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

2021 ◽  
Vol 22 (14) ◽  
pp. 7335
Author(s):  
Yuika Oouchi ◽  
Megumi Watanabe ◽  
Yosuke Ida ◽  
Hiroshi Ohguro ◽  
Fumihito Hikage
Keyword(s):  
Extracellular Matrix ◽  
Electrical Resistance ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Coiled Coil ◽  
Three Dimension ◽  
Muscle Actin ◽  
Transendothelial Electrical Resistance ◽  
Down Regulation ◽  
Two Dimension

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.

scholarly journals The Role of Epigenetics in the Fibrotic Processes Associated with Glaucoma

2014 ◽  
Vol 2014 ◽  
pp. 1-13 ◽  
Author(s):  
Fiona McDonnell ◽  
Colm O’Brien ◽  
Deborah Wallace
Keyword(s):  
Extracellular Matrix ◽  
Risk Factor ◽  
Transforming Growth Factor ◽  
Smooth Muscle Actin ◽  
Lamina Cribrosa ◽  
Epigenetic Mechanisms ◽  
Muscle Actin ◽  
Main Risk Factor ◽  
Hypoxic Environment

Glaucoma is an optic neuropathy that affects 60 million people worldwide. The main risk factor for glaucoma is increased intraocular pressure (IOP), this is currently the only target for treatment of glaucoma. However, some patients show disease progression despite well-controlled IOP. Another possible therapeutic target is the extracellular matrix (ECM) changes in glaucoma. There is an accumulation of ECM in the lamina cribrosa (LC) and trabecular meshwork (TM) and upregulation of profibrotic factors such as transforming growth factorβ(TGFβ), collagen1α1 (COL1A1), andα-smooth muscle actin (αSMA). One method of regulating fibrosis is through epigenetics; the study of heritable changes in gene function caused by mechanisms other than changes in the underlying DNA sequence. Epigenetic mechanisms have been shown to drive renal and pulmonary fibrosis by upregulating profibrotic factors. Hypoxia alters epigenetic mechanisms through regulating the cell’s response and there is a hypoxic environment in the LC and TM in glaucoma. This review looks at the role that hypoxia plays in inducing aberrant epigenetic mechanisms and the role these mechanisms play in inducing fibrosis. Evidence suggests that a hypoxic environment in glaucoma may induce aberrant epigenetic mechanisms that contribute to disease fibrosis. These may prove to be relevant therapeutic targets in glaucoma.

scholarly journals Diverse effects of pan-ROCK and ROCK2 inhibitors on 2 D and 3D cultured human trabecular meshwork (HTM) cells treated with TGFβ2

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Megumi Watanabe ◽  
Yosuke Ida ◽  
Hiroshi Ohguro ◽  
Chiaki Ota ◽  
Fumihito Hikage
Keyword(s):  
Trabecular Meshwork ◽  
Smooth Muscle Actin ◽  
Three Dimensional ◽  
Coiled Coil ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Muscle Actin ◽  
Transendothelial Electrical Resistance ◽  
Gene Expressions ◽  
Rock Inhibitor ◽  
2D And 3D

AbstractA pan-ROCK-inhibitor, ripasudil (Rip), and a ROCK2 inhibitor, KD025, were used To study the effects of Rho-associated coiled-coil containing protein kinase (ROCK)1 and 2 on two-dimensional (2D) and three-dimensional (3D) cultures of a TGFβ2-treated human trabecular meshwork (HTM) cells. In the presence of 5 ng/mL TGFβ2, the effects of these inhibitors were characterized by transendothelial electrical resistance (TEER), FITC-dextran permeability, and the size and stiffness of 3D sphenoids, the expression of extracellular matrix (ECM) including collagen1, 4 and 6, and fibronectin, α-smooth muscle actin, a tissue inhibitor of metalloproteinase (TIMP)1–4, and matrix metalloproteinase (MMP)2, 9 and 14. TGFβ2 caused a significant increase in the TEER values, and decrease in FITC-dextran permeability, as well as a decrease in the sizes and stiffness of the 3D sphenoids. In the presence of ROCK inhibitors, the TGFβ2-induced effects of the TEER and FITC-dextran permeability were inhibited, especially by KD025. Rip induced a significant increase in sizes and a decrease in the stiffness of the TGFβ2-treated 3D sphenoids, although the effects of KD025 were weaker. Gene expressions of most of the ECMs, TIMP2 and MMP9 of 2D and 3D HTM cells were significantly up-regulated by TGFβ2. Those were significantly and differently modulated by Rip or KD025.

