Differential expression of VEGF mRNA splice variants in
newborn and adult hyperoxic lung injury
Richard H.
Watkins1,
Carl T.
D'Angio1,
Rita M.
Ryan1,
Alka
Patel2, and
William M.
Maniscalco1
1 Division of Neonatology,
Department of Pediatrics, Strong Children's Research Center,
University of Rochester School of Medicine, Rochester 14642; and
2 Department of Pharmacology and
Toxicology, State University of New York at Buffalo, Buffalo, New York
14214
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ABSTRACT |
Lung development and repair of hyperoxic injury
require closely regulated growth and regeneration of alveolar
capillaries. Vascular endothelial growth factor (VEGF), a mitogen for
endothelial cells, is expressed by alveolar epithelial cells.
Alternative splicing of VEGF mRNA results in isoforms of varying
mitogenicity and solubility. We examined changes in the proportions of
the VEGF splice variant mRNAs in rabbit lung development and in
control, oxygen-injured, and recovering newborn and adult rabbit lungs. The proportion of the 189-amino acid VEGF mRNA, which codes for an
isoform that binds to the extracellular matrix, increased fivefold during development (from 8% of total VEGF message at 22 days gestation to 40% in 10-day newborn lungs; P < 0.001). During neonatal oxygen injury, its expression declined from 38 to 8% of VEGF message (P < 0.002)
and returned to the control value in recovery. A similar pattern was
observed in adults. VEGF protein in lung lavage fluid increased
slightly during hyperoxia, declined to barely detectable levels at the
50% lethal dose time point, and increased 10-fold (newborn) or up to
40-fold (adult) in recovering animals. We conclude that alternative
splicing may have important roles in the regulation of VEGF activity in
developing and injured lungs.
vascular endothelial growth factor; messenger ribonucleic acid; angiogenesis; alveolar type II cells; oxygen; growth factors
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INTRODUCTION |
ANGIOGENESIS, the growth of new blood vessels, is
important in the development and repair of the lung. The gas-exchange
function of the lung requires the development and maintenance of an
extensive capillary network of endothelial cells in close proximity to
a thin layer of alveolar epithelial cells. Capillary regeneration is
also essential for repair of oxygen-induced lung injury. Acute hyperoxic lung injury is characterized by damage to endothelial and
epithelial cells, interstitial edema, and inflammation (8, 13, 18, 32).
Impaired function or loss of microvascular endothelial cells
contributes to serum protein leakage and edema. Without timely repair
and replacement of endothelial cells, alveolar fibrosis or death may result.
Numerous regulators of angiogenesis have been identified, including
basic fibroblast growth factor, transforming growth factor-
, interleukin-8, and vascular endothelial growth factor (VEGF) (12, 22).
Of these, VEGF, a homodimeric glycoprotein of 34-45 kDa, appears
to play a pivotal role. The loss of even a single VEGF allele is lethal
to the mouse embryo, indicating an essential role in the development of
the vascular system (10). VEGF is mitogenic almost exclusively for
endothelial cells. Besides being a mitogen for endothelial cells, VEGF
is a vascular permeability factor that induces fenestrations in
endothelial cells (30). VEGF also induces endothelial cell expression
of several proteolytic enzymes that promote extracellular matrix (ECM)
degradation, essential for endothelial cell migration and sprouting
(11).
In the normal lung, VEGF is quite abundant, similar to other highly
vascularized tissues (3). Lung VEGF is expressed primarily by alveolar
epithelial cells, which are in close proximity to microvascular
endothelial cells (24, 25). After hyperoxic injury, the expression of
VEGF message declines in both newborn and adult lungs (23), coincident
with the loss of endothelial cells (14). During recovery, when
endothelial cells proliferate, VEGF mRNA becomes very abundant in
alveolar type II cells (24). The level of VEGF protein in the
parenchyma of the newborn lung follows the same time course as type II
cell VEGF expression (23).
VEGF protein exists as several isoforms that are produced by
alternative splicing of the primary transcript or by limited proteolysis. The precise functional differences among the isoforms are
not known, but they differ in solubility, receptor affinity, and
mitogenic potency. The primary VEGF transcript derives from a single
VEGF gene that contains eight exons (35). Variable splicing involving
exons 6 and 7 results in up to five isoforms containing 121, 145, 165, 189, and 206 amino acids after removal of a common signal peptide (11,
29). Exons 6 and 7 each contain a region of basic amino acids with a
high affinity for heparin. The presence or absence of these
heparin-binding regions influences the ECM binding and solubility of
each isoform. Thus isoforms containing both exons
(VEGF206 and
VEGF189, where the subscript number is the number of amino acids) bind tightly to the ECM or cell
surface heparan sulfates. VEGF121,
which lacks both exons, does not bind heparin and is highly diffusable.
VEGF165, with one heparin-binding
region, is moderately diffusable (26). Limited diffusability may
spatially limit the action of ECM-binding isoforms such as
VEGF189, whereas highly diffusable
isoforms such as VEGF121 may have
widespread action. Affinity for heparin may play an important role in
VEGF binding to its receptors (15, 16, 29), accounting for the lower
receptor affinity of VEGF121
compared with that of VEGF165. The
isoforms also differ in their mitogenicity.
