Research ArticleReproductive Biology

Nrf2 inactivation enhances placental angiogenesis in a preeclampsia mouse model and improves maternal and fetal outcomes

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Sci. Signal.  16 May 2017:
Vol. 10, Issue 479, eaam5711
DOI: 10.1126/scisignal.aam5711
  • Fig. 1 Accumulation of oxidative stress in placentas from patients with preeclampsia and improvements in maternal and fetal outcomes in PAH mice with Nrf2 deficiency.

    (A) Lipid peroxidation status of human placentas. Immunohistochemical detection of malondialdehyde (brown) was performed using placentas at 32-week gestation from normal pregnancies and those affected by preeclampsia. Hematoxylin (blue) was used to stain nuclei. The pictures show representative data from six normal pregnancy and six preeclampsia patients. (B) Maternal mortality rate of PAH mice between E13.5 and delivery. n = 10, 24, 26, and 25 dams for NCP-WT, PAH-Keap1KD, PAH-WT, and PAH-Nrf2KO, respectively. (C) Neonatal mortality rate in 0.5 to 7.5 days after birth. n = 38, 45, 66, and 77 neonates for NCP-WT, PAH-Keap1KD, PAH-WT, and PAH-Nrf2KO, respectively. (D and E) Fetal body weight at E16.5 (D) and E18.5 (E). n = 49, 69, 72, and 50 fetuses at E16.5 for NCP-WT, PAH-Keap1KD, PAH-WT, and PAH-Nrf2KO, respectively; n = 43, 53, 60, and 62 fetuses at E18.5 for NCP-WT, PAH-Keap1KD, PAH-WT, and PAH-Nrf2KO, respectively. (F and G) Representative photomicrograph of fetuses from pregnant dams of each genotype at E16.5 (F) and E18.5 (G). Scale bars, 50 μm (A) and 5 mm (F and G). Data in bar graphs are means ± SD (B to E). *P < 0.05 and **P < 0.01 (B to E).

  • Fig. 2 Nrf2 deficiency improves placental angiogenesis in PAH mice.

    (A) ILB4 immunostaining (brown) of labyrinth zone sections at E16.5. Hematoxylin (blue) was used for counterstaining. Lower panels are magnified images of upper panels. # indicates junctional zones. The pictures show representative data from n = 4, 5, 3, and 4 of placentas from NCP-WT, PAH-Keap1KD, PAH-WT, and PAH-Nrf2KO mice, respectively. (B) Quantification of ILB4-positive vascular areas in sections from E16.5 placentas. ILB4-positive areas in labyrinth zone sections were measured using ImageJ software (National Institutes of Health), and the relative positive area in PAH mouse samples is shown, with the average value from NCP-WT mouse samples set as 1.0. n = 4, 5, 3, and 4 placentas from NCP-WT, PAH-Keap1KD, PAH-WT, and PAH-Nrf2KO mice, respectively. (C) Endothelial cell proliferation in the labyrinth zone. Immunoreactivity for pHH3 was used to detect cellular proliferation in sections from the E16.5 labyrinth zone. Lower panels are merged images of pHH3 (red), ILB4 (green), and DAPI (blue, nucleus) staining. White arrowheads indicate pHH3 and ILB4 double-positive (yellow) proliferating endothelial cells. The pictures show representative data from five placentas for each group. (D) Number of pHH3-positive nuclei in a section from the E16.5 labyrinth zone. n = 5 placentas for each group. Scale bars, 100 μm (A) and 50 μm (C). Data in bar graphs are means ± SD (B and D). *P < 0.05 and **P < 0.01 (B and D).

  • Fig. 3 Nrf2 deficiency augments oxidative DNA damage in the labyrinth layer of PAH mice.

