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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 in vitro 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. In the simplest single cell type-derived spheroid cultures, there are also various scaffold-assisted methods such as hydrogel-assisted cultures, biofilm-assisted cultures, particle-assisted cultures, and magnet particle-assisted cultures, as well as non-assisted methods, such as static suspension cultures, floating cultures, and hanging drop cultures. Since each method can be differently influenced by various factors such as gravity force, buoyant force, centrifugal force, and magnetic force, in addition to non-physiological scaffolds, each method has its own advantages and disadvantages, and the methods have different suitable applications. We have been focusing on the use of a hanging drop culture method for modeling various non-cancerous and cancerous diseases because this technique is affected only by gravity force and buoyant force and is thus the simplest method among the various single cell type-derived spheroid culture methods. We have found that the biological natures of spheroids generated even by the simplest method of hanging drop cultures are completely different from those of 2D cultured cells. In this review, we focus on the biological aspects of single cell type-derived spheroid culture and its applications in in vitro models for various diseases.

Fatty acid-binding proteins (FABPs), a family of lipid chaperone molecules that are involved in intracellular lipid transportation to specific cellular compartments, stimulate lipid-associated responses such as biological signaling, membrane synthesis, transcriptional regulation, and lipid synthesis. Previous studies have shown that FABP4, a member of this family of proteins that are expressed in adipocytes and macrophages, plays pivotal roles in the pathogenesis of various cardiovascular and metabolic diseases, including diabetes mellitus (DM) and hypertension (HT). Since significant increases in the serum levels of FABP4 were detected in those patients, FABP4 has been identified as a crucial biomarker for these systemic diseases. In addition, in the field of ophthalmology, our group found that intraocular levels of FABP4 (ioFABP4) and free fatty acids (ioFFA) were substantially elevated in patients with retinal vascular diseases (RVDs) including proliferative diabetic retinopathy (PDR) and retinal vein occlusion (RVO), for which DM and HT are also recognized as significant risk factors. Recent studies have also revealed that ioFABP4 plays important roles in both retinal physiology and pathogenesis, and the results of these studies have suggested potential molecular targets for retinal diseases that might lead to future new therapeutic strategies.

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. RNA sequencing and bioinformatic analyses of the differentially expressed genes (DEGs) using an ingenuity pathway analysis (IPA) suggested that various modulatory factors related to epithelial -mesenchymal transition (EMT) including STAT3, GLI1, ZNF367, NKX3-2, ZIC2, IFIT2, HEY1 and FBLX, may be the possible upstream regulators and/or causal network master regulators responsible for the acquirement of drug resistance in MIA PaCa-2-GR and -PR. In addition, among the prominently altered DEGs (Log2 fold changes more than 6 or less than -6), FABP5, IQSEC3, and GASK1B were identified as unique genes associated with their antisense RNA or pseudogenes, and among these, FABP5 and GASK1B are known to function as modulators of cancerous EMT. Therefore, the observations reported herein suggest that modulations of cancerous EMT may be key molecular mechanisms that are responsible for inducing chemoresistance against GEM or PTX in MIA PaCa-2 cells.

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., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors. Published by Elsevier Ltd.)

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.

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.

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.

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.

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 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.

The objective of the current study was to examine the effects of fibroblast growth factor-2 (FGF-2) on conjunctival fibrogenesis that was induced by the presence of transforming growth factor-β2 (TGF-β2). Two-dimension (2D) and three-dimension (3D) cultured human conjunctival fibroblasts (HconF) were used for this purpose. The 2D and 3D cultured HconF were characterized by transendothelial electrical resistance (TEER) and FITC dextran permeability measurements (2D), real-time metabolic analyses (2D), size and stiffness measurements (3D), and the mRNA expression of extracellular matrix molecules, their modulators, Tissue inhibitor of metalloproteinases and matrix metalloproteinases and ER-stress related genes (2D and 3D). FGF-2 significantly increased planar proliferation, as evidenced by TEER values and FITC dextran permeability, and shifted glucose metabolism to the energetic phenotype of 2D HconF cells, and the stiffness of the 3D spheroids, and these effects were further enhanced in the presence of TGF-β2. Analyses of the expression of possible candidate molecules involved in cell architecture and stress indicated that some additive effects caused by both factors were also recognized in some of these molecules. The findings reported herein indicate that the FGF-2, either along or additively with TGF- β2 increased the fibrogenetic changes on the plane as well as in the spatial space of HconF cells., (© 2022. The Author(s).)

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.

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-β2. 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-β2-treated cells, whereas the TGF-β2-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-β2 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-β2-induced changes.

We report herein on the effects of all- trans retinoic acid (ATRA) on two-dimensional (2D) and three-dimensional (3D) cultures of human trabecular meshwork (HTM) cells that were treated with transforming growth factor β2 (TGF-β2). In the presence of 5 ng/mL TGF-β2, the effects of ATRA on the following were observed: (1) the barrier function of the 2D HTM monolayers, as determined by trans -endothelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC) dextran permeability measurements; (2) a Seahorse cellular bio-metabolism analysis; (3) physical properties, including the size and stiffness, of 3D spheroids; (4) the gene expression of extracellular matrix (ECM) molecules, ECM modulators including tissue inhibitor of metalloproteinases (TIMPs), matrix metalloproteinases (MMPs), tight junction (TJ)-related molecules, and endoplasmic reticulum (ER)-stress-related factors. ATRA significantly inhibited the TGF-β2-induced increase in the TEER values and FITC dextran permeability of the 2D monolayers, while an ATRA monotreatment induced similar effects as TGF-β2. A real-time metabolic analysis revealed that ATRA significantly inhibited the TGF-β2-induced shift in metabolic reserve from mitochondrial oxidative phosphorylation to glycolysis in 2D HTM cells, whereas ATRA alone did not induce significant metabolic changes. In contrast, ATRA induced the formation of substantially downsized and softer 3D spheroids in the absence and presence of TGF-β2. The different effects induced by ATRA toward 2D and 3D HTM cells were also supported by the qPCR analysis of several proteins as above. The findings reported here indicate that ATRA may induce synergistic and beneficial effects on TGF-β2-treated 2D- and 3D-cultured HTM cells; those effects varied significantly between the 2D and 3D cultures.

