as vasculogenesis is restricted to the embryonic period of
development. Vasculogenesis involves the formation of the
Olofsson et al., 1996). Both share a striking structural similar-
ity with VEGF, thus expanding the family of known VEGF-
produced as a disulfide-linked dimer of 415 amino acid expressed in adult mouse lung, heart and kidney, where
iearliest blood vessels by in situ differentiation of endothelial
cells from mesodermal precursor cells known as angioblasts
(Risau et al., 1988). Angiogenesis is the subsequent formation
of blood vessels via sprouting and intussusception from pre-
existing ones. This mechanism also occurs where neovascu-
larization is required in adults, and is of particular significance
in wound healing, maturation of ovarian follicles and tumor
development. The mechanisms regulating the latter processes
are of particular interest as they may generate targets for the
therapeutic control of pathological processes dependent on
angiogenesis (Folkman and Shing, 1992; Hanahan and
Folkman, 1996).
Several inhibitors and stimulators of angiogenesis have been
like growth factors.
VEGF-C protein was purified and its cDNA cloned from
human prostatic carcinoma cells (Joukov et al., 1996). While
being homologous with other members of the VEGF/platelet
derived growth factor (PDGF) family, the C-terminal half of
VEGF-C contains extra cysteine-rich motifs characteristic of
the protein component of silk produced by the larval salivary
glands of the midge, Chironomus tentans. Human VEGF-C is
proteolytically processed, binds the Flt4 receptor tyrosine
kinase, which we have renamed the VEGF receptor-3
(VEGFR-3), and induces tyrosine autophosphorylation of
VEGFR-3 and VEGFR-2 (Joukov et al., 1996). In addition,
VEGF-C stimulates the migration of bovine capillary endo-INTRODUCTION
The cardiovascular system is the first organ system to begin
functioning in the developing embryo. The inner layer of blood
and lymphatic vessels as well as the endocardium are formed
by endothelial cells that play a critical role in physiological and
pathological processes of the vasculature. The process known
growth factor (VEGF) is currently held to be the major endo-
thelial-cell-specific angiogenesis and permeability factor,
whereas the related placenta growth factor is expressed only in
a restricted set of tissues (for a review, see (Dvorak et al., 1995;
Ferrara et al., 1992; Neufeld et al., 1994). Recently two new
endothelial-cell-specific growth factors VEGF-B and VEGF-C
have been cloned (Joukov et al., 1996; Lee et al., 1996;
residue polypeptides, sharing an 85% identity with the
human VEGF-C amino acid sequence. The recombinant
mouse VEGF-C protein was secreted from transfected cells
as VEGFR-3 (Flt4) binding polypeptides of 30-32· 103 Mr
and 22-23· 103 Mr which preferentially stimulated the
autophosphorylation of VEGFR-3 in comparison with
VEGFR-2 (KDR). In in situ hybridization, mouse VEGF-
C mRNA expression was detected in mesenchymal cells of
postimplantation mouse embryos, particularly in the
VEGFR-3 was also prominent. The pattern of expression
of VEGF-C in relation to its major receptor VEGFR-3
during the sprouting of the lymphatic endothelium in
embryos suggests a paracrine mode of action and that one
of the functions of VEGF-C may be in the regulation of
angiogenesis of the lymphatic vasculature.
Key words: VEGF-C receptor, VEGFR-3, vascular system,
endothelial cell, mouse, lymphatic system, angiogenesisDevelopment 122, 3829-3837 (1996)
Printed in Great Britain © The Company of Biologists Limited 1996
DEV4752
The vascular endothelial growth factor family has recently
been expanded by the isolation of two new VEGF-related
factors, VEGF-B and VEGF-C. The physiological functions
of these factors are largely unknown. Here we report the
cloning and characterization of mouse VEGF-C, which is
SUMMARY
VEGF-C receptor binding and pattern o
role in lymphatic vascular development
Eola Kukk*, Athina Lymboussaki*, Suvi Taira, Arja Ka
and Kari Alitalo†
Molecular/Cancer Biology Laboratory, Haartman Institute, PL21 (
*Both first authors have contributed equally to this work
†Author for correspondencedescribed, but only a few of them appear to be endothelial cell-
specific. Many factors affect the proliferation and differen-
tiation of the endothelium indirectly. Vascular endothelial3829
regions where the lymphatic vessels undergo sprouting
from embryonic veins, such as the perimetanephric,
axillary and jugular regions. In addition, the developing
mesenterium, which is rich in lymphatic vessels, showed
strong VEGF-C expression. VEGF-C was also highly
f expression with VEGFR-3 suggests a
painen, Michael Jeltsch, Vladimir Joukov
Haartmaninkatu 3), 00014 University of Helsinki, Finlandthelial cells in collagen gels. VEGF-C is thus a novel regulator
of endothelia.
