Title

Effects of hypoxia and hyperglycemia on proliferation and expression of glucose-related signaling molecules in extravillous trophoblast cell line in vitro Chan, Yuk-ling; sss

Author(s)

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

Abstract of thesis entitled

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 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 trophoblast cell line in vitro 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)

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 Figure 3.1

Distribution of Glut-1 in the placenta 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 under normoxic and hypoxic conditions

Figure 3.5 Figure 3.6

Immunofluorescent staining of VEGF expression in TEV-1 Effect of glucose concentration on Glut-1 mRNA expression under normoxic and hypoxic conditions

Figure 3.7

Effect of glucose concentration on Glut-1 protein expression under normoxic and hypoxic conditions

Figure 3.8 Figure 3.9

Immunofluorescent staining of Glut-1 expression in TEV-1 Effect of glucose concentration on IR mRNA expression under normoxic and hypoxic conditions

Figure 3.10 Effect of glucose concentration on IRS-1 mRNA expression under normoxic and hypoxic conditions Figure 3.11 Effect of glucose concentration on IRS-2 mRNA expression under normoxic and hypoxic conditions Figure3.12 Effect of glucose concentration on IR protein expression under normoxic and hypoxic conditions
viii

Figure 3.13 Effect of glucose concentration on IRS-1 protein expression under normoxic and hypoxic conditions Figure 3.14 Effect of glucose concentration on IRS-2 protein expression under normoxic and hypoxic conditions 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 Table 2.2 Table 2.3 Table 2.4

Chemicals that were used in the study Composition of Ham's F-10

Primary antibodies used in Western Blot Analysis Primary antibodies used in immunofluorescent staining

Table of Abbreviation

HCG IUGR DNA VEGF PIGF TNFalpha HLA-G ROS HIF PAS ARNT mRNA Epo Glut-1 PDK-1 HRE MAPK PI3K mmHg ATP HepG2 kDa GDM OGTT FPG

Human chorionic gonadotrophin Intrauterine growth restriction Deoxyribonucleic acid Vascular endothelial growth factor Placental growth factor Tumor necrosis factor alpha Human leukocyte antigen Reactive oxygen species Hypoxia-inducible factor PER-ARNT-SIM Aryl hydrocarbon receptor nuclear translocator Messenger ribonucleic acid Erythropiotein Glucose transporter pyruvate dehydrogenase kinase-1 Hypoxia-responsive elements Mitogen-activated protein kinase Phosphatidylinositol 3-kinase Millimeter of mercury Adenosine Triphosphate Human hepatocellular liver carcinoma cell line Kilodalton Gestational diabetes mellitus Oral glucose tolerance test Fasting plasma glucose level
xi

mg/dl g EGF PDGF PKB IRS PH PTB SDS PDK IMA HPV ml
o

milligrams/deciliter gram Epidermal growth factor platelet-derived growth factor Protein kinase B Insulin receptor substrate Pleckstrin homology Phosphotyrosine-binding Sodium Dodecyl Sulfate 3-Phosphoinositide-dependent kinase Ischemia-modified albumin Human papillomavirus milliliter Degree Celcius Carbon dioxide Ethylenediaminetetraacetic acid Minute Revolutions per minute Phosphate buffer saline Dimethyl sulfoxide microliter Minimolar Molar Ultraviolet nanometer Ribonucleic acid diethypyrocarbonate

CO2 EDTA min rpm PBS DMSO l mM M UV nm RNA DEPC

xii

g/l TBE bp RT cDNA dNTP MgCl2 MQ H2O PCR HCL NaCl DOC NP BSA SDS-PAGE DH2O APS TEMED mA PVDF V PBST ECL IR VEGF

Microgram per microliter Tris-borate-EDTA Base pair Reverse Transcription Complementary dioxribonucleic acid Deoxyribonucleotide triphosphate Magnesium chloride
Milli-q water

Water Polymerase chain reaction Hydrochloride Sodium chloride Sodium deoxycholate Nonyl phenoxylpolyethoxylethanol Bovine serum albumin SDS Polyacrylamide Gel Electrophoresis Distilled water Ammonium peroxodisulphate N,N,N',N'-Tetramethylethylenediamine milliampere Polyvinylidene Fluoride Voltage Phosphate Buffered Saline solution with Tween 20 Enhanced chemiluminescence Insulin receptor Vascular endothelial growth factor

xiii

Contents
Abstract Declaration Acknowledgements List of Figures List of Tables List of Abberviations iii vi vii viii x xi

Chapter 1

Literature Review

1 1 2 2 3 4 5 6 6 8 9 10 11 14 14 15 16

1.1 Placental development 1.2 Structure of Placenta 1.3 Functions of placenta 1.4 Nutrients transporation in the placenta 1.5 Trophoblast 1.6 Extravillous trophoblast cell line 1.7 Gaseous exchange in the placenta 1.8 Oxygen tension and hypoxic condition 1.9 Translocation of HIF-1 1.10 Hypoxic environment in placenta and trophoblast 1.11 Glucose transport in placenta 1.12 Glucose transporter-1 (GLUT-1) 1.13 Abnormal glucose metabolism 1.14 Uteroplacental Insufficiency 1.15 Gestational diabetes mellitus 1.16 Insulin signalling pathway

xiv

1.17 Insulin receptor 1.18 Cascade of signalling 1.19 Insulin receptor substrate-1 (IRS-1) 1.20 Insulin receptor substrate-2 (IRS-2) 1.21 Roles of IRS-1 and IRS-2 in IR pathway on glucose metabolism 1.22 Placental changes in hyperglycaemia and hypoxia 1.23 Aims of study 1.24 Hypothesis

16 17 18 18 19 21 21 22

Chapter 2

Materials and Methods

24 25 25 25 25 25 26 26 27 28 28 29 32 32 33

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

xv

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

33 33

34 34 35 35 35 36 37 37

37

38 39

Chapter 3

Results

40 41

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 3.2.2 3.2.3 Expression of VEGF mRNA Expression of VEGF protein Immunofluorescent staining of VEGF

45

45 47 49

xvi

3.3

Effect of different concentrations of glucose and oxygen on GLUT-1 expression in TEV-1 cells 3.3.1 3.3.2 3.3.3 Expression of GLUT-1 mRNA Expression of GLUT-1 protein Immunofluorescent staining of GLUT-1

51

51 54 56 58

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 3.4.2 3.4.3 Expression of IR, IRS-1 and IRS-2 mRNA Expression of IR, IRS-1 and IRS-2 proteins Immunofluorescent staining of IR, IRS-1 and IRS-2

58 63 68

Chapter 4

Discussion

72

Conclusion

90

References

91

xvii

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

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

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, maternofetal 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

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

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-1 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.

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

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- subunits (HIF-1, HIF-2 and HIF-3) (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-1 subunit (Licht et al., 2006) giving rise to three different heterodimers. All subunits were encoded by distinct gene loci and using alternative promoters and splicing pattern. The domain architecture of HIF-1 and HIF2 are similar, but the latter has more limited tissue expression, while HIF-3 was least studied (Weidemann and Johnson, 2008). HIF-3 lacks structures for transactivation in the C-termini as other HIF- isoforms, supporting a role of HIF-3 in forming inactive

heterodimers with HIF-1 to inhibit HIF response (Weidemann and Johnson, 2008;Nangaku et al., 2008). While HIF-1 is constitutively expressed (Salceda and Caro, 1997), the expression of HIF-1 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-1 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-1 HIF-1 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

hypoxic condition, HIF-1

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-1 dimerizes with HIF-1, 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 signalregulated kinase 1 and 2, and phosphatidylinositol 3-kinase (PI3K) pathways are responsible for the activation of the downstream molecules induced by HIF1 (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

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 (BrahimiHorn 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 fetalfacing 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 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 -subunits, each of which is linked to a -subunit by disulfide bonds. The -subunits are located outside the cell and bind to insulin, while the -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 -subunits, insulin activates the tyrosine kinase activity of insulin receptor -subunits, and phosphorylates the adjacent -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 PI3KPKB 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) IRS-2 is expressed in various cell types, including fibroblasts, liver, lung, spleen, heart, kidney, 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

compensate insulin resistance by islet hyperplasia. IRS-1 and IRS-2 have differential roles. IRS-1 is needed for proper insulin production 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 Glut-4 is the predominant insulin-mediated glucose transporter expressed in muscle 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 mice 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 the pathophysiology and clinical 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 various hormones such as estrogen, progesterone, 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 In vitro study showed that proliferation of cultured human undifferentiated placental 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).

1.23 Aims of study It is widely accepted that maternal diabetes significantly alters the expression of molecules 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 The hypothesis of the project was that high glucose up-regulated the expression of 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 2.11 Reverse transcription and quantitative real time PCR (qPCR) analysis of 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 121C 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 (37C) for culture.

