Search Results (8)

Background: Although lysophosphatidic acid (LPA) is known to have multiple pathophysiological roles, its contributions to ocular tissues, especially conjunctival fibrogenesis, remain to be elucidated. Methods: To study this issue, the effects of LPA on transforming growth factor-β2 (TGF-β2)-induced fibrogenesis of two-dimensional (2D) and three-dimensional (3D) cultures of human conjunctival fibroblasts (HconF) were examined by the following analyses: (1) planar proliferation determined by transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran permeability measurements, (2) real-time metabolic analyses, (3) measurements of the size and stiffness of 3D spheroids, and (4) mRNA expression of extracellular matrix (ECM) molecules and their modulators. Results: LPA had no effect on TGF-β2-induced increase in the planar proliferation of HconF cells. LPA induced a more quiescent metabolic state in 2D HconF cells, but this metabolic suppression by LPA was partially blunted in the presence of TGF-β2. In contrast, LPA caused a substantial decrease in the hardness of 3D HconF spheroids independently of TGF-β2. In agreement with these different LPA-induced effects between 2D and 3D cultured HconF cells, mRNA expressions of ECM and their modulators were differently modulated. Conclusion: The findings that LPA induced the inhibition of both TGF-β2-related and -unrelated subepithelial proliferation of HconF cells may be clinically applicable. Full article

The current study’s objective was to elucidate some currently unknown biological indicators to evaluate the biological nature of cancer-associated fibroblasts (CAFs). For this purpose, four different CAFs, CAFS1, CAFS2, SCC17F and MO-1000, were established using surgical specimens from oral squamous cell carcinomas (OSCC) with different clinical malignant stages (CAFS1 and CAFS2, T2N0M0, stage II; SCC17F and MO-1000, T4aN2bM0, stage IVA). Fibroblasts unrelated to cancer (non-CAFs) were also prepared and used as controls. Initially, confirmation that these four fibroblasts were indeed CAFs was obtained by their mRNA expression using positive and negative markers for the CAF or fibroblasts. To elucidate possible unknown biological indicators, these fibroblasts were subjected to a cellular metabolic analysis by a Seahorse bioanalyzer, in conjugation with 3D spheroid cultures of the cells and co-cultures with a pancreas ductal carcinoma cell line, MIA PaCa-2. The mitochondrial and glycolytic functions of human orbital fibroblasts (HOF) were nearly identical to those of Graves’-disease-related HOF (GOF). In contrast, the characteristics of the metabolic functions of these four CAFs were different from those of human conjunctival fibroblasts (HconF), a representative non-CAF. It is particularly noteworthy that CAFS1 and CAFS2 showed markedly reduced ratios for the rate of oxygen consumption to the extracellular acidification rate, suggesting that glycolysis was enhanced compared to mitochondrial respiration. Similarly, the physical aspects, their appearance and stiffness, of their 3D spheroids and fibroblasts that were induced effects based on the cellular metabolic functions of MIA PaCa-2 were also different between CAFs and non-CAFs, and their levels for CAFS1 or SCC17F were similar to those for CAFS2 or MO-1000 cells, respectively. The findings reported herein indicate that cellular metabolic functions and the physical characteristics of these types of 3D spheroids may be valuable and useful indicators for estimating potential biological diversity among various CAFs. Full article

Three highly homologous isoforms of TGF-β, TGF-β-1~3, are involved in the regulation of various pathophysiological conditions such as wound healing processes in different manners, despite the fact that they bind to the same receptors during their activation. The purpose of the current investigation was to elucidate the contributions of TGF-β-1 ~3 to the pathology associated with conjunctiva. For this purpose, the biological effects of these TGF-β isoforms on the structural and functional properties of two-dimensional (2D) and three-dimensional (3D) cultured human conjunctival fibroblasts (HconF) were subjected to the following analyses: 1) transendothelial electrical resistance (TEER), a Seahorse cellular metabolic measurement (2D), size and stiffness measurements of the 3D HTM spheroids, and the qPCR gene expression analyses of extracellular matrix (ECM) components (2D and 3D). The TGF-β isoforms caused different effects on the proliferation of the HconF cell monolayer evaluated by TEER measurements. The differences included a significant increase in the presence of 5 ng/mL TGF-β-1 and -2 and a substantial decrease in the presence of 5 ng/mL TGF-β-3, although there were no significant differences in the response to the TGF-β isoforms for cellular metabolism among the three groups. Similar to planar proliferation, the TGF-β isoforms also induced diverse effects toward the mechanical aspects of 3D HconF spheroids, where TGF-β-1 increased stiffness, TGF-β-2 caused no significant effects, and TGF-β-3 caused the downsizing of the spheroids and stiffness enhancement. The mRNA expression of the ECMs were also modulated in diverse manners by the TGF-β isoforms as well as the culture conditions for the 2D vs. 3D isoforms. Many of these TGF-β-3 inducible effects were markedly different from those caused by TGF-β1 and TGF-β-2. The findings presented herein suggest that the three TGF-β isoforms induce diverse and distinctly different effects on cellular properties and the expressions of ECM molecules in HconF and that these changes are independent of cellular metabolism, thereby inducing different effects on the epithelial and subepithelial proliferation of human conjunctiva. Full article

