Dysregulation of angiopoietins is associated with placental malaria and low birth weight

Abstract

Background: Placental malaria (PM) is associated with adverse pregnancy outcomes including low birth weight (LBW). However, the precise mechanisms by which PM induces LBW are poorly defined. Based on the essential role of angiopoietin (ANG)-1 and -2 in normal placental vascular development, we hypothesized that PM may result in the dysregulation of angiopoietins and thereby contribute to LBW outcomes.

Methods and findings: In a mouse model of PM, we show that Plasmodium berghei ANKA infection of pregnant mice resulted in dysregulated angiopoietin levels and fetal growth restriction. PM lead to decreased ANG-1, increased ANG-2, and an elevated ratio of ANG-2/ANG-1 in the placenta and the serum. These observations were extended to malaria-exposed pregnant women: In a study of primigravid women prospectively followed over the course of pregnancy, Plasmodium falciparum infection was associated with a decrease in maternal plasma ANG-1 levels (P = 0.031) and an increase in the ANG-2:ANG-1 ratio (P = 0.048). ANG-1 levels recovered with successful treatment of peripheral parasitemia (P = 0.010). In a cross-sectional study of primigravidae at delivery, angiopoietin dysregulation was associated with PM (P = 0.002) and LBW (P = 0.041). Women with PM who delivered LBW infants had increased ANG-2:ANG-1 ratios (P = 0.002) compared to uninfected women delivering normal birth weight infants.

Conclusions: These data support the hypothesis that dysregulation of angiopoietins is associated with PM and LBW outcomes, and suggest that ANG-1 and ANG-2 levels may be clinically informative biomarkers to identify P. falciparum-infected mothers at risk of LBW deliveries.

Figure 1. Experimental model recapitulates the spontaneous abortion and low birth weight outcomes characteristic of PM.

(A) Pregnant BALB/c females infected (dark bars) with P. berghei on gestational day (G)13 have an increased rate of abortion and decreased proportion of viable pups per litter compared to uninfected mice (light bars) by G19, day 6 of infection/control injection (D6). Proportion of viable fetuses depicted by solid bars; aborted fetuses, by striped bars. (B) Body weight of viable fetuses is decreased with maternal malaria infection (dark symbols) as compared to weight of fetuses from uninfected mice (light symbols). Dots are individual viable fetuses; bars represent the median of each group. 20–83 fetuses were collected per group (from 4–13 pregnant females per group). *** P<0.001 (Mann-Whitney).

Figure 2. Placental Angpt2 mRNA expression is increased by malaria infection (P = 0.0063, 2-way ANOVA).

(A) Angpt1 and (B) Angpt2 transcripts were measured by real-time quantitative PCR using cDNA templates reverse transcribed from placental RNA from pregnant mice uninfected (light bars) and infected with P. berghei (dark bars). Copy number was normalized to housekeeping gene expression as described in Materials and Methods. (C) The relative expression of Angpt2/Angpt1 is also increased in placentas associated with viable fetuses of infected mice (P = 0.0032, 2-way ANOVA on log-transformed data). Dots are individual placentas associated with viable fetuses; bars represent the median of each group. 4–6 mice are represented per group. *P<0.01 (Bonferonni post-test). D, day post infection/control injection; G, gestational day.

Figure 3. Placental ANG-1 and ANG-2 protein levels are dysregulated by malaria infection.

(A) ANG-1 and (B) ANG-2 protein levels in placentas from pregnant mice uninfected (light bars) and infected with P. berghei (dark bars). Protein levels were detected by Western blot, quantified by densitometry and normalized to intensity of β-actin bands. Dots are individual placentas associated with viable fetuses; bars represent the median of each group. 3–13 mice are represented per group. **P<0.01, ∧ P = 0.053 (unpaired t-test). D, day post infection/control injection; G, gestational day.

Figure 4. Malaria infection decreases maternal serum ANG-1 in pregnant mice (P <0.001, 2-way ANOVA).

Serum ANG-1 levels from uninfected (light bars) and P. berghei infected (dark bars) pregnant mice as measured by ELISA. Dots are individual mice; bars represent the median of each group. *** P<0.001 (Bonferonni post-test). D, day post infection/control injection; G, gestational day.

Figure 5. The presence of peripheral parasitemia during pregnancy correlates with decreased plasma ANG-1 levels (P = 0.031, mixed linear model).

(A) Peripheral plasma ANG-1 levels (± SEM) for uninfected primigravid women who had no detectable peripheral or placental parasitemia during the course of the study (n = 8). (B & C) Peripheral plasma ANG-1 levels from two representative primigravid women with PM. The mean levels for uninfected women are shown as reference (in light shade). Boxed data points represent visits where women were peripheral blood-smear positive for P. falciparum.

Figure 6. Angiopoietin-1 levels are increased upon resolution of peripheral parasitemia.

Paired peripheral plasma (A) ANG-1 and (B) ANG-2/ANG-1 levels of primigravid women at consecutive visits: the first, when parasitemic by peripheral blood smear microscopy, and the next, when successfully treated and blood-smear negative. To account for physiological variation in angiopoietin levels with gestational age, values were normalized to mean value of aparasitemic controls at the corresponding gestational age. n = 13 pairs. ** P<0.01, ^∧ P>0.05 (paired t-test).

Figure 7. Plasma angiopoietin levels at delivery are dysregulated in PM and with LBW outcomes.

(A–C) Peripheral plasma and (D) matched placental plasma obtained at delivery of normal birth weight (NBW) or LBW infants from primigravid women with (PM+) or without (PM-) PM were measured for ANG-1 and ANG-2 by ELISA. (A) Mean maternal peripheral plasma ANG-1 is elevated with LBW deliveries in PM- but not PM+ women. Statistical analyses by Mann-Whitney test. (B) Maternal peripheral plasma ANG-2 is elevated with PM. Statistical analyses by Mann-Whitney test. (C) Elevated maternal peripheral plasma ANG-2/ANG-1 ratio at delivery is associated with PM (P = 0.0016) and LBW (P = 0.0406); 2-way ANOVA on log-transformed data. Statistical analyses between groups by t-test on log-transformed data. (D) Placental plasma ANG-2/ANG-1 ratio levels are elevated compared to peripheral plasma levels. Statistical analysis by t-test on log-transformed data. Dots represent individual women, lines represent the median of each group. Peripheral plasma, PM- NBW (n = 47), PM- LBW (n = 44), PM+ NBW (n = 51), PM+ LBW (n = 35). Placental plasma, PM- NBW (n = 41), PM- LBW (n = 34), PM+ NBW (n = 44), PM+ LBW (n = 28). * P<0.05, ** P<0.01, *** P<0.001, # P = 0.06, ^∧ P = 0.486.