Corneal stromal stem cell-derived extracellular vesicles inhibit corneal neovascularization.

PURPOSE: Corneal neovascularization (CNV) threatens corneal transparency and is a leading cause of vision loss. This study evaluates the anti-angiogenic effects of extracellular vesicles (EVs) derived from human corneal stromal stem cells (CSSCs).

METHODS

Cultured human umbilical vein endothelial cells (HUVECs) were treated with CSSC-EVs to examine the influence on cell growth and modulation of angiogenesis using xCELLigence and tube formation assays. In vivo, a mouse model of CNV was induced by silver nitrate (AgNO₃) cauterization, and the injured corneas were treated with CSSC-EV eye drops twice daily for four days. On day 10, new vessel formation on mouse corneas was graded, and the expression of angiogenic markers was assessed using immunostaining and qPCR. Results were analyzed using one-way ANOVA.

RESULTS

HUVEC cultures treated with CSSC-EVs showed reduced growth with a significant increase in cell doubling time. On Geltrex-coated culture surface, HUVECs incubated with CSSC-EVs generated reduced numbers of vascular tube structures, with significantly reduced junctions and nodes. In vivo, CNV developed in AgNO₃-cauterized mouse corneas by day 10 post-injury. Eye drop treatment with CSSC-EVs attenuated the formation of new vessels expressing vascular marker CD31 and lymphatic marker LYVE1. Compared to injured controls treated with phosphate-buffered saline (PBS) eye drops, the expression of angiogenic markers, including ANGPT1 and 2, CD31, VEGFA, VEGFR1, and R2, was significantly downregulated in EV-treated corneas (P < 0.05).

CONCLUSION

The anti-angiogenic effect of CSSC-EVs demonstrated their potential to inhibit CNV. Overall, the topical application of CSSC-EVs was safe and effective for addressing CNV-related pathologies.