This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Galambos, C. Articles by deMello, D. E. Search for Related Content PubMed PubMed Citation Articles by Galambos, C. Articles by deMello, D. E.

Defective Pulmonary Development in the Absence of Heparin-Binding Vascular Endothelial Growth Factor Isoforms

Departments of Pathology and Pediatrics, Saint Louis University Health Sciences Center and Cardinal Glennon Children's Hospital, Saint Louis, Missouri; and Schepens Eye Research Institute and Harvard Medical School, Boston, Massachusetts

Address correspondence to: Daphne E. deMello, M.D., Department of Pathology, Cardinal Glennon Children's Hospital, 1465 South Grand Boulevard, Saint Louis, MO 63104. E-mail: demellde{at}slu.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Development of the airways, alveoli, and the pulmonary vasculature in the fetus is a process that is precisely controlled. One of the growth factors involved, vascular endothelial growth factor (VEGF), is so critical for embryonic development that in the mouse, elimination of just a single allele is lethal. In the early stages of lung development, the mouse VEGF gene expresses three isoforms (120, 164, and 188) in a distinct temporo-spatial pattern, suggesting a specific function for each. We engineered mice that express only VEGF 120, to study the role of VEGF isoforms in lung development. Lung vessel development in these mice was studied by scanning electron microscopy of Mercox casts of lung vasculature. Airway and air–blood barrier development was analyzed by light microscopy, transmission electron microscopy, immunohistochemistry, and morphometry. In all VEGF120/120 fetuses and pups, lung vascular casts were smaller and less dense compared with 120/+ and wild-type littermates. Although the generation count of pre-acinar vessels was similar in all three genotypes, the most peripheral vessels were dilated and were more widely separated in 120/120 fetuses of all ages, compared with 120/+ and wild-type littermates. In addition, 120/120 animals had fewer air-blood barriers and a decreased airspace-parenchyma ratio compared with 120/+ and wild-type littermates. We concluded that the absence of VEGF 164 and 188 isoforms impairs lung microvascular development and delays airspace maturation, indicating an essential role for heparin-binding VEGF isoforms in normal lung development.

Abbreviations: charge couple device, CCD • diaminobenzidine, DAB • embryonic day 0, E0 • extracellular matrix, ECM • fetal liver kinase receptor, Flk-1 • fms-like tyrosine kinase receptor, Flt-1 • high power fields, hpf • osmium tetroxide, OsO₄ • phosphate-buffered saline, PBS • postnatal day 1, PN1 • Type I, T1 • Tris-Na-Blocking buffer, TNB • vascular endothelial growth factor, VEGF

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lung development is a carefully orchestrated process consisting of complex interactions among growth factors, matrix proteins, and cytokines (1). The molecular dialogue that occurs between epithelial and endothelial cells is reflected in the intimate physical association of pre-acinar arteries with conducting airways. This communication also takes place within alveoli, where fusion occurs between the basement membranes of the epithelial and endothelial cells to produce the air–blood barrier.

Previously, we described three processes that contribute to the formation of the lung's vasculature: angiogenesis, which gives rise to the central vessels via the sprouting of new vessels from preexisting ones; vasculogenesis, which provides the peripheral vessels via the formation of capillaries from blood lakes; and fusion between the central and peripheral systems to establish the pulmonary circulation (2). Studies in the Flk-Lac Z mouse model have shown that lung vessel development occurs at all stages and directly corresponds to overall lung growth (3). These authors identified endothelial cell precursors of the developing pulmonary vasculature before vessel lumen formation and suggested that pulmonary vascular development occurs predominantly by vasculogenesis (3). A likely candidate as a regulator for the formation of the lung's vasculature is vascular endothelial growth factor (VEGF). High levels of VEGF messenger RNA and protein are expressed by epithelial cells of the lung, suggesting that VEGF plays a role in the epithelial-endothelial interactions that are critical to normal lung development (4–6). The deletion of even a single copy of the VEGF gene leads to embryonic death from failure of blood vessel development, indicating that the expression level of VEGF is tightly controlled (7). As a result of the embryonic lethality, study of early fetal lung vascular development in the absence of the VEGF gene is not feasible.

The VEGF gene comprises eight exons, which are alternately spliced to produce a number of isoforms, depending on the animal species. The human VEGF gene, for example, yields five isoforms (VEGF120, 145, 164, 188, and 206) (8), whereas alternate splicing of the murine VEGF mRNA results in three major isoforms (VEGF120, 164, and 188) (9). VEGF120 is freely diffusible, whereas VEGF164 and 188 isoforms are significant for their ability to bind to heparan sulfate on the cell surface and in the extracellular matrix (10) as well as neuropilin on the endothelial cell surface (11). VEGF isoforms are expressed in distinct temporo-spatial patterns, suggesting that each may serve a specific function (6, 12). For instance, in the adult, VEGF188 expression is the highest in lung and heart, whereas brain has very low levels (6, 13). During rabbit and murine lung development, a specific temporal expression pattern of VEGF isoforms is observed (6, 12). During rabbit lung development, VEGF120 expression remains constant, and VEGF 164 expression peaks in early gestation and declines at birth, while the expression of VEGF188 is low in early gestation and high at birth. The same isoform distribution pattern is observed in adult and newborn rabbit lungs after exposure to hyperoxic stress (12). In the mouse, the levels of VEGF120 and 164 increased only slightly during embryonic lung development, and their levels remained relatively low in the adult lung. On the other hand, the levels of VEGF188 in the developing lung increase continuously from E13 to just before birth, and remain high during the postnatal period through adulthood (6). These findings suggest that among the three VEGF isoforms VEGF188 may play the most active role in lung development.

