Evidence of impaired mitochondrial cellular bioenergetics in ocular fibroblasts derived from glaucoma patients.

Glaucoma is a progressive optic neuropathy characterized by the neurodegeneration of the retinal ganglion cells (RGCs) resulting in irreversible visual impairment and eventual blindness. RGCs are extremely susceptible to mitochondrial compromise due to their marked bioenergetic requirements and morphology. There is increasing interest in therapies targeting mitochondrial health as a method of preventing visual loss in managing glaucoma. The bioenergetic profile of Tenon's ocular fibroblasts from glaucoma patients and controls was investigated using the Seahorse XF24 analyser. Impaired mitochondrial cellular bioenergetics was detected in glaucomatous ocular fibroblasts including basal respiration, maximal respiration and spare capacity. Spare respiratory capacity levels reflect mitochondrial bio-energetic adaptability in response to pathophysiological stress. Basal oxidative stress was elevated in glaucomatous Tenon's ocular fibroblasts and hydrogen peroxide (H2O2) induced reactive oxygen species (ROS) simulated the glaucomatous condition in normal Tenon's ocular fibroblasts. This work supports the role of therapeutic interventions to target oxidative stress or provide mitochondrial energetic support in glaucoma.

Anti-glaucoma medications lowered decorin and altered profibrotic proteins in human tenon's fibroblasts.

Long term exposure to anti-glaucoma medications (AGMs) leads to an increase in extracellular matrix (ECM) accumulation in primary glaucoma patients. This study aims to evaluate the effect of topical AGMs in primary human tenon's fibroblasts (HTFs) and analyze the expression of profibrotic and anti-fibrotic proteins. Primary HTFs were cultured from patients undergoing cataract (control) and trabeculectomy. The different types of AGMs in single/multiple combinations (BB, PG, AA, CAI, CH, combinations of 3- PG + AA + CAI, 4A- BB + PG + AA + CAI, 4B- BB + PG + CAI + CH and 5- BB + PG + AA + CAI + CH) on chronic exposure were tested for cell viability using MTT assay and morphological alterations. Profibrotic proteins mainly SPARC, LOXL2, COL1A1 and anti-fibrotic DCN were analyzed in treated HTFs using q-PCR and ELISA. Sirius red staining and collagen gel contraction (CGC) assay were performed to assess collagen synthesis and the contractility of HTFs, respectively. Except for AA and CH, the other AGMs at a higher concentration were found to decrease the cell viability of HTFs. The morphology of HTFs were altered on exposure to BB, CH and AA; Profibrotic proteins i.e., SPARC, LOXL2 and COL1A1 were significantly increased (p < 0.05) on exposure to a combination of AGMs with TGF-ß1, whereas the anti-fibrotic DCN expression was significantly lowered (p < 0.05) in single/multiple AGM exposure. Sirius red staining showed increased collagen synthesis with combinations of AGMs with TGF-ß1. Meanwhile, HTFs showed increased collagen gel contraction with TGF-ß1, CAI and CH. This study reveals that altered profibrotic proteins, with significantly lowered DCN on chronic exposure of AGMs in HTFs.

Understanding the cellular response of human tenon fibroblast on polycaprolactone-Aloe vera blend fiber.

The signalling response is determined by the cell's reaction to different biochemical and biophysical inputs such as stiffness, topological, and structural alignment. The surface patterns at the nano-scale can be an influential factor in cell signalling behaviour. It is important to understand the cellular response to the biophysical cues for biomedical applications. Biomaterials have an important role in regenerative tissue engineering. In this study, we have fabricated electrospun polycaprolactone (PCL) and PCL-Aloe vera (PCL-AV) nanofibrous matrix and studied its effect on the human tenon fibroblast (HTF) cellular and morphological changes. The electrospun fibers were characterized using Scanning Electron Microscope (SEM), Fourier Transform Infrared spectroscopy (FTIR), Atomic Force Microscopy (AFM) and Brunaur, Emette and Teller (BET) analysis for their morphology, composition, topography, surface area and porosity. The results revealed fiber size, roughness and porosity has been altered by addition of AV. The HTF cell viability, proliferation and expression of focal adhesion proteins, such as FAK, Ezrin, Vasp and Cofilin on the PCL-AV fiber matrix were examined. The results showed a change in cellular morphology and a significant change in the cofilin phosphorylation on PCL-AV nanofiber. The influence of Aloe vera composition on the nano-dimension of the PCL has made a significant impact on the cellular morphology at both gene and protein levels. This observation suggests that AV composition in the nanofiber can significantly influence the HTF cellular adhesions.

