Sequential expression of VEGF and its receptors in human placental villi during very early pregnancy: differences between placental vasculogenesis and angiogenesis

Vascularization within the human placenta is the result of the de novo formation of vessels derived from pluripotent precursor cells in the mesenchymal core of the villi. Vascularization of placental villi starts at around day 21 post conception (p.c.) with a four somite embryo. At this stage progenitors of haemangiogenic cells differentiate to form first vessels. These progenitor cells are thought to be directly derived from mesenchymal cells rather than originating from fetal blood cells. We investigated the relation between differentiation of stromal cells towards endothelial cells and vascular structures and the expression pattern of the respective growth factors. Using transmission electron microscopy and immunohistochemistry (for VEGF, Flt-1, Flk-1, CD14, CD34, and CD68) the development of placental vasculogenesis during very early stages of pregnancy (days 22-48 p.c.) was studied. We found that VEGF is strongly expressed in villous cytotrophoblast cells and subsequently in Hofbauer cells while its receptors Flt-1 and Flk-1 are found on vasculogenic and angiogenic precursor cells. The developmental expression and secretion of VEGF suggests its involvement in recruitment, maintenance and formation of first angiogenic cells and vessels. Interactions between VEGF and Flk-1 and Flt-1 may regulate placental vasculogenesis and angiogenesis in a paracrine and autocrine manner. The sequential expression of growth factors in different cell types may point to the fact that placental vasculogenesis and angiogenesis are clearly distinct events.     

Differential expression and the anti-apoptotic effect of human placental neurotrophins and their receptors

Neurotrophin (NT) is important in the survival, maintenance and differentiation of neuronal tissue, and functions in follicle maturation, tumor growth, angiogenesis and immunomodulation; however, the expression of NT and its receptors (NTR) in human placenta and their influence on fetal growth are unclear. Here we investigated the correlation of NT and NTR in human placenta with uterine environment and fetal growth. TrkB, a NTR, mRNA was expressed on decidual and villous tissue and increased with gestational age, localizing in the trophoblast layer and endothelium by immunohistochemistry. Villous TrkB mRNA was significantly increased in preeclampsia (PE) than in controls and was higher in the normotensive small for gestational age (SGA) placenta, although it was not significant. It was also significantly increased in the small twin of discordant twin pregnancies. Brain-derived neurotrophic factor (BDNF), the main ligand of TrkB, was expressed in membranous chorion and villous tissue and was significantly higher in maternal plasma in normotensive SGA and PE than in controls. TrkB mRNA expression was up-regulated on cultured villous tissue explants and on JEG-3, a choriocarcinoma cell line, by H₂O₂ treatment. BDNF decreased apoptotic cells in H₂O₂-treated JEG-3, indicating that BDNF/TrkB signaling had anti-apoptotic effects against oxidative stress in JEG-3, suggesting a protective role of BDNF/TrkB in human villous tissue under unfavorable conditions in utero.     

Identification of arginase in human placental villi

l-Arginine is the common substrate for arginase and nitric oxide synthase (NOS). Arginase converts l-arginine to urea and ornithine, which is the principal precursor for production of polyamines required for cell proliferation. Human placenta expresses endothelial NOS (eNOS) in syncytiotrophoblasts, but the expression of arginase has not been fully elucidated. Our aim was to investigate the expression and distribution patterns of arginase-I (A-I) and arginase-II (A-II) in human placental villi in the first trimester and at term using immunohistochemistry, RT-PCR and Western blot analysis. The arginase enzyme activity in placental villi was also measured. Immunohistochemistry showed different distribution patterns of the arginase isoforms during gestation: A-I was observed only in cytotrophoblasts, while A-II was observed in both cytotrophoblasts and syncytiotrophoblasts. RT-PCR and Western blot analysis showed expression of A-I and A-II in the first trimester and at term in human placental villi. Expression of A-II and arginase activity was greater in the first trimester than at term. Differentiation of cytotrophoblasts into syncytiotrophoblasts may be associated with l-arginine metabolism through modulation of l-arginine availability for eNOS and A-I. And elevated arginase activity in the early gestational period may be responsible for proliferation of trophoblasts by increasing polyamines production. These results suggest that the l-arginine-ornithine-polyamine and l-arginine-nitric oxide pathways play a role in placental growth and development.     

Cross-sectional features and three-dimensional structure of human placental villi

Human placental villi from both normal and complicated pregnancies were examined by both light and scanning electron microscopy. The findings provide evidence that histological features such as syncytial sprouts, bridges, and a net-like arrangement of villi represent tangential sections of irregularly shaped villi rather than proliferative activity of the villous surface. Hence the two-dimensional appearance of paraffin and semithin sections has to be interpreted three-dimensionally in comparison with the respective scanning electron micrographs. In the light of these findings the various types of villous maldevelopment are summarized in a diagram which may be used as an aid for pathological diagnosis.     

Immunohistochemical demonstration of epidermal growth factor (EGF) receptors in normal human placental villi

With an avidin-biotin-peroxidase (or glucose oxidase) complex method using anti-epidermal growth factor receptor monoclonal antibody (528 IgG), the tissue and cellular distribution of the receptors for epidermal growth factors (EGF) in normal human placental villi, from 6 to 42 weeks of gestation, were studied. EGF receptors were mainly localized on the free surface of the syncytiotrophoblast that directly faced to intervillous space of the maternal circulation. The cell surface of cytotrophoblasts, except for the region that was adjacent to the basal lamina, was also positive for EGF receptors. The receptors were in close contact to the fetal vessels in the villous stroma. The EGF receptors on the syncytiotrophoblast were thought to be involved in the production and secretion of human chorionic gonadotropin and placental lactogen, probably under the control of maternal EGF. The receptors on cytotrophoblasts may play a role in trophoblastic proliferation, possibly mediated by EGF in the fetal circulatory system.     

