From the Ludwig Institute for Cancer Research, Post
Office Box 2008, Royal Melbourne Hospital, Victoria 3050 Australia and
the § Molecular/Cancer Biology Laboratory, Haartman
Institute and Helsinki University Hospital, P. O. Box 63 (Haartmaninkatu 8) SF 00014 University of Helsinki,
Helsinki, Finland
Received for publication, January 5, 2001, and in revised form, February 19, 2001
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ABSTRACT |
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Human vascular endothelial growth factor-D
(VEGF-D) binds and activates VEGFR-2 and VEGFR-3, receptors expressed
on vascular and lymphatic endothelial cells. As VEGFR-2 signals for
angiogenesis and VEGFR-3 is thought to signal for lymphangiogenesis, it
was proposed that VEGF-D stimulates growth of blood vessels and
lymphatic vessels into regions of embryos and tumors. Here we report
the unexpected finding that mouse VEGF-D fails to bind mouse VEGFR-2 but binds and cross-links VEGFR-3 as demonstrated by biosensor analysis
with immobilized receptor domains and bioassays of VEGFR-2 and VEGFR-3
cross-linking. Mutation of amino acids in mouse VEGF-D to those in the
human homologue indicated that residues important for the VEGFR-2
interaction are clustered at, or are near, the predicted
receptor-binding surface. Coordinated expression of VEGF-D and VEGFR-3
in mouse embryos was detected in the developing skin where the
VEGF-D gene was expressed in a layer of cells beneath the
developing epidermis and VEGFR-3 was localized on a network of vessels
immediately beneath the VEGF-D-positive cells. This suggests that
VEGF-D and VEGFR-3 may play a role in establishing vessels of the skin
by a paracrine mechanism. Our study of receptor specificity suggests
that VEGF-D may have different biological functions in mouse and man.
Blood vessel development in embryos and tumors depends on members
of the vascular endothelial growth factor
(VEGF)1 family of proteins
(for review see (1)). VEGF (also known as VEGF-A and vascular
permeability factor) is essential for vascular development
during embryogenesis (2, 3) and is crucial for angiogenesis in solid
tumors (4) that facilitates tumor growth and metastasis (4, 5). VEGF
binds the receptor tyrosine kinases VEGFR-1 (Flt-1) (6, 7) and VEGFR-2
(Flk-1/KDR) (8, 9) that are specifically expressed on the surface of
endothelial cells in embryos and tumors (10). As signaling from VEGFR-2 is crucial for both embryonic (11) and tumor angiogenesis (12, 13),
other ligands that activate this receptor, such as VEGF-D and VEGF-C
(14, 15), could also stimulate angiogenesis. VEGF-C binds both VEGFR-2
and VEGFR-3 (Flt-4) (14), is angiogenic (16, 17) and lymphangiogenic
(18), and may play a role in the development of lymphatic vessels (19).
The effects of VEGF-C on lymphatic vessels are probably mediated by
activation of VEGFR-3 (20), a receptor expressed on venous endothelial
cells during early embryogenesis that subsequently becomes restricted
to the endothelium of lymphatic vessels (21, 22). VEGFR-3 signaling is
thought to induce lymphangiogenesis (20) and hyperplasia of lymphatic vessels2, although this
receptor can be up-regulated on tumor blood vessels (23) and is
involved in signaling for tumor angiogenesis (24).
The most recently discovered mammalian member of the VEGF family,
VEGF-D (also known as c-fos-induced growth factor) (25), is
angiogenic (26), mitogenic for endothelial cells in vitro (15), is expressed at many sites in the developing embryo (27, 28), and
is localized in human tumors (29). VEGF-D also induced lymphangiogenesis and metastatic spread via the lymphatics in a mouse
tumor model (30). VEGF-D is initially synthesized as a precursor
protein containing N- and C-terminal propeptides in addition to the
VEGF homology domain (VHD), the region of the protein that shares
homology with all VEGF family members and contains receptor-binding
epitopes (15, 27). The N- and C-terminal propeptides are
proteolytically cleaved from the VHD during biosynthesis to generate a
mature, secreted form consisting of dimers of the VHD (27). The mature
form of human VEGF-D binds both VEGFR-2 and VEGFR-3 with much higher
affinity than does unprocessed VEGF-D (27). Therefore proteolytic
processing is important for activating human VEGF-D. As human VEGF-D
activates VEGFR-2 and VEGFR-3 (15), it has been proposed that VEGF-D
can stimulate the growth of blood vessels and lymphatic vessels into
regions of developing embryos and tumors.
