Differential Effects of Benzalkonium Chloride on Human Trabecular Meshwork Cells Not Treated or Treated with Transforming Growth Factor-ß2 or Dexamethasone.

Purpose: The objective of the present study was to evaluate the effects of low concentrations of benzalkonium chloride (BAC) (10-7%, 10-6%, or 10-5%) on healthy and glaucomatous human trabecular meshwork (HTM) cells. For this purpose, we used in vitro models replicating a healthy HTM and HTM with primary open-angle glaucoma (POAG) or steroid-induced glaucoma (SG) using two-dimensional (2D) cultures of HTM cells not treated or treated with a 5 ng/mL solution of transforming growth factor-ß2 or 250 nM dexamethasone (DEX). Methods: Analyses were carried out for (1) the intercellular affinity function of 2D HTM monolayers, as determined by transepithelial electrical resistance (TEER) measurements; (2) cell viability; (3) cellular metabolism by using a Seahorse bioanalyzer; and (4) expression of extracellular matrix (ECM) molecules, an ECM modulator, and cell junction-related molecules. Results: In the absence and presence of BAC (10-7% or 10-5%), intercellular affinity function determined by TEER and cellular metabolic activities were significantly and dose dependently affected in both healthy and glaucomatous HTM cells despite the fact that there was no significant decrease in cell viabilities. However, the effects based on TEER values were significantly greater in the healthy HTM. The mRNA expression of several molecules that were tested was not substantially modulated by these concentrations of BAC. Conclusions: The findings reported herein suggest that low concentrations of BAC may have unfavorable adverse effects on cellular metabolic capacity by inducing increases in the intercellular affinity properties of the HTM, but those effects of BAC were different in healthy and glaucomatous HTM cells.

TGF-ß-3 Induces Different Effects from TGF-ß-1 and -2 on Cellular Metabolism and the Spatial Properties of the Human Trabecular Meshwork Cells.

To compare the effects among three TGF-ß isoforms (TGF-ß-1, TGF-ß-2, and TGF-ß-3) on the human trabecular meshwork (HTM), two-dimensional (2D) and three-dimensional (3D) cultures of commercially available certified immortalized HTM cells were used, and the following analyses were conducted: (1) trans-endothelial electrical resistance (TEER) and FITC dextran permeability measurements (2D); (2) a real-time cellular metabolic analysis (2D); (3) analysis of the physical property of the 3D HTM spheroids; and (4) an assessment of the gene expression levels of extracellular matrix (ECM) components (2D and 3D). All three TGF-ß isoforms induced a significant increase in TEER values and a relative decrease in FITC dextran permeability in the 2D-cultured HTM cells, but these effects were the most potent in the case of TGF-ß-3. The findings indicated that solutions containing 10 ng/mL of TGF-ß-1, 5 ng/mL of TGF-ß-2, and 1 ng/mL of TGF-ß-3 had nearly comparable effects on TEER measurements. However, a real-time cellular metabolic analysis of the 2D-cultured HTM cells under these concentrations revealed that TGF-3-ß induced quite different effects on the metabolic phenotype, with a decreased ATP-linked respiration, increased proton leakage, and decreased glycolytic capacity compared with TGF-ß-1 and TGF-ß-2. In addition, the concentrations of the three TGF-ß isoforms also caused diverse effects on the physical properties of 3D HTM spheroids and the mRNA expression of ECMs and their modulators, in many of which, the effects of TGF-ß-3 were markedly different from TGF-ß-1 and TGF-ß-2. The findings presented herein suggest that these diverse efficacies among the TGF-ß isoforms, especially the unique action of TGF-ß-3 toward HTM, may induce different effects within the pathogenesis of glaucoma.

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.

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 ß2 (TGF-ß2). In the presence of 5 ng/mL TGF-ß2, 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-ß2-induced increase in the TEER values and FITC dextran permeability of the 2D monolayers, while an ATRA monotreatment induced similar effects as TGF-ß2. A real-time metabolic analysis revealed that ATRA significantly inhibited the TGF-ß2-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-ß2. 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-ß2-treated 2D- and 3D-cultured HTM cells; those effects varied significantly between the 2D and 3D cultures.

Hypoxia Differently Affects TGF-ß2-Induced Epithelial Mesenchymal Transitions in the 2D and 3D Culture of the Human Retinal Pigment Epithelium Cells.

The hypoxia associated with the transforming growth factor-ß2 (TGF-ß2)-induced epithelial mesenchymal transition (EMT) of human retinal pigment epithelium (HRPE) cells is well recognized as the essential underlying mechanism responsible for the development of proliferative retinal diseases. In vitro, three-dimensional (3D) models associated with spontaneous O2 gradients can be used to recapitulate the pathological levels of hypoxia to study the effect of hypoxia on the TGF-ß2-induced EMT of HRPE cells in detail, we used two-dimensional-(2D) and 3D-cultured HRPE cells. TGF-ß2 and hypoxia significantly and synergistically increased the barrier function of the 2D HRPE monolayers, as evidenced by TEER measurements, the downsizing and stiffening of the 3D HRPE spheroids and the mRNA expression of most of the ECM proteins. A real-time metabolic analysis indicated that TGF-ß2 caused a decrease in the maximal capacity of mitochondrial oxidative phosphorylation in the 2D HRPE cells, whereas, in the case of 3D HRPE spheroids, TGF-ß2 increased proton leakage. The findings reported herein indicate that the TGF-ß2-induced EMT of both the 2D and 3D cultured HRPE cells were greatly modified by hypoxia, but during these EMT processes, the metabolic plasticity was different between 2D and 3D HRPE cells, suggesting that the mechanisms responsible for the EMT of the HRPE cells may be variable during their spatial spreading.