Abstract
Purpose: :
To determine the influence of n-acetylcysteine (NAC) on the production of Reactive Oxygen Species (ROS) by CECs following VEGF exposure.
Methods: :
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% FBS. For reactive oxygen species (ROS) production assays, CECs were plated on 96-well plates at a concentration of 3,000 cells per well and allowed to grow in complete growth media (EGM-2) to 80% confluency. Prior to running the ROS assay, cells were pre-treated for 6 hours with EGM-2 media supplemented with 25 mM of n-acetylcysteine (NAC). The ROS production was detected with a 5-(and-6)-chloromethyl-2',7'-dichlorodihydro-fluorescein diacetate, acetyl ester (DCF-DA) based assay (Invitrogen, Carlsbad, CA). 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 CECs. Following a 30 min incubation with 100 µL of serum free EBM-2 media, cells were incubated for 15 minutes with 10, 50, or 100 ng/ml of VEGF165 to induce ROS production. As a control, cells were incubated with EGM-2 media supplemented with 200 µM hydrogen peroxide to serve as an exogenous source of ROS. Fluorescent measurements were taken on a Wallac 1420 VICTOR2 plate reader using an excitation wavelength of 485 nm and a reading wavelength of 520 nm. Background fluorescence, as determined by cells untreated with the DCF-DA reagent, was subtracted from all data points.
Results: :
There was an 85% reduction in ROS for cells treated with 25 mM of NAC compared to untreated controls. CECs incubated with 10, 50 and 100 ng/ml of VEGF for 15 minutes displayed a 53%, 61% and 74% increase in ROS, respectively (p<.03). When incubated with VEGF and 25 mM of NAC, the increase in ROS was reduced by 63%, 44%, and 47% (p<.02)
Keywords: choroid • vascular endothelial growth factor • antioxidants