Differential Effects of Benzalkonium Chloride on Human Trabecular Meshwork Cells Not Treated or Treated with Transforming Growth Factor-ß2 or Dexamethasone.

Purpose: The objective of the present study was to evaluate the effects of low concentrations of benzalkonium chloride (BAC) (10-7%, 10-6%, or 10-5%) on healthy and glaucomatous human trabecular meshwork (HTM) cells. For this purpose, we used in vitro 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-ß2 or 250 nM dexamethasone (DEX). Methods: 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. Results: In the absence and presence of BAC (10-7% or 10-5%), intercellular affinity function determined by TEER and cellular metabolic activities were significantly and dose dependently affected in both healthy and glaucomatous HTM cells despite the fact that there was no significant decrease in cell viabilities. However, the effects based on TEER values were significantly greater in the healthy HTM. The mRNA expression of several molecules that were tested was not substantially modulated by these concentrations of BAC. Conclusions: The findings reported herein suggest that low concentrations of BAC may have unfavorable adverse effects on cellular metabolic capacity by inducing increases in the intercellular affinity properties of the HTM, but those effects of BAC were different in healthy and glaucomatous HTM cells.

Significant and Various Effects of ML329-Induced MITF Suppression in the Melanoma Cell Line.

To study the inhibitory effects on microphthalmia-associated transcription factor (MITF)-related biological aspects in malignant melanomas (MMs) in the presence or absence of the low-molecular MITF specific inhibitor ML329, cell viability, cellular metabolic functions, and three-dimensional (3D) spheroid formation efficacy were compared among MM cell lines including SK-mel-24, A375, dabrafenib- and trametinib-resistant A375 (A375DT), and WM266-4. Upon exposure to 2 or 10 µM of ML329, cell viability was significantly decreased in WM266-4, SK-mel-24, and A375DT cells, but not A375 cells, in a dose-dependent manner, and these toxic effects of ML329 were most evident in WM266-4 cells. Extracellular flux assays conducted using a Seahorse bioanalyzer revealed that treatment with ML329 increased basal respiration, ATP-linked respiration, proton leakage, and non-mitochondrial respiration in WM266-4 cells and decreased glycolytic function in SK-mel-24 cells, whereas there were no marked effects of ML329 on A375 and A375DT cells. A glycolytic stress assay under conditions of high glucose concentrations also demonstrated that the inhibitory effect of ML329 on the glycolytic function of WM266-4 cells was dose-dependent. In addition, ML329 significantly decreased 3D-spheroid-forming ability, though the effects of ML329 were variable among the MM cell lines. Furthermore, the mRNA expression levels of selected genes, including STAT3 as a possible regulator of 3D spheroid formation, KRAS and SOX2 as oncogenic-signaling-related factors, PCG1a as the main regulator of mitochondrial biogenesis, and HIF1a as a major hypoxia transcriptional regulator, fluctuated among the MM cell lines, possibly supporting the diverse ML329 effects mentioned above. The findings of diverse ML329 effects on various MM cell lines suggest that MITF-associated biological activities are different among various types of MM.

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 (10-8%, 10-6%, or 10-4%) 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 10-8%, 10-6%, or 10-4% concentrations of BAC, cell viability deteriorated and this deterioration was dose-dependent. The results showed that maximal mitochondrial respiration was decreased, the mRNA expression of most of ECM proteins was decreased, and ER stress-related molecules were substantially and dose-dependently down-regulated in HCSFs by the BAC treatment. CONCLUSIONS: The findings reported herein indicate that the presence of BAC, even at such low concentrations, is capable of causing the deterioration of cellular metabolic functions and negatively affecting the response to ER stress in HCSF cells resulting in a substantially decreased cellular viability.

