Proc. Natl. Acad. Sci. USA
Vol. 95, pp. 548–553, January 1998
Cell Biology
Vascular endothelial growth factor D (VEGF-D) is a ligand for the
tyrosine kinases VEGF receptor 2 (Flk1) and VEGF
receptor 3 (Flt4)
MARC G. ACHEN*
†
,MICHAEL JELTSCH
‡
,EOLA KUKK
‡
,TAIJA MA¨KINEN
‡
,ANGELA VITALI*, ANDREW F. WILKS*,
KARI ALITALO
‡
, AND STEVEN A. STACKER*
*Ludwig Institute for Cancer Research, Post Office Box 2008, Royal Melbourne Hospital, Victoria 3050, Australia; and
‡
MolecularyCancer Biology Laboratory
Haartman Institute, PL21 (Haartmaninkatu 3), 00014 University of Helsinki, Finland
Edited by Donald Metcalf, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia, and approved November 18, 1997
(received for review August 4, 1997)
ABSTRACT We have identified a member of the VEGF
family by computer-based homology searching and have des-
ignated it VEGF-D. VEGF-D is most closely related to VEGF-C
by virtue of the presence of N- and C-terminal extensions that
are not found in other VEGF family members. In adult human
tissues, VEGF-D mRNA is most abundant in heart, lung,
skeletal muscle, colon, and small intestine. Analyses of
VEGF-D receptor specificity revealed that VEGF-D is a ligand
for both VEGF receptors (VEGFRs) VEGFR-2 (Flk1) and
VEGFR-3 (Flt4) and can activate these receptors. However,
VEGF-D does not bind to VEGFR-1. Expression of a truncated
derivative of VEGF-D demonstrated that the receptor-binding
capacities reside in the portion of the molecule that is most
closely related in primary structure to other VEGF family
members and that corresponds to the mature form of
VEGF-C. In addition, VEGF-D is a mitogen for endothelial
cells. The structural and functional similarities between
VEGF-D and VEGF-C define a subfamily of the VEGFs.
The formation of blood vessels occurs either by the in situ
differentiation of endothelial cell precursors (angioblasts) and
association of these cells to form primitive vessels, a process called
vasculogenesis, or by growth of preexisting vessels, a process called
angiogenesis (for review, see ref. 1). Vasculogenesis establishes the
primary vascular plexus of the early embryo, whereas development
of blood vessels during later embryogenesis and adult life occurs
primarily by angiogenesis. Angiogenesis in the adult is tightly
controlled; under normal circumstances it occurs almost exclu-
sively in the female reproductive system (2). However, angiogen-
esis can be activated in the adult in response to tissue damage and
is important in certain pathological conditions such as tumorigen-
esis, rheumatoid arthritis, and diabetic retinopathy (2). Once blood
vessels have been established, endothelial cells undergo tissue-
specific changes to generate numerous types of functionally dis-
tinct vessels as organs differentiate (3). These processes require
that endothelial cells respond to a variety of extracellular signals
that activate receptors responsible for growth and differentiation.
VEGF is a homodimeric glycoprotein that is mitogenic for
endothelial cells and is an angiogenic factor that acts via the
endothelial-specific receptor tyrosine kinases (VEGF recep-
tors, VEGFRs) VEGFR-1 (Flt1) and VEGFR-2 (Flk1) (for
review, see ref. 4). Development of blood vessels in the embryo
is dependent on VEGF as the formation of vessels in mouse
embryos heterozygous for a disrupted VEGF gene was aber-
rant and resulted in embryonic lethality (5, 6). VEGF is also
a potent inducer of vascular permeability (7).
