The chrondroitin sulfate proteoglycan (CSPG4) regulates human trophoblast function

Introduction

Trophoblast growth and invasion of the uterine endometrium are critical events during placentation and are tightly regulated by locally produced factors. Abnormal placentation can result in early miscarriage or preeclampsia and intrauterine growth restriction, leading to impaired fetal and/or maternal health. Chondroitin sulfate proteoglycan 4 (CSPG4) is involved in cancer cell migration and invasion, processes which are critical during placentation but unlike in cancer, trophoblast invasion is highly regulated. CSPG4 expression and function in trophoblast is unknown. We determined CSPG4 expression in human first trimester placenta and implantation sites, and investigated whether CSPG4 influenced proliferation, migration and invasion of a human extravillous trophoblast (EVT) cell line (HTR8/SVneo cells) as a model for extravillous trophoblast (EVT).

Methods and results

Immunoreactive CSPG4 localized to EVT cells in the trophoblast shell, subpopulations of interstitial EVT cells within the decidua and cytotrophoblast cells in placental villi. In HTR8/SVneo cells, siRNA knockdown of CSPG4 stimulated proliferation and decreased migration/invasion. In primary first trimester placental villi explants two cytokines, interleukin 11 (IL11) and leukemia inhibitory factor (LIF) with known roles in trophoblast function, stimulated CSPG4 mRNA expression and immunoreactive protein in the cyotrophoblast.

Discussion and conclusion

This is the first demonstration of the production and function of CSPG4 in human placentation. These data suggest that locally produced CSPG4 stimulates human EVT migration and invasion and suggests that IL11 and LIF regulate villous cytotrophoblast differentiation towards the invasive phenotype at least in part via CSPG4.     

The role of interleukin-1β in human trophoblast motility

The pleiotropic cytokine interleukin-1β (IL-1β) can promote physiological cell migration, as well as cancer cell invasion and metastasis. Its role in human trophoblast invasion, however, has not been satisfactorily answered since direct, indirect as well as no effects on trophoblast motility have been published. Therefore, the role of IL-1β has been re-evaluated by exclusively using human primary trophoblast model systems. Immunofluorescence of first trimester placentae indicated IL-1 receptor 1 (IL-1R1) protein expression in first trimester villous cytotrophoblasts (vCTB) and extravillous trophoblasts (EVT). The latter expressed higher mRNA levels of the receptor as shown by comparative gene chip data of vCTB and EVT. Similarly, Western blot analyses and immunofluorescence revealed a time- and differentiation-dependent increase of IL-1R1 in primary EVT seeded on fibronectin. IL-1β dose-dependently elevated migration of isolated first trimester EVT through fibronectin-coated transwells, which was inhibited in the presence of IL-1R antagonist (IL-1Ra), whereas proliferation of these cells was not affected. Similarly, the interleukin did not alter proliferation of vCTB and cell column trophoblasts in floating villi of early pregnancy, but promoted migration in villous explant cultures seeded on collagen I. Western blot analyses of supernatants of primary EVT and first trimester villous explant cultures revealed IL-1β induced secretion of urokinase plasminogen activator (uPA), plasminogen activator inhibitor (PAI)-1 and PAI-2, which was diminished upon combined IL-1β/IL-1Ra treatment. In conclusion, these data suggest that IL-1β directly promotes trophoblast motility of first trimester EVT involving the uPA/PAI system.     

Expression of endothelial nitric oxide synthase by extravillous trophoblast cells in the human placenta

Previous reports have documented the expression of endothelial nitric oxide synthase (eNOS) expression by the syncytiotrophoblast layer of the villus in the human placenta. In contrast, the underlying villous cytotrophoblast cells do not express the enzyme. Because extravillous cytotrophoblasts have not been as extensively investigated, our objective was to test whether these cells express eNOS. Using both a mouse monoclonal and a rabbit polyclonal antibody, we demonstrated immunoreactive eNOS in trophoblast cell columns emanating from anchoring villi in second trimester placentae. Cytokeratin positive trophoblast cells lying beneath remnant anchoring villi, lining decidual blood vessels and scattered throughout the basal plate of normal term and pre-eclamptic placentae also expressed immunoreactive eNOS. By Western analysis, the monoclonal and polyclonal antibodies were shown to be absolutely and relatively specific for eNOS, respectively. The finding of immunoreactive eNOS expression by extravillous trophoblast cells was substantiated by in situ hybridization. Using riboprobes generated from a bovine eNOS cDNA, we demonstrated specific hybridization in the endothelium of blood vessels in the umbilical cord, thus validating the in situ hybridization methodology, as well as specific hybridization in the extravillous trophoblast cells of the basal plate in normal term placenta. In conclusion, several different populations of extravillous trophoblast cells in the basal plate of the human placenta express eNOS.     

