Extravillous trophoblast: proliferation and invasion during pregnancy.
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Cell Division↗
Biomedical subjects
B Huppertz
Publications and source records attributed to B Huppertz.
Pathogenesis of intrauterine growth restriction (IUGR)-conclusions derived from a European Union Biomed 2 Concerted Action project 'Importance of Oxygen Supply in Intrauterine Growth Restricted Pregnancies'-a workshop report.
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Adult↗
Low-dose-rate ionizing irradiation for inhibition of secondary cataract formation.
INTRODUCTION: Secondary cataract formation limits visual function after cataract surgery. Various experimental methods utilizing the pharmacologic inhibition of lens epithelial cell proliferation have been proposed. However, diffusion into the anterior chamber may lead to damage of corneal endothelial cells. This study evaluated the inhibition of lens epithelial cell proliferation with a capsular bag ring, labeled with a beta-emitting radioisotope. METHODS AND MATERIALS: In vitro studies using rabbit lens epithelial cells were performed to investigate the dose-dependent effect of irradiation. Based on these results, P-32-labeled PMMA rings were implanted into the capsular bag of NZW rabbits in vivo after phacoemulsification. Animals were examined for development of posterior capsule opacification over a period of 12 weeks following surgery. Radiation damage to the surrounding ocular tissue was subsequently analyzed in histologic sections using TUNEL assay and proliferation marker. RESULTS: Irradiation of lens epithelial cells in vitro with >5 Gy resulted in a dose-dependent decrease in the number of cells. BrdU testing demonstrated a near complete inhibition of cell proliferation. In vivo, implantation of P-32-labeled PMMA rings led to inhibition of epithelial cell proliferation and secondary cataract formation but was not able to fully inhibit aberrant differentiation of some remaining cells. Histologic examination showed no evidence of radiation damage of the ciliary body or the corneal endothelium. CONCLUSIONS: Low-dose beta irradiation exhibits the potential for inhibition of lens epithelial cell proliferation both in vitro and in vivo. Further investigation of various nuclides and their radiation profiles is needed to optimize the prevention of posterior capsule opacification due to epithelial cell proliferation.
Animals↗
Apoptosis and syncytial fusion in human placental trophoblast and skeletal muscle.
Skeletal muscle fibers and placental villous trophoblast are the main representatives of syncytia in the human. Both syncytia are derived from fusion of mononucleated stem cells, show a high degree of differentiation, and have lost their generative potency. Consequently, for their growth both depend on fusion of additional stem cells. There is evidence that syncytial fusion is directly or indirectly related to apoptotic events: As early as in the differentiated stages of the mononucleated stem cells, initiation stages of the apoptosis cascade have been observed. After syncytial fusion progression of the cascade is retarded or blocked by a variety of mechanisms. In this review we emphasize the links between apoptosis cascade, differentiation pathways and syncytial fusion. It needs to be elucidated whether these processes simply take place in parallel, both temporally and spatially, or whether there are causal connections between apoptosis cascade and syncytial fusion. Based on recent data obtained for placental villous trophoblast, it is tempting to speculate that early molecular mechanisms of the apoptosis cascade are involved in differentiation and syncytial fusion. Data obtained in skeletal muscles support this assumption and reveal a considerable degree of homology in genesis, maintenance and turnover of both tissues.
Animals↗
An improved and rapid method to construct skin equivalents from human hair follicles and fibroblasts.
To produce sufficient amounts of high quality skin equivalents (SE), either allogenic for dermatopharmacological and dermatotoxicological studies or autologous for transplantation purposes, we established a rapid, easy and cost effective three-dimensional SE model on the basis of human dermal fibroblasts, collagen and freshly plucked hair follicles. Acidic liquid collagen was polymerized with sodium hydroxide in the presence of fibroblasts to form a dermal equivalent (DE) resembling normal human dermis. At 24 h later, freshly plucked hair follicles were implanted into the surface of these DEs after cutting their bulbs off. Another 48 h later, the surface of the SEs was lifted to the air-liquid interface. Fourteen days after implantation, outgrowing keratinocytes from the outer root sheath of the hair follicles completely covered the surface of the SE and built a fully developed, multi-layered and cornified epidermis. Histology and immunofluorescence studies with specific antibodies directed against components of keratinocytes, fibroblasts, cell-adhesion molecules, different extracellular matrix and basement membrane proteins revealed the similarity of our three-dimensional SEs to the in vivo situation in normal human skin. Using autologous cell sources and cell culture media enriched with serum from the respective cell donor, it will be possible to use these SEs for autologous transplantation, thereby reducing the risk of transplant rejection.
