Hypoxia and Angiogenesis in IUGR

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Hypoxia and Angiogenesis in IUGR

Hanna S. M. Wahlquist

B.Sc. Biomedical science

May 2006

School of Biomedical Sciences, Dublin Institute of Technology, Kevin Street. Dublin 8

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ABSTRACT

BACKGROUND: Intrauterine growth restriction (IUGR) is a condition where the infant

fails to reach its genetic growth potential due to numerous factors. IUGR foetuses are associated with high perinatal mortality and morbidity. This study investigated if hypoxia might be involved during gestation, and whether if hypoxia may have an impact on the development of the placenta, through regulation of various angiogenic factors, such as HIF-1α, VEGF, PIGF, VEGFR-1 and CD31.

METHOD: Each sample of placental tissue was stained by immunohistochemisty for

HIF-1α, VEGF, PIGF, VEGFR-1 and CD31. The intensity of staining was graded and the difference between the normal term placentas and the IUGR term placentas were evaluated.

RESULTS: HIF-1α was found to be upregulated in the normal term placental tissue, and

VEGFR-1 was found to be upregulated in the IUGR term placental tissue. The other antibodies did not show any significant difference and PIGF failed to show any positive staining.

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ACKNOWLEDGEMENTS

Special thanks to my supervisor, Mr. Joe Vaughan, for providing me with such an interesting project.

My sincerest thanks to Cathal McCarthy for his invaluable help and advice, and for his the provision of the antibodies and the placenta for this project.

Thanks also to all in the postgrad lab and in laboratory 210.

My thanks to Pia Ek for letting me go to Dublin for my project even though I did not have all the credits required to do so.

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Fig. 1A

1.0 Introduction

1.1 The development of the placenta

After fertilization of an oocyte the cell divides rapidly until a hollow blastocyst has formed. The blastocyst has a peripheral layer of blastomeres forming the trophoblast, with a mass of cells at one aspect, called the polar trophoblast, bulging into the central lumen, known as the inner cell mass (seen in Fig. 1A). Together with maternal

contribution the trophoblast eventually develops into the placenta. The inner cell mass gives rise to the foetus. (1, 2)

Implantation occurs when the blastocyst has been in the uterine cavity for 2-3 days. The endometrium is invaded by the polar trophoblast and after 10 days the blastocyst is completely buried in the endometrium. The penetration of the endometrium is facilitated by a decrease of desmosomes (spot-adhering junctions) linking the endometrial cells that undergo apoptosis. (2)

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syncytiotrophoblast layer occurs at the embryonic pole facing the endometrium. The syncytiotrophoblast rapidly surrounds the entire embryo, while invading the

endometrium. A sponge-like network of spaces called the lacunae develops within a short time. In the beginning the lacunae is filled with uterine secretion and tissue fluid. Later intermediate trophoblasts force the endometrial capillaries to disintegrate resulting in maternal blood leakage into the lacunae. The envelopment of maternal capillaries by trophoblasts establishes an arterial supply and a venous drainage system. At the end of the 4th month the placenta is essentially formed and after this the placenta only grows in diameter, complementing growth in the size of the uterus. (1)

1.2 IUGR

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Fig. 1.2A

circumference ratio and an estimation of the foetuses weight is then calculated using these measurements. (5)

Morbidity and mortality increases markedly if birth weight falls from the 10th percentile to the first percentile, and infants born between 38-42 weeks’ gestation with a birth weight between 1500-2500g have a perinatal morbidity and mortality 5-30 times

compared to those infants whose weights are in the 10-90th percentile. If the birth weight is lower than 1500g the risk of morbidity significantly increases. (6)

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Fig. 1.2B

The mother’s pregnancy weight gain can indicate the size of the foetus. Estimating the amount of amniotic fluid is another important use of the ultrasound, since a decrease in amniotic fluid is closely associated with IUGR.

IUGR is classified as asymmetric and symmetric. Asymmetric growth restriction implies that the foetus is undernourished and the energy is directed to maintain growth of vital organs such as heart and brain, at the expense of muscle, liver and fat. Usually

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A foetus with symmetric growth restriction will have a body that is proportionally small, which often is a result of a decrease in cell reproduction resulting in fewer cells. This could emphasise an infection or chromosomal abnormalities in the foetus.

