Philipp Velicky

Control of human endometrial stromal cell motility by PDGF-BB, HB-EGF and trophoblast-secreted factors.

Human implantation involves extensive tissue remodeling at the fetal-maternal interface. It is becoming increasingly evident that not only trophoblast, but also decidualizing endometrial stromal cells are inherently motile and invasive, and likely contribute to the highly dynamic processes at the implantation site. The present study was undertaken to further characterize the mechanisms involved in the regulation of endometrial stromal cell motility and to identify trophoblast-derived factors that modulate migration. Among local growth factors known to be present at the time of implantation, heparin-binding epidermal growth factor-like growth factor (HB-EGF) triggered chemotaxis (directed locomotion), whereas platelet-derived growth factor (PDGF)-BB elicited both chemotaxis and chemokinesis (non-directed locomotion) of endometrial stromal cells. Supernatants of the trophoblast cell line AC-1M88 and of first trimester villous explant cultures stimulated chemotaxis but not chemokinesis. Proteome profiling for cytokines and angiogenesis factors revealed neither PDGF-BB nor HB-EGF in conditioned media from trophoblast cells or villous explants, while placental growth factor, vascular endothelial growth factor and PDGF-AA were identified as prominent secretory products. Among these, only PDGF-AA triggered endometrial stromal cell chemotaxis. Neutralization of PDGF-AA in trophoblast conditioned media, however, did not diminish chemoattractant activity, suggesting the presence of additional trophoblast-derived chemotactic factors. Pathway inhibitor studies revealed ERK1/2, PI3 kinase/Akt and p38 signaling as relevant for chemotactic motility, whereas chemokinesis depended primarily on PI3 kinase/Akt activation. Both chemotaxis and chemokinesis were stimulated upon inhibition of Rho-associated, coiled-coil containing protein kinase. The chemotactic response to trophoblast secretions was not blunted by inhibition of isolated signaling cascades, indicating activation of overlapping pathways in trophoblast-endometrial communication. In conclusion, trophoblast signals attract endometrial stromal cells, while PDGF-BB and HB-EGF, although not identified as trophoblast-derived, are local growth factors that may serve to fine-tune directed and non-directed migration at the implantation site.

Notch Signaling Plays a Critical Role in Motility and Differentiation of Human First-Trimester Cytotrophoblasts

Failures in human extravillous trophoblast (EVT) development could be involved in the pathogenesis of pregnancy diseases. However, the underlying mechanisms have been poorly characterized. Here, we provide evidence that Notch signaling could represent a key regulatory pathway controlling trophoblast proliferation, motility, and differentiation. Immunofluorescence of first-trimester placental tissues revealed expression of Notch receptors (Notch2 and Notch3) and membrane-anchored ligands (delta-like ligand [DLL] 1 and -4 and Jagged [JAG] 1 and -2) in villous cytotrophoblasts (vCTBs), cell column trophoblasts (CCTs), and EVTs. Notch4 and Notch1 were exclusively expressed in vCTBs and in CCTs, respectively. Both proteins decreased in Western blot analyses of first-trimester, primary cytotrophoblasts (CTBs) differentiating on fibronectin. Luciferase reporter analyses suggested basal, canonical Notch activity in SGHPL-5 cells and primary cells that was increased upon seeding on DLL4-coated dishes and diminished in the presence of the Notch/γ-secretase inhibitors N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT) or L-685,458. Bromodeoxyuridine labeling, cyclin D1 mRNA expression, and cell counting indicated that chemical inhibition of Notch signaling elevated proliferation in the different primary trophoblast model systems. Notch inhibition also increased motility of SGHPL-5 cells through uncoated and fibronectin-coated Transwells, motility of primary CTBs, as well as migration in villous explant cultures on collagen I. Accordingly, small interfering RNA-mediated gene silencing of Notch1 also elevated SGHPL-5 cell migration. In contrast, motility of primary cultures and SGHPL-5 cells was diminished in the presence of DLL4. Moreover, DAPT increased markers of differentiated EVT, ie, human leukocyte antigen G1, integrin α5, and T-cell factor 4, whereas DLL4 provoked the opposite. In summary, the data suggest that canonical Notch signaling impairs motility and differentiation of first-trimester CTBs.

Function and control of human invasive trophoblast subtypes: Intrinsic vs. maternal control.