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 in vitro 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. In 2D culture, 565 up-regulated genes and 391 down-regulated genes were identified as differentially expressed genes (DEGs) by adipogenic induction of 3T3-L1 preadipocytes, whereas only 69 up-regulated genes and 59 down-regulated genes were identified as DEGs in 3D culture. Ingenuity Pathway Analysis (IPA) revealed that genes associated with lipid metabolism were identified as 2 out of the top 3 causal networks related to diseases and function in 3D spheroids, whereas only one network related to lipid metabolism was identified within the top 9 of these causal networks in the 2D planar cells, suggesting that adipogenic induction in the 3D culture condition exhibits a more adipocyte-specific gene expression pattern in 3T3-L1 preadipocytes. Real-time metabolic analysis revealed that the metabolic capacity shifted from glycolysis to mitochondrial respiration in differentiated 3T3-L1 cells in the 3D culture condition but not in those in the 2D cultured condition, suggesting that adipogenic differentiation in 3D culture induces a metabolic phenotype of well-differentiated adipocytes. Consistently, expression levels of mitochondria-encoded genes including mt-Nd6, mt-Cytb, and mt-Co1 were significantly increased by adipogenic induction of 3T3-L1 preadipocytes in 3D culture compared with those in 2D culture. Taken together, the findings suggest that induction of adipogenesis in 3D culture provides a more adipocyte-specific gene expression pattern and enhances mitochondrial respiration, resulting in more adipocyte-like cellular properties.

TGF-ß2 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 (COL) 1, 4 and 6, and fibronectin (FN), a tissue inhibitor of metalloproteinase (TIMP) 1-4, matrix metalloproteinase (MMP) 2, 3, 9 and 14, and several endoplasmic reticulum (ER) stress-related factors. In the 2D HCSFs, TGF-ß2 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 COL1, COL4, FN, αSMA, TIMP1, TIMP, and most ER stress-related genes and the downregulation of COL6 and MMP3. In the case of 3D spheroids, TGF-ß2 induced the downsizing and stiffening of 3D spheroids and the upregulation of COL6, MMP14, and most ER stress-related genes. These findings suggest that TGF-ß2 significantly induced a number of EMT-associated biological events including planar proliferation, cellular metabolic functions, and the production of ECM molecules in the 2D cultured HCSF cells, but these effects were significantly less pronounced in the case of 3D HCSF spheroids.

Physical Properties and Cellular Metabolic Characteristics of 3D Spheroids Are Possible Definitive Indices for the Biological Nature of Cancer-Associated Fibroblasts.

The current study's objective was to elucidate some currently unknown biological indicators to evaluate the biological nature of cancer-associated fibroblasts (CAFs). For this purpose, four different CAFs, CAFS1, CAFS2, SCC17F and MO-1000, were established using surgical specimens from oral squamous cell carcinomas (OSCC) with different clinical malignant stages (CAFS1 and CAFS2, T2N0M0, stage II; SCC17F and MO-1000, T4aN2bM0, stage IVA). Fibroblasts unrelated to cancer (non-CAFs) were also prepared and used as controls. Initially, confirmation that these four fibroblasts were indeed CAFs was obtained by their mRNA expression using positive and negative markers for the CAF or fibroblasts. To elucidate possible unknown biological indicators, these fibroblasts were subjected to a cellular metabolic analysis by a Seahorse bioanalyzer, in conjugation with 3D spheroid cultures of the cells and co-cultures with a pancreas ductal carcinoma cell line, MIA PaCa-2. The mitochondrial and glycolytic functions of human orbital fibroblasts (HOF) were nearly identical to those of Graves'-disease-related HOF (GOF). In contrast, the characteristics of the metabolic functions of these four CAFs were different from those of human conjunctival fibroblasts (HconF), a representative non-CAF. It is particularly noteworthy that CAFS1 and CAFS2 showed markedly reduced ratios for the rate of oxygen consumption to the extracellular acidification rate, suggesting that glycolysis was enhanced compared to mitochondrial respiration. Similarly, the physical aspects, their appearance and stiffness, of their 3D spheroids and fibroblasts that were induced effects based on the cellular metabolic functions of MIA PaCa-2 were also different between CAFs and non-CAFs, and their levels for CAFS1 or SCC17F were similar to those for CAFS2 or MO-1000 cells, respectively. The findings reported herein indicate that cellular metabolic functions and the physical characteristics of these types of 3D spheroids may be valuable and useful indicators for estimating potential biological diversity among various CAFs.

TGF-ß Isoforms Affect the Planar and Subepithelial Fibrogenesis of Human Conjunctival Fibroblasts in Different Manners.

