Anti-allergic drugs inhibit the proliferation of human Tenon's capsule fibroblast and maintain the experimental filtering blebs on rabbit eyes

PURPOSE: To examine whether tranilast and ketotifen fumarate inhibit the growth of human Tenon's capsule fibroblasts and maintain experimental filtering blebs on rabbit eyes. METHODS: Human Tenon's capsule fibroblasts were grown in Dulbecco's modified Eagle's medium with 10% fetal calf serum and growth was measured with 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) and direct counting of cell number. Production of collagen and transforming growth factor-beta 1 (TGF-beta 1) was measured with corresponding enzyme-linked immunosorbent assay (ELISA). In experimental filtering surgery performed on rabbit eyes, the effects of the topical drugs on intraocular pressure and on maintenance of the filtering blebs were observed. RESULTS: Both tranilast and ketotifen inhibited the growth of the fibroblasts and half-inhibitory concentrations were 200 microM and 70 microM, respectively. Low concentration (10 microM) of tranilast and ketotifen decreased the synthesis of collagen but neither drug had any obvious effect on TGF-beta 1 production. Both drugs prolonged the life of the experimental filtering bleb significantly [12.5 +/- 3.2 (mean +/- standard deviation) days, 13.9 +/- 3.4 days, respectively; control, 10.5 +/- 2.4 days]. CONCLUSION: Tranilast and ketotifen inhibited the growth of human Tenon's capsule proliferation and are capable of maintaining experimental filtering blebs in rabbit eyes.     

Effects of tranilast on cultured rabbit corneal keratocytes and corneal haze after photorefractive keratectomy

PURPOSE: In vitro and in vivo studies were performed to elucidate the effects of tranilast on cellular proliferation and collagen synthesis. METHODS: Subculturing was carried out using keratocytes from rabbits that underwent photorefractive keratectomy (PRK) and developed corneal haze, and keratocytes from normal rabbit cornea. RESULTS: Tranilast suppressed proliferation in cultured keratocytes from the corneal haze region at doses of 30 and 300 micromol/L and collagen synthesis at doses of 3, 30, and 300 micromol/L. Normal corneal cultures showed suppression of keratocyte proliferation and collagen synthesis only at a high dose of tranilast (300 micromol/L). Betamethasone suppressed proliferation of keratocytes in both haze and normal cornea at a dose of 10 micromol/L, as well as collagen synthesis at respective doses of 1 and 10 micromol/L. Diclofenac sodium suppressed collagen synthesis of keratocytes in haze cornea at a high dose of 100 micromol/L, and in keratocytes in normal cornea, at doses of 10 and 100 micromol/L. In an in vivo study, either 0.5% tranilast, 0.1% betamethasone phosphate eye drops, or a tranilast base solution (control) was instilled four times daily to rabbits that had undergone PRK. Weekly evaluation of the inhibitory effect of these drugs on the development of haze was performed 2 weeks after surgery. Tranilast suppressed haze 6-13 weeks after PRK, but betamethasone phosphate showed no effect. CONCLUSION: These results indicate that tranilast is potentially effective for inhibiting the corneal haze that occurs after PRK.     

Effect of tranilast on proliferation,collagen gel contraction,and transforming growth factor beta secretion of retinal pigment epithelial cells and fibroblasts

The efficiency of tranilast for the treatment of proliferative vitreoretinopathy (PVR) was investigated in vitro. A tetrazolium-based colorimetric assay showed that the 300-microM concentration of tranilast inhibited proliferation of bovine retinal pigment epithelial (RPE) cells and rabbit dermal fibroblasts with no toxicity. The contraction of collagen gels embedded with these cells was evaluated in the cultures. Compared with the gel incubated with minimal essential medium and 0.35% bovine serum albumin and/or fetal calf serum, tranilast inhibited gel contraction. Enzyme-linked immunosorbent assay revealed that a 300-microM concentration of tranilast inhibited transforming growth factor-beta(1) (TGF-beta(1)) secretion significantly (p < 0.01). These results suggest that tranilast may inhibit the proliferation of RPE cells and fibroblasts and contraction of intraocular fibrous membranes by suppressing TGF-beta(1) secretion from these cells with a potential to treat PVR.     

