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TitleEffects of hypoxia and hyperglycemia on proliferation andexpression of glucose-related signaling molecules inextravillous trophoblast cell line in vitro

Author(s) Chan, Yuk-ling; 陳玉玲

Citation

Issue Date 2008

URL http://hdl.handle.net/10722/52239

Rights Creative Commons: Attribution 3.0 Hong Kong License

Effects of hypoxia and hyperglycemia on

proliferation and expression of glucose-related

signaling molecules in extravillous

trophoblast cell line in vitro

By

CHAN YUK LING

B.Sc. (Hons) H.K.U.

A thesis submitted in partial fulfillment of the requirements for

the Degree of Master of Philosophy

in Department of Obstetrics and Gynaecology, Faculty of Medicine

The University of Hong Kong

Temporary Binding for Examination Purposes

October, 2008

i

Abstract of thesis entitled

Effects of hypoxia and hyperglycemia on

proliferation and expression of glucose-related

signaling molecules in extravillous


Submitted by

CHAN YUK LING

for the degree of Master of Philosophy

at The University of Hong Kong

in October 2008

ii

Abstract of thesis entitled

Effects of hypoxia and hyperglycemia on proliferation and expression of

glucose-related signaling molecules in extravillous


Submitted by

CHAN YUK LING

For the degree of Master of Philosophy

at The University of Hong Kong

in October 2008

Placenta is an important transient fetal organ that governs material exchange

between the fetus and the mother. These exchange processes are mediated by the

trophoblast cells of the placenta. It is widely accepted that maternal diabetes

significantly alters the expression of molecules regulating glucose transport. Diabetes

is also associated with uteroplacental insufficiency resulting in the hypoxia of the

placental tissue. There is no information in the literature on the response of

trophoblast cells under simultaneous hypoxic and hyperglycemic conditions.

The hypothesis of the project was that high glucose environment as occurred

in diabetic pregnancy disturbed placental glucose transport, and hypoxia as occurred

during uteroplacental insufficiency would augment the effect of hyperglycemia,

resulting in dysfunction of the placenta in the long run. Due to time constraint of the

curriculum, only the effects of hyperglycemia and hypoxia alone or in combination on

the biology of a trophoblast cell line were studied.

iii

In this study, a trophoblast cell line, TEV-1 was used. The cells were treated

with two different concentrations of glucose and oxygen for 8, 24 and 48 hours. The

glucose concentrations used were 5.5 (euglycemia) and 25 mmol/L (hyperglycemia).

The oxygen tensions used were 20% (normoxia) and 1% oxygen (hypoxia). After

treatment, the proliferation, the mRNA and protein expression of vascular endothelial

growth factor (VEGF), glucose transporter-1 (GLUT-1) and insulin signaling

molecules including insulin receptor (IR), insulin receptor substrate-1 (IRS-1) and

IRS-2 were examined. Western Blot analysis and immunofluoresence were used for

protein expression analyses while quantitative polymerase chain reaction was used to

evaluate expression at the mRNA level.

Hyperglycemic treatment did not have significant effect in the proliferation of

TEV-1 cells when compared with cells cultured in physiological concentration of

glucose. On the other hand, hypoxia induced proliferation of the cells. The effect

could be detected after 8 hours of culture. The effects were independent of the

concentration of glucose in the culture medium.

Similar to cell proliferation, VEGF production of the trophoblast cells was not

affected by hyperglycemic treatment. However, hypoxic condition significantly

increased the expression of VEGF mRNA and protein in TEV-1 cells. Hyperglycemia

did not affect hypoxia-induced VEGF expression

Results revealed that the GLUT-1 protein expression was suppressed under

hyperglycemic condition when compared with that in euglycemic condition. An

up-regulation of GLUT-1 mRNA and protein expressions were observed in TEV-1

cells cultured in hypoxic condition after 48 hours of culture. The effect of hypoxia

was dominant over that of hyperglycemia as the kinetics and magnitude of GLUT-1

expression in hypoxic and hyperglycemic conditions are similar to those cultured in

hypoxic condition alone.

iv

The results of the insulin signaling molecules fluctuated within the treatment

period. After 48 hour of culture, hyperglycemia up-regulated IR immunoreactivities

and mRNA expression, while hypoxia down-regulated IRS-2 immunoreactivities and

mRNA expression. Hypoxia and hyperglycemic treatment had no effect on IRS-1

expression.

In conclusion, hyperglycemia and hypoxia had differential effects on

proliferation and gene expression of the TEV-1 cells. The present data suggested that

the two conditions acted on the cell independently.

(497 words)

v

Declaration

I declare that this thesis represents my own work, except where due

acknowledgement is made, and that it has not been previously included in a

thesis, dissertation or report submitted to this university or to any other

institution for a degree, diploma or other quantifications.

Signed…………………………

CHAN Yuk Ling

vi

Acknowledgements

I would wish to express my deepest gratitude to my current supervisors, Prof.

W.S.B. Yeung and Dr. P.C.N. Chiu for their guidance, constant encouragement and

invaluable advice throughout my study. And I would also thank my former

co-supervisor Dr. M.Y. Choy for granting me the opportunity to take part in this

project. I would also appreciate the Department of Obstetrics and Gynaecology for

providing the facilities for my study.

I would like to thank all my colleagues in the Department of Obstetrics and

Gynecology, University of Hong Kong. Especially thanks should be given to Dr.

C.Y.L. Lee, Dr. V.W.S Liu, Dr. K.F. Lee, Dr. W.M. Shek, Mr. K.L. Kwok, Ms. K.M.

Chow and Mr. K.W. Lam for their assistance and guidance. Special

acknowledgements should also be given to my labmates, they are Mr. W.N. Chow,

Mr. M.K. Chung, Mr. C.L. Lee and Mr. Andrew Liu for their continuous assistance

and encouragement. Thanks should also be given to my dearest friends, Ms. Daisy

Chan, Ms. May Leung, Ms. Butterfly Cheung, Ms. Maggie Wong, Ms. Sharon

Kwong for their encouragement. Without their endless support and encouragement, it

is impossible for me to finish this project.

Finally, I would like to express my greatest thanks to my dearest parents and my

sister, Eunice, as well as my beloved companion Steven for their continuous support

and caring.

vii

List of Figures

Figure 1. 12 Distribution of Glut-1 in the placenta

Figure 3.1 Effect of different glucose concentration

on the proliferation of TEV-1

Figure 3.2 Effect of 20% and 1 % oxygen on the proliferation of TEV-1

under euglycemic and hyperglycemic conditions

Figure 3.3 Effect of glucose concentration on VEGF mRNA expression

under normoxic and hypoxic conditions

Figure 3.4 Effect of glucose concentration on VEGF protein expression


Figure 3.5 Immunofluorescent staining of VEGF expression in TEV-1

Figure 3.6 Effect of glucose concentration on Glut-1 mRNA expression


Figure 3.7 Effect of glucose concentration on Glut-1 protein expression


Figure 3.8 Immunofluorescent staining of Glut-1 expression in TEV-1

Figure 3.9 Effect of glucose concentration on IR mRNA expression


Figure 3.10 Effect of glucose concentration on IRS-1 mRNA expression


Figure 3.11 Effect of glucose concentration on IRS-2 mRNA expression


Figure3.12 Effect of glucose concentration on IR protein expression


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Figure 3.13 Effect of glucose concentration on IRS-1 protein expression


Figure 3.14 Effect of glucose concentration on IRS-2 protein expression


Figure 3.15 Immunofluorescent staining of IR expression in TEV-1

Figure 3.16 Immunofluorescent staining of IRS-1 expression in TEV-1

Figure 3.17 Immunofluorescent staining of IRS-2 expression in TEV-1

ix

List of Tables

Table 2.1 Chemicals that were used in the study

Table 2.2 Composition of Ham's F-10

Table 2.3 Primary antibodies used in Western Blot Analysis

Table 2.4 Primary antibodies used in immunofluorescent staining

x

Table of Abbreviation

HCG Human chorionic gonadotrophin

IUGR Intrauterine growth restriction

DNA Deoxyribonucleic acid

VEGF Vascular endothelial growth factor

PIGF Placental growth factor

TNFalpha Tumor necrosis factor alpha

HLA-G Human leukocyte antigen

ROS Reactive oxygen species

HIF Hypoxia-inducible factor

PAS PER-ARNT-SIM

ARNT Aryl hydrocarbon receptor nuclear translocator

mRNA Messenger ribonucleic acid

Epo Erythropiotein

Glut-1 Glucose transporter

PDK-1 pyruvate dehydrogenase kinase-1

HRE Hypoxia-responsive elements

MAPK Mitogen-activated protein kinase

PI3K Phosphatidylinositol 3-kinase

mmHg Millimeter of mercury

ATP Adenosine Triphosphate

HepG2 Human hepatocellular liver carcinoma cell line

kDa Kilodalton

GDM Gestational diabetes mellitus

OGTT Oral glucose tolerance test

FPG Fasting plasma glucose level

xi

mg/dl milligrams/deciliter

g gram

EGF Epidermal growth factor

PDGF platelet-derived growth factor

PKB Protein kinase B

IRS Insulin receptor substrate

PH Pleckstrin homology

PTB Phosphotyrosine-binding

SDS Sodium Dodecyl Sulfate

PDK 3-Phosphoinositide-dependent kinase

IMA Ischemia-modified albumin

HPV Human papillomavirus

ml milliliter oC Degree Celcius

CO2 Carbon dioxide

EDTA Ethylenediaminetetraacetic acid

min Minute

rpm Revolutions per minute

PBS Phosphate buffer saline

DMSO Dimethyl sulfoxide

microliter μl

mM Minimolar

M Molar

UV Ultraviolet

nm nanometer

RNA Ribonucleic acid

DEPC diethypyrocarbonate

xii

μg/μl Microgram per microliter

TBE Tris-borate-EDTA

bp Base pair

RT Reverse Transcription

cDNA Complementary dioxribonucleic acid

dNTP Deoxyribonucleotide triphosphate

MgCl2 Magnesium chloride

Milli-q water MQ

H2O Water

PCR Polymerase chain reaction

HCL Hydrochloride

NaCl Sodium chloride

DOC Sodium deoxycholate

NP Nonyl phenoxylpolyethoxylethanol

BSA Bovine serum albumin

SDS-PAGE SDS Polyacrylamide Gel Electrophoresis

DH2O Distilled water

APS Ammonium peroxodisulphate

TEMED N,N,N',N'-Tetramethylethylenediamine

mA milliampere

PVDF Polyvinylidene Fluoride

V Voltage

PBST Phosphate Buffered Saline solution with Tween 20

ECL Enhanced chemiluminescence

IR Insulin receptor

VEGF Vascular endothelial growth factor

xiii

Contents Abstract iii

Declaration vi

Acknowledgements vii

List of Figures viii

List of Tables x

List of Abberviations xi

Chapter 1 Literature Review 1

1.1 Placental development 1

1.2 Structure of Placenta 2

1.3 Functions of placenta 2

1.4 Nutrients transporation in the placenta 3

1.5 Trophoblast 4

1.6 Extravillous trophoblast cell line 5

1.7 Gaseous exchange in the placenta 6

1.8 Oxygen tension and hypoxic condition 6

1.9 Translocation of HIF-1α 8

1.10 Hypoxic environment in placenta and trophoblast 9

1.11 Glucose transport in placenta 10

1.12 Glucose transporter-1 (GLUT-1) 11

1.13 Abnormal glucose metabolism 14

1.14 Uteroplacental Insufficiency 14

1.15 Gestational diabetes mellitus 15

1.16 Insulin signalling pathway 16

xiv

1.17 Insulin receptor 16

1.18 Cascade of signalling 17

1.19 Insulin receptor substrate-1 (IRS-1) 18

1.20 Insulin receptor substrate-2 (IRS-2) 18

1.21 Roles of IRS-1 and IRS-2 in IR pathway on glucose metabolism 19

1.22 Placental changes in hyperglycaemia and hypoxia 21

1.23 Aims of study 21

1.24 Hypothesis 22

Chapter 2 Materials and Methods 24

2.1 Autoclave (Model ASB930, Astell, U.K) 25

2.2 Laminar Flow Hood 25

2.3 Carbon Dioxide Incubator 25

2.4 Hypoxic chamber 25

2.5 Disposable Plastic ware for media preparation and cell culture 25

2.6 Microscope 26

2.7 Hemacytometer 26

2.8 Chemicals 27

2.9 Immortalized first trimester extracellular trophoblast cell line: TEV-1 28

2.9.1 Source of TEV-1 28

2.9.2 Medium for TEV-1 cell culture 29

2.9.3 TEV-1 cell culture 32

2.10 Determination of TEV-1 viability and proliferation 32

2.11 Reverse transcription and quantitative real time PCR (qPCR) analysis of

mRNA expression

33

xv

2.11.1 Total RNA extraction and absorbance measurement 33

2.11.2 Determination of extracted RNA quality by denaturing

agarose gel electrophoresis

33

2.11.3 Reverse Transcription of RNA 34

2.11.4 Quantitative real time PCR (qPCR) 34

2.12 Immunoblotting analyses of solubilized TEV-1 35

2.12.1 Protein extraction 35

2.12.2 SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE) 35

2.12.3 Immunoblotting analyses of TEV-1 cell lysate 36

2.13 Experimental Protocol 37

2.13.1 The effect of hypoxia on the viability and proliferation of

TEV-1

37

2.13.2 The effect of glucose concentrations and/or hypoxia on

mRNA and protein expression

37

2.13.3 Immunofluorescent staining 38

2.14 Data analysis 39

Chapter 3 Results 40

3.1 Effect of concentration of glucose and oxygen on the viability and

proliferation of TEV-1

41

3.2 Effect of different concentrations of glucose and oxygen on VEGF

expression in TEV-1 cells

45

3.2.1 Expression of VEGF mRNA 45

3.2.2 Expression of VEGF protein 47

3.2.3 Immunofluorescent staining of VEGF 49

xvi

xvii

3.3 Effect of different concentrations of glucose and oxygen on GLUT-1


51

3.3.1 Expression of GLUT-1 mRNA 51

3.3.2 Expression of GLUT-1 protein 54

3.3.3 Immunofluorescent staining of GLUT-1 56

3.4 Effect of different concentrations of glucose and oxygen on the

expression of proteins implicated in insulin signaling in TEV-1 cells

58

3.4.1 Expression of IR, IRS-1 and IRS-2 mRNA 58

3.4.2 Expression of IR, IRS-1 and IRS-2 proteins 63

3.4.3 Immunofluorescent staining of IR, IRS-1 and IRS-2 68

Chapter 4 Discussion 72

Conclusion 90

References 91

Thesis Title: Effects of hypoxia and hyperglycemia on proliferation and expression

of glucose-related signaling molecules in extravillous trophoblast cell line in vitro

Submitted by

CHAN YUK LING

For the degree of Master of Philosophy

1.1 Placental development

Placenta is a specially differentiated organ that develops in pregnancy. It is a

unique organ found only in mammals, and is the only organ composing of cells derived

from two individuals, namely the fetus and the mother (Taylor and others 1997).

Nevertheless, it is regarded as a fetal organ as it is originated from fetal trophoblast cells

(Pardi and Cetin, 2006). Five days after fertilization, the preimplantation embryo

develops into a blastocyst with an outer layer of cells known as trophectoderm (Taylor

and others 1997). The trophectoderm cells attach, transform into trophoblast cells and

begin to invade the endometrium of the uterus on day 6 - 7 after fertilization (Myren et al.,

2007). The trophoblast cells proliferate and differentiate into the placenta.

