Papers - FURUHASHI Masato

Machine learning-based analyses of contributing factors for the development of hypertension: a comparative study.

Marenao Tanaka, Yukinori Akiyama, Kazuma Mori, Itaru Hosaka, Keisuke Endo, Toshifumi Ogawa, Tatsuya Sato, Toru Suzuki, Toshiyuki Yano, Hirofumi Ohnishi, Nagisa Hanawa, Masato Furuhashi

Clinical and experimental hypertension (New York, N.Y. : 1993)   47 ( 1 ) 2449613 - 2449613  2025.12  [International journal]

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OBJECTIVES: Sufficient attention has not been given to machine learning (ML) models using longitudinal data for investigating important predictors of new onset of hypertension. We investigated the predictive ability of several ML models for the development of hypertension. METHODS: A total of 15 965 Japanese participants (men/women: 9,466/6,499, mean age: 45 years) who received annual health examinations were randomly divided into a training group (70%, n = 11,175) and a test group (30%, n = 4,790). The predictive abilities of 58 candidates including fatty liver index (FLI), which is calculated by using body mass index, waist circumference and levels of γ-glutamyl transferase and triglycerides, were investigated by statistics analogous to the area under the curve (AUC) in receiver operating characteristic curve analyses using ML models including logistic regression, random forest, naïve Bayes, extreme gradient boosting and artificial neural network. RESULTS: During a 10-year period (mean period: 6.1 years), 2,132 subjects (19.1%) in the training group and 917 subjects (19.1%) in the test group had new onset of hypertension. Among the 58 parameters, systolic blood pressure, age and FLI were identified as important candidates by random forest feature selection with 10-fold cross-validation. The AUCs of ML models were 0.765-0.825, and discriminatory capacity was significantly improved in the artificial neural network model compared to that in the logistic regression model. CONCLUSIONS: The development of hypertension can be simply and accurately predicted by each ML model using systolic blood pressure, age and FLI as selected features. By building multiple ML models, more practical prediction might be possible.

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Can small dense LDL cholesterol be estimated from the lipid profile?

Tatsuya Sato, Marenao Tanaka, Masato Furuhashi

Current opinion in lipidology    2025.04  [International journal]

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PURPOSE OF REVIEW: Small dense low-density lipoprotein cholesterol (sdLDL-C) is recognized for its strong atherosclerogenic potential. However, its direct measurement remains impractical in clinical settings due to its high cost, time constraints, and labor-intensive nature. This review discusses the benefits and limitations of estimating sdLDL-C using conventional lipid fractions, highlighting recent advancements in estimation methods. RECENT FINDINGS: Sampson et al. proposed a novel equation for estimating sdLDL-C based on conventional lipid parameters, offering a more accessible alternative to direct measurement. Recent studies, including ours, demonstrated that this estimation method achieves sufficiently high accuracy for overall application. However, its accuracy can be improved by incorporating machine learning. Furthermore, sdLDL-C estimated by Sampson's equation has been shown to be a superior risk marker for hypertension, an intermediate phenotype of atherosclerosis, and ischemic heart disease, a major cardiovascular event, compared to conventional lipid profiles alone, although further research is needed to determine whether estimated sdLDL-C is equivalent to directly measured sdLDL-C in risk assessment. SUMMARY: Estimated sdLDL-C presents a promising alternative to direct measurement. While estimated sdLDL-C levels can serve a risk marker for cardiovascular diseases, further research is needed to refine estimation models and explore their integration into clinical practice.

