Tumor Necrosis Factor-alpha Blockade Improves Uterine Artery Resistance, Maternal Blood Pressure, and Fetal Growth in Placental Ischemic Rats

Abstract

We recently reported that adoptive transfer of cytolytic Natural Killer cells (cNKs) from the Reduced Uterine Perfusion Pressure (RUPP) rat induces a preeclampsia (PE)-like phenotype in pregnant rats, accompanied by increased TNF-α. The purpose of this study was to investigate a role for increased TNF-α to induce oxidative stress (ROS), decrease nitric oxide (NO) bioavailability, and induce vascular dysfunction as mechanisms of hypertension (HTN) and intrauterine growth restriction (IUGR) in RUPPs. Pregnant Sprague Dawley rats underwent the RUPP or a Sham procedure on gestation day (GD) 14. On GDs 15 and 18, a subset of Sham and RUPP rats received i.p.injections of vehicle or 0.4 mg/kg of Etanercept (ETA), a soluble TNF-α receptor (n = 10/group). On GD18, Uterine Artery Resistance Index (UARI) was measured, and on GD19, mean arterial pressure (MAP), fetal and placental weights were measured, and blood and tissues were processed for analysis. TNF-α blockade normalized the elevated MAP observed RUPP. Additionally, both fetal and placental weights were decreased in RUPP compared to Sham, and were normalized in RUPP + ETA. Placental ROS was also increased in RUPP rats compared to Sham, and remained elevated in RUPP + ETA. Compared to Sham, UARI was elevated in RUPPs while plasma total nitrate was reduced, and these were normalized in ETA treated RUPPs. In conclusion, TNF-α blockade in RUPPs reduced MAP and UARI, improved fetal growth, and increased NO bioavailability. These data suggest that TNF-α regulation of NO bioavailability is a potential mechanism that contributes to PE pathophysiology and may represent a therapeutic target to improve maternal outcomes and fetal growth.

Keywords: Inflammation; Intrauterine Growth Restriction; Preeclampsia; Tumor Necrosis Factor-alpha.

Figure 1:. Effect of TNF-α Blockade on Mean Arterial Pressure and Intrauterine Growth Restriction in Pregnant Rats.

On Gestation Day (GD) 14, either the Reduced Uterine Perfusion Pressure (RUPP) procedure or a Sham procedure was performed on pregnant Sprague Dawley rats. On GD 15 and 18, vehicle or 0.4 mg/kg Etanercept (ETA) was injected i.p. in a subset of Sham and RUPP rats. On GD 19, conscious Mean Arterial Pressure (A) was measured and fetal weights, (B) placental weights (C),and fetal resorptions (D) were recorded under isoflurane anesthesia. Sham n=10; RUPP n=10; Sham+ETA n=10; RUPP+ETA n=10. All data are expressed as mean ± SEM. Statistical analyses were performed using two-way ANOVA with multiple comparisons followed by Tukey’s post-hoc test. *p<0.05 versus Sham; #p<0.05 vs RUPP.

Figure 2:. Effect of TNF-α Blockade on Uterine Artery Resistance Index (UARI), Circulating Total Nitrate and 8-Isoprostane, and Placental cGMP and Reactive Oxygen Species (ROS).

On Gestation Day (GD) 14, either the Reduced Uterine Perfusion Pressure (RUPP) procedure or a Sham procedure was performed on pregnant Sprague Dawley rats. On GD 15 and 18, vehicle or 0.4 mg/kg Etanercept (ETA) was injected i.p. in a subset of Sham and RUPP rats. On GD18, UARI (A) was measured via Doppler Ultrasound and on GD19, blood and placentas were collected under isoflurane anesthesia and frozen for analysis. Plasma was used to measure circulating total nitrate (B) and circulating 8-isoprostane (C) levels. Additionally, placental cGMP (D) and placental ROS (E) were measured. Sham: n=5–10; RUPP: n=5–10; Sham+ETA: n=5–10; RUPP+anti-IFNγ: n=5–10. All data are expressed as mean ± SEM. Statistical analyses were performed using two-way ANOVA with multiple comparisons followed by Tukey’s post-hoc test. *p<0.05 versus Sham; #p<0.05 vs RUPP.