There are three distinct periods of placental development. The first period (early

pregnancy) starts from the beginning of gestation, during which proliferation and

differentiation of the trophoblasts occur, leading to the formation of villous and

extravillous structure. The extravillous trophoblasts anchor the placenta to the uterus and

remodel the uterine spiral arteries into vessels of low resistance. In the second period, the

trophoblast cells grow and differentiate continuously to become mature. In the final

period, a mass expansion of the placenta at the end of gestation occurs (Desoye and

1

Hauguel-de, 2007). Among these periods, early pregnancy is the most crucial period of

feto-placental growth and placental function in response to maternal metabolism, which

significantly affect fetal development (Ericsson et al., 2007).

1.2 Structure of Placenta

The placenta has two interfaces, the maternal surface and the fetal surface. The

former is dark maroon in color and is divided into cotyledons, while the later is shiny,

gray in color and appears translucent (American Academy of Family Physicians, 1998).

The fetal part of the placenta possesses chorionic villi to increase the surface area of

absorption. It contains a capillary network of umbilical blood vessels and fetal capillaries.

The maternal side is composed of projections from decidua of the uterus. Between these

structures are the intervillous spaces, which are supplied with maternal blood from the

arteries and venule of the uterine wall (Taylor and others 1997). Thus the maternal and

fetal circulations are separated, though material exchanges can occur across the interfaces.

This arrangement prevents the fetus from exposing to the relatively high blood pressure

of the maternal circulation, and avoids mixing of different blood groups of the mother

and fetus (Taylor and others 1997)

1.3 Functions of Placenta

The placenta anchors the embryo in the uterus during early gestation, and

produces hormones such as human chorionic gonadotrophin (HCG) to prevent the

breakdown of the corpus luteum, thereby maintaining the thickness of the endometrium

with ovarian steroids (Taylor and others 1997). The placenta plays a central role in fetal

2

nutrition (Schneider, 1991). It regulates nutrients and oxygen supply to the fetus through

controlling the blood flow in the placenta mediated by secretion of placental hormones,

angiogenic factors and vasodilators, etc. (Cross, 2006). Nutrient supply to the fetus can

be influenced by numerous factors, such as maternal nutrition and metabolism, materno-

fetal concentration gradient, uteroplacental blood flow, placental size and its capabilities

of transfer (Pardi and Cetin, 2006).

1.4 Nutrients transportation in the placenta

Nutrients, respiratory gases and water are transferred from the mother to the fetus

through the placenta (Owens and Fall, 2008;Cleal and Lewis, 2008). The fetal demand of

nutrients increases along with the gestation period. Thus the placenta has to cope with the

increasing fetal demand. There are three major mechanisms for nutrient exchange in the

placenta, including direct transfer of nutrients through the placenta, metabolism and

consumption of nutrients by the placenta itself and conversion of nutrients to alternative

forms for absorption (Hay, Jr., 1994). The fetus may take up the nutrients through passive

diffusion, carrier-mediated facilitated diffusion, active transport and pinocytosis (Myren

et al., 2007). For example, glucose is transported by facilitated diffusion, while protein is

broken down into amino acids which are then carried by specific amino acid transporters

(Hay, Jr., 1994). The transport is affected by nutrient concentration gradients, placental

blood flow and metabolism (Jones et al., 2007). Failure in regulating the transport

systems may affect fetal growth, or together with complications such as diabetes mellitus

(Jansson et al., 2006), result in conditions such as reduced fetal growth in intrauterine

3

growth restriction (IUGR) (Jansson et al., 2003), preeclampsia (Scioscia et al., 2008), or

fetal death (Froen et al., 2004;2008).

1.5 Trophoblast

The trophoblast is the building blocks of the placenta. It possesses some special

properties. For instances, it expresses endogenous retrovirus products, oncofetal proteins

and imprinted genes, and its DNA is comparatively unmethylated (Trundley and Moffett,

2004). There are different sub-types of trophoblast cells serving different roles to ensure

proper placental operation. In the first trimester, placental implantation occurs. It involves

two types of trophoblast cells, namely villous trophoblasts and extra-villous trophoblasts.

(Feng et al., 2005).

The villous trophoblasts consist of the multinucleated syncytiotrophoblast and the

cytotrophoblast. The former is derived from the latter, forms the epithelium of the

chorionic villi for anchoring function (Gude et al., 2004), and interacts with the maternal

blood in the intervillous spaces for nutrient exchange (Evain-Brion, 2001). The

syncytiotrophoblast layer is greatly reduced while the cytotrophoblast layer beomes

discontinuous as pregnancy progresses.

The extra-villous trophoblast cells (EVT) is originated from the cytotrophoblast

stem cells. About Day 14 of pregnancy, some cytotrophoblast cells break through the

syncytiotrophoblast layer and are transformed to the EVT cells (Lunghi et al., 2007a).

These cells are invasive and invade through the deciduas towards the myometrium for

subsequent remodeling of the uterine spiral arteries (Evain-Brion, 2001;Gude et al.,

2004), which involves destruction of the muscular walls of the arteries and replacement

4

of the endothelial cells by the extravillous endovascular trophoblasts. The remodeling

enables blood flow to the placenta in a low-resistance, dilated and compliant way

(Gude et al., 2004;Trundley and Moffett, 2004). The degree of invasion is decisive in

pregnancy success. Either too invasive or inadequate invasion is associated with

complications such as pre-eclampsia, intrauterine growth retardation or uterine rupture

and hemorrhage (Trundley and Moffett, 2004). Oxygen tension is one of the

indispensable factors activating the invasive potential of trophoblasts (Caniggia et al.,

2000b;James et al., 2006).

1.6 Extravillous trophoblast cell line

Since trophoblast cells are directly involved in placental function, they are always

the focus of study in placentation. However, first trimester trophoblast tissue has limited

availability. The limited life span of trophoblast in culture and contamination with other

cell types of the placenta/decidua are other obstacles for research studies using primary

placental tissue culture. Therefore, Choy and coworkers (Choy et al., 2000), established

the first trimester extravillous trophoblast cell line (TEV-1) from placental tissue

obtained from legal abortion of normal first trimester pregnancies. The placental cells

were cultured and retroviral infected by human papilloma virus E6 and E7. TEV-1

retains the characteristics of extravillous trophoblasts, including possession of invasive

potential and responsiveness to TGF-β1 treatment, a trophoblast invasion, migration and

proliferation regulator (Feng et al., 2005). This cell line was used in this research project

because of it was derived from normal pregnancy and was available in the laboratory.

5

Nowadays, there are several first trimester human trophoblast cell lines, e.g.

BeWo, JAR and JEG3. However, they are derived from choriocarcinoma exhibiting

cancerous phenotype. They differ from primary trophoblast in the expression level of

HLA-G and hCG (Kawata et al., 1984;Mochizuki et al., 1998). The others

cytotrophoblast cell lines, such as NPC, L10, are either not yet well developed nor not

well characterized in terms of trophoblastic markers (Hiden et al., 2007;Rong-Hao et al.,

1996;Choy and Manyonda, 1998).

1.7 Gaseous Exchange in the placenta

Aerobic respiration of the fetus depends completely on the maternal oxygen

supply through the placenta. The availability of oxygen, not only supports the essential

metabolic activities but also acts as also a source of reactive oxygen species (ROS)

affecting activity of many genes, like transcription factors AP-1 and Smad (Wagner,

2008;Wu, 2006). The transfer of oxygen from the maternal blood to the fetus in the

placenta is mostly by simple diffusion. Thus, the maintenance of a lower oxygen

concentration in the fetal blood within the villous capillaries than the maternal blood in

the intervillous space is important to keep the concentration gradient necessary for the

unidirectional flow of oxygen towards the fetal side. The placenta itself consumes about

10%-30% of oxygen passing through it (Fox and Sebire, 2007).

1.8 Oxygen tension and Hypoxic condition

Hypoxia refers to a condition of insufficient oxygen supply to support metabolism.

In human, hypoxia occurs when vascular supply is interrupted, or when a tumor offshoots

6

its vascular supply (Krohn et al., 2008). Exposure to hypoxia is a normal part of fetal life,

and the response of the fetus towards hypoxia is important for its development (Ream et

al., 2008). These responses may be acute or chronic in nature that may last from several

seconds to minutes, or even hours (Semenza, 1999). Longer responses such as changes in

gene expressions regulating process like erythropoiesis, angiogenesis and glucose

metabolism are important in the maintenance of cell survival (De Marco and Caniggia,

2002). However, exposure to altered oxygen tension may be life-threatening (Windham

et al., 1992), or lead to fetal abnormalities and postnatal deficits such as pulmonary

oedema in lung (Weissmann et al., 2006) and cell apoptosis (Antonova et al.,

2007;Heazell et al., 2008).

Hypoxia induces adaptive cellular changes mediated by pathways involving

members of hypoxia-inducible factor (HIF) family of transcription factors (Breier et al.,

2007). They belong to the subfamily of basic helix-loop-helix transcription factors with a

PAS (PER-ARNT-SIM) motif (Wang et al., 1995). There are three members in the HIF

family, namely, HIF-1, HIF-2 and HIF-3. They differ from each other by having three

different HIF-α subunits (HIF-1α, HIF-2α and HIF-3α) (Licht et al., 2006;Wang et al.,

1995;Ema et al., 1997a;Flamme et al., 1997;Tian et al., 1997;Gu et al., 1998;Makino et

al., 2001) and share a common HIF-1β subunit (Licht et al., 2006) giving rise to three

different heterodimers. All α subunits were encoded by distinct gene loci and using

alternative promoters and splicing pattern. The domain architecture of HIF-1α and HIF-

2α are similar, but the latter has more limited tissue expression, while HIF-3α was least

studied (Weidemann and Johnson, 2008). HIF-3α lacks structures for transactivation in

the C-termini as other HIF-α isoforms, supporting a role of HIF-3α in forming inactive

7

heterodimers with HIF-1α to inhibit HIF response (Weidemann and Johnson,

2008;Nangaku et al., 2008). While HIF-1β is constitutively expressed (Salceda and Caro,

1997), the expression of HIF-1α subunits is regulated at the translation level and by

protein degradation (Salceda et al., 1996); the proteins are rapidly degraded under

normoxic condition via ubiquitin-proteasome system, though their half life is prolonged

in hypoxia (Huang et al., 1996). Thus, the formation of the HIF-1 complex is highly

dependent on the abundance of the HIF-1α subunits (Salceda and Caro, 1997).

Hypoxia increases cellular concentration of HIF-1 (Nangaku and Eckardt, 2007),

which and in turn induces the expression of a number of genes related to glucose

metabolism, glycolysis, erythropoiesis, catecholamine metabolism and angiogenesis

(Ema et al., 1997b;Semenza, 2000b;Weidemann and Johnson, 2008;Shih and Claffey,

1998). In general, there are 3 classes of hypoxia-induced genes: 1) those involved in

metabolic adaption; such as glucose transporters; 2) those ensure cell survival during

hypoxia, for example, the vascular endothelial growth factor (VEGF); and 3) those favor

adaption of the whole organism towards hypoxia, like erythropiotein (Epo) (Shih and

Claffey, 1998). Among the glucose transporters, Glucose transporters 1 and 3 are induced

by hypoxia. Other enzymes involved in regulating glucose metabolism, such as

cytochrome c oxidase and pyruvate dehydrogenase kinase-1 (PDK-1) are also induced in

hypoxia (Weidemann and Johnson, 2008).

1.9 Translocation of HIF-1α

HIF-1α heterodimer interacts with a specific DNA sequence 5’ – TACGTGCT-3’,

and the half-life of the interaction is about 1 minute (Wang and Semenza, 1993). In

8

hypoxic condition, HIF-1α is stabilized and translocated from the cytosol into the

nucleus due to the presence of a nuclear localization signal at its C-terminus. In the

nucleus, HIF-1α dimerizes with HIF-1β, recrutining the coactivators p300/CBP, and

induces expression of its transcriptional targets such as Glut-1, Glut-3, VEGF and Epo

genes via binding to hypoxia-responsive elements (HREs) (Kaluz et al., 2008;Srinivas et

al., 2001). Signaling pathways such as the p38 mitogen-activated protein kinase, p42/p44

extracellular signal–regulated kinase 1 and 2, and phosphatidylinositol 3-kinase (PI3K)

pathways are responsible for the activation of the downstream molecules induced by HIF-

1α (Wenger et al., 2005).

1.10 Hypoxic Environment in placenta and trophoblast

Oxygen is an important regulator of placentation. Relative to the maternal side,

the fetal side is always in hypoxia condition in mammals (Ream et al., 2008). Low

oxygen environment is crucial to the development of placenta in early pregnancy by

preventing trophoblast from differentiation into the invasive phenotype before 10 -12

weeks of gestation (Caniggia et al., 2000a). Greater oxygen tension would direct

differentiation of cytotrophoblast into villous trophoblast instead of extravillous

trophoblasts (Robins et al., 2007). Only about 11% of oxygen is present in the rat

placenta between days 6.5 and 13.5 of gestation (Rosario et al., 2008). The arterial

oxygen partial pressure in normal human female is about 80-100 mmHg. The highest

partial pressure of oxygen in the fetus is only about 22-32 mmHg in late pregnancy

(Ream et al., 2008). There is continuous remodeling of blood vessels in the placenta

resulting in the increase in oxygen tension from about 10 weeks to 24 weeks (Kingdom

9

and Kaufmann, 1999;Caniggia et al., 2000b).

In addition to its leading role of trophoblast differentiation, glucose metabolism is

also altered by hypoxia (Baumann et al., 2007). Hypoxia altered the expression of several

glucose transporters have expression in hypoxia (Ebert et al., 1995). Cellular glucose

uptake mediated by glucose transporters depends on the glucose concentration gradient

(Shih and Claffey, 1998). The increased glucose transporter expression and glucose

consumption rate (Bashan et al., 1992) enable the cells to maintain a level of ATP by

anaerobic respiration sufficient for survival and proliferation in hypoxic stress (Brahimi-

Horn and Pouyssegur, 2007a). This adaption to hypoxia results in accumulation of lactic

acid in the cells. (Brahimi-Horn and Pouyssegur, 2007a).

1.11 Glucose Transport in Placenta

Glucose is one of the most important nutrients transported across the placenta,

and is the primary substrate for metabolism of the fetus and the placenta. Maternal

glucose concentration is always higher than that in the fetal plasma. The requirement of

glucose increases with the development of the fetus (Morriss and Boyd 1988;Battaglia

and Meschia 1986). Glucose is transport across the placenta by transporter proteins by

facilitative diffusion (Hay, Jr., 1995;Johnson and Smith, 1980;Johnson and Smith,

1985;Rice et al., 1976;Rice et al., 1979). The facilitative glucose transporter family

(GLUT) consists of thirteen members (Zhao and Keating, 2007;Wood and Trayhurn,

2003). Each of them has its distinctive localization, tissue-specific and kinetic

characteristics (Mueckler, 1994;McGowan et al., 1995).

Facilitated diffusion transport systems are also called passive carriers, which

10

transport substrate down a concentration gradient without energy consumption. It works

most efficiently when the concentration of the substrate is moderate. The primary

function of these carriers is to mediate exchange of glucose between the blood and the

cellular cytoplasm. As the transporters carry substrate passively down a concentration

gradient, they can promote both uptake of glucose into or efflux of glucose out of the

cells. Each member of the transporter family is tissue specific and usually two or more

different family members exist in a single cell type (Mueckler, 1994). The family

members can be divided into three classes that share common sequence motifs. (Joost and

Thorens, 2001). Class 1 includes glucose transporters Glut-1-4, class 2 includes GLUT5,

GLUT7, GLUT9 and GLUT11 and Class 3 is composed of GLUT6, 8, 10, 12 and

HMIT1 (Joost and Thorens, 2001). The 3 classes exhibit different substrate specificities,

with class one mainly transports glucose, class two mainly transports fructose and

members of class 3 transport various kinds of hexoses (Manolescu et al., 2007).

