help button home button
AJRCMB
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Published ahead of print on February 18, 2005, doi:10.1165/rcmb.2004-0287OC
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2004-0287OCv1
32/5/420    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by McGrath-Morrow, S. A.
Right arrow Articles by Tuder, R. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by McGrath-Morrow, S. A.
Right arrow Articles by Tuder, R. M.
American Journal of Respiratory Cell and Molecular Biology. Vol. 32, pp. 420-427, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0287OC

Vascular Endothelial Growth Factor Receptor 2 Blockade Disrupts Postnatal Lung Development

Sharon A. McGrath-Morrow, Cecilia Cho, Chung Cho, Lijie Zhen, Daniel J. Hicklin and Rubin M. Tuder

Department of Pediatrics, Eudowood Division of Pediatric Respiratory Sciences; Division of Cardiopulmonary Pathology, Department of Pathology; and Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins University, Baltimore, Maryland; and Imclone Systems Inc., New York, New York

Correspondence and requests for reprints should be addressed to Dr. Sharon McGrath-Morrow, Department of Pediatric Pulmonary, Johns Hopkins Hospital, Park 316 N. Wolfe St., Baltimore, MD 21287-2533. E-mail address: smorrow{at}jhmi.edu


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Vascular endothelial growth factor (VEGF) is necessary for normal postnatal lung development and may underlie the structural lung damage that follows hyperoxic exposure. To determine the individual roles of VEGF receptors (VEGFR) 2 and 1 on postnatal lung growth, neonatal mice were treated with neutralizing antibodies to VEGFR-2 (DC101) or VEGFR-1 (MF1) in the perinatal period. At 1 wk of age, mice treated with DC101 on Days 2 and 4 of life had significantly larger mean alveolar diameters consistent with impaired alveolization. By 2 wk of age, however, perinatally treated DC101 mice had normal-appearing alveolar structure. Mice exposed to perinatal hyperoxia (O2) also had larger mean alveolar diameters at 1 wk of age, but unlike DC101-treated mice, their mitotic index was decreased at 1 wk of age and they had persistent alveolar enlargement beyond the first 2 wk of life. The O2-treated lung also had an increase in caspase 3 at 1 wk of age and significantly greater expression of nitrotyrosine at 2 wk of age. Therefore, VEGFR-2 blockade in the perinatal period disrupts early alveolar development, but the effect is reversible with time, whereas hyperoxic lung injury is associated with ongoing lung structural impairment.

Key Words: VEGF receptor 2 • postnatal lung growth • hyperoxia


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The majority of alveolar lung growth occurs in the postnatal period. In the rodent, most growth occurs during the first 2 wk of postnatal life, whereas alveolar growth in the human extends to about the first 2 yr of life (1–3). The perinatal period is critical for proper lung adaptation to postnatal life. Indeed, perinatal lung injury caused by prematurity, infections, or hyperoxia can interfere with lung growth, leading to abnormalities in lung structure and function that may persist throughout the pediatric and adult ages (4–6). Some of the interventions used to facilitate proper oxygenation, such as oxygen administration, may worsen the underlying lung injury and impair alveolar growth by interfering with essential growth factors such as vascular endothelial growth factor (VEGF) (7).

VEGF plays a critical role in the lung, because it is essential for lung development and alveolar structural maintenance in the adult lung (8). In the mouse, treatment with a soluble truncated VEGF receptor (mFlt(1–3)-IgG), led to organ growth failure, oversimplification of lung alveolar structures, renal failure, and death within the first week of life (9). Rats treated with the VEGF receptor blocker, SU5416, developed emphysema-like changes and pulmonary hypertension, which persisted up to 3 mo after initial treatment (10).

Both VEGFR-2 (KDR in humans and Flk-1 in mice) and VEGFR-1 (Flt-1) have a high affinity for the VEGF ligand and are required for fetal blood vessel formation (11–13). VEGFR-2 signaling regulates many of the effects of VEGF, including its mitogenic, angiogenic, prosurvival, and permeability actions. VEGFR-2 blockade with the rat monoclonal antibody DC101 interrupts VEGF signaling and decreases angiogenic blood vessel growth associated with tumors (14–19). The role of VEGFR-1 in VEGF signaling is far less clear, because it may relay growth or inhibitory signals depending on the experimental system, animal age, and target endothelial cell bed. VEGFR-1 is expressed in both a membrane and truncated soluble form. The soluble form of VEGFR-1 (sVEGFR-1) has been shown to negatively regulate VEGF activity during fetal life, and increased sVEGFR-1 have been found in the serum, amniotic fluid, and placenta of women with pre-eclampsia (20–23). In turn, membrane-bound VEGFR-1 can enhance VEGF-induced VEGFR-2 phosphorylation and pathologic angiogenesis via placental growth factor (PLGF) (24, 25). VEGFR-1 mediates mobilization of bone marrow derived hematopoietic stem cell precursors and monocytes, and enhances monocyte migration and binding to endothelial cells (11, 26). Furthermore VEGFR-1 may be critical in the establishment of metastasis in the lungs (27).

The importance of VEGFR-1 or VEGFR-2 in the perinatal lung remains undetermined. Although early increases in VEGF lavage can occur with hyperoxia exposure, a decrease in VEGF and VEGFR expression has most recently been linked to the pathogenesis of hyperoxic lung injury (28–30). Also, the deletion of HIF-2{alpha} transcription factor causes a VEGF-dependent respiratory distress associated with immaturity of alveolar type II cells (31). These results suggest that in perinatal lung, impairment in VEGF signaling may interfere with normal alveolization, whereas in adult lung, impairment in VEGF signaling may interfere with VEGF's role as a maintenance factor (8).

