Torin 2

Effects of mammalian target of rapamycin inhibitors on fibrosis after trabeculectomy

Nozomi Igarashi, Megumi Honjo *, Makoto Aihara
Department of Ophthalmology, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan

A B S T R A C T

Glaucoma, the second leading cause of blindness worldwide, is characterized by aberrant elevations of intra- ocular pressure (IOP), which can damage the optic nerve. IOP reduction is the only effective therapy for pre- vention of visual impairment and blindness in both hypertensive and normotensive individuals, and in some cases, trabeculectomy is a major surgical procedure that can lower IOP in patients with glaucoma. No matter how surgical technique and postoperative care advances, excessive scarring and tissue fibrosis could result from increased human conjunctival fibroblast (HCF) proliferation and extracellular matriX (ECM) deposition of the subconjunctival tissue and scleral flaps would persist after trabeculectomy. And these issues are major impedi- ments to IOP reduction and filtering of bleb formations, so the modulation of the factors which can induce fibrosis could used as a novel strategy to control scarring after trabeculectomy. In this study, we examined the effects of mammalian target of rapamycin (mTOR) inhibitors (rapamycin or Torin1) on the fibrotic response induced by transforming growth factor-beta 1 (TGF-β1) in cultured human conjunctival fibroblast (HCF) cells.
The study also examined the effects of mTOR inhibitor on fibrosis after trabeculectomy in rabbit eyes. In in vitro studies, we stimulated HCFs with TGF-β1, and confirmed that the expression levels of fibronectin, collagen type I alpha 1 chain (COL1A1), and α-smooth muscle actin (SMA) were significantly upregulated in HCFs with TGF-β1, by means of quantitative real-time polymerase chain reaction and immunocytochemistry. And those TGF-β1- induced changes were significantly attenuated with mTOR inhibitors, rapamycin or Torin1. Additionally the migration rate of HCFs was examined under conditions of TGF-β1 induction, TGF-β1-induced changes were significantly attenuated with mTOR inhibitors. A rabbit model of trabeculectomy was examined in vivo, and the effects of topical mTOR inhibitor were also examined, and found that topical treatment with mTOR inhibitor significantly suppressed collagen deposition in rabbit eyes after trabeculectomy. These results have demonstrated that mTOR inhibitors may provide a novel treatment modality for reducing the fibrotic response in HCFs and improving bleb scarring after filtration surgery.

Keywords:
Human conjunctival fibroblasts mTOR Rapamycin Torin 1 Trabeculectomy

1. Introduction

Glaucoma, the second leading cause of blindness worldwide, is characterized by aberrant elevations of intraocular pressure (IOP), which can damage the optic nerve (Quigley and Broman., 2006; Kwon et al., 2009; Weinreb and Khaw., 2004). IOP reduction is the only effective therapy for prevention of visual impairment and blindness in both hypertensive and normotensive individuals. Trabeculectomy is a major surgical procedure that can lower IOP in patients with glaucoma (Cairns, 1968; Ramulu et al., 2007). However, despite advances in surgical technique and postoperative care, excessive scarring and tissue fibrosis—resulting from increased human conjunctival fibroblast (HCF) proliferation and extracellular matriX (ECM) deposition of the subcon- junctival tissue and scleral flaps—persist after this procedure; these is- sues are major impediments to IOP reduction and filtering of bleb formations (Skuta and Parrish., 1987; Broadway and Chang., 2001).
Various aqueous mediators (e.g., transforming growth factor-beta [TGF-β], vascular endothelial growth factor, connective tissue growth factor, monocyte chemoattractant protein-1, and members of the matriX metalloproteinase family) are involved in ECM production and HCF fibrosis; the modulation of these factors has been used as a novel strategy to control scarring after trabeculectomy (Cordeiro et al., 2000; Li et al., 2009; Mathes and Barton., 2011; Lei et al., 2016; Inoue et al., 2014; Wong et al., 2003). However, these treatments often fail to maintain tissue health and can lead to thin-walled blebs with a risk for bleb leakage and infection; they do not facilitate the successful formation of glaucoma filtration blebs. Mammalian target of rapamycin (mTOR) is a downstream factor in the TGF-β pathway; mTOR is responsible for many types of bioactive changes, including fibrosis (Lawrence and Nho., 2018; Gui et al., 2015; Zhao et al., 2015). It is known that there exists two enzyme complexes; mTOR complex 1 and 2 (mTORC1 and mTORC2). Rapamycin is a mTORC1 inhibitor and mTORC2 is known to be insen- sitive to rapamycin. Torin1 is an ATP-competitive mTOR inhibitor which directly inhibits both mTORC1 and mTORC (Lawrence and Nho., 2018).
Antifibrotic effects of mTOR inhibitors have been observed in many contexts, including hepatic fibrosis, pulmonary fibrosis, and rheumatic diseases(Lawrence and Nho., 2018; Reilly et al., 2017; Peng et al., 2017; Perl, 2016). To the best of our knowledge, there have been no in- vestigations of the potential effects of mTOR inhibitors after trabecu- lectomy. In this study, we evaluated the abilities of mTOR inhibitors to prevent TGF-β-induced fibrosis in HCFs in vitro and after trabeculectomy in an in vivo rabbit model.

