Screening of the Drug-Induced Effects of Prostaglandin EP2 and FP Agonists on 3D Cultures of Dexamethasone-Treated Human Trabecular Meshwork Cells.

Megumi Watanabe,Yosuke Ida,Masato Furuhashi,Yuri Tsugeno,Fumihito Hikage,Hiroshi Ohguro

doi:10.3390/biomedicines9080930

Abstract

The objective of the current study was to perform a screening of the drug-induced effects of the prostaglandin F2α (PGF2α) and EP2 agonist, omidenepag (OMD), using two- and three-dimensional (2D and 3D) cultures of dexamethasone (DEX)-treated human trabecular meshwork (HTM) cells. The drug-induced effects on 2D monolayers were characterized by measuring the transendothelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)–dextran permeability, the physical properties of 3D spheroids, and the gene expression of extracellular matrix (ECM) molecules, including collagen (COL) 1, 4 and 6, and fibronectin (FN), α smooth muscle actin (αSMA), a tissue inhibitor of metalloproteinase (TIMP) 1–4, matrix metalloproteinase (MMP) 2, 9 and 14 and endoplasmic reticulum (ER) stress-related factors. DEX induced a significant increase in TEER values and a decrease in FITC–dextran permeability, respectively, in the 2D HTM monolayers, and these effects were substantially inhibited by PGF2α and OMD. Similarly, DEX also caused decreased sizes and an increased stiffness in the 3D HTM spheroids, but PGF2α or OMD had no effects on the stiffness of the spheroids. Upon exposure to DEX, the following changes were observed: the upregulation of COL4 (2D), αSMA (2D), and TIMP4 (2D and 3D) and the downregulation of TIMP1 and 2 (3D), MMP2 and 14 (3D), inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6) (2D), and glucose regulator protein (GRP)78 (3D). In the presence of PGF2α or OMD, the downregulation of COL4 (2D), FN (3D), αSMA (2D), TIMP3 (3D), MMP9 (3D) and the CCAAT/enhancer-binding protein homologous protein (CHOP) (2D), and the upregulation of TIMP4 (2D and 3D), MMP2, 9 and 14 (2D), respectively, were observed. The findings presented herein suggest that 2D and 3D cell cultures can be useful in screening for the drug-induced effects of PGF2α and OMD toward DEX-treated HTM cells.

Highlights

The mechanism responsible for the intraocular pressures (IOPs) elevation has been proposed to involve an increase in the resistance to the outflow of aqueous media from the trabecular meshwork (TM), presumably caused by the deposition of higher than normal levels of extracellular matrix (ECM) molecules such as collagen molecules (COLs), fibronectin (FN), and others leading to the development of both primary open-angle glaucoma (POAG) and steroid-induced glaucoma (SG) [4,5]
Monolayers may be FP- and EP2-dependent, but the effects caused by the EP2 agonist were cultured human trabecular meshwork (HTM) monolayers may be FP- and EP2-dependent, but the effects caused by the greater thatwere those caused by the
A number of 3D culture methods have been developed recently and include scaffoldassisted techniques such as gel matrix and micro-carriers, or liquid cultures on lowattachment plates, in hanging drops or in rotation [31,32]. These 3D culture methods have many advantages compared to conventional 2D cultures, there are practical problems associated with each method [31,32,33,34,35,36]

Summary

Introduction

It is well recognized that glaucomatous optic neuropathy (GON), a progressive and chronic optic neuropathy caused by elevated intraocular pressures (IOPs), leads to the development of irreversible blindness [1,2,3]. The mechanism responsible for the IOP elevation has been proposed to involve an increase in the resistance to the outflow of aqueous media from the trabecular meshwork (TM), presumably caused by the deposition of higher than normal levels of extracellular matrix (ECM) molecules such as collagen molecules (COLs), fibronectin (FN), and others leading to the development of both primary open-angle glaucoma (POAG) and steroid-induced glaucoma (SG) [4,5]. Biomedicines 2021, 9, 930 to the administration of anti-glaucoma medication as the initial therapy, laser treatment or surgical intervention are sometimes required for the treatment of GON [6].

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Conclusion