To study the pathophysiological roles of the fatty acid-binding proteins (FABPs) within the retina, we performed; (1) immunolabeling of human retinas, wild type (WT) rat and mouse retinas, rat models for diabetic retinopathy (DR) and retinitis pigmentosa (RP) with anti-FABP3, FABP4, FABP5, FABP7, FABP8 and FABP12, (2) electroretinogram (ERG) measurements of WT and FABP4-deficient (Fabp4 -/- ) mice, (3) ELISA or gas chromatography measurements of plasma (P-) and vitreous (V-) levels of FABP4 and vascular endothelial growth factor A (VEGFA), and fatty acids (FAs) from patients with retinal vascular disease (RVD) including proliferative DR (PDR, n = 30) and retinal vein occlusion (RVO, n = 18) and non-RVD (n = 18). Within the human retina, diverse expressions of FABP3, FABP4, FABP7 and FABP8 were identified. In contrast, positive immunoreactivities toward only FABP4 and FABP12 were detected in the cases of rat and mouse retinas, and interestingly, the FABP4 labeling patterns for the WT, DR and RP rat retinas were different. The ERG amplitudes of Fabp4 -/- mice were enhanced compared with those of WT mice. The concentrations of V-FABP4, V-VEGFA and total FAs were significantly higher in RVD patients than in non-PDR patients (P < 0.05). The V-FAs levels of each were significantly and positively correlated with V-FABP4 and V-VEGFA, although no significant correlation between vitreous (V-) and plasma (P-) FABP4, VEGFA and FAs were detected. The current study reveals that V-FAs appear to have significant roles in both retinal physiology as well as the pathogenesis of RVD with FABP4, which is commonly expressed within the retina in most species., (Copyright © 2022. Published by Elsevier Ltd.)

The additive effects of an α2-adrenergic agonist, brimonidine (BRI), on the pan-ROCK inhibitor (ROCK-i), ripasudil (Rip), and the ROCK2-I, KD025, on adipogenic differentiation (DIF+) were examined using two- or three-dimension (2D or 3D) cultures of 3T3-L1 cells. The following analyses were carried out: (1) lipid staining (2D and 3D), (2) real-time measurements of cellular metabolism (2D), (3) mRNA expression of DIF+ related genes and extracellular matrix molecules (ECMs) including collagen (Col)-1, -4, and -6, and fibronectin (Fn), and (4) the sizes and physical properties of the 3D spheroids. The findings indicate that DIF+ induced (1) a substantial enhancement in lipid staining and enhanced expression of the Pparγ and Fabp4 genes, (2) significantly larger and softer 3D spheroids, and (3) down-regulation of Col1 and Fn and up-regulation of Col4 and Col6 genes. Treatment with Rip alone caused a significant enhancement in adipogenesis of both the 2D and 3D cultured 3T3-L1 cells and in the physical properties of the 3D spheroids; these effects were substantially inhibited by BRI, and the effects induced by BRI or KD025 were not insignificant. These collective findings indicate that the addition of BRI inhibited the Rip-induced enhancement of DIF+ in 3T3-L1 cells, presumably by modulating ROCK1 signaling.

We report herein on the effects of brimonidine (BRI), an α2-adrenergic agonist, on two-dimensional (2D) and three-dimensional (3D) cell-cultured TGF-β2-untreated and -treated human trabecular meshwork (HTM) cells. In the presence of TGF-β2 (5 ng/mL), (1) the effects of BRI on (1) the 2D HTM monolayers' barrier function were investigated as estimated using trans-endothelial electrical resistance (TEER) measurement and FITC dextran permeability; (2) real-time analyses of cellular metabolism using a Seahorse Bioanalyzer; (3) the largeness and hardness of 3D spheroids; and (4) the expression of genes that encode extracellular matrix (ECM) proteins, including collagens (COL) 1, 4, and 6; fibronectin (FN) and α-smooth muscle actin (α-SMA); ECM modulators, including a tissue inhibitor of matrix proteinase (TIMP) 1-4; matrix metalloproteinase (MMP) 2, 9, and 14; and several endoplasmic reticulum (ER) stress-related genes, including the X-box-binding protein 1 (XBP1), the spliced XBP1 (sXBP1), glucose-regulated protein (GRP)78, GRP94, and CCAAT-enhancer-binding protein homologous protein (CHOP). BRI markedly inhibited the TGF-β2-induced increase in the values of TEER of the 2D cell monolayer and the hardness of the 3D spheroids, although it had no effect on their sizes. BRI also cancelled the TGF-β2-induced reduction in mitochondrial maximal respiration but had no effect on the glycolytic capacity. In addition, the gene expression of these molecules was quite different between the 2D and 3D cultures of HTM cells. The present observations found in this study indicate that BRI may beneficially affect TGF-β2-induced changes in both cultures, 2D and 3D, of HTM cells, although their structural and functional properties that were altered varied significantly between both cultures of HTM cells.

To characterize our recently established in vitro glaucomatous human trabecular meshwork (HTM) models using dexamethasone (DEX)- or TGF-β2-treated HTM cells, (1) two-dimensional (2D) cultured HTM cells were characterized by means of the real-time cellular metabolism analysis using a Seahorse analyzer, and (2) the effects of mechanical compression stresses toward the three-dimensional (3D) HTM spheroids were evaluated by analyzing the gene expression of several ECM proteins, inflammatory cytokines, and ER stress-related factors of those 3D HTM spheroid models. The results indicated that (1) the real-time cellular metabolism analysis indicated that TGF-β2 significantly induced an energy shift from mitochondrial oxidative phosphorylation (OXPHOS) into glycolysis, and DEX induced similar but lesser effects. In contrast, ROCK2 inhibition by KD025 caused a substantial reverse energy shift from glycolysis into OXPHOS. (2) Upon direct compression stresses toward the untreated control 3D HTM spheroids, a bimodal fluctuation of the mRNA expressions of ECM proteins was observed for 60 min, that is, initial significant upregulation (0-10 min) and subsequent downregulation (10-30 min) followed by another upregulation (30-60 min); those of inflammatory cytokines and ER stress-related factors were also bimodally changed. However, such compression stresses for 30 min toward TGF-β2- or DEX-treated 3D HTM spheroids induced downregulation of most of those of inflammatory cytokines and ER stress-related factors in addition to upregulation of COL1 and downregulation of FN. The findings presented herein indicate that (1) OXPHOS of the HTM cells was decreased or increased by TGF-β2 or DEX stimulation or ROCK2 inhibition, and (2) mechanical compression stresses toward 3D HTM spheroids may replicate acute, subacute, and chronic HTM models affected by elevated intraocular pressures.

