Effect Of Low Oxygen Culture On Secretion Of Trophic And Angiogenic Factors By RPE Cells.

Abstract

Purpose: Age-related macular degeneration (AMD) is the leading cause of blindness affecting people over 65 years old. The “dry” form results from atrophy of the retinal pigment epithelium (RPE). Regenerative medicine aims at restoring this layer using in vitro cell culture. In vivo, the RPE is exposed to 4.5-11% of oxygen. However, RPE cell culture is usually performed under atmospheric conditions (21% O2). This study was undertaken in order to evaluate the influence of low oxygen level on the expression of secreted trophic and angiogenic factors by RPE cells.

Methods: Primary cultures of RPE cells were established from human donor globes and cultured in DMEM-Ham’s F12 (3:1) medium supplemented with 10% serum and antibiotics under physiological (<5% O2) or atmospheric (21% O2) oxygen conditions. When the cells reached confluency, they were grown without serum and the conditioned medium was collected after 48h. Trophic factors secreted by RPE cells were analyzed using proteome profiler array kits (human cytokines and angiogenesis arrays). Scanned films were analyzed using ImageJ software.

Results: A higher secretion of IL-6, IL-8/CXCL8, C5/C5a, slCAM-1/CD54, INF-gamma, IL-23, MCP1/CCL2, TSP-1, IGFBP-3, PAI-1, PEDF and VEGF was observed in RPE cells cultured at low oxygen level. Inversely, this condition resulted in lower protein expression of CXCL16, Endothelin-1, uPA and PDGF-AA.

Conclusions: This study showed a different secretome in RPE cells grown under physiological oxygen conditions. Further analysis will be necessary to understand the extent of those changes on RPE phenotype as well as on other retinal cell types in order to develop a regenerative therapy for dry AMD.

Keywords: 701 retinal pigment epithelium • 635 oxygen • 490 cytokines/chemokines