It may be considered that IUGR is a consequence of a disease process in one or more of the three compartments that regulate and sustain foetal growth, the placenta, the maternal compartment, or the foetus. (3)

Placental factors:

Decreased blood flow in the placenta and uterus Abruptio placentae Placenta previa Infarction Preeclampsia Haemangioma Maternal factors:

High blood pressure Chronic kidney disease Diabetes mellitus

Heart or respiratory disease Malnutrition

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Anaemia

Substance abuse (alcohol, cigarettes, drugs) Low socioeconomic status

Foetal factors:

Multiple gestation (twins, triplets, etc.) Infections

Birth defects

Chromosomal abnormalities (trisomy 13, 18, 21 or triploidy) Turner’s syndrome (some cases)

In IUGR, foetuses may have problems at birth such as:

Decreased oxygen levels Meconium aspiration Hypoglycemia Low Apgar scores

Difficulties to maintain a normal body temperature

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1.3 Angiogenesis

Angiogenesis is the physiological process involving the growth of new blood vessels from already existing vessels. (7) Growth of new blood vessels is an important natural process in the body. Angiogenesis occurs during wound healing and after injury or insult to restore blood flow to tissues. It also occurs during pregnancy especially in the

formation of placenta. To regulate angiogenesis in the body, there are a number of factors that are either stimulating (angiogenic growth factors) or inhibiting. In a healthy individual there is a balance between growth factors and inhibitors. (8)

1.3.1 The process of angiogenesis:

Diseased or injured tissues produce and release angiogenic growth factors (proteins) that diffuse into the nearby tissues. The angiogenic growth factors bind to specific receptors located on the endothelial cells (EC) of nearby pre-existing blood vessels. Once growth factors bind to their receptors, the endothelial cells become activated. Signals are

transmitted from the cell's surface to the nucleus. The endothelial cell's machinery begins to produce new molecules including enzymes. Enzymes dissolve tiny holes in the sheath-like covering basement membrane surrounding all existing blood vessels. The endothelial cells begin to divide (proliferate), and migrate out through the dissolved holes of the existing vessel towards the diseased tissue. Specialized molecules called adhesion molecules, or integrins (avb3, avb5) serve as grappling hooks to help pull forward the sprouting of the new blood vessel. Additional enzymes (matrix metalloproteinases) are produced to dissolve the tissue in front of the sprouting vessel tip in order to

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vessel. Sprouting endothelial cells roll up to form a blood vessel tube. Individual blood vessel tubes connect to form blood vessel loops that can circulate blood. Finally, newly formed blood vessel tubes are stabilized by specialized muscle cells (smooth muscle cells, pericytes) that provide structural support. Blood flow is then initiated. (7)

1.4 Factors involved in angiogenesis

1.4.1 VEGF (Vascular endothelial growth factor)

VEGF binds to VEGFR-1 and VEGFR-2 and activates them; the receptors are expressed by endothelial cells within the blood vessel wall and are found on the cell membrane. VEGF is essential in the establishment of a functional vascular system, embryogenesis and early postnatal development. For endothelial cells to survive in immature blood vessels, VEGF is needed. If VEGF is absent the endothelial cells undergo apoptosis. When blood vessels have matured they can survive without VEGF. (9)

When VEGF binds to VEGFR-1 and VEGFR-2 a signalling cascade is initiated, which has an impact on survival, migration and proliferation of endothelial cells; this ultimately leads to angiogenesis. (10)

1.4.2 VEGFR-1 (Vascular endothelial growth factor receptor 1, also known as Flt-1) This is a cell membrane receptor kinase found on the cell surface of vascular endothelial cells, and has a high affinity to VEGF. It is involved in angiogenesis and the growth of endothelial cells.

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1.4.3 CD31, also known as PECAM-1 (Platelet Endothelial Cell Adhesion Molecule-1)

CD31 is a membrane protein and a member of the immunoglobulin superfamily. It

mediates cell-to-cell adhesion and is expressed constitutively on the surface of embryonic and adult endothelial cells and weakly expressed on many platelets and peripheral

leukocytes. It is also detected on embryonic stem cells and on bone marrow-derived haemopoietic stem cells. During early post-implantation embryonic development multiple alternatively spliced isoforms of CD31 are detected. It also has a role in neutrophils transendothelial migration. Endothelial cell-to-cell interactions that are mediated by CD31 are involved in angiogenesis. (12)

1.4.4 HIF-1α (Hypoxia inducible factor-1α)

Hypoxia is defined as an abnormal and pathological low level of oxygen at a defined point in time and at a given site. HIF-1α is a transcription factor, and together with other hypoxia inducible factors it represents the link between oxygen effectors and sensors at a local, cellular and systemic level. Many genes have an adaptive response to hypoxia, such as those involved in glycolysis, erythropoiesis, angiogenesis and glucose transport. HIF-1α can be found in most human tissue such as heart, brain, lung, liver, skeletal muscle, kidney, placenta, pancreas, spleen, thymus, prostate, testis, ovary, small intestines, colon, leukocytes and skin.

