1 Pediatric Heart Lung Center and Section of Pediatric Pulmonary Medicine, University of Colorado School of Medicine, Denver, Colorado 80218; and 2 Department of Anatomy, Physiology, and Cell Biology, School of Veterinary Medicine, University of California, Davis, California 95616
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In addition to its vasodilator properties,
nitric oxide (NO) promotes angiogenesis in the systemic circulation and
tumors. However, the role of NO in promoting normal lung vascular
growth and its impact on alveolarization during development or in
response to perinatal stress is unknown. We hypothesized that NO
modulates lung vascular and alveolar growth and that decreased NO
production impairs distal lung growth in response to mild hypoxia.
Litters of 1-day-old mouse pups from parents that were heterozygous for endothelial nitric oxide synthase (eNOS) deficiency were placed in a
hypobaric chamber at a simulated altitude of 12,300 ft
(FIO2 = 0.16). After 10 days,
the mice were killed, and lungs were fixed for morphometric and
molecular analysis. Compared with wild-type controls, mean linear
intercept (MLI), which is inversely proportional to alveolar surface
area, was increased in the eNOS-deficient (eNOS /
) mice [51 ± 2 µm (eNOS
/
) vs. 41 ± 1 µm (wild type); P < 0.01]. MLI was also increased in the eNOS
heterozygote (+/
) mice (44 ± 1 µm; P < 0.03 vs. wild type). Vascular volume density was decreased in the eNOS
/
mice compared with wild-type controls (P < 0.03). Lung
vascular endothelial growth factor (VEGF) protein and VEGF receptor-1
(VEGFR-1) protein content were not different between the study groups.
In contrast, lung VEGFR-2 protein content was decreased from control
values by 63 and 34% in the eNOS
/
and eNOS +/
mice,
respectively (P < 0.03). We conclude that exposure to
mild hypoxia during a critical period of lung development impairs alveolarization and reduces vessel density in the eNOS-deficient mouse.
We speculate that NO preserves normal distal lung growth during hypoxic
stress, perhaps through preservation of VEGFR-2 signaling.
pulmonary hypertension; persistent pulmonary hypertension of the newborn; lung hypoplasia; congenital diaphragmatic hernia; angiogenesis
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PREMATURE BIRTH, PERINATAL STRESS, and the treatment of neonatal respiratory distress syndrome (RDS) can cause lung injury, leading to the development of chronic lung disease of infancy, known as bronchopulmonary dysplasia (BPD) (37). Although mechanisms that cause BPD are not completely understood, surfactant deficiency, ventilator-induced lung injury, oxygen toxicity, inflammation, and infection have been identified as important pathogenic factors (21). More recently, the routine use of exogenous surfactant therapy has reduced the severity of acute lung disease in premature neonates, but the incidence of BPD continues to rise (22). Lung histology of infants and older children who die from BPD demonstrate striking inhibition of distal lung growth (1, 19, 32, 42, 44). Impaired alveolarization and dysmorphic vascular growth decrease lung surface area, resulting in abnormal gas exchange, exercise intolerance, and pulmonary hypertension in patients with BPD (1, 22). Mechanisms that disrupt alveolarization and angiogenesis resulting in the development of BPD are only beginning to be understood.
The saccular and alveolar phases of lung development are characterized by rapid increases in vessel growth and septation within the distal air space. These phases of lung development occur from 24 wk of gestation through the first 3 yr of life in humans and during the first 3 wk of postnatal life in rodents (7, 33). Lung development is dependent on interactions between developing vessels and air spaces (4, 15, 40) and is a highly complex process dependent on a temporal and spatial expression of multiple cytokines, growth factors, growth factor receptors, and other signals (35, 39, 45). Septation and the formation of alveoli late in gestation are intricately linked with angiogenesis as reflected by the findings that inhibition of angiogenesis impairs alveolarization (20). Likewise, hypoxic exposure during critical periods of lung development can disrupt both alveolar and pulmonary vascular growth (33, 46). Mechanisms that link alveolar development and vessel growth are uncertain.
Vascular endothelial growth factor (VEGF) is a potent mitogen for angiogenesis (13) that plays an important role during fetal lung development. VEGF exists as multiple isoforms and induces vascular growth and angiogenesis through the activation of its receptors VEGFR-1 (flt-1) and VEGFR-2 (KDR/flk-1) (36, 49). Lung VEGF mRNA and protein are decreased in human neonates who die with BPD (5, 27), and treatment of newborn rats with a VEGFR inhibitor reduces vessel density and alveolarization in infant and adult rats (20, 30). These studies suggest that disruption of VEGF signaling can impair vessel growth and alveolarization and may contribute to abnormal lung structure in infants with BPD.