Two VEGF-specific receptors, fetal liver kinase receptor (Flk-1) and fms-like tyrosine kinase receptor (Flt-1), have been localized to endothelial cells of lung capillaries (14, 15). Expression of these receptors is present throughout lung development, in the perinatal period and persists in the adult lung (14, 15). These findings suggest a role for VEGF not only in capillary growth but in endothelial cell maintenance as well.

Mice engineered by exon deletion to express only VEGF120 (120/120) develop to term, but die shortly after birth. VEGF120/120 mice displayed a variety of vascular defects, including impaired postnatal cardiac angiogenesis, which results in severe myocardial ischemia and early postnatal lethality (13). To investigate the roles of VEGF164 and VEGF188 isoforms in mouse pulmonary vascular development, we examined lung development in VEGF120/120 mice. We found that mice expressing only VEGF120 had impaired peripheral lung vascular development, a reduced number of air-blood barriers, and delayed airspace formation. Our findings indicate an essential role for the heparan sulfate-binding VEGF isoforms in lung vascular and airspace development.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Animals
The generation of the VEGF120 isoform-specific mice has been described previously (13). VEGF120/120 mice have been shown by quantitative RNase protection to express levels of VEGF120 mRNA that are equivalent to the total VEGF mRNA levels of their VEGF+/+ littermates (13). Quantification of protein levels is not possible because isoform-specific antiserum is not available. To obtain VEGF120 isoform-specific embryos and newborn pups, VEGF120/+ heterozygous female mice were crossed with VEGF120/+ heterozygous male mice to obtain timed-pregnant female mice. The plug date was defined as embryonic day 0 (E0) and the day of birth was defined postnatal day 1 (PN1). For harvesting of embryos, the timed-pregnant female mice were euthanized by exposure to CO₂ for approximately 1 min. The gravid uterus was suspended in a bath of cold 0.9% NaCl, and the fetuses were delivered after amnionectomy and removal of the placenta. The postnatal animals were anesthetized by placement on ice for 15 min and then decapitated before dissection. Fetal pup crown/rump length and body weight was determined for comparison with published standards (16). Approximately 400 fetuses and pups from 44 litters, ranging in age from E9 to PN1, were harvested for this study.

Genotyping
Genomic DNA isolated from the whole embryos (younger than E15) or the tail portions (E15 and older embryos) were used for VEGF genotyping. Genotyping was done by polymerase chain reaction (PCR) with the following three primers: 5' CAG TCT ATT GCC TCC TGA CCT TCA GGG TC 3' (forward primer A: intron 5), 5' CTT GCG TCC ACA CCG TCA CAT TAA GTC AC 3' (reverse primer B: intron 7), and 5'TTC AGA GCG GAG AAA GCA TTT GTT TGT CCA 3' (forward primer C: intron 7), using standard PCR protocol. The PCR product from the wild-type VEGF gene is 400 base pairs (bp) (intron 7 from primers B and C), and 230 bp (intron 5 to intron 7 from primers A and C, upon exons 6 and 7 deletion) for the mutant allele.

Light and Transmission Electron Microscopy
Fetuses, aged E9 and E10, were fixed by immersion in 2% glutaraldehyde. For fetuses and pups aged E15–PN1, the lungs were removed from the thoracic cavity and immersed in 2% glutaraldehyde. Blocks of 2–3 mm from the fixed upper lobes were postfixed in osmium tetroxide (OsO₄). After dehydration in acetone, the blocks were embedded in Spurr (Polysciences Inc., Warrington, PA). Toludine blue stained 1-µm–thick sections were screened for those containing predominantly peripheral lung tissue and not conducting airways, and these were sectioned for ultrastructural examination. Sections, 100-nm–thick, were mounted on copper grids, stained with aqueous uranyl-acetate and lead citrate, and examined with a JEOL 100 CX (JEOL USA, Inc., Peabody, MA) electron microscope.

For light microscopy, the specimens were fixed in 10% formaldehyde or 4% paraformaldehyde, dehydrated in graded alcohols and embedded in paraffin. 6-µm–thick sections were stained with hematoxylin-eosin, or collected on Super Frost plus slides (Fisher Laboratories, Pittsburgh, PA) for immunohistochemical stains.

Immunostaining
Identification of Type I (T1) pneumocytes was performed using a monoclonal antibody to T1, a protein transcript generously provided by Mary Williams Ph.D., Boston University, School of Medicine (17). Immunohistochemistry was performed in combination with Tyramide Signal Amplification (Tyramide Signal Amlification kit (NEN Life Science Products Inc., Boston, MA). Briefly, paraffin sections were deparaffinized, rehydrated, and subjected to antigen retrieval using the Dako Antigen Retrieval kit (DAKO Corp., Carpinteria, CA) and a declocking chamber (Biocare Medical, Walnut Creek, CA). After washing, the sections were incubated in 3% H₂O₂ in methanol for 3 min at room temperature, washed, blocked with normal goat serum for 60 min at room temperature, and incubated with a 1:5 dilution of the primary antibody in PBS overnight at 4°C. After washing, the sections were incubated with the secondary antibody, goat anti-hamster IgG-HRP conjugated (ICN Pharmaceuticals, Costa Mesa, CA), and diluted 1:100 in TNB (Tris-Na-Blocking buffer, 0.1 M Tris-HCl, pH: 7.4, 0.15 M NaCl, 0.1% blocking agent: this agent is the company's secret; they are not willing to identify it) buffer for 30 min at room temperature. After washing, the sections were incubated in a 1:50 dilution of biotinyl tyramide in amplification diluent (Tyramide Signal Amplification kit; NEN Life Science Products Inc.), for 6.5 min at room temperature. After another wash, the sections were incubated in diaminobenzidine (DAB) (Sigma, St. Louis, MO) for 10 min at room temperature, washed, counterstained in 1% Fast Green (Fisher Scientific, Fair Lawn, NJ) mounted in Permount (Fisher Scientific, Pittsburgh, PA), and coverslipped.

