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

Wenshi Chen,Xuejiao Yang,Wei Zhu,Yuqing Zhang,Xian Yang,Jingwang Fang

doi:10.3389/fphar.2020.00302

Wenshi Chen, Xuejiao Yang + Show 4 more

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https://doi.org/10.3389/fphar.2020.00302

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Abstract

PurposeContinuous reductions in trabecular meshwork (TM) cellularity inhibit aqueous humor (AH) outflow, which is the main cause of primary open-angle glaucoma. Rho-associated 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.MethodsImmortalized human TM (iHTM) cells, glaucomatous TM (GTM3) 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-MYOCY437H 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.ResultsY-27632 (100 μM) significantly promoted the proliferation of both immortal TM cells and pTM cells. In GTM3 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-MYOCY437H 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-MYOCY437H mice was significantly improved after Y-27632 administration.ConclusionY-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.

Highlights

Glaucoma is the leading cause of irreversible blindness worldwide, and pathological intraocular pressure (IOP) is the main underlying pathogenic factor
Our results suggest that the Rho-associated protein kinase inhibitor (ROCKi) Y-27632 promotes Trabecular meshwork (TM) cell proliferation and phagocytosis and significantly reduces long-term IOP and increases aqueous humor (AH) outflow
We compared the expression of myocilin, a glucocorticoid-inducible gene in the TM cells

Summary

Introduction

Glaucoma is the leading cause of irreversible blindness worldwide, and pathological intraocular pressure (IOP) is the main underlying pathogenic factor. Trabecular meshwork (TM) cells are the primary cells in the conventional outflow pathway of AH (Stamer and Clark, 2017). Accelerated loss of TM cells hinders the activity of the conventional outflow pathway, leading to increased IOP via a decreased outflow facility, which is the main risk factor for primary open-angle glaucoma (POAG) (Stamer et al, 2015). The functions of ROCKs are not entirely understood, the Rho/ROCK signaling pathway has been confirmed to participate in many processes, such as cell contraction, adhesion, migration and invasion, transformation, phagocytosis, and proliferation (Riento and Ridley, 2003). An important study demonstrated that the Rho/ROCK signaling pathway plays an essential role in TM function, AH outflow, and IOP (Rao et al, 2017)

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