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:
    10371073
  • 负责人:
  • 金额:
    $ 38.03万
  • 依托单位:
  • 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 2024-03-31
  • 项目状态:
    已结题

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.
翻译后摘要:目前,没有任何药物治疗可以改善血流~ 1200万PAD患者(仅在美国)。 VEGF-A是一种有效的血管生成生长因子,在PAD临床试验中测试失败。我们最近展示了 不能解释选择性剪接的抗血管生成VEGF-A(VEGF 165 b)同种型表达和 功能是VEGF-A临床试验失败的原因之一。外显子8 C- VEGF-A同种型的末端导致促血管生成VEGF 165 a(V165 a-WT)和抗血管生成VEGF 165 a(V165 a-WT)的形成。 VEGF 165 b(V165 b-WT)同种型。这2种同种型之间的唯一差异是从 V165 a-WT同种型中的“CDKPRR”与V165 b-WT同种型中的“SLTRKD "。我们最近已经证明,1)的分数 与对照相比,在人PAD肌肉活检中,V165 b-WT的总VEGF-A比V165 a-WT高2.5倍, 2)在内皮细胞(EC)中,V165 b-WT阻断V165 a-WT诱导的R1激活,即使在V165 a-WT以10倍低的浓度存在时, 比V165 a-WT高。V165 b-WT的净结果是效力比V165 a-WT高10倍,并且效力比V165 a-WT高2.5倍。 比V165 a-WT丰富的是抗血管生成相对于促血管生成VEGF同种型25倍的功能摩尔过量 在缺血的肌肉中。使用单克隆抗体的V165 b-WT抑制允许V165 a-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VEGFR 1(R1)信号通路 PAD模型。因此,我们的中心假设指出,“R1结合的V165 b-WT的置换是必要的, 允许配体诱导的R1-自磷酸化和下游信号传导增强灌注恢复 在PAD '。调节V165 b-WT的R1沉默能力的分子过程尚不清楚。关键残基 V165 a-WT和V165 b-WT之间的改变是V165 a-WT和V165 b-WT中高度带正电荷的精氨酸残基的替换。 V165 a-WT(CDKPRR),V165 b-WT(SLTRKD)中含有中性赖氨酸-天冬氨酸。我们假设由于 由于“KD”残基赋予的净中性电荷,V165 b-WT结合不能诱导细胞内的强内旋转, R1的胞内结构域,其是二聚化、自磷酸化和激活下游信号传导所必需的。 为了检验我们的假设,我们将V165 b-WT的C-末端中的“KD”残基转换为“RR”(V165 bKD →RR), 观察V165 bKD →RR诱导的体外缺血EC中R1活化的变化。我们的初步数据显示 即使在V165 b-WT被诱导的条件下,V165 bKD →RR也诱导R1活化,而V165 a-WT未能诱导R1活化。 R1激活。此外,V165 bKD →RR可显著诱导缺血EC血管生成和存活 与V165 a-WT相比更高,表明PAD的潜在治疗。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、结构变化和受体二聚化 V165 b-WT和V165 bKD →RR在体外调控R1的活化。在Aim-2中,我们将确定细胞- V165 bKD →RR诱导的特异性R1信号转导对EC和巨噬细胞表型的调节在目标3中,我们 将在临床前PAD模型中使用VEGF-A缺陷小鼠、2型糖尿病小鼠和EC特异性R1缺陷小鼠 确定V165 bKD →RR的翻译潜能是否为R1依赖性。

项目成果

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Vijay Chaitanya Ganta其他文献

Vijay Chaitanya Ganta的其他文献

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{{ truncateString('Vijay Chaitanya Ganta', 18)}}的其他基金

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
  • 批准号:
    10589112
  • 财政年份:
    2019
  • 资助金额:
    $ 38.03万
  • 项目类别:
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
  • 批准号:
    9892024
  • 财政年份:
    2019
  • 资助金额:
    $ 38.03万
  • 项目类别:
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
  • 批准号:
    10023048
  • 财政年份:
    2019
  • 资助金额:
    $ 38.03万
  • 项目类别:

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