Numerous proteins closely related in primary structure to
VEGF have been reported in recent years that may also play
roles in vascular biology. Placenta growth factor (PlGF) is
approximately 46% identical in amino acid sequence to VEGF
(8), binds to VEGFR-1 (9), and can form heterodimers with
VEGF (10). Although the biological function of PlGF is
unknown, it can significantly potentiate the action of low
concentrations of VEGF in vitro and in vivo (9). VEGF-B,
which is approximately 43% identical in amino acid sequence
to VEGF, is mitogenic for endothelial cells, can form het-
erodimers with VEGF, and may be involved in angiogenesis in
muscle and heart (11). As yet, the receptors for VEGF-B are
uncharacterized. Another member of the VEGF family,
VEGF-C, was isolated as a ligand for the tyrosine kinase
VEGFR-3 (Flt4) (12), a receptor that is expressed in endo-
thelial cell precursors in day 8.5 mouse embryos and later in
development is expressed in venous and lymphatic endothe-
lium (13). The pattern of VEGF-C gene expression in mouse
embryos suggests that VEGF-C may regulate angiogenesis of
the lymphatic vasculature (14). VEGF-C is also a ligand for
VEGFR-2 (12), but the functional significance of this potential
interaction in vivo is unknown. The amino acid sequence of
VEGF-C has a central region that is related to other members
of the VEGF family and exhibits approximately 30% identity
to VEGF. In addition, the VEGF-C sequence has N-terminal
and C-terminal extensions that are not present in VEGF,
PlGF, or VEGF-B (12, 15). The biosynthesis of VEGF-C
involves proteolytic processing that gives rise to a mature
secreted protein that essentially consists of the VEGF homol-
ogy domain (16), i.e., the portion of the molecule that is related
in primary structure to all other members of the VEGF family
and that contains the cystine knot motif that is found in VEGF
family members and in other growth factors (17).
We report herein the characterization of a human cDNA for a
fifth member of the VEGF family, designated VEGF-D, that is
most closely related to VEGF-C in primary structure. The mouse
homologue of VEGF-D was recently designated as the c-fos-
induced growth factor (18). We show that VEGF-D binds to and
induces tyrosine phosphorylation of the endothelial cell receptors
VEGFR-2 and VEGFR-3 and that, as for VEGF-C, the capacity
of VEGF-D to bind to these receptors is associated with the VEGF
homology domain. In addition, VEGF-D is mitogenic for endo-
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© 1998 by The National Academy of Sciences 0027-8424y98y95548-6$2.00y0
PNAS is available online at http:yywww.pnas.org.
This paper was submitted directly (Track II) to the Proceedings office.
Abbreviations: BAE, bovine aortic endothelial cell; EpoR, erythro-
poietin receptor; EST, expressed sequence tag; IL-3, interleukin 3;
PlGF, placenta growth factor; VEGF, vascular endothelial growth
factor; VEGFR, VEGF receptor.
Data deposition: The sequence reported in this paper has been
deposited in the GenBank database (accession no. AJ000185).
†
To whom reprint requests should be addressed. e-mail: Marc.achen@
ludwig.edu.au.
548

thelial cells. Our results demonstrate the existence of a subfamily
of the VEGFs that has VEGF-C and VEGF-D as founding
members.
MATERIALS AND METHODS
Cloning of cDNA for VEGF-D. Computer searches for
VEGF-related sequences were carried out by using the FASTA
search algorithm (19). The expressed sequence tag (EST)
encoding the C-terminal region of VEGF-D (GenBank acces-
sion no. H24828) had been isolated by the Integrated Molec-
ular Analysis of Genome Expression Consortium as part of the
Washington University–Merck EST Project (20). The EST was
obtained from the American Type Culture Collection and was
used as hybridization probe for isolation of cDNA from a
human lung cDNA library (Stratagene).
Northern Blot Analysis. A 1.1-kb fragment of the human
VEGF-D cDNA, containing the region encoding amino acid
residues 168–354 (Fig. 1) and approximately 500 nt of the 39
untranslated region, was used to screen human multiple-tissue
Northern blots (CLONTECH) according to manufacturer’s in-
structions.
Expression of VEGF-DDNDC in COS Cells and Purification
by Affinity Chromatography. A DNA fragment encoding the
portion of the human VEGF-D polypeptide from residues 93
to 201 was inserted into the expression vector pEFBOSSFLAG
immediately downstream from DNA sequence encoding the
interleukin 3 (IL-3) signal sequence (25) and the FLAG
octapeptide (IBIyKodak), so that protein synthesis would give
rise to a truncated secreted VEGF-D polypeptide that was
tagged with the FLAG octapeptide at its N terminus. This
protein was designated VEGF-DDNDC. VEGF-DDNDC was
expressed in COS cells that had been transiently transfected by
the DEAE-dextran method (26). The resulting conditioned
cell culture medium (approximately 150 ml), collected after 7
days of incubation, was subjected to affinity chromatography
using a resin to which the M2 (anti-FLAG) mAb (IBIyKodak)
had been coupled according to the manufacturer. An identical
affinity purification was carried out in parallel by using con-
ditioned culture medium from cells that had been transfected
with pEFBOSSFLAG (i.e., without VEGF-D coding se-
quences) to generate negative control samples for bioassays.
