thesis_final

The expression of the relaxin receptor in human reproductive tissues.
A comparison between normotensive and preeclamptic women.
Tahnee Saunders
A thesis submitted in partial fulfillment of the requirements for the Research Project as part of
the Master of Biotechnology degree.
Department of Zoology
The University of Melbourne
November 2014

Table of Contents
ABSTRACT
..............................................................................................................................................................
3

1.
INTRODUCTION
................................................................................................................................................
4

1.1
PREECLAMPSIA
..............................................................................................................................................................
4

1.2
DEVELOPMENT
OF
THE
PLACENTA
................................................................................................................................
5

1.3
STRUCTURE
OF
UMBILICAL
CORD
..................................................................................................................................
6

1.4
FACTORS
THAT
REGULATE
THE
PROCESSES
OF
THE
PLACENTA
AND
UMBILICAL
CORD
........................................
7

1.5
RELAXIN
..............................................................................................................................................................................
8

1.5.1
Vasodilation
.................................................................................................................................................................
8

1.5.2
Vascularisation
...........................................................................................................................................................
9

1.5.3
Implications
in
Preeclampsia
............................................................................................................................
10

1.6
ACTION
THROUGH
RXFP1
............................................................................................................................................
10

1.7
AIMS
AND
HYPOTHESES
................................................................................................................................................
11

2.
MATERIALS
AND
METHODS
......................................................................................................................
12

2.1
PATIENTS
AND
SAMPLE
COLLECTION
.........................................................................................................................
12

2.2
ISOLATION
OF
HUMAN
VEIN
ENDOTHELIAL
CELLS
.................................................................................................
12

2.3
FIRST
STRAND
CDNA
SYNTHESIS
................................................................................................................................
13

2.4
REVERSE
TRANSCRIPTION
POLYMERASE
CHAIN
REACTION
(RT-‐PCR)
.............................................................
13

2.5
QUANTITATIVE
REAL
TIME
PCR
.................................................................................................................................
14

2.6
IMMUNOHISTOCHEMISTRY
............................................................................................................................................
14

2.7
BIO-‐INFORMATICS
..........................................................................................................................................................
15

2.8
STATISTICAL
ANALYSIS
.................................................................................................................................................
15

3.
RESULTS
..........................................................................................................................................................
16

3.1
RXFP1
GENE
EXPRESSION
.............................................................................................................................................
16

HUVECs
...................................................................................................................................................................................
16

Placenta
.................................................................................................................................................................................
16

3.2
RXFP1
LOCALISATION
IN
THE
UMBILICAL
CORD
....................................................................................................
17

3.3
BIOINFORMATIC
ANALYSIS
...........................................................................................................................................
17

4.
DISCUSSION
....................................................................................................................................................
24

4.1
FUTURE
DIRECTIONS
.....................................................................................................................................................
27

5.
CONCLUSION
..................................................................................................................................................
27

ACKNOWLEDGEMENTS
...................................................................................................................................
28

3
Abstract

Relaxin has been shown to have a significant role in multiple functions necessary to achieve the
adaptations observed in pregnancy. Preeclampsia occurs when these adaptations are inadequate
and a vicious cycle of placental hypoxia and endothelial dysfunction ensue that results in the
maternal symptoms of high blood pressure and proteinuria, which characterise preeclampsia.
While there is no significant decrease in relaxin levels of normotensive women in comparison to
preeclamptic women, relaxin acts through its receptor RXFP1 and therefore changes in
expression of RXFP1 may contribute to preeclampsia. My study investigated RXFP1 expression
in human tissues and cells obtained from normotensive and preeclamptic women, including
umbilical vein endothelial cells (HUVECs), umbilical cord and placenta. I demonstrated Rxfp1
expression in HUVECs and found no significant change in Rxfp1 or Rln expression in the
placenta of preeclamptic women compared to normotensive women. In addition, I detected a
change in the correlation between Rxfp1 and Rln expression in the placenta, which suggests it is
the ratio of Rln to Rxfp1 that is of relevance and not the absolute values of expression. RXFP1
was also localised to the umbilical cord, with positive staining in a specific cell type in the
Wharton’s jelly, possibly fibroblasts. This finding uncovers a potential new role for relaxin in the
turnover of extracellular matrix of the umbilical cord and relaxin may influence blood flow to
the foetus. My bioinformatics analysis corroborated my findings, which outline the relevance of
RXFP1 expression in understanding the actions of relaxin in pregnancy.

4
1. Introduction
Pregnancy is a unique physiological condition in which the mother is required to adapt to
balance maternal-foetal demands. These adaptations include profound changes to the
endocrine, cardiovascular, renal and respiratory systems to accommodate the increasing
demands of the growing foetus. Adaptations of particular importance occur in the
cardiovascular system and uteroplacental vasculature; for which remodeling and expansion of
begin soon after fertilisation (Longo, 1983). A reduction in peripheral vascular resistance is
observed soon after conception as well as changes in vascular endothelial function. The
reduction in peripheral vascular resistance activates volume-changing mechanisms, which
contribute to the 50% increase in plasma achieved by term (Chapman et al., 1998). Cardiac
output gradually increases throughout pregnancy through increases in heart rate and stroke
volume until partway through the second trimester (Chapman et al., 1998). Renal adaptations
mirror those observed in the cardiovascular system (Weissgerber and Wolfe, 2006). The
explained adaptations achieve a high-flow low-resistance circulation (Spaanderman et al.,
2000) allowing for an improved transport capacity to the foetus (Bauer et al., 1998) and the
movement of foetal waste from the placenta to relevant maternal organs (Chapman et al.,
1998). Increased transport capacity also facilitates enhanced circulation of placental hormones
(Chapman et al., 1998).
1.1 Preeclampsia

Preeclampsia and intrauterine growth restriction result due to a failure of the vascular system
to adapt effectively to the physiological pressures of pregnancy. In particular, preeclampsia is
characterised by hypertension and development of proteinuria after 20 weeks gestation
(Mutter and Karumanchi, 2008). Despite being one of the leading causes of preterm delivery
and maternal death, the causal mechanisms for preeclampsia still remain to be elucidated.
Currently the only cure for preeclampsia is the delivery of the placenta, thus a trade-off is
made between maternal and foetal health in the decision of the time for delivery (Roberts and
Gammill, 2005). Complications for the mother and foetus stem from the premature delivery,
as well as the impacts of the pathophysiology of the disease. Specifically, complications for
the foetus may include low birth weight, prematurity and death. There are multiple
implications for the mother’s health including renal failure, liver failure, cerebral edema and
sometimes death (Mutter and Karumanchi, 2008). Risk factors for preeclampsia include

5
obesity, diabetes, hypertension, and kidney disease, all of which can be accompanied by
underlying vascular disease, suggesting that preeclampsia is in fact due to circulating
angiogenic factors that are imbalanced and causing endothelial dysfunction. Preeclampsia can
develop in absence of a developing foetus (Powe et al., 2011) suggesting the placenta is the
primary source of the pathogenesis of preeclampsia (Matsuo, 2007). Preeclampsia is
associated with reduced placental perfusion and placental ischemia, potential initiating events
of preeclampsia, and are linked to endothelial dysfunction (Gilbert et al., 2008), depicting a
complex pathogenesis.
1.2 Development of the Placenta

