Abstract
Purpose :
The efficacy in decreasing vascularization and macular edema with vascular endothelial growth factor (VEGF) inhibitors is unquestionable. Despite their beneficial aspects, neuronal cell death, fibrosis and atrophy in retina have been reported. Müller cell derived-VEGF plays an essential role in retinal neurons and glial cell maintenance. We therefore evaluated the effects of aflibercept (AFL) and bevacizumab (BVZ) on Müller cell death, autophagy and structural-related proteins, as well as in cell contraction under normoxia and hypoxia conditions.
Methods :
The human Müller cell-line MIO-M1 (license agreement with UCLB-XIP, London, UK; Limb et al 2002) was subjected to the following treatments for 24, 48, or 72 hours: a) only culture media; b) 400 µM cobalt chloride (CoCl2); c) AFL or BVZ 0.5 mg/mL; d) 400 µM CoCl2+ AFL or BVZ 0.5 mg/mL. Immunofluorescence and Western Blotting analysis were performed to detect the level of caspase-3 (CASP3, apoptosis), beclin-1 (BECN1, autophagy), glutathione synthetase (GS, glial cell marker), glial fibrillary acidic protein (GFAP, gliosis and cytoskeletal) and alpha smooth muscle actin (α-SMA, cytoskeletal). Collagen gel contraction assay was also performed.
Results :
Hypoxia increased BECN1, and had little effect in CASP3 levels. An upregulation of CASP3 was observed in BVZ-treated cells under normoxia and hypoxia in short-term exposure. Hypoxia decreased GS levels, and no changes were detected with anti-VEGFs alone. However, GS reduction in cells under long-term hypoxia was not fully restored by anti-VEGF treatments. Although long-term treatments with both anti-VEGFs in cells under normoxia increased GFAP levels, they were not able to restore their levels under hypoxia, which marked decreased GFAP and α-SMA. Hypoxia or anti-VEGF treatments did not affect Müller cell contraction.
Conclusions :
BVZ, but not AFL, induced glial cell death in short-term exposure. Both AFL and BVZ seem to induce glia activation in MIO-M1 cells under normoxic condition in long-term exposure. This could be related to fibrosis and atrophy emergence in the retina of patients along the anti-VEGF treatments in some retinal diseases. Hypoxia can alter the microenvironment of the retina, rendering it unable to respond to the effects of anti-VEGFs.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.