In this study, we asked whether VEGFR-1 and/or VEGFR-2 are required for proper lung development in the immediate perinatal life. We reasoned that if blockade of VEGF signaling reproduces the critical cellular events of hyperoxia, then the lung phenotype caused by VEGFR-1 and/or VEGFR-2 blockade would resemble that caused by hyperoxia. Our studies uncovered a differential role for VEGFR-2 versus VEGFR-1 for proper perinatal lung development during the first week of life. We also found that interruption of VEGF signaling caused lung injury that only partly reproduced that observed by hyperoxia.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
All animal experiments were performed according to animal protocols approved by the Animal Care Use Committee of the Johns Hopkins University School of Medicine. CD-1 mice (Charles River, Wilmington, MA) were used for all experiments.

VEGF Receptor Antibodies
Mice treated with VEGFR antibodies were kept in room air at all times. Mice received either DC101, an antibody to VEGFR-2 (0.08 mg/dose) or MF1, an antibody to VEGFR-1 (0.1 mg/dose). The specificity and neutralizing properties of these antibodies have been validated in numerous prior studies (32). This dosing has been developed in prior studies aimed at growth arrest of tumor blood vessels (33). Control animals received an equivalent dose of endotoxin-free rat IgG serum or no treatment. One group of mice received DC101 on Days 2, 4, and 6 of life by intraperitoneal injection (designated as DC101(3)). Because of the increase lethality with three injections, a second group of mice received intraperitoneal injections of DC101 on Days 2 and 4 of life (designated as DC101(2)). Animals treated with MF1 or IgG, received injections on Days 2, 4, and 6 of life. The volume per injection was 50 µl. Mice that received either rat serum IgG or no treatment displayed similar lung phenotypes. Mice were killed and evaluated at weekly intervals up to 3 wk of age.

Hyperoxia
Neonatal mice were exposed to 85–90% hyperoxia for the first 5 d of life. Nursing mothers were rotated every 24 h to prevent injury from acute oxygen toxicity. Excess CO2 was absorbed using anhydrous calcium sulfate (Drierite # 23,001; W.A. Hammond Drierite Co. Ltd., Xenia, OH). At the end of 5 d, neonatal mice were removed from hyperoxia and allowed to recover in room air.

Isolation of Lung for Analysis
The left lung was infused through the trachea with 0.5% agarose at 60°C under 25 cm of H2O pressure and then cooled on ice, before serial sectioning (34). The right side of the lung was tied off and frozen in liquid nitrogen, stored at –80°C.

Tissue Processing and Lung Morphometry
Left lung and kidneys were placed in 10% formalin overnight and then paraffin-embedded in toto. Five-micrometer histologic sections were cut and deparaffinized using xylene. H&E staining was performed. From lung sections of 1- and 2-wk-old mice, the mean alveolar diameters (MADs) were calculated using the Image Pro Plus program (Media Cybernetics, Silver Spring, MD) as previously described (4, 35). Mice with impaired alveolization would be expected to have significantly larger MADs. Each slide contained lung from an individual animal. Ten random sections from each slide were acquired with a x20 lens. This was performed in a blinded fashion so that the operator would not know the identity of the specimen. Large airways and vessels were excluded from analysis. From each section of lung, the number of alveoli per section was calculated along with the mean diameter of each continuous alveolus. By averaging the mean diameters of the alveoli from each section, the MAD was calculated. The data were normalized to alveolar septal length (which is an approximation of the alveolar surface area).

Immunohistochemistry
For detection of nitrotyrosine, antigen retrieval was performed with citrate buffer for 20 min, followed by washing, and blockade with 3% H2O2 for 15 min. Antinitrotyrosine antibody (Abcam Ab7048–50, 1:1,000 dilution; Abcam Inc., Cambridge, MA) was applied for 30 min, then washed in TBS. Secondary antibody, using InnoGenex mouse-on-mouse Iso-IHC kit (HC-3119–06; BioGenex, San Ramon, CA), was applied for 10 min at room temperature, washed, processed as per the Vectastain Universal Quick Kit (PK-8800; Vector Laboratories, Burlingame, CA) and developed with diaminobenzidine (DAB, #K3468; DakoCytomation California Inc., Carpinteria, CA), counter-stained, dehydrated, and mounted with permount. Quantification of DAB staining was performed using the Image Pro Plus software (Media Cybernetics). Staining was normalized to lung perimeter.

Mitotic Index
Five-micrometer deparaffinized hydrated sections were rinsed and blocked with avidin/biotin block for 10 min and washed with 1x PBS. Anti-PCNA antibody (cat# SC-7907, 1:50 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) was applied for 1 h at room temperature. Sections were washed and a secondary antibody (goat anti-rabbit Alexa Floura 488, #A11001, Green 1:200 dilution; Molecular Probes Inc., Eugene, OR) was applied for 30 min at room temperature and washed. DAPI was then applied to the tissue sections for 5 min (#D1306, 1:1,000 dilution; Molecular Probes), then washed in phosphate-buffered saline (PBS). Sections were mounted with VECTOR Hard-set mount (Vector Laboratories). From each slide, 10 random sections were viewed using a fluorescent microscope, and DAPI-stained cells were counted. Next, PCNA-positive (+) cells (fluorescein-labeled) that merged with the DAPI+ cells were counted. The mitotic index was calculated by dividing the PCNA+ cells by DAPI+ cells, and then multiplying by 100.

Western Blot Analysis
Whole lung homogenate was used for Western blot analysis. Protein concentrations were determined using the Biorad DC protein assay kit (Bio-Rad Laboratories, Hercules, CA). Lysates were loaded and run on a 12% SDS-polyacrylamide gel. Each protein gel contained 60 µg of protein per lane and transferred to nitrocellulose. Western blot analysis was performed using active caspase 3 (sc-7272, 1:1,000 dilution; Santa Cruz), as recommended in 5% blotto in PBS and 0.05% Tween for 1 h at room temperature. The blots were then washed three times in PBS-Tween, incubated with a anti-mouse Ig, horseradish peroxidase–linked whole antibody (NA931, 1:10,000 dilution; Amersham, Arlington Heights, IL) for 1 h, then washed and developed using chemiluminescence (ECL) (RPN 2106; Amersham). For normalization of loading, blots were stripped using Western blot stripping buffer (Prod#21059; Pierce Biotechnology, Inc., Rockford, IL) as recommended and reprobed with actin mouse monoclonal antibody (sc-8432, 1:1,000 dilution; Santa Cruz) and processed as above.

