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PubMedDate not supplied

In vitro sensitivity of human Tenon's capsule fibroblasts to mitomycin C and its correlation with outcome of glaucoma filtration surgery.

Dose-inhibition studies of proliferating fibroblasts grown from Tenon's capsule biopsy specimens obtained during trabeculectomy from 17 glaucoma patients were performed using varying concentrations of mitomycin C (MMC), with exposure of cells to the drug for either 5 days or 5 minutes. Surgical outcomes were determined 3 months postoperatively. Based on in vitro results, fibroblasts were classified as either MMC-"resistant" or MMC-"sensitive." Thirteen surgically successful cases were associated with 10 "sensitive" and three "resistant" fibroblasts; four surgical failures with one "sensitive" and three "resistant" fibroblasts. The in vitro studies were positively correlated with the clinical outcomes, the former correctly predicting the latter in 76.5% of the surgeries.

Adolescent↗
PubMed1998

Antimetabolite-induced apoptosis in Tenon's capsule fibroblasts.

PURPOSE: To determine whether treatment with mitomycin-c and 5-fluorouracil induces apoptotic death in cultured subconjunctival fibroblasts. METHODS: Cultured human subconjunctival Tenon's capsule fibroblasts were exposed to 5-minute applications of mitomycin-C (up to 1 mg/ml) or 5-fluorouracil (up to 50 mg/ml) or phosphate-buffered saline solution (PBS). Fibroblast apoptosis was determined by cell morphology, apoptosis-specific protein expression, and DNA fragmentation by TdT-mediated dUTP nick-end labeling (TUNEL). In addition, apoptosis was quantified by direct cell counts based on morphology or lactate dehydrogenase release. RESULTS: Morphologic changes characteristic of apoptosis included nuclear and cytoplasmic condensation and occasional nuclear fragmentation while the plasma membrane remained intact. Apoptosis-specific protein expression and DNA fragmentation was observed in fibroblasts 48 hours after mitomycin-C treatment but not in control PBS-treated fibroblasts. The amount of apoptosis induced was dose dependent and partially inhibited by the addition of fetal calf serum to growth medium immediately after treatment. CONCLUSIONS: Mitomycin-C and high-dose 5-fluorouracil induce apoptosis in cultured Tenon's fibroblasts. Mitomycin-C-induced apoptosis is inhibited by fetal calf serum, indicating that exogenous factors influence the susceptibility of a fibroblast population to apoptosis. The induction and regulation of fibroblast apoptosis provides a novel target for the potential regulation of scarring.

Antimetabolites↗
PubMed1999

Periocular accumulation of timolol and betaxolol in glaucoma patients under long-term therapy.

PURPOSE: To determine if betaxolol or timolol is present in measurable concentration in the Tenon capsule in patients under long-term topical therapy. METHODS: Small (1-cc) specimens of Tenon capsule were removed at the time of filtering surgery from 15 glaucoma patients under long-term preoperative topical therapy, nine of whom had been treated with timolol and six of whom had been receiving betaxolol. Methanol extracts of these tissue samples were analyzed quantitatively for the presence of either beta-adrenergic antagonist by high-performance liquid chromatography. RESULTS: Drug was detected in every specimen. A mean total of 2.6 (range, 0.1-30.0) microg of betaxolol was detected per 1-cc specimen. CONCLUSION: Timolol and betaxolol penetrate the conjunctiva and accumulate in the Tenon capsule. In patients under long-term therapy, the periocular tissue can accumulate a greater quantity of beta-antagonist than is present in a daily dosage of applied eyedrops, manyfold higher than the maximal intraocular concentration.

Adrenergic beta-Antagonists↗
PubMed1990

The effect of 5-fluorouracil and cytarabine on human fibroblasts from Tenon's capsule.