The Effect of Irradiated Riboflavin in Human Tenon's Fibroblast - A Study on Cellular Viability.

PURPOSE/ AIM: The main purpose of this work is to study the cellular viability effect of irradiated riboflavin in cultured human tenon fibroblasts. MATERIALS AND METHODS: The tenon tissue was harvested from a patient undergoing strabismus surgery. The human tenon fibroblast cell culture and isolation were performed according to the standard laboratory cell culturing protocol. The cells were divided into three groups: control, treatment with irradiated and non-irradiated riboflavin. There were five different concentrations (0.00156%, 0.003125%, 0.00625%, 0.0125%, 0.025%) in each group of riboflavin. The fibroblasts were treated with riboflavin and the cellular viability was assessed at 24-hour and 48-hour post treatment with MTT 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide colorimetric assay. The absorbance values were analysed using Magellan microplate reader data analysis. A triplicate of readings was taken. The data were presented as mean ± standard deviation of the triplicates. Statistical analysis was performed with Statistical Package for Social Sciences (SPSS) analysis version 23. RESULTS: Irradiated riboflavin caused a concentration-dependent cell death in human tenon fibroblast cell culture (p < .05). The antiproliferative difference between irradiated and non-irradiated riboflavin was significant up to 48 hours (p < .05). Post hoc multiple comparisons showed higher concentrations of irradiated riboflavin (0.0125% and 0.025%) caused more reduction in cellular viability in human tenon fibroblast cells (p < .05). The duration of treatment is not a causative factor in this study. CONCLUSIONS: This pilot experiment demonstrated that irradiated riboflavin induced cell death in human tenon fibroblast culture in a concentration-dependent manner, but is not time-dependent. Further exploratory investigations should be performed to determine the mechanism of cell death. We postulate that apoptosis occurred in these irradiated riboflavin-treated cells.

Zinc oxide nanoparticle-triggered oxidative stress and autophagy activation in human tenon fibroblasts.

Glaucoma is a leading cause of irreversible blindness worldwide due to elevated intraocular pressure, and filtering surgery can efficiently control intraocular pressure of glaucoma patients. However, failure of filtering surgery commonly results from scarring formation at the surgical site, in which fibroblast proliferation plays an essential role in the scarring process. Our previous study has demonstrated that zinc oxide (ZnO) nanoparticles could efficiently inhibit human tenon fibroblasts (HTFs) proliferation. The present study aimed to explore the underlying mechanism involved in oxidative stress and autophagy signaling in zinc oxide (ZnO) nanoparticles-induced inhibition of HTFs proliferation. In this study, we investigated the effect of ZnO nanoparticles on HTFs proliferation, mitochondrial function, ATP production and nuclear morphology. Moreover, we also explored the interactions between ZnO nanoparticles and HTFs, investigated the influence of ZnO nanoparticles on the autophagosome formation, the expression of autophagy-related 5 (Atg5), Atg12 and Becn1 (Beclin 1), and the level of light chain 3 (LC3). The results suggested that ZnO nanoparticles can efficiently inhibit HTFs proliferation, disrupt the mitochondrial function, attenuate the adenosine triphosphate (ATP) generation, and damage the nuclear morphology of HTFs. Exposure of HTFs to ZnO nanoparticles can also induce the shifted peak, elevate the expression of Atg5, Atg12 and Becn1, enhance the autophagosome formation, and promote the LC3 expression, and thus activate autophagy signaling. Overall, ZnO nanoparticles can apparently trigger oxidative stress and activate autophagy signaling in HTFs, and thus inhibit HTFs proliferation and mediate HTFs apoptosis.

