Benzalkonium chloride greatly deteriorates the biological activities of human corneal stroma fibroblasts in a concentration-dependent manner.

BACKGROUND: Corneal tissues indirectly obtain nutritional needs and oxygen to maintain their homeostasis, and therefore, benzalkonium chloride (BAC) containing ocular instillations for medical therapy may, in turn, induce toxic effects more than expected in corneal tissues, especially the inside stroma layer. METHODS: To evaluate the effects of very low concentrations (10-8%, 10-6%, or 10-4%) of BAC on human corneal stroma, we used two-dimensional (2D) cultures of human corneal stromal fibroblast (HCSF) cells and carried out the following analyses: (1) cell viability measurements, (2) Seahorse cellular bio-metabolism analysis, and (3) the expression of ECM molecules and endoplasmic reticulum (ER) stress-related molecules. RESULTS: In the absence and presence of 10-8%, 10-6%, or 10-4% concentrations of BAC, cell viability deteriorated and this deterioration was dose-dependent. The results showed that maximal mitochondrial respiration was decreased, the mRNA expression of most of ECM proteins was decreased, and ER stress-related molecules were substantially and dose-dependently down-regulated in HCSFs by the BAC treatment. CONCLUSIONS: The findings reported herein indicate that the presence of BAC, even at such low concentrations, is capable of causing the deterioration of cellular metabolic functions and negatively affecting the response to ER stress in HCSF cells resulting in a substantially decreased cellular viability.

3D culture induction of adipogenic differentiation in 3T3-L1 preadipocytes exhibits adipocyte-specific molecular expression patterns and metabolic functions.

Adipose tissues are closely related to physiological functions and pathological conditions in most organs. Although differentiated 3T3-L1 preadipocytes have been used for in vitro adipose studies, the difference in cellular characteristics of adipogenic differentiation in two-dimensional (2D) culture and three-dimensional (3D) culture remain unclear. In this study, we evaluated gene expression patterns using RNA sequencing and metabolic functions using an extracellular flux analyzer in 3T3-L1 preadipocytes with and without adipogenic induction in 2D culture and 3D culture. In 2D culture, 565 up-regulated genes and 391 down-regulated genes were identified as differentially expressed genes (DEGs) by adipogenic induction of 3T3-L1 preadipocytes, whereas only 69 up-regulated genes and 59 down-regulated genes were identified as DEGs in 3D culture. Ingenuity Pathway Analysis (IPA) revealed that genes associated with lipid metabolism were identified as 2 out of the top 3 causal networks related to diseases and function in 3D spheroids, whereas only one network related to lipid metabolism was identified within the top 9 of these causal networks in the 2D planar cells, suggesting that adipogenic induction in the 3D culture condition exhibits a more adipocyte-specific gene expression pattern in 3T3-L1 preadipocytes. Real-time metabolic analysis revealed that the metabolic capacity shifted from glycolysis to mitochondrial respiration in differentiated 3T3-L1 cells in the 3D culture condition but not in those in the 2D cultured condition, suggesting that adipogenic differentiation in 3D culture induces a metabolic phenotype of well-differentiated adipocytes. Consistently, expression levels of mitochondria-encoded genes including mt-Nd6, mt-Cytb, and mt-Co1 were significantly increased by adipogenic induction of 3T3-L1 preadipocytes in 3D culture compared with those in 2D culture. Taken together, the findings suggest that induction of adipogenesis in 3D culture provides a more adipocyte-specific gene expression pattern and enhances mitochondrial respiration, resulting in more adipocyte-like cellular properties.

TGF-ß2 Induces Epithelial-Mesenchymal Transitions in 2D Planer and 3D Spheroids of the Human Corneal Stroma Fibroblasts in Different Manners.

