R. Graf

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.

Proteases in the human full-term placenta

SummaryAminopeptidase A, not yet defined aminopeptidases and endopeptidases, dipeptidyl peptidase I, II and IV, γ-glutamyl transferase and oxytocinase were investigated in the normal human full-term placenta using qualitative (catalytic) cytochemistry, isoelectric focusing, immunocytochemistry and kinetic fluorometry. Aminopeptidase A could be visualized cytochemically in the smooth muscle cells of the chorionic plate, stem villi and basal plate blood vessels. Aminopeptidases were found in connective tissue fibres of the chorionic plate, villous stroma, basal plate and paraplacenta. Dipeptidyl peptidase IV was detected at the same sites as the aminopeptidases and, in addition, in amniotic epithelial cells, fibroblasts of the villous stroma, endothelium of chorinic plate and villous blood vessels as well as in the basophilic cytotrophoblast cells (x-cells) of the basal plate and paraplacenta, and it possibly also occurred in some domains of the plasma membrane of the syncytiotrophoblast and cytotrophoblast cells. The x-cells surrounded the fetus in the form of a dipeptidyl peptidase IV-positive shell at the border to the mother. The enzyme represented the first specific marker for x-cells. Dipeptidyl peptidase I and II were primarily found in Hofbauer cells (macrophages) of the villous stroma, but also in the syncytiotrophoblast, other villous stromal cells and cells of the chorionic and basal plate. γ-Glutamyl transferase was present in some connective tissue elements of the chorionic plate. Oxytocinase and endopeptidases were not detected. Isoclectric focusing of proteases revealed different molecular forms of dipeptidyl peptidase IV in the paraplacenta and villous tree, while the aminopeptidases shared the same pattern in both regions. Immunocytochemical staining of dipeptidyl peptidase IV in the villous tree resembled the pattern obtained by catalytic cytochemistry except for the blood vessel endothelium and the x-cells of the basal plate. Fluorometrically, all proteases were more active in the villous tree than in the paraplacenta. The kinetic measurements revealed the highest hydrolysis rates for dipeptidyl peptidase IV followed by the aminopeptidases. In contrast to eatalytic cytochemistry all proteases were detectable when using fluorometry.

Molecular anatomy of the perivascular sheath in human placental stem villi: the contractile apparatus and its association to the extracellular matrix

Abstract. In previous studies, we have shown that smooth muscle cells and myofibroblast subpopulations of the perivascular stem villous sheath of the human placenta contain focal adhesion plaques and talin immunoreactivity. The close association of these cells to elastic and collagen fibres have led to the assumption of a functional myofibroelastic unit within the perivascular stem villous sheath. Interactions between the extracellular matrix and smooth muscle cells depend on a variety of structural protein assemblies. In the present study, we examined, by immunocytochemistry, whether the molecular assembly of extracellular matrix proteins and molecules of focal adhesions, known to be essential for signal transduction in smooth muscle cells, are also found in smooth muscle cells of the perivascular stem villous sheath of the human placenta. Vascular and extravascular smooth muscle cells were immunoreactive for α-actinin, vinculin, paxillin and tensin, the integrin chains α1 and β1, and the basement membrane components laminin and heparan/-chondroitin sulfate proteoglycan perlecan. pp125FAK did not react. In the extracellular matrix of blood vessel walls and the perivascular stem villous sheath, we found immunoreactivity of fibronectin and collagen types I, VI and undulin (collagen type XIV). From our data we conclude that within the perivascular stem villous sheath, there exists a system of signal transduction molecules, indicating a cross talk between the smooth muscle cells of this sheath and their surrounding extracellular matrix.

The extravascular contractile system in the human placenta

In the human placenta, besides the fetal blood vessel system a second extravascular contractile system exists. It is localized in the chorionic plate and runs in a longitudinal direction and adjacent to fetal blood vessels into the stem villi, where it forms perivascular contractile sheaths. Characteristically, cells of the extravascular contractile system are extremely long and spindle-shaped and give rise to fine cell processes, by which they obviously contact each other or insert into the basement membrane of the trophoblast. They show immunoreactivity with desmin, vimentin, α-actin, myosin, nitric oxide synthase type I (brain form) and dipeptidyl peptidase IV. The ultrastructure suggests that cells of the extravascular contractile system are related to smooth muscle cells, including subpopulations with myofibroblastic features. In stem villi a few cells are nitric oxide synthase type I immunoreactive. These cells are thought to be specialized smooth-muscle-like cells of the extravascular contractile system or cells of the extravascular contractile system related to paraneurons that generate nitric oxide, which, in turn, may modulate the tone of perivascular contractile sheaths. The high dipeptidyl peptidase IV activity suggests that modulation of the extravascular contractile system may also occur by substance P.

