MiR-146a reduces fibrosis after glaucoma filtration surgery in rats.

PURPOSE: To explore the impact of microRNA 146a (miR-146a) and the underlying mechanisms in profibrotic changes following glaucoma filtering surgery (GFS) in rats and stimulation by transforming growth factor (TGF)-ß1 in rat Tenon's capsule fibroblasts. METHODS: Cultured rat Tenon's capsule fibroblasts were treated with TGF-ß1 and analyzed with microarrays for mRNA profiling to validate miR-146a as the target. The Tenon's capsule fibroblasts were then respectively treated with lentivirus-mediated transfection of miR-146a mimic or inhibitor following TGF-ß1 stimulation in vitro, while GFS was performed in rat eyes with respective intraoperative administration of miR-146a, mitomycin C (MMC), or 5-fluorouracil (5-FU) in vivo. Profibrotic genes expression levels (fibronectin, collagen Iα, NF-KB, IL-1ß, TNF-α, SMAD4, and α-smooth muscle actin) were determined through qPCR, Western blotting, immunofluorescence staining and/or histochemical analysis in vitro and in vivo. SMAD4 targeting siRNA was further used to treat the fibroblasts in combination with miR-146a intervention to confirm its role in underlying mechanisms. RESULTS: Upregulation of miR-146a reduced the proliferation rate and profibrotic changes of rat Tenon's capsule fibroblasts induced by TGF-ß1 in vitro, and mitigated subconjunctival fibrosis to extend filtering blebs survival after GFS in vivo, where miR-146a decreased expression levels of NF-KB-SMAD4-related genes, such as fibronectin, collagen Iα, NF-KB, IL-1ß, TNF-α, SMAD4, and α-smooth muscle actin(α-SMA). Additionally, SMAD4 is a key target gene in the process of miR-146a inhibiting fibrosis. CONCLUSIONS: MiR-146a effectively reduced TGF-ß1-induced fibrosis in rat Tenon's capsule fibroblasts in vitro and in vivo, potentially through the NF-KB-SMAD4 signaling pathway. MiR-146a shows promise as a novel therapeutic target for preventing fibrosis and improving the success rate of GFS.

Effects of atorvastatin on the function of Tenon's capsule fibroblasts in human eyes.

PURPOSE: This study aimed to explore the role of atorvastatin (ATO) in the prevention and treatment of the scarring of filtration channels after glaucoma surgery. METHODS: Human Tenon's capsule fibroblasts (HTFs) were co-cultured with various concentrations of ATO. First, Cell Counting Kit-8 assay was performed to evaluate the effects of various concentrations of ATO on the viability of HTFs. Then, after the ATO stimulated the HTFs for 24 h, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to evaluate the apoptosis of HTFs. Transwell assay was also performed to evaluate the migration of HTFs. Moreover, enzyme-linked immunosorbent assay (ELISA) was performed to detect the protein expression levels of transforming growth factor-ß1 (TGF-ß1) and TGF-ß2 in the cell culture supernatant of HTFs. Western blot was carried out to detect the protein expression levels of smooth muscle actin (SMA), p38, Smad3, fibronectin, collagen I and collagen III in different groups. RESULTS: The results revealed that ATO could inhibit the proliferation and migration of HTFs. Based on the TUNEL assay, 100 µM and 150 µM ATO could induce cell apoptosis. The ELISA results indicated that ATO could down-regulate the expression level of TGF-ß2, and western blot analysis revealed that the protein expression levels of SMA, p38, Smad3, fibronectin, collagen I and collagen III in the TGF-ß2 group were all up-regulated compared with the control group, whereas the addition of ATO could reverse this up-regulation. CONCLUSIONS: ATO could inhibit the proliferation and migration of HTFs and induce their apoptosis. It was preliminary proven that ATO could inhibit the signaling pathway induced by TGF-ß. It is suggested that ATO could be a basis for the treatment of the scarring of filtration channels after glaucoma surgery.

ALK5 Inhibition of Subconjunctival Scarring From Glaucoma Surgery: Effects of SB-431542 Compared to Mitomycin C in Human Tenon's Capsule Fibroblasts.