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Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) Is an Independent Risk Factor for the Development of Ischemic Heart Disease ― A 10-Year Cohort Study ―

Toshifumi Ogawa, Tatsuya Sato, Marenao Tanaka, Yukinori Akiyama, Kei Nakata, Hidemichi Kouzu, Kazuma Mori, Hiroki Aida, Wataru Kawaharata, Itaru Hosaka, Toru Suzuki, Nagisa Hanawa, Masato Furuhashi

Circulation Reports ( Japanese Circulation Society )   2025.04

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High Level of Estimated Small Dense Low-Density Lipoprotein Cholesterol as an Independent Risk Factor for the Development of Ischemic Heart Disease Regardless of Low-Density Lipoprotein Cholesterol Level ― A 10-Year Cohort Study ―

Keisuke Endo, Marenao Tanaka, Tatsuya Sato, Masafumi Inyaku, Kei Nakata, Wataru Kawaharata, Hiroki Aida, Itaru Hosaka, Yukinori Akiyama, Nagisa Hanawa, Masato Furuhashi

Circulation Journal ( Japanese Circulation Society )   2025.03

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Elevated urinary fatty acid-binding protein 4 level predicts future renal dysfunction and poor prognosis in Japanese patients with diabetes: a longitudinal cohort study

Marenao Tanaka, Tatsuya Sato, Tomohito Gohda, Nozomu Kamei, Maki Murakoshi, Erika Ishiwata, Kei Nakata, Yukinori Akiyama, Keisuke Endo, Wataru Kawaharata, Hiroki Aida, Toru Suzuki, Mitsunobu Kubota, Michiyoshi Sanuki, Yusuke Suzuki, Masato Furuhashi

Clinical Kidney Journal ( Oxford University Press (OUP) )   2025.03

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Abstract Background Fatty acid-binding protein 4 (FABP4) is an adipokine secreted from adipocytes and macrophages and is also expressed in injured, but not normal, glomerular endothelial cells. Elevated levels of urinary FABP4 (U-FABP4) have been reported to be associated with glomerular damage and increased proteinuria. Methods The associations of levels of U-FABP4 at baseline with future events including renal dysfunction defined by a 30% decline in estimated glomerular filtration rate (eGFR) and all-cause death were investigated in 660 patients with diabetes (type 1/2: 57/603). Results During a follow-up period (median: 62 months), 90 patients (13.6%) developed renal dysfunction, and 66 patients (10.0%) died (median follow-up period: 65 months). Kaplan-Meier survival curves showed that there were significant differences in cumulative incidences for a 30% decline in eGFR and all-cause death in patients divided by the tertiles of U-FABP4 level. Furthermore, multivariable Cox proportional hazard models with a restricted cubic spline showed that hazard ratios for a 30% decline in eGFR and all-cause death increased with a higher level of logarithmically transformed (Log) U-FABP4 after adjustment for age, sex, type of diabetes, body mass index, current smoking habit, duration of diabetes, comorbidities of hypertension and dyslipidemia, eGFR and the categorical classification of urinary albumin-creatinine ratio. The addition of Log U-FABP4 to traditional risk factors significantly increased the discriminatory capacities for renal dysfunction in net reclassification improvement (NRI) and integrated discrimination improvement and for all-cause death in NRI. Conclusion U-FABP4 is a predictive biomarker for future renal dysfunction and poor prognosis in patients with diabetes.

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The Combination of PPARα Agonist GW7647 and Imeglimin Has Potent Effects on High-Glucose-Induced Cellular Biological Responses in Human Retinal Pigment Epithelium Cells.

Nami Nishikiori, Megumi Watanabe, Megumi Higashide, Araya Umetsu, Toshifumi Ogawa, Masato Furuhashi, Hiroshi Ohguro, Tatsuya Sato

Bioengineering (Basel, Switzerland)   12 ( 3 )  2025.03  [International journal]

