The Anti-angiogenic VEGF165b and VEGFR1 Signaling in Peripheral Artery Disease

点击翻译按钮获取中文摘要

Peripheral arterial disease (PAD) is caused by atherosclerosis that reduce blood flow, causing pain with walking, amputation, a poor quality of life, and death. Current medical therapies in PAD have no effect on blood flow. Vascular endothelial growth factor receptor (VEGFR)-2- mediated endothelial nitric oxide synthase (eNOS) is the dominant pathway promoting hypoxia-dependent angiogenesis. Many pre-clinical studies have focused on VEGFR2 for therapeutic angiogenesis but human studies have failed. VEGFR1+/- and mice with a deleted VEGFR1 intracellular domain (tyrosine kinase dead, TK-/-) have impaired angiogenesis and perfusion recovery compared to wild-type. The mechanisms by which VEGFR1 promotes hypoxia-dependent angiogenesis are poorly understood and vastly understudied compared to VEGFR2. Alternative splicing of VEGF-A results in a 6 amino acid switch that changes the pro-angiogenic VEGFxxxa (xxx = the number of amino acids) to the “anti-angiogenic” VEGFxxxb (VEGF165b for 165 amino acids) isoform. Of critical importance is our unexpected demonstration that the anti-angiogenic effects of the VEGFxxxb are directly linked to VEGFR1 signaling; a finding that may shift the paradigm of hypoxia-dependent angiogenesis in general and VEGFR signaling in PAD in particular. Using in vitro, preclinical, and human tissue studies, we showed that VEGF165b is up-regulated with ischemia and preferentially binds to VEGFR1, limiting phosphorylation at Y1333. A VEGF165b-specific inhibitory antibody increased hypoxia-dependent angiogenesis by increasing binding of VEGF165a to VEGFR1, but the VEGF165b antibody had no effect on the binding of either VEGF165b or VEGF165a to VEGFR2. With increased pVEGFR1Y1333 (but not VEGFR2 activation) and VEGFR1-pSTAT3 association, and the angiogenic effects of anti-VEGF165b were VEGFR1-dependent. Both VEGF165a and VEGF165b bind VEGFR1 with similar affinity but VEGF165b inhibited VEGF165a-mediated VEGFR1 activation at 10-fold lower concentrations, despite being an equimolar activator of VEGFR2. We show VEGF165b induces an inflammatory, anti-angiogenic M1 phenotype whereas anti-VEGF165b induces a reparative, pro-angiogenic M2 phenotype. VEGF165b+ cells were up-regulated in human monocytes found in PAD patients. Taken together, these findings point to critical but previously unrecognized pro-angiogenic roles for VEGFR1 that are dictated by VEGF165b. We pose that the anti-angiogenic effects of VEGF165b in hypoxia and PAD are mediated by inhibition of VEGFR1 signaling in both ECs and macrophages. Aim 1 will determine whether antibody- mediated VEGF165b inhibition induces hypoxia-dependent angiogenesis via a distinct VEGFR1-dependent pathway. Aim 2 will determine the mechanisms by which VEGF165b promotes VEGFR1-mediated M1 macrophage polarization in mouse bone marrow derived macrophages (mBMDM) and human, peripheral blood mononuclear cells. Aim 3 will determine the VEGFR1-mediated signaling pathways that promote hypoxia-dependent angiogenesis in ECs and macrophages.