Editing VEGFR2 Blocks VEGF-Induced Activation of Akt and Tube Formation

Abstract

PurposeVascular 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.MethodsFour 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.ResultsIn 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.ConclusionsThe CRISPR-SpCas9 technology is a potential novel approach to prevention of pathologic angiogenesis.

Apratoxin S4 Inspired by a Marine Natural Product, a New Treatment Option for Ocular Angiogenic Diseases

“…1i). As expected, aflibercept was only able to affect the VEGF induced phosphorylation of VEGFR2 24 ( Supplementary Fig. S2).…”

AAV-CRISPR/Cas9–Mediated Depletion of VEGFR2 Blocks Angiogenesis In Vitro

“…The four 20-nt target DNA sequences preceding a 5′-NGG PAM sequence at exon 18 in the genomic VEGFR2 locus (NC_000071.6) were selected for generating sgRNA for SpCas9 targets using the CRISPR design Web site ( http://crispr.mit.edu ; in the public domain). 13 , 19 The four target sequences were 5′-TCAGTTCCCCTTCATTGGCC-3′ (K7), 5′-AGGCTACTTGTCTATTGTCA-3′ (K8), 5′-TTCATCTGGATCCATGACGA -3′ (K9), and 5′-GGATCCAGATGAACTCCCAT-3′ (K10). The control sgRNA sequence (5′-TGCGAATACGCCCACGCGATGGG-3′) was designed to target the lacZ gene from Escherichia coli .…”

Vitreous Fibronectin and Fibrinogen Expression Increased in Eyes With Proliferative Diabetic Retinopathy After Intravitreal Anti-VEGF Therapy

“…36 However, the ELISA for VEGFR2 revealed no significant difference (P ¼ 0.13) in VEGFR2 concentrations among the three groups, and showed that the VEGFR2 concentration was numerically greater in the control group (2.33 6 1.87 ng/mL) than in the PRP group (1.97 6 1.00 ng/mL) and IVI group (1.21 6 0.66 ng/mL). Possible mechanisms causing downregulation of VEGFR2 by IVI should be assessed in a larger number of samples from PDR patients, ideally in a randomized controlled fashion, with or without IVI.…”