Figure legends
Fig. 1. CBX treatment suppresses placental 11β-HSD2 expression
and activity and induces the features of PE in pregnant rats. Pregnant
rats were administrated (s.c) with CBX at dosage of 0.6,1.2 and 2.4mg/kg
from GD7.5 to GD17.5. Control rats were injected with same volume of
saline. Urine was collected from GD18.5 to GD19.5. After determination
of arterial BP, the rats were sacrificed on GD20.5 for collection of
blood and tissues. A, 11β-HSD2 expression and activity in placentas. B,
corticosterone levels in placentas. C, corticosterone levels in maternal
circulation. D, MAP measured from GD 7.5 until GD 19.5. E, SBP measured
on GD20.5. F, protein/creatinine in urine. F, morphology of glomeruli
stained by hematoxylin and eosin (400ⅹ). G, histopathological score of
glomerular pathology. H, fetal weight measured on GD 20.5. I, the
circulatory sFlt-1 level and sFlt-1/PIGF. Data are expressed as mean±SEM
(n=8). Statistic was performed by one-way ANOVA(A,C,D,E,F,H&J) and
Kruskal-Wallis test (B&I). MAP: mean arterial pressure; SBP: systolic
blood pressure.
Fig. 2. Placenta-targeted delivery of CBX leads to hallmark of
PE in pregnant rats. Pregnant rats were intravenously injected with
CBX, CBX-NPs, CBX-SCR and CBX-CSA every two days from GD7.5 to GD17.5.
Control rats were injected with same volume of saline. The dosage of CBX
was 0.6mg/kg. Urine of all the animals was collected from GD18.5 to
GD19.5. After determination of arterial BP, the rats were sacrificed on
GD20.5 for collection of blood and tissues. A, 11β-HSD2 expression and
activity in placentas. B, corticosterone level in placentas. C,
corticosterone levels in maternal circulation. D, MAP measured from GD
7.5 until GD 19.5 in pregnant rats. E, SBP measured on GD20.5. F,
protein/creatinine in urine. G, morphology of glomeruli stained by
hematoxylin and eosin and periodic acid-Schiff (400ⅹ). H,
histopathological score of glomerular pathology. I, fetal weight on GD
20.5. J, the circulatory sFlt-1 level and sFlt-1/PIGF in the pregnant
rats. Data are expressed as mean±SEM(n=6). Statistic was performed by
one-way ANOVA (A-F,H&J) and Kruskal-Wallis test (I). MAP: mean arterial
pressure; SBP: systolic blood pressure.
Fig.3. DEX administration induces the features of PE in pregnant
rats. Pregnant rats were administrated (s.c) with DEX at dosage of
0.1mg/kg from GD7.5 to GD17.5. Control rats were injected with same
volume of saline. Urine was collected from GD18.5 to GD19.5. After
determination of arterial BP, the animals were sacrificed on GD20.5 for
collection of blood and tissues. A, SBP measured on GD20.5. B,
protein/creatinine ratio in urine. C, morphology of glomeruli stained by
hematoxylin and eosin and periodic acid-Schiff (400×).
D,histopathological score of glomerular pathology. E, the circulatory
sFlt-1 level. F, fetal weight measured on GD 20.5. Data are expressed as
mean±SEM (n=8). Statistic was performed by two-tailed Student’s ttest. SBP: systolic blood pressure.
Fig. 4. CBX treatment leads to reduced interstitial trophoblast
invasion,impaired endovascular SA remodeling and abnormal placentation.Pregnant rats were administrated (s.c) with CBX at a dosage of 0.6, 1.2
and 2.4mg /kg/d from GD7.5 to GD17.5. The rats were sacrificed on
GD20.5, and placental tissues were collected for histological analysis.
A, the representative image of the total area occupied by CK-positive
interstitial trophoblast cells in MT (20×). B, cumulative data of
interstitial trophoblast invasion in MT of the pregnant rats with
administration of CBX. C, representative CK staining of trophoblast
invasion, and α-actin staining of vascular smooth muscle in spiral
arteries of the pregnant rats with systemic administration (200×). D,
H&E staining were performed to evaluated the morphology of placental
labyrinth of the pregnant rats with systemic administration (upper
panel:400×, lower panel:100× ). E, immunofluorescence analysis of
laminin staining (400×). Data are expressed as mean±SEM (n=8). Statistic
was performed by one-way ANOVA. TBC: trophoblast cells.
Fig.5. Placenta-targeted delivery of CBX results in deficient
interstitial trophoblast invasion, impaired endovascular SA remodeling
and abnormal placentation in pregnant rats. Pregnant rats were
intravenously injected with CBX, CBX-NPs, CBX-SCR and CBX-CSA every two
days from GD7.5 to GD17.5. Control rats were injected with same volume
of saline. The dosage of CBX was 0.6mg/kg. The rats were sacrificed on
GD20.5, and placental tissues were collected for histological analysis.