Isolation and characterization of Hofbauer cells from human placental villi

Summary Hofbauer cells are a major cell type of the human placental villous core and they are particularly numerous at the beginning of pregnancy. In the present study we describe a method suitable to obtain HC suspensions in a highly purified form. These suspensions have been analyzed for surface markers using a battery of monoclonal antibodies. Of all the surface markers used, Hofbauer cells were only positive for 4F2, LeuM2 and LeuM3 monoclonals which mainly detect cells of the monocyte-macrophage lineage. Hofbauer cells were consistently negative for HLA-DR antigens, C3bR and T- or B-cell markers. Hofbauer cells appeared capable of phagocytosing latex beads, adhering to and spreading over plastic surface and secreting lysozyme. In contrast, they failed to originate an efficient respiratory burst in response to appropriate stimulation. Hofbauer cells were positive for ANAE with a perinuclear localization of the enzyme activity, but consistently negative for peroxidase. These observations suggest that they share a number of features with cells of the monocyte-macrophage lineage and yet have some distinctive properties.     

Lectin binding of membrane proteins from human placental villi

The binding of membrane proteins from human placental villi to lectins such as concanavalin A (Con A), wheat germ agglutinin (WGA) and Riccinus communis agglutinin (RCA) was examined. Five major components of 66K, 69K, 100K, 130K and 170K dalton showed specific binding to WGA, whereas two other major components of 43K and 40K dalton had only weak binding. There were no membrane proteins which bound specifically to either Con A or RCA. The WGA-Sepharose column is considered useful for the purification of human placental membrane proteins. Although the function of these proteins is still unknown, a diagnostic method not only for determining the pathological state of the placenta, but also for cancer will possibly be developed by taking advantage of them.     

A comparative study of human placental and fetal liver catalase during development

The activity and a few properties of catalase have been compared in the developing human placenta and fetal liver. The presence of the enzyme in both the tissues is discernible as early as in the 6th wk of gestation and the activity increases gradually with the advancement of pregnancy. Maximum enzyme activity in both placenta and fetal liver is found to be associated with the soluble supernatant fraction obtained by centrifuging the tissue homogenates at 105 000 × g. Kinetic studies reveal the enzymatic decomposition of H₂O₂ to follow first-order kinetics at lower substrate concentrations, and then to deviate from the original linearity, demonstrating mixed-order kinetics. Thermostability of placental catalase increases with prenatal development, while the enzyme from fetal liver remains moderately heat-stable throughout the gestation. Treatment of the homogenates with Triton X-100 is found to be most effective in increasing catalase activity in each of these tissues.     

Expression of Fas-ligand in first trimester and term human placental villi

The expression of Fas-ligand (FasL) on trophoblast cells is thought to play a role in immune regulation during human pregnancy. However, there are some discrepancies in the published data concerning the cell types expressing FasL in the placental villi. Therefore, we examined the expression of FasL on cryosections of first trimester and term placental tissue with three different anti-sera against FasL, which are in common use. By immunohistochemistry, all three anti-sera principally gave the same staining result. In the first trimester of pregnancy, villous cytotrophoblast cells underlying the syncytium, as well as all extravillous trophoblast cells of cell columns and cell islands, gave a clear, mainly membrane-located staining, whereas the syncytiotrophoblast, which forms the borderline to the maternal blood flow, only gave a spot-like reaction in distinct areas. The same result was obtained with term placental villi; however, in this tissue, the staining of the villous cytotrophoblast cells was less pronounced. From our results, we suggest that in placental villi, an important role of FasL in immune regulation is not very conclusive because this molecule is mainly expressed on trophoblast with no access to maternal blood or tissue. This is in contrast to the uterine part of the placenta, where FasL expressing trophoblast cells are in close contact with apoptotic maternal leukocytes.     

Quantitative evidence for the spatial dispersal of trophoblast nucleic in human placental villi during gestation

A common assertion in the literature is that Langhans cells in placental villi decline in number during gestation but this is a misinterpretation which may be caused by the greater growth of villous surface area compared with trophoblast volume. To test this possibility, human placentae were collected at 12–41 weeks of gestation for a cross-sectional study on the packing density of nuclei within villous trophoblast. Numbers of nuclei in the cyto- and syncytiotrophoblast were estimated using a design-based stereological device, the physical disector (parallel pairs of sections). Surface areas were estimated in order to assess the overall growth of villous arborizations. Packing densities of nuclei were calculated and expressed as numbers/ 1000 μm² of villous surface. Densities decreased during gestation and this can be explained by expansion of villous surface area and thinning of trophoblast. The biggest drop in packing density of cytotrophoblast nuclei (30 per cent) occurred between 17–21 and 22–26 weeks and this period coincided with the largest changes in villous surface area (62 per cent increase) and trophoblast thickness (30 per cent decrease). Results are consistent with the notion of an epithelial proliferative unit of constant volume and comprising about nine syncytiotrophoblast nuclei per Langhans cell.