Angiogenic factors and alveolar vasculature: development and alterations by injury in very premature baboons

William M. Maniscalco, Richard H. Watkins, Gloria S. Pryhuber, Abhay Bhatt, Colleen Shea, and Heidie Huyck

Division of Neonatology, Strong Children's Research Center, Department of Pediatrics, University of Rochester, Rochester, New York 14642


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Proper formation of the pulmonary microvasculature is essential for normal lung development and gas exchange. Lung microvascular development may be disrupted by chronic injury of developing lungs in clinical diseases such as bronchopulmonary dysplasia. We examined microvascular development, angiogenic growth factors, and endothelial cell receptors in a fetal baboon model of chronic lung disease (CLD). In the last third of gestation, the endothelial cell marker platelet endothelial cell adhesion molecule (PECAM)-1 increased 7.5-fold, and capillaries immunostained for PECAM-1 changed from a central location in airspace septa to a subepithelial location. In premature animals delivered at 67% of term and supported with oxygen and ventilation for 14 days, PECAM-1 protein and capillary density did not increase, suggesting failure to expand the capillary network. The capillaries of the CLD animals were dysmorphic and not subepithelial. The angiogenic growth factor vascular endothelial growth factor (VEGF) and its receptor fms-like tyrosine kinase receptor (Flt-1) were significantly decreased in CLD. Angiopoietin-1, another angiogenic growth factor, and its receptor tyrosine kinase with immunoglobulin and epidermal growth factor homology domains were not significantly changed. These data suggest that CLD impairs lung microvascular development and that a possible mechanism is disruption of VEGF and Flt-1 expression.

lung; microvasculature; bronchopulmonary dysplasia


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

PREMATURE INFANTS TREATED with ventilation and supplemental oxygen frequently develop bronchopulmonary dysplasia (BPD), a chronic lung disease (CLD) that has significant mortality and morbidity. The combination of immature lungs, oxidant injury, volutrauma, and chronic inflammation are probably important etiologic factors in BPD. The pathology of BPD, including poor alveolarization and a dysmorphic microvasculature, suggests an arrest in lung development (9, 22). Proper formation of alveolar capillaries, a key step in lung development, is required for efficient gas exchange. Alveolar capillary development begins in early fetal lung and continues through the alveolar stage (7, 37). The complex process of endothelial differentiation and organization into the alveolar microvasculature might be disrupted after premature birth and injury from supplemental oxygen and ventilation. In an autopsy study of human premature newborns dying with BPD, we found an abnormal alveolar capillary network and decreased expression of an endothelial cell marker (5).

The lung endothelial cell population increases continuously during gestation (37). Vessel formation occurs by two processes: vasculogenesis, the development of vessels from endothelial cells that differentiate from mesenchymal precursors, and angiogenesis, the sprouting of new vessels from the preexisting vasculature. Both processes occur in the developing lung (14, 38). During lung differentiation, the distal microvasculature, which probably forms by vasculogenesis, transforms from loosely organized capillaries imbedded in the mesenchyme to an extensive network of subepithelial alveolar capillaries. In the canalicular stage (18-26 wk gestation in the human), the capillaries are located centrally in thickened distal airspace septa (7). As the septa become thin, the capillaries become closely aligned with the distal epithelium, which is differentiating into type I and type II cells.

The regulation of pulmonary microvascular development is not known, but epithelial and mesenchymal interactions may be essential (20). The processes necessary for vascular development, including endothelial cell differentiation, migration, interaction with the basement membrane, formation of cell-cell junctions, and attraction of supporting pericytes, can be regulated by several growth factors. Of particular interest are the vascular endothelial growth factor family (VEGF A-D) and the angiopoietin family (Ang-1-4). Because their receptors are largely restricted to endothelial cells, the actions of these factors are mainly endothelial cell specific (46). VEGF (also known as VEGF-A) is mitogenic for endothelial cells, induces capillary permeability, and regulates endothelial cell migration and tube formation. VEGF may also be an endothelial cell survival factor (2). Loss of a single VEGF allele is embryo lethal, underscoring its critical role in vascular development (8). VEGF has several isoforms that are produced by alternative splicing of the primary mRNA. The splice variants are differentially expressed in lung development and injury (43). The VEGF receptors fms-like tyrosine kinase receptor (Flt-1, VEGFR-1) and fetal liver kinase (Flk-1, VEGFR-2) have different functions: Flt-1 may mediate vascular organization, and Flk-1 mediates endothelial differentiation and proliferation (17, 39). VEGF is expressed by distal airspace epithelial cells in fetal and postnatal lung (4, 47). Hypoxia, growth factors, and cell differentiation, which are key in lung development and injury, regulate VEGF expression (4, 6, 28).

Ang-1 ligation to its receptor tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (TIE-2) mediates vascular remodeling and endothelial interactions with supporting cells (41). A related endothelial receptor, TIE-1, regulates endothelial cell integrity and is essential for vessel network formation, particularly in the later stages of embryonic development (36). The ligand for TIE-1 is not known. The VEGF and Ang-1 families work in concert during vessel formation (3).

