Inhibition of angiogenesis decreases alveolarization in the
developing rat lung
Malathi
Jakkula1,
Timothy D.
Le Cras1,
Sarah
Gebb1,
K. Peter
Hirth2,
Rubin M.
Tuder3,
Norbert F.
Voelkel4, and
Steven H.
Abman1
1 Pediatric Pulmonary Medicine, Pediatric Heart Lung Center,
Department of Pediatrics, 3 Department of Pathology, and
4 Pulmonary Hypertension Center, University of Colorado
School of Medicine, Denver, Colorado 80218; and 2 Sugen
Incorporated, South San Francisco, California 94080
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ABSTRACT |
To determine whether angiogenesis is necessary for
normal alveolarization, we studied the effects of two
antiangiogenic agents, thalidomide and fumagillin, on
alveolarization during a critical period of lung growth in infant rats.
Newborn rats were treated with daily injections of fumagillin,
thalidomide, or vehicle during the first 2 wk of life. Compared with
control treatment, fumagillin and thalidomide treatment reduced lung
weight-to-body weight ratio and pulmonary arterial density by 20 and
36%, respectively, and reduced alveolarization by 22%.
Because these drugs potentially have nonspecific effects on lung
growth, we also studied the effects of Su-5416, an inhibitor of
the vascular endothelial growth factor receptor known as kinase insert
domain-containing receptor/fetal liver kinase (KDR/flk)-1. As observed
with the other antiangiogenic agents, Su-5416 treatment decreased
alveolarization and arterial density. We conclude that treatment with
three different antiangiogenic agents attenuated lung vascular growth
and reduced alveolarization in the infant rat. We speculate that
angiogenesis is necessary for alveolarization during normal lung
development and that injury to the developing pulmonary circulation
during a critical period of lung growth can contribute to lung hypoplasia.
bronchopulmonary dysplasia; congenital diaphragmatic hernia; fumagillin; lung growth; lung hypoplasia; pulmonary circulation; pulmonary hypertension; thalidomide; vascular endothelial growth
factor
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INTRODUCTION |
NORMAL
DEVELOPMENT of the human lung can be divided into five stages:
embryonic (3-7 wk gestation), pseudoglandular (5-17 wk),
canalicular (16-26 wk), saccular (24-38 wk), and alveolar (up
to 2-3 yr of age) (7, 13, 16, 24, 29, 41, 42). Although these periods of lung development are similar across mammalian
species, the relative timing and length of each stage varies between
species. In the rat, alveolarization begins during late gestation but
primarily occurs during the first 2 wk after birth (29).
Alveoli are formed by the septation of large saccules that
constitute the gas-exchange region of the immature lung during this
critical period of lung growth. Secondary septae form as ridges that
grow into distal air spaces, thereby increasing lung surface area and
enhancing the capacity for gas exchange (7). Mechanisms
that regulate alveolarization are poorly understood, but multiple
stimuli modulate distal lung growth, including genetic factors, oxygen
tension, nutrition, hormones, and autocrine and paracrine growth
factors (5-7, 25, 29-31, 41, 42).
During the period of alveolarization, the lung also undergoes marked
vascular growth as reflected by the 20-fold increase in alveolar and
capillary surface areas from birth to adulthood (49). Lung
vascular growth involves two basic processes: vasculogenesis, the
formation of new blood vessels from endothelial cells within the
mesenchyme, and angiogenesis, the formation of new blood vessels from
sprouts of preexisting vessels (7, 14, 40, 41). Mechanisms
that increase vascular surface area during late gestation and the early
postnatal period are poorly understood, but it is clear that
coordination of distal air space and vascular growth is essential for
normal lung development. For example, the production and timing of the
release from respiratory epithelial cells of angiogenic factors such as
vascular endothelial growth factor (VEGF) and basic fibroblast growth
factor are likely to play key roles in normal lung vascular growth
(2-4, 17, 33, 36, 38, 41, 42, 48, 51).
