Int6/eIF3e Silencing Promotes Placenta Angiogenesis in a Rat Model of Pre-eclampsia

Abstract

We investigated whether stable eukaryotic translation initiation factor 3e/inter 6 (eIF-3e/Int6) RNA-silencing (siRNA-Int6) can ameliorate pre-eclampsia (PE) by promoting angiogenesis in an N-nitro-L-arginine methyl ester (L-NAME)-induced rat pre-eclampsia (PE) model. Twenty-four pregnant female Sprague-Dawley rats were allocated into 4 groups, including controls (Con) without any treatment, and 18 from gestational day (GD) 7 to GD17 L-NAME-treated rats, which were divided into stable siRNA-Int6 transfected (siRNA-Int6), negative vector control siRNA (NC-siRNA) and PE control (PE-Con) groups. All adenovirus siRNA transfections were performed on GD7 via intravenous tail injection. On GD0, GD11 and GD17, blood pressure, and on GD6 and GD17, protein estimations in 24 h urine samples were conducted. All animals were sacrificed on GD18. In the PE-Con group, placental Int6 was expressed to a significantly greater level than in the Con group, which was reversed by the application of siRNA-Int6. Blood pressure and proteinuria were significantly lower in the siRNA-Int6 group than in the PRE-Con group. As shown by CD31 and IB4 expression, placental micro-vascular density (MVD) was significantly higher in the siRNA-Int6 group than in the PE-Con and NC-siRNA groups, which has accompanied by enhanced trophoblast invasion. Int6 silencing alleviated the maternal clinical manifestations of pre-eclampsia and promoted placental angiogenesis in pregnant L-NAME-treated rats.

Figure 1

Flowchart of the present study.

Figure 2

Scheme of the placental tissue sampling for immunohistochemistry (IHC) analyses. Section A is located close to the fetal, section B in the center and section C close to the maternal side of the placenta.

Figure 3

Blood serum concentrations of Int6 and HIF2α. **P < 0.01, *P < 0.05. Con = control without pre-eclampsia; PE-Con = pre-eclampsia control without treatment; siRNA-Int6 = pre-eclampsia rats treated with siRNA against Int6; NC-siRNA = pre-eclampsia rats treated with siRNA vector negative control.

Figure 4

Blood serum concentrations of HIF2α related cytokines IL-6 and IL-8 and bFGF. ***P < 0.001, **P < 0.01, *P < 0.05. Con = control without pre-eclampsia; PE-Con = pre-eclampsia control without treatment; siRNA-Int6 = pre-eclampsia rats treated with siRNA against Int6; NC-siRNA = pre-eclampsia rats treated with siRNA vector negative control.

Figure 5

(a) SBP of each indicated group was measured non-invasively using the tail-cuff method on GD0, GD11 and GD17. (b) The proteinuria in each group was detected using CBB kits on GD6 and GD17. ***P < 0.001, **P < 0.01, *P < 0.05. Con = control without pre-eclampsia; PE-Con = pre-eclampsia control without treatment; siRNA-Int6 = pre-eclampsia rats treated with siRNA against Int6; NC-siRNA = pre-eclampsia rats treated with siRNA vector negative control.

Figure 6

Int6 expression in placental rat tissues derived from (A) close to the fetal side, (B) in the center and (C) close to the maternal side of the placentas. PE-Con = pre-eclampsia control without treatment; siRNA-Int6 = pre-eclampsia rats treated with siRNA against Int6; NC-siRNA = pre-eclampsia rats treated with siRNA vector negative control.

Figure 7

Immunohistochemical staining of Int-6 in placental tissues (section B) (blue is DAPI dye of the nuclei and brown is the Int-6 dye). (a–d) Represent the Con, PE-Con, siRNA-Int6 and NC-siRNA groups. Black arrows in (b) indicate cytosolic Int-6 and the red arrow indicate nucleic Int-6. (e) Immunoreactivity scores of the indicated groups. *P < 0.001. Con = control without pre-eclampsia; PE-Con = pre-eclampsia control without treatment; siRNA-Int6 = pre-eclampsia rats treated with siRNA against Int6; NC-siRNA = pre-eclampsia rats treated with siRNA vector negative control.

Figure 8

CD31 immunostaining in Con, PE-Con, NC-siRNA and siRNA-Int6 in slices derived from (A) close to the maternal side, (B) in the center and (C) close to the fetal side of rat placentas. Con = control without pre-eclampsia; PE-Con = pre-eclampsia control without treatment; siRNA-Int6 = pre-eclampsia rats treated with siRNA against Int6; NC-siRNA = pre-eclampsia rats treated with siRNA vector negative control.

Figure 9

CD31 immunostaining of placental tissue to reveal microvessels. A greater degree of small vessel neoformation (arrows) is visible in the siRNA-Int6 group compared to the other groups. (a–d) Represent the Con, PE-Con, siRNA-Int6 and NC-siRNA groups. E. Statistical analysis revealed that there was a clear trend toward increased MVD in the siRNA-Int6 group. *P < 0.05. Con = control without pre-eclampsia; PE-Con = pre-eclampsia control without treatment; siRNA-Int6 = pre-eclampsia rats treated with siRNA against Int6; NC-siRNA = pre-eclampsia rats treated with siRNA vector negative control.

Figure 10

IB4 (A,B) cytokeratin immunostaining in close to maternal (a) center (b) and close to the fetal sides (c) of placentas from si-RNA-Int6 and NC-siRNA treated rats. siRNA-Int6 = pre-eclampsia rats treated with siRNA against Int6; NC-siRNA = pre-eclampsia rats treated with siRNA vector negative control.

Figure 11

Influence on pregnancy outcomes. (a) The number of alive pups, (b) The weight of the pups (gr), (c) The placenta weights (gr), (d) Ratio of placenta and pup weights. *P < 0.05. Con = control without pre-eclampsia; PE-Con = pre-eclampsia control without treatment; siRNA-Int6 = pre-eclampsia rats treated with siRNA against Int6; NC-siRNA = pre-eclampsia rats treated with siRNA vector negative control.