Vascular endothelial growth factor gene 460 polymorphism is associated with pterygium formation in female patients

PURPOSE: Pterygium is composed of proliferating fibrovascular tissue, and its formation and progression require neovascularization. Vascular endothelial growth factor (VEGF) is a specific mitogen for vascular endothelial cells and is essential for neovascularization. Recently, the VEGF-460 polymorphism was reported to be associated with increased VEGF basal promoter activity and with several fibrovascular diseases, such as proliferative diabetic retinopathy, endometriosis, and chronic renal disease. Therefore, this study evaluated the potential association between pterygium formation and VEGF-460 polymorphism. METHODS: One hundred twenty-seven pterygium patients and 102 volunteers without pterygium were enrolled in this study. Polymerase chain reaction-restriction fragment length polymorphism analysis was used to resolve the VEGF-460 genotypes of pterygium patients and normal controls. RESULTS: There were no significant differences between pterygium and control groups in age, sex, and distribution of genotype and allelic frequency of VEGF-460 polymorphism. However, when results were stratified by sex, there were significant differences between female patients and controls in the distribution of genotype and allelic frequency of VEGF-460 polymorphism. Females who carried at least 1 C allele (C/C and C/T genotypes) had about a 2.5-fold increased risk of developing pterygium compared with those who carried the T/T genotype, and there was a significantly higher frequency of C/C and C/T genotypes in younger female patients than in older female patients. There were no differences between male patients and controls in the distribution of genotype and allelic frequency of the VEGF-460 polymorphism. CONCLUSIONS: VEGF-460C polymorphism is associated with pterygium formation in female patients. Females who carried the C allele have increased risk of developing pterygium at a younger age.     

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.     

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.     

Connective tissue growth factor in pterygium: simultaneous presence with vascular endothelial growth factor – possible contributing factor to conjunctival scarring

BACKGROUND: Various growth factors have been detected in pterygium and been associated with its vasculogenesis. The basic pathophysiological mechanisms responsible especially for the fibrotic activity in pterygium are, however, not yet known. Connective tissue growth factor (CTGF) has been shown to be substantially involved in various processes of fibrosis. We report on the presence of CTGF in pterygium and its simultaneous presence with vascular endothelial growth factor (VEGF). METHODS: Samples of pterygium were collected during surgery with informed consent of the patients. Specific, non-commercial primary antibodies against CTGF were used to detect CTGF using immunohistochemistry. Specificity of antibodies was confirmed with Western-blot analysis. The same specimens were stained with commercial antibodies for VEGF. Additionally RT-PCR analysis was performed from pterygium samples. RESULTS: CTGF was detected in the epithelium of all samples as well as in some stromal keratocytes. The RT-PCR confirmed the identity of CTGF in these samples. The staining pattern differed slightly from that of VEGF, which was detected in all samples. The control sections were negative. CONCLUSION: CTGF is present in the epithelium of a majority of pterygia and probably contributes to fibrosis. Simultaneous presence with VEGF suggests growth factor interaction and possible involvement in apoptotic dysregulation.     

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     

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.     

The relation between expression of basic fibroblast growth factor and mast cells in pterygium

OBJECTIVE: To examine the expression and distribution of basic fibroblast growth factor (bFGF) and mast cells in pterygium, and evaluate its effects in the pterygium formation and progression. METHOD: The expression of bFGF and mast cell tryptase in 17 primary pterygia, 6 recurrent pterygia and 6 normal conjunctival specimens were studied. The mast cell count and bFGF expression situation were observed. RESULTS: The bFGF was specifically localized in the epithelium, blood vessels and a subset of connective tissue cells. The bFGF expression was increased in the recurrent pterygium. The numbers of infiltrating mast cells (five 400 x sights) were (45.47 +/- 5.50) cells and (48.83 +/- 3.19) cells in the primary and recurrent pterygium respectively. In the comparisons between the cells in the pterygium (primary and recurrent) and (4.24 +/- 2.36) cells in the normal connective tissue, there were significant differences (F = 200.3128; q = 26.6762, 23.7341; P < 0.05). The shape and distribution of all the tryptase-positive cells (mast cells) in the pterygium tissues were similar to that of the cells with bFGF expression in the connective tissue. And the majority of bFGF-positive cells (87.54 +/- 3.60)% were similar to that of mast cells in the connective tissue. CONCLUSIONS: All infiltrating mast cells in pterygium have bFGF-positive expression. The bFGF expression is increased in the epithelium, blood vessels and infiltrating mast cells of the pterygium, and may contribute to the formation and progression of a pterygium.     

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.     

Growth factors in cultured pterygium fibroblasts: immunohistochemical and ELISA analysis

· Background: In order to study growth factors in the pathogenesis and recurrence of pterygium, we grew pterygium tissues in culture and compared fibroblasts from primary and from recurrent pterygia with reference to the fibroangiogenic growth factors basic fibroblast growth factor (b-FGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β) and tumor necrosis factor α (TNF-α). · Methods: We used indirect immunohistochemical procedures against human b-FGF, PDGF, TGF-β and TNF-α. As controls, we used cultured normal human conjunctival fibroblasts. A serum-free conditioned medium (CM) from confluent fibroblasts derived from primary and recurrent explants was assessed by enzyme-linked immunosorbent assay to determine the level of the above-mentioned growth factors. · Results: Immunoreactivity of b-FGF was stronger in recurrent than in primary pterygium fibroblasts. PDGF immunolabeling was stronger in primary than in recurrent pterygium fibroblasts. TGF-β and TNF-α immunolabeling was weak in both pterygia. All these growth factors were very sparse in normal conjunctival fibroblasts. Basic-FGF and TGF-β₁ were found in the CM from both primary and recurrent pterygium, while PDGF and TNF-α were not detectable. · Conclusion: The strong immunoreactivity and the release of b-FGF in cultured fibroblasts of recurrent pterygia suggest that fibroblasts may play an important role in the recurrence of pterygium. Received: 7 May 1997 Revised version received: 3 November 1997 Accepted: 8 December 1997     

Immunohistochemical localization of vascular endothelial growth factor, transforming growth factor alpha, and transforming growth factor beta1 in human corneas with neovascularization

PURPOSE: To analyze presence and distribution of vascular endothelial growth factor (VEGF), transforming growth factor (TGF)alpha, and TGFbeta1 in human corneas with neovascularization due to different corneal diseases. METHODS: Indirect immunohistochemistry for VEGF, TGFalpha, and TGFbeta1, was performed on paraffin-embedded corneas obtained by keratoplasty. Corneas from each of the four main groups of histopathologic diagnoses associated with corneal neovascularization were analyzed (scarring after keratitis, graft rejection/insufficiency, acute necrotizing keratitis, scarring after mechanical/chemical injury). Subclassification of inflammatory infiltrates was done using immunohistochemistry for CD3 (T-lymphocytes) and CD68 (macrophages). RESULTS: The analyzed angiogenic factors were detectable in corneas from all four histopathologic groups in a similar distribution; capillary endothelial cells, stromal and intravascular inflammatory cells (T-lymphocytes, macrophages), and basal corneal epithelial cells stained positive for the tested angiogenic factors. CONCLUSION: The angiogenic factors VEGF, TGFalpha, and TGFbeta1 are detectable in human corneas with neovascularization. Their distribution is quite uniform in different corneal diseases, resulting in corneal angiogenesis. An antiangiogenic therapy inhibiting corneal neovascularization by antagonizing angiogenic factors would have to counteract several angiogenic factors.