In studies of systemic vessels and tumors, VEGF stimulates angiogenesis by the elaboration of nitric oxide (NO) through activation of endothelial nitric oxide synthase (eNOS) (12). VEGF activates eNOS in endothelial cells via its interaction with VEGFR-2 (26, 41), and the proangiogenic effect of VEGF in the systemic circulation is partly dependent on eNOS activity (14). Whether VEGF-induced angiogenesis is dependent on eNOS activity in the developing lung is uncertain. In different settings, NO has been reported as having both proangiogenic (14, 34) and antiangiogenic effects (38). The role of eNOS during postnatal lung vascular growth and its role in the response to injury, such as hypoxia, has not been directly studied.
On the basis of these past studies, we hypothesized that NO modulates lung vascular and alveolar growth and that decreased NO production impairs distal lung growth in response to mild postnatal hypoxia. To test this hypothesis, we studied the effects of mild hypobaric hypoxia (16% oxygen) in mice that are genetically deficient in eNOS. We report that although eNOS deficiency did not affect alveolarization in infant mice raised in room air, mild hypoxia impaired alveolarization and reduced vessel density in eNOS-deficient mice.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Study animals and protocols. All procedures and protocols were reviewed and approved by the Animal Care and Use Committee at the University of Colorado Health Sciences Center. Mice (C57/B6) that were genetically engineered to be heterozygous for a deficiency of eNOS (18) were obtained from Jackson Laboratories. Genomic DNA was isolated from tails of experimental animals. Genotyping was done by PCR, utilizing previously described primers to identify the presence of the Neo gene insert in the eNOS gene (18).
Exposure of neonatal mice to mild hypoxia. Males and females known to be heterozygous for eNOS deficiency were bred to ensure that the generation of litters were homozygous and heterozygous for eNOS deficiency and wild type. The litters of heterozygote × heterozygote matings were delivered and allowed to recover for 24 h. Mothers and litters were either treated in hypobaric chambers at a simulated altitude of 12,300 ft (16% O2; PO2 = 90 mmHg) for 10 days or maintained in room air. Exposure to hypoxia was continuous with less than a 1-h/day interruption for animal care. After 10 days, total body weight was measured on each animal, and lung tissue was collected from the studies below.
Tissue for histological analysis. Animals were killed with intraperitoneal injections of pentobarbital (100 mg/kg). A catheter was placed into the trachea, and the lungs were inflated at 30 cmH2O pressure with 4% paraformaldehyde in RNase-free phosphate-buffered saline (PBS) and maintained under constant pressure for at least 45 min. A ligature was tightened around the trachea to maintain pressure, and then tracheal cannula was removed. The lungs were immersed in paraformaldehyde solution overnight. The right lower lobe was embedded in paraffin, and sections were obtained with a microtome set at 5 µm. Sections were mounted on RNase-free slides for histochemical analysis.
Morphometric analysis. Six lung sections from each animal were selected for study in an unbiased fashion. The orientation of these samples was at random, creating isotropic uniform random plane sections of the lung tissue. Each section was stained with hematoxylin and eosin. Images of each section were captured using the ×20 objective as a high-resolution PICT image by a ProgRes 3008 digital camera (JenOptik; 1,928 × 1,450 pixel resolution) and were analyzed with the use of Stereology Toolbox software (Davis, CA). The intra-alveolar distance was measured as the mean linear intercept (MLI), which was determined by dividing the total length of 42 lines drawn across the lung section by the number of intercepts encountered as determined by the investigator (48). The investigator was blinded to the identity of the sections at the time of analysis. Lines that crossed large airways or vessels were excluded from analysis. MLI is inversely proportional to the surface area of the lung (47, 48). Radial alveolar counts (RAC) were assessed by the methods of Cooney and Thurlbeck and Emery and Mithal (11). Sections were also stained for the presence of factor VIII (Dako), an endothelial-specific marker. A point-counting method was used to determine the volume fraction [or vessel volume density (Vv)] of immunoreactive sites in which the lung parenchyma served as the volume of reference (10, 17). A grid of 100 points was superimposed on color photographs taken using the ×20 objective from seven random noncontiguous fields per animal. The number of points falling on immunoreactive sites and on lung parenchyma was recorded. Lung parenchyma was defined as parenchymal tissue excluding large vessels and airways. The Vv was calculated as the ratio of the number of points falling on factor VIII sites to points on lung parenchyma.
