impact of glucocorticoids on placental growth and ... · glucocorticoids and placental growth 7...

Impact of Glucocorticoids on

Placental Growth and Vascularisation

by

Damien Phillip Hewitt, BSc (Hons)

This thesis is presented for the degree of

DOCTOR OF PHILOSOPHY

of The University of Western Australia,

School of Anatomy and Human Biology

Submitted: April, 2007

2 Glucocorticoids and Placental Growth

Glucocorticoids and Placental Growth 3

To my family


Preface

The experimental work in this thesis was undertaken in the School of Anatomy and

Human Biology, The University of Western Australia under the supervision of

Professor Brendan Waddell and Dr Peter Mark, with the financial assistance of a

University Postgraduate Award.

The work described is original and was carried out by myself except where the specific

contributions of other persons are acknowledged.

Damien Phillip Hewitt

April 2007


ABSTRACT

Glucocorticoids are critical for the maturation of the fetus late in pregnancy. Indeed,

clinical administration of glucocorticoids is used to accelerate fetal lung maturation in

mothers at risk of pre-term delivery. Increased glucocorticoid exposure, however, can

have detrimental effects on fetal and placental growth and increase the risk of disease in

later life. Many studies have focused on the effect of an increase in the transplacental

passage of glucocorticoids on both fetal growth and subsequent postnatal development.

But there is a growing body of evidence to suggest that the impact of glucocorticoids on

fetal growth is mediated, in part, via their direct effects on the placenta. The regulation

of placental growth and development involves a complex balance of numerous

signalling mechanisms, and among an extensive list of potential candidates affected by

glucocorticoids, the studies in this thesis have focused on the role of three related

pathways.

Firstly, the nuclear receptor, peroxisome proliferator-activated receptor γ (PPARG),

initially identified for its role in insulin sensitivity, has been implicated in placental

development from gene deletion studies in the mouse. Most notably, the placentas of

Pparg null mice displayed impaired vascularisation, raising the possibility that PPARG

may also be important in promoting the growth of fetal vessels late in pregnancy.

Secondly, the secreted frizzled related protein-4 (SFRP4) is a secreted form of the

frizzled receptor that inhibits the normal action of wnt molecules. The wnt signalling

pathway is a highly conserved pathway that plays a crucial role in the regulation of cell

growth, differentiation and apoptosis. Accordingly, gene deletion studies involving a

range of signalling molecules within the wnt pathway show that wnt signalling is

essential for placental development. Furthermore, the expression of SFRP4 in particular

is associated with anti-proliferative and pro-apoptotic signalling in other reproductive

tissues. Finally, the endothelial cell-specific mitogen, vascular endothelial growth factor

(VEGF) plays a crucial role in the establishment and maintenance of a functional

placenta, with gene deletion studies demonstrating that the loss of even a single allele of

VEGF results in an embryonic lethal phenotype due to a failure in the development of

placental vasculature. Thus, placental expression of the major VEGF isoforms was also

investigated in relation to detailed stereological analysis of placental labyrinthine

vascularisation in normal pregnancy and after increased glucocorticoid exposure. Each

parameter was assessed in the two functionally- and morphologically-distinct regions of


the rat placenta, the basal and labyrinth zones across the final third of normal pregnancy

(day 16 to 22; term = day 23), and in response to increased glucocorticoid exposure (1

µg/ml dexamethasone acetate in maternal drinking water, days 13-22).

Expression and activation of PPARG was upregulated (65% increase) specifically in the

rapidly growing labyrinth zone over the final third of normal pregnancy, but this

increase was inhibited (37% reduction) after maternal treatment with dexamethasone. In

contrast, the expression of SFRP4 was markedly upregulated (14-fold increase) in the

regressing basal zone near term, whereas labyrinthine expression of SFRP4 remained

low over the final third of pregnancy. Dexamethasone treatment increased expression of

SFRP4 in both basal (1.2-fold increase) and labyrinth zones (2.8 fold increase) and was

associated with increased nuclear translocation of β-catenin in giant trophoblasts of the

basal zone. These data suggest that dexamethasone inhibits wnt-mediated growth

signalling in the placenta, consistent with the well-recognised, anti-proliferative effects

of glucocorticoids.

Unbiased stereological analyses of the labyrinth zone showed a 4-5 fold increase in the

volume and surface area of maternal and fetal blood spaces, including vascular

remodeling of the fetal capillary network near term. In accordance with the observed

vascular development, quantitative PCR analysis demonstrated that trophoblastic

expression of each VEGF isoform was markedly upregulated (2- to 5-fold increases),

specifically in the labyrinth zone over the final third of normal pregnancy.

Immunohistochemical analyses confirmed expression of VEGF protein within

labyrinthine trophoblast. Dexamethasone treatment reduced normal vascular

development within the labyrinth zone near term (50-70% reduction in maternal and

fetal blood space volume and surface areas). Most notably, dexamethasone impaired the

normal increase in fetal vessel density (30-50% reduction in volume and surface area

compared to untreated controls) over the final third of pregnancy, but had no effect on

the density of maternal blood spaces. Expression of the Vegf120 and Vegf188 isoforms

were similarly reduced (40% and 64%, respectively) following maternal dexamethasone

treatment.

Overall, these studies quantify the labyrinth zone-specific increases in placental

expression of PPARG and VEGF in association with a marked increase in

vascularisation observed near term. Furthermore, this study demonstrates for the first

time that these increases in gene expression are prevented by maternal dexamethasone


treatment which also inhibits growth of the fetal capillary network. Elevated expression

of SFRP4 in the regressing basal zone late in gestation and in both placental zones after

dexamethasone-induced placental growth restriction is consistent with a role for SFRP4

in glucocorticoid-mediated inhibition of wnt signalling. Collectively, the data presented

in this thesis show that glucocorticoid inhibition of fetal growth is mediated in large part

via effects on the placenta, specifically through inhibition of signals that promote

proliferation and vascularisation.


ACKNOWLEDGEMENTS

Firstly, I would like to thank Professor Brendan Waddell and Dr Peter Mark for their

supervision, guidance and patience. There is no doubt that the constant questioning had

to take its toll. You have both stood up admirably when lesser supervisors may have

tried to change the locks.

To my laboratory neighbours, Caitlin Wyrwoll, Greg Cozens, Melissa Berg, Kayty

Plastow and Sue Hisheh, I thank you for your support and friendship. You have stood

by me and created a prosperous research environment.

I would also like to thank Lutz Slomianka and Adrian Baddeley for their discussion on

stereological principles.

I gratefully acknowledge the support of the University Postgraduate Award for the

duration of my candidacy.

And finally, I would like to thank my wife, Sarah, and family for both their

encouragement and their shared catch-phrase, “maybe I shouldn’t have asked”.


TABLE OF CONTENTS

Abstract .........................................................................................................................7

Acknowledgements ......................................................................................................11

Table of Contents ........................................................................................................13

List of Figures .............................................................................................................19

List of Tables ...............................................................................................................23

Thesis Format .............................................................................................................25

Abbreviations ..............................................................................................................27

Publications arising from this and related work ........................................................31

Chapter 1 Introduction.................................................................................................33

Chapter 2 Literature Review .......................................................................................36

2.1 Reproductive biology of the laboratory rat.....................................................36

2.1.1 Oestrus cycle and pregnancy...................................................................36

2.1.2 The rat placenta .......................................................................................36

2.1.2.1 Placental development ............................................................................38

2.1.2.2 Utero-placental morphology ...................................................................39

2.1.2.3 Placental function....................................................................................43

2.1.2.4 Regulation of placental development......................................................43

2.2 Glucocorticoids ...............................................................................................45

2.2.1 Synthesis and control ..............................................................................45

2.2.2 Glucocorticoid receptors and action........................................................47

2.2.3 Glucocorticoids during pregnancy ..........................................................47

2.2.4 Effects of glucocorticoids on fetal growth and programming the adult

phenotype ................................................................................................50

2.2.5 Glucocorticoids and the placenta ............................................................51

2.3 Peroxisome proliferator-activated receptors..................................................52

2.3.1 Retinoid X receptors and PPAR response elements ...............................52

2.3.2 PPAR activation......................................................................................53

2.3.3 Regulation of PPAR expression..............................................................53


2.3.4 PPARs and the placenta .......................................................................... 54

2.4 The wnt signalling pathway ............................................................................ 56

2.4.1 Canonical wnt signalling......................................................................... 56

2.4.2 Function of wnts ..................................................................................... 58

2.4.3 Wnts and the placenta .............................................................................58

2.4.4 Frizzled and frizzled related proteins...................................................... 58

2.4.5 Glucocorticoid regulation of wnt signalling ........................................... 59

2.5 Vascular endothelial growth factor ................................................................ 61

2.5.1 VEGF isoforms ....................................................................................... 61

2.5.2 VEGF receptors....................................................................................... 64

2.5.3 Regulation of VEGF expression ............................................................. 66

2.5.4 VEGF and the placenta ........................................................................... 66

Chapter 3 Experimental Objectives ............................................................................69

Chapter 4 Materials and Methods............................................................................... 73

4.1 Animals ........................................................................................................... 73

4.1.1 Rat maintenance...................................................................................... 73

4.1.2 Mating and pregnancy determination ..................................................... 73

4.2 Manipulation of glucocorticoid exposure ....................................................... 74

4.3 Surgical procedures ........................................................................................ 74

4.3.1 Preparation and anesthesia ...................................................................... 74

4.3.2 Tissue collection and separation of placental zones ............................... 74

4.4 Immunohistochemistry .................................................................................... 75

4.4.1 Reagents ..................................................................................................75

4.4.2 Tissue fixation and processing................................................................ 75

4.4.3 Poly-L-Lysine coating of microscope slides........................................... 76

4.4.4 Sectioning................................................................................................ 76

4.4.5 Dewaxing ................................................................................................ 76

4.4.6 Blocking for non-specific staining.......................................................... 77

4.4.7 Immunostaining ...................................................................................... 77

4.5 Real time reverse transcription-polymerase chain reaction........................... 78

4.5.1 Background ............................................................................................. 78


4.5.2 Reagents ..................................................................................................78

4.5.3 RNA sample preparation.........................................................................79

4.5.4 Reverse transcription...............................................................................80

4.5.5 Primers ....................................................................................................80

4.5.6 Polymerase chain reaction (PCR) ...........................................................82

4.5.7 Quantification of transcripts....................................................................82

4.5.8 Melt curve analysis .................................................................................82

4.6 Stereological measures ...................................................................................86

4.6.1 Sample preparation and sectioning .........................................................86

4.6.2 Frame sampling.......................................................................................86

4.6.3 Statistical corrections ..............................................................................86

4.6.4 Volume estimates ....................................................................................86

4.6.5 Surface area estimates .............................................................................87

4.6.6 Estimates of fetal capillary density, length and mean diameter..............88

4.7 Statistical analyses ..........................................................................................88

Chapter 5 Placental expression of peroxisome proliferator-activated receptors in

rat pregnancy and the effect of increased glucocorticoid exposure .........................91

5.1 Abstract ...........................................................................................................91

5.2 Introduction.....................................................................................................92

5.3 Materials and Methods ...................................................................................94

5.3.1 Animals ...................................................................................................94

5.3.2 Glucocorticoid manipulations .................................................................94

5.3.3 Tissue collection .....................................................................................94

5.3.4 RNA sample preparation.........................................................................94

5.3.5 Real-time RT-PCR..................................................................................95

5.3.6 Immunohistochemistry............................................................................95

5.3.7 Statistical analysis ...................................................................................97

5.4 Results .............................................................................................................98

5.4.1 Gestational changes in fetal, placental and placental zone weights........98

5.4.2 Gestational changes in Ppara, Ppard, Pparg and Rxra expression ..........98

5.4.3 Fetal and placental weights after dexamethasone treatment .................102

5.4.4 Effect of dexamethasone on expression of PPAR isoforms and Rxra ..102


5.5 Discussion ..................................................................................................... 105

Chapter 6 Placental expression of secreted frizzled related protein-4 in the rat and

the impact of glucocorticoid-induced fetal and placental growth restriction........ 109

6.1 Abstract ......................................................................................................... 109

6.2 Introduction................................................................................................... 110

6.3 Materials and Methods ................................................................................. 112

6.3.1 Animals ................................................................................................. 112

6.3.2 Glucocorticoid treatment....................................................................... 112

6.3.3 Tissue collection ................................................................................... 112

6.3.4 RNA sample preparation....................................................................... 112

6.3.5 Real-time RT-PCR................................................................................ 113

6.3.6 Immunohistochemistry ......................................................................... 113

6.3.7 In situ hybridization .............................................................................. 113

6.3.8 Statistical analysis ................................................................................. 114

6.3.9 In situ hybridization .............................................................................. 116

6.3.10 Statistical analysis ................................................................................. 116

6.4 Results ........................................................................................................... 117

6.4.1 Expression of SFRP4 in the rat placenta during normal pregnancy ..... 117

6.4.2 Expression of CTNNB1 in the rat placenta during normal pregnancy .117

6.4.3 Expression of SFRP4 and CTNNB1 after maternal dexamethasone

treatment................................................................................................ 123

6.5 Discussion ..................................................................................................... 127

Chapter 7 Glucocorticoids prevent the normal increase in placental vascular

endothelial growth factor expression and placental vascularity during late

pregnancy in the rat.................................................................................................... 131

7.1 Abstract ......................................................................................................... 131

7.2 Introduction................................................................................................... 132

7.3 Methods......................................................................................................... 134

7.3.1 Animals ................................................................................................. 134

7.3.2 Glucocorticoid manipulations ............................................................... 134

7.3.3 Tissue collection ................................................................................... 134


7.3.4 RNA isolation .......................................................................................134

7.3.5 Real-time RT-PCR................................................................................135

7.3.6 Immunohistochemistry..........................................................................135

7.3.7 Stereological measures..........................................................................137

7.3.8 Statistical analysis .................................................................................137

7.4 Results ...........................................................................................................138

7.4.1 Gestational changes in VEGF expression.............................................138

7.4.2 Vascular changes in the rat placenta over the final third of pregnancy 138

7.4.3 Changes in VEGF expression following dexamethasone treatment .....144

7.4.4 Vascular changes in the rat placenta following dexamethasone treatment

144

7.5 Discussion .....................................................................................................148

Chapter 8 General Discussion....................................................................................151

Chapter 9 References..................................................................................................157

Appendix....................................................................................................................173


LIST OF FIGURES

Figure 2.1 Development of the rat placenta on (A) day 14 and

(B) day 18 Page 41

Figure 2.2 Zones of the rat placenta and blood space

arrangement at day 22 of rat pregnancy Page 42

Figure 2.3 Regulation of glucocorticoid secretion Page 46

Figure 2.4 Placental glucocorticoid barrier Page 49

Figure 2.5 Activation of PPAR/RXR heterodimers Page 55

Figure 2.6 The canonical wnt pathway Page 57

Figure 2.7 SFRP inhibition of wnt signalling Page 60

Figure 2.8 VEGF isoforms Page 63

Figure 2.9 Receptor activation by VEGF isoforms Page 65

Figure 4.1 Standard curve generation for Ppara mRNA Page 83

Figure 4.2 Real time PCR amplification plot for Ppara mRNA

standards Page 84

Figure 4.3 Melt curve analysis for Ppara mRNA Page 85

Figure 4.4 A grid of cycloid arcs and test points superimposed

on the placental labyrinth Page 89

Figure 5.1 Fetal, placental and placental zone weights at days 16

and 22 of rat pregnancy Page 99


Figure 5.2 Zonal expression of (A) Ppara; (B) Ppard; (C) Pparg

and; (D) Rxra mRNA at day 16 and 22 of rat

pregnancy Page 100

Figure 5.3 Zonal distribution of PPARG and RXRA compared

to negative controls (no primary) at day 22 in the rat

placenta by immunohistochemistry Page 101

Figure 5.4 Fetal, placental and placental zone weights after

dexamethasone (Dex) treatment compared to

untreated controls (Con) Page 103

Figure 5.5 Zonal expression of Pparg mRNA at day 22 of rat

pregnancy following treatment with dexamethasone

(Dex) compared to untreated controls (Con) Page 104

Figure 6.1 Zonal expression of (A) Sfrp4 and (B) Ctnnb1 mRNA

measured by real time RT-PCR at days 16 and 22 of

rat pregnancy Page 118

Figure 6.2 Zonal distribution of Sfrp4 mRNA at days 16 and 22

of rat pregnancy by in situ hybridization Page 119

Figure 6.3 Zonal distribution of SFRP4 protein at days 16 and

22 of rat pregnancy by immunohistochemistry Page 120

Figure 6.4 Subcellular distribution of CTNNB1 protein at days

16 and 22 of rat pregnancy by immunohistochemistry Page 121

Figure 6.5 Labyrinthine distribution of CTNNB1 at day 22 of rat

pregnancy by immunohistochemistry Page 122

Figure 6.6 Zonal expression of (A) Sfrp4 and (B) Ctnnb1 mRNA

measured by real time RT-PCR at day 22 of rat

pregnancy following treatment with dexamethasone Page 124


Figure 6.7 Zonal distribution of Sfrp4 mRNA by in situ

hybridization following maternal treatment with

dexamethasone Page 125

Figure 6.8 Zonal distribution of SFRP4 protein and subcellular

localization of the CTNNB1 protein at day 22 after

treatment with dexamethasone Page 126

Figure 7.1 Expression of (A) Vegf120, (B) Vegf164 and (C) Vegf188

mRNA in the basal and labyrinth zones of the rat

placenta on days 16 and 22 of pregnancy Page 140

Figure 7.2 Spatial distribution of VEGF by

immunohistochemistry at day 22 of rat pregnancy Page 141

Figure 7.3 Stereological analysis of labyrinth zone components

on days 16 and 22 showing changes in (A) absolute

volume and (B) absolute surface area of the

vasculature, and changes in (C) relative vascular

volumes and (D) relative surface areas of the

vasculature Page 142

Figure 7.4 Stereological analysis of fetal capillary network on

days 16 and 22 of pregnancy showing changes in (A)

total combined capillary length, (B) mean capillary

diameter, and (C) capillary density Page 143

Figure 7.5 Effects of dexamethasone treatment from day 13 of

pregnancy on zonal expression of (A) Vegf120, (B)

Vegf164 and (C) Vegf188 mRNA at day 22 Page 145


pregnancy on the (A) absolute volume, (B) absolute

surface area, (C) relative volume, and (D) relative

surface area of the labyrinthine vasculature at day 22 Page 146



pregnancy on (A) total combined capillary length, (B)

mean capillary diameter, and (C) relative capillary

density of the labyrinthine vasculature at day 22 Page 147

Figure 8.3 Proposed regulation of trophoblastic VEGF

expression via PPARG Page 156


LIST OF TABLES

Table 2.1 The phases of the estrus cycle in the adult Wistar rat Page 37

Table 4.1. Processing protocol Page 76

Table 4.2 Dewaxing protocol Page 77

Table 4.3 Rehydration protocol Page 78

Table 4.4 Primers used for real time RT-PCR Page 81

Table 5.1 Primers used for Ppara, Ppard, Pparg, Rxra and L19

real time RT-PCR Page 96

Table 6.1 Primers and conditions used for Sfrp4, Ctnnb1 and

L19 real time RT-PCR. Page 115

Table 7.1 Primers and conditions used for Vegf isoforms and

Rpl19 real time RT-PCR. Page 136

Table 7.2 Fetal and placental zone weights following maternal

dexamethasone treatment. Page 139


THESIS FORMAT

General

This thesis is presented as three published manuscripts resulting from work conducted

exclusively for this thesis. These manuscripts form the three Results chapters, which is

preceded by the Introduction, Literature Review, Experimental Objectives and Materials

and Methods chapters. The literature review and materials and methods chapters expand

on the relevant background that was not included in each of the papers including

detailed calculations and rationale behind the methods chosen. The final two chapters

provide a General Discussion of the overall findings and list of all the References cited

in this thesis.

Presentation of data

Most of the data is presented in graphical format, but for data of lesser importance,

tabular presentations have been included. Immunohistochemical data are presented as

colour images of typical sections. Figures are generally presented on separate pages,

wherever possible they are placed after the text in which they are cited, otherwise they

are presented at the end of the results section of each experimental chapter.

References

Published work referred to in the text is cited by indication of authors and the year of

publication. When the number of authors exceed two, only the first are mentioned

followed by et al and the year of publication.


ABBREVIATIONS

11BHSD 11β-hydroxysteroid dehydrogenase

A adenine

aa amino acids

ACTH adrenocorticotrophic hormone

ANOVA analysis of variance

APC adenomatous polyposis coli

AVP arginine vasopressin (a.k.a. antidiuretic hormone)

Bas basal zone

Bcl-2 B-cell lymphoma/leukaemia-2

bp base pairs

BSA bovine serum albumin

BZ basal zone

C cytosine

CBX carbenoxolone

CD31 platelet/endothelial cell adhesion molecule (a.k.a. PECAM)

cDNA complementary deoxyribonucleic acid

Con control

CRH corticotrophin releasing hormone

CTNNB1 beta-catenin

CV choriovitelline placenta

DAB diaminobenzidine

DB decidua basalis

DC decidua capsularis

ddH20 distilled, deionised water

Dex dexamethasone

dH20 distilled water

DHC 11-dehydrocorticosterone

DKK1 dickkopf-1

DNA deoxyribonucleic acid

dNTPs deoxynucleotide triphosphates


DSH human dishevelled

DTT dithiothreitol

DVL murine dishevelled

ECM extra cellular matrix

EDTA ethylenediamine tetra-acetate

FA fatty acids

Fgf fibroblast growth factor

Flk-1 fetal liver kinase 1

Flt-1 fms-like tyrosine kinase 1

FM fetal mesenchyme

Fzd frizzled receptor

G guanine

GC glucocorticoids

Gcm1 glial cells missing-1 protein

GR glucocorticoid receptor

GSK3B glycogen sythase kinase-3-beta

GTr giant trophoblast

HIF1 hypoxia-inducible factor-1

HPA hypothalamic-pituitary adrenal (axis)

HRP horseradish peroxidase

IUGR intrauterine growth restriction

kb kilobases

kDa kilodalton

Kdr kinase insert domain-containing receptor

Kg kilograms


L litres

Lab labyrinth zone

Lef lymphoid enhancer-binding factor

LSD least significant difference

LZ labyrinth zone

M maternal myometrium

MBS maternal blood space

MJ megajoules

M-MLV moloney murine leukaemia virus

MMTV mouse mammary tumour virus

mRNA messenger ribonucleic acid

MUC1 mucin-1

n number

NaCl sodium chloride

NP1 neurophillin-1

PBS phosphate buffered saline

PCP planar cell polarity

PCR polymerase chain reaction

PG prostaglandins

PlGF placental growth factor

PPAR peroxisome proliferator-activated receptor

PPRE PPAR response element

RNA ribonucleic acid

RNase ribonuclease

rpm revolutions per minute

RT reverse transcriptase

RXRA retinoid X receptor alpha


SEM standard error of the mean

SFLT-1 soluble FLT-1

SFRP4 secreted frizzled related protein-4

T thymine

TAE tris acetic acid buffer

TBS-T tris buffered saline plus Tween 20

Tcf transcription factor

Tm melting temperature

Tr trophoblast

Triton X iso-octylphenoxypolyethoxyethanol

Tween 20 polyoxyethylene sorbitane monolaurate

TZD thiazolidinediones

VEGF vascular endothelial growth factor

VEGF-R VEGF receptor

Wg wingless

wt/vol weight/volume (concentration)

x g times gravity

XIAP X-linked inhibitor of apoptosis


PUBLICATIONS ARISING FROM THIS AND RELATED WORK

Chapter 5

Hewitt DP, Mark, PJ, Waddell, BJ. (2006). Placental expression of peroxisome

proliferator-activated receptors in rat pregnancy and the effect of increased

glucocorticoid exposure. Biol Reprod 74(1): 23-8.

Chapter 6

Hewitt DP, Mark, PJ, Waddell, BJ. (2006). Placental expression of secreted frizzled

related protein-4 in the rat and the impact of glucocorticoid-induced fetal and placental

growth restriction. Biol Reprod 75(1): 75-81.

Chapter 7

Hewitt DP, Mark, PJ, Waddell, BJ. (2006). Glucocorticoids prevent the normal increase

in placental vascular endothelial growth factor expression and placental vascularity

during late pregnancy in the rat. Endocrinology 147(12): 5568-74.

Abstracts

Hewitt DP, Mark, PJ, Waddell, BJ. Glucocorticoids prevent the normal increase in

placental VEGF expression and placental vascularity in the rat. Endocrine and

Reproductive Sciences Symposium. Perth, Aug 2006.

Hewitt DP, Mark, PJ, Waddell, BJ. Changes in placental expression of PPARγ in rat

pregnancy are associated with altered expression of MUC1 and VEGF. Proceedings of

the Endocrine Society of Australia. Perth, Sept 2005.