VEGF165 is the most mitogenic, and
VEGF121 is much less mitogenic
(21). A receptor that is specific for
VEGF165, which may enhance binding to the mitogenic VEGF receptor Flk-1, has been described (33). VEGF189 does not bind to Flk-1 and
may be an ECM storage form (28). Thus although the specific roles of
VEGF isoforms are not known, their biological properties suggest
differing functions.
The solubility and mitogenicity of VEGF are also regulated by limited
posttranslational cleavage of the larger isoforms (20). For example,
VEGF189 can be cleaved by the
urokinase-type plasminogen activator (uPA) to create a peptide that is
similar to VEGF165 in its
solubility, mitogenicity, and receptor binding (28). Both
VEGF189 and
VEGF165 can be cleaved by plasmin
to create a peptide that is similar to
VEGF121 in solubility and
mitogenicity (21). Settings with increased plasmin activity, such as
tissue injury, may favor conversion of ECM-bound isoforms to soluble isoforms that can affect more distant endothelial cells.
Little is known about the relative abundance of the different VEGF
splice variants in the normal lung or during development, injury, and
recovery. Whereas VEGF189 mRNA was
noted to be relatively abundant in the adult rat lung,
VEGF165 mRNA has the highest level of expression in most other rat tissues (2). We hypothesized that
during development or recovery from injury, i.e., times of active
endothelial cell proliferation, the messages for isoforms with high
mitogenicity or solubility
(VEGF165 and
VEGF121) would be most abundant.
Conversely, in the normal adult lung, we hypothesized that the messages
for the storage isoforms (VEGF189
and VEGF206) would be prevalent.
Our findings show that the relative proportion of
VEGF189 mRNA is greater in adult
lungs than in several other tissues. During lung development, the
relative proportion of VEGF165 declines significantly, whereas the proportion of
VEGF189 mRNA increases. The
relative proportion of VEGF189
mRNA declines significantly during hyperoxic injury in both newborn and
adult lungs but returns to control values during recovery. We also
found that immunoreactive VEGF protein in lung lavage fluid increases
up to 40-fold during recovery.
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MATERIALS AND METHODS |
Animals and hyperoxic exposure. The
use of animals for this study was approved by the University of
Rochester (Rochester, NY) Committee on Animal Resources. Newborn New
Zealand White rabbits were separated from their mothers within 24 h of
birth, placed in Plexiglas chambers, and exposed to either humidified
>95% oxygen or air at 5 l/min as previously described (23). Newborns
were maintained in hyperoxia for 2, 4, 6, 8, or 9 [50% lethal
dose (LD50)] days.
Additional newborns were exposed to >95% oxygen for 9 days and then
allowed to recover from the acute lung injury in 60% oxygen for 1, 3, 5, or 13 days. Attempts at recovering newborns in air resulted in high
mortality. Both the hyperoxia-exposed and age-matched control animals
were killed with an intraperitoneal injection of 200 mg/kg of
pentobarbital sodium.
Adult male New Zealand White rabbits were exposed to >95% oxygen in
Plexiglas chambers for up to 64 h and allowed to recover in air for up
to 7 days as previously described (24). The animals were killed with an
intravenous injection of pentobarbital sodium after 0, 24, 48, and 64 h
of oxygen exposure and at 1, 2, 3, 5, and 7 days of recovery.
Approximately 80% of the animals survived recovery in air.
Fetal rabbits were obtained by hysterotomy from timed-pregnant New
Zealand White rabbits at 22, 25, and 28 days gestation (term = 31 days). The lungs were removed and flash-frozen at
70°C for
later RNA isolation.
RNA and lavage preparation. In animals
used for RNA preparations, a thoracotomy was performed, the right main
stem bronchus was clamped, and the right lung was removed and
flash-frozen in liquid nitrogen. Samples of skin, kidney, and placenta
were also obtained from normal adult rabbits and flash-frozen. Total
RNA was isolated from each rabbit lung with the method of Chomczynski and Sacchi (7).
The lungs for lavage were exposed by thoracotomy, perfused in situ with
balanced salt solution (BSS), and removed. Newborn lungs were lavaged
with five aliquots of ice-cold BSS of sufficient volume to fully
distend the lung. Volume of the lavage fluid for newborns was 250 ml/kg, and absolute volumes varied between 5 and 40 ml depending on
weight. For each time point, age-matched control animals were done for
reference. Adult rabbits were lavaged with eight 50-ml aliquots of BSS.
The lavage fluids for each animal were then pooled, and the cells were
sedimented at 300 g for 6 min. The
lavage supernatants were stored at
70°C.
Alveolar type II cell isolation. Type
II cells from 4-day-old normal rabbits were isolated as previously
described (31). Briefly, the pulmonary vasculature was perfused in
situ, and the lungs were removed and lavaged with BBS. The lungs were
digested with protease solution (trypsin, DNase I, and elastase),
minced, and filtered to obtain a single cell suspension. Type II cells were purified on a discontinuous Percoll gradient and counted with a
hemocytometer, and viability was assessed by trypan blue exclusion.
Purity and viability were >90%. Cells were flash-frozen at
70°C for future RNA isolation.