The development of the placenta is complex and is achieved through interaction of many
factors, and involves changes to maternal vasculature and endometrium. These changes are
accompanied by production of new vessels on the foetal side, achieved either through de novo
production of vessels (vasculogenesis) or branching of existing vessels (angiogenesis).
Normal placentation begins with the cytotrophoblast anchoring the embryo in the wall of the
uterus, and invading the interstitium of the decidua. Cytotrophoblasts differentiate to form
primary villi, which are surrounded in a layer of syncytiotrophoblast cells. Cytotrophoblast
cells within the villi differentiate into extravillous trophoblast (EVT) cells, and when the villi
anchor to the maternal endometrium, the syncytiotrophoblast layers disappear allowing EVT
cells to migrate to the maternal spiral arteries. The EVT cells then play a key role in the
remodeling of the maternal spiral arteries by replacing the normal muscular and elastic
structures of the spiral arteries resulting in a change in phenotype of the epithelium to the
phenotype of an endothelium. The EVT cells cause the widening and strengthening of the
arterial walls, resulting in a larger lumen and low resistance phenotype which allows optimal
blood supply to the developing embryo (Silasi et al., 2010). In preeclampsia there is shallow
or even absent remodeling of the maternal spiral arteries, which maintain their epithelium
phenotype, and high-resistance nature to blood flow (Brosens et al., 1970). This creates a
hypoxic environment that may be the first insult in the development of preeclampsia.
Normal development of the placenta continues through vasculogenesis and angiogenesis.
Angiogenesis occurs normally during the menstrual cycle to rebuild the uterine wall
following menstruation, and the process during placental development follows similar steps.
Up to 24 weeks gestation angiogenesis occurs primarily through branching and the formation

6
of new vessels by sprouting, after which and until term the nonbranching angiogenic process
becomes more prevalent, with the formation of new vessels by elongation (Chen and Zheng,
2014). The normal growth of the placenta through angiogenesis requires a balance of
angiogenic and antiangiogenic factors. An abnormal level of these factors can lead to the
development of preeclampsia.
1.3 Structure of Umbilical Cord

The umbilical cord is the link between mother and foetus, and consists of a helical structure of
two arteries and one vein that are surrounded by a mucous connective tissue called Wharton’s
jelly (Benirschke and Kaufmann, 2000). The Wharton’s jelly is thought to prevent
compression, torsion and bending and makes up 70% of the cross-sectional area of the
umbilical cord by the middle of gestation (Skulstad et al., 2006). The most abundant cell type
found in Wharton’s jelly are called myofibroblasts (Takechi et al., 1993). These are immersed
in a complex extracellular matrix with bundles of fibrils surrounded by a slender network of
microfibrils (Franc et al., 1998). Myofibroblasts are an intermediate between smooth muscle
cells and fibroblasts (Gabbiani et al., 1972), and are present at various stages of differentiation
from fibroblasts to myofibroblasts within the umbilical cord. These stages of differentiation
form a radial pattern within the umbilical cord and can be defined as three zones: vessels
adventitia, Wharton’s jelly and subamniotic area (Nanaev et al., 1997). Myofibroblasts are
thought to play a role in the synthesis and accumulation of the extracellular matrix, while
extracellular matrix components are capable of influencing the differentiation of fibroblasts.
Therefore the context of cell differentiation is important when considering the extracellular
matrix structure of the umbilical cord (Nanaev et al., 1997).
The complex extracellular matrix of the Wharton’s jelly has the vital function of preventing
the umbilical cord from twisting. Furthermore, Wharton’s jelly is speculated to play a role in
the regulation of umbilical cord blood flow, as small for gestational age infants are linked to
‘lean’ umbilical cords (Raio et al., 2003). The artery lumen of lean umbilical cords has no
change in area, whereas the umbilical vein and Wharton’s jelly decrease in area (Di Naro et
al., 2001). It is therefore suggested that a potential decrease in blood flow to the infant is
consequence of the shrinking of the Wharton’s jelly (Gebrane-Younes et al., 1986). The area
of Wharton’s jelly is also reduced in preeclampsia (Raio et al., 2002), with changes in
biochemical factors such as gelatinase. A reduction in gelatinase activity leads to an

7
accumulation of collagen and a decrease in elastin (Galewska et al., 2000), therefore leading
to a decline in elasticity of the umbilical arterial wall which may cause the decrease in blood
flow to the foetus of preeclamptic women. The changes seen in the umbilical cord during
preeclampsia therefore warrant further investigation, as a potential source for understanding
the changes in blood flow to the foetus.
1.4 Factors that regulate the processes of the placenta and umbilical cord

The development of the placenta is highly dependent on vascular endothelial growth factor
(VEGF) which plays a pivotal role in the processes of vasculogenesis and angiogenesis (Chen
and Zheng, 2014). VEGF is a homodimeric glycoprotein that acts through two protein kinase
receptors: VEGFR-1 and VEGFR-2 (Silasi et al., 2010). Both receptors are transmembrane
tyrosine kinase receptors, which act through phosphorylation of a cytoplasmic substrate to
initiate a cascade of cellular responses, including mitogen activated protein kinases (MAPK),
posphoinositade-3-kinase (PI3k) / Akt1 and nitric oxide (NO) signaling pathways (Chen and
Zheng, 2014) that cause proliferation, migration and tube formation processes of
angiogenesis. The soluble form of the VEGF receptor is called soluble fms-like protein kinase
1 (sFlt-1) and circulates in the blood as a potent antagonist to VEGF and proangiogenic
proteins including, placental growth factor (PIGF). sFlt-1 binding to these proteins prevents
them from binding to their endogenous receptors (Shibuya, 2001, Kendall et al., 1996).
Another factor that is less well understood is endoglin, which is a co-receptor for
transforming growth factors (TGF) found on cell surfaces. TGF-ß1 and TGF-ß3 are inhibitors
of trophoblast differentiation (Wang et al., 2009), and it is thought endoglin may regulate the
inhibition of EVT differentiation (Caniggia et al., 1997). TGF-ß1 is present in the Wharton’s
jelly at seven times the concentration present in the umbilical cord artery. It is speculated that
TGF-ß1 may stimulate the production of the large amounts of collagen that makes up the
extracellular matrix of Wharton’s jelly (Malkowski et al., 2008). TGF-ß1 may play a critical
role in the structure of the umbilical cord and is implicated in placental development through
its inhibition of trophoblast differentiation and therefore the remodeling of the maternal spiral
arteries. A delicate balance is achieved between these factors to allow for normal
development of the placenta, and possibly the umbilical cord.
However during preeclampsia there is an imbalance in the prior mentioned factors. A soluble
form of endoglin (sEng) is increased in the circulation of preeclamptic women, and binds to