Experimental studies (5, 7, 29-31,
39) have shown that adverse stimuli such as hypoxia,
hyperoxia, or glucocorticoid treatment during the critical period of
postnatal lung growth in the rat can disrupt alveolarization and cause
lung hypoplasia. These models of lung hypoplasia are also associated
with abnormalities of the pulmonary circulation (25, 26, 39, 44a). In the clinical setting, lung hypoplasia is
frequently associated with abnormalities of the pulmonary circulation
in diverse diseases, including bronchopulmonary dysplasia (BPD),
congenital diaphragmatic hernia, primary lung hypoplasia, and Down's
syndrome (1, 8, 11, 28, 45, 47). It is generally presumed
that disruption of alveolarization is likely to cause failure of lung
vascular growth; whether injury to the developing pulmonary
circulation or disruption of normal angiogenesis can also contribute to
impaired alveolarization is unknown.
Antiangiogenic therapy with agents such as thalidomide and
fumagillin has been studied in diverse experimental settings in order
to examine basic mechanisms of angiogenesis and their potential role in
pathological settings such as tumor angiogenesis (17, 50).
Because fumagillin and thalidomide block angiogenesis by inhibiting
endothelial cell proliferation (12, 17, 21, 22, 34, 37),
which is critical for angiogenesis, these drugs provide useful
pharmacological tools for studying the physiological roles of
angiogenesis in different experimental settings. Therefore, to study
the role of angiogenesis in postnatal lung growth, we proposed the
hypothesis that disruption of angiogenesis during a critical period of
lung growth would decrease alveolarization. To test this hypothesis,
we studied the effects of fumagillin and thalidomide
treatment during a critical period of postnatal lung growth in neonatal
rats and report that these agents decrease lung weight, pulmonary
arterial density, and alveolarization. Because these agents may
have nonspecific effects on lung growth independent of their effects on
endothelial cell proliferation, we further tested this hypothesis
by treating neonatal rats with an antiangiogenic agent, Su-5416,
an inhibitor of the VEGF receptor kinase insert domain-containing/fetal
liver kinase (KDR/flk)-1 (18, 43). As observed with
fumagillin and thalidomide, we report that Su-5416 treatment also
decreased alveolarization and arterial density at 2 wk of age. These
findings suggest that angiogenesis is necessary for normal
alveolarization during a critical period of lung development in the rat.
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METHODS |
Study Animals
All procedures and protocols used in this study were approved by
the Animal Care and Use Committee at the University of Colorado Health
Sciences Center. Timed-pregnant, pathogen-free Sprague-Dawley rats were
purchased from Harlan Laboratories (Indianapolis, IN). The pregnant
rats were maintained at Denver's altitude for at least 1 wk before
giving birth. Animals were fed ad libitum and exposed to 12:12-h
light-dark cycles.
Experimental Design
Daily injections of fumagillin, thalidomide, or vehicle
(DMSO) were started 1 day after birth and continued until 13 days of
age. Study group assignment was randomized for each litter. All drugs
were freshly prepared on the day of study and stored at
20°C for
the remainder of the study period. Fumagillin and thalidomide (Biomol
Technologies, Plymouth Meeting, PA) were dissolved in DMSO before
treatment. In the fumagillin group (n = 22 animals), fumagillin was administered by subcutaneous injection at a dose of 2 mg/kg body wt. Thalidomide (n = 14 animals) was
administered by intraperitoneal injection at a dose of 10 mg/kg. An
equivalent volume of 100% DMSO (Sigma, St. Louis, MO) was administered
to the vehicle control study group (n = 18 animals) by
intraperitoneal injection. Doses and routes of administration were
based on published studies (12, 21, 22) on tumor angiogenesis
and preliminary data from our laboratory. Study rats were killed on
day 14 by intraperitoneal injection of pentobarbital sodium
(0.3 mg/g body weight). Body, lung, and cardiac weights were measured,
and the lungs were prepared for histology and morphometric analysis. At death, the hearts were removed and dissected to isolate the free wall
of the right ventricle from the left ventricle and septum. The ratio of
right ventricle weight to left ventricle plus septum weight (RV/LV+S)
was used as an index of right ventricular hypertrophy.
The same protocol was used to study the effects of Su-5416 (Sugen,
South San Francisco, CA), a selective inhibitor of the VEGF receptor
KDR/flk-1 (18, 43). Newborn rats (n = 30 animals) from each litter were randomly assigned to treatment with
Su-5416 (20 mg/kg) or vehicle control (carboxymethylcellulose),
administered by subcutaneous injections on alternate days, beginning on
day 1 and continuing through day 13. The dose of
Su-5416 was based on a previous study (18) of tumor
antiangiogenesis in mice. Study animals were killed, and the lungs were
studied as outlined above.
