Abstract
Purpose :
Calcium-phosphate minerals are essential contributors both to drusen development and progression to end-stage disease in Age Related Macular Degeneration (AMD). Our aim was to investigate the effects of calcium-phosphate minerals on cell differentiation and maturation of human retinal pigment epithelial (RPE) cells.
Methods :
Human RPE cells (hRPE; 85,000 cells/cm2 seeding density) were cultured in the presence or absence of 25 µg/cm2 hydroxyapatite (HAP), whitlockite (WHT) or zinc-doped HAP (10ZnHAP) in the Geltrex coating of the 0.33cm2 Transwell membranes. Human RPE cells were maintained in culture for 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, WHT or 10ZnHAP was significantly lower than that of control cells (50.7±3.1; 51.0±1.3; 52.3±1.5 vs 74.1±5.8 Ω*cm2, respectively; p<0.05). Cells cultured on HAP, WHT or 10ZnHAP had significantly higher ECAR values at baseline compared to control cells (41.4±6.01; 39.2±3.2; 42.9±6.8 vs 30.1±3.2 mpH/min/10,000 cells, respectively; p<0.05). After stress induction the cells grown on HAP, WHT or 10ZnHAP had significantly lower OCR values compared to control cells (207.8±24.8; 217.4±31.2; 211.9±30.7 vs 314.2±24.9 pmol/min/10,000 cells, respectively; p < 0.05).
Conclusions :
These results show that RPE cell differentiation and metabolism is likely to be altered when cells are exposed to calcium-phosphate mineral deposits in the human eye. Based on our data, the RPE appears to utilise the glycolytic respiratory pathway more under resting conditions, but show reduced mitochondrial respiration under stress when there are sub-RPE minerals present. 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 2020 ARVO Annual Meeting abstract.