Statistics
Statistical calculations were performed using the SPSS 12.0 statistical package for Windows (Chicago, IL). Differences in measured variables between experimental and control groups were determined using comparison of the means using a one-way ANOVA (Bonferroni and Tukey post hoc analysis of statistical differences, as appropriate) and Student's t test (two-tailed, equal variance). Statistical difference was accepted at P < 0.05.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Neutralization of VEGFR-2 Disrupts Lung Growth during the Perinatal Period
Mice treated with VEGFR-2 antibody on Days 2, 4, and 6 of life (DC101(3)) developed enlarged and fewer alveoli at 1 wk of age when compared with control lungs. These abnormalities continued to be present at 2 wk of age and were associated with a high mortality (Figure 1a). DC101(3)-treated mice also had areas of marked hemorrhage and macrophage infiltration in the alveolar spaces (Figure 1b). Overall, these findings were consistent with endothelial cell damage and altered alveolar growth caused by impaired VEGFR-2 signaling during a critical period of perinatal lung development.




View larger version (191K):
[in this window]
[in a new window]
  		Figure 1. Alveolar airspace enlargement in 1- and 2-wk-old lung from mice treated with three doses of DC101 in the perinatal period. (a) Upper panels represent lung from DC101(3)-treated mice. Note fewer alveoli and enlarged alveolar spaces ({leftrightarrow}) compared with control lung (lower panels). (b) Left panel demonstrates alveolar macrophages in DC101(3)-treated mice (black arrow). Right panel shows extensive alveolar hemorrhage in DC101(3)-treated mice (blue arrow).

 
Because most of the DC101(3)-treated mice died between 1 and 3 wk of age (Figure 2), we then treated mice with only two doses of DC101 (DC101(2)), on Days 2 and 4 of life. This dosing allowed us to study the effects of perinatal VEGFR-2 blockade on lung development as the mice matured during the first weeks of life. The DC101(2) mice had a less severe pulmonary phenotype (Figure 3a) and greater survival at 2 and 3 wk of age than DC101(3)-treated mice. The mice treated with VEGFR-1 antibodies (MF1) on Days 2, 4, and 6 of life had a normal lung phenotype and no increase in mortality when compared with control mice.



View larger version (11K):
[in this window]
[in a new window]
  		Figure 2. Increased mortality in mice treated with Dc101 in the perinatal period. At 2 wk of age, mice treated with three doses of DC101 had significantly greater mortality then control, MF1, and Dc101(2) mice (*P < 0.0001, 0.001, and 0.02, respectively). Comparisons of the means were done by one-way ANOVA. At 3 wk of age, mice treated with two doses of DC101 had significantly greater mortality then control mice (**P < 0.04) by Student's t test. Each data point represents at least 11 animals pooled from two to three litters. The mean survival of each litter is plotted.

 



View larger version (143K):
[in this window]
[in a new window]
  		Figure 3. Alveolar airspace enlargement in 1-wk-old DC101(2)-treated lung is reversible at 2 wk of age. (a) DC101(2) lung demonstrates increased airspace enlargement at 1 wk of age (left upper panel) that improves by 2 wk of age (right upper panel). No histologic differences are noted between 1-wk-old control (left lower panel) and MF1-treated lung (left middle panel). (b) In 1-wk-old lung, MAD is significantly less in control and MF1-treated lung compared with DC101(3)- and oxygen-treated lung (P < 0.001). MAD of control lung is significantly less than DC101(2) (P < 0.02). (c) In 2-wk-old lung, MAD of control and MF1-treated lung is significantly less than DC101(3)- and oxygen-treated lung (P < 0.0001). MADs were calculated for each individual animal, from 10 random sections using the program Image Pro Plus. Comparisons of the means were done by one-way ANOVA. The horizontal line in each group represents the mean. Each square represents an individual animal (n = 3–8).

 
When DC101 antibody was given to mice that were 2 wk old, for a total of three injections (every other day/weight adjusted dose), lung phenotype was normal at 4 wk of age and there was no increase in mortality (data not shown), thus indicating that the perinatal effects of VEGFR-2 inhibition on lung growth were restricted to the first week of postnatal life.

Effect of VEGFR Antibodies on Alveolar Growth
To quantify the effect of perinatal administration of VEGFR antibodies on postnatal lung growth, we measured the mean alveolar diameters (MADs) from the lungs of individual animals. Larger MADs have been shown to be associated with impaired alveolization in postnatal lung (4).

MAD measurements were calculated from the lungs of 1- and 2-wk-old mice (Figures 3b and 3c). Mice treated with two (DC101(2)) or three (DC101(3)) doses of DC101 were compared with control, MF1-treated and mice exposed to perinatal hyperoxia (O2). At 1 wk of age, DC101(2)-, DC101(3)-, and O2-exposed mice had significantly greater MADs than control mice. At 2 wk of age, DC101(3)- and O2-treated, but not DC101(2)-treated mice, had significantly greater MADs than control mice. These findings suggest that the lung recovery that occurs after VEGFR-2 blockade in the perinatal period is to a large extent dependent on the extent of VEGFR-2 blockade and the ensuing lung injury. Furthermore, these data also suggest that hyperoxia-induced lung injury is partly modeled by longer VEGFR-2 signaling blockade.

