Abstract
Since 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.
Highlights
Since 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
We found that the effects of PGF2α agonists and EP2 agonists on adipogenesis as well as the expression of extracellular matrix (ECM) in 3T3-L1 cells were significantly different between their 2D and 3D cultures[22,24], and the findings suggested that 3D cultures may become a reliable ex vivo model for use in this research field
In the presence of 10 μM Y27632, both DIF+ induced up-regulated or down-regulated genes expression except Col[1] were increased (Fig. 2). These findings indicated that Rho-associated coiled-coil containing protein kinase inhibitor (ROCK-i), Rip and Y27632 have a significant effect on adipogenesis and ECM expression in 2D cultured 3T3-L1 cells, and these effects were similar regarding adipogenesis, but different with respect to ECM expression between the Rho-associated coiled-coil containing protein kinases (ROCKs)-is
Summary
Since 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. 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 conducted. In the present study, to report on the effects of the ROCK-is, Rip and Y27632 on the adipogenesis of the 3T3-L1 cells, a most widely used as adipocyte cell line for adipogenesis related research, we studied adipogenesis by lipid staining as well as adipogenesis related gene expressions, their ECM expressions (2D and 3D cell cultures), and sizes and physical stiffness by a micro-squeezer of the 3D organoid. During the course of the 3D 3T3-L1 organoid culture, their physical properties including the 3D organoid size, appearance by phase contrast and electron microscope images, and stiffness were investigated
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