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@med.tohoku.ac.jp (N.S.); masiyamamoto@med.tohoku.ac.jp

(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.