The specificity of receptor binding by vascular endothelial growth factor-d is different in mouse and man.

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 vessels 2 , 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 * 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. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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 website 3 ). 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 g/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 Ci of [ 3 H]thymidine and further incubation for 4 h prior to harvesting using an automated cell harvester (Tomtec®). Incorporated [ 3 H]thymidine was measured by ␤-counting (Canberra Packard "Top Count NXT " scintillation counter, Meriden, CT).
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 SMART 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 k d , 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 (GenBank TM 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 5Ј untranslated region immediately upstream from the translation start codon.
Immunohistochemistry-Cryostat sections of embryonic day (E) 15.5mouse embryos were fixed in cold acetone for 10 min, and endogenous peroxidase activity was quenched by incubation in 0.2% H 2 O 2. 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 antigoat Ig-horseradish peroxidase (DAKO Corp., Carpinteria, CA) for 45 min. VEGFR-3 staining was detected using 3,3Ј-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 IgG 1 (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 Ile 94 -Pro 197 of VEGF-D and Arg 122 -Gly 220 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 ϳ20 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 spe-cific 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 crosslinking 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 ϳ10-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, Gly 155 -Val 156 -Met 157 , 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, Ser 150 -Leu 151 -Ile 152 , 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, Gly 200 in the mouse protein, corresponding to Ala 195 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 Gly 200 was altered to Ala, exhibited greatly enhanced activity in the VEGFR-2 bioassay in comparison with the wild-type mouse protein but ϳ13-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 Gly 200 to Ala, did not exhibit activity in the bioassay (data not shown), indicating that the Gly 200 to Ala alteration is required in combination with the SLI mutation to restore activity. A derivative of VEGF-D(ASLI), in which Thr 101 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 ϳ8-fold less activity than human VEGF-D. As Ile 96 in human VEGF-D is distant from the predicted receptorbinding 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 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.  (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.

DISCUSSION
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 crosslinked mouse VEGFR-2 4 . Given that VEGFR-2 signals for angiogenesis (11)(12)(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 upregulated on blood vessels during wound healing (45).
The predicted structure of the VHD of human VEGF-D indi-cated that three of the amino acid residues that differ between human and mouse VEGF-D, which occur sequentially in the amino acid sequence (residues Ser 150 -Leu 151 -Ile 152 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 receptorbinding surface, including Ser 150 , Leu 151 , Ile 152 , and Ala 195 , 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 ϳ13-fold . 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. 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 Ser 150 ,Leu 151 , Ile 152 , and Ala 195 in human VEGF-D, which are important for the VEGFR-2 interaction, are different from those for the residues in VEGF (Arg 82 , Lys, 84 and His 86 ), 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-3positive 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.