Purpose: The gold standard for managing postoperative ocular fibrosis in glaucoma surgery is the chemotherapeutic mitomycin C (MMC) despite its association with significant adverse effects. This study compares in vitro the antifibrotic efficacy and cytotoxicity of the small-molecule TGFß1 inhibitor SB-431542 (SB) to MMC. Methods: To measure collagen contraction, human Tenon's capsule fibroblasts (HTCFs) embedded in a three-dimensional collagen lattice were exposed to 0.2 mg/mL MMC or 20 µM SB followed by incubation with 2 ng/mL TGFß1. Total protein extracted from experimentally treated HTCFs underwent immunoblotting for α-smooth muscle actin (α-SMA), matrix metallopeptidase 9 (MMP-9), and EDA splice-variant fibronectin (EDA-FN) expression. Cytotoxicity and cell metabolism were assessed using LIVE/DEAD staining, lactate dehydrogenase (LDH) assay, and methylthiazole tetrazolium (MTT) assay. Results: Collagen lattice contraction in TGFß1-induced HTCFs was significantly lowered by SB and MMC. Pretreatment with SB and MMC significantly lowered protein expression of α-SMA, MMP-9, and EDA-FN in HTCFs relative to TGFß1 alone. HTCF viability in collagen lattices was significantly reduced with MMC pretreatment but not SB pretreatment. MMC-pretreated HTCFs had a significant increase in LDH release after 3 hours and a decrease in MTT activity after 20 minutes, while SB-pretreated HTCFs showed no significant changes via MTT or LDH assay during the same treatment period. Conclusions: SB shows comparable efficacy to MMC in reducing expression of fibrosis-promoting proteins in HTCFs and in vitro scarring activity. SB distinguishes itself from MMC by exhibiting less cytotoxicity in both two-dimensional and three-dimensional in vitro assays. Translational Relevance: This study demonstrates in vitro the potential of SB as a safer alternative ocular antifibrotic agent.

ZD6474 attenuates TGF-ß1-induced fibrosis in human Tenon fibroblasts and inhibits neovascularization via AKT-mTOR signaling pathway.

PURPOSE: To investigate the anti-fibrotic effect of ZD6474 (a novel inhibitor of VEGF and EGF) in TGF-ß1 stimulated human Tenon's capsule fibroblasts (HTFs) and the anti-angiogenetic role in HUVECs, compared to that of mitomycin C (MMC). METHODS: The effects of ZD6474 on cell proliferation or migration in TGF-ß1-stimulated HTFs and HUVECs were determined, using CCK8 or wound healing assay, respectively. The typical markers of fibrosis in TGF-ß1-stimuated HTFs were detected, vimentin by immunofluorescence, α-SMA and snail by western blot. Tube formation was applied to validate the anti-angiogenesis effect in HUVECs following ZD6474 treatment. Furthermore, phosphorylated AKT and mTOR (p-AKT and p-mTOR) were evaluated, compared to the standardized total AKT and mTOR, using western blot. RESULTS: There was almost no decreased cell viability in HTFs following ZD6474 (≤ 1 µM/mL) treatment, but MMC (> 50 µg/mL) significantly impaired cell viability. ZD6474 significantly inhibited TGF-ß1-stimulated proliferation and migration in HTFs, compared to control group (**P < 0.01). ZD6474 also significantly attenuated the TGF-ß1-stimulated expression of vimentin, α-SMA and snail in HTFs. Tube formation was notably interrupted in HUVECs following ZD6474 treatment (**P < 0.01). P-AKT and p-mTOR were significantly decreased in response to ZD6474 treatment in TGF-ß1- induced HTFs and HUVECs. CONCLUSIONS: ZD6474 exerts anti-proliferation and anti-fibrotic effects in TGF-ß1-stimulated HTFs perhaps via regulating AKT-mTOR signaling pathway. ZD6474 also inhibited proliferation, migration and tube formation in HUVECs via the same signaling pathway. We concluded that ZD6474 may be potentially a novel agent in preventing bleb dysfunction following glaucoma filtration surgery (GFS).

