Tom Hahn

Heterogeneity of microvascular endothelial cells isolated from human term placenta and macrovascular umbilical vein endothelial cells

The present study compares some phenotypic and physiologic characteristics of microvascular and macrovascular endothelial cells from within one human organ. To this end microvascular endothelial cells from human full-term placenta (PLEC) were isolated using a new method and compared with macrovascular human umbilical vein endothelial cells (HUVEC) and an SV40-transformed placental venous endothelial cell line (HPEC-A2). PLEC were isolated by enzymatic perfusion of small placental vessels, purified on a density gradient and cultured subsequently. Histological sections of the enzyme-treated vessels showed a selective removal of the endothelial lining in the perfused placental cotyledons. The endothelial identity of the cells was confirmed by staining with the endothelial markers anti-von Willebrand factor, Ulex europaeus lectin and anti-QBEND10. The cells internalized acetylated low-density lipoprotein and did not show immunoreactivity with markers for macrophages, smooth muscle cells and fibroblasts. The spindle-shaped PLEC grew in swirling patterns similar to that described for venous placental endothelial cells. However, scanning electron microscopic examination clearly showed that PLEC remained elongated at the confluent state, in contrast to the more polygonal phenotype of HPEC-A2 and HUVEC that were studied in parallel. The amount of vasoactive substances (endothelin-1,2, thromboxane, angiotensin II, prostacyclin) released into the culture medium and the proliferative response to cytokines was more similar to human dermal microvessels (MIEC) derived from non-fetal tissue than to HUVEC. Potent mitogens such as vascular endothelial growth factors (VEGF121, VEGF165) and basic fibroblast growth factor (FGF-2) induced proliferation of all endothelial cell types. Placental growth factors PIGF-1 and PIGF-2 effectively stimulated cell proliferation on PLEC (142 +/- 7% and 173 +/- 10%) and MIEC (160 +/- 20% and 143 +/- 28%) in contrast to HUVEC (9 +/- 8% and 15 +/- 20%) and HPEC-A2 (15 +/- 7% and 24 +/- 6%) after 48 h incubation time under serum-free conditions. These data support evidence for (1) the microvascular identity of the isolated PLEC described in this study, and (2) the phenotypic and physiologic heterogeneity of micro- and macrovascular endothelial cells within one human organ.

Sustained hyperglycemia in vitro down-regulates the GLUT1 glucose transport system of cultured human term placental trophoblast: a mechanism to protect fetal development?

The trophoblast of human placenta is directly exposed to the maternal circulation. It forms the main barrier to maternal–fetal glucose transport. The present study investigated the effect of sustained hyperglycemia in vitro on the glucose transport system of these cells. Trophoblasts isolated from term placentas and immunopurified were cultured for 24, 48, and 96 h in DMEM containing either 5.5 (normoglycemia) or 25 mmol/l D‐glucose (hyperglycemia), respectively. Initial uptake of glucose was measured using 3‐O‐[14C]methyl‐D‐glucose. Kinetic parameters were calculated as KM = 73 mmol/l and Vmax = 29 fmol s–1 per trophoblast cell. Uptake rates of cells cultured under hyperglycemic conditions did not differ at exogenous D‐glucose concentrations in the physiological range (1, 5.5, 10, and 15 mmol/l), but were significantly decreased by 25% (P < 0.05) at diabetes‐like concentrations (20 and 25 mmol/l) as compared to normoglycemic conditions. This effect was due to a decrease in Vmax (–50%), whereas KM remained virtually unaffected. GLUT1 mRNA levels were lower by 50% (P < 0.05; Northern blotting) and GLUT1 protein was reduced by 16% (P < 0.05; Western blotting) in trophoblast cells cultured under hyperglycemic vs. normoglycemic conditions. We conclude that prolonged hyperglycemia in vitro reduces trophoblast glucose uptake at substrate concentrations corresponding to blood levels of poorly controlled diabetic gravidas. This effect is due to diminished GLUT1 mRNA and protein expression in the trophoblast.—Hahn, T., Barth, S., Weiss, U., Mosgoeller, W., Desoye, G. Sustained hyperglycemia in vitro down‐regulates the GLUT1 glucose transport system of cultured human term placental trophoblast: a mechanism to protect fetal development? FASEB J. 12, 1221–1231 (1998)