Previous studies of receptor binding by VEGF-D were carried out using
the human protein (15, 27). Here we characterize the receptor-binding
specificity of mouse VEGF-D. Unlike human VEGF-D, that binds both
VEGFR-2 and VEGFR-3, mouse VEGF-D is specific for VEGFR-3 in the mouse;
it does not bind mouse VEGFR-2. This finding suggests that the
biological functions of VEGF-D in mouse and man may differ.
VEGF-D Derivatives, VEGF Receptors, and Antibodies--
Mouse
VEGF-D consists of the VHD, amino acid residues 97-206 tagged at the N
terminus with the FLAG octapeptide (Scientific Imaging Systems)
(31). Mouse VEGF-D-Cterm-FLAG is identical to mouse VEGF-D except that
it is tagged at the C terminus with FLAG instead of at the N terminus.
Human VEGF-D consists of amino acid residues 93-201, the region
exactly homologous to residues 98-206 of mouse VEGF-D, and is tagged
at the N terminus with FLAG (15). Mutants of mouse VEGF-D were
generated using polymerase chain reaction with oligonucleotides
encoding the mutated residues essentially as described previously (32).
Expression plasmids derived from pEFBOSSFLAG (C. McFarlane, Walter and
Eliza Hall Institute, Melbourne, Australia) encoding human, mouse, and
mutant VEGF-D were transiently transfected into 293EBNA cells using
FuGENE 6 (Roche Molecular Biochemicals, Indianapolis, IN) and the
proteins purified by affinity chromatography with M2 (anti-FLAG)
monoclonal antibody as described elsewhere (27). Expression and
purification of the extracellular domain of mouse VEGFR-2
(VEGFR-2-FLAG) has been described previously (33). Chimeric proteins
consisting of the extracellular domain of human VEGFR-2 (the first four
Ig-like domains), human VEGFR-3 (the first three Ig-like domains), or mouse VEGFR-3 (the first three Ig-like domains) and the Fc portion of
IgG (hVEGFR-2-Ig, hVEGFR-3-Ig, and mVEGFR-3-Ig, respectively) were
expressed by transient transfection of 293EBNA cells with expression
plasmids for these Ig fusion proteins (kind gifts from K. Pajusola and
Y. Gunji, Helsinki, Finland). Ig fusion proteins were purified by
affinity chromatography with protein A-Sepharose (Amersham Pharmacia
Biotech) according to the manufacturer's instructions.
SDS-Polyacrylamide Gel Electrophoresis, Western Blot Analysis,
and Protein Quantitation--
SDS-polyacrylamide gel electrophoresis
and Western blotting with M2 monoclonal antibody were carried out as
described previously (27). The extinction coefficient for mouse and
human VEGF-D was estimated based on amino acid composition using the
ProtParam tool program at the ExPASy
website3).
Quantitation of mouse and human VEGF-D was by spectrophotometry at 280 nm. The relative abundance of mouse, human, and mutants of VEGF-D was
confirmed by SDS-polyacrylamide gel electrophoresis followed by silver
staining of serial dilutions of these proteins.
