{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,16]],"date-time":"2026-10-16T22:09:57Z","timestamp":1792188597820,"version":"4.4.0"},"reference-count":51,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2020,9,9]],"date-time":"2020-09-09T00:00:00Z","timestamp":1599609600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Grimshaw-Gudewicz Charitable Foundation","award":["AMD grant"],"award-info":[{"award-number":["AMD grant"]}]},{"DOI":"10.13039\/100006312","name":"BrightFocus Foundation","doi-asserted-by":"publisher","award":["M2015214"],"award-info":[{"award-number":["M2015214"]}],"id":[{"id":"10.13039\/100006312","id-type":"DOI","asserted-by":"publisher"}]},{"name":"National Council of Scientific and Technological Development - Brazil","award":["PDE 210477\/2014-8"],"award-info":[{"award-number":["PDE 210477\/2014-8"]}]},{"DOI":"10.13039\/100000053","name":"National Eye Institute","doi-asserted-by":"publisher","award":["Core Grant P30EY003790"],"award-info":[{"award-number":["Core Grant P30EY003790"]}],"id":[{"id":"10.13039\/100000053","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Many patients with wet age-related macular degeneration do not respond well to anti- vascular endothelial growth factor A (VEGFA) therapy for choroidal neovascularization (CNV), and the efficacy of anti-VEGFA decreases over time. We investigated the hypothesis that fibrotic changes, in particular via endothelial-to-mesenchymal transition (EndoMT), play a role in CNV and alter the therapeutic effects of VEGFA pathway blockage. Induction of EndoMT of primary human retinal endothelial cells led to a significantly reduced response to VEGFA at the level of gene expression, cellular proliferation, migration, and tube formation. Suppression of EndoMT restored cell responsiveness to VEGFA. In a mouse model of spontaneous CNV, fibrotic changes and EndoMT persisted as the CNV lesions became more established over time. VEGFA receptor-2 (VEGFR2) antagonism further induced fibrosis and EndoMT in the CNV. The combination of VEGFR2 antagonism and fibrosis\/EndoMT inhibition was more effective than either individual treatment in reducing CNV. Our data indicate that fibrosis and EndoMT are involved in the progression of CNV, are exacerbated by VEGFR2 inhibition, and could provide an explanation for the reduced efficacy of anti-VEGFA treatment over time.<\/jats:p>","DOI":"10.3390\/cells9092057","type":"journal-article","created":{"date-parts":[[2020,9,10]],"date-time":"2020-09-10T22:58:01Z","timestamp":1599778681000},"page":"2057","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":31,"title":["Fibrotic Changes and Endothelial-to-Mesenchymal Transition Promoted by VEGFR2 Antagonism Alter the Therapeutic Effects of VEGFA Pathway Blockage in a Mouse Model of Choroidal Neovascularization"],"prefix":"10.3390","volume":"9","author":[{"given":"Franco Aparecido","family":"Rossato","sequence":"first","affiliation":[{"name":"Department of Ophthalmology, Schepens Eye Research Institute of Mass Eye and Ear, and Harvard Medical School, 20 Staniford Street, Boston, MA 02114, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yu","family":"Su","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Schepens Eye Research Institute of Mass Eye and Ear, and Harvard Medical School, 20 Staniford Street, Boston, MA 02114, USA"},{"name":"Ophthalmic Center, Renmin Hospital of Wuhan University, Wuhan 430000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ashley","family":"Mackey","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Schepens Eye Research Institute of Mass Eye and Ear, and Harvard Medical School, 20 Staniford Street, Boston, MA 02114, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4982-1999","authenticated-orcid":false,"given":"Yin Shan Eric","family":"Ng","sequence":"additional","affiliation":[{"name":"Department of Ophthalmology, Schepens Eye Research Institute of Mass Eye and Ear, and Harvard Medical School, 20 Staniford Street, Boston, MA 02114, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2020,9,9]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"160","DOI":"10.1111\/cge.12206","article-title":"Age-related macular degeneration-clinical review and genetics update","volume":"84","author":"Ratnapriya","year":"2013","journal-title":"Clin. Genet."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"55","DOI":"10.1016\/j.ophtha.2018.07.028","article-title":"Safety and efficacy of anti-vascular endothelial growth factor therapies for neovascular age-related macular degeneration: A report by the American Academy of Ophthalmology","volume":"126","author":"Bakri","year":"2019","journal-title":"Ophthalmology"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1671","DOI":"10.1167\/iovs.09-4496","article-title":"Effects on choroidal neovascularization after anti-VEGF Upload using intravitreal ranibizumab, as determined by spectral domain-optical coherence tomography","volume":"51","author":"Framme","year":"2010","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1016\/j.ophtha.2008.10.018","article-title":"Ranibizumab versus verteporfin photodynamic therapy for neovascular age-related macular degeneration: Two-year results of the ANCHOR study","volume":"116","author":"Brown","year":"2009","journal-title":"Ophthalmology"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1419","DOI":"10.1056\/NEJMoa054481","article-title":"Ranibizumab for neovascular age-related macular degeneration","volume":"355","author":"Rosenfeld","year":"2006","journal-title":"N. Engl. J. Med."