1.12 Glucose Transporter-1 (Glut-1)

Glut-1 to Glut-4 belong to class I facilitative glucose transporters which have

similar function, structure and tissue distribution (Wood and Trayhurn, 2003). Glut-1 is

the first transporter to be isolated and cloned from a HepG2 cell line. Thus it is also

called the erythrocyte, brain or HepG2 transporter (Mueckler, 1994;Mueckler et al.,

1985). Glut-1 is widely expressed at low level in most tissue for constitutive glucose

transport. It has been localized to the brain, muscle, adipose tissue and endothelium

(Mueckler, 1994), and is the chief transporters responsible for glucose transport across

the placenta (Hay, Jr., 1994;Illsley, 2000;Takata et al., 1990). In fact, its mRNAs are

11

detectable in early mouse embryos and oocytes (Hogan et al., 1991;Aghayan et al., 1992).

Glut-1 serves as the basal glucose transporter isoform for cellular metabolism and

glucose transport (Illsley, 2000). Compared the sequence between human Glut-1, 2, 3, 4

and 5, 39-65% of their sequences are identical and 50-76% of the sequences are similar,

demonstrating that they evolutionary related. (Bell et al., 1990). Glut-1 is the most

conservative isoform among different animal species. Its protein sequence among rat,

mouse, rabbit, pig and human share a homology of 87-98% (Mueckler, 1994).

Eight facilitated glucose transporters have been found in the human placenta.

These molecules transport glucose bidirectionally with the direction of transport

determined by the transmembrane glucose gradient (Bhatia edited. 2005). GLUT-1 is the

only placental glucose transporter that can be found on both the maternal- and fetal-

facing surfaces of the syncytiotrophoblast (Barros et al., 1995), with relatively higher

abundance of the transporter on the side of microvilli than that of basal membrane. Such

asymmetric distribution of Glut-1 governs the rate of glucose transport across the

placenta (Refer to Fig. 1), and alteration of Glut-1 expression on the basal membrane

have great impact on glucose transfer and uptake to the fetus (Baumann et al., 2002).

Excess expression of Glut-1 on the basal membrane may lead to surplus influx of glucose

into the placenta and to the fetus.

12

Fig. 1 Distribution of Glut-1 in the human placenta, showing that there is an asymmetry

localization of Glut-1, with a higher abundance on microvilli side. Originated from IIIsey,

Glucose Transporters in the human placenta, Placenta, 2000, 21, 17p. Modified on 20th Aug 2008

Several treatments affect the expression of Glut-1 in cells. These include phorbol

ester (Hiraki et al., 1988), hypoxia (Loike et al., 1992), insulin (Tordjman et al., 1989)

and glucose (Tordjman et al., 1990;Walker et al., 1988;Wertheimer et al., 1991) treatment.

Some of these treatments exert effects on Glut-1 at the basal membrane only, but not

those on the microvillus side of the membrane (Baumann et al., 2002), indicating a

complicated mechanism of regulation of Glut-1 expression.

Another transporters, the facilitated glucose transporters 3 (Glut-3) was also

found to be expressed in the placenta (Sciullo et al., 1997). It has a molecular size of

49kDa (Hauguel-de et al., 1997). Compared with Glut-1 in the placenta, Glut-3 is less

abundant and is expressed in a different localization (Jansson et al., 1995); it is found in

the villous tissue and the vascular endothelium (Hauguel-de et al., 1997). Its expression

13

also decreases over gestation period (Sakata et al., 1995) . Glut-3 together with Glut-1

also can be found in maternal leukocytes and platelets (Korgun et al., 2002;Korgun et al.,

2005), suggesting their roles in providing energy for immunological tolerance in the

deciduas (Korgun et al., 2005).

1.13 Abnormal Glucose Metabolism

Inadequate supply of oxygen to the placenta and the fetus would cause maternal

and neonatal complications, such as preeclampsia. Failure of glucose uptake in the

placenta and therefore abnormal glucose transport to the fetus would have adverse effects

on pregnancy. Such impaired or imbalanced glucose transport may be resulted from

increased or decreased expression of placental glucose transporters that may lead to

clinical complications such as fetal intrauterine growth restriction (IUGR) and gestational

diabetes (Hay, 2006;Sun and Yang, 2007;Yang et al., 2005;Lesage et al., 2002).

1.14 Uteroplacental Insufficiency

Uteroplacental insufficiency means insufficient blood flow to the placenta during

pregnancy. And it is regarded as one of the underlying causes of intrauterine growth

restriction (IUGR) (Hendrix and Berghella, 2008). This defect at the uteroplacental

interface can be caused by inadequate vascular adaptation leading to increased uterine

vascular resistance, reduced capacitance and total blood flow to the placenta that result in

placental insufficiency at (Henriksen and Clausen, 2002;Salafia, 1997). Uteroplacental

insufficiency occur in normal condition like twinning, or abnormal conditions such as

reduced placental size, chronic maternal hypoxia, or uterine ischemia (DeSesso, 1987).

14

Under these conditions, the intrauterine environment is altered, and the fetus would

experience stresses like acidosis and hypoxia (Economides et al., 1991;O'Brien et al.,

2007). The outcome of uteroplacental insufficiency includes fetal malnutrition and hence

low birth weight (Henriksen and Clausen, 2002). Previous researches had reported that

uteroplacental blood velocity was reduced in diabetic rats, suggesting a possible link

between diabetes and IUGR (Chartrel et al., 1990). Thus it is interesting to see whether

maternal hyperglycaemic environment would further affect the nutrient supply to the

IUGR fetus.

1.15 Gestational diabetes mellitus

Gestational diabetes mellitus (GDM) is defined as glucose intolerance with its

onset or first recognition during pregnancy (Magee et al., 1993). It is a common

pregnancy complication worldwide and occurs in about 5-7% of all pregnancies in the

States affecting about 200,000 pregnancies annually (Engelgau et al., 1995). For Asia or

even Africa, its prevalence is even higher when compared with western countries (Holan

et al., 2008). During pregnancy, glucose intolerance, if any, can be assessed by carrying

out oral glucose tolerance test (OGTT) during 24-28 weeks of gestation (Kautzky-Willer

and Bancher-Todesca, 2003). GDM is diagnosed when the fasting plasma glucose level

(FPG) is over 126 mg/dl (Tominaga, 1999), or when the blood glucose level is over

155mg/dl after a 2-hour 75 g glucose tolerance test. Both genetic and environment

factors contribute to the development of GDM, (Shaat and Groop, 2007), These factors

include obesity, maternal age over 35, family history of diabetes and smoking (Dye et al.,

1997;Ross, 2006;England et al., 2004). GDM have adverse effect on both the mother and

15

the fetus, inducing pregnancy complications such as increased rate of cesarean delivery

(Sermer et al., 1995), preeclampsia, preterm birth, macrosomia and hypoglycemia,

diabetes and obesity in long term. (Elchalal, 2004;Schaefer-Graf and Kleinwechter, 2006).

Insulin Resistance

1.16 Insulin signaling pathway

Insulin signaling pathway is a highly conserved pathway in many organisms

regulating nutrient availability. The pathway influence glucose uptake and utilization,

which in turn may affect cellular metabolism and growth. Insulin is a hormone produced

by the pancreatic β cells. In the target tissues, it binds to its specific cell-surface receptor

and trigger cascade of down-streaming signaling pathway regulating gene expression

(DeBosch and Muslin, 2008).

1.17 Insulin receptor

Insulin receptor is present in all vertebrate and has variable expression level in

different tissues, promoting uptake of glucose in insulin responsive tissues such as

adipocytes and hepatocyes. In humans, insulin receptor gene is located on chromosome

19 with 22 exons. It consists of two α-subunits, each of which is linked to a β-subunit by

disulfide bonds. The α-subunits are located outside the cell and bind to insulin, while the

β-subunits are located inside the cells and possess intrinsic protein tyrosine kinase

activity as the receptor of epidermal growth factor (EGF) and platelet-derived growth

factor (PDGF). The insulin receptor-complex is internalized by endocytosis for

degradation. This attenuates the insulin signal and serves as an auto-regulation in

16

response to prolonged insulin stimulation (White and Kahn, 1994).

1.18 Cascade of Signalling

After binding to the α-subunits, insulin activates the tyrosine kinase activity of insulin

receptor β-subunits, and phosphorylates the adjacent β-subunit of the dimer (Nelson and

Cox, 2000)(Ward et al., 2008). The activated receptor then phosphorylates the insulin

receptor substrates (IRS) which serve as docking proteins for cellular kinases to activate

various signaling pathways, including the Grb2-Sos-Ras-MAPK pathway and the PI3K-

PKB pathway (Nelson and Cox,2000). The translocation of Glut-4 to the plasma

membrane surface is regulated by activating the PI3K pathway through IRS-1 (Cheatham

et al., 1994;Kotani et al., 1995;Okada et al., 1994).

At least four insulin receptor substrates have been discovered (IRS-1 to IRS-4).

IRS-1 and IRS-2 are the major insulin receptor substrates for regulating glucose

homeostasis. The IRS-1 and IRS-2 have similar structure but show distinct and

differential roles in various organs. They have no intrinsic catalytic activity but with

several domains that can interact with the receptor and downstream molecules. IRS-1 and

IRS-2 have two conserved domains, the amino-terminal pleckstrin homology (PH)

domain and the adjacent phosphotyrosine-binding (PTB) domain; the latter domain

interacts with insulin receptor. The carboxy-terminal domain encodes numerous tyrosine

and serine residues for phosphorylation (Sun et al., 1991;Sun et al., 1995;White, 2002).

There are more than 20 tyrosine phosphorylation residues for the IRS proteins that serve

as docking sites for downstream signaling molecules (Thirone et al., 2006).

17

1.19 Insulin Receptor Substrate -1 (IRS-1)

IRS-1 is a single exon gene located in human chromosome 2 (Araki et al., 1993).

It is present in muscle, heart, liver, adipocytes, and kidney (White et al., 1985). Its

predicted molecular size is 131 kDa. However, its size in SDS-polyacrylamide gel is 185

kDa probably due to high level of phosphorylation (Thirone et al., 2006;White, 1997).

The PH domain in different species is highly specific for IRS-1 and is important for

insulin-mediated phosphorylation (Thirone et al., 2006). Other than the insulin receptor,

it has been reported that some other activators such as interleukin-4 receptor also uses

IRS-1 as an intermediate for signal transduction (Wang et al., 1993). IRS-1 is believed to

play an important role in insulin-stimulated glucose transport (Okada et al., 1994). Since

IRS-1 was the major phosphorylated IRS-protein found in adipocytes upon insulin

stimulation , it may be the chief isoform responsible for transmitting metabolic signals

(Rosen et al., 1978). 1.20 Insulin Receptor Substrate-2 (IRS-2)

es, including fibroblasts, liver, lung, spleen,

heart, k

IRS-2 is expressed in various cell typ

idney, skeletal muscles and brain (Sun et al., 1997;Thirone et al., 2006). It is

located on human chromosome 13 and is about 10kDa larger than IRS-1(White, 1998).

There is 75% amino acid sequence homology at the amino termini of IRS-1 and IRS-2,

while the carboxy-termini shows only 35% homology (Thirone et al., 2006).

IRS-2 null mice have reduced pancreatic cell mass and therefore fail to

compen r

-1 is needed for proper insulin

produc

sate insulin resistance by islet hype plasia.

IRS-1 and IRS-2 have differential roles. IRS

tion in beta-cells, while IRS-2 is involved in the regulation of beta-cell mass; the

18

total functional beta-cell mass determines the amount of insulin to be produced (Niessen,

2006). Disruption of IRS-1 in mice leads to development of mild insulin resistance and

increase in insulin secretion for compensation. On the contrary, IRS-2 null mice have

reduced pancreatic cell mass, and therefore fail to compensate insulin resistance by

islet hyperplasia, resulting in progressive development of diabetes (Withers et al., 1998).

Upon interaction with the IR, IRS-2 uses the PTB domain as for IRS-1, as well as a

central domain located between amino acids 591 and 738, which was absent in IRS-1, to

tyrosine phosphorylate its downstream target. Such difference may contribute to the

variation the duration of tyrosine phosphorylation between IRS-1 and IRS-2: IRS-1

tyrosine phosphorylation is sustained whereas phosphorylation of IRS-2 is transient

(Valverde et al., 1998) . In addition, IRS-2 has relatively weaker interaction with Grb2

than IRS-1 in activating the ras-MAPK pathway. These differences imply the signaling

specificity of the two IRSs in metabolic regulation.

1.21 Roles of IRS-1 and IRS-2 in IR pathway on glucose metabolism

r expressed in

muscle

Glut-4 is the predominant insulin-mediated glucose transporte

and fat tissues, where dietary glucose uptake mainly occurs (Thirone et al., 2006).

Insulin induces translocation of Glut-4 from intracellular storage sites to the plasma

membrane (Marette et al., 1992;Satoh et al., 1993). The primary adipocytes of IRS-1

knockout mice have decreased glucose transport and impaired GLUT-4 translocation to

the plasma membrane in response to insulin (Tamemoto et al., 1994). IRS-1 null mice

have marked defects in insulin content and in insulin secretory response to glucose

(Kulkarni et al., 1999).

19

On the other hand, insulin-stimulated glucose uptake in isolated muscle of IRS-2

null m

the pathophysiology and

clinical

s hormones such as estrogen,

progest

ice is not different from that of the wildtype mice (Baumann et al., 2007),

suggesting IRS-2 stimulation may not be essential in stimulating glucose transport

(Thirone et al., 2006). IRS-2 null mice show no suppression of hepatic glucose

production. This together with reduced hepatic glycogen synthesis and stores and

dysregulation of lipid metabolism suggest a role of IRS-2 in regulating glucose

metabolism rather than glucose transport (Previs et al., 2000).

GDM resembles diabetes mellitus type 2 in terms of

signs (Kautzky-Willer and Bancher-Todesca, 2003). During pregnancy, glucose

tolerance deteriorates, which will usually restores to normal level after delivery (Kuhl et

al., 1985) and only 10% < would develop into GDM (Kuhl, 1991). Pathogenesis leading

to the impaired glucose tolerance is associated with decreasing insulin sensitivity in

which the production by pancreatic beta cells are no longer able to compensate for the

increased insulin resistance. (Shaat and Groop, 2007),

In normal pregnancy, the levels of variou

erone, prolactin, cortisol, human chorionic gonadotrophin, placental growth

hormone and human placental lactogen increase. The fuel utilization is also increased for

the development of the fetus (Barbour, 2003). With increased in insulin resistance that

prohibits insulin-mediated glucose uptake by peripheral tissues of the mother in later

gestational stage, more glucose is available to the fetus (Kernan et al., 2002;Lain and

Catalano, 2007). The change in insulin resistance is apparently due to a combination of

increased maternal adiposity and the insulin-desensitizing effects of placental hormones

(Buchanan and Xiang, 2005). On the other hand, the insulin level rises to compensate for

20

the insulin resistance, enable blood glucose level to be under control (Sivan and Boden,

2003). The beta-cell function starts to decline with prolonged increase in insulin

production (Gastaldelli et al., 2004). Patients with type 2 diabetes and gestational

diabetes have reduction in insulin sensitivity and beta cells function (Kahn, 2003). If

insulin resistance progresses, suppression of hepatic glucose production and lipolysis

ceases due to down-regulation and impaired function of IRS proteins. (Bajaj and

Defronzo, 2003;Catalano et al., 2002), causing impaired glucose tolerance, and

eventually hyperglycaemia and gestational diabetes or type 2 (Vannini, 1994).

