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To establish appropriate ex vivo models for a glaucomatous trabecular meshwork (TM), two-dimensional (2D) and three-dimensional (3D) cultures of human trabecular meshwork cells (HTM) were prepared in the presence of 250 nM dexamethasone (DEX) or 5 ng/mL TGFβ2, and characterized by the following analyses; transendothelial electrical resistance (TEER) measurements, FITC dextran permeability, scanning electron microscopy and the expression of the extracellular matrix (ECM) including collagen (COL)1, 4 and 6, and fibronectin (FN), α-smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinase (TIMP)1–4, and matrix metalloproteinase (MMP)2, 9 and 14. DEX and TGFβ2 both caused a significant increase or decrease in the TEER values and FITC dextran permeability. During the 3D spheroid culture, DEX or TGFβ2 induced a mild and significant down-sizing and an increase in stiffness, respectively. TGFβ2 induced a significant up-regulation of COL1 and 4, FN, α-SMA, and MMP 2 and 14 (2D) or COL1 and 6, and TIMP2 and 3 (3D), and DEX induced a significant up-regulation of FN (3D) and TIMP4 (2D and 3D). The findings presented herein indicate that DEX or TGFβ2 resulted in mild and severe down-sized and stiff 3D HTM spheroids, respectively, thus making them viable in vitro HTM models for steroid-induced and primary open angle glaucoma.

A pan-ROCK-inhibitor, ripasudil (Rip), and a ROCK2 inhibitor, KD025, were used To study the effects of Rho-associated coiled-coil containing protein kinase (ROCK)1 and 2 on two-dimensional (2D) and three-dimensional (3D) cultures of a TGFβ2-treated human trabecular meshwork (HTM) cells. In the presence of 5 ng/mL TGFβ2, the effects of these inhibitors were characterized by transendothelial electrical resistance (TEER), FITC-dextran permeability, and the size and stiffness of 3D sphenoids, the expression of extracellular matrix (ECM) including collagen1, 4 and 6, and fibronectin, α-smooth muscle actin, a tissue inhibitor of metalloproteinase (TIMP)1–4, and matrix metalloproteinase (MMP)2, 9 and 14. TGFβ2 caused a significant increase in the TEER values, and decrease in FITC-dextran permeability, as well as a decrease in the sizes and stiffness of the 3D sphenoids. In the presence of ROCK inhibitors, the TGFβ2-induced effects of the TEER and FITC-dextran permeability were inhibited, especially by KD025. Rip induced a significant increase in sizes and a decrease in the stiffness of the TGFβ2-treated 3D sphenoids, although the effects of KD025 were weaker. Gene expressions of most of the ECMs, TIMP2 and MMP9 of 2D and 3D HTM cells were significantly up-regulated by TGFβ2. Those were significantly and differently modulated by Rip or KD025.

Purpose: The effects of Rho-associated coiled-coil containing protein kinase (ROCK) 1 and 2 inhibitor, ripasudil hydrochloride hydrate (Rip), ROCK2 inhibitor, KD025 or rosiglitazone (Rosi) on two-dimension (2D) and three-dimension (3D) cultured human conjunctival fibroblasts (HconF) treated by transforming growth factor (TGFβ2) were studied. Methods: Two-dimension and three-dimension cultured HconF were examined by transendothelial electrical resistance (TEER, 2D), size and stiffness (3D), and the expression of the extracellular matrix (ECM) including collagen1 (COL1), COL4 and COL6, fibronectin (FN), and α-smooth muscle actin (αSMA) by quantitative PCR (2D, 3D) in the presence of Rip, KD025 or Rosi. Results: TGFβ2 caused a significant increase in (1) the TEER values (2D) which were greatly reduced by Rosi, (2) the stiffness of the 3D organoids which were substantially reduced by Rip or KD025, and (3) TGFβ2 induced a significant up-regulation of all ECMs, except for COL6 (2D) or αSMA (3D), and down-regulation of COL6 (2D). Rosi caused a significant up-regulation of COL1, 4 and 6 (3D), and down-regulation of COL6 (2D) and αSMA (3D). Most of these TGFβ2-induced expressions in the 2D and αSMA in the 3D were substantially inhibited by KD025, but COL4 and αSMA in 2D were further enhanced by Rip. Conclusion: The findings reported herein indicate that TGFβ2 induces an increase in fibrogenetic changes on the plane and in the spatial space, and are inhibited by Rosi and ROCK inhibitors, respectively.

