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

Molecules ◽  
2021 ◽  
Vol 26 (21) ◽  
pp. 6382
Author(s):  
Megumi Watanabe ◽  
Yosuke Ida ◽  
Masato Furuhashi ◽  
Yuri Tsugeno ◽  
Fumihito Hikage ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Physical Properties ◽  
Trabecular Meshwork ◽  
Electrical Resistance ◽  
Transendothelial Electrical Resistance ◽  
Gene Expressions ◽  
Rock Inhibitor ◽  
Stiffness Analysis ◽  
3D Cell Cultures ◽  
2D And 3D

Effects of a pan-ROCK-inhibitor, ripasudil (Rip), and a ROCK2 inhibitor, KD025 on dexamethasone (DEX)-treated human trabecular meshwork (HTM) cells as a model of steroid-induced glaucoma were investigated. In the presence of Rip or KD025, DEX-treated HTM cells were subjected to permeability analysis of 2D monolayer by transendothelial electrical resistance (TEER) and FITC–dextran permeability, physical properties, size and stiffness analysis (3D), and qPCR of extracellular matrix (ECM), and their modulators. DEX resulted in a significant increase in the permeability, as well as a large and stiff 3D spheroid, and those effects were inhibited by Rip. In contrast, KD025 exerted opposite effects on the physical properties (down-sizing and softening). Furthermore, DEX induced several changes of gene expressions of ECM and their modulators were also modulated differently by Rip and KD025. The present findings indicate that Rip and KD025 induced opposite effects toward 2D and 3D cell cultures of DEX-treated HTM cells.

scholarly journals Establishment of appropriate glaucoma models using dexamethasone or TGFβ2 treated three-dimension (3D) cultured human trabecular meshwork (HTM) cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Megumi Watanabe ◽  
Yosuke Ida ◽  
Hiroshi Ohguro ◽  
Chiaki Ota ◽  
Fumihito Hikage
Keyword(s):  
Trabecular Meshwork ◽  
Ex Vivo ◽  
Open Angle Glaucoma ◽  
Smooth Muscle Actin ◽  
Three Dimensional ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Three Dimension ◽  
Trabecular Meshwork Cells ◽  
2D And 3D

AbstractTo establish appropriate ex vivo models for a glaucomatous trabecular meshwork (TM), two-dimensional (2D) and three-dimensional (3D) cultures of human trabecular meshwork cells (HTM) were prepared in the presence of 250 nM dexamethasone (DEX) or 5 ng/mL TGFβ2, and characterized by the following analyses; transendothelial electrical resistance (TEER) measurements, FITC dextran permeability, scanning electron microscopy and the expression of the extracellular matrix (ECM) including collagen (COL)1, 4 and 6, and fibronectin (FN), α-smooth muscle actin (α-SMA), tissue inhibitor of metalloproteinase (TIMP)1–4, and matrix metalloproteinase (MMP)2, 9 and 14. DEX and TGFβ2 both caused a significant increase or decrease in the TEER values and FITC dextran permeability. During the 3D spheroid culture, DEX or TGFβ2 induced a mild and significant down-sizing and an increase in stiffness, respectively. TGFβ2 induced a significant up-regulation of COL1 and 4, FN, α-SMA, and MMP 2 and 14 (2D) or COL1 and 6, and TIMP2 and 3 (3D), and DEX induced a significant up-regulation of FN (3D) and TIMP4 (2D and 3D). The findings presented herein indicate that DEX or TGFβ2 resulted in mild and severe down-sized and stiff 3D HTM spheroids, respectively, thus making them viable in vitro HTM models for steroid-induced and primary open angle glaucoma.

scholarly journals Screening of the Drug-Induced Effects of Prostaglandin EP2 and FP Agonists on 3D Cultures of Dexamethasone-Treated Human Trabecular Meshwork Cells

Biomedicines ◽  
2021 ◽  
Vol 9 (8) ◽  
pp. 930
Author(s):  
Megumi Watanabe ◽  
Yosuke Ida ◽  
Masato Furuhashi ◽  
Yuri Tsugeno ◽  
Hiroshi Ohguro ◽  
...  
Keyword(s):  
Trabecular Meshwork ◽  
Smooth Muscle Actin ◽  
Three Dimensional ◽  
Prostaglandin F2α ◽  
Fluorescein Isothiocyanate ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Drug Induced ◽  
Related Factors ◽  
3D Cultures ◽  
2D And 3D

