Expression of VEGF and its receptors Flt-1 and Flk-1/KDR is altered in lambs with increased pulmonary blood flow and pulmonary hypertension
Eugenia Mata-Greenwood,1
Barbara Meyrick,2
Scott J. Soifer,3,4
Jeffrey R. Fineman,3,4 and
Stephen M. Black1,5
Departments of 1Pediatrics and
5Molecular Pharmacology, Northwestern University,
Chicago, Illinois 60611-3008; 2Department of
Pathology, Vanderbilt University Medical Center, Nashville, Tennessee
37232-2650; and 3Department of Pediatrics and
4Cardiovascular Research Institute, University of
California San Francisco, San Francisco, California 94143-0106
Submitted 15 November 2002
; accepted in final form 24 March 2003
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ABSTRACT
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Utilizing in utero aortopulmonary vascular graft placement, we developed a
lamb model of congenital heart disease and increased pulmonary blood flow. We
showed previously that these lambs have increased pulmonary vessel number at 4
wk of age. To determine whether this was associated with alterations in VEGF
signaling, we investigated vascular changes in expression of VEGF and its
receptors, Flt-1 and KDR/Flk-1, in the lungs of shunted and age-matched
control lambs during the first 8 wk of life. Western blot analysis
demonstrated that VEGF, Flt-1, and KDR/Flk-1 expression was higher in shunted
lambs. VEGF and Flt-1 expression was increased at 4 and 8 wk of age
(P <0.05). However, KDR/Flk-1 expression was higher in shunted
lambs only at 1 and 4 wk of age (P <0.05). Immunohistochemical
analysis demonstrated that, in control and shunted lambs, VEGF localized to
the smooth muscle layer of vessels and airways and to the pulmonary epithelium
while increased VEGF expression was localized to the smooth muscle layer of
thickened media in remodeled vessels in shunted lambs. VEGF receptors were
localized exclusively in the endothelium of pulmonary vessels. Flt-1 was
increased in the endothelium of small pulmonary arteries in shunted animals at
4 and 8 wk of age, whereas KDR/Flk-1 was increased in small pulmonary arteries
at 1 and 4 wk of age. Our data suggest that increased pulmonary blood flow
upregulates expression of VEGF and its receptors, and this may be important in
development of the vascular remodeling in shunted lambs.
vascular graft placement; shunted lambs
VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) is a cell-specific
angiogenic and vasculogenic mediator
(11,
29). It is observed
ubiquitously at sites of angiogenesis, and its levels correlate closely with
the spatial and temporal events of blood vessel growth
(36). Some of the most
prominent roles of VEGF include induction of endothelial cell proliferation
and migration, induction of endothelial expression of proteases, stimulation
of microvascular leakage, and increase in nascent endothelial cell survival
(9,
11,
29). These effects are
mediated by two transmembrane tyrosine kinase endothelial-specific receptors:
fms-like tyrosine kinase-1 (Flt-1) and kinase insert domain-containing
receptor/fetal liver kinase-1 (KDR/Flk-1)
(9,
11,
29). Molecular cloning of the
cDNAs for VEGF have revealed that alternative mRNA splicing results in the
generation of at least six isoforms that vary between 120 and 205 amino acids
in length (19,
38).
Recent evidence has demonstrated alterations in VEGF expression in a
variety of pulmonary hypertensive disorders. For example, increased VEGF
expression has been reported in newborns with congenital diaphragmatic hernia
and pulmonary hypertension
(34), and increased VEGF
protein has been reported in the tracheal aspirates and type II pneumocytes of
neonates with persistent pulmonary hypertension (PPHN)
(23). Similarly, increased
VEGF expression is reported in the lungs of adults with advanced pulmonary
vascular disease secondary to congenital heart disease, with more pronounced
expression in areas of advanced plexiform lesions
(13,
17,
39). These observations
correlate VEGF dysregulation with altered endothelial phenotype and abnormal
vascularization. In addition, various clinical and in vivo studies on VEGF
expression in models of increased blood flow have been reported. For instance,
increased blood flow induced by bradycardia, or in response to exercise,
induces a temporary increase in VEGF mRNA expression and protein secretion
(24,
40). In vitro studies also
support the concept that increased biomechanical forces, such as cyclic
stretch and laminar shear stress, lead to increased VEGF expression
(12,
15,
31,
37).
