Evaluation of senescence in RPE and choriocapillaris in young, age matched, and AMD human donor eyes.

Abstract

Purpose : Cellular senescence is a key feature of the aging process, contributing to both normal aging and diseases of aging. Research on the role of senescence in human eyes with age-related macular degeneration (AMD) pathogenesis is limited. We have conducted preliminary studies to test the hypothesis that telomere length in cells of the choriocapillaris and retinal pigment epithelium (RPE) are shorter in AMD affected eyes compared to young and age matched controls and that senescence contributes to endothelial cell loss in AMD.

Methods : Donor eyes were categorized as young (< 40 yo), age matched controls (age >71 years, without AMD) or AMD (age >71 years with early atrophic AMD). Average telomere lengths of genomic DNA were determined from 5 young, 6 aged, and 4 AMD bulk RPE/Choroid punches using a commercially available telomere length RT-qPCR assay. To address cell type specific telomere lengths, we performed a pilot study utilizing laser capture microdissection (LCM) to collect choriocapillaris and RPE cells separately from fixed cryosections of young (n=5), aged (n=6), and AMD (n=5) donor maculas. Finally, immortalized and primary choroidal endothelial cells were treated with H2O2 to induce senescence, followed by exposure to complement-intact or complement-depleted serum, and deposition of the C5b-9 membrane attack complex (MAC) was evaluated.

Results : No statistically significant difference in telomere length was observed between bulk RPE-choroid or LCM-isolated RPE and choriocapillaris of young, aged or AMD donors. Overall, telomere lengths of RPE cells were longer than those of cells in choriocapillaris from the same donors, although this did not reach statistical significance. Senescence induction resulted in cells with larger surface area and an increased frequency of nuclei positive for senescence-associated heterochromatin foci markers (MacroH2A and HMGA2). Senescent cells and their subendothelial matrix showed increased MAC deposition.

Conclusions : No obvious differences in telomere lengths of LCM-extracted RPE and choriocapillaris were observed between young, age-matched, and AMD human donor eyes. H2O2 treatment induced senescence-associated changes in immortalized and primary choroidal endothelial cells. Senescent cells and subendothelial matrix also displayed increased MAC deposition, suggesting that senescence may contribute to vascular susceptibility to MAC in AMD.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.