ABSTRACT The establishment of a functional placenta is pivotal for normal fetal development and the maintenance of pregnancy. In the course of early placentation, trophoblast precursors differentiate into highly invasive trophoblast subtypes. These cells, referred to as extravillous trophoblasts (EVTs), penetrate the maternal uterus reaching as far as the inner third of the myometrium. One of the most fundamental functions of EVTs is the transformation of spiral arteries to establish the uteroplacental blood circulation assuring an adequate nutrient and gas supply to the developing fetus. To achieve this, specialized EVT subpopulations interact with maternal immune cells, provoke elastolysis in the arterial wall and replace the endothelial cells lining the spiral arteries to induce intraluminal vascular remodeling. These and other trophoblast-mediated processes are tightly controlled by paracrine signals from the maternal decidua and furthermore underlie an intrinsic cell-type specific program. Various severe pregnancy complications such as preeclampsia or intrauterine growth retardation are associated with abnormal EVT function, shallow invasion, and decreased blood flow to the placenta. Hence a better understanding of human trophoblast invasion seems mandatory to improve therapeutic intervention. This approach, however, requires a profound knowledge of the human placenta, its various trophoblast subtypes and in particular a better understanding of the regulatory network that controls the invasive phenotype of EVTs.

Extravillous Trophoblast-Associated ADAM12 Exerts Pro-Invasive Properties, Including Induction of Integrin Beta 1-Mediated Cellular Spreading

ABSTRACT ADAM12, consisting of a membrane-bound (ADAM12L) and a secreted (ADAM12S) form, is expressed exclusively in regenerating and developing tissue as well as in certain cancer types. Strong ADAM12 expression levels have been noticed in the human placenta, and deregulated ADAM12S levels were associated with various pregnancy-related disorders including pre-eclampsia and intrauterine growth restriction. However, the role of ADAM12 in trophoblast motility has not been investigated so far. Hence, the present study aimed to investigate the specific function of the protease by using different primary trophoblast cell models. Immunofluorescence and Western blot analyses of first trimester placental tissue and differentiating primary first trimester cytotrophoblasts (CTBs) indicated strong upregulation of both of the ADAM12 isoforms during extravillous trophoblast differentiation. Functional assays involving short interfering RNA (siRNA)-mediated knockdown studies in primary CTBs and first trimester explant cultures revealed a significant repression of trophoblast motility upon partial loss of ADAM12. Conversely, isoform-specific overexpression in the ADAM12-negative trophoblast cell line SGHPL-5 enhanced the invasive capacity of these cells. We further confirmed proteolytic activity of trophoblast-derived ADAM12S by demonstrating its potential to degrade insulin-like growth factor-binding protein 3. Finally, we suggest that ADAM12S exerts its pro-migratory function in trophoblasts by inducing integrin beta 1-mediated cellular spreading.

Chymase-producing cells of the innate immune system are required for decidual vascular remodeling and fetal growth

Intrauterine growth restriction (IUGR) is caused by insufficient remodeling of spiral arteries (SAs). The mechanism underlying the relevance of natural killer cells (NKs) and mast cells (MCs) for SA remodeling and its effects on pregnancy outcome are not well understood. We show that NK depletion arrested SA remodeling without affecting pregnancy. MC depletion resulted in abnormally remodeled SAs and IUGR. Combined absence of NKs and MCs substantially affected SA remodeling and impaired fetal growth. We found that α-chymase mast cell protease (Mcpt) 5 mediates apoptosis of uterine smooth muscle cells, a key feature of SA remodeling. Additionally, we report a previously unknown source for Mcpt5: uterine (u) NKs. Mice with selective deletion of Mcpt5+ cells had un-remodeled SAs and growth-restricted progeny. The human α-chymase CMA1, phylogenetic homolog of Mcpt5, stimulated the ex vivo migration of human trophoblasts, a pre-requisite for SA remodeling. Our results show that chymases secreted by uMCs and uNKs are pivotal to the vascular changes required to support pregnancy. Understanding the mechanisms underlying pregnancy-induced vascular changes is essential for developing therapeutic options against pregnancy complications associated with poor vascular remodeling.

Notch2 controls prolactin and insulin-like growth factor binding protein-1 expression in decidualizing human stromal cells of early pregnancy.

