VEGF165b Modulates Endothelial VEGFR1-STAT3 Signaling Pathway and Angiogenesis in Human and Experimental Peripheral Arterial Disease.

RATIONALE Atherosclerotic-arterial occlusions decrease tissue perfusion causing ischemia to lower limbs in patients with peripheral arterial disease (PAD). Ischemia in muscle induces an angiogenic response, but the magnitude of this response is frequently inadequate to meet tissue perfusion requirements. Alternate splicing in the exon-8 of vascular endothelial growth factor (VEGF)-A results in production of proangiogenic VEGFxxxa isoforms (VEGF165a, 165 for the 165 amino acid product) and antiangiogenic VEGFxxxb (VEGF165b) isoforms. OBJECTIVE The antiangiogenic VEGFxxxb isoforms are thought to antagonize VEGFxxxa isoforms and decrease activation of VEGF receptor-2 (VEGFR2), hereunto considered the dominant receptor in postnatal angiogenesis in PAD. Our data will show that VEGF165b inhibits VEGFR1 signal transducer and activator of transcription (STAT)-3 signaling to decrease angiogenesis in human and experimental PAD. METHODS AND RESULTS In human PAD versus control muscle biopsies, VEGF165b: (1) is elevated, (2) is bound higher (versus VEGF165a) to VEGFR1 not VEGFR2, and (3) levels correlated with decreased VEGFR1, not VEGFR2, activation. In experimental PAD, delivery of an isoform-specific monoclonal antibody to VEGF165b versus control antibody enhanced perfusion in animal model of severe PAD (Balb/c strain) without activating VEGFR2 signaling but with increased VEGFR1 activation. Receptor pull-down experiments demonstrate that VEGF165b inhibition versus control increased VEGFR1-STAT3 binding and STAT3 activation, independent of Janus-activated kinase-1)/Janus-activated kinase-2. Using VEGFR1+/- mice that could not increase VEGFR1 after ischemia, we confirm that VEGF165b decreases VEGFR1-STAT3 signaling to decrease perfusion. CONCLUSIONS Our results indicate that VEGF165b prevents activation of VEGFR1-STAT3 signaling by VEGF165a and hence inhibits angiogenesis and perfusion recovery in PAD muscle.