HIF-1α levels are low or absent under normal oxygen conditions, if oxygen

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In placentas of all gestational ages HIF-1α is expressed by the villous cytotrophoblast, syncytiotrophblast and fetoplacental vasculature. In the first 10 weeks of pregnancy the human placenta is in a relatively hypoxic environment resulting in an increase in HIF-1α expression. As gestation progresses the expression of HIF-1α decreases as the levels of oxygen increases.

1.4.5 PIGF (Placental growth factor)

PIGF is a type of vascular endothelial growth factor and is expressed mainly in

trophoblasts, but also in endothelial cells. PIGF acts through VEGFR-1 and VEGFR-2 (only as VEGF/PIGF heterodimers), tyrosine kinase receptors, and plays a role in

placental vascular development. The expression of PIGF increases early in gestation and peaks around 26-30 weeks, as term approaches the expression is decreased.

PIGF affects trophoblast proliferation, protects cells from apoptosis and has a direct vascular effect that promotes successful placentation. (14)

1.5 Immunohistochemistry (IHC)

Immunohistochemistry is a method used to identify and analyze different cell types and is based on the binding of antibodies to specific components of the cell.

IHC combines immunological, biochemical and anatomical techniques for identification of specific tissue components by specific antigen/antibody reaction tagged with a visible label. This makes it possible to visualize the localization and distribution of specific cellular components within tissues or cells.

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biotin has a very strong affinity to each other and is therefore widely used in

immunohistochemical staining. Avidin is a large glycoprotein and can be labeled with fluorescein or peroxidase, whereas the biotin is a vitamin with a low molecular weight and can be conjugated to different biological molecules such as antibodies.

It is a three layer technique with an unlabeled primary antibody as the first layer, biotinylated secondary antibody as the second layer. The third layer is a complex of avidin-biotin peroxidase. To produce different colorimetric end products the peroxidase is developed by DAB or other substrates. (15)

1.6 Antigen retrieval

Many antigen demonstrations can be significantly improved by pre-treatment with antigen retrieval reagents. Antigen retrieval will break the protein cross-links that were formed during formalin fixation and thereby unmask hidden antigen sites and restoring immunoreactivity to the tissue antigen.

Antigen retrieval methods used for formalin-fixed, paraffin-embedded tissues include heat inducing sections for varying lengths of time in solutions such as citric buffer or EDTA-buffer. Heating methods used include the microwave oven and the pressure cooker.

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1.7 Aims of the project

The first aim of the project is to examine about the structure of the placenta by staining with haematoxylin and eosin. Placentas from different stages of normal gestation will be stained, 24-28 weeks, 30-35 weeks and 37+ weeks to examine morphological differences during the development of the placenta. Term IUGR placentas will also be examined morphologically by H&E staining and compared with normal term placentas.

The second aim of the project will be to stain 10 IUGR term placental samples and 10 normal term placental samples with 5 different antibodies (VEGF, CD31, HIF-1α, VEGFR-1 and PIGF) and to optimise the immunohistochemical protocol for these

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2.0 Materials and Methods

2.1 Materials

Distilled water

PBS (Phosphate Buffered Saline)

DAB (3, 3’-diaminobenzidine tetrahydrochloride) Methanol

Xylene

Absolute alcohol Ethanol (70%) DPX

Vectastain ABC-kit Elite PK-6200 Universal

Anti-CD31, monoclonal mouse antibody, Dako Cytomation Anti-HIF1α, monoclonal mouse antibody, Novus Biologicals

Anti-PIGF, purified polyclonal goat antibody, Santa Cruz Biotechnology Anti-VEGF, purified rabbit polyclonal antibody, Calbiochem

Anti-VEGFR-1, epitope specific rabbit antibody, Lab vision Corporation Electrical pressure cooker, Princess model: DYB350

