mTOR inhibitors potentially reduce TGF-β2-induced fibrogenic changes in trabecular meshwork cells

We examined the effects of mTOR inhibitors on the fibrotic response induced by transforming growth factor-beta2 (TGF-β2) in cultured human trabecular meshwork (hTM) cells. TGF-β2-induced expression of fibronectin, collagen type I, alpha 1 chain (COL1A1), and alpha-smooth muscle actin (αSMA) in hTM cells was examined in the presence or absence of mTOR inhibitors using quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry. The migration rates of hTM cells were examined in the presence of TGF-β2 with or without mTOR inhibitors. An in vitro study showed that the expression of fibronectin, COL1A1, and αSMA was upregulated by TGF-β2 treatment of hTM cells; such upregulation was significantly suppressed by mTOR inhibitors. The inhibitors significantly reduced the migration rate of TGF-β2-stimulated hTM cells. mTOR inhibitors may usefully reduce the fibrotic response of hTM cells and we may have to explore if it is also effective in in vivo model.


mTOR inhibitors potentially reduce TGF-β2-induced fibrogenic changes in trabecular meshwork cells Nozomi Igarashi, Megumi Honjo * & Makoto Aihara
We examined the effects of mTOR inhibitors on the fibrotic response induced by transforming growth factor-beta2 (TGF-β2) in cultured human trabecular meshwork (hTM) cells. TGF-β2-induced expression of fibronectin, collagen type I, alpha 1 chain (COL1A1), and alpha-smooth muscle actin (αSMA) in hTM cells was examined in the presence or absence of mTOR inhibitors using quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry. The migration rates of hTM cells were examined in the presence of TGF-β2 with or without mTOR inhibitors. An in vitro study showed that the expression of fibronectin, COL1A1, and αSMA was upregulated by TGF-β2 treatment of hTM cells; such upregulation was significantly suppressed by mTOR inhibitors. The inhibitors significantly reduced the migration rate of TGF-β2-stimulated hTM cells. mTOR inhibitors may usefully reduce the fibrotic response of hTM cells and we may have to explore if it is also effective in in vivo model.
Glaucoma is known to be the second leading cause of blindness worldwide. Aberrant increases in intraocular pressure (IOP) characterize glaucoma, and such an increase in IOP can damage the optic nerve [1][2][3] . IOP reduction is the only established effective therapy that suppresses visual impairment and blindness in both hypertensive and normotensive individuals. IOP elevation is reported to be principally the result of increased aqueous humor (AH) outflow resistance within the conventional outflow pathway 1,4,5 . Damage to the collector channels is implicated as a cause of resistance within the distal pathway in patients with advanced glaucoma 6 . In the conventional pathway, cellular responses is reported to be involved in the pathophysiology of glaucoma. Remodeling and excess accumulation of extracellular matrix (ECM) materials, adhesive interactions, regulation of the contractile properties of trabecular meshwork cells, and decreased permeability in stratum corneum have been associated with glaucomatous degeneration of outflow pathway tissues [7][8][9] .
Various aqueous soluble mediators (i.e. vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF), transforming growth factor-beta (TGF-β), monocyte chemotactic protein-1 (MCP-1), and members of the matrix metalloproteinase (MMP) family), are reported to have important roles in the ECM production and induction of fibrogenic changes in human trabecular meshwork (hTM) cells. The materials act in both autocrine and paracrine manners 7,10-12 .
Mammalian target of rapamycin (mTOR) is a downstream factor affected by TGF-β, and it triggers many bioactive changes, including fibrosis 13 . Anti-fibrotic effects of mTOR inhibitors have been extensively reported in patients with hepatic or pulmonary fibrosis and rheumatic diseases [14][15][16][17] ; however, to the best of our knowledge, no report has yet discussed the potential effects of mTOR inhibitors on hTM cells. The PI3K/mTOR pathway has been implicated in initiating fibroblast proliferation, survival and differentiation. The downstream cascades of PI3K is known to be regulated by the lipid phosphatase and tensin homolog (PTEN). In fibroblasts derived from lung or hepatic fibrosis patients' tissue, they are known to show aberrant PI3K activity [18][19][20][21] . And the issue if this cascade is upregulated in glaucomatous hTM needs further exploration. In normal human tissue, the β1 integrin binds polymerised collagen and activates PTEN activity, leading to the inhibition of their proliferation. As such, targeting mTOR in those diseases could be the novel treatment in such disease, and for the glaucoma patients, we speculated that inhibition of mTOR could be a novel treatment in glaucoma patients. TGF-β-mTOR pathway is implicated in fibrotic diseases. PI3K-mTOR pathway could be involved in fibrotic changes in glaucomatous lamina cribrosa cells. Given the role of TM's profibrotic changes in contributing to elevated intraocular pressure, this study investigated whether TGF-β2-PI3K-mTOR pathway is involved in hTM cell profibrosis' .
Here, we evaluated if mTOR inhibitors might attenuate hTM cells' fibrosis induced by TGF-β2 in vitro.

