Abstract
Purpose :
Cigarette smoking is a known risk factor for the development of proliferative vitreoretinopathy (PVR). The underlying pathogenesis of PVR involves retinal pigment epithelium (RPE) cells undergoing epithelial mesenchymal transition (EMT). We examined the effect of cigarette smoke extract (CSE) on RPE cell EMT in vitro to study one of the potential mechanisms through which cigarette smoke impacts PVR formation
Methods :
An immortalized human RPE cell line (ARPE19), was used for all in vitro experiments. CSE was made with research grade cigarettes by bubbling smoke through 10 ml 0.1% FBS medium for 2 min. The resulting solution was used as a stock (10%) and diluted further. The pH was adjusted to 7.2, the solution was filtered through a pore filter for sterilization, and then standardized by measuring the absorbance at a wavelength of 320 nm. The resulting CSE solution was used within 30 min of preparation. ARPE19 cells were cultured in 0.1% FBS media overnight and were then treated for 4 or 24 hours in 1 % CSE or control (0.1% FBS) media. A subset of cells underwent 24 hour recovery in control media after CSE exposure. Cell morphology was examined prior to and after all experimental conditions. RPE cell EMT was measured using Western blotting and immunofluorescence microscopy for alpha smooth muscle actin (αSMA), a key marker for EMT
Results :
CSE exposed ARPE19 cells acquired a spindle-like mesenchymal appearance which was not present in the control media ARPE-19 cells. Exposure to CSE caused a time and dose-dependent relative increase in αSMA expression in ARPE19 cells compared to ARPE19 cells in control media, by Western blotting. ARPE19 cells in 1% CSE for 24 hours had a 2-fold increase in αSMA expression compared to cells in control media. ARPE19 cells treated with CSE exhibited higher αSMA fluorescence compared to cells in control media
Conclusions :
ARPE19 cells exposed to CSE develop a mesenchymal appearance and undergo EMT, measured by an increase in αSMA expression by Western blotting and immunofluorescence. These findings support the potential mechanism of CSE-induced EMT and the higher rates of PVR development in smokers. Future studies on the mechanism of CSE-induced EMT will provide more insight into the role of cigarette smoke exposure and PVR development
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.