Synthetic Antiangiogenic Vascular Endothelial Growth Factor-A Splice Variant Revascularizes Ischemic Muscle in Peripheral Artery Disease.

BACKGROUND: Alternative splicing in the eighth exon C-terminus of VEGF-A (vascular endothelial growth factor-A) results in the formation of proangiogenic VEGF(165)a and antiangiogenic VEGF(165)b isoforms. The only known difference between these 2 isoform families is a 6-amino acid switch from CDKPRR (in VEGF(165)a) to SLTRKD (in VEGF(165)b). We have recently shown that VEGF(165)b can induce VEGFR2-activation but fails to induce VEGFR1 (VEGF receptor 1)-activation. The molecular mechanisms that regulate VEGF(165)b's ability toward differential VEGFR2 versus VEGFR1 activation/inhibition are not yet clear. METHODS AND RESULTS: Hypoxia serum starvation was used as an in vitro peripheral artery disease model. Unilateral single ligation of the femoral artery was used as a preclinical peripheral artery disease model. VEGFR1 activating ligands have 2 arginine (RR) residues in their eighth exon C-terminus, that were replaced by lysine-aspartic acid (KD) in VEGF(165)b. A synthetic anti-angiogenic VEGF(165)b splice variant in which the KD residues were switched to RR (VEGF(165)b(KD→RR)) activated both VEGFR1- and VEGFR2-signaling pathways to induce ischemic-endothelial cell angiogenic capacity in vitro and enhance perfusion recovery in a severe experimental-peripheral artery disease model significantly higher than VEGF(165)a. Phosphoproteome arrays showed that the therapeutic efficacy of VEGF(165)b(KD→RR) over VEGF(165)a is due to its ability to induce P38-activation in ischemic endothelial cells. CONCLUSIONS: Our data shows that the KD residues regulate VEGF(165)b's VEGFR1 inhibitory property but not VEGFR2. Switching these KD residues to RR resulted in the formation of a synthetic/recombinant VEGF(165)b(KD→RR) isoform that has the ability to activate both VEGFR1- and VEGFR2-signaling and induce ischemic-endothelial cell angiogenic and proliferative capacity that matched the angiogenic requirement necessary to achieve perfusion recovery in a severe experimental-peripheral artery disease model.