To examine the epithelial-mesenchymal transition (EMT) that is induced on the human corneal stroma, two- and three-dimensional (2D and 3D) cultures of human corneal stroma fibroblasts (HCSFs) were used. In this study, HCSF 2D monolayers and 3D spheroids were characterized by (1) scanning electron microscopy (SEM), (2) trans-endothelial electrical resistance (TEER) measurements and fluorescein isothiocyanate (FITC)-dextran permeability, (3) cellular metabolic measurements, (4) the physical properties of 3D HCSF spheroids, and (5) the extracellular matrix (ECM) molecule gene expressions, including collagen (COL) 1, 4 and 6, and fibronectin (FN), a tissue inhibitor of metalloproteinase (TIMP) 1-4, matrix metalloproteinase (MMP) 2, 3, 9 and 14, and several endoplasmic reticulum (ER) stress-related factors. In the 2D HCSFs, TGF-ß2 concentration-dependently generated (1) a considerable increase in ECM deposits revealed by SEM, (2) an increase in TEER values and a decrease in FITC-dextran permeability, (3) increases in both mitochondrial and glycolytic functions, and a substantial upregulation of COL1, COL4, FN, αSMA, TIMP1, TIMP, and most ER stress-related genes and the downregulation of COL6 and MMP3. In the case of 3D spheroids, TGF-ß2 induced the downsizing and stiffening of 3D spheroids and the upregulation of COL6, MMP14, and most ER stress-related genes. These findings suggest that TGF-ß2 significantly induced a number of EMT-associated biological events including planar proliferation, cellular metabolic functions, and the production of ECM molecules in the 2D cultured HCSF cells, but these effects were significantly less pronounced in the case of 3D HCSF spheroids.

Plasma free amino acid profiles are associated with serum high molecular weight adiponectin levels in Japanese medical check-up population without type 2 diabetes mellitus.

To prevent the progression of type 2 diabetes mellitus (T2DM), early detection and intervention are important. Several studies have already shown that the serum adiponectin level could be useful for evaluating the future risk of T2DM. Recently, plasma free amino acid (PFAA) concentrations have also emerged as potential biomarkers that predict the future onset of T2DM. In this study, we aimed to further characterise PFAA profiles by elucidating the association with the serum high molecular weight (HMW) adiponectin level in this cross-sectional study. A total of 1000 Japanese subjects who underwent medical check-ups were enrolled, and their plasma concentrations of 21 amino acids and clinical parameters were measured. The subjects without T2DM were divided into quartiles (Q1-4) by serum HMW adiponectin level, and the association with between PFAA concentrations was analysed. Concentrations of glutamate, alanine, proline, tyrosine, histidine, methionine, lysine, branched-chain amino acids (BCAAs) and tryptophan varied significantly according to the adiponectin quartile. Furthermore, serum adiponectin levels showed significant inverse correlations with these amino acids. The change in the PFAA profile in the group with the lowest adiponectin concentrations (Q1) was similar to that of T2DM patients. Although both adiponectin levels and PFAA concentrations are known to be altered by the accumulation of visceral fat and insulin resistance, the levels of glutamate, BCAA, lysine and tryptophan remain significantly associated with adiponectin level after adjustment for age, body mass index and homeostasis model assessment of insulin resistance, showing the direct association between PFAA concentrations and the serum HMW adiponectin level. Registration number: University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR) UMIN000029920, registered on Nov 13th 2017 (prospectively registered).

Unexpected Crosslinking Effects of a Human Thyroid Stimulating Monoclonal Autoantibody, M22, with IGF1 on Adipogenesis in 3T3L-1 Cells.