VEGF-DDNDC arising from affinity chromatography was
analyzed by Western blot analysis using mAb M2 (IBIyKodak)
or control antibody as described by the manufacturer.
Bioassay to Monitor Binding of Ligands to the Extracellular
Domain of VEGFR-2. A derivative of the pre-B cell line BayF3
(27), expressing a chimeric receptor consisting of the extra-
cellular domain of mouse VEGFR-2 and the transmembrane
and cytoplasmic domains of the mouse erythropoietin receptor
(EpoR) (28), was generated and designated BayF3-VEGFR-
2-EpoR (S.A.S., unpublished results). The BayF3-VEGFR-2-
EpoR cells were washed three times in PBS to remove all IL-3,
resuspended in cell culture medium without IL-3, and distrib-
uted into 96-well microtiter plates at a concentration of 10,000
cells per well. Fractions arising from affinity purification of
VEGF-DDNDC were then diluted into the cell culture me-
dium. Cells expressing a chimeric receptor, consisting of the
extracellular domain of the mouse endothelial cell receptor
Tie2 (29) and the transmembrane and cytoplasmic domains of
mouse EpoR (S.A.S. and A.S. Runting, unpublished results),
were used as a nonresponding control cell line. Cells were
incubated for 48 h, during which the cells in the cell culture
medium alone had died, and DNA synthesis occurring in the
other wells (i.e., in the presence of VEGF-DDNDC) was
determined by addition of 1 mCi of [
3
H]thymidine and quan-
titating incorporation over a 4-h period by b-counting.
Expression of Proteins in Baculovirus and Analysis of Receptor
Stimulation. Derivatives of human VEGF-D and VEGF-C
cDNA were cloned into the baculoviral vector pFASTBAC1
(GIBCOyBRL) for generation of viral stocks. The supernatants
of High Five cells (Invitrogen) were harvested 48 h after infection
with virus stocks, adjusted to pH 7 with NaOH, diluted with 1 vol
of DMEM containing 0.2% fetal calf serum and used to stimulate
NIH 3T3 cells expressing human VEGFR-3 (30) or porcine aortic
endothelial cells expressing human VEGFR-2 (31). Stimulation
of cells and analysis of phosphorylated receptors were carried out
as described (12).
Binding Assays with Soluble VEGFR Extracellular Do-
mains. For binding experiments, 293T cells were transfected
with plasmids encoding the soluble receptor-Ig fusion proteins
VEGFR-1-Ig (E. Korpelainen, Haartman Institute, Helsinki),
VEGFR-2-Ig (Y. Gunji, Haartman Institute, Helsinki), or
VEGFR-3-Ig (K. Pajusola, Biotechnology Institute, Helsinki)
by using the calcium phosphate method. The cells were
incubated for 24 h after transfection, washed with DMEM
containing 0.2% BSA, and starved for 24 h. Medium was then
collected and clarified by centrifugation, and fusion proteins
were precipitated by using protein A-Sepharose beads. The
Sepharose beads were then incubated at room temperature for
FIG. 1. Comparison of human VEGF-D with other members of the
VEGF family. Alignment of the deduced amino acid sequences of
human VEGF-D, mouse VEGF-D (18), human VEGF-C (15), human
VEGF
165
(21), human VEGF-B
167
(11), and human PlGF-2 (22) is
shown. Residues that match the sequence of human VEGF-D are
boxed. The asterisks above the hVEGF-D sequence denote the eight
cysteine residues that are conserved in all VEGF family members.
Arrows denote positions of proteolytic cleavage that give rise to
mature VEGF-C (16). The line above the hVEGF-D sequence denotes
a putative signal sequence for protein secretion (23). Potential N-
linked glycosylation sites in human VEGF-D are marked by brackets
above the sequence. Solid circles above the hVEGF-D sequence
denote cysteine residues involved in motifs that resemble those of
Balbiani ring 3 protein (CX
10
CXCXC) (24).
Cell Biology: Achen et al. Proc. Natl. Acad. Sci. USA 95 (1998) 549