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INTRODUCTION |
Angiogenesis is an important developmental process and is also
critically involved in several pathological conditions such as
rheumatoid arthritis, diabetic retinopathy, and growth of solid tumors
(1, 2). VEGF1 is a major
mediator of normal and abnormal angiogenesis (1). VEGF is secreted by
tumor cells and their surrounding stroma and is causally involved in
the progression of the majority of solid tumors (1). Inhibition of VEGF
activity using various inhibitors results in suppression of tumor
growth in vivo (3-6). VEGF is also up-regulated in ischemic
tissues and has been implicated as a potential therapy for peripheral
and myocardial ischemia (7).
VEGF is expressed as at least four different splice isoforms (1). The
most abundantly expressed variant is VEGF165, which contains a heparin-binding domain. Two VEGF receptor tyrosine kinases,
Flt-1 (VEGFR-1) and KDR (VEGFR-2), are expressed on proliferating and
quiescent endothelial cells (8). Although Flt-1 exhibits higher
affinity for VEGF165, its function in the adult vasculature is still poorly understood. Mice lacking the complete Flt-1 gene display an increased number of endothelial progenitors and vascular disorganization and die in utero at embryonic day 9 (9, 10). However, animals homozygous for a deletion of the cytoplasmic domain are fertile and do not display any obvious defects (11). Mice in
which the flk-1/KDR gene has been
inactivated also die at embryonic day 9. They are deficient in
vasculogenesis and also lack blood island formation (12).
Placenta growth factor (PlGF) shares 53% identity with the
platelet-derived growth factor-like domain of VEGF (13). It binds Flt-1
with high affinity but is unable to interact with KDR (14). PlGF exerts
minimal effects on cell growth and migration, suggesting that binding
to Flt-1 alone is not sufficient to mediate these activities. In some
instances, however, effects of PlGF on mitogenicity and MAP kinase
activation have been reported (15). In recent studies, the effects of
PlGF on endothelial cells have been compared with those of another
VEGF-like molecule, VEGForf. Members of the
VEGForf gene family have been isolated from the Orf
parapoxyvirus, and the encoded proteins bind to KDR but not Flt-1 (16,
17). VEGForf mediates migration of KDR-expressing PAE cells
and corneal angiogenesis to an extent that is comparable with VEGF (17, 18). However, the residues mediating VEGForf binding to KDR are unknown because the three basic amino acids comprising the major
determinants of VEGF binding to the KDR receptor are absent in
VEGForf. In addition, VEGForf does not possess
the heparin-binding domain found on VEGF. These differences may
complicate conclusions about VEGF function, drawn from studies using
VEGForf proteins. VEGF binding to KDR leads to receptor
phosphorylation and activation of MAP kinases as well as tyrosine
phosphorylation of PI3K and PLC
. Although Flt-1 becomes
phosphorylated and can activate several signaling molecules when
expressed in heterologous cells (19), it is unclear whether these
signaling events also occur in endothelial cells in response to Flt-1 engagement.
Recent studies have shown that KDR signaling results in endothelial
nitric-oxide synthase up-regulation and activation (20). Nitric oxide
plays a critical role in the VEGF-induced endothelial cell
proliferation, migration, and tube formation, as well as increased
vascular permeability, hypotension, and angiogenesis in vivo
(21-24). VEGF stimulates the formation of vasodilator prostaglandins, which have been implicated as mediators of VEGF-induced vascular permeability but not new blood vessel formation (21, 25). Recently,
it was shown that a VEGF mutant chimeric protein that is unable to
trigger KDR activation can cause vascular permeability to an extent
that is similar to that of wild type VEGF (26). This led the
authors to conclude that either Flt-1 or another as yet unknown
receptor is responsible for the increase in vascular permeability in
response to VEGF administration.
We sought to address these issues using novel, highly selective, VEGF
mutants generated by phage-display technology (27). Structure-function
studies have demonstrated that VEGF interacts differently with its
cognate receptors KDR and Flt-1. Hence, it has been possible to
generate VEGF mutants that show a strong preference for binding either
one or the other receptor. Compared with an earlier generation of
receptor specific mutants (28), the novel mutants display a
substantially increased selectivity and thus are more likely to yield
relevant results pertaining to the function of each individual VEGF
receptor. By selectively activating Flt-1 or KDR in primary endothelial
cells, we have studied the involvement of each individual receptor in
mediating VEGF signaling and cell migration. KDR activation alone is
sufficient for the activation of signal transducers involved in
mitogenesis and cell migration. Selective KDR engagement also induces
in vivo angiogenesis and vascular permeability.
