Angiogenesis, proteases and angiogenic factors during the inception of pregnancy. Crucial contributors or trivial bystanders?

Vascularised, receptive endometrium is essential for implantation and for the success of the embryo-maternal interaction. Disturbances in vascular development may play an important role in frequently occurring pathologies during pregnancy, such as early pregnancy wastage, pre-eclampsia and intrauterine growth restriction. Adaptation to implantation starts during the menstrual cycle by stromal decidualisation and the induction of angiogenesis, the formation of new vessels from existing vasculature. These processes continue throughout the 1st trimester. Angiogenesis is a multi-step process involving degradation of the basal membrane, activation, migration of endothelial cells and finally the formation of capillaries and the recruitment of pericellular smooth muscle cells. Numerous factors are involved in these processes. Three distinct groups of angiogenic factors, namely membrane bound proteases, vascular growth factors and its receptors and angiopoietins and its receptor, were analysed in this thesis. Pericellular proteolysis plays an important role in angiogenesis being required for endothelial cell migration, invasion and tube formation. In chapter 2 we studied the expression of proteases, matrix-metalloproteinase (MMPs) and uPA, by human endometrial microvascular endothelial cells (hEMVEC) and their involvement in the formation of capillary tubes in vitro. Inhibition of uPA and MMPs both reduced tube formation. hEMVEC expressed various MMPs mRNAs and proteins; in particular MMP-1, MMP-2, MT1-, MT3- and MT4-MMP. Immunohistochemistry confirmed the presence of MT3-MMP in endothelial cells of endometrial tissue. Overexpression of TIMP-1 or TIMP-3 by adenoviral transduction of hEMVEC reduced tube formation to the same extent. In addition, tube formation by hEMVEC was partly inhibited by the presence of anti-MT-3-MMP IgG. Thus, tube formation by hEMVEC is, at least in part, regulated by MT3-MMP. The study described in chapter 3 analyses the presence of MT-MMPs in human endometrium and their correlation with endometrial neovascularisation. The data show that all MT-MMP antigens are expressed in endometrium in a cycle-dependent pattern. The vascular expression of MT2- MT3- and MT4-MMP correlated with angiogenic episodes of the cycle. Since MT2- and MT3-MMP are known to regulate capillary-like tube formation, these findings support earlier in vitro data on the regulatory role of MT3-MMP in endometrial angiogenesis. Additionally, MT2-MMP appears to be associated with endometrial neovascularisation as well. Chapter 4 focuses on the role of proteases in decidual angiogenesis and remodelling in general. The expression of membrane-type matrix metalloproteinases (MT-MMPs) and urokinase/urokinase receptor (uPA/uPAR) were studied in decidua basalis (DB), decidua parietalis (DP) and decidual secretory endometrium (DSE) of human 1st trimester pregnancies. This enabled evaluation of decidual expression of the proteases, their regulation by pregnancy-induced hormones and/or the extra-villous trophoblast (EVT) and their relationship with decidual remodelling, trophoblast invasion and vascularisation. Pericellular-acting proteases appeared to be regulated by both pregnancy-induced hormones and EVT; uPAR and MT1-MMP by pregnancy-induced hormones and uPA, MT2-, MT3-, and MT5-MMP by EVT. All proteases were expressed by the EVT themselves and might be involved in trophoblast invasion. MT2- and MT3-MMP protein expression in endothelium was reduced in DB. Since these MT-MMPs are known regulators of angiogenesis, they may not only support decidual remodelling and trophoblast invasion but also partially regulate vascularisation at the embryonic implantation site. Chapter 5 describes the effect of human embryo-conditioned medium on in vitro endometrial angiogenesis. The conditioned media were shown to contain significant amounts of VEGF-A and stimulated in vitro tube formation by hEMVEC. This inducing effect was counteracted by adding soluble VEGF-R1. The other mediators, which have been described as produced by the early embryo/trophoblast, for instance Il-10, TGF_, PlGF, hCG and TGF_ had no effect on tube formation by hEMVEC. In conclusion, we show that the human embryo is able, via VEGF-A, to stimulate in vitro endometrial angiogenesis at the time of implantation. Chapter 6 evaluates vascular adaptation to implantation. We studied vascularisation and the expression of angiogenic factors in decidua basalis (DB), decidua parietalis (DP) and decidual secretory endometrium (DSE) of 1st trimester pregnancies. This enabled not only evaluation of decidual vascularisation and the expression of VEGF-A, PlGF, Flt-1, KDR, Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2) and TIE-2, but also their regulation by pregnancy-induced hormones and/or the extra-villous trophoblast (EVT). Both pregnancy-induced hormones and EVT influence vascularisation by enhancing vascular and luminal surface and reducing vessel density at the implantation site. These changes correlate with differences in gene and protein expression. PlGF mRNA and PlGF and Flt-1 protein expressions were elevated in DB under influence of EVT. Also the angiopoietins were differentially expressed, in favour of Ang-2, in DB. These data suggest that PlGF, Flt-1, Ang-1 and Ang-2 may be regulators of angiogenesis at the implantation site. Chapter 7 describes vascular adaptation to implantation in Missed Abortions by studying vascularisation and the expression of angiogenic factors and proteases in decidua basalis (DB), decidua parietalis (DP) and decidual secretory endometrium (DSE) of 1st trimester pregnancies which are complicated by a missed abortion. This enabled evaluation of decidual vascularisation, expression of VEGF-A, PlGF, Flt-1, KDR, Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2), TIE-2, uPA, uPAR, MT1-, MT2-, MT3- and MT5-MMP and their regulation by pregnancy-induced hormones and/or the extra-villous trophoblast (EVT). In Chapter 8, the expression of angiogenic factors in the 1st trimester, obtained during chorion villus sampling, were correlated with the pregnancy outcome. This was performed to study the hypothesis that early imperfections in the formation of the placental bed may result in pre-eclampsia, eclampsia or intra uterine growth retardation (IUGR). The mRNA levels of VEGF-A, PlGF, Flt-1, KDR, Angiopoietin-1 (Ang-1), Angiopoietin-2 (Ang-2) and TIE-2 did not show a correlation with pregnancy outcome. This chapter is incorporated as a preliminary report since the provided samples were not sufficient but we do think this study is of value to the thesis. In Chapter 9 the results of the studies of this thesis are discussed and Chapter 10 gives a final conclusion and recommendations for future research.