(Received for publication, September 12, 1994; and in revised form, January 20, 1995)
From the
Vascular endothelial growth factor (VEGF) is a potent and
selective mitogen for endothelial cells that is angiogenic in vivo and induced by hypoxia. A homologous protein, placenta growth
factor (PlGF), is also reported to be mitogenic for endothelial cells
in culture. The rat GS-9L glioma cell line produces not only VEGF
homodimers but also PlGF homodimers and a novel heterodimer composed of
VEGF and PlGF subunits. All three dimeric forms were purified to
apparent homogeneity, and their structures and mitogenic activities
were compared. VEGFPlGF heterodimers are vascular endothelial
cell mitogens nearly as potent as VEGF homodimers. Therefore, some of
the biological activities attributed to VEGF homodimers might be
mediated by VEGF
PlGF heterodimers. In contrast, pure PlGF
homodimers are mitogenic for endothelial cells only at high, possibly
non-physiologic concentrations; thus the biological relevance of their
mitogenic activity for these cells is not obvious. However, the
existence of not only homodimers but also heterodimers clearly extends
the similarity between the VEGF/PlGF and the homologous
platelet-derived growth factor systems.
Vascular endothelial cells form a luminal non-thrombogenic
monolayer throughout the vascular system. Mitogenesis of these cells is
required for embryonic vascular development, growth, and repair.
Although multiple growth factors have been reported to be mitogenic for
vascular endothelial cells(1) , only vascular endothelial
growth factor (VEGF) ()has been established to be highly
selective for these
cells(2, 3, 4, 5, 6, 7, 8) .
Furthermore, in contrast to the broad spectrum acidic and basic
fibroblast growth factors, which do not contain classical secretory
leader sequences(9, 10) , the full-length translation
products of VEGF and the homologous protein, placenta growth factor
(PlGF) include amino-terminal signal sequences consistent with their
active secretion.
VEGF (11, 12, 13, 14) and PlGF (15) are glycosylated homodimers that share 46% amino acid sequence identity with each other (16) and are 18-20% identical to the amino acid sequences of platelet-derived growth factor (PDGF) A and B subunits. As in the PDGF system, alternative splicing generates multiple forms of VEGF (17) and PlGF(18) . Human VEGF exists in four mature processed forms containing 206, 189, 165, and 121 amino acids, the most prevalent being the 165-amino acid form(17) . The 206- and 189-amino acid forms each contain a highly basic 24-amino acid insert that promotes tight binding to heparin and, presumably, heparan proteoglycans on cellular surfaces and within extracellular matrices (19) . Differential splicing of human PlGF mRNA leads to either 132- or 153-amino acid residue mature proteins(18) . The 21-amino acid basic insert near the COOH terminus of the longer form also confers heparin binding(16) . Analogous to VEGF, this basic insert could partition the protein onto cell surface and extracellular matrix heparan proteoglycans and, perhaps, might facilitate nuclear binding. It is presently not clear, however, if any biologically functional differences exist among the alternatively spliced forms of VEGF and PlGF.
VEGF appears to be relevant for vascular growth and function. VEGF mRNA expression has been shown to be related temporally and spatially to the development of the embryonic vascular system (20, 21) and to physiological angiogenesis in the female reproductive system(22) . Hypoxia has been shown to increase VEGF mRNA levels in cultured cells (23, 24, 25) , and ischemic cardiac myocytes express elevated VEGF levels both in vitro and in vivo(26) . In addition to its angiogenic properties, VEGF can under certain circumstances induce vascular permeability; hence it is also known as vascular permeability factor(2) .
During purification of VEGF from rat GS-9L glioma cell-conditioned media, we observed that two chromatographic peaks of vascular endothelial cell mitogenic activity eluted from a cation exchange HPLC column(6) . The latter purer peak was the focus of the original purification effort that yielded homodimeric VEGF(14) . We now report the purification to apparent homogeneity of another vascular endothelial cell mitogen from the earlier eluting peak of mitogenic activity. This newly identified protein is a heterodimer composed of both VEGF and PlGF subunits. In addition, we have purified a PlGF homodimer from these same conditioned media. We compare the biological and physical characteristics of this pure heterodimer with VEGF and PlGF homodimers purified from the same source.