2. Materials and methods

2.1. Cell culture and passage

Primary HCF cells were isolated from human donor eyes and char- acterized as described previously (Aoyama et al., 2017). The cells were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum and Antibiotic-Antimycotic solution (100×) (Sigma-Aldrich, St. Louis, MO, USA) at 37 ◦C in 5% CO2. Cells from passages 3–6 were used in all experiments. The cells were treated with 10 ng/mL TGF-β1 for 24 h, with or without pretreatment for 30 min with 10 or 100 nM rapamycin (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) or 10 or 100 nM Torin 1 (Selleck Chemicals, Houston, TX, USA). 3 independent cell strains attained from patients were used. And each cell culture experiments were technically replicated at least for 3 times. TGF-β1 was dissolved in MQ, and both rapamycin and Torin1 were dissolved in DMSO. Both rapamycin or Torin1 were then diluted with PBS to the target concentration. According to the data attained with MTT assay (Fig. 1), there were no significant difference among those compounds for the potential toXicity against HCFs, we used MQ as vehicle.

2.2. MTT assay

We used MTT Cell Proliferation Assay Kit (BioAssay Systems, CA, USA) to evaluate the cell proliferation. MTT assay was performed ac- cording to the manufacture’s protocol. Briefly, cells were plated in a 96- well plate and 80 μL of culture medium with or without TGF-β1 (10 ng/mL) along with or without rapamycin (10 nM or 100 nM), Torin1 (10 nM or 100 nM) or 0.00995% DMSO in a CO2 incubator at 37 ◦C overnight. Then 15 μL of MTT reagent was added to each well and miXed gently, and incubated for 4 h in a CO2 incubator at 37 ◦C. 100 μL of solubilizer was added to each well, and miXed gently on an orbital shaker for 1 h at room temperature, and absorbance at OD570nm was recorded.

2.3. Quantitative polymerase chain reaction

Cells were lysed using ISOGEN (NIPPON GENE Ltd., Tokyo, Japan), and mRNA was isolated using chloroform and isopropyl alcohol. mRNA was then used to synthesize cDNA by means of a PrimeScript RT Reagent Kit (Takara Bio, Shiga, Japan). Quantitative real-time polymerase chain reaction (qPCR) was performed as previously described (Igarashi et al., 2020). For qPCR, primer sequences for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and fibronectin were taken from previously published sequences; the primers were purchased from Hokkaido Sys- tem Science (Hokkaido, Japan).