We have developed an animal model of pulmonary hypertension by inserting an
aortopulmonary vascular graft in the late-gestational fetal lamb
(3,
4,
28). Postnatally, these lambs
have increased pulmonary blood flow and pressure. In addition, they display
pulmonary vascular remodeling characterized by increased medial wall thickness
of the small muscular pulmonary arteries and abnormal extension of muscle to
peripheral pulmonary arteries
(18,
27,
28). Last, at 4 wk of age,
these lambs have a transient increase in the number of barium-filled
intra-acinar pulmonary arteries, which may represent an early adaptive
angiogenesis and/or vessel recruitment
(28). We hypothesized that
increased VEGF expression participates in pulmonary vascular remodeling
secondary to increased pulmonary blood flow. Therefore, in the present study,
we investigated the developmental changes in expression of VEGF and its two
main receptors: Flt-1 and KDR/Flk-1 in 1-day-old and 1-, 4-, and 8-wk-old
lambs with increased pulmonary blood flow.
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METHODS
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Surgical preparation and care. Forty mixed-breed, pregnant Western
ewes (137141 days gestation, term = 145 days) were operated on under
sterile conditions as previously described
(28). After spontaneous
delivery of the lambs (27 days after surgery), antibiotics
[106 units of penicillin G potassium and 25 mg of gentamicin
sulfate intramuscularly (im)] were administered for 2 days. The lambs were
weighed daily, and respiratory rate and heart rate were measured. Furosemide
(1 mg/kg im) was administered daily, and elemental iron (50 mg im) was given
weekly. At 1 day and at 1, 4, or 8 wk of age, the lambs were anesthetized with
intravenous infusions of ketamine hydrochloride (
1
mg·kg-1·min-1) and diazepam (0.002 mg
· kg-1 · h-1), intubated with a 5.5-mm
outer diameter endotracheal tube, and mechanically ventilated with a
pediatric, time-cycled, pressure-limited ventilator (Healthdyne, Marietta,
GA). Ventilation with 21% O2 was adjusted to maintain an arterial
PCO2 (PaCO2) between 35
and 45 Torr. Via a midsternotomy incision, the lambs were instrumented to
measure pulmonary and systemic arterial pressure, right and left atrial
pressure, heart rate, left pulmonary blood flow, and oxygen saturations as
previously described (28).
Then, at 1 day and at 1, 4, or 8 wk after birth, the lambs were euthanized
with an intravenous injection of pentobarbital sodium (Euthanasia CII; Central
City Medical, Union City, CA) and subjected to bilateral thoracotomy. An
autopsy was performed to confirm patency of the vascular graft. The lungs were
removed and prepared for RNA preparation, Western blot analysis, and
immunohistochemistry. All procedures and protocols were approved by the
Committee on Animal Research of the University of California, San Francisco
and Northwestern University.
Tissue preparation for immunohistochemistry. The heart and lungs
were removed en bloc. The lungs were dissected with care to preserve the
integrity of the vascular endothelium. Sections (23 g) from each lobe
of the lung were removed. These tissues were snap-frozen in liquid
N2 and stored at -70°C until analysis.
For immunohistochemistry, the pulmonary vascular tree was rinsed with cold
(4°C) PBS to remove blood and was fixed by perfusion with cold (4°C)
4% paraformaldehyde. The pulmonary artery was then clamped. The airways were
fixed at 20 cm of H2O pressure by filling the trachea with cold
(4°C) 4% paraformaldehyde. When the lungs were distended at this pressure,
the trachea was clamped. The lungs were fixed for 24 h at 4°C by immersion
in 4% paraformaldehyde. Representative slices from each lobe were removed,
placed in 30% sucrose until they sank, placed in optimum cutting temperature
compound, frozen on dry ice, and stored at -70°C until they were
sectioned. Five- to ten-micrometer sections were cut using a cryostat,
transferred to aminoalkylsilane-treated slides (Superfrost Plus; Fisher
Scientific, Santa Clara, CA), and stored at -70°C
(5).
Generation of an ovine VEGF antiserum. The ovine VEGF (oVEGF)
protein sequence differs greatly from human VEGF. Therefore, an oVEGF-specific
antiserum was prepared by injecting rabbits with a highly antigenic protein
fragment corresponding to oVEGF120 (sequence:
NH2-GCRIKPHQSQHIGEMSFLQHNK-COOH). Rabbits were bled at 6, 8, and 10
wk, and the 8-wk bled was immunopurified (BioSynthesis, Louisville, TX). The
specificity of the antiserum was assessed by Western blot analysis using
positive control generated from COS-7 cells transfected with an oVEGF-pCDNA3
construct. The cell media provided a secreted protein around 25 kDa. The
rabbit anti-oVEGF sera did not cross-react with human VEGF.