MIST (Magnetic Immuno Staining Tray) APES-Slides

Slide racks

EDTA (Ethylenediaminetetraacetic acid disodium salt) VWR International ltd. BDH Microprocessor pH Meter, Hanna instruments pH210

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50 ml tubes, Sarstedt

Citric acid, BDH Laboratory Supplies

Water bath, RA Lamb, MSC Medical Supply C. LTD model E65 Microtome, Microm HM325, Syntec Scientific, Base Enterprise Centre Windsor Incubator, MSC Medical Supply Co. LTD

Centrifuge 5417 C, eppendorf Eppendorf tubes

Electronic Balance MP-300, Chyo, Medical Supply Co. Paraffin imbedded placental tissues

Cover slips

1L Volumetric flasks Beakers (1L)

Glass staining bath (400 ml) Mayer’s Haematoxylin Harris’ Heamatoxylin Eosin (1%)

Acid-alcohol (1%)

2.2 Preparation of the samples

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2.3 Haematoxylin and eosin staining

Sections were collected from the water after dewaxing and placed on the MIST. They were covered with Harris’s haematoxylin for 5 min and then washed in running water for 3 min to “blue” the sections. The sections were then differentiated in 1% Acid-alcohol for 2 seconds and then blued in water. The sections were covered with 1% eosin for 3 min and then rinsed well with tap water. Sections were dehydrated through ethanol (70%), absolute alcohol and xylene and then mounted in DPX.

2.4 Antigen retrieval methods

Microwave oven, pressure cooker and protease retrieval methods were used. EDTA-buffer (pH 8.0), and citrate EDTA-buffer (pH 6.0) were used for both microwave and pressure cooker, retrieval methods.

The EDTA-buffer was made with 3.7 g of EDTA in 1L of water pH to 8.0. The citrate buffer was made with 2.1 g of citric acid in 1L of water, pH to 6.0.

Using the microwave retrieval method 500ml of either EDTA or citrate buffer was poured in a plastic box in which the slides were immersed. The box was placed into the microwave on a high setting for 18 min. The buffer was allowed to cool for 20 min before moving the slides into distilled water.

Using the pressure cooker retrieval method the slides were immersed in 500ml of citrate or EDTA buffer in a plastic box, which were sealed in the pressure cooker. The cooker was set to a high pressure mode for 12min. The buffer was allowed to cool for 20 min before the slides were moved into distilled water.

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slides were placed on a tray in the incubator. The protease had been allowed to dissolve at 37C in the incubator, prior to its application.

The slides were then covered with the protease and left in the incubator for 5 min. After incubation the slides were rinsed in distilled water.

2.5 Immunohistochemical protocol

The sections were collected from the water and placed on the MIST. To block

endogenous peroxidase the sections were covered with 3% Hydrogen peroxide for 10 min. Sections were rinsed with distilled water, circled with dako-pen and then covered with PBS for 5 min. Sections were covered with 2-3 drops of normal serum for 5 min. Sections were then covered with primary antibody for 30 min. Following the removal of the primary antibody the sections were rinsed in PBS for 1min, 3 changes.

The slides were covered with biotinylated secondary antibody for 15 min, and then rinsed in PBS for 1 min, 3 changes. The slides were covered with ABC-reagent for 15 min, and then again rinsed in PBS for 1 min, 3 changes.

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Fig. 2.6A Fig. 2.6B

Fig. 2.6C

2.6 Scoring of slides

All slides were visualised blind using a Leica DM L52 microscope. The sections were scored with +1, +2 or +3 depending on the intensity and quantity of staining.

Scoring +1 Scoring +2

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Fig. 3.1A Fig. 3.1B

Fig. 3.1C Fig. 3.1D

3.0 Results

3.1 Haematoxylin and eosin staining

24-28 weeks of gestation 30-35 weeks of gestation

37+ weeks of gestation IUGR 37+ weeks of gestation

There were a number of morphological differences seen in the range of placental tissues stained by H&E. In the placenta from 24-28 weeks of gestation the number of

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endothelial cells and the syncytiotrophoblast is large. Later in gestation the distance decreases as more nutrients and oxygen are needed for foetal growth and the vessels are required to be close to the surface.

In placental tissue from 24-28 weeks of gestation the amount of maternal blood is low and there are lower numbers of blood vessels in the villi. Later in gestation more maternal blood between villi was seen and the quantity of foetal vessels was increased. The IUGR term placental tissue had a lower level of maternal blood and more foetal blood vessels could be seen in the villi.