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BACKGROUND: Hyperglycemic changes in the cellular biological properties of retinal pigment epithelium cells are involved in the pathophysiology of diabetic retinopathy (DR). To assess the effects of the new anti-diabetic agent imeglimin (Ime) on DR, the pharmacological effects of Ime and those of metformin (Met) in combination with the PPARα agonist GW7646 (GW) on adult retinal pigment epithelium (ARPE19) cells cultured in high-glucose conditions were compared. METHODS: Cell viability, levels of reactive oxygen species (ROS), monolayer barrier function measured by transepit very much helial electrical resistance (TEER), and metabolic functions determined by an extracellular flux analyzer were evaluated. RESULTS: While glucose concentrations did not alter cell viability regardless of the presence of Met or Ime, levels of ROS were significantly increased by the high-glucose conditions, and increased levels of ROS were significantly alleviated by the combination of Ime and GW but not by Met alone. Similarly, TEER values were increased by high-glucose conditions, but the effects of high-glucose conditions were dramatically enhanced by the combination of Ime and GW. Furthermore, a metabolic assay showed that an energetic shift was induced by the combination of Ime and GW, whereas energy status became quiescent with Met or Ime alone. CONCLUSIONS: The collective results suggest that Ime in combination with GW has synergetic effects on high-glucose-induced cellular biological changes in ARPE19 cells.

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Construction of Predictive Models for Cardiovascular Mortality by Machine Learning Approaches in Patients Who Underwent Transcatheter Aortic Valve Implantation

Shunsaku Otomo, Itaru Hosaka, Marenao Tanaka, Naoto Murakami, Nobuaki Kokubu, Atsuko Muranaka, Ryo Nishikawa, Naoki Hachiro, Ryota Kawamura, Jun Nakata, Nobutaka Nagano, Yukinori Akiyama, Tatsuya Sato, Yutaka Iba, Toshiyuki Yano, Nobuyoshi Kawaharada, Masato Furuhashi

Circulation Reports ( Japanese Circulation Society )   2025.03

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Expression and Secretion of Intraocular Fatty Acid-Binding Protein 4 (ioFABP4) and 5 (ioFABP5) Are Regulated by Glucose Levels and Fatty Acids.

Hiroshi Ohguro, Megumi Higashide, Erika Ishiwata, Fumihito Hikage, Megumi Watanabe, Nami Nishikiori, Tatsuya Sato, Masato Furuhashi

International journal of molecular sciences   26 ( 5 )  2025.02  [International journal]

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Intraocularly, fatty acid-binding protein 4 (FABP4) and 5 (FABP5) mainly originate from human ocular choroidal fibroblasts (HOCF), and human nonpigmented ciliary epithelium (HNPCE) cells have been suggested to be pivotally involved in intraocular pathophysiology. To elucidate the unidentified regulatory mechanisms of the gene expression and protein secretion of FABPs, the effects of glucose levels, fatty acids (FAs), and peroxisome proliferator-activated receptor (PPAR) modulators were studied. To elucidate the additional biological role of FABPs, laser choroidal neovascularization (CNV) in Fabp4-/- and Fabp4/5-/- mice was analyzed by fluorescein angiography. By changing glucose levels, the secretion and expression of FABP4 in HOCF were significantly upregulated, whereas the secretion and expression of FABP5 in HNPCE decreased. The administration of various FAs, particularly docosahexaenoic acid (DHA), markedly increased the expression and secretion of both FABPs. PPAR modulators also influenced the secretion and expression of FABPs. In vivo, wild-type retina exhibited evident CNV with high fluorescein intensity, while Fabp4-/- retina showed reduced CNV formation and Fabp4/5-/- retina displayed evident CNV along with vitreous leakage. These findings suggest that (1) the production and secretion of intraocular FABP4 and FABP5 are distinctly regulated by glucose levels, FAs, and PPARs; and (2) intraocular FABP4 and FABP5 are critical for inducing retinal neovascularization and maintaining the blood-aqueous barrier.

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Unraveling Novel Subsets of Lymphocytes Involved in Sac Expansion in the Tertiary Lymphoid Structure Within an Abdominal Aortic Aneurysm.