    (A) 8-OHdG staining (dark purple) of placentas at E18.5. The lower panels are magnified views of the dotted rectangles in the upper panels. Nuclear Fast Red (pink) was used for counterstaining. Red arrows indicate 8-OHdG–positive foci, which are distributed in the labyrinth zone. Scale bar, 50 μm. The pictures are representative images from n = 4, 5, 3, and 4 of placentas from NCP-WT, PAH-Keap1KD, PAH-WT, and PAH-Nrf2KO mice, respectively. (B) Number of 8-OHdG–positive foci per section in E18.5 placentas. n = 4, 5, 3, and 4 placentas for NCP-WT, PAH-Keap1KD, PAH-WT, and PAH-Nrf2KO, respectively. (C) Nrf2 and Nqo1 protein abundance in placentas at E18.5 was examined by Western blotting. n = 3 placentas for each group. Lamin B and α-tubulin were used as internal controls for nuclear extracts (upper) and cytoplasmic extracts (lower), respectively. The pictures show representative data from three independent experiments, each of which used three placentas per group. (D) Relative quantification of Nrf2 and Nqo1 protein abundance by densitometry analysis of the Western blotting data in (C) after normalization to the internal controls. n = 3 placentas for each group. (E) Expression of Nrf2 target genes in the labyrinth zone at E18.5 were determined by qRT-PCR. n = 5, 5, 6, and 6 for NCP-WT, PAH-Keap1KD, PAH-WT, and PAH-Nrf2KO, respectively. Data in bar graphs are means ± SD (B, D, and E). *P < 0.05, **P < 0.01 (B, D, and E).

  • Fig. 4 Nrf2 deficiency induces the expression of angiogenic and inflammatory factors in the PAH placenta.

    (A) Heat map of the microarray data for the labyrinth zone samples from NCP-WT, PAH-Keap1KD, PAH-WT, and PAH-Nrf2KO mice (n = 2 placentas for each group) at E16.5 shows the relative expression of genes related to angiogenesis or inflammation, which were selected from the list of genes in the Nrf2-dependent PAH-specific gene signature (data file S1). Blue letters indicate genes for which multiple probes are included in this signature. (B) qRT-PCR analyses of the expression of mRNAs encoding angiogenic chemokines [Ccl2, Ccl5, Cxcl9, and Cxcl10; highlighted in red letters in (A)] and inflammation-related genes (Il1a, Il1b, Cxcl1, and Tnfa) in the labyrinth zone of PAH mice at E16.5. n = 5, 5, 6, and 5 placentas for NCP-WT, PAH-Keap1KD, PAH-WT, and PAH-Nrf2KO, respectively. Data in bar graphs are means ± SD. *P < 0.05, **P < 0.01.

  • Fig. 5 Pharmacological activation of Nrf2 worsens perinatal complications in PAH mice.

    (A) Maternal mortality rate of NCP-WT and PAH-WT mice treated with CDDO-Im every other day from E13.5 to E17.5. n = 3 NCP-WT dams with or without CDDO-Im administration; n = 7 PAH-WT dams with or without CDDO-Im administration. (B) Fetal body weight of E16.5 and E18.5 embryos in NCP-WT or PAH-WT mice treated with CDDO-Im. n = 18 and 9 fetuses at E16.5 in NCP-WT and PAH-WT dams treated with vehicle, respectively; n = 15 and 23 fetuses at E16.5 in NCP-WT and PAH-WT dams treated with CDDO-Im, respectively. n = 18 and 28 fetuses at E18.5 in NCP-WT and PAH-WT dams treated with vehicle, respectively; n = 22 and 33 fetuses at E18.5 in NCP-WT and PAH-WT dams treated with CDDO-Im, respectively. (C) Placental abortion and vaginal bleeding in PAH-WT mice treated with CDDO-Im. These features were observed in every PAH mouse after CDDO-Im administration. Arrows and arrowheads indicate the fetus and placenta, respectively. Data in bar graphs are means ± SD. *P < 0.05, **P < 0.01.

  • Fig. 6 Schematic of Nrf2-mediated angiogenic suppression in the PAH placenta.