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Fig. 3.2A Fig. 3.2B Fig. 3.2C Fig. 3.2D Endothelial cells 3.2 CD31

Positive control (tonsil) for CD31 Negative control for CD31

CD31 normal placenta (37+ weeks) CD31 IUGR placenta

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Fig. 3.3A Fig. 3.3B

Fig. 3.3C Fig. 3.3D

Stroma

3.3 HIF-1α

Positive control for HIF-1α (Colon Negative control for HIF-1α Adenocarcinoma)

HIF-1α normal placenta (37+ weeks) HIF-1α IUGR placenta

The microwave oven with citrate buffer was the first antigen retrieval method applied for this antibody, with an antibody dilution of 1:500. The intensity of staining was of good quality but came out very strong. The second antigen retrieval method applied was the citrate buffer in the pressure cooker, which also produced staining of good intensity. Repeating the retrieval methods with EDTA buffer the intensity of staining was weak. The best retrieval method was the citrate buffer in the microwave oven.

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After antibody optimization with different dilutions, 1:500, 1:700 1:1000, 1:2000 and 1:5000, the best dilution seemed to be the 1:700. Stromal cells, vascular endothelial cells and the syncytiotrophoblast layer all stained positive with this antibody.

In the normal term placentas the syncytiotrophoblast layer had the strongest intensity of staining with an average mark of 3. Endothelial cells in the normal term placentas were stained with an average mark of 2, also the stroma had an average staining mark of 2. The syncytiotrophoblast layer of the IUGR term placentas was marked 1-2, with the majority of samples having a score of 1. For the endothelial cells and the stroma the average mark were 0 in the IUGR term placentas. (See Appendix I, Table 2)

In both groups the syncytiotrophoblast layer had the highest intensity of staining of all cell types. The stroma and the endothelial cells were equal in their staining within the two different groups.

This illustrates a significant difference between the two groups for HIF-1α, where the normal placental group had a stronger intensity of staining than the IUGR placental group.

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3.4 PIGF

This antibody had not been previously evaluated on paraffin embedded tissue. The antigen retrieval methods with microwave oven and pressure cooker using both EDTA and citrate buffer illustrated no positivity on either the placental or positive control tissue. The protease retrieval method also failed to produce any positivity.

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Fig. 3.5A Fig. 3.5B Fig. 3.5C Fig. 3.5D Syncytiotrophoblast layer 3.5 VEGFR-1

Positive control VEGFR-1 Negative control VEGFR-1 (Normal placenta)

VEGFR-1 normal placenta (37+ weeks) VEGFR-1 IUGR placenta

Antigen retrieval methods used for this antibody included the microwave with citrate buffer and EDTA buffer, and the pressure cooker with citrate buffer and EDTA buffer. There was no major difference in staining intensity between the methods used, but the microwave oven with EDTA buffer had a better intensity and higher quality of staining. Positive cells included the syncytiotrophoblast layer and stromal cells. In the

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normal term placentas the average score was 1. No positively stained stromal cells were visualised in the normal term placentas but four IUGR term placentas had positive staining in the stromal area.

This illustrates a difference in staining between the two groups, the syncytiotrophoblast layer illustrated the highest intensity of staining. (See Appendix I, Table 3)

Antibody dilution used was 1:50.

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Fig. 3.6A Fig. 3.6B Fig. 3.6C Fig. 3.6D Fig. 3.6E Stromal cells Syncytiotrophblast layer Syncytiotrophblast layer 3.6 VEGF

Positive control for VEGF Negative VEGF (Colon adenocarcinoma)

VEGF normal placenta (37+ weeks) VEGF normal placenta (37+ weeks)

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For the VEGF antibody the antigen retrieval methods with microwave oven and pressure cooker using citrate buffer and EDTA buffer failed to produce positivity. The protease method using different dilutions (1:20, 1:50, and 1:100) illustrated positivity in 1:20 and in 1:50 but none in 1:100. The 1:20 produced the strongest intensity but quality of the staining still wasn’t optimal. After reading a review (16) of a paper using this particular antibody we tried antigen retrieval with citrate buffer in the microwave and incubated the primary antibody for 12 hours at 4C. The same time antibody dilutions (1:20, 1:50, and 1:100) were also performed. Positivity was illustrated in 1:20 and 1:50 but not in 1:100. An antibody dilution of 1:20 produced the strongest intensity and compared with the protease retrieval method this staining was more specific.

