Complement activation is critical for placental ischemia-induced hypertension in the rat

Abstract

Preeclampsia is a major obstetric problem defined by new-onset hypertension and proteinuria associated with compromised placental perfusion. Although activation of the complement system is increased in preeclampsia compared to normal pregnancy, it remains unclear whether excess complement activation is a cause or consequence of placental ischemia. Therefore, we hypothesized that complement activation is critical for placental ischemia-induced hypertension. We employed the reduced utero-placental perfusion pressure (RUPP) model of placental ischemia in the rat to induce hypertension in the third trimester and evaluated the effect of inhibiting complement activation with a soluble recombinant form of an endogenous complement regulator, human complement receptor 1 (sCR1; CDX-1135). On day 14 of a 21-day gestation, rats received either RUPP or Sham surgery and 15 mg/kg/day sCR1 or saline intravenously on days 14-18. Circulating complement component 3 decreased and complement activation product C3a increased in RUPP vs. Sham (p<0.05), indicating complement activation had occurred. Mean arterial pressure (MAP) measured on day 19 increased in RUPP vs. Sham rats (109.8±2.8 mmHg vs. 93.6±1.6 mmHg). Treatment with sCR1 significantly reduced elevated MAP in RUPP rats (98.4±3.6 mmHg, p<0.05) and reduced C3a production. Vascular endothelial growth factor (VEGF) decreased in RUPP compared to Sham rats, and the decrease in VEGF was not affected by sCR1 treatment. Thus, these studies have identified a mechanistic link between complement activation and the pregnancy complication of hypertension apart from free plasma VEGF and have identified complement inhibition as a potential treatment strategy for placental ischemia-induced hypertension in preeclampsia.

Figure 1

sCR1 significantly inhibits placental ischemia-induced increase in mean arterial pressure (MAP). The increase in MAP in RUPP Saline (n=22) compared to Sham Saline rats (n=19) was significantly inhibited by daily iv administration of 15 mg/kg sCR1 (RUPP sCR1, n=9). MAP did not differ between Sham sCR1 (n=6) and Sham Saline groups. Values represent mean ± SE of MAP measured day 19 of gestation (term=21 days). *p<0.05 for indicated comparisons.

Figure 2

Complement component C3 is reduced in serum obtained day 19 of gestation in RUPP Saline (n=10) compared to Sham Saline rats (n=12). Units of C3 are relative to a rat serum standard as described in Materials and Methods. *p<0.05 vs Sham.

Figure 3

sCR1 significantly inhibits placental ischemia-induced increase in C3a. C3a concentrations increased in RUPP Saline (n=13) compared to Sham Saline dams (n=12). Treatment with 15 mg/kg/day sCR1 decreased C3a serum concentrations in both RUPP (n=5) and Sham (n=6) groups compared to RUPP Saline. Values represent geometric mean ± SE of C3a units/μl in serum obtained day 19 of gestation. Units of C3a are relative to a standard pool of yeast activated rat serum as described in Materials and Methods. *p<0.05 for indicated comparisons, #p<0.05 comparing Sham Saline to Sham sCR1.

Figure 4

Treatment with 15 mg/kg/day sCR1 for 5 days significantly reduced CH₅₀ in serum of RUPP and Sham animals. The CH₅₀ in RUPP Saline (n=12) and Sham Saline (n=12) groups were not different. Treatment with sCR1 significantly decreased CH₅₀ in serum collected on day 19 from RUPP sCR1 (n=5) and Sham sCR1 (n=6) compared to controls. Values represent geometric mean ± SE of CH₅₀ in serum collected day 19 of gestation. *p<0.05 for indicated comparisons, #p<0.05 comparing Sham Saline to Sham sCR1. Sham sCR1 vs RUPP sCR1 was also tested for this outcome and was statistically different, p<0.05.

Figure 5

sCR1 treatment did not reverse RUPP-induced decrease in free VEGF concentrations in plasma. RUPP Saline animals (n= 21) had a decrease in VEGF compared to Sham Saline animals (n= 19). VEGF concentrations did not change in RUPP sCR1 animals (n=9) compared to RUPP Saline animals. Concentration of VEGF did not differ in Sham Saline and Sham sCR1 (n=6) animals. Values represent geometric mean ± SE of free VEGF measured by ELISA in plasma obtained from animals day 19 of gestation. *p<0.05 for indicated comparisons.

Figure 6

Effect of placental ischemia and sCR1 on relaxation responses of carotid artery in vitro. Values represent mean ± SE of the fraction relaxation of arteries pre-contracted with thromboxane mimetic U46619. A. Carotid arteries isolated from RUPP Saline (n=16) or Sham Saline (n=13) animals relaxed to increasing concentrations of acetylcholine with no significant treatment effect. B. sCR1 enhances endothelial-independent relaxation in carotid arteries isolated from RUPP and Sham animals. Carotid arteries isolated from animals either treated with saline or sCR1 were pre-contracted with U46619 and the fraction relaxation to increasing concentrations of sodium nitroprusside (SNP) determined. *Repeated measures ANOVA demonstrated a significant sCR1 effect in carotid arteries from RUPP and Sham animals. Values represent geometric mean ± SE of 6–16 animals is each group.

Figure 7

sCR1 treatment did not affect RUPP-induced fetal resorptions. RUPP Saline animals (n= 22) had an increase in resorptions compared to Sham Saline animals (n= 19). The fraction of fetal resorptions did not change in RUPP sCR1 animals (n=9) compared to RUPP Saline animals. Values for Sham Saline did not differ compared to Sham sCR1 (n=6). Values represent mean ± SE of the fraction of resorbed fetuses on day 19 of gestation. *p<0.05 for indicated comparisons.