Western blot analysis. Frozen lung samples were homogenized in ice-cold buffer containing 50 mM Tris · HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 0.1% 2-mercaptoethanol, 1 mM 4-(2-aminoethyl)benzenesulfonyl fluoride, 1 µM leupeptin, and 1 µM pepstatin A. The 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 (6) using bovine serum albumin as the standard. Briefly, 25 µg of protein sample per lane were resolved by SDS-PAGE, and proteins from the gel were transferred to nitrocellulose membrane. Blots were blocked 1 h in 5% nonfat dry milk in Tris-buffered saline (TBS) with 0.1% Tween 20. These blots were incubated for 1 h at room temperature with either rabbit anti-human polyclonal VEGF antibody (sc-152; Santa Cruz Biotechnology), rabbit anti-mouse polyclonal VEGFR-1 (Flt-1) antibody (sc-504; Santa Cruz Biotechnology), or rabbit anti-human polyclonal VEGFR-2 antibody (KDR/flk-1; sc-316; Santa Cruz Biotechnology) diluted 1:200 in 5% nonfat dry milk in TBS with 0.1% Tween 20. Blots were incubated for 1 h at room temperature with a goat anti-rabbit IgG-horseradish peroxidase antibody (Santa Cruz Biotechnology). After being washed, bands were visualized by enhanced chemiluminescence (ECL Plus kit; Amersham Pharmacia Biotech, Little Chalfont, UK). Adult mouse lung homogenate was run as a control, and the band that comigrated with the molecular size as identified by the manufacturer for the protein of interest was quantified by densitometry for VEGFR-1 and VEGFR-2. For Western blot analysis of VEGF, recombinant mouse VEGF was used as a control. Densitometry was performed using NIH Image (v. 1.61).
Immunohistochemical staining. Lungs were fixed in 4% paraformaldehyde for 24 h and then stored in 70% ethanol. The left lower lobe was embedded in paraffin, cut into 5-µm-thick sections, and mounted on "plus" slides. Slides were deparaffinized in HemoDe and rehydrated by serial immersions in 100% ethanol, 95% ethanol, 70% ethanol, and 100% water. Proteinase K (50 µg/ml) was placed on the sections for 5 min. The sections were washed with 1× PBS (2.7 mM KCl, 1.2 mM KH2PO4, 138 mM NaCl, 8.1 mM Na2HPO4). Endogenous peroxidase activity was quenched by immersion in 3% hydrogen peroxide in methanol. The slides were rinsed with 1× PBS. The sections were incubated with 10% goat/2% mouse serum and rabbit anti-human polyclonal factor VIII antibody (DAKO, A0082) or mouse IgG diluted 1:1,000 in 1× PBS with 1% BSA and 0.1% sodium azide for 1 h at room temperature. After incubation, the sections were rinsed with 1× PBS, incubated in 10% goat/2% mouse serum for 5 min, and then incubated with biotin-labeled goat anti-mouse secondary antibody diluted 1:200 in 10% goat/2% sheep serum for 15 min at room temperature. After incubation with the secondary antibody, sections were rinsed with 1× PBS. Sections were incubated with ABC complex (Vector) for 30 min at room temperature, rinsed in 1× PBS, and developed with diaminobenzidine (DAB) and hydrogen peroxide. Washing with water stopped the DAB reaction. A light hematoxylin counterstain was applied. Sections were dehydrated by sequential immersion in 70% ethanol, 95% ethanol, and 100% ethanol and then in HemoDe before a coverslip was placed on the section.
Statistical analysis. Data are presented as means ± SE. Statistical analysis was performed with the Statview software package (SAS Institute, Cary, NC). Statistical comparisons were made using analysis of variance and Fisher's protected least significant differences test. P < 0.05 was considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Body weight.
At 11 days postnatal age, body weight was not different between
wild-type, eNOS heterozygote (eNOS +/), and eNOS-deficient (eNOS
/
) mice raised in room air. Of the mice raised in 16% oxygen, body
weight of the eNOS
/
was significantly lower than eNOS +/
and
wild-type mice (Table 1,
P < 0.01). Within each study group, body
weights of the animals raised in 16% oxygen were significantly lower
than those raised in room air (Table 1, P < 0.01).
|
Lung histology and morphometric studies.
There was no significant change in MLI between wild-type, eNOS +/, or
eNOS
/
animals raised in room air. Compared with wild-type animals,
eNOS
/
animals raised in 16% oxygen had enlarged distal airspaces
(Fig. 1). After exposure to mild hypoxia,
the MLI in the eNOS
/
mice was increased by 25% compared with the MLI measurements in the wild-type mice (52 ± 2.0 µm vs. 41 ± 1 µm; P < 0.01; Fig.