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"2537","DOI":"10.1016\/j.ophtha.2012.09.006","article-title":"Intravitreal aflibercept (VEGF trap-eye) in wet age-related macular degeneration","volume":"119","author":"Heier","year":"2012","journal-title":"Ophthalmology"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Comparison of Age-Related Macular Degeneration Treatments Trials Research Group, Maguire, M.G., Martin, D.F., Ying, G.S., Jaffe, G.J., Daniel, E., Grunwald, J.E., Toth, C.A., Ferris, F.L., and Fine, S.L. (2016). Five-Year Outcomes with Anti-Vascular Endothelial Growth Factor Treatment of Neovascular Age-Related Macular Degeneration: The Comparison of Age-Related Macular Degeneration Treatments Trials. Ophthalmology, 123, 1751\u20131761.","DOI":"10.1016\/j.ophtha.2016.03.045"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"2292","DOI":"10.1016\/j.ophtha.2013.03.046","article-title":"Seven-year outcomes in ranibizumab-treated patients in ANCHOR, MARINA, and HORIZON: A multicenter cohort study (SEVEN-UP)","volume":"120","author":"Rofagha","year":"2013","journal-title":"Ophthalmology"},{"key":"ref_9","first-page":"1857","article-title":"Resistance to anti-VEGF therapy in neovascular age-related macular degeneration: A comprehensive review","volume":"10","author":"Yang","year":"2016","journal-title":"Drug Des. Devel. Ther."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"656","DOI":"10.1016\/j.ophtha.2013.10.019","article-title":"Risk of scar in the comparison of age-related macular degeneration treatments trials","volume":"121","author":"Daniel","year":"2014","journal-title":"Ophthalmology"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"451","DOI":"10.1016\/j.oret.2017.08.019","article-title":"Tomographic biomarkers predicting progression to fibrosis in treated neovascular age-related macular degeneration: A multimodal imaging study","volume":"2","author":"Casalino","year":"2018","journal-title":"Ophthalmol. Retin."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"6","DOI":"10.3928\/15428877-20100924-01","article-title":"Development of subretinal fibrosis after anti-VEGF treatment in neovascular age-related macular degeneration","volume":"42","author":"Hwang","year":"2011","journal-title":"Ophthalmic. Surg. Lasers Imaging"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"14361","DOI":"10.1038\/ncomms14361","article-title":"The molecular basis of endothelial cell plasticity","volume":"8","author":"Dejana","year":"2017","journal-title":"Nat. Commun."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1074","DOI":"10.1016\/j.ajpath.2011.06.001","article-title":"Role of endothelial-mesenchymal transition (EndoMT) in the pathogenesis of fibrotic disorders","volume":"179","author":"Li","year":"2011","journal-title":"Am. J. Pathol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"3439","DOI":"10.1038\/s41467-018-05982-z","article-title":"PDGF-mediated mesenchymal transformation renders endothelial resistance to anti-VEGF treatment in glioblastoma","volume":"9","author":"Liu","year":"2018","journal-title":"Nat. Commun."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"932","DOI":"10.1016\/j.athoracsur.2017.01.112","article-title":"Vascular Endothelial Growth factor prevents endothelial-to-mesenchymal transition in hypertrophy","volume":"104","author":"Illigens","year":"2017","journal-title":"Ann. Thorac. Surg."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Kuiper, E.J., Van Nieuwenhoven, F.A., De Smet, M.D., Van Meurs, J.C., Tanck, M.W., Oliver, N., Klaassen, I., Van Noorden, C.J., Goldschmeding, R., and Schlingemann, R.O. (2008). The angio-fibrotic switch of VEGF and CTGF in proliferative diabetic retinopathy. PLoS ONE, 3.","DOI":"10.1371\/journal.pone.0002675"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1403","DOI":"10.1002\/bit.25905","article-title":"High-throughput investigation of endothelial-to-mesenchymal transformation (EndMT) with combinatorial cellular microarrays","volume":"113","author":"Wang","year":"2016","journal-title":"Biotechnol. Bioeng."