Comparison of the Drug-Induced Efficacies between Omidenepag Isopropyl, an EP2 Agonist and PGF2α toward TGF-β2-Modulated Human Trabecular Meshwork (HTM) Cells

Abstract

To compare the drug-induced efficacies between omidenepag (OMD), an EP2 agonist, and prostaglandin F2α (PGF2α) on glaucomatous trabecular meshwork (TM) cells, two- and three-dimensional (2D and 3D) cultures of TGF-β2-modulated human trabecular meshwork (HTM) cells were used. The following analyses were performed: (1) transendothelial electrical resistance (TEER) and FITC-dextran permeability measurements (2D), (2) the size and stiffness of the 3D spheroids, and (3) the expression (both 2D and 3D) by several extracellular matrix (ECM) molecules including collagen (COL) 1, 4 and 6, and fibronectin (FN), and α smooth muscle actin (αSMA), tight junction (TJ)-related molecules, claudin11 (Cldn11) and ZO1, the tissue inhibitor of metalloproteinase (TIMP) 1-4, matrix metalloproteinase (MMP) 2, 9 and 14, connective tissue growth factor (CTGF), and several endoplasmic reticulum (ER) stress-related factors. TGF-β2 significantly increased the TEER values and decreased FITC-dextran permeability, respectively, in the 2D HTM monolayers, and induced the formation of downsized and stiffer 3D HTM spheroids. TGF-β2-induced changes in TEER levels and FITC-dextran permeability were remarkably inhibited by PGF2α. PGF2α induced increases in the sizes and stiffness of the TGF-β2-treated 3D spheroids, but OMD enhanced only spheroid size. Upon exposure to TGF-β2, the expression of most of the molecules that were evaluated were significantly up-regulated, except some of ER stress-related factors were down-regulated. TJ-related molecules or ER stress-related factors were significantly up-regulated (2D) or down-regulated (3D), and down-regulated (2D) by PGF2α and OMD, while both drugs altered the expression of some of the other genes in the 3D spheroids in a different manner. The findings presented herein suggest that PGF2α and OMD differently modulate the permeability of the TGFβ2-modulated 2D monolayers and the physical properties of the 3D HTM spheroids.

Keywords: 3D spheroid cultures; PGF2α; TGFβ2; human trabecular meshwork; omidenepag; omidenepag isopropyl.

Figure 1

Effects of PGF2α and the EP2 agonist, omidenepag (OMD), on barrier functions of TGFβ2-treated 2D HTM monolayers by transendothelial electrical resistance (TEER) (A) and FITC-dextran permeability (B). To compare the effects of 100 nM PGF2α (PG) and omidenepag (OMD) on the barrier functions of 5 ng/mL TGF-β2 (TGFβ)-treated 2D HTM monolayers, TEER values (Ωcm²) and FITC-dextran permeability were measured in addition to the TGFβ untreated control, and these values were plotted in panel A and B, respectively. “+” is reagents addition. All experiments were performed using fresh preparations (n = 3–4). All data are presented as the arithmetic mean ± standard error of the mean (SEM). *** p < 0.005 (ANOVA followed by a Tukey’s multiple comparison test).

Figure 2

Effects of PGF2α and the EP2 agonist, omidenepag (OMD), on sizes of TGFβ2-treated 3D HTM spheroids at days 1, 3, or 6.To compare the effects of 100 nM PGF2α (PG) and omidenepag (OMD) on the mean sizes of the 5 ng/mL TGF-β2 (TGFβ) HTM 3D spheroids, measurements were made on days 1, 3, or 6 in addition to TGFβ untreated controls, and plotted (n = 10–12 3D spheroids for each experimental condition). “+” is reagents addition. Data that are presented are the arithmetic mean ± standard error of the mean (SEM), and statistical differences were determined by ANOVA followed by a Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01.

Figure 3

Effects of PGF2α and the EP2 agonist, omidenepag (OMD), on stiffness of TGFβ2-treated 3D HTM spheroids.To compare the effects of 100 nM PGF2α (PG) and omidenepag (OMD) on the stiffness of the 5 ng/mL TGF-β2 (TGFβ) HTM 3D spheroids, those at day 6 were subjected to a micro-squeezer analysis in addition to TGFβ untreated controls. The required force (μN) was measured and force/displacement (μN/μm) were plotted (n = 10–12 3D spheroids in each experimental condition). “+” is reagents addition. Data presented are the arithmetic mean ± standard error of the mean (SEM), and statistical differences were determined by ANOVA followed by a Tukey’s multiple comparison test. ** p < 0.01.

Figure 4

Effects of PGF2α and the EP2 agonist, omidenepag (OMD), on the mRNA expression of ECM proteins in 2D- and 3D-cultured HTM cells. At Day 6, HTM 2D cells and 3D spheroids (NT: non-treated control) and those treated with a 5 ng/mL solution of TGF-β2 (TGFβ) in the absence and presence of 100 nM PGF2α (PG) or the EP2 agonist, omidenepag (OMD) were subjected to qPCR analysis to estimate the expression of mRNA in ECMs (COL1, COL4, COL6, FN and αSMA). Analyses were performed in triplicate using fresh preparations (n = 12–15 3D spheroids each). “+” is reagents addition. Data presented are the arithmetic mean ± standard error of the mean (SEM), and statistical differences were determined by ANOVA followed by a Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.005.

Figure 5

Immunofluorescence images of the expressed COL6 in 3D HTM spheroids. At Day 6, 3D spheroids (NT: non-treated control) and those treated with a 5 ng/mL solution of TGF-β2 (TGFβ) in the absence and presence of 100 nM PGF2α (PG) or the EP2 agonist, omidenepag (OMD) were immunostained with specific antibodies against COL6 (green), DAPI (blue) and phalloidin (Phal, red). Representative immunolabeling by anti-COL6 are shown in panel (A) (Scale bar: 100 µm) and the intensities of staining are plotted in panel (B). All experiments were performed in duplicate using fresh preparations consisting of 10 spheroids each. Data presented (total n = 20 different 3D spheroids’ images) are the arithmetic mean ± standard error of the mean (SEM), and statistical differences were determined by ANOVA followed by a Tukey’s multiple comparison test. ** p < 0.01.

Figure 6

Effects of PGF2α and the EP2 agonist, omidenepag (OMD), on the mRNA expressions of tight junction related proteins in 2D- and 3D-cultured HTM cells. At Day 6, 2D and 3D cultured HTM cells (NT: non-treated control) and those treated with a 5 ng/mL solution of TGF-β2 (TGFβ) in the absence and presence of 100 nM PGF2α (PG) or the EP2 agonist, omidenepag (OMD) were subjected to mRNA expression analysis of mRNA in claudin11 (Cldn11) and ZO1. Analyses were performed in triplicate using fresh preparations (n = 12–15 3D spheroids each). Data presented are the arithmetic mean ± standard error of the mean (SEM), and statistical differences were determined by ANOVA followed by a Tukey’s multiple comparison test. * p < 0.05, ** p < 0.01, *** p < 0.005.