RT-PCR amplification and cloning of rabbit VEGF
cDNA. cDNA was synthesized from total RNA with murine
leukemia virus reverse transcriptase and oligo
d(T)16 primers (GeneAmp RNA PCR
Kit, Perkin-Elmer Cetus, Norwalk, CT) according to the manufacturer's
instructions. Amplification for the purpose of cloning and sequencing
the coding region of the different splice variants was performed with
human VEGF-specific primers for the first and eighth exons: sense
primer 5'-TGGAT
AACTTTCTGCTGTCTT-3'
(including the underlined translation start site) and antisense primer
5'-CCTGGAAT
CCGCCTCGGCTTGTCAC-3' (including the underlined translation stop site). Restriction enzyme
sites for BamH I and
EcoR I were added to the primers for ease of cloning. Amplification was performed through 35 cycles (30 s at
94°C, 1 min at 59°C, and 1 min at 72°C). Amplified
sequences were cloned into pBluescript SK II(+) (Stratagene,
La Jolla, CA) and sequenced with the ABI Prism Dye Terminator Cycle
Sequencing Ready Reaction Kit (Perkin-Elmer Cetus).
Semiquantitative RT-PCR of VEGF splice
variants. RT-PCR was performed to determine the
relative proportions of each splice variant in different tissue and
cell samples. We used a sense primer located in exon 4 at nucleotide
355 from the translation start site
(5'-CAGTGAAT
AGATGAGCTTCCTACAGCAC-3')
and the same antisense primer as described in RT-PCR
amplification and cloning of rabbit VEGF
cDNA. PCR products from these primers were
smaller, but the relative size differences were maximized, resulting in improved separation on gel electrophoresis.
[32P]dCTP (2 × 106 dpm) was added to each PCR
reaction. To determine whether the relative amplification efficiency of
different VEGF splice variant cDNAs by PCR was equivalent, we amplified
with PCR three VEGF clones (coding for
VEGF189,
VEGF165, and
VEGF121) individually and mixed
in varying ratios. The three clones amplified with equal efficiency,
and the ratios of the amplified products reflected the starting ratios.
We therefore concluded that differences in the relative ratios of the
amplified splice variant products would reflect actual differences in
the relative ratios of the splice variant mRNAs. We also amplified
rabbit lung cDNA through 25, 30, and 35 cycles to determine whether
nearing an amplification plateau would have any effect on the relative
ratios of the splice variants present in a sample. The ratios remained
consistent through the different levels of amplification. In all
subsequent samples, we used 30 cycles of amplification. Amplified
products were electrophoresed on a denaturing 8 M urea-40%
formamide-5% polyacrylamide gel. Highly denaturing conditions were
found to be essential to prevent cross-hybridization among the splice
variants. The products were quantified by phosphorimaging and computer
image analysis (ImageQuant, Molecular Dynamics, Sunnyvale, CA). To
account for the differences in signal intensity due to size
differences, the relative amounts of the amplified splice variants were
converted to molar ratios. All semiquantitative RT-PCR data are
expressed in terms of relative molar proportions of the splice variants.
RNase protection assay. Antisense cRNA
probe was synthesized from the linearized DNA clone of rabbit
VEGF189 and labeled with [
-32P]UTP to a
specific activity of 6.7 × 108 dpm/µg. The length of the
probe sequence complementary to the VEGF189 mRNA was 649 bases. The
probe also included ~60 bp of nonhybridizing vector sequence.
Unlabeled sense RNAs were synthesized from the linearized DNA clones of
VEGF121,
VEGF165, and
VEGF189. These were used to check
the size of the RNase protection assay (RPA) products generated from
hybridization of the antisense
VEGF189 probe and the splice variants.
RPAs were performed according to the manufacturer's instructions (RPA
II Kit, Ambion, Austin, TX). Samples of total RNA (10 µg) from
newborn rabbit lungs were hybridized overnight to a molar excess
(105 dpm) of gel-purified,
full-length riboprobe. Negative controls with yeast tRNA and positive
controls with the synthesized sense strand RNAs were also hybridized to
a molar excess of probe. Single-strand RNA was removed by digestion
with the manufacturer's RNase A/T1 solution (1:500) for 30 min at
37°C. The RNA samples were then ethanol precipitated in the
manufacturer's buffer, denatured, and separated on a 5%
polyacrylamide-8 M urea denaturing gel. The gels were dried and
quantified by phosphorimaging and computer analysis. Molar ratios were calculated.
Enzyme-linked immunosorbent assay.
Lavage supernatants were tested for immunoreactive VEGF protein with a
commercial anti-human VEGF ELISA according to the manufacturer's
directions (Quantikine, R&D Systems, Minneapolis, MN). The antibodies
in the kit recognize all VEGF isoforms. The kit detected serial
dilutions of rabbit samples in a linear fashion. Rabbit VEGF results
are expressed as the picogram per milliliter value indicated from the
human VEGF standard curve generated by the assay. The lower limit of detection of the assay was 5 pg/ml. Samples that yielded values above
the standard range of the assay were diluted with Hanks' balanced salt
solution and remeasured.
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RESULTS |
Cloning and sequencing of rabbit VEGF splice
variants. RT-PCR amplification of RNA was used to
examine VEGF splice variants in rabbit lungs. For quantification of the
relative ratios of the splice variants, we used primers from exons 4 and 8, which are in all splice variants. Amplification yielded cDNAs of
predicted sizes that were consistent with alternative splicing of the
sixth and seventh exons (Fig.
