(Received for publication, October 4, 1995; and in revised form, November 10, 1995)
From the
Here we show that the Escherichia coli expressed
monomers of placenta growth factor (PLGF) and vascular
endothelial growth factor (VEGF)
can be re-folded in
vitro to form PLGF/VEGF heterodimers. The purified recombinant
PLGF/VEGF heterodimers and VEGF homodimers have potent mitogenic and
chemotactic effects on endothelial cells. However, PLGF/VEGF
heterodimers display 20-50-fold less mitogenic activity than
VEGF
homodimers. In contrast, PLGF
homodimers have little or no effect in these in vitro assays. We also demonstrate the presence of natural PLGF/VEGF
heterodimers in the conditioned media of various human tumor cell
lines. While PLGF/VEGF heterodimers bind with high affinity to a
soluble Flk-1/KDR receptor, PLGF
homodimers fail to bind
to this receptor. Cross-linking of
I-ligands to human
umbilical vein endothelial cells reveals that PLGF/VEGF heterodimers
and VEGF
homodimers, but not PLGF
homodimers, form complexes with membrane receptors. VEGF
homodimers and PLGF/VEGF heterodimers stimulate tyrosine
phosphorylation of a 220-kDa protein, the expected size for the KDR
receptor in human umbilical vein endothelial cells, whereas
PLGF
homodimers are unable to induce tyrosine
phosphorylation of this protein. These data indicate that PLGF may
modulate VEGF-induced angiogenesis by the formation of PLGF/VEGF
heterodimers in cells producing both factors.
Angiogenesis, the growth of new capillary vessels, is a multi-step process that involves enzymatic degradation of the basement membrane of a local venule, capillary endothelial cell proliferation, migration, tissue infiltration, and lumen formation(1, 2) . It is required for many physiological and pathological processes such as embryonic development, wound healing, tissue and organ regeneration, diabetic retinopathy, and tumor growth(3) . Neovascularization is essential for solid tumor growth and is thought to be regulated by tumor cell-produced factors that have mitogenic and chemotactic effects on vascular endothelial cells(4, 5) .
A variety of growth
factors can stimulate angiogenesis in vitro and in
vivo(6) . Among these angiogenic factors, vascular
endothelial growth factor (VEGF), ()also known as vascular
permeability factor(7, 8) , has been characterized as
an endothelial cell specific growth factor. VEGF/vascular permeability
factor is a homodimeric 34-42-kDa glycoprotein with potent
endothelial cell mitogenic, chemotactic, and vascular
permeability-enhancing
activities(9, 10, 11, 12) . The gene
for human VEGF is organized into 8 exons. As a result of alternative
splicing, at least four transcripts encoding mature monomeric VEGF
containing 121, 165, 189, and 206 amino acid residues
(VEGF
, VEGF
, VEGF
, and
VEGF
) have been identified(13, 14) .
While VEGF
and VEGF
are diffusible proteins
that are secreted into the medium, VEGF
and VEGF
are mainly bound to heparan sulfate proteoglycans on the cell
surface and in the extracellular matrix(15, 16) . VEGF
contains a potential N-linked glycosylation site; the natural
protein is a glycoprotein. Recombinant VEGF expressed in Escherichia coli is indistinguishable from the natural protein
in its in vitro biological actions, suggesting that the
carbohydrate moiety may not be required for activity. VEGF is a highly
conserved protein that has a cross-species activity.
A cDNA encoded protein having 53% amino acid sequence identity in the PDGF-like region of VEGF has recently been isolated from a human placenta cDNA library(17) . This protein, named placenta growth factor (PLGF), is now recognized as a member of the family of vascular endothelial growth factors. Based on its homology with VEGF, PLGF was also proposed to be an angiogenic factor although little is known about its biological functions in vivo(17) . As a result of alternative splicing of the same gene, at least two different mRNAs coding for monomeric PLGF precursors with 149 and 170 amino acids have been described similar to VEGF(18) . The smaller isoform, also called PLGF-1, has a 20-amino acid signal peptide that is cleaved to yield a 129-residue mature protein. The large isoform (PLGF-2) is identical to PLGF-1 except for the insertion of a highly basic 21-amino acid sequence encoded by exon 6. This positively charged sequence increases the binding affinity of PLGF-2 to heparin or heparin-like molecules (18, 19) . Thus, PLGF-1 is readily secreted into the conditioned medium, whereas PLGF-2 is most likely sequestered on the cell surface or in the extracellular matrix.
