighing less than 2500g, although this was not significantly when compared with one way ANOVA with post hoc bonferroni correction. Placental growth factor showed a similar pattern of secretion in mothers of baby boys, however was not altered in a statistically significant manner.
This data is consistent with previous reports suggesting that kisspeptin is lower in women with intra-uterine growth restriction and provides further evidence that plasma kisspeptin may be representative of placental function.
150 Figure 33: Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum
BHCG, Serum Endoglin, Serum FLT-1 and Serum PLGF with Birth weight in boys:
Mean ±SEM of Multiple of Median (MOM) of plasma PK1, plasma Kisspeptin, Serum
BHCG, Serum Endoglin, Serum FLT-1 and Serum PLGF was compared in mothers of male neonates by birthweight categories (<2500g, 2500-4000g and >4000g) by one way
ANOVA with post hoc bonferroni correction. ** P<0.01, *** P<0.001.
** 2.5 1.4 *** 1.2 2.0 MOM of MOM of 1.0 1.5 plasma 0.8 plasma PK1 Kisspeptin 1.0 0.6 0.4 0.5 0.2 0.0 0.0 <2500g 2500g-4000g >4000g <2500g 2500g-4000g >4000g birth weight in boys (g) birth weight in boys (g)
1.2 1.2 1.0 1.0 0.8 MOM of MOM of 0.8 serum HCG 0.6 serum endoglin 0.6 0.4 0.4 0.2 0.2 0.0 0.0 <2500g 2500g-4000g >4000g <2500g 2500g-4000g >4000g birth weight in boys (g) birth weight in boys (g)
1.4 * 1.4 1.2 1.2 1.0 1.0 MOM of MOM of serum FLT-1 0.8 serum PLGF 0.8 0.6 0.6 0.4 0.4 0.2 0.2 0.0 0.0 <2500g 2500g-4000g >4000g <2500g 2500g-4000g >4000g birth weight in boys (g) birth weight in boys (g)
151 Figure 34: Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum
BHCG, Serum Endoglin, Serum FLT-1 and Serum PLGF with Birth weight in girls.
Mean ±SEM of Multiple of Median (MOM) of plasma PK1, plasma Kisspeptin, Serum
BHCG, Serum Endoglin, Serum FLT-1 and Serum PLGF was compared in mothers of female neonates by birthweight categories (<2500g, 2500-4000g, and >4000g) by one way
ANOVA with post hoc bonferroni correction.
3.5 1.2 3.0 1.0 2.5 MOM of MOM of 0.8 2.0 plasma plasma PK1 0.6 1.5 Kisspeptin 1.0 0.4 0.5 0.2 0.0 0.0 <2500g 2500g-4000g >4000g <2500g 2500g-4000g >4000g birth weight in girls (g) birth weight in girls (g)
1.4 1.2 1.2 1.0 1.0 MOM of 0.8 MOM of 0.8 serum endoglin 0.6 serum HCG 0.6 0.4 0.4 0.2 0.2 0.0 0.0 <2500g 2500g-4000g >4000g <2500g 2500g-4000g >4000g birth weight in girls (g) birth weight in girls (g)
1.4 1.2 1.2 1.0 1.0 MOM of 0.8 0.8 MOM of serum FLT-1 0.6 0.6 serum PLGF 0.4 0.4 0.2 0.2 0.0 0.0 <2500g 2500g-4000g >4000g <2500g 2500g-4000g >4000g birth weight in girls (g) birth weight in girls (g)
152 Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG, Serum
Endoglin, Serum FLT-1 and Serum PLGF with head circumference:
Another commonly used anthropometric measurement of neonatal size is head circumference. Therefore I analysed whether the pregnancy markers evaluated were able to identify women bearing neonates with alteration in head circumference. As previously, I evaluated the pregnancy markers separately in women bearing male and women bearing female foetuses (see Figure 35 and 36).
Again kisspeptin was the only marker which was significantly altered, being higher in baby boys with larger head circumference than those with smaller head circumference.
None of the other pregnancy markers were significantly altered by category of neonatal head circumference.
153 Figure 35: Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum
BHCG, Serum Endoglin, Serum FLT-1 and Serum PLGF with head circumference in boys.
Mean ±SEM of Multiple of Median (MOM) of plasma PK1, plasma Kisspeptin, Serum
BHCG, Serum Endoglin, Serum FLT-1 and Serum PLGF was compared in mothers of male neonates by categories of head circumference (<33cm, 34-36cm, and >36cm) by one way
ANOVA with post hoc bonferroni correction.
