Departments of 1Physiology, 2Obstetrics and Gynaecology, and 3Pediatrics, Canadian Institutes of Health Research Group in Perinatal Health and Disease, The Perinatal Research Centre, The University of Alberta, Edmonton, Alberta, Canada T6G 2S2
Submitted 19 August 2002 ; accepted in final form 6 March 2003
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ABSTRACT |
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alveolarization; oxygen; septation
Vascular endothelial growth factor (VEGF) is a key regulator of lung development. It acts through two distinct tyrosine kinase receptors, VEGFR1 (Flt-1) and VEGFR2 (KDR/flk-1), and is essential for endothelial cell differentiation as well as the sprouting of new capillaries from preexisting vessels (angiogenesis; see Refs. 12 and 29). Angiogenesis has been shown to be necessary for the development of the alveoli in the newborn rat. Jakkula et al. (15) administered inhibitors of endothelial cell proliferation (thalidomide and fumagillin) to rat pups between days 3 and 14 and demonstrated decreased capillary density along with inhibited septation of the lungs. In the same study, VEGF action was blocked by the VEGFR2 receptor blocker SU-5416, and similar results were observed (15). Further evidence that VEGF and angiogenesis are associated with the development of the alveoli comes from a study showing that pulmonary cell stretch, which stimulates capillary and alveolar septal growth in an in vitro system, upregulated VEGF mRNA and protein expression (26).
VEGF and its receptors have previously been shown to respond to O2 in neonatal animals. Maniscalco et al. (21) showed that neonatal rabbits exposed to 100% O2 for 9 days had decreased VEGF mRNA abundance, decreased alveolar epithelial cell VEGF expression, and decreased VEGF immunostaining. In later work, it was demonstrated that the reduction in VEGF mRNA was mainly because of a decrease in expression of the VEGF189 splice variant (34). However, Watkins and colleagues (34) actually showed an increase in lavage fluid VEGF protein on days 4, 6, and 8 during exposure of neonatal rabbits to 100% O2 and only a decrease after 9 days of hyperoxia. It has further been demonstrated that the expression of VEGF and its receptors is decreased after exposure to >95% O2 for 48 h in the adult rat, but nothing is known of the response of VEGF receptors to hyperoxia in neonatal rabbits or rats (18).
The transduction of an O2 signal to the level of gene expression
requires the nuclear translocation and activation of redox-responsive
transcription factors over specific ranges of PO2. VEGF
expression is induced when most cell types are exposed to hypoxia as a result
of increased transcriptional activation and mRNA stabilization of
hypoxia-inducible factor-1 (HIF-1
; see Ref.
31). HIF-2
(or
HIF-1
-like factor) has close sequence similarity to HIF-1
(10); however, the modes of
expression of HIF-1
and HIF-2
vary greatly, as HIF-2
was
first reported to be abundantly expressed in the adult lung of mice in the
normoxic state, whereas HIF-1
is ubiquitous at much lower O2
levels (10). Both HIF-2
and VEGF mRNA were found to be highly expressed in alveolar epithelial cells
in the parturient newborn lung, whereas the low levels of HIF-1
during
gestation did not change. It is therefore postulated that HIF-2
regulates VEGF expression under normoxic conditions and during lung and
vascular development. Recent experiments further demonstrated that loss of
HIF-2
impaired lung maturation and created a subtle deficit in
vascularization of the alveolar septa
(7).
There may be differences in lung maturation between the rat and the rabbit;
dexamethasone has been shown to inhibit septation in the neonatal rat;
however, recent data show that there is no difference in lung structure after
administration of dexamethasone to neonatal rabbits
(19,
24). No experiments have as
yet addressed the effects of O2 on VEGF expression or protein
levels in the neonatal rat lung or VEGF receptor expression or levels in any
model of inhibited alveolar development. The initial aim of these experiments,
therefore, was to determine changes in VEGF, VEGFR1, and VEGFR2 mRNA
expression and protein levels, both during the normal period of alveolar
development in the neonatal rat lung and during exposure to a hyperoxic
environment. Additionally, we studied one of the potential regulators of VEGF
expression in this system, HIF-2, which has been previously shown to
respond to high O2 concentrations.
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METHODS |
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Sprague-Dawley albino rat pups (Charles River Laboratories, St. Constant, Quebec, Canada) of both sexes were used. They were housed in the Health Sciences Animal Laboratory Service Department of the University of Alberta under veterinary supervision. The approval of the University of Alberta Animal Care Committee was obtained, and the guidelines of the Canadian Council of Animal Care were followed in all experimental procedures. Dams were maintained on regular laboratory rodent pellets and water ad libitum and were kept on a 12:12-h light-dark cycle.
