Investigating the metabolic effects of basal mineral calcification on human RPE cell culture

Abstract

Purpose : Calcification is increasingly becoming recognized as an important contributor to the clinical appearance of age-related macular degeneration (AMD). In this study, we studied the effects of various calcium minerals on human retinal pigment epithelial (RPE) cell.

Methods : Human RPE cells (hRPE; 85,000 or 125,000 cells/cm² seeding density) were cultured in the presence or absence of 25 µg/cm² hydroxyapatite (HAP) or whitlockite (WHT), embedded in the Geltrex coating of 0.33cm² Transwell membrane inserts, and were maintained in culture for up to 4 weeks. Cell phenotype was assessed by qualitative monitoring of cell pigmentation and measuring trans-epithelial electrical resistance (TEER). Metabolic alterations were assessed using the Seahorse XF Cell Energy Phenotyping Kit to determine the mitochondrial (oxygen consumption rate (OCR)) and glycolytic (extracellular acidification rate (ECAR)) respiratory capacity of the hRPE cells.

Results : Based on visual assessment of pigmentation, control cells began to differentiate earlier than cells cultured on any of the seeded minerals (2 vs 3 weeks, respectively). TEER for HAP and WHT was significantly lower than that of control cells at both 2 weeks and 4 weeks (45.7±0.9 and 49.6±0.8; 44.6±1.6 and 46.9±0.4 vs 53.1±0.9 and 63.5±2.7 Ω*cm², respectively; p<0.05). Cells cultured on HAP and WHT had significantly higher ECAR values at baseline compared to control cells at 2 and 4 weeks (36.6±3.6 and 39.4±2.1; 35.4±2.3 and 36.9±1.8; vs 24.9±1.7 and 28.7±1.1 mpH/min/10,000 cells, respectively; p<0.05). After stress induction the cells grown on HAP and WHT had significantly lower OCR values compared to control cells, at 4 weeks only (219.0±22.2; 203.3±11.6 vs 299.2±9.2 pmol/min/10,000 cells, respectively; p < 0.05) with no significant difference observed at 2 weeks.

Conclusions : When sub-RPE minerals are present, the RPE appears to utilise the glycolytic respiratory pathway under resting conditions, and continues to do so after differentiation is initiated. Cells grown in the presence of calcium-phosphate minerals also show reduced mitochondrial respiration under stress compared to cells grown naturally, but only after differentiation appears to have started. These indicate that the mitochondrial respiration pathway is detrimentally affected by the presence of calcification in vivo, which is consistent with the notion that mitochondrial changes are involved in AMD.

This is a 2021 ARVO Annual Meeting abstract.