Inhibiting Anti-angiogenic VEGF165b Activates a Novel miR-17-20a-Calcipressin-3 Pathway that Revascularizes Ischemic Muscle in Peripheral Artery Disease

BACKGROUND: VEGF(165)a increases the expression of microRNA-17-92 cluster, promoting developmental, retinal, and tumor angiogenesis. We have previously shown that VEGF(165)b, an alternatively spliced VEGF-A isoform, inhibits the VEGFR-STAT3 pathway in ischemic endothelial cells (ECs) to decrease their angiogenic capacity. In ischemic macrophages (Møs), VEGF(165)b inhibits VEGFR1 to induce S100A8/A9 expression, which drives M1-like polarization. Our current study aims to determine whether VEGF(165)b inhibition promotes perfusion recovery by regulating the miR-17-92 cluster in preclinical PAD. METHODS: Hind limb ischemia (HLI) induced by femoral artery ligation and resection was used as a preclinical PAD model. Hypoxia serum starvation (HSS) was used as an in vitro PAD model. VEGF(165)b was inhibited/neutralized by an isoform-specific VEGF(165)b antibody. RESULTS: Systematic analysis of miR-17-92 cluster members (miR-17-18a-19a-19b-20a-92) in experimental-PAD models showed that VEGF(165)b-inhibition induces miRNA-17-20a (within miR-17-92 cluster) in HSS-ECs and HSS-bone marrow derived macrophages (BMDMs) vs. respective normal and/or isotype matched IgG controls to enhance perfusion-recovery. Consistent with the bioinformatics analysis that revealed RCAN3 as a common target of miR-17 and miR-20a, Argonaute-2 pull-down assays showed decreased miR-17-20a expression and higher RCAN3 expression in the RISC complex of HSS-ECs and HSS-BMDMs vs. the respective controls. Inhibiting miR-17-20a induced RCAN3 levels to decrease ischemic angiogenesis and promoted M1-like polarization to impair perfusion recovery. Finally, using STAT3 inhibitors, S100A8/A9 silencers and VEGFR1-deficient ECs and Møs, we show that VEGF(165)b inhibition activates the miR-17-20a-RCAN3 pathway independent of VEGFR1-STAT3 or VEGFR1-S100A8/A9 in ischemic ECs and ischemic Møs, respectively. CONCLUSION: Our data revealed a hereunto unrecognized therapeutic ‘miR-17-20a-RCAN3’ pathway in the ischemic vasculature that is VEGFR1-STAT3/S100A8/A9 independent and is activated only upon VEGF(165)b inhibition in PAD.