2). MLI in the eNOS +/
mice raised in
16% oxygen was increased by 6% compared with wild-type mice (44 ± 1 µm; P < 0.03; Fig. 2). MLI of wild-type mice
raised in room air was not different compared with those raised in 16%
oxygen (42 ± 1 µm; P = not significant; Fig.
2). There was no difference in the RAC between the three groups of
animals raised in room air. There was a 42% decrease in RAC in the
eNOS
/
mice raised in mild hypoxia compared with wild-type mice
(4.13 ± 0.24 vs. 7.14 ± 0.14; P < 0.01;
Fig. 3). The RAC of eNOS +/
mice was
reduced by 13% compared with wild-type mice (6.23 ± 0.19;
P < 0.01; Fig. 3). There was no difference in the RAC
of wild-type animals raised in room air compared with those raised in
16% oxygen. There were no differences in Vv between the
three groups of animals raised in room air. Lung Vv in the
eNOS
/
mice raised in 16% oxygen was decreased compared with
values measured in wild-type mice (69 ± 3 vs. 75 ± 2%;
P < 0.03; Fig. 4).
|
|
|
|
Effects of mild hypoxia on lung VEGF, VEGFR-1, and VEGFR-2
expression.
As determined by Western blot analysis, we detected no differences in
lung VEGF protein in the eNOS /
, eNOS +/
, and wild-type mice when
raised in room air or 16% oxygen (Fig.
5). Lung VEGFR-1 protein expression in
the eNOS
/
and eNOS +/
mice was not different from measurements
in wild-type mice that were raised in room air or 16% oxygen (Fig.
6). In addition, lung VEGFR-2 expression
was not different between genotypes raised in room air. However,
compared with wild-type controls, 16% oxygen exposure decreased
VEGFR-2 protein by 63 and 33% in eNOS
/
and +/
mice,
respectively (P < 0.03 for each vs. controls; Fig.
7).
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We report that in contrast to wild-type mice, exposure to mild
hypoxia (16% oxygen) impairs alveolarization in eNOS-deficient mice,
as evidenced by an increase in MLI, which is inversely proportional to
the internal lung surface area, and reduced RAC, in addition to
decreased vascular volume density. In infant mice raised in room air,
alveolarization is not reduced in eNOS-deficient mice, and mild hypoxia
(16% oxygen) has no apparent effect on alveolarization in wild-type
mice. We also report that mild hypoxia does not affect lung VEGF
protein expression but reduces VEGFR-2 protein expression by 63% in
eNOS /
mice. These findings suggest that with mild hypoxic stress
in the early postnatal period, eNOS deficiency impairs angiogenesis and
alveolar development. These findings also suggest that NO production
plays an important role in modulating distal lung growth in response to
mild stress.
This is the first study to report the effects of mild neonatal hypoxia on pulmonary vascular and alveolar development in animals with primary eNOS deficiency. Although alveolarization and lung vascular growth are not apparently affected by mild postnatal hypoxia, mice with a genetic deficiency of eNOS have an increased susceptibility for abnormal lung growth in response to mild postnatal hypoxia. Our findings are consistent with a recent report of impaired compensatory lung growth after pneumonectomy in adult mice with eNOS deficiency (31). These changes consist of alveolar simplification and decreased vessel density, which are similar to the lung pathology of infants with BPD (1, 22). Interestingly, the expression of a potent angiogenic growth factor, VEGF, is unchanged in the hypoxic eNOS-deficient mouse, but lung expression of VEGFR-2, the primary receptor involved in VEGF-stimulated angiogenesis, is decreased. These results suggest that eNOS contributes to the maintenance of normal lung vascular and alveolar growth in response to mild postnatal hypoxic stress. We further speculate that this mechanism may be partially through the preservation of VEGF signaling via increased VEGFR-2 expression.
We have observed a decrease in body weight in eNOS /
mice exposed
to mild hypoxia, which correlates with previous studies that have
observed a reduction in size (both weight and crown-rump length) of
eNOS
/
mice (16). This previous study suggests that
the reduction in size of the eNOS
/
mice was not from malnutrition but may be secondary to abnormalities of somatic growth from the eNOS
deficiency. The role of malnutrition on alveolar growth and development
is well described (2, 23-25). If the abnormalities of
alveolarization that we report were solely due to a reduction in body
weight as a consequence of the eNOS deficiency, then we would expect
that these abnormalities in alveolarization would be seen in eNOS
/
animals raised in room air. This study did not quantify other measures
of growth, such as crown-rump length, and we cannot exclude that
undernutrition may play a role in the abnormalities of lung growth that
we have observed.