Itaru Hosaka, Ippei Ikegami, Takuma Mikami, Tatsuya Sato, Toshifumi Ogawa, Kei Mukawa, Marenao Tanaka, Keisuke Endo, Yukinori Akiyama, Akihito Ohkawa, Junji Nakazawa, Tsuyoshi Shibata, Tomohiro Nakajima, Yutaka Iba, Chikara Shiiku, Satoshi Sumino, Ryuji Koshima, Kenichi Takano, Shingo Ichimiya, Nobuyoshi Kawaharada, Masato Furuhashi

Journal of the American Heart Association     e040279  2025.02  [International journal]

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BACKGROUND: Chronic inflammation is involved in the development of abdominal aortic aneurysm (AAA). A tertiary lymphoid structure (TLS) within vascular lesions has recently been focused on for its role in modulation of inflammation in local tissues. We aimed to elucidate the relationships between TLS and pathophysiology of AAA. METHODS: Abdominal aortic samples obtained from 37 patients with AAA (men/women: 34/3, age: 72.8±9.9 years) and 15 autopsied patients who died from non-aortic events (men/women: 11/4, age: 65.5±9.8 years) were investigated. RESULTS: TLSs in AAA lesions were confirmed by focal infiltration of CD3-positive cells surrounding germinal center-like structures containing CD20-positive cells between the tunica adventitia and tunica media layers. The formation of a TLS was significantly more prevalent in AAA patients than in autopsied patients. The number of TLSs in AAA lesions was positively correlated with sac diameter (r=0.357, P=0.035) and the amount of intraluminal thrombosis (r=0.466, P=0.005). T cells and B cells were predominant cellular populations among CD45+ cells in AAA lesions. There was a significantly positive correlation between the proportions of interfollicular T follicular helper (CD3+CD4+CD45RA-CXCR5+PD-1+) cells and double negative B (CD3-CD19+IgD-CD27-) cells, and they were positively correlated with sac diameter, intraluminal thrombosis, and serum lipids. Deposited single-cell RNA-sequencing data for AAA showed that T follicular helper cells and double negative B cells were associated with lipid metabolism, T cell activation/proliferation and inflammation. CONCLUSIONS: The formation of a TLS in AAA lesions is associated with sac diameter and intraluminal thrombosis in connection with interfollicular T follicular helper cells and double negative B cells, which may contribute to the pathophysiology of AAA and might be novel therapeutic targets for the development of AAA.

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High-Glucose-Induced Metabolic and Redox Alterations Are Distinctly Modulated by Various Antidiabetic Agents and Interventions Against FABP5/7, MITF and ANGPTL4 in Melanoma A375 Cells

Nami Nishikiori, Hiroshi Ohguro, Megumi Watanabe, Megumi Higashide, Toshifumi Ogawa, Masato Furuhashi, Tatsuya Sato

International Journal of Molecular Sciences ( MDPI AG )  26 ( 3 ) 1014 - 1014  2025.01

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Hyperglycemia-induced effects on cellular metabolic properties and reactive oxygen species (ROS) generation play pivotal roles in the pathogenesis of malignant melanoma (MM). This study assessed how metabolic states, ROS production, and related gene expression are modulated by antidiabetic agents. The anti-diabetic agents metformin (Met) and imeglimin (Ime), inhibitors of fatty acid-binding proteins 5/7 (MF6) and microphthalmia-associated transcription factor (MITF) (ML329), and siRNA-mediated knockdown of angiopoietin-like protein 4 (ANGPTL4), which affect mitochondrial respiration, ROS production, and related gene expression, were tested in A375 (MM cell line) cells cultured in low (5.5 mM) and high glucose (50 mM) conditions. Cellular metabolic functions were significantly and differently modulated by Met, Ime, MF6, or ML329 and knockdown of ANGPTL4. High glucose significantly enhanced ROS production, which was alleviated by Ime but not by Met. Both MF6 and ML329 reduced ROS levels under both low and high glucose conditions. Knockdown of ANGPTL4 enhanced the change in glucose-dependent ROS production. Gene expression related to mitochondrial respiration and the pathogenesis of MM was significantly modulated by different glucose conditions, antidiabetic agents, MF6, and ML329. These findings suggest that glucose-dependent changes in cellular metabolism and redox status are differently modulated by antidiabetic agents, inhibition of fatty acid-binding proteins or MITF, and ANGPTL4 knockdown in A375 cells.