To determine peripheral vessel density within the lung, platelet-endothelial cell adhesion molecule (PECAM) staining of frozen sections of the lungs from E 18.5 fetuses of each of the three genotypes was done as follows: lungs were fixed in 4% paraformaldehyde overnight at 4°C and cryoprotected with 15% sucrose followed by 30% sucrose solution washes. The fixed lungs were then embedded in frozen tissue-embedding media (Fisher, Cat# SH75–1250). Sections 5 µm thick were mounted on aminopropyltriethoxysilane (Silane; Sigma Cat# A3684)-coated slides and stored at -80°C. Before staining, the slides were air dried for at least 30 min and then rehydrated in phosphate-buffered saline (PBS). The sections were treated with 36% urea solution at 95 ± 50°C in a microwave oven for 10 min for antigen retrieval. Endogenous peroxidase activity was blocked by 0.75% H₂O₂ for 30 min at 37°C, followed by incubation with 10% donkey serum, 3% BSA, and 50 mM NH₄Cl for 1 h at room temperature. The sections were incubated at 4°C overnight with the primary antibody against CD-31 (PECAM-1, Cat# 550274; BD Pharmingen, San Diego, CA) at a 1:40 dilution (38 ng/ml). After washing in PBS, sections were incubated in biotinylated anti-rat IgG (BD Pharmingen) at a 1:250 dilution for 30 min at 37°C, and subsequently with 1:100 avidin-biotin–linked peroxidase for 30 min at 37°C (ABC Kit; Vector Laboratories, Burlingame, CA). The substrate used for detection was 0.1% 3.3'-diaminobenzidine, and Fast Green was used as a counterstain. Control sections were run in parallel and these included replacements of the specific antibody with PBS. Positive immunostaining for CD-31 was revealed by brown staining of endothelial cells in the lumen of blood vessels.

Determination of Airspace–Parenchyma Ratios
The ratio of air spaces to parenchyma was determined by the Chalkley point-counting method as described previously (18). Briefly, toluidine blue-stained, 1-µm–thick sections of peripheral lung tissue from E15, and E16 fetuses and PN1 pups were digitized with a charge couple device (CCD) camera and the images displayed on a computer monitor. A transparency of a 315-point grid was superimposed on the monitor screen and the points overlying airspaces and lung parenchyma were counted respectively. The investigator (D.E.deM.) was unaware of the genotype during this analysis. The airspace-parenchyma ratio was calculated and Student's t test was used to determine statistical significance between genotypes.

Quantitation of Air–Blood Barriers
Electron micrographs of 10 random fields of the lung from PN1 pups of each genotype were used to determine the number of air-blood barriers per alveolus. During the analysis, the investigator (C.G.) was unaware of the genotype. An air–blood barrier was defined as an oval or round space, either empty or containing blood cells abutting the airspace lumen and bounded on one side by an endothelial cell. The Student's t test was used to determine statistical significance between genotypes.

Quantitation of Peripheral Vessels
PECAM labeling was used as a marker of endothelial cells within the walls of peripheral lung vessels. Under the light microscope, the total number of PECAM-stained cells in 11 high-power fields of the lung sections from each genotype was counted. The quantitation was conducted in a masked fashion and the observer (D.E.deM.) did not know the genotype during the analysis.

Vascular Casts and Scanning Electron Microscopy
To obtain lung vascular casts, the anterior chest wall was removed, and a freshly prepared mixture of Mercox catalyst and resin (Ladd Research Industries, Burlington, VT) at a 50:1 ratio was injected into the right cardiac ventricle through a 30-gauge needle, under a dissecting microscope, applying gentle continuous pressure. The injection was stopped when the left cardiac atrium was filled with Mercox, indicating that the pulmonary arteries and veins that are connected to the cardiac chambers would also be filled. Generally, injections were completed within 1 min. The resin hardened within 10 min. To clear soft tissue, the injected fetuses and pups were placed in 20% KOH for 7–10 d with daily changes of solution. Under a dissecting microscope, the pulmonary vascular casts were separated from the whole-body vascular casts. The casts were then dehydrated in graded alcohols for 10 min and transferred to liquid carbon dioxide for critical point drying. Dried specimens were mounted on aluminum stubs with conductive carbon cement, coated with platinum, and examined using a JEOL JSM-5800 scanning electron microscope (JEOL USA, Inc., Peabody, MA). Scanning electron microscopy was performed on lung vascular casts of fetuses aged E15–18, and PN1 pups. Because of the small size of the fetuses and the fragility of the tissues, not all cast preparation was successful, but for each time period, one complete cast was chosen for detailed scanning electron microscopy. The analysis was done without knowledge of the animal's genotype.

Generation Counts of Pre-Acinar Vessels
In E15 fetuses, the number of generations of pre-acinar vessels was determined under scanning electron microscopy at low magnification (x43). At this early age, the development of the peripheral vasculature is not dense enough to obscure the proximal vessels, so that generation counts could be made. At later ages, the density of the peripheral vasculature obscures the pre-acinar vessels and precludes such study.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Genotype of each fetus or pup was determined by PCR using genomic DNA. The PCR genotyping results were further confirmed by Southern blot analysis (13) for the initial few litters of embryos/pups.