3.0 1.2 ** 2.5 MOM of 1.0 2.0 MOM of plasma 0.8 1.5 plasma PK1 0.6 1.0 Kisspeptin 0.4 0.5 0.2 0.0 0.0 <33 34-36 >36 <33 34-36 >36 Head circumference in boys (cm) Head circumference in boys (cm)
1.2 1.2 1.0 MOM of 1.0 0.8 MOM of serum 0.8 0.6 serum BHCG 0.6 0.4 endoglin 0.4 0.2 0.2 0.0 0.0 <33 34-36 >36 <33 34-36 >36 Head circumference in boys (cm) Head circumference in boys (cm)
1.2 1.2 1.0 MOM of 1.0 0.8 MOM of serum 0.8 0.6 serum FLT-1 0.6 0.4 PLGF 0.4 0.2 0.2 0.0 0.0 <33 34-36 >36 <33 34-36 >36 Head circumference in boys (cm) Head circumference in boys (cm)
154 Figure 36: Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum
BHCG, Serum Endoglin, Serum FLT-1 and Serum PLGF with head circumference in girls.
Mean ±SEM of Multiple of Median (MOM) of plasma PK1, plasma Kisspeptin, Serum BHCG,
Serum Endoglin, Serum FLT-1 and Serum PLGF was compared in mothers of female neonates by categories of head circumference (<33cm, 34-36cm, and >36cm) by one way ANOVA with post hoc bonferroni correction.
3.0 1.2 2.5 MOM of 1.0 2.0 MOM of plasma 0.8 1.5 plasma PK1 0.6 1.0 Kisspeptin 0.4 0.5 0.2 0.0 0.0 <33 34-36 >36 <33 34-36 >36 Head circumference in girls (cm) Head circumference in girls (cm)
1.2 1.2 1.0 1.0 MOM of MOM of 0.8 0.8 serum serum 0.6 BHCG 0.6 endoglin 0.4 0.4 0.2 0.2 0.0 0.0 <33 34-36 >36 <33 34-36 >36 Head circumference in girls (cm) Head circumference in girls (cm)
1.2 1.4 1.0 1.2 MOM of 0.8 MOM of 1.0 serum 0.6 serum 0.8 FLT-1 PLGF 0.6 0.4 0.4 0.2 0.2 0.0 0.0 <33 34-36 >36 <33 34-36 >36 Head circumference in girls (cm) Head circumference in girls (cm)
155 Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum BHCG, Serum
Endoglin, Serum FLT-1 and Serum PLGF with prematurity:
Premature delivery is frequently defined as giving birth prior to 37 weeks of gestation.
Premature delivery is associated with increased neonatal morbidity and could be due to abnormal placentation. Therefore I evaluated whether these markers were altered in women who gave birth at less than 37 weeks of gestation when compared with women who gave birth when they were due (see Figure 37).
Only PK-1 was statistically significantly altered being lower in women who gave birth prematurely than women who gave birth when due.
156 Figure 37: Changes in plasma levels of plasma PK1, plasma Kisspeptin, Serum
BHCG, Serum Endoglin, Serum FLT-1 and Serum PLGF with premature delivery.
Mean ±SEM of Multiple of Median (MOM) of plasma PK1, plasma Kisspeptin, Serum
BHCG, Serum Endoglin, Serum FLT-1 and Serum PLGF in women with premature delivery (<36 weeks) and in women with delivery which was not premature (>37 weeks).
Each pair of multiples of median was compared by unpaired t test; * P<0.05.
2.5 * 2.0 Multiples 1.5 of Median 1.0 0.5 0.0 <36 >37 . <36 >37 . <36 >37 . <36 >37 . <36 >37 . <36 >37 PK1 KP HCG ENG FLT-1 PLGF Gestation at Delivery
157 Discussion
A fifth of pregnancies end in miscarriage, and the damaging emotional impact can lead to depression and anxiety which may last for several months (Lee et al. 1996).
PK-1 did not appear to change with gestation, although did appear to be reduced in women with premature delivery. Of the markers evaluated, kisspeptin showed the most promise as a screening test for identifying asymptomatic women at increased risk of pregnancy complications due to abnormal placentation. Women with lower kisspeptin levels corrected for gestation had an increased risk of subsequently suffering miscarriage, and women with imminent miscarriage had even lower levels of kisspeptin. Kisspeptin had the greatest area under a ROC curve for identification of asymptomatic women at increased risk of subsequent miscarriage.
