{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,20]],"date-time":"2026-10-20T15:46:45Z","timestamp":1792511205524,"version":"4.5.0"},"reference-count":53,"publisher":"MDPI AG","issue":"16","license":[{"start":{"date-parts":[[2021,8,18]],"date-time":"2021-08-18T00:00:00Z","timestamp":1629244800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Neovascular age-related macular degeneration (nAMD) featuring choroidal neovascularization (CNV) is the principal cause of irreversible blindness in elderly people in the world. Integrated stress response (ISR) is one of the intracellular signals to be adapted to various stress conditions including endoplasmic reticulum (ER) stress. ISR signaling results in the upregulation of activating transcription factor 4 (ATF4), which is a mediator of ISR. Although recent studies have suggested ISR contributes to the progression of some age-related disorders, the effects of ATF4 on the development of CNV remain unclear. Here, we performed a murine model of laser-induced CNV and found that ATF4 was highly expressed in endothelial cells of the blood vessels of the CNV lesion site. Exposure to integrated stress inhibitor (ISRIB) reduced CNV formation, vascular leakage, and the upregulation of vascular endothelial growth factor (VEGF) in retinal pigment epithelium (RPE)-choroid-sclera complex. In human retinal microvascular endothelial cells (HRMECs), ISRIB reduced the level of ATF4 and VEGF induced by an ER stress inducer, thapsigargin, and recombinant human VEGF. Moreover, ISRIB decreased the VEGF-induced cell proliferation and migration of HRMECs. Collectively, our findings showed that pro-angiogenic effects of ATF4 in endothelial cells may be a potentially therapeutic target for patients with nAMD.<\/jats:p>","DOI":"10.3390\/ijms22168890","type":"journal-article","created":{"date-parts":[[2021,8,18]],"date-time":"2021-08-18T10:54:35Z","timestamp":1629284075000},"page":"8890","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Role of Activating Transcription Factor 4 in Murine Choroidal Neovascularization Model"],"prefix":"10.3390","volume":"22","author":[{"given":"Hiroto","family":"Yasuda","sequence":"first","affiliation":[{"name":"Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Gifu 501-1196, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Miruto","family":"Tanaka","sequence":"additional","affiliation":[{"name":"Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Gifu 501-1196, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anri","family":"Nishinaka","sequence":"additional","affiliation":[{"name":"Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Gifu 501-1196, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shinsuke","family":"Nakamura","sequence":"additional","affiliation":[{"name":"Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Gifu 501-1196, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Masamitsu","family":"Shimazawa","sequence":"additional","affiliation":[{"name":"Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Gifu 501-1196, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hideaki","family":"Hara","sequence":"additional","affiliation":[{"name":"Department of Biofunctional Evaluation, Gifu Pharmaceutical University, Gifu 501-1196, Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,8,18]]},"reference":[{"key":"ref_1","first-page":"74","article-title":"Age-Related Macular Degeneration","volume":"59","author":"Gheorghe","year":"2015","journal-title":"Rom. J. Ophthalmol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"e106","DOI":"10.1016\/S2214-109X(13)70145-1","article-title":"Global prevalence of age-related macular degeneration and disease burden projection for 2020 and 2040: A systematic review and meta-analysis","volume":"2","author":"Wong","year":"2014","journal-title":"Lancet Glob. Health"},{"key":"ref_3","first-page":"1929","article-title":"Subfoveal fibrovascular membranes in age-related macular degeneration express vascular endothelial growth factor","volume":"37","author":"Kvanta","year":"1996","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_4","first-page":"855","article-title":"Transdifferentiated retinal pigment epithelial cells are immunoreactive for vascular endothelial growth factor in surgically excised age-related macular degeneration-related choroidal neovascular membranes","volume":"37","author":"Lopez","year":"1996","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1144","DOI":"10.1136\/bjophthalmol-2014-305702","article-title":"Guidelines for the management of neovascular age-related macular degeneration by the European Society of Retina Specialists (EURETINA)","volume":"98","author":"Chong","year":"2014","journal-title":"Br. J. Ophthalmol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"376","DOI":"10.1016\/j.ajpath.2012.06.006","article-title":"A brief history of anti-VEGF for the treatment of ocular angiogenesis","volume":"181","author":"Kim","year":"2012","journal-title":"Am. J. Pathol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"100906","DOI":"10.1016\/j.preteyeres.2020.100906","article-title":"Incomplete response to Anti-VEGF therapy in neovascular AMD: Exploring disease mechanisms and therapeutic opportunities","volume":"82","author":"Mettu","year":"2021","journal-title":"Prog. Retin. Eye Res."