The role of placental alkaline phosphatase in the regulation of insulin-like growth factor binding

213 Pages·2011·2.43 MB·English

The role of placental alkaline phosphatase in the
regulation of insulin-like growth factor binding
protein-1 in pregnancy complicated by diabetes

A thesis submitted to the University of Manchester for the degree of
PhD in the Faculty of Medical and Human Sciences

2011

Alexandra Lubina Solomon

School of Medicine
Table of Contents
List of figures………………………………………………………………………..12
List of tables………………………………………………………………………....15
Abstract….………………………………..………………………………………...16
Abbreviations……………...……………………….………………………………..20

Chapter 1 General introduction
1.1 Diabetes Mellitus (DM) and pregnancy………………………….…25
1.2 Abnormal fetal growth………………………………………………26
1.2.1 Macrosomia...…………………………………………………………26
1.2.2 Small for gestational age (SGA) and intrauterine growth
restriction (IUGR)……………………………………….…………..26
1.3 Human placental function and structure…………………………..28
1.3.1 Normal pregnancy……………………………………………………28
1.3.2 Pregnancy complicated by maternal DM…………………………….28
1.4 Insulin-like growth factor (IGF) axis: introduction……………….30
1.4.1 IGFs and fetal growth in normal pregnancy…………………….…..31
1.4.1.1 In vitro studies……….……………………………………………………….31
1.4.1.2 Animal models……………………………………………………………….32
1.4.1.3 Human pregnancy: fetal IGFs…..…………………………………………32
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1.4.1.4 Human pregnancy: maternal IGFs………………………………………..33
1.4.1.5 Human pregnancy: placental IGFs………………..………………………33
1.4.1.6 IGFs in patients with diabetes………………………………………..…….33
1.4.2 IGFBPs in pregnancy……………………………………………..….34
1.4.2.1 Expression of IGFBPs on human placenta….………………………….34
1.4.2.2 IGFBP-1 in human maternal/cord/fetal serum and amniotic fluid…... 35
1.4.2.3 IGFBP-1 in pregnancy complicated by maternal diabetes…………….35
1.4.2.4 IGFBP-1 in animal models……………...………………………………….36
1.4.3 Phosphoforms of human IGFBP-1………….……………………….36
1.4.4 Regulation of IGFBP-1………………………………...…………….39
1.4.4.1 IGFBP-1 regulation in vitro….…………………………………………….39
1.4.4.2 IGFBP-1 and glucose homeostasis in animal studies…………………..40
1.4.4.3 IGFBP-1 regulation in clinical studies….………………………………..40
1.4.4.4 IGFBP-1 proteolysis……………………….………………………………..40
1.4.4.5 Summary of the regulation of IGF/IGFBP-1 on the maternal/fetal
interface…..………………………………………………….………………..40
1.5 Alkaline Phosphatase (AP): introduction…………………….…….43
1.5.1 Total AP and PLAP in human pregnancy….…….………………….44
1.5.1.1 Placenta, its membranes and amniotic fluid……………………………..44
1.5.1.2 Cord blood……………………………………………………………………45
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1.5.1.3 Maternal serum AP and PLAP……….…………………………………….45
1.5.2 Total AP and PLAP in complicated human pregnancy.…………….46
1.5.3 Total AP and PLAP in human pregnancy with diabetes……………46
1.5.3.1 Maternal serum PLAP………………………………………………………47
1.5.3.2 Placenta……………………………………………………………………….47
1.6 Project hypothesis……………………………………………………49
1.7 Aims for PhD project………………………………………………..50
Chapter 2 Materials and methods