The objective of the current study was to perform a screening of the drug-induced effects of the prostaglandin F2α (PGF2α) and EP2 agonist, omidenepag (OMD), using two- and three-dimensional (2D and 3D) cultures of dexamethasone (DEX)-treated human trabecular meshwork (HTM) cells. The drug-induced effects on 2D monolayers were characterized by measuring the transendothelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)–dextran permeability, the physical properties of 3D spheroids, and the gene expression of extracellular matrix (ECM) molecules, including collagen (COL) 1, 4 and 6, and fibronectin (FN), α smooth muscle actin (αSMA), a tissue inhibitor of metalloproteinase (TIMP) 1–4, matrix metalloproteinase (MMP) 2, 9 and 14 and endoplasmic reticulum (ER) stress-related factors. DEX induced a significant increase in TEER values and a decrease in FITC–dextran permeability, respectively, in the 2D HTM monolayers, and these effects were substantially inhibited by PGF2α and OMD. Similarly, DEX also caused decreased sizes and an increased stiffness in the 3D HTM spheroids, but PGF2α or OMD had no effects on the stiffness of the spheroids. Upon exposure to DEX, the following changes were observed: the upregulation of COL4 (2D), αSMA (2D), and TIMP4 (2D and 3D) and the downregulation of TIMP1 and 2 (3D), MMP2 and 14 (3D), inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6) (2D), and glucose regulator protein (GRP)78 (3D). In the presence of PGF2α or OMD, the downregulation of COL4 (2D), FN (3D), αSMA (2D), TIMP3 (3D), MMP9 (3D) and the CCAAT/enhancer-binding protein homologous protein (CHOP) (2D), and the upregulation of TIMP4 (2D and 3D), MMP2, 9 and 14 (2D), respectively, were observed. The findings presented herein suggest that 2D and 3D cell cultures can be useful in screening for the drug-induced effects of PGF2α and OMD toward DEX-treated HTM cells.

scholarly journals ROCK inhibitors beneficially alter the spatial configuration of TGFβ2-treated 3D organoids from a human trabecular meshwork (HTM)

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Chiaki Ota ◽  
Yosuke Ida ◽  
Hiroshi Ohguro ◽  
Fumihito Hikage
Keyword(s):  
Trabecular Meshwork ◽  
Kinase Inhibitors ◽  
Spatial Configuration ◽  
Coiled Coil ◽  
Protein Kinase Inhibitors ◽  
Molecular Pharmacology ◽  
Transendothelial Electrical Resistance ◽  
Beneficial Effects ◽  
2D And 3D ◽  
Aqueous Outflow

AbstractTo elucidate molecular pharmacology of Rho-associated coiled-coil containing protein kinase inhibitors (ROCK-i, Ripasudil and Y27632) on their efficiency for aqueous outflow, 2D or 3D cultures of a human trabecular meshwork (HTM) were prepared in the presence of TGFβ2. Those were examined by transendothelial electrical resistance (TEER, 2D), electronic microscopy (EM, 2D and 3D), expression of the extracellular matrix (ECM) including collagen1 (COL1), COL4 and COL6, and fibronectin (FN) by immunolabeling and/or quantitative PCR (3D), and solidity of 3D organoids by a micro-squeezer. TGFβ2 significantly increased the TEER values in 2D cultures, and the ECM expression indicated that the 3D organoids assumed a more densely packed shape. ROCK-i greatly reduced the TGFβ2-induced enhancement of TEER and the immunolabeled ECM expression of the 3D organoids. In contrast, the mRNA expression of COL1 was increased, and those of COL4 and FN were unchanged. EM revealed that TGFβ2 caused the HTM cells to become more compact and abundant ECM deposits within the 3D organoids were observed. These were significantly inhibited by ROCK-i. The dense solids caused by the presence of TGFβ2 were significantly suppressed by ROCK-i. Current study indicates that ROCK-i cause beneficial effects toward the spatial configuration of TGFβ2-induced HTM 3D organoids.

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.