Hewitt DP, Mark, PJ, Dharmarajan, AM, Waddell, BJ. Placental expression of secreted

frizzled related protein (sFRP4) in the rat: association with β-catenin localization and

regulation by glucocorticoids. Proceedings of the Society for Reproductive Biology.

Perth, Sept 2005.

Hewitt DP, Mark, PJ, Waddell, BJ. Placental Expression of PPARalpha, delta, gamma

and RXRalpha in rat pregnancy and the effect of increased glucocorticoid exposure.

Proceedings of the International Congress of Endocrinology. Lisbon, Aug 2004.

Hewitt DP, Mark, PJ, Waddell, BJ. Placental Expression of PPARalpha, delta, gamma

and RXRalpha in rat pregnancy and the effect of increased glucocorticoid exposure.

Proceedings of the Endocrine Society of Australia. Sydney, Aug 2004.

Hisheh S, Hewitt DP, Mark, PJ, Waddell, BJ. Marked upregulation of COX-2

expression in rat placenta during late pregnancy. Proceedings of the Endocrine Society

of Australia. Sydney, Aug 2004.


Chapter 1 Introduction

Glucocorticoids are a class of steroid hormones that function via the glucocorticoid

receptor to control energy metabolism and contribute to the stress response. During

pregnancy, maternal and fetal glucocorticoids are critical for the maturation of the fetus.

Thus, synthetic glucocorticoids are commonly given to mothers at risk of preterm

delivery to accelerate fetal lung maturation. Increased fetal glucocorticoid exposure can,

however, restrict fetal growth and increase rates of fetal morbidity and mortality.

Moreover, there is growing concern that restricted fetal growth, which is commonly

indicative of a poor intrauterine environment, can increase the risk of adult diseases

such as type II diabetes and hypertension.

In addition to direct effects on the fetus, it appears likely that increased glucocorticoid

exposure may also impact on fetal well-being indirectly via effects on the placenta.

Indeed, maternal administration of glucocorticoids increases the rate of trophoblast

apoptosis and restricts placental growth in rodent models. Despite the wealth of

knowledge on the effect of glucocorticoids on fetal growth and organ development,

little is known about the role of the placenta in this process. Therefore, the major

objectives of this thesis were to investigate the expression pattern of potential key

regulators of placental growth and vascularisation in normal pregnancy and after

increased maternal glucocorticoid exposure.

In total, three major studies were undertaken in this thesis (Chapters 5-7), each

investigating rat placental gene expression during normal gestation and following

glucocorticoid-induced fetal and placental growth restriction. The first two studies

investigated the placental expression of the peroxisome proliferator-activated receptors

(PPARs) and the secreted inhibitor of the Wnt pathway, secreted frizzled related

protein-4 (SFRP4). These two molecules represent new candidates that may function in

the regulation of placental vascularisation and the balance between trophoblastic

growth, differentiation and apoptosis. The third study investigated the vascular growth

that occurs over the final third of normal pregnancy in the rat in association with the

expression the endothelial cell-specific mitogen, vascular endothelial growth factor

(VEGF). As some of the results of initial studies relate to findings of subsequent work, a

brief introduction and rationale of each study follows.


The first study (Chapter 5) was based on previous findings that deletion of the nuclear

receptor, Pparg, results in an embryonic lethal phenotype due to a failure of placental

vascular development. Most notably, Pparg-null mice exhibited a reduction in fetal

vessel growth of the placenta suggestive of a role for placental PPARG in fetal capillary

development. Therefore, Chapter 5 investigated the expression of the three PPAR

isoforms and their heterodimeric partner, RXRA, in the rat placenta over the final third

of pregnancy, the period of maximal fetal and placental growth. The expression of each

gene was assessed separately in the two functionally- and morphologically-distinct

regions of the placenta, the basal and labyrinth zones. Expression data for each isoform

during normal gestation was then compared to the zonal expression observed after

glucocorticoid-induced fetal and placental growth restriction.

For the second study (Chapter 6) the focus shifted to the role of the wnt pathway in

placental development and specifically, the expression of the secreted frizzled relative,

SFRP4. Previous data on wnt signalling had implicated individual components of the

pathway in the growth and vascularisation of the placenta, as well as regulation of key

growth factor expression. Therefore, the expression pattern of SFRP4 was determined in

the basal and labyrinth zones across normal gestation and after maternal glucocorticoid

treatment. Assessment was also made of the cytoplasmic accumulation and subsequent

nuclear translocation of β-catenin in trophoblast cells, since this is a critical endpoint of

wnt signalling.

Recent reports indicate that glucocorticoid-mediated inhibition of placental growth is

associated with the reduced transplacental passage of both glucose and leptin without a

change in transporter expression, suggesting that glucocorticoids may reduce placental

transfer via inhibition of placental vascularisation. Therefore, the final study quantified

the normal development of maternal and fetal blood spaces in terms of volumes and

surface areas over the final third of rat pregnancy and after maternal glucocorticoid

treatment. Associated changes in expression of the major VEGF isoforms found in the

placenta were also assessed over the final third of pregnancy and following maternal

glucocorticoid treatment.


Chapter 2 Literature Review

2.1 Reproductive biology of the laboratory rat

The laboratory rat is a non-seasonal, spontaneously ovulating, polyestrous mammal

whose basic reproductive physiology is conserved across most mammalian species

(Rowlinds and Weir 1984; Ojeda and Urbanski 1988; Wooding and Flint 1994).

Frequent reproduction and a short gestation period mean the rat provides a convenient

model for the study of placental and fetal development.

2.1.1 Oestrus cycle and pregnancy

The female rat has an average lifespan of about three years with estrus cycles

commencing around one and a half months and maximum fertility occurring between

three and ten months of age (Long and Evans 1922). The rat oestrus cycle lasts between

four and five days and is divided into four periods: proestrus, oestrus, metestrus, and

diestrus (Freeman 1988). Each of the periods is characterised by detectable changes in

vaginal cytology, with a predominance of cornified (denucleated) cells at oestrus (see

Table 2.1). Of the four periods, oestrus is the only time the female is sexually receptive.

During each cycle, between 3 and 10 ova are released from each ovary which results in

approximately 12 to 15 conceptuses in each pregnancy. Following fertilization, the

blastocysts implant in the uterine lining on day 6, with the onset of parturition on the

morning of the 23rd day (based on the first day of pregnancy being the day on which the

presence of spermatozoa is detected in a vaginal smear).

2.1.2 The rat placenta

In mammals the growth of the embryo quickly exceeds the point at which the direct

exchange of nutrients can be accomplished across the outer membranes. The formation

of the placenta, a transient apposition of fetal and maternal tissues, facilitates the

exchange of nutrients, metabolic wastes and respiratory gases. The following discussion

on placental development and morphology is based on previous extensive descriptions

(Long and Evans 1922; Enders 1965; Davies and Glasser 1968; Steven and Morriss

1975; Wooding and Flint 1994).


Table 2.1 The phases of the estrus cycle in the adult Wistar rat (Adapted from Mandl, 1951).

Phases of the Rat Oestrus Cycle

Proestrus

Oestrus

Metestrus

Diestrus

Stage Vaginal SmearLength (hrs)

12-14

25-27

6-8

55-57

Thick smear contains mainly leukocytes and nucleated epithelial cells, cornified cells present approaching oestrus

Almost exclusively cornified cells

Begins with similar smear to oestrus, ends with a thick smear and the appearance of leukocytes and nucleated epithelial cells

Thin smear containing leukocytes and nucleated epithelial cells, absence of cornified cells

Leukocytes Epithelial CellsCornified Cells


2.1.2.1 Placental development

The loss of the zona pellucida and subsequent implantation of the blastocyst in the

uterine wall take place around day 6 (Enders and Schlafke 1967) whereby the polar

trophoblast cells immediately superficial to the inner cell mass invade and engulf the

uterine epithelium. These cells continue to divide to become the ectoplacental cone and

eventually become the labyrinth zone of the chorioallantoic placenta (Enders 1965;

Steven and Morriss 1975). Implantation and subsequent invasion of the rat blastocyst

converts uterine cells into decidual cells by what is known as the decidual reaction

(Steven and Morriss 1975). The rat, in particular, has a massive decidual reaction when

compared to other haemochorial mammals (Wooding and Flint 1994). Mural

trophoblast cells do not form part of the ectoplacental cone, but rather increase their

DNA content to become giant trophoblast cells of the basal zone (Steven and Morriss

1975).

A unique feature of the rodent (and insectivores) is that the developing embryo is

surrounded by an extraembryonic membrane called ‘Reichert’s membrane’. Reichert’s

membrane develops from day 6 of gestation, persisting until day 18 when it ruptures

and retracts to the margins of the placenta. This membrane becomes necessary due to

the complete inversion of the embryo, and the subsequent lack of a surrounding chorion

(Steven and Morriss 1975).

Before the chorioallantoic placenta is formed, nutrient exchange is facilitated by the

choriovitelline (CV) placenta. The CV placenta functions to acquire nutrients taken up

by the extraembryonic endoderm and an inner layer of mesoderm deposited within the

yolk sac. On day 11-12, blood vessels coalesce in the yolk sac wall to form the vitelline

circulation (Steven and Morriss 1975).

The chorioallantoic placenta, which is created from the fusion of the inner allantoic

mesenchyme and the adjacent chorion at days 9-10, is the primary site of feto-maternal

exchange after day 11 (Steven and Morriss 1975). The outgrowth and confluence of

fetal villi within the ectoplacental cone erode the decidua basalis as deep as the uterine

myometrium. The subsequent fusion of chorionic villi creates a labyrinth of maternal

blood spaces, initially lined with maternal endothelium, later replaced with fetal

trophoblast (Wooding and Flint 1994).


2.1.2.2 Utero-placental morphology

The non-pregnant rat uterus consists of two horns (bicornuate) 30-40 mm in length with

a caudally fused portion 7-10 mm long. During pregnancy, the uterus undergoes marked

morphological changes with a shift towards estrogen-driven endometrial cell

proliferation (Tachi et al. 1972) and increased vascular permeability (Johnson and Dey

1980; Phillips and Poyser 1981). The site of implantation along the uterine horn can

account for slight differences in fetal anatomy and physiology due to the sex of adjacent

fetuses (Sachs and Meisel 1988) and proximal-distal variations in uterine blood flow

(Gorodeski et al. 1995). Thus, random sampling along the uterine horn is required to

reduce the bias of positional variations on fetal measures.

In the absence of all maternal tissues (except free blood) the human and rat placenta are

classified as haemochorial (Steven and Morriss 1975). However, unlike the two layers

of trophoblast found in the human placenta, the rat placenta has three layers of

trophoblast in direct contact with maternal blood (Steven and Morriss 1975).

Furthermore, fetal-maternal exchange is facilitated by a labyrinth arrangement of fetal

tissues rather than the fetal villous tree seen in the human, although both species have

retained a discoid structure (Hogarth 1978; Wooding and Flint 1994).

The mature chorioallantoic placenta of the rodent contains two functionally- and

morphologically-distinct zones: the basal and labyrinth zones (Fig. 2.1). The basal zone

contains giant trophoblast cells, spongiotrophoblast cells and glycogen cells, of which

only the giant trophoblast cells are maintained until term. Unlike the labyrinth zone, the

basal zone contains no fetal vasculature (Enders 1965; Wooding and Flint 1994). The

labyrinth zone contains trophoblastic septa which surround fetal mesenchyme and

contain fetal blood vessels (Fig. 2.2). Within the labyrinth zone there appears to be no

obvious direction in the structure of fetal tissue or maternal blood spaces (Enders 1965;

Wooding and Flint 1994). The relative proportions of the two zones differ markedly

across pregnancy (Waddell et al. 1998). The volume of the basal zone peaks at

approximately day 16, and then remains at a similar size until term. In contrast, the

labyrinth zone undergoes a 3-fold increase from day 16 to 22 (Waddell et al. 1998),

presumably to provide for the increased demands of the fetus near term (Davies and

Glasser 1968).


An often overlooked structure of the mature rodent placenta is the intraplacental yolk

sac or sinus of Duval (Ogura et al. 1998; Kovacs et al. 2002; Pijnenborg and Vercruysse

2006). This endodermal sinus is an invagination of the yolk sac into the labyrinthine

placenta that is thought to play a role in maternal-fetal calcium exchange (Kovacs et al.

2002; Brasier et al. 2004) and regulation of placental blood flow (Matejevic et al. 1996).


DB

DC

CVM

BZ

LZ

DBMBZ

LZ

A

B

Figure 2.1 Development of the rat placenta on (A) day 14 and (B) day 18. Growth of the labyrinth

zone contributes the majority of total placental growth after day 14 of pregnancy to facilitate the

increased demands of the fetus late in pregnancy. M: maternal myometrium, DC: decidua capsularis, CV:

choriovitelline placenta, LZ: labyrinth zone, BZ: basal zone, DB: decidua basalis.


BZ

LZ

FM

FC

GTr

Tr

MBS

FM Tr

GTr

BZ

LZ

Figure 2.2 Zones of the rat placenta and blood space arrangement at day 22 of rat pregnancy. The

placental labyrinth (LZ) is characterised by a network of fetal capillaries (FC) surrounded by fetal

mesenchyme (FM) awash in maternal blood contained within trophoblast (Tr)-lined maternal blood

spaces (MBS). The basal zone (BZ) contains fewer maternal blood spaces, no fetal vessels and a

predominance of giant trophoblast cells (GTr).


2.1.2.3 Placental function

The labyrinth zone serves as the site of feto-maternal exchange allowing the

transplacental passage of nutrients, wastes and respiratory gases. In addition, the rodent

placenta has been shown to be an active steroidogenic organ. The basal zone functions

as the major site of hormone synthesis during early pregnancy with a shift towards a

more balanced production across the two zones near term (Hogarth 1978). The giant

cells of the basal zone have been identified as the primary source of progesterone and

androgens (Matt and Macdonald 1985; Wooding and Flint 1994) while the terminally

differentiated syncytiotrophoblast of the labyrinth zone provides the major source of

prolactin (Dai et al. 2002). By contrast, cytotrophoblastic cells of the labyrinth zone

exhibit limited steroidogenic properties (Soares et al. 1996).

2.1.2.4 Regulation of placental development

The ability to produce transgenic and mutant mice has allowed functional investigation

of the distinct roles of individual genes involved in placental development.

Development of the early placenta from the outer layer of trophoblast stem cells of the

blastocyst involves a number of critical steps which are regulated by a various genes,

notably fibroblast growth factor (FGF) and its downstream effectors (Rossant and Cross

2001; Cross et al. 2003; Charnock-Jones et al. 2004). The following discussion,

however, will focus on those factors that affect the development of the mature

chorioallantoic placenta.

Among those genes known to be critical for labyrinthine development, glial cells

missing-1 protein (GCM1), in particular, is critical for trophoblast differentiation and

branching of the villous tree (Anson-Cartwright et al. 2000). FGF is also required for

chorioallantoic branching as evidenced by the deletion of the FGF receptor gene, Fgfr2

(Xu et al. 1998) and several downstream effectors of FGF signalling including vascular

endothelial growth factor (VEGF; see section 2.5), growth-factor-receptor-bound

protein and growth-factor-receptor-bound protein-associated protein 1 (Rossant and

Cross 2001).

Mutations in various components of the wnt-β-catenin signalling pathway have also

been implicated in the development of mature placental morphology. Notably, the


deletion of Wnt7b or Tcf/Lef1 genes disrupts chorioallantoic fusion, while the absence

of Wnt2 or Fzd5 genes impairs normal vascular development (see section 2.4).

After the initial formation of the labyrinthine placenta several other transcription factors

are also critical for its vascular development including the homeobox protein, distal-less

3 (Morasso et al. 1999), extra-embryonic tissue-spermatogenesis-homeobox gene 1 (Li

and Behringer 1998) and the peroxisome proliferator-activated receptor γ (PPARG; see

section 2.3) (Barak et al. 1999). Placental development is also regulated by a range of

other factors including insulin-like growth factor II, the deletion of which leads to fetal

and placental growth restriction (Constancia et al. 2002), and estrogen via its effects on

the expression of VEGF (see section 2.5.3).


2.2 Glucocorticoids

Glucocorticoids are a class of steroid hormones produced by the adrenal glands under

the control of the hypothalamic-pituitary-adrenal (HPA) axis. Glucocorticoids act via

the glucocorticoid receptor (GR) which is expressed ubiquitously and regulates cell

differentiation, metabolism and stress-related homeostasis (Reul et al. 1989; Sun et al.

1997; Speirs et al. 2004). During pregnancy, endogenous glucocorticoids from both the

mother and the fetus are of particular importance in the regulation of fetal and placental

growth (Seckl 2004). Indeed, clinical use of synthetic glucocorticoids is commonly used

to accelerate the maturation of fetal lungs in mothers at risk of preterm birth (Sloboda et

al. 2005). However, increased glucocorticoid exposure during pregnancy is known have

detrimental effects on placental and fetal growth (Benediktsson et al. 1993; Waddell et

al. 2000; Seckl 2004; Seckl and Meaney 2004; Sloboda et al. 2005). The following

discussion on glucocorticoids serves to introduce the biological properties of

glucocorticoids and relate physiological functions to glucocorticoid-mediated fetal and

placental pathologies.

2.2.1 Synthesis and control

Glucocorticoids, primarily in the form of cortisol in the human and corticosterone in the

rat, are synthesized in the adrenal cortex from cholesterol via a series of reactions

catalysed by specific steroidogenic enzymes (Fraser 1992). Glucocorticoids are secreted

in a pulsatile fashion from the zona fasciculata following stimulation by

adrenocorticotrophic hormone (ACTH) from corticotrophs of the anterior pituitary

gland. ACTH secretion is regulated by corticotrophin releasing hormone (CRH) and

arginine vasopressin (AVP) from the hypothalamus, with both the hypothalamic and

pituitary components of the axis subject to negative feedback by glucocorticoids (Fig.

2.3; Nussey and Whitehead 2001). Plasma glucocorticoid levels exhibit a diurnal

circadian rhythm whereby maximal levels occur early upon waking in both the human

(Esteban et al. 1991) and the rat (Dallman et al. 1993). This HPA axis can be disturbed,

however, by stimuli that challenge internal homeostasis including disease, aging and

stress.


Anterior PituitaryACTH

HypothalamusCRH/AVP

Adrenal CortexGC

NegativeFeedback

Figure 2.3 Regulation of gluccorticoid secretion. Under the influence of low glucocorticoids (GC) or in

response to stress, the hypothalamus releases corticotrophin releasing hormone (CRH) and arginine

vasopressin (AVP) which stimulates the production of adrenocorticotrophic hormone (ACTH) from the

anterior pituitary and GC from the adrenal cortex. Both the hypothalamus and pituitary are subject to

negative feedback from circulating GC.


2.2.2 Glucocorticoid receptors and action

Glucocorticoid action in target tissues is dependent on the expression of two main

receptors, the GR and the mineralocorticoid receptor. Cortisol activates the cytoplasmic

GR by displacement of accompanying heat shock proteins, and subsequent

phosphorylation which allows nuclear translocation of the ligand-bound, dimerised

receptor (for review see Duma et al. 2006). The activated GR then binds to

glucocorticoid response elements to alter transcription of specific target genes (Nussey

and Whitehead 2001; Duma et al. 2006). Although glucocorticoids exhibit potent

metabolic effects in a range of tissues, they primarily increase blood glucose levels via

anabolic effects in the liver and catabolic effects in fat and muscle. Other actions of

glucocorticoids include maintenance of blood pressure, neuronal excitability,

glomerular filtration rate, fetal organ maturation, as well as inflammatory and immune

responses (Fraser 1992; Nussey and Whitehead 2001).

2.2.3 Glucocorticoids during pregnancy

Glucocorticoids play an essential role during pregnancy in the maternal adaptation to

fetal nutrient demands, the anticipation of future lactation and the promotion of fetal

organ maturation late in gestation (Liggins 1994). In the human, circulating

glucocorticoids increase in the fetal compartment late in pregnancy to facilitate fetal

lung maturation and surfactant synthesis (Pasqualini et al. 1991). Plasma corticosterone,

the primary glucocorticoid in the rat, increases to a peak at day 22, presumably to

mobilise maternal energy stores (Atkinson and Waddell 1995). Placental growth and

function are also regulated by glucocorticoids (for reviews see Burton and Waddell

1999; Seckl and Meaney 2004) with expression of the GR increasing in the labyrinth

zone of the rat placenta late in pregnancy (Mark et al. 2002).

Although glucocorticoids are required for normal fetal development, excess

glucocorticoid exposure can restrict fetal growth and increase the risk of disease in later

life. Control of fetal glucocorticoid exposure is therefore tightly regulated by the

expression of two, 11β-hydroxysteroid dehydrogenase (11BHSD) enzymes, types 1 and

2, in the placenta. 11BHSD1 catalyses the interconversion of active glucocorticoids

(cortisol in the human and corticosterone in the rat) with their inactive forms (cortisone

and 11-dehydrocorticosterone respectively). By contrast, 11BHSD2 catalyses the

unidirectional conversion of active glucocorticoids to their respective inactive forms


(White et al. 1997; Seckl 2004). Each of the two 11BHSD enzymes are expressed in

both the human and rodent placenta (Brown et al. 1996a; Waddell et al. 1998), however,

11BHSD2 expression and the resultant inactivation of cortisol appears to be more

physiologically significant particularly late in human pregnancy (for review see Burton

and Waddell 1999). The impact of glucocorticoids on fetal growth and development is

therefore regulated by the ‘placental glucocorticoid barrier’(Fig. 2.4; Benediktsson et al.

1997). In the rodent, however, although basal zone expression of 11BHSD2 increases

over the final third of pregnancy, labyrinthine expression falls over the same period

(Burton et al. 1996; Waddell et al. 1998). Moreover, the high expression of 11BHSD1

in the labyrinth zone near term (Burton et al. 1996; Waddell et al. 1998) creates a

potentially deleterious environment that may be balanced by the expression of

11BHSD2 in fetal organs and the development of a functional fetal HPA axis late in

pregnancy (Brown et al. 1996b). Preterm maternal administration has utilised the

synthetic glucocorticoids, betamethasone and dexamethasone, which are a poor

substrates for the 11BHSD enzymes and therefore exhibit higher potency in fetal organ

maturation (see below).


FM

FC

TrMBSDEX

DEXDEX

DEXGR

GR

GC

GC

GR

11BHSD2

(Active)

GC(Inactive)

Figure 2.4 Placental glucocorticoid barrier. Active glucocorticoids (GC) are converted to into their

inactive metabolites by the 11BHSD2 enzyme. Dexamethasone (DEX) is a poor substrate for 11BHSD2,

and thus facilitates glucocorticoid receptor (GR) activation in placental cells and transplacental passage in

significant quantities.


2.2.4 Effects of glucocorticoids on fetal growth and programming the adult

phenotype

Clinical glucocorticoid administration is used for mothers at risk of preterm delivery to

accelerate fetal lung maturation (Ward 1994; Sloboda et al. 2005). Historically,

betamethasone and dexamethasone have been used based on their pharmacological

properties, including being poor substrates for 11BHSD, which allows access to

placental GR and passage to the fetal compartment. Excess glucocorticoid exposure,

however, reduces birth weight in both human and animal models (Benediktsson et al.

1993; Burton and Waddell 1994; Smith and Waddell 2000; Seckl 2004) and has been

shown to increase fetal mortality rates in the rodent (Gunberg 1957; Yang et al. 1969).

Growth restriction is most notable when exposure occurs during the period of maximal

fetal and placental growth late in pregnancy (for review see Seckl and Meaney 2004).

Fetal exposure to excess endogenous maternal glucocorticoids can also affect fetal

growth as evidenced by inhibition of the placental glucocorticoid barrier (11BHSD

activity) by carbenoxolone (Lindsay et al. 1996; Smith and Waddell 2000) and

mutations in the gene encoding 11BHSD also reducing birth weight (Holmes et al.

2006). Moreover, maternal treatment with the 11β-hydroxylase inhibitor, metyrapone,

decreases glucocorticoid synthesis and increases fetal growth (Burton and Waddell

1994; Smith and Waddell 2000), suggesting that normal fetal growth is limited by

maternal glucocorticoids. Interestingly, recent data indicates that chronic maternal stress

also impairs fetal growth in rats, with a concurrent inhibition of 11BHSD2 expression

(Mairesse et al. 2007).

It is now clear the early life events can influence the pathophysiology of the adult, with

a poor intrauterine environment linked to increased risk of a range of diseases, including

hypertension (Barker et al. 1990) and cardiovascular disease (Barker et al. 1993).

Excess glucocorticoid exposure, in particular, has been implicated in impaired glucose

and insulin responses to an oral glucose load at six months in the rat (Lindsay et al.

1996). In addition to glucose signalling, maternal glucocorticoid treatment in the rodent

has been shown to impact upon postnatal testicular growth and function (Shishkina and

Dygalo 1994), aberrant male copulatory behaviour (Politch and Herrenkohl 1984),

delayed puberty onset (Smith and Waddell 2000), and elevated leptin levels and blood

pressure (Wyrwoll et al. 2006).


