All- trans Retinoic Acids Synergistically and Beneficially Affect In Vitro Glaucomatous Trabecular Meshwork (TM) Models Using 2D and 3D Cell Cultures of Human TM Cells

Abstract

We report herein on the effects of all-trans retinoic acid (ATRA) on two-dimensional (2D) and three-dimensional (3D) cultures of human trabecular meshwork (HTM) cells that were treated with transforming growth factor β2 (TGF-β2). In the presence of 5 ng/mL TGF-β2, the effects of ATRA on the following were observed: (1) the barrier function of the 2D HTM monolayers, as determined by trans-endothelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC) dextran permeability measurements; (2) a Seahorse cellular bio-metabolism analysis; (3) physical properties, including the size and stiffness, of 3D spheroids; (4) the gene expression of extracellular matrix (ECM) molecules, ECM modulators including tissue inhibitor of metalloproteinases (TIMPs), matrix metalloproteinases (MMPs), tight junction (TJ)-related molecules, and endoplasmic reticulum (ER)-stress-related factors. ATRA significantly inhibited the TGF-β2-induced increase in the TEER values and FITC dextran permeability of the 2D monolayers, while an ATRA monotreatment induced similar effects as TGF-β2. A real-time metabolic analysis revealed that ATRA significantly inhibited the TGF-β2-induced shift in metabolic reserve from mitochondrial oxidative phosphorylation to glycolysis in 2D HTM cells, whereas ATRA alone did not induce significant metabolic changes. In contrast, ATRA induced the formation of substantially downsized and softer 3D spheroids in the absence and presence of TGF-β2. The different effects induced by ATRA toward 2D and 3D HTM cells were also supported by the qPCR analysis of several proteins as above. The findings reported here indicate that ATRA may induce synergistic and beneficial effects on TGF-β2-treated 2D- and 3D-cultured HTM cells; those effects varied significantly between the 2D and 3D cultures.

Keywords: TGF-β2; brimonidine; human trabecular meshwork (HTM); three-dimensional spheroid cultures; α2-adrenergic agonist.

Figure 1

Effects of all-trans retinoic acid (ATRA) on trans-endothelial electrical resistance (TEER) values and FITC dextran permeability of TGF-β2-treated 2D cultures of HTM cell monolayers. To evaluate the effects of 10 μM ATRA on the barrier function (Ωcm²) of 2D-cultured HTM monolayers, untreated cultures, and cultures that had been treated with 5 ng/mL TGF-β2 (TGFβ), measurements by TEER (A) and FITC dextran permeability (B) were performed. “+” is reagents addition. “-” is reagents non-addition. All experiments were performed in triplicate using fresh preparations (n = 4, total 12). Data are presented as the mean ± standard error of the mean (SEM). *** p < 0.005; ANOVA followed by a Tukey’s multiple comparison test.

Figure 2

Effects of all-trans retinoic acid (ATRA) on a real-time cellular metabolism analysis of 2D-cultured HTM cells. Two-dimensionally cultured HTM cells untreated (NT—nontreated control) or treated with TGF-β2 (TGF, 5 ng/mL) and/or ATRA (10 μM) were subjected to a real-time metabolic function analysis using a Seahorse XFe96 Bioanalyzer. Panel (A,B): simultaneous measurements of OCR (A) and ECAR (B) at baseline and those subsequently supplemented with Oligo (a complex V inhibitor), FCCP (a protonophore), rotenone/antimycin A (R/A, complex I/III inhibitors), and 2DG (a hexokinase inhibitor). (C,D) Key parameters of mitochondrial respiration (C) and glycolytic flux (D). Basal OCR value was obtained by subtracting OCR with rotenone/antimycin A from baseline OCR. The adenosine triphosphate (ATP)-linked respiration was estimated by subtracting OCR with Oligo from baseline OCR. Proton leak was calculated by subtracting OCR with rotenone/antimycin A from OCR with Oligo. Maximal respiration was calculated by subtracting OCR with rotenone/antimycin A from OCR with FCCP. Spare respiratory capacity was calculated by subtracting baseline OCR from OCR with FCCP. Nonmitochondrial respiration was defined as OCR with rotenone/antimycin A. Basal ECAR was calculated by subtracting ECAR with 2DG from baseline ECAR. Glycolytic capacity was calculated by subtracting ECAR with 2DG from ECAR with Oligo. Glycolytic reserve was calculated by subtracting baseline ECAR from ECAR with oligomycin. Non-glycolytic acidification was defined as the final value of ECAR with 2DG. All experiments were performed in triplicated using fresh preparations (n = 5, total 15). Data are presented as the mean ± SEM. * p < 0.05; ANOVA followed by a Tukey’s multiple comparison test.

