Receptor-mediated effects of VEGF and PIGF on the mitogenic and angiogenic actkivity of human placental microvascular endothelial cells

The human placenta develops within 9 months to an organ with a high capacity for maternal-fetal exchange of gases and nutrients and, in the reverse direction, for waste products. The fully developed placenta at term comprises an impressive length of fetal capillaries of about 320km with a total inner capillary surface area of 12m 2 . It is obvious that during placental development very active vasculogenesis (de novo formation of vessels by differentiation of progenitor cell types) and angiogenesis (sprouting of capillaries from existing blood vessels) must occur. Many peptide growth factors, in particular the heparin-binding growth factors, promote angiogenesis in vitro and in vivo, but the only growth factors known to act directly on endothelial cells are the vascular endothelial growth factors (VEGF) and the placental growth factors (PIGF). There is evidence that these growth factors as well as their receptors KDR and fit-1 are expressed in die human placenta, but the sites of expression are controversially discussed. In the present project the role of the placenta-specific angiogenic growth factors VEGF and PIGF in mitogenesis and angiogenesis with the resulting development of fetal-placental vessels shall be investigated. We postulate that VEGFs and PIGFs are produced by different cell types of human fall term placenta and that they regulate the proliferation of placental endothelial cells and the development of fetal placental capillaries (angiogenesis) via KDR- and fit-1 receptors. This essential step for placental development is regulated by paracrine and autacrine mechanisms. The project involves work with endothelial cells isolated from the placenta proper. Micro- and macrovascular endothelial cells from human foreskin and umbilical cord, respectively, serve as control cell lines. Because several lines of evidence indicate cells within the placenta as source for these growth factors suggesting paracrine regulation, all cell types of the placenta will be isolated separately and conditioned media will be prepared. The in vitro effects of these media on endothelial cell proliferation and angiogenesis are to be tested. The receptors mediating any effects will be determined after their downregulation with antisense-oligonucleotides and by using neutralizing antibodies. Data to support the working hypothesis would represent the first direct experimental evidence of an angiogenic process in the human placenta and demonstrate the autonomy of this complex process from maternal and fetal factors.

The human placenta plays a central role for the intrauterine growth of the fetus. It develops within 9 months to a multifunctional organ. The manifold and important functions are the result of a complex interplay between several cell types within the placenta and with signals that come from both the maternal and fetal circulation. While in the past the tissue that exposes the placenta to the maternal circulation has received considerable interest, the surface facing the fetal circulation ie, the endothelial cells, had not been studied at the time when the project begun. The aim of the project was to investigate how the endothelial cells that line the placental vessels develop, to identify the role of some factors that govern regulation of this process and to characterise some properties of these cells. In a first step methods had to be developed that allowed the isolation and culture of the cells. We succeeded in establishing procedures that made possible the separate isolation form cells of arteries and veins within the vascular tree. These cells were rigorously and comprehensively characterised in order to identify molecules that are related to or involved in the differentiation to either the arterial or the venous cell lineage. The cells can be used by the wider scientific community that is particularly interested in angiogenesis and vascular development as well as by all those who want to study the effects of oxygenation and shear stress on cell properties, because these parameters are different in both legs of the vascular tree. Currently, negotiations are ongoing with a commercial company that is interested in buying the technology. In addition to scrutinising the cells we studied the influence of various endothelial specific growth factors such as placenta growth factors, vascular endothelial growth factors and fibroblast specific growth factor and compared them in different endothelial cells from large and small vessels. The data obtained confirm the phenotypic difference between the macro- and microvasculature and demonstrate that some of these growth factors are more potent in large vessels and some more in small vessels.