Anti-allergic drugs inhibit the proliferation of bovine lens epithelial cells in culture

PURPOSE: To examine the inhibitory effects of tranilast, ketotifen fumarate, and disodium cromoglycate which are used clinically as anti-allergic agents, on the growth of bovine lens epithelial cells (LE) in culture. METHODS: LE was grown in Dulbecco's modified Eagle's medium(DMEM) with 10% fetal calf serum and growth was measured with 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide(MTT) and 5-bromo-2'deoxy-uridine(BrdU). Production of collagen, transforming growth factor-beta 1(TGF-beta 1), and basic-fibroblast growth factor(b-FGF) were measured with corresponding enzyme-linked immunosorbent assay(ELISA). Apoptotic cell death was detected by TdT-mediated dUTP-biotin nick end labelling method(TUNEL technique) and the DNA ladder method. RESULTS: Both ketotifen and tranilast inhibited the growth of LE, and half-inhibitory concentrations were 200 microM and 1,000 microM, respectively. Disodium cromoglycate did not inhibit LE proliferation significantly. Ketotifen and tranilast decreased the synthesis of collagen but had no obvious effect on TGF-beta 1 and b-FGF production. Apoptotic cell death was detected in LE treated with ketotifen or tranilast. CONCLUSION: Ketotifen and tranilast may be clinically useful for the prevention of aftercataract. Apoptotic cell death may be involved in the process.     

Inhibitory effects of tranilast on the proliferation and functions of human pterygium-derived fibroblasts

PURPOSE: We studied the possibility that tranilast, an antiallergic and antiproliferative drug, may be beneficial for the treatment of pterygium. METHODS: Pterygium-derived cells were identified by immunohistochemical methods. Growth rate of pterygium-derived cells was determined by using a hemocytometer. Chemotaxis was determined in a microchemotaxis chamber. Pterygium-derived cells were cultured on floating collagen gel, and the contracted diameter was measured. Collagen synthesis by pterygium-derived cells was determined by the collagenase digestive method. Tranilast was added to the culture medium at final concentrations of 0, 12.5, 25, 50, and 100 microg/ml. RESULTS: Pterygium-derived cells were stained with anti-prolylhydroxylase and anti-alpha-smooth muscle actin, and identified as fibroblasts. Tranilast inhibited the proliferation and chemotaxis of pterygium-derived fibroblasts, and the collagen-gel contraction induced by these cells, but it exerted no inhibitory action on collagen synthesis by pterygium-derived fibroblasts. CONCLUSION: Tranilast may be useful for suppressing the recurrence and, possibly, the development of pterygium.     

The effect of viper venom on the attachment, migration and proliferation of Tenon's capsular fibroblasts

OBJECTIVES: To observe the effect of viper (Ahylysantipfarciasi) venom on attachment to collagen, migration and proliferation of rabbit Tenon's capsular fibroblast (TFs) in tissue culture. METHODS: Anti-adhesion experiment: The 3 -- 5 passage TFs suspended in DMEM medium were pre-incubated for 30 minutes in the presence of various concentrations of viper venom (0, 2.5 x 10(-4), 5.0 x 10(-4), 1.0 x 10(-3) and 5.0 x 10(-3) U/ml) at 37C, 5% CO(2), then inoculated in 24-well plate coated with rat-tail collagen. After incubation for 90 minutes, the floating cells were removed. The attached cells in each well were enumerated microscopically and determined by the value of absorption (A) of 3-(4, 5-dimethylthiazolzyl)-2, 5-diphenyl tetrazodium bromide (MTT). Anti-migration experiment: A denuded area was made when the 3rd passage of TFs was confluent into a monolayer. Cells were then exposed to viper venom (0 -- 5.0 x 10(-3) U/ml) at 37C, 5% CO(2), and the cells having migrated into the denuded area were enumerated every 6 hours. Anti-proliferative experiment: Two hours after the 3rd passage of TFs was incubated in the 24-well plate at 37C, 5% CO(2), they were exposed to different concentrations of viper venom drug (0 -- 5.0 x 10(-3) U/ml). Twenty-four and 48 hours later, the number of cells was determined by MTT method. RESULTS: Viper venom inhibited TFs from attaching to collagen in a dose-dependent manner and the ID(50) was 1.0 x 10(-3) U/ml. Only did 5.0 x 10(-3) U/ml of viper venom show significant difference from the control during 6 -- 48 hours. The difference of A value was not significant among all groups at 24 and 48 hr. CONCLUSIONS: In vitro, viper venom (> 1.0 x 10(-3) U/ml) can significantly inhibit the attachment of TFs to collagen, and 5.0 x 10(-3) U/ml can inhibit the migration, but can not affect their proliferation.     