In normal
tissues, VEGF has been found to be expressed in a variety of cell types
such as activated macrophages, keratinocytes, pancreatic cells,
hepatocytes, smooth muscle cells, and embryonic fibroblasts (14) , whereas the expression of PLGF is limited to the
placental tissue (18) . Both VEGF and PLGF are expressed at
high levels in many tumor tissues. An overlapping expression pattern
was observed in tumors such as renal carcinomas, hepatomas,
glioblastomas, and choriocarcinomas (18, 20, 21) . The expression of VEGF mRNA
can be further up-regulated by hypoxia, phorbol ester, and transforming
growth factor-
(22, 23, 33) . The
regulation of PLGF expression has not yet been reported.
Two receptor tyrosine kinases have been described as putative VEGF receptors. Flt-1 (Fms-like tyrosine kinase) (24) and KDR (kinase insert domain-containing receptor) proteins (25, 26) (the mouse homologue of the latter is called Flk-1; see (27) ), bind VEGF with high affinity. Both KDR/Flk-1 and Flt-1 contain seven immunoglobulin-like domains in the extracellular regions and large insert sequences in their intracellular kinase domains(28) . These receptors are almost exclusively expressed on endothelial cells(29) . In addition to the membrane-spanning Flt-1, a cDNA coding for a truncated soluble form of Flt-1 has been cloned from a human vascular endothelial cell library (30) . PLGF bind with a high affinity to Flt-1, but not to KDR/Flk-1 (31) .
The amino acid sequences of VEGF and PLGF reveal limited but significant (approximately 18%) homologies with A and B chains of PDGF. All eight cysteine residues involved in intra- and interchain disulfides are conserved among these growth factors(14) . In order to study the possible interactions between PLGF and VEGF, we have developed both in vitro and in vivo heterodimerization assays of these two factors. We report here that PLGF forms a heterodimer with VEGF. We show that PLGF/VEGF heterodimers exert overlapping but different biological activities on endothelial cells and on the receptor-mediated signal transductions as compared to PLGF and VEGF homodimers.
For quantitation of PLGF homodimers, 200 µl of an affinity-purified goat anti-human PLGF polyclonal antibody at a concentration of 2 µg/ml was coated onto each well of 96-well microtiter plates. After an overnight incubation, the solution was removed and wells were washed and blocked as mentioned above. At the end of incubation, wells were washed and color development was performed as described above.
For quantitation of PLGF/VEGF heterodimers, 96-well microtiter plates identical to those used in the PLGF homodimer-ELISA were used. However, instead of the horseradish peroxidase-conjugated anti-PLGF polyclonal antibody, an enzyme-conjugated anti-VEGF polyclonal antibody was used for detection. This ELISA assay is specific for detection of PLGF/VEGF heterodimers and shows no cross-reactivity with PLGF or VEGF homodimers.
The I-labeled
PLGF
homodimers, VEGF
homodimers, and
PLGF/VEGF heterodimers were immunoprecipitated by a mouse monoclonal
antibody raised against human VEGF and by a goat anti-human PLGF
antibody. One nanogram of each iodinated protein was incubated at 4
°C with 2 µg of each antibody in a solubilization buffer (150
mM NaCl, 20 mM Tris-HCl, pH 8.0, 5 mM EDTA,
1% Triton X-100) for 2 h. To remove nonspecific binding proteins, 2 ml
of conditioned medium from [
S]methionine-labeled
human JAR choriocarcinoma cells (see above) was preincubated at 4
°C with 50 µl of protein A-Sepharose for 2 h. The supernatants
were incubated at 4 °C for 2 h with 10 µg of the mouse
anti-human VEGF monoclonal antibody and 10 µg of the goat
anti-human PLGF polyclonal antibody, respectively. The immunocomplexes
were precipitated by protein A-Sepharose (Pharmacia). For
co-immunoprecipitation, the
S-labeled materials
precipitated by the anti-VEGF antibody were eluted by the SDS Laemmli
sample buffer(36) . The eluted materials were further
immunoprecipitated with the anti-PLGF specific antibody in the
solubilization buffer containing a final concentration of 0.003% SDS.
