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

外周动脉疾病(PAD)是由动脉粥样硬化引起的，动脉粥样硬化会减少血液流动，导致疼痛 行走、截肢、低质量的生活和死亡。目前PAD的药物治疗对血液没有影响 流。血管内皮生长因子受体-2介导的内皮型一氧化氮合酶 内皮型一氧化氮合酶(ENOS)是促进缺氧依赖性血管生成的主要途径。许多临床前研究都有 专注于VEGFR2用于治疗性血管生成，但人类研究失败了。VEGFR1/-和患有 VEGFR1胞内结构域缺失(酪氨酸激酶死亡，TK-/-)损害了血管生成和灌流 恢复期与野生型相比。血管内皮生长因子受体促进低氧依赖的机制 与VEGFR2相比，人们对血管生成知之甚少，也远未得到充分的研究。可选的拼接 血管内皮生长因子-A导致6个氨基酸的开关，改变促血管生成的血管生成因子xxxa(xxx= 氨基酸)到“抗血管生成”的VEGFxxxb(165个氨基酸对应的VEGF165b)异构体。至关重要的 我们意想不到的证据表明，VEGFxxxb的抗血管生成作用直接与 VEGFR1信号；这一发现可能改变通常情况下缺氧依赖的血管生成的范式和 尤其是PAD中的VEGFR信号。使用体外、临床前和人体组织研究，我们表明 VEGF165b在缺血时上调，并优先与VEGFR1结合，限制Y1333的磷酸化。 血管内皮生长因子165b特异性抑制性抗体通过增加血管内皮生长因子的结合增加缺氧依赖的血管生成 VEGF165a与VEGFR1结合，但VEGF165b抗体对VEGF165b或VEGF165a的结合均无影响 致VEGFR2。随着pVEGFR1Y1333(但不是VEGFR2激活)和VEGFR1-pSTAT3关联的增加， 抗VEGF165b的血管生成作用依赖于VEGFR1。VEGF165a和VEGF165b都绑定 VEGFR1具有相似的亲和力，但VEGF165b对VEGF165a介导的VEGFR1激活的抑制作用降低了10倍 浓度，尽管是VEGFR2的等摩尔激活剂。我们发现VEGF165b诱导了一个 炎症性、抗血管生成的M1表型，而抗VEGF165b诱导修复性的、促血管生成的M2 表型。PAD患者单核细胞中VEGF165b细胞表达上调。加在一起， 这些发现指出了VEGFR1的关键但之前未被认识到的促血管生成作用 由VEGF165b。我们认为VEGF165b在低氧和PAD中的抗血管生成作用是通过 内皮细胞和巨噬细胞中VEGFR1信号的抑制。目标1将确定抗体是否- VEGF165b抑制通过不同的VEGFR1依赖诱导缺氧依赖的血管生成 路径。目标2将确定VEGF165b促进VEGFR1介导的M1的机制 小鼠骨髓源性巨噬细胞(MBMDM)和人外周血巨噬细胞极化 血液单个核细胞。AIM 3将确定VEGFR1介导的信号通路促进 内皮细胞和巨噬细胞低氧依赖的血管生成。

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.