Figure 3:. Effect of TNF-α Blockade on Circulating and Placental Angiogenic Factors.

On Gestation Day (GD) 14, either the Reduced Uterine Perfusion Pressure (RUPP) procedure or a Sham procedure was performed on pregnant Sprague Dawley rats. On GD 15 and 18, vehicle or 0.4 mg/kg Etanercept was injected i.p. in a subset of Sham and RUPP rats. On GD 19, blood and placentas were collected under isoflurane anesthesia and processed for further analysis. Circulating sFlt-1(A) and VEGF(B) were measured via ELISA and the sFlt-1:VEGF ratio (C) is shown. Placental VEGF (D) was measured using the Bio-Plex Pro Rat Cytokine Immunoassay Kit. Sham: n=9–10; RUPP: n=10; Sham+ETA: n=9–10; RUPP+ETA: n=9–10. All data are expressed as mean ± SEM. Statistical analyses were performed using two-way ANOVA with multiple comparisons followed by Tukey’s post-hoc test. *p<0.05 versus Sham; #p<0.05 vs RUPP.

Figure 4:. Effect of TNF-α Blockade on Circulating and Placental Cytokines.

On Gestation Day (GD) 14, either the Reduced Uterine Perfusion Pressure (RUPP) procedure or a Sham procedure was performed on pregnant Sprague Dawley rats. On GD 15 and 18, vehicle or 0.4 mg/kg Etanercept was injected i.p. in a subset of Sham and RUPP rats. On GD19, blood and placentas were collected under isoflurane anesthesia and frozen for analysis of circulating TNF-α (A), circulating IL-6 (B), circulating IFNγ (C), placental TNF-α (D), placental IL-6 (E), and placental IFNγ (F). Sham: n=8–9; RUPP: n=8–10; Sham+ETA: n=7–9; RUPP+ETA: n=7–10. All data are expressed as mean ± SEM. Statistical analyses were performed using two-way ANOVA with multiple comparisons followed by Tukey’s post-hoc test. *p<0.05 versus Sham; #p<0.05 vs RUPP.

Figure 5:. Western Blot analysis of placental eNOS expression.

On Gestation Day (GD) 14, either the Reduced Uterine Perfusion Pressure (RUPP) procedure or a Sham procedure was performed on pregnant Sprague Dawley rats. On GD 15 and 18, vehicle or 0.4 mg/kg Etanercept was injected i.p. in a subset of Sham and RUPP rats. On GD19, placentas were collected following sacrifice and processed for analysis of placental eNOS expression. (A).Quantified fold change of placental eNOS expression normalized to β-actin. (B) A representative blot of eNOS and β-actin expression in all groups is shown. Sham: n=6; RUPP: n=6; Sham+ETA: n=6; RUPP+ETA: n=6. All data are expressed as mean ± SEM. Statistical analyses were performed using two-way ANOVA with multiple comparisons followed by Tukey’s post-hoc test. *p<0.05 versus Sham; #p<0.05 vs RUPP.

Figure 6:. Western Blot analysis of placental eNOS S1176 phosphorylation.

On Gestation Day (GD) 14, either the Reduced Uterine Perfusion Pressure (RUPP) procedure or a Sham procedure was performed on pregnant Sprague Dawley rats. On GD 15 and 18, vehicle or 0.4 mg/kg Etanercept was injected i.p. in a subset of Sham and RUPP rats. On GD19, placentas were collected following sacrifice and processed for analysis of placental eNOS expression. (A).Quantified fold change of placental eNOS expression normalized to β-actin. (B) A representative blot of eNOS pS1176 and β-actin expression in all groups is shown. Sham: n=6; RUPP: n=6; Sham+ETA: n=5; RUPP+ETAγ: n=6. All data are expressed as mean ± SEM. Statistical analyses were performed using two-way ANOVA with multiple comparisons followed by Tukey’s post-hoc test. *p<0.05 versus Sham; #p<0.05 vs RUPP.