Novel expression of EGFL7 in placental trophoblast and endothelial cells and its implication in preeclampsia

Abstract

The mammalian placenta is the site of nutrient and gas exchange between the mother and fetus, and is comprised of two principal cell types, trophoblasts and endothelial cells. Proper placental development requires invasion and differentiation of trophoblast cells, together with coordinated fetal vasculogenesis and maternal vascular remodeling. Disruption in these processes can result in placental pathologies such as preeclampsia (PE), a disease characterized by late gestational hypertension and proteinuria. Epidermal Growth Factor Like Domain 7 (EGFL7) is a largely endothelial-restricted secreted factor that is critical for embryonic vascular development, and functions by modulating the Notch signaling pathway. However, the role of EGFL7 in placental development remains unknown. In this study, we use mouse models and human placentas to begin to understand the role of EGFL7 during normal and pathological placentation. We show that Egfl7 is expressed by the endothelium of both the maternal and fetal vasculature throughout placental development. Importantly, we uncovered a previously unknown site of EGFL7 expression in the trophoblast cell lineage, including the trophectoderm, trophoblast stem cells, and placental trophoblasts. Our results demonstrate significantly reduced Egfl7 expression in human PE placentas, concurrent with a downregulation of Notch target genes. Moreover, using the BPH/5 mouse model of PE, we show that the downregulation of Egfl7 in compromised placentas occurs prior to the onset of characteristic maternal signs of PE. Together, our results implicate Egfl7 as a possible factor in normal placental development and in the etiology of PE.

Keywords: EGFL7; Endothelium; Notch signaling; Placenta; Preeclampsia; Trophoblast.

Figure 1. EGFL7 is expressed by maternal and fetal endothelial cells in the mouse placenta

In situ hybridization was performed to determine Egfl7 mRNA localization, using an Egfl7 riboprobe on 100μm thick vibratome sections of E10.5 C57Bl/6 placentas (A–E). Higher magnification images of boxes in (A) demonstrating Egfl7 transcript is highly expressed in the maternal decidua (B) and fetal labyrinth (C). Egfl7 sense controls (A inset) show specificity of Egfl7 riboprobe. Paraffin sections of the vibratome sections showing cellular morphology after Egfl7 riboprobe staining in the maternal decidua (D) and fetal labyrinth (E). To determine the cell types expressing EGFL7 protein in the placenta, double immunofluorescent staining was performed on E12.5 C57BL/6 placentas for EGFL7 (red), CD31 (green) and nuclear DAPI (blue) (F–M). EGFL7 colocalizes with the endothelial cell marker, CD31, in the maternal decidua (F–I) and the fetal labyrinth (J–M). Dec-Maternal Decidua, JZ-Junctional Zone, Lab-Fetal Labyrinth. Scale bar (F–M) = 20μm.

Figure 2. EGFL7 is expressed in the trophoblast cell lineage of the mouse

In situ hybridization was performed to determine Egfl7 mRNA localization, using an Egfl7 riboprobe on 100μm thick vibratome sections of E10.5 C57Bl/6 placentas (A). Shown is the junctional zone of the placenta (A). Paraffin sections of the vibratome section showing cellular morphology of Egfl7 riboprobe staining in the junctional zone of the placenta (B). Arrows indicate positive Egfl7 transcript signal in the junctional zone trophoblast cells. (TGC=Trophoblast Giant Cell). To determine the cell type expressing EGFL7 protein in the placenta, double immunofluorescent staining was performed on E10.5 C57BL/6 placentas (C–F) for the pan-trophoblast marker, CYTOKERATIN (CK, red), EGFL7 (green), and nuclear DAPI (blue). Cross-section of E10.5 placenta showing the fetal labyrinth (Lab), junctional zone (JZ, demarked by white lines) and maternal decidua (Dec). High magnification images (D–F) of the junctional zone demonstrating colocalization of EGFL7 with CK in the JZ trophoblast cells (arrows). Whole mount immunofluorescent staining of blastocyst-stage C57BL/6 embryos (G–J) for DAPI (blue), EGFL7 (red), and trophectoderm cell marker, CDX2 (green) reveals EGFL7 protein localization to both inner cell mass (ICM) cells and trophectoderm cells (arrowheads). Real Time RT-PCR data for Egfl7 transcript expression in trophoblast stem cells (TSC), embryonic stem cells (ESC), and mouse embryonic fibroblasts (MEF), shows a significantly higher level of Egfl7 expression in TSC,**P<0.01 (K, top). Agarose gel with products of semi-quantitative RT-PCR for TSC, ESC and MEFs are shown, using Egfl7 and β-actin specific primers (K, bottom). RNA in situ hybridization analysis of Egfl7 mRNA in TSC (L–O) defines Egfl7 transcript expression in TSC. Phase-contrast and bright-field images of TSC incubated with an Egfl7 sense control probe (L–M), and an Egfl7 antisense probe (N–O). Double immunofluorescent staining of TSC (P–T) for DAPI, EGFL7, and CDX2 demonstrating EGFL7 protein localization to TSC. Phase-contrast image of TSC colony is shown in (P). Scale bars in D–F=25μm, G–J=20μm, C and L–T=100μm.