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2208","DOI":"10.1016\/j.ajpath.2017.06.015","article-title":"A proinflammatory function of toll-like receptor 2 in the retinal pigment epithelium as a novel target for reducing choroidal neovascularization in age-related macular degeneration","volume":"187","author":"Feng","year":"2017","journal-title":"Am. J. Pathol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1850","DOI":"10.1016\/j.ajpath.2015.03.019","article-title":"Endothelial to mesenchymal transition contributes to endothelial dysfunction in pulmonary arterial hypertension","volume":"185","author":"Good","year":"2015","journal-title":"Am. J. Pathol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"3709","DOI":"10.1167\/iovs.14-13989","article-title":"Spontaneous CNV in a novel mutant mouse is associated with early VEGF-A-driven angiogenesis and late-stage focal edema, neural cell loss, and dysfunction","volume":"55","author":"Nagai","year":"2014","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"5424","DOI":"10.1167\/iovs.15-17264","article-title":"IL-18 Immunotherapy for neovascular AMD: Tolerability and efficacy in nonhuman primates","volume":"56","author":"Doyle","year":"2015","journal-title":"Investig. Ophthalmol. Vis. Sci."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"2534","DOI":"10.1016\/j.ajpath.2015.04.029","article-title":"Novel CCR3 antagonists are effective mono- and combination inhibitors of choroidal neovascular growth and vascular permeability","volume":"185","author":"Nagai","year":"2015","journal-title":"Am. J. Pathol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1265","DOI":"10.15252\/emmm.201505889","article-title":"Targeting key angiogenic pathways with a bispecific CrossMAb optimized for neovascular eye diseases","volume":"8","author":"Regula","year":"2016","journal-title":"EMBO Mol. Med."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"6392","DOI":"10.1073\/pnas.0802047105","article-title":"Notch signaling mediates hypoxia-induced tumor cell migration and invasion","volume":"105","author":"Sahlgren","year":"2008","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"170","DOI":"10.1016\/j.canlet.2013.01.027","article-title":"Snail2 promotes osteosarcoma cell motility through remodelling of the actin cytoskeleton and regulates tumor development","volume":"333","author":"Sharili","year":"2013","journal-title":"Cancer Lett."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1827","DOI":"10.1016\/j.ajpath.2011.06.032","article-title":"Twist1 is up-regulated in gastric cancer-associated fibroblasts with poor clinical outcomes","volume":"179","author":"Sung","year":"2011","journal-title":"Am. J. Pathol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"33787","DOI":"10.1038\/srep33787","article-title":"Endothelial to mesenchymal transition contributes to arsenic-trioxide-induced cardiac fibrosis","volume":"6","author":"Zhang","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2660","DOI":"10.1016\/j.ajpath.2011.07.042","article-title":"Inflammation-induced endothelial-to-mesenchymal transition: A novel mechanism of intestinal fibrosis","volume":"179","author":"Rieder","year":"2011","journal-title":"Am. J. Pathol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"11853","DOI":"10.1038\/ncomms11853","article-title":"Endothelial to mesenchymal transition is common in atherosclerotic lesions and is associated with plaque instability","volume":"7","author":"Evrard","year":"2016","journal-title":"Nat. Commun."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1018","DOI":"10.1038\/s41419-018-1049-0","article-title":"Novel engineered, membrane-localized variants of vascular endothelial growth factor (VEGF) protect retinal ganglion cells: A proof-of-concept study","volume":"9","author":"Shen","year":"2018","journal-title":"Cell Death Dis."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"729","DOI":"10.1083\/jcb.201105103","article-title":"Snail1 controls epithelial-mesenchymal lineage commitment in focal adhesion kinase-null embryonic cells","volume":"195","author":"Li","year":"2011","journal-title":"J. Cell Biol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"14309","DOI":"10.1073\/pnas.1001520107","article-title":"Inhibition of Wnt\/beta-catenin\/CREB binding protein (CBP) signaling reverses pulmonary fibrosis","volume":"107","author":"Henderson","year":"2010","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"25754","DOI":"10.1038\/srep25754","article-title":"SARS coronavirus papain-like protease induces Egr-1-dependent up-regulation of TGF-beta1 via ROS\/p38 MAPK\/STAT3 pathway","volume":"6","author":"Li","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1038\/eye.2010.196","article-title":"The role of anti-inflammatory agents in age-related macular degeneration (AMD) treatment","volume":"25","author":"Wang","year":"2011","journal-title":"Eye"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"717","DOI":"10.1002\/iub.1059","article-title":"The role of endothelial-mesenchymal transition in development and pathological process","volume":"64","author":"Lin","year":"2012","journal-title":"IUBMB Life"},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"303","DOI":"10.1161\/ATVBAHA.114.303220","article-title":"A role for partial endothelial-mesenchymal transitions in angiogenesis?","volume":"35","author":"Wu","year":"2015","journal-title":"Arterioscler. Thromb. Vasc. Biol."