Western blot analysis. Immunoblotting was performed as previously
described (3,
4). Protein extracts (100
µg) were separated on 420% (VEGF) and 7.5% (for VEGF receptors Flt-1
and KDR/Flk-1) SDS-polyacrylamide gel and electrophoretically transferred to
polyvinylidene difluoride membranes (Amersham, Arlington Heights, IL). The
membranes were blocked with 5% nonfat dry milk in Tris-buffered saline
containing 0.1% Tween 20 (TBS-T). After 1 h of blocking, the membranes were
incubated at 4°C for two consecutive days with 1:500 (
3.4 µg/ml)
dilution of rabbit polyclonal anti-oVEGF in blocking solution. Alternatively,
membranes were probed with goat polyclonal antibodies against rabbit
polyclonal antibody against mouse Flt-1 (2 µg/ml, Santa Cruz Laboratories)
and goat polyclonal antibody against human KDR/Flk-1 (1 µg/ml, R&D
Systems) for 2 h at room temperature. Membranes were washed 3 x 15 min
with TBS-T and then hybridized with anti-rabbit horseradish peroxidase
antibody for 45 min. After 3 x 15 min washes, bands were visualized with
chemiluminescence using a Kodak Digital Science Image Station (NEN, Boston,
MA). The following size bands were obtained: 25 kDa for monomeric VEGF,
200220 kDa for KDR/Flk-1, and 130 and 180 kDa for Flt-1. For Flt-1, the
130-kDa band was found to be nonspecific.
To compare the various protein levels obtained from controls and shunts
(n = 5, total of 10 samples per age) of various ages (1-day-old and
1-, 4-, and 8-wk-old, total of 40 samples), four different gels were run (1
for each age containing protein samples from both control and shunted lambs).
Also, included on each gel was an internal control (a lung extract prepared
from a 1-day-old shunted lamb). Each of the four membranes was probed for the
particular protein of interest and then reprobed the next day for
-actin. Each densitometric value was divided by its
-actin control
to obtain a relative value for VEGF, Flt-1, and Flk-1 protein levels. Relative
values were averaged and then corrected using a factor (relative protein level
of internal control obtained from that particular blot divided by relative
protein level of internal control from the original day 1 membrane).
In this way, differences in time exposure leading to different protein levels
were observed and corrected for. Standard deviations were also corrected by
using the same factor.
Immunohistochemistry. Immunohistochemistry was performed as
previously described (3,
4). Studies were done on serial
sections of control and shunted ovine lung using rabbit anti-oVEGF sera
(BioSynthesis) and rabbit polyclonal anti-Flt-1 or goat polyclonal
anti-KDR/Flk-1 (Santa Cruz Laboratories). Frozen tissue sections (7 µm)
were thawed at room temperature. Samples were fixed for 10 min in cold acetone
and then washed 3x with PBS. To eliminate nonspecific binding of the
primary antiserum to tissue proteins, tissue sections were incubated with 1%
horse serum in PBS (blocking solution) for 1 h. Tissue sections were then
incubated with anti-oVEGF (1:100), anti-human KDR/Flk-1, or Flt-1 (5
µg/ml), in the presence of monoclonal smooth muscle cell-actin antibody
(1:400, Sigma) in blocking solution at 4°C overnight. After three washes
with PBS x 5 min, samples were visualized with Rhodamine Red-X goat
anti-rabbit and Oregon Green 488 goat anti-mouse secondary antibodies
(Molecular Probes) at a concentration of 1:400 in blocking solution for 45 min
at room temperature. After three further washes with PBS, an anti-fading
solution was added, and samples were visualized by fluorescence microscopy.
For each tissue section, a parallel experiment was carried out in which the
primary antibody was omitted. This served as the negative control. A minimum
of three different sets of control and shunt lung tissues were prepared and
examined. Because it is difficult to utilize immunohistochemistry for
qualitative measurements on protein expression, we used this technique only to
determine protein localization and to determine whether there were differences
between shunt and control lambs in the numbers of vessels expressing VEGF,
KDR/Flk-1, and Flt-1. To carry out this procedure, small muscularized
pulmonary arteries were visualized next to airways, and at least 30 vessels
(representing at least 10 fields) were counted as positively immunoreactive or
nonimmunoreactive for a particular protein. The number of vessels
immunoreactive for each protein as a proportion of the total vessels counted
was determined. Results were then calculated as the average number of
positively stained vessels ± SD (n = at least 3 different
lambs from each age group), and statistical significance was calculated as
described below.
