Small Extracellular Vesicle Treatment of Trabecular Meshwork Fibrosis: 2D/3D In Vitro and In Vivo Analyses.

Purpose: Fibrosis of the trabecular meshwork (TM) is a key pathological mechanism in POAG. Small extracellular vesicles (sEVs), a type of extracellular secretion, have various functions, such as antifibrotic effects and injury repair. This study investigated the antifibrotic effects of human bone marrow mesenchymal stem cell-derived (hBMMSC) sEVs on TM cells in two-dimensional (2D)/three-dimensional (3D) cultures in vitro and in vivo. Methods: Primary human TM cells were isolated from corneal rings and characterized. We generated 3D TM cultures via scaffoldless 3D culture. sEVs were extracted from hBMMSC supernatants via gradient ultracentrifugation and characterized via electron microscopy and nanometer flow analysis. A TGFβ2-induced fibrosis model was established in 2D/3D TM cell cultures, and the effects of sEVs treatment were assessed via Western blot, immunofluorescence, and morphological analyses. A chronic ocular hypertension mouse model was constructed by injecting the TGFβ2-overexpressing adenovirus Ad-TGFβ2C226/228S. The hBMMSC sEVs were injected into the anterior chamber 2 weeks later. The intraocular pressure (IOP) and changes in fibronectin (FN) and α-smooth muscle actin (α-SMA) in the iridocorneal angle were determined. Results: The hBMMSC sEVs significantly reduced FN and α-SMA expression in both the 2D and 3D TM fibrosis models. sEVs also mitigated the TGFβ2-induced reductions in 3D cultured TM volume, porosity, and pore density. In vivo, sEVs injection effectively reduced TGFβ2-induced IOP elevation and decreased FN and α-SMA expression in the iridocorneal angle. Conclusions: hBMMSC sEVs significantly attenuate TGFβ2-induced TM fibrosis via both protein expression and morphological changes. In addition, hBMMSC sEVs have therapeutic potential in alleviating the TGFβ2-induced increase in IOP linked to TM fibrosis.