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PubMed2003

Class II transactivator (CIITA) promoter methylation does not correlate with silencing of CIITA transcription in trophoblasts.

Trophoblast cells are unique because they do not express major histocompatibility complex (MHC) class II antigens, either constitutively or after exposure to interferon-gamma (IFN-gamma). The absence of MHC class II antigens on trophoblasts is thought to play a critical role in preventing rejection of the fetus by the maternal immune system. The inability of trophoblasts to express MHC class II genes is primarily due to lack of the class II transactivator (CIITA), a transacting factor that is required for constitutive and IFN-gamma-inducible MHC class II transcription. We, therefore, investigated the silencing of CIITA expression in trophoblasts. In transient transfection assays, transcription from the IFN-gamma-responsive CIITA type IV promoter was upregulated by IFN-gamma in trophoblasts, which suggests that CIITA is silenced by an epigenetic mechanism in these cells. Polymerase chain reaction analysis demonstrated that the CIITA type IV promoter is methylated in both the human choriocarcinoma cell lines JEG-3 and Jar and in 2fTGH fibrosarcoma cells, which are IFN-gamma inducible for CIITA. Conversely, methylation of the CIITA type IV promoter was not observed in human primary cytotrophoblasts isolated from term placentae or in mouse or rat trophoblast cell lines. Simultaneous treatment with IFN-gamma and the histone deacetylase inhibitor trichostatin A weakly activated CIITA transcription in mouse trophoblasts. Stable hybrids between human choriocarcinoma and fibrosarcoma cells and between mouse trophoblasts and fibroblasts expressed CIITA following treatment with IFN-gamma. These results suggest that silencing of CIITA transcription is recessive in trophoblasts and involves an epigenetic mechanism other than promoter methylation. The fact that CIITA is expressed in the stable hybrids implies that trophoblasts may be missing a factor that regulates chromatin structure at the CIITA promoter.

Animals↗
PubMed1997

Monoclonal antibody against phosphatidylserine inhibits in vitro human trophoblastic hormone production and invasion.

Naturally occurring antiphospholipid (aPL) antibodies against cardiolipin (CL)- and phosphatidylserine (PS)-dependent antigens are associated with placental dysfunction and unsuccessful pregnancy. Murine monoclonal aPL antibodies react with placental trophoblast and may interfere with normal trophoblastic function. In this study, we evaluated the expression of phospholipid-dependent antigens during trophoblast differentiation and measured the effects of monoclonal aPL antibodies on two in vitro aspects of trophoblast differentiation: hormone production and invasion into filters coated with extracellular matrix. Murine monoclonal IgM aPL antibodies that differentiated between PS and CL were used: 3SB9b reacted only with PS (CL-/PS+), D11A4 reacted only with CL (CL+/PS-), and BA3B5C4 reacted with both CL and PS (CL+/PS+). Isolated trophoblasts were cultured for 4 days, and reactivity with monoclonal aPL antibodies was evaluated daily. BA3B5C4 (CL+/PS+) reacted strongly with most trophoblasts that were freshly isolated (Day 0) and through 2 days of culture, after which time the percentage of cells reactive with BA3B5C4 decreased steadily. 3SB9b (CL-/PS+) reactivity increased during incubation; about 8% of cells reacted initially, but after 1 day of incubation 100% reacted, and this percentage remained stable throughout the 4-day incubation. D11A4 (CL+/PS-) reacted only minimally and at the level of the negative control monoclonal antibody (mAb) with 1- and 2-day cultures. Both mAbs that reacted with PS-dependent antigens completely prevented invasion of matrigel-coated filters by isolated trophoblasts. These mAbs also inhibited trophoblastic hCG and human PL production by more than 45%. Thus, as trophoblasts undergo differentiation, they are reactive with mAbs against PS. These antibodies are inhibitory in vitro to trophoblastic hormone production and invasion.

Antibodies, Monoclonal↗
PubMed1997

Signaling pathways controlling trophoblast cell differentiation: Src family protein tyrosine kinases in the rat.

