Keratocyte-Derived Exosomes Suppress Fibrogenic and EMT-Associated Gene Expression in Tenon’s Fibroblasts: An In Vitro and In Silico Network-Based Analysis
Description
Aim: To evaluate the potential of activated keratocyte-derived exosomes (aKExo) to modulate fibrogenic responses and epithelial-to-mesenchymal transition (EMT)-related pathways in Tenon’s fibroblasts. Methods: aKExo were isolated from human limbal stromal keratocytes (passages 5-6), and characterized via transmission electron microscopy (TEM), dynamic light scattering (DLS), and flow cytometry (positive for CD9/CD63/CD81). Primary Tenon’s fibroblasts, isolated from a glaucoma patient undergoing tube-shunt surgery, were treated with aKExo at three concentrations (3.5×10⁻¹ - 3.5×10⁻³ µg/mL) for 24 hours. The level of mRNA expression of fibrogenic/EMT-related genes (ACTA2, TGFB1, THY1, ZEB1, CTNNB1, SNAI1) was quantified using real-time PCR. A bioinformatic analysis was subsequently performed to identify the hub genes and protein-protein interaction networks using Reactome Cytoscape sofwares. Results: aKExo at the concentration of 3.5×10⁻1 µg/mL significantly downregulated the expression of ACTA2, TGFB1, THY1, ZEB1, CTNNB1, and SNAI1. Protein network analysis revealed significant enrichment in processes related to EMT, cadherin-mediated adhesion, TGFB1 signaling, and IL-4/IL-13-mediated immune modulation. Conclusion: aKExo effectively attenuate fibrogenic and EMT-related gene networks in Tenon’s fibroblasts. This molecular and pathway-based evidence supports aKExo potential as anti-fibrotic agents for safer modulation of wound healing following glaucoma surgery.