The fatty acid-binding protein4 (FABP4) and vascular endothelial growth factor A (VEGFA) play key roles in the metabolic and cardiovascular diseases, and proliferative diabetic retinopathy (PDR), respectively. To identify FABP4 in vitreous fluid in PDR, vitreous concentrations of FABP4 (V-FABP4) and VEGFA (V-VEGFA) from PDR (n = 20) and non-PDR (n = 20) patients were determined by Enzyme-Linked ImmunoSorbent Assays. The data, which included height and weight, systemic blood pressures, several blood biochemical parameters and blood flow at the optic nerve head (ONH) by laser speckle flowgraphy (LSFG) were collected. The levels of V-FABP4 and V-VEGFA were significantly higher in PDR patients than in non-PDR patients (P < 0.001) with a high positive correlation (r = 0.72, P < 0.001) between them. The findings were not affected by body mass index values and the presence of vitreous hemorrhaging. Among the clinical parameters, V-FABP4 correlated positively with creatinine and negatively with age and aspartate transaminase (AST) levels, while V-VEGFA correlated positively with fasting plasma glucose and hemoglobin A1c (HbA1c) levels but negatively with AST. Multiple regression analyses indicated that V-VEGFA, or V-FABP4, AST and HbA1c were independent predictors of V-FABP4 or V-VEGFA, respectively. Both were negatively correlated, but more evident in V-FABP4, with the ONH ocular blood flow.

To establish an appropriate in vitro model for the local environment of cardiomyocytes, three-dimensional (3D) spheroids derived from H9c2 cardiomyoblasts were prepared, and their morphological, biophysical phase contrast and biochemical characteristics were evaluated. The 3D H9c2 spheroids were successfully obtained, the sizes of the spheroids decreased, and they became stiffer during 3–4 days. In contrast to the cell multiplication that occurs in conventional 2D planar cell cultures, the 3D H9c2 spheroids developed into a more mature form without any cell multiplication being detected. qPCR analyses of the 3D H9c2 spheroids indicated that the production of collagen4 (COL4) and fibronectin (FN), connexin43 (CX43), β-catenin, N-cadherin, STAT3, and HIF1 molecules had increased and that the production of COL6 and α-smooth muscle actin (α-SMA) molecules had decreased as compared to 2D cultured cells. In addition, treatment with rapamycin (Rapa), an mTOR complex (mTORC) 1 inhibitor, and Torin 1, an mTORC1/2 inhibitor, resulted in significantly decreased cell densities of the 2D cultured H9c2 cells, but the size and stiffness of the H9c2 cells within the 3D spheroids were reduced with the gene expressions of several of the above several factors being reduced. The metabolic responses to mTOR modulators were also different between the 2D and 3D cultures. These results suggest that as unique aspects of the local environments of the 3D spheroids, the spontaneous expression of GJ-related molecules and hypoxia within the core may be associated with their maturation, suggesting that this may become a useful in vitro model that replicates the local environment of cardiomyocytes.

