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 VEGFstimulated 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.

Antagonistic VEGF variants engineered to simultaneously bind to and inhibit VEGFR2 and α v β 3 integrin

“…Interestingly, this mutant contained the sequence SPAS immediately following the RGD tripeptide motif, similar to the RGDSPAS sequence found in fibronectin. As expected, yeast-displayed scVEGFwt and scVEGFmut bound with high affinity to VEGFR1, consistent with previous studies on wild-type VEGF and similar protein mutants (29,40). VEGFR1 is thought to modulate the activity of VEGFR2 and also plays a role in a number of human diseases (2); thus it will be interesting in future studies to explore the biological effects of known point mutations that diminish VEGFR1 binding.…”

“…The α v β 3 integrin recognizes numerous ECM ligands, and binding leads to cell adhesion to the ECM and activation of similar growth and proliferation pathways (25,27). In previous work, VEGF was converted to a receptor antagonist by introducing several point mutations into one of its VEGFR binding epitopes (28,29). These mutations blocked one receptor binding site while leaving the other intact, preventing receptor dimerization.…”

“…An antagonist variant (scVEGFmut) was also created that contained four amino acid mutations corresponding to residues critical for VEGFR recognition at one pole of the molecule: chain 1 F17A, E64A; chain 2 I46A, I83A ( Fig. 1 A and B and Table S1) (28)(29)(30). Both scVEGFwt and scVEGFmut were well expressed on the yeast cell surface and bound to soluble VEGFR2 extracellular domain (Fig.…”

Crystal Structure of the Orf Virus NZ2 Variant of Vascular Endothelial Growth Factor-E

“…VE L13 is, to our knowledge, the first artificially engineered VEGF mutant where such minimal sequence replacement established new receptor specificity, documenting the progress in protein engineering technology in the past decade. Our data are reminiscent of earlier published work where structure-based design of receptor antagonists and of ligands with improved receptor specificity have been described (51)(52)(53). Similar approaches have been described for the design of enzymes with new substrate specificity or functionality (54 -56).…”

Rat strain-dependent susceptibility to ischemia-induced retinopathy associated with retinal vascular endothelial growth factor regulation

“…The C(-634)G polymorphism reversed from the translational start site in the 5 0 UTR of the VEGF gene is strongly associated with an increased risk of retinopathy (Awata et al 2002). VEGF variants with an intact receptor binding interface inhibit VEGF-stimulated receptor phosphorylation and proliferation of human endothelial cells (Siemeister et al 1998).…”