Three highly homologous isoforms of TGF-ß, TGF-ß-1~3, are involved in the regulation of various pathophysiological conditions such as wound healing processes in different manners, despite the fact that they bind to the same receptors during their activation. The purpose of the current investigation was to elucidate the contributions of TGF-ß-1 ~3 to the pathology associated with conjunctiva. For this purpose, the biological effects of these TGF-ß isoforms on the structural and functional properties of two-dimensional (2D) and three-dimensional (3D) cultured human conjunctival fibroblasts (HconF) were subjected to the following analyses: 1) transendothelial electrical resistance (TEER), a Seahorse cellular metabolic measurement (2D), size and stiffness measurements of the 3D HTM spheroids, and the qPCR gene expression analyses of extracellular matrix (ECM) components (2D and 3D). The TGF-ß isoforms caused different effects on the proliferation of the HconF cell monolayer evaluated by TEER measurements. The differences included a significant increase in the presence of 5 ng/mL TGF-ß-1 and -2 and a substantial decrease in the presence of 5 ng/mL TGF-ß-3, although there were no significant differences in the response to the TGF-ß isoforms for cellular metabolism among the three groups. Similar to planar proliferation, the TGF-ß isoforms also induced diverse effects toward the mechanical aspects of 3D HconF spheroids, where TGF-ß-1 increased stiffness, TGF-ß-2 caused no significant effects, and TGF-ß-3 caused the downsizing of the spheroids and stiffness enhancement. The mRNA expression of the ECMs were also modulated in diverse manners by the TGF-ß isoforms as well as the culture conditions for the 2D vs. 3D isoforms. Many of these TGF-ß-3 inducible effects were markedly different from those caused by TGF-ß1 and TGF-ß-2. The findings presented herein suggest that the three TGF-ß isoforms induce diverse and distinctly different effects on cellular properties and the expressions of ECM molecules in HconF and that these changes are independent of cellular metabolism, thereby inducing different effects on the epithelial and subepithelial proliferation of human conjunctiva.

mTOR Inhibitors Modulate the Physical Properties of 3D Spheroids Derived from H9c2 Cells.

To establish an appropriate in vitro model for the local environment of cardiomyocytes, three-dimensional (3D) spheroids derived from H9c2 cardiomyoblasts were prepared, and their morphological, biophysical phase contrast and biochemical characteristics were evaluated. The 3D H9c2 spheroids were successfully obtained, the sizes of the spheroids decreased, and they became stiffer during 3-4 days. In contrast to the cell multiplication that occurs in conventional 2D planar cell cultures, the 3D H9c2 spheroids developed into a more mature form without any cell multiplication being detected. qPCR analyses of the 3D H9c2 spheroids indicated that the production of collagen4 (COL4) and fibronectin (FN), connexin43 (CX43), ß-catenin, N-cadherin, STAT3, and HIF1 molecules had increased and that the production of COL6 and α-smooth muscle actin (α-SMA) molecules had decreased as compared to 2D cultured cells. In addition, treatment with rapamycin (Rapa), an mTOR complex (mTORC) 1 inhibitor, and Torin 1, an mTORC1/2 inhibitor, resulted in significantly decreased cell densities of the 2D cultured H9c2 cells, but the size and stiffness of the H9c2 cells within the 3D spheroids were reduced with the gene expressions of several of the above several factors being reduced. The metabolic responses to mTOR modulators were also different between the 2D and 3D cultures. These results suggest that as unique aspects of the local environments of the 3D spheroids, the spontaneous expression of GJ-related molecules and hypoxia within the core may be associated with their maturation, suggesting that this may become a useful in vitro model that replicates the local environment of cardiomyocytes.

Prostanoid FP and EP2 Receptor Agonists Induce Epithelial and Subepithelial Fibrogenetic Changes in Human Conjunctival Fibroblasts in Different Manners.