Cell Proliferation, Apoptosis, and Oxidative Stress
To determine why lung growth is only transiently impaired in DC101(2)-treated mice when compared with O2-exposed mice, studies of cell proliferation, apoptosis, and oxidative stress were performed. At 1 and 2 wk of age, lungs of DC101(2)-treated mice had a mitotic index similar to that of lungs in control and MF1-treated mice (Figures 4a and 4b). O2-exposed lung, however, had a significantly decreased mitotic index at 1 wk of age compared to control, MF1 and DC101(2)-treated lung but mitotic index was not significantly different from the other groups at 2 wk of age. These findings suggest that in the perinatal period, brief blockade of VEGFR-2 signaling does not significantly decrease lung cell proliferation, as occurs in lung exposed to hyperoxia. Because it has been shown that perinatal lung exposed to hyperoxia may develop impaired alveolar growth secondary to an increase in apoptosis (36, 37), we measured caspase 3 protein levels from 1-wk-old lung to determine if DC101(2)-treated lung had evidence of increased apoptosis. Caspase 3 protein levels were elevated in O2-exposed lungs at 1 wk of age (Figure 5), but not in lung from DC101(2)-treated mice.



View larger version (77K):
[in this window]
[in a new window]
  		Figure 4. Decreased cell proliferation in oxygen-exposed lung at 1 wk of age. (a) At 1 wk of age control, MF1-, and Dc101(2)-treated lung have significantly more PCNA-positive cells than lung exposed to hyperoxia (white arrow points to PCNA staining). (b) The mitotic index of control, MF1-, and Dc101(2)-treated lung was significantly higher then hyperoxia-exposed lung (P < 0.03, 0.01, and 0.05, respectively). Comparisons of the means were done by one-way ANOVA (n = 3–6).

 


View larger version (14K):
[in this window]
[in a new window]
  		Figure 5. Increase in caspase 3 staining in 1-wk hyperoxia-treated lung. Equal amounts of whole lung homogenates were loaded and run on a 12% SDS-polyacrylamide gel. Increased protein levels of caspase 3 were found in lung exposed to perinatal hyperoxia.

 
We then measured nitrotyrosine staining as a marker for oxidative stress, due to formation of peroxynitrite by superoxide and nitric oxide. This was done because markers of oxidative stress have been found to be increased in response to lung injury and may be associated with impaired lung growth (4, 38). We found that the lungs of mice exposed to perinatal O2 had increased nitrotyrosine staining at 2 wk of age compared with control, MF1-, and DC101(2)-treated lung (Figures 6a–6c).



View larger version (48K):
[in this window]
[in a new window]
  		Figure 6. Increase in nitrotyrosine staining in 2-wk-old hyperoxia-treated lung. (a) At 2 wk of age, lungs from mice treated with perinatal O2 had significantly more nitrotyrosine staining than control, MF1-, and DC101(2)-treated lung (P < 0.04, 0.003, and 0.001, respectively). Comparisons of the means were done by one-way ANOVA; n = 4. (b) Representative lung sections from 2-wk-old mice treated with either O2, MF1, or DC101(2) in the perinatal period. Note increased nitrotyrosine staining in O2-treated lung (black arrows). (c) Arrow points to nitrotyrosine staining in alveolar septum.

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Our findings reveal that VEGF is critical for early postnatal lung growth. We have shown, using a neutralizing monoclonal antibody to VEGFR-2, that blockade of VEGF signaling during the perinatal period impairs postnatal alveolar growth. This effect, however, is dose- and age-dependent. Mice that received three doses of DC101 antibody in the perinatal period had impaired alveolar growth at 1 and 2 wk of age and evidence of vascular leak and increased mortality. Mice that received only two doses of DC101 antibody in the perinatal period had impairment of alveolar growth at 1 wk that was reversible by 2 wk of age. Furthermore, the perinatal lung injury observed due to VEGFR-2 blockade differed from that of hyperoxia, in that hyperoxia-induced injury was persistent and associated with decreased alveolar cell proliferation, and increased apoptosis and oxidative stress.

Our studies also revealed that in contrast to VEGFR-2 blockade, VEGFR-1 blockade did not adversely impact perinatal alveolar growth, thus suggesting that the VEGFR-2 signaling accounts for most of the VEGF protective effect in the perinatal lungs. Lungs of DC101(3) mice had an increase in alveolar macrophages and hemorrhage consistent with severe lung injury. This was associated with markedly enlarged and fewer alveoli, indicating poor alveolar growth and decreased lung surface area. The protective role of VEGFR-2 signaling was underscored by the milder phenotype obtained with two doses of DC101. Our results indicate that VEGF is not only required during lung morphogenesis and late fetal development, but also in the first week of rodent adaptation to postnatal life.

It was recently described that overexpression of VEGF164 in newborn mice resulted in vascular leak, pulmonary hemorrhage, and emphysematous changes in the adult lung possibly secondary to increased nitric oxide (39). In our study, VEGF/VEGFR-2 signaling blockade in the perinatal period also led to increased pulmonary hemorrhage in DC101(3) mice and impaired alveolization in the DC101(2) and DC101(3) mice. These studies suggest that alteringVEGF/VEGFR-2 signaling by either overexpression of VEGF or VEGFR-2 blockade can detrimentally affect postnatal alveolar growth by disrupting normal endothelial cell development.

Our observation that VEGFR-1 blockade in the perinatal period had no effect on lung growth or survival was surprising, particularly because VEGFR-1 has been shown to be important for macrophage migration and may be necessary for the development of metastatic lung lesions in certain malignancies via induction of metalloprotease 9 (40, 41). Furthermore, it is possible that VEGFR-1 activation by placental growth factor (PlGF) might enhance the lung protective effects of VEGF–VEGFR-2 signaling, as demonstrated in pathologic angiogenesis (25). Alternatively, VEGFR-1 overstimulation may lead to emphysema-like changes as described in the PlGF-overexpressing mouse (42).