蛇毒制剂对体外培养的兔结膜下成纤维细胞的影响

目的　观察蛇毒制剂对体外培养的兔结膜下成纤维细胞与胶原黏附及细胞移行和增殖的影响。方法　将培养的第3～5代兔结膜下成纤维细胞于不同浓度的蛇毒制剂孵育后,接种于涂有鼠尾胶原的24孔板中,去除未黏附的细胞,用目镜网格器计数法和四甲基偶氮唑盐法记录贴壁细胞量。在第3代生长融合细胞的培养板上划线,造成无细胞的裸露区,在不同浓度的蛇毒制剂下孵育,用目镜网格器每6h观察记录移行至裸露区的细胞数。第3代兔结膜下成纤维细胞接种于24孔板与不同浓度的蛇毒制剂共育24、48h后,用四甲基偶氮唑盐法记录各孔细胞数量的吸光度(A)值。结果蛇毒制剂抑制结膜下成纤维细胞黏附呈剂量依赖性。抑制成纤维细胞黏附的半数有效量(ID50)为1.0×10     

曲尼司特对青光眼患者体外培养的眼部Tenon囊成纤维细胞增殖和移行的影响

目的 探讨曲尼司特(tranilast)对青光眼患者体外培养的眼部Tenon囊成纤维细胞增殖及移行的影响。方法 取青光眼患者滤过术中剪下的Tenon囊组织,在体外进行成纤维细胞原代及传代培养。分别采用MTT法、细胞计数法、免疫组化加图像分析法及划线法,研究不同浓度的曲尼司特对体外培养的成纤维细胞增殖及移行的影响及其与蛋白激酶C(PKC)表达的关系。结果 当浓度在12．5～100．0mg／L之间变化时,曲尼司特能明显抑制成纤维细胞的增殖,强度呈剂量依赖性;50．0mg／L和100．0mg／L的曲尼司特能明显抑制成纤维细胞的移行,并下调细胞内PKC的表达。结论 曲尼司特能抑制成纤维细胞的增殖及移行,这种作用可能与下调细胞内PKC的表达有关。     

Partial restoration of the keratocyte phenotype to bovine keratocytes made fibroblastic by serum

PURPOSE: To determine whether keratocytes made fibroblastic in vitro by addition of fetal bovine serum to the medium regain the keratocyte phenotype after culture in serum-free medium. METHODS: Collagenase-isolated keratocytes from bovine corneas were plated in DMEM/F-12 containing 1% horse plasma, to allow cell attachment, and then cultured until day 4 in either DMEM/F-12 alone, to retain the keratocyte phenotype, or in DMEM containing 10% fetal bovine serum, to cause the keratocytes to become fibroblastic. Medium for the fibroblastic cells was replaced on day 4 with serum-free medium, and cells were cultured until day 12. Cell phenotypes were determined on days 4 to 5 and 11 to 12 of culture as follows: (1) by the morphologic appearance on phase-contrast microscopy; (2) by the levels of aldehyde dehydrogenase in the cells, determined by SDS-PAGE and Coomassie blue staining; (3) by the relative synthesis of collagen types I and V, determined by (14)C-proline radiolabeling; (4) by pepsin digestion and analysis of collagen types by SDS-PAGE autoradiography; (5) by relative synthesis of cornea-specific proteoglycan core proteins determined by analysis of chondroitinase- or endo-beta-galactosidase-generated radiolabeled core proteins by SDS-PAGE autoradiography; and (6) by the relative synthesis of keratan sulfate and chondroitin sulfate determined by (35)SO(4) radiolabeling and measuring the sensitivity to endo-beta-galactosidase and chondroitinase ABC. RESULTS: Keratocytes cultured in serum-free medium appeared dendritic and became fibroblastic in appearance when exposed to medium containing serum. Keratocytes and fibroblasts synthesized a similar proportion of collagen types I and V. However, compared with the keratocytes, the fibroblasts possessed no aldehyde dehydrogenase and synthesized significantly higher levels of decorin and significantly lower levels of prostaglandin D synthase (PGDS) and keratan sulfate. Subsequent culture of the fibroblasts in serum-free medium did not restore aldehyde dehydrogenase to keratocyte levels but did restore the cell morphology to a more dendritic appearance and returned the synthesis of decorin, PGDS, and keratan sulfate to keratocyte levels. CONCLUSIONS: The results of these studies indicate that primary cultures of keratocytes made fibroblastic by exposure to serum can return to their keratocyte phenotype in synthesizing extracellular matrix. These results also indicate that the differences in the organization of the collagenous matrix produced by keratocytes and fibroblasts may be related more to the different proteoglycan types than to the collagen types produced.     