The hypoxia associated with the transforming growth factor-β2 (TGF-β2)-induced epithelial mesenchymal transition (EMT) of human retinal pigment epithelium (HRPE) cells is well recognized as the essential underlying mechanism responsible for the development of proliferative retinal diseases. In vitro, three-dimensional (3D) models associated with spontaneous O 2 gradients can be used to recapitulate the pathological levels of hypoxia to study the effect of hypoxia on the TGF-β2-induced EMT of HRPE cells in detail, we used two-dimensional-(2D) and 3D-cultured HRPE cells. TGF-β2 and hypoxia significantly and synergistically increased the barrier function of the 2D HRPE monolayers, as evidenced by TEER measurements, the downsizing and stiffening of the 3D HRPE spheroids and the mRNA expression of most of the ECM proteins. A real-time metabolic analysis indicated that TGF-β2 caused a decrease in the maximal capacity of mitochondrial oxidative phosphorylation in the 2D HRPE cells, whereas, in the case of 3D HRPE spheroids, TGF-β2 increased proton leakage. The findings reported herein indicate that the TGF-β2-induced EMT of both the 2D and 3D cultured HRPE cells were greatly modified by hypoxia, but during these EMT processes, the metabolic plasticity was different between 2D and 3D HRPE cells, suggesting that the mechanisms responsible for the EMT of the HRPE cells may be variable during their spatial spreading.

The objective of the current study was to examine the roles of ROCK1 and 2 on the spatial architecture of human corneal stroma. We examined the effects of a pan-ROCK inhibitor (pan-ROCK-i), ripasudil, and a ROCK2 inhibitor (ROCK2-i), KD025 on the expression of genes that encode for ECM proteins including collagen (COL) 1, 4, 6, and fibronectin (FN), their regulators, a tissue inhibitor of metalloproteinase (TIMP) 1-4, matrix metalloproteinase (MMP) 2, 9 and 14, and ER stress-related factors of two- and three-dimensional (2D and 3D) cultures of human corneal stroma fibroblasts (HCSFs), and the physical properties of 3D HCSF spheroids. A gene expression analysis using ROCK-is indicated that KD025 (ROCK2 selective ROCK inhibitor) induced more significant changes than Rip (ripasudil, pan-ROCK inhibitor), suggesting that ROCK2 might be more extensively involved in the metabolism of ECM proteins and cell architectures of the 2D cultured HCSFs than ROCK1. In terms of the physical properties, size and stiffness of the 3D HCSFs spheroids, Rip caused a significant enlargement and this enhancement was concentration-dependent while KD025 also exerted a similar but less pronounced effect. In contrast, Rip and KD025 modulated physical stiffness differently, in that Rip caused a substantial decrease and KD025 caused an increase. Such diverse effects between Rip and KD025 were also observed for the gene expressions of ECM proteins, their regulators, and ER-stress related factors. The findings presented herein suggest that the ROCK1 and 2 influence the spatial architecture of 3D HCFS spheroids in different manners., (© 2022. The Author(s).)

This study reports on the pathological significance of the vitreous fatty acid-binding protein (Vt-FABP) 4 and 5, and vascular endothelial growth factor A (Vt-VEGFA) in patients with retinal vascular diseases (RVDs) including proliferative diabetic retinopathy (PDR) and retinal vein occlusion (RVO). Subjects with PDR (n = 20), RVO (n = 10), and controls (epiretinal membrane, n = 18) who had undergone vitrectomies were enrolled in this study. The levels of Vt-FABP4, Vt-FABP5, and Vt-VEGFA were evaluated by enzyme-linked immunosorbent assays (ELISA). Retinal circulation levels were measured by a laser-speckle flow analyzer (LSFA) and other relevant data were collected. The Vt-FABP5 levels were significantly (p < 0.05) elevated in patients with RVDs compared to control patients. This elevation was more evident in patients with RVO than with PDR. Log Vt-FABP5 was significantly correlated negatively or positively with all the LSFA retinal circulation indexes or Log triglycerides (r = 0.31, p = 0.031), respectively. However, the elevations in the Vt-FABP4 and Vt-VEGFA levels were more evident in the PDR group (p < 0.05) and these factors were correlated positively with Log fasting glucose and negatively with some of the LSFA retinal circulation indexes. Multivariable regression analyses indicated that the LSFA blood flows of the optic disc at baseline was an independent effector with Log Vt-FABP5 other than several possible factors including age, gender, Log triglycerides, Log Vt-FABP4 and Log Vt-VEGFA. These current findings suggest that Vt-FABP5 is involved in the pathogenesis of RVD in a manner that is different from that for Vt-FABP4 and Vt-VEGFA, presumably by regulating retinal circulation.

To compare the drug-induced efficacies between omidenepag (OMD), an EP2 agonist, and prostaglandin F2α (PGF2α) on glaucomatous trabecular meshwork (TM) cells, two- and three-dimensional (2D and 3D) cultures of TGF-β2-modulated human trabecular meshwork (HTM) cells were used. The following analyses were performed: (1) transendothelial electrical resistance (TEER) and FITC-dextran permeability measurements (2D), (2) the size and stiffness of the 3D spheroids, and (3) the expression (both 2D and 3D) by several extracellular matrix (ECM) molecules including collagen (COL) 1, 4 and 6, and fibronectin (FN), and α smooth muscle actin (αSMA), tight junction (TJ)-related molecules, claudin11 (Cldn11) and ZO1, the tissue inhibitor of metalloproteinase (TIMP) 1-4, matrix metalloproteinase (MMP) 2, 9 and 14, connective tissue growth factor (CTGF), and several endoplasmic reticulum (ER) stress-related factors. TGF-β2 significantly increased the TEER values and decreased FITC-dextran permeability, respectively, in the 2D HTM monolayers, and induced the formation of downsized and stiffer 3D HTM spheroids. TGF-β2-induced changes in TEER levels and FITC-dextran permeability were remarkably inhibited by PGF2α. PGF2α induced increases in the sizes and stiffness of the TGF-β2-treated 3D spheroids, but OMD enhanced only spheroid size. Upon exposure to TGF-β2, the expression of most of the molecules that were evaluated were significantly up-regulated, except some of ER stress-related factors were down-regulated. TJ-related molecules or ER stress-related factors were significantly up-regulated (2D) or down-regulated (3D), and down-regulated (2D) by PGF2α and OMD, while both drugs altered the expression of some of the other genes in the 3D spheroids in a different manner. The findings presented herein suggest that PGF2α and OMD differently modulate the permeability of the TGFβ2-modulated 2D monolayers and the physical properties of the 3D HTM spheroids.

