Human placental expression of SLIT/ROBO signaling cues: effects of preeclampsia and hypoxia

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.

FIG. 1

mRNA Expression of SLIT/ROBO molecules in the human healthy term placenta. Total RNA (2 μg) samples were reversed transcribed (RT), and polymerase-chain reaction (PCR) was used for measuring mRNA levels. Total RNA without RT served as the no-RT control (not shown). The ribosome protein L19 (RPL19) served as a loading control. Lanes: 1, 5-mo-old rat cortex; 2, Postnatal Day 1 rat brain; 3, human term placental villi; and 4, HUVEC.

FIG. 2

Immunolocalization of SLIT/ROBO molecules in human placentas. Paraffin embedded sections (6 μm) of human healthy term placentas were processed for immunohistochemical analysis of SLIT2, SLIT3, ROBO1, and ROBO4 with specific anti-SLIT2 (goat, 10 μg/ml), anti-SLIT3, anti-ROBO1, and anti-ROBO4 antibodies (rabbit, 1 μg/ml) using the Zymed/Invitrogen SuperPicture kit. Negative controls were run in parallel with goat (10 μg/ml) or rabbit (1 μg/ml) IgGs. All the panels are in the same magnification. Arrowheads denote capillary endothelial cells, and arrows represent syncytiotrophoblasts. Bar = 30 μm.

FIG. 3

Messenger RNA and protein expressions of SLIT/ROBO molecules in human placentas from healthy term and PE pregnancies. A) Steady-state mRNA levels were measured by real-time q-PCR. Serial dilutions (5×) of HUVEC cDNA were used as internal controls. The mRNA levels were normalized to RPL19 and presented as ratios relative to the controls. The data are expressed as means ± SD, *P < 0.05, **P < 0.01. B) Protein levels were determined by immunoblotting analysis. The bar graph summarizes the data (mean ± SD) from five healthy term (NP) and six preeclamptic (PE) placentas. *P < 0.05, **P < 0.01, ***P < 0.001.

FIG. 4

Hypoxia regulation of SLIT/ROBO mRNA expression in BeWo and HUVEC. BeWo (A and C) and HUVEC (B and D) were exposed to hypoxia (2% vs. 10% O₂, upper panels) or deferoxamine mesylate (100 μM DFO, lower panels) for 24 h. Total RNA was subjected to q-PCR analysis (A and B) as indicated in Figure 3A. The bar graph summarizes the immunoblotting data (mean ± SD) relative to normaxia (10% O₂). *P < 0.05, **P < 0.01, and ***P < 0.001 vs. controls.

FIG. 5

Hypoxia regulation of SLIT/ROBO protein expression in BeWo and HUVEC. BeWo (A) and HUVEC (B) were exposed to hypoxia (2% O₂) or normoxia (10% O₂) for 48 h. Total protein extracts were immunoblotted to determine the levels of SLIT/ROBO proteins. HIF1A protein was determined as a positive control of hypoxia treatment. The bar graph summarizes the immunoblotting data (mean ± SD) relative to normoxia (10% O₂). **P < 0.01 and ***P < 0.001 vs. controls.