The in vitro cellular inhibitory effects of two pyrimidine antimetabolites, 5-fluorouracil (5-FU) and cytarabine (ara-C), on the attachment and proliferation of human Tenon's capsule fibroblasts after 1, 2, 3, 5, 7, and 10 days of growth were measured with a Coulter counter, a colorimetric method using the endogenous enzyme hexosaminidase, and 3H-thymidine uptake. Neither 5-FU nor ara-C affected cell attachment. The 50% inhibitory dose (ID50) for 5-FU, as measured by the Coulter counter and hexosaminidase assay, was 0.2 and 0.4 micrograms/ml, respectively, at day 5 and decreased to 0.01 and 0.10 micrograms/ml, respectively, on later days. The ID50 for ara-C as measured by the Coulter counter and hexosaminidase assay was 0.01 and 0.1 micrograms/ml at day 3 and remained constant over time. Much lower ID50s were measured by thymidine uptake for both drugs. These findings may indicate that 5-FU has a delayed effect on cellular proliferation due to conversion into more active metabolites. The ara-C has a direct and constant inhibitory effect on cellular proliferation and is ten times more potent than 5-FU as an antiproliferative drug. Thus ara-C may have clinical utility in preventing failure of glaucoma filtering surgery.

Anterior Eye Segment↗
PubMed1987

The effect of beta irradiation on monkey Tenon's capsule fibroblasts in tissue culture.

Fibroblast proliferation with subsequent bleb scarring is a major cause of filtering surgery failure. To investigate possible antiproliferative effects of beta radiation, owl monkey Tenon's capsule fibroblasts in tissue culture were irradiated to doses of 30, 100, 300, 1,000, and 3,000 rads with a linear accelerator and to doses of 95,285, and 950 rads with a Strontium-90 applicator. The irradiated cell proliferation expressed as the percentage of the non-irradiated control growth on the third and seventh days, respectively, after beta irradiation was: 97% and 96% after 30 rads; 72% and 90% after 100 rads; 48% and 44% after 300 rads; 39% and 14% after 1,000 rads; and 39% and 14% after 3,000 rads. Similar effects on cell proliferation were observed with the Strontium-90 applicator. The inhibitory effect of beta irradiation on fibroblast proliferation in tissue culture suggests that beta irradiation after filtering surgery may reduce postoperative bleb scarring.

Animals↗
PubMed1997

In vitro effects of doxorubicin and mitomycin C on human Tenon's capsule fibroblasts.

Doxorubicin is an antiproliferative agent, that also inhibits prolyl 4-hydroxylase in vitro. We evaluated the effects of doxorubicin and mitomycin C (MMC) on the proliferation of human Tenon's capsule fibroblasts and the secretion of type I collagen in these cells to explore the potential use of doxorubicin as an antifibrotic agent after glaucoma filtering surgery. Standard immunoassays were used to determine the concentrations of type I procollagen COOH-terminal peptide (PIP), laminin and vitronectin receptor in conditioned media and cell lysates in the presence or absence of doxorubicin or MMC. The mitotic activity and viability of these cells were also determined. Cellular ultrastructure was evaluated by transmission electron microscopy. Doxorubicin and MMC decreased the cellular viability, the mitotic activity, and the production of the peptides measured. The inhibition of PIP secretion into the culture medium was higher in doxorubicin-treated cultures than in MMC-treated cultures at the tested concentrations (5-100 microM). The decrease in PIP levels in cell lysates was less in doxorubicin-treated culture (25 microM) than in MMC-treated culture (25 microM), suggesting that procollagen synthesis and secretion might be attenuated by doxorubicin. Ultrastructural studies revealed increased numbers of lysosomes in the cytoplasm of doxorubicin-treated cells relative to MMC-treated cells. Doxorubicin and MMC reduced cell viability and inhibited the proliferation and synthesis of PIP, laminin, and vitronectin receptor peptide. Inhibitory effects of doxorubicin on PIP secretion were more potent than those of MMC. Doxorubicin may be useful for inhibiting the fibrotic response at the site of ocular filtering surgery.

Adult↗
PubMed1996

Effect of fibrostatin C, an inhibitor of prolyl 4-hydroxylase, on collagen secretion by human Tenon's capsule fibroblasts in vitro.