Determination of circulating plasma VEGF levels. Because of
inherent differences between human VEGF and oVEGF, the commercially available
ELISA does not adequately detect oVEGF. Thus we developed a competitive ELISA
that specifically detects oVEGF. This ELISA uses a rabbit polyclonal antibody
against oVEGF that was synthesized using the sequence corresponding to
oVEGF120 (NH2-GCRIKPHQSQHIGEMSFLQHNK-COOH) as the
capture antibody (10 ng/ml in PBS). The methodology for measuring oVEGF is as
follows: a 96-well plate (Costar) is coated with 100 µl of capture antibody
at room temperature overnight. The plate is then washed three times with 0.05%
Tween in PBS and blocked with 300 µl/well of 5% sucrose and 1% BSA in PBS
for 1 h. The blocking solution is then aspirated and the plates washed as in
PBS/Tween. For the assay, plasma (150 µl) or various dilutions of standard
oVEGF (0.032 µg) are added together with biotinylated oVEGF (50
µl at 200 ng/ml) and allowed to bind to the capture antibody for 2 h at
room temperature. The wells are then aspirated and washed in PBS/Tween, and
streptavidin-horseradish peroxidase (100 µl, R&D Systems) is added for
20 min. The wells are then aspirated and washed, and a detection solution for
biotin (100 µl of tetramethylbenzidine, Sigma) is used to detect bound
biotinylated oVEGF. After 20 min, a stop solution of 2N
H2SO4 (50 µl) is used, and the plate is read at 450
nm. Unknown values for VEGF are then calculated with the aid of Tablecurve
software using the standard curve for known amounts of oVEGF. Cross-reaction
with human VEGF was found to be 20%, <1% with transforming growth factor
(TGF)-
1 and nerve growth factor, and <5% with basic fibroblast growth
factor (bFGF).
Data analysis. Quantitation of protein expression was performed
using a Kodak Image Station 440CF and KDS1D imaging software. This allows a
pixel density from 1103 instead of 256 gray scale of
autoradiographic film. The response is linear within this range.
In all experiments, means ± SE were calculated, and comparisons
between control and shunted lambs were made by ANOVA. When differences were
present among study groups, Student-Newman-Keuls post hoc testing was
performed. P < 0.05 was considered statistically significant.
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RESULTS
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Analysis of VEGF protein expression by immunoblotting in the lungs of
normal lambs (controls) showed a progressive decline: 1-wk-olds expressed
0.44, 4-wk-olds expressed 0.25, and 8-wk-olds expressed 0.3 of day 1
values (Fig. 1). In the shunt
model, VEGF protein expression was equivalent to controls at 1 day and 1 wk.
However, VEGF expression was significantly increased in the peripheral lung
tissue of shunted lamb lung at 4 and 8 wk of age (459% and 149%, P
< 0.05, Fig. 1, A and
B). Analysis of VEGF mRNA by RNase protection assays
showed a similar pattern, where VEGF mRNA was significantly increased in
shunted lambs at 4 and 8 wk of age (147% and 98% of controls, respectively,
P < 0.05) but not at either 1 day or 1 wk of age
(Fig. 1, C and
D). Analysis of circulating levels of VEGF in plasma
samples from 1- to 8-wk-old control and shunted lambs did not reveal any
significant increases compared with controls (data not shown).
Immunohistochemical analysis of VEGF in conjunction with smooth muscle actin
revealed that VEGF was highly expressed in the epithelium and smooth muscle
layer of airways from control and shunted lambs. However, 4- and 8-wk-old
shunted lambs showed an increased VEGF expression in the smooth muscle layer
of small pulmonary vessels, especially in those possessing a thickened media
(Fig. 2, AH).
There was no major change in expression or localization of VEGF at 1 day and 1
wk between shunted and control lambs.

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Fig. 1. Western blot and RNase protection analyses for vascular endothelial growth
factor (VEGF) in peripheral lung tissue from 1-day-old and 1-, 4-, and
8-wk-old lambs (control, and after insertion of an aorta-to-pulmonary artery
vascular graft in utero, shunt). A: representative Western blots are
shown from protein extracts (100 µg) prepared from lung tissue from
1-day-old and 1-, 4-, and 8-wk-old lambs (1 control and 1 shunt from each
age), separated on a 420% SDS-polyacrylamide gradient gel,
electrophoretically transferred to Hybond membranes, and analyzed using a
specific antiserum raised against VEGF. VEGF protein expression was increased
in shunted lambs only at 4 and 8 wk. The band shown for VEGF is 25 kDa and
represents the monomeric form of VEGF. Also included on each gel was an
internal control [peripheral lung extract (50 µg) prepared from a 1-day-old
shunt]. Each membrane was also reprobed for -actin to normalize for
differences in protein loading. Con, control; Sh, shunt. B:
densitometric values for relative VEGF protein from 5 control and 5 shunted
lambs at each age was determined as described in METHODS. In
shunted lambs, relative VEGF protein is increased by 459% at 4 wk and 149% at
8 wk (P < 0.05). Values are means ± SE.
