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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: [email protected] (N.S.); [email protected]
(M.Y.)
Published 16 May 2017, Sci. Signal. 10, eaam5711 (2017)
DOI: 10.1126/scisignal.aam5711
The 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
Other Supplementary Material for this manuscript includes the following:
(available at www.sciencesignaling.org/cgi/content/full/10/479/eaam5711/DC1)
www.sciencesignaling.org/cgi/content/full/10/479/eaam5711/DC1
Data file S1 (Microsoft Excel format). Microarray data revealed candidate genes
for an Nrf2-dependent PAH-specific gene signature.
Fig. S1. Mouse models used in this study. Female mice homozygous for the human
AGT transgene (hAGTTg/Tg genotype) were bred with male mice homozygous for the
human REN transgene (hRENTg/Tg genotype), and the pregnant dam (PAH mice) showed
preeclampsia-like phenotypes due to overexpression of human AGT and renin in the
liver and embryonic placenta, respectively, as reported previously (16). Normal control
pregnancy (NCP) mice were obtained by intercrossing hAGTTg/Tg mice and served as
controls. To investigate the roles of the Keap1-Nrf2 system in preeclampsia,
Keap1-knockdown (Keap1KD) alleles (Keap1KD/KD) and Nrf2-knockout (Nrf2KO)
alleles (Nrf2-/-) were integrated into PAH or NCP mice. Each gene-modified mouse line
was backcrossed to C57BL/6J mice for more than 5 generations.
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. (A)
Fetal body weight at E13.5, E16.5, and E18.5. n = 23, 37, 34 and 43 fetuses at E13.5 in
NCP-WT, PAH-Keap1KD, PAH-WT and PAH-Nrf2KO, respectively; n = 39, 49 and
19 fetuses at E16.5 in NCP-Keap1KD, NCP-WT and NCP-Nrf2KO, respectively; n =
28, 43 and 37 fetuses at E18.5 in NCP-Keap1KD, NCP-WT and NCP-Nrf2KO,
respectively. (B) Systolic blood pressure of non-pregnancy (left), NCP (middle) and
PAH (right) mice. (left) n= 5, 6 and 5 non-pregnant hAgtTg/Tg female mice for Keap1KD,
WT and Nrf2KO, respectively. (middle) n = 3, 5 and 4 dams at E13.5 for
NCP-Keap1KD, NCP-WT and NCP-Nrf2KO, respectively; n =3, 5 and 5 dams at E16.5
for NCP-Keap1KD, NCP-WT and NCP-Nrf2KO, respectively; n = 5 dams at E18.5 for
each genotype. (right) n = 5 dams at E13.5 for each group; n = 5, 5, 6 and 5 dams at
E16.5 for NCP-WT, PAH-Keap1KD, PAH-WT and PAH-Nrf2KO, respectively; n = 5,
6, 5 and 5 dams at E18.5 for NCP-WT, PAH-Keap1KD, PAH-WT and PAH-Nrf2KO,
respectively. Data are shown as the mean ± SD. *P < 0.05 and **P < 0.01 compared to
NCP-WT (middle) and PAH-WT (right).
Fig. S3. Genetic deletion of Nrf2 improves vascular plexus selectively in the PAH
placenta. (A) Quantification of ILB4-positive vascular areas in sections from the E18.5
labyrinth zone. ILB4-positive areas in a labyrinth zone section were measured using
NIH Image software, and the relative positive area in PAH mouse samples is shown,
with the average area in NCP-WT mouse samples set as 1.0. n = 4, 4, 4 and 5 placentas
for NCP-WT, PAH-Keap1KD, PAH-WT and PAH-Nrf2KO, respectively. (B)
pHH3-positive proliferating endothelial cells per section from E18.5 placentas. n = 4, 5,
6 and 5 placentas for NCP-WT, PAH-Keap1KD, PAH-WT and PAH-Nrf2KO,
respectively. (C) ILB4 staining (brown) of labyrinth zone sections from NCP mice at
E16.5. Hematoxylin (blue) was used for counterstaining. Lower panels are magnified
images of upper panels. # indicates junctional zones. Scale bars: 100 μm. The pictures
are representative of 3 placentas for each group. (D) Quantification of ILB4-positive
vascular areas in a placental section from NCP mice at E16.5. ILB4-positive areas in a
labyrinth zone section were measured using NIH Image software, 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 = 3 placentas for each group. Data in bar graphs are shown
as the mean ± SD (A, B, and D). *P < 0.05, **P < 0.01 (A, B, and D).