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Glomerular diameter is associated with a reduction in urinary protein by treatment with dapagliflozin in patients with chronic kidney disease

Arata Osanami, Hiroaki Komatsu, Yufu Gocho, Keitaro Nishizawa, Marenao Tanaka, Yuichi Nakamura, Masato Furuhashi

Clinical and Experimental Nephrology ( Springer Science and Business Media LLC )   2025.01

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

Fumihito Hikage, Megumi Suzuki, Tatsuya Sato, Araya Umetsu, Toshifumi Ogawa, Nami Nishikiori, Masato Furuhashi, Hiroshi Ohguro, Megumi Watanabe

Scientific reports   15 ( 1 ) 384 - 384  2025.01  [International journal]

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To elucidate the role of IGF1R inhibition in the pathogenesis of Graves' orbitopathy (GO), the effects of linsitinib (Lins) on a recombinant human TSHR antibody (M22) and IGF1 to activate TSHR and IGF1R of human orbital fibroblasts (HOFs) obtained from patients without GO (HOFs) and patients with GO (GHOFs) were studied using in vitro three-dimensional (3D) spheroid models in addition to their 2D planar cell culture. For this purpose, we evaluated 1) cellular metabolic functions by using a seahorse bioanalyzer (2D), 2) physical properties including size and stiffness of 3D spheroids, and mRNA expression of several extracellular matrix (ECM) proteins, their modulators (CCL2 LOX, CTGF, MMPs), ACTA2 and inflammatory cytokines (IL1β, IL6). Administration of IGF1 and M22 induced increases of cellular metabolic functions with the effect on HOFs being much more potent than the effect on GHOFs, suggesting that IGF1R and TSHR of GHOFs may already be stimulated. Lins had effects similar to those of IGF1/M22 on cellular biological functions of HOFs but not on those of GHOFs. As for physical properties of 3D GHOFs spheroids, stiffness but not size was significantly increased by IGF1 and/or M22. In contrast, Lins significantly inhibited the M22-induced increase in stiffness despite the fact that Lins alone had no effect. The mRNA expression levels of several genes of ECM proteins and most of the other genes also fluctuated similarly to the changes in stiffness of 3D spheroids despite the fact that Lins induced up-regulation of inflammatory cytokines and MMP3. The findings presented herein indicate that IGF1R inhibition by Lins may beneficially affect GO-related fibrogenesis.

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Prognostic implication of sarcopenia diagnosed by updated Asian Working Group for Sarcopenia criteria in older patients with heart failure: Utility and limitation

Satoshi Katano, Kotaro Yamano, Toshiyuki Yano, Ryo Numazawa, Ryohei Nagaoka, Suguru Honma, Yusuke Fujisawa, Yasuhiro Miki, Yuhei Takamura, Hayato Kunihara, Hiroya Fujisaki, Hidemichi Kouzu, Katsuhiko Ohori, Masaki Katayose, Akiyoshi Hashimoto, Masato Furuhashi

The Journal of nutrition, health and aging ( Elsevier BV )  29 ( 1 ) 100434 - 100434  2025.01

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The serum γ-Glutamyltransferase Level is Associated with the Development of Hypertension in Alcohol Infrequent Drinkers but not in Frequent Drinkers

Kazuma Mori, Marenao Tanaka, Tatsuya Sato, Yukinori Akiyama, Hirofumi Ohnishi, Nagisa Hanawa, Masato Furuhashi

Internal Medicine ( Japanese Society of Internal Medicine )   2025

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Urinary fatty acid‐binding protein 4 is a promising biomarker for glomerular damage in patients with diabetes mellitus