*P <0.05 for control vs. shunt; P
<0.05 vs. 1 day control. C: representative RNase protection assays
are shown from a cRNA probe for ovine VEGF that was hybridized overnight to 50
µg of total lung RNA prepared from 1-day-old and 1-, 4-, and 8-wk-old lambs
(1 control and 1 shunt from each age). VEGF mRNA expression was increased in
shunted lambs only at 4 and 8 wk. There were no protected fragments detected
in the lanes where the probe was hybridized without RNA (PA) or in the
presence of tRNA. VEGF is undigested probe. A cRNA for ovine 18S was also
hybridized to serve as a control for RNA loading. D: densitometric
values for relative VEGF mRNA (normalized to 18S mRNA and to control values)
from 5 control and 5 shunted lambs at each age. In shunted lambs, relative
VEGF mRNA increased by 147% at 4 wk and 98% at 8 wk (P <0.05).
Values are means ± SE. *P <0.05 control vs.
shunt.
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Fig. 2. VEGF protein expression in vivo in lung sections prepared from 1-day-old
and 1-, 4-, and 8-wk-old lambs (control, and after insertion of an
aorta-to-pulmonary artery vascular graft in utero, shunt). Immunohistochemical
localization of VEGF expression in the lung in vivo from 1-day-old (A
and B), 1-wk-old (C and D), 4-wk-old (E
and F), and 8-wk-old (G and H) lambs: control
(A, C, E, G) and shunt (B, D, F, H). Polyclonal rabbit
anti-VEGF antiserum and monoclonal mouse anti-smooth muscle cell-actin
antibody were used to localize expression of VEGF. VEGF expression is shown in
red, whereas smooth muscle cell-actin expression is shown in green.
Colocalization is shown in yellow. Magnification is x200. Micrographs
shown are representative of at least 3 different sets of twin matches (control
and shunt). AW, airway; V, vessel. Smooth muscle layers from pulmonary
arteries from 4- and 8-wk-old shunts, but not controls, show intense VEGF
staining.
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VEGF signals through two tyrosine kinase membrane receptors: KDR/Flk-1 and
Flt-1. Therefore, we analyzed the changes in protein expression of KDR/Flk-1
by Western blotting and immunohistochemistry. Its pattern of expression in the
lungs of control lambs correlated well with that of VEGF where expression was
higher at 1 day, then dropped to 25% at 1 wk, then 9% at 4 wk, and finally 3%
at 8 wk of age compared with 1-day average values
(Fig. 3, P < 0.05
relative to day 1 values). Relative to average values of control
lambs, KDR/Flk-1 protein expression was significantly increased in the lungs
of shunt animals at 1 wk and 4 wk of age (397% and 88%, respectively,
P < 0.05, Fig. 3)
and nonsignificantly decreased at 8 wk of age (57% of control values,
Fig. 3). This is of interest
since the increase in this receptor preceded the increase in VEGF expression.
It is important to note that although VEGF expression in the shunt model
showed a biphasic pattern (with 2 peaks of expression), KDR/Flk-1 expression
in the shunt showed a delayed decline in values (Figs.
1 and
3). Immunohistochemistry
studies showed that KDR/Flk-1 was present exclusively in the endothelium of
small vessels (diameter <200 µm) and in capillaries (diameter <10
µm) of both control and shunted lambs
(Fig. 4). Compared with control
samples, shunt lung sections showed a nonsignificant increase in KDR/Flk-1
expression in the endothelium of small arteries at 1 wk (62.6 ± 11.6%
positively stained vessels in the shunt compared with 29.6 ± 7.9% in
control, P = 0.087). At 4 wk of age, shunt lung sections showed a
significant increase in KDR/Flk-1 expression in the endothelium of small
arteries (52.3 ± 10.2% stained vessels in the shunt compared with 16
± 1.2% in control, P < 0.05;
Fig. 4, AH). In
addition, there were more capillaries positively stained with KDR/Flk-1 in the
4-wk-old samples compared with control samples
(Fig. 4,
AH).