2.4. Immunocytochemistry

Cells were grown in chamber slides. Following serum starvation for 24 h, the cells were treated with 10 ng/mL TGF-β1 for 24 h, with or without mTOR inhibitors; each inhibitor was added 30 min before TGF- β1 treatment. The cells were fiXed in ice-cold 4% paraformaldehyde for 15 min, permeabilized with 0.3% Triton X-100 for 5 min, and blocked in 3% bovine serum albumin for 30 min. Immunocytochemistry was per- formed as described previously (Igarashi et al., 2020). The primary antibodies were anti-fibronectin (1:400; Abcam, Cambridge, MA, USA), anti-collagen type I (1:400; Cell Signaling Technology, Danvers, MA, USA), anti-rhodamine phalloidin (7:1000; Thermo Fisher Scientific, Waltham, MA, USA), and anti-α-SMA (1:500, Sigma-Aldrich). Alexa Fluor 488 and 594 secondary antibodies (1:1000) were purchased from Thermo Fisher Scientific.

2.5. Western blotting

Cells were first starved by incubation for 24 h in serum-free medium. The cells were then treated with 10 ng/mL TGF-β1 for 24 h, with or without mTOR inhibitors; the inhibitors were added 30 min before TGF- β1 treatment. Subsequently, cells were collected in radio- immunoprecipitation assay buffer (RIPA buffer, Thermo Fisher Scientific K.K., Kanagawa, Japan) containing protease inhibitors (Roche Diagnostics, Basel, Switzerland); they were sonicated and centrifuged. Protein concentrations in the supernatant were determined by the bicinchoninic acid assay, using a BCA Protein Assay Kit (Thermo Fisher Scientific K.K.). Western blotting was performed as previously described (Igarashi et al., 2020). Protein bands were detected by ImageQuant LAS 4000 mini (GE Healthcare, Chicago, IL, USA). Primary antibodies were anti-α-SMA (1:1000, Sigma-Aldrich), anti-fibronectin (1:1000; Abcam), and anti-collagen type I (1:1000; Cell Signaling Technology). Horse- radish peroXidase (HRP)-conjugated secondary antibody (1:10,000) was purchased from Thermo Fisher Scientific. β-tubulin served as the loading control. All membranes were stripped of antibodies using WB Stripping solution, then incubated with mouse monoclonal antibody β-tubulin (1:1000) and subsequently with HRP-conjugated goat anti-mouse IgG antibody (1:2000). Densitometry of scanned films was performed using ImageJ 1.49 (National Institutes of Health, Bethesda, MD, USA); protein expression levels are expressed relative to the level of β-tubulin.

2.6. Migration assay

The cells were starved by incubation for 24 h in serum-free medium. Four scratches in each well were made with a pipette tip, then photo- graphed using a microscope (BZ-9000, Keyence Corp., Osaka, Japan).
The starvation medium was changed to medium containing TGF-β1 alone or TGF-β1 with an mTOR inhibitor at the indicated concentration. The cells were then incubated at 37 ◦C; scratches were photographed at 1, 2, 4, 6, and 24 h after stimulation. The scratch widths were measured at the shortest distances between the edges of migrated cells (including their protrusions) from both sides. And the same points were measured with ImageJ 1.49 (National Institutes of Health), at the same points. Changes in scratch widths were recorded and used to calculate migra- tion distances.

2.7. Animals and rabbit model of glaucoma filtration surgery

All animals used in this study were handled in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, as well as the requirements of the Institutional Animal Research Committee of the University of Tokyo. All investigations involving rabbits (i.e., surgery, IOP measurements, and assessments of histologic features) were conducted in a masked manner.
Ten eyes of male Japanese white rabbits (mean weight, 2.5 kg) were used in this study. The animals were maintained in conventional animal rooms and individually housed in plastic cages in a room with controlled temperature and relative humidity (22 3 ◦C and 55 10%, respectively), with a 12-h light/dark cycle and access to food and water ad libitum. Rabbits were acclimated for 2 weeks prior to inclusion in this study. Rabbits were anesthetized with an intramuscular injection of ketamine hydrochloride (5 mg/kg body weight), and xylazine hydro- chloride (5 mg/kg body weight). A limbus-based flap of the conjunctiva and the Tenon capsule was made at a distance of 5 mm from the limbus in the superior nasal quadrant of the right eye. A sclerostomy was per- formed; a fistula was constructed toward the anterior chamber. The conjunctiva was closed with three 8-0 polyglactin 910 sutures.

