Nanoparticle-mediated transgene expression of insulin-like growth factor 1 in the growth restricted guinea pig placenta increases placenta nutrient transporter expression and fetal glucose concentrations

Abstract

Fetal growth restriction (FGR) significantly contributes to neonatal and perinatal morbidity and mortality. Currently, there are no effective treatment options for FGR during pregnancy. We have developed a nanoparticle gene therapy targeting the placenta to increase expression of human insulin-like growth factor 1 (hIGF1) to correct fetal growth trajectories. Using the maternal nutrient restriction guinea pig model of FGR, an ultrasound-guided, intraplacental injection of nonviral, polymer-based hIGF1 nanoparticle containing plasmid with the hIGF1 gene and placenta-specific Cyp19a1 promotor was administered at mid-pregnancy. Sustained hIGF1 expression was confirmed in the placenta 5 days after treatment. Whilst increased hIGF1 did not change fetal weight, circulating fetal glucose concentration were 33%-67% higher. This was associated with increased expression of glucose and amino acid transporters in the placenta. Additionally, hIGF1 nanoparticle treatment increased the fetal capillary volume density in the placenta, and reduced interhaemal distance between maternal and fetal circulation. Overall, our findings, that trophoblast-specific increased expression of hIGF1 results in changes to glucose transporter expression and increases fetal glucose concentrations within a short time period, highlights the translational potential this treatment could have in correcting impaired placental nutrient transport in human pregnancies complicated by FGR.

Keywords: fetal growth restriction; insulin-like 1 growth factor; placenta; pregnancy; therapeutic.

FIGURE 1

Representative images of in situ hybridization (ISH) for plasmid-specific mRNA expression in the guinea pig placenta 5 days after intra-placental nanoparticle treatment. (a) ISH (first panel) confirmed plasmid-specific hIGF1 expression in the guinea pig sub-placenta/decidua 5 days after nanoparticle treatment. Serial sectioning and immunohistochemistry (second panel) confirmed plasmid-specific hIGF1 was localized to trophoblast cells. (b) Saline injected tissue was used as a negative control and no positive staining for plasmid-specific hIGF1 was observed. Representative images at low magnification (top row) and high magnification (bottom row). Arrows = the presence of red dots indicating plasmid-specific hIGF1. n = 2 sham dams and 4 nanoparticle dams. Scale bar top row = 200 μm, scale bar bottom row = 50 μm. hIGF1, human insulin-like growth factor 1; mRNA, messenger RNA

FIGURE 2

Effect of maternal nutrient restriction (MNR) diet and hIGF1 nanoparticle treatment on mid-pregnancy maternal, fetal and maternal-fetal interface weight. (a) At time of sacrifice, maternal carcass weight (maternal weight minus total fetal and maternal-fetal interface weight) was lower in the MNR group compared to control diet group. (b) Maternal liver weight as a percentage of carcass weight was not different with either diet or hIGF1 nanoparticle treatment. (c) Maternal spleen weight at a percentage of carcass weight was not different with either diet or hIGF1 nanoparticle treatment. (d) MNR resulted in approximately 22%–25% reduction in fetal weight at mid-pregnancy, and there was no effect of placental hIGF1 nanoparticle treatment in MNR fetuses. (e) MNR reduced maternal-fetal interface weight at mid-pregnancy, which was also not affected by hIGF1 nanoparticle treatment. (f) There was no difference in maternal-fetal interface efficiency (fetal to maternal-fetal interface weight ratio) with either MNR diet or hIGF1 nanoparticle treatment. Fetal sex was not a significant factor for any of the fetal/placental outcomes. n = 7 control dams (21 fetuses/placentas), 5 MNR dams (14 fetuses/placentas) and 7 MNR + hIGF1 dams (19 fetuses/placentas). Data are estimated marginal means ± 95% confidence interval. All p values calculated using generalized estimating equations with Bonferroni post hoc analysis. Asterix denote a significant difference of p ≤ 0.05. hIGF1, human insulin-like growth factor 1

