Rho-Associated Protein Kinase Inhibitor Treatment Promotes Proliferation and Phagocytosis in Trabecular Meshwork Cells

Abstract

Purpose: Continuous reductions in trabecular meshwork (TM) cellularity inhibit aqueous humor (AH) outflow, which is the main cause of primary open-angle glaucoma. Rhoassociated protein kinase inhibitor (ROCKi) targets the TM to reduce intraocular pressure (IOP) and increase AH outflow facility. However, the underlying mechanisms are not entirely clear. Here, we aimed to investigate the effect of a ROCKi (Y-27632) on TM cell proliferation and phagocytosis. Methods: Immortalized human TM (iHTM) cells, glaucomatous TM (GTM 3) cells, and primary human TM (pTM) cells were cultured and identified. The effects of various concentrations of Y-27632 on F-actin cytoskeleton were assessed using immunofluorescence. Cell proliferation effects were evaluated using a cell counting kit-8 (CCK8), cell counting, and Ki67 immunostaining. Cell phagocytosis was evaluated using immunofluorescence and flow cytometry in immortalized TM cells. C57BL/6J and Tg-MYOC Y437H mice were used to investigate the proliferative effects of Y-27632 on TM cells in vivo. The effect of Y-27632 on IOP was monitored for 2 weeks, and the outflow facility was detected 2 weeks after IOP measurement. TM cells in mice were counted using immunohistochemistry. Results: Y-27632 (100 µM) significantly promoted the proliferation of both immortal TM cells and pTM cells. In GTM 3 cells, phagocytosis was significantly greater in the Y-27632 group than in the control group, nearly reaching the level of phagocytosis in iHTM, as determined using immunofluorescence and flow cytometry. In Tg-MYOC Y437H mice, treatment with Y-27632 significantly decreased IOP and increased outflow facility, which greatly influenced the long-term IOP-lowering effect. The number of TM cells in Tg-MYOC Y437H mice was significantly improved after Y-27632 administration. Conclusion: Y-27632 promoted cell proliferation and phagocytosis of TM cells, and its proliferative effect was demonstrated in a transgenic mouse model. These results revealed a new IOP-lowering mechanism of Y-27632 through effects on TM cells, suggesting the potential for a correlation between TM cellularity and long-term recovery of IOP.

Cell–Matrix Interactions in the Eye: From Cornea to Choroid

“…Similarly, the expression and/or induction of numerous other proteoglycans and matricellular proteins, such as tenascin C, thrombospondin-1 and -2, SPARC (secreted protein, acidic and rich in cysteine), connective tissue growth factor (CTGF), fibronectin, various integrins, and periostin, have also been associated with changes in intraocular pressure [ 30 , 41 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 ]. These mechanisms are not limited to ECM turnover, and also include modulation of such processes as cellular contraction, adhesion and migration, proliferation, and phagocytosis [ 60 , 68 , 69 , 70 , 71 ] ( Figure 5 ). It has been suggested that cell–matrix interactions like these are mechanisms by which the trabecular meshwork can regulate intraocular pressure homeostasis [ 30 ], and elements in these interactive pathways, such as Rho kinase inhibitors, have shown promise as therapeutic targets [ 71 , 72 , 73 ].…”

“…These mechanisms are not limited to ECM turnover, and also include modulation of such processes as cellular contraction, adhesion and migration, proliferation, and phagocytosis [ 60 , 68 , 69 , 70 , 71 ] ( Figure 5 ). It has been suggested that cell–matrix interactions like these are mechanisms by which the trabecular meshwork can regulate intraocular pressure homeostasis [ 30 ], and elements in these interactive pathways, such as Rho kinase inhibitors, have shown promise as therapeutic targets [ 71 , 72 , 73 ].…”

Topical Medication Therapy for Glaucoma and Ocular Hypertension

“…Y-27632 is used in the treatment of OHT since it modulates the cytoskeletal alterations in TM cells to increase the conventional outflow, and relax the ciliary muscle contraction ( Chen et al, 2020 ; Luo et al, 2021 ). At present, there is no related clinical study, but glaucomatous transgenic mice experiment demonstrated that Y-27632 can significantly decreased IOP and increased outflow facility, which greatly influenced the long-term IOP-lowering effect ( Chen et al, 2020 ).…”

RhoA/ROCK-YAP/TAZ Axis Regulates the Fibrotic Activity in Dexamethasone-Treated Human Trabecular Meshwork Cells

“…In addition, RhoA induced ECM accumulation and activated myofibroblast-like cells. These findings indicated that DEX may induce the fibrotic activity in HTM cells through the RhoA/ROCK pathway and that RhoA probably serves as an intervening factor for lowering IOP ( Rao et al, 2017 ; Tan et al, 2018 ; Tan et al, 2019 ; Chen et al, 2020 ; Tan et al, 2020 ).…”

“…RhoAG14V, a constitutively active variant of RhoA, has been reported to induce ECM synthesis ( Pattabiraman and Rao, 2010 ) and decrease AH outflow through the TM in anterior chambers ( Pattabiraman et al, 2015 ). Other studies have suggested that the RhoA/ROCK plays an important role in regulating IOP, which was reduced in the presence of RhoA inhibitor C3 transferase ( Tan et al, 2018 ; Tan et al, 2019 ; Tan et al, 2020 ) and ROCK inhibitor Y-27632 ( Pattabiraman et al, 2015 ; Chen et al, 2020 ). Based on these findings, it was demonstrated that DEX not only increased the expression level of RhoA but also activated ROCK.…”