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G. Byfield, A. Parker, S. Budd, M. Hartnett; In the Presence of VEGF, Choroidal Endothelial Cells Increase Production of Reactive Oxygen Species in a Dose and Time Dependent Manner. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1187.
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© ARVO (1962-2015); The Authors (2016-present)
To determine the effect of VEGF165 on the production of ROS by CECs.
Primary human choroidal endothelial cells (CECs) were isolated from donor eyes obtained from the North Carolina Eye Bank. CECs were grown in endothelial growth media (EGM-2) supplemented with 10% fetal bovine serum (FBS). For reactive oxygen specie (ROS) production assays, CECs were plated (3,000/well) on 96-well plates and allowed to grow to 80% confluency. ROS production was detected with a 5-(and-6)-chloromethyl-2',7'-dichlorodihydro-fluorescein diacetate, acetyl ester (DCF-DA) based assay (Invitrogen, Carlsbad, CA). Briefly, cells were incubated with 100 µL of the EBM-2/DCF (5 µM) solution for 10 minutes to allow the ROS indicator to permeate the cells. This was followed by a 30 min incubation with 100 µL of media, and vascular endothelial cell growth factor 165 (VEGF165) to induce ROS production. Different concentrations and time points for VEGF165 incubation were used. Positive controls for ROS production were cells incubated with EGM-2 media supplemented with 200 µM hydrogen peroxide. Fluorescence was measured using an excitation wavelength of 485 nm and a reading wavelength of 520 nm. Background fluorescence, as determined by cells not incubated with the DCF-DA reagent and was subtracted from all data points. Subsequently, fluorescence and thus ROS production were set relative to untreated cells (ROS production: 1.00)
In comparison to untreated cells, CECs incubated with 10 ng/ml VEGF had a 53% increase in ROS production. Cells incubated in higher concentrations of VEGF (30 ng/ml or 45 ng/ml) produced more ROS (61% and 74% respectively) compared to controls. There was also a 2-fold or higher significant increase in ROS production (p<0.01) following the incubation of cells with VEGF from 30 to 60 minutes.
Addition of VEGF to cells incubated with growth media showed a dose dependent increase of ROS production. These data provide evidence of a connection between the role of VEGF and production of oxidative compounds which has been proposed in the development of AMD.
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