Bioassays for Receptor Binding and Cross-linking--
Bioassays
for monitoring the binding and cross-linking of VEGFR-2 and VEGFR-3
involved the use of cell lines expressing chimeric receptors consisting
of the extracellular, ligand-binding domain of mouse VEGFR-2 or human
VEGFR-3 and the transmembrane and cytoplasmic domains of the
erythropoietin receptor (EpoR). In addition, cells expressing chimeric
receptors consisting of the extracellular domain of the mouse
endothelial cell receptor Tie2 and the transmembrane and cytoplasmic
domains of EpoR were used as a non-responding cell line. The chimeric
receptors had been transfected into the Ba/F3 cell line, a line of
pre-B cells, which survives and proliferates in the presence of
interleukin-3 (IL-3) but which dies after IL-3 deprivation. It has been
shown previously that signaling from the cytoplasmic EpoR domain of
chimeric receptors upon ligand binding is capable of rescuing these
cells in the absence of IL-3 (34). The expression of the VEGFR-2/EpoR,
VEGFR-3/EpoR, and Tie2/EpoR chimeric receptors in Ba/F3 cells allows
detection of specific ligand binding and cross-linking of the
extracellular domains of these receptors, which results in signaling
from the cytoplasmic domain of the EpoR and cell survival and
proliferation in the absence of IL-3. The cell lines expressing the
chimeric receptors are designated VEGFR-2-EpoR-Ba/F3 (15, 33),
VEGFR-3-EpoR-Ba/F3 (31), and Tie2-EpoR-Ba/F3 (35).
Samples of purified mouse and human VEGF-D were diluted in cell culture
medium (Dulbecco's modified Eagle's medium containing 10% (v/v)
fetal bovine serum, 50 mM L-glutamine, 50 µg/ml gentamicin, 1.2 mg/ml G418) deficient in IL-3. Bioassay cell
lines were then incubated in the media for 48 h at 37 °C, and
DNA synthesis was then quantitated by the addition of 1 µCi of
[3H]thymidine and further incubation for 4 h
prior to harvesting using an automated cell harvester
(Tomtec®). Incorporated [3H]thymidine was
measured by Biosensor Analysis--
All protein preparations were analyzed
for homogeneity, and buffer was exchanged into the appropriate buffers
by micropreparative size exclusion high pressure liquid chromatography
using a Superose 12 (3.2/30) column installed in a SMARTTM system
(Amersham Pharmacia Biotech) immediately prior to use (36). Receptor
domains were coupled to the carboxymethylated dextran layer of a CM5
sensor chip using standard amine coupling chemistry (36) for analysis of ligand binding using a BIAcore 3000 optical biosensor (BIAcore, Uppsala, Sweden). Automatic targeting of immobilization levels was
achieved using BIAcore 3.1 control software (37). Following immobilization, residual activated ester groups were blocked by treatment with 1 M ethanolamine hydrochloride, pH 8.5, followed by washing with 10 mM diethylamine to remove
non-covalently bound material. The 10 mM diethylamine was
also used to regenerate the sensor surface between analyses. Samples
for assay were diluted in running buffer (10 mM HEPES, pH
7.4, 150 mM NaCl, 3.4 mM EDTA, 0.005% Tween
20). The integrity of the surface was assessed by binding of purified
mouse and human VEGF-D. The apparent binding affinities of mouse and
human VEGF-D to receptor domains were determined by analysis of the
initial dissociation phase to obtain the kd, which was
then used to constrain a global analysis of the association region of
the curves assuming a 1:1 Langmuirian model. Data were analyzed using
BIAevaluation 3.0 (BIAcore, Uppsala, Sweden) as described previously
(38).
In Situ Hybridization--
In situ hybridization was
carried out as described elsewhere (27). Two non-overlapping antisense
RNA probes, homologous to the regions of mouse VEGF-D cDNA
(GenBankTM accession number X99572) encoding from
amino acid residues 1-85 (probe A) and 199-317 (probe B), were used
in this study. In addition to encoding the N-terminal 85 amino acids of
VEGF-D, probe A also contained 80 nucleotides of the 5' untranslated
region immediately upstream from the translation start codon.
Immunohistochemistry--
Cryostat sections of embryonic day (E)
15.5-mouse embryos were fixed in cold acetone for 10 min, and
endogenous peroxidase activity was quenched by incubation in 0.2%
H2O2. Sections were blocked for 15 min in
phosphate-buffered saline/0.5% bovine serum albumin and incubated with
goat anti-human VEGFR-3 (Flt-4) affinity-purified antiserum (R&D
Systems, Minneapolis, MN) for 1.5 h at room temperature. Sections
were then incubated with rabbit anti-goat Ig-horseradish peroxidase
(DAKO Corp., Carpinteria, CA) for 45 min. VEGFR-3 staining was detected
using 3,3'-diaminobenzidine (DAKO Corp.). As an adsorption control, the VEGFR-3 anti-serum was incubated for 1 h at room temperature with a 10-fold molar excess of a chimeric protein consisting of the extracellular domain of human VEGFR-3 fused to the Fc
region of human IgG1 (R&D) before being used for immunohistochemistry.