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Fig. 3. Western blot analysis for kinase insert domain-containing receptor/fetal
liver kinase-1 (KDR/Flk-1) in peripheral lung tissue from 1-day-old and 1-,
4-, and 8-wk-old lambs (control, and after insertion of an aorta-to-pulmonary
artery vascular graft in utero, shunt). A: representative Western
blots are shown from protein extracts (100 µg) prepared from lung tissue
from 1-day-old and 1-, 4-, and 8-wk-old lambs (1 control and 1 shunt from each
age), separated on a 7.5% SDS-polyacrylamide gradient gel, electrophoretically
transferred to Hybond membranes, and analyzed using a goat polyclonal
anti-human KDR/Flk-1. KDR/Flk-1 protein expression was increased in shunted
lambs only at 1 and 4 wk. Internal controls were used to compare relative
amounts among the developmental ages. B: densitometric values for
relative KDR/Flk-1 protein from 5 control and 5 shunted lambs at each age. In
shunted lambs, relative KDR/Flk-1 protein is increased by 397% at 1 wk and 88%
at 4 wk (P < 0.05). Values are means ± SE.
*P <0.05 for control vs. shunt; P <
0.05 vs. 1-day control.
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Fig. 4. KDR/Flk-1 protein expression in vivo in lung sections prepared from
1-day-old and 1-, 4-, and 8-wk-old lambs (control, and after insertion of an
aorta-to-pulmonary artery vascular graft in utero, shunt). Immunohistochemical
localization of KDR/Flk-1 expression in the lung in vivo from 1-day-old
(A and B), 1-wk-old (C and D), 4-wk-old
(E and F), and 8-wk-old (G and H) lambs:
control (A, C, E, G) and shunt (B, D, F, H). Polyclonal goat
anti-KDR/Flk-1 antibody and monoclonal mouse anti-smooth muscle cell-actin
antibody were used to localize expression of KDR/Flk-1. KDR/Flk-1 expression
is shown in red, whereas smooth muscle cell-actin expression is shown in
green. Colocalization is shown in yellow. Magnification is x200.
Micrographs shown are representative of at least 3 different sets of twin
matches (control and shunt). Arrows, pulmonary arteries. Pulmonary arteries
from 1- and 4-wk-old shunts, but not controls, show intense KDR/Flk-1 staining
in the endothelium of pulmonary arteries.
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In contrast with the normal developmental pattern of VEGF and KDR/Flk-1
expression, Flt-1 (VEGF-R1) protein expression in the normal lamb increased
with age: 1-, 4-, and 8-wk-old average protein levels were 58, 49, and 174%
higher than 1-day-old controls, respectively
(Fig. 5). Compared with control
values, average protein levels of Flt-1 in the shunt model were significantly
elevated only at 4 wk of age (157% higher than control values, P <
0.05, Fig. 5). However, Flt-1
expression was nonsignificantly elevated at 1 and 8 wk of age (23 and 27%
higher than control values, respectively,
Fig. 5). Immunohistochemical
analysis demonstrated that Flt-1 is abundantly expressed in the endothelium of
pulmonary vessels (both veins and arteries) and of capillaries, and expression
was found to be highest in both 8-wk-old control and shunted lambs. In
contrast with the normal developmental pattern, expression of Flt-1 was
elevated in medium- to small-sized pulmonary vessels in the 4-wk-old shunt
(63.4 ± 6.7% stained vessels in the shunt compared with 30.7 ±
3% in controls, P < 0.05) but was similar to control lambs in
microvessels (Fig. 6,
AH).

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Fig. 5. Western blot analysis for fms-like tyrosine kinase (Flt-1) in peripheral
lung tissue from 1-day-old and 1-, 4-, and 8-wk-old lambs (control, and after
insertion of an aorta-to-pulmonary artery vascular graft in utero, shunt).
A: representative Western blots are shown from protein extracts (100
µg) prepared from lung tissue from 1-day-old and 1-, 4-, and 8-wk-old lambs
(1 control and 1 shunt from each age), separated on a 7.5% SDS-polyacrylamide
gradient gel, electrophoretically transferred to Hybond membranes, and
analyzed using a specific antiserum raised against Flt-1. The band shown for
Flt-1 is 180 kDa as the 130-kDa band was nonspecific. Flt-1 protein expression
was increased in shunted lambs only at 4 wk. B: average densitometric
values for Flt-1/ -actin protein from 5 control and 5 shunted lambs at
each age. In shunted lambs, relative Flt-1 protein is increased by 157% at 4
wk (P < 0.05). Values are means ± SE.
*P < 0.05 for control vs. shunt; P
< 0.05 vs. 1-day control.