A previous study by Le Cras and coworkers demonstrated that SU5416, an inhibitor of VEGF receptors 1 and 2, caused impaired lung growth and development of pulmonary hypertension when given to neonatal rats (10). Several reasons may explain the differences between the study by Le Cras and colleagues and our study. In their study, although only one dose of SU5416 was given to the perinatal rat, the inhibitory effects of SU5416 have been shown to last longer then 72 h (43). SU5416 may also accumulate in cells, and subcutaneous administration of SU5416 may allow for prolonged VEGFR-2 blockade through slow release of the drug. Furthermore, SU5416 also causes VEGFR-1 blockade, exerts an inhibitory effect against the platelet-derived growth factor (PDGF) receptor, and is a weak inhibitor of fibroblast growth factor receptor (44). Therefore, by treating perinatal mice with DC101 or MF1 antibodies, we were able to avoid interactions with other receptor-signaling pathways, thus allowing us to study the specific effects of VEGFR-1 or VEGFR-2 blockade on postnatal lung growth.

A potential limitation of our study is that VEGFR-2 blockade in the perinatal period may affect kidney function. This, in turn, could have an effect on lung function independent of a primary pulmonary abnormality. Acute kidney failure has been shown to alter lung function through a macrophage-mediated increase in pulmonary permeability and dysregulation of salt and water channels in the lung (45–47). Although we did not perform permeability studies, DC101(2)-treated mice showed histologic evidence of lung recovery at 2 wk of age despite persistent abnormalities in the kidney (data not shown), suggesting that kidney dysregulation did not interfere with alveolar growth.

The effect of hyperoxia on postnatal lung may vary according to the length of exposure, degree of hyperoxia, and age and species of the model used. In a model using perinatal rabbit, 9 d of hyperoxia exposure caused downregulation of VEGF in the lung, whereas there was no decrease in VEGF mRNA after 4 d of hyperoxia (48). The effect of hyperoxia on VEGFR-2 autophosphorylation, however, was not studied at either time point; therefore, it is unknown whether functional VEGF/VEGFR-2 signaling was impaired at the 4-d time point. In our model we found that blocking VEGFR-2 signaling resulted in impaired alveolar growth in the first week of life; however, the lungs of mice treated with two doses of DC101 recovered at 2 wk of age, unlike lungs exposed to 5 d of hyperoxia. We found that although both DC101(2)- and O2-treated lung had impaired alveolization at 1 wk of age, only the O2-treated lung had a decrease in mitotic index. Another study, supporting our results, also found that VEGF inactivation in the lung did not decrease alveolar proliferation, even though the mice developed emphysematous changes (49). In that study a marked reduction in pulmonary VEGF was achieved by giving VEGFloxP mice intratracheal adeno-associated cre recombinase virus. Although they had increased TUNEL staining, they did not have decreased cell proliferation by PCNA staining. We were surprised that we did not find an increase in apoptosis in the 1-wk-old DC101(2) lung as found in other models of VEGFR-2 blockade (49). Although caspase 3 was not increased in DC101(2) lung, an increase in apoptosis may have been missed by not sampling an earlier time point. The O2-treated lung had both an increase in caspase 3 protein levels at 1 wk of age and an increase in nitrotyrosine (a marker of oxidative stress) at 2 wk of age. These findings may account for the persistent impairment in alveolar growth in lung exposed to perinatal hyperoxia (4). Furthermore, the difference in the extent of lung destruction, between VEGFR-2 blockade and hyperoxia, might also be explained by the requirement of prolonged VEGFR-2 neutralization. Furthermore, acute hyperoxia exposure may affect growth factors other than VEGF (50).

In conclusion, our results indicate that disruption of VEGF–VEGFR-2 signaling, by DC101 in the perinatal period, impairs postnatal alveolar growth in a dose-dependent manner. DC101(2) disruption of postnatal lung growth resembles that of hyperoxia-induced injury; however, unlike hyperoxia, its effect on alveolar growth can be reversible by 2 wk of age. Finally, we also found that blockade of VEGFR-1 in the perinatal period, by MF1, does not alter lung growth or survival, suggesting that VEGFR-1 is not essential for postnatal lung growth or survival.


   Acknowledgments
 
The authors thank Fang Liao for her generous technical support. Antibodies from Imclone Systems Inc. were studied in the present work.


   Footnotes
 
This study was supported in part by an NIH grant, 2201 HL066554 to R.M.T.

Conflict of Interest Statement: S.A.M.-M. has no declared conflicts of interest; C.C. has no declared conflicts of interest; C.C. has no declared conflicts of interest; L.Z. has no declared conflicts of interest; D.J.H. is an employee of Imclone Systems Incorporated and holds share options in it; and R.M.T. has no declared conflicts of interest.

Received in original form September 13, 2004

Received in final form January 12, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