Promotion by fibronectin of collagen gel contraction mediated by human corneal fibroblasts

Collagen contraction mediated by corneal fibroblasts (CFs) is implicated in the maintenance of corneal shape. Given that fibronectin is expressed at sites of corneal stromal wounding, we investigated the effect of fibronectin on CF-mediated collagen gel contraction. Human CFs were cultured in a three-dimensional gel of type I collagen in the absence or presence of various extracellular matrix (ECM) components. The contraction of collagen gels mediated by CFs was evaluated by measurement of changes in gel diameter. The formation of stress fibers and focal adhesions in CFs was examined by fluorescence microscopy. The abundance of paxillin, phosphorylated paxillin, integrins alpha5, beta1, and alpha2, and alpha-smooth muscle actin in CFs was examined by immunoblot analysis. Fibronectin promoted CF-mediated collagen gel contraction in a concentration- and time-dependent manner. Other ECM proteins or glycosaminoglycans did not exhibit such an effect. Fibronectin also induced cell spreading, the formation of stress fibers, and the establishment of focal adhesions containing paxillin in CFs cultured in three-dimensional collagen gels. In addition, it increased the amounts of paxillin, phosphorylated paxillin, and integrins alpha5 and beta1 in these cells. The expression of integrin alpha2 and alpha-smooth muscle actin was not affected by fibronectin, however. Furthermore, the peptide GRGDSP (an antagonist of fibronectin receptors) blocked the stimulatory effect of fibronectin on CF-mediated collagen gel contraction. These results suggest that fibronectin promoted CF-mediated collagen gel contraction in a manner dependent on the formation of stress fibers and focal adhesions, the activation of paxillin, and the up-regulation of integrin alpha5beta1. Fibronectin may therefore contribute to the maintenance of corneal shape by CFs during the healing of stromal wounds.     

Evaluation for safety of cultured corneal fibroblasts with cotreatment of alcohol and mitomycin C

