RHO-Associated Coiled-Coil-Containing Protein Kinase Inhibitors Significantly Modulate the Epithelial-Mesenchymal Transition Induced by TGF-β2 in the 2-D and 3-D Cultures of Human Corneal Stroma Fibroblasts.

Background/objectives: The objective of the present study was to examine the unidentified effects that RHO-associated coiled-coil-containing protein kinase 1 and 2 antagonists exert on the transforming growth factor beta2-induced epithelial-mesenchymal transition of the human corneal stroma.

Methods: In the presence or absence of pan-RHO-associated coiled-coil-containing protein kinase inhibitors, ripasudil or Y27632 and RHO-associated coiled-coil-containing protein kinase 2 inhibitor, KD025, we analyzed the following: (1) planar proliferation caused by trans-endothelial electrical resistance and the cellular metabolic characteristics of the two-dimensional cultures of human corneal stroma fibroblasts; (2) the physical properties of a three-dimensional human corneal stroma fibroblasts spheroid; and (3) the gene expressions and their regulators in the extracellular matrix, along with the tissue inhibitors of metalloproteinases and matrix metalloproteinases and the endoplasmic reticulum stress-related factors of the two-dimensional and three-dimensional cultures in human corneal stroma fibroblasts.

Results: Exposure to 5 nM of the transforming growth factor beta2 markedly increased the trans-endothelial electrical resistance values as well as the metabolic function in two-dimensional cultures of human corneal stroma fibroblasts.

Effects of linsitinib on M22 and IGF:1-treated 3D spheroids of human orbital fibroblasts.

To elucidate the role of IGF1R inhibition in the pathogenesis of Graves' orbitopathy (GO), the effects of linsitinib (Lins) on a recombinant human TSHR antibody (M22) and IGF1 to activate TSHR and IGF1R of human orbital fibroblasts (HOFs) obtained from patients without GO (HOFs) and patients with GO (GHOFs) were studied using in vitro three-dimensional (3D) spheroid models in addition to their 2D planar cell culture. For this purpose, we evaluated 1) cellular metabolic functions by using a seahorse bioanalyzer (2D), 2) physical properties including size and stiffness of 3D spheroids, and mRNA expression of several extracellular matrix (ECM) proteins, their modulators (CCL2 LOX, CTGF, MMPs), ACTA2 and inflammatory cytokines (IL1β, IL6). Administration of IGF1 and M22 induced increases of cellular metabolic functions with the effect on HOFs being much more potent than the effect on GHOFs, suggesting that IGF1R and TSHR of GHOFs may already be stimulated.

Unexpected and Synergistical Effects of All-Trans Retinoic Acid and TGF-β2 on Biological Aspects of 2D and 3D Cultured ARPE19 Cells.

To study the effects of all-trans retinoic acid (ATRA) on TGF-β2-induced effects of human retinal pigment epithelium cells under normoxia and hypoxia conditions. Two-dimensionally (2D) and three-dimensionally (3D) cultured ARPE19 cells were subjected to cellular functional analyses by transepithelial electrical resistance (TEER) and an extracellular flux assay (2D), measurement of levels of reactive oxygen species (ROS), gene expression analyses of , , , , and (2D), and physical property analyses (3D). Under a normoxia condition, treatment with 100 nM ATRA substantially decreased barrier function regardless of the presence of 5 ng/mL TGF-β2 in 2D ARPE19 monolayer cells.

TGF-β effects on adipogenesis of 3T3-L1 cells differ in 2D and 3D cell culture conditions.

The TGF-β superfamily plays a pivotal role in the regulation of adipogenesis, but little is known about the potential differential role of the three isoforms of TGF-β, TGF-β-1~3. To further elucidate their role, two-dimensionally (2D) and three-dimensionally (3D) cultured 3T3-L1 mouse preadipocytes were subjected to the following analyses: (a) qPCR analysis of adipogenesis-related factors and major extracellular matrix protein (2D and /or 3D), (b) lipid staining by Oil Red O (2D) or BODIPY (3D), (c) Seahorse cellular metabolic measurement (2D), and (d) size and stiffness measurements of 3D 3T3-L1 spheroids. In the 2D cultured 3T3-L1 cells, mRNA expression levels of adipogenesis-related genes and Oil Red O lipid staining intensity were significantly increased by adipogenesis and they were substantially decreased following treatment with 0.

Application of Single Cell Type-Derived Spheroids Generated by Using a Hanging Drop Culture Technique in Various Disease Models: A Narrow Review.