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1857","DOI":"10.2147\/DDDT.S97653","article-title":"Resistance to anti-VEGF therapy in neovascular age-related macular degeneration: A comprehensive review","volume":"10","author":"Sun","year":"2016","journal-title":"Drug Des. Dev. Ther."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"473","DOI":"10.2147\/CIA.S2777","article-title":"Age-related macular degeneration and the aging eye","volume":"3","author":"Ehrlich","year":"2008","journal-title":"Clin. Interv. Aging"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1","DOI":"10.7554\/eLife.62048","article-title":"Small molecule cognitive enhancer reverses age-related memory decline in mice","volume":"9","author":"Krukowski","year":"2020","journal-title":"eLife"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"648","DOI":"10.1523\/JNEUROSCI.0628-17.2017","article-title":"Local Inhibition of PERK Enhances Memory and Reverses Age-Related Deterioration of Cognitive and Neuronal Properties","volume":"38","author":"Sharma","year":"2018","journal-title":"J. Neurosci."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"707","DOI":"10.1016\/j.bbrc.2007.01.043","article-title":"Activation of the unfolded protein response in Parkinson\u2019s disease","volume":"354","author":"Hoozemans","year":"2007","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41467-020-19352-1","article-title":"ATF4 leads to glaucoma by promoting protein synthesis and ER client protein load","volume":"11","author":"Kasetti","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1202","DOI":"10.1080\/02713683.2017.1297998","article-title":"Elevated Activating Transcription Factor 4 and Glucose-Regulated 78 Kda Protein Levels Correlate with Inflammatory Cytokines in the Aqueous Humor and Vitreous of Proliferative Diabetic Retinopathy","volume":"42","author":"Wang","year":"2017","journal-title":"Curr. Eye Res."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"5314","DOI":"10.1126\/science.aat5314","article-title":"The integrated stress response: From mechanism to disease","volume":"368","author":"Walter","year":"2020","journal-title":"Science"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1374","DOI":"10.15252\/embr.201642195","article-title":"The integrated stress response","volume":"17","author":"Koryga","year":"2016","journal-title":"EMBO Rep."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1099","DOI":"10.1016\/S1097-2765(00)00108-8","article-title":"Regulated Translation Initiation Controls Stress-Induced Gene Expression in Mammalian Cells","volume":"6","author":"Harding","year":"2000","journal-title":"Mol. Cell"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1227","DOI":"10.1084\/jem.20071460","article-title":"ATF4 is an oxidative stress\u2013inducible, prodeath transcription factor in neurons in vitro and in vivo","volume":"205","author":"Lange","year":"2008","journal-title":"J. Exp. Med."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"3493","DOI":"10.1007\/s00018-012-1252-6","article-title":"The eIF2\u03b1 kinases: Their structures and functions","volume":"70","author":"Donnelly","year":"2013","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Cho, H., Wu, M., Zhang, L., Thompson, R., Nath, A., and Chan, C. (2013). Signaling dynamics of palmitate-induced ER stress responses mediated by ATF4 in HepG2 cells. BMC Syst. Biol., 7.","DOI":"10.1186\/1752-0509-7-9"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1870","DOI":"10.1002\/jbmr.1958","article-title":"ATF4 promotes bone angiogenesis by increasing vegf expression and release in the bone environment","volume":"28","author":"Zhu","year":"2013","journal-title":"J. Bone Miner. Res."