Trophoblast giant cell differentiation is characterized by endoreduplication and expression of members of the prolactin (PRL) gene family and can be simulated in vitro via manipulations of the Rcho-1 trophoblast cell line. The regulation of trophoblast cell proliferation and differentiation involves tyrosine protein kinase signaling pathways. Treatment of Rcho-1 trophoblast cells with tyrosine kinase inhibitors disrupted differentiation-dependent expression of members of the PRL gene family and cytoskeletal organization. Activated p60c-src, p62c-yes, and p53/56lyn were present in the Rcho-1 rat trophoblast cell line and in differentiated trophoblast cells isolated from the developing rat placenta. p60c-src and p62c-yes were active in proliferating and differentiating trophoblast cells. During proliferation, p62c-yes exhibited distinct associations with other phosphoproteins (34, 66, 76, and 150 kDa). p53/56lyn was activated only in differentiating trophoblast cells. p53/56lyn showed a differentiation-dependent accumulation in cytoskeletal and membrane fractions, whereas p60c-src levels were virtually invariant in both fractions. Expression patterns of csk, a negative regulator of Src family kinase activities, were not consistent with its involvement in the differentiation-dependent activation of p53/56lyn; however, there was some indication of the participation of a tyrosine phosphatase in the regulation of p53/56lyn. In conclusion, p60c-src, p62c-yes, and p53/56lyn patterns of activation in trophoblast cells are consistent with their involvement in the control of trophoblast cell proliferation and differentiation.

Animals↗
PubMed1999

Signal transduction and biological function of placenta growth factor in primary human trophoblast.

Placenta growth factor (PlGF), a member of the vascular endothelial growth factor family of angiogenic factors, is prominently expressed by trophoblast. In addition to its role as a paracrine angiogenic factor within the placenta and endometrium, presence of its receptor, Flt-1, on trophoblast suggests that PlGF also may have an autocrine role(s) in regulating trophoblast function. To elucidate its role in trophoblast, we examined the signal transduction and functional responses of primary human trophoblast to PlGF. Exogenous PlGF induced specific activation of the stress-activated protein kinase (SAPK) pathways, c-Jun-N terminal kinase (JNK) and p38 kinase, in primary term trophoblast with little to no induction of the extracellular signal regulated kinase (ERK-1 and -2) pathways. In contrast, PlGF induced significant ERK-1 and -2 activity in human umbilical vein endothelial cells but did not induce JNK or p38 activity. PlGF-induced activation of the SAPK signaling pathways protected trophoblast from growth factor withdrawal-induced apoptosis, but it did not protect trophoblast from apoptosis induced by the pro-inflammatory cytokines, interferon gamma and tumor necrosis factor alpha. These results provide the first direct evidence of a biochemical and functional role for PlGF/Flt-1 in normal trophoblast and suggest that aberrant PlGF expression during pregnancy may impact upon trophoblast function as well as vascularity within the placental bed.

Apoptosis↗
PubMed2002

Control of expression of major histocompatibility complex genes in horse trophoblast.

In most mammals, the fetus limits its presentation of paternal antigens to the mother by suppressing the cell-surface expression of proteins of the major histocompatibility complex (MHC) on trophoblast. In the horse, however, functional, polymorphic MHC class I antigens are expressed at high levels on the invasive trophoblast cells of the chorionic girdle between Days 32 and 36 of pregnancy, although not on the adjacent noninvasive trophoblast of the chorion and allantochorion membranes. In this study, the control of MHC class I gene expression was investigated in invasive and noninvasive horse trophoblast, and the MHC class I loci expressed by invasive trophoblast were identified. Northern blot hybridization of Day 33-34 conceptus tissue revealed both transcriptional and posttranscriptional regulation of cell-surface MHC class I expression in horse trophoblast. The invasive MHC class I-positive trophoblast showed levels of steady-state mRNA nearly as high as those in lymphoid tissues from adult horses, whereas noninvasive MHC class I-negative trophoblast also contained transcripts for MHC class I, but at lower levels similar to those present in adult horse nonlymphoid tissue. We also cloned and sequenced polymerase chain reaction products from the transmembrane and cytoplasmic regions of MHC class I transcripts in chorionic girdle and lymphocytes, and determined that horse invasive trophoblast appears to transcribe the same MHC class I loci transcribed in lymphocytes, including both polymorphic and nonpolymorphic loci. These data from the horse demonstrate that functional alloantigen presentation by trophoblast can be a normal part of early pregnancy.