To elucidate the currently unknown mechanisms responsible for the diverse biological aspects between two-dimensional (2D) and three-dimensional (3D) cultured 3T3-L1 preadipocytes, RNA-sequencing analyses were performed. During a 7-day culture period, 2D- and 3D-cultured 3T3-L1 cells were subjected to lipid staining by BODIPY, qPCR for adipogenesis related genes, including peroxisome proliferator-activated receptor γ ( Pparγ ), CCAAT/enhancer-binding protein alpha ( Cebpa ), Ap2 ( fatty acid-binding protein 4; Fabp4 ), leptin , and AdipoQ ( adiponectin ), and RNA-sequencing analysis. Differentially expressed genes (DEGs) were detected by next-generation RNA sequencing (RNA-seq) and validated by a quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Bioinformatic analyses were performed on DEGs using a Gene Ontology (GO) enrichment analysis and an Ingenuity Pathway Analysis (IPA). Significant spontaneous adipogenesis was observed in 3D 3T3-L1 spheroids, but not in 2D-cultured cells. The mRNA expression of Pparγ , Cebpa, and Ap2 among the five genes tested were significantly higher in 3D spheroids than in 2D-cultured cells, thus providing support for this conclusion. RNA analysis demonstrated that a total of 826 upregulated and 725 downregulated genes were identified as DEGs. GO enrichment analysis and IPA found 50 possible upstream regulators, and among these, 6 regulators-transforming growth factor β1 (TGFβ1), signal transducer and activator of transcription 3 (STAT3), interleukin 6 (IL6), angiotensinogen (AGT), FOS, and MYC-were, in fact, significantly upregulated. Further analyses of these regulators by causal networks of the top 14 predicted diseases and functions networks (IPA network score indicated more than 30), suggesting that STAT3 was the most critical upstream regulator. The findings presented herein suggest that STAT3 has a critical role in regulating the unique biological properties of 3D spheroids that are produced from 3T3-L1 preadipocytes.

The objective of the current study was to examine the drug-induced effects of the EP2 agonist, omidenapag (OMD), on human corneal stroma, two- and three-dimensional (2D and 3D) cultures of human corneal stroma fibroblasts (HCSFs). The drug-induced effects on 2D monolayers and 3D spheroids were characterized by examining the ultrastructures by scanning electron microscope (SEM), transendothelial electrical resistance (TEER) measurements, and fluorescein isothiocyanate (FITC)-dextran permeability. The physical properties of 3D spheroids with respect to size and stiffness were also examined. In addition, the gene expressions of extracellular matrix (ECM) molecules, including collagen (COL) 1, 4, and 6, and fibronectin (FN), a tissue inhibitor of metalloproteinase (TIMP) 1-4, matrix metalloproteinase (MMP) 2, 9, and 14, aquaporin1 (AQP1), and several endoplasmic reticulum (ER) stress-related factors were evaluated. In the 2D HCSFs, OMD induced (1) a significant increase in ECM deposits, as evidenced by SEM, the mRNA expression of COL4 and FN, and (2) a decrease in TEER values and a concentration-dependent increase in FITC-dextran permeability. In the case of 3D spheroids, OMD had no effect on size but a substantial increase in stiffness was observed. Furthermore, such OMD-induced effects on stiffness were dramatically modulated by the osmotic pressure of the system. In contrast to the above 2D cultures, among the ECM molecules and the modulators of 3D spheroids, namely, TIMPS and MMPs, the down-regulation of COL1, TIMP1 and 2 and the up-regulation of MMP9 were observed. Interestingly, such diversity in terms of OMD-induced gene expressions between 2D and 3D cultures was also recognized in AQP1 (2D; no significant change, 3D; significant up-regulation) and ER stress-related genes. The findings presented herein suggest that the EP2 agonist, OMD, alters the physical stiffness of 3D spheroids obtained from human corneal stroma fibroblasts and this alteration is dependent on the osmotic pressures. 2D and 3D cell cultures may be useful for evaluating the drug induced effects of OMD toward human corneal stroma., (© 2021 Federation of American Societies for Experimental Biology.)

To study the additive effects of Rho-associated coiled-coil containing protein kinase inhibitors, ripasudil (Rip) to bimatoprost acid (BIM-A) on orbital adipose tissue, three-dimensional (3D) cultures of human orbital fibroblasts (HOFs) were prepared and the physical properties including the 3D spheroid size and stiffness, lipid staining by BODIPY and the mRNA expression of adipogenesis-related genes, PPARγ and AP2, and extracellular matrix (ECM) including collagen (COL)1, 4 and 6, and fibronectin (FN) were analyzed. Adipogenesis (DIF+) induced (1) enlargement and increasing stiffness of the 3D HOFs spheroid, (2) increased lipid staining, the expression of adipogenesis-related gene expressions, and (3) the down-regulation of COL1 and FN and up-regulation of COL4 and COL6. In the presence of BIM-A, (1) such DIF+-induced changes in 3D spheroid size and stiffness were significantly inhibited or enhanced, respectively, (2) the lipid staining and its related gene expressions were significantly down-regulated, and (3) the expression of COL1 and COL6 were up-regulated. By the addition of Rip to BIM-A, the above BIM-A-induced effects were all inhibited, except for the up-regulation of COL6 and FN expression, that is, enlarging and decreasing stiffness, enhancement of lipid staining and its related gene expression, and down-regulation of COL1 expression. Our present study indicates that Rip significantly suppressed BIM-A-induced effects toward 3D HOFs spheroids., (© 2021. Japan Human Cell Society.)

