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

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.

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The Effect of the H-1152P, a Potent Rho-Associated Coiled Coil-Formed Protein Kinase Inhibitor, in Rabbit Normal and Ocular Hypertensive Eyes

Current Eye Research ◽

10.1080/02713680902783763 ◽

2009 ◽

Vol 34 (4) ◽

pp. 282-286 ◽

Cited By ~ 24

Author(s):

Masahiro Nishio ◽

Takaki Fukunaga ◽

Masahiko Sugimoto ◽

Kengo Ikesugi ◽

Kengo Sumi ◽

...

Keyword(s):

Protein Kinase ◽

Kinase Inhibitor ◽

Coiled Coil ◽

Protein Kinase Inhibitor

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Fasudil, a Rho-Associated Coiled Coil-Forming Protein Kinase Inhibitor, Recovers Methylmercury-Induced Axonal Degeneration by Changing Microglial Phenotype in Rats

Toxicological Sciences ◽

10.1093/toxsci/kfy281 ◽

2018 ◽

Vol 168 (1) ◽

pp. 126-136 ◽

Cited By ~ 7

Author(s):

Masatake Fujimura ◽

Fusako Usuki ◽

Atsushi Nakamura

Keyword(s):

Protein Kinase ◽

Axonal Degeneration ◽

Kinase Inhibitor ◽

Coiled Coil ◽

Protein Kinase Inhibitor ◽

Microglial Phenotype

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

International Journal of Molecular Sciences ◽

10.3390/ijms22094648 ◽

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.

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