  1. Thurlbeck WM. Postnatal human lung growth. Thorax 1982;37:564–571.[Abstract]
  2. Massaro D, Massaro GD. Invited Review: pulmonary alveoli: formation, the "call for oxygen," and other regulators. Am J Physiol Lung Cell Mol Physiol 2002;282:L345–L358.[Abstract/Free Full Text]
  3. Wert SE. Fetal and neonatalogy physiology, 3rd ed. Polin RA, Fox WW, Abman SH, editors. Philadelphia:W. B. Saunders; 2004.
  4. McGrath-Morrow SA, Cho C, Soutiere S, Mitzner W, Tuder R. The effect of neonatal hyperoxia on the lung of p21(Waf1/Cip1/Sdi1)-deficient mice. Am J Respir Cell Mol Biol 2004;30:635–640.[Abstract/Free Full Text]
  5. Dauger S, Ferkdadji L, Saumon G, Vardon G, Peuchmaur M, Gaultier C, Gallego J. Neonatal exposure to 65% oxygen durably impairs lung architecture and breathing pattern in adult mice. Chest 2003;123:530–538.[Abstract/Free Full Text]
  6. Warner BB, Stuart LA, Papes RA, Wispe JR. Functional and pathological effects of prolonged hyperoxia in neonatal mice. Am J Physiol 1998;275:L110–L117.
  7. D'Angio CT, Maniscalco WM. The role of vascular growth factors in hyperoxia-induced injury to the developing lung. Front Biosci 2002;7:d1609–d1623.[Medline]
  8. Tuder RM, Kasahara Y, Voelkel NF. Inhibition of vascular endothelial growth factor receptors causes emphysema in rats. Chest 2000;117:281S.[Free Full Text]
  9. Gerber HP, Hillan KJ, Ryan AM, Kowalski J, Keller GA, Rangell L, Wright BD, Radtke F, Aguet M, Ferrara N. VEGF is required for growth and survival in neonatal mice. Development 1999;126:1149–1159.[Abstract]
  10. Le Cras TD, Markham NE, Tuder RM, Voelkel NF, 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 2002;283:L555–L562.[Abstract/Free Full Text]
  11. Ferrara N, Gerber HP, LeCouter J. The biology of VEGF and its receptors. Nat Med 2003;9:669–676.[CrossRef][Medline]
  12. Fong GH, Zhang L, Bryce DM, Peng J. Increased hemangioblast commitment, not vascular disorganization, is the primary defect in flt-1 knock-out mice. Development 1999;126:3015–3025.[Abstract]
  13. Shalaby F, Rossant J, Yamaguchi TP, Gertsenstein M, Wu XF, Breitman ML, Schuh AC. Failure of blood-island formation and vasculogenesis in Flk-1-deficient mice. Nature 1995;376:62–66.[CrossRef][Medline]
  14. Bruns CJ, Shrader M, Harbison MT, Portera C, Solorzano CC, Jauch KW, Hicklin DJ, Ellis LM. Effect of the vascular endothelial growth factor receptor-2 antibody DC101 plus gemcitabine on growth, metastasis and angiogenesis of human pancreatic cancer growing orthotopically in nude mice. Int J Cancer 2002;102:101–108.[CrossRef][Medline]
  15. Jung YD, Mansfield PF, Akagi M, Takeda A, Liu W, Bucana CD, Hicklin DJ, Radinsky R, Ellis LM. Effects of combination anti-vascular endothelial growth factor receptor and anti-epidermal growth factor receptor therapies on the growth of gastric cancer in a nude mouse model. Eur J Cancer 2002;38:1133–1140.
  16. Shaheen RM, Ahmad SA, Liu W, Reinmuth N, Jung YD, Tseng WW, Drazan KE, Bucana CD, Hicklin DJ, Ellis LM. Inhibited growth of colon cancer carcinomatosis by antibodies to vascular endothelial and epidermal growth factor receptors. Br J Cancer 2001;85:584–589.[CrossRef][Medline]
  17. Shaheen RM, Tseng WW, Vellagas R, Liu W, Ahmad SA, Jung YD, Reinmuth N, Drazan KE, Bucana CD, Hicklin DJ, et al. Effects of an antibody to vascular endothelial growth factor receptor-2 on survival, tumor vascularity, and apoptosis in a murine model of colon carcinomatosis. Int J Oncol 2001;18:221–226.[Medline]
  18. Zhang L, Yu D, Hicklin DJ, Hannay JA, Ellis LM, Pollock RE. Combined anti-fetal liver kinase 1 monoclonal antibody and continuous low-dose doxorubicin inhibits angiogenesis and growth of human soft tissue sarcoma xenografts by induction of endothelial cell apoptosis. Cancer Res 2002;62:2034–2042.[Abstract/Free Full Text]
  19. Sweeney P, Karashima T, Kim SJ, Kedar D, Mian B, Huang S, Baker C, Fan Z, Hicklin DJ, Pettaway CA, et al. Anti-vascular endothelial growth factor receptor 2 antibody reduces tumorigenicity and metastasis in orthotopic prostate cancer xenografts via induction of endothelial cell apoptosis and reduction of endothelial cell matrix metalloproteinase type 9 production. Clin Cancer Res 2002;8:2714–2724.[Abstract/Free Full Text]
  20. Kearney JB, Ambler CA, Monaco KA, Johnson N, Rapoport RG, Bautch VL. Vascular endothelial growth factor receptor Flt-1 negatively regulates developmental blood vessel formation by modulating endothelial cell division. Blood 2002;99:2397–2407.[Abstract/Free Full Text]
  21. Sugimoto H, Hamano Y, Charytan D, Cosgrove D, Kieran M, Sudhakar A, Kalluri R. Neutralization of circulating vascular endothelial growth factor (VEGF) by anti-VEGF antibodies and soluble VEGF receptor 1 (sFlt-1) induces proteinuria. J Biol Chem 2003;278:12605–12608.[Abstract/Free Full Text]
  22. Levine RJ, Maynard SE, Qian C, Lim KH, England LJ, Yu KF, Schisterman EF, Thadhani R, Sachs BP, Epstein FH, et al. Circulating angiogenic factors and the risk of preeclampsia. N Engl J Med 2004;350:672–683.[Abstract/Free Full Text]
  23. Zhou Y, McMaster M, Woo K, Janatpour M, Perry J, Karpanen T, Alitalo K, Damsky C, Fisher SJ. Vascular endothelial growth factor ligands and receptors that regulate human cytotrophoblast survival are dysregulated in severe preeclampsia and hemolysis, elevated liver enzymes, and low platelets syndrome. Am J Pathol 2002;160:1405–1423.[Abstract/Free Full Text]
  24. Autiero M, Waltenberger J, Communi D, Kranz A, Moons L, Lambrechts D, Kroll J, Plaisance S, DeMol M, Bono F, et al. Role of PlGF in the intra- and intermolecular cross talk between the VEGF receptors Flt1 and Flk1. Nat Med 2003;9:936–943.[CrossRef][Medline]
  25. Carmeliet P, Moons L, Luttun A, Vincenti V, Compernolle V, De Mol M, Wu Y, Bono F, Devy L, Beck H, et al. Synergism between vascular endothelial growth factor and placental growth factor contributes to angiogenesis and plasma extravasation in pathological conditions. Nat Med 2001;7:575–583.[CrossRef][Medline]