Cell Division↗
Macrophage-induced apoptosis limits endovascular trophoblast invasion in the uterine wall of preeclamptic women.
Impaired invasion of uteroplacental arteries by extravillous trophoblast cells is a key pathogenic mechanism of preeclampsia. We previously demonstrated that reduced trophoblast invasion into uteroplacental spiral arteries was associated with an excess of macrophages in and around these arteries. To explore the significance of these observations, we correlated the extent of extravillous trophoblast apoptosis in placental bed biopsy specimens with macrophage distribution and studied the effect of macrophages upon trophoblast apoptosis in vitro. Extravillous trophoblast hybrid cells were cocultured with activated macrophages exposed to exogenous tumor necrosis factor alpha (TNFalpha), anti-tumor necrosis factor receptor I (TNF-RI), and tryptophan depletion, and the rates of trophoblast apoptosis were measured. Extravillous trophoblast hybrid cells showed increased rates of apoptosis following exposure to exogenous TNFalpha, with tryptophan depletion, and when cocultured with activated macrophages. The proapoptotic effects of macrophages in vitro were completely inhibited only by simultaneous addition of tryptophan and anti-TNF-RI. Our data indicate that macrophages, residing in excess in the placental bed of preeclamptic women, are able to limit extravillous trophoblast invasion of spiral arterial segments through apoptosis mediated by the combination of TNFalpha secretion and tryptophan depletion. The mechanisms by which macrophages are activated and recruited to the placental bed are presently unknown but are likely central to the pathogenesis of preeclampsia.
Adult↗
Expression of a cytokeratin 18 neo-epitope is a specific marker for trophoblast apoptosis in human placenta.
In epithelial cells the caspase-mediated cleavage of cytokeratin 18 during apoptosis leads to the formation of a specific neo-epitope, recognized by the antibody M30. To test whether this antibody can be used as a specific marker for apoptotic trophoblast, we have stained serial sections of villi and junctional zone of first and third trimester human placenta with antibodies against cytokeratins 7 and 18, and against active caspase 3, with M30 and with the TUNEL reaction. Comparison of M30 immunoreactivities with TUNEL positivity and immunoreactivities for cytokeratins 7 and 18 clearly demonstrates that M30 specifically labels late apoptotic trophoblast cells. This finding is supported by the fact that in trophoblast, M30 immunoreactivities largely overlap with those for active caspase 3. As compared to the TUNEL test, the M30 immune reaction appears to be a highly reproducible marker for apoptotic trophoblast. This antibody stains a larger number of cells within the apoptosis cascade as compared to the TUNEL reaction, since cytokeratin 18 cleavage starts earlier than cleavage of DNA and since endonuclease activation can be bypassed in some trophoblast cells. The data suggest that M30 is superior to the TUNEL reaction as a marker for the detection of trophoblast apoptosis since it is easier to handle, more specific for apoptosis and less prone to artifacts.
Adult↗
Apoptosis: molecular control of placental function--a workshop report.
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Adult↗
Human trophoblast contains an intracellular protein reactive with an antibody against CD133--a novel marker for trophoblast.
CD133 is a protein expressed on the cell membrane of a subfraction of haematopoietic stem and progenitor cells, as well as on some epithelial cells. Previously available antibodies against CD133 recognized only the glycosylated protein, localized to membrane protrusions or microvilli. Due to this, immature intracellular stages of the CD133 protein could not be visualized using these antibodies. We describe reactivity of a commercially available antibody against CD133, called AC133-2, with an intracellular protein in trophoblast. Both villous and extravillous cytotrophoblast, as well as syncytiotrophoblast were stained by AC133-2 in cryostat sections of first trimester and term placenta. Villous stroma was not stained. AC133-2 reactivity was seen in methanol-fixed primary trophoblast cells and trophoblast-derived cell lines, and was coexpressed with cytokeratin-7. CD133 messenger RNA was present in trophoblast and trophoblast-derived cell lines, but also in cells not displaying any reactivity with CD133 antibodies. AC133-2 recognized a 55-60 kDa protein on Western blots of cell extracts including trophoblast. The exact nature of this protein is not yet understood. However, AC133-2 is applicable as a positive marker for the characterization of all subtypes of trophoblast and for trophoblast cell lines.
AC133 Antigen↗
Cellular localization of human relaxin-like factor in the cyclic endometrium and placenta.
