Abstract 17454: Inhibition of Anti-angiogenic VEGF-A Isoform Activates a Novel VEGFR1 Signaling That Induces Therapeutic Angiogenesis and Perfusion Recovery in Experimental PAD

Abstract

Introduction: Classic VEGF-A induced angiogenesis involves VEGFR2 (VR2)-PI3K-AKT-eNos activation. Ischemia induces VEGF165b (V165b, an anti-angiogenic VEGF-A isoform) levels in Peripheral Arterial Disease (PAD) muscle. Since V165b competes with pro-angiogenic VEGF-A isoforms to bind and block VEGF-A dependent activation of VR2 and angiogenesis, we hypothesized that “V165b inhibition removes the anti-angiogenic brakes on VR2-PI3K-Akt signaling to promote therapeutic angiogenesis and perfusion recovery in PAD”. Methods and Results: Hind limb ischemia (HLI), an experimental PAD model was performed by femoral artery ligation and resection in worse recovery animal strains (Balbc and type 2-Diabetes (T2D), n=10/group). Mice were treated i.m. with isoform specific V165b blocking antibody or IgG immediately post-surgery. V165b inhibition significantly increased perfusion recovery (Balbc: V165b-Ab=75.35±4.7 vs. IgG=50.13±2.4%, T2D: 65.70±5.8 vs. 44.04±6.08%) assessed by laser Doppler. Furthermore, V165b inhibition increased vascular density by ~2 fold in ischemic muscle and significantly decreased necrosis scores in both animal strains compared to IgG. In vitro, HUVECs treated with V165b-Ab showed significantly higher capillary like tube formation on matrigel suggesting that endogenous V165b inhibition is sufficient to promote angiogenesis. In stark contrast to our hypothesis, V165b inhibition significantly induced VR1 activation but not VR2, along with Stat3 activation, decreased Akt, Erk1/2 activation and decreased P53 levels (in d3 post HLI tissue samples) compared to IgG. In vitro, HUVECs treated with V165b-Ab also showed significant increase in VR1 and Stat3 activation compared to IgG. Moreover, ligand-receptor binding experiments showed that V165b inhibition significantly increased the binding of not only VEGF-A but also VEGF-B and PlGF to VR1 in ischemic muscle as well as in HUVECs in vitro (P<0.05 is considered significant). Conclusion: We conclude that V165b not only competes with VEGF-A but also with VEGF-B and PlGF to bind and block VR1 activation in ischemic muscle. Our data point towards a novel VR1 signaling that activates Stat3 and inhibits P53 to promote perfusion recovery in ischemic muscle post V165b inhibition.