To elucidate the effects of the new antidiabetic agent, imeglimin (Ime, 2 mM), on high-glucose-induced cellular stress in cardiac cells, its effects were compared with those of the conventional antidiabetic agent metformin (Met, 2 mM) based on various cellular pathophysiological functions. H9c2 cardiomyoblasts were cultured under normal-glucose (5.5 mM, N-Glu) or high-glucose (50 mM, H-Glu) conditions. Cellular metabolic function was evaluated using a Seahorse XFe96 Bioanalyzer, along with measurements of reactive oxygen species (ROS) production, expression levels of the autophagy-related marker LC3, and intercellular adhesion properties measured based on transepithelial electrical resistance (TEER). Cells cultured under H-Glu conditions showed enhanced mitochondrial and glycolytic activities, which were suppressed by Met or Ime. Under H-Glu conditions, total cellular ROS (t-ROS) levels were significantly increased. Met had little effect on t-ROS under H-Glu conditions, whereas Ime markedly reduced both t-ROS and mitochondrial ROS (m-ROS) levels under H-Glu conditions. The LC3-II/LC3-I ratio, a marker of autophagic activity, decreased under H-Glu conditions; however, this reduction was not significantly affected by treatment with either Met or Ime. Regarding intercellular adhesion properties, TEER values were elevated under H-Glu conditions compared to N-Glu conditions, and those under H-Glu conditions were further increased by Ime but not Met. In support of these results, the mRNA levels of cell-adhesion-related molecules, including β-catenin and N-cadherin, were also altered by Ime. Collectively, Ime modulated high-glucose-induced alterations in the biological properties of H9c2 cardiomyoblasts, independent of changes in autophagic activity.

The purpose of the current investigation was to elucidate what kinds of responsible mechanisms induce elongation of the sclera in myopic eyes. To do this, two-dimensional (2D) cultures of human scleral stromal fibroblasts (HSSFs) obtained from eyes with two different axial length (AL) groups, <26 mm (low AL group, n = 2) and >27 mm (high AL group, n = 3), were subjected to (1) measurements of Seahorse mitochondrial and glycolytic indices to evaluate biological aspects and (2) analysis by RNA sequencing. Extracellular flux analysis revealed that metabolic indices related to mitochondrial and glycolytic functions were higher in the low AL group than in the high AL group, suggesting that metabolic activities of HSSF cells are different depending the degree of AL. Based upon RNA sequencing of these low and high AL groups, the bioinformatic analyses using gene ontology (GO) enrichment analysis and ingenuity pathway analysis (IPA) of differentially expressed genes (DEGs) identified that sterol regulatory element-binding transcription factor 2 (SREBF2) is both a possible upstream regulator and a causal network regulator. Furthermore, SREBF1, insulin-induced gene 1 (INSIG1), and insulin-like growth factor 1 (IGF1) were detected as upstream regulators, and protein tyrosine phosphatase receptor type O (PTPRO) was detected as a causal network regulator. Since those possible regulators were all pivotally involved in lipid metabolisms including fatty acid (FA), triglyceride (TG) and cholesterol (Chol) biosynthesis, the findings reported here indicate that FA, TG and Chol biosynthesis regulation may be responsible mechanisms inducing AL elongation via HSSF.

To elucidate the role of IGF1R inhibition in the pathogenesis of Graves’ orbitopathy (GO), the effects of linsitinib (Lins) on a recombinant human TSHR antibody (M22) and IGF1 to activate TSHR and IGF1R of human orbital fibroblasts (HOFs) obtained from patients without GO (HOFs) and patients with GO (GHOFs) were studied using in vitro three-dimensional (3D) spheroid models in addition to their 2D planar cell culture. For this purpose, we evaluated 1) cellular metabolic functions by using a seahorse bioanalyzer (2D), 2) physical properties including size and stiffness of 3D spheroids, and mRNA expression of several extracellular matrix (ECM) proteins, their modulators ( CCL2 LOX, CTGF, MMPs ), ACTA2 and inflammatory cytokines ( IL1β, IL6 ). Administration of IGF1 and M22 induced increases of cellular metabolic functions with the effect on HOFs being much more potent than the effect on GHOFs, suggesting that IGF1R and TSHR of GHOFs may already be stimulated. Lins had effects similar to those of IGF1/M22 on cellular biological functions of HOFs but not on those of GHOFs. As for physical properties of 3D GHOFs spheroids, stiffness but not size was significantly increased by IGF1 and/or M22. In contrast, Lins significantly inhibited the M22-induced increase in stiffness despite the fact that Lins alone had no effect. The mRNA expression levels of several genes of ECM proteins and most of the other genes also fluctuated similarly to the changes in stiffness of 3D spheroids despite the fact that Lins induced up-regulation of inflammatory cytokines and MMP3 . The findings presented herein indicate that IGF1R inhibition by Lins may beneficially affect GO-related fibrogenesis.