Despite their homology, the VEGFs probably have different

3830
functional roles that may overlap, as regulatory factors of the
endothelium. In order to clarify of the function of VEGF-C in
vivo (Joukov et al., 1996), we isolated the mouse VEGF-C
cDNA and analysed its protein product and mRNA expression
pattern in developing mouse embryos.
MATERIALS AND METHODS
Isolation of mouse cDNA clones for VEGF-C
To isolate mouse VEGF-C cDNAs, approximately 1· 106 bacterio-
phage lambda clones from a 12 day p.c. mouse embryo cDNA library
(EXlox library, Novagen # 69632-1) were screened with a radiola-
beled PCR fragment of human VEGF-C cDNA containing nucleotides
495 to 1661 (sequence accession number X94216). One positive clone
was isolated, a 1.3 kb EcoRI/HindIII fragment of the insert was
subcloned to the corresponding sites of the pBluescript II SK+ vector
(Stratagene) and sequenced. The cDNA sequence of this clone was
homologous with the human VEGF-C sequence, except that about 710
bp of sequence present in the human clone was missing from the 5¢ -
end. For further screening of mouse cDNA libraries, a HindIII-BstXI
(HindIII site is from the pBluescript II SK+ polylinker) fragment of
881 bp from the coding region of the mouse cDNA clone was used
as a probe to isolate two additional cDNA clones from an adult mouse
heart l ZAP II cDNA library (Stratagene # 936306). Three further
clones were isolated from a mouse heart 5¢ -stretch-plus cDNA library
in l gt11 (Clontech #ML5002b). Of the latter clones, one was found
to contain an insert of about 1.8 kb. The insert of this cDNA clone
was subcloned into the EcoRI sites of the pBluescript II SK+ vector
and both strands were sequenced.
Analysis of mRNA expression in tissues
Mouse embryo multiple tissue northern blot (Clontech) containing 2
m g of polyadenylated RNAs from 7, 11, 15 and 17 day postcoital (p.c.)
embryos was hybridized with mouse VEGF-C cDNA fragment (base
pairs 499-656) and washed in stringent conditions. A mouse adult
tissue northern blot was hybridized with the same probe for VEGF-
C and with a VEGFR-3 cDNA fragment (nucleotides 1-595; accession
number X68203). Mouse b -actin probe (Clontech) was used as a
control.
In situ hybridization of mouse embryos
In situ hybridization of tissue sections was performed as described
previously (Västrik et al., 1995). The mouse VEGF-C antisense RNA
probe was generated from linearized pBluescript II SK+ plasmid
(Stratagene), containing an EcoRI/HindIII fragment corresponding to
nt 558-979 of mouse VEGF-C cDNA, where the 3¢ noncoding region
and the BR3P repeats had been removed by exonuclease III treatment.
Radiolabeled RNA was synthesized using T7 polymerase and
[35S]UTP (Amersham). Mouse VEGFR-3 antisense and sense RNA
probes were synthesized in a similar manner from linearized pGEM-
3Z(f+) plasmid containing the mouse VEGFR-3 cDNA insert
described previously (Kaipainen et al., 1993). The high stringency
wash was for 45 minutes at 65°C in a solution containing 30 mM DTT
and 4· SSC. The slides were exposed for 28 days, developed and
stained with Haematoxylin.