Most studies of vascular changes in CLD of infancy have relied on autopsy samples (9), which are limited by the decreasing mortality from BPD and the inherent variability of tissue from human patients. Most of these studies did not focus on the alveolar microvasculature. Studies of postnatal animals treated with hyperoxia or ventilation cannot address the effects of these injuries at critical stages of lung development. Premature baboons delivered at 140 days' gestation (75% of term) and treated with 100% oxygen and ventilation for 10 days had decreased endothelial cells in hyperexpanded lung (10), consistent with the toxic effects of oxygen on pulmonary endothelial cells (13). Using baboons that were delivered at 125 days (67% of term) and treated with surfactant, appropriate oxygen, and ventilation for 1-2 mo, Coalson et al. (11) found alveolar hypoplasia, decreased vascularization, and dysmorphic alveolar capillaries compared with term animals.

In previous work, we found that acute hyperoxic injury decreased lung VEGF expression (25). We hypothesized that injury to premature lungs would lead to disrupted vascular development, possibly by altering the normal genetic program for angiogenic factor expression. The present work reports the temporal and spatial expression of an endothelial cell marker [platelet endothelial cell adhesion molecule (PECAM)-1], VEGF and Ang-1, and their receptors in normal fetal baboon lung and in 125-day-gestation animals after 6 and 14 days of oxygen and ventilation. These animals had decreased PECAM-1 expression, decreased capillary density, and abnormal development of alveolar capillaries, compared with gestational controls. Expression of VEGF, its receptor Flt-1, and TIE-1 were decreased in injured lung. Flk-1, Ang-1, and TIE-2 were unchanged. These data show that development of CLD rapidly results in abnormal alveolar capillaries, which may be due to disruption of the normal genetic program for angiogenic factor/receptor expression.


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
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Animals and treatment protocols. Frozen baboon lung tissue and slides from paraffin-imbedded tissue were obtained from the Southwest Foundation for Biomedical Research (San Antonio, TX). Detailed protocols for baboon pregnancy, delivery, and postnatal management have been described (11). All animal protocols conformed with American Association for Accreditation of Animal Care guidelines. Briefly, timed gestations were confirmed by serial ultrasounds. Baboons were delivered by hysterotomy at 125, 140, 160, and 175 days' gestation (term is 185 days). Animals killed immediately after delivery were gestational controls. Some animals were killed at 1-3 days after term delivery. Study animals were delivered at 125 days' gestation (67% of term), intubated, and immediately treated with exogenous surfactant. Infant animals were treated with ventilation (pressure limited, time cycled) to maintain tidal volumes of 4-6 ml/kg and arterial partial pressure of CO2 between 45 and 55 mmHg. Supplemental oxygen was given to maintain arterial partial pressure of O2 55-70 mmHg. Positive end-expiratory pressure was adjusted to maintain normal lung volumes. Nutritional and fluid support was delivered to maintain urine output and caloric intake via intravenous protein and lipid solutions. All animals were treated with antibiotics. Some animals were killed after 6 days of support (125d 6dPRN), and others were killed after 14 days (125d 14dPRN). The 125d 14dPRN animals were compared with 140-day gestational controls to determine the effects of premature delivery and postnatal treatment on the progress of normal in utero lung development.

Tissue preparation. At necropsy the lungs were prepared as described by Coalson et al. (11). Briefly, lungs were inflation fixed with 4% paraformaldehyde at 20 cmH2O pressure for 24 h. Lung tissue blocks were dehydrated and imbedded in paraffin, and 4-µm sections were cut. Other lung tissue samples were immediately frozen in liquid nitrogen.

Western immunoblot. As described previously (34), ~100 mg of frozen lung tissue was homogenized in 50 mM Tris · HCl, pH 7.4, 150 mM NaCl, 2 mM EDTA, 25 mM sodium fluoride, alpha -glycerophosphate, 0.1 mM sodium vanadate, 1 mM phenylmethylsufonyl fluoride, 0.2% Triton X-100, 0.3% Igepal CA-630, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 10 µg/ml aprotinin (A6279; Sigma). Proteins (10 µg) in Laemmli buffer were separated on polyacrylamide-SDS gels and transferred to polyvinylidene difluoride membrane. Membranes were blocked in PBS with 5% nonfat dry milk (NFDM) at 4°C and incubated with either a goat anti-PECAM-1 polyclonal IgG (0.1 µg/ml in PBS, 5% NFDM, 0.05% Tween 20) or a rabbit anti-actin polyclonal IgG (1:5,000 in PBS, 5% NFDM, 0.05% Tween 20). The antibodies detect human PECAM-1 (sc-1506; Santa Cruz Biotechnology) or human actin (A2066; Sigma). The membranes were incubated in peroxidase-conjugated anti-goat or anti-rabbit secondary antibody (1:5,000; Santa Cruz Biotechnology). Immunodetection was by enhanced chemiluminescence (ECL Plus; Amersham, Arlington Heights, IL). The bands were quantified by image analysis.