Role of lncRNA in the proliferation mechanism of Tenon fibroblasts / 中华实验眼科杂志

At present, the mechanism of scar formation in filtering area after glaucoma filtering surgery has not been fully understood.It is believed that stimulation of cytokines and growth factors during postoperative wound healing causes excessive activation of fibroblasts in the Tenon capsule, resulting in the deposition of extracellular matrix and scar formation.Long non-coding RNA (lncRNA) is a kind of RNA sequence that does not encode proteins.It can regulate cell proliferation, apoptosis and become a precursor of microRNA, and its specific expression has been proved and plays an important regulatory role in various fibrotic tissue.With the deepening of research, lncRNA has also been found to be involved in the formation and development of filtering bleb scarring after glaucoma filtering surgery.Previous studies have shown that lncRNA is specifically expressed in Tenon capsule tissues after glaucoma filtering surgery, and different lncRNAs can affect the proliferation of fibroblasts in Tenon capsule or the synthesis of extracellular matrix through different ways, which can affect the formation of fibroblasts scarring.In this article, the role of different lncRNAs in the proliferation of Tenon fibroblasts and its potential in treating bleb scarring were summarized so as to provide a new direction for the treatment of postsurgical scarring.

Zinc oxide nanoparticles induce human tenon fibroblast apoptosis through reactive oxygen species and caspase signaling pathway.

Glaucoma is the leading cause of irreversible blindness in the world and trabeculectomy remains still the most commonly performed filtration surgery. Failure of trabeculectomy is due to the formation of scarring, which is associated with the increased fibroblast proliferation, activation, and collagen deposition at the site of the drainage channel with subconjunctival fibrosis. Our previous study has revealed that zinc oxide (ZnO) nanoparticles could efficiently decrease the expressions of TGF-ß1 and inhibit fibroblast-mediated collagen lattice contraction. However, the mechanism underlying ZnO nanoparticle-induced fibroblast apoptosis is still unclear. In the present study, we investigated the effect of ZnO nanoparticles on the reactive oxygen species (ROS) and mitochondrial membrane potential (Δψm) in human Tenon fibroblasts (HTFs). Moreover, we also explored the influence of ZnO nanoparticles on the expression of Caspase-3, Caspase-9, apoptotic protease-activating factor-1 (Apaf-1), fibroblast-specific protein-1 (FSP-1), collagen III, and E-cadherin. The results indicated that ZnO nanoparticles markedly inhibit HTFs viability and decrease the Δψm in a concentration-dependent pattern. Exposure of HTFs to ZnO nanoparticles could also induce the elevated Caspase-3, Caspase-9, and Apaf-1 expression, decrease the levels of FSP-1, collagen III, and E-cadherin expression, leading to HTFs apoptosis. Our results suggested that elevated ROS and activated Caspase signaling play a fundamental role in ZnO nanoparticle-induced HTFs apoptosis.

Zinc oxide nanoparticles ameliorate collagen lattice contraction in human tenon fibroblasts.

Glaucoma is a major cause of irreversible blindness in the world and filtering surgery is commonly carried out to control intraocular pressure. Failure of filtering surgery is usually due to postoperative scarring, and fibroblast proliferation, collagen production and subconjunctival fibrosis play a prominent role in obstructing aqueous humor from the anterior chamber to the subconjunctival space. Zinc oxide (ZnO) nanoparticles have been widely applied in biomedical fields. However, the influence of ZnO nanoparticles on human tenon fibroblasts (HTFs) is still unclear. In the present study, we first explored the effects of various concentrations of ZnO nanoparticles on HTFs proliferation, reactive oxygen species (ROS) generation, cell cycle arrest, and apoptosis. Further, we determined the changes of transforming growth factor-ß (TGF-ß1), fibronectin (FN) extra domain A (ED-A), and procollagen I carboxyterminal propeptide (PICP) at mRNA and protein levels, explored the effect of ZnO nanoparticles on the collagen lattice contraction in HTFs. The results indicated that ZnO nanoparticles can efficiently inhibit HTFs proliferation, elevate ROS production level, and induce cell cycle arrest at G2/M phase, leading to HTFs apoptosis. ZnO nanoparticles can also decrease the expressions of TGF-ß1, ED-A, and PICP at mRNA and protein levels; significantly prevent fibroblast-mediated collagen lattice contraction. Taken together, ZnO nanoparticles can efficiently ameliorate collagen lattice contraction in HTFs, and may be a promising antifibrotic agent in glaucoma filtration surgery. Our findings provide a new insight on anti-scar formation after glaucoma filtration surgery by using ZnO nanoparticles.

Effect of cytoglobin overexpression on extracellular matrix component synthesis in human tenon fibroblasts.

