Interactions between the complement and endothelin systems in normal pregnancy and following placental ischemia

Abstract

Preeclampsia is characterized by new onset hypertension and fetal growth restriction and is associated with aberrant activation of the innate immune complement system and stressed or ischemic placenta. Previous studies have suggested a role for both endothelin and complement system activation products in new onset hypertension in pregnancy, but inter-relationships of the pathways are unclear. We hypothesized that complement activation following placental ischemia stimulates the endothelin pathway to cause hypertension and impair fetal growth. The Reduced Uterine Perfusion Pressure (RUPP) model results in hypertension and fetal growth restriction in a pregnant rat due to placental ischemia caused by mechanical obstruction of blood flow to uterus and placenta. The effect of inhibitor of complement activation soluble Complement Receptor 1 (sCR1) and endothelin A receptor (ET_A) antagonist atrasentan on hypertension, fetal weight, complement activation (systemic circulating C3a and local C3 placental deposition) and endothelin [circulating endothelin and message for preproendothelin (PPE), ET_A and endothelin B receptor (ET_B) in placenta] in the RUPP rat model were determined. Following placental ischemia, sCR1 attenuated hypertension but increased message for PPE and ET_A in placenta, suggesting complement activation causes hypertension via an endothelin independent pathway. With ET_A antagonism the placental ischemia-induced increase in circulating C3a was unaffected despite inhibition of hypertension, indicating systemic C3a alone is not sufficient. In normal pregnancy, inhibiting complement activation increased plasma endothelin but not placental PPE message. Atrasentan treatment increased fetal weight, circulating endothelin and placental ET_A message, and unexpectedly increased local complement activation in placenta (C3 deposition) but not C3a in circulation, suggesting endothelin controls local placental complement activation in normal pregnancy. Atrasentan also significantly decreased message for endogenous complement regulators Crry and CD55 in placenta and kidney in normal pregnancy. Results of our study indicate that complement/endothelin interactions differ in pregnancies complicated with placental ischemia vs normal pregnancy, as well as locally vs systemically. These data clearly illustrate the complex interplay between complement and endothelin indicating that perturbations of either pathway may affect pregnancy outcomes.

Keywords: Complement; Endothelin; Placental ischemia; Pregnancy.

Fig. 1.

Effect of inhibitor of complement activation, sCR1, on average fetal and placental weight and the endothelin system. Animals were treated daily with 15 mg/kg sCR 1 or saline (Veh) iv from GD14-18 and plasma endothelin measured by ELISA in plasma collected from the abdominal aorta at GD19. mRNA was isolated from GD19 placenta and the Delta delta Ct method of relative quantification was used to determine fold change in mRNA expression compared to ß actin with change in Sham Veh defined as 1. **p<0.05 for main surgery effect by ANOVA, *p<0.05 for indicated post hoc comparisons. Values represent the mean ± SE in Veh (n=10-20) or sCR1 treated animals (n=5-11). A. RUPP surgery significantly decreased fetal and placental weight and sCR1 exacerbated the effect following placental ischemia. B. sCR1 treatment increased PPE and ET_A message following placental ischemia. C. Post hoc comparisons demonstrated a significant increase in plasma endothelin in Sham animals treated with sCR1.

Fig. 2.

ET_A antagonist atrasentan inhibits placental ischemia induced increase in mean arterial pressure, increases circulating endothelin and increases ET_A message. Animals received drinking water ad lib either with or without 50 or 75 ug/ml atrasentan from GD13-19 and mean arterial pressure and average fetal and placental weight determined on GD19. Plasma endothelin was measured by ELISA in plasma collected from the abdominal aorta at GD19. mRNA was isolated from GD19 placenta and the Delta delta Ct method of relative quantification was used to determine fold change in mRNA expression compared to ß actin with change in Sham Water defined as 1. **p<0.05 for main surgery effect by ANOVA, *p<0.05 for indicated post hoc comparisons. Values represent the mean ± SE of mean arterial pressure or average fetal weight measured GD19 in water (n=7-10) or atrasentan treated animals (n=5-7). A. The increase in mean arterial pressure in RUPP animals was decreased by treatment with atrasentan. Atrasentan also significantly decreased the mean arterial pressure in Sham animals. B. RUPP surgery significantly decreased average fetal and placental weight as determined by ANOVA analysis. Post hoc comparison indicated increased fetal weight in Sham animals. C. By ANOVA RUPP surgery did not significantly increase PPE message in placenta. Post hoc comparisons indicated that PPE message was significantly increased in RUPP animals treated with atrasentan. For ET_A., ANOVA analysis demonstrated a significant surgery and treatment effect, with increased ET_A in Sham animals detected post hoc.

Fig. 3.

Effect of the ET_A antagonist atrasentan on complement activation. Animals were treated with either 50 or 75 ug/ml atrasentan in drinking water from GD13-19 and placenta, kidney cortex and serum obtained at GD19. Values represent the mean ± SE in water (n=7-12) or atrasentan treated animals (n=5-6). Serum C3a was determined by Western blot with units of C3a relative to a standard pool of yeast activated rat serum as described in Methods. C3 deposition in GD19 placenta was determined by immunohistochemistry with staining graded by two blinded observers from 0 to 3, negative to strongly positive vs isotype control. **p<0.05 for main surgery effect. *p<0.05 for indicated comparisons. The RUPP-induced increase in C3a was not significantly altered by atrasentan. Atrasentan treatment significantly increased C3 deposition in Sham animals, and C3 deposition was significantly increased comparing Sham Water to RUPP Water animals.

Fig 4

Effect of the ET_A antagonist atrasentan on message for complement regulators. A. Animals were treated with either 50 or 75 ug/ml atrasentan in drinking water from GD13-19 and placenta and kidney cortex obtained at GD19. Values represent the mean ± SE in water (n=7-12) or atrasentan treated animals (n=5-6). Atrasentan treatment in vivo significantly decreased message for complement regulators Crry and CD55 in placenta and kidney cortex of Sham animals. B. IEC-6 cells were treated with either 0, 10, 20 or 40 ug/ml atrasentan in media for 24 hours and the change in message for complement regulators Crry and CD55 determined in cells harvested after 24 hours of treatment. mRNA was isolated from cells and the Delta-delta Ct method of relative quantification used to determine fold change in mRNA expression compared to ß actin with change in media alone defined as 1. Values represent the mean ± SE of the change from media alone in 4-5 different experiments. *p<0.05 for indicated comparisons.