The involvement of eukaryotic translation initiation factor 4E in extravillous trophoblast cell function

Extravillous trophoblast cells (EVT) are major players in placental implantation. They differentiate in the villous cell column, invade to the uterus and remodel the uterine spiral arteries. Trophoblast and tumor cells have similar invasion mechanisms, share similar biochemical mediators (e.g. c-myc, MMP9) and growth-factors (e.g. VEGF). The mRNA of these proteins has extremely structured 5-UTR and their translation is highly dependent on eukaryotic-translation-initiation-factor-4E (eIF4E). Cancer cells have elevated eIF4E and are more vulnerable to its silencing than normal cells. We speculated that like cancer, trophoblast function is highly eIF4E dependent.ObjectiveAnalyze eIF4E involvement in EVT differentiation and function.Study designEIF4E levels were assessed in first-trimester human placentae and in placental explants before and after EVT differentiation. The effect of eIF4E knockdown (siRNA, ribavirin) on the phenotype of placental explant and EVT cell lines (HTR-8/SVNEO) was evaluated. Tested parameters included eIF4E and its target levels, migration, invasion, cell death, cell cycle and cell count.ResultsHigh eIF4E levels were found in cytotrophoblast and especially EVT cells during their differentiation in the villi, compared to other placental cell types. EIF4E silencing increased cell death and cell cycle arrest in placental explants and HTR-8/SVNEO cells. Although it induced EVT outgrowth in the placental explants, it reduced HTR-8/SVNEO motility, reflecting the importance of using ex vivo models that include an intact placental microenvironment in its original architecture.ConclusionsOur results suggest that eIF4E prevents final EVT differentiation and supports placental cell proliferation and survival. A balance between cell proliferation and differentiation is crucial for placental development and implantation.     

Characterization and role of NUMB in the human extravillous trophopblast

NUMB is a multifunctional protein involved in asymmetric cell differentiation, proliferation and maintenance. Four mammalian NUMB isoforms have been identified, which utilize the phosphotyrosine binding (PTB) domain and the proline rich region (PRR) domain to regulate cell growth and differentiation in the developing nervous system. The observation that a decrease in spongiotrophoblast number and thickness of placentae of null (Numb^−/−) mouse embryos, which died at E10.5, suggests NUMB may play a role in placental development. In this study, we demonstrated for the first time, that NUMB isoforms 1, 2, 3, and 4 are present in the human placenta and the human extravillous trophoblast (EVT) cell line HTR8/SVneo. We report three novel isoforms, NUMB 7, 8, and 9, identified by cloning of RT-PCR products and sequencing. Corresponding sequences of novel isoforms were submitted to genebank (accession numbers for each new isoform: NUMB 7- EU265736, NUMB 8- EU265737 and NUMB 9-EU265738). Western blot analysis confirmed the presence of all NUMB isofoms in human placental samples in all trimesters and in EVT cells. NUMB immunosignals were extensively localized in human extravillous trophoblasts and decidual cells at the maternal-fetal interface. NUMB 8 appeared to be the predominant isoform in placental villi. Furthermore, cell migration studies revealed NUMB isoform 1 to be involved in EVT cell migration and NUMB isoforms 2 and 4 to induce EVT apoptosis.     

Detection of class I MHC mRNA in subpopulations of first trimester cytotrophoblast cells by in situ hybridization

Class I MHC mRNA has been identified in both villous and extravillous cytotrophoblast cells in first trimester placentas by in situ hybridization. In this report, we expand those observations to additional morphologically and anatomically distinct subpopulations of trophoblast cells in early placentas using the same experimental approach. In the transition zone of first trimester placental villi, where cytotrophoblast cells are proliferating to form new villi or to migrate into adjacent tissue, both cytotrophoblast cells beneath the uninterrupted syncytium and the proliferating cytotrophoblast cells contained class I mRNA whereas a layer of cytotrophoblast cells proximal to the villus core did not contain class I mRNA. In the placental bed, migrating cytotrophoblast cells contained high levels of class I mRNA as determined by the intensity of staining. In contrast, multinucleated giant trophoblast cells contained little specific message. Alterations in levels of class I mRNA seem therefore to be associated with trophoblast proliferation, migration and differentiation.     