The Effect of CHIR 99021, a Glycogen Synthase Kinase-3ß Inhibitor, on Transforming Growth Factor ß-Induced Tenon Fibrosis.

Purpose: This study investigated the effect of glycogen synthase kinase-3ß (GSK-3ß) inhibition on the fibrosis of human Tenon's fibroblasts (HTFs) induced by transforming growth factor-ß (TGF-ß). Methods: Quantitative real-time PCR and Western blot analyses were performed to determine the expression levels of molecules associated with the fibrosis of HTFs by TGF-ß (fibronectin, collagen Iα, and α-smooth muscle actin) and GSK-3ß. The levels of phosphorylated Smad2 and Smad3 were also analyzed in the presence of the GSK-3ß inhibitor CHIR 99021. The wound healing assay was performed to determine the effect of CHIR 99021 on the migration of HTFs. All experiments were conducted using primary cultured HTFs or human tenon tissues obtained from normal subjects and patients with glaucoma. Results: Treatment with TGF-ß resulted in an increase in the levels of molecules associated with the fibrosis of HTFs. The expression levels of these molecules were higher in the tenon tissues obtained from patients with glaucoma than those from normal subjects. When the HTFs were treated with TGF-ß, a significant increase in the active form of GSK-3ß (Y216) was observed. A significant decrease in the active form of GSK-3ß and molecules associated with fibrosis by TGF-ß was noted in HTFs treated with CHIR 99021. CHIR 99021 treatment reduced the phosphorylated Smad2/Smad2 and phosphorylated Smad3/Smad3 ratios in HTFs and attenuated HTF migration. Conclusions: Our results demonstrated the effect of GSK-3ß inhibition on the regulation of TGF-ß-mediated fibrosis of HTFs, suggesting GSK-3ß to be a potential target for maintaining bleb function after glaucoma filtration surgery.

Dihydroartemisinin Inhibits TGF-ß-Induced Fibrosis in Human Tenon Fibroblasts via Inducing Autophagy.

BACKGROUND: The formation of hypertrophic scars (HS) can result in the failure of glaucoma surgery, and fibrosis is known to be closely associated with the progression of HS. Dihydroartemisinin (DHA) has been reported to inhibit the progression of fibrosis; however, whether DHA can alleviate the formation of HS remains unclear. METHODS: In the present study, in order to examine the effects of DHA on the progression of HS, human Tenon's capsule fibroblasts (HTFs) were isolated from patients who underwent glaucoma surgery. In addition, Western blot analysis, microtubule associated protein 1 light chain 3 α staining and reverse transcription-quantitative PCR were performed to detect protein and mRNA expression levels in the HTFs, respectively. Cell proliferation was detected by Ki67 staining. Flow cytometry was used to examine apoptosis and reactive oxygen species (ROS) levels in the HTFs. RESULTS: The results revealed that TGF-ß promoted the proliferation and fibrosis of HTFs; however, DHA significantly reversed the effects of TGF-ß by increasing cell autophagy. In addition, DHA notably induced the apoptosis of TGF-ß-stimulated HTFs by increasing the ROS levels, while these increases were partially reversed by 3-methyladenine. Furthermore, DHA notably increased the expression of microRNA (miR)-145-5p in HTFs in a dose-dependent manner. CONCLUSION: The present study demonstrated that DHA inhibits the TGF-ß-induced fibrosis of HTFs by inducing autophagy. These findings may aid in the development of novel agents for the prevention of the formation of HS following glaucoma surgery.

The Expression and Role of Hypoxia-induced Factor-1α in Human Tenon's Capsule Fibroblasts under Hypoxia.