Homology Modeling--
Models of human VEGF-D and human VEGFR-2,
based on the crystal structure of VEGF in complex with VEGFR-1 (PDB
entry 1FLT (39)), were created using MODELLER (40). The model complex includes residues Ile94-Pro197 of VEGF-D
and Arg122-Gly220 of VEGFR-2, encompassing the
second Ig-like domain that interacts with VEGF (41). The 1:2 complex of
VEGF-D with Ig-like domain 2 of VEGFR-2 was generated by superimposing
the coordinates of the VEGF-D and the VEGFR-2 models onto the
co-crystal structure. Identification of the highest quality model was
achieved using a combination of the MODELLER and ProsaII scores
(42).
Mouse VEGF-D Does Not Bind Mouse VEGFR-2--
We have previously
shown that mature human VEGF-D, consisting of the VHD, binds,
cross-links, and activates VEGFR-2 and VEGFR-3 (15, 27, 31). In
preliminary studies to analyze the interaction of mouse VEGF-D with
VEGFR-2, we observed a lack of binding (data not shown). This was
surprising given that the VHDs of mouse and human VEGF-D are closely
related in primary structure (Fig.
1A) and that human VEGF-D
binds VEGFR-2 (15). Therefore we used biosensor analysis to investigate
the binding of the VHDs of mouse and human VEGF-D to immobilized
receptor extracellular domains of VEGFR-2 and VEGFR-3.
Mouse and human VEGF-D, each consisting of the VHD tagged at the N
terminus with the FLAG octapeptide, were expressed and purified as
described under "Experimental Procedures." The identity of these
proteins was confirmed by silver staining and Western blot analysis. As
expected, the apparent molecular masses of the subunits of these
proteins were ~20 kDa (Fig. 1B). Analysis of the biosensor
binding curves for the interactions of mouse and human VEGF-D with
mouse and human VEGFR-2 and VEGFR-3 demonstrated that mouse VEGF-D did
not bind the extracellular domain of mouse VEGFR-2 but bound to mouse
VEGFR-3 (Fig. 1C); human VEGF-D bound both human VEGFR-2 and
human VEGFR-3 (Fig. 1D). These findings indicate that mouse
VEGF-D is specific for VEGFR-3 in the mouse but that human VEGF-D
interacts with both VEGFR-2 and VEGFR-3 in human. Interestingly, mouse
VEGF-D bound human VEGFR-2 and human VEGF-D bound mouse VEGFR-2,
indicating that the inability of mouse VEGF-D to bind mouse VEGFR-2 may
be a consequence of differences in both growth factor and receptor in
comparison to the human homologues. The kinetics of the
interactions of human VEGF-D with mouse VEGFR-2 and human VEGFR-3 were
similar to those documented previously (27).
