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

Abstract

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).

Figure 1.

Effects of bimatoprost acid (BIM-A), and omidenepag (OMD) on adipogenesis in 3D cultured human orbital fibroblasts (HOFs). The mean sizes of 3D organoids of HOFs preadipocytes (DIF-, closed circles) and their adipogenic differentiation (DIF+, closed squares) in the presence and absence of 100 nM BIM-A (closed triangles), or OMD (10 nM: open circles, 100 nM: open squares, or 10,000 nM: open triangles) were measured. Representative phase contrast images of each organoid at day 10 under several conditions as above are shown in (A) (scale bar: 100 µm). Fluctuations in organoid size during a 10-day culture were plotted (B) and those at day 10 were compared among the experimental groups (C). All experiments were performed in triplicate using fresh preparations consisting of 16 organoids each. Data are presented as arithmetic means ± the standard error of the mean (SEM). ***P < 0.005 (ANOVA followed by a Tukey's multiple comparison test).

Figure 2.

Representative confocal images of lipid staining (BODIPY) and mRNA expression of adipogenesis related genes, PPARγ, AP2, and ADIPO Q of 3D HOFs organoids under various conditions. The 3D HOFs organoids at day 10 under various conditions; preadipocytes (DIF-) and their adipogenic differentiation (DIF+) without or with 100 nM of BIM-A or 100 nM OMD were prepared for immunostaining by BODIPY (red), DAPI (blue), and phalloidin (green) (A), and their staining intensities by BODIPY were plotted (B). For qPCR analysis to estimate the mRNA expression of adipogenesis related genes, PPARγ, AP2, and ADIPO Q, 10 nM OMD was also included in addition to the above conditions (C 1-3). All experiments were performed in duplicate using fresh preparations consisting of 10 organoids each for immunostaining or 16 organoids each for qPCR analysis. Data are presented as the arithmetic mean ± the standard error of the mean (SEM). *P < 0.05, ***P < 0.005, ****P < 0.001 (ANOVA followed by a Tukey's multiple comparison test).

Figure 3.

The mRNA expression of ECM in 3D HOFs organoids. At day 10, 3D HOFs organoids of preadipocytes (DIF-) and their adipogenic differentiation (DIF+) without or with 100 nM BIM-A, or 10, or 100 nM OMD were subjected to qPCR analysis to estimate the mRNA expression of ECM (COL 1: collagen 1, COL 4: collagen 4, COL 6: collagen 6, and FN: fibronectin). All experiments were performed in duplicate using fresh preparations consisting of 16 organoids each. Data are presented as arithmetic means ± the standard error of the mean (SEM). *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001 (ANOVA followed by a Tukey's multiple comparison test).

Figure 4.

Physical solidity of 3D HOFs organoids. Among the experimental groups under several conditions; preadipocytes of the HOFs (DIF-) and their adipogenic differentiation (DIF+) in the absence and presence of 100 nM BIM-A, or 100 nM OMD, physical solidity of the 3D organoids at day 10 were measured by means of a micro-squeezer (µN/µm force/displacement). All experiments were performed in duplicate using fresh preparations consisted of 12 to 19 organoids. Data are presented as arithmetic means ± the standard error of the mean (SEM). ns (not significant), ***P < 0.005 (ANOVA followed by a Tukey's multiple comparison test).