Homodimeric PlGF was initially
fractionated by sequential chromatography on CM-Sephadex C-50,
concanavalin A-Sepharose lectin affinity, and poly(aspartic acid) WCX
HPLC columns as described for the VEGF homodimer and VEGFPlGF
heterodimer purifications. Further fractionation of homodimeric PlGF
relied on the identification of immunocross-reactive protein by Western
analysis using antisera made to a synthetic polypeptide corresponding
to amino acid residues 31-50 of the longer mature form of rat
PlGF conjugated to a tuberculin-purified protein derivative as
described(27) . A second antiserum was made to a synthetic
polypeptide identical in sequence to residues 1-24 of mature rat
VEGF. These two antisera were used to identify chromatographic
fractions containing either one or both of the PlGF and VEGF subunits.
Fractions from the poly(aspartic acid) column containing PlGF but not
VEGF immunocross-reactive bands were pooled and chromatographed by
sequential fractionation on a 5
0.46-cm Vydac C
reversed phase HPLC column followed by a 5
0.1-cm C
microbore column as described for the heterodimer purification.
Figure 1:
Purification of the VEGFPlGF.
Serum-free conditioned media were fractionated by CM-Sephadex C-50 and
concanavalin A-Sepharose as described(6) . A, active
fractions were pooled, loaded onto a poly(aspartic acid) WCX HPLC
column in 50 mM sodium phosphate, pH 6.0, and eluted at 0.75
ml/min with a linear gradient of 0-1 M NaCl. Two peaks
of HUVE cell mitogenic activity were observed, and each was pooled (horizontalbars). Material in pool 2 was purified to
yield homodimeric VEGF(6) . B, fractions from the
first active peak were combined (pool 1), loaded onto a C
reversed phase HPLC column equilibrated in 10 mM trifluoroacetic acid, and eluted with a linear gradient of
0-67% acetonitrile. C, mitogenically active fractions
were again pooled and rechromatographed using a microbore C
column equilibrated in 10 mM trifluoroacetic acid and
eluted with a linear gradient of 0-50%
acetonitrile.
Compared with homodimeric VEGF that has an
apparent nonreduced mass of 43.5 kDa, the newly purified nonreduced
VEGFPlGF mitogen migrates as major 49.5-kDa and trace
40-kDa
bands (Fig. 2A, lanes1 and 2). On
reducing SDS-PAGE, pure VEGF homodimer migrates at its subunit mass of
27 kDa, whereas this novel mitogen separates into two principal bands
of 27 and 31 kDa (Fig. 2B, lanes1 and 2). Immunocross-reactive VEGF was demonstrated to
exist in both nonreduced proteins (Fig. 3A, lanes1 and 2) and to correspond to the reduced 27-kDa but not
the 31-kDa band (Fig. 3B, lanes1 and 2). Barely detectable lower mass immunocross-reactive VEGF
bands present in the nonreduced (Fig. 3A, lane1,
29 kDa) and reduced (Fig. 3B, lane1,
21 kDa) VEGF lanes are presumable trace
degradation products not detectable on the corresponding silver-stained
gels (Fig. 2A, lane1; Fig. 2B, lane1). Traces of
incompletely reduced immunocross-reactive dimers are also occasionally
detected on long exposures (e.g.
50-kDa bands in Fig. 3B, lanes1 and 5). The
masses of the dimers, calculated from the more accurate reduced subunit
masses, are 54 kDa for the VEGF homodimer and 58 kDa for VEGF
PlGF
heterodimer.
Figure 2:
Purity of VEGF, VEGFPlGF, and PlGF.
Purities of VEGF, VEGF
PlGF, and PlGF (100 ng each) were
determined by electrophoresis through SDS-14% polyacrylamide gels under
nonreducing (A) and reducing (B) conditions followed
by silver staining. Left side, molecular mass standards; lane 1, VEGF; lane 2, VEGF
PlGF; lane
3, PlGF.
Figure 3:
Western analysis of VEGF, VEGFPlGF,
and PlGF. Pure VEGF, VEGF
PlGF, and PlGF (100 ng each) were
analyzed using subunit-specific antisera after electrophoresis through
SDS-14% polyacrylamide gels under nonreducing (A) and reducing (B) conditions, followed by electrophoretic transfer to
Immobilon-P polyvinylidene difluoride membranes. Pure VEGF (lane
1), VEGF
PlGF (lane 2), and PlGF (lane 3)
were probed with a 1:1000 dilution of VEGF-specific antiserum.
