scholarly journals Pan-ROCK and ROCK2 Inhibitors Affect Dexamethasone-Treated 2D- and 3D-Cultured Human Trabecular Meshwork (HTM) Cells in Opposite Manners

Molecules ◽  
2021 ◽  
Vol 26 (21) ◽  
pp. 6382
Author(s):  
Megumi Watanabe ◽  
Yosuke Ida ◽  
Masato Furuhashi ◽  
Yuri Tsugeno ◽  
Fumihito Hikage ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Physical Properties ◽  
Trabecular Meshwork ◽  
Electrical Resistance ◽  
Transendothelial Electrical Resistance ◽  
Gene Expressions ◽  
Rock Inhibitor ◽  
Stiffness Analysis ◽  
3D Cell Cultures ◽  
2D And 3D

Effects of a pan-ROCK-inhibitor, ripasudil (Rip), and a ROCK2 inhibitor, KD025 on dexamethasone (DEX)-treated human trabecular meshwork (HTM) cells as a model of steroid-induced glaucoma were investigated. In the presence of Rip or KD025, DEX-treated HTM cells were subjected to permeability analysis of 2D monolayer by transendothelial electrical resistance (TEER) and FITC–dextran permeability, physical properties, size and stiffness analysis (3D), and qPCR of extracellular matrix (ECM), and their modulators. DEX resulted in a significant increase in the permeability, as well as a large and stiff 3D spheroid, and those effects were inhibited by Rip. In contrast, KD025 exerted opposite effects on the physical properties (down-sizing and softening). Furthermore, DEX induced several changes of gene expressions of ECM and their modulators were also modulated differently by Rip and KD025. The present findings indicate that Rip and KD025 induced opposite effects toward 2D and 3D cell cultures of DEX-treated HTM cells.

scholarly journals Diverse effects of pan-ROCK and ROCK2 inhibitors on 2 D and 3D cultured human trabecular meshwork (HTM) cells treated with TGFβ2

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Megumi Watanabe ◽  
Yosuke Ida ◽  
Hiroshi Ohguro ◽  
Chiaki Ota ◽  
Fumihito Hikage
Keyword(s):  
Trabecular Meshwork ◽  
Smooth Muscle Actin ◽  
Three Dimensional ◽  
Coiled Coil ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Muscle Actin ◽  
Transendothelial Electrical Resistance ◽  
Gene Expressions ◽  
Rock Inhibitor ◽  
2D And 3D

AbstractA 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.

scholarly journals Simultaneous Use of ROCK Inhibitors and EP2 Agonists Induces Unexpected Effects on Adipogenesis and the Physical Properties of 3T3-L1 Preadipocytes

2021 ◽  
Vol 22 (9) ◽  
pp. 4648
Author(s):  
Yosuke Ida ◽  
Megumi Watanabe ◽  
Hiroshi Ohguro ◽  
Fumihito Hikage
Keyword(s):  
Extracellular Matrix ◽  
Adipose Tissue ◽  
Protein Kinase ◽  
Physical Properties ◽  
Coiled Coil ◽  
Three Dimension ◽  
Additive Effects ◽  
Rock Inhibitor ◽  
Pparγ Expression ◽  
2D And 3D

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.

scholarly journals ROCK inhibitors beneficially alter the spatial configuration of TGFβ2-treated 3D organoids from a human trabecular meshwork (HTM)

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Chiaki Ota ◽  
Yosuke Ida ◽  
Hiroshi Ohguro ◽  
Fumihito Hikage
Keyword(s):  
Trabecular Meshwork ◽  
Kinase Inhibitors ◽  
Spatial Configuration ◽  
Coiled Coil ◽  
Protein Kinase Inhibitors ◽  
Molecular Pharmacology ◽  
Transendothelial Electrical Resistance ◽  
Beneficial Effects ◽  
2D And 3D ◽  
Aqueous Outflow

AbstractTo elucidate molecular pharmacology of Rho-associated coiled-coil containing protein kinase inhibitors (ROCK-i, Ripasudil and Y27632) on their efficiency for aqueous outflow, 2D or 3D cultures of a human trabecular meshwork (HTM) were prepared in the presence of TGFβ2. Those were examined by transendothelial electrical resistance (TEER, 2D), electronic microscopy (EM, 2D and 3D), expression of the extracellular matrix (ECM) including collagen1 (COL1), COL4 and COL6, and fibronectin (FN) by immunolabeling and/or quantitative PCR (3D), and solidity of 3D organoids by a micro-squeezer. TGFβ2 significantly increased the TEER values in 2D cultures, and the ECM expression indicated that the 3D organoids assumed a more densely packed shape. ROCK-i greatly reduced the TGFβ2-induced enhancement of TEER and the immunolabeled ECM expression of the 3D organoids. In contrast, the mRNA expression of COL1 was increased, and those of COL4 and FN were unchanged. EM revealed that TGFβ2 caused the HTM cells to become more compact and abundant ECM deposits within the 3D organoids were observed. These were significantly inhibited by ROCK-i. The dense solids caused by the presence of TGFβ2 were significantly suppressed by ROCK-i. Current study indicates that ROCK-i cause beneficial effects toward the spatial configuration of TGFβ2-induced HTM 3D organoids.

