Proliferative response of cultured human tenon's capsule fibroblasts to platelet-derived growth factor isoforms

Background: Although platelet-derived growth factor (PDGF) has been thought to be critical in the wound-healing response of Tenon's capsule fibroblasts after glaucoma filtration surgery, no information is currently available concerning the proliferative effect of PDGF isoforms on this cell type. The aim of the present study was to evaluate the proliferative effect of PDGF-AB heterodimer and PDGF-AA and -BB homodimers on cultured human Tenon's capsule fibroblasts. Methods: Human Tenon's capsule fibroblasts, cultured under serum-free conditions, were stimulated with PDGF-AA, -AB and -BB isoforms in concentrations ranging from 1 to 100 ng/ml. Cell numbers were determined on days 1, 3, 5 and 7, using a cell counter. Results: Addition of PDGF-AB and -BB led to a dosedependent increase in cell proliferation. A maximal response (79.9% over control) was obtained after 7 days with 30 ng/ml of PDGF-BB, with an EC₅₀ of 8.9 ng/ml. The maximal increase in cell proliferation caused by PDGF-AB (30 ng/ml) was 54.9%, with an EC₅₀ of 12.5 ng/ml. Stimulation with PDGF-AA revealed a significant effect only with concentrations higher than 30 ng/ml. Conclusion: Our results indicate that PDGF-AB and -BB isoforms are potent stimulators of proliferation of human Tenon's capsule fibroblasts, suggesting that PDGF-AB and -BB isoforms play an important role in the wound-healing response after glaucoma filtration surgery.Presented in part at the annual meeting of the Deutsche Ophthalmologische Gesellschaft, Mannheim, September 1996     

Interferon-alpha and interferon-gamma sensitize human tenon fibroblasts to mitomycin-C

PURPOSE: To investigate the effect of interferon (IFN)-alpha and IFN-gamma pretreatment on mitomycin C (MMC)-induced cell death in human Tenon fibroblasts (HTFs) and the mechanisms by which IFN-alpha and IFN-gamma modulate the susceptibility of HTFs to MMC. METHODS: HTFs were pretreated with IFN-alpha and IFN-gamma for 48 hours before 5-minute application of 0.4 mg/mL MMC. Cell death after 48 hours was determined by Annexin V/propidium iodide (PI) staining and lactate dehydrogenase (LDH) release assay. Fas, Fas-ligand, and Bcl-2 expression were determined by flow cytometry. Fas associated death domain (FADD), Bax, cytochrome c, and caspase expression were determined by Western blot analysis and immunofluorescence staining. RESULTS: MMC treatment increased cell death and upregulated Fas and FADD expression, but had no effect on Fas-Ligand, Bax, Bcl-2, or cytochrome c. Neither IFN-alpha nor IFN-gamma alone induced HTF death, but each increased cell death 2 days after MMC treatment in a dose-dependent fashion. Combination IFN-alpha and IFN-gamma had a synergistic effect. IFN-alpha and IFN-gamma pretreatment increased Fas expression. Fas upregulation was associated with increased sensitivity to MMC. IFN pretreatment increased procaspase-8, procaspase-9, and procaspase-3 expression, and caspase-3 activation. Caspase-8, caspase-3, and broad caspase inhibitors, but not caspase-9 inhibitor, inhibited MMC-induced cell death in nonpretreated and IFN-pretreated cells. CONCLUSIONS: IFN-alpha and IFN-gamma enhance the susceptibility of HTFs to MMC-induced cell death through a Fas-mediated and a caspase-3-dependent pathway. Pretreatment with IFN primed HTFs to MMC, providing a potential means for initially slowing the healing response with IFN and subsequently terminating fibroblast activity through MMC-induced cell death.     

Effects of timolol, betaxolol, and levobunolol on human tenon's fibroblasts in tissue culture.

Evidence has been found suggesting that long-term therapy with topical antiglaucoma medications may decrease the success of glaucoma filtering surgery. To investigate this question further, the antiproliferative effects of the preservative benzalkonium chloride and three pure and commercially available beta-adrenergic antagonist preparations (timolol, betaxolol, and levobunolol) were studied on tissue cultures of human Tenon's capsule fibroblasts. Each drug preparation was tested on three different cell lines. Fibroblast growth was measured with tritiated thymidine uptake and hexosaminidase assays. Trypan blue uptake was used to assess cell viability microscopically. The commercially available preparations containing benzalkonium chloride and those of betaxolol and levobunolol without the preservative had similar inhibitory doses for 50% of cells. The timolol preparation without preservative was significantly less toxic than its commercially available one. The three tested beta-adrenergic blockers did not stimulate fibroblast proliferation directly in this in vitro model. Even when the cultures were washed free of the drugs, growth continued to be suppressed, suggesting that the inhibition was not reversible. An increase in fibroblasts and inflammatory cells after long-term antiglaucoma medical therapy thus may be caused not by a direct stimulation of cell proliferation but by chronic inflammation from the irritating effects of antiglaucoma medications and/or their preservatives.     