Previous reports of the effects of hypoxia on lung structure have generally studied the response of the developing lung to more severe hypoxia (33, 46). Postnatal exposure of infant rats to 13% oxygen during the first 2 wk of life results in abnormal lung development, characterized by impaired septation and decreased surface area for gas exchange (33). Brief perinatal hypoxia (10% oxygen) during the last 3 days of gestation and for the first 3 days of life decreases alveolar septation and vessel density in infant rats, which persists into adulthood (46). These studies illustrate that hypoxic exposure during critical periods of lung development disrupts alveolar and pulmonary vascular structure.
A link between vascular growth and alveolarization has been previously demonstrated through studies with inhibitors of angiogenesis, including fumagillin, thalidomide, and SU-5416 (a VEGFR inhibitor). Treatment with these agents impairs alveolarization in infant and adult rats (20, 30). We have also observed a reduction in alveolarization and pulmonary arterial density in response to mild hypoxia in a genetic model of pulmonary hypertension, the Fawn-Hooded rat (FHR) (28). The FHR strain is characterized by decreased lung eNOS expression in fetal and neonatal life (29). Interestingly, treatment of FHR with low levels of supplemental oxygen improves vessel density and alveolarization, suggesting that the genetic abnormality in the FHR increases the susceptibility for abnormal lung development with stress (29). Still, the exact genetic abnormality in the FHR is unknown, and the FHR has been known to have abnormalities in serotonin and endothelin pathways (3, 43, 50, 52), which may also contribute to the abnormal adaptation to hypoxia in the FHR.
Angiogenesis is a complex process that requires endothelial cell proliferation, migration, and vascular tube formation (9, 39). VEGF is a potent growth factor for endothelial cell proliferation, survival, and angiogenesis (36). Previous studies have shown that specific inhibition of VEGFRs during the neonatal period resulted in a decrease in alveolarization and pulmonary arterial density (20, 30). NO is a downstream effector of VEGF via VEGF/VEGFR-2 phosphorylation and activation of eNOS (12, 14, 26, 41). In the systemic circulation, VEGF-mediated angiogenesis in wound healing is dependent on functional eNOS, as shown by a reduction in VEGF-induced angiogenesis in the eNOS-deficient mouse (14). In the ischemic hindlimb model, eNOS-deficient mice did not increase angiogenesis in response to VEGF, further evidence that eNOS is a downstream mediator of VEGF (34). VEGF signaling that results in endothelial cell proliferation is via an upregulation of VEGFR-2 that is dependent on NO (51). NO plays a role in VEGF/VEGFR-1 signaling that is responsible for the formation of mature vascular structures. Selective inhibition of VEGFR-1 results in the formation of abnormal aneurysm-like structures and accumulation of endothelial cells, and this effect was rescued by NO donors (8). These studies suggest that normal endothelial cell proliferation and differentiation into vascular structures in response to VEGF is dependent on functional eNOS. Whereas our study suggests that the eNOS is not essential for normal lung vascular and alveolar development at ambient oxygen tension, functional eNOS is critical for normal lung vascular and alveolar development during hypoxic stress.
Potential limitations of our study include that abnormalities in expression of other growth factors and receptors may be affected by eNOS deficiency, and these may contribute to abnormalities of lung angiogenesis and alveolarization. We did not measure absolute changes in NO production in these animals, and it is possible that there were compensatory increases in the other NOS isoforms (type I and type II) that may have compensated for the lack of eNOS. Further studies will need to be performed to see whether exogenous NO can rescue this phenotype. We were not able to measure lung compliance and elastic recoil in these animals, and changes in these could lead to abnormal air space size and a reduction in secondary septal height. Future studies are needed to assess these measures of lung function in these animals. Further studies will need to be performed to elucidate the specific mechanisms by which a deficiency of eNOS affects endothelial cell growth, migration, and differentiation. Finally, a deficiency of eNOS may have effects on prenatal lung vascular and air space development that may predispose this genetic model from adapting to mild hypoxic exposure.