1A).
To clone and sequence the coding regions of the splice variants, we
used primers from the first and eighth exons, which generated major
products of 648, 630, 576, and 444 bp in length. Sequencing showed the
cDNAs to be the coding regions of
VEGF189,
VEGF183,
VEGF165, and
VEGF121, respectively (Fig.
1B). Sequence analysis of the rabbit
VEGF189 cDNA revealed 94%
nucleotide and amino acid homology with human
VEGF189 (Fig. 1C). Similar to other species,
rabbit VEGF165 lacks the sixth of
eight exons spanning the coding region and
VEGF121 lacks the sixth and
seventh exons. Rabbit VEGF183 is
identical to VEGF189 except it is
lacking 18 bp at the 3'-end of exon 6. It is expressed at a low
level in the lung. A fifth, minor cDNA seen on RT-PCR (Fig.
1A) may represent the splice
variant VEGF145 as previously reported (19, 29). This product constituted ~5% of the total variants and did not appear to change with hyperoxia. Clones for this
minor cDNA were not isolated, and, therefore, we cannot confirm its
identity by sequence analysis. We detected no
VEGF206 mRNA, consistent with the
very restricted expression of this splice variant.

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Fig. 1.
Rabbit lung vascular endothelial growth factor (VEGF) splice variants
and nucleotide and deduced amino acid sequences of coding region of
rabbit 189-amino acid VEGF
(VEGF189).
A: RT-PCR amplification of rabbit lung
VEGF mRNA with primers from 4th and 8th exons yielded 4 primary splice
variant cDNAs of 314, 296, 242, and 110 bp, corresponding to
VEGF189,
VEGF183,
VEGF165, and
VEGF121, respectively. A low level
of a 5th variant was also detected at 182 bp. This product was ~5%
of the total and was not sequenced, but size is consistent with
VEGF145.
B: exon composition of VEGF splice
variants cloned from rabbit lung. Boxes, various exons not drawn to
scale. Nos. on top, length in bases.
C: sequence of coding region of rabbit
VEGF189. First amino acid of
signal peptide is numbered 26. First amino acid of mature
polypeptide is numbered +1. PCR primer sites (human) are underlined.
Vertical lines divide exons. Arrow, alternative splice site in exon 6 for VEGF183. Sequences for RT-PCR
bands corresponding to VEGF183,
VEGF165, and
VEGF121 were consistent with exon
sequences noted here.
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VEGF189 mRNA is more abundant in the lung
than in other rabbit tissues.
RT-PCR performed on total RNA from adult rabbit lung, liver, spleen,
and kidney demonstrated tissue-specific variations in the relative
proportions of the VEGF splice variants (Fig.
2). As noted in Cloning
and sequencing of rabbit VEGF splice variants, the
primary splice variants detected in the normal lung were
VEGF189, VEGF165, and
VEGF121. Because
VEGF189 is difficult to quantify apart from VEGF183 on a gel, we
have included both signals in quantifying
VEGF189. For the purpose of
studying changes in the bioavailability of VEGF, it is reasonable to
group the measurement of these variants because they contain both of
the heparin-binding regions present in the sixth and seventh exons.
Their bioavailability characteristics are therefore distinct from those
of VEGF165 and VEGF121. The relative percentage
of each splice variant present was determined with the sum of the
measured variants as the denominator. In the normal adult lung, the
proportion of total VEGF mRNA that was composed of
VEGF189 [41 ± 1% (SE)] was significantly greater than that in the liver,
spleen, or kidney, which had 16% or less VEGF189
(P < 0.001 by Student's
t-test with Bonferroni correction). In
contrast, the proportion of
VEGF165 was significantly less in
the lung than in the other tissues (39 ± 1 vs. 60-63%;
P < 0.001).

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Fig. 2.
RT-PCR amplification of rabbit RNA. A:
representative gel from RT-PCR of rabbit tissues. Lung (Lu), liver
(Li), spleen (S), and kidney (K) RNAs from adult rabbits were RT-PCR
amplified and run on a 5% polyacrylamide denaturing gel.
B: quantification of RT-PCR results
showing relative molar ratios of
VEGF189,
VEGF165, and
VEGF121 in each of the 4 tissues.
Tissue samples from 3 separate animals were analyzed. Data are means ± SE. Ratio of VEGF189 was
higher in lung than in other tissues.
VEGF189 mRNA comprised 41 ± 1% of total VEGF in lung, whereas in the other tissues,
VEGF189 averaged <16% of total
VEGF message. Difference in proportion of
VEGF189 between lung and each of
the other tissues was significant (P < 0.001 by Student's t-test with
Bonferroni correction for multiple comparisons).
VEGF165 was 39 ± 1% in lung
but 60% or greater in the other tissues
(P < 0.001). Proportion of
VEGF121 was 20-25% in all
tissues.
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Relative abundance of lung VEGF splice variants
changes during development. Lung VEGF mRNA increases
approximately threefold during gestation (Watkins and Maniscalco,
unpublished observations) and is abundant in newborns.