PURPOSE: To investigate the effects of alcohol and mitomycin C (MMC) on cultured corneal fibroblast of the rabbit to determine the safety of this compound for clinical use. METHOD: Corneal fibroblasts of New Zealand rabbits were cultured. Various concentrations (0%, 10%, 20%, 30%, 40%, and 60%) of ethanol were applied for 10, 20, 30, and 40 seconds to estimate dose- and time-dependent responses of cultured corneal fibroblasts. Cell viability was assessed using the MTT assay. Treated cells were additionally stained with Hoechst and annexin V for the identification of apoptosis. To investigate the coeffects of ethanol and MMC, dose and time dependency were evaluated after treatment with various concentrations of ethanol and MMC at different exposure times, and cell viability was established. To determine the latent effects of ethanol and MMC, cultured corneal fibroblasts were cotreated with various concentrations of ethanol and 0.02% MMC for various periods and washed out, and then one group was incubated for 24 hours and another group was not incubated. Cell viability was estimated, and Hoechst and annexin V staining were performed before and after incubation. To establish the pathway of cell death, caspase-3 activities were measured in cultured corneal fibroblasts treated with ethanol or MMC. RESULTS: Cell viability after ethanol treatment was dose and time dependent. After application of ethanol for 10 seconds, cell viability was significantly reduced with 20% ethanol (P = 0.001). At 20, 30, and 40 seconds of treatment with 10% ethanol, cell viability was significantly reduced (P < 0.01). Hoechst and annexin V staining revealed typical characteristics of apoptosis, such as bright fluorescent chromatin condensation, low fluorescence of nuclear fragmentation, and cell membrane shrinkage. Cell viability was more significantly reduced after cotreatment with alcohol and MMC, compared with treatment with alcohol alone. Moreover, cell viability was considerably decreased in the incubated group, compared with the nonincubated group. After 24 hours of incubation, cultured corneal fibroblasts cotreated with 10% ethanol and 0.02% MMC were stained with Hoechst and annexin V. Results were similar to data obtained with ethanol-treated cells. However, after application of 20% alcohol and MMC, a significant number of cells were not viable and were detached from the well walls. Caspase-3 activity significantly increased after treatment with 30% ethanol only and 30% ethanol in conjunction with 0.02% MMC. CONCLUSIONS: Alcohol and MMC reduced cell viability in cultured corneal fibroblasts in a dose- and time-dependent manner and had synergistic effects. This is related to the caspase-3 pathway, especially with concentrations of ethanol over 30%. Cotreatment with these reagents may significantly damage cultured corneal fibroblasts.     

晶状体和玻璃体提取物及地塞米松影响Tenon囊成纤维细胞增殖和胶原合成的实验研究

目的　探讨晶状体和玻璃体提取物对猪眼筋膜囊(Tenon囊)成纤维细胞增殖和胶原合成的影响及其在糖皮质激素作用下的变化。方法　体外培养猪眼Tenon囊成纤维细胞,应用噻唑蓝(MTT)比色法和逆转录聚合酶链反应(RT -PCR)法观察在晶状体和玻璃体提取物及地塞米松单独或联合作用下,Tenon囊成纤维细胞增殖和胶原合成的变化。结果　与对照细胞吸光度(A值)(0 .305±0.013)比较, 10mg/L晶状体提取物( 0. 411±0. 000 )和10mg/L玻璃体提取物( 0. 349±0 .027)作用2d即具有促进细胞生长作用,差异有统计学意义(P=0. 00);且随着晶状体提取物、玻璃体提取物浓度的增高,A值相应增加,差异均有统计学意义(P=0. 00)。Ⅰ型胶原的相对含量在晶状体提取物作用细胞内为12 290±231,在玻璃体提取物作用细胞内为10 .853±231,与对照细胞(9389±178)比较,差异均有统计学意义(P<0. 01)。地塞米松对125mg/L晶状体提取物和125mg/L玻璃体提取物作用的Tenon囊成纤维细胞增殖分别具有一定抑制作用,差异有统计学意义(P=0. 00)。地塞米松和提取物联合作用细胞内Ⅰ型胶原的相对含量与提取物单独作用细胞比较,差异均无统计学意义(P>0 .05)。结论　晶状体和玻璃体提取物均可明显刺激Tenon囊成纤维细胞增殖,促进Tenon囊成纤维细胞内胶原合成,     

Platelet-derived growth factor receptor kinase inhibitor AG1295 and inhibition of experimental proliferative vitreoretinopathy

PURPOSE: Receptor tyrosine kinase (RTK) activation is critical for growth factor-mediated cell proliferation. The present study was designed to determine the effect of tyrphostin AG1295, a selective blocker of platelet-derived growth factor (PDGF) RTK, on proliferative vitreoretinopathy (PVR) development. METHODS: Rabbit conjunctival fibroblasts cells (1 x 10(4)) were seeded into 96-well plates and maintained in Dulbecco's modified essentialmedium (DMEM) with 0.5% fetal bovine serum. The cells were exposed to 50 ng/mL PDGF-AAor PDGF-BBor phosphate-buffered saline with or without AG1295 (1 microM, 10 microM, and 100 microM). After 3 days, the viable cells in each well were measured by 3,(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Homologous rabbit conjunctival fibroblasts were injected intravitreally, followed by injection of 100 microM of AG1295. The development of tractional retinal detachment (TRD) was assessed to evaluate the effect of AG1295 in vivo. Electroretinography and histologic studies were performed after intravitreal injection of AG1295 into untreated eyes to evaluate toxicity. RESULTS: Two concentrations of AG1295 (10 and 100 microM) significantly inhibited rabbit conjunctival fibroblast cell growth stimulated by PDGF-AA or PDGF-BB in vitro. Development of TRD was significantly attenuated (P <.01) with 100 microM of AG1295 until day 21. No significant histologic or retinal functional damage was found in the AG1295-treated group. CONCLUSIONS: PDGF receptor specific inhibitor AG1295 attenuated PVR without significant side effects in rabbits. This reagent could be a useful treatment to prevent PVR.     