BACKGROUND: Conjunctival filtering bleb scar formation is the main reason for the failure of glaucoma filtration surgery. Cytoglobin (Cygb) has been reported to play an important role in extracellular matrix (ECM) remodeling, fibrosis and tissue damage repairing. This study aimed to investigate the role of Cygb in anti-scarring during excessive conjunctival wound healing after glaucoma filtration surgery. METHODS: Cygb was overexpressed in human tenon fibroblasts (hTFs) by transfecting hTFs with lentiviral particles encoding pLenti6.2-FLAG-Cygb. Changes in the mRNA and protein levels of fibronectin, collagen I, collagen III, TGF-ß1, and HIF1α were determined by RT-PCR and western blotting respectively. RESULTS: After Cygb overexpression, hTFs displayed no significant changes in visual appearance and cell counts compared to controls. Whereas, Cygb overexpression significantly decreased the mRNA and protein expression levels of collagen I, collagen III and fibronectin compared with control (p < 0.01). There was also a statistically significant decrease in the mRNA and protein levels of TGF-ß1 and HIF-1α in hTFs with overexpressed Cygb compared with control group (p < 0.05). CONCLUSION: Our study provided evidence that overexpression of Cygb decreased the expression levels of fibronectin, collagen I, collagen III, TGF-ß1 and HIF-1α in hTFs. Therefore, therapies targeting Cygb expression in hTFs may pave a new way for clinicians to solve the problem of post-glaucoma surgery scarring.

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.

Effect of cytoglobin overexpression on extracellular matrix component synthesis in human tenon fibroblasts

BACKGROUND: Conjunctival filtering bleb scar formation is the main reason for the failure of glaucoma filtration surgery. Cytoglobin (Cygb) has been reported to play an important role in extracellular matrix (ECM) remodeling, fibrosis and tissue damage repairing. This study aimed to investigate the role of Cygb in anti-scarring during excessive conjunctival wound healing after glaucoma filtration surgery. METHODS: Cygb was overexpressed in human tenon fibroblasts (hTFs) by transfecting hTFs with lentiviral particles encoding pLenti6.2-FLAG-Cygb. Changes in the mRNA and protein levels of fibronectin, collagen I, collagen III, TGF-ß1, and HIF1α were determined by RT-PCR and western blotting respectively. RESULTS: After Cygb overexpression, hTFs displayed no significant changes in visual appearance and cell counts compared to controls. Whereas, Cygb overexpression significantly decreased the mRNA and protein expression levels of collagen I, collagen III and fibronectin compared with control (p < 0.01). There was also a statistically significant decrease in the mRNA and protein levels of TGF-ß1 and HIF-1α in hTFs with overexpressed Cygb compared with control group (p < 0.05). CONCLUSION: Our study provided evidence that overexpression of Cygb decreased the expression levels of fibronectin, collagen I, collagen III, TGF-ß1 and HIF-1α in hTFs. Therefore, therapies targeting Cygb expression in hTFs may pave a new way for clinicians to solve the problem of post-glaucoma surgery scarring.

Effects of vascular endothelial growth factor on the fibrosis and the expression of secreted protein acidic and rich in cysteine in cultured human Tenon's fibroblast / 上海交通大学学报（医学版）

Objective:To investigate the effects of vascular endothelial growth factor (VEGF) on the expression of secreted protein acidic and rich in cysteine (SPARC) and the fibrosis in cultured human Tenon's fibroblast (HTF) in vitro. Methods:HTF cells were obtained from Tenon's capsule tissues of patients undergoing strabismus surgery. Immunofluorescence was used to identify the HTF cells. HTF cells were cultured with different concentrations of VEGF, and which were divided into four groups, i.e., 0 ng/mL group, 25 ng/mL group, 50 ng/mL group and 100 ng/mL group. The expression of SPARC, collagen- , and matrix metalloprotein 9 (MMP-9) and the activity of extracellular signal-regulated kinase (ERK) pathway were analyzed by Western blotting and real-time quantitative PCR (qPCR). The abilities of proliferation and migration of HTF cells were detected by MTS assay and scratch test, respectively. Results:HTF cells were observed and identified by inverted phase contrast microscope and immunofluorescence. Under the stimulation of VEGF, the expression of protein and mRNA of SPARC, collagen-I and MMP-9 of HTF cells in other three groups were increased compared with 0 ng/mL group; the phosphorylation activities of ERK pathway were up-regulated, and the proliferation and migration abilities of HTF cells were up-regulated. And the effect was the most obvious in the 50 ng/mL group. Conclusion:VEGF is involved in promoting the fibrosis of HTF cells accompanied by the up-regulation of the SPARC, which suggests SPARC may become a potential regulatory site.

