mTOR Inhibitors Modulate the Biological Nature of TGF-β2-Treated or -Untreated Human Trabecular Meshwork Cells in Different Manners
<b>Background/Objectives:</b> 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, transfor...
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2024-11-01
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| author | Megumi Watanabe Tatsuya Sato Toshiyuki Yano Megumi Higashide Toshifumi Ogawa Nami Nishikiori Masato Furuhashi Hiroshi Ohguro |
| author_facet | Megumi Watanabe Tatsuya Sato Toshiyuki Yano Megumi Higashide Toshifumi Ogawa Nami Nishikiori Masato Furuhashi Hiroshi Ohguro |
| author_sort | Megumi Watanabe |
| collection | DOAJ |
| description | <b>Background/Objectives:</b> 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. <b>Methods:</b> 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. <b>Results:</b> 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. <b>Conclusions:</b> 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. |
| format | Article |
| id | doaj-art-7d6afdd7daa54d8bb6dc856c439e73b5 |
| institution | Kabale University |
| issn | 2227-9059 |
| language | English |
| publishDate | 2024-11-01 |
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| spelling | doaj-art-7d6afdd7daa54d8bb6dc856c439e73b52024-11-26T17:53:23ZengMDPI AGBiomedicines2227-90592024-11-011211260410.3390/biomedicines12112604mTOR Inhibitors Modulate the Biological Nature of TGF-β2-Treated or -Untreated Human Trabecular Meshwork Cells in Different MannersMegumi Watanabe0Tatsuya Sato1Toshiyuki Yano2Megumi Higashide3Toshifumi Ogawa4Nami Nishikiori5Masato Furuhashi6Hiroshi Ohguro7Departments of Ophthalmology, School of Medicine, Sapporo Medical University, S1W17, Sapporo 060-8556, JapanDepartments of Cardiovascular, Renal and Metabolic Medicine, Sapporo Medical University, S1W17, Sapporo 060-8556, JapanDepartments of Cardiovascular, Renal and Metabolic Medicine, Sapporo Medical University, S1W17, Sapporo 060-8556, JapanDepartments of Ophthalmology, School of Medicine, Sapporo Medical University, S1W17, Sapporo 060-8556, JapanDepartments of Cardiovascular, Renal and Metabolic Medicine, Sapporo Medical University, S1W17, Sapporo 060-8556, JapanDepartments of Ophthalmology, School of Medicine, Sapporo Medical University, S1W17, Sapporo 060-8556, JapanDepartments of Cardiovascular, Renal and Metabolic Medicine, Sapporo Medical University, S1W17, Sapporo 060-8556, JapanDepartments of Ophthalmology, School of Medicine, Sapporo Medical University, S1W17, Sapporo 060-8556, Japan<b>Background/Objectives:</b> 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. <b>Methods:</b> 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. <b>Results:</b> 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. <b>Conclusions:</b> 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.https://www.mdpi.com/2227-9059/12/11/2604TGF-β2human trabecular meshwork3D culturerapamycinmTORautophagy |
| spellingShingle | Megumi Watanabe Tatsuya Sato Toshiyuki Yano Megumi Higashide Toshifumi Ogawa Nami Nishikiori Masato Furuhashi Hiroshi Ohguro mTOR Inhibitors Modulate the Biological Nature of TGF-β2-Treated or -Untreated Human Trabecular Meshwork Cells in Different Manners Biomedicines TGF-β2 human trabecular meshwork 3D culture rapamycin mTOR autophagy |
| title | mTOR Inhibitors Modulate the Biological Nature of TGF-β2-Treated or -Untreated Human Trabecular Meshwork Cells in Different Manners |
| title_full | mTOR Inhibitors Modulate the Biological Nature of TGF-β2-Treated or -Untreated Human Trabecular Meshwork Cells in Different Manners |
| title_fullStr | mTOR Inhibitors Modulate the Biological Nature of TGF-β2-Treated or -Untreated Human Trabecular Meshwork Cells in Different Manners |
| title_full_unstemmed | mTOR Inhibitors Modulate the Biological Nature of TGF-β2-Treated or -Untreated Human Trabecular Meshwork Cells in Different Manners |
| title_short | mTOR Inhibitors Modulate the Biological Nature of TGF-β2-Treated or -Untreated Human Trabecular Meshwork Cells in Different Manners |
| title_sort | mtor inhibitors modulate the biological nature of tgf β2 treated or untreated human trabecular meshwork cells in different manners |
| topic | TGF-β2 human trabecular meshwork 3D culture rapamycin mTOR autophagy |
| url | https://www.mdpi.com/2227-9059/12/11/2604 |
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