Marenao Tanaka, Tatsuya Sato, Tomohito Gohda, Nozomu Kamei, Maki Murakoshi, Erika Ishiwata, Keisuke Endo, Wataru Kawaharata, Hiroki Aida, Kei Nakata, Yukinori Akiyama, Mitsunobu Kubota, Michiyoshi Sanuki, Toru Suzuki, Yusuke Suzuki, Masato Furuhashi

Journal of Diabetes Investigation ( Wiley )   2024.12

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ABSTRACT Aims/Introduction Fatty acid‐binding protein (FABP) 4, which acts as an adipokine secreted by adipocytes, macrophages, and capillary endothelial cells, is expressed in injured glomerular cells. It has been reported that urinary (U‐) FABP4 is associated with renal dysfunction and proteinuria in several glomerular kidney diseases. However, the clinical significance of U‐FABP4 in diabetic kidney disease (DKD) remains undetermined. Materials and Methods Immunohistological analyses of FABP4 and FABP1 (liver‐type FABP), an established biomarker for impaired proximal tubules, were performed in the kidneys of patients with DKD and nonobese diabetic mice (KK‐Ta/Akita mice). The associations between U‐FABP4 and U‐FABP1 with kidney function and metabolic indices were also investigated in patients with type 1 diabetes (n = 57, mean age: 61 years) and patients with type 2 diabetes (n = 608, mean age: 65 years). Results In both patients with diabetes and diabetic mice, FABP4 was expressed in injured glomeruli with increased markers of endoplasmic reticulum stress in addition to peritubular capillaries, whereas FABP1 was mainly expressed in proximal tubules. Levels of U‐FABP4 and U‐FABP1 were independently associated with each other, and both levels were independently associated with estimated glomerular filtration rate (eGFR) and urinary albumin‐to‐creatinine ratio (UACR) after adjustment of age, sex, type of diabetes, duration of diabetes, and systolic blood pressure in patients with diabetes. Conclusions Urinary level of FABP4 derived from injured glomeruli with increased endoplasmic reticulum stress is independently associated with eGFR and UACR, suggesting a promising biomarker for glomerular damage in patients with diabetes.

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A silent interplay between elevated intraocular pressure, glaucoma, and hypertension.

Tatsuya Sato, Araya Umetsu, Marenao Tanaka, Hiroshi Ohguro, Masato Furuhashi

Hypertension research : official journal of the Japanese Society of Hypertension    2024.12  [International journal]

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セマグルチド注射薬により20年間のインスリン強化療法から離脱できた2型糖尿病の1例

寺沢 誠, 中田 圭, 大久保 武志, 小川 俊史, 隅田 健太郎, 佐藤 達也, 神津 英至, 矢野 俊之, 古橋 眞人

糖尿病 ( (一社)日本糖尿病学会 )  67 ( 12 ) 511 - 511  2024.12

腹腔鏡下胃スリーブ状切除術によりインスリン抵抗性-インスリン必要量増加-体重増加の悪循環を脱し、肥満関連合併症の改善が得られた1例

小川 俊史, 佐藤 達也, 大久保 武志, 寺沢 誠, 中田 圭, 隅田 健太郎, 神津 英至, 矢野 俊之, 矢島 諒人, 伊東 竜哉, 竹政 伊知朗, 古橋 眞人

日本内分泌学会雑誌 ( (一社)日本内分泌学会 )  100 ( 4 ) 805 - 805  2024.12

LINC02154 promotes cell cycle and mitochondrial function in oral squamous cell carcinoma.