PURPOSE: To determine the expression of hypoxia-induced factor-1α (HIF-1α) and its downstream factors in human Tenon's capsule fibroblasts (HTFs) and changes in HTFs biological functions, we explored the role of HIF-1α in HTFs under hypoxia to provide a basis for studying the regulation of HIF-1α in wound healing after glaucoma surgery. MATERIALS AND METHODS: we established HTFs hypoxia model in vitro, meanwhile the HIF-1α agonist VH298 or inhibitor KC7F2 was added to HTFs, and the normoxia group was used as a control. Western blot, immunofluorescence and ELISA were used to detect the expression of HIF-1α, vascular endothelial growth factor (VEGF), transforming growth factor-ß (TGF-ß), Smads and collagen I. The proliferation of HTFs was quantified by cell counting kit-8, and cell migration was tested by healing scratch test. RESULTS: HIF-1α protein expression increased under hypoxia, peaked from 4-24 h, and then decreased. The secretion of VEGF and TGF-ß increased with prolonged hypoxia time. VH298 and KC7F2 upregulated and downregulated the levels of VEGF and TGF-ß, respectively, suggesting that HIF-1α upregulates and downregulates the levels of VEGF and TGF-ß in HTFs under hypoxia, respectively. HIF-1α upregulated the proliferation, migration and collagen synthesis of HTFs under hypoxia. CONCLUSIONS: Regulating HIF-1α and its downstream factors effectively regulated HTFs proliferation, migration and collagen synthesis. HIF-1α is a promising regulator in the study of wound healing after glaucoma surgery.

A Tenon's capsule/bulbar conjunctiva interface biomimetic to model fibrosis and local drug delivery.

Glaucoma filtration surgery is one of the most effective methods for lowering intraocular pressure in glaucoma. The surgery efficiently reduces intra-ocular pressure but the most common cause of failure is scarring at the incision site. This occurs in the conjunctiva/Tenon's capsule layer overlying the scleral coat of the eye. Currently used antimetabolite treatments to prevent post-surgical scarring are non-selective and are associated with potentially blinding side effects. Developing new treatments to target scarring requires both a better understanding of wound healing and scarring in the conjunctiva, and new means of delivering anti-scarring drugs locally and sustainably. By combining plastic compression of collagen gels with a soft collagen-based layer, we have developed a physiologically relevant model of the sub-epithelial bulbar conjunctiva/Tenon's capsule interface, which allows a more holistic approach to the understanding of subconjunctival tissue behaviour and local drug delivery. The biomimetic tissue hosts both primary human conjunctival fibroblasts and an immune component in the form of macrophages, morphologically and structurally mimicking the mechanical proprieties and contraction kinetics of ex vivo porcine conjunctiva. We show that our model is suitable for the screening of drugs targeting scarring and/or inflammation, and amenable to the study of local drug delivery devices that can be inserted in between the two layers of the biomimetic. We propose that this multicellular-bilayer engineered tissue will be useful to study complex biological aspects of scarring and fibrosis, including the role of inflammation, with potentially significant implications for the management of scarring following glaucoma filtration surgery and other anterior ocular segment scarring conditions. Crucially, it uniquely allows the evaluation of new means of local drug delivery within a physiologically relevant tissue mimetic, mimicking intraoperative drug delivery in vivo.

Epigallocatechin-3-gallate (EGCG) inhibits myofibroblast transformation of human Tenon's fibroblasts.

Myofibroblast transformation of human Tenon's fibroblasts severely challenges the outcome of glaucoma filtration surgery. epigallocatechin-3-gallate (EGCG) is considered as a potential reagent to overcome this issue for its anti-fibrosis effect on various human diseases, but it is unclear on the fibrosis of Tenon's fibroblasts. This study was conducted to investigate the effect of EGCG on TGF-ß1-induced myofibroblast transformation of human Tenon's fibroblasts. The human Tenon's fibroblasts were incubated in the medium containing 10 ng/mL TGF-ß1, and subsequently treated with EGCG or mitomycin C (MMC). The cell proliferation and migration were analyzed. The expression of alpha-smooth muscle actin (α-SMA), type I collagen (Col-I), and p-Smad2/3 were also evaluated. It showed that EGCG and MMC strongly inhibited the elevation in cell number in tissue explants compared to the tissues treated with TGF-ß1 alone. Scratch-Wound assay showed that 48 h after TGF-ß1 induction, only 10% of the wound width remained. But cells treated with EGCG still showed over 93% wound width. Further, EGCG effectively inhibited TGF-ß1-induced expression of α-SMA and Col-I as well as phosphorylation of Smad2/3 in Tenon's fibroblasts. Altogether, we concluded that EGCG suppressed the myofibroblast transformation in Tenon's fibroblasts through inactivating TGF-ß1/Smad signaling. These findings demonstrate that EGCG can be considered as one of the possible antifibrotic reagents for preventing postoperative scarring in glaucoma filtration surgery.