Allantois↗
PubMed2002

Differential regulation and function of the Fas/Fas ligand system in human trophoblast cells.

Trophoblast rejection, which is characterized by increased apoptosis, is mediated by T helper (Th)-1, or proinflammatory, cytokines, whereas Th-2, or anti-inflammatory, cytokines confer immune protection and facilitate implantation. We investigated the role of both types of cytokines on the expression and function of the Fas/Fas ligand (FasL) apoptotic pathway in trophoblast cells. First-trimester human trophoblast primary-culture cells as well as A3 and HTR/8 trophoblast cell lines were treated with proinflammatory cytokines such as interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNFalpha) and with the anti-inflammatory cytokines interleukin (IL)-6 and IL-10. Sensitivity to Fas-mediated apoptosis was measured using an activating anti-Fas monoclonal antibody. Cell viability was evaluated using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and CellTiter 96 assay. Fas/FasL mRNA and protein expression levels were determined using reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. Trophoblast cells normally express FasL, but low levels of Fas, and they are resistant to Fas-mediated apoptosis. IFN-gamma and TNFalpha promote Fas expression and sensitivity, whereas IL-6 and IL-10 increase the resistance of trophoblast cells to Fas-mediated apoptosis. Furthermore, IL-10 treatment activates FLICE-like inhibitory protein (FLIP), a downstream inhibitor of Fas apoptotic signaling. Although trophoblast cells express Fas, susceptibility to Fas does not necessarily correlate with its expression. In this study, we demonstrate that Th-2 cytokines increase the resistance of trophoblast cells to Fas-mediated apoptosis either by inhibiting Fas expression or by inducing FLIP activation. This "trophoblast-cytokine-Fas/FasL triad" determines the ability of the Fas/FasL system to regulate trophoblast viability and, consequently, the success or failure of pregnancy.

Apoptosis↗
PubMed1991

Further studies of lectin binding by villous and extravillous trophoblast in normal and pathological pregnancy.

Examination of human pregnancy tissues with a panel of lectins provides the opportunity to probe different aspects of carbohydrate structure. Nine biotinylated lectins [concanavalin A (con A), wheat germ agglutin (WGA), Lens culinaris A (LCH-A), Pisum sativum (PSA), Phaseolus vulgaris (PHA-E and PHA-L), Ulex europaeus 1 (UEA1), Griffonia simplicifolia (GSI and GSII)] were used to investigate the lectin binding of human trophoblast in normal, tubal, and molar pregnancy. All lectins except UEA1 bound to normal villous syncytiotrophoblast. Binding of lectins to extravillous trophoblast was more restricted than to villous trophoblast, occurring predominantly with con A, PHA-E, PHA-L, WGA, GSI, and GSII. LCH-A reacted with cyto-trophoblastic columns but not with interstitial or endovascular trophoblast. Con A and GSII were the only lectins that bound to trophoblastic giant cells. GSI and GSII bound preferentially to extravillous trophoblast, showing only focal reactivity with villous trophoblast. Lectin binding in ectopic pregnancy was similar to that in normal first-trimester intrauterine pregnancy. Reactivity in molar pregnancy also generally mirrored that observed in normal pregnancy; however, reactivity of GSII with villous trophoblast was more consistent than that observed in normal pregnancy, and GSI showed uniform binding to proliferating syncytial areas. Thus, lectin binding studies allow definition of surface carbohydrates, which may play a role in the controlled trophoblast proliferation and invasion that occurs in normal pregnancy.