1.22 Placental changes in hyperglycaemia and hypoxia

ured human undifferentiated

placent

.23 Aims of study

In vitro study showed that proliferation of cult

al cells from placenta was decreased in the presence of high glucose, suggesting

that diabetes may lead to abnormal development of the placenta (Nelson and Curran,

1989). Both high glucose and hypoxia alter the expression of glucose transporters in

trophoblasts respectively (Baumann et al., 2007;Hahn et al., 1998).

1

ccepted that maternal diabetes significantly alters the expression of

molecu

It is widely a

les regulating glucose transport. Apart from hyperglycemia, other associated

changes such as higher arterial blood pressure, heart rate, serum triglycerides, insulin and

increased insulin resistance also occur in maternal diabetes (Szymanska et al., 2008).

These changes together with hyperglyceimia may affect other metabolic process in the

body. For example, recent researches have shown that ischemia-modified albumin (IMA)

21

molecules are modified in diabetic patients and in chronic hypoxic conditions (Piwowar

et al., 2008). Diabetes is associated with uteroplacental insufficiency. This situation could

be worsen when the mother has previous surgical uterine artery ligation for management

of uterine fibroids or myomas, possibly causing reduced oxygen and nutrient transport

across the placenta (O'Dowd et al., 2008) as well as ischemic hypoxia in vivo (Das et al.,

1998). In sheep and rat, hyperglycaemia alone alters the expression of glucose

transporters in placenta, and hypoxia will modify the effect of hyperglycaemia (Das et al.,

1998). Hyperglycaemia was found to first increase in placental Glut-1 and decline in

Glut-1 concentration gradually in 48 hours, whereas hypoxia up-regulates Glut-1

expression (Das et al., 1998). Thus, ischemic hypoxia in the placenta due to placental

insufficiency or uterine ligation with a maternal hyperglycaemia condition would

probably occur and alter the function of the glucose transporters and hence the placental

functions. Such alternations may lead to the potential development of type 2 diabetes of

fetus in later life, just like those IUGR rats (Simmons et al., 2001). Information of these

conditions in human are hardly found at this moment. The aim of the project is to

investigate whether each of these events, hyperglycaemia and hypoxia, or their

combination, would have a differential effect on glucose metabolism in the human

placenta.

1.24 Hypothesis

f the project was that high glucose up-regulated the expression of The hypothesis o

glucose transporters in the human placenta, leading to increase in glucose uptake to the

fetus through the placenta. Hypoxia, as one of the factors perturbing the

22

microenvironment of placenta, would augment the effect of hyperglycaemia, resulting in

insulin resistance and dysfunction of the placenta in the long run. In this project, the

expression profiles of insulin signaling molecules of the placenta were also examined, in

order to have a better understanding of their roles in glucose homeostasis in the placenta.

23

Chapter 2. Materials and Methods

2.1 Autoclave (Model ASB930, Astell, U.K)

2.2 Laminar Flow Hood

2.3 Carbon Dioxide Incubator

2.4 Hypoxic chamber

2.5 Disposable Plastic ware for media preparation and cell culture

2.6 Microscope

2.7 Hemacytometer

2.8 Chemicals

2.9 Immortalized first trimester extracellular trophoblast cell line: TEV-1

2.9.1 Source of TEV-1

2.9.2 Medium for TEV-1 cell culture

2.9.3 TEV-1 cell culture

2.10 Determination of TEV-1 viability and proliferation


mRNA expression

2.11.1 Total RNA extraction and absorbance measurement

2.11.2 Determination of extracted RNA quality by denaturing agarose gel

electrophoresis

2.11.3 Reverse Transcription of RNA

2.11.4 Quantitative real time PCR (qPCR)

2.12 Immunoblotting analyses of solubilized TEV-1

2.12.1 Protein extraction

2.12.2 SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)

2.12.3 Immunoblotting analyses of TEV-1 cell lysate

2.13 Experimental Protocol

2.13.1 The effect of hypoxia on the viability and proliferation of TEV-1

2.13.2 The effect of glucose concentrations and/or hypoxia on mRNA and protein

expression

2.13.3 Immunofluorescent staining

2.14 Data analysis

24

2.1 Autoclave (Model ASB930, Astell, U.K)

An autoclave (Model ASB930, Astell, U.K) was used to sterilize all the glassware

and pipette tips under 1kg/cm2 pressure at 121°C for 20 minutes.

2.2 Laminar Flow Hood

All the culture media were prepared in a laminar flow hood (NU-425-300, Nuaire,

Plymouth, U.S.A.). The manipulation of the trophoblast cell for culture was also

performed in the hood.

2.3 Carbon Dioxide Incubator

A carbon dioxide incubator (3548, Forma Scientific, Ohio, U.S.A.) was used for

incubating of trophoblast cell. It provided a humidified atmosphere with a constant

carbon dioxide concentration (5% in air) and a stable temperature (37°C) for culture.

2.4 Hypoxic chamber

The hypoxic environment was established and maintained in a hypoxic chamber (C-

374, BioSpherix Ltd., Lacona, N.Y., USA) which located inside the incubator. The

hypoxic chamber was supplied with pre-mixed gas (1% oxygen, 5% carbon dioxide, 94%

nitrogen) and was monitored by an oxygen sensor (P-110-E702, BioSpherix Ltd.).

2.5 Disposable Plastic ware for media preparation and cell culture

Fifteen milliliter polystyrene centrifuge tubes (Corning, New York, U.S.A.) were

used for standard preparation of trophoblast cells. The pipette tips for micro-dispensers

25

were autoclaved before use. All culture media were sterilized with 0.22 μm pore size low

protein binding cellulose acetate membrane filters (Corning). Sterilized media were

stored in 250 ml disposable culture flask (Cellstar, Frickenhausen, Germany) and kept in

a cold room at 4°C. Trophoblast cells were cultured on sterile tissue culture flask (IWAKi,

Japan).

2.6 Microscope

A Zeiss Axioskop Microscope (Zeiss, Oberkochen, Germany) was used to count the

number, and assess the vitality and morphology of trophoblast cell.

2.7 Hemacytometer

The concentrations of trophoblast cell were determined by a hemacytometer

(Neubauer, Mariendfeld, Germany).

26

2.8 Chemicals

The chemicals used in this study were of molecular biology grade or cell culture

grade and are shown in Table 2.1.

Table 2.1: Chemicals that were used in the study

Reagents Suppliers Catalogue Number

0.05% trypsin/EDTA solution Invitrogen, Carlsbad, CA, USA 15400-054

1% [100U] of penicillin/streptomycin Invitrogen 15070-063

10%APS Sigma-Aldrich A9164

30% Polyacrylamide Bio-Rad, Hercules, CA, USA 161-0156

5% skimmed milk Carnation, Nestlé S.A., Vevey, Switzerland –

Acetone Merck KGaA, Darmstadt, Germany 100034

Agarose Invitrogen 15510-027

Antibody diluent Dako, Glostrup, Denmark S3022

Beta-mercaptoethanol Sigma-Aldrich M3148

Boric acid USB 76324

Bovine serum albumin [BSA] USB Corp. U510857-100G

Bromophenol Blue Bio-Rad, Hercules, CA, USA 161-0404

Chloroform Sigma-Aldrich C2432

Coomassie Plus Pierce PIE-23236

Diethyl pyrocarbonate Sigma-Aldrich D5758

DMSO Sigma-Aldrich D2650

ECL Western Blotting Reagents GE Healthcare Bio-Sciences RPN2106

EDTA USB Corp. US15701

Ethanol Merck KGaA 100983

Ethidium bromide Sigma-Aldrich E1510

F-10 Nutrient Mixture (Ham) Invitrogen 12390-035

Fetal bovine serum Invitrogen 10270-106

Full-Range Rainbow Molecular Weight Markers

GE Healthcare Bio-Sciences, Piscataway, USA RPN800E

Glucose Sigma-Aldrich G6125/G7773

27

Glycerol BDH, Poole, England 101185L

Glycine USB Corp. US16407-5kg

Horse serum Sigma-Aldrich H1270-100ml

Isopropanol Merck KGaA 109634

Methanol Merck KGaA 106009

NaCl Sigma-Aldrich, St. Louis, Missouri, USA S6150

NP-40 Sigma-Aldrich, St. Louis, Missouri, USA N6507

Penicillin-G/Streptomycin Invitrogen 15070

Phosphate buffer saline (PBS) Sigma-Aldrich, St. Louis, Missouri, USA P4417

Propidium iodide Sigma-Aldrich, St. Louis, Missouri, USA P4864

Protease Inhibitor Roche Diagnostics 11697498001

Sodium deoxycholate [DOC] Sigma-Aldrich D6750

Sodium dodecyl sulfate [SDS] BDH 444464T

TEMED Bio-Rad 161-0801

Tris USB Corp US75825-5kg

Triton X-100 Sigma-Aldrich, St. Louis, Missouri, USA T8532

TRIzol® reagent Invitrogen 15596-018

Trypan blue Invitrogen 15250-061

Tween-20 BDH 663684B

2.9 Immortalized first trimester extracellular trophoblast cell line: TEV-1

2.9.1 Source of TEV-1

Immortalized human first trimester extravillous trophoblast cell line, TEV-1, was

used in this study. Immortalized TEV-1 was developed by transfection of human

Papilloma virus pLXSN-E6/E7 open-reading frames into primary culture of trophoblasts

from human placentas (Feng et al., 2005). The immortalized TEV-1 cell line retains most

of the characteristics of the normal extravillous trophoblast cells (Feng et al., 2005).

TEV-1 cells expressed extravillous trophoblast markers including cytokeratin 7, human

28

leucocyte antigen G (HLA-G), and cluster of differentiation antigen 9 (CD9). It also

produces active matrix metalloproteinase (MMP) - 2 and -9, two most studied MMPs in

extravillous trophoblast cell invasion (Cohen et al., 2006).

2.9.2 Medium for TEV-1 cell culture

Ham’s F-10 (Invitrogen) was used for trophoblast cell culture. The composition of

Ham’s F-10 is shown in Table 2.2. The working Ham’s F-10 medium contained 5% heat-

inactivated fetal bovine serum (Invitrogen), 1% of penicillin-G, and 1% of streptomycin

sulphate. The solution was filtered through 0.22 μm filters (Corning, U.S.A.) and stored

at 4oC until use. The medium was equilibrated overnight to pH 7.5 in a carbon dioxide

incubator before use.

29

Table 2.2: Composition of Ham’s F-10

COMPONENTS Molecular Weight

Concentration (mg/L) Molarity

Amino Acids

Glycine 75 7.5 0.1

L-Alanine 89 9 0.101124

L-Arginine hydrochloride 211 211 1

L-Asparagine-H2O 150 15 0.1

L-Aspartic acid 133 13 0.097744

L-Cysteine 121 25 0.206612

L-Glutamic Acid 147 14.7 0.1

L-Glutamine 146 146 1

L-Histidine hydrochloride-H2O 210 23 0.109524

L-Isoleucine 131 2.6 0.019847

L-Leucine 131 13 0.099237

L-Lysine hydrochloride 183 29 0.15847

L-Methionine 149 4.5 0.030201

L-Phenylalanine 165 5 0.030303

L-Proline 115 11.5 0.1

L-Serine 105 10.5 0.1

L-Threonine 119 3.6 0.030252

L-Tryptophan 204 0.6 0.002941

L-Tyrosine disodium salt dihydrate 261 2.62 0.010038

L-Valine 117 3.5 0.029915

Vitamins

Biotin 244 0.024 0.000098

Choline chloride 140 0.7 0.005

D-Calcium pantothenate 477 0.7 0.001468

30

Folic Acid 441 1.3 0.002948

Niacinamide 122 0.6 0.004918

Pyridoxine hydrochloride 206 0.2 0.000971

Riboflavin 376 0.4 0.001064

Thiamine hydrochloride 337 1 0.002967

Vitamin B12 1355 1.4 0.001033

i-Inositol 180 0.5 0.002778

Inorganic Salts

Calcium Chloride (CaCl2) (anhyd.) 111 33.3 0.3

Cupric sulfate (CuSO4-5H2O) 250 0.0025 0.00001

Ferric sulfate (FeSO4-7H2O) 278 0.834 0.003

Magnesium Sulfate (MgSO4) (anhyd.) 120 74.62 0.621833

Potassium Chloride (KCl) 75 285 3.8

Potassium Phosphate monobasic (KH2PO4) 136 83 0.610294

Sodium Bicarbonate (NaHCO3) 84 1200 14.285714

Sodium Chloride (NaCl) 58 7400 127.586205

Sodium Phosphate dibasic (Na2HPO4) anhydrous 142 153.7 1.082394

Zinc sulfate (ZnSO4-7H2O) 288 0.03 0.000104

Other Components

D-Glucose (Dextrose) 180 1100 6.111111

Hypoxanthine Na 159 4.7 0.02956

Lipoic Acid 206 0.2 0.000971

Phenol Red 376.4 1.2 0.003188

Sodium Pyruvate 110 110 1

Thymidine 242 0.7 0.002893

31

2.9.3 TEV-1 cell culture

Unless otherwise specified, the cell line was seeded in 250 ml tissue culture flasks

(IWAKi, Japan) at a concentration of 2 x 106/ml and cultured in Ham’s F-10 with 5%

fetal calf serum at 37°C in 5% CO2. To avoid contamination, all the steps of TEV-1

culture were done inside a laminar flow hood (Nuaire, USA). Trypan blue exclusion test

was preformed to determine the viability of the cells. The concentration of viable cells

was determined with the use of a Neubauer hemocytometer (Marienfeld, Germany).

2.10 Determination of TEV-1 viability and proliferation

TEV-1 viability and proliferation was determined by the Cell Proliferation Kit II

(Roche, Germany) according to the manufacturer’s instructions. The assay is based on the

cleavage of the tetrazolium salt, sodium 3´-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-

bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate (XTT) in the presence of an

electron-coupling reagent, producing a soluble formazan salt. This conversion only

occurs in viable cells. In brief, 50 μl of working solution was added to the TEV-1 cell

culture. XTT working solution was prepared just prior to use according to manufacturer’s

protocol by mixing 1 ml of XTT stock solution with 20 μl of electron coupling reagent.

The cells were then incubated further at 37°C in 5% CO2 for 4 hours, and the absorbency

was measured using micro-plate reader at 450 nm (TECAN Infinite F200, Tecan Group

Ltd., Männedorf, Switzerland) accompanied with the application software Magellan V6.3

(Tecan Group Ltd.). Trypan blue exclusion test was also preformed to determine the

TEV-1 viability.

32


mRNA expression

2.11.1 Total RNA extraction and absorbance measurement

After collecting and washing the cell pellets from treatments, total cellular RNA was

extracted from cell pellets using the TRIzol® reagent (Invitrogen) according to the

manufacturer’s instruction. In brief, 1 ml of TRIzol reagent was added to the cell pellet

and homogenized by vortex for 15 seconds. After 3 mins of incubation at room

temperature, 0.2 ml of chloroform (Merck KGaA, Darmstadt, Germany) was added. The

sample was vigorously shaken for 2 to 3 min at room temperature until milky layer

appeared on the top layer of the mixture. It was then centrifuged at 12 000 × g for 15 min

at 4°C in a microcentrifuge (Eppendorf 5417R Centrifuge, Eppendorf, Hamburg,

Germany). RNA was precipitated from the aqueous phase with equal volume of

isopropanol (Merck KGaA), washed with 500μl 75% ethanol and dissolved in 50 μl of

diethylpyrocarbonate (DEPC) treated water (Sigma). The amount of nucleic acid in the

sample was quantified by measuring the absorbance at 260 nm (Beckman DU 650,

Beckman Coulter, Fullerton, CA, USA).