Isoliquiritigenin protects against angiotensin II-induced fibrogenesis by inhibiting NF-κB/PPARγ inflammatory pathway in human Tenon's capsule fibroblasts.

PURPOSE: To examine the protective effects of Isoliquiritigenin (ISL) in angiotensin II (ANG II)-induced inflammation and fibrosis on Human Tenon's capsule Fibroblasts (HTFs) and Mouse Peritoneal Macrophages (MPMs). This study also investigated the potential mechanism of action of ISL. METHOD: Methyl-thiazolyl tetrazolium (MTT) assay was used to test ISL toxicity. An ELISA and an RT-qPCR assay detected the inflammatory cytokines (TNF-α, IL-6, COX-2, and ICAM-1). A Western blot investigated the expression levels of inflammation-related signals [nuclear factor-κB (NF-κB), peroxisome proliferator-activated receptor γ (PPARγ)], and fibrogenesis, including fibronectin and alpha-smooth muscle actin (α-SMA)]. Protein expressions of α-SMA were measured by immunofluorescence. RESULTS: Pre-treatment with ISL (10 or 20 µM) dose-dependently decreased the mRNA levels of TNF-α, IL-6, ICAM-1, and COX-2 induced by ANG II (1 µg/ml) in both MPMs and HTFs. ANG II remarkably increased the amount of P65 in the nuclei and decreased the amount of P65 in the cytoplasm. Additionally, ANG II reduced PPARγ expression levels in a time-dependent manner. Furthermore, these effects which were induced by ISL were remarkably neutralized by ISL pre-treatment. Finally, ANG II markedly elevated the expression of fibronectin and α-SMA. CONCLUSION: ISL could alleviate ANG II-induced fibrogenesis by inhibiting the NF-κB/PPARγ inflammatory pathway. In addition, ISL may be a potential agent for the treatment of conjunctival fibrosis. Most importantly, the NF-κB/PPARγ signaling pathway could be an effective therapeutic target for the prevention and treatment of conjunctival fibrosis after glaucoma surgery.

Silencing of p53 reduces cell migration in human Tenon's fibroblasts induced by TGF-ß.

PURPOSE: Growth factors are considered as key molecules that participating in fibrosis formation. This research aimed to clarify potential effects of p53 on regulation of transforming growth factor ß (TGF-ß) and fibrosis formation and investigate the associated mechanisms. METHODS: Vimentin was examined to identify human Tenon's fibroblasts (HTFs). p53-targeting small interfere RNA (siRNA) was synthesis and transfected into HTFs. Real-time PCR assay was utilized to evaluate p53 and microRNA-29b (miR-29b) expression. Immunocytochemical assay was used to observe TGF-ß expression. The wound healing assay was conducted to evaluate migration of HTFs. Dual-luciferase assay was employed to identify interaction between p53 and miR-29b in HTFs. RESULTS: Vimentin was extensively distributed in HTFs cells. HTFs at density of 5 × 104 cells/ml and 6 days exhibited the best growth. The p53 level in TGF-ß treatment group was significantly higher compared to that in blank group (p < 0.01). miR-29b level in siRNA targeting p53 group was significantly increased compared to that in blank group (p < 0.01). siRNA targeting p53 could significantly inhibit HTFs migration compared to that in single TGF-ß treating HTFs group (p < 0.01). Relative luciferase activity was significantly increased in p53 overexpressed HTFs compared to that in cells transfected with empty pcDNA3.0 plasmid (p < 0.01). CONCLUSIONS: p53 inhibited expression of TGF-ß, suppressed HTFs migration and inhibited HTFs growth, by reducing miR-29b expression and interacting with miR29b gene in HTFs.

