Vascular endothelial growth factor D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) and VEGF receptor 3 (Flt4)

Abstract

We have identified a member of the VEGF family by computer-based homology searching and have designated 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.

Figure 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 VEGF₁₆₅ (21), human VEGF-B₁₆₇ (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 (CX₁₀CXCXC) (24).

Figure 2

Northern blot analyses for detection of VEGF-D mRNA in polyadenylated RNA from human tissues. (Upper) Results of hybridizations with a human VEGF-D cDNA probe. (Lower) Results with a β-actin cDNA probe after VEGF-D probe had been stripped from the filters. The sizes of RNA molecular size markers, in kb, for the VEGF-D hybridizations are shown to the left. Tissues used as sources of RNA are indicated. SK. MUSCLE, skeletal muscle; S. INTESTINE, small intestine (mucosal lining).

Figure 3

Analysis of VEGF-DΔNΔC by silver staining, Western blotting, and a bioassay to assess binding to VEGFR-2. (A) Silver stain (Left) and Western blot analysis (Right) of VEGF-DΔNΔC arising from affinity purification (fraction 3). Samples analyzed by silver staining were the fraction containing VEGF-DΔNΔC (+) and a control fraction arising from affinity chromatography of the conditioned medium from cells transfected with expression vector lacking VEGF-D coding sequences (−). Western blot analysis was carried out by using the fraction containing VEGF-DΔNΔC with mAb M2 or a control isotype-matched antibody (Neg). Molecular mass markers (kDa) are indicated. (B) Analysis of VEGF-DΔNΔC using the VEGFR-2 bioassay to assess binding to the extracellular domain of VEGFR-2. Bioassay cells (10⁴ cells) were washed to remove IL-3 and incubated with the recombinant VEGF-DΔNΔC in fraction 3 from the M2 affinity chromatography (VEGF-DΔNΔC). The negative controls were cell culture medium without added growth factor (Medium Alone) and fraction 3 from affinity chromatography of the conditioned medium from cells transfected with expression vector lacking VEGF-D coding sequences (Vector). The positive control was a series of doubling dilutions of mouse VEGF₁₆₄ from an initial concentration of 100 ng/ml (VEGF₁₆₄). VEGF-DΔNΔC was also tested against Ba/F3 cells expressing a chimeric receptor consisting of the extracellular domain of Tie2 and the transmembrane and cytoplasmic domains of EpoR (VEGF-DΔNΔC: Tie2/EpoR). All of the fractions used for the assays were tested at an initial concentration of 10% in cell culture medium followed by doubling dilutions. The concentration of VEGF-DΔNΔC at 10% dilution of fraction 3 was 300 ng/ml. Cells were incubated for 48 h, and cell proliferation was then quantitated by the addition of [³H]thymidine and measuring the amount incorporated over a 4-h period. Assays were carried out in duplicate and error bars denote 1 SD.

Figure 4

Activation of the receptor tyrosine kinases VEGFR-3 and VEGFR-2 by VEGF-D and precipitation of VEGF-D by soluble VEGFR-Ig fusion proteins. (A) Activation of VEGFR-2 and VEGFR-3 by VEGF-D. Recombinant proteins expressed in Baculovirus were used to stimulate NIH 3T3 cells expressing VEGFR-3 or porcine aortic endothelial cells expressing VEGFR-2. After stimulation, cells were lysed and receptors were immunoprecipitated with receptor-specific antibodies and analyzed by Western blotting with phosphotyrosine-specific antibodies. (Upper) Activation of VEGFR-3 with VEGF-CΔNΔC (lane 1), supernatant from uninfected cells (lane 2), VEGF-DΔNΔC-H₆ (lane 3), and full-length VEGF-D-H₆ (lane 4). Arrows denote the positions of the phosphorylated proteolytically processed 125-kDa form and the unprocessed 195-kDa form of VEGFR-3. (Lower) Activation of VEGFR-2 with VEGF-CΔNΔC (lane 1), supernatant from uninfected cells (lane 2), VEGF-DΔNΔC-H₆ (lane 3), and full-length VEGF-D-H₆ (lane 4). The arrow denotes the position of the phosphorylated VEGFR-2. The positions of molecular mass markers, in kDa, are shown to the left. (B) Precipitation of VEGF-D by soluble VEGFR-Ig fusion proteins. Precipitation of labeled VEGF₁₆₅, VEGF-CΔNΔC, and VEGF-DΔNΔC by VEGFR-1-Ig, VEGFR-2-Ig, and VEGFR-3-Ig was carried out. The fusion proteins used for the precipitations, denoted by the name of the VEGFR involved, are shown to the right. The lane marked Vector denotes results of precipitations from medium derived from cells transfected with expression vector lacking sequence encoding VEGFs.

Figure 5

Mitogenic effect of VEGF-DΔNΔC on BAEs. BAEs were treated with VEGF-DΔNΔC arising from affinity chromatography or a negative control fraction from affinity chromatography of the conditioned medium from cells transfected with expression vector lacking VEGF-D coding sequences. The fraction containing VEGF-DΔNΔC was diluted in cell culture medium containing 5% fetal bovine serum to give the concentrations of growth factor shown. The VEGF-DΔNΔC concentration of 300 ng/ml was achieved by a 1:10 dilution. The positive control was HPLC-purified mouse VEGF₁₆₄ in cell culture medium at the concentrations shown. Dotted line A indicates the response to a 1:10 dilution of the negative control fraction. Dotted line B indicates the response of the cells to the cell culture medium alone. BAEs were seeded at a density of 10⁴ cells per well for all assays. The results are expressed as the mean ± 1 SD.