2.11.2 Determination of extracted RNA quality by denaturing agarose gel

electrophoresis

The quality of RNA is checked by denaturing agarose gel electrophoresis and

ethidium bromide staining. In brief, 2 μl of RNA samples were run in a 1% Agarose

(Invitrogen) gel using 1X TBE buffer (89 mM Tris; 89 mM Boric acid; 2 mM EDTA at

pH 8.0) (USB Corp., Cleveland, Ohio, USA) and stained with ethidium bromide (Sigma).

RNA bands are visualized and captured by a Gel Documentation System (AlphaImager

33

HP Imaging System with AlphaEaseFC software, Alpha Innotech, San Leandro, CA,

USA). The respective ribosomal bands should appear as sharp bands on the stained gel.

28S ribosomal RNA band (~5000bp) should be present with intensity approximately

twice that of the 18S RNA band (~2000bp).

2.11.3 Reverse Transcription of RNA

The total RNA was reverse transcribed using the TaqMan reverse transcription

reagent kit (Applied Biosystems, Foster City, USA) with the final solution containing:

Components Per Sample (μl)10X Taqman RT Buffer 3

Deoxy NTPs mixture 6 25mM MgCl2 6.6

Random Hexamers 1.5 RNase inhibitor 0. 6

Multisucible RTase 0.75 Total 18.45

The mixture was incubated for 10 min at 25°C, for 40 mins at 48°C, then for 5 min at

95°C. The processes were performed using a PTC-100 Thermal Cycler (MJ Research Inc.,

South San Francisco, CA, USA).

2.11.4 Quantitative real time PCR (qPCR)

The resulting cDNA were subjected to qPCR analysis. The real-time

quantification of the mRNA was performed using an Applied Biosystems 7500 Real-

Time PCR System (Applied Biosystems, Foster City, CA). Multiplex quantitative

polymerase chain reaction using beta-actin as an internal control for the normalization of

RNA loading was performed in a 20 μl reaction mixture containing 5 μl of sample DNA;

34

10μl 2X TaqMan Universal PCR Master Mix and 1μl of 20X Gene Expression Assay for

the targets (Applied Biosystems). Water was used as the no RNA template control. The

thermal cycling condition was as follow: an activation step at 95oC for 10min, followed

by 45 cycles of denaturation, annealing and amplification (94oC for 15sec, 60oC for 30sec

and 76oC for 30sec, respectively). Fluorescence data was collected during the annealing

step.

2.12 Immunoblotting analyses of solubilized TEV-1

2.12.1 Protein extraction

10×106 TEV-1 was washed in PBS for three times, centrifuged and pelleted by

centrifugation at 1000×g for 10 minutes, and resuspended in 150 μl lysis buffer (0.5M

Tris HCL, 5M NaCl, 1% Sodium deoxycholate, 0.5M Ethylenediaminetetraacetic acid,

1% NP-40) in the presence of a cocktail of protease inhibitors (Roche) for 60 minutes.

The insoluble fraction was discarded after centrifugation at 12000g for 15 minutes. The

protein concentration was measured by Braford assay (Coomassie Plus- Bradford Asaay

Kit, Pierce, Rockford, IL, USA).

2.12.2 SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)

SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), is used to

separate different proteins according to their electrophoretic mobility. Before SDS-PAGE,

cell lysates were denatured by boiling at 95°C for 5 minutes with 2-mercaptoethanol.

These denatured proteins were then loaded onto a 10% of SDS-polyacrylamide gel (Bio-

Rad, Hercules, CA, USA). SDS-PAGE was performed in a Mini-protein III system (Bio-

35

Rad) at a constant current of 30 mA until the dye front just ran out of the gel. Protein

molecular weight marker (Full-Range Rainbow Molecular Weight Markers, GE

Healthcare Bio-Sciences, Piscataway, USA) was used to determine the molecular size of

the separated proteins.

2.12.3 Imunoblotting analyses of TEV-1 cell lysate

After SDS-PAGE, the gel was blotted on a polyvinylidene fluoride (PVDF)

membrane (Amersham Hybond™-P, GE Healthcare Bio-Sciences) with pore size of 4.5

µm. Protein transfer was performed at a constant voltage of 110V at room temperature in

transfer buffer (15.6 mM Tris at pH 8.0; 120 mM glycine; 20% methanol). Blots were

then blocked for 1 hour with 5% skimmed milk (Carnation, Nestlé S.A., Vevey,

Switzerland) in PBST (PBS with 0.05% Tween-20) with slow shaking. The blots were

washed five times (10 mins each) with PBST between each of the following steps. The

blots were incubated with different primary antibodies (Tables 2.3) overnight at 4°C,

followed by the incubation with horseradish peroxidase-conjugated secondary antibody

(GE Healthcare Bio-Sciences) at a dilution of 1:5000. The protein that bound the antibody

was visualized with enhanced chemiluminescence with ECL Western Blotting Reagents

(GE Healthcare Bio-Sciences) according to the manufacturer’s instructions.

Autoradiography for signal detection was performed with Lumi-Film Chemiluminescent

Detection Films (Roche Diagnostics) using the X-ray Film Processor (FUJIFILM FMP-

100A, FUJI Photo Film Co. Ltd., Tokyo, Japan) for film development. Beta-actin was

used as the control for normalization. Quantification of protein bands was carried out

with Image J 1.36b software (http://rsbweb.nih.gov/ij/index.html).

36

Table 2.3: Primary antibodies used in Western Blot analysis.

Antibody Company Dilution Source

Beta-Actin Sigma-Aldrich 1:100000 Mouse Glut-1 Santa Cruz 1:500 Goat

IR CST 1:1000 Mouse IRS-1 CST 1:1000 Rabbit IRS-2 CST 1:1000 Rabbit VEGF Zymed 1:500 Mouse

2.13 Experimental Protocol

2.13.1 The effect of hypoxia on the viability and proliferation of TEV-1

This study aimed to study the tolerance of TEV-1 to hyperglycemia and/or hypoxic

stress. Confluent TEV-1 cells were subjected to varying glucose concentrations

(Euglycemia: 5.5mM; Hyperglycemia: 25 mM) and/or hypoxia for 8, 24, 48 or 72 hours.

Cellular hypoxia was attained by maintaining the cells in an atmosphere of 1% oxygen,

5% carbon dioxide and 94% nitrogen using a hypoxic chamber. The cell viability and

proliferation were then determined as described above.

2.13.2 The effect of glucose concentrations and/or hypoxia on mRNA and protein

expression

In vitro studies were conducted using TEV-1 human extravillous trophoblast cell

line. Cells in 250 ml disposable culture flask were grown to confluence in Ham’s F-10

medium contained 5% heat-inactivated fetal bovine serum. Confluent TEV-1 cells were

then subjected to varying glucose concentrations (Euglycemia: 5.5mM; Hyperglycemia:

25 mM) and/or hypoxia for 8, 24 or 48 hours. After the incubation, the cells were

collected by centrifugation at 4°C and resuspended in cold PBS. This wash cycle was

37

repeated three times. The mRNA and protein expression of glucose transporter-1, VEGF

and insulin signaling molecules including (IR, IRS-1, IRS-2) were then determined by

qPCR and immunobloting respectively as described above.

2.13.3 Immunofluorescent staining

TEV-1 cells were cultured on chamber slides (IWAKI) under varying glucose

concentrations and/or hypoxia as described in section 2.13.2 for 48 hours. After

incubation, the cells were washed three times with PBS to remove cell debris, and fixed

with methanol and acetone mixture (1:1 v/v) at -80 °C for 7 minutes. The slides were air

dried at room temperature. They were then permeablized by 0.1% Triton X-100 for 5

mins at room temperature and washed three times before blocking by 1 % horse serum

for 30 minutes. Afterward, they were washed and incubated with primary antibodies

(Table 2.4) overnight at 4°C. Bound antibodies were detected by FITC-conjugated

secondary antibody (Invitrogen) in a dilution of 1:300 for 30 minutes. After staining, the

slides are washed in PBS and mounted in DAKO fluorescent mounting medium (Dako).

for photo capturing. The fluorescence patterns of TEV-1 in randomly selected fields were

determined under a confocal microscope (Zeiss, Germany) with x200 magnification. The

filter set for FITC-conjugated secondary antibody consisted of an excitation/emission of

490/520 nM.

38

Table 2.4: Primary antibodies used in Immunofluorescent staining.

Antibody Company Dilution Source

Glut-1 Santa Cruz 1:100 Goat IR CST 1:50 Mouse

IRS-1 CST 1:50 Rabbit IRS-2 CST 1:50 Rabbit VEGF Zymed 1:50 Mouse

2.14 Data analysis

All the data were expressed as mean and standard error of mean (SEM). They were

analyzed by statistical software (SigmaPlot 10.0 and SigmaStat 2.03; Jandel Scientific,

San Rafael, CA, USA). For all experiments, the non-parametric ANOVA on Rank test for

multiple comparisons was used. Parametric Student t-test or non-parametric Mann

Whitney U test were used where appropriate as the post-test. A probability value <0.05

was considered to be statistically significant.

39

Chapter 3. Results 3.1 Effect of concentration of glucose and oxygen on the viability and proliferation

of TEV-1

3.2 Effect of different concentrations of glucose and oxygen on VEGF expression

in TEV-1 cells

3.2.1 Expression of VEGF mRNA

3.2.2 Expression of VEGF protein

3.2.3 Immunofluorescent staining of VEGF

3.3 Effect of different concentrations of glucose and oxygen on GLUT-1


3.3.1 Expression of GLUT-1 mRNA

3.3.2 Expression of GLUT-1 protein

3.3.3 Immunofluorescent staining of GLUT-1

3.4 Effect of different concentrations of glucose and oxygen on the expression of

proteins implicated in insulin signaling in TEV-1 cells

3.4.1 Expression of IR, IRS-1 and IRS-2 mRNA

3.4.2 Expression of IR, IRS-1 and IRS-2 proteins

3.4.3 Immunofluorescent staining of IR, IRS-1 and IRS-2

40

3.1 Effect of concentration of glucose and oxygen on the viability and proliferation

of TEV-1

The effect of glucose at concentrations of 5.5 mM (euglycemia) and 25 mM

(hyperglycemia) and oxygen at concentrations of 20% (normoxia) and 1% (hypoxia) on

the viability and proliferation of TEV-1 cells were compared by performing the XTT

proliferation assay. The experiments were repeated five times. The results are shown in

Figure 3.1 and 3.2.

In the XTT assay, the number of cells in the culture well was proportional to the

optical density. The number of TEV-1 cells increased continuously with increase in

culture time in both normoxic (Figure 3.1A) and hypoxic (Figure 3.1B) conditions. After

culturing the cells for more than 48 hours, the growth rate of the cells become slower.

This was shown by comparing the optical density between 72- and 48-hour and between

48- and 24-hour. There was significant difference in OD450 in the comparison between

72- and 48-hour but not between 48- and 24-hour (Figure 3.1 A and B).

The concentration of glucose in the culture medium did not affect the viability and

proliferation of TEV-1 cells in normoxic (Figure 3.1A) and hypoxic (Figure 3.1B)

conditions. There was no significant difference in OD450 between cells cultured in

medium containing 25 mM glucose and those in 5.5 mM glucose at all the time point

studied within 72-hour of incubation (Figure 3.1A and B).

In contrast, hypoxic condition significantly (P<0.05) enhanced the proliferation of

TEV-1 cells after 8 and 24 hours of culture in both euglycemia (Figure 3.2A) and

hyperglycemia (Figure 3.2B) conditions. At both time points, the OD450 of the well

containing cells cultured in hypoxic condition were significantly higher than that of wells

with cells in normoxic condition. For instance, at 24-hour, the OD450 of the wells

41

increased from 0.56 ± 0.02 (normoxia) to 0.78 ± 0.03 (hypoxia) (Figure.3.2A) and from

0.70 ± 0.05 (normoxia) to 0.87 ± 0.04 (hypoxia) (Figure 3.2B) in hyperglycemic and

euglycemic condition respectively. There were no differences in the optical density

between the normoxic group and hypoxic group at the 48- and 72-hour time points in

both the hyperglycemic and euglycemic conditions, suggesting the growth of the cells in

normoxia gradually caught up with those in hypoxia.

42

Figure 3.1

The effect of different glucose concentrations on the proliferation of TEV-1 under (A)

normoxic (20% oxygen) and (B) hypoxic (1% oxygen) condition as determined by XTT

proliferation assay. The values are mean ± S.E.M. of five independent experiments.

B

Hours0 20 40 60 80

OD

450

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

5.5 mM25 mM

P>0.05 P<0.05

P>0.05 P<0.05

25 mM

5.5 mM

A

Hours0 20 40 60 80

OD

450

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.45.5 mM25 mM

P>0.05 P<0.05

P>0.05 P<0.05 5.5 mM

25 mM

43

Figure 3.2 The effects of 20% (normoxia) and 1% oxygen (hypoxia) on the proliferation

of TEV-1 under (A) euglycemic (5.5 mM glucose) and (B) hyperglycemic (25 mM

glucose) condition as determined by XTT proliferation assay. The values are mean ±

S.E.M. of five independent experiments. * P<0.05 when compared with normoxia at the

same time point.

Hours0 20 40 60 80

OD

450

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4NormoxiaHypoxia

* *A

Hours0 20 40 60 80

OD

450

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4NormoxiaHypoxia

**

B

44

3.2 Effect of different concentrations of glucose and oxygen on VEGF expression in

TEV-1 cells

3.2.1 Expression of VEGF mRNA

Oxygen is a major regulator of VEGF (Ahmed et al., 2000). Hypoxia has been

demonstrated to upregulate VEGF expression in placental and related chorioallantoic

tissues (Wheeler et al., 1995; Gleadle et al., 1995; Shore et al., 1997). In this study, the

VEGF mRNA expression of TEV-1 was compared in conditions having different glucose

and oxygen concentrations. The results are shown in Figure 3.3. The level of VEGF

mRNA in the TEV-1 cells cultured in medium supplemented with glucose 5.5 mM and in

atmospheric oxygen was used as a reference (control) for normalization in analyzing the

result, i.e. their levels of expression were regarded as 100%.

Consistent with previous studies, hypoxia significantly increased (P<0.05) the

expression of the VEGF mRNA when compared with the control at the 8- and 24-hour

time points. A doubling of the VEGF expression levels was detected as early as 8 hour of

treatment. The increase in VEGF expression was detected irrespective of the glucose

treatment, i.e. the treatment with 5.5 mM and 25 mM of glucose under hypoxic condition

had similar levels of VEGF expression, though both are significantly higher than the

control. The magnitude of increase due to hypoxic treatment decreased thereafter. At the

48-hour time point, the levels were similar to the control. High glucose treatment under

atmospheric oxygen tension did not affect VEGF expression, which was not different

(P>0.05) from that of the control at the same time point.

45

Figure 3.3 The effect of glucose at concentrations of 5.5 mM and 25 mM on the VEGF

mRNA expression of TEV-1 under normoxia or hypoxia conditions. The levels of VERG

mRNA were determined by qPCR. The values are mean ± S.E.M. of five independent

experiments. VEGF mRNA level in TEV-1 cells under normoxic (20% oxygen) and

euglycemia (5.5 mM) was used as control. #P<0.05 when compared with the control at

the same time point. a-bP<0.05 when compared with cells treated in different conditions at

the same time point.