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Fig. 6. Flt-1 protein expression in vivo in lung sections prepared from 1-day-old
and 1-, 4-, and 8-wk-old lambs (control, and after insertion of an
aorta-to-pulmonary artery vascular graft in utero, shunt). Immunohistochemical
localization of Flt-1 expression in the lung in vivo from 1-day-old
(A and B), 1-wk-old (C and D), 4-wk-old
(E and F), and 8-wk-old (G and H) lambs:
control (A, C, E, G) and shunt (B, D, F, H). Polyclonal
rabbit anti-Flt-1 antibody and monoclonal mouse anti-smooth muscle cell-actin
antibody were used to localize expression of Flt-1. Flt-1 expression is shown
in red, whereas smooth muscle cell-actin expression is shown in green.
Colocalization is shown in yellow. Magnification is x200. Micrographs
shown are representative of at least 3 different sets of twin matches (control
and shunt). Arrows, pulmonary arteries. Four-week-old pulmonary arteries from
shunt, but not controls, show intense Flt-1 staining.
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DISCUSSION
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In an effort to begin to unravel the molecular mechanisms involved in the
process of vascular remodeling due to increased pulmonary blood flow and/or
pressure, we investigated the expression of VEGF and its two main receptors,
KDR/Flk-1 and Flt-1, in our lamb model of pulmonary hypertension with
increased pulmonary blood flow. We observed that VEGF and its receptors are
upregulated in the pulmonary circulation of shunted lambs compared with
age-matched controls. The increase in VEGF expression observed in the shunt
model correlated with an increase in blood vessel number, as we have reported
previously (28). This
observation is in accordance with the potent mitogenic effect of VEGF on
endothelial cells (9,
11). The angiogenic process
involves endothelial proliferation and migration, matrix degradation and
invasion, and capillary/vessel morphogenesis
(25). VEGF has been shown to
participate in this process mainly in the initial steps of inducing
endothelial proliferation and migrations
(25). VEGF has been shown to
synergize with bFGF, which has a role in endothelial cell reorganization and
vessel morphogenesis, i.e., in late angiogenesis
(8). Tissue invasion is
achieved, in part, by an increase in extracellular matrix proteases like
tissue plasminogen activator, urokinase plasminogen activator and its
receptor, and plasmin (8,
25). VEGF upregulates the
expression of these proteases at the transcriptional level
(8). In addition, VEGF also
possesses other roles involved in an adaptive response of the vascular system
to an insult. For instance, VEGF has been shown to stimulate endothelial
recovery from injury (1,
21) and to stimulate
endothelial nitric oxide synthase expression and activity leading to
vasodilation (35). Therefore,
upregulation of VEGF might be an adaptive response to increased blood
flow.
Several clinical and in vivo studies have shown a transient increase in
local and circulating VEGF levels in models of increased blood flow, as occurs
in response to increased exercise, pregnancy, and induced bradycardia
(24,
40). In addition, increased
local expression of VEGF and KDR/Flk-1 has been observed in vascular lesions
occurring in various pulmonary hypertensive disorders
(23,
39). For instance, increased
VEGF expression has been observed in the tracheal aspirates and type II
pneumocytes of neonates with PPHN
(23). Similarly, VEGF levels
are increased in the lungs of newborns with congenital diaphragmatic hernia
and pulmonary hypertension
(34) and in the lungs of
adults with advanced pulmonary vascular disease secondary to congenital heart
disease (13,
39). Our studies, using a
model of increased pulmonary blood flow, suggest that selective upregulation
of VEGF and its receptors in small pulmonary arteries indicates that
dysregulation of this angiogenesis leads to altered endothelial behavior. In
our studies, we were unable to observe a significant increase in VEGF
circulating levels in the shunt compared with control values. This suggests
that changes in the expression of VEGF may occur selectively in the pulmonary
vessel walls of small arteries that become remodeled. Similarly, other studies
have observed a selective upregulation of VEGF and other angiogenic factors in
pulmonary vascular lesions with unchanging circulating levels of these growth
factors, as in the plexiform lesions of severe primary pulmonary hypertension
(13,
39).
We also observed increased expression of VEGF in the smooth muscle layer of
small pulmonary arteries, in particular those that become remodeled, together
with an increased expression of its receptors KDR/Flk-1 and Flt-1 in adjacent
endothelium. Of interest is the fact that we observed a sequential increase in
VEGF receptors, in which KDR/Flk-1 was increased at earlier stages (14
wk) and Flt-1 showed increases at later stages (4 and 8 wk of age). On VEGF
binding, KDR/Flk-1 becomes rapidly phosphorylated at tyrosine residues, and
this leads to endothelial mitogenesis, migration, and changes in cell
morphology (degradation of stress fibers)
(30). Therefore, KDR/Flk-1
mediates most VEGF angiogenic effects, thereby showing an important role in
the activation phase of angiogenesis
(2,
29).
