The Effects of VEGF Over- expression on the Utero-placental Circulation Vedanta Mehta University College London 2011 A thesis submitted for the degree of Doctor of Philosophy 1 Declaration I, Vedanta Mehta, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract Impaired uterine blood flow (UBF) leads to fetal growth restriction (FGR), one of the most challenging obstetric complications. FGR is associated with stillbirth, long-term neurological impairment and adult onset cardiovascular disease; there is no treatment currently available. Previously, it was shown that sustained local over-expression of Vascular Endothelial Growth Factor (VEGF) in the uterine arteries (UAs) of pregnant sheep using an adenoviral vector results in increased UBF as measured by Doppler sonography, reduced vascular contractility and increased vascular relaxation 4-7 days after administration. The aim of this thesis is to examine the long-term effects on UBF, UA vascular reactivity, and the possible mechanism of action. Telemetric transit-time flow probes were implanted around the UAs of mid-gestation pregnant sheep (n=10), and a telemetric blood-pressure sensitive catheter was inserted in the maternal (n=5) or fetal (n=4) carotid arteries. After obtaining baseline values for 7 days, we injected adenovirus vectors (5x1011 particles) encoding the VEGF-A165 gene (Ad.VEGF) into one UA, and a reporter β-galactosidase gene (Ad.LacZ), contra-laterally. UBF and maternal haemodynamics were measured daily until term, when the UAs were harvested and their vascular reactivity studied. There was a significant increase in UBF in the Ad.VEGF transduced side (36.53% v/s 20.08%, p=0.02), a reduction in UA contractility but no difference in relaxation. A significantly greater number of adventitial blood vessels were observed in the Ad.VEGF treated UA. There were no significant changes in maternal or fetal blood pressure post-injection. Similar effects were observed with injection of an adenovirus encoding another member of the VEGF gene family, VEGF-DΔNΔC (n=5). 3 Endothelial cells (ECs) were isolated from the UAs of control mid- gestation pregnant sheep, cultured and infected with Ad.VEGF or Ad.LacZ vector. Protein extracted from UAECs infected ex vivo was assayed for eNOS and phosphorylated eNOS (Ser1177) levels by Western blotting. eNOS and phospho- eNOS levels increased with rising Ad.VEGF concentrations in UAECs; Ad.LacZ vector transduction had no effect. Local over-expression of VEGF effects a long-term increase in UBF by upregulation of eNOS, with neovascularization of the adventitia and reduced UA contractility. These changes may benefit pregnancies complicated by severe FGR. With clinical translation in mind, the last section of this thesis describes the optimization of a technique of gene targeting to the utero-placental circulation of growth-restricted guinea pigs, using a thermo-sensitive pluronic gel. Having optimized this technique, VEGF over-expression in the uteroplacental circulation is now being tested in this animal model of FGR. 4 Table of Contents Title Page………………………………………………………………………….1 Declaration………………………………………………………………………...2 Abstract……………………………………………………………………………3 Table of Contents.................................................................................................... 5 List of Figures....................................................................................................... 14 List of Tables ........................................................................................................ 21 Publications........................................................................................................... 24 Acknowledgements............................................................................................... 25 Selected abbreviations .......................................................................................... 27 Chapter 1 Introduction........................................................................................................... 30 1.1 Maternal Factors resulting in FGR ................................................................. 33 1.1.1 Maternal Hypertensive Conditions .......................................................... 33 1.1.2 Maternal Autoimmune Disorders ............................................................ 33 1.1.3 Maternal Lifestyle.................................................................................... 34 1.1.4 Therapeutic Agents .................................................................................. 35 1.1.5 Malnutrition ............................................................................................. 35 1.1.6 Environmental Pollution .......................................................................... 36 1.1.7 Uterine and placental vascular insufficiency ........................................... 36 1.2 Fetal Factors resulting in FGR........................................................................ 36 1.2.1 Aneuploidy.............................................................................................. 36 1.2.2 Genomic Imprinting and Uniparental Disomy ........................................ 37 1.2.3 Fetal Malformations................................................................................. 38 1.2.4 Perinatal infections................................................................................... 39 1.2.5 Preterm birth ............................................................................................ 39 1.3 Utero-placental vascular insufficiency ........................................................... 40 1.3.1 Development of human uteroplacental vessels........................................ 40 1.3.2 Uterine blood flow in normal human pregnancies................................... 41 1.3.3 Uterine blood flow in pregnancies affected by uteroplacental insufficiency...................................................................................................... 42 1.3.3.1 Fetal growth restriction due to uteroplacental insufficiency................. 42 1.3.3.2 Pre-eclampsia........................................................................................ 43 1.3.3.3 Histological and morphological differences of human PET and FGR . 44 1.4 Diagnosis and Management of pregnancies complicated by FGR ................. 48 1.5 Perinatal Consequences of Fetal Growth Restriction ..................................... 53 1.5.1 Mortality .................................................................................................. 53 1.5.2 Prematurity and Associated Complications............................................ 53 1.5.3 Hypothermia ............................................................................................ 54 1.5.4 Hypoglycemia.......................................................................................... 54 1.5.5 Polycythemia............................................................................................ 54 1.5.6 Immune Deficiency and Sepsis................................................................ 54 5 1.6 Long-term consequences of Fetal Growth Restriction ................................... 55 1.6.1 Coronary Heart Disease ........................................................................... 56 1.6.2 Type II Diabetes....................................................................................... 56 1.6.3 Hypertension............................................................................................ 57 1.7 Prenatal Therapies for FGR caused by utero-placental insufficiency ............ 58 1.7.1 Bed rest .................................................................................................... 59 1.7.2 Oxygen supplementation ......................................................................... 59 1.7.3 Aspirin...................................................................................................... 60 1.7.4 β-adrenergic receptor agonists ................................................................. 61 1.7.5 Viagra....................................................................................................... 61 1.7.6 Growth Factor treatment.......................................................................... 62 1.7.7 Treatment with melatonin and vitamin C ................................................ 63 1.7.8 Nitric oxide donors .................................................................................. 63 1.7.9 Pravastatin................................................................................................ 64 1.7.10 Japanese Herbal Medicine ..................................................................... 64 1.8 Types of placentation...................................................................................... 65 1.8.1 Epitheliochorial placenta ......................................................................... 66 1.8.2 Syndesmochorial placenta ....................................................................... 67 1.8.3 Endotheliochorial placenta....................................................................... 67 1.8.4 Haemochorial placenta............................................................................. 67 1.9 Animal Models of FGR .................................................................................. 67 1.9.1 Naturally occurring models.....................................................................