Expression and analysis of recombinant VEGF-C
The 1.8 kb mouse VEGF-C cDNA was cloned as an EcoRI fragment
into the retroviral expression vector pBabe-puro (a kind gift from Dr
Hartmut Land, ICRF, London) containing the SV40 early promoter
region (Morgensternand and Land, 1990) and transfected into the
BOSC23 packaging cell line by the calcium-phosphate precipitation
method (Pear et al., 1994). For comparison, these cells were also trans-
fected with the previously described human VEGF-C construct in the
E. Kukk and others pREP7 expression vector (Joukov et al., 1996). The transfected cellswere cultured for 48 hours prior to metabolic labelling. Cells were
changed into DMEM medium devoid of cysteine and methionine and
after 45 minutes of preincubation and medium change, about 120
m Ci/ml of Pro-mixTM L-[35S] in vitro cell labelling mix (Amersham)
in the same medium was added. After 6 hours of incubation, the culture
medium was collected and clarified by centrifugation.
For immunoprecipitation, 1 ml aliquots of the media from meta-
bolically labelled BOSC23 cells transfected with empty vector or
mouse or human recombinant VEGF-C, respectively, were incubated
overnight on ice with 2 m l of rabbit polyclonal antiserum against an
N-terminal 17 aa peptide of mature human VEGF-C (EETIK-
FAAAHYNTEILK; V. J. et al., unpublished data). Incubation with
protein A sepharose was carried out for 40 minutes at 4°C with gentle
agitation. The sepharose beads were then washed twice with immuno-
precipitation buffer, four times with 20 mM Tris-HCl pH 7.4, samples
were boiled in Laemmli buffer and analysed by 12.5 % sodium
dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE).
For receptor binding experiments, 1 ml aliquots of media from
metabolically labelled BOSC23 cells were incubated with VEGFR-3
extracellular domain covalently coupled to sepharose for 4 hours at
4°C with gentle mixing. The sepharose beads were washed four times
with ice-cold phosphate-buffered saline (PBS) and the samples were
analysed as described in (Joukov et al., 1996).
VEGFR-3 and VEGFR-2 stimulation experiments
For the VEGFR-3 receptor stimulation experiments, subconfluent
NIH3T3-VEGFR-3 (Flt4) cells (Pajusola et al., 1994) were starved
overnight in serum-free medium containing 0.2% BSA. The cells
were stimulated with the conditioned medium from VEGF-C vector-
transfected cells for 5 minutes, washed three times with cold PBS con-
taining 200 m M vanadate and lysed in RIPA buffer (10 mM Tris pH
7.5, 50 mM NaCl, 0.5% sodium deoxycholate, 0.5% Nonidet P40,
0.1% SDS, 0.1 U/ml Aprotinin, 200 m M vanadate) for immunopre-
cipitation analysis. The lysates were centrifuged for 25 minutes at
16 000 g and the resulting supernatants were incubated for 2 hours on
ice with the specific antisera, followed by immunoprecipitation using
protein A-sepharose and analysis in 7% SDS-PAGE. Polypeptides
were transferred onto nitrocellulose and analyzed by immunoblotting
using anti-phosphotyrosine (Transduction Laboratories) and anti-
receptor antibodies, as described by Pajusola et al. (1994). Stripping
of the filter was carried out at 50°C for 30 minutes in 100 mM 2-mer-
captoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7 with occasional
agitation.
VEGFR-2 stimulation was studied in subconfluent porcine aortic
endothelial (PAE) cells expressing VEGFR-2 (PAE-KDR) (Wal-
tenberger et al., 1994) which were starved overnight in serum-free
medium containing 0.2% BSA. Stimulation was carried out and the
lysates prepared as described above. For receptor immunoprecipita-
tion, specific antiserum for VEGFR-2 (Waltenberger et al., 1994) was
used (a kind gift from Dr Lena Claesson-Welsh, Ludwig Institute for
Cancer Research, Uppsala, Sweden) and the immunoprecipitates were
analyzed as described for VEGFR-3 in 7% SDS-PAGE followed by
western blotting with anti-phosphotyrosine antibodies, stripping the
filter and re-probing it with anti-VEGFR-2 antibodies (Santa Cruz).
RESULTS
Cloning and analysis of mouse VEGF-C cDNA
Three different cDNA libraries were screened, first using
probes made from the human VEGF-C cDNA (Joukov et al.,
1996) and then partial mouse VEGF-C cDNAs as hybridiz-
ation probes, as detailed in the Materials and Methods section.
Fig. 1A shows schematic structures of the clones obtained in
comparison with human VEGF-C cDNA. Examination of the
1822 bp mouse VEGF-C cDNA and its predicted protein