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Viren Govindaraju, Neeraj Vij; Cigarette Smoke induced autophagy-impairment regulates AMD pathogenesis mechanisms in RPE cells. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5238.
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Cigarette smoke and aging are leading risk factors for age-related macular degeneration (AMD) pathogenesis. Based on our recent studies, we hypothesize that cigarette smoke exposure and aging mediated proteostasis/autophagy-impairment serves as a central mechanism for AMD pathogenesis and autophagy-inducing drugs could mitigate these AMD pathogenesis mechanisms in retinal pigment epithelial (RPE) cells.
ARPE-19 cells were incubated with cigarette smoke extract (CSE), cysteamine (250µM) and/or fisetin (40µM). Ubiquitin (Ub) and SQSTM1/p62 expression and peri-nuclear co-localization was evaluated via western blot analysis and immunocytochemistry respectively to quantify changes in proteostasis/autophagy. CSE/drug induced changes in autophagy activity were quantifying by co-localization of LC3-GFP/Ub-RFP reporter. Cell proliferation was analyzed via MTS assay and reactive oxygen species (ROS) production was assessed via CMH2DCFDA-dye based assay. Cellular senescence was measured by counting SA-b-galactosidase positive (blue) cells under the light microscope.
Increases in insoluble ubiquitinated-proteins and p62 (impaired-autophagy marker) were observed on western blot in response to 5% (p<0.01-Ub, p<0.05-p62) or 10% (p<0.01-Ub, p<0.05-p62) CSE treatments, which was reduced with cysteamine (p<0.01-Ub) or fisetin (p<0.05-Ub) treatment. Next, we found that 10%-CSE treatment (p<0.05) induces peri-nuclear co-localization of p62 (red) and Ub (green) that was also ameliorated by cysteamine (p<0.05) or fisetin (p<0.05). Furthermore, CSE induced autophagy-impairment (p<0.01) was significantly decreased with cysteamine (p<0.05) or fisetin (p<0.05) treatment, which was quantified by a significant (p<0.05) decrease in LC3-GFP/Ub-RFP co-localization. In addition, cysteamine (p<0.05) or fisetin (p<0.001) were also shown to mitigate CSE induced ROS production (p<0.001). However, only cysteamine (p<0.05) rescued CSE impaired RPE cell proliferation (p<0.0001). Lastly, CSE induced RPE-cellular senescence (p<0.0001) that was ameliorated by cysteamine (p<0.0001) or fisetin (p<0.0001) treatment.
Cigarette smoke induced proteostasis/autophagy-impairment regulates mechanisms associated with AMD pathogenesis in RPE cells. Moreover, autophagy-inducing drugs such as cysteamine and fisetin can ameliorate AMD pathogenesis mechanisms that warrant further investigation in pre-clinical models.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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