8
and blocks TGF-ß1 signaling (ten Dijke et al., 2008). This leads to endothelial dysfunction
and may influence inadequate remodeling of the maternal spiral arteries. The hypoxic
condition that results from inadequate maternal spiral artery remodeling stimulates the
production of sFlt-1 which binds to and inactivates PIGF and VEGF, suppressing normal
placental angiogenesis (Seki, 2014). Therefore the factors stimulated by low levels of oxygen,
act in a counterintuitive manner and worsen the hypoxic state in the placenta, inducing a
vicious cycle of placental hypoxia (Seki, 2014).
1.5 Relaxin

A 6kDa peptide hormone, relaxin is expressed by the corpus luteum during the secretory
phase and early pregnancy, with circulating levels of relaxin peaking at the end of the first
trimester (Sherwood, 1994). Further research found that relaxin is expressed by the human
endometrium at different stages during the menstrual cycle (Weiss et al., 2001), as well as
human endometrial cells in vitro (Palejwala et al., 2002). Trophoblasts, with prior mentioned
key roles in the developing placenta and spiral artery remodeling also express relaxin
(Conrad, 2011). Relaxin is expressed by maternal and foetal cells of the placenta, specifically
decidual cells, intermediate trophoblasts and syncytiotrophoblasts (Goh et al., 2013). The
relaxin produced by the decidua and placenta (dRln) does not enter the circulation, and its
actions are purely autocrine and paracrine, where as the relaxin produced by the corpus
luteum (sRln) enters maternal circulation and therefore functions systemically (Rocha et al.,
2013). The molecular mechanisms of relaxin are complex, and implicated in multiple
signaling pathways of different cell types.
1.5.1 Vasodilation
Relaxin is primarily known for its vasodilatory function, in which it has two categorised
responses: the rapid vasodilatory response and the sustained vasodilatory response. The rapid
response is seen within minutes and is transduced by endothelial G protein coupling to
PI3k/Akt dependent phosphorylation, and activation of nitric oxide (NO) synthase (NOS)
(Bathgate et al., 2006). The sustained vasodilatory response takes hours or days of prolonged
exposure to relaxin, and acts via matrix metalloproteinase (MMP) -9 or -2 depending on the
duration of the exposure (hours or days respectively) causing an increase in gelatinase
activity. The gelatinase in turn hydrolyses big endothelin (ET) to form ET1-32, which then
activates endothelial ETb receptor and initiates the NO vasodilatory pathway (Novak et al.,

9
2006, Novak et al., 2001, Jeyabalan et al., 2007). Local production of relaxin in vessels,
demonstrates a signaling system that increases arterial compliance (Novak et al., 2006).
Specifically to pregnancy adaptations, during the first trimester increased circulating relaxin
results in vasodilation. In rats, immunoneutralised relaxin results in the absence of the
vasodilation normally observed in early pregnancy (Debrah et al., 2006). However, there are
no reports of decrease vasodilation in women who become pregnant through egg donation and
therefore lack the circulating relaxin normally produced by the corpus luteum (Conrad and
Baker, 2013, Johnson et al., 1991). Yet, relaxin administration to virgin female rats causes
decreased systemic vascular resistance and increases cardiac output (Conrad et al., 2004),
mimicking pregnancy adaptations with a magnitude similar to that of a midterm pregnant rat
(Slangen et al., 1996). Finally, circulating relaxin levels are positively correlated with uterine
flow in early pregnancy (Jauniaux et al., 1994). While the absence of circulating relaxin in
ovum-donated pregnancies is not linked to a lack of vasodilation, there is a body of evidence
to support the role of relaxin in the vasodilation of early pregnancy.
1.5.2 Vascularisation
Many vasodilators also have angiogenic functions (Conrad and Novak, 2004). Vascularisation
at wound sites has been linked to relaxin mediated VEGF angiogenesis (Unemori et al.,
2000). In human endometrial cells, relaxin has been found to increase expression of VEGF in
in vitro (Unemori et al., 1999), specifically in the stromal and epithelial glandular cells
(Palejwala et al., 2002) suggesting a role for relaxin in the regulation of vascularisation of
reproductive tissues. Furthermore, human endometrial cells cultured and treated with relaxin,
show vascular development in vitro (Parry and Vodstrcil, 2007). Exogenous relaxin produced
conceptus-like mediated changes in the vasculature of non-human primate in vivo
(Blankenship and Enders, 2003) and female monkeys show increased endometrial thickness
and implantation bleeding when treated with human relaxin during the period of peri-
implantation (Hayes, 2004). In the context of humans, observations have been made in vivo
with treatment of relaxin being associated with an increase in menometrorrhagia in women
(Unemori et al., 1999). Despite abundant reports of relaxin’s role in the angiogenesis and
reproduction in non-human species, the role of relaxin in human reproduction is still not clear.
However in conjunction, work on animal-models and observations in humans suggest that
relaxin stimulates neovascularisation that is mediated by the VEGF (Unemori et al., 1999).

10
1.5.3 Implications in Preeclampsia
Relaxin has a functional role in a multitude of signaling pathways that are implicated in
placental development and early pregnancy vascular adaptations and its function may play a
role in the development of preeclampsia. However when comparing circulating relaxin levels
between normotensive and preeclamptic women no significant difference was observed
(Lafayette et al., 2011, Szlachter et al., 1982). It has been shown that invading trophoblasts
express gelatinase activity, facilitating migration and invasion into the uterus and data
suggests ETb-receptor-endothelial NOS system promotes trophoblast invasion (Martin and
Conrad, 2000, Chakraborty et al., 2003) implicating relaxin in this process. Also in a non-
human primate model, relaxin treatment was found to stimulate endometrial angiogenesis and
increase endometrial lymphocyte number – implicating a role for relaxin in key processes
involved in the placental development and successful spiral artery remodelling (Goldsmith et
al., 2004, Goldsmith and Weiss, 2013). While there is no significant decrease in circulating
relaxin in preeclamptic pregnancies (Szlachter et al., 1982), this measure is not indicative of
bioavailability of relaxin and signaling may still be impaired in preeclampsia. Relaxin acts
through its receptor RXFP1, therefore bioavailability and action of relaxin is dependent on
expression levels of Rxfp1 and an antagonist to relaxin signaling, a truncated version of the
relaxin receptor (Scott et al., 2006, Kern and Bryant-Greenwood, 2009). The function of
relaxin in vasodilation, trophoblast invasion, maternal spiral artery remodeling and placental
development suggest a potential role in preeclampsia, and the expression of Rxfp1 is key to
understanding the bioavailability and signaling of relaxin in normal pregnancy and
preeclampsia.
1.6 Action through RXFP1

RXFP1 is the main receptor for relaxin in reproductive tissues (Anand-Ivell et al., 2007),
however the majority of information regarding Rxfp1 expression in human reproductive tissue
is obtained from tissues from the non-pregnanct cycle (Vodstrcil et al., 2007). Rxfp1 is
expressed on myometrial cells, endometrial stromal and epithelial cells, uterine epithelial cells
as well as blood vessel endothelial cells and smooth muscle (Ivell et al., 2007, Heng et al.,
2008). Rxfp1 expression was low in undifferentiated endometrial stromal cells in comparison
with decidual cells in vitro (Mazella et al., 2004). In vivo human chorion and decidua also
express Rxfp1 (Bryant-Greenwood et al., 2005). Relaxin signaling through RXFP1 in
endometrial stromal cells is the critical factor to induce intracellular cyclic adenosine 5’-