Women who gave birth to boys with low birth weight and small head circumference also had lower levels of kisspeptin than in women with babies with normal anthropometric parameters. Furthermore, women with risk factors for pregnancy complications due to abnormal placentation such as women with increased BMI, women who smoke during pregnancy and women with recurrent miscarriage, also had lower levels of plasma kisspeptin than gestation-corrected values found in mothers without these risk factors.
Miscarriage occurs most commonly during early pregnancy, however due to the logistics of confirmation of pregnancy and referral for the first antenatal pregnancy visit, most women attend their first antenatal screening visit at 8 weeks of gestation or more. At 8 weeks of gestation many miscarriages may have already occurred. However miscarriage in early pregnancy is predominantly due to foetal chromosomal abnormality and is hence inevitable and less amenable to intervention. Miscarriage in later pregnancy is more likely to be
158 associated with treatable causes of miscarriage such as autoimmune or thrombotic disorders. It is therefore possible that a safe and mimimally invasive test such as a blood kisspeptin level could be used to identify pregnancies at increased risk of pregnancy complications such as miscarriage, and this risk stratification could allow for targeted obstetric surveillance or intervention in identified women.
In summary, I have shown that plasma kisspeptin measurement in asymptomatic women at antenatal booking visit provides a highly predictive marker for identifying asymptomatic women at risk of miscarriage and other pregnancy complications.
159
Chapter 4
General Discussion
160 Prokineticin-1 (PK-1) and prokineticin-2 (PK-2) are two closely related cysteine-rich peptides which both bind to the prokineticin-1 and prokineticin-2 receptors (PKR-1 and
PKR-2). The prokineticins were originally named due to their ability to stimulate gastrointestinal motility. Since that time, the prokineticins have been found to play a role in other biological processes including reproduction and nociception. Prokineticin-2 is known to be expressed in regions of the central nervous system involved in food intake. Consistent with this, previous work from our own department has shown that administration of prokineticin-2 to rodents reduces food intake both in lean and obese rodents.
However Prokineticin-2 is predominantly expressed within the central nervous system, whereas prokineticin-1 is heavily expressed within the gut. I therefore hypothesised that prokineticin 1 may be a novel gut hormone. In this thesis, I investigated the physiological role of prokineticin-1 in controlling appetite in rodents and also compared its efficacy with prokineticin-2. I also measured the changes in plasma levels of prokineticin in humans during feeding to establish the potential physiological relevance of prokineticin-1 as an anorectic gut hormone.
Central administration of PK-1 was as effective as equimolar doses of PK-2 at reducing food intake. Central PK-1 or PK-2 administration both significantly reduced food intake during the first hour following administration down to 22% following 1500 pmol. However there were not significant reductions in food intake beyond this time frame suggesting a short duration of action.
If PK-1 were to be used as a therapeutic to combat obesity, it is be important to assess the peripheral effects of PK-1 administration. Peripheral IP injection of PK-1 reduced food intake by just over half during the first hour following administration. Doses of 20 and 60 nmol/kg of both PK-1 and PK-2 similarly reduced food intake over the first hour by approximately half when compared with saline. Impressively a dose of 540 nmol/kg of 161 either PK-1 or PK-2 reduced food intake by over 90% during the first hour. However the effect on food intake had worn off by 4 hrs post administration, suggesting a short duration of action.
I also measured the serum PK-1 levels by a commercial ELISA kit (Peprotech) in mice.
Serum PK-1 levels were highest at 20 minutes following injection at 40 pg/ml, before falling to 10 pg/ml at 60 minutes following administration suggesting a short half life of approximately half an hour. This would be consistent with the duration of action of food intake during the experiments. Given the short duration of action it would seem reasonable that more frequent administration may have more profound effects on body weight over a longer period and similar experiments with PK-2 have suggested that tachyphylaxis to the prokineticins with repeated administration does not occur.