O2 Exposure
Parallel litters of randomly divided rat pups and their dams were placed in 0.14-m3 Plexiglas exposure chambers containing >95% O2 or 21% O2 (room air/normoxia), as previously published (4, 6, 23), from day 4 to day 14 of postnatal life. O2 concentrations were monitored daily (Ventronic O2 analyzer no. 5517, Temecula, CA). O2 and room air were filtered through barium hydroxide lime (Baralyme; Chemeton Medical Division, St. Louis, MO) to keep CO2 levels below 0.5% and through charcoal to remove odors. Temperature and humidity were maintained at 26°C and 7580%, respectively. Chambers were opened for <15 min daily to switch dams between air and O2 environments and to clean dirty cages.
Preparation of Lung Samples
Pups from each exposure group were killed on days 4, 6, 9, 12, and 14 with an intraperitoneal overdose of pentobarbital sodium (100 mg/kg Euthanyl; MIC Pharmaceuticals, Cambridge, ON, Canada). Lung vasculature was washed by perfusion with 5 ml of ice-cold PBS injected in the right ventricle. The lungs were removed and snap frozen.
RT and Real-Time Quantitative PCR
Total RNA was isolated using TRIzol reagent according to the manufacturer's instructions (Life Technologies, Burlington, Ontario, Canada). Samples were further treated with DNase I (DNA-free; Ambion, Austin, TX) to ensure that no DNA contamination existed. Quality of RNA was assessed by formaldehyde agarose gel electrophoresis.
RT. Total RNA (100 ng) was added to a reaction mixture containing 100 ng random nanomers (Stratagene, La Jolla, CA), 1x cDNA first-strand buffer, 500 mM DTT, 0.4 U/µl RNase inhibitor, 1 mM each deoxy-NTPs, and 0.75 U/µl Superscript II RT (GIBCO-BRL Life Technologies). Negative RT (no enzyme) and no-template (no RNA) controls were also included. The RT thermal cycle was 25°C for 10 min, 50°C for 45 min, and 85°C for 5 min.
Real-time PCR. For real-time PCR, total VEGF and HIF-2 were
detected using SYBR-green. VEGFR1 and VEGFR2 were detected using a
fluorescently labeled molecular beacon. Primers and fluorescent beacons used
are shown in Table 1. Primers
were purchased from Sigma Genosys (Oakville, Ontario, Canada), and the
fluorescent molecular beacons were from Stratagene. Primers were optimized for
annealing temperature and RT RNA concentration. The correct product size and
sequence were then confirmed by electrophoresis and DNA sequencing,
respectively.
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The PCR mixture (50 µl total volume) consisted of 0.2 µM of each
primer, 0.2 µM of each molecular beacon (VEGF receptors and cyclophilin
only), 10x PCR buffer (including SYBR green for VEGF and HIF-2;
Perkin-Elmer-Applied Biosystems, Warrington, UK), 1.9 mM MgCl2, 0.2
mM each dNTPs, 0.04 U/µl Taq polymerase (GIBCO-BRL Life
Technologies), and 2 µl cDNA. Amplification and detection were performed
using the iCycler iQ real-time PCR detection system (Bio-Rad Laboratories,
Hercules, CA) with the following cycle profile: 50°C for 2 min, 95°C
for 10 min, and 45 cycles of 95°C for 15 s, 56°C for 1 min, and
72°C for 30 s.
Analysis of real-time PCR results. The mRNA of day 4
animals was pooled and used as a control group to allow analysis between PCR
plates. All results have been normalized to cyclophilin, a cytoskeletal
protein, which is expressed constitutively in all tissues, and did not change
during these experiments. The mean value was 2.13 ± 0.13 relative
fluorescent units. Quantification was performed by determining the threshold
cycle (CT). CT is proportional to the
amplified starting copy number of cDNA (or RNA). All reactions were performed
in triplicate and controlled by a no-template reaction. The quantity of mRNA
was calculated by normalizing the CT of genes of interest
to the CT of the housekeeping protein cyclophilin of the
same RNA probe, according to the following formula:
CT = CTGene of interest
mRNA - CTcyclophilin mRNA
(27).