Alveolar epithelial type II cells are the primary VEGF-expressing cells
in newborn rabbits (23). To investigate further VEGF
expression in lung development, we measured the relative ratios of the
splice variants in fetal and newborn lungs. RNA from 22-, 25-, and 28-day fetal rabbit lungs (term = 31 days) and from 1-, 4- and 10-day-old newborn rabbit lungs was isolated and amplified by
RT-PCR (Fig. 3). The relative abundance of VEGF189 steadily
increased from 8 ± 1% at 22 days gestation to 40 ± 2% in
10-day-old newborns (P < 0.0001;
r2 = 0.84 by
linear regression). The proportion of
VEGF165 was high in the 22-day
fetal lung (70 ± 2%) and decreased significantly during
development, declining to 42 ± 5% by 10 days postnatal age
(P < 0.003;
r2 = 0.51 by
linear regression). There were no consistent changes in the proportion
of VEGF121 in lung development. In
RNA isolated from 4-day newborn type II cells, the molar ratios of the
splice variants were similar to those in the whole lung at this time. At 10 days postnatal age, the relative proportions of the VEGF splice
variants were similar to the values in the adult lung. Thus the
proportion of VEGF189 increased
fivefold from 22-day fetal lungs to 10-day-old newborn lungs, and
VEGF165 decreased proportionately.

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Fig. 3.
Proportion of VEGF189 message
increases as VEGF165 decreases
during lung development. A:
representative gels from RT-PCR of rabbit lung RNA at various fetal
and postnatal ages. d, Day. B:
representative RT-PCR comparing proportion of VEGF splice variants in
4d postnatal lung and type II (TII) cells isolated from 4d rabbits.
C: quantification of RT-PCR data. Data
are means ± SE from 2-3 animals/age. Data for TII cells are
from 2 separate preparations. At 22 days gestation, molar ratio of
VEGF189 was 8 ± 1% of total
VEGF mRNA, but proportion of this variant increased significantly to 40 ± 2% by 10d postnatal (P < 0.001; r2 = 0.84 by linear regression). Decrease in proportion of
VEGF165 from 70 ± 2% in 22d
fetal lung to 42 ± 5% at 10d of age was also significant
(P < 0.003). There were no
significant changes in molar ratio of
VEGF121. Ratio of splice variants
in 4d whole lung was very similar to that in TII cells from this age
animal. The 10d postnatal lung had splice variant ratios that are
similar to those in adult lung.
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VEGF189 variant differentially decreases
during hyperoxic lung injury in newborn rabbit and increases during
recovery.
After 9 days of exposure to >95% oxygen, newborn rabbit lungs
exhibit a decrease in total VEGF mRNA that is then reversed during 5 days of recovery (23). To determine whether hyperoxic injury also
changes the ratio of VEGF mRNA splice variants, we performed RT-PCR on
RNA from 9-day oxygen-exposed newborn rabbit lungs, age-matched control
lungs, and 1-, 3-, and 5-day recovered lungs. We found that the
proportion of VEGF189 to total
VEGF splice variant mRNA dropped from 38 ± 2 to 8 ± 1% with
exposure to hyperoxia (P < 0.005 by
Student's t-test). The proportions of
both VEGF165 and
VEGF121 increased significantly at
this time (P < 0.01). During recovery, a time of endothelial cell proliferation,
VEGF189 increased to 30 ± 2% of the total VEGF at 1 day and to 35 ± 1% at 3 days and reached the control value (39 ± 3%) at 5 days of recovery (Fig. 4). During this time, the proportions
of VEGF165 and
VEGF121 declined to control
values. Although we have not measured the absolute amounts of the VEGF
variants present, it is clear that with hyperoxic injury the level of
VEGF189 message declines
substantially because both the amount of total VEGF mRNA and the
proportion that is VEGF189
decrease.

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Fig. 4.
Ratio of VEGF189 to total VEGF
mRNA in newborn lung declines during hyperoxic injury and increases
during recovery. A: results of a
representative RT-PCR of rabbit lung RNA from control (C), 9d
hyperoxia-exposed (O2), and
newborn animals recovering in 60%
O2 for 1d (1dR), 3d (3dR), and 5d
(5dR). B: quantification of relative
molar ratios of VEGF189,
VEGF165, and
VEGF121 in C,
O2-injured, and recovering lungs.
Data are means ± SE for 3 animals/time point.
VEGF189 declined from 38 ± 2%
of total VEGF message in C animals to 8 ± 1% after 9d of hyperoxia
(P < 0.005 by Student's
t-test), whereas
VEGF165 and
VEGF121 increased significantly
(P < 0.01). Normal ratios were
reestablished during recovery.
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To confirm these results, we also performed RPAs to measure changes in
the splice variant ratios during hyperoxic exposure and recovery in
newborn rabbits. Antisense
32P-labeled
VEGF189 hybridized to total RNA
was digested with a mixture of RNases A and T1 at a dilution that
digested all single-stranded overhangs and loops but did not
significantly nick RNA opposite a loop site. It was anticipated that
the VEGF189 riboprobe would generate protected fragments of 649 bases when hybridized to
VEGF189 RNA, 477 and 154 bases
when hybridized to VEGF183, 423 and 154 bases when hybridized to
VEGF165, and 423 and 22 bases when
hybridized to VEGF121. To test our
digestion conditions and demonstrate that the anticipated fragments are
in fact generated, we did RPAs with an antisense
VEGF189 riboprobe and sense RNA
generated from three clones for
VEGF189,
VEGF165, and
VEGF121 (Fig.
