Am J Physiol Lung Cell Mol Physiol 302: L36–L46, 2012.
First published October 14, 2011; doi:10.1152/ajplung.00294.2011.
Excess soluble vascular endothelial growth factor receptor-1 in amniotic fluid impairs
lung growth in rats: linking preeclampsia with bronchopulmonary dysplasia
Jen-Ruey Tang,1 S. Ananth Karumanchi,2 Gregory Seedorf,1 Neil Markham,1 and Steven H. Abman1
1
Department of Pediatrics, Pediatric Heart-Lung Center, University of Colorado at Denver and Health Sciences Center,
Aurora, Colorado; and 2Howard Hughes Medical Institute, Beth Israel Deaconess Medical Center and Harvard Medical
School, Boston, Massachusetts
Submitted 6 September 2011; accepted in final form 9 October 2011
bronchopulmonary dysplasia; preeclampsia; vascular endothelial
growth factor; soluble fms-like tyrosine kinase 1; pulmonary hypertension
BRONCHOPULMONARY DYSPLASIA (BPD) is the chronic lung disease of infancy that follows premature birth and is characterized by impaired lung growth due to early lung injury (53).
BPD is a common and severe complication of preterm birth
(19, 59, 66). Infants with BPD have prolonged respiratory
insufficiency and are at high risk for pulmonary hypertension
(39, 65), lung infections (51, 78), recurrent hospitalizations
(63), exercise intolerance (30, 74, 75), and adverse neurodevelopmental outcome (3). Past studies have identified signifi-
Address for reprint requests and other correspondence: J. R. Tang, Pediatric
Heart Lung Center, Dept. of Pediatrics, Univ. of Colorado Health Sciences
Center, P15-4460A, Mail Stop 8614, 12700 East 19th Ave., Aurora, CO 80045
(e-mail: [email protected]).
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cant contributions to the pathogenesis of BPD from prematurity, side effects of respiratory support (9, 18, 52), and complications of neonatal illness (5, 27, 44). Although the use of
antenatal steroids, surfactant, and improvements in ventilator
and oxygen therapies have markedly altered the clinical course
of preterm infants, the incidence of BPD has not decreased in
the very premature infants during the past decade (59, 66). Late
respiratory complications of premature birth and BPD generally reflect the effects of persistent abnormalities in distal lung
structure, which include decreased alveolarization and a dysmorphic pulmonary vascular bed (14).
Recently, the impact of an adverse intrauterine environment
on impairing neonatal lung growth and worsening respiratory
outcomes of preterm infants has been recognized by both
clinical (8, 10, 28, 31, 41, 54, 55) and experimental studies (29,
47, 69, 71). Among diverse antenatal factors, preeclampsia
(PE) is shown to be independently associated with a high risk
for BPD, but the underlying mechanism is unknown (28, 31,
40, 79).
PE is the most common complication of pregnancy and is
due to maternal endothelial dysfunction that leads to hypertension, proteinuria, and other complications (61, 62). Moreover,
maternal PE is a frequent cause of preterm birth (7), accounting
for nearly 20% of delivery before 28 wk gestational age (49),
and significantly increases perinatal and neonatal morbidities
(61, 62). Placental overproduction of soluble vascular endothelial growth factor (VEGF) receptor-1 (soluble fms-like tyrosine kinase-1, also known as sFlt-1), which inhibits VEGF
signaling mainly through trapping free VEGF (38, 60), causes
maternal endothelial dysfunction and plays a central role in the
pathogenesis of PE (37, 43, 46, 48). The magnitude of excess
sFlt-1 in maternal serum not only correlates with the severity of
PE (48, 73), but serum levels of sFlt-1 in the mother are also
inversely related to gestational age and birth weight of the
newborn (73). In addition to high maternal serum levels, sFlt-1
is markedly increased in amniotic fluid during the second and
third trimesters of pregnancies complicated by PE (64, 76, 77).
Whether exposure of the fetal lung to excess intra-amniotic
sFlt-1 can impair lung VEGF signaling and contribute to the
pathogenesis of BPD is unknown. VEGF, a key regulator of
angiogenesis (12, 56), plays a critical role in directing vascular
and alveolar growth in the developing lung. Previous studies
have shown that VEGF inhibition during the early neonatal
period results in persistent abnormalities of alveolar and pulmonary vascular structures into and beyond infancy, which are
characteristic of pathological changes in human BPD (32, 42,
67). However, whether transient impairment of VEGF signaling in the fetus is sufficient to cause sustained disruption in
postnatal lung growth has not been studied.
