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 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 Reactivities of a Prostanoid EP2 Agonist, Omidenepag, Are Useful for Distinguishing between 3D Spheroids of Human Orbital Fibroblasts without or with Graves’ Orbitopathy

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3196
Author(s):  
Yosuke Ida ◽  
Hanae Ichioka ◽  
Masato Furuhashi ◽  
Fumihito Hikage ◽  
Megumi Watanabe ◽  
...  
Keyword(s):  
Physical Properties ◽  
Coiled Coil ◽  
Thyroid Stimulating Hormone ◽  
Three Dimension ◽  
Rock Inhibitor ◽  
Related Factors ◽  
Graves Orbitopathy ◽  
Orbital Fibroblasts ◽  
Receptor Antibodies ◽  
3D Spheroids

Background. To obtain new insights into the activation of the thyroid-stimulating hormone (TSH) and insulin-like growth factor 1 (IGF-1) receptors in human orbital fibroblasts (n-HOFs), the effects of the prostanoid EP2 agonist, omidenepag (OMD), and a rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, ripasudil (Rip) were evaluated using three-dimension (3D) n-HOFs spheroids in the absence and presence of the recombinant human TSH receptor antibodies, M22 and IGF-1. Methods. The effects of 100 nM OMD or 10 μM Rip on the physical properties, size, stiffness, and mRNA expression of several extracellular matrix (ECM) molecules, their regulator, inflammatory cytokines, and endoplasmic reticulum (ER) stress-related factors were examined and compared among 3D spheroids of n-HOFs, M22-/IGF-1-activated n-HOFs and GO-related human orbital fibroblasts (GHOFs). Results.The physical properties and mRNA expressions of several genes of the 3D n-HOFs spheroids were significantly and diversely modulated by the presence of OMD or Rip. The OMD-induced effects on M22-/IGF-1-activated n-HOFs were similar to the effects caused by GHOHs, but quite different from those of n-HOFs. Conclusions. The findings presented herein indicate that the changes induced by OMD may be useful in distinguishing between n-HOFs and GHOFs.

scholarly journals ROCK inhibitors enhance the production of large lipid-enriched 3D organoids of 3T3-L1 cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yosuke Ida ◽  
Fumihito Hikage ◽  
Hiroshi Ohguro
Keyword(s):  
Electron Microscopy ◽  
Extracellular Matrix ◽  
Side Effects ◽  
Protein Kinase ◽  
Mrna Expression ◽  
Kinase Inhibitor ◽  
Coiled Coil ◽  
Protein Kinase Inhibitor ◽  
Fatty Tissue ◽  
Three Dimension

AbstractSince the recent discovery of prostaglandin-associated peri-orbitopathy, a great deal of interest has developed concerning the side effects of anti-glaucoma medications toward periocular fatty tissue, especially their adipogenesis. Two- or three-dimension (2D or 3D) cultures of the 3T3-L1 cells were employed to elucidate the effects of the Rho-associated coiled-coil containing protein kinase inhibitor (ROCK-i) the anti-glaucoma drug, Ripasudil, and other ROCK-i, such as Y27632 on adipogenesis. Ultrastructure by electron microscopy and physical stiffness measurements by a micro-squeezer demonstrated the 3D organoids had essentially matured during the 7-day culture. The effects of ROCK-i on 3D organoid sizes, lipid staining, the mRNA expression of adipogenesis related genes, Pparγ, Cebpa and Leptin, and extracellular matrix (ECM) including collagen (COL) 1, 4 and 6, and fibronectin, and physical stiffness were then conducted. Upon adipogenesis, the sizes, lipid staining and mRNA expressions of adipogenesis related genes, Col 4 and Col 6 were dramatically increased, and were further enhanced by ROCK-i. Micro-squeezer analysis demonstrated that adipogenesis resulted in a marked less stiffed 3D organoid and this was further enhanced by ROCK-i. Our present study indicates that ROCK-i significantly enhanced the production of large lipid-enriched 3T3-L1 3D organoids.

A visible light-controllable Rho kinase inhibitor based on a photochromic phenylazothiazole

2021 ◽  
Author(s):  
Kazuya Matsuo ◽  
Sampreeth Thayyil ◽  
Mitsuyasu Kawaguchi ◽  
Hidehiko Nakagawa ◽  
Nobuyuki Tamaoki
Keyword(s):  
Protein Kinase ◽  
Visible Light ◽  
Kinase Inhibitor ◽  
Coiled Coil ◽  
Rho Kinase ◽  
Rock Inhibitor ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Rho Kinase Inhibitor

Rho-associated coiled-coil-containing protein kinase (ROCK) is a serine-threonine kinase, whose inhibitors are useful for the regulation of actomyosin system. Here, we developed a photoswitchable ROCK inhibitor based on a phenylazothiazole...