Cells stained positive with this antibody included the syncytiotrophoblast layer, vascular endothelial cells and stromal cells. The scores given for the syncytiotrophoblast in both normal term placentas and IUGR term placentas were 1. The vascular endothelial cells and stromal cells in both normal term placenta and IUGR term placentas were given score of 0-1, illustrating no difference in expression of VEGF between the two groups. (See Appendix I, Table 4)

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4.0 Discussion

Angiogenesis is defined as the sprouting of new blood vessels from existing vessels; it is a tightly regulated process and dependent on numerous factors. The balance between pro-angiogenic factors and pro-angiogenic inhibitors is important and if the balance is shifted angiogenesis is either “switched off” or “switched on”. Embryogenesis and wound healing result from the angiogenic process being switched on so that new blood vessels can develop. One of major angiogenic factors is VEGF which binds to VEGFR-1 (Flt-1), VEGFR-2 and also to sFlt-1(soluble fms-like tyrosine kinase 1).

In IUGR, foetuses are smaller than expected for the number of weeks of pregnancy. IUGR is considered to be a consequence of a disease process in the placenta, the foetus or the maternal compartment, resulting in a lack of development of the foetus.

Placental infarcts are often seen histologically in IUGR placentas, hypoxia seems to be responsible for these infarcts, indicating a role for hypoxia in IUGR. In early gestation the placenta is in a hypoxic environment, which leads to an increase in HIF-1α, a transcription factor. This increased level of HIF-1α ultimately leads to angiogenesis through regulation of proteins such as VEGF. A hypoxic environment is essential at the beginning of pregnancy, as it initiates angiogenesis which results in the formation of new blood vessels to deliver O2 and nutrients to the growing foetus.

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It has been shown that sFlt-1 inhibits angiogenesis in preeclampsia. (17) In vitro analysis of preeclamptic conditioned medium showed that the angiogenesis was restored to levels comparable with that in normal conditioned medium, if the sFlt-1 was removed. (17) This illustrates that sFlt-1 has a very strong influence on the angiogenic process and that, in this obstetric complication, angiogenesis is distorted. As sFlt-1 is regulated by hypoxia we proposed that hypoxia increased sFlt-1 in IUGR. We measured protein levels of HIF-1α, VEGF, VEGFR-1 and PIGF to examine if hypoxia was upregulated in IUGR term placentas and if hypoxia affected angiogenic factors in IUGR, resulting in defective placentation and the development of IUGR.

The results for CD31 concluded that the protein expression of the vascular endothelial cells showed no difference between the normal term placenta and the IUGR term placental tissue. This suggests that the cell-to-cell adhesion in the vascular endothelial cells in the foetal vessels is not affected by the state of IUGR.

Our results for HIF-1α suggested that it was upregulated in the normal term placentas, having a higher expression than in the IUGR term placentas. A study by Rajakumar et al (18) examining normal placentas illustrated that HIF-1α expression was high in early gestation and decreased as gestation proceeds. They used two different antibodies for HIF-1α., one of them was the same antibody as we used in our study (Novus biological). However our results differ as expression was still quite high at term in the normal

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that the expression of HIF-α is regulated by the IL-β in normal human cytotrophoblasts. Serum-starved normal human cytotrophblasts were exposed to IL-1β, and the result showed an induction of the HIF-1α protein expression that was concentration dependent. However the molecular mechanism that regulates this expression would need further investigation.

Studies (20) on HIF-2α imply that HIF-2α might be involved in increased placental apoptosis in IUGR, as its expression was increased in the studied IUGR cohort. Increased apoptosis in IUGR would be an indicator of placental insufficiency.

The results for VEGF protein expression showed no significant difference between the two groups. In several studies it has been shown that VEGF is up regulated by hypoxia in both tumours and the human placenta. (21, 22, 23, 24) In a study by Gurel D. et al (25), with 19 IUGR term placental samples and 27 normal term placental samples, there was no upregulation of VEGF in IUGR term placentas, which complies with our results. A study by Jenny Y. M. et al (16) also failed to show any upregulation of VEGF in IUGR term placentas compared to normal term placentas.

VEGFR-1 was up regulated in IUGR term placentas compared to the normal term placentas. This upregulation has also been shown by Satu Helske et al (26).They studied the vascular endothelial growth factors in normal and complicated pregnancies, such as IUGR, preeclampsia and foetal alcohol syndrome. Their results showed that the increased expression of VEGFR-1 could be associated with hypoxia and abnormal placental

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hypoxia.