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Tang JR, Karumanchi SA, Seedorf G, Markham N, Abman
SH. Excess soluble vascular endothelial growth factor receptor-1
in amniotic fluid impairs lung growth in rats: linking preeclampsia
with bronchopulmonary dysplasia. Am J Physiol Lung Cell Mol
Physiol 302: L36 –L46, 2012. First published October 14, 2011;
doi:10.1152/ajplung.00294.2011.—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 intraamniotic 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.
PREECLAMPSIA IMPAIRS INFANT LUNG GROWTH
To determine mechanisms that may link PE with a high risk
for BPD, we hypothesized that antenatal exposure to excess
sFlt-1 disrupts normal lung development through impaired
VEGF signaling in utero. We further hypothesized that fetal
exposure to elevated levels of intra-amniotic sFlt-1 is sufficient
to cause sustained abnormalities of lung structure into infancy.
We report that intra-amniotic sFlt-1 treatment during late
gestation impaired lung VEGF signaling and increased apoptosis of lung cells in newborn rats, which were followed by
reductions in alveolarization and lung vascular growth along
with biventricular hypertrophy during infancy. These findings
demonstrate biological plausibility underlying epidemiological
evidence that links PE with BPD.
MATERIALS AND METHODS
All procedures and protocols were approved by the Animal Care
and Use Committee at the University of Colorado Health Sciences
Center. Pregnant Sprague-Dawley rats were purchased from Charles
River Laboratories (Wilmington, MA) and maintained in room air at
Denver’s altitude (1,600 meters; barometric pressure, 630 mmHg;
inspired oxygen tension, 122 mmHg) for at least 1 wk before giving
birth. Animals were fed ad libitum and exposed to day-night cycles
alternatively every 12 h. Rats were killed with an intraperitoneal
injection of pentobarbital sodium (0.3 mg/g body wt; Fort Dodge
Animal Health, Fort Dodge, IA).
Study Design
Intra-amniotic sFlt-1administration. At 20 days gestation (term: 22
days), pregnant rats were prepared for receiving intra-amniotic injections. The timing of injection during the late canalicular stage of lung
development in the rat was selected to parallel a similar stage of
human lung development in 24- to 26-wk premature newborns who
are at the highest risk for BPD. After premedication with buprenorphine (0.01– 0.05 mg/kg, intramuscular injection), laparotomy was
preformed on pregnant rats under general anesthesia with 1–2%
isoflurane inhalation via a face mask (Anesthesia machine: Matrx by
Midmark, model VIP3000). During anesthesia and laparotomy, pregnant rats were kept on a heating pad for preventing hypothermia.
Pregnant rats were randomly assigned to saline control or sFlt-1
group; the saline group received 50 ␮l of normal saline per amniotic
sac, and the sFlt-1 group received 1 ␮g of recombinant human
sFlt-1-Fc (R&D Systems, Minneapolis, Minnesota) diluted to 50 ␮l
with normal saline per sac. We established the dose of intra-amniotic
sFlt-1 from preliminary studies in which sFlt-1 at lower doses (0.125–
0.25 ␮g/sac) had a small but significant decrease in radial alveolar
counts (RAC, 23% decrease; P ⬍ 0.05) in infant rats. However, right
ventricle-to-body weight ratios are not increased at a dose ⬍0.5
␮g/sac (data not shown). As a result, the dose of intra-amniotic sFlt-1
at 1 ␮g/sac was chosen for this study. Under sterile preparation, a
midline abdominal incision of 3– 4 cm in length was made to expose
the amniotic sacs for intra-amniotic injections. The amniotic sac
closest to the right ovary was first identified and injected, and then in
a counterclockwise sequence each sac was identified and injected with
a maximum of 10 sacs injected per dam. Limiting sFlt-1 injections to
10 sacs per pregnant rat was to achieve a consistent total dose of sFlt-1
on the individual mother rats, given intra-amniotic sFlt-1 is absorbed
into the maternal circulation through an intramembranous pathway,
which is characterized by a microscopic network of fetal vasculature
on the fetal surface of the placenta to mediate the transfer of intraamniotic substances into fetal and maternal circulations (23–26).