To elucidate the additive effects of an EP2 agonist, omidenepag (OMD) or butaprost (Buta) on the Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, ripasudil (Rip) on adipose tissue, two- or three-dimension (2D or 3D) cultures of 3T3-L1 cells were analyzed by lipid staining, the mRNA expression of adipogenesis-related genes, extracellular matrix (ECM) molecules including collagen (Col) -1, -4 and -6, and fibronectin (Fn), and the sizes and physical properties of 3D organoids, as measured by a micro-squeezer. The results indicate that adipogenesis induced (1) an enlargement of the 3D organoids; (2) a substantial enhancement in lipid staining as well as the expression of the Pparγ, Ap2 and Leptin genes; (3) a significant softening of the 3D organoids, the effects of which were all enhanced by Rip except for Pparγ expression; and (4) a significant downregulation in Col1 and Fn, and a significant upregulation in Col4, Col6, the effects of which were unchanged by Rip. When adding the EP2 agonist to Rip, (1) the sizes of the 3D organoids were reduced substantially; (2) lipid staining was increased (OMD), or decreased (Buta); (3) the stiffness of the 3D organoids was substantially increased in Buta; (4-1) the expression of Pparγ was suppressed (2D, OMD) or increased (2D, Buta), and the expressions of Ap2 were downregulated (2D, 3D) and Leptin was increased (2D) or decreased (3D), (4-2) all the expressions of four ECM molecules were upregulated in 2D (2D), and in 3D, the expression of Col1, Col4 was upregulated. The collective findings reported herein indicate that the addition of an EP2 agonist, OMD or Buta significantly but differently modulate the Rip-induced effects on adipogenesis and the physical properties of 2D and 3D cultured 3T3-L1 cells.

Grape ( Vitis vinifera L.) is an important fruit crop in the world. It is used as a table grape and is also used for raisin and wine production. Grape berries accumulate secondary metabolites, such as anthocyanins, tannins, and resveratrol, which are known as functional compounds for human health. Multidrug and toxic compound extrusion transporter (MATEs) transport secondary metabolites. MATEs also transport other solutes, including organic acids, and toxic xenobiotics, depending on cation gradient and play various roles in plants. MATE comprises 300–500 amino acid residues and possesses a MATE domain and 8–12 transmembrane domains. In the present study, 59 MATE genes were identified in the grape genome, and phylogenetic analysis revealed the presence of four groups of grape MATEs (Group 1–4). Their information, such as gene structures, protein motifs, predicted subcellular localizations, and gene IDs of four genome annotations, that is, CRIBI v1, CRIBI v2, Genoscope, and Vcost v3, were annotated. The transport substrates and physiological functions of grape MATEs were estimated based on their homology with the analyzed MATEs in other plant species. Group 1 may transport toxic compounds and alkaloids, Group 2 may transport polyphenolic compounds, Group 3 may transport organic acids, and Group 4 may transport plant hormones related to signal transduction. In addition to the known anthocyanin transporters, VvMATE37 and VvMATE39, a novel anthocyanin transporter, VvMATE38 in Group 2, was suggested as a key transporter for anthocyanin accumulation in grape berry skin. VvMATE46, VvMATE47, and VvMATE49 in Group 3 may contribute to Al 3+ detoxification and Fe 2+ /Fe 3+ translocation via organic acid transport. This study provides helpful and fundamental information for grape MATE studies and resolves the confusion of gene IDs in different genome annotations.