Enhancement of trophoblast differentiation and survival by low molecular weight heparin requires heparin-binding EGF-like growth factor.

Does low molecular weight heparin (LMWH) require heparin-binding epidermal growth factor (EGF)-like growth factor (HBEGF) signaling to induce extravillous trophoblast differentiation and decrease apoptosis during oxidative stress? LMWH increased HBEGF expression and secretion, and HBEGF signaling was required to stimulate trophoblast extravillous differentiation, increase invasion in vitro and reduce trophoblast apoptosis during oxidative stress. Abnormal trophoblast differentiation and survival contribute to placental insufficiency syndromes, including preeclampsia and intrauterine growth restriction. Preeclampsia often manifests as a pro-thrombotic state, with unsuccessful transformation of the spiral arteries that reduces oxygen supply and can produce placental infarction. LMWH improves placental function by increasing blood flow. Recent data suggest that the actions of LMWH transcend its anti-coagulative properties, but the molecular mechanism is unknown. There is evidence that LMWH alters the expression of human HBEGF in trophoblast cells, which regulates human trophoblast pathophysiology. HBEGF, itself, is capable of increasing trophoblast survival and invasiveness. First-trimester placental explants and the HTR-8/SVneo cell line, established using extravillous trophoblast outgrowths from first-trimester villous explants, were treated in vitro with LMWH to examine the effects on HBEGF signaling and trophoblast function under normal physiological and pathological conditions. A highly specific antagonist of HBEGF and other inhibitors of HBEGF downstream signaling were used to determine the relationship between LMWH treatment and HBEGF. Placental tissues (n = 5) were obtained with IRB approval and patient consent from first-trimester terminations. Placental explants and HTR-8/SVneo cells were cultured on plastic or Matrigel™ and treated with a therapeutic dose of LMWH (Enoxaparin; 10 IU/ml), with or without CRM197, pan Erb-B2 Receptor Tyrosine Kinase (ERBB) inhibitor, anti-ERBB1 or ERBB4 blocking antibodies, or pretreatment of cells with heparitinase I. Extravillous differentiation was assessed by immunocytochemistry to determine the relative levels of integrins α6β4 and α1β1. Trophoblast invasiveness was assessed in villous explants by measuring outgrowth from villous tips cultured on Matrigel, and by invasion assays with HTR-8/SVneo cells cultured on Matrigel-coated transwell insert. Placental explants and HTR-8/SVneo cells were exposed to oxidative stress in a hypoxia-reoxygenation (H-R) model, measuring cell death by TUNEL assay, caspase 3 cleavage, and BCL-2α expression. LMWH induced extravillous differentiation, according to trophoblast invasion assays and integrin (α6β4-α1β1) switching. Treatment with LMWH rescued cytotrophoblasts and HTR-8/SVneo cells from apoptosis during exposure to reoxygenation injury, based on TUNEL, caspase 3 cleavage and BCL-2α expression. Experiments using CRM197, ERBB1 and ERBB4 blocking antibodies, pan-ERBB inhibitor and removal of cell surface heparin demonstrated that the effects of LMWH on trophoblast invasion and survival were dependent upon HBEGF signaling. N/A. The primary limitation of this study was the use of only in vitro experiments. Patient demographics from elective terminations were not available. These data provide new insights into the non-coagulation-related aspects of perinatal LMWH treatment in the management of placental insufficiency disorders. This research was supported by grants from the National Institutes of Health (HD071408 and HL128628), the March of Dimes, and the W. K. Kellogg Foundation. There were no conflicts or competing interests.

Bolnick AD ，Bolnick JM ，Kohan-Ghadr HR ，Kilburn BA ，Pasalodos OJ ，Singhal PK ，Dai J ，Diamond MP ，Armant DR ，Drewlo S ... -  《-》

Trophoblast subtype-specific EGFR/ERBB4 expression correlates with cell cycle progression and hyperplasia in complete hydatidiform moles.