Ribonuclease protection assay. Tissues were lysed in 4 M guanidinium isothiocyanate (Kodak Chemical, Rochester, NY), 0.5% N-lauryl sarcosine, and 25 mM sodium citrate (Sigma), and stored at -80°C. Total cell RNA was extracted by Phase Lock Gel II columns (Eppendorf; 5 Prime, Boulder, CO). The ribonuclease protection assays (RPAs) were performed with commercial reagents and protocols (Riboquant; PharMingen). Radiolabeled, single-strand RNA probes for human PECAM-1, VEGF, Ang-1, Flt-1, TIE-1, TIE-2, and L32 were synthesized at room temperature using [alpha -32P]uridine 5'-triphosphate (3,000 Ci/mmol; EasyTides; DuPont-New England Nuclear, Boston, MA) and T7 polymerase. RNA samples (5 µg), including human RNA and yeast transfer RNA (2 µg) as positive and negative controls, were dried, then resuspended in hybridization buffer with radiolabeled probe [2 µl, 3 × 105 counts · min-1 (cpm) · µl-1]. The samples were denatured at 90°C and incubated overnight at 56°C. Single-stranded RNA was digested in an RNase A/T1 cocktail, followed by proteinase K digestion. The remaining radiolabeled RNA fragments were resolved on a 6% acrylamide/urea gel (GIBCO-BRL), using radiolabeled probe (1,000-2,000 cpm) as size markers. The gels were dried and analyzed by phosphorimaging (Molecular Dynamics, Sunnyvale, CA). Abundance of each mRNA was expressed relative to mRNA for L32, a ribosomal protein.

Northern hybridization. Analysis of Flk-1 (VEGFR-2) mRNA was conducted as described previously (4). The cDNA, which was made by RT-PCR of human RNA, is 591 bp and corresponds to bases 2,837-3,427 of the coding region. The Northern hybridization was quantified by phosphorimaging using the ribosomal 18S band for normalization.

Immunohistochemistry. For PECAM-1 immunohistochemistry, slides were rinsed in xylenes and ethanol and then rehydrated in an ethanol series. Antigen retrieval was performed in 100 mM Tris · HCl, pH 10. Slides were heated in a microwave for 4 min and then on 50% power for 10 min. The slides were blocked in TBS (Tris · HCl, 150 mM NaCl, pH 7.5)/2% horse serum. Primary goat anti-PECAM-1 polyclonal antibody (sc-1506; Santa Cruz) diluted 1:1,500 in TBS/1% horse serum was added and incubated overnight at 4°C. Biotinylated horse anti-goat IgG diluted 1:200 in TBS/1.5% horse serum was added for 45 min, followed by ABC-AP reagent. Vector red substrate was added, and the slides were incubated for 15-20 min. The slides were lightly counterstained in methyl green before being dehydrated and mounted. The negative control was goat IgG in TBS/1% horse serum.

For VEGF immunohistochemistry, the slides were digested with 0.1% trypsin in PBS and rinsed in PBS. They were then incubated with 1% H2O2 in methanol for 30 min to eliminate endogenous peroxidase. The slides were rinsed, blocked with 1% casein in TBS, and incubated overnight at 4°C with 0.5 µg/ml of polyclonal rabbit anti-human VEGF (sc-152; Santa Cruz) in TBS/1% casein. Slides were incubated with biotinylated goat anti-rabbit IgG diluted in TBS/1% casein. After being rinsed in TBS, slides were incubated in streptavidin-horseradish peroxidase (HRP) 1:100, rinsed, and treated with tyramide (New England Nuclear) 1:50, following the manufacturer's protocol. The slides were incubated with streptavidin-HRP 1:100, and stained using 3,3'-diaminobenzidine (DAB). The slides were then counterstained with hematoxylin. Nonimmune rabbit IgG diluted in TBS/1% casein was the negative control.

Quantitative morphometry of PECAM-1 immunostaining. We quantified capillary density by measuring the area of PECAM-1 immunostaining relative to the total area of parenchymal cells. This measurement quantified the proportion of parenchymal lung area that was occupied by PECAM-1-positive endothelial cells. Images from each slide were captured with a ×40 objective and differential interference contrast. Images that contained large vessels or airways were excluded, and new images were obtained for a total of six images per slide. Color thresholding of the brown DAB stain was performed using Metamorph software (Universal Imaging, Downingtown, PA) to determine the area of PECAM-1-positive cells. The threshold range was set to include the entire area of brown-staining endothelial cells but to exclude random light-brown background staining. Auto threshold for dark objects was used with minor adjustments to measure the total area of all parenchymal cells. The ratio of PECAM-1-positive area/total cell area was calculated for each image, and average ratios were determined for each slide. Three to five different baboon lungs for each time point were measured. Reproducibility of the measurements was documented by repeat analysis of the PECAM-1-positive cell area and the total cell area in the same six images and in new sets of six images from the same lungs. Both methods demonstrated >95% reproducibility of the measurements.

In situ hybridization. Protocols for in situ hybridization were described previously (25). The VEGF cDNA is 94% homologous to human VEGF and hybridizes to all VEGF mRNA splice variants (26). The cDNA for Ang-1 was obtained by RT-PCR of human lung mRNA and is 1,046 bp. Briefly, lung tissue sections on TES-treated slides were treated with proteinase K and equilibrated in triethanolamine (pH 8.0) and acetic anhydride. Prehybridization was at 55°C for 2 h. Hybridization was performed at 53°C using 33P-labeled probes. After hybridization, the slides were rinsed and digested with RNases, and stringent washes were performed in 0.1× saline sodium citrate (SSC) at 64°C for VEGF and 56°C for Ang-1. The slides were dipped in NTB-2 emulsion and exposed at 4°C.

