Antagonism of the Sphingosine 1-Phosphate (S1P) Signaling Pathway in Pregnant Ewes Alters Placentome Architecture, Amino Acid Transport, and Fetal Growth.

Abstract

Angiogenesis is a critical factor in the process of placentation. In ruminants, angiogenesis occurs primarily in placentomes, formed by interdigitation and branching morphogenesis of placental cotyledons and maternal endometrial caruncles, which supply hematotrophic nutrition to the developing conceptus. We hypothesized that the lysosphingolipid sphingosine-1-phosphate (S1P) stimulates angiogenesis within developing sheep placentomes through activation of the S1P receptors (S1P1-S1P5) and that their antagonism would alter placentomal angiogenesis and subsequent uteroplacental nutrient exchange during early to mid pregnancy. In Study 1, passage 4-5 primary ovine uterine artery endothelial cells (oUAEC) from 3 late pregnant sheep were seeded as monolayers onto three dimensional (3D) collagen matrices containing angiogenic growth factors and S1P. S1P dose-dependently (1–1000 nM) enhanced endothelial cell invasion compared to VEGF and FGF2 alone. Using RT-PCR to detect S1P1-S1P5, only S1P1 and S1P3 receptors were found to be expressed in oUAEC. Antagonism of these receptors with FTY720 and VPC 23019 blocked S1P-induced endothelial cell invasion in vitro. In Study 2, we examined potential physiological mechanisms for actions of endogenous S1P in the control of ovine placental angiogensis. Ewes with a single fetus were given daily intravenous injections of FTY720 (n=6; 1.5 mg/day) or vehicle (n=5) from Days 30 to 60 prior to collection of tissues on Day 60. FTY720 treatment altered the histoarchitecture of the placentome as evidenced by decreased stromal area of caruncular crypts and diminished thickness of connective tissue forming the placentomal capsule. Concomitant with these effects on interdigitation of uterine and placental tissues, the total volume of cotyledonary tissue per placentome was greater in FTY720-treated ewes. Immunofluorescence localization of the intermediate filament proteins, cytokeratin and vimentin indicated no overt change in synctial epithelia, whereas stromal tissue thickness and density were decreased with FTY720 treatment. Immunofluorescence localization of alpha-smooth muscle actin and the endothelial marker, von Willebrand Factor, in placentomes revealed that FTY720 treatment increased diameter of the lumen, thickness of the tunica muscularis of vessels, but decreased the total number of vessels in caruncles as compared to controls. FTY720 treatment also affected transport of amino acids across the placentae and fetal development as evidenced by decreases in methionine levels in allantoic fluid (P<0.05), aspartate and glutamate levels in amniotic fluid (P<0.005), and crown-rump length of fetuses (P<0.05), however, total fetal weight was not affected (P>0.05). Results of this study indicates that antagonism of the S1P signaling pathway using FTY720 interfered with placentomal development, including angiogenesis, transport of micronutrients from the uterus to the placenta and fetal growth. We conclude that S1P is a key regulatory molecule responsible for mediating development of the angiogenic network in placentomes of sheep that provide hematatrophic support for the developing conceptus. (Research supported by National Research Initiative Competitive Grants Program, Cooperative State Research, Education, and Extension Service, USDA award 2008-35203-18830 to KAD and AHA Scientist Development Grant #053020N to KJB) (poster)