2.8. Evaluation of postoperative effects of mTOR inhibitor in a rabbit model of glaucoma filtration surgery

IOP measurements were performed with a Pneumatonometer Model 30 Classic (RE Medical Inc., Osaka, Japan) under topical anesthesia. The rabbits were divided into two groups: a treatment group (treated with rapamycin dissolved in DMSO and diluted to 100 nM with phosphate- buffered saline) and a control group (treated with dimethylsulfoXide in phosphate-buffered saline). Three measurements were obtained, and the mean difference in IOP between treated and control eyes was recorded. After surgery, 1 cm of 3 mg/g ofloXacin ointment was applied to the eye. Topical rapamycin or dimethylsulfoXide was applied daily for 5 days postoperatively.

2.9. Histologic evaluation

For this analysis, rabbits were euthanized 5 days postoperatively. Whole eyes were enucleated and fiXed in 4% paraformaldehyde in phosphate-buffered saline for 48 h at 4 ◦C, then embedded in Tissue-Tek medium (Sakura Finetechnical, Tokyo, Japan). An incision was made at a 90◦ angle from the surgical site. Serial sections were cut through the sclerostomy site. ApproXimately every fifth section was stained with standard hematoXylin-eosin (HE) and Elastica van Gieson (EVG; for collagen). The extent of fibroproliferation and scar formation in each section was evaluated in a masked manner.