# #

#

#

a

a

b

a a

b

46

3.2.2 Expression of VEGF protein

The effects of the oxygen and glucose treatments on VEGF protein expression was

determined by western blotting analysis (Figure 3.4A). Semi-quantitative analyses of the

images were then performed for comparison (Figure 3.4B). The experiment was repeated

three times. The amounts of protein were normalized with reference to that of β-actin. As

for analysis done for VEGF mRNA, VEGF protein levels from cells cultured in medium

supplemented with glucose 5.5 mM and in atmospheric oxygen were used as a reference

for normalization (control).

Similar to the mRNA results, VEGF protein levels were increased significantly

(P<0.05) by more than 150% irrespective of the glucose treatment when compared with

the control. The higher levels of VEGF protein expression were detected at all the time

points studied. This differed from that of VEGF mRNA, which decreased gradually after

an initial increase at the 8-hour time point. The difference probably represented a much

half-life for VEGF protein than mRNA. The VEGF protein levels in cells treated with 25

mM glucose under normoxic condition were not different (P>0.05) from the control at all

the time points studied.

47

Figure 3.4 The effects of different glucose conditions (N: 5.5 mM; M: 15 mM; H: 25

mM) on the VEGF protein expression of TEV-1 under normoxic (20% oxygen) and

hypoxic (1% oxygen) condition. The protein expression was studied by western blot

using specific anti-VEGF antibody. (A) Representative images of the western blot result;

(B) Semi-quantitative comparison of the VEGF expression. The data were mean ± S.E.M.

of densitometric measurements from three independent experiments. VEGF level in

TEV-1 cells under normoxic (20% oxygen) and euglycemia (5.5 mM) was used as

control. #P<0.05 when compared with the control at the same time point. a-bP<0.05 when

compared with cells treated in different conditions at the same time point.

A

N M H N M H N M H

Normoxia

8 hours 24 hours 48 hours

Hypoxia

N M H N M H N M H


B

#

# # #

#

#

a

a a

a

b b

48

3.2.3 Immunofluorescent staining of VEGF

The expression of VEGF immunoreactivities in TEV-1 on day 2 of culture was

determined by immunofluorescent staining and examined under a confocal microscope.

The experiments were repeated for 3 times. Figure 3.5 shows representative images of the

VEGF immunoreactivities in TEV-1 under different culture conditions. At the start of the

experiment (time zero), VEGF immunoreactivity was localized to the cytoplasm of the

TEV-1 cells. Upon treatment of the cells with 1% oxygen, the intensity of VEGF

immunoreactivities significantly increased. Similar to the mRNA results, there was no

difference in immunoreactvities of VEGF in cells cultured in euglycemic and

hypogycemic condition. The VEGF immunoreactivities of TEV-1 cells under normoxia

condition were similar to that at time zero irrespective of the glucose treatment.

49

Figure 3.5 Immunofluorescent staining of VEGF expression in TEV-1 cells on day 2

after treatment cultured in different oxygen and glucose concentrations. VEGF

immunoreactivities were stained green. The cell nucleus was counterstained with

propidium iodide (PI) and appeared as red fluorescence. The level of VEGF signals at

time zero (Time 0) was used as the control.

50

3.3 Effect of different concentrations of glucose and oxygen on GLUT-1 expression

in TEV-1 cells

3.3.1 Expression of GLUT-1 mRNA

The primary glucose transporter in the placental tissue is GLUT1, one of the

facilitative (GLUT) glucose transporters (Illsey, 2000;; Barros et al., 1995;(Jansson et al.,

1993;Devaskar et al., 1994;Takata et al., 1994)). Previous studies have demonstrated

significant changes in the expression and activity of human placental GLUT-1 in diabetic

pregnancy (Gaither et al., 1999). The aim of this experiment was to investigate the time-

dependent effects of hyperglycemia and/or hypoxic conditions on GLUT-1 mRNA

expression in TEV-1 by qPCR. The results are shown in Figure 3.6. The experiment was

repeated for five times. Similar to that of VEGF analyses, the levels of GLUT1 mRNA

cultured in normoxic and euglycemic conditions were used as the control and as

reference for normalization.

In atmospheric oxygen, the GLUT-1 mRNA expression of cells treated with 25 mM

glucose was decreased significantly (P<0.05); the level was half of that of the control

value at the 8-hour time point. The level of GLUT-1 under this culture condition

gradually increase and by 48-hour, the level was not different (P>0.05) from that of the

control.

In an atmosphere containing 1% oxygen, 5.5 mM glucose treatment tended to

increase the GLUT-1 expression above the control, though the differences had not yet

reached statistical significance. However, the levels were significantly higher (P<0.05)

than that from culture cultured in normoxic and hyperglycemic conditions at the 8- and

24-hour time points.

51

The above observation suggested that hyperglycemic treatment decreased whereas

hypoxic treatment tended to increase the mRNA expression of GLUT-1. Therefore it was

not surprised to see that the GLUT-1 mRNA levels in cells cultured in hypoxic and

hyperglycemic conditions had values that were intermediate between the above two

groups. The mRNA levels were similar in all the treatment at the 48-hour time point.

52

Figure 3.6 The effect of glucose at concentrations of 5.5 mM and 25 mM on the GLUT-1

mRNA expression of TEV-1 under normoxia or hypoxia conditions. The levels of

GLUT-1 mRNA were determined by qPCR. The values are mean ± S.E.M. of five

independent experiments. GLUT-1 mRNA level in TEV-1 cells under normoxic (20%

oxygen) and euglycemia (5.5 mM) was used as control. #P<0.05 when compared with the

control at the same time point. a-bP<0.05 when compared with cells treated in different

conditions at the same time point.

#

#

a

b

a

b

53

3.3.2 Expression of GLUT-1 protein

The expression of GLUT-1 protein treated with different concentrations of oxygen

(1% and 20%) and glucose (5.5 mM, 15 mM, 25 mM) was determined by western

blotting analysis (Figure 3.7A). Semi-quantitative analyses of the images from three

independent experiments were then performed for comparison (Figure 3.7B). The

amounts of protein were normalized with reference to that of β-actin. The GLUT1 protein

levels in normoxic and euglycemic conditions were used as the control and as reference

for normalization.

In normoxic environment, GLUT-1 protein expression in TEV-1 cells was

significantly (P<0.05) down-regulated when the cells were treated with 25 mM glucose;

the values at the 8-, 24- and 48-hour time points were decreased by 40-50% when

compared with the control. On the other hand, the GLUT-1 protein levels in cells cultured

in hypoxic conditions irrespective of the glucose treatment increased gradually with the

time of culture. The levels in hyperglycemic condition was similar to the control at 8-

hour but was significantly higher (P<0.05) than the control 48-hour of culture. The

protein levels of GLUT-1 in cells cultured in hypoxic and hyperglycemic condition were

significantly higher (P<0.05) than that of cells cultured in normoxic and hyperglycemic

condition.

54


mM) on the GLUT-1 protein expression of TEV-1 under normoxic (20% oxygen) and


using specific anti-VEGF antibody. (A) Representative images of the western blot result;

(B) Semi-quantitative comparison of the GLUT-1 expression. The data were mean ±

S.E.M. of densitometric measurements from three independent experiments. GLUT-1

level in TEV-1 cells under normoxic (20% oxygen) and euglycemia (5.5 mM) was used

as control. #P<0.05 when compared with the control at the same time point. a-bP<0.05

when compared with cells treated in different conditions at the same time point.



Hypoxia

N M H N M H N M H

Normoxia

N M H N M H N M H

(A)

(B)

# # #

#

a

b

a

b

a a

b

55

3.3.3 Immunofluorescent staining of GLUT-1

The expression of GLUT-1 immunoreactivities in TEV-1 cells after 48 hours of

culture was determined by immunofluorescent staining and examined under a confocal

microscope. The experiments were repeated for 3 times. Typical images of the

immunofluorescent staining are shown in Figure 3.8. At the start of the treatment (Time

0), only weak signal of GLUT-1 was detected in TEV-1 cells (Figure 3.8). After culturing

in normoxic condition for 48 hours, a slight increase in the immunoreactivities was found

in cells treated with 5.5 mM glucose. On the other hand, no difference in the intensity of

the signal was observed between the time zero samples and those cultured in

hyperglycemia environment.

Hypoxia up-regulated GLUT-1 immunoreactivities of the cultured TEV-1 cells

significantly. The increase in signal seemed to be more in the hyperglycemic than in the

euglycemic conditions (Figure 3.8).

56

Figure 3.8 Immunofluorescent staining for GLUT-1 expression in TEV-1 cells after

treating the cells with different concentrations of oxygen (1%, 20%) and glucose (5.5

mM, 25 mM) for two days. The GLUT-1 expression was stained green. The cell nucleus

was counterstained with propidium iodide (PI) and appeared as red fluorescence. The

level of GLUT-1 signals at time zero (Time 0) was used as the control.

57

3.4 Effect of different concentrations of glucose and oxygen on the expression of

proteins implicated in insulin signaling in TEV-1 cells

3.4.1 Expression of IR, IRS-1 and IRS-2 mRNA

Insulin initiates a wide variety of growth and metabolic effects by binding to the

insulin receptor (IR) and activating its intrinsic tyrosine kinase. This event leads to

phosphorylation of the tyrosine residues in a variety of docking proteins including insulin

receptor substrate (IRS) proteins (White, 1997). Proteins implicated in insulin signaling

including IR, IRS-1 and IRS-2 have been identified in human placenta (Catalano et al.,

2002;(Desoye et al., 1997;Jones et al., 1993;Laviola et al., 2005;Rui et al., 2001;Scioscia

et al., 2006). Dysregulation of the insulin receptor and IRS proteins have been

demonstrated in gestational diabetes mellitus (Alonso et al., 2006;Osmond et al.,

2000;Sesti et al., 2001). This experiment evaluated the effect of glucose treatment and/or

hypoxia on IR, IRS-1 and IRS-2 mRNA expression in TEV-1 cells. The results of the

experiments are shown in Figure 3.9 to 3.11. Similar to the two experiments reported

above, the mRNAs were quantified by qPCR. The data shown in Figure 3.9 to 3.11 are

derived from 5 independent experiments.

There was no difference in the levels of IR mRNA in the TEV-1 cells among all the

conditions tested in this experiment at time points earlier than 48-hour. High glucose (25

mM) treatment significantly (P<0.05) increased the mRNA expression of IR after 48

hours of culture in both normoxic environments. In hypoxic condition, the level of IR

mRNA in cells treated with 5.5 mM glucose was significantly lower (P<0.05) than the

control and those treated in normoxic and hyperglycemic conditions.

58

The expression of IRS-1 mRNA was not affected by 1% and 20% oxygen treatment

in euglycemic condition. High glucose treatment also did not affect IRS-1 mRNA

expression in normoxic condition. However, treatment with 25 mM glucose in hypoxic

condition significantly decreased (P<0.05) the IRS-1 mRNA level at the 24- and 48-hour

time points when compared with the control; the value was 60% of the control values.

The value at the 24-hour time point was also significantly lower (P<0.05) than cells

cultured in normoxic and hyperglycemic conditions.

Similar to that of IRS-1, the mRNA level of IRS-2 was not affected by 1% and 20%

oxygen treatment in euglycemic condition. Hypoxia and hyperglycemia decreased the

expression of IRS-2 mRNA gradually, such that by 48 hour of culture, the level was

significantly lower (P<0.05) from the control. On the other hand, normoxia with the same

glucose treatment induced a transient increase in IRS-2 mRNA at the 24-hour time point.

59

Figure 3.9 The effect of glucose at concentrations of 5.5 mM and 25 mM on the IR

mRNA expression of TEV-1 under normoxia or hypoxia conditions. The levels of IR


experiments. IR mRNA level in TEV-1 cells under normoxic (20% oxygen) and




#

a

b

60

Figure 3.10 The effect of glucose at concentrations of 5.5 mM and 25 mM on the IRS-1

mRNA expression of TEV-1 under normoxia or hypoxia conditions. The levels of IRS-1


experiments. IRS-1 mRNA level in TEV-1 cells under normoxic (20% oxygen) and




# #

a

b

61

Figure 3.11 The effect of glucose at concentrations of 5.5 mM and 25 mM on the IRS-2

mRNA expression of TEV-1 under normoxia or hypoxia conditions. The levels of IRS-2


experiments. IRS-2 mRNA level in TEV-1 cells under normoxic (20% oxygen) and



#

#

62

3.4.2 Expression of IR, IRS-1 and IRS-2 proteins

3.4.2. Expression of IR protein

Figure 3.12A shows representative images of western blotting for determination of

the expression of IR protein in TEV-1 cells treated with different concentrations of

glucose and oxygen. The semi-quantitative comparisons of the protein expression in

normoxic and hypoxic condition are shown in Figure 3.12B. The data were derived from

three independent experiments. The amounts of protein were normalized with reference

to that of β-actin. Cells cultured in normoxic and euglycemic conditions were used as

control and reference for normalization.

In hypoxic condition, the protein level of IR in cells cultured in medium containing

5.5 mM glucose was significantly down-regulated (P<0.05) at the 8- and 48-hour time

points when compared with the control values; their values were decreased by about 40%.

Hyperglycemic treatment under atmospheric oxygen significantly decreased (P<0.05) IR

protein expression at the 8-hour time point. The level gradually increased thereafter and

at the 24-hour time point, it was not different (P>0.05) from the control. The levels of IR

protein under hypoxic and hyperglycemic condition were similar to the control at all the

time points studied.

The protein expression of IRS-1 fluctuated widely. There were no statistical

differences among the groups at all the time points studied in the Western blot analyses.

Compared with cells cultured in normoxic and euglycemic conditions (control),

those cultured in hypoxic and hyperglycaemic conditions tended to have lower IRS-2

protein expression; statistical significant differences (P<0.05) were found at the 8- and

48-hour time points. These levels were also significantly lower (P<0.05) than those in

63

normoxic and hyperglycaemic conditions at the same time points. On the other hand,

normoxic and hyperglycaemic conditions induced a transient increase of more than 2

folds of IRS-2 protein expression when compared with the control at the 24-hour time

point. The level dropped to the control level by 48-hour of culture.

64


mM) on the IR protein expression of TEV-1 under normoxic (20% oxygen) and hypoxic

(1% oxygen) condition. The protein expression was studied by western blot using

specific anti-IR antibody. (A) Representative images of the western blot result; (B) Semi-

quantitative comparison of the IR expression. The data were mean ± S.E.M. of

densitometric measurements from three independent experiments. IR level in TEV-1 cells

under normoxic (20% oxygen) and euglycemia (5.5 mM) was used as control. #P<0.05

when compared with the control at the same time point.

N M H N M H N M H


hypoxia

N M H N M H N M H


normoxia

(A)

(B)

# #

#

65


mM) on the IRS-1 protein expression of TEV-1 under normoxic (20% oxygen) and


using specific anti-IRS-1 antibody. (A) Representative images of the western blot result;

(B) Semi-quantitative comparison of the IRS-1 expression. The data were mean ± S.E.M.

of densitometric measurements from three independent experiments. IRS-1 level in TEV-

1 cells under normoxic (20% oxygen) and euglycemia (5.5 mM) was used as control.

hypoxia N M H N M H N M H


N M H N M H N M H


normoxia

(A)

(B)

66


mM) on the IRS-2 protein expression of TEV-1 under normoxic (20% oxygen) and


using specific anti-IRS-2 antibody. (A) Representative images of the western blot result;

(B) Semi-quantitative comparison of the IRS-2 expression. The data were mean ± S.E.M.

of densitometric measurements from three independent experiments. IRS-2 level in TEV-

1 cells under normoxic (20% oxygen) and euglycemia (5.5 mM) was used as control. #P<0.05 when compared with the control at the same time point. a-bP<0.05 when

compared with cells treated in different conditions at the same time point.