Adhesion Molecules in Human Trophoblast – A Review. II. Extravillous Trophoblast

At the tips of anchoring villi, cytotrophoblast (CTB) proliferation leads to a process of multilayering in which cells lose their attachment to the villous basement membrane and develop into columns, within which they adhere to one another using desmosomes, with associated intermediate filament bundles. Non-desmosomal cadherins, tight junction proteins and other adhesion molecules are also present, suggesting that actin-associated adhesions contribute to placental anchorage. In the distal columns, cell–cell interactions diminish, cells upregulate β1 integrins and bind to a provisional fibrinoid extracellular matrix, eventually detaching to migrate into the decidual stroma and myometrium, where interstitial and endovascular extravillous trophoblast (EVT) populations show distinct repertoires of adhesion molecules.     

Immunohistochemical localization of HLA antigens and placental proteins (alpha hCG, beta hCG CTP, hPL and SP1 in villous and extravillous trophoblast in normal human pregnancy: a distinctive pathway of differentiation of extravillous trophoblast

Immunohistochemical localization of HLA antigens and placental proteins (alpha hCG, beta hCG CTP, hPL and SP1) in villous and extravillous trophoblast at various stages of normal human gestation were studied, using hysterectomy specimens. In the chorionic villi, the capacity for synthesizing placental proteins seemed to develop in parallel with the morphological change from mononuclear cells to multinucleated syncytiotrophoblast and no villous trophoblast expressed HLA antigens. In contrast, extravillous trophoblast, including the multinucleated trophoblastic cells at the deciduomuscular junction, expressed HLA-A, -B, and -C, and their capacity for synthesizing placental proteins did not seem to correspond with the degree of morphological change: the location of alpha hCG, beta hCG CTP and SP1 was restricted to mononuclear trophoblast in the superficial decidua, while hPL was present extensively in extravillous trophoblast. These findings strongly suggest that extravillous trophoblast possesses many distinctive biological features and differentiates in an independent manner. Mononuclear trophoblast forming the cell columns was also positive for HLA-A, -B, and -C, and no placental protein was demonstrated in these cells; this, together with previous morphological observations, may indicate the germinative nature of these cells.     

妊娠期及妊高征滋养细胞E-钙粘附素的表达

目的 :研究妊娠各期胎盘床绒毛外细胞滋养细胞 (EVT)E 钙粘附素 (E cad herin)表达的变化 ,并与妊高征胎盘床中的表达进行比较。方法 :用免疫组化法 ,对 4 0例标本进行染色 (孕早期 9份 ,孕中期 4份 ,孕晚期 2 7份 ,其中妊高征组 8份 ,非妊高征组 19份 )。结果 :孕 10周前绒毛的细胞滋养细胞仅小部分E cadherin染色呈弱阳性。孕 10周后 ,绒毛的细胞滋养细胞染色均为强阳性 ,细胞柱区域从近端到远端染色逐渐减弱。孕中期EVT染色几乎为阴性 ,孕晚期又稍有增强。妊高征时EVT染色减弱不明显。妊高征组和非妊高征组染色阳性的细胞分别为 2 7.4 5 %和 12 .74 % (P <0 .0 1)。结论 :E cadherin表达减弱或消失有利于细胞滋养细胞的浸润。妊高征时 ,E cadherin表达减弱不明显 ,细胞滋养细胞浸润能力减弱 ,以至螺旋动脉缺乏妊娠期生理性变化 ,导致发生妊高征。     

ADAM28 localizes to HLA-G+ trophoblasts and promotes column cell outgrowth

IntroductionTrophoblast progenitor cell differentiation towards the extravillous trophoblast (EVT) lineage initiates within proximal regions of anchoring columns of first trimester placental villi. While molecular processes controlling the initial stages of progenitor cell differentiation along the EVT pathway have been described, much remains unknown about factors important in distal column cell differentiation into invasive EVTs. ADAMs are proteases that regulate growth factor signaling, cell-matrix adhesion, and matrix proteolysis, and thus impact many processes relevant in placentation. Global gene expression studies identified the ADAM subtype, ADAM28, to be highly expressed in EVT-like trophoblasts, suggesting that it may play a role in EVT function. This study aims to test the functional importance of ADAM28 in column cell outgrowth and maintenance.MethodsADAM28 mRNA levels and protein localization were determined by qPCR and immunofluorescence microscopy analyses in purified placental villi cell populations and tissues. ADAM28 function in trophoblast column outgrowth was examined using ADAM28-targetting siRNAs in Matrigel-imbedded placental explant cultures.ResultsWithin placental villi, ADAM28 mRNA levels were highest in HLA-G⁺ column trophoblasts, and consistent with this, ADAM28 was preferentially localized to HLA-G⁺ trophoblasts within distal anchoring columns and decidual tissue. siRNA-directed loss of ADAM28 impaired trophoblast column outgrowth and resulted in increased apoptosis in matrix-invading trophoblasts.DiscussionOur findings suggest that ADAM28 promotes column outgrowth by providing survival cues within anchoring column cells. This study also provides insight into a possible role for ADAM28 in driving differentiation of column trophoblasts into invasive HLA-G⁺ EVT subsets.     