To elucidate the effects of switching a PGF2α agonist, bimatoprost acid (BIM-A), to an EP2 agonist (Omidenepag-OMD; butaprost-Buta) or reversing the switching on adipose tissue, two-dimensional (2D) and three-dimensional (3D) cultures of 3T3-L1 cells were analyzed by lipid staining and according to the mRNA expression of adipogenesis-related genes ( Pparγ, Ap2 , and Leptin ), components of the extracellular matrix (ECM; collagen1 ( Col1 ), Col4 , Col6 , and fibronectin ( Fn )), and the sizes and stiffness of the 3D spheroids. Switching from BIM-A to EP2 agonists caused (1) suppression of lipid staining and downregulation of most adipogenesis-related genes, (2) smaller and stiffer 3D spheroids, and (3) upregulation of Col1 and Fn , downregulation of Col4 (2D), or up-regulation of all ECM genes (3D, BIM-A to OMD), as well as downregulation of Col6 (3D, BIM-A to Buta). In contrast, reversing the switching resulted in (1) an enhancement in lipid staining (2D) and a significant upregulation of adipogenesis-related genes (2D, 3D Buta to BIM-A), (2) larger and slightly stiffer 3D spheroids, and (3) upregulation of Col1 and Fn (2D). These collective findings indicate that the switching orders of BIM-A and EP2 agonists have a significant effect on lipid metabolism, ECM expression, and the physical stiffness of 3T3-L1 cells.

Background: To obtain new insights into the activation of the thyroid-stimulating hormone (TSH) and insulin-like growth factor 1 (IGF-1) receptors in human orbital fibroblasts (n-HOFs), the effects of the prostanoid EP2 agonist, omidenepag (OMD), and a rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, ripasudil (Rip) were evaluated using three-dimension (3D) n-HOFs spheroids in the absence and presence of the recombinant human TSH receptor antibodies, M22 and IGF-1., Methods: The effects of 100 nM OMD or 10 μM Rip on the physical properties, size, stiffness, and mRNA expression of several extracellular matrix (ECM) molecules, their regulator, inflammatory cytokines, and endoplasmic reticulum (ER) stress-related factors were examined and compared among 3D spheroids of n-HOFs, M22-/IGF-1-activated n-HOFs and GO-related human orbital fibroblasts (GHOFs)., Results: The physical properties and mRNA expressions of several genes of the 3D n-HOFs spheroids were significantly and diversely modulated by the presence of OMD or Rip. The OMD-induced effects on M22-/IGF-1-activated n-HOFs were similar to the effects caused by GHOHs, but quite different from those of n-HOFs., Conclusions: The findings presented herein indicate that the changes induced by OMD may be useful in distinguishing between n-HOFs and GHOFs.

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.

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.

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.

To 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., (© 2021. The Author(s).)

The additive effects of prostaglandin (PG)-EP2 agonists on a PG-FP agonist toward adipogenesis in two- or three-dimension (2D or 3D) cultures of 3T3-L1 cells was examined by lipid staining, the mRNA expression of adipogenesis related genes, and extracellular matrixes (ECMs) including collagen molecules (Col) -1, -4 and -6, and fibronectin (Fn), and the sizes and physical properties of 3D sphenoids, as measured by a micro-squeezer. The results indicate that adipogenesis induced 1) an enlargement in the sizes of 3D sphenoids, 2) a substantial enhancement in lipid staining, the expression of the PParγ, Ap2 and Leptin genes, and 3) a significant decrease in the stiffness of the 3D sphenoids. These effects were inhibited by bimatoprost acid (BIM-A), but 4) adipogenesis induced significant down-regulation of Col1 and Fn, and the significant up-regulation of the Col4 and Col6 genes were unchanged by BIM-A. On the addition of an EP2 agonist, such as omidenepag (OMD) or butaprost (Buta), to BIM-A, 1) the sizes of the 3D sphenoids were further decreased, 2) lipid staining was decreased (2D; OMD, 3D; Buta) 3) the stiffness of the 3D sphenoids was increased by Buta, 4) the expression of PParγ was up-regulated (2D; Buta) or unchanged (3D), the expression of Ap2 was down-regulated (2D; OMD) or up-regulated (3D; Buta), and the expression of Leptin was increased (2D), 5) the expression of all four (OMD) or all except Col4 (buta) in 2D, and Col1and Col4 (OMD) in 3D were up-regulated. These collective findings indicate that the addition of an EP2 agonist, OMD or Buta significantly modulated the BIM-A induced suppression of adipogenesis as well as physical properties of 2D and 3D cultured 3T3-L1 cells in different manners., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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), α SMA (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), α SMA (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.

A 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., (© 2021. The Author(s).)

To elucidate the pharmacological effects of Rho-associated coiled-coil containing protein kinase inhibitors (ROCK-is), ripasudil (Rip), Y27632, and KD025, on human orbital fatty tissue, the human orbital fibroblasts (HOFs) were three-dimensional (3D) cultured for 12 days. The effects of ROCK-is on the physical properties of the 3D-cultured HOF spheroids, including their sizes and physical stiffness, their adipogenesis by lipid staining, and the mRNA expression of adipogenesis-related genes, PPARγ and AP2 , and extracellular matrix (ECM) including collagen (COL) 1, 4, and 6, and fibronectin were analyzed. A significant increase in the sizes, physical stiffness, lipid staining, and mRNA expression of adipogenesis-related genes, COL4 and COL6 , and a decrease in COL1 expression were observed with adipogenesis (DIF+). In the presence of ROCK-is, such DIF+-induced effects were differently modulated as follows: (1) the sizes were not affected or significantly enhanced by Rip, Y27632, or KD025, (2) the physical stiffness was significantly decreased in Rip and Y27632, but was substantially increased in KD025, (3) the lipid staining was further enhanced or significantly suppressed by Rip, Y27632, or KD025, and both PPARγ and AP2 expression were significantly downregulated or upregulated by KD025 or Rip, and (4) Rip upregulated the expression of COL4 , Y27632 upregulated the expression of COL1 , COL4 , and COL6 , and KD025 upregulated the expression of COL1 and COL4 . This study indicates that ROCK-is significantly and differently modulate physical properties of the 3D HOF spheroids as well as their adipogenesis., (© 2021 The Authors. FASEB BioAdvances published by Wiley Periodicals LLC on behalf of The Federation of American Societies for Experimental Biology.)

