Effects of topical TGF-β1, TGF-β2, ATX, and LPA on IOP elevation and regulation of the conventional aqueous humor outflow pathway

Purpose: The effects of aqueous mediators possibly increasing the outflow resistance, transforming growth factor-β1 (TGF-β1), TGF-β2, autotaxin (ATX), and lysophosphatidic acid (LPA) on human trabecular meshwork (hTM) cells and monkey Schlemm’s canal endothelial (SCE) cells were characterized and compared, and the effects of intracameral application of these mediators on intraocular (IOP) elevation were also examined. Methods: Cells were treated with TGF-β1, TGF-β2, ATX, LPA, or vehicle, and mRNA and protein expression levels of α-SMA, COL1A1, fibronectin, β-catenin, and ZO-1 were examined with real-time quantitative PCR (RT-qPCR) or immunofluorescence analyses or both. The permeability of cell monolayers was measured by determining the transendothelial electrical resistance (TEER) or with the fluorescein isothiocyanate (FITC)-dextran permeability assay. IOP was evaluated in rabbit eyes after intracameral administration of the mediators. Results: All mediators induced upregulation of α-SMA, COL1A1, and fibronectin in hTM cells. The effect of TGF-β2 on mRNA expression of fibrotic markers was statistically significantly greater than that of TGF-β1. The effects of ATX and LPA indicated the time-dependent difference in the upregulation of α-SMA, COL1A1, and fibronectin. The TEER and FITC-dextran permeability of the SCE cells was evaluated after treatment with TGF-β1 and TGF-β2, but no statistically significant change was observed within 24 h. ATX and LPA also reduced permeability statistically significantly after 3 h and 0.5 h, respectively, and the effect of LPA was more rapid compared to that of ATX. Statistically significant IOP elevation was observed in rabbit eyes as early as 0.5–2.0 h after ATX and LPA treatment and at 24 h after treatment with TGF-β2. Conclusions: TGF-β2 and ATX and LPA regulate aqueous outflow by modulation of hTM cells and SCE cells, and differences in timing between the effects of each mediator were observed. ATX and LPA showed more rapid effects on IOP elevation than TGF-β2. It was suggested that TGF-β2 and ATX/LPA are involved in increases of IOP, but the timing and sustainability differ between mediators, and they may play specific roles in different glaucoma subtypes.