Insulin receptors in syncytitrophoblast and fetal endothelium of human placenta. Immunohistochemical evidence for developmental changes in distribution pattern

The localisation of insulin receptors (IR) was investigated on cryosections of human non-pathologic first trimester and full term placentae by indirect immunohistochemistry with three different monoclonal antibodies (MABS). In placentae from 6 to 10 weeks post-menstruation (p-m.), only syncytiotrophoblast was stained, predominantly that of mesenchymal villi and syncytial sprouts, which are areas of high proliferative activity. In placentae from 11 to 14 weeks p-m., endothelial cells commenced to react with the IR MABS and the syncytiotrophoblast was less intensely labelled than at weeks 6 to 10 p-m. In term placentae, the microvillous membrane of the syncytiotrophoblast showed only patches of weak immunoreactivity. In contrast, the endothelial cells in the placenta but not in the umbilical cord were strongly stained. The amniotic epithelium in the chorionic plate and fibroblasts in the stroma were conspicuously labelled. The data indicate: (1) the receptor density on villous syncytiotrophoblast decreases and that of fetal endothelium increases throughout gestation; (2) syncytiotrophoblast of human term placentae expresses a low level per unit area of surface IR; and (3) the majority of IR in human term placentae is located in fetal endothelium. Apart from yet unknown functional effects of maternal and fetal insulin at the placental barrier, the results suggest a growth promoting effect on the trophoblast of maternal insulin in first trimester as well as developmental effects of fetal insulin on the feto-placental vessels at term.

Localisation of the high affinity factilitative glucose transporter protein GLUT 1 in the placenta of human, marmoset monkey (Callithrix jacchus) and rat at different developmental stages

In the present study, the facilitative D-glucose transporter protein GLUT 1 was localised by immunohistochemistry in the placenta of human, marmoset (Callithrix jacchus) and rat at different developmental stages. A polyclonal antiserum agains a 13-amino-acid peptide of the GLUT 1 carboxy terminus was used. It identified a protein of around 50 kDa molecular weight in immunoblotting of the placental tissues. GLUT 1 was located in the syncytiotrophoblast, in cytotrophoblast cells and in fetal endothelium. Similar staining patterns, except in human extravillous cytotrophoblast cells, were observed at all differentiation stages, despite differences in the internal placental architecture of the species. In the marmoset placenta, GLUT 1 was undetectable in endothelial cells of maternal vessels. In rat placentae, trophoblastic giant cells, epithelial cells of both visceral and parietal yolk sac, yolk sac vessels and the stratum spongiosum were stained. Reicherts membrane did not immunoreact. Preadsorption of the antiserum with a 13-amino-acid peptide resulted in the loss of immunoreactivity. The results suggest that GLUT 1 is a prominent isoform of glucose transporters in mammalian placentae. It is generally abundant in placental cell populations bordering on the maternal and fetal circulations and may therefore facilitate an effective glucose supply to the fetus and placenta.

Glucose Transporter Expression in Rat Embryo and Uterus During Decidualization, Implantation, and Early Postimplantation

Abstract Efficient transfer of glucose from the mother to the embryonic compartment is crucial to sustain the survival and normal development of the embryo in utero, because the embryos production of this primary substrate for oxidative metabolism is minimal. In the present study, the temporal sequence of expression of the sodium-independent facilitative glucose transporter isoforms GLUTs 1, 3, 4, and 5 was investigated in the developing rat uteroembryonic unit between conception and Gestational Day 8 using immunohistochemistry. The GLUTs 1, 3, and 4 were expressed in the embryonic tissues after the start of implantation, being colocalized in the parietal endoderm, visceral endoderm, primary ectoderm, extraembryonic ectoderm, and the ectoplacental cone. In the uterus, a faint GLUT1 labeling emerged, but not until Gestational Day 3, in the luminal epithelium, endometrial stroma, and decidual cells. The intensity of GLUT1 staining increased in the latter population with progressing decidualization. Endometrial glands and myometrial smooth muscle cells stained neither for GLUT1 nor for GLUT3 until postimplantation. During all developmental stages examined, GLUT4 was visualized throughout the pregnant rat uterus, as was GLUT3 (with the above-mentioned exceptions). The density of GLUT5 was generally less than the sensitivity of the immunohistochemical detection method in all tissues investigated. In conclusion, the data point to a significant expression of the high-affinity glucose transporters GLUTs 1, 3, and 4 in the rat uteroembryonic unit, providing supportive evidence for an important role of facilitative glucose diffusion during peri-implantation development.

