Omidenepag, a non-prostanoid EP2 receptor agonist, induces enlargement of the 3D organoid of 3T3-L1 cells

Ida, Yosuke; Hikage, Fumihito; Umetsu, Araya; Ida, Haruka; Ohguro, Hiroshi

doi:10.1038/s41598-020-72538-x

Cited by 28 publications

(74 citation statements)

References 36 publications

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“…To compare effects of FP-Rag, BIM-A, and EP2 agonist on physical property of the 3D HOFs organoid, changes of their sizes were evaluated. Similar to the 3T3-L1 cells, 14 , 18 uniform round-shaped 3D organoids were obtained from 20,000 HOFs cells ( Fig. 1 A).…”

Section: Resultssupporting

confidence: 67%

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Omidenepag, a Selective, Prostanoid EP2 Agonist, Does Not Suppress Adipogenesis in 3D Organoids of Human Orbital Fibroblasts

Hikage

Ida

Ouchi

et al. 2021

Trans. Vis. Sci. Tech.

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Purpose The purpose of this study was to present the effects of the prostanoid EP2 agonist, omidenepag (OMD) on human orbital fibroblasts (HOFs) using a three-dimension (3D) cell culture. Methods During adipogenesis of 3D HOFs organoids, changes in size, lipids staining, mRNA expression of adipogenesis related genes, PPARγ , AP2 , and ADIPOQ , and extracellular matrix, collagen 1 (COL 1), COL 4, COL 6, and fibronectin (FN), and stiffness by a micro-squeezer were examined in the presence and absence of either 100 nM bimatoprost acid (BIM-A) or 10, 100, or 10,000 nM OMD. Results The size of the 3D organoids increased dramatically during adipogenesis, and these were further enhanced in the presence of OMD in contrast to the BIM-A induced suppression effect. The intensity of lipid staining and the mRNA expression of PPARγ were significantly increased upon adipogenesis, and both or latter was markedly inhibited in the presence of OMD or BIM-A, respectively. AP2 expression was also upregulated by adipogenesis, and was further enhanced by BIM-A. The adipogenesis-induced downregulation of COL 1 and FN, or the upregulation of the expression of COL 4 and COL 6 were all suppressed in the presence of BIM-A. In contrast, OMD caused similar effects on COL 4, COL 6, or FN expression, but caused a significant increase in COL 1 expression. Stiffness was significantly increased upon adipogenesis, and was further increased or substantially decreased by BIM-A or OMD, respectively. Conclusions The present study indicates that the FP2 agonist, OMD, had different effects on 3D HOFs organoids, as compared to BIM-A. Translational Relevance The current study suggests that OMD may not induce deepening of upper eyelid sulcus (DUES).

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Section: Resultssupporting

confidence: 67%

“… 2 , 11 , 14 Furthermore, our recent pilot study indicated that OMD induced different effects toward 3T3-L1 cells as compared to BIM-A. 18 This prompted us to perform a similar study based on 3D organoid cultures using HOFs rather than 3T3-L1 cells to determine the effects of OMD on human orbital fatty tissues.…”

Section: Introductionmentioning

confidence: 99%

Omidenepag, a Selective, Prostanoid EP2 Agonist, Does Not Suppress Adipogenesis in 3D Organoids of Human Orbital Fibroblasts

Hikage

Ida

Ouchi

et al. 2021

Trans. Vis. Sci. Tech.

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“…The objective of this current study was to evaluate the effects of ROCK-is toward adipogenesis and ECM expression in 3T3-L1 cells. In our previous studies, we found that the effects of PGF2α agonists and EP2 agonists on adipogenesis as well as the expression of 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. However, our 3D drop-culture method has not yet fully characterized so far, especially in terms of the physical properties, sizes and stiffness of organoids during the period of their culture.…”

Section: Resultsmentioning

confidence: 91%

“…Oil Red O lipid staining of the 2D cultured 3T3-L1 cells and BODIPY lipid staining of the 3D 3T3-L1 organoids were performed as described previously 24 . To quantify their staining levels, measurements of optical density of the extracted Oil Red O dye, and the fluorescence intensity of the BODIPY-stained lipid droplets were used, respectively 24 .…”

Section: Methodsmentioning

confidence: 99%

“…The RNA extracted from 16 organoids of each groups were processed to reverse transcription, and each respective gene expression was quantified by real-time PCR as described previously 24 . In terms of assay specificity for qPCR, to guarantee a high quality of the assay, predesigned primers shown in the supplemental Table 1 from Integrated DNA Technologies, Inc. Iowa USA, were used.…”

Section: Methodsmentioning

confidence: 99%

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

Ida

Hikage

Ohguro

2021

Sci Rep

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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.

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TGF‐β effects on adipogenesis of 3T3‐L1 cells differ in 2D and 3D cell culture conditions

Umetsu,

Watanabe,

Sato

et al. 2024

FEBS Open Bio

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The TGF‐β superfamily plays a pivotal role in the regulation of adipogenesis, but little is known about the potential differential role of the three isoforms of TGF‐β, TGF‐β‐1~3. To further elucidate their role, two‐dimensionally (2D) and three‐dimensionally (3D) cultured 3T3‐L1 mouse preadipocytes were subjected to the following analyses: (a) qPCR analysis of adipogenesis‐related factors and major extracellular matrix protein (2D and /or 3D), (b) lipid staining by Oil Red O (2D) or BODIPY (3D), (c) Seahorse cellular metabolic measurement (2D), and (d) size and stiffness measurements of 3D 3T3‐L1 spheroids. In the 2D cultured 3T3‐L1 cells, mRNA expression levels of adipogenesis‐related genes and Oil Red O lipid staining intensity were significantly increased by adipogenesis and they were substantially decreased following treatment with 0.1 nm TGF‐β isoforms, with TGF‐β2 having the greater effects. Consistent with these results, treatment with TGF‐β2 resulted in suppression of mitochondrial and glycolytic functions in 2D cultured 3T3‐L1 cells. However, the inhibitory effect of TGF‐β on adipogenesis decreased under 3D spheroid culture conditions and TGF‐β isoforms did not affect adipogenesis‐induced (a) enlargement and downsizing of 3T3‐L1 spheroids, (b) increase in BODIPY lipid staining intensity, and (c) up‐regulation of the mRNA expression of adipogenesis‐related genes. The findings presented herein suggest that the three TGF‐β isoforms have different suppressive effects on adipogenesis‐related cellular properties of 2D cultured 3T3‐L1 cells and that their effects decrease under 3D spheroid culture conditions.

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Omidenepag, a non-prostanoid EP2 receptor agonist, induces enlargement of the 3D organoid of 3T3-L1 cells

Cited by 28 publications

References 36 publications

Omidenepag, a Selective, Prostanoid EP2 Agonist, Does Not Suppress Adipogenesis in 3D Organoids of Human Orbital Fibroblasts

Omidenepag, a Selective, Prostanoid EP2 Agonist, Does Not Suppress Adipogenesis in 3D Organoids of Human Orbital Fibroblasts

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

TGF‐β effects on adipogenesis of 3T3‐L1 cells differ in 2D and 3D cell culture conditions

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