scholarly journals Autotaxin May Have Lysophosphatidic Acid-Unrelated Effects on Three-Dimension (3D) Cultured Human Trabecular Meshwork (HTM) Cells

2021 ◽  
Vol 22 (21) ◽  
pp. 12039
Author(s):  
Megumi Watanabe ◽  
Masato Furuhashi ◽  
Yuri Tsugeno ◽  
Yosuke Ida ◽  
Fumihito Hikage ◽  
...  
Keyword(s):  
Er Stress ◽  
Lysophosphatidic Acid ◽  
Trabecular Meshwork ◽  
Electrical Resistance ◽  
Three Dimensional ◽  
Three Dimension ◽  
Two Dimensional ◽  
Transendothelial Electrical Resistance ◽  
Related Factors ◽  
Signaling Axis

Purpose: The objective of the current study was to evaluate the effects of the autotaxin (ATX)–lysophosphatidic acid (LPA) signaling axis on the human trabecular meshwork (HTM) in two-dimensional (2D) and three-dimensional (3D) cultures of HTM cells. Methods: The effects were characterized by transendothelial electrical resistance (TEER) and FITC-dextran permeability (2D), measurements of size and stiffness (3D), and the expression of several genes, including extracellular matrix (ECM) molecules, their modulators, and endoplasmic reticulum (ER) stress-related factors. Results: A one-day exposure to 200 nM LPA induced significant down-sizing effects of the 3D HTM spheroids, and these effects were enhanced slightly on longer exposure. The TEER and FITC-dextran permeability data indicate that LPA induced an increase in the barrier function of the 2D HTM monolayers. A one-day exposure to a 2 mg/L solution of ATX also resulted in a significant decrease in the sizes of the 3D HTM spheroids, and an increase in stiffness was also observed. The gene expression of several ECMs, their regulators and ER-stress related factors by the 3D HTM spheroids were altered by both ATX and LPA, but in different manners. Conclusions: The findings presented herein suggest that ATX may have additional roles in the human TM, in addition to the ATX–LPA signaling axis.

scholarly journals Rosiglitasone and ROCK Inhibitors Modulate Fibrogenetic Changes in TGF-β2 Treated Human Conjunctival Fibroblasts (HconF) in Different Manners

2021 ◽  
Vol 22 (14) ◽  
pp. 7335
Author(s):  
Yuika Oouchi ◽  
Megumi Watanabe ◽  
Yosuke Ida ◽  
Hiroshi Ohguro ◽  
Fumihito Hikage
Keyword(s):  
Extracellular Matrix ◽  
Electrical Resistance ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Coiled Coil ◽  
Three Dimension ◽  
Muscle Actin ◽  
Transendothelial Electrical Resistance ◽  
Down Regulation ◽  
Two Dimension

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.

MONEL ALLOY 401

Alloy Digest ◽  
1970 ◽  
Vol 19 (6) ◽  
Keyword(s):  
Corrosion Resistance ◽  
Physical Properties ◽  
Tensile Properties ◽  
Temperature Coefficient ◽  
Nickel Alloy ◽  
Electrical Resistance ◽  
Heat Treating ◽  
High Electrical Resistance ◽  
Good Corrosion Resistance ◽  
Copper Nickel Alloy

Abstract MONEL alloy 401 is a copper-nickel alloy with high electrical resistance and is used primarily in specialized electrical and electronic applications. It has a negligible temperature coefficient of electrical resistance and good corrosion resistance. This datasheet provides information on composition, physical properties, hardness, and tensile properties. It also includes information on forming, heat treating, and machining. Filing Code: Cu-216. Producer or source: Huntington Alloy Products Division.

