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).

To elucidate the currently unknown mechanisms responsible for the diverse biological aspects between two-dimensional (2D) and three-dimensional (3D) cultured 3T3-L1 preadipocytes, RNA-sequencing analyses were performed. During a 7-day culture period, 2D- and 3D-cultured 3T3-L1 cells were subjected to lipid staining by BODIPY, qPCR for adipogenesis related genes, including peroxisome proliferator-activated receptor γ (Pparγ), CCAAT/enhancer-binding protein alpha (Cebpa), Ap2 (fatty acid-binding protein 4; Fabp4), leptin, and AdipoQ (adiponectin), and RNA-sequencing analysis. Differentially expressed genes (DEGs) were detected by next-generation RNA sequencing (RNA-seq) and validated by a quantitative reverse transcription–polymerase chain reaction (qRT–PCR). Bioinformatic analyses were performed on DEGs using a Gene Ontology (GO) enrichment analysis and an Ingenuity Pathway Analysis (IPA). Significant spontaneous adipogenesis was observed in 3D 3T3-L1 spheroids, but not in 2D-cultured cells. The mRNA expression of Pparγ, Cebpa, and Ap2 among the five genes tested were significantly higher in 3D spheroids than in 2D-cultured cells, thus providing support for this conclusion. RNA analysis demonstrated that a total of 826 upregulated and 725 downregulated genes were identified as DEGs. GO enrichment analysis and IPA found 50 possible upstream regulators, and among these, 6 regulators—transforming growth factor β1 (TGFβ1), signal transducer and activator of transcription 3 (STAT3), interleukin 6 (IL6), angiotensinogen (AGT), FOS, and MYC—were, in fact, significantly upregulated. Further analyses of these regulators by causal networks of the top 14 predicted diseases and functions networks (IPA network score indicated more than 30), suggesting that STAT3 was the most critical upstream regulator. The findings presented herein suggest that STAT3 has a critical role in regulating the unique biological properties of 3D spheroids that are produced from 3T3-L1 preadipocytes.

To characterize our recently established in vitro glaucomatous human trabecular meshwork (HTM) models using dexamethasone (DEX)- or TGF-β2-treated HTM cells, (1) two-dimensional (2D) cultured HTM cells were characterized by means of the real-time cellular metabolism analysis using a Seahorse analyzer, and (2) the effects of mechanical compression stresses toward the three-dimensional (3D) HTM spheroids were evaluated by analyzing the gene expression of several ECM proteins, inflammatory cytokines, and ER stress-related factors of those 3D HTM spheroid models. The results indicated that (1) the real-time cellular metabolism analysis indicated that TGF-β2 significantly induced an energy shift from mitochondrial oxidative phosphorylation (OXPHOS) into glycolysis, and DEX induced similar but lesser effects. In contrast, ROCK2 inhibition by KD025 caused a substantial reverse energy shift from glycolysis into OXPHOS. (2) Upon direct compression stresses toward the untreated control 3D HTM spheroids, a bimodal fluctuation of the mRNA expressions of ECM proteins was observed for 60 min, that is, initial significant upregulation (0–10 min) and subsequent downregulation (10–30 min) followed by another upregulation (30–60 min); those of inflammatory cytokines and ER stress-related factors were also bimodally changed. However, such compression stresses for 30 min toward TGF-β2- or DEX-treated 3D HTM spheroids induced downregulation of most of those of inflammatory cytokines and ER stress-related factors in addition to upregulation of COL1 and downregulation of FN. The findings presented herein indicate that (1) OXPHOS of the HTM cells was decreased or increased by TGF-β2 or DEX stimulation or ROCK2 inhibition, and (2) mechanical compression stresses toward 3D HTM spheroids may replicate acute, subacute, and chronic HTM models affected by elevated intraocular pressures.

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The objective of the current study was to examine the roles of ROCK1 and 2 on the spatial architecture of human corneal stroma. We examined the effects of a pan-ROCK inhibitor (pan-ROCK-i), ripasudil, and a ROCK2 inhibitor (ROCK2-i), KD025 on the expression of genes that encode for ECM proteins including collagen (COL) 1, 4, 6, and fibronectin (FN), their regulators, a tissue inhibitor of metalloproteinase (TIMP) 1–4, matrix metalloproteinase (MMP) 2, 9 and 14, and ER stress-related factors of two- and three-dimensional (2D and 3D) cultures of human corneal stroma fibroblasts (HCSFs), and the physical properties of 3D HCSF spheroids. A gene expression analysis using ROCK-is indicated that KD025 (ROCK2 selective ROCK inhibitor) induced more significant changes than Rip (ripasudil, pan-ROCK inhibitor), suggesting that ROCK2 might be more extensively involved in the metabolism of ECM proteins and cell architectures of the 2D cultured HCSFs than ROCK1. In terms of the physical properties, size and stiffness of the 3D HCSFs spheroids, Rip caused a significant enlargement and this enhancement was concentration-dependent while KD025 also exerted a similar but less pronounced effect. In contrast, Rip and KD025 modulated physical stiffness differently, in that Rip caused a substantial decrease and KD025 caused an increase. Such diverse effects between Rip and KD025 were also observed for the gene expressions of ECM proteins, their regulators, and ER-stress related factors. The findings presented herein suggest that the ROCK1 and 2 influence the spatial architecture of 3D HCFS spheroids in different manners.