Tranilast inhibits cell proliferation and collagen synthesis by rabbit corneal and Tenon's capsule fibroblasts

PURPOSE: To determine whether tranilast, N-(3,4-dimethoxycinnamoyl) anthranilic acid, influences cell proliferation and collagen synthesis by rabbit Tenon's capsule fibroblasts (TFs) and corneal stromal fibroblasts (CFs). METHODS: Rabbit TFs and CFs (7000 cells/well) were cultured in F-12 nutrient mixture supplemented with 1% FBS, plus 0, 3, 30, or 300 microM tranilast, and the number of cells was counted 72 hrs later. To determine the effect of tranilast on collagen synthesis, cells at confluence were cultured in a medium containing 0, 3, 30, or 300 microM tranilast and labeled with 3H-proline, and the amount of radioactivity incorporated into collagenase-sensitive proteins was measured. RESULTS: At 300 microM, tranilast decreased the number of TFs by about 27% and the number of CFs by about 45%, but had no effect on cell viability. The same concentration of tranilast reduced TFs collagen synthesis and CFs collagen synthesis. CONCLUSIONS: Tranilast may inhibit scar formation after trabeculectomy for glaucoma and after excimer laser photorefractive keratectomy.     

Proliferative effects of heparin-binding epidermal growth factor-like growth factor on pterygium epithelial cells and fibroblasts

PURPOSE: To investigate the growth promoting and chemotactic effects of heparin binding epidermal growth factor-like growth factor (HB-EGF), recently shown to be upregulated by ultraviolet irradiation in pterygium-derived epithelium cells (PECs) and pterygium fibroblasts (PFs). METHODS: PECs and PFs were incubated with various concentrations of HB-EGF. Cell proliferation was evaluated by measurement of [3H]thymidine incorporation. The potential chemotactic effect of HB-EGF on these two cell lines was assessed with migration assays, using modified Boyden chambers and checkerboard analysis. RESULTS: Incubation of PECs and PFs with HB-EGF resulted in a significant increase in [3H]thymidine incorporation compared with that of control cells. HB-EGF stimulated chemotaxis of both PECs and PFs. Maximum stimulation occurred at 1 ng/mL for PFs and 10 ng/mL for PECs. These effects were abolished by the addition of a neutralizing antibody to HB-EGF. CONCLUSIONS: The findings demonstrate the potential proliferative and chemotactic effects of HB-EGF on both PECs and PFs. This is the first study to illustrate the positive effect of a specific growth or chemotactic factor on the cellular elements of a pterygium.     

Overexpression of Insulin-like growth factor-binding protein-2 in pterygium body fibroblasts

PURPOSE: To examine the expression pattern of insulin-like growth factor-binding protein (IGFBP)-2 in cultured primary pterygium fibroblasts and compare it with expression in normal conjunctival fibroblasts. METHODS: Profile of gene expression by normal conjunctival and primary pterygium fibroblasts was performed by using a cDNA microarray. The overexpression of IGFBP-2 thus identified was further confirmed by RT-PCR and Western blot analysis of cultured cells and by immunohistochemistry on primary pterygium and normal conjunctival tissue sections. RESULTS: A dramatically increased expression of IGFBP-2 mRNA was demonstrated in cDNA microarray membranes from two different pterygium fibroblasts. This finding was confirmed by RT-PCR in four additional different pterygium fibroblasts and by Western blot analysis of their culture supernatants. Immunohistochemistry of frozen sections from primary pterygium demonstrated increased staining in extracellular matrix of the stroma, compared with that of the normal conjunctiva. IGFBP-2 was also found in goblet cells of both normal conjunctival and pterygium epithelia. CONCLUSIONS: The increased expression of IGFBP-2 mRNA and protein in pterygium fibroblasts is further strong evidence to support the transformed phenotype of these cells and helps explain why there is increased growth of fibrovascular tissue. This phenotype may be used as a marker to assess the malignant nature of pterygium growth and recurrence.     