Similarly, considering the communicaiton between the amniotic cavity and maternal and fetal circualtions through the intramembranous
pathway, intra-amniotic saline was given in separate litters to serve as
controls. The total number of amniotic sacs in each mother rat was
examined and recorded during laparotomy. The abdominal incision
was closed with nylon sutures. Bupivacaine (1–2 mg/kg, subcutaneous injection) was applied over the incision wound for postoperative
pain control. Pregnant rats were monitored closely to ensure arousal
within 10 min after surgery, and rats were placed back to the cages
and were monitored for activity, ability to drink and eat, and for signs
of bleeding or infection.
Cesarean section. Two days after intra-amniotic injections, cesarean section was performed on pregnant rats under general anesthesia
with isoflurane inhalation, as described above. The fetus in the
amniotic sac closest to the right ovary was first delivered, which was
followed by delivery of the rest of the fetuses in a counterclockwise
sequence, to identify fetuses exposed to intra-amniotic injections. The
total number of amniotic sacs in each mother rat was further verified
at the time of delivery. The main reason for performing cesarean
section instead of allowing vaginal delivery is to identify the fetuses
exposed to intra-amniotic injections, based on the order of the amniotic sacs and their anatomic locations related to the ovaries. All of the
rat pups in the injected amniotic sacs were delivered within 5 min
after the onset of anesthesia. Maternal rats were then killed with
pentobarbital sodium. Newborn rats were immediately placed on a
heating pad to avoid hypothermia and were dried manually with gauze
sponges. Pups received no supplemental oxygen or artificial ventilation at birth. The survival rate at birth was recorded. Within 30 min
after birth, the pups were weighed and placed with foster mother rats
in regular cages. For the first 24 h of life, the newborn pups were
monitored closely for mortality or signs of respiratory distress.
Rat lungs were harvested at birth for Western blot analysis and at birth
and 14 days of age for histological assessment. Hearts were dissected and
weighed at birth and 7 and 14 days of age. Three to nine rats were studied
in each group for each measurement at each time point. Survival of the
infant rats was monitored and recorded daily from birth throughout the
study period. Survival rate was calculated as the number of survived pups
divided by the number of sacs that received intra-amniotic injection in
each given litter. Body weight was measured at birth and at the time of
being killed for study measurements.
Study Measurements
Tissue for histological analysis. Animals were killed with intraperitoneal pentobarbital sodium. A catheter was placed in the trachea,
and the lungs were inflated with 4% paraformaldehyde and maintained at 20 cmH2O pressure for 60 min. A ligature was tightened
around the trachea to maintain pressure, and then the tracheal cannula
was removed. Lungs were then immersed in 4% paraformaldehyde at
room temperature for 24 h for fixation. A 2-mm-thick transverse
section was taken from the midplane of the right lower lobe and left
lobe of the fixed lungs per animal, respectively, to process and embed
in paraffin wax.
Immunohistochemistry. Slides with 5-␮m paraffin sections were
stained with hematoxylin and eosin for assessing alveolar structures
and with von Willebrand Factor (vWF), an endothelial cell-specific
marker, for quantifying vessel density.
Morphometric analysis. RAC, mean linear intercept (MLI), and
other indexes of alveolar structure and pulmonary vessel density were
determined by standard morphometric techniques, as outlined below.
In each animal, at least 5 measurements were obtained for RAC, at
least 10 images were processed for computer-assisted image analysis
of alveolar structure, and at least 10 high-power fields were examined
for measuring pulmonary vessel density.
RADIAL ALVEOLAR COUNTS. Alveolarization was assessed by the
RAC method of Emery and Mithal as described (16, 17). Respiratory
bronchioles were identified as bronchioles lined by epithelium in one
part of the wall. From the center of the respiratory bronchiole, a
perpendicular line was dropped to the edge of the acinus connective
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Animals
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PREECLAMPSIA IMPAIRS INFANT LUNG GROWTH
images were captured on a Zeiss Axioscope2 microscope, using the ⫻10
objective and captured as a high-resolution TIFF image by a MicroPublisher digital camera (2,560 ⫻ 1,940 pixel resolution; Qimaging,
Burnaby, Canada). The fields with large airways or vessels were avoided.