The precipitated proteins were harvested by protein A-Sepharose and
were analyzed on a 15% SDS-polyacrylamide gel as described previously.
For chemical cross-linking of iodinated factors to
endothelial cells, monolayers of HUVE cells were grown to confluence in
six-well plates. The conditioned medium was removed, and cells were
washed twice with phosphate-buffered saline. Twenty nanograms of I-PLGF
homodimers,
I-VEGF
homodimers, and
I-PLGF/VEGF heterodimers were added to each well in a
total volume of 1 ml of Dulbecco's modified Eagle's medium
without serum. The mixture was incubated at 4 °C for 1 h, followed
by addition of 20 µl of 10 mM disuccinimidyl suberate
cross-linker and incubated at room temperature for 30 min. Cells were
washed three times with PBS and then lysed with 0.5% Nonidet P-40, 1
mM EDTA, 1 mM phenylmethylsulfonyl fluoride in PBS.
After centrifugation, the soluble fraction of each lysate was analyzed
by a 12% SDS gel.
Figure 1:
Generation of PLGF/VEGF
heterodimers in vitro. A, SDS-PAGE analysis of dimers (lanes 1-4) and monomers (lanes 5-8) of
recombinant PLGF (lanes 2 and 6),
VEGF
(lanes 1 and 5), and
PLGF
/VEGF
under reducing (lanes
5-8) and non-reducing (lanes 1-4) conditions.
The PLGF/VEGF heterodimers (lanes 3 and 4) were
prepared from a mixture of equimolar amounts of PLGF
and
VEGF
monomers in the presence of DTT followed by dialysis
(see ``Experimental Procedures''). About 300 ng of each
protein purified by affinity chromatographies was loaded onto a 12% SDS
gel, followed by silver staining. B, Western immunoblot
analysis of monomers (lanes 5-8) and dimers (lanes
1-4) of recombinant PLGF
, VEGF
,
and PLGF
/VEGF
using a goat anti-human PLGF
polyclonal antibody. Approximately, 50 ng of each protein was separated
on a 12% SDS gel, transferred onto nitrocellulose membrane, and blotted
by the anti-PLGF antibody. The antibody specifically recognized
PLGF
homodimers (lanes 2 and 3),
PLGF
monomers (lanes 6-8), and PLGF/VEGF
heterodimers (lanes 3 and 4). No cross-reactivities
with either VEGF
homodimers (lane 1) or monomers (lane 5) were detected by the antibody. C, Western
blot analysis using a goat anti-human VEGF antibody. The antibody
reacted with either VEGF
homodimers (lanes 1 and 3) or monomers (lanes 5, 7, and 8)
and PLGF/VEGF heterodimers (lanes 3 and 4), but
failed to recognize PLGF homodimers (lane 2) and monomers (lane 6). VV, VEGF
homodimers; PP, PLGF
homodimers; M, mixture of
PLGF
homodimers and VEGF
homodimers and
PLGF
/VEGF
heterodimers; VP,
VEGF
/PLGF
heterodimers. Molecular markers
in kilodaltons (kDa) are indicated on the left side of each
panel.
Western immunoblotting analysis showed that a polyclonal antibody
raised against PLGF specifically recognized both monomers (Fig. 1B, lane 6) and homodimers of
PLGF (lane 2), but not VEGF (lanes 1 and 5). Similarly, an anti-VEGF polyclonal antibody
specifically reacted with VEGF
homodimers (Fig. 1C, lane 1) or monomers (lane
5), but not with PLGF (lanes 2 and 6). The
heterodimeric form of PLGF/VEGF could be detected by the anti-PLGF
antibody (Fig. 1B, lanes 3 and 4) and
the anti-VEGF antibody (Fig. 1C, lanes 3 and 4). From these results, we conclude that the PLGF
and VEGF
monomers expressed from E. coli,
similarly to PDGF A chain and B chain monomers, can be re-folded in
vitro to form PLGF/VEGF heterodimers in addition to their
homodimers.