2.4 Hypoxic chamber The hypoxic environment was established and maintained in a hypoxic chamber (C374, 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 m 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 4C. 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 0.05% trypsin/EDTA solution 1% [100U] of penicillin/streptomycin 10%APS 30% Polyacrylamide 5% skimmed milk Acetone Agarose Antibody diluent Beta-mercaptoethanol Boric acid Bovine serum albumin [BSA] Bromophenol Blue Chloroform Coomassie Plus Diethyl pyrocarbonate DMSO ECL Western Blotting Reagents EDTA Ethanol Ethidium bromide F-10 Nutrient Mixture (Ham) Fetal bovine serum Full-Range Rainbow Molecular Weight Markers Glucose Suppliers Invitrogen, Carlsbad, CA, USA Invitrogen Sigma-Aldrich Bio-Rad, Hercules, CA, USA Carnation, Nestl S.A., Vevey, Switzerland Merck KGaA, Darmstadt, Germany Invitrogen Dako, Glostrup, Denmark Sigma-Aldrich USB USB Corp. Bio-Rad, Hercules, CA, USA Sigma-Aldrich Pierce Sigma-Aldrich Sigma-Aldrich GE Healthcare Bio-Sciences USB Corp. Merck KGaA Sigma-Aldrich Invitrogen Invitrogen GE Healthcare Bio-Sciences, Piscataway, USA Sigma-Aldrich 100034 15510-027 S3022 M3148 76324 U510857-100G 161-0404 C2432 PIE-23236 D5758 D2650 RPN2106 US15701 100983 E1510 12390-035 10270-106 RPN800E G6125/G7773 Catalogue Number 15400-054 15070-063 A9164 161-0156

27

Glycerol Glycine Horse serum Isopropanol Methanol NaCl NP-40 Penicillin-G/Streptomycin Phosphate buffer saline (PBS) Propidium iodide Protease Inhibitor Sodium deoxycholate [DOC] Sodium dodecyl sulfate [SDS] TEMED Tris Triton X-100 TRIzol reagent Trypan blue Tween-20

BDH, Poole, England USB Corp. Sigma-Aldrich Merck KGaA Merck KGaA Sigma-Aldrich, St. Louis, Missouri, USA Sigma-Aldrich, St. Louis, Missouri, USA Invitrogen Sigma-Aldrich, St. Louis, Missouri, USA Sigma-Aldrich, St. Louis, Missouri, USA Roche Diagnostics Sigma-Aldrich BDH Bio-Rad USB Corp Sigma-Aldrich, St. Louis, Missouri, USA Invitrogen Invitrogen BDH

101185L US16407-5kg H1270-100ml 109634 106009 S6150 N6507 15070 P4417 P4864 11697498001 D6750 444464T 161-0801 US75825-5kg T8532 15596-018 15250-061 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 Hams F-10 (Invitrogen) was used for trophoblast cell culture. The composition of Hams F-10 is shown in Table 2.2. The working Hams F-10 medium contained 5% heatinactivated fetal bovine serum (Invitrogen), 1% of penicillin-G, and 1% of streptomycin sulphate. The solution was filtered through 0.22 m 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 Hams F-10

COMPONENTS Amino Acids Glycine L-Alanine L-Arginine hydrochloride L-Asparagine-H2O L-Aspartic acid L-Cysteine L-Glutamic Acid L-Glutamine L-Histidine hydrochloride-H2O L-Isoleucine L-Leucine L-Lysine hydrochloride L-Methionine L-Phenylalanine L-Proline L-Serine L-Threonine L-Tryptophan L-Tyrosine disodium salt dihydrate L-Valine Vitamins Biotin Choline chloride D-Calcium pantothenate

Molecular Weight 75 89 211 150 133 121 147 146 210 131 131 183 149 165 115 105 119 204 261 117

Concentration (mg/L) 7.5 9 211 15 13 25 14.7 146 23 2.6 13 29 4.5 5 11.5 10.5 3.6 0.6 2.62 3.5

Molarity

0.1 0.101124 1 0.1 0.097744 0.206612 0.1 1 0.109524 0.019847 0.099237 0.15847 0.030201 0.030303 0.1 0.1 0.030252 0.002941 0.010038 0.029915

244 140 477

0.024 0.7 0.7

0.000098 0.005 0.001468

30

Folic Acid Niacinamide Pyridoxine hydrochloride Riboflavin Thiamine hydrochloride Vitamin B12 i-Inositol Inorganic Salts Calcium Chloride (CaCl2) (anhyd.) Cupric sulfate (CuSO4-5H2O) Ferric sulfate (FeSO4-7H2O) Magnesium Sulfate (MgSO4) (anhyd.) Potassium Chloride (KCl) Potassium Phosphate monobasic (KH2PO4) Sodium Bicarbonate (NaHCO3) Sodium Chloride (NaCl) Sodium Phosphate dibasic (Na2HPO4) anhydrous Zinc sulfate (ZnSO4-7H2O) Other Components D-Glucose (Dextrose) Hypoxanthine Na Lipoic Acid Phenol Red Sodium Pyruvate Thymidine

441 122 206 376 337 1355 180

1.3 0.6 0.2 0.4 1 1.4 0.5

0.002948 0.004918 0.000971 0.001064 0.002967 0.001033 0.002778

111 250 278 120 75 136 84 58 142 288

33.3 0.0025 0.834 74.62 285 83 1200

0.3 0.00001 0.003 0.621833 3.8 0.610294 14.285714

7400 127.586205 153.7 0.03 1.082394 0.000104

180 159 206 376.4 110 242

1100 4.7 0.2 1.2 110 0.7

6.111111 0.02956 0.000971 0.003188 1 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 Hams F-10 with 5% fetal calf serum at 37C 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 manufacturers 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 l of working solution was added to the TEV-1 cell culture. XTT working solution was prepared just prior to use according to manufacturers protocol by mixing 1 ml of XTT stock solution with 20 l of electron coupling reagent. The cells were then incubated further at 37C in 5% CO2 for 4 hours, and the absorbency was measured using micro-plate reader at 450 nm (TECAN Infinite F200, Tecan Group Ltd., Mnnedorf, 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

2.11 Reverse transcription and quantitative real time PCR (qPCR) analysis of 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 manufacturers 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 4C 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 500l 75% ethanol and dissolved in 50 l 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 l 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 (l) 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 18.45 Total The mixture was incubated for 10 min at 25C, for 40 mins at 48C, then for 5 min at 95C. 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 RealTime 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 l reaction mixture containing 5 l of sample DNA;

34

10l 2X TaqMan Universal PCR Master Mix and 1l 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 10106 TEV-1 was washed in PBS for three times, centrifuged and pelleted by centrifugation at 1000g for 10 minutes, and resuspended in 150 l 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 95C for 5 minutes with 2-mercaptoethanol. These denatured proteins were then loaded onto a 10% of SDS-polyacrylamide gel (BioRad, Hercules, CA, USA). SDS-PAGE was performed in a Mini-protein III system (Bio35

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 m. 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, Nestl 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 4C, 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 manufacturers instructions.

Autoradiography for signal detection was performed with Lumi-Film Chemiluminescent Detection Films (Roche Diagnostics) using the X-ray Film Processor (FUJIFILM FMP100A, 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 Beta-Actin Glut-1 IR IRS-1 IRS-2 VEGF Company Sigma-Aldrich Santa Cruz CST CST CST Zymed Dilution 1:100000 1:500 1:1000 1:1000 1:1000 1:500 Source Mouse Goat Mouse Rabbit Rabbit 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 Hams 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 4C 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 4C. 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 Glut-1 IR IRS-1 IRS-2 VEGF Company Santa Cruz CST CST CST Zymed Dilution 1:100 1:50 1:50 1:50 1:50 Source Goat Mouse Rabbit Rabbit 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 3.2 Effect of concentration of glucose and oxygen on the viability and proliferation of TEV-1 Effect of different concentrations of glucose and oxygen on VEGF expression in TEV-1 cells 3.2.1 3.2.3 3.3 Expression of VEGF mRNA Immunofluorescent staining of VEGF 3.2.2 Expression of VEGF protein Effect of different concentrations of glucose and oxygen on GLUT-1 expression in TEV-1 cells 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. 25 mM P<0.05
1.4 1.2 1.0 5.5 mM 25 mM

P>0.05

OD450

0.8 0.6 0.4

P<0.05
0.2 0.0 0 20 40

5.5 mM

P>0.05

60

80

Hours
25 mM

P<0.05
1.4 1.2 1.0

P>0.05

OD450

0.8 0.6 0.4 0.2 0.0 0 20 40 60 80

P<0.05

5.5 mM

P>0.05

5.5 mM 25 mM

Hours

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.

1.4 1.2 1.0 Normoxia Hypoxia

OD450

0.8 0.6 0.4 0.2 0.0 0

20

40

60

80

Hours

1.4 1.2 1.0 Normoxia Hypoxia

OD450

0.8 0.6 0.4 0.2 0.0 0

20

40

60

80

Hours

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# a# #a

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 -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
H Normoxia

48 hours M N

24 hours H M N

8 hours H M N

48 hours H Hypoxia M N H

24 hours M N H

8 hours M N

B # a # # # a # a b

# a 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 timedependent 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-b

P<0.05 when compared with cells treated in different

conditions at the same time point.

a 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 -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 8hour 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

Figure 3.7 The effects of different glucose conditions (N: 5.5 mM; M: 15 mM; H: 25 mM) on the GLUT-1 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 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. when compared with cells treated in different conditions at the same time point.
a-b

P<0.05

(A)
H Normoxia

48 hours M N H

24 hours M N H

8 hours M N

48 hours H Hypoxia M N H

24 hours M N H

8 hours M N

(B)
# a a a a # b

# b

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

# 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 mRNA were determined by qPCR. The values are mean S.E.M. of five independent experiments. IRS-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.

# # 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 mRNA were determined by qPCR. The values are mean S.E.M. of five independent experiments. IRS-2 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.

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 -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

Figure 3.12 The effects of different glucose conditions (N: 5.5 mM; M: 15 mM; H: 25 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) Semiquantitative 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.

(A)
H normoxia

48 hours M N H

24 hours M N H

8 hours M N

48 hours H hypoxia M N H

24 hours M N H

8 hours M N

(B)

# #

65

Figure 3.13 The effects of different glucose conditions (N: 5.5 mM; M: 15 mM; H: 25 mM) on the IRS-1 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-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 TEV1 cells under normoxic (20% oxygen) and euglycemia (5.5 mM) was used as control.

(A)
H normoxia

48 hours M N H

24 hours M N H

8 hours M N

48 hours H hypoxia M N H

24 hours M N H

8 hours M N

(B)

66

Figure 3.14 The effects of different glucose conditions (N: 5.5 mM; M: 15 mM; H: 25 mM) on the IRS-2 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-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 TEV1 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-b

P<0.05 when

compared with cells treated in different conditions at the same time point. 48 hours 24 hours N H M N H 8 hours M N

(A)
normoxia

48 hours H hypoxia M N H

24 hours M N H

8 hours M N

(B)

a #

a b # 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 treating the cells with different concentrations of oxygen (1%, 20%) and glucose (5.5 mM, 25 mM) for two days. The IRS-1 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.

70

Figure 3.17 Immunofluorescent staining for IRS-2 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 IRS-2 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.

