Angiogenesis, the growth of new vessels, is required for a wide
variety of physiologic and pathologic proliferative processes. Recent
evidence implicates vascular endothelial growth factor (VEGF), (
)an endothelial-cell specific mitogen and angiogenesis
inducer(1, 2) , as a critical regulator of normal and
pathologic angiogenesis(3) . VEGF mRNA expression is temporally
and spatially related to proliferation of blood vessels in the ovarian
corpus luteum or in the developing embryo(4, 5) .
Furthermore, recent studies have shown that monoclonal antibodies
specific for VEGF are able to suppress the growth of human tumor cell
lines in nude mice, suggesting that VEGF is an important mediator of
tumor angiogenesis(6) . VEGF mRNA undergoes alternative
splicing events that lead to the production of four mature homodimeric
proteins, each monomer having 121, 165, 189, or 206 amino acids
(VEGF
, VEGF
, VEGF
, and
VEGF
, respectively)(7, 8, 9) .
VEGF
does not bind to heparin; in contrast, VEGF
and VEGF
bind to heparin with increasingly greater
affinity. Cells transfected with cDNA encoding VEGF
or
VEGF
secrete bioactive VEGF into the medium. In contrast,
when VEGF
and VEGF
were expressed in
mammalian cells, little or no VEGF can be found in the
medium(9) . In previous studies, we demonstrated that
VEGF
or VEGF
are secreted but are almost
completely bound to heparan sulfate containing proteoglycans in the
extracellular matrix(10, 11) . However, a diffusible
fragment having qualitatively the same activity as intact VEGF could be
released by plasmin. Extracellular matrix derived from cells expressing
VEGF
or VEGF
and, to a lesser extent,
VEGF
, promotes the growth of vascular endothelial cells,
demonstrating that matrix-bound VEGF is bioactive(11) .
In
the present study, we studied the interaction with heparin to address
the biologic significance of the larger molecular forms of VEGF. We
isolated and characterized plasmin-generated fragments of VEGF
and compared these fragments to native VEGF
or
VEGF
with respect to various biochemical and biological
functions. Our studies demonstrate that loss of the carboxyl-terminal
domain, whether due to proteolysis or alternative splicing, correlates
with a substantial decrease in endothelial cell mitogenic activity of
VEGF.
EXPERIMENTAL PROCEDURES
Materials
Iodine-125 radionuclide was purchased
from DuPont NEN. Chloramine T hydrate (N-chloro-p-toluene sulfonamide) and sodium
metabisulfite were purchased from Aldrich. Tris and sodium phosphate
salts (monobasic and dibasic) were obtained from Calbiochem and
Scientific Products/Mallinckrodt, respectively. Hydrochloric acid,
trichloroacetic acid, and Tween 20 (polyethylene-20-sorbitan
monolaurate) were from Fisher Scientific. Bovine serum albumin and
aprotinin were purchased from Sigma (St. Louis, MO). Gel filtration
columns (PD-10), S-Sepharose columns, metal-chelating Sepharose and
heparin-Sepharose were from Pharmacia Biotech Inc. Reverse phase and
heparin columns were from Vydac (Hesperia, CA) and PerSeptive
Biosystems (Cambridge, MA), respectively. Acetonitrile was HPLC-grade
from J.T. Baker. The sodium salt of porcine intestinal heparin at 1000
USP units/ml was purchased from Elkins-Sinn, Inc. (Cherry Hill, NJ).
Plasmin was from Helena Laboratories (Beaumont, TX). Trypsin,
chymotrypsin, Pronase and elastase were from Calbiochem, and
collagenase, clostripain, proteinase K, and Staphylococcus aureus V8 protease were from Worthington Biochemical (Freehold, NJ).
Bromelain, subtilisin, pepsin, and thermolysin were from Boehringer
Mannheim. Thrombin was kindly provided by Dr. Walter Kisiel of the
University of New Mexico. Affinity-purified rabbit anti-human IgG, Fc
specific antibodies and polystyrene 96-well breakaway microtiter plates
were purchased from Cappel Laboratories (West Chester, PA) and Dynatech
(Chantilly, VA), respectively. Fetal calf serum was from HyClone
(Logan, UT). The construction, expression and purification of FLT-1 and
KDR receptor-IgG chimeras was as described by Park et
al.(12) .
VEGF
Expressed and Purified from Chinese
Hamster Ovary Cells
Recombinant human VEGF
was
purified (1) from media conditioned by transfected Chinese
hamster ovary cells (7, 13) , as described previously.
Harvested cell culture fluid was collected after 7 days and
concentrated 10-fold by ultrafiltration. The concentrate was
diafiltered into 20 mM Na phosphate at pH 7. VEGF was purified
by cation exchange chromatography on S-Sepharose and eluted with a
gradient of NaCl. Further purification was obtained by metal-chelating
chromatography, followed by hydrophobic interaction chromatography
using gradient elutions in imidazole and ammonium sulfate,
respectively. VEGF
was formulated by gel filtration on
G25 Sephadex in 125 mM NaCl and 10 mM sodium citrate
at pH 6. Identity was established by SDS-PAGE, reverse phase HPLC,
amino-terminal sequencing and amino acid composition.