The role of substance P in the pathogenesis of pterygia

PURPOSE: Pterygium is a prevalent ocular surface disorder thought to be triggered by chronic ultraviolet damage to the limbus. One of the enigmatic features of pterygium is its wing-like shape, and the mechanism(s) supporting its centripetal growth remain to be elucidated. Because the growth pattern of pterygia mirrors the radial arrangement of corneal nerves, the authors propose that neuropeptides may facilitate its directional growth. This hypothesis prompted an investigation of the role of the sensory neuropeptide substance P (SP) and its receptor (NK(1) receptor) in directing cell migration in pterygia that may explain the characteristic growth pattern. METHODS: Immunohistochemical analysis for SP and the NK(1) receptor was performed on five pterygium specimens with corresponding autologous conjunctiva and limbus. Migration of pterygium epithelium, fibroblasts, and vascular endothelial cells toward SP was assessed by using a modified Boyden chamber. RESULTS: SP and NK(1) receptors were localized to infiltrating fibroblasts, mononuclear cells and the epithelia of pterygium, conjunctiva, and limbus, with elevated NK(1) receptor staining observed in pterygia. SP at nanomolar concentrations induced cell migration in pterygium fibroblasts and vascular endothelium in a dose-dependent fashion, which was inhibited by an NK(1) receptor antagonist. Pterygium epithelial cells were not migratory in these experiments. CONCLUSIONS: For the first time, this study showed the presence of NK(1) receptor in pterygia and that SP is a potent chemoattractant for pterygium fibroblasts and vascular endothelial cells, implying that SP may contribute to the shape of pterygia through its profibrogenic and angiogenic action.     

翼状胬肉组织中胶原纤维的病理学改变

目的从病理学角度探讨翼状胬肉发生发展的机制。方法选择临床切除的复发及原发胬肉材料共50例,分头、体、尾部行苏木素-伊红染色及透射电镜观察。结果进行期胬肉表面复层鳞状上皮化生明显,杯状细胞数量多于静止期。浅层毛细血管较静止期胬肉增多,可见新生毛细血管内皮细胞异常连接,胶原纤维排列形态紊乱,成纤维细胞胞核见分裂相,胶原纤维及弹性纤维大量变性,可见中电子密度均质物及大量空泡变性胶原。空泡变性的成纤维细胞内可见线粒体肿胀,各种细胞器结构不清。结论翼状胬肉有明显的胶原纤维变性,研究提示氧自由基可能参与胬肉组织发生发展进程。     

Differential expression of matrix metalloproteinases and their tissue inhibitors at the advancing pterygium head

PURPOSE: Pterygia are a proliferative and inflammatory growth of limbal epithelial stem cell origin, characterized by corneal tissue invasion and extensive matrix remodeling including the destruction of Bowman's layer (BL). The purpose of this study was to determine the expression of matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) at the advancing pterygium edge. METHODS: Formalin-fixed, paraffin-embedded whole eyes (n = 11) with pterygia attached, were serially sectioned and analyzed immunohistochemically to determine the spatial distribution of four MMPs and three TIMPs. Tear samples were collected from other patients with pterygia (n = 11) and displayed by gelatin zymography. RESULTS: Collagenase-1 was expressed by pterygium epithelial cells, corneal stromal fibroblasts and pterygium fibroblasts that had migrated between the epithelium and BL at the advancing pterygium edge. Collagenase-3 and gelatinases A and B were detected in all pterygia, intensely staining columnar epithelial cells directly adjacent to the denatured BL. In addition, gelatinase A immunoreactivity was observed on BL. Immunoreactivity for TIMP-1 and -3 paralleled that of the gelatinases, with more intense staining in epithelial cells and fibroblasts where BL was absent. TIMP-2 was faintly detected in pterygium epithelial cells but intensely stained pterygium fibroblasts. Gelatinase B was the most abundant gelatinolytic enzyme present in tears, elevated approximately twofold in eyes with pterygia versus the contralateral control eyes. CONCLUSIONS: This investigation is the first to identify the expression pattern of MMPs and TIMPs at the advancing pterygium edge in specimens of human eyes and in tears derived from patients with pterygia. These enzymes may be responsible for the destruction of BL, and their pattern of differential expression suggests that each may play a selective role in the pathogenesis of pterygia.     

