ROCK inhibitors modulate the physical properties and adipogenesis of 3D spheroids of human orbital fibroblasts in different manners

Abstract

To elucidate the pharmacological effects of Rho-associated coiled-coil containing protein kinase inhibitors (ROCK-is), ripasudil (Rip), Y27632, and KD025, on human orbital fatty tissue, the human orbital fibroblasts (HOFs) were three-dimensional (3D) cultured for 12 days. The effects of ROCK-is on the physical properties of the 3D-cultured HOF spheroids, including their sizes and physical stiffness, their adipogenesis by lipid staining, and the mRNA expression of adipogenesis-related genes, PPARγ and AP2, and extracellular matrix (ECM) including collagen (COL) 1, 4, and 6, and fibronectin were analyzed. A significant increase in the sizes, physical stiffness, lipid staining, and mRNA expression of adipogenesis-related genes, COL4 and COL6, and a decrease in COL1 expression were observed with adipogenesis (DIF+). In the presence of ROCK-is, such DIF+-induced effects were differently modulated as follows: (1) the sizes were not affected or significantly enhanced by Rip, Y27632, or KD025, (2) the physical stiffness was significantly decreased in Rip and Y27632, but was substantially increased in KD025, (3) the lipid staining was further enhanced or significantly suppressed by Rip, Y27632, or KD025, and both PPARγ and AP2 expression were significantly downregulated or upregulated by KD025 or Rip, and (4) Rip upregulated the expression of COL4, Y27632 upregulated the expression of COL1, COL4, and COL6, and KD025 upregulated the expression of COL1 and COL4. This study indicates that ROCK-is significantly and differently modulate physical properties of the 3D HOF spheroids as well as their adipogenesis.

Keywords: ROCK; ROCK inhibitor; Rho kinase; human orbital fibroblasts (HOFs); three‐dimensional (3D) tissue culture.

FIGURE 1

Effects of ROCK‐is on the area sizes of the 3D human orbital fibroblast (HOF) spheroids during adipogenesis. At Day 12, the mean area sizes (μm²) of the 3D spheroids of HOFs preadipocytes (DIF−) and their adipogenic differentiation (DIF+) without or with 1 or 10 µM ripasudil (Rip), Y27632, or KD025 are plotted in panel (A). Among the different ROCK‐is forms, to compare their effects toward HOFs adipogenesis, the mean area sizes (μm²) of 10 µM of each ROCK‐is were replotted in panel (B), and their representative phase contrast microscopic images are shown in panel (C). All experiments were performed in triplicate using fresh preparations, each of which consisted of 16 spheroids. Data are presented as arithmetic means ±standard error of the mean (SEM). **p < 0.01, ***p < 0.005, ****p < 0.001 (ANOVA followed by Tukey's multiple comparison test). ROCK, Rho‐associated coiled‐coil containing protein kinase

FIGURE 2

Effects of ROCK‐is on physical stiffness of the 3T3‐L1 3D spheroids. At Day 12, the 3D human orbital fibroblast (HOF) spheroids of preadipocytes (DIF−) and their adipogenic differentiation (DIF+) without or with 1 or 10 µM ripasudil (Rip), Y27632, or KD025 were subjected to a physical solidity analysis using a microsqueezer. The force required to induce deformation until half diameter was reached (μN/μm force/displacement) were measured and the data are potted in panel A. Among the different ROCK‐is forms, to compare their effects toward HOFs adipogenesis, the physical stiffness of 10 µM of each ROCK‐is were replotted in panel B. All experiments were performed using freshly prepared 12–20 spheroids. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 (ANOVA followed by Tukey's multiple comparison test). ROCK, Rho‐associated coiled‐coil containing protein kinase

FIGURE 3

Effects of ROCK‐is on adipogenesis of 3D human orbital fibroblast (HOF) spheroids. The 3D HOF spheroids from Day 12 was prepared under several conditions: preadipocytes (DIF−) and their adipogenic differentiation (DIF+) without or with 10 µM ripasudil (Rip), Y27632, or KD025. These samples were immunostained with DAPI (blue), phalloidin (green), and BODIPY (red) (panel A, scale bar: 100 μm) and their staining intensities were plotted (panel B). The mRNA expressions of adipogenesis‐related genes including Pparγ and Ap2 under above conditions were plotted in panel ©. All experiments were performed in duplicate using fresh preparations, each consisting of 16 spheroids. Data are presented as arithmetic means ± SEM. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 (ANOVA followed by Tukey's multiple comparison test). ROCK, Rho‐associated coiled‐coil containing protein kinase

FIGURE 4

Effects of ROCK‐is on mRNA expressions of mRNA expression of ECMs in 3D human orbital fibroblast (HOF) spheroids. At Day 12, the 3D HOF spheroids of preadipocytes (DIF−) and their adipogenic differentiation (DIF+) without or with 10 µM ripasudil (Rip), Y27632, or KD025 were subjected to a quantitive PCR analysis to estimate the mRNA expression of ECMs. All experiments were performed in duplicate using fresh preparations, each of which consisted of 16 spheroids. Data are presented as arithmetic means ± SEM. COL1, collagen 1; COL4, collagen 4; COL6, collagen 6; ECM, extracellular matrix; FN, fibronectin. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 (ANOVA followed by Tukey's multiple comparison test). ROCK, Rho‐associated coiled‐coil containing protein kinase