Cell culture methods are indispensable strategies for studies in biological sciences and for drug discovery and testing. Most cell cultures have been developed using two-dimensional (2D) culture methods, but three-dimensional (3D) culture techniques enable the establishment of models that replicate various pathogenic conditions and they provide valuable insights into the pathophysiology of various diseases as well as more precise results in tests for drug efficacy. However, one difficulty in the use of 3D cultures is selection of the appropriate 3D cell culture technique for the study purpose among the various techniques ranging from the simplest single cell type-derived spheroid culture to the more sophisticated organoid cultures.

FABP5 Is a Possible Factor for the Maintenance of Functions of Human Non-Pigmented Ciliary Epithelium Cells.

To elucidate the possible biological roles of fatty acid-binding protein 5 (FABP5) in the intraocular environment, the cells from which FABP5 originates were determined by using four different intraocular tissue-derived cell types including human non-pigmented ciliary epithelium (HNPCE) cells, retinoblastoma (RB) cells, adult retinal pigment epithelial19 (ARPE19) cells and human ocular choroidal fibroblast (HOCF) cell lines, and the effects of FABP ligand 6, a specific inhibitor for FABP5 and FABP7 were analyzed by RNA sequencing and seahorse cellular metabolic measurements. Among these four different cell types, qPCR analysis showed that FABP5 was most prominently expressed in HNPCE cells, in which no mRNA expression of FABP7 was detected. In RNA sequencing analysis, 166 markedly up-regulated and 198 markedly down-regulated differentially expressed genes (DEGs) were detected between non-treated cells and cells treated with FABP ligand 6.

Intraocular fatty acids induce reinforcement of barrier functions on the outer blood-retinal barrier.

The aim of the present study was to elucidate unknown effects of intraocular fatty acids (ioFAs) including palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), arachidonic acid (C20:4), eicosapentaenoic acid (EPA, C20:5) and docosahexaenoic acid (DHA, C22:6) on the outer blood-retinal barrier (oBRB). For this purpose, human retinal pigment epithelium cell line ARPE19 was subjected to analyses for evaluating the following biological phenotypes: (1) cell viability, (2) cellular metabolic functions, (3) barrier functions by trans-epithelial electrical resistance (TEER), and (4) expression of tight junction (TJ) molecules. In the presence of 100 nM ioFAs, no significant effects on cell viability of ARPE19 cells was observed.

Metabolic dysfunction-associated steatotic liver disease (SLD) and alcohol-associated liver disease, but not SLD without metabolic dysfunction, are independently associated with new onset of chronic kidney disease during a 10-year follow-up period.

Aims: The new nomenclature of steatotic liver disease (SLD) including metabolic dysfunction-associated SLD (MASLD), MASLD and increased alcohol intake (MetALD), and alcohol-associated liver disease (ALD) has recently been proposed. We aimed to elucidate the relationship between each category of SLD and chronic kidney disease (CKD).

Methods: We investigated the effects of various SLDs on the development of CKD, defined as estimated glomerular filtration rate (eGFR) <60 mL/min/1.

Inhibition of mTOR differently modulates planar and subepithelial fibrogenesis in human conjunctival fibroblasts.

Purpose: In the current investigation, the effects of the mTOR inhibitors, Rapa and Torin1 on the TGF-β2-induced conjunctival fibrogenesis were studied.

Study Design: Experimental research.

Methods: 2D and 3D cultures of HconF were subjected to the following analyses; (1) planar proliferation evaluated by TEER (2D), (2) Seahorse metabolic analyses (2D), (3) subepithelial proliferation evaluated by the 3D spheroids' size and hardness, and (4) the mRNA expression of ECM proteins and their regulators (2D and 3D).

Lysophosphatidic Acid Modulates TGF-β2-Induced Biological Phenotype in Human Conjunctival Fibroblasts.

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.

FABP4 Is an Indispensable Factor for Regulating Cellular Metabolic Functions of the Human Retinal Choroid.

The purpose of the current study was to elucidate the physiological roles of intraocularly present fatty acid-binding protein 4 (FABP4). Using four representative intraocular tissue-derived cell types, including human non-pigmented ciliary epithelium (HNPCE) cells, retinoblastoma (RB) cells, adult retinal pigment epithelial19 (ARPE19) cells and human ocular choroidal fibroblast (HOCF) cells, the intraocular origins of FABP4 were determined by qPCR analysis, and the intracellular functions of FABP4 were investigated by seahorse cellular metabolic measurements and RNA sequencing analysis using a specific inhibitor for FABP4, BMS309403. Among these four different cell types, FABP4 was exclusively expressed in HOCF cells.