Arterial Density Measurements After Barium Sulfate-Gelatin
Infusions
Pulmonary arterial density was measured from lungs that were
infused with barium sulfate according to previously established methods
(13, 14). Immediately after death, PBS was infused through
a main pulmonary artery catheter to flush the pulmonary circulation
free of blood. A barium sulfate-gelatin mixture was heated to 70°C
and infused into the main pulmonary artery at 74 mmHg. Pressure
was maintained for at least 5 min to ensure penetration of the barium
mixture. Lung tissue was subsequently fixed for histology as
described in Lung Histology and Radial Alveolar
Counts or used for arteriograms. Pulmonary arterial
density was measured by counting (by two blinded observers)
barium-filled arteries per high-power field (×100 magnification) in
8-10 randomly selected fields taken from at least two blocks of tissue.
Lung Histology and Radial Alveolar Counts
Lungs were fixed for histology by tracheal instillation of 10%
buffered Formalin under constant pressure (20 cmH2O). The
tracheae were ligated after sustained inflation, and the lungs were
excised and immersed in Formalin overnight. Formalin-fixed lung tissue was cut into 4- to 5-mm-thick sections, placed in 10% buffered Formalin, and embedded in paraffin. Paraffin sections (5 µm thick) were serially mounted onto Superfrost Plus slides (Fisher Scientific, Fair Lawn, NJ) and stained with hematoxylin and eosin. At least three
lung sections from each animal were assessed for morphometric analysis.
Alveolarization was measured by the radial alveolar counts (RAC)
methods of Cooney and Thurlbeck (9-11) and Emory and
Mithal (16). Briefly, radial counts were
performed by determining the number of septae that intersected a
perpendicular line drawn from the center of a respiratory bronchiole to
the distal acinus (connective tissue septum or pleura). At least 10 counts were performed on each lung section.
Statistical Analysis
Data are presented as means ± SE. Statistical analysis was
performed with the Statview software package (Abacus Concepts, Berkeley, CA). Statistical comparisons were made with the use of ANOVA
and Fisher's protected least significant difference test. P < 0.05 was considered significant.
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RESULTS |
Effects of Thalidomide and Fumagillin Treatment
Lung, body, and cardiac weights.
Body weights were not reduced by fumagillin or thalidomide treatment
compared with weights of control rats (25.3 ± 2.0 g with fumagillin; 30.7 ± 0.6 g with thalidomide; 26.3 ± 1.8 g with vehicle). Lung weights were 0.44 ± 0.02 g
for the fumagillin-treated, 0.41 ± 0.03 g for the
thalidomide-treated, and 0.55 ± 0.06 g for control rats.
Compared with the control group, lung-to-body weight ratios were
reduced by 30 and 38% in the fumagillin and thalidomide treatment groups, respectively (0.017 ± 0.001 with fumagillin; 0.015 ± 0.001 with thalidomide; 0.023 ± 0.002 with vehicle control;
P < 0.05 for control vs. other groups; Fig.
1). Right ventricular mass or hypertrophy
as determined by the RV/LV+S was not different between the drug
treatment and vehicle control groups (0.39 ± 0.01 with
fumagillin; 0.38 ± 0.02 with thalidomide; 0.38 ± 0.02 with
vehicle).

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Fig. 1.
Effects of fumagillin and thalidomide treatment on
lung-to-body weight ratios in 14-day-old Sprague-Dawley rats. Compared
with control rats, the ratio of lung-to-body weight was reduced in the
treated rats. * P < 0.05 for treated vs. nontreated
groups.
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Pulmonary arterial density after barium sulfate-gelatin infusion.
To determine whether treatment with antiangiogenic agents during a
critical period of lung development could decrease arterial density, we
infused the main pulmonary artery with a barium sulfate-gelatin suspension. Arteriograms from the left lungs of thalidomide- and fumagillin-treated rats showed a decrease in the filling of small pulmonary vessels (a decrease in background haze) compared with arteriograms of control animals (Fig. 2).
