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:
  • 金额:
    $ 37.99万
  • 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 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,没有任何药物疗法可以改善约1200万PAD患者的血流(仅在美国)。 血管内皮生长因子-A是一种有效的血管生成生长因子,在PAD临床试验中未获成功。我们最近展示了 未能解释选择性剪接的抗血管生成血管内皮生长因子-A(VEGF165b)异构体的表达和 功能是导致血管内皮生长因子-A临床试验失败的原因之一。外显子-8 C-中的交替剪接 血管内皮生长因子-A亚型末端导致促血管生成因子165a(V165a-WT)的形成和抗血管生成作用 VEGF165b(V165b-WT)亚型。这两种异构体之间的唯一区别是6个氨基酸从 V165a-WT中的‘CDKPRR’亚型与V165b-WT中的‘SLTRKD’亚型相同。我们最近已经证明了1)分数 在人PAD肌肉活检组织中,V165b-WT比V165a-WT的总血管内皮生长因子-A高2.5倍 2)在内皮细胞中,V165b-WT可阻断V165a-WT诱导的R1激活,即使在低10倍时也是如此 水平高于V165a-WT。V165b-WT的净效果是V165a-WT的10倍,是V165a-WT的2.5倍 比V165a-WT丰富的是功能摩尔过量的抗血管生成与促进血管生成的VEGF亚型的25倍 在缺血肌中。使用单抗抑制V165b-WT允许V165a-WT与R1结合并激活 缺血内皮细胞和巨噬细胞中促进临床前血流灌注的新的VEGFR1(R1)信号通路 垫子模型。因此,我们的中心假设是:“R1结合的V165b-Wt的位移是必要的 允许配体诱导的R1-自动磷酸化和下游信号转导以促进血流灌注恢复 在Pad‘中。调节V165b-WT的R1沉默能力的分子过程尚不清楚。密钥残留物 V165a-WT和V165b-WT之间的变化是取代了高正电荷的精氨酸残基 V165b-WT(SLTRKD)中含有中性赖氨酸-天冬氨酸的V165a-WT(CDKPRR)。我们假设由于一个 由‘KD’残基赋予的净中性电荷,V165b-WT结合不能引起强烈的内旋转 R1的胞内结构域,它是二聚化、自动磷酸化和激活下游信号所必需的。 为了验证我们的假设,我们将V165b-WT C末端的‘KD’残基转换为‘RR’(V165bKD→RR)和 检测V165bKD→RR对体外培养的缺血内皮细胞R1激活的影响。我们的初步数据显示 即使在V165b-WT被诱导的情况下,V165bKD→RR也能诱导R1激活,而V165a-WT不能诱导 激活了R1。此外,V165bKD→RR诱导的缺血内皮细胞血管生成潜能和存活显著 与V165a-WT相比更高,表明PAD有潜在的治疗作用。根据这些数据,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 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体外诱导特异性R1信号调节EC和巨噬细胞表型在AIM-3中,我们 将在临床前PAD模型中使用血管内皮生长因子-A缺陷小鼠、2型糖尿病小鼠和EC特异性R1缺陷小鼠 为了确定V165bKD→RR的翻译潜力是否依赖于R1。

项目成果

期刊论文数量(2)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
MicroRNA-30b Is Both Necessary and Sufficient for Interleukin-21 Receptor-Mediated Angiogenesis in Experimental Peripheral Arterial Disease.
  • DOI:
    10.3390/ijms23010271
  • 发表时间:
    2021-12-27
  • 期刊:
  • 影响因子:
    5.6
  • 作者:
    Wang T;Yang L;Yuan M;Farber CR;Spolski R;Leonard WJ;Ganta VC;Annex BH
  • 通讯作者:
    Annex BH
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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:
    10371073
  • 财政年份:
    2019
  • 资助金额:
    $ 37.99万
  • 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:
    $ 37.99万
  • 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:
    $ 37.99万
  • 项目类别:

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