scholarly journals Effectiveness of Ripasudil, a Rho-Associated Coiled/Coil-Containing Protein Kinase Inhibitor, in Improving Retinoschisis and Cystic-Like Foveal Cavities in Eyes with X-Linked Retinoschisis

2020 ◽  
Vol 11 (2) ◽  
pp. 411-417
Author(s):  
Hitomi Suimon ◽  
Masahiko Sugimoto ◽  
Hisashi Matsubara ◽  
Mineo Kondo
Keyword(s):  
Protein Kinase ◽  
Chart Review ◽  
Kinase Inhibitor ◽  
Coiled Coil ◽  
Clinical Information ◽  
Rock Inhibitor ◽  
First Case ◽  
Juvenile Retinoschisis ◽  
Successful Resolution ◽  
Visual Disturbances

This is the first reported case of a successful resolution of cystic-like foveal cavities in eyes with X-linked juvenile retinoschisis (XLRS) treated with topical ripasudil hydrochloride hydrate, a Rho-associated coiled/coil-containing protein kinase (ROCK) inhibitor. A chart review was performed on 1 patient to collect all relevant clinical information and the optical coherence tomographic (OCT) images. A healthy 18-year-old young man presented with bilateral visual disturbances. The patient was diagnosed with XLRS from the spoke-wheel pattern around the macula, negative electroretinograms, and retinoschisis with cystic-like foveal cavities in the OCT images. Significant reductions of the retinoschisis and cystic-like cavities were observed after treatment with topical ripasudil. This is the first case of XLRS that had a resolution of cystic-like foveal cavities after topical ripasudil, a ROCK inhibitor. Since many XLRS patients have a worsening of their visual acuities due to the progressive nature of retinoschisis and cystic-like foveal cavities, topical ripasudil offers a potential treatment option.

scholarly journals MK2 SUMOylation regulates actin filament remodeling and subsequent migration in endothelial cells by inhibiting MK2 kinase and HSP27 phosphorylation

Blood ◽  
2011 ◽  
Vol 117 (8) ◽  
pp. 2527-2537 ◽  
Author(s):  
Eugene Chang ◽  
Kyung-Sun Heo ◽  
Chang-Hoon Woo ◽  
Hakjoo Lee ◽  
Nhat-Tu Le ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Actin Filament ◽  
Coiled Coil ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Laminar Shear Stress ◽  
Sumoylation Site ◽  
Rock Inhibitor ◽  
Hsp27 Phosphorylation ◽  
Tnf Α

Abstract Actin filament remodeling regulates several endothelial cell (EC) processes such as contraction, migration, adhesion, and shape determination. Mitogen-activated protein kinase (MAPK)–activated protein kinase 2 (MK2)–mediated phosphorylation of heat-shock protein 27 kDa (HSP27) promotes actin filament remodeling, but little is known about the regulation of this event in ECs. We found that tumor necrosis factor-α (TNF-α) SUMOylated MK2 at lysine (K)-339 affected EC actin filament organization and migration. Loss of the MK2 SUMOylation site (MK2-K339R) increased MK2 kinase activity and prolonged HSP27 phosphorylation, enhancing its effects on actin filament-dependent events. Both TNF-α–mediated EC elongation and steady laminar shear stress–mediated EC alignment were increased by MK2-K339R. Moreover, kinase-dead dominant-negative MK2 (DN-MK2) inhibited these effects. Cell migration is a dynamic process regulated by actin filament remodeling. Both wild-type MK2 (WT-MK2) and DN-MK2 significantly enhanced TNF-mediated inhibition of EC migration, and MK2-K339R further augmented this effect. Interestingly, the p160-Rho–associated coiled-coil kinase (ROCK) inhibitor Y-27632 reversed this effect by MK2-K339R, which strongly suggests that both excessive and insufficient levels of actin filament remodeling can block EC migration. Our study shows that MK2 SUMOylation is a new mechanism for regulating actin filament dynamics in ECs.

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 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.

701-P: Effects of Exercise on Extracellular Matrix Modifiers in Subcutaneous Adipose Tissue of Obese Adults

Diabetes ◽  
2020 ◽  
Vol 69 (Supplement 1) ◽  
pp. 701-P
Author(s):  
PALLAVI VARSHNEY ◽  
BENJAMIN J. RYAN ◽  
CHIWOON AHN ◽  
MICHAEL W. SCHLEH ◽  
JEFFREY F. HOROWITZ
Keyword(s):  
Extracellular Matrix ◽  
Adipose Tissue ◽  
Subcutaneous Adipose Tissue ◽  
Obese Adults ◽  
Subcutaneous Adipose
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