Abstract
Purpose: :
The over expression of the serpin anti-angiogenic neurotrophic Pigment Epithelium Derived Factor (PEDF) protects photoreceptors from light damage, and ARPE-19 cells from H2O2-induced apoptosis. We have shown that PEDF is an agonist of docosahexaenoic acid (DHA)-derived neuroprotectin D1 (NPD1) synthesis, and that this docosanoid, in turn, elicits potent anti-inflammatory and anti-apoptotic actions. Therefore, the purpose of this study was to determine if PEDF’s mediated protection, which is known to engage NPD1, persists after removal of PEDF treatment, and if DHA supplementation potentiates this bioactivity.
Methods: :
ARPE-19 cells where cultured for 72 h to achieve ~100% confluencey. Cells were serum starved for 18 hours in 0.5% FCS, DMEM/F12 1:1 media, after which 50 ng/ml PEDF, 200nM DHA + 50ng/ml PEDF, or ETOH (equal volume) was added to the media. After 6 hours of incubation, media was replaced with fresh 0.5% FCS, DMEM/F12 1:1 media for 24h. Cells were then challenged with 675, 700, and 725 µM H2O2 for 16 hours. Cells were fixed with neutral buffered formalin and stained with Hoestch 33258 in 1% triton X100. Cells were counted using an automated unbiased image analysis of nuclear morphology. Cell death was determined via nuclear chromatin condensation (µm2).
Results: :
Control cells display a survival level of 92 ± 2%. Cells treated with 675, 700, and 725µM H2O2 for 16 hours resulted in nuclear chromatin condensation and a cell survival level of 87± 4%, 45 ± 5, and 42%±9%, respectively. Cells pretreated with 50ng/ml PEDF for 6 hours, then DMEM/F12 for 24 hours prior to stress, exhibit protection with a survival level of 93 ± 6%, 90% ± 7%, and 50%±12%, respectively. Cells pretreated with PEDF plus 200nM DHA show protection with a survival level of 97±2%, 94±3%, and 74±10%, respectively.
Conclusions: :
Our results demonstrate that PEDF pretreatment elicits protection of human-ARPE-19 cells from oxidative stress-induced cell death up to 24 hours post attenuation of the treatment with PEDF, and when DHA is supplemented simultaneously this protection is bolstered. This data suggests protection via DHA-derived NPD1 synthesis. Thus, application of DHA/NPD1 and/or PEDF could be used as a potential therapeutic approach for the prevention or attenuation of the initiation or early progression of retinal degeneration.
Keywords: neuroprotection • retinal pigment epithelium