Effect of conjunctival structure and inflammatory cell counts on intraocular pressure after trabeculectomy

Purpose: To evaluate the conjunctival structure and inflammatory cell counts and to determine the predictive value of these histological parameters for postoperative intraocular pressure (IOP) levels after trabeculectomy. Methods: A clinical and histological study was performed on consecutive patients. Postoperative (mean 32. 8 +/- 18.4 months; range 6-60 months) conjunctival biopsies of 36 eyes of 28 primary open-angle glaucoma patients who had trabeculectomy between 1992 and 1995 were included in the study. According to postoperative pressure control, patients with < or = 16 mm Hg and those with >16 mm Hg were taken as groups 1 and 2, respectively. The control group (group 3) consisted of 15 age-matched patients without glaucoma, who had received no topical therapy. We compared the conjunctival structure and cell counts within these groups. Goblet cells, acute inflammatory cells, chronic inflammatory cells, fibroblasts, epithelial thickness, vascular density, mucopolysaccharides and collagen compositions were determined in groups 1, 2 and 3. Results: The number of goblet cells was significantly higher in group 1 (6.74 +/- 7.23) than group 2 (3. 09 +/- 2.77; p = 0.017). No statistical difference was observed in the number of acute inflammatory cells, chronic inflammatory cells, fibroblasts, epithelial thickness, vascular density, mucopolysaccharide or collagen compositions between groups 1 and 2 (p > 0.05). In addition, when groups 1 and 2 were compared with the control group, there was a significant decrease in goblet cells (p < 0.001), an increase in acute inflammatory cells, chronic inflammatory cells, fibroblasts, epithelial thickness and vascular density (p < 0.001), but there was no significant difference in mucopolysaccharide and collagen compositions (p > 0.05). Conclusions: This study suggests that at the time of surgery a high number of goblet cells may be a predictor of lowered IOP (< or = 16 mm Hg) following trabeculectomy without antimetabolite. Copyright Copyright 1999 S.Karger AG, Basel     

Effect of tranilast eyedrops in preventing posterior capsule opacification: preliminary report.

PURPOSE: To evaluate the effect of tranilast eyedrops in preventing fibrous opacification of the posterior lens capsule after cataract extraction and intraocular lens (IOL) implantation. SETTING: The Second Department of Ophthalmology, Toho University School of Medicine, Tokyo, and Shohzankai Medical Foundation, Miyake Eye Hospital, Nagoya, Japan. METHODS: This study comprised eyes having continuous curvilinear capsulorhexis and phacoemulsification/aspiration followed by implantation of a posterior chamber IOL in the capsular bag. In this prospective, randomized, controlled, and double-masked trial, tranilast 0.5% (Rizaben) eyedrops (15 eyes) or its placebo eyedrops (20 eyes) were given 4 times a day for 3 months after surgery. An anterior eye segment analysis system (EAS 1000, Nidek Co., Ltd.) was used to evaluate the degree of fibrous posterior capsule opacification (PCO) 1 week and 1 and 3 months after surgery. RESULTS: The mean PCO density in the tranilast group was 17.1 cct +/- 4.6 (SD), 20.0 +/- 3.6 cct, and 23.0 +/- 7.7 cct (cct = computer compatible tape) at 1 week and 1 and 3 months, respectively. In the control group, it was 18.2 +/- 5.3, 30.2 +/- 7.8, and 38.4 +/- 8.0 cct, respectively. There was a significant difference in the 1 and 3 month findings between the 2 groups (P < .001). CONCLUSION: Tranilast was effective in preventing fibrous PCO at an early postoperative stage. The possible mechanisms of its effect may be prevention of collagen synthesis by minimizing transforming growth factor type beta released during lens epithelial cell metaplasia.     