  26. Clauss M, Weich H, Breier G, Knies U, Rockl W, Waltenberger J, Risau W. The vascular endothelial growth factor receptor Flt-1 mediates biological activities. Implications for a functional role of placenta growth factor in monocyte activation and chemotaxis. J Biol Chem 1996;271:17629–17634.[Abstract/Free Full Text]
  27. Hiratsuka S, Nakamura K, Iwai S, Murakami M, Itoh T, Kijima H, Shipley JM, Senior RM, Shibuya M. MMP9 induction by vascular endothelial growth factor receptor-1 is involved in lung-specific metastasis. Cancer Cell 2002;2:289–300.[CrossRef][Medline]
  28. Perkett EA, Klekamp JG. Vascular endothelial growth factor expression is decreased in rat lung following exposure to 24 or 48 hours of hyperoxia: implications for endothelial cell survival. Chest 1998;114:52S–53S.[Medline]
  29. Hosford GE, Olson DM. Effects of hyperoxia on VEGF, its receptors, and HIF-2alpha in the newborn rat lung. Am J Physiol Lung Cell Mol Physiol 2003;285:L161–L168.[Abstract/Free Full Text]
  30. Corne J, Chupp G, Lee CG, Homer RJ, Zhu Z, Chen Q, Ma B, Du Y, Roux F, McArdle J, et al. IL-13 stimulates vascular endothelial cell growth factor and protects against hyperoxic acute lung injury. J Clin Invest 2000;106:783–791.[Medline]
  31. Compernolle V, Brusselmans K, Acker T, Hoet P, Tjwa M, Beck H, Plaisance S, Dor Y, Keshet E, Lupu F, et al. Loss of HIF-2alpha and inhibition of VEGF impair fetal lung maturation, whereas treatment with VEGF prevents fatal respiratory distress in premature mice. Nat Med 2002;8:702–710.[Medline]
  32. Hicklin DJ, Witte L, Zhu Z, Liao F, Wu Y, Li Y, Bohlen P. Monoclonal antibody strategies to block angiogenesis. Drug Discov Today 2001;6:517–528.[CrossRef][Medline]
  33. Prewett M, Huber J, Li Y, Santiago A, O'Connor W, King K, Overholser J, Hooper A, Pytowski B, Witte L, et al. Antivascular endothelial growth factor receptor (fetal liver kinase 1) monoclonal antibody inhibits tumor angiogenesis and growth of several mouse and human tumors. Cancer Res 1999;59:5209–5218.[Abstract/Free Full Text]
  34. Halbower AC, Mason RJ, Abman SH, Tuder RM. Agarose infiltration improves morphology of cryostat sections of lung. Lab Invest 1994;71:149–153.[Medline]
  35. Tuder RM, Zhen L, Cho CY, Taraseviciene-Stewart L, Kasahara Y, Salvemini D, Voelkel NF, Flores SC. Oxidative stress and apoptosis interact and cause emphysema due to vascular endothelial growth factor receptor blockade. Am J Respir Cell Mol Biol 2003;29:88–97.[Abstract/Free Full Text]
  36. McGrath-Morrow SA, Stahl J. Apoptosis in neonatal murine lung exposed to hyperoxia. Am J Respir Cell Mol Biol 2001;25:150–155.[Abstract/Free Full Text]
  37. Barazzone C, Horowitz S, Donati YR, Rodriguez I, Piguet PF. Oxygen toxicity in mouse lung: pathways to cell death. Am J Respir Cell Mol Biol 1998;19:573–581.[Abstract/Free Full Text]
  38. Kooy NW, Royall JA, Ye YZ, Kelly DR, Beckman JS. Evidence for in vivo peroxynitrite production in human acute lung injury. Am J Respir Crit Care Med 1995;151:1250–1254.[Abstract]
  39. Le Cras TD, Spitzmiller RE, Albertine KH, Greenberg JM, Whitsett JA, Akeson AL. VEGF causes pulmonary hemorrhage, hemosiderosis, and air space enlargement in neonatal mice. Am J Physiol Lung Cell Mol Physiol 2004;287:L134–L142.[Abstract/Free Full Text]
  40. Sawano A, Iwai S, Sakurai Y, Ito M, Shitara K, Nakahata T, Shibuya M. Flt-1, vascular endothelial growth factor receptor 1, is a novel cell surface marker for the lineage of monocyte-macrophages in humans. Blood 2001;97:785–791.[Abstract/Free Full Text]
  41. An P, Lei H, Zhang J, Song S, He L, Jin G, Liu X, Wu J, Meng L, Liu M, et al. Suppression of tumor growth and metastasis by a VEGFR-1 antagonizing peptide identified from a phage display library. Int J Cancer 2004;111:165–173.[Medline]
  42. Tsao PN, Li H, Wei SC, Ko ML, Chou HC, Hsieh WS, Hsieh FJ. Expression of angiogenic factors and their receptors in postnatal mouse developing lung. J Formos Med Assoc 2004;103:137–143.[Medline]
  43. Mendel DB, Schreck RE, West DC, Li G, Strawn LM, Tanciongco SS, Vasile S, Sawyer LK, Cherrington JM. The angiogenesis inhibitor SU5416 has long-lasting effects on vascular endothelial growth factor receptor phosphorylation and function. Clin Cancer Res 2000;6:4848–4858.[Abstract/Free Full Text]
  44. Fong TA, Shawver LK, Sun L, Tang C, App H, Powell TJ, Kim YH, Schreck R, Wang X, Risau W, et al. SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor receptor (Flk-1/KDR) that inhibits tyrosine kinase catalysis, tumor vascularization, and growth of multiple tumor types. Cancer Res 1999;59:99–106.[Abstract/Free Full Text]
  45. Kramer AA, Postler G, Salhab KF, Mendez C, Carey LC, Rabb H. Renal ischemia/reperfusion leads to macrophage-mediated increase in pulmonary vascular permeability. Kidney Int 1999;55:2362–2367.[CrossRef][Medline]
  46. Rabb H, Wang Z, Nemoto T, Hotchkiss J, Yokota N, Soleimani M. Acute renal failure leads to dysregulation of lung salt and water channels. Kidney Int 2003;63:600–606.[CrossRef][Medline]
  47. Rabb H, Chamoun F, Hotchkiss J. Molecular mechanisms underlying combined kidney-lung dysfunction during acute renal failure. Contrib Nephrol 2001;132:41–52.
  48. Maniscalco WM, Watkins RH, D'Angio CT, Ryan RM. Hyperoxic injury decreases alveolar epithelial cell expression of vascular endothelial growth factor (VEGF) in neonatal rabbit lung. Am J Respir Cell Mol Biol 1997;16:557–567.[Abstract]
  49. Tang K, Rossiter HB, Wagner PD, Breen EC. Lung-targeted VEGF inactivation leads to an emphysema phenotype in mice. J Appl Physiol 2004;97:1559–1566.[Abstract/Free Full Text]
  50. Bustani P, Kotecha S. Role of cytokines in hyperoxia mediated inflammation in the developing lung. Front Biosci 2003;8:s694–s704.



