Abstract
Purpose: :
Cigarette smoke (CS), through oxidative damage, is the strongest risk factor for AMD. Nitric Oxide (NO), a major component of CS, is itself, a potent oxidant. NO is a key mediator for several physiologic processes, including activation of the transcription factor Nrf-2, which regulates a cytoprotective transcriptional program against oxidative and nitrosative damage. NO from either CS or neural nitric oxide synthase (nNOS), which is increased in the RPE in AMD, could potentially injure the RPE cell if it is not neutralized by the cell’s cytoprotective response. The response by the Nrf2 system in RPE cells on NO in CS or nNOS is unknown, and was therefore tested in this study.
Methods: :
NO was measured in ARPE-19 cells after treatment with 50-250 ug/mL Cigarette smoke condensate (CSE) or 100uM SNAP, a NO donor, using a DAF-2DA fluorescent assay. Using two novel polymeric nanoparticles which encapsulate the plasmid (2-methylpentane-1,5-diamine terminated and (PEO)4-bisamine terminated poly(1,4- butanediol diacrylate-co-5-amino-1-pentanol) denoted 454 and 455) or FuGeneHD, ARPE-19 cells were transfected with 3 ug of pcDNA 2.1 nNOS FL Express Tag HIS plasmid or GFP (control) plasmid for 4h. After 24-48h, total RNA was extracted, reverse transcribed, and assessed for expression of Nrf2 and two Nrf2 responsive genes, GCLM and NQO1, using Taqman RT-qPCR.
Results: :
CSE increased NO in a dose dependent manner (all conditions, p<0.001), CSE 50ug/mL (5.2 fold at 1h, 9.6 fold at 4h, and 12.1 fold at 6h), 125ug/mL (5.9 fold at 1h, 11 fold at 4h, 14.5 fold at 6h), 250 ug/mL(7.2 fold at 1h, 12 fold at 4h, 16.1 fold at 6h) and SNAP 100uM (4.3 fold at 1h, 8.3 fold at 4h, 10.7 fold at 6h). nNOS transfected cells showed an increase in NO after 24 h: nNOS 454 (2.1 fold, p=0.003), nNOS 455 (2.3 fold, p=0.01) and nNOS fugene (2.2 fold, p=0.01). RPE cells overexpressing nNOS at 24h showed increase expression of NQO1 when transfected with nNOS 454 (vs. untreated p=0.04, vs. GFP454 p=0.06), nNOS 455 (vs untreated p<0.02, vs. GFP455 p<0.02) and GCLM when transfected with nNOS 454 (vs. untreated p<0.02). RPE cells overexpressing nNOS at 48h showed increased NQO1 when transfected with nNOS 454 (vs. untreated p=0.05) and nNOS 455 (vs. GFP 455 p<0.02), GCLM when transfected with nNOS 454 (vs. untreated p=0.02) and Nrf2 when transfected with nNOS 455 (vs GFP455 p=0.02).
Conclusions: :
NO induced Nrf2, NQO1, and GCLM in RPE cells. These changes suggest that the Nrf2 pathway is important for protecting from NO related stress from either an endogenous (nNOS) or exogenous source (CS).