Abstract
Purpose: The effects of Rho-associated coiled-coil containing protein kinase (ROCK) 1 and 2 inhibitor, ripasudil hydrochloride hydrate (Rip), ROCK2 inhibitor, KD025 or rosiglitazone (Rosi) on two-dimension (2D) and three-dimension (3D) cultured human conjunctival fibroblasts (HconF) treated by transforming growth factor (TGFβ2) were studied. Methods: Two-dimension and three-dimension cultured HconF were examined by transendothelial electrical resistance (TEER, 2D), size and stiffness (3D), and the expression of the extracellular matrix (ECM) including collagen1 (COL1), COL4 and COL6, fibronectin (FN), and α-smooth muscle actin (αSMA) by quantitative PCR (2D, 3D) in the presence of Rip, KD025 or Rosi. Results: TGFβ2 caused a significant increase in (1) the TEER values (2D) which were greatly reduced by Rosi, (2) the stiffness of the 3D organoids which were substantially reduced by Rip or KD025, and (3) TGFβ2 induced a significant up-regulation of all ECMs, except for COL6 (2D) or αSMA (3D), and down-regulation of COL6 (2D). Rosi caused a significant up-regulation of COL1, 4 and 6 (3D), and down-regulation of COL6 (2D) and αSMA (3D). Most of these TGFβ2-induced expressions in the 2D and αSMA in the 3D were substantially inhibited by KD025, but COL4 and αSMA in 2D were further enhanced by Rip. Conclusion: The findings reported herein indicate that TGFβ2 induces an increase in fibrogenetic changes on the plane and in the spatial space, and are inhibited by Rosi and ROCK inhibitors, respectively.
Highlights
It is well known that the human conjunctiva plays pivotal roles in serving as a physical protecting barrier and maintaining the homeostasis of the ocular surface [1]
Upon exposure to a variety of stimuli, transforming growth factor (TGF)-β, fibroblasts can be transdifferentiated into myofibroblasts [10,12,13], which are associated with smooth muscle cell characteristics and the expressed α-smooth muscle actin (α-SMA)
We reported on the development of a suitable in vivo model that replicates the glaucomatous human trabecular meshwork (HTM) by a three-dimension (3D) drop culture method [36,37,38] using TGF-β2-treated HTM cells in addition to the conventional two-dimension (2D) culture, and successfully evaluated the drug efficacy of Rho-associated coiled-coil containing protein kinase (ROCK)-is on their TGF-β2-induced fibrogenetic changes [38]
Summary
It is well known that the human conjunctiva plays pivotal roles in serving as a physical protecting barrier and maintaining the homeostasis of the ocular surface [1]. In terms of the clinical aspects of subconjunctival fibrosis, it has been suggested that the regulation of wound healing in the conjunctiva is of great importance in terms of the surgical outcomes of ocular surface-related diseases, such as pterygium and glaucoma [3,4,5,6,7]. It is generally recognized that the fibroblast is the responsible cell in the normal wound healing process, as well as in the development of fibrosis [10]. It is known that several cytokines and growth factors are involved in this wound healing process. During the normal wound repair process, myofibroblasts undergo apoptosis and are removed from the wound area If such a wound repairing process fails, progressive fibrosis by myofibroblasts may cause additional scar formation [12,14]. Anti-VEGF agents have the potential for clinical use in regulating TGF-β2 expression in conjunctival fibroblasts
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