Triptolide inhibits TGF-ß-induced matrix contraction and fibronectin production mediated by human Tenon fibroblasts.

AIM: To determine if triptolide influences the contractility and fibronectin production in human Tenon fibroblasts (HTFs). METHODS: HTFs were cultured in type I collagen gels with or without transforming growth factor beta (TGF-ß) and/or triptolide. The diameter of the collagen gel was used to measure contraction. Immunoblot analysis was used to quantify myosin light chain (MLC) phosphorylation and integrin expression. Laser confocal fluorescence microscopy was used to monitor the formation of actin stress fibers. Fibronectin production was measured with an enzyme immunoassay. RESULTS: Triptolide inhibition of contraction in TGF-ß-induced collagen gel mediated by HTFs was dose-dependent and statistically significant at 3 nmol/L (P<0.05) and maximal at 30 nmol/L and significantly time dependent at 2d (P<0.05). Triptolide reduced TGF-ß-induced expression of integrins α5 and ß1, phosphorylation of MLC, and formation of stress fibers in HTFs. Furthermore, the inhibition of triptolide on the attenuated TGF-ß-induced production of fibronectin by HTFs was concentration-dependent and significant at 1 nmol/L (P<0.05) and maximal at 30 nmol/L. CONCLUSION: Triptolide suppress the contractility of HTFs induced by TGF-ß and the production of fibronectin by these cells. It is promising that triptolide treatment may possibly inhibit scar formation after glaucoma filtration surgery.

Inhibitory Effects of Angiotensin II Receptor Blockade on Human Tenon Fibroblast Migration and Reactive Oxygen Species Production in Cell Culture.

PURPOSE: We investigate the effect of angiotensin receptor blockade on the migration of human Tenon fibroblasts (HTF), using irbesartan, an angiotensin II receptor type 1 (AT1R) blocker (ARB) as a potential antifibrotic agent in glaucoma filtration surgery. METHODS: Confluent HTF cultures were scratched with a 1 mL pipette tip and treated with either irbesartan (10, 50, and 100 µg/mL) or angiotensin II (2 µg/mL). The extent of HTF migration up to 30 hours, and cell number and morphology at 72 hours was evaluated. To assess the effect on reactive oxygen species (ROS) level, HTF were treated with either irbesartan (10 µg/mL) or angiotensin II (2 µg/mL) for 24 hours after scratching, and then stained with dihydroethidium (DHE) before evaluation by confocal microscopy. RESULTS: Irbesartan inhibited HTF migration by 50% to 70% compared to controls (P < 0.05). Levels of ROS were almost completely attenuated by irbesartan (DHE fluorescence intensity of 5.68E-09) (P < 0.05). Irbesartan reduced cell numbers by 50% and induced morphologic changes with loss of pseudopods (P < 0.05). Conversely, angiotensin II increased cell numbers up to 4-fold while retaining cell viability. CONCLUSIONS: Irbesartan inhibited HTF migration and ROS production. It also reduced cell numbers and altered HTF morphology. Angiotensin II increased cell number without altering morphology. This initial study warrants future investigations for further potential antifibrotic effects of this drug. TRANSLATIONAL RELEVANCE: This in vitro study focused on investigations of irbesartan's effects on HTF migration, ROS production, as well as HTF cell numbers and morphology. It suggests a potential therapeutic strategy worth further exploration with a view towards postoperative wound healing modulation in glaucoma filtration surgery.

Effect of Lignocaine Concentration on Human Fibroblasts Growth in Eyes Undergoing Trabeculectomy: An in vitro Study.

PURPOSE: To evaluate the effect of lignocaine on growth and apoptosis indication of primary human Tenon's capsule fibroblast (HTFs) in an in vitro model. PATIENTS AND METHODS: Tenon's capsule tissue obtained from patients undergoing trabeculectomy were grown in cell culture medium. The effect of different concentrations of lignocaine (0.5, 1.0, 1.5, and 2%) on the morphology and growth of the fibroblasts was studied using microscopy, cell viability, and proliferation assay, and apoptosis was detected using the FITC Annexin V Apoptosis Kit. RESULTS: Morphological changes similar to those of apoptotic cells, including cytoplasmic vacuolation, shrinkage, and rounding were visualized in the cells treated with concentrations greater than 1.0% (i.e., 1.5, 2.0%). Though proliferation inhibition was found with all four concentrations (0.5-2.0%), the viability of cells decreased from 1.0% lignocaine. CONCLUSION: 0.5% lignocaine prevents proliferation of fibroblasts without causing apoptosis in vitro.