Purpose: To examine the effects of prostanoid FP and EP2 receptor agonists, PGF2α and Omidenepag (OMD), respectively, on the transforming growth factor beta (TGF-ß2) induced conjunctival fibrogenesis. Methods: Two-dimension (2D) and three-dimension (3D) cultures of these fibroblasts were subjected to following analyses: (1) planar proliferation evaluated by transendothelial electron resistance (TEER) measurements, (2) real-time metabolic analyses, (3) subepithelial proliferation evaluated by 3D spheroid' size and stiffness measurements, and (4) the mRNA expression of extracellular matrix (ECM) molecules and their modulators. Results: TGF-ß2 induced increase in the planar proliferation was significantly decreased or enhanced by PGF2α or OMD, respectively. The proportion of oxygen consumption required to drive ATP synthesis compared with that driving proton leakage was increased by PGF2α and OMD independently with TGF-ß2. In contrast, maximal mitochondrial respiration was decreased by PGF2α and OMD, and the OMD-induced effect was further enhanced by the presence of TGF-ß2. In addition, the TGF-ß2 dependent increase in the glycolytic capacity was cancelled by PGF2α and/or OMD. Alternatively, subepithelial proliferation, as evidenced by the stiffness of the 3D spheroids, was substantially increased by both PGF2α and OMD, and these were differently modulated by TGF-ß2. The expression of several related factors as above fluctuated among the conditions for both 2D and 3D and TGF-ß2 untreated or treated cultures. Conclusion: The present findings indicate that the prostanoid FP or the EP2 receptor agonist may solely and differently induce the planar and subepithelial proliferation of HconF cells and these were also modulated by TGF-ß2.

Simultaneous Effects of a Selective EP2 Agonist, Omidenepag, and a Rho-Associated Coiled-Coil Containing Protein Kinase Inhibitor, Ripasudil, on Human Orbital Fibroblasts.

Purpose: To assess the combined effects of omidenepag (OMD), a selective EP2 agonist, and ripasudil (Rip), an inhibitor of rho-associated coiled-coil containing protein kinases, on the human orbital adipose tissue, two-dimensional (2D) or three-dimensional (3D) cultures of human orbital fibroblasts (HOFs) were employed. Methods: Cellular metabolic functions (2D), physical (3D), lipid staining (3D), and quantitative polymerase chain reaction for adipogenesis-related genes, PPARγ and AP2, and extracellular matrix (ECM) molecules, including collagen (COL)1, 4, and 6, and fibronectin (FN) (3D) were evaluated in the presence of OMD (100 nM) and/or Rip (10 µM). Results: Real-time metabolic analyses revealed that the adipogenic differentiation (DIF+) with OMD significantly shifted an energetic state toward energetic, whereas DIF+ with Rip significantly shifted that toward quiescent. In the case of both drugs upon DIF+, the metabolic effect of OMD was predominant. DIF+ induced enlargement and stiffed 3D spheroid with increased lipid staining and mRNA expression of adipogenesis-related genes, COL4 and COL6, and decreased the expression of COL1. In the presence of OMD and/or Rip to DIF+, (1) the sizes were further increased by Rip and the stiffness was significantly decreased by OMD or Rip and (2) COL4 or AP2 expression was substantially increased by OMD or Rip, respectively. Conclusion: The results presented herein indicate that the metabolic effects of OMD and Rip exerted opposing effects and the effects of OMD toward Ap2 and ECM expressions were distinct from those of Rip, but the effects of OMD toward the physical aspects and adipogenesis of the 3D cultured HOFs were similar to the effects of Rip.

Three-Dimensional Spheroid Configurations and Cellular Metabolic Properties of Oral Squamous Carcinomas Are Possible Pharmacological and Pathological Indicators.

The objective of the current study was to elucidate the clinicopathological significance and appearance of in vitro three-dimension (3D) spheroid models of oral malignant tumors that were prepared from four pathologically different squamous cell carcinoma (OSCC; low-grade; SSYP and MO-1000, intermediate-grade; LEM2) and oral adenosquamous carcinoma (OASC; high-grade; Mesimo) obtained from patients with different malignant stages. To characterize the biological significance of these cell lines themselves, two-dimensional (2D) cultured cells were subjected to cellular metabolic analysis by a Seahorse bioanalyzer alongside the measurement of the cytotoxicity of cisplatin (CDDP). The appearance of their 3D spheroids was then observed by phase contrast microscopy, and both 2D and 3D cultured cells were subject to trypsin digestion and qPCR analysis of factors related to oncogenic signaling and other related analyses. ATP-linked respiration and proton leaking were significantly different among the four cell lines, and the malignant stages of these cultures were significantly associated with increased ATP-linked respiration and decreased proton leakage. Alternatively, the appearances of these 3D spheroids were also significantly diverse among them, and their differences increased in the order of LEM2, MO-1000, SSYP, and Mesimo. Interestingly, these orders were exactly the same in that the efficacies of CDDP-induced cytotoxicity increased in the same order. qPCR analysis indicated that the levels of expression of oncogenic signaling-related factors varied among these four cell lines, and the values for fibronectin and a key regulator of mitochondrial biogenesis, PGC-1α, were prominently elevated in cultures of the worst malignant Mesimo cells. In addition, although 0.25% trypsin-induced destruction was comparable among all four 2D cultured cells, the values for the 3D spheroids were also substantially varied among these cultures. The findings reported herein indicate that cellular metabolic functions and 3D spheroid architectures may be valuable and useful indicators for estimating the pathological and drug-sensitive aspects of OSCC and OASC malignancies.