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

72

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 (ForsbachSanchez 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

80

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 sevenfold 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 agematched 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-1 (aryl hydrocarbon nuclear translocator, ARNT) and the inducible HIF-1. In conditions of low oxygen, the stable HIF-1 subunit dimerizes with HIF-1 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-1 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 proteinmediated 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 upregulated 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 IRS1 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 coworkers (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

References

1. 2008. [Changes in the variables of coagulation and fibrinolysis at preeclampsia and spontaneous abortion]. Akush. Ginekol. (Sofiia) 47:9-13.

2. Aghayan, M., L.V.Rao, R.M.Smith, L.Jarett, M.J.Charron, B.Thorens, and S.Heyner. 1992. Developmental expression and cellular localization of glucose transporter molecules during mouse preimplantation development. Development 115:305-312.

3. Ahmed, A. 1995. Colocalisation of vascular endothelial growth factor and its Flt1 receptor in human placenta. Growth Factors 12:235-243.

4. Ahmed, A. 1997. Role of VEGF receptor-1 (Flt-1) in mediating calciumdependent nitric oxide release and limiting DNA synthesis in human trophoblast cells. Lab Invest 76:779-791.

5. Ahmed, A. 2000. Regulation of placental vascular endothelial growth factor (VEGF) and placenta growth factor (PIGF) and soluble Flt-1 by oxygen--a review. Placenta 21 Suppl A:S16-24:S16-S24.

6. Airley and, R.E. 2007. Hypoxic regulation of glucose transport, anaerobic metabolism and angiogenesis in cancer: novel pathways and targets for anticancer therapeutics. Chemotherapy 53:233-256.

91

7. al-Lamki, R.S., J.N.Skepper, and G.J.Burton. 1999. Are human placental bed giant cells merely aggregates of small mononuclear trophoblast cells? An ultrastructural and immunocytochemical study. Hum. Reprod. 14:496-504.

8. al-Okail, M.S. and O.S.al-Attas. 1994. Histological changes in placental syncytiotrophoblasts of poorly controlled gestational diabetic patients. Endocr. J. 41:355-360.

9. Alonso, A., C.G.Del Rey, A.Navarro, J.Tolivia, and C.G.Gonzalez. 2006. Effects of gestational diabetes mellitus on proteins implicated in insulin signaling in human placenta. Gynecol. Endocrinol. 22:526-535.

10. American Academy Family Physician [ homepage on the Internet]. Examination of the Placenta. Vol 57. No.5; [ Rev 1 Mar 1998; cited 8 Aug 2008]. Available from: http://www.aafp.org/afp/980301ap/yetter.html

10. Antonova, O.A., S.A.Loktionova, N.V.Golubeva, Y.A.Romanov, and A.V.Mazurov. 2007. Damage and activation of endothelial cells during in vitro hypoxia. Bull. Exp. Biol. Med. 144:504-506.

11. Araki, E., X.J.Sun, B.L.Haag, III, L.M.Chuang, Y.Zhang, T.L.Yang-Feng, M.F.White, and C.R.Kahn. 1993. Human skeletal muscle insulin receptor substrate-1. Characterization of the cDNA, gene, and chromosomal localization. Diabetes 42:1041-1054.

92

12. Bajaj, M. and R.A.Defronzo. 2003. Metabolic and molecular basis of insulin resistance. J. Nucl. Cardiol. 10:311-323.

13. Barbour, L.A. 2003. New concepts in insulin resistance of pregnancy and gestational diabetes: long-term implications for mother and offspring. J. Obstet. Gynaecol. 23:545-549.

14. Barros, L.F., D.L.Yudilevich, S.M.Jarvis, N.Beaumont, and S.A.Baldwin. 1995. Quantitation and immunolocalization of glucose transporters in the human placenta. Placenta 16:623-633.

15. Bashan, N., E.Burdett, H.S.Hundal, and A.Klip. 1992. Regulation of glucose transport and GLUT1 glucose transporter expression by O2 in muscle cells in culture. Am. J. Physiol 262:C682-C690.

16. Battaglia FC, Meschia G. An Introduction to fetal physiology. Orlando, FL:Academic Press, 1986.

17. Baumann, C.A. 2000. CAP defines a second signalling pathway required for insulin-stimulated glucose transport. Nature 407:202-207.

18. Baumann, M.U., S.Deborde, and N.P.Illsley. 2002. Placental glucose transfer and fetal growth. Endocrine. 19:13-22.

93

19. Baumann, M.U., S.Zamudio, and N.P.Illsley. 2007. Hypoxic upregulation of glucose transporters in BeWo choriocarcinoma cells is mediated by hypoxiainducible factor-1. Am. J. Physiol Cell Physiol 293:C477-C485.

20. Bell, G.I., T.Kayano, J.B.Buse, C.F.Burant, J.Takeda, D.Lin, H.Fukumoto, and S.Seino. 1990. Molecular biology of mammalian glucose transporters. Diabetes Care 13:198-208.

21. Bloxam, D.L., B.E.Bax, and C.M.Bax. 1997. Culture of syncytiotrophoblast for the study of human placental transfer. Part II: Production, culture and use of syncytiotrophoblast. Placenta 18:99-108.

22. Boileau, C. 2000. Regulation of extrathymic T cell development and turnover by oncostatin M. J. Immunol. 164:5713-5720.

23. Bose, P. 2006. Aberrations of early trophoblast differentiation predispose to pregnancy failure: lessons from the anti-phospholipid syndrome. Placenta 27:869-875.

24. Boyd, J.D.a.H.W.J. 1970. The Human Placenta. W. Heffer, Cambridge..

25. Bhatia J. edited, Perinatal Nutrition Optimizing infant Health and Development, USA: Dekker, 2005, 81p.

94

26. Brahimi-Horn, M.C. and J.Pouyssegur. 2007a. Hypoxia in cancer cell metabolism and pH regulation. Essays Biochem. 43:165-178.

27. Brahimi-Horn, M.C. and J.Pouyssegur. 2007b. Hypoxia in cancer cell metabolism and pH regulation. Essays Biochem. 43:165-178.

28. Breier, G., A.H.Licht, A.Nicolaus, A.Klotzsche, B.Wielockx, and Z.Kirsnerova. 2007. HIF in vascular development and tumour angiogenesis. Novartis. Found. Symp. 283:126-133.

29. Brown, L.F. 1993. Increased expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in kidney and bladder carcinomas. Am. J. Pathol. 143:1255-1262.

30. Buchanan, T.A. and A.H.Xiang. 2005. Gestational diabetes mellitus. J. Clin. Invest 115:485-491.

31. Burton, G.J. 2002. Uterine glands provide histiotrophic nutrition for the human fetus during the first trimester of pregnancy. J. Clin. Endocrinol. Metab 87:29542959.

32. Caniggia, I. 2000. Hypoxia-inducible factor-1 mediates the biological effects of oxygen on human trophoblast differentiation through TGFbeta(3). J. Clin. Invest 105:577-587.

95

33. Caniggia, I., J.Winter, S.J.Lye, and M.Post. 2000b. Oxygen and placental development during the first trimester: implications for the pathophysiology of pre-eclampsia. Placenta 21 Suppl A:S25-S30.

34. Caniggia, I., J.Winter, S.J.Lye, and M.Post. 2000a. Oxygen and placental development during the first trimester: implications for the pathophysiology of pre-eclampsia. Placenta 21 Suppl A:S25-S30.

35. Catalano, P.M., S.E.Nizielski, J.Shao, L.Preston, L.Qiao, and J.E.Friedman. 2002. Downregulated IRS-1 and PPARgamma in obese women with gestational diabetes: relationship to FFA during pregnancy. Am. J. Physiol Endocrinol. Metab 282:E522-E533.

36. Challier, J.C. 1986. Effect of insulin on glucose uptake and metabolism in the human placenta. J. Clin. Endocrinol. Metab 62:803-807.

37. Chartrel, N.C., M.T.Clabaut, F.A.Boismare, and J.C.Schrub. 1990. Uteroplacental hemodynamic disturbances in establishment of fetal growth retardation in streptozocin-induced diabetic rats. Diabetes 39:743-746.

38. Cheatham, B. and C.R.Kahn. 1995. Insulin action and the insulin signaling network. Endocr. Rev. 16:117-142.

96

39. Cheatham, B., C.J.Vlahos, L.Cheatham, L.Wang, J.Blenis, and C.R.Kahn. 1994. Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation. Mol. Cell Biol. 14:4902-4911.

40. Choy, M.Y. and I.T.Manyonda. 1998. The phagocytic activity of human first trimester extravillous trophoblast. Hum. Reprod. 13:2941-2949.

41. Choy, M.Y., W.G.St, and I.T.Manyonda. 2000. Efficient, rapid and reliable establishment of human trophoblast cell lines using poly-L-ornithine. Early Pregnancy. 4:124-143.

42. Clarson, L.H. 1997. Expression of the facilitated glucose transporters (GLUT1 and GLUT3) by a choriocarcinoma cell line (JAr) and cytotrophoblast cells in culture. Placenta 18:333-339.

43. Cleal, J.K. and R.M.Lewis. 2008. The mechanisms and regulation of placental amino acid transport to the human foetus. J. Neuroendocrinol. 20:419-426.

44. Cohen, M., A.Meisser, L.Haenggeli, and P.Bischof. 2006. Involvement of MAPK pathway in TNF-alpha-induced MMP-9 expression in human trophoblastic cells. Mol. Hum. Reprod. 12:225-232.

97

45. Coughlan, M.T., M.Permezel, H.M.Georgiou, and G.E.Rice. 2004. Repression of oxidant-induced nuclear factor-kappaB activity mediates placental cytokine responses in gestational diabetes. J. Clin. Endocrinol. Metab 89:3585-3594.

46. Crocker, I.P. 2003. Differences in apoptotic susceptibility of cytotrophoblasts and syncytiotrophoblasts in normal pregnancy to those complicated with preeclampsia and intrauterine growth restriction. Am. J. Pathol. 162:637-643.

47. Cross, J.C. 2006. Placental function in development and disease. Reprod. Fertil. Dev. 18:71-76.

48. Das, U.G., H.F.Sadiq, M.J.Soares, W.W.Hay, Jr., and S.U.Devaskar. 1998. Timedependent physiological regulation of rodent and ovine placental glucose transporter (GLUT-1) protein. Am. J. Physiol 274:R339-R347.