11
monophosphate (cAMP), which are necessary for phenotypic changes of early pregnancy and
decidualisation (Tseng et al., 1992, Telgmann and Gellersen, 1998, Bartsch et al., 2004). The
link between relaxin signaling and cAMP supports the traditional G-protein coupled receptor
(GPCR) activation of G8 however induction of tyrosine phosphorylation in human uterine
fibroblasts (Palejwala et al., 2001) and activation of the MAPK pathway in human
endometrial cells (Zhang et al., 2002) suggests a non-traditional GPCR signaling cascade
downstream of RXFP1. Relaxin signaling through RXFP1 also increases in intracellular
cyclic guanine monophosphate (cGMP) levels in human vascular smooth muscle, associated
with the NO pathway for vasodilation discussed previously (Bani et al., 1998).
The molecular signaling mechanisms of relaxin through RXFP1 are still not completely
understood, however it is clear that multiple pathways are involved in relaxin signaling and
are implicated in adaptations to pregnancy. The majority of information regarding the
expression of RXFP1 is limited to animal models in vivo and the understanding of human
expression, limited to in vitro cell cultures, with the exception of the work of Bryant-
Greenwood et al. (2007) on the placenta and fetal membrane. The umbilical cord has not yet
been investigated for RXFP1 expression and little is known about the expression of RXFP1 in
the disease state of preeclampsia. This highlights a gap in the knowledge in regards to where
RXFP1 is expressed in the reproductive tissues of pregnant women, and if there are any
differences in expression in women who experience preeclampsia.
1.7 Aims and Hypotheses

My project aimed to examine RXFP1 expression in tissues of late pregnant normotensive
(NT) and preeclamptic (PE) women, collected during caesarean section. The first aim was to
examine the gene expression of Rxfp1 in Human Umbilical Vein Endothelial Cells
(HUVECs) of normotensive women through Reverse Transcription Polymerase Chain
Reaction (RT-PCR). Further immunohistochemistry studies were conducted to localise
RXFP1 to the umbilical cord of NT women. The secondary aim of this study was to
investigate any changes in Rxfp1 receptor expression in PE women, through quantitative real-
time PCR (qPCR) of placenta and further RT-PCR of PE HUVECs. The second component of
the project comprised of a bioinformatic analysis of microarray data. All in aim to investigate
the hypothesis that Rxfp1 is downregulated in PE compared to NT.

12
2. Materials and Methods
2.1 Patients and Sample Collection
All samples were collected by our collaborators at the Mercy Hospital (Heidelberg,
Melbourne), with the assistance of Professor Stephen Tong. Women gave written consent for
umbilical cord and placental tissue collection. Tissues were collected from women with
preeclampsia and normotensive women during caesarean section delivery of the infant.
Preeclampsia was defined using the American College of Obstetricians and Gynaecologists
(ACOG) guidelines: systolic blood pressure equal to or greater than 140mmHg, or diastolic
blood pressure equal to or greater than 90mmHg after 20 weeks gestation in a women with
previously normal blood pressure; and proteinuria: urinary excretion of equal to or greater
than 0.3g protein in a 24hour urine specimen (ACOG et al., 2002). Placental tissue was
obtained immediately following delivery, washed in sterile phosphate-buffered saline (PBS),
snap frozen and stored at -80°C. RNA was extracted from the frozen placental samples by our
collaborators at the Mercy Hospital, and samples were provided to me as cDNA. Umbilical
cords were immediately washed following delivery with ice-cold sterile PBS. HUVECs
isolation was performed immediately following delivery. Umbilical cords to be analysed by
immunohistochemistry were kept in sterile PBS solution until embedded in paraffin by the
Anatomy Department, Faculty of Medicine, Dentistry and Health Sciences, University of
Melbourne. Human ethics approval was obtained for this study from the Mercy Health
Human Research Ethics Committee.
2.2 Isolation of Human Umbilical Vein Endothelial Cells
HUVECs were isolated by our collaborators at the Mercy Hospital in Melbourne, as
previously described (Ganguly et al., 2012). Collagenase was perfused into the umbilical vein
and incubated for 10 minutes. The cord was gently massaged to loosen endothelial cells from
the lumen of the vein; the solution was then drained into a tube containing endothelial cell
media (ECM). The cord was washed twice with cord buffer to remove any remaining cells,
and solution was drained into the same tube. Cells were then centrifuged for 10 minutes at
350 x g. The supernatant was removed and 10ml of ECM added to the pellet of endothelial
cells to gently resuspend cells. Cells were then transferred to a T75 flask that had been pre-
coated in 0.2% gelatin. The cells were grown at 37°C and 5% CO2. The next day the flask
was gently shaken to dislodge any red blood cells, and supernatant was removed. The cells
were washed with warm Hank’s balanced salt solution (HBSS) and removed. Cells were then

13
fed with 10ml of ECM and examined under the microscope to confirm that red blood cells
were successfully removed, and to check the degree of confluence and morphology. The
media was changed every three days until the cells reached confluency. Cells were then split
using 0.08% solution of trypsin containing 1mM EDTA. Endothelial cells were plated in
dishes pre-coated with 0.02% gelatin (Ganguly et al., 2012).
2.3 First Strand cDNA synthesis
HUVECs RNA was provided by the Stephen Tong group and first strand cDNA synthesis
was performed using 1µg RNA diluted in 11µl of nucleotide free water (NFW), in a 20µl
reaction with 1µl of random hexamers (100ng/µl) (Invitrogen, Lifetechnologies, Carlsbad,
CA, USA), 4µl of 5xRT Buffer with MgCl2 (Invitrogen), 1µl of 0.1M dTT (Invitrogen), 1µl
of RNase inhibitor (Invitrogen) and 1µl of Superscript RT III (Invitrogen). A control solution
was also prepared to perform a negative control, with all components mentioned previously
with the exception of the 1µl of Superscript RT III (Invitrogen), which was replaced with 1µl
of NFW. PCR cycle was performed using a MyCycler thermal cycler (BIO-RAD, Gladesville,
NSW, Australia) and run for 10 minutes at 25°C, 50 minutes at 50°C and then 5 minutes at
85°C and finally held at 4°C.
2.4 Reverse Transcription Polymerase Chain Reaction
The expression of Rxfp1 in HUVECs was examined through RT-PCR of cDNA synthesized
from RNA provided by the Stephen Tong group. A 25µl reaction was prepared with 9.5µl of
NFW, 12.5µl of Green Go Taq (Promega, Annadale, SA, Australia), 1µl of forward and 1µl
of reverse oligonucleotide primers for Rxfp1 and 1µl of cDNA. For the negative control,
NFW was substituted for the 1µl of cDNA, to verify a lack of contamination. The PCR
reaction was performed in a MyCycler thermal cycler (BIO-RAD) for an initial 2 minutes at
85°C, then run for 40 cycles of 1 minute at 94°C, 1 minute at 60°C then 1 minute at 72°C,
after the completion of the cycles, a further 72°C for 10 minutes was completed and the
samples were held at 15°C. The PCR products were then subjected to agarose gel
electrophoresis. A 2% agarose gel was prepared, using 3g of agarose in 150ml of 0.5x Buffer
(TBE), and 3 µl of ethidium bromide. The gel was loaded with samples and a DNA
hyperladder (Bioline, Alexandria, NSW, Australia) and run for approximately 60 minutes on
100V. The gel was then examined and imaged under UV light.

