Proc. Natl. Acad. Sci. USA
Vol. 95, pp. 11709–11714, September 1998
Cell Biology
Vascular endothelial growth factor B (VEGF-B) binds to VEGF
receptor-1 and regulates plasminogen activator activity in
endothelial cells
BIRGITTA OLOFSSON*
†
,EIJA KORPELAINEN
†‡
,MICHAEL S. PEPPER
§
,STEFANO J. MANDRIOTA
§
,KARIN AASE*,
VIJAY KUMAR
‡
,YUJI GUNJI
‡
,MICHAEL M. JELTSCH
‡
,MASABUMI SHIBUYA
¶
,KARI ALITALO
†‡i
,
AND ULF ERIKSSON*
†i
*Ludwig Institute for Cancer Research, Stockholm Branch, Box 240, S-171 77 Stockholm, Sweden;
‡
MolecularyCancer Biology Laboratory, Haartman Institute,
P.O. Box 21 (Haartmaninkatu 3), 00014 Helsinki, Finland;
§
Department of Morphology, University Medical Center, 1211 Geneva 4, Switzerland; and
¶
Institute
for Medical Science, University of Tokyo, Minato-ku, Tokyo 108, Japan
Edited by M. Judah Folkman, Harvard Medical School, Boston, MA, and approved August 10, 1998 (received for review January 9, 1998)
ABSTRACT The vascular endothelial growth factor
(VEGF) family has recently expanded by the identification and
cloning of three additional members, namely VEGF-B, VEGF-C,
and VEGF-D. In this study we demonstrate that VEGF-B binds
selectively to VEGF receptor-1yFlt-1. This binding can be
blocked by excess VEGF, indicating that the interaction sites on
the receptor are at least partially overlapping. Mutating the
putative VEGF receptor-1yFlt-1 binding determinants Asp
63
,
Asp
64
, and Glu
67
to alanine residues in VEGF-B reduced the
affinity to VEGF receptor-1 but did not abolish binding. Muta-
tional analysis of conserved cysteines contributing to VEGF-B
dimer formation suggest a structural conservation with VEGF
and platelet-derived growth factor. Proteolytic processing of the
60-kDa VEGF-B
186
dimer results in a 34-kDa dimer containing
the receptor-binding epitopes. The binding of VEGF-B to its
receptor on endothelial cells leads to increased expression and
activity of urokinase type plasminogen activator and plasmino-
gen activator inhibitor 1, suggesting a role for VEGF-B in the
regulation of extracellular matrix degradation, cell adhesion,
and migration.
Vascular endothelial growth factor (VEGF) has been impli-
cated as a key regulator of blood vessel formation (1). It is
required for both vasculogenesis, where mesoderm-derived
angioblasts form tubes, and for angiogenesis, where capillaries
form by sprouting or intussusception from existing vessels (2).
While vasculogenesis is restricted to embryonic development,
angiogenesis continues to operate throughout life when neo-
vascularization is required. Physiological angiogenesis is
mainly restricted to the female reproductive cycle and wound
healing, but the angiogenic machinery can also be recruited by
pathological processes such as tumor growth (3).
VEGF exerts its functions through binding to two receptor
tyrosine kinases, VEGFR-1yFlt-1 and VEGFR-2yKDR (1)
.
These receptors are expressed almost exclusively on endothe-
lial cells, although VEGFR-1 is also found in monocytes,
where it mediates migration (4, 5). Targeted homozygous null
mutations of both receptor genes result in arrest of embryonic
development (6, 7). Disruption of the VEGFR-1 gene inter-
feres with the organization of the vascular endothelium (6),
whereas VEGFR-2 is required for endothelial cell differenti-
ation and definitive hematopoiesis (7, 8). VEGF levels are
critical for normal development, as inactivation of even one
allele results in embryonic death (9, 10).