Decidualization, the transformation of the human uterine mucosa into the endometrium of pregnancy, is critical for successful implantation and embryonic development. However, key regulatory factors controlling differentiation of uterine stromal cells into hormone-secreting decidual cells have not been fully elucidated. Hence, we herein investigated the role of the Notch signaling pathway in human decidual stromal cells (HDSC) isolated from early pregnancy samples. Immunofluorescence of first trimester decidual tissues revealed expression of Notch2 receptor and its putative, membrane-anchored interaction partners Jagged1, Delta-like (DLL) 1 and DLL4 in stromal cells whereas other Notch receptors and ligands were absent from these cells. During in vitro differentiation with estrogen/progesterone (E2P4) and/or cyclic adenosine monophosphate (cAMP) HDSC constitutively expressed Notch2 and weakly downregulated Jagged1 mRNA and protein, measured by quantitative PCR (qPCR) and Western blotting, respectively. However, increased levels of DLL1 and DLL4 were observed in the decidualizing cultures. Transfection of a Notch luciferase reporter and qPCR of the Notch target gene hairy and enhancer of split 1 (HES1) revealed an induction of canonical Notch activity during in vitro differentiation. In contrast, treatment of HDSC with a chemical Notch/γ-secretase inhibitor decreased cAMP/E2P4-stimulated Notch luciferase activity, HES1 transcript levels and mRNA expression of the decidual marker genes prolactin (PRL) and insulin-like growth factor binding protein 1 (IGFBP1). Similarly, siRNA-mediated gene silencing or antibody-mediated blocking of Notch2 diminished HES1, PRL and IGFBP1 mRNA levels as well as secreted PRL protein. In summary, the data suggest that canonical, Notch2-dependent signaling plays a role in human decidualization.

Notch-dependent RBPJκ inhibits proliferation of human cytotrophoblasts and their differentiation into extravillous trophoblasts

Abnormal development of invasive trophoblasts has been implicated in the pathogenesis of human pregnancy diseases such as pre-eclampsia. However, critical signalling pathways controlling formation and differentiation of these cells have been poorly elucidated. Here, we provide evidence that the canonical Notch pathway, operating through Notch-dependent activation of its key regulatory transcription factor RBPJκ, controls proliferation and differentiation in villous explant cultures and primary trophoblasts of early pregnancy. Immunofluorescence of first trimester placental tissue revealed expression of RBPJκ and its co-activators, the MAML proteins, in nuclei of proliferative cell column trophoblasts (CCT) and differentiated, extravillous trophoblasts (EVTs). However, RBPJκ expression, transcript levels of the Notch target gene HES1 and activity of a Notch/RBPJκ-dependent luciferase reporter decreased during in vitro differentiation of primary cytotrophoblasts on fibronectin. Silencing of RBPJκ using silencing RNAs (siRNAs) increased proliferation of CCTs in floating villous explant cultures analysed by outgrowth and BrdU labelling. Similarly, down-regulation of the transcription factor enhanced BrdU incorporation in isolated primary cultures. However, motility of these cells was not affected. In addition, gene silencing of RBPJκ increased cyclin D1 expression in the two trophoblast model systems as well as markers of the differentiated, EVT, i.e. integrin α1, ADAM12 and T-cell factor 4. In summary, the data suggest that Notch-dependent RBPJκ activity could be required for balanced rates of trophoblast proliferation and differentiation in human placental anchoring villi preventing exaggerated trophoblast overgrowth as well as premature formation of EVTs.

Endothelin-1 down-regulates matrix metalloproteinase 14 and 15 expression in human first trimester trophoblasts via endothelin receptor type B

