Placental Expression of Slit/Robo Signaling: Implication in Angiogenesis and Preeclampsia.

Abstract

The Slit/Robo signaling system was initially identified as an extracellular cue to guide axon pathfinding, to promote axon branching, and to control neuronal migration. Recent studies showed that it also regulates tumor angiogenesis. However, the role of this system in physiological angiogenesis especially in the placenta is not known. We are interested in the systemic expression and function of this signaling pathway in placental biology, with an emphasis on placental angiogenesis. We first determined the expression pattern of ligands (Slits) and receptors (Robo) in term human placental tissues and cultured primary ovine fetal placental artery endothelial cells (oFPAEC), human umbilical cord vein endothelial cells (HUVEC), human primary villous trophoblasts, and the human transformed trophoblast cell lines (i.e., BeWo and JEG3). Expression of Slits/Robo mRNA was determined by reverse-transcription-polymerase chain reaction (RT-PCR) and protein levels were determined by immunoblotting with specific antibodies. Paraffiln-embedded sections of term human placental tissues were used for immunohistochemical (IHC) analysis. Among the known mammalian Slits (1-3) and Robos (1-4), Slit3, Robo1 and Robo4 mRNAs are highly expressed in oFPAEC and HUVEC, and Slit2 mRNA was mainly expressed in human primary villous trophoblastic and the transformed BeWo, but not JEG3, cells. Immunoblotting and IHC confirmed their protein expression patterns. The co-expression of Slit and Robo in the placental vasculature and the neighboring trophoblast cells suggests that this signaling pathway may participate in the regulation of endothelial cell function including angiogenesis in the placenta. Furthermore, we measured the relative mRNA expression levels of the Slit/Robo molecules in term placental tissues of normal and severe preeclamptic pregnancies (n = 6/gorup). Real time RT-PCR revealed that the mRNA levels of Slit2, Robo 1 and Robo4 in human placental tissues are 30-35% lower in preeclamptic than normal pregnancy (P= 0.187, 0.136, 0.342, respectively), whereas no difference was observed in Slit3 mRNA (P=0.903). Both Slit2 and Robo1 were immunolocalized in human placental villous capillary endothelial and trophoblast cells. Thus, the differential expression patterns of the Slit and Robo molecules in the placental cells suggest that this signaling system may participate in placental endothelial and trophoblast cell cross-talk towards angiogenesis. Dysfunctional regulation of this signaling pathway may contribute to the eitiology of preeclampsia (Supported by NIH grants and a UCSD Academic Senate Grant).