Human Placental Expression of SLIT/ROBO Signaling Cues: Effects of Preeclampsia and Hypoxia1

Abstract

Preeclampsia is characterized by dysfunctional endothelium and impaired angiogenesis. Recent studies suggest that the neuronal guidance SLIT/ROBO system regulates tumor angiogenesis. This study investigated if SLIT and ROBO are differentially expressed in healthy term and preeclamptic placentas and if hypoxia regulates SLIT and ROBO expression in placental trophoblast and endothelial cells. Total RNA and protein were extracted from placental tissues of healthy term (n = 5) and preeclamptic (n = 6) pregnancies and used for SLIT/ROBO expression analyses with reverse transcription-polymerase chain reaction (RT-PCR), real-time quantitative-PCR, and immunoblotting. Paraffin-embedded tissues were processed to localize SLIT/ROBO proteins in placental villi by immunohistochemistry. BeWo choriocarcinoma cells and human umbilical vein endothelial cells (HUVEC) were treated with 2% or 10% oxygen or the hypoxia mimetic deferoxamine mesylate (100 μM) to test if hypoxia regulates SLIT/ROBO expression. SLIT2, SLIT3, ROBO1, and ROBO4 mRNA and proteins were detected in the placenta. SLIT2 and ROBO1 proteins localized in the syncytiotrophoblast, and SLIT3, ROBO1, and ROBO4 in capillary endothelium of the placental villi. Levels of ROBO1 and ROBO4 as well as sFLT1 (soluble fms-like tyrosine kinase-1) proteins were significantly greater in preeclamptic placentas compared to normal controls. Hypoxia significantly increased both mRNA and protein levels of SLIT2 in BeWo cells and of SLIT3, ROBO1, and ROBB4 in HUVEC. Thus, trophoblast and endothelial coexpression of SLIT/ROBO suggests an autocrine/paracrine regulatory system for regulating placental function. Differential expression of SLITs and ROBOs in healthy term and preeclamptic placentas and hypoxia regulation of their expressions in placental cells implicate a potential pathophysiological role for this system in preeclampsia.

Copy Number Variation Is a Fundamental Aspect of the Placental Genome

“…Downregulation of cell adhesion genes is necessary for trophoblast invasion in both mice and humans [40], [41]. Further—and quite remarkably—Liao et al [42] found that upregulation of genes in the SLIT/ROBO neuronal guidance system in the human placenta is associated with the pregnancy disease pre-eclampsia. UR domain formation could also enable TGCs to simply save materials and time, a hypothesis that has been proposed for polyploidy in general [43].…”

Regulation of Placental Angiogenesis

“…Knockdown or overexpression of Robo4 leads to either lack of or misdirected intersomitic vessels . In human placenta, Slit2 and Robo1 proteins are expressed in the syncytiotrophoblast, while Slit3 and Robo1 and Robo4 are detected in capillary endothelium of the placental villi . Moreover, levels of Robo1 and Robo4 are significantly greater in preeclamptic placentas compared to normal controls; hypoxia significantly increased both mRNA and protein levels of Slit2 in the trophoblast cell line BeWo and Slit3 and Robo1/4 in human umbilical cord endothelial cells .…”

Polyteny: still a giant player in chromosome research

“…Why should those genes in particular be targeted? Hannibal et al suggest that the known requirement for down-regulation of cell adhesion and neurogenesis genes during placental development means that underreplication could act as an additional layer of control in TGCs (Kokkinos et al 2010; Liao et al 2012). Yarosh and Spradling instead argue that the stochastic DNA breaks, which can lead to deletions, inversions, and translocations, in cell surface genes and immunoglobulin genes are themselves advantageous for the organism by generating genetic diversity (Fig.…”