Amnion↗
PubMed2004

Effects of low-molecular-weight and unfractionated heparin on trophoblast function.

OBJECTIVE: Unfractionated and low-molecular-weight heparin and low-dose aspirin are used for the prevention of pregnancy loss in pregnant women with thrombophilia. We investigated the effect of these drugs on in vitro models of human extravillous trophoblast motility and differentiation. METHODS: Chorion from term placentas was digested and extravillous trophoblast isolated. Extravillous trophoblast formed giant multinuclear cells that were counted after 24, 36, and 48 hours of culture. This model was then used to investigate the effect of unfractionated, low-molecular-weight heparin and aspirin on in vitro extravillous trophoblast differentiation at both therapeutic and supratherapeutic doses. In addition, the effect of unfractionated and low-molecular-weight heparin on hepatocyte growth factor-stimulated SGHPL-4 cell (extravillous trophoblast cell line) motility was determined by time-lapse microscopy. RESULTS: At therapeutic doses unfractionated heparin promoted extravillous trophoblast differentiation. However, low-molecular-weight heparin inhibited giant multinuclear cells formation. At supratherapeutic doses, both low-molecular-weight and unfractionated heparin promoted extravillous trophoblast differentiation. Low-dose aspirin had minimal effects on the extravillous trophoblast differentiation. Both unfractionated and low-molecular-weight heparin inhibited hepatocyte growth factor-stimulated extravillous trophoblast motility at supratherapeutic doses. At a therapeutic dose of 0.25 IU/mL, only unfractionated heparin inhibited hepatocyte growth factor-stimulated motility, whereas low-molecular-weight heparin had no effect. CONCLUSION: Our data suggest that unfractionated and low-molecular-weight heparin have differing effects on trophoblast differentiation and motility at therapeutic doses. This finding may be one of many factors that contribute to the clinical scenario.

Anticoagulants↗
PubMed1996

Evaluation of trophoblast invasion in placental bed biopsies of the baboon, with immunohistochemical localisation of cytokeratin, fibronectin, and laminin.

Biopsies of placentas (n = 21), placental bed (n = 17) and decidua (n = 26) of various gestation periods (30 to 140 days) were used to study trophoblast invasion in the baboon. Application of immunohistochemical staining for cytokeratin allowed proper identification of trophoblast. Earlier reports showing restricted trophoblast invasion in this species were confirmed by the finding that endovascular trophoblast was present in only one third of biopsies containing spiral arteries. Moreover, immunostaining for cytokeratin revealed that in several arteries only a few isolated trophoblastic cells were present, while the vessel had not undergone the normal physiological change. Trophoblast invasion could only be detected within decidual, but not in myometrial, segments of spiral arteries. Interstitial trophoblast invasion was very limited and multinuclear giant cells were absent. Immunohistochemical staining suggested a contribution of laminin to the fibrinoid deposition within the physiologically changed spiral arteries, while fibronectin was present intracellularly in the invaded trophoblast. Because of differences in the trophoblast invasion pattern, the baboon cannot be regarded as a satisfactory experimental model to explore results of inadequate endovascular trophoblast invasion which, in the human, leads to pregnancy complications such a preeclampsia.

Animals↗
PubMed1996

Functional characteristics of human trophoblast interferons.

Human trophoblast populations from first-and third-trimester placentas produce interferons (IFNs) in the presence of growth factors (CSF and PDGF) or when infected with virus. The highly invasive extravillous trophoblast population produced a higher level of IFNs (three- to eightfold, P < 0.05) than the noninvasive villous trophoblast population when stimulated with growth factors and/or virus. The level of IFN produced was dependent on the type of trophoblast population, the type of inducer and the stage of differentiation of the trophoblasts. Tandem immunoaffinity chromatography of the virus-induced trophoblast IFNs resulted in the isolation of trophoblast IFN-alpha and -beta types. The purified trophoblast IFNs have antiviral, antiproliferative and immunoregulatory properties. Furthermore, the trophoblast IFNs inhibited the expression of proto-oncogenes such as EGF-R, c-erbB2 and c-fms reported to be involved in normal trophoblast growth and differentiation. These data suggest essential roles of interferons in normal human development during pregnancy.

