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Ana M. de Lucas-Cerrillo, Jena J. Steinle, Tonia Rex; The Effect Of Epo On Human Retinal Endothelial Cell Migration, Proliferation, And Apoptosis. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3002. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To determine if EPO or EPO-R76E induce angiogenesis in primary human retinal endothelial cells (RECs) in vitro.
Serum deprived HEK293 cells were transfected with plasmids containing wild type EPO (EPO) or EPO-R76E. The medium was collected and concentrated, and EPO or EPO-R76E was quantified by ELISA and used in the cell death, proliferation and migration assays. REC death was induced by starving cells overnight in the presence or absence of 0.5 to 5 U/ml EPO or EPO-R76E beginning 6 hours prior to serum deprivation. The Roche Cell Death Detection ELISA-Plus kit was used according to manufactures protocol. REC proliferation was assessed using the Millipore Cell Proliferation Assay Kit. RECs were plated in a 96-well plate, starved overnight, and incubated with: no serum, 10% serum, or 0.5 to 5 U/ml EPO or EPO-R76E. Absorbance was measured at 24 and 48 hr following drug treatments. Cell migration was assayed using the BD BioCoat Angiogenesis System Endothelial Cell Invasion kit. RECs were plated in the top chambers and the bottom chambers contained media with: no serum, 10% serum, or 0.5 to 5 U/ml EPO or EPO-R76E. Plates were incubated 24 hr at 37°C and then labeled with Calcein AM. Fluorescence was read at 485/528 nm.
Serum deprivation induced a 5-fold increase in cell death as compared to the positive control, this was unchanged by treatment with 0.5 to 5U/ml EPO or 0.5U/ml EPO-R76E. Treatment with 1.5 or 5U/ml EPO-R76E induced a 4-fold increase in cell death compared to the positive control, however this decrease was not statistically significant. Serum deprivation inhibited cell proliferation and addition of EPO or EPO-R76E had no effect at any dose or time point. Serum treatment induced migration of the RECs. No migration was detected in serum-deprived RECs with or without treatment of EPO or EPO-R76E at any dose tested.
Our results indicate that neither EPO nor EPO-R76E induce angiogenesis in vitro in serum-deprived RECs. There was a trend toward a slight anti-apoptotic effect in cells treated with EPO-R76E, but more experiments need to be performed to determine if this was a real effect. Additional future studies will assess the ability of EPO and EPO-R76E to induce angiogenesis in the presence of 1% serum.
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