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.

The fatty acid-binding protein4 (FABP4) and vascular endothelial growth factor A (VEGFA) play key roles in the metabolic and cardiovascular diseases, and proliferative diabetic retinopathy (PDR), respectively. To identify FABP4 in vitreous fluid in PDR, vitreous concentrations of FABP4 (V-FABP4) and VEGFA (V-VEGFA) from PDR (n = 20) and non-PDR (n = 20) patients were determined by Enzyme-Linked ImmunoSorbent Assays. The data, which included height and weight, systemic blood pressures, several blood biochemical parameters and blood flow at the optic nerve head (ONH) by laser speckle flowgraphy (LSFG) were collected. The levels of V-FABP4 and V-VEGFA were significantly higher in PDR patients than in non-PDR patients (P < 0.001) with a high positive correlation (r = 0.72, P < 0.001) between them. The findings were not affected by body mass index values and the presence of vitreous hemorrhaging. Among the clinical parameters, V-FABP4 correlated positively with creatinine and negatively with age and aspartate transaminase (AST) levels, while V-VEGFA correlated positively with fasting plasma glucose and hemoglobin A1c (HbA1c) levels but negatively with AST. Multiple regression analyses indicated that V-VEGFA, or V-FABP4, AST and HbA1c were independent predictors of V-FABP4 or V-VEGFA, respectively. Both were negatively correlated, but more evident in V-FABP4, with the ONH ocular blood flow.

Purpose: To evaluate Nuclear Factor NF-κB (NF-κB) signaling on microglia activation, migration, and angiogenesis in laser-induced choroidal neovascularization (CNV)., Methods: Nine-week-old C57BL/6 male mice were randomly assigned to IMD-0354 treated or untreated groups (5 mice, 10 eyes per group). CNV was induced with a 532-nm laser. Laser spots (power 250 mW, spot size 100 μm, time of exposure 50 ms) were created in each eye using a slit-lamp delivery system. Selective inhibitor of nuclear factor kappa-B kinase subunit beta (IKK2) inhibitor IMD-0354 (10 μg) was delivered subconjunctivally; vehicle-treated mice were the control. The treatment effect on CNV development was assessed at five days post-CNV induction in vivo in C57BL/6 and Cx3cr1 gfp/wt mice by fluorescent angiography, fundus imaging, and ex vivo by retinal flatmounts immunostaining and Western blot analysis of RPE/Choroidal/Scleral complexes (RCSC) lysates. In vitro evaluations of IMD-0354 effects were performed in the BV-2 microglial cell line using lipopolysaccharide (LPS) stimulation., Results: IMD-0354 caused a significant reduction in the fluorescein leakage and size of the laser spot, as well as a reduction in microglial cell migration and suppression of phospho-IκBα, Vascular endothelial growth factor (VEGF-A), and Prostaglandin-endoperoxide synthase 2 (COX-2). In vivo and ex vivo observations demonstrated reduced lesion size in mice, CD68, and Allograft inflammatory factor 1 (IBA-1) positive microglia cells migration to the laser injury site in IMD-0354 treated eyes. The data further corroborate with GFP-positive cells infiltration of the CNV site in Cx3cr1 wt/gfp mice. In vitro IMD-0354 (10-25 ng/ml) treatment reduced NF-κB activation, expression of COX-2, caused decreased Actin-F presence and organization, resulting in reduced BV-2 cells migration capacity., Conclusion: The present data indicate that NF-κB activation in microglia and it's migration capacity is involved in the development of laser CNV in mice. Its suppression by NF-κB inhibition might be a promising therapeutic strategy for wet AMD., (Copyright © 2021 Elsevier Inc. All rights reserved.)

To elucidate the additive effects of an EP2 agonist, omidenepag (OMD) or butaprost (Buta) on the Rho-associated coiled-coil-containing protein kinase (ROCK) inhibitor, ripasudil (Rip) on adipose tissue, two- or three-dimension (2D or 3D) cultures of 3T3-L1 cells were analyzed by lipid staining, the mRNA expression of adipogenesis-related genes, extracellular matrix (ECM) molecules including collagen (Col) -1, -4 and -6, and fibronectin (Fn), and the sizes and physical properties of 3D organoids, as measured by a micro-squeezer. The results indicate that adipogenesis induced (1) an enlargement of the 3D organoids; (2) a substantial enhancement in lipid staining as well as the expression of the Ppar γ, Ap2 and Leptin genes; (3) a significant softening of the 3D organoids, the effects of which were all enhanced by Rip except for Ppar γ expression; and (4) a significant downregulation in Col1 and Fn , and a significant upregulation in Col4 , Col6 , the effects of which were unchanged by Rip. When adding the EP2 agonist to Rip, (1) the sizes of the 3D organoids were reduced substantially; (2) lipid staining was increased (OMD), or decreased (Buta); (3) the stiffness of the 3D organoids was substantially increased in Buta; (4-1) the expression of Ppar γ was suppressed (2D, OMD) or increased (2D, Buta), and the expressions of Ap2 were downregulated (2D, 3D) and Leptin was increased (2D) or decreased (3D), (4-2) all the expressions of four ECM molecules were upregulated in 2D (2D), and in 3D, the expression of Col1 , Col4 was upregulated. The collective findings reported herein indicate that the addition of an EP2 agonist, OMD or Buta significantly but differently modulate the Rip-induced effects on adipogenesis and the physical properties of 2D and 3D cultured 3T3-L1 cells.