Determination of Airspace–Parenchyma Ratios
In E15 and E16 fetuses, and PN1 pups, airspace maturation in VEGF120 homozygous mice was delayed. In E15 fetuses, there was a delay of at least one developmental stage compared with the wild-type littermate (Figure 1) . Lungs of VEGF120 homozygous fetuses were in the glandular phase of development. The conducting airways were small and separated by broad expanses of mesenchyme. The lungs of the wild-type littermates were in the canalicular phase so that distal air spaces had developed, and these were lined by cuboidal cells and separated by thin bands of mesenchyme (Figure 1). A similar developmental delay was observed in E16 fetuses (Figure 2 , top). To assess the degree of airway or airspace development, the ratio of airway/airspace to parenchyma was determined. In E15 VEGF120 homozygous animals, this ratio was significantly less than in wild-type littermates, 0.05 versus 0.1 (P < 0.007, Table 1) . In heterozygous animals, the airspaces were larger than in homozygous littermates, the airspace–parenchyma ratio being 0.0858 (P < 0.08). The difference between heterozygous and wild-type littermates did not reach statistical significance (P < 0.19). A reduction in the airspace–parenchyma ratios of VEGF 120 homozygous animals compared with wild-type animals was also present in E16 fetuses, 0.175 versus 0.322 (P < 0.006), and PN1 pups, 1.359 versus 2.478 (P < 0.016), Table 1. In these older animals also, the airspace–parenchyma ratios of heterozygous E16 fetuses and PN1 pups were lower compared with their respective wild-type littermates (Table 1).

View larger version (127K): [in this window] [in a new window]   Figure 1. Toluidine-blue stained, 1-µm–thick sections of peripheral lung tissue from E15 fetuses are compared. Left, wild-type animal; right, homozygous littermate. Airspace maturation in the homozygous animal is delayed by at least one developmental phase compared with the wild-type littermate. Asterisks mark peripheral airspaces, lined by cuboidal cells; arrows point to proximal airways lined by columnar cells. Airspaces lined by cuboidal cells have not yet appeared in the homozygous animal.

View larger version (155K): [in this window] [in a new window]   Figure 2. Left, wild-type; middle, heterozygous; right, homozygous. Top: photomicrogaphs of lung from E16 VEGF 120/120 fetal mice. In the homozygous animal, lung development is in the canalicular stage whereas in the wild-type and heterozygous littermates, development has progressed to the saccular phase (H&E stain). Bottom: immunoperoxidase stain of paraffin sections of PN1 mouse lungs. Primary incubation with anti-T1 monoclonal antibody–specific for TI pneumocytes. Linear black HRP-reaction product (arrows) labels TI pneumocytes within alveoli. There is no difference in staining pattern between the three genotypes. Counterstain Fast Green.

View larger version (119K): [in this window] [in a new window]   Figure 3. Electron micrographs of peripheral lung from PN1 pup. Left, wild-type; right, homozygous littermate. The number of air–blood barriers per airspace (arrows) is significantly decreased in the homozygous pup. On the right, white double-headed arrow traverses cytoplasmic processes and nuclei of cells separating capillary from lumen of airspace.

Quantitation of Peripheral Vessel Density
In the homozygous E 18.5 fetus, peripheral lung vessel density was significantly reduced when compared with both the wild-type (384 versus 499/11 high-power fields [hpf]; P < 0.03), and the heterozygous littermates (488/11 hpf; P < 0.03). The vessel density in the heterozygous fetus did not differ from that in the wild-type littermate (488 versus 499/11 hpf; P < 0.4).

Vascular Casts, Scanning Electron Microscopy, and Generation Count of Pre-Acinar Vessels
The earliest gestational age at which a successful complete pulmonary vascular cast could be obtained for a VEGF120 homozygous fetus was E15. At all time points studied (E15, 17, 18, and P1), VEGF120 homozygous fetuses had casts that were smaller and less dense compared with the heterozygous and wild-type littermates (Figure 4) . Analysis by SEM showed that the proximal vessel branches (pre-acinar vessels) were not significantly different in number or caliber (Figure 5 , top), but the density of the peripheral vasculature was strikingly sparse in VEGF120 homozygous fetuses of all ages studied compared with heterozygous and wild-type littermates (Figure 5, bottom, and Figure 6) . In VEGF120 homozygous fetuses, the luminal diameter of these peripheral end-vessels was larger, and the morphologic profiles coarse compared with the heterozygous or wild-type littermates (Figure 5, bottom, and Figure 6). In heterozygous fetuses, the overall size of the pulmonary vascular casts were not obviously different compared with the wild-type littermates, suggesting that growth of the larger vessels was not affected, but the smaller peripheral vasculature is decreased in density. These differences persisted through PN1, the latest age for which a pulmonary cast was examined. The size of the vascular casts reflects overall lung size. Throughout gestation, lung size was similar between wild-type and heterozygous littermates. However, the lungs of homozygous littermates were smaller by 0.5–1 mm in apex to base length compared with heterozygous and wild-type littermates. In addition, in E18.5 animals, the homozygous fetal lungs were relatively bloodless compared with wild-type and heterozygous littermates, reflecting the decreased peripheral vessel density seen in the casts. The size of the trachea and main bronchi however did not differ among the genotypes (Figure 7) .

View larger version (106K): [in this window] [in a new window]   Figure 4. Lung mercox vascular casts of VEGF 120/120 fetal mouse littermates. (A) wild-type; (B) heterozygous; (C) homozygous; top, E17; bottom, E18. The homozygous fetal casts are smaller, and fewer peripheral vessels make the casts less dense than those of the wild-type or heterozygous littermates.

