Abstract
2D and 3D cultures of 3T3-L1 cells were employed in a study of the effects of Omidenepag (OMD), interacting with a non-prostanoid EP2 receptor, on adipogenesis. Upon adipogenesis, the effects on lipid staining, the mRNA expression of adipogenesis-related genes (Pparγ, CEBPa, Ap2, and Glut4) and the extracellular matrix (ECM) including collagen type 1, 4 and 6, and fibronectin, and the size and physical property of 3D organoids were compared between groups that had been treated with EP2 agonists (butaprost and OMD) and PGF2α. Upon adipogenesis, these significantly suppressed lipid staining and the mRNA expression of related genes. EP2 agonists and PGF2α influenced the mRNA expression of ECM in different manners, and these effects were also different between 2 and 3D cultures. Examining the physical properties by a microsqueezer indicated that the solidity of the 3D organoids became significantly lowered upon adipogenesis and these effects were not affected by EP2 agonists. In contrast, 3D organoid stiffness was markedly enhanced by the presence of PGF2α. These observations indicate that EP2 agonists affect the adipogenesis of 3T3-L1 cells in different manners, as compared to PGF2α, suggesting that OMD may not induce PGF2α related orbital fat atrophy, called the deepening of the upper eyelid sulcus (DUES).
Highlights
Omidenepag isopropyl (OMDI), a prodrug, is hydrolyzed in the eye to the active form (Omidenepag, OMD) which functions as a selective, nonprostaglandin, prostanoid EP2 agonist, was recently developed and is being used in the treatment of patients with OH and glaucoma[11,12]
Using the 3D organoid culture technique, in our previous studies, we found that Prostaglandin F2α (PGF2α) significantly affected adipogenesis and the extracellular matrixes (ECMs) in 3T3-L1 c ells[16] as well as human orbital fibrobrasts (HOFs)[17]
To compare pharmacological effects between PGF2α and EP receptor agonists toward adipogenesis of the 2D cultured 3T3-L1 cells, lipid staining by Oil Red O, and the mRNA expression of adipogenesis related genes including Pparγ, Cebpα, Ap2 and Glut[4], and major ECM (Col 1: collagen 1, Col 4: collagen 4, Col 6: collagen 6, Fn: fibronectin) were evaluated in the presence of PGF2α or EP receptor agonists (Buta: butaprost, OMD: Omidenepag)
Summary
Omidenepag isopropyl (OMDI), a prodrug, is hydrolyzed in the eye to the active form (Omidenepag, OMD) which functions as a selective, nonprostaglandin, prostanoid EP2 agonist, was recently developed and is being used in the treatment of patients with OH and glaucoma[11,12]. Studies using OH monkeys demonstrated that the dynamics of the pharmacokinetics of OMD in the aqueous humor (AH) are quite different from those of FP agonists they are categorized as members of the prostanoid receptor family of agonists[11]. Based upon these facts, it would be of great interest to examine the issue of whether OMD may cause DUSE or not. Yamamoto et al recently examined the effects of OMD on adipogenesis in 3T3-L1 cells in conventional 2D cultures, and found that OMD had no effect on adipocyte differentiation[12]. In the current study, using a replicated pathogenic DUES model by 2D and 3D cultures using 3T3-L1 cells, we analyzed and compared the effects of FP and EP agonists, OMD and butaprost (Buta), on adipogenesis, ECM expression, and the sizes and physical properties of the 3D organoids
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