Fig. S4. Expression profiles of Nrf2 target genes in the placenta of PAH mice. (A)
Nrf2 and Nqo1 protein abundance in placentas at E16.5 were examined by Western
blotting. n = 3 placentas for each group. Lamin B and -Tubulin were used as internal
controls for nuclear extracts (upper) and cytoplasmic extracts (lower), respectively. The
pictures show representative data from 3 independent experiments, each of which used
3 placentas per group. Right panels: Relative quantification of protein abundance of
Nrf2 and Nqo1 by densitometry analysis of the Western blotting data (left) after
normalization to the internal controls. n = 3 placentas for each group. (B) Expression of
Nrf2 target genes in the labyrinth zone at E16.5 was determined by qRT-PCR. n = 5, 5,
6 and 5 placentas for NCP-WT, PAH-Keap1KD, PAH-WT and PAH-Nrf2KO,
respectively. Data in bar graphs are shown as the mean ± SD. *P < 0.05, **P < 0.01.
Fig. S5. Microarray analysis to identify an Nrf2-dependent PAH-specific gene
signature. (A) Strategy for the microarray analysis of labyrinth zone samples from
PAH and NCP mice at E16.5. n = 2 placentas for each group. Microarray probes with a
greater than 1.5-fold change in average intensity in NCP-WT and PAH-Nrf2KO
samples compared to PAH-WT samples were included in the gene expression profiles
changed by PAH (gray) and Nrf2 (blue), respectively. The shared transcriptional
changes in the gray and blue categories were identified as the Nrf2-dependent
PAH-specific gene signature. (B) The genes in the Nrf2-dependent PAH-specific gene
signature were subjected to DAVID pathway analysis (https://david.ncifcrf.gov/), and
the cytokine-cytokine receptor interaction pathway was identified as the top (P =
2.3e-8) overrepresented KEGG pathway (http://www.genome.jp/kegg/pathway.html).
Red stars indicate genes in the list. (C) qRT-PCR analysis of mRNA encoding classic
angiogenic factors in the labyrinth zone from PAH and NCP mice at E16.5. n = 5, 5, 6
and 5 placentas for NCP-WT, PAH-Keap1KD, PAH-WT and PAH-Nrf2KO,
respectively. Data are shown as the mean ± SD. No significant changes were observed.
Fig. S6. Genetic modification of the Keap1-Nrf2 system in PAH mice does not
significantly change the plasma concentrations of sFLT1 and PLGF2. Plasma
concentrations of PLGF2 (left panels) and sFLT1 (right panels) were measured in NCP
and PAH mice at E16.5 and E18.5. For PLGF2 at E16.5, n = 5, 5, 4 and 4 dams for
NCP-WT, PAH-Keap1KD, PAH-WT and PAH-Nrf2KO, respectively; for PLGF2 at
E18.5, n = 6, 5, 5 and 5 for NCP-WT, PAH-Keap1KD, PAH-WT and PAH-Nrf2KO,
respectively. For sFLT1 at E16.5, n = 5, 5, 4 and 4 for NCP-WT, PAH-Keap1KD,
PAH-WT and PAH-Nrf2KO, respectively; for sFLT1 at E18.5, n = 5, 5, 4 and 4 for
NCP-WT, PAH-Keap1KD, PAH-WT and PAH-Nrf2KO, respectively. Data are shown
as the mean ± SD.
Fig. S7. Induction of Nrf2 target gene expression in the labyrinth zone of PAH-WT
mice by pharmacological activation of Nrf2. qRT-PCR analysis of Nrf2 target genes
in the E18.5 labyrinth zone from NCP-WT and PAH-WT mice treated with CDDO-Im
or vehicle. n = 3, 3, 3 and 4 placentas for vehicle-treated NCP-WT, CDDO-Im-treated
NCP-WT, vehicle-treated PAH-WT and CDDO-Im-treated PAH-WT mice, respectively.
Data are shown as the mean ± SD. *P < 0.05, **P < 0.01.
Table S1. Primer sequences for genotyping mouse lines.