Figure 3

Effects of all-trans retinoic acid (ATRA) on the physical properties, size (A), and stiffness (B) of TGF-β2-treated 3D HTM spheroids. Using nontreated control (NT) cultures and cultures treated with 5 ng/mL TGF-β2 (TGFβ) in the absence or presence of 10 μM ATRA, the mean size areas in the 3D HTM spheroids are plotted in (A). Their hardness was estimated by measurement of the force (μN) inducing a 50% semidiameter using a micro-squeezer, and force/displacement (μN/μm) values are plotted in (B). “+” is reagents addition. “-” is reagents non-addition. These experiments were performed in triplicate using fresh preparations (n = 10, 30 and 15, total 45, for size measurement and stiffness analysis, respectively). Data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005; ANOVA followed by a Tukey’s multiple comparison test.

Figure 4

Effects of ATRA on the gene expression of ECM molecules in TGF-β2-treated 2D- and 3D-cultured HTM cells. Two- and three-dimensionally cultured HTM cells untreated (NT) or treated with 5 ng/mL TGF-β2 (TGFβ) and/or ATRA (10 μM) were subjected to quantitative polymerase chain reaction (qPCR) analysis to estimate the expression of mRNA in ECMs (COL1, COL4, COL6, FN, and αSMA). All experiments were performed in triplicate each using freshly prepared 2D HTM cells and 3D HTM spheroids (n = 15–20, total 45–60) in each experimental condition. Data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005; ANOVA followed by a Tukey’s multiple comparison test.

Figure 5

Effects of ATRA on the mRNA expression of TIMPs and MMPs in TGF-β2-treated 2D- and 3D-cultured HTM cells. Two- and three-dimensionally cultured HTM cells untreated (NT; nontreated control) or treated with 5 ng/mL TGF-β2 (TGFβ) and/or ATRA (10 μM) in the absence or presence of 10 μM ATRA were subjected to qPCR analysis to estimate the expression of mRNA in TIMP1–4, and MMP 2, 9, and 14. All experiments were performed in triplicate each using freshly prepared 2D HTM cells and 3D HTM spheroids (n = 15–20, total 45–60) in each experimental condition. Data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005; ANOVA followed by a Tukey’s multiple comparison test.

Figure 6

Effects of ATRA on the mRNA expression of TJ-related molecules and major ER-stress-related genes in TGF-β2-treated 2D- and 3D-cultured HTM cells. Two- and three-dimensionally cultured HTM cells untreated (NT) or treated with 5 ng/mL TGF-β2 (TGFβ) and/or ATRA (10 μM) were subjected to qPCR analysis to estimate the expression of mRNA in TJ-related molecules, ZO1 and CLDN11, and major ER-stress-related genes including the X-box binding protein (XBP), sXBP, glucose regulator protein (GRP)78, GRP94, and CCAAT/enhancer-binding protein homologous protein (CHOP). All experiments were performed in triplicate each using freshly prepared 2D HTM cells and 3D HTM spheroids (n = 15–20, total 45–60) in each experimental condition. Data are presented as the mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.005; ANOVA followed by a Tukey’s multiple comparison test.