Human tenon's fibroblast-produced ifnbeta and the prevention of t-cell apoptosis

PURPOSE: Fibroblast-T-cell interactions may contribute to the development of chronic inflammation, a risk factor for trabeculectomy failure. This study was undertaken to determine whether normal and growth-arrested human Tenon's fibroblasts (HTF) can prevent cytokine deprivation-mediated T-cell apoptosis through the secretion of interferon (IFN)beta. METHODS: HTF were used either untreated or pretreated with mitomycin-C (MMC; 0.1 or 0.4 mg/ml) or 5-fluorouracil (5FU; 25 or 50 mg/ml). IL2-deprived T cells were cocultured with HTF. T-cell viability was measured at specific time points. Human Tenon's fibroblast-conditioned medium was used either untreated or treated with a neutralizing antibody against IFNbeta to block its action, after which IL2-deprived T cells were added and T-cell viability was measured. An image analysis system was used to determine the production of IFNbeta by either untreated or MMC-treated HTF. RESULTS: T-cell viability was significantly greater when T cells were cocultured with both untreated and growth-arrested HTF than when T cells were cultured alone (day 7, P = 0.0001). Neutralizing the action of IFNbeta blocked HTF-mediated T-cell rescue from apoptosis. Both untreated and growth-arrested HTF secrete IFNbeta, and MMC at 0.4 mg/ml appeared to increase IFNbeta production. CONCLUSIONS: Cytokine deprivation-mediated T-cell apoptosis can be prevented by the action of IFNbeta secreted by both normal and growth-arrested HTF, which suggests that growth-arrested HTF can still participate in an aggressive wound-healing reaction by mediating a persistent inflammatory phase. This may partly explain why some trabeculectomies fail in high-risk patients, despite the use of antimetabolites.     

The effect of ICG on mitomycin C cytotoxicity in human tenon fibroblasts

PURPOSE: To examine the effects of indocyanine green (ICG) with and without mitomycin C (MMC) on proliferation of cultured human Tenon fibroblasts. METHOD: Fibroblast monolayers were exposed to either MMC [0.4 mg/mL in phosphate buffered saline (PBS)] or PBS containing ICG (0.0625%, 0.125%, 0.25%, and 0.5% in 200 microL PBS) or a combination of MMC (0.4 mg/mL in PBS) and ICG (0.25% and 0.5%) for 5 minutes. Controls were exposed for 5 minutes to MMC, PBS, or culture medium containing no ICG. After treatment, the monolayers were washed and incubated in culture medium for 24, 48, 72 hours, and 1 week periods after which the number of viable cells was quantified. RESULTS: The presence of ICG alone, at concentrations ranging from 0.0625% to 0.5%, had no effect on the rate of fibroblast proliferation measured at any of the incubation periods. As expected, MMC treatment resulted in a significant reduction in viable fibroblast number (8.4+/-0.13x10(3)). ICG in combination with MMC did not significantly alter fibroblast numbers (8.5+/-0.05x10(3)) up to 1 week compared with MMC alone (8.4+/-0.12x10(3)). CONCLUSIONS: ICG at concentrations of 0.5% and below do not reduce proliferation of Tenon capsule fibroblasts. ICG did not potentiate or diminish the effect of MMC on Tenon capsule fibroblast proliferation.     

Co-treatment of suberoylanilide hydroxamic acid and mitomycin-C induces the apoptosis of rabbit tenon's capsule fibroblast and improves the outcome of glaucoma filtration surgery

PURPOSE: This study was undertaken to develop a new treatment modality that would be able to minimize fibrosis and provide better outcome with glaucoma filtration surgery (GFS). METHODS: We examined whether co-treatment with mitomycin-C (MMC) and histone deacetylase inhibitor (HDACi) suberoylanilide hydroxamic acid (SAHA) efficiently induces apoptosis on rabbit Tenon's capsule fibroblasts (TCF) in vitro. We further examined the effect of co-treatment with SAHA and MMC on the alteration of IOP and the bleb survival in rabbits following GFS. RESULTS: Co-treatment of MMC and SAHA efficiently induces apoptosis in TCFs via the up-regulation of p53 and increased phosphorylation of p53 on serine 15 and 392. Also, co-treatment of SAHA and low-dose MMC decreases IOP, prolongs bleb survival, and induces apoptosis of cells under the bleb area following GFS. CONCLUSION: This study shows that a co-treatment of SAHA and MMC could improve the outcome of GFS.     