Figure 2:
Mitogenic activity of recombinant
PLGF homodimers, VEGF
homodimers, and
PLGF/VEGF heterodimers on HUVE cells. Pure PLGF
homodimers, VEGF
homodimers, and PLGF/VEGF
heterodimers were assayed for their abilities to stimulate DNA
synthesis by incorporation of [
H]thymidine. These
factors were serially diluted and added to duplicate wells of 96-well
plates, which were seeded with 5
10
HUVE
cells/well. Forty hours later, [
H]thymidine was
added to each well, and radioactivity incorporated into DNA was
measured 16-24 h later.
) PLGF
homodimers;
, VEGF
homodimers;
,
VEGF
/PLGF
heterodimers. Data represent
(means ± S.D.) of duplicate of each
concentration.
Figure 3:
Chemotactic effects of recombinant
PLGF homodimers, VEGF
homodimers, and
PLGF/VEGF heterodimers on HUVE cells. HUVE cells (1
10
) were seeded in the upper wells of a 48-well plates (1
10
cells/well) of a Boyden chamber and incubated
for 5 h at 37 °C in M199 medium containing different concentrations
of PLGF
homodimers (&cjs2108;), VEGF
homodimers (
), and PLGF/VEGF heterodimer (&cjs2113;) in the
lower wells. Cells migrating through a polycarbonate filter with pore
size of 8 µm were scored. Media without added growth factors
(
) serve as controls. Data represent (means ± S.E.) of
triplicate for each concentration.
Figure 4:
Sensitive ELISA assays for PLGF
homodimers, VEGF homodimers, and PLGF/VEGF heterodimers. A,
analysis of antibody specificity. Thirty nanograms (30,000 cpm/ng) of I-PLGF
homodimers (PP),
I-VEGF
homodimers (VV), and
I-PLGF/VEGF heterodimers (VP) were
immunoprecipitated by a goat anti-human PLGF polyclonal antibody (lanes 1-3) and by an anti-human VEGF monoclonal
antibody (lanes 4-6). Serial dilutions of pure
VEGF
homodimers (B), PLGF
homodimers (C), and PLGF
/VEGF
heterodimers (D) were added to 96-well microtiter
plates, which were precoated overnight with 200 µl of 2 µg/ml
anti-VEGF (A) polyclonal antibody and anti-PLGF polyclonal
antibody (B and C). The bound growth factors were
detected by color development using the horseradish
peroxidase-conjugated secondary anti-VEGF polyclonal antibody (A and C) and/or the horseradish peroxidase-conjugated
anti-PLGF polyclonal antibody (B).
``Sandwich'' enzyme
detection methods were established with these specific antibodies, and
each assay was designed to detect a specific dimer. Typical standard
curves of the sensitive immunoassays for VEGF homodimers (B),
PLGF homodimers (C), and PLGF/VEGF heterodimers (D)
are shown in Fig. 4. As described under ``Experimental
Procedures,'' VEGF homodimer and PLGF homodimer detection assays
revealed less than 10% cross-reactivity with PLGF/VEGF heterodimers,
while the PLGF/VEGF heterodimer immunoassay exhibited no
cross-reactivity with either VEGF homodimers or PLGF homodimers. These
sensitive immunoassays were used to measure concentrations of PLGF
homodimers, VEGF homodimers, and PLGF/VEGF heterodimers secreted into
the conditioned medium from various normal and tumor cell lines (Table 1). Of note, values reported for VEGF and PLGF homodimers
in Table 1have been adjusted for the contributions of
heterodimers as a result of their cross-reactivities in the VEGF
homodimer immunoassay. High concentrations (4.0-8.9 ng/ml) of
PLGF/VEGF heterodimers were detected in conditioned media derived from
the 2-day cultured JAR and JE-3 choriocarcinoma cell lines (Table 1). Equivalent quantities of PLGF homodimers
(4.4-7.1 ng/ml) were also found in the conditioned media of these
cell lines. Approximately, 10-fold lower quantities of VEGF homodimers
were detected in the same conditioned media derived from these cell
lines. These findings are consistent with our data from Northern blot
analysis, namely that the amount of VEGF mRNA synthesized was
10-20-fold smaller than that of PLGF. ()Based on
relative concentrations of PLGF/VEGF heterodimers and VEGF homodimers
in the conditioned media, it appeared that, at least in these cell
lines, there was preference for VEGF to exist as PLGF/VEGF heterodimers
and the excess amount of PLGF protein is present as homodimers.