The major conduit pulmonary arteries and their branches also appeared
narrow compared with those in control rats, and lung histology revealed
a reduction in the number of barium-filled arteries in the fumagillin-
and thalidomide-treated rats (Fig.
3A). Pulmonary arterial
density, as determined by barium-filled arteries per high-power field, was reduced by 20 and 36% in the fumagillin- and thalidomide-treated rats, respectively, compared with that in control animals (32.0 ± 0.2 with fumagillin; 29.0 ± 0.8 with thalidomide; and 44 ± 0.3 with vehicle; P < 0.05 vs. control group; Fig.
3B).

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Fig. 2.
Barium arteriograms of the left lungs of fumagillin- and
thalidomide-treated 14-day-old rats. Lumen diameters of large pulmonary
arteries and barium filling of small pulmonary arteries ("background
haze") were reduced in treated (middle and
right) compared with vehicle control rats
(left).
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Fig. 3.
Effects of fumagillin and thalidomide treatment on the
density of small pulmonary arteries in 14-day-old rats. A:
barium-filled arteries appear sparse after antiangiogenic treatment of
the developing rat (magnification, ×100). B: vessel density
counts were reduced after antiangiogenic treatment. Counts were
performed per high-power field (HPF; original magnification, ×100).
* P < 0.05 for treated vs. control rats.
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Lung histology and RAC.
Lung histology revealed striking differences in lung structure between
the fumagillin and thalidomide groups and the control group. Compared
with vehicle control rat lungs, fumagillin and thalidomide treatment
caused a histological pattern of alveolar simplification
characterized by the presence of larger and fewer distal air spaces
(Fig. 4A). To quantify the
apparent decreases in alveolar number, we measured RAC in the
fumagillin-, thalidomide-, and vehicle-treated groups. Compared with
lungs from control rats, RAC were reduced by 22% in both the
fumagillin- and thalidomide-treated rats (Fig. 4B).


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Fig. 4.
Effects of fumagillin and thalidomide treatment on lung
histology and alveolarization in 14-day-old rats. A: in
contrast with the distal lung of the vehicle control rats, lung
histology demonstrates a pattern of alveolar simplification in both of
the treatment groups (magnification, ×200). B: compared
with that in control animals, alveolar number as determined by
measurements of radial alveolar counts (RAC) was reduced after
antiangiogenic treatment. * P < 0.05 for treated
vs. control rats.
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Effects of Su-5416 Treatment
Compared with vehicle-treated control group, treatment of neonatal
rats with Su-5416, a specific inhibitor of the VEGF receptor KDR/flk-1,
reduced both lung and body weight (see Table
1). Lung weight was decreased in the
Su-5416-treated rats versus control animals (0.49 ± 0.02 vs.
0.33 ± 0.01 g; P < 0.05) as was body weight
(33.74 ± 1.36 vs. 21.42 ± 0.77 g; P < 0.05). As demonstrated by light microscopy, Su-5416 treatment altered
lung architecture, causing a histological pattern of enlarged distal
air spaces ("alveolar simplification") with decreased arterial
density (Fig. 5). Su-5416 treatment
reduced RAC by 30% (9.8 ± 0.8 in control lungs vs. 6.8 ± 0.7 in treated lungs; P < 0.02; Fig.
6). Barium arteriograms from the left
lungs of Su-5416-treated rats showed a decrease in the filling of small
pulmonary vessels (Fig. 7). The major conduit pulmonary arteries and their branches were more narrowed compared with those in control animals, and pulmonary arterial density
was reduced by Su-5416 treatment (Fig.
8). At the doses used in this study,
Su-5416 had a more striking effect on the reduction of arterial density
than fumagillin or thalidomide.

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Fig. 5.
Effects of Su-5416 treatment on lung histology in
14-day-old rats. Compared with lungs from vehicle-treated control rats,
Su-5416 treatment decreased alveolarization and vessel density.
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Fig. 6.
Effects of Su-5416 treatment on alveolarization in the
developing rat lung as assessed by RAC. RAC were markedly reduced after
Su-5416 treatment.
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Fig. 7.
Barium arteriograms of the left lungs of Su-5416-treated
14-day-old rats. Compared with lungs from vehicle-treated control rats,
the lumen diameters of large pulmonary arteries and barium filling of
small pulmonary arteries ("background haze") were reduced in
Su-5416-treated rats.