TENON CAPSULE-VITREOUS CAVITY FISTULA AFTER HYDROGEL SCLERAL BUCKLE REMOVAL.

PURPOSE: To report a rare case of vitreous cavity-Tenon capsule fistula formation after removal of a symptomatic hydrogel scleral buckle. METHODS: Case report. RESULTS: A 43-year-old man presented with chronic headache and involuntary gaze deviation for over 1 year after hydrogel scleral buckle surgery 25 years prior. After removal of the scleral buckle, the patient developed a fluid-filled inflation of the buckle capsule, surrounding a previously noted area of severe scleral thinning. Ocular ultrasonography suggested a fistulous connection between the vitreous cavity and the sub-Tenon space in the area of scleral thinning. There was resolution of diplopia and headache postoperatively, with stability of the fluid collection on clinical examination. Because of high risk of further surgery and resolution of the patient's symptoms, conservative management was elected. CONCLUSION: This is the first report, to the best of our knowledge, of Tenon capsule-vitreous cavity fistula formation after scleral buckle explantation. Because of innate ability to expand, as well as tendency to become friable, hydrogel buckles have a higher risk of requiring removal and of complications from explantation, respectively. Our patient experienced relief of symptoms, without complication from the fistula, and was successfully managed conservatively.

Tube Shunt Revision With Excision of Fibrotic Capsule Using Mitomycin C With and Without Ologen-a Collagen Matrix Implant: A 3-Year Follow-up Study.

PRECIS: Tube revision with capsule excision in failed glaucoma drainage devices (GDDs) has good medium-term success effectively reducing the intraocular pressure (IOP) and medication burden. Implantation of Ologen may limit the complications, particularly erosion. PURPOSE: To evaluate the 36-month outcomes of tube shunt revision with capsule excision using Mitomycin C (MMC) versus MMC with Ologen-a collagen matrix implant. MATERIALS AND METHODS: Twenty-three eyes with failed GDD underwent tube revision with fibrotic capsule excision. 12 of them received a MMC application whereas the other 11 also received an Ologen implant. Qualified success, changes in IOP, medication burden, and complication rates were evaluated and compared. RESULTS: Three years post-revision, qualified success for the whole cohort was 58% with no significant difference between the MMC group (52%) and MMC+Ologen group (67%; P=0.606). Mean survival time for each group was 27.4 and 29.8 months, respectively. With no intergroup differences through 3 years, capsule excision leads to a significant decrease in IOP from 28.6±6.5 to 15.1±4.3 mm Hg (47% reduction) and in antiglaucoma medications, from 3.6±1.2 to 2.5±1.3 mm Hg (30% reduction; P<0.001). Complication rates were significantly lower in the MMC+Ologen group (27%) compared with the MMC group (75%; P=0.022). Plate erosion happened in 25% of the eyes in the MMC group which required excision of the tube and plate, but no such complication was observed in the MMC+Ologen group. CONCLUSIONS: Revision of a failed tube shunt by excision of the encapsulated bleb offers good medium-term outcomes by reducing the IOP and glaucoma medications. Although the addition of Ologen did not affect the medium-term success, IOP, or medication burden, its implantation yielded significantly lower complication rates.

LncRNA NR_003923 promotes cell proliferation, migration, fibrosis, and autophagy via the miR-760/miR-215-3p/IL22RA1 axis in human Tenon's capsule fibroblasts.

Noncoding RNAs (ncRNAs), including long ncRNAs (lncRNA) have manifested an important role in the pathophysiology of many diseases. Glaucoma is a primary cause of irreversible blindness worldwide. However, the involvement of lncRNAs in glaucoma remains largely unknown. Here, we performed the lncRNA expression assay based on clinical tissues and identified a specific functional lncRNA, NR_003923, and investigated its potential role in glaucoma. Knockdown of NR_003923 in human Tenon's capsule fibroblast cells (HTFs) inhibited TGF-ß-induced cell migration, proliferation, fibrosis, and autophagy. The dual luciferase reporter assay confirmed that miR-760 and miR-215-3p interacted with NR_003923. miR-760 and miR-215-3p inhibitor reversed the effects of NR_003923 and TGF-ß-induced cell apoptosis. Moreover, the expression of miR-760 and miR-215-3p was decreased in glaucoma comparing with control. Furthermore, through microarray we found IL22RA1 was increased in glaucoma and both of miR-760 and miR-215-3p bound to the 3' UTR of IL22RA1. Overexpression of IL22RA1 enhanced HTFs migration and proliferation, while miR-760 and miR-215-3p mimics reversed these promotive biological roles induced by IL22RA1. In conclusion, NR_003923 and IL22RA1 might contribute to glaucoma progression and be a novel and potential biomarkers and therapeutic targets for glaucoma.