We have studied the cellular localization of the relaxin-like factor (RLF) in the histologically normal cyclic endometrium collected from days 3--26 of the menstrual cycle. RLF transcripts and protein were detected in the luminal and glandular epithelium and in stromal cells at all stages of the cyclic endometrium. Increased expression of RLF was observed in endometrial tissues in the proliferative as compared to the secretory phase, suggesting that oestrogens affect RLF gene activity in the human endometrium. The cellular localization of RLF transcripts and protein was also determined in first trimester placental tissues obtained from normal and ectopic tubal implantation sites and in third trimester placentae of normal and pre-eclamptic pregnancies. In first trimester placenta, weaker expression of RLF was observed in the syncytiotrophoblast as compared to the underlying cytotrophoblast. Extravillous trophoblast cells constitutively expressed RLF. Trophoblast cells were the main source of RLF in the human placenta and trophoblastic RLF gene activity was unaffected by either the site of implantation or the invasive properties of the cytotrophoblast as demonstrated by samples from patients with tubal implantation and pre-eclampsia respectively. Decidual cells weakly expressed RLF. The presence of unprocessed and cleaved immunoreactive RLF in term placenta was determined by Western analysis. The above results suggest a functional role for both RLF isoforms within normal placental tissue.
Adult↗
Cytogenetic and DNA-fingerprint characterization of choriocarcinoma cell lines and a trophoblast/choriocarcinoma cell hybrid.
We report the successful fusion of human choriocarcinoma cells with normal human trophoblast cells to a choriocarcinoma/trophoblast hybrid. The hybrid cells ACH1P were derived from fusion of primary male trophoblast cells with the HGPRT-defective choriocarcinoma cell line AC1-1. The karyotypes of the parental choriocarcinoma cell line JEG-3, its HGPRT-defective mutant clones AC1-1, AC1-5, and AC1-9, and the choriocarcinoma/trophoblast hybrid ACH1P are presented, together with a detailed characterization of the AC1-specific chromosomal marker add(X)(q26) using conventional cytogenetic banding techniques and multiplex-fluorescence in situ hybridization (M-FISH). To our knowledge, this is the first report of a stably proliferating human cell hybrid of trophoblastic origin, providing a unique cell culture model to study trophoblast-related invasion and its underlying genetic mechanisms.
Cell Fusion↗
Development of the placental villous tree and its consequences for fetal growth.
Co-ordinated development of the fetal villous tree of the placenta is necessary for continued fetal growth and well-being. Before fetal viability, blood vessel development within the developing immature intermediate villi (IIV) is characterized by branching angiogenesis, such that the placenta expands to produce 10-16 generations of stem villi. Once fetal viability is attained, a developmental switch occurs to form large numbers of gas-exchanging terminal villi (TV) by non-branching angiogenesis in mature intermediate villi (MIV). Several growth factors, including vascular endothelial growth factor (VEGF), placenta growth factor (PlGF), angiopoietins, and angiostatins are produced within the villi and act locally, via their receptors, to control angiogenesis. Their relative contributions to placental vascular development are not fully understood at the present time. Severe early-onset intrauterine growth restriction (IUGR) is characterized by absent/reversed end-diastolic flow velocity (ARED) in the umbilical arteries, leading to fetal hypoxia, acidosis and a substantial rise in perinatal mortality and morbidity. The placentas from such cases show a deficit in peripheral villous development, which may be perpetuated by the effects of oxygen (delivered by maternal blood into the intervillous space) upon VEGF-directed angiogenesis, the so-called 'placental hyperoxia' theory of villous maldevelopment. Trophoblast apoptosis is a significant feature of early-onset IUGR and may explain poor flow-independent transfer of nutrients to the fetus. Finally, since transgenic mouse studies highlight the importance of trophoblast-derived transcription factors for placental villous (labyrinth) development, it is possible that the villous trophoblast controls the orderly development of the underlying mesoderm and blood vessels into the fetal villi.
Animals↗
The human placenta is encircled by a ring of smooth muscle cells.
The marginal zone of the human term placenta was studied by transmission electron microscopy and immunohistochemistry using antibodies against cytoskeletal filaments, extracellular matrix molecules and endothelial markers. The marginal sinus of the intervillous space is separated from the chorionic and basal plates by a layer of cells expressing vimentin, desmin, alpha- and gamma-smooth muscle actins, and smooth muscle myosin. Also ultrastructurally, these cells share all features with smooth muscle cells. This muscular ring is continuous with the media of uteroplacental veins entering the marginal sinus. In the basal plate the muscle cells may extend far into the central parts of the placenta. The muscular ring is separated from the intervillous space by a layer of endothelial cells. They are continuous with the maternal endothelium of the marginal uteroplacental veins. Moreover this endothelium covers neighbouring parts of the chorionic and basal plates, locally extending to the surfaces of large stem villi. The data suggest (1) that the marginal zone of the intervillous space ('marginal sinus') represents the dilated and merged parts of uteroplacental veins and (2) that lateral growth of the human placenta partly takes place by expansion into the uteroplacental veins. The functional importance of this muscular ring remains unknown.