5A). The
anticipated bands were seen, and RPA with a mixture of all three sense
RNAs gave the combination of bands expected.

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Fig. 5.
RNase protection assay (RPA) with
VEGF189 antisense probe confirms
decrease in VEGF189 message
relative to total VEGF mRNA during hyperoxic injury.
A: RPA was performed to assess banding
patterns generated by hybridization of antisense
VEGF189 probe to synthesized sense
RNA of VEGF121,
VEGF165, and
VEGF189. M, size markers in bp.
121, Bands of 22 (data not shown) and 423 bp of
VEGF121; 165, bands of 154 and 423 bp of VEGF165; 189, band of 649 bp
of VEGF189; Mix, hybridization to
all 3 primary splice variants yields main bands of 649, 423, and 154 bp. B: RPA with
VEGF189 probe and RNA from C,
O2-injured, and recovering newborn
lungs. Each lane represents a separate animal. Band at 649 bp indicates
amount of VEGF189 present. Band at
423 bp indicates amount of VEGF165
and VEGF121. Light band at 477 bp
indicates presence of a small amount of
VEGF183. Relative amount of
VEGF189 declined during
O2 injury and returned to C levels
during recovery.
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RPAs performed with RNA samples from newborn rabbit lungs demonstrated
a change in the ratios of the splice variants during injury and
recovery that closely matched the changes seen with RT-PCR (Fig.
5B). We compared the density of the
band at 649 bases (generated by
VEGF189) with that of the band
at 423 bases (generated by VEGF165
and VEGF121). Relative molar
percentages were calculated with the sum of the two densities as the
denominator after adjustment for the differences in band size. Scanning
densitometry indicated that the 649-base fragment was 35% of the total
density in control lungs but only 9% in the injured lungs. During
recovery, the molar percentage of
VEGF189 increased gradually from
24% at 1 day of recovery to 35% at 5 days of recovery. These data are
consistent with those obtained with RT-PCR.
Hyperoxic injury in the adult rabbit lung results in
changes in the VEGF variants similar to those seen in newborn hyperoxic lung injury. Adult rabbits were exposed to >95%
oxygen for 64 h and allowed to recover in room air for up to 7 days.
VEGF RNA from the lungs was amplified by RT-PCR and quantified (Fig.
6). VEGF189 mRNA comprised 41 ± 1% of the total VEGF present in control lungs, but after 64 h of
oxygen, the proportion of VEGF189
dropped to 20 ± 3% of the total VEGF
(P < 0.002 by Student's
t-test). The relative proportions of
both VEGF165 and
VEGF121 significantly increased at
this time (P < 0.05). During
recovery, VEGF189 increased from
16 ± 2% at 1 day to 39 ± 5% at 7 days. The decline in the proportion of VEGF189 and the
increase in VEGF165 and
VEGF121 during hyperoxic injury
are similar to changes in the ratios of the splice variants in the
newborn lung.

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Fig. 6.
Ratio of VEGF189 to total VEGF
mRNA decreases during hyperoxic injury in adult rabbit lung and
increases during recovery. A: results
from a representative RT-PCR of RNA from adult C, 64-h
O2-exposed, and 3d, 5d, and 7d
adult animals recovering in air. Note that this gel does not contain
samples from animals recovered for 1d.
B: quantification of RT-PCR data. Data
are means ± SE from 2-4 animals/time point.
VEGF189 as a molar percentage of
total VEGF message declined from 41 ± 1% in C animals to 20 ± 3% in hyperoxic animals (P < 0.002 by Student's t-test), whereas both
VEGF165 and
VEGF121 increased significantly
(P < 0.05). Values returned to
control level by 7dR.
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VEGF protein in lung lavage fluids increases during
recovery from hyperoxic injury. A previous study (23)
demonstrated by immunohistochemical analysis that VEGF protein in the
parenchyma of the newborn lung declined to nearly undetectable levels
during hyperoxic injury but reappeared during recovery, coincident with increased VEGF mRNA. Because lung VEGF is expressed primarily by
alveolar epithelial cells, we reasoned that it could be detected in
lung lavage fluid. An anti-human VEGF ELISA was used to measure immunoreactive VEGF protein in the lavage fluid of newborn and adult
lungs. The antibodies used recognized all isoforms of VEGF. Lavage
fluids of hyperoxic lungs yielded VEGF protein concentrations slightly
greater than those in control lungs (Fig.
7A). By
9 days of hyperoxia (LD50),
however, VEGF was barely detectable. During the first 5 days of
recovery, the lavage fluid VEGF increased 10-fold over age-matched
control levels. By 13 days of recovery, the level declined to the
control value. In adult rabbits, a similar pattern was observed (Fig.
7B). Low levels of VEGF protein were measured in the control lungs. During hyperoxic exposure, the VEGF
level rose approximately twofold. The lowest level of VEGF was seen at
1 day of recovery, but subsequent concentrations at 3 and 5 days of
recovery increased up to 40-fold over the control level. By 7 days of
recovery, the VEGF concentration decreased to just twofold above the
control value.

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|
Fig. 7.