Analysis of Receptor Binding and Cross-linking at the Cell
Surface--
To further analyze the interaction of mouse VEGF-D with
VEGFR-2 and VEGFR-3, we used bioassays of receptor binding and
cross-linking. The assays involved the use of Ba/F3 pre-B cell lines
designated VEGFR-2-EpoR-Ba/F3 (33) and VEGFR-3-EpoR-Ba/F3 (31), which survive and proliferate only in the presence of growth factors capable
of binding and cross-linking the extracellular domains of mouse VEGFR-2
and human VEGFR-3, respectively. In previous studies, all of the
activating ligands for VEGFR-2 or VEGFR-3 (i.e. VEGF,
VEGF-C, VEGF-D, and viral VEGFs) stimulated the VEGFR-2-EpoR-Ba/F3 or
VEGFR-3-EpoR-Ba/F3 bioassay cell lines (15, 31, 33, 43). Both mouse and
human VEGF-D were tested in the mouse VEGFR-2 and human VEGFR-3
bioassays (Fig. 2). Mouse VEGF-D did not
promote significant proliferation of the VEGFR-2-EpoR-Ba/F3 cells
indicating that this ligand cannot bind and cross-link mouse VEGFR-2 at
the cell surface (Fig. 2A). This result is consistent with
the biosensor data demonstrating that mouse VEGF-D does not bind
immobilized mouse VEGFR-2. Surprisingly, mouse VEGF-D was more potent
than human VEGF-D in the human VEGFR-3 bioassay, an ~10-fold greater concentration of human VEGF-D was required to give comparable stimulation of the VEGFR-3 cell line (Fig. 2B). Mouse
VEGF-D-Cterm-FLAG, a derivative of mature mouse VEGF-D tagged with FLAG
at the C terminus rather than at the N terminus, exhibited comparable
activity to the N-terminal-tagged derivative in both bioassays (data
not shown), indicating that the position of FLAG did not influence receptor interactions.
To identify amino acid residues important for the interaction of VEGF-D
with its receptors, a mutant of mouse VEGF-D, designated VEGF-D(SLI),
was generated in which the three sequential residues, Gly155-Val156-Met157, were altered
to the corresponding sequence of Ser-Leu-Ile found in human VEGF-D (see
Fig. 1A for positions of these altered residues in the
primary structure). These three residues in mouse VEGF-D were chosen
for alteration because a predicted three-dimensional structure of human
VEGF-D, based on the crystal structure of VEGF in complex with VEGFR-1
(39), indicated that the corresponding residues in human VEGF-D,
Ser150-Leu151-Ile152, are near or
part of a putative receptor-binding surface (Fig. 2C). Mouse
VEGF-D(SLI) exhibited reduced activity compared with wild-type mouse
VEGF-D in the VEGFR-3 bioassay, comparable with the activity of human
VEGF-D (Fig. 2B). However, this mutant did not exhibit
significantly enhanced activity in the VEGFR-2 bioassay (Fig.
2A). As the three altered residues in mouse VEGF-D(SLI) did
not confer activity in the VEGFR-2 bioassay, further mutants were
generated to recapitulate human VEGF-D-like activity. Of the
differences in amino acid sequence between mouse VEGF-D(SLI) and human
VEGF-D, Gly200 in the mouse protein, corresponding to
Ala195 in the human protein, is most closely located to the
predicted receptor-binding surface (Fig. 2C). A mutant of
VEGF-D(SLI), designated VEGF-D(ASLI), in which residue
Gly200 was altered to Ala, exhibited greatly enhanced
activity in the VEGFR-2 bioassay in comparison with the wild-type mouse
protein but ~13-fold less activity than human VEGF-D (Fig.
2A). In contrast, mouse mutant VEGF-D(A), in which the only
alteration from wild-type was Gly200 to Ala, did not
exhibit activity in the bioassay (data not shown), indicating that the
Gly200 to Ala alteration is required in combination with
the SLI mutation to restore activity. A derivative of VEGF-D(ASLI), in
which Thr101 was altered to the corresponding Ile residue
at position 96 in human VEGF-D, was designated VEGF-D(AISLI) and
exhibited marginally more activity than VEGF-D(ASLI) but ~8-fold less
activity than human VEGF-D. As Ile96 in human VEGF-D is
distant from the predicted receptor-binding surface (Fig.
2C), the enhanced activity of mouse VEGF-D(AISLI) in
comparison with the ASLI mutant indicates that the residues distant
from the receptor-binding surface, which differ between mouse and human
VEGF-D, can influence the VEGFR-2 receptor interaction.
Expression of VEGF-D and VEGFR-3 Is Coordinated in Developing Mouse
Skin--
The finding that mouse VEGF-D binds mouse VEGFR-3 but not
mouse VEGFR-2 suggests that the vessels in the mouse that respond to
mouse VEGF-D are those expressing VEGFR-3. To determine whether VEGF-D
and VEGFR-3 expression is coordinated during embryogenesis, we analyzed
E15.5 mouse embryos for these markers. A strong signal for VEGF-D
mRNA was detected by in situ hybridization in a layer of
cells in the developing skin, beneath the developing epidermis (Fig.