VEGF has been shown to regulate most steps of the
angiogenic process, including endothelial cell degradation of
extracellular matrix (ECM), migration, proliferation, and tube
formation (1). In keeping with its ability to induce ECM
degradation, VEGF increases the expression and activity of
plasminogen activators, urokinase type plasminogen activator
(uPA) and tissue type plasminogen activator (tPA) (11). These
serine proteases convert plasminogen to plasmin and are
thereby involved in tissue remodeling, cell invasion, and throm-
bolysis (reviewed in ref. 12). Whereas tPA is a fibrin-
dependent intravascular enzyme, uPA functions as a receptor
(uPAR)-bound cell surface activator. Both proteases are spe-
cifically inhibited by plasminogen activator inhibitor type 1
(PAI-1), the expression of which is also up-regulated by VEGF
(11). This inhibition may serve to protect ECM from excessive
proteolysis, as concerted expression of PAI-1 and uPA has
been observed during physiological angiogenesis in vivo (13).
Interestingly, both PAI-1 and uPAyuPAR have recently been
implicated in regulation of cell adhesion and migration (12).
uPAR and PAI-1 compete for binding to vitronectin, and
PAI-1 regulates adhesion also directly by competing with
integrin a
v
b
3
for vitronectin binding. Taken together, the
uPAyuPARyPAI-1 system may have a dual role: it can regulate
proteolysis and cellular adhesiveness, the latter being inde-
pendent of the enzymatic function.
The VEGF family of growth factors comprises at present
five members—i.e., VEGF, placenta growth factor (PlGF)
(14), VEGF-ByVRF (15, 16), VEGF-CyVRP (17, 18), and
VEGF-DyFlGF (19, 20). While PlGF binds selectively to
VEGFR-1 (21), VEGF-C and VEGF-D bind both VEGFR-
3yFlt-4 and VEGFR-2 (17, 22). The corresponding receptor(s)
for VEGF-B has not been reported. VEGF-B resembles PlGF
in two aspects: it exists as two alternatively spliced forms,
VEGF-B
167
and VEGF-B
186
, which differ in their affinity for
heparin and thus release and bioavailability, and it forms
heterodimers with VEGF (15, 23), a property likely to alter its
receptor specificity and biological effects. In contrast to PlGF,
however, VEGF-B is widely expressed and is most prominent
in heart and skeletal muscle (15).
Alanine-scanning mutagenesis of VEGF has implicated the
negatively charged amino acid residues Asp
63
, Glu
64
, and Glu
67
in VEGFR-1 binding (24). These acidic amino acid residues
are conserved in VEGF-B and to lesser extent in PlGF.
The publication costs of this article were defrayed in part by page charge
payment. This article must therefore be hereby marked ‘‘advertisement’’ in
accordance with 18 U.S.C. §1734 solely to indicate this fact.
© 1998 by The National Academy of Sciences 0027-8424y98y9511709-6$2.00y0
PNAS is available online at www.pnas.org.
This paper was submitted directly (Track II) to the Proceedings office.
Abbreviations: ECM, extracellular matrix; PAI-1, plasminogen acti-
vator inhibitor type 1; PlGF, placenta growth factor; tPA, tissue type
plasminogen activator; uPA, urokinase type plasminogen activator;
VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor;
h-, human; m-, mouse; bFGF, basic fibroblast growth factor.
†
B.O. and E.K. contributed equally to this work, as did K.A. and U.E.
i
To whom reprint requests should be addressed. e-mail: ueri@licr.ki.se
or Kari.Alitalo@helsinki.fi.
11709

Covalent dimerization of VEGF has been shown to be required
for its biological activity (25), and recently the determination
of the crystal structure verified the antiparallel arrangement of
the two subunits covalently linked by two disulfide bridges
between Cys
51
and Cys
60
(26). The eight conserved cysteine
residues characteristic of the PDGFyVEGF growth factor
family imply structural conservation between the members
(for recent sequence alignment see ref. 20).
In this work we report that VEGF-B binds specifically to
VEGFR-1. Mutation of the putative receptor-binding deter-
minants to alanine residues reduced the affinity to VEGFR-1
but did not abolish receptor binding, and mutations in con-
served cysteine residues predict that VEGF-B forms antipar-
allel dimers. Furthermore, we show that VEGF-B
186
is pro-
teolytically processed, and we analyze the ability of VEGF-B
to regulate the uPAyPAI-1 system in endothelial cells.
