A two amino acid switch in the anti-angiogenic VEGF165b isoform creates a novel agent for therapeutic angiogenesis in peripheral artery disease

抗血管生成 VEGF165b 亚型中的两个氨基酸开关创造了一种用于治疗外周动脉疾病血管生成的新型药物

基本信息

批准号：
10589112
负责人：
Vijay Chaitanya Ganta
金额：
$ 37.99万
依托单位：
AUGUSTA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-12-15 至 2025-03-31
项目状态：
未结题

项目摘要

Abstract: Currently, no medical therapies can improve blood flow to ~12 million PAD patients (in the US alone). VEGF-A, a potent angiogenic growth factor was tested unsuccessfully in PAD clinical trials. We recently showed that failure to account for alternatively spliced anti-angiogenic VEGF-A (VEGF165b) isoform expression and function is one of the contributing factors behind VEGF-A clinical trial failure. Alternate splicing in exon-8 C- terminus of VEGF-A isoform results in the formation of pro-angiogenic VEGF165a (V165a-WT) and anti-angiogenic VEGF165b (V165b-WT) isoforms. The only difference between these 2 isoforms is a 6 amino acid shift from ‘CDKPRR’ in V165a-WT isoforms to ‘SLTRKD’ in V165b-WT isoforms. We have recently shown that 1) the fraction of V165b-WT is 2.5X higher than V165a-WT in total VEGF-A in human PAD muscle biopsies compared to controls and 2) in endothelial cells (ECs), V165b-WT blocked V165a-WT induced R1 activation even when present at 10X lower levels than V165a-WT. The net consequence of V165b-WT being 10X more potent than V165a-WT and being 2.5X more abundant than V165a-WT is a 25 fold functional molar excess of anti-angiogenic vs. pro-angiogenic VEGF isoforms in ischemic muscle. V165b-WT inhibition using a monoclonal antibody allowed V165a-WT to bind to R1 and activate novel VEGFR1 (R1) signaling pathways in ischemic ECs and macrophages that promoted perfusion in preclinical PAD models. Hence, our central hypothesis states that ‘displacement of R1 bound V165b-WT is necessary to allow ligand-induced R1-autophosphorylation and downstream signaling to enhance perfusion recovery in PAD’. Molecular processes that regulate R1 silencing ability of V165b-WT are not yet clear. Key residue alterations between V165a-WT and V165b-WT are the replacement of highly positively charged arginine residues in V165a-WT (CDKPRR) with neutral lysine-aspartic acid acids in V165b-WT (SLTRKD). We hypothesized that due to a net neutral charge conferred by ‘KD’ residues, V165b-WT binding cannot induce a strong internal rotation in the intracellular domain of R1 that is necessary to dimerize, autophosphorylate, and activate downstream signaling. To test our hypothesis, we switched the ‘KD’ residues in the C-terminus of V165b-WT to ‘RR’ (V165bKD→RR) and examined V165bKD→RR induced changes in R1 activation in ischemic ECs in vitro. Our preliminary data showed that V165bKD→RR induced R1 activation even in conditions where V165b-WT is induced, while V165a-WT failed to induce R1 activation. Furthermore, V165bKD→RR induced ischemic EC angiogenic potential and survival significantly higher compared to V165a-WT indicating a potential therapeutic for PAD. Based on these data, In Aim-1, we will determine the molecular processes (including binding affinities, structural changes, and receptor dimerization processes) by which V165b-WT and V165bKD→RR regulate R1 activation in vitro. In Aim-2, we will determine the cell- specific R1 signaling induced by V165bKD→RR to regulate EC and macrophage phenotypes in vitro. In Aim-3, we will use VEGF-A deficient mice, type-2 diabetic mice and EC-specific R1 deficient mice in preclinical PAD models to establish whether the translational potential of V165bKD→RR is R1 dependent.

摘要：目前，没有医疗疗法可以改善约1200万PAD患者的血液（仅在美国）。 VEGF-A，在PAD临床试验中未能成功测试潜在的血管生成因子。我们最近显示未能考虑剪接的抗血管生成VEGF-A（VEGF165B）同工型表达和功能是VEGF-A临床试验失败背后的因素之一。 Exon-8 C-中的替代剪接 VEGF-A同工型的末端导致形成促血管生成VEGF165A（V165A-WT）和抗血管生成 VEGF165B（V165B-WT）同工型。这两种同工型之间的唯一区别是从 V165A-WT同工型中的“ CDKPRR”在V165B-WT同工型中为“ Sltrkd”。我们最近表明1）与对照组相比 2）在内皮细胞（EC）中，V165B-WT阻断V165A-WT诱导R1激活，即使存在于10倍下水平比V165A-WT。 V165B-WT的净后果比V165A-WT高10倍，而2.5倍的可能性更高比V165A-WT丰富的是25倍的抗血管生成与亲血管生成VEGF同工型的功能摩尔在缺血性肌肉中。使用单克隆抗体的V165B-WT抑制允许V165A-WT与R1结合并激活缺血性EC和巨噬细胞中促进临床前灌注的新型VEGFR1（R1）信号通路垫模型。因此，我们的中心假设指出，“ R1结合V165B-WT的位移是必要的允许配体诱导的R1辅助磷酸化和下游信号传导，以增强灌注恢复在垫子中’。调节V165B-WT的R1沉默能力的分子过程尚不清楚。关键居住 V165A-WT和V165B-WT之间的变化是替代高阳性的精氨酸。 V165A-WT（CDKPRR）在V165B-WT（SLTRKD）中具有中性赖氨酸 - 天冬氨酸酸。我们假设由于净中性电荷由“ KD”残差赋予，V165B-WT结合不能引起强烈的内部旋转 R1的细胞内结构域是二聚，自磷酸化和激活下游信号所必需的。为了检验我们的假设，我们将V165B-WT的C末端中的“ KD”残基切换为“ RR”（V165BKD→RR）和检查了V165BKD→RR在体外缺血性EC中诱导的R1激活变化。我们显示的初步数据即使在诱导V165B-WT的情况下，V165BKD→RR也会诱导R1激活，而V165A-WT未能诱导 R1激活。此外，V165BKD→RR诱导的缺血性EC血管生成潜力和生存显着与V165A-WT相比，较高的是PAD的潜在理论。基于这些数据，在AIM-1中，我们将确定分子过程（包括结合亲和力，结构变化和接收器二聚化 v165b-wt和v165bkd→RR在体外调节R1激活的过程）。在AIM-2中，我们将确定细胞 - V165BKD→RR诱导的特定R1信号传导在体外调节EC和巨噬细胞表型。在AIM-3中，我们将在临床前垫模型中使用VEGF-A缺乏小鼠，2型糖尿病小鼠和EC特异性R1缺陷小鼠确定V165BKD→RR的翻译电势是否取决于R1。