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First published online 17 August 2005
doi: 10.1242/dev.01971

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EPHB4 regulates chemokine-evoked trophoblast responses: a mechanism for incorporating the human placenta into the maternal circulation

¹ Biomedical Sciences Graduate Program, University of California San Francisco, San Francisco, CA 94143, USA
² Department of Cell and Tissue Biology, University of California San Francisco, San Francisco, CA 94143, USA
³ Department of Physiology, Universiti Sains Malaysia, Kelantan, Malaysia
⁴ Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA 94143, USA
⁵ Department of Anatomy, University of California San Francisco, San Francisco, CA 94143, USA

View larger version (93K): [in a new window]   Fig. 1. Placental cytotrophoblasts invade the uterine wall where they breach veins and extensively remodel maternal spiral arterioles. The bulk of the placenta is composed of numerous tree-like projections termed chorionic villi where maternal-fetal exchange occurs. These structures mediate the passage of nutrients, gases and wastes between fetal blood, which circulates through the villous core, and maternal blood, which circulates through the intervillous space. The uteroplacental circulation is established by cytotrophoblasts that acquire an invasive/endothelial phenotype as they leave the placenta and enter the uterine wall. Differentiation begins when cytotrophoblast progenitors that reside in a single layer surrounding the stromal core of anchoring villi proliferate and form a cell column. These structures attach to the uterine wall and give rise to cells that invade the underlying decidual stroma. Invasive cytotrophoblasts breach uterine blood vessels connecting both the arterial and the venular circulation to the intervillous space. However, once this connection is made, remodeling of the venous side is halted. By contrast, cytotrophoblasts migrate up the lumina of spiral arterioles, eventually replacing the endothelial lining of the vessels and part of the muscular wall. This process encompasses the decidual and inner third of the myometrial segments of these vessels. NK, natural killer; m, macrophage.

View larger version (73K): [in a new window]   Fig. 2. Chemokines stimulate cytotrophoblast migration. (A) Cytokeratin 7 (CK7) (red) and CXCL16 (green) expression in the region of the maternal-fetal interface shown in Fig. 1. (A) CXCL16-specific antibody stained invasive and endovascular cytotrophoblasts (CTBs), identified by their cytokeratin 7 expression. The nuclei of cells, which were stained with DAPI, appear blue. IVS, intervillous space; AV, anchoring villi. (B) Pertussis toxin (PTX) or a CXCR4-blocking antibody decreased cytotrophoblast migration required for aggregation in vitro. (C) In a migration assay, the addition of individual chemokines (CXCL12, CXCL16 and CCL21) stimulated cytotrophoblast migration in a dose-dependent manner, and the effects were additive. Error bars indicate s.d. (D) Checkerboard analysis in which the chemokines were added to either the upper, lower or both chambers revealed that these molecules stimulated cytotrophoblast migration by increasing chemokinesis. Scale bars: 100 µm.

View larger version (109K): [in a new window]   Fig. 3. Coincident with exit from the placental compartment, cytotrophoblasts switch from a venous to an arterial phenotype, as shown by their modulation of EPH and ephrin family members. (A-F) In situ hybridization; adjacent sections were stained with cytokeratin 7 (CK7), which identified trophoblasts. Venular and arterial endothelial cells (ECs) that lined uterine vessels expressed EPHB4 (A) and ephrin B2 (B). (C) Cytotrophoblast progenitors (CTB prog.) and syncytiotrophoblasts (STB) within the placenta expressed EPHB4. Commitment to the differentiation pathway that gives rise to invasive cytotrophoblasts (iCTBs) is associated with an abrupt downregulation of EPHB4 and a concomitant upregulation of ephrin B1 (D) and EPHB2 (E) mRNAs. Subsequently, within the uterine stroma, ephrin B2 expression is induced (F). The endovascular subpopulation of invasive cytotrophoblasts that line maternal arteries also expressed high levels of these molecules (D-F). (G) Schematic diagram of the human maternal-fetal interface highlighting important aspects of cytotrophoblast interactions with uterine vessels. The pattern of cytotrophoblast EPH and ephrin expression is shown in the colors indicated. (H) Cytotrophoblasts modulated EPH and ephrin expression during differentiation along the invasive pathway in vitro. Immediately after isolation from placental chorionic villi, the progenitors expressed EPHB4, but upregulated ephrin B1, B2 and EPHB2 after 12 hours in culture. IVS, intervillous space; T, trimester. Scale bars: 50 µm in A,B,F; 100 µm in C-E.