Antiviral Agents↗
PubMed1999

First trimester human endovascular trophoblast cells express both HLA-C and HLA-G.

PROBLEM: In human pregnancies, trophoblasts, in contrast to placental connective tissue and the fetus itself, come into direct contact with the maternal allorecognizing system at special sites. Villous syncytiotrophoblasts washed around by maternal blood lack HLA class I proteins, whereas extravillous trophoblasts, which deeply invade maternal uterine tissues, express high amounts of HLA-G and also HLA-C, the latter to a lesser degree, however. A subpopulation of extravillous trophoblasts, the endovascular trophoblast, enters maternal spiral artery lumen and, like syncytiotrophoblast, comes into direct contact with maternal blood. Less is known about HLA class I distribution on this endovascular trophoblast subpopulation. METHOD OF STUDY: A comparative immununohistochemical analysis was done on decidual cryo-sections containing trophoblast-invaded spiral arteries using different anti-HLA class I monoclonal antibodies (mAbs) and a peroxidase-labeled streptavidinbiotin detection system. RESULTS: MAbs W6/32 (anti-HLA-A, -B, -C, -G), HCA2 (anti-HLA-A, -G) G233 and 87G (both anti-HLA-G) resulted in strong positivity on endovascular trophoblasts. L31 (anti-HLA-C) and HC10 (anti-HLA-B, -C) revealed clear positivity, whereas TU149 (anti-HLA-B, -C, some -A) produced a heterogeneous staining pattern, faintly positive on some endovascular trophoblastic cells and negative on others. MAb LA45 (anti-HLA-A, -B) did not bind to any endovascular trophoblast, neither did BFL.1 (anti-HLA-G) nor 16G1 (anti-HLA-G, soluble). CONCLUSION: This study shows that trophoblastic cells belonging to the endovascular subpopulation express considerable amounts of HLA-G and slightly less HLA-C.

Antibodies, Monoclonal↗
PubMed2007

NOD protein expression and function in first trimester trophoblast cells.

PROBLEM: Through the expression of pattern recognition receptors, the trophoblast can recognize and respond to infectious microorganisms and, therefore, participate in the control of pathogens that may compromise fetal well-being. We hypothesize that the trophoblast has the ability to sense invasive intracellular bacteria through the cytoplasmic-based nucleotide-binding oligomerization domain (NOD) proteins. The aim of this study was to characterize the expression and function of NOD proteins in first trimester trophoblast cells. METHOD OF STUDY: NOD1 and NOD2 expressions by first trimester trophoblast cells were evaluated by immunohistochemistry, Western blot analysis and reverse transcription-polymerase chain reaction. The effect of NOD2 activation on trophoblast cells was determined by analyzing the cytokine response following treatment with muramyl dipeptide (MDP). RESULTS: Both NOD1 and NOD2 were expressed by first trimester placental villi and localized to trophoblast cells. Moreover, NOD1, NOD2 and the signaling effector protein, RIP-like interacting CLARP kinase (RICK), were all expressed by isolated trophoblast cells. Following exposure to the NOD2 ligand, MDP, trophoblast cells generated a pro-inflammatory cytokine response. This response was confirmed to be specific, as an NOD2-deficient trophoblast cell line failed to respond to MDP unless transfected with NOD2. CONCLUSION: These findings suggest that, through the expression and function of NOD proteins, first trimester trophoblast cells are able to recognize and respond to invasive intracellular pathogens that may have evaded other forms of pattern recognition.

Acetylmuramyl-Alanyl-Isoglutamine↗
PubMed1997

Placental trophoblasts resist infection by multiple human immunodeficiency virus (HIV) type 1 variants even with cytomegalovirus coinfection but support HIV replication after provirus transfection.

