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The Levels of Angiogenic and Anti-Angiogenic Molecule Concentrations in Pregnancy Based Disorders in the Maternal and Fetal Circ

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The Levels of Angiogenic and Anti-Angiogenic Molecule Concentrations in Pregnancy Based Disorders in the Maternal and Fetal Circ Master of Philosophy THE LEVELS OF ANGIOGENIC AND ANTI-ANGIOGENIC MOLECULE CONCENTRATIONS IN PREGNANCY BASED DISORDERS IN THE MATERNAL AND FETAL CIRCULATION Islam Afzal Supervisor: Professor Asif Ahmed Department of Reproductive and Vascular Biology November 2012 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Islam Afzal Contents Table of Figures ............................................................................................... 5 Abbreviations ................................................................................................... 7 Acknowledgements .......................................................................................... 9 Abstract .......................................................................................................... 10 Introduction .................................................................................................... 11 Preeclampsia ................................................................................................. 12 Vascular Growth Factors and Preeclampsia .................................................. 15 VEGF and VEGF Receptors .......................................................................... 16 Intracellular Signalling by VEGF .................................................................... 19 Vascular Endothelial Growth Factors ............................................................. 20 VEGFR-1 ....................................................................................................... 21 Placental Growth Factor (PlGF) ..................................................................... 22 Aims and Objectives ...................................................................................... 27 Methods ......................................................................................................... 28 Clinical Data of Maternal and Fetal Samples ................................................. 29 Statistical Analysis ......................................................................................... 30 Tissue and Cell Culture .................................................................................. 31 Maintenance of primary cells and cell lines .................................................... 31 Cell cryopreservation ..................................................................................... 32 Details of Primary Cells and Cell Lines .......................................................... 32 Human Umbilical Vein Endothelial Cell Culture (HUVEC) ............................. 32 In-vitro Cell Function Analysis ........................................................................ 33 General ELISA Protocol ................................................................................. 33 Results ........................................................................................................... 35 3 Islam Afzal Clinical Data ................................................................................................... 36 Clinical Characteristics of the study population .............................................. 36 Sandwich ELISA to detect sVEGFR-1 in human plasma samples ................. 39 Analysis of Maternal and Fetal Plasma Samples for soluble Endoglin ........... 43 Analysis of Maternal and Fetal Plasma Samples for VEGF ........................... 46 Analysis of Maternal and Fetal Plasma Samples for PlGF ............................. 49 Free sVEGFR-1 presence in the plasma ....................................................... 52 ELISA following the Insertion of siRNA of Heme Oxygenase ........................ 54 The effect of Honokiol of sFlt and sEng release ............................................ 59 Conclusion ..................................................................................................... 64 sVEGFR-1...................................................................................................... 65 Soluble Endoglin ............................................................................................ 66 VEGF ............................................................................................................. 68 PlGF ............................................................................................................... 69 Free VEGFR-1 ............................................................................................... 70 Heme-oxygenase ........................................................................................... 71 Honokiol ......................................................................................................... 71 Future Work ................................................................................................... 73 Bibliography ................................................................................................... 77 Appendix ........................................................................................................ 88 4 Islam Afzal Table of Figures Figure 1: The differences between normal and PE placental invasion. In the impaired preeclamptic state, invasion of the spiral arteries is shallow, and they remain small, highly resistant vessels (Lam et al., 2005). ................................................................................................................... 15 Figure 2 VEGF family and ligands. .................................................................................................... 17 Figure 3. Diagrammatic representation showing the interaction of the various angiogenic factors resulting in preeclampsia. ....................................................................................................... 23 Figure 4. Schematic showing the degredation of Heme by HO in an NADPH dependant manner releasing CO, Fe2+ and biliverdin. Fe2+, which is cytotoxic, is stored by ferritin. ....................... 25 Figure 5 Characteristics of women in the different groups and of their infants. ............................... 38 Figure 6. Maternal plasma concentrations of sVEGFR-1 of women in the different groups. ............. 40 Figure 7. Plasma concentration of sVEGFR-1 present in the fetal circulation at delivery in the different groups. ..................................................................................................................... 42 Figure 8. Maternal plasma concentrations of soluble endoglin (sEng) of women in the different groups. .................................................................................................................................... 44 Figure 9. Plasma concentration of soluble endoglin (sEng) present in the fetal circulation at delivery in the different groups. ........................................................................................................... 45 Figure 10. Maternal plasma concentrations of vascular endothelial growth factor (VEGF) of women in the different groups. ........................................................................................................... 47 Figure 11. Plasma concentration of vascular endothelial growth factor (VEGF) present in the fetal circulation at delivery in the different groups. ........................................................................ 48 Figure 12. Maternal plasma concentrations of placental growth factor (PlGF) of women in the different groups. ..................................................................................................................... 50 Figure 13. Plasma concentration of placental growth factor (PlGF) present in the fetal circulation at delivery in the different groups. ............................................................................................. 51 Figure 14. Plasma concentration of free sVEGFR-1 present in the maternal circulation at delivery in the different groups. ............................................................................................................... 53 5 Islam Afzal Figure 15. Bar chart showing the levels of anti-angiogenic factor sVEGFR-1 detected by ELISA in human umbilical vein endothelial cells following siRNA knockdown of heme-oxygenase 1 (HO- 1) and following 24 hour treatment with VEGF. ...................................................................... 56 Figure 16. Bar chart showing the levels of anti-angiogenic factor sVEGFR-1 detected by ELISA in human umbilical vein endothelial cells following siRNA knockdown of heme-oxygenase 2 (HO- 2) and following 24 hour treatment with VEGF. ...................................................................... 57 Figure 17. Bar chart showing the levels of anti-angiogenic factor sVEGFR-1 detected by ELISA in human umbilical vein endothelial cells following siRNA knockdown of heme-oxygenase 1 and heme-oxygenase 2 (HO-1 and HO-2) and following 24 hour treatment with VEGF. ................. 59 Figure 18. Bar chart showing the dose dependant decrease in sVEGFR-1 release from
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