Do trophoblast subtypes differ in their expression of erythroblastic leukaemia viral oncogene homologue (ERBB) receptor family members and responsiveness towards specific growth factor ligands? Our data reveal a reciprocal expression pattern of epidermal growth factor receptor (EGFR)/ERBB4 in proliferative and ERBB2/ERBB3 in invasive trophoblast subtypes, as well as a restricted responsiveness to epidermal growth factor (EGF) and heparin-binding (HB)-EGF. EGFR is expressed by villous cytotrophoblasts (vCTBs), but absent from extravillous trophoblasts (EVTs), which specifically up-regulate ERBB2. Tissue samples of human first trimester placentae (n = 50) and deciduae (n = 5) obtained from elective pregnancy terminations were used to study trophoblast subtype-specific ERBB receptor expression and responsiveness to recombinant human EGF and HB-EGF. Age-matched complete hydatidiform mole (CHM) placentae (n = 12) were assessed for EGFR and ERBB4 expression in proliferation-competent regions. ERBB receptor expression was analysed in primary trophoblast cell isolates by means of microarray, quantitative real-time PCR and western blotting, as well as immunofluorescence stainings of placental and decidual tissue sections. EGF and HB-EGF were tested for their potential to activate ERBB receptors in purified EGFR(+) and HLA-G(+) trophoblasts. 5-Ethynyl-2'-deoxyuridine incorporation assays were performed to study the effect of both ligands on the proliferative capacity of primary trophoblasts as well as of vCTBs and proximal cell column trophoblasts (pCCTs) in placental floating explants. Finally, the average number of EGFR(+) vCTB and pCCT layers was determined in CHM placentae and compared with healthy age-matched controls. Proliferative vCTBs and pCCTs co-express EGFR and ERBB4, but are devoid of ERBB2 and ERBB3. In contrast, HLA-G(+) trophoblast subtypes exhibit an EGFR/ERBB4(-) and ERBB2/ERBB3(+) phenotype. EGF and HB-EGF activate EGFR, ERBB4, AKT and extracellular signal-regulated kinase 1/2 in EGFR(+) primary trophoblasts; however, they do not show an effect on HLA-G(+) EVTs. Both ligands strongly induce cell cycle progression in primary trophoblasts (P < 0.05) and placental explant-associated vCTBs (P < 0.05) and pCCTs (P < 0.05). Notably, EGFR(+) vCTB (P < 0.0001) and pCCT (P < 0.0001) layers are significantly expanded in CHM placentae when compared with healthy controls. Cells were removed from their physiological context and may therefore respond differently to various stimuli. In this study we define EGFR as a marker for proliferative trophoblast subtypes within the human placenta. Manipulation of EGFR signalling might thus offer a promising therapeutic avenue for the treatment of molar pregnancies associated with trophoblast hyperplasia. This study was supported by the Austrian Science Fund (grant P-25187-B13 to J.P.). There are no competing interests to declare.

Fock V ，Plessl K ，Fuchs R ，Dekan S ，Milla SK ，Haider S ，Fiala C ，Knöfler M ，Pollheimer J ... -  《-》

ADAM8 localizes to extravillous trophoblasts within the maternal-fetal interface and potentiates trophoblast cell line migration through a β1 integrin-mediated mechanism.

Does A Disintegrin And Metalloproteinase 8 (ADAM8) control extravillous trophoblast (EVT) differentiation and migration in early human placental development? ADAM8 mRNA preferentially localizes to invasive HLA-G-positive trophoblasts, associates with the acquirement of an EVT phenotype and promotes trophoblast migration through a mechanism requiring β1-integrin. Placental establishment in the first trimester of pregnancy requires the differentiation of progenitor trophoblasts into invasive EVTs that produce a diverse repertoire of proteases that facilitate matrix remodeling and activation of signaling pathways important in controlling cell migration. While multiple ADAM proteases, including ADAM8, are highly expressed by invasive trophoblasts, the role of ADAM8 in controlling EVT-related processes is unknown. First trimester placental villi and decidua (6-12 weeks' gestation), primary trophoblasts and trophoblastic cell lines (JEG3, JAR, Bewo, HTR8/SVNeo) were used to examine ADAM8 expression, localization and function. All experiments were performed on at least three independent occasions (n = 3). Placental villi and primary trophoblasts derived from IRB approved first trimester placental (n = 24) and decidual (n = 4) were used to examine ADAM8 localization and expression by in situ RNAScope hybridization, flow cytometry, quantitative PCR and immunoblot analyses. Primary trophoblasts were differentiated into EVT-like cells by plating on fibronectin and were assessed by immunofluorescence microscopy and immunoblot analysis of keratin-7, vimentin, epidermal growth factor receptor (EGFR), HLA-G and ADAM8. ADAM8 function was examined in primary EVTs and trophoblastic cell lines utilizing siRNA-directed silencing and over-expression strategies. Trophoblast migration was assessed using Transwell chambers, cell-matrix binding was tested using fibronectin-adhesion assays, and ADAM8-β1-integrin interactions were determined by immunofluorescence microscopy, co-immunoprecipitation experiments and function-promoting/inhibiting antibodies. Within first trimester placental tissues, ADAM8 preferentially localized to HLA-G+ trophoblasts residing within anchoring columns and decidua. Functional experiments in primary trophoblasts and trophoblastic cell lines show that ADAM8 promotes trophoblast migration through a mechanism independent of intrinsic protease activity. We show that ADAM8 localizes to peri-nuclear and cell-membrane actin-rich structures during cell-matrix attachment and promotes trophoblast binding to fibronectin matrix. Moreover, ADAM8 potentiates β1-integrin activation and promotes cell migration through a mechanism dependent on β1-integrin function. The primary limitation of this study was the use of in vitro experiments in examining ADAM8 function, as well as the implementation of immortalized trophoblastic cell lines. Histological localization of ADAM8 within placental and decidual tissue sections was limited to mRNA level analysis. Further, patient information corresponding to tissues obtained by elective terminations was not available. The novel non-proteolytic pro-migratory role for ADAM8 in controlling trophoblast migration revealed by this study sheds insight into the importance of ADAM8 in EVT biology and placental development. This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC-Discovery Grant) and the Canadian Institutes of Health Research (CIHR-Open Operating Grant). There are no conflicts or competing interests. NA.

