Effects of bevacizumab, ranibizumab, and aflibercept on phagocytic properties in human RPE cybrids with AMD versus normal mitochondria.

ABSTRACT

PURPOSE: A critical biological function of retina pigment epithelium (RPE) cells is phagocytosis of photoreceptor outer segment (POS) disc membranes. Mitochondrial damage and dysfunction are associated with RPE cells of age-related macular degeneration (AMD) retinas. In this study, we use a transmitochondrial cybrid model to compare the phagocytic properties of RPE cells that contain AMD mitochondria versus age-matched normal mitochondria and their response to treatment with anti-vascular endothelial growth factor (VEGF) drugs bevacizumab, ranibizumab, and aflibercept. METHODS: Cybrids, which are cell lines with identical nuclei but mitochondria (mt) from different subjects, are created by fusing mtDNA depleted ARPE-19 cells with platelets from AMD or age-matched normal patients. AMD (n = 5) and normal (n = 5) cybrids were treated with 1 µm fluorescent latex beads (1.52 × 107 beads/mL) and either 2.09 µM of bevacizumab, 2.59 µM of ranibizumab, or 5.16 µM of aflibercept. These doses of anti-VEGF drugs are equivalent to intravitreal injections given to AMD patients with choroidal neovascularization. Flow cytometry was performed using the ImageStreamX Mark II to assess phagocytic bead-uptake. The average fold values for bead-uptake and SEM were calculated using GraphPad Prism software. RESULTS: Normal cybrids showed decreased bead-uptake with a fold value of 0.65 ±â€¯0.103 (p = 0.01) after treatment with bevacizumab, 0.80 ±â€¯0.034 (p = 0.0003) with ranibizumab, and 0.81 ±â€¯0.053 (p = 0.007) with aflibercept compared to the untreated normal cybrids (baseline fold of 1). The bevacizumab-treated, ranibizumab-treated, and aflibercept-treated AMD cybrids had decreased bead-uptake with a fold value of 0.71 ±â€¯0.061 (p = 0.001), 0.70 ±â€¯0.101 (p = 0.02), and 0.74 ±â€¯0.125 (p = 0.07), respectively, compared to the untreated AMD cybrids (baseline fold of 1). CONCLUSIONS: Our initial findings showed that when treated with bevacizumab and ranibizumab, both AMD cybrids and age-matched normal cybrids had a significant decrease in bead-uptake. A similar decrease in bead-uptake was found in normal cybrids treated with aflibercept and while the AMD values trended lower, they were not significant. This data suggests that anti-VEGF drugs can cause loss of phagocytic function.