Does plasminogen activator inhibitor-1 (PAI-1) control trophoblast invasion? A study of fetal and maternal tissue in intrauterine, tubal and molar pregnancies

Urokinase plasminogen activator, its receptor and the inhibitor PAI-1 are believed to control proteolysis and remodelling of maternal tissue during trophoblast invasion. This system appears to be strictly regulated in normal intrauterine pregnancies whereas tubal and molar pregnancies seem to be characterized by an uncontrolled excessive placental invasion. This study evaluates subcellular PAI-1 by immunohistochemistry in the villous placenta, in the basal plate and placental bed, and in the decidual compartments of normal, tubal and molar pregnancies. PAI-1 was present in villous syncytiotrophoblasts and co-localized focally with fibrin-type fibrinoid on the surface of the chorionic villi. Basal plate and placental bed extravillous interstitial trophoblasts, as well as vascular trophoblasts, were also PAI-1 positive. In the decidua parietalis, PAI-1 was observed in the cytoplasm of the non-invaded decidual cells. In the decidua basalis comprising the basal plate, PAI-1 was seen to be membrane-associated or confined to the extracellular matrix (ECM) facing the invasive front of anchoring villi. The ECM of decidua capsularis and chorion laeve displayed the most pronounced PAI-1 expression towards the maternal interface. In contrast, the majority of placental bed decidual cells adjacent to the interstitial and vascular trophoblasts were PAI-1 negative. Only a few stromal cells distant from the implantation site were PAI-1 positive in the tubal pregnancies and decidualization was not present. Likewise, excessive decidual necrosis and fibrinoid deposition devoid of PAI-1 was a common finding in complete molar pregnancies. These results suggest that PAI-1 defines specific extravillous invasive trophoblasts within the maternal decidua. Moreover, maternal cellular lack of PAI-1 in tubal pregnancies and excessive decidual necrosis in molar pregnancies indicate an uncontrolled placental invasion. The present data indicate that trophoblast invasion is primarily regulated by signals from decidual cells.     

TNF-alpha/TNFRI in primary and immortalized first trimester cytotrophoblasts

During the first trimester of pregnancy endogenous expression of tumour necrosis factor (TNF)-alpha has been detected in villous, as well as in proliferating and invading extravillous, trophoblasts suggesting that the protein could be involved in trophoblast differentiation. To gain insights into the putative role of the TNF-alpha signalling pathway, we investigated expression of its receptors, TNFR I and II, in first trimester placentae and early trophoblasts, and studied the influence of the cytokine on cell proliferation and apoptosis. ELISA and RT-PCR revealed secretion/expression of TNFRI protein/mRNA in immortalized ED27 cells and purified first trimester cytotrophoblasts, while soluble TNFRII was undetectable in cell culture supernatants. In agreement, immunohistochemical analyses of first trimester placentae showed that TNFRI is localized to the villous cyto- and syncytiotrophoblast, to the proliferating cytotrophoblasts of the cell islands and cell columns, as well as to extravillous cells invading decidual tissue. TNFRII, however, was absent in early trophoblast populations. Interleukin (IL)-1 and phorbol 12-myristate 13-acetate (PMA) induced shedding of TNFRI from ED27 and primary cells suggesting that under inflammatory conditions the soluble receptor protein may protect from cytotoxic effects of TNF-alpha. Upon incubation with increasing amounts of TNF-alpha no significant changes in DNA-content or cell numbers were found, suggesting that the cytokine does not augment proliferation of primary cytotrophoblasts. High doses of TNF-alpha, however, provoked growth arrest in ED27 cells as evaluated by cell counting, but did not induce necrosis/apoptosis as was assessed by TUNEL assay. In first trimester cells addition of elevated amounts of TNF-alpha resulted in the appearance of TUNEL-positive cells and an increase in caspase-3 enzyme activity suggesting that the TNF-alpha-dependent apoptotic cascade is executed in a portion of the early cytotrophoblasts.