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 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.

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 VEGF165b. A synthetic anti-angiogenic VEGF165b splice variant in which the KD residues were switched to RR (VEGF165bKD→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 VEGF165bKD→RR over VEGF165a is due to its ability to induce P38-activation in ischemic endothelial cells.

CONCLUSIONS

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 VEGF165bKD→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.