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

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.

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

Scientific Reports ◽

10.1038/s41598-021-98766-3 ◽

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.

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

Scientific Reports ◽

10.1038/s41598-021-94791-4 ◽

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.

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2D- and 3D-cultures of human trabecular meshwork cells: A preliminary assessment of an in vitro model for glaucoma study

PLoS ONE ◽

10.1371/journal.pone.0221942 ◽

2019 ◽

Vol 14 (9) ◽

pp. e0221942 ◽

Cited By ~ 10

Author(s):

Stefania Vernazza ◽

Sara Tirendi ◽

Sonia Scarfì ◽

Mario Passalacqua ◽

Francesco Oddone ◽

...

Keyword(s):

Trabecular Meshwork ◽

In Vitro Model ◽

Preliminary Assessment ◽

3D Cultures ◽

Trabecular Meshwork Cells ◽

Vitro Model ◽

2D And 3D

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

Current Issues in Molecular Biology ◽

10.3390/cimb43030121 ◽

2021 ◽

Vol 43 (3) ◽

pp. 1715-1725

Author(s):

Hiroyasu Katayama ◽

Masato Furuhashi ◽

Araya Umetsu ◽

Fumihito Hikage ◽

Megumi Watanabe ◽

...

Keyword(s):

Physical Properties ◽

Er Stress ◽

Molecular Mechanisms ◽

Lysyl Oxidase ◽

Hypoxia Inducible Factor ◽

Three Dimensional ◽

Tissue Inhibitor Of Metalloproteinase ◽

Down Regulation ◽

Related Factors ◽

Stress Related Genes

In the current study, to elucidate the pathological characteristics of myopic scleral stroma, three-dimensional (3D) cultures of human scleral stroma fibroblasts (HSSFs) with several axial lengths (AL, 22.80–30.63 mm) that were obtained from patients (n = 7) were examined. Among the three groups of ALs, <25 mm (n = 2), 25–30 mm (n = 2), and >30 mm (n = 3), the physical properties of the 3D HSSFs spheroids with respect to size and stiffness, the expressions of extracellular matrix (ECM) molecules, including collagen (COL) 1, 4, and 6 and fibronectin (FN) by qPCR and immunohistochemistry (IHC), and the mRNA expression of ECM metabolism modulators including hypoxia-inducible factor 1A (HIF 1A), HIF 2A, lysyl oxidase (LOX), tissue inhibitor of metalloproteinase (TIMP) 1–4, and matrix metalloproteinase (MMP) 2, 9, and 14 as well as several endoplasmic reticulum (ER) stress-related factors were compared. In the largest AL group (>30 mm), the 3D HSSFs spheroids were (1) significantly down-sized and less stiff compared to the other groups, and (2) significant changes were detected in the expression of some ECMs (qPCR; the up-regulation of COL1 and COL4, and the down-regulation of FN, IHC; the up-regulation of COL1 and FN, and down-regulation of COL4). The mRNA expressions of ECM modulators and ER stress-related genes were also altered with increasing AL length (up-regulation of HIF2A, MMP2, XBP1, and MMP14, down-regulation of LOX, TIMP 2 and 3, GRP78, GRP94, IRE1, and ATF6). In addition, a substantial down-regulation of some ER stress-related genes (ATF4, sXPB1 and CHOP) was observed in the 25–30 mm AL group. The findings presented herein suggest that small and stiffer 3D HSSFs spheroids in the largest AL group may accurately replicate the pathological significance of scleral thinning and weakening in myopic eyes. In addition, the modulation of several related factors among the different AL groups may also provide significant insights into our understanding of the molecular mechanisms responsible for causing myopic changes in the sclera.