Actins↗
The apoptosis cascade--morphological and immunohistochemical methods for its visualization.
Apoptosis is involved in morphogenesis of embryonic tissues as well as in homeostasis of adult organs and tissues. It is the main process by which organs maintain cell mass and at the same time eliminate excess and aged cells that have lost their functional importance. The typical morphological signs of apoptosis (cellular shrinkage, membrane blebbing, nuclear condensation and fragmentation) are the final results of a complex biochemical cascade of events, some of which are inextricably linked to the process of differentiation. Studies that analyze all stages of this cascade, rather than the final morphological stages of apoptotic death, are essential in order that specific link(s) between differentiation and apoptosis are appreciated. This review outlines the main stages of the apoptosis cascade together with current methods for their morphological visualization. Starting with (a) receptors and ligands known to induce apoptosis, we continue with (b) early initiator stages of apoptosis, and (c) proteins regulating and potentially inhibiting further progression of the cascade, into (d) irreversible execution stages of the cascade, and finally (d) the morphological events of apoptotic death. For each stage we present those aspects of the biochemical background that are morphologically relevant, together with proven methods for their visualization. We offer technical advice at each stage based upon our experience of studying differentiation and apoptosis in human placental trophoblast.
Animals↗
Nucleic acid sequence of feline preprorelaxin and its localization within the feline placenta.
The cat placenta is known to secrete large amounts of relaxin. We employed uteroplacental tissue at approximately Day 35 of gestation to determine the nucleic acid sequence of feline preprorelaxin using reverse transcription- and rapid amplification of cDNA ends-polymerase chain reaction. Feline preprorelaxin cDNA was found to consist of 540 base pairs encoding a protein of 180 amino acids (aa). We identified a signal peptide of 25 aa, a B domain of 33 aa, a C domain of 98 aa, and an A domain of 24 aa. The putative receptor binding region in the N'-terminal part of the B domain contained one substitution from the classical GRELVR motif (L-->F). Feline preprorelaxin shared highest homology with porcine and equine preprorelaxin. Northern analysis revealed a specific 1-kilobase transcript present in total RNA of feline uteroplacental tissue but not of liver tissue. Nonradioactive in situ hybridization was used to localize relaxin mRNA, and immunohistochemistry was used to localize the relaxin hormone and cytokeratin, in tissues of the feto-maternal interface recovered from two queens at Day 35 of gestation. Specific hybridization signals for relaxin mRNA were exclusively detected in cells located in the lamellar placental labyrinth but were absent from other placental and nonplacental uterine parts. The cells expressing relaxin mRNA also displayed immunoreactivity for cytokeratin and were, therefore, identified as trophoblast cells. Immunoreactive relaxin colocalized in those placental areas expressing relaxin mRNA. Trophoblast cells located at the villous chorioallantoic tips invading the endometrium and extravillous trophoblast cells in the junctional placental zone were devoid of relaxin.
Amino Acid Sequence↗
Apoptosis cascade progresses during turnover of human trophoblast: analysis of villous cytotrophoblast and syncytial fragments in vitro.
Growth and survival of the trophoblast layer of the human placenta depends on continuous incorporation of villous trophoblast stem cells (cytotrophoblast), by syncytial fusion, into the syncytiotrophoblast. Descriptive studies suggest that this process may be intimately related to apoptosis. We have analyzed the expression and activation of initiator and execution caspases, critical effectors of apoptosis, in relation to trophoblast turnover (differentiation) in human placental villi. We used immunohistochemistry, caspase enzyme histochemistry, caspase activity assays, Western blots, and autoradiography techniques on placental tissue sections, trophoblast lysates, villous explants, and isolated trophoblast fragments (villous cytotrophoblast and mononuclear syncytial elements) in vitro. Our data demonstrate expression of initiator caspases 8 and 10 and activity of caspase 8 in villous cytotrophoblast. Proforms of the execution caspases 3, 6, and 7 were also expressed in villous cytotrophoblast, but activation of execution caspases 3 and 6 could only be demonstrated in the syncytiotrophoblast after syncytial fusion. Down-regulation of the general transcription level (reduced incorporation of [3H]uridine) as well as cleavage of the execution caspase substrates poly-(ADP-ribose)polymerase and lamin B was confined to syncytiotrophoblast and preceded the final events of apoptotic death (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling reactivity and nuclear collapse). Our data confirm that the apoptosis cascade in villous trophoblast is regulated in parallel with trophoblast differentiation, syncytial fusion, and trophoblast turnover.