小檗碱调节翼状胬肉成纤维细胞增殖能力的机制研究

目的:探讨小檗碱对体外培养翼状胬肉成纤维细胞增殖能力的影响及可能的作用机制。方法:通过对手术获取的翼状胬肉组织进行培养,获取翼状胬肉成纤维细胞。采用不同终浓度(0、20、40、80μmol/L)小檗碱对细胞进行诱导,检测小檗碱诱导后翼状胬肉成纤维细胞凋亡水平、线粒体膜电位和凋亡相关因子mRNA与蛋白表达水平变化。结果:小檗碱能够以剂量依赖的方式提高体外培养翼状胬肉成纤维细胞线粒体去极化水平、细胞凋亡比率、促凋亡基因Bax和Bad mRNA与蛋白的表达水平,降低Bcl-2 mRNA和蛋白的表达水平。结论:小檗碱可能通过提高体外培养翼状胬肉细胞线粒体去极化水平诱导胬肉细胞发生凋亡。     

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,     

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.     

锗132对体外培养翼状胬肉成纤维细胞的增殖抑制作用观察

观察锗１３２对体外培养翼状胬肉成纤维细胞的抗增殖作用,寻找辅助治疗翼状胬肉和预防翼状胬肉复发的新方法。方法将翼状胬肉成纤维细胞行体外原代和传代培养;观察不同浓度锗１３２及丝裂霉素Ｃ对成纤维细胞生长曲线的影响及培养的翼状胬肉成纤维细胞增殖抑制作用和毒性作用;采用免疫组织化学方法检测增殖细胞核抗原（ｐｒｏｌｉｆｅｒａｔｉｎｇ ｃｅｌｌ ｎｕｃｌｅａｒ ａｎｔｉｇｅｎ,ＰＣＮＡ）的表达,结果锗１３２可抑     

CD34在翼状胬肉中表达的意义

目的研究翼状胬肉组织中CD34的表达及意义。方法对50例手术切除的翼状胬肉标本行苏木精-伊红染色、过碘酸希夫染色，并进行形态学、免疫组织化学研究和免疫荧光共聚焦显微镜下观察CD34、波形蛋白（VIM）、血管内皮生长因子（VEGF）在翼状胬肉中的表达。结果在翼状胬肉标本中，CD34在血管内皮细胞、血管周细胞以及一些散在的星形、梭形细胞中表达阳性，而在血管壁上及成熟的胶原细胞上呈阴性表达；VIM阳性表达于大部分翼状胬肉组织中；组织中部分疏松排列的网状及裂隙样、管样区结构过碘酸希夫染色呈阳性的紫红色反应；网状区的梭形细胞VEGF阳性表达。结论翼状胬肉组织中CD34阳性细胞来源于间充质干细胞的分化。翼状胬肉除以血管发生和血管新生的方式生成血管外，血管拟态可能是另外一种供血途径。     

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.     

Capillaries in the epithelium of pterygium

AIM—To present new morphological observations of intraepithelial capillaries in pterygium and to provide some explanations for this phenomenon.
METHODS—The ultrastructural features of pterygia from 26 patients were examined. Surgically excised tissue was processed for conventional light and transmission electron microscopy.
RESULTS—Individual capillaries within the epithelium of the anterior half towards the head of pterygia were identified in 11 specimens out of 26 pterygia examined (42.3%). The perivascular connective tissue of the intraepithelial capillaries contained fibroblasts, collagen fibrils, and elastin-like material. Epithelial cells surrounding these capillaries showed defects in the basal lamina in contrast with the continuous basal lamina of the endothelium. In the intercellular space of the epithelium an amorphous substance, occasional fibroblast processes, and collagen fibrils were frequently observed.
CONCLUSION—Capillaries in the epithelium of pterygia are rare, but not exceptional. The ingrowth of these vessels from the stroma into the epithelium can be interpreted as a reaction to hypoxia or deficiency of any other substance transported via the bloodstream. Apparently, the perivascular connective tissue can be used by ingrowing fibroblasts as a migration pathway. The migrating fibroblasts appear to use the defects of the epithelial basal lamina (whether partially or complete) in order to reach the intercellular space. It is possible that collagen fibrils in the epithelial intercellular space have been laid down by fibroblasts which contribute to the pathological dedifferentiation of the conjunctival epithelium.