VEGF Immunoassay
Samples were homogenized in lysis buffer (50 mM Tris · HCl, 3 mM sucrose, 0.1% Triton X-100, and 1 mM protease inhibitor cocktail; Calbiochem-Novabiochem, La Jolla, CA), supernatant was removed, and protein content was estimated using a Micro BCA Protein Assay Reagent Kit (Pierce, Rockford, IL).
Assay was performed using the Quantikine M, mouse VEGF Immunoassay kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. This assay recognizes both the 164- and 120-amino acid residue forms of VEGF. Starting sample concentration was 2 µg total protein/µl; samples were diluted fivefold before analysis. Results are expressed as picograms VEGF per microliter total protein.
Western Immunoblotting
The presence and relative abundance of VEGFR1 and VEGFR2 were determined
using Western immunoblotting, as described by Laemmli
(20). Aliquots from lung
homogenates (prepared as above) were diluted in reducing sample buffer (0.5 M
Tris-Cl, 2% -mercaptoethanol, 87% glycerol, 10% SDS, and 1% bromphenol
blue). Protein (40 µg/well) was loaded in 6% polyacrylamide gels. Proteins
were separated by electrophoresis, transferred to nitrocellulose membranes
(Bio-Rad, Mississauga, Ontario, Canada), and then blocked for nonspecific
binding in a 7% skimmed milk solution. Membranes were incubated with primary
antibodies raised in rabbit against VEGFR1 and VEGFR2 (Alpha-Diagnostics) for
2 h at a 1:500 dilution. Membranes were washed and incubated for 1 h with
peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch, Bio/Can
Scientific, Mississauga, ON, Canada). After repeated washing, membranes were
incubated with enhanced chemiluminescence reagent (Amersham Pharmacia Biotech)
and placed in a Fluor-X Max Imager (Bio-Rad, Mississauga, Ontario, Canada)
where the image was captured, and bands were analyzed by densitometric
analysis. Each gel contained a day 4 sample from a pool of extracted
tissues, and results on all subsequent days were normalized to the
densitometric value of this sample. Only normalized values from days
612 are shown in Figs.
1,
2,
3,
4.
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Statistical Analysis
Western immunoblotting results were calculated as a ratio to day 4 values for comparison, and PCR results used day 4 lungs as control values. All results were normally distributed and were analyzed by two-way ANOVA, where variance was distributed according to treatment and time. When a significant F value was found, Tukey's post hoc test was used to determine significance. Statistical significance was achieved at P < 0.05.
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RESULTS |
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Changes in total mRNA levels for the angiogenic growth factor, VEGF, were examined. The primers used for the real-time PCR analysis amplified a sequence that was common to all VEGF splice variants. Expression of message for total VEGF tended to increase slightly between days 6 and 12 in the animals raised in room air (Fig. 1A), and this increase reached significance by day 14 (P < 0.05) where values were two times those of day 6 air-exposed pups. Exposure to O2 abolished the increase in mRNA levels seen in the normoxic pups so that O2-exposed pups exhibited significantly lower VEGF mRNA levels than those from lungs of normoxic pups on days 12 and 14 (P < 0.001; Fig. 1A).
An increase, similar to that of the total VEGF mRNA, was seen in VEGF protein mass between days 4 and 12 (P < 0.05). This increase was maintained through day 14 at 0.61 ± 0.04 pg/µg total lung protein in the lungs of normoxic pups (Fig. 1B). Contrary to the inhibition seen in total VEGF mRNA, O2 exposure resulted in a biphasic effect on VEGF protein levels, stimulating an increase from day 4 (0.41 ± 0.03 pg/µg protein) to day 9 (0.57 ± 0.03 pg/µg protein; P < 0.001) and then a decrease on day 12 to lower than normoxic control values (0.40 ± 0.06 compared with 0.59 ± 0.02 pg/µg total lung protein, respectively; P < 0.001). This trend was maintained on day 14, although values were not significant at this age (Fig. 1B).
VEGFR1
We were interested in investigating whether O2 exposure had an effect on the VEGF receptor mRNA expression or protein levels. VEGFR1 mRNA increased from day 6 to 14 when levels were three times higher than those of the day 6 group (P < 0.05; Fig. 2A). Rat pups exposed to hyperoxia inhibited this age-dependent increase in VEGFR1 mRNA as levels remained similar to day 6 throughout the course of the experiment. This resulted in VEGFR1 mRNA levels of the O2-exposed group being significantly lower than the air-exposed group on days 9, 12, and 14 (P < 0.001; Fig. 2A).