Whether cell-free human immunodeficiency virus type 1 (HIV-1) can productively infect placental trophoblasts (which in turn could transmit the virus into the fetal circulation) is controversial but essential to know for the evaluation of alternative routes (such as cell-mediated infection or trophoblast damage). We have addressed infection factors such as cell purity, source, culture methods, and activation states as well as virus variant and detection methods to conclusively determine the outcome of trophoblast challenge by free virus. Pure (> 99.98%) populations of trophoblasts from 11 different placentas were challenged at a multiplicity of infection (MOI) as high as 6 with five different HIV-1 variants, three of which are non-syncytium-forming, macrophage-tropic isolates from infected infants, with and without coinfection with cytomegalovirus; these preparations were monitored for productive infection for up to 3 weeks after challenge by five different criteria, the most sensitive of which were cocultivation with target cells that can detect virus at an MOI of 10(-7) and HIV DNA PCR that detects 30 virus copies per 10(5) cells. Infection was never detected. However, molecularly cloned T-cell (pNL4-3)- and macrophage (pNLAD8)-tropic provirus plasmids, when transfected into primary trophoblasts, yielded productive infections, indicating that trophoblasts do not suppress late-stage virus replication and assembly. Because of the purity of the trophoblast preparations, the extended length of the infection culture period, the number of trophoblast preparations and virus types examined, the sensitivity of the bioassays and molecular detection assays, and the observations that trophoblasts can support virus replication from provirus, the results of this study strongly argue that free virus cannot infect primary villous trophoblasts.

Cell Membrane↗
PubMed1995

Transition from lacunar to villous stage of implantation in the macaque, including establishment of the trophoblastic shell.

Adhesion of trophoblast of the blastocyst to and penetration into the uterine epithelium, invasion into the maternal vessels and endometrial stroma, and establishment of the basic organization of the placenta all occur within the first week following the initiation of implantation in the human, macaques and several other primates. The cellular rearrangements and interactions of trophoblast with endometrial epithelial cells and stroma were studied during this preiimplantation stage in macaques. At the early lacunar stage, 1-2 days after the initiation of implantation, both cytotrophoblast and syncytial trophoblast can be found at the maternal surface of lacunae. As the lacunar stage advances, both syncytial trophoblast and cytotrophoblast are found throughout the implantation site including in septae partitioning lacunae, but syncytial trophoblast lines most of the lacunae and forms the confluence with the maternal vessels. Indentations of fetal mesenchyme and accumulation of cytotrophoblast cells within the septae occur rapidly. Over a period of about 2 days, clusters of cytotrophoblast cells pass beyond the syncytial trophoblast at the maternal surface as the anchoring villi, establishing the trophoblastic shell. The bypassing of epithelial plaque cells by cytotrophoblast cells and elimination of stromal matrix between such clusters verifies that this is a progressive invasion rather than simply superficial growth. Concomitant with establishment of the trophoblastic shell there is extravasation of blood, necrosis of plaque cells, and massive invasion of vessels by cytotrophoblast resulting in a necrotic zone between the trophoblastic shell and endometrium. It is concluded that only in the brief period of time of establishment of the trophoblastic shell is the full invasive potential of cytotrophoblast realized and that only at this stage does cytotrophoblast demonstrate the type of invasive and migratory behavior which has been achieved with isolated human cytotrophoblast cells in vitro.

Animals↗
PubMed2005

Phagocytosis of necrotic but not apoptotic trophoblasts induces endothelial cell activation.