2.10. Statistical analysis

Data were statistically analyzed using EZR software (Saitama Medi- cal Center, Hidaka, Japan) (Kanda., 2013). The results are expressed as the mean standard deviation (SD). Differences among groups were analyzed by one-way analysis of variance, followed by post hoc Tukey test. Values of P < 0.05 were considered statistically significant. 3. Results 3.1. Effects of TGF-β1 and mTOR inhibitors on cell proliferation Firstly, we confirmed the potential toXicity of DMSO (0.00995%), rapamycin (10 nM or 100 nM) and Torin1 (10 nM or 100 nM) to HCF with MTT assay. Fig. 1A shows that there was no significant difference among the groups concerning the potential toXicity to HCF. And also, as shown in Fig. 1B, the measured OD570nm value was significantly upregulated when the cells were treated with TGF-β1 (10 ng/mL) compared to control or DMSO groups. And the TGF-β1-induced upregulated OD570nm value was significantly attenuated with 100 nM rapamycin, 10 nM Torin1 and 100 nM Torin1. And there were no sig- nificant difference between control and DMSO treated groups. 3.2. Effects of TGF-β1 and mTOR inhibitors on mRNA expression of α—SMA, fibronectin and COL1A1 in HCFs mRNA levels in HCFs were assessed by qPCR (Fig. 2). The relative mRNA expression levels of COL1A1, fibronectin, and α-SMA were significantly higher in cells treated with TGF-β1, compared with the control group; these expression levels were reduced by treatment with an mTOR inhibitor (Fig. 2). 3.3. Effects of TGF-β1 and mTOR inhibitors on fibrotic responses in HCFs Immunocytochemistry and Western blotting were used to assess fibrotic responses to TGF-β1, as well as the effects of an mTOR inhibitor (rapamycin or Torin 1). Fig. 3A–D shows the results of immunocyto- chemistry results: the protein expression levels of fibronectin, COL1A1, phalloidin, and α-SMA were upregulated in HCFs after TGF-β1 treatment; these fibrotic responses were significantly suppressed by treat- ment with rapamycin or Torin 1. Fig. 4A–D shows the data from Western blotting (n = 3): fibronectin, COL1A1, and α-SMA protein expression levels were upregulated in HCFs after TGF-β1 treatment; these fibrotic responses were significantly suppressed by treatment with mTOR inhibitors. 3.4. Effects of TGF-β1 and mTOR inhibitors on cell migration A cell migration assay was used to examine the effect of TGF-β1 on HCF motility (Fig. 5). The migration of HCFs after 1, 2, 4, 6, and 24 h was recorded; the migration significantly accelerated in cells that had been treated with TGF-β1 for 4, 6, and 24 h (P < 0.05, P < 0.01, and P < 0.001, respectively) and was suppressed by treatment with an mTOR inhibitor for 4, 6, and 24 h (P < 0.05, P < 0.01, and P < 0.001, respectively). 3.5. Effects of mTOR inhibitor on filtration blebs Topical instillation of rapamycin significantly reduced fibrogenic changes after glaucoma filtration surgery in our rabbit model. Fig. 6A shows the typical appearance of blebs following glaucoma filtration surgery. IOP did not significantly differ between rapamycin-treated and control groups (Supplemental Fig. 1). The mean preoperative IOP was 23.4 1.3 mmHg; the lowest IOP was recorded at postoperative day 2 (mean, 17.1 2.6 mmHg), while the mean IOP on the final day was 21.6 2.0 mmHg. No significant postoperative changes in pupil dilation or corneal morphology were observed in either group. Surgical sites were examined 5 days postoperatively and stained with HE and EVG. HE staining of control eyes revealed nearly complete scarring over the sclerostomy site (Fig. 6B); EVG staining revealed new collagen deposition in the scleral gap and bleb area (Fig. 6D). In contrast, HE staining of rapamycin-treated eyes showed significantly reduced scar formation at postoperative day 5 (Fig. 6C). EVG staining also revealed that rapamycin-treated eyes contained bleb cavities of moderate size with minimal deposition of additional collagen in the scleral gap (Fig. 6E). Higher magnification analysis of EVG staining showed dense collagen fiber formation and scar tissue in both the con- junctiva and the subconjunctival scar (Fig. 6D), as well as the area around the failed bleb (Fig. 6F), in the control group. In contrast, the surviving rapamycin-treated bleb showed a much looser architecture with a visible conjunctiva (Fig. 6E) and bleb formation with fewer collagen deposits (Fig. 6G). 