2.2.5 Glucocorticoids and the placenta

Placental development is a critical determinant of fetal growth and it appears that the

impact of glucocorticoids may be mediated, in part, via direct effects on placental

growth and function. Specifically, rodent models suggest that placental development is

more sensitive to altered glucocorticoid exposure than fetal growth, the former being

compromised by increased levels of trophoblast apoptosis (Waddell et al. 2000) and

reduced growth (Benediktsson et al. 1993). Moreover, maternal glucocorticoid

treatment reduces the transplacental passage of glucose (Langdown and Sugden 2001)

and leptin (Smith and Waddell 2003) without a reduction in the expression of associated

placental transporter proteins. This suggests that increased glucocorticoid exposure is

likely to impact on the vascular exchange network of the placenta. Indeed,

glucocorticoids downregulate expression of the potent endothelial cell-specific mitogen,

vascular endothelial growth factor (VEGF) in various tissues (Heiss et al. 1996;

Koedam et al. 2002; Smink et al. 2003; Alagappan et al. 2005; Alonso et al. 2005)

which is likely to impact on placental vascular development (see section 2.5).


2.3 Peroxisome proliferator-activated receptors

PPARs are a subclass of the nuclear hormone receptor superfamily of ligand-regulated

transcription factors. Activation of the PPARs regulates a diverse range of biological

processes including cellular growth, development, differentiation and homeostasis

(Desvergne and Wahli 1999; Michalik et al. 2002; Evans et al. 2004). To date, three

PPAR subtypes (PPARA, PPARD and PPARG) encoded by separate genes have been

described, each with distinct tissue distribution patterns and groups of target genes

(Desvergne and Wahli 1999; Wang et al. 2002).

The human Ppara gene is located on chromosome 22, with the mouse homologue on

chromosome 15 (Desvergne and Wahli 1999). PPARA is predominantly expressed in

the liver, heart, brown adipose tissue, kidney and intestine and has a primary role in

lipid metabolism (Latruffe and Vamecq 2000). Ppard (also known as Pparb) is found

on human chromosome 6 and mouse chromosome 17 (Desvergne and Wahli 1999) and

although it appears to be ubiquitously expressed, recent studies indicate a possible role

in fat burning and adaptive thermogenesis (Evans et al. 2004). PPARG, originally

characterised for its role in adipocyte differentiation, has since been linked to various

physiological processes including insulin sensitivity and tumour growth (Koeffler 2003;

Evans et al. 2004). The Pparg gene is located on human chromosome 3 and mouse

chromosome 6 (Desvergne and Wahli 1999).

2.3.1 Retinoid X receptors and PPAR response elements

Unlike other nuclear hormone receptors, PPARs cannot bind DNA as either a monomer

or homodimer, but instead require the formation of a heterodimer with the retinoid X

receptor (RXR; Fig. 2.5) (Desvergne and Wahli 1999; Feige et al. 2005) of which three

distinct isoforms (RXRA, RXRB and RXRG) have been identified (Mangelsdorf et al.

1992). Upon ligand activation, PPAR:RXR heterodimers bound to PPAR response

elements (PPRE) upregulate specific gene transcription in the presence of appropriate

co-activators (Desvergne and Wahli 1999; Berger and Moller 2002; Michalik et al.

2002; Tan et al. 2005). In the absence of ligand, PPARG:RXR heterodimers in

particular are also able to inhibit gene expression in combination with particular co-

repressor complexes (Pascual et al. 2005; Tan et al. 2005). PPREs are characterised by a

direct repeat of two core recognition motifs AGGTCA spaced by a single adenine

nucleotide, and an extended 5’-half site (AACT) (Kliewer et al. 1992; Desvergne and


Wahli 1999; Feige et al. 2005). RXRs are common binding partners for other members

of the nuclear receptor superfamily and thus competition for a heterodimeric partner

may play a role in regulating the activity of PPARs. In vivo competition, however, is

likely to be the result of limited RXR expression. Indeed, due to the similar binding

properties of PPARs to RXRs and PPREs, the possibility exists that differential

expression of each isoform may play a role in individual isoform activation in situations

of limited RXR expression (Desvergne and Wahli 1999).

2.3.2 PPAR activation

Part of the diversity in the actions of each PPAR isoform stems from the large range

ligands that activate them (Desvergne and Wahli 1999). Indeed, rapid expansion of

knowledge regarding PPAR activity is, in part, due to their ease of use as therapeutic

targets. Amongst the naturally occurring ligands, each of the three PPAR isoforms are

bound and activated by numerous fatty acids and prostaglandins suggesting that the

metabolic effects of PPARs may reflect self regulation of fatty acid levels. Common

synthetic ligands include the hypolipidemic agents, such as fibrates, which

preferentially bind the PPARA isoform, and insulin sensitizing agents, such as the

thiazolidinediones, that selectively bind PPARG (Desvergne and Wahli 1999). In

addition, Mukherjee et al. (1997) suggest that RXR agonists can upregulate the

expression of PPAR target genes as observed in the insulin sensitisation of diabetic

mice, a notable effect of PPARG activation. Although ligand binding is the dominant

form of PPAR activation, notable activation can occur in the absence of ligand as

evidenced by inhibition of transcriptional activity by dominant negative mutants of

PPARA and PPARG (Gurnell et al. 2000). In support of this, PPAR:RXR heterodimers

will bind to PPREs in the absence of a specific ligand (Feige et al. 2005).

2.3.3 Regulation of PPAR expression

The regulation of PPAR expression appears to be tissue- and isoform-specific with

confounding results observed in different contexts. For example, the expression of

Ppara and Pparg are downregulated by gonadotrophins in mouse ovarian macrophages

(Minge et al. 2006). The expression of Pparg is down regulated by PPARG-specific

ligands, such as troglitazone, as observed in ovarian macrophages (Minge et al. 2006)

and by troglitazone and tumour necrosis factor α in differentiated human adipocytes

(Perrey et al. 2001). This effect was reversed in skeletal muscle (Park et al. 1998), and


isolated rat hepatocytes (Davies et al. 1999; Davies et al. 2002) with troglitazone

upregulating PPARG expression in vitro. Glucocorticoids also upregulate Pparg

expression during adipocyte differentiation in vitro (Vidal-Puig et al. 1997; Zilberfarb et

al. 2001). Reduced free fatty acid concentration and exercise increases the expression of

PPARA and PPARD in human skeletal muscle (Watt et al. 2004).

2.3.4 PPARs and the placenta

Gene deletion studies have shown that PPARG is critically important for development

with Pparg-null mice dying by mid-gestation due to a failure in placental vascular

development (Barak et al. 1999). Moreover, recent human studies have revealed that

several aspects of early placental development are associated with PPARG expression,

including trophoblast differentiation and invasion (Schaiff et al. 2000; Tarrade et al.

2001; Fournier et al. 2002). Ppard-null mice also exhibit placental defects leading to

embryonic death by mid-gestation in the majority of cases (Barak et al. 2002), and Rxra

and/or Rxrb knockouts exhibit a similar phenotype to that seen in Pparg-null mice

(Wendling et al. 1999).

Each of the three PPAR isoforms is also known to be expressed in the mature rat

placenta (Langdown and Sugden 2001; Wang et al. 2002; Asami-Miyagishi et al. 2004)

but their roles in placental function remain unclear. Activation of PPARG has been

shown to promote angiogenesis in other tissues (Yamakawa et al. 2000; Verma et al.

2004; Yasuda et al. 2005) and thus may contribute to the marked increase in placental

vascularisation observed late in pregnancy. In addition to effects on vascularisation,

PPARs may play important roles in placental transport and local metabolism of fatty

acids (Wang et al. 2002). Activation of PPARD has been shown to suppress 11BHSD2

expression in human trophoblast cells (Julan et al. 2005), possibly affecting the

placental glucocorticoid barrier and growth of the placenta (see section 2.2.3 and 2.2.4).


RXRPPAR

FA

PGTZD

FA

Figure 2.5 Activation of PPAR/RXR heterodimers. Activation of PPARG heterodimers results in the

binding of the activated heterodimer to PPAR response elements on the DNA and activation of specific

target genes. Each of the PPARs are activated by a range of prostaglandins (PG) and fatty acids (FA),

while PPARG is also activated by the thiazolidinedione (TZD) group of drugs commonly used in the

treatment of type II diabetes.


2.4 The wnt signalling pathway

The wnt pathway has been under detailed investigation for the last 20 years, in which

time a number of signalling molecules have been identified. The term ‘wnt’ is derived

from a combination of the wingless gene (wg) in Drosophila and the MMTV proto-

oncogene (int-1) in mammalian cells, that later proved to be homologous genes. To

date, 19 wnt proteins have been described and all are highly conserved across

mammalian species. The wnt gene family encodes a group of secreted glycoproteins

usually 350-400 amino acids long with a conserved region containing 23-24 cystine

residues (Cadigan and Nusse 1997).

The wnt-initiated signalling cascade is generally associated with a canonical pathway

which involves the accumulation of cytoplasmic and/or nuclear β-catenin (CTNNB1),

and the transcription of specific target genes via the T-cell factor/lymphoid enhancer-

binding factor (TCF/LEF) family of DNA-binding proteins. It has recently become

apparent that wnt molecules may also act through non-canonical pathways such as the

wnt/Ca2+ pathway or the planar cell polarity pathway involved in the alignment and

bundling of actin filaments (Bejsovec 2005).

2.4.1 Canonical wnt signalling

The following discussion on the canonical wnt pathway is based on extensive

descriptions by Nusse and Varmus (1992), Cadigan (2002), and Nelson and Nusse

(2004). Canonical wnt signaling is initiated by binding of the secreted wnt glycoprotein

to a transmembrane frizzled (FZD)/lipoprotein receptor-related protein complex

resulting in the activation of the cytoplasmic protein disheveled (DSH). DSH inactivates

glycogen synthase kinase-3β (GSK3B) which results in the accumulation and

subsequent nuclear translocation of β-catenin (CTNNB1). In the presence of appropriate

co-activators, CTNNB1 binds the T-cell factor/lymphoid enhancer-binding factor

(TCF/LEF) family of DNA-binding proteins and upregulates transcription of various

target genes that drive proliferation and cell fate determination including cyclin-D1, the

c-MYC oncogene, survivin and various members of the wnt pathway. In the absence of

wnt signaling, active GSK3B forms an intracellular complex with adenomatous

polyposis coli (APC) and axin, targeting CTNNB1 for degradation via the ubiquitin-

proteosome pathway, thus removing the trophic stimulus (Fig. 2.6).


TCF

TCF

β-catenin

Axin

GSK3

APC β-catenin

β-catenin

β-catenin

Frizzled Frizzled

Dsh

Arrow/LRP5/6

Arrow/LRP5/6

Dsh

Wnt

Axin GSK3

APC

β-catenin

Figure 2.6 The canonical wnt pathway. Adapted from Logan and Nusse (2004). In the absence of wnt

signalling (left) CTNNB1 is targeted for degradation by an Axin/GSK3B/APC complex. In contrast, the

presence of a wnt ligand (right) activates dishevelled, which inhibits the formation of the

Axin/GSK3B/APC complex allowing cytoplasmic and nuclear accumulation of CTNNB1 and activation

of TCF transcription factors.


2.4.2 Function of wnts

The wnt signalling pathway involves ligand mediated cell-cell communication that

controls a wide range of biological processes through alteration of gene transcription.

The large number of ligand, receptor and associated signalling molecule variants add to

the complexity of wnt function. Numerous mouse knockout models exemplify the

function of wnts in controlling embryonic patterning as well as brain, retinal, heart,

adipose, bone, lung, placental and reproductive tract development. In the adult, loss of

specific wnt genes also leads to defects in kidney, respiratory and reproductive function

(for reviews see Logan and Nusse, 2004; Moon et al, 2004).

2.4.3 Wnts and the placenta

Wnt ligands and FZD receptors are expressed in almost all tissue types and appear to

play a role in female fertility and placental function. Active wnt signaling has

previously been demonstrated in human cytotrophoblasts (Eberhart and Argani 2001),

and recent reports demonstrate a critical role for the wnt pathway in placental

development. Specifically, Wnt7b and the Tcf/Lef1 genes are required for normal

chorioallantoic fusion (Galceran et al. 1999; Parr et al. 2001) and Wnt2 null mice

display impaired placental angiogenesis, reduced birth weight, and a higher rate of

mortality (Monkley et al. 1996). Interestingly, Fzd5 knockout mice also display defects

in placental vascularisation possibly implicating a role for Wnt5a and Wnt10b as the

physiological ligands of FZD5 in the placenta (Ishikawa et al. 2001).

2.4.4 Frizzled and frizzled related proteins

Frizzled proteins are approximately 700 amino acids in length with an amino terminal

end rich in cystine residues which serves as the binding site for the wnt ligand. This is

followed by seven trans-membrane domains anchoring the frizzled receptor to the cell

membrane (Wodarz and Nusse 1998). The FZD receptor was originally identified in

Drosophila whereby transfection of DFz2 conferred responsiveness to wg via direct

binding (Bhanot et al. 1996). Subsequent research has determined that individual wnts

are specific for certain FZD receptors, though current knowledge suggests that each wnt

ligand may bind to several receptors and vice-versa (Wodarz and Nusse 1998). Secreted

frizzled related proteins (SFRPs) are structurally similar to the frizzled receptors (30-

50% homology) but lack the transmembrane and intracellular components necessary for


signal transduction (Rattner et al. 1997; Kawano and Kypta 2003). To date there are 5

proteins identified (SFRP1, SFRP2, SFRP3, SFRP4 and SFRP5) each containing the

cystine rich domain found in the FZD receptors (Kawano and Kypta 2003). It has been

proposed that SFRPs inhibit the wnt pathway by binding to either the wnt ligand or

frizzled receptor (Guo et al. 1998; Bafico et al. 1999), potentially inhibiting the entire

canonical pathway (Suzuki et al. 2004). Inhibition of the wnt pathway results in

continual degradation of CTNNB1 and the absence of TCF/LEF-mediated gene

expression (Fig 2.7). Previous data have established a role for one of these secreted

proteins, SFRP4, in association with apoptosis of corpus luteum, ventral prostate and

mammary gland during involution in the rodent (Wolf et al. 1997; Marti et al. 1999;

Lacher et al. 2003). Additional studies have also identified an association between

SFRP4 and apoptosis in the thecal cells of the rat ovary during ovulation (Drake et al.

2003; Hsieh et al. 2003) and failing human myocardial cells (Schumann et al. 2000).

Furthermore, levels of SFRP4 expression inversely correlate with tumor progression in

human endometrial stromal and undifferentiated endometrial sarcomas (Hrzenjak et al.

2004). The precise role of SFRP4 in placental wnt signaling, however, remains to be

elucidated.

2.4.5 Glucocorticoid regulation of wnt signalling

There is a growing list of factors that affect wnt signalling in a variety of in vivo and in

vitro models, and among these a significant number relate to regulation by the nuclear

receptor superfamily, including the GR (Mulholland et al. 2005). Specifically, Ohnaka

et al (2005) have shown that the synthetic glucocorticoid, dexamethasone, can inhibit

wnt signalling in human osteoblasts via upregulation of the wnt-inhibitor, Dickkopf-1

(DKK1). More recent studies have demonstrated that glucocorticoids can inhibit

osteoblast differentiation and increase apoptosis via upregulation of the secreted frizzled

relative, SFRP1 (Wang et al. 2005). Glucocorticoids have also been shown to activate

GSK3B in osteoblasts, which promotes the degradation of CTNNB1 thus inhibiting the

wnt signalling pathway (Smith et al. 2002).


TCF

Frizzled Arrow/LRP5/6

Dsh

Axin GSK3

APC

β-catenin

SFRP

WntSFRP

Figure 2.7 SFRP inhibition of wnt signalling. Binding of SFRP to either the wnt ligand or Frizzled

receptor allows the formation of an Axin-GSK3B-APC complex that targets β-catenin (CTNNB1) for

degradation via the proteosome-ubiquitination pathway. CTNNB1 degradation thus reduces nuclear

translocation and transcription of wnt target genes.


2.5 Vascular endothelial growth factor

The cardiovascular system in the embryo is the first system to develop to a functional

state, providing a supply of nutrients and gases while enabling the removal of metabolic

wastes. Blood vessels develop via vasculogenesis, whereby new vessels are formed

directly from the differentiation and reorganization of mesenchymal stem cells, and

angiogenesis, which involves the formation of new blood vessels from the branching of

existing endothelium. Although the control of vascular growth is a complex process, the

presence of vascular endothelial growth factor (VEGF) appears to be a critical element

in the formation of new blood vessels. VEGF, also known as VEGFA or vascular

permeability factor, is a heparin binding, homodimeric glycoprotein of approximately

46 kDa that induces migration and proliferation of endothelial cells (Leung et al. 1989).

The Vegf gene family includes VEGFB, VEGFC, VEGFD and placental growth factor

(PLGF), however, the current review of the literature focuses on the biology of the

potent endothelial mitogen VEGF, its isoforms and receptors.

While VEGF is known to play a role in physiological vasculogenesis and angiogenesis,

it has also been implicated in the pathology of proliferative retinopathies, age related

macular degeneration, tumors, rheumatoid arthritis and psoriasis (Ferrara 1999; Ferrara

2004). Indeed, anti-VEGF therapy has recently been approved as a first line treatment in

the control of metastatic colorectal cancer (Tonra and Hicklin 2007). Gene deletion

studies demonstrate the importance of VEGF in developmental vasculogenesis and

angiogenesis, with a complete abrogation of vascular development in homozygous Vegf

knockout mice. Such is the critical nature of VEGF that even heterozygotes show an

embryonic lethal phenotype which is associated with a failure of placental vascular

development (Carmeliet et al. 1996; Ferrara et al. 1996). In addition to the role of VEGF

as an endothelial cell mitogen, it is also known to promote cell survival via upregulation

of anti-apoptotic proteins such as, Bcl-2, XIAP and survivin (for review see Ferrara

2004).

2.5.1 VEGF isoforms

The human and rodent Vegf genes are both encoded from eight exons with seven

introns. Alternate splicing of the sixth and seventh exons results in the generation of

four separate isoforms (termed VEGF121, VEGF165, VEGF189 and VEGF206 in the

human) with 121, 165, 189, 206 amino acids respectively. Similar isoforms have been


found in rodent tissues, but these isoforms generally code for one amino acid less than

their corresponding human homologue (VEGF120, VEGF164, VEGF188 and VEGF205).

Although the regulation of the relative abundance of splice variants is unclear, the

VEGF121, VEGF165 and VEGF189 appear to be the more highly expressed isoforms in

most human tissues (Fig. 2.8).

VEGF165, the predominant isoform in the majority of tissues, and the prototype isoform

in terms of functional capacity, lacks the residues encoded by exons 6 and 7. This

isoform retains the heparin binding properties of the less functional isoforms and thus, a

significant portion of the VEGF165 isoform is bound to the extracellular matrix (ECM).

However, because it is freely secreted in significant quantities the VEGF165 isoform is

still biologically active. VEGF121 is an acidic polypeptide that fails to bind to heparin

but is a freely diffusible and highly potent endothelial cell mitogen. In contrast,

VEGF189 and VEGF206 are highly basic and are primarily sequestered to the ECM where

they have little impact on mitogenic or angiogenic signalling. The ECM bound isoforms

can be released by heparin or heparinase-mediated cleavage into a 110 amino acid

isoform which may play a role in the regulation of local VEGF bioavailability and

activity. Importantly, however, the loss of the heparin binding domain reduces the

mitogenic activity of VEGF. A less frequent, but freely secreted splice variant

(VEGF145) has also been identified in human placenta and uterus (Charnock-Jones

1993), as well as rat and human penile erectile tissue (Burchardt, 1999) and ovine

placenta (Cheung 1995). The impact of this isoform on vascular development, however,

remains to be determined (Ferrara 2004).


Exons 1-5Exons 1-5 6B6B6A6A 77 88VEGF206

Exons 1-5Exons 1-5 6A6A 77 88VEGF189

Exons 1-5Exons 1-5 77 88VEGF165

Exons 1-5Exons 1-5 6A6A 88VEGF145

Exons 1-5Exons 1-5 88VEGF120

Exons 1-3Exons 1-3 7B7B 88VEGF110

342 Nucleotides 51 13272 18

234 Nucleotides 78 18

Figure 2.8 Human VEGF isoforms. Each VEGF isoform is the result of differentially spliced variants

which can be discriminated by PCR, whereby positioning of primers spans unique exon boundaries

(Adapted from Burchardt et al, (1999)).


2.5.2 VEGF receptors

VEGF exerts its biological action via two tyrosine kinase receptors, VEGF receptor 1

(VEGFR1) and VEGFR2 (also known as fms-like tyrosine kinase 1 (Flt1) and fetal liver

kinase 1 (Flk1) or kinase insert domain-containing receptor (KDR), respectively; Fig

2.9). Each contains seven extracellular immunoglobulin-like domains, a single

transmembrane region and a consensus intracellular tyrosine kinase sequence

interrupted by a kinase insert domain (for review see Ferrara 2004).

Despite being the first VEGF receptor identified (de Vries et al. 1992), the role of

VEGFR1 remains unclear. VEGF, and indeed PLGF, have been shown to directly bind

to the second Ig-like domain of VEGFR1 (Park et al. 1994). Furthermore, VEGFR1 is

upregulated in response to hypoxia (Gerber et al. 1997), but has limited mitogenic

properties. In the majority of cases, VEGFR1 appears to act as a decoy receptor,

sequestering VEGF from the more potent VEGFR2. An alternatively spliced, soluble

variant (SFLT-1) has also been identified with similar inhibitory properties (Kendall

and Thomas 1993). Thus, VEGFR1 may exhibit a dual function in different

circumstances. Although VEGFR2 binds VEGF with lower affinity than VEGFR1, it is

critical for developmental angiogenesis and haematopoiesis (Shalaby et al. 1995) and is

therefore the primary receptor for the mitogenic, angiogenic and permeability-

enhancing effects of VEGF. A third receptor, originally identified in neuronal guidance,

and unrelated to the tyrosine receptor kinases, termed neurophilin-1 (NP1), was found to

contain a VEGF binding site and to increase endothelial cell proliferation and

angiogenesis (Soker et al. 1998). Interestingly, there is no evidence of subsequent

signalling after VEGF-NP1 binding, although NP1 does enhance the binding of

VEGF165 to VEGFR2, potentially via the formation of a NP1/VEGFR1 complex (for

review see Ferrara 2004).

The expression of VEGF receptors is almost exclusively limited to endothelial cells,

although Clark et al (1996) have localized VEGFR1 to the trophoblast of first and third

trimester human placenta. Notably, the VEGFR2 isoform is only expressed in

endothelial cells. Interestingly, the localization of VEGFR1 was commonly associated

with VEGF indicating that VEGF may signal through this receptor in trophoblast.

Alternatively, the VEGF bound to VEGFR1 (or potentially the soluble form SFLT1)

may serve as an inactive pool of VEGF that can be released by the presence of PLGF

(Autiero et al. 2003; Ferrara 2004).


VEGFR1VEGFR2

VEGF206

VEGF189

VEGF121

EndothelialCell

ECM

VEGF110

VEGF165

NP1

(Soluble)VEGFR1

Figure 2.9 Receptor activation by VEGF isoforms. VEGF isoforms 121 and 165 act via VEGFR1 and

VEGFR2 to stimulate mitotic activity in endothelial cells. VEGF isoforms 189 and 206 are primarily

bound to the extracellular matrix (ECM), however, cleavage of ECM-bound isoforms results in a 110

amino acid isoform that is able to activate VEGF-R1 and R2 receptors.


2.5.3 Regulation of VEGF expression

The control of VEGF expression primarily relies on oxygen tension in combination with

a variety of hormonal and growth factor signals (for review see Ferrara 2004). Hypoxia

activates the hypoxia-inducible factor 1 (HIF1) which binds to a 28-base sequence in

the VEGF promoter to directly upregulate gene transcription (Levy, 1995; Liu 1995). Of

the major growth factors, epidermal growth factor, transforming growth factor α and β,

keratinocyte growth factor, insulin-like growth factor-I, fibroblast growth factor and

platelet-derived growth factor are all known to upregulate VEGF expression (for review

see Ferrara 2004). Hormonal factors, including thyroid stimulating hormone, ACTH,

gonadotrophins, estrogens, progestins and androgens also upregulate expression

(Albrecht et al. 2004; Ferrara 2004; Robb et al. 2004). In addition, activation of PPARG

(Yamakawa et al. 2000; Verma et al. 2004; Yasuda et al. 2005) and certain oncogenic

mutations in the wnt signaling pathway (Zhang et al. 2001) increase VEGF expression.