3D Spheroid Configurations Are Possible Indictors for Evaluating the Pathophysiology of Melanoma Cell Lines.

To study the molecular mechanisms responsible for inducing the spatial proliferation of malignant melanomas (MM), three-dimension (3D) spheroids were produced from several MM cell lines including SK-mel-24, MM418, A375, WM266-4, and SM2-1, and their 3D architectures and cellular metabolisms were evaluated by phase-contrast microscopy and Seahorse bio-analyzer, respectively. Several transformed horizontal configurations were observed within most of these 3D spheroids, and the degree of their deformity was increased in the order: WM266-4, SM2-1, A375, MM418, and SK-mel-24. An increased maximal respiration and a decreased glycolytic capacity were observed within the lesser deformed two MM cell lines, WM266-4 and SM2-1, as compared with the most deformed ones. Among these MM cell lines, two distinct cell lines, WM266-4 and SK-mel-24, whose 3D appearances were the closest and farthest, respectively, from being horizontally circular-shaped, were subjected to RNA sequence analyses. Bioinformatic analyses of the differentially expressed genes (DEGs) identified KRAS and SOX2 as potential master regulatory genes for inducing these diverse 3D configurations between WM266-4 and SK-mel-24. The knockdown of both factors altered the morphological and functional characteristics of the SK-mel-24 cells, and in fact, their horizontal deformity was significantly reduced. A qPCR analysis indicated that the levels of several oncogenic signaling related factors, including KRAS and SOX2, PCG1α, extracellular matrixes (ECMs), and ZO1 had fluctuated among the five MM cell lines. In addition, and quite interestingly, the dabrafenib and trametinib resistant A375 (A375DT) cells formed globe shaped 3D spheroids and showed different profiles in cellular metabolism while the mRNA expression of these molecules that were tested as above were different compared with A375 cells. These current findings suggest that 3D spheroid configuration has the potential for serving as an indicator of the pathophysiological activities associated with MM.

TGF-ß-3 Induces Different Effects from TGF-ß-1 and -2 on Cellular Metabolism and the Spatial Properties of the Human Trabecular Meshwork Cells.

To compare the effects among three TGF-ß isoforms (TGF-ß-1, TGF-ß-2, and TGF-ß-3) on the human trabecular meshwork (HTM), two-dimensional (2D) and three-dimensional (3D) cultures of commercially available certified immortalized HTM cells were used, and the following analyses were conducted: (1) trans-endothelial electrical resistance (TEER) and FITC dextran permeability measurements (2D); (2) a real-time cellular metabolic analysis (2D); (3) analysis of the physical property of the 3D HTM spheroids; and (4) an assessment of the gene expression levels of extracellular matrix (ECM) components (2D and 3D). All three TGF-ß isoforms induced a significant increase in TEER values and a relative decrease in FITC dextran permeability in the 2D-cultured HTM cells, but these effects were the most potent in the case of TGF-ß-3. The findings indicated that solutions containing 10 ng/mL of TGF-ß-1, 5 ng/mL of TGF-ß-2, and 1 ng/mL of TGF-ß-3 had nearly comparable effects on TEER measurements. However, a real-time cellular metabolic analysis of the 2D-cultured HTM cells under these concentrations revealed that TGF-3-ß induced quite different effects on the metabolic phenotype, with a decreased ATP-linked respiration, increased proton leakage, and decreased glycolytic capacity compared with TGF-ß-1 and TGF-ß-2. In addition, the concentrations of the three TGF-ß isoforms also caused diverse effects on the physical properties of 3D HTM spheroids and the mRNA expression of ECMs and their modulators, in many of which, the effects of TGF-ß-3 were markedly different from TGF-ß-1 and TGF-ß-2. The findings presented herein suggest that these diverse efficacies among the TGF-ß isoforms, especially the unique action of TGF-ß-3 toward HTM, may induce different effects within the pathogenesis of glaucoma.

