Editing VEGFR2 Blocks VEGF-Induced Activation of Akt and Tube Formation.
Invest Ophthalmol Vis Sci
; 58(2): 1228-1236, 2017 02 01.
Article
em En
| MEDLINE
| ID: mdl-28241310
ABSTRACT
Purpose:
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.Methods:
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.Results:
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.Conclusions:
The CRISPR-SpCas9 technology is a potential novel approach to prevention of pathologic angiogenesis.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Receptor 2 de Fatores de Crescimento do Endotélio Vascular
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Fator A de Crescimento do Endotélio Vascular
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Proliferação de Células
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Proteínas Proto-Oncogênicas c-akt
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Edição de Genes
Tipo de estudo:
Qualitative_research
Limite:
Humans
Idioma:
En
Ano de publicação:
2017
Tipo de documento:
Article