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5.0 Conclusion

Hypoxia ultimately leads to angiogenesis and the findings from the H&E staining showed an increased amount of foetal vessels in the IUGR term placentas, compared to the

normal term placenta, which would suggest that hypoxia could be a factor involved in angiogenesis in the IUGR cohort.

Some of our results failed to indicate that hypoxia was upregulated in IUGR. It would have been expected that if hypoxia was involved there would be an upregulation of the HIF-1α in the IUGR compared to the normal term placentas, and in that extent also VEGF expression would be increased, as HIF-1α is a regulator of VEGF. If VEGF is increased it would suggest that VEGFR-1 would also be increased.

A decrease in PIGF would also suggest a hypoxic environment, but unfortunately staining for PIGF was unsuccessful and we can’t establish whether it is up or downregulated.

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in the IUGR cohort, from the microarray study.

In conclusion our results imply that there must be factors other than hypoxia which regulate the angiogenic process or that hypoxia affects factors that we have not examined in this study. Further studies on PIGF and hypoxia would be beneficial.

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6.0 Bibliography

(1) Barabara Young, John W. heath. Chapter 19. Female reproductive system: The placenta. In Wheater’s Functional Histology. Churchill Livingstone, London 2000: 359-361.

(2) Abraham L. Kierszenbaum. Chapter 23. Fertilization, placentation, and lactation. In Histology and cell biology an introduction to pathology. Mosby, USA. 2002: 589-594. (3) University of Virginia: High risk pregnancy 2004

http://www.healthsystem.virginia.edu/uvahealth/peds_hrpregnant/iugr.cfm (4) Hygeia Foundation Inc & Institute of perinatal loss and bereavement Giancarlo Mari, M.D. Intrauterine Growth Retardation, Vol.1 No.8 The Yale University School of Medicine

http://hygeia.org/poems8.htm

(5) American Academy of Family Physicians, News & Publications. Intrauterine Growth Restriction: Identification and Management

David Peleg, M.D., Colleen M. Kennedy, M.D., and Stephen K. Hunter, M.D., PH.D. University of Iowa Hospitals and Clinics, Iowa City, Iowa 1998

http://www.aafp.org/afp/980800ap/peleg.html

(6) Resnik R, MD Intrauterine growth restriction. The American College of Obstetricians and Gynecologists 2002; 99: 490-496

(7) The angiogenesis Foundation; Understanding angiogenesis http://www.angio.org/understanding/understanding.html

(8) Aboudola S. and Kini R A. Angiogenesis in lymphproliferative disorders: a therapeutic target? Current Opinion in Hematology 12:279-283 2005

(9) Research VEGF; VEGF functions

http://www.researchvegf.com/researchvegf/vegf-overview/vegf-functions/index.m (10) Ferrara N, Gerber H-P and LeCouter J. The Biology of Vegf and its receptors. Nature medicine Vol. 9 2003

(11) Cancer Research Institute

Larry Witte, ImClone Systems Incorporated, New York, NY http://www.cancerresearch.org/antibodies2002/larry_witte.html (12) IHC World Online Center for Immunohistochemistry

CD31 (PECAM-1) Immunohistochemical Staining Protocol on Formalin Fixed, Paraffin Embedded Mouse Tissues

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(13) EntreMed Hypoxia-inducible factor 1A (HIF-1A) http://www.entremed.com/go.cfm?do=Page.View&id=172

(14) Torry D S, Mukherjea D, Arroyo J, and Torry R J. Expression and Function of placenta growth factor: Implications for abnormal placentation.

Department of Medical microbiology and immunology and obstetrics and gynecology, Southern Illinois University School of medicine, Springfield, Illinois; and College of Pharmacy and health sciences Drake University, Des Moines, Iowa. 2003

(15) IHC World Online Center for Immunohistochemistry Introduction Immunohistochemistry

http://www.ihcworld.com/introduction.htm#intro

(16) Jenny Y M et al. Expression of vascular endothelial growth factor in third-trimester placentas is not increased in growth-restricted fetuses.

Department of Obstetrics & Gynaecology, and Department of Pathology. The University of Hong Kong. Queen Mary Hospital. Hong Kong. People’s Republic of China.

Published by Elsevier Science Inc.

(17) Ahmad S and Ahmed A. Elevated placental soluble vascular endothelial growth factor receptor-1 inhibits angiogenesis in preeeclampsia.

