Abstract
Purpose :
Antagonists of Vascular Endothelial Growth Factor (VEGF) are repeatedly intravitreally injected for elevated periods, e.g. for treatment of age-related macular degeneration. Aim of the study was to investigate effects of repetitive long-term stimulation with VEGF-antagonists on RPE cells in vitro, focussing on premature aging.
Methods :
Primary porcine RPE cells were treated up to 12 weeks with weekly doses of 125 µg/ml ranibizumab, bevacizumab, aflibercept or CD20-antibody rituximab. Toxicity was evaluated with MTT and trypan blue exclusion assay, activity of ß-galaktosidase in a commercial assay, expression of mTOR, phospho-mTor, ß-amyloid and Cathepsin D in Western blot. Area and number of mitochondria and autophagosomes were assessed in electron microscopy. Statistical analysis was conducted using a mixed linear model (assessment of organelles), and a student’s t-test (toxicity, galactosidase assay, Western blots).
Results :
Weekly treatment up to 12 weeks displayed no toxic effects. Long-term treatment reduced ß-galactosidase activity in cells treated with aflibercept or rituximab (both p<0.05). A trend toward reduction was also seen after ranibizumab (p=0.061) and bevacizumab (p=0.052) treatment. No changes in expression of any tested protein was found after long-term treatment (4 weeks, 12 weeks), except for a slight reduction of Cathepsin expression in rituximab treated cells. Ultrastructural analysis showed no alterations in mitochondria after long-term treatment with any substance. However, area of autophagosomes in bevacizumab and aflibercept treated cells was significantly reduced compared to both ranibizumab (p<0.05 each) and rituximab (p< 0.05 for bevacizumab and p<0.01 for aflibercept).
Conclusions :
Weekly treatment with VEGF-antagonists up to 3 months does not induce premature aging in primary RPE cells. However, a significant difference can be found between bevacizumab and aflibercept on the one hand, and ranibizumab on the other hand, with less autophagosomal area in bevacizumab and aflibercept. This is consistent with our findings on reduced phagocytic activity in aflibercept and bevacizumab, but not ranibizumab, treated RPE cells. The influence of these compounds on degradative pathways should be further investigated.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.