To study the effects of the crosslinking of IGF1 and/or the human thyroid-stimulating monoclonal autoantibody (TSmAb), M22 on mouse adipocytes, two- and three-dimensional (2D or 3D) cultures of 3T3-L1 cells were prepared. Each sample was then subjected to the following analyses: (1) lipid staining, (2) a real-time cellular metabolic analysis, (3) analysis of the mRNA expression of adipogenesis-related genes and extracellular matrix (ECM) molecules including collagen (Col) 1, 4 and 6, and fibronectin (Fn), and (4) measurement of the size and physical properties of the 3D spheroids with a micro-squeezer. Upon adipogenic differentiation (DIF+), lipid staining and the mRNA expression of adipogenesis-related genes in the 2D- or 3D-cultured 3T3-L1 cells substantially increased. On adding IGF1 but not M22 to DIF+ cells, a significant enhancement in lipid staining and gene expressions of adipogenesis-related genes was detected in the 2D-cultured 3T3-L1 cells, although some simultaneous suppression or enhancement effects by IGF1 and M22 against lipid staining or Fabp4 expression, respectively, were detected in the 3D 3T3-L1 spheroids. Real-time metabolic analyses indicated that monotherapy with IGF1 or M22 shifted cellular metabolism toward energetic states in the 2D 3T3-L1 cells upon DIF+, although no significant metabolic changes were induced by DIF+ alone in 2D cultures. In addition, some synergistical effects on cellular metabolism by IGF1 and M22 were also observed in the 2D 3T3-L1 cells as well as in cultured non-Graves' orbitopathy-related human orbital fibroblasts (n-HOFs), but not in Graves' orbitopathy-related HOFs (GHOFs). In terms of the physical properties of the 3D 3T3-L1 spheroids, (1) their sizes significantly increased upon DIF+, and this increase was significantly enhanced by the presence of both IGF1 and M22 despite downsizing by monotreatment, and (2) their stiffness increased substantially, and no significant effects by IGF-1 and/or M22 were observed. Regarding the expression of ECM molecules, (1) upon DIF+, significant downregulation or upregulation of Col1 and Fn (3D), or Col4 and 6 (2D and 3D) were observed, and (2) in the presence of IGF-1 and/or M22, the mRNA expression of Col4 was significantly downregulated by M22 (2D and 3D), but the expression of Col1 was modulated in different manners by monotreatment (upregulation) or the combined treatment (downregulation) (3D). These collective data suggest that the human-specific TSmAb M22 induced some unexpected simultaneous crosslinking effects with IGF-1 with respect to the adipogenesis of 2D-cultured 3T3-L1 cells and the physical properties of 3D 3T3-L1 spheroids.

G-Protein-Coupled Receptors Mediate Modulations of Cell Viability and Drug Sensitivity by Aberrantly Expressed Recoverin 3 within A549 Cells.

To elucidate the currently unknown molecular mechanisms responsible for the aberrant expression of recoverin (Rec) within cancerous cells, we examined two-dimensional (2D) and three-dimensional (3D) cultures of Rec-negative lung adenocarcinoma A549 cells which had been transfected with a plasmid containing human recoverin cDNA (A549 Rec) or an empty plasmid as a mock control (A549 MOCK). Using these cells, we measured cytotoxicity by several anti-tumor agents (2D), cellular metabolism including mitochondrial and glycolytic functions by a Seahorse bio-analyzer (2D), the physical properties, size and stiffness of the 3D spheroids, trypsin sensitivities (2D and 3D), and RNA sequencing analysis (2D). Compared with the A549 MOCK, the A549 Rec cells showed (1) more sensitivity toward anti-tumor agents (2D) and a 0.25% solution of trypsin (3D); (2) a metabolic shift from glycolysis to oxidative phosphorylation; and (3) the formation of larger and stiffer 3D spheroids. RNA sequencing analysis and bioinformatic analyses of the differentially expressed genes (DEGs) using Gene Ontology (GO) enrichment analysis suggested that aberrantly expressed Rec is most likely associated with several canonical pathways including G-protein-coupled receptor (GPCR)-mediated signaling and signaling by the cAMP response element binding protein (CREB). The findings reported here indicate that the aberrantly expressed Rec-induced modulation of the cell viability and drug sensitivity may be GPCR mediated.