Investigation of Conjunctival Fibrosis Response Using a 3D Glaucoma Tenon's Capsule + Conjunctival Model.

Purpose: Surgical techniques such as trabeculectomy aim to treat glaucoma by making an incision into the scleral tissue, to create an alternative drainage pathway for aqueous to flow into the sub-Tenon's/subconjunctival space. However, tissue fibrosis and wound healing occurring after the procedures can reduce the success rate. This study aims to investigate the synergistic effects of aqueous humor in combination with shear stress on the fibrosis response occurring in Tenon's capsule and conjunctival tissue (TCCT) after glaucoma surgery. Methods: Two-dimensional (2D) and 3D in vitro TCCT models were constructed by seeding porcine Tenon's capsule + conjunctival fibroblasts in collagen gel. These were used to investigate key growth factors (singular and natural form) with shear stress, which are believed to influence tissue fibrosis after glaucoma surgery. In addition to cell proliferation assessments, a nondestructive assay to quantify neocollagen synthesis in TCCT models, in response to these factors, has been applied up to 14 days. Results: TCCT fibroblast proliferation increased significantly with doses of TGF-ß, TNF-α, and VEGF, in comparison with the control. Furthermore, fibroblasts exposed to 50% aqueous humor had significantly increased proliferation and actin expression. Shear stress-induced mechanotransduction was also found to promote metabolic activity across experimental conditions. Neocollagen labeling cross validated the fibrosis process. Conclusions: Shear stress appeared to enhance the influence of key growth factors and further promoted fibrotic response within the model. These findings offer a useful insight for further study into the wound-healing response triggered by aqueous fluid outflow after glaucoma surgery.

An in vitro study of scarring formation mediated by human Tenon fibroblasts: Effect of Y-27632, a Rho kinase inhibitor.

Scar formation is the most common cause for failure of glaucoma filtration surgery because of increased fibroblast proliferation and activation. We have now examined the effect of Y-27632, a Rho-associated protein kinase (ROCK) inhibitor, on postsurgical scarring formation in human Tenon fibroblasts (HTFs). Collagen gel contraction assay was used to compare contractility activity of Y-27632 with several antiglaucoma drugs. Immunofluorescence and western blotting were used to examine expression of scar formation-related factors. We found that Y-27632 inhibited collagen gel contraction, as well as α-smooth muscle actin and vimentin expression; these were promoted by treatment with latanoprost, timolol, or transforming growth factor (TGF)-ß. To investigate the effect of Y-27632 in postsurgical scarring, we mimicked TGF-ß secretion by stimulating HTFs with TGF-ß prior to Y-27632 treatment. HTFs cultured in the presence of TGF-ß significantly increased gel contraction. In contrast, when HTFs were treated with 10µM Y-27632, contraction was significantly inhibited. Furthermore, Y-27632 reduced TGF-ß-induced phosphorylation of mitogen-activated protein kinase signalling. These results suggest that ROCK inhibitors may inhibit fibrosis by inhibiting transdifferentiation of Tenon fibroblasts into myofibroblasts and by inhibiting TGF-ß signalling after surgery through mitogen-activated protein kinase pathway suppression. These results implicate that ROCK inhibitors may improve outcomes after filtering surgery with a potential antiscarring effect, while latanoprost and timolol may induce fibrosis. SIGNIFICANCE OF THE STUDY: Scar formation is the primary cause of failure after glaucoma filtration surgery. A ROCK inhibitor, Y-27632, has been introduced as a novel potential antiglaucoma treatment to reduce intraocular pressure. The aim of our study was to elucidate the effect of Y-27632 on scarring formation after glaucoma filtration surgery, in direct comparison with other antiglaucoma drugs. Our findings thus suggested that Y-27632 may inhibit fibrosis and improve outcome after glaucoma filtration surgery through inhibition of transdifferentiation of Tenon fibroblasts into myofibroblasts, and the TGF-ß and MAPK signalling after surgery, while latanoprost and timolol may induce fibrosis.