Pre-eclampsia associated alterations of the elastic fibre system in umbilical cord vessels.

Although pre-eclampsia (PE) is often associated with fetal hypoxia, hypertension and/or disturbed function of the fetal circulation, the effect of these altered hemodynamic parameters on the structure and composition of umbilical vessels has not been systematically investigated before. Therefore, this study focuses on PE-associated changes of the elastic fibre system in umbilical cord vessels investigated by light and electron microscopy, immunocytochemistry and biochemistry. In umbilical cord veins, no changes in thickness of the vessel wall or of any sublayer were observed. However, the internal elastic lamina of the veins was split in 80% of the PE-group in contrast to 20% in uncomplicated pregnancies. This effect was significant (α <0.01) from 36 weeks of gestation onwards. In umbilical cord arteries, the entire arterial vessel wall was found to be 15% thicker in PE than in uncomplicated pregnancies. The enlargement was caused by an increase of both the tunica intima and tunica media. The thickening of the tunica intima was attributed to a migration of smooth muscle cells towards the endothelium, accompanied by a splitting of the internal elastic lamina. Compared to uncomplicated pregnancies, smooth muscle cells of arteries and veins in PE showed a metabolic activation demonstrated by highly dilated endoplasmic reticulum. A semiquantitative score method as well as a quantitative dot blot assay indicated a PE-associated reduction of elastin expression in the arterial vessel walls. In summary, PE obviously induces a decrease of the elastin content accompanied by a thickening of the vessel wall in umbilical cord arteries. This remodeling of the elastic fibre system, together with an increased migration of smooth muscle cells, might represent part of the functional adaptation system of the umbilical cord arteries on the altered hemodynamic conditions in PE.

Elastic fibres are an essential component of human placental stem villous stroma and an integrated part of the perivascular contractile sheath.

Abstract.The stroma of human placental stem villi is believed to consist only of reticular and collagen fibres. In the present study we were able to show for the first time by light (orcein staining) and electron microscopy large amounts of elastic fibres in the stem villous stroma. Electron microscopically, homogeneous elastin was found alone or in association with microfibrils. In addition, microfibrils were observed forming long bands. These three structures, generally known to form elastic connective tissue, were seen in close connection with placental extravascular smooth muscle cells, which belong to the perivascular contractile sheath (PVCS) of stem villi. Elastin was associated with these smooth muscle cells and connected to collagen fibres via microfibrils. Collagen fibres were additionally interconnected by spike-like structures. Extravascular smooth muscle cells revealed numerous adhesion plaques which occupied conspicuously long cytoplasmic faces of the plasma membrane. In cryostat sections, immunoreactivity of talin, an attachment protein of adhesion plaques linking intracellular α-actin filaments with extracellular fibronectin, was detected in extravascular and vascular (media) smooth muscle cells. The arrangement of placental extravascular smooth muscle cells, elastic and collagen fibres suggests a functional myofibroelastic unit within the PVCS, which surrounds the large foetal blood vessels possibly contributing to elasticity and supporting tensile and/or contracting forces within the stem villi.

Histochemical evaluation of placental angiotensinase A in pre-eclampsia: Enzyme activity in villous trophoblast indicates an enhanced likelihood of gestational proteinuric hypertension

The aim of this study was to examine whether differences in placental angiotensinase A (glutamyl aminopeptidase, EC 3.4.11.7) activities occurred in hypertensive complications of pregnancy compared with uncomplicated pregnancies. Biochemical and semiquantitative histochemical methods were used and compared for their applicability. Angiotensinase A activity was detected using L-alpha glutamyl-4-methoxy-2-naphthylamide (alpha-Glu-MNA) as substrate and Fast Blue B salt for simultaneous azo-coupling in cryostat sections of placental tissue samples from 32 patients with pre-eclampsia, 11 patients with pregnancy-induced hypertension and 44 participants with uncomplicated pregnancies. The graduated intensity of reaction product in the villous trophoblast and in fetal blood vessels was evaluated semiquantitatively in a double-blind study by light microscopy (semiquantitative score method). Score levels were related to relative frequencies of hypertensive disorders (proportional odds model) and correlated to the severity of gestational hypertension (Spearmans rank correlation). After detection of enzyme activity, the same tissue samples were homogenized and used for kinetic fluorometric measurements under the same substrate and buffer conditions as in enzyme histochemistry. Enhanced villous trophoblastic angiotensinase A activity was significantly associated with an increased frequency of pre-eclampsia in pregnant women (cumulative odds ratio x 0(1) 6.37; P < 0.001) and showed significant correlations with the severity of gestational hypertensive disorders, represented by systolic (r = 0.31; P < 0.05) and diastolic (r = 0.34; P < 0.05 blood pressure and by concomitant proteinuria (r = 044; P < 0.01). Histochemical evaluation of fetal blood vessels and biochemical measurements revealed no statistically significant results. In conclusion this study demonstrates for the first time that increased villous trophoblastic angiotensinase A activity indicates an increased likelihood of the presence of pre-eclampsia and the severity of hypertensive disorders in pregnancy.

