Abstract
Purpose :
To investigate how nitric oxide (NO) release and mitochondrial function contribute to the protective action of anti-VEGF (Ranibizumab and Aflibercept) on retinal pigmented epithelium (RPE), performing co-culture experiments between RPE and umbilical vascular endothelial cells (HUVEC)
Methods :
Knowing that RPE and HUVEC cross-talk is of fundamental relevance for the maintenance of outer retina structure and function, co-cultures RPE/HUVEC were performed and exposed to Ranibizumab/Aflibercept in the absence or presence of NO synthase (NOS) inhibitor and phosphatidylinositol-3-kinase/protein kinase B (PKB), extracellular-signal-regulated kinases 1/2 (ERK1/2) and p38 mitogen activated protein kinase (p38 MAPK) blockers. Following intra and inter-cellular mechanisms were examined: NO release, cellular proliferation/migration, cell viability, mitochondrial membrane potential, apoptosis and PKB, p38 MAPK and ERK1/2 expression/activation. Specific kits were used for cell viability, NO and reactive oxygen species detection, apoptosis and mitochondrial membrane potential measurement. Western Blot was performed for detection of apoptotic markers and other kinases. Cell migration was measured by wound healing assay and cell proliferation using XCELLigence.
Results :
In physiologic conditions, Aflibercept/Ranibizumab increased NO release in a dose and time-dependent way in RPE cells co-cultured with HUVEC. Opposite results were obtained in RPE cells pretreated with hydrogen peroxide. Both anti-VEGF prevented the fall of cell viability and of mitochondrial membrane potential and increased cell migration. Those actions were inhibited in presence of specific blockers. Aflibercept/Ranibizumab counteracted the changes of apoptotic markers, NOS, PKB and ERK1/2 caused by peroxidation.
Conclusions :
In the present study, the potential role of NO and mitochondria in the protective action of Aflibercept/Ranibizumab on RPE was described. In particular, after stimulation with both anti-VEGF, HUVEC seem to release paracrine factors involved in RPE response to anti-VEGF.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.