Progesterone-induced blocking factor blockade causes hypertension in pregnant rats

Abstract

Preeclampsia (PE) is a multisystem disorder characterized by new onset hypertension in mid-late gestation and can include multi-organ dysfunction with or without proteinuria. It affects 5%-7% of all pregnancies in the U.S., making PE a major contributor to maternal and fetal morbidity and mortality. Currently, there is no cure for this pregnancy complication except for early delivery of the placenta and fetus. Moreover, the therapeutic options to treat PE are very limited. One potential trigger for the development of PE is progesterone deficiency-induced imbalance between T Helper 1(Th1)/Th2 cells, an increase in cytolytic natural killer (NK) cells and inflammatory cytokines that in turn leads to endothelial dysfunction, intrauterine growth restriction (IUGR) and hypertension. Importantly, progesterone signals the synthesis of progesterone-induced blocking factor (PIBF) which has anti-inflammatory effects and could promote the regulation of inflammation balance during pregnancy. However, the role of progesterone and PIBF in the pathophysiology of PE is still not fully understood. Thus, this current study was designed to test the hypothesis that inhibition of PIBF causes signs of PE in pregnant Sprague Dawley rats. In order to address our hypothesis, rabbit anti-PIBF IgG (0.25, low dose-LD or 0.50 mg/mL, high dose-HD) was administered intraperitoneally on gestation day (GD) 15 to normal pregnant Sprague Dawley (NP) rats. On GD 18, carotid catheters were inserted and on GD 19 mean blood pressure (MAP) and samples were collected for further analysis. MAP in normal pregnant rats (NP) rats (n = 7) was 99 ± 3 mmHg, which increased to 116 ± 2 mmHg in NP+ anti-PIBF LD (n = 10) and 113 ± 4 mmHg in NP+ anti-PIBF HD (n = 4), p <0 .05. Plasma TNF-alpha levels were 35 ± 8 pg/mL in NP rats and increased to 84 ± 21 pg/mL in NP+ Anti-PIBF HD (n = 4), p <0 .05. Plasma IL-4 and IL-10 levels were 22 ± 5 and 25+6 pg/mL in NP (n = 5), which decreased to 6 ± 1 and 8 ± 1 pg/mL in NP+ Anti-PIBF LD (n = 6, p < 0.05) and 16 ± 4 and 15 ± 5 pg/mL in NP+ Anti-PIBF HD (n = 4). Circulating total NK cells were 67 ± 11 % gate in NP rats (n = 3), which decreased to 28 ± 7% gate in NP+ Anti-PIBF LD and 45 ± 6% gate in NP+ Anti-PIBF HD. Cytolytic NK cells were increased in NP+ Anti-PIBF HD, p <0 .05. Moreover, circulating NO levels were significantly decreased while renal cortex PPET-1 levels increased NP+ Anti-PIBF HD. Our study demonstrates that PIBF blockade causes hypertension, inflammation and signs of endothelial dysfunction, all of which are associated with PE, thus indicating the importance of progesterone signalling pathways during a healthy pregnancy.

Keywords: inflammation; preeclampsia; pregnancy; progesterone-induced blocking factor.

Figure 1:

Gate Strategy for NK cells. Lymphocytes were gated in a forward (FSC) and side scatter (SSC) plot, and then doublets were excluded. Cells that stained as ANK61+ were designated as NK cells. Cells that stain as ANK44+ were designated as cytolytic NK cells.

Figure 2:

Gate Strategy for Th2 cells. Lymphocytes were gated in a forward (FSC) and side scatter (SSC) plot, and then doublets were excluded.

Figure 3.

PIBF blockade causes hypertension in pregnant Sprague Dawley rats. Statistical differences were established using a one-way ANOVA. Results were reported as means ± SEM and considered statistically significant when p<0.05. * p<0.05 vs. NP group.

Figure 4.

PIBF blockade increases inflammation in pregnant Sprague Dawley rats. (A) PIBF blockade high dose increases TNF-alpha levels compared to NP. (B) PIBF blockade low dose reduces IL-4 levels compared to NP group. (C) PIBF blockade low dose reduces IL-10 levels compared to NP group. Statistical differences were established using a one-way ANOVA. Results were reported as means ± SEM and considered statistically significant when p<0.05. * p<0.05 vs. NP control.

Figure 5.

PIBF blockade reduces placental Th2 cells in pregnant Sprague Dawley rats. Statistical differences were established using a one-way ANOVA. Results were reported as means ± SEM and considered statistically significant when p<0.05. * p<0.05 vs. NP control.

Figure 6.

PIBF blockade increases cytolytic Nk cells in in pregnant Sprague Dawley rats. Statistical differences were established using a one-way ANOVA. Results were reported as means ± SEM and considered statistically significant when p<0.05. * p<0.05 vs. NP control.

Figure 7.

PIBF blockade increases renal cortex PPET-1 and reduces nitrate nitrite levels in pregnant Sprague Dawley Rats. Statistical differences were established using Student t test. Results were reported as means ± SEM and considered statistically significant when p<0.05. * p<0.05 vs. NP control.