PURPOSE: To investigate the effect of fibrostatin C, a prolyl 4-hydroxylase (PH) inhibitor produced by Streptomyces catenulae subsp. griseospora, on type I collagen secretion by human Tenon's capsule fibroblasts (TCFs) in vitro, as an indication of the potential therapeutic efficacy of this compound in antifibrotic therapy after glaucoma filtering surgery. METHODS: The concentrations of type II procollagen COOH-terminal peptide (PIP) in culture medium or cell lysate were determined by immunoassays. For comparison with its effect on PIP secretion, we determined the effects of the agent on the secretion of other peptides, including laminin, vitronectin receptor, and a metalloproteinase inhibitor. The expression of collagens I and III, fibronectin, and PH by TCFs was examined by immunohistochemistry, and cellular ultrastructure was evaluated by transmission electron microscopy. RESULTS: Fibrostatin C (50 microM) significantly reduced the concentration of PIP in culture medium and increased its concentration in the cell lysate in a dose-dependent manner, but it had no effect on the secretion of other peptides. Cell viability and proliferation were not affected by fibrostatin C. Fibrostatin C also increased the number of cytoplasmic granules immunoreactive with antibodies to collagen I or III, but had no effect on fibronectin or PH immunoreactivity. Ultrastructurally, cisternae of the endoplasmic reticulum were dilated in fibrostatin C-treated TCFs, consistent with the retention of underhydroxylated collagen precursors in this organelle. CONCLUSIONS: Fibrostatin C inhibits the secretion of type I collagen by cultured TCFs. This agent may thus prove therapeutically beneficial for inhibiting the excess fibrosis in the wound of filtering surgery.

Aged↗
PubMed1992

Effect of brief exposure to mitomycin C on viability and proliferation of cultured human Tenon's capsule fibroblasts.

PURPOSE: The purpose of this study is to determine why a single intraoperative dose of mitomycin C (MMC) appears to promote the success of glaucoma filtration surgery. METHODS: Human Tenon's capsule fibroblasts were exposed to MMC and 5-fluorouracil (5-FU) in vitro at the concentrations and durations of exposure used clinically. Cell proliferation was assessed by quantification of 3H-thymidine uptake. Cell viability was studied using a sulfarhodamine B cell protein stain and by trypan blue exclusion. RESULTS: Neither MMC (0.4 mg/ml) nor 5-FU (40 mg/ml) were cytocidal. Both 1- and 5-minute exposures to MMC were antiproliferative. A 1-minute exposure to 0.4 mg/ml inhibited 3H-thymidine uptake by 77%. For the 5-minute exposure, 3H-thymidine uptake was inhibited by 50% at 0.06 mg/ml and by 90% at 0.4 mg/ml. For 5-FU, 3H-thymidine uptake was inhibited by 50% at 10 mg/ml and by 64% at 40 mg/ml. CONCLUSIONS: Mitomycin C probably does not improve the success of filtration surgery by killing fibroblasts. The ability of a brief exposure to MMC to improve filtration surgery may be due to an almost complete inhibition of proliferation. Alternatively, it may be due to sustained tissue binding, effects on other components of wound healing, such as cell migration and extracellular matrix production, or effects on the vasculature. A 1-minute exposure may be as effective as a 5-minute exposure.

Cell Division↗
PubMedDate not supplied

The features of Tenon's capsule at the limbus.

Glaucomatous optic neuropathy is a multifactorial disease, whose most important risk is high intraocular pressure. Filtering surgery, i.e. trabeculectomy, is the operation of choice to lower pressure, allowing the aqueous to outflow from the anterior chamber under the conjuctiva and Tenon's capsule. Failure to establish a filtration bleb is due in most cases to subconjunctival fibrosis, leading to bleb encapsulation (Tenon's cyst). Our study aimed to locate the best cleavage plane for aqueous drainage. Based on histological data, the subtenonian space appears to satisfy this requirement, due to low scarring aptitude and low resistance.

Adult↗
PubMed1998

OPC-15161 suppresses the proliferation of Tenon's capsule fibroblasts and the production of type I collagen and fibronectin stimulated by TGF-beta1 in vitro.

PURPOSE: We investigated the effects of OPC-15161 on the growth of cultured human Tenon's capsule fibroblasts (TCFs), as well as on the production of type I procollagen, fibronectin, and laminin. These effects were examined in the presence or absence of TGF-beta1. METHODS: Cell proliferation was assayed by counting cell number and assay of DNA synthesis. Cytotoxicity was determined by the MTT method. Matrix components were assayed by enzyme immunoassay of material in the medium and in the cell lysate with or without OPC-15161. Total protein content was determined. Cellular ultrastructure was also evaluated. RESULTS: Treatment with OPC-15161 (up to 100.0 microg ml(-1)) significantly reduced the proliferation and DNA synthesis of TCFs. No significant decrease in MTT values was observed in confluent TCF cultures with OPC-15161 (up to 100.0 microg ml(-1)). TGF-beta1 enhanced the TCF production of procollagen I and fibronectin. OPC-15161 significantly decreased the procollagen I content in both the medium, in the cell lysate of TGF-beta1-stimulated cells, and fibronectin content in the lysate. OPC-15161 did not affect the laminin or total protein content, either with or without TGF-beta1. No ultrastructural evidence of cytotoxicity was observed. CONCLUSIONS: OPC-15161 inhibited the proliferation of TCFs, and reduced their production of procollagen I and fibronectin in the presence of TGF-beta1 without evidence of cytotoxicity. OPC-15161 may be useful in inhibiting the excessive fibrosis produced in the wound in response to filtering surgery.