These images were processed as previously described (4).
PULMONARY VESSEL DENSITY. Pulmonary vessel density was determined by counting vWF-stained vessels with an external diameter
at 50 ␮m or less per high-power field. The fields containing large
airways or vessels with external diameter ⬎50 ␮m were avoided.
Indexes of Right Ventricular Hypertrophy and Left Ventricular
Hypertrophy
tissues or septum or pleura, and the number of septae intersected by
this line was counted.
MLI AND OTHER INDEXES OF ALVEOLAR STRUCTURE. Measurements of MLI, nodal point density, and interstitial thickness were performed with a computer-assisted image analysis program. Briefly, the
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 ␮m. 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.
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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.
The right ventricle (RV) and left ventricle plus septum (LV⫹S)
were dissected and weighed. The ratios of RV to LV⫹S weights
(RV/LV⫹S%) and RV/body weights (RV/BW%) were determined to
evaluate right ventricular hypertrophy (RVH). The ratio of LV⫹S to
body weight (LV⫹S/BW%) was determined to evaluate left ventricular hypertrophy (LVH).
Western blot analysis. Frozen lung samples were homogenized in
ice-cold radioimmunoprecipitation buffer (PBS, 1% Nonidet P-40,
0.5% sodium deoxycholate, 0.1% SDS, and 10 mg/ml phenylmethylsulfonyl fluoride) with protease inhibitor (Pierce, Rockford, IL) and
phosphatase inhibitor cocktail (Calbiochem, Gibbstown, NJ). Samples
were centrifuged at 1,500 g for 20 min at 4°C to remove cellular
debris. Protein content in the supernatant was determined by the
Bradford method (11), using BSA as the standard. Next, 25 ␮g [for
total VEGF, endothelial nitric oxide synthase (eNOS), and active
caspase-3] or 50 ␮g [for total and phosphorylated vascular endothelial
growth factor receptor 2 (VEGFR2)] of protein sample per lane was
resolved by SDS-PAGE, and proteins from the gel were transferred to
polyvinylidene difluoride membrane. Blots were blocked for 1 h or
overnight in 5% nonfat dry milk in PBS with 0.1% Tween 20. For
total VEGF and eNOS, the blots were incubated for 1 h at room
temperature with rabbit anti-human polyclonal VEGF antibody (sc-
PREECLAMPSIA IMPAIRS INFANT LUNG GROWTH
Lung Histology and Morphometric Analyses
Figure 2 shows the effects of intra-amniotic sFlt-1 treatment
on alveolarization in 14-day-old rats. The distal airspace appeared simplified with decreased septation and alveoli in sFlt-1
rats as compared with saline control (Fig. 2A). By morphometric analysis, sFlt-1 rats had decreased RAC (P ⬍ 0.0001),
increased MLI (P ⬍ 0.0001), and decreased nodal point density (P ⬍ 0.0001) compared with saline control; interstitial
thickness was not different between the two groups (Fig. 2B).
The effects of intra-amniotic sFlt-1 treatment on pulmonary
vascular growth in 14-day-old rats are shown in Fig. 3. Pulmonary vessel density was decreased by 38% in sFlt-1 rats
compared with saline control (P ⬍ 0.0001). There was no
difference found between the male and female in the sFlt-1
group regarding lung histology and morphometric analyses.
Statistical Analysis
Statistical analysis was performed with the InStat 3.0 software
package (GraphPad Software, San Diego, CA). Statistical comparisons were made between groups using t-test or ANOVA with Newman-Keuls post hoc analysis for significance. P ⬍ 0.05 was considered significant.
RESULTS
Survival Rate and Body Weight
A total of three litters of fetal rats received intra-amniotic
saline injections, and four litters received intra-amniotic sFlt-1
treatment. Both control and sFlt-1 groups had 100% survival at
birth (Fig. 1A). In both groups, newborn rats appeared pink and
vigorous without evidence of cyanosis or respiratory distress,
as closely monitored for the first 24 h of life. In the two litters
kept through day 14 in each group, the survival rate of sFlt-1
rats decreased to 80% at day 1, statistically not different from
the 95% of survival rate in saline control. No mortality was
found at and beyond day 2 in either group. There was no
difference in body weights between sFlt-1 and saline rats at
birth, and days 7 and 14 (Fig. 1B). The rate of body weight gain
was similar between the two groups from birth through day 14.