3 A, B, and
D). Immunohistochemical analysis revealed a network of
VEGFR-3-positive vessels immediately beneath the layer of
VEGF-D-positive cells (Fig. 3E). This finding is consistent with previous studies in which VEGFR-3 mRNA was detected on a network of vessels immediately beneath the skin of E12.5 mouse embryos
(21). Although vessels in the E15.5 skin were also immunopositive for
VEGFR-2, one of the ligands for this receptor, VEGF, was undetectable and the other, VEGF-C, was only very weakly positive in hair follicles (data not shown). The localization of a VEGFR-3-positive network of
vessels in the developing skin immediately beneath a layer of cells
expressing VEGF-D suggests that the interaction of VEGF-D with VEGFR-3
could occur in vivo and may have a role in the development of these vessels.
The finding that VEGF-D is a specific ligand for
VEGFR-3 in the mouse was unexpected as it is often assumed that
homologous ligands exhibit similar receptor-binding characteristics in
different species. This is the first report of a VEGF family member
with different receptor-binding specificity in mouse and man. This difference in specificity was not observed for the closely related growth factor VEGF-C, as human VEGF-C activated human VEGFR-2 (14) and
mouse VEGF-C bound and cross-linked mouse
VEGFR-24. Given that VEGFR-2
signals for angiogenesis (11-13), whereas VEGFR-3 is thought to signal
for lymphangiogenesis (20), the data reported here suggest that
VEGF-D may induce both angiogenesis and lymphangiogenesis in man but
only lymphangiogenesis in the mouse. However, this issue is complicated
by the findings that VEGFR-3, although specific to lymphatic
endothelium in normal adult tissues (21, 22), is up-regulated on
angiogenic blood vessels in cancer (23, 44), and signaling via this
receptor appears to be required for tumor angiogenesis (24).
Furthermore, VEGFR-3 can also be up-regulated on blood vessels during
wound healing (45).
The predicted structure of the VHD of human VEGF-D indicated that three
of the amino acid residues that differ between human and mouse VEGF-D,
which occur sequentially in the amino acid sequence (residues
Ser150-Leu151-Ile152 in the human
protein), were located near, or were part of, a putative
receptor-binding surface. This putative receptor-binding surface in
human VEGF-D is likely to interact with both VEGFR-2 and VEGFR-3 as it
is known that VEGF binds both VEGFR-1 and VEGFR-2 using the same
binding interface (39). Furthermore, a neutralizing antibody to human
VEGF-D that blocks the interaction with VEGFR-2 also blocks binding to
VEGFR-3 (31). Mutation of these three residues in mouse VEGF-D to the
homologous residues in human VEGF-D decreased the potency of binding
and cross-linking of human VEGFR-3 in the Ba/F3 bioassay to that
exhibited by the human protein, indicating that this region is
important for the VEGFR-3 interaction. Although mouse VEGF-D binds and
cross-links human VEGFR-3 more potently than human VEGF-D, the kinetics
for the binding of mouse VEGF-D measured with a biosensor were not
significantly different from those observed for human VEGF-D. This
suggests that the greater activity of mouse VEGF-D in the bioassay may
be due to more efficient dimerization of the receptor or differences in
the efficiency of receptor internalization and recycling of receptors
to the cell surface, phenomena that can be dramatically altered by
single amino acid substitutions in growth factors (46, 47). The greater activity of mouse VEGF-D in the human VEGFR-3 bioassay also indicates that mouse VEGF-D may be a better therapeutic for inducing signaling via VEGFR-3 and has a more appropriate structure on which to base design of small molecule VEGFR-3 agonists.