14
2.5 Quantitative Real Time PCR
The comparative cycle threshold method (2-∆CT
) was used to quantify the expression of Rxfp1
and relaxin (Rln) in the placenta, with glyceryde 3-phosphate dehydrogenase (GAPDH) as the
endogenous control. The PCR reactions were carried out in triplicate using 96-well plate in
10µL volumes, consisting of 5µL 2X SensiFast probe Lo-Rox mix (Bioline), forward and
reverse primers and probe (0.5µL primers and 0.25µL probe for Rxfp1 and Rln, and 0.2µL
primers and 0.1µL probe for GAPDH) with PCR water (2.75µL for Rxfp1 and Rln, and 3.5µL
for GAPDH) and 1uL cDNA (1:10 dilution for GAPDH). Three plates were run, one for each
gene for which expression was to be analysed. The PCR reactions were carried out in a ViiA7
Real Time PCR system (Applied Biosystems, Life Technologies, Carlsbad, CA, USA) under
the following conditions: 95°C for 1 minute then 40 cycles of 95°C for 10 seconds and 60°C
for 30 seconds.
2.6 Immunohistochemistry
To examine the localisation of Rxfp1 protein in the umbilical cord by immunohistochemistry,
paraffin-embedded umbilical cord sections (7µm) first underwent a de-waxing procedure.
Slides were immersed in histolene for 10 minutes, and transferred to a second histolene for a
further 10 minutes. To rehydrate, slides were immersed in descending ethanols, each for 5
minutes (two 100%, next 90% and finally 70%). Slides were then transferred to 1X TBS.
Antigen retrieval solution (ARS) was prepared by adding 1.12g of 10mM Tris to 800ml of
distilled water (ddH2O), and 0.19g 0.5mM ethylene glycol tetraacetic acid (ETGA). 1ml of
10N NaOH and 9ml of ddH2O was added to the solution with 0.38g of
ethylenediaminetetraacetic acid (EDTA), pH was adjusted to 9.0 and finally the solution was
topped up with ddH2O to make 1L of ARS. The ARS was brought up to 90-100°C and slides
were immersed for 10 minutes and then allowed to cool at room temperature (RT) for 20
minutes. Slides were rinsed in 1X TBS and endogenous peroxidase was blocked using a
blocking solution (3% H2O2 in 1X TBS prepared by adding 5ml 30% H2O2 to 45ml 1x TBS)
for 60 minutes at RT. Slides were again rinsed in 1X TBS and then incubated in Background
Sniper (Biocare Medical, Concord, CA, USA) for 20 minutes. Primary antibodies were
prepared at 1:500 concentrations in antibody diluent (5ml 10X TBS, 45ml MilliQ water,
25mg BSA (0.05%), and Triton-X 100 (3.75µl in 1ml)). Three different primary antibodies
were used on separate sections for comparison: LGR7 Monoclonal antibody (H00059350-
M01, Abnova, Taipei City, Taiwan), LGR7 735-757 (H-001-53, Phoenix Pharmaceuticals

15
Inc., Belmont, CA, USA) and anti-LGR7 extracellular LDL domain (Sigma-Genosys, The
Woodlands, TX). Mouse IgG (Dako, North Sydney, NSW, Australia) was used as a negative
control serum. Sections were incubated overnight in approximately 75µl of primary antibody
or negative control serum. On the second day, slides were washed in TTBS and incubated
with MACH 4 Mouse Probe (Biocare Medical) for 10 minutes and then incubated with
MACH 4 HRP-Polymer (Biocare Medical) for 20 minutes. Slides were washed in TTBS and
incubated in DAB Chromagen for 2-5 minutes depending which antibody until a brown
colour developed, then washed with distilled water. Finally, slides were DAB enhanced for 1
minute, using DAB Sparkle (Biocare Medical) and washed with MilliQ water. The sections
were then counter stained with hemotoxylin (45 seconds, then rinsed with water and
immersed in ascending ethanols for 2 minutes each: 70%, 95%, and 2x 100%, with a final 2
minutes in histolene) and mounted in Cytoseal, and viewed and imaged under brightfield
microscopy.
2.7 Bio-informatics
A search was conducted using the National Centre for Biotechnology Information (NCBI)
database called GEO Datasets (http://www.ncbi.nlm.nih.gov/geo), keywords utilised in the
search included: ‘preeclampsia’ ‘umbilical cord’ ‘placenta’ ‘RXFP1’ and ‘LGR7’. A total of
ten relevant studies were found in a variety of preeclamptic disease states, all utilising
microarray expression analysis. These datasets were then downloaded, and the specific
expression data for Rxfp1 isolated for statistical analysis.
2.8 Statistical Analysis
Statistical analysis was performed using Graphpad Prism 6 for Mac. Data are expressed as
means ± standard error of the mean (SEM) and were analysed using an unpaired student’s t-
test, assuming non-equal standard deviations (Welch corrected) due to the difference in
variance between samples from preeclamptic women and normotensive women. The
difference in variance was detected through an F-TEST. A Pearson product-moment
correlation test was also performed to assess the linear correlation between expressions of
different genes, with a strong correlation found when r > 0.70 or < -0.70 (positive r = positive
correlation, and negative r = negative correlation). Results were considered statistically
different if p value < 0.05, with statistical confidence level set at 95%.

16
3. Results
3.1 Rxfp1 Gene Expression

HUVECs
I first assessed Rxfp1 expression in HUVECs obtained from the umbilical cords of
normotensive women and women with preeclampsia through RT-PCR. One gene transcript
for Rxfp1 of approximately 450bp was detected in HUVECs obtained from NT women. This
corresponds to the full-length form of Rxfp1 (Siebel et al. 2003, Scott et al. 2005). Treatment
with relaxin for 48hrs resulted in a stronger band, in comparison the 24hrs treatment and no
relaxin treatment (Figure 1A). Although lanes 1 and 3, and 13 and 14 were replicates, in this
reaction there was no PCR product in lanes 1 and 13 whereas the corresponding samples in
lanes 3 and 14 has a PCR product, indicating the presence of Rxfp1. All lanes except negative
control NFW showed a strong positive band for GAPDH at 250bp, therefore negative results
for Rxfp1 gene expression were not due to poor quality cDNA synthesis. Having established
the presence of Rxfp1 in HUVECs from NT women, expression of Rxfp1 and GAPDH as the
endogenous control was then assessed in HUVECs from two PE women compared to a
positive control of HUVECs from a NT woman (Figure 1B). Strong positive PCR bands at
250bp indicated the expression of GAPDH in both PE and NT HUVECs. However a strong
positive band was also present in the reverse transcription negative control in which NFW
was used instead of superscript enzyme during cDNA synthesis. This suggests contamination
of the RNA sample, as the negative controls with NFW showed no band. It is most likely the
negative control was contaminated with superscript enzyme. In addition, no PCR products for
Rxfp1 were detected in any of the PE samples as well as the positive control HUVECs from
NT woman, suggesting that PCR reaction was unsuccessful.
Placenta
As I had a greater number of samples of cDNA from the placenta (n=15 NT, n=15 PE), I was
able to quantify the expression of both Rxfp1 and Rln, using GAPDH as the endogenous
control. GAPDH expression (t = 1.437, df = 27.26, p = 0.1620) was not significantly different
between NT and PE placentas therefore supporting its use as an endogenous control.
Comparing PE and NT women (Figure 2), there was no significant difference in expression of
Rxfp1 (t = 0.9202, df = 27.89, p = 0.3654) or Rln (t = 1.330, df = 23.98, p = 0.1960)