Addition of ROCK Inhibitors Alleviates Prostaglandin-Induced Inhibition of Adipogenesis in 3T3L-1 Spheroids.

To elucidate the additive effects of the ROCK inhibitors (ROCK-i), ripasudil (Rip) and Y27632 on bimatoprost acid (BIM-A), a prostaglandin analog (PG), on adipose tissue, two- and three-dimensional (2D or 3D) cultures of 3T3-L1 cells, the most well characterized cells in the field of lipid research, were used. The cells were subjected to a variety of analyses including lipid staining, real-time cellular metabolic analysis, the mRNA expressions of genes related to adipogenesis and extracellular matrices (ECMs) as well as the sizes and physical properties of the 3D spheroids by a micro-squeezer. BIM-A induced strong inhibitory effects on most of the adipogenesis-related changes in the 2D and 3D cultured 3T3-L1 cells, including (1) the enlargement and softening of the 3D spheroids, (2) a dramatic enhancement in lipid staining and the expression of adipogenesis-related genes, and (3) a decrease in mitochondrial and glycolytic metabolic function. By adding ROCK-i to the BIM-A, most of these BIM-A-induced effects were cancelled. The collective findings reported herein suggest that ROCK-i eliminated the PG-induced suppression of adipogenesis in the 3T3-L1 cells, accompanied by the formation of enlarged 3D spheroids. Such effects of adding ROCK-i to a PG in preadipocytes on cellular properties appear to be associated with the suppression of PG-induced adverse effects, and provide additional insight into our understanding of lipid-related research.

The Selective α1 Antagonist Tamsulosin Alters ECM Distributions and Cellular Metabolic Functions of ARPE 19 Cells in a Concentration-Dependent Manner.

The purpose of the present study was to examine the effect of the selective α1 antagonist tamsulosin (TAM) on human retinal pigment epithelium cells, ARPE 19. Two-dimension (2D) and three-dimension (3D) cultured ARPE 19 cells were used in the following characterizations: (1) ultrastructure by scanning electron microscopy (SEM) (2D); (2) barrier functions by transepithelial electrical resistance (TEER) measurements, and FITC-dextran permeability (2D); (3) real time cellular metabolisms by Seahorse Bioanalyzer (2D); (4) physical properties, size and stiffness measurements (3D); and (5) expression of extracellular matrix (ECM) proteins, including collagen1 (COL1), COL4, COL6 and fibronectin (FN) by qPCR and immunohistochemistry (2D and 3D). TAM induced significant effects including: (1) alteration of the localization of the ECM deposits; (2) increase and decrease of the TEER values and FITC-dextran permeability, respectively; (3) energy shift from glycolysis into mitochondrial oxidative phosphorylation (OXPHOS); (4) large and stiffened 3D spheroids; and (5) down-regulations of the mRNA expressions and immune labeling of most ECM proteins in a concentration-dependent manner. However, in some ECM proteins, COL1 and COL6, their immunolabeling intensities were increased at the lowest concentration (1 µM) of TAM. Such a discrepancy between the gene expressions and immunolabeling of ECM proteins may support alterations of ECM localizations as observed by SEM. The findings reported herein indicate that the selective α1 antagonist, TAM, significantly influenced ECM production and distribution as well as cellular metabolism levels in a concentration-dependent manner.

Benzalkonium Chloride, Even at Low Concentrations, Deteriorates Intracellular Metabolic Capacity in Human Conjunctival Fibroblasts.