VEGF mRNA splice variant analysis. VEGF mRNA splice variants were analyzed by RT-PCR as described previously (43). Briefly, primers located in VEGF exons 5 and 8 were used to amplify the splice variants (VEGF189, VEGF165, and VEGF121) in the presence of [32P]dCTP. Amplified products were separated on highly denaturing polyacrylamide gels and quantified by phosphorimaging and image analysis. To account for differences in signal intensity due to the differences in splice variant sizes, the data were expressed as molar ratios.


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ABSTRACT
INTRODUCTION
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DISCUSSION
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Lung PECAM-1 protein and mRNA increase during gestation and are decreased in CLD. PECAM-1 is a marker for endothelial cells. Lung PECAM-1 protein increased 7.5-fold between 125 days' gestation and 1-3 days' postnatal life as demonstrated by Western immunoblot (Fig. 1, A and B), indicating a substantial expansion of the endothelial cell population. Expression of PECAM-1 mRNA also increased steadily during lung development (Fig. 1C). Animals delivered on day 125 of gestation and supported for 6 days with appropriate supplemental oxygen and ventilation (125d 6dPRN) had no increase in PECAM-1 protein or mRNA (Fig. 1, D and E). By 14 days of supplemental oxygen and ventilation (125d 14dPRN), PECAM-1 protein had not changed from 125 days' gestation and was reduced by nearly 70% compared with the 140-day gestational controls (Fig. 1D). PECAM-1 mRNA was also significantly reduced in the 125d 14dPRN animals. (Representative RPA lanes are shown in Fig. 10.) These data suggest that 6 or 14 days of appropriate oxygen and ventilation prevented the normal increase in endothelial cells that would have occurred in utero.


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Fig. 1.   Expression of platelet endothelial cell adhesion molecule (PECAM)-1 protein and mRNA during lung development and in chronic lung disease (CLD). A: Western immunoblot for PECAM-1 protein (arrow) and actin during lung development and in the groups of 125-day-gestation animals killed after 6 days of support (125d 6dPRN) and after 14 days (125d 14dPRN). Each lane represents a separate animal. B: quantification of PECAM-1 immunoblot shows a 7.5-fold increase in PECAM-1 protein between 125 days' gestation and the 1 to 3-day postnatal animals. C: quantification of ribonuclease protection assay (RPA) for PECAM-1/L32. (The RPA gel is not shown.) During gestation, the relative abundance of PECAM-1 mRNA increases steadily (n = 4-6 animals for each point). D: quantification of PECAM-1 protein in CLD. PECAM-1 protein does not increase from 125 days' gestation in the 126d 6dPRN or 125d 14dPRN animals. The 125d 14dPRN animals have significantly less PECAM-1 protein than the 140-day gestational controls (n = 4-6 animals at each point; *P < 0.005 by Student's t-test). E: quantification of PECAM-1 mRNA in CLD. Compared with 140-day gestational controls, the 124d 14dPRN animals have significantly less PECAM-1 mRNA relative to L32 (n = 4-6 animals at each point; #P < 0.05 by Student's t-test). All data are means ± SE. (Representative RPA lanes are shown in Fig. 10.)

Abnormal immunostaining for PECAM-1 in CLD animals. Immunohistochemistry for lung PECAM-1 in 125-day gestational controls showed isolated capillaries imbedded in the thick septa of the distal potential airspaces (Fig. 2A). The septa frequently had several capillaries, but few capillaries had a subepithelial location, and intercapillary connections were sparse. By 140 days' gestation, more extensive capillary development was noted in thinner septa (Fig. 2B). Rather than isolated capillaries, long segments of capillaries were identified, suggesting an interconnecting network. Many capillary segments were immediately subepithelial (Fig. 2B, arrows). By day 160 (Fig. 2C), the septa were very thin and the endothelium appeared to constitute an increasing proportion of septal cells. The 175-day gestational controls had an extensive capillary network that extended into the secondary septa (Fig. 2D).


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Fig. 2.   PECAM-1 immunohistochemistry. A: at 125 days' gestation, PECAM-1 (brown staining) is detected in isolated capillaries in the potential distal airspace septa. Relatively few capillaries are subepithelial, and the capillary segments are relatively short and lack extensive interconnections. B: by 140 days of gestation, the capillary segments are longer and are frequently subepithelial (arrows). Fewer isolated capillaries are noted, implying formation of a capillary network. At 160 (C) and 175 (D) days of gestation, extensive PECAM-1 immunostaining is noted in the primary and secondary septa, suggesting an intricate capillary network. E: the 125d 6dPRN animals had isolated capillary segments in thickened airspace septa. F: the 125d 14dPRN animals continued to have isolated capillary segments located centrally in the thickened septa. Overall, immunostaining is decreased and few capillary segments are subepithelial, compared with the 140-day gestational controls (B), suggesting failure to develop a capillary network. The distal airspaces are very large and have few secondary septa compared with the 140-day gestational controls. Control antibody gave little background immunostaining (not shown). Original magnification = ×300.

The 125d 6dPRN animals (Fig. 2E) had thick septa and endothelial cells of isolated capillaries imbedded centrally in the septa. By 14 days of appropriate oxygen and ventilation (Fig. 2F), the distal airspaces were large and the septa remained thickened and hypercellular. Isolated capillaries were centrally located with few capillaries found in subepithelial locations. Compared with the 140-day gestational controls, the 125d 14dPRN animals had decreased PECAM-1 staining and lacked an extensive capillary network, suggesting disruption of normal microvascular development.

Decreased endothelial cell area in CLD lung. To quantify capillary density, we used computer-assisted measurement of the PECAM-1-immunostained area compared with total parenchymal cell area, using differential interference contrast (Fig. 3). The tissue sections analyzed contained mainly the alveolar parenchyma and excluded any large airways or blood vessels. Between 125 and 140 days' gestation, the area of PECAM-1 immunostaining increased from 31 ± 2% to 39 ± 2% (mean ± SE) of total parenchymal area (P < 0.05). In the CLD animals, however, the relative PECAM-1-immunostained area did not increase above the value at 125 days. The 125d 14dPRN animals had a significantly decreased proportion of PECAM-1-immunostained area compared with the 140-day gestational controls (27 ± 1% vs. 39 ± 2%; P < 0.001). These data suggest that the relative capillary density did not increase in the CLD animals.


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Fig. 3.   Decreased capillary density in CLD. The area of PECAM-1-immunostained endothelial cells as a proportion of total lung parenchymal cell area was measured by computer-assisted analysis of differential interference contrast images. Six random fields were chosen from each slide for 3-5 animals at each time point. The capillary area did not increase in the PRN animals compared with the 140-day gestational control (140 GC). *P < 0.001.

Lung VEGF expression increases during gestation but is disrupted with CLD. To evaluate potential mechanisms of normal and abnormal capillary development, we investigated expression of VEGF, Ang-1, and their receptors. In situ hybridization for VEGF mRNA during lung development (Fig. 4, A-D) showed that the 125-day gestational controls had VEGF message scattered in the thick interairspace septa. A high concentration of VEGF mRNA was noted in some cells on the surface of the septa. At 140 days' gestation (Fig. 4B), VEGF message was concentrated in isolated cells of the septal epithelium (arrow), although the interior septal cells continued to have some message. By 160 days' gestation (Fig. 4C), the message was almost exclusively in isolated septal epithelial cells. As the septa thinned in more mature animals (Fig. 4D), VEGF mRNA remained in isolated septal epithelial cells, which resembled cuboidal type II cells on higher power (not shown). Little VEGF mRNA was noted in airway epithelial cells. The relative abundance of total lung VEGF mRNA increased during development and remained relatively high in the adult (Fig. 4E). The proportions of the alternatively spliced variants of VEGF mRNA changed during lung development, with VEGF189 increasing from 14% at 125 days' gestation to 30% at 3 days postnatal life (P < 0.05; Fig. 4F).


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Fig. 4.   Changes in cell-specific VEGF mRNA during lung development. A: in situ hybridization at 125 days' gestation showed VEGF mRNA (black grains) scattered in septal cells of the potential distal airspaces. Some cells had a concentration of grains. B: by 140 days' gestation, individual septal cells (arrow) had abundant VEGF mRNA. Scattered grains were also seen in interior septal cells. At gestational days 160 (C) and 175 (D) and in 1- to 3-day-old postnatal animals (not shown), the individual VEGF-expressing cells become more prominent compared with other parenchymal cells that do not have VEGF mRNA. The sense control hybridization gave very little background (not shown). E: quantification of RPA for VEGF mRNA (representative lanes are in Fig. 10) showed that the relative abundance of this message increased during gestation and remained high postnatally. F: the relative proportion of VEGF mRNA splice variants changes during lung development. Of total VEGF mRNA, the molar ratio of VEGF189 doubled between 125 days' gestation and 1-3 days' postnatal life (P < 0.05; Student's t-test). Black bars, VEGF189; gray bars, VEGF165; open bars, VEGF121. The data are means ± SE of 4 separate animals at each point.

In the 125-day-gestation animals that received appropriate oxygen and ventilation for 6 days, VEGF mRNA was scattered in the interairspace septa (Fig. 5A). By 14 days of oxygen and ventilation (Fig. 5B), VEGF message was decreased in many areas of lung compared with the 140-day gestational controls. Fewer epithelial cells with intense VEGF signal were noted in these areas. However, some areas of lung had relatively well-preserved VEGF expression, particularly where airspace septa were not thick (not shown), suggesting heterogeneity of the injury. Total lung VEGF mRNA was significantly decreased in the 125d 14dPRN animals compared with the 140-day gestational controls (Fig. 5C). There were no changes in the relative proportions of the mRNA splice variants (data not shown).


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Fig. 5.   VEGF mRNA decreases in CLD. In situ hybridizations for VEGF mRNA in 125d 6dPRN animals (A) and 125d 14dPRN animals (B) show a paucity of cells with abundant VEGF mRNA in the CLD animals, particularly compared with the 140-day gestational controls (compare to Fig. 4B). C: quantification of RPA for VEGF mRNA showed the message did not increase in the animals treated with appropriate oxygen and ventilation. The 125d 14dPRN animals had VEGF mRNA that was significantly less than the 140-day gestational controls (*P = 0.025). Data are means ± SE of 4-6 animals at each point. (Representative RPA lanes are in Fig. 10.)

Decreased VEGF immunohistochemistry in CLD. Immunostaining for VEGF during lung development followed a pattern similar to the in situ hybridizations (Fig. 6, A-D). The 125-day-gestation animals had VEGF protein detected in the distal potential airspace septa. By 175 days' gestation, the protein was localized in cuboidal epithelial cells, likely type II cells, in the septa. Little VEGF protein was detected in airway cells (not shown). The 125 6dPRN animals had VEGF immunostaining in the septa (Fig. 6E). However, the 125d 14dPRN animals had substantially decreased VEGF immunostaining, particularly in areas where the distal airspace septa were thickened (Fig. 6F). Together, these data suggest that 125-day-gestation lungs treated with appropriate oxygen and ventilation have decreased expression of VEGF mRNA and protein compared with the 140-day gestational controls.


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Fig. 6.   VEGF immunohistochemistry. At 125 (A) and 140 (B) days' gestation, VEGF protein was detected in septa between potential distal airspaces (brown staining). Little immunostaining was noted in large airway cells or endothelial cells of large vessels (not shown). By 160 days' gestation (C), when septa are thin, VEGF immunostaining was mainly in septal cells and became restricted to cuboidal septal cells by 175 days' gestation (D, arrow). The 125d 6dPRN animals (E) had detectable VEGF protein in the interairspace septa. F: VEGF immunostaining was very reduced in the 125d 14dPRN animals compared with the 140-day gestational controls (C). Control antibody gave very little nonspecific immunostaining (not shown).

Ang-1 and its receptor TIE-2 are not altered in CLD. In situ hybridization for Ang-1 in the 125- and 140-day-gestation animals showed mRNA scattered among cells in the potential distal airspace septa (Fig. 7, A and B). Unlike VEGF, which was expressed in epithelial cells, Ang-1 message was not concentrated in any particular cell type. Little Ang-1 message was detected in airway cells or smooth muscle. A similar pattern was observed in the 125d 6dPRN and 125d 14dPRN animals: Ang-1 message was detected mainly in the septal mesenchymal cells (Fig. 7, C and D). RPA for Ang-1 and TIE-2 showed that the messages increased in whole lung during development and did not change during the course of exposure to appropriate oxygen and ventilation (Fig. 8, A-D). (Representative lanes for the RPAs are shown in Fig. 10.)


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Fig. 7.   In situ hybridization for angiopoietin (Ang)-1 in fetal lung and CLD. A: at 125 days' gestation, Ang-1 mRNA (black grains) was at relatively low abundance and found uniformly in the septal cells of potential distal airspaces. B: in the 140-day-gestation lung the mRNA was in septal cells, but unlike VEGF, no particular cell had abundant Ang-1 message. C: no major changes in Ang-1 abundance or cell-specific location occurred in the 125d 6dPRN animals or the 125d 14dPRN animals (D). The sense control had low nonspecific hybridization (not shown).



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Fig. 8.   Quantification of RPA for Ang-1 and tyrosine kinase with immunoglobulin and epidermal growth factor homology domains (TIE-2) relative to L32 in lung development and CLD. A: Ang-1 mRNA increased in lung development and was relatively high in postnatal lung. B: message for TIE-2, the Ang-1 receptor, followed a pattern similar to Ang-1. Neither Ang-1 (C) nor TIE-2 (D) changed significantly in CLD. Data are means ± SE of 4-6 animals at each time point. (Representative RPA lanes are in Fig. 10.)

Endothelial cell receptors Flt-1 and TIE-1 mRNAs decrease in CLD. Flt-1 (VEGFR-1) mRNA increased during fetal lung development, with a peak at 175 days' gestation, but declined in postnatal lung (Fig. 9A). Flk-1 (VEGFR-2) mRNA increased 3.7-fold between 125 and 160 days' gestation (Fig. 9B). TIE-1 mRNA, an orphan tyrosine kinase receptor that mediates vascular network formation in latter stages of development, increased during lung development, had maximal message abundance toward the end of gestation, but declined in the postnatal lung (Fig. 9C). During the course of 6 or 14 days of appropriate oxygen and ventilation, messages for both Flt-1 and TIE-1 did not increase from the 125-day-gestation values (Fig. 9, D and F). The 125d 14dPRN animals had significantly less mRNA for these two receptors than the 140-day gestational controls. There was no effect of injury on Flk-1 expression (Fig. 8E). (Representative lanes for the RPAs are shown in Fig. 10.)


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Fig. 9.   Quantification mRNAs for the VEGF receptors fms-like tyrosine kinase receptor (Flt-1, A and D) and fetal liver kinase (Flk)-1 (B and E), and the orphan receptor TIE-1 (C and F). During lung development, Flt-1 mRNA (A) peaked in late gestation and declined postnatally. Flk-1 mRNA (B) increased 3.7-fold between 125 and 160 days of gestation. TIE-1 mRNA (C) peaked in late gestation and declined in postnatal lung. Development of CLD resulted in significantly decreased expression of Flt-1 (D) and TIE-1 (F) in the 125d 14dPRN animals compared with the 140-day gestational controls. Flk-1 mRNA (E) did not change in the CLD animals. Data are means ± SE from 4-6 animals. (*P < 0.01; #P < 0.005) (Representative RPA lanes are in Fig. 10.)



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Fig. 10.   Representative lanes for the RPAs. The original RPA gels contained lanes from multiple animals for each time point. Shown is a composite of representative lanes from several gels. Lanes: 1 (125-day gestational control), 2 (140-day gestational control), 3 (175-day gestational control), 4 (3 days postnatal), 5 (adult), 6 (125d 6d PRN), 7 (125d 14d PRN). Bands: a (FLT-1), b (Flt-4), c (TIE-1), x (noncontributory band), d (TIE-2), e (PECAM-1), x (noncontributory band), f (Ang-1), g (VEGF), h (L32), I (glyceraldehyde-3-phosphate dehydrogenase).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Development of the alveolar microvasculature is a key step in lung development. Many studies have investigated alveolar epithelial cell differentiation, but little is known about regulation of pulmonary microvascular development. Premature infants are born with an underdeveloped microvasculature and lung injury, hyperoxia, and inflammatory cytokines may be toxic to endothelial cells (13, 40). Hyperoxic lung injury also destroys alveolar epithelial cells and impairs epithelial-mesenchymal interactions that are necessary for lung vascular development (20). As shown in this study and others (4, 25, 47), VEGF is expressed by fetal distal airspace epithelial cells, suggesting that altered lung epithelial cell function may disrupt normal microvascular development.

We found that the endothelial marker, PECAM-1, increased more than sevenfold from 125 days' gestation to term, consistent with expansion of the pulmonary vasculature. As noted in this study in primates and by others in rodents (7), the morphology of the distal airspace capillaries changed from isolated capillaries imbedded in thick septa at 125 days' gestation to a more complex capillary network that was largely subepithelial at 140 days' gestation. This time is equivalent to ~24-26-wk gestation in the human. Our finding of increased lung VEGF and Ang-1 expression during gestation and the localization of VEGF to the distal airspace epithelium supports the hypothesis that these angiogenic factors contribute to pulmonary microvascular development. Extending work by Coalson et al. (11) in a similar baboon model, we found that CLD interrupted microvascular development, as measured by PECAM-1 protein, mRNA and area of PECAM-1 immunostaining. Our data suggest that alterations in the genetic program of VEGF, Flt-1, and TIE-1 expression contribute to disordered microvasculature in CLD.

Vascular development is a complex process that requires cell differentiation, migration, cell-to-cell contacts, tube formation and branching, interaction with basement membrane, and recruitment of supporting cells. The mechanisms that regulate these processes are largely unknown, but genetic knockout experiments suggest that disruption of VEGF, Ang-1, or their receptors leads to embryo-lethal vascular abnormalities (16, 17, 36, 39, 41). VEGF and Ang-1 have distinct roles in vascular development. Whereas VEGF is mitogenic and induces endothelial cell differentiation and migration, Ang-1 may be required for stabilization of developing vascular networks. Interactions between VEGF and angiopoietin family members may be necessary for normal vascular development (19, 30). We found a temporal association between increased PECAM-1 and increased VEGF and Ang-1 expression, suggesting that these factors may have a role in lung vasculogenesis, although their precise roles in this organ are not known.

The mechanisms regulating VEGF or Ang-1 expression in fetal lung are not clear. VEGF is induced by hypoxia, which may mediate its expression in normal development (1, 24). Many growth factors, including transforming growth factor-beta and platelet-derived growth factor, can induce VEGF (18, 32), but their role in lung development is not known. We showed previously that VEGF expression is localized to type II alveolar epithelial cells in mature lung (26). In the current study, septal cells of the potential distal airspace diffusely expressed VEGF mRNA at 125 days' gestation. By 140 days' gestation, individual distal epithelial cells had abundant VEGF mRNA. It is likely that VEGF expression is linked to distal epithelial cell differentiation. The increase in VEGF189 splice variant we found during baboon lung development is consistent with findings in the rabbit and mouse (29, 43). This variant, which is strongly heparin binding, is relatively rare in most other tissues. A specific role for the VEGF isoforms has not been determined, but the developmental increase in VEGF189 in lung supports a specialized function. Together, these data suggest that alveolar epithelial cells have a major role in regulating the temporal and spatial development of the alveolar microvasculature.

Very little is known about regulation of Ang-1 or its receptor TIE-2. Our findings show that mesenchymal cells rather than epithelial cells in the distal airspace express Ang-1. Lung cell-specific expression of Ang-1 has not been reported previously. Explants of large veins and arteries and some endothelial cells express Ang-1 (45). Ang-1 mRNA is downregulated by several growth factors in vitro (15), whereas hypoxia, tumor necrosis factor-alpha , and interleukin (IL)-beta increase endothelial TIE-2 (44). TIE-1, the orphan receptor that increased in fetal baboon lung but declined postnatally, is induced by VEGF and is essential for latter stages of vascular development (36).

Few studies have examined microvascular development in immature lung with CLD. Coalson et al. (11) found that 1-2 mo of appropriate oxygen and ventilation in 125-day-gestation baboons resulted in decreased alveolarization, dysmorphic capillaries, and decreased volume density of capillaries. Newborn rats exposed to 100% oxygen for 6 days had decreased number of capillaries (35), consistent with the toxic effect of oxygen on endothelial cells. We found decreased PECAM-1 expression in human infants who died with BPD compared with infants without BPD (5). In the present study, we found that 125-day-gestation baboons treated with appropriate oxygen and ventilation to maintain normal arterial blood gases had a 70% decrease in PECAM-1 protein and a 27% decrease in capillary density compared with 140-day-gestation controls. The 125d 14dPRN animals also had dysmorphic capillaries that frequently were in the center of thickened septa rather than subepithelial as in the gestational controls. Together, these findings support the hypothesis that CLD results in arrested lung microvascular development. Formation of normal alveolar capillaries may be necessary for alveolar development. For example, inhibition of angiogenesis by blocking a VEGF receptor during the first 2 wk of life resulted in decreased alveolarization in neonatal rats (21). It is possible that impaired microvascular development contributed to decreased alveolarization in the premature baboon model. Alternatively, impaired alveolarization may have resulted in decreased capillary formation separate from effects of decreased VEGF expression.