Adult↗
PubMed1996

The effect of TGF-beta 1 and TGF-beta 2 on the proliferation of human Tenon's capsule fibroblasts in tissue culture.

This study examines the effect of transforming growth factor beta 1 and 2 (TGF-beta 1 and TGF-beta 2) on the proliferation of human Tenon's capsule fibroblasts in tissue culture. Both TGF-beta 1 and TGF-beta 2 were shown to stimulate proliferation at 1000 and 100 pg/ml concentrations. We discuss the significance of these findings with respect to wound healing following trabeculectomy, and postulate why some eyes are more at risk of failure than others.

Cell Division↗
PubMed1997

In vitro effects of mitomycin C on human Tenon's capsule fibroblasts & correlation with surgical outcome in glaucoma patients.

The effects of mitomycin C (MMC, 1 microgram/ml for 5 min) on growth and morphology of six MMC 'sensitive' and four MMC 'resistant' human Tenon's capsule fibroblasts grown from 10 patients who had had trabeculectomy for glaucoma were studied. The surgical outcome of trabeculectomy in these patients were evaluated at the end of 8-10 wk. Both types of fibroblasts showed increase of growth up to 7 days. MMC 'sensitive' fibroblasts degenerated and declined to insignificant numbers in 47 days. MMC 'resistant' fibroblasts after a decline in growth up to 27 days showed remarkable recovery in morphology and growth by day 37. Surgical outcome positively correlated with in vitro results of 'sensitive' fibroblasts. A few clones of MMC 'resistant' fibroblasts survived and multiplied as MMC effect had worn off.

Antibiotics, Antineoplastic↗
PubMed1993

Interferon-alpha 2b inhibits proliferation of human Tenon's capsule fibroblasts.

To evaluate the potential of interferon-alpha 2b (IFN-alpha) as an antifibrotic treatment after glaucoma filtering surgery, we studied its ability to inhibit mitogen-induced proliferation of human Tenon's capsule fibroblasts in vitro, as shown by [3H]-thymidine incorporation. IFN-alpha inhibited fetal calf serum-induced proliferation in a dose-dependent manner in concentrations ranging from 10 to 10,000 IU/ml (P < 0.001). Inhibition was also found when IFN-alpha was added prior to the mitogen (P = 0.001); this inhibitory effect lasted at least 3 days. We suggest that IFN-alpha deserves further evaluation as a possible agent for the control of ocular fibrosis.

Cell Division↗
PubMed2004

APC0576 decreases production of pro-inflammatory chemokine and extracellular matrix by human Tenon's capsule fibroblasts.

PURPOSE: To control intraocular pressure in patients treated by trabeculectomy, progressive inflammation, fibroblast proliferation, and the enhanced expression of extracellular matrix (ECM), causes of scar formation at the bleb, must be prevented. Using human Tenon's capsule fibroblasts (TCFs), we examined the effect of APC0576, a suppressor of NF-kappaB-dependent gene activation of human vascular endothelial cells that does not adversely affect cell viability, on the production of pro-inflammatory chemokine and ECM. Its effect on TCF proliferation was also assessed. METHODS: Enzyme-linked immunosorbent assay was used to detect the pro-inflammatory cytokines IL-8 and MCP-1 in the supernatant of TCFs stimulated with IL-1alpha in the presence or absence of APCO576 and ECM proteins, COOH-terminal peptide of type I procollagen (PIP), fibronectin (FN), and laminin (LN) in supernatants and lysates of TCFs stimulated with IL-1alpha or TGF-beta. The proliferative response of IL-1alpha-stimulated TCFs was examined using the SF formazan solution reaction. RESULTS: APC0576 significantly suppressed the production by TCFs of IL-8 (p<0.0001), MCP-1(p<0.0001), PIP (supernatants: p<0.0001, cell lysates: p<0.0001), LN (supernatants: p<0.0001, lysates: p<0.0001), and FN (supernatants: p<0.0001, lysates: p<0.0001) as well as their proliferation (p<0.0001). CONCLUSIONS: APC0576 suppressed pro-inflammatory chemokine and ECM production in TCFs as well as their proliferation. It may represent a novel candidate for the postoperative management of patients treated by trabeculectomy.

