Modulation of Anti-angiogenic VEGFxxxb Reveals Novel VEGF Signaling in Peripheral Arterial Disease

Despite extensive research no therapies exist for Critical limb ischemic Peripheral Arterial Disease (CLI-PAD) patients and often the outcome is amputation. VEGF-A, a pro-angiogenic molecule has been widely sorted for use as PAD therapy. Classically, VEGF-A binds to VEGFR1 (R1) and VEGFR2 (R2) to promote physiological and pathological angiogenesis. While data on outcomes/signaling due to VEGF-A binding to R1 are extremely limited, VEGF-A binding to R2 activates PI3K-Akt-eNos signaling to induce potent angiogenesis. Clinical trials aimed to induce VEGF-A directly/indirectly to activate R2 dependent angiogenesis showed no success suggesting inadequate knowledge of VEGF family and signaling in PAD. Recent discovery of an alternatively spliced VEGF-A isoform family termed VEGFxxxb (VEGF165b) that was predicted to bind to R1 and R2 and block pro-angiogenic VEGF-A function suggested that VEGF165b induction in ischemic muscle was not taken into account in designing the VEGF-A clinical trials, probably resulting in miscalculating the dosage and strategies needed to increase VEGF-A function to promote perfusion. Since, VEGF165b has been shown to be the most abundant isoform among VEGFxxxb members, we hypothesized that VEGF165b inhibition is necessary to increase the bioavailability of VEGF-A to R1 and R2 to promote angiogenesis in PAD. Based on this hypothesis, we used a specific blocking antibody to inhibit VEGF165b function in experimental CLI-PAD models and found that VEGF165b inhibition improved perfusion vs. IgG. However, this did not occur through R2 signaling but through less characterized R1 signaling. We observed that VEGF165b inhibition induced R1 activation followed by Stat3 activation and P53 inhibition indicating a novel R1 signaling is activated post VEGF165b inhibition that enhanced perfusion recovery. Importantly, VEGF165b inhibition not only increased the bioavailability of VEGF-A, but also VEGF-B and PlGF (R1 specific ligands whose functions are also less characterized) to R1 in ischemic muscle. In the current proposal we will identify the VEGF ligand (VEGF-A, VEGF-B or PlGF) that activates this novel R1 signaling in ischemic muscle and will determine whether we can further enhance this signaling by combining VEGF165b inhibition with VEGF ligand treatments. We will also confirm whether VEGF165b induction correlates with decreased R1 and/or R2 activation in human PAD biopsies vs. normal that can serve as a point of modulation for human therapies. (AHA Program: Scientist Development Grant)