PK-1 expression was also reduced in the stomachs of fasted mice. This would be consistent with the pattern of expression which would be expected if PK-1 was a physiological gut hormone, being low in fasted mice and increasing with food intake to signal satiety to the appetite centres in the brain. However if PK-1 was a physiological gut hormone then it would be expected that plasma PK-1 levels would be low during fasting and rise following food intake signalling satiety. However I was not able to demonstrate a clear effect on circulating PK-1 levels in humans following feeding, which may either be due to technical issues or due to the existence of other currently unidentified more dominant factors on circulating PK-1 levels. Possible technical reasons for not seeing a rise in plasma PK-1 levels may be due to difficulties in measuring circulating PK-1 with the antibodies used in current commercially available ELISA kits, as many participants had levels which were towards the lower end of the sensitivity of the assay. Another possibility is that other unidentified factors may influence plasma PK-1 levels to a greater degree than those seen following feeding. Reliability of the assay may have been compromised by interfering 162 factors, binding proteins or non-specificity of the antibody, making it difficult to draw firm conclusions regarding circulating levels of PK-1 with feeding. We are in the process of developing our own in-house prokineticin assay in order to be able to more accurately characterise the circulating levels of PK-1 with feeding and during pregnancy.
In summary, I have demonstrated that both central and peripheral administration of PK-1 potently reduces food intake and that experession of PK-1 levels in the stomachs of mice change in manner consistent with that of a physiological gut hormone. PK-1 therefore represents a novel target for the development of anti-obesity therapies. The efficacy of some anorectic hormones in obesity is limited by resistance in obesity models, such as seen with leptin. However prokineticin-2 has been shown to be effective in diet induced obese mice (Gardiner et al. 2010). Furthermore, some anti-obesity therapies can be susceptible to tachyphylaxis with repeated administration such that seen with melanocortin agonists
(Gardiner et al. 2010). However the prokineticins do not appear to be susceptible to tachyphylaxis with repeated dosing (Gardiner et al. 2010). The discovery of novel gut hormones is important, in that there is likely to be redundancy in the system with multiple gut hormones being involved in controlling appetite. It is likely that multiple agents with a small individual effect will need to be used in combination at small doses in order to minimise side effects whilst having additive effects on reducing food intake. Prokineticin-1 may therefore represent an important target for the development of anti-obesity therapies.
Prokineticin-1 is also a potent angiogenic mitogen on endocrine vascular epithelium and hence it is also known as ‘Endocrine Gland-Vascular Endothelial Factor’. Placental angiogenesis plays an important role in placental function and risk of pregnancy complications such as miscarriage. I therefore hypothesised that prokineticin-1 is a biomarker of pregnancy complications in humans. In this thesis, I have measured 163 circulating levels of prokineticin-1 found in pregnancy and correlated these with the occurrence of pregnancy complications. I have also compared the utility of prokineticin 1 with other potential novel markers of pregnancy complications.
Circulating PK-1 levels did not appear to be the best performing marker for identifying complications associated with pregnancy. PK-1 did not change with gestation, although did appear to be reduced in women with premature delivery. Of the markers evaluated, plasma kisspeptin showed the most promise as a screening test for identifying asymptomatic women at increased risk of pregnancy complications due to abnormal placentation. Women with lower kisspeptin levels corrected for gestation had an increased risk of subsequently suffering miscarriage, and women with imminent miscarriage had even lower levels of kisspeptin. Kisspeptin also had the greatest area under a ROC curve for identification of asymptomatic women at increased risk of subsequent miscarriage.
Women who gave birth to boys with low birth weight and small head circumference also had lower levels of kisspeptin than in women with babies with normal anthropometric parameters. Furthermore, women with risk factors for pregnancy complications due to abnormal placentation such as women with increased BMI, women who smoke during pregnancy and women with recurrent miscarriage, also had lower levels of plasma kisspeptin than gestation-corrected values found in mothers without these risk factors.
Miscarriage occurs most commonly during early pregnancy, however due to the logistics of confirmation of pregnancy and referral for the first antenatal pregnancy visit, most women attend their first antenatal screening visit at 8 weeks of gestation or more. At 8 weeks of gestation many miscarriages may have already occurred. However miscarriage in early pregnancy is predominantly due to foetal chromosomal abnormality and is hence inevitable and less amenable to intervention. Miscarriage in later pregnancy is more likely to be associated with treatable causes of miscarriage such as autoimmune or thrombotic 164 disorders. It is therefore possible that a safe and mimimally invasive test such as a blood kisspeptin level could be used to identify pregnancies at increased risk of pregnancy complications such as miscarriage, and this risk stratification could allow for targeted obstetric surveillance or intervention in identified women.