This article has been cited by other articles:


Home page
 Physiol. GenomicsHome page
O. Boucherat, M.-L. Franco-Montoya, C. Thibault, R. Incitti, B. Chailley-Heu, C. Delacourt, and J. R. Bourbon
Gene expression profiling in lung fibroblasts reveals new players in alveolarization
Physiol Genomics, December 19, 2007; 32(1): 128 - 141.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
R. D. Bland, L. M. Mokres, R. Ertsey, B. E. Jacobson, S. Jiang, M. Rabinovitch, L. Xu, E. S. Shinwell, F. Zhang, and M. A. Beasley
Mechanical ventilation with 40% oxygen reduces pulmonary expression of genes that regulate lung development and impairs alveolar septation in newborn mice
Am J Physiol Lung Cell Mol Physiol, November 1, 2007; 293(5): L1099 - L1110.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
J.-R. Tang, G. Seedorf, V. Balasubramaniam, A. Maxey, N. Markham, and S. H. Abman
Early inhaled nitric oxide treatment decreases apoptosis of endothelial cells in neonatal rat lungs after vascular endothelial growth factor inhibition
Am J Physiol Lung Cell Mol Physiol, November 1, 2007; 293(5): L1271 - L1280.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
R. D. Bland, L. Xu, R. Ertsey, M. Rabinovitch, K. H. Albertine, K. A. Wynn, V. H. Kumar, R. M. Ryan, D. D. Swartz, K. Csiszar, et al.
Dysregulation of pulmonary elastin synthesis and assembly in preterm lambs with chronic lung disease
Am J Physiol Lung Cell Mol Physiol, June 1, 2007; 292(6): L1370 - L1384.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Lung Cell. Mol. Physiol.Home page
M. Yee, P. F. Vitiello, J. M. Roper, R. J. Staversky, T. W. Wright, S. A. McGrath-Morrow, W. M. Maniscalco, J. N. Finkelstein, and M. A. O'Reilly
Type II epithelial cells are critical target for hyperoxia-mediated impairment of postnatal lung development
Am J Physiol Lung Cell Mol Physiol, November 1, 2006; 291(5): L1101 - L1111.
[Abstract] [Full Text] [PDF]

Home page
 Proc Am Thorac SocHome page
R. M. Tuder, T. Yoshida, W. Arap, R. Pasqualini, and I. Petrache
State of the Art. Cellular and Molecular Mechanisms of Alveolar Destruction in Emphysema: An Evolutionary Perspective
Proceedings of the ATS, August 1, 2006; 3(6): 503 - 510.
[Abstract] [Full Text] [PDF]

Home page
 Proc Am Thorac SocHome page
S. D. Shapiro
Extracellular matrix in lung disease. Proceedings of the Twenty-First Transatlantic Airway Conference.
Proceedings of the ATS, July 1, 2006; 3(5): 397 - 455.
[Full Text] [PDF]

Home page
 Am. J. Respir. Crit. Care Med.Home page
I. Petrache, I. Fijalkowska, L. Zhen, T. R. Medler, E. Brown, P. Cruz, K.-H. Choe, L. Taraseviciene-Stewart, R. Scerbavicius, L. Shapiro, et al.
A Novel Antiapoptotic Role for {alpha}1-Antitrypsin in the Prevention of Pulmonary Emphysema
Am. J. Respir. Crit. Care Med., June 1, 2006; 173(11): 1222 - 1228.
[Abstract] [Full Text] [PDF]

Home page
 CirculationHome page
K. R. Stenmark and V. Balasubramaniam
Angiogenic Therapy for Bronchopulmonary Dysplasia: Rationale and Promise
Circulation, October 18, 2005; 112(16): 2383 - 2385.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
2004-0287OCv1
32/5/420    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by McGrath-Morrow, S. A.
Right arrow Articles by Tuder, R. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by McGrath-Morrow, S. A.
Right arrow Articles by Tuder, R. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 2005 American Thoracic Society.