Results
Characterization of hTM cells. Primary hTM cells were isolated from human donor eyes, characterized as described previously by Keller et al. 22 Supplemental Fig. 1 shows the result of immunocytochemistry to characterize the hTM cells, and the cells were positive for AQP (Aquaporin)-1, Vimentin, TIMP (Tissue Inhibitor of Metalloproteinase)-3, COL4A1 (Collagen IV Alpha 1 Chain) and MGP (Matrix Gla Protein) but were negative for desmin. And in Supplementary Fig. 2, hTM cells treated with 100 nM and 500 nM Dexamethasone showed significant upregulation of myocilin mRNA with RT-qPCR (Supplemental Fig. 2A; P < 0.01 between control and 100 nM or 500 nM Dexamethasone treated groups, and P < 0.05 between 100 and 500 nM Dexamethasone treated groups), and also significant upregulation of myocilin was confirmed with WB when hTM cells were treated with 100 nM Dexamethasone (P < 0.01, Supplemental Fig. 2B,C).
Effects of TGF-β2 and the mTOR inhibitors on cell proliferation. First, we explored the potential toxicity of DMSO (0.00995% v/v), rapamycin (10 nM or 100 nM), and Torin 1 (10 nM or 100 nM) toward hTM cells using the MTT assay. Figure 1A shows that there was no significant difference among the groups, and also no significant difference between the control and DMSO-treated groups. As shown in Fig. 1B, the OD 570 value increased significantly when the cells were treated with TGF-β2 (10 ng/mL) compared to the control group (P < 0.001). The TGF-β2-induced upregulated OD 570 value was significantly attenuated in the presence of 100 nM rapamycin (P < 0.05) or 100 nM Torin 1 (P < 0.01).

The effects of TGF-β2 and mTOR inhibitors on the expression of mRNAs encoding alpha-smooth muscle actin (αSMA) and COL1A1 (Collagen I Alpha 1 Chain) in hTM cells. The mRNA levels in
hTM cells were assessed by qPCR (Fig. 1E, F). The mRNA expression levels of αSMA and COL1A1 were significantly higher after the addition of TGF-β2 than those of the control group (P < 0.001), but these rises were reduced by the mTOR inhibitors ( Fig. 1E; P < 0.01 for 100 nM Rapamycin and 10 nM Torin1, and P < 0.001 for 100 nM Torin1, Fig. 1F; P < 0.05 for 100 nM Rapamycin, P < 0.01 for 10 nM Torin1, P < 0.001 for 100 nM Torin1).

The effects of TGF-β2 and mTOR inhibitors on the fibrotic responses of hTM cells as assessed via immunohistochemistry and Western blotting. Immunocytochemistry and Western blotting were
used to assess the fibrotic responses to TGF-β2 and the effects of the mTOR inhibitors (rapamycin and Torin 1). Figure 2 shows the immunocytochemical data. The levels of COL1A1, fibronectin, phalloidin, and αSMA increased in hTM cells after TGF-β2 treatment, and these fibrotic responses were significantly suppressed by rapamycin or Torin 1. Figure 3A, B shows the Western blotting data (n = 3), which also indicate that the levels of fibronectin, COL1A1, and αSMA were upregulated in hTM cells after TGF-β2 treatment (P < 0.01 for Fibronectin, P < 0.05 for COL1A1 and αSMA), and that these fibrotic responses were significantly suppressed by Torin1 (P < 0.01 for Fibronectin, P < 0.05 for COL1A1 and αSMA). Also, the expression of fibronectin upregulated with TGF-β2 treatment was significantly attenuated with rapamycin ( Fig. 3A, B; P < 0.05).
Upregulation of mTOR and ROCK (Rho-Associated Kinase) 1 + ROCK2 induced by TGF-β2 and its attenuation with rapamycin or Torin1. Figure 3C, D shows the expression of mTOR, phosphorylated-mTOR, ROCK1 + ROCK2 when hTM cells were stimulated under TGF-β2 with or without rapamycin or Torin1. As shown in Fig. 3C, D, the expression of phosphotylated-mTOR and ROCK1 + ROCK2 was significantly upregulated with TGF-β2 stimulation (Fig. 3C, D; P < 0.05 for phosphorylated-mTOR and P < 0.01 for ROCK1 + ROCK2), and the change was attenuated with rapamycin or Torin1 ( Fig. 3D; P < 0.05 for both Rapamycin and Torin1 for phosphorylated-mTOR and, P < 0.05 for Rapamycin and P < 0.01 for Torin1 for ROCK).

