The purpose of this study is to examine if aqueous autotaxin (ATX) and TGF-β levels could be used for differentiating glaucoma subtypes. This prospective observational study was performed using aqueous humor samples obtained from 281 consecutive patients. Open angle glaucoma patients were classified into three groups: primary open-angle glaucoma (POAG), secondary open-angle glaucoma (SOAG), and exfoliation glaucoma (XFG). Aqueous levels of ATX and TGF-βs were quantified. The AUC as well as sensitivity and specificity for the classification into normal and glaucoma subtypes using four indicators-ATX, TGF-β1, TGF-β2, and TGF-β3, upon the application of three machine learning methods. ATX, TGF-β1, and TGF-β3 were positively correlated with IOP, and ATX was significantly and negatively correlated with the mean deviation. From least absolute shrinkage and selection operator regression analysis, the AUC values to distinguish each subgroup [normal, POAG, SOAG, and XFG] ranged between 0.675 (POAG vs. normal) and 0.966 (XFG vs. normal), when four variables were used. High AUC values were obtained with ATX for discriminating XFG from normal eyes and with TGF-β3 for discriminating XFG from normal eyes, POAG, or SOAG. Aqueous TGF-β and ATX exhibited high diagnostic performance in detecting glaucoma subtypes, and could be promising biomarkers for glaucoma.

Trabecular meshwork constitutes the conventional outflow pathway and controls intraocular pressure by regulating aqueous outflow. Mechanical stimulation has been studied as one of the triggers to regulate aqueous outflow in trabecular meshwork, but it is not well understood. We investigated that how transient receptor potential cation channel subfamily V member 4 (TRPV4) functions in human trabecular meshwork cells (HTMC) and affects intraocular pressure (IOP). HTMC were treated with TRPV4 siRNA, followed by incubation for 24 hours. We confirmed the suppression of TRPV4 mRNA expression and the reduction of Ca²⁺ influx by the TRPV4 agonist GSK1016790A in TRPV4 siRNA-treated HTMC. TRPV4 siRNA-treated HTMC exhibited a significant reduction in Ca²⁺ influx and production of arachidonic acid and prostaglandin (PG) E₂ induced by mechanical stretch, and direct activation of TRPV4 by GSK1016790A increased production of arachidonic acid, PGE₂, and PGD₂ and inhibited gel contraction. Furthermore, TRPV4-deficient mice had higher IOP than wild-type mice, and GSK1016790A administration lowered IOP. These results suggest that TRPV4 mediates the cellular response induced by trabecular meshwork stretch, leading to IOP reduction through the production of prostaglandins and inhibition of cell contraction. Targeting TRPV4 may have therapeutic benefits that lead to lowering IOP in glaucoma patients.

This study investigated the effects of omidenepag (OMD), a novel selective EP2 receptor agonist, on human trabecular meshwork (HTM) cells, monkey Schlemm’s canal endothelial (SCE) cells, and porcine ciliary muscle (CM) to clarify the mechanism of intraocular pressure (IOP) reduction involving conventional outflow pathway. In HTM and SCE cells, the effects of OMD on transforming growth factor-β2 (TGF-β2)-induced changes were examined. The expression of actin cytoskeleton and extracellular matrix (ECM) proteins, myosin light chain (MLC) phosphorylation in HTM cells were evaluated using real-time quantitative PCR, immunocytochemistry, and western blotting. The expression of barrier-related proteins, ZO-1 and β-catenin, and permeability of SCE cells were evaluated using immunocytochemistry and transendothelial electrical resistance. The CM contraction was determined by contractibility assay. OMD significantly inhibited expression of TGF-β2 induced mRNA, protein, and MLC-phosphorylation on cytoskeletal and ECM remodeling in the HTM dose dependently. In SCE cells, OMD suppressed TGF-β2-induced expression of the barrier-related proteins and decreased SCE monolayer permeability. OMD at 3 µM significantly inhibited CM contraction, however, the effect was not significant at lower concentrations. IOP lowering effect of OMD through conventional outflow pathway is exerted by increasing outflow facilities with the modulation of TM cell fibrosis and SCE cell permeability.