Additionally, VEGF (lane 4), VEGF
PlGF (lane 5),
and PlGF (lane 6) were probed with a 1:1000 dilution of
PlGF-specific antiserum. Bound antibody was incubated with
I-protein A followed by overnight exposure and
visualization using a PhosphorImager.
Despite unequal silver staining intensity (Fig. 2B, lane2), these two reduced
and carboxymethylated polypeptides derived from the heterodimeric
protein are chromatographically resolved as peaks (Fig. 4, peaks 1 and 2) of virtually identical absorbance
(area ratio of 1.00:1.03) at 210 nm eluted from a reversed phase
C HPLC column. The second of the two eluted protein peaks
is confirmed by NH
-terminal (APTTEGEQKAHEVV) and
polypeptide sequencing to be the previously identified (14) 164-amino acid subunit present in VEGF homodimers (data
not shown). However, the first peak, subsequently identified to be a
rat homologue of PlGF, is distinct by both NH
-terminal
amino acid sequencing (ALSAGNXSTEMEVVPFNEV and an equal amount
of this sequence missing the first 3 residues) and sequence analysis of
a family of polypeptides purified on a C
reversed phase
HPLC column from a Lys-C digest. Therefore, this newly purified mitogen
appears to be a heterodimer composed of VEGF and PlGF subunits. Amino
acid analysis of each reduced and carboxymethylated subunit peak (Fig. 4) combined with their polypeptide masses calculated from
the VEGF and subsequently determined PlGF amino acid sequences confirms
a very similar molar ratio (1.00:1.05), demonstrating that the
heterodimer is composed of one VEGF and one PlGF subunit.
Figure 4:
Subunit composition of VEGFPlGF
heterodimer. Reduced and carboxymethylated heterodimer subunits were
purified on a microbore C
reversed phase HPLC column
equilibrated in 10 mM trifluoroacetic acid and eluted with a
0-50% linear gradient of acetonitrile. The third major peak,
eluting at an effluent volume of
9.3 ml, was present in the
reagent blank. Peak 1 was subsequently determined by amino-terminal and
peptide sequencing to correspond to the PlGF subunit, and peak 2 was
identified as the VEGF subunit.
Figure 5: Amino acid sequence homology between rat and human PlGF. Complete amino acid sequences of rat and human PlGF were compared. Identical residues are enclosed in boxes. The locations of the mature amino termini of the polypeptides are identified by singlearrowheads, and asparagine residues in potential glycosylation sequences are denoted by asterisks. The location of the polybasic insert is designated by the two-headed arrow.
The fully processed rat subunit amino acid
sequence is 60% identical (Fig. 5) to human PlGF (15) increasing to 66% within the putative receptor-binding
core structure corresponding to the PDGF homologue v-sis. By
both Southern blot analysis and determination of cDNA sequences of
several species ()this second subunit is identified as the
rat counterpart of PlGF. Therefore, we adopted the PlGF nomenclature
for consistency despite the glioma cell source of the protein.
The
presence of VEGF but not PlGF in the VEGF homodimer and both VEGF and
PlGF bands in the VEGFPlGF heterodimer were subsequently
confirmed by Western blots of nonreducing (Fig. 3A, lanes1, 2, 4, and 5) and
reducing (Fig. 3B, lanes1, 2, 4, and 5) gels using individual
peptide-based antisera specific to VEGF and PlGF subunits.
Immunostaining in the region of the trace
40-kDa band seen on
silver-stained nonreduced SDS-PAGE (Fig. 2, lane2) is also detected on nonreduced Western gels (Fig. 3A, lane2). A corresponding
minor band was not detected after reduction, so it might reflect a
minor form generated by disulfide shuffling under nonreduced denaturing
conditions that is more efficiently recognized by the anti-VEGF than
the anti-PlGF antisera. Consistent with the amino acid sequence data,
the slower and faster migrating major bands on the blot of the reducing
gel of the heterodimer cross-react with the anti-PlGF and anti-VEGF
antisera, respectively.
Figure 6:
Purification of the PlGF homodimer.
Initial purification of PlGF homodimer by sequential chromatography on
CM-Sephadex C-50 and concanavalin A-Sepharose was performed as
described for the VEGF homodimer and VEGFPlGF heterodimer
purification. A, fractions specifically eluted from the lectin
affinity column were chromatographed on a poly(aspartic acid) WCX HPLC
column in 50 mM sodium phosphate, pH 6.0, and eluted at 0.75
ml/min with a linear gradient of 0-1 M NaCl over 60 min.
