UVB-elicited induction of MMP-1 expression in human ocular surface epithelial cells is mediated through the ERK1/2 MAPK-dependent pathway

PURPOSE: Pterygia are common, frequently recurring ocular surface lesions characterized by tissue remodeling, cellular proliferation, angiogenesis, and inflammation. The increased incidence of pterygia in persons exposed to excessive solar radiation suggests that ultraviolet (UV) light may play a critical role in the pathogenesis of this disease. These investigations were focused on the expression of collagenase-1 (matrix metalloproteinase [MMP]-1) in pterygia and cultured pterygium epithelial cells, to determine whether the expression of this protease could be modified after exposure to UVB. METHODS: Pterygium, conjunctival, and limbal epithelial cells were subcultured and exposed to various amounts of UVB. The conditioned medium and RNA were harvested for analysis by gelatin zymography, Western blot analysis, ELISA, and RT-PCR. Furthermore, whole pterygium specimens were irradiated to determine secreted MMP-1 levels. RESULTS: Immunohistochemical analysis revealed enhanced MMP-1 expression in pterygia that corresponded precisely with p63-positive epithelial cells. In contrast, significantly less MMP-1 reactivity was found in normal conjunctiva, limbus, and cornea. A dose- and time-dependent increase in MMP-1 was observed when pterygium epithelial cells were exposed to UVB with no significant modulation of inhibitor activity. MMP-1 was not affected in irradiated normal conjunctival epithelial cells or in pterygium fibroblasts but was induced in limbal epithelial cells. Although the induction of MMP-1 after UVB was not mediated by an intermediate soluble factor, the extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein kinase (MAPK) intracellular pathway was involved. CONCLUSIONS: Collectively, these data support the hypothesis of the involvement of UV light and MMPs in the development of pterygia and may assist in devising new therapeutic approaches for the treatment and prevention of pterygia.     

基质细胞衍生因子及其受体CXCR4在翼状胬肉的异常表达

目的探讨基质细胞衍生因子(stromal cell-derived factor-1,SDF-1)及其受体CXCR4在翼状胬肉的表达及意义。方法用免疫组织化学染色方法对4例正常人结膜组织、11例初发性翼状胬肉和9例复发性翼状胬肉组织中SDF-1/CXCR4蛋白的表达进行检测。结果在正常结膜组织中SDF-1/CXCR4仅表达于基底部上皮细胞;胬肉组织中有17例,全层上皮细胞均有SDF-1/CXCR4的表达,胬肉基质层中浸润的炎性细胞、血管内皮细胞均有CXCR4的蛋白表达;3例初发性翼状胬肉表达方式与正常结膜相似。结论翼状胬肉组织SDF-1及其受体CXCR4蛋白表达增加;上皮细胞分泌的SDF-1通过与其受体CXCR4的相互作用可能参与了翼状胬肉的血管形成和炎性细胞浸润。     

Angiogenesis in pterygium: morphometric and immunohistochemical study

OBJECTIVE: To evaluate the role of angiogenesis in the pathogenesis of pterygium by comparing the expression of von-Willebrand factor (vWF) and vascular endothelial growth factor (VEGF) in pterygium, and in normal superior bulbar conjunctiva. METHODS: 23 human samples from pterygium and the superior bulbar conjunctiva were stained using rabbit anti-vWF and anti-VEGF antibodies. The density of vWF and VEGF positive vessels, VEGF staining intensity and the number of VEGF positive stromal, epithelial and vascular endothelial cells were evaluated. RESULTS: Pterygium specimens had higher average vWF and VEGF positive microvascular counts per high power field (P = 0.0012), higher average VEGF staining intensity scores in epithelial, stromal and endothelial cells (p < 0.0001) and higher VEGF positive cell counts (P < 0.0001) than normal conjuctiva. CONCLUSIONS: Over-expression of VEGF in pterygium tissue, together with the abundance of vWF-stained new vessels, may support previous suggestions that angiogenesis may play a role in the formation of pterygium.     

Relations between lymphangiogenesis and the size of pterygium

AIM: To examine the relations between lymphangiogenesis and the size of pterygium. METHODS: Tissues from 88 primary and 34 recurrent pterygia were evaluated, and those from 7 nasal epibulbar conjunctiva segments were used as controls. Pterygium slices from each patient were stained with LYVE-1 monoclonal antibodies to identify lymphatic microvessel for calculating lymph-vascular area (LVA), lymph-microvascular density (LMD) and lymph-vascular luminal diameter (LVL). Also, the relations between lymphangiogenesis (measuring by LVA, LMD and LVL) and the size of pterygium (extension, width and area) were explored. RESULTS: There were a few LYVE-1(+) lymphatic vessels in normal epibulbar conjunctiva segments. However, the number of lymphatic vessels slightly increased in primary pterygia and dramatically increased in recurrent pterygia. LVA, LMD and LVL significantly increased in recurrent pterygia in comparison with primary pterygia (all P<0.05). Both LMD and LVA were correlated with the width and area of pterygia (both P< 0.05), and LVA was also correlated with the extension of pterygia(P<0.05). CONCLUSION: Lymphangiogenesis is correlated with the size of pterygium. The outgrowth of lymphatic vessels might contribute to the development of pterygia.     