Unexpected Crosslinking Effects of a Human Thyroid Stimulating Monoclonal Autoantibody, M22, with IGF1 on Adipogenesis in 3T3L-1 Cells.

To study the effects of the crosslinking of IGF1 and/or the human thyroid-stimulating monoclonal autoantibody (TSmAb), M22 on mouse adipocytes, two- and three-dimensional (2D or 3D) cultures of 3T3-L1 cells were prepared. Each sample was then subjected to the following analyses: (1) lipid staining, (2) a real-time cellular metabolic analysis, (3) analysis of the mRNA expression of adipogenesis-related genes and extracellular matrix (ECM) molecules including collagen (Col) 1, 4 and 6, and fibronectin (Fn), and (4) measurement of the size and physical properties of the 3D spheroids with a micro-squeezer. Upon adipogenic differentiation (DIF+), lipid staining and the mRNA expression of adipogenesis-related genes in the 2D- or 3D-cultured 3T3-L1 cells substantially increased. On adding IGF1 but not M22 to DIF+ cells, a significant enhancement in lipid staining and gene expressions of adipogenesis-related genes was detected in the 2D-cultured 3T3-L1 cells, although some simultaneous suppression or enhancement effects by IGF1 and M22 against lipid staining or Fabp4 expression, respectively, were detected in the 3D 3T3-L1 spheroids. Real-time metabolic analyses indicated that monotherapy with IGF1 or M22 shifted cellular metabolism toward energetic states in the 2D 3T3-L1 cells upon DIF+, although no significant metabolic changes were induced by DIF+ alone in 2D cultures. In addition, some synergistical effects on cellular metabolism by IGF1 and M22 were also observed in the 2D 3T3-L1 cells as well as in cultured non-Graves' orbitopathy-related human orbital fibroblasts (n-HOFs), but not in Graves' orbitopathy-related HOFs (GHOFs). In terms of the physical properties of the 3D 3T3-L1 spheroids, (1) their sizes significantly increased upon DIF+, and this increase was significantly enhanced by the presence of both IGF1 and M22 despite downsizing by monotreatment, and (2) their stiffness increased substantially, and no significant effects by IGF-1 and/or M22 were observed. Regarding the expression of ECM molecules, (1) upon DIF+, significant downregulation or upregulation of Col1 and Fn (3D), or Col4 and 6 (2D and 3D) were observed, and (2) in the presence of IGF-1 and/or M22, the mRNA expression of Col4 was significantly downregulated by M22 (2D and 3D), but the expression of Col1 was modulated in different manners by monotreatment (upregulation) or the combined treatment (downregulation) (3D). These collective data suggest that the human-specific TSmAb M22 induced some unexpected simultaneous crosslinking effects with IGF-1 with respect to the adipogenesis of 2D-cultured 3T3-L1 cells and the physical properties of 3D 3T3-L1 spheroids.

G-Protein-Coupled Receptors Mediate Modulations of Cell Viability and Drug Sensitivity by Aberrantly Expressed Recoverin 3 within A549 Cells.

To elucidate the currently unknown molecular mechanisms responsible for the aberrant expression of recoverin (Rec) within cancerous cells, we examined two-dimensional (2D) and three-dimensional (3D) cultures of Rec-negative lung adenocarcinoma A549 cells which had been transfected with a plasmid containing human recoverin cDNA (A549 Rec) or an empty plasmid as a mock control (A549 MOCK). Using these cells, we measured cytotoxicity by several anti-tumor agents (2D), cellular metabolism including mitochondrial and glycolytic functions by a Seahorse bio-analyzer (2D), the physical properties, size and stiffness of the 3D spheroids, trypsin sensitivities (2D and 3D), and RNA sequencing analysis (2D). Compared with the A549 MOCK, the A549 Rec cells showed (1) more sensitivity toward anti-tumor agents (2D) and a 0.25% solution of trypsin (3D); (2) a metabolic shift from glycolysis to oxidative phosphorylation; and (3) the formation of larger and stiffer 3D spheroids. RNA sequencing analysis and bioinformatic analyses of the differentially expressed genes (DEGs) using Gene Ontology (GO) enrichment analysis suggested that aberrantly expressed Rec is most likely associated with several canonical pathways including G-protein-coupled receptor (GPCR)-mediated signaling and signaling by the cAMP response element binding protein (CREB). The findings reported here indicate that the aberrantly expressed Rec-induced modulation of the cell viability and drug sensitivity may be GPCR mediated.