A
high concentration of PLGF/VEGF heterodimers (1 ng/ml) was also
detected in the conditioned medium of 4MBr Rhesus monkey bronchus
epithelial cells. Smaller amounts of PLGF/VEGF heterodimers (25 pg/ml)
were found in the conditioned media of human HepG2 hepatoma and A431
squamous carcinoma cells. It seems that the amount of PLGF/VEGF
heterodimers produced by a cell line directly correlates with the ratio
of expression levels between PLGF and VEGF. As a control, we also
analyzed the conditioned medium derived from HUVE cells, which
contained a small amount of PLGF homodimers but undetectable levels of
VEGF homodimers and PLGF/VEGF heterodimers. In addition, an overlapping
but distinct pattern of multiple bands with molecular masses ranging
from 36 to 54 kDa was immunoprecipitated by the anti-VEGF monoclonal
antibody, the anti-PLGF antibody, and anti-VEGF/anti-PLGF
antibodies. These results are consistent with previous
findings that both VEGF and PLGF exist as multiple, alternatively
spliced isoforms that may constitute various homodimers as well as
heterodimers via interchain disulfide bridge
formations(14, 18, 19) . From these data, we
conclude that PLGF and VEGF proteins exist as homodimers and PLGF/VEGF
heterodimers in tumor cells that produce both factors.
Figure 5:
Co-immunoprecipitation of the I-labeled PLGF
homodimers, VEGF
homodimers, and PLGF/VEGF heterodimers by a soluble Flk-1-AP
receptor. a, the structure of the Flk-1-AP soluble receptor
fusion protein, consisting of the seven immunoglobulin-like domains of
the Flk extracellular region fused to a human placenta AP tag. b, specificity of the anti-human placenta AP monoclonal
antibody. Approximately 30 ng of
I-labeled PLGF
homodimers (lane 5), VEGF
homodimers (lane 4), and PLGF/VEGF (lane 6) heterodimers were
subjected to immunoprecipitation by the anti-AP antibody. As controls,
I-labeled PLGF
homodimers (lane
2), VEGF
homodimers (lane 1) and PLGF/VEGF
heterodimers (lane 3) without antibody precipitation were
loaded on the same SDS gel. c, NIH-3T3 fibroblast cells
producing the soluble Flk-1-AP receptor were grown to confluence in
Dulbecco's modified Eagle's medium for 3 days and
conditioned medium containing approximately 2.5 µg/ml Flk-AP were
collected. About 30 ng of
I-labeled PLGF
homodimers (PP), VEGF
homodimers (VV), and PLGF/VEGF heterodimers (VP) were incubated
with 500 µl of conditioned medium, and ligand-receptor complexes
were immunoprecipitated by the anti-AP antibody. The precipitated
immunocomplexes were analyzed by SDS-PAGE under reducing
(+DTT) and non-reducing (-DTT)
conditions.
To further
investigate the interaction between VEGF/PLGF heterodimers and Flk-1
receptor, cross-linking experiments were performed using I-labeled factors and the soluble Flk-1-AP receptor.
Cross-linked complexes migrating at positions of 220 and 215 kDa, the
expected sizes of VEGF-Flk-1-AP and PLGF/VEGF-Flk-1-AP complexes, were
detected in the
I-PLGF/VEGF heterodimer (Fig. 6a, VP) and
I-VEGF
homodimer (VV) reactions, but not in the
I-PLGF
homodimer reaction (PP).