Contrary to mRNA expression, protein levels of VEGFR1 did not change significantly between days 6 and 14 in normoxic-exposed pups (Fig. 2B). However, exposure of rat pups to a hyperoxic environment did cause a significant decrease in overall VEGFR1 protein levels (P < 0.05 between air- and O2-exposed groups; Fig. 2B). A significant interaction between group (air/O2) and postnatal day (age) was not observed.
VEGFR2
Message transcribing for VEGFR2 protein increased between days 9 and 14 in animals exposed to a normoxic environment so that by day 14 mRNA levels were 2.3 times those on day 6 (P < 0.05; Fig. 3A). Exposure to O2 had a significant effect by day 12 when VEGFR2 mRNA levels decreased to one-half the day 6 air values and were only 27% of the mRNA levels of the day 12 air-exposed pups (P < 0.001). The effect of O2 exposure was even greater by day 14, when mRNA levels from the O2 group were merely 8% of the air group (P < 0.001; Fig. 3A).
Protein levels for VEGFR2 in the normoxic-raised pups did not demonstrate the increase observed for VEGFR2 mRNA (Fig. 3B). However, exposure to O2 did have a similar effect of decreasing protein levels. VEGFR2 mRNA levels of the hyperoxic-exposed pups were 60% of those from the air-exposed group by day 12 (P < 0.001); this decrease was maintained through day 14 (P < 0.001; Fig. 3B).
HIF-2
To identify a possible mechanism whereby VEGF mRNA is decreased after
exposure of rat pups to hyperoxia, O2-induced changes in mRNA for
the transcription factor HIF-2 were determined. HIF-2
mRNA
followed a similar pattern to that of VEGF. There was an age-dependent
increase in HIF-2
mRNA between days 9 and 14, with
day 14 values increasing to 2.4 times those on day 9
(P < 0.05; Fig.
4A). Again, similar to total VEGF mRNA levels,
O2 exposure inhibited the increase in HIF-2
mRNA seen in the
control animals; there was a significant difference between air and
O2 groups on days 12 and 14 (P <
0.001; Fig. 4A). By
day 14, mRNA levels of hyperoxic-exposed pups were 62% of those in
the normoxic-exposed animals (P < 0.001;
Fig. 4A). There was a
strong correlation between VEGF mRNA and HIF-2
mRNA in the normoxic
pups; the correlation coefficient was 0.799 (P < 0.001;
Fig. 4B).
Interestingly, when the pups were exposed to a hyperoxic environment, this
correlation was lost (correlation coefficient, 0.355).
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DISCUSSION |
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Our work is in agreement with that of Maniscalco and colleagues (21) who showed that neonatal rabbits exposed to 100% O2 for 9 days from birth had decreased VEGF mRNA abundance (21) because of a significant decline in VEGF189 (34). In our experiments, although total VEGF mRNA was decreased because of hyperoxia, VEGF164 protein levels were found to increase on day 9 before decreasing below normoxic values on day 12. Interestingly, Watkins et al. (34) found a similar trend measuring total VEGF protein from lavage fluid of newborn rabbits exposed to 100% O2; they found increased VEGF on days 4, 6, and 8 before levels decreased to below those of air-exposed animals on day 9 (34). Because total mRNA decreased and protein levels of combined VEGF164 and VEGF120 initially increased in our experiments, it is likely that the 188 splice variant was decreased during the first 6 days of hyperoxic exposure.
VEGF expression is induced when most cell types are exposed to hypoxia as a
result of increased transcriptional activation and mRNA stabilization of
HIF-1 and HIF-2
(1,
31). This study demonstrates
increasing levels of HIF-2
mRNA expression from day 9 to
day 14 in the postnatal rat lung, which is in agreement with earlier
work showing a concurrent increase in HIF-2
and VEGF in the neonatal
mouse lung between days 5 and 14
(10). The cellular location of
HIF-2
in fetal and newborn mice and adult rats is the alveolar
epithelial cell (type II; see Refs.
7,
10, and
35) where it has been found in
the nucleus and also in the pulmonary artery endothelium
(35). Normobaric hypoxia
(created by perfusion with carbon monoxide) is a strong stimulus for
HIF-2
expression in adult rat lung
(35). It is unknown how
hyperoxia decreases HIF-2
levels, although the hypoxia-induced
increased expression may be via hypoxia-dependent stabilization of the protein
or through a multistep process leading to interaction with cAMP response
element B-binding protein
(9).