The objective of this study was to clarify the effects of benzalkonium chloride (BAC) on two-dimensional (2D) and three-dimensional (3D) cultures of human conjunctival fibroblast (HconF) cells, which are in vitro models replicating the epithelial barrier and the stromal supportive functions of the human conjunctiva. The cultured HconF cells were subjected to the following analyses in the absence and presence of 10-5% or 10-4% concentrations of BAC; (1) the barrier function of the 2D HconF monolayers, as determined by trans-endothelial electrical resistance (TEER) and FITC dextran permeability, (2) real-time metabolic analysis using an extracellular Seahorse flux analyzer, (3) the size and stiffness of 3D HconF spheroids, and (4) the mRNA expression of genes that encode for extracellular matrix (ECM) molecules including collagen (COL)1, 4 and 6, and fibronectin (FN), α-smooth muscle actin (α-SMA), ER stress related genes including the X-box binding protein-1 (XBP1), the spliced XBP1 (sXBP1) glucose regulator protein (GRP)78, GRP94, and the CCAAT/enhancer-binding protein homologous protein (CHOP), hypoxia inducible factor 1α (HIF1α), and Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α). In the presence of BAC, even at low concentrations at 10-5% or 10-4%, the maximal respiratory capacity, mitochondrial respiratory reserve, and glycolytic reserve of HconF cells were significantly decreased, although the barrier functions of 2D HconF monolayers, the physical properties of the 3D HconF spheroids, and the mRNA expression of the corresponding genes were not affected. The findings reported herein highlight the fact that BAC, even such low concentrations, may induce unfavorable adverse effects on the cellular metabolic capacity of the human conjunctiva.

All Trans-Retinoic Acids Facilitate the Remodeling of 2D and 3D Cultured Human Conjunctival Fibroblasts.

Vitamin A derivative, all-trans-retinoic acid (ATRA), is known to be a potent regulator of the growth and differentiation of various types of cells. In the present study, the unidentified effects of ATRA on superficial and vertical spreading conjunctival scarring were examined. The study involved the use of two-dimensional (2D) and three-dimensional (3D) cultures of human conjunctival fibroblast (HconF) cells in the presence or absence of TGF-ß2. The effects of ATRA (1 µM) on superficial or vertical spreading conjunctival scarring were evaluated by the barrier function by trans-endothelial electrical resistance (TEER) and FITC dextran permeability measurements and real-time metabolic analysis, as well as the physical properties, namely, the size and stiffness, of 3D spheroids, respectively. In addition, the expressions of several related molecules, including extracellular matrix (ECM) molecules, ECM modulators including a tissue inhibitor of metalloproteinases (TIMPs), matrix metalloproteinases (MMPs), and ER stress-related factors, were examined. ATRA significantly induced (1) an increase in TEER values and a decrease in FITC dextran permeability, respectively, in the 2D monolayers, and (2) relatively and substantially increased the size and stiffness, respectively, of the 3D spheroids. These ATRA-induced effects were further enhanced in the TGF-ß2-treated cells, whereas the TGF-ß2-induced enhancement in glycolytic capacity was canceled by the presence of ATRA. Consistent with these physical and morphological effects, the mRNA expressions of several molecules were significantly but differently induced between 2D and 3D cultures by ATRA, although the presence of TGF-ß2 did not substantially affect these gene expression levels. The findings reported in this study indicate that ATRA may exacerbate both superficial and vertical conjunctival fibrosis spreading independently of TGF-ß2-induced changes.

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.

FGF-2 enhances fibrogenetic changes in TGF-ß2 treated human conjunctival fibroblasts.

The objective of the current study was to examine the effects of fibroblast growth factor-2 (FGF-2) on conjunctival fibrogenesis that was induced by the presence of transforming growth factor-ß2 (TGF-ß2). Two-dimension (2D) and three-dimension (3D) cultured human conjunctival fibroblasts (HconF) were used for this purpose. The 2D and 3D cultured HconF were characterized by transendothelial electrical resistance (TEER) and FITC dextran permeability measurements (2D), real-time metabolic analyses (2D), size and stiffness measurements (3D), and the mRNA expression of extracellular matrix molecules, their modulators, Tissue inhibitor of metalloproteinases and matrix metalloproteinases and ER-stress related genes (2D and 3D). FGF-2 significantly increased planar proliferation, as evidenced by TEER values and FITC dextran permeability, and shifted glucose metabolism to the energetic phenotype of 2D HconF cells, and the stiffness of the 3D spheroids, and these effects were further enhanced in the presence of TGF-ß2. Analyses of the expression of possible candidate molecules involved in cell architecture and stress indicated that some additive effects caused by both factors were also recognized in some of these molecules. The findings reported herein indicate that the FGF-2, either along or additively with TGF- ß2 increased the fibrogenetic changes on the plane as well as in the spatial space of HconF cells.