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An Automated Multiplexed Hepatotoxicity and CYP Induction Assay Using HepaRG Cells in 2D and 3D

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555217701058 ◽

2017 ◽

Vol 22 (5) ◽

pp. 614-625 ◽

Cited By ~ 6

Author(s):

Lindsey M. Ott ◽

Karthik Ramachandran ◽

Lisa Stehno-Bittel

Keyword(s):

Enzyme Activity ◽

Cytochrome P450 ◽

Early Stage ◽

Three Dimensional ◽

Atp Depletion ◽

Drug Induced ◽

Cyp Induction ◽

Heparg Cells ◽

Cyp3a4 Enzyme ◽

2D And 3D

Drug-induced liver injury (DILI) and drug–drug interactions (DDIs) are concerns when developing safe and efficacious compounds. We have developed an automated multiplex assay to detect hepatotoxicity (i.e., ATP depletion) and metabolism (i.e., cytochrome P450 1A [CYP1A] and cytochrome P450 3A4 [CYP3A4] enzyme activity) in two-dimensional (2D) and three-dimensional (3D) cell cultures. HepaRG cells were cultured in our proprietary micromold plates and produced spheroids. HepaRG cells, in 2D or 3D, expressed liver-specific proteins throughout the culture period, although 3D cultures consistently exhibited higher albumin secretion and CYP1A/CYP3A4 enzyme activity than 2D cultures. Once the spheroid hepatic quality was assessed, 2D and 3D HepaRGs were challenged to a panel of DILI- and CYP-inducing compounds for 7 days. The 3D HepaRG model had a 70% sensitivity to liver toxins at 7 days, while the 2D model had a 60% sensitivity. In both the 2D and 3D HepaRG models, 83% of compounds were predicted to be CYP inducers after 7 days of compound exposure. Combined, our results demonstrate that an automated multiplexed liver spheroid system is a promising cell-based method to evaluate DILI and DDI for early-stage drug discovery.

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Differential reprogramming of breast cancer subtypes in 3D cultures and implications for sensitivity to targeted therapy

Scientific Reports ◽

10.1038/s41598-021-86664-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Esmee Koedoot ◽

Liesanne Wolters ◽

Marcel Smid ◽

Peter Stoilov ◽

Gerhard A. Burger ◽

...

Keyword(s):

Breast Cancer ◽

Cell Lines ◽

Three Dimensional ◽

Culture Conditions ◽

Aurora Kinases ◽

Cancer Subtypes ◽

Cyclin Dependent Kinases ◽

3D Cultures ◽

Tnbc Cell ◽

2D And 3D

AbstractScreening for effective candidate drugs for breast cancer has shifted from two-dimensional (2D) to three-dimensional (3D) cultures. Here we systematically compared the transcriptomes of these different culture conditions by RNAseq of 14 BC cell lines cultured in both 2D and 3D conditions. All 3D BC cell cultures demonstrated increased mitochondrial metabolism and downregulated cell cycle programs. Luminal BC cells in 3D demonstrated overall limited reprogramming. 3D basal B BC cells showed increased expression of extracellular matrix (ECM) interaction genes, which coincides with an invasive phenotype not observed in other BC cells. Genes downregulated in 3D were associated with metastatic disease progression in BC patients, including cyclin dependent kinases and aurora kinases. Furthermore, the overall correlation of the cell line transcriptome to the BC patient transcriptome was increased in 3D cultures for all TNBC cell lines. To define the most optimal culture conditions to study the oncogenic pathway of interest, an open source bioinformatics strategy was established.

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

International Journal of Molecular Sciences ◽

10.3390/ijms222112039 ◽

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.

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Homogeneous 2D and 3D alignment of cardiomyocyte in dilated cardiomyopathy revealed by intravital heart imaging

Scientific Reports ◽

10.1038/s41598-021-94100-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Kiyoshi Masuyama ◽

Tomoaki Higo ◽

Jong-Kook Lee ◽

Ryohei Matsuura ◽

Ian Jones ◽

...

Keyword(s):

Heart Failure ◽

Dilated Cardiomyopathy ◽

Cardiac Dysfunction ◽

Three Dimensional ◽

Single Layer ◽

Specific Pattern ◽

Two Dimensional ◽

Heart Imaging ◽

Novel Method ◽

2D And 3D

AbstractIn contrast to hypertrophic cardiomyopathy, there has been reported no specific pattern of cardiomyocyte array in dilated cardiomyopathy (DCM), partially because lack of alignment assessment in a three-dimensional (3D) manner. Here we have established a novel method to evaluate cardiomyocyte alignment in 3D using intravital heart imaging and demonstrated homogeneous alignment in DCM mice. Whilst cardiomyocytes of control mice changed their alignment by every layer in 3D and position twistedly even in a single layer, termed myocyte twist, cardiomyocytes of DCM mice aligned homogeneously both in two-dimensional (2D) and in 3D and lost myocyte twist. Manipulation of cultured cardiomyocyte toward homogeneously aligned increased their contractility, suggesting that homogeneous alignment in DCM mice is due to a sort of alignment remodelling as a way to compensate cardiac dysfunction. Our findings provide the first intravital evidence of cardiomyocyte alignment and will bring new insights into understanding the mechanism of heart failure.