View larger version (122K): [in this window] [in a new window]   Figure 5. SEM of lung mercox vascular casts from VEGF 120/120 fetal mouse littermates. Left, wild-type; middle, heterozygous; right, homozygous. Top, E15; bottom, E17. Top: pre-acinar (larger, central) vessels are of similar caliber and branching pattern in all three genotypes. Bottom: in the homozygous fetus, small peripheral vessels are fewer and of larger caliber compared with heterozygous or wild-type littermates.

View larger version (106K): [in this window] [in a new window]   Figure 6. SEM of lung mercox vascular casts from VEGF 120/120 PN1 littermate mouse pups. Left, wild-type; middle, heterozygous; right, homozygous. Top: pre-acinar (larger, central) vessels are of similar caliber and branching pattern in all three genotypes. Bottom: in the homozygous fetus, small peripheral vessels are fewer and of larger caliber compared with heterozygous or wild-type littermates.

View larger version (49K): [in this window] [in a new window]   Figure 7. Photographs of fetal mouse lungs from three gestational ages: top, E13.5; middle, E15.5; and bottom, E18.5. Lungs of wild-type (left) and heterozygous (middle) fetuses do not differ in size. The lungs of homozygous fetuses (right) are smaller compared with wild-type and heterozygous littermate lungs at all gestational ages.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

As lung development progresses during gestation, the branching of airways and formation of airspaces gradually encroaches upon the enveloping mesenchyme so that the ratio of airspace to parenchyma increases. A decrease in the airspace-parenchyma ratio reflects a developmental delay. In our study, E15 fetuses had airspace-parenchyma ratios of 0.05, 0.08, and 0.1 in VEGF120 homozygous, heterozygous, and wild-type littermates, respectively (Table 1). This reflects a significant decrease in airspace-parenchyma ratio in the VEGF120 homozygous pups, pointing to a significant delay in lung development. This delay in lung development persisted through PN1 (Table 1). Interestingly, at E15, heterozygous littermates had no significant alteration in airspace-parenchyma ratio, suggesting that in this early stage, the presence of a single intact VEGF allele for the generation of VEGF164 and 188 isoforms is sufficient for normal lung airspace development. However, later in development, at E16 and in PN1 pups, there is a delay in lung maturation even in the heterozygous animals. The impaired lung development in the VEGF120 homozygous and heterozygous animals compared with wild-type littermates indicates a role for the heparin-binding VEGF isoforms and suggests that an optimal balance of isoforms is necessary for normal lung development.

In newborn mice, impaired organ development and growth retardation occurred when VEGF was partially inhibited by Cre-loxP–mediated gene targeting. More complete VEGF inhibition such as that caused by the administration of mFlt(1–3)-IgG, a soluble VEGF receptor chimeric protein, resulted in death (21). The importance of a critical level of VEGF expression during development is further supported by a recent study in which overexpression of VEGF in the respiratory epithelium of transgenic mice using the promoter from the human surfactant protein C (SP-C) gene, resulted in disruption of acinar structure (22).

The formation of the air-blood barrier is a seminal event in lung development. The air-blood barrier is a membrane formed at the alveolar lumen by fusion of the basement membrane of the capillary endothelial cell with the basement membrane of the overlying TI epithelial cell. Gas exchange, the prime function of the lung, occurs across this interface. In alveolar capillary dysplasia, a rare human disorder, air-blood barriers fail to form, and the affected infant dies soon after birth because of hypoxemia (23, 24). The mechanisms/factors controlling this important step in lung development are unknown and, as yet, a molecular basis for this disease has not been identified. In the present study, VEGF120/120 pups had a significant reduction in the number of air-blood barriers (1.15 in VEGF120 homozygous pups compared with 3.45 and 4.65 in heterozygous and wild-type littermates, respectively). The number of air-blood barriers in both VEGF120 homozygous and heterozygous animals was significantly reduced (Table 2), suggesting an exquisite dose dependence on VEGF isoforms 164 and/or 188 for air-blood barrier formation. Another possible reason for the failure of air-blood barrier formation is the failure of conversion of Type II cells to TI cells. However, immunostaining for TI cells revealed normal numbers of TI cells in lungs from all three genotypes. We conclude therefore, that the VEGF isoforms 164 and/or 188 are important for air-blood barrier formation.

The pre-acinar vessel generation counts did not differ among the different genotypes, suggesting that proximal lung vessel development is not critically dependent upon VEGF164 and 188. Peripheral lung vessel development, however, was significantly impaired in VEGF120 homozygous animals of all gestational ages studied. The homozygous fetuses and pups had a sparse peripheral vasculature with end-vessels that were short and coarse. Peripheral vessel density determined by PECAM staining was significantly reduced, suggesting aberrant development.

We have previously reported that the levels of VEGF188 increase dramatically during the late canalicular and the late saccular phases of lung development (6). The increase in VEGF188 correlates temporally and spatially with the formation of primitive alveolar structures, suggesting that VEGF188 may be involved in the formation of the air-blood barrier. The proximity of the VEGF-producing pulmonary epithelium and the target vasculature (6) create a spatial association that permits intercellular communication. The importance of this relationship was demonstrated in studies in which lung mesenchyme (vascular precursors) was cultured in the presence or absence of lung epithelium (25). In the absence of lung epithelium, lung mesenchyme degenerated and few Flk-1–positive cells remained. By contrast, in the presence of lung epithelium, the mesenchyme yielded a number of Flk-1–positive cells, which associated with the epithelium in a manner similar to that observed in vivo.