2.1 Placental sample collection and processing…….…………………..52
2.2 Cell culture…………………………………………………………...53
2.3 Sources of IGFBP-1………………...……………………………….54
2.3.1 HepG2-CM and its collection…………………………..…………….54
2.3.2 Purified pIGFBP-1…………………..……………………………….54
2.3.3 Human plasma obtained from non-pregnant subjects………………55
2.4 PLAP and total AP activity assays………………………………….55
2.4.1 Protocol 1……………………………………………………………..55
2.4.1.1 Explants co-cultured with IGFBP-1 (PLAP activity assay)……….…...55
2.4.1.2 Explant-CM co-cultured with IGFBP-1 (soluble PLAP activity assay)56
2.4.2 Protocol 2: explants co-cultured with pIGFBP-1 (PLAP activity
assay)…………………………………...……………………………..56
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2.4.3 Total alkaline phosphatase (AP) activity assay….…………………..57
2.5 PLAP inhibition by PLAP-specific monoclonal antibody……...….57
2.6 Western blotting …………………….………………………………57
2.6.1 Preparation of tissue lysates…………………………………………57
2.6.2 Protein quantification………………………………………………...58
2.6.3 Preparation of samples for electrophoresis…...……………………..58
2.6.3.1 IGFBP-1 analysis (non-reducing conditions)…………………………....58
2.6.3.1.1 Analysis of tissue samples……………………….………………………….58
2.6.3.1.2 Analysis of IGFBP-1 present in solution….………………………………58
2.6.3.2 PLAP analysis (reducing conditions)……………………………………..59
2.6.4 Protein separation…………………………………………………….59
2.6.4.1 SDS-electrophoresis….………………………………………….…………..59
2.6.4.2 nog electrophoresis………………………………………………………….59
2.6.5 Protein transfer.………………………………………………………60
2.6.6 Protein detection………………..……………………………………60
2.6.6.1 Western immunoblotting (western blot, WB)……….…………………….60
2.6.6.2 Western ligand blotting……………………………………………………..61
2.6.7 Development…………….……………………………………….……62
2.6.8 Re-probing…………………………………………………………….62
2.6.9 Densitometry…………………………………………………...……..62
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2.7 Immunostaining………………………………………...……………62
2.8 Real time polymerase chain reaction (RT-PCR)……….………….63
2.8.1 RNA extraction……………………………………………………….63
2.8.2 RNA quantification…….…………………………………………….64
2.8.3 cDNA generation…………………………………………………….64
2.8.4 Real- time PCR…………...…………………………………………..64
2.9 Lactate dehydrogenase (LDH) assay……………………………….65
2.10 Statistical analysis……………..……………………………………..66
2.11 Clinical study methods………………………………………………66
Chapter 3: results Selection of a suitable model
for in vitro study of the IGF system/PLAP

interrelation-ship in pregnancy

3.1 Introduction……………………………………..……………….…..68
3.2 Results and discussion……………………………………………….69
3.2.1 Tissue morphology analysis………………….………………………69
3.2.2 Lactate dehydrogenize (LDH) assay…………………………………73
3.2.3 Analysis of PLAP in term human placenta…….……………………75
3.2.3.1 Placental PLAP expression (RT-qPCR)………….……………………….75
3.2.3.2 Placental PLAP expression (western blot)………………………………77
3.2.3.3 Placental PLAP expression (immunohistochemistry)….……………….79
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Chapter 4: results De-phosphorylation of IGFBP-
1 is mediated by placenta via PLAP activity

4.1 Introduction…………………………………...……………………..83
4.2 Results……..…………………………….……………………………84
4.2.1 IGFBP-1 culture and its in vitro de-phosphorylation….……………84
4.2.1.1 IGFBP-1 derived from Hep G2-CM……………………………………….84
4.2.1.2 IGFBP-1 of human plasma origin……..…………………………………..87
4.2.2 Optimization of IGFBP-1 incubation conditions…..………………..89
4.2.3 Placenta de-phosphorylates IGFBP-1 (human serum)………...….93
4.2.4 IGFBP-1 binds to the placenta following exposure to explants.…..95
4.2.5 Placenta de-phosphorylates IGFBP-I due to PLAP activity…….…..95
4.3 Discussion…………………………………………………………...101
4.3.1 Placenta, but not placental-CM de-phosphorylates IGFBP-1….….101
4.3.2 PLAP governs IGFBP-1 de-phosphorylation………………………102
Chapter 5: results Regulation of PLAP
expression/ activity in the placenta