4. Discussion It is important to prevent postoperative subconjunctival scarring after trabeculectomy, because fibrosis of blebs often leads to surgical failure, which has been reported in 35–43% of patients due to post- operative scarring and subconjunctival fibrosis (CAT-152 0102 Trabeculectomy Study Group et al., 2007; Landers et al., 2012). Many procedures including adjunctive treatment with antiproliferative agents (e.g., mitomycin C, 5-fluorouracil, or anti-TGF-β antibodies) have been used in attempts to prevent scarring and bleb failure. However, these treatments often fail to maintain tissue health and can lead to thin-walled blebs with a risk for bleb leakage and infection; they do not facilitate the successful formation of glaucoma filtration blebs (CAT-152 0102 Trabeculectomy Study Group et al., 2007; Landers et al., 2012; Al et al., 2015). Various mediators (e.g., TGF-β, vascular endothelial growth factor, connective tissue growth factor, monocyte chemoattractant protein-1, and members of the metalloproteinase family) are involved in the pathogenesis of scarring after trabeculectomy (Cordeiro et al., 2000; Li et al., 2009; Mathew and Barton., 2011; Lei et al., 2016; Inoue et al., 2014; Wong et al., 2003; Browne et al., 2011). Among these growth factors, the levels of several factors (i.e., TGF-β, vascular endothelial growth factor, connective tissue growth factor, and monocyte chemoattractant protein-1) have been shown to enhance the aqueous humor levels in patients with primary open-angle glaucoma, exfoliation glaucoma, or neovascular glaucoma; thus, these factors may stimulate signaling pathways that lead to myofibroblast transdifferentiation of HCFs, monocyte-derived cell infiltration, and ECM remodeling after trabeculectomy(Cordeiro et al., 2000; Mathew and Barton., 2011; Lei et al., 2016; Inoue et al., 2014; Browne et al., 2011). The results of several in vivo and in vitro studies have suggested that modulation of these factors could be used as a novel strategy to control scarring after trabeculectomy (Mathew and Barton., 2011; Lei et al., 2016; .Chong et al., 2017). After tissue damage, activated fibroblasts migrate to the damage site, where they differentiate into α-SMA-positive myofibroblasts, which synthesize ECM components (e.g., collagen) and contribute to the formation of contractile stress fibers that bind to the ECM (Hinz et al., 2007). TGF-β is considered a major bioactive lipid mediator that in- fluences fibrosis and induces many types of cellular responses that include downstream mTOR signaling. mTOR is a downstream factor of the TGF-β cascade; notably, mTOR is involved in fibrotic changes in many organs and diseases (Lawrence and Nho., 2018; Gui et al., 2015; Zhao et al., 2015; Reilly et al., 2017). In this study, we speculated that mTOR inhibitors could be useful for the inhibition of excessive fibro- genic changes and inflammation, as well as improvement in the surgical outcomes of filtering surgery. We conducted in vitro studies to determine the effects of mTOR in- hibitors on fibrotic changes in HCFs (Figs. 2, 3A–D, and 4A–D). TGF-β1 treatment significantly enhanced α-SMA, fibronectin, and COL1A1 mRNA expression levels in HCFs, as determined by qPCR (Fig. 2). Additionally, immunocytochemistry and Western blotting (Fig. 3A–D and 4A–D) revealed that treatment with mTOR inhibitors (i.e., rapamycin or Torin 1) significantly suppressed these fibrotic responses, which suggested that the inhibition of mTOR could serve as a target for prevention of TGF-β-mediated fibrosis after trabeculectomy. As shown in Fig. 5, TGF-β1 treatment significantly accelerated cell migration after 4, 6, and 24 h; this cell migration was significantly attenuated by treatment with an mTOR inhibitor. As the mTOR inhibitors are known to regulate the cell proliferation, we also performed the MTT assay and confirmed that the TGF-β1-induced cell proliferation was also signifi- cantly