We suggest that the intimate association between the epithelial cell and the capillary endothelium, that leads to the formation of the air-blood barrier in the lung, is determined by the epithelial expression of VEGF188, which in turns directs the juxtaposition of the growing capillary. Because the pulmonary epithelial cells also make diffusible VEGF 120 and VEGF 164, these isoforms could function to attract the endothelial cells to the site of the developing alveolus. Subsequently, VEGF188, which is tightly bound to heparan sulfate proteoglycans on the epithelial cell surface and in the extracellular matrix (ECM), may serve a trophic or survival role. Immunohistochemical analyses of the developing mouse lung revealed that in the early stages (E11.5), VEGF is distributed uniformly throughout the airway epithelium and in the subepithelial matrix, whereas later, its expression is restricted to the growing tips of airway branches in the distal lung, i.e., at the site of new vessel formation (26). The importance of local VEGF expression for normal lung development is demonstrated by our study in which VEGF120/120 animals have impaired peripheral lung vascular development, in addition to reduced numbers of air-blood barriers and delayed air space maturation.

Thus, in addition to its importance in lung development, VEGF probably also plays an important role in maintenance of lung structure. This is strongly indicated by studies in the rat (27), in which Su-5416, a selective inhibitor of the VEGF receptor VEGFR2 was administered from PN1–13. The treated animals, in which VEGF signaling was thus blocked, had enlarged airspaces, decreased alveolar number, and decreased arterial density. Administration of the VEGFR2 inhibitor to adult rats for 3 wk also resulted in enlarged airspaces because of disrupted alveolar septa due to septal cell apoptosis. This septal damage could be blocked by the inhibition of caspase (28).

The phenotype of defective lung development in these VEGF120/120 mice, which have been genetically engineered to express only the freely diffusible VEGF120 isoform, supports our hypothesis because the mice exhibit defective air-blood barrier formation in addition to delayed airspace maturation. Therefore, we propose that the VEGF164 or VEGF188 isoforms bind to the ECM with high affinity and thus, provide the critical localized VEGF needed for normal pulmonary vascular development and maintenance.

	Acknowledgments

 
The authors thank Dan He, M.D, Ph.D., The Schepens Eye Research Institute, Boston, MA, for her invaluable help with the PECAM-1 immunostaining of lung sections. This work was supported in part by grants from the NIH #HL55600 (D. deM.), NIH #CA45548 (P. D'A.), and from the Fleur de Lis Foundation of Cardinal Glennon Children's Hospital (A. A.). P. D'A. is the Jules and Doris Stein Professor to Prevent Blindness.