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, n = 2) and >27 mm (high AL group, n = 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. Based upon RNA sequencing of these low and high AL groups, the bioinformatic analyses using gene ontology (GO) enrichment analysis and ingenuity pathway analysis (IPA) of differentially expressed genes (DEGs) identified that sterol regulatory element-binding transcription factor 2 (SREBF2) is both a possible upstream regulator and a causal network regulator. Furthermore, SREBF1, insulin-induced gene 1 (INSIG1), and insulin-like growth factor 1 (IGF1) were detected as upstream regulators, and protein tyrosine phosphatase receptor type O (PTPRO) was detected as a causal network regulator. Since those possible regulators were all pivotally involved in lipid metabolisms including fatty acid (FA), triglyceride (TG) and cholesterol (Chol) biosynthesis, the findings reported here indicate that FA, TG and Chol biosynthesis regulation may be responsible mechanisms inducing AL elongation via HSSF.

Addition of ROCK Inhibitors Alleviates Prostaglandin-Induced Inhibition of Adipogenesis in 3T3L-1 Spheroids.

To elucidate the additive effects of the ROCK inhibitors (ROCK-i), ripasudil (Rip) and Y27632 on bimatoprost acid (BIM-A), a prostaglandin analog (PG), on adipose tissue, two- and three-dimensional (2D or 3D) cultures of 3T3-L1 cells, the most well characterized cells in the field of lipid research, were used. The cells were subjected to a variety of analyses including lipid staining, real-time cellular metabolic analysis, the mRNA expressions of genes related to adipogenesis and extracellular matrices (ECMs) as well as the sizes and physical properties of the 3D spheroids by a micro-squeezer. BIM-A induced strong inhibitory effects on most of the adipogenesis-related changes in the 2D and 3D cultured 3T3-L1 cells, including (1) the enlargement and softening of the 3D spheroids, (2) a dramatic enhancement in lipid staining and the expression of adipogenesis-related genes, and (3) a decrease in mitochondrial and glycolytic metabolic function. By adding ROCK-i to the BIM-A, most of these BIM-A-induced effects were cancelled. The collective findings reported herein suggest that ROCK-i eliminated the PG-induced suppression of adipogenesis in the 3T3-L1 cells, accompanied by the formation of enlarged 3D spheroids. Such effects of adding ROCK-i to a PG in preadipocytes on cellular properties appear to be associated with the suppression of PG-induced adverse effects, and provide additional insight into our understanding of lipid-related research.

The Selective α1 Antagonist Tamsulosin Alters ECM Distributions and Cellular Metabolic Functions of ARPE 19 Cells in a Concentration-Dependent Manner.

The purpose of the present study was to examine the effect of the selective α1 antagonist tamsulosin (TAM) on human retinal pigment epithelium cells, ARPE 19. Two-dimension (2D) and three-dimension (3D) cultured ARPE 19 cells were used in the following characterizations: (1) ultrastructure by scanning electron microscopy (SEM) (2D); (2) barrier functions by transepithelial electrical resistance (TEER) measurements, and FITC-dextran permeability (2D); (3) real time cellular metabolisms by Seahorse Bioanalyzer (2D); (4) physical properties, size and stiffness measurements (3D); and (5) expression of extracellular matrix (ECM) proteins, including collagen1 (COL1), COL4, COL6 and fibronectin (FN) by qPCR and immunohistochemistry (2D and 3D). TAM induced significant effects including: (1) alteration of the localization of the ECM deposits; (2) increase and decrease of the TEER values and FITC-dextran permeability, respectively; (3) energy shift from glycolysis into mitochondrial oxidative phosphorylation (OXPHOS); (4) large and stiffened 3D spheroids; and (5) down-regulations of the mRNA expressions and immune labeling of most ECM proteins in a concentration-dependent manner. However, in some ECM proteins, COL1 and COL6, their immunolabeling intensities were increased at the lowest concentration (1 µM) of TAM. Such a discrepancy between the gene expressions and immunolabeling of ECM proteins may support alterations of ECM localizations as observed by SEM. The findings reported herein indicate that the selective α1 antagonist, TAM, significantly influenced ECM production and distribution as well as cellular metabolism levels in a concentration-dependent manner.

