Effect of Long-term Anti-VEGF Treatment on Viability and Function of RPE Cells.

PURPOSE/AIM OF THE STUDY: Vascular endothelial growth factor (VEGF)-antagonists are given over long time periods in the clinic, but the long-term effects on retinal pigment epithelium (RPE) cells are not fully investigated. This study aims to investigate these effects with two clinical relevant VEGF antagonists, bevacizumab and aflibercept, on the function of primary RPE cells. MATERIALS AND METHODS: All tests were conducted with primary porcine RPE. Cells were stimulated with bevacizumab or aflibercept (both 250 µg/ml) for 1 day, 7 days or 4 weeks. Cell viability was tested in MTT Assay. Secretion of TGF-ß was tested in ELISA, phagocytosis in a microscopic assay, migration in a scratch assay, and expression of RPE65 in Western blot. Barrier function was tested for bevacizumab in transwell-cultured cells by measuring transepithelial electrical resistance for up to 3 days. RESULTS: Viability was reduced by both antagonists at all time points tested. TGF-ß secretion was not altered by any treatment. Phagocytosis was not significantly reduced by any treatment. Wound healing ability was not significantly altered by any treatment. The expression of RPE65 was reduced by bevacizumab but not aflibercept after 4 weeks. Transepithelial electrical resistance was not altered. CONCLUSIONS: Long-term treatment with anti VEGF may affect viability of RPE cells, and treatment with bevacizumab may have effects on RPE function in long-term treatment.

Pro-inflammatory activation changes intracellular transport of bevacizumab in the retinal pigment epithelium in vitro.

PURPOSE: Bevacizumab 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. METHODS: ARPE-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-1ß, TNFα) secretion was investigated with ELISA. RESULTS: Poly 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. CONCLUSIONS: Pro-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.

Effect of long-term inflammation on viability and function of RPE cells.

PURPOSE: Degenerative ocular disorders like age-related macular degeneration (AMD) are associated with long-term pro-inflammatory signals on retinal pigment epithelial (RPE) cells. In this study, we investigated the effect of long term treatment of RPE cells with agonists of toll-like receptor (TLR) -3 (Polyinosinic:polycytidylic acid, Poly I:C), TLR-4 (lipopolysaccharide, LPS) and the pro-inflammatory cytokine TNFα. METHODS: All tests were conducted with primary porcine RPE. Cells were stimulated with Poly I:C (1, 10, 100 µg/ml), LPS (0.1, 1, 10 µg/ml) or TNFα (12.5, 25 or 50 ng/ml) for 1 day, 7 days or 4 weeks. Cell viability tests (MTT) were additionally tested in ARPE-19 cells. Cytokine secretion (IL-6, IL-1ß, IL-8, TNFα, TGF-ß) was tested in ELISA, phagocytosis in a microscopic assay, and expression of RPE65 in Western blot. Barrier function was tested in transwell-cultured cells by measuring transepithelial resistance for up to 3 days. RESULTS: LPS and TNFα significantly reduce cell viability after 1 day and 7 days, Poly I:C after 7 days and 4 weeks. LPS, Poly I:C and TNFα significantly induce the secretion of IL-6 and IL-8 at all tested time points. IL-1ß is increased by LPS and Poly I:C after 1 day, but not by TNFα. TNFα secretion is increased by Poly I:C and LPS after 1 day but not at later time points. TGF-ß secretion is not influenced by any stimulus. Concerning RPE function, LPS decreased phagocytosis after 7 days, while Poly I:C and TNFα showed no effect. RPE65 expression was strongly reduced by TNFα and LPS after 4 weeks. Wound healing capacity was reduced by Poly I:C but induced by LPS after 7 d and 4 w. Barrier function was not affected by Poly I:C or LPS, while TNFα reduced barrier function after 1 h, 4 h and 3 days. CONCLUSION: Long term pro-inflammatory stimuli reduce RPE viability, barrier properties and cellular function and induce pro-inflammatory cytokines and therefore may contribute directly to atrophic changes in AMD.