(Received for publication, June 15, 1995; and in revised form, October 27, 1995)
From the
VEGF and VEGF
are vascular
endothelial growth factor splice variants that promote the
proliferation of endothelial cells and angiogenesis. VEGF
contains the 44 additional amino acids encoded by exon 7 of the
VEGF gene. These amino acids confer upon VEGF
a heparin
binding capability which VEGF
lacks.
I-VEGF
bound to three vascular endothelial
growth factor (VEGF) receptors on endothelial cells, while
I-VEGF
bound selectively only to the flk-1 VEGF receptor which corresponds to the larger of the
three VEGF receptors. The binding of
I-VEGF
to flk-1 was not affected by the removal of cell surface
heparan sulfates or by heparin. Both VEGF
and
VEGF
inhibited the binding of
I-VEGF
to a soluble extracellular domain of
the flk-1 VEGF receptor in the absence of heparin. However,
heparin potentiated the inhibitory effect of VEGF
by
2-3-fold. These results contrast with previous observations which
have indicated that the binding of
I-VEGF
to the flk-1 receptor is strongly dependent on
heparin-like molecules. Further experiments showed that the receptor
binding ability of VEGF
is susceptible to oxidative
damage caused by oxidants such as H
O
or
chloramine-T. VEGF
was also damaged by oxidants but to a
lesser extent. Heparin or cell surface heparan sulfates restored the flk-1 binding ability of damaged VEGF
but not
the receptor binding ability of damaged VEGF
. These
observations suggest that alternative splicing can generate a diversity
in growth factor signaling by determining receptor recognition
patterns. They also indicate that the heparin binding ability of
VEGF
may enable the restoration of damaged VEGF
function in processes such as inflammation or wound healing.
Four vascular endothelial growth factor (VEGF) ()forms are produced by alternative splicing from the VEGF
gene(1, 2, 3) . The 121-amino acid form
(VEGF
) lacks a heparin binding ability, while
VEGF
, VEGF
, and VEGF
bind
efficiently to heparin. All forms are mitogenic to vascular endothelial
cells and induce permeabilization of blood
vessels(4, 5) . VEGF
induces
angiogenesis in vivo(6) and plays a central role in
the process of tumor angiogenesis(7, 8, 9) .
Binding and cross-linking experiments have shown that VEGF
binds to three VEGF receptors on the cell surface of vascular
endothelial cells(10, 11, 12) . Two VEGF
receptors have recently been identified and
cloned(13, 14, 15, 16) . These
include the mouse flk-1 receptor and its human homologue KDR,
and the human flt-1 receptor. The flk-1 receptor was
shown to transduce a VEGF
mitogenic
signal(9, 17) , while activation of the flt-1 receptor does not seem to result in a similar mitogenic
response(16, 18) .
The binding of I-VEGF
to various VEGF receptors and the
effects of heparin on the binding have been characterized
extensively(10, 11, 12, 19) , while
the interaction of VEGF
with the VEGF receptors of
vascular endothelial cells has not yet been studied. We show that
VEGF
binds selectively to the larger of the three VEGF
receptors of human umbilical vein-derived endothelial cells (HUE) and
that this receptor probably corresponds to the human homologue of the flk-1 VEGF receptor. We also show that both VEGF
and VEGF
are susceptible to oxidative damage and
that heparin restores the receptor binding ability of damaged
VEGF
, but not the receptor binding ability of damaged
VEGF
.
The binding and the cross-linking of I-VEGF
to endothelial cells was done as described
previously(10, 11) , and the binding of
I-VEGF
was done similarly. Nonspecific
binding was determined in the presence of 1-2 µg/ml unlabeled
VEGF. The level of nonspecific binding ranged between 10 and 20% of the
total binding. The binding and cross-linking of
I-VEGF
to a flk-1/SEAP fusion
protein containing the extracellular domain of the flk-1 receptor was done as described previously for
I-VEGF
(19, 23) .
Figure 1:
Iodination of VEGF and
VEGF
. VEGF variants were iodinated to a specific activity
of
10
cpm/ng using the chloramine-T method.
I-VEGF
(121) or
I-VEGF
(165) were reduced by
boiling for 3 min in the presence of 0.1 M dithiothreitol. The
iodinated proteins were chromatographed on a 12% SDS-PAGE gel and
autoradiographed.