Figure 3. EGFL7 is expressed in endothelial cells and trophoblast cells of the human placenta

Double immunofluorescent staining of normal human placentas (A–D) for EGFL7 (red), CD31 (green), and nuclear DAPI (blue) showing localization of EGFL7 protein on endothelial cells. Double immunofluorescent staining of human chorionic villi from placentas at 40-weeks of gestation (E–H) for nuclear DAPI (blue), EGFL7 (red), and pan-trophoblast marker CYTOKERATIN (CK) (green) demonstrates EGFL7 protein localization to trophoblast cells (arrows-fetal vessels, arrowheads-trophoblasts). Control staining of a close-by section (insets, E–H) using IgG and secondary antibodies only and nuclear Hoechst (blue) shows specificity of the antibodies. Expression of EGFL7 protein is found in human cytotrophoblast cells isolated from term placentas (I–L). Cells were stained for Hoechst (blue), EGFL7 (red) and CYTOKERATIN (green) (I–L). Scale bars in E–H and insets =100μm, and I–L=30μm.

Figure 4. EGFL7 is downregulated in the placentas of the BPH/5 mouse model of preeclampsia

Real Time RT-PCR data for Egfl7 transcript levels in E10.5 placentas from C57BL/6 (WT) and BPH/5 mice reveal a significant decrease in Egfl7 in BPH/5 mice *P<0.05 (A). Immunofluorescent staining for EGFL7 protein (red), CD31 (green), and nuclear DAPI (blue) on the fetal labyrinth of E10.5 placentas from C57BL/6 (B–E) and BPH/5 mice (F–I) demonstrating a decrease in EGFL7 in BPH/5 placentas. Scale bar=100μm.

Figure 5. EGFL7 is significantly reduced in human preeclamptic placentas

Double immunofluorescent staining of normal and preeclamptic (PE) human placentas (A–F) for EGFL7 protein (red), CD31 (green), and nuclear Hoechst (blue) revealing a decrease in EGFL7 protein in PE placentas (arrows-fetal vessels, arrowheads-trophoblasts). Real Time RT-PCR data for EGFL7 (G), CD31 (H), and VEGF (I) on normal and preeclamptic (PE) human placentas demonstrating a significant decrease in EGFL7 transcript levels in PE placentas, even further than the known angiogenic factor, VEGF (n=10, *P<0.05). Scale bars = 50μm.

Figure 6. Expression of NOTCH target genes and NOTCH receptors are downregulated in PE, concomitant with EGFL7

Real Time RT-PCR data for NOTCH pathway members on normal and preeclamptic (PE) human placentas (A–H). Gene expression analysis for HES1 (A), HEY1 (B), and HEY2 (C)demonstrating a significant decrease in all NOTCH target gene transcripts in PE. Gene expression analysis for NOTCH receptors NOTCH1 (D), NOTCH2 (E), and NOTCH4 (F) demonstrating a significant downregulation in NOTCH1 and NOTCH4, and a downward trend in NOTCH2 in PE patients. Gene expression analysis indicating no change in NOTCH ligands DLL4 (G) and JAGGED1 (H). (n=3–7, *P<0.05). Immunofluorescent staining of a cross section of human villi (I–K) for EGFL7 (red), NOTCH4 (green), and Hoechst (blue) (arrowheads-trophoblasts) shows colocalization of EGFL7 protein and NOTCH4. (*P<0.05) Scale bars = 50 μm.