title: Single- and repeated-dose toxicity study of bevacizumab, ranibizumab, and aflibercept in ARPE-19 cells under normal and oxidative stress conditions.
abstract: We assessed the effect of single and repeated doses of bevacizumab, ranibizumab, and aflibercept on cell viability, proliferation, permeability, and apoptosis of ARPE-19 cells. MTT and BrdU assays were used to determine viability and proliferation after single or repeated doses of anti-VEGF drugs under normal and oxidative stress conditions. Caspase-3 expression after single and repeated doses of the 3 drugs was assessed using immunofluorescence. Transepithelial-electrical-resistance (TER) was measured to study the effect of anti-VEGFs on retinal pigment epithelium (RPE) permeability under normal and oxidative stress conditions. Flow cytometry was used to detect intracellular accumulation of the drugs. Finally, a wound healing assay was performed to investigate the effect of the drugs on RPE cell migration. Single and multiple doses of anti-VEGF drugs had no effect on cell viability and proliferation. The oxidative effect of H2O2 decreased cell viability and proliferation; however, no difference was observed between anti-VEGF treatments. Immunofluorescence performed after single and repeated doses of the drugs revealed some caspase-3 expression. Interestingly, anti-VEGFs restored the increased permeability induced by H2O2. The 3 drugs accumulated inside the cells and were detectable 5 days after treatment. Finally, none of the drugs affected migration. In conclusion, no measureable toxic effect was observed after single or repeated doses of VEGF antagonists under normal and oxidative stress. Intracellular accumulation of the drugs does not seem to be toxic or affect cell functions. Our study suggests that anti-VEGFs could have a preventive effect on the maintenance of the RPE barrier under oxidative stress.
authors.affiliation: Experimental Ophthalmology Laboratory, School of Medicine, University of Navarra, 1 Irunlarrea Street, 31008 Pamplona, Spain; Department of Ophthalmology, Clínica Universidad de Navarra, School of Medicine, University of Navarra, 36 Pio XII Avenue, 31008 Pamplona, Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: Saenz-de-Viteri
authors.affiliation: Experimental Ophthalmology Laboratory, School of Medicine, University of Navarra, 1 Irunlarrea Street, 31008 Pamplona, Spain; IdiSNA, Navarra Institute for Health Research, 31008 Pamplona, Spain.
authors.forename: Patricia
authors.initials: P
authors.lastname: Fernández-Robredo
authors.affiliation: Experimental Ophthalmology Laboratory, School of Medicine, University of Navarra, 1 Irunlarrea Street, 31008 Pamplona, Spain; IdiSNA, Navarra Institute for Health Research, 31008 Pamplona, Spain. Electronic address: mahersan@unav.es.
authors.forename: María
authors.initials: M
authors.lastname: Hernández
authors.affiliation: Experimental Ophthalmology Laboratory, School of Medicine, University of Navarra, 1 Irunlarrea Street, 31008 Pamplona, Spain.
authors.forename: Jaione
authors.initials: J
authors.lastname: Bezunartea
authors.affiliation: Experimental Ophthalmology Laboratory, School of Medicine, University of Navarra, 1 Irunlarrea Street, 31008 Pamplona, Spain.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Reiter
authors.affiliation: Experimental Ophthalmology Laboratory, School of Medicine, University of Navarra, 1 Irunlarrea Street, 31008 Pamplona, Spain; IdiSNA, Navarra Institute for Health Research, 31008 Pamplona, Spain.
authors.forename: Sergio
authors.initials: S
authors.lastname: Recalde
authors.affiliation: Experimental Ophthalmology Laboratory, School of Medicine, University of Navarra, 1 Irunlarrea Street, 31008 Pamplona, Spain; Department of Ophthalmology, Clínica Universidad de Navarra, School of Medicine, University of Navarra, 36 Pio XII Avenue, 31008 Pamplona, Spain; IdiSNA, Navarra Institute for Health Research, 31008 Pamplona, Spain.
authors.forename: Alfredo
authors.initials: A
authors.lastname: García-Layana
mesh_terms: D000068258:Bevacizumab / Q000378:metabolism / Q000633:toxicity*; D053148:Caspase 3 / Q000378:metabolism; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D017097:Electric Impedance; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D018384:Oxidative Stress; D000069579:Ranibizumab / Q000378:metabolism / Q000633:toxicity*; D040262:Receptors, Vascular Endothelial Growth Factor / Q000378:metabolism / Q000633:toxicity*; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000633:toxicity*; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors*
chemical_list: D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A; C533178:aflibercept; D000068258:Bevacizumab; D040262:Receptors, Vascular Endothelial Growth Factor; D053148:Caspase 3; D000069579:Ranibizumab
keywords: Age-related-macular degeneration; Diabetic macular edema; Retinal pigmented epithelium; VEGF-antagonists; Vascular endothelial growth factor