Background: Elevated transforming growth factor (TGF)-β2 in aqueous humor (AH) has been suggested to contribute to trabecular meshwork (TM) fibrosis and intraocular pressure (IOP) regulation in primary open-angle glaucoma (POAG), but TGF-β2 is downregulated in secondary open-angle glaucoma (SOAG). Because autotaxin (ATX) is upregulated in SOAG, we investigated the relationships and trans-signaling interactions of these mediators. Methods: The level of ATX in AH was determined using a two-site immunoenzymetric assay, and TGF-β levels were measured using the Bio-Plex Pro TGF-β Assay. RNA scope was used to assess the expression of ATX and TGF-β2 in human’s eye specimen. And in vitro studies were performed using hTM cells to explore if trans-signaling of TGF-β2 regulates ATX expressions. Results: TGF-β2/ATX ratio was significantly high in AH of control or POAG compared with SOAG, and negatively correlated with IOP. RNA scope revelated positive expressions of both TGF-β2 and ATX in ciliary body (CB) and TM in control, but ATX expressions was significantly enhanced in SOAG. In hTM cells, ATX expressions were regulated by TGF-β2 with concentration-dependent manner. In counter, ATX also induced TGF-β1, TGF-β2 and TGFBI upregulations and activation of the Smad-sensitive promoter, as well as upregulation of fibrotic markers, and these upregulation was significantly suppressed by both TGF-β and ATX inhibition. Conclusions: Trans-signaling of TGF-β2 regulates ATX expressions and thereby induced upregulations of TGF-βs or fibrosis of hTM. TGF-β2 trans-signaling potently regulate ATX transcription and signaling in hTM cells, which may reflect different profile of these mediators in glaucoma subtypes. Trial Registration This prospective observational study was approved by the Institutional Review Board of the University of Tokyo and was registered with the University Hospital Medical Information Network Clinical Trials Registry of Japan (ID: UMIN000027137). All study procedures conformed to the Declaration of Helsinki. Written informed consent was obtained from each patient.

Purpose: To evaluate the histological changes associated with, and the potential mechanisms of, intraocular pressure (IOP) reduction by micropulse cyclophotocoagulation (MP-CPC) in rabbit eyes. Methods: MP-CPC was performed on the right eyes of Dutch belted rabbits, whereas the left eyes served as controls. The laser power settings were 250, 500, 750, 1000, 1500, and 2000 mW, 10 seconds per sweep, 100 seconds in total. IOP, outflow facility, and uveoscleral outflow tract imaging, using a fluorescent tracer, were examined at one week after MP-CPC. Changes of morphology and protein expressions in the outflow tissues, conjunctiva, and sclera were also evaluated. Results: Significant reductions in IOP after MP-CPC were observed at 500 to 1000 mW (P = 0.036 and P = 0.014, respectively). The pre-MP-CPC IOP was 11.35 ± 0.41 mm Hg. At one week after surgery, the respective IOP values in the eyes treated at 500 mW and 1000 mW were 9.45 ± 0.49 mm Hg and 7.4 ± 0.27 mm Hg, respectively. Severe ciliary body damage was observed at 1500 to 2000 mW. MMP1–3 and fibronectin expression levels in the outflow tract and ciliary body were upregulated after MP-CPC. The α-smooth muscle actin (α-SMA) was upregulated at higher power levels. MP-CPC significantly increased uveoscleral outflow, whereas the outflow facility did not change. The α-SMA, collagen, and fibronectin were significantly upregulated in the subconjunctiva and sclera. Conclusions: Reactive fibrotic responses were observed in the outflow tract, conjunctiva, and sclera after MP-CPC. A potential mechanism of IOP reduction by MP-CPC in pigmented rabbit eyes may involve increased uveoscleral outflow related to MMP upregulation.