An antagonistic vascular endothelial growth factor (VEGF) variant inhibits VEGF-stimulated receptor autophosphorylation and proliferation of human endothelial cells

Abstract

Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

Figure 1

Competition binding of VEGF variants to VEGF receptors and stimulation of endothelial cells. Recombinant extracellular domains of VEGF receptors Flt-1 (A) and KDR (B), respectively, coated onto Maxisorb plates were incubated with biotinylated VEGF₁₆₅ (10 ng/ml for Flt-1 or 30 ng/ml for KDR) in the presence of increased concentrations of various VEGF₁₂₁ variants expressed in E. coli. (C) Quiescent HUVE cells were stimulated with increased concentrations of various VEGF₁₂₁ variants, and growth factor-stimulated DNA synthesis was measured as [³H]thymidine incorporation. VEGF variants used were wild-type VEGF₁₂₁ (•), VEGF₁₂₁-L2 (□), VEGF₁₂₁-L3 (◊), and VEGF₁₂₁-C₆₁S (▴).

Figure 2

Scheme of receptor recognition by wild-type VEGF and by the heterodimeric VEGF₁₆₅-L2/VEGF₁₂₁-L3 variant. Major determinants for recognition of Flt-1 and KDR are located on loop/turn II (L2) and loop/turn III (L3). A heterodimeric VEGF molecule consisting of a loop/turn II mutant (L2 m) chain and of a loop/turn III mutant (L3 m) chain displays only one intact receptor binding interface.

Figure 3

SDS/PAGE analysis of the heterodimeric VEGF₁₆₅-L2/VEGF₁₂₁-L3 variant. Aliquots of the refolded mixture of VEGF₁₆₅-L2 and VEGF₁₂₁-L3 before heparin chromatography (lanes 1 and 5) and of the pooled VEGF₁₆₅-L2/VEGF₁₂₁-L3 heterodimer containing fractions after heparin chromatography (lanes 3 and 7) were electrophoresed on a SDS/15% gel along with VEGF₁₂₁-L3 (lanes 2 and 6) and VEGF₁₆₅-L2 (lanes 4 and 8) homodimers under reducing (lanes 1–4) and under nonreducing (lanes 5–8) conditions.

Figure 4

Competition binding of the VEGF₁₆₅-L2/VEGF₁₂₁-L3 heterodimer to VEGF receptors. Recombinant extracellular domains of VEGF receptors Flt-1 (A) and KDR (B), respectively, coated onto Maxisorb plates were incubated with biotinylated VEGF₁₆₅ (10 ng/ml for Flt-1 or 30 ng/ml for KDR) in the presence of increased concentrations of the VEGF₁₆₅-L2/VEGF₁₂₁-L3 heterodimer (⧫) and of VEGF₁₂₁ (•), respectively.

Figure 5

Inhibition of VEGF-stimulated KDR autophosphorylation. (A) Quiescent human umbilical artery endothelial cells (lane 1) were stimulated with VEGF₁₂₁ (10 ng/ml; lane 2) in the presence of increased concentrations of the VEGF₁₆₅-L2/VEGF₁₂₁-L3 heterodimer (HD; lanes 3–6). KDR was immunoprecipitated and the blot was probed with an anti-phosphotyrosine antibody (αpY), and with an anti-KDR antibody (αKDR). (B) KDR phosphorylation was calculated by videodensitometry and normalized on KDR contents.

Figure 6

Competition of VEGF₁₂₁-stimulated proliferation of endothelial cells. Quiescent HUVE cells were stimulated either with increased concentrations of VEGF₁₂₁ (•) and of the VEGF₁₆₅-L2/VEGF₁₂₁-L3 heterodimer (⧫), respectively, or with of VEGF₁₂₁ (10 ng/ml) and increased concentrations of VEGF₁₆₅-L2 homodimer (▪), of VEGF₁₂₁-L3 homodimer (□), and of VEGF₁₆₅-L2/VEGF₁₂₁-L3 heterodimer (◊), respectively, were added. Growth factor-stimulated DNA synthesis was measured as [³H]thymidine incorporation. The dotted line represents level of DNA synthesis achieved by stimulation with VEGF₁₂₁ (10 ng/ml).