TGF-β Isoforms and Local Environments Greatly Modulate Biological Nature of Human Retinal Pigment Epithelium Cells.

To characterize transforming growth factor-β (TGF-β) isoform (TGF-β1~3)-b's biological effects on the human retinal pigment epithelium (RPE) under normoxia and hypoxia conditions, ARPE19 cells cultured by 2D (two-dimensional) and 3D (three-dimensional) conditions were subjected to various analyses, including (1) an analysis of barrier function by trans-epithelial electrical resistance (TEER) measurements; (2) qPCR analysis of major ECM molecules including , , and ; ; ; and , a master regulator for mitochondrial respiration;, tight junction-related molecules, and ; and ; (3) physical property measurements of 3D spheroids; and (4) cellular metabolic analysis. Diverse effects among TGF-β isoforms were observed, and those effects were also different between normoxia and hypoxia conditions: (1) TGF-β1 and TGF-β3 caused a marked increase in TEER values, and TGF-β2 caused a substantial increase in TEER values under normoxia conditions and hypoxia conditions, respectively; (2) the results of qPCR analysis supported data obtained by TEER; (3) 3D spheroid sizes were decreased by TGF-β isoforms, among which TGF-β1 had the most potent effect under both oxygen conditions; (4) 3D spheroid stiffness was increased by TGF-β2 and TGF-β3 or by TGF-β1 and TGF-β3 under normoxia conditions and hypoxia conditions, respectively; and (5) the TGF-β isoform altered mitochondrial and glycolytic functions differently under oxygen conditions and/or culture conditions. These collective findings indicate that the TGF-β-induced biological effects of 2D and 3D cultures of ARPE19 cells were substantially diverse depending on the three TGF-β isoforms and oxygen levels, suggesting that pathological conditions including epithelial-mesenchymal transition (EMT) of the RPE may be exclusively modulated by both factors.

The Specific ROCK2 Inhibitor KD025 Alleviates Glycolysis through Modulating STAT3-, CSTA- and S1PR3-Linked Signaling in Human Trabecular Meshwork Cells.

To investigate the biological significance of Rho-associated coiled-coil-containing protein kinase (ROCK) 2 in the human trabecular meshwork (HTM), changes in both metabolic phenotype and gene expression patterns against a specific ROCK2 inhibitor KD025 were assessed in planar-cultured HTM cells. A seahorse real-time ATP rate assay revealed that administration of KD025 significantly suppressed glycolytic ATP production rate and increased mitochondrial ATP production rate in HTM cells. RNA sequencing analysis revealed that 380 down-regulated and 602 up-regulated differentially expressed genes (DEGs) were identified in HTM cells treated with KD025 compared with those that were untreated.

Modulation of Epithelial-Mesenchymal Transition Is a Possible Underlying Mechanism for Inducing Chemoresistance in MIA PaCa-2 Cells against Gemcitabine and Paclitaxel.

To elucidate the currently unknown molecular mechanisms responsible for the similarity and difference during the acquirement of resistance against gemcitabine (GEM) and paclitaxel (PTX) in patients with pancreatic carcinoma, we examined two-dimensional (2D) and three-dimensional (3D) cultures of parent MIA PaCa-2 cells (MIA PaCa-2-PA) and their GEM resistance cell line (MIA PaCa-2-GR) and PTX resistance (MIA PaCa-2-PR). Using these cells, we examined 3D spheroid configurations and cellular metabolism, including mitochondrial and glycolytic functions, with a Seahorse bio-analyzer and RNA sequencing analysis. Compared to the MIA PaCa-2-PA, (1) the formation of the 3D spheroids of MIA PaCa-2-GR or -PR was much slower, and (2) their mitochondrial and glycolytic functions were greatly modulated in MIA PaCa-2-GR or -PR, and such metabolic changes were also different between their 2D and 3D culture conditions.

Differential Effects of Benzalkonium Chloride on Human Trabecular Meshwork Cells Not Treated or Treated with Transforming Growth Factor-β2 or Dexamethasone.

The objective of the present study was to evaluate the effects of low concentrations of benzalkonium chloride (BAC) (10%, 10%, or 10%) on healthy and glaucomatous human trabecular meshwork (HTM) cells. For this purpose, we used models replicating a healthy HTM and HTM with primary open-angle glaucoma (POAG) or steroid-induced glaucoma (SG) using two-dimensional (2D) cultures of HTM cells not treated or treated with a 5 ng/mL solution of transforming growth factor-β2 or 250 nM dexamethasone (DEX). Analyses were carried out for (1) the intercellular affinity function of 2D HTM monolayers, as determined by transepithelial electrical resistance (TEER) measurements; (2) cell viability; (3) cellular metabolism by using a Seahorse bioanalyzer; and (4) expression of extracellular matrix (ECM) molecules, an ECM modulator, and cell junction-related molecules.