VEGF
and VEGF
Expressed,
Refolded, and Purified from Escherichia coli
We have recently
developed a procedure for preparing unglycosylated VEGF from bacterial
expression. Transformed E. coli cells were lysed by
sonication, and the VEGF
(or VEGF
) protein
was recovered in an insoluble pellet after centrifugation. The pellet
was washed with 4 M urea in 20 mM Tris buffer at pH 8
with 5 mM EDTA before solubilization by addition of 25 mM dithiothreitol to the wash buffer. The extraction was allowed to
continue for 2 h with stirring at 4 °C before centrifugation to
remove insoluble bacterial components. The extract was then dialyzed
overnight against 0.4 M NaCl, 20 mM Tris-HCl, pH 8 at
4 °C during which time the extracted protein was allowed to
re-fold. The dialyzed, re-folded VEGF
was purified by
adsorption to a cation exchange resin (S-Sepharose) and elution with a
gradient of 0.4-1.0 M NaCl. Fractions containing dimeric
VEGF
(or VEGF
), as determined by SDS-PAGE,
were pooled and the protein further purified by C4 reverse phase
chromatography in 0.1% trifluoroacetic acid with elution by an
acetonitrile gradient. VEGF
and VEGF
eluted
in approximately 30% acetonitrile. Identity was established by SDS-PAGE
under both reducing and non-reducing conditions, by peptide sequencing,
amino acid analysis, and mass spectrometry. Refolding of E.
coli-derived VEGF was evaluated by comparison with CHO-derived
VEGF
using monoclonal antibody-based enzyme-linked
immunosorbent assay, heparin affinity, and receptor binding as
described under ``Results.''
Enzyme Digests of VEGF
Initial screening of
plasmin, thrombin, elastase, collagenase, trypsin, chymotrypsin,
pepsin, subtilisin, clostripain, bromelain, Pronase, proteinase K,
thermolysin, and S. aureus V8 protease for digestion of VEGF
was done at enzyme to substrate ratios of 1:100 (by weight) at 25
°C for 24 h at pH 7. Digests were stopped by freezing the samples
prior to analysis by reverse phase HPLC and SDS-PAGE under reducing and
non-reducing conditions.
Preparation of VEGF
,
VEGF
, and the Carboxyl-terminal polypeptide
(111-165) from Plasmin Digestion of
VEGF
Plasmin was added to purified VEGF
(1:200 ratio by weight) and incubated at 25 °C, pH 7.4, for 4
h for the isolation of VEGF
or 24 h for the isolation
of VEGF
and carboxyl-terminal polypeptide
(111-165). A schematic diagram depicting the plasmin cleavage of
VEGF is shown in Fig. 1. The plasmin digests were stopped at the
appropriate time by the addition of aprotinin at a 10-fold molar excess
with respect to plasmin. Partially digested VEGF (4 h digest) were
separated on a POROS heparin HE2 column as described below. The limit
digest of VEGF (24 h) was applied to a heparin-Sepharose column (1.5
10 cm) pre-equilibrated in 0.15 M NaCl, 50 mM phosphate buffer at pH 7 (PBS). VEGF
eluted from the
heparin column in the void volume. After the column was washed (with 10
column volumes of PBS), the carboxyl-terminal polypeptide
(111-165) was eluted with 1 M NaCl, 50 mM phosphate at pH 7. The protein containing fractions were dialyzed
against PBS. Purity and identity was assessed by SDS-PAGE, amino acid
sequence and composition, reverse phase HPLC, and mass spectrometry.
Figure 1:
Plasmin cleavage of human
VEGF
. This schematic diagram depicts the sequential
cleavage of the Arg
-Ala
bond in each of the
VEGF monomers. The monomers of VEGF are shown in anti-parallel
orientation, with the carboxyl termini in distant positions, according
to homology with the platelet-derived growth factor
dimer crystal
structure(35) . The monomers of a VEGF
dimer are
covalently linked via two interchain disulfide bonds shown as black
bars. Within the 1-110 and 111-165 domains of each
monomer, there are three and four intrachain disulfide bonds,
respectively (not shown). There are no interdomain disulfides linking
the receptor binding domain (1-110) to the carboxyl-terminal
domain (111-165); such that following plasmin cleavage, the
55-amino acid carboxy polypeptides are released from the VEGF
dimer. VEGF
, VEGF
, and
VEGF
dimers are shown in the top, middle, and bottom,
respectively.
Radiolabeling of VEGF
VEGF
was
radiolabeled using a modification of the chloramine T-catalyzed
iodination method described by Hunter and Greenwood(14) . In a
typical reaction, 50 µl of 1 M Tris-HCl, 0.01% Tween 20 at
pH 7.5 was added to 5 µl of sodium iodide-125 (0.5 mCi) in a capped
reaction vessel. An aliquot of VEGF (10 µg/10 µl) in 125 mM NaCl, 10 mM sodium citrate at pH 6 was added to the
reaction vessel. Iodination was initiated by addition of 12.5 µl of
1 mg/ml chloramine T in 0.1 M sodium phosphate, pH 7.5. After
60 s, iodination was terminated by addition of sodium metabisulfite (25
µl, 1 mg/ml) in 0.1 M sodium phosphate, pH 7.5. The
reaction vessel was vortexed after each addition. The reaction mixture
was immediately applied to a PD-10 column (G25 Sephadex) that was
pre-equilibrated with 0.5% bovine serum albumin, 0.01% Tween 20 in
phosphate-buffered saline. Fractions were collected and counted for
iodine-125 radioactivity with a
scintillation counter (LKB model
1277). Typically, the specific radioactivity of the iodinated VEGF was
26 ± 2.5 µCi/µg, which corresponded to approximately 1
I/2 molecules of VEGF
dimer. There are four
tyrosines in VEGF
at positions 21, 25, 39, and 45.