Flt-1 has been implicated in upregulated endothelial expression of tissue
and urokinase plasminogen activator expression and plasminogen activator
inhibitor-1 (10,
20); these processes are
needed for vessel maturation and vessel wall integrity. Flt-1 has also been
reported to be expressed in vascular smooth muscle cells where it enhances
matrix metalloproteinase expression
(10,
29). Because of its higher
binding affinity with VEGF, Flt-1 has been suggested to sequester VEGF to
prevent its binding to KDR/Flk-1 and, therefore, act in the resolution phase
of angiogenesis (20). However,
studies on abnormal angiogenic processes indicate that Flt-1 can also mediate
positive angiogenic responses of VEGF, since it occurs in carcinogenic
processes (16). Our
observations on the initial increase in KDR/Flk-1 levels followed by a latter
increase in Flt-1 in the shunt model correlates with our previous observations
on increased blood vessel number at 4 wk of age and a subsequent decrease to
normal values at 8 wk of age. Moreover, our data suggest that increased
pulmonary blood flow, as occurs in our shunt model, dysregulates the process
of angiogenesis by upregulating VEGF and its receptors. However, our data also
indicate that these effects are temporal, with the increases in Flt-1 possibly
being involved in the resolution of these angiogenic events. Our data also
indicate that Flt-1 upregulation in the shunt at 4 wk of age correlates with
VEGF upregulation. This is of interest since both Flt-1 and VEGF
transcriptional regulation occur via similar transcription factors, i.e.,
hypoxia-inducible factor 1
and activator protein-1
(14).
The mechanisms by which increased pulmonary blood flow increase VEGF
expression remain unknown. It has been hypothesized that the increase in
angiogenic growth factors TGF-
1 and VEGF observed in models of increased
blood flow is due to an increase in biomechanical forces, i.e., cyclic stretch
and laminar shear stress (15,
24,
41). In accordance with this
theory, various in vitro results have shown that cyclic stretch and laminar
shear stress increase VEGF expression in smooth muscle cells and
cardiomyocytes in a time-dependent fashion
(15,
24,
31,
37,
41). Many signaling molecules
have been proposed to mediate this effect, including indirect proangiogenic
molecules such as TGF-
1, IL-1, and PDGF
(7). We have previously shown
an increased expression of TGF-
1 and its proangiogenic receptors in our
shunt model.
Our previous data indicate that TGF-
1 is upregulated as early as 1 wk
of age in the lungs of shunted lambs, preceding the increase in VEGF
expression. Furthermore, the data presented here indicate that increased VEGF
expression persists after TGF-
1 expression returns to control levels,
suggesting that TGF-
1 regulates the increases in VEGF observed at later
ages. Moreover, these molecular changes correlated with structural changes.
Together, these data suggest a role for coordinated expression of TGF-
1
and VEGF in the alterations of pulmonary vascular morphology induced by
increased pulmonary blood flow secondary to congenital heart disease.
Alterations in pulmonary vascular remodeling and growth are a major source
of morbidity and mortality for children and adults with congenital heart
disease. Although early surgical correction of many defects has significantly
reduced the incidence of irreversible pulmonary vascular disease, reversible
remodeling may still be associated with altered reactivity that results in
perioperative morbidity. In addition, mild pulmonary vascular remodeling in
infants with single ventricle physiology and diminished pulmonary vascular
growth in infants with pulmonary atresia may eliminate corrective surgical
options. Therefore, the status of the pulmonary vasculature is often the
principal determinant of outcome in congenital heart disease. Although the
morphology is well described, the mechanisms of abnormal vascular growth are
not well understood. In the present study, we describe novel alterations in
VEGF expression in an animal model of congenital heart disease with increased
pulmonary blood flow. A better understanding of these and other mechanisms
could profoundly affect the timing and feasibility of surgical and medical
treatments for congenital heart disease and, therefore, warrants further
investigations.
 |
FOOTNOTES
|
---|
Address for reprint requests and other correspondence: S. M. Black,
Northwestern Univ. Medical School, Ward 12-191, 303 E. Chicago Ave., Chicago,
IL 60611-3008 (E-mail:
steveblack{at}northwestern.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.
 |
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