The objective of this study was to clarify the effects of benzalkonium chloride (BAC) on two-dimensional (2D) and three-dimensional (3D) cultures of human conjunctival fibroblast (HconF) cells, which are in vitro models replicating the epithelial barrier and the stromal supportive functions of the human conjunctiva. The cultured HconF cells were subjected to the following analyses in the absence and presence of 10⁻⁵% or 10⁻⁴% concentrations of BAC; (1) the barrier function of the 2D HconF monolayers, as determined by trans-endothelial electrical resistance (TEER) and FITC dextran permeability, (2) real-time metabolic analysis using an extracellular Seahorse flux analyzer, (3) the size and stiffness of 3D HconF spheroids, and (4) the mRNA expression of genes that encode for extracellular matrix (ECM) molecules including collagen (COL)1, 4 and 6, and fibronectin (FN), 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 1α (PGC1α). In the presence of BAC, even at low concentrations at 10⁻⁵% or 10⁻⁴%, the maximal respiratory capacity, mitochondrial respiratory reserve, and glycolytic reserve of HconF cells were significantly decreased, although the barrier functions of 2D HconF monolayers, the physical properties of the 3D HconF spheroids, and the mRNA expression of the corresponding genes were not affected. The findings reported herein highlight the fact that BAC, even such low concentrations, may induce unfavorable adverse effects on the cellular metabolic capacity of the human conjunctiva. Full article

Vitamin A derivative, all-trans-retinoic acid (ATRA), is known to be a potent regulator of the growth and differentiation of various types of cells. In the present study, the unidentified effects of ATRA on superficial and vertical spreading conjunctival scarring were examined. The study involved the use of two-dimensional (2D) and three-dimensional (3D) cultures of human conjunctival fibroblast (HconF) cells in the presence or absence of TGF-β2. The effects of ATRA (1 μM) on superficial or vertical spreading conjunctival scarring were evaluated by the barrier function by trans-endothelial electrical resistance (TEER) and FITC dextran permeability measurements and real-time metabolic analysis, as well as the physical properties, namely, the size and stiffness, of 3D spheroids, respectively. In addition, the expressions of several related molecules, including extracellular matrix (ECM) molecules, ECM modulators including a tissue inhibitor of metalloproteinases (TIMPs), matrix metalloproteinases (MMPs), and ER stress-related factors, were examined. ATRA significantly induced (1) an increase in TEER values and a decrease in FITC dextran permeability, respectively, in the 2D monolayers, and (2) relatively and substantially increased the size and stiffness, respectively, of the 3D spheroids. These ATRA-induced effects were further enhanced in the TGF-β2-treated cells, whereas the TGF-β2-induced enhancement in glycolytic capacity was canceled by the presence of ATRA. Consistent with these physical and morphological effects, the mRNA expressions of several molecules were significantly but differently induced between 2D and 3D cultures by ATRA, although the presence of TGF-β2 did not substantially affect these gene expression levels. The findings reported in this study indicate that ATRA may exacerbate both superficial and vertical conjunctival fibrosis spreading independently of TGF-β2-induced changes. Full article

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. Full article

Scar formation can cause the failure of glaucoma filtration surgery. We investigated the effect of AR12286, a selective Rho-associated kinase inhibitor, on myofibroblast transdifferentiation and intraocular pressure assessment in rabbit glaucoma filtration surgery models. Cell migration and collagen contraction were used to demonstrate the functionality of AR12286-modulated human conjunctival fibroblasts (HConFs). Polymerase chain reaction quantitative analysis was used to determine the effect of AR12286 on the production of collagen Type 1A1 and fibronectin 1. Cell migration and collagen contraction in HConFs were activated by TGF-β1. However, compared with the control group, rabbit models treated with AR12286 exhibited higher reduction in intraocular pressure after filtration surgery, and decreased collagen levels at the wound site in vivo. Therefore, increased α-SMA expression in HConFs induced by TGF-β1 could be inhibited by AR12286, and the production of Type 1A1 collagen and fibronectin 1 in TGF-β1-treated HConFs was inhibited by AR12286. Overall, the stimulation of HConFs by TGF-β1 was alleviated by AR12286, and this effect was mediated by the downregulation of TGF-β receptor-related SMAD signaling pathways. In vivo results indicated that AR12286 thus improves the outcome of filtration surgery as a result of its antifibrotic action in the bleb tissue because AR12286 inhibited the TGF-β receptor-related signaling pathway, suppressing several downstream reactions in myofibroblast transdifferentiation. Full article

Recent evidence suggests that chloride channels are critical for cell proliferation, migration, and differentiation. We examined the effects of transforming growth factor (TGF)-β1 on chloride channel expression and associations with human conjunctival fibroblast (HConF) biology. To investigate the potential role of chloride channel (CLC)-2 in migration, transition to myofibroblasts and extracellular matrix (ECM) synthesis of HconF, a small interfering RNA (siRNA) approach was applied. TGF-β1-induced migration and transition of fibroblasts to myofibroblasts characterized by α-smooth muscle actin (α-SMA) expression, supported by increased endogenous expression of CLC-2 protein and mRNA transcripts. ECM (collagen I and fibronectin) synthesis in HConF was enhanced by TGF-β1. CLC-2 siRNA treatment reduced TGF-β1-induced cell migration, transition of fibroblasts to myofibroblasts, and ECM synthesis of HConF. CLC-2 siRNA treatment in the presence of TGF-β1 inhibited phosphorylation of PI3K and Akt in HConF. These findings demonstrate that CLC-2 chloride channels are important for TGF-β1-induced migration, differentiation, and ECM synthesis via PI3K/Akt signaling in HConF. Full article