Benzalkonium Chloride, Even at Low Concentrations, Deteriorates Intracellular Metabolic Capacity in Human Conjunctival Fibroblasts.

The objective of this study was to clarify the effects of benzalkonium chloride (BAC) on two-dimensional (2D) and three-dimensional (3D) cultures of human conjunctival fibroblast (HconF) cells, which are in vitro models replicating the epithelial barrier and the stromal supportive functions of the human conjunctiva. The cultured HconF cells were subjected to the following analyses in the absence and presence of 10-5% or 10-4% concentrations of BAC; (1) the barrier function of the 2D HconF monolayers, as determined by trans-endothelial electrical resistance (TEER) and FITC dextran permeability, (2) real-time metabolic analysis using an extracellular Seahorse flux analyzer, (3) the size and stiffness of 3D HconF spheroids, and (4) the mRNA expression of genes that encode for extracellular matrix (ECM) molecules including collagen (COL)1, 4 and 6, and fibronectin (FN), α-smooth muscle actin (α-SMA), ER stress related genes including the X-box binding protein-1 (XBP1), the spliced XBP1 (sXBP1) glucose regulator protein (GRP)78, GRP94, and the CCAAT/enhancer-binding protein homologous protein (CHOP), hypoxia inducible factor 1α (HIF1α), and Peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α). In the presence of BAC, even at low concentrations at 10-5% or 10-4%, the maximal respiratory capacity, mitochondrial respiratory reserve, and glycolytic reserve of HconF cells were significantly decreased, although the barrier functions of 2D HconF monolayers, the physical properties of the 3D HconF spheroids, and the mRNA expression of the corresponding genes were not affected. The findings reported herein highlight the fact that BAC, even such low concentrations, may induce unfavorable adverse effects on the cellular metabolic capacity of the human conjunctiva.

All Trans-Retinoic Acids Facilitate the Remodeling of 2D and 3D Cultured Human Conjunctival Fibroblasts.

Vitamin A derivative, all-trans-retinoic acid (ATRA), is known to be a potent regulator of the growth and differentiation of various types of cells. In the present study, the unidentified effects of ATRA on superficial and vertical spreading conjunctival scarring were examined. The study involved the use of two-dimensional (2D) and three-dimensional (3D) cultures of human conjunctival fibroblast (HconF) cells in the presence or absence of TGF-ß2. The effects of ATRA (1 µM) on superficial or vertical spreading conjunctival scarring were evaluated by the barrier function by trans-endothelial electrical resistance (TEER) and FITC dextran permeability measurements and real-time metabolic analysis, as well as the physical properties, namely, the size and stiffness, of 3D spheroids, respectively. In addition, the expressions of several related molecules, including extracellular matrix (ECM) molecules, ECM modulators including a tissue inhibitor of metalloproteinases (TIMPs), matrix metalloproteinases (MMPs), and ER stress-related factors, were examined. ATRA significantly induced (1) an increase in TEER values and a decrease in FITC dextran permeability, respectively, in the 2D monolayers, and (2) relatively and substantially increased the size and stiffness, respectively, of the 3D spheroids. These ATRA-induced effects were further enhanced in the TGF-ß2-treated cells, whereas the TGF-ß2-induced enhancement in glycolytic capacity was canceled by the presence of ATRA. Consistent with these physical and morphological effects, the mRNA expressions of several molecules were significantly but differently induced between 2D and 3D cultures by ATRA, although the presence of TGF-ß2 did not substantially affect these gene expression levels. The findings reported in this study indicate that ATRA may exacerbate both superficial and vertical conjunctival fibrosis spreading independently of TGF-ß2-induced changes.