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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.

SUBMITTER: Oouchi Y

PROVIDER: S-EPMC8307967 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Human Trabecular Meshwork (HTM) Cells Treated with TGF-β2 or Dexamethasone Respond to Compression Stress in Different Manners.

Project description:To characterize our recently established in vitro glaucomatous human trabecular meshwork (HTM) models using dexamethasone (DEX)- or TGF-β2-treated HTM cells, (1) two-dimensional (2D) cultured HTM cells were characterized by means of the real-time cellular metabolism analysis using a Seahorse analyzer, and (2) the effects of mechanical compression stresses toward the three-dimensional (3D) HTM spheroids were evaluated by analyzing the gene expression of several ECM proteins, inflammatory cytokines, and ER stress-related factors of those 3D HTM spheroid models. The results indicated that (1) the real-time cellular metabolism analysis indicated that TGF-β2 significantly induced an energy shift from mitochondrial oxidative phosphorylation (OXPHOS) into glycolysis, and DEX induced similar but lesser effects. In contrast, ROCK2 inhibition by KD025 caused a substantial reverse energy shift from glycolysis into OXPHOS. (2) Upon direct compression stresses toward the untreated control 3D HTM spheroids, a bimodal fluctuation of the mRNA expressions of ECM proteins was observed for 60 min, that is, initial significant upregulation (0-10 min) and subsequent downregulation (10-30 min) followed by another upregulation (30-60 min); those of inflammatory cytokines and ER stress-related factors were also bimodally changed. However, such compression stresses for 30 min toward TGF-β2- or DEX-treated 3D HTM spheroids induced downregulation of most of those of inflammatory cytokines and ER stress-related factors in addition to upregulation of COL1 and downregulation of FN. The findings presented herein indicate that (1) OXPHOS of the HTM cells was decreased or increased by TGF-β2 or DEX stimulation or ROCK2 inhibition, and (2) mechanical compression stresses toward 3D HTM spheroids may replicate acute, subacute, and chronic HTM models affected by elevated intraocular pressures.

| S-EPMC9219943 | biostudies-literature

ROCK 1 and 2 affect the spatial architecture of 3D spheroids derived from human corneal stromal fibroblasts in different manners.

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Pan-ROCK and ROCK2 Inhibitors Affect Dexamethasone-Treated 2D- and 3D-Cultured Human Trabecular Meshwork (HTM) Cells in Opposite Manners

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TGF-β-induced activation of conjunctival fibroblasts is modulated by FGF-2 and substratum stiffness.

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| S-EPMC7673499 | biostudies-literature

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mTOR inhibitors potentially reduce TGF-β2-induced fibrogenic changes in trabecular meshwork cells.

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| S-EPMC8266857 | biostudies-literature

Antifibrotic effects of eupatilin on TGF-β1-treated human vocal fold fibroblasts.

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Knockdown of lncRNA-ATB suppresses autocrine secretion of TGF-β2 by targeting ZNF217 via miR-200c in keloid fibroblasts.

Project description:Abnormally high activation of transforming growth factor-β (TGF-β) signaling has been demonstrated to be involved in the initiation and progression of keloids. However, the functional role of long non-coding RNA (lncRNA)-activated by TGF-β (lncRNA-ATB) in keloids has not been documented. Here we investigated the role of lncRNA-ATB in the autocrine secretion of TGF-β in keloid fibroblasts (KFs) and explored the underlying molecular mechanism. Using immunohistochemistry and quantitative RT-PCR analysis, we showed that lncRNA-ATB and ZNF217, a transcriptional activator of TGF-β, were overexpressed and miR-200c, which targets ZNF217, was under-expressed in keloid tissue and keloid fibroblasts. Through gain- and loss-of-function studies, we demonstrated that knockdown of lncRNA-ATB decreased autocrine secretion of TGF-β2 and ZNF217 expression but upregulated expression of miR-200c in KFs. Stable downregulation of ZNF217 expression decreased the autocrine secretion of TGF-β2. miR-200c was endogenously associated with lncRNA-ATB, and inhibition of miR-200c overcame the decrease in ZNF217 expression in KFs. Taken together, these findings indicate that lncRNA-ATB governs the autocrine secretion of TGF-β2 in KFs, at least in part, by downregulating the expression level of ZNF217 via miR-200c, suggesting a signaling axis consisting of lncRNA-ATB/miR-200c/ZNF217/TGF-β2. These findings may provide potential biomarkers and targets for novel diagnostic and therapeutic approaches for keloids.

| S-EPMC4835760 | biostudies-literature

m6A Epitranscriptomic Microarray of primary mouse RPE cells treated with TGF-β2

Project description:m6A-mRNA&lncRNA Epitranscriptomic Microarray of primary mouse RPE cells comparing control untreated RPE cells with RPE cells treated with TGF-β2 at a concentration of 10 ng/ml. The goal was to determine the effects of RNA m6a methylation on primary mouse RPE cells undergoing epithelial-mesenchymal transition induced by TGF-β2.

2021-06-26 | GSE178946 | GEO

Endothelial cells from different anatomical origin have distinct responses during SNAIL/TGF-β2-mediated endothelial-mesenchymal transition.

Project description:BackgroundEndothelial-mesenchymal transition (EndMT) is a complex process whereby differentiated endothelial cells undergo phenotypic transition to mesenchymal cells. EndMT can be stimulated by several factors and the most common are the transforming growth factor-beta (TGF-β) and SNAIL transcription factor. Given the diversity of the vascular system, it is unclear whether endothelial cells lining different vessels are able to undergo EndMT through the same mechanisms. Here we evaluate the molecular and functional changes that occur in different types of endothelial cells following induction of EndMT by overexpression of SNAIL and TGF-β2.ResultsWe found that responses to induction by SNAIL are determined by cell origin and marker expression. Human coronary endothelial cells (HCAECs) showed the greatest EndMT responses evidenced by significant reciprocal changes in the expression of mesenchymal and endothelial markers, effects that were potentiated by a combination of SNAIL and TGF-β2. Key molecular events associated with EndMT driven by SNAIL/TGF-β2 involved extracellular-matrix remodeling and inflammation (IL-8, IL-12, IGF-1, and TREM-1 signaling). Notch signaling pathway members DLL4, NOTCH3 and NOTCH4 as well as members of the Wnt signaling pathway FZD2, FZD9, and WNT5B were altered in the combination treatment strategy, implicating Notch and Wnt signaling pathways in the induction process.ConclusionOur results provide a foundation for understanding the roles of specific signaling pathways in mediating EndMT in endothelial cells from different anatomical origins.

| S-EPMC6325528 | biostudies-literature

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