A physiological concentration of anandamide promotes the migration of human endometrial fibroblast and the interaction with endothelial cells invitro.

INTRODUCTION: The mechanisms that govern fibroblast behavior during the vascular adaptations of the uterus at early pregnancy remain unknown. Anandamide, an endocannabinoid, binds to cannabinoid receptors (CBs), and regulates gestation and angiogenesis. Its tone is regulated by fatty acid amide hydrolase (FAAH) within the uterus. We investigated the role of anandamide in endometrial fibroblasts migration and whether anandamide modulates fibroblasts-endothelial crosstalk. METHODS: T-hESC and EA.hy926 cell lines were used as models of endometrial stromal and endothelial cells, respectively. T-hESC were incubated with anandamide plus different agents. Migration was tested (wound healing assay and phalloidin staining). Protein expression and localization were studied by Western blot and immunofluorescence. To test fibroblast-endothelial crosstalk, EA.hy926 cells were incubated with fibroblast conditioned media obtained after T-hESC migration. RESULTS: Anandamide 1 nM increased T-hESC migration via CB1 and CB2. Cyclooxygenase-2 participated in anandamide-stimulated fibroblast migration. Prostaglandin F2alpha, and not prostaglandin E2, increased fibroblast wound closure. CB1, CB2, cyclooxygenase-2 and FAAH were expressed in T-hESC. Anandamide did not alter cyclooxygenase-2 localization but induced its cytoplasmic and nuclear expression through CB1 and CB2. URB-597, a FAAH selective inhibitor, also increased T-hESC migration via both CBs, and augmented cyclooxygenase-2 expression. Conditioned media from anandamide-induced T-hESC wound healing closure stimulated endothelial migration and did not alter their proliferation. Soluble factors from cyclooxygenase-2 were secreted by T-hESC and participated in T-hESC-induced EA.hy926 migration. Although anandamide-conditioned media augmented in EA.hy926 the expression of γH2AX, a marker of DNA damage, cyclooxygenase-2 was not involved in this effect. DISCUSSION: Our results provide novel evidence about an active role of anandamide on endometrial fibroblast behavior as a mechanism regulating uterine vascular adaptations in early gestation.

Novel role for lysophosphatidic acid in vascular remodeling at the maternal-fetal interface.

Lysophosphatidic acid (LPA) belongs to the group of phosphorylated lipids reported as crucial mediators in the physiology of reproduction. LPA binds to G-protein-coupled receptors and regulates a wide range of female reproductive functions. This bioactive lipid has also been implicated in vascular functions during physiological and pathological conditions. In this regard, the establishment of a successful pregnancy requires proper coordination of vascular processes and remodeling of maternal blood vessels during early gestation. During this process, first trimester cytotrophoblast changes from an invasive to an endovascular phenotype and transforms uterine spiral arteries which are the nutrient supply for placenta and fetus. Here we present an overview of LPA participation in vascular remodeling and highlight the importance of LPA-LPA3 signaling during early gestation at the maternal-fetal interface.

Lysophosphatidic acid induces the crosstalk between the endovascular human trophoblast and endothelial cells in vitro.

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 trophoblast-endothelial 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.

Steroid hormones induce in vitro human first trimester trophoblast tubulogenesis by the lysophosphatidic acid pathway.

Successful implantation and placentation requires that extravillous cytotrophoblast acquires an endovascular phenotype and remodels uterine spiral arteries. Progesterone (P4) and estradiol (E2) control many of the placental functions, but their role in vascular remodeling remains controversial. Here, we investigated whether P4 and E2 regulate the acquisition of the human first trimester trophoblast endovascular phenotype, and the participation of the lysophosphatidic acid pathway. For this purpose, human first trimester HTR-8/SVneo cells were seeded on Geltrex and assayed for capillary-like tube formation. P4 and E2 increased HTR-8/SVneo tube formation in a concentration-dependent manner and this effect is mediated by the LPA3 receptor. Moreover, sex steroids increased the mRNA levels of the main enzyme that produce lysophosphatidic acid (lysophospholipase-D) but did not regulate LPA3 mRNA levels. Overall, we demonstrate that steroid hormones regulate HTR-8/SVneo trophoblast capillary-like structures formation and we propose that this process could be modulated directly or indirectly by mechanisms associated to the LPA/LPA3 pathway.

Lysophosphatidic acid-triggered pathways promote the acquisition of trophoblast endovascular phenotype in vitro.

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, LPA1 and LPA3 , were expressed in HTR-8/SVneo. By using selective antagonists, we showed that enhanced tubulogenesis was mediated by LPA3 . 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 LPA-triggered regulatory pathways promote trophoblast endovascular response in vitro, suggesting a new role for LPA during spiral artery remodeling at the maternal-fetal interface.