In summary, exposure to mild hypoxia in the eNOS-deficient mouse results in abnormalities of pulmonary angiogenesis and alveolarization, as evidenced by a reduction in alveolar surface area for gas exchange and a decreased vascular volume density. Furthermore, this phenomenon is not observed during development at normal ambient oxygen tension. These mice show no change in VEGF and VEGFR-1 expression but show a decrease in VEGFR-2 expression compared with wild-type mice. We speculate that NO is essential in pulmonary angiogenesis and alveolarization in the postnatal lung during mild hypoxic stress through the preservation of VEGF signaling via increasing VEGFR-2 expression.
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: V. Balasubramaniam, Pediatric Pulmonology, Dept. of Pediatrics, Univ. of Colorado Health Sciences Center, C-218, 4200 E. 9th Ave., Denver, CO 80262 (E-mail: vivek.balasubramaniam{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. Section 1734 solely to indicate this fact.
First published February 14, 2003;10.1152/ajplung.00421.2002
Received 9 December 2002; accepted in final form 1 February 2003.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abman, S.
Pulmonary hypertension in chronic lung disease of infancy: pathogenesis, pathophysiology, and treatment.
In: Chronic Lung Disease in Early Infancy, edited by Bland RD,
and Coalson JJ.. New York: Dekker, 1999, p. 619-668.
2.
Atkinson, SA.
Special nutritional needs of infants for prevention of and recovery from bronchopulmonary dysplasia.
J Nutr
131:
942S-946S,
2001
3.
Aulakh, CS,
Wozniak KM,
Hill JL,
Devane CL,
Tolliver TJ,
and
Murphy DL.
Differential neuroendocrine responses to the 5-HT agonist m-chlorophenylpiperazine in Fawn-Hooded rats relative to Wistar and Sprague-Dawley rats.
Neuroendocrinology
48:
401-406,
1988[ISI][Medline].
4.
Baldwin, HS.
Early embryonic vascular development.
Cardiovasc Res
31:
E34-E45,
1996[ISI][Medline].
5.
Bhatt, AJ,
Amin SB,
Chess PR,
Watkins RH,
and
Maniscalco WM.
Expression of vascular endothelial growth factor and Flk-1 in developing and glucocorticoid-treated mouse lung.
Pediatr Res
47:
606-613,
2000
6.
Bradford, MM.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.
Anal Biochem
72:
248-254,
1976[ISI][Medline].
7.
Burri, P.
Structural aspects of prenatal and postnatal development and growth for the lung.
In: Lung Growth and Development, edited by McDonald JA.. New York: Dekker, 1997, p. 1-35.
8.
Bussolati, B,
Dunk C,
Grohman M,
Kontos CD,
Mason J,
and
Ahmed A.
Vascular endothelial growth factor receptor-1 modulates vascular endothelial growth factor-mediated angiogenesis via nitric oxide.
Am J Pathol
159:
993-1008,
2001
9.
Carmeliet, P.
Mechanisms of angiogenesis and arteriogenesis.
Nat Med
6:
389-395,
2000[ISI][Medline].
10.
Coalson, JJ,
Winter VT,
Siler-Khodr T,
and
Yoder BA.
Neonatal chronic lung disease in extremely immature baboons.
Am J Respir Crit Care Med
160:
1333-1346,
1999
11.
Cooney, TP,
and
Thurlbeck WM.
The radial alveolar count method of Emery and Mithal: a reappraisal 2-intrauterine and early postnatal lung growth.
Thorax
37:
580-583,
1982[Abstract].
12.
Dimmeler, S,
Dernbach E,
and
Zeiher AM.
Phosphorylation of the endothelial nitric oxide synthase at Ser-1177 is required for VEGF-induced endothelial cell migration.
FEBS Lett
477:
258-262,
2000[ISI][Medline].
13.
Ferrara, N.
Role of vascular endothelial growth factor in regulation of physiological angiogenesis.
Am J Physiol Cell Physiol
280:
C1358-C1366,
2001
14.
Fukumura, D,
Gohongi T,
Kadambi A,
Izumi Y,
Ang J,
Yun CO,
Buerk DG,
Huang PL,
and
Jain RK.
Predominant role of endothelial nitric oxide synthase in vascular endothelial growth factor-induced angiogenesis and vascular permeability.
Proc Natl Acad Sci USA
98:
2604-2609,
2001
15.
Gebb, SA,
and
Shannon JM.
Tissue interactions mediate early events in pulmonary vasculogenesis.
Dev Dyn
217:
159-169,
2000[ISI][Medline].
16.
Hefler, LA,
Reyes CA,
O'Brien WE,
and
Gregg AR.
Perinatal development of endothelial nitric oxide synthase-deficient mice.
Biol Reprod
64:
666-673,
2001
17.