17
comparative to GAPDH. However a Pearson product-moment correlation test between the
expression of Rxfp1 and Rln in NT compared to PE women yielded interesting results (Figure
2D, E). In NT women, there was a positive correlation between expression levels of Rxfp1
and Rln (r = 0.984, p < 0.001) whereas in PE women there was a negative correlation between
expression levels of Rxfp1 and Rln (r = -0.968, p < 0.001). Therefore, while there are no
statistical differences in the expression levels of Rxfp1 and Rln alone, there is a significant
correlation between the expression of the two genes.
3.2 RXFP1 Localisation in the Umbilical Cord
I localised RXFP1 protein in the umbilical cord of obtained from NT women because samples
from PE women were difficult to obtain. Strong positive immunostaining for RXFP1 was
present in the umbilical cord, umbilical vein and Wharton’s jelly (Figure 3A). In comparison
there was no positive staining in the negative control in which mouse IgG replaced the
primary antibody (Figure 3B). A higher power image shows RXFP1 in the endothelial cells,
and smooth muscle of the umbilical vein (Figure 3C) and displays an interesting staining
pattern in the Wharton’s jelly (Figure 3E). The staining of the Wharton’s jelly is absent in the
negative control (Figure 3F) and appears to be non-random and specific to one cell type. The
umbilical artery also showed positive staining, however as the artery lumen collapsed I was
unable to image the artery effectively and therefore was unable to localise RXFP1 expression.
3.3 Bioinformatic Analysis
The bioinformatic search produced 10 studies of relevance, all outlined in Table 1. Eight of
the studies analysed placental tissues. With one sampling the chorionic villus, in which the
expression of Rxfp1 did not significantly differ between NT and PE (Figure 4A). The
remaining studies collected and cultured cells from the umbilical cord, one the fibroblasts and
the other HUVECs and Human Umbilical Artery Endothelial Cells (HUAECs). The
HUVECs and HUAECs were obtained from a healthy NT woman. A significant increase (t =
4.462, df = 6.000, p = 0.0043) in Rxfp1 expression was detected in the artery compared to the
vein (Figure 4B). Finally, the cultured fibroblasts derived from the umbilical cords of NT and
PE women were raised in two oxygen levels (4% and 20%). There was no significant
difference in Rxfp1 expression between NT and PE at the same oxygen level, however Rxfp1
expression was significantly decreased in fibroblasts cultured in 20% oxygen compared to 4%
oxygen, of both NT (t = 8.302, df = 12.51, p < 0.0001) and PE (t = 9.359, df = 20.85, p <
0.0001).

18
Figure 1. Reverse transcription-polymerase chain reaction expression of Rxfp1 and GAPDH
in HUVECs. Amplicon expected size is indicated. A) Expression of Rxfp1 was confirmed in
HUVECs obtained from normotensive (NT) women first. Lanes 11 and 12 were treated with
relaxin (10nM, 24h) as well as lanes 23 and 24 (10nm, 48h). Lanes 1 and 2 (24h) and 13 and
14 (48h) received no treatment. B) Expression of Rxfp1 was then analysed in HUVECS
obtained from preeclamptic women (PE1 and PE2) compared to a positive control (HUVECs
from NT woman (NT1). Negative controls included the cDNA synthesis reaction with NFW
replacing the superscript enzyme (CT1), cDNA synthesis with NFW in place of RNA (CT2)
and NFW in place of cDNA.

19

Figure 2. Expression of Rxfp1, Rln and GAPDH in the placenta of normotensive women (NT)
and women with preeclampsia (PE), measured by quantitative real-time polymerase chain
reaction. A-C) Values are the mean ± standard error of the mean. D-E) Values are the mean of
the triplicate measured. A) GAPDH expression (Ct) level in PE and NT. B) Rxfp1 expression
relative to GAPDH expressed as 2-ΔCT
. C) Rln expression relative to GAPDH expressed as 2-ΔCT
.
D) Positive correlation between levels of Rln and Rxfp1 expression in NT. E) Negative correlation
between levels of Rln and Rxfp1 expression in PE. * p < 0.05 for correlation test.

20

Figure 3. Immunohistochemistry localisation of RXFP1 in the human umbilical cord derived
from a normotensive woman. A) Staining evidence for the presence of RXFP1 in the
umbilical cord. B) A lack of staining indicates the negative control is clean. C) RXFP1
localised to the endothelial cells and smooth muscle. D) Negative controls some slight
background stain in the smooth muscle, but none in the endothelial cells. E) Specific pattern
of staining indicating RXFP1 localisation to a specific cell type in the Wharton’s jelly of the
umbilical cord. F) A lack of staining indicates the negative control is clean. (L = lumen, SM =
smooth muscle, WJ = Wharton’s jelly, EC = endothelial cells, FB = fibroblast).
A" B"
C" D"
E" F"
L" L"
L"L"
SM" SM"
SM" SM"
EC"
EC"
WJ" WJ"
WJ" WJ"
FB"
FB"

21
Table 1. A summary of studies found in the bioinformatics search of the NCBI GEO
database, with the tissue and specific condition investigated. Statistical tests of variance (F
TEST) and difference between means (T TEST) values indicated, and key findings
summarised.
Study
(Accession Number)
Tissue Condition RXFP1 Expression
Key Findings
Keijser R
Afink GB
Ris-Stalpers C
(GSE54618)
Placenta Preeclampsia No significant difference in expression
between NT and PE. A p < 0.05 was
obtained for the F TEST of variance,
therefore NT and PE variances significantly
differ.
TTEST
t = 1.124 df = 14.49 p value = 0.2731
FTEST
f = 6.143 Dfd = 11 p value = 0.0055
Yang P, Ezashi
T, Schust D,
Roberts MR
(GSE54400)
Umbilical
cord
fibroblasts
Preeclampsia No significant difference in expression
between NT and PE at both levels of O2. A
significant difference between levels of O2
for both NT and PE groups.
TTEST comparing NT to PE 4% O2
t = 0.8041 df = 19.59 p value = 0.4310
TTEST comparing NT to PE 20% O2
t = 0.09691 df = 20.45 p value = 0.9237
GSE44711 /
(Blair et al., 2013)
Placenta Early-Onset
Preeclampsia
No significant difference in expression
between NT and PE.
TTEST
t = 1.152 df = 12.52 p = 0.2710
GSE35574 /
(Guo et al., 2013)
Placenta Preeclampsia
and IUGR
between NT and PE.
TTEST
t = 0.2458 df = 30.85 p = 0.8075
GSE30186 / (Tao
Meng et al., 2012)
between NT and PE.
TTEST
t = 0.2950 df = 9.541 p = 0.7743
GSE25906 /
(Shengdar Tsai,
2011)
between NT and PE.
TTEST
t = 0.5897 df = 42.06 p = 0.5586
GSE12767 /
(Founds et al.,
2009)
Placenta
(first trimester
– chorionic
villus
sampling)
Preeclampsia
(pre-
symptomatic)
differ.
TTEST
t = 0.4427 df = 7.742 p = 0.6701
FTEST
f = 36.10, Dfd = 3 p = 0.0136