Previous our study reported that three-dimension (3D) cultures of human orbital fibroblasts (HOFs) replicated the etiology of deepening of the upper eyelid sulcus (DUES) caused by prostaglandin F2α analogues (PGF2α-ags). To examine this further, the effects of PGF2α-ags on HOFs were characterized by (1) lipid staining (2D; two-dimension, 3D), (2) comparison of the 3D organoid sizes of preadipocytes (DIF-) or adipocytes (DIF+) that had been treated with various concentrations of several PGF2α-ags, (3) physical stiffness (3D), and (4) the mRNA expression of adipogenic related genes, extracellular matrix (ECM), tissue inhibitors of metalloproteinases (TIMPs) and matrix metalloproteinases (MMPs) (3D). PGF2α-ags caused a dramatic down-sizing of the 3D DIF+ organoids and this reduction was concentration dependent. The effects caused by PGF2α-ags were also observed in 3D preadipocytes. Micro-squeezer analysis clearly indicated that PGF2α-ags induced an increase in their physical solidity. The size of each organoid under several conditions was inversely correlated with the mRNA expression profile of collagen1 (COL1), TIMP2, and MMP2 and 9. These findings indicate that PGF2α-ags affect the expression of COL1, TIMP2, and MMP2 and 9 which, in turn, modulate the 3D ECM network within the organoids, thus resulting in their downsizing.

3D organoid cultures were used to elucidate the periocular effects of several anti-glaucoma drugs including a prostaglandin F2α analogue (bimatoprost acid; BIM-A), EP2 agonist (omidenepag; OMD) or a Rho-associated coiled-coil containing protein kinase (ROCK) inhibitor (ripasudil; Rip) on Grave's orbitopathy (GO) related orbital fatty tissue. 3D organoids were prepared from GO related human orbital fibroblasts (GHOFs) obtained from patients with GO. The effects of either 100 nM BIM-A, 100 nM OMD or 10 μM Rip on the 3D GHOFs organoids were examined with respect to organoid size, physical properties by a micro-squeezer, and the mRNA expression of extracellular matrix (ECM) proteins including collagen (COL) 1, COL 4, COL 6, and fibronectin (FN), ECM regulatory genes including lysyl oxidase (LOX), Connective Tissue Growth Factor (CTGF) and inflammatory cytokines including interleukin-1β (IL1β) and interleukin-6 (IL6). The size of the 3D GHOFs organoids decreased substantially in the presence of BIM-A, but also increased substantially in the presence of the others (OMD or Rip). The physical stiffness of the 3D GHOFs organoids was significantly decreased by Rip. BIM-A caused significantly the down-regulation of three ECM genes, Col 1, Col 6 and Fn, and two ECM regulatory genes and the up-regulation of IL6. In the presence of OMD, two ECM genes, Col 1 and Fn, and LOX were significantly down-regulated but IL1β and IL6 were significantly up-regulated. In the case of Rip, Col 1, FN and CTGF were significant down-regulated. Our present findings indicate that anti-glaucoma drugs modulate the structures and physical properties 3D GHOFs organoids in different manners by modifying the gene expressions of ECM, ECM regulatory factors and inflammatory cytokines. The results indicate that the benefits and demerits of anti-glaucoma medications need to be scrutinized carefully, in cases of patients with GO., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Purpose: The purpose of this study was to present the effects of the prostanoid EP2 agonist, omidenepag (OMD) on human orbital fibroblasts (HOFs) using a three-dimension (3D) cell culture., Methods: During adipogenesis of 3D HOFs organoids, changes in size, lipids staining, mRNA expression of adipogenesis related genes, PPARγ, AP2, and ADIPOQ, and extracellular matrix, collagen 1 (COL 1), COL 4, COL 6, and fibronectin (FN), and stiffness by a micro-squeezer were examined in the presence and absence of either 100 nM bimatoprost acid (BIM-A) or 10, 100, or 10,000 nM OMD., Results: The size of the 3D organoids increased dramatically during adipogenesis, and these were further enhanced in the presence of OMD in contrast to the BIM-A induced suppression effect. The intensity of lipid staining and the mRNA expression of PPARγ were significantly increased upon adipogenesis, and both or latter was markedly inhibited in the presence of OMD or BIM-A, respectively. AP2 expression was also upregulated by adipogenesis, and was further enhanced by BIM-A. The adipogenesis-induced downregulation of COL 1 and FN, or the upregulation of the expression of COL 4 and COL 6 were all suppressed in the presence of BIM-A. In contrast, OMD caused similar effects on COL 4, COL 6, or FN expression, but caused a significant increase in COL 1 expression. Stiffness was significantly increased upon adipogenesis, and was further increased or substantially decreased by BIM-A or OMD, respectively., Conclusions: The present study indicates that the FP2 agonist, OMD, had different effects on 3D HOFs organoids, as compared to BIM-A., Translational Relevance: The current study suggests that OMD may not induce deepening of upper eyelid sulcus (DUES).

Since the recent discovery of prostaglandin-associated peri-orbitopathy, a great deal of interest has developed concerning the side effects of anti-glaucoma medications toward periocular fatty tissue, especially their adipogenesis. Two- or three-dimension (2D or 3D) cultures of the 3T3-L1 cells were employed to elucidate the effects of the Rho-associated coiled-coil containing protein kinase inhibitor (ROCK-i) the anti-glaucoma drug, Ripasudil, and other ROCK-i, such as Y27632 on adipogenesis. Ultrastructure by electron microscopy and physical stiffness measurements by a micro-squeezer demonstrated the 3D organoids had essentially matured during the 7-day culture. The effects of ROCK-i on 3D organoid sizes, lipid staining, the mRNA expression of adipogenesis related genes, Pparγ, Cebpa and Leptin, and extracellular matrix (ECM) including collagen (COL) 1, 4 and 6, and fibronectin, and physical stiffness were then conducted. Upon adipogenesis, the sizes, lipid staining and mRNA expressions of adipogenesis related genes, Col 4 and Col 6 were dramatically increased, and were further enhanced by ROCK-i. Micro-squeezer analysis demonstrated that adipogenesis resulted in a marked less stiffed 3D organoid and this was further enhanced by ROCK-i. Our present study indicates that ROCK-i significantly enhanced the production of large lipid-enriched 3T3-L1 3D organoids.