Hyperglycaemia-induced subcellular redistribution of GLUT1 glucose transporters in cultured human term placental trophoblast cells.

Aims/hypothesis. We have recently shown that hyperglycaemia down-regulates the GLUT1 glucose transport system of term placental trophoblast. The reduction in GLUT1 protein alone was, however, not sufficient to explain the decrease in net glucose uptake, suggesting additional mechanisms. Therefore, we hypothesised that hyperglycaemia in vitro leads to a GLUT1 translocation from the trophoblast surface to intracellular sites.¶Methods. This was tested in our study by determining the subcellular distribution of GLUT1 in human term placental trophoblast (n = 5 placentas) cultured for 48 h with 5 compared with 25 mmol/l d-glucose in vitro using immunogold labelling.¶Results. Electron microscopic examination of cell profiles showed that 73 % of total GLUT1 molecules reside in the trophoblast plasma membrane under basal conditions. The reduced GLUT1 expression (–20 %; p < 0.05) after culture of the cells with 25 mmol/l glucose was accompanied by an internalisation of plasma membrane GLUT1, resulting in a loss of 40 % (p < 0.05) in cell surface transporter labelling. Western blotting identified a characteristically broad band between 55–65 kDa, confirming the specificity of the GLUT1 antiserum.¶Conclusion/interpretation. We postulate that in addition to down-regulating human GLUT1 protein concentrations, glucose exerts its autoregulatory effect on hexose transport in term placental trophoblast by altering GLUT1 partitioning between the plasma membrane and intracellular sites in favour of the latter. [Diabetologia (2000) 43: 173–180]

Hyperglycemia regulates the glucose‐transport system of clonal choriocarcinoma cells in vitro. A potential molecular mechanism contributing to the adjunct effect of glucose in tumor therapy

Glucose is taken up by tumor cells via sodium‐independent facilitated diffusion along a concentration gradient. To examine the regulation of this process by substrate concentration, we investigated the effect of hyperglycemia on the glucose‐transport system of choriocarcinoma‐derived JAR and JEG‐3 cells by culturing them for 24, 48 and 96 hr in medium containing either 5.5 (normoglycemia) or 25 (hyperglycemia) mM d‐glucose, respectively. Immunocytochemically, choriocarcinoma cells expressed the high‐affinity glucose transporter isoforms GLUT1 and GLUT3. Based on initial uptake measurements using 3‐O‐[14C]methyl‐d‐glucose, kinetic parameters were calculated as Km= 15 mM and Vmax= 95 fmol/sec per cell for JAR and Km= 9 mM and Vmax= 64 fmol/sec per cell for JEG‐3 cells. In JAR cells cultured under hyperglycemic conditions, uptake rates were significantly increased at 15, 20 and 25 mM exogenous d‐glucose concentrations as compared with normoglycemic conditions. This effect was due to an increase in Vmax, whereas Km remained unchanged. Using Northern blotting, GLUT1 mRNA levels were higher but GLUT3 transcripts were reduced upon hyperglycemia. Western blotting revealed elevated GLUT1 and GLUT3 expression under hyperglycemic conditions. Hyperglycemia did not significantly influence the glucose‐transport system of JEG‐3 cells. We conclude that sustained hyperglycemia stimulates the glucose‐transport system of JAR, but not of JEG‐3, choriocarcinoma cells in vitro due to changes in GLUT1 and GLUT3 expression levels. We speculate that this mechanism may contribute to the beneficial effects of induced hyperglycemia as an adjuvant in tumor therapy. Int. J. Cancer 78:353–360, 1998.© 1998 Wiley‐Liss, Inc.