A, the representative image of the total area occupied by CK-positive
interstitial trophoblast cells in MT (20×). B, cumulative data of
interstitial trophoblast invasion in MT of the pregnant rats with
administration of CBX. C, representative CK staining of trophoblast
invasion, and α-actin staining of vascular smooth muscle in spiral
arteries of the pregnant rats with systemic administration (200×). D,
H&E staining were performed to evaluated the morphology of placental
labyrinth of the pregnant rats with systemic administration (upper
panel:400×, lower panel:100× ). E, immunofluorescence analysis of
laminin staining (400×). Data are expressed as mean±SEM (n=6). Statistic
was performed by one-way ANOVA. TBC: trophoblast cells.
Fig.6. Systemic administration and placenta-targeted delivery of
CBX lead to reduced uteroplacental perfusion in pregnant rats. Pregnant
rats were administrated (s.c) with CBX every day from GD7.5 to GD17.5,
or injected with CBX, CBX-NPs, CBX-SCR and CBX-CSA every two days from
GD7.5 to GD17.5. The rats were anesthetized for ultrasound
biomicroscopy. A, the representative images of the vasculatures in
implantation sites (spiral arteries and maternal channels) and fetal
umbilical artery visualized by ultrasound biomicroscopy. B, the
representative images of doppler flow waveform of umbilical artery,
maternal channel, spiral artery. C, cumulative data of the PSV of the
umbilical artery, maternal channel and spiral artery in the pregnant
rats with systemic administration of CBX. D, cumulative data of the PSV
of the umbilical artery, maternal channel and spiral artery in the
pregnant rats with placenta-targeted administration of CBX. Data are
expressed as mean±SEM (n=6). Statistic was performed by Kruskal-Wallis
test (C) and one-way ANOVA (D).
Fig.7. 11β-HSD2 modulates sFlt-1 release via modulation of
ADAM17 expression in placentals. A, 11β-HSD2 modulates sFlt1 release in
cultured human placental explants. Placental explants were treated with
cortisol (10-6M) in the presence or absence of CBX
(10-5 or 10-6M) for 24h. The media
were then collected for determination of sFlt1 concentration. B&C,
11β-HSD2 modulates sFlt1 release in primary syncytiotrophoblasts. Human
primary syncytiotrophoblasts were treated with
cortisol(10-9-10-6M) in the presence
or absence of CBX(10-5-10-6M) for
24h (B), or the cells were transfected with control siRNA and 11β-HSD2
siRNA, and then treated with cortisol (10-6M) for 24h
(C). The level of sFlt-1 in culture supernatant was determined by ELISA.
D, 11β-HSD2 modulates ADAM17 expression in primary syncytiotrophoblasts.
Primary syncytiotrophoblasts were transfected with control siRNA
and11β-HSD2 siRNA, and then treated with cortisol
(10-6M) for 24h. Cells were harvested for
determination of ADAM17 expression by western blotting. Representative
bands of ADAM17 were shown on the top of the cumulative data. E, the
role of ADAM17 in sFlt-1 release in primary syncytiotrophoblasts. Cells
were transfected with control siRNA and ADAM17 siRNA for 24h. Culture
media were harvest for determination of sFlt-1 content. F, GCs
stimulates ADAM17 gene transcriptive activity in syncytiotrophoblasts.
Placental trophoblasts were transfected with pGL3-luciferase reporter
containing ADAM17 promoter or mutant ADAM17 promoter and combination
with pRL-TK-Renilla-luciferase plasmid for 12hrs. Cells were then
treated with DEX (10-6M) for 24hrs. Luciferase assays
were performed using the dual luciferase assay kit. Data are expressed
as mean±SEM (n=5 independent cultures in each figure). Statistic was
performed by two-way ANOVA (A,C,D,F), two-tailed Student’s t test(E) and
one-way and two-way ANOVA (B).
Fig.8. The effects of CBX administration on ADAM17 expression in
placentas of pregnant rats and ADAM17 and11β-HSD2 levels in placentas of
PE patients. A, pregnant rats were administrated (s.c) with CBX at a
dosage of 0.6,1.2 and 2.4mg /kg/d from GD7.5 to GD17.5. Placental
tissues were collected on GD 20.5 for determination of ADAM17 protein
levels by western blotting. Representative bands of ADAM17 were shown on
the top of the cumulative data. Data are expressed as mean±SEM (n=8).
Statistic was performed by one-way ANOVA. B, the rats were intravenously
injected with CBX, CBX-NPs, CBX-SCR and CBX-CSA every two days from
GD7.5 to GD17.5. The rats were sacrificed on GD20.5, and placental
tissues were collected for determination of ADAM17 protein levels by
western blotting. Representative bands of ADAM17 were shown on the top
of the cumulative data. Data are expressed as mean±SEM (n=6). Statistic
was performed by and Kruskal-Wallis test. C, ADAM17 and 11β-HSD2 in
normal and PE placentas recruited from a Shanghai hospital.
Representative bands of ADAM17 and 11β-HSD2 were shown on the top of the
cumulative data. Data are expressed as mean±SEM (n=43 in each group).
Statistic was performed by Mann-Whitney U test.