By contrast, glucocorticoids are potent inhibitors of VEGF expression as observed in

brain tumors (Heiss et al. 1996), chondrocytes (Koedam et al. 2002; Smink et al. 2003),

human airway smooth muscle (Alagappan et al. 2005) and hypothalamic magnocellular

neurons (Alonso et al. 2005).

2.5.4 VEGF and the placenta

Placental growth and placental blood flow are critical determinants of fetal growth.

Indeed, numerous pathologies that reduce blood flow or placental exchange capacity are

known to impact on fetal growth and survival. Amongst the factors that contribute to

placental angiogenesis it is not surprising that VEGF plays a central role (Reynolds et

al. 2005). The importance of VEGF was made abundantly clear when gene deletion

studies demonstrated that even heterozygotes show an embryonic lethal phenotype

associated with a failure of placental vascular development (Carmeliet et al. 1996;

Ferrara et al. 1996).

Following the establishment of a functional placenta, VEGF remains crucial in the rapid

growth and vascularisation that normally occurs thereafter. Placental expression of Vegf

mRNA increases during late pregnancy in the baboon (Hildebrandt et al. 2001; Albrecht

et al. 2004; Robb et al. 2004) and the rodent (Ni et al. 1997) in association with

increased vascularisation of the placenta (Hildebrandt et al. 2001; Lewis et al. 2001;

Doherty et al. 2003; Albrecht et al. 2004; Coan et al. 2004; Robb et al. 2004). In the


rodent study, however, no distinction was made between the two morphologically- and

functionally-distinct zones of the placenta (Ni et al. 1997). By contrast, a reduction in

circulating levels of VEGF (Lyall et al. 1997) and pharmacological inhibition of VEGF

(Rutland et al. 2005) are both associated with the impaired vascular growth of

intrauterine growth restriction. Interestingly, increased circulating levels of the soluble

form of the VEGFR1 receptor (SFLT1) have been observed in preeclampsia (Maynard

et al. 2003) and may also contribute to the apparent endothelial dysfunction of this

condition by inhibition of VEGF activity.


Chapter 3 Experimental Objectives

The overall aim of the studies presented in this thesis was to investigate the impact of

glucocorticoids on placental development over the final third of pregnancy. From the

review of the literature is it clear that glucocorticoids, either from the mother or via

exogenous administration, can restrict fetal growth. Previous data suggest that

glucocorticoids reduce placental growth while increasing the rate of trophoblast

apoptosis and reducing transplacental passage of crucial growth factors such as glucose

and leptin. The exact mechanism by which glucocorticoids directly impact on the

growth and function of the placenta, however, remains to be determined. Placental

vascularisation is known to be controlled by a complex balance of numerous signalling

pathways. Gene deletion studies have identified a range of such pathways that function

in the control of placental development, including the PPAR nuclear receptors, the wnt

signalling pathway and the potent angiogenic factor, VEGF. Using the rat placenta, the

spatial and temporal expression of the PPAR nuclear receptors, the secreted wnt

antagonist, SFRP4, and the potent angiogenic factor, VEGF, were analysed in

conjunction with detailed stereological quantification of placental vascularity during the

final third of normal pregnancy and after maternal glucocorticoid administration. To this

end, three separate studies were undertaken and the specific experimental objectives of

each are outlined below.

Study 1: Placental expression of peroxisome proliferator-activated receptors in rat

pregnancy and the effect of increased glucocorticoid exposure

The objective of this study was to quantify the spatial and temporal distribution of the

three PPAR isoforms, and their heterodimeric partner RXRA, across the final third of

normal rat pregnancy and after increased maternal glucocorticoid exposure. Placental

expression of each gene was quantified by real-time RT-PCR and combined with

immunohistochemical localisation at day 16 and 22 of normal pregnancy, the period of

maximal fetal and placental growth. Separate analyses were made for the two

morphologically- and functionally-distinct regions of the placenta, the basal and

labyrinth zone, since only the latter undergoes significant growth over the final third of

pregnancy. The results from day 22 of normal pregnancy were compared with those


obtained after administration of dexamethasone, a synthetic glucocorticoid that is poorly

metabolised by the 11BHSD glucocorticoid barrier, thus allowing relatively unrestricted

access to the GR. These analyses provided an insight into the zone-specific expression

of the PPAR isoforms, highlighting the individual variation in expression patterns of

each isoform across normal pregnancy and in the context of glucocorticoid-mediated

inhibition of placental growth.

Study 2: Placental expression of secreted frizzled related protein-4 in the rat and the

impact of glucocorticoid-induced fetal and placental growth restriction.

Recent studies have shown that glucocorticoids impact on placental development via

reduced growth and increased rates of trophoblast apoptosis. One potential candidate in

the mediation of cell fate signalling in the placental milieu is the secreted frizzled

related protein, SFRP4. This inhibitor of the wnt pathway has been shown to regulate

cell fate signalling and may play a role in wnt-mediated apoptotic and/or anti-

proliferative signalling in placental trophoblast. Indeed, increased expression of SFRP4

is associated with apoptotic and anti-proliferative signalling in other reproductive

tissues, while wnt signalling also plays a critical role in placental vascularisation. The

expression of SFRP4 was investigated during normal placental development and after

glucocorticoid-mediated placental growth restriction. Quantification of SFRP4 was

carried out by real-time RT-PCR and combined with localisation of Sfrp4 mRNA (in

situ hybridisation) and protein (immunohistochemistry). Spatial and temporal analyses

of SFRP4 were analysed in conjunction with the tissue and sub-cellular distribution of

placental CTNNB1, a marker of functional wnt signalling. Taken together, these

experiments suggest a role for SFRP4 in both normal gestation and glucocorticoid-

mediated placental growth restriction, emphasizing the link between SFRP4 and the

mediation of wnt signalling via expression and distribution of CTNNB1.


Study 3: Glucocorticoids prevent the normal increase in placental vascular

endothelial growth factor expression and placental vascularity during late pregnancy

in the rat.

The first two studies provided profiles of two novel regulators of placental growth and

development and indeed, potential regulators of placental VEGF expression over the

final third of pregnancy. Study 1 (Chapter 5) showed that PPARG, in particular, is

specifically upregulated in the labyrinth zone near term during normal pregnancy, but is

inhibited following maternal dexamethasone treatment. The labyrinthine expression of

SRFP4 (Chapter 6), however, remains low over the course of normal pregnancy,

increasing only after maternal dexamethasone treatment. In combination, these results

are consistent with glucocorticoid-mediated inhibition of placental angiogenesis via

modulation of VEGF expression.

Therefore, the objective of Study 3 was to quantify the expression pattern of the major

placental VEGF isoforms in conjunction with the vascular development that occurs over

the course of normal pregnancy. Unbiased stereological analyses facilitated the

quantification of changes in normal placental vascularisation which was compared to

placentas from mothers treated with dexamethasone. The experiments of Study 3 thus

quantified the normal growth of the labyrinthine placental vasculature, with separate

analyses of fetal and maternal compartments allowing comparison with patterns of gene

expression. Placental expression of VEGF was determined by real-time RT-PCR and

localised by immunohistochemistry at days 16 and 22 of normal pregnancy. These data

were compared to the expression and distribution of VEGF after maternal

dexamethasone treatment.


Chapter 4 Materials and Methods

4.1 Animals

4.1.1 Rat maintenance

Nulliparous albino Wistar rats aged between 8 and 12 weeks were obtained from

Animal Resources Centre (Murdoch, WA, Australia) and maintained under controlled

conditions. At all times rats were maintained in environmentally controlled rooms

(mean temperature 21°C, mean humidity 43%) where lighting was based on a strict 12

hour light: 12 hour dark cycle.

Rats were normally housed three per cage, with rat and mouse cubes (18.9% protein,

5.2% fat, 5.0% crude fibre, 0.77% calcium, 0.57% phosphate, 0.41% NaCl, 14.5 MJ/kg

digestible energy; Glen Forrest Stockfeeders, WA, Australia) and acidified tap water

(tap water plus 0.026% HCl) available ad libitum. All procedures involving animals

were conducted after approval by the Animal Ethics Committee of The University of

Western Australia.

4.1.2 Mating and pregnancy determination

Estrous cycle stage was determined by the use of a moistened wire loop inserted to a

depth of approximately 5 mm into the vagina. Collected cells were then smeared onto a

glass slide for examination under a light microscope. Estrus was characterised by the

presence of cornified cells and the absence of leukocytes and epithelial cells (Table 2.1).

Female rats then were mated by placing 1 male in a cage of three to five females before

1600 hours and left overnight. The first day of pregnancy was designated as the day in

which spermatozoa were present in a vaginal smear.


4.2 Manipulation of glucocorticoid exposure

Dexamethasone is a synthetic glucocorticoid that is able to increase circulating

glucocorticoid levels. Moreover, dexamethasone is a relatively poor substrate for the

enzyme 11BHSD, which enables relatively unrestricted access to the GR in each of the

maternal, fetal and placental compartments. Increased fetal and placental glucocorticoid

exposure was therefore achieved by administration of, the highly water soluble,

structural analogue, dexamethasone-21-acetate (1 μg/ml; Sigma, St Louis, MO, USA) in

the drinking water from day 13 to 22 of pregnancy (term = 23 days). Previous studies

have shown that these treatments reduce fetal weight by approximately 30% at day 22

(Waddell et al. 2000) which is consistent with the reduced birth weight observed in

human cases of intrauterine growth restriction. Solutions were stored in 2.5 L containers

at room temperature.

4.3 Surgical procedures

4.3.1 Preparation and anesthesia

Prior to surgical procedures, all surfaces were wiped with 70% ethanol and instruments

autoclaved to ensure sterility. Rats were anesthetized with a mixture of halothane

(induction 5%, maintenance 2.5%, Rhone Merieux, VIC, Austraila), nitrous oxide (0.8

L/min, Medical EP grade, BOC, NSW, Australia) and oxygen (0.2 L/min, Medical EP

grade, BOC, NSW, Australia) using a Floutec 3 inhalation anesthetic apparatus

(Cyprane, Yorkshire, England).

4.3.2 Tissue collection and separation of placental zones

A 3 cm abdominal incision along the midline was used to expose the entire length of

both uterine horns. After determining litter size, three fetuses and their placentas were

chosen randomly, excluding the two fetuses on either side of the caudally fused portion

of the uterus. Each of the three chosen fetuses were dissected free from the uterus and

fetal membranes and weighed. The corresponding placenta was initially weighed as a

whole, and again after blunt dissection into basal and labyrinth zones. Placental tissues

were either snap frozen in liquid nitrogen for nucleic acid analysis, fixed in

HistochoiceTM MB (Amresco, Solon, OH, USA) for immunohistochemistry, or in fresh

4% paraformaldehyde (Merck, Darmstadt, Germany) in PBS for in situ hybridisation.


4.4 Immunohistochemistry

4.4.1 Reagents

The fixative for all immunohistochemical procedures was Histochoice ‘MB’ fixative

(Amresco, Solon, OH, USA). The mouse monoclonal primary antibodies to PPARG

(sc-7273) and VEGF (sc-7269), and rabbit polyclonal primary antibody to RXRA (sc-

553) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The

mouse monoclonal primary antibody to CTNNB1 (C7082) was purchased from Sigma

(St Louis, MO, USA). The rabbit polyclonal primary antibody to SFRP4 was a kind gift

from Dr Robert Friis, University of Bern, Switzerland. The mouse monoclonal primary

antibody to CD31 (#550300) was purchased from BD Biosciences (NSW, Australia).

The avidin-biotin enzyme kit (Vectastain ABC Kit) was purchased from Vector

Laboratories (Burlingame, CA, USA). Ethanol, methanol, toluene, Triton-X and Depex

mounting medium were all purchased from BDH Chemicals (Kilsyth, Vic, Australia)

and Paraplast wax was from Oxford Labware (St Louis, MO, USA). All other reagents

were purchased from Sigma Chemical Company (St Louis, MO, USA). Phosphate

buffered saline (PBS) contained 8 g NaCl, 0.2 g KCl, 1.4 g Na2HPO4 and 0.24 g

KH2PO4 made up to 1 L with dH2O, pH 7.5.

4.4.2 Tissue fixation and processing

After excision, whole placentas were immediately immersed in ice-cold ‘MB’ fixative

and incubated overnight with agitation. Following fixation, samples were transferred to

70% ethanol and stored at 4°C until processing. Tissue processing was carried out in a

Shandon Citidell 1000 tissue processor (Shandon Southern Processor Products Ltd,

Cheshire, England) according to the protocol in Table 4.1.


Table 4.1. Processing protocol

Solution Time

90% Ethanol 2 hours (h)

100% Ethanol 2 h

100% Ethanol 2 h

100% Ethanol 2 h

Toluene 2 h

Toluene 2 h

Wax (65°C) 2 h

Wax (65°C) 2 h

Wax (65°C) 30 minutes (vacuum)

4.4.3 Poly-L-Lysine coating of microscope slides

To promote the adhesion of paraffin sections, slides were treated with poly-L-lysine.

Slides were initially cleaned with an acid alcohol solution (1% HCl in 70% ethanol) and

dried before a 5 minute immersion in poly-L-lysine (1:10 dilution in dH2O). Slides were

then drained and allowed to dry completely.

4.4.4 Sectioning

4 μm transverse sections from paraffin-embedded placental blocks were cut on a rotary

microtome (RM 2125 RT) from Leica Microsystems Pty Ltd (North Ryde, NSW,

Australia) then floated on a glass slide with approximately 10 ml of 25% ethanol before

floating on a water bath containing dH2O (45°C). Sections were then applied to poly-L-

lysine coated slides and incubated at 45°C overnight to encourage adhesion.

4.4.5 Dewaxing

Prior to immunohistochemical analysis, sections were deparaffinised in two 5 minute

changes of toluene then hydrated through graded alcohols according to Table 4.2.


Table 4.2. Dewaxing protocol

Solution Time

100% ethanol 2 min

100% ethanol 2 min

90% ethanol 2 min

70% ethanol 2 min

ddH2O 2 x 5 min

4.4.6 Blocking for non-specific staining

Immediately following dewaxing, each section was incubated for 10 minutes in 3%

hydrogen peroxide (H2O2; in methanol) to quench endogenous peroxidase activity.

Residual H2O2 was removed by two 5 min rinses in PBS before subsequent application

of any primary antibodies.

4.4.7 Immunostaining

Mouse monoclonal primary antibody to PPARG, and rabbit polyclonal primary

antibody to RXRA were each used at a dilution of 1:100 (in PBS, 0.1% BSA, 0.1%

Triton X) overnight at 4°C. Mouse monoclonal primary antibodies to CTNNB1, CD31

and VEGF and rabbit polyclonal primary antibody to SFRP4 were each used at a

dilution of 1:50 (in PBS, 0.1% BSA, 0.1% Triton X) overnight at 4°C. Unbound

primary antibody was removed with two 5 minute washes in PBS.

The removal of primary antibody was followed by a 30 min room temperature

incubation with biotinylated anti-mouse or anti-rabbit secondary at 1:200 (in PBS, 0.1%

BSA Triton X) as appropriate. Unbound secondary antibody was removed with two 5

minute washes in PBS before application of freshly made streptavidin-peroxidase

complex at a 1:50 dilution in PBS followed by Vectastain ABC Kit for 30 min at room

temperature. Positive staining was visualized by application of diaminobenzidine

substrate (0.7 mg in 1 ml of PBS) for 30 seconds, or until the desired contrast was

achieved, whereby the reaction was stopped by washing in water. Each section was

counterstained with hematoxylin for a further 30 sec before dehydration in graded

alcohols (Table 4.3) and mounting in Depex mounting medium.


Table 4.3. Rehydration protocol

Solution

50% Ethanol 10 agitations




Toluene 10 agitations

Toluene 10 agitations

Toluene until mounting

4.5 Real time reverse transcription-polymerase chain reaction

4.5.1 Background

The specific method of real time RT-PCR used in these studies utilizes the properties of

the fluorescent dye, SYBR green, which binds exclusively to the minor groove of

double stranded DNA. Importantly, SYBR green only fluoresces when bound

facilitating the exclusive detection of double stranded DNA. Amplification of each

specific product is therefore able to be measured by levels of fluorescence at the end of

the elongation phase of each cycle. The cycle at which the sample fluorescence

increases above background levels is known as the threshold cycle (Cτ) and this is

proportional to the original starting concentration of transcript in the cDNA.

4.5.2 Reagents

Analyses of all mRNA transcript expression levels were performed by real time RT-

PCR on the Rotorgene 3000 (Corbett Industries, Sydney, Australia) using iQ SYBR

Green Supermix (BioRad, Hercules, CA, USA). Gel extraction was performed with

QIAEX II gel extraction kit (Qiagen, Melbourne, Australia). RNAzap and DNA-free

reagent were obtained from Ambion (Austin, TX, USA). Other reverse transcription

reagents including 5X first strand buffer, dNTPs, M-MLV Reverse Transcriptase RNase

H Point Mutant, random hexamer primers, RNasin and agarose were all purchased from

Promega Corp. (Madison, WI, USA). Tri-Reagent was obtained from Molecular

Resources Centre (Cincinnati, OH, USA). Ethidium bromide (Roche Molecular


Biochemicals, Castle Hill, NSW, Australia) was made to a 10 mg/ml solution in RNase

free ddH2O. Ultraclean PCR Cleanup kit was purchased from MoBio Industries (Solana

Beach, CA, USA). All other reagents used for RT-PCR were purchased from Sigma

Chemical Company (St Louis, MO, USA). TAE buffer (10 x) was prepared as follows:

48.4 g Tris, 11.4 ml glacial acetic acid, 20 ml 0.5 M EDTA (pH 8.0), made up to 1 L

with ddH2O.

4.5.3 RNA sample preparation

Tissue destined for RNA extraction was snap frozen in sterile microfuge tubes by

immersion in liquid nitrogen and stored at -80°C until extraction. Total RNA was

isolated from placental zones by homogenizing approximately 50 mg of tissue in 1 ml

of ice cold Tri-Reagent homogenization buffer. Sterility of homogenization equipment

was achieved via cleaning relevant surfaces with RNAzap.

The resultant homogenate was added to a 1.5 ml microfuge tube containing 200 µl of

chloroform and vortexed for 15 seconds. Samples were stored on ice for 15 minutes and

then centrifuged at 12,000 x g for 15 minutes. All centrifugations were carried out at

4°C in an Eppendorf refrigerated centrifuge (Hamburg, Germany). The upper, aqueous

phase was then transferred to a clean tube containing an equal volume of isopropanol,

mixed and incubated on at room temperature for 15 minutes. Each tube was then

centrifuged for another 15 minutes at 12,000 x g. The supernatant was removed and the

resultant pellet was washed with 1 ml of 75% ethanol (in RNase free ddH2O) and

centrifuged for 8 min at 7,600 x g. The pellet was air dried for approximately 10

minutes or until translucent, then dissolved in 50 μl RNase free water.

Quantification of total RNA was carried out using a spectrophotometer (Beckman Pty.

Ltd., Highgate, WA, Australia) at a wavelength of 260 nm. The spectrophotometer was

blanked against RNase free water and then the absorbance of 50 μl of each RNA sample

diluted 20-fold in RNase free water was measured. RNA integrity was assessed by

agarose gel (2% in 1 x TAE) electrophoresis prior to ethidium bromide staining of the

nucleic acids.


4.5.4 Reverse transcription

Total RNA was treated to remove contaminating genomic DNA using the DNA-free

reagent (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. For

reverse transcription, DNase-treated total RNA (1 μg) was added to 0.5 μl random

hexamer primers (100 pmol/μl), made up to 12 μl with RNase free ddH2O and

incubated for 70°C to remove secondary structure then placed on ice for 5 minutes.

First strand buffer (M-MLV reverse transcriptase 5x reaction buffer) 4 μl, dNTPs (5

mM) and 1.2 μl of RNasin were then added. The solution was mixed gently and

incubated for 2 min at 42°C before the addition of 1 μl of M-MLV Reverse

Transcriptase RNase H Point Mutant. Reactions were mixed and incubated at 42°C for

60 min in a thermal cycler (MJ Research Inc. Watertown, MA). The reaction was then

inactivated by heating to 70°C for 15 min. The resultant cDNAs were purified using the

Ultraclean PCR Cleanup kit (MoBio Industries, Solana Beach, CA, USA) and stored at

-20°C.

4.5.5 Primers

The primers for quantitative PCR analysis of target transcripts and their empirically

determined annealing temperatures are detailed in Table 4.4. Primers for Ppard and

total Pparg were adapted from Hoekstra et al (2003) whereas those for Ppara, Rxra,

Muc1, Sfrp4 and Ctnnb1 were designed using Primer 3 software (MIT/Whitehead

Institute; http://www-genome.wi.mit.edu) (Rozen and Skaletsky 2000). No distinction

was made between Pparg1 and Pparg2 on the basis that the rodent placenta expresses

only the Pparg1 isoform (Barak et al. 1999). Primers for Vegf isoform-specific

amplification were also designed using Primer 3 software (MIT/Whitehead Institute;

http://www-genome.wi.mit.edu) (Rozen and Skaletsky 2000) with the reverse primers

for Vegf120, Vefg164 and Vegf188 positioned at distinct exon boundaries for each isoform

(Fig 2.5). Each of the selected primer pairs were positioned to span introns to ensure no

product was amplified from contaminating genomic DNA. Primer specificity was

determined initially by melt curve analysis (see section 4.5.9) followed by separation of

products on an agarose gel to determine size and to confirm the presence of a single

band. The resulting amplicons were also sequenced to confirm specificity.


Table 4.4. Primers used for real time RT-PCR

Gene Forward/Reverse Primer (5` to 3`) Annealing Temp.

Amplicon Size

F AAT CCA CGA AGC CTA CCT GA Ppara

R GTC TTC TCA GCC ATG CAC AA 54°C 132 bp

F GAG GGG TGC AAG GGC TTC TT Ppard

R CAC TTG TTG CGG TTC TTC TTC TG 60°C 101 bp

F CAT GCT TGT GAA GGA TGC AAG Pparg

R TTC TGA AAC CGA CAG TAC TGA CAT63°C 131 bp

F GTC AAG CAG CAG ACA AGC AG Rxra

R GAG AAG GAG GCA ATC AGC AG 57°C 136 bp

F CTT CCC ACT GTG TGG ACC TT

Sfrp4 R CTA TCC CTC GAA CGC AAG TC 62°C 180 bp

F TGA AGG TGC TGT CTG TCT GC

Ctnnb1 R GCT GCA CTA GAG TCC CAA GG 62°C 192 bp

F ATG AAG TGG TGA AGT TCA TGG A Vegf120

R TTG TCA CAT TTT TCT GGC TTT G 59°C 319 bp

F ATG AAG TGG TGA AGT TCA TGG A Vegf164

R AGG CTC ACA GTG ATT TTC TGG 59°C 323 bp

F AGG AAA GGG AAA GGG TCA AA Vegf188

R AAA TGC TTT CTC CGC TCT GA 60°C 96 bp

F CCC AAC TAC CTG GTC TGC AT Muc1

R ACT GTG GGT AGC CGT GGT AG 60°C 145 bp

F CTG AAG GTC AAA GGG AAT GTG Rpl19/L19 R GGA CAG AGT CTT GAT GAT CTC

52°C 195 bp


4.5.6 Polymerase chain reaction (PCR)

Real time RT-PCR was carried out using the Rotorgene 3000 instrument and iQ SYBR

Green Supermix. Each 10 µL reaction contained 5 µl of 2 x Supermix, 0.5 µL of each of

the forward and reverse primers at a final concentration of 0.5 µM (except for Ppara

primers which were at 0.25 µL) and was made up to 8 µL with nuclease free water.

Template (2 μl of either sample or standard) was added to each tube. Cycling conditions

for all assays included an initial 94°C denaturation for 3 minutes, followed by 30-35

cycles of 94°C denaturation, specific annealing temperatures (Table 4.4), and extension

at 72°C. The timing within PCR cycles varied between assays, whereby Ppara, Ppard,

Pparg, Rxra, Sfrp4, Ctnnb1 and Muc1 had denaturation times of 0 sec, annealing times

of 15 sec and extension times of 5 sec; whereas Vegf120, Vegf164, Vegf188 and Rpl19 had

denaturation, annealing and extension times of 30 sec each per cycle.

4.5.7 Quantification of transcripts

All samples were standardised against the internal control, Rpl19, as previously

described (Orly et al. 1994). Values were determined within the log-linear phase of the

amplification curve. The point at which each sample exceeded the cycle threshold was

compared to the Cτ values for the standard curve. Standard curves for each product were

generated using the Rotorgene 3000 software from gel extracted (QIAEX II gel

extraction kit) PCR products amplifying 10-fold serial dilutions and (Fig 4.1 and 4.2).