Keywords: pterygium; ultrastructure; epithelium; blood vessels     

Involvement of bone marrow-derived stem and progenitor cells in the pathogenesis of pterygium

AIMS: To evaluate the involvement of multipotential stem and progenitor cells in the pathogenesis of pterygium. METHODS: Paraffin-embedded and snap-frozen primary pterygium (n = 10) were serially sectioned and analysed immunohistochemically to determine the expression level of AC133 (marker for the primitive haematopoietic progenitors), CD34 (marker for the haematopoietic progenitor cells and endothelium), c-Kit (marker for haematopoietic and stromal progenitor cells), and STRO-1 (a differentiation antigen present on bone marrow fibroblast cells and on various nonhaematopoietic progenitor cells). RESULTS: In all the primary pterygium, immunoreactivity of AC133 and STRO-1 was found in some of the epithelial and stromal cells, CD34 was observed in the vascular endothelium, and some scattered ovoidal cells were found in the subepithelial connective tissue. C-Kit was expressed mainly in the basal epithelium of the head portions, and some spindle-shaped stromal cells. There is no immunoreactivity of AC133, c-Kit, and STRO-1 in normal conjunctiva, whereas CD34 was mildly stained with vessel wall. CONCLUSION: Multipotential stem and progenitor cells may be involved in the pathogenesis of pterygium through its differentiation into fibroblasts and vascular endothelial cells.     

Role of Heat Shock Protein 47 in Transdifferentiation of Human Tenon's Fibroblasts to Myofibroblasts

ABSTRACT: BACKGROUND: Heat shock protein 47 (Hsp47) is a well-known molecular chaperone in collagen synthesis and maturation. The aim of this study is to investigate its putative role in the transdifferentiation of Tenon's fibroblasts to myofibroblasts. METHODS: Primary cultured human Tenon's fibroblasts were exposed to transforming growth factor-beta1 (TGF-beta1) for up to 48 hours. The mRNA levels of Hsp47 and alpha smooth muscle actin (alphaSMA) were determined by quantitative real time RT-PCR. After delivery of small interfering RNA (siRNA) molecules targeting Hsp47 into the cells, the expression of Hsp47 and alphaSMA proteins was determined by western immunoblotting. RESULTS: TGF-beta1 increased the mRNA expressions of both Hsp47 and alphaSMA in human Tenon's fibroblasts, as determined by quantitative real time RT-PCR. However, it induced the protein expression of only alphaSMA but not Hsp47, as determined by western immunoblots. When siRNAs specific for Hsp47 were introduced into those cells, the TGF-beta1-induced expression of alphaSMA was significantly attenuated on western immunoblots; after 48 hours of exposure to TGF-beta1, the relative densities of immunobands were 11.58 for the TGF-beta1 only group and 2.75 for the siRNA treatment group, compared with the no treatment control group (p < 0.001). CONCLUSIONS: Our data suggest that Hsp47 may be related to the TGF-beta1-induced transdifferentiation of human Tenon's fibroblasts to myofibroblasts.     

β-catenin在翼状胬肉成纤维细胞中的表达

目的:β-catenin在正常的细胞粘附和wnt信号通路中发挥重要功能,本文探讨其在翼状胬肉组织及成纤维细胞中的表达及TGF-β1对其的影响。方法:通过组织块培养获取正常球结膜和翼状胬肉成纤维细胞并传至第4代,用10μg/LTGF-β1作用于细胞。抗β-catenin抗体对体内取出组织和培养的细胞行免疫组化染色;免疫印迹法检测培养成纤维细胞中β-catenin的表达情况。结果:免疫组化显示翼状胬肉成纤维细胞中β-catenin阳性着色强于正常结膜成纤维细胞。β-catenin在胬肉组织中较多,且多分布于上皮下纤维血管组织,上皮染色较少。体外培养见翼状胬肉成纤维细胞的生长较正常结膜成纤维细胞快、增殖能力强。免疫印迹法显示在翼状胬肉成纤维细胞蛋白中β-catenin的特异性阳性条带灰度值高于正常球结膜成纤维细胞蛋白。TGF-β1能促进翼状胬肉成纤维细胞中的β-catenin蛋白表达。结论:β-catenin在翼状胬肉组织及其成纤维细胞中的表达增高,提示β-catenin与翼状胬肉的发病有关。