MATERIALS AND METHODS
Cell Culture and Materials. Sf9 cells were maintained in
Sf-900 II SFM (GIBCOyBRL Life Technologies) supple-
mented with 0.1% pluronic f-68 for suspension growth, High
Five cells (Invitrogen) in Ex-cell 400 medium (JHR Bio-
science; Lenexa, KS), and Schneider 2 (S2) cells (Invitrogen)
were grown in DES-medium (Invitrogen) supplemented with
10% fetal calf serum (FCS). 293-EBNA, 293-T, and NIH
3T3-Flt1 cells (27) were grown in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% FCS. NIH 3T3-
Flt1 cells were kept under continuous selection with 200 mgyml
neomycin. Bovine adrenal cortex-derived microvascular endo-
thelial (BME) cells were kindly provided by M. B. Furie and
S. C. Silverstein (28) and were grown in MEMa (GIBCO;
Basel, Switzerland) supplemented with 15% donor calf serum
on 1.5% gelatin-coated tissue culture flasks. Plasmin was
purchased from Boehringer Mannheim, anti-VEGF (MAB
293) fromR&DSystems, and human (h)VEGF
165
fromR&
D Systems or from Preprotech (Rocky Hill, NJ). Recombinant
mouse (m)VEGF
164
and human basic fibroblast growth factor
(bFGF) (155 amino acid form) were kind gifts from H. Weich
(GBF, Braunschweig Germany) and P. Sarmientos (Farmitalia
Carlo Erba, Milan), respectively.
Construction of Expression Plasmids and Generation of
VEGF-B Mutants. The expression plasmid pIg-VEGFR-1
coding for the first five Ig-like domains of VEGFR-1 fused to
human IgG1 Fc was constructed by ligating a HindIII fragment
(coding for amino acids 1–549 of VEGFR-1) from pLTR-Flt1
into pIgplus vector (Ingenius; Novagen). Prior to the cloning
the latter had been digested with XhoI and XbaI, blunted, and
religated to correct the reading frame for the fusion protein
production. For the spIg-VEGFR-2 construct, cDNA encod-
ing the first four Ig-like domains of VEGFR-2 was amplified
by PCR using human fetal lung cDNA library (CLONTECH)
as a template. The primers 59-ATGGTACCCCCAGGCTCA-
GCATACAAAAAGAC-39 and 59-GCGTCTAGAGGGTG-
GGACATACACAACCAG-39were used and the amplified
fragment was cleaved with KpnI and XbaI and inserted into
corresponding sites of signal pIg vector (Ingenius). mVEGF-
B
186
cDNA (23) was cleaved by EcoRI and subcloned in
pFASTBAC1 (GIBCOyBRL Life Technologies). A (His)
6
tag
and an enterokinase site were introduced at the N terminus,
devoid of signal sequence, using PCR with mVEGF-B
186
pSG5
as a template and the primers 59-ATCGAGATCTTCATCA-
CCATCACCATCACGGAGATGACGATGACAAACCTG-
TGTCCCAGTTT-39 and 59-CAAGGGCGGGGCTTAGAG-
ATCTAGCT-39 (both containing BglII sites). The amplified
fragment was cleaved with BglII and inserted into the BamHI
site, in frame with the signal sequence of gp67 of pAcGP67A
(PharMingen). Human VEGF-B
186
cDNA was amplified by
PCR using the forward primer 59-GGAATTCCCCGCCCA-
GGCCCCTGTC-39 and the reverse primer 59-GGAATTCA-
ATGATGATGATGATGATGAGCCCCGCCCTTGGC-39.
The amplified product containing a C-terminal (His)
6
tag was
inserted into the EcoRI site of pPIC-9 (Invitrogen), in frame
with the a mating factor signal sequence. The cysteine-to-
serine mutants and the alanine mutants in mVEGF-B
167
pSG5
as well as mVEGF-B exon 1–5 mutant containing a C-terminal
Kemptide motif (29), VEGF-B
kEx1–5
pSG5, were generated by
M13-based in vitro single-stranded mutagenesis employing the
helper phage M13KO7 (30) and the dut
2
ung
2
Escherichia coli
strain RZ1032 (31). The primers 59-ACGTAGATCTCCTG-
TGTCCCAG-39 and 59-ACGTGAATTCTCAGCTGTCTG-
GCTTCAC -39 (introducing a stop codon after exon 5) were
used to PCR-amplify the alanine mutants, which were sub-
cloned in pMTyBipyV5-HisC (Invitrogen). All constructs
were verified by sequencing.
