Abstract
Purpose :
Dry age-related macular degeneration (AMD) is a leading cause of blindness in the world, with no effective treatment. Cigarette smoke is the leading environmental risk factor for AMD and is an external toxin that triggers autophagy in the RPE. While autophagy-modulated processes, including epithelial-mesenchymal transition (EMT) and cell death, have been observed in AMD RPE genetic models, the role environmental factors may play in contributing to both EMT and cell death is not well understood. Here we aim to investigate the effect of varying cigarette smoke extract (CSE) dosages on autophagic flux in RPE cells, through using autophagy modulators LC3 and P62, EMT transcription factors ZEB1 and SNAIL1, and calcein/ethidium homodimer-1 cell viability assay staining.
Methods :
To evaluate the extent of autophagic flux in CSE-treated cells, ARPE-19 cells were treated with low (100ug/ml), medium (250 ug/ml), or high (500 ug/ml) dose CSE for 4 hours. Cells were harvested for RNA and protein that were used for RT-qPCR and Western blot analysis, respectively, using autophagy markers LC3 and P62. To evaluate the effect of CSE on inducing EMT or cell death in CSE-treated ARPE-19 cells, EMT levels were measured using RT-qPCR for EMT TFs ZEB1 and SNAIL1; cell viability was evaluated using calcein/ethidium homodimer-1 cell viability assay staining.
Results :
LC3B-II was significantly increased as CSE dosage increased, indicating a dose-dependent relationship between CSE dose and LC3-modulated autophagy. P62 significantly increased from 0 ug/ml to 100 ug/ml and significantly decreased as CSE dosage increased from 250 ug/ul to 500 ug/ul. SNAIL1 and ZEB1 expression increased from 0 ug/ml to 250 ug/ml CSE, but significantly decreased at 500 ug/ml, suggesting cells adopted EMT at lower dosages before unable to evade cell death at higher dosages. Cells treated with 100 ug/ml and 250 ug/ml CSE showed elongated morphology indicative of Type II EMT, while 500 ug/ml CSE-treated cells showed less elongation and a higher number of dead cells as indicated by the viability assay.
Conclusions :
CSE treatment at lower dosages induced lower levels of autophagic flux and EMT while higher CSE dosages induced greater autophagic flux and cell death. These results expand potential for development of novel treatment interventions providing personalized care through stratification of patients by smoking level.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.