Aged↗
PubMed1999

Serum-free cultivation of human Tenon's capsule fibroblasts.

PURPOSE: In the present study, the effect of three different serum-free and one serum-containing control medium on adhesion, proliferation, cryopreservation and PDGF-induced effects on cell proliferation of human Tenon's capsule fibroblasts (HTF) was compared. MATERIALS AND METHODS: Third passage HTF were suspended in four different culture media (WM/F12, WM/F12/FCS 1%, LR-1, DMEM) and plating efficiency was determined after 24h using a cell counter system. Subsequently, cells were seeded at a density of 50/mm2 and cultured for ten days using the different culture media. Cell number was determined at day 2, 4, 7 and 10 after seeding. Furthermore, HTF cultured under the different conditions were stimulated by PDGF-BB [50 ng/ml]. Additionally, cell vitality after two weeks cryopreservation in five different culture media (WM/F12, WM/F12/FCS 1%, WM/F12/FCS 20%, LR-1, DMEM) was determined. RESULTS: The plating efficiency of HTF when seeded in serum-free medium ranged from 55.3% to 59.6%. Using serum containing WM/F12/FCS 1% a slightly higher plating efficiency of 74.8% was obtained. Proliferation assays revealed population doublings of 0.77 with WM/F12/FCS 1% after an incubation period of 10 days. Cultivation of HTF using serum-free conditions did not cause significant cell proliferation but a slight cell loss which ranged from 23.1% to 34%. Addition of PDGF-BB resulted in a significant increase in cell proliferation with WM/F12/FCS 1%, WM/F12 and DMEM. After two weeks of cryopreservation in WM/F12, LR-1, DMEM, WM/F12/FCS 1% and WM/F12/FCS 20%, only the application of high serum concentrations led to sufficient preservation of cell vitality with a plating efficiency of 82.9%. DISCUSSION: The results of the present study demonstrate that the use of serum-containing media is mandatory for cryopreservation of HTF. Seeding of cells can be performed either with serum or without serum. HTF cultured under serum-free conditions can be maintained quiescent with a sufficient number of cells remaining vital. The serum-free media used in this study can be applied for the investigation of cytokine effects on HTF.

Cell Adhesion↗
PubMedDate not supplied

Effect of diabetes mellitus and hyperglycemia on the proliferation of human Tenon's capsule fibroblasts: implications for wound healing after glaucoma drainage surgery.

Glaucoma drainage surgery in diabetic patients is associated with a relatively poor prognosis due to increased scarring at the site of surgery, secondary to increased proliferation of human Tenon's capsule fibroblasts (hTCF). This is in marked contrast to diabetic wound healing at other sites, where it is generally impaired. The aim of this study was to determine why diabetics show an increased ocular scarring response in comparison to that found at other sites. Under normoglycemic conditions, hTCF isolated from diabetics showed a mean reduction in short-term proliferation (95% CI) of 45 +/- 12% compared with normal controls (p < 0.001). Under hyperglycemic conditions, proliferation of diabetic hTCF was reduced by 21 +/- 11% (p < 0.01) compared with nondiabetic controls. When exposed to transforming growth factor-beta2 (1-10,000 pg/ml) and platelet-derived growth factor-BB (0.5-500 ng/ml) under both normo- and hyperglycemic conditions, there was a dose-related increase in proliferation of both diabetic and nondiabetic controls. There was no significant difference in response to cytokine stimulation between the two groups at any of the cytokine concentrations used. Western blot analysis did not show any apparent difference in the expression of platelet-derived growth factor receptor alpha, mitogen-activated protein kinase/ERK2, or transforming growth factor-beta receptor II to account for the reduced proliferation of diabetic hTCF. These results suggest that hTCF behave in a manner similar to fibroblasts from other nonocular sites and that the increased proliferation and scarring response found in vivo may be secondary to the previously noted elevated cytokine concentrations in the aqueous and vitreous of diabetics.

Adult↗