Editing VEGFR2 Blocks VEGF-Induced Activation of Akt and Tube Formation.
Editing VEGFR2 Blocks VEGF-Induced Activation of Akt and Tube Formation.
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DOI：
10.1167/iovs.16-20537
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发表时间：
2017-02-01
影响因子：
4.4
通讯作者：
Lei H
中科院分区：
文献类型：
--
作者：
Huang X;Zhou G;Wu W;Ma G;D'Amore PA;Mukai S;Lei H
血管内皮生长因子受体2（VEGFR 2）在VEGF诱导的血管生成中起关键作用。该项目的目标是测试以下假设：使用成簇规则间隔回文重复序列（CRISPR）相关DNA内切核酸酶（Cas）9技术编辑化脓链球菌（SpCas 9）中的基因组VEGFR 2基因座能够阻断VEGF诱导的Akt激活和管形成。选择用于基于人基因组VEGFR 2外显子3基因座合成单向导RNA的四个20个核苷酸，并分别克隆到lentiCRISPR v2载体中。通过桑格DNA测序、Surveyor核酸酶分析和下一代测序（NGS）分析来自转导的原代人视网膜微血管内皮细胞（HREC）的基因组VEGFR 2外显子3的DNA片段。在转导的细胞中，VEGFR 2的表达和VEGF刺激的信号传导事件（例如，Akt磷酸化）通过Western印迹分析确定;检查VEGF诱导的细胞反应（增殖、迁移和管形成）。在VEGFR 2-sgRNA/SpCas 9转导的HREC中，桑格DNA测序表明存在突变，并且NGS证明在基因组VEGFR 2基因座中存在83.57%的插入和缺失; VEGFR 2的表达在VEGFR 2-sgRNA/SpCas 9转导的HREC中耗尽。此外，在具有VEGFR 2-sgRNA/SpCas 9的HREC中存在比具有LacZ-sgRNA/SpCas 9的那些更低水平的Akt磷酸化，并且在VEGFR 2耗尽的HREC中存在比用阿柏西普或雷珠单抗处理的那些更少的VEGF刺激的Akt活化、增殖、迁移或管形成。CRISPR-SpCas 9技术是预防病理性血管生成的潜在新方法。
Vascular endothelial growth factor receptor 2 (VEGFR2) plays a key role in VEGF-induced angiogenesis. The goal of this project was to test the hypothesis that editing genomic VEGFR2 loci using the technology of clustered regularly interspaced palindromic repeats (CRISPR)-associated DNA endonuclease (Cas)9 in Streptococcus pyogenes (SpCas9) was able to block VEGF-induced activation of Akt and tube formation. Four 20 nucleotides for synthesizing single-guide RNAs based on human genomic VEGFR2 exon 3 loci were selected and cloned into a lentiCRISPR v2 vector, respectively. The DNA fragments from the genomic VEGFR2 exon 3 of transduced primary human retinal microvascular endothelial cells (HRECs) were analyzed by Sanger DNA sequencing, surveyor nuclease assay, and next-generation sequencing (NGS). In the transduced cells, expression of VEGFR2 and VEGF-stimulated signaling events (e.g., Akt phosphorylation) were determined by Western blot analyses; VEGF-induced cellular responses (proliferation, migration, and tube formation) were examined. In the VEGFR2-sgRNA/SpCas9–transduced HRECs, Sanger DNA sequencing indicated that there were mutations, and NGS demonstrated that there were 83.57% insertion and deletions in the genomic VEGFR2 locus; expression of VEGFR2 was depleted in the VEGFR2-sgRNA/SpCas9–transduced HRECs. In addition, there were lower levels of Akt phosphorylation in HRECs with VEGFR2-sgRNA/SpCas9 than those with LacZ-sgRNA/SpCas9, and there was less VEGF-stimulated Akt activation, proliferation, migration, or tube formation in the VEGFR2-depleted HRECs than those treated with aflibercept or ranibizumab. The CRISPR-SpCas9 technology is a potential novel approach to prevention of pathologic angiogenesis.