The band of
I-PLGF/VEGF-Flk-1-AP complexes on the SDS gel
was less intensive than that of
I-VEGF-Flk-1-AP complex,
which perhaps is due to various amounts of complexes recovered during
experimental procedures. These results are consistent with
immunoprecipitation data showing that PLGF/VEGF heterodimers bind to
Flk-1 receptor (Fig. 5). Our preliminary results using the
soluble Flk-1-AP receptor suggest that PLGF/VEGF heterodimers and VEGF
homodimers bind to KDR/Flk-1 receptor with a similar high affinity as
described previously(23) .
Figure 6:
Cross-linking of I-labeled
PLGF
homodimers, VEGF
homodimers, and
PLGF/VEGF heterodimers to the soluble Flk-1-AP receptor and to the
membrane receptors of HUVE cells. a,
I-PLGF
homodimers (PP),
I-VEGF homodimers (VV), and
I-PLGF/VEGF heterodimers (VP)
(30 ng/ml) were incubated with 5 µg of Flk-1-AP in the presence of
bis(sulfosuccinimidyl) suberate chemical cross-linker. The cross-linked
complexes were immunoprecipitated using the anti-AP antibody and were
analyzed by a 12% SDS gel. The arrow indicates the position of
cross-linked complexes. b, HUVE cells were incubated in
Dulbecco's modified Eagle's medium containing
I-PLGF
homodimers (PP),
I-VEGF
homodimers (VV), and
I-PLGF/VEGF heterodimers (VP) for 2 h. Cells
were washed and incubated with 10 mM disuccinimidyl suberate
cross-linker. Following cross-linking, the reaction was quenched,
washed with PBS, and extracted with 1% Triton X-100. The extracts
corresponding to 10
cells were loaded on a 12% SDS
gel.
Figure 7:
Tyrosine phosphorylation of the membrane
and intracellular proteins of HUVE cells. HUVE cells grown to
confluence were stimulated at 37 °C for 5 min with 100 ng/ml
PLGF homodimers (PP), VEGF
homodimers (VV), and PLGF/VEGF heterodimers (VP). Cells incubated with M199 medium alone were used as
controls (no factor). Cell lysates were separated by a 12% SDS gel and
transferred to a nitrocellulose membrane. The membrane was
immunoblotted with an anti-phosphotyrosine monoclonal antibody. The upper arrow points to the position of a phosphorylated
membrane protein corresponding to the size of KDR receptor. The lower arrow indicates a 38-kDa protein phosphorylated by both
PLGF
homodimers (PP) and PLGF/VEGF heterodimers (VP), but not VEGF homodimers (VV).
VEGF has been identified as a specific mitogen and
chemotactic factor for endothelial cells and also induces blood vessel
permeability (12, 14, 39) . Other effects of
VEGF include induction of plasminogen activator, synthesis of urokinase
receptor and plasminogen activator inhibitor-1, stimulation of hexose
transport in endothelial cells, mobilization of intracellular
Ca, and stimulation of endothelial cell and monocyte
migration in vitro(23) . The recently identified PLGF
shares some of the structural and functional properties of VEGF. For
example, PLGF has significant sequence homology to VEGF including eight
conserved cysteine residues in the PDGF-like domain. Both PLGF and VEGF
can exist as alternatively spliced
multi-isoforms(18, 39) . In addition, PLGF was
originally reported as a potent endothelial cell-specific mitogen in in vitro studies (17) , although subsequent reports
showed that PLGF was only a weak mitogen for endothelial
cells(31, 40) . While biological activities of VEGF
have been well characterized, little is known about functions of PLGF
on angiogenesis both in vitro and in vivo.