4.5.8 Melt curve analysis

The thermal dissociation properties of PCR products provide an additional method to

determine the identity of the amplified product, since DNA species of identical

composition denature at the same temperature. Conversely, multiple peaks within the

melt curve indicate the presence of non-specific amplification products. By heating each

of the PCR products between 70 and 99°C, the level of fluorescence emitted can

therefore describe the composition of amplified products in each sample. Each

particular species of DNA is represented as a peak in a graph of the negative derivative

of fluorescence plotted against temperature (Fig 4.3).


Figure 4.1 Real time amplification plot for Ppara mRNA standards. Amplification profiles of seven

standards (10-fold dilutions) where fluorescence is plotted against cycle number. The threshold line

indicates the point at which all samples fall within the log-linear phase of amplification and is the point

used to determine the threshold cycle for each sample.


Concentration107106105104103102101

Thre

shol

d cy

cle

25

20

15

10

R = 0.99991M = -3.388Efficiency = 0.97

Ppara mRNA

Figure 4.2 Standard curve generated for Ppara mRNA. Amplification profiles of seven

standards (10-fold dilutions) were used to generate standard curves for each gene. The

threshold cycle at which unknown samples reached their respective log-linear phase of

amplification is used to determine the concentration of unknown samples.


Figure 4.3 Melt curve analysis for Ppara mRNA. Denaturation of a single DNA product is represented

by the single peak (at approximately 84°C) in the derivative of fluorescence plotted against temperature.


4.6 Stereological measures

Stereological measurements were based on methods described previously in Coan et al.

(2004) in the mouse placenta and described in detail below.

4.6.1 Sample preparation and sectioning

Placentas taken for stereological measurements were subjected to the same fixation and

processing schedule described earlier for immunohistochemistry (sections 4.2.2 and

4.4.2). Three placentas from each of four separate litters within each time point or

treatment group were assessed for placental volumes, surface areas and fetal capillary

density. Sections were cut at 4 µm thickness close to the midline and arranged vertically

with the chorionic plate providing the theoretical horizontal plane.

4.6.2 Frame sampling

Frames were sampled after the whole tissue was overlaid with a theoretical grid

whereby numbered frames covered the entire section. Random number generation

selected a single frame within each section, of three placentas from each of four

separate litters within each time point or treatment group (i.e. 12 fields of view). Frames

were viewed at 400x magnification (objective lens = 40x magnification) to allow

accurate identification of placental morphology.

4.6.3 Statistical corrections

Volume, surface area and length densities were adjusted to compensate for tissue

processing-associated shrinkage of the tissue by measurement of average maternal

erythrocyte diameter before and after processing (Burton and Palmer 1988; Ali et al.

1996). Measures of density were converted to absolute measures by multiplying by the

total volume of the labyrinth zone assuming a specific gravity of 1g/cm3.

4.6.4 Volume estimates

Relative volumes of labyrinth zone components were calculated by a 35 point grid

overlaid on each field of view obtained as described above (Fig. 4.4). Points that fell in

each placental structure were counted and converted to a fraction of total placental

volume according to the Cavalieri principle (Gundersen and Osterby 1981). Absolute


volume of labyrinthine components (maternal blood space, fetal capillary and remaining

tissue volume) were determined by multiplying each component’s fraction by the total

placental volume according to the equation:

V(structure) = P(structure)/P(total)

where V(structure) is the volume of the structure (e.g. the volume of fetal capillaries)

within the reference space (e.g. the labyrinth zone); P(structure) is the number of points that

fell within the structure; and P(total) is the total number of points that fell within the

reference space (Fig. 4.4) (Coan et al. 2004).

4.6.5 Surface area estimates

Surface area measurements were estimated by using the same fields obtained for

volume measurements and overlaying a grid of cycloid arcs as previously reported

(Coan et al. 2004). Points of intersection of cycloid arcs with the lumen of maternal

blood spaces and fetal capillaries were used to calculate surface area densities in

vertically oriented sections (Fig. 4.5). The use of cycloid arcs was used correct for the

bias associated with vertically oriented sections randomly rotated around the horizontal

axis (Baddeley et al. 1986). CD31 immunohistochemistry (Section 4.4) was also used to

aid in the distinction between maternal and fetal blood spaces. Surface areas were

therefore estimated according to the equation:

S(structure) = (2 x ΣI(structure)/L(test line) x ΣP(ref)) x V(ref)

whereby S(structure) is the surface area of the structure (e.g. surface area of the fetal

capillaries); ΣI(structure) is the sum of intersection points of this surface area; L(test line) is

the length of the test line associated with each of the points; ΣP(ref) is the total number of

points that hit the reference space (e.g. the labyrinth zone), and; V(ref) is the volume of

the reference space (Coan et al. 2004).


4.6.6 Estimates of fetal capillary density, length and mean diameter

Combined length density, mean diameter and overall density of fetal capillaries were all

estimated by counting the number of capillary profiles in a counting square containing

two contiguous forbidden lines (Coan et al. 2004). The density of fetal capillaries was

expressed as the number of capillary profiles counted per unit of area (see below).

Capillary length density was then determined by the equation:

Lv(capillaries) = 2QA

whereby Lv(capillaries) is the length density of fetal capillaries, and QA is the number of

capillary profiles counted per counting frame. The length density is adjusted to give a

total combined length of the fetal capillaries by multiplying by the volume of the

labyrinth zone. The mean diameter of fetal capillaries was then estimated by the

following equation:

d = 2 x ([Vv(structure)/Lv(capillaries)]/π)1/2

4.7 Statistical analyses

Routine statistical analyses were performed according to the methods as previously

described (Snedecor and Cochran 1989). All group values were expressed as means ±

SEM, standardized by Rpl19 in the case of real time RT-PCR data. Comparisons

between or among groups were made by unpaired t-tests (Microsoft Excel, 2003) or

two-way analyses of variance (ANOVA; GenStat7, Hemel Hempstead, UK)) as

appropriate. Where the F test for the ANOVA reached statistical significance (P < 0.05),

differences were assessed by least significant difference (LSD) test. Details of specific

tests are included in the “Methods” section of each study.


Figure 4.4 A grid of cycloid arcs superimposed on the placental labyrinth. Volume is estimated by

the relative number of points that fall within each component of the tissue. Surface area is estimated in

vertical sections by the number of intersections that each blood space makes with the cycloid arcs. FC:

fetal capillaries; MBS: maternal blood space; scale bar = 10 µm.


Chapter 5 Placental expression of peroxisome proliferator-

activated receptors in rat pregnancy and the effect of

increased glucocorticoid exposure

Reprint of published manuscript, Biol Reprod 74(1): 23-8.

5.1 Abstract

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear

hormone receptor superfamily of ligand-activated transcription factors. Recent gene

deletion studies indicate that PPARG and PPARD play critical roles in rodent

development, including effects on placental vascularization. In this study we

investigated the expression of the PPAR isoforms and their heterodimeric partner,

RXRA, in the two functionally and morphologically distinct zones of the rat placenta

during normal gestation and after glucocorticoid-induced fetal and placental growth

restriction. Real-time reverse transcription-polymerase chain reaction and

immunohistochemical analysis demonstrated markedly higher expression of Ppara,

Pparg, and Rxra mRNA in labyrinth zone trophoblast as compared with basal zone near

term. There was also a marked increase in Pparg (65%, P < 0.05) and Ppara (91%, P <

0.05) mRNA specifically in the labyrinth zone over the final third of pregnancy. In

contrast, expression of Ppard mRNA fell (P < 0.001) in both placental zones over the

same period. Maternal dexamethasone treatment (1 µg/ml in drinking water; Days 13–

22, term = 23 days) reduced placental (44%) and fetal (31%) weights and resulted in a

fall in Pparg (37%, P < 0.05) mRNA expression specifically in the labyrinth zone at

Day 22. Placental expression of Ppara, Ppard, and Rxra was unaffected by

dexamethasone treatment. These data suggest that PPARG:RXRA heterodimers play

important roles in labyrinth zone growth late in pregnancy, possibly supporting vascular

development. Moreover, glucocorticoid inhibition of placental growth appears to be

mediated, in part, via a labyrinth-zone-specific suppression of PPARG.


5.2 Introduction

Intrauterine growth restriction (IUGR) is a major problem confronting obstetric and

paediatric medicine, with low birth weight increasing the rate of neonatal morbidity and

mortality (Barker et al. 1989). IUGR associated with a compromised environment in

utero can also increase the risk of adult onset diseases due to fetal programming (Seckl

and Meaney 2004). The control of fetal growth involves a range of genetic and

environmental factors mediated, in part, via the placenta (Fowden 1995; Bauer et al.

1998), and although a relationship between placental function and fetal well-being is

well established (Godfrey 2002; Pardi et al. 2002; Regnault et al. 2002; Kaufmann et al.

2003), the complex regulation of placental growth and development remains poorly

understood. Recent gene deletion studies (Barak et al. 1999; Barak et al. 2002) suggest

that the peroxisome proliferator-activated receptors (PPARs) may be key players in this

regulation, together with downstream effectors that impact on placental growth and

vascularity.

PPARs are a subclass of the nuclear hormone receptor superfamily of ligand-regulated

transcription factors. Activation of the PPARs regulates a diverse range of biological

processes including cellular growth, development, differentiation and homeostasis

(Desvergne and Wahli 1999; Michalik et al. 2002; Evans et al. 2004). To date, three

PPAR subtypes (PPARA, PPARD and PPARG) encoded by separate genes have been

described, each with distinct tissue distribution patterns and groups of target genes

(Desvergne and Wahli 1999; Wang et al. 2002). PPARA is predominantly expressed in

the liver, with a primary role in lipid metabolism (Latruffe and Vamecq 2000). PPARD

is ubiquitously expressed, and recent studies indicate a possible role in fat burning and

adaptive thermogenesis (Evans et al. 2004). PPARG, although originally characterised

for its role in adipocyte differentiation, has since been linked to various physiological

processes including insulin sensitivity and tumour growth (Koeffler 2003; Evans et al.

2004). Ligand-activated PPARs form a functional heterodimer with another nuclear

receptor, the retinoid X receptor (RXR) (Feige et al. 2005), of which three distinct RXR

isoforms (RXRA, RXRB and RXRG) have been described (Mangelsdorf et al. 1992).

Gene deletion studies have shown that PPARG is critically important for placental

development with Pparg-null mice dying by mid-gestation due to a failure in placental

vascular development (Barak et al. 1999). Consistent with this aspect of the Pparg-null

phenotype, activation of PPARG has been shown to promote angiogenesis in other


tissues (Yamakawa et al. 2000; Verma et al. 2004; Yasuda et al. 2005). Moreover,

recent studies reveal several aspects of human placental development have been linked

to PPARG activation, including trophoblast differentiation and invasion (Schaiff et al.

2000; Tarrade et al. 2001; Fournier et al. 2002). Ppard-null mice also exhibit placental

defects leading to embryonic death by mid-gestation in the majority of cases (Barak et

al. 2002), and Rxra and/or Rxrb knockouts exhibit a phenotype similar to that seen in

Pparg-null mice (Wendling et al. 1999). In addition to these vascular effects, it has also

been proposed that PPARs may play important roles in relation to placental transport

and metabolism of fatty acids (Wang et al. 2002).

Although each of the three PPAR isoforms is known to be expressed in the rat placenta

(Langdown and Sugden 2001; Wang et al. 2002; Asami-Miyagishi et al. 2004), their

temporal and zone-specific patterns of expression have not been quantified. The current

study, therefore, investigated the expression of the three PPAR isoforms and RXRA (the

predominant binding partner of the PPARs) over the final third of pregnancy, the period

of maximal fetal and placental growth. Separate analyses were conducted for the two

functionally and morphologically distinct zones of the rat placenta, the basal and

labyrinth zones, because only the latter grows in association with fetal growth in the

period of maximal fetal growth (Waddell et al. 1998), consistent with its role in fetal-

maternal exchange. The expression patterns of the PPARs and RXRA were also

examined in a model of increased glucocorticoid exposure known to restrict fetal and

placental growth (Burton and Waddell 1994), since previous reports indicate that

glucocorticoids can modulate PPAR expression in various tissues (Vidal-Puig et al.

1997; Zilberfarb et al. 2001; Mouthiers et al. 2005).


5.3 Materials and Methods

5.3.1 Animals


Animal Resources Centre (Murdoch, Australia) and maintained under controlled

conditions as described previously (Waddell et al. 1998). Rats were mated overnight

with the first day of pregnancy designated as the day in which spermatozoa were present

in a vaginal smear. All procedures involving animals were conducted after approval by

the Animal Ethics Committee of The University of Western Australia.

5.3.2 Glucocorticoid manipulations

Increased fetal and placental glucocorticoid exposure was achieved by administration of

dexamethasone acetate (1 μg/ml; Sigma, St Louis, MO, USA) in the drinking water

from day 13 to 22 of pregnancy (term = 23 days). We have previously shown that these

treatments reduce fetal weight by approximately 30% at day 22 (Waddell et al. 2000).

5.3.3 Tissue collection

For collection of placental zones, pregnant rats were anaesthetized with

halothane/nitrous oxide at either day 16 or 22 of gestation and 3 fetuses and placentas

were removed from each mother and weighed. Placental zones were separated by blunt

dissection, individually weighed, and either snap frozen in liquid nitrogen for real-time

quantitative RT-PCR or fixed in Histochoice MB (Amresco, Solon, OH, USA) for

immunohistochemistry.


Total RNA was isolated from placental zones at days 16 and 22 of pregnancy using Tri-

Reagent (Molecular Resources Centre, Cincinnati, OH, USA) as per the manufacturer’s

instructions. RNA integrity was assessed by ethidium bromide staining of the nucleic

acids prior to agarose gel electrophoresis (data not shown). Total RNA was treated to

remove contaminating genomic DNA using DNA-free reagent (Ambion, Austin TX,

USA). DNase-treated total RNA (1 μg) was used to synthesize cDNA using M-MLV

Reverse Transcriptase RNase H Point Mutant and random hexamer primers (Promega,

Madison, WI, USA) as per the manufacturer’s instructions. The resultant cDNAs were


purified using the Ultraclean PCR Cleanup kit (MoBio Industries, Solana Beach, CA,

USA).

5.3.5 Real-time RT-PCR

Analyses of expression levels for the three PPAR isoforms, Rxra, and L19 transcripts

were performed by real time RT-PCR on the Rotorgene 3000 (Corbett Industries,

Sydney, Australia) using iQ SYBR Green Supermix (BioRad, Hercules, CA, USA).

Primers for Ppard and total Pparg (Table 5.1) were adapted from Hoekstra et al

(Hoekstra et al. 2003) whereas those for Ppara and Rxra were designed using Primer 3

software (MIT/Whitehead Institute; http://www-genome.wi.mit.edu) (Rozen and

Skaletsky 2000). Each of the selected primer pairs were positioned to span introns to

ensure no product was amplified from genomic DNA, and the resulting amplicons were

sequenced to confirm specificity (data not shown). All samples were standardised

against L19 as previously described (Orly et al. 1994). Standard curves for each product

were generated from gel extracted (QIAEX II, Qiagen, Melbourne, Australia) PCR

products amplifying 10-fold serial dilutions and the Rotorgene 3000 software.

5.3.6 Immunohistochemistry

Sections were cut at 4 μm, deparaffinised and rehydrated before incubation for 10 min

in 3% H2O2 to block endogenous peroxidases. Mouse monoclonal primary antibody to

PPARG (sc-7273), and rabbit polyclonal primary antibody to RXRA (sc-553) were

obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA) and each used at a

dilution of 1:100 overnight at 4°C. This was followed by a 30 min room temperature

incubation with biotinylated anti-mouse or anti-rabbit secondary at 1:200 (BA2001 and

BA1000; Vector Laboratories, Burlingame, CA, USA) and streptavidin-peroxidase

complex at 1:50 (Vectastain ABC Kit, Vector Laboratories, CA, USA). Sections were

visualised by application of diaminobenzidine substrate (Sigma, St Louis, MO, USA)

and counterstained with haematoxylin before dehydration in graded alcohols and

mounting in DPX.


Table 5.1. Primers used for Ppara, Ppard, Pparg, Rxra and L19 real time RT-PCR

Gene Forward/Reverse Primer Annealing Temp.

Amplicon Size

AAT CCA CGA AGC CTA CCT GA Ppara

GTC TTC TCA GCC ATG CAC AA 54°C 132 bp

GAG GGG TGC AAG GGC TTC TT Ppard

CAC TTG TTG CGG TTC TTC TTC TG 60°C 101 bp

CAT GCT TGT GAA GGA TGC AAG Pparg

TTC TGA AAC CGA CAG TAC TGA CAT 63°C 131 bp

GTC AAG CAG CAG ACA AGC AG Rxra

GAG AAG GAG GCA ATC AGC AG 57°C 136 bp

CTG AAG GTC AAA GGG AAT GTG L19

GGA CAG AGT CTT GAT GAT CTC 52°C 195 bp


5.3.7 Statistical analysis

Fetal, total placental and placental zone weight values were determined for each mother

as the average of the three estimates. All group values are expressed as means (± SEM)

of gene expression standardised by L19. Two-way ANOVAs (GenStat7, Hemel

Hempstead, UK) were used to assess variation in expression levels for each of the

PPAR isoforms and Rxra, with gestational age and placental zone as sources of

variance. Where the F test for the ANOVA reached statistical significance (P < 0.05),

differences were assessed by least significant difference (LSD) tests (Snedecor and

Cochran 1989). The effects of dexamethasone on fetal and placental weights were

assessed by unpaired t-tests.


5.4 Results

5.4.1 Gestational changes in fetal, placental and placental zone weights

Fetal and placental weight both increased markedly from day 16 to 22 of pregnancy

(fetus: 18-fold increase, P < 0.001; placenta: 2-fold increase, P < 0.01). This increase in

placental weight was attributable entirely to growth of the labyrinth zone (3-fold

increase, P < 0.001), with no change evident basal zone weight (Fig. 5.1).

5.4.2 Gestational changes in Ppara, Ppard, Pparg and Rxra expression

The mRNAs for all PPAR isoforms and Rxra were readily detectable in both zones of

the rat placenta (Fig. 5.2). Expression of Ppara and Pparg mRNA increased in the

labyrinth zone (91%, P < 0.05; and 65%, P < 0.05, respectively) from day 16 to 22 of

pregnancy but remained unchanged in the basal zone over the same period. In contrast,

expression of Ppard mRNA decreased in both the basal (76%, P < 0.001) and labyrinth

zones (63%, P < 0.001) from day 16 to 22. Expression of Rxra mRNA was higher (P <

0.05) in the labyrinth zone compared to the basal zone at both day 16 (2.1-fold) and day

22 (4.8-fold).

Immunohistochemical analysis confirmed the overall pattern of PPARG and RXRA

distribution between the two placental zones. Moreover, PPARG and RXRA were both

localized to the nuclei of the labyrinth zone trophoblast, with no immunostaining

present in fetal endothelial cells of the labyrinth zone or giant trophoblast cells of the

basal zone (Fig. 5.3). No change in distribution of PPARG or RXRA was observed

between days 16 and 22.


16 2216 220.00.10.20.30.40.50.6

Fetal (/10) Placental Basal Labyrinth

16 2216 22

Wei

ght (

g)

**

*

FIG. 5.1. Fetal, placental and placental zone weights at days 16 and 22 of rat pregnancy. Values are

the mean ± SEM (n = 6-11 per group). * P < 0.01, compared with corresponding day 16 value (unpaired

t-test).


a

b

aaa

b b

a

a

b

aa a

bb

a

0.0

0.5

1.0

1.5

Basal Labyrinth

Rel

ativ

e ex

pres

sion

A

0

20

40

60

80

Basal Labyrinth

B

05

101520253035

Basal Labyrinth

C

0

2

4

6

8

Basal Labyrinth

D

Rel

ativ

e ex

pres

sion

16 2216 22 16 22 16 22

16 2216 22 16 22 16 22

Ppara Ppard

Pparg Rxra

FIG. 5.2. Zonal expression of (A) Ppara; (B) Ppard; (C) Pparg and; (D) Rxra mRNA at day 16 and

22 of rat pregnancy. Values are the mean ± SEM (n = 6-11 per group). Those values without common

notation differ (P < 0.05, one-way ANOVA and t-test).


PPARG

RXRA

FIG. 5.3. Zonal distribution of PPARG and RXRA compared to negative controls (no primary) at

day 22 in the rat placenta by immunohistochemistry. (A) and (D) PPARG and RXRA negative

controls; (B) and (E) PPARG and RXRA displaying nuclear expression in labyrinth zone trophoblast,

with no expression detected in either fetal endothelial cells of the labyrinth zone or giant trophoblast of

the basal zone; (C) and (F) higher power view of labyrinth zone trophoblast nuclei stained positive for

PPARG and RXRA (arrowheads) with no localisation to fetal endothelial cell nuclei. LZ: labyrinth zone;

BZ: basal zone; scale bar = 5μm.


5.4.3 Fetal and placental weights after dexamethasone treatment

Dexamethasone treatment from day 13 of pregnancy reduced fetal weight at day 22 by

24% (P < 0.01) and placental weight by 32% (P < 0.01), with the latter due to

comparable effects on both basal and labyrinth zone weights (Fig. 5.4).

5.4.4 Effect of dexamethasone on expression of PPAR isoforms and Rxra

Expression of all the PPAR isoforms and Rxra were assessed following treatment with

dexamethasone. Expression of Pparg mRNA at day 22 was reduced by 37% (P < 0.05)

in the labyrinth zone after dexamethasone treatment compared with placentas from the

untreated mothers (Fig. 5.5). In contrast, expression of the other PPAR isoforms and

Rxra were all unaffected by dexamethasone treatment (data not shown).

Immunohistochemical analysis revealed no change in the cellular distribution of either

PPARG or RXRA following treatment with dexamethasone.


Con DexCon Dex

***

0.00.10.20.30.40.50.6

Fetal (/10) Placental Basal Labyrinth

Con DexCon Dex

Wei

ght (

g)

**

FIG. 5.4. Fetal, placental and placental zone weights after dexamethasone (Dex) treatment

compared to untreated controls (Con). Values are the mean ± SEM (n = 9-11 per group). * P < 0.01, **

P < 0.001, compared with corresponding control value (unpaired t-test).


05

101520253035

Rel

ativ

e Ex

pres

sion

*

Basal Labyrinth

Con DexCon Dex

Pparg

FIG. 5.5. Zonal expression of Pparg mRNA at day 22 of rat pregnancy following treatment with

dexamethasone (Dex) compared to untreated controls (Con). Values are the mean ± SEM (n = 9-11

per group). * P < 0.05, compared with corresponding control value (one-way ANOVA and t-test).


5.5 Discussion

This study investigated the expression of PPARA, PPARD, PPARG and their

heterodimeric partner, RXRA, in the basal and labyrinth zones of the rat placenta over

the final third of pregnancy. The major findings were that Ppara, Pparg and Rxra

mRNA expression were all higher in the rapidly-growing labyrinth zone near term, and

Ppara and Pparg mRNA increased from day 16 to 22 specifically in the labyrinth zone.

In contrast, Ppard expression fell in both the basal and labyrinth zones over the final

third of pregnancy. Maternal dexamethasone treatment restricted fetal and placental

growth and resulted in a marked reduction in Pparg mRNA expression specifically

within the labyrinth zone, but had no effect on the expression of Rxra or the other PPAR

isoforms. These data are consistent with the previously reported obligatory role for

PPARG in placental development, and further suggest that PPARG is particularly

important in the labyrinth zone during the period of maximal fetal and placental growth.

The higher expression of PPARG and RXRA in the labyrinth zone compared with the

basal zone observed in this study is consistent with a role for PPARG in promoting

placental growth, possibly through increased trophoblast differentiation and/or

angiogenesis of fetal blood vessels. Accordingly, the increase observed in labyrinth

zone expression of PPARG occurred in association with the major growth of this zone

during the final third of rodent pregnancy, which includes a marked increase in

placental vascularisation (Coan et al. 2004). Immunohistochemical localisation of both

PPARG and RXRA to the nuclei of labyrinth zone trophoblasts is consistent with the

proposed role of these transcription factors acting as a heterodimer to alter gene

transcription. In vitro human studies suggest that PPARG promotes trophoblast invasion

(Fournier et al. 2002) and differentiation (Schaiff et al. 2000) and thus, PPARG

expression may influence these key developmental events of the rat placenta. Gene

deletion studies in the mouse have also established a role for PPARG in placental

development. Barak et al. (Barak et al. 1999) demonstrated that Pparg knockout mice

die by mid-gestation due to inadequate development of the placental vasculature. In

particular, fetal vessels appeared not to invade the labyrinth of Pparg-null placentas.

More recently, Asami-Miyagishi et al. (2004) investigated the role of PPARG during

mid-pregnancy by maternal administration of PPARG agonists between days 9 and 11.