Tryptic mapping of RCM VEGF
indicated approximately 30%
and 70% of the radioactivity co-eluted with tryptic peptides T2 (amino
acids 17-23) and T3 (amino acids 24-56), respectively.
Dodecyl Sulfate Gel Electrophoresis
Radiolabeled
samples were either reduced, or reduced and carboxymethylated (RCM)
prior to gel electrophoresis. The reduced samples were denatured in 1%
SDS with 10 mM dithiothreitol (DTT) and heated at 37 °C
for 30 min. Samples for reduction and carboxymethylation were treated
by a modified procedure of Crestfield et al.(15) .
Those samples were dialyzed overnight in 8 M urea, 0.5 M Tris-HCl at pH 8.3 with 5 mM EDTA, then reduced with 10
mM DTT at 37 °C for 30 min. Iodoacetic acid (1 M IAA in 1 M NaOH) was added to a final concentration of 25
mM and incubated at 25 °C for 15 min. The alkylation
reaction was quenched by addition of 25 mM DTT, followed by
overnight dialysis at 4 °C with 50 mM NH
CO
. Electrophoretic analysis was by the
method of Laemmli (16) on a SDS gradient acrylamide gel
(10-20%), followed by either Coomassie Blue or ammoniacal silver
staining(17) . After the stained gels were dried, the
radiolabeled proteins were visualized by autoradiography.
Amino Acid Analysis and Ultraviolet Spectroscopy
A
Beckman 6300 amino acid analyzer was used with a sodium citrate program
and ninhydrin detection. Aliquots (10 µl each) of VEGF samples were
hydrolyzed in constant boiling HCl for 24 h at 110 °C. Quantitation
was based on the yields of alanine and leucine. VEGF samples were
diluted in 125 mM NaCl, 10 mM sodium citrate at pH 6
and scanned for UV absorption from 190 to 800 nm on a Hewlett Packard
8452A diode array spectrophotometer. The molar extinction coefficient
for VEGF
was determined as 0.37 absorbance units for a 1
mg/ml solution at 276 nm.
Mass Spectrometry
The molecular weights of VEGF
variants were analyzed using a Sciex API III triple quadrupole mass
spectrometer. The data was obtained by scanning from 300 to 2000 Da
with a 0.8-ms dwell time per mass step. A Harvard infusion pump was
used to introduce the samples into the mass spectrometer at a flow rate
of 3-5 µl/min. The data was collected in the data summing
mode and analyzed using MacBioSpec software.
Reverse Phase HPLC
Analytical separations of VEGF
fragments were done using a Vydac C
, 5-µm bead,
300-Å pore size column (4.6
250 mm) pre-equilibrated in
0.1% trifluoroacetic acid, 5% acetonitrile at 40 °C on a Hewlett
Packard 1090 liquid chromatograph with diode array detection.
Typically, an aliquot of VEGF (10 µl) containing 30 µg was
injected onto the HPLC column with a flow rate of 1 ml/min. Reverse
phase separation of VEGF and VEGF fragments was done with a two-step
gradient from 5% to 25% acetonitrile with 0.1% trifluoroacetic acid for
20 min, followed by a shallow gradient from 25% to 33% in 32 min. The
effluent was monitored for absorbance (at 210 and 280 nm).
Heparin Affinity Chromatography
Analytical and
preparative affinity chomatography was performed on a HP1090 liquid
chromatograph using a POROS HE2 heparin column (4.6
100 mm).
The column was pre-equilibrated with 50 mM Na
PO
buffer at pH 7.4 using a 0.5 ml/min
flow rate at 40 °C. Samples of VEGF were injected and eluted with a
salt gradient from 0 to 1 M NaCl in 40 min (25
mM/min). The effluent was monitored for absorbance at 210 and
280 nm. Fractions (0.5 ml each) were collected and further analyzed as
described under ``Results.''