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"34631","DOI":"10.1038\/srep34631","article-title":"Detection of aqueous VEGF concentrations before and after intravitreal injection of anti-VEGF antibody using low-volume sampling paper-based ELISA","volume":"6","author":"Hsu","year":"2016","journal-title":"Sci. Rep."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"1351","DOI":"10.1093\/ndt\/gfr550","article-title":"Inhibition of the p38 MAPK pathway ameliorates renal fibrosis in an NPHP2 mouse model","volume":"27","author":"Sugiyama","year":"2012","journal-title":"Nephrol. Dial. Transplant."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"31053","DOI":"10.18632\/oncotarget.8842","article-title":"Inhibition of myocyte-specific enhancer factor 2A improved diabetic cardiac fibrosis partially by regulating endothelial-to-mesenchymal transition","volume":"7","author":"Chen","year":"2016","journal-title":"Oncotarget"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"515","DOI":"10.1042\/BJ20101500","article-title":"Transforming growth factor-beta2 promotes Snail-mediated endothelial-mesenchymal transition through convergence of Smad-dependent and Smad-independent signalling","volume":"437","author":"Medici","year":"2011","journal-title":"Biochem. J."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"297","DOI":"10.1042\/BJ20070797","article-title":"The selectivity of protein kinase inhibitors: A further update","volume":"408","author":"Bain","year":"2007","journal-title":"Biochem. J."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1358","DOI":"10.1016\/j.bbamcr.2007.03.010","article-title":"p38 MAP-kinases pathway regulation, function and role in human diseases","volume":"1773","author":"Cuenda","year":"2007","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1046\/j.1523-1747.1998.00262.x","article-title":"Increased microvascular density and enhanced leukocyte rolling and adhesion in the skin of VEGF transgenic mice","volume":"111","author":"Detmar","year":"1998","journal-title":"J. Investig. Dermatol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"7614","DOI":"10.1074\/jbc.M009705200","article-title":"Vascular endothelial growth factor expression of intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), and E-selectin through nuclear factor-kappa B activation in endothelial cells","volume":"276","author":"Kim","year":"2001","journal-title":"J. Biol. Chem."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"3811","DOI":"10.1182\/blood.V93.11.3811","article-title":"Vascular endothelial cell growth factor-induced tissue factor expression in endothelial cells is mediated by EGR-1","volume":"93","author":"Mechtcheriakova","year":"1999","journal-title":"Blood"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"992","DOI":"10.1038\/nm0996-992","article-title":"During angiogenesis, vascular endothelial growth factor and basic fibroblast growth factor regulate natural killer cell adhesion to tumor endothelium","volume":"2","author":"Melder","year":"1996","journal-title":"Nat. Med."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"1765","DOI":"10.1007\/s00018-016-2147-8","article-title":"Inflammation and its role in age-related macular degeneration","volume":"73","author":"Kauppinen","year":"2016","journal-title":"Cell. Mol. Life Sci."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"294","DOI":"10.3389\/fimmu.2018.00294","article-title":"Endothelial to Mesenchymal Transition Represents a Key Link in the Interaction between Inflammation and Endothelial Dysfunction","volume":"9","author":"Cho","year":"2018","journal-title":"Front. Immunol."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"3463","DOI":"10.1038\/s41467-018-05926-7","article-title":"NCK-dependent pericyte migration promotes pathological neovascularization in ischemic retinopathy","volume":"9","author":"Dubrac","year":"2018","journal-title":"Nat. Commun."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"141","DOI":"10.1007\/s10456-007-9085-x","article-title":"RGS5 expression is a quantitative measure of pericyte coverage of blood vessels","volume":"11","author":"Mitchell","year":"2008","journal-title":"Angiogenesis"}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/9\/9\/2057\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T10:08:26Z","timestamp":1760177306000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/9\/9\/2057"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,9,9]]},"references-count":51,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2020,9]]}},"alternative-id":["cells9092057"],"URL":"https:\/\/doi.org\/10.3390\/cells9092057","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,9,9]]}}}