FIGURE 3

Effect of maternal nutrient restriction (MNR) diet and hIGF1 nanoparticle treatment on mid-pregnancy placenta microstructure. (a) Compared to control, trophoblast volume density was increased in the MNR placentas but not different in the MNR + hIGF1 placentas. (b). There was no change to the maternal blood space (MBS) volume density with either diet or hIGF1 nanoparticle treatment. (c) hIGF1 nanoparticle treatment of MNR placentas increased fetal capillary volume density compared to both control and MNR placentas. (d) There was a reduction in the interhaemal distance (distance between MBS and fetal circulation) in MNR + hIGF1 placentas compared to control and MNR placentas. There was no effect of fetal sex on any of the outcomes assessed. n = 7 control dams (21 placentas), 5 MNR dams (14 placentas) and 7 MNR + hIGF1 dams (19 placentas). Data are estimated marginal means ± 95% confidence interval. All p values calculated using generalized estimating equations with Bonferroni post hoc analysis. Asterix denote a significant difference of p ≤ 0.05. hIGF1, human insulin-like growth factor 1

FIGURE 4

Effect of maternal nutrient restriction (MNR) diet and hIGF1 nanoparticle treatment on maternal and fetal blood glucose concentrations. (a) There was no difference in maternal blood glucose concentrations at mid pregnancy with either diet or hIGF1 nanoparticle treatment. (b) Glucose concentrations were increased in MNR + hIGF1 fetuses compared to control and MNR fetuses. n = 6 control dams (14 fetuses), 5 MNR dams (10 fetuses) and 7 MNR + hIGF1 dams (17 fetuses). Data are estimated marginal means ± 95% confidence interval. All p values calculated using generalized estimating equations with Bonferroni post hoc analysis. Asterix denote a significant difference of p ≤ 0.05. hIGF1, human insulin-like growth factor 1

FIGURE 5

Effect of maternal nutrient restriction (MNR) diet and hIGF1 nanoparticle treatment on placental glucose transporter expression. (a) mRNA expression of Slc2A1 was reduced in MNR + hIGF1 placentas compared to MNR, but not different to control. (b) There was no difference in protein expression of Slc2A1 in control, MNR or MNR + hIGF1 placentas. Representative western blot: lanes 1 and 2 = control, lanes 3 and 4 = MNR, lanes 5 and 6 = MNR+ hIGF1. (c) Representative images of immunohistochemical staining for Slc2A1 showing relocalization of Slc2A1 to the apical membrane of the placental syncytium (arrow head). Image 1 = control, image 2 = MNR, image 3 = MNR + hIGF1. Asterix represents maternal blood space. (d) mRNA expression of Slc2A3 was reduced in MNR placentas compared to control, but increased back to normal levels in MNR + hIGF1 placentas. (e) There was also increased protein expression of Slc2A3 in MNR + hIGF1 placentas compared to control and MNR. Representative western blot: lanes 1 and 2 = control, lanes 3 and 4 = MNR, lanes 5 and 6 = MNR + hIGF1. n = 6 control dams (14 fetuses), 5 MNR dams (10 fetuses) and 7 MNR + hIGF1 dams (17 fetuses). Data are estimated marginal means ± 95% confidence interval. All p values calculated using generalized estimating equations with Bonferroni post hoc analysis. Asterix denote a significant difference of p ≤ 0.05. hIGF1, human insulin-like growth factor 1

FIGURE 6

Effect of maternal nutrient restriction (MNR) diet and hIGF1 nanoparticle treatment placenta amino acid transporter mRNA and protein expression. (a) mRNA expression of Slc38A1 was increased in MNR + hIGF1 placentas compared to control. (b) mRNA expression of Slc38A2 was reduced in MNR placentas compared to control, but increased to normal levels in the MNR + hIGF1 placentas. (c) There was no difference in protein expression of Slc38A1 between the groups. Representative western blot: lanes 1 and 2 = control, lanes 3 and 4 = MNR, lanes 5 and 6 = MNR + hIGF1. n = 6 control dams (14 placentas), 5 MNR dams (10 placentas) and 7 MNR + hIGF1 dams (17 placentas). Data are estimated marginal means ± 95% confidence interval. All p values calculated using generalized estimating equations with Bonferroni post hoc analysis. Asterix letters denote a significant difference of p ≤ 0.05. hIGF1, human insulin-like growth factor 1