49. de Grauw, T.J., R.E.Myers, and W.J.Scott. 1986. Fetal growth retardation in rats from different levels of hypoxia. Biol. Neonate 49:85-89.

50. De Marco, C.S. and I.Caniggia. 2002. Mechanisms of oxygen sensing in human trophoblast cells. Placenta 23 Suppl A:S58-S68.

51. DeBosch, B.J. and A.J.Muslin. 2008. Insulin signaling pathways and cardiac growth. J. Mol. Cell Cardiol. 44:855-864.

98

52. DeSesso, J.M. 1987. Maternal factors in developmental toxicity. Teratog. Carcinog. Mutagen. 7:225-240.

53. Desoye and Myatt. 2004. Oxidative stress in the placenta. Histochem. Cell Biol. 122:369-382.

54. Desoye, G., M.Hartmann, C.J.Jones, H.J.Wolf, G.Kohnen, G.Kosanke, and P.Kaufmann. 1997. Location of insulin receptors in the placenta and its progenitor tissues. Microsc. Res. Tech. 38:63-75.

55. Desoye, G. and M.S.Hauguel-de. 2007. The human placenta in gestational diabetes mellitus. The insulin and cytokine network. Diabetes Care 30 Suppl 2:S120-S126.

56. Desoye, G. and E.Shafrir. 1994. Placental metabolism and its regulation in health and diabetes. Mol. Aspects Med. 15:505-682.

57. Devaskar, S.U., U.P.Devaskar, R.E.Schroeder, D.deMello, F.T.Fiedorek, Jr., and M.Mueckler. 1994. Expression of genes involved in placental glucose uptake and transport in the nonobese diabetic mouse pregnancy. Am. J. Obstet. Gynecol. 171:1316-1323.

58. Dye, T.D., K.L.Knox, R.Artal, R.H.Aubry, and M.A.Wojtowycz. 1997. Physical activity, obesity, and diabetes in pregnancy. Am. J. Epidemiol. 146:961-965.

99

59. Ebert, B.L., J.D.Firth, and P.J.Ratcliffe. 1995. Hypoxia and mitochondrial inhibitors regulate expression of glucose transporter-1 via distinct Cis-acting sequences. J. Biol. Chem. 270:29083-29089.

60. Economides, D.L., K.H.Nicolaides, and S.Campbell. 1991. Metabolic and endocrine findings in appropriate and small for gestational age fetuses. J. Perinat. Med. 19:97-105.

61. Elchalal, U. 2004. Gestational diabetes mellitus: what else is new? Isr. Med. Assoc. J. 6:268-270.

62. Ema, M., S.Taya, N.Yokotani, K.Sogawa, Y.Matsuda, and Y.Fujii-Kuriyama. 1997a. A novel bHLH-PAS factor with close sequence similarity to hypoxiainducible factor 1alpha regulates the VEGF expression and is potentially involved in lung and vascular development. Proc. Natl. Acad. Sci. U. S. A 94:4273-4278.

63. Ema, M., S.Taya, N.Yokotani, K.Sogawa, Y.Matsuda, and Y.Fujii-Kuriyama. 1997b. A novel bHLH-PAS factor with close sequence similarity to hypoxiainducible factor 1alpha regulates the VEGF expression and is potentially involved in lung and vascular development. Proc. Natl. Acad. Sci. U. S. A 94:4273-4278.

64. Engelgau, M.M., W.H.Herman, P.J.Smith, R.R.German, and R.E.Aubert. 1995. The epidemiology of diabetes and pregnancy in the U.S., 1988. Diabetes Care 18:1029-1033.

100

65. England, L.J., R.J.Levine, C.Qian, L.M.Soule, E.F.Schisterman, K.F.Yu, and P.M.Catalano. 2004. Glucose tolerance and risk of gestational diabetes mellitus in nulliparous women who smoke during pregnancy. Am. J. Epidemiol. 160:12051213.

66. Ericsson, A., K.Saljo, E.Sjostrand, N.Jansson, P.D.Prasad, T.L.Powell, and T.Jansson. 2007. Brief hyperglycaemia in the early pregnant rat increases fetal weight at term by stimulating placental growth and affecting placental nutrient transport. J. Physiol 581:1323-1332.

67. Esterman, A., M.A.Greco, Y.Mitani, T.H.Finlay, F.Ismail-Beigi, and J.Dancis. 1997. The effect of hypoxia on human trophoblast in culture: morphology, glucose transport and metabolism. Placenta 18:129-136.

68. Evain-Brion, D. 2001. [The 2 differentiation pathways of the human trophoblast]. Gynecol. Obstet. Fertil. 29:497-502.

69. Farrell, P.A. 1992. Effect of training on insulin secretion from single pancreatic beta cells. Med. Sci. Sports Exerc. 24:426-433.

70. Feng, H.C., M.Y.Choy, W.Deng, H.L.Wong, W.M.Lau, A.N.Cheung, H.Y.Ngan, and S.W.Tsao. 2005. Establishment and characterization of a human firsttrimester extravillous trophoblast cell line (TEV-1). J. Soc. Gynecol. Investig. 12:e21-e32.

101

71. Flamme, I., T.Frohlich, R.M.von, A.Kappel, A.Damert, and W.Risau. 1997. HRF, a putative basic helix-loop-helix-PAS-domain transcription factor is closely related to hypoxia-inducible factor-1 alpha and developmentally expressed in blood vessels. Mech. Dev. 63:51-60.

72. Forsbach-Sanchez, G., H.E.Tamez-Perez, and J.Vazquez-Lara. 2005. Diabetes and pregnancy. Arch. Med. Res. 36:291-299.

73. Forsythe, J.A. 1996. Activation of vascular endothelial growth factor gene transcription by hypoxia-inducible factor 1. Mol. Cell Biol. 16:4604-4613.

74. Fox. H, N.J.Sebire. Pathology of the Placenta, major problems in pathology. 3rd ed. China: Elsevier; 2007.63p.

75. Froen, J.F., J.O.Gardosi, A.Thurmann, A.Francis, and B.Stray-Pedersen. 2004. Restricted fetal growth in sudden intrauterine unexplained death. Acta Obstet. Gynecol. Scand. 83:801-807.

76. Gaither, K., A.N.Quraishi, and N.P.Illsley. 1999. Diabetes alters the expression and activity of the human placental GLUT1 glucose transporter. J. Clin. Endocrinol. Metab 84:695-701.

102

77. Gastaldelli, A., E.Ferrannini, Y.Miyazaki, M.Matsuda, and R.A.DeFronzo. 2004. Beta-cell dysfunction and glucose intolerance: results from the San Antonio metabolism (SAM) study. Diabetologia 47:31-39.

78. Genbacev, O. and R.K.Miller. 2000. Post-implantation differentiation and proliferation of cytotrophoblast cells: in vitro models--a review. Placenta 21 Suppl A:S45-S49.

79. Genbacev, O., Y.Zhou, J.W.Ludlow, and S.J.Fisher. 1997. Regulation of human placental development by oxygen tension. Science 277:1669-1672.

80. Glazier, J.D. and T.Jansson. 2004. Placental transport in early pregnancy--a workshop report. Placenta 25 Suppl A:S57-S59.

81. Gleadle, J.M. 1995. Regulation of angiogenic growth factor expression by hypoxia, transition metals, and chelating agents. Am. J. Physiol 268:C1362-C1368.

82. Gletsu, N., W.Dixon, and M.T.Clandinin. 1999. Insulin receptor at the mouse hepatocyte nucleus after a glucose meal induces dephosphorylation of a 30-kDa transcription factor and a concomitant increase in malic enzyme gene expression. J. Nutr. 129:2154-2161.

103

83. Gordon, M.C. 1995. Insulin and glucose modulate glucose transporter messenger ribonucleic acid expression and glucose uptake in trophoblasts isolated from firsttrimester chorionic villi. Am. J. Obstet. Gynecol. 173:1089-1097.

84. Graham, C.H. 1998. Hypoxia stimulates urokinase receptor expression through a heme protein-dependent pathway. Blood 91:3300-3307.

85. Gu, Y.Z., S.M.Moran, J.B.Hogenesch, L.Wartman, and C.A.Bradfield. 1998. Molecular characterization and chromosomal localization of a third alpha-class hypoxia inducible factor subunit, HIF3alpha. Gene Expr. 7:205-213.

86. Gude, N.M., C.T.Roberts, B.Kalionis, and R.G.King. 2004. Growth and function of the normal human placenta. Thromb. Res. 114:397-407.

87. Hahn, T., S.Barth, U.Weiss, W.Mosgoeller, and G.Desoye. 1998. Sustained hyperglycemia in vitro down-regulates the GLUT1 glucose transport system of cultured human term placental trophoblast: a mechanism to protect fetal development? FASEB J. 12:1221-1231.

88. Hauguel-de, M.S., J.C.Challier, A.Kacemi, M.Cauzac, A.Malek, and J.Girard. 1997. The GLUT3 glucose transporter isoform is differentially expressed within human placental cell types. J. Clin. Endocrinol. Metab 82:2689-2694.

104

89. Hay, W.W., Jr. 1995. Regulation of placental metabolism by glucose supply. Reprod. Fertil. Dev. 7:365-375.

90. Hay, W.W. 2006. Placental-fetal glucose exchange and fetal glucose metabolism. Trans. Am. Clin. Climatol. Assoc. 117:321-340.

91. Hay, W.W., Jr. 1994. Placental transport of nutrients to the fetus. Horm. Res. 42:215-222.

92. Heazell, A.E., H.A.Lacey, C.J.Jones, B.Huppertz, P.N.Baker, and I.P.Crocker. 2008. Effects of oxygen on cell turnover and expression of regulators of apoptosis in human placental trophoblast. Placenta 29:175-186.

93. Hendrix, N. and V.Berghella. 2008. Non-placental causes of intrauterine growth restriction. Semin. Perinatol. 32:161-165.

94. Henriksen, T. and T.Clausen. 2002. The fetal origins hypothesis: placental insufficiency and inheritance versus maternal malnutrition in well-nourished populations. Acta Obstet. Gynecol. Scand. 81:112-114.