N M H N M H N M H


normoxia

hypoxia


N M H N M H N M H

(A)

(B)

#

#

# #

#

a

b

a

b b

67

3.4.3 Immunofluorescent staining of IR, IRS-1 and IRS-2

Before experimentation (Time 0), no or only weak signal of IR was detected in

TEV-1 cells (Figure 3.15). After 48 hours of culture in medium containing 5.5 mM

glucose, IR immunoreactivity was detected primarily in nucleus of TEV-1 irrespective of

oxygen concentration in the culture environment. High glucose treatment upregulated IR

immunoreactivities significantly. Some of the cells also showed cytoplasmic IR

immunoreactivities with the treatment.

The immunreactivities of IRS-1 were mainly localized to the cytoplasm of TEV-1

(Figure 3.16). No observable difference was found between different treatments for 48

hours and the time zero control.

As shown in Figure 3.17, IRS-2 immunoreactivities are manly localized to the

cytoplasm and/or plasma membrane of TEV-1 cells. Hypoxia treatment for 48 hours

suppressed the expression of IRS-2 immunoreactivities in both the euglycemic and

hyperglycemic group. On the other hand, glucose concentration had no observable effect

on the immunoreactivity of IRS-2 in both hypoxic and normoxic environment.

68

Figure 3.15 Immunofluorescent staining for IR expression in TEV-1 cells after treating

the cells with different concentrations of oxygen (1%, 20%) and glucose (5.5 mM, 25

mM) for two days. The IR expression was stained green. The cell nucleus was

counterstained with propidium iodide (PI) and appeared as red fluorescence. The level of

IR signals at time zero (Time 0) was used as the control.

69

Figure 3.16 Immunofluorescent staining for IRS-1 expression in TEV-1 cells after


mM, 25 mM) for two days. The IRS-1 expression was stained green. The cell nucleus


level of IR signals at time zero (Time 0) was used as the control.

70

Figure 3.17 Immunofluorescent staining for IRS-2 expression in TEV-1 cells after


mM, 25 mM) for two days. The IRS-2 expression was stained green. The cell nucleus


level of IR signals at time zero (Time 0) was used as the control.

71

Chapter 4 Discussion

4.1 Human placental development

4.2 Human placenta in hypoxic environment

4.3 Human placenta in hyperglycemia

4.4 Use of TEV-1 for studying trophoblast functions

4.5 Effect of hyperglycemia on trophoblast proliferation

4.6 Effect of hypoxia on trophoblast proliferation

4.7 Effect of hyperglycemia on VEGF expression

4.8 Effect of hypoxia on VEGF expression

4.9 Combine effect of hyperglycemia and hypoxia on VEGF expression

4.10 Effect of hyperglycemia on GLUT-1 expression

4.11 Effect of hypoxia on GLUT-1 expression

4.12 Combine effect of hyperglycemia and hypoxia on GLUT-1 expression

4.13 Effect of hyperglycemia and hypoxia on insulin signaling molecules

4.14 Conclusion

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4.1 Human placental development

The placenta is a highly specialized organ and a transient organ that occurs only in

pregnancy. It mediates the physiological exchange between the mother and the fetus

(Boyd, 1970). After successful implantation and initiation of placentation, trophoblast

cells undergo extensive proliferation and differentiation (Staun-Ram and Shalev,

2005;Lunghi et al., 2007b;al-Lamki et al., 1999). The differentiation of the trophoblast

follows two main pathways, i.e. the villous and the extravillous pathway. The villous

(non-migratory) cytotrophoblast cells proliferate, differentiate and fuse to form the

syncytiotrophoblast of the chorionic villi. By days 13 to 14 of pregnancy, some

cytotrophoblast cells penetrate the syncytiotrophoblast surrounding the early conceptus to

form columns of extravillous cytotrophoblast cells. Some of these contiguous cells form

the cytotrophoblastic shell at the interface of the feto-maternal compartments, while the

others migrate and penetrate the decidua, and eventually invade and remodel the maternal

blood vessels in the uterine decidua to form the endovascular trophoblasts. Extravillous

cytotrophoblast cells also invade interstitially (interstitial trophoblast). These invasive

cells cause circumferential expansion of the placenta and recruit maternal arterioles,

allowing subsequent expansion of the villous region of the placenta.

4.2 Human placenta in hypoxic environment

Several lines of evidence suggest that the early stage of placental development takes

place in a hypoxic environment. The intrauterine oxygen tension during early pregnancy

at 8 weeks prior to establishment of maternal blood flow into the intervillous space is

extremely low and ranged from 1-5% of oxgyen (Maltepe and Simon, 1998;Jauniaux et

73

al., 2000). Blood flow to the human intervillous space does not begin until 10 to 12

weeks of pregnancy (Maltepe and Simon, 1998;Jauniaux et al., 2000). Furthermore,

assessment by Doppler ultrasound demonstrated that women who had premature onset of

blood flow to the intervillous space had a higher incidence of miscarriage (Jauniaux et al.,

2003).

Although low oxygen is essential for proper early placentation, it is deleterious for

term placental tissue. Hypoxia hinders differentiation and induces apoptosis in isolated

term trophoblast cells (Levy, 2000). Severe hypoxic condition was believed to be

associated with pregnancy complications such as preeclampsia and intrauterine growth

restriction(Rajakumar et al., 2008). Studies using term villous explants have shown that

hypoxia causes morphological changes characteristics of trophoblast from preeclampsic

pregnancies, including increased syncytial degeneration and excessive shedding,

increased cytotrophoblast proliferation and altered expression of stage-specific antigens

(Ong and Burton, 1991). In addition, hypoxia has been demonstrated to lead to

hypertrophy of placenta making the transport of oxygen and nutrients to the inner cells of

the placenta more difficult and time consuming (de Grauw et al., 1986).

4.3 Human placenta in hyperglycemia

Diabetes mellitus is a syndrome of disordered metabolism, usually due to a

combination of hereditary and environmental causes, resulting in abnormally high blood

sugar levels (hyperglycemia). Diabetic pregnancy can be divided into pregestational

diabetes mellitus (PGDM, diabetes diagnosed before pregnancy) and gestational diabetes

mellitus (GDM; glucose intolerance detected during pregnancy). In diabetes, the placenta

74

undergoes a variety of structural and functional changes (Desoye and Myatt,

2004;Desoye and Hauguel-de, 2007), including increased placental weight (Makhseed et

al., 2004), oxidative stress (Coughlan et al., 2004), and nutrient transport and filtration

problems (Jansson et al., 2002). Pathological studies of term placenta from diabetes

revealed patchy syncytial necrosis, dilated rough endoplasmic reticulum,

cytotrophoblastic hyperplasia, narrowing of the small vessels, focal thickening of the

basement membranes, and extracellular matrix alterations (Jansson et al., 2002;Coughlan

et al., 2004;al-Okail and al-Attas, 1994; Pietryga et al., 2004). Diabetic pregnancy is also

associated with major obstetric complications including spontaneous abortion (Forsbach-

Sanchez et al., 2005;Luerssen and Winsch, 2005) and placental alternation (Pedersen,

1998) in the first trimester.

4.4 Use of TEV-1 for studying trophoblast functions

Malignant trophoblast cells, i.e. choriocarcinoma cells are well-accepted and

frequently used model for studying trophoblast behavior. Although these cell lines (e.g.

Bewo, Jeg3 and JAR) have some similarity to trophoblast, there are differences between

these cell lines and trophoblasts probably because they are derived from tumors. They are

different in terms of gene expression (Vegh et al., 1999), proliferation (Lash et al., 2008)

and regulation of invasiveness/differentiation (Hohn et al., 1998). Immortalized TEV-1

was developed by transfection of human Papilloma virus pLXSN-E6/E7 open-reading

frames into primary culture of trophoblast cells from human placenta (Feng et al., 2005).

The immortalized TEV-1 cell line retains most of the characteristics of the normal

extravillous trophoblast cells (Feng et al., 2005). In this project, TEV-1 was used as a

75

human first trimester trophoblast model to study the in vitro effect of hyperglycemia

and/or hypoxia on proliferation, GLUT-1 and insulin signaling molecules expression of

trophoblast. The experimental design was to mimic the in vivo condition of ischemic

hypoxia that was encountered in uteroplacental insufficiency of maternal diabetes

(Chartrel et al., 1990) and on uterine artery ligation (Ogata et al., 1986).

There are four reasons for not using primary trophoblast cells in this study. First, the

trophoblast compartment of the human placenta comprises a variety of trophoblast types,

which differ in physiological function and degree of differentiation. Despite some success

in isolating trophoblast cells from first trimester pregnancies (Bloxam et al., 1997), the

low cell yield of the reported methods still makes comprehensive investigations very

difficult. Second, the availability of first trimester placenta from termination of

pregnancy with the current clinical practice of medical abortion was limited, making the

use of these primary tissues for the research within the restricted timeframe of this Master

of Philosophy program impossible. Third, results generated from cell lines are more

reproducible. This is in contrast with the use of explant cultures, which although provides

advantage of allowing trophoblast outgrowth in a manner similar to that occurs in vivo,

the responses of the explants to environmental challenges tend to vary greatly among

individual samples (James et al., 2006). In order to obtain meaningful data from explant

culture study, a high number of replicates are required, which is unlikely to be possible in

view of the restrictions of this research curriculum (see above). Finally, although term

placentas are available and much of the known data about human placental transport is

derived from studies of term placenta, they are not used for this project because there is

increasing evidence that placental transport in early pregnancy may differ from that at

76

term in many respects (Glazier and Jansson, 2004). For instance, nutrition is histiotrophic

with trophoblast phagocytosis of endometrial glandular secretions including glycogen in

the first trimester before flow of maternal blood into the intervillous space, (Burton,

2002). This mode of nutrient transport is altered after ~12 weeks when maternal blood

gets into the terminal villi of the placenta, and transfer of respiratory gases, nutrients and

waste products occurs across the placental membrane mainly by diffusion and facilitated

transport. The differences in cellular distribution of GLUTs between the first trimester

and the term placentas have also been demonstrated (Illsley, 2000;Korgun et al., 2005).

4.5 Effect of hyperglycemia on trophoblast proliferation

During human implantation and placentation, adequate proliferation and

differentiation of trophoblast cells are the basic requirements for a normal pregnancy. In

a diabetic pregnancy, the altered maternal milieu may affect these processes.

Disturbances of trophoblast and placental growth and differentiation in the first trimester

by inadequate glycemic control can have long-term consequences for placental structure

and function throughout gestation (Reece et al., 1994). Although the placenta in diabetes

at term pregnancy has been intensively studied (Desoye and Shafrir, 1994), little is

known about the effects of diabetes on first-trimester placentas.

In this study, hyperglycemic treatment did not have significant effect in the

proliferation of TEV-1 cells. To the best of my knowledge, there was only one study in

the literature investigating the effect of glucose concentrations on three first trimester

choriocarcinoma cell lines, namely Bewo, JAR and JEG-3 (Weiss et al., 2001). The

results of the study showed that hyperglycemic challenge for 24 hours suppressed the

77

proliferation of the Bewo and the JEG-3, but not the JAR cells. However, such

suppression effect was not observed after 48 hours in all the three cell lines. One possible

reason for the discrepancy between Weiss and coworkers’ study and the present report is

difference in the experimental cell models used. Although they are all derived from first

trimester trophoblast tissue, the three choriocarcinoma cell lines and TEV-1 cells may

have different characteristics (see section 4.4). In addition, the choriocarcinoma cell lines

can be distinguished by their degree of differentiation, from the least differentiated, such

as JAR, to the most differentiated such as BeWo (Wice, 1990;Hochberg, 1992;Moe,

1994;Mitchell, 1995;Hohn et al., 1998).

4.6 Effect of hypoxia on trophoblast proliferation

As extravillous trophoblasts move away from the placenta they differentiate into an

invasive phenotype in a process which is tightly regulated both spatially and temporally.

Only the extravillous trophoblasts close to the villi proliferate in vivo (Irving, 1995). As

the extravillous trophoblasts migrate away from the villi, they progressively develop an

invasive phenotype invading into the maternal decidua, and lose their proliferative

potential (Genbacev et al., 1997;Genbacev and Miller, 2000).

It has been shown in vitro that the proliferation and differentiation of human

extravillous cytotrophoblasts is regulated by oxygen tension (Gude et al., 2004;Genbacev

et al., 1997). There are two opposing schools of thought on the effect of hypoxia on

trophoblast differentiation in the first trimester human pregnancy. One school of thought

believes that hypoxia enhances the trophoblast proliferation while the other proposes that

hypoxia suppresses such event and promotes an invasive phenotype.

78

The first school of thought is supported by a number of observations including:

(1) HTR-8/SVneo cells, a Simian Virus 40 transfected human first trimester

cytotrophoblast cell line, have increased proliferation and reduced invasion

through Matrigel when cultured in 2% oxygen conditions (Kilburn, 2000);

(2) isolated first trimester cytotrophoblasts have increased rates of DNA synthesis

when cultured in 2% oxygen in comparison to 20% oxygen (Jiang, 2000);

(3) the ratio of cytotrophoblast : syncytiotrophoblast nuclei is abruptly decreased in

placenta over 8 weeks of gestation despite the number of cytotrophoblast cells

per unit area remains constant, suggesting that cytotrophoblast proliferation is

greater in early pregnancy when the growth environment is believed to be

hypoxic (Bose, 2006)

(4) in comparison to explants cultured in 20% oxygen, first trimester villous

explants cultured in 2 or 3% oxygen have increased 5-bromo-2'-deoxyuridine

(thymidine analogue) incorporation (a proliferation marker), increased

extravillous trophoblast outgrowth and increased total number of cells in the

outgrowth (Genbacev et al., 1997;Caniggia, 2000;Sferruzzi-Perri, 2003)

(5) hypoxia has been shown to reduce the invasive capacity of trophoblasts and the

expression of molecules associated with an invasive trophoblast phenotype such

as integrin and matrix metalloprotease-2 (MMP-2) (Genbacev et al.,

1997;Kilburn, 2000;Crocker, 2003).

However, a smaller body of indirect and contradictory evidences exist suggesting

that hypoxia promotes an invasive trophoblast phenotype that may be important in

achieving sufficient depth and extent of trophoblast invasion (James, 2004;Graham,

79

1998;James, 2006). For example, extravillous trophoblast outgrowths formed under

hypoxic conditions contained fewer cells (i.e. reduce proliferation) than those produced

in normoxia (James, 2004). Graham and colleagues, (1998) in contrast to the results from

Kilburn and coworkers (2000), found that culture of HTR-8/SVneo trophoblast cells in

1% oxygen increased the invasion of these cells through Matrigel in comparison to

culture in 20% oxygen, by up-regulating uPA receptors.

In the present study, the proliferation of TEV-1 cells was enhanced under an

atmosphere of 1% oxygen when compared with those cultured under 20% oxygen. The

response of the cells to hypoxia was quick with a statistical significant difference

detected after 8 hours of culture irrespective of the glycemic treatment. Trophoblast cell

lines show differential response to low oxygen concentrations in terms of proliferation,

which is dependent on culture period (Lash, 2007). For example, Fitzpatrick and Graham

(1998) reported no change in the number of HTR-8/SVneo cells after 24 h culture in

hypoxia. However, Kilburn et al. (2000) reported increased proliferation of this cell type

after 72 h culture in 2% oxygen. Interestingly, another group (Lash, 2007) observed a

decrease in proliferation after 48 and 72 hours in hypoxia using the same cell line. One

possible explanation for the discrepancies among studies is the differences in the culture

condition and original seeding density of the cell lines. In addition, the proliferation

response of TEV-1 to hypoxia may reflect the ability of these cells to sense oxygen, and

their ability to regulate the HIF (hypoxia inducible factor) system.