VEGF protein in lung lavage fluid increases during recovery from
hyperoxic injury. A: concentrations of
immunoreactive VEGF were measured in C,
O2-injured, and recovering newborn
lung lavage fluids by ELISA. Data are means ± SE for 2-3
animals at each time point. Normalizing data to pg/g body wt gave very
similar results. Amount of VEGF in lavage fluid during injury increased
twofold compared with C level, dropped to barely detectable levels at
50% lethal dose time point, and increased by 8-fold compared with C
level during 1st 5 days of recovery. By 2 wk of recovery, VEGF
concentration returned to normal range. No major differences in
recovered lavage volumes were noted between
O2-exposed and age-matched control
animals. B: VEGF concentrations in
adult rabbit lung lavage fluid show a pattern of change during
hyperoxic injury and recovery similar to that seen in newborn lung.
However, drop in lavage fluid VEGF occurs more quickly in adult lung.
Data are means ± SE for 2-3 animals/time point. During
recovery, concentration of VEGF in adult lavage fluid increases
40-fold. No major differences were noted in recovered lavage volumes
between C and O2-exposed
animals.
|
|
 |
DISCUSSION |
The proliferation and migration of endothelial cells, the sprouting and
formation of new blood vessels, and the remodeling of extracellular
matrix characterize the complex process of angiogenesis, essential for
the development and repair of the lung. Several growth factors,
including basic fibroblast growth factor, transforming growth
factor-
, and VEGF, can modulate this process. VEGF is particularly
important because it is mitogenic mainly for endothelial cells.
Significant variation in VEGF mitogenicity, receptor affinity, and ECM
binding result from alternative splicing of the primary VEGF
transcript. Unlike several other organs, adult lungs had a
significantly greater proportion of
VEGF189 mRNA, which codes for a
highly ECM-bound isoform, whereas the other tissues had a significantly
greater proportion of VEGF165,
which codes for a more soluble isoform. In 22-day-gestation fetal
rabbit lungs, the proportion of
VEGF189 was low (8%), whereas
VEGF165 mRNA was highly expressed
(>70%). VEGF189 increased
significantly during fetal rabbit development, and
VEGF165 became less prominent.
With oxygen injury, the relative proportion of lung VEGF mRNA splice variants changed: VEGF189 mRNA
declined and the proportions of VEGF165 and
VEGF121 increased. Control values
were reestablished during recovery. Total VEGF protein in lung lavage
fluid increased up to 40-fold during recovery.
The sequences of VEGF splice variants found in the rabbit lung are
quite similar to those found in human mRNA. Rabbit
VEGF121, VEGF165, and
VEGF189 share ~94% homology
with their human counterparts. We also cloned a
VEGF183 message that lacks 18 bp
from the 3'-end of exon 6. The 5'-end of this 18-bp
fragment begins with GT, the consensus sequence for the 5' splice
donor necessary for splicing. Thus the variability of the 5'
donor splice site between exons 6 and 7 may generate several isoforms,
including VEGF183,
VEGF189, and
VEGF206.
VEGF183, although missing 18 bp,
still contains the heparin-binding site of exon 6. It seems likely,
therefore, that its affinity for heparan sulfate is high and that it
would be similar in its binding characteristics to
VEGF189. For this reason, we
included the contributions of
VEGF183 in our measurements of VEGF189.
Because the pulmonary endothelium in the normal adult lung has a low
proliferation rate, the production of VEGF by the normal lung suggests
that its role may be endothelial cell maintenance. The intact
VEGF189 isoform may be inactive as
a mitogen because of its inability to bind efficiently to the
high-affinity VEGF receptor Flk-1, which transmits the signals for
mitogenesis and chemotaxis (28).
VEGF189 is likely a storage form,
but it may be activated by proteolytic cleavage. Two serine proteases
that cleave and activate VEGF189
are uPA and plasmin (28). Although these proteases may be present in
the normal lung (17), it is not known whether stored
VEGF189 is released by proteolytic
cleavage in the normal lung or whether intact
VEGF189 has a function in the
maintenance of the pulmonary vasculature. Intact
VEGF189 binds to a second
high-affinity VEGF receptor, flt-1, which is present in endothelial
cells (28). The role of flt-1 is not clear, although it does not appear
to transduce mitogenic signals in endothelial cells (36).
Our findings that VEGF189
increases in lung development and is more abundant in the lung than in
several other tissues suggest that it may have a unique role in the
development and maintenance of the normal pulmonary endothelium. For
example, alveolar capillaries in the mature lung are directly subjacent
to the alveolar epithelial basement membrane, whereas the alveolar
capillaries in fetal lung are located centrally in the alveolar septa
(4). Increased expression of a highly ECM-bound VEGF such as
VEGF189 by developing alveolar
epithelial cells may regulate the spatial orientation of the microvasculature.
The mechanisms governing the changes we observed in the relative
amounts of the VEGF splice variants during lung development and injury
are unknown. Although different cell types may express different VEGF
splice variants (6) and organ-specific differences in the splice
variants exist, there are no data on the differences among cells within
an organ. It is possible that a change in the total VEGF mRNA level of
one cell type in the lung may result in a change in the ratio of VEGF
variants observed in the whole lung. For example, a previous study
(23) showed that VEGF-expressing cells, including neonatal
type II cells, were most prominent in the alveolar epithelium. The
present study showed that whole lung splice variant ratios were very
similar to type II cells from 4-day-old rabbits. It is possible that
the increase in whole lung VEGF189
during fetal development represents an increase in the number or
differentiation state of type II cells. Similarly, a shift in the
pattern of whole lung VEGF splice variant expression during lung injury
and recovery may reflect changes in the contribution of type II cells.
