Abstract
Purpose :
The integrity of the retinal pigment epithelium (RPE) is essential for the proper functioning of the neural retina, especially the photoreceptors. Serving as a polarized monolayer barrier between photoreceptors and the choriocapillaris, the RPE is crucial for nutrient transport, waste removal, and synthesizing proteins vital for retinal homeostasis. Directionally secreted RPE proteins play a key role in normal retinal function, with their imbalance contributing to age-related macular degeneration (AMD). This study investigates how aging influences the proteome dynamics of the RPE, with a focus on understanding the role of directionally secreted RPE proteins and their potential contribution to AMD, aiming to provide insights for future therapeutic strategies.
Methods :
Porcine primary RPE cells were grown on transwells (n=2) for 1 month (young) and 10 months (old) in regular growth media then switched to serum free media for 7 days. Media was collected from both apical and basal chambers, along with lysed cells, and naïve media (not used on cells). Media was concentrated using Amicon filters (3,000 MWCO) then loaded onto an SDS-PAGE gel. Samples were run into the stacking gel and then stained with Coomassie Blue for protein visualization. The gel was destained and individual bands cut out for label-free proteomics analyses.
Results :
Intracellular as well as apical and basal proteome changes between young and old RPE cultures were examined to understand how the RPE responds to aging. We observed that as the RPE aged, there were significant differences in abundance of proteins involved in the complement cascade (c3 [log2FC=2], c3/c5 convertase [log2FC=5]), epithelial to mesenchymal transition (TGFβ1 [log2FC=3], IGF2[log2FC=3], vimentin [log2FC=2)], ECM remodeling (A1AT [log2FC=10], MMP2 [log2FC=-4], HTRA1 [log2FC=-2]), and drusen-related protein deposition (clusterin [log2FC=1], TIMP3 [log2FC=1], APOE [log2FC=4]). These changes are also accompanied by sub-RPE deposit accumulation in aged cultures as visualized by electron microscopy.
Conclusions :
Through these studies we aimed to address both the temporal and spatial remodeling of the RPE proteome associated with aging. We expect these data will serve as an important resource for future mechanistic and therapeutic studies aimed at ameliorating RPE health and integrity.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.