Takeshi Niinuma, Hiroshi Kitajima, Tatsuya Sato, Toshifumi Ogawa, Kazuya Ishiguro, Masahiro Kai, Eiichiro Yamamoto, Yui Hatanaka, Iyori Nojima, Mutsumi Toyota, Akira Yorozu, Shohei Sekiguchi, Noritsugu Tohse, Masato Furuhashi, Hiroshi Ohguro, Akihiro Miyazaki, Hiromu Suzuki

Cancer science    2024.11  [International journal]

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Long noncoding RNAs (lncRNAs) play pivotal roles in the development of human malignancies, though their involvement in oral squamous cell carcinoma (OSCC) remains incompletely understood. Using The Cancer Genome Atlas (TCGA) dataset, we analyzed expression of 7840 lncRNAs in primary head and neck squamous cell carcinoma (HNSCC) and found that upregulation of LINC02154 is associated with a poorer prognosis. LINC02154 knockdown in OSCC cell lines induced cell cycle arrest and apoptosis, and significantly attenuated tumor growth in vitro and in vivo. Notably, depletion of LINC02154 downregulated FOXM1, a master regulator of cell cycle-related genes. RNA pulldown and mass spectrometry analyses identified a series of proteins that could potentially interact with LINC02154, including HNRNPK and LRPPRC. HNRNPK stabilizes FOXM1 expression by interacting with the 3'-UTR of FOXM1 mRNA, which suggests LINC02154 and HNRNPK promote cell cycling by regulating FOXM1 expression. Additionally, LINC02154 positively regulates HNRNPK expression by inhibiting microRNAs targeting HNRPNK. Moreover, LINC02154 affects mitochondrial function by interacting with LRPPRC. Depletion of LINC02154 suppressed expression of mitochondrial genes, including MTCO1 and MTCO2, and inhibited mitochondrial respiratory function in OSCC cells. These results suggest that LINC02154 exerts its oncogenic effects by modulating the cell cycle and oxidative phosphorylation in OSCC, highlighting LINC02154 as a potential therapeutic target.

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mTOR Inhibitors Modulate the Biological Nature of TGF-β2-Treated or -Untreated Human Trabecular Meshwork Cells in Different Manners.

Megumi Watanabe, Tatsuya Sato, Toshiyuki Yano, Megumi Higashide, Toshifumi Ogawa, Nami Nishikiori, Masato Furuhashi, Hiroshi Ohguro

Biomedicines   12 ( 11 )  2024.11  [International journal]

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Background/Objectives: Mammalian target of rapamycin (mTOR) inhibition may have been suggested to have a beneficial effect on the glaucomatous human trabecular meshwork (HTM). To study the effects of the mTOR inhibitors rapamycin (Rapa) and Torin1 on the glaucomatous HTM, transforming growth factor-β2 (TGF-β2)-treated two-dimensionally (2D) and three-dimensionally (3D) cultured HTM cells were used. Methods: We evaluated (1) the levels of autophagy via Western blot analysis using a specific antibody against microtubule-associated protein 1 light chain 3 (LC3), (2) barrier capacity based on transepithelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC) permeability (2D), (3) cellular metabolic functions (2D), (4) the size and stiffness of spheroids, and (5) the mRNA expression of ECM proteins. Results: TGF-β2-induced inhibition of autophagy was significantly inhibited by Rapa and Torin1. Rapa and Torin1 substantially decreased barrier capacity in both TGF-β2-untreated and TGF-β2-treated HTM cells. Cellular metabolic analysis indicated that Rapa, but not Torin1, substantially enhanced both mitochondrial and glycolytic functions of TGF-β2-untreated HTM cells. In the physical properties of spheroids, TGF-β2 resulted in the formation of down-sized and stiffened spheroids. mTOR inhibitors decreased the size but not the stiffness of TGF-β2-untreated spheroids and significantly reduced the TGF-β2-related increase in the stiffness but not the size of spheroids. The diverse effects of mTOR inhibitors on TGF-β2-untreated and TGF-β2-treated spheroids were also observed in the mRNA expression of extracellular matrix proteins. Conclusions: The results taken together suggest that mTOR inhibitors significantly influence the biological aspects of both a single layer and multiple layers of the TGF-β2-treated HTM and untreated HTM.

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