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AAV-CRISPR/Cas9–Mediated Depletion of VEGFR2 Blocks Angiogenesis In Vitro
PURPOSE: Pathologic angiogenesis is a component of many diseases, including neovascular age-related macular degeneration, proliferation diabetic retinopathy, as well as tumor growth and metastasis. The purpose of this project was to examine whether the system of adeno-associated viral (AAV)–mediated...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
The Association for Research in Vision and Ophthalmology
2017
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5714046/ https://www.ncbi.nlm.nih.gov/pubmed/29204648 http://dx.doi.org/10.1167/iovs.17-21902 |
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author | Wu, Wenyi Duan, Yajian Ma, Gaoen Zhou, Guohong Park-Windhol, Cindy D'Amore, Patricia A. Lei, Hetian |
author_facet | Wu, Wenyi Duan, Yajian Ma, Gaoen Zhou, Guohong Park-Windhol, Cindy D'Amore, Patricia A. Lei, Hetian |
author_sort | Wu, Wenyi |
collection | PubMed |
description | PURPOSE: Pathologic angiogenesis is a component of many diseases, including neovascular age-related macular degeneration, proliferation diabetic retinopathy, as well as tumor growth and metastasis. The purpose of this project was to examine whether the system of adeno-associated viral (AAV)–mediated CRISPR (clustered regularly interspaced short palindromic repeats)–associated endonuclease (Cas)9 can be used to deplete expression of VEGF receptor 2 (VEGFR2) in human vascular endothelial cells in vitro and thus suppress its downstream signaling events. METHODS: The dual AAV system of CRISPR/Cas9 from Streptococcus pyogenes (AAV-SpGuide and -SpCas9) was adapted to edit genomic VEGFR2 in primary human retinal microvascular endothelial cells (HRECs). In this system, the endothelial-specific promoter for intercellular adhesion molecule 2 (ICAM2) was cloned into the dual AAV vectors of SpGuide and SpCas9 for driving expression of green fluorescence protein (GFP) and SpCas9, respectively. These two AAV vectors were applied to production of recombinant AAV serotype 5 (rAAV5), which were used to infect HRECs for depletion of VEGFR2. Protein expression was determined by Western blot; and cell proliferation, migration, as well as tube formation were examined. RESULTS: AAV5 effectively infected vascular endothelial cells (ECs) and retinal pigment epithelial (RPE) cells; the ICAM2 promoter drove expression of GFP and SpCas9 in HRECs, but not in RPE cells. The results showed that the rAAV5-CRISPR/Cas9 depleted VEGFR2 by 80% and completely blocked VEGF-induced activation of Akt, and proliferation, migration as well as tube formation of HRECs. CONCLUSIONS: AAV-CRISRP/Cas9–mediated depletion of VEGFR2 is a potential therapeutic strategy for pathologic angiogenesis. |
format | Online Article Text |
id | pubmed-5714046 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2017 |
publisher | The Association for Research in Vision and Ophthalmology |
record_format | MEDLINE/PubMed |
spelling | pubmed-57140462017-12-06 AAV-CRISPR/Cas9–Mediated Depletion of VEGFR2 Blocks Angiogenesis In Vitro Wu, Wenyi Duan, Yajian Ma, Gaoen Zhou, Guohong Park-Windhol, Cindy D'Amore, Patricia A. Lei, Hetian Invest Ophthalmol Vis Sci Biochemistry and Molecular Biology PURPOSE: Pathologic angiogenesis is a component of many diseases, including neovascular age-related macular degeneration, proliferation diabetic retinopathy, as well as tumor growth and metastasis. The purpose of this project was to examine whether the system of adeno-associated viral (AAV)–mediated CRISPR (clustered regularly interspaced short palindromic repeats)–associated endonuclease (Cas)9 can be used to deplete expression of VEGF receptor 2 (VEGFR2) in human vascular endothelial cells in vitro and thus suppress its downstream signaling events. METHODS: The dual AAV system of CRISPR/Cas9 from Streptococcus pyogenes (AAV-SpGuide and -SpCas9) was adapted to edit genomic VEGFR2 in primary human retinal microvascular endothelial cells (HRECs). In this system, the endothelial-specific promoter for intercellular adhesion molecule 2 (ICAM2) was cloned into the dual AAV vectors of SpGuide and SpCas9 for driving expression of green fluorescence protein (GFP) and SpCas9, respectively. These two AAV vectors were applied to production of recombinant AAV serotype 5 (rAAV5), which were used to infect HRECs for depletion of VEGFR2. Protein expression was determined by Western blot; and cell proliferation, migration, as well as tube formation were examined. RESULTS: AAV5 effectively infected vascular endothelial cells (ECs) and retinal pigment epithelial (RPE) cells; the ICAM2 promoter drove expression of GFP and SpCas9 in HRECs, but not in RPE cells. The results showed that the rAAV5-CRISPR/Cas9 depleted VEGFR2 by 80% and completely blocked VEGF-induced activation of Akt, and proliferation, migration as well as tube formation of HRECs. CONCLUSIONS: AAV-CRISRP/Cas9–mediated depletion of VEGFR2 is a potential therapeutic strategy for pathologic angiogenesis. The Association for Research in Vision and Ophthalmology 2017-12 /pmc/articles/PMC5714046/ /pubmed/29204648 http://dx.doi.org/10.1167/iovs.17-21902 Text en Copyright 2017 The Authors http://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License. |
spellingShingle | Biochemistry and Molecular Biology Wu, Wenyi Duan, Yajian Ma, Gaoen Zhou, Guohong Park-Windhol, Cindy D'Amore, Patricia A. Lei, Hetian AAV-CRISPR/Cas9–Mediated Depletion of VEGFR2 Blocks Angiogenesis In Vitro |
title | AAV-CRISPR/Cas9–Mediated Depletion of VEGFR2 Blocks Angiogenesis In Vitro |
title_full | AAV-CRISPR/Cas9–Mediated Depletion of VEGFR2 Blocks Angiogenesis In Vitro |
title_fullStr | AAV-CRISPR/Cas9–Mediated Depletion of VEGFR2 Blocks Angiogenesis In Vitro |
title_full_unstemmed | AAV-CRISPR/Cas9–Mediated Depletion of VEGFR2 Blocks Angiogenesis In Vitro |
title_short | AAV-CRISPR/Cas9–Mediated Depletion of VEGFR2 Blocks Angiogenesis In Vitro |
title_sort | aav-crispr/cas9–mediated depletion of vegfr2 blocks angiogenesis in vitro |
topic | Biochemistry and Molecular Biology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5714046/ https://www.ncbi.nlm.nih.gov/pubmed/29204648 http://dx.doi.org/10.1167/iovs.17-21902 |
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