In our experiments, both VEGFR1 and VEGFR2 were decreased during exposure of the neonatal lung to a high-O2 environment. However, these receptors are known to have different modes of action. Targeted disruption of VEGFR1 leads to mice with mature, differentiated endothelial cells but with large, disorganized vessels thought to result from overproduction of endothelial progenitor cells rather than vascular disorganization (13, 14). It is now thought that VEGFR1 plays a negative regulatory role by binding to VEGF (11). VEGFR2 knockout mice produce neither differentiated endothelial cells nor organized blood vessels and also possess no hematopoietic precursors (29, 32). This receptor is responsible for endothelial mitogenesis and migration as well as regulating vascular permeability (2). Our results suggest that endothelial cell differentiation and migration, as stimulated through the VEGFR2, and endothelial cell maintenance and possibly spatial organization, through VEGFR1, are both necessary for postnatal lung development. However, if, as suggested above, VEGF188 is diminished early in the experiments, decreased signaling through VEGFR1 may be an important early result of hyperoxic exposure, since VEGF188 is unable to bind VEGFR2 in its intact form but does bind VEGFR1. This early response could affect different aspects of alveolar development than later in development when all VEGF splice variants appear to be decreased. Indeed events do occur at different critical periods during alveolarization (23); maximal lung cell proliferation occurs during the first half of alveolarization, maximal endothelial cell proliferation peaks on day 7 (17), and the concentration of pulmonary arteries doubles between days 8 and 11 (25).
It has been shown that it is VEGFR2, rather than VEGFR1, that mediates the advanced lung maturation seen when VEGF is exogenously administered to premature mouse fetuses in vivo (7). Furthermore, the VEGFR2 receptor blocker SU-5416 caused decreased capillary density and inhibited septation, suggesting that angiogenesis occurring by VEGF signaling through VEGFR2 receptor is essential for the maturation of the neonatal lung. Because VEGF189 can be cleaved by urokinase-type plasminogen activator forming a peptide similar to VEGF165, which also has similar receptor binding and mitogenic properties (28), the early lower levels of VEGF188 may decrease signaling through VEGFR2 by this mechanism.
Recent evidence suggests that VEGF affects more than just endothelial
cells. Overexpression of VEGF in the developing pulmonary epithelium of
transgenic mice not only increased growth of pulmonary vessels but disrupted
branching morphogenesis and inhibited type I cell differentiation as well
(36). There is also evidence
that VEGF may be involved in epithelial cell growth and proliferation in the
human fetal lung in vitro (5).
VEGFR2 protein is found in the distal airway epithelial cells of the
midtrimester human lung, and exogenous VEGF added to explants caused increased
epithelial proliferation and volume density
(5). A recent study by
Compernolle et al. (7)
demonstrated that type II cells expressed VEGFR2 transcripts and responded to
VEGF by increasing their production of surfactant protein (SP)-B and SP-C. The
same paper demonstrated that, as well as creating a subtle deficit in
vascularization of the alveolar septa, loss of HIF-2 impaired lung
maturation (7).
This study has important implications in the pathology of chronic lung disease of prematurity (CLD), which in its severest form develops into bronchopulmonary dysplasia (BPD). The pathology of BPD in recent years suggests that the condition is mainly the result of arrested development of the premature lung (16), resulting in a lack of alveolarization and dysmorphic vasculature. Therefore, the process of endothelial differentiation and organization in the alveolar microvasculature may be disrupted after premature birth and injury from O2 and ventilation. Recently, a study by Bhatt et al. (3) of premature human infants dying of BPD showed a disrupted pulmonary vasculature along with decreased expression of VEGF and VEGFR1. Further work from the same laboratory demonstrated a failure of the normal increase in capillary density or the endothelial cell marker platelet endothelial cell adhesin molecule-1 in very premature baboons; VEGF and VEGFR1 were again decreased in this model of CLD (22).
We conclude that hyperoxic exposure during the period of alveolarization in
the rat pup causes decreased VEGF levels, possibly through decreased levels of
the transcription factor HIF-2, and decreased VEGF receptor levels.
Because it is evident that the vascular growth is a fundamental part of normal
alveolar development, we speculate that hyperoxic-induced changes in VEGF may
be an important element in the pathology of BPD.
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ACKNOWLEDGMENTS |
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The research was funded by the Canadian Institutes of Health Research and the Alberta Heritage Foundation for Medical Research. G. E. Hosford was funded by a studentship from the Alberta Heritage Foundation for Medical Research.
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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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