Inhibition of the AT1R agonistic autoantibody in a rat model of preeclampsia improves fetal growth in late gestation

Abstract

Placenta ischemia, the initiating event in preeclampsia (PE), is associated with fetal growth restriction. Inhibition of the agonistic autoantibody against the angiotensin type 1 receptor AT₁-AA, using an epitope-binding inhibitory peptide ('n7AAc') attenuates increased blood pressure at gestational day (G)19 in the clinically relevant reduced uterine perfusion pressure (RUPP) model of PE. Thus we tested the hypothesis that maternal administration of 'n7AAc' does not transfer to the fetus, improves uterine blood flow and fetal growth, and attenuates elevated placental expression of miRNAs implicated in PE and FGR. Sham or RUPP surgery was performed at G14 with vehicle or 'n7AAc' (144 µg/day) administered via an osmotic pump from G14 to G20. Maternal plasma levels of the peptide on G20 were 16.28 ± 4.4 nM, and fetal plasma levels were significantly lower at 1.15 ± 1.7 nM (P = 0.0007). The uterine artery resistance index was significantly elevated in RUPP (P < 0.0001) but was not increased in 'n7AAc'-RUPP or 'n7AAc'-Sham versus Sham. A significant reduction in fetal weight at G20 in RUPP (P = 0.003) was not observed in 'n7AAc'-RUPP. Yet, percent survival was reduced in RUPP (P = 0.0007) and 'n7AAc'-RUPP (P < 0.0002). Correlation analysis indicated the reduction in percent survival during gestation was specific to the RUPP (r = 0.5342, P = 0.043) and independent of 'n7AAc'. Placental miR-155 (P = 0.0091) and miR-181a (P = 0.0384) expression was upregulated in RUPP at G20 but was not elevated in 'n7AAc'-RUPP. Collectively, our results suggest that maternal administration of 'n7AAc' does not alter fetal growth in the RUPP implicating its potential as a therapeutic for the treatment of PE.NEW & NOTEWORTHY The seven amino acid inhibitory peptide to the AT₁-AA ('n7AAc') has limited transfer to the fetus at gestational day 20, improves uterine blood flow and fetal growth in the reduced uterine perfusion pressure model of preeclampsia (PE), and does not impair fetal survival during gestation in sham-operated or placental ischemic rats. Collectively, these findings suggest that maternal administration of 'n7AAc' as an effective strategy for the treatment of PE is associated with improved outcomes in the fetus.

Keywords: 'n7AAc'; AT1-AA blockade; fetal growth restriction; intrauterine growth restriction; microRNAs; preeclampsia.

Graphical abstract

Figure 1.

Biodistribution of the 7 amino acid inhibitory peptide. A: plasma levels of a NH₂-terminal rhodamine-labeled [5(6)-carboxytetramethyrhodamine] seven amino acid (7aa) inhibitory peptide to the angiotensin type II receptor agonistic antibody (AT₁-AA) from gestational day 14 (G14) to gestational day 20 (G20) in infused pregnant rats (n = 4). B: maternal and fetal plasma concentration of the rhodamine-labeled AT₁-AA 7aa inhibitory peptide from infused pregnant rats (n = 4) euthanized on G20. C: representative ex vivo fluorescence images of the placenta and fetal pups euthanized on G20 from saline or rhodamine-labeled AT₁-AA 7aa inhibitory peptide (n = 4)-infused pregnant rats (top, full litter; bottom, close up of representative placenta and fetus) at G20. For A and B, each scatter plot represents means ± SD. P = 0.0007, statistical significance.

Figure 2.

Uterine artery resistance index (UARI) measurement by Doppler ultrasound. UARI was measured in sham-operated and reduced uterine perfusion pressure (RUPP)-pregnant rats treated with saline or the novel seven amino acid inhibitory peptide against the agonistic autoantibody to the angiotensin type II receptor ('n7AAc') on gestational day 20 (G20) by Doppler sonography. A–D: representative UARI figures. Thin white arrow in B represents an early diastolic notch in the flow velocity waveform. Sham, n = 8; RUPP, n = 10; 'n7AAc'-Sham, n = 9; 'n7AAc'-RUPP, n = 10. For E, each scatter plot represents mean ± SE. EDV, end-diastolic velocity; PSV, peak systolic velocity. Two-way ANOVA with Dunnett’s multiple comparison test was used for comparisons. P < 0.05, statistical significance.

Figure 3.

Average fetal weight and morphometrics of viable pups at G20. A–D: average fetal weight (A), average placental weight (B), average brain weight (C), and average liver weight (D) of viable pups per litter at gestational day 20 (G20) in sham-operated and reduced uterine perfusion pressure (RUPP) dams treated with saline or the novel seven amino acid inhibitory peptide against the agonistic autoantibody to the angiotensin type II receptor ('n7AAc'). Sham, n = 8; RUPP, n = 10; 'n7AAc'-Sham, n = 9; 'n7AAc'-RUPP, n = 10. Each data point represents an average of viable pups per litter. Scatter plots represent means ± SE. Two-way ANOVA with Dunnett’s multiple comparison test was used for comparisons. P < 0.05, statistical significance.

Figure 4.

Number of viable pups and percentage of survival. A: number of viable pups per litter at gestational day 20 (G20). B: percent survival of viable pups [percent survival = number of viable pups at G20/number of viable embryos at gestational day 14 (G14) × 100]. Groups include Sham-operated and reduced uterine perfusion pressure (RUPP) dams treated with saline or the novel seven amino acid inhibitory peptide against the agonistic autoantibody to the angiotensin type II receptor ('n7AAc'). Sham, n = 8; RUPP, n = 10; 'n7AAc'-Sham, n = 9; 'n7AAc'-RUPP, n = 10 on G20. Each data point represents an average of viable pups per litter. Scatter plots represent means ± SE. Two-way ANOVA with Dunnett’s multiple comparison test was used for comparisons. P < 0.05, statistical significance.

Figure 5.

Correlation of fetal weight of viable pups at gestational day 20 (G20) and percent survival. Relationship between fetal weight of viable offspring at G20 and percent survival at G20. Sham-operated (A) or reduced uterine perfusion pressure (RUPP) (B) dams treated with saline or the novel seven amino acid inhibitory peptide against the agonistic autoantibody to the angiotensin type II receptor ('n7AAc'). Pearson’s correlation analysis: r = 0.3097, P= 0.2110 (A); r = 0.5342, P = 0.0403 (B). P < 0.05, statistical significance.

Figure 6.

miRNA-181a and miRNA-155 expression. Total RNA extracted from the placenta of viable pups from sham-operated and reduced uterine perfusion pressure (RUPP) dams at gestational day 20 (G20) that were treated with saline or the novel seven amino acid inhibitory peptide against the agonistic autoantibody to the angiotensin type II receptor ('n7AAc') was used to quantify miRNA expression. Sham, n = 8; RUPP, n = 10; 'n7AAc'-Sham, n = 9; 'n7AAc'-RUPP, n = 10. Placental expression levels for miRNA 181a (A) and miRNA-155 (B) were measured using quantitative RT-PCR. Scatter plots represent means ± SE. Two-way ANOVA with Dunnett’s multiple comparison test was used for comparisons. P < 0.05, statistical significance.