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"5995","DOI":"10.1167\/iovs.13-12117","article-title":"Modulation of angiogenesis by genetic manipulation of atf4 in a mouse modulation of angiogenesis by genetic manipulation","volume":"54","author":"Wang","year":"2013","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"989","DOI":"10.1593\/neo.13262","article-title":"Amino Acid Deprivation Promotes Tumor Angiogenesis through the GCN2\/ATF4 Pathway","volume":"15","author":"Wang","year":"2013","journal-title":"Neoplasia"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"581","DOI":"10.1210\/er.2003-0027","article-title":"Vascular Endothelial Growth Factor: Basic Science and Clinical Progress","volume":"25","author":"Ferrara","year":"2004","journal-title":"Endocr. Rev."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1248","DOI":"10.1016\/j.cell.2019.01.021","article-title":"VEGF in Signaling and Disease: Beyond Discovery and Development","volume":"176","author":"Apte","year":"2019","journal-title":"Cell"},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Zhou, H., Zhao, X., Yuan, M., and Chen, Y. (2020). Comparison of cytokine levels in the aqueous humor of polypoidal choroidal vasculopathy and neovascular age-related macular degeneration patients. BMC Ophthalmol., 20.","DOI":"10.1186\/s12886-019-1278-8"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1167\/iovs.61.13.39","article-title":"Complement Activation Products and Cytokines in Pachychoroid Neovasculopathy and Neovascular Age-Related Macular Degeneration","volume":"61","author":"Kato","year":"2020","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"559","DOI":"10.1016\/j.molcel.2014.03.022","article-title":"VEGF Signals through ATF6 and PERK to Promote Endothelial Cell Survival and Angiogenesis in the Absence of ER Stress","volume":"54","author":"Karali","year":"2014","journal-title":"Mol. Cell"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"88","DOI":"10.1016\/j.molcel.2020.10.031","article-title":"ISRIB Blunts the Integrated Stress Response by Allosterically Antagonising the Inhibitory Effect of Phosphorylated eIF2 on eIF2B","volume":"81","author":"Zyryanova","year":"2021","journal-title":"Mol. Cell"},{"key":"ref_30","first-page":"1","article-title":"Pharmacological brake-release of mRNA translation enhances cognitive memory","volume":"2013","author":"Sidrauski","year":"2013","journal-title":"eLife"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"e1672","DOI":"10.1038\/cddis.2015.49","article-title":"Partial restoration of protein synthesis rates by the small molecule ISRIB prevents neurodegeneration without pancreatic toxicity","volume":"6","author":"Halliday","year":"2015","journal-title":"Cell Death Dis."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"1407","DOI":"10.1002\/acn3.50826","article-title":"Vanishing white matter: Deregulated integrated stress response as therapy target","volume":"6","author":"Abbink","year":"2019","journal-title":"Ann. Clin. Transl. Neurol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"2599","DOI":"10.1167\/iovs.07-1508","article-title":"Increased Choroidal Neovascularization following Laser Induction in Mice Lacking Lysyl Oxidase-like 1","volume":"49","author":"Yu","year":"2008","journal-title":"Investig. Opthalmol. Vis. Sci."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"7847","DOI":"10.1038\/ncomms8847","article-title":"IL10-driven STAT3 signalling in senescent macrophages promotes pathological eye angiogenesis","volume":"6","author":"Nakamura","year":"2015","journal-title":"Nat. Commun."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"23705","DOI":"10.1073\/pnas.1908355116","article-title":"RIP1 kinase mediates angiogenesis by modulating macrophages in experimental neovascularization","volume":"116","author":"Ueta","year":"2019","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"535","DOI":"10.2119\/molmed.2010.00070","article-title":"Endoplasmic Reticulum Stress in Age-Related Macular Degeneration: Trigger for Neovascularization","volume":"16","author":"Salminen","year":"2010","journal-title":"Mol. Med."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1016\/0042-6989(85)90076-8","article-title":"A schematic eye for the mouse, and comparisons with the rat","volume":"25","author":"Remtulla","year":"1985","journal-title":"Vision Res."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"629","DOI":"10.1016\/j.molcel.2018.06.038","article-title":"Coordination between Two Branches of the Unfolded Protein Response Determines Apoptotic Cell Fate","volume":"71","author":"Chang","year":"2018","journal-title":"Mol. Cell"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"397","DOI":"10.1038\/s41419-020-2601-2","article-title":"Fine tuning of the unfolded protein response by ISRIB improves neuronal survival in a model of amyotrophic lateral sclerosis","volume":"11","author":"Bugallo","year":"2020","journal-title":"Cell Death Dis."