title: Intracellular pathways following uptake of bevacizumab in RPE cells.
abstract: The anti-VEGF antibody bevacizumab is widely used off-label for the treatment of various ocular diseases, most commonly in age-related macular degeneration and diabetic macular edema. Bevacizumab is able to penetrate the retina and is found in the choroid after intravitreal injection in a time dependent manner. It has previously been shown to be taken up by the retinal pigment epithelium (RPE). In this study, we have investigated the intracellular pathway following uptake of bevacizumab in RPE cells, tested both in primary porcine RPE cells and in the human cell line ARPE19. Bevacizumab displays a characteristic, time-dependent pattern of intracellular distribution, as detected by immunofluorescence and pulse chase experiments. In both primary cells and the cell line, intracellular bevacizumab can be found after seven days, as detected by immunofluorescence and Western blotting. Immediately after application, bevacizumab partially colocalizes with Rab5, indicating some uptake in early endosomes. Intracellularly, bevacizumab is detected in the cytoskeletal fraction, aligning with actin filaments, as revealed by subcellular fractioning and immunofluorescence. Bevacizumab seems to travel along actin filaments by myosin7a, as determined by triple staining immunofluorescence. Interestingly, over a period of seven days, bevacizumab seems to accumulate in certain storage areas, as observed by immunofluorescence. Furthermore, results obtained with immunocytochemistry, Western blotting and flow cytometry indicate that bevacizumab may be released from the RPE cells via exosomes. In conclusion, bevacizumab is taken up by and transported in the retinal pigment epithelial cells in a characteristic, time-dependent manner, where it seems to move along actin filaments by myosin7a and seem to be partially released from the cells via exosomes.
authors.affiliation: University of Kiel, University Medical Center, Department of Ophthalmology, Kiel, Germany; University of Cairo, Kasr Al-Aini Faculty of Medicine, Cairo, Egypt.
authors.forename: Shereen Hassan
authors.initials: SH
authors.lastname: Aboul Naga
authors.affiliation: University of Kiel, University Medical Center, Department of Ophthalmology, Kiel, Germany.
authors.forename: Michaela
authors.initials: M
authors.lastname: Dithmer
authors.affiliation: University of Kiel, University Medical Center, Institute of Immunology, Kiel, Germany.
authors.forename: Guranda
authors.initials: G
authors.lastname: Chitadze
authors.affiliation: University of Kiel, University Medical Center, Institute of Immunology, Kiel, Germany.
authors.forename: Dieter
authors.initials: D
authors.lastname: Kabelitz
authors.affiliation: University of Kiel, Institute of Anatomy, Kiel, Germany.
authors.forename: Ralph
authors.initials: R
authors.lastname: Lucius
authors.affiliation: University of Kiel, University Medical Center, Department of Ophthalmology, Kiel, Germany.
authors.forename: Johann
authors.initials: J
authors.lastname: Roider
authors.affiliation: University of Kiel, University Medical Center, Department of Ophthalmology, Kiel, Germany. Electronic address: aklettner@auge.uni-kiel.de.
authors.forename: Alexa
authors.initials: A
authors.lastname: Klettner
mesh_terms: D020533:Angiogenesis Inhibitors / Q000493:pharmacokinetics; D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000493:pharmacokinetics*; D000068258:Bevacizumab; D015153:Blotting, Western; D002478:Cells, Cultured; D004195:Disease Models, Animal; D055354:Exosomes / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007424:Intracellular Fluid / Q000378:metabolism; D008268:Macular Degeneration / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D055213:Retinal Pigment Epithelium / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D013552:Swine; D042461:Vascular Endothelial Growth Factor A / Q000037:antagonists & inhibitors
chemical_list: D020533:Angiogenesis Inhibitors; D061067:Antibodies, Monoclonal, Humanized; D042461:Vascular Endothelial Growth Factor A; D000068258:Bevacizumab
keywords: Actin-filament; Bevacizumab; Exosomes; Myosin7a; Retinal pigment epithelium