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.