Fatty acid metabolism is involved in both retinal physiology and the pathology of retinal vascular diseases.

To study the pathophysiological roles of the fatty acid-binding proteins (FABPs) within the retina, we performed; (1) immunolabeling of human retinas, wild type (WT) rat and mouse retinas, rat models for diabetic retinopathy (DR) and retinitis pigmentosa (RP) with anti-FABP3, FABP4, FABP5, FABP7, FABP8 and FABP12, (2) electroretinogram (ERG) measurements of WT and FABP4-deficient (Fabp4-/-) mice, (3) ELISA or gas chromatography measurements of plasma (P-) and vitreous (V-) levels of FABP4 and vascular endothelial growth factor A (VEGFA), and fatty acids (FAs) from patients with retinal vascular disease (RVD) including proliferative DR (PDR, n = 30) and retinal vein occlusion (RVO, n = 18) and non-RVD (n = 18). Within the human retina, diverse expressions of FABP3, FABP4, FABP7 and FABP8 were identified. In contrast, positive immunoreactivities toward only FABP4 and FABP12 were detected in the cases of rat and mouse retinas, and interestingly, the FABP4 labeling patterns for the WT, DR and RP rat retinas were different. The ERG amplitudes of Fabp4-/- mice were enhanced compared with those of WT mice. The concentrations of V-FABP4, V-VEGFA and total FAs were significantly higher in RVD patients than in non-PDR patients (P < 0.05). The V-FAs levels of each were significantly and positively correlated with V-FABP4 and V-VEGFA, although no significant correlation between vitreous (V-) and plasma (P-) FABP4, VEGFA and FAs were detected. The current study reveals that V-FAs appear to have significant roles in both retinal physiology as well as the pathogenesis of RVD with FABP4, which is commonly expressed within the retina in most species.

An α2-Adrenergic Agonist, Brimonidine, Beneficially Affects the TGF-ß2-Treated Cellular Properties in an In Vitro Culture Model.

We report herein on the effects of brimonidine (BRI), an α2-adrenergic agonist, on two-dimensional (2D) and three-dimensional (3D) cell-cultured TGF-ß2-untreated and -treated human trabecular meshwork (HTM) cells. In the presence of TGF-ß2 (5 ng/mL), (1) the effects of BRI on (1) the 2D HTM monolayers' barrier function were investigated as estimated using trans-endothelial electrical resistance (TEER) measurement and FITC dextran permeability; (2) real-time analyses of cellular metabolism using a Seahorse Bioanalyzer; (3) the largeness and hardness of 3D spheroids; and (4) the expression of genes that encode extracellular matrix (ECM) proteins, including collagens (COL) 1, 4, and 6; fibronectin (FN) and α-smooth muscle actin (α-SMA); ECM modulators, including a tissue inhibitor of matrix proteinase (TIMP) 1-4; matrix metalloproteinase (MMP) 2, 9, and 14; and several endoplasmic reticulum (ER) stress-related genes, including the X-box-binding protein 1 (XBP1), the spliced XBP1 (sXBP1), glucose-regulated protein (GRP)78, GRP94, and CCAAT-enhancer-binding protein homologous protein (CHOP). BRI markedly inhibited the TGF-ß2-induced increase in the values of TEER of the 2D cell monolayer and the hardness of the 3D spheroids, although it had no effect on their sizes. BRI also cancelled the TGF-ß2-induced reduction in mitochondrial maximal respiration but had no effect on the glycolytic capacity. In addition, the gene expression of these molecules was quite different between the 2D and 3D cultures of HTM cells. The present observations found in this study indicate that BRI may beneficially affect TGF-ß2-induced changes in both cultures, 2D and 3D, of HTM cells, although their structural and functional properties that were altered varied significantly between both cultures of HTM cells.