The present report supports the first school of thought. It is believed that the

proliferative response of the trophoblast to hypoxia would create a large pool of

trophoblast cells in early pregnancy, thereby providing a sufficient numbers of cells for

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subsequent invasion into the maternal deciduas (Jiang, 2000;Kilburn, 2000;Genbacev et

al., 1997;Caniggia, 2000;Crocker, 2003;Sferruzzi-Perri, 2003).

4.7 Effect of hyperglycemia on VEGF expression

Many studies have identified VEGF as a key angiogenic factor in both physiological

and pathological conditions. In human placenta, VEGF expression in trophoblast has

been demonstrated by in situ hybridization and immunohistochemical studies (Sharkey,

1993;Ahmed, 1995;Vuorela, 1997). It has also been demonstrated that trophoblast

secretes VEGF in vitro (Shore et al., 1997). It is hypothesized that VEGF may regulate

trophoblast differentiation and invasion and may promote fetoplacental vascular

development and stabilization (Wulff, 2003;Ahmed, 2000). However, the data available

on the relationship between hyperglycemia and VEGF expression in trophoblast is not

available. Previous researches done on retinal pigment epithelial cells have found that

VEGF mRNA and protein were up-regulated by high glucose (Xiao et al., 2006). On the

other hand, VEGF expression in endothelial cells was either decreased (Pinter, 2001) or

not affected (Larger, 2004) by hyperglycemic treatment. In diabetic fibroblast, a seven-

fold decrease in VEGF production has also been observed (Lerman, 2003).

This is the first report demonstrating that hyperglycemic treatment had no effect on

VEGF production of extravillous trophoblast cells. Similar response in kinetics and

magnitude on VEGF expression are found in cells treated with 5.5 and 25 mM glucose.

Therefore the effect of high glucose on VEGF production is cell-type specific.

81

4.8 Effect of hypoxia on VEGF expression

Oxygen is thought to be a major regulator of the balance between VEGF and

placental growth factor (PlGF) function. Hypoxia has been demonstrated to up-regulate

VEGF expression in tumors (Plate, 1992;Brown, 1993;Takahashi, 1994) and vascular

smooth muscle cells (Stavri, 1995). In placental and related chorioallantoic tissues,

VEGF expression is up-regulated by hypoxia (Wheeler, 1995;Gleadle, 1995;Shore et al.,

1997)and down-regulated by hyperoxia (Shore et al., 1997). In situation with impaired

uteroplacental circulation such as in pre-eclampsia with preserved end diastolic flow

when the placenta and the fetus are in hypoxic environment, the peripheral placental villi

form rich branching networks, and the fetal blood flow is normal or even reduced

(Kiserud, 1994). Semi-quantitative Western blotting analysis of these placentas

demonstrated an increased expression of VEGF when compared to gestational age-

matched normal placentas (Ahmed, 1997), suggesting that placental hypoxia up-regulated

VEGF production in vivo and induced angiogenesis.

Consistent with previous studies, results of this study showed that hypoxic condition

significant induced the expression of VEGF mRNA and protein in TEV-1 cells. The

discrepancies observed between the kinetics of mRNA and protein expressions may be

explained by the longer half-life of proteins. The gradual down-regulation of VEGF

mRNA after 8 hours in hypoxia might also be explained by inappropriate time points of

analysis (8, 24 and 48 h). Upregulation of VEGF expression by hypoxia, for example, has

been showed to peak after 6 h of exposure in rat calvarial osteoblasts (Warren, 2001).

The exact mechanism by which oxygen modulates VEGF expression is unknown,

but hypoxia-inducible factor-1 (HIF-1) is likely to play a key role. HIF-1 is known to

82

activate the transcription of genes in response to hypoxia (Wang and Semenza,

1993;Safran, 2003). HIF-1 itself is a transcription factor that binds DNA as a heteromeric

complex composing of two subunits, the constitutively expressed HIF-1β (aryl

hydrocarbon nuclear translocator, ARNT) and the inducible HIF-1α. In conditions of low

oxygen, the stable HIF-1α subunit dimerizes with HIF-1β to form an active HIF-1

complex, which binds to a hypoxia-responsive promoter element (5’-TACGTG-3’)

present in a variety of hypoxia-related genes and up-regulate their transcription (Wang,

1995). In conditions of normoxia, the HIF-1α protein is rapidly ubiquitinated and

degraded by the proteasome (Maxwell, 1999). HIF-1 has been shown to regulate the

expression of more than 20 genes in response to changing oxygen tension (Semenza,

2000a), including VEGF (Forsythe, 1996). In fact, HIF-1 is the major regulator of VEGF

expression in hypoxic condition. However, the precise mechanisms by which oxygen

levels are detected by cells remain unclear.

4.9 Combined effect of hyperglycemia and hypoxia on VEGF expression

There is no previous study examining the combined effect of hypoxia and

hyperglycaemia on VEGF expression of the first trimester trophoblasts. Results from the

present study, the VEGF mRNA and protein expression in cells treated with 25 mM

glucose and 1% oxygen were similar to those treated with 1% oxygen alone, suggesting

that hyperglycemia did not affect hypoxia-induced VEGF expression. This response of

TEV-1 cells is different from that of proximal tubular kidney cells; high glucose

suppressed hypoxia-induced VEGF mRNA and protein response in cultured proximal

tubular cells (Katavetin, 2006). The effect of hyperglycemic treatment was also

83

demonstrated in vivo using the ischemia-reperfusion model and streptozotocincin-induced

hyperglycemia in hypoxia-responsible element (HRE)-luciferase transgenic rats

(Katavetin, 2006). Thus the suppressive effect of high glucose on hypoxia-induced VEGF

expression in the proximal tubular cells was suggested to be mediated, at least in part, by

alteration of the HIF-HRE pathway. However, the results was not supported in another

study on the same cell type showing that both VEGF mRNA and protein expressions

were enhanced with no additive effects of the two treatments (Kim et al., 2002).

4.10 Effect of hyperglycemia on GLUT-1 expression

Glucose is the primary source of energy for the human fetus and the placenta. The

fetus produces minimal amounts of glucose. Therefore, glucose supply needs to come

from the mother. Transport of glucose across the placenta is generally via protein-

mediated facilitated diffusion involving a number of glucose transporters (Marconi,

1996). The primary glucose transporter present in placental villous tissue is GLUT-1,

which is a ubiquitous isoform of the facilitated-diffusion glucose transporter family,

expressing in almost all tissues examined thus far and is regarded as the constitutive form

of the transporter. The presence of GLUT-1 in the placenta has been confirmed by

Western blotting (Jansson et al., 1993;Barros et al., 1995), immunohistochemical

observation (Farrell, 1992;Takata, 1992;Jansson et al., 1993)and expression of mRNA

(Jansson et al., 1995;Clarson, 1997).

Glucose transporters in various cells and tissues have been shown to be regulated by

a diverse range of factors, the most common being glucose and glycolytic substrates

(Klip, 1994;Sasson, 1997). Several groups have examined the response of placental

84

glucose transporters to varying extracellular glucose concentrations. Hyperglycaemia

treatment at extracellular glucose concentrations ≥20 mM substantially reduces the

expression of GLUT-1 mRNA, and to lesser extent the protein expression and activity of

GLUT-1 (Hahn et al., 1998; Gordon, 1995; Illsley, 1998). However, at glucose

concentrations <20 mM, some reports showed a decrease in activity when compared to

control (Gordon, 1995; Illsley, 1998), whereas others indicated no change in activity

(Illsley, 1998; Hahn et al., 1998).

In TEV-1 cells, expression of the GLUT-1 was suppressed by treatment with

exogenous glucose at concentration of 25 mM. Overall, the evidence suggests that that

the protein expression of the transporter was suppressed under hyperglycemic condition

when compared with that in euglycemic condition. Thus the cells are expected to take up

less glucose. This decrease in glucose transport can be a mechanism to protect the fetus

for uptaking excessive glucose in hyperglycemic condition. It has been suggested that

prolonged hyperglycaemia downregulates GLUT-1 transporter in human trophoblasts

(Hahn et al., 1998). However, the decrease in GLUT-1 mRNA is transient and return to

the control value after 48 hours. This changing pattern of Glut-1 expression in response

to hyperglycemia demonstrates the ability of TEV-1 to normalize Glut-1 expression and

recover from hyperglycemia after 48 hours. Similar kinetics has also been observed in

heart tissue (Joyner, 2004).

However, these outcomes from in vitro studies are contrary to the in vivo

observations in women with diabetes. The expression of basal membrane GLUT-1

protein and the glucose transport activity of syncytial basal membranes are significant

increased in purified microvillous and basal membranes from term pregestational and

85

gestational diabetes (Gaither et al., 1999). This observation is supported by the results of

another investigation looking at the expression of GLUT-1 in syncytial membranes from

the placental tissue of long-term (>20 year) insulin-dependent diabetics (Jansson, 1999).

The discrepancies between in vitro and in vivo observations could be due to the

involvement of complex regulatory mechanisms of glucose transport in vivo involving

multiple factors, such as maternal placental growth hormone level (Baumann et al., 2002),

whereas a chemically defined condition was used in in vitro study. The other possibility

is the difference in the physiology of the trophoblast cells in first trimester and in term

pregnancy.

4.11 Effect of hypoxia on GLUT-1 expression

Regulation of GLUT-1 by hypoxia has been extensively studied in a variety of

tissues. In hypoxic conditions, GLUT-1 gene transcription in tissues is increased through

the HIF pathway as a result of decreased degradation of the HIF-1 (Semenza, 2000a).

Furthermore, hypoxia and inhibitors of oxidative phosphorylation decrease degradation

of GLUT-1 transcripts (Ebert et al., 1995). Although there are a number of studies

investigating the response of trophoblasts to low oxygen tension, most of them study the

role of hypoxia on the differentiation and invasive potential of trophoblasts. Only a few

reports on the regulation of placental or trophoblast glucose transport by hypoxia have

been published (Esterman et al., 1997;Illsley, 1984;Das et al., 1998).

An up-regulation of GLUT-1 mRNA and protein expressions were observed in

TEV-1 cells cultured in hypoxic condition after 48 hours (Chapter 3). In BeWo cells,

GLUT-1 protein was found up-regulated following exposure to cobalt, desferroxamine (a

86

hypoxia inducing agent) or 1% O2 for 12 hours, and remained elevated for at least 72

hour (Baumann et al., 2002;Baumann et al., 2007). These data confirm that isolated

trophoblast cells respond to hypoxia in a manner similar to many other cells types,

increasing the GLUT-1 levels in both the acute and chronic hypoxic conditions. Indeed,

another glucose transporter found in the placenta, GLUT-3, has also reported to be up-

regulated by low oxygen levels (Baumann, 2000).

Intracellular glucose is accumulated in two distinct pools; the metabolic pool is used

for glycolysis and the structural pool is used for the synthesis of glycoproteins and

extracellular matrix macromolecules. In aerobic conditions, oxidative phosphorylation

breaks down glucose efficiently to produce maximum amount of ATP. However, in the

absence of oxidative phosphorylation in anaerobic conditions glycolysis dominates and

significantly higher levels of lactic acid are produced (Airley and, 2007). This metabolic

switch to anaerobic glycolysis is through to be regulated by HIF-1 transcription factor, as

indicated by the decreased growth rates, lower lactic acid production and decreased

acidosis in mammalian cells lacking HIF-1 during hypoxia (Seagroves, 2001). Cancer

cells also produce a high level of lactic acid in hypoxic environment (Brahimi-Horn and

Pouyssegur, 2007b).

The increase in GLUT-1 expression in hypoxic condition is thought to be a

compensatory response in order to maintain glucose homeostasis. Although glycolysis

functions to produce ATP during hypoxia, energy production by the process is far less

than that by oxidative phosphorylation in normoxic environment. Thus, in order to keep

up with the energy demand for metabolic activities, the cells increase the expression of

87

GLUT-1 and probably the other glucose transporter as well to increase glucose uptake for

energy production by a less efficient process, glycolysis.

4.12 Combine effect of hyperglycemia and hypoxia on GLUT-1 expression

In this study, we demonstrated that, the expression levels of GLUT-1 in TEV-1

cultured in hypoxic and hyperglycemic condition were significantly higher (P<0.05) than

that of cells cultured in normoxic and hyperglycemic condition. However, the kinetics

and magnitude are similar to those cultured in hypoxia condition alone. No similar study

have been performed in human trphoblast. The results also suggest that hypoxia can

overcome the suppressing effect of hyperglycemia on GLUT-1 expression.

4.13 Effect of hyperglycemia and hypoxia on insulin signaling molecules

The present results showed that insulin signaling molecules including IR, IRS-1 and

-2 were expressed in the first trimester trophoblast cell line. Autophosphorylation of IR

upon insulin binding triggers a cascade of phosphorylation of protein substrates inside a

cell, allowing them to enhance the biological effects of insulin. IRSs are the chief

substrates for the IR tyrosine kinase. The other main substrates include Src and collagen

homologous protein Shc (White and, 1998).

IR is expressed on the plasma membrane of human placental cells (Desoye et al.,

1997). Interestingly, this study demonstrated the expression of IR in the nuclei of the

TEV-1 cells. It has been reported that insulin receptors are translocated to the nucleus

after a glucose meal in mouse hepatocytes via internalization process (Gletsu et al., 1999).

Such translocation of IR was proposed to alter the phosphorylation state of DNA binding

88

proteins in the nucleus enhancing the transcription rate of related genes (Gletsu et al.,

1999). Another possibility was that the insulin receptor itself in the nucleus may induce

insulin signaling pathway directly (Gletsu et al., 1999).

Although the functions and properties of insulin signaling molecules have been

extensively characterized in many cell types, the information of these molecules in

trophoblasts and placenta are contradictory. In term placentas, the amount of IR and IRS-

1 are higher from GDM women with adequate glycaemic control than placentas from

normal pregnant women (Alonso et al., 2006). In contrast, the results of Desoye and co-

workers (1992) show that trophoblast plasma membranes from GDM women expressed

less IR than that from a healthy control group in vitro. In mouse embryonic fibroblast,

hypoxia leads to caspase-mediated cleavage of IRS-1 (Kang, 1997). Although several

reports failed to document insulin-stimulated glucose transport in the human placenta

(Challier, 1986;Rankin, 1986;Schmon, 1991), insulin induced phosphorylation of

mitogen associated protein kinase (Boileau, 2000) and a number of DNA-binding

proteins (Cheatham and Kahn, 1995) in trophoblast cells leading to enhanced DNA

synthesis.

Virtually nothing is known about how oxygen tension and hyperglycemia

condition affects the expression of insulin signaling molecules in human placenta. The

results of the insulin signaling molecules fluctuated widely within the treatment period

making a definitive conclusion difficult. Nevertheless, the results showed that after 48

hour of culture, hyperglycemia up-regulated IR immunoreactivities and mRNA

expression, while hypoxia down-regulated IRS-2 immunoreactivities and mRNA

89

expression. Hypoxia and hyperglycemic treatment had no effect on IRS-1 expression.

The physiological implication of these observation remains to be investigated.

4.14 Conclusion

The first trimester trophoblast cell line, TEV-1 responded to hyperglycemic and

hypoxic treatment in terms of cell proliferation and gene expression. The responses of the

cells to VEGF and GLUT-1 could be explained as means of the cells to maintain its

oxygen supply and glucose homeostasis respectively. The effects of the hypoxic and

hypoglycemic treatments were distinct from each other. Apart from GLUT-1 mRNA

expression, there was no additive or synergistic effect on all the parameters measured in

the combined hypoxic and hyperglycemic treatment. The data indicated that

hyperglycemia and hypoxia act independently to affect the biology of the TEV-1 cells.

90

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