View larger version (91K): [in a new window]   Fig. 4. Cytotrophoblasts avoid substrates containing EPHB4, but not ephrin B2. Laminin with either ephrin B2-Fc (A) or EPHB4-Fc (B) and fluorescent anti-human Fc was spotted on the plate before the entire surface was covered with laminin alone. (C,D) Cytokeratin 7 staining showed cytotrophoblast distribution on the tissue culture substrate. Initially, cytotrophoblasts were plated as a monolayer. (C) By 12 hours, the cells continued to associate with ephrin B2-Fc substrates, but (D) few cells were attached to EPHB4-Fc spots. Scale bar: 200 µm.

View larger version (34K): [in a new window]   Fig. 5. Cytotrophoblast migration is reduced by co-culture with 3T3 cells that express the venous receptor EPHB4. (A) Staining of stably transfected 3T3 cell lines with antibodies specific for EPHB4 and ephrin B2 showed that they expressed the respective proteins. (B,D) Our previous work (Librach et al., 1991) showed that isolated cytotrophoblasts cultured on laminin or Matrigel substrates rapidly migrate toward one another, forming numerous aggregates. Similar behavior was observed when the cytokeratin-positive (CK7) cytotrophoblasts (green) were co-cultured with control 3T3 cells. (C,E) By contrast, when cytotrophoblasts were cultured with 3T3 cells expressing EPHB4 (3T3-EPHB4), aggregate formation was markedly inhibited, and the cells appeared to spread on the substrate. Tracking cell movement for 15 hours showed that, in comparison with controls (F), cytotrophoblasts co-cultured with 3T3-EPHB4 cells migrated much shorter distances (G). (H) Quantifying the average linear distance traveled showed that migration was significantly reduced when the cytotrophoblasts were co-cultured with 3T3-EPHB4 cells on either laminin or Matrigel substrates. Error bars indicate s.d. Scale bars: 20 µm in A; in B, 50 µm for B,C; in D, 10 µm for D,E.

View larger version (44K): [in a new window]   Fig. 6. EPHB4 decreases chemokine-stimulated cytotrophoblast migration. (A) RT-PCR detected expression of PDZ-RGS3 in RNA samples isolated from first trimester (1T) and second trimester (2T) cytotrophoblasts (CTB). PBMCs, peripheral blood mononuclear cells. +, positive control; –, negative control. (B) Cytokeratin 7 staining of cytotrophoblasts that had migrated to the underside of the transwell filter after overnight culture. EPHB4 decreased cytotrophoblast migration towards chemokines, while baseline and growth factor-stimulated migration was not significantly affected. (C) Quantification of migration assays showed that EPHB4 downregulated chemokine-induced migration by an average of 60%. Error bars indicate s.d.

View larger version (26K): [in a new window]   Fig. 7. Model describing the role of EPHs and ephrins in patterning chemokine-induced cytotrophoblast invasion. (A) A rapid switch in EPH and ephrin expression generates repulsive signals that orient cytotrophoblast (CTB) invasion away from the placenta and towards the uterine wall. (B) Within the uterine stroma, invasive cytotrophoblasts distinguish veins from arterioles based on their expression of EPHB4 and ephrin B2, respectively. Our data suggest that cytotrophoblast interactions with EPHB4-expressing cells inhibit their chemokine-induced migration, one mechanism that could restrict their remodeling of veins. By contrast, interactions with ephrin B2-expressing cells permit migration. As a result, cytotrophoblast remodeling of the uterine vasculature is biased toward the arterial side of the circulation.