Direct/indirect protective effects elicited by anti-vascular endothelial growth factor drugs on retinal pigment epithelium cells (ARPE-19 cell line)

The Age-Related Macular Degeneration (AMD) is the leading cause of irreversible loss of vision. In the mechanisms of action of the anti-VEGF agents the involvement of nitric oxide (NO), the mitochondria function and apoptosis have not been examined. In the present study, in order to analyze the cross-talk between retinal pigment epithelium (RPE; ARPE-19) cells and endothelial cells, we conducted co-culture experiments between RPE and human umbilical vascular endothelial cells (HUVEC). RPE and HUVEC/RPE co-culture, were exposed to Ranibizumab/Aflibercept in the absence/presence of NO synthase (NOS) inhibitor, phosphatidylinositol 3′-kinase (PI3K), extracellular signal regulated kinases 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (p38 MAPK) blockers. Specific kits were used for cell viability, NO, ROS detection, apoptosis and mitochondrial membrane potential measurement. Western blot was performed for apoptosis markers, NOS isoforms, and other kinases detection. Cell migration was measured by wound healing assay and cell proliferation was analyzed by using xCELLigence. In RPE alone or in co-culture with HUVEC, Aflibercept/Ranibizumab increased NO release in a dose and time-dependent way, in physiologic conditions. Opposite results were obtained in RPE pretreated with hydrogen peroxide. Both anti-VEGF drugs were able to prevent the fall of cell viability and of mitochondrial membrane potential. Those effects were reduced by inhibitors of NOS, PI3K, ERK1/2 and p38MAPK. Both drugs were able to increase cell viability and migration. Finally, Aflibercept/Ranibizumab counteracted the changes of apoptosis markers, NOS expression/activation, protein kinase B (PKB or Akt) and ERK1/2 activation caused by peroxidation. This study has shown for the first time new mechanisms involving NO and mitochondria, in the actions of Aflibercept/Ranibizumab in RPE.