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 ␮m. 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.
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507, 1:500; Santa Cruz Biotechnology) and mouse anti-human polyclonal eNOS (BD610297; BD Biosciences), respectively, in 5%
nonfat dry milk in PBS with 0.1% Tween 20. For active caspase-3, the
blots were incubated overnight at 4°C with rabbit cleaved caspae-3
antibody (Asp175; Cell Signaling). For VEGFR2, the blots were
incubated overnight at 4°C with rabbit phosphorylated VEGFR2
antibody (Tyr1175; no. 2478; Cell Signaling) in 5% BSA; after
finishing the detection of phosphorylated VEGFR2, the blots were
incubated with stripping buffer (no. 46430; Thermo Scientific) and
then incubated for 1 h at room temperature with rabbit VEGFR2
antibody (no. 2479; Cell Signaling) in 5% nonfat dry milk in PBS with
0.1% Tween 20. For secondary antibody, blots were incubated for 1
h at room temperature with a goat anti-rabbit IgG horseradish peroxidase (HRP) antibody (sc-2054; Santa Cruz Biotechnology) or goat
anti-mouse HRP-conjugated antibody (no. 172–1011; Bio-Rad). After
being washed, bands were visualized by enhanced chemiluminescence
(ECL Advance kit; Amersham Pharmacia Biotech, Buckinghamshire,
UK). For Western analysis of VEGF, recombinant mouse VEGF
protein (Santa Cruz Biotechnology) was used as a positive control. On
each blot, ␤-actin was detected as a housekeeping protein to compare
expression between samples; mouse monoclonal ␤-actin antibody
(Sigma A-5316; Sigma-Aldrich) was used for detecting ␤-actin.
Densitometry was performed using Quantity One (Bio-Rad). Three to
five animals were analyzed per study group.
Immunofluorescent colocalization. To identify lung cells undergoing apoptosis, immunofluorescence staining was performed on 5-␮m
cryosections cut from the optimum cutting temperature-embedded
lung blocks. The sections were blocked with BSA in PBS for 1 h and
washed with PBS and then were incubated with anti-active caspase-3
antibody (1:50; AB3623; Chemicon International) along with either
anti-vWF antibody (1:50; sc-8068; Santa Cruz Biotechnology), antivimentin antibody (1:50; sc-6260; Santa Cruz Biotechnology), antibody to ␣-smooth muscle actin (␣-SMA) (1:50; ab18147; Abcam), or
antibody to surfactant protein-C (SP-C) precursor (1:100; sc-7706;
Santa Cruz Biotechnology) at 4°C overnight. Next, the sections were
incubated with secondary antibodies at 1:500 (donkey anti-rabbit
Alexa Fluor 594; donkey anti-goat or donkey anti-mouse 488; Molecular Probes, Eugene, OR) for 2 h at room temperature and washed.
The sections were then mounted with 4=,6-diamidino-2-phenylindole
(Vector Laboratories) and imaged with an Olympus IX71 fluorescence microscope (Olympus America, Center Valley, PA).
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PREECLAMPSIA IMPAIRS INFANT LUNG GROWTH
RVH and LVH Indexes
The effects of intra-amniotic sFlt-1 treatment on heart
weights in infant rats are shown in Fig. 4. The RV/LV⫹S and
RV/BW ratios (Fig. 4, A and B), both of which are proportional
to the degree of RVH, were comparable between sFlt-1 rats
and saline control at birth and at day 7, respectively. Both
parameters in sFlt-1 rats increased above control values at day
14 (P ⬍ 0.05). Similarly, the LV⫹S/BW ratio (Fig. 4C), an
index of LVH, was similar between sFlt-1 and saline groups at
birth and at day 7, respectively, but then increased in sFlt-1 rats
at day 14 to 19% above control values (p ⬍ 0.05). From birth
through day 7, the RVH and LVH indexes decreased in both
groups (Fig. 4, A–C). From day 7 to day 14, the RV/BW ratio
tended to further decrease in the control, whereas it trended up
in sFlt-1 rats (Fig. 4B). During this period, the LV⫹S/BW ratio
nearly further decreased in the control, but it remained unchanged in sFlt-1 rats (Fig. 4C). The time course suggests
neonatal onsets of progressive RVH and LVH in sFlt-1 rats. At
day 14, the effect of intra-amniotic sFlt-1 treatment on increasing RV/BW% was more striking in female infant rats than male
(Fig. 4D). There was no apparent gender-related difference on
LV⫹S/BW% in sFlt-1 rats (Fig. 4E).