scholarly journals The effects of tamoxifen and estradiol on myometrial differentiation and organization during early uterine development in the CD1 mouse

Reproduction ◽  
2009 ◽  
Vol 138 (2) ◽  
pp. 341-350 ◽  
Author(s):  
Mohamed K Mehasseb ◽  
S C Bell ◽  
M A Habiba
Keyword(s):  
Extracellular Matrix ◽  
Image Analysis ◽  
Steroid Receptors ◽  
Smooth Muscle Actin ◽  
Estrogen Receptor Α ◽  
Matrix Proteins ◽  
Muscle Actin ◽  
Uterine Adenomyosis ◽  
Circular Arrangement ◽  
Uterine Development

We used a neonatal mouse model to examine the histogenesis of uterine adenomyosis, and to test whether adenomyosis is due to an abnormality in myometrial differentiation, or in extracellular matrix proteins expression. We also studied the effects of tamoxifen and estradiol on uterine development, myometrial differentiation, and organization. Female CD1 pups were treated with oral tamoxifen (1 mg/kg) (n=27) or estradiol (0.1 mg/kg) (n=24) from age 1 to 5 days. Uteri from control (n=27) and treated mice were obtained on days 2, 5, 10, 15, and 42 of age. We examined the sections histologically, using image analysis and immunohistochemistry for α-smooth muscle actin (α-SMA), desmin, vimentin, laminin, fibronectin, and estrogen receptor-α. Following tamoxifen exposure, all uteri showed adenomyosis by 6 weeks of age (seen as early as day 10). The inner myometrium showed thinning, lack of continuity, disorganization, and bundling. α-SMA expression was normal. Desmin expression normally showed a wave of maturation that was absent in tamoxifen-treated mice. In the estradiol group, adenomyosis was not observed. All uterine layers were normally developed, but hypertrophied. The inner myometrium retained its circular arrangement. There was no difference in the localization of laminin or fibronectin between groups (laminin expression was reduced in the tamoxifen treated uteri). Vimentin could not be detected in all groups. Our results suggest that the development of the inner myometrium is particularly sensitive to estrogen antagonism, and can be affected by steroid receptors modulation. Disruption of the inner myometrium may play a role in the development of uterine adenomyosis.

scholarly journals Collagen content and extracellular matrix cause cytoskeletal remodelling in pancreatic fibroblasts

2019 ◽  
Vol 16 (154) ◽  
pp. 20190226 ◽  
Author(s):  
Andreas Stylianou ◽  
Vasiliki Gkretsi ◽  
Maria Louca ◽  
Lefteris C. Zacharia ◽  
Triantafyllos Stylianopoulos
Keyword(s):  
Extracellular Matrix ◽  
Stromal Cells ◽  
Smooth Muscle Actin ◽  
Coiled Coil ◽  
Substrate Stiffness ◽  
Tumour Invasion ◽  
Alpha Smooth Muscle Actin ◽  
Desmoplastic Reaction ◽  
Young’S Moduli ◽  
Ecm Proteins

In many solid tumours a desmoplastic reaction takes place, which results in tumour tissue stiffening due to the extensive production of extracellular matrix (ECM) proteins, such as collagen, by stromal cells, mainly fibroblasts (FBs) and cancer-associated fibroblasts (CAFs). In this study, we investigated the effect of collagen stiffness on pancreatic FBs and CAFs, particularly on specific cytoskeleton properties and gene expression involved in tumour invasion. We found that cells become stiffer when they are cultured on stiff substrates and express higher levels of alpha-smooth muscle actin (α-SMA). Also, it was confirmed that on stiff substrates, CAFs are softer than FBs, while on soft substrates they have comparable Young's moduli. Furthermore, the number of spread FBs and CAFs was higher in stiffer substrates, which was also confirmed by Ras-related C3 botulinum toxin substrate 1 ( RAC1 ) mRNA expression, which mediates cell spreading. Although stress fibres in FBs become more oriented on stiff substrates, CAFs have oriented stress fibres regardless of substrate stiffness. Subsequently, we demonstrated that cells' invasion has a differential response to stiffness, which was associated with regulation of Ras homologue family member ( RhoA ) and Rho-associated, coiled-coil containing protein kinase 1 ( ROCK-1 ) mRNA expression. Overall, our results demonstrate that collagen stiffness modulates FBs and CAFs cytoskeleton remodelling and alters their invasion properties.

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