Received in original form August 15, 2001

Received in final form April 23, 2002

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Price, W. A., and A. D. Stiles. 1996. New insights into lung growth and development. Curr. Opin. Pediatr. 8:202–208.[Medline]
2. deMello, D. E., D. Sawyer, N. Galvin, and L. M. Reid. 1997. Early fetal development of lung vasculature. Am. J. Respir. Cell Mol. Biol. 16:568–581.[Abstract]
3. Schachtner, S. K., Y. Wang, and S. H. Baldwin. 2000. Qualitative and quantitative analysis of embryonic pulmonary vessel formation. Am. J. Respir. Cell Mol. Biol. 22:157–165.[Abstract/Free Full Text]
4. Carmeliet, P., and Jain R. K. 2000. Angiogenesis in cancer and other diseases. Nature 407:249–257[Medline]
5. Acarregui, M. J., S. T. Penisten, K. L. Goss, K. Ramirez, and J. M. Snyder. 1999. Vascular endothelial growth factor gene expression in human fetal lung in vitro. Am. J. Respir. Cell Mol. Biol. 20:14–23.[Abstract/Free Full Text]
6. Ng, Y. S., R. Rohan., M. E. Sunday, D. E. deMello, and P. A. D'Amore. 2001. Differential expression of VEGF isoforms in mouse during development and in the adult. Dev. Dyn. 220:112–121.[Medline]
7. Carmeliet, P., V. Ferreira, G. Breier, S. Pollefeyt, L. Kieckens, M. Gertsenstein, M. Fahrig, A. Vandenhoeck, K. Harpal, C. Eberhardt, C. Declercq, J. Pawling, L. Moons, D. Collen, W. Risau, and A. Nagy. 1996. Abnormal blood vessel development and lethality in embryos lacking a single VEGF allele. Nature 380:435–439.[Medline]
8. Tischer, E., R. Mitchell, T. Hartman, M. Silva, D. Gospodarowicz, J. C. Fiddes, and J. A. Abraham 1991. The human gene for vascular endothelial growth factor: multiple protein forms are encoded through alternative exon splicing. J. Biol. Chem. 266:11947–11954.[Abstract/Free Full Text]
9. Ferrara, N. 1999. Role of vascular endothelial growth factor in the regulation of angiogenesis. Kidney Int. 56:794–814.[Medline]
10. Neufeld, G., T. Cohe, O. Gitay, H. Coren, S. Poltorak, S. Tessler, R. Sharon, S. Gengrinovitch, and B. Leci. 1996. Similarities and differences between the vascular endothelial growth factor (VEGF) splice variants. Cancer Metastasis Rev. 15:153–158.[Medline]
11. Soker, S., S. Takishima, H. Q. Miao, G. Neufeld, and M. Klagsbrun. 1998. Neuropilin-1 is expressed by endothelial and tumor cells as an isoforms-specific receptor for vascular endothelial growth factor. Cell 92:735–745.[Medline]
12. Watkins, R., T. D'Angio, R. Ryan, A. Patel, and W. Maniscaclo. 1999. Differential expression of VEGF mRNA splice variants in newborn and adult hyperoxic lung injury. Am. J. Physiol. 276:L858–867.[Abstract/Free Full Text]
13. Carmeliet, P., Y. S. Ng, D. Nuyens, G. Theilmeier, K. Brusselmans, I. Cornelissen, E. Ehler, V. V. Kakkar, I. Stalmans, V. Mattot, J. C. Perriard, M. Dewerchin, W. Flameng, A. Nagy, F. Lupu, L. Moons, D. Collen, P. A. D'Amore, and D. T. Shima. 1999. Impaired myocardial angiogenesis and ischemic cardiomyopathy in mice lacking the vascular endothelial growth factor isoforms VEGF164 and VEGF188. Nat. Med. 5:495–502.[Medline]
14. Marszalek, A., T. Daa, K. Kashima, I. Nakayama, and S. Yokoyama. 2001. Expression of vascular endothelial growth factor and its receptor in the developing rat lung. Jpn. J. Physiol. 51:313–318.[Medline]
15. Bhatt, A. J., S. B. Amin, P. R. Chess, R. H. Watkins, and W. M. Maniscalco. 2000. Expression of vascular endothelial growth factor and Flk-1 in developing and glucocorticoid-treated mouse lung. Pediatr. Res. 47:606–613.[Medline]
16. Crispen, G., Jr. 1975. Handbook on Laboratory Mouse. Charles C. Thomas, Springfield, Il. 65
17. Williams, M. C., Y. Cao, A. Hinds, A. K. Rishi, and A. Wetterwald. 1996. T1 alpha protein is developmentally regulated and expressed by alveolar type I cells, choroid plexus, and ciliary epithelia of adult rats. Am. J. Respir. Cell Mol. Biol. 14:577–585.[Abstract]
18. Chalkley, H. W. 1943. Method for the quantitative morphologic analysis of tissues. J. Natl. Cancer Inst. 4:47–53
19. deMello, D. E., and L. Reid. 2000. Embryonic and early fetal development of human lung vasculature and its functional implications. Pediatr. Dev. Pathol. 3:439–449.[Medline]
20. Gerber, H. P., K. J. Hillan, A. M. Ryan, J. Kowalski, G. A. Keller, L. Rangell, B. D. Wright, F. Radtke, M. Aguet, and N. Ferrara. 1999. VEGF is required for growth and survival in neonatal mice. Development 126: 1149–1159.[Abstract]
21. Ferrara, N., K. Carver-Moore, H. Chen, M. Dowd, L. Lu, K. S. O'Shea, L. Powell-Braxton, K. J. Hillan, and M. W. Moore. 1996. Heterozygous embryonic lethality induced by targeted inactivation of the VEGF gene. Nature 380:439–442.[Medline]
22. Zeng, X., S. E. Wert, R. Federici, K. G. Peters, and J. A. Whitsett. 1998. VEGF enhances pulmonary vasculogenesis and disrupts lung morphogenesis in vivo. Dev. Dyn. 211:215–227.[Medline]
23. Cullinane, C., P. N. Cox, and M. M. Silver. 1992. Persistent pulmonary hypertension of the newborn due to alveolar capillary dysplasia. Pediatr. Pathol. 12:499–514.[Medline]
24. Janney, C. G., F. B. Askin, and C. Kuhn. 1981. Congenital alveolar capillary dysplasia-an unusual cause of respiratory distress in the newborn. Am. J. Clin. Pathol. 76:722–727.[Medline]
25. Gebb, S. A., and J. M. Shannon. 2000. Tissue interactions mediate early events in pulmonary vasculogenesis. Dev. Dyn. 217:159–69[Medline]
26. Healy, A. M., L. Morgenthau, X. Zhu, H. W. Farber, and W. V. Cardoso. 2000. VEGF is deposited in the subepithelial matrix at the leading edge of branching airways and stimulates neovascularization in the murine embryonic lung. Dev. Dyn. 219:341–352.[Medline]
27. Jakkula, M., T. D. Le Cras, S. Gebb, K. P. Hirth, R. M. Tuder, N. F. Voelkel, and S. H. Abman. 2000. Inhibition of Vascular Endothelial Growth Factor (VEGF) Receptor (KDR/Flk-1) decreases alveolarization and alters vascular growth and structures in the developing rat lung. Ped. Res. 47:71A
28. Kasahara, Y., R. M. Tuder, L. Taraseviciene-Stewart, T. D. Le Cras, S. Abman, P. K. Hirth, J. Waltenberger, and N. F. Voelkel. 2000. Inhibition of VEGF receptors causes lung cell apoptosis and emphysema. J. Clin. Invest. 106:1311–1319.[Medline]

This article has been cited by other articles:

				S. H. Abman The Dysmorphic Pulmonary Circulation in Bronchopulmonary Dysplasia: A Growing Story Am. J. Respir. Crit. Care Med., July 15, 2008; 178(2): 114 - 115. [Full Text] [PDF]

				M. E. De Paepe, C. Patel, A. Tsai, S. Gundavarapu, and Q. Mao Endoglin (CD105) Up-regulation in Pulmonary Microvasculature of Ventilated Preterm Infants Am. J. Respir. Crit. Care Med., July 15, 2008; 178(2): 180 - 187. [Abstract] [Full Text] [PDF]

				V. Muehlethaler, A. M. Kunig, G. Seedorf, V. Balasubramaniam, and S. H. Abman Impaired VEGF and nitric oxide signaling after nitrofen exposure in rat fetal lung explants Am J Physiol Lung Cell Mol Physiol, January 1, 2008; 294(1): L110 - L120. [Abstract] [Full Text] [PDF]

				A. C. White, K. J. Lavine, and D. M. Ornitz FGF9 and SHH regulate mesenchymal Vegfa expression and development of the pulmonary capillary network Development, October 15, 2007; 134(20): 3743 - 3752. [Abstract] [Full Text] [PDF]