    Overproduction of human ANGII (hANGII) stimulates AT1R signaling and creates an anti-angiogenic milieu in the labyrinth zone of PAH mice. ROS accumulation counteracts the anti-angiogenic milieu by inducing the expression of genes encoding angiogenic chemokines and cytokines. Because Nrf2 decreases ROS concentrations, Nrf2 inactivation ameliorates the intra-uterine growth retardation (IUGR) and PAH complications by inducing ROS-mediated angiogenesis in PAH-Nrf2KO mice. In PAH-Keap1KD mice, placental angiogenesis is further worsened due to ROS quenching by activated Nrf2.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/479/eaam5711/DC1

    Fig. S1. Mouse models used in this study.

    Fig. S2. Fetal body weight, systolic blood pressure, and gene expression profiles of RAS components in NCP and PAH mice with Keap1 or Nrf2 genetic mutation.

    Fig. S3. Genetic deletion of Nrf2 improves vascular plexus selectively in the PAH placenta.

    Fig. S4. Expression profiles of Nrf2 target genes in the placenta of PAH mice.

    Fig. S5. Microarray analysis to identify an Nrf2-dependent PAH-specific gene signature.

    Fig. S6. Genetic modification of the Keap1-Nrf2 system in PAH mice does not significantly change the plasma concentrations of sFLT1 and PLGF2.

    Fig. S7. Induction of Nrf2 target gene expression in the labyrinth zone of PAH-WT mice by pharmacological activation of Nrf2.

    Table S1. Primer sequences for genotyping mouse lines.

    Table S2. Primer sequences for qRT-PCR analysis.

    Data file S1. Microarray data revealed candidate genes for an Nrf2-dependent PAH-specific gene signature.

  • Supplementary Materials for:

    Nrf2 inactivation enhances placental angiogenesis in a preeclampsia mouse model and improves maternal and fetal outcomes

    Masahiro Nezu, Tomokazu Souma, Lei Yu, Hiroki Sekine, Nobuyuki Takahashi, Andrew Zu-Sern Wei, Sadayoshi Ito, Akiyoshi Fukamizu, Zsuzsanna K. Zsengeller, Tomohiro Nakamura, Atsushi Hozawa, S. Ananth Karumanchi, Norio Suzuki,* Masayuki Yamamoto*

    *Corresponding author. Email: sunorio{at}med.tohoku.ac.jp (N.S.); masiyamamoto{at}med.tohoku.ac.jp (M.Y.)

    This PDF file includes:

    • Fig. S1. Mouse models used in this study.
    • Fig. S2. Fetal body weight, systolic blood pressure, and gene expression profiles of RAS components in NCP and PAH mice with Keap1 or Nrf2 genetic mutation.
    • Fig. S3. Genetic deletion of Nrf2 improves vascular plexus selectively in the PAH placenta.
    • Fig. S4. Expression profiles of Nrf2 target genes in the placenta of PAH mice.
    • Fig. S5. Microarray analysis to identify an Nrf2-dependent PAH-specific gene signature.
    • Fig. S6. Genetic modification of the Keap1-Nrf2 system in PAH mice does not significantly change the plasma concentrations of sFLT1 and PLGF2.
    • Fig. S7. Induction of Nrf2 target gene expression in the labyrinth zone of PAH-WT mice by pharmacological activation of Nrf2.
    • Table S1. Primer sequences for genotyping mouse lines.
    • Table S2. Primer sequences for qRT-PCR analysis.
    • Legend for data file S1

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    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Microarray data revealed candidate genes for an Nrf2-dependent PAH-specific gene signature.

    Citation: M. Nezu, T. Souma, L. Yu, H. Sekine, N. Takahashi, A. Z.-S. Wei, S. Ito, A. Fukamizu, Z. K. Zsengeller, T. Nakamura, A. Hozawa, S. A. Karumanchi, N. Suzuki, M. Yamamoto, Nrf2 inactivation enhances placental angiogenesis in a preeclampsia mouse model and improves maternal and fetal outcomes. Sci. Signal. 10, eaam5711 (2017).

    © 2017 American Association for the Advancement of Science

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