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Fig. 8.
Effects of Su-5416 treatment on arterial density measured
as vessels/HPF in the 14-day-old rat lung.
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DISCUSSION |
Extensive vascular growth accompanies the increase in
alveolarization during the critical period of postnatal lung
development in young rats, but mechanisms that coordinate
alveolarization with growth of the pulmonary circulation are uncertain.
We hypothesized that if vascular growth were necessary for the increase
in alveolarization during this time period, then disruption of
angiogenesis should cause lung hypoplasia. To test this hypothesis, we
treated neonatal rats during the critical period of lung growth with
three different antiangiogenic agents: thalidomide, fumagillin, and
Su-5416. We report that treatment with thalidomide or fumagillin caused
a histological pattern of alveolar simplification, reduced RAC and pulmonary arterial density, and decreased lung-to-body weight ratios.
These findings support the hypothesis that angiogenesis may be
necessary for alveolarization during normal lung development and that
adverse stimuli that disrupt vascular growth may contribute to lung
hypoplasia. Because thalidomide and fumagillin may have additional
effects on lung growth that are independent of their well-established
antiangiogenic activities and to determine the potential role of the
VEGF receptor KDR/flk-1 during this critical period of postnatal lung
growth, we performed a similar protocol with a KDR/flk-1 inhibitor,
Su-5416. As observed with the other antiangiogenic agents, Su-5416
treatment reduced alveolarization as determined by RAC and decreased
arterial density. These findings suggest that the KDR/flk-1 receptor
contributes significantly to vascular growth in the postnatal rat lung
and provide additional support for the hypothesis that angiogenesis is
necessary for normal alveolarization during the postnatal period of
rapid lung growth.
Previous studies (7, 29, 30) have demonstrated that
alveoli are formed by the septation of large saccules that constitute the gas-exchange region of the immature lung. Secondary septae are
formed by alternate upfolding of one of the two capillary layers on
either side of the primary septum (7). The
double-capillary network persists until later in maturation when focal
fusions lead to the development of a single-capillary layer and
thinning of the interstitium (7). This mechanism implies
that the failure of capillary growth and maturation or disruption of
the upfolding of the double-capillary network could limit
alveolarization. Thus we speculate that failure of growth or maturation
of the pulmonary circulation during the critical period of
alveolarization could potentially decrease septation and cause lung
hypoplasia. However, few studies have directly addressed the effects of
disrupted angiogenesis on lung development during the neonatal period.
Because alveolarization primarily occurs during late gestation and the
early postnatal period (30), it is likely that disruption
of alveolarization by abnormal intrauterine stimuli, premature birth,
postnatal injury, or other mechanisms contributes to this problem. Past
studies (5, 6, 29-31, 39) have shown that exposure to
dexamethasone, hyperoxia, or hypoxia decreases alveolar number in
neonatal rats. Premature infants with BPD and children with lung
hypoplasia have distal air spaces that fail to septate, resulting in
fewer alveoli and a reduced surface area for gas exchange (28,
32, 47). In BPD, lung injury occurs in premature newborns before
the alveolar period of lung development. Several morphometric studies
of older infants and children dying with BPD have demonstrated impaired alveolarization and abnormal lung vascular development (28, 47). Because hyperoxia, ventilator-induced stretch injury,
inflammation, and hypoxia can directly injure the pulmonary circulation
(15, 31, 39), we speculate that disruption of vascular
growth early in development may contribute to the subsequent failure of
alveolarization during infancy and childhood.
