Placental productions and expressions of soluble endoglin, soluble fms-like tyrosine kinase receptor-1, and placental growth factor in normal and preeclamptic pregnancies

Abstract

Context: Increased production of antiangiogenic factors soluble endoglin (sEng) and soluble fms-like tyrosine kinase receptor-1 (sFlt-1) by the placenta contributes to the pathophysiology in preeclampsia (PE).

Objective: Our objective was to determine the differences in endoglin (Eng), fms-like tyrosine kinase receptor-1 (Flt-1), and placental growth factor (PlGF) expressions between normal and PE placentas and sEng, sFlt-1, and PlGF production by trophoblast cells (TC) cultured under lowered oxygen conditions.

Methods: TCs isolated from normal and PE placentas were cultured under regular (5% CO2/air) and lowered (2% O2/5% CO2/93% N2) oxygen conditions. sEng, sFlt-1, and PlGF productions were determined by ELISA. Protein expressions for Eng, Flt-1, and PlGF in the placental tissues were accessed by immunohistochemical staining and Western blot analysis. Deglycosylated Eng, Flt-1, and PlGF protein expressions in placental tissues were also examined.

Results: PE TCs produced significantly more sEng, sFlt-1, and PlGF compared with those from normal TCs (P < 0.05). Under lowered oxygen conditions, PE TCs, but not normal TCs, released more sEng and sFlt-1. In contrast, both normal and PE TCs released less PlGF (P < 0.05). Enhanced expressions of Eng and Flt-1, as well as glycosylated Eng and Flt-1, were observed in PE placentas. Immunoblot also revealed that TCs released glycosylated sFlt-1, but not sEng, in culture.

Conclusions: PE TCs produce more sEng, sFlt-1, and PlGF than normal TCs. Lowered oxygen conditions promote sEng and sFlt-1, but reduce PlGF, productions by PE TCs. More glycosylated sEng and sFlt-1 are present in PE placentas. Trophoblasts release glycosylated sFlt-1, but unglycosylated sEng, in culture.

Figure 1

sEng (top panel), sFlt-1 (middle panel), and PlGF (bottom panel) productions by placental trophoblasts. Trophoblasts from normal (n = 7) and PE (n = 7) placentas were cultured under regular (21%) or lowered (2%) O₂ conditions over 48 h. Trophoblasts from PE placentas produced more sEng, sFlt, and PlGF when cultured under regular O₂ condition. *, P < 0.05, PE vs. normal under 21% O₂. #, P < 0.05, Two percent O₂ vs. 21% O₂.

Figure 2

Representative immunostaining of Eng (A and D), Flt-1 (B and E), and PlGF (C and F) in normal (A–C) and PE (D–F) placental tissue sections. Bar, 20 μm.

Figure 3

Placental tissue expressions for Eng, Flt-1, and PlGF by Western blot analyses. Placental tissues were homogenized, and total tissue proteins were run on SDS-PAGE under reducing conditions. Protein expressions for Eng (A) and Flt-1 (B) were up-regulated in preeclamptic tissue samples compared with normal tissue samples. In contrast, protein expression for PlGF (C) was down-regulated in preeclamptic tissue samples. The patterns of total tissue protein expressions for Eng, Flt-1, and PlGF are remarkably different between normal and preeclamptic placentas. M, Protein marker.

Figure 4

Eng and Flt-1 expressions in placental tissues, trophoblast culture medium, and placental tissues after being treated with N-glycosidase (N-Gly-F). Upper panels, Eng expression. Lower panels, Flt-1 expression. A, Tissue expression. B, Trophoblast culture medium expression. C, Preeclamptic placental tissues treated with or without N-glycosidase for 48 h. Compared with tissue expression, only a single band with approximately 65 kD for Eng and diffused bands with approximately 110–150 kD are shown in the cell culture medium. After N-glycosidase treatment, the higher molecular mass bands for Eng at 90 kD and Flt-1 at approximately 140–150 kD disappeared, which suggests that glycosylated Eng and Flt-1 are present in the preeclamptic placental tissue, and trophoblasts are more likely able to release glycosylated sFlt-1, but not Eng, in culture. M, Protein marker.