In summary, I have shown that plasma kisspeptin measurement in asymptomatic women at antenatal booking visit provides a highly predictive marker for identifying asymptomatic women at risk of miscarriage and other pregnancy complications.
165
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201
Appendices
202 Appendix 1: Solutions
Bovine serum albumin (BSA) solution (2mg/ml): Dissolve 2g of BSA in 1l of 0.01M
PBS.
0.5M ethylenediaminetetra-acetic acid (EDTA): In 800ml autoclaved water dissolve
186.1g C10H14H2O8Na2.2H2O and adjust to pH 8.0 with 1M NaOH. Make up to 1L with water.
Phosphate buffer (RIA buffer): 48g of Na2HPO4.2H2O, 4.13g KH2PO4, 18.61g
C10H14H2O8Na2.2H2O, 2.5g NaN3 were dissolved in 5L of GDW, which had been boiled and allowed to cool, the pH was measured to confirm it was 7.4 ± 0.1 and the buffer was stored at 4oC.
0.01M phosphate buffered saline (PBS): Carry out a 1:100 dilution of 1M PBS as prepared above.
203 Appendix 2: Amino Acid Codes
Amino Acids Codes
Three letter Single letter
Glycine Gly G
Proline Pro P
Alanine Ala A
Valine Val V
Leucine Leu L
Isoleucine Ile I
Methionine Met M
Cysteine Cys C
Phenylalanine Phe F
Tyrosine Tyr Y
Tryptophan Trp W
Histidine His H
Lysine Lys K
Arginine Arg R
Glutamine Gln Q
Asparagine Asn N
Glutamic Acid Glu E
Aspartic Acid Asp D
Serine Ser S
Threonine Thr T
204 Appendix 3: List of Suppliers
Abnova, Taiwan
Abbott Animal Health, Kent, UK
Adam Equipment, Milton Keynes, UK.
Advanced Biotechnology Centre, Imperial College London, UK
Ambion, Warrington, UK)
Amersham International plc, Little Chalfont, Buckinghamshire, UK.
Associated Dental Products Ltd, Kemdent Works, Purton, Swindon, UK
ATCC, Middlesex, UK
Bachem St. Helens, UK
Bayer UK, Bury St. Edmonds, UK
BOC Gases, Guildford, Surrey, UK
Bright Instruments, Huntingdon, UK
Charles River, Margate, UK.
Clark Electromedial Instruments, Pangbourne, Reading, UK
Columbus Instruments, Columbus, Ohio, USA
CrystalChem, Illinois, USA
Dako UK Ltd., Cambridgeshire, UK
Durect Corporation, Cupertino, CA, USA
Fisher Scientific, Leicestershire, UK
Gibco BRL (Life Technologies Ltd.), Paisley, Renfrewshire, UK.
GraphPad, La Jolla, CA, USA
Harlan, Bicester, Oxon, UK
Harvard Apparatus, Massachusetts, USA
205 Helena Biosciences, Sunderland, UK
Invitrogen, Paisley, UK
Johnson and Johnson, Livingston, UK
Life Sciences Technology, Eggenstein, Germany
Merck Chemicals Ltd., Nottingham, UK
Millipore, Watford, Herts, UK
New England Bioloabs, Hitchin, Herts, UK
Novagen, Nottingham, UK
Parke-Davis, Pontypool, Gwent, UK.
Peninsula Laboratories, St Helen’s, UK
PeproTech Ltd., London, UK
Peptide Institute, Kyoto, Japan.
Perkin Elmer, Massachusetts, USA
Pharmacia Biotechnology Ltd., St. Albans, Hertsfordshire, UK.
Plastics One Inc, Roanoke, VA, USA.
Promega, Southampton, UK
Qiagen, Hilden, Germany.
Reno, NV, USA
Research Diets Inc, New Brunswick, NJ, USA
Roanoke, VA, USA
Santa Cruz Biotechnology Inc., California, USA
Schering-Plough Corporation, Welwyn Garden City, Herts, UK
SDS UK Ltd, Witham, Essex, UK.
Seton Healthcare, Oldham, Lancs, UK
Shandon Southern Products, Runcorn, Cheshire, UK 206 Sigma-Aldrich Company Ltd., Poole, Dorset, UK.
StataCorp, College Station, Texas, USA
Systat Ltd., Evanston, Illinois, USA.
VetTech Solutions, Cheshire, UK
VWR, Poole, UK
207