Discussion
We explored whether mTOR inhibitors reduced ECM remodeling and TM cell fibrosis, both of which are thought to play roles in the pathogenesis of glaucoma. Many soluble mediators affect ECM remodeling and TM fibrosis; in particular, elevated levels of TGF-β2 play a critical role in glaucoma pathogenesis. Although the fibrogenic changes induced by TGF-β2 are thought to be crucial in terms of IOP elevation and downregulation of AH drainage, the detailed mechanism is not fully understood. Many efforts have been made to reduce fibrogenic changes, and ROCK inhibitors are currently reported as a new treatment to reduce ECM remodeling or TM fibrosis. And alongside with the ROCK inhibitors, novel treatments or medications are urgently needed for those who are refractive to the existing treatments. We focused on the PI3K-mTOR pathway, which is involved    n = 3). The various components were upregulated in hTM cells exposed to TGF-β2, and the fibrotic responses were significantly suppressed by the mTOR inhibitors rapamycin and Torin 1. One-way analysis of variance followed by the Tukey post-hoc test. *P < 0.05, **P < 0.01. (C) and (D) Western blotting for mTOR, phosphorylated mTOR and ROCK1 + ROCK2 in hTM cells. The representative bands for fibronectin, mTOR, phosphorylated-mTOR and ROCK1 + ROCK2 are shown in (C), and the relative expression of fibronectin, mTOR, phosphorylated-mTOR and ROCK1 + ROCK2 compared to the loading control (β-tubulin) are shown in (D) (n = 3). The various components were upregulated in hTM cells exposed to TGF-β2, and the fibrotic responses were significantly suppressed by the mTOR inhibitors rapamycin and Torin 1. One-way analysis of variance followed by the Tukey post-hoc test. *P < 0.05, **P < 0.01. . The baseline indicates the migration distance of cells not exposed to TGF-β2 or the mTOR inhibitors. TGF-β2 significantly accelerated migration to 24 h (P < 0.001); this was suppressed by mTOR inhibitors (P < 0.001). One-way analysis of variance followed by the Tukey post-hoc test. ***P < 0.001. www.nature.com/scientificreports/ in many biological processes, including cell proliferation and fibrosis, to explore if inhibition of the cascade of PI3K-mTOR pathway could aid glaucoma treatment. In our in vitro study, TGF-β2 stimulation upregulated the expression of phosphorylated-PI3K (Fig. 1C, D, P < 0.05), and the change was attenuated with TGF-β inhibitor (Fig. 1C, D, P < 0.05), supporting the involvement of the PI3K-mTOR pathway in glaucoma pathogenesis. PI3K-mTOR pathway dysfunction has been implicated in many fibrotic diseases, including cancer and pulmonary or cardiac fibrosis. Overexpression of components of the PI3K-mTOR pathway often dysregulate cell proliferation, apoptosis, inflammation, and autophagy, triggering fibrosis progression 23,24 . Recently, Irnated et al. 25 reported elevated levels of mRNA encoding PI3K-mTOR in glaucomatous lamina cribrosa cells. To the best of our knowledge, ours is the first study to report the involvement of the PI3K-mTOR pathway in the glaucomatous changes of hTM cells.