In the
present study, we demonstrate that PLGF and VEGF, similar to the A and
B chains of PDGF, can be readily dimerized into homodimers as well as
heterodimers both in cell-free and intracellular systems. The
PLGF/VEGF
heterodimers obtained from in
vitro assays were purified to homogeneity using specific
antibody-coupled affinity chromatographies. Similar to VEGF homodimers,
PLGF/VEGF heterodimers display a mitogenic response for vascular
endothelial cells in vitro. However, the mitogenic activity of
PLGF/VEGF heterodimers with an ED
of approximately 100
ng/ml is 20-50-fold lower than that of VEGF
homodimers (ED
of 2-6 ng/ml). DiSalvo et
al.(40) have shown that PLGF/VEGF heterodimers are nearly
as potent endothelial cell mitogens as are VEGF homodimers. The
discrepancies between these findings and our data may reflect the
differences between endothelial cells used in each assay, which may
express different types and/or levels of VEGF receptors (see below). In
contrast, PLGF
homodimers even at high concentrations
(
1 µg/ml) have little or no effect on stimulation of DNA
synthesis of endothelial cells. Likewise, while both VEGF
homodimers and PLGF/VEGF heterodimers are potent chemotactic
factors for vascular endothelial cells, PLGF homodimers failed to
induce a chemotactic response in these cells. These findings indicate
that PLGF by formation of heterodimers with VEGF may down-regulate the
mitogenic activities but up-regulate the chemotactic effects of VEGF on
endothelial cells. Thus, it is possible that PLGF and VEGF interact
with each other in vivo by a similar pathway, especially in
cells producing both growth factors. This interaction may be important
in the regulation of in vivo angiogenesis, a process that is
involved in many physiological and pathological events such as
embryonic development, tumor growth, wound healing, and diabetic
retinopathy(2) . A large body of work obtained from recent
studies has demonstrated that VEGF is a key regulator of in vivo angiogenesis(41) .
PLGF is expressed in tissues
switched to angiogenic phenotypes such as the placenta and some tumor
tissues, but not in normal adult tissues (18, 21, 42) . To investigate if PLGF and
VEGF form heterodimers in vivo, we have developed two specific
antibodies that react with both monomeric and dimeric forms of PLGF and
VEGF. These specific antibodies allow us to quantitatively detect PLGF
and VEGF homodimers as well as heterodimers secreted by various tumor
cell lines. Interestingly, PLGF and VEGF polypeptides preferentially
form heterodimers in tumor cells such as JAR and JE-3 choriocarcinoma
and 4MBr bronchus cell lines expressing both VEGF and PLGF proteins. In
addition, a recent study reported that PLGF/VEGF heterodimers were also
present in the conditioned medium derived from rat GS-9L glioma cells (40) . These findings suggest that PLGF/VEGF heterodimers may
play an important role in this tumor cell-induced angiogenesis. Our
recent study has shown that JE-3 choriocarcinoma cells develop highly
angiogenic tumors when implanted subcutaneously in immunodeficient
mice, ()although these cells produce low quantities of VEGF
homodimers (Table 1).
Four VEGF and two PLGF isoforms encoded by distinct mRNA splice variants may all participate in various combinatorial homo- and heterodimerizations. These combinations could increase the complexity of secretions, heparin binding affinities, receptor interactions and biological functions of PLGF homodimers, VEGF homodimers, and PLGF/VEGF heterodimers. Co-immunoprecipitation of PLGF/VEGF heterodimers in the conditioned medium of JAR tumor cells with the anti-VEGF monoclonal antibody and the anti-PLGF polyclonal antibody reveals multiple bands with molecular masses ranging from 39 to 55 kDa (data not shown). These experiments support the hypothesis that various isoforms of PLGF and VEGF may be involved in the formation of multiple homo- and heterodimers.
Two high affinity receptors for
VEGF have been characterized, Flt-1 (VEGFR-1) and Flk-1/KDR
(VEGFR-2)(23) . Recently, generation of transgenic mice
deficient in Flk-1 by disruption of the gene indicates that the Flk-1
receptor-mediated signals are essential for yolk sac blood island
formation and vasculogenesis in the mouse embryo(43) .
Transfection and signal transduction studies have shown that Flk-1
mediates signals causing striking changes in cell morphology,
ligand-induced mitogenicity, and chemotaxis, whereas Flt-1-transfected
cells lack such responses to VEGF(28) . To date, the cellular
events that signal through Flt-1 receptor have not been characterized.
However, the fact that Flt-1-knock-out mice are embryonic-lethal due to
a malformation of the vascular system suggests that Flt-1 is an
important angiogenic receptor for endothelial cell differentiation and
vascular organization during embryonic development(44) . In the
present paper, we demonstrate that the PLGF/VEGF heterodimer binds to a
soluble Flk-1 receptor fused to the alkaline phosphatase. The binding
affinity of PLGF/VEGF heterodimers to Flk-1 is similar to that of
VEGF. As VEGF
homodimers,
I-PLGF/VEGF heterodimers can be cross-linked to the
membrane of HUVE cells, which express both Flk-1 and Flt-1 receptors.
