Characterizing extracellular vesicles from diabetic keratopathy and keratoconus.

Abstract

Purpose : Numerous diseases threaten the human corneal integrity and function including diabetic keratopathy (DK) and keratoconus (KC). DK causes corneal manifestations, scarring, and epithelial erosions. KC is a disorder wherein the cornea undergoes progressive thinning and steepening, often associated with scarring and loss of visual acuity. Recent studies suggest that extracellular vesicles (EVs) may play a role in cellular communication in the cornea as well as during the wound healing cascade. EVs are small, sphere-like structures produced from cells and can facilitate cell-cell interactions. We sought to reveal the unique fingerprints of EVs derived from DK and KC corneas.

Methods : Primary human corneal stromal fibroblasts were isolated and cultured from healthy (HCF), Type 1 (T1DM) & Type 2 (T2DM) diabetes, and keratoconic (HKC) donor corneas. The cells were seeded on constructs at a cell density of 1x10⁶ cells/well and stimulated with Vitamin C (0.5 mM). The cells were grown for 4 weeks and their media was collected, and analyzed twice weekly using the ExoView R100. Statistical analysis was performed using One-Way ANOVA.

Results : HCFs secreted more total EVs (2009 average particles), while T1DMs produced the least total EVs (1239 average particles). The EV phenotypes had similar surface protein expressions in all cell types in which they favored CD63, CD81/CD9 and CD63/CD81/CD9. The surface proteins were compared between each cell type and HCFs showed upregulation of CD81, CD63/CD9, and CD81/CD9. T1DMs had upregulation of CD9 and CD63/CD81/CD9. T2DMs had upregulation of CD63, CD81 and CD63/CD81. Lastly, HKCs showed upregulation of CD63, CD63/CD9 and CD63/CD81/CD9. The tetraspanin expressions were then compared weekly for 4 weeks within each cell type. HKCs had a significant increase in CD63 after week 1, while T1DMs had a significant decrease in CD9, and T2DMs had a higher expression of CD63/CD81 during week 4.

Conclusions : For the first time, we were able to characterize EVs derived from DK and KC corneal stromal fibroblasts cultured on 3D constructs. This study revealed unique phenotypes in KC- and DK-derived EVs in vitro when compared to healthy samples. These findings may help guide researchers and clinicians for future ameliorative techniques and potential therapeutic targets for DK and KC.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.