It is hypothesized that preeclampsia is caused by factors from the placenta that induce endothelial cell activation. Trophoblasts are cells that may be shed from the placenta, then deported in the maternal blood, and finally become trapped in the pulmonary capillaries. The ultimate fate of deported trophoblasts is unknown, but to prevent clogging of the pulmonary circulation they must be cleared from the capillary beds. We examined the hypothesis that endothelial cells phagocytose deported trophoblasts and also examined the consequent effects of the trophoblasts on endothelial cells. Fluorescently labeled trophoblast-derived choriocarcinoma cells were induced to become apoptotic or necrotic and exposed to endothelial cell monolayers. Confocal microscopy demonstrated uptake of both apoptotic and necrotic trophoblasts, and this phagocytosis could be inhibited by cytochalasin B. Phagocytosis of necrotic but not apoptotic trophoblasts induced increased endothelial intercellular adhesion molecule 1 (ICAM-1) expression, as well as increased adhesion of monocytes to endothelial cell monolayers. Inhibiting the phosphatidylinositol 3-kinase and p38 mitogen-activated protein kinase pathways blocked both expression of ICAM-1 and phagocytosis, whereas inhibition of the P42/44 mitogen-activated protein kinase pathway blocked only ICAM-1 expression. This work suggests that endothelial cells can phagocytose deported trophoblasts and that the mechanism of trophoblast death (apoptotic or necrotic) could have major effects on the maternal vascular response to shed trophoblasts.

Apoptosis↗
PubMed2000

Human placental trophoblasts secrete a disintegrin metalloproteinase very similar to the insulin-like growth factor binding protein-3 protease in human pregnancy serum.

During the course of human pregnancy, there is a marked increase in insulin-like growth factor (IGF) binding protein (IGFBP)-3 protease activity in maternal serum that is first evident at 6 weeks of gestation, persists through term, and returns to nonpregnancy levels by day 5 postpartum. This protease activity cleaves IGFBP-3 into smaller fragments that have markedly reduced affinity for the IGFs. To date, the precise identity and cellular origin of the pregnancy-associated serum IGFBP-3 protease have not been established. To investigate whether placental and/or decidual tissues, which uniquely develop during pregnancy, may be sources of the pregnancy-associated serum IGFBP protease, we examined the secretion of IGFBP-3 protease in vitro by isolated human cytotrophoblasts or fibroblasts from second trimester placentae and by in vitro decidualized human endometrial stromal cells. Cytotrophoblasts were either cultured alone, which favors aggregation and fusion, or cocultured with decidualized endometrial stromal cells, which favors differentiation to an invasive phenotype. IGFBP-3 protease activity was detected in trophoblast, but not in placental fibroblast or decidualized endometrial cultures, and was also present in trophoblast-endometrial cocultures. Western ligand blot and Western immunoblot analyses showed that most of the endogenous IGFBP-3 in trophoblast cultures was in the form of low molecular weight fragments with reduced IGF binding affinity. The substrate specificity of the trophoblast-derived protease was identical to that in pregnancy serum, showing activity against IGFBP-2, -3, and -4, but being inactive against IGFBP-1. IGFBP-3 proteolysis by both pregnancy serum and trophoblast conditioned medium showed a major peak of activity at neutral pH. The trophoblast-derived activity caused time-and temperature-dependent proteolysis of IGFBP-3 into fragments of identical size as those produced by pregnancy serum, and also shared its sensitivity to protease inhibitors: highly sensitive to EDTA and o-phenanthroline, partially sensitive to the serine protease inhibitors AEBSF and aprotinin, and insensitive to alpha2-antiplasmin, and to aspartic and cysteine protease inhibitors. IGFBP-3 proteolysis by both pregnancy serum and trophoblast conditioned medium was also insensitive to tissue inhibitor of metalloproteinase-1, precluding the involvement of the matrix metalloproteinases. In contrast, both the pregnancy serum- and trophoblast-derived proteases were preferentially inhibited by a hydroxamic acid derivative with selective activity against the disintegrin-metalloproteinase tumor necrosis factor-alpha converting enzyme. This study shows that placental trophoblasts produce an IGFBP-3 protease with characteristics very similar to the activity found in pregnancy serum and indicates these cells at the maternal-fetal interface are a potential source of the pregnancy-associated serum IGFBP-3 protease. The findings further suggest that the main IGFBP-3 protease activity in both pregnancy serum and trophoblast conditioned medium may correspond to a disintegrin-metalloproteinase type enzyme.