This treatment decreased the rate of spontaneous fetal mortality by half, consistent with

PPARG support of placental vascular development. Interestingly, in contrast to the


marked changes in spatial or temporal expression of placental PPARs and RXRA

observed in the present study, Wang et al (Wang et al. 2002) previously reported

consistent expression of all three PPARs between the basal and labyrinth zones and

across the second half of rat pregnancy. In addition, the expression of Rxra mRNA was

noted to be higher in the basal zone when compared to the labyrinth zone, increasing

throughout the placenta from day 13 to day 19 (Wang et al. 2002). These

inconsistencies presumably reflect the far more accurate assessment of mRNA

expression possible by real time, quantitative RT-PCR as used in the present work.

Localisation of PPARG to the labyrinth trophoblast (and its absence from fetal

endothelium) suggests that activation of PPARG may result in trophoblastic secretion of

angiogenic factors to stimulate the growth of fetal blood vessels. Consistent with this

proposal, several studies have suggested that PPARG exerts a positive effect on

angiogenesis by stimulating expression of vascular endothelial growth factor (VEGF)

(Bamba et al. 2000; Jozkowicz et al. 2000; Yamakawa et al. 2000; Yasuda et al. 2005).

Indeed, activation of PPARG by the endogenous PPARG ligand, 15-deoxy-Δ12, 14-

prostaglandin J2 (15∆PGJ2), has demonstrated a role for 15∆PGJ2 in the PPARG-

mediated upregulation of VEGF in a human macrophage cell line (Bamba et al. 2000).

Interestingly, 15∆PGJ2 appears to be synthesised in the rat placenta (Asami-Miyagishi

et al. 2004; Capobianco et al. 2005) and so may serve as the endogenous ligand for

placental PPARG activation. Further studies are required to determine whether

activation of PPARG in trophoblast cells influences VEGF expression and downstream

effects on fetal vasculature.

The current study also shows that maternal dexamethasone treatment, which clearly

restricted both fetal and placental growth, resulted in reduced expression of Pparg

mRNA specifically in the labyrinth zone. These data suggest that dexamethasone-

induced placental growth restriction may be due, at least in part, to a down regulation of

PPARG expression specifically within trophoblast cells of the labyrinth zone. This

reduction in PPARG expression may reduce the expression of secreted growth or

angiogenic factors from labyrinthine trophoblasts inhibiting fetal vascular development.

In a previous report, total placental PPARG expression at day 21 of rat pregnancy was

found to be unaffected by maternal dexamethasone treatment (day 15-21; 100-

200 μg/kg/day) (Langdown and Sugden 2001). In this case, however, placental

expression of PPARG was measured in whole placentas rather than individual zones,


thus highlighting the need for individual examination of the two distinct zones of the

rodent placenta.

PPARD expression is also critical for placental development as shown by gene deletion

studies, with limited survival of homozygous null pups (Barak et al. 2002). However,

unlike Pparg-null placentas, the labyrinth zone of Ppard-null mice displayed normal

vascular development. Our data show that placental Ppard mRNA expression falls

during the last third of rat pregnancy, possibly a reflection of reduced functional

importance for PPARD at this time. Recent work in human trophoblast cells suggests

that PPARD may suppress expression of 11β-hydroxysteroid dehydrogenase-2

(11BHSD2), the enzyme responsible for metabolism of active glucocorticoids within

the placenta (Julan et al. 2005). Such a role appears unlikely in the rat, however, since

we have previously shown that labyrinthine expression of 11BHSD2 falls between days

16 and 22 of pregnancy (Burton et al. 1996; Waddell et al. 1998). Alternatively, the

reduction in PPARD expression near term may facilitate the actions of PPARG, since

all of the PPARs competitively bind RXRA as determined by their relative abundance

and binding affinities. Therefore lower expression of PPARD would be expected to

enhance PPARG interaction with RXRA and thus facilitate its downstream effects on

placental vascularisation.

This study also confirmed that the rat placenta expresses PPARA, and as with PPARG,

this expression increased specifically within the labyrinth zone towards term. Although

the role of placental PPARA remains to be determined, it may impact on fatty acid

transport and metabolism either alone, or in combination with PPARG. Wang et al.

(Wang et al. 2002) suggested the presence of PPARA and PPARG late in pregnancy

may facilitate the necessary increase in transplacental fatty acid transfer stimulated by

increased fetal demand. Consistent with this hypothesis, the reduction in labyrinthine

PPARG expression observed following glucocorticoid-induced placental growth

restriction may reduce transfer of maternal fatty acids to the fetus, and possibly

contribute to the restricted fetal growth. In addition, placental PPARA may be important

in relation to placental steroid production, since previous studies indicate that PPARA

activation may promote steroidogeneisis in human trophoblasts by increasing the pool

of available cholesterol through fatty acid oxidation (Hashimoto et al. 2004). Although

the rodent placenta exhibits only limited steroidogenic capacity relative to other species

(Chan and Leathem 1975), the rat placenta is a significant local source of progesterone

within the intrauterine compartment (Benbow and Waddell 1995).


In conclusion, our data suggest that in addition to promoting early placental

development, PPARG is important for placental growth and development late in

pregnancy, particularly in the rapidly-growing labyrinth zone. Moreover, inhibition of

placental and fetal growth by dexamethasone treatment was associated with reduced

PPARG expression specifically within the labyrinth zone, suggesting that placental

vascular development may be impeded in this model of IUGR. Further ligand binding

studies are required to assess whether PPARs may provide a therapeutic target in the

treatment of IUGR.


Chapter 6 Placental expression of secreted frizzled related

protein-4 in the rat and the impact of glucocorticoid-induced

fetal and placental growth restriction

Reprint of published manuscript, Biol Reprod 75(1): 75-81.

6.1 Abstract

Wnt genes regulate a diverse range of developmental processes, including placental

formation. Activation of the WNT pathway results in translocation of beta-catenin

(CTNNB1) into the nucleus and the subsequent activation of transcription factors that

promote proliferation. The secreted frizzled related proteins (SFRPs) are thought to

inhibit WNT signaling by binding to the WNT ligand or its frizzled receptor. In this

study, we compared the expression patterns of one of these secreted molecules, SFRP4,

in the two morphologically and functionally distinct regions of the rat placenta during

the last third of pregnancy. In addition, we assessed whether placental SFRP4

expression is altered in a model of glucocorticoid-induced placental growth restriction.

Temporal analyses of the rat placenta by quantitative RT-PCR, in situ hybridization, and

immunohistochemistry during the final third of pregnancy demonstrated elevated levels

of Sfrp4 mRNA and SFRP4 protein near term, specifically in trophoblast cells of the

basal zone. This increase in expression of SFRP4 in basal zone trophoblasts was

associated with a reduction in CTNNB1 nuclear translocation, consistent with inhibition

of the WNT pathway. Maternal dexamethasone treatment (1 µg/ml of drinking water,

Days 13–22), which has previously been shown to reduce placental growth, further

increased the expression of Sfrp4 mRNA in both the basal and labyrinth zones of the

placenta at Day 22. Collectively, these data demonstrate that increased expression of

SFRP4 is associated with reduced growth of placental regions in normal pregnancy and

after glucocorticoid-induced growth retardation. These observations, together with

associated changes in CTNNB1 localization, support the hypothesis that increased

placental expression of SFRP4 inhibits the WNT pathway and thereby influences

placental growth via effects on cell fate signaling.


6.2 Introduction

Intrauterine growth restriction (IUGR) is a major health concern because it increases the

rate of neonatal morbidity and mortality (Barker et al. 1989), and the subsequent

incidence of adult onset diseases due to fetal programming (Seckl and Meaney 2004).

The control of fetal growth involves a range of genetic and environmental factors which

are mediated, in part, via the placenta (Fowden 1995; Bauer et al. 1998). Thus, while a

strong relationship between placental function and fetal well-being is well recognised

(Godfrey 2002; Pardi et al. 2002; Regnault et al. 2002; Kaufmann et al. 2003), the

complex regulation of trophoblast proliferation and apoptosis remains poorly

understood. Gene deletion studies have established a critical role for WNT signaling in

placental development (Monkley et al. 1996; Galceran et al. 1999; Ishikawa et al. 2001;

Parr et al. 2001) and thus, modulation of WNT action may impact on proliferation and

apoptosis of placental trophoblast and consequently fetal growth.

WNT signaling is initiated by binding of WNT ligands to a transmembrane frizzled

(FZD)/lipoprotein receptor-related protein complex resulting in the activation of the

cytoplasmic protein disheveled (DVL). DVL inactivates glycogen synthase kinase-3β

(GSK3B) that results in the accumulation and subsequent nuclear translocation of β-

catenin (CTNNB1). In the presence of appropriate co-activators, CTNNB1 binds the T-

cell factor/lymphoid enhancer-binding factor (TCF/LEF) family of DNA-binding

proteins and upregulates transcription of various target genes that drive proliferation and

cell fate determination. In the absence of WNT signaling, the activated GSK3B forms

an intracellular complex with adenomatous polyposis coli and axin, targeting CTNNB1

for degradation via the ubiquitin-proteosome pathway removing the trophic stimulus

(Nusse and Varmus 1992; Cadigan 2002; Nelson and Nusse 2004).

WNT signaling has been previously demonstrated in human cytotrophoblasts (Eberhart

and Argani 2001), and recent reports demonstrate a critical role for the WNT pathway

in placental development. Specifically, Wnt7b and the Tcf/Lef1 genes are required for

normal chorioallantoic fusion (Galceran et al. 1999; Parr et al. 2001) and Wnt2 null

mice display impaired placental angiogenesis, reduced birth weight, and a higher rate of

mortality (Monkley et al. 1996). Moreover, Fzd5 knockout mice also display defects in

placental vascularisation implicating a role for WNT5A and WNT10B, the

physiological ligands of FZD5, in placental development (Ishikawa et al. 2001). A role

for CTNNB1, the key mediator of WNT signaling, has also been implicated in


trophoblast adhesion (Nakano et al. 2005), survival (Dash et al. 2005) and

differentiation (Getsios et al. 2000).

Secreted frizzled related proteins (SFRPs) are structurally similar to the frizzled

receptors but lack the transmembrane and intracellular components necessary for signal

transduction (Rattner et al. 1997). It is proposed that SFRPs inhibit the WNT pathway

by binding to either the WNT ligand or frizzled receptor (Guo et al. 1998; Bafico et al.

1999), potentially inhibiting the entire canonical pathway (Suzuki et al. 2004).

Inhibition of the WNT pathway results in continual degradation of CTNNB1 and the

absence of TCF/LEF-mediated gene expression. Previous data have established a role

for one of these secreted proteins, SFRP4, in involution of reproductive tissues, possibly

due to a pro-apoptotic action (Wolf et al. 1997; Marti et al. 1999; Lacher et al. 2003),

and in apoptosis of thecal cells during ovulation (Drake et al. 2003; Hsieh et al. 2003).

Furthermore, levels of SFRP4 expression inversely correlate with tumor progression in

human endometrial stromal and undifferentiated endometrial sarcomas (Hrzenjak et al.

2004). Thus, placental expression of SFRP4 is likely to impact on placental growth via

effects on the balance between cellular proliferation and apoptosis.

The present study, therefore, investigated the placental expression and distribution of

SFRP4 and CTNNB1 over the final third of pregnancy, the period of maximal fetal and

placental growth. Separate analyses were made of the two functionally- and

morphologically-distinct zones of the rat placenta, the basal and labyrinth zones, since

the latter grows extensively between days 16 and 22 (Waddell et al. 1998; Hewitt et al.

2006b), whereas the basal zone does not grow and undergoes a notable increase in

apoptosis during the same period (Waddell et al. 2000). These analyses showed marked

upregulation of Sfrp4 mRNA in the basal zone near term, but not in the rapidly-growing

labyrinth zone. Therefore, because glucocorticoids can induce placental growth

restriction and trophoblast apoptosis (Waddell et al. 2000; Sugden and Langdown 2001;

Ain et al. 2005; Hewitt et al. 2006b) and are known to inhibit WNT signaling in other

cell types (Ohnaka et al. 2005), we also tested the hypothesis that maternal

dexamethasone treatment stimulates placental SFRP4.


6.3 Materials and Methods

6.3.1 Animals



conditions as described previously (Waddell et al. 1998). Rats were mated overnight

and the day in which spermatozoa were present in a vaginal smear was designated day 1

of pregnancy. All procedures involving animals were conducted after approval by the

Animal Ethics Committee of The University of Western Australia.

6.3.2 Glucocorticoid treatment

Increased fetal and placental glucocorticoid exposure was achieved by administration of

dexamethasone acetate (1 μg/ml; Sigma, St Louis, MO) in the drinking water from day

13 to 22 of pregnancy (term = 23 days). We have previously shown that this treatment

reduces fetal and placental weights at day 22 by 25-30% (Waddell et al. 2000; Hewitt et

al. 2006b).


For collection of placental zones, pregnant rats were anaesthetized with

halothane/nitrous oxide at either day 16 or 22 of gestation and 3 fetuses and placentas

were removed from each mother. Placental zones were separated by blunt dissection

and snap frozen in liquid nitrogen for real-time quantitative RT-PCR or alternatively

whole placentas were fixed in Histochoice MB (Amresco, Solon, OH) for

immunohistochemistry.


Total RNA was isolated from placental zones at days 16 and 22 of pregnancy using Tri-

Reagent (Molecular Resources Centre, Cincinnati, OH) as per the manufacturer’s

instructions. RNA integrity was assessed by ethidium bromide staining of the nucleic

acids prior to agarose gel electrophoresis (data not shown). Total RNA was treated to

remove contaminating genomic DNA using DNA-free reagent (Ambion, Austin, TX).

DNase-treated total RNA (1 μg) was used to synthesize cDNA using M-MLV Reverse

Transcriptase RNase H Point Mutant and random hexamer primers (Promega, Madison,


WI) as per the manufacturer’s instructions. The resultant cDNAs were purified using the

Ultraclean PCR Cleanup kit (MoBio Industries, Solana Beach, CA).


Analyses of expression levels for Sfrp4, Ctnnb1 and Rpl19 transcripts were performed

by real time RT-PCR on the Rotorgene 3000 (Corbett Industries, Sydney, Australia)

using iQ SYBR Green Supermix (BioRad, Hercules, CA). Primers for Sfrp4 and Ctnnb1

(Table 6.1) were designed using Primer 3 software (MIT/Whitehead Institute;

http://www-genome.wi.mit.edu) (Rozen and Skaletsky 2000). Each of the selected

primer pairs were positioned to span introns to ensure no product was amplified from

genomic DNA, and the resulting amplicons were sequenced to confirm specificity (data

not shown). All samples were standardized against Rpl19 as previously described (Orly

et al. 1994). Standard curves for each product were generated from gel extracted

(QIAEX II, Qiagen, Melbourne, Australia) PCR products using 10-fold serial dilutions

and the Rotorgene 3000 software.


Sections of placenta were cut at 4 μm, deparaffinised and rehydrated, then incubated for

10 min in 3% H2O2 to block endogenous peroxidases. Mouse monoclonal primary

antibody to CTNNB1 (C7082, Sigma, MO) and rabbit polyclonal primary antibody to

SFRP4 (1:50; a gift from Dr Robert Friis, University of Bern, Switzerland) were each

applied overnight at 4°C followed by a 30 min room temperature incubation in

biotinylated anti-mouse or anti-rabbit secondary at 1:200 (BA2001 and BA1000; Vector

Laboratories, Burlingame, CA) and streptavidin-peroxidase complex at 1:50 (Vectastain

ABC Kit, Vector Laboratories). Positive staining was visualized by application of

diaminobenzidine substrate (Sigma) and counterstained with haematoxylin before

dehydration in graded alcohols and mounting in DPX.

6.3.7 In situ hybridization

In situ hybridization with a digoxigenin-labeled riboprobe to localize the expression of

Sfrp4 mRNA was performed as described previously by our laboratory (Guo et al. 1998;

Drake et al. 2003; Lacher et al. 2003). Briefly, 5 μm sections were dewaxed and

rehydrated in PBS before pre-treatment with proteinase K (10 mg/ml) for 15 min at


37°C in a humidifying chamber. Slides were acetylated with fresh acetic anhydride

followed by incubation in pre-hybridization solution for 2 h at 55°C and hybridization

in 200 ng/ml of labeled sense or antisense probe (16 h at 55°C). The following day the

slides were washed extensively, then blocked (0.5% w/v blocking agent in TBS) before

addition of anti-digoxigenin antibody (1:1000) for 1 h at room temperature. Sfrp4

mRNA was visualized by addition of NBT/BCIP solution (20 μl/ml) and incubation in

darkness until sufficient contrast was obtained, halting the color reaction with ddH20.


All data are expressed as means ± SEM. Two-way analysis of variance (ANOVA;

GenStat7, Hemel Hempstead, UK) was used to assess variation in expression levels of

Sfrp4 and Ctnnb1 mRNA (standardized to Rpl19 expression) for gestational age and

placental zone. Where the F test for the ANOVA reached statistical significance (P <

0.05), differences were assessed by least significant difference (LSD) test (Snedecor and

Cochran 1989).


Table 6.1. Primers and conditions used for Sfrp4, Ctnnb1 and L19 real time RT-PCR.

Gene Forward/Reverse Primer Annealing

Temp.

Amplicon

Size

CTT CCC ACT GTG TGG ACC TT

Sfrp4 CTA TCC CTC GAA CGC AAG TC

62°C 180 bp

TGA AGG TGC TGT CTG TCT GC

Ctnnb1 GCT GCA CTA GAG TCC CAA GG

62°C 192 bp

CTG AAG GTC AAA GGG AAT GTG L19



6.3.9 In situ hybridization

In situ hybridization with a digoxigenin-labeled riboprobe to localize the expression of

Sfrp4 mRNA was performed as described previously by our laboratory (Guo et al. 1998;

Drake et al. 2003; Lacher et al. 2003). Briefly, 5 μm sections were dewaxed and

rehydrated in PBS before pre-treatment with proteinase K (10 mg/ml) for 15 min at

37°C in a humidifying chamber. Slides were acetylated with fresh acetic anhydride

followed by incubation in pre-hybridization solution for 2 h at 55°C and hybridization

in 200 ng/ml of labeled sense or antisense probe (16 h at 55°C). The following day the

slides were washed extensively, then blocked (0.5% w/v blocking agent in TBS) before

addition of anti-digoxigenin antibody (1:1000) for 1 h at room temperature. Sfrp4

mRNA was visualized by addition of NBT/BCIP solution (20 μl/ml) and incubation in

darkness until sufficient contrast was obtained, halting the color reaction with ddH20.


All data are expressed as means ± SEM. Two-way analysis of variance (ANOVA;

GenStat7, Hemel Hempstead, UK) was used to assess variation in expression levels of

Sfrp4 and Ctnnb1 mRNA (standardized to Rpl19 expression) for gestational age and

placental zone. Where the F test for the ANOVA reached statistical significance (P <

0.05), differences were assessed by least significant difference (LSD) test (Snedecor and

Cochran 1989).


6.4 Results

6.4.1 Expression of SFRP4 in the rat placenta during normal pregnancy

The mRNA for Sfrp4 was readily detectable by real time RT-PCR in both zones of the

placenta (Fig. 6.1A). Expression of Sfrp4 mRNA increased in the basal zone over the

final third of pregnancy (14-fold from day 16 to 22; P < 0.01) and was markedly higher

in the basal compared to the labyrinth zone at day 22 (26-fold; P < 0.001). Localization

of Sfrp4 mRNA by in situ hybridization (Fig. 6.2) reflected the zonal changes in

expression observed by RT-PCR and showed that trophoblast cells of the basal zone

were the major source of Sfrp4 mRNA expression at day 22. Weak expression was also

observed in the labyrinth zone trophoblast cells near term but not at day 16.

Immunohistochemical analysis detected the presence of the SFRP4 protein in the basal

zone trophoblast with minimal expression in the labyrinth zone (Fig. 6.3A and B). The

SFRP4 protein at day 16 was demonstrated to be weakly expressed throughout all cell

types of the placenta, consistent with mRNA distribution obtained by real time RT-PCR

analysis, and its intracellular distribution was primarily cytoplasmic (Fig. 6.3B). By day

22, however, expression of the SFRP4 protein was exclusively limited to the cell

membrane or extracellular space surrounding basal zone trophoblasts (Fig. 6.3C and D).

6.4.2 Expression of CTNNB1 in the rat placenta during normal pregnancy

The expression and distribution of CTNNB1 was used as an endpoint of WNT

signaling, and thus the functional capacity of the SFRP4 protein. Real time RT-PCR

analysis demonstrated the expression of Ctnnb1 mRNA did not change in either zone

between day 16 and 22 (Fig. 6.1B). There was however, 3-fold higher expression of

Ctnnb1 mRNA in the labyrinth as compared to the basal zone of day 22 placentas (P <

0.001).

Expression of the CTNNB1 protein at day 16 was localized primarily to the nuclei of

basal zone trophoblast cells, with relatively weak cytoplasmic expression (Fig. 6.4A and

B). At day 22, while most cells still exhibited nuclear expression of CTNNB1, there

were now substantial numbers that were entirely CTNNB1 negative (Fig 6.4C and D).

Notably, intense nuclear, cytoplasmic and membranous staining of CTNNB1 was also

observed the intra-placental yolk sac of the labyrinth zone near term (Fig. 6.5).


Sfrp4 Ctnnb1

0

1

2

3

4

Basal Labyrinth

Rel

ativ

e Ex

pres

sion

0

1

2

3

Basal Labyrinth

Rel

ativ

e Ex

pres

sion

*

16 22 16 22 16 22 16 22

**A B

Figure 6.1. Zonal expression of (A) Sfrp4 and (B) Ctnnb1 mRNA at days 16 and 22 of rat

pregnancy. Values are the mean ± SEM (n = 6-11 per group). * P < 0.01, compared with corresponding

day 16 value, ** P < 0.001, compared to corresponding basal zone value (two way ANOVA, unpaired t-

test).


Figure 6.2. Zonal distribution of Sfrp4 mRNA at days 16 and 22 of rat pregnancy by in situ

hybridization. (A) Absence of Sfrp4 mRNA expression at day 16; (B) cytoplasmic staining of basal zone

trophoblast cells (arrowhead) at day 22. LZ: labyrinth zone; BZ: basal zone; scale bar = 10 μm.


Figure 6.3. Zonal distribution of SFRP4 protein at days 16 and 22 of rat pregnancy by

immunohistochemistry. (A) and (B) Ubiquitous cytoplasmic expression of SFRP4 (brown stain) was

evident at day 16; (C) and (D) membranous expression of SFRP4 in basal zone trophoblast cells at day 22

(arrowheads). LZ: labyrinth zone; BZ: basal zone; scale bar = 10 μm.


Figure 6.4. Subcellular distribution of CTNNB1 protein at days 16 and 22 of rat pregnancy by

immunohistochemistry. (A) and (B) Intense nuclear localization of CTNNB1 protein to trophoblast cells

of the basal zone (arrowhead) at day 16; (C) and (D) absence of CTNNB1 nuclear localization in many

cells at day 22 as demonstrated by haematoxylin counterstain (blue). Scale bar = 10 μm.


Figure 6.5. Labyrinthine distribution of CTNNB1 at day 22 of rat pregnancy by

immunohistochemistry. Intense nuclear, cytoplasmic and membranous distribution of the CTNNB1

protein in the intraplacental yolk sac of the labyrinth zone. Scale bar = 10 μm.


6.4.3 Expression of SFRP4 and CTNNB1 after maternal dexamethasone

treatment

Maternal dexamethasone treatment resulted in increased expression of Sfrp4 mRNA at

day 22 (1.2-fold and 2.8-fold higher in the basal and labyrinth zones, respectively, P <

0.05; Fig. 6.6A). The expression of Sfrp4 was 15-fold higher (P < 0.001) in the basal

zone as compared to the labyrinth zone of dexamethasone treated mothers, consistent

with the pattern of expression observed in control placentas.

In situ hybridization for sfrp4 and immunohistochemistry for SFRP4 demonstrated

similar patterns of placental distribution to those observed in the untreated controls.

Sfrp4 mRNA was primarily localized to the basal zone trophoblast cells (Fig. 6.7),

whereas SFRP4 protein was localized to the cell membrane and the extracellular space

around basal zone trophoblasts (Fig. 6.8A and B).

Real time RT-PCR analysis of Ctnnb1 mRNA revealed reduced expression in the

labyrinth zone of placentas from dexamethasone-treated mothers (42%, P < 0.01; Fig.

6.6B). No change was observed in Ctnnb1 mRNA expression in the basal zone of

dexamethasone-treated placentas. Intracellular distribution of the CTNNB1 protein was

unchanged by dexamethasone treatment as determined by immunohistochemistry (Fig.

6.8C and D).


Sfrp4 Ctnnb1

0123

456

Basal Labyrinth

Rel

ativ

e Ex

pres

sion

0

1

2

3

4

Basal Labyrinth

Rel

ativ

e Ex

pres

sion

* *

*Con Dex Con Dex

A B

Con Dex Con Dex

Figure 6.6. Zonal expression of (A) Sfrp4 and (B) Ctnnb1 mRNA measured by real time RT-PCR at

day 22 of rat pregnancy following treatment with dexamethasone. Values are the mean ± SEM (n = 6-

11 per group). * P < 0.05, compared with corresponding control value (two way ANOVA, unpaired t-

test).