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‐β1 (TGF‐β1) 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‐β 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‐β1 elevated the expression of α‐SMA and vimentin in HTFs; this elevation was inhibited by silibinin. TGF‐β1 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‐β1‐treated HTFs was inhibited by silibinin. The effects of silibinin on TGF‐β1‐stimulated HTFs were mediated via the down‐regulation of TGF‐β 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‐β receptor–related signalling pathway in TGF‐β‐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.     

Cellular effects of mitomycin-C on human corneas after photorefractive keratectomy

PURPOSE: To investigate the effects of mitomycin-C (MMC) on epithelial and keratocyte cell kinetics after photorefractive keratectomy (PRK) using an in vitro human cornea model. SETTING: Department of Academic Ophthalmology, Rayne Institute, St. Thomas' Hospital, London, United Kingdom. METHODS: Twenty-four human eye-bank corneas were placed in a specially designed acrylic corneal holder and cultured using the air-interface organ culture technique for up to 4 weeks. The corneas were divided into 3 groups. Group 1 consisted of 8 human corneas that had -9.00 diopter (D) myopic PRK without MMC application. Group 2 consisted of 8 corneas that had -9.00 D PRK with MMC (0.2 microg/mL) application for 1 minute on the stromal surface after ablation. Group 3 consisted of 8 corneas that had -9.00 D PRK with 2-minute exposure to MMC (0.2 microg/mL). Temporal events in epithelial and keratocyte cell kinetics were evaluated using digital imaging, confocal microscopy, and light microscopy. RESULTS: Epithelial latency was significantly delayed with MMC application in Groups 2 and 3 (P<.001). Epithelial migration was delayed in Group 3 (2-minute exposure) compared to migration in Group 2 (P<.04), with a consequent delay in epithelial closure (P<.001). Group 3 corneas had poorly differentiated epithelium that was significantly thinner than in Groups 1 and 2 (P<.0001). A significant delay in keratocyte regeneration occurred after MMC application (P<.0005). At 4 weeks, the anterior stromal cell density was significantly lower in Group 3 than Group 2 (P<.001). There were no significant differences in the mid- and posterior stromal keratocyte density between the groups. CONCLUSIONS: Results suggest that epithelial healing after MMC is characterized by prolonged latency and decreased migration rate dependent on exposure time. Mitomycin C application did not result in increased loss of keratocytes, but it significantly delayed keratocyte repopulation in the anterior stroma. The use of MMC 0.2 microg/mL for 1 minute resulted in optimum modulation of healing characterized by reduced keratocyte activation with normal epithelial differentiation.     

Trabeculectomies in fellow eyes have an increased risk of tenon's capsule cysts

OBJECTIVE: To assess outcomes and complications of primary trabeculectomies in fellow eyes in a large group of patients. The assumption was that first and fellow eyes undergoing fistulizing surgical procedures behave similarly in the postoperative period. DESIGN: Retrospective nonrandomized comparative trial (paired eye study). PARTICIPANTS: Over a 4-year period, 566 consecutive patients underwent primary trabeculectomy, all without antifibrotic agents. One hundred thirty-eight of these patients underwent bilateral surgery. INTERVENTION: A primary trabeculectomy was performed. MAIN OUTCOME MEASURES: Preoperative data, postoperative intraocular pressure, and visual acuity were monitored. In addition, complications and the need for consecutive surgical procedures were noted. RESULTS: The mean follow-up period of these trabeculectomies was 27.4 months (range, 7-62 months). Of the 124 bilateral adult cases, no statistical difference was found in intraocular pressures, number of antiglaucomatous medications, and success or failure rates between the two eyes. The need for enhancement procedures, such as needling or surgical excision of Tenon's capsule cysts, was significantly higher in fellow eyes than in first eyes (16 vs. 6 cases; P = 0.03; Mann-Whitney U test). Hypotony as a complication occurred more frequently in fellow eyes. CONCLUSIONS: Primary trabeculectomies performed in both eyes of patients have a remarkably similar clinical course. Failures of first eyes may be a reason to use antimetabolites in primary trabeculectomy of the fellow eye. The present data suggest that fellow eyes have a greater risk of Tenon's capsule cyst formation. This may be important for patient counseling before surgery. These results may additionally be important for clinical studies. Given that first and fellow eyes do not behave in an absolutely similar manner, study designs making intraindividual comparisons may not be feasible.     