STUDY QUESTION Does endothelin-1 (ET-1) regulate matrix metalloproteinase (MMP) 14 and 15 production and invasion of human first trimester trophoblasts? SUMMARY ANSWER ET-1 in pathophysiological concentrations down-regulates MMP14 and MMP15 expression via endothelin receptor (ETR) type B and decreases trophoblast migration and invasion. WHAT IS KNOWN ALREADY MMP14 and MMP15 are involved in trophoblast invasion. Impairment of invasion has been linked to pregnancy complications such as pre-eclampsia (PE). ET-1 is up-regulated in PE. STUDY DESIGN, SIZE, DURATION In vitro study using primary human trophoblasts from 50 first trimester placentas (gestational week 7–12). PARTICIPANTS/MATERIALS, SETTING, METHODS Trophoblasts were cultured in the absence or presence of 10–100 nM ET-1. MMP14 and MMP15 mRNA and protein were quantified by RT-qPCR and Western blotting, respectively. Selective antagonists for ETRA (BQ-123) or ETRB (BQ-788) were used to identify ETR subtypes involved. Functional ET-1 effects were tested in first trimester chorionic villous explants and transwell invasion assays. The roles of tumor necrosis factor (TNF)-&agr; (25 ng/ml) and oxygen (1%) in ET-1 regulation of MMP14 and 15 expression were assessed by Western blotting. MAIN RESULTS AND THE ROLE OF CHANCE ET-1 down-regulated MMP14 and MMP15 mRNA (−21% and −26%, respectively, P < 0.05) and protein levels (–18% and –22%, respectively, P < 0.05). This effect was mediated via ETRB. ET-1 decreased trophoblast outgrowth in placental explants (−24%, P < 0.05) and trophoblast invasion (−26%, P ⩽ 0.01). TNF-&agr; enhanced ET-1 mediated MMP15 down-regulation (by 10%, P < 0.05), whereas hypoxia abolished the effect of ET-1 on both MMPs. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION Only primary trophoblasts were used in this study. Since trophoblast yield from first trimester placental material is limited, further aspects of MMP14 and 15 regulation could not be characterized. Other anti-invasive factors may be altered by ET-1 in trophoblasts and, thus, contribute to the reduced invasion, but have not been investigated. Oxygen levels similar to those found in the decidua (5–8% O2) were not analyzed in this study. WIDER IMPLICATIONS OF THE FINDINGS ET-1 modifies placental function already during the first trimester of pregnancy, the time-window when the placental changes implicated in PE occur. Thus, our results improve the understanding of the placental mechanisms underlying trophoblast invasion and PE. STUDY FUNDING/COMPETING INTEREST(S) The study was funded by the Oesterreichische Nationalbank (Anniversary Fund, project number: 14796) and the Herzfeldersche Familienstiftung (to J.P.; number: 00685). AMM received funding from the Austrian Science Fund FWF (W1241) and the Medical University Graz through the PhD Program Molecular Fundamentals of Inflammation (DK-MOLIN). The authors have no conflict of interest.

Wingless ligand 5a is a critical regulator of placental growth and survival

The maternal uterine environment is likely critical for human placental morphogenesis and development of its different trophoblast subtypes. However, factors controlling growth and differentiation of these cells during early gestation remain poorly elucidated. Herein, we provide evidence that the ligand Wnt5a could be a critical regulator of trophoblast proliferation and survival. Immunofluorescence of tissues and western blot analyses of primary cultures revealed abundant Wnt5a expression and secretion from first trimester decidual and villous stromal cells. The ligand was also detectable in decidual glands, macrophages and NK cells. Wnt5a increased proliferation of villous cytotrophoblasts and cell column trophoblasts, outgrowth on collagen I as well as cyclin A and D1 expression in floating explant cultures, but suppressed camptothecin-induced apoptosis. Similarly, Wnt5a stimulated BrdU incorporation and decreased caspase-cleaved cytokeratin 18 neo-epitope expression in primary cytotrophoblasts. Moreover, Wnt5a promoted activation of the MAPK pathway in the different trophoblast models. Chemical inhibition of p42/44 MAPK abolished cyclin D1 expression and Wnt5a-stimulated proliferation. Compared to controls, MAPK phosphorylation and proliferation of cytotrophoblasts declined upon supplementation of supernatants from Wnt5a gene-silenced decidual or villous stromal cells. In summary, non-canonical Wnt5a signalling could play a role in early human trophoblast development by promoting cell proliferation and survival.

IFPA Meeting 2012 Workshop Report III: Trophoblast deportation, gestational trophoblastic disease, placental insufficiency and fetal growth restriction, trophoblast over-invasion and accreta-related pathologies, placental thrombosis and fibrinolysis

Workshops are an important part of the IFPA annual meeting as they allow for discussion of specialized topics. At IFPA meeting 2012 there were twelve themed workshops, five of which are summarized in this report. These workshops related to various aspects of placental biology but collectively covered areas of clinical research and pregnancy disorders: 1) trophoblast deportation; 2) gestational trophoblastic disease; 3) placental insufficiency and fetal growth restriction; 4) trophoblast overinvasion and accreta-related pathologies; 5) placental thrombosis and fibrinolysis.