Effects of Intra-Amniotic sFlt-1 on VEGF Signaling in the
Newborn Rat Lung
To assess the effects of intra-amniotic sFlt-1treatment on
lung VEGF signaling at birth, phosphorylated VEGFR2, total
VEGFR2, total VEGF, and eNOS proteins were measured by
Western blot analyses on newborn rat lung homogenates. Lung
phosphorylated VEGFR2 protein was decreased in sFlt-1 rats
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.
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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.
PREECLAMPSIA IMPAIRS INFANT LUNG GROWTH
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compared with saline control (P ⬍ 0.05; Fig. 5A), whereas
lung total VEGFR2 protein was comparable between the two
groups (Fig. 5B). The ratio of phosphorylated VEGFR2 to total
VEGFR2 proteins in the sFlt-1 group was 46% below control
values in the newborn rat lungs (P ⬍ 0.01; Fig. 5C). Total lung
VEGF (Fig. 6) and eNOS protein contents (Fig. 7) did not
differ between sFlt-1 and control groups at birth.
Effects of Intra-Amniotic sFlt-1 on Apoptosis in the Newborn
Rat Lung
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.
broblasts. Active caspase-3 staining was not found in lung cells
positive for SP-C (Fig. 10C).
DISCUSSION
We found that excess sFlt-1 in amniotic fluid during the late
canalicular and early saccular stage of lung development reduces VEGF signaling and increases apoptosis in the newborn
rat lung, which is followed by sustained reductions in alveolarization and pulmonary vascular growth during infancy. In
addition, infant rats that are exposed to excess intra-amniotic
sFlt-1 develop RVH and LVH over time. These findings
suggest that the increased risk for BPD and vascular dysfunction in infants with maternal PE may result from fetal exposure
to elevated levels of intra-amniotic sFlt-1.
For more than a decade, epidemiological studies have shown
an increased risk of BPD in preterm infants born to preeclamptic mothers (28, 31, 40, 79), but the underlying mechanism was
unknown. The present study shows that excess intra-amniotic
sFlt-1 is sufficient to cause sustained abnormalities of lung
structure during infancy. This effect is likely mediated through
impairing lung VEGF signaling in the fetus, leading to increased endothelial and mesenchymal apoptosis in the distal
airspace of the newborn. These findings further demonstrate
the critical role of VEGF signaling in directing vascular and
alveolar growth in the developing lung. Moreover, the present
study reveals that intact VEGF signaling is required to maintain both endothelial and mesenchymal cell survival during
lung development. Our finding confirms and extends past
studies that endothelial survival relies on VEGF signaling
through phosphorylation of VEGFR2 (20) and that inhibition
of VEGFR2 injures pulmonary endothelium in the fetus (80)
and neonates (68). A recent study shows that VEGF mediates
endothelial-mesenchymal cross-talk to drive morphogenesis of
testis (15). Future work is needed to determine whether VEGF
signaling directly regulates mesenchymal cells or through endothelial cells in the developing lung.
This is the first study demonstrating that primary inhibition
of VEGF in the fetus results in sustained abnormalities of lung
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To evaluate to the effects of intra-amniotic sFlt-1 treatment
on activity of apoptosis in newborn rat lungs, active caspase-3
protein was analyzed by Western blot on lung homogenates,
and showed elevation by 76% in sFlt-1 rats (P ⬍ 0.01;
Fig. 8A). Consistent with Western blot analysis, active caspase-3
signals remarkably increased in sFlt-1 rats as compared with
saline control, in immunofluorescence staining on newborn rat
lung sections (Fig. 8B).
