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

Alternative splicing in the eighth exon C-terminus of VEGF-A (vascular endothelial growth factor-A) results in the formation of proangiogenic VEGF₁₆₅a and antiangiogenic VEGF₁₆₅b isoforms. The only known difference between these 2 isoform families is a 6-amino acid switch from CDKPRR (in VEGF₁₆₅a) to SLTRKD (in VEGF₁₆₅b). We have recently shown that VEGF₁₆₅b can induce VEGFR2-activation but fails to induce VEGFR1 (VEGF receptor 1)-activation. The molecular mechanisms that regulate VEGF₁₆₅b's ability toward differential VEGFR2 versus VEGFR1 activation/inhibition are not yet clear.

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 VEGF₁₆₅b. A synthetic anti-angiogenic VEGF₁₆₅b splice variant in which the KD residues were switched to RR (VEGF₁₆₅b_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 VEGF₁₆₅a. Phosphoproteome arrays showed that the therapeutic efficacy of VEGF₁₆₅b_KD→RR over VEGF₁₆₅a is due to its ability to induce P38-activation in ischemic endothelial cells.

Our data shows that the KD residues regulate VEGF₁₆₅b's VEGFR1 inhibitory property but not VEGFR2. Switching these KD residues to RR resulted in the formation of a synthetic/recombinant VEGF₁₆₅b_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.