Abstract
Purpose :
Extracellular vesicle (EV) release from retinal pigment epithelium (RPE) cells may drive hypoxia induced angiogenesis of the choroid. Neovascular age-related macular degeneration (nAMD) and RPE are often studied in conjunction, however, little is known about the release of EVs from the adjacent choroidal cells and their participation in the disease. We tested the hypothesis that hypoxia alters EV release from human melanocytes.
Methods :
Human melanocytes were cultured normoxic (20% O2) or hypoxic (3% O2) to model nAMD pathology. EVs were isolated by differential ultracentrifugation (1000xg/4oC/10’, 25000xg/4oC/30’, 115,000xg/4oC/90’) then characterized by nanoparticle tracking analysis (ZetaView ParticleMetrix®) yielding EV number, size, and population characteristics. To standardize EV yields, the cell number from each culture was counted to determine the total EV release per cell. Statistical analysis was performed using a two-sample t-test.
Results :
Mean EV size from normoxic melanocytes was 128.2nm, SD +/- 7.67nm. Mean number of EVs per cell was 721.31 particles/cell, SD +/- 221.98 particles/cell. Mean EV size from hypoxic melanocytes was 127.78nm, SD +/- 3.78nm. Mean number of EVs released per cell was 54.87 particles/cell, SD +/- 15.13 particles/cell. The normal versus hypoxic melanocyte EV populations did not differ significantly in mean size (p=0.46), however the quantity of EVs released per cell was significantly reduced from hypoxic melanocytes (p<0.001).
Conclusions :
Our results show that hypoxia significantly reduces EV release from melanocytes, which is the opposite of what we have found previously for RPE cells. Our results suggest that EV release is cell-type specific and may relate to participation in choroidal neovascularization. The next steps towards determining the significance of choroidal melanocytic EV release will be to examine to potential of melanocyte EV on angiogenic activity by probing the EVs for proteins that have shown to play a strong role in neovascularization, for example vascular endothelial growth factor and pigment-epithelium derived factor.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.