attenuated by treatment with an mTOR inhibitor (Fig. 1). In past reports of filtration surgery in animal models, obstruction of the sclerostomy site has been observed, due to excessive ECM deposition and contraction (Darby and Hewitson., 2007; Miller et al., 1989). In the present study, histologic analysis of rabbit tissues showed that topical instillation of rapamycin led to significant reduction of subconjunctival collagen deposition, compared with controls (Fig. 6). These observations were consistent with the in vitro findings that mTOR inhibitors signifi- cantly reduced fibrogenic changes in HCFs, suggesting that the benefi- cial effects of mTOR inhibitors in the rabbit model of glaucoma may be mediated by the reduction of cell contraction and inhibition of HCF transdifferentiation. mTOR inhibitors are already in clinical use and have been proven to be effective and clinically safe for treatment of cancer, allograft rejection, and tuberous sclerosis complex or lym- phangioleiomyomatosis; thus, they presumably modulate the immune system (Li et al., 2014). Clinically available drugs such as rapamycin or its derivative, everolimus, may be appliable for management of scarring after glaucoma filtration surgery. This study had several limitations. First, it did not determine optimal dosage for anti-scarring effects of mTOR inhibitors following trabecu- lectomy surgery; further studies are needed, including animal models for assessment of longer treatment periods and clinical trials to determine clinical applicability of these findings. Second, no direct comparisons were made regarding the effects of simultaneous treatment with mTOR inhibitors and mitomycin C; a future study in our institute will compare surgical outcomes with respect to concurrent use of mTOR inhibitors and mitomycin C concerning the long-term prognosis. Third, this study only focused on the short-term prognosis after trabeculectomy under topical application of mTOR inhibitor, so further study to evaluate the long-term prognosis including IOP evaluation using mTOR inhibitors would be needed. 5. Conclusions In glaucoma surgery, postoperative fibrosis or scarring at the surgical site and subconjunctival spaces persists as a major obstacle to achieve- ment of sustained IOP reduction. In the present study, we found that mTOR could serve as a target for downregulation of the fibrogenic cascade. The modulation of mTOR could be used as a novel approach for inhibition of excessive fibrogenic changes in filtration blebs and improvement of surgical outcomes after glaucoma surgery. References Al Habash, A., Aljasim, L.A., Owaidhah, O., Edward, D.P., 2015. A review of the efficacy of mitomycin C in glaucoma filtration surgery. Clin. Ophthalmol. 9, 1945–1951. Aoyama-Araki, Y., Honjo, M., Uchida, T., Yamagishi, R., Kano, K., Aoki, J., Aihara, M., 2017. Sphingosine-1-Phosphate (S1P)-Related response of human conjunctival fibroblasts after filtration surgery for glaucoma. Invest. Ophthalmol. Vis. Sci. 58 (4), 2258–2265. Broadway, D.C., Chang, L.P., 2001. Trabeculectomy, risk factors for failure and the preoperative state of the conjunctiva. J. Glaucoma 10, 237–249. Browne, J.G., Ho, S.L., Kane, R., Oliver, N., Clark, A.F., O’Brien, C.J., Crean, J.K., 2011. Connective tissue growth factor is increased in pseudoexfoliation glaucoma. Invest. Ophthalmol. Vis. Sci. 52 (6), 3660–3666. Cairns, J.E., 1968. Trabeculectomy. Preliminary report of a new method. Am. J. Ophthalmol. 66, 673–679. CAT-152 0102 Trabeculectomy Study Group, Khaw, P., Grehn, F., Hollo´, G., Overton, B., Wilson, R., Vogel, R., Smith, Z., 2007. A phase III study of subconjunctival human anti-transforming growth factor beta(2) monoclonal antibody (CAT-152) to prevent scarring after first-time trabeculectomy. Ophthalmology 114, 1822–1830. Chong, R.S., Lee, Y.S., Chu, S.W.L., Toh, L.Z., Wong, T.T.L., 2017. Inhibition of monocyte chemoattractant protein 1 prevents conjunctival fibrosis in an experimental model of glaucoma filtration surgery. Invest. Ophthalmol. Vis. Sci. 58 (9), 3432–3439. Cordeiro, M.F., Chang, L., Lim, K.S., Daniels, J.T., Pleass, R.D., Siriwardena, D., Khaw, P.T., 2000. Modulating conjunctival wound healing. Eye 14, 536–547. Darby, I.A., Hewitson, T.D., 2007. Fibroblast differentiation in wound healing and fibrosis. Int. Rev. Cytol. 