Relations between lymphangiogenesis and the size of pterygium

AIM: To examine the relations between lymphangiogenesis and the size of pterygium. METHODS: Tissues from 88 primary and 34 recurrent pterygia were evaluated, and those from 7 nasal epibulbar conjunctiva segments were used as controls. Pterygium slices from each patient were stained with LYVE-1 monoclonal antibodies to identify lymphatic microvessel for calculating lymph-vascular area (LVA), lymph-microvascular density (LMD) and lymph-vascular luminal diameter (LVL). Also, the relations between lymphangiogenesis (measuring by LVA, LMD and LVL) and the size of pterygium (extension, width and area) were explored. RESULTS: There were a few LYVE-1(+) lymphatic vessels in normal epibulbar conjunctiva segments. However, the number of lymphatic vessels slightly increased in primary pterygia and dramatically increased in recurrent pterygia. LVA, LMD and LVL significantly increased in recurrent pterygia in comparison with primary pterygia (all P<0.05). Both LMD and LVA were correlated with the width and area of pterygia (both P< 0.05), and LVA was also correlated with the extension of pterygia(P<0.05). CONCLUSION: Lymphangiogenesis is correlated with the size of pterygium. The outgrowth of lymphatic vessels might contribute to the development of pterygia.     

Evaluation of transforming growth factor and vascular endothelial growth factor polymorphisms in Taiwan Chinese patients with pterygium

PURPOSE: Pterygium is an invasive and highly vascularized growth, thought to arise from activated and proliferating limbal epithelial stem cells. Epidemiologic studies have found the increase of active angiogenic and epithelial growth factors in pterygia, and implicated that these molecules could be involved directly or indirectly in the pathogenesis of pterygia as causative factors. The aim of this study was to investigate the association of polymorphisms of transforming growth factor (TGF) and vascular endothelial growth factor (VEGF) with pterygium. METHODS: A total of 133 pterygium patients and 105 volunteers without pterygium were enrolled in this study. Polymerase chain reaction based restriction fragment length polymorphism analysis was used to resolve the TGF-Beta1-509 and VEGF-460 genotypes. RESULTS: There was no significant difference in the allele frequency or genotype of TGF-Beta1-509 or VEGF-460 between total pterygium and the control group. No interaction between TGF-Beta1-509 and VEGF-460 was found either. CONCLUSIONS: These results indicate that TGF-Beta1-509 and VEGF-460 polymorphisms were not highly associated with the pathology of pterygium. However, it may still be worthwhile to continue to search for angiogenic gene polymorphisms in order to predict the development of pterygium.     

Initial experience with autologous conjunctiva/limbus transplantation in pterygium

The present paper reports on the technique and results of conjunctiva/limbus transplantation in 13 eyes with primary or cicatricial pterygium (12 cases and 1 case, respectively). In all cases the pterygia were located nasally. The operation was performed by bare sclera excision, covered with a free graft from the superotemporal conjunctiva and limbus. After a mean follow-up time of 5 months there was only one recurrence. On the basis of a new understanding of limbal diseases the authors believe that conjunctiva/limbus transplantation is an encouraging and appropriate technique for ideal anatomic reconstruction and low recurrence rates in pterygia.     

Pterygium with bulbar conjunctival hemorrhages

· Background: This study was carried out to describe the clinical features and electron-microscopic characteristics of the capillaries in pterygium with conjunctival hemorrhage. · Methods: We compared the clinical findings in Japanese and Tunisian primary pterygia and in pterygium with and without bulbar conjunctival hemorrhages. The capillary fine structures of pterygium with bulbar conjunctival hemorrhages were studied by electron microscopy and compared with those without bulbar conjunctival hemorrhages. · Results: Conjunctival hemorrhages were noted in 16.4% of Tunisian patients but not in any Japanese patients. The former pterygia were significantly larger, more vascular and more frequently hyperemic than the latter. Pterygia with bulbar conjunctival hemorrhages were more hyperemic than without hemorrhages. Electron microscopy revealed interruptions of the endothelial cells and basement membrane of the capillaries in the pterygia with bulbar conjunctival hemorrhages, with blood cells escaping through the endothelial interruptions. · Conclusion: Conjunctival hemorrhage in pterygia may be caused by fragility of the endothelial cells and basement membrane in the capillaries, which easily induces hemorrhage when the eyes are irritated by rubbing or by conjunctival foreign bodies. Pterygium with conjunctival hemorrhage is more frequently found in Tunisians than in Japanese. Received: 6 April 1998 Revised version received: 29 September 1998 Accepted: 5 October 1998     