To identify which cells express active caspase-3 in the
newborn lung, immunofluorescent staining was performed
with active caspase-3 and one of the markers of variable lung
cell types on newborn rat lung sections. Compared with scarce
signals of active caspase-3 on vWF-positive cells in saline
control (Fig. 9A), sFlt-1 rats demonstrated colocalization of
vWF and active caspase-3 in microvessels (Fig. 9B) but not in
larger pulmonary vessels (Fig. 9C). To further identify nonendothelial cells stained with active caspase-3 in the distal airspace of sFlt-1 rats (Fig. 9B), immunofluorescence staining
was performed for active caspase-3 with vimentin, ␣-SMA,
and SP-C precursor, respectively. Vimentin is expressed in
mesenchymal and endothelial cells, ␣-SMA is a marker of
myofibroblasts in distal airspace, and SP-C precursor is produced exclusively by type-2 alveolar epithelial cells. Many
cells that were positive for active caspase-3 coexpressed vimentin in the interstitium of distal airspace (Fig. 10A), indicating the localization of active caspase-3 in mesenchymal
cells in addition to endothelial cells. There was no colocalization of active caspase-3 and ␣-SMA (Fig. 10B), indicating that
those active caspas-3 (⫹) mesenchymal cells are not myofi-
L42
PREECLAMPSIA IMPAIRS INFANT LUNG GROWTH
structure during infancy. Previous studies have shown that
intra-amniotic endotoxin or antenatal dexamethasone treatment
downregulates lung VEGF or VEGFR-2 expression at birth
and impairs alveolarization through late infancy in rats (57,
69). The present study highlights that antenatal impairment of
lung VEGF signaling may link the sustained disruption of
infant lung growth with preceding intrauterine stimuli, such as
chorioamnionitis, antenatal dexamethasone, and PE, in animal
models. Past studies have shown that neonatal VEGF inhibition reduces angiogenesis as well as alveolar growth in the
developing lung, but the mechanisms are poorly understood
(22, 42, 50, 67, 81). With impaired VEGF signaling in utero, as
shown in the current study, mesenchymal cell apoptosis could
further compromise vascular growth in the developing lung,
given mesenchymal cells are capable of differentiating into
perivascular cells to stabilize newly forming endothelial channels (2, 45). Future study is needed to better characterize the
␣-SMA-negative mesenchymal lung cells that undergo apoptosis under suppression of VEGFR2 signaling, in order to delineate whether mesenchymal injury due to VEGF inhibition is
directly involved in the impaired formation of secondary septae
in the developing lung.
This rat model of PE demonstrates that excess sFlt-1 in
amniotic fluid impairs lung VEGF signaling in the fetus. Following intra-amniotic sFlt-1 treatment, the reduction in phosphorylated VEGFR2 in the presence of intact total VEGFR2
indicates a shortage of lung VEGF to activate VEGFR2 in the
newborn lung. As in clinical PE, excess intra-amniotic sFlt-1
may directly reach the fetal lung via fetal breathing, or through
an intramembranous pathway and fetal swallowing to enter the
fetal circulation and then arrive in the fetal lung. Bidirectional
flow of amniotic fluid across human fetal trachea has been
proven by Doppler velocimetry during normal fetal breathing
movements (36). Moreover, fetal pulmonary aspiration of intra-amniotic sFlt-1 is supported by the finding that muscle
paralysis inhibits lung deposition of intra-amniotic iron dextran
in fetal rabbits, whereas iron accumulates in control fetal lungs
(21). In addition, substances in the amniotic cavity can be
transferred into the fetal circulation through an intramembranous pathway (23–26), which is characterized by a micro-
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).
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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.
PREECLAMPSIA IMPAIRS INFANT LUNG GROWTH
L43
scopic network of fetal vasculature on the fetal surface of the
placenta (23, 24).
This study also reports the late development of RVH in
infant rats treated with intra-amniotic sFlt-1, suggesting the
progressive onset of pulmonary hypertension after exposure to
the fetal environment of PE. Our findings are interesting not
only because pulmonary hypertension can complicate BPD
(39, 65) but also because offspring of mothers with PE are at
risk for pulmonary vascular dysfunction later in life (33).
