Objective: The specific signaling events by which circulating endothelial progenitor cells (EPCs) responding 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 as well as the effects of these factors on the EPCs.
Methods: Primary cultures of human retinal glial cells were incubated under hypoxic or normoxic conditions. Effects of hypoxia were confirmed by analysis of HIF-lalpha and VEGF expression. Circulating EPCs (CD34 +, CD133+ double-positive cells) were isolated by FACS Vantage from the human periphery blood. EPCs were treated with conditioned media from retinal glial cells incubated under either hypoxic or normoxic conditions. Effects of conditioned media on the proliferation and differentiation of EPCs were evaluated.
Results: Incubation of human retinal glial cells at low oxygen concentration for 24 hours lead to HIF-lalpha expression and a significant increase of VEGF released to the media[ (319. 16 +/-34. 12) pg/ml versus (220. 28+/-24. 33) pg/ml] as compared to the control cultures incubated at normoxic conditions (P <0. 01). Treatment of EPCs with conditioned medium from retinal glial cells incubated at low oxygen concentration resulted in an increase of proliferative activity (day 3: 0. 83+/-0. 10 versus 0. 61+/-0. 22,P <0. 05; day 7: 1. 53+/-0. 21 versus 0. 88+/-0. 25, P <0. 05) and presence of differentiated endothelial cells as compared to cells cultured with conditioned media from normoxic cultures.
Conclusions: Conditioned media from retinal glial cells contain factors capable of activating the proliferation and differentiation of circulating EPCs. Analysis of the effects of conditioned medium from neuronal glial cells incubated under hypoxic conditions on circulating human EPCs might be useful in providing an effective experimental method for the identification of the specific signaling molecules involved in the activation of circulating EPCs for the regeneration of retinal blood vessels.