Possible limitations of this study include the potential for
nonspecific effects of thalidomide and fumagillin on the developing lung. Fumagillin, an antibiotic secreted by Aspergillus
fumigatus, and thalidomide, an immunomodulatory drug with sedative
and teratogenic effects, have been shown to inhibit angiogenesis
(12, 17, 21, 22, 34, 37). Thalidomide, known as a
teratogen for its disruption of fetal limb formation (phocomelia), has
been reintroduced as a therapeutic agent for use in macular
degeneration, leprosy, Crohn's disease, Behcet's disease, arthritis,
acquired immunodeficiency syndrome (AIDS), graft versus host lung
disease after bone marrow transplantation, and metastatic cancer
(17). Although thalidomide may inhibit the effects of
tumor necrosis factor-
, recent studies (12, 22) have
also shown that it can inhibit VEGF- and basic fibroblast growth
factor-induced neovascularization in a mouse corneal model. In addition
to thalidomide, fumagillin and its synthetic analog TNP-470 are also
antiangiogenic, and effects on vascular growth have been extensively
studied in diverse experimental settings. Fumagillin may act by binding
endothelial methionine aminopeptidase II (21). Whether
both of these drugs can also reduce alveolarization by direct
inhibition of epithelial growth, elastin production in developing
septae, or related nonvascular mechanisms is unknown.
In contrast, Su-5416 is a potent antagonist of the KDR/flk-1 receptor,
has potent inhibitory effects on tumor angiogenesis, and is currently
undergoing clinical trials in patients with cancer (18,
43). When used in similar treatment protocols as the other antiangiogenic agents, Su-5416 treatment also inhibits
alveolarization. The exact mechanism by which inhibition of endothelial
cell proliferation leads to decreased alveolar septation is uncertain;
we speculate that these effects may be due to a trophic effect
on vascular endothelium, altered production of endothelium-derived
products such as nitric oxide or platelet-derived growth factor (PDGF), or other mechanisms (19, 23, 33, 35, 38, 46, 51). In
addition to its effects on the KDR/flk-1 receptor, Su-5416 can also
block the platelet-derived growth factor receptor at higher
concentrations (IC50: 1.5 µm for KDR; 20 µM for PDGF
receptor) (44). Whether part of the response to Su-5416 in
this study is also in part due to inhibition of PDGF receptors is
uncertain. However, a recent study (20) demonstrated that
VEGF inhibition with the use of an inducible
Cre-loxP-mediated gene-targeting approach or by
treatment with a soluble VEGF receptor protein caused growth failure
and high mortality in neonatal mice. In addition to reduced body and
organ weights, VEGF inhibition also decreased lung weight and altered
lung histology. A further study (20) revealed a
marked alteration in endothelial cell appearance as viewed by electron
microscopy and increased apoptosis, suggesting that VEGF is necessary
for endothelial survival as well as for proliferation.
Recently, our laboratory has observed abnormal lung development in the
Fawn-Hooded rat (FHR), a genetic strain that develops severe pulmonary
hypertension with modest decreases in alveolar PO2 (25). Although the FHR strain
has been generally considered as a genetic model of "primary"
pulmonary hypertension, our laboratory (25)
previously reported that FHR lungs showed a pattern of alveolar
simplification and reduced pulmonary arterial density early during
maturation. Similarly, in other rat models of lung hypoplasia caused by
neonatal dexamethasone treatment (29) or hypoxia (5,
6, 31), pulmonary arterial density is also reduced, and there is
an increased risk for late development of pulmonary hypertension (26, 44a). Whether decreased angiogenesis plays a significant role in the
development of lung hypoplasia in these models is unknown, but data
from this current report support the concept that inhibition of
angiogenesis during a critical period of lung growth may contribute to
impaired alveolarization.
In summary, we report that treatment of neonatal rats with two
different inhibitors of angiogenesis during a critical period of
postnatal lung growth caused lung hypoplasia as demonstrated by reduced
lung-to-body weight ratios, alveolarization, and pulmonary arterial
density. We also report that an inhibitor of the VEGF receptor
KDR/flk-1 also reduced alveolarization, further suggesting that
disruption of angiogenesis attenuates normal lung development. Based on
these findings, we speculate that disruption of angiogenesis impairs
alveolarization, suggesting that primary or acquired abnormalities of
lung vascular growth can cause or contribute to lung hypoplasia. We
further speculate that new strategies that stimulate vascular growth
may provide an alternate approach to the treatment of abnormal lung
growth in clinical disorders such as BPD and congenital diaphragmatic hernia.
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FOOTNOTES |
Address for reprint requests and other correspondence: S. H. Abman, Dept. of Pediatrics, B-395, The Children's Hospital, 1056 E. Nineteenth Ave., Denver, CO 80218-1088 (E-mail:
steven.abman{at}uchsc.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 7 January 2000; accepted in final form 10 April 2000.
 |
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