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Do 3D Face Images Capture Cues of Strength, Weight, and Height Better than 2D Face Images do?

Adaptive Human Behavior and Physiology ◽

10.1007/s40750-021-00170-8 ◽

2021 ◽

Vol 7 (3) ◽

pp. 209-219

Author(s):

Iris J Holzleitner ◽

Alex L Jones ◽

Kieran J O’Shea ◽

Rachel Cassar ◽

Vanessa Fasolt ◽

...

Keyword(s):

Three Dimensional ◽

Physical Characteristics ◽

Two Dimensional ◽

3D Images ◽

3D Face ◽

Face Images ◽

Similar Accuracy ◽

2D And 3D ◽

2D Images ◽

Better Than

Abstract Objectives A large literature exists investigating the extent to which physical characteristics (e.g., strength, weight, and height) can be accurately assessed from face images. While most of these studies have employed two-dimensional (2D) face images as stimuli, some recent studies have used three-dimensional (3D) face images because they may contain cues not visible in 2D face images. As equipment required for 3D face images is considerably more expensive than that required for 2D face images, we here investigated how perceptual ratings of physical characteristics from 2D and 3D face images compare. Methods We tested whether 3D face images capture cues of strength, weight, and height better than 2D face images do by directly comparing the accuracy of strength, weight, and height ratings of 182 2D and 3D face images taken simultaneously. Strength, height and weight were rated by 66, 59 and 52 raters respectively, who viewed both 2D and 3D images. Results In line with previous studies, we found that weight and height can be judged somewhat accurately from faces; contrary to previous research, we found that people were relatively inaccurate at assessing strength. We found no evidence that physical characteristics could be judged more accurately from 3D than 2D images. Conclusion Our results suggest physical characteristics are perceived with similar accuracy from 2D and 3D face images. They also suggest that the substantial costs associated with collecting 3D face scans may not be justified for research on the accuracy of facial judgments of physical characteristics.

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Fucoxanthin Holds Potential to Become a Drug Adjuvant in Breast Cancer Treatment: Evidence from 2D and 3D Cell Cultures

Molecules ◽

10.3390/molecules26144288 ◽

2021 ◽

Vol 26 (14) ◽

pp. 4288

Author(s):

Fernanda Malhão ◽

Ana Catarina Macedo ◽

Carla Costa ◽

Eduardo Rocha ◽

Alice Abreu Ramos

Keyword(s):

Breast Cancer ◽

Cell Lines ◽

3D Models ◽

Anticancer Activities ◽

Cytotoxic Effects ◽

3D Cultures ◽

3D Cell Cultures ◽

Tumoral Cell ◽

Microscopy Techniques ◽

2D And 3D

Fucoxanthin (Fx) is a carotenoid derived from marine organisms that exhibits anticancer activities. However, its role as a potential drug adjuvant in breast cancer (BC) treatment is still poorly explored. Firstly, this study investigated the cytotoxic effects of Fx alone and combined with doxorubicin (Dox) and cisplatin (Cis) on a panel of 2D-cultured BC cell lines (MCF7, SKBR3 and MDA-MB-231) and one non-tumoral cell line (MCF12A). Fucoxanthin induced cytotoxicity against all the cell lines and potentiated Dox cytotoxic effects towards the SKBR3 and MDA-MB-231 cells. The combination triggering the highest cytotoxicity (Fx 10 µM + Dox 1 µM in MDA-MB-231) additionally showed significant induction of cell death and genotoxic effects, relative to control. In sequence, the same combination was tested on 3D cultures using a multi-endpoint approach involving bioactivity assays and microscopy techniques. Similar to 2D cultures, the combination of Fx and Dox showed higher cytotoxic effects on 3D cultures compared to the isolated compounds. Furthermore, this combination increased the number of apoptotic cells, decreased cell proliferation, and caused structural and ultrastructural damages on the 3D models. Overall, our findings suggest Fx has potential to become an adjuvant for Dox chemotherapy regimens in BC treatment.

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