Howard, V,
and
Reed MG.
Unbiased Stereology: Three-Dimensional Measurement in Microscopy. Oxford, UK: Springer in association with the Royal Microscopical Society, 1998.
18.
Huang, PL,
Huang Z,
Mashimo H,
Bloch KD,
Moskowitz MA,
Bevan JA,
and
Fishman MC.
Hypertension in mice lacking the gene for endothelial nitric oxide synthase.
Nature
377:
239-242,
1995[ISI][Medline].
19.
Husain, AN,
Siddiqui NH,
and
Stocker JT.
Pathology of arrested acinar development in postsurfactant bronchopulmonary dysplasia.
Hum Pathol
29:
710-717,
1998[ISI][Medline].
20.
Jakkula, M,
Le Cras TD,
Gebb S,
Hirth KP,
Tuder RM,
Voelkel NF,
and
Abman SH.
Inhibition of angiogenesis decreases alveolarization in the developing rat lung.
Am J Physiol Lung Cell Mol Physiol
279:
L600-L607,
2000
21.
Jobe, AH,
and
Bancalari E.
Bronchopulmonary dysplasia E.
Am J Respir Crit Care Med
163:
1723-1729,
2001
22.
Jobe, AJ.
The new BPD: an arrest of lung development.
Pediatr Res
46:
641-643,
1999[ISI][Medline].
23.
Kalenga, M,
Tschanz SA,
and
Burri PH.
Protein deficiency and the growing rat lung. I. Nutritional findings and related lung volumes.
Pediatr Res
37:
783-788,
1995[Abstract].
24.
Kalenga, M,
Tschanz SA,
and
Burri PH.
Protein deficiency and the growing rat lung. II. Morphometric analysis and morphology.
Pediatr Res
37:
789-795,
1995[Abstract].
25.
Kotecha, S.
Lung growth: implications for the newborn infant.
Arch Dis Child Fetal Neonatal Ed
82:
F69-F74,
2000
26.
Kroll, J,
and
Waltenberger J.
VEGF-A induces expression of eNOS and iNOS in endothelial cells via VEGF receptor-2 (KDR).
Biochem Biophys Res Commun
252:
743-746,
1998[ISI][Medline].
27.
Lassus, P,
Turanlahti M,
Heikkila P,
Andersson LC,
Nupponen I,
Sarnesto A,
and
Andersson S.
Pulmonary vascular endothelial growth factor and Flt-1 in fetuses, in acute and chronic lung disease, and in persistent pulmonary hypertension of the newborn.
Am J Respir Crit Care Med
164:
1981-1987,
2001
28.
Le Cras, TD,
Kim DH,
Gebb S,
Markham NE,
Shannon JM,
Tuder RM,
and
Abman SH.
Abnormal lung growth and the development of pulmonary hypertension in the Fawn-Hooded rat.
Am J Physiol Lung Cell Mol Physiol
277:
L709-L718,
1999
29.
Le Cras, TD,
Kim DH,
Markham NE,
and
Abman AS.
Early abnormalities of pulmonary vascular development in the Fawn-Hooded rat raised at Denver's altitude.
Am J Physiol Lung Cell Mol Physiol
279:
L283-L291,
2000
30.
Le Cras, TD,
Markham NE,
Tuder RM,
Voelkel NF,
and
Abman SH.
Treatment of newborn rats with a VEGF receptor inhibitor causes pulmonary hypertension and abnormal lung structure.
Am J Physiol Lung Cell Mol Physiol
283:
L555-L562,
2002
31.
Leuwerke, SM,
Kaza AK,
Tribble CG,
Kron IL,
and
Laubach VE.
Inhibition of compensatory lung growth in endothelial nitric oxide synthase-deficient mice.
Am J Physiol Lung Cell Mol Physiol
282:
L1272-L1278,
2002
32.
Margraf, LR,
Tomashefski JF, Jr,
Bruce MC,
and
Dahms BB.
Morphometric analysis of the lung in bronchopulmonary dysplasia.
Am Rev Respir Dis
143:
391-400,
1991[ISI][Medline].
33.
Massaro, GD,
Olivier J,
and
Massaro D.
Short-term perinatal 10% O2 alters postnatal development of lung alveoli.
Am J Physiol Lung Cell Mol Physiol
257:
L221-L225,
1989
34.
Murohara, T,
Asahara T,
Silver M,
Bauters C,
Masuda H,
Kalka C,
Kearney M,
Chen D,
Symes JF,
Fishman MC,
Huang PL,
and
Isner JM.
