Introduction:
Extracellular (ectopic) calcification is associated with age‑related macular degeneration (AMD), the leading cause of irreversible vision loss in the Western world (1). A key pathological feature is the accumulation of drusen between the retinal pigment epithelium (RPE) and the Bruch’s membrane, disrupting exchange of nutrients and oxygen, and the clearance of waste products (2). Drusen contain proteins, lipids, and calcium phosphate minerals, primarily hydroxyapatite (HAP) and, less frequently, whitlockite (WHT) (3–5). Although mineralisation is linked to the progression to end-stage AMD, the mechanisms involved in this process are not well understood.

 

Aims and Objectives:
This project aims to elucidate the intra- and extra-cellular factors that contribute to ectopic calcification in the sub-RPE space during the onset and progression of AMD.

 

Methods:
Human primary RPE cells (hRPE) were cultured on transwell inserts and treated with conditions that promote calcification. Long‑term (LT, glycerophosphate, 2 weeks) or short‑term (ST, high concentration sodium phosphate dibasic, 72 hours) calcification‑inducing media, or pre-seeded synthetic calcium phosphate mineral were used. Transepithelial electrical resistance (TEER) was used to measure cell barrier functionality across multiple time points (n = 4). Gene expression changes were quantified using qPCR to examine markers associated with calcification and metabolism (n = 4).

Resipher assays and Seahorse measurements assessed RPE metabolic capacity measured as oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). We compared values obtained from cells grown on HAP and WHT, with calcium diphosphate (CPD) and no‑crystal used as controls (n = 6).

Data were analysed using t-test when comparing two groups, and one-way or two‑way ANOVA when comparing three or more groups. Statistical significance was set at p < 0.05.

 

Results:
LT calcification inducing media induced a significant TEER decline over time compared with controls (p < 0.05), indicating progressive disruption of tight junction integrity. ST treatment similarly reduced TEER (p < 0.05), with the majority of TEER loss occurring within the first 24 hours (p < 0.05).

qPCR revealed that LT treatment significantly decreased expression of calcification‑ and glycolysis‑related genes (ABCC6, HK2, PFKB3; p < 0.05), while ST treatment did not induce significant changes. This suggests LT treatment as a model for gene expression changes in sub-RPE calcification

Metabolic assays showed a significant metabolic shift towards glycolysis in crystal‑treated hRPE, when compared to control cells at 4‑week (p < 0.05) and 6‑week (p < 0.05) differentiation. These results suggest impaired mitochondrial respiration and calcification-induced metabolic impairment.

 

Conclusions:

Here, we showed that extracellular calcification impacts RPE cell functionality, and metabolic capacity. These results indicate that calcification may directly influence retinal function, supporting clinical observations that link calcification to rapid progression toward end-stage AMD.