Lipid Metabolism Regulators Are the Possible Determinant for Characteristics of Myopic Human Scleral Stroma Fibroblasts (HSSFs).

The purpose of the current investigation was to elucidate what kinds of responsible mechanisms induce elongation of the sclera in myopic eyes. To do this, two-dimensional (2D) cultures of human scleral stromal fibroblasts (HSSFs) obtained from eyes with two different axial length (AL) groups, <26 mm (low AL group, = 2) and >27 mm (high AL group, = 3), were subjected to (1) measurements of Seahorse mitochondrial and glycolytic indices to evaluate biological aspects and (2) analysis by RNA sequencing. Extracellular flux analysis revealed that metabolic indices related to mitochondrial and glycolytic functions were higher in the low AL group than in the high AL group, suggesting that metabolic activities of HSSF cells are different depending the degree of AL.

Benzalkonium chloride greatly deteriorates the biological activities of human corneal stroma fibroblasts in a concentration-dependent manner.

Background: Corneal tissues indirectly obtain nutritional needs and oxygen to maintain their homeostasis, and therefore, benzalkonium chloride (BAC) containing ocular instillations for medical therapy may, in turn, induce toxic effects more than expected in corneal tissues, especially the inside stroma layer.

Methods: To evaluate the effects of very low concentrations (10%, 10%, or 10%) of BAC on human corneal stroma, we used two-dimensional (2D) cultures of human corneal stromal fibroblast (HCSF) cells and carried out the following analyses: (1) cell viability measurements, (2) Seahorse cellular bio-metabolism analysis, and (3) the expression of ECM molecules and endoplasmic reticulum (ER) stress-related molecules.

Results: In the absence and presence of 10%, 10%, or 10% concentrations of BAC, cell viability deteriorated and this deterioration was dose-dependent.

3D culture induction of adipogenic differentiation in 3T3-L1 preadipocytes exhibits adipocyte-specific molecular expression patterns and metabolic functions.

Adipose tissues are closely related to physiological functions and pathological conditions in most organs. Although differentiated 3T3-L1 preadipocytes have been used for adipose studies, the difference in cellular characteristics of adipogenic differentiation in two-dimensional (2D) culture and three-dimensional (3D) culture remain unclear. In this study, we evaluated gene expression patterns using RNA sequencing and metabolic functions using an extracellular flux analyzer in 3T3-L1 preadipocytes with and without adipogenic induction in 2D culture and 3D culture.

A high level of thyroid-stimulating hormone is a risk factor for the development of chronic kidney disease in men: a 10-year cohort study.

Hypothyroidism has been reported to be associated with chronic kidney disease (CKD). However, the impact of thyroid-stimulating hormone (TSH) on new onset of CKD and its gender dependence remain undetermined. We investigated the association of serum TSH level and the development of CKD defined by estimated glomerular filtration rate (eGFR) < 60 mL/min/1.

TGF-β2 Induces Epithelial-Mesenchymal Transitions in 2D Planer and 3D Spheroids of the Human Corneal Stroma Fibroblasts in Different Manners.

To examine the epithelial-mesenchymal transition (EMT) that is induced on the human corneal stroma, two- and three-dimensional (2D and 3D) cultures of human corneal stroma fibroblasts (HCSFs) were used. In this study, HCSF 2D monolayers and 3D spheroids were characterized by (1) scanning electron microscopy (SEM), (2) trans-endothelial electrical resistance (TEER) measurements and fluorescein isothiocyanate (FITC)-dextran permeability, (3) cellular metabolic measurements, (4) the physical properties of 3D HCSF spheroids, and (5) the extracellular matrix (ECM) molecule gene expressions, including collagen () , , and fibronectin (), a tissue inhibitor of metalloproteinase () , matrix metalloproteinase () , , , and several endoplasmic reticulum (ER) stress-related factors. In the 2D HCSFs, TGF-β2 concentration-dependently generated (1) a considerable increase in ECM deposits revealed by SEM, (2) an increase in TEER values and a decrease in FITC-dextran permeability, (3) increases in both mitochondrial and glycolytic functions, and a substantial upregulation of , , , , , TIMP, and most ER stress-related genes and the downregulation of and .