				M C Pustovrh, A Jawerbaum, V White, E Capobianco, R Higa, N Martinez, J J Lopez-Costa, and E Gonzalez The role of nitric oxide on matrix metalloproteinase 2 (MMP2) and MMP9 in placenta and fetus from diabetic rats Reproduction, October 1, 2007; 134(4): 605 - 613. [Abstract] [Full Text] [PDF]

				F. A. Groenman, M. Rutter, J. Wang, I. Caniggia, D. Tibboel, and M. Post Effect of chemical stabilizers of hypoxia-inducible factors on early lung development Am J Physiol Lung Cell Mol Physiol, September 1, 2007; 293(3): L557 - L567. [Abstract] [Full Text] [PDF]

				B. Thebaud and S. H. Abman Bronchopulmonary Dysplasia: Where Have All the Vessels Gone? Roles of Angiogenic Growth Factors in Chronic Lung Disease Am. J. Respir. Crit. Care Med., May 15, 2007; 175(10): 978 - 985. [Abstract] [Full Text] [PDF]

				B. Wojciak-Stothard, L. Y. F. Tsang, E. Paleolog, S. M. Hall, and S. G. Haworth Rac1 and RhoA as regulators of endothelial phenotype and barrier function in hypoxia-induced neonatal pulmonary hypertension Am J Physiol Lung Cell Mol Physiol, June 1, 2006; 290(6): L1173 - L1182. [Abstract] [Full Text] [PDF]

				M. E. De Paepe, Q. Mao, J. Powell, S. E. Rubin, P. DeKoninck, N. Appel, M. Dixon, and F. Gundogan Growth of Pulmonary Microvasculature in Ventilated Preterm Infants Am. J. Respir. Crit. Care Med., January 15, 2006; 173(2): 204 - 211. [Abstract] [Full Text] [PDF]

				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]

				B. Thebaud, F. Ladha, E. D. Michelakis, M. Sawicka, G. Thurston, F. Eaton, K. Hashimoto, G. Harry, A. Haromy, G. Korbutt, et al. Vascular Endothelial Growth Factor Gene Therapy Increases Survival, Promotes Lung Angiogenesis, and Prevents Alveolar Damage in Hyperoxia-Induced Lung Injury: Evidence That Angiogenesis Participates in Alveolarization Circulation, October 18, 2005; 112(16): 2477 - 2486. [Abstract] [Full Text] [PDF]

				F. Ladha, S. Bonnet, F. Eaton, K. Hashimoto, G. Korbutt, and B. Thebaud Sildenafil Improves Alveolar Growth and Pulmonary Hypertension in Hyperoxia-induced Lung Injury Am. J. Respir. Crit. Care Med., September 15, 2005; 172(6): 750 - 756. [Abstract] [Full Text] [PDF]

				T. R. Grover, T. A. Parker, V. Balasubramaniam, N. E. Markham, and S. H. Abman Pulmonary hypertension impairs alveolarization and reduces lung growth in the ovine fetus Am J Physiol Lung Cell Mol Physiol, April 1, 2005; 288(4): L648 - L654. [Abstract] [Full Text] [PDF]

				M. van Tuyl, J. Liu, J. Wang, M. Kuliszewski, D. Tibboel, and M. Post Role of oxygen and vascular development in epithelial branching morphogenesis of the developing mouse lung Am J Physiol Lung Cell Mol Physiol, January 1, 2005; 288(1): L167 - L178. [Abstract] [Full Text] [PDF]

				C. Galambos Modeling Alveolar Capillary Dysplasia Circ. Res., September 3, 2004; 95(5): e35 - e35. [Full Text] [PDF]

				T. D. Le Cras, R. E. Spitzmiller, K. H. Albertine, J. M. Greenberg, J. A. Whitsett, and A. L. Akeson VEGF causes pulmonary hemorrhage, hemosiderosis, and air space enlargement in neonatal mice Am J Physiol Lung Cell Mol Physiol, July 1, 2004; 287(1): L134 - L142. [Abstract] [Full Text] [PDF]

				K. Ihida-Stansbury, D. M. McKean, S. A. Gebb, J. F. Martin, T. Stevens, R. Nemenoff, A. Akeson, J. Vaughn, and P. L. Jones Paired-Related Homeobox Gene Prx1 Is Required for Pulmonary Vascular Development Circ. Res., June 11, 2004; 94(11): 1507 - 1514. [Abstract] [Full Text] [PDF]

				S. Bornes, M. Boulard, C. Hieblot, C. Zanibellato, J. S. Iacovoni, H. Prats, and C. Touriol Control of the Vascular Endothelial Growth Factor Internal Ribosome Entry Site (IRES) Activity and Translation Initiation by Alternatively Spliced Coding Sequences J. Biol. Chem., April 30, 2004; 279(18): 18717 - 18726. [Abstract] [Full Text] [PDF]

				R. N.N. Han, S. Babaei, M. Robb, T. Lee, R. Ridsdale, C. Ackerley, M. Post, and D. J. Stewart Defective Lung Vascular Development and Fatal Respiratory Distress in Endothelial NO Synthase-Deficient Mice: A Model of Alveolar Capillary Dysplasia? Circ. Res., April 30, 2004; 94(8): 1115 - 1123. [Abstract] [Full Text] [PDF]

				S. Santos, V. I. Peinado, J. Ramirez, J. Morales-Blanhir, R. Bastos, J. Roca, R. Rodriguez-Roisin, and J. A. Barbera Enhanced Expression of Vascular Endothelial Growth Factor in Pulmonary Arteries of Smokers and Patients with Moderate Chronic Obstructive Pulmonary Disease Am. J. Respir. Crit. Care Med., May 1, 2003; 167(9): 1250 - 1256. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar