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Pediatric Heart Lung Center, Department of Pediatrics, University of Colorado Health Sciences Center, The Children’s Hospital, Denver, Colorado
Submitted 7 September 2004 ; accepted in final form 12 May 2005
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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bronchopulmonary dysplasia; lung development; vascular endothelial growth factor; angiogenesis
Mechanisms that impair lung growth and cause persistent abnormalities in lung structure in premature infants with BPD remain poorly understood. Recently, experimental studies have shown that growth of the pulmonary circulation and alveolarization are closely coordinated, as demonstrated by findings that disruption of angiogenesis impairs lung structure (20). Treatment of neonatal rats with antiangiogenic agents, including fumagillin and thalidomide, decreases alveolarization and lung growth in infant rats, which is similar to the lung histology of BPD (20). Treatment of newborn rats with the vascular endothelial growth factor (VEGF) receptor inhibitor, SU-5416, also decreases alveolarization and vascular growth, suggesting that impaired VEGF signaling may contribute to abnormal lung structure after neonatal lung injury (20, 25). Interestingly, lung VEGF expression is decreased after hyperoxia in rabbits and in a primate model of BPD (30, 32). Clinical studies further support the hypothesis that decreased VEGF expression contributes to the pathogenesis of BPD (4, 24).
Previous animal studies have shown that exposure to hyperoxia in the neonatal period causes lung structural changes that are similar to the histology seen in human infants with BPD (39, 45, 46). Lung histology after hyperoxia is characterized by reduced complexity of the distal lung with decreased alveolar number and vascular growth (39, 40, 45, 46). Mechanisms by which hyperoxia inhibits lung growth in BPD and potential therapeutic strategies to improve lung growth in BPD remain unknown. Neonatal hyperoxia reduces lung VEGF mRNA and protein expression, suggesting that downregulation of VEGF may contribute to the simplified lung structure seen in this model (23, 30, 31). Recent studies further suggest that the reduction in VEGF protein persists beyond the hyperoxic injury and into the recovery period (27). However, whether late treatment with VEGF after hyperoxia can improve lung growth during recovery is unknown.
Therefore, we hypothesized that treatment with recombinant human VEGF (rhVEGF) protein would enhance alveolarization and vascular growth in infant rats during recovery from neonatal hyperoxia. To address this question, we studied the effects of rhVEGF treatment on lung structure during recovery from hyperoxia. We report that rhVEGF treatment improves alveolarization and vessel growth in infant rats during recovery after neonatal hyperoxia.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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All procedures and protocols were approved by the Animal Care and Use Committee at the University of Colorado Health Sciences Center. Pregnant Sprague-Dawley rats were purchased (Harlan Laboratories, Indianapolis, IN) and maintained at Denver’s altitude (1,600 m; barometric pressure, 630 mmHg; inspired oxygen pressure, 122 mmHg) for at least 1 wk before giving birth. Pups were delivered naturally at term gestation. Litter size was standardized to 10 pups. Animals were fed ad libitum and exposed to day-night cycles alternatively every 12 h throughout the study period.
Study Design
Four groups of animals were used in these experiments with 10 animals in each group (Fig. 1). All pups were maintained in room air for the first 24–36 h of life to allow successful transition to postnatal life before randomization to study groups. On postnatal day 3, half of the animals were placed in 75% oxygen, and half remained in room air for the next 12 days. On postnatal day 14, all animals were placed in room air. The animals were randomly assigned to treatment with rhVEGF or saline. Half the animals in each litter were treated with intramuscular injections of rhVEGF at a dose of 20 mg/kg, and half were treated with normal saline injections for a total of 7 days. The four study groups included hyperoxia + VEGF (H+V), hyperoxia + normal saline (H+NS), room air + VEGF (RA+V), and room air + normal saline (RA+NS). Litters of randomly divided rat pups and their dams were placed in Plexiglas chambers containing 75% oxygen or room air. Oxygen concentrations were continuously monitored, and gas lines were filtered with charcoal and barium hydroxide to maintain CO2 levels <0.05%. Chambers were opened briefly (<10–15 min) for cleaning and injections. Animals were killed on postnatal day 22 with intraperitoneal injections of pentobarbital (3 mg/kg body wt), and lung tissue was harvested for analysis.
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Body weight, lung weight, lung volume, right ventricular hypertrophy. Each animal was weighed before death. At autopsy, half the animals had lungs harvested for frozen tissue. Before freezing, wet lung weights were determined. The lungs of the remaining animals were fixed with paraformaldehyde, and subsequently lung volume was determined by displacement of water. Additionally, the heart was resected through a midline sternotomy. The right ventricle (RV) was dissected from the left ventricle and septum (LV+S). The RV and LV+S were weighed separately and the ratios of RV to LV + S weights were determined as a standard assessment of right ventricular hypertrophy (RVH) (12).
Fixation of lung tissue. Rat lungs were prepared and fixed in situ at the time of death on day 22. A midline sternotomy was made. Lungs and heart were exposed. The left atrium was incised to allow drainage of perfusate. Phosphate-buffered saline (PBS) was then injected into the right ventricle to flush the pulmonary circulation free of blood. The trachea was cannulated, and 4% paraformaldehyde was instilled into the lungs under constant pressure (30 cmH2O) for 30–60 min. This inflation pressure was chosen based on a previous study evaluating optimal inflation pressure (3, 15). The trachea was then ligated with the lungs under continued distending pressure. Lungs were removed and submersed in fixative for 24 h at room temperature.
Lung morphometric analysis. Transverse sections were obtained from the midplane of the upper, middle, and lower lobes of the formalin-fixed right lung for morphometric analysis. Sections from each animal were processed and embedded in paraffin wax. Paraffin sections (5 µm thick) were cut from each block and stained with hematoxylin and eosin. Analysis of each section was carried out in a blinded fashion. Alveolarization was assessed by performing radial alveolar counts (RAC), according to the method of Emery and Mithal (9, 10). From the center of the respiratory bronchiole, a perpendicular was drawn to the edge of the acinus (as defined by a connective tissue septum or the pleura), and the number of septa intersected by this line was counted. Six counts were performed for each animal.
For assessment of the mean linear intercept (MLI), six lung sections were selected in an unbiased fashion. Images of each section were captured with a Magnafire digital camera through an Olympus IX 81 microscope and were saved as PICT files. The images were then analyzed with the use of Stereology Toolbox software (Morphometrix, Davis, CA). The mean interalveolar distance was measured as the MLI, by dividing the total length of lines drawn across the lung section by the number of intercepts encountered, as described by Thurlbeck (10).
For assessment of skeletonization, six lung sections were selected in unbiased fashion. Images of each section were captured with a Magnafire digital camera through an Olympus IX 81 microscope and were saved as PICT files. Using digital image processing, SoveaPro (Reindeer Graphics), we transformed the lung parenchyma into a skeleton of curved and straight line segments with nodal and end points as described by Tschanz and Burri (43). The number of nodal points per high power field (hpf) was counted.
Immunohistochemistry and vessel volume density. Immunohistochemistry for factor VIII was performed to identify vessels for morphometric assessment. Paraffin-embedded slides from paraformaldehyde-fixed tissue were deparaffinized in CitriSolv (Fisher Scientific, Pittsburgh, PA). The sections were rehydrated by serial immersions in 100% ethanol, 95% ethanol, 70% ethanol, and water. Sections were digested with Proteinase K at a concentration of 500 µg/ml for 10 min at room temperature and then washed with PBS with 2.7 mM KCl, 1.2 mM KH2PO4, 138 mM NaCl, and 8.1 mM Na2HPO4. Endogenous peroxidase activity was reduced by immersion in 3% hydrogen peroxide in methanol. After rinsing, sections were covered in 10% goat serum for 30 min, and incubated with rabbit antifactor VIII antibody (1:1,000) diluted in PBS with 1% BSA and 0.1% sodium azide for 60 min. After incubation, the sections were rinsed with PBS and incubated with biotin-labeled secondary antibody (diluted 1:200 in PBS with 2% goat serum) for 30 min. After incubation with the secondary antibody, the sections were rinsed with PBS, incubated in ABC complex (Vector) for 30 min at room temperature, rinsed in PBS and developed with diaminobenzidine and hydrogen peroxide. Slides were lightly counterstained with hematoxylin. We then dehydrated the slides by sequential immersion in 70% ethanol, 95% ethanol, 100% ethanol, and CitriSolv before applying coverslips. Four lung sections were selected and captured by digital camera for analysis. The cells positive for factor VIII were stained brown. The number of factor VIII-positive vessels per high power field was counted. The magnification used was x20. Vessels immediately adjacent to large airways were excluded.
Statistical Analysis
Statistical comparison was made using analysis of variance and Fisher’s protected least-significant-difference test with Statview software package (Abacus Concepts, Berkeley, CA). Differences were considered significant at P < 0.05. The results were presented as means ± SE.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Ten animals were in each study group. Body weights of the hyperoxia group were higher than body weights of the RA group at the beginning and at the end of the experiment, reflecting differences between birth weights of the pups from the different litters. However, VEGF treatment did not alter body weight within either the hyperoxia or RA groups. Lung weights were also higher in the hyperoxia group than in the RA group, but there was no difference in lung weights after VEGF or saline treatment within the hyperoxia or RA groups. The lung-body weight ratios were not different between VEGF saline treatments in either the hyperoxia- or RA-exposed groups. (Table 1).
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In comparison with control animals (RA+NS), lung histology of animals exposed to hyperoxia (H+NS) was characterized by decreased septation, distal air space enlargement, and a reduction in complexity (Fig. 2). rhVEGF treatment during recovery after hyperoxia (H+VEGF) restored normal lung structure, which appears similar to the lung histology of RA-raised controls. rhVEGF treatment of animals raised in RA (RA+VEGF) had no apparent effect on distal lung structure. Additionally, there was no histological evidence of inflammation or edema in the VEGF-treated animals.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Animal models of hyperoxic lung injury have been used to study the mechanisms that contribute to the development of BPD (39, 45, 46). Although many pathogenic factors contribute to the development of BPD, impaired VEGF signaling may play a prominent role (20, 23, 30). Hyperoxia reduces lung VEGF expression in adult rats as well as neonatal rabbits (23, 30). A recent study has reported a sustained reduction in lung VEGF and VEGF receptor (VEGFR)-2 expression during prolonged hyperoxia and during the recovery period in infant rats (17). Treatment of neonatal rats with antiangiogenic agents, including SU-5416, a VEGF receptor inhibitor, decreases alveolarization and vascular growth (20), providing direct evidence that impaired VEGF signaling contributes to abnormal lung structure after neonatal lung injury. Clinical studies have shown that lung VEGF expression is decreased in human infants with fatal BPD (4), and VEGF protein levels are reduced in tracheal fluid of premature infants with BPD (24).
Mechanisms by which VEGF treatment improves lung growth during recovery from hyperoxia are uncertain but may be due to effects on endothelial cells, epithelial cells, or both. VEGF treatment may enhance endothelial cell growth, function, and survival, thereby increasing vascular growth that is necessary to sustain normal alveolarization (20). Endothelial cells are prone to oxidant injury, and the absence of VEGF, an important endothelial cell survival factor, may further increase susceptibility of the endothelium to hyperoxic stress. VEGF may have a direct effect on angiogenesis and alveolarization through liberation of nitric oxide (NO) (11, 14, 16, 20, 38). In addition, studies have shown a persistent reduction in VEGF protein following neonatal hyperoxic lung injury despite recovery in RA and that inhaled nitric oxide (iNO) preserves normal lung growth after disruption of VEGF signaling (27, 41). Several studies have also shown that lung endothelial nitric oxide synthase is also reduced in animal models of BPD (2, 28, 41). Previous studies have demonstrated that iNO during hyperoxia exposure may reduce acute lung injury in several animal models (13, 19, 34, 36, 44). These studies collectively suggest a critical role for the VEGF-NO signaling pathway during normal early postnatal lung development.
Alternatively, VEGF may have a direct effect on the pulmonary epithelium in promoting alveolarization. Studies have shown that VEGF may be involved in epithelial growth in fetal human lung explants in vitro and that exogenous VEGF can increase epithelial proliferation (5). A recent study has further demonstrated that VEGFR-2 transcripts are expressed by type II cells and that type II cells respond to VEGF treatment with increased surfactant protein (SP)-B and SP-C production (8).
In addition to the important role of VEGF signaling during normal lung development, recent studies have implicated VEGF in the pathophysiology of acute lung injury in various models (35). Genetic overexpression of VEGF causes pulmonary hemorrhage and edema in mice; however, these models are associated with dramatic increases in VEGF content (22, 26). Lung VEGF levels are reduced early in acute respiratory distress syndrome (ARDS) (29), and humans recovering from ARDS have increasing VEGF levels in epithelial lining fluid (42). These findings suggest that VEGF may have diverse roles including that of "permeability factor" by promoting early lung edema after injury, whereas VEGF may enhance recovery later in the course. Thus the roles of VEGF may depend on timing and degree of endothelial-epithelial cell injury, as well as the degree of altered VEGF expression. This study examined the effects of exogenous rhVEGF treatment only during the recovery phase after hyperoxia. Although we did not directly measure lung edema, we found no difference in lung weight, body weight, and lung-body weight ratio after VEGF treatment. Body weights and lung weights were higher in the hyperoxic group compared with the RA group at the beginning and end of the study due to differences in initial birth weights between the hyperoxia and RA litters at the onset of the experiment.
Interestingly, MLI was increased and surface area, which is calculated from MLI, was reduced in RA animals treated with VEGF compared with RA animals treated with NS. This may reflect the differences between morphometric methods. RACs and nodal points by skeletonization did not reflect this difference. Alternatively, VEGF treatment in RA animals may alter lung structure, as suggested in previous studies, which showed that huge increases in lung VEGF gene expression in newborn mice can cause lung injury (26).
There are several potential limitations of this study. First, the body weights of the hyperoxia group were higher than the RA group at the start of the study due to differences between litters. However, VEGF did not alter body weight in either hyperoxia or RA. Second, term animals rather than premature animals were used in this model of BPD. However, the alveolar phase of lung development in the newborn rat occurs during the first 3 wk of postnatal life (6, 7, 33), making this a useful time period during which to study mechanisms that impair lung growth. In addition, we studied the effects of VEGF treatment occurred during recovery after hyperoxia. Whether VEGF treatment during acute lung injury would have similar effects remains unknown. Mechanisms through which VEGF increases vessel and alveolar growth in this model are uncertain and are currently under investigation. Finally, rhVEGF protein administration may have adverse systemic effects but were not studied in this protocol.
In summary, we found that exposure of neonatal rats to hyperoxia results in impairment of alveolarization and vascular growth, which persists despite recovery in RA. We also report that rhVEGF treatment during the recovery period after neonatal hyperoxia restores normal lung architecture. Further studies are needed to determine the exact mechanisms by which VEGF treatment improves alveolarization and lung growth after neonatal hyperoxia, but these findings suggest that VEGF therapy may play a role in enhancing distal lung growth in BPD.
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FOOTNOTES |
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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.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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