Abstract
Purpose :
Electronic cigarettes (e-cigs), battery-powered devices that produce aerosols by heating e-liquid solution, are a popular alternative to smoking. It is well-established that smoking is a major modifiable risk factor for age-related macular degeneration (AMD). Notably, like cigarette smoke, e-cig vapor exposure stimulates pro-inflammatory and angiogenic responses in the mouse retina. Based on these findings, our purpose in this study was to utilize induced pluripotent stem cell (iPSC)-based disease modeling studies and evaluate the direct relevance of e-cig vapor for AMD pathology development.
Methods :
Parallel cultures of control iPSC-derived retinal pigment epithelium (RPE) cells grown as polarized monolayer in transwell inserts were supplemented daily with either varied concentrations of freshly prepared e-cig vapor extract (ECVE, 1%, 5% or 10%) or vehicle (cell culture media;untreated) for 14 days and evaluated longitudinally for cell viability (Calcein AM), and epithelial barrier integrity (transepithelial measurements). At the end of the treatment duration (day 14), quantitative real-time PCR and Western blotting was used to assess expression/levels of inflammation-related genes/proteins (e.g., C3, IL-6). Furthermore, rate of phagocytosis of photoreceptor outer segment (POS) post-feeding of POS (~20 POS/RPE cell) for 2h was determined by measuring levels of RHO, a POS-specific protein, by using Western blotting. Lastly, immunocytochemistry was used to evaluate count and area of TIMP3/NileRed/APOE co-localizing sub-RPE deposits on the transwell membrane.
Results :
Chronic exposure (14 days) of control RPE to 1%, 5% and 10% ECVE did not adversely impact cell viability and epithelial barrier integrity. In contrast, compared to untreated iPSC-RPE; ECVE-treated iPSC-RPE showed i) reduced uptake of POS, ii) elevated levels of extracellular HMGB1, a prototypic damage associated molecular pattern (DAMP) molecule, iii) increased levels of sPLA2-IIa, a pro-inflammatory enzyme that promotes inflammation by generation of reactive lipids and iv) increased count and area of sub-RPE TIMP3/Nile Red/APOE- positive drusen-like deposits.
Conclusions :
Using iPSC-based disease modeling studies, we show that ECVE exposure promotes sterile inflammation and induces several pro-maculopathy changes in RPE cells, including decreased POS phagocytosis and formation of sub-RPE drusen-like deposits.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.