Figure 6.7. Zonal distribution of Sfrp4 mRNA by in situ hybridization following maternal

treatment with dexamethasone. (A) Localization of Sfrp4 mRNA to trophoblast cells (arrowhead) of the

basal zone at day 22; (B) high expression of Sfrp4 mRNA in the basal zone from dexamethasone-treated

mothers. LZ: labyrinth zone; BZ: basal zone; scale bar = 10 μm.


Figure 6.8. Zonal distribution of SFRP4 protein and subcellular localization of the CTNNB1

protein at day 22 after treatment with dexamethasone. (A) and (B) Membranous expression of the

SFRP4 protein in the extracellular space and at the membrane of basal zone trophoblast cells (arrowhead).

(C) and (D) Absence of CTNNB1 nuclear localization in many cells at day 22 as demonstrated by

haematoxylin counterstain (blue). LZ: labyrinth zone; BZ: basal zone; scale bar = 10 μm.


6.5 Discussion

This study investigated the expression of SFRP4 and CTNNB1, both key regulators of

cellular proliferation and apoptosis, in the basal and labyrinth zones of the rat placenta

during the period of maximal fetal and placental growth. Sfrp4 mRNA expression

increased markedly in basal zone trophoblasts near term but remained minimal in the

rapidly-growing labyrinth zone. The elevation in basal zone expression of Sfrp4 mRNA

was associated with increased localization of the SFRP4 protein to the extracellular

space and plasma membrane of these cells. During the same period, the expression of

Ctnnb1 mRNA remained unchanged but nuclear localization of the CTNNB1 protein

was reduced, consistent with the absence of basal zone growth. Maternal

dexamethasone treatment increased expression of SFRP4 in both the basal and labyrinth

zones, an effect that may contribute to the reduction in placental growth (Hewitt et al.

2006b) and the increase in apoptosis previously observed in this model (Waddell et al.

2000). Collectively, these data are consistent with inhibition of the WNT pathway by

SFRP4, with downstream effects on cell fate signaling that influence placental

proliferation and apoptosis.

The control of placental growth involves a delicate balance of proliferative and

apoptotic signaling. In the current study we demonstrate that SFRP4 expression

increases markedly in the non-proliferative basal zone near term but remains very low

in the rapidly-growing labyrinth zone. Moreover, SFRP4 expression was also stimulated

by dexamethasone in both the basal and labyrinth zones, both of which exhibit reduced

growth in this model (Hewitt et al. 2006b). These observations are consistent with the

anti-proliferative action of SFRP4 in mesothelioma (Lee et al. 2004), uterine (Hrzenjak

et al. 2004) and colorectal (Suzuki et al. 2004) cancer, and support the concept that

glucocorticoid-mediated inhibition of the WNT pathway (Ohnaka et al. 2005) may

involve, in part, increased SFRP4 expression. Previous studies have also linked

expression of SFRP4 to increased apoptosis in a range of reproductive tissues (Wolf et

al. 1997; Marti et al. 1999; Schumann et al. 2000; Drake et al. 2003; Lacher et al. 2003),

and so the rise in SFRP4 specifically within the basal zone between days 16 and 22 of

pregnancy is in accordance with our previous observation that only the basal zone

shows increased apoptosis over this period (Waddell et al. 2000). Interestingly, Fujita et

al. (Fujita et al. 2002) have demonstrated increased expression of Sfrp4 mRNA

corresponding with decidual cell proliferation between days 9 and 15 of rat pregnancy.


This suggests that SFRP4 may influence proliferation and apoptosis in a tissue-specific

manner, but further studies are required to assess this possibility.

The function of the frizzled related proteins is thought to involve inhibition of the

canonical WNT pathway via competitive binding to the WNT ligand or the

transmembrane frizzled receptor (Bafico et al. 1999). The membranous and extracellular

distribution of the SFRP4 protein in the basal zone of the rat placenta observed in this

study is consistent with its binding to either the frizzled receptor or the WNT ligand. A

similar distribution was recently noted for SFRP4 in prostate cancer in association with

reduced rates of proliferation (Horvath et al. 2004), and a loss of membranous SFRP4

expression also occurs in the transition from normal esophageal epithelium to

esophageal adenocarcinoma (Zou et al. 2005). In contrast, the SFRP4 protein has also

been localized to the cytoplasm of a colorectal cancer cell line (Feng Han et al. 2006) in

association with increased proliferation. Collectively, these data indicate that the

subcellular localization of SFRP4 may determine its major function, and in the case of

the basal zone trophoblast the membranous localization of SFRP4 suggests that it

inhibits the WNT pathway.

The effects of WNT pathway inhibition by frizzled related proteins, including SFRP4,

are likely to be mediated via effects on CTNNB1 localization (Guo et al. 1998; Bafico

et al. 1999). Thus, the canonical WNT pathway promotes proliferation via the nuclear

accumulation of CTNNB1 and subsequent activation of TCF/LEF family of

transcription factors (Nusse and Varmus 1992; Cadigan 2002), and has been implicated

specifically in trophoblast differentiation (Getsios et al. 2000; Nakano et al. 2005) and

survival (Dash et al. 2005). The present study shows that increased expression of

SFRP4 in basal zone trophoblasts is accompanied by reduced CTNNB1 nuclear

localization, consistent with inhibition of the WNT pathway by SFRP4. The association

between the expression of SFRP4, CTNNB1 and the relative amounts of proliferation

and apoptosis, however, is still unclear. Feng Han et al. (Feng Han et al. 2006) found

increased expression of both SFRP4 and CTNNB1 in human colorectal carcinoma

suggesting that SFRP4 may not inhibit the nuclear accumulation of CTNNB1.

Similarly, recent reports on the expression of SFRP1, 2, 4 and 5 indicate that of the

isoforms examined, only SFRP4 did not reduce CTNNB1 nuclear expression or induce

apoptosis in a colorectal cancer cell line (Suzuki et al. 2004). In the same model,

however, SFRP4 reduced colony formation (Suzuki et al. 2004), and so the SFRP4-

associated reduction in CTNNB1 observed in the basal zone trophoblasts may relate


primarily to an inhibition of proliferation as well as induction of apoptosis. Further in

vitro studies are required to confirm a causal link between SFRP4 action and inhibition

of WNT-mediated trophoblast proliferation.

Normal placental development requires rapid growth and vascularization of maternal

and fetal blood spaces, whereas in IUGR this process is compromised. Zhang et al

(Zhang et al. 2001) proposed that the WNT pathway may play a role in promoting

angiogenesis, specifically by upregulation of vascular endothelial growth factor (VEGF)

expression in colonic epithelial cells. Therefore, the upregulation of SFRP4 following

dexamethasone treatment may result in inhibition of the WNT pathway and a

subsequent reduction in VEGF expression in the labyrinth zone. Indeed, in recent

preliminary studies we observed that dexamethasone markedly reduces the expression

of Vegf mRNA in the placental labyrinth zone (Hewitt, Mark and Waddell, unpublished

data). Moreover, gene deletion studies involving various components of the WNT

pathway (Monkley et al. 1996; Ishikawa et al. 2001; Parr et al. 2001) all show defective

placentation, particularly in relation to angiogenesis, suggesting that vascular

development of the placenta is reliant on the WNT signaling pathway.

The distribution of CTNNB1 within the placental labyrinth was predominantly within

the nucleus and cytoplasm of trophoblast cells lining the intra-placental yolk sac, an

extension of the yolk sac into the labyrinth zone (Pijnenborg and Vercruysse 2006),

known to play a role in maternal-fetal calcium transfer (Kovacs et al. 2002; Brasier et

al. 2004). While CTNNB1 may influence cell fate signaling in these cells via the WNT

pathway as discussed above, its membranous localization suggests that it may also play

a role in cell-cell adhesion and migration as previously demonstrated in various cell

types (Nelson and Nusse 2004). Whatever the function of CTNNB1 in the labyrinth

zone, it is likely to have been compromised by dexamethasone treatment, since the latter

reduced the expression of Ctnnb1 mRNA specifically in the labyrinth zone at day 22.

In conclusion, this study shows that expression of SFRP4 increases markedly near term

in the non-proliferative placental basal zone, but not in the rapidly growing labyrinth

zone. Associated changes in CTNNB1 localization suggest that SFRP4 inhibits the

WNT pathway in trophoblast cells. Dexamethasone treatment further increased

placental SFRP4 expression at term, consistent with the hypothesis that SFRP4 plays an

anti-proliferative and/or pro-apoptotic role in glucocorticoid-induced placental growth

restriction.


Chapter 7 Glucocorticoids prevent the normal increase in

placental vascular endothelial growth factor expression and

placental vascularity during late pregnancy in the rat

Reprint of published manuscript, Endocrinology 147(12): 5568-74.

7.1 Abstract

Increased glucocorticoid exposure reduces fetal growth and predisposes to an increased

risk of disease in later life. In addition to direct effects on fetal growth, glucocorticoids

also compromise fetal growth indirectly via detrimental effects on placental growth and

function. The current study investigated the effects of dexamethasone-induced

intrauterine growth restriction on placental vascular development and expression of the

endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF). Separate

analyses were conducted for the three main VEGF isoforms (VEGF120, VEGF164, and

VEGF188) in the two functionally and morphologically distinct regions of the rat

placenta, the basal and labyrinth zones. Quantitative PCR and immunohistochemical

analysis demonstrated that expression of VEGF was markedly up-regulated specifically

in the rapidly growing labyrinth zone over the final third of normal pregnancy.

Unbiased stereological analyses showed an associated increase in the volume and

surface area of maternal and fetal blood spaces, including vascular remodeling of the

fetal capillary network near term. In contrast, dexamethasone-induced fetal and

placental growth restriction reduced expression of the Vegf120 and Vegf188 isoforms and

prevented normal labyrinthine vascular development near term. Most notably,

dexamethasone impaired the normal increase in fetal vessel density over the final third

of pregnancy, with no effect on the density of maternal blood spaces. Overall, this study

quantifies the labyrinth zone-specific increases in placental VEGF expression and

vascular development during normal pregnancy, and shows that these increases are

prevented by maternal dexamethasone treatment. Our data suggest that glucocorticoid-

induced restriction of fetal and placental growth is mediated, in part, via inhibition of

placental VEGF expression and an associated reduction in placental vascularization.


7.2 Introduction

Glucocorticoids are crucial for maturation of the fetus late in pregnancy, and clinical

glucocorticoid administration is used to accelerate fetal lung development in mothers at

risk of preterm delivery (Ward 1994; Sloboda et al. 2005). Increased fetal

glucocorticoid exposure, however, reduces birth weight, and is associated with an

increased risk of cardiovascular and metabolic disease in later life (Seckl 2004; Seckl

and Meaney 2004). Placental development is a critical determinant of fetal growth and it

appears that the effects of glucocorticoids on fetal growth may be mediated in part, via

effects on placental growth and function. Specifically, rodent models suggest that

placental growth is more sensitive to altered glucocorticoid exposure than fetal growth,

the former being compromised by increased levels of trophoblast apoptosis (Waddell et

al. 2000). Moreover, maternal glucocorticoid treatment appears to reduce the

transplacental passage of glucose (Langdown and Sugden 2001) and leptin (Smith and

Waddell 2003) without a reduction in the expression of associated placental transporter

proteins. This suggests that increased glucocorticoid exposure is likely to impact on the

vascular exchange network of the placenta. Indeed, glucocorticoids downregulate

expression of the endothelial cell-specific mitogen, vascular endothelial growth factor

(VEGF) in various tissues (Heiss et al. 1996; Koedam et al. 2002; Smink et al. 2003;

Alagappan et al. 2005; Alonso et al. 2005), but the impact of glucocorticoids on

placental VEGF expression remains unknown.

VEGF (also referred to as VEGF-A) is a potent angiogenic factor belonging to a gene

family that includes VEGF-B, -C, -D, and placental growth factor (Ferrara 2004).

VEGF promotes angiogenensis through induction of endothelial cell proliferation and

increased vascular permeability (Ferrara 2004), and gene deletion studies show that

VEGF is essential for normal embryonic development. Indeed, even heterozygotes show

an embryonic lethal phenotype associated with a failure of placental vascular

development (Carmeliet et al. 1996; Ferrara 2004). In normal pregnancy placental

VEGF expression increases near term in the rodent (Ni et al. 1997), baboon

(Hildebrandt et al. 2001; Albrecht et al. 2004; Robb et al. 2004) and sheep (Reynolds et

al. 2005) in association with increased placental vascularization (Hildebrandt et al.

2001; Lewis et al. 2001; Doherty et al. 2003; Albrecht et al. 2004; Coan et al. 2004;

Robb et al. 2004). In contrast, reduced placental expression of VEGF has been observed

in cases of intrauterine growth restriction (Lyall et al. 1997) and pharmacological


inhibition of VEGF is associated with impaired placental vascularity (Rutland et al.

2005).

The rodent Vegf gene is encoded from eight exons with alternate splicing of the sixth

and seventh exons resulting in the generation of five alternate splice variants (VEGF120,

VEGF144, VEGF164, VEGF188, VEGF205) with 120, 144, 164, 188 and 205 amino acids

respectively. VEGF120 is a freely secreted isoform that does not bind heparin and is thus

a highly potent endothelial cell mitogen. The VEGF188 and VEGF205 isoforms display

reduced bioactivity due to their sequestration within the extracellular matrix. VEGF164

has intermediate activity between the small and large isoforms because it is secreted and

is therefore functionally active, but significant quantities are retained bound to the cell

surface and extracellular matrix (Ferrara 2004). Four of the VEGF isoforms (120, 164,

188 and 205) have been identified in the rat placenta with the 120 and 164 isoforms

increasing near term (Ni et al. 1997). Homologues of rodent VEGF144 have also been

identified in human (Anthony et al. 1994) and sheep (Matsumoto et al. 2002) placenta.

In the current study we investigated the impact of increased glucocorticoid exposure on

the expression of the major VEGF isoforms (VEGF120, VEGF164 and VEGF188) and

vascularization of the rat placenta over the final third of pregnancy, the period of

maximal fetal and placental growth. Increased glucocorticoid exposure was achieved

via maternal administration of dexamethasone in a model known to restrict fetal and

placental growth (Waddell et al. 1998; Hewitt et al. 2006b). Separate analyses were

conducted for the two functionally and morphologically distinct regions of the placenta,

the basal and labyrinth zones, because only the latter is involved in fetal-maternal

transport and undergoes dramatic growth and vascular development over the final third

of pregnancy.


7.3 Methods

7.3.1 Animals

Nulliparous albino Wistar rats aged between 8 and 12 weeks were obtained from the


conditions as described previously (Benbow and Waddell 1995). Rats were mated

overnight and the day on which spermatozoa were present in a vaginal smear was

designated day 1 of pregnancy. All procedures involving animals were conducted only

after approval by the Animal Ethics Committee of The University of Western Australia.

7.3.2 Glucocorticoid manipulations

Increased fetal and placental glucocorticoid exposure was achieved by maternal

administration of dexamethasone acetate (1 μg/ml maternal drinking water; Sigma, St

Louis, MO) from day 13 to 22 of pregnancy (term = 23 days). We have previously

shown that this treatment reduces fetal weight by 25-30% at day 22 (Waddell et al.

2000; Hewitt et al. 2006b).


Rats were anaesthetized with halothane/nitrous oxide at either day 16 or 22 of gestation

and 3 fetuses and placentas were removed from each mother. Placental zones were

separated by blunt dissection and snap frozen in liquid nitrogen for real-time

quantitative RT-PCR or alternatively whole placentas were fixed in Histochoice MB

(Amresco, Solon, OH) and processed for routine paraffin histology for subsequent

immunohistochemical or stereological analyses.

7.3.4 RNA isolation

Total RNA was isolated from placental samples using Tri-Reagent (Molecular

Resources Centre, Cincinnati, OH) as per the manufacturer’s instructions. RNA

integrity was assessed by ethidium bromide staining of the nucleic acids following

agarose gel electrophoresis (data not shown). Total RNA was treated to remove

contaminating genomic DNA using DNA-free reagent (Ambion, Austin, TX). DNase-

treated total RNA (1 μg) was used to synthesize cDNA using Moloney Murine

Leukemia Virus Reverse Transcriptase RNase H Point Mutant and random hexamer


primers (Promega, Madison, WI) as per the manufacturer’s instructions. The resultant

cDNAs were purified using the Ultraclean PCR Cleanup kit (MoBio Industries, Solana

Beach, CA).


Analyses of expression levels for Vegf transcripts were performed by real time RT-PCR

on the Rotorgene 3000 (Corbett Industries, Sydney, Australia) using iQ SYBR Green

Supermix (BioRad, Hercules, CA) and the amplification conditions outlined in Table

7.1. Primers for Vegf isoform amplification were designed using Primer 3 software

(MIT/Whitehead Institute; http://www-genome.wi.mit.edu) (Rozen and Skaletsky 2000)

and the reverse primers for VEGF120, VEGF164 and VEGF188 were positioned at distinct

exon boundaries for each isoform. Each of the selected primer pairs were positioned to

span introns to ensure no product was amplified from genomic DNA, and the resulting

amplicons were sequenced to confirm specificity (data not shown). All samples were

standardized against Rpl19 as previously described (Orly et al. 1994). Standard curves

for each assay were generated from gel extracted (QIAEX II, Qiagen, Melbourne,

Australia) PCR products using 10-fold serial dilutions and the Rotorgene 3000 software.


Sections were cut at 4 μm, deparaffinized and rehydrated, then incubated for 10 min in

3% H2O2 to block endogenous peroxidases. Mouse monoclonal primary antibody to

CD31 (BD Biosciences, NSW, Australia) and VEGF (Santa Cruz Biotechnology, Santa

Cruz, CA) were each used at a dilution of 1:50 overnight at 4 C followed by a 30 min

room temperature incubation in biotinylated anti-mouse secondary at 1:200 (Vector

Laboratories, Burlingame, CA, USA) and streptavidin-peroxidase complex at 1:50

(Vectastain ABC Kit, Vector Laboratories). Sections were visualized by application of

diaminobenzidine substrate (Sigma) and counterstained with hematoxylin before

dehydration in graded alcohols and mounting in DPX.


Table 7.1. Primers and conditions used for Vegf isoforms and Rpl19 real time RT-PCR.

Gene Forward/Reverse Primer Annealing Temp.

Amplicon Size

ATG AAG TGG TGA AGT TCA TGG A Vegf120

TTG TCA CAT TTT TCT GGC TTT G 59°C 319 bp

ATG AAG TGG TGA AGT TCA TGG A Vegf164

AGG CTC ACA GTG ATT TTC TGG 59°C 323 bp

AGG AAA GGG AAA GGG TCA AA Vegf188

AAA TGC TTT CTC CGC TCT GA 60°C 96 bp

CTG AAG GTC AAA GGG AAT GTG Rpl19



7.3.7 Stereological measures

Detailed analysis of the labyrinth zone vasculature was achieved by random sampling of

12 fields of view (objective lens = 40x magnification) of placentas collected from

untreated mothers at either day 16 or 22, or from dexamethasone-treated mothers at day

22. Relative volumes of labyrinth zone components were calculated by a 35 point grid

overlaid on each field of view, counting points that fell in each placental structure

according to the Cavalieri principle (Gundersen and Osterby 1981). Surface area

measurements were estimated by using the same fields obtained for volume

measurements and overlaying a grid of cycloid arcs as previously reported in Coan et al.

(Coan et al. 2004). Points of intersection of cycloid arcs with the lumen of maternal

blood spaces and fetal capillaries were used to calculate surface area densities in

vertically oriented sections (Coan et al. 2004), with CD31 immunostaining used to aid

in the distinction between maternal and fetal blood spaces. Combined length density,

mean diameter and overall density of fetal capillaries were all estimated by counting the

number of capillary profiles in a counting square containing two contiguous forbidden

lines (Coan et al. 2004). Volume, surface area and length densities were adjusted for

shrinkage of the tissue by measurement of average maternal erythrocyte diameter before

and after tissue processing (Burton and Palmer 1988; Ali et al. 1996). Measures of

density were converted to absolute measures by multiplying by the total volume of the

labyrinth zone assuming a specific gravity of 1g/cm3.


All group values were expressed as means ± SEM, standardized by Rpl19 in the case of

real time RT-PCR data. Two-way ANOVAs (GenStat7, Hemel Hempstead, UK) were

used to assess variation in expression levels of Vegf mRNA, volume and surface area

estimates for gestational age and placental zone. Where the F test for the ANOVA

reached statistical significance (P < 0.05), differences were assessed by least significant

difference (LSD) test. Changes in mean capillary diameter and length of fetal capillaries

over gestation and following dexamethasone treatment were assessed by unpaired t-

tests.


7.4 Results

7.4.1 Gestational changes in VEGF expression

Vegf mRNA was readily detectable in both zones of the rat placenta over the final third

of pregnancy (Fig. 7.1). Increased labyrinthine expression of Vegf120 (3.2-fold increase;

P < 0.01), Vegf164 (2.4-fold increase; P < 0.01) and Vegf188 (4.7-fold increase; P <

0.001) was observed from day 16 to 22 of normal rat pregnancy, but no changes were

observed in basal zone expression. Expression of the VEGF protein was localized

predominantly to the cytoplasm of the labyrinth zone trophoblast and some basal

trophoblast cells, with no expression observed in fetal endothelial cells (Fig. 7.2).

Localization of the VEGF protein was consistent at days 16 and 22 (data not shown).

7.4.2 Vascular changes in the rat placenta over the final third of pregnancy

Stereological analyses of the labyrinth zone during normal pregnancy (Figs. 7.3 and 7.4)

revealed that this region of the placenta underwent a marked increase (P < 0.001) in the

absolute volumes and surface areas of both maternal blood spaces (5-fold increase in

volume and surface area, Figs. 7.3A & B) and fetal capillaries (volume: 4-fold increase,

surface area: 5-fold increase, Figs. 7.3A & B) over the final third of pregnancy. The

increases in volume and surface area were accompanied by a marked rise in the total

combined length of the fetal capillary network (7-fold increase, P < 0.001, Fig. 7.4A).

These changes were due in part to a higher density of maternal blood spaces (volume:

46% increase, P < 0.001, and surface area: 49% increase, P < 0.01, Figs. 7.3C & D) and

fetal capillaries (volume: 48% increase, and surface area: 54% increase, P < 0.01, Figs.

7.3C & D) at day 22, including an increase in the relative density of fetal capillaries

(2.2-fold increase, P < 0.001, Fig. 7.4C) and a reduction in the mean diameter of fetal

vessels (19% reduction, P < 0.05, Fig. 7.4B). Tissue volume (i.e. labyrinthine volume

excluding vascular lumen) also increased (2.2-fold, P < 0.01, Fig. 7.3A) from day 16 to

22 although its proportional volume fell by 30% (P < 0.01, Fig. 7.3C) over this period.


Table 7.2. Fetal and placental zone weights following maternal dexamethasone treatment. Values

are the mean ± SEM (n = 6). * P < 0.01, compared with corresponding Control value (unpaired t-test).

Control Dexamethasone

Fetus (g) 5.56 ± 0.12 4.34 ± 0.25*

Basal Zone (mg) 135 ± 15 93 ± 5

Labyrinth Zone (mg) 307 ± 13 197 ± 21*


0

1

2

3

4

0

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16

Basal Labyrinth

16 2216 220

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25

30

Basal Labyrinth

16 22 16 22

Basal Labyrinth

16 22 16 22

Rel

ativ

e ex

pres

sion

A B CVegf120 Vegf164 Vegf188 ****

FIG. 7.1. Expression of (A) Vegf120, (B) Vegf164 and (C) Vegf188 mRNA in the basal and labyrinth

zones of the rat placenta on days 16 and 22 of pregnancy. Values are the mean ± SEM (n = 5-6 per

group). * P < 0.01, ** P < 0.001, compared with corresponding day 16 value (two-way ANOVA and

LSD-test).


FIG. 7.2. Spatial distribution of VEGF by immunohistochemistry at day 22 of rat pregnancy. (A)

No primary antibody (negative control). (B) VEGF expression primarily localized to the cytoplasm of

labyrinthine trophoblast cells with some basal zone trophoblast cells exhibiting weak expression. (C)

Higher power view of labyrinthine VEGF distribution showing positive trophoblast cells (black

arrowheads) and negative fetal endothelium (white arrowheads). LZ: labyrinth zone, BZ: basal zone;

scale bar 10 μm.


