Abstract
Purpose:
Oxidative stress is hypothesized to contribute to RPE apoptosis in Age Macular Degeneration (AMD). CS a strong oxidant and the strongest risk factor for AMD, yet how smoking affects the RPE is unclear. Nrf2 is a transcriptional factor that controls a cascade of antioxidant genes and could protect the RPE from CS, but it decreases with age and chronic oxidative stress. The purpose herein was to determine the effect of CS and Nrf2 deficiency on RPE cellular function and apoptosis.
Methods:
ARPE-19 cells were treated with CS extract (Murty Pharmaceutical) for 24h and then Nrf2 or control siRNA(Applied Biosystems). Viability was determined by MTS. Total RNA and protein were extracted and used for microarray, RT-qPCR and Western blot analysis.
Results:
Cells exposed to 100-500 ug/ml CS for 24h remained viable. Since microarray analysis of cells exposed to 100-250 ug/ml CS showed an increase in the antioxidant response, unfolded protein response (UPR), and mitochondrial function, we explored the extent of each pathway’s contribution to cellular dysfunction. Despite a CS induced increase in antioxidant gene expression, we observed a dose dependent increase in superoxide (p≤ 0.01) and protein carbonylation, suggestive of oxidative damage. CS induced the UPR (IRE-1a, PERK, ATF6; p≤0.05), CHOP was increased by 500ug/ml CS (p≤0.05), but the pro-apoptotic ER resident caspase 4 was not induced. When evaluating mitochondrial function, we found a dose dependent decrease in ATP production (p≤0.01), but no release of cytochrome C. We conclude that CS induces oxidative damage and impaired mitochondrial function, but the dysfunction was not sufficient to cause apoptosis. We next knocked down (KD) Nrf2 and found that CS decreased cell viability by 20% (100ug/ml) to 32% (500ug/ml CS; p≤0.01). Superoxide production was magnified with Nrf2 KD (p≤0.05). The UPR was increased (P≤0.05), but caspase-4 was not altered. Nrf2 KD with CS further decreased ATP production, and promoted cytochrome C release, indicative of intrinsic apoptosis activation.
Conclusions:
CS generates a protective antioxidant and UP Responses. Impaired Nrf2 signaling, such as that associated with age or chronic CS, decreases the antioxidant response and generates mitochondrial dysfunction, leading to intrinsic apoptosis. Further studies will determine the impact of mitochondrial damage in dry AMD.
Keywords: 412 age-related macular degeneration •
424 antioxidants •
701 retinal pigment epithelium