Figure 2:
The effect of heparin and the effect of
heparinase treatment on the binding and cross-linking of I-VEGF
and
I-VEGF
to HUE cells. HUE cells were grown to confluence on
gelatin-precoated 10-cm dishes. Cells were washed once with
phosphate-buffered saline at 37 °C and were incubated in binding
buffer (20 mM HEPES, pH 7.2, 0.1% gelatin in Dulbecco's
modified Eagle's medium) for 1 h at 37 °C with (lanes
5, 6, 11, and 12) or without (lanes
1-4 and 7-10) 0.05 unit/ml heparinase 1. The
cells were subsequently washed twice with cold phosphate-buffered
saline, and 2.4 ml of cold binding buffer containing 10 ng/ml
I-VEGF
(lanes 1-6) or 5
ng/ml
I-VEGF
(lanes 7-12)
were added to respective dishes. Other additions were: heparin (1
µg/ml), lanes 4, 6, 8, 10, and 12; unlabeled VEGF
(2 µg/ml), lanes
2, 9, and 10; and unlabeled VEGF
(2 µg/ml), lane 3. The binding, the subsequent
cross-linking of bound growth factor to the cells using 0.25 mM disuccinimidyl suberate, SDS-PAGE of cross-linked samples, and the
visualization of cross-linked products were done as described. Equal
amounts of protein from cell lysates were chromatographed in each
lane.
Figure 3:
VEGF inhibits selectively
the binding of
I-VEGF
to the 225-kDa VEGF
receptor of HUE cells.
I-VEGF
(5 ng/ml) was
bound to confluent HUE cells grown in 10-cm dishes in the presence of 1
µg/ml heparin and the following concentrations of unlabeled
VEGF
(µg/ml): lane 1, 0; lane 2,
0.05; lane 3, 0.1; lane 4, 2. After 2 h at 4 °C,
the cells were washed, bound
I-VEGF
was
cross-linked to cell surface receptors, and
I-VEGF
-receptor complexes were visualized
as described in the legend to Fig. 2.
The binding of I-VEGF
to the larger VEGF
receptor of the endothelial cells was not affected by the removal of
cell surface heparin-like molecules (Fig. 2, lane 5).
This was perhaps to be expected as
I-VEGF
does not bind to heparin. This observation also indicates that
heparin-like molecules do not affect the VEGF
binding
ability of the larger VEGF receptor of the endothelial cells. However,
not all of the VEGF receptors that are capable of
I-VEGF
binding behave similarly. The
I-VEGF
binding ability of the VEGF
receptors of YU-ZAZ6 melanoma cells is inhibited upon the removal of
cell surface heparin-like molecules by heparinase digestion or by the
addition of exogenous heparin, suggesting that heparin-like molecules
can modulate the VEGF
binding ability of the YU-ZAZ6 VEGF
receptors (21) .
Figure 4:
Immunoprecipitation of I-VEGF-receptor cross-linked complexes with
anti-flk-1 or with anti-flt-1 antibodies. A,
I-VEGF
(5 ng/ml) was bound and cross-linked
to confluent HUE cells grown in 6-cm dishes. The cells were lysed, and
aliquots containing
I-VEGF
-receptor
complexes were examined by SDS-PAGE (lane 1) or
immunoprecipitated using various antibodies as described under
``Experimental Procedures'' (lanes 2-4). The
antibodies used (1 µg/ml each) were: anti-flt-1 (lane
2), anti- HB-EGF (lane 3), and anti-flk-1 (lane 4).
Antibody-
I-VEGF
-receptor complexes were
precipitated for 1 h at 4 °C using protein G-Sepharose.
Immunoprecipitated
I-VEGF
-receptor
complexes were detached from the beads by boiling in SDS-PAGE sample
buffer, separated on a 6% gel, and visualized as described under
``Experimental Procedures.'' B,
I-VEGF
(10 ng/ml) was bound and
cross-linked to confluent HUE cells as described above for
I-VEGF
. Following cross-linking, the cells
were lysed.
I-VEGF
-receptor complexes in
cell lysates were visualized immediately (lane 1) or
immunoprecipitated using anti-flk-1 antibodies (lane
2), anti-flt-1 antibodies (lane 3), or
anti-HB-EGF antibodies (lane 4) as described under Fig. A.
I-VEGF
-receptor complexes were
separated using SDS-PAGE and visualized as described under Fig. A.