Because type II cells have increased VEGF expression during recovery
from hyperoxia (24), it is likely that these cells make a major
contribution to the splice variant patterns during recovery. It is
unlikely that our data resulted from dying animals because extremely
ill animals (cyanotic or lethargic) were culled and not used for analysis.
Another explanation for the changes in the splicing patterns of VEGF is
that extracellular and intracellular signals cause changes in VEGF
variant ratios within expressing cells. Molecules that play a role in
splicing are regulated in a variety of ways. For example, the activity
and selectivity of serine-arginine-rich (SR) proteins, which mediate
the selection and joining of splice sites, are influenced by
phosphorylation, concentration of pre-mRNA, regulator proteins, and RNA
enhancer regions (5). Although any of the SR proteins efficiently
splice constitutive splice sites (such as those in VEGF exons
1-5), specific SR proteins in concert with other factors determine
which variable splice sites (as in VEGF exons 6-8) are utilized.
It remains to be determined what factors cause the changes in the VEGF
splice variant ratios during development and injury.
The decline in total VEGF expression or a particular splice variant
during hyperoxic injury may contribute to the endothelial loss
associated with hyperoxic lung injury. For example, hyperoxia in the
rat retina results in decreased VEGF expression and the subsequent
apoptosis of endothelial cells (1). Intraocular addition of exogenous
VEGF before the hyperoxic period prevents the apoptosis. In vitro, VEGF
ameliorates the apoptotic effects of tumor necrosis factor-
on
endothelial cells (34). These data imply that VEGF may have a
maintenance function for endothelial cells. A decrease in normal VEGF
expression or a change in normal splice variant ratios may contribute
to endothelial cell loss in hyperoxic or inflammatory injury. It is not
known, however, whether different VEGF isoforms have different effects
on endothelial cell survival.
Our original hypothesis was that there would be an increase in the
proportion of mRNA for soluble VEGF isoforms during recovery. We
observed that the relative proportions of the VEGF mRNA splice variants
returned to control values in recovery. However, we also found that
total VEGF protein in lung lavage fluid increased up to 40-fold during
recovery. Although the relative proportions of the splice variants
returned to control values in recovery, the large increase in VEGF
protein suggests that both soluble and ECM-binding isoforms would be
highly abundant in recovering lung tissue. Another potential mechanism
that may increase VEGF solubility in recovery is proteolytic
processing. With injury and inflammation, proteases such as uPA and
plasmin are induced in endothelial cells, alveolar epithelial cells,
and activated macrophages (9, 17). Limited proteolysis results in the
conversion of VEGF189 and
VEGF165 to an active, soluble
isoform similar to VEGF121 (21,
28). Proof that VEGF is processed in lung injury, however, will require
further study.
A limitation of this study was the inability to verify that the VEGF
mRNA splice variants are translated into corresponding proteins. Cells
transfected with individual splice variant cDNAs express the
appropriate protein isoform in vitro (27), but correlation of splice
variant message abundance with isoform levels for organs or tissues in
vivo has not been reported. There are no data suggesting that any of
the splice variants are not translated or that they have differing
translational efficiencies. Because all VEGF isoforms can be
proteolytically processed to a smaller, non-heparin-binding isoform
(20, 28), determining the total amount of protein translated from a
splice variant mRNA would be very difficult, particularly in a setting
of plasmin activation. Such an analysis would require measurement of
both the intact isoform and any proteolytic products. Currently, there
is no method to trace the lineage of a cleavage product.
In summary, we found that the rabbit VEGF splice variant sequences are
quite similar to human VEGF variants. An additional variant,
VEGF183, was identified. We found
that the lung, unlike some other tissues, had
VEGF189 as a major splice variant.
The proportions of VEGF mRNA splice variants changed in lung
development: VEGF189 increased
significantly, whereas VEGF165
declined. In hyperoxic injury of both newborn and adult rabbits, the
relative proportion of VEGF189
decreased, whereas that of VEGF165
increased. Normal proportions were reestablished in recovery. VEGF
protein levels in lung lavage fluid from recovering animals increased manyfold over the levels in control animals. These results are suggestive of differing and flexible roles for VEGF splice variants in
the development, maintenance, and repair of the lung. The precise roles
of the VEGF splice variants in developing and injured tissues, however,
remain to be clarified.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge the expert technical work of Michael
LoMonaco and Anna Paxhia.
 |
FOOTNOTES |
This work was supported by National Heart, Lung, and Blood Institute
(NHLBI) Specialized Center of Research (SCOR) Grant HL-36543 (to W. M. Maniscalco); NHLBI Grant RO1-HL-54632 (to W. M. Maniscalco); NHLBI National Research Service Award 5F32-HL-09022 (to C. T. D'Angio); a March of Dimes grant (to R. M. Ryan); and NHLBI Clinical Investigator Award HL-02630 (to R. M. Ryan).
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. §1734 solely to indicate this fact.
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).
Received 23 March 1998; accepted in final form 25 January 1999.
 |
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