激光共焦显微镜与组织病理学对翼状胬肉组织中微血管密度研究的比较

背景 研究表明,翼状胬肉是眼表的一种异常细胞增生性疾病,血管因素在它的生长和复发中起重要作用.多种抗新生血管治疗方法被应用于翼状胬肉,但活体观察翼状胬肉血管情况的研究尚少见.目的 比较角膜激光共焦显微镜与免疫组织化学染色对翼状胬肉组织中微血管密度研究的结果. 方法 采用前瞻性病例对照研究方法,收集原发性翼状胬肉患者20例20眼为翼状胬肉组,手术中获取翼状胬肉组织;另收集年龄和性别匹配的行内眼手术及斜视手术的患者20例20眼为正常球结膜组,手术中获取正常鼻侧球结膜组织作为对照,翼状胬肉组患者均于术前用海德堡角膜激光共焦显微镜采集5幅翼状胬肉头部的微血管图像,同时采集正常球结膜组正常球结膜微血管图像.对两组患者手术中获得的标本进行常规组织病理学检查和CD31免疫组织化学染色,计算组织标本中微血管密度值,对翼状胬肉组患者激光共焦显微镜所测的微血管密度值以及手术标本中的微血管密度值分别与正常球结膜组进行比较,并分别将两组用两种方法测得的血管密度值进行相关分析.结果 角膜激光共焦显微镜下翼状胬肉基质中的微血管密度( MVD)值为( 8929±2993) μm/mm2,正常球结膜中的微血管密度值为( 4202±692) μm/mm2,二者比较差异有统计学意义(t=6.881,P＜0.01).翼状胬肉的组织标本中CD31染色阳性的微血管数为(21.00±4.06)个/高倍视野,正常球结膜组为(6.07±1.75)个/高倍视野,二者比较差异有统计学意义(t=12.312,P＜0.01).翼状胬肉组和正常球结膜组激光共焦显微镜活体测得的MVD值与免疫组织化学离体测得的MVD值均呈显著正相关(翼状胬肉组:r=0.649,P＜0.01;正常球结膜组:r=0.572,P＜0.01).结论 角膜激光共焦显微镜活体动态观察翼状胬肉组织微血管的方法优于离体的免疫组织化学检测法,角膜激光共焦显微镜对翼状胬肉的血管形态进行活体研究有助于进一步研究翼状胬肉组织的生物学行为及其机制.     

The pterygium, Autologous conjunctiva-limbus transplantation as treatment]

Based on a new concept of the limbus as a junctional zone for separating the vascularized conjunctiva from the avascular cornea, this study presents conjunctival/limbal autograft transplantation for 22 cases of pterygium. The pterygia were primary in 17 eyes, cicatricial in 1 and recurrent in 4. In all cases a free transplant of the superotemperal limbus with an adjacent piece of thin conjunctiva was placed in the excision area. Postoperative follow-up ranged from 1 1/2 to 17 months, with a mean of 8.7 months. Only two pterygia recurred. In all other cases ideal anatomic reconstruction was achieved without any side effects. The authors believe that conjunctival/limbal transplantation is an encouraging technique for treating a pterygium surgically.     

Angiogenesis in pterygium: study of microvessel density, vascular endothelial growth factor, and thrombospondin-1

PURPOSE: This retrospective study aims to elucidate the role of angiogenesis in the pathogenesis of pterygium. We evaluated microvessel density (MVD), and expression of vascular endothelial growth factor (VEGF) and thrombospondin-1 (TSP-1). METHODS: Fifty-two surgically excised pterygia and seven normal conjunctivae were immunohistochemically studied applying the streptavidin-biotin method in paraffin-embedded tissue sections. Monoclonal antibodies were targeted against CD31, VEGF, and TSP-1 proteins. Results: Pterygium presented with statistically significant higher average count of microvessels compared to normal conjunctivae (17.97+/-8.5 vs5.72+/-5 per high power field, P=0.001). In 24/52 (46.2%) cases of pterygium, high expression levels for VEGF were demonstrated, whereas the mean percentage of VEGF-positive epithelial cells was 58.03%. Furthermore, normal conjunctival presented statistically significant higher expression levels for VEGF in epithelial cells (83.14+/-36.08 vs58.03+/-31.23%, P=0.007). On the contrary, the presence of VEGF immunoreactivity in vascular endothelial and stromal cells was significantly higher in pterygium tissues (P<0.0001). Stromal staining for TSP-1 was detected in only 29/52 (55.8%) of the cases and no correlation with normal conjunctivae was found. Finally, statistically significant positive correlation between MVD values and stromal VEGF expression was found (P=0.049). CONCLUSION: The angiogenesis-related factors that were studied proved to be highly expressed in pterygium tissue. On the contrary, TSP expression level was low, allowing inducers of angiogenesis to act uninhibited. This phenomenon could provide the pathogenic basis of pterygium formation.     