Similarly, we found LVH in infant rats exposed to excess
sFlt-1 in utero. Interestingly, a high incidence of systemic
hypertension has been recognized in infants with BPD, but its
etiology is unclear (1). Moreover, past studies have identified
an increased risk of systemic vascular dysfunction in offspring
of mothers with PE, but the mechanism remains unknown (33,
35, 58, 70, 72). As shown in a previous study, neonatal
treatment with truncated sFlt-1 impairs the growth of renal
glomerular capillaries in mice (22), suggesting that renal vascular injury may contribute to systemic hypertension or LVH
following disruption of VEGF signaling during development.
Future studies would determine whether systemic vascular
abnormalities have existed early in those with maternal PE,
contributing to hypertension in infants with BPD and predisposing to cardiovascular diseases later in life.
This study provides mechanistic insights underlying the risk
of preterm infants for BPD in the setting of maternal PE. In one
study on preterm infants with very low birth weight, maternal
PE is associated with increased BPD at 36 wk corrected age
although it did not increase oxygen requirement at 28 days
postnatal age (40). These findings suggest that progressive
worsening of lung disease can occur later in the neonatal
course of preterm infants born to preeclamptic mothers, which
is a pattern observed in clinical BPD (6, 13, 34). Moreover, the
present study demonstrates that the fetal environment of PE is
sufficient to cause sustained and late pulmonary structural
abnormalities characteristic of human BPD, despite the absence of adverse postnatal stimuli, such as hyperoxia, mechanical ventilation, infection, or related problems. Collectively,
these findings suggest the need for new strategies to prevent
BPD, of which the incidence seems to have reached a plateau
in the very premature infants (59, 66). Future research is
required to investigate whether early intervention, before those
high-risk infants become symptomatic for clinical deterioration, may rescue the preterm lung that has been injured before
birth.
A potential limitation of this rat model is the lack of direct
placental production of sFlt-1 to more comprehensively reproduce the fetal environment of PE. We speculate that it explains
why the sFlt-1 rats did not have signs of intrauterine growth
restriction (IUGR), which is a common complication of PE. On
the other hand, the current study demonstrates impaired neonatal lung growth in the absence of reduced birth weight or
decreased postnatal somatic growth, indicating that impaired
lung growth following intra-amniotic sFlt-1 treatment is not
due to IUGR or neonatal malnutrition in this model. Moreover,
the present study highlights that excess intra-amniotic sFlt-1
per se is sufficient to impair lung VEGF signaling at birth.
Considering the pharmacokinetics of sFlt-1 as administered via
single intra-amniotic injections in this study, the concentrations
of intra-amniotic sFlt-1 immediately after injections cannot be
directly compared with the amniotic levels of sFlt-1 measured
in patients diagnosed with PE. In brief, sFlt-1 injected into the
amniotic cavity is absorbed through an intramembranous pathway into the fetal circulation and maternal circulation, whereby
excess sFlt-1 is metabolized and excreted in the maternal
compartment, gradually decreasing the concentrations of intra-
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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 ␣-smooth muscle actin (␣-SMA)
(green; B) nor surfactant protein-C (SP-C) (green; C). The areas demonstrating positive (vimentin) and negative (␣-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.
L44
PREECLAMPSIA IMPAIRS INFANT LUNG GROWTH
amniotic sFlt-1 after single injections in this rat model. In
patients diagnosed with PE, the levels of sFlt-1 in amniotic
fluid are rather relatively steady, since sFlt-1 is constantly
overproduced in the placenta.
We conclude that intra-amniotic sFlt-1 treatment during
late gestation impairs VEGF signaling and increases apoptosis in the newborn rat lung, which is followed by persistent reductions in alveolarization and pulmonary vascular
growth and the subsequent development of biventricular
hypertrophy during infancy. We speculate that disruption of
VEGF signaling in utero due to antenatal exposure of excess
sFlt-1 contributes to an increased risk for BPD and the
predisposition for pulmonary and systemic vascular dysfunction in infants with maternal PE.
This work was partly supported by grants from the National Heart, Lung,
and Blood Institute, including T32 HL-07670 (for J. R. Tang) and RO1
HL-68702 (S. H. Abman).
DISCLOSURES
Dr. Karumanchi is a co-inventor on multiple patents held by the Beth Israel
Deaconess Medical Center that are related to angiogenic factors for the use in
diagnosis and therapy of preeclampsia. Dr. Karumanchi has financial interest
in Aggamin LLC.
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