Protein Expression and Purification. For production of
recombinant baculoviral protein in Sf9 and High Five cells,
mVEGF-B recombinant plaques were purified and amplified
(32), and the corresponding expressed proteins as well as
hVEGF-B
186
expressed in Pichia pastoris (strain GS115) were
purified by using Ni-NTA Superflow resin (Qiagen). For
ligand competition assay, High Five cells were infected with
mVEGF-B
186
pFASTBAC1 virus or with a mock virus, and the
media were harvested 48 hr after infection and immediately
used or frozen at 270°C. The S2 cells were transfected and the
expression was induced according to the supplier. The condi-
tioned media were collected 72 hr after induction.
Antibodies. Purified m(His)
6
VEGF-B
186
protein was used for
immunization of rabbits according to standard procedures. The
obtained antiserum and the antiserum to mVEGF-B N-terminal
peptide (23) were affinity purified with m(His)
6
VEGF-B
186
co-
valently bound to CNBr-activated Sepharose CL-4B (Pharma-
cia). For quantitative immunoblots, media from infected or
transfected insect cells were electrophoresed together with 1–30
ng of purified m(His)
6
VEGF-B
186
as a standard and detected by
using the affinity-purified antibodies.
Transfections, Immunoprecipitations, and Soluble Recep-
tor Binding. 293-T cells were transfected with hVEGF
165
pSG5,
mVEGF-B
167
pSG5, VEGF-B
kEx1–5
pSG5, mVEGF-B
186
pSG5, VEGFR-1 pIg, and VEGFR-2 pIg by using calcium
phosphate precipitation. VEGFR-3 EC-Ig pREP7 (a kind gift
from K. Pajusola, Biotechnology Institute, Helsinki), and
hVEGF-CDNDC (His)
6
pREP7 (33) were similarly expressed
in 293-EBNA cells. Cells expressing the growth factors were
metabolically labeled 48 hr after transfection with 100 mCiyml
Pro-mix L-[
35
S] (Amersham) for 5–6 hr (1 mCi 5 37 kBq), and
the media were collected. Heparin was added to the labeling
medium of VEGF-B
167
and VEGF at 10 mgyml, unless oth-
erwise stated. Metabolically labeled media (except from the
VEGF transfection) were immunodepleted of endogenous
expressed VEGF and heterodimers by absorption for 2 hr with
2 mgyml VEGF antibody MAB 293 on staphylococcal protein
A-Sepharose. Media of the cells expressing receptor Igs were
replaced 48 hr after transfection by DMEM containing 0.1%
BSA and incubated for an additional 12 hr. About 50 ng of
receptor-Ig fusions and the corresponding volume of media
from mock-transfected cells were absorbed to protein A-
Sepharose. The metabolically labeled growth factors were
incubated with the receptor-Igs for 3 hr at 14°C and washed
with ice-cold binding buffer (PBSy0.5% BSAy0.02% Tween
20y1 mM phenylmethylsulfonyl fluoride) three times and
twice with PBS containing 1 mM phenylmethylsulfonyl fluo-
ride. For competition studies 2 mg of recombinant hVEGF
165
was added to the binding reaction. Equal volumes of media
containing the metabolically labeled factors were immunopre-
cipitated with the affinity-purified N-terminal peptide
VEGF-B antibody, VEGF-C antiserum 882 (33), or VEGF
MAB 293 for 2 hr, washed twice with ice-cold 10 mM TriszHCl,
pH 8.0y1% Triton X-100y25 mM EDTAy1 mM phenylmeth-
ylsulfonyl fluoride and twice with PBS containing 1 mM
phenylmethylsulfonyl fluoride, and analyzed by SDSyPAGE.
11710 Cell Biology: Olofsson et al. Proc. Natl. Acad. Sci. USA 95 (1998)