22
GSE4707 /
(Nishizawa et al.,
2007)
Placenta Preeclampsia
(early and late
onset)
differ.
TTEST (early and late onset grouped as PE)
t = 1.555 df = 10.85 p = 0.1487
FTEST
f = 20.48 Dfd = 3 p = 0.0304
GSE6573 /
(Herse et al.,
2007)
Uteroplacental
– fat, decidua
and placenta
Preeclampsia No significant difference in expression
between NT and PE for all tissue types,
however there was a significant difference in
expression between tissue types, with
significantly increased expression in the
decidua.
TTEST (fat) n=3
t = 1.448 df = 2.115 p = 0.2782
TTEST (decidua) n=3
t = 1.685 df = 3.791 p = 0.1711
TTEST (placenta) n=3
t = 1.628 df = 2.576 p = 0.2167
GSE43475 /
(Aranguren et al.,
2013)
Umbilical
cord arterial
and venous
endothelial
cells
Healthy Significant difference in expression between
artery and vein, with an increase of
expression in the artery.
TTEST
t = 4.462 df = 6.000 p = 0.0043

23

Figure 4. Bioinformatic database search, Rxfp1 expression obtained through microarray
analysis. A) Placental cDNA obtained in pre-symptomatic women through chorionic villus
sampling, and later diagnosed with preeclampsia (PE) and then matched with normotensive
(NT) women for controls. B) Increase in the expression of Rxfp1 in the umbilical artery
compared to the vein of a NT woman * p < 0.05. C) Cultured fibroblasts cells obtained from
umbilical cord from NT and PE women, raised in two levels of O2 (4% and 20%). a: p < 0.05
4% O2 compared to 20% O2 for NT. b: p < 0.05 4% O2 compared to 20% O2 for PE.

24
4. Discussion

The first aim of this study was to demonstrate expression of Rxfp1 in HUVECs obtained from
NT women. Through RT-PCR, I showed Rxfp1 transcripts in HUVECS, which was supported
by the immunolocalisation of RXFP1 to the endothelial cells of the umbilical vein as well as
the bioinformatics analysis. The latter study reported expression of Rxfp1 in HUVECs as well
as HUAECs (Aranguren et al., 2013). The second aim of my study was to compare the
expression of Rxfp1 in tissue obtained from NT women compared to PE women. Although
my RT-PCR data were inconclusive, qPCR analysis showed no significant difference in the
expression of Rxfp1 or Rln in PE placenta compared to NT placenta. This was further
supported by the bioinformatics analysis, with no significant differences in Rxfp1 expression
observed in any study (Tao Meng et al., 2012, Shengdar Tsai, 2011, Nishizawa et al., 2007,
Herse et al., 2007, Guo et al., 2013, Founds et al., 2009, Blair et al., 2013). Finally, the
localisation of RXFP1 to the umbilical cord illustrated expression of RXFP1 in the Wharton’s
jelly, possibly in fibroblasts. This possibility is also supported by the bioinformatics analysis
in which cultured fibroblast cells obtained from human umbilical cord, expressed Rxfp1.
Initially, I demonstrated Rxfp1 expression in HUVECs but in a second RT-PCR experiment in
which I assessed HUVECs from PE and NT women, there were no PCR products. The second
RT-PCR experiment may have lacked PCR products due to an unsuccessful PCR reaction, or
more likely the mRNA expression levels were too low to be detected by the RT-PCR method.
The expression of Rxfp1 and localisation of RXFP1 to the endothelial cells of the umbilical
cord I demonstrated in HUVECs, is supported by actions of relaxin on HUVECs which
include a prolonged increase in ERK 1/2 phosphorylation (Dschietzig et al., 2003) and the
mediation of iNOS expression (Quattrone et al., 2004). Furthermore, Rxfp1 expression has
been demonstrated in endothelial cells of aorta, vena cava, and mesenteric artery (Jelinic et
al., 2014) in which relaxin causes vasodilation ex vivo (Li et al., 2005). The expression of
Rxfp1 in the endothelial cells of the umbilical vein and artery therefore suggest a vasodilation
function for relaxin in the umbilical vessels. The significantly higher expression of Rxfp1 in
the umbilical artery compared to the umbilical vein shown in the bioinformatics analysis,
suggests that the vasodilation function of relaxin is more potent in the artery and therefore
important in maintaining blood flow to the foetus.

25
The localisation of RXFP1 to the umbilical vein demonstrated a darker positive stain in the
endothelial cells in comparison to the smooth muscle, which may suggest a higher expression
of RXFP1 in the endothelial cells compared to the smooth muscle cells, however it is
important to acknowledge the edge effect that can occur in immunohistochemistry techniques
(True, 2008, Werner et al., 2000). Jelinic et al. (2014) reported a higher RXFP1 expression in
endothelial cells of rat aorta, vena cava and mesenteric artery compared to smooth muscle,
while also demonstrating RXFP1 expression to be greater in smooth muscle cells than
endothelial cells in rat femoral artery and vein. As semi-quantitative analysis was not
completed on my immunohistochemistry data, any difference in the expression of RXFP1
between endothelial cells and smooth muscle cannot be quantitatively confirmed. The
vasodilatory physiologically relevant RXFP1 expression location, whether endothelial or
smooth muscle, is still not understood in systemic vessels (Conrad, 2011). It was
hypothesised that relaxin binds to RXFP1 in smooth muscles cells which initiates NO
signaling of endothelial cells to cause vasodilation (McGuane et al., 2011). The differential
expression of RXFP1 in endothelial cells compared to smooth muscle may be physiologically
relevant and represents diverse signaling pathways for relaxin acting in particular vessels.
The hypothesis that Rxfp1 expression would be downregulated in PE women compared to NT
women was not supported by the analysis of placental tissue. My experiment demonstrated no
significant difference in Rxfp1 or Rln expression in the placenta of PE compared to NT, and
this finding was further supported by eight of the bioinformatics studies analysed. It is
important to acknowledge that tissue collection occurred near term and therefore any
differences that may have been present in early pregnancy and play a role in the development
of preeclampsia were not observed. However, in lieu of the difficulties in accessing tissue
from early pregnancy, microarray analysis of chorionic villus samples, also did not show a
significant difference in Rxfp1 expression. This therefore suggests that a decrease in Rxfp1 or
Rln is not a cause or effect of preeclampsia. However, a correlation analysis revealed a strong
positive correlation between Rln and Rxfp1 expression in NT placenta that switches to a
strong negative correlation in PE placenta. Parallels can be drawn between the positive
correlation found in NT placenta, with the stronger bands for Rxfp1 expression present in
HUVECs treated with relaxin for 48hrs compared to 24hrs and no relaxin treatment, therefore
suggesting that an increase in relaxin causes an upregulation of Rxfp1. In isolated decidual
cells, relaxin treatment increases the expression of Rxfp1 (Mazella et al., 2004), therefore
supporting the positive correlation observed in NT placenta. However a negative correlation

