........................................................................................................................................... ii Co-Authorship ................................................................................................................................ iv Acknowledgements .......................................................................................................................... v List of Figures ................................................................................................................................. xi List of Abbreviations .................................................................................................................... xiii Chapter 1 General Introduction ....................................................................................................... 1 1.1 Endometriosis ........................................................................................................................ 1 1.1.1 Genetics of endometriosis ............................................................................................... 2 1.1.2 Lifestyle and environmental risk factors for endometriosis ............................................ 2 1.2 Pathogenesis of endometriosis ............................................................................................... 3 1.2.1 Transplantation theory .................................................................................................... 3 1.2.2 Celomic metaplasia ......................................................................................................... 4 1.2.3 Lymphatic and vascular dissemination ........................................................................... 5 1.3 Ovarian steroid hormone-inflammation-immune axis in endometriosis disease progression 5 1.3.1 Estrogen formation in endometriosis .............................................................................. 5 1.3.2 Prostaglandins in endometriosis...................................................................................... 6 1.3.3 Cytokine and chemokine expression in endometriosis ................................................... 7 1.3.4 Immune cell function in endometriosis ........................................................................... 9 1.4 Angiogenesis in endometriosis ............................................................................................ 11 1.4.1 Angiogenesis in the female reproductive system .......................................................... 12 1.4.2 Mechanisms of angiogenesis ........................................................................................ 13 1.4.3 Stromal cell derived factor-1 and endothelial progenitor cells in angiogenesis ........... 14 1.4.4 Anti-angiogenic therapy in endometriosis .................................................................... 17 1.5 Models of anti-angiogenic therapy in endometriosis ........................................................... 17 1.5.1 In vitro models to investigate potential anti-angiogenic compounds ............................ 17 1.5.2 Autologous or syngeneic rodent models ....................................................................... 18 1.5.3 Xenotransplantation of human endometrium into immunodeficient mice model ......... 21 1.6 Role of Thrombospondin-1 in angiogenesis ........................................................................ 22 1.6.1 Thrombospondin-1mimetic, ABT-898 ......................................................................... 24 1.7 Hypothesis and Objectives ................................................................................................... 26 Chapter 2 Microscopic anatomy of the pregnant mouse uterus throughout gestation ................... 28 2.1 Abstract ................................................................................................................................ 28 viii 2.2 Background .......................................................................................................................... 28 Chapter 3 A peptide inhibitor of synuclein-γ reduces neovascularization of human endometriotic lesion .............................................................................................................................................. 55 3.1 Abstract ................................................................................................................................ 55 3.2 Introduction .......................................................................................................................... 56 3.3 Materials and methods ......................................................................................................... 58 3.3.1 Ethics............................................................................................................................. 58 3.3.2 Acquisition of endometriosis and endometrium tissue ................................................. 58 3.3.3 Immunohistochemistry of SNCG in ectopic and eutopic endometrium ....................... 58 3.3.4 Synthetic peptides ......................................................................................................... 59 3.3.5 Co-immunoprecipitation – detection of SNCG association with internalized peptides 60 3.3.6 Human umbilical vein endothelial cell SNCG immunoflourescence ........................... 60 3.3.7 In vitro angiogenesis assay ........................................................................................... 61 3.3.8 Intravital fluorescent microscopy .................................................................................. 61 3.3.9 Alymphoid xenograft mouse model of endometriosis .................................................. 62 3.3.10 Isolectin-B4 fluorescence ............................................................................................ 63 3.3.11 Statistical analysis ....................................................................................................... 63 3.4 Results .................................................................................................................................. 64 3.4.1 Expression of SNCG is elevated in endometriosis lesions ........................................... 64 3.4.2 SP012 is uptaken by human endometriotic lesions in vivo ........................................... 64 3.4.3 SP012 inhibits endothelial cell tube formation in vitro ................................................ 65 3.4.4 SP012 reduces endometriotic lesion vascularity in a mouse model.............................. 65 3.5 Discussion ............................................................................................................................ 66 Chapter 4 Thrombospondin-1 mimetic peptide ABT-898 affects neovascularization and survival of human endometriotic lesions in a xenograft mouse model........................................................ 75 4.1 Abstract ................................................................................................................................ 75 4.2 Introduction .......................................................................................................................... 76 4.3 Materials and methods ......................................................................................................... 78 4.3.1 Human umbilical vein endothelial cells proliferation assay ......................................... 78 4.3.2 Matrigel angiogegenic tube formation assay ................................................................ 79 4.3.3 Mouse model of endometriosis ..................................................................................... 80 4.3.4 Treatment with ABT-898 .............................................................................................. 80 4.3.5 Detection of circulating endothelial progenitor cells .................................................... 81 4.3.6 Cytokine profiles in mouse peripheral blood ................................................................ 81 ix 4.3.7 Ultrasound image acquisition of endometriotic lesions in vivo .................................... 82 4.3.8 Necroscopy and lesion assessment in a mouse model .................................................. 83 4.3.9 Endometriotic lesion histology and CD31 immunofluoresence ................................... 83 4.3.10 Effects of ABT-898 on reproductive outcomes in mice ............................................. 84 4.3.11 Implantation site histology and isolectin fluorescence ............................................... 85 4.3.12 Stastical analysis ......................................................................................................... 86 4.4 Results .................................................................................................................................. 86 4.4.1 Effects of ABT-898 on human umbilical vein endothelial cell proliferation and tube formation in vitro ................................................................................................................... 86 4.4.2 Characterization of circulating endothelial progenitor cells in peripheral blood of experimental and control mice ............................................................................................... 87 4.4.3 Plasma cytokine profile in experimental and control mice ........................................... 88 4.4.4 Effects of ABT-898 on neovascularization of human endometriotic lesions in a mouse model ..................................................................................................................................... 89 4.4.5 Effects of ABT-898 on reproductive outcomes in mice ............................................... 90 4.5 Discussion ....................................................................................................