MAGNIFER 36K

Alloy Digest ◽  
1996 ◽  
Vol 45 (6) ◽  
Keyword(s):  
Corrosion Resistance ◽  
Physical Properties ◽  
Tensile Properties ◽  
Electrical Resistance ◽  
Iron Alloy ◽  
Heat Treating ◽  
Soft Magnetic ◽  
Nickel Iron ◽  
Saturation Induction ◽  
Nickel Iron Alloy

Abstract Magnifer 36K is a soft-magnetic nickel-iron alloy with 36% nickel. It has a saturation induction of 1.3 T and a rather high electrical resistance of 0.75 ohm mm2/m. This datasheet provides information on composition, physical properties, hardness, and tensile properties. It also includes information on corrosion resistance as well as forming, heat treating, machining, and joining. Filing Code: FE-110. Producer or source: VDM Technologies Corporation.

DRIVER 180 ALLOY

Alloy Digest ◽  
1978 ◽  
Vol 27 (2) ◽  
Keyword(s):  
Corrosion Resistance ◽  
Physical Properties ◽  
Tensile Properties ◽  
Nickel Alloy ◽  
Electrical Resistance ◽  
Operating Temperature ◽  
Heat Treating ◽  
Specific Resistivity ◽  
Copper Nickel ◽  
Copper Nickel Alloy

Abstract DRIVER 180 ALLOY is a copper-nickel alloy for use where moderate electrical resistance is required. The number designation refers to its specific resistivity (180 ohms/cir mil/ft) which is combined with a fairly low coefficient of resistance (180 x 10^-6 per C). Its maximum recommended operating temperature is 1000 F. This datasheet provides information on composition, physical properties, and tensile properties. It also includes information on corrosion resistance as well as forming, heat treating, machining, and joining. Filing Code: Cu-348. Producer or source: Wilbur B. Driver Company.

DRIVER 90 ALLOY

Alloy Digest ◽  
1977 ◽  
Vol 26 (11) ◽  
Keyword(s):  
Physical Properties ◽  
Tensile Properties ◽  
Nickel Alloy ◽  
Electrical Resistance ◽  
Operating Temperature ◽  
Heat Treating ◽  
Specific Resistivity ◽  
Copper Nickel ◽  
Copper Nickel Alloy

Abstract DRIVER 90 ALLOY is a copper-nickel alloy for use where only moderately low electrical resistance is required. The number designations refers to its specific resistivity (90 ohms/cir mil/ft) which is combined with a moderate coefficient of resistance. Its maximum recommended operating temperature is 800 F. This datasheet provides information on composition, physical properties, and tensile properties. It also includes information on forming and heat treating. Filing Code: Cu-343. Producer or source: Wilbur B. Driver Company.

scholarly journals Fucoxanthin Holds Potential to Become a Drug Adjuvant in Breast Cancer Treatment: Evidence from 2D and 3D Cell Cultures

Molecules ◽  
2021 ◽  
Vol 26 (14) ◽  
pp. 4288
Author(s):  
Fernanda Malhão ◽  
Ana Catarina Macedo ◽  
Carla Costa ◽  
Eduardo Rocha ◽  
Alice Abreu Ramos
Keyword(s):  
Breast Cancer ◽  
Cell Lines ◽  
3D Models ◽  
Anticancer Activities ◽  
Cytotoxic Effects ◽  
3D Cultures ◽  
3D Cell Cultures ◽  
Tumoral Cell ◽  
Microscopy Techniques ◽  
2D And 3D

Fucoxanthin (Fx) is a carotenoid derived from marine organisms that exhibits anticancer activities. However, its role as a potential drug adjuvant in breast cancer (BC) treatment is still poorly explored. Firstly, this study investigated the cytotoxic effects of Fx alone and combined with doxorubicin (Dox) and cisplatin (Cis) on a panel of 2D-cultured BC cell lines (MCF7, SKBR3 and MDA-MB-231) and one non-tumoral cell line (MCF12A). Fucoxanthin induced cytotoxicity against all the cell lines and potentiated Dox cytotoxic effects towards the SKBR3 and MDA-MB-231 cells. The combination triggering the highest cytotoxicity (Fx 10 µM + Dox 1 µM in MDA-MB-231) additionally showed significant induction of cell death and genotoxic effects, relative to control. In sequence, the same combination was tested on 3D cultures using a multi-endpoint approach involving bioactivity assays and microscopy techniques. Similar to 2D cultures, the combination of Fx and Dox showed higher cytotoxic effects on 3D cultures compared to the isolated compounds. Furthermore, this combination increased the number of apoptotic cells, decreased cell proliferation, and caused structural and ultrastructural damages on the 3D models. Overall, our findings suggest Fx has potential to become an adjuvant for Dox chemotherapy regimens in BC treatment.

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