胶原相关整合素对人巩膜成纤维细胞增殖和胶原合成的影响

目的 观察胶原相关整合素对人巩膜成纤维细胞增殖和胶原合成的影响.方法 首先进行人巩膜成纤维细胞原代培养与鉴定,RT-PCR检测胶原相关整合素α1、α2、β1亚单位mRNA表达,Western blot法检测胶原相关整合素α1、α2、β1亚单位蛋白表达,CCK-8检测胶原相关整合素α1β1和α2β1对人巩膜成纤维细胞增殖的影响,实时荧光定量PCR检测胶原相关整合素α1β1和α2β1对人巩膜成纤维细胞Ⅰ型胶原mRNA表达水平的影响.结果 成功进行了人巩膜成纤维细胞的原代培养.人巩膜成纤维细胞存在胶原相关整合素α1、α2、β1亚单位mRNA及蛋白水平的表达.胶原相关整合素α1β1 1 mg·L-1和4 mg·L-1抗体组细胞存活率分别为(86.3±4.5)％和(74.3±6.8)％,与对照组的100％相比,细胞存活率均显著下降,差异均有统计学意义(均为P＜0.05),且呈浓度依赖性的抑制作用.胶原相关整合素α2β1抗体在1mg ·L-1时有抑制作用,细胞存活率为(89.3±12.1)％,但与对照组的100％相比差异无统计学意义(P＞0.05),4mg· L-1抗体组细胞存活率下降为(74.5±6.6)％,与对照组相比差异有统计学意义(P＜0.05).胶原相关整合素α1β1(4 mg·L-1)抗体组Ⅰ型胶原mRNA表达明显减低,是对照组的0.48倍(P＜0.05).胶原相关整合素α2β1抗体1 mg·L-1组和4 mg· L-1组Ⅰ型胶原mRNA表达分别是对照组的0.94倍和0.87倍,与对照组相比差异均无统计学意义(均为P ＞0.05).结论 人巩膜成纤维细胞存在胶原相关整合素α1、α2、β1亚单位的表达.胶原相关整合素α1β1和α2β1影响人巩膜成纤维细胞的增殖.胶原相关整合素α1β1参与人巩膜成纤维细胞胶原的合成.     

丝裂霉素在LASEK术中的应用

目的：观察中高度近视在准分子激光上皮下角膜磨镶术(laser-assisted subepithelial keratomileusis,LASEK)中应用0.1g/L丝裂霉素(mitomycin-C,MMC)抑制术后角膜上皮下雾状混浊(haze)的疗效。

方法：对78例156眼患者行LASEK,随机分成MMC组(86眼)及对照组(70眼),术中两组分别采用含0.1g/L MMC与平衡盐溶液(BSS)的吸水棉签点蘸激光切削后的基质床。观察患者术后1,3,5,7d; 2wk; 1,3,6mo的症状,裸眼视力,角膜上皮愈合时间及haze形成情况。

结果：术后MMC组haze发生率低于对照组,两组haze形成差异有统计学意义(P<0.05); 术后角膜上皮愈合时间及裸眼视力均无明显差异。

结论：LASEK术中点蘸法应用0.1g/L MMC可抑制haze的形成。     

Quaternary ammonium cytotoxicity in a human conjunctival cell line

PURPOSE: Ophthalmic preparations can induce conjunctival toxicity, often caused by preservatives. The aim of this study was to evaluate in vitro cytotoxicity of quaternary ammonium. METHODS: Cytotoxicity tests were done on a continuous human conjunctival cell line using microplate cold light cytofluorimetry. Membrane integrity (neutral red test), DNA condensation (Hoechst 33342 test) and reactive oxygen species (ROS) production (dichlorofluorescein diacetate and hydroethidine tests) were evaluated on living cells treated with different concentrations of benzalkonium chloride, benzododecinium bromide and cetrimide (0.00001 to 0.01%) after 15 minutes of treatment or 15 minutes and 24 hours of cell recovery. RESULTS: All the compounds tested showed similar in vitro effects. Using the neutral red test, we observed a decrease in membrane integrity even at 0.005% and 0.01% (p < 0.001) and after a short time (15 minutes). A stimulation of ROS production (H2O2 and O2) was observed at 0.00001% and above (p < 0.001), associated with a chromatine condensation due to an apoptotic phenomenon. CONCLUSION: A necrotic phenomenon is suggested at high concentrations of quaternary ammonium preservatives whereas an apoptotic mechanism exists for lower concentrations. This toxicity observed in vitro can explain some of the ocular surface damage caused by long-term use of preserved eye-drops.