Effects of titanium dioxide nanoparticles on the inhibition of cellular activity in human Tenon's fibroblasts under UVA exposure.

PURPOSE: To investigate the effect of titanium dioxide (TiO2) nanoparticles on the inhibition of the in vitro cellular activity of human Tenon's fibroblasts (HTFs) under UVA exposure. METHODS: The effects of TiO2 nanoparticles on human Tenon's fibroblasts were evaluated after 1, 4, 6, and 24 h of exposure to UVA at levels of 2.5, 5.0, and 10 J/cm2. The methyl thiazolyl tetrazolium (MTT) assay was performed to measure the suppression of cellular metabolic activity. The lactate dehydrogenase (LDH) assay was performed to determine the extent of cell membrane damage. Flow cytometric analysis and inverted phase-contrast and electron microscopy were performed. The scratch wound assay was performed to visualize suppression of cellular migration. RESULTS: MTT assay values were similar between the UVA-exposed groups and the control group without UVA exposure. However, the combined exposure of TiO2 nanoparticles and UVA exposure induced significant dose-dependent inhibition of cellular viability and damage to HTFs, especially at concentrations of TiO2 equal to or greater than 100 µg/mL and 2.5 J/cm2 of UVA irradiation. Changes in cellular morphology increased in a dose-dependent pattern with a TiO2 concentration greater than 100 µg/mL under UVA exposure. At a TiO2 concentration of 150 µg/mL, damage to the cellular morphology of the HTFs was significantly increased, and nanoparticles were seen inside of the cytoplasm in the affected HTFs exposed to UVA. There was a significant reduction of cellular migration at TiO2 concentrations higher than 150 µg/mL. CONCLUSION: TiO2 nanoparticles inhibited the cellular activity of HTFs under UVA irradiation and showed potential for use to prevent the wound scarring of Tenon's fibroblasts. Further studies will be necessary to determine the optimal concentration of TiO2 nanoparticles and UVA exposure dose for clinical applications.

Tacrolimus inhibits proliferation and induces apoptosis by decreasing survivin in scar fibroblasts after glaucoma surgery.

OBJECTIVE: To investigate the effect of tacrolimus on the proliferation of fibroblasts after glaucoma surgery. MATERIALS AND METHODS: Biopsy was applied in this study. Under aseptic conditions, tissues were collected from rabbits, cut into small pieces and cultured. Morphology of fibroblasts was observed under a microscope. Features of fibroblasts were identified via immunocytochemistry and reverse transcription-polymerase chain reaction (RT-PCR). Western blotting and RT-PCR were performed to detect the expressions of related proteins after treatment. Flow cytometry and cell counting kit-8 (CCK-8) assay were employed to examine the proliferation of human Tenon's capsule fibroblasts (HTFs) after tacrolimus treatment. RESULTS: Tacrolimus decreased the levels of survivin and α-smooth muscle actin (α-SMA) after transforming growth factor-ß (TGF-ß) treatment. Besides, it inhibited proliferation and induced apoptosis of HTFs. CONCLUSIONS: Tacrolimus reduces proliferation and promotes apoptosis of HTFs by inhibiting the expression of survivin, which may be a strategy for treating hypertrophic scar after glaucoma surgery.

Is Autophagy Dysfunction a Key to Exfoliation Glaucoma?

In this short report we review previous work toward the identification of the protein and cellular sources of exfoliation glaucoma and described our recent finding on dysfunction of autophagy in Tenon capsule fibroblasts obtained from exfoliation syndrome glaucoma patients at the time of surgery and discuss the potential implications of these findings for understanding the cellular sources of the disease.