95. Hiden, U., C.Wadsack, N.Prutsch, M.Gauster, U.Weiss, H.G.Frank, U.Schmitz, C.Fast-Hirsch, M.Hengstschlager, A.Potgens, A.Ruben, M.Knofler, P.Haslinger, B.Huppertz, M.Bilban, P.Kaufmann, and G.Desoye. 2007. The first trimester human trophoblast cell line ACH-3P: a novel tool to study autocrine/paracrine

105

regulatory loops of human trophoblast subpopulations--TNF-alpha stimulates MMP15 expression. BMC. Dev. Biol. 7:137.

96. Hiraki, Y., O.M.Rosen, and M.J.Birnbaum. 1988. Growth factors rapidly induce expression of the glucose transporter gene. J. Biol. Chem. 263:13655-13662.

97. Hochberg, A. 1992. Differentiation of choriocarcinoma cell line (JAr). Cancer Res. 52:3713-3717.

98. Hogan, A., S.Heyner, M.J.Charron, N.G.Copeland, D.J.Gilbert, N.A.Jenkins, B.Thorens, and G.A.Schultz. 1991. Glucose transporter gene expression in early mouse embryos. Development 113:363-372.

99. Hohn, H.P., M.Linke, B.Ugele, and H.W.Denker. 1998. Differentiation markers and invasiveness: discordant regulation in normal trophoblast and choriocarcinoma cells. Exp. Cell Res. 244:249-258.

100. Holan, S., S.Vangen, K.Hanssen, and B.Stray-Pedersen. 2008. [Pregnancy in diabetes among Asian, African and Norwegian women]. Tidsskr. Nor Laegeforen. 128:1289-1292.

101. Huang, L.E., Z.Arany, D.M.Livingston, and H.F.Bunn. 1996. Activation of hypoxia-inducible transcription factor depends primarily upon redox-sensitive stabilization of its alpha subunit. J. Biol. Chem. 271:32253-32259.

106

102. Illsley, N.P. 1984. Mechanical and metabolic viability of a placental perfusion system in vitro under oxygenated and anoxic conditions. Placenta 5:213-225.

103. Illsley, N.P. 1998. Glycaemic regulation of glucose transporter expression and activity in the human placenta. Placenta 19:517-524.

104. Illsley, N.P. 2000. Glucose transporters in the human placenta. Placenta 21:14-22.

105. Irving, J.A. 1995. Characteristics of trophoblast cells migrating from first trimester chorionic villus explants and propagated in culture. Placenta 16:413-433.

106. James, J. 2004. Cellular and molecular characterization of early and late retinal stem cells/progenitors: differential regulation of proliferation and context dependent role of Notch signaling. J. Neurobiol. 61:359-376.

107. James, J.L. 2006. The effects of oxygen concentration and gestational age on extravillous trophoblast outgrowth in a human first trimester villous explant model. Hum. Reprod. 21:2699-2705.

108. James, J.L., P.R.Stone, and L.W.Chamley. 2006. The regulation of trophoblast differentiation by oxygen in the first trimester of pregnancy. Hum. Reprod. Update. 12:137-144.

107

109. Jansson, T. 1999. Placental glucose transport and GLUT 1 expression in insulindependent diabetes. Am. J. Obstet. Gynecol. 180:163-168.

110. Jansson, T., I.Cetin, T.L.Powell, G.Desoye, T.Radaelli, A.Ericsson, and C.P.Sibley. 2006. Placental transport and metabolism in fetal overgrowth -- a workshop report. Placenta 27 Suppl A:S109-S113.

111. Jansson, T., E.A.Cowley, and N.P.Illsley. 1995. Cellular localization of glucose transporter messenger RNA in human placenta. Reprod. Fertil. Dev. 7:1425-1430.

112. Jansson, T., Y.Ekstrand, C.Bjorn, M.Wennergren, and T.L.Powell. 2002. Alterations in the activity of placental amino acid transporters in pregnancies complicated by diabetes. Diabetes 51:2214-2219.

113. Jansson, T., M.Wennergren, and N.P.Illsley. 1993. Glucose transporter protein expression in human placenta throughout gestation and in intrauterine growth retardation. J. Clin. Endocrinol. Metab 77:1554-1562.

114. Jansson, T., M.Wennergren, and T.Powell. 2003. [Pregnancy complications can impair placental transport systems. Aberrant fetal growth, for example in diabetes, is only now beginning to be understood]. Lakartidningen 100:2508-2511.

108

115. Jauniaux, E., B.Gulbis, and G.J.Burton. 2003. The human first trimester gestational sac limits rather than facilitates oxygen transfer to the foetus--a review. Placenta 24 Suppl A:S86-S93.

116. Jauniaux, E., G.Pahal, C.Gervy, and B.Gulbis. 2000. Blood biochemistry and endocrinology in the human fetus between 11 and 17 weeks of gestation. Reprod. Biomed. Online. 1:38-44.

117. Jiang, B. 2000. Hypoxia prevents induction of aromatase expression in human trophoblast cells in culture: potential inhibitory role of the hypoxia-inducible transcription factor Mash-2 (mammalian achaete-scute homologous protein-2). Mol. Endocrinol. 14:1661-1673.

118. Johnson, L.W. and C.H.Smith. 1980. Monosaccharide transport across microvillous membrane of human placenta. Am. J. Physiol 238:C160-C168.

119. Johnson, L.W. and C.H.Smith. 1985. Glucose transport across the basal plasma membrane of human placental syncytiotrophoblast. Biochim. Biophys. Acta 815:44-50.

120. Jones, C.J., M.Hartmann, A.Blaschitz, and G.Desoye. 1993. Ultrastructural localization of insulin receptors in human placenta. Am. J. Reprod. Immunol. 30:136-145.

109

121. Jones, H.N., T.L.Powell, and T.Jansson. 2007. Regulation of placental nutrient transport--a review. Placenta 28:763-774.

122. Joost, H.G. and B.Thorens. 2001. The extended GLUT-family of sugar/polyol transport facilitators: nomenclature, sequence characteristics, and potential function of its novel members (review). Mol. Membr. Biol. 18:247-256.

123. Joyner, N.T. 2004. In vivo hyperglycemia and its effect on Glut-1 expression in the embryonic heart. Birth Defects Res. A Clin. Mol. Teratol. 70:438-448.

124. Kahn, S.E. 2003. The relative contributions of insulin resistance and beta-cell dysfunction to the pathophysiology of Type 2 diabetes. Diabetologia 46:3-19.

125. Kaluz, S., M.Kaluzova, and E.J.Stanbridge. 2008. Regulation of gene expression by hypoxia: Integration of the HIF-transduced hypoxic signal at the hypoxiaresponsive element. Clin. Chim. Acta 395:6-13.

126. Kang, J.X. 1997. Mannose-6-phosphate/insulin-like growth factor-II receptor is a receptor for retinoic acid. Proc. Natl. Acad. Sci. U. S. A 94:13671-13676.

127. Katavetin, P. 2006. High glucose blunts vascular endothelial growth factor response to hypoxia via the oxidative stress-regulated hypoxia-inducible factor/hypoxia-responsible element pathway. J. Am. Soc. Nephrol. 17:1405-1413.

110

128. Kautzky-Willer, A. and D.Bancher-Todesca. 2003. [Gestational diabetes]. Wien. Med. Wochenschr. 153:478-484.

129. Kawata, M., K.Sizer, S.Sekiya, J.R.Parnes, and L.A.Herzenberg. 1984. Limitation of differential expression of HLA-A,B,C antigens on choriocarcinoma cell lines by messenger RNA for HLA heavy chain but not by beta 2-microglobulin. Cancer Res. 44:4011-4016.

130. Kernan, W.N., S.E.Inzucchi, C.M.Viscoli, L.M.Brass, D.M.Bravata, and R.I.Horwitz. 2002. Insulin resistance and risk for stroke. Neurology 59:809-815.

131. Kilburn, B.A. 2000. Extracellular matrix composition and hypoxia regulate the expression of HLA-G and integrins in a human trophoblast cell line. Biol. Reprod. 62:739-747.

132. Kim, B.S., J.Chen, T.Weinstein, E.Noiri, and M.S.Goligorsky. 2002. VEGF expression in hypoxia and hyperglycemia: reciprocal effect on branching angiogenesis in epithelial-endothelial co-cultures. J. Am. Soc. Nephrol. 13:20272036.

133. Kingdom, J.C. and P.Kaufmann. 1999. Oxygen and placental vascular development. Adv. Exp. Med. Biol. 474:259-275.

111

134. Kiserud, T. 1994. Ductus venosus blood velocity and the umbilical circulation in the seriously growth-retarded fetus. Ultrasound Obstet. Gynecol. 4:109-114.

135. Klip, A. 1994. Regulation of expression of glucose transporters by glucose: a review of studies in vivo and in cell cultures. FASEB J. 8:43-53.

136. Korgun, E.T., C.Celik-Ozenci, Y.Seval, G.Desoye, and R.Demir. 2005. Do glucose transporters have other roles in addition to placental glucose transport during early pregnancy? Histochem. Cell Biol. 123:621-629.

137. Korgun, E.T., R.Demir, P.Sedlmayr, G.Desoye, G.Arikan, P.Puerstner, M.Haeusler, G.Dohr, G.Skofitsch, and T.Hahn. 2002. Physiological leukocytosis during pregnancy is associated with changes in glucose transporter expression of maternal peripheral blood granulocytes and monocytes. Am. J. Reprod. Immunol. 48:110-116.

138. Kotani, K., A.J.Carozzi, H.Sakaue, K.Hara, L.J.Robinson, S.F.Clark, K.Yonezawa, D.E.James, and M.Kasuga. 1995. Requirement for phosphoinositide 3-kinase in insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. Biochem. Biophys. Res. Commun. 209:343-348.

139. Krohn, K.A., J.M.Link, and R.P.Mason. 2008. Molecular imaging of hypoxia. J. Nucl. Med. 49 Suppl 2:129S-148S.

112

140. Kuhl, C. 1991. Insulin secretion and insulin resistance in pregnancy and GDM. Implications for diagnosis and management. Diabetes 40 Suppl 2:18-24.