Cells, Cultured↗
PubMed2004

Placental trophoblast-derived factors diminish endothelial barrier function.

Although increased vascular permeability is an important event in the pathogenesis of preeclampsia, the origin of the circulating factor(s) that elicits this endothelial barrier dysfunction is not known. In this study, we use coculture of endothelial cells and placental trophoblast cells to determine whether placental trophoblasts are a potential source of the factor(s) that mediate the increased vascular permeability of preeclampsia. Human umbilical vein endothelial cells grown in Transwell inserts or on coverslips were cocultured with trophoblast cells isolated from normal and preeclamptic placentas or placenta conditioned media. Endothelial cell barrier function was determined by: 1). measurements of electrical resistance and leakage of horseradish peroxidase, and 2). immunofluorescent staining of vascular endothelial-cadherin, pan-cadherin, and occludin. Uterine myometrium endothelial cells were also studied for comparison. We observed the following: 1). electrical resistance was significantly (P < 0.01) decreased (compared with control endothelial cells) in endothelial cell monolayers cocultured with normal trophoblast cells and further reduced in endothelial cells cocultured with preeclamptic trophoblast cells; 2). an increased horseradish peroxidase leakage that was correlated with the decreased electrical resistance in cocultured cells; and 3). disorganized tight junction proteins and an altered distribution of vascular endothelial-cadherin and occludin in monolayers of endothelial cells cocultured with preeclamptic trophoblast cells. Similar responses were noted in uterine myometrium endothelial cells. We conclude that: 1). placental trophoblast cells produce factors that diminish the barrier function of endothelial cells; 2). endothelial tight junctions are more susceptible to factors released from preeclamptic trophoblast cells than from normal trophoblast cells; and 3). these results implicate trophoblast-derived factors in the increased vascular permeability associated with preeclampsia.

Adherens Junctions↗
PubMed1990

Purified first and third trimester placental trophoblasts differ in in vitro hormone secretion.

In vitro studies with cytotrophoblasts obtained from term placentas have shown low levels of placental protein hormone secretion during the first 2 days in culture, followed by a marked increase during days 3 and 4. Since maternal serum placental hormone levels at term and during the first trimester differ, it is conceivable that cytotrophoblasts from first trimester placentas will differ in endocrine function from those derived from term placentas. Therefore, we examined the secretion of hCG, hCG alpha, human placental lactogen (hPL), and progesterone (P) both in the basal state and after exposure to 8-bromo-cAMP or endogenous cAMP stimulation with cholera toxin in cytotrophoblasts purified by enzymatic dispersion and Percoll gradient centrifugation from four first trimester and four third trimester placentas. At the time of seeding, all cells were mononuclear, and the degrees of aggregation and syncytia formation were similar in first and third trimester trophoblasts during the 4 days in culture. First trimester trophoblasts secreted greater quantities of hCG than did term trophoblasts, while basal secretion of hCG alpha, hPL, and progesterone were similar. Qualitative differences in the hormone secretory patterns were apparent. hCG secretion by first trimester trophoblasts decreased over the 4 days in culture, while the amounts secreted by third trimester trophoblasts increased. hCG alpha levels increased for 2-3 days in first trimester trophoblasts and then decreased, while hCG alpha increased in term trophoblast medium over the 4 days. The ratio of hCG alpha to hCG in media from first and third trimester cultures reflected the relative ratios of these hormones in placental tissue and maternal serum at analogous stages of pregnancy. hPL concentrations in the medium declined between days 3-4 in first trimester cultures, while they increased between days 3-4 in third trimester cultures. The secretory pattern of P was somewhat more erratic. cAMP stimulation led to a similar rise in hCG, hCG alpha, and P secretion in first and third trimester trophoblasts, and a variable response for hPL secretion. These results indicate that the functional activity of placental trophoblasts in culture depends in part upon the age of the placenta from which the cells are derived. The differences may represent intrinsic differences in function or the presence of inhibitory or stimulatory factors of maternal, fetal, or trophoblast origin.

Adult↗