Changes in keratins and alpha-smooth muscle actin during three-dimensional reconstitution of eccrine sweat glands

2016 ◽  
Vol 365 (1) ◽  
pp. 113-122 ◽  
Author(s):  
Haihong Li ◽  
Xuexue Li ◽  
Bingna Zhang ◽  
Mingjun Zhang ◽  
Wenlong Chen ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Actin ◽  
Three Dimensional ◽  
Sweat Glands ◽  
Muscle Actin ◽  
Alpha Smooth Muscle Actin ◽  
Eccrine Sweat Glands ◽  
Eccrine Sweat

scholarly journals Microvascular fragment spheroids: Three-dimensional vascularization units for tissue engineering and regeneration

2021 ◽  
Vol 12 ◽  
pp. 204173142110355
Author(s):  
Lisa Nalbach ◽  
Danièle Müller ◽  
Selina Wrublewsky ◽  
Wolfgang Metzger ◽  
Michael D Menger ◽  
...  
Keyword(s):  
Tissue Engineering ◽  
Cell Function ◽  
Smooth Muscle Actin ◽  
Three Dimensional ◽  
Stromal Vascular Fraction ◽  
Muscle Actin ◽  
Tissue Constructs ◽  
First Time ◽  
Tissue Defects

Adipose tissue-derived microvascular fragments (MVF) serve as vascularization units in tissue engineering and regenerative medicine. Because a three-dimensional cellular arrangement has been shown to improve cell function, we herein generated for the first time MVF spheroids to investigate whether this further increases their vascularization potential. These spheroids exhibited a morphology, size, and viability comparable to that of previously introduced stromal vascular fraction (SVF) spheroids. However, MVF spheroids contained a significantly higher number of CD31-positive endothelial cells and α-smooth muscle actin (SMA)-positive perivascular cells, resulting in an enhanced angiogenic sprouting activity. Accordingly, they also exhibited an improved in vivo vascularization and engraftment after transplantation into mouse dorsal skinfold chambers. These findings indicate that MVF spheroids are superior to SVF spheroids and, thus, may be highly suitable to improve the vascularization of tissue defects and implanted tissue constructs.

scholarly journals Autotaxin May Have Lysophosphatidic Acid-Unrelated Effects on Three-Dimension (3D) Cultured Human Trabecular Meshwork (HTM) Cells

2021 ◽  
Vol 22 (21) ◽  
pp. 12039
Author(s):  
Megumi Watanabe ◽  
Masato Furuhashi ◽  
Yuri Tsugeno ◽  
Yosuke Ida ◽  
Fumihito Hikage ◽  
...  
Keyword(s):  
Er Stress ◽  
Lysophosphatidic Acid ◽  
Trabecular Meshwork ◽  
Electrical Resistance ◽  
Three Dimensional ◽  
Three Dimension ◽  
Two Dimensional ◽  
Transendothelial Electrical Resistance ◽  
Related Factors ◽  
Signaling Axis

Purpose: The objective of the current study was to evaluate the effects of the autotaxin (ATX)–lysophosphatidic acid (LPA) signaling axis on the human trabecular meshwork (HTM) in two-dimensional (2D) and three-dimensional (3D) cultures of HTM cells. Methods: The effects were characterized by transendothelial electrical resistance (TEER) and FITC-dextran permeability (2D), measurements of size and stiffness (3D), and the expression of several genes, including extracellular matrix (ECM) molecules, their modulators, and endoplasmic reticulum (ER) stress-related factors. Results: A one-day exposure to 200 nM LPA induced significant down-sizing effects of the 3D HTM spheroids, and these effects were enhanced slightly on longer exposure. The TEER and FITC-dextran permeability data indicate that LPA induced an increase in the barrier function of the 2D HTM monolayers. A one-day exposure to a 2 mg/L solution of ATX also resulted in a significant decrease in the sizes of the 3D HTM spheroids, and an increase in stiffness was also observed. The gene expression of several ECMs, their regulators and ER-stress related factors by the 3D HTM spheroids were altered by both ATX and LPA, but in different manners. Conclusions: The findings presented herein suggest that ATX may have additional roles in the human TM, in addition to the ATX–LPA signaling axis.

Sign in / Sign up

Export Citation Format

Share Document