141. Kuhl, C., P.J.Hornnes, and O.Andersen. 1985. Etiology and pathophysiology of gestational diabetes mellitus. Diabetes 34 Suppl 2:66-70.

142. Kulkarni, R.N., J.N.Winnay, M.Daniels, J.C.Bruning, S.N.Flier, D.Hanahan, and C.R.Kahn. 1999. Altered function of insulin receptor substrate-1-deficient mouse islets and cultured beta-cell lines. J. Clin. Invest 104:R69-R75.

143. Lain, K.Y. and P.M.Catalano. 2007. Metabolic changes in pregnancy. Clin. Obstet. Gynecol. 50:938-948.

144. Larger, E. 2004. Normal insulin sensitivity during the late preclinical stage of type 1 diabetes. Diabetes Care 27:1842-1843.

145. Lash, G.E. 2007. Effect of low oxygen concentrations on trophoblast-like cell line invasion. Placenta 28:390-398.

146. Lash, G.E., S.Quenby, G.J.Burton, A.Nakashima, B.R.Kamat, J.Ray, and J.N.Bulmer. 2008. Gestational diseases -- a workshop report. Placenta 29 Suppl A:S92-S94.

113

147. Laviola, L., S.Perrini, G.Belsanti, A.Natalicchio, C.Montrone, A.Leonardini, A.Vimercati, M.Scioscia, L.Selvaggi, R.Giorgino, P.Greco, and F.Giorgino. 2005. Intrauterine growth restriction in humans is associated with abnormalities in placental insulin-like growth factor signaling. Endocrinology 146:1498-1505.

148. Lerman, O.Z. 2003. Cellular dysfunction in the diabetic fibroblast: impairment in migration, vascular endothelial growth factor production, and response to hypoxia. Am. J. Pathol. 162:303-312.

149. Lesage, J., D.Hahn, M.Leonhardt, B.Blondeau, B.Breant, and J.P.Dupouy. 2002. Maternal undernutrition during late gestation-induced intrauterine growth restriction in the rat is associated with impaired placental GLUT3 expression, but does not correlate with endogenous corticosterone levels. J. Endocrinol. 174:3743.

150. Levy, R. 2000. Apoptosis in human cultured trophoblasts is enhanced by hypoxia and diminished by epidermal growth factor. Am. J. Physiol Cell Physiol 278:C982-C988.

151. Licht, A.H., F.Muller-Holtkamp, I.Flamme, and G.Breier. 2006. Inhibition of hypoxia-inducible factor activity in endothelial cells disrupts embryonic cardiovascular development. Blood 107:584-590.

114

152. Loike, J.D., L.Cao, J.Brett, S.Ogawa, S.C.Silverstein, and D.Stern. 1992. Hypoxia induces glucose transporter expression in endothelial cells. Am. J. Physiol 263:C326-C333.

153. Luerssen, M.A. and A.L.Winsch. 2005. Diabetes under control: identifying and treating gestational diabetes mellitus: advances in screening and current interventions. Am. J. Nurs. 105:65-71.

154. Lunghi, L., M.E.Ferretti, S.Medici, C.Biondi, and F.Vesce. 2007b. Control of human trophoblast function. Reprod. Biol. Endocrinol. 5:6.

155. Lunghi, L., M.E.Ferretti, S.Medici, C.Biondi, and F.Vesce. 2007a. Control of human trophoblast function. Reprod. Biol. Endocrinol. 5:6.

156. Magee, M.S., C.E.Walden, T.J.Benedetti, and R.H.Knopp. 1993. Influence of diagnostic criteria on the incidence of gestational diabetes and perinatal morbidity. JAMA 269:609-615.

157. Makhseed, M.A., M.A.Ahmed, and V.M.Musini. 2004. Impaired gestational glucose tolerance. Its effect on placental pathology. Saudi. Med. J. 25:1241-1244.

158. Makino, Y., R.Cao, K.Svensson, G.Bertilsson, M.Asman, H.Tanaka, Y.Cao, A.Berkenstam, and L.Poellinger. 2001. Inhibitory PAS domain protein is a negative regulator of hypoxia-inducible gene expression. Nature 414:550-554.

115

159. Maltepe, E. and M.C.Simon. 1998. Oxygen, genes, and development: an analysis of the role of hypoxic gene regulation during murine vascular development. J. Mol. Med. 76:391-401.

160. Manolescu, A.R., K.Witkowska, A.Kinnaird, T.Cessford, and C.Cheeseman. 2007. Facilitated hexose transporters: new perspectives on form and function. Physiology. (Bethesda. ) 22:234-240.

161. Marconi, A.M. 1996. The impact of gestational age and fetal growth on the maternal-fetal glucose concentration difference. Obstet. Gynecol. 87:937-942.

162. Marette, A., J.M.Richardson, T.Ramlal, T.W.Balon, M.Vranic, J.E.Pessin, and A.Klip. 1992. Abundance, localization, and insulin-induced translocation of glucose transporters in red and white muscle. Am. J. Physiol 263:C443-C452.

163. Maxwell, P.H. 1999. The tumour suppressor protein VHL targets hypoxiainducible factors for oxygen-dependent proteolysis. Nature 399:271-275.

164. McGowan, K.M., S.D.Long, and P.H.Pekala. 1995. Glucose transporter gene expression: regulation of transcription and mRNA stability. Pharmacol. Ther. 66:465-505.

165. Mitchell, A.M. 1995. Characterization of cell polarity and epithelial junctions in the choriocarcinoma cell line, JAR. Placenta 16:31-39.

116

166. Mochizuki, M., T.Maruo, H.Matsuo, T.Samoto, and N.Ishihara. 1998. Biology of human trophoblast. Int. J. Gynaecol. Obstet. 60 Suppl 1:S21-S28.

167. Moe, A.J. 1994. Functional characterization of L-alanine transport in a placental choriocarcinoma cell line (BeWo). Placenta 15:797-802.

168. Mueckler, M. 1994. Facilitative glucose transporters. Eur. J. Biochem. 219:713725.

169. Mueckler, M., C.Caruso, S.A.Baldwin, M.Panico, I.Blench, H.R.Morris, W.J.Allard, G.E.Lienhard, and H.F.Lodish. 1985. Sequence and structure of a human glucose transporter. Science 229:941-945.

170. Myren, M., T.Mose, L.Mathiesen, and L.E.Knudsen. 2007. The human placenta-an alternative for studying foetal exposure. Toxicol. In Vitro 21:1332-1340.

171. Nangaku, M. and K.U.Eckardt. 2007. Hypoxia and the HIF system in kidney disease. J. Mol. Med. 85:1325-1330.

172. Nangaku, M., R.Inagi, T.Miyata, and T.Fujita. 2008. Hypoxia and HypoxiaInducible Factor in Renal Disease. Nephron Exp. Nephrol. 110:e1-e7.

117

173. Nelson, D.M. and E.M.Curran. 1989. High glucose levels decrease proliferation of cultured human fetal cells from placenta. Am. J. Obstet. Gynecol. 161:15531558.

174. Nelson, D.L. and M.M. Cox. Lehninger Principles of Biochemistry. 3rd ed. USA:Worth Publishers, 2000, 445-47pp

175. Niessen, M. 2006. On the role of IRS2 in the regulation of functional beta-cell mass. Arch. Physiol Biochem. 112:65-73.

176. O'Brien, E.A., V.Barnes, L.Zhao, R.A.McKnight, X.Yu, C.W.Callaway, L.Wang, J.C.Sun, M.J.Dahl, A.Wint, Z.Wang, T.M.McIntyre, K.H.Albertine, and R.H.Lane. 2007. Uteroplacental insufficiency decreases p53 serine-15 phosphorylation in term IUGR rat lungs. Am. J. Physiol Regul. Integr. Comp Physiol 293:R314-R322.

177. O'Dowd, R., J.C.Kent, J.M.Moseley, and M.E.Wlodek. 2008. Effects of uteroplacental insufficiency and reducing litter size on maternal mammary function and postnatal offspring growth. Am. J. Physiol Regul. Integr. Comp Physiol 294:R539-R548.

178. Ogata, E.S., M.E.Bussey, and S.Finley. 1986. Altered gas exchange, limited glucose and branched chain amino acids, and hypoinsulinism retard fetal growth in the rat. Metabolism 35:970-977.

118

179. Okada, T., Y.Kawano, T.Sakakibara, O.Hazeki, and M.Ui. 1994. Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes. Studies with a selective inhibitor wortmannin. J. Biol. Chem. 269:3568-3573.

180. Ong, P.J. and G.J.Burton. 1991. Thinning of the placental villous membrane during maintenance in hypoxic organ culture: structural adaptation or syncytial degeneration? Eur. J. Obstet. Gynecol. Reprod. Biol. 39:103-110.

181. Osmond, D.T., C.J.Nolan, R.G.King, S.P.Brennecke, and N.M.Gude. 2000. Effects of gestational diabetes on human placental glucose uptake, transfer, and utilisation. Diabetologia 43:576-582.

182. Owens, S. and C.H.Fall. 2008. Consequences of poor maternal micronutrition before and during early pregnancy. Trans. R. Soc. Trop. Med. Hyg. 102:103-104.

183. Pardi, G. and I.Cetin. 2006. Human fetal growth and organ development: 50 years of discoveries. Am. J. Obstet. Gynecol. 194:1088-1099.

184. Pedersen, J.F. 1998. Serum levels of human placental lactogen, pregnancyassociated plasma protein A and endometrial secretory protein PP14 in first trimester of diabetic pregnancy. Acta Obstet. Gynecol. Scand. 77:155-158.

119

185. Pietryga, M., W.Biczysko, E.Wender-Ozegowska, J.Brazert, E.Bieganska, and R.Biczysko. 2004. [Ultrastructural examination of the placenta in pregnancy complicated by diabetes mellitus]. Ginekol. Pol. 75:111-118.

186. Pinter, E. 2001. Hyperglycemia-induced vasculopathy in the murine conceptus is mediated via reductions of VEGF-A expression and VEGF receptor activation. Am. J. Pathol. 158:1199-1206.

187. Piwowar, A., M.Knapik-Kordecka, and M.Warwas. 2008. Ischemia-modified albumin level in type 2 diabetes mellitus - Preliminary report. Dis. Markers 24:311-317.