Brimonidine Modulates the ROCK1 Signaling Effects on Adipogenic Differentiation in 2D and 3D 3T3-L1 Cells.

The additive effects of an α2-adrenergic agonist, brimonidine (BRI), on the pan-ROCK inhibitor (ROCK-i), ripasudil (Rip), and the ROCK2-I, KD025, on adipogenic differentiation (DIF+) were examined using two- or three-dimension (2D or 3D) cultures of 3T3-L1 cells. The following analyses were carried out: (1) lipid staining (2D and 3D), (2) real-time measurements of cellular metabolism (2D), (3) mRNA expression of DIF+ related genes and extracellular matrix molecules (ECMs) including collagen (Col)-1, -4, and -6, and fibronectin (Fn), and (4) the sizes and physical properties of the 3D spheroids. The findings indicate that DIF+ induced (1) a substantial enhancement in lipid staining and enhanced expression of the Pparγ and Fabp4 genes, (2) significantly larger and softer 3D spheroids, and (3) down-regulation of Col1 and Fn and up-regulation of Col4 and Col6 genes. Treatment with Rip alone caused a significant enhancement in adipogenesis of both the 2D and 3D cultured 3T3-L1 cells and in the physical properties of the 3D spheroids; these effects were substantially inhibited by BRI, and the effects induced by BRI or KD025 were not insignificant. These collective findings indicate that the addition of BRI inhibited the Rip-induced enhancement of DIF+ in 3T3-L1 cells, presumably by modulating ROCK1 signaling.

Human Trabecular Meshwork (HTM) Cells Treated with TGF-ß2 or Dexamethasone Respond to Compression Stress in Different Manners.

To characterize our recently established in vitro glaucomatous human trabecular meshwork (HTM) models using dexamethasone (DEX)- or TGF-ß2-treated HTM cells, (1) two-dimensional (2D) cultured HTM cells were characterized by means of the real-time cellular metabolism analysis using a Seahorse analyzer, and (2) the effects of mechanical compression stresses toward the three-dimensional (3D) HTM spheroids were evaluated by analyzing the gene expression of several ECM proteins, inflammatory cytokines, and ER stress-related factors of those 3D HTM spheroid models. The results indicated that (1) the real-time cellular metabolism analysis indicated that TGF-ß2 significantly induced an energy shift from mitochondrial oxidative phosphorylation (OXPHOS) into glycolysis, and DEX induced similar but lesser effects. In contrast, ROCK2 inhibition by KD025 caused a substantial reverse energy shift from glycolysis into OXPHOS. (2) Upon direct compression stresses toward the untreated control 3D HTM spheroids, a bimodal fluctuation of the mRNA expressions of ECM proteins was observed for 60 min, that is, initial significant upregulation (0-10 min) and subsequent downregulation (10-30 min) followed by another upregulation (30-60 min); those of inflammatory cytokines and ER stress-related factors were also bimodally changed. However, such compression stresses for 30 min toward TGF-ß2- or DEX-treated 3D HTM spheroids induced downregulation of most of those of inflammatory cytokines and ER stress-related factors in addition to upregulation of COL1 and downregulation of FN. The findings presented herein indicate that (1) OXPHOS of the HTM cells was decreased or increased by TGF-ß2 or DEX stimulation or ROCK2 inhibition, and (2) mechanical compression stresses toward 3D HTM spheroids may replicate acute, subacute, and chronic HTM models affected by elevated intraocular pressures.

ROCK 1 and 2 affect the spatial architecture of 3D spheroids derived from human corneal stromal fibroblasts in different manners.