American Heart Association 2004

(18) Rajakumar A and Conrad K P. Expression, ontogeny, and regulation of hypoxia inducible transcription factors in the human placenta

Biology of Reproduction 63: 559-569. 2000

(19) Qian D, Lin H-Y et al. Normoxic induction of the hypoxic-inducible factor-1α by interleukin-1β involves the extracellular signal-regulated kinase ½ pathway in normal human cytotrophblast cells.

Biology of Reproduction 70: 1822-1827. 2004

(20) Hypoxia-inducible factor-2alpha is involved in enhanced apoptosis in the placenta from pregnancies with foetal growth restriction.

Pathology Int. 54: 843-849. 2004

(21) Schweiki D et al. Vascular endothelial growth factor induced by hypoxia may mediate hypoxia initiated angiogenesis.

Nature 359: 843-845. 1992

(22)Plate KH et al. Vascular endothelial growth factor is a potential tumour angiogenesis factor in human gliomas in vivo.

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(23) Wheeler T et al. Angiogenesis in the placental environment. Placenta 16: 289-296. 1995

(24) Gleadle JM et al. Regulation of angiogenic growth factor expression by hypoxia, transition metals & chelating agents.

Am. J. Physiol. 268: C1362-C1368 1995

(25) Gurel D, et al. Expression of IGR-IR and VEGF and trophoblastic proliferative activity in placentas from pregnancies complicated by IUGR.

Pathol. Res. Pract. 199: 803-809 2003

(26) Helske S et al. Expression of vascular endothelial factor receptors 1, 2 and 3 in placentas from nor mal and complicated pregnancies.

Molecular Human Reproduction 7: 205-210 2001

(27) Greber et al. Differential transcriptional regulation in the two VEGF receptor genes. Flt-1, but not Flk-1/KDR is up-regulated by hypoxia.

Journal of Biol. Chem. 272: 23659-23667 1997

(28) Lam PM et al. Upregulation of mRNA expression of vascular endothelial growth factors and its receptors by exogenous human chorionic gonadotropin in cultured oviduct mucosal cells.

(43)

APPENDIX I–TABLES

Table 1 CD31 expression in normal term placental tissue and IUGR term placental tissue

Degree of positivity

Samples Vascular endothelial cells

(44)

Table 2 HIF-1α expression in normal term placental tissue and IUGR term placental

tissue

Degree of positivity Samples Syncytiotrophoblasts Vascular endothelial

(45)

Table 3 VEGFR-1 expression in normal term placental tissue and IUGR term placental

tissue

Degree of positivity

Samples Syncytiotrophoblast Stroma

(46)

Table 4 VEGF expression in normal term placental tissue and IUGR term placental

tissue

Degree of positivity Samples Syncytiotrophoblasts Vascular endothelial

(47)

APPENDIX II–REAGENT PREPARATIONS

Citrate buffer (pH 6.0)

2.1g citric acid 1L distilled water 2M NaOH

Citric acid was diluted in 950ml of distilled water. The pH was adjusted to 6.0 using 2M NaOH, changes in pH were monitored on the electronic pH meter. After adjustment the solution were transferred to a 1L volumetric flask and filled with distilled water to the 1L mark.

EDTA buffer (pH 8.0)

3.7g of EDTA 1L of distilled water 2M NaOH

(48)

3% Hydrogen peroxide (H202)

1ml 30% H202

9ml Methanol

9 ml of methanol was measured into a 50ml tube using a pipette. 1ml of H202 was added

to the methanol and the solution was kept at 4C when not used. All work was performed in a fume hood.

DAB reagent

0.5ml concentrated DAB (frozen) 4.5ml PBS

5 drops of H202

The 0.5ml glass tube of DAB was allowed to defrost in room temperature. The PBS was then added followed by the H202. The solution was applied to the slides as quickly as

(49)

APPENDIX III–ANTIBODIES

Human antigen Species Control Dilution Source

CD31 Mouse Tonsil 1:50 Dako

Cytomation

HIF-1α Mouse Colon

Adenocarcinoma

1:700 Novus

Biologicals

VEGF Rabbit Colon

Adenocarcinoma

1:20 Calbiochem

PIGF Goat Colon

Adenocarcinoma

1:20 Santa Cruz Biotechnology

VEGFR-1 Rabbit Placenta 1:50 Lab Vision

Hypoxia and Angiogenesis in IUGR