Abstract
Purpose: :
To determine and characterize the differences in the binding of bevacizumab and ranibizumab to human RPE and human vascular endothelium cell membranes.
Methods: :
The expression of VEGF and the receptor for Fc (FcR), on human umbilical vein endothelial cells (HUVEC), ARPE-19 and primary human RPE (phRPE) cell membranes was determined under normoxic (21% O2) and hypoxic (10% O2/10 nM, PMA for 12 hours) conditions using Western blotting and immunohistochemistry. Effect of bevacizumab (625 µg/ml) and ranibizumab (125 µg/ml) on cell viability and complement activation was determined with FACS analysis. Confocal microscopy and iC3b ELISA was used to confirm the results. VEGF-expressing SKOV cells were used as a positive control.
Results: :
phRPE, ARPE-19 and HUVECs express VEGF on cell membrane; however Fc expression was observed only on phRPE and HUVEC. Hypoxia increases membrane-bound VEGF expression in HUVEC (2.3±0.6x) and RPE (2.6±0.4x) cells, but FcR expression remained unchanged. Bevacizumab was able to bind to HUVEC and RPE cell membranes resulting in RPE cell death (3.8%), whereas ranibizumab bound weakly on RPE without causing cell death. Bevacizumab binding to HUVEC cells was completely blocked by FcR-antibodies indicating that its binding is mediated through Fc-FcR interaction. Additionally, bevacizumab binding to RPE cells increased in hypoxia along with an increase in membrane-bound VEGF expression.
Conclusions: :
Bevacizumab binds to different ligands on HUVEC (FcR) and human RPE (membrane-bound VEGF) cells. Binding of bevacizumab is associated with complement activation and cell death. In contrast, Ranibizumab can only weakly bind to RPE and does not activate complement
Keywords: age-related macular degeneration • receptors: pharmacology/physiology • retinal pigment epithelium