The objective of the current study was to examine the roles of ROCK1 and 2 on the spatial architecture of human corneal stroma. We examined the effects of a pan-ROCK inhibitor (pan-ROCK-i), ripasudil, and a ROCK2 inhibitor (ROCK2-i), KD025 on the expression of genes that encode for ECM proteins including collagen (COL) 1, 4, 6, and fibronectin (FN), their regulators, a tissue inhibitor of metalloproteinase (TIMP) 1-4, matrix metalloproteinase (MMP) 2, 9 and 14, and ER stress-related factors of two- and three-dimensional (2D and 3D) cultures of human corneal stroma fibroblasts (HCSFs), and the physical properties of 3D HCSF spheroids. A gene expression analysis using ROCK-is indicated that KD025 (ROCK2 selective ROCK inhibitor) induced more significant changes than Rip (ripasudil, pan-ROCK inhibitor), suggesting that ROCK2 might be more extensively involved in the metabolism of ECM proteins and cell architectures of the 2D cultured HCSFs than ROCK1. In terms of the physical properties, size and stiffness of the 3D HCSFs spheroids, Rip caused a significant enlargement and this enhancement was concentration-dependent while KD025 also exerted a similar but less pronounced effect. In contrast, Rip and KD025 modulated physical stiffness differently, in that Rip caused a substantial decrease and KD025 caused an increase. Such diverse effects between Rip and KD025 were also observed for the gene expressions of ECM proteins, their regulators, and ER-stress related factors. The findings presented herein suggest that the ROCK1 and 2 influence the spatial architecture of 3D HCFS spheroids in different manners.

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.

Fatty Acid-Binding Proteins 4 and 5 Are Involved in the Pathogenesis of Retinal Vascular Diseases in Different Manners.

This study reports on the pathological significance of the vitreous fatty acid-binding protein (Vt-FABP) 4 and 5, and vascular endothelial growth factor A (Vt-VEGFA) in patients with retinal vascular diseases (RVDs) including proliferative diabetic retinopathy (PDR) and retinal vein occlusion (RVO). Subjects with PDR (n = 20), RVO (n = 10), and controls (epiretinal membrane, n = 18) who had undergone vitrectomies were enrolled in this study. The levels of Vt-FABP4, Vt-FABP5, and Vt-VEGFA were evaluated by enzyme-linked immunosorbent assays (ELISA). Retinal circulation levels were measured by a laser-speckle flow analyzer (LSFA) and other relevant data were collected. The Vt-FABP5 levels were significantly (p < 0.05) elevated in patients with RVDs compared to control patients. This elevation was more evident in patients with RVO than with PDR. Log Vt-FABP5 was significantly correlated negatively or positively with all the LSFA retinal circulation indexes or Log triglycerides (r = 0.31, p = 0.031), respectively. However, the elevations in the Vt-FABP4 and Vt-VEGFA levels were more evident in the PDR group (p < 0.05) and these factors were correlated positively with Log fasting glucose and negatively with some of the LSFA retinal circulation indexes. Multivariable regression analyses indicated that the LSFA blood flows of the optic disc at baseline was an independent effector with Log Vt-FABP5 other than several possible factors including age, gender, Log triglycerides, Log Vt-FABP4 and Log Vt-VEGFA. These current findings suggest that Vt-FABP5 is involved in the pathogenesis of RVD in a manner that is different from that for Vt-FABP4 and Vt-VEGFA, presumably by regulating retinal circulation.

Comparison of the Drug-Induced Efficacies between Omidenepag Isopropyl, an EP2 Agonist and PGF2α toward TGF-ß2-Modulated Human Trabecular Meshwork (HTM) Cells.

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-ß2-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-ß2 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-ß2-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-ß2-treated 3D spheroids, but OMD enhanced only spheroid size. Upon exposure to TGF-ß2, 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 TGFß2-modulated 2D monolayers and the physical properties of the 3D HTM spheroids.