Ultrastructural characterization of the gonads of Triops cancriformis (Crustacea, Notostraca) from populations containing both females and males: no evidence for hermaphroditic reproduction

Abstract The gonads of female and male Triops cancriformis specimens from populations of the northern part of the species distribution area were examined by conventional transmission electron microscopy in order to gain insight into the complicated reproductive mode of the species. Ovarian follicles consisted of an oocyte and three nurse cells and were surrounded by a thin layer of follicular cells. Oocytes are initially smaller than nurse cells and contained mitochondria of the cristae type as well as many free ribosomes. The prominent nucleus contained a nucleolus. The cytoplasm of oocytes was filled with yolk globules that were surrounded by membranes of the endoplasmic reticulum. Nurse cells also contained yolk globules. The follicle duct walls consisted of closely packed elongated cells covered by a lamellar basal lamina. No distinct Golgi apparatus was found in the follicle duct cells. The passage of oocytes through the duct was accompanied by a marked flattening of the follicle duct cells. Simultaneously, the oocytes were wrapped in eggshell material before entering the longitudinal oviduct. Testicular lobes were not found in any of the investigated female specimens. In male animals, the epithelial wall of the testicular tubules consisted of both germinal and vegetative cells. Maturing spermatids formed multicellular nests located in clearly delimited cysts in the intercellular space of the testicular epithelium. The lumen of the testicular tubules contained, exclusively, free mature spermatozoa. These characteristics point to the cystic type of spermiogenesis. Mature spermatozoa were non-flagellate, had a regular ovoid shape of 4–5 μm diameter, and an acrosome was not found. Only a negligible amount of spermatozoa (< 5%) showed signs of degeneration. In conclusion, the results provide evidence for a parthenogenetic and/or bisexual reproduction mode rather than a hermaphroditic one in the populations examined.

Heterogeneous histochemical reaction pattern of the lectin Bandeiraea (Griffonia) simplicifolia with blood vessels of human full-term placenta

Bandeiraea simplicifolia lectin (BS-I) stains vascular endothelium in various species. In humans, less than 10% of the specimens studied exhibit a reaction with BS-I. In the present histochemical study, the reactivity of BS-I with placental blood vessels and its correlation with the blood group from mother and newborn child was investigated. Acetone-fixed cryosections of representative tissue segments of human full-term placenta and umbilical cord were stained with BS-I. The staining pattern of tissues from patients with different blood groups was identical, although the reaction of BS-I in the placenta was heterogeneous. BS-I did not react with the umbilical cord. Vascular smooth muscle cells at the insertion site of the umbilical cord into the chorionic plate, and endothelium deeper in the chorionic plate, became progressively stained. The endothelial cells and tunica muscularis of smaller arteries and veins in stem villi lost their reactivity in parallel with decreasing vessel size. Arterioles and venules reacted heterogeneously. Capillaries, trophoblastic basement membranes, especially epithelial plates, and sometimes the syncytiotrophoblast were labelled in several terminal villi. The data indicate that 1) the placenta binds BS-I to fetal endothelium independent of the blood group, 2) cell-surface antigens on placental endothelial cells are expressed heterogeneously and 3) cell-surface glycans are constituted in an organ-specific manner on human endothelial cells.

Self-regulation of the endothelin receptor system in choriocarcinoma cells.

The human trophoblast secretes endothelin-1 (ET-1) and expresses ET receptors. The present study tested whether the transformed BeWo, JAR and JEG-3 choriocarcinoma cells: (1) secrete endothelin-1 (ET-1); (2) express both ET-A and ET-B receptor subtypes; and (3) have the potential to allow for autologous regulation of ET-receptor proteins. The cells were cultured for 24/48 h with or without 10% FCS and, in experiments on receptor regulation, with ET-1 (5-20 nM and 10 microM). ET-1 secretion was measured by RIA and receptor levels by immunoblotting. All cell types secreted ET-1 albeit at different levels and sensitivity to FCS. All cell lines expressed both ET-A (JEG-3>BeWo=JAR) and ET-B (JEG-3=JAR>BeWo) receptor subtypes, which could be up- and downregulated depending on ET-1 concentration, culture time and FCS presence. It is concluded that BeWo, JAR and JEG-3 choriocarcinoma cells secrete ET-1 and express both ET-A and ET-B receptor subtypes. The receptor levels can be regulated by ET-1. This provides the molecular basis for an autocrine system with the potential of autologous regulation of yet unidentified ET-1-induced functions.