The capacity of the mouse VEGF-D mutants generated in this study to
bind and cross-link VEGFR-2 indicated that residues in human VEGF-D at,
or near, the putative receptor-binding surface, including
Ser150, Leu151, Ile152, and
Ala195, are important for the VEGFR-2 interaction. Although
the ASLI mutant of mouse VEGF-D, containing these four residues of
human VEGF-D, had much greater activity than wild-type mouse VEGF-D in
the VEGFR-2 bioassay, it still had ~13-fold less activity than human
VEGF-D. This indicates that residues distant from the receptor-binding interface do play a role. The locations of the residues
Ser150,Leu151, Ile152, and
Ala195 in human VEGF-D, which are important for the VEGFR-2
interaction, are different from those for the residues in VEGF
(Arg82, Lys,84 and His86), which
were shown to be critical for the binding of this receptor by
alanine-scanning mutagenesis (48). This suggests that different sets of
residues in the receptor-binding surfaces of these two growth factors
are critical for the VEGFR-2 interaction.
Previous studies indicated that mouse VEGF-D induced proliferation of
endothelial cells in vitro and was angiogenic both in vivo and in vitro (26). The angiogenesis activity
in vivo was demonstrated in the rabbit corneal assay, and
in vitro studies of angiogenesis and mitogenic activity were
performed using human umbilical vein endothelial cells. As we have
shown that mouse VEGF-D binds human VEGFR-2, the mitogenic effects of
mouse VEGF-D on human umbilical vein endothelial cells observed
previously could indeed have been mediated by VEGFR-2, although the
involvement of VEGFR-3 cannot be discounted. The angiogenic activity
observed in the rabbit corneal assay may have been a consequence of the capacity of mouse VEGF-D to activate rabbit VEGFR-2, although it is not
known if this ligand binds VEGFR-2 in the rabbit.
Our finding that VEGF-D binds VEGFR-3 but not VEGFR-2 in the mouse
suggests that expression of genes for VEGFR-3 and VEGF-D should be
coordinated at sites in the mouse embryo during development to
establish a paracrine mode of action that is typical of VEGF family
members (49, 50). Indeed, expression of VEGFR-3 and VEGF-D is
coordinated in developing mouse skin, as a layer of cells positive for
VEGF-D mRNA is immediately adjacent to a network of
VEGFR-3-positive vessels. Therefore, it will be important to monitor
the effect of VEGF-D deficiency on the development of vessels in the
skin. The unexpected finding that mouse VEGF-D does not bind mouse
VEGFR-2 has important implications for the interpretation of biological
models and pharmacological approaches for monitoring the function and
utility of VEGF-D.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-counting (Canberra Packard "Top Count NXTTM " scintillation counter, Meriden, CT).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Alignment of human and mouse VEGF-D, analysis
of purified proteins, and binding to immobilized receptors.
A, alignment of the amino acid sequences of the VHDs of
human and mouse VEGF-D. The derivatives of human and mouse VEGF-D used
in this study consisted of these VHDs tagged at the N terminus with the
FLAG octapeptide, but the FLAG sequence is not shown here. The
sequences are numbered from the VEGF-D initiation methionines (15, 25).
The sequence in bold font represents residues of mouse
VEGF-D that differ from human VEGF-D. Cysteine residues involved in the
disulfide bridges that establish the cystine knot are
shaded. B, silver staining (top panel)
and Western blot analysis using anti-FLAG antibody (bottom
panel) of human VEGF-D (hVEGF-D) and mouse VEGF-D (mVEGF-D). The
positions of molecular mass markers (in kDa) are shown to the
left. C, biosensor analysis of the interaction of
mouse VEGF-D with mouse VEGFR-2 (top panel) and mouse
VEGFR-3 (middle panel) and of human VEGF-D with mouse
VEGFR-2 (bottom panel). Chimeric receptor proteins were
immobilized onto a carboxymethylated dextran surface as described under
"Experimental Procedures." Growth factors (30 µl) were injected
over the surface at a flow rate of 10 µl/min at the following
concentrations: top panel: 1520, 760, 380, 190, 95, and 48 nM; middle panel: 1100, 550, 275, 138, 69, and
34 nM; bottom panel; 650, 325, 163, 81, 41, and
20 nM. The sensorgrams shown have been subtracted with the
corresponding signal obtained when the same sample was passed over a
blank control channel. D, kinetic data derived from the
biosensor analysis. Kinetic data were extracted by global fitting using
BIAevaluation 3.0 assuming a 1:1 Langmuirian model with mass transfer.
ka = association rate constant;
kd = dissociation rate constant;
KD =
ka/kd = affinity
constant.