MBS FC Tissue

0

5

10

15

20

25

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10

20

30

40

16 22 16 22 16 22

MBS FC Tissue16 22 16 22 16 22

MBS16 22

FC16 22

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ume

(mm

3 )R

elat

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ace

Are

a (c

m2 )

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**

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020

406080

100120

0

20

40

60

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FIG. 7.3. Stereological analysis of labyrinth zone components on days 16 and 22 showing changes in

(A) absolute volume and (B) absolute surface area of the vasculature, and changes in (C) relative

vascular volumes and (D) relative surface areas of the vasculature. Values are the mean ± SEM (n = 4

per group). * P < 0.01; ** P < 0.001, compared with corresponding day 16 value (two-way ANOVA and

LSD-test).


0

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th (m

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apill

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sity

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m) *

B

FIG. 7.4. Stereological analysis of fetal capillary network on days 16 and 22 of pregnancy showing

changes in (A) total combined capillary length, (B) mean capillary diameter, and (C) capillary

density. Values are the mean ± SEM (n = 4 per group). * P < 0.05, ** P < 0.001, compared with

corresponding day 16 values (unpaired t-test).


7.4.3 Changes in VEGF expression following dexamethasone treatment

Administration of dexamethasone over the final third of pregnancy reduced fetal weight

(22% reduction; P < 0.01) and the weight of the placental labyrinth zone (35%; P <

0.01) (Table 7.2). Maternal dexamethasone treatment also reduced labyrinthine

expression of Vegf120 (40% reduction; P < 0.05) and Vegf188 (64% reduction; P < 0.01),

but had no effect on the expression of Vegf164 (Fig. 7.5). No change was observed in

basal zone expression of any isoform. Immunolocalization of the VEGF protein did not

change following treatment with dexamethasone (data not shown).

7.4.4 Vascular changes in the rat placenta following dexamethasone

treatment

Stereological analyses of the placental labyrinth zone showed that dexamethasone

treatment reduced (P < 0.01) absolute volumes and surface areas associated with

maternal (volume: 48% reduction; surface area: 46% reduction, Figs. 7.6A & B) and

fetal blood spaces (volume: 73% reduction, surface area: 61% reduction, Figs. 7.6A &

B). Absolute placental tissue volume was also reduced (36%, P < 0.05, Fig. 7.6A) by

dexamethasone, despite a relatively higher density (18% increase, P < 0.05, Fig. 7.6C).

Dexamethasone also impaired the normal increase in fetal vessel density (volume: 51%

reduction; surface area: 30% reduction, P < 0.01, Figs. 7.6C & D) and total combined

fetal capillary length (66% reduction, P < 0.01, Fig. 7.7A) over the final third of

pregnancy, but had no effect on the density of maternal blood space (i.e. relative

volume) or its surface area (Figs. 7.6C & D). In addition, treatment with dexamethasone

slightly reduced the size of fetal capillaries (12% reduction, P < 0.05, Fig. 7.7B) and the

relative density of fetal capillary profiles (37% reduction, P < 0.01, Fig. 7.7C).


0

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Basal Labyrinth

Con Dex Con Dex

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Con DexDexCon

Rel

ativ

e ex

pres

sion

A B CVegf120 Vegf164 Vegf188

**

*

FIG. 7.5. Effects of dexamethasone treatment from day 13 of pregnancy on zonal expression of (A)

Vegf120, (B) Vegf164 and (C) Vegf188 mRNA at day 22. Values are the mean ± SEM (n = 5-6 per group)

for placentas obtained from dexamethasone-treated (Dex) and untreated control (Con) mothers. * P <

0.05, ** P < 0.01, compared with corresponding control value (two-way ANOVA and LSD-test).


0

4

8

12

16

20

MBS FC TissueCon Dex Con Dex Con Dex

Rel

ativ

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olum

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Are

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m2 )

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FCCon Dex

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ume

(mm

3 )

***

**

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40

60

80

100

FIG. 7.6. Effects of dexamethasone treatment from day 13 of pregnancy on the (A) absolute volume,

(B) absolute surface area, (C) relative volume, and (D) relative surface area of the labyrinthine

vasculature at day 22. Values are the mean ± SEM (n = 4 per group) for placentas obtained from

dexamethasone-treated (Dex) and untreated control (Con) mothers. * P < 0.05; ** P < 0.01, compared

with corresponding control value (two-way ANOVA and LSD-test).


0

100

200

300

Con Dex

Leng

th (m

)

**

A

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illar

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C

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Con Dex

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er (µ

m)

*B

FIG. 7.7. Effects of dexamethasone treatment from day 13 of pregnancy on (A) total combined

capillary length, (B) mean capillary diameter, and (C) relative capillary density (QA) of the

labyrinthine vasculature at day 22. Values are the mean ± SEM (n = 4 per group) for placentas obtained

from dexamethasone-treated (Dex) and untreated control (Con) mothers. * P < 0.05, ** P < 0.001,

compared with corresponding control values (unpaired t-test).


7.5 Discussion

This study investigated the impact of increased glucocorticoid exposure on the spatial

and temporal expression of the endothelial cell-specific mitogen, VEGF, and associated

changes in placental vascularization over the final third of rat pregnancy. The

expression of Vegf mRNA increased specifically in the rapidly-growing labyrinth zone

between days 16 and 22 of normal pregnancy, and this change coincided with a marked

increase in vascularization of this placental zone. In contrast, treatment with

dexamethasone, which reduced fetal and placental growth, prevented the normal rise in

Vegf expression and the associated increase in labyrinthine vascularity over the final

third of pregnancy. Most notably, dexamethasone-induced reductions in absolute

vascular volume and surface area were accompanied by a reduction in the density of the

fetal vasculature, but there was no effect on the density of maternal blood spaces. Thus,

dexamethasone appears to reduce placental growth by preventing the normal

development of the fetal vasculature within the labyrinthine placenta.

Unbiased stereological analyses of the rat placenta in this study clearly demonstrated

rapid vascular development within the labyrinth zone, the site of fetal-maternal

exchange, over the final third of pregnancy. This increase in vascularization would be

expected to enhance the efficiency of fetal-maternal transport and is thus consistent with

the greater demands of the fetus late in pregnancy. In absolute terms, fetal vessels and

maternal blood spaces exhibited a 4 to 5-fold increase over the final third of pregnancy,

whereas relative densities (i.e. luminal volume expressed as a proportion of total

labyrinth volume) increased by approximately 50% over the same period. Previous

studies in the rodent have reported comparable increases in placental vascularization

(Lewis et al. 2001; Doherty et al. 2003; Coan et al. 2004), however, the results of this

study include a wider range of pregnancy, and demonstrate a similar rate of vascular

growth in the rat to that observed in the mouse (Coan et al. 2004). In addition, the

increase in the total combined length of fetal capillaries as well as the reduction in mean

capillary diameter observed near term suggest that fetal vascular remodeling is involved

in the development of the fetal capillary network of the rat placenta in a similar manner

to that observed in the mouse (Coan et al. 2004).

While these vascular changes are potentially due to the effect of various angiogenic

factors, the parallel increase in the expression of the endothelial cell-specific mitogen,

VEGF, in labyrinth zone trophoblast cells, suggests that this is likely to be a major


stimulus. Thus, a 2.4 to 4.7-fold increase in the expression of Vegf isoforms was

associated with a 4-fold increase in absolute volume, a 5-fold increase in absolute

surface area and a 7-fold increase in total combined length of fetal capillaries. In

addition to the current data, prior studies have demonstrated the importance of VEGF in

the development of placental vasculature and subsequent fetal growth via promotion of

endothelial cell proliferation and differentiation in the mouse and human (Carmeliet et

al. 1996; Ferrara 2004). In particular, gene deletion studies have shown that the loss of

even one VEGF allele results in impaired placental vasculature and embryonic lethality

by day 13 of pregnancy (Carmeliet et al. 1996; Ferrara 2004). Our observation that the

major Vegf mRNA isoforms and fetal vascularity both increase in the labyrinth zone

towards term, together with similar observations in other species (Hildebrandt et al.

2001; Albrecht et al. 2004; Robb et al. 2004), points to a key role for VEGF in

stimulating the major vascular remodeling that occurs in the placenta to ensure

continued fetal growth in late pregnancy.

In contrast to the increase in placental vascularity and Vegf mRNA expression during

normal pregnancy, maternal dexamethasone treatment between days 13 and 22 reduced

Vegf120 and Vegf188 mRNA expression and absolute volumes and surface areas of both

maternal blood spaces and fetal capillaries. Previous reports show that increased

glucocorticoid exposure reduces transplacental passage of glucose (Langdown and

Sugden 2001) and leptin (Smith and Waddell 2003) with no reduction in the expression

of placental transporter proteins. It seems likely that these effects on transport are non-

specific, reflecting a glucocorticoid-induced reduction in the effective vascular

exchange area within the labyrinth zone. A key finding of this study was that the effects

of dexamethasone were specific to the fetal vasculature, with no apparent effect on the

density of maternal blood spaces. In effect, it appears that dexamethasone treatment

suppressed the normal increase in the density of fetal capillary volume and surface area

over the final third of pregnancy, since the vascularity of the dexamethasone-treated

placentas at day 22 was comparable to that at day 16. Therefore, these data suggest that

dexamethasone treatment suppresses fetal vessel growth, consistent with the prevention

of the normal increase in Vegf mRNA observed over the final third of pregnancy. With

respect to total placental volume, a recent study suggests that the reduction in non-

vascular tissue volume observed in cases of IUGR may reflect the reliance on adequate

fetal vascularity (Rutland et al. 2005). Thus, the dexamethasone-mediated effects on


labyrinth zone growth observed in the present study may be a consequence of the

primary effects on VEGF-mediated fetal capillary growth.

While the present study is the first to report an inhibitory effect of glucocorticoids on

placental VEGF expression, similar effects have been observed in other tissues

including airway smooth muscle cells and chondrocytes (Heiss et al. 1996; Koedam et

al. 2002; Smink et al. 2003; Alagappan et al. 2005; Alonso et al. 2005). Exactly how

glucocorticoids suppress Vegf expression remains uncertain, but potentially it could

involve reduced transcription of the Vegf gene due to interaction of the activated

glucocorticoid receptor with the Vegf promoter, and/or indirect effects via other

placental genes that regulate VEGF expression. For example, we recently demonstrated

that labyrinth zone expression of PPARG, activation of which upregulates VEGF

expression in various cell types (Yamakawa et al. 2000; Yasuda et al. 2005), is

suppressed by dexamethasone (Hewitt et al. 2006b). In addition, dexamethasone

upregulates the expression of SFPR4 in rat trophoblast (Hewitt et al. 2006a), which

could reduce Vegf gene transcription via inhibition of WNT signaling (Zhang et al.

2001). Interestingly, dexamethasone did not affect labyrinthine expression of the Vegf164

isoform, raising the possibility that glucocorticoids regulate VEGF by means of an

isoform-specific degradation of Vegf mRNA. Indeed, a previous report indicates that

dexamethasone decreases Vegf mRNA stability in cultured keratinocytes (Gille et al.

2001). Further studies are required to elucidate the specific mechanisms by which

dexamethasone suppresses placental VEGF expression in our model of fetal and

placental growth restriction.

In conclusion, we have shown that VEGF expression in the labyrinth zone of the rat

placenta is upregulated near term, and that maternal dexamethasone treatment prevents

this increase. These changes are consistent with the marked increase in labyrinthine

vascular development observed over the final third of normal pregnancy and impaired

vascularization observed during dexamethasone-induced fetal and placental growth

restriction. Although the control of VEGF expression and vascular growth is likely to

involve a complex balance of diverse molecular signals, the results of the current study

suggest that glucocorticoids restrict fetal and placental growth via inhibition of placental

VEGF expression and subsequent placental vascularization.


Chapter 8 General Discussion

The overall objective of the studies described in this thesis was to quantify the impact of

increased glucocorticoid exposure on placental vascularisation and expression of some

of the key regulators of placental growth and vascular development. Three major studies

were carried out, and the findings of each have been presented and discussed in detail

within their respective chapters. In this final chapter, the major findings will be

summarised, integrated and discussed in relation to their biological significance.

The first study (Chapter 5) quantified changes in the expression of the PPAR nuclear

receptors over the final third of normal rat pregnancy and after glucocorticoid-induced

fetal and placental growth restriction. This study was formulated on the basis that mouse

knockout models indicated a critical role for both Pparg and Ppard in normal placental

development (Barak et al. 1999; Barak et al. 2002). Pparg-null mutants, in particular,

exhibited reduced invasion of fetal blood vessels within the developing placenta, raising

the possibility that PPARG may also function in the development of the placental

vasculature late in pregnancy. Furthermore, glucocorticoid-mediated regulation of

PPARG activity has been observed in other tissues (Vidal-Puig et al. 1997; Zilberfarb et

al. 2001; Mouthiers et al. 2005) and so it was considered possible that changes in

PPARG may contribute to the inhibitory effects of glucocorticoids on placental growth

and function (Benediktsson et al. 1993; Waddell et al. 2000).

Several key findings emerged from these experiments; most notably, labyrinthine

expression of Pparg was upregulated near term in normal rat pregnancy, and

glucocorticoid-induced placental growth restriction inhibited this rise. These results are

consistent with a role for PPARG in the development of the vascular network within the

placenta over the final third of pregnancy. Moreover, subsequent unpublished analyses

conducted since the publication of this work suggest that placental PPARG expression

leads to parallel changes in levels of receptor activation. Specifically, the tightly

regulated PPARG target, Muc1 (Shalom-Barak et al. 2004), the expression of which is

absent in PPARG knockouts and is upregulated after treatment with PPARG agonists,

was also upregulated near term and inhibited by glucocorticoids (see Appendix). Thus,

using Muc1 mRNA expression as a surrogate indicator of PPARG activity, one can

speculate that activity of PPARG plays a role in placental development during normal

pregnancy, and that this role is compromised after maternal glucocorticoid treatment. In


particular, the established links between PPARG and formation of the early placental

vasculature (Barak et al. 1999), suggest that PPARG might not only be responsible for

the establishment of fetal vessels in the first half of pregnancy, but may also function in

the rapid growth and vascularisation of the placenta observed late in pregnancy.

Although the specific functions of PPARG and indeed each of the PPARs remain

unclear, the results of Chapter 5 establish the basis for the investigation of PPARG as a

potential therapeutic target in the promotion of placental growth and vascularisation.

PPARG is the specific target for the thiazolidinedione (TZD) group of drugs which are

commonly used to increase insulin sensitivity in type II diabetes. Therefore, TZD

treatment in pregnancies affected by vascular anomalies could potentially attenuate the

glucocorticoid-induced inhibition of normal vascular growth, particularly late in

pregnancy. In support of this proposition, Asami-Miyagishi et al. (2004) investigated

the effects of maternal administration of a TZD between days 9 and 11 of rat pregnancy

and showed a marked reduction in the normal spontaneous abortion rate. Taken

together, these data suggest that TZD treatment and subsequent PPARG activation may

be able to stimulate placental angiogenesis which, in turn, would promote fetal growth

and survival. Furthermore, it remains possible that TZD treatment may serve as a

potential therapy to stimulate placental vascularisation in pregnancies affected by high

levels of maternal glucocorticoids or as a combined therapy for women receiving

glucocorticoids to accelerate fetal organ maturation. Detailed analysis of placental

development after TZD treatment, however, is still required to determine the precise

relationship between PPARG, the development of the vascular network, and the role of

glucocorticoids in its inhibition.

The exclusive localisation of both PPARG and RXRA to trophoblast cells suggests that

activation of PPARG may result in trophoblastic secretion of angiogenic factors to

stimulate the growth of fetal blood vessels. In accordance with this, recent data suggests

that PPARG exerts a positive effect on angiogenesis by stimulating expression of VEGF

(Bamba et al. 2000; Jozkowicz et al. 2000; Yamakawa et al. 2000; Yasuda et al. 2005).

Moreover, the results of Chapter 7 indicate that the placental expression and activity of

PPARG are consistent with the pattern of change observed in VEGF expression. It

would be of interest, therefore, to examine specifically whether activation of PPARG

(via treatment with TZDs) increases trophoblastic expression of VEGF and associated

placental angiogenesis.


Glucocorticoids are commonly associated with normal cellular differentiation and

growth inhibition. Indeed, glucocorticoids are essential for fetal organ maturation late in

pregnancy. Excess glucocorticoid exposure, however, can reduce placental and fetal

growth (Benediktsson et al. 1993) and increase rates of trophoblast apoptosis (Waddell

et al. 2000). The mechanisms by which glucocorticoids affect this process, however,

remain unclear. The second study (Chapter 6), therefore, investigated the expression of

the secreted wnt inhibitor, SFRP4, in normal pregnancy and following maternal

glucocorticoid treatment on the basis that this particular isoform is associated with

apoptotic (Marti et al. 1999; Drake et al. 2003) and anti-proliferative signalling (Suzuki

et al. 2004) in other tissues, and that the wnt pathway, a common driver of cellular

growth, is inhibited by glucocorticoids (Ohnaka et al. 2005). Gene deletion studies have

already established a critical role for wnt signaling in placental vascular development

(Monkley et al. 1996; Galceran et al. 1999; Ishikawa et al. 2001; Parr et al. 2001) and so

it was considered likely that glucocorticoid-mediated inhibition of the wnt pathway may

also impact on placental development late in pregnancy.

Placental expression of SFRP4 was markedly upregulated in the basal zone near term

consistent with previously observed increases in trophoblast apoptosis and reduced

growth of this zone over the final third of pregnancy (Waddell et al. 2000). Maternal

glucocorticoid treatment further increased expression of SRFP4 in both the basal and

labyrinth zones, suggestive of a role for SFRP4 in glucocorticoid-mediated placental

growth restriction. Interestingly, while previous reports demonstrate that glucocorticoids

upregulate basal zone apoptosis, this increase is not observed in the labyrinth zone

(Waddell et al. 2000). The role of labyrinthine SFRP4 therefore, may involve anti-

proliferative or pro-differentiation signalling rather than pro-apoptotic effects.

To investigate whether SFRP4-mediated inhibition of the wnt pathway is involved in

glucocorticoid-induced inhibition of placental growth, the relationship between SRPF4

expression and intracellular localisation of CTNNB1 as a marker of wnt pathway

activation was also examined. CTNNB1 provided a convenient target because of the

ability to observe nuclear translocation, a process that indicates functional wnt

signalling. Nuclear CTNNB1 then binds to transcription factors and upregulates specific

wnt target genes. Thus, a marked increase in expression of SFRP4 in the non-

proliferating basal zone near term was accompanied by an increase in the nuclear

translocation of CTNNB1.


In addition to the potential anti-proliferative effects of placental SFRP4, wnt signalling

has also been linked to an upregulation of VEGF (Zhang et al. 2001). Thus, the

glucocorticoid-mediated upregulation of SFRP4 and subsequent inhibition of the wnt

pathway may play some role in glucocorticoid-induced inhibition of VEGF expression

(Chapter 7). Labyrinthine expression of SFRP4, however, is relatively low and although

maternal glucocorticoid treatment more than doubles this expression, it is uncertain

whether this level of SFRP4 expression is sufficient to impact on placental

vascularisation and growth of the labyrinth zone.

Wnt signalling has also been shown to increase the expression of PPARD (He et al.

1999). This is consistent with the observed reduction in placental expression of PPARD

near term in the basal zone where SFRP4 appears to inhibit wnt-mediated signalling. In

the context of the labyrinth zone, activation of PPARG can increase wnt function (Saez

et al. 2004) and, therefore, the high expression of labyrinthine PPARG near term during

normal pregnancy is consistent with a role for PPARG in wnt-mediated placental

growth and vascular development. Furthermore, inhibition of PPARG by maternal

glucocorticoid treatment may also inhibit wnt function independently of SFRP4, since

SFRP4 expression is relatively low in the labyrinth zone.

Previous studies have determined that glucocorticoids also reduce transplacental

passage of glucose (Langdown and Sugden 2001) and leptin (Smith and Waddell 2003)

without a reduction in the expression of placental transporter proteins. Therefore,

glucocorticoid-mediated effects on placental exchange may be non-specific, reflecting a

general inhibition of placental vascularisation rather than specific transporter activity.

Indeed, the results of Chapter 5 suggest that glucocorticoids affect the expression and

activation of PPARG, which is critical for fetal vessel growth during the establishment

of the placenta (Barak et al. 1999). Thus, the final study (Chapter 7) quantified placental

vascularisation in association with changes in VEGF expression over the final third of

normal rat pregnancy and in a model of glucocorticoid-induced fetal and placental

growth restriction.

Over the final third of pregnancy the rodent placenta doubles in size, primarily due to a

3-fold increase in labyrinthine weight. In Chapter 7 it was shown that this growth is

accompanied by a marked increase in both the volume and surface area associated with

maternal blood spaces and fetal capillaries of the labyrinth zone. In contrast, maternal

dexamethasone administration suppressed the normal increase in placental


vascularisation. Most notably, dexamethasone reduced the normal increase in fetal

vessel volume and surface area density but had no effect on the density measures of

maternal blood spaces. These findings are consistent with a role for glucocorticoids in

the inhibition of placental transport via a reduction in placental vascularisation.

In accordance with an increase in placental vascular development, normal expression of

VEGF was upregulated specifically in the proliferating labyrinth zone over the final

third of pregnancy. Maternal dexamethasone treatment, however, inhibited the normal

rise in labyrinthine expression of VEGF. This finding is consistent with the suppression

of VEGF expression by glucocorticoids observed in other tissues (Heiss et al. 1996;

Koedam et al. 2002; Smink et al. 2003; Alagappan et al. 2005; Alonso et al. 2005). The

glucocorticoid-mediated reduction in VEGF expression is also consistent with impaired

vascularisation of the endothelial-lined fetal capillaries and the restricted fetal growth

observed after glucocorticoid treatment. Therefore, the reduced growth of fetal vessels

observed after maternal treatment with dexamethasone may be the result of

glucocorticoid-mediated inhibition of PPARG expression, which in turn reduces the

trophoblastic expression of VEGF (Fig. 8.3).

Overall the studies presented in this thesis provide insight into placental vascular

development and potential mechanisms of glucocorticoid-mediated fetal and placental

growth restriction. Over the final third of pregnancy, glucocorticoids restrict the normal

increase in PPARG and VEGF expression in the rapidly growing labyrinth zone in

association with marked inhibition of fetal vessel growth within the mature placenta. In

contrast, glucocorticoids upregulate the expression of SFRP4 in basal zone trophoblast

cells, potentially affecting wnt-mediated trophoblast growth, differentiation and

apoptotic signals. It is possible that PPARG may function as a therapeutic target in the

upregulation of VEGF expression because of the convenience of currently proven

pharmaceuticals available as specific PPARG agonists.


PPARγ Ligands:• Fatty Acids• Prostaglandins• Thiazolidinediones

PPARγ Ligands:• Fatty Acids• Prostaglandins• Thiazolidinediones

MaternalBlood Space

Trophoblast

FetalMesenchyme

Fetal Capillary

Fetal Endothelium

ActivatedPPARγ/RXRαHeterodimer

ActivatedPPARγ/RXRαHeterodimer

VEGFVEGFVEGFVEGFVEGFVEGF

Figure 8.3 Proposed regulation of trophoblastic VEGF expression via PPARG. VEGF expression

during normal pregnancy may be upregulated via activation of PPARG:RXRA heterodimers. In contrast,

glucocorticoid mediated inhibition of PPARG expression would reduce VEGF expression and

subsequently reduce angiogenic activity of fetal vessels.


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APPENDIX

The data contained within this appendix were completed after the publication of the

second manuscript (Chapter 6) but provide important information regarding the

activation of PPARG. The rationale is based on previous findings by Shalom-Barak et

al. (2004) in Pparg-null mice, indicating that expression of the mucin gene (Muc1) is

tightly controlled by PPARG in the rodent placenta. The results in this appendix are

consistent with the proposition that Muc1 can serve as a marker of PPARG activation in

rodent trophoblast.


Rel

ativ

e Ex

pres

sion

Basal Labyrinth

16 22 16 22

Muc1

0

2

4

6 *

Figure 8.1 Zonal expression of Muc1 mRNA at days 16 and 22 of rat pregnancy. Expression of Muc1

mRNA was 2.4-fold higher at day 22 compared to corresponding day 16 value. There was no change in

basal zone expression. Values are the mean ± SEM (n = 5-6 per group). * P < 0.01, compared with

corresponding day 16 values (two-way ANOVA and LSD-test).


R

elat

ive

Expr

essi

on

Basal LabyrinthCon Dex Con Dex

Muc1

2

4

6

*

Figure 8.2 Effects of dexamethasone treatment from day 13 of pregnancy on zonal expression of

Muc1 mRNA at day 22 of rat pregnancy. Maternal dexamethasone treatment reduced labyrinthine

expression of Muc1 by 51%, with no change in basal zone expression. Values are the mean ± SEM (n =

5-6 per group) for placentas obtained from dexamethasone-treated (Dex) and untreated control (Con)

mothers. * P < 0.01, compared with corresponding control value (two-way ANOVA and LSD-test).

impact of glucocorticoids on placental growth and ... · glucocorticoids and placental growth 7...

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