26
between Rln and Rxfp1 expression has been shown in the rat myometrium where relaxin
downregulated expression of Rxfp1 (Mercado-Simmen et al., 1982), furthermore in the KO
Rln-/- mouse model, the lack of relaxin results in an upregulation of Rxfp1. In addition, cell
type specific regulation of Rxfp1 may occur, as after 30 weeks gestation there is an increase in
Rxfp1 expression in the basal plate, however no change in expression in the villous
trophoblast (Lowndes et al., 2006). The ability for relaxin to regulate the expression of its
receptor is therefore multifaceted, and the change in correlation of Rln and Rxfp1 expression
between NT and PE placenta, supports a changing mechanism of the ligand-receptor
interaction that regulates expression of Rxfp1. Further, this finding proposes that it is in fact
the ratio of Rln : Rxfp1, which is important in possibly causing a change to the relaxin
signaling in PE, and changes to the absolute values of Rxfp1 and Rln expression do not give
insight into deviations from normal relaxin signaling of NT women.
The immunolocalisation of RXFP1 to the umbilical cord illustrated an interesting positive
staining in the Wharton’s jelly, which appears to be cell-type specific. Comparison of this
staining pattern to histological analyses of Wharton’s jelly shows a similarity with
immunolocalisation of alpha smooth muscle actin that has been shown to be expressed by
myofibroblast cells (Kobayashi et al., 1998). Myofibroblast cells show phenotypic features of
both fibroblasts and smooth muscle cells and produce extracellular matrix (Becker and
Hewitson, 2000). Myofibroblast differentiation into fibroblasts is inhibited by relaxin in vitro
(Samuel et al., 2009, Mookerjee et al., 2009). Furthermore, relaxin has been shown interact
with fibroblast cells, interfering with the synthesis of collagen and other components of the
extracellular matrix (McMurtry et al., 1980), therefore modulating connective tissue
composition. The effect of relaxin on fibroblasts and inhibition of fibrosis is well
characterized in non-reproductive tissues such as the heart, lungs and liver (Unemori et al.,
1996, Samuel et al., 2007, Bennett et al., 2003); less is known about human reproductive
tissues and cells, with exception of the following findings. Cultured human fibroblast cells
derived from lower uterine segment, respond to relaxin by increased MMP-1, MMP-2 and
MMP-3 expression as well as the inhibition of the production of tissue inhibitor of
metalloproteinase-1 (TIMP1), an inhibitor of MMP action (Palejwala et al., 2001). In
addition, relaxin induces direct inhibition of TGFβ1 in vitro, in fibroblasts derived from the
human vagina (Yan Wen et al., 2008). The pathway in which relaxin binding to Rxfp1
achieves inhibition of TGF-β1 in human and rodent renal cells is well understood (Mookerjee

27
et al., 2009). This pathway of TGF-β1 inhibition was also demonstrated in rat ventricular
fibroblasts (Hossain et al., 2011), suggesting that relaxin’s ability to inhibit signaling via the
TGF-β1 / Smad2 axis is species and organ independent (Chow, 2013). Inhibition of TGF-β1
via this pathway is therefore a potential action of relaxin in the umbilical cord, where TGF-β1
expression is seven times higher than in the umbilical artery (Sobolewski et al., 2005).
RXFP1 localisation to the Wharton’s jelly, hypothesised to be specific to fibroblast cells
therefore unlocks a potential new role for relaxin in the umbilical cord that ultimately may
affect the flow of blood to the foetus.
4.1 Future Directions
I would like to repeat the analysis of Rxfp1 expression in HUVECs, utilising qPCR to
quantify and confirm Rxfp1 expression in NT HUVECs, and compare this to PE HUVECs. In
addition, further investigation into the mechanisms under which relaxin regulates the
expression of Rxfp1 is necessary, with the aim of understanding the correlation between Rln
and Rxfp1 expression in the placenta, and the change from positive correlation (NT) to
negative correlation in PE. Finally, I would like to colocalise RXFP1 in the umbilical cord
with a marker for myofibroblasts and fibroblasts to confirm the cell type expressing RXFP1 in
the Wharton’s jelly. This would confirm the ability of relaxin to bind to these cells types and
take us one step closer to understanding the potential role for relaxin in the Wharton’s jelly.
5. Conclusion

The hypothesis that there is a decrease in RXFP1 expression in PE women compared to NT
women was not supported by my results of Rxfp1 expression of HUVECs, placenta and
umbilical cord obtained by varied techniques and bioinformatics. Suggesting that the
inadequate vascular adaptations of preeclampsia are not the result of a lack of receptor for
relaxin signaling in the placenta. The change in correlation between Rxfp1 and Rln expression
from a positive correlation in NT, to a negative correlation in PE raises questions about
continued potential for a change in relaxin signaling in preeclampsia, that is more complex
than a simple downregulation of Rxfp1. In fact, the ratio of Rln to Rxfp1 may be of more
relevance. Furthermore, a potential new functional role for relaxin in the umbilical cord
structure warrants further investigation to confirm Rxfp1 expression in the fibroblasts of
Wharton’s jelly as well as the role of relaxin signaling in these specific cell types.

28
Acknowledgements
I would like to thank the Stephen Tong group for their assistance in tissue collection and their
ongoing collaboration. Without their hard work, this project would not have been possible.
A big thank you to the members of the lab for welcoming me into their space, Chen, Hooi,
Scott, Dhanushke and especially Maria for her support in the immunohistochemistry. A very
special thanks to Sev for her help with the early writing of this thesis and personal contact
with the Stephen Tong group at the Mercy Hospital. To Kelly I extend my appreciation for
her patience in teaching me, without her I would not have any results. Finally, Sarah, thank
you for being so supportive, passing on your wisdom in primer design, escorting me to the
Royal Women’s Hospital to image my slides and for answering my never ending emails and
countless questions.
Most of all, I am grateful to Laura for giving me the opportunity to experience research in an
area I am passionate about, with the added bonus of working in a lab with amazing people;
and for her constant enthusiasm and encouragement.
Finally, thank you to Matthew for his support over the past two years. I, and others,
appreciate the amount of effort Matthew puts into the Master of Biotechnology, it definitely
would not be the same course without him.

29
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