Abstract
Purpose :
Alterations to extracellular matrix (ECM) and inflammatory cytokines have been associated with many retinal diseases. Normal production of ECM by retinal pigmented epithelial cells (RPE) helps to maintain the basement membrane of the RPE and the underlying Bruch’s membrane. However, dysregulated inflammatory signaling may trigger ECM remodeling and contribute to retinal pathology. Our aim was to further investigate the link between inflammation and ECM using a high content imaging assay to detect fibronectin deposition by RPE cells.
Methods :
ARPE-19 cells were cultured in 384-well plates for durations ranging from one day to 4 weeks. Cells were then challenged with either single or chronic treatments with TGF-β or TNF-α followed by staining with Oregon Green-gelatin, which binds fibronectin. Cells were fixed with 4% paraformaldehyde, counter-stained for additional proteins of interest, and imaged on the Molecular Devices ImageXpress Micro 4. Oregon Green-gelatin staining intensity was quantified using the MetaXpress Multi Wavelength Cell Scoring analysis module.
Results :
The deposition of fibronectin by ARPE-19 cells in response to the different cytokines depended on how long the cells had been in culture. At short culture times (1 day), RPE cells deposit fibronectin in response to TGF-β1 & 2, but not TNF-α. Conversely, at longer culture times (2-4 weeks), cells exhibit a robust deposition of fibronectin in response to TNF-α, but little change is observed following TGF-β treatment. Long term cultures of ARPE-19 cells exhibit contact inhibition and develop tight junctions as determined by ZO-1 staining. When long term ARPE-19 cultures were treated chronically with TNF-α the deposition of fibronectin was greatly enhanced, indicating that repeated exposure, as may occur in a disease state, can impact cellular response. Studies are on-going to determine if there is any effect of chronic TGF-β treatment on fibronectin production.
Conclusions :
We have shown that ARPE-19 cells exhibit different responses to inflammatory stimuli depending on their time in culture. Given that RPE cells in vivo are quiescent with intact tight junctions, the results following cytokine treatment by long term ARPE-19 cells may be more indicative of the response in vivo. Moreover, conditions of chronic low-grade inflammation may impact the responsiveness of RPE cells.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.