Binding Assays with Soluble Receptors
Polystyrene
96-well breakaway microtiter plates were coated overnight at 4 °C
with 100 µl of affinity-purified rabbit anti-human IgG, Fc-specific
antibodies at 10 µg/ml in 50 mM Na
CO
at pH 9.6. The microtiter plates were blocked with 200 µl of
10% fetal calf serum in phosphate-buffered saline (FBS/PBS) for 1 h at
25 °C. Blocking buffer was removed, and 100 µl of a solution
containing receptor-IgG chimeric protein (FLT1-IgG or KDR-IgG) at 15
ng/ml (70 pM final concentration),
I labeled
VEGF
from CHO cells (5000 cpm per well, 20 pM final), and cold competitor at varying concentrations in PBS with
10% FBS was added to the microtiter wells. Binding was carried out at
ambient temperature for 4 h with gentle agitation, after which the
wells were washed four times with 10% FBS in PBS. The bound
radioactivity was quantitated with a
scintillation counter (LKB
model 1277). Binding data was analyzed using a four-parameter fitting
program (Kaleidagraph, Adelbeck Software). The receptor binding studies
were repeated in the presence of heparin (10 µg/ml) to observe the
maximal effect of heparin on the receptor-VEGF interaction. In these
studies, we have used heparin as a commercial reagent to help address
the biologic significance of the various forms of VEGF. Similar to the
results observed by Tessler et al.(18) and
Gitay-Goren et al.(26) , dose-response studies with
increasing heparin indicated the optimal concentration to achieve
maximal KDR receptor binding.
Endothelial Cell Proliferation Assay for
VEGF
Bovine adrenal cortical capillary endothelial cells were
maintained in low glucose Dulbecco's modified Eagle's
medium supplemented with 10% calf serum, 2 mM glutamine, and
antibiotics (growth medium) as described previously(1) . For
bioassay, cells were sparsely seeded in 12-well plates with 7
10
cells/well in 1 ml of growth medium. Samples of VEGF or
VEGF variants (1 ml) were diluted in the culture media at
concentrations from 1 µg/ml to 10 pg/ml (final) and layered onto
the seeded cells. After 5 days, the cells were dissociated with trypsin
and quantified using a Coulter Counter (Miami, FL).
RESULTS
Limited Proteolysis of VEGF
The susceptibility
of VEGF to proteolytic digestion was explored using the glycosylated
form of VEGF
as expressed and purified from CHO cell
conditioned media. As observed in Fig. 2A (lane
2), VEGF
appeared on SDS-PAGE as a doublet of
proteins at 43 and 45 kDa. Tryptic mapping of reduced and
carboxymethylated VEGF indicated approximately 75% of the protein
contained N-linked glycosylation at Asn
; the
remaining 25% of VEGF was unglycosylated (data not shown). The doublet
of protein observed on SDS-PAGE was due to partial glycosylation. As
seen in other studies, VEGF
from E. coli appeared as a single protein band of 38 kDa. A variety of enzyme
digests of VEGF
were prepared under similar conditions
(1:100, enzyme:VEGF at 25 °C and for 20 h). Relatively complete
digestion was observed on reverse phase HPLC with broad-specificity
enzymes, such as subtilisin, Pronase, proteinase K, and thermolysin.
Enzymes such as plasmin, trypsin, chymotrypsin, clostripain, and
bromelain yielded varying extents of partial digestion resulting in a
``core protein,'' which was resistant to further proteolysis.
No proteolysis was observed with thrombin, collagenase, elastase, S. aureus V8 protease, or pepsin as indicated by HPLC. The
digestion profiles were similar for CHO- and E. coli-derived
VEGF
. A series of enzyme digests were analyzed by
non-reducing SDS-PAGE, which indicated that trypsin and plasmin cleaved
VEGF, but thrombin, elastase, chymotrypsin, or pepsin did not (Fig. 2A). These enzyme digests were reduced,
carboxymethylated, and evaluated by SDS-PAGE (Fig. 2B)
to observe additional proteolysis that was not apparent under
non-reducing conditons. Some proteolysis of VEGF by chymotrypsin was
observed (lane 7), in addition to the cleavage of VEGF by
plasmin and trypsin (lanes 4 and 6). VEGF
is relatively resistant to proteolysis by enzymes such as
collagenase, thrombin, and elastase. However, plasmin rapidly cleaves
VEGF into discrete, non-disulfide-linked fragments.
Figure 2:
Enzymatic digestion of CHO-derived
VEGF
. Digests with thrombin, plasmin, elastase, trypsin,
chymotrypsin, and pepsin are evaluated in panel A, by
non-reducing SDS-PAGE. In panel A, lanes 1, blank; lane 2, non-reduced CHO-derived VEGF
; lane
3, thrombin; lane 4, plasmin; lane 5, elastase; lane 6, trypsin; lane 7, chymotrypsin; lane
8, pepsin. In panel B, the samples were RCM after the
enzyme digestion. Lane 1, non-reduced CHO-derived
VEGF
; lane 2, RCM CHO-derived
VEGF
; lane 3, thrombin; lane 4,
plasmin; lane 5, elastase; lane 6, trypsin; lane
7, chymotrypsin; lane 8, pepsin digests of RCM
CHO-derived VEGF
.
Isolation and Identification of Plasmin-cleaved VEGF
Fragments
To eliminate the apparent heterogeneity due to
glycosylation of CHO cell expressed VEGF
, we studied the
activity of plasmin on E. coli-derived VEGF
. The
absence of carbohydrate on this form of VEGF was confirmed by tryptic
mapping. A plasmin digest of VEGF
from E. coli (1:200 enzyme:substrate) was initiated and samples removed at
various times for analysis by reverse phase HPLC. After 2 h of plasmin
treatment, four protein peaks with absorption at 210 nm were resolved
by HPLC (Fig. 3). The first peak, eluting at 12 min, exhibited
minimal absorption at 280 nm, consistent with the carboxyl-terminal
region of VEGF, which has only one aromatic amino acid, phenylalanine
128. Protein containing HPLC fractions were collected, pooled and
identified by amino acid sequence, composition, and mass spectrometry.
