Rosiglitasone and ROCK Inhibitors Modulate Fibrogenetic Changes in TGF-β2 Treated Human Conjunctival Fibroblasts (HconF) in Different Manners

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.

Modulation of the Physical Properties of 3D Spheroids Derived from Human Scleral Stroma Fibroblasts (HSSFs) with Different Axial Lengths Obtained from Surgical Patients

“…To elucidate the pathological involvement of human scleral stromal fibroblasts (HSSFs) within the myopia etiology, 3D spheroid cultures of HSSFs obtained from seven patients with several axial lengths (ALs, 22.80-30.63 mm) were employed. Consistent with our previous studies using human orbital fibroblasts (HOFs) [11,12] and human conjunctival fibroblasts (HconFs) [17], uniform, round-shaped 3D spheroids of HSSFs were successfully produced during the 5-day culture (Figure 1A). Nuclear staining with DAPI indicated (1) no artifacts or cell death, and (2) multiple layers of concentrically aligned HSSFs cells were observed within the interiors of the 3D HSSFs spheroids (Figure 1B).…”

Hypoxia Differently Affects TGF-β2-Induced Epithelial Mesenchymal Transitions in the 2D and 3D Culture of the Human Retinal Pigment Epithelium Cells

“…Experiments were taken in six parallel wells and repeated in dupicate. The TEER values for the HRPE cell monolayers were determined using a TEER plate (0.4 μm pore size and 12 mm diameter; Corning Transwell, Sigma-Aldrich, Burlington, MA, USA) and an electrical resistance system (KANTO CHEMICAL CO. INC., Tokyo, Japan) as described in a previous study [ 32 , 53 ]. Alternatively, HRPE cells prepared as above were further processed for a 3D spheroid preparation, as described below.…”

“…Therefore, to examine these issues in more detail, the TGF-β2-induced EMT of HRPE cells under normoxia and hypoxia conditions were subjected to the following analyses, employing our recently developed 3D cell cultures [ 29 , 30 , 31 , 32 ], in addition to conventional 2D cultures: (1) the barrier functions of 2D HRPE monolayers, (2) cellular mitochondrial and glycolytic functions (2D), physical properties of the 3D spheroids, and (3) the expression of ECM proteins, HIF1α and 2α, with the possible up-stream regulators requiring the generation of the 3D spheroids including STAT3 , IL6 , FOS , TGFb1 , AGT and MYC , and a master regulator of mitochondrial functions, PGC1a (2D and 3D).…”

Benzalkonium Chloride, Even at Low Concentrations, Deteriorates Intracellular Metabolic Capacity in Human Conjunctival Fibroblasts

“…As a negative control, the same diluent was used. In the presence 10 −5 % or 10 −4 % BAC, or only diluent, HconF cells (ScienCell Research laboratories, CA U.S.A.) were 2D cultured and further maintained or subjected to 3D spheroid culturing for 6 days using hanging drop culture plates (# HDP1385, Sigma-Aldrich, St Louis, MO, USA), as described in our previous study [ 26 ]. Briefly, 2D cultured HconF cells in 150 mm 2D culture dishes at 37 °C in the Fibroblast Medium (FM, Cat.…”

“…This information rationally suggests that BAC-induced risk may be evoked, even when much lower concentrations (5 × 10 −5 % ~ or 10 −3 %) of BAC are used as compared with the above estimated threshold levels. Therefore, to study the cytotoxic effects of BAC at much lower concentrations (10 −5 % and 10 −4 %) toward conjunctival tissues, we employed recently established in vitro models for the epithelial barrier and the stromal supportive functions of the human conjunctiva using two-dimension (2D) and three-dimension (3D) spheroid cultures of the human conjunctival fibroblasts (HconF) cells [ 26 ], and the following analyses were carried out: (1) barrier functions of 2D cultured HconF monolayers by trans-endothelial electron resistance (TEER) and FITC dextran permeability measurements, (2) measurements of real-time mitochondrial and glycolytic cellular function, (3) measurements of the size and hardness of the 3D HconF spheroids, and (4) quantitative PCR of major extracellular matrix (ECM) molecules, including collagen (COL)1, 4 and 6, and fibronectin, α-smooth muscle actin (α-SMA), ER stress related genes including the X-box binding protein-1 (XBP1), the spliced XBP1 (sXBP1) glucose regulator protein (GRP)78, GRP94, and the CCAAT/enhancer-binding protein homologous protein (CHOP), hypoxia inducible factor 1α (HIF1α), and Peroxisome proliferator-activated receptor gamma coactivator 1 (PGC1α).…”