Oxidative stress (OS) induces the loading of oxidized phospholipids (OxPLs) in exosomes derived from human non-pigmented ciliary epithelium (NPCE)

Purpose : Exosomes are nanovesicles that serve as extracellular biological communicators modulating the cells that take up the exosomes. The aim of the study was to evaluate the changes in the lipidome of the exosomes produced by NPCE when exposed to acute and chronic OS.

Methods : NCPE was exposed to OS via a free radical-generating azo compound 2,2′-Azobis (2-amidinopropane) dihydrochloride (AAPH) - 15 mM AAPH for 90 min for acute OS (A-OS) or 1.5 mM AAPH for 24 h as chronic OS (C-OS) and exosomes were isolated post treatment. Lipid extraction from exosomes was accomplished using the Bligh and Dyer method. MRM-based untargeted lipidomics was performed. Data analysis was accomplished using Metaboanalyst 6.0. Data were auto-scaled to obtain a normal distribution, and unpaired t-tests and volcano plot analyses were conducted. Lipids were analyzed according to treatment group, class, fatty acyl residue chain length, and unsaturation level.

Results : A total of 566 phospholipid species were identified in the oxidative stressed groups, falling within five subcategories: Phosphatidylcholines (PC), Phosphatidylethanolamines (PE), Phosphatidylglycerols (PG), Phosphatidylinositols (PI), and Phosphatidylserines (PS). When considering the OxPLs in the A-OS group when compared to control, and using a p-value < 0.05, 41 total species were found to increase. Chronic exposure induced 28 species to increase. Eleven of these species were found to be in common between the two groups. When considering volcano plot analysis, with significance being defined as a greater than 1.2-fold change and a p < 0.05, 26 species were found to be significantly increased in the A-OS group while 21 increased in the C-OS group. Eight of these were found to be in common.

Conclusions : OxPLs were found in the exosomes produced by NCPE cells in response to OS. Acute stress induces more loading of OxPLs in exosomes than chronic stress. This suggests that either the cell adapts and responds to this stress over time, or the higher intensity of A-OS induces more OxPLs to be loaded into exosomes. This experiment has demonstrated for the first time that OxPLs are loaded into NPCE derived exosomes following OS.