The early eluting peak of plasmin-cleaved VEGF
was
identified as the polypeptide (111-165) by the amino-terminal
sequence: Ala
-Arg-Gln-Glu-Asn-Pro
. The
amino acid composition and minimal retention on reverse phase HPLC was
consistent with the highly charged, hydrophilic 55-amino acid,
carboxyl-terminal polypeptide. Mass spectral analysis indicated a
molecular weight of 6473 atomic mass units for the (111-165)
polypeptide, which compared well with the expected value of 6474 atomic
mass units based on the amino acid sequence. The later eluting HPLC
peaks, with retention times of 32, 35, and 38 min were identified as
homodimer of (1-165), heterodimer of (1-165, 1-110)
and homodimer of (1-110), respectively. All of the later eluting
peaks shared the amino-terminal sequence:
Ala
-Pro-Met-Ala-Glu
. The homodimer of
(1-165) and the homodimer of (1-110) exhibited molecular
weights of 38,306 and 25,385 atomic mass units, which compared well
with the theoretical values of 38,300 and 25,389 atomic mass units,
respectively. Retention times of these plasmin cleavage products are
consistent with the loss of the hydrophilic carboxyl-terminal
polypeptide (111-165), such that the heterodimer (1-165,
1-110) and homodimer (1-110) are progressively more
hydrophobic and later eluting on reverse phase HPLC.
Figure 3:
Reverse phase HPLC separation of plasmin
digest products of E. coli-derived VEGF
. After 4
h of proteolysis at 25 °C with plasmin (1:200, enzyme:substrate),
the partial digest products were resolved by HPLC. The plasmin digest
products include: the 55-amino acid carboxyl-terminal polypeptide (55), VEGF
homodimer (165/165),
VEGF
heterodimer (165/110), VEGF
homodimer (110/110), which eluted at 12, 32, 35, and 38
min, respectively.
Kinetic Analysis of Plasmin Cleavage
Fig. 4shows the kinetics for plasmin cleavage of E.
coli-derived VEGF
(enzyme:substrate 1:100) at pH 7.
The digest was analyzed at various times by reverse phase HPLC to
quantify the amount of each form of VEGF. Cleavage of intact homodimer
(1-165) occurred with a concomitant increase in the heterodimer
(1-165, 1-110) followed by the appearance of the homodimer
(1-110). The amount of (111-165) polypeptide increased over
time and achieved a maximal level, which was approximately 40% of that
observed for the (1-110) homodimer. Additional proteolysis of the
cleaved polypeptide (111-165) by plasmin leads to lower molecular
weight species, which eluted at 11 min on HPLC (Fig. 3). The
plasmin catalyzed cleavage of VEGF
homodimer was
approximately 3-fold faster than that observed for VEGF
cleavage, as indicated by the rate of disappearance for each
protein observed on HPLC. The observation that VEGF
homodimer was cleaved faster than the VEGF
heterodimer was unexpected, since the two plasmin cleavage sites
(Arg
-Ala
in each monomer) are identical in
the dimer. We considered that the lysine-rich polypeptide
(111-165) of VEGF may act as an additional recognition site for
plasmin on VEGF and that the kringles of plasmin (kringles 1, 4, and 5)
may function as lysine-binding modules for binding VEGF. Fig. 1shows schematically the sequential cleavage of VEGF by
plasmin. In the first cleavage, the intact carboxyl-terminal region of
VEGF presented by the opposing VEGF
monomer may increase
binding and cleavage of the full-length monomer, but does not
participate in the binding of the heterodimer resulting in reduced rate
for the second cleavage of VEGF
.
Figure 4:
Kinetics of plasmin cleavage of
VEGF
. The progress of plasmin digestion was followed by
repetitive reverse phase HPLC analysis at hourly intervals. The HPLC
eluate was monitored for absorbance at 210 nm, and the amount of
VEGF
homodimer, VEGF
heterodimer,
VEGF
homodimer, and the 55-amino acid carboxyl-terminal
polypeptide at the indicated times were determined by peak area
integration.
Heparin Binding of Homo- and Heterodimeric
VEGF
The heparin binding function of VEGF
was
studied by analytical affinity chromatography. Samples of E.
coli-derived VEGF
, before and after treatment with
plasmin, were applied to a POROS heparin column and eluted with an
increasing gradient of NaCl. The eluate was monitored for optical
density at 210 nm, and appropriate pooled fractions were analyzed by
reverse phase HPLC. The results, shown in Table 1, indicate that
the heparin binding function of VEGF
is completely
mediated by the carboxyl-terminal domain (111-165). The heparin
affinity of VEGF
and the polypeptide (111-165) are
nearly equivalent as determined by the concentration of NaCl required
for elution (680 and 690 mM, respectively). Heterodimeric
VEGF
eluted at 420 mM NaCl, indicating a
significant decrease in heparin affinity associated with the loss of
one carboxyl-terminal domain. No heparin binding was observed for
homodimeric VEGF
and VEGF
. Various
conditions for plasmin digestion were used to prepare hetero- and
homodimeric VEGF, followed by preparative heparin chromatography. Fig. 5shows E. coli VEGF
, lacking
carbohydrate modification, which yields a single band on SDS-PAGE with
an apparent molecular mass of 38 kDa (lane 1). The SDS-PAGE
analysis of a plasmin digest of E. coli VEGF
appears in lane 2. VEGF
homodimer was not
retained on heparin-agarose (lane 3), while the
(111-165) polypeptide with trace amounts of homo- and
heterodimeric VEGF were eluted with 1 M NaCl (lane
4). Gradient elution of partial plasmin digests on a POROS-heparin
column yielded highly purified preparations of VEGF
and VEGF
as indicated by reverse phase HPLC (data
not shown).
