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，动脉粥样硬化减少血流，引起疼痛， 行走，截肢，生活质量差，甚至死亡目前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）是促进低氧依赖性血管生成的主要途径。许多临床前研究已经 专注于VEGFR 2用于治疗性血管生成，但人体研究失败。VEGFR 1 +/-小鼠和 缺失的VEGFR 1胞内结构域（酪氨酸激酶死亡，TK-/-）具有受损的血管生成和灌注 与野生型相比，VEGFR 1促进缺氧依赖性 与VEGFR 2相比，血管生成知之甚少，研究也非常不足。的选择性剪接 VEGF-A导致6个氨基酸的开关，其改变促血管生成VEGFxxxa（xxx =促血管生成VEGFxxxa的数目）。 氨基酸）转化为“抗血管生成”VEGFxxxb（165个氨基酸的VEGF 165 b）同种型。至关重要 是我们意想不到的证明，VEGFxxxb的抗血管生成作用直接与 VEGFR 1信号传导：这一发现可能会改变缺氧依赖性血管生成的模式， 尤其是PAD中的VEGFR信号传导。通过体外、临床前和人体组织研究，我们发现， VEGF 165 b在缺血时上调，并优先与VEGFR 1结合，限制Y1333的磷酸化。 VEGF 165 b特异性抑制性抗体通过增加VEGF 165 b的结合来增加缺氧依赖性血管生成。 VEGF 165 a与VEGFR 1的结合，但VEGF 165 b抗体对VEGF 165 b或VEGF 165 a的结合没有影响 VEGFR2随着pVEGFR 1 Y1333（但不是VEGFR 2活化）和VEGFR 1-pSTAT 3结合的增加， 抗VEGF 165 b的血管生成作用是依赖于VEGF 1的。VEGF 165 a和VEGF 165 b均结合 VEGFR 1具有相似的亲和力，但VEGF 165 b抑制VEGF 165 a介导的VEGFR 1活化的能力低10倍。 尽管是等摩尔的VEGFR 2激活剂，但其浓度是相同的。我们发现VEGF 165 b可以诱导 炎性、抗血管生成M1表型，而抗VEGF 165 b诱导修复性、促血管生成M2 表型在PAD患者中发现的人单核细胞中，VEGF 165 b+细胞上调。综合起来看， 这些发现指出了VEGFR 1的重要但以前未被认识的促血管生成作用， VEGF 165 b的我们认为VEGF 165 b在缺氧和PAD中的抗血管生成作用是由以下因素介导的： 抑制EC和巨噬细胞中的VEGFR 1信号传导。目标1将确定抗体是否- 介导的VEGF 165 b抑制通过不同的VEGFR 1依赖性血管生成诱导缺氧依赖性血管生成 通路目的2将确定VEGF 165 b促进VEGFR 1介导的M1的机制。 小鼠骨髓源性巨噬细胞（mBMDM）和人外周血中的巨噬细胞极化 血单核细胞目的3将确定VEGFR 1介导的信号通路， 内皮细胞和巨噬细胞中的低氧依赖性血管生成。

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.