These cross-linked complexes are probably due to KDR/Flk-1 receptor
because
I-PLGF
known to bind to Flt-1
receptor is unable to be cross-linked to these cells. One possible
explanation for these observations could be that the HUVE cells used in
our experiments do not express significant amounts of Flt-1 receptor.
Both VEGF homodimers and PLGF/VEGF heterodimers stimulated tyrosine
phosphorylation of a 220-kDa protein, the expected size for the KDR
receptor in HUVE cells. Interestingly, PLGF/VEGF heterodimers and PLGF
homodimers, but not VEGF homodimers induce phosphorylation of a 38-kDa
protein, although PLGF homodimers failed to form cross-linked complexes
with membrane proteins of HUVE cells. These observations suggest that
although VEGF homodimers and PLGF/VEGF heterodimers share the same
KDR-mediated signal pathways, the latter is also able to induce signals
mediated by PLGF homodimers. It is, however, also possible that
PLGF/VEGF heterodimers may interact with an unknown receptor. Our
recent studies have shown that the PLGF/VEGF heterodimer does not bind
to Flt-4 receptor whose ligand has not yet been reported.
The presence of PLGF homodimers, VEGF homodimers, and
PLGF/VEGF heterodimers is analogous to the AA, BB, and AB dimers of the
PDGF family(45) . Interestingly, PDGF-BB homodimers have also
been characterized as an angiogenic factor, which especially induces a
potent chemotactic response from endothelial cells(46) .
Because the eight cysteine residues involving intra- and interchain
disulfide bond formations are all conserved within PDGF and VEGF/PLGF
families, it is possible that PLGF and VEGF may also form various
heterodimers with PDGF-AA and PDGF-BB. The similarity between PDGF and
PLGF/VEGF systems also extends to their high affinity membrane
receptors. Two types of PDGF receptors, and
, have been well
characterized(47) . All PDGF isoforms can induce the formation
of
receptor homodimers, whereas AB and BB, but not AA, are
able to induce the dimerization of
receptor, and only BB is
able to induce
receptor dimerization. Similar to the PDGF
receptor system, Flk-1 and Flt-1 perhaps also form three types of homo-
and heterodimeric receptors upon binding to their corresponding ligands
as represented in Fig. 8. So far, many of these possible
interactions are not known and remain to be characterized. From the
PDGF system, one can speculate that PLGF/VEGF heterodimers are able not
only to induce the homodimerization of Flk-1/Flk-1, but also to induce
the formation of Flt-1/Flt-1 and Flk-1/Flt-1 receptor homodimers and
heterodimers. Thus, further functional characterization of these
receptor dimers that mediate various angiogenic responses would help us
to better understand their roles in the regulation of angiogenesis in vivo.
Figure 8: Schematic representation of possible interactions of PLGF homodimers, VEGF homodimers, and PLGF/VEGF heterodimers with their membrane receptors. VEGF homodimers bind to both Flt-1 (VEGFR-1) and Flk-1 (VEGFR-2) receptors. PLGF/VEGF heterodimers bind to the Flk-1 receptor that mediates mitogenic, chemotactic, and membrane ruffling signals for endothelial cells. The Flt-1 receptor does not mediate such signaling pathways. PLGF homodimers bind only to Flt-1 receptors. Solid arrows represent the characterized interactions between these ligands with their receptors. Dashed arrows indicate the unknown possible interactions. Upon ligand binding, VEGF receptors may form Flk-1/Flk-1 homodimers, Flt-1/Flt-1 homodimers, and Flk-1/Flt-1 heterodimers. Whether the PLGF/VEGF heterodimer is the only ligand for the Flk-1/Flt-1 heterodimeric receptor and what type of biological signals are transduced by the Flk-1/Flt-1 receptor heterodimers by the Flt-1/Flt-1 homodimers remain to be studied.