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 ... -  《-》

Extravillous trophoblast invasion of venous as well as lymphatic vessels is altered in idiopathic, recurrent, spontaneous abortions.

Do extravillous trophoblasts (EVTs) invade non-arterial decidual vessels in healthy and pathological pregnancies? Our results reveal that trophoblast invasion of venous and lymphatic vessels is a frequent event during the first trimester of pregnancy and is compromised in  recurrent spontaneous abortion (RSA). In addition, the present data suggest that EVTs populate regional lymph nodes during pregnancy. Human trophoblasts remodel and invade decidual spiral arteries. In addition, a recent report demonstrates that trophoblasts contact and invade decidual veins. Tissue samples of human first trimester deciduae basalis (n = 54, 6th-13th weeks of gestation) obtained from elective pregnancy terminations were used to study trophoblast invasion into veins and lymphatics, in comparison to arteries. Age-matched cases of idiopathic, recurrent spontaneous abortions tissue samples (n = 23) were assessed for cell numbers of EVTs in these decidual vessels. In addition, lymph nodes of four pregnant women were analysed for the presence of EVTs. Localization, frequency and EVT-mediated targeting and invasion of arterial, venous as well as lymphatic vessels were determined in first trimester decidua basalis tissue sections using immunofluorescence staining with antibodies against CD31, CD34, ephrin B2 (EFNB2), ephrin receptor B4 (EPHB4), HLA-G, podoplanin, prospero-related homeobox 1 (Prox-1), alpha-smooth muscle actin 2 (ATCTA2), von willebrand factor (vWF) and proteoglycan 2 (PRG2). Arterial, venous and lymphatic-associated EVTs were further characterized according to their position in the vascular structure and classified as intramural (im) or intraluminal (il). EVTs, specifically expressing PRG2, target and invade veins and lymphatics in first trimester decidua basalis since HLA-G+ trophoblast were detected in the vascular wall (intramural EVT, imEVTs) and in the lumen of these vessels (intraluminal EVT, ilEVTs). In total, 276 arteries, 793 veins and 113 lymphatics were analysed. While EVTs contact and invade arteries and veins to a similar extent we found that lymphatics are significantly less affected by EVTs (P = 0.001). Moreover, ilEVTs were detected in the lumen of venous and lymphatic vessels, whereas ilEVTs were only found occasionally in the lumen of arteries. Interestingly, RSA tissue sections contained significantly more arterial (P = 0.037), venous (P = 0.002) and lymphatic vessels (P < 0.001), compared to healthy controls. However, while RSA-associated arterial remodeling was unchanged (P = 0.39) the ratios of EVT-affected versus total number of veins (P = 0.039) and lymphatics (P < 0.001) were significantly lower in RSA compared to age-matched healthy decidual sections. Finally, HLA-G+/PRG2+/CD45-EVTs can be detected in regional lymph nodes of pregnant women diagnosed with cervical cancer. N/A. In this study, first trimester decidual tissues from elective terminations of pregnancies have been examined and used as a reference for healthy pregnancy. However, this collective may also include pregnancies which would have developed placental disorders later in gestation. Due to limitations in tissue availability our staining results for EVT-specific marker expression in regional lymph nodes of pregnant women are based on four cases only. In this study, we propose migration of HLA-G+ cells into regional lymph nodes during pregnancy suggesting that the human EVT is capable of infiltrating maternal tissues via the blood stream. Moreover, the description of compromised EVT invasion into the venous and lymphatic vasculature in RSA may help to better understand the pathological characteristics of idiopathic recurrent pregnancy loss. This study was supported by the Austrian Science Fund (grant P-25187-B13 to J.P. and grant P-28417-B30 to M.K.). There are no competing interests to declare.

Windsperger K ，Dekan S ，Pils S ，Golletz C ，Kunihs V ，Fiala C ，Kristiansen G ，Knöfler M ，Pollheimer J ... -  《-》

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 ... -  《-》

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

Trophoblast 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. ADAM28 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. Within 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. Our 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.

De Luca LC ，Le HT ，Mara DL ，Beristain AG ... -  《-》

Dynamic changes in hyperglycosylated human chorionic gonadotrophin throughout the first trimester of pregnancy and its role in early placentation.

What is the in situ localization and function of hyperglycosylated hCG (hCG-H) in first trimester pregnancy tissues? HCG-H localizes to the syncytiotrophoblast, cytotrophoblast and invasive extravillous trophoblast within the maternal decidua and promotes invasion during the first trimester of pregnancy. Serum levels of hCG-H decline dramatically throughout the first trimester of pregnancy. As hCG-H is produced by choriocarcinoma cells, it is proposed to regulate trophoblast invasion. Tissues were collected from elective first trimester pregnancy terminations. Placental villous and decidua basalis were collected from Week 6 to Week 12 of gestation (n = 49). Tissues were collected from elective first trimester surgical pregnancy terminations to determine localization, abundance and function of hCG-H. Placental villous outgrowth studies determined the impact of neutralizing endogenous hCG-H on trophoblast function. Real-time proliferation, migration and invasion assays using JEG-3 choriocarcinoma cells further elucidated the role of hCG-H in trophoblast function. HCG-H localized to syncytiotrophoblast layer of the placental villous from gestational weeks 6-9; thereafter hCG-H localized as a discrete layer between syncytio- and cyto-trophoblast layers. Immunoreactive hCG-H was also observed within the cytotrophoblast layer in Week 7-8 of gestation. HCG-H abundance decreased within placental villous from Weeks 6-12 of gestation (n = 3 placentas per gestational weeks 6-12). HCG-H also localized to anchoring villi within maternal decidua, extravillous trophoblasts invading into the maternal decidua and endovascular trophoblasts remodeling maternal blood vessels. Treatment of primary first trimester villous explants with hCG-H neutralizing antibody reduced trophoblast outgrowth (n = 3 placentas, P < 0.05). Treatment of a trophoblast cell line with neutralizing antibody reduced trophoblast invasion (n = 4, P < 0.05) but did not affect migration or proliferation. Functional invasion and migration assays performed using cell lines. Not possible to perform such assays with primary human material. HCG-H is an important autocrine factor facilitating trophoblast invasion in the first trimester of pregnancy. Targeting hCG-H may prove useful in the treatment of pathologic pregnancies, such as ectopic pregnancies, or pregnancy complications including pre-eclampsia and gestational trophoblast diseases. This work was supported by the Victorian Government Operational Infrastructure Support Program. J.E. is supported by NHMRC project grant #1047756, L.A.S. and E.D. by NHMRC Fellowships #1002018 and #550905 respectively and E.M. by an NHMRC Early Career Fellowship #611827. The authors have no conflicts of interest relating to this work.

Evans J ，Salamonsen LA ，Menkhorst E ，Dimitriadis E ... -  《-》