Paclitaxel inhibits cell proliferation and collagen lattice contraction via TGF-ß signaling pathway in human tenon's fibroblasts in vitro.

As an anti-microtubule agent, paclitaxel has been widely applied clinically. However, the effects of paclitaxel on human tenon's fibroblast (HTF) proliferation and migration in vitro was still unclear. In the present study, we explored the influences of paclitaxel on HTF cell proliferation, cell viability, cell cycle phase distribution under various concentrations of paclitaxel (i.e., 0, 10(-8), 10(-7), 10(-6)mol/l) via real-time cell electronic system and flow cytometry, further determined the expression of TGF-ß1 and connective tissue growth factor (CTGF) after treatment with different concentrations of paclitaxel. Moreover, extra cellular matrix production and collagen lattice contraction assay were also explored. The results indicate that paclitaxel could apparently inhibit the cell viability, induces the elevation of S and G2/M phases of HTFs, and downregulates the expression of both TGF-ß1 and CTGF. Meanwhile, the levels of fibronectin extra domain A (EDA), collagen and collagen lattice contraction were apparently reduced after treatment with paclitaxel. Overall, paclitaxel could apparently inhibit the proliferation of HTFs and leads to cell cycle arrest at both S and G2/M phases, attenuates the generation of collagen and collagen lattice contraction, decreases the expressions of TGF-ß1, CTGF and fibronectin EDA. The inhibitory mechanism of paclitaxel on HTFs is involved in TGF-ß1 signaling pathway.

The evaluation of human tenon's fibroblasts and endothelial cell responses to antifibrotics alone and in combination with α-tocopherol.

PURPOSE: We aimed to evaluate the influence of current antifibrotic agents as well as the possible results obtained by combining these agents. This study included α-tocopherol, a strong antifibrotic and an efficient neuromediator of pathways used by other agents. MATERIALS AND METHODS: Mitochondrial Bcl-2, Bax, cytochrome c and cytoplasmic caspase-3 expression, as well as toxic effect patterns, mitosis and cellular reactions due to α-tocopherol alone or combined with paclitaxel, mitomycin C and 5-flurouracil (5-FU), was studied in series obtained from human endothelial and primary Tenon's fibroblast cell cultures. RESULTS: The strongest apoptotic effect in both cell groups belonged to paclitaxel, followed by mitomycin C, and despite the overall suppressive effect of the α-tocopherol combination, mitomycin C increased its efficiency on the endothelial cells. The apoptosis/necrosis ratio was highest in α-tocopherol and lowest in paclitaxel, with α-tocopherol generally decreasing necrosis. Bax was observed at a high level with mitomycin C. Cytotoxicity was the highest with paclitaxel, and the caspase-3 reaction was markedly higher with mitomycin C in both cell types. In the α-tocopherol and 5-FU slides, mitosis and a layered formation were observed. The addition of α-tocopherol reduced the cytotoxicity of all antifibrotic agents in both cell series by decreasing the cell numbers, leading to necrosis. CONCLUSIONS: Alone or in combination, the use of α-tocopherol and 5-FU is safer than other agents. By suppressing the cytotoxic effects of other antifibrotic agents, α-tocopherol is a promising drug for improving the effects of antifibrotics in many aspects of medicine. In addition, it has the potential to play a role beyond its antioxidant and antifibrotic activity in ocular surgery.

Effects of ranibizumab on the extracellular matrix production by human Tenon's fibroblast.

