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.
Abstract
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.
To compare the effects among three TGF-β isoforms (TGF-β-1, TGF-β-2, and TGF-β-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-β 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-β-3. The findings indicated that solutions containing 10 ng/mL of TGF-β-1, 5 ng/mL of TGF-β-2, and 1 ng/mL of TGF-β-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-β induced quite different effects on the metabolic phenotype, with a decreased ATP-linked respiration, increased proton leakage, and decreased glycolytic capacity compared with TGF-β-1 and TGF-β-2. In addition, the concentrations of the three TGF-β 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-β-3 were markedly different from TGF-β-1 and TGF-β-2. The findings presented herein suggest that these diverse efficacies among the TGF-β isoforms, especially the unique action of TGF-β-3 toward HTM, may induce different effects within the pathogenesis of glaucoma.
Abstract
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.
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,
= 2) and >27 mm (high AL group,
= 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 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
γ,
and
genes; (3) a significant softening of the 3D organoids, the effects of which were all enhanced by Rip except for
γ expression; and (4) a significant downregulation in
and
, and a significant upregulation in
,
, 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
γ was suppressed (2D, OMD) or increased (2D, Buta), and the expressions of
were downregulated (2D, 3D) and
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
,
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.
Fatty acid-binding proteins (FABPs), a family of lipid chaperone molecules that are involved in intracellular lipid transportation to specific cellular compartments, stimulate lipid-associated ...responses such as biological signaling, membrane synthesis, transcriptional regulation, and lipid synthesis. Previous studies have shown that FABP4, a member of this family of proteins that are expressed in adipocytes and macrophages, plays pivotal roles in the pathogenesis of various cardiovascular and metabolic diseases, including diabetes mellitus (DM) and hypertension (HT). Since significant increases in the serum levels of FABP4 were detected in those patients, FABP4 has been identified as a crucial biomarker for these systemic diseases. In addition, in the field of ophthalmology, our group found that intraocular levels of FABP4 (ioFABP4) and free fatty acids (ioFFA) were substantially elevated in patients with retinal vascular diseases (RVDs) including proliferative diabetic retinopathy (PDR) and retinal vein occlusion (RVO), for which DM and HT are also recognized as significant risk factors. Recent studies have also revealed that ioFABP4 plays important roles in both retinal physiology and pathogenesis, and the results of these studies have suggested potential molecular targets for retinal diseases that might lead to future new therapeutic strategies.
The current global water body maps have a resolution of approximately 30 m depending on the available remote sensing data. A water body map with higher spatial resolution is required to distinguish ...smaller rivers for advanced applications involving global carbon cycle and real-time flood predictions. Conventional water extraction methods use water indices that combine visible and infrared spectra. State-of-the-art remote sensing data including aerial photography, offers a few-meter spatial resolution but only contains the visible spectrum. Here, we established a water extraction method at 60-cm resolution via Bayesian inference, using aerial photography’s visible spectrum combining the Landsat-based Global 1-second Water Body Map and the Open Street Map (OSM). The Japan Flow Direction Map (J-FlwDir) was used to link the water bodies. Our method detected the main streams of the Tsurumi and Tama rivers and their tributaries, which were not resolved by the Landsat-based dataset. With our approach, rivers with widths >10 m were detected and water extent was obtained for 37% of small rivers, represented as lines on the OSM. These findings indicate that our method, solely based on visible spectra with hydrography data and existing water body maps, improves the spatial resolution of water mapping without requiring infrared.
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.
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