PEDF和VEGF在翼状胬肉组织中的不平衡表达

目的 检测翼状胬肉组织中血管生长因子VEGF、FGF-2和抑制因子PEDF的表达，以研究PEDF在翼状胬肉发展中所起作用。方法 用免疫荧光组化法及免疫印迹法对9例原发性翼状胬肉组织进行PEDF、VEGF及FGF-2的检测、分析，并与8例正常人结膜、角膜组织进行对照研究。结果 PEDF在正常人结膜、角膜上皮中高表达，而在翼状胬肉组织中没有或弱表达。VEGF和FGF-2在翼状胬肉组织中高表达，而在正常人结膜、角膜组织中表达较弱。结论 在翼状胬肉组织中血管生长抑制因子PEDF没有或弱表达，而血管生长因子VEGF和FGF-2高表达，提示两者不平衡表达引起纤维血管增生。     

自体角膜缘干细胞移植治疗翼状胬肉

目的探讨翼状胬肉切除加自体角膜缘干细胞移植术治疗翼状胬肉的疗效。方法为120例（139眼）翼状胬肉在局麻下行胬肉切除，将同侧眼颞上方带有角膜缘干细胞的结膜瓣移植到胬肉处的巩膜上。结果术后反应轻，移植片均存活，降低了复发率。结论自体角膜缘干细胞移植治疗翼状胬肉可行性好。     

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.     

EphrinB2 and EphB4 expression in pterygia: new insights and preliminary results

Objective: Pterygium is a growth of fibrovascular tissue onto the cornea, in which the mechanisms of cell proliferation and vascularization are unknown. The ephrin-Eph system, especially ephrinB2 and its receptor EphB4, has been shown to play an important role in tumor angiogenesis. EphrinB2 and EphB4 have also been reportedly involved in the pathogenesis of ocular angiogenesis. This study was designed to investigate the expression of ephrinB2 and its receptor EphB4 in pterygia.Design: Experimental study of the expression of ephrinB2 and its receptor EphB4 in pterygia.Participants: Twenty-three primary pterygia, 5 recurrent pterygia, and 11 normal conjunctiva were studied.Methods: Immunohistochemistry studies were used to assess ephrinB2 and EphB4 protein expression levels and the tissue distribution in the samples.Results: EphrinB2 and EphB4 staining was present at a dense level in the total epithelium of the head portions of both primary and recurrent pterygial specimens, although just in the basal and parabasal layer of the epithelium of most of the normal conjunctivae.Conclusions: EphrinB2 and EphB4 appear to be overexpressed in pterygium, and they may play important roles in its development.     

表皮生长因子受体家族在翼状胬肉上皮内的异常表达

目的 了解表皮生长因子受体（ｅｐｉｄｅｒｍａｌ ｇｒｏｗｔｈ ｆａｃｔｏｒ ｒｅｃｅｐｔｏｒ,ＥＧＦＲ）家族的ＥＧＦＲ、ＥｒｂＢ２及ＥｒｂＢ３蛋白在翼状胬肉上皮内的表达。方法 用免疫荧光组织化学及Ｗｅｓｔｅｒｎ ｂｌｏｔ地１５例初发期翼状胬肉患者切除的翼状胬肉组织进行ＥＧＦＲ、ＥｒｂＢ２及ＥｒｂＢ３蛋白的检测,并与正常人结膜组织进行对照。结果 免疫荧光组织化学染色显示,在正常结膜上皮中,ＥＧＦＲ蛋     

翼状胬肉组织中环氧化酶-2及p21-ras的表达研究

目的 通过免疫组织化学方法检测翼状胬肉组织中环氧化酶-2（COX-2）和p21ras的表达，进一步证实氧自由基在翼状胬肉形成和发展中的作用。方法 选择临床切除的静止期、进行期和复发期胬肉材料共40例，行苏木精-伊红染色及COX-2和p21ras免疫组织化学染色。结果 COX-2及p21ras在正常球结膜中表达微弱，静止期胬肉组织中COX-2和p21ras阳性染色主要位于复层鳞状上皮化生细胞，在进行期及复发期翼状胬肉组织中COX-2及p21ras阳性染色弥漫性分布于复层鳞状上皮化生细胞的胞浆和杯状细胞的胞浆中，胬肉组织中的炎性细胞和血管内皮细胞胞浆可见少量COX-2及p21ras表达。结论 p21ras and COX-2的过度表达在翼状胬肉的发生和发展过程中可能起重要作用。