Anti-Vascular Endothelial Growth Factors (Anti-VEGF) agents have received recent interest as potential anti-fibrotic agents for their concurrent use with trabeculectomy. Preliminary cohort studies have revealed improved bleb morphology following trabeculectomy augmented with ranibizumab. The effects of this humanized monoclonal antibody on human Tenon's fibroblast (HTF), the key player of post trabeculectomy scar formation, are not fully understood. This study was conducted to understand the effects of ranibizumab on extracellular matrix production by HTF. The effect of ranibizumab on HTF proliferation and cell viability was determined using MTT assay (3-(4,5-dimethylthiazone-2-yl)-2,5-diphenyl tetrazolium). Ranibizumab at concentrations ranging from 0.01 to 0.5 mg/mL were administered for 24, 48 and 72 h in serum and serum free conditions. Supernatants and cell lysates from samples were assessed for collagen type 1 alpha 1 and fibronectin mRNA and protein level using quantitative real time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). After 48-h, ranibizumab at 0.5 mg/mL, significantly induced cell death under serum-free culture conditions (p < 0.05). Ranibizumab caused significant reduction of collagen type 1 alpha 1 (COL1A1) mRNA, but not for fibronectin (FN). Meanwhile, COL1A1 and FN protein levels were found upregulated in treated monolayers compared to control monolayers. Ranibizumab at 0.5 mg/mL significantly reduced cell viability in cultured HTF. From this study, we found that single application of ranibizumab is inadequate to induce the anti-fibrotic effects on HTF, suggesting the importance of adjunctive therapy. Further studies are underway to understand mechanism of actions of ranibizumab on HTF.

Silibinin inhibits myofibroblast transdifferentiation in human tenon fibroblasts and reduces fibrosis in a rabbit trabeculectomy model.

PURPOSE: To investigate the effect of silibinin in myofibroblast transdifferentiation and in animal trabeculectomy models. METHODS: The effect of silibinin on the expression of α-smooth muscle actin (α-SMA) and vimentin in response to transforming growth factor-ß1 (TGF-ß1) was determined in human tenon fibroblasts (HTFs). Cell migration and collagen contraction arrays were used to demonstrate the functionality of silibinin-modulated HTFs. ELISA analysis was used to determine the effect of silibinin on the release of type 1 collagen and connective tissue growth factor (CTGF). The effect of silibinin on the activation of the TGF-ß receptor-related pathway was evaluated by Western blotting. A rabbit model of trabeculectomy was established to assess the effect of silibinin in vivo. RESULTS: TGF-ß1 elevated the expression of α-SMA and vimentin in HTFs; this elevation was inhibited by silibinin. TGF-ß1 increased cell migration and collagen contraction of HTFs, which were also suppressed by silibinin. The production of both CTGF and type 1 collagen in TGF-ß1-treated HTFs was inhibited by silibinin. The effects of silibinin on TGF-ß1-stimulated HTFs were mediated via the down-regulation of TGF-ß receptor-related SMAD signalling pathways. In the rabbit model of trabeculectomy, silibinin increased the period of decreasing intraocular pressure after trabeculectomy and reduced the production of collagen and α-SMA at the site of blebs in vivo. CONCLUSION: Silibinin inhibited the TGF-ß receptor-related signalling pathway in TGF-ß-treated HTFs and several of the downstream events associated with myofibroblast transdifferentiation. Silibinin also improved the outcome of trabeculectomies by reducing the fibrotic response in the bleb tissue in vivo.

Matrix metalloproteinase and cytokine expression in Tenon fibroblasts during scar formation after glaucoma filtration or implant surgery in rats.

Failure of surgery for glaucoma is usually due to post-surgical scarring (fibrosis), a process in which fibroblasts play a prominent role. We investigated the molecular mechanisms of such scarring by examining the expression of matrix metalloproteinases and cytokines in Tenon fibroblasts isolated from rats after glaucoma surgery. Filtration surgery was performed in one eye and implant surgery in the other; and Tenon fibroblasts were isolated from the tissue surrounding the bleb after surgery. The cells were cultured and examined for the expression of matrix metalloproteinases (MMPs) by reverse transcription-polymerase chain reaction, immunoblot and gelatin zymographic analyses. Culture supernatants were also assayed for cytokines with a multiplex array. The amounts of MMP-1 and MMP-3 mRNAs and proteins were greater in cells isolated after implant surgery than in those isolated after filtration surgery, with the progression of scar formation being more complete after the former surgery. The secretion of interleukin-6 (IL-6) by cells isolated after filtration surgery was greater than that for cells isolated after implant surgery. Depletion of IL-6 by RNA interference in cells isolated after filtration surgery increased the expression of MMP-1 and MMP-3 in these cells. These results thus suggest that the expression of MMP-1 and MMP-3 in Tenon fibroblasts is regulated by IL-6 during, and may play an important role in, scar formation after glaucoma surgery.