Gene Sense Primer Antisense Primer
hAGT Tg CTTCTCAGTGAGCGCTCCTCC GAACTCCTGGCCTCAAGCGATCC
hREN Tg CTCACAGGTGGGTGGAATCAC CCTCTTGCTCCAGAGTGGCGCTGTC
Agt GGGCTCTTCACTGCTCCAGGC CCACCGTAGTCTCAGGAGTATG
Ren1 TGTGGCCAGTCCCTGGGGACCG CTCACAGGTGGGTGGAATCAC
Nrf2 TGGACGGGACTATTGAAGGCTG and
GCGGATTGACCGTAATGGGATAGG GCCGCCTTTTCAGTAGATGGAGG
Keap1 CAGCAGTTAAGGGCACCAATGC and
CAACGAGAAGCGCGATCACA CCTGCCTCAGCTTCCCATCA
Table S2. Primer sequences for qRT-PCR analysis.
For qRT-PCR with SYBR Green
Gene Sense Primer Antisense Primer
Srxn1 AGGGGCTTCTGCAAACCTA TGGCATAGCTACCTCACTGCT
Ccl2 GGCTCAGCCAGATGCAGTTAA CCTACTCATTGGGATCATCTTGCT
Ccl5 TGCAGAGGACTCTGAGACAGC GAGTGGTGTCCGAGCCATA
Cxcl9 CTTTTCCTCTTGGGCATCAT GCATCGTGCATTCCTTATCA
Cxcl10 GCTGCCGTCATTTTCTGC TCTCACTGGCCCGTCATC
Il1a ATTGCGGATTCCATGTTGTT TCAAAGCCCAAAGGAAGCTA
Il1b TGTAATGAAAGACGGCACACC TCTTCTTTGGGTATTGCTTGG
Cxcl1 ATCCAGAGCTTGAAGGTGTTG GTCTGTCTTCTTTCTCCGTTACTT
Agt GGCAAATCTGAACAACATTGG TTCCTCCTCTCCTGCTTTGA
Ace AAAGAAGCTTCAGAACCTGGAC GTCTAGCAGGATCTGGTTGTACTCT
Agtr1a ACTCACAGCAACCCTCCAAG CTCAGACACTGTTCAAAATGCAC
Ren1 CCCGACATTTCCTTTGACC TGTGCACAGCTTGTCTCTCC
Angpt1 GGGGGAGGTTGGACAGTAA CATCAGCTCAATCCTCAGC
Angpt2 GATCTTCCTCCAGCCCCTAC TTTGTGCTGCTGTCTGGTTC
Vegfa CAGGCTGCTGTAACGATGAA CTATGTGCTGGCTTTGGTGA
Kdr GGCGGTGGTGACAGTATCTT GTCACTGACTGAGGCGATGA
Flt1 TTCGGAAGACAGAAGTTCTCGTT GACCTCGTAGTCACTGAGGTTTTG
Tek TGGAGTCAGCTTGCTCCTTT ACCTCCAGTGGATCTTGGTG
hREN TTTGACACTGGTTCGTCCAA CGAAGCATCGAAGAGCTTGT
hAGT AGACTGGCTGCTCCCTGAT GGAGAAGCCCTTCATCTTCC
For qRT-PCR with TaqMan Probes
Gene Sense Primer Antisense Primer
rRNA CGGCTACCACATCCAAGGAA GCTGGAATTACCGCGGCT
(FAM-labeled probe: TGCTGGCACCAGACTTGCCCTC)
Nqo1 AGCTGGAAGCTGCAGACCTG CCTTTCAGAATGGCTGGCA
(FAM-labeled probe: ATTTCAGTTCCCATTGCAGTGGTTTGGG)
Txnrd1 AGAAAGTGCTGGTCTTGGATTTTG ACACGTTCCTCCGAGACCC
(FAM-labeled probe: TCTGGTCCCAAGAGGAGTCGGTGTG)
Gstm1 CCTATGATACTGGGATACTGGAACG GGAGCGTCACCCATGGTG
(FAM-labeled probe: CGCGGACTGACACACCCGATCC)
Data file S1. Microarray data revealed candidate genes for an Nrf2-dependent
PAH-specific gene signature. Microarray analysis of the labyrinth zone from PAH and
NCP mice at E16.5 (n = 2 placentas for each group) identified 473 probes (223 decreased
expression and 250 with increased probes in PAH-WT samples compared to both
NCP-WT and PAH-Nrf2KO samples; see Fig. S5A) as an Nrf2-dependent PAH-specific
gene signature. Among the 473 probes, non-annotated probes were removed, and probes
with a greater than 1.5-fold change in average intensity of the duplicate samples between
the PAH-WT samples and the NCP-WT samples are shown. The expression profiles of
genes in red letters were verified by qRT-PCR in Fig. 4B.