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bioRxiv · 10.64898/2026.03.27.714594

Hyperosmolar stress promotes the release of small extracellular vesicles containing metabolic proteins from corneal epithelial cells

Hernandez, B. J.·Morakis, V.·Lemoff, A.·Mondal, A.·Robertson, D. M.
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Abstract

PurposeHyperosmolar stress (HOS) is a major contributor to corneal epithelial cell damage in dry eye disease. We have previously shown that HOS damages mitochondria and impairs cell metabolism in corneal epithelial cells. Small extracellular vesicles (sEVs) are cell-derived lipid envelopes that are present in all body fluids, including tears. Prior studies suggest that sEV release and composition may be linked with changes in cell metabolism. In this study, we tested the effects of HOS on sEV release and composition, and found that sEV cargo may reflect early, underlying changes in dry eye disease. MethodsTelomerase-immortalized human corneal epithelial (hTCEpi) cells were treated with 450 mOsm NaCl for five days to induce chronic HOS. sEVs were isolated using differential centrifugation followed by iodixanol density gradient flotation. Particle number was determined using Nanoparticle Tracking Analysis (NTA). Mass spectrometry was used to assess the sEV proteome, and selected proteins were validated by immunoblot. Proteome pathways were analyzed using KEGG and CORUM. ResultsPathway analysis revealed an increase in metabolic proteins and proteasome components in sEV cargo released from hTCEpi cells exposed to HOS. These proteins were increased more than fourfold in HOS-sEVs. Examination of proteins involved in the endosomal pathway and NTA further confirmed an increase in HOS-sEV release. ConclusionOur findings suggest a potential mechanism whereby corneal epithelial cells exposed to HOS retain proteins involved in maintaining tissue integrity, while simultaneously releasing unneeded proteins involved in cell metabolism. The presence of metabolic proteins in sEVs may serve as early indicators of dry eye disease.

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Hernandez, B. J., Morakis, V., Lemoff, A., Mondal, A., Robertson, D. M.. 2026-03-28. Hyperosmolar stress promotes the release of small extracellular vesicles containing metabolic proteins from corneal epithelial cells. https://doi.org/10.64898/2026.03.27.714594

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