Le HT ，Atif J ，Mara DL ，Castellana B ，Treissman J ，Baltayeva J ，Beristain AG ... -  《-》

Emerging nonanticoagulant role of low molecular weight heparins on extravillous trophoblast functions and on heparin binding-epidermal growth factor and cystein-rich angiogenic inducer 61 expression.

To examine the effects of low molecular weight heparins (LMWHs) on extravillous trophoblast (EVTC) invasiveness and on EVTC expression/secretion of heparin binding-EGF (HB-EGF) and cystein-rich angiogenic inducer 61 (Cyr61), both of which are involved in the process of EVTC invasion. Furthermore, to investigate the intracellular DNA binding activity of activator protein (AP)-1. Experimental study. Department of Obstetrics Gynecology, Università Cattolica del Sacro Cuore, Rome, Italy. Cultures of primary EVTC cells isolated from patients with first trimester unexplained recurrent miscarriage. The effects of LMWHs on EVTC invasiveness were examined by an in vitro matrigel invasion assay. Matrix metalloprotease-2 activity (MMP-2) was examined by gelatin zimography. HB-EGF and Cyr61 expression and secretion were studied by Western blot analysis and ELISA assay. AP-1 activity was measured through a multiwell colorimetric assay. The EVTC invasiveness, the expression/secretion of HB-EGF and Cyr61 proteins, and the AP-1 DNA binding activity in the presence of increasing concentrations of LMWHs were investigated. Both LMWHs, and primarily tinzaparin, increased EVTC invasiveness, by enhancing the MMP-2 proteolytic activity, and induced the expression/secretion of HB-EGF and Cyr61 in EVTC. This effect was mediated by an increased DNA binding activity of AP-1. Both LMWHs are able to promote EVTC development because they are able to stimulate the EVTC invasive properties. Our results may provide a possible biological rationale for the clinical use of LMWH for placental-mediated pregnancy complications unrelated to prothrombotic disorders.

D'Ippolito S ，Di Nicuolo F ，Marana R ，Castellani R ，Stinson J ，Tersigni C ，Scambia G ，Di Simone N ... -  《-》

Trophoblast survival signaling during human placentation requires HSP70 activation of MMP2-mediated HBEGF shedding.

Survival of trophoblast cells in the low oxygen environment of human placentation requires metalloproteinase-mediated shedding of HBEGF and downstream signaling. A matrix metalloproteinase (MMP) antibody array and quantitative RT-PCR revealed upregulation of MMP2 post-transcriptionally in human first trimester HTR-8/SVneo trophoblast cells and placental villous explants exposed to 2% O. Specific MMP inhibitors established the requirement for MMP2 in HBEGF shedding and upregulation. Because α-amanitin inhibited the upregulation of HBEGF, differentially expressed genes were identified by next-generation sequencing of RNA from trophoblast cells cultured at 2% O for 0, 1, 2 and 4 h. Nine genes, all containing HIF-response elements, were upregulated at 1 h, but only HSPA6 (HSP70B') remained elevated at 2-4 h. The HSP70 chaperone inhibitor VER 155008 blocked upregulation of both MMP2 and HBEGF at 2% O, and increased apoptosis. However, both HBEGF upregulation and apoptosis were rescued by exogenous MMP2. Proximity ligation assays demonstrated interactions between HSP70 and MMP2, and between MMP2 and HBEGF, supporting the concept that MMP2-mediated shedding of HBEGF, initiated by HSP70, contributes to trophoblast survival at the low O concentrations encountered during the first trimester, and is essential for successful pregnancy outcomes. Trophoblast survival during human placentation, when oxygenation is minimal, required HSP70 activity, which mediated MMP2 accumulation and the transactivation of anti-apoptotic ERBB signaling by HBEGF shedding.

Jain CV ，Jessmon P ，Barrak CT ，Bolnick AD ，Kilburn BA ，Hertz M ，Armant DR ... -  《-》