188. Plate, K.H. 1992. Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo. Nature 359:845-848.

189. Previs, S.F., D.J.Withers, J.M.Ren, M.F.White, and G.I.Shulman. 2000. Contrasting effects of IRS-1 versus IRS-2 gene disruption on carbohydrate and lipid metabolism in vivo. J. Biol. Chem. 275:38990-38994.

190. Rajakumar, A., H.M.Michael, A.Daftary, A.Jeyabalan, C.Gilmour, and K.P.Conrad. 2008. Proteasomal activity in placentas from women with preeclampsia and intrauterine growth restriction: implications for expression of HIF-alpha proteins. Placenta 29:290-299.

120

191. Rankin, J.H. 1986. Maternal insulin and placental 3-O-methyl glucose transport. J. Dev. Physiol 8:247-253.

192. Ream, M.A., A.M.Ray, R.Chandra, and D.M.Chikaraishi. 2008. Early Fetal Hypoxia Leads to Growth Restriction and Myocardial Thinning. Am. J. Physiol Regul. Integr. Comp Physiol.

193. Reece, E.A., C.J.Homko, and A.Wiznitzer. 1994. Doppler velocimetry and the assessment of fetal well-being in normal and diabetic pregnancies. Ultrasound Obstet. Gynecol. 4:508-514.

194. Rice, P.A., J.E.Rourke, and E.L.Nesbitt, Jr. 1976. In vitro perfusion studies of the human placenta. IV. Some characteristics of the glucose transport system in the human placenta. Gynecol. Invest 7:213-221.

195. Rice, P.A., J.E.Rourke, and R.E.Nesbitt, Jr. 1979. In vitro perfusion studies of the human placenta. VI. Evidence against active glucose transport. Am. J. Obstet. Gynecol. 133:649-655.

196. Robins, J.C., A.Heizer, A.Hardiman, M.Hubert, and S.Handwerger. 2007. Oxygen tension directs the differentiation pathway of human cytotrophoblast cells. Placenta 28:1141-1146.

121

197. Rong-Hao, L., S.Luo, and L.Z.Zhuang. 1996. Establishment and characterization of a cytotrophoblast cell line from normal placenta of human origin. Hum. Reprod. 11:1328-1333.

198. Rosario, G.X., T.Konno, and M.J.Soares. 2008. Maternal hypoxia activates endovascular trophoblast cell invasion. Dev. Biol. 314:362-375.

199. Rosen, O.M., C.J.Smith, C.Fung, and C.S.Rubin. 1978. Development of hormone receptors and hormone responsiveness in vitro. Effect of prolonged insulin treatment on hexose uptake in 3T3-L1 adipocytes. J. Biol. Chem. 253:7579-7583.

200. Ross, G. 2006. Gestational diabetes. Aust. Fam. Physician 35:392-396.

201. Rui, L., T.L.Fisher, J.Thomas, and M.F.White. 2001. Regulation of insulin/insulin-like growth factor-1 signaling by proteasome-mediated degradation of insulin receptor substrate-2. J. Biol. Chem. 276:40362-40367.

202. Safran. 2003. HIF hydroxylation and the mammalian oxygen-sensing pathway. J. Clin. Invest 111:779-783.

203. Sakata, M., H.Kurachi, T.Imai, C.Tadokoro, M.Yamaguchi, Y.Yoshimoto, Y.Oka, and A.Miyake. 1995. Increase in human placental glucose transporter-1 during pregnancy. Eur. J. Endocrinol. 132:206-212.

122

204. Salafia, C.M. 1997. Placental pathology of fetal growth restriction. Clin. Obstet. Gynecol. 40:740-749.

205. Salceda, S., I.Beck, and J.Caro. 1996. Absolute requirement of aryl hydrocarbon receptor nuclear translocator protein for gene activation by hypoxia. Arch. Biochem. Biophys. 334:389-394.

206. Salceda, S. and J.Caro. 1997. Hypoxia-inducible factor 1alpha (HIF-1alpha) protein is rapidly degraded by the ubiquitin-proteasome system under normoxic conditions. Its stabilization by hypoxia depends on redox-induced changes. J. Biol. Chem. 272:22642-22647.

207. Sasson, S. 1997. Substrate autoregulation of glucose transport: hexose 6phosphate mediates the cellular distribution of glucose transporters. Diabetologia 40:30-39.

208. Satoh, S., H.Nishimura, A.E.Clark, I.J.Kozka, S.J.Vannucci, I.A.Simpson, M.J.Quon, S.W.Cushman, and G.D.Holman. 1993. Use of bismannose photolabel to elucidate insulin-regulated GLUT4 subcellular trafficking kinetics in rat adipose cells. Evidence that exocytosis is a critical site of hormone action. J. Biol. Chem. 268:17820-17829.

209. Schaefer-Graf, U.M. and H.Kleinwechter. 2006. Diagnosis and new approaches in the therapy of gestational diabetes mellitus. Curr. Diabetes Rev. 2:343-352.

123

210. Schmon, B. 1991. Insulin and glucose do not affect the glycogen content in isolated and cultured trophoblast cells of human term placenta. J. Clin. Endocrinol. Metab 73:888-893.

211. Schneider, H. 1991. The role of the placenta in nutrition of the human fetus. Am. J. Obstet. Gynecol. 164:967-973.

212. Scioscia, M., K.Gumaa, S.Kunjara, M.A.Paine, L.E.Selvaggi, C.H.Rodeck, and T.W.Rademacher. 2006. Insulin resistance in human preeclamptic placenta is mediated by serine phosphorylation of insulin receptor substrate-1 and -2. J. Clin. Endocrinol. Metab 91:709-717.

213. Scioscia, M., M.A.Paine, K.Gumaa, C.H.Rodeck, and T.W.Rademacher. 2008. Release of inositol phosphoglycan P-type by the human placenta following insulin stimulus: A multiple comparison between preeclampsia, intrauterine growth restriction, and gestational hypertension. J. Matern. Fetal Neonatal Med.1-5.

214. Sciullo, E., G.Cardellini, M.G.Baroni, P.Torresi, A.Buongiorno, P.Pozzilli, and F.Fallucca. 1997. Glucose transporter (Glut1, Glut3) mRNA in human placenta of diabetic and non-diabetic pregnancies. Early Pregnancy. 3:172-182.

215. Seagroves, T.N. 2001. Transcription factor HIF-1 is a necessary mediator of the pasteur effect in mammalian cells. Mol. Cell Biol. 21:3436-3444.

124

216. Semenza, G.L. 2000a. HIF-1: mediator of physiological and pathophysiological responses to hypoxia. J. Appl. Physiol 88:1474-1480.

217. Semenza, G.L. 2000b. Hypoxia, clonal selection, and the role of HIF-1 in tumor progression. Crit Rev. Biochem. Mol. Biol. 35:71-103.

218. Semenza, G.L. 1999. Perspectives on oxygen sensing. Cell 98:281-284.

219. Sermer, M., C.D.Naylor, D.J.Gare, A.B.Kenshole, J.W.Ritchie, D.Farine, H.R.Cohen, K.McArthur, S.Holzapfel, A.Biringer, and . 1995. Impact of increasing carbohydrate intolerance on maternal-fetal outcomes in 3637 women without gestational diabetes. The Toronto Tri-Hospital Gestational Diabetes Project. Am. J. Obstet. Gynecol. 173:146-156.

220. Sesti, G., M.Federici, D.Lauro, P.Sbraccia, and R.Lauro. 2001. Molecular mechanism of insulin resistance in type 2 diabetes mellitus: role of the insulin receptor variant forms. Diabetes Metab Res. Rev. 17:363-373.

221. Sferruzzi-Perri, A.N. 2003. Interleukin-5 transgene expression and eosinophilia are associated with retarded mammary gland development in mice. Biol. Reprod. 69:224-233.

222. Shaat, N. and L.Groop. 2007. Genetics of gestational diabetes mellitus. Curr. Med. Chem. 14:569-583.

125

223. Sharkey, A.M. 1993. Expression of mRNA for vascular endothelial growth factor in human placenta. J. Reprod. Fertil. 99:609-615.

224. Shih, S.C. and K.P.Claffey. 1998. Hypoxia-mediated regulation of gene expression in mammalian cells. Int. J. Exp. Pathol. 79:347-357.

225. Shore, V.H., T.H.Wang, C.L.Wang, R.J.Torry, M.R.Caudle, and D.S.Torry. 1997. Vascular endothelial growth factor, placenta growth factor and their receptors in isolated human trophoblast. Placenta 18:657-665.

226. Simmons, R.A., L.J.Templeton, and S.J.Gertz. 2001. Intrauterine growth retardation leads to the development of type 2 diabetes in the rat. Diabetes 50:2279-2286.

227. Sivan, E. and G.Boden. 2003. Free fatty acids, insulin resistance, and pregnancy. Curr. Diab. Rep. 3:319-322.

228. Srinivas, V., I.Leshchinsky, N.Sang, M.P.King, A.Minchenko, and J.Caro. 2001. Oxygen sensing and HIF-1 activation does not require an active mitochondrial respiratory chain electron-transfer pathway. J. Biol. Chem. 276:21995-21998.

229. Staun-Ram, E. and E.Shalev. 2005. Human trophoblast function during the implantation process. Reprod. Biol. Endocrinol. 3:56.

126

230. Stavri, G.T. 1995. Basic fibroblast growth factor upregulates the expression of vascular endothelial growth factor in vascular smooth muscle cells. Synergistic interaction with hypoxia. Circulation 92:11-14.

231. Sun, W.J. and H.X.Yang. 2007. [Maternal and fetal outcomes in pregnant women with abnormal glucose metabolism]. Zhonghua Fu Chan Ke. Za Zhi. 42:377-381.

232. Sun, X.J., S.Pons, L.M.Wang, Y.Zhang, L.Yenush, D.Burks, M.G.Myers, Jr., E.Glasheen, N.G.Copeland, N.A.Jenkins, J.H.Pierce, and M.F.White. 1997. The IRS-2 gene on murine chromosome 8 encodes a unique signaling adapter for insulin and cytokine action. Mol. Endocrinol. 11:251-262.