Nitric oxide synthase modulates angiogenesis in response to tissue ischemia.
J Clin Invest
101:
2567-2578,
1998
35.
Mustonen, T,
and
Alitalo K.
Endothelial receptor tyrosine kinases involved in angiogenesis.
J Cell Biol
129:
895-898,
1995[ISI][Medline].
36.
Neufeld, G,
Cohen T,
Gengrinovitch S,
and
Poltorak Z.
Vascular endothelial growth factor (VEGF) and its receptors.
FASEB J
13:
9-22,
1999
37.
Northway, WH, Jr,
Rosan RC,
and
Porter DY.
Pulmonary disease following respirator therapy of hyaline-membrane disease. Bronchopulmonary dysplasia.
N Engl J Med
276:
357-368,
1967[ISI][Medline].
38.
Pipili-Synetos, E,
Sakkoula E,
Haralabopoulos G,
Andriopoulou P,
Peristeris P,
and
Maragoudakis ME.
Evidence that nitric oxide is an endogenous antiangiogenic mediator.
Br J Pharmacol
111:
894-902,
1994[Abstract].
39.
Risau, W.
Mechanisms of angiogenesis.
Nature
386:
671-674,
1997[ISI][Medline].
40.
Shannon, J,
and
Deterding R.
Epithelial-mesenchymal interactions in lung development.
In: Lung Growth and Development, edited by McDonald JA.. New York: Dekker, 1997, p. 88-118.
41.
Shen, B,
Lee DY,
and
Zioncheck TF.
Vascular endothelial growth factor governs endothelial nitric oxide synthase expression via a KDR/Flk-1 receptor and a protein kinase C signaling pathway.
J Biol Chem
274:
33057-33063,
1999
42.
Sobonya, RE,
Logvinoff MM,
Taussig LM,
and
Theriault A.
Morphometric analysis of the lung in prolonged bronchopulmonary dysplasia.
Pediatr Res
16:
969-972,
1982[Abstract].
43.
Stelzner, TJ,
O'Brien RF,
Yanagisawa M,
Sakurai T,
Sato K,
Webb S,
Zamora M,
McMurtry IF,
and
Fisher JH.
Increased lung endothelin-1 production in rats with idiopathic pulmonary hypertension.
Am J Physiol Lung Cell Mol Physiol
262:
L614-L620,
1992
44.
Stocker, JT.
Pathologic features of long-standing "healed" bronchopulmonary dysplasia: a study of 28 3- to 40-month-old infants.
Hum Pathol
17:
943-961,
1986[ISI][Medline].
45.
Tallquist, MD,
Soriano P,
and
Klinghoffer RA.
Growth factor signaling pathways in vascular development.
Oncogene
18:
7917-7932,
1999[ISI][Medline].
46.
Tang, JR,
Le Cras TD,
Morris KG, Jr,
and
Abman SH.
Brief perinatal hypoxia increases severity of pulmonary hypertension after reexposure to hypoxia in infant rats.
Am J Physiol Lung Cell Mol Physiol
278:
L356-L364,
2000
47.
Thurlbeck, WM.
The internal surface area of nonemphysematous lungs.
Am Rev Respir Dis
95:
765-773,
1967[ISI][Medline].
48.
Thurlbeck, WM.
Measurement of pulmonary emphysema.
Am Rev Respir Dis
95:
752-764,
1967[ISI][Medline].
49.
Tischer, E,
Mitchell R,
Hartman T,
Silva M,
Gospodarowicz D,
Fiddes JC,
and
Abraham JA.
The human gene for vascular endothelial growth factor. Multiple protein forms are encoded through alternative exon splicing.
J Biol Chem
266:
11947-11954,
1991
50.
Tschopp, B,
and
Weiss HJ.
Decreased ATP, ADP and serotonin in young platelets of fawn-hooded rats with storage pool disease.
Thromb Diath Haemorrh
32:
670-677,
1974[ISI][Medline].
51.
Wang, J,
Morita I,
Onodera M,
and
Murota S.
Induction of KDR expression in bovine arterial endothelial cells by thrombin: involvement of nitric oxide.
J Cell Physiol
190:
238-250,
2002[ISI][Medline].
52.
Zamora, MR,
Stelzner TJ,
Webb S,
Panos RJ,
Ruff LJ,
and
Dempsey EC.
Overexpression of endothelin-1 and enhanced growth of pulmonary artery smooth muscle cells from fawn-hooded rats.
Am J Physiol Lung Cell Mol Physiol
270:
L101-L109,
1996