April 2010
Volume 51, Issue 13
ARVO Annual Meeting Abstract  |   April 2010
Nrf2 Signaling is Activated After Cigarette Smoke Extract Exposure in Rpe Cells in vitro and in vivo
Author Affiliations & Notes
  • N. Kondo
    Ophthalmology, Johns Hopkins University, Baltimore, Maryland
  • M. Cano
    Ophthalmology, Johns Hopkins University, Baltimore, Maryland
  • K. Ebrahimi
    Ophthalmology, Johns Hopkins University, Baltimore, Maryland
  • J. Handa
    Ophthalmology, Johns Hopkins University, Baltimore, Maryland
  • Footnotes
    Commercial Relationships  N. Kondo, None; M. Cano, None; K. Ebrahimi, None; J. Handa, None.
  • Footnotes
    Support  RPB R01 EY019904; the Robert Bond Welch Professorship
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 4104. doi:
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      N. Kondo, M. Cano, K. Ebrahimi, J. Handa; Nrf2 Signaling is Activated After Cigarette Smoke Extract Exposure in Rpe Cells in vitro and in vivo. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4104.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Cigarette smoking (CS) is a powerful chemical oxidant and a strong epidemiologic risk factor for the development of Age-related Macular Degeneration (AMD). Previously, we showed that CS induces phenotypic changes to the retinal pigmented epithelium (RPE) in a mouse model. Nrf2 is a transcriptional factor that controls a cascade of antioxidant genes, and could protect the RPE from CS induced oxidative insult. We hypothesize that Nrf2 will be activated upon CS by RPE as apart of the cytoprotective response. The purpose of this project is to examine the Nrf2 cytoprotective response by the RPE after CS exposure.

Methods: : ARPE-19 cells were exposed to cigarette smoke extract for 24 hours. Nrf2 responsiveness was evaluated by nuclear translocation of Nrf2 protein by western analysis, Nrf2 response gene expression by RT-qPCR, and complement activation by western analysis. Nrf2+/+ and Nrf2-/- mice were exposed to CS for 1-21 days. The RPE/choroid was extracted and analyzed by RT-qPCR for Nrf2 responsive gene expression.

Results: : Using the MTS viability assay, visually confluent ARPE-19 cells exposed to 0-250mcg/ml CSE for up to 24 hours showed no evidence of toxicity while 500-1000mcg/ml CSE caused a 20% and 80% reduction, respectively, in viability compared to DMSO control cultures. To demonstrate Nrf2 responsiveness in human RPE cells, Western blot of cells exposed to 100 and 250 ng/ml CS Extract for 18 hours showed a 2.25- and 2.50-fold increase, respectively, in nuclear Nrf2 protein compared to vehicle treated cells (p<0.01 for each dose of CS extract vs. vehicle). At this time point, a dose response effect of CS on Nrf2 responsive gene expression (NQO1, HO-1, and GCLM) was also observed (ANOVA, p<0.001 compared to control, and 100 mcg/ml vs 250 mcg/ml; expression was normalized to β-actin.). CS can influence the complement cascade. Using similar culture conditions, while CSE 250mcg/ml did not induce C3 by Western analysis, CD46, an inhibitor of C3, was reduced by 2-fold compared to untreated controls. Nrf2+/+ mice (n=3 ) exposed for either 1 or 21 days of CS, had induction of NQO-1 and GCLM in the RPE/choroid compared to mice raised in air. There was no induction in Nrf2-/- mice raised in CS or air.

Conclusions: : RPE cells in vitro and in vivo show evidenc e of Nrf2 nuclear translocation and is associated with increased expression of antioxidant genes. This acute response could be an important protective response by RPE cells to CS exposure.

Keywords: age-related macular degeneration 

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