Successful implantation and placentation requires that extravillous cytotrophoblast acquires an endovascular phenotype and remodels uterine spiral arteries. Defects in this mechanism correlate with severe obstetric complications as implantation failure and preeclampsia. Lysophosphatidic acid (LPA) participates in embryo implantation and contributes to vascular physiology in different biological systems. However, the role of LPA on trophoblast endovascular transformation has not been studied. Due to difficulties in studying human pregnancy in vivo, we adopted a pharmacological approach in vitro to investigate LPA action in various aspects of trophoblast endovascular response, such as the formation of endothelial capillary-like structures, migration, and proliferation. The HTR-8/SVneo cell line established from human first trimester cytotrophoblast was used to model the acquisition of the endovascular phenotype by the invading trophoblast. LPA increased HTR-8/SVneo tube formation, migration (wound healing assay and phalloidin staining) and proliferation (MTT assay). LPA G protein-coupled receptors, LPA 1 and LPA 3 , were expressed in HTR-8/SVneo. By using selective antagonists, we showed that enhanced tubulogenesis was mediated by LPA 3 . In addition, cyclooxygenase-2 and inducible nitric oxide synthase pathways participated in LPA-stimulated tubulogenesis. Inducible nitric oxide synthase was activated downstream cyclooxygenase-2. Furthermore, prostaglandin E2 and a nitric oxide donor (SNAP) increased trophoblast tube formation in a concentration-dependent manner. Finally, we observed that cyclooxygenase-2 and inducible nitric oxide synthase were localized in the nucleus, and LPA did not modify their cellular distribution. Our results show that LPAtriggered regulatory pathways promote trophoblast endovascular response in vitro, suggesting a new role for LPA during spiral artery remodeling at the maternal-fetal interface. K E Y W O R D Scyclooxygenase-2, endovascular trophoblast, inducible nitric oxide synthase, LPA 3 receptor, lysophosphatidic acid 758 |

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Spiral artery remodeling at the maternal-fetal interface is crucial for successful pregnancy and requires the interaction between the first trimester trophoblast and the endothelial cells of the maternal vessels. However, the precise mechanism of this dialog has yet to be determined. The current study investigated whether lysophosphatidic acid (LPA) modulates trophoblast-endothelial crosstalk in vitro. HTR-8/ SVneo trophoblast cell line (H8) was seeded on top of Geltrex, incubated with LPA or LPA + NS-398 (selective cyclooxygenase-2 inhibitor), LPA + 1400W (selective inducible nitric oxide synthase inhibitor) or LPA + IL-6 neutralizing antibody and assayed for tube formation to model the acquisition of trophoblast endovascular phenotype. The supernatants were collected and used as conditioned media (CM). To test trophoblast-endothelial crosstalk, the endothelial cell line EA.hy926 was incubated with trophoblast CM. The CM from LPA-induced tubulogenesis stimulated endothelial cells migration and did not modify the apoptosis. Soluble factors derived from cyclooxygenase-2 and IL-6 pathways were involved in H8-EA.hy926 interaction under the LPA effect. Moreover, LPA increased the levels of IL-6 mRNA by cyclooxygenase-2 pathway in H8 cells. Collectively, LPA promotes trophoblastendothelial crosstalk in vitro and induces the release of trophoblast soluble factors that stimulate endothelial cells migration without changes in apoptosis. The evidence presented here provides new insights about an active role of LPA as a lipid mediator regulating vascular remodeling at the maternal-fetal interface. K E Y W O R D S cyclooxygenase-2, endothelial cells, human first trimester trophoblast, IL-6, LPA, metalloproteases 1 | INTRODUCTIONThe coordination of vascular processes at the maternal-fetal interface is crucial for the maintenance of gestation and requires a profound reorganization of uterine and fetal tissues. In spiral artery remodeling, the extravillous cytotrophoblast induces the loss of the endothelium of the maternal blood vessels through a mechanism that is coordinated spatio-temporally (Choudhury et al., 2017;Whitley & Cartwright, 2009. This process begins with a trophoblast independent stage and prepares the endometrium for trophoblast invasion. Then, the extravillous cytotrophoblast invades the maternal spiral arteries, and replaces endothelial and muscle layers, whereas acquiring an endovascular phenotype. These dramatic changes conclude when the endovascular trophoblast encloses the vessels and surrounds them with fibrin deposits, temporarily replacing the endothelium with a trophoblast layer.