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"5396","DOI":"10.1158\/0008-5472.CAN-12-0474","article-title":"The Unfolded Protein Response Induces the Angiogenic Switch in Human Tumor Cells through the PERK\/ATF4 Pathway","volume":"72","author":"Wang","year":"2012","journal-title":"Cancer Res."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"285","DOI":"10.1369\/0022155417694872","article-title":"Association of ATF4 Expression with Tissue Hypoxia and M2 Macrophage Infiltration in Infantile Hemangioma","volume":"65","author":"Xia","year":"2017","journal-title":"J. Histochem. Cytochem."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"12741","DOI":"10.1073\/pnas.0605457103","article-title":"Identification of inflammatory gene modules based on variations of human endothelial cell responses to oxidized lipids","volume":"103","author":"Gargalovic","year":"2006","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1186\/s12950-015-0076-1","article-title":"ATF4 is a novel regulator of MCP-1 in microvascular endothelial cells","volume":"12","author":"Huang","year":"2015","journal-title":"J. Inflamm."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"20991","DOI":"10.1074\/jbc.M111.226266","article-title":"Relationship between Complement Membrane Attack Complex, Chemokine (C-C Motif) Ligand 2 (CCL2) and Vascular Endothelial Growth Factor in Mouse Model of Laser-induced Choroidal Neovascularization","volume":"286","author":"Liu","year":"2011","journal-title":"J. Biol. Chem."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"777","DOI":"10.1007\/s00417-004-0910-2","article-title":"Fibrinogen stimulates in vitro angiogenesis by choroidal endothelial cells via autocrine VEGF","volume":"242","author":"Shiose","year":"2004","journal-title":"Graefe\u2019s Arch. Clin. Exp. Ophthalmol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"330","DOI":"10.1182\/blood-2007-09-112870","article-title":"ATF4-dependent transcription is a key mechanism in VEGF up-regulation by oxidized phospholipids: Critical role of oxidized sn-2 residues in activation of unfolded protein response","volume":"112","author":"Oskolkova","year":"2008","journal-title":"Blood"},{"key":"ref_47","first-page":"116","article-title":"TNF-\u03b1 mediates choroidal neovascularization by upregulating VEGF expression in RPE through ROS-dependent \u03b2-catenin activation","volume":"22","author":"Wang","year":"2016","journal-title":"Mol. Vis."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1051","DOI":"10.1080\/02713680601100459","article-title":"The Dynamic Conduct of Bone Marrow-Derived Cells in the Choroidal Neovascularization Microenvironment","volume":"31","author":"Hou","year":"2006","journal-title":"Curr. Eye Res."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"3747","DOI":"10.1167\/iovs.18-23892","article-title":"Lactic Acid Upregulates VEGF Expression in Macrophages and Facilitates Choroidal Neovascularization","volume":"59","author":"Song","year":"2018","journal-title":"Investig. Opthalmol. Vis. Sci."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"196","DOI":"10.1016\/j.exer.2016.03.020","article-title":"Edaravone is a free radical scavenger that protects against laser-induced choroidal neovascularization in mice and common marmosets","volume":"146","author":"Masuda","year":"2016","journal-title":"Exp. Eye Res."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"9898","DOI":"10.1038\/srep09898","article-title":"The effect of triamcinolone acetonide on laser-induced choroidal neovascularization in mice using a hypoxia visualization bio-imaging probe","volume":"5","author":"Takata","year":"2015","journal-title":"Sci. Rep."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"1406","DOI":"10.1016\/j.bbrc.2020.10.032","article-title":"ANKFY1 is essential for retinal endothelial cell proliferation and migration via VEGFR2\/Akt\/eNOS pathway","volume":"533","author":"Tanaka","year":"2020","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_53","doi-asserted-by":"crossref","unstructured":"Nakamura, S., Takizawa, H., Shimazawa, M., Hashimoto, Y., Sugitani, S., Tsuruma, K., and Hara, H. (2013). Mild Endoplasmic Reticulum Stress Promotes Retinal Neovascularization via Induction of BiP\/GRP78. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0060517"}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/16\/8890\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:46:36Z","timestamp":1760165196000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/22\/16\/8890"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,8,18]]},"references-count":53,"journal-issue":{"issue":"16","published-online":{"date-parts":[[2021,8]]}},"alternative-id":["ijms22168890"],"URL":"https:\/\/doi.org\/10.3390\/ijms22168890","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,8,18]]}}}