Antibodies to cytokeratins bind to epitopes in human uterine smooth muscle cells in normal and pathological pregnancies

Cytokeratin antibodies have been widely used for the identification of trophoblast cells in the placental bed, following their invasion from the developing conceptus. Their identification centres upon the expression of cytokeratin in epithelial cells, from which trophoblast cells are derived. Our recent observations indicate that this strict relationship may be more complex than was thought. Cryostat and paraffin sections of human decidua and myometrium, taken from the placental bed and the uterotomy cut, were examined immunocytochemically for cytokeratins using ten antibody clones selected to identify different cytokeratin proteins and antigenic epitopes. Biopsy specimens were obtained from normal and pathological pregnancies (pre‐eclampsia, fetal retardation, amnioninfection, hysterorrhexis, placenta praevia) at the time of caesarean section (26–41 weeks of pregnancy). Antibodies against nine clones, CAM 5.2, MNF 116, AE1/AE3, CK5, KS‐B17.2, CY‐90, M20, E3, and 34βE12 identified, as expected, syncytial giant cells and mononuclear trophoblasts within the placental bed and glandular epithelial cells throughout the uterus. In addition, they stained numerous fusiform cells that were classified by established criteria to represent smooth muscle cells, both within blood vessels and myometrium. No staining differences were observed between normal and pathological disorders. These results indicate that cytokeratin antibodies CAM 5.2, MNF 116 and AE1/AE3, and other antibodies targeting proteins 8 and 18, cross‐react with epitopes expressed in cells other than giant trophoblastic cells and mononuclear trophoblasts in the uterus and, thus, caution has to be used when such antibodies are used for the diagnostic characterization of tissues related to the placental bed.

Enzyme histochemical evidence for the presence of potential blood pressure regulating proteases in cultured villous explants from human first trimester placentae

The proteases dipeptidyl peptidase IV, angiotensinase A and microsomal alanyl aminopeptidase are present in the human term placenta where they may be involved in the local modulation of placental blood pressure. In order to establish an in vitro model system to study the significance of these proteases in disorders related to pregnancy-induced hypertension, the activity of the proteases was localized histochemically in cultured explants of villi from human first trimester placentae. These studies revealed a similar distribution pattern of the activity of the proteases of cryostat sections of first trimester placental villi and in cultured tissue of the same placentae. Dipeptidyl peptidase IV and angiotensinase A activity were present in cytotrophoblast cells and dipeptidyl peptidase IV activity was found in the syncytiotrophoblast, respectively. Additionally, the activity of the proteases was visualized in various populations of stromal cells. Comparing our results with former studies, the protease activity pattern in first trimester placentae was found to be the same as in term placentae. Despite morphological changes of the tissue after 14 d in culture the localization of the proteases remained unchanged up to 52 d of culture. The results suggest that placental explants may serve as a suitable in vitro model for experimental studies on the role of proteases in pregnancy-induced hypertension.

Localization of hyaluronan with a hyaluronan-specific hyaluronic acid binding protein in the placenta in pre-eclampsia.

Hyaluronan (HA), a high molecular weight polysaccharide, is a major component of connective tissue and is thus present in the extracellular matrix of most tissues. Increased serum concentrations have been reported in association with pre-eclampsia and liver malfunction, amongst other disorders. We have performed histochemical investigations with a HA-specific hyaluronic acid binding protein in placentas from uncomplicated pregnancies and from patients with pre-eclampsia. Staining for HA was found in the stroma and blood vessel walls of stem villi in all the placentas investigated. The syncytiotrophoblast and cytotrophoblast cells usually remained unstained. In addition, reactivity for HA was found within and on the surface of intervillous and perivillous fibrinoid deposits. Since fibrinoid deposits are increased in pre-eclampsia, our findings suggest that the increased HA serum concentrations in cases of pre-eclampsia could result from the stroma of the infarcted villi and from the fibrinoid deposits. HA may reach the maternal blood through fibrinoid gaps.