The main objective of current study was to identify the fatty acid-binding protein 4 (FABP4) expressed in both adipocytes and macrophages in vitreous fluid from patients with retinal vein occlusion (RVO). Patients with RVO (n = 14, CRVO; central RVO n = 5, BRVO; branch RVO n = 9) and non-RVO (macular hole or epiretinal membrane, n = 18) were surgically treated by a 25 or 27G vitrectomy. Undiluted vitreous fluid samples obtained as the result of surgery were subjected to enzyme-linked immunosorbent assays to measure the levels of FABP4 and vascular endothelial growth factor A (VEGFA).Data including ocular blood flow by laser speckle flow graphy (LSFG), height and weight, systemic blood pressures and several blood biochemistry values were collected. Among the LSFG mean blur rate (MBR) values of the optic nerve head (ONH) at baseline, MA (MBR of all area), MV (MBR of the vascular area), and MV-MT (MBR of the tissue area) were significantly decreased in patients with CRVO. The levels of V-FABP4 and V-VEGFA were relatively or significantly (P< 0.05) higher in the BRVO or CRVO patients compared to the non-RVO patients, respectively. A positive correlation (r = 0.36, P = 0.045) or a negative correlation (r = -0.51, P = 0.006) was observed between Log V-FABP4 and Log V-VEGF, or Log V-FABP4 and MV-MT at post-operative 1-week, respectively. Furthermore, neither of these factors were affected with respect to sex, body mass index and several clinical parameters that were collected, except that a positive correlation was observed for Log V-FABP4 with blood urea nitrogen. Stepwise multivariable regression analyses indicated that MV-MT at post-operative 1week was independently associated with Log V-FABP4 after adjustment for age and gender, and gender and Log V-FABP4 were independently associated with Log V-VEGFA after adjustment for age. The findings reported herein suggest that an independent factor, FABP4 may be synergistically involved in the pathogenesis of RVO with VEGFA., Competing Interests: The authors have declared that no competing interests exist.

To 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.

2D and 3D cultures of 3T3-L1 cells were employed in a study of the effects of Omidenepag (OMD), interacting with a non-prostanoid EP2 receptor, on adipogenesis. Upon adipogenesis, the effects on lipid staining, the mRNA expression of adipogenesis-related genes (Pparγ, CEBPa, Ap2, and Glut4) and the extracellular matrix (ECM) including collagen type 1, 4 and 6, and fibronectin, and the size and physical property of 3D organoids were compared between groups that had been treated with EP2 agonists (butaprost and OMD) and PGF2α. Upon adipogenesis, these significantly suppressed lipid staining and the mRNA expression of related genes. EP2 agonists and PGF2α influenced the mRNA expression of ECM in different manners, and these effects were also different between 2 and 3D cultures. Examining the physical properties by a microsqueezer indicated that the solidity of the 3D organoids became significantly lowered upon adipogenesis and these effects were not affected by EP2 agonists. In contrast, 3D organoid stiffness was markedly enhanced by the presence of PGF2α. These observations indicate that EP2 agonists affect the adipogenesis of 3T3-L1 cells in different manners, as compared to PGF2α, suggesting that OMD may not induce PGF2α related orbital fat atrophy, called the deepening of the upper eyelid sulcus (DUES).

Purpose: To elucidate the molecular etiology of deepening of the upper eyelid sulcus (DUES) induced by prostaglandin (PG) analogs, a three-dimensional (3D) tissue culture system was employed using human orbital fibroblasts (HOFs)., Methods: During adipogenesis, changes in HOF 3D organoid sizes, as well as their lipids stained by BODIPY and expression of the extracellular matrix (ECM) by immunolabeling and/or quantitative PCR, were studied in the presence or absence of either 100-nM bimatoprost acid or 100-nM prostaglandin F2α., Results: The size of the 3D organoids increased remarkably during adipogenesis, but such increases were significantly inhibited by the presence of PG analogs. Staining intensities by BODIPY and mRNA expression of peroxisome proliferator-activated receptor gamma were significantly increased upon adipogenesis but were not influenced by the presence of PG analogs. Unique changes in ECM expression observed with or without adipogenic differentiation were significantly modified by the presence of PG analogs., Conclusions: Our present study indicates that PG analogs have the potential to modulate the ECM network within HOF 3D organoids. Thus, a 3D tissue culture system may be a suitable strategy for understanding the disease etiology of DUES.

To establish a deepening of the upper eyelid sulcus (DUES) model that can be induced by prostaglandin (PG) analogues, a three-dimension (3D) tissue culture was employed. Upon adipogenesis of the 3T3-L1 organoid, the effects of either Bimatoprost acid (BIM-A), or PGF2α were examined. During the adipogenesis, organoid size, lipid staining by BODIPY and expression of the extracellular matrix (ECM) by immunocytochemistry and/or quantitative PCR were employed. The size of the organoid increased remarkably during the adipogenesis, while such increases were significantly inhibited by the presence of PGF2α or BIM-A. BODIPY positive lipid-laden cells significantly increased during the adipogenesis, while in contrast they were greatly suppressed by the presence of PGF2α. Characteristic and spatial changes in ECM expressions observed upon adipogenesis were greatly modified by the presence of PGs. Our present study using a 3D tissue culture may be a suitable strategy toward understanding disease etiology of DUES.

Thyroid-associated orbitopathy (TAO) is a disfiguring periocular connective tissue disease associated with autoimmune thyroid disorders. It is a potentially blinding condition, for which no effective pharmacological treatment has been established. Despite a suggested role played by autoimmune thyrotropin receptor activation in the pathogenesis of TAO, the cellular and molecular events contributing to the fibrotic and inflammatory disease process of TAO are not fully defined. By developing a three-dimensional organoid culture of human orbital fibroblasts (OFs), we sought to determine the molecular mechanism underlying the fibrotic disease process of TAO. In this ex vivo model, we have demonstrated that hypoxia-inducible factor (HIF) 2α (HIF2A), but not its paralog HIF1A, accelerates extracellular matrix (ECM) deposition by inducing a collagen-cross-linking enzyme, lysyl oxidase (LOX). Inhibiting HIF2A and LOX with short hairpin RNA or small molecular antagonists effectively ameliorated fibrotic disease process within TAO organoids. Conversely, the overexpression of a constitutively active HIF2A in mouse OFs was sufficient to initiate LOX-dependent fibrotic tissue remodeling in OF organoids. Consistent with these findings, HIF2A and LOX were highly expressed in human TAO tissues paralleling excess ECM deposition. We propose that the HIF2A-LOX pathway can be a potential therapeutic target for the prevention and treatment of TAO.