Preeclampsia: 2-methoxyestradiol induces cytotrophoblast invasion and vascular development specifically under hypoxic conditions

Abstract

Inadequate invasion of the uterus by cytotrophoblasts is speculated to result in pregnancy-induced disorders such as preeclampsia. However, the molecular mechanisms that govern appropriate invasion of cytotrophoblasts are unknown. Here, we demonstrate that under low-oxygen conditions (2.5% oxygen), 2-methoxyestradiol (2-ME), which is a metabolite of estradiol and is generated by catechol-o-methyltransferase (COMT), induces invasion of cytotrophoblasts into a naturally-derived, extracellular matrix. Neither low-oxygen conditions nor 2-ME alone induces the invasion of cytotrophoblasts in this system; however, low-oxygen conditions combined with 2-ME result in the appropriate invasion of cytotrophoblasts into the extracellular matrix. Cytotrophoblast invasion under these conditions is also associated with a decrease in the expression of hypoxia-inducible factor-1alpha (HIF-1alpha), transforming growth factor-beta3 (TGF-beta3), and tissue inhibitor of metalloproteinases-2 (TIMP-2). Pregnant COMT-deficient mice with hypoxic placentas and preeclampsia-like features demonstrate an up-regulation of HIF-1alpha, TGF-beta3, and TIMP-2 when compared with wild-type mice; normal levels are restored on administration of 2-ME, which also results in the resolution of preeclampsia-like features in these mice. Indeed, placentas from patients with preeclampsia reveal lower levels of COMT and higher levels of HIF-1alpha, TGF-beta3, and TIMP-2 when compared with those from normal pregnant women. We demonstrate that low-oxygen conditions of the placenta are a critical co-stimulator along with 2-ME for the proper invasion of cytotrophoblasts to facilitate appropriate vascular development and oxygenation during pregnancy.

Figure 1

Hypoxic exposure and imaging. A: Schematic of the controlled-atmosphere and glass chamber we used to culture cells in hypoxia. B: Enlarged view of the area enclosed by the dotted line in A. C: Concentration of oxygen within the media. We used an oxygen probe to track the concentration of oxygen and plotted the depletion as a function of time. The concentration of oxygen in the media in which trophoblasts were cultured decreased with time over 7 hours until it equilibrated with the mixture of gas that was injected into the chamber. D: Examples of the circularity ratio and the tracing by using Image J software are shown.

Figure 2

Differentiation of trophoblasts to an invasive phenotype by 2-ME and hypoxia. Phase contrast images (A–K) of trophoblasts cultured on a layer of MG, in 17% O₂ or 2.5% O₂, and in the presence or absence of 2-ME, are shown. A–D: After 15 hours of culture, the trophoblasts adopted a spherical morphology where the cells have clustered. E and F: In 17% O₂ for 95 hours, HTR-8 cells with or without 2-ME are shown. G and H: 2.5% O₂ for 95 hours, trophoblasts with or without 2-ME are shown. In the presence of 2-ME under hypoxia, trophoblasts exhibit dendrites (highlighted by arrows in H) extended into the MG. We repeated experiments four independent times and measured a total of 10 clusters for circularity ratio assessment. I–K: Loss of invasive phenotype after returning trophoblasts to 17% O₂. Trophoblasts cultured with 2-ME under 17% O₂ (I) or 2.5% O₂ (J) for 72 hours are shown. Similar to H, 72 hours incubation under 2.5% O₂ with 2-ME showed extension of dendrites, but after an additional 24 hours of culture under 17% O₂, the cells lost the invasive phenotype (K). L: The results of the Boyden chamber assay are shown. Incubation that is longer than 48 hours (72 hours), results in the death of most cells in this system. The results are shown as mean ± SE. n = 5 in each group. *P < 0.05.

Figure 3

Endothelial cell-specific CD31 expression induced by 2-ME under hypoxic conditions. A: A representative western blot picture of CD31 (135 kDa) is shown. B: Densitometric analysis of a 135 kDa CD31 protein expression is shown. Results are shown as the relative expression against 17% O₂ without 2-ME as in each set of western blots. Data are shown as the mean ± SE in the graph (n = 3). *P < 0.05. C: No alteration in the expression of the epithelial cell marker E-cadherin by the presence of 2-ME. Representative results from three independent experiments are shown.

Figure 4

Suppression of HIF-1α, TGF-β3, and TIMP-2 by 2-ME under hypoxic condition. A: Trophoblasts cultured for 3 hours or 95 hours in 17% or 2.5% O₂ were harvested, and HIF-1α levels were analyzed by using Western blot analysis. The representative results are shown from at least three independent experiments. B: Densitometric analysis of a HIF-1α protein expression at 95 hours is shown. Results are shown as the relative expression against 17% O₂ without 2-ME as in each set of western blots. Data are shown as the mean ± SE in the graph (n = 3). *P < 0.05. C: Total RNA was extracted from HTR-8 cultures after 95 hours of incubation with or without 2-ME under 17% or 2.5% O₂. Complementary cDNA was synthesized by using 0.5 μg of total RNA, and TGF-β3 expression levels were analyzed by PCR. The experiment was performed twice and showed the same results. D: Western blot analysis for TIMP-2 by using cultured trophoblast after 95 hours of incubation with or without 2-ME under 17% or 2.5% O₂is shown. The representative results are shown in three independent experiments.

Figure 5

Down-regulation of TGF-β3 and TIMP-2 by 2-ME in placentas of COMT^−/− mice. A–C: The immunohistochemical labeling of TGF-β3 in placenta/decidua from COMT^+/+ (A), COMT^−/− (B), and 2-ME-administered COMT^−/− mice (C) is shown. D–F: The immunohistochemical labeling of TIMP-2 in placenta/decidua from COMT^+/+ (D), COMT^−/− (E), and 2-ME-administered COMT^−/− mice (F) is shown. Formalin-fixed paraffin embedded sections were used in the analysis. Brownish staining represents positive immunostaining (arrows in B and E). The solid lines indicate the border between placenta and decidua. The dotted lines indicate the border between the spongiotrophoblast layer and the labyrinth layer of placenta (P, placenta; D, decidua; SP, spongiotrophoblast layer; L, labyrinth layer). Wild-type mice are designated as COMT^+/+ mice in the figure. The representative results are shown from three independent experiments.

Figure 6

Down-regulation of COMT is associated with up-regulation of HIF-1α, TGF-β3, and TIMP-2 in placentas of preeclampsia patients. Immunohistochemical analysis of villous COMT (A and B), HIF −1α (D and E), TGF-β3 (G and H), TIMP-2 (J and K), and nonvillous COMT (M and N) in placentas from patients with preeclampsia and normal pregnancy is shown. Brownish staining represents positive immunoreactivity. Each sample was analyzed by immunostaining intensity in six different areas (indicated as dot), scores were averaged, and statistical analyses were performed (C, F, I, L and O). Three normotensive control placentas and three severe cases of preeclamptic placentas were analyzed. Formalin-fixed paraffin-embedded sections are used in the analysis. *P < 0.05.

Figure 7

Schematic illustration of extravillous cytotrophoblast invasion into the uterus. A: With sufficient 2-ME, cytotrophoblast with an invasive phenotype can invade up to the proximal third of the myometrium. B: With less 2-ME, the cytotrophoblasts are less invasive; therefore, shallow invasion of cytotrophoblasts develops.