Scientific Reports
To explore if inhibition of the cascade of PI3K-mTOR pathway reduced the fibrogenic changes in hTM cells, we conducted additional in vitro studies to determine the effects of mTOR inhibitors on the fibrotic changes in hTM cells (Figs. 1E, F, 2, 3A, B). TGF-β2 significantly induced the expression of αSMA, COL1A1, and fibronectin by hTM cells, as revealed by qPCR (Fig. 1E, F, P < 0.001) and immunocytochemistry (Fig. 2) and Western blotting (Fig. 3A, B; P < 0.01 for Fibronectin, P < 0.05 for COL1A1 and αSMA). The mTOR inhibitors (rapamycin and Torin 1) significantly suppressed these fibrotic responses in qPCR ( Fig. 1E; P < 0.01 for 100 nM Rapamycin and 10 nM Torin1, and P < 0.001 for 100 nM Torin1, Fig. 1F; P < 0.05 for 100 nM Rapamycin, P < 0.01 for 10 nM Torin1, P < 0.001 for 100 nM Torin1), in immunocytochemistry (Fig. 2), and western blotting (Fig. 3A, B; Torin1 showed significance of P < 0.01 for Fibronectin, P < 0.05 for COL1A1 and αSMA and rapamycin showed significance in inhibiting the expression of fibronectin by P < 0.05), suggesting that inhibition of mTOR reduces hTM fibrosis, ECM remodeling, and the associated fibrogenic changes. There exists two enzyme complexes; mTOR complex 1 and 2 (mTORC1 and mTORC2), and Rapamycin is a mTORC1 inhibitor and mTORC2 is known to be insensitive to rapamycin. Torin1 is an ATP-competitive mTOR inhibitor which directly inhibits both mTORC1 and mTORC 15 . As such, we speculated that rapamycin didn't show enough effect on attenuation of TGF-β2 induced fibrogenic changes compared to Torin1.
In our in vitro study, TGF-β2 stimulation upregulated the expression of phosphorylated-mTOR and ROCK1 + ROCK2 in parallel with the expression of αSMA (Fig. 3C, D; P < 0.05 for phosphorylated-mTOR and P < 0.01 for ROCK1 + ROCK2), and the change was also attenuated with rapamycin or Torin1 ( Fig. 3D; P < 0.05 for both Rapamycin and Torin1 for phosphorylated-mTOR and, P < 0.05 for Rapamycin and P < 0.01 for Torin1 for ROCK).
Also, as shown in Fig. 4, TGF-β2 significantly accelerated cell migration to 24 h (P < 0.001); such migration was significantly attenuated by the mTOR inhibitors (P < 0.001). The mechanisms regulating AH outflow and IOP are heavily dependent on physiological changes in the cytoskeleton and ECM surrounding hTM cells. In glaucoma patients, impaired cell shape, actin cytoskeleton, and cell adhesive interactions, are observed in TM cells 7 . And in in vivo rat model, such impairment observed in TM cells it induced an increase in TM contractile activity, a decrease in AH outflow facility, and elevated the IOP and fibrogenic activity of TM 7 . As such, aberrant migration or proliferation of the hTM cells may contribute to hTM cell dysfunction and cause upregulation of IOP. Referring to those reports in the past 7,11,12 , inhibition of cell migration or proliferation could benefit in glaucoma treatment.
Our work has several limitations. First, we produced only in vitro data; in vivo studies to assess the effectiveness of mTOR inhibitors in a glaucoma model are required. Second, mTOR inhibitors should be tested in clinical trials. Third, this is an in vitro study to modify the pathogenesis of POAG by stimulating normal hTM cells with TGF-β2, further in vitro studies using glaucomatous hTM cells without using any glaucomatous stimuli like TGF-β2.
When treating glaucoma patients, the attainment of a sustained reduced IOP is the only known method, but some patients are refractive to existing glaucoma eyedrops. In addition to further exploration of the pathogenesis of glaucoma, new glaucoma eyedrops which can modulate those proven mechanisms are required. It has been suggested that glaucomatous fibrotic hTM changes are associated with a number of factors that drive fibrosis, including TGF-β2, as explored in the present study. The various factors may affect hTM cells via a multitude of downstream signaling pathways, including the PKCα, MAPK, Rho-Rho kinase, JNK, and (possibly) PI-3K/ Akt-mTOR pathways. An understanding of the roles played by these pathways, and their crosstalk, might slow or prevent excessive pro-fibrotic ECM gene induction and the resulting fibrosis that is characteristic of glaucoma. We found that mTOR targeting downregulated the fibrogenic cascade; modulation of mTOR activity might inhibit excessive fibrogenic changes in hTM cells and could be a novel treatment in reducing elevated IOP.

Materials and methods
Cell culture and passage. The hTM cells from three donor eyes (46 years old, 52 years old, and 55 years old, without glaucoma) were used in this study, and hTM cells were cultured in growth medium containing 10% fetal bovine serum (FBS) and Antibiotic Antimycotic Solution (100 ×) (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in 5% CO 2 . Cells were seeded at 5000 cells/cm 2 and grown to confluence. Cells from passages 3-6 were used in the experiments. The cells were treated with TGF-β2 for 24 h with or without pretreatment for 30 min with rapamycin (Fujifilm Wako Pure Chemical Corp., Osaka, Japan) or Torin 1 (Selleck Chemicals, Houston, TX, USA) as an mTOR inhibitor. This study is not deemed a Human Subject Research because cells were acquired post-mortem from de-identified donor tissues and is thus considered exempt by University of Tokyo's Institutional Review Board (IRB). Nevertheless, all experiments were done in accordance with the tenets of the Declaration of Helsinki. And this study does not include in vivo data, but all the in vitro studies were performed in accordance with ARRIVE guideline. www.nature.com/scientificreports/ Reprints and permissions information is available at www.nature.com/reprints.
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