233. Sun, X.J., P.Rothenberg, C.R.Kahn, J.M.Backer, E.Araki, P.A.Wilden, D.A.Cahill, B.J.Goldstein, and M.F.White. 1991. Structure of the insulin receptor substrate IRS-1 defines a unique signal transduction protein. Nature 352:73-77.

234. Sun, X.J., L.M.Wang, Y.Zhang, L.Yenush, M.G.Myers, Jr., E.Glasheen, W.S.Lane, J.H.Pierce, and M.F.White. 1995. Role of IRS-2 in insulin and cytokine signalling. Nature 377:173-177.

235. Szymanska, M., D.A.Bomba-Opon, and M.Wielgos. 2008. Blood pressure and lipid changes in gestational diabetes mellitus. Neuro. Endocrinol. Lett. 29:328333.

127

236. Takahashi, A. 1994. Markedly increased amounts of messenger RNAs for vascular endothelial growth factor and placenta growth factor in renal cell carcinoma associated with angiogenesis. Cancer Res. 54:4233-4237.

237. Takata, K., T.Kasahara, M.Kasahara, O.Ezaki, and H.Hirano. 1994. Immunolocalization of glucose transporter GLUT1 in the rat placental barrier: possible role of GLUT1 and the gap junction in the transport of glucose across the placental barrier. Cell Tissue Res. 276:411-418.

238. Takata, K., T.Kasahara, M.Kasahara, O.Ezaki, and H.Hirano. 1990. Erythrocyte/HepG2-type glucose transporter is concentrated in cells of bloodtissue barriers. Biochem. Biophys. Res. Commun. 173:67-73.

239. Takata, Y. 1992. Intracellular signaling by a mutant human insulin receptor lacking the carboxyl-terminal tyrosine autophosphorylation sites. J. Biol. Chem. 267:9065-9070.

240. Tamemoto, H., T.Kadowaki, K.Tobe, T.Yagi, H.Sakura, T.Hayakawa, Y.Terauchi, K.Ueki, Y.Kaburagi, S.Satoh, and . 1994. Insulin resistance and growth retardation in mice lacking insulin receptor substrate-1. Nature 372:182-186.

241. Thirone, A.C., C.Huang, and A.Klip. 2006. Tissue-specific roles of IRS proteins in insulin signaling and glucose transport. Trends Endocrinol. Metab 17:72-78.

128

242. Tian, H., S.L.McKnight, and D.W.Russell. 1997. Endothelial PAS domain protein 1 (EPAS1), a transcription factor selectively expressed in endothelial cells. Genes Dev. 11:72-82.

243. Tominaga, M. 1999. [Diagnostic criteria for diabetes mellitus]. Rinsho Byori 47:901-908.

244. Tordjman, K.M., K.A.Leingang, D.E.James, and M.M.Mueckler. 1989. Differential regulation of two distinct glucose transporter species expressed in 3T3-L1 adipocytes: effect of chronic insulin and tolbutamide treatment. Proc. Natl. Acad. Sci. U. S. A 86:7761-7765.

245. Tordjman, K.M., K.A.Leingang, and M.Mueckler. 1990. Differential regulation of the HepG2 and adipocyte/muscle glucose transporters in 3T3L1 adipocytes. Effect of chronic glucose deprivation. Biochem. J. 271:201-207.

246. Trundley, A. and A.Moffett. 2004. Human uterine leukocytes and pregnancy. Tissue Antigens 63:1-12.

247. Valverde, A.M., M.Lorenzo, S.Pons, M.F.White, and M.Benito. 1998. Insulin receptor substrate (IRS) proteins IRS-1 and IRS-2 differential signaling in the insulin/insulin-like growth factor-I pathways in fetal brown adipocytes. Mol. Endocrinol. 12:688-697.

129

248. Vannini, P. 1994. [Pregnancy and diabetes: physiopathological aspects]. Minerva Endocrinol. 19:45-50.

249. Vegh, G.L., V.Fulop, Y.Liu, S.W.Ng, Z.S.Tuncer, D.R.Genest, P.Paldi-Haris, J.Foldi, S.C.Mok, and R.S.Berkowitz. 1999. Differential gene expression pattern between normal human trophoblast and choriocarcinoma cell lines: downregulation of heat shock protein-27 in choriocarcinoma in vitro and in vivo. Gynecol. Oncol. 75:391-396.

250. Vuorela, P. 1997. Expression of vascular endothelial growth factor and placenta growth factor in human placenta. Biol. Reprod. 56:489-494.

251. Wagner, P.D. 2008. The biology of oxygen. Eur. Respir. J. 31:887-890.

252. Walker, P.S., J.A.Donovan, B.G.Van Ness, R.E.Fellows, and J.E.Pessin. 1988. Glucose-dependent regulation of glucose transport activity, protein, and mRNA in primary cultures of rat brain glial cells. J. Biol. Chem. 263:15594-15601.

253. Wang, G.L. 1995. Effect of protein kinase and phosphatase inhibitors on expression of hypoxia-inducible factor 1. Biochem. Biophys. Res. Commun. 216:669-675.

130

254. Wang, G.L., B.H.Jiang, E.A.Rue, and G.L.Semenza. 1995. Hypoxia-inducible factor 1 is a basic-helix-loop-helix-PAS heterodimer regulated by cellular O2 tension. Proc. Natl. Acad. Sci. U. S. A 92:5510-5514.

255. Wang, G.L. and G.L.Semenza. 1993. Characterization of hypoxia-inducible factor 1 and regulation of DNA binding activity by hypoxia. J. Biol. Chem. 268:2151321518.

256. Wang, L.M., M.G.Myers, Jr., X.J.Sun, S.A.Aaronson, M.White, and J.H.Pierce. 1993. IRS-1: essential for insulin- and IL-4-stimulated mitogenesis in hematopoietic cells. Science 261:1591-1594.

257. Ward, C., M.Lawrence, V.Streltsov, T.Garrett, N.McKern, M.Z.Lou, G.Lovrecz, and T.Adams. 2008. Structural insights into ligand-induced activation of the insulin receptor. Acta Physiol (Oxf) 192:3-9.

258. Warren, S.M. 2001. Hypoxia regulates osteoblast gene expression. J. Surg. Res. 99:147-155.

259. Weidemann, A. and R.S.Johnson. 2008. Biology of HIF-1alpha. Cell Death. Differ. 15:621-627.

260. Weiss, U., M.Cervar, P.Puerstner, O.Schmut, J.Haas, R.Mauschitz, G.Arikan, and G.Desoye. 2001. Hyperglycaemia in vitro alters the proliferation and

131

mitochondrial activity of the choriocarcinoma cell lines BeWo, JAR and JEG-3 as models for human first-trimester trophoblast. Diabetologia. 44:209-219.

261. Weissmann, N., F.Grimminger, and W.Seeger. 2006. Hypoxia in lung vascular biology and disease. Cardiovasc. Res. 71:618-619.

262. Wenger, R.H., D.P.Stiehl, and G.Camenisch. 2005. Integration of oxygen signaling at the consensus HRE. Sci. STKE. 2005:re12.

263. Wertheimer, E., S.Sasson, E.Cerasi, and Y.Ben-Neriah. 1991. The ubiquitous glucose transporter GLUT-1 belongs to the glucose-regulated protein family of stress-inducible proteins. Proc. Natl. Acad. Sci. U. S. A 88:2525-2529.

264. Wheeler, T. 1995. Angiogenesis and the placental environment. Placenta 16:289296.

265. White and, M.F. 1998. The IRS-signaling system: a network of docking proteins that mediate insulin and cytokine action. Curr. Top. Microbiol. Immunol. 228:179-208:179-208.

266. White, M.F. 1997. The insulin signalling system and the IRS proteins. Diabetologia 40 Suppl 2:S2-17.

132

267. White, M.F. 2002. IRS proteins and the common path to diabetes. Am. J. Physiol Endocrinol. Metab 283:E413-E422.

268. White, M.F. 1998. The IRS-signalling system: a network of docking proteins that mediate insulin action. Mol. Cell Biochem. 182:3-11.

269. White, M.F. and C.R.Kahn. 1994. The insulin signaling system. J. Biol. Chem. 269:1-4.

270. White, M.F., R.Maron, and C.R.Kahn. 1985. Insulin rapidly stimulates tyrosine phosphorylation of a Mr-185,000 protein in intact cells. Nature 318:183-186.

271. Wice, B. 1990. Modulators of cyclic AMP metabolism induce syncytiotrophoblast formation in vitro. Exp. Cell Res. 186:306-316.

272. Windham, G.C., S.H.Swan, and L.Fenster. 1992. Parental cigarette smoking and the risk of spontaneous abortion. Am. J. Epidemiol. 135:1394-1403.

273. Withers, D.J., J.S.Gutierrez, H.Towery, D.J.Burks, J.M.Ren, S.Previs, Y.Zhang, D.Bernal, S.Pons, G.I.Shulman, S.Bonner-Weir, and M.F.White. 1998. Disruption of IRS-2 causes type 2 diabetes in mice. Nature 391:900-904.

274. Wood, I.S. and P.Trayhurn. 2003. Glucose transporters (GLUT and SGLT): expanded families of sugar transport proteins. Br. J. Nutr. 89:3-9.

133

275. Wu, W.S. 2006. The signaling mechanism of ROS in tumor progression. Cancer Metastasis Rev. 25:695-705.

276. Wulff, C. 2003. Angiogenesis during primate placentation in health and disease. Reproduction. 126:569-577.

277. Xiao, Q., S.Zeng, S.Ling, and M.Lv. 2006. Up-regulation of HIF-1alpha and VEGF expression by elevated glucose concentration and hypoxia in cultured human retinal pigment epithelial cells. J. Huazhong. Univ Sci. Technolog. Med. Sci. 26:463-465.

278. Yang, H.X., M.H.Zhang, W.J.Sun, and Y.Dong. 2005. [Associated factors of preeclampsia complicated in pregnant women with abnormal glucose metabolism]. Zhonghua Fu Chan Ke. Za Zhi. 40:577-580.

279. Zhao, F.Q. and A.F.Keating. 2007. Functional properties and genomics of glucose transporters. Curr. Genomics 8:113-128.

134