5.1 Introduction…………….…………………………………………..110
5.2 Methods……….…………………………………………………….112
5.3 Results……………………………………………………………….112
5.3.1 The influence of glucose on PLAP…………………………………112
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5.3.1.1 Glucose concentration in culture media.............…….………………..112
5.3.1.2 Analysis of the effect of glucose on PLAP protein expression……….114
5.3.1.3 Effect of glucose on PLAP activity……………………………………….116
5.3.2 The influence of insulin, IGF-I and IGF-II on PLAP activity…….118
5.3.3 The influence of hypoxia on IGFBP-1 de-phosphorylation.……...121
5.3.4 Summary of the results…………………………………………..….121
5.4 Discussion…………………………………………...………………123
5.4.1 The effect of glucose on PLAP………………………..…………….123
5.4.2 The effect of insulin and IGFs on PLAP activity…….…………….125
5.4.3 The effect of hypoxia on IGFBP-1 de-phosphorylation………...…127
Chapter 6: results PLAP and the IGF system in
pregnancy complicated by diabetes and

accelerated fetal growth

6.1 Introduction……………….……………….……………………….131
6.2 Methods and study design………………………………………….131
6.2.1 Study design…………………………………………………………131
6.2.2 Recruitment……………………………………….…………………132
6.2.2.1 Patients with diabetes…….……….……………………….………………132
6.2.2.2 Healthy controls…………………………………………………………….134
6.2.3 Collection and analysis of clinical data and samples………………134
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6.2.3.1 Clinical data collection……..……………………………………………..134
6.2.3.2 Anthropometric measurements of the neonates….……………………..135
6.2.3.3 Placenta………..…………………………………………………………….135
6.2.3.4 Maternal and cord blood…….……………………………………………136
6.2.3.4.1 Blood collection……..……….……………………………………………..136
6.2.3.4.2 IGF-I assay……….…………………………………………………………136
6.2.3.4.3 IGF-II assay…….…………………………………………………………..137
6.2.3.4.4 Measurement of total IGFBP-1 concentration…………………………137
6.2.3.4.5 Analysis of the phosphorylation status of IGFBP-1……………………137
6.2.3.5 Statistical analysis………………………………………………………….138
6.3 General characteristics of recruited subjects……………………..140
6.3.1 Characteristics of the subjects enrolled into the study……………..140
6.3.2 Past and present medical history of enrolled subject………………140
6.3.3 Treatment and follow up of recruited subjects…….…………….....143
6.3.3.1 Treatment before pregnancy………………………….…………………..143
6.3.3.2 Treatment during pregnancy………………….…………………………..143
6.3.4 Pregnancy clinical outcome………………………...………………144
6.3.4.1 General infant outcome………………..…………………………………..144
6.3.4.2 Infant birth weight……………..…………………………………………...144
6.3.4.2.1 Infant birth weight in relation to maternal characteristics…….....…145
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6.3.4.2.2 Infant weight in relation to diabetes control and treatment………..…147
6.3.4.3 Maternal outcome (whole study group)…………………………………147
6.4 Results…………….…………………………………………………149
6.4.1 Infant and placental weight…………………………………………149
6.4.2 Infant weight and maternal IGF system…………………………..151
6.4.2.1 IGF-I/ II in maternal blood……………………………………………….151
6.4.2.2 Maternal total IGFBP-1…….……………………………………………..154
6.4.2.3 PLAP…………..……………………………………………………………..156
6.4.2.3.1 PLAP expression…….……………………………………………………..156
6.4.2.3.2 Phosphorylation status of IGFBP-1 in maternal blood………………158
6.4.2.3.3 Placental PLAP activity (ex-vivo)……………………………...……162
6.4.3.1 Infant growth and cord IGF system……….……………………………..164
6.4.3.1 IGF-I/II in cord blood………..…………………….……………………..164
6.4.3.2 Total cord IGFBP-1…………………..………………………………..….169
6.4.3.3 Phosphorylation status of cord IGFBP-1……………...………………..171
6.5 Discussion……………………………………………………….…..173
6.5.1 Maternal factors and fetal growth………………………………….173
6.5.2 Placental and fetal growth……………………………….………….176
6.5.3 Maternal IGF system in relation to placental and fetal growth…...176
6.5.3.1 Maternal IGFs………………………………………………………………176
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