Fractions containing PlGF homodimer were identified by Western analysis
probing with both VEGF and PlGF antisera (inset). Fractions
from the poly(aspartic acid) column that contained PlGF but not VEGF
immunocross-reactive bands were pooled (solidbar)
and loaded onto a C
reversed phase HPLC column (B), and protein was eluted as described for heterodimer
purification. Fractions containing PlGF immunocross-reactive bands (B, inset) were pooled and rechromatographed on the
same column (C), and the major peak was chromatographed on a
microbore C
column (D) as described under
``Experimental Procedures.''
Figure 7:
Mitogenic activity of purified VEGF and
PlGF homodimers and heterodimer. Pure VEGF (), VEGF
PlGF
(
), and PlGF (
) were assayed as a function of
dose-monitoring [methyl-
H]thymidine
incorporation into the DNA of nearly confluent HUVE
cells.
We previously identified two resolved peaks of vascular
endothelial cell mitogenic activity eluting from a cation exchange
column of partially purified conditioned medium from rat GS-9L glioma
cells(6) . Active VEGF homodimer in the later eluting peak was
purified to homogeneity, sequenced, cloned, and shown to be homologous
to PDGF A and B subunits. Similar human VEGFs were concurrently
purified and cloned by several other groups(11, 12) .
Although we originally purified and partially characterized limited
amounts of VEGF PlGF heterodimer from conditioned media of cells
grown under ambient oxygen,
based on reports of hypoxic
induction of VEGF expression (23, 24, 25) we
subsequently lowered the O
levels used for conditioning
media to only 3%. Both previously identified peaks of mitogenic
activity eluted from an anion exchange HPLC column (6) increased
5-fold, although the second VEGF homodimer
peak typically contained more activity than the preceding
VEGF
PlGF heterodimer peak.
We have purified the growth factor
in the first detectable endothelial cell mitogenic peak eluted from the
anion exchange HPLC column. The nonreduced protein migrates with an
apparent mass of 49.5 kDa on SDS-PAGE. Upon reduction, the constituent
subunits migrate on SDS-PAGE as polypeptides of 27 and 31 kDa,
consistent with the possibility that the newly purified mitogen is
dimeric. Although the larger mass polypeptide does not stain as
intensely with silver as the lower mass band, both reduced and
alkylated polypeptides exhibit equal absorbance at 210 nm that,
confirmed by mass from amino acid analysis, demonstrates 1:1
stoichiometry of the two chains. Amino-terminal and peptide
microsequencing of the purified polypeptides confirms the presence of
VEGF and a second distinct subunit. Western blots of nonreduced and
reduced protein using specific peptide-based antisera unique for rat
VEGF and, after determination of its sequence, PlGF confirm that this
molecule contains both subunits. Therefore, based on its mass, subunit
content, and stoichiometry, this newly identified endothelial cell
mitogen is a heterodimer composed of VEGF and PlGF subunits. Like VEGF
homodimers, the VEGFPlGF heterodimer is mitogenic for vascular
endothelial cells although with an approximately 3-fold higher
ED
.
Reverse transcriptase PCR cloning, using primers
based on the peptide sequences, establishes rat PlGF as a full-length
158-amino acid residue chain that, based on NH-terminal
sequencing, is processed to a 135-residue form that is 60% identical to
human PlGF. While lower than the 89% identity between rat and human
VEGF, the PlGF homology is still within the range exhibited between rat
and human growth factors. We were not able to find a rat gene more
closely related to human PlGF by Southern analysis.
Furthermore, on the basis of cDNA sequence from several mammalian
species, this gene appears to accommodate mutations at a higher
frequency than the homologous VEGF gene.
Two
amino-terminal rat PlGF sequences were observed following removal of
either a 23- or a 26-amino acid residue leader sequence. Either these
two mature NH termini are generated by alternative signal
peptidase cleavage sites or subsequent NH
-terminal
proteolysis removes 3 amino acid residues after the primary processing
event. Despite the shorter polypeptide chain length, the PlGF subunit
migrates at a higher mass (31 kDa) than the VEGF subunit (27 kDa). This
discrepancy is presumably attributable to more extensive PlGF
glycosylation at multiple locations, including at least 2 of 3
consensus N-glycosylation sites, compared with the single N-glycosylation site present in rat VEGF.