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Fig. 2.
Receptor binding and cross-linking at
the cell surface and predicted structure of VEGF-D. The capacity
of mouse and human VEGF-D and mouse VEGF-D mutants to bind and
cross-link mouse VEGFR-2 and human VEGFR-3 was assessed using
bioassays. VEGFR-2-EpoR-Ba/F3 (A) and VEGFR-3-EpoR-Ba/F3
(B) cell lines were incubated in IL-3-deficient medium
containing serially diluted human VEGF-D (hVEGF-D), mouse VEGF-D
(mVEGF-D), or mouse VEGF-D mutants SLI, ASLI, or AISLI for 48 h at
37 °C. DNA synthesis was then quantitated by
[3H]thymidine incorporation. Each point on the
graphs represents the mean cpm incorporated from duplicate
assays, and error bars denote the range of cpm measured. The
graph for the VEGFR-3 bioassay shows data from a negative control in
which mouse VEGF-D was incubated with a non-responding Ba/F3 cell line
expressing the extracellular domain of the Tie2 receptor. C,
predicted three-dimensional structures of human VEGF-D (top
panel) and of VEGF-D complexed with VEGFR-2 (bottom
panel). The VEGF-D structure is a homodimer of the VHD, one
subunit of which is yellow and the other orange.
The predicted receptor-binding interface of VEGF-D is indicated with
black diagonal lines. The region of human VEGFR-2 shown here
is the second Ig-like domain (blue). The structure for the
VEGF-D/VEGFR-2 complex depicts the interaction of the VEGF-D dimer with two receptor subunits. The panels display surface
rendering of these proteins. Residues that differ between the VHDs of
human and mouse VEGF-D are shown in purple. The positions of
residues altered in mouse VEGF-D mutants are indicated.
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Fig. 3.
VEGF-D and VEGFR-3 expression in the
developing skin of E15.5 day mouse embryos. A, light
field micrograph of a tissue section containing the skin of the upper
dorsal surface hybridized with the VEGF-D antisense probe A (see
"Experimental Procedures"). Identical results were obtained with
non-overlapping VEGF-D antisense RNA probe B (not shown). The square
denotes the area shown at high magnification in D. B, dark field micrograph of A showing
distribution of VEGF-D mRNA beneath the epidermis of the skin.
C, dark field micrograph of a serial section to that in
B hybridized with a VEGF-D sense RNA probe. D,
light field micrograph at high magnification showing VEGF-D
gene expression (arrowheads) beneath the epidermis of the
developing skin of the back of the embryo. E, immunostaining
of vessels beneath the skin of the back using anti-VEGFR-3 antiserum
(brown denotes positive signal). F, adsorption
control on a section serial to that in E in which VEGFR-3
antiserum was preincubated with VEGFR-3-Fc chimeric protein prior to
immunostaining.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Prof. Antony Burgess for critical reading of this manuscript and Dr. Tanya Petrova for providing the mVEGFR-3-Ig construct.
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FOOTNOTES |
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* This work was supported by the National Health and Medical Research Council of Australia and the Anti-Cancer Council of Victoria.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence and reprint requests should be addressed. Tel.: 613-9341-3155; Fax: 613-9341-3107; E-mail: Marc.achen@ludwig.edu.au.
Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M100097200
2 Veikkola, T., Jussila, L., Makinen, T., Karpanen, T., Jeltsch, M., Petrova, T. V., Kubo, H., Thurston, G., McDonald, D. M., Achen, M. G., Stacker, S. A., and Alitalo, K. (2001) EMBO J. 20, 1223-1231.
3 Contact corresponding author for Website address.
4 M. E. Baldwin and M. G. Achen, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: VEGF, vascular endothelial growth factor; VEGFR, VEGF receptor; VHD, VEGF homology domain; EpoR, erythropoietin receptor; IL-3, interleukin-3; E, embryonic day; h, human; m, mouse.
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