Excess soluble vascular endothelial growth factor receptor-1 in amniotic fluid impairs lung growth in rats: linking preeclampsia with bronchopulmonary dysplasia

Abstract

Epidemiological studies have shown that maternal preeclampsia (PE) increases the risk of bronchopulmonary dysplasia (BPD), but the underlying mechanism is unknown. Soluble vascular endothelial growth factor receptor-1 (soluble VEGFR1, known as soluble fms-like tyrosine kinase 1, or sFlt-1), an endogenous antagonist of vascular endothelial growth factor (VEGF), is markedly elevated in amniotic fluid and maternal blood in PE. Therefore, we hypothesized that antenatal exposure to excess sFlt-1 disrupts lung development through impaired VEGF signaling in utero, providing a mechanistic link between PE and BPD. To determine whether increased sFlt-1 in amniotic fluid is sufficient to cause sustained abnormalities of lung structure during infancy, sFlt-1 or saline was injected into amniotic sacs of pregnant Sprague-Dawley rats at 20 days of gestation (term, 22 days). After birth, pups were observed through 14 days of age for study. We found that intra-amniotic sFlt-1 treatment decreased alveolar number, reduced pulmonary vessel density, and caused right and left ventricular hypertrophy in 14-day-old rats. In addition, intra-amniotic sFlt-1 treatment suppressed activation of lung VEGF receptor-2 and increased apoptosis in endothelial and mesenchymal cells in the newborn lung. We conclude that exposure to excess sFlt-1 in amniotic fluid during late gestation causes sustained reductions in alveolarization and pulmonary vascular growth during infancy, accompanied by biventricular hypertrophy suggesting pulmonary and systemic hypertension. We speculate that impaired VEGF signaling in utero due to exposure of high amniotic fluid levels of sFlt-1 in PE disrupts lung growth and contributes to the increased risk of BPD in infants born to mothers with PE.

Fig. 1.

Effects of intra-amniotic soluble fms-like tyrosine kinase 1 (sFlt-1) treatment on survival rate and body weight (BW) of infant rats, from birth through 14 days of age. A: both saline and sFlt-1 groups had 100% survival at birth; from day 1 to day 14, 80% of rats survived in the sFlt-1 group and 95% survived in the control (p > 0.05). B: there was no difference in body weight between sFlt-1 rats and saline controls at birth, and days 7 and 14.

Fig. 2.

Effects of intra-amniotic sFlt-1 treatment on distal lung growth in 14-day-old rats; lung histology was stained with hematoxylin and eosin. Micrographs are representative and were obtained at the same magnification. Internal scale bar = 100 μm. sFlt-1 rats demonstrated simplified alveoli (A), decreased radial alveolar counts, increased mean linear intercept, and reduced nodal point density (B), compared with the control; n = 5–9 animals/group.

Fig. 3.

Effects of intra-amniotic sFlt-1 treatment on pulmonary vessel density in lung histology stained with von Willebrand Factor (vWF) from 14-day-old rats. Micrographs are representative and were obtained at the same magnification. Internal scale bar = 100 μm. Pulmonary vessel density was reduced in sFlt-1 rats as shown by histology (A) and documented by morphometric analysis (P < 0.0001; B); n = 5–9 animals/group.

Fig. 4.

Effects of intra-amniotic sFlt-1 treatment on heart weights in infant rats. The ratios of the right ventricle (RV)/left ventricle + septum (LV + S) (A), RV/body weight (RV/BW) (B), and LV/BW (C) are shown at birth, and days 7 and 14. These indexes from the sFlt-1 group were at control values at birth and day 7 but then rose above control levels by day 14. RV/BW% in the sFlt-1 group was higher in female infant rats than males at day 14 (D), but there was no gender-related difference in LV+S/BW% at day 14 in sFlt-1 rats (E); n = 4–8 animals/group.

Fig. 5.

Effects of intra-amniotic sFlt-1 treatment on phosphorylated (ph) vascular endothelial growth factor receptor-2 (VEGFR2) and total VEGFR2 protein contents in the newborn rat lung. Compared with saline controls, sFlt-1 rats showed decreased ph-VEGFR2 (A), unchanged total VEGFR2 (B), and a decreased ratio of ph-VEGFR2 to total VEGFR2 proteins (C) in the lung at birth; n = 3–5 animals/group. NS, not significant.

Fig. 6.

Effects of intra-amniotic sFlt-1 treatment on protein contents of total VEGF in the newborn rat lung. Lung vascular endothelial growth factor (VEGF) protein contents were similar between sFlt-1 and control groups at birth; n = 5 animals/group.

Fig. 7.

Effects of intra-amniotic sFlt-1 treatment on protein contents of endothelial nitric oxide synthase (eNOS) in the newborn rat lung. Lung eNOS protein contents were similar between sFlt-1 and control groups at birth; n = 5 animals/group.

Fig. 8.

Effects of intra-amniotic sFlt-1 treatment on active caspase-3 in the newborn rat lung. sFlt-1 rats showed increased protein contents of active caspase-3 in lung homogenates by Western blot analysis (P < 0.01; A) and increased active caspase-3 by immunofluorescence staining on lung sections in comparison with the controls (B); n = 5 animals/group in Western blot analysis. Micrographs of immunofluorescence staining are representative and were obtained at the same magnification, ×200.

Fig. 9.

Immunofluorescence staining for vWF (green) and active caspase-3 (red) on lung sections from newborn rats. Micrographs are representative and were obtained at the same magnification, ×200. Compared with scarce signals of active caspase-3 (red) in the control (A), sFlt-1 rats demonstrated colocalization of vWF (green) and active caspase-3 (red) in microvessels (B, arrow; higher magnification in the inset) but not in larger pulmonary vessels (C). Some of those active casapse-3-postive cells were not stained with vWF (B).

Fig. 10.

Immunofluorescence staining on lung sections from newborn rats. Micrographs are representative and were obtained at the same magnification, ×200. In sFlt-1 rats, active caspase-3 (red) was localized in cells stained with vimentin (green; A) but not localized in cells stained with α-smooth muscle actin (α-SMA) (green; B) nor surfactant protein-C (SP-C) (green; C). The areas demonstrating positive (vimentin) and negative (α-SMA; SP-C) colocalization with active caspase-3 are indicated with arrows and shown in higher magnification in the insets. Control rats showed few cells positive for active caspase-3 in each set of immunofluorescence staining.