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X. Liu, M. Zeng, Y. Liu; Effects of Conditioned Media from Human Retinal Cell Cultures Incubated under Hypoxic Conditions on Circulating Endothelial Progenitor Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1766.
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The use of genetically modified EPCs expressing antiangiogenic factors has been proposed as a potential therapeutic tool for the treatment of diabetic retinopathy. Circulating EPCs may provide a practical source for autologous transplants. However, the specific signaling events by which circulating EPC populations may sense and respond to alterations of the retinal vasculature induced by hypoxia are not yet completely understood. Our goal was to generate a suitable experimental model for the identification of such factors using human retinal and EPC cells.
Primary cultures of human retinal cells were incubated under hypoxic (5% O2) or normoxic (21% O2) conditions for 48 hours. Effects of hypoxia were confirmed by analysis of VEGF induction using ELISA. Circulating EPCs (CD34/CD133 double–positive cells) were isolated by FACS from the human periphery blood. Enriched EPCs were treated with media from retinal cells incubated under either hypoxic or normoxic conditions. Effects of conditioned media on proliferation and differentation of EPCs were evaluated by a colorimetric assay (WST–1, Chiemcon) and staining with Ulex europaeus–lectin and Dil–LDL uptake assay.
Incubation of human retinal cells at low oxygen concentration lead to a significant increase of VEGF released to the media of 32.5% (319.16±17.14 pg/ml vs 241.95 ±18.38 pg/ml, p<0.01) compared to the control cultures incubated at normoxic conditions. Treatment of EPCs with conditioned medium from retinal cells incubated at low oxygen concentration resulted in an increase of proliferative activity (0.83±0.1 vs0.61±0.22,p<0.05) and presence of differentiated endothelila cells (29±5 cells/mm2 vs 18±8/mm2, p<0.05) compared to conditioned media from normoxic cultures
Conditioned media from retinal cells contains factor capable of activating proliferation and differentiation of circulating EPCs. Analysis of the effects of conditioned media from neuronal cells incubated under hypoxic conditions on circulating human EPCs might be useful in providing effective experimental means for the identification of the specific signaling molecules involved in the activation of circulating EPCs for the regeneration of retinal blood vessels.
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