The second of the two newly characterized rat proteins, a homodimer of PlGF, was only identified and purified after specific anti-PlGF antisera were generated since it was not detectably active at the concentrations present in partially purified conditioned media. Once purified, detectable but low specific mitogenic activity for HUVE cells was observed. Human PlGF was cloned and expressed, and crude conditioned medium containing recombinant protein was reported to be mitogenic for a bovine aortic endothelial cell-derived line in culture (15, 16) . However, since the protein was not purified, its potency could not be determined and compared with that of VEGF. Furthermore, the presence of some of the recombinant human PlGF in more active heterodimers with host cell VEGF was not excluded.
The low activity of the rat PlGF homodimers is consistent with the
very weak mitogenic activity of purified human recombinant homodimeric
PlGF(34) compared with recombinant human
VEGF
PlGF heterodimers and VEGF homodimers. The low mitogenic
activity of human recombinant PlGF does not appear to be the
consequence of inappropriate folding or denaturation since it binds
purified soluble FLT VEGF receptors with high affinity(35) .
PlGF does not, however, bind tightly to the KDR receptor. In contrast,
VEGF binds to extracellular recognition domains of both FLT and KDR.
Taken together, these binding and activity data indicate that FLT is
not abundant on these endothelial cells, is selectively inactivated by
heterodimer formation with a dominant negative soluble FLT expressed by
endothelial cells (36) , or, perhaps, cannot efficiently
transduce a mitogenic signal. PlGF homodimers might be better mitogens
for other types of vascular endothelial cells. If so, then the
endothelial cell range of the heterodimers could be broader than that
of either of the homodimers. Alternatively, PlGF homodimers might more
efficiently stimulate as yet unidentified non-endothelial cell targets.
These data clearly demonstrate that rat glioma cells produce the two
related VEGF and PlGF subunits and that all three possible combinations
of homodimers and heterodimers are produced. The presence of PlGF and
VEGF homodimers and heterodimers is formally analogous to the presence
of AA, BB, and AB dimers in the homologous PDGF system. The choice of
the type of PDGF dimer predominantly expressed in platelets exhibits
species variation. In human platelets AB heterodimers are principally
expressed(37) , whereas BB homodimers are most abundant in
pigs(38) . The physiological consequences of the type of PDGF
dimers that are expressed are not obvious. Similarly, given the nearly
equivalent mitogenic activities of VEGF homodimers and VEGFPlGF
heterodimers, a substantial advantage of expressing one or the other
dimer is not apparent. VEGF has been shown to be very specific for
vascular endothelial cells, being inactive on fibroblasts, vascular
smooth muscle cells, corneal endothelial cells, adrenal cortical cells,
lens epithelial cells, baby hamster kidney cells, granulosa cells,
Balb/MK keratinocytes, and Balb/c 3T3
fibroblasts(3, 4, 5, 6) . Although
VEGF
PlGF is also inactive on Balb/c 3T3 fibroblasts,
neither it nor PlGF homodimers have yet been assayed on a wide
variety of cells in culture.
The similarity between the PDGF and
VEGF/PlGF systems extends to their receptors. Two types of homologous
PDGF receptors, and
, have been described and characterized.
Whereas
receptors bind all three isoforms with high affinity,
receptors only recognize PDGF BB with high affinity and PDGF AB
with lower affinity but do not bind PDGF AA. In addition, PDGF binding
induces receptor dimerization such that PDGF AA dimerizes
receptors, AB promotes formation of
and
dimers,
and BB induces formation of all three receptor dimers(39) . It
is likely that the analogous situation occurs in the VEGF
PlGF
family that also consists of two known receptors, FLT and KDR, which
are as homologous to each other as are the PDGF
and
receptors. We can then speculate that VEGF
PlGF heterodimers might
be able to induce the formation of not only FLT
FLT receptor
homodimers but also KDR
FLT receptor heterodimers analogous to
that observed in the PDGF system. Given that both VEGF and PlGF can
exist as alternatively spliced forms and that both homo- and
heterodimeric ligands and receptors are possible, the potential exists
for extensive modulation of response in the VEGF system. Further
functional characterization of the multiple dimeric VEGFs and their
interactions with their receptors will be necessary to determine if
differential responses to these mitogens occur.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) L40030[GenBank].