In Vitro Effects of Bevacizumab Treatment on Newborn Rat Retinal Cell Proliferation, Death, and Differentiation

Abstract

PURPOSE. Vascular endothelial growth factor (VEGF) is an important signal protein in vertebrate nervous development, promoting neurogenesis, neuronal patterning, and glial cell growth. Bevacizumab, an anti-VEGF agent, has been extensively used for controlling pathological retinal neovascularization in adult and newborn patients, although its effect on the developing retina remains largely unknown. The purpose of this study was to investigate the effect of bevacizumab on cell death, proliferation, and differentiation in newborn rat retina. METHODS.Retinal explants of sixty 2-day-old Lister hooded rats were obtained after eye enucleation and maintained in culture media with or without bevacizumab for 2 days. Immunohistochemical staining was assessed against proliferating cell nuclear antigen (PCNA, to detect cell proliferation); caspase-3 and beclin-1 (to investigate cell death); and vimentin and glial fibrillary acidic protein (GFAP, markers of glial cells). Gene expressions were quantified by real-time reverse-transcription polymerase chain reaction. Results from treatment and control groups were compared.RESULTS. No significant difference in the staining intensity (on immunohistochemistry) of PCNA, caspase-3, beclin-1, and GFAP, or in the levels of PCNA, caspase-3, beclin-1, and vimentin mRNA was observed between the groups. However, a significant increase in vimentin levels and a significant decrease in GFAP mRNA expression were observed in bevacizumab-treated retinal explants compared with controls.CONCLUSIONS. Bevacizumab did not affect cell death or proliferation in early developing rat retina but appeared to interfere with glial cell maturation by increasing vimentin levels and downregulating GFAP gene expression. Thus, we suggest anti-VEGF agents be used with caution in developing retinal tissue. (Invest Ophthalmol Vis Sci. 2012;53:7904-7911)

Progress in Small Molecule Therapeutics for the Treatment of Retinoblastoma

“…[176] There is some limited data to suggest that anti-VEGF agents may cautiously be used in developing retinae: bevacizumab did not induce any changes in cell death or proliferation in early developing rat retinal explants maintained in organotypic culture, [176] and no electroretinographic or morphological abnormalities were detected in 11- or 35- day old rabbits who received intravitreal injections of bevacizumab. [177] Given the importance of vasculature in retinal function, more thorough investigation of the developmental toxicology of angiogenesis inhibitors is certainly warranted before they are pursued as retinoblastoma therapeutics.…”

Bevacizumab Reduces Neurocan Content and Gene Expression in Newborn Rat Retina In Vitro

“…48 Neurocan is synthesized normally by neurons 49 but also by reactive glial cells. 50,51 Since Miguel et al 26 found changes in retinal glial maturation produced by bevacizumab, it is possible that such abnormality contributed to the reduced neurocan content observed in our study.…”

“…The subject is important since VEGF, which is expressed by astrocytes in the retinal tissue during its development, 44,45 apparently acts as a neuroprotective and neurotrophic factor for retinal cells, influencing neuronal growth, differentiation, and survival. 7,23 Thus, Miguel et al 26 observed changes in glial maturation with upregulation of vimentin and downregulation of GFAP mRNA in retinal explants of newborn rats treated with bevacizumab for 2 days in vitro compared with controls.…”

“…[16][17][18][19][20] On the other hand, other experimental in vivo and in vitro studies have shown ultrastructural abnormalities in the retina, including increased apoptotic activity [21][22][23][24] and increased reactivity of glial cells 25 in adult retinal tissues. Furthermore, Miguel et al 26 evaluated newborn rat retinal explants exposed to bevacizumab for 48 hours, and found increased vimentin labeling and a decrease of glial fibrillary acidic protein mRNA levels in treated tissue compared with controls, suggesting that the anti-VEGF activity of bevacizumab may interfere with glial cell maturation in the early stages of retinal development. During retinal development, cellular migration, dendrites, and axon outgrowth occur through the extracellular matrix (ECM).…”

Optical coherence tomography and multifocal electroretinography of patients with advanced neovascular age-related macular degeneration before, during, and after treatment with ranibizumab

“…As AMD affected mainly the central region, changes were expected in R1, which had the largest concentration of retinal neurons affected by the disease and was the place where the drug's anti-angiogenic effect was expectedly higher (4) . Some experimental studies suggested a possible toxic effect of high-dose intravitreal VEGF inhibitors on retinal neurons (29,30) . The lower retinal electrical activity observed immediately after treatment raised the possibility of a deleterious effect of the medication on macular function, which fortunately recovered over time.…”