Suppressive effects of tranilast on eotaxin-1 production from cultured conjunctival fibroblasts

PURPOSE: Eotaxin, a CC-chemokine with selective chemotactic effects for eosinophils, has been reported to play an important role in allergic conjunctival diseases. We previously reported that eotaxin is produced by conjunctival fibroblasts and keratocytes on stimulation with Th2 cytokines. Tranilast is known to have anti-allergic properties. In this study, we examined the inhibitory effect of tranilast, an anti-allergic drug, on eotaxin-1 production from cultured conjunctival fibroblasts. METHODS:Conjunctival fibroblasts obtained from normal patients were cultured in DMEM/F12 medium. On the fifth passage, the cells were transferred to a 96-well plate and, after starvation for 24 hr, TNF-alpha, IL-4, and tranilast or dexamethasone were added. After 48 hr, the concentrations of eotaxin-1 in the supernatants were measured by ELISA, and the cells were tested for eotaxin-1 expression by real-time PCR. RESULTS:Eotaxin-1 production was observed on simultaneous stimulation with TNF-alpha and IL-4. This production was inhibited by both tranilast and dexamethasone. Inhibition of eotaxin-1 expression was also observed by real-time PCR. CONCLUSIONS: Eotaxin-1 production from conjunctival fibroblasts was inhibited by both tranilast and dexamethasone. These results suggest that the anti-allergic effect of tranilast may be partly due to the inhibition of eotaxin-1 production from conjunctival fibroblasts.     

Injured corneal epithelial cells promote myodifferentiation of corneal fibroblasts

PURPOSE: To determine whether injured corneal epithelial cells stimulate myodifferentiation in corneal fibroblasts and whether transforming growth factor (TGF)-beta is involved. METHODS: Rabbit corneal fibroblasts were cultured on collagen gel, with or without cocultured corneal epithelial cells or with partially scraped epithelial cells, on a companion plate separated by a permeable membrane. To evaluate fibroblast-induced gel contraction, gel thickness was measured daily relative to the original thickness. Total fibroblasts on the gel were counted. Myofibroblasts were counted by using immunocytochemical identification with anti-alpha-smooth muscle actin (alpha-SMA). TGF-beta was assayed in the media on days 3 and 6. These procedures also were performed in the presence of anti-TGF-beta antibody. RESULTS: Gel contraction, alpha-SMA-positive cells, and total cell number were significantly greater on gels with injured epithelial cells than on gels without epithelial cells or with uninjured epithelial cells, as was TGF-beta concentration in the media. Anti-TGF-beta antibody eliminated these differences. CONCLUSIONS: Injured epithelial cells stimulate myodifferentiation in fibroblasts through one or more soluble factors, including TGF-beta.     

Connective tissue growth factor expression and action in human corneal fibroblast cultures and rat corneas after photorefractive keratectomy

PURPOSE: Connective tissue growth factor (CTGF) has been linked to fibrosis in several tissues. In this study, the interactions between CTGF and transforming growth factor (TGF)-beta were assessed in human corneal fibroblasts, and the levels and location of CTGF protein and mRNA were measured during healing of excimer laser ablation wounds in rat corneas. METHODS: Human corneal fibroblasts were incubated with TGF-beta1, -beta2, and -beta3 isoforms, and CTGF mRNA and protein were measured. CTGF was immunolocalized in the cultured fibroblasts by using a specific antibody. Regulation of collagen synthesis by TGF-beta and CTGF was assessed in human corneal fibroblasts with a neutralizing antibody and an antisense oligonucleotide to CTGF. CTGF mRNA and protein were measured in rat corneas up to day 21 after excimer ablation of the cornea. CTGF protein was immunolocalized in rat corneas after photorefractive keratectomy (PRK), and the presence of CTGF mRNA and protein in ex vivo rat corneal scrapings was established. RESULTS: All three TGF-beta isoforms stimulated expression of CTGF in human corneal fibroblasts, and CTGF was immunolocalized in the cells. Both TGF-beta and CTGF increased collagen synthesis in corneal fibroblasts. Furthermore, CTGF antibody or antisense oligonucleotide blocked TGF-beta-stimulated collagen synthesis. CTGF protein and mRNA increased in rat corneas through day 21 after PRK. CTGF expression was also detected in ex vivo scrapings of rat corneas. CONCLUSIONS: These data demonstrate that CTGF is expressed by corneal cells after stimulation by TGF-beta, that CTGF expression increases significantly during corneal wound healing, and that CTGF mediates the effects of TGF-beta induction of collagen synthesis by corneal fibroblasts. These data support the hypothesis that CTGF promotes corneal scar formation and imply that regulating CTGF synthesis and action may be an important goal for reducing corneal scarring.     

