Departments of Ophthalmology, Sapporo Medical University School of Medicine, Sapporo, Japan.
Cardiovascular, Renal and Metabolic Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan.
Sci Rep. 2022 May 6;12(1):7419. doi: 10.1038/s41598-022-11407-1.
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.
本研究旨在探讨 ROCK1 和 ROCK2 在人眼角膜基质空间结构中的作用。我们研究了一种泛 ROCK 抑制剂（pan-ROCK-i）、利帕舒地尔和 ROCK2 抑制剂（ROCK2-i）KD025 对编码细胞外基质蛋白（包括胶原 COL1、4、6 和纤维连接蛋白 FN）及其调节剂、基质金属蛋白酶（MMP）抑制剂、基质金属蛋白酶（MMP）2、9 和 14 以及二维和三维（2D 和 3D）培养的人眼角膜基质成纤维细胞（HCSFs）的两种和三种组织抑制剂（TIMP）1-4 的基因表达的影响，以及 3D HCSF 球体的物理特性。使用 ROCK-i 的基因表达分析表明，KD025（ROCK2 选择性 ROCK 抑制剂）比 Rip（利帕舒地尔，泛 ROCK 抑制剂）诱导更显著的变化，这表明 ROCK2 可能比 ROCK1 更广泛地参与 ECM 蛋白和 2D 培养的 HCSFs 细胞结构的代谢。就 3D HCSFs 球体的物理特性，大小和硬度而言，Rip 导致明显的增大，这种增强作用呈浓度依赖性，而 KD025 也产生类似但不那么明显的作用。相反，Rip 和 KD025 对物理硬度的调节方式不同，Rip 导致大幅度下降，而 KD025 导致上升。Rip 和 KD025 之间也观察到对 ECM 蛋白及其调节剂和 ER 应激相关因子的基因表达的这种不同影响。本研究结果表明，ROCK1 和 ROCK2 以不同的方式影响 3D HCFS 球体的空间结构。