257, 143–179. Gui, Y.S., Wang, L., Tian, X., Li, X., Ma, A., Zhou, W., Zeng, N., Zhang, J., Cai, B., Zhang, H., Chen, J.Y., Xu, K.F., 2015. mTOR overactivation and compromised autophagy in the pathogenesis of pulmonary fibrosis. PloS One 10 (9), e0138625. Hinz, B., Phan, S.H., Thannickal, V.J., Galli, A., Bochaton-Piallat, M.L., Gabbiani, G., 2007. The myofibroblast: one function, multiple origins. Am. J. Pathol. 170 (6), 1807–1816. Igarashi, N., Honjo, M., Yamagishi, R., Kurano, M., Yatomi, Y., Igarashi, K., Kaburaki, T., Aihara, M., 2020. Involvement of autotaxin in the pathophysiology of elevated intraocular pressure in Posner-Schlossman syndrome. Sci. Rep. 10 (1), 6265. Inoue, T., Kawaji, T., Tanihara, H., 2014. Monocyte chemotactic protein-1 level in the aqueous humour as a prognostic factor for the outcome of trabeculectomy. Clin. EXp. Ophthalmol. 42, 334–341. Kanda, Y., 2013. Investigation of the freely available easy-to-use software ’EZR’ for medical statistics. Bone Marrow Transplant. 48 (3), 452–458. Kwon, Y.H., Fingert, J.H., Kuehn, M.H., Alward, W.L., 2009. Primary open-angle glaucoma. N. Engl. J. Med. 360, 1113–1124. Landers, J., Martin, K., Sarkies, N., Bourne, R., Watson, P., 2012. A twenty-year Torin 2 follow- up study of trabeculectomy: risk factors and outcomes. Ophthalmology 119, 694–702.
Lawrence, J., Nho, R., 2018. The role of the mammalian target of rapamycin (mTOR) in pulmonary fibrosis. Int. J. Mol. Sci. 19 (3), 778.
Lei, D., Dong, C., Wu, W.K., Dong, A., Li, T., Chan, M.T., Zhou, X., Yuan, H., 2016.
Lentiviral delivery of small hairpin RNA targeting connective tissue growth factor blocks profibrotic signaling in tenon’s capsule fibroblasts. Invest. Ophthalmol. Vis. Sci. 57, 5171–5180.
Li, Z., Van Bergen, T., Van de Veire, S., Van de Vel, I., Moreau, H., Dewerchin, M., Maudgal, P.C., Zeyen, T., Spileers, W., Moons, L., Stalmans, I., 2009. Inhibition of vascular endothelial growth factor reduces scar formation after glaucoma filtration surgery. Invest. Ophthalmol. Vis. Sci. 50, 5217–5225.
Li, J., Kim, S.G., Blenis, J., 2014. Rapamycin: one drug, many effects. Cell Metabol. 19 (3), 373–379.
Mathew, R., Barton, K., 2011. Anti-vascular endothelial growth factor therapy in glaucoma filtration surgery. Am. J. Ophthalmol. 152, 10–15.
Miller, M.H., Grierson, I., Unger, W.I., Hitchings, R.A., 1989. Wound healing in an animal model of glaucoma fistulizing surgery in the rabbit. Ophthalmic Surg. 20 (5), 350–357.
Peng, R., Wang, S., Wang, R., Wang, Y., Wu, Y., Yuan, Y., 2017. Antifibrotic effects of tanshinol in experimental hepatic fibrosis by targeting PI3K/AKT/mTOR/p70S6K1 signaling pathways. Discov. Med. 23 (125), 81–94.
Perl, A., 2016. Activation of mTOR (mechanistic target of rapamycin) in rheumatic diseases. Nat. Rev. Rheumatol. 12 (3), 169–182.
Quigley, H.A., Broman, A.T., 2006. The number of people with glaucoma worldwide in 2010 and 2020. Br. J. Ophthalmol. 90, 262–267.
Ramulu, P.Y., Corcoran, K.J., Corcoran, S.L., Robin, A.L., 2007. Utilization of various glaucoma surgeries and procedures in Medicare beneficiaries from 1995 to 2004. Ophthalmology 114, 2265–2270.
Reilly, R., Mroz, M.S., Dempsey, E., Wynne, K., Keely, S.J., McKone, E.F., Hiebel, C., Behl, C., Coppinger, J.A., 2017. Targeting the PI3K/Akt/mTOR signalling pathway in Cystic Fibrosis. Sci. Rep. 7 (1), 7642.
Skuta, G.L., Parrish 2nd, R.K., 1987. Wound healing in glaucoma filtering surgery. Surv. Ophthalmol. 32, 149–170.
Weinreb, R.N., Khaw, P.T., 2004. Primary open-angle glaucoma. Lancet 363, 1711–1720.
Wong, T.T., Mead, A.L., Khaw, P.T., 2003. MatriX metalloproteinase inhibition modulates postoperative scarring after experimental glaucoma filtration surgery. Invest. Ophthalmol. Vis. Sci. 44, 1097–1103.
Zhao, Q.D., Viswanadhapalli, S., Williams, P., Shi, Q., Tan, C., Yi, X., Bhandari, B., Abboud, H.E., 2015. NADPH oXidase 4 induces cardiac fibrosis and hypertrophy through activating Akt/mTOR and NFκB signaling pathways. Circulation 131 (7), 643–655.