Colchicine reverts cell shape but not collagen phenotypes in corneal endothelial cells modulated by polymorphonuclear leukocytes

The authors have shown previously that polymorphonuclear leukocytes (PMN) modulate rabbit corneal endothelial cells (CEC) into cells that irreversibly acquire the characteristics of fibroblasts, including multilayering of spindle-shaped cells and deposition of interstitial extracellular matrix composed predominantly of type I collagen. In an attempt to determine if the changes in cell shape caused by the disruption of cytoskeleton are correlated with the alteration of collagen phenotypes in fibroblastic corneal endothelial cells (FCEC), colchicine and cytochalasin B (CB) were used. A series of dose-response studies were performed, and correlated with exposure time. When cells were exposed to the drugs (ranging from 0.01-4.0 micrograms/ml) 24 hr after plating, the majority of cells treated with colchicine dramatically changed from fibroblastic to polygonal shape: cells became flattened and cytoplasmic processes disappeared. Conversely, no apparent changes were observed in the CB-treated cells. On removal of colchicine, the cells resumed fibroblastic morphology within 24 hr; most of the cells again developed cytoplasmic processes. When collagen phenotypes were analyzed by electrophoresis, types I, III, and V collagen were present in either the colchicine or CB-treated cells, regardless of the concentration of drug used. However, synthesis of type I trimer and type III collagen was significantly increased in the cells treated with colchicine at concentrations greater than or equal to 1.0 microgram/ml; the alpha 1:alpha 2 ratio was approximately 4.5, and type III accounted for 35-40% of the total collagen. CB did not induce a similar alteration. These observations indicate that changes in cell shape are not related to the switch of collagen phenotypes in FCEC.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Rho-kinase inhibitor H-1152P suppresses the wound-healing activities of human Tenon's capsule fibroblasts in vitro

PURPOSE: To analyze the role of Rho-kinase signaling in the wound-healing activities of human Tenon's capsule fibroblasts by using H-1152P, a potent inhibitor of this kinase, in vitro. METHODS: The optimal concentration of H-1152P was determined by MTT test. Cell proliferation was measured by BrdU incorporation and Ki-67 immunostaining, whereas cell viability was investigated by ethidium homodimer-1 dye exclusion. The actin cytoskeleton organization was demonstrated by alpha-smooth muscle actin (SMA) immunostaining and Alexa 488-phalloidin staining. Cell migration was studied on restrained collagen gels and in a scratch-wound assay followed by Ki-67 and fibronectin immunostaining. The effect of H-1152P on contraction was analyzed in floating collagen gels populated with fibroblasts, which were subsequently processed for fibronectin immunostaining. The levels of adducin and the protein kinase A (PKA)-dependent phosphorylation of this protein were detected by immunoblot analysis, to rule out interference with PKA. RESULTS: Incorporation of BrdU and upregulation of Ki-67 were reduced by 80% to 90% in cells incubated with 10 microM of this inhibitor for 4 days (P < 0.01). H-1152P caused the disassembly of stress fibers in a dose-dependent manner without exerting toxic effects and without a considerable interference with the PKA-pathway. H-1152P also significantly suppressed cell migration 3- to 3.5-fold and the contraction of collagen lattices fivefold with a dose-dependent impairment in the assembly of the fibronectin network. CONCLUSIONS: These findings imply a role for Rho-kinase in the wound-healing activities of human Tenon's capsule fibroblasts and show the potential of H-1152P as a safe and specific means to suppress these events.