Abstract
PurposeBevacizumab is taken up and transported through the retinal pigment epithelium. Inflammatory signaling may influence this interaction. In the present study, we have investigated the effect of pro-inflammatory stimuli on the uptake, intracellular localization, and transepithelial transport of bevacizumab.MethodsARPE-19 cell line or primary porcine RPE cells were treated with clinical relevant concentrations of bevacizumab (250 µg/ml). Pro-inflammatory signaling was induced by TLR-3 agonist polyinosinic:polycytidylic acid (Poly I:C). Viability was investigated with MTT and trypan-blue exclusion assay, and cell number, uptake, and intracellular localization were investigated with immunofluorescence, investigating also actin filaments, the motor protein myosin 7a and lysosomes. Immunofluorescence signals were quantified. Intracellular bevacizumab was additionally detected in Western blot. Barrier function was investigated with transepithelial resistant measurements (TER). The transepithelial transport of bevacizumab and its influence on cytokine (IL-6, IL-8, IL-1β, TNFα) secretion was investigated with ELISA.ResultsPoly I:C in combination with bevacizumab reduced the viability of the cells. Treatment with Poly I:C reduced the uptake of bevacizumab, changed the intensity of the actin filaments, and reduced the colocalization with myosin 7a. In addition, Poly I:C reduced the capacity of RPE cells to transport bevacizumab over the barrier. In addition, bevacizumab reduced the secretion of IL-8 and TNFα after Poly I:C stimulation at selected time points.ConclusionsPro-inflammatory activation of RPE cells with TLR-3 agonist Poly I:C changes the interaction of RPE cells with the anti-VEGF compound bevacizumab, reducing its uptake and transport. On the other hand, bevacizumab might influence pro-inflammatory cytokine release. Our data indicate that inflammation may influence the pharmacokinetic of bevacizumab in the retina.
Highlights
Age-related macular degeneration (AMD) is the main cause for vision loss in the Western world and its prevalence is worldwide on the rise [1, 2]
After treatment for 1 day, the combined treatment of ARPE-19 cells with 100 μg/ml polycytidylic acid (Poly I):C and 250 μg/ml bevacizumab significantly reduced the number of cell nuclei compared to control (Fig. 1d)
We have investigated the transepithelial transport of bevacizumab in primary Retinal pigment epithelium (RPE) cells, cultivated on transwell plates which showed a stable barrier, assessed by transepithelial resistant measurements (TER) measurement
Summary
Age-related macular degeneration (AMD) is the main cause for vision loss in the Western world and its prevalence is worldwide on the rise [1, 2]. It presents in an early form, which is asymptomatic for the patient, and two late forms. In exudative AMD, anti-VEGF therapy is the state-of-the art treatment [9]. Despite the official approval of anti-VEGF therapies such as ranibizumab or aflibercept for treatment of AMD [10, 11], off-label application of bevacizumab is common due to the lower price and the comparable outcomes [12, 13]
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