Apoptosis↗
Neuropilin-1 is a placenta growth factor-2 receptor.
Placenta growth factor (PlGF) belongs to the family of vascular endothelial growth factors (VEGFs). It binds to the flt-1 VEGF receptor but not to the KDR/flk-1 receptor which is thought to mediate most of the angiogenic and proliferative effects of VEGF. Three PlGF isoforms are produced by alternative splicing. PlGF-1 and PlGF-3 differ from PlGF-2 since they lack the exon 6 encoded peptide which bestows upon PlGF-2 its heparin binding properties. Cross-linking experiments revealed that 125I-PlGF-2 binds to two endothelial cell surface receptors in a heparin dependent fashion. The binding of 125I-PlGF-2 to these receptors was inhibited by an excess of PlGF-2 and by the 165-amino acid form of VEGF (VEGF165), but not at all by VEGF121 and very marginally if at all by PlGF-1. The apparent molecular weight and the binding characteristics of these receptors correspond to those of the recently identified VEGF165 specific receptor neuropilin-1, and we therefore conclude that neuropilin-1 is a receptor for PlGF-2. The binding of 125I-PlGF-2 as well as the binding of 125I-VEGF165 to these receptors was inhibited by a synthetic peptide derived from exon 6 of PlGF. Furthermore, the binding of 125I-PlGF-2, but not that of 125I-VEGF165, was also inhibited by a synthetic peptide derived from exon 7 of PlGF. These observations indicate that the peptides encoded by these exons probably participate in the formation of the domain which mediates the binding of PlGF-2 to these receptors. We have also determined, using chemically modified heparin species, that the presence of sulfate moieties on the glucosamine-O-6 and on the iduronic acid-O-2 groups of heparin was required for the potentiation of 125I-PlGF-2 binding to these receptors. To determine if PlGF-2 is able to induce biological responses that are not induced by PlGF-1, we compared the effects of PlGF-1 and PlGF-2 on the migration and proliferation of endothelial cells. Both PlGF forms induced migration of endothelial cells. However, there was no quantitative difference between the response to PlGF-2 and the response to PlGF-1. Furthermore, neither PlGF-1 nor PlGF-2 had any effect upon the proliferation of the endothelial cells.
Animals↗
Villous cytotrophoblast regulation of the syncytial apoptotic cascade in the human placenta.
Villous trophoblast in the human placenta consists of a population of proliferating stem cells which differentiate and individually fuse into the syncytiotrophoblast. We studied the apoptotic cascade in this complex epithelial layer by immunohistochemical localization of Fas, FasL, Bcl-2, Mcl-1, pro-caspase-3 and caspase-3, T-cell-restricted intracellular antigen-related protein (TIAR), poly(ADP-ribose) polymerase (PARP), lamin B, topoisomerase IIalpha, and transglutaminase II in cryostat and paraffin-fixed tissue sections from normal human first-trimester and term placental villi. The relationship between the apoptotic cascade and syncytial fusion was studied by coincubation of intact villi with FITC-coupled annexin-V, to detect the phosphatidylserine flip, and propidium iodide, to detect plasma membrane permeability. The final events of the apoptotic cascade were studied by the TUNEL reaction and ultrastructural appearance of the trophoblast. The phosphatidylserine flip was identified in some of the villous cytotrophoblastic cells, but the presence of both Bcl-2 and Mcl-1 proteins presumably prevented continuation of the apoptotic cascade. The syncytiotrophoblast demonstrated heterogeneous findings, suggesting variable progression along the apoptotic cascade. In some areas Bcl-2 and Mcl-1 predominated, with preservation of the nuclear proteins PARP, lamin B, and topoisomerase IIalpha; in other areas, especially in and around syncytial sprouts, Bcl-2 and Mcl-1 were absent, accompanied by loss of nuclear proteins, presence of phosphatidylserine flip, and TUNEL positivity. These data suggest that the apoptotic cascade is initiated in the villous cytotrophoblast, which in turn promotes syncytial fusion. Donation of anti-apoptotic proteins into the syncytium, such as Bcl-2 and Mcl-1, focally inhibits further progression along this cascade. Completion of the apoptotic cascade takes place in and around syncytial sprouts, providing further evidence that these are the sites of trophoblast shedding into the maternal circulation.
Apoptosis↗