Abnormal microvascular development in CLD may result from disruption of the normal genetic program for angiogenic factor or endothelial cell receptor expression. Our finding that VEGF mRNA and immunostaining are decreased in the 125d 14dPRN animals compared with the 140-day gestational controls supports this hypothesis. Lack of epithelial VEGF may impair correct spatial formation of capillaries, preventing subepithelial location. VEGF is decreased in tracheal aspirates from human infants with BPD (23), and we found decreased VEGF immunostaining in human autopsy specimens with BPD (5). VEGF is required for normal vascular development and functions as a survival factor for endothelial cells, particularly in hyperoxia (2). Supporting a critical role for VEGF in preservation of endothelial cells, Corne et al. (12) found that induction of VEGF by IL-13 protected mice from hyperoxic lung injury. Ang-1 expression did not change in baboons with CLD, suggesting that this angiogenic factor alone is insufficient for normal microvascular development in lungs exposed to oxygen and ventilation. However, because VEGF and Ang-1 work in concert, normal Ang-1 expression with decreased VEGF expression may contribute to abnormal vascular development.

The mechanism of decreased VEGF expression in CLD is not known. Hyperoxic lung injury results in decreased VEGF in neonatal and adult animals (25, 31) and may have contributed to our findings. Hyperoxia may directly inhibit VEGF gene expression, possibly by decreased hypoxia-inducible factor-1 activation, but data supporting this hypothesis are lacking. Individual distal airspace epithelial cells develop VEGF expression by the 140th day of gestation, and damage to these cells or interruption in their differentiation may impair VEGF expression. In previous studies we found that VEGF is expressed by type II cells with low surfactant protein-C (SP-C) expression and is not expressed by proliferating cells (25, 26). Therefore, a change in distal epithelial cell phenotype to proliferative type II cells or to cells with high SP-C might result in decreased VEGF expression. Proof of these speculations is lacking.

We found that expression of the VEGF receptor, Flt-1, and the orphan receptor TIE-1 were decreased in the 125d 14dPRN animals. A decreased endothelial cell population, as suggested by decreased PECAM-1, might explain these results. However, decreased VEGF in CLD may also contribute to the decline in TIE-1 mRNA, because VEGF induces TIE-1 expression (27). In addition, TIE-1 is induced by hypoxia (27) and may be inhibited by hyperoxia. Both Flt-1 and TIE-1 regulate vascular network formation (17, 36), and loss of these endothelial receptors may result in the disruption of the capillary network in CLD lung. Our finding that Flt-1 but not Flk-1 was decreased in injury suggests independent regulation of these VEGF receptors.

These data have important limitations. The morphometric measurement of capillary density was based on the PECAM-1-immunostained area. Although PECAM-1 is widely used as a marker of endothelial cells, it can be up- or downregulated in certain conditions (33, 42). Threshold gating the computer analysis to count cells that had PECAM-1-immunostaining signal just above background allowed measurement of capillary area that was nearly independent of the absolute amount of PECAM-1. Endothelial cells that were devoid of any PECAM-1 would not be counted, however. We expressed the data as area of PECAM-1 immunostaining per area of parenchymal cells, rather than capillary area per field, to avoid variations resulting from differences in lung inflation. Microvascular morphometrics of the baboons at 1-2 mo of age also suggest disrupted alveolar capillary development in CLD (11). We found VEGF mRNA and protein in individual distal airspace epithelial cells that resembled type II cells. VEGF message and protein may be easier to detect in cuboidal cells than in flattened type I cells, and our data do not exclude type I cell expression of VEGF. The premature baboons used in this study are a model of CLD, and we cannot dissect the individual contributions of oxygen, ventilation, or other factors on our findings. Some other elements of the care of these animals may have affected vascular development or angiogenic factor expression.

In summary, appropriate oxygen and ventilation in very premature baboons disrupt pulmonary microvascular development. A potential mechanism for impaired microvascular development is an altered genetic program for distal epithelial cell expression of VEGF and the endothelial receptors Flt-1 and TIE-1. We speculate that a poorly developed pulmonary microvasculature may contribute to abnormal alveolar development and impaired gas exchange in CLD.


    ACKNOWLEDGEMENTS

We authors thank Dr. Raymond Baggs for help in quantifying capillary density.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grants HL-63400 (W. M. Maniscalco) and HL-63039 (G. S. Pryhuber).

Address for reprint requests and other correspondence: W. M. Maniscalco, Box 651, Dept. of Pediatrics, Univ. of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642 (E-mail: William_Maniscalco{at}URMC.Rochester.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajplung.00325.2001

Received 14 August 2001; accepted in final form 10 November 2001.


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DISCUSSION
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