title: PKR promotes choroidal neovascularization via upregulating the PI3K/Akt signaling pathway in VEGF expression.
abstract: The aim of this study was to investigate the functions of dsRNA-activated protein kinase (PKR) in choroidal neovascularization (CNV) and related signaling pathways in the production of vascular endothelial growth factor (VEGF).
  
  
  
  A chemical hypoxia model of in vitro RF/6A cells, a rhesus choroid-retinal endothelial cell line, was established by adding cobalt chloride (CoCl2) to the culture medium. PKR, phosphophosphatidylinositol 3-kinase (p-PI3K), phosphoprotein kinase B (p-Akt), and VEGF protein levels in RF/6A cells were detected with western blotting. PKR siRNA and the PI3K inhibitor LY294002 were used to evaluate the roles of the PKR and PI3K signaling pathways in VEGF expression with western blotting. In an ARPE-19 (RPE cell line) and RF/6A cell coculture system, proliferation, migration, and tube formation of RF/6A cells under hypoxic conditions were measured with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Transwell, and Matrigel Transwell assays, respectively. In vivo CNV lesions were induced in C57BL/6J mice using laser photocoagulation. The mice were euthanized in a timely manner, and the eyecups were dissected from enucleated eyes. PKR, p-PI3K, p-Akt, and VEGF protein levels in tissues were detected with western blotting. To evaluate the leakage area, fundus fluorescein angiography and choroidal flat mount were performed on day 7 after intravitreal injection of an anti-PKR monoclonal antibody.
  
  
  
  The in vitro RF/6A cell chemical hypoxia model showed that PKR expression was upregulated in parallel with p-PI3K, p-Akt, and VEGF expression, peaking at 12 h. PKR siRNA downregulated PKR, p-PI3K, p-Akt, and VEGF expression. In addition, the PI3K inhibitor LY294002 greatly decreased the p-PI3K, p-Akt, and VEGF protein levels, but PKR expression was unaffected, indicating that Akt was a downstream molecule of PKR that upregulated VEGF expression. In the ARPE-19 (RPE cell line) and RF/6A cell coculture system, PKR siRNA reduced the migration and tube formation of the RF/6A cells. In vivo, PKR, p-PI3K, p-Akt, and VEGF expression increased and peaked at 7 days in the mouse CNV model induced by laser photocoagulation. Furthermore, on the RPE and choroid cryosections, PKR colocalized with CD31, suggesting that PKR was expressed by the vascular endothelium. The intravitreal injection of an anti-PKR monoclonal antibody decreased the progression and leakage area of CNV in mice.
  
  
  
  PKR promotes CNV formation via the PI3K/Akt signaling pathway in VEGF expression. Additionally, the anti-PKR monoclonal antibody significantly decreased CNV in a mouse model, showing the antibody may have therapeutic potential in human CNV.
authors.affiliation: Department of Ophthalmology, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China; Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Medical College, Nantong University, Nantong, Jiangsu, PR China.
authors.forename: Manhui
authors.initials: M
authors.lastname: Zhu
authors.affiliation: Department of Pathogen Biology, Medical College, Nantong University, Nantong, Jiangsu, China; Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Medical College, Nantong University, Nantong, Jiangsu, PR China.
authors.forename: Xiaojuan
authors.initials: X
authors.lastname: Liu
authors.affiliation: Laboratory Animals Centre, Nantong University, Nantong, Jiangsu, China; Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Medical College, Nantong University, Nantong, Jiangsu, PR China.
authors.forename: Shengcun
authors.initials: S
authors.lastname: Wang
authors.affiliation: Laboratory Animals Centre, Nantong University, Nantong, Jiangsu, China; Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Medical College, Nantong University, Nantong, Jiangsu, PR China.
authors.forename: Jin
authors.initials: J
authors.lastname: Miao
authors.affiliation: Laboratory Animals Centre, Nantong University, Nantong, Jiangsu, China; Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Medical College, Nantong University, Nantong, Jiangsu, PR China.
authors.forename: Liucheng
authors.initials: L
authors.lastname: Wu
authors.affiliation: Department of Ophthalmology, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China; Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Medical College, Nantong University, Nantong, Jiangsu, PR China.
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Yang
authors.affiliation: Department of Ophthalmology, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China; Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Medical College, Nantong University, Nantong, Jiangsu, PR China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Ophthalmology, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China; Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Medical College, Nantong University, Nantong, Jiangsu, PR China.
authors.forename: Lihua
authors.initials: L
authors.lastname: Kang
authors.affiliation: Department of Ophthalmology, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China; Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Medical College, Nantong University, Nantong, Jiangsu, PR China.
authors.forename: Wendie
authors.initials: W
authors.lastname: Li
authors.affiliation: Department of Ophthalmology, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China; Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Medical College, Nantong University, Nantong, Jiangsu, PR China.
authors.forename: Chen
authors.initials: C
authors.lastname: Cui
authors.affiliation: Department of Ophthalmology, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China; Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Medical College, Nantong University, Nantong, Jiangsu, PR China.
authors.forename: Hui
authors.initials: H
authors.lastname: Chen
authors.affiliation: Department of Ophthalmology, Affiliated Hospital of Nantong University, Nantong, Jiangsu, China; Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Medical College, Nantong University, Nantong, Jiangsu, PR China.
authors.forename: Aimin
authors.initials: A
authors.lastname: Sang
mesh_terms: D000818:Animals; D015153:Blotting, Western; D015687:Cell Hypoxia; D002465:Cell Movement; D049109:Cell Proliferation; D002829:Choroid / Q000098:blood supply; D020256:Choroidal Neovascularization / Q000209:etiology / Q000378:metabolism* / Q000473:pathology; D004730:Endothelium, Vascular / Q000378:metabolism*; D005451:Fluorescein Angiography; D019084:Fluorescent Antibody Technique, Indirect; D020868:Gene Silencing; D006801:Humans; D058449:Intravitreal Injections; D008253:Macaca mulatta; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D034741:RNA, Small Interfering; D012171:Retinal Vessels / Q000166:cytology; D015398:Signal Transduction / Q000502:physiology*; D015854:Up-Regulation; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism*; D019892:eIF-2 Kinase / Q000502:physiology*
chemical_list: D034741:RNA, Small Interfering; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D019892:eIF-2 Kinase; C000605978:protein kinase R, mouse