Region-Specific Proteomic Profiles of Extracellular Vesicles (EVs) Derived from Human Macular and Peripheral...

Background: Regional heterogeneity of the macula and peripheral retinas contributes to the differences in retinal physiology, metabolic demand and susceptibility to macular diseases such as age-related macular degeneration (AMD), diabetic macular oedema (DME) and Macular telangiectasia type 2 (MacTel). Extracellular vesicles (EVs) released from the retinal pigment epithelium (RPE)-choroid are increasingly recognised as mediators of extracellular communication and correlate with the molecular biological states of their tissue of origin. However, regional variation in EV composition in human RPE-Choroid tissues remains poorly characterised. Methods: Human macular and peripheral RPE-choroid explants from non-diseased donor eyes (n = 4) were cultured ex vivo using a transwell system. EV and EV-depleted conditioned media were collected by differential ultracentrifugation, respectively. EVs were characterised by transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA). Proteomic profiling was performed using LC-MS/MS followed by multivariate, pathway, and differential expression analyses. Results: TEM and NTA confirmed the presence of vesicle-like particles in a 119-140 nm size range, indicating small EVs in both regions. Proteomic analysis demonstrated a region-associated separation between the macular and peripheral samples in both EV proteome and EV-depleted soluble secretome datasets. GO enrichment analysis revealed that macular EV proteomes were enriched for wound healing, cell-substrate adhesion, and focal adhesion-related terms with a high abundance of integrin and annexin family members, whereas peripheral EV proteomes were enriched for retinoid- and vitamin-binding terms. In the EV-depleted soluble secretome, macular-enriched proteins were associated with actin binding and extracellular matrix-related terms, while peripheral-enriched proteins were correlated with RNA localisation and nuclear compartment terms. Comparative analysis identified EV-specific, secretome-specific, and shared extracellular protein pools. Several EV-specific markers and membrane proteins such as annexins and integrins, showed a relatively high enrichment in macular EV proteomes, whereas CD63 is more abundant in peripheral EV proteomes. In contrast, EV-depleted soluble secretome contains cytokine- and ligand-associated proteins, including MIF, SPP1, and IL6, which may suggest that EVs and soluble secreted proteins represent partially distinct extracellular signalling compartments. Conclusions: Human macular and peripheral RPE-choroid explants released secretory proteins in a regional differentiated manner, supported by PLS-DA and GO enrichment analyses of both EV proteomes and EV-depleted soluble secretome. Further analysis of the EV proteomes may show that such differences were also able to be reflected in protein categories such as EV-specific markers, mitochondrial and membrane proteins. These findings provide a foundation for future investigations into the role of EV-mediated communication in macular diseases and may support the development of region-specific extracellular biomarkers and therapeutic targets.