Figure 5:
Heparin restores the receptor binding
ability of oxidized VEGF, but not the receptor binding
ability of oxidized VEGF
. A,
I-VEGF
(25 ng/ml) was bound to ELISA dishes
coated with flk-1/SEAP fusion protein in a final volume of 50
µl as described under ``Experimental Procedures.'' The
binding was performed in the presence (
) or absence (
,
) of heparin (1 µg/ml) and in the presence of increasing
concentrations of either untreated VEGF
(
) or
VEGF
which was treated with chloramine-T as described
under ``Experimental Procedures'' (
,
). The
binding was performed for 2 h at room temperature, after which the
dishes were washed 3 times with buffer containing 0.1% Tween 20 as
described. Bound
I-VEGF
was solubilized
using 0.5 N NaOH, and aliquots were counted in a
-counter. 100% of
I-VEGF
binding
corresponds to 5500 cpm. B,
I-VEGF
(25 ng/ml) was bound to ELISA dishes coated with flk-1/SEAP fusion protein in a final volume of 50 µl as
described. The binding was performed in the presence (
) or
absence (
,
) of heparin (1 µg/ml) and in the
presence of increasing concentrations of either untreated VEGF
(
) or VEGF
which was treated with
chloramine-T as described (
,
). The binding was performed
as described under A. 100% of
I-VEGF
binding corresponds to 10,000 cpm. C,
I-VEGF
(25 ng/ml) was bound to ELISA dishes
coated with flk-1/SEAP fusion protein as described. The
binding was performed in the presence (hatched columns) or in
the absence (empty columns) of 1 µg/ml heparin. The
VEGF
and VEGF
concentration used for
competition was 1 µg/ml. VEGF
and VEGF
were treated or not with H
O
(1%) as
described under ``Experimental Procedures.'' D,
I-VEGF
(20 ng/ml) was bound and
cross-linked to confluent heparinase 1-digested HUE cells grown in
10-cm dishes in the absence (lanes 1 and 3) or in the
presence (lanes 2 and 4) of 1 µg/ml heparin.
Unlabeled VEGF
was added to a final concentration of 2
µg/ml to some of the binding reactions (lanes 3 and 4). The heparinase digestion and the visualization of
cross-linked complexes were done as described in Fig. 2.
These experiments imply that the receptor binding ability of
VEGF may be impaired during iodination. Subsequent
experiments have indicated that the flk-1/SEAP binding ability
of VEGF
is sensitive to oxidants. VEGF
damaged by oxidizing agents such as chloramine-T or
H
O
was not able to compete with
I-VEGF
for binding to flk-1/SEAP
in the absence of heparin (Fig. 5, A and C).
However, the ability to compete with
I-VEGF
for binding to flk-1/SEAP was partially restored by the
addition of 1 µg/ml heparin to the binding reaction (Fig. 5, A and C). Similar results were also obtained in
analogous experiments performed with heparinase-treated HUE cells (not
shown). Oxidized VEGF
also lost some of its ability to
compete with
I-VEGF
for binding to flk-1/SEAP, although VEGF
seemed to be somewhat
more resistant than VEGF
to oxidation (Fig. 5, B and C). However, in contrast to VEGF
,
the addition of heparin did not restore the flk-1/SEAP binding
ability of damaged VEGF
(Fig. 5, B and C). The relative insensitivity of VEGF
to
oxidative damage may explain why
I-VEGF
, in
contrast to
I-VEGF
, is still able to bind
to flk-1/SEAP and to the flk-1 receptor of the
vascular endothelial cells in the absence of heparin-like molecules.
The potentiating effect that heparin exerts on the receptor binding
ability of untreated VEGF could reflect oxidative damage
sustained before or during VEGF
purification.
Alternatively, it could mean that heparin has a real ability to
potentiate the binding of undamaged VEGF
to the flk-1 VEGF receptor and perhaps to other types of VEGF receptors as
well. Our results imply that the heparin binding ability of
VEGF
may be required under conditions in which oxidizing
agents and free radicals are produced. Such conditions can be
encountered in biological processes such as wound healing,
hypoxia-induced angiogenesis, or inflammation, processes in which
VEGF
was shown to play an important
role(24, 25, 26, 27, 28) .
Under such conditions, cell surface heparin-like molecules could
restore the activity of damaged VEGF
molecules. This
restorative function of heparin-like molecules could be of critical
importance under conditions in which the initial concentration of
VEGF
is low to begin with. Heparin-like molecules are
also able to restore the activity of damaged bFGF and aFGF and to
protect them from inactivation by heat and
oxidation(29, 30) . The protective and restorative
effects of heparin could perhaps account for some of the opposing
conclusions that were obtained in experiments designed to assess the
importance of heparin-like molecules in the interaction of bFGF with
FGF receptors(31, 32, 33) . Such a
restorative effect would be harder to detect in the case of bFGF since
an active bFGF homologue lacking a heparin binding ability (like
VEGF
) is unavailable.
In conclusion, our experiments
indicate that both VEGF and VEGF
bind to
the 180-kDa VEGF receptor of HUE cells forming 220-230-kDa
complexes after covalent cross-linking. However, only VEGF
is capable of binding to the two smaller VEGF receptors of the
endothelial cells. To the best of our knowledge, this is the first time
that splice variants of a growth factor are found to differ in receptor
recognition patterns. We have also performed immunoprecipitation
experiments which indicate that the 220-kDa
I-VEGF-receptor complex contains the KDR/flk-1 VEGF receptor. Competition experiments using a soluble fusion
protein containing the extracellular domain of flk-1 have also
revealed that heparin is not essential for the binding of VEGF
or VEGF
to flk-1 receptors. These
experiments also suggest that the ability to bind heparin-like
molecules may help to preserve the biological function of VEGF
under conditions in which oxidants and free radicals are
produced.