H. Neudeck

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.

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.

Binding of antibodies against high and low molecular weight cytokeratin proteins in the human placenta with special reference to infarcts, proliferation and differentiation processes

Recent immunocytochemical studies have shown that placental villous trophoblasts contain the high molecular weight cytokeratin (CK) proteins 5/6 and 17. In the case of CK 17, trophoblastic immunostaining was positive in villi covered by fibrinoid. CKs 5/6 and 17 are expressed by hyperproliferative cells. The aim of this investigation was to examine the location of these CKs in placental infarcts, known to be demarcated by fibrinoid and hyperproliferative trophoblasts. The results were compared with those obtained by immunostaining against Ki-67, tenascin and α1-, α6- and β1- integrins, which are involved in cell proliferation, differentiation and regenerative processes. Furthermore, the expression of the single CKs 7, 8, 10, 13, 14, 18 and 19 was investigated by immunocytochemistry and immunoblotting. While low and high molecular weight CKs were present in villous and extravillous trophoblasts, only low molecular weight CKs were detected in vascular and extravascular placental smooth muscle cells. Placental infarcts revealed different immunoreactivities in the infarct margin and centre: high molecular CKs, tenascin, Ki-67 and oncofoetal fibronectin predominated in the infarct margin, low molecular CKs, fibrin and integrins in the centre. The expression of tenascin and a defined change in the expression of CK 17 indicates villous repair and hyperproliferative mechanisms in placental infarcts.

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.

Decrease of elastic tissue fibres in stem villus blood vessels of the human placenta during IUGR and IUGR with concomitant pre-eclampsia

In a recent study we described an increase of elastic tissue fibres in blood vessel walls of placental stem villi during pre-eclampsia when compared to uncomplicated pregnancies. Furthermore, the thickness of these blood vessel walls was enhanced in pre-eclampsia. Since it is known that elastic tissue fibres increase in systemic hypertension, it may be assumed that the enhancement of elastic tissue fibres in placental stem villi during pre-eclampsia may be induced by the hypertension. To get further insight into this assumption, we examined the amount of elastic tissue fibres in stem villus blood vessels of placentae of pregnancies complicated by intrauterine growth retardation (isolated IUGR, fourteen cases), a disease without hypertension of the mother and such with pre-eclampsia and concomitant IUGR (IUGR+PE, nine cases). Each study group was compared with uncomplicated pregnancies (twenty-six cases). Unfixed cryostat serial sections were processed for conventional orcein staining and for the demonstration of α-actin-immunoreactivity. The intensity of orcein staining of stem villus blood vessel walls was evaluated by a semiquantitative score method. Significant lower intensities of orcein staining were calculated for blood vessel walls of placentae of isolated IUGR (P=0.0007) and IUGR+PE (P=0.0039) when compared to uncomplicated pregnancies each. Additionally, the blood vessel wall thickness of stem villi of isolated IUGR (P=0.0081) and IUGR+PE (P=0.0007) was significantly reduced. In comparison to the above mentioned investigation, our results show that, in contrast to isolated pre-eclampsia, elastic tissue fibres are decreased during pregnancies complicated by IUGR, independently of the occurrence of concomitant pre-eclampsia when compared to uncomplicated pregnancies. From our studies it may be considered that the increase of elastic tissue fibres in placentae of patients with isolated pre-eclampsia may be induced by systemic hypertension. Furthermore, our study underline arguments that IUGR may be an independent disease of the fetus.

Increase of segments of elastic-type blood vessel walls in fetal placental stem villi during pre-eclampsia at term

In recent studies we described the presence of elastic-type blood vessels within trunci and rami chorii of human placental stem villi. For systemic and pulmonary hypertension it is known that elastic fibres are enhanced in arteries. The aim of our study was, therefore, to examine whether pre-eclampsia may lead to an increase of elastic tissue fibres in blood vessel walls of placental stem villi and whether there are differences in the thickness of blood vessel walls within these villi when compared to normotensive pregnant women. Twenty-six women with uncomplicated pregnancies and 25 patients with pre-eclampsia were investigated. Unfixed cryostat serial sections were processed for conventional orcein staining and for the demonstration of α-actin-immunoreactivity. The intensity of orcein staining of stem villus blood vessel walls was evaluated by a semiquantitative score method. Significant higher intensities of orcein staining (P<0.00001) were calculated for blood vessel walls of placentae with pre-eclampsia. The amount of thick stem villus vessels (>41 µm) increased during pre-eclampsia from 39 gestational weeks onwards. Our study demonstrates that segments of thick blood vessel walls and elastic-type vessel walls are increased in placental stem villi of patients with pre-eclampsia. This reaction may protect the fetal placental vessels and avert an increase of the fetal hypertension.

The elastic fiber system in the human placenta with special reference to elastic type blood vessels

Summary Generally, elastic fibers are part of a so-called elastic fiber system. This system includes elastin (a homogenous component), elaunin (elastin connected to microfibrils) and oxytalan fibers. Recently, we have demonstrated elastic tissue fibers as an essential component of the perivascular contractile sheath (PVCS) also in human placental stem villi. In fetal placental blood vessels, the findings about elastic tissue fibers are contradictory and no information exists about oxytalan fibers in the human placenta. Therefore, we reinvestigated the distribution of the elastic fiber system in the blood vessels of the umbilical cord, chorionic plate and stem villi. Using unfixed orcein-stained cryostal sections of term placentae, we have found a well developed elastic fiber system in fetal blood vessels. It is not possible to discriminate between arteries and veins by morphological criteria and/or the distribution of elastic fibers. After oxidation with peracetic acid additional orcein-stained fibers, were detected in Whartons jelly and in the placental stroma. The high amount of elastic fibers throughout umbilical and placental blood vessels makes it reasonable to postulate the existence of elastic type blood vessels. Nevertheless, the great range of distribution varieties and the differing amount of elastic fibers in the blood vessels of all investigated placentae was striking. Within stem villi, there exists a longitudinal and circular vascular (inner) and a longitudinal extravascular (outer=PVCS) elastic fiber system. These two systems are ‘connected’ with the smooth muscle cells in both systems, thus forming two contractile myofibroelastic subsystems of a large overall placental myofibroelastic fiber system, in which tightly regulated cellmatrix interactions exists.

Histochemical Evaluation of Placental Dipeptidyl Peptidase IV (CD26) in Pre‐eclampsia: Enzyme Activity in Villous Trophoblast Indicates an Enhanced Likelihood of Gestational Hypertensive Disorders

PROBLEM: To determine whether differences in placental dipeptidyl peptidase IV (DPP IV, CD26) activities occurred in hypertensive complicated pregnancies as compared with uncomplicated pregnancies.