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EXPERIMENTAL PROCEDURES |
Reagents--
HUVEC were purchased from Cell Systems (Kirkland,
WA). LY 294002 was purchased from Biomol. The construction and
characterization of Flt-1- and KDR-selective VEGF are described by Li
et al. (27). The mutations are as follows: KDR-selective
VEGF D63S/G65M/L66R and Flt-selective VEGF I43A/I46A/Q79A/I83A. The
mutations were introduced into VEGF165 and wild type VEGF,
and the receptor-selective mutants were expressed in Escherichia
coli and purified as described (28). The endotoxin content of the
purified material did not exceed 0.2 Eu/mg.
Binding Assays--
The determination of binding affinities of
the receptor-selective VEGF mutants was carried in solution as
described (29) utilizing soluble IgG-Fc fusions of KDR and Flt-1 (14).
To further characterize the activities of the Flt-sel mutant,
125I-VEGF binding assays were performed in PAE cells
transfected with the mutant Flt-1 receptor Flt(ANGG), as described
previously (30). The PAE cell line lacks endogenous VEGF receptors
(31).
Kinase Receptor Activation Assay--
KDR phosphorylation was
analyzed in CHO cells stably expressing KDR with a C-terminal epitope
tag. Kinase receptor activation assay assays were performed as
described recently (32).
Cell Culture--
Passage 4-7 HUVEC were maintained in
CS-C medium (Cell Systems, Kirkland, WA) containing 10% fetal
bovine serum and growth factors on gelatin-coated dishes and made
quiescent by 14 h of starvation in 0.2% fetal bovine serum. Cells
were treated as indicated and washed once in ice-cold
phosphate-buffered saline containing 0.1 mM sodium
orthovanadate. Cells were lysed in 0.5-1 ml of RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 50 mM Tris, pH 8.0) containing 0.1 mM sodium
orthovanadate, 5 mM para-nitrophenylphosphate,
10 mM sodium fluoride, 0.5 µM okadaic acid,
and a protease inhibitor mixture (Roche MB 1836145). Culture of PAE
cells was performed as described previously (30, 31).
Immunoprecipitation--
Protein A/G beads (Pierce) were blocked
for nonspecific protein binding in 50 mM HEPES, pH 7.2, 0.1% Triton X-100, 150 mM NaCl, and 1 mg/ml ovalbumin for
30 min. Antibodies were precoupled in the same buffer for 1 h at
4 °C with head-over-end rotation, and beads were washed three times
in lysis buffer. Beads were added to the lysates and rotated overnight.
Beads were washed sequentially in 50 mM Tris, pH 7.6, 150 mM NaCl, 1% Triton X-100, 1 mM
CaCl2; 50 mM Tris, pH 7.6, 500 mM
NaCl, 0.1% Triton X-100, 1 mM CaCl2; and 50 mM Tris, pH 7.6, 150 mM NaCl, 0.05% Triton X-100, 1 mM CaCl2. Beads were resuspended in
2× sample buffer and boiled. Supernatants were applied directly to
4-12% Tris-glycine gradient gels (Novex).
Antisera--
Anti-phospho-ERK antiserum was purchased from
Promega. Anti-phospho-p38 antibodies were from New England Biolabs. p85
was immunoprecipitated with antibodies from Transduction Labs (P13020) and Neomarkers (MS 424-P). A monoclonal antibody from Upstate Biotechnologies, Inc. was used for the immunoprecipitation of PLC
. The phosphotyrosine antibodies PY20 or E120H from Transduction Labs were used.
HUVEC and PAE Cell Migration Assays--
Migration assays were
performed essentially as described previously (30). Falcon 8.0-µm
filter inserts (Falcon 3097) were coated with type 1 collagen
(VITROGEN, COHESION). PAE clones were grown in Ham's F-12 medium, 10%
fetal calf serum, 0.25 mg/ml G418. HUVEC (obtained from Cell Systems,
<p8) were grown in Cell Systems complete medium (4ZO-500) with 10%
fetal calf serum. Cells were trypsinized and transferred to endothelial
basal medium (Clonetics) with 0.1% bovine serum albumin for the assay.
Cells were plated at 5 × 104/upper chamber. Growth
factors were placed in the lower chamber, and inhibitors were in the
upper chamber. Cells were allowed to migrate at 37 °C for 14-16 h.
For the LY294002 inhibitor experiments, cells were allowed to adhere
for 30 min prior to addition of the inhibitor. 20 min after inhibitor
addition, VEGF was added to the bottom well, and the assay was allowed
to proceed for only 4 h to avoid the occurrence of apoptosis
associated with the treatment of these primary cells with LY294002.
Cells were removed from the upper side of the membrane by scraping with
a polyurethane swab, and then the remaining cells on the bottom side of
the membrane were fixed with methanol. Cells were stained with Yo-Pro
Iodide nuclear stain (Molecular Probes) and counted under low power
fluorescence using an Image-Pro cell recognition program.
Corneal Pocket Angiogenesis Assay--
Assays were performed as
described previously (33), with the following modifications. Harlan
Sprague-Dawley rats are anesthetized using a gas
(isoflurane)/injectable ketamine (80 mg/kg) xylazine (15 mg/kg)
combination. The eyes are gently proptosed and secured in place using
nontraumatic forceps. With a number 15 blade, a 1.5-mm incision was
made slightly below the center of the cornea. Using a microspatula
(ST80017, ASSI), the incision was carefully blunt-dissected through the
stroma toward the outer canthus of the eye. A hydron-coated pellet
(2 × 2 mm) containing growth factor (200 ng), methylcellulose,
and aluminum sucralfate (100 µg) was inserted into the base of the
pocket. After surgery the eyes were coated with gentamicin ointment. At
day 6 the animals were injected with high molecular weight fluorescein
isothiocyanate-dextran and euthanized to allow for visualization of the
vasculature. Corneal whole mounts were made of the enucleated eyes and
measurements of neovascular area completed using computer-assisted
image analysis (Image-Pro Plus).
Vascular Permeability Assay--
Vascular permeability was
assessed using a modified Miles assay described previously (21, 25,
34-36). Hairless guinea pigs (600-800 g) were anesthetized by
intramuscular administration of xylazine (5 mg/kg) and ketamine (75 mg/kg). The animals then received an intracardiac injection of 1 ml of
1% Evans blue dye. Sixty minutes later, intradermal injections (0.1 ml) of VEGF or the appropriate VEGF mutant were made in a grid, which
allowed us to construct a dose-response curve for each compound
(n = 4/molecule). VEGF165 was diluted in
phosphate-buffered saline/bovine serum albumin (1 mg/ml) and used at
doses of 0, 0.3, 1, 3, 10, 30, and 100 ng/site. The dose of all other
proteins was adjusted to inject equimolar amounts of protein. Sixty
minutes after injections were made, animals were euthanized with
pentobarbital (160 mg/kg IV), and their skin was removed, cleaned from
connective tissues, and photographed. Quantification of the dye
extravasation area was carried out on the pictures using an image
analysis software (NIH Image®). For each dose, measurements were made
in triplicate and averaged to minimize measurement errors.
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RESULTS |
Characterization of Receptor-selective Mutants--
We set out to
determine the different contributions of KDR and Flt-1 to VEGF
signaling in primary endothelial cells. Because endothelial cells
express both VEGF receptors, we made use of two novel VEGF mutants
engineered by the use of a phage display selection process to bind
selectively to either KDR or Flt-1 (27). These mutations were
introduced into the VEGF165 cDNA and recombinant proteins were produced in E. coli. The full-length VEGF
mutant proteins were analyzed for their ability to bind to either Flt-1 or KDR in solution. Fig. 1A
demonstrates the relative inability of the Flt-1-selective VEGF mutant
(Flt-sel) to compete efficiently with wild type 125I-VEGF
for binding to KDR. The reduction of binding of the KDR-selective mutant (KDR-sel2) to Flt-1 is shown in Fig. 1B. Table
I summarizes the Kd
values and relative affinities of the individual mutants to Flt-1 and
KDR. Binding of the KDR-selective variant to Flt-1 is reduced by a
factor of 2000-fold, whereas Flt-selective VEGF binds KDR 128-fold less
well. To further characterize the specificity of the mutants, we tested
them with respect to their ability to induce KDR phosphorylation in
intact cells (Fig. 1C). KDR-selective VEGF was essentially
as potent and effective as wild type VEGF. Surprisingly, the ability of
Flt-sel VEGF to induce KDR phosphorylation was reduced by at least 4 orders of magnitude. However, as illustrated in Fig. 1D,
Flt-sel VEGF was fully capable of competing for 125I-VEGF
binding to the Flt-1 receptor even in the context of intact cells and
was as potent and effective as VEGF165 in promoting motility of PAE cells expressing Flt(ANGG). Unlike wild type Flt-1, this receptor mutant is able to mediate a KDR-like motility response to
VEGF because of the removal of a repressor motif in the juxtamembrane region (30). These findings argue against the possibility that the
inability of Flt-sel VEGF to induce KDR phosphorylation may be due to
inherent instability and/or lack of activity of the mutant protein.

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Fig. 1.
Specificity and activity of KDR- and
Flt-selective VEGF mutants. Competition of VEGF wild type and
mutant protein binding with 125I-VEGF to KDR. Binding of
125I-labeled VEGF was performed in the presence of the
indicated concentrations of cold ligand. A, KDR binding
activities. B, Flt-1 binding activities. C, KDR
phosphorylation. The ability of mutant and wild type VEGF proteins to
induce KDR phosphorylation was analyzed over the indicated
concentration rage. D, Flt-sel VEGF is biologically active.
This mutant protein displays the same potency as VEGF165 in
a competitive displacement of 125I-VEGF from transfected
PAE cells expressing Flt(ANGG) (top panel). Flt-sel VEGF
also promotes chemotaxis of PAE-Flt(ANGG) cells as efficiently as wild
type VEGF, while KDR-sel VEGF is ineffective (bottom panel).
Growth factors were added at the indicated concentrations as described
under "Experimental Procedures." Cells were allowed to migrate for
14-16 h. Flt(ANGG) is a mutant Flt-1 receptor that, unlike wild type
Flt-1, is able to mediate a motility response to VEGF (30). Data are
expressed as stimulation index of quadruplicate wells relative to
basal, factor-independent migration. Bars represent the
means ± S.E.
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Activation of MAP Kinases--
Earlier work by this and other
laboratories has generally shown that PlGF binding to Flt-1 is not able
to cause marked mitogenesis in endothelial cells (14, 19), although
evidence to the contrary has also been reported in transfected cell
lines (15). We therefore tested whether KDR-selective VEGF was capable
of mediating mitogenic signaling. As expected, activation by
KDR-selective VEGF triggered phosphorylation of ERK1 and ERK2 in HUVEC
(Fig. 2A). The extent of
phosphorylation was indistinguishable from that obtained using wild
type VEGF. Flt-1-selective VEGF at the highest concentration used
resulted in minimal phosphorylation of ERK2. The homodimeric VEGF
mutants utilized in this study are not expected to promote receptor
heterodimer formation. Hence Flt-1 does not contribute to MAP kinase
activation.

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Fig. 2.
Activation of MAP kinases. Quiescent
HUVEC were either left untreated or stimulated with VEGF or VEGF mutant
proteins for 5 min. A, Western blots were probed for
phosphorylated ERK1 and ERK2. B, the phosphorylation state
of p38 stress-activated MAP kinase was assessed with a phospho-specific
antiserum. wt, wild type.
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VEGF has been reported to stimulate the stress-activated p38 MAP kinase
(37, 38). To determine which VEGF receptor is involved, the
phosphorylation status of p38 was analyzed after stimulation with wild
type Flt- and KDR-selective VEGF. Fig. 2B demonstrates that
only KDR-selective VEGF was able to stimulate p38 phosphorylation.
However, as above indicated, Flt-selective VEGF was as effective as
wild type VEGF165 in inducing migration of PAE cells
expressing Flt(ANGG), demonstrating that this mutant is biologically
active. Furthermore, in agreement with a previous report that
implicates Flt-1 activation in metalloproteinase release (39),
Flt-selective VEGF stimulated the release of increased amounts of
matrix metalloprotease-9 proteolytic activity from human vascular
smooth muscle cells (27).
PI3K and PLC
Phosphorylation--
PLC
phosphorylation and
activation has been implicated in VEGF signaling. PLC
binding to
both KDR (40, 41) and Flt-1 (19, 42, 43) has been reported. To
determine which VEGF receptor(s) are involved in PLC
activation in
primary endothelial cells, HUVEC were treated with VEGF or VEGF
receptor-selective mutants, and PLC
phosphorylation was assessed
after immunoprecipitation (Fig.
3A). Both wild type and
KDR-selective VEGF were able to stimulate PLC
phosphorylation to a
similar extent. Flt-selective VEGF did not increase PLC
phosphorylation over background levels, arguing against a role for
Flt-1 in PLC
activation in HUVEC.

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Fig. 3.
KDR stimulates PLC
and PI3K phosphorylation. Quiescent HUVEC were stimulated
with 20 ng/ml of the indicated growth factors. A, PLC was
immunoprecipitated from whole cell lysates and analyzed for tyrosine
phosphorylation. B, lysates were immunoprecipitated
(IP) with monoclonals antibodies against p85 PI3K and tested
for phosphotyrosine. wt, wild type.
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PI3K has been demonstrated to transmit survival signals through the
activation of Akt in several cell types (44). VEGF also acts as a
survival factor for endothelial cells, and this signal requires PI3K
and Akt kinase activity (45). In a variety of cell types, PI3K activity
has been demonstrated to be involved in cytoskeletal changes following
growth factor stimulation as well as cell migration (46, 47).
Therefore, the ability of the VEGF proteins to cause phosphorylation of
the p85 regulatory subunit of PI3K was assessed after
immunoprecipitation. Only wild type and KDR-selective VEGF were capable
of causing phosphorylation the PI3K regulatory subunit (Fig.
3B).
Effect of Receptor-selective VEGF Mutants on Endothelial Cell
Migration--
One of the central aspects of VEGF action on
endothelial cells is its ability to act as a chemoattractant and
stimulate the migration of endothelial cells. Fig.
4A shows the effect of
receptor-selective VEGF on HUVEC in a modified Boyden chamber assay. In
several independent experiments, VEGF caused a 4-5-fold increase in
HUVEC migration. KDR-selective VEGF is as effective as wild type VEGF
in the promotion of HUVEC migration. Flt-selective VEGF is unable to
increase cell migration over background levels.

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Fig. 4.
Analysis of HUVEC migration. HUVEC
migration was analyzed in a modified Boyden chamber assay. Experiments
were performed in triplicate. Error bars represent the
standard error. A, HUVEC migration in response to the
indicated concentrations of wild type, Flt-selective, and KDR-selective
VEGF. B, PI3K inhibition impairs HUVEC migration in response
to wild type VEGF. wt, wild type.
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To determine the contribution of PI3K to endothelial cell migration,
different concentrations of the inhibitor LY 294002 were added to the
assay after the cells had been allowed to attach to the membrane.
Because of the deleterious effects of PI3K inhibition on endothelial
cell survival a short-term assay was performed (see "Experimental
Procedures"). Fig. 4B shows that at its highest concentration, and LY 294002 caused a 56% inhibition of HUVEC migration. Thus, PI3K activity contributes significantly to endothelial cell migration.
KDR, but Not Flt-1, Signaling Causes in Vivo
Angiogenesis--
Because endothelial cells in adult organisms express
both KDR and Flt-1 and previous approaches that interfere with in
vivo angiogenesis have been targeted at interfering with VEGF
binding to both receptors, we sought to identify the receptor(s)
responsible for in vivo angiogenesis. Hydron pellets
containing 200 ng of growth factors were implanted into rat corneas,
and the angiogenic areas were evaluated after 1 week (Fig.
5A). KDR-selective VEGF was as
efficient as wild type VEGF in inducing corneal angiogenesis. Although
Flt-selective VEGF occasionally induced marginal angiogenesis (Fig.
5A), analysis of the angiogenic surface areas in several animals showed that Flt-selective VEGF was unable to stimulate angiogenesis over control levels. PlGF gave only a marginal response (Fig. 5B). Therefore, only KDR and not Flt-1 is capable of
promoting angiogenesis in vivo.

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Fig. 5.
Angiogenic effects of VEGF mutants in the rat
cornea. A, representative examples of the extent of
corneal angiogenesis in response to wild type, Flt-selective, and
KDR-selective VEGF. B, quantitative analysis of the surface
areas of corneal angiogenesis caused by wild type and mutant VEGF
proteins as well as PlGF.
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Increased Vascular Permeability Is Mediated through KDR but Not
Flt-1 Activation--
Similarly, we sought to determine the relative
importance of KDR and Flt-1 receptors for the VEGF-induced vascular
permeability. Vascular permeability was assessed by the Miles assay,
and responses were evaluated 60 min following the intradermal
administration of VEGF and VEGF receptor-selective mutants.
KDR-selective VEGF induced vascular permeability to a comparable extent
as wild type VEGF, whereas Flt-1 receptor-selective VEGF caused
essentially no leakage (Fig.
6A). The results obtained in
four animals are summarized in Fig. 6B. In additional
experiments, we tested the Flt-1-selective mutant at much higher
concentrations (up to 30 µg/site), and still no extravasation was
observed (data not shown). Therefore, VEGF-induced vascular
permeability is mediated by KDR receptor binding. To further confirm
these results, we compared the vascular permeability of
VEGF165 and the Flt-1-selective ligand PlGF. To rule out
the possibility that differences in Flt-1 receptor binding affinity
between VEGF and PlGF could explain the lack of vascular permeability
induced by PlGF, we used PlGF at equimolar doses and at doses 20-fold
higher relative to those of VEGF. Whereas VEGF induced a
dose-dependent dye extravasation, PlGF did not induce any
vascular permeability regardless of whether we used equimolar or
20-fold higher doses relative to those of VEGF consistent with previous
reports (Refs. 14 and 48 and Fig. 6C). These results
demonstrate the critical role of KDR receptor activation in the
VEGF-induced vascular permeability response.

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Fig. 6.
VEGF-induced vascular permeability is
mediated by binding to KDR receptors. A, representative
pictures of skin of guinea pigs used in the Miles assay showing the
vascular permeability in response to wild type VEGF165, and
KDR receptor-selective VEGF mutant, and the lack of effect of the Flt
receptor-selective VEGF mutant. B, dose-response curves for
VEGF and receptor-selective VEGF mutants. Error bars
represent S.E. C, comparison of the vascular permeabilty
response obtained with equimolar doses of VEGF and PlGF and a 20-fold
higher dose of PlGF over VEGF.
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DISCUSSION |
The expression of at least two VEGF receptor tyrosine
kinases on endothelial cells has made it difficult to elucidate the individual contributions of each receptor to VEGF signaling. Although PlGF binds preferentially to Flt-1, its use has sometimes led to
conflicting results especially when heterologous cell types such as
NIH3T3 fibroblasts were used (15, 19).
This study explores whether the activities of both Flt-1 and KDR are
required to mediate VEGF functions in primary endothelial cells and
in vivo. To this end, we employed homodimers of
receptor-selective VEGF mutants. Flt-selective VEGF was unable to
generate a mitogenic signal, as evidenced also by its inability to
stimulate ERK activity. This result is in agreement with previous work
that examined the abilities of earlier receptor-selective mutants to
promote endothelial cell proliferation (28). Interestingly, although
the novel Flt-1-selective mutant demonstrated a 128-fold selectivity
using soluble IgG chimeric receptors, in a kinase receptor activation
assay format it was virtually devoid of the ability to induce KDR
phosphorylation, even at concentrations exceeding 10,000 ng/ml.
Therefore, the receptor binding assays largely underestimated the
extent of specificity of this mutant that may be obtained in a
biological context. There are various examples of lack of good
correlation between binding affinity and biological activity of mutant
proteins (49). In the case of our mutant, one of the possible
explanations for the discrepancy is that the reduced affinity leads to
only a transient existence of ligand-receptor complexes, below a
critical threshold for effective receptor phosphorylation and
dimerization. Interestingly, a "minimum residency time threshold"
for effective phosphorylation has been recently described for the
prolactin receptor (50). However, it was important to verify that
Flt-sel VEGF is capable of exerting the appropriate biological
activity. This mutant is able to compete for 125I-VEGF
binding in PAE cells expressing Flt(ANGG) with a dose response nearly
identical to that of VEGF165, demonstrating its ability to
bind Flt-1 not only in soluble receptor assays but also in the context
of intact cells. Furthermore, this binding results in stable receptor
activation, as assessed by the ability of Flt-sel VEGF to effectively
promote migration of PAE cells expressing Flt(ANGG) in an overnight
assay. Although wild type Flt-1 fails to transmit a motility signal
(30, 31), the juxtamembrane mutant Flt(ANGG) is able to mediate
VEGF-dependent migration in transfected PAE cells, similar
to that mediated by KDR (30). Additionally, as previously mentioned,
Flt-selective VEGF has been shown to stimulate the release of matrix
metalloprotease-9 proteolytic activity from cultured human vascular
smooth muscle cells (27).
Activation of the KDR receptor was also sufficient to cause cell
migration in HUVEC. Therefore, we also examined the activation of
proteins previously reported to be involved in the migratory response
of normal endothelial cells. Both PI3K and PLC
phosphorylation were
stimulated to the same extent by wild type VEGF and KDR-selective VEGF.
HUVEC migration was inhibited by treatment with the PI3K inhibitor
LY294002, demonstrating a prominent role for PI3K in endothelial cell
migration. This is consistent with results obtained for other cell
types, such as epithelial cells and fibroblasts, whereas PI3K activity
is required for cytoskeletal changes and cell migration (46, 47).
Recently, the properties of novel VEGF family members present in
Orf parapoxyviruses have been analyzed (VEGForf).
VEGForf is a distant relative of VEGF-A. It lacks a
heparin-binding domain and binds preferentially to KDR. Additionally,
VEGForf features a threonine- and proline-rich C-terminal
domain of unknown function that is not present in any other mammalian
VEGF. VEGForf mediates endothelial cell mitogenesis and
migration of KDR-expressing PAE cells as well as corneal angiogenesis
to an extent that is comparable with VEGF165 (18, 43). Our
rationally designed KDR-selective VEGF should be more representative
for the functions of mammalian VEGFs, because it is largely unchanged
from wild type VEGF, and only its ability to interact with Flt-1 has
been removed.
Because our Flt-1-selective VEGF mutant shows substantially reduced
binding and little or no ability to activate KDR, we tested whether
this highly selective protein could induce dye extravasation. The
absence of extravasation following administration of the Flt-1 receptor-selective mutant and the comparable extent of vascular permeability caused by wild type VEGF and the KDR receptor-selective mutant indicates that VEGF-induced vascular permeability is solely mediated through binding of VEGF to the KDR receptor.
However, a chimeric mutant of mouse VEGF, which shows preferential
binding to Flt-1, has recently been reported to stimulate vascular
permeability, leading the authors (26) to conclude that KDR
activation is not required to mediate this biological activity of VEGF.
These conflicting conclusions may be explained, at least in part, by
the fact that the chimeric murine VEGF mutant was obtained by
site-directed mutagenesis in which amino acids 83-89 were substituted
by analogous regions of PlGF (26). The differences in structure and/or
signal transduction between VEGF and PlGF may be responsible in part
for this discrepancy. Furthermore, it is possible that the high local
concentrations of the injected protein employed in the vascular
permeability assay (up to 1 µg/ml) require a greater degree of
specifity of the mutant VEGF protein than that afforded by such
chimeric mutant, to achieve meaningful results. In support of this
possibility, our earlier generation Flt-1-selective mutants (28), which
display a lower degree of receptor selectivity than the mutant used in
the present study, are able to cause as much as 50% of the vascular
permeability response as wild type VEGF (data not shown). Furthermore,
the finding that our KDR-selective mutant, which exhibits 2000-fold selectivity for KDR versus Flt-1, is fully competent to
cause vascular permeability provides strong evidence in favor of the involvement of KDR. Interestingly, a KDR-selective VEGF mutant in the
background of the receptor-binding domain (27) was also fully competent
in promoting vascular permeability (data not shown), indicating that
the heparin-binding domain has little or no contribution to this
activity. The role of the KDR receptor in mediating vascular permeability is supported by other studies that describe a mutant VEGF-C, which has lost both KDR receptor binding and vascular permeability activities (34) and a VEGF molecule produced by Orf
viruses, which binds KDR but not Flt-1 receptors and induces vascular
permeability (16, 18). Additionally, PlGF fails to induce vascular
permeability (14, 48). Therefore, we conclude that binding of VEGF to
KDR is necessary and sufficient to induce vascular permeability.
Similar to the results obtained in the in vivo angiogenesis
experiments, a contribution from Flt-1 is not required to elicit this
biological effect.
Our findings ascertain that KDR can be the sole mediator of
VEGF-induced migration, angiogenesis and permeability in endothelial cells. Furthermore, these VEGF mutants should provide especially valuable tools to further dissect Flt-1 and KDR function in
vivo through the use in transgenic models, where the protein is
overexpressed. Their high selectivity over a broad concentration range
may be critical to correctly define the VEGF receptor biology in such circumstances.