Figure 5:
SDS-PAGE of plasmin digested E.
coli-derived VEGF
. Lane 1, native E.
coli-derived VEGF
; lane 2, plasmin digest
at 20 h; lane 3, heparin-Sepharose column flow-though
containing VEGF
homodimer; lane 4, 1 M NaCl eluate containing predominantly (111-165) polypeptide,
and trace amounts of VEGF
homodimer, VEGF
heterodimer, and VEGF
homodimer.
Binding of VEGF Isoforms to Soluble Receptors in the
Presence and Absence of Heparin
Binding of radioiodinated
CHO-derived VEGF
to KDR-IgG and FLT1-IgG was analyzed
using a competitive displacement radioreceptor assay ( Fig. 6and Fig. 7). Various concentrations of unglycosylated (expressed in E. coli) VEGF
, VEGF
,
VEGF
, and VEGF
were tested for displacement
of labeled glycosylated (expressed in CHO cells) VEGF
.
The 55-amino acid, carboxyl-terminal domain of VEGF (111-165) was
tested at 1000-fold molar excess and did not inhibit VEGF binding to
the soluble receptors. At concentrations greater than 1000-fold molar
excess, the isolated carboxyl-terminal domain appeared to partially
displace VEGF
from receptor. Radiolabeled VEGF binding to
the soluble form of KDR was half-maximally displaced at 38 pM VEGF
in the absence of heparin (Fig. 6A). Glycosylation of VEGF
did not
affect KDR binding, as indicated by the similar affinity exhibited by
CHO- and E. coli-derived VEGF
(Table 2).
Loss of one or both carboxyl-terminal domain(s) had no effect on the
affinity of VEGF for KDR as indicated by the IC
values for
VEGF
heterodimer and VEGF
and
VEGF
homodimers (38, 29, and 30 pM,
respectively). In the presence of heparin, the amount of VEGF bound to
KDR was increased more than 3-fold; however, the apparent affinity of
VEGF for KDR was unchanged (Fig. 6B). Glycosylated and
unglycosylated VEGF
bound KDR with equivalent IC
values (31 and 28 pM, respectively). Loss of
carboxyl-terminal domains resulted in 2-fold decreased KDR affinity (in
the presence of heparin), as observed in competition studies with
VEGF
heterodimer and VEGF
and
VEGF
homodimers (IC
values of 45, 60, and 63
pM, respectively).
Figure 6:
Binding of VEGF isoforms to soluble
KDR-IgG receptor. The competitive displacement of
I-labeled CHO-derived VEGF
binding to KDR
receptor (15 ng/ml) with various concentrations of CHO-derived
VEGF
(
), E. coli-derived VEGF
(
), E. coli-derived VEGF
heterodimer (
), VEGF
homodimer (
),
VEGF
homodimer (
), and 55-amino acid
carboxyl-terminal polypeptide (
). Panels A and B show the results of the binding studies in the absence and
presence of heparin, respectively. These binding curves are the result
of triplicate assays.
Figure 7:
Binding of VEGF isoforms to soluble FLT-1
IgG receptor. The competitive displacement of
I-labeled
CHO-derived VEGF
binding to FLT-1 receptor (5 ng/ml) with
various concentrations of CHO-derived VEGF
(
), E. coli-derived VEGF
(
), E.
coli-derived VEGF
heterodimer (
),
VEGF
homodimer (
), VEGF
homodimer
(
), and 55-amino acid carboxyl-terminal polypeptide (
). Panels A and B show the results of the binding
studies in the absence and presence of heparin, respectively. These
binding curves are the result of duplicate
assays.
Glycosylated and unglycosylated
VEGF
exhibited similar affinity for soluble FLT-1
receptor, approximately 10 pM in the absence of heparin (Fig. 7A). Loss of one carboxyl-terminal domain was
associated with 3-fold decreased FLT-1 affinity as indicated by the
binding of VEGF
heterodimer. The loss of both
carboxyl-terminal domains resulted in approximately 10-fold reduced
FLT-1 affinity as exhibited by the VEGF
homodimer.
Similarly, the values obtained for the natural splice variant
VEGF
indicated greater than 20-fold decreased binding to
FLT-1 compared with that observed for VEGF
(Table 2). The carboxyl-terminal domain itself (polypeptide
111-165) exhibited no binding to FLT-1 (as was observed with
KDR). In the presence of heparin, the differences in FLT-1 binding
observed for the VEGF variants were diminished (Fig. 7B). Glycosylated and unglycosylated VEGF 165
bound soluble FLT-1 with values for IC
of 15 and 19
pM, respectively. VEGF
also exhibited
similar binding affinity. The binding values for VEGF
and
VEGF
were reduced approximately 2-3-fold,
respectively, compared to those observed for VEGF
.
Differential Stimulation of Endothelial Cell Growth
Induced by VEGF Isoforms
Stimulation of endothelial cell
proliferation by VEGF variants was evaluated with bovine adrenal
cortical capillary endothelial cells (Fig. 8). The effective
concentration to induce 50% of maximal stimulation (EC
)
was determined in vitro by incubation of endothelial cells
with VEGF or VEGF variants at concentrations that varied from 0.3
pM to 40 µM. Glycosylated and unglycosylated
VEGF
exhibited similar EC
values of 5.8 and
5.2 pM, respectively. The loss of one carboxyl-terminal domain
resulted in approximately 7-fold decrease in potency for
VEGF
as indicated by the higher EC
value
of 40 pM. Lower molecular weight variants, VEGF
and VEGF
, displayed greater than 100-fold reduced
potency with values of 2.58 and 2.56 nM for EC
,
respectively. The isolated carboxyl-terminal domain (111-165) had
no stimulatory effect on endothelial cell proliferation even with 4
orders of magnitude molar excess of polypeptide compared to the
half-maximally effective concentration for VEGF
. Similar
results were observed with fetal bovine aortic endothelial cells with
respect to a 10-fold and 100-fold loss in mitogenic potency with
VEGF
and VEGF
(or VEGF
),
respectively (data not shown). These results demonstrate the critical
role of the 111-165 region of VEGF in the stimulation of both
large and small vessel endothelial cell proliferation.
Figure 8:
Endothelial cell proliferation in response
to VEGF isoforms. Bovine adrenal cortical endothelial cells were
cultured for 5 days in the presence of varying concentrations of
CHO-derived VEGF
(
), E. coli-derived
VEGF
(
), E. coli-derived VEGF
heterodimer (
), VEGF
homodimer (
),
VEGF
homodimer (
), and 55-amino acid
carboxyl-terminal polypeptide (
). The cells were trypsinized and
counted as described under ``Experimental Procedures.'' These
results represent the average of duplicate
assays.
DISCUSSION
Extracellular proteolysis and remodeling of the extracellular
matrix play key roles in a variety of developmental
processes(19) . In addition, plasminogen activation and
generation of plasmin have been shown to be important for the
angiogenesis cascade(20) . Such processes also play a major
role in the local invasiveness and metastasis of tumor cells. Strong
experimental evidence supports the concept that growth factors stored
in the extracellular matrix and released in the course of its
degradation are major mediators of such inductive processes. Numerous
growth factors including fibroblast growth factor, platelet-derived
growth factor, granulocyte/macrophage colony-stimulating factor,
transforming growth factor-
, and leukemia inhibitory factor have
been shown to be associated with the extracellular
matrix(21, 22, 23, 24, 25) .
Alternatively spliced molecular species of VEGF are differentially
localized to heparan sulfate containing proteoglycans of the
extracellular matrix or released as diffusible
proteins(10, 11) . In addition to this
transcriptionally regulated heterogeneity, there appears the potential
for proteolytic processing which may further regulate the
bioavailability of VEGF. In the present study, we have demonstrated
that plasmin readily cleaves VEGF
to yield a non-heparin
binding isoform, VEGF
, and a heparin-binding fragment
composed of the carboxyl-terminal domain (111-165). In contrast
to plasmin, neither thrombin, elastase, nor collagenase efficiently
cleaves VEGF. Like VEGF
, VEGF
has no
affinity for heparin and is a diffusible molecule(11) .
Therefore, the carboxyl-terminal domain (amino acids 111-165) is
completely responsible for the observed heparin binding of VEGF in
vitro. Interestingly, a partially cleaved form, VEGF
was isolated that exhibited intermediate heparin affinity.
We
have used the multiple forms of VEGF obtained by plasmin cleavage or
alternative splicing to evaluate the role of the carboxyl-terminal
domain on VEGF receptor affinity and endothelial cell growth. The
55-amino acid peptide (111-165) of VEGF did not bind the soluble
KDR receptor, as indicated by the complete lack of inhibition observed
with
I-labeled VEGF
binding. Binding to the
KDR receptor is mediated by determinants in the 1-110 region of
VEGF
. The lower molecular weight diffusible forms,
VEGF
and VEGF
, bind KDR with similar
affinity as VEGF
in the absence of heparin. A modest
effect of heparin is the 3-4-fold increased capacity of KDR for
binding VEGF. The heparin-induced potentiation of VEGF binding to KDR
has also been observed by other investigators. Gitay-Goren et al.(26) observed an increased number of high affinity VEGF
binding sites on endothelial cells in the presence versus the
absence of heparin, but heparin did not significantly alter the
dissocation constant. Tessler et al.(18) also
reported increased binding of VEGF
to Flk-1/KDR type
receptors transfected into NIH3T3 cells as a function of heparin.
FLT-1 binding is significantly different for the long and short
forms of VEGF. In contrast to the results observed with KDR, the
shorter forms of VEGF (110 and 121 forms) bound FLT-1 receptor with
10-20-fold decreased affinity compared to VEGF
.
VEGF
displayed FLT-1 binding intermediate to the 165
and 110 forms of VEGF. Taken together, these results demonstrate that
the 55-amino acid domain of VEGF mediates, in part, the binding to
FLT-1. This domain appears to enhance FLT-1 binding in addition to the
receptor binding determinants contained in VEGF
, but by
itself the 55-amino acid domain cannot compete with VEGF
binding to FLT-1, as indicated by the lack of competition with
greater than 1000-fold molar excess of (111-165) polypeptide (see Fig. 7). As such, the 55-amino acid, carboxyl-terminal domain of
VEGF
plays a different role with KDR as compared to FLT-1
in mediating receptor binding. For example, there is no heparin-induced
potentiation of FLT-1 binding of VEGF as is observed with KDR. Cohen et al.(27) have observed the inhibition of heparin on
VEGF binding to FLT-1 receptors on human melanoma cells. These
investigators observed significantly decreased melanoma cell binding of
I-labeled VEGF
and VEGF
in
the presence of exogenous heparin (1 µg/ml) compared to that
observed in the absence of heparin.
VEGF interaction with cell
surface heparan-sulfate containing proteoglycans on endothelial cell
growth has been examined by numerous
investigators(18, 25, 26, 27, 28, 29, 30) .
Sasisekharan et al.(30) have indicated a role for
heparin-like molecules by inhibiting endothelial cell proliferation and in vivo neovascularization with heparinase. The binding of
I-labeled VEGF
to endothelial cells was
completely inhibited by pretreatment with heparinase and could be
restored by the addition of exogenous heparin (25) . To
evaluate the significance of the relative heparin affinity of VEGF
isoforms, we tested the mitogenic activity of the 165, 165/110, 110,
and 121 forms of VEGF on primary cultured bovine adrenal cortical
capillary cells and fetal bovine aortic endothelial cells. Loss of one
or both carboxyl-terminal domains of VEGF significantly reduced the
proliferation of endothelial cells. The mitogenic potency of
VEGF
and VEGF
was substantially decreased
(>100-fold) compared to VEGF
. VEGF
exhibited 7-10-fold reduced activity on endothelial cell
growth. These results are most interesting in light of the similarities
and differences observed with various VEGF isoforms binding soluble KDR
or FLT-1 receptors. The modestly decreased affinity observed with
soluble KDR and FLT-1 receptors and the 110 or 121 isoforms of VEGF
does not account for the drastically decreased endothelial cell
mitogenic potency. This discrepancy suggests that the stability of
VEGF-heparan sulfate-receptor complexes probably contributes to
effective signal transduction and stimulation of endothelial cell
proliferation. Further studies are clearly warranted to explore the
regulatory function of heparin-like molecules in VEGF receptor
interaction, signal transduction, and mitogenesis of endothelial cells.
Our findings indicate that VEGF, by alternative splicing and/or
limited proteolysis, has the potential to express structural and
functional heterogeneity that yields a graded biological response. Our
current understanding of VEGF biology suggests the following sequence
of events may occur during angiogenesis. Initially, cells respond to
hypoxia or other stimuli by inducing VEGF transcription(31) ,
resulting in increased expression of long and short forms of VEGF,
although VEGF
is probably the most abundant isoform.
Compared to the longer forms of VEGF, the diffusible forms would
migrate a greater distance, bind VEGF receptors, and trigger
endothelial cell proliferation and migration. The intensity of the
angiogenic signal would be weakest at the most distant sites, given the
lesser mitogenic potency of VEGF
and VEGF
.
Closer to the site of VEGF synthesis (i.e. ischemic tissues),
the concentration of the 165 isoform is expected to be increased due to
extracellular matrix binding and the effect of the biochemical gradient
would be enhanced with the associated greater mitogenic potency of
VEGF
. In the most ischemic areas, matrix-associated VEGF
is localized to the cells of origin with the highest concentration and
potency. The heterogeneity of VEGF structure and function allows the
formation of a biochemical gradient for the migration and chemotaxis of
proliferating endothelial cells. In circumstances where plasminogen
activation occurs (e.g. tumors and wounds), the presence of
plasmin may serve to release stored forms of matrix-bound VEGF to
amplify the angiogenic signal.
Placental growth factor (PlGF) is a
protein with 53% homology to VEGF (32, 33) . PlGF
binds FLT-1 with similar affinity as VEGF, but does not bind to the KDR
receptor(12) . Interestingly, recent studies describing the
existence of VEGF
PlGF heterodimers further extend the concept
that structural heterogeneity may be responsible for a graded
biological response. While the PlGF homodimer had minimal endothelial
cell mitogenic activity compared to that of the VEGF homodimer, the
heterodimer displayed intermediate activity(34) . With respect
to